Background: Preclinical rodent models for Parkinson’s disease (PD) based on viral human alpha-synuclein (h-αSyn) overexpression recapitulate some of the pathological hallmarks as it presents in humans, such as progressive cell loss and additional synucleinopathy in cortical and subcortical structures. Recent studies have combined viral vector-based overexpression of human wild-type αSyn with the sequential or simultaneous inoculation of preformed fibrils (PFFs) derived from human αSyn. Objective: The goal of the study was to investigate whether sequential or combined delivery of the AAV vector and the PFFs are equipotent in inducing stable neurodegeneration and behavioral deficits. Methods: Here we compare between four experimental paradigms (PFFs only, AAV-h-αSyn only, AAV-h-αSyn with simultaneous PFFs, and AAV-h-αSyn with sequential PFFs) and their respective GFP control groups. Results: We observed reduction of TH expression and loss of neurons in the midbrain in all AAV (h-αSyn or GFP) injected groups, with or without additional PFFs inoculation. The overexpression of either h-αSyn or GFP alone induced motor deficits and dysfunctional dopamine release/reuptake in electrochemical recordings in the ipsilateral striatum. However, we observed a substantial formation of insoluble h-αSyn aggregates and inflammatory response only when h-αSyn and PFFs were combined. Moreover, the presence of h-αSyn induced higher axonal pathology compared to control groups. Conclusion: Simultaneous AAV and PFF injections are equipotent in the presented experimental setup in inducing histopathological and behavioral changes. This model provides new and interesting possibilities for characterizing PD pathology in preclinical models and means to assess future therapeutic interventions.

Adeno Associated Virus (AAV)-mediated gene expression in the brain is widely applied in the preclinical setting to investigate the therapeutic potential of specific molecular targets, characterize various cellular functions, and model central nervous system (CNS) diseases. In therapeutic applications in the clinical setting, gene therapy offers several advantages over traditional pharmacological based therapies, including the ability to directly manipulate disease mechanisms, selectively target disease-afflicted regions, and achieve long-term therapeutic protein expression in the absence of repeated administration of pharmacological agents. Next to the gold-standard iodixanol-based AAV vector production, we recently published a protocol for AAV production based on chloroform-precipitation, which allows for fast in-house production of small quantities of AAV vector without the need for specialized equipment. To validate our recent protocol, we present here a direct side-by-side comparison between vectors produced with either method in a series of in vitro and in vivo assays with a focus on transgene expression, cell loss, and neuroinflammatory responses in the brain. We do not find differences in transduction efficiency nor in any other parameter in our in vivo and in vitro panel of assessment. These results suggest that our novel protocol enables most standardly equipped laboratories to produce small batches of high quality and high titer AAV vectors for their experimental needs.