Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson’s Disease in Rodents

Abstract

Parkinson’s disease (PD) is the second most common neurodegenerative disease, affecting approximately one-percent of the population over the age of sixty. Although many animal models have been developed to study this disease, each model presents its own advantages and caveats. A unique new model has arisen to study the role of alpha-synuclein (aSyn) in the pathogenesis of PD. This model involves the conversion of recombinant monomeric aSyn protein to a fibrillar form—the aSyn pre-formed fibril (aSyn PFF)—which is then injected into the brain or introduced to the media in culture. Although many groups have successfully adopted and replicated the aSyn PFF model, issues with generating consistent pathology have been reported by investigators. To improve the replicability of this model and diminish these issues, The Michael J. Fox Foundation for Parkinson’s Research (MJFF) has enlisted the help of field leaders who performed key experiments to establish the aSyn PFF model to provide the research community with guidelines and practical tips for improving the robustness and success of this model. Specifically, we identify key pitfalls and suggestions for avoiding these mistakes as they relate to generating the aSyn PFFs from monomeric protein, validating the formation of pathogenic aSyn PFFs, and using the aSyn PFFs in vivo or in vitro to model PD. With this additional information, adoption and use of the aSyn PFF model should present fewer challenges, resulting in a robust and widely available model of PD.

The center of olfactory bulb‐seeded α‐synucleinopathy is the limbic system and the ensuing pathology is higher in male than in female mice

“…These facts contradict the argument that shorter survival periods per se and different ages at induction underlie the topographically restricted pathology in our hands. Rather, it seems more probable that fibril sonication protocols explain the differences across studies, as stated above . Our findings also differ significantly from a recent report that olfactory bulb α‐synucleinopathy causes similar pathology as in prodromal PD , but their model was generated by α‐synuclein overexpression.…”

A simple, versatile and robust centrifugation‐based filtration protocol for the isolation and quantification of α‐synuclein monomers, oligomers and fibrils: Towards improving experimental reproducibility in α‐synuclein research

“…Figure illustrates how the protocol described above can be used to ensure reproducible preparations of PFF seeds for use in seeding‐based aggregation assays (Polinski et al, ; Tarutani et al, ) or in cellular and animal studies to investigate aSyn pathology formation and spreading (Abdelmotilib et al, ; Luk et al, , ; Shimozawa et al, ; Volpicelli‐Daley et al, ). This is achieved by assessing the following parameters for each PFF preparation: (1) the percentage of fibril formation; (2) the percentage of remaining soluble α‐syn species in each preparation and (3) the relative stability of the fibrils as determined by the amount of soluble α‐syn species released after sonication of the fibrils.…”

“…Figure 1 illustrates how the protocol described above can be used to ensure reproducible preparations of PFF seeds for use in seeding-based aggregation assays (Polinski et al, 2018;Tarutani et al, 2016) or in cellular and animal studies to investigate aSyn pathology formation and spreading (Abdelmotilib et al, 2017;Luk et al, 2012Luk et al, , 2009Shimozawa et al, 2017;Volpicelli-Daley et al, 2011). This is achieved by assessing the following parameters for each PFF preparation: (1) Figure S3).…”

Stereotaxic Targeting of Alpha-Synuclein Pathology in Mouse Brain Using Preformed Fibrils

“…As PFFs can settle over time, gently flick the closed Eppendorf tube to mix contents with a vortex before drawing into the syringe for injection. The sonicated PFF solution should be used on the day of sonication [46]. Extended storage is not recommended as this increases the risk of microbial contamination.…”