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.
The protein α-synuclein, a key player in Parkinson’s disease (PD) and other synucleinopathies, exists in different physiological conformations: cytosolic unfolded aggregation-prone monomers and helical aggregation-resistant multimers. It has been shown that familial PD-associated missense mutations within the α-synuclein gene destabilize the conformer equilibrium of physiologic α-synuclein in favor of unfolded monomers. Here, we characterized the relative levels of unfolded and helical forms of cytosolic α-synuclein in post-mortem human brain tissue and showed that the equilibrium of α-synuclein conformations is destabilized in sporadic PD and DLB patients. This disturbed equilibrium is decreased in a brain region-specific manner in patient samples pointing toward a possible “prion-like” propagation of the underlying pathology and forms distinct disease-specific patterns in the two different synucleinopathies. We are also able to show that a destabilization of multimers mechanistically leads to increased levels of insoluble, pathological α-synuclein, while pharmacological stabilization of multimers leads to a “prion-like” aggregation resistance. Together, our findings suggest that these disease-specific patterns of α-synuclein multimer destabilization in sporadic PD and DLB are caused by both regional neuronal vulnerability and “prion-like” aggregation transmission enabled by the destabilization of local endogenous α-synuclein protein.
In neurodegenerative proteinopathies, intracellular inclusions are histopathologically and ultrastructurally heterogeneous but the significance of this heterogeneity is unclear. Patient- derived iPSC models, while promising for disease modeling, do not form analogous inclusions in a reasonable timeframe and suffer from limited tractability and scalability. Here, we developed an iPSC toolbox that utilizes piggyBac-based or targeted transgenes to rapidly induce CNS cells with concomitant expression of misfolding-prone proteins. The system is scalable and amenable to screening and longitudinal tracking at single-cell and single-inclusion resolution. For proof-of- principle, cortical neuron alpha-synuclein inclusionopathy models were engineered to form inclusions spontaneously or through exogenous seeding by alpha-synuclein fibrils. These models recapitulated known fibril- and lipid-rich inclusion subtypes in human brain, shedding light on their formation and consequences. Genetic-modifier and protein-interaction screens identified sequestered proteins in these inclusions, including RhoA, that were deleterious to cells when lost. This new iPSC platform should facilitate biological and drug discovery for neurodegenerative proteinopathies.
The α-synuclein protein (αS) is the major constituent of pathological neuronal inclusions both in Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB) with differential brain region-specific pathology patterns and clinical presentations. Two hypotheses that were recently put forward were either that of brain-region specific vulnerability or that of different αS aggregates, “strains”, that would affect different brain regions via “prion-like” spread. What governs these patterns, their hypothesized “prion-like” progression and region-specific vulnerability to αS aggregation in different synucleinopathies is still largely unknown. Data collected in the last decade suggests that αS can exhibit in different conformations under physiological and/or pathological conditions, which in turn help govern aggregation propensity. The cytosolic unfolded, monomeric form of αS (αSCU) is aggregation-prone and can misfold into soluble, toxic oligomers, protofilaments, and amyloid fibrils with self-templating properties, while the cytosolic helically folded, multimeric form (αSCH) resists disease-associated changes.
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