Summary Inclusions comprised of α-synuclein (α-syn), i.e. Lewy bodies (LBs) and Lewy neurites (LNs), define synucleinopathies including Parkinson’s Disease (PD) and dementia with Lewy Bodies (DLB). Here, we demonstrate that pre-formed fibrils generated from full length and truncated recombinant α-syn enter primary neurons, likely by adsorptive-mediated endocytosis and promote recruitment of soluble endogenous α-syn into insoluble PD-like LBs and LNs. Remarkably, endogenous α-syn was sufficient for formation of these aggregates, and overexpression of wild type or mutant α-syn was not required. LN-like pathology first developed in axons and propagated to form LB-like inclusions in perikarya. Accumulation of pathologic α-syn led to selective decreases in synaptic proteins, progressive impairments in neuronal excitability and connectivity, and eventually, neuron death. Thus, our data contribute important insights into the etiology and pathogenesis of PD-like α-syn inclusions, their impact on neuronal functions, and provide a model for discovering therapeutics targeting pathologic α-syn- mediated neurodegeneration.

This protocol describes a primary neuronal model of formation of α-synuclein (α-syn) aggregates that recapitulate features of Lewy Bodies and Lewy Neurites found in Parkinson’s disease brains and other synucleinopathies. This model allows investigation of aggregate formation, their impact on neuron function, and development of therapeutics. Addition of pre-formed fibrils (PFFs) synthesized from recombinant α-syn to neurons seeds recruitment of endogenous α-syn into aggregates characterized by detergent-insolubility, and hyperphosphorylation. Aggregate formation follows a lag phase of 2–3 days, followed by formation in axons by days 4–7, spread to somatodendritic compartments by days 7–10, and neuron death around 14 days post-PFF. Here, we provide methods and highlight critical steps for PFF formation, addition to cultured hippocampal neurons, and confirmation of aggregate formation. Neurons derived from various brain regions from non-transgenic and genetically-engineered mice and rats can be used, allowing interrogation of the impact of specific genes on aggregate formation.

Parkinson's disease (PD) is a common neurodegenerative disease caused by genetic and environmental factors. We analyzed induced pluripotent stem cell (iPSC)-derived neural cells from PD patients and presymptomatic individuals carrying mutations in the PINK1 and LRRK2 genes, and healthy control subjects. We measured several aspects of mitochondrial responses in the iPSC-derived neural cells including production of reactive oxygen species, mitochondrial respiration, proton leakage and intraneuronal movement of mitochondria. Cellular vulnerability associated with mitochondrial function in iPSC-derived neural cells from PD patients and at-risk individuals could be rescued with coenzyme Q10, rapamycin or the LRRK2 kinase inhibitor GW5074. Analysis of mitochondrial responses in iPSC-derived neural cells from PD patients carrying different mutations provides insights into convergence of cellular disease mechanisms between different familial forms of PD and highlights the importance of oxidative stress and mitochondrial dysfunction in PD.