Pathogenic aggregation of α-synuclein (αSyn) is implicated in Parkinson's disease and related disorders. αSyn has long been regarded as a natively unfolded monomer that acquires secondary structure only when it binds to its target. This model is the basis for a number of published studies, but Bartels et al. report that endogenous αSyn isolated under entirely non-denaturing conditions from brain tissue, cell lines and living human cells occurs principally as a folded tetramer. This finding suggests that destabilization of the αSyn tetramer precedes misfolding and aggregation in synucleinopathies, and that agents that stabilize the normal tetramer may reduce αSyn pathogenicity. Parkinson’s disease is the second most common neurodegenerative disorder1,2. Growing evidence indicates a causative role of misfolded forms of the protein α-synuclein in the pathogenesis of Parkinson’s disease3,4. Intraneuronal aggregates of α-synuclein occur in Lewy bodies and Lewy neurites5, the cytopathological hallmarks of Parkinson’s disease and related disorders called synucleinopathies4. α-Synuclein has long been defined as a ‘natively unfolded’ monomer of about 14 kDa (ref. 6) that is believed to acquire α-helical secondary structure only upon binding to lipid vesicles7. This concept derives from the widespread use of recombinant bacterial expression protocols for in vitro studies, and of overexpression, sample heating and/or denaturing gels for cell culture and tissue studies. In contrast, we report that endogenous α-synuclein isolated and analysed under non-denaturing conditions from neuronal and non-neuronal cell lines, brain tissue and living human cells occurs in large part as a folded tetramer of about 58 kDa. Several methods, including analytical ultracentrifugation, scanning transmission electron microscopy and in vitro cell crosslinking confirmed the occurrence of the tetramer. Native, cell-derived α-synuclein showed α-helical structure without lipid addition and had much greater lipid-binding capacity than the recombinant α-synuclein studied heretofore. Whereas recombinantly expressed monomers readily aggregated into amyloid-like fibrils in vitro, native human tetramers underwent little or no amyloid-like aggregation. On the basis of these findings, we propose that destabilization of the helically folded tetramer precedes α-synuclein misfolding and aggregation in Parkinson’s disease and other human synucleinopathies, and that small molecules that stabilize the physiological tetramer could reduce α-synuclein pathogenicity.

α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation

α-Synuclein is a presynaptic neuronal protein that is linked genetically and neuropathologically to Parkinson's disease (PD). α-Synuclein may contribute to PD pathogenesis in a number of ways, but it is generally thought that its aberrant soluble oligomeric conformations, termed protofibrils, are the toxic species that mediate disruption of cellular homeostasis and neuronal death, through effects on various intracellular targets, including synaptic function. Furthermore, secreted α-synuclein may exert deleterious effects on neighboring cells, including seeding of aggregation, thus possibly contributing to disease propagation. Although the extent to which α-synuclein is involved in all cases of PD is not clear, targeting the toxic functions conferred by this protein when it is dysregulated may lead to novel therapeutic strategies not only in PD, but also in other neurodegenerative conditions, termed synucleinopathies.

/pdf/a-synuclein-in-parkinson-s-disease-jswzbre492.pdf

α-Synuclein in Parkinson’s Disease

Parkinson's disease (PD), one of the most frequent neurodegenerative disorders, is no longer considered a complex motor disorder characterized by extrapyramidal symptoms, but a progressive multisystem or—more correctly—multiorgan disease with variegated neurological and nonmotor deficiencies. It is morphologically featured not only by the degeneration of the dopaminergic nigrostriatal system, responsible for the core motor deficits, but by multifocal involvement of the central, peripheral and autonomic nervous system and other organs associated with widespread occurrence of Lewy bodies and dystrophic Lewy neurites. This results from deposition of abnormal α-synuclein (αSyn), the major protein marker of PD, and other synucleinopathies. Recent research has improved both the clinical and neuropathological diagnostic criteria of PD; it has further provided insights into the development and staging of αSyn and Lewy pathologies and has been useful in understanding the pathogenesis of PD. However, many challenges remain, for example, the role of Lewy bodies and the neurobiology of axons in the course of neurodegeneration, the relation between αSyn, Lewy pathology, and clinical deficits, as well as the interaction between αSyn and other pathologic proteins. Although genetic and experimental models have contributed to exploring the causes, pathomechanisms, and treatment options of PD, there is still a lack of an optimal animal model, and the etiology of this devastating disease is far from being elucidated. © 2011 Movement Disorder Society

Neuropathology of sporadic Parkinson's disease: Evaluation and changes of concepts

Heterozygous mutations in GBA1, the gene encoding lysosomal glucocerebrosidase, are the most frequent known genetic risk factor for Parkinson’s disease. Reduced glucocerebrosidase and α-synuclein accumulation are directly related in cell models of Parkinson’s disease. We investigated relationships between Parkinson’s disease-specific glucocerebrosidase deficits, glucocerebrosidase-related pathways, and α-synuclein levels in brain tissue from subjects with sporadic Parkinson’s disease without GBA1 mutations. Brain regions with and without a Parkinson’s disease-related increase in α-synuclein levels were assessed in autopsy samples from subjects with sporadic Parkinson’s disease (n = 19) and age- and post-mortem delay-matched controls (n = 10). Levels of glucocerebrosidase, α-synuclein and related lysosomal and autophagic proteins were assessed by western blotting. Glucocerebrosidase enzyme activity was measured using a fluorimetric assay, and glucocerebrosidase and α-synuclein messenger RNA expression determined by quantitative polymerase chain reaction. Related sphingolipids were analysed by mass spectrometry. Multivariate statistical analyses were performed to identify differences between disease groups and regions, with non-parametric correlations used to identify relationships between variables. Glucocerebrosidase protein levels and enzyme activity were selectively reduced in the early stages of Parkinson’s disease in regions with increased α-synuclein levels although limited inclusion formation, whereas GBA1 messenger RNA expression was non-selectively reduced in Parkinson’s disease. The selective loss of lysosomal glucocerebrosidase was directly related to reduced lysosomal chaperone-mediated autophagy, increased α-synuclein and decreased ceramide. Glucocerebrosidase deficits in sporadic Parkinson’s disease are related to the abnormal accumulation of α-synuclein and are associated with substantial alterations in lysosomal chaperone-mediated autophagy pathways and lipid metabolism. Our data suggest that the early selective Parkinson’s disease changes are likely a result of the redistribution of cellular membrane proteins leading to a chronic reduction in lysosome function in brain regions vulnerable to Parkinson’s disease pathology.

/pdf/reduced-glucocerebrosidase-is-associated-with-increased-a-48u1p1nhu6.pdf

Reduced glucocerebrosidase is associated with increased α-synuclein in sporadic Parkinson's disease.

Parkinson’s disease (PD) is characterized by the accumulation of misfolded and aggregated α-synuclein (α-syn) into intraneuronal inclusions named Lewy bodies (LBs). Although it is widely believed that α-syn plays a central role in the pathogenesis of PD, the processes that govern α-syn fibrillization and LB formation remain poorly understood. In this work, we sought to dissect the spatiotemporal events involved in the biogenesis of the LBs at the genetic, molecular, biochemical, structural, and cellular levels. Toward this goal, we further developed a seeding-based model of α-syn fibrillization to generate a neuronal model that reproduces the key events leading to LB formation, including seeding, fibrillization, and the formation of inclusions that recapitulate many of the biochemical, structural, and organizational features of bona fide LBs. Using an integrative omics, biochemical and imaging approach, we dissected the molecular events associated with the different stages of LB formation and their contribution to neuronal dysfunction and degeneration. In addition, we demonstrate that LB formation involves a complex interplay between α-syn fibrillization, posttranslational modifications, and interactions between α-syn aggregates and membranous organelles, including mitochondria, the autophagosome, and endolysosome. Finally, we show that the process of LB formation, rather than simply fibril formation, is one of the major drivers of neurodegeneration through disruption of cellular functions and inducing mitochondria damage and deficits, and synaptic dysfunctions. We believe that this model represents a powerful platform to further investigate the mechanisms of LB formation and clearance and to screen and evaluate therapeutics targeting α-syn aggregation and LB formation.

https://www.pnas.org/content/pnas/117/9/4971.full.pdf

The process of Lewy body formation, rather than simply α-synuclein fibrillization, is one of the major drivers of neurodegeneration

α-Synuclein is a major component of Lewy bodies and glial cytoplasmic inclusions, pathological hallmarks of idiopathic Parkinson's disease and multiple system atrophy, and it is assumed to be aetiologically involved in these conditions. However, the quantitative status of brain α-synuclein in different Parkinsonian disorders is still unresolved and it is uncertain whether α-synuclein accumulation is restricted to regions of pathology. We compared membrane-associated, sodium dodecyl sulfate-soluble α-synuclein, both the full-length 17 kDa and high molecular weight species, by western blotting in autopsied brain of patients with Parkinson's disease (brainstem-predominant Lewy body disease: n = 9), multiple system atrophy ( n = 11), progressive supranuclear palsy ( n = 16), and of normal controls ( n = 13). Brain of a patient with familial Parkinsonism-dementia due to α-synuclein locus triplication (as positive control) showed increased membrane-associated, sodium dodecyl sulfate-soluble α-synuclein levels with abundant high molecular weight immunoreactivity. In multiple system atrophy, a massive increase in 17 kDa membrane-associated, sodium dodecyl sulfate-soluble α-synuclein was observed in highly pathologically affected regions, including putamen (+1760%, range +625–2900%), substantia nigra [+1000% (+356–1850%)], and white matter of internal capsule [+2210% (+430–6830%)] together with numerous high molecular weight species. Levels of 17 kDa membrane-associated, sodium dodecyl sulfate-soluble α-synuclein were only modestly increased in less affected areas (cerebellar cortex, +95%; caudate, +30%; with both also showing numerous high molecular weight species) and were generally normal in cerebral cortices. In both Parkinson's disease and progressive supranuclear palsy, membrane-associated, sodium dodecyl sulfate-soluble α-synuclein levels were normal in putamen and frontal cortex whereas a trend was observed for variably increased 17 kDa membrane-associated, sodium dodecyl sulfate-soluble α-synuclein concentrations [+184% (−60% to +618%)] with additional high molecular weight species in Parkinson's disease substantia nigra. No obvious correlation was observed between nigral membrane-associated, sodium dodecyl sulfate-soluble α-synuclein accumulation and Lewy body density in Parkinson's disease. Two progressive supranuclear palsy cases had membrane-associated, sodium dodecyl sulfate-soluble α-synuclein accumulation in substantia nigra similar to multiple system atrophy. Several Parkinson's disease patients had very modest high molecular weight membrane-associated, sodium dodecyl sulfate-soluble α-synuclein accumulation in putamen. Levels of 17-kDa membrane-associated, sodium dodecyl sulfate-soluble α-synuclein were generally positively correlated with those of high molecular weight membrane-associated, sodium dodecyl sulfate-soluble α-synuclein and there was a trend for a positive correlation between striatal dopamine loss and 17-kDa membrane-associated, sodium dodecyl sulfate-soluble α-synuclein concentrations in multiple system atrophy. Brain membrane-associated, sodium dodecyl sulfate-soluble α-synuclein accumulations in Parkinson's disease and multiple system atrophy are regionally specific, suggesting that these sporadic α-synucleinopathies, unlike familial Parkinsonism-dementia, are not associated with a simple global over-expression of the protein. Despite a similar extent of dopamine depletion, the magnitude of brain membrane-associated, sodium dodecyl sulfate-soluble α-synuclein changes is disease specific, with multiple system atrophy clearly having the most severe accumulation. Literature discrepancies on α-synuclein status in ‘Parkinson's disease’ might be explained by inclusion of cases not having classic brainstem-predominant Lewy body disease and by variable α-synuclein accumulation within this diagnostic classification.

/pdf/brain-a-synuclein-accumulation-in-multiple-system-atrophy-404xl2qnyu.pdf

Lee C. Ang

Papers

Brain α-synuclein accumulation in multiple system atrophy, Parkinson's disease and progressive supranuclear palsy: a comparative investigation