The Toxicologist - Society of Toxicology

164 TRANSLOCATOR PROTEIN (18 KDA) (TSPO): A PRE-
CLINICAL BIOMARKER OF NEURODEGENERATION
IN SANDHOFF DISEASE MICE.
J. Choi 1 , H.Wang 2 , C.J.Endres 2 , J.J.Fox 2 , G. Green 2 , M.G.Pomper 2 and
T. R. Guilarte 1 . 1 Environmental Health Sciences, Columbia University Mailman
School of Public Health, New York, NY and 2 Radiology, Johns Hopkins Hospital,
Baltimore, MD.
Translocator protein (18 kDa) (TSPO), formerly known as the peripheral benzodiazepine
receptor (PBR), has been widely used as a sensitive biomarker of brain injury
and inflammation. Sandhoff disease is an autosomal recessive neurodegenerative
disease characterized by excess glycolipid storage due to the lack of lysosomal
β-hexosaminidase. A deficiency of this enzyme results in impaired degradation of
G M2 and G A2 gangliosides and other glycolipids, leading to severe neurodegenerative
changes in the brain. In the present study, we used a mouse model of Sandhoff
disease to assess the longitudinal expression of TSPO as a function of disease progression.
Using a novel TSPO-specific ligand [ 125 I]Iodo-DPA-713, we found that
TSPO levels were significantly increased in relevant brain regions prior to the behavioral
expression of disease using ex vivo quantitative autoradiography. These
findings were confirmed using in vivo [ 125 I]Iodo-DPA-713 microSPECT imaging.
We also show that the increase in TSPO levels was associated with neurodegenerative
changes using silver staining and activation of microglia and astrocytes visualized
by immunohistochemistry. In brain regions known to be undergoing neurodegenerative
changes, there was differential expression of microglia and astrocytes
with microglia being activated early in the neurodegenerative process and astrocytes
activated at a later time point. These findings provide strong evidence that TSPO
can be used as an early pre-clinical biomarker of neurodegenerative changes prior to
the behavioral manifestation of disease. Further, the novel TSPO radioligand
[ 125 I]Iodo-DPA-713 appears to be a useful SPECT ligand for the quantitation and
visualization of TSPO levels in the rodent brain.
165 THE CYANOBACTERIAL NEUROTOXIN β-N-
METHYLAMINO-L-ALANINE (BMAA) INDUCES
UNFOLDED PROTEIN RESPONSE (UPR) AND
CHANGES OF SOD ACTIVITY LEVELS IN HUMAN
NEURONAL CELLS.
O. Okle 1 , M. Helmer 1 , K. Stemmer 1, 2 and D. R. Dietrich 1 . 1 Human &
Environmental Toxicology, University of Konstanz, Konstanz, Germany and 2 Obesity
Research Centre at the Metabolic Disease Institute, University of Cincinnati,
Cincinnati, OH.
The cyanobacterial β-N-methylamino-L-alanine (BMAA) is described as an excitotoxin
and assumed to be involved in the development of amyotrophic lateral sclerosis/
Parkinsonism-dementia complex (ALS/ PDC) on Guam. Although potential
biomagnification of BMAA in the food chain of the indigenous Chamorro of
Guam may lead to the observed ALS/ PDC, BMAA was also reported outside of
the Pacific area, i.e. in brain tissue of Canadian patients with Alzheimer’s disease.
The toxic effect of BMAA in in vivo and in vitro studies, mouse and rat models, is
primarily characterized by its acute high dose excitotoxicity which, however, does
not explain the chronic exposure associated effects e.g. ALS/ PDS. It is therefore
plausible that in the observed human neuropathy intracellular mechanisms, other
than excitotoxicity, are involved in the progressive degradation of motor neurons.
Within 3 hours of exposure of the human neuroblastoma cell line SH-SY5Y,
BMAA induced an unfolded protein response (UPR) and an alteration of additional
stress response proteins. A dose and time dependent increase of ROS, formerly
detected in neuronal rodent models, was also observed in this human neuronal
model. Increased ROS was also linked to a modified expression and activity of
SOD1. As changes in SOD activity are closely related to the pathogenesis of familial
ALS patients, both, changed SOD activity and UPR, as observed in the human
neuronal cell model, could provide for a slow demise of motor neurons and thus
contribute to the onset or progression of ALS/PDC in humans.
166 CYTOTOXICITY OF β-N-METHYLAMINO-L-ALANINE
(L-BMAA) AND METHYLAZOXYMETHANOLACETATE
(MAMAC) AND ASSOCIATED DIFFERENTIAL MRNA
EXPRESSION IN HUMAN NEURONAL CELLS.
M. Helmer 1 , O. Okle 1 , K. Stemmer 1, 2 and D. R. Dietrich 1 . 1 Human &
Environmental Toxicology, University of Konstanz, Konstanz, Germany and 2 Obesity
Research Centre at the Metabolic Disease Institute, University of Cincinnati,
Cincinnati, OH.
The cyanobacterial toxin β-N-methylamino-l-alanine (L-BMAA) is neurotoxic
amino acid acting via excitotoxic pathways. The cycad plant toxin cycasin and its
aglykon methylazoxymethanol (MAM) and acetate methylazoxymethanolacetate
(MAMAc) have been described to be genotoxic via formation of DNA adducts.
There is a possible link between the consumption of cycas plants containing both
toxins and the high incidence of the neurodegenerative disease amyotrophic lateral
sclerosis/ Parkinsonism dementia complex (ALS/PDC) among the indigenous
Chamorro people on the Mariana Islands. The aim of the current in vitro study was
to characterize the effects of L-BMAA and MAMAc on the growth, cell viability
and mRNA expression in the human neuroblastoma cell line SH SY5Y.
We observed a time and dose dependent cytotoxicity of MAMAc, determined via
the MTT reduction assay, neutral red uptake test and non-differential cell counts.
The susceptibility of cells to L-BMAA cytotoxicity appeared to be increased by pretreatment
of the cells with MAMAc. Exposure to non-cytotoxic concentrations of
MAMAc and L-BMAA (increased/decreased) mRNA expression, examined by
cDNA synthesis and qPCR, of the cellular stress markers O6-methylguanine-DNA
methyltransferase (MGMT), 70 kd Heat shock Protein (Hsp70) and BCL2-associated
athanogene 1 (BAG1). Thus cycasin and L-BMAA exposure could induce an
increased dysfunctionality of cellular homeostasis in human neuronal cells and thus
contribute to the onset or progression of ALS/PDC.
167 PERTURBATION OF REGIONAL BRAIN METABOLISM
IN F344 RATS AFTER LOW LEVEL EXPOSURE TO
DIISOPROPYLFLUOROPHOSPHATE (DFP).
D. Mahle 1 and N. V. Reo 2 . 1 AFRL/RHPB, Wright-Patterson Air Force Base, OH
and 2 Wright State University, Fairborn, OH.
The mechanism of organophosphate (OP) induced inhibition of acetylcholinesterase
and subsequent excitotoxicity is well described. However, exposure
to OPs at non-cholinergic doses has been reported to cause aberrations in neuronal
development, cellular signaling and deficits in cognitive behavior and spatial memory.
Little is known about the mechanisms of action for this noncholinergic toxicity
of OPs. Using multinuclear NMR techniques the metabolic effects of DFP in
rat brain can be investigated and possible correlations with acetylcholinesterase
(AChE) inhibition can be explored. Adult male F344 rats were administered 1
mg/kg DFP or saline via subcutaneous injection at 10 mL/kg and were euthanized
at 0.5, 1, 2, 12, 24 and 48 hr post dose. Brains were removed and cortex, cerebellum,
hippocampus and brainstem were collected. Lipid and aqueous extracts were
prepared from each brain region, and profiles of small molecule metabolites, lipids
and phospholipids were measured using 1H, 13C and 31P NMR spectroscopy.
Whole blood AChE activity in DFP-treated rats was 77% of control values at 2 hr
post dose and continued to decline to 65% at 48 hr. All brain regions reached a
minimum of AChE activity (40-55%) at 1-2 hr post dose with the exception of cortex
which had a minimum of activity at 12 hr post dose. AChE activity returned to
60-80% of control by 48 hr. At 2 hr post dose, cortex showed the most changes in
the lipid profile with significant decreases in cholesterol, ω-3 and ω-6 fatty acids, as
well as phosphatidylcholine, phosphatidylethanolamine and ethanolamine plasmalogen.
The mitochondrial phospholipid cardiolipin was significantly decreased
after 2 hr in brainstem. Niacinamide was increased in brainstem and hippocampus
2 hr post DFP, while succinate was increased in cerebellum 2 hr post DFP. By evaluating
the impact of low level OP exposure on the metabolic profile of specific
brain regions, we hope to gain a greater understanding of the noncholinergic mechanisms
of action and sensitive target areas of OPs.
168 INVESTIGATING GENE-ENVIRONMENT
INTERACTIONS RELEVANT TO PARKINSON’S
DISEASE USING DROSOPHILA.
C. A. Remillard, H. O. Lawal and D. E. Krantz. University of California, Los
Angeles, Los Angeles, CA.
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the selective
loss of dopaminergic (DA) neurons. The vast majority of PD cases are sporadic
and their etiology is poorly understood. Exposure to environmental toxins increases
the risk of PD but individual susceptibility varies suggesting additional genetic effects.
We are using Drosophila to investigate these potential gene-environment interactions.
Since DA itself may be neurotoxic, genes that regulate cellular DA levels
may be particularly important. We tested the effects of two genes that regulate levels
of cellular dopamine and its metabolites: the Drosophila vesicular monoamine
transporter (dVMAT), which packages and transports dopamine into synaptic vesicles,
and Drosophila aldehyde dehydrogenase (dALDH), which converts the highly
toxic dopamine metabolite 3,4dihydroxyphenylacetaldehyde (DOPAL) into the
less toxic species 3,4dihydroxyphenyl acetic acid (DOPAC). We exposed dVMAT
and dALDH mutant flies to pesticides, including paraquat and rotenone, both of
which are established models of PD. We then assayed organismal survival, locomotor
behaviors related to DA signaling and the number of DA neurons that remained
SOT 2011 ANNUAL MEETING 35