Acetylated α-Tubulin and α-Synuclein: Physiological Interplay and Contribution to α-Synuclein Oligomerization.

Emerging evidence supports that altered α-tubulin acetylation occurs in Parkinson's disease (PD), a neurodegenerative disorder characterized by the deposition of α-synuclein fibrillary aggregates within Lewy bodies and nigrostriatal neuron degeneration. Nevertheless, studies addressing the interplay between α-tubulin acetylation and α-synuclein are lacking. Here, we investigated the relationship between α-synuclein and microtubules in primary midbrain murine neurons and the substantia nigra of post-mortem human brains. Taking advantage of immunofluorescence and Proximity Ligation Assay (PLA), a method allowing us to visualize protein-protein interactions in situ, combined with confocal and super-resolution microscopy, we found that α-synuclein and acetylated α-tubulin colocalized and were in close proximity. Next, we employed an α-synuclein overexpressing cellular model and tested the role of α-tubulin acetylation in α-synuclein oligomer formation. We used the α-tubulin deacetylase HDAC6 inhibitor Tubacin to modulate α-tubulin acetylation, and we evaluated the presence of α-synuclein oligomers by PLA. We found that the increase in acetylated α-tubulin significantly induced α-synuclein oligomerization. In conclusion, we unraveled the link between acetylated α-tubulin and α-synuclein and demonstrated that α-tubulin acetylation could trigger the early step of α-synuclein aggregation. These data suggest that the proper regulation of α-tubulin acetylation might be considered a therapeutic strategy to take on PD.

α-Synuclein oligomers in skin biopsy of idiopathic and monozygotic twin patients with Parkinson's disease.

A variety of cellular processes, including vesicle clustering in the presynaptic compartment, are impaired in Parkinson's disease and have been closely associated with α-synuclein oligomerization. Emerging evidence proves the existence of α-synuclein-related pathology in the peripheral nervous system, even though the presence of α-synuclein oligomers in situ in living patients remains poorly investigated. In this case-control study, we show previously undetected α-synuclein oligomers within synaptic terminals of autonomic fibres in skin biopsies by means of the proximity ligation assay and propose a procedure for their quantification (proximity ligation assay score). Our study revealed a significant increase in α-synuclein oligomers in consecutive patients with Parkinson's disease compared to consecutive healthy controls (P < 0.001). Proximity ligation assay score (threshold value > 96 using receiver operating characteristic) was found to have good sensitivity, specificity and positive predictive value (82%, 86% and 89%, respectively). Furthermore, to disclose the role of putative genetic predisposition in Parkinson's disease aetiology, we evaluated the differential accumulation of oligomers in a unique cohort of 19 monozygotic twins discordant for Parkinson's disease. The significant difference between patients and healthy subjects was confirmed in twins. Intriguingly, although no difference in median values was detected between consecutive healthy controls and healthy twins, the prevalence of healthy subjects positive for proximity ligation assay score was significantly greater in twins than in the consecutive cohort (47% versus 14%, P = 0.019). This suggests that genetic predisposition is important, but not sufficient, in the aetiology of the disease and strengthens the contribution of environmental factors. In conclusion, our data provide evidence that α-synuclein oligomers accumulate within synaptic terminals of autonomic fibres of the skin in Parkinson's disease for the first time. This finding endorses the hypothesis that α-synuclein oligomers could be used as a reliable diagnostic biomarker for Parkinson's disease. It also offers novel insights into the physiological and pathological roles of α-synuclein in the peripheral nervous system.

The Association between α-Synuclein and α-Tubulin in Brain Synapses.

α-synuclein is a small protein that is mainly expressed in the synaptic terminals of nervous tissue. Although its implication in neurodegeneration is well established, the physiological role of α-synuclein remains elusive. Given its involvement in the modulation of synaptic transmission and the emerging role of microtubules at the synapse, the current study aimed at investigating whether α-synuclein becomes involved with this cytoskeletal component at the presynapse. We first analyzed the expression of α-synuclein and its colocalization with α-tubulin in murine brain. Differences were found between cortical and striatal/midbrain areas, with substantia nigra pars compacta and corpus striatum showing the lowest levels of colocalization. Using a proximity ligation assay, we revealed the direct interaction of α-synuclein with α-tubulin in murine and in human brain. Finally, the previously unexplored interaction of the two proteins in vivo at the synapse was disclosed in murine striatal presynaptic boutons through multiple approaches, from confocal spinning disk to electron microscopy. Collectively, our data strongly suggest that the association with tubulin/microtubules might actually be an important physiological function for α-synuclein in the synapse, thus suggesting its potential role in a neuropathological context.

Neuronal microtubules and proteins linked to Parkinson's disease: a relevant interaction?

Neuronal microtubules are key determinants of cell morphology, differentiation, migration and polarity, and contribute to intracellular trafficking along axons and dendrites. Microtubules are strictly regulated and alterations in their dynamics can lead to catastrophic effects in the neuron. Indeed, the importance of the microtubule cytoskeleton in many human diseases is emerging. Remarkably, a growing body of evidence indicates that microtubule defects could be linked to Parkinson's disease pathogenesis. Only a few of the causes of the progressive neuronal loss underlying this disorder have been identified. They include gene mutations and toxin exposure, but the trigger leading to neurodegeneration is still unknown. In this scenario, the evidence showing that mutated proteins in Parkinson's disease are involved in the regulation of the microtubule cytoskeleton is intriguing. Here, we focus on α-Synuclein, Parkin and Leucine-rich repeat kinase 2 (LRRK2), the three main proteins linked to the familial forms of the disease. The aim is to dissect their interaction with tubulin and microtubules in both physiological and pathological conditions, in which these proteins are overexpressed, mutated or absent. We highlight the relevance of such an interaction and suggest that these proteins could trigger neurodegeneration via defective regulation of the microtubule cytoskeleton.

Microtubule defects in mesenchymal stromal cells distinguish patients with Progressive Supranuclear Palsy.

Progressive Supranuclear Palsy (PSP) is a rare neurodegenerative disease whose etiopathogenesis remains elusive. The intraneuronal accumulation of hyperphosphorylated Tau, a pivotal protein in regulating microtubules (MT), leads to include PSP into tauopathies. Pathological hallmarks are well known in neural cells but no word yet if PSP-linked dysfunctions occur also in other cell types. We focused on bone marrow mesenchymal stromal cells (MSCs) that have recently gained attention for therapeutic interventions due to their anti-inflammatory, antiapoptotic and trophic properties. Here, we aimed to investigate MSCs biology and to disclose if any disease-linked defect occurs in this non-neuronal compartment. First, we found that cells obtained from patients showed altered morphology and growth. Next, Western blotting analysis unravelled the imbalance in α-tubulin post-translational modifications and in MT stability. Interestingly, MT mass is significantly decreased in patient cells at baseline and differently changes overtime compared to controls, suggesting their inability to efficiently remodel MT cytoskeleton during ageing in culture. Thus, our results provide the first evidence that defects in MT regulation and stability occur and are detectable in a non-neuronal compartment in patients with PSP. We suggest that MSCs could be a novel model system for unravelling cellular processes implicated in this neurodegenerative disorder.

A distal CCAAT/NUCLEAR FACTOR Y complex promotes chromatin looping at the FLOWERING LOCUS T promoter and regulates the timing of flowering in Arabidopsis.

For many plant species, reproductive success relies on the proper timing of flowering, and photoperiod provides a key environmental input. Photoperiod-dependent flowering depends on timely expression of FLOWERING LOCUS T (FT); however, the coordination of various cis-regulatory elements in the FT promoter is not well understood. Here, we provide evidence that long-distance chromatin loops bring distal enhancer elements into close association with the proximal promoter elements bound by CONSTANS (CO). Additionally, we show that NUCLEAR FACTOR Y (NF-Y) binds a CCAAT box in the distal enhancer element and that CCAAT disruption dramatically reduces FT promoter activity. Thus, we propose the recruitment model of photoperiod-dependent flowering where NF-Y complexes, bound at the FT distal enhancer element, help recruit CO to proximal cis-regulatory elements and initiate the transition to reproductive growth.

Key Role of the Adenylate Moiety and Integrity of the Adenylate-Binding Site for the NAD(+)/H Binding to Mitochondrial Apoptosis-Inducing Factor.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with pro-life and pro-death activities, which plays critical roles in mitochondrial energy metabolism and caspase-independent apoptosis. Defects in AIF structure or expression can cause mitochondrial abnormalities leading to mitochondrial defects and neurodegeneration. The mechanism of AIF-induced apoptosis was extensively investigated, whereas the mitochondrial function of AIF is poorly understood. A unique feature of AIF is the ability to form a tight, air-stable charge-transfer (CT) complex upon reaction with NADH and to undergo a conformational switch leading to dimerization, proposed to be important for its vital and lethal functions. Although some aspects of interaction of AIF with NAD(+)/H have been analyzed, its precise mechanism is not fully understood. We investigated how the oxidized and photoreduced wild-type and G307A and -E variants of murine AIF associate with NAD(+)/H and nicotinamide mononucleotide (NMN(+)/H) to determine the role of the adenylate moiety in the binding process. Our results indicate that (i) the adenylate moiety of NAD(+)/H is crucial for the association with AIF and for the subsequent structural reorganization of the complex, but not for protein dimerization, (ii) FAD reduction rather than binding of NAD(+)/H to AIF initiates conformational rearrangement, and (iii) alteration of the adenylate-binding site by the G307E (equivalent to a pathological G308E mutation in human AIF) or G307A replacements decrease the affinity and association rate of NAD(+)/H, which, in turn, perturbs CT complex formation and protein dimerization but has no influence on the conformational switch in the regulatory peptide.

Linking microtubules to Parkinson's disease: the case of parkin.

Microtubules (MTs) are dynamic polymers consisting of α/ß tubulin dimers and playing a plethora of roles in eukaryotic cells. Looking at neurons, they are key determinants of neuronal polarity, axonal transport and synaptic plasticity. The concept that MT dysfunction can participate in, and perhaps lead to, Parkinson's disease (PD) progression has been suggested by studies using toxin-based and genetic experimental models of the disease. Here, we first learn lessons from MPTP and rotenone as well as from the PD related genes, including SNCA and LRRK2, and then look at old and new evidence regarding the interplay between parkin and MTs. Data from experimental models and human cells point out that parkin regulates MT stability and strengthen the link between MTs and PD paving the way to a viable strategy for the management of the disease.

Comparing GBA1-Parkinson's disease and idiopathic Parkinson's disease: α-Synuclein oligomers and synaptic density as biomarkers in the skin biopsy.

The main genetic risk factors for Parkinson's disease (PD) are presently represented by variants in GBA1 gene encoding for the ß-glucocerebrosidase (GCase). Searching for a peripheral biomarker that can be used for selecting and monitoring patients in clinical trials targeting GBA1-associated PD (GBA1-PD) is a current challenge. We previously demonstrated that α-synuclein oligomers expressed as proximity ligation assay (PLA) score in synaptic terminals of skin biopsy are a reliable biomarker for distinguishing idiopathic PD (iPD) from healthy controls (HC). This cross-sectional study investigates an unexplored cohort of GBA1-PD (n = 27) compared to 28 HC, and 36 iPD cases to (i) analyze α-synuclein oligomers and quantify them throughout PLA score, (ii) investigate GCase expression in brain and synaptic terminals targeting the sweat gland, (iii) unravel indicators that could differentiate patients with specific GBA1 mutations. PLA score discriminates GBA1-PD from HC with sensitivity = 88.9% (95% CI 70.84-97.65), specificity = 88.5% (95% CI 69.85-97.55), and PPV = 88.9% (95% CI 73.24-95.90), AUC value = 0.927 (95% CI 0.859-0.996). No difference was found between GBA1-PD patients and iPD, suggesting a common pathological pathway based on α-synuclein oligomers. GCase score did not differ in GBA1-PD, iPD, and HC in the synaptic terminals, whereas a positive correlation was found between PLA score and GCase score. Moreover, a significant increase in synaptic density was observed in GBA1-PD compared to iPD and HC (P < 0.0001). Employing ROC curve to discriminate GBA1-PD from iPD, we found an AUC value for synaptic density = 0.855 (95% CI 0.749-0.961) with sensitivity = 85.2% (95% CI 66.27%-95.81%), specificity = 77.1% (95% CI 59.86%-89.58%), and PPV = 74.19% (60.53%-84.35%). The highest synaptic density values were observed in p.N409S patients. This work points out to the value of both PLA score and synaptic density in distinguishing GBA1-PD from iPD and to their potential to stratify and monitor patients in the context of new pathway-specific therapeutic options.

Linking acetylated α-Tubulin redistribution to α-Synuclein pathology in brain of Parkinson's disease patients.

Highly specialized microtubules in neurons are crucial to both health and disease of the nervous system, and their properties are strictly regulated by different post-translational modifications, including α-Tubulin acetylation. An imbalance in the levels of acetylated α-Tubulin has been reported in experimental models of Parkinson's disease (PD) whereas pharmacological or genetic modulation that leads to increased acetylated α-Tubulin successfully rescues axonal transport defects and inhibits α-Synuclein aggregation. However, the role of acetylation of α-Tubulin in the human nervous system is largely unknown as most studies are based on in vitro evidence. To capture the complexity of the pathological processes in vivo, we analysed post-mortem human brain of PD patients and control subjects. In the brain of PD patients at Braak stage 6, we found a redistribution of acetylated α-Tubulin, which accumulates in the neuronal cell bodies in subcortical structures but not in the cerebral cortex, and decreases in the axonal compartment, both in putamen bundles of fibres and in sudomotor fibres. High-resolution and 3D reconstruction analysis linked acetylated α-Tubulin redistribution to α-Synuclein oligomerization and to phosphorylated Ser 129 α-Synuclein, leading us to propose a model for Lewy body (LB) formation. Finally, in post-mortem human brain, we observed threadlike structures, resembling tunnelling nanotubes that contain α-Synuclein oligomers and are associated with acetylated α-Tubulin enriched neurons. In conclusion, we support the role of acetylated α-Tubulin in PD pathogenesis and LB formation.

Cross-talk between α-synuclein and the microtubule cytoskeleton in neurodegeneration.

Looking at the puzzle that depicts the molecular determinants in neurodegeneration, many pieces are lacking and multiple interconnections among key proteins and intracellular pathways still remain unclear. Here we focus on the concerted action of α-synuclein and the microtubule cytoskeleton, whose interplay, indeed, is emerging but remains largely unexplored in both its physiology and pathology. α-Synuclein is a key protein involved in neurodegeneration, underlying those diseases termed synucleinopathies. Its propensity to interact with other proteins and structures renders the identification of neuronal death trigger extremely difficult. Conversely, the unbalance of microtubule cytoskeleton in terms of structure, dynamics and function is emerging as a point of convergence in neurodegeneration. Interestingly, α-synuclein and microtubules have been shown to interact and mediate cross-talks with other intracellular structures. This is supported by an increasing amount of evidence ranging from their direct interaction to the engagement of in-common partners and culminating with their respective impact on microtubule-dependent neuronal functions. Last, but not least, it is becoming even more clear that α-synuclein and tubulin work synergically towards pathological aggregation, ultimately resulting in neurodegeneration. In this respect, we supply a novel perspective towards the understanding of α-synuclein biology and, most importantly, of the link between α-synuclein with microtubule cytoskeleton and its impact for neurodegeneration and future development of novel therapeutic strategies.

Glucocerebrosidase is imported into mitochondria and preserves complex I integrity and energy metabolism.

Mutations in GBA1, the gene encoding the lysosomal enzyme ß-glucocerebrosidase (GCase), which cause Gaucher's disease, are the most frequent genetic risk factor for Parkinson's disease (PD). Here, we employ global proteomic and single-cell genomic approaches in stable cell lines as well as induced pluripotent stem cell (iPSC)-derived neurons and midbrain organoids to dissect the mechanisms underlying GCase-related neurodegeneration. We demonstrate that GCase can be imported from the cytosol into the mitochondria via recognition of internal mitochondrial targeting sequence-like signals. In mitochondria, GCase promotes the maintenance of mitochondrial complex I (CI) integrity and function. Furthermore, GCase interacts with the mitochondrial quality control proteins HSP60 and LONP1. Disease-associated mutations impair CI stability and function and enhance the interaction with the mitochondrial quality control machinery. These findings reveal a mitochondrial role of GCase and suggest that defective CI activity and energy metabolism may drive the pathogenesis of GCase-linked neurodegeneration.

Astrocytes expressing Vitamin D-activating enzyme identify Parkinson's disease.

INTRODUCTION: Astrocytes are involved in Parkinson's disease (PD) where they could contribute to α-Synuclein pathology but also to neuroprotection via α-Synuclein clearance. The molecular signature underlying their dual role is still elusive. Given that vitamin D has been recently suggested to be protective in neurodegeneration, the aim of our study was to investigate astrocyte and neuron vitamin D pathway alterations and their correlation with α-Synuclein aggregates (ie, oligomers and fibrils) in human brain obtained from PD patients. METHODS: The expression of vitamin D pathway components CYP27B1, CYP24A1, and VDR was examined in brains obtained from PD patients (Braak stage 6; n = 9) and control subjects (n = 4). We also exploited proximity ligation assay to identified toxic α-Synuclein oligomers in human astrocytes. RESULTS: We found that vitamin D-activating enzyme CYP27B1 identified a subpopulation of astrocytes exclusively in PD patients. CYP27B1 positive astrocytes could display neuroprotective features as they sequester α-Synuclein oligomers and are associated with Lewy body negative neurons. CONCLUSION: The presence of CYP27B1 astrocytes distinguishes PD patients and suggests their contribution to protect neurons and to ameliorate neuropathological traits.

Microtubule acetylation: A reading key to neural physiology and degeneration.

Neurons are the perfect example of cells where microtubules are essential to achieve an extraordinary degree of morphological and functional complexity. Different tubulin isoforms and associated post-translational modifications are the basis to establish the diversity in biochemical and biophysical properties of microtubules including their stability and the control of intracellular transport. Acetylation is one of the key tubulin modifications and it can influence important structural, mechanical and biological traits of the microtubule network. Here, we present the emerging evidence for the essential role of microtubule acetylation in the control of neuronal and glial function in healthy and degenerative conditions. In particular, we discuss the pathogenic role of tubulin acetylation in neurodegenerative disorders and focus on Parkinson's disease. We also provide a critical analysis about the possibility to target tubulin acetylation as a novel therapeutic intervention for neuroprotective strategies.

N-terminal interaction domain implicates PAK4 in translational regulation and reveals novel cellular localization signals.

The serine/threonine kinase PAK4 is a Rho GTPases effector protein implicated in many critical biological processes, including regulation of cell morphology and motility, embryonic development, cell survival, response to infection, and oncogenic transformation. Consistently with its pro-oncogenic features, PAK4 was found to be overexpressed in many cancer cell lines and tissues, and to be necessary to promote activation of survival pathways. PAK4, like other Paks, is now considered a promising target for specific therapy. Little is known on its modes of regulation, molecular partners, and substrates. Because the N-terminal regulatory moiety plays important roles in PAK4 activity and functions, even independently of GTPase interactions, in this study we employed an affinity chromatography approach to identify N-terminal domain binding partners. Within this protein region we identified a novel interaction domain involved in association with ribonucleoprotein (RNP) complexes, suggesting PAK4 implications in translational regulation. Indeed, we found that active PAK4 can affect (cap-independent) translation from specific IRES sequences in vivo, and that the N-terminal domain is critical for this regulation. Further, we could establish that within the RNP interacting sequence PAK4 regulatory domain contains targeting elements that drive cytoplasmic localization and act as nuclear export signal. Functional implication of endogenous PAK4 protein, which was found in both cytoplasmic and nuclear fractions, in IRES-mediated translation further underlines the significance of the reported findings. Our data reveal novel means for PAK4 regulation of gene expression, and provide new elements to understand the molecular mechanisms that determine PAK4 cellular localization and functions.

The imbalance between dynamic and stable microtubules underlies neurodegeneration induced by 2,5-hexanedione.

Exposure to environmental toxins, including hydrocarbon solvents, increases the risk of developing Parkinson's disease. An emergent hypothesis considers microtubule dysfunction as one of the crucial events in triggering neuronal degeneration in Parkinson's disease. Here, we used 2,5-hexanedione (2,5-HD), the toxic metabolite of n-hexane, to analyse the early effects of toxin-induced neurodegeneration on the cytoskeleton in multiple model systems. In PC12 cells differentiated with nerve growth factor for 5 days, we found that 2,5-HD treatment affected all the cytoskeletal components. Moreover, we observed alterations in microtubule distribution and stability, in addition to the imbalance of post-translational modifications of α-tubulin. Similar defects were also found in vivo in 2,5-HD-intoxicated mice. Interestingly, we also found that 2,5-HD exposure induced significant changes in microtubule stability in human skin fibroblasts obtained from Parkinson's disease patients harbouring mutations in PRKN gene, whereas it was ineffective in healthy donor fibroblasts, suggesting that the genetic background may really make the difference in microtubule susceptibility to this environmental Parkinson's disease-related toxin. In conclusion, by showing the imbalance between dynamic and stable microtubules in hydrocarbon-induced parkinsonism, our data support the crucial role of microtubule defects in triggering neurodegeneration.

Phospho-HDAC6 Gathers Into Protein Aggregates in Parkinson's Disease and Atypical Parkinsonisms.

HDAC6 is a unique histone deacetylase that targets cytoplasmic non-histone proteins and has a specific ubiquitin-binding activity. Both of these activities are required for HDAC6-mediated formation of aggresomes, which contain misfolded proteins that will ultimately be degraded via autophagy. HDAC6 deacetylase activity is increased following phosphorylation on serine 22 (phospho-HDAC6). In human, HDAC6 localizes in neuronal Lewy bodies in Parkinson's disease (PD) and in oligodendrocytic Papp-Lantos bodies in multiple system atrophy (MSA). However, the expression of phospho-HDAC6 in post-mortem human brains is currently unexplored. Here, we evaluate and compare the distribution of HDAC6 and its phosphorylated form in human brains obtained from patients affected by three forms of parkinsonism: two synucleinopathies (PD and MSA) and a tauopathy (progressive supranuclear palsy, PSP). We find that both HDAC6 and its phosphorylated form localize with pathological protein aggregates, including α-synuclein-positive Lewy bodies in PD and Papp-Lantos bodies in MSA, and phospho-tau-positive neurofibrillary tangles in PSP. We further find a direct interaction of HDAC6 with α-synuclein with proximity ligation assay (PLA) in neuronal cell of PD patients. Taken together, our findings suggest that both HDAC6 and phospho-HDAC6 regulate the homeostasis of intra-neuronal proteins in parkinsonism.

2-Phenyloxazole-4-carboxamide as a Scaffold for Selective Inhibition of Human Monoamine Oxidase†B.

A series of 2-phenyloxazoles bearing an amide group at position 4 were designed and synthesized for evaluation as potential inhibitors of human recombinant monoamine oxidases (hrMAOs). Results of kinetics experiments demonstrated that all compounds behave as competitive MAO inhibitors, with good selectivity toward the MAO-B isoform. The most potent and selective derivatives are characterized by inhibition constant (Ki ) values in the sub-micromolar range and a good selectivity index (Ki MAO-A /Ki MAO-B >50). Some derivatives were also found to be able to inhibit MAO activity in nerve growth factor (NGF)-differentiated PC12 cells, taken as a model of neuronal cells. In particular, 2-(2-hydroxyphenyl)-N-phenyloxazole-4-carboxamide (compound 4 a) may be a promising new scaffold, exerting the highest selectivity and inhibitory effect toward MAOs in NGF-differentiated PC12 cell lysates, without compromising cell viability. Molecular docking analysis allowed a rationalization of the experimentally observed binding affinity and selectivity.

Parkin absence accelerates microtubule aging in dopaminergic neurons.

Loss-of-function caused by mutations in the parkin gene (PARK2) lead to early-onset familial Parkinson's disease. Recently, mechanistic studies proved the ability of parkin in regulating mitochondria homeostasis and microtubule (MT) stability. Looking at these systems during aging of PARK2 knockout mice, we found that loss of parkin induced an accelerated (over)acetylation of MT system both in dopaminergic neuron cell bodies and fibers, localized in the substantia nigra and corpus striatum, respectively. Interestingly, in PARK2 knockout mice, changes of MT stability preceded the alteration of mitochondria transport. Moreover, in-cell experiments confirmed that loss of parkin affects mitochondria mobility and showed that this defect depends on MT system as it is rescued by paclitaxel, a well-known MT-targeted agent. Furthermore, both in PC12 neuronal cells and in patients' induced pluripotent stem cell-derived midbrain neurons, we observed that parkin deficiencies cause the fragmentation of stable MTs. Therefore, we suggest that parkin acts as a regulator of MT system during neuronal aging, and we endorse the hypothesis that MT dysfunction may be crucial in the pathogenesis of Parkinson's disease.