A One-Step Mouse Model of Parkinson's Disease Combining rAAV-α-Synuclein and Preformed Fibrils of α-Synuclein

Developing preclinical animal models that faithfully mimic the progressive nature of Parkinson's disease (PD) is crucial for advancing mechanistic insights as well as therapeutic discovery. While recombinant adeno-associated virus (rAAV)-driven α-synuclein overexpression is widely used, its reliance on high viral titers introduces nonspecific toxicity and limits physiological relevance. The SynFib model, which combines modest rAAV-driven α-synuclein expression (Syn) with α-synuclein preformed fibril (PFF) seeding (Fib), has shown promise in reproducing PD-like pathology. However, current implementations of this SynFib model have largely been confined to rats and require sequential surgeries, which increase animal distress and reduce reproducibility. Here, we present a streamlined protocol to generate a SynFib mouse model of PD that integrates rAAV-α-synuclein delivery and PFF injection into a single stereotaxic surgery. Using fine glass capillaries, this method prevents backflow of injected material, reduces injection-induced trauma, minimizes neuroinflammation, and ensures robust lesion development. This streamlined mouse model provides a reproducible and practical system to investigate α-synuclein-associated pathology and serves as a versatile platform for preclinical testing of potential therapeutics for PD. Key features • This protocol optimizes the SynFib model of PD in mice and combines rAAV-driven α-synuclein expression and PFF injection in one stereotaxic surgery, reducing animal distress. • Uses modest α-synuclein expression levels targeted to cells in the substantia nigra region of the brain. • The mouse model recapitulates hallmark PD-like features, including α-synuclein aggregation, progressive neurodegeneration, and neuroinflammation. • This mouse model can be a useful tool for α-synuclein-related mechanistic studies and preclinical therapeutic testing for PD.