Glycine-to-aspartic acid mutation at codon 51 in Snca disrupts the synaptic localisation of α-synuclein and enhances its propensity for synucleinopathy.

Point mutations in the SNCA gene, which encodes α-synuclein (αSyn), are a known cause of familial Parkinson's disease. The glycine-51-aspartic acid (G51D) mutation causes early-onset neurodegeneration with complex, wide-spread αSyn pathology. We used CRISPR/Cas9 gene editing to introduce the G51D point mutation into the endogenous rat Snca gene. Our goal was to investigate whether the G51D αSyn mutation gives rise to synucleinopathy and neurodegenerative phenotypes in rats. Co-localisation immunostaining studies with synaptic proteins revealed that αSynG51D protein fails to efficiently localise to synapses. Furthermore, biochemical isolation of synaptosomes from rat cortex demonstrated a significant depletion of αSyn in SncaG51D/+ and SncaG51D/G51D rats. Unbiased proteomic investigation of the cortex identified significant synaptic dysregulation in SncaG51D/G51D animals. Finally, we compared the propensity for synucleinopathy of Snca+/+ and SncaG51D/G51D rats by stereotaxically delivering αSyn pre-formed fibrils (PFFs) into the pre-frontal cortex. At an early time-point, 6 weeks post-injection, we observed discrete Lewy pathology-like structures positive for phosphoserine-129-αSyn (pS129-αSyn) only in SncaG51D/G51D brains. At 26 weeks post-injection of PFFs, SncaG51D/G51D brains exhibited intense, discrete pS129-αSyn-positive structures, while Snca+/+ brains exhibited diffuse pS129-αSyn immunostaining. In summary, G51D mutagenesis of the endogenous Snca rat gene caused reduced synaptic localisation of αSyn, proteomic evidence of early synaptic dysfunction, and enhanced propensity for αSyn pathology.