Miki Yasuo, Shimoyama Shuji, Tanaka Makoto T, Kushibiki Hanae, Nakahara Asa, Wen Xiaopeng, Hijioka Masanori, Kon Tomoya, Murthy Megha, Furukawa Tomonori, Bettencourt Conceição, Mori Fumiaki, Mizukami Hiroki, Takahashi Shirushi, Tada Mari, Kitamura Yoshihisa, Kakita Akiyoshi, Warner Thomas T, Wakabayashi Koichi
Department of Neuropathology, Biomedical Research Center, Hirosaki University Graduate School of Medicine, Hirosaki, 036-8562, Japan.
Department of Neurophysiology, Biomedical Research Center, Hirosaki University Graduate School of Medicine, Hirosaki, 036-8562, Japan.
Transl Neurodegener. 2025 Jun 23;14(1):32. doi: 10.1186/s40035-025-00493-6.
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.
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.
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.
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.
尽管体外研究不断增加，但仍缺乏关于异常α-突触核蛋白（α-Syn）如何失调人脑海泡运输和突触功能的直接证据。
我们使用多系统萎缩（MSA）小鼠模型的脑组织进行了转录组分析，该模型在注射他莫昔芬后会形成人α-Syn阳性胶质细胞质包涵体样结构和神经元细胞质包涵体样结构。然后，我们使用来自71例人类病例（帕金森病，n = 10；路易体痴呆[DLB]，n = 19；MSA，n = 15；对照：n = 27）的脑样本、一份人类血液样本（对照：n = 1）以及培养细胞进行了组织学和生化分析。
基于MSA小鼠模型的转录组，我们鉴定出10种可能与α-Syn相互作用的囊泡运输蛋白，包括突触结合蛋白13（SYT13）。使用人脑样本的免疫组织化学表明，在转录组分析中鉴定出的10种囊泡运输蛋白中，只有SYT13被纳入路易体和胶质细胞质包涵体中。邻近连接分析显示，SYT13与磷酸化α-Syn的相互作用程度高于与内源性α-Syn的相互作用程度。免疫沉淀证实SYT13主要与磷酸化α-Syn、SYT1以及可溶性N-乙基马来酰亚胺敏感附着蛋白受体（SNARE）复合物结合。滤膜捕获分析揭示了SYT13与富含β-折叠的可溶性毒性α-Syn寡聚体之间的相互作用。此外，分级分析显示DLB和MSA中突触处SYT13蛋白水平显著升高。值得注意的是，观察到SYT13水平与突触处聚集的α-Syn之间存在相关性。在SH-SY5Y细胞中观察到SYT13与SYT1和SNARE复合物共同调节细胞外囊泡释放。SH-SY5Y细胞中SYT13过表达会损害细胞外囊泡释放。同样，与对照组相比，DLB和MSA病例脑匀浆中的细胞外囊泡数量显著减少。
在突触核蛋白病中，异常α-Syn通过与SYT13相互作用损害细胞外囊泡释放。我们的研究结果为缓解突触核蛋白病患者囊泡运输和突触功能失调的治疗策略提供了见解。
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