Construct optimization for AAV-mediated human α-syn overexpression, and validation across research settings: Development of a shared tool for the research community.

The adeno-associated virus (AAV) alpha-synuclein (α-syn) overexpression model of Parkinson's disease (PD) shares many etiopathological features of the human disease, including the formation of aggregated α-syn, neuroinflammation, and nigral neurodegeneration. However, despite the high face validity of this model, it is notoriously variable, yielding inconsistent results with seemingly similar methodologies. In an effort to streamline the utility of this model for the broader research community, we partnered with The Michael J. Fox Foundation for Parkinson's Research (MJFF) to reduce variability and provide a validated tool to be distributed to the research community. Herein, the intent was to assess numerous vector batches in two distinct research groups, sharing tissue and cross-analyzing data to ensure rigor and reproducibility of this model. We assessed several variables, including AAV serotype, genome structure, and promoter construct. Our final selection consisted of a self-complementary AAV genome, carrying the hybrid chicken beta-actin promoter, packaged into AAV5. The resultant construct provides for dose-dependent nigrostriatal degeneration and associated indices such as neuroinflammation and behavioral impairments. Moreover, as reported by others, we observed significant toxicity using a reporter fluorophore as a control; instead, we generated and validated a null vector as the accompanying control. These vectors and plasmid genomes are available for distribution via MJFF.