Abnormal α-synuclein binds to synaptotagmin 13, impairing extracellular vesicle release in synucleinopathies.

BACKGROUND: Despite increasing in vitro research, direct evidence of how abnormal α-synuclein (α-Syn) dysregulates vesicular transport and synaptic function in the human brain is lacking. METHODS: We performed a transcriptome analysis using brain tissues from a multiple system atrophy (MSA) mouse model, which develops human α-Syn-positive glial cytoplasmic inclusion-like structures and neuronal cytoplasmic inclusion-like structures after tamoxifen injection. We then performed histologic and biochemical analyses using brain samples from 71 human cases (Parkinson's disease, n = 10; dementia with Lewy bodies [DLB], n = 19; MSA, n = 15; control: n = 27), a human blood sample (control: n = 1), and cultured cells. RESULTS: Based on the transcriptome of the MSA mouse model, we identified 10 vesicular transport proteins, including synaptotagmin 13 (SYT13), that might interact with α-Syn. Immunohistochemistry using human brain samples demonstrated that of the 10 vesicular transport proteins identified in the transcriptome analysis, only SYT13 was incorporated into both Lewy bodies and glial cytoplasmic inclusions. Proximity ligation assays revealed that SYT13 exhibited a higher degree of interactions with phosphorylated α-Syn than with endogenous α-Syn. Immunoprecipitation confirmed that SYT13 bound predominantly to phosphorylated α-Syn, SYT1, and the soluble N-ethylmaleimide-sensitive attachment protein receptor (SNARE) complexes. Filter trap assays revealed interactions between SYT13 and soluble toxic β-sheet-rich α-Syn oligomers. Furthermore, fraction analysis showed a significant increase of SYT13 protein levels at the synapses in DLB and MSA. Notably, a correlation was observed between the levels of SYT13 and aggregated α-Syn at the synapses. SYT13 was observed to regulate extracellular vesicle release in association with SYT1 and the SNARE complexes in SH-SY5Y cells. SYT13 overexpression in SH-SY5Y cells impaired extracellular vesicle release. Consistently, the numbers of extracellular vesicles were significantly reduced in the brain homogenates of DLB and MSA cases compared with those in controls. CONCLUSIONS: Abnormal α-Syn impairs extracellular vesicle release through interactions with SYT13 in synucleinopathies. Our findings provide insights into therapeutic strategies for alleviating dysregulations of vesicular transport and synaptic function in patients with synucleinopathies.