Ranjita Betarbet

Chronic systemic pesticide exposure reproduces features of Parkinson's disease

The cause of Parkinsons disease (PD) is unknown, but epidemiological studies suggest an association with pesticides and other environmental toxins, and biochemical studies implicate a systemic defect in mitochondrial complex I. We report that chronic, systemic inhibition of complex I by the lipophilic pesticide, rotenone, causes highly selective nigrostriatal dopaminergic degeneration that is associated behaviorally with hypokinesia and rigidity. Nigral neurons in rotenone-treated rats accumulate fibrillar cytoplasmic inclusions that contain ubiquitin and α-synuclein. These results indicate that chronic exposure to a common pesticide can reproduce the anatomical, neurochemical, behavioral and neuropathological features of PD.

Mechanism of Toxicity in Rotenone Models of Parkinson's Disease

Exposure of rats to the pesticide and complex I inhibitor rotenone reproduces features of Parkinsons disease, including selective nigrostriatal dopaminergic degeneration and α-synuclein-positive cytoplasmic inclusions (Betarbet et al., 2000; Sherer et al., 2003). Here, we examined mechanisms of rotenone toxicity using three model systems. In SK-N-MC human neuroblastoma cells, rotenone (10 nm to 1 μm) caused dose-dependent ATP depletion, oxidative damage, and death. To determine the molecular site of action of rotenone, cells were transfected with the rotenone-insensitive single-subunit NADH dehydrogenase of Saccharomyces cerevisiae (NDI1), which incorporates into the mammalian ETC and acts as a “replacement” for endogenous complex I. In response to rotenone, NDI1-transfected cells did not show mitochondrial impairment, oxidative damage, or death, demonstrating that these effects of rotenone were caused by specific interactions at complex I. Although rotenone caused modest ATP depletion, equivalent ATP loss induced by 2-deoxyglucose was without toxicity, arguing that bioenergetic defects were not responsible for cell death. In contrast, reducing oxidative damage with antioxidants, or by NDI1 transfection, blocked cell death. To determine the relevance of rotenone-induced oxidative damage to dopaminergic neuronal death, we used a chronic midbrain slice culture model. In this system, rotenone caused oxidative damage and dopaminergic neuronal loss, effects blocked by α-tocopherol. Finally, brains from rotenone-treated animals demonstrated oxidative damage, most notably in midbrain and olfactory bulb, dopaminergic regions affected by Parkinsons disease. These results, using three models of increasing complexity, demonstrate the involvement of oxidative damage in rotenone toxicity and support the evaluation of antioxidant therapies for Parkinsons disease.

Subcutaneous rotenone exposure causes highly selective dopaminergic degeneration and α-synuclein aggregation

Previous studies demonstrated that chronic systemic exposure to the pesticide and mitochondrial toxin rotenone through jugular vein cannulation reproduced many features of Parkinsons disease (PD) in rats, including nigrostriatal dopaminergic degeneration and formation of alpha-synuclein-positive cytoplasmic inclusions in nigral neurons (R. Betarbet et al., 2000, Nat. Neurosci. 3, 1301-1306). Although novel and conceptually important, the rotenone model of PD suffered from being extremely labor-intensive. The current paper demonstrates that these same features of PD can be reproduced by chronic, systemic exposure to rotenone following implantation of subcutaneous osmotic pumps. Chronic subcutaneous exposure to low doses of rotenone (2.0-3.0 mg/kg/day) caused highly selective nigrostriatal dopaminergic lesions. Striatal neurons containing DARPP-32 (dopamine and cAMP-regulated phosphoprotein) remained intact with normal morphology, and NeuN staining revealed normal neuronal nuclear morphology. Neurons of the globus pallidus and subthalamic nucleus were spared. Subcutaneous rotenone exposure caused alpha-synuclein-positive cytoplasmic aggregates in nigral neurons. This new protocol for chronic rotenone administration is a substantial improvement in terms of simplicity and throughput.

Complex I and Parkinson's Disease

Complex I of the mammalian electron transfer chain is composed of at least 43 protein subunits, of which 7 are encoded by mtDNA. It catalyzes the transfer of electrons from NADH to ubiquinone and translocates protons from the mitochondrial matrix to the intermembrane space. It may also play direct roles in the mitochondrial permeability transition and in cell death pathways. Despite the limitations of current complex I assays, biochemical studies have suggested the presence of a mild, systemic defect of complex I in Parkinsons disease (PD). Recent experimental work has modeled this abnormality using rotenone to systemically inhibit complex I. Chronic rotenone exposure accurately recapitulated the pathological, biochemical, and behavioral features of PD. Thus, relatively subtle complex I abnormalities‐‐either genetic or acquired‐‐may be central to the pathogenesis of PD.

Selective microglial activation in the rat rotenone model of Parkinson's disease

Chronic rotenone exposure reproduces features of Parkinsons disease (PD) (Nat. Neurosci. 3 (2000) 1301; Exp. Neurol. 179 (2003) 9). We investigated the role of glial activation in rotenone toxicity in vivo. Male Lewis rats received 2-3 mg/kg rotenone per day for up to 4 weeks. In 50% of surviving rotenone-treated animals, there was nigrostriatal dopaminergic degeneration, marked by reduced tyrosine hydroxylase immunoreactivity). Extensive microglial activation, determined by OX-42-ir, occurred in striatum and nigra of rotenone-treated animals, and was prominent before anatomical evidence of dopaminergic lesions. Microglia enlarged and developed short, stubby processes in rotenone-treated animals. Rotenone-induced microglial activation was less pronounced in cortex. Reactive astrocytosis was minimal and limited to a thin rim around the lesion. Marked microglial activation with minimal astrocytosis is another pathological feature of PD reproduced by rotenone treatment.

Ubiquitin–proteasome system and Parkinson's diseases

Parkinsons disease (PD) is a progressive neurodegenerative disorder characterized by nigrostriatal dopaminergic degeneration and development of cytoplasmic inclusions known as Lewy bodies. To date, the mechanisms involved in PD pathogenesis are not clearly understood. Clues from genetic studies including identification of mutations in genes for alpha-synuclein, parkin, and ubiquitin carboxy hydrolase L1 associated with familial PD and the presence of proteinaceous cytoplasmic inclusions in spared dopaminergic nigral neurons in sporadic cases of PD have suggested an important role for ubiquitin-proteasome system (UPS) and aberrant protein degradation. In vivo and in vitro studies have linked parkin, alpha-synuclein, and oxidative stress to a compromised UPS and PD pathogenesis suggesting novel therapeutic targets.

Environment, Mitochondria, and Parkinson's Disease

Parkinsons disease (PD) is a common and disabling neurodegenerative disease marked by progressive motor dysfunction, which results from selective degeneration of the nigrostriatal pathway. Epidemiological studies indicate that exposure to pesticides, rural living, farming, and drinking well water are associated with an increased risk of developing PD. Rare cases of PD are caused by mutations in nuclear genes, and there is increasing evidence for susceptibility genes that alter disease risk. Parkinsons disease is also associated with a systemic defect in mitochondrial complex I activity. Animal models indicate that exposure to inhibitors of mitochondrial complex I, including pesticides, is sufficient to reproduce the features of PD, but genetic factors clearly modulate susceptibility. Complex I defects may result in oxidative stress and increase the susceptibility of neurons to excitotoxic death. In this way, environmental exposures and mitochondrial dysfunction may interact and result in neurodegeneration.

The rotenone model of Parkinson's disease: genes, environment and mitochondria

Parkinsons disease (PD) is occasionally caused by single gene mutations or by single toxic exposures, but most cases of PD are probably caused by some combination of genetic susceptibility and environmental exposure. Using rotenone as a prototype for an environmental toxicant, we argue here that genetic and environmental causes of PD converge on common pathogenic mechanisms. If so, protective strategies devised for one type of PD may be broadly useful for other forms of the disease.

Role of External Pallidal Segment in Primate Parkinsonism: Comparison of the Effects of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Parkinsonism and Lesions of the External Pallidal Segment

These experiments re-examined the notion that reduced activity in the external pallidal segment (GPe) results in the abnormalities of neuronal discharge in the subthalamic nucleus (STN) and the internal pallidal segment (GPi) and in the development of parkinsonian motor signs. Extracellular recording in two rhesus monkeys, which had been rendered parkinsonian by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), revealed that the average neuronal discharge rate decreased in GPe but increased in STN and GPi. After MPTP, neurons in all three nuclei tended to discharge in oscillatory bursts. In addition, GABA release in STN (measured with microdialysis) was reduced, indicative of reduced activity along the GPe-STN pathway. Finally, the concentration of glutamic acid dehydrogenase (GAD; measured with autoimmunoradiography) was increased in GPe and GPi, likely reflecting increased striatal input and increased activity of local axon collaterals, respectively. Surprisingly, GAD protein in STN remained unchanged, indicating that the usual assumption that GAD levels are determined primarily by the overall activity of GABAergic elements may be too simplistic. The results from the MPTP-treated animals were compared with results obtained in a second group of three animals with ibotenic acid lesions of GPe. GPe lesions resulted in increased discharge in STN and GPi, comparable with the changes seen after MPTP but did not induce oscillatory bursting and had no behavioral effects. The results indicate that a mere reduction of GPe activity does not produce parkinsonism. Other changes, such as altered discharge patterns in STN and GPi, may play an important role in the generation of parkinsonism.

Dopaminergic and GABAergic interneurons of the olfactory bulb are derived from the neonatal subventricular zone.

Earlier studies in our laboratory have demonstrated that a discrete region of the anterior part of the neonatal subventricular zone (SVZa) contains exclusively neuronal progenitor cells. The descendants of the SVZa progenitor cells are destined for the granule cell and glomerular layers of the olfactory bulb, where they differentiate into granule and periglomerular cells, the interneurons of the olfactory bulb, respectively. In the present set of experiments we examined the neurotransmitter phenotype of the SVZa‐derived cells. In order to label SVZa‐derived cells, the cell proliferation marker bromodeoxyuridine (BrdU) was injected into the SVZa of postnatal day 2 (P2) rats. After 3 weeks, by which time most of the SVZ‐aderived cells have migrated to their final destination in the bulb, the animals were perfused and their brains processed for immunohistochemistry. To identify the neurotransmitter phenotype of the SVZa‐derived cells, sagittal sections of the forebrain, including the olfactory bulb, were double‐labeled with an antibody to BrdU in conjunction with an antibody to γ‐amino‐butyric acid (GABA) or tyrosine hydroxylase (TH), the rate limiting enzyme in the synthesis of dopamine. Using simultaneous indirect immunofluorescence to detect the presence of single‐ and double‐labeled cells, we found that 59% and 51% of the BrdU‐positive cells were immunoreactive for GABA in the granule cell and glomerular layers, respectively. In addition, 10% of the BrdU‐positive periglomerular cells were immunoreactive for TH. The presence of double‐labeled (BrdU‐positive/GABA‐positive and BrdU‐positive/TH‐positive) cells in the olfactory bulb, demonstrates that the SVZa is a source of the GABAergic and dopaminergic interneurons of the olfactory bulb during postnatal development.