The Toxicologist - Society of Toxicology

1278 PINK1 AND MITOCHONDRIAL DYNAMICS: A NEW
PERSPECTIVE OF MITOCHONDRIAL DYSFUNCTION
IN PARKINSON’S DISEASE?
K. Tieu 1, 2, 3 , M. Cui 1, 2 , X. Tang 1, 2 , W. V. Christian 3 and Y. Yoon 4 . 1 Neurology,
University of Rochester, Rochester, NY, 2 Center for Translational Neuromedicine,
University of Rochester, Rochester, NY, 3 Environmental Medicine, University of
Rochester, Rochester, NY and 4 Anesthesiology, University of Rochester, Rochester, NY.
Mutations in the mitochondrial encoded protein PTEN-induced putative kinase 1
(PINK1) cause autosomal recessive Parkinson’s disease (PD). In mammalian cells,
mutant PINK1 has been reported to promote fission or inhibit fusion on mitochondria;
however, the mechanism by which this process occurs remains elusive.
Using an inducible expression system in mammalian dopaminegic neurons, we report
here that human mutant PINK1 (L347P and W437X) mediates an overall fission
effect by increasing the ratio of mitochondrial fission over fusion proteins,
leading to excessive dysfunctional fragmented mitochondria. Knocking down endogenous
Pink1 produces similar effects. In contrast, over-expressing human wild
type PINK1 produces a pro-fusion effect by increasing the ratio of mitochondrial
fusion/fission proteins, without resulting in functionally compromised mitochondria.
Parkin knockdown blocks the imbalance in fission/fusion proteins –suggesting
PINK1 and parkin maintain proper mitochondrial function and integrity via this
mitochondrial machinery. Through genetic manipulations and pharmaceutical
treatment, which inhibit mitochondrial fission, both structural and functional mitochondrial
defects induced by mutant PINK1 were attenuated. This study suggests
that targeting the mitochondrial fission/fusion machinery either through genetic
approach or treatment with small molecules may represent a novel therapeutic
avenue for PD.
1279 FUNCTIONAL ROLE OF THE NEUROTOXICITY
BIOMARKER PROTEIN TSPO/PBR IN PRIMARY
MICROGLIA.
J. Choi 1 , M. Ifuku 2 , M. Noda 2 and T. R. Guilarte 1 . 1 Environmental Health
Sciences, Johns Hopkins School of Public Health, Baltimore, MD and 2 Lab of
Pathophysiology, Kyushu University, Fukuoka, Japan.
Translocator protein-18 kDa (TSPO), previously called the peripheral benzodiazepine
receptor (PBR), is a glia protein that has been extensively used as a biomarker
of brain injury in a variety of animals models of neurotoxicity and in
human neurodegenerative disease (Chen&Guilarte, Pharm Ther 118:1-17,2008).
While TSPO is a validated biomarker of neurotoxicity, the functional role of TSPO
in glial cells is not known. In this study, we examine the effect of TSPO activation
by the TSPO-specific ligands R-PK11195 (PK) and Ro5-4864 (Ro) in primary microglia.
Exposure to different concentrations (1-1000 nM) of PK for 24 hrs resulted
in increased production of intracellular reactive oxygen species and increased microglia
proliferation and phagocytosis. No effect on intracellular levels of reactive
nitrogen species was measured. A similar effect was observed with Ro. These ligands
did not affect microglia migration, but PK produced mitochondria hyperpolarization
while Ro did not. We also examine whether these ligands could alter expression
of pro-inflammatory genes and cytokine release in lipopolysaccharide (LPS)-activated
microglia. At a 10 nM concentration, neither PK nor Ro had an effect on
LPS-induced increase in COX-2, IL-1β, or TNF-α gene expression. Ro but not PK
did enhance the LPS-induced release of IL-1β. Consistent with this finding, Ro but
not PK increased the number of apoptotic microglia. These studies showed that
TSPO activation regulates cellular processes that are essential to microglia function.
Further, when microglia are activated by an inflammogen, TSPO activation enhances
IL-1β release and increases microglia apoptosis. These findings suggest that
TSPO plays a critical role in cellular functions that are essential to mount a microglia
response to the disruption of brain homeostasis. They also indicate that
under pathological conditions, TSPO activation may be responsible for the elimination
of activated microglia via apoptosis. [Supported by NIEHS-ES07062 to
TRG and NIEHS-T32 ES07141]
1280 OVARIECTOMY POTENTIATES REDUCTIONS IN
STRIATAL NERVE TERMINAL DOPAMINE LEVELS
AFTER CHRONIC PCB EXPOSURE.
A. Dreiem 1, 2 , V. M. Miller 1, 2 , S. Sanchez-Morrissey 1 , K. L. Andrews 1 , N. Neu 1 ,
K. O. Brosch 1 and R. F. Seegal 1, 2 . 1 Wadsworth Center, Albany, NY and 2 School of
Public Health, University at Albany, Albany, NY.
Estrogen is suggested to protect against Parkinson’s disease (PD) because reductions
in estrogen levels by oophorectomy increases the risk of PD in women, and hormone
replacement therapy protects against PD. Estrogen also protects against loss
of dopaminergic function in animal models of PD. Polychlorinated biphenyls
(PCBs) are dopaminergic toxicants implicated in PD, and women occupationally
exposed to PCBs have higher PD mortality than males (Epidemiology (2006),
17:8-13). We have investigated whether reductions in female ovarian hormones, induced
by ovariectomy (OVX), modulate the effects of chronic exposure to PCBs
(A1254) on dopamine function in C57BL/6 mice. Female mice underwent OVX
or sham surgery at 11 weeks of age. After 2 weeks’ recovery mice were exposed to
500 ppm A1254 or corn oil (vehicle) for 70 days. After sacrifice, DAT activity was
assessed by [ 3 H]-DA uptake and levels of DA and DA metabolites were analyzed by
HPLC in striatal synaptosomes.
A1254 significantly reduced synaptosomal DA levels relative to vehicle exposed animals
in OVX, but not in sham operated females, suggesting that ovariectomy sensitizes
female mice to A1254. DA turnover, assessed as DOPAC/DA ratios, was increased
by ovariectomy, indicating that loss of gonadal hormones increased DA
metabolism. In contrast, DA turnover was not altered by A1254 exposure. A1254
exposure competitively inhibited DA reuptake via DAT, indicated by increased DAT
K M
values, relative to controls in both sham and OVX animals. These observations
suggest that the ‘double hit’ of OVX and A1254 exposure, increasing DA turnover
and inhibiting DA reuptake into the nerve terminal, respectively, lead to reduced
DA levels in striatal nerve terminals. These findings are in agreement with the hypothesis
that loss of ovarian hormones increases sensitivity to environmental contaminants,
and suggests that postmenopausal women may be more sensitive to PCBs
than premenopausal women. Supported by NIH grant R01ES014675 to RFS.
1281 TANESPIMYCIN BLOCKS BORTEZOMIB-INDUCED
PERIPHERAL NEUROPATHY IN RATS.
J. Oberdoerster 1 , B. D. Car 2 , O. P. Flint 2 , T. P. Reilly 1 , J. Arezzo 4 , M. Litwak 4 ,
A. M. Fletcher 1 , G. E. Schulze 1 , R. Westhouse 2 and D. M. Berman 3 . 1 Drug
Safety Evaluation, Bristol-Myers Squibb Company, East Syracuse, NY, 2 Discovery
Toxicology, Bristol-Myers Squibb Company, Princeton, NJ, 3 Global Clinical Research,
Bristol-Myers Squibb Company, Princeton, NJ and 4 Neuroscience and Neurology,
Albert Einstein College of Medicine, Bronx, NY.
Tanespimycin (TAN) is a Heat-Shock Protein 90 inhibitor currently in Phase 3
clinical trials with bortezomib (BTZ) for the treatment of multiple myeloma
(MM). While BTZ-induced severe peripheral neuropathy is observed in 8-12% of
patients with MM, no severe peripheral neuropathy was observed in a Phase 1/2
trial in MM patients treated with BTZ and TAN. The potential neuroprotective effect
of TAN was confirmed in an initial TAN/BTZ combination study in rats utilizing
a sensory threshold endpoint (ie, mechanical allodynia) wherein BTZ-induced
hypernociception, consistent with a peripheral neuropathy, was ameliorated
by co-administration of TAN. We extend these findings in the same model by exploring
exploring electrophysiologic and histopathologic endpoints. Male Sprague-
Dawley rats were intravenously administered BTZ and/or TAN at clinically relevant
doses (ie, 0.22 mg/kg and 20 mg/kg, respectively) twice weekly for 5 to 8
weeks; both preventative and reversal paradigms were explored. After 5 weeks of
dosing with BTZ alone, nerve conduction velocity (NCV) in the caudal nerve was
significantly reduced compared to age-matched control values (p < 0.05). However,
when BTZ and TAN were co-administered simultaneously, the deficit in caudal
nerve NCV was prevented and velocities were indistinguishable from findings in
age-matched controls. Histopathologic evaluations, including electronic microscopy
and immunohistochemistry, are ongoing. These findings confirm that
TAN prevents BTZ-induced neuropathy in rats, consistent with the observation of
less severe neuropathy in humans. To our knowledge, this is the first drug demonstrated
to protect against BTZ-induced neuropathy in animal models, with clinical
evidence to support a bench-to-bedside correlation.
1282 THE PROGRESSION OF EXPERIMENTAL
AUTOIMMUNE ENCEPHALITIS IS DEPENDENT ON
NOD2 AND RICK ACTIVATION IN MICROGLIA
REPRESENTING A NOVEL THERAPEUTIC TARGET
FOR MULTIPLE SCLEROSIS.
P. J. Shaw, J. R. Lukens, S. Burns, H. Chi, M. A. McGargill and T. Kanneganti.
Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN.
The innate immune system is required for host defense to recognize invading
pathogens and to eliminate them. Recent work has identified a group of cytosolic
pattern recognition receptors called the NOD-like receptor (NLR) family of proteins.
NOD1 and NOD2 are NLR proteins which are activated by fragments of
peptidoglycan. Following direct or indirect activation of NOD2 by muramyl
dipeptide (MDP), a CARD domain is exposed, recruiting the kinase RICK via homotypic
CARD interactions. RICK activation results in MAPK pathway and NFκB
activation. Multiple sclerosis (MS) is an autoimmune disease in which T cells
attack oligodendrocytes and strip neurons of the myelin sheath required to propagate
electrical signals. Peptidoglycan from a bacterial infection or gut microflora is
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