Downregulation of α-Synuclein Protein Levels by an Intracellular Single-Chain Antibody.

Background: Accumulation of -synuclein (Syn) in the dopaminergic neurons is a common pathology seen in patients with Parkinson's disease (PD). Overproduction of Syn potentiates the formation of oligomeric Syn aggregates and enhances dopaminergic neuron degeneration. Downregulating intracellular monomeric Syn prevents the formation of Syn oligomers and is a potential therapeutic strategy to attenuate the progression of PD. Objective: The purpose of this study is to investigate the efficacy of gene delivery of Syn-specific single-chain antibodies in vitro and in vivo. Methods and Results: The plasmids for Syn and selective antibodies (NAC32, D10, and VH14) were constructed and were transfected to HEK293 and SH-SY5Y cells. Co-expression of Syn with NAC32, but not D10 or VH14, profoundly downregulated Syn protein, but not Syn mRNA levels in these cells. The interaction of Syn and NAC32 antibody was next examined in vivo. Adeno-associated virus (AAV)-Syn combined with AAV-NAC32 or AAV-sc6H4 (a negative control virus) were stereotactically injected into the substantia nigra of adult rats. AAV-NAC32 significantly reduced AAV-encoded Syn levels in the substantia nigra and striatum and increased tyrosine hydroxylase immunoreactivity in the striatum. Also, in the animals injected with AAV-NAC32 alone, endogenous Syn protein levels were significantly downregulated in the substantia nigra. Conclusion: Our data suggest that AAV-mediated gene transfer of NAC32 is a feasible approach for reducing the expression of target Syn protein in brain.