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Studies on postmortem brains from Parkinson's patients reveal elevated iron in the substantia nigra (SN). Selective cell death in this brain region is associated with oxidative stress, which may be exacerbated by the presence of excess iron. Whether iron plays a causative role in cell death, however, is controversial. Here, we explore the effects of iron chelation via either transgenic expression of the iron binding protein ferritin or oral administration of the bioavailable metal chelator clioquinol (CQ) on susceptibility to the Parkinson's-inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrapyridine (MPTP). Reduction in reactive iron by either genetic or pharmacological means was found to be well tolerated in animals in our studies and to result in protection against the toxin, suggesting that iron chelation may be an effective therapy for prevention and treatment of the disease.

The vesicular monoamine transporter 2 (VMAT2; SLC18A2) is responsible for packaging dopamine into vesicles for subsequent release and has been suggested to serve a neuroprotective role in the dopamine system. Here, we show that mice that express ϳ5% of normal VMAT2 (VMAT2 LO) display age-associated nigrostriatal dopamine dysfunction that ultimately results in neurodegeneration. Elevated cysteinyl adducts to L-DOPA and DOPAC are seen early and are followed by increased striatal protein carbonyl and 3-nitrotyrosine formation. These changes were associated with decreased striatal dopamine and decreased expression of the dopamine transporter and tyrosine hydroxylase. Furthermore, we observed an increase in ␣-synuclein immunoreactivity and accumulation and neurodegeneration in the substantia nigra pars compacta in aged VMAT2 LO mice. Thus, VMAT2 LO animals display nigrostriatal degeneration that begins in the terminal fields and progresses to eventual loss of the cell bodies, ␣-synuclein accumulation, and an L-DOPA responsive behavioral deficit, replicating many of the key aspects of Parkinson's disease. These data suggest that mishandling of dopamine via reduced VMAT2 expression is, in and of itself, sufficient to cause dopamine-mediated toxicity and neurodegeneration in the nigrostriatal dopamine system. In addition, the altered dopamine homeostasis resulting from reduced VMAT2 function may be conducive to pathogenic mechanisms induced by genetic or environmental factors thought to be involved in Parkinson's disease.

␣-Synuclein-containing aggregates represent a feature of a variety of neurodegenerative disorders, including Parkinson's disease (PD). However, mechanisms that promote intraneuronal ␣-synuclein assembly remain poorly understood. Because pesticides, particularly the herbicide paraquat, have been suggested to play a role as PD risk factors, the hypothesis that interactions between ␣-synuclein and these environmental agents may contribute to aggregate formation was tested in this study. Paraquat markedly accelerated the in vitro rate of ␣-synuclein fibril formation in a dosedependent fashion. When mice were exposed to the herbicide, brain levels of ␣-synuclein were significantly increased. This up-regulation followed a consistent pattern, with higher ␣-synuclein at 2 days after each of three weekly paraquat injections and with protein levels returning to control values by day 7 post-treatment. Paraquat exposure was also accompanied by aggregate formation. Thioflavine S-positive structures accumulated within neurons of the substantia nigra pars compacta, and dual labeling and confocal imaging confirmed that these aggregates contained ␣-synuclein. The results suggest that up-regulation of ␣-synuclein as a consequence of toxicant insult and direct interactions between the protein and environmental agents are potential mechanisms leading to ␣-synuclein pathology in neurodegenerative disorders. Parkinson's disease (PD)1 is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons in the nigrostriatal pathway and the formation of intraneuronal inclusions (called Lewy bodies) in different brain regions. Although the etiology of PD remains unknown, several lines of evidence suggest a pathogenetic role of the protein ␣-synuclein. In particular, ␣-synuclein is a major component of Lewy bodies in all PD patients (1, 2), and ␣-synuclein mutations have been associated with clinical and pathological parkinsonism in rare autosomal dominant familial cases (3, 4). It has been hypothesized that a tendency of ␣-synuclein to aggregate may underlie its involvement in Lewy body formation and neurodegeneration. In individuals with ␣-synuclein mutations, abnormal forms of the protein could trigger pathological processes as a result of their enhanced propensity to self-assemble (5-7). However, in the vast majority of patients with idiopathic (nonfamilial) PD, the lack of ␣-synuclein mutations (8, 9) indicates that additional mechanisms may lead to conformational changes and consequent protein aggregation. One such mechanism could be the interaction of ␣-synuclein with other chemical species.The association and fibrillation of ␣-synuclein appear to involve a shift in equilibrium from the natively unfolded to a partially folded protein conformation (10). In a recent study, Uversky and colleagues (11) have shown that incubating ␣-synuclein in the presence of paraquat or other pesticides dramatically accelerates the rate of ␣-synuclein fibrillation in vitro, probably due to the preferential binding of these compound...

The aggregation of alpha-synuclein is believed to play an important role in the pathogenesis of Parkinson's disease as well as other neurodegenerative disorders ("synucleinopathies"). However, the function of alpha-synuclein under physiologic and pathological conditions is unknown, and the mechanism of alpha-synuclein aggregation is not well understood. Here we show that alpha-synuclein forms a tight 2:1 complex with histones and that the fibrillation rate of alpha-synuclein is dramatically accelerated in the presence of histones in vitro. We also describe the presence of alpha-synuclein and its co-localization with histones in the nuclei of nigral neurons from mice exposed to a toxic insult (i.e., injections of the herbicide paraquat). These observations indicate that translocation into the nucleus and binding with histones represent potential mechanisms underlying alpha-synuclein pathophysiology.

Pathologic accumulation of ␣-synuclein is a feature of human parkinsonism and other neurodegenerative diseases. This accumulation may be counteracted by mechanisms of protein degradation that have been investigated in vitro but remain to be elucidated in animal models. In this study, lysosomal clearance of ␣-synuclein in vivo was indicated by the detection of ␣-synuclein in the lumen of lysosomes isolated from the mouse midbrain. When neuronal ␣-synuclein expression was enhanced as a result of toxic injury (i.e. treatment of mice with the herbicide paraquat) or transgenic protein overexpression, the intralysosomal content of ␣-synuclein was also significantly increased. This effect was paralleled by a marked elevation of the lysosome-associated membrane protein type 2A (LAMP-2A) and the lysosomal heat shock cognate protein of 70 kDa (hsc70), two essential components of chaperonemediated autophagy (CMA). Immunofluorescence microscopy revealed an increase in punctate (lysosomal) LAMP-2A staining that co-localized with ␣-synuclein within nigral dopaminergic neurons of paraquat-treated and ␣-synuclein-overexpressing animals. The data provide in vivo evidence of lysosomal degradation of ␣-synuclein under normal conditions and, quite importantly, under conditions of enhanced protein burden. In the latter, increased lysosomal clearance of ␣-synuclein was mediated, at least in part, by CMA induction. It is conceivable that these neuronal mechanisms of protein clearance play an important role in neurodegenerative processes characterized by abnormal ␣-synuclein buildup.Several lines of clinical and experimental evidence support a pathogenetic role of ␣-synuclein in Parkinson disease (PD) 2 and other neurodegenerative disorders. The precise mechanisms by which this endogenously expressed protein becomes involved in pathologic processes have yet to be fully elucidated. However, data from both clinical and laboratory studies indicate that enhanced ␣-synuclein expression is itself capable of triggering a parkinsonian syndrome in humans and PD-like pathology in animal models. Indeed, multiplication mutations of the ␣-synuclein gene that result in increased expression of the wild-type protein are causally associated with autosomal dominant parkinsonism (1, 2). From the experimental standpoint, evidence of a gain of toxic function of elevated ␣-synuclein includes the observation of neurodegeneration and neuronal inclusions in the substantia nigra of rats and monkeys that overexpress ␣-synuclein after viral-mediated neuronal transduction (3, 4).An important corollary to the concept that enhanced ␣-synuclein may have deleterious consequences is that intraneuronal mechanisms of protein homeostasis, such as degradation pathways, could well play a key role in maintaining "non-toxic" levels of ␣-synuclein. Although ␣-synuclein clearance is likely to occur through different mechanisms, recent reports have underscored the important contribution of lysosomal pathways of protein degradation and, in particular, chaperone-mediated autopha...

Protein deposition diseases involve the aggregation of normally soluble proteins, leading to both fibrillar and amorphous deposits. The aggregation of alpha-synuclein is associated with Parkinson's disease, and the aggregation of the Abeta peptide is associated with Alzheimer's disease. Here we show that L-dopa, dopamine, and other catecholamines dissolve fibrils of alpha-synuclein and Abeta peptide generated in vitro. The catecholamines also inhibited the fibrillation of these proteins. In addition, intraneuronal alpha-synuclein deposits formed in a mouse model were dissolved by incubation of tissue slices with L-dopa. These catecholamines are susceptible to oxidative breakdown, and we show that oxidation products are more effective than the parent compounds in inhibition. The ability to dissolve fibrils provides a new approach for studying mechanisms and consequences (e.g., the relationship between fibril formation and neurodegeneration) of protein aggregation. It is also likely to help in the development of strategies for the prevention and treatment of protein deposition diseases.

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