Age-Progressive Synaptic and Axonal Dysregulation Induced by Purkinje Cell-Targeted AAV Expression of SCA14 PKCγ in...

Spinocerebellar ataxia type 14 (SCA14) is an autosomal-dominant disorder caused by more than 80 PRKCG missense variants encoding protein kinase Cγ (PKCγ), a serine/threonine kinase highly enriched in Purkinje cells (PCs). Despite typically late onset and slow progression, the molecular basis of age-related decline remains unclear. We used a somatic in vivo approach to express wild-type (WT) PKCγ-GFP or the prototypical G128D PKCγ-GFP selectively in PCs of neonatal mice via an adeno-associated virus (AAV) under a PC-specific promoter. G128D PKCγ-GFP formed cytoplasmic aggregates, mislocalized PCs during development, and produced gait deficits by 4 weeks that worsened with age, despite preserved PC counts and overall cerebellar volume at 1.5 years. Immunohistochemistry revealed a selective vulnerability of climbing-fiber (CF) input: vesicular glutamate transporter 2 (VGLUT2), a marker of CF synapses, declined significantly from 12 to 60 weeks in G128D mice, and the VGLUT2-positive innervation field was narrower than age-matched WT at both time points. By contrast, the glutamate/aspartate transporter GLAST in Bergmann-glial radial processes was reduced predominantly at 12 weeks in G128D mice. The δ2 glutamate receptor (GluD2) at parallel fibers and glial fibrillary acidic protein (GFAP) decreased with age but were comparable between expression conditions. Notably, aggregated mutant PKCγ accumulated within the axon initial segment (AIS), whose architecture progressively deteriorated; the altered AIS excluded PKCγ-GFP from distal axons and, by 60 weeks, was associated with reduced delivery of the vesicular GABA transporter (VGAT) to deep cerebellar nuclei (DCN), consistent with impaired anterograde transport. Hence, rather than overt neuronal loss, the cumulative burden of G128D-specific CF/axonal deficits and age-accentuated circuit and glial changes-reduced GLAST function and decreased GluD2-accounts for the worsening motor phenotype. This AAV-based system provides a practical platform to dissect late-onset pathogenic mechanisms and evaluate therapeutic strategies in vivo.