AAV-mediated intracerebral expression of an α-synuclein-targeting intrabody improves motor functions in aged rats.

Abnormal accumulation of alpha-synuclein (αSyn) in axons and presynaptic terminals plays a critical role in αSyn-mediated dopaminergic neurodegeneration. A strong correlation between aging and elevated αSyn levels in the substantia nigra has been identified in both humans and non-human primates. Aging is the most prominent risk factor for Parkinson's disease (PD), contributing to nigrostriatal dopamine degeneration and motor impairments. Therefore, reducing intracellular αSyn accumulation in affected neurons may help prevent nigrostriatal degeneration, preserve dopaminergic function, and delay PD onset in aging individuals. Our previous works demonstrated that adeno-associated virus 1 (AAV1)-mediated NAC32 intrabody expression effectively reduced αSyn accumulation and alleviated bradykinesia in young adult rats overexpressing αSyn in the substantia nigra. This study aimed to investigate whether AAV-mediated NAC32 intrabody expression in the substantia nigra could ameliorate αSyn-associated dopaminergic dysfunction and improve age-related motor deficits in aged rats. We first investigated the mechanism by which NAC32 reduces αSyn levels. Comparisons of αSyn burden, tyrosine hydroxylase (TH) expression, and locomotor activity were made between young and aged rats. In aged rats, we evaluated behavioral performance, dopaminergic markers, and synaptic markers following AAV1-NAC32 gene delivery into the substantia nigra. Our results showed that the NAC32-mediated αSyn reduction was not prevented by inhibition of proteasomal, lysosomal, or autophagic pathways and was associated with reduced αSyn mRNA levels. Aged rats exhibited decreased locomotor activity, elevated αSyn levels, and reduced TH expression in the substantia nigra. NAC32 intrabody expression in the substantia nigra significantly reduced αSyn accumulation, restored TH expression, increased synaptic markers and striatal dopamine levels, and improved locomotor performance in aged rats. These effects occurred without detectable elevation of pro-inflammatory cytokine levels in bulk striatal tissue. Our findings suggest that AAV-mediated NAC32 intrabody expression in the substantia nigra may serve as a therapeutic strategy to mitigate αSyn-induced dopaminergic dysfunction and motor impairments associated with aging. These results highlight the therapeutic potential of intrabody-based gene therapy for PD and other αSyn-related disorders.