Effects of Excess Brain-Derived Human α-Synuclein on Synaptic Vesicle Trafficking.

α-Synuclein is a presynaptic protein that regulates synaptic vesicle trafficking under physiological conditions. However, in several neurodegenerative diseases, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, α-synuclein accumulates throughout the neuron, including at synapses, leading to altered synaptic function, neurotoxicity, and motor, cognitive, and autonomic dysfunction. Neurons typically contain both monomeric and multimeric forms of α-synuclein, and it is generally accepted that disrupting the balance between them promotes aggregation and neurotoxicity. However, it remains unclear how distinct molecular species of α-synuclein affect synapses where α-synuclein is normally expressed. Using the lamprey reticulospinal synapse model, we previously showed that acute introduction of excess recombinant monomeric or dimeric α-synuclein impaired distinct stages of clathrin-mediated synaptic vesicle endocytosis, leading to a loss of synaptic vesicles. Here, we expand this knowledge by investigating the effects of native, physiological α-synuclein isolated from the brain of a neuropathologically normal human subject, which comprised predominantly helically folded multimeric α-synuclein with a minor component of monomeric α-synuclein. After acute introduction of excess brain-derived human α-synuclein, there was a moderate reduction in the synaptic vesicle cluster and an increase in the number of large, atypical vesicles called "cisternae." In addition, brain-derived α-synuclein increased synaptic vesicle and cisternae sizes and induced atypical fusion/fission events at the active zone. In contrast to monomeric or dimeric α-synuclein, the brain-derived multimeric α-synuclein did not appear to alter clathrin-mediated synaptic vesicle endocytosis. Taken together, these data suggest that excess brain-derived human α-synuclein impairs intracellular vesicle trafficking and further corroborate the idea that different molecular species of α-synuclein produce distinct trafficking defects at synapses. These findings provide insights into the mechanisms by which excess α-synuclein contributes to synaptic deficits and disease phenotypes.

Multi-Dimensional, Short-Timescale Quantification of Parkinson's Disease and Essential Tremor Motor Dysfunction.

Introduction: Parkinson's disease (PD) is a progressive movement disorder characterized by heterogenous motor dysfunction with fluctuations in severity. Objective, short-timescale characterization of this dysfunction is necessary as therapies become increasingly adaptive. Objectives: This study aims to characterize a novel, naturalistic, and goal-directed tablet-based task and complementary analysis protocol designed to characterize the motor features of PD. Methods: A total of 26 patients with PD and without deep brain stimulation (DBS), 20 control subjects, and eight patients with PD and with DBS completed the task. Eight metrics, each designed to capture an aspect of motor dysfunction in PD, were calculated from 1-second, non-overlapping epochs of the raw positional and pressure data captured during task completion. These metrics were used to generate a classifier using a support vector machine (SVM) model to produce a unifying, scalar "motor error score" (MES). The data generated from these patients with PD were compared to same-day standard clinical assessments. Additionally, these data were compared to analogous data generated from a separate group of 12 patients with essential tremor (ET) to assess the task's specificity for different movement disorders. Finally, an SVM model was generated for each of the eight patients with PD and with DBS to differentiate between their motor dysfunction in the "DBS On" and "DBS Off" stimulation states. Results: The eight metrics calculated from the raw positional and force data captured during task completion were non-redundant. MES generated by the SVM analysis protocol showed a strong correlation with MDS-UPDRS-III scores assigned by movement disorder specialists. Analysis of the relative contributions of each of the eight metrics showed a significant difference between the motor dysfunction of PD and ET. Much of this difference was attributable to the homogenous, tremor-dominant phenotype of ET motor dysfunction. Finally, in individual patients with PD with DBS, task performance and subsequent SVM classification effectively differentiated between the "DBS On" and "DBS Off" stimulation states. Conclusion: This tablet-based task and analysis protocol correlated strongly with expert clinical assessments of PD motor dysfunction. Additionally, the task showed specificity for PD when compared to ET, another common movement disorder. This specificity was driven by the relative heterogeneity of motor dysfunction of PD compared to ET. Finally, the task was able to distinguish between the "DBS On" and "DBS Off" states within single patients with PD. This task provides temporally-precise and specific information about motor dysfunction in at least two movement disorders that could feasibly correlate to neural activity.

Analysis of α-synuclein species enriched from cerebral cortex of humans with sporadic dementia with Lewy bodies.

Since researchers identified α-synuclein as the principal component of Lewy bodies and Lewy neurites, studies have suggested that it plays a causative role in the pathogenesis of dementia with Lewy bodies and other 'synucleinopathies'. While α-synuclein dyshomeostasis likely contributes to the neurodegeneration associated with the synucleinopathies, few direct biochemical analyses of α-synuclein from diseased human brain tissue currently exist. In this study, we analysed sequential protein extracts from a substantial number of patients with neuropathological diagnoses of dementia with Lewy bodies and corresponding controls, detecting a shift of cytosolic and membrane-bound physiological α-synuclein to highly aggregated forms. We then fractionated aqueous extracts (cytosol) from cerebral cortex using non-denaturing methods to search for soluble, disease-associated high molecular weight species potentially associated with toxicity. We applied these fractions and corresponding insoluble fractions containing Lewy-type aggregates to several reporter assays to determine their bioactivity and cytotoxicity. Ultimately, high molecular weight cytosolic fractions enhances phospholipid membrane permeability, while insoluble, Lewy-associated fractions induced morphological changes in the neurites of human stem cell-derived neurons. While the concentrations of soluble, high molecular weight α-synuclein were only slightly elevated in brains of dementia with Lewy bodies patients compared to healthy, age-matched controls, these observations suggest that a small subset of soluble α-synuclein aggregates in the brain may drive early pathogenic effects, while Lewy body-associated α-synuclein can drive neurotoxicity.

Immunohistochemical Detection of Alpha-Synuclein in Unfixed Human Brain Tissue.

In both research and diagnostics, immunohistochemistry is an essential method for assessing pathology in neurodegenerative diseases. Typically, at autopsy, one hemisphere of the brain is formalin fixed for sectioning and histochemical analysis, while the other hemisphere is flash frozen for biochemical studies. While this process ensures the generation of high-quality slides, it is important to assess the pathology of tissue near pieces used for biochemical analyses, since pathology burden can be unevenly distributed throughout the brain (Fig. 1). Here, we describe a method for assessing alpha-synuclein (αSyn) pathology in unfixed human brain tissue using a novel monoclonal antibody.

Refolding of helical soluble α-synuclein through transient interaction with lipid interfaces.

α-Synuclein (αSyn) is a key player in the pathogenesis of Parkinson's disease and other synucleinopathies. Here, we report the existence of a novel soluble α-helical conformer of αSyn, obtained through transient interaction with lipid interfaces, and propose dynamic oligomerization as the mechanism underlying its stability. The conformational space of αSyn appears to be highly context-dependent, and lipid bilayers might thus play crucial roles as molecular chaperones in a cellular environment.

Familial knockin mutation of LRRK2 causes lysosomal dysfunction and accumulation of endogenous insoluble α-synuclein in neurons.

Missense mutations in the multi-domain kinase LRRK2 cause late onset familial Parkinson's disease. They most commonly with classic proteinopathy in the form of Lewy bodies and Lewy neurites comprised of insoluble α-synuclein, but in rare cases can also manifest tauopathy. The normal function of LRRK2 has remained elusive, as have the cellular consequences of its mutation. Data from LRRK2 null model organisms and LRRK2-inhibitor treated animals support a physiological role for LRRK2 in regulating lysosome function. Since idiopathic and LRRK2-linked PD are associated with the intraneuronal accumulation of protein aggregates, a series of critical questions emerge. First, how do pathogenic mutations that increase LRRK2 kinase activity affect lysosome biology in neurons? Second, are mutation-induced changes in lysosome function sufficient to alter the metabolism of α-synuclein? Lastly, are changes caused by pathogenic mutation sensitive to reversal with LRRK2 kinase inhibitors? Here, we report that mutation of LRRK2 induces modest but significant changes in lysosomal morphology and acidification, and decreased basal autophagic flux when compared to WT neurons. These changes were associated with an accumulation of detergent-insoluble α-synuclein and increased neuronal release of α-synuclein and were reversed by pharmacologic inhibition of LRRK2 kinase activity. These data demonstrate a critical and disease-relevant influence of native neuronal LRRK2 kinase activity on lysosome function and α-synuclein homeostasis. Furthermore, they also suggest that lysosome dysfunction, altered neuronal α-synuclein metabolism, and the insidious accumulation of aggregated protein over decades may contribute to pathogenesis in this late-onset form of familial PD.

Parkinson-causing α-synuclein missense mutations shift native tetramers to monomers as a mechanism for disease initiation.

ß-Sheet-rich α-synuclein (αS) aggregates characterize Parkinson's disease (PD). αS was long believed to be a natively unfolded monomer, but recent work suggests it also occurs in α-helix-rich tetramers. Crosslinking traps principally tetrameric αS in intact normal neurons, but not after cell lysis, suggesting a dynamic equilibrium. Here we show that freshly biopsied normal human brain contains abundant αS tetramers. The PD-causing mutation A53T decreases tetramers in mouse brain. Neurons derived from an A53T patient have decreased tetramers. Neurons expressing E46K do also, and adding 1-2 E46K-like mutations into the canonical αS repeat motifs (KTKEGV) further reduces tetramers, decreases αS solubility and induces neurotoxicity and round inclusions. The other three fPD missense mutations likewise decrease tetramer:monomer ratios. The destabilization of physiological tetramers by PD-causing missense mutations and the neurotoxicity and inclusions induced by markedly decreasing tetramers suggest that decreased α-helical tetramers and increased unfolded monomers initiate pathogenesis. Tetramer-stabilizing compounds should prevent this.