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Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mice and nonhuman primates causes a parkinsonian disorder characterized by a loss of dopamine-producing neurons in the substantia nigra and corresponding motor deficits. MPTP has been proposed to exert its neurotoxic effects through a variety of mechanisms, including inhibition of complex I of the mitochondrial respiratory chain, displacement of dopamine from vesicular stores, and formation of reactive oxygen species from mitochondrial or cytosolic sources. However, the mechanism of MPTP-induced neurotoxicity is still a matter of debate. Recently, we reported that the yeast single-subunit nicotinamide adenine dinucleotide (reduced) dehydrogenase (NDI1) is resistant to rotenone, a complex I inhibitor that produces a parkinsonian syndrome in rats, and that overexpression of NDI1 in SK-N-MC cells prevents the toxicity of rotenone. In this study, we used viral-mediated overexpression of NDI1 in SK-N-MC cells and animals to determine the relative contribution of complex I inhibition in the toxicity of MPTP. In cell culture, NDI1 overexpression abolished the toxicity of 1-methyl-4-phenylpyridinium, the active metabolite of MPTP. Overexpression of NDI1 through stereotactic administration of a viral vector harboring the NDI1 gene into the substantia nigra protected mice from both the neurochemical and behavioral deficits elicited by MPTP. These data identify inhibition of complex I as a requirement for dopaminergic neurodegeneration and subsequent behavioral deficits produced by MPTP. Furthermore, combined with reports of a complex I defect in Parkinson's disease (PD) patients, the present study affirms the utility of MPTP in understanding the molecular mechanisms underlying dopaminergic neurodegeneration in PD.

Sporadic Parkinson's disease (PD) is most likely caused by a combination of environmental exposures and genetic susceptibilities, although there are rare monogenic forms of the disease. Mitochondrial impairment at complex I, oxidative stress, alpha-synuclein aggregation, and dysfunctional protein degradation, have been implicated in PD pathogenesis, but how they are related to each other is unclear. To further evaluated PD pathogenesis here, we used in vivo and in vitro models of chronic low-grade complex I inhibition with the pesticide rotenone. Chronic rotenone exposure in vivo caused oxidative modification of DJ-1, accumulation of alpha-synuclein, and proteasomal impairment. Interestingly, the effects become more regionally restricted such that systemic complex I inhibition eventually results in highly selective degeneration of the nigrostriatal pathway. DJ-1 modifications, alpha-synuclein accumulation, and proteasomal dysfunction were also seen in vitro and these effects could be prevented with alpha-tocopherol. Thus, chronic exposure to a pesticide and mitochondrial toxin brings into play three systems, DJ-1, alpha-synuclein, and the ubiquitin-proteasome system, and implies that mitochondrial dysfunction and oxidative stress link environmental and genetic forms of the disease.

Parkinson's disease (PD) is a progressive neurological disorder marked by nigrostriatal dopaminergic degeneration and development of cytoplasmic proteinaceous aggregates known as Lewy bodies. Although the pathogenic mechanisms responsible for PD are not completely understood, many clues have come from biochemical, epidemiological, and genetic studies. Mutations in certain genes found in rare, familial cases of PD, such as alpha-synuclein and parkin, suggest a role for the ubiquitin-proteosome system and aberrant protein aggregation. Biochemical analyses have implicated mitochondrial dysfunction in PD. Epidemiological and animal model studies point to a role for environmental toxins, some of which are mitochondrial inhibitors. Mitochondrial dysfunction, resulting from either genetic defects, environmental exposures or an interaction between the two, may cause alpha-synuclein aggregation or neurodegeneration through oxidative stress or excitotoxicity. A better understanding of the mechanisms underlying PD should reveal novel therapeutic targets.

Chronic infusion of rotenone (Rot) to Lewis rats reproduces many features of Parkinson disease. Rot (3 mg/kg/day) was infused subcutaneously to male Lewis rats for 6 days using Alzet minipumps. Control rats received the vehicle only. Presence of 0.1% bovine serum albumin during the isolation procedure completely removed rotenone bound to the mitochondria. Therefore all functional changes observed were aftereffects of rotenone toxicity in vivo. In Rot rat brain mitochondria (Rot-RBM) there was a 30-40% inhibition of respiration in State 3 and State 3U with Complex I (Co-I) substrates and succinate. Rot did not affect the State 4Δψ of RBM and rat liver mitochondria (RLM). However, Rot-RBM required two times less Ca2+ to initiate permeability transition (mPT). There was a 2-fold increase in or H2O2 generation in Rot-RBM oxidizing glutamate. Rot infusion affected RLM little. Our results show that in RBM, the major site of reactive oxygen species generation with glutamate or succinate is Co-I. We also found that Co-II generates substantial amounts of reactive oxygen species that increased 2-fold in the Rot-RBM. Our data suggest that the primary mechanism of the Rot toxic effect on RBM consists in a significant increase of generation that causes damage to Co-I and Co-II, presumably at the level of 4Fe-4S clusters. Decreased respiratory activity diminishes resistance of RBM to Ca2+ and thus increases probability of mPT and apoptotic cell death. We suggest that the damage to Co-I and Co-II shifts generation from the CoQ10 sites to more proximal sites, such as flavines, and makes it independent of the RBM functional state.

Paraquat, MPTP, and rotenone reproduce features of Parkinson's disease (PD) in experimental animals. The exact mechanisms by which these compounds damage the dopamine system are not firmly established, but selective damage to dopamine neurons and inhibition of complex I are thought to be involved. We and others have previously documented that the toxic metabolite of MPTP, MPP+, is transported into dopamine neurons through the dopamine transporter (DAT), while rotenone is not transported by DAT. We have also demonstrated the requirement for complex I inhibition and oxidative damage in the dopaminergic neurodegeneration produced by rotenone. Based on structural similarity to MPP+, it has been proposed that paraquat exerts selective dopaminergic toxicity through transport by the DAT and subsequent inhibition of mitochondrial complex I. In this study we report that paraquat is neither a substrate nor inhibitor of DAT. We also demonstrate that in vivo exposure to MPTP and rotenone, but not paraquat, inhibits binding of 3H-dihydrorotenone to complex I in brain mitochondria. Rotenone and MPP+ were both effective inhibitors of complex I activity in isolated brain mitochondria, while paraquat exhibited weak inhibitory effects only at millimolar concentrations. These data indicate that, despite the apparent structural similarity to MPP+, paraquat exerts its deleterious effects on dopamine neurons in a manner that is unique from rotenone and MPTP.

Parkinson's 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 α-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, α-synuclein, and oxidative stress to a compromised UPS and PD pathogenesis suggesting novel therapeutic targets.

Chronic rotenone exposure reproduces features of Parkinson's 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.

Sporadic, non-familial Parkinson's disease is characterized by a 15-30% reduction in complex I activity of the electron transport chain. A pharmacological model of reduced complex I activity was created by prolonged treatment of SH-SY5Y cells with low doses (5-20 nM) of rotenone, a selective inhibitor of complex I. Short-term (less than 2 week) exposure to rotenone did not influence calcium signaling, production of reactive oxygen species, or mitochondrial morphology. However, following 2 weeks of rotenone exposure, SH-SY5Y cells showed unusual calcium dynamics, specifically multiple calcium responses to carbachol, a muscarinic agonist. These secondary calcium responses were not seen in control SH-SY5Y cells and were dependent upon calcium influx. Mitochondrial membrane potential was also reduced in low dose rotenone-treated cells. These results demonstrate that a chronic, partial reduction in complex I activity, such as that seen in Parkinson's disease, can alter cell signaling events and perhaps increase the susceptibility of cells to calcium overload and subsequent cell death.

Altered nerve growth factor (NGF) regulation has been linked to the pathophysiology of hypertension. Vascular smooth muscle cells from an inbred hypertensive, but normoactive rat strain (WKHT) secreted NGF at a greater rate than from a hyperactive, normotensive strain (WKHA). Exposure to phorbol ester increased NGF secretion rates from WKHT by 400-800% but not from WKHA vascular muscle. NGF secretion rates from both WKHT and WKHA vascular cells were elevated by co-application of platelet-derived growth factor (PDGF) and transforming growth factor-beta1 (TGF-beta1) by 300-1000%. This response was partially attenuated by actinomycin D, an inhibitor of RNA transcription. These results suggest that regulation of NGF production does not occur solely at the level of transcription and post-transcriptional mechanisms operate. Analysis of NGF mRNA stability in the two strains following PDGF and TGF-beta1 treatment showed that NGF mRNA in WKHT had a half-life of 126.2+/-11.68 min while in WKHA vascular smooth muscle cells, the half-life was 47. 33+/-11.98 min. In addition to increased NGF mRNA stability in WKHT vascular muscle, these cells have an increased translational efficiency of NGF protein; elevated synthesis of NGF protein per unit NGF mRNA. Differences in signaling pathways may result in increased NGF mRNA stability and translational efficiency that may account for the elevated NGF protein in WKHT vascular smooth muscle cells.

Rotenone, a pesticide and complex I inhibitor, causes nigrostriatal degeneration similar to Parkinson disease pathology in a chronic, systemic, in vivo rodent model [M. Alam, W.J. Schmidt, Rotenone destroys dopaminergic neurons and induces parkinsonian symptoms in rats, Behav. Brain Res. 136 (2002) 317-324; R. Betarbet, T.B. Sherer, G. MacKenzie, M. Garcia-Osuna, A.V. Panov, J.T. Greenamyre, Chronic systemic pesticide exposure reproduces features of Parkinson's disease, Nat. Neurosci. 3 (2000) 1301-1306; S.M. Fleming, C. Zhu, P.O. Fernagut, A. Mehta, C.D. DiCarlo, R.L. Seaman, M.F. Chesselet, Behavioral and immunohistochemical effects of chronic intravenous and subcutaneous infusions of varying doses of rotenone, Exp. Neurol. 187 (2004) 418-429; T.B. Sherer, J.H. Kim, R. Betarbet, J.T. Greenamyre, Subcutaneous rotenone exposure causes highly selective dopaminergic degeneration and alpha-synuclein aggregation, Exp. Neurol. 179 (2003) 9-16.]. To better investigate the role of mitochondria and complex I inhibition in chronic, progressive neurodegenerative disease, we developed methods for long-term culture of rodent postnatal midbrain organotypic slices. Chronic complex I inhibition over weeks by low dose (10-50 nM) rotenone in this system lead to dose- and time-dependent destruction of substantia nigra pars compacta neuron processes, morphologic changes, some neuronal loss, and decreased tyrosine hydroxylase (TH) protein levels. Chronic complex I inhibition also caused oxidative damage to proteins, measured by protein carbonyl levels. This oxidative damage was blocked by the antioxidant alpha-tocopherol (vitamin E). At the same time, alpha-tocopherol also blocked rotenone-induced reductions in TH protein and TH immunohistochemical changes. Thus, oxidative damage is a primary mechanism of mitochondrial toxicity in intact dopaminergic neurons. The organotypic culture system allows close study of this and other interacting mechanisms over a prolonged time period in mature dopaminergic neurons with intact processes, surrounding glia, and synaptic connections.

Exposure of rats to the pesticide and complex I inhibitor rotenone reproduces features of Parkinson's 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 nmto 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 ofSaccharomyces 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 Parkinson's 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 Parkinson's disease.

Parkinson's 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.

Parkinson's disease (PD) is associated with complex compensatory changes in the functional and neurochemical anatomy of the basal ganglia resulting from striatal dopamine deficiency. Available evidence suggests that glutamatergic corticostriatal pathway becomes overactive following nigrostriatal dopaminergic denervation. Since N-methyl-d-aspartate (NMDA) receptors are abundant and functionally important in the striatum, we examined the effects of striatal dopamine deficiency on the distribution and density of NMDA receptor subunit 1 (NR1) and serine phosphorylation (ser897) of NR1 in the striatum. Phosphorylation at this residue is believed to be dependent on protein kinase A. In both 6-hydroxydopamine-lesioned rats and MPTP-treated monkeys, striatal dopamine denervation resulted in down-regulation of NR1 expression and an increase in the expression of ser897-phosphorylated NR1 by striatal neurons. The decreased NR1 expression may be a compensatory response to overactive glutamatergic inputs, which appear to occur in response to striatal dopamine denervation in animal models of PD. Furthermore, the alterations observed in protein kinase A-dependent phosphorylation of NR1 are suggestive of receptor internalization and subcellular trafficking of NR1 in response to increased striatal glutamatergic activity.

Previous studies demonstrated that chronic systemic exposure to the pesticide and mitochondrial toxin rotenone through jugular vein cannulation reproduced many features of Parkinson's 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.

Chronic systemic complex I inhibition caused by rotenone exposure induces features of Parkinson's disease (PD) in rats, including selective nigrostriatal dopaminergic degeneration and formation of ubiquitin- and alpha-synuclein-positive inclusions (Betarbet et al., 2000). To determine underlying mechanisms of rotenone-induced cell death, we developed a chronic in vitro model based on treating human neuroblastoma cells with 5 nm rotenone for 1-4 weeks. For up to 4 weeks, cells grown in the presence of rotenone had normal morphology and growth kinetics, but at this time point, approximately 5% of cells began to undergo apoptosis. Short-term rotenone treatment (1 week) elevated soluble alpha-synuclein protein levels without changing message levels, suggesting that alpha-synuclein degradation was retarded. Chronic rotenone exposure (4 weeks) increased levels of SDS-insoluble alpha-synuclein and ubiquitin. After a latency of >2 weeks, rotenone-treated cells showed evidence of oxidative stress, including loss of glutathione and increased oxidative DNA and protein damage. Chronic rotenone treatment (4 weeks) caused a slight elevation in basal apoptosis and markedly sensitized cells to further oxidative challenge. In response to H2O2, there was cytochrome c release from mitochondria, caspase-3 activation, and apoptosis, all of which occurred earlier and to a much greater extent in rotenone-treated cells; caspase inhibition provided substantial protection. These studies indicate that chronic low-grade complex I inhibition caused by rotenone exposure induces accumulation and aggregation of alpha-synuclein and ubiquitin, progressive oxidative damage, and caspase-dependent death, mechanisms that may be central to PD pathogenesis.

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 Parkinson's 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.

Parkinson’s disease (PD) is a late-onset, progressive disease characterized by motor and postural defects. Pathologically, PD is marked by specific nigrostriatal dopaminergic degeneration and the formation of Lewy bodies, cytoplasmic inclusions containing ubiquitin and a-synuclein.l Current therapeutic approaches for PD focus on alleviating symptoms that occur in the late-stages. An understanding of PD pathogenesis and the development of accurate models of the disease are essential for the development of novel therapies targeted at preventing the progression of PD.

The cause of Parkinson's 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 alpha-synuclein. These results indicate that chronic exposure to a common pesticide can reproduce the anatomical, neurochemical, behavioral and neuropathological features of PD.

Ht30/=Ht10/=Ht5). Cells with reduced mitochondrial activity also showed abnormal responses to the stimulation of NGF output. Thrombin and phorbol ester elevated NGF production from Ht100, Ht30 and Ht10 cells, but not from Ht5 cells. Ht30 cells, despite secreting less NGF basally than Ht100 cells, reached a similar or greater NGF output upon stimulation. Mitogens increased NGF output and NGF mRNA levels with the largest effect on NGF protein in Ht30 cells. Free radical production and the ability of cells to respond to NGF-inducing agents were related. These data suggest that chronic impairment of mitochondrial function associates with disturbances in cellular production of a signaling protein.

The spontaneously hypertensive rat (SHR) was developed as a genetic model of essential hypertension. In vivo and in vitro evidence demonstrates that vascular smooth muscle cells (VSMCs) from the SHR produce more nerve growth factor (NGF) than the normotensive Wistar-Kyoto (WKY) control strain. This increased NGF production is accompanied by excessive innervation of target tissues in the SHR. In the present study, a sensitive, competitive, quantitative, reverse-transcriptase polymerase chain reaction (C Q RT-PCR) assay is characterized and used to analyze levels of NGF mRNA in cultured VSMCs derived from the SHR and WKY strains as well as bladder tissue. Differences in NGF secretion rates between SHR and WKY VSMCs were partially due to an increased stability of NGF mRNA in SHR VSMCs. Following treatment with platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta 1) to elevate NGF production, the half-life of the NGF mRNA was 104.5 +/- 18.0 min in SHR VSMCs, compared to only 36.5 +/- 11.6 min in WKY VSMCs. Sequence analysis of the 3' untranslated region (UTR) revealed no strain differences in cis-acting sequences potentially involved in determining mRNA stability. Thus, it seems unlikely to be a 3'UTR mutation that prolongs mRNA lifetime. Rather, differential regulation of an RNA-binding protein may play a role in the abnormal NGF mRNA stability in SHR VSMCs. SHR VSMCs also demonstrate an increased translational efficiency of NGF protein; more NGF protein is synthesized per unit of NGF mRNA. The use of a C Q RT-PCR assay has allowed the determination that abnormal NGF mRNA stabilization as well as altered translational efficiency may contribute to excess NGF synthesis and progressive hypertension in the SHR.

The production of nerve growth factor (NGF) in peripheral organs may play a role in the pathophysiology of hypertension and in obstructive disorders of the bladder outlet. We have been examining the cellular processes of NGF delivery and secretion in smooth muscle. NGF secretion from vascular smooth muscle cells (VSMCs) cultured from genetically hypertensive (WKHT), hyperactive (WKHA), and a control Wistar rat strain were assayed using a two-site ELISA of the culture media. Bladder smooth muscle cells (BSMCs) from the Wistar strain were also studied. The serine protease, thrombin, increased NGF secretion from all types of VSMCs but had no effect on Wistar BSMCs. The thrombin-mediated increase in NGF secretion was prevented by actinomycin D and cycloheximide, suggesting that RNA transcription and protein synthesis are required. The effect of thrombin was additive with a phorbol ester-induced elevation in NGF secretion rates from 4 to 6 h and was attenuated by a 24-h downregulation of protein kinase C. These results suggest that extracellular protease activity may regulate NGF secretion in smooth muscle. Thrombin may act in response to vascular injury, increasing NGF secretion from VSMCs, initiating VSMC migration, and preparing the VSMCs for reinnervation following an insult.

Exposure of rats to the pesticide and complex I inhibitor rotenone reproduces features of Parkinson’s 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 1μM) 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, orbyNDI1 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 Parkinson’s 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 Parkinson’s disease.

We appreciate Jenner's thoughtful comments about our recent work on a new model of Parkinson's disease (PD) 1xChronic systemic pesticide exposure reproduces features of Parkinson's disease. Betarbet, R et al. Nat. Neurosci. 2000; 3: 1301–1306Crossref | PubMed | Scopus (2021)See all References1. He raises many of the same issues with which we have been struggling for some time. To put our work into proper perspective, it should be pointed out that we did not set out to establish a link between pesticides and PD. Instead, we were interested in whether a systemic defect in complex I of the mitochondrial respiratory chain could produce the selective nigrostriatal dopaminergic degeneration that characterizes PD. As Jenner points out, several groups have reported a systemic complex I defect in PD; our question was whether it was relevant to PD pathogenesis. To model this systemic mitochondrial abnormality, we used the classical lipophilic complex I inhibitor, rotenone, because it crosses biological membranes easily and independent of transporters. To our surprise, systemic rotenone administration produced highly selective degeneration of precisely the neurones that degenerate in PD, and many dying neurones contained cytoplasmic inclusions similar to the Lewy bodies of PD. Our results clearly suggest that a modest systemic defect in a ubiquitous mitochondrial enzyme might be central to PD pathogenesis.The real question, of course, is how commonly might complex I defects contribute to cases of PD? Jenner suggests that ‘the lack of a detectable change in complex I in many individuals with PD suggests that it cannot underlie nigral degeneration in all individuals with the illness.’ This might or might not be true. Although we certainly would not argue that all ‘idiopathic’ PD is caused by a complex I defect, we believe typical complex I assays are relatively insensitive to all but gross defects. The enzyme assays are usually performed with single, saturating concentrations of NADH and coenzyme Q, and therefore cannot detect altered substrate or co-factor affinities or reduced activity as a result of depressed levels of endogenous substrate or co-factor 2xCoenzyme Q10 levels correlate with the activities of complexes I and II/III in mitochondria from parkinsonian and nonparkinsonian subjects. Shults, C.W et al. Ann. Neurol. 1997; 42: 261–264Crossref | PubMed | Scopus (189)See all References2. Oxygraphic measurements of respiration using complex I substrates might be even less sensitive as a result of threshold effects 3xThreshold effects and control of oxidative phosphorylation in nonsynaptic rat brain mitochondria. Davey, G.P and Clark, J.B. J. Neurochem. 1996; 66: 1617–1624Crossref | PubMedSee all References3. As Jenner highlights, we do not know which cell populations in substantia nigra have a complex I defect; however, if in some individuals it is selective for the dopamine neurons (which comprise 1–2% of the local cells), this would certainly not be detectable with conventional assays. Moreover, minimal inhibition of complex I with a concentration of rotenone (5 nm) that reduces enzyme activity very little 4xMitochondria deficient in complex I activity are depolarized by hydrogen peroxide in nerve terminals: relevance to Parkinson's disease. Chinopoulos, C and Adam-Vizi, V. J. Neurochem. 2001; 76: 302–306Crossref | PubMed | Scopus (53)See all References4 (and respiration not at all 1xChronic systemic pesticide exposure reproduces features of Parkinson's disease. Betarbet, R et al. Nat. Neurosci. 2000; 3: 1301–1306Crossref | PubMed | Scopus (2021)See all References1) is sufficient to produce over a period of weeks progressive oxidative damage to protein and DNA and, eventually, apoptotic cell death 5xAn in vitro model of Parkinson's disease: chronic complex I inhibition. Sherer, T.B et al. Soc. Neurosci. Abstr. 2000; 26: 280See all References5. Thus, low-grade complex I defects might be undetectable with conventional assays, yet have catastrophic consequences in the long run. In summary, our results 1xChronic systemic pesticide exposure reproduces features of Parkinson's disease. Betarbet, R et al. Nat. Neurosci. 2000; 3: 1301–1306Crossref | PubMed | Scopus (2021)See all References1 indicate that complex I dysfunction is sufficient to reproduce the features of PD; however, the degree to which complex I defects contribute to typical cases of PD is uncertain, and in our opinion, probably underestimated.Because epidemiological studies have repeatedly implicated pesticides in the pathogenesis of PD, the fact that rotenone is a pesticide was also of interest to us. Our study gives the pesticide hypothesis ‘biological plausibility’. As Jenner notes, several more common pesticides are also potent inhibitors of complex I, and we do not yet know whether these also cause selective nigrostriatal degeneration. However, it is becoming clear that other types of pesticides might also damage the dopamine system 6xThe nigrostriatal dopaminergic system as a preferential target of repeated exposures to combined paraquat and maneb: implications for Parkinson's disease. Thiruchelvam, M et al. J. Neurosci. 2000; 20: 9207–9214PubMedSee all References6. Nonetheless, we recognize that James Parkinson described the ‘shaking palsy’ long before the widespread use of pesticides. (Interestingly however, rotenone, as crushed derris root, has been used in Europe for hundreds of years.) In this regard, it is essential to recognize that many natural compounds are complex I inhibitors. For example, the piericidins from Streptomyces strains, and the acetogenins from Annonacae plants (such as the custard apple) inhibit complex I more potently than rotenone. The extent to which our food contains – or is contaminated by – such substances is unknown. Even rhubarb contains a weak complex I inhibitor. Thus, there may be reasons other than pesticide exposure to stay out of the garden. Better yet, we should figure out what's in the garden.

To investigate the role of chronic mitochondrial dysfunction on intracellular calcium signaling, we studied basal and stimulated cytosolic calcium levels in SH-SY5Y cells and a derived cell line devoid of mitochondrial DNA (Rho degrees ). Basal cytosolic calcium levels were slightly but significantly reduced in Rho degrees cells. The impact of chronic depletion of mitochondrial DNA was more evident following exposure of cells to carbachol, a calcium mobilizing agent. Calcium transients generated in Rho degrees cells following application of carbachol were more rapid than those in SH-SY5Y cells. A plateau phase of calcium recovery during calcium transients was present in SH-SY5Y cells but absent in Rho degrees cells. The rapid calcium transients in Rho degrees cells were due, in part, to increased reliance on Na(+)/Ca(2+) exchange activity at the plasma membrane and the plateau phase in calcium recovery in SH-SY5Y cells was dependent on the presence of extracellular calcium. We also examined whether mitochondrial DNA depletion influenced calcium responses to release of intracellular calcium stores. Rho degrees cells showed reduced responses to the uncoupler, FCCP, and the sarcoplasmic reticulum calcium ATPase inhibitor, thapsigargin. Acute exposure of SH-SY5Y cells to mitochondrial inhibitors did not mimic the results seen in Rho degrees cells. These results suggest that cytosolic calcium homeostasis in this neuron-like cell line is significantly altered as a consequence of chronic depletion of mitochondrial DNA.

Parkinson's 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. Parkinson's 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.