Mechanisms of aluminium neurotoxicity in oxidative stress-induced ...

Universidade de Santiago de Compostela
Facultade de Medicina
Departamento de Bioquímica e Bioloxía Molecular
Mechanisms of aluminium neurotoxicity in
oxidative stress-induced degenerative
processes in relation to Parkinson's disease
Tesis doctoral
Sofía Sánchez Iglesias
2009

Universidade de Santiago de Compostela
Facultade de Medicina
Departamento de Bioquímica e Bioloxía Molecular
Mechanisms of aluminium neurotoxicity in
oxidative stress-induced degenerative processes in
relation to Parkinson’s disease
Estudio de los mecanismos de neurotoxicidad del aluminio
en procesos degenerativos por estrés oxidativo
relacionados con la enfermedad de Parkinson
MEMORIA presentada por
SOFÍA SÁNCHEZ IGLESIAS
para optar al Grado de Doctor en Bioquímica
Santiago de Compostela
2009

Don Ramón Soto Otero y Doña Estefanía Méndez Álvarez, profesores titulares del
Departamento de Bioquímica y Biología Molecular de la Universidad de Santiago de
Compostela,
INFORMAN:
Que la presente memoria titulada Estudio de los Mecanismos de Neurotoxicidad
del Aluminio en Procesos Degenerativos por Estrés Oxidativo Relacionados con la
Enfermedad de Parkinson, presentada por Doña Sofía Sánchez Iglesias para optar al
grado de Doctor en Bioquímica, ha sido realizada bajo nuestra dirección en los
Laboratorios del Departamento de Bioquímica y Biología Molecular de esta
Universidad, y considerando que constituye trabajo de tesis y reúne los requisitos
necesarios para ser valorado por el tribunal correspondiente, autorizamos su
presentación al Consejo de Departamento correspondiente.
Y para que así conste y surta los efectos oportunos, firmamos el presente
informe en Santiago de Compostela a 15 de octubre de 2009.
El Director de la Tesis La Directora de la Tesis
Ramón Soto Otero
Profesor titular
Dpto. Bioquímica y Biología Molecular
El Doctorando
Sofía Sánchez Iglesias
Estefanía Méndez Álvarez
Profesora titular
Dpto. Bioquímica y Biología Molecular

Esta Tesis Doctoral forma parte de los Proyectos de Investigación:
“Estudio de los mecanismos de neurotoxicidad del aluminio en procesos degenerativos
por estrés oxidativo relacionados con la enfermedad de Parkinson” de la Xunta de
Galicia (PGIDIT03PXIB20804PR; 2003-2006).
“Búsqueda de un nuevo fármaco para el tratamiento de la enfermedad de Parkinson
con una doble acción farmacológica: inhibidora MAO-B y neuroprotectora frente al
estrés oxidativo inducido por dopamina” del Ministerio de Ciencia y Tecnología con la
contribución del Fondo Europeo de Desarrollo Regional (BFI2003-00493; 2004-2007).
“Búsqueda de un nuevo fármaco para el Parkinson: Inhibidor MAO-B y protector de los
efectos inducidos por la dopamina sobre la cadena de transporte electrónico
mitocondrial” del Ministerio de Ciencia e Innovación (SAF2007-66114; 2007-2010).
La realización de esta Tesis ha sido posible gracias a la concesión de las becas y ayudas
de la Xunta de Galicia y de Roche Research Foundation (Suiza).

Acknowledgements
The work of this thesis could not have been accomplished if it wasn’t for the people
below.
First and foremost I wish to express my sincere gratitude and appreciation to
my two excellent directors of the thesis, Prof. Ramón Soto-Otero and Prof. Estefanía
Méndez-Álvarez. It has been an honour to work with you and learn from you. Thank
you Ramón for given me the opportunity to work within the Neurochemistry research
group and for providing me an exciting research project. Your insightful advice,
guidance, and support make my Ph.D. experience productive and stimulating. Thank
you Fanny for your continuous encouragements, patience, and valuables feedbacks.
I wish to express special thanks to my colleague and friend Javier Iglesias-
González who shared with me a lot of ideas and provided a dynamic and stimulating
environment. Javi, thank you for your professional and personal support that made my
graduate experience more enjoyable. I don’t forget our valuable discussions and our
numerous memorable giggles; it has been a pleasure working together.
I wish to express my appreciation to all members past or present of the
Neurochemistry research group that I have had the pleasure to work with or alongside,
especially Dr. Álvaro Hermida-Ameijeiras for his guidance and assistance in the first
years of this work.
Grateful acknowledgements are also made to Prof. José Luis Labandeira-García
for constructive criticism and detailed review during the preparation of the
publications, Prof. Jannette Rodriguez Pallares for excellent advice, Dr. Ana Muñoz-
Patiño and Pablo Rey for immunohistochemistry studies, and Prof. Pilar Bermejo-
Berrera and Prof. María del Carmen Barciela-Alonso for electrothermal atomic
absorption spectrometry experiments.

Now life is not only science, I would therefore use this opportunity to thank my
best friend Rosi for her patience and care during good and tough times in the Ph.D.
pursuit. Always beside me.
Finally, I would like to dedicate this work to my family: my grandmother
Carmen and my grandfather Jesús always in my mind, my brother Jesús for his way of
being, and very especially my parents María del Carmen and Alfonso for their unending
love and support from ever. Thank you.

Abbreviations
AA ascorbic acid
AD Alzheimer’s disease
ADH aldehyde dehydrogenase
BBB blood-brain barrier
BG basal ganglia
BHT butylated hydroxytoluene
BioH4
5,6,7,8-tetrahydrobiopterin
BSA bovine serum albumine
CaM calmodulin
CAT catalase
CBD corticobasal degeneration
Cdk5 cyclin-dependent kinase 5
CMA chaperone mediated autophagy
CNS central nervous system
COMT catechol-O-methyl transferase
COX-2 cyclooxygenase-2
CSF cerebrospinal fluid
CT computarized tomography
CySH cysteine
DA 3-hydroxytyramine hydrochloride (dopamine)
DAergic dopaminergic
DAT dopamine transporter
DES dialysis encephalopathy syndrome
DLB dementia with Lewy bodies
DMV dorsal motor nucleus of the vagus
DOPAC dihydroxyphenylacetic acid
DOPAL 3,4-dihydroxyphenylacetaldehyde
eNOS endothelial nitric oxide synthase
EOPD early onset of Parkinson’s disease
ER endoplasmic reticulum
FeBAD fetal basis of adult disease
GABA �-aminobutyric acid
GPCR G-protein coupled receptor
GPe external globus pallidus
GPi internal globus pallidus
GPx glutathione peroxidase
GR glutathione reductase
GSH reduced glutathione

Abbreviations (continuation)
GSSH oxidized glutathione
H2O2
hydrogen peroxide
HVA homovanillic acid
IL interleukin
iNOS inducible nitric oxide synthase
IP3 inositol triphosphate
JP juvenile parkinsonism
KO knock-out
LB Lewy body
L-DOPA 3,4-dihydroxy-L-phenylalanine
LN Lewy neurite
LPS lipopolysaccharide
Maneb manganese ethylenebisadithiocarbamate
MAO monoamine oxidase
MAPK mitogen-activated protein kinase
MDA malonodialdehyde
MEF2 myocyte enhancer factor 2
MFB median forebrain bundle
mGLUR metabotropic glutamate receptor
Mit mitochondria
MPP + 1-methyl-4-phenyl pyridinium ion
MPPP 1-methyl-4-phenyl-4-propionoxy-piperidine
MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
MRI magnetic resonance imaging
MSA multiple system atrophy
mtDNA mitochondrial DNA
MTP mitochondrial transition pore
NBM nucleus basalis of Meynert
NINDS National Institute of Neurological Disorders and Stroke
NMDAR glutamate N-methyl-D-aspartate receptor
nNOS neuronal nitric oxide synthase
NO ● / NO nitric oxide
oxygen
O2
O2 ●─ superoxide anion
oatp organic anion transporting polypeptide
● OH hydroxyl radical
6-OHDA 6-hydroxydopamine
ONOO ●─ peroxynitrite

Abbreviations (continuation)
PCC protein carbonyl content
PCD programmed cell death
PD Parkinson’s disease
PET positron emission tomography
PI phosphatidylinositol
PIP2
phosphatidylinositol biphosphate
PI-PLC phosphatidylinositol-specific phospholipase C
PKC protein kinase C
PQ 1,1’-dimethyl-4,4’-bipyridinium, paraquat
Prx2 peroxiredoxin 2
PSP progressive supranuclear palsy
PTC protein thiol content
RNS reactive nitrogen species
ROOH hydroperoxides
ROS reactive oxygen species
SN substantia nigra
SNpc substantia nigra pars compacta
SNpr substantia nigra pars reticularis
SOD superoxide dismutase
SPECT single photon emission computarized tomography
STN subthalamic nucleus
t½
half life
TBA thiobarbituric acid
TBARS thiobarbituric acid reactive substances
TEP 1,1,3,3-tetraethoxypropane
TH tyrosine hydroxylase, tyrosine 3-monooxygenase
TH-ir tyrosine hydroxylase immunoreactivity
TNF-� tumour necrosis factor-�
TPN total parenteral nutrition
UPS ubiquitin-proteasome system
VTA ventral tegmental area
wt wild type
YOPD young-onset PD

List of figures
Introduction
Figure 1 An essay of the shaking palsy 1
Figure 2 The Parkinson’s complex 3
Figure 3 Dopamine synthesis 12
Figure 4 Dopamine catabolism 13
Figure 5 Dopamine pathways 14
Figure 6 Distribution of DA receptors in normal brain 14
Figure 7 The basal ganglia 15
Figure 8 Direct and indirect pathway in normal BG circuits 16
Figure 9 SNpc depigmentation 17
Figure 10 Human brain sections immunostained for DAT 18
Figure 11 Basal ganglia: normal and parkinsonian state 19
Figure 12 LB and LN in PD patients and localization of LB and LN 22
Figure 13 Six stages Braak’s system 24
Figure 14 Immunosections of stages 3-6 of Braak’s system 24
Figure 15 MPTP metabolism 28
Figure 16 The multiple hit model of PD 31
Figure 17 Pathologic mechanisms in the etiology of PD 32
Figure 18 The ubiquitin-proteasome system 34
Figure 19 Generation of ROS and RNS in SNpc neuron 43
Figure 20 Interaction between microglia and DAergic neurons 47
Figure 21 Soluble aluminium ions as a function of solution pH 54
Figure 22 Effects of aluminium and iron on the membrane peroxidation 68
Figure 23 PIP2 hydrolisis altered by aluminium 69
Chapter 1
Figure 1 Changes in TBARS levels in the ipsilateral and contralateral side of
both striatum and ventral midbrain after stereotaxic, unilateral,
intrastriatal injection of 6-OHDA to rats
Figure 2 Changes in PCC in the ipsilateral and contralateral side of both
striatum and ventral midbrain after stereotaxic, unilateral,
intrastriatal injection of 6-OHDA to rats
Figure 3 Changes in PTC in the ipsilateral and contralateral side of both
striatum and ventral midbrain after stereotaxic, unilateral,
intrastriatal injection of 6-OHDA to rats
Chapter 2
Figure 1 Levels of aluminium in the different areas of the rat brain 113
97
98
99

List of figures (continuation)
Chapter 3
Figure 1 Levels of TBARS, protein carbonyls, and protein thiols in different
brain areas of rats i.p. treated with saline or aluminium chloride
for ten days
Figure 2 Enzyme activity of SOD, GPx, and CAT in different brain areas of
rats i.p. treated with saline or aluminium chloride for ten days
Figure 3 Microphotographs showing changes in the striatal TH-ir one week
post-lesion in rats injected intraventricularly
Figure 4 Density of striatal DAergic terminals estimated as optical density,
one week post-surgery in the different experimental groups.
Figure 5 Levels of TBARS, protein carbonyls, and protein thiols in both
ventral midbrain and striatum of different groups of rats
Figure 6 In vitro effects of aluminium on TBARS formation, protein
carbonyl content, and protein thiol content induced by the
autoxidation of 6-OHDA in mitochondrial preparations from rat
brain
138-139
140-141
142
143
144-145
146-147

List of tables
Introduction
Table 1 Differential diagnostic in parkinsonian disorders 9
Table 2 UK Parkinson’s Disease Society Brain Bank’s diagnostic criteria for
the diagnosis of probable PD
10
Table 3 National Institute of Neurological Disorders (NINDS) diagnostic
criteria for PD
11
Table 4 Gene loci associated with PD 26
Table 5 Lipid composition of normal adult human brain 65
Chapter 2
Table 1 Graphite furnace programme for aluminium determination by
ETAAS
Chapter 3
Table 1 In vitro effects of the presence of aluminium on the enzyme
activities of SOD, GPx, CAT, and MAO
113
137

TABLE OF CONTENTS
INTRODUCTION ....................................................................................... 1
PARKINSON’S DISEASE ...................................................................................................... 1
An overview ............................................................................................................................................ 1
Prevalence and incidence of PD .............................................................................................................. 2
Clinical characteristics of PD ................................................................................................................... 3
Resting tremor ....................................................................................................................................................................... 4
Rigidity ................................................................................................................................................................................... 4
Bradykinesia ........................................................................................................................................................................... 5
Postural instability ................................................................................................................................................................. 5
Progression of motor symptoms ............................................................................................................................................ 6
Non-motor symptoms ............................................................................................................................................................ 6
Diagnosis of PD ....................................................................................................................................... 7
Neurochemical and neuropathological features of PD ......................................................................... 12
Dopamine ............................................................................................................................................................................ 12
The basal ganglia .................................................................................................................................................................. 15
Degeneration of all DAergic pathways and other systems ................................................................................................... 20
Lewy bodies, Lewy neurites and pale bodies ....................................................................................................................... 21
The six-stage system: a novel neuropathological concept of neurodegeneration ............................................................... 23
Etiology and pathogenesis of PD .......................................................................................................... 25
Genetics of PD ...................................................................................................................................................................... 25
Ageing .................................................................................................................................................................................. 27
Role of environmental exposure .......................................................................................................................................... 27
An example of environmental toxin: MPTP................................................................................................................... 27
Other environmental factors ........................................................................................................................................ 29
Metals ........................................................................................................................................................................... 29
Potential theories ................................................................................................................................................................ 30
The “dual-hit” hypothesis ............................................................................................................................................. 30
The “multiple hit or multihit” hypothesis ...................................................................................................................... 30
The connection between PD and the FeBAD hypothesis .............................................................................................. 31
Parkinson diseases: a syndrome of different disorders ................................................................................................. 32
Misfolding and aggregation of proteins ............................................................................................................................... 33
Oxidative stress .................................................................................................................................................................... 38
Transition metals: the example of iron ......................................................................................................................... 40
DA metabolism as a source of ROS ............................................................................................................................... 41
Nitric oxide .................................................................................................................................................................... 42
Contribution of oxidative and nitrosative stress to PD pathogenesis ............................................................................ 43
Excitotoxicity ........................................................................................................................................................................ 45
Cell death pathways ............................................................................................................................................................. 48
Experimental models of PD ................................................................................................................... 49
Toxin-induced models .......................................................................................................................................................... 49
6-OHDA ......................................................................................................................................................................... 49
MPTP ............................................................................................................................................................................. 50
Rotenone, paraquat and maneb ................................................................................................................................... 50
Inflammation-based models ................................................................................................................................................ 51
Gene-based models ............................................................................................................................................................. 51
ALUMINIUM .................................................................................................................... 53
General features ................................................................................................................................... 53
Aluminium speciation .......................................................................................................................................................... 53
Sources of aluminium exposure ............................................................................................................ 54
Environmental exposure ...................................................................................................................................................... 54
Dietary exposure .................................................................................................................................................................. 55
Iatrogenic exposure ............................................................................................................................................................. 56
Occupational exposure ........................................................................................................................................................ 56
Aluminium toxicokinetics ...................................................................................................................... 57
Absorption of aluminium ..................................................................................................................................................... 57
Oral absorption .................................................................................................................................................................... 57

Intranasal absorption ........................................................................................................................................................... 58
Dermal absorption ............................................................................................................................................................... 59
Distribution of aluminium in the body ................................................................................................................................. 60
Excretion .............................................................................................................................................................................. 61
Elimination rate ................................................................................................................................................................... 61
Aluminium influx and efflux into the brain ........................................................................................... 62
Putative mechanisms of aluminium neurotoxicity ................................................................................ 63
Aluminium and oxidative stress ........................................................................................................................................... 64
The brain: a target for aluminium ................................................................................................................................. 64
Aluminium catalyses iron-driven biological oxidations ................................................................................................. 65
Aluminium stimulates superoxide-/non-iron-driven biological oxidations ................................................................... 65
Aluminium-superoxide complex theory ........................................................................................................................ 66
Aluminium inhibits the antioxidant enzymes and others .............................................................................................. 66
Aluminium as a cell membrane menace .............................................................................................................................. 67
Aluminium affects cell signaling ........................................................................................................................................... 68
AIMS OF THE PRESENT THESIS ............................................................... 75
OBJETIVOS DE LA PRESENTE TESIS ......................................................... 79
CHAPTER 1 Time-course of brain oxidative damage caused
by intrastriatal administration of 6-hydroxydopamine in a rat
model of Parkinson’s disease ................................................................ 83
ABSTRACT ....................................................................................................................... 85
INTRODUCTION .............................................................................................................. 86
MATERIALS AND METHODS ............................................................................................ 88
Chemicals .............................................................................................................................................. 88
Animal treatment.................................................................................................................................. 88
Brain samples ........................................................................................................................................ 89
Determination of TBARS ....................................................................................................................... 89
Determination of protein carbonyl content (PCC) ................................................................................. 89
Determination of protein thiol content (PTC) ....................................................................................... 90
Statistical analysis ................................................................................................................................. 91
RESULTS .......................................................................................................................... 92
Effects of 6-OHDA administration on TBARS concentration ................................................................. 92
Effects of 6-OHDA administration on PCC ............................................................................................. 92
Effects of 6-OHDA administration on PTC ............................................................................................. 93
DISCUSSION .................................................................................................................... 94
CHAPTER 2 Analysis of brain regional distribution of
aluminium in rats via oral and intraperitoneal administration ............. 103
ABSTRACT ..................................................................................................................... 105
INTRODUCTION ............................................................................................................ 106
MATERIALS AND METHODS .......................................................................................... 107
Chemicals ............................................................................................................................................ 107
Animal treatment................................................................................................................................ 107
Brain samples ...................................................................................................................................... 108
Electrothermal atomic absorption spectrometry (ETAAS) .................................................................. 108
Statistical analysis ............................................................................................................................... 109
RESULTS ........................................................................................................................ 110
Aluminium distribution in the brain after oral and i.p. administration ............................................... 110
DISCUSSION .................................................................................................................. 111

CHAPTER 3 Brain oxidative stress and selective behaviour of
aluminium in specific areas of rat brain: potential effects in a 6-
OHDA-induced model of Parkinson’s disease ...................................... 117
ABSTRACT ..................................................................................................................... 119
INTRODUCTION ............................................................................................................ 120
MATERIALS AND METHODS .......................................................................................... 122
Chemicals ............................................................................................................................................ 122
Animal treatment................................................................................................................................ 122
Brain samples ...................................................................................................................................... 123
Preparation of brain mitochondria ..................................................................................................... 124
Determination of TBARS, PCC, and PTC .............................................................................................. 124
Measurement of SOD activity ............................................................................................................. 125
Measurement of GPx activity .............................................................................................................. 125
Measurement of CAT activity .............................................................................................................. 126
Determination of MAO activity ........................................................................................................... 126
Immunohistochemistry ....................................................................................................................... 126
Statistical analysis ............................................................................................................................... 127
RESULTS ........................................................................................................................ 128
In vivo effects of aluminium administration on brain lipid peroxidation and protein
oxidation ....................................................................................................................................... 128
In vivo effects of aluminium administration on the brain activity of different antioxidant
enzymes ......................................................................................................................................... 128
Effects of aluminium administration on the degeneration of DA terminals in the
striatum ......................................................................................................................................... 129
Effects of aluminium administration on brain lipid peroxidation and protein oxidation in
6-OHDA-lesioned rats and controls ............................................................................................... 130
In vitro effects of aluminium on the lipid peroxidation and protein oxidation induced by
6-OHDA autoxidation in brain mitochondrial preparations .......................................................... 131
In vitro effects of aluminium on the enzyme activities of GPx, SOD, CAT, and MAO .......................... 131
DISCUSSION .................................................................................................................. 132
SUMMARY .......................................................................................... 151
CONCLUSIONS ..................................................................................... 157
RESUMEN ............................................................................................ 163
CONCLUSIONES ................................................................................... 169
PUBLICATIONS AS A DIRECT RESULT FROM THIS THESIS ............................................. 175
PUBLICATIONS ARISING FROM COLLABORATIVE WORK DURING THIS THESIS ........... 176
REFERENCES ........................................................................................ 179

INTRODUCTION

INTRODUCTION
PARKINSON’S DISEASE
An overview
INTRODUCTION
Parkinson‟s disease (PD) was first formally described in modern times in a
famous monograph entitled “An essay on the shaking palsy” published in 1817 by an
English physician called James Parkinson (Parkinson 1817; Figure 1). In this report, the
disease was initially defined as shaking palsy (paralysis agitans) and the syndromes
observed were described as “involuntary tremulous motion, with lessened muscular
power, in parts not in action and even when supported; with a propensity to bend the
trunk forward, and to pass from a walking to a running pace: the senses and intellects
being uninjured”.
Figure 1: An essay of the shaking palsy (Parkinson 1817)
1

INTRODUCTION
2
PD, also called idiopathic PD or primary parkinsonism, is the most common
movement disorder besides essential tremor and the second most common age-related
neurodegenerative disease after Alzheimer's disease (AD) (Fahn and Przedborski 2000,
Tanner and Aston 2000, Mayeux 2003, Fahn and Sulzer 2004). Moreover, PD is the
main cause of chronic progressive Parkinsonism accounting for ~80% of cases.
Parkinsonism, also known as Parkinson‟s syndrome, atypical Parkinson‟s or secondary
Parkinson‟s, is a term which refers to the neurological syndrome characterised by three
cardinal motor symptoms: bradykinesia, rigidity and tremor.
Prevalence and incidence of PD
More than 4.5 million people worldwide were affected by PD in 2007. Due to
increasing life expectancy of the general population and multiplication of effective
therapies, the number of individuals over 50 years of age with PD was expected to
double to around 9 million patients in the world‟s 10 most populous nations between
2007 and the year 2030 (Dorsey et al. 2007). At the time of writing, PD is affecting
approximately 6 million people worldwide.
In regards to disease epidemiology, the mean age onset of sporadic PD (also
known as idiopathic PD) is 55 years, but is now thought to be in the early-to-mid 60s
(Inzelberg et al. 2002). Early onset of PD (EOPD) is rare, about 4% of patients develop
clinical signs of the disease before 50 years (Van Den Eeden et al. 2003). Within
EOPD, there appear to be two groups namely: young-onset PD (YOPD), with onset
between 21 and 40 years, and juvenile Parkinsonism (JP), with onset at

INTRODUCTION
PD is found in all ethnic groups, but with geographical differences in
prevalence. Men seem to be slightly more prone to the disorder (Baldereschi et al. 2000,
Lai et al. 2003, Benito-León et al. 2004). Incidence rates range from 8.6 to 19.0 per
100,000 inhabitants in the USA and Europe when strict diagnostic criteria of PD are
applied (Twelves et al. 2003). Direct comparison of prevalence and incidence estimates
are difficult and complicated because of methodological differences between studies (de
Rijk et al. 1995, de Rijk et al. 1997b, Benito-León et al. 2003). Stricter diagnosis
criteria, record-based studies or studies done in clinical settings yielded lower incidence
or prevalence rates than door-to-door surveys or studies with in person examination
(Schoenberg et al. 1988, Morgante et al. 1992, Tison et al. 1994, de Rijk et al. 1995,
Claveria et al. 2002, Benito-León et al. 2003, Benito-León et al. 2004, de Lau et al.
2004).
Clinical characteristics of PD
PD exhibits great variability in clinical presentation as it presents with a range of
motor, psychiatric, cognitive, sensory and autonomic symptoms. The three cardinal
clinical symptoms of PD include rest tremor, rigidity, and bradykinesia. Often presented
as “preclinical PD”, motor and non-motor features that may precede the cardinal motor
symptoms by long periods of time should be taken more critically into consideration as
they are part of the disease process (Figure 2; Langston 2006).
Figure 2: The Parkinson’s complex (Langston 2006)
3

INTRODUCTION
Resting tremor
4
Resting tremor is the most commonly recognized feature of PD and the first
motor symptom at disease onset in approximately 75% of PD patients (Jankovic et al.
1990, Hughes et al. 1993, Schrag et al. 2007). However, 25 % of patients with PD never
develop tremor (Hughes et al. 1993). Rest tremor has a frequency of 4–6 Hz and
classically resembles pill-rolling (descriptive of the rhythmic alternating motion of the
forefinger and thumb). This motor manifestation/symptom is usually a supination-
pronation tremor, asymmetric, and most prominent in the distal upper extremity as
resting foot tremor is much less common than hand tremor as a presenting sign. Tremor
at rest is lost during sleep and markedly decreased or abolished with voluntary
movement (so typically it does not impair activities of daily living). It should be noted
that PD tremor can also be seen to have a postural component. Nevertheless, rest tremor
is worsened by excitement, anxiety, apprehension, contralateral motor activity, and is
most easily seen when the patient is asked to ambulate with arms hanging at their sides.
Observation of the patient during ambulation reveals much about the patient‟s
symptoms, including rigidity and bradykinesia.
Rigidity
Rigidity is the raised resistance appreciated during the passive movement of the
limb about a joint (often of a “lead pipe” quality). A “ratchety” sensation can be noted,
indicating the contraction and relaxation of the muscles underlying the tremor. It can
have a cogwheel quality even without tremor, but is usually more pronounced in the
more tremulous limb. Rigidity is enhanced by contralateral motor activity or mental task
performance. Bradykinesia and rigidity are less common then rest tremor but are still
frequently seen at onset of PD.

Bradykinesia
INTRODUCTION
Bradykinesia, the most disabling symptom of PD, is easily observed. It results in
the slowness in movement and as a consequence significantly impairs quality of life as
everyday tasks performance is delayed (eating, dressing, arising from a chair, getting in
and out of a car or bed). It initially manifests by difficulties with fine motor tasks
(reduced arm swing in walking, speed of handwriting, doing up buttons) and often by
decreased blink rate. Various tests can be performed to assess limb bradykinesia, such
as fist closing and opening, finger and foot tapping, alternating forearm pronation and
supination (Martinez-Martin et al. 1994). Additional motor symptoms are also
noticeable as a result of hypokinesia (reduction in movement amplitude) and akinesia
(absence of normal unconscious movements) in combination with rigidity, such as
micrographia (decreased size of handwriting), reduced stride length in walking,
hypophonia (diminished voice volume), dysarthia (slurred speech), hypomimia (facial
masking of normal facial expression), and dysphagia (problems swallowing), and
sialorrhea (drooling, failure to swallow without thinking about it).
Postural instability
Postural instability or the impairment of the righting reflex, sometimes judged a
cardinal feature of PD, is non-specific and is less responsive to treatment compared with
the others cardinal motor symptoms. Postural instability or gait disturbances refer to the
gradual development of poor balance, leading to a high risk for falls and injuries, and
sometimes, confinement to a wheelchair. It can be test with the retropulsion test (pulling
the patient backward to check for balance recovery). This feature, in combination with
rigidity and bradykinesia, results in important disability to the patient. Gait becomes
slower, with shuffling, and turning is en bloc (as opposed to pivoting). Freezing is
characterised by the inability to begin a voluntary movement such as walking or striking
gait hesitation on turning or arriving at a real or perceived obstacle. Postural instability
and gait abnormality are not typical at onset of PD. They are usually absent in early
5

INTRODUCTION
disease, especially in younger patients, but become a common complication of
advanced PD.
Progression of motor symptoms
6
The motor features of tremor and bradykinesia are usually localized to the upper
extremities (Uitti et al. 2005). Within one to three years, they extend to the other
ipsilateral limb and affect the contralateral limbs in three to eight years (Poewe and
Wenning 1998). Despite the fact that PD is almost always a bilateral disease, this
asymmetrical pattern remains so throughout most of the course of the illness, even in
advances stages (Hughes et al. 2001). As tremor gravity is rather stable over time,
whereas severity of bradykinesia and rigidity evolve similarly, some authors
hypothesized the existence of different underlying pathophysiological processes (Louis
et al. 1999).
Non-motor symptoms
Although motor features define PD, various non-motor symptoms, equally
significant and potentially disabling, are typically seen. They include autonomic
dysfunction (including orthostatic hypotension, bowel and bladder difficulties,
abdominal bloating, constipation, urinary urgency and dysfunction, flushing, excessive
sweating, temperature dysregulation, and impotence/sexual dysfunction; Jost 2003,
Poewe 2008), sensory symptoms (eg. pain, paresthesiae; Quinn et al. 1986), sleep
disturbances (REM sleep behaviour disorder, periodic limb movements, etc.; Stacy
2002), psychiatric changes (depression, hallucinations, anxiety symptoms), and
cognitive dysfunction (memory deficits, bradyphrenia, executive function difficulty,
dementia; Levin and Katzen 2005, Rippon and Marder 2005, Caviness et al. 2007).
Depression is common in PD, as it develops in about 50% of individuals with PD
(McDonald et al. 2003). Dementia is significantly more frequent in PD, especially in
older patients, ranging from 40-80% of patients (Cedarbaum and McDowell 1987, Emre
2003, Poewe 2008). Non-motor symptoms, present with considerable variability, may

INTRODUCTION
predate the occurrence of motor symptoms and should be recognized and treated as
soon as possible to maximize functionality as well as quality of life.
Diagnosis of PD
The diagnosis of PD is essentially made on clinical criteria. Whereas these
clinical criteria lead, at best, to a diagnosis of probable PD, post-mortem confirmation is
required to establish a definite diagnosis of PD (Hughes et al. 1992a, Koller 1992).
Historically, the identification of Lewy bodies (LB) on autopsy was considered the
criterion standard for diagnosis (Gibb and Lees 1988).
A diagnosis of primary parkinsonism requires at least the presence of two of the
primary cardinal motor symptoms (resting tremor, bradykinesia, rigidity) and exclusion
of potential causes of secondary parkinsonism. The following features are considered by
most experts as essential and the most important to discriminate PD from other
diagnoses: a unilateral or asymmetrical onset of motor symptoms and a consistent
response of the symptoms to an adequate dose of a dopaminergic (DAergic) agent such
as levodopa given for a sufficient period. If it is not the case, an alternative diagnosis
should be assumed. The differential diagnosis of PD includes normal ageing, essential
tremor, and other parkinsonian syndromes such as progressive supranuclear palsy
(PSP), multiple system atrophy (MSA) and vascular parkinsonism, amongst others
(Stoessl and Rivest 1999; Table 1).
The diagnosis of PD is difficult especially on early-stages of the disease, because
atypical features may be absent or insignificant and there is no information on
levodopa-responsiveness. A long-term follow-up of patients is needed to revise
diagnoses on the basis of information on disease progression, physical examination,
improvement/emergence of other symptoms and responsiveness to DAergic treatment.
Post-mortem studies demonstrated misdiagnosis in up to 25% of patients diagnosed
with PD by general neurologists as their brain reveal no pathological sign of the disease
(Rajput et al. 1991, Hughes et al. 1992a). It is worth noting that accuracy of PD
7

INTRODUCTION
diagnosis was substantially higher in patients diagnosed by experts in movement
disorders (Hughes et al. 2002).
8
In order to improve diagnostic accuracy, revised and more stringent diagnostic
criteria were developed by the UK Parkinson‟s Disease Society Brain Bank (Table 2)
(Hughes et al. 1992b) and the National Institute of Neurological Disorders and Stroke
(NINDS) (Table 3) (Gelb et al. 1999). Nevertheless they still remain controversial as
clinicopathological studies suggest that about 10-20% of PD cases remain as false
positives (Hughes et al. 2001, Litvan et al. 2003).
The problem is that a reliable and easily valid diagnostic test for PD is not yet
available. When there is doubt about the diagnosis or when the patient‟s history or
clinical findings are atypical, sophisticated neuroimaging techniques may be useful to
differentiate PD from other diseases (Piccini and Brooks 2006). Conventional brain
imaging, such as magnetic resonance imaging (MRI) or computarized tomography
(CT), are helpful when normal pressure hydrocephalus or vascular parkinsonism are
suspected (Demirkiran et al. 2001). Sophisticated imaging techniques of the DA
pathways such as positron emission tomography (PET) or single photon emission
computarized tomography (SPECT), can make a distinction of PD from essential
tremor, dystonic tremor, neuroleptic-induced parkinsonism and psychogenic
parkinsonism/postsynaptic forms of parkinsonism in specialised settings (Vingerhoets et
al. 1994, Lee et al. 2000, Winogrodzka et al. 2001, Piccini and Brooks 2006). Although
these promising techniques have become more widely available and easier to use, much
controversy exists (because of the lack of universally accepted neuropathological
criteria for PD) (Clarke and Gutman 2002, Marek et al. 2002, Litvan et al. 2003).
Usefulness for population epidemiological studies is still limited as sensitivity and
resolution of these neuroimaging techniques need to be improved and refined.

Table 1: Differential diagnostic in parkinsonian disorders (Alves et al. 2008)
Parkinson’s disease a
Idioptahic
Familial
Symptomatic parkinsonism Drug-induced
Neuroleptics, antidepressants, lithium
Antiemetics
Antihypertensive agents, antiarrhythmics
Vascular disease
Intoxication (MPTP, rotenone, others)
Traumatic
Post-infectious
Neoplasm
Normal pressure hydrocephalus
Parkinsonism due to other
neurodegenerative disorders
a synucleinopathy; b tauopathy
Atypical parkinsonism
Multiple system atrophy (MSA) a
Progressive supranuclear palsy (PSP) b
Corticobasal degeneration (CBD) b
Dementia with Lewy bodies (DLB) a
Alzheimer’s disease b (AD)
Others
INTRODUCTION
9

INTRODUCTION
Table 2: UK Parkinson’s Disease Society Brain Bank’s diagnostic criteria for the
diagnosis of probable PD (Jankovic 2008, Davie 2008)
Step 1: Diagnosis of a parkinsonian syndrome
10
Bradykinesia, and at least one of the following criteria:
Muscular rigidity
4-6 Hz rest tremor
Postural instability (not caused by primary visual, vestibular, cerebellar
or propioceptive dysfunction)
Step 2: Exclusion of other causes of parkinsonism
i History of repeated strokes with stepwise progression of parkinsonian features
ii History of repeated head injury
iii History of definite encephalitis
iv Oculogyric crises
v Neuroleptic treatment at the onset of symptoms
vi More than one affected relative
vii Sustained remission
viii Strictly unilateral features after 3 years
ix Supranuclear gaze palsy
x Cerebellar signs
xi Early severe autonomic involvement
xii Early severe dementia with disturbances of memory, language and praxis
xiii Babinski’s sign
xiv Presence of cerebral tumour or communicating hydrocephalus on CT scan
xv Negative response to large doses of levodopa (if malabsorption excluded)
Step 3: Supportive (prospective) criteria for PD (at least three or more required for
diagnosis of definite PD)
i Unilateral onset
ii Rest tremor present
iii Progressive disorder
iv Persistent asymmetry affecting side of onset most
v Excellent response (70–100%) to levodopa
vi Severe levodopa-induced chorea (dyskinesia)
vii Levodopa response for 5 years or more
viii Clinical course of 10 years or more

INTRODUCTION
Table 3: National Institute of Neurological Disorders (NINDS) diagnostic
criteria for PD (Jankovic 2008)
Group A features (characteristic of PD)
Resting tremor
Bradykinesia
Rigidity
Asymmetric onset
Group B features (suggestive of alternative diagnoses)
Features unusual early in the clinical course
Prominent postural instability in the first 3 years after symptom onset
Freezing phenomenon in the first 3 years
Hallucinations unrelated to medications in the first 3 years
Dementia preceding motor symptoms or in the first year
Supranuclear gaze palsy (other than restriction of upward gaze) or slowing of vertical
saccades
Severe, symptomatic dysautonomia unrelated to medications
Documentation of condition known to produce parkinsonism and plausibly connected
to the patient’s symptoms (such as suitably located focal brain lesions or neuroleptic
use within the past 6 months)
Criteria for definite PD
All criteria for probable Parkinson’s are met and
Histopathological confirmation of the diagnosis is obtained at autopsy
Criteria for probable PD
At least three of the four features in group A are present and
None of the features in group B is present (note: symptom duration >3 years is
necessary to meet this requirement) and
Substantial and sustained response to levodopa or a dopamine agonist has been
documented
Criteria for possible PD
At least two of the four features in group A are present; at least one of these is tremor
or bradykinesia and
Either none of the features in group B is present or symptoms have been present (3
years and none of the features in group B is present and
Either substantial and sustained response to levodopa or a dopamine agonist has
been documented or the patient has not had an adequate trial of levodopa or a
dopamine agonist
11

INTRODUCTION
Neurochemical and neuropathological features of PD
Dopamine
12
Dopamine (DA) plays important roles in control movement, motivation and
reward, behaviour and cognition, mood, sleep, attention and learning. This biogenic
monoamine is synthesized in the catecholaminergic neurons from L-tyrosine. First,
tyrosine hydroxylase (tyrosine 3-monooxygenase, TH) converts L-tyrosine to 3,4-
dihydroxy-L-phenylalanine (L-DOPA). TH requires 5,6,7,8-tetrahydrobiopterin (BioH4)
as a coenzyme (Agid 1991). The second step is catalyzed by the DOPA decarboxylase
(aromatic L-amino acid decarboxylase, AADC), L-DOPA is decarboxylated to DA
(Figure 3). In some neurones, beta-hydroxylase processes DA into noradrenaline, which
is metabolized to adrenaline by phenylethanolamine N-methyltransferase. DA is
degraded by catechol-O-methyl transferase (COMT), aldehyde dehydrogenase (ADH)
and monoamine oxidase (MAO) (Figure 4). MAO-A and -B also degrade other
monoamines including serotonin, noradrenaline, and adrenaline.
Figure 3: Dopamine synthesis (Méndez-Álvarez and Soto-Otero 2004)
L-tyrosine
tyrosine
hydroxylase
HO
COO
HO HO
H 2N
HN
N
O
H
N
N
H
NH 3
O 2
CH CH CH3 OH OH
5,6,7,8-tetrahydrobiopterin
(BioH 4)
NAD
dihydrobiopterin
reductase
NADH + H
HN
H 2O
NH 3
COO
3,4-dihydroxy-L-phenylalanine
(L-DOPA)
HN
N
O
H
N
N
CH CH CH3 OH OH
quinonoid dihydrobiopterin
(BioH 2)
CO 2
aromatic L-amino acid
decarboxylase
(pyridoxal phosphate)
HO NH 3
HO
dopamine
(DA)

Figure 4: Dopamine catabolism (Méndez-Álvarez and Soto-Otero 2004)
HO NH 3
HO
dopamine
(DA)
O 2
INTRODUCTION
H2O2 HO
CHO H2O + NAD
monoamine oxidase
HO
3,4-dihydroxyphenylacetaldehyde
(DOPAL)
aldehyde dehydrogenase
NADH + H
CH 3
H 2O
O
HO
NH 4
catechol-O-methyltransferase
COO
homovanillic acid
(HVA)
S-adenosyl-homocysteine
(SAH)
S
CH 2
CH2 CH NH3 COO
CH 2
OH
O
N
N
OH
NH 2
N
N
HO
HO
COO
3,4-dihydroxyphenylacetic acid
(DOPAC)
CH3 S
CH2 CH2 CH NH3 COO
CH 2
OH
O
N
N
OH
NH 2
N
S-adenosyl-methionine
(SAM)
DA is a neurotransmitter widely distributed throughout the whole brain, but
mostly localized in the striatum. It is produced by mesencephalic neurons of the
substantia nigra (SN) and ventral tegmental area (VTA), and by hypothalamic neurons
of the arcuate and periventricular nuclei (Cooper et al. 1996). DAergic neurons located
in these areas project their axons to the striatum (nigrostriatal pathway), neocortex
(mesocortical pathway), limbic system (mesolimbic pathway) and hypophysis
(tuberoinfundibular pathway) (Figure 5). DA receptor family consists of five members
divided into two subfamilies: the D1-like family comprises D1 and D5 receptors,
whereas the D2-like family includes D2, D3 and D4 receptors (Cooper et al. 1996,
Jackson and Westlind-Danielsson 1994). D1 receptors are widely expressed in the brain,
whereas D2 receptors are principally seen in the striatum and nucleus accumbens
(Figure 6). As we will discuss later, DA plays a central role in the pathology of PD as
this neurotransmitter is crucial for basal ganglia regulation.
N
13

INTRODUCTION
Figure 5: Dopamine pathways (CNSforum www.cnsforum.com)
Figure 6: Distribution of DA receptors in normal brain (CNSforum www.cnsforum.com)
14

The basal ganglia
INTRODUCTION
The basal ganglia (BG) constitute mainly five subcortical nuclei located deep in
the hemispheres: the substantia nigra with its pars compacta (SNpc) and pars reticularis
(SNpr), the striatum (caudate nucleus and putamen), the internal and external segments
of globus pallidus (GPi and GPe), and the subthalamic nucleus (STN) (Figure 7). The
BG form a complex network of anatomically and functionally segregated loops
involved in the regulation of emotional and cognitive functions and particularly in the
control of cortically initiated motor activity, which if disturbed leads to some form of
movement disorder.
Figure 7: The basal ganglia (Society for Neuroscience www.sfn.org)
These parallel loops have been functionally subdivided into motor, oculomotor,
limbic, association and orbitofrontal loops (Alexander et al. 1986, Alexander and
Crutcher 1990, Middleton and Strick 2000). The striatum is the primary input structure
of BG as it receives excitatory glutamatergic signals from many areas of the sensory
motor cortex and thalamus, as well as a dense innervation from midbrain DA neurons.
The most abundant striatal neurons are striatonigral and striatopallidal neurons
15

INTRODUCTION
accounting each for approximately 49% (Rymar et al. 2004). The firsts exhibit high
expression of D1 receptors and send axons to the main output nuclei of BG for
movement control, GPi and SNpr, while the latters contain high expression of D2
receptors and innervate directly GPe.
16
In the classical model of BG circuitry, the motor loop integrates two distinct
routes identified as “direct” and “indirect” pathways which proceed from the striatum,
especially from the putamen, and that act in opposing ways to control movement (Albin
et al. 1989, Alexander and Crutcher 1990, DeLong 1990). The “direct” pathway
facilitates voluntary movement. Striatonigral neurons project to GABAergic neurons in
GPi and SNpr, which in turn send axons to the motor nuclei of the thalamus. As the
neurotransmitter of both projections (striatum to GPi/Snpr, and Gpi/SNpr to thalamus)
is �-aminobutyric acid (GABA), which is inhibitory, the net effect of the direct pathway
activity is a disinhibition of excitatory thalamocortical projections, resulting in
activation of cortical premotor circuits and facilitation of movement. On the other hand,
the “indirect” pathway originates in striatopallidal neurons that project to GPe providing
an inhibitory effect. This region in turn sends axons to the STN (disinhibition) which
provides outflow to GPi and SNpr (excitation) enhancing the inhibitory effect of the BG
output nuclei on thalamocortical neurons, thus reducing movement (Figure 8).
Figure 8: Direct and indirect pathway in normal BG circuits, sagittal view of a mouse brain (Kreitzer
and Malenka 2008)

The death of SNpc DAergic neurons
INTRODUCTION
The main pathological biochemical hallmark of PD is the death of DAergic
neurons residing in the ventral midbrain nucleus, called the SNpc. The cell bodies of the
SNpc neurons project primarily to the putamen and upward to the caudate nucleus
composing the nigrostriatal pathway. The loss of these neurons which contain
considerable amounts of neuromelanin leads to the neuropathological finding of SNpc
depigmentation (Figure 9; Marsden 1983). Consequently the death of nigrostriatal
DAergic neurones results in a loss of the neurotransmitter DA in the striatum, more
precisely in the dorsolateral putamen where the depletion of DA is most pronounced
(Bernheimer et al. 1973). This leads to the main motor features of PD as the striatum
integrates signals from the motor cortex and regulates neural circuits within the BG to
control movement. Furthermore this agrees well with the correlation between the pattern
of SNpc cell loss and the expression levels of the DA transporter (DAT) mRNA (Figure
10; Uhl et al. 1994).
Figure 9: SNpc depigmentation, appearance of normal (left) and Parkinsonian (right) human midbrain
(National Center for Biotechnology Information www.ncbi.nlm.nih.org)
At the onset of symptoms, approximately 60% of the SNpc DAergic neurones
and 80% of DA content in putamen have already been lost (Fearnley and Lees 1991,
Deumens et al. 2002). This suggests that neuronal death in PD may result from a “dying
back” process as the striatal DAergic nerve terminals seem to be the primary target of
neurodegeneration before subsequent cell body effects (Dauer and Przedborski 2003).
DA depletion causes a cascade of functional changes in BG circuitry as it leads to
17

INTRODUCTION
reduced activity in the “direct” pathway and increased activity in the “indirect”
pathway, resulting in excessive GPi/SNpr inhibitory output to the thalamus (Figure 11).
These changes lead to a hypokinetic disorder characterized by inhibition of movement
(Kreitzer and Malenka 2008).
Figure 10: Human brain sections immunostained for DAT of normal person (A) and PD patient (B)
(Betarbet et al. 2002)
18
Neuroimaging and pathological studies suggest that there is a long latency
between nigral degeneration begin and symptoms onset. The presymptomatic latent
phase was estimated to be approximately 6 years in idiopathic PD and longer in familial
PD (Fearnley et al. 1991, Morrish et al. 1996, Hilker et al. 2005). This long
asymptomatic period indicates that compensatory mechanisms are able to deal with
progressively lower DA levels in order to preserve apparent normal BG function. These
compensatory mechanisms which comprise increased striatal DA turnover and receptor
sensitivity, upregulation of striatopallidal enkephalin levels, increased subthalamic
excitation of GPe, and maintenance of cortical motor area activation (Bezard et al. 1999,
Bezard et al. 2001), might be potentially damaging to nigral neurons through rising
cellular metabolic demand, oxidative stress, and excitotoxicity (Rodriguez et al. 1998,
Jenner 2003).
The PD-associated cell loss has a characteristic topology distinct from the
pattern seen in the course of normal aging of the human organism as the SN is
characterized by a low level of neuronal degeneration (Kubis et al. 1995). Furthermore,
normal aging-related morphological modifications were found to be most intensive in

INTRODUCTION
the dorsomedial part of SNpc while in PD the ventrolateral and caudal portions of SNpc
are particularly affected (Fearnley and Lees 1991).
Figure 11: Classical model of the BG in the normal state (A) and parkinsonian state (B) (Obeso et al.
2008)
19

INTRODUCTION
Degeneration of all DAergic pathways and other systems
20
Although the nigrostriatal pathway is the most severely affected in PD, all
ascending DAergic pathways in the central nervous system (CNS) do degenerate
although to variable degrees (reviewed by Hornykiewicz and Kish 1987). The
mesolimbic DAergic neurons which reside adjacent to the SNpc in the VTA and
connect with the ventral striatum are less affected in PD: they are decreased between
37-50% of that in healthy subjects (Price et al. 1978, Javoy-Agid and Agid 1980, Uhl et
al. 1985, Dymecki et al. 1996). As a result, there is significantly less DA depletion in
the caudate nucleus (Price et al. 1978), the main site of projection for these neurons.
It is important to stress that PD not only affects the DAergic nerve cells of the
SN but also other areas and neurotransmitter systems: noradrenergic (locus coeruleus
(LC), where cell loss may exceed that seen in SNpc; Zarow et al. 2003), cholinergic
(nucleus basalis of Meynert, NBM; dorsal motor nucleus of vagus, DMV), and
serotonergic (dorsal raphe nuclei). Pathological changes are also observed in
glutaminergic, tryptaminergic, GABAergic, and adrenergic neurons (Braak and Braak
2000). Besides, cell loss is seen in other regions: postganglionic sympathetic neurons
and cortex (especially cingulate and entorhinal cortices), olfactory bulb, and autonomic
nervous system (Braak et al. 1995, Braak and Braak 2000, Levy 2009). Interestingly,
the loss of non-DAergic neurons, especially in the caudal brainstem, may even become
involved before the DAergic neurons (Braak et al. 2006).
Thus, PD symptoms are the consequence of progressive degeneration of the
DAergic neuronal BG system but also of other ascending subcortical neuronal systems.
Whereas the main motor manifestations of PD are linked to neurodegeneration in the
DAergic systems, the non-DAergic pathways degeneration likely contributes as a major
cause of many of the non-motor symptoms of PD, such as depression, cognitive decline,
constipation and autonomic dysfunction. As an example, degeneration of cholinergic
cortical inputs and hippocampal structures are part of the cause of the high rate of
dementia that accompanies PD, especially in older persons. As the clinical correlates of
lesions of these non-DAergic neurotransmitters pathways are not as clearly

INTRODUCTION
characterized as are lesions in the DAergic systems, the temporal relationship of
damage to specific neurochemical systems is not well established. Although, for
example, some patients develop depression months or years prior to the onset of motor
symptoms, the non-motor symptoms are generally thought to dominate the more severe
or later stages of the disease (Chaudhuri et al. 2006).
Lewy bodies, Lewy neurites and pale bodies
In addition to neuronal loss, another important pathological feature observed in
PD is the presence of fibrillar aggregates, called LB, Lewy neurites (LN) and pale
bodies, in the few remaining surviving nigral DAergic neurons (Dauer and Przedborski
2003, Probst et al. 2008).
There are two morphologic types of LB: the brainstem classical type and the
cortical type. Brainstem-type LB are intraneuronal cytoplasmic inclusions, spherical or
elongated, 5-25 μm in diameter, and possessing a dense eosinophilic core and a clear
peripheral halo (Figure 12A; Duffy and Tennyson 1965, Roy and Wolman 1969,
Pappolla 1986). Cortical LB are also intracytoplasmic inclusions but often irregular in
shape and lacking a distinctive core and conspicuous halo Figure 12B. LN correspond
to abnormal dystrophic neurites that contain filaments similar to those found in LB
(Figure 12C; Dickson et al. 1991). Whereas LB are developed in the perikarya, LN are
located in the neuronal processes (Figure 12D). Pale bodies are rounded well-defined
areas without halos, less eosinophilic, found in melanized cells of the SNpc and the
locus coeruleus. They contain vesicular and granular structures and filaments identical
to those seen in LB. As pale bodies often co-occur with one or more LB in the same
neurons, they have been proposed to represent a stage in the formation of LB.
LB are composed of more than 76 molecules, including numerous proteins, such
as α-synuclein, parkin, ubiquitin, and neurofilaments (Wakabayashi et al. 2007).
Whereas the SNpc appears to be the first region to be affected by neuronal loss, LB are
not confined to the SNpc but can also be found within many of the spared neurons in
21

INTRODUCTION
other brain areas and in the peripheral autonomic nervous system as the disease
progresses (Forno et al. 1996, Wakabayashi and Takahashi 1997, Spillantini et al. 1998,
Braak et al. 2003, Wakabayashi et al. 2007). The extensive distribution of LB could
account for a variety of motor and non-motor symptoms of PD. Interestingly it has been
proposed that LB appear first in both the olfactory bulb and lower brain stem, following
then a foreseeable progression which contributes to explain the loss of olfaction which
might precede motor symptoms.
Figure 12: LB and LN in PD patients (A, B: Wakabayashi and Takahashi 2007, C: Braak and Del Tredici
2008) and localization of LB and LN (D: Braak et al. 2003)
22
A B
C D
The presence of LB is not specific for PD as they occur also in AD, dementia
with LB (DLB) and in healthy people of advanced age at a greater frequency than the
prevalence of PD (Gibb and Lees 1988). The role of LB in neuronal cell death is
controversial and under debate: LB may be toxic by interfering with normal cellular
processes and/or by sequestering important proteins for cell survival or on the other
hand, they could be cytoprotective as they may sequester and degrade the toxic proteins

INTRODUCTION
(Olanow et al. 2004). Most likely, LB seem to be symptomatic in PD pathogenesis, not
causative (Wakabayashi et al. 2007).
The six-stage system: a novel neuropathological concept of neurodegeneration
Recently, Braak and colleagues established a staging procedure based on the
distribution of LB and LN (Braak et al. 2003, Braak et al. 2006). According to the
pathological changes observed during the cross-sectional study originally performed on
autopsy cases, the authors proposed that the formation of proteinaceous intraneuronal
LB and LN follows a topographically predictable sequence in six stages with ascending
progression from medullary and olfactory nuclei to the cortex (Figure 13). In early
stages 1 and 2, patients are pre-symptomatic. LB pathology first appears in the dorsal
motor nucleus of the vagal nerve and is confined to the medulla oblongata, pontine
tegmentum and anterior olfactory structures. As the disease advances, in stages 3 and 4,
pathological changes appear in the SN and in other areas of the basal midbrain and
forebrain. Motor symptoms become clinically manifest during this phase. Finally the
final stages 5 and 6 are frequently associated with cognitive impairment as the
neocortex becomes involved (Figure 14).
Many groups give strong support to this neuropathological concept which states
that neurodegeneration in PD starts in non-DAergic areas, notably the enteric nervous
system and then rises via spinal cord and brainstem to nigral and subsequent cortical
neurons (Abbott et al. 2001, Przuntek et al. 2004, Abbotth et al 2005, Ross et al. 2005,
Ross et al. 2006). Nevertheless, it has also been brought into question as some
neuropathological studies showed brains with important neurodegeneration and α-
synuclein pathology in the SNpc and higher brain structures but lacking medullary
involvement (Parkinnen et al. 2003, Jellinger 2004, Parkinnen et al. 2005, Attems and
Jellinger 2008, Dickson et al. 2008, Kalaitzakis et al. 2008a, 2008b, Parkinnen et al.
2008).
23

INTRODUCTION
Figure 13: Six stages of brain pathology in idiopathic PD (Braak et al. 2003)
Figure 14: Stages 3–6 of pathological changes associated with sporadic PD in hemisphere sections
immunostained for �-synuclein (Braak et al. 2006)
24

Etiology and pathogenesis of PD
INTRODUCTION
The cause of most cases of PD remains unclear. By the date several pathogenic
mechanisms based on environmental, genetic and histopathological findings have been
implicated in this disease. They may be primary activating events for the disease and
secondary events involved in promoting progression of PD over time. The contributions
of genetic and environmental risk factors and the impact of compensatory mechanisms
on PD progression are still unanswered questions. Some forms of PD have been
associated to specific environmental causes (Langston et al. 1983, Thrash et al. 2007,
Jang et al. 2008), while a few familial forms of PD and related parkinsonism are related
to apparent genetic causes (Biskup et al. 2008, Gasser 2009).
Genetics of PD
In the majority of PD cases, there is no apparent genetic linkage and the disease
is referred to as “sporadic” or idiopathic PD. Thus, familial cases only represent a 5-
10% of the whole affected population. The disease is inherited and defined by at least
one relative (parent or sibling) also having PD. It is not always clear if this is due to
heritable genetic factors or shared environment. But an inherited genetic contribution
was put forward by various studies, mono- and dizygotic twins and families with PD
history (Tanner 1999, Thacker and Ascherio 2008).
An increasing number of single gene mutations causing familial PD have been
identified (Table 4). They show both autosomal dominant and recessive as well as X-
linked modes of inheritance (Vila and Przedborski 2004, Wood-Kaczmar et al. 2006).
Many of these have also been found in apparently sporadic cases (Warner and Schapira
2003) suggesting that they may act as susceptibility factors. Chromosomal regions have
been designated as “PARK” genes. These include two autosomal dominant genes: �-
synuclein (SNCA) (Polymeropoulos et al. 1997, Krüger et al. 1998) and leucine-rich
repeat kinase 2 LRRK2 (Paisán-Ruiz et al. 2004, Zimprich et al. 2004), and three
autosomal recessive genes: parkin (PRKN) (Kitada et al. 1998), PTEN-induced putative
25

INTRODUCTION
kinase 1 (PINK1) (Valente et al. 2004), and DJ-1 (Bonifati et al. 2003). New and crucial
insights into the PD pathogenesis have been supplied by the discovery of these genetic
mutations and the improved comprehension of dysfunction of their abnormally encoded
proteins.
Table 4: Gene loci associated with PD ( * Toulouse and Sullivan 2008, # Schapira 2008)
Locus
(MIM#)
PARK1
(168601)
PARK2
(600116)
PARK3
(602404)
PARK4
(605543)
26
Inheritance
Chromosomal
location
Gene
AD 4q21 SNCA
(mutations)
Onset
Clinical features
Mid-late Idiopathic PD, low
incidence of tremor,
fast progression
AR 6q25.2–q27 Parkin Early Drug induced
dyskinesia, dystonia,
slow progression
AD 2p13 Not known Late Idiopathic PD,
dementia, fast
progression
AD 4q21 SNCA
(duplication /
triplication)
Late Dementia,
autonomic
dysfunction,
postural tremor,
fast progression
Neuropathology a
Nigral degeneration,
with LB
Nigral degeneration,
without LB
Nigral degeneration,
with LB, NFT and
plaques
Nigral degeneration,
with LB, vacuoles in
neurons
PARK5 AD * 4p14
UCHL1 Mid-late Idiopathic PD * Not reported
(191342)
# 4p15
# Yes
PARK6 AR * 1p36
PINK1 Early Slow progression, Not reported
(605909)
# 1p35
drug-induced
dyskinesia
PARK7 AR 1p36 DJ1 Early Slow progression, Not reported
(606324)
psychiatric
symptoms
PARK8 AD * 12q12
LRRK2 Late Idiopathic PD Nigral degeneration,
(607060)
# 12p12
variable features
including LB, NFT and
ubiquitinated
inclusions
PARK9 b
AR * 1p36
ATP13A2 Juvenile Spasticity,
Striatal atrophy
(606693)
# 1p32
supranuclear gaze
paralysis, dementia
PARK10 AD * 1p
Not known Late Not reported Not reported
(606852)
# 1p32
PARK11
(607688)
AD 2q36–q37 Not known Late Not reported Not reported
PARK12 X-linked Xq21–q25 Not known Not
Not reported Not reported
(300557)
reported
PARK13
(610297)
AD 2p12 Htra2 Late Idiopathic PD Not reported
(601828) 2q22–q23 NR4a2 Late Idiopathic PD Not reported
(603779) 5q23.1–q23.3 Synphilin1 Late Idiopathic PD Not reported
a LB: Lewy bodies, NFT: neurofibrillary tangles, plaques: amyloid plaques.
b PARK9 initially presented as idiopathic PD but is now known as Kufor–Rakeb syndrome.

Ageing
INTRODUCTION
Ageing remains an important risk factor of PD. It is associated with a decline of
pigmented neurons in the SNpc (McGeer et al. 1977) and a decline in striatal DA
transporters (Van Dyck et al. 2002) but no difference between the caudate and putamen
as seen in PD. Even if the incidence of PD is known to increase with age, this disease is
not a simple acceleration of ageing as the pattern of neuronal loss in SN in PD patients
and in the normal aging population differs substantially (Kish et al. 1992).
Role of environmental exposure
Exposure to environmental factors may strongly influence risk of PD.
Environmental factors have also been implicated in the pathogenesis of PD with varying
degrees of association. These include environmental toxins, occupation, dietary factors,
drug use and metals amongst others.
An example of environmental toxin: MPTP
The environmental toxin hypothesis considers that PD-related neurodegeneration
is a consequence from exposure to a DAergic neurotoxin. MPTP (1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine) is a prototypic example of how an exogenous toxin can
mimic the clinical and pathological features of PD. Davis et al. (1979) and Langston et
al. (1983) were the first to convincingly demonstrate the link between environmental
toxin exposure and the development of PD. They observed that, after illicit drug
consumption, a series of patients developed physical symptoms consistent with PD, and
acute levodopa-responsive parkinsonism. In one case, substantial damage to nigral
neurones was observed (Davis et al. 1979). Analysis of the drug used revealed a
contamination with two side products, MPTP and MPPP (1-methyl-4-phenyl-4-
propionoxy-piperidine) in the synthesis of a meperidine analogue (Langston and Ballard
1983). The causative link between MPTP and the parkinsonian syndrome observed in
27

INTRODUCTION
the drug users was further confirmed by the development of symptoms very similar to
PD following injection of MPTP in an animal model (Burns et al. 1983).
28
Studies showed that MPTP readily crosses the blood-brain barrier (BBB) and is
oxidized by MAO-B in astrocytes within glia to a pyridinium derivative, MPP + (1-
methyl-4-phenyl pyridinium ion) (Ransom et al. 1987). MPP + is then selectively taken
up by the DAergic cells and accumulated in nigrostriatal neurons (Chiba et al. 1985).
After entering the mitochondria by the passive cationic gradient, MPP + inhibits the
NADH-dehydrogenase at complex I in the respiratory chain preventing ATP synthesis
and leading to reactive oxygen species (ROS) production and cell death in DAergic
neurons (Chiba et al. 1984, Heikkila et al. 1985, Nicklas et al. 1985, Vyas et al. 1986,
Singer and Ramsay 1990) (Figure 15). Studies showed that pre-treatment of animals
with two selective MAO-B inhibitors (deprenyl or pargyline), blocked MPTP toxicity
most likely by blocking MPTP oxidation to MPP + (Heikkila et al. 1984, Langston et al.
1984, Markey et al. 1984).
Figure 15: MPTP metabolism (Dauer and Przedborski 2003)

INTRODUCTION
The finding of MPTP toxicity not only established a link between environmental
exposure and the development of PD, but also introduced MPTP as experimental model
for the study of PD. Other DAergic neurotoxins have been identified, such as the
insecticide rotenone and the herbicide paraquat (Brooks et al. 1999, Betarbet et al. 2000,
Dhillon et al. 2008, Cannon et al. 2009). As MPTP, paraquat and rotenone are selective
complex I inhibitors and induce DA diminution in animal studies. This will be
discussed later in the section “Experimental models of PD”.
Other environmental factors
Since the discovery of MPTP-induced parkinsonism, many epidemiological
studies have been performed to examine the link between the risk of PD and other
environmental factors. Lifestyle-associated factors can have positive or negative
influence on the risk of developing PD. Living in a rural environment (in industrialised
countries), farming, exposure to pesticide use and wood preservatives, and drinking
well water (Hertzman et al. 1990, Engel et al. 2001, Priyadarshi et al. 2001, Baldiet al.
2003, Gorell et al. 2004, Dick 2006) seem to confer an increased risk of PD but it
remains debatable (Koller et al. 1990, Gorell et al. 1998). On the contrary, some
environmental factors appear to exhibit a negative correlation with PD. Caffeine intake,
cigarette smoking, and high vitamin intake are inversely associated with the risk of
developing this disease (Baron 1986, Morens et al. 1995, Ross et al. 2000, Hernan et al.
2002, Lai et al. 2002, Landrigan et al. 2005, de Lau and Breteler 2006).
Metals
Exposure to some specific metals such as aluminium, amalgam, copper, iron,
lead, manganese, mercury, zinc, and a combination of metals have also been
hypothesised to be a risk factor in PD through the accumulation of metals in the SN and
increased oxidative stress (Ngim and Devathasan 1989, Zayed et al. 1990, Seidler et al.
1996, Gorell et al. 1997, Gorell et al. 1999, Rybicki et al. 1999, Kuhn et al. 1998, Lai et
al. 2002). During the past few years, special attention was paid to aluminium. As it will
29

INTRODUCTION
be largely discussed later, its ubiquity and extensive use in products and processes
coupled with its reported ability to cause neurodegeneration have made aluminium a
cause of health concern (Exley 1999, Yokel 2000, Zatta et al. 2003, Kawahara 2005).
Potential theories
30
Experimental models of PD, epidemiologic studies, neuropathologic
investigations, and genetic analyses, which implicated both environmental and genetic
factors in the pathogenesis of PD, provided important insights into the pathogenesis of
PD (Beal 2001, Betarbet et al. 2002, Dawson and Dawson 2003, Siderowf and Stern
2003). But the exact causative pathogenic pathway(s) for the development of PD
remain(s) hard to demonstrate. Several hypotheses aiming to explain the cause and
beginnings of PD have been proposed.
The “dual-hit” hypothesis
The “dual-hit” hypothesis proposed that a hitherto unknown neurotrophic
pathogen induces the pathological process causing PD and triggering the sequential
involvement of vulnerable regions (Hawkes et al. 2007). This pathogen may enter the
brain via two routes: nasal, with anterograde progression to the amygdala and temporal
lobe; or gastric, by subsequent retrograde and transneuronal transport.
The “multiple hit or multihit” hypothesis
The “multiple hit or multihit” hypothesis points to the idea that PD, originally
identified as a DA-deficit disorder, is probably a multietiological disease resulting of the
combination of multiple interlinking combinated factors or insults acting together
(Sulzer 2007). As assumed by Lang and colleagues, a disease with various contributing
etiologic issues could have several diverse ways of onset and progression (Lang et al.
2007) and this could explain the interindividual clinical and neuropathological
heterogeneity observed in the course of PD. It is important to note that this supposition

INTRODUCTION
may clarify why many neuropathologic studies did not conform to the six-stage Braak
system and why some patients present obvious non-DAergic features while others have
little difficulty with these over a prolonged disease course.
The connection between PD and the FeBAD hypothesis
Also, Barlow and colleagues recently explored the possibility to link PD with the
fetal basis of adult disease (FeBAD) hypothesis. They proposed that environmental
stressors encountered very early in life (in utero or during the perinatal period) may
decrease the number of DAergic neurons making the nigrostriatal system vulnerable to
subsequent insults or to a failure of normal compensatory mechanisms (Figure 16;
Barlow et al. 2007). Indeed, even if PD is considered as an age-related disorder, the
biology of this disease may start decades prior to the critical treshold of 60% neuronal
loss or clinical manifestations.
Figure 16: The multiple hit model of PD (Barlow et al. 2007)
31

INTRODUCTION
Parkinson diseases: a syndrome of different disorders
32
There is a large body of evidence indicating that impairment in protein biology,
mitochondrial dysfunction, neuroinflammation, cell death pathways, excitotoxicity, and
oxidative stress, to cite only some of the most salient, are key pathologic mechanisms in
the etiology of PD, all of which are tightly linked (Figure 17; Mattson 2000, Chung et
al. 2001, Vila and Przedborski 2003, Eriksen et al. 2005, Gandhi and Wood 2005,
Abou-Sleiman et al. 2006, Olanow 2007, Tansey et al. 2007). But none of these
pathogenic mechanisms alone has been proven to be the exact causative and unifying
cytotoxic pathway leading to neurodegeneration in PD (Sulzer 2007). It is now widely
recognized that PD is not a single homogenous disease but to a certain extent a
multifaceted syndrome of different neurological disorders, caused by a complex
interaction between genetic, environmental, and other factors leading to a common final
pathology (Moore et al. 2005, Klein and Schlossmacher 2007, Schapira 2008, Zhou et
al. 2008). Actually, the term Parkinson diseases was proposed to reassess this syndrome
of multiple “parkinsonism types” and remind that multiple etiologies are possible to
explain the patient‟s neurologic syndrome (Weiner 2008). In the future, PD will be
probably subdivided into different varieties and ethiologies, maybe assigning numbers
to each different “parkinsonism types”.
Figure 17: Pathologic mechanisms in the etiology of PD

Misfolding and aggregation of proteins
INTRODUCTION
Dysfunction at any stages of protein production (transcription and translation,
post-translational modification, trafficking and degradation) can lead to malformed
proteins and aggregation. There is now compelling evidence that protein aggregation
and abnormal processing of proteins are involved in the pathogenesis of PD (Schulz and
Dichgans 1999). Despite the rarity of the familial forms of PD, the identification of
single genes linked to this disease has provided decisive insights into probable
mechanisms of its pathogenesis. Mutations in the genes α-synuclein, parkin or UCH-L1
are thought to impair the protein degradation pathway involving ubiquitination and the
proteasome (Figure 18B).
The protein α-synuclein is the major constituent of the LB. In its native state,
monomeric α-synuclein is a soluble unfolded protein that under certain conditions is
prone to misfold and to aggregate forming oligomeric species. These oligomers, termed
protofibrils, are soluble and can then become amyloid-like fibrils which are insoluble.
These latter are the major components of LB (Spillantini et al. 1998) and can also form
LN in neuronal processes. Mutations on SNCA, the gene encoding α-synuclein, cause a
rare autosomant dominant form of familial PD (Polymeropoulos et al. 1997, Kruger et
al. 1998). Genomic rearrangement in the form of duplication and triplication of the wild
type (wt) SNCA gene cause typical and atypical PD, respectively (Singletton et al.
2003, Chartier-Halin et al. 2004, Ibañez et al. 2004, Fuchs et al. 2007) and the presence
of increased copy numbers of the wt α-synuclein gene causes PD (Ibañez et al. 2004).
Moreover, α-synuclein aggregates are also found in various other neurodegenerative
diseases termed synucleinopathies, such as MSA, DLB, and pure autonomic failure
(Trojanowski and Lee 2002).
33

INTRODUCTION
Figure 18: The ubiquitin-proteasome system (A) and possible sites where different forms of PD might
interfere with the UPS (B) (Olanow and McNaught 2006)
34
The discovery of ubiquitin as a major constituent of LB (Kuzuhara et al. 1988)
was the first evidence that suggest the involvement of the ubiquitin-proteasome system
(UPS) dysfunction in the pathogenesis of PD. This was later supported by the
identification of parkin mutations associated with young onset autosomal recessive PD
(Kitada at al. 1988). The UPS is the major mechanism used to degrade abnormal
proteins, or proteins at the ends of their life cycles. Misfolded or unwanted proteins
targeted for degradation are covalently modified via polyubiquitination on lysine
residues and directed to the proteasome. Three enzymes are required: ubiquitin-
activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases

INTRODUCTION
(E3) (Figure 18A). Loss-of-function mutations in parkin, an E3 ubiquitin ligase, abolish
its activity leading to proteasomal dysfunction and subsequent accumulation of
aggregated proteins (Cookson 2005, Yang et al. 2009). Parkin was also shown to
interact with PINK1 (PARK6 gene) and DJ-1 (PARK7 gene) (Shiba et al. 2009, Um et
al. 2009). These three PD-related proteins physically interact and form a functional E3
ligase complex to regulate UPS-mediated protein degradation (Xiong et al. 2009). The
role of the UPS in neurodegeneration was also enhanced with the identification of
mutation I93M in UCH-L1 (Leroy et al. 1998) in autosomal recessive PD. The UCH-L1
protein is a de-ubiquitinating enzyme, it cleaves ubiquitin from the ubiquitin-protein
adduct enhabling the protein to enter the proteasome. UCH-L1 mutations may prejudice
proteasomal degradation and also lower the availability of ubiquitin monomers required
for the removal of additional misfolded proteins. Some studies demonstrated another
mutation within this gene (S18Y) that decreases risk for PD, but this remains
controversial (Maraganore et al. 2004, Healy et al. 2006). In brief, when a component of
the UPS required for degrading proteins is altered or the capacity of UPS has been
exceeded as an outcome of the excessive production of misfolded proteins, or a
combination of both, proteolytic stress happens (Figure 18B, Olanow 2007). This leads
to the accumulation and aggregation of proteins with subsequent damage in a wide
range of cellular functions and apoptosis.
35

INTRODUCTION
Mitochondrial dysfunction
36
Many data support now a role for aberrant mitochondrial form and function in
the pathogenesis of PD (Schapira 2008). Mitochondria are essential for the generation
of cellular energy through oxidative phosphorylation. They are also the major cellular
source of free radicals and are implicated in the regulation and initiation of cell death
pathways and in calcium homeostasis. Mitochondria dysfunction can lead to insufficient
ATP production hence impairing all ATP-dependent cellular processes and generate and
accumulate ROS, rendering then cells more vulnerable to oxidative stress and other
interconnected processes, including excitotoxicity. Originally, MPTP toxicity and
reduction of complex I activity in the SNpc of PD patients provided evidence for the
participation of mitochondrial pathways in PD (Schapira et al. 1992, Mann et al. 1994).
Moreover, demonstration of the involvement of PD-associated genes products in
mitochondrial function reinforced the connection between PD and mitochondrial
biology (Dodson and Guo 2007). Mutations of these PD-associated genes will likely
disrupt mitochondrial function and affect the cellular response to oxidative stress.
PINK1, a mitochondrial serine-threonine kinase that shares homology with
calmodulin (CaM)-dependent protein kinase I, localizes principally within
mitochondria, in particular at the outer mitochondrial membrane (Zhou et al. 2008)
while others suggested an inner mitochondrial membrane localization (Silvestri et al.
2005, Gandhi et al. 2006) or a cytoplasmic pool (Weihofen et al. 2007, Haque et al.
2008). PINK1 seems to provide protection against oxidative stress (Valente et al. 2004,
Yang et al. 2006). Its overexpression defends cell from mitochondrially-induced
apoptosis caused by staurosporine, and from mitochondrial depolarization and apoptosis
generated by the proteasomal inhibitor MG132 (Wood-Kaczmar et al. 2008). PINK1 is
thought to interact with HTRA2 (Plun-Favreau et al. 2007), a mitochondrial serine
protease, that is released from the mitochondrial intermembrane space into the cytosol,
where it induces apoptotic cell death in addition to the permeabilization of the
mitochondrial membrane leading to cytochrome c release (Suzuki et al. 2001, Hegde et
al. 2002, Suzuki et al. 2004). Furthermore, recent studies suggest that PINK1 could act

INTRODUCTION
in connection with Parkin, maybe controlling the balance between mitochondrial fission
and fusion (Deng et al. 2008), and also with α-synuclein (Marongui et al. 2009). Parkin
is located partially to the outer mitochondrial membrane (Darios et al. 2003) and also in
the mitochondrial matrix. Parkin protects against oxidative stress and has a
hypothesized role in mitochondrial gene transcription and biogenesis in proliferating
cells, but the precise mechanism is unknown (Jiang et al. 2004). A recent study
demonstrated a primary function for Parkin in the regulation of mitochondrial turnover.
Actually, Parkin was shown to be specifically recruited to damaged or uncoupled
mitochondria with low potential membrane to promote their clearance through the
autophagosome (Narendra et al. 2008). Another PD and mitochondrial function related
protein is DJ-1, which is mainly expressed in the cytosol, in the nucleus and in the
mitochondria (a pool located in the mitochondrial intermembrane space and in the
matrix). DJ-1 is thought to be neuroprotective (Abou-Sleiman et al. 2003, Zhang et al.
2005, Abeliovich and Flint Beal 2006, Zhou et al. 2006, Andres-Mateos et al. 2007,
Schapira 2008) as it was recently reported to translocate to the mitochondria under
conditions of oxidative stress (Junn et al. 2008). DJ-1 is then relocalized into the
nucleus, where it is proposed to bind multiple RNAs and regulate p53‟s transcriptional
activity.
37

INTRODUCTION
Oxidative stress
38
Oxygen is necessary for life, but paradoxically, it is also toxic as a by-product of
its metabolism produces ROS (Reaction 1). ROS are defined as molecular entities that
react with cellular components, resulting in detrimental effects on their function as they
are highly toxic to cells. ROS include both molecular species, such as hydrogen
peroxide (H2O2), and free radicals (a term used to designate any chemical species
containing highly reactive unpaired electrons), such as superoxide anion (O2 ●─ ), and
hydroxyl radical ( ● OH). The latter is the most potentially dangerous of ROS, short-lived
but highly reactive, which causes enormous damage to biological molecules. In addition
to ROS, reactive nitrogen species (RNS) are also generated notably nitric oxide (NO ● ,
NO) and peroxynitrite (ONOO ●─ ).
Reaction 1: Metabolism of O2 and production of ROS
O2 + e ─ ●─ ─ +
→ O2 + e + 2H → H2O2 + e ─ → ● OH + OH ─ + e ─ + 2H + → 2H2O
ROS can interact with nearby critical cellular components such as membrane
lipids, proteins, and DNA and oxidize them, leading to a wide range of damaging
effects. In the case of proteins, ROS may directly oxidize amino acids leading to loss of
functions of proteins and disruption of the active sites of enzymes. Exposure of
membranes of fatty acids to ROS may result in the formation of alkoxyl (RO ● ), peroxyl
(ROO ● ), and lipid epoxides and hydroperoxides (ROOH). The latter can additionally be
further oxidized close by unsaturated fatty acids in a chain reaction event stimulated by
ROO ● and RO ● , leading to disruption of both plasma and mitochondrial membranes.
Oxidation of nucleic acids may provoke strand breakage, nucleic acid-protein
crosslinking, and nucleic base modifications which can have an effect on DNA
transcription, translation and replication. Within neurons ROS may also interfere with
signal transduction and gene expression affecting cell survival, thus inducing cell death.

INTRODUCTION
Oxidative damage happens in all our tissues all the time. There is a basal level of
oxidative damage to DNA, lipids and proteins (Halliwell and Gutteridge 2006) and
under normal conditions free radicals will be quickly detoxified by the body‟s defence
systems. Mechanisms that resist against oxidative damage comprise antioxidant
scavengers such as glutathione (GSH), ascorbic acid (AA), alfa-tocopherol, carotenoids,
flavonoids, polyphenols and antioxidant enzymes. These latter, SOD, GPx, CAT, are
known to be responsible for the detoxification of ROS: SOD catalysing the dismutation
of two molecules of O2 ●� to give H2O2 and O2, and GPx and CAT participating in the
removal of H2O2 (Reactions 2 and 3). Nevertheless, under certain circumstances, greater
amounts of ROS and RNS are produced, which finish up by overcoming the cellular
defence mechanisms. Failures in the systems that repair and replace oxidized
biomolecules contribute to a situation of oxidative stress, defined as “an imbalance
between oxidants and antioxidants, in favour of the oxidants, leading to a disruption of
redox signaling and control and/or molecular damage” (Sies and Jones 2007).
Reaction 2: Dismutation of O2 by SOD
●─ +
2 O2 + 2H H2O2 + O2
Reaction 3: Removal of H2O2 by GPx
2 GSH + H2O2
SOD
GPx
GSSG + 2H2O
In the past decades, oxidative stress was the first common pathogenic factor to
be considered as contributing to the degeneration of DAergic neurons in PD (Jenner
1998, Lang and Lozano 1998, Schapira 1999). Increased SOD, iron, lipid peroxidation
markers and nitrated proteins levels, and decreased GSH levels in SNpc of PD patients
suggested that oxidative stress may play a significant function in PD neurodegeneration
(Saggu et al. 1989, Sofic et al. 1991, Sofic et al. 1992, Ilic et al. 1999, Agil et al. 2006).
39

INTRODUCTION
Transition metals: the example of iron
40
Iron is a redox-active metal able to generate O2 ●─ from its interaction with
molecular oxygen by auto-oxidating (Reaction 4). The reverse reaction is also possible.
As a result of the Fenton reaction (Reaction 5), Fe 2+ may also be oxidized in the
presence of H2O2 and generate ● OH and Fe 3+ which provokes lipid oxidation through its
reaction with hydroperoxides normally present in the biological systems. In biological
environment, other reactions as the Haber-Weiss reaction also occurs and requires the
catalytic contribution of redox metal ions (Reaction 7).
Reaction 4 Fe 2+ + O2 → Fe 3+ + O2 ●─
●─ +
2 O2 + 4H → 2H2O2
Reaction 5 Fe 2+ + H2O2 → Fe 3+ + ● OH + OH ─ (Fenton reaction)
Reaction 6 Fe 3+ + O2 ●─ → Fe 2+ + O2
Reaction 7 O2 ●─ + H2O2 → O2 + ● OH + OH ─ (Haber Weiss reaction)
In PD, iron content of the SNpc is elevated with an increase of the Fe 3+ /Fe 2+
ratio from 2:1 to almost 1:2 (Riederer et al. 1988, 1989, Sofic et al. 1988, Dexter et al.
1993). These levels of iron increase the conversion of H2O2 to ● OH via the Fenton
reaction and promote a greater turnover in the Haber-Weiss reaction, leading to an
amplification of oxidative stress (Riederer and Youdim 1993). Alternatively, oxidative
stress may increase the levels of free iron (Halliwell and Gutteridge 2003) which may
interact with α-synuclein promoting its aggregation (Schipper et al. 1998, Hochstrasser
et al. 2004).

DA metabolism as a source of ROS
INTRODUCTION
The selective neurodegeneration of SNpc DAergic in PD seems to suggest that
these neurons are more vulnerable to oxidative stress, but the reason behind this is not
completely understood. One potential explaination was based, at least in part, on DA
metabolism either by autoxidation or catalyzed by MAO. Actually, this neurotransmitter
can react with molecular oxygen generating peroxides, active quinones, � OH and other
ROS that, along with those created from the respiratory chain failure, may lead to both
modifications of proteins and depletion of GSH generating a highly oxidative
intracellular environment.
The MAO catalyzed oxidation of DA to 3,4-dihydroxyphenylacetaldehyde
(DOPAL) leads to H2O2 formation, which is normally cleared by GSH. As GSH levels
are decreased in SN of PD patients (Sian et al. 1994) H2O2 may be converted into � OH
which may trigger lipid peroxidation causing the death of DAergic neurons.
Furthermore, DA autoxidation results in the formation of the semiquinone radical which
can be directly toxic (Stokes et al. 1999, Danielson and Andersen 2008) or which can
lead to the formation of ROS (Olanow 1990). O2 ●─ is either metabolized into H2O2 or
reacts with NO, generating the strongly reactive ONOO ●─ . After a complex process of
polymerization, this autoxidation pathway leads finally to the generation of the dark-
coloured, insoluble pigment called neuromelanin (Graham 1978). This compound was
implicated in the vulnerability and susceptibility of DAergic neurons to
neurodegeneration (Abou-Sleiman et al. 2006) but its precise mechanism remains
uncertain. Several hypotheses tried to explain its contribution to the DAergic neurons
death, through a macromolecule crowding effect or as an intraneuronal toxic reservoir
by binding transition metals like iron or neurotoxic coumpounds like MPP + (Chung et
al. 2001).
41

INTRODUCTION
Nitric oxide
42
There is evidence that not only ROS but also the metabolism of NO, a free
radical, may play a part in oxidative damage in PD (Gerlach et al. 1999, Torreilles et al.
1999, Boje 2004). NO is synthesized from L-arginine by three isoforms of nitric oxide
synthase (NOS): inducible (iNOS), endothelial (eNOS), and neuronal NOS (nNOS),
using NADPH and molecular oxygen (Kavya et al. 2006). NO has long been recognized
as a signaling molecule for vasodilatation and neurotransmission but it has many
important functions in other physiologic systems, such as in the immune, respiratory,
neuromuscular and nervous systems. Moreover, NO also participates in pathogenic
pathways. It can react with other ROS to generate the highly toxic RNS (Reynolds et al.
2007, Szabo et al. 2007). As depicted in Figure 19 NO can react with O2 ●─ to form the
more reactive ONOO ●─ . Peroxynitrite is known to promote cellular damages by means
of lipid peroxidation, DNA fragmentation, protein nitration, and activation of caspase
dependent and/or independent cell death pathways (Beckman and Koppenol 1996, Hong
et al. 2004, Szabo et al. 2007). Additionnally, when reacted with H + or CO2, ONOO ●─
can further convert to nitrogen dioxide (NO2) and ● OH, two highly toxic intermediates.
Protein modifications by NO and/or ONOO ●─ such as S-nitrosylation and nitration may
affect cell survival. Protein nitration by NO or ONOO ●─ usually inserts a nitro (-NO2)
group onto one of the two carbons of the aromatic ring of tyrosine residues to form
nitrotyrosine (Gow et al. 2004). On the other hand S-nitrosylation, which also happens
under both physiologic and pathogenic conditions, regulates gene transcription,
vesicular trafficking, receptor mediated signal transduction, and apoptosis (Chung
2007). Many enzymes, receptors and neuroprotective proteins may be modified by NO
through their reactive cysteine (CySH) thiols to form the corresponding nitrosothiols
(Stamler et al. 1992, Ahern et al. 2002, Hess et al. 2005, Chung 2007).

Contribution of oxidative and nitrosative stress to PD pathogenesis
INTRODUCTION
As we have previously seen, genes linked to familial PD are important in
mitochondrial function or in the handling of misfolded proteins (Abou-Sleiman et al.
2006, Savitt et al. 2006, Sulzer 2007). Besides, oxidative and nitrosative stress had a
significant effect on the normal function of familial PD-related gene products in the
process of neurodegeneration.
Figure 19: Generation of ROS and RNS in SNpc neuron (Tsang and Chung 2009)
Oxidative stress is known to promote protein misfolding and aggregation. For
example, α-synuclein can undergo oxidative modifications such as the addition of a DA
adduct on α-synuclein (Conway et al. 2001). This modification stabilizes the toxic α-
synuclein protofibrils and makes it resistant to chaperone mediated autophagy (CMA)
leading to a complete blockade of other proteins degradation via this pathwaw
(Martinez-Vicente et al. 2008). As well, α-synuclein protofibrils can permeabilize
synaptic vesicles (Volles et al. 2001, Mazzulli et al. 2006, Mosharov et al. 2006) leading
to an increase of more α-synuclein protofibrils. Nitrosative modifications of α-synuclein
43

INTRODUCTION
have also been demonstrated in all tyrosine residues (Takahashi et al. 2002). Nitrated α-
synuclein is more prone to aggregation (Giasson et al. 2000, Zhang et al. 2000), more
resistant to proteolysis and more immunogenic explaining why inflammatory responses
are frequent in PD patients (Goedert 2001, Benner et al. 2008). We have previously also
commented that PINK-1 and DJ-1 are known to possess protective function against
oxidative stress (Savitt et al. 2006, Thomas and Beal 2007).
44
Oxidative and nitrosative stress can also impair cellular systems that protect
against misfolded or aggregated proteins. As an example, parkin can be S-nitrosylated
on a critical CySH residue within its catalytic RING domains impairing its E3 ligase
activity and neuroprotective functions (Chung et al. 2004, Yao et al. 2004).
Additionally, DA can covalently modify parkin leading to parkin aggregation and
weakening its ligase activity (LaVoie et al. 2005). S-nitrosylated and DA-modified
parkin were observed in the SNpc of PD patients. Moreover, normal UPS function can
also be compromised by the tendency of oxidized proteins to be more resistant to
proteasomal degradation probably by means of the formation of aggregates (Friguet and
Szweda 1997, Davies 2001). Protein aggregates may also lead to the UPS impairment
by blocking the access of the proteasome to other proteins (Grune et al. 2004).
Despite being the major site of ROS formation, the respiratory chain itself is
targeted by oxidative stress. Complex I and various enzymes in the Krebs cycle can be
inactived by oxidation (Cadenas and Davies 2000, Cecarini et al. 2007) contributing to
the reduction of the ATP biosynthesis. Moreover, mitochondrial DNA (mtDNA) that is
transitorily connected to the inner mitochondrial membrane is particularly vulnerable to
oxidative damage (Goetz and Gerlach 2004). Consequently ROS-induced mtDNA
injury may play a part in mitochondrial dysfunction (Stewart and Heales 2003).

Excitotoxicity
INTRODUCTION
Glutamate is the major excitatory neurotransmitter in the CNS, but it can be
toxic to cells. Excitotoxicity is a pathological process that may occur as a consequence
of an excessive stimulation of the glutamate N-methyl-D-aspartate receptor (NMDAR)
and other receptors associated with glutamatergic signalling, such as metabotropic
glutamate receptors (mGLURs), calcium channels and other G-protein coupled
receptors (GPCR), and the subsequent massive influx of extracellular calcium. This
toxic and pathological increase in cytoplasmic calcium activates a number of calcium-
dependent enzymes involved in the catabolism of proteins, phospholipids and nucleic
acids, and in the synthesis of NO leading in turn to mitochondrial damage, formation of
oxidizing species, and downstream activation of cell death pathways.
There is some evidence implicating excitotoxicity as one contributing
mechanism to the pathogenesis in PD. As the SN receives rich glutamatergic inputs
from neocortex and the STN, the demise of nigrostriatal DA in PD leads to disinhibition
of striatal neurons, and consequently to disruption of the neurotransmitter balance in
striatum resulting in glutamatergic overactivity, whereas under physiological conditions
there is equilibrium between the activation of striatal neurons through NMDAR and
inhibition by the D2 receptors. It was hypothesized that weak excitotoxicity may happen
secondary to a mitochondrial defect with decreased ATP formation leading to an ATP
dependent magnesium-blockade of the NMDAR or to disinhibition of glutamatergic
STN neurons resulting from DA depletion (Beal 1998, Rodriguez et al. 1998).
Furthermore, NMDAR antagonists were shown to provide protection against MPP + -
induced neurotoxicity and obtain antiparkinsonian like activity in animal models
(Turski et al. 1991, Uitti et al. 1996, Schmidt and Kretschmer 1997) whereas
compounds that enhance NMDAR function worsens parkinsonian symptoms (Giuffra et
al. 1993). In addition, NO has been associated with NMDAR excitotoxicity. During
over-activation of the NDMAR, nNOS is recruited to the glutamate receptor by a
postsynaptic density protein called PSD-95 and synthesizes then NO which can react
with other ROS to form the highly toxic ONOO ●─ (Sattler et al. 1999, Aarts et al. 2002).
45

INTRODUCTION
46
Another pathway has been linked to excitotoxicity. In PD there is evidence for
abnormal activation of cyclin-dependent kinase 5 (Cdk5), a serine/threonine kinase
which needs activating partners, p35 and p39 (Cheung et al. 2004, Cheung 2006). Over-
activation of NMDAR was shown to provoke cleavage of p35 to p25, a protein that
cause a more robust and prolonged activation of Cdk5. Excessive Cdk5 activity has
been linked to PD pathogenesis (O‟Hare et al. 2005, Smith et al. 2006, Wang et al.
2007, Qu et al. 2007) and is known to contribute to neuronal death by phosphorylating
the transcription factor myocyte enhancer factor 2 (MEF2) and the antioxidative stress
enzyme peroxiredoxin 2 (Prx2), leading to attenuation of their normal prosurvival
functions. Actually, Prx2 breaks down H2O2 to H2O and O2 protecting the cells against
oxidative stress (Abou-Sleiman et al. 2006), whereas MEF2 is an important
transcription factor of prosurvival genes (Potthoff and Olson 2007).
Neuroinflammation and glial cells
It is well known that the brain can initiate injury responses, such as
neuroinflammation, as a way to clean up injured brain tissues. The main cellular
responders are microglia, specialized macrophages capable of producing cytokines and
other protective factors to regulate the injury response. Although neuroinflammation
may have initial protective effects, it has been hypothesized to be a potential contributor
to PD pathogenesis (Hartmann et al. 2003). Its exact role is unclear: it may act as an
initial event of PD pathogenesis or be a downstream result of another triggering
pathogenic factor.
Immunohistochemical studies have demonstrated the presence of activated
microglia, increased expression of iNOS, cytokines like tumour necrosis factor-α (TNF-
α) and interleukin (IL)-1β, and pro-inflammatory signalling cascades such as
cyclooxygenase-2 (COX-2) and NF-κB in striatum, SN and cerebrospinal fluid (CSF) of
PD patients (Boka et al. 1994, Mogi et al. 1994, Blum-Degen et al. 1995, Hunot et al.
1996, Hirsh et al. 1998, Muller et al. 1998, Hunot et al. 1999, Knott et al. 2000, Nagatsu
et al. 2000, Nagatsu 2002). Animal models of PD using rotenone, MPTP, and 6-OHDA
showed levels of microglial activation similar to what is found in PD (Czlonkowska et

INTRODUCTION
al. 1996, Cicchetti et al. 2002, Sherer et al. 2003). In post-mortem examination of brains
from humans exposed to MPTP, activated microglia was present up to 16 years after
exposure indicating a long-lasting and ongoing inflammatory response (Langston et al.
1999). Indeed, the inflammatory cytokines seem to intensify and perpetuate the
neuroinflammation (McGeer et al. 2001, Orr et al. 2002, McGeer and McGeer 2004,
Bartels and Leenders 2007). Furthermore, neuromelanin, which is released by dying
DAergic neurons, was shown to activate microglia in vitro (Wilms et al. 2003). As large
amounts of superoxide radicals are produced by activated microglia, chronic
neuroinflammation may then promote the degeneration of DAergic neurons once
activated, probably through increased oxidative stress (Figure 20; Whitton 2007). This
may lead to the creation of a vicious cycle that further increases DAergic toxicity in the
SNpc.
Figure 20: Interaction between microglia and DAergic neurons (Whitton 2007)
47

INTRODUCTION
Cell death pathways
48
There is substantial indication that cell death in PD occurs by way of a signal-
mediated apoptotic process (Hirsch et al. 1999, Mattson et al. 2000). Apoptosis is a
mechanism of programmed cell death (PCD) carried out by caspases during which a cell
goes through various morphological changes including membrane blebbing, changes to
the cell membrane such as loss of membrane asymmetry and attachment, cell shrinkage,
nuclear condensation, and DNA fragmentation. A normal functioning of apoptosis
avoids triggering harmful responses, such as inflammation. A great number of apoptotic
pathways are activated in response to PD-induced dysfunction, such as JNK signaling,
induction of Bax, p53 activation, cell cycle re-entry, bcl-2 family signalling, and
caspase activation (Hirsch et al. 1999, Tatton et al. 2003, Nair et al. 2006, Levy et al.
2009). As an example, mutations in LRRK2 are linked to activation of the intrinsic
apoptotic pathway, with cytochrome c released into the cytosol, activation of caspase 3
and nuclear condensation (Iaccarino et al. 2007).

Experimental models of PD
INTRODUCTION
Animal models are invaluable tools to understand the pathophysiology and
motor deficits occurring in PD disease. Furthermore, they are also fundamental to create
new neuroprotective strategies and therapeutic interventions to improve symptomatic
management. Since PD does not develop spontaneously in animals, various models
have been developed in distinct species in order to mimic the characteristic functional
changes observed in PD. These include toxin-based models, gene-based models and
inflammation-based models.
Toxin-induced models
6-OHDA, the first animal toxin-based model of PD associated with loss of
DAergic neurons in the SNpc was introduced more than 40 years ago (Ungerstedt
1968). Since then many different neurotoxins, such as MPTP, paraquat and rotenone
have been used to induce DAergic neurodegeneration in animal models.
6-OHDA
The discovery of 6-OHDA toxicity toward catecholaminergic neurons initiated
the epoch of toxin-based models of PD (Ungerstedt 1968, Ungerstedt and Arbuthnott
1970). As 6-OHDA does not cross the BBB it must be administered directly in the brain
by local stereotaxic injection into the SN, median forebrain bundle (MFB), or striatum,
with the contralateral side serving as control, or by peripheral administration in neonatal
rats (Perese et al. 1989, Przedborski et al. 1995, Rodriguez-Pallares et al. 2007,
Sánchez-Iglesias et al. 2007a). 6-OHDA injection into the striatum leads to a more
progressive and slower degeneration of nigrostriatal neurons, which lasts for 1-3 weeks
(Sauer and Oertel 1994, Przedborski et al. 1995, Fleming et al. 2005), while injections
into SN or the nigrostriatal tract result in striatal DA depletion 2 to 3 days later (Faull
and Laverty 1969). The quantity of neurotoxin used, the site of injection, and the
different sensitivity between animal species determines the extent of the lesion.
49

INTRODUCTION
Although the 6-OHDA model does not replicate many features of PD, such as
extranigral pathology or LB-like inclusions (Dauer and Przedbroski 2003, Lane and
Dunnett 2008), it is still extensively used. This model is particularly useful to
quantitatively assay the anti-PD properties of compounds for pharmacological
screening. Other 6-OHDA models, like bilateral intrastriatal (Roedter et al. 2001) or
intraventricular administration of 6-OHDA (Rodriguez et al. 2001, Rodríguez-Díaz et
al. 2001, Rey et al. 2007, Sánchez-Iglesias et al. 2009), were successfully developed
with methods meant to cause a bilateral and more slowly arising PD.
MPTP
50
MPTP is one of the most common toxins used to mimic the hallmarks of PD. Its
metabolite MPP + is a potent complex I inhibitor in DAergic neurons and an excellent
substrate for DAT, a fact that explains its selectivity towards DAergic neurons. Acute,
subacute and chronic MPTP protocols are generally used with some variations in mice
(Lau et al. 1990, Rozas et al. 1998, Petroske et al 2001, Muñoz et al. 2006, Joglar et al.
2009) as rats are not susceptible to MPTP. Loss of DAergic neurons, bradykinesia,
rigidity, and posture abnormalities developed in systemic MPTP treatment (Sedelis et
al. 2000). As well, small granular inclusions containing �-synuclein were seen in SN
DAergic neurons, so as an extra-nigral pathology (Wallace et al. 1984, Hallman et al.
1985, Meredith et al. 2002, 2004). Recently, a technically challenging intraventricular
MPP + chronic rat model has also been developed (Yazdani et al. 2006)
Rotenone, paraquat and maneb
Rotenone, a natural cytotoxic coumpound widely used as pesticide, is highly
lipophilic and can easily cross cell membranes and the BBB. Chronic exposure of
rotenone potently and uniformly inhibits mitochondrial complex I binding at the same
site as MPP + throughout the rat brain. The herbicide 1,1‟-dimethyl-4,4‟-bipyridinium or
paraquat (PQ) can also crosses the BBB and shares structural similarity to MPP + but has
no selectivity for DAT. PQ is reduced by complex I leading to formation of a PQ radical
capable of disrupting mitochondrial function. Treatments with rotenone and paraquat

INTRODUCTION
induce selective DAergic neuron degeneration and have been used to model sporadic
Parkinson's disease (Brooks et al. 1999, Peng et al. 2004, Hsuan et al. 2006, Inden et al.
2007). Animal models using paraquat generally also co-administer maneb (manganese
ethylenebisadithiocarbamate) to enhance toxicity. Nevertheless, general toxicity of PQ
and rotenone make their use difficult. As MPTP they produce nigrostriatal degeneration
but their effects are more widespread. A recent study suggested that rotenone and
paraquat do not induce neuroinflammation and microglial activation directly but rather
indirectly as a result of DAergic neuron damage or through factors released by neurons
or astrocytes (Klintworth et al. 2009).
Inflammation-based models
Neuroinflammation is present in PD and is believed to contribute to PD
pathogenesis. Therefore, inflammation-based models have been developed.
Lipopolysaccharide (LPS) is a bacterial coat protein and has been used to model
neuroinflammation in PD as it is a potent activator of microglial cells and inducer of
inflammation (Whitton 2007). Distinct protocols have been used by the date: acute
intracerebral (Castano et al. 1998, Herrera et al. 2000, Kim et al. 2000, Iravani et al.
2005), chronic intracerebral (Gao et al. 2002), acute systemic (Quin et al. 2007), and
intrauterine (Ling et al. 2002, 2004, 2006) administration of LPS. LPS causes DAergic
cell death and decreases locomotor activity in rodents (Iravani et al. 2005).
Gene-based models
More recently, the identification of genetic mutations of PD has enabled the
creation of etiologic-specific PD animal models. These include genetically modified
mice with null mutations of parkin, DJ-1 and PINK1 genes (knock-out mice), an extra
gene copy of �-synuclein and LRRK2, or point mutations of genes located in different
PARK loci (Goldberg et al. 2003, Itier et al. 2003, Palacino et al. 2004, Von Coelln et
al. 2004, Smith et al. 2006, Chesselet 2007, Dodson and Guo 2007, Kitada et al. 2007,
Sang et al. 2007, Yamaguchi et al. 2007, Yang et al. 2007, Zhu et al. 2007). Additional
51

INTRODUCTION
mouse models, such as Pitx3-aphakia mouse or Engrailed double knock-out mouse,
were developed on the basis of genes crucial for development and survival of DAergic
neurons (Nunes et al. 2003, Sgado et al. 2006, Ding et al. 2007). Recently, tissue-
specific knock-out mouse models targeting the expression of genes of interest in a
region- or neuron-specific manner were created (Gelman et al. 2003, Ekstrand et al.
2004, Lindeberg et al. 2004, Backet et al. 2005, Zhuang et al. 2005, Backman et al.
2006, Borgkvist et al. 2006, Turiault et al. 2007).
52
As there is no experimental PD model to date that mimics exactly the
pathogenesis and progression happening in PD, one priority at this time is to develop an
experimental model of PD that reproduces the clinical and pathological features of PD,
such as progressive loss of DAergic neurons in the striatum and SN, LB-like inclusion
in the brain, and L-DOPA-responsive movement disorder.

ALUMINIUM
General features
INTRODUCTION
The human organism is constantly and inevitably exposed to aluminium, a
ubiquitous metal which is the third most abundant element in the Earth‟s crust (after
oxygen and silicon), representing 8.3% of total components (Becaria et al. 2002). Its
atomic number is 13 and its electronic configuration is [Ne]3s 2 3p 1 . Aluminium has two
isotopes, Al 27 which has a natural abundance of 99.9% and Al 26 with a half-life (t1/2) of
7.2 � 10 5 years. In nature, aluminium does not occur in the elemental state but is found
in combination with oxygen, fluorine, silicon, sulphur and other species (Brusewitz
1984, Wagner 1999). The physical, chemical and biological properties of the aluminium
complexes will determine the toxicokinetics of this metal (Harris et al. 1997).
Aluminium speciation
Aluminium forms a wide range of hydroxyl complexes in water solution,
evolving from Al 3+ towards [Al(OH)4] - within the 3-8 pH range (Reaction 8; Figure 21).
Reaction 8:
[Al(H2O)6] 3+ � [Al(H2O)5(OH)] 2+ � [Al(H2O)4(OH)2] + � Al(OH)3 � [Al(OH)4] -
Actually, in aqueous solution at pH < 5.0, the prevalent mononuclear Al species is the
octahedral hexahydrate [Al(H2O)6] 3+ (generally abbreviated as Al 3+ ). In less acidic
solutions with pH values greater than 5.0, [Al(H2O)6] 3+ undergoes hydrolysis that yield
different species such as [Al(H2O)5(OH)] 2+ (abbreviated as [Al(OH)] 2+ ) and
[Al(H2O)4(OH)2] + (abbreviated as [Al(OH)2] + ). But not only mononuclear aluminium
species exist in aqueous solution, as it is inclined to polymerize forming the unstable
dimer [Al2(OH)2(H2O)8] 4+ and the more stable polymers [Al6(OH)12] 6+ , [Al10(OH)22] 8+
53

INTRODUCTION
and [Al13(OH)30] 9+ . The solid Al(OH)3 is the predominant species in the neutral pH
range, whereas the soluble tetrahedral aluminate [Al(OH)4] - predominates above pH 8.
Figure 21: Mole fraction of soluble aluminium ions as a function of solution pH in aqueous solution
(Priest 2004)
Sources of aluminium exposure
Environmental exposure
54
Aluminium and its compounds are widely distributed in the environment
although most naturally occurring aluminium is bound and not readily bioavailable. As
example, this metal is present in a variety of minerals, such as silicates (feldspars and
micas), complexed with fluorine and sodium as cryolite, and is chiefly mined as
bauxite, a mineral containing 40-60% aluminium oxide (alumina) (Ganrot 1986).
Aluminium is a major constituent of atmospheric particulates, primarily found as
alumino-silicates, originating from natural soil erosion, mining or agricultural activities,
volcanic eruptions, or coal combustion. Atmospheric aluminium concentrations were
reported to range generally from 5 to 180 µg/m 3 (Sorenson et al. 1974) whereas
concentrations in industrial areas are often in the milligram per cubic meter range.

INTRODUCTION
Varying amounts of aluminium are present naturally in groundwater and surface
water, including those used as sources of drinking water. Acidification of lakes and
streams by acid rain mobilize aluminium from the soil to the aquatic environment
increasing the amount of this metal (Cronan and Schoefield 1979, Harris et al. 1996).
Aluminium concentrations in natural water normally are small but may vary in the
urban areas (Constantini and Giordano 1991) depending if aluminium flocculents (most
commonly alum or aluminium sulphate) are used during the treatment process for
purification purposes for clarifying turbid drinking water (Martin 1986, Lévesque et al.
2000).
Dietary exposure
Aluminium is found in the tissues of many plants and animals. The
concentration in foods varies widely, depending upon the product, the type of
processing, and the geographical origin (means range from

INTRODUCTION
Iatrogenic exposure
56
In short, the major route of aluminium exposure for an adult is food
(approximately 95%), drinking water only contributes

INTRODUCTION
and antiperspirants containing aluminium chlorohydrate (Flarend et al. 2001) represent
other possible sources of exposure.
Aluminium toxicokinetics
Absorption of aluminium
Aluminium may be absorbed by several routes: oral, intranasal, transdermal, and
parenteral pathways. The major sites of aluminium absorption are the gastrointestinal
tract (Ittel 1993), the skin (Exley 1998, 2004), and the olfactory and oral epithelia
(Roberts 1986).
Oral absorption
The gastrointestinal tract is one of the main sites of aluminium absorption (Ittel
1993). The proportion of absorbed aluminium following oral intake is very low ranging
from 0.06 to 1.5% (Moore et al. 2000, Flaten et al. 2001, Yokel et al. 2001a). The
amount of aluminium absorbed across the gastrointestinal tract depends on many
factors, including pH, aluminium speciation and dietary agents (Partridge et al. 1989,
Deng et al. 1998). Moreover, the intestinal absorption of aluminium was shown to
increase in various pathological conditions, such as AD (Moore et al. 2000). As an
example, a low pH will enhance the solubility of aluminium species then increasing
aluminium absorption. Small organic acids also present in the diet of man, including
citrate, lactate, and ascorbic, gluconic, malic, oxalic, and tartaric acids, are able to
prevent its precipitation during transit as they complex with the metal, increasing its
absorption from the gastrointestinal tract (Deng et al. 2000, Venturini-Soriano and
Berthon 2001, Whitehead et al. 1997), and to aument tissue retention of aluminium in
rats orally dosed with aluminium (Domingo et al. 1991, 1994). On the other hand
phosphorus, silicone and other dietary factors such as phytate and polyphenols seem to
decrease absorption (Yokel and O‟Callaghan 1998, Powell and Thompson 1993, Powell
57

INTRODUCTION
et al. 1993), probably through formation of insoluble complexes with aluminium ion in
the gut (Schaefer et al. 1988). Three mechanisms, reviewed by Greger and Sutherland
(1997), were proposed to explain the increase in aluminium absorption provoked by
citrate: enhanced aluminium solubility in the gastrointestinal tract, transport of
aluminium citrate into mucosal cells, and opening of epithelial tight junctions that are
present between mucosal cells (Taylor et al. 1998).
58
Aluminium absorption does not seem to happen in the stomach where most
aluminium is converted to soluble monomolecular species at low pH (Froment et al.
1989). At near-neutral pH aluminium precipitation occurs in the intestine.
Consequently, the small portion of aluminium accessible for transport is the part that
has been complexed with organic molecules in the stomach, allowing it to remain
soluble at higher pH of the small intestine (Reiber et al. 1995). The primary site of
aluminium absorption is the proximal intestine (Greger and Sutherland 1997). The
precise mechanism of gastrointestinal absorption is not yet fully known. It has been
suggested that intestinal absorption of aluminium occurs paracellularly along
enterocytes and through tight junctions by passives processes (Exley et al. 1996) and
transcellularly through enterocytes involving passive facilitated and active transport
processes, such as calcium uptake and sodium transport processes, and a role for
transferrin (Greger 1993, Greger and Sutherland 1997). Interestingly, it was suggested
that each aluminium species likely has its own absorption mechanism (Van der Voet
1992).
Intranasal absorption
Inhalation exposure to aluminium occurs from cosmetic (aerosols),
environmental and occupational sources (fumes, dusts, flakes). Inhaled aluminium was
suggested to accumulate in the brain through absorption via the olfactory system
(Roberts 1986, Exley et al. 1996) or through systemic absorption via the lung epithelia
(Gitelman et al. 1995) and through the gastrointestinal tract as particulates are
swallowed (Rollin et al. 1993). Pulmonary absorption seems to be more efficient than
gastrointestinal absorption. Actually, Jones and Benett (1986) estimated that

INTRODUCTION
approximately 3% of aluminium is absorbed into the blood from the lung. Although
there is still controversy regarding the ability of aluminium to enter the brain from nasal
cavity, it has been suggested that aluminium may be able of directly entering the brain
from the nose through olfactory neurons. These latter, which are the only part of the
CNS with direct contact to external milieu, are located in the roof of the nasal cavity
and project to the olfactory bulb. These neurons synapse with complex pathways in the
olfactory bulb which subsequently project to other cerebral areas, such as the olfactory
cortex, cortex, and hippocampus. A few studies tried to demonstrate that aluminium
distribution in the brain occurs through olfactory nerve uptake, axonal and trans-
synaptic transport. Absorption of aluminium from the olfactory pathway has been
studied in rabbits exposed to aluminium lactate application in the upper nasal cavity for
one month. This resulted in aluminium accumulation in the olfactory bulb, pyriform
cortex, hippocampus and cerebral cortex (Perl and Good 1987) although this prolonged
exposure may probably have led to mechanical disruption of the olfactory epithelia
(Lewis et al. 1994). Rats exposed to aluminium acetylacetonate had aluminium deposits
in the olfactory bulb, ponsmedulla and hippocampus (Zatta et al. 1993). One nose-only
exposure study showed that aluminium was also distributed to the brain stem of rats
using aluminium chlorohydrate (Divine et al. 1999).
Dermal absorption
Aluminium chloride was shown to be absorbed through the skin when applied to
the skin of mice, and to accumulate in both serum and brain, especially in the
hippocampus (Anane et al. 1995). Nevertheless, systemic absorption of the metal may
have been enhanced by mice shaving prior to its application. As aluminium
chlorhydrate is the active ingredient extensivey used in antiperspirants, the skin was
predicted to be another route of aluminium entry into the systemic circulation (Exley
2004b). Aluminium chlorhydrate is thought to precipitate inside the eccrine sweat
glands and to form insoluble aluminium hydroxyde, which then physically blocks the
sweat duct (Quartrale 1988, Teagraden et al. 1982) but its efficacy in reducing
perspiration may also be due to chemical inhibition of the sweat gland (Strassburger and
59

INTRODUCTION
Coble 1987). Transdermal absorption of only 0.012% has been reported after a one-time
underarm application of 26 Al chlorhydrate (Flarend et al. 2001) while a case report
showed that transdermal uptake of aluminium may also be important (Guillard et al.
2004).
Distribution of aluminium in the body
60
Once absorbed into the bloodstream, aluminium circulates bound to various
plasma proteins: 93% is bound to transferrin, 6% to citrate and the remaining to
hydroxide and phosphate (Harris et al. 2003). The metal is distributed unequally to all
tissues within the body throughout normal and aluminium-intoxicated individuals
(Alfrey et al. 1980, Di Paolo et al. 1997), and aluminium-treated experimental animals
(Greger and Sutherland 1997). The highest levels of aluminium in mammalian tissues
are found in the skeleton and lungs, approximately 50% and 25% of the 30 to 50 mg
aluminium body burden in the healthy human subject (Alfrey 1984, Ganrot 1986,
ATSDR 1999). Aluminium also accumulates in human brain, skin, lower
gastrointestinal tract, lymph nodes, adrenals, and parathyroid glands (Tipton and Cook
1963, Hamilton et al. 1973, Cann et al. 1979, Alfrey 1980).
Aluminium accumulation in different target organs varies with the aluminium
salt administered, species studied and route used, dose and duration of exposure (Ding
and Zhu 1997, Yokel and McNamara 1988), but also with age, kidney function, disease
status, and dietary compounds (Greger 1993). The human brain contains lower levels of
aluminium when compared to other organs (Walker et al. 1994, Andrási et al. 2005,
Yokel and McNamara 2001). In the case of dialysis encephalopathy, this concentration
may aument drastically (Alfrey et al. 1976).

Excretion
INTRODUCTION
As insoluble aluminium hydroxyl coumpounds are formed at neutral pH most of
the dietary aluminium is excreted in the faeces without ever being absorbed. If renal
functions are not compromised approximately 95% of the absorbed aluminium is
quickly excreted in the urine by kidneys, presumably as aluminium citrate. Biliary
system accounts for less than 2% of total aluminium elimination (Kovalchik et al. 1978,
Priest et al. 1995, Yokel et al. 1996). Decreased renal functionality increases the risk of
aluminium accumulation and toxicity (Greger and Sutherland 2007). Indeed, dialysis
encephalopathy syndrome (DES) was developed by renal-impaired people who received
aluminium in dialysis fluids or parenterally (Alfrey et al. 1976).
Elimination rate
Aluminium accumulation is not only due to impaired renal functions or exposure
to high quantities of aluminium but also occurs physiologically with aging (McDermott
et al. 1979). Aluminium contents of brain, serum, lungs, blood, liver, kidneys, and bone
have been demonstrated to increase with age (Markesbery et al. 1984, Zapatero et al.
1995, Greger and Sutherland 1997, Shimizu et al. 1994, U. S. Public Health Service
1992, Stitch 1957, Tipton and Shafer 1964, Roider and Drasch 1999, Markesbery et al.
1981) and younger individuals absorbe less aluminium than older people. Actually, it
has been estimated that aluminium deposits in the brain at a rate of 6 μg per year of life
(Edwardson 1991). This increasing body burden with age in the brain may be produced
by a slow, or no, elimination of aluminium coupled to a continued exposure to the metal
and a decreased ability to remove the metal from the brain with age. Different
aluminium half-lives (t½) have been reported suggesting that there is more than one
compartment of aluminium storage from which the metal is slowly eliminated (Wilhelm
et al. 1990, Ljunggren et al. 1991, Priest et al. 1995). The t½ of aluminium elimination
positively correlates with the duration of the exposure as longer t½ were observed when
the duration of sampling after exposure was increased. Bone stores about 58% of the
human aluminium body burden and its aluminium clearance is more rapid than from
brain, which is logical regarding to bone turnover and lack of neuron turnover.
61

INTRODUCTION
Aluminium influx and efflux into the brain
62
The brain has lower aluminium concentrations (about 1%) than many other
tissues (Yokel and McNamara 2001), but it is an important target organ for aluminium-
toxicity. Even as the BBB is highly selective to a great variety of substances, including
metals, aluminium is able to cross it and enter the brain from blood (Yokel 2002b) and
to accumulate in nerve and glial cells. Although the mechanism(s) responsible for
aluminium transport at the BBB has not been clarified yet, it has been reported that
aluminium can penetrate into the brain as a complex with transferrin by a receptor-
mediated endocytosis (Roskams and Connor 1990) and also bound to citrate via a
specific transporter, being the system Xc � (L-glutamate/L-CySH exchanger) the most
recently accepted candidate (Nagasawa et al. 2005). There is also evidence for
transporter-mediated efflux from the brain, the organic anion transporting polypeptide
(oatp) family was suggested to be a candidate (Yokel 2006).
The existence of a long apparent half-life of aluminium in brain tissue has been
used to explain its easy accumulation into the brain following aluminium administration
(Yokel et al. 2001b, Baydar et al. 2003, Sánchez-Iglesias et al. 2007b). The elimination
half-life of aluminium from human brain is calculated to be seven years (Yokel and
McNamara 2001). This assumption and the long life of neurons could be involved in the
elevated levels of aluminium found in the brain of some patients suffering PD (Hirsh et
al. 1991, Good et al. 1992, Yasui et al. 1992) and AD (Crapper et al. 1973, Perl and
Brody 1980), a statement that, on the other hand, will not be understoo as the principal
cause of those disorders.
Since aluminium and its compounds are widely distributed in the environment
(food, drinking water, airborne contaminants) and extensively used in a variety of
products and processes (antiperspirants, antacids, intravenous solutions), it has aroused
considerable health concern, due to both its common exposure and its particularly
reported ability to cause neurodegeneration (Exley 1999, Yokel 2000, Zatta et al. 2003,
Kawahara 2005). As we have previously seen, the daily reported mean dietary intake of

INTRODUCTION
3.5 mg may be increased by the frequent use of aluminium-containing antiperspirants
and non-prescription drugs (Weburg and Berstad 1986, Flarend et al. 2001), as well as
by occupational exposure to this metal (Meyer-Baron et al. 2007).
Putative mechanisms of aluminium neurotoxicity
No biological function has yet been attributed to aluminium, for which reason it
is considered a nonessential metal (Yokel 2002a). However, aluminium neurotoxicity
was first outlined by Dölken in 1897. Since then, aluminium accumulation in the brain
has been repetitively linked to various neurodegenerative diseases (Yokel 2000, Zatta et
al. 2003, Kawahara 2005). As a matter of fact, this non-redox active metal has been
demonstrated to be a toxicant that is implicated in dialysis encephalopathy (Alfrey et al.
1976), osteomalacia (Parkinson et al. 1979), non-iron responsible anemia (Elliot et al.
1978), and also linked to many other diseases including AD (Exley 1999, Flaten 2001,
Gupta et al. 2005), amyotrophic lateral sclerosis (Kurland 1988), and PD (Dexter et al.
1989, Yasui et al. 1992, Good et al. 1992). Aluminium was shown to be
overaccumulated in the brain of post-mortem patients compared to healthy individuals
(Perl et al. 1982, Yasui et al. 1992, Ward and Mason 1987)
Many reviews have been published on the toxic effects of aluminium.
Nevertheless, and despite all hitherto reported data concerning aluminium neurotoxicity,
no single unifying mechanism responsible for its neurotoxicity has been identified.
Therefore, several interlinked hypotheses have been proposed, highlighting distinct
events as a basis for the beginning of aluminium toxic brain damage.
63

INTRODUCTION
Aluminium and oxidative stress
64
The model involving aggravation of oxidative stress is among the most
recognized hypothesis of aluminium neurotoxicity but the precise molecular mechanism
by which it causes oxidative damage into the brain remains uncertain. Actually, a large
number of studies confirmed the existence of a state of oxidative stress in most of the
neurodegenerative disorders in which aluminium is present in relative high quantities.
Numerous publications have detailed an increase in the formation of ROS after
aluminium exposure both in vivo and in vitro (Katyal et al. 1997, Flora et al. 2003,
Ogasawara et al. 2003, Nehru and Anand 2005). However, the reported results as a
whole appear controversial, to such an extent that both pro-oxidant (Zatta et al. 2002,
Exley 2004a) and antioxidant (Oteiza et al. 1993a, Abukadar et al. 2004a) properties
have been attributed to this metal.
The brain: a target for aluminium
The brain is a main target for aluminium neurotoxicity as it is highly susceptible
to oxidative insult for several reasons: (a) a high oxygen turnover as the brain consumes
a proportionally larger amount of oxygen than other organs, around 20% of the body‟s
oxygen (Halliwell 1992), (b) a low mitotic rate, (c) low concentrations/activities of
brain antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD), and
glutathione peroxidase (GPx), (d) (Halliwell and Gutteridge 1985) a large amount of
highly oxidizable polyunsaturated fatty acids in neuronal cell membranes (Table 5), and
(e) a high brain content of iron (Youdim 1988).
Several hypotheses have been given to explain the possible link between
aluminium and the promotion of oxidative stress (Exley 2004a). Actually, even though
aluminium is not a transition metal, and consequently does not undergo redox reactions,
it can cause oxidative stress both in vivo and in vitro through multiple mechanisms. As
examples, it has been shown to stimulate in vitro iron-induced and non-iron-induced
lipid peroxidation (Gutteridge et al. 1985, Verstraeten and Oteiza 2000, Meglio and

INTRODUCTION
Oteiza 1999), non-iron-mediated oxidation of NADH (Meglio and Oteiza 1999, Kong et
al. 1992), and non-iron-mediated formation of the � OH (Méndez-Álvarez et al. 2002).
Table 5: Lipid composition of normal adult human brain (dry weight)
(Zatta et al. 2002)
Gray matter (%) White matter (%)
Cholesterol 22 27.5
Total phospholipids 69.5 45.9
Phosphatidylserine 8.7 7.9
Galactocerebroside 5.4 19.8
Galactocerebroside sulphate 1.7 5.4
Aluminium catalyses iron-driven biological oxidations
Aluminium was also shown to potentiate the capacity of pro-oxidants transition
metals, such as iron and copper which are present in most cell compartments, to
produce oxidative stress (Bondy and Kirstein 1996, Bondy et al. 1998). It was
hypothesized that colloidal aluminium may complex these pro-oxidant metals and
permit them to participate in Fenton reaction for an extended time (Yang et al. 1999,
Campbell et al. 2001).
Aluminium stimulates superoxide-/non-iron-driven biological oxidations
We have seen that O2 ●─ is the ROS with the lowest oxidant capacity but it can
undergo a one-electron transfer to generate H2O2 which can in turn through the Fenton
reaction and in the presence of reduced metal ions, be converted to ● OH, increasing its
redox potential. Various studies proposed that aluminium could enhance the O2 ●─ -
mediated oxidation in different systems that generate this ROS: the photochemical
decomposition of rose Bengal (Kong et al. 1992, Meglio and Oteiza 1999), the
autoxidation of 6-OHDA (Méndez-Álvarez et al. 2002), the vanadium-mediated
oxidation of NADH (Adler et al. 1995), and the xanthine/xanthine oxidase system
65

INTRODUCTION
(Kong et al. 2002). In the same way, trivalent cations chemically and physically related
to aluminium (lanthanum, gallium and scandium) were shown to increase O2 ●─
oxidative capacity (Meglio and Oteiza 1999).
Aluminium-superoxide complex theory
66
It was hypothesized that the formation of a complex between Al 3+ and O2 ●─ may
explain the pro-oxidant activity of aluminium and also the catalytic activity connecting
both superoxide-driven and iron-driven biological oxidations. Aluminium is a strong
Lewis acid which may react with the superoxide anion and form an aluminium
superoxide anion, which is a more potent oxidant than the superoxide anion on its own
(Kong et al. 1992, Exley 2004a).
Al 3+ + O2 ●─ ↔ AlO2 ●2+
As an example, the iron-supported oxidation of DA to melanin was shown to be
facilitated by aluminium (Di and Bi 2003). The subsequent binding of melanin with
aluminium leads to an increased melanin-induced lipid oxidation in a process mediated
by an Al–O2 ●─ complex (Meglio and Oteiza 1999, Verstraeten et al. 2008).
Aluminium inhibits the antioxidant enzymes and others
Additionally, aluminium also appears to affect the activities of several
antioxidant enzymes in different parts of the brain and cause oxidative damage (Nehru
and Anand 2005, Julka and Gill 1996a, Atienzar et al. 1998, Moumen et al. 2001,
Gómez et al. 2005, Sánchez-Iglesias et al. 2009). The decrease of the activity of
antioxidative enzymes may facilitate the propagation of lipid peroxidation. In addition,
the ability of aluminium to affect the functionality of mitochondria has also been
documented (Niu et al. 2005).

Aluminium as a cell membrane menace
INTRODUCTION
Numerous studies performed both in vitro and in vivo considered that the
principal consequences of aluminium on the cerebral functions are mediated through
damage to cell membranes. Metals without redox capacity as aluminium were suggested
to make fatty acids more available to the action of free radicals and therefore ease the
spread of lipid peroxidation (Oteiza et al. 1993a, Ohyashiki et al. 1998).
Actually, aluminium binds to membrane components and modifies both
membrane biophysical properties and dynamics. Binding of aluminium is enhanced by
the presence of polar head groups with negative charge and happens via formation of
both cis and trans complexes (Oteiza 1994, Verstraeten 2000). This metal was shown to
bind through electrostatic interactions preferentially to the phosphatidylserine,
phosphatidic acid, and poliphosphoinositides found in biological membranes because of
their overall negative charge due to their carboxylate or phosphate moieties. Trans-
interactions or intervesicle interactions are not specifically implicated in lipid oxidation
as they result in membrane aggregation and fusion (Verstaeten and Oteiza 1995).
Actually, superficial charges are neutralized by aluminium which causes the
intercrossing and dehydration of neighboring phosphatidylserine molecules. On the
other hand, cis-interactions within the same vesicle lead to the lateral reorganization of
these phospholipids (lipid clustering) and formation of distinct microdomains enriched
in acidic phospholipids with lower membrane fluidity (Verstraeten and Oteiza 1995,
Verstraeten et al. 1997a, 1998). The local accumulation of poly-unsaturated fatty acids
caused by aluminium makes the phospholipids higly susceptible to ROS, leading to the
propagation of lipid peroxidation.
Aluminium was also shown to modify the biophysical properties of membranes
containing galactocerebrosides or polyphosphoinositides (Verstraeten et al. 1998, 2003,
Verstraeten and Oteiza 2002), myelin and synaptosomal membranes, which may result
in injurious effects for neurotransmission (Ohba et al. 1994, Julka and Gill 1996b,
Verstraeten et al. 1997b, Silva et al. 2002, Pandya et al. 2004).
67

INTRODUCTION
68
As mentioned before, aluminium is able to stimulate iron-induced oxidations. In
the peroxidation process both metals are thought to act synergistically: while the
aluminium binding to neuronal membranes is attended to facilitate its attack by iron-
mediated oxidative damage, the subsequent oxidation of the membrane will
consecutively increase its binding to aluminium, hence exacerbating peroxidation
(Figure 22).
Figure 22: Effects of aluminium and iron on the membrane peroxidation (Zatta et al. 2002,
Oteiza et al. 2004)
Lipid peroxidation is particularly active in the brain where it induces: a) changes
in the structure of biological membranes resulting in the loss of membrane fluidity, b)
changes of the structural order of membrane lipids (Palmeira and Oliveira 1992), c)
variations in membrane potential, d) an increase in membrane permeability to ions
(Marshansky et al. 1983), e) modifications in the activity of membrane-bound enzymes
(Pereira et al. 1996), and f) alterations in receptor functions.
Aluminium affects cell signaling
Another mechanism that has been suggested to be involved in aluminium
toxicity is the alteration of specific cell signaling cascades. Aluminium interferes with
phosphoinositide signal transduction pathway (Nostrandt et al. 1996, Quintal-Tun et al.
2007). This metabolic pathway mediated by the enzyme phosphatidylinositol-specific
phospholipase C (PI-PLC) renders inositol triphosphate (IP3) and diacylglycerol, two
important intracellular second messengers. IP3 is in charge of the intracellular

INTRODUCTION
mobilization of Ca 2+ with the consequent activation of multiple signals, whereas
diacylglycerol activates enzymes such as protein kinase C (PKC). Aluminium leads to a
decrease in the hydrolysis of phosphatidylinositol biphosphate (PIP2) both in vivo and in
vitro (McDonald and Mamrack 1988, Shafer et al. 1993, McDonald and Mamrack 1995,
Shafer and Mundy 1995, Nostrandt et al. 1996). As we have seen before, aluminium
preferential binding to negative charges of polyphosphoinositides causes partial
neutralization of negative charge, clustering, and increased local concentration of these
lipids. All these together result in a limited accessibility of PI-PLC to its substrates and
the subsequent decreased in the enzyme activity (Figure 23) (Verstraeten and Oteiza
2002, Verstraeten et al. 2003). Signaling cascades involving either binding of regulatory
proteins to polyphosphoinositides in the membranes or involving phosphatidylinositol
(PI)-derived second messengers may be negatively altered by aluminium.
Figure 23: PIP2 hydrolysis altered by aluminium (Oteiza et al. 2004)
Inflammation generally accompanies neurodegenerative diseases. One of the
initial events in the cascade leading to inflammatory responses is the activation of
transcription factors. The cytokine TNF-α activates transcription factor NF-κB which
consecutively accelerates the transcription of specific genes involved in inflammation,
such as other cytokines, iNOS, and complement factors by translocating to the nucleus
and binding to their promoter regions. Aluminium was reported to cause an
inflammatory response in the brain both in vivo and in vitro (Yokel and O‟Callaghan
1998, Ghribi et al. 2001a, Platt et al. 2001, Campbell et al. 2002, Becaria et al. 2003,
Johnson and Sharma 2003). The metal activates NF-κB and TNF-α expression leading
to cell death and proliferation of reactive glial cells rising tissue damage (Campbell et
al. 2002, 2004).
69

INTRODUCTION
70
The signal transduction pathway of mitogen-activated protein kinase (MAPK)
plays also an important role in the apoptosis in neurons or astrocytes. Aluminium was
demonstrated to activate in cultured cortical neurons one of the important members in
MAPK family, the stress-activated protein kinase c-jun N-terminal kinase (SAPK/JNK)
(Fu et al. 2003), whose phosphorylation and dephosphorylation are considered as the
molecular key in stress signal transduction. SAPK/KNK activates its downstream
transcription factor c-jun, which is the important component of AP-1. This latter
modulates the expression of target genes and participates in the regulation of cell
differentiation and apoptosis.
Neuronal accumulation of aluminium affects mitochondria integrity and
functionality (reviewed by Kumar and Gill 2009). The metal enters into the neuron
upon cell depolarization and inhibits Na + /Ca 2+ exchange thus inducing an extreme
accumulation of mitochondrial Ca 2+ . This triggers the opening of the mitochondrial
transition pore (MTP) and the consequent release of cytochrome c leading finally to
activation of the caspase family proteases. Studies that used intracisternal injection of
the aluminium-maltolate complex have shown that aluminium targets mitochondria.
Aluminium-maltolate is a lipophilic complex stable at physiological pH and proposed to
be formed in the gastrointestinal tract. This neurotoxic complex produces cytoskeletal
alterations (Klatzo et al. 1965, Savory et al. 1996, 1998) leading to tangle formation and
cell apoptosis (Johnson et al. 2005). Aluminium-maltolate induces cytochrome c
translocation, increased expression of the proapoptotic protein Bax, and decreased
expression of the antiapoptotic protein Bcl-2, finally leading to cell apoptosis (Ghribi et
al. 2001a, 2002).
In addition, aluminium also affects the endoplasmic reticulum (ER), the major
storage location for calcium, which also contains members of the Bcl-2 family, such as
Bcl-2 and Bcl-XL. The stress induced by aluminium-maltolate activates caspase 12
leading to a specific type of ER-mediated cell death by apoptosis (Ghribi et al. 2001b,
2001c, 2002).

Aluminium impairs neurotransmission
INTRODUCTION
Aluminium has a negative impact on the main steps of the neurotransmission
event which takes place at the synapse, such as synthesis and storage of
neurotransmitters, triggered release from the presynaptic terminal, interaction with
receptors on the postsynaptic cell, and inactivation of neurotransmitters (Gonçalves and
Silva 2007). The metal may alter the physical properties of synaptic membranes which
could influence the release and/or uptake of neurotransmitters, or directly inhibit the
enzymes in charge of the synthesis, utilization and degradation of these molecules.
There are evidences demonstrating that this metal disturbs some crucial events of DA
neurotransmission. The DA metabolism appears to be altered in animal models of
aluminium: DA levels are reduced by 40% in the striatum (Ravi et al. 2000) and the
concentration of DA metabolites such as dihydroxyphenylacetic acid (DOPAC) and
homovanillic acid (HVA) are lower in the hypothalamus of treated animals (Tsunoda
and Sharma 1999). Besides, the ability of aluminium to affect the expression of DA
receptors D1 and D2 (Kim et al. 2007) and to inhibit DA-β-hydroxylase, the enzyme in
charge of converting DA into noradrenaline (Milanese et al. 2001), has also been
documented. Aluminium interferes also with glutamatergic, cholinergic, and
GABAergic metabolisms. Actually, aluminium exposure in rats leads to decreased
acetylcholine synthesis and degradation, reduction of muscarinic acetylcholine receptors
(Julka et al. 1995), increased contents of glutamine and glutamate, and decreased
GABA level (El-Rahman 2003, Nayak and Chatterjee 2003). Alterations of calcium
metabolism may also be of importance for aluminium neurotoxicity as calcium has a
crucial role in neurotransmitter release. As an example, aluminium inhibits GABA
synaptosomal release and uptake as a consequence of the inhibition of Ca 2+ /CaM-
dependent calcineurin activity (Cordeiro et al. 2003).
71

AIMS

AIMS OF THE PRESENT THESIS
The main objectives of this thesis were distributed in three chapters as follows:
Chapter 1
AIMS
The first principal aim is to determine the kinetics of the oxidative damage induced in a
6-OHDA model of PD and quantify the resulting oxidative effects in order to set up the
accurate post-injection time when studying the oxidative effects of aluminium. To
achieve this aim we have stablished the following specific objectives:
1) To analyse the time-course of the oxidative damage caused by unilateral and
intrastriatal administration of 6-OHDA (6 μg in 5 μl of sterile saline containing
0.2% ascorbic acid) in both the ipsilateral and contralateral sides of both striatum
and ventral midbrain of the rat.
2) To quantify the changes observed in the indices of lipid peroxidation (TBARS) and
oxidative status of proteins (PCC and PTC) in the time-course of brain oxidative
damage induced by 6-OHDA injection in the experimental model of PD mentioned
above.
Chapter 2
The second main objective is to elaborate a dosage procedure that ensures a significant
accumulation of aluminium into the brain and to establish its exact distribution in
different areas of the rat brain. In this case, the specific objective is:
3) To evaluate the precise regional accumulation of the absorbed aluminium in
cerebellum, ventral midbrain, cortex, hippocampus and striatum of rats following
the use of two different administration routes, oral and intraperitoneal.
75

AIMS
Chapter 3
The third major aim of this thesis is to clarify the capacity of aluminium to modify the
oxidant level of certain brain regions and to augment the striatal DAergic
neurodegeneration in a 6-OHDA model of PD. The specific objectives to reach this aim
are:
4) To investigate the in vivo effects induced by aluminium on indices of oxidative
76
stress by quantification of lipid peroxidation (TBARS) and the oxidant status of
proteins (PCC, PTC), in the cerebellum, ventral midbrain, cortex, hippocampus, and
striatum of rats.
5) To assess the in vivo effects of aluminium on the activity of certain antioxidant
enzymes, such as SOD, GPx and CAT, in the cerebellum, ventral midbrain, cortex,
hippocampus, and striatum of rats.
6) To measure the in vitro effects of aluminium on the activity of some antioxidant
enzymes (SOD, GPx, CAT) and the monoamine oxidase activity (MAO-A and
MAO-B).
7) To study the ability of aluminium to potentiate the capacity of 6-OHDA
administered in the third ventricle in an experimental model of PD to cause
oxidative stress (lipid peroxidation and protein oxidation) in ventral midbrain and
striatum and to induce neurodegeneration in striatal DAergic terminals.

OBJETIVOS

OBJETIVOS DE LA PRESENTE TESIS
OBJETIVOS
Los objetivos principales de esta tesis fueron distribuidos en tres capítulos de la
siguiente manera:
Capítulo 1
El primer objetivo principal es determinar la cinética de las lesiones oxidativas
producidas en un modelo experimental de Parkinson con 6-OHDA y cuantificar los
efectos oxidativos resultantes. Este estudio nos permitirá establecer el tiempo exacto
después de la inyección con 6-OHDA en el que estudiar los efectos oxidativos del
aluminio. Los objetivos concretos son:
1) Analizar el curso temporal del daño oxidativo causado por la administración
unilateral e intrastriatal de 6-OHDA (6 µg en 5 µl de salino estéril con 0.2% ácido
ascórbico) en los lados ipsilaterales y contralaterales del estriado y mesencéfalo
ventral de la rata.
2) Cuantificar los cambios observados en los níveles de peroxidación lipídica
(TBARS) y del estado oxidativo de las proteínas (contenidos de grupos carbonilo y
tiol en proteínas) durante el curso temporal del daño oxidativo cerebral inducido
por la inyección de 6-OHDA en el modelo experimental de la enfermedad de
Parkinson mencionado anteriormente.
Capítulo 2
El segundo objetivo principal consiste en elaborar un régimen de dosis en el que se
garantice una acumulación significativa de aluminio en el cerebro y establecer la
distribución exacta del metal en las regiones cerebrales de las ratas. En este caso, el
objetivo concreto es:
79

OBJETIVOS
3) Evaluar de forma precisa la acumulación regional del aluminio absorbido en el
80
cerebelo, mesencéfalo ventral, corteza, hipocampo y estriado de las ratas después
de la utilización de dos vías distintas de administración, oral e intraperitoneal.
Capítulo 3
El tercer objetivo principal de esta tesis consiste en esclarecer la capacidad del aluminio
para alterar el estado oxidativo de ciertas regions cerebrales y para aumentar la
neurodegeneración dopaminérgica estriatal en un modelo experimental de Parkinson
con 6-OHDA, siendo los objetivos concretos:
4) Investigar los efectos in vivo inducidos por el aluminio en los índices de estrés
oxidativo, mediante la cuantificación de la peroxidación lipídica (TBARS) y el
estado oxidativo de las proteínas (contenidos en grupos carbonilo y tiol en
proteínas), en cerebelo, mesencéfalo ventral, corteza, hipocampo y estriado de rata.
5) Evaluar los efectos in vivo del aluminio sobre la actividad de determinados enzimas
antioxidantes, tales como la superóxido dismutasa, la glutatión peroxidasa y la
catalasa, en cerebelo, mesencéfalo ventral, corteza, hipocampo y estriado de rata.
6) Cuantificar los posibles efectos in vitro del aluminio sobre la actividad de los
enzimas antioxidantes estudiados (superóxido dismutasa, glutatión peroxidasa y
catalasa) y sobre la actividad de las monoamino oxidasas A y B.
7) Estudiar la capacidad del aluminio para aumentar la capacidad de la 6-OHDA
administrada en el tercer ventrículo en un modelo experimental de la enfermedad de
Parkinson para causar estrés oxidativo (peroxidación lipídica y oxidación protéica)
en el mesencéfalo ventral y estriado y para inducir neurodegeneración en las
terminales dopaminérgicas estriatales.

CHAPTER 1

CHAPTER 1 Time-course of brain oxidative damage
caused by intrastriatal administration of 6-hydroxydopamine in
a rat model of Parkinson’s disease
Sofía Sánchez-Iglesias,* Pablo Rey,† Estefanía Méndez-Álvarez,* José
Luis Labandeira-García† and Ramón Soto-Otero*
*Laboratory of Neurochemistry, Department of Biochemistry and Molecular Biology, Faculty of Medicine,
University of Santiago de Compostela, Santiago de Compostela, Spain
†Laboratory of Neuroanatomy and Experimental Neurology, Department of Morphological Sciences,
Faculty of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain
Neurochem. Res. (2007) 32, 99–105.
83

ABSTRACT
CHAPTER 1
The unilateral and intrastriatal injection of 6-OHDA is commonly used to
provide a partial lesion model of PD in the investigation of the molecular mechanisms
involved in its pathogenesis and to assess new neuroprotective treatments. Its capacity
to induce neurodegeneration has been related to its ability to undergo autoxidation in the
presence of oxygen and consequently to generate oxidative stress. The aim of the
present study was to investigate the time course of brain oxidative damage induced by
6-OHDA (6 μg in 5 μl of sterile saline containing 0.2% ascorbic acid) injection in the
right striatum of male Sprague-Dawley rat. The results of this study show that the
indices of both lipid peroxidation (TBARS) and protein oxidation (PCC and PTC)
increase simultaneously in the ipsilateral striatum and ventral midbrain, reaching a peak
value at 48-h post-injection for both TBARS and PCC, and at 24 h for PTC. A lower but
significant increase was also observed in the contralateral side (striatum and ventral
midbrain). The indices of oxidative stress returned to values close to those found in
controls at 7-day postinjection. These data show that the oxidative stress is a possible
triggering factor for the neurodegenerative process and the retrograde
neurodegeneration observed after the first week post-injection seems to be a
consequence of the cell damage caused during the first days post-injection. Finally, the
optimal time to assess brain indices of oxidative stress (TBARS, PCC and PTC) in this
model is 48-h post-injection.
Keywords: Parkinson‟s disease, 6-hydroxydopamine, oxidative damage, lipid
peroxidation, protein oxidation.
85

CHAPTER 1
INTRODUCTION
86
6-OHDA (2,4,5-trihydroxyphenylethylamine; 6-OHDA) is a selective
catecholaminergic neurotoxin widely used to produce DAergic lesions in the
nigrostriatal system (Dauer and Przedborski 2003). The main use of these lesions is to
create experimental models of PD that provide insight into the molecular mechanisms
involved in the development of PD in order to test new strategies for DAergic
neuroprotection, and to experiment with transplantation approaches (Aebischer et al.
1991, He et al. 2001, Soto-Otero et al. 2002, Muñoz et al. 2004, López-Real et al.
2005). The structural similarity of 6-OHDA with both DA and noradrenaline makes it
an appropriate ligand for the plasma membrane transporter systems of DA (DAT) and
noradrenaline (Luthman et al. 1989). This feature, together with the inability shown by
6-OHDA to cross the BBB (Garver et al. 1975), makes the protocol chosen for a
specific DAergic lesion in the nigrostriatal system extremely important. The main
protocol used consists of the stereotaxic intracerebral injection of the toxin into a
specific area of the nigrostriatal systems, which includes the stria tum, the medial
forebrain bundle or the SN (Javoy et al. 1976). The intrastriatal injection of 6-OHDA is
generally performed unilaterally, using the contralateral side as a control (Ungerstedt
1971). It has been shown that the intrastriatal administration of 6-OHDA causes a rapid
degeneration of nigrostriatal terminals as early as 24 h, and the loss of tyrosine
hydroxylase immunoreactivity (TH-ir) increases for up to 5 days following the lesion
(Ichitani et al. 1991).
Presumably, the neurotoxicity of 6-OHDA is attributed to its ability to generate
ROS, and it is a well-known fact that under physiologyical conditions this compound is
rapidly oxidized by molecular oxygen to give H2O2, � OH and the corresponding p-
quinone. Although, the Fenton reaction could be involved in the formation of the
hazardous � OH during the autoxidation of 6-OHDA, this is done without the
involvement of the ferrous ion or any other transitionmetal ion (Marti et al. 1997). At
this point, it seems interesting to highlight the reported ability of ascorbate to enhance
the production of ROS by 6-OHDA (Soto-Otero et al. 2000, Méndez-Álvarez et al.

CHAPTER 1
2001), particularly due to the fact that 6-OHDA is always administered in a saline
solution containing about 2% of ascorbate. In these cases, the presence of ascorbate sets
a redox-cycling, which regenerates 6-OHDA from its p-quinone leading to a continuous
production of ROS. Moreover, the presence of the enzyme dehydroascorbate reductase
in the brain may contribute to sustaining this redox-cycling. Although, this is the
molecular mechanism generally accepted to explain the ability of 6-OHDA to produce
oxidative stress and consequently responsible for its neurotoxicity, it has been also
reported that 6-OHDA can act directly by inhibiting the mitochondrial respiratory chain
at the level of complex I (Glinka and Youdim 1995, Glinka et al. 1996, Glinka et al.
1998). Assuming the reported capacity of complex I inhibitors to increase the leakage of
superoxide anions from the electron transport chain (Brand et al. 2004), this latter
mechanism could also contribute to increase the ability of 6-OHDA to produce ROS
and consequently to generate oxidative stress. Recent reports refer to the potential
contribution of ER stress to the cell death induced by 6-OHDA (Yamamuro et al. 2006).
Despite the widespread use of the intrastriatal injection of 6-OHDA to obtain an
experimental model of parkinsonism and an awareness of the morphological changes
following its administration (Jonsson 1983, Jeon et al. 1995), no data has been reported
on the kinetics of the oxidative damage it induces in the nigrostriatal system. However,
this information is crucial when this model is exploited to assess the anti-parkinsoniam
properties of new drugs (Jiang et al. 1993) or the benefit of transplantation or gene
therapy to repair the damaged pathways (He et al. 2001, Björklund et al. 2002). In the
light of this, the aim of the present study was to investigate the time-course of the
oxidative damage caused by unilateral and intrastriatal administration of 6-OHDA in the
ipsilateral and contralateral side of both striatum and ventral midbrain, and also includes
a quantification of the changes observed in the indices of lipid peroxidation (TBARS)
and oxidative status of proteins (PCC and PTC).
87

CHAPTER 1
MATERIALS AND METHODS
Chemicals
88
6-OHDA hydrochloride, ascorbic acid, thiobarbituric acid (TBA), butylated
hydroxytoluene crystalline (BHT), 2,4-dinitrophenylhydrazine hydrochloride,
desferrioxamine, 1,1,3,3-tetramethoypropane, 5,5‟-dithiobis-(2-nitrobenzoic acid),
sodium dodecylsulfate, EDTA, and bovine serum albumin (BSA) were purchased from
Sigma Chemical Co. (St. Louis, MO, USA). Guanidine hydrochloride was from Aldrich
Chemical Co. (Milwaukee, WI, USA). The water used for the preparations of solutions
was of 18.2 MΩ (Milli-RiOs/Q-A10 grade, Millipore Corp., Bedford, MA, USA). All
remaining chemicals used were of analytical grade and were purchased from Fluka
Chemie AG (Buchs, Switzerland).
Animal treatment
A total of 32 male Sprague-Dawley rats, each weighing about 200 g, were used.
All the experiments were carried out in accordance with the „„Principles of laboratory
animal care‟‟ (NIH publication No. 86–23, revised 1985) and approved by the
corresponding committee at the University of Santiago de Compostela. Rats were
stereotaxically injected in the right striatum with 6 μg of 6-OHDA in 5 μl of sterile
saline containing 0.2% ascorbic acid. Stereotaxic coordinates were 1.0 mm anterior to
bregma, 2.7 mm right of midline, 5.5 mm ventral to the dura, and tooth bar at -3.3. The
solution was injected with a 5 μl Hamilton syringe couple to a monitorized injector
(Stoelting, Wood Dale, IL, USA) and the cannula was left in situ for 5 min after
injection. All surgery was performed under equithesin anesthesia (3 ml/kg i.p.). Groups
of four rats were decapitated at the following times after injection: 5 min, 1 h, 12 h, 24
h, 48 h, 3 days, and 7 days. A group of four rats (control) was sacrificed immediately
after the administration of the saline.

Brain samples
CHAPTER 1
After decapitation, the brain was removed, the striatum and ventral midbrain
dissected, and the resulting samples frozen on dry ice. Each sample was immediately
sonicated (250 Digital sonifer, Branson Ultrasonic Co., Danbury, CT, USA) with four
volumes (w/v) a Na2PO4/KH2PO4 buffer (pH 7.4) isotonized with KCl and containing
200 μM BHT and 200 μM desferrioxamine. These compounds were used to prevent
amplification of lipid peroxidation during the progression of the analysis.
Determination of TBARS
The TBARS determination was performed spectrophotometrically using a
previously published method (Soto-Otero et al. 2002). Briefly, an aliquot of the sample
(200 μl) was treated with SDS (8%, w/v) followed by acetic acid (20%) and the mixture
vortexed for 1 min. Then, TBA (0.8%) was added and the resulting mixture incubated at
95°C for 60 min. After cooling to room temperature, 3 ml of n-butanol were added and
the mixture shaken vigorously. After centrifugation at 4,000 rpm for 5 min, the
absorbance of the supernatant (organic layer) was measured at 532 nm using an UV-
VIS spectrophotometer, model Lambda 35 (Perkin-Elmer Inc., Norwalk, CT, USA). For
calibration, a standard curve (5–150 nM) was generated using the malonodialdehyde
(MDA) derived by the acid hydrolysis (SO4H2; 1.5%, v/v) of 1,1,3,3-tetraethoxypropane
(TEP) and the TBARS results expressed as nmol MDA/mg protein. The protein
concentration of the sample was determined according to the method of Markwell et al.
(1978), using BSA as the standard.
Determination of protein carbonyl content (PCC)
The PCC was assessed spectrophotometrically according to a procedure
previously published (Hermida-Ameijeiras et al. 2004). Briefly, an aliquot of the sample
was submitted to precipitation of nucleic acids with 1% streptomycin sulfate (1:9, v/v)
89

CHAPTER 1
followed by centrifugation at 13,000 rpm. The pellet was then discarded and the
supernatant treated with trichloroacetic acid (1 M) followed consecutively by sonication
and centrifugation in a microcentrifuge (model E, Beckman Instruments, Palo Alto, CA,
USA) at 13,000 rpm for 5 min. The resulting pellet was reconstituted in NaOH (0.5 M)
with vigorous vortexing for 3 min. Then, 10 mM 2,4-dinitrophenylhydrazine in 2 M
chloric acid was added and the mixture incubated at room temperature for 1 h, in
darkness, and with continuous agitation. After the addition of trichloroacetic acid (1 M),
the resulting mixture was centrifuged at 13,000 rpm for 5 min. The resulting pellet was
washed twice with ethyl acetate : ethanol (1:1, v/v). Then, the washed pellet was
reconstituted with 6 M guanidine in a 20 mM KH2PO4 buffer (pH 2.3) and the
absorbance of the solution measured at 370 nm. The PCC was calculated from the
absorbance data using as absorption coefficient for dinitrophenylhydrazone ε = 22,000
M –1 cm –1 and expressing this parameter as nmol carbonyls/mg protein. Because of the
numerous washing steps, protein content in the final pellet was estimated on an HCl
blank pellet processed simultaneously using a BSA standard curve in 6 M guanidine,
and reading the absorbance at 280 nm.
Determination of protein thiol content (PTC)
90
The PTC of proteins was estimated spectrophotometrically using a modification
introduced to a standard assay (Hermida-Ameijeiras et al. 2004). Briefly, an aliquot of
the sample (200 μl) was treated with trichloroacetic acid (0.5 M) for protein
precipitation. After vortexing and centrifugation at 13,000 rpm for 5 min, the resulting
pellet was reconstituted in an 80 mM Na3PO4 and 2 mM EDTA buffer (pH 8.0)
containing 70 mM sodium dodecylsulfate. Then, 100 μM 5,5‟-dithiobis-(2-nitrobenzoic
acid) in a buffer 100 mM Na3PO4 (pH 8.0) were added and the mixture incubated at
room temperature for 20 min with continuous mixing. After centrifugation at 13,000
rpm for 5 min, the absorbance of the resulting solution was measured at 412 nm and the
PTC calculated from this data using as absorption coefficient for 2-nitro-5-
mercaptobenzoic acid ε = 13,600 M –1 cm –1 and expressing the corresponding parameter
as nmol thiols/mg protein.

Statistical analysis
CHAPTER 1
Results were expressed as the mean ± SD from five animals. Statistical
differences were tested using one-way ANOVA followed by LSD for multiple
comparisons (p < 0.05). All statistical analyses were performed using Sigmastat 3.0
from Jandel Scientific (San Diego, CA, USA).
91

CHAPTER 1
RESULTS
Effects of 6-OHDA administration on TBARS concentration
92
As shown in Fig. 1, the intrastriatal administration of 6 μg of 6-OHDA in the
right striatum caused a significant and progressive increase of TBARS concentration in
both right striatum (F(7,24) = 51.73, p < 0.001) and right ventral midbrain (F(7,24) =
72.81, p < 0.001), obtaining a peak-effect 48 h after the injection. Although, the
increase in the right striatum was significant at 5 min versus 1 h for ventral midbrain,
the augmentation observed after 48 h was higher in the right ventral midbrain (+83%)
than in the right striatum (+26%). At 7-day post-injection, the values returned to those
of the controls. A significant and progressive increase in the TBARS concentration was
also observed in the contralateral side, affecting again to both left striatum (F(7,24) =
10.39, p < 0.001) and left ventral midbrain (F(7,24) = 6.30, p < 0.001). The peak-effect
was also observed at 48 h, but the increase was lower (+23% in the left striatum and
+26% in the left ventral midbrain). At 7-day post-injection the values returned to the
concentrations found in the corresponding controls.
Effects of 6-OHDA administration on PCC
The PCC also exhibited a significant and progressive time course increase (Fig.
2), which affected both right striatum (F(7,24) = 57.55, p < 0.001) and right ventral
midbrain (F(7,24) = 54.76, p < 0.001). In this case, the increase observed in PCC was
not significant till 1-h post-injection. However, the peak-effect was observed once again
48 h after injection, and higher in the right ventral midbrain (+42%) than in the right
striatum (+38%). A significant and progressive increase in PCC was also observed in
the contralateral side, affecting to both left striatum (F(7,24) = 5.95, p < 0.001) and left
ventral midbrain (F(7,24) = 3.48, p < 0.05). However, in this case, although the peak-
effect was also observed at 48-h post-injection, the increase found was lower (+12% in

CHAPTER 1
the left striatum and +11% in the left ventral midbrain). The corresponding
concentrations achieved values close to those found in the controls at 7-day post-
injection.
Effects of 6-OHDA administration on PTC
Figure 3 shows the time-course of the changes observed for PTC in proteins
following 6-OHDA administration. As can be seen, a significant and progressive
reduction in PTC was found in both right striatum (F(7,24) = 5.06, p < 0.05) and right
ventral midbrain (F(7,24) = 25.87, p < 0.001). The maximal decrease for both right
striatum (–7%) and right ventral midbrain (–20%) was found at 24 h. Although the
peak-effect did not happen at the same time for both TBARS concentration and PCC,
the value obtained for PTC at 24 h was not significantly different when compared with
that observed 48-h post-injection. The PTC in the contralateral side only exhibited a
significant reduction in the left ventral midbrain (F(7,24) = 5.06, p < 0.05), because the
changes observed in the left striatum did not reach statistical significance (F(7,24) =
0.87, p > 0.05). Once again, the minimum value found in the left striatum for PTC was
observed 24-h post-injection and the decrease was of –8%, a value lower than that
found in the ipsilateral side. No significant differences were found when the value
obtained at 24 h was compared with that obtained at 48 h.
93

CHAPTER 1
DISCUSSION
94
As shown by the data here reported, the intrastriatal injection of 6-OHDA causes
a continuous increase in the indices of oxidative stress in the ipsilateral side (striatum
and ventral midbrain), which expands over 48 h for both lipid peroxidation and PCC,
and over 24 h for PTC. This quick evolution of the indices of oxidative stress agrees
with both morphological observations showing that DAergic neurons start to die within
the first 24 h (Jonsson 1983, Ichitani et al. 1991, Jeon et al. 1995) and that rapid
autoxidation of 6-OHDA takes place under physiological conditions (Soto-Otero et al.
2000, Méndez-Álvarez et al. 2001, Méndez-Álvarez et al. 2002). Once peak-values are
reached, each of the indices begins a slow decline to values very close to those found in
controls after 7-day post-injection. Taking into account the fact that the degeneration of
the nigrostriatal system can last for 1–2 weeks (Sauer and Oertel 1994, Przedborski et
al. 1995), our data appears to show that the here reported oxidative damage is the cause
of the DAergic lesion and not a consequence of this process, but this is still open to
question (Andersen 2004). Furthermore, the fact that the neurodegenerative process
continues when the indices of oxidative stress returned to the initial values (control
values) appears to prove that the oxidative stress generated by 6-OHDA autoxidation
causes irreversible damage in DAergic neurons and endangers their survival. Similar
findings of early oxidative stress have been observed after MPTP application
(Przedborski et al. 2004) as well as after chronic treatment of rats with rotenone
(Giasson et al. 2000). However, the fact that in our study the indices of oxidative stress
increase simultaneously in both striatum and ventral midbrain seems to discard previous
suggestions involving a chemical axotomic action of 6-OHDA in the delay and gradual
degeneration of DAergic neurons found in this model of DAergic neurodegeneration
(Sauer and Oertel 1994). At this point, it is interesting to note that the maximum
increase in the indices of oxidative stress is higher in the ventral midbrain than in the
striatum and occurs simultaneously. Assuming the accepted involvement of oxidative
stress in apoptosis (Giasson et al. 2000), the apoptotic-like features observed 1–3 weeks
after lesion (Marti et al. 1997), and the ability shown by caspase inhibitors to protect

CHAPTER 1
against degeneration (Trojanowski and Lee 2003), our results corroborate the
involvement of the oxidative stress caused by 6-OHDA autoxidation as a triggering
factor in the development of neurodegeneration. In view of both the recently reported
ability of oxidative stress to act as a primary event for α-synuclein polimerization and
the involvement of these aggregates in neurodegenerative processes (Cutillas et al.
1999, Krantic et al. 2005), the fibrillization of α-synuclein could also be responsible for
the slow rate of neurodegeneration observed after unilateral, intrastriatal administration
of 6-OHDA. Evidently, both our results and the suggested hypothesis do not discard the
suggested involvement of microglia in the corresponding neuronal death (Rodrigues et
al. 2001, Przedborski and Goldman 2004), because the formation of certain uncharged
ROS during the 6-OHDA autoxidation, with a relatively non short half-life, makes these
substances highly diffusible through biological membranes and suitable for cellular
signaling (Vroegop et al. 1995, Kamsler and Segal 2004).
It is also important to emphasize that the unilateral injection of 6-OHDA also
caused a significant increase in the indices of oxidative stress of the contralateral side,
affecting both the striatum and the ventral midbrain. Our results show that the
magnitude of these increases was lower than that found in the ipsilateral side, which
agree with the reported inability of intrastriatal and unilateral injections of 6-OHDA to
cause loss of cell bodies in the contralateral side (Lee et al. 1996). However, these data
clearly preclude the use of the contralateral side as a control to assess neurochemical
changes induced by 6-OHDA in this experimental model of PD. This may be explained
by the above-mentioned ability of certain ROS generated by 6-OHDA autoxidation to
diffuse through biological membranes.
In summary, our data confirm that intrastriatal and unilateral injections of 6-
OHDA cause oxidative stress (lipid peroxidation and protein oxidation), which
increases during the first 2-day post-injection and returns to approximate control levels
at the 17-day post-injection. This appears to be the triggering factor for the
neurodegenerative process, and the retrograde neurodegeneration following the first
week post-injection seems to be a consequence of the cell damage caused within the
first days post-injection. Finally, when this model is used to assess new neuroprotective
95

CHAPTER 1
strategies or to study the oxidative potential of a specific factor, the measurement of
brain indices of oxidative stress (TBARS and the content of oxidized groups in proteins)
should be performed at 48-h post-injection, which is when maximum values are
obtained.
Acknowledgments
This study was supported by Grant BFI2003-00493 from the Ministerio de Ciencia y
Tecnología with the contribution of the European Regional Development Found
(Madrid, Spain) and Grant PGIDIT03PXIB20804PR from the Xunta de Galicia
(Santiago de Compostela, Spain). The authors are indebted to Dr. J. L. Otero-Cepeda
for statistical analysis of the results.
96

CHAPTER 1
Fig. 1 Changes in TBARS levels in the ipsilateral and contralateral side of both striatum
(St) and ventral midbrain (VM) after stereotaxic, unilateral (right), intrastriatal injection
of 6-OHDA (6 �g in 5 �l of sterile saline containing 0.2% ascorbic acid) to rats. Each
point represents the mean ± SD from five animals (n = 5). Asterisks denote values
significantly different from the corresponding control (LSD test; p < 0.05).
97

CHAPTER 1
Fig. 2 Changes in PCC in the ipsilateral and contralateral side of both striatum (St) and
ventral midbrain (VM) after stereotaxic, unilateral (right), intrastriatal injection of 6-
OHDA (6 �g in 5 �l of sterile saline containing 0.2% ascorbic acid) to rats. Each point
represents the mean ± SD from five animals (n = 5). Asterisks denote values
significantly different from the corresponding control (LSD test; p < 0.05).
98

CHAPTER 1
Fig. 3 Changes in PTC in the ipsilateral and contralateral side of both striatum (St) and
ventral midbrain (VM) after stereotaxic, unilateral (right), intrastriatal injection of 6-
OHDA (6 �g in 5 �l of sterile saline containing 0.2% ascorbic acid) to rats. Each point
represents the mean ± SD from five animals (n = 5). Asterisks denote values
significantly different from the corresponding control (LSD test; p < 0.05).
99

CHAPTER 2

CHAPTER 2 Analysis of brain regional distribution of
aluminium in rats via oral and intraperitoneal administration
Sofía Sánchez-Iglesias,* Ramón Soto-Otero,* Javier Iglesias-González,*
M. Carmen Barciela-Alonso,† Pilar Bermejo-Barrera† and Estefanía
Méndez-Álvarez*
*Laboratory of Neurochemistry, Department of Biochemistry and Molecular Biology, Faculty of Medicine,
University of Santiago de Compostela, Santiago de Compostela, Spain
†Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Chemistry, University of
Santiago de Compostela, Santiago de Compostela, Spain
J. Trace Elem. Med. Biol. (2007) 21, S1, 31–34.
103

ABSTRACT
CHAPTER 2
In the present work, accumulation and distribution of aluminium in the rat brain
following both intraperitoneal and oral administration were studied. Male Sprague-
Dawley rats received a daily i.p. injection of aluminium chloride (10 mg Al 3+ /kg/day)
for one week or oral and progressively increasing administration of aluminium chloride
and citric acid up to 100 and 356 mg/kg/day, respectively. Electrothermal atomic
absorption spectrometry was used to determine aluminium concentration in different
brain areas (cerebellum, ventral midbrain, cortex, hippocampus, and striatum). The
results we obtained provided us insights about the most efficiency way for aluminium
administration in neurochemical studies focused to investigate the ability of this metal
to induce brain oxidative stress and consequently to cause neurodegeneration. Most of
the brain areas showed accumulation of aluminium, but a greater and more significant
increase was noted in the group receiving aluminium via intraperitoneal administration.
Aluminium distribution was also dependent on the administration route. According to
the here reported results, the intraperitoneal administration of aluminium was most
effective in improving aluminium accumulation in the different areas of the rat brain.
Our data suggest that aluminium neurotoxicity may be mediated by its particular
distribution in specific brain areas.
Keywords: Aluminium, brain distribution, intraperitoneal, oral, rat.
105

CHAPTER 2
INTRODUCTION
106
Aluminium is one of the most abundant metals in the earth‟s crust and all around
present in our domestic environment. Although, it is considered a non-essential element
for living organisms, the medical interest in this metal comes from the reported
neurotoxicity of aluminium (Yokel 2000). The bioavailabilility of aluminium, its ability
to cross the BBB, and the relatively slow rate of elimination from the brain, contribute
to guarantee the accumulation of aluminium into the brain (Priest 2004), which
represents an enhanced neurotoxicological risk. Thus, aluminium has been implicated in
aging-related changes (Deloncle et al. 2001) and several neurodegenerative diseases
(Kawahara 2005). Although, it is generally accepted that the neurotoxicity of aluminium
is caused by its ability to increase oxidative damage in the brain (Moumen et al. 2001),
the molecular mechanisms by which it causes neuronal damage are not fully understood
(Zatta et al. 2002). Evidently, the non-redox nature of this metal, so as the extensive
range of values reported in the literature concerning its accumulation in the brain,
contribute greatly to this uncertain situation. The purpose of this study was to elucidate
the precise localization of the absorbed aluminium in the brain following the use of two
different administration routes, oral and intraperitoneal, in order to clarify its
distribution in the brain and in this way contribute to understand all the factors involved
in its neurotoxicity.

MATERIALS AND METHODS
Chemicals
CHAPTER 2
Nitric acid 69% Hiperpur� from Panreac SA (Barcelona, Spain) was used to
perform microwave digestions. H2O2 30% Suprapur� and a dogfish muscle DORM-2
certified reference material were purchased from Merck (Darmstadt, FRG) and from the
National Research Council (Otawa, Canada), respectively. Argon N50 (99.999% purity)
was used as a sheath gas for the atomizer and to purge internally. Aluminium chloride
hexahydrate was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The water
used for the preparations of solutions was of 18.2 M� (Milli-RiOs/Q-A10 grade,
Millipore Corp., Bedford, MA, USA). All remaining chemicals used were of analytical
grade and were purchased from Fluka Chemie AG (Buchs, Switzerland).
Animal treatment
Forty male Sprague-Dawley rats (200-250 g) were used in this study. All
experiments were performed in accordance with the NIH publication “Principles of
laboratory animal care” and approved by the Ethics Committee of the University of
Santiago de Compostela. Animals were randomly divided into four experimental group:
the first group was daily i.p. injected with aluminium chloride (Sigma Chemical Co.) in
saline (0.9% NaCl) at a dose of 10 mg aluminium/kg/day (pH 4) for one week. The
second group was i.p. injected with saline over the same period. The third group was
given orally 25 mg aluminium/kg/day and 89 mg citric acid/kg/day in saline for one
week. After this period, aluminium and citric acid doses were increased to 50
mg/kg/day and 178 mg/kg/day, respectively, for one more week. Finally, doses were
adjusted to 100 mg aluminium/kg/day and 356 mg citric acid/kg/day for two additional
weeks. Citric acid was added to enhance the gastrointestinal absorption of aluminium
(Gómez et al. 1999). The fourth group was given saline orally during the entire
107

CHAPTER 2
experimental period of four weeks. Aluminium dosage was adjusted according to the
animal‟s body weight just before experiment. All rats received a standard diet (A04,
Panlab, Barcelona, Spain) and the corresponding drinking (saline or aluminium
salt+citric acid) ad libitum. Body weight and fluid intake were measured three times a
week to adjust the doses in order to achieve a constant aluminium and citric acid intake.
Brain samples
108
After treatment, animals were sacrificed by decapitation and brains quickly
excised. Regional brain segments [cerebellum (CE), ventral midbrain (VM), cortex
(CO), hippocampus (H), striatum (ST)] were rapidly dissected out on ice plate,
according to Paxinos and Watson (2007). Weighed brain regions were stored at –40°C
until trace element analysis.
Electrothermal atomic absorption spectrometry (ETAAS)
The samples were homogenised and digested with nitric acid (HNO3) and H2O2,
using microwave energy. A portion of 500 mg of sample was weighed into a digestion
vessel and 2 mL of HNO3 were added. Afterwards, vessels were introduced into the
microwave oven at 100 W during 12 min. Then, 1 mL of H2O2 was added into digestion
vessel and introduced into the microwave oven during 10 min at 300 W. Subsequently,
the sample was adjusted to its final volume (5 mL) by addition of ultrapure water. A
portion of 20 µL of the digested sample was introduced into the graphite furnace tube
for its analysis by ETAAS. Measurements were performed using an atomic absorption
spectrometer Model 1100 B (Perkin-Elmer, Norwalk, CT, USA), equipped with an
HGA-700 graphite furnace atomizer and AS-70 autosampler. A hollow cathode lamp
operating at 25 mA, which provided a 309 nm line with a spectral bandwidth of 0.7 nm,
was used. Deuterium background correction and pyrolytic graphite coated tubes with
L´vov platforms were employed. All measurements made during this study used
integrated absorbance with an integration time of 5 s. The mineralization and

CHAPTER 2
atomization used were 1,500 and 2,800 ºC respectively (Table 1). The volume injected
was 20 µL. Each sample was digested by triplicate and measured by duplicate.
Aluminium concentrations in samples were calculated using a standard addition method
in an analytical range of 0-20 µg/L. The limit of detection was 0.12 µg/g. The accuracy
of the method was investigated using the Reference Material DORM-2 with a
certificated aluminium concentration of 10.9±1.7 µg/g, being the aluminium
concentration obtained 11.4±2.2 µg/g. Reagents for aluminium determination were of
the highest available purity in order to avoid any risk of sample contamination. All
glassware was washed and kept in 10% (v/v) HNO3 for at least 48 hours and then rinsed
with ultrapure water (Milli-RiOs/Q-A10).
Statistical analysis
Results were expressed as the mean ± SD. Statistical differences were tested using
one-way ANOVA followed by Bonferroni‟s test for multiple comparisons. The
accepted level of statistical significance was p < 0.05. All statistical procedures were
carried out using the Statgraphics Plus 5.0 statistical package (Manugistics, Rockville,
MD, USA).
109

CHAPTER 2
RESULTS
Aluminium distribution in the brain after oral and i.p.
administration
110
As can be seen in Fig. 1, aluminium content in brain tissue was significantly
increased in both aluminium-exposed groups (i.p. and oral) when compared to the
corresponding control, except in VM of the i.p. aluminium-treated group.
All brain regions of oral aluminium-treated animals showed significant increased
levels of aluminium in relation to their controls: +69% CE, +200% VM, +116% CO,
+66% H, +143% ST, with highest levels in the ventral midbrain>striatum>cortex,
cerebellum and hippocampus. In orally-treated control animals, all areas showed a
similar accumulation of aluminium.
In i.p. aluminium-treated animals, aluminium content was significantly increased
compared to the respective i.p. controls (+727% CE, +667% CO, +877% H and +294%
ST). Notwithstanding, the level of aluminium in VM was significantly reduced
compared to that of the control group (–83%). Aluminium in i.p. metal-treated animals
seemed to accumulate preferentially in the following cerebral areas:
hippocampus>cortex>striatum>cerebellum>ventral midbrain.
When comparing the two different methods of aluminium administration, we
noted a greater and significant increase of aluminium in the i.p. aluminium-treated
groups compared to the orally-treated group: +77% H, +73% CO, +51% CE and +39%
ST. In turn, aluminium concentration in VM was significantly reduced (–1963%).

DISCUSSION
CHAPTER 2
Aluminium enters the brain through the BBB. Previous studies suggest that there
are carrier-mediated mechanisms that allow uptake of aluminium into the brain and
efflux into blood (Yokel 2002b). It was also proposed that aluminium distribution
depends on the animal species in question and the chemical form of aluminium
administered (Baydar et al. 2003). The aim of the present work was to evaluate the brain
regional accumulation of aluminium in rats following the administration of aluminium
chloride through two distinct administration routes: intraperitoneal and oral.
Oral administration of aluminium chloride for four weeks resulted in a
significant increase in brain aluminium concentration in all the investigated brain areas.
These results agree with previous studies reporting an increase of aluminium
concentration in both whole brain (Sahin et al. 1994) and specific areas of the brain
(Gupta and Shukla 1995, Roig et al. 2006), which contribute to explain the impairment
in motor coordination observed by some authors (Sahin et al. 1995). However, it has
also been reported no significant accumulation of aluminium in the whole brain (Gómez
et al. 1999, Colomina et al. 2002) and even a paradoxical reduction of aluminium
concentration in the whole brain of mice chronically treated with a diet containing
aluminium (Golub et al. 2000). This wide variety of results could be related with the
aluminium salt used and/or the extent of brain area selected.
Our results show that i.p aluminium chloride exposure for one week also caused
a significant and greater aluminium accumulation in cerebellum, cortex, hippocampus
and striatum, a finding that is consistent with previous literature concerning particular
brain areas (Julka and Gill 1996a, Nayak and Chatterjee 2003, Abubakar et al. 2004b,
Sakamoto et al. 2004). However, most of these publications used extensive cerebral
regions. It has been also reported a no significant aluminium accumulation following
i.p. administration of aluminium chloride (Moumen et al. 2001). The observation that
i.p. administration of aluminium predominantly accumulates in the hippocampus is in
accordance with a previous work (Julka and Gill 1996a). Our present study make the
interesting point that, while oral administration of aluminium chloride resulted in a
111

CHAPTER 2
significant increase of the metal concentration in the ventral midbrain, i.p.
administration led to a decrease in aluminium concentration in this cerebral region. In
our opinion, this curious observation could be related to the specific composition of this
area and the reported ability of aluminium to change the permeability of plasma
membranes in function of their particular composition (Silva et al. 2002).
112
Our results clearly show that aluminium accumulation in the brain not only
varied with the administration route used but also in its distribution in the distinct brain
areas. Evidently, both the presence of citric acid in the oral dose of aluminium and the
difference in the time of treatment also could contribute to the reported differences.
However, in view of the reported variance in aluminium distribution in different
cerebral regions following both treatments, it can be argued that the effects of
aluminium cannot be generalized to the whole brain and a study of the kinetics of
aluminium distribution in specific cerebral areas is necessary to understand the
neurotoxicity of this metal and its contribution to a particular neurological disorder.
Acknowledgments
This study was supported by the Xunta de Galicia (Santiago, Spain; Grant
PGIDIT03PXIB20804PR).

Table 1. Graphite furnace programme for aluminium determination by ETAAS
Step
Temperature
(ºC)
Ramp
(s)
Hold
(s)
Ar flow
(mL min -1 )
Dry 150 15 20 300
Pyrolisis 1500 15 20 300
Atomization 2800 0 5 50 (read)
Cleaning 2200 1 5 300
CHAPTER 2
Fig. 1 Levels of aluminium in the different areas of the rat brain. Data are expressed as
mean ± SD. Asterisks denote values significantly different (one-way ANOVA followed
by a Bonferroni‟s test) for treatment versus control group (*, p < 0.01, **, p < 0.001)
and for the oral-treated group versus intraperitoneally-treated group (***, p < 0.001).
�g Al 3+ / g
20
15
10
5
0
Control
Al-treated
**
***
*
*
***
**
**
i.p. oral i.p. oral i.p. oral i.p. oral i.p. oral
CE VM CO H ST
**
***
** ***
*
**
***
**
113

CHAPTER 3

CHAPTER 3 Brain oxidative stress and selective
behaviour of aluminium in specific areas of rat brain: potential
effects in a 6-OHDA-induced model of Parkinson’s disease
Sofía Sánchez-Iglesias,* Estefanía Méndez-Álvarez,* , § Javier Iglesias-
González,* Ana Muñoz-Patiño,† , § Inés Sánchez-Sellero,‡ José Luís
Labandeira-García,† , § and Ramón Soto-Otero,* , §
*Laboratory of Neurochemistry, Department of Biochemistry and Molecular Biology, Faculty of Medicine,
University of Santiago de Compostela, Santiago de Compostela, Spain
†Laboratory of Neuroanatomy and Experimental Neurology, Department of Morphological Sciences,
Faculty of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain
‡Department of Pathological Anatomy and Forensic Sciences, Faculty of Veterinary Medicine, University
of Santiago de Compostela, Lugo, Spain
§Networking Research Center on Neurodegenerative Diseases (CIBERNED), Spain
J. Neurochem. (2009) 109, 879–888.
117

ABSTRACT
CHAPTER 3
The ability of aluminium to affect the oxidant status of specific areas of the brain
(cerebellum, ventral midbrain, cortex, hippocampus, striatum) was investigated in male
Sprague-Dawley rats intraperitoneally treated with aluminium chloride (10 mg
Al 3+ /kg/day) for ten days. The potential of aluminium to act as an etiological factor in
PD was assessed by studying its ability to increase oxidative stress in ventral midbrain
and striatum and the striatal DAergic neurodegeneration induced by 6-OHDA
administered intraventricularly in an experimental model of PD. The results showed that
aluminium caused an increase in oxidative stress (TBARS, PCC, and PTC) for most of
the brain regions studied, which was accompanied by a decrease in the activity of some
antioxidant enzymes (SOD, CAT, GPx). However, studies in vitro confirmed the
inability of aluminium to affect the activity of those enzymes. The reported effects
exhibited a regional-selective behaviour for all the cerebral structures studied.
Aluminium also enhanced the ability of 6-OHDA to cause oxidative stress and
neurodegeneration in the DAergic system, which confirms its potential as a risk factor
in the development of PD.
Keywords: 6-hydroxydopamine, aluminium, antioxidant enzymes, lipid peroxidation,
Parkinson‟s disease, protein oxidation.
119

CHAPTER 3
INTRODUCTION
120
The human organism is constantly and inevitably exposed to aluminium, a
ubiquitous metal which is the third most abundant element in the Earth‟s crust,
representing 8% of total components (Martin 1997). Although, no biological function
has yet been attributed to it (Yokel 2002a), aluminium is a toxicant implicated in
dialysis encephalopathy (Alfrey et al. 1976), osteomalacia (Parkinson et al. 1979), non-
iron responsible anemia (Elliot et al. 1978), and also linked to many other diseases
including AD (Exley 1999, Gupta et al. 2005), PD (Yasui et al. 1992), and amyotrophic
lateral sclerosis (Kurland 1988).
Its ubiquity and extensive use in products and processes coupled with its ability
to cause neurodegeneration have made aluminium a cause of health concern (Exley
1999, Yokel 2000, Zatta et al. 2003). The daily reported mean dietary intake of 3.5 mg
may be increased by the frequent use of aluminium-containing antiperspirants and non-
prescription drugs (Weburg and Berstad 1986, Flarend et al. 2001), as well as by
occupational exposure (Meyer-Baron et al. 2007). Aluminium entry into the brain
occurs mainly through the BBB. Although the mechanism(s) responsible for aluminium
transport at the BBB remains unclear, it has been reported that aluminium can penetrate
into the brain as a complex with transferrin by a receptor-mediated endocytosis
(Roskams and Connor 1990) and bound to citrate via a specific transporter, the system
Xc � (L-glutamate/L-CySH exchanger) is the most recently accepted candidate
(Nagasawa et al. 2005). The apparently long half-life of aluminium in brain tissue has
been used to explain its easy accumulation in the brain (Yokel et al. 2001b, Sánchez-
Iglesias et al. 2007b), which together with the long life of neurons could be related to
the elevated levels of aluminium found in the brain of some patients suffering PD
(Yasui et al. 1992) and AD (Perl and Brody 1980). However this fact should not be
interpreted as the primary cause of those disorders.
Nevertheless, and despite all hitherto reported data concerning aluminium
neurotoxicity, the precise molecular mechanisms responsible for its neurotoxicity
remain largely unknown. Even though it is not a transition metal, and consequently does

CHAPTER 3
not undergo redox reactions, numerous publications have detailed an increase in the
formation of ROS after aluminium exposure (Nehru and Anand 2005). Most of the
studies performed both in vitro and in vivo have attributed its neurotoxicity to the lipid
peroxidation caused by the interaction between ROS and cell membranes (Gutteridge et
al. 1985, Zatta et al. 2002), thus considering the latter as the main targets of the oxidant-
mediated damage. Furthermore, aluminium appears to affect the brain activities of
several antioxidant enzymes (Julka and Gill 1996a). In addition, its ability to affect the
expression of DA receptors D1 and D2 (Kim et al. 2007) as well as the functionality of
mitochondria (Niu et al. 2005) has also been documented. However, the controversy
surrounding these findings is such that both pro-oxidant (Zatta et al. 2002, Exley 2004a)
and antioxidant (Oteiza et al. 1993a, Abubakar et al. 2004a) properties have been
attributed to this metal. The usefulness of the studies to clarify the accepted
involvement of aluminium in the pathogenesis of some neurodegenerative process has
also been brought into question (Savory and Ghribi 2007).
Consequently, the aim of this present study was to investigate the in vivo effects
induced by aluminium on indices of oxidative stress in the cerebellum, ventral
midbrain, cortex, hippocampus, and striatum of rat brain. We quantified the levels of
lipid peroxidation (thiobarbituric acid reactive substances, TBARS) and the oxidant
status of proteins (PCC, PTC), as well as an in vivo assessment of the effects of
aluminium on the activity of certain antioxidant defence enzymes, which included SOD,
GPx, and CAT. To shed some light on the molecular mechanisms involved, the effects
of aluminium on the in vitro activity of antioxidant enzymes have also been
investigated. Finally, taking into account the high levels of aluminium found in the SN
of some patients suffering PD (Yasui et al. 1992), we investigated its ability to modify
the capacity of 6-OHDA administered intraventricularly in an experimental model of
PD (Soto-Otero et al. 2002, Sánchez-Iglesias et al. 2007a) to cause oxidative stress in
ventral midbrain and striatum and to induce neurodegeneration in striatal DAergic
terminals.
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CHAPTER 3
MATERIALS AND METHODS
Chemicals
122
Aluminium chloride hexahydrate, BSA, CAT, cytochrome c, desipramine
hydrochoride, 3,3‟-diaminobenzidine, 2,4-dinitrophenylhydrazine hydrochloride, 5,5‟-
dithiobis-(2-nitrobenzoic acid), EDTA, GPx, GR, H2O2, ketamine/xylazine, mouse
monoclonal antibody to TH, TBA, xanthine, and xanthine oxidase were purchased from
Sigma Chemical Co. (St. Louis, MO, USA). Avidin-biotin-peroxidase complex and
biotinylated secondary antibody were purchased from Vector (Burlingame, CA, USA).
The water used for the preparations of solutions was Milli-RiOs/Q-A10 grade,
(Millipore Corp., Bedford, MA, USA). All remaining chemicals used were of analytical
grade and were purchased from Fluka Chemie AG (Buchs, Switzerland).
Animal treatment
Adult male Sprague-Dawley rats (200-250 g) were used to perform the in vivo
studies. All animals were housed individually in polypropylene cages to reduce
extraneous trace element contamination in a room equipped with 12 h light/dark
automatic light cycles, maintained at 22�1°C with a relative humidity of 65%. All
experiments were carried out in accordance with the “Principles of laboratory animal
care” (NIH publication No. 86-23, revised 1996) and approved by the corresponding
committee at the University of Santiago de Compostela. Aluminium dosage was
adjusted according to animal‟s body weight just before each experiment. All rats were
allowed a standard maintenance diet (A04, Panlab S.L., Barcelona, Spain) and water ad
libitum. Animals were randomly assigned to two experimental groups. The first group
was subdivided into two subgroups, each consisting of ten animals: rats in subgroup A
were daily i.p. injected with aluminium chloride in saline (NaCl 0.9%) at a dose of 10
mg Al 3+ /kg for 10 days; rats in subgroup B were injected with the same volume of

CHAPTER 3
saline over the same period. The second group was subdivided into four subgroups of
11 animals each: rats in subgroup C were used as normal (i.e. non-lesioned) controls,
and received the corresponding injections of vehicle for 10 days; rats in subgroup D
were i.p. injected with aluminium chloride at a dose of 10 mg Al 3+ /kg/day for 10
consecutive days; rats in subgroup E received i.p. injections of saline for 10 days and
were lesioned on the 8th day (two hours after i.p. injection) with 200 µg 6-OHDA in 3
µl sterile saline containing 0.2% ascorbic acid injected in the third ventricle; rats in
subgroup F were i.p. injected with aluminium chloride (10 mg Al 3+ /kg/day) for 10 days
and were lesioned with 6-OHDA in the same way as subgroup E. Injection solutions
containing aluminium were prepared by dissolving aluminium chloride in saline,
adjusting to pH 4.6 with sodium hydroxide, and waiting the equilibration time necessary
to obtain a clear solution. In all cases the volume injected was 0.5 ml.
Stereotaxic coordinates for intraventricular injection of 6-OHDA were 0.8 mm
posterior to bregma, midline, 6.5 mm ventral to the dura, and tooth bar at 0. The
solution was injected with a 10 µl Hamilton syringe coupled to a motorized injector
(Stoelting, Wood Dale, IL, USA), at 0.5 µl/min, and the cannula was left in situ for 5
min after injection. All surgery was performed under ketamine/xylazine anesthesia, and
30 min prior to injection of 6-OHDA, rats received desipramine (25 mg/kg i.p.) to
prevent uptake of 6-OHDA by noradrenergic terminals. The accuracy of the lesions and
cannula placement were confirmed by post-mortem analysis with cresyl violet staining.
Brain samples
At the end of the experimental period animals of subgroups A and B were
stunned with carbon dioxide and sacrificed by decapitation. Brains were quickly
removed and rinsed with ice-cold saline. Regional brain segments (cerebellum, ventral
midbrain, cortex, hippocampus, striatum) according to Paxinos and Watson (2007) were
immediately dissected out on an ice plate. For biochemical assays, weighed brain
samples were individually homogenized in a Na2PO4/KH2PO4 buffer (pH 7.4)
isotonized with KCl (1:6 w/v for cerebellum, 1:2 for cortex; 1:30 for ventral midbrain;
123

CHAPTER 3
1:15 for hippocampus and 1:10 for striatum) in a Dounce tissue grinder (Kontes,
Vineland, NJ, USA). The homogenates were then centrifuged at 14,000 g for 20
minutes at 4°C. The supernatants were aliquoted and stored at –80°C to determine
protein content and the activity of GPx, SOD, and CAT. The resulting pellets were
resuspended with cold Na2PO4/KH2PO4 buffer (pH 7.4) isotonized with KCl and
containing 20 �M BHT and 20 �M desferrioxamine. These compounds were used to
prevent amplification of lipid peroxidation during the progression of the analysis. Then,
homogenates were immediately sonicated for 5 seconds (250 Digital Sonifer, Branson
Ultrasonic Co., Danbury, CT, USA). These samples were used for the quantification of
TBARS, PCC, PTC, and protein concentration.
124
Forty-eight hours after lesion with 6-OHDA, five rats of subgroups C, D, E, and
F were killed according to the above cited procedure in order to perform biochemical
studies because it has been previously reported that the peak for oxidative stress is
reached at this time (Sánchez-Iglesias et al. 2007a). In these rats, the levels of the
oxidative stress caused by 6-OHDA were estimated by quantification of both lipid
peroxidation and protein oxidation in ventral midbrain and striatum.
Preparation of brain mitochondria
Brain mitochondria from Sprague-Dawley rats weighing 200-250 g were
obtained by differential centrifugation according to a previously published method
(Méndez-Álvarez et al. 1997) and protein concentration determined according to
Markwell et al. (1978), using BSA as the standard.
Determination of TBARS, PCC, and PTC
The TBARS, PCC, and PTC determinations for in vitro and in vivo experiments
were performed spectrophotometrically as previously described in Hermida-Ameijeiras
et al. (2004) and Sánchez-Iglesias et al. (2007a), respectively. 2,4-
Dinitrophenylhydrazine hydrochloride and 5,5‟-dithiobis-(2-nitrobenzoic acid) were

CHAPTER 3
used as chemical dosimeters for PCC and PTC determinations, respectively. For in vitro
experiments, AlCl3 was preincubated with brain mitochondria at a final Al 3+
concentration of 5 �M.
Measurement of SOD activity
SOD activity was spectrophotometrically determined by the method reported by
McCord and Fridovich (1969). The assay was performed at 25°C in 2.8 ml of 50 mM
potassium phosphate buffer (pH 7.8) containing 0.1 mM EDTA, 0.05 mM xanthine and
0.01 mM cytochrome c. Then, 100 �l xanthine oxidase (0.02 U) were added in order to
produce a rate of reduction of cytochrome c of 0.025 absorbance unit per minute,
followed by 100 �l of brain sample. Increase in absorption was monitored at 550 nm for
5 minutes. SOD activity was expressed in U/mg protein. To evaluate the potential in
vitro effect of aluminium on SOD, different concentrations of Al 3+ (10, 50, 100 �M)
were added, followed by 100 �l of SOD (1 U) instead of brain sample.
Measurement of GPx activity
GPx activity was spectrophotometrically measured by a modified version of the
method reported by Flohe and Gunzler (1984). Briefly, the reaction mixture consisted of
500 �l phosphate buffer (60 mM, 0.6 mM EDTA, 1 mM NaN3, pH 7.0), 100 �l GR (0.5
U), and 100 �l GSH (1 mM). Brain sample (100 �l) was added to the reaction mixture
and preincubated at 37°C for 10 min. Then, 100 �l NADPH (150 μM in 0.1% NaHCO3)
were added to reaction mixture and the hydroperoxide-independent consumption of
NADPH monitored for 3 min. The reaction was started by adding 100 �l H2O2 (150
μM) and the decrease in absorption at 340 nm monitored for 5 min. Molar extinction
coefficient of 6.22·10 3 M -1 ·cm -1 of NADPH at 340 nm was used to calculate GPx
activity which was expressed in mU/mg protein. When assessing the in vitro effect of
aluminium on GPx activity, different concentrations of Al 3+ (10, 50, 100 �M) were
125

CHAPTER 3
initially incorporated to the reaction mixture, followed by 100 �l of GPx (0.2 U) instead
of brain sample.
Measurement of CAT activity
126
CAT activity was polarographically measured following the formation of O2
using a Clark-type oxygen electrode, according to a slight modification to a previous
published method (Méndez-Álvarez et al. 1998). Briefly, 100 �l brain sample were
incubated in the electrode chamber and the reaction started with the addition of 20 �l of
a 500 �M solution of H2O2. CAT activity was expressed as U/mg protein. For in vitro
studies, 100 �l of CAT (20 U) were first added to the reaction mixture, followed by
different concentrations of Al 3+ (10, 50, 100 �M) in order to estimate the effect of the
metal on CAT activity.
Determination of MAO activity
MAO activity was spectrophotometrically measured in mitochondria
preparations as previously described (Soto-Otero et al. 2001), using kynuramine as a
non-selective substrate and (-)-deprenyl and clorgyline as irreversible inhibitors for
MAO-A and MAO-B estimation, respectively. Different concentrations of Al 3+ (10, 50,
100 �M) were incorporated to the incubation in order to assess the effect of aluminium
on both MAO-A and MAO-B activity.
Immunohistochemistry
The remaining six rats of subgroups C, D, E, and F were processed for
immunohistochemistry. Animals were killed by a chloral hydrate overdose one week
post-lesion and then processed for TH immunohistochemistry according to a previously
published methodology (Rey et al. 2007).

Statistical analysis
CHAPTER 3
Data were expressed as the mean ± SD. Statistical differences were tested using one-
way ANOVA followed by Bonferroni‟s test for multiple comparisons and by post hoc
Student-Newman-Keuls test for immunohistochemistry studies. The statistical
significance was set at p < 0.05. Normality of populations and homogeneity of variances
were verified before each ANOVA. All statistical procedures were carried out using the
Statgraphics Plus 5.0 statistical package (Manugistics, Rockville, MD, USA).
127

CHAPTER 3
RESULTS
In vivo effects of aluminium administration on brain lipid
peroxidation and protein oxidation
128
Lipid peroxidation was assessed by the determination of TBARS concentration,
and protein oxidation was estimated by both PCC and PTC. As shown in Fig. 1A,
animals exposed to aluminium (10 mg Al 3+ /kg/day for 10 days) exhibited a significant
increase in lipid peroxidation in the regions of cerebellum (+159%), ventral midbrain
(+54%), cortex (+20%) and striatum (+33%), while there were no significant changes in
hippocampus. TBARS levels in the striatum were particularly high when compared with
those found in other cerebral regions and in both aluminium-treated and control rats.
Following aluminium treatment, both PCC (Fig. 1B) and PTC (Fig. 1C) were
significantly elevated as compared to controls in cerebellum (+26%, +19%,
respectively), ventral midbrain (+135%, +15%, respectively) and striatum (+26%,
+16%, respectively), while there was a significant decrease in the cortex region (–12%,
–16%, respectively) and in the hippocampus (–20%, –26%, respectively). When
compared to other areas of non treated animals, PCC and PTC control levels were
significantly higher in cerebral cortex and lower in the ventral midbrain (Figs. 1B and
1C).
In vivo effects of aluminium administration on the brain activity of
different antioxidant enzymes
The effects of aluminium administration (10 mg Al 3+ /kg/day for 10 days) on the
activity of different antioxidant enzymes (SOD, GPx, CAT) were also studied in several
brain regions, and the results showed that the i.p. administration of aluminium
influenced SOD activity (Fig. 2A). Thus, there was a significant decrease in cerebellum
(–26%) and cortex (–21%) of the aluminium-treated group, while a significant increase

CHAPTER 3
was noted in hippocampus (+22%). No significant changes were detected in ventral
midbrain and striatum as compared with the controls. As depicted in Fig. 2B, exposure
to aluminium caused depletion in the activity of GPx in cerebellum (–26%), ventral
midbrain (–28%), cortex (–28%), and striatum (–11%). By contrast, GPx activity
increased in the hippocampus (+40%). Exposure of rats to aluminium decreased the
levels of CAT activity (Fig. 2C) in the cerebellum (–41%), ventral midbrain (–29%),
and striatum (–25%) compared to respective controls. CAT activity was not
significantly modified in the cortex, but showed a significant increase in the
hippocampus (+35%).
Effects of aluminium administration on the degeneration of DA
terminals in the striatum
In control rats, i.e. rats not lesioned with 6-OHDA (subgroup C) and rats treated
with aluminium alone (subgroup D), the DAergic neurons in the pars compacta of the
SN were intensely immunoreactive to TH, and a dense and evenly distributed TH
immunoreactivity (TH-ir) was observed throughout the striatum, which indicated the
presence of a dense network of nigrostriatal DAergic terminals (Fig. 3A-B). No
significant changes in the density of DA striatal terminals were observed in control rats
(i.e. not injected with 6-OHDA; subgroups C and D; Fig. 4). TH-ir terminal density was
higher in the control groups (subgroups C and D; Fig. 3A-B) than in rats that were
intraventricularly injected with 6-OHDA (subgroups E and F; Fig. 3C-D). We observed
a significant decrease in TH-ir terminal density (–27%) in rats intraventricularly
injected with 6-OHDA when compared to control rats (subgroup-C rats). In rats i.p.
treated with aluminium and subjected to intraventricular injection of 6-OHDA
(subgroup F), the reduction in the density of DA striatal terminals with respect to the
control rats (subgroup D) and rats injected with 6-OHDA (subgroup E) was statistically
significant (–48% and –28%, respectively; Fig. 4).
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CHAPTER 3
Effects of aluminium administration on brain lipid peroxidation and
protein oxidation in 6-OHDA-lesioned rats and controls
130
Once again, the concentration of TBARS was used as an index of lipid
peroxidation in striatum and ventral midbrain (Fig. 5A). Both regions of animals i.p.
injected with aluminium alone (subgroup D) showed a statistical difference in TBARS
production compared to non-lesioned (subgroup C) control rats (+40% in ventral
midbrain and +16% in striatum). Intraventricular injection of 6-OHDA resulted in a
significant increase in TBARS concentration in both striatum and ventral midbrain
when compared to control rats (+29% and +76%, respectively) and to animals treated
with aluminium (+12% and +26%, respectively)). Ventral midbrain and striatum
TBARS levels in rats i.p. treated with aluminium and intraventricularly injected with 6-
OHDA were +107% and +40% (respectively) increased in respect to control animals.
When compared to rats only treated with aluminium (i.e. without 6-OHDA; subgroup
D), the TBARS levels of subgroup-F rats were markedly increased in both regions
(+47% in ventral midbrain and +21% in striatum) and there were also significant
differences when compared with subgroup E, rats only lesioned with 6-OHDA (+17%
in ventral midbrain and +8% in striatum). Protein oxidation was assessed by the
determination of PCC and PTC in the samples of striatum and ventral midbrain. No
significant changes were noticed in the PTC (Fig. 5C) except for rats lesioned with 6-
OHDA when compared with animals treated only with aluminium (–11%). By contrast,
as shown in Fig. 5B, intraventricular injection of 6-OHDA induced a significant
increase in the PCC in striatum and ventral midbrain (+33% and +22%, respectively) as
compared with control rats. Treatment with aluminium (subgroups D) revealed a
significant difference in ventral midbrain and striatum as compared to control rats
(+11% and +18%, respectively). On the other hand, in subgroup F (animals treated with
aluminium and lesioned with 6-OHDA) the PCC was reduced in both regions (–16% in
ventral midbrain and striatum) as compared to the rats lesioned with 6-OHDA.

CHAPTER 3
In vitro effects of aluminium on the lipid peroxidation and protein
oxidation induced by 6-OHDA autoxidation in brain mitochondrial
preparations
As depicted in Fig. 6A, the incubation of 6-OHDA (10 �M) with brain
mitochondria at 37°C for 20 min induced a significant production of TBARS when
compared to the mitochondria (Mit) and Mit+Al controls (+91% and +116%,
respectively). Significant changes in TBARS production (+100%, +127%) were
observed when the autoxidation of 6-OHDA occurred in the presence of Al 3+ (5 �M) as
compared to the Mit and Mit+Al controls, respectively. However, the presence of
aluminium did not significantly affect the level of TBARS found after the incubation of
6-OHDA in mitochondrial preparations when compared to the Mit+6-OHDA control.
The incubation of 6-OHDA (10 �M) with brain mitochondria at 37°C for 20 min
induced a significant increase in the PCC (+7%; Fig. 6B) as compared to the Mit
control. However, the results obtained in the presence of Al 3+ (5 �M) were not
significantly different from control values. As can be seen in Fig. 6C, the PTC exhibited
a significant reduction (–9%) in the incubation of 6-OHDA (10 �M) with brain
mitochondria at 37°C for 20 min when compared with that obtained in the control with
mitochondria alone and Mit+Al 3 . The presence of aluminium had no significant effect
on the PTC found with mitochondrial incubations when compared to controls.
In vitro effects of aluminium on the enzyme activities of GPx, SOD,
CAT, and MAO
The results regarding the effect of aluminium on the in vitro activities of GPx,
CAT, SOD, MAO-A and MAO-B enzymes are displayed in Table 1. In all cases, no
significant differences were detected between assays performed in the presence and in
the absence of Al 3+ (10, 50, 100 �M)
131

CHAPTER 3
DISCUSSION
132
Aluminium is a non-redox metal whose accumulation in the brain has been
linked to various neurodegenerative diseases (Yokel 2000, Zatta et al. 2003). Several
hypotheses have been given to explain its reported ability to promote biological
oxidations (Exley 2004a). Thus, it has been shown to facilitate iron-induced lipid
peroxidation (Gutteridge et al. 1985); non-iron-induced lipid peroxidation (Verstraeten
and Oteiza 2000); non-iron-mediated oxidation of NADH (Kong et al. 1992); and non-
iron-mediated formation of � OH (Méndez-Álvarez et al. 2002). Additionally, it also
appears to inhibit several antioxidant enzymes in different parts of the brain (Nehru and
Anand 2005).
In the present study aluminium was administered at a dosage regimen to
guarantee its accumulation in different areas of rat brain (Sánchez-Iglesias et al. 2007b).
Aluminium-treated animals remained healthy and showed no signs of toxicity during
the treatment. However, certain tiny, white inclusions were observed floating in the
abdominal cavity, probably caused by a partial precipitation of the aluminium and
previously reported (Abubakar et al. 2004a).
Previous studies have shown results ranging from no significant changes in
TBARS levels (Oteiza et al. 1993b), to an increase in TBARS levels (Sharma and
Mishra 2006), and a significant decrease in TBARS levels (Abubakar et al. 2004b). Our
data clearly demonstrate that aluminium exposure caused a significant increase in the
levels of TBARS in most of the brain areas studied, but particularly so in the
cerebellum. Although, some authors have reported a decrease (Esparza et al. 2003) or
no changes (Deloncle et al. 1999) in the levels of TBARS, our results are in total
(Esparza et al. 2005, Nehru and Anand 2005, Dua and Gill 2001, Jyoti et al. 2007) or
partial agreement (Julka and Gill 1996a) with most previously published data.
Curiously, we found no significant changes in the levels of TBARS in the hippocampus
following aluminium administration. In our opinion, the apparent contradictory results
found in the literature are due to the use of different chemical forms for aluminium
exposure (Esparza et al. 2003, Julka and Gill 1996a) and/or to the use of different

CHAPTER 3
administration routes (Deloncle et al. 1999, Jyoti and Sharma 2006) as we have
demonstrated recently (Sánchez-Iglesias et al. 2007b).
Another index to assess oxidative stress is the oxidant status of proteins. We
found that the effects caused by aluminium on both PCC and PTC were not
homogenous for all areas studied. Indeed, our data showed that aluminium provoked an
increase in both PCC and PTC in the cerebellum, ventral midbrain, and striatum,
whereas a decrease was found in the cortex and hippocampus. The most noteworthy
effect of these results is the lack of correlation between the increase observed in the
PCC, ostensibly a consequence of a situation of oxidative stress, and the apparently
contradictory increase in PTC. The increase in the PTC may be a consequence of
increased GSH production (Khanna and Nehru 2007) and its subsequent capacity to
reduce disulfide groups in proteins. Our results contrast with previous findings that
aluminium exposure affects neither PCC (Oteiza et al. 1993b) nor PTC (Oteiza et al.
1993b) in whole brain. Others report a significant increase of PCC in both hippocampus
(Jyoti and Sharma 2006) and cortex (Jyoti et al. 2007) and a significant loss of PTC in
whole brain (Dua and Gill 2001). Once again, this variability in relation to previous data
appears to corroborate the importance of the chemical speciation of aluminium and the
route of administration. Interestingly, the aluminium salt used not only affects the
accumulation of this metal in different cells but also modulates its toxic effects
(Lévesque et al. 2000). Despite these findings and taking into account the complexity of
the aluminium speciation chemistry (Bharathi et al. 2008), it is very difficult to gain
insight into the relation between the particular chemical speciation of aluminium in the
brain and its biological effects. Nevertheless, our results clearly show that aluminium
can create a situation of oxidative stress in most brain areas. This is mainly a
consequence of the fact that aluminium is a strong Lewis acid which allows it to react
with the superoxide anion and form a metal-superoxide complex (AlO2 ●2+ ), which is a
more potent oxidant than the superoxide anion and putatively has a high catalytic
potential, as suggested by Kong et al. (1992) and later discussed by Exley (2004a).
These changes were accompanied by significant variations in the activity of
SOD, GPx, and CAT. Our results indicate that all the neural tissues examined, except
133

CHAPTER 3
hippocampus, showed similar behaviour patterns and emphasize a significant decrease
in the enzyme activity of most antioxidant systems, and agree with previous studies
(Julka and Gill 1996a, Dua and Gill 2001, Abubakar et al. 2004a, Nehru and Anand
2005, Jyoti et al. 2007). Our in vitro experiments show no significant modification in
the enzyme activity of either of SOD, GPx, and CAT, which we interpret as a
declination of the gene expression of antioxidant enzymes (González-Muñoz et al.
2008).This reduction in antioxidant enzyme activity helps to explain the brain oxidative
stress observed following aluminium administration. Furthermore, the reduction in GPx
activity also explains the increase in the ratio GSH/GSSG previously reported by other
authors (Esparza et al. 2003, Khanna and Nehru 2007), which helps explain the increase
in the PTC found after aluminium exposure. The inability of aluminium to affect both
MAO-A and MAO-B activity discards their involvement in the aluminium-induced
brain oxidative stress. Data showing a partial disagreement with our findings have also
been reported (Esparza et al. 2005), but is possibly due to the regimen of aluminium
administration. A putative explanation for the increase in the activity of SOD, GPx, and
CAT in the hippocampus (Sánchez-Iglesias et al. 2007b), may be the existence of a
compensatory mechanism due to the ability of cells to increase the expression of
antioxidant enzymes when exposed to high levels of toxicants (Gechev et al. 2002), an
effect which could overcome the ability of moderate levels of aluminium to inhibit gen
expression of antioxidant enzymes. Another interpretation could be the confinement of
aluminium in granulovacuoles of neurofibrillar tangles due to its high capacity to
interact with tau proteins in hippocampal cells (Walton 2006), thus reducing the
presence of free aluminium and consequently its disposition to affect the activity of
antioxidant enzymes and generate oxidative stress. Although, our findings contrast with
those of Julka and Gill (1996a), similar results were also found by Esparza et al. (2003).
134
We used an animal model of PD induced by 6-OHDA administration in the third
ventricle to assess the ability of aluminium to affect both the index of oxidative stress in
the nigrostriatal system and the extent of lesions of the DA system in the striatum. Our
results showed that aluminium causes an enhancement in 6-OHDA-induced lipid
peroxidation and protein oxidation (except for PTC in ventral midbrain). The
administration of aluminium alone (i.e. without 6-OHDA) also caused a significant

CHAPTER 3
difference in the levels of both lipid peroxidation and protein oxidation for PCC.
Although, these results are in partial agreement with the results obtained with non-
lesioned rats, they do not agree with the in vitro results obtained using brain
mitochondria preparations, in which we found no significant effect due to the presence
of aluminium. Evidently, these findings corroborate the importance of the in vivo
studies in this kind of investigation, which appears to be a consequence of the tight
relationship existing among the different molecular mechanisms coexisting within the
brain. Our immunochemical study showed that aluminium administration alone caused
no significant change in TH-ir striatal terminals. In contrast, lesion with 6-OHDA
caused a loss of TH-ir striatal terminals, which was significantly increased with the
administration of aluminium. Obviously, these data show the ability of aluminium to
increase the capacity of 6-OHDA to cause nigrostriatal neurodegeneration. Evidently,
the importance of these data is increased by the ability of neuromelanin to bind
aluminium which may be the cause of the high concentration of aluminium found in the
brain of certain patients suffering PD (Yasui et al. 1992). Furthermore, our results also
help us understand the reported ability of aluminium to potentiate etiological agents and
accelerate the progression of a disease (Exley and Esiri 2006). In conclusion, aluminium
acts mainly as a pro-oxidant probably by reducing the activity of defence antioxidant
enzymes. In addition, the increase in oxidative stress together with enhanced 6-OHDA-
induced neurodegeneration, suggest the participation of free radical-induced oxidative
cell injury in mediating aluminium neurotoxicity. Interestingly, we demonstrated that
aluminium neither enhanced lipid peroxidation nor decreased antioxidant enzyme
activities in the hippocampus. Given that aluminium content was markedly increased in
the hippocampus (Sánchez-Iglesias et al. 2007b), this dual effect could be related to the
biphasic behaviour of aluminium previously reported by Abubakar et al. (2004a). In our
opinion, the high concentration of aluminium in the hippocampus may possibly induce
an increase in defence enzyme levels, which could cause the steady state of TBARS
levels after aluminium exposure. However, in the remaining cerebral areas, antioxidant
enzymes have a lower activity, which is interpreted as a consequence of the postulated
oxidant potential of AlO2 ●2+ (Kong et al. 1992, Exley 2004a) and consequently the
factor responsible for the oxidative impairment found in those brain areas. Finally, we
135

CHAPTER 3
assume that the distinct cerebral areas exhibit different sensitivities to aluminium, which
can be partly explained by the differences in the brain barrier mechanisms. In fact, our
data lean towards the concept of the blood-brain regional barrier introduced by Zheng et
al. (2003). Evidently, the hypothesis that it would be more accurate to talk about a
blood-hippocampal barrier and a blood-striatum barrier, for example, needs to be
corroborated and characterised by future research.
Acknowledgments
This study was supported by grants PGIDIT03PXIB20804PR (to R.S.-O.) and
PGIDIT07CSA005208PR (to J.L.L.-G.) from XUGA (Santiago de Compostela, Spain),
and grants SAF2007-66114 (to R.S.-O.) and BFU2006-07414 (to J.L.L.-G.) from
Ministerio de Ciencia e Innovación (Madrid, Spain).
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Table 1. In vitro effects of the presence of aluminium on the enzyme activities of SOD,
GPx, CAT, and MAO
Al 3+ (�M) SOD (U) GPx (U) CAT (U)
MAO
(nmol 4-HQ/mg protein/min)
MAO-A MAO-B
control 10.8 � 0.86 1.96 � 0.194 24.7 � 1.77 1.15 � 0.077 2.19 � 0.057
10 11.2 � 0.78 2.14 � 0.131 22.8 � 0.42 1.14 � 0.054 2.15 � 0.064
50 11.3 � 0.41 2.25 � 0.115 23.4 � 1.43 1.13 � 0.083 2.17 � 0.089
100 11.0 � 0.92 2.17 � 0.257 24.0 � 2.75 1.14 � 0.095 2.16 � 0.052
SOD, GPx, and CAT activities were determined using commercial enzymes, while
MAO activity (MAO-A and MAO-B) was determined using mitochondrial preparation
obtained from rat brain. All the incubations were performed at 37ºC. Abbreviations:
SOD, superoxide dismutase; GPx, glutathione peroxidase; CAT, catalase; MAO,
monoamine oxidase; 4-HQ, 4-hydroxyquinoline. Data are expressed as means � SD
from four determinations (n = 4). Significance of differences among groups was
assessed by a one-way ANOVA followed by a Bonferroni‟s test. No significant
differences were obtained for p < 0.05.
137

CHAPTER 3
Fig. 1 Levels of TBARS (A), protein carbonyls (B), and protein thiols (C) in different
brain areas of rats i.p. treated with saline (control) or aluminium chloride (10 mg
Al 3+ /kg/day) for ten days. Abbreviation: Al, aluminium. Data are expressed as means ±
SD from five rats (n = 5). Significance of differences among groups was assessed by a
one-way ANOVA followed by a Bonferroni‟s test. Asterisks denote values significantly
different from the corresponding control (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
138

CHAPTER 3
139

CHAPTER 3
Fig. 2 Enzyme activity of: SOD (A), GPx (B), and CAT (C) in different brain areas of
rats i.p. treated with saline (control) or aluminium chloride (10 mg Al 3+ /kg/day) for ten
days. Abbreviation: Al, aluminium. Data are expressed as means ± SD from five rats (n
= 5). Significance of differences among groups was assessed by a one-way ANOVA
followed by a Bonferroni‟s test. Asterisks denote values significantly different from the
corresponding control (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
140

CHAPTER 3
141

CHAPTER 3
Fig. 3 Microphotographs showing changes in the striatal TH-ir one week post-lesion in
rats injected intraventricularly with vehicle (i.e. controls; A), i.p. treated with
aluminium (10 mg Al 3+ /kg/day) for ten days (B), lesioned intraventricularly on the 8th
day with 200 µg 6-OHDA (C), or i.p. treated with aluminium for 10 days and lesioned
on the 8th day with 6-OHDA (D). Scale bar = 1 mm.
142

CHAPTER 3
Fig. 4 Density of striatal DAergic terminals estimated as optical density, one week post-
surgery in the different experimental groups. Abbreviation: TH-ir, tyrosine hydroxylase
immunoreactivity. Optical densities are expressed as percentages of the values obtained
in the control groups. Data are expressed as means ± SD obtained from six animals (n =
6). Means that differ significantly are indicated by a different letter (p < 0.05, one-way
ANOVA and post-hoc Student-Newman-Keuls test).
143

CHAPTER 3
Fig. 5 Levels of TBARS (A), protein carbonyls (B), and protein thiols (C) in both
ventral midbrain and striatum of different groups of rats: control, i.p. treated with saline
for 10 days and injected intraventricularly with saline; control+Al, i.p. treated with
aluminium for 10 days and injected intraventricularly with saline; 6-OHDA, i.p. treated
with saline for 10 days and lesioned with 6-OHDA on the 8th day; and finally 6-
OHDA+Al, i.p. treated with aluminium for 10 days and lesioned on the 8th day with 6-
OHDA. Doses used: aluminium (10 mg Al 3+ /kg/day), 6-OHDA (200 µg). Abbreviation:
Al, aluminium. Rats were sacrificed two days after lesioning. Data are expressed as
means ± SD from five rats (n = 5). Significance of differences among groups was
assessed by a one-way ANOVA followed by a Bonferroni‟s test. Asterisks, pads and
section sign denote values significantly different from the corresponding controls:
control, control+Al, 6-OHDA respectively (*, # , § , p < 0.05; **, # # , §§ , p < 0.01; *** p <
0.001).
144

CHAPTER 3
145

CHAPTER 3
Fig. 6 In vitro effects of Al 3+ (5 �M) on TBARS formation (A), protein carbonyl
content (B), and protein thiol content (C) induced by the autoxidation of 6-OHDA (10
�M) in mitochondrial preparations (1 mg protein/ml) from rat brain. Mitochondria
incubations were performed in phosphate buffer (pH 7.4, KCl isotonic) at 37°C for 20
min. Abbreviation: Al, aluminium. Data are expressed as means � SD from five
independent experiments. Significance of differences among groups was assessed by a
one-way ANOVA followed by a Bonferroni‟s test. Asterisks, pads and section sign
denote values significantly different from the corresponding controls: Mit, Mit+Al, and
Mit+6-OHDA, respectively (*, # , p < 0.05; ***, # # # , p < 0.001).
146

CHAPTER 3
147

SUMMARY

SUMMARY
SUMMARY
Aluminium has become an important health concern due to both the frequent
exposure to this metal and the suggested ability of aluminium to cause
neurodegeneration. Pathological conditions such as PD have been associated with the
accumulation of aluminium in brain. Although, aluminium is not a redox metal, it has
been reported to be able to enhance brain oxidative stress. However, the molecular
mechanism of its neurotoxicity remains not well understood. Consequently, we resolved
to gain insight into the mechanisms of aluminium neurotoxicity in oxidative stress-
induced degenerative processes in relation with PD.
Before starting the whole procedures with aluminium, it was crucial to establish
the kinetics of the oxidative damage induced in a 6-OHDA model of PD and also to
quantify the changes observed in the indices of lipid peroxidation and oxidant status of
proteins in striatum and ventral midbrain. These results would thereafter enable us to
decide at which exact post-injection time should be performed the measurement of
indices of brain oxidative stress when assessing the oxidative effects of aluminium. To
accomplish the first part of this thesis, we chose the unilateral and intrastriatal injection
of 6-OHDA to lesion the DAergic nigrostriatal pathway system in male Sprague-
Dawley rats. This procedure has been extensively used to examine the degeneration of
the DAergic neurons characteristics of PD. When compared to others 6-OHDA models
with injections into the SN or the nigrostriatal tract, this method mimics more closely
the progression of PD as it leads to a slower retrograde degeneration of the nigrostriatal
system over a period of several weeks (Sauer and Oertel 1994, Przedborski et al. 1995,
Lee et al. 1996, Shimohama et al. 2003). In fact, this allowed us to use different groups
of rats sacrificed at distinct post-injection times (from 5 min to 7 days). Our results
clearly indicated that unilateral and intrastriatal injection of 6-OHDA results in
increased levels of lipid peroxidation and protein oxidation in the ventral midbrain and
in the striatum. We observed for both areas an aument during the first 2 days post-
injection and values returned to near control levels at the 7 day post-injection. Peak
151

SUMMARY
values were attained at 48 hours post-injection for both TBARS and PCC, and at 24
hours for PTC. Interestingly, lower but significant increases in oxidative stress levels
were also seen in the contralateral side (ventral midbrain and striatum) excluding this
zone as a control to determine neurochemical alterations caused by 6-OHDA in this
experimental model of PD. At last and according to the obtained results, we decided to
establish the optimal time of 48 hours post-injection for quantification of brain
oxidative stress indices when assessing the oxidative potential of aluminium in this
experimental model of PD.
152
The second part of this thesis consisted in developing a dosage regimen of
aluminium to rats which would guarantee a significant accumulation of this metal in
brain areas and also to determine its precise distribution in the brain. As it was
previously suggested that aluminium distribution depends on the animal species in
question and the chemical form of aluminium administered, we opted for two distinct
administration routes: oral and intraperitoneal. Sprague-Dawley rats were either daily
i.p. injected with aluminium chloride (10 mg Al 3+ /kg/day) for 1 week, or given orally
progressive increasing doses of aluminium chloride (25, 50, 100 mg Al 3+ /kg/day)
supplemented with citrate (89, 178, 356 mg/kg/day) during 4 weeks. Our results showed
that both administration routes led to aluminium accumulation. A greater and more
significant increase was noted in the group receiving aluminium via intraperitoneal
administration for most brain areas except in ventral midbrain. Distribution also varied
with the administration route used. In accordance to these results we resolved to use the
intraperitoneal administration route to clarify the brain oxidative stress provoked by
aluminium.
Finally the third phase of this thesis was dedicated to determine the ability of
aluminium to alter the oxidant status of specific brain areas, such as cerebellum, ventral
midbrain, cortex, hippocampus, and striatum. Male Sprague-Dawley rats were
intraperitoneally administered with aluminium chloride (10 mg Al 3+ /kg/day) in saline
for 10 days. As we demonstrated before, this dosage procedure was sufficient to insure
aluminium accumulation in brain areas. Animals were sacrificed 48 hours after lesion to
perform lipid peroxidation and protein oxidation studies because the peak for oxidative

SUMMARY
stress is reached at this time as we previously reported in the first phase of this thesis.
Our results showed that, except for hippocampus, the metal triggered an increase in
oxidative stress levels (determined as TBARS, PCC, and PTC) for most of the brain
regions studied, which was accompanied by decreased activities of the antioxidant
enzymes (SOD, CAT, and GPx). However, studies in vitro confirmed the inability of
aluminium to affect the activity of those enzymes and also of MAO-A and MAO-B. The
reported effects exhibited a regional-selective behaviour for all the cerebral structures
studied. Worthy of note is the case of the hippocampus, as aluminium exposure resulted
in increased antioxidant enzymes activities, no significant alterations of lipid
peroxidation and decreased protein oxidations. These results might be explained by the
high aluminium accumulation and the promotion of compensatory mechanism(s) in this
brain area.
Lastly, and to shed some light on the potential of aluminium to act as an
etiological factor in PD, we studied the ability of this metal to increase the striatal
DAergic neurodegeneration and various indices of oxidative stress in ventral midbrain
and striatum of rats injected intraventricularly with 6-OHDA. This animal model was
known to induce a more slowly ensuing parkinsonian syndrome exhibiting similar
topographic depletion of DAergic neurons to that observed in PD (Rodriguez et al.
2001, Rodríguez-Díaz et al. 2001) and to avoid the focal lesion around the cannula tip
produced when 6-OHDA is directly injected in the brain tissues. We followed the same
dosage procedure as previously mentioned (daily intraperitoneal injection of 10 mg mg
Al 3+ /kg/day in saline for ten days) except that rats were lesioned on the 8 th day with 6-
OHDA injected in the third ventricle. Animals were killed 1 week post-lesion for
immunohistochemistry studies as it was reported that progressive degeneration of
mesencephalic DAergic cells produced by intraventricular injection of 6-OHDA
reached the definitive lesion pattern at the end of the first week postinjection (Rodriguez
et al. 2001). Our results indicated that aluminium enhanced the ability of 6-OHDA to
cause lipid peroxidation and protein oxidation (except for PTC in ventral midbrain). In
addition, the metal was able to increase the capacity of 6-OHDA to cause
153

SUMMARY
neurodegeneration in the DAergic system as the loss of TH-ir striatal terminals was
significantly increased.
Note: It is also important to note that all experiments carried out during this thesis
were in accordance with the “Principles of laboratory animal care” (NIH
publication No. 86-23, revised 1996) and approved by the corresponding Ethics
Committee at the University of Santiago de Compostela. Furthermore, all efforts
were made to reduce the number of animals used and to minimize animal
suffering.
154

CONCLUSIONS

CONCLUSIONS
The results obtained in the present thesis led us to the following conclusions:
Chapter 1
CONCLUSIONS
1) Intrastriatal and unilateral injections of 6-OHDA cause a continuous and
simultaneous increase in the indices of oxidative stress in the ipsilateral side,
reaching a peak value at the 48 hours post-injection for both TBARS and PCC, and
at 24 hours for PTC, and returning slowly to approximate control levels after 7-day
post-injection in the case of TBARS, PCC and PTC.
2) The contralateral side should not be used as a control to assess neurochemical
changes induced by 6-OHDA in this experimental model of PD as the unilateral
injection of 6-OHDA causes a significant increase in the indices of oxidative stress,
affecting both the striatum and the ventral midbrain, although lower than that found
in the ipsilateral side.
3) The measurement of the brain indices of oxidative stress (TBARS and the content
of oxidized groups in proteins) should be performed at 48-h following the
intracerebral administration of 6-OHDA (peak-values time) when this model is
used to assess new neuroprotective strategies or to study the oxidative potential of a
specific etiological factor.
Chapter 2
4) Oral administration of aluminium chloride to rats for 4 weeks results in a
significant increase in brain aluminium concentration in all the investigated brain
areas (cerebellum, ventral midbrain, cortex, hippocampus and striatum).
157

CONCLUSIONS
5) Intraperitoneal aluminium chloride exposure to rats for one week causes a
158
significant and greater aluminium accumulation in cerebellum, cortex,
hippocampus and striatum, whereas a decrease in aluminium concentration is
reported in ventral midbrain.
6) Aluminium administered i.p. predominantly accumulates in the hippocampus,
whereas aluminium administered orally principally accumulates in the ventral
midbrain.
7) Aluminium accumulation in the brain and its distribution in the different areas of
the brain varie with the administration route used.
Chapter 3
8) Aluminium i.p. exposure to rats can create a situation of oxidative stress in most of
the brain areas studied.
9) Aluminium causes a significant increase in the levels of TBARS in ventral
midbrain, cortex and striatum, but particularly so in the cerebellum, while no
significant changes is noted in the hippocampus.
10) Aluminium provokes a significant increase in both PCC and PTC in the cerebellum,
ventral midbrain, and striatum, whereas a decrease is reported in the cortex and
hippocampus. The molecular mechanism involved in the lack of a direct correlation
between the increase observed in the PCC, and the “apparently” contradictory
increase in PTC has not been found out.
11) All the brain regional areas examined show similar behaviour patterns as
aluminium leads to a decrease of the enzyme activity of SOD, GPx, and CAT,
except in hippocampus where antioxidant enzymes activities increase.
12) Aluminium does not significantly alter the enzyme activities of either of SOD,
GPx, and CAT in vitro.

13) Aluminium does affect neither MAO-A nor MAO-B activity in vitro.
CONCLUSIONS
14) In this experimental model of PD aluminium causes an enhancement in 6-OHDA-
induced lipid peroxidation and protein oxidation in vivo, except for PTC in ventral
midbrain.
15) The administration of aluminium to control rats that were given intraventricularly
saline instead of 6-OHDA also causes a significant difference in the levels of both
lipid peroxidation and protein oxidation for PCC.
16) Immunohistochemical studies show that aluminium administration alone causes no
significant change in TH-ir striatal terminals. In contrast, lesion with 6-OHDA
causes a loss of TH-ir striatal terminals, which is significantly increased with the
administration of aluminium.
According to the previously mentioned conclusions, we establish that the results
here reported confirm the potential of aluminium as a risk factor in the development of
PD. This metal acts mainly as a pro-oxidant and is able to reduce the activities of
antioxidant enzymes. Its toxicity in the different brain areas studied (cerebellum, ventral
midbrain, cortex, and striatum) is probably mediated by the contribution of AlO2 ●2+ and
other free radical-induced oxidative cell damage. Interestingly, the results observed in
the hippocampus (high aluminium content and not increased oxidant status) may be
caused by specific compensatory mechanism(s) of the blood-hippocampal barrier but
this idea still needs to be corroborated and characterized by future research.
159

RESUMEN

RESUMEN
RESUMEN
Desde el punto de vista sanitario, el interés social que despierta el aluminio es
cada vez mayor. Esto se debe a que la sociedad actual sufre una alta exposición a este
metal y a que diversos estudios llevaron a considerar al aluminio como una sustancia
capaz de causar neurodegeneración. Condiciones patológicas como la enfermedad de
Parkinson han sido frecuentemente relacionadas con la acumulación de aluminio en el
cerebro. Además, aunque el aluminio sea un metal inactivo desde el punto de vista
redox, varios estudios han puesto de manifiesto que en determinadas circunstancias este
metal potencia el estrés oxidativo cerebral. Sin embargo, el mecanismo molecular de su
neurotoxicidad por el momento no es bien conocido. Por este motivo, decidimos
profundizar en el conocimiento de los mecanismos de la neurotoxicidad del aluminio en
procesos degenerativos inducidos por estrés oxidativo relacionados con la enfermedad
de Parkinson.
Para abordar esta investigación, resultaba imprescindible un conocimiento
previo de la cinética del daño oxidativo inducido por la 6-OHDA en un modelo
experimental de Parkinson, así como cuantificar los cambios observados en los índices
de peroxidación lipídica y estado oxidativo de las proteínas en estriado y mesencéfalo
ventral. Estos resultados nos permitirían conocer el momento exacto tras la
administración intracerebral de 6-OHDA en el que efectuar la medida de estrés
oxidativo cerebral para el estudio de los efectos oxidativos del aluminio. Para este
estudio, elegimos la inyección unilateral e instraestriatal de 6-OHDA para lesionar la
vía dopaminérgica nigroestriatal en ratas macho Sprague-Dawley. Este procedimiento
ha sido frecuentemente utilizado para estudiar la degeneración de las neuronas
dopaminérgicas características de la enfermedad de Parkinson. Si lo comparamos a
otros modelos, con inyecciones de 6-OHDA en sustancia negra o tracto nigroestriatal,
este método se aproxima más a la progresión de la enfermedad de Parkinson, puesto que
conlleva una degeneración retrógrada del sistema nigroestriado más lenta, con un
período de varias semanas (Sauer and Oertel 1994, Przedborski et al. 1995, Lee et al.
163

RESUMEN
1996, Shimohama et al. 2003). De hecho, esto nos permitió hacer un seguimiento de
cinética con distintos grupos de ratas que abarcó un período de 7 días. Nuestros
resultados indicaron claramente que la inyección unilateral e intraestriatal de 6-OHDA
produce elevados niveles de peroxidación lipídica y de oxidación protéica en el
mesencéfalo ventral y en el estriado. Para ambas regiones observamos un aumento
durante los 2 primeros días después de la inyección y un retorno de los valores a niveles
aproximados de control a los 7 días de la inyección. Los valores más altos fueron
alcanzados a las 48 horas de la inyección para TBARS y para el contenido de grupos
carbonilo en proteínas, y a las 24 horas para el contenido de grupos tiol en proteínas.
Resultó interesante observar que en el lado contralateral (mesencéfalo ventral y
estriado) se produce también un aumento significativo, aunque en menor cuantía, de los
niveles de estrés oxidativo, hecho que desaconseja el uso de esa zona como control para
determinar alteraciones neuroquímicas producidas por la 6-OHDA en este modelo
experimental de Parkinson. Finalmente, y de acuerdo con los resultados obtenidos
decidimos establecer el tiempo óptimo en 48 horas después de la inyección para
cuantificar los niveles de estrés oxidativo cerebral en los ensayos sobre la capacidad
oxidativa del aluminio en este modelo experimental de Parkinson.
164
La segunda parte de esta tesis consistió en desarrollar un régimen de
dosificación del aluminio en ratas, que garantizase una acumulación significativa de este
metal en el cerebro, y con ello un conocimiento preciso de la distribución del aluminio
en las regiones cerebrales. Puesto que previamente había sido demostrado que la
distribución del aluminio depende de la especie animal en cuestión y de la fórmula
química del aluminio administrado, optamos por utilizar dos vías distintas de
administración: oral e intraperitoneal. Se usaron ratas Sprague-Dawley que fueron
inyectadas diariamente por vía intraperitoneal con cloruro de aluminio (10 mg
Al 3+ /kg/día) durante una semana, o bien recibieron por vía oral una dosis progresiva de
cloruro de aluminio (25, 50, 100 mg Al 3+ /kg/día) y citrato (89, 178, 356 mg/kg/día)
durante 4 semanas. Nuestros resultados indicaron que ambas vías de administración
produjeron una acumulación de aluminio en cerebro. La acumulación fue mayor y más
significativa para el grupo que recibía el aluminio por vía intraperitoneal en todas las

RESUMEN
zonas cerebrales excepto en el mesencéfalo ventral. La distribución también varió según
el modo de administración. De acuerdo con estos resultados decidimos utilizar la vía de
administración intraperitoneal para estudiar el estrés oxidativo cerebral producido por el
aluminio.
Finalmente dedicamos la tercera fase de esta tesis a determinar la capacidad del
aluminio para alterar el estado oxidativo en áreas cerebrales específicas, tales como
cerebelo, mesencéfalo ventral, corteza, hipocampo y estriado. Ratas macho Sprague-
Dawley fueron inyectadas intraperitonealmente con cloruro de aluminio (10 mg
Al 3+ /kg/día) en salino durante 10 días. Como demostramos previamente, esta
dosificación era suficiente para asegurar una acumulación del aluminio en las regiones
cerebrales estudiadas. Para realizar estudios de peroxidación lipídica y de oxidación
protéica, los animales fueron sacrificados a las 48 horas después de la administración
intraventricular de 6-OHDA porque, como comentamos anteriormente, el valor álgido
de estrés oxidativo ocurre en ese momento. Nuestros resultados indicaron que, excepto
para el hipocampo, el metal produjo un aumento en los niveles de estrés oxidativo
(formación de TBARS y contenido de grupos carbonilo y tiol en proteínas) para la
mayoría de las zonas cerebrales estudiadas, acompañado de un descenso en las
actividades de las enzimas antioxidantes (superóxido dismutasa, catalasa y glutatión
peroxidasa). Sin embargo, estudios in vitro confirmaron la incapacidad del aluminio
para alterar la actividad de esos enzimas, así como también para afectar a la actividad de
las monoamino oxidasas A y B. Los efectos observados presentan un comportamiento
selectivo y regional para todas las estructuras estudiadas. Cabe destacar el caso del
hipocampo, ya que la exposición al aluminio produjo un aumento de las actividades de
las enzimas antioxidantes, una disminución de las oxidaciones protéicas y no alteró de
manera significativa la peroxidación lipídica. Estos resultados pueden ser explicados
por la alta acumulación de aluminio y el fomento de uno o varios mecanismos
compensatorios en esa zona cerebral.
165

RESUMEN
166
Por último y con el fin de esclarecer el papel del aluminio como un posible
factor etiológico de la enfermedad de Parkinson, hemos estudiado la capacidad de ese
metal para aumentar la neurodegeneración dopaminérgica estriatal y los índices de
estrés oxidativo en el mesencéfalo ventral y el estriado de ratas inyectadas
intraventricularmente con 6-OHDA. Este modelo animal es conocido por inducir un
síndrome parkinsoniano más lento, mostrando una depleción de neuronas
dopaminérgicas topográficamente similar (Rodriguez et al. 2001, Rodríguez-Díaz et al.
2001), y evitando la lesión focal alrededor de la punta de la cánula, producida cuando la
6-OHDA es introducida directamente en los tejidos cerebrales. Para la dosificación
seguimos el procedimiento mencionado anteriormente (inyección i.p. diaria de 10 mg
Al 3+ /kg/día durante 10 días), excepto que las ratas fueron lesionadas el octavo día con 6-
OHDA inyectada en el tercer ventrículo. Para realizar los estudios
inmunohistoquímicos, los animales fueron sacrificados una semana después de la
lesión, porque la degeneración progresiva de las células dopaminérgicas mesencefálicas
producida por inyección intraventricular de 6-OHDA llega a un nivel definitivo de
lesión una semana después de la inyección (Rodriguez et al. 2001). Nuestros resultados
indicaron que el aluminio aumenta la capacidad de la 6-OHDA para causar
peroxidación lipídica y oxidación protéica (excepto para el contenido de grupos tiol en
proteínas en el mesencéfalo ventral). Además, el metal aumentó la capacidad de la 6-
OHDA para producir la neurodegeneración del sistema dopaminérgico, ya que la
pérdida de terminales estriatales inmunoreactivas para la tirosina hidroxilasa aumentó
de manera significativa.
Nota: Cabe destacar que todos los experimentos realizados a lo largo de esta tesis
fueron conformes a la publicación del NIH, nº 86-23, revisada en 1996,
“Principles of laboratory animal care”, y aprobados por el correspondiente
Comité de Ética de la Universidad de Santiago de Compostela. Además, todos
los esfuerzos fueron realizados para reducir la cantidad de animales utilizados y
para minimizar el sufrimiento de los mismos.

CONCLUSIONES

CONCLUSIONES
CONCLUSIONES
Los resultados obtenidos a lo largo de esta tesis permitieron establecer las siguientes
conclusiones:
Capítulo 1
1) Las inyecciones intraestriatales y unilaterales de 6-OHDA provocan un aumento
continuo y simultáneo de los índices de estrés oxidativo en el lado ipsilateral,
alcanzando un valor máximo 48 horas después de la inyección para TBARS y para
el contenido de grupos carbonilo en proteínas, y a las 24 horas para el contenido de
grupos tiol en proteínas. Los valores de TBARS, así como el contenido de grupos
carbonilo y tiol en proteínas vuelven lentamente a los valores control después de 7
días tras la administración de 6-OHDA.
2) En este modelo experimental de Parkinson, el lado contralateral no debe ser
utilizado como control para evaluar los cambios neuroquímicos inducidos por 6-
OHDA, dado que la inyección unilateral de 6-OHDA provoca un aumento
significativo en los índices de estrés oxidativo, aunque en menor cuantía que los
índices observados en el lado ipsilateral, afectando tanto el estriado como el
mesencéfalo ventral.
3) Cuando este modelo experimental es utilizado para evaluar nuevas estrategias
neuroprotectoras o para estudiar el potencial oxidativo de un posible factor
etiológico, la cuantificación de niveles cerebrales de estrés oxidativo (TBARS y
contenido de grupos oxidados en proteínas) debe llevarse a cabo 48 horas después
de la administración intracerebral de 6-OHDA, que es el tiempo en el que se
alcanzan los valores máximos.
169

CONCLUSIONES
Capítulo 2
4) La administración oral de cloruro de aluminio a ratas durante 4 semanas provoca un
170
aumento significativo en la concentración cerebral de aluminio en todas las zonas
estudiadas (cerebelo, mesencéfalo ventral, corteza, hipocampo y estriado).
5) La administración intraperitoneal de cloruro de aluminio a ratas durante una
semana da lugar a una destacada e importante acumulación de aluminio en
cerebelo, corteza, hipocampo y estriado, mientras que en mesencéfalo ventral se
observa una disminución en la concentración de aluminio.
6) El aluminio administrado vía intraperitoneal se acumula de manera predominante
en el hipocampo, mientras que el aluminio administrado oralmente se acumula
principalmente en el mesencéfalo ventral.
7) Tanto la concentración de aluminio en cerebro como su distribución en las distintas
áreas cerebrales varía dependiendo de la vía de administración usada para el
aluminio.
Capítulo 3
8) La administración intraperitoneal de aluminio a ratas lleva a una situación de estrés
oxidativo en la mayoría de las zonas cerebrales estudiadas.
9) El aluminio provoca un aumento destacado de los niveles de TBARS en
mesencéfalo ventral, corteza y estriado, y sobre todo en cerebelo, mientras que no
se hallan cambios significativos en hipocampo.
10) El aluminio ocasiona un aumento significativo en el contenido de grupos carbonilo
y tiol en proteínas en cerebelo, mesencéfalo ventral y estriado, mientras que en
corteza e hipocampo se observa una disminución. No se ha podido establecer el
mecanismo molecular responsable de la falta de correlación directa entre el

CONCLUSIONES
aumento de grupos carbonilo observado en proteínas y el “aparentemente”
contradictorio aumento en el contenido de grupos tiol.
11) Todas las regiones cerebrales examinadas muestran patrones similares, puesto que
el aluminio induce una disminución de la actividad enzimática de la superóxido
dismutasa, la glutatión peroxidasa y la catalasa, excepto en el hipocampo en el que
la actividad de estos enzimas antioxidantes aumenta.
12) In vitro, el aluminio no modifica de manera significativa las actividades
enzimáticas de la superóxido dismutasa, glutatión peroxidasa y catalasa.
13) In vitro, el aluminio no afecta a las actividades enzimáticas de la monoamino
oxidasa A (MAO-A) ni de la monoamino oxidasa B (MAO-B).
14) In vivo, el aluminio provoca un aumento de la peroxidación lipídica y de la
oxidación protéica inducidas por 6-OHDA en el modelo experimental de Parkinson
utilizado, excepto para el contenido de grupos tiol en mesencéfalo ventral.
15) La administración de aluminio a ratas control a las que se administró
intraventricularmente salino en lugar de 6-OHDA también provocó una variación
significativa, tanto en el índice de peroxidación lipídica como en el estado
oxidativo de proteínas en lo que respecta a contenido de grupos carbonilo.
16) Los estudios inmunohistoquímicos demuestran que la administración de aluminio
solo no provoca cambio significativo de inmunoreactividad para tirosina
hidroxilasa en las terminales estriatales. Sin embargo, la lesión con 6-OHDA causa
una pérdida de inmunoreactividad de tirosina hidroxilasa en las terminales
estriatales, que aumenta de manera significativa con la administración de aluminio.
171

CONCLUSIONES
172
En base a las conclusiones que se acaban de enumerar, establecemos, que los
resultados expuestos confirman el potencial del aluminio como factor de riesgo en el
desarrollo de la enfermedad de Parkinson. Este metal actúa principalmente como un
pro-oxidante y es capaz de disminuir las actividades de los enzimas antioxidantes. Su
toxicidad observada en las diferentes áreas cerebrales estudiadas (cerebelo, mesencéfalo
ventral, corteza y estriado) probablemente se halla mediada por la contribución del
anión superóxido de aluminio AlO2 ●2+ y por daños oxidativos a células inducidos por
otros radicales libres. Cabe destacar que los resultados observados en el hipocampo
(alto contenido en aluminio y estado oxidante no aumentado) pueden ser causados por
uno o varios mecanismos compensatorios de la barrera hemato-hipocampal, pero esta
idea necesita ser corroborada y caracterizada por investigaciones futuras.

APPENDIX

Publications as a direct result from this thesis
APPENDIX
Sánchez-Iglesias S., Méndez-Álvarez E., Iglesias-González J., Muñoz-Patiño A.,
Sánchez-Sellero I., Labandeira-García J. L. and Soto-Otero R. (2009) Brain oxidative
stress and selective behaviour of aluminium in specific areas of rat brain: potencial
effects in a 6-OHDA-induced model of Parkinson‟s disease. J. Neurochem. 109, 879–
888. PMID: 19425176.
Sánchez-Iglesias S., Soto-Otero R., Iglesias-Gónzalez J., Barciela-Alonso M. C.,
Bermejo-Barrera P. and Méndez-Álvarez E. (2007) Analysis of brain regional
distribution of aluminium in rats via oral and intraperitoneal administration. J. Trace
Elem. Med. Biol. 21 Suppl. 1, 31–34. PMID: 18039493.
Sánchez-Iglesias S., Rey P., Méndez-Álvarez E., Labandeira-García J. L. and Soto-
Otero, R. (2007) Time course of brain oxidative damage caused by intrastriatal
administration of 6-hydroxydopamine in a rat model of Parkinson‟s disease.
Neurochem. Res. 32, 99–105. PMID: 17160721.
175

APPENDIX
Publications arising from collaborative work during this thesis
Soto-Otero R., Méndez-Álvarez E., Sánchez-Iglesia S., Zubko F. I., Voskressensky L.
G., Varlamov A. V., De Candia M. and Altomare, C. (2008) Inhibition of 6-
hydroxydopamine-induced oxidative damage by 4,5-dihydro-3H-2-benzazepine N-
oxides. Biochem. Pharmacol. 75, 1526–37. PMID: 18275937.
Rey P., López-Real A., Sánchez-Iglesias S., Muñoz A., Soto-Otero R. and Labandeira-
García J. L. (2007) Angiotensin type-1-receptor antagonists reduce 6-hydroxydopamine
toxicity for dopaminergic neurons. Neurobiol. Aging 28, 555–567. PMID: 16621167.
Soto-Otero R., Sanmartín-Suárez C., Sánchez-Iglesias S., Hermida-Ameijeiras A.,
Sánchez-Sellero I. and Méndez-Álvarez E. (2006) Study on the ability of 1,2,3,4-
tetrahydropapaveroline to cause oxidative stress: mechanisms and potential implications
in relation to Parkinson‟s disease. J. Biochem. Mol. Toxicol. 20, 209–220. PMID:
17009235.
Hermida-Ameijeiras A., Méndez-Álvarez E., Sánchez-Iglesias S., Sanmartín-Suárez C.
and Soto-Otero R. (2004) Autoxidation and MAO-mediated metabolism of dopamine as
a potential cause of oxidative stress: role of ferrous and ferric ions. Neurochem. Int. 45,
103–116. PMID: 15082228.
176

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