Background Despite increasing in vitro research,direct evidence of how abnormalα-synuclein(α-Syn)dysregulates vesicular transport and synaptic function in the human brain is lacking.Methods We performed a transcript...Background Despite increasing in vitro research,direct evidence of how abnormalα-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 humanα-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 withα-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 phosphorylatedα-Syn than with endogenousα-Syn.Immunoprecipitation confirmed that SYT13 bound predominantly to phosphorylatedα-Syn,SYT1,and the soluble N-ethylmaleimide-sensitive attachment protein receptor(SNARE)complexes.Filter trap assays revealed interactions between SYT13 and soluble toxicβ-sheet-richα-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 aggregatedα-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 Abnormalα-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.展开更多
基金supported by Hirosaki University Priority Research Grant for Future Innovation(YM),JSPS KAKENHI(24K10654(YM),20K16592(TK),23K06802(FM)and 23K24209(KW))the Collaborative Research Project of the Brain Research Institute,Niigata University(YM,AK),AMED(JP23wm0425019 and 24zf0127012(AK))+1 种基金Grants-in-Aid from the Research Committee of CNS Degenerative Diseases,Research on Policy PlanningEvaluation for Rare and Intractable Diseases,Health,Labour and Welfare Sciences Research Grants,the Ministry of Health,Labour and Welfare,Japan(AK).CB is supported by Alzheimer’s Research UK and the MSA Trust.
文摘Background Despite increasing in vitro research,direct evidence of how abnormalα-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 humanα-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 withα-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 phosphorylatedα-Syn than with endogenousα-Syn.Immunoprecipitation confirmed that SYT13 bound predominantly to phosphorylatedα-Syn,SYT1,and the soluble N-ethylmaleimide-sensitive attachment protein receptor(SNARE)complexes.Filter trap assays revealed interactions between SYT13 and soluble toxicβ-sheet-richα-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 aggregatedα-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 Abnormalα-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.
