Characterization of pathology-inducing α-synuclein species from human diseased brain tissue

Synucleinopathies are a group of neurodegenerative diseases characterized by the presence of pathological accumulations of misfolded, phosphorylated α-synuclein (αSyn) protein. Multiple lines of evidence indicate that synucleinopathy disease progression is driven by a prion-like process of transmission of a pathologic form of αSyn. One potential therapeutic approach to prevent cell-to-cell propagation is to target this transmissible species with selective antibodies. In this study, a rodent primary neuronal culture reporter system was developed to monitor induction of detergent-insoluble, phosphorylated (pS129) aggregates of αSyn. Induction of pS129 αSyn pathology was observed with both synthetic αSyn fibrils (PFFs) and brain lysates from multiple system atrophy (MSA) patients but not αSyn monomers or human brain lysate controls. The induction-competent species in MSA lysates could be enriched by high-speed centrifugation suggesting that it is present as a high molecular weight aggregate. Furthermore, samples derived from brain lysates from Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB) patients also induced pS129 αSyn pathology, but required longer incubation times. Lastly, the potential of αSyn selective antibodies to immunodeplete induction-competent forms of αSyn from both PFF and synucleinopathy brain samples is described. The results demonstrate that antibodies targeting the C-terminal of αSyn are most effective for immunodepletion of pathology-inducing forms of αSyn from samples derived from human synucleinopathy brains. Furthermore, the data support the hypothesis that antibodies that recognize a C-terminal epitope and exhibit selectivity for oligomeric forms over monomeric forms of αSyn represent a desirable target for immunotherapy for synucleinopathy patients.

Disulfide Bond Mediates Aggregation, Toxicity, and Ubiquitylation of Familial Amyotrophic Lateral Sclerosis-linked Mutant SOD1

Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene cause familial amyotrophic lateral sclerosis (ALS) through the gain of a toxic function; however, the nature of this toxic function remains largely unknown. Ubiquitylated aggregates of mutant SOD1 proteins in affected brain lesions are pathological hallmarks of the disease and are suggested to be involved in several proposed mechanisms of motor neuron death. Recent studies suggest that mutant SOD1 readily forms an incorrect disulfide bond upon mild oxidative stress in vitro, and the insoluble SOD1 aggregates in spinal cord of ALS model mice contain multimers cross-linked via intermolecular disulfide bonds. Here we show that a non-physiological intermolecular disulfide bond between cysteines at positions 6 and 111 of mutant SOD1 is important for high molecular weight aggregate formation, ubiquitylation, and neurotoxicity, all of which were dramatically reduced when the pertinent cysteines were replaced in mutant SOD1 expressed in Neuro-2a cells. Dorfin is a ubiquityl ligase that specifically binds familial ALS-linked mutant SOD1 and ubiquitylates it, thereby promoting its degradation. We found that Dorfin ubiquitylated mutant SOD1 by recognizing the Cys6- and Cys111-disulfide cross-linked form and targeted it for proteasomal degradation.