Familial Parkinson’s point mutation abolishes multiple system atrophy prion replication

Abstract

In the neurodegenerative disease multiple system atrophy (MSA), α-synuclein misfolds into a self-templating conformation to become a prion. To compare the biological activity of α-synuclein prions in MSA and Parkinson's disease (PD), we developed nine α-synuclein−YFP cell lines expressing point mutations responsible for inherited PD. MSA prions robustly infected wild-type, A30P, and A53T α-synuclein-YFP cells, but they were unable to replicate in cells expressing the E46K mutation. Coexpression of the A53T and E46K mutations was unable to rescue MSA prion infection in vitro, establishing that MSA α-synuclein prions are conformationally distinct from the misfolded α-synuclein in PD patients. This observation may have profound implications for developing treatments for neurodegenerative diseases.ultiple system atrophy (MSA) is a rapidly progressing neurodegenerative disease resulting in dysfunction of the autonomic nervous system and disrupted motor function. The pathological hallmark of MSA is the presence of α-synuclein aggregates that coalesce into glial cytoplasmic inclusions (GCIs) in oligodendrocytes (1). MSA is one of four synucleinopathies, the other three being Parkinson's disease (PD), Parkinson's disease with dementia (PDD), and dementia with Lewy bodies (DLB). In PD, PDD, and DLB patients, α-synuclein accumulates in neurons as Lewy bodies and Lewy neurites (2). Recent studies demonstrate that MSA is unequivocally caused by α-synuclein prions, which are misfolded proteins that undergo self-propagation and are capable of transmitting disease to transgenic (Tg) mice (3-6). Using TgM83 +/− mice, which express human α-synuclein with the familial A53T mutation, both intracranial (3, 5) and peripheral inoculation (6) of brain homogenate from a total of 17 MSA patients induced neurological disease in the animals. In addition, α-synuclein prions isolated from MSA patients propagated in cultured HEK cells that express mutant human α-synuclein*A53T fused to YFP (α-syn140*A53T−YFP) (4). While PD transmission studies have induced α-synuclein neuropathology (7-9), attempts to transmit disease to TgM83 +/− mice or to infect α-syn140*A53T−YFP cells have been unsuccessful (5), suggesting that distinct conformations of misfolded α-synuclein or α-synuclein prion strains may be responsible for these diseases.To study the strain-specific properties of α-synuclein prions in MSA patients and to reconcile the differences between PD and MSA prions, we conducted a series of studies to investigate the effect of inherited PD point mutations on MSA prion replication. In addition to the original WT (α-syn140-YFP) and α-syn140*A53T-YFP cells first reported in 2015 (4), we generated seven additional HEK cell lines expressing single mutations (A30P and E46K), double mutations (A30P,A53T and E46K,A53T), and truncated α-synuclein*A53T (residues 1-95, 1-97, and 29-97). After measuring MSA prion infection in the WT, A30P, and A53T cell lines, we were surprised to observe that the E46K mutation prevented the replication of MSA prions in vi...

Kinetics of α-synuclein prions preceding neuropathological inclusions in multiple system atrophy

“…Contributing to this growing field of investigation, we compared strain differences in the synucleinopathies Parkinson's disease (PD) and multiple system atrophy (MSA), two movement disorders characterized by the neuropathological accumulation of α-synuclein in the brain. Using a cell-based assay, we demonstrated that distinct conformations of α-synuclein are present in PD and MSA patient samples [15]. Similar results have been reported by others [16,17].…”

Localized Induction of Wild-Type and Mutant Alpha-Synuclein Aggregation Reveals Propagation along Neuroanatomical Tracts

“…patients are incubated in a cell line expressing this particular mutation (33,48). Although it is tempting to suggest that the distinct characteristics of this particular ␣S mutant arise due to the distinct structural conformer the E46K point mutation produces, as suggested by numerous in vitro findings (49)(50)(51)(52)(53)(54), in our studies these differences may have been due to variations in the mouse versus human ␣S protein or in differences in the amount of the ␣S seeding factor in each sample.…”

Antibodies against alpha‐synuclein: tools and therapies

“…As a previous study reported that antibodies generated against synthetic forms of α‐syn characterized by high molecular weight species have little predictive and, presumably, therapeutic value (Woerman et al . ), future approaches may better approach this question by focusing on lower molecular weight species or isolated species from post‐mortem brain tissue. Most previous studies have employed antibodies targeting monomeric or fibrillar α‐syn.…”

“…In LB disease research, there are a paucity of studies fully characterizing the diversity and complexity of a-syn aggregates in the diseased brain with the aim of understanding those that are of greatest pathogenic relevance. As a previous study reported that antibodies generated against synthetic forms of a-syn characterized by high molecular weight species have little predictive and, presumably, therapeutic value (Woerman et al 2018), future approaches may better approach this question by focusing on lower molecular weight species or isolated species from post-mortem brain tissue. Most previous studies have employed antibodies targeting monomeric or fibrillar a-syn.…”