Amanda L. Woerman

Synucleinopathies are a group of neurodegenerative diseases characterized by the presence of misfolded α-synuclein inclusions which cause progressive disease by spreading throughout the brain in a prion-like manner. Throughout the neurodegenerative disease field, the ability of a single protein to give rise to multiple distinct clinical disorders is explained by the strain hypothesis, or the idea that the misfolded protein conformation determines the resulting disease. This was initially shown using transmission studies in cell lines and mouse models; more recently cryo-electron microscopy (cryo-EM) validated this idea by identifying distinct α-synuclein filament folds in brain tissues from patients with Parkinson’s disease, multiple system atrophy (MSA), and juvenile-onset synucleinopathy. However, very little is known about the α-synuclein filament structures that form in animal models of these disorders, and thus their relevance to human disease and suitability as models for therapeutic development remains a question. Here we report the first atomic resolution cryo-EM structures of α-synuclein fibrils from an MSA patient sample before and after transmission to a transgenic mouse model of disease. Our findings indicate that while distinct adaptations occur during fibril replication in the mouse host, key structural facets are maintained, validating the merits of this transmission model for supporting preclinical research on MSA.

In synucleinopathies, the protein α-synuclein misfolds into Lewy bodies (LBs) in patients with Lewy body disease (LBD) or into glial cytoplasmic inclusions (GCIs) in patients with multiple system atrophy (MSA). The ability of a single misfolded protein to cause disparate diseases is explained by the prion strain hypothesis, which argues that protein conformation is a major determinant of disease. We recently reported the unexpected finding of a novel α-synuclein strain in a Parkinson’s disease with dementia patient sample containing GCI-like co-pathology along with widespread LB pathology, which led us to question if two α-synuclein strains can interact with one another in a patient and, if so, can strain competition occur. To test this possibility, we used the strain interference model developed in the prion field, in which a slower replicating strain—in this study, mouse-passaged MSA—is used to compete with a faster replicating strain—here, recombinant preformed fibrils (PFFs)—following sciatic nerve (sc.n.) inoculation. Unexpectedly, we found that PFFs generated using the same method differed in their ability to neuroinvade following sc.n. inoculation based on α-synuclein monomer source. Using a PFF preparation that does spread from the periphery, we conducted strain competition studies by first injecting TgM83^+/− mice with mouse-passaged MSA into the sc.n. followed by a second injection with PFFs at 30, 45, and 60% of the MSA incubation period. We found that the two α-synuclein strains exhibited a synergistic effect during neuroinvasion, which was characterized by a decrease in incubation period along with evidence of the mouse-passaged MSA strain in the brain of terminal animals. These findings indicate that two α-synuclein strains can synergize with one another to accelerate the progression of clinical disease, representing a novel outcome in mixed infection studies.

In synucleinopathies, the protein α-synuclein misfolds into Lewy bodies (LBs) in patients with Lewy body disease (LBD) or into glial cytoplasmic inclusions (GCIs) in patients with multiple system atrophy (MSA). The ability of a single misfolded protein to cause disparate diseases is explained by the prion strain hypothesis, which argues that protein conformation is a major determinant of disease. While structural, biochemical, and biological studies show that LBD and MSA patient samples contain distinct α-synuclein strains, we recently reported the unexpected finding of a novel α-synuclein strain in a Parkinson’s disease with dementia patient sample containing GCI-like co-pathology along with widespread LB pathology. This finding led us to question if two α-synuclein strains can interact with one another in a patient and, if so, can strain competition occur. Notably, this would not only impact the clinical presentation of disease but would also have profound impacts on successful therapeutic development. To test this possibility, we used the strain interference superinfection model developed in the prion field, in which a slower replicating strain—in this study, mouse-passaged MSA—is used to compete with a faster replicating strain—here, recombinant preformed fibrils (PFFs)— following sciatic nerve (sc.n.) inoculation. Unexpectedly, we found that PFFs generated using the same method differed in their ability to neuroinvade following sc.n. inoculation based on α-synuclein monomer source. Using a PFF preparation that does spread from the periphery, we conducted strain competition studies by first injecting TgM83 ^+/− mice with mouse-passaged MSA into the sc.n. followed by a second injection with PFFs at 30, 45, and 60% of the MSA incubation period. Unlike in the prion field, where the faster replicating strain inhibits the slower strain at the 30 and 45% time points, we found that the two α-synuclein strains exhibited a synergistic effect during neuroinvasion. Notably, disease onset across the three cohorts was shortened compared to MSA inoculation alone, and brains from terminal animals showed evidence of both the PFF and mouse-passaged MSA strains, suggesting the two strains worked together to accelerate neuroinvasion in the mice. These findings have important implications for disease progression in patients with α-synuclein co-pathologies. The finding that two strains can synergize with one another to accelerate the progression of clinical disease represents a novel outcome in mixed infection studies and more broadly expands our understanding of the effect of prion strain biology on disease pathogenesis.

Multiple system atrophy (MSA) and the Lewy body diseases (LBDs) are caused by α-synuclein misfolding into distinct conformations, or strains, with unique biological properties. MSA patient samples readily transmit disease following intracranial (i.c.) inoculation into humanized mice, whereas LBD samples typically do not. Unexpectedly, we identified one LBD patient sample that, following i.c. inoculation, transmitted neurological disease to four out of six mice over an extended incubation period. In light of these unexpected results, we sought to identify the α-synuclein strain responsible for disease onset. Using immunohistochemistry, we identified both Lewy bodies and oligodendroglial inclusions with a glial cytoplasmic inclusion-like appearance in the substantia nigra of the patient sample. To determine if these glial inclusions were due to the presence of low titer MSA α-synuclein in the sample, we performed a secondary passage of two terminal mouse brains from the primary passage and found that the humanized mice developed disease with a shortened incubation period. Unexpectedly, using our panel of mutant α-syn140–YFP cells to analyze the primary and secondary passage samples showed that the strain isolated in the in vivo studies has unique biological properties compared to the MSA and LBD strains. These data suggest that the oligodendroglial pathology in the LBD patient sample was not caused by MSA co-pathology, and provide evidence for the isolation of a novel distinct α-synuclein strain.

Rodent models that accurately recapitulate key aspects of human disease have long been fundamental to the successful development of clinical interventions. This is greatly underscored in the neurodegenerative disease field, where preclinical testing of anti‐prion therapeutics against rodent‐adapted prions resulted in the development of small molecules effective against rodent‐adapted prions but not against human prions. These findings provided critical lessons for ongoing efforts to develop treatments for patients with neurodegenerative diseases caused by misfolding and accumulation of the proteins tau and α‐synuclein, or tauopathies and synucleinopathies, respectively. To avoid the potential pitfalls previously identified in the prion field, this review focuses on rodent models currently available to study tau and α‐synuclein disease pathogenesis, emphasizing the strengths and limitations of each with the particular goal of better supporting preclinical research. image

In patients with synucleinopathies, the protein α-synuclein misfolds into multiple conformations, each of which determines whether a patient develops multiple system atrophy (MSA) or one of three Lewy body diseases (LBDs). However, patients may also first present with pure autonomic failure, which strictly impacts autonomic nerves in the periphery, which can then phenoconvert into MSA or a LBD. When neuroinvasion happens, it remains unknown if strain properties are retained or if strain adaptation occurs, even though neuroinvasion of some prion protein (PrP) strains is known to result in the emergence of novel PrP strain variants. To investigate this question in synucleinopathies, we inoculated TgM83^+/- mice, which express human α-synuclein with the A53T mutation, with a mouse-passaged MSA patient sample either intracranially (i.c.) or into the sciatic nerve (sc.n.), and compared the biochemical and biological properties of α-synuclein prions in the brains of terminal mice. Importantly, while i.c. and sc.n. transmission studies generated pathogenic α-synuclein with similar properties, both the primary and secondary passaged MSA samples had different infectivity profiles in a panel of α-syn140-YFP cells than the starting MSA patient sample, indicating that MSA prions adapt during initial passage in TgM83^+/- mice. Similarly, using i.c. inoculation of A53T preformed fibrils to study strain selection, we found both biochemical and biological evidence that mouse passage exerts a selective pressure on α-synuclein prions in which a sub-population of starting conformations emerges in terminal animals. Together, these findings demonstrate that similar conformational selective pressures known to impact PrP prion replication also impact replication of α-synuclein prions.

Background: Multiple system atrophy (MSA) and Parkinson's disease (PD) are caused by misfolded α-synuclein spreading throughout the central nervous system. While familial PD is linked to several α-synuclein mutations, no mutations are associated with MSA. We previously showed that the familial PD mutation E46K inhibits replication of MSA prions both in vitro and in vivo , providing key evidence to support the hypothesis that α-synuclein adopts unique strains in patients. Objective: Here we sought to further interrogate α-synuclein misfolding to identify the structural determinants that contribute to MSA strain biology. Methods: We engineered a panel of cell lines harbouring both PD-linked and novel mutations designed to identify key residues that facilitate α-synuclein misfolding in MSA. We also used Maestro in silico analyses to predict the effect of each mutation on α-synuclein misfolding into one of the reported MSA cryo-electron microscopy conformations. Results: In many cases, our modelling accurately identified mutations that facilitated or inhibited MSA replication. However, Maestro was occasionally unable to predict the effect of a mutation, demonstrating the challenge of using computational tools to investigate intrinsically disordered proteins. Finally, we used our cellular models to determine the mechanism underlying the E46K-driven inhibition of MSA replication, finding that the E46/K80 salt bridge is necessary to support α-synuclein misfolding. Conclusions: Our studies used a structure-based approach to investigate α-synuclein misfolding, resulting in the creation of a powerful panel of cell lines that can be used to interrogate MSA strain biology.

Pre-formed fibrils (PFFs) made from recombinant α-synuclein are broadly used throughout the field in cellular and animal models of Parkinson’s disease. However, their ability to successfully recapitulate disease biology is a controversial topic. In this article, two researchers debate this issue with Amanda Woerman taking the view that PFFs are a model of synucleinopathy but not Parkinson’s disease, while Kelvin Luk defends their use as an important tool in the field.