Evaluating alpha-synuclein proteinopathy and consequences for birdsong in zebra finch basal ganglia area X

Lewy body pathology is a major hallmark of Parkinson's Disease (PD) and other dementias. The process of Lewy body formation is largely driven by the aggregation of alpha-synuclein (αsyn), an abundant presynaptic chaperone protein that has been shown to propagate between neurons when misfolded. Preclinical animal models inducing αsyn aggregation in the brain have demonstrated a range of behavioral consequences, though studies connecting behavioral changes to specific features of pathology are lacking. Considering vocal impairment manifests early in individuals with PD and fails to resolve upon dopamine replacement, we examined the effect of αsyn proteinopathy on birdsong in adult male zebra finches to investigate these early mechanisms of PD-related vocal dysfunction. In this study, we describe a novel tool for measuring αsyn expression called the Border Expression Ratio (BER) based on the discrete physiological distribution of αsyn surrounding basal ganglia song center, Area X. Following overexpression of human αsyn in Area X using bilateral injections of adeno-associated virus, we show that BER can be used to measure regional αsyn proteinopathy, revealing a positive correlation between right hemisphere pathology and a reduction in the variation of harmonic syllable duration. Finally, we provide evidence of serine 129 phosphorylation-a biomarker for aggregated αsyn-in Area X and cortical song nucleus LMAN of αsyn-overexpressing finches, despite this residue not being conserved in zebra finch αsyn, indicating modification of the human transgene. Together, these findings provide a framework for future analyses investigating αsyn propagation over time and the effects on vocal behavior.