ABSTRACT

Macroautophagy/autophagy is vital for neuronal homeostasis and functions. Accumulating evidence suggest that autophagy is impaired during cerebral ischemia, contributing to neuronal dysfunction and neurodegeneration. However, the outcomes after transient modification in autophagy machinery are not fully understood. This study investigated the effects of ischemic stress on autophagy and synaptic structures using a rat model of oxygen-glucose deprivation (OGD) in hippocampal neurons and a mouse model of middle cerebral artery occlusion (MCAO). Upon acute ischemia, an initial autophagy modification occurred in an upregulation manner. Following, the number of lysosomes increased, as well as lysosomal volume, indicating dysfunctional lysosomal storage. These changes were prevented by inhibiting autophagy via 3-methyladenine (3-MA) treatment or ATG7 (autophagy related 7) knockdown, or were mimicked by rapamycin (RAPA), a known activator of autophagy. This suggests that dysfunctional lysosomal storage is associated with the early burst of autophagy. Dysfunctional lysosomal storage contributed to autophagy dysfunction because the basal level of MTOR-dependent lysosomal biogenesis in the reperfusion was not sufficient to clear undegraded cargoes after transient autophagy upregulation. Further investigation revealed that impairment of synaptic ultra-structures, accompanied by dysfunctional lysosomal storage, may result from a failure in dynamic turnover of synaptic proteins. This indicates a vital role of autophagy-lysosomal machinery in the maintenance of synaptic structures. This study supports previous evidence that dysfunctional lysosomal storage may occur following the upregulation of autophagy in neurons. Appropriate autophagosome-lysosomal functioning is vital for maintenance of neuronal synaptic function and impacts more than the few known synaptic proteins.

Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AD: Alzheimer disease; ALR: autophagic lysosome reformation; ATG7: autophagy related 7; CTSB: cathepsin B; CTSD: cathepsin D; DAPI: 4ʹ,6-diamidino-2-phenylindole; DEGs: differentially expressed genes; DMEM: Dulbecco’s modified Eagle’s medium; DMSO: dimethyl sulfoxide; GO: Gene Ontology; HBSS: Hanks’ balanced salt solution; HPCA: hippocalcin; i.c.v: intracerebroventricular; KEGG: kyoto encyclopedia of genes and genomes; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; LSDs: lysosomal storage disorders; MAP2: microtubule-associated protein 2; MCAO: middle cerebral artery occlusion; mCTSB: mature CTSB; mCTSD: mature CTSD; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; OGD/R: oxygen-glucose deprivation/reoxygenation; PBS: phosphate-buffered saline; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; proCTSD: pro-cathepsin D; RAPA: rapamycin; RNA-seq: RNA sequencing; RPS6KB/p70S6K: ribosomal protein S6 kinase; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SIM: Structured Illumination Microscopy; SNAP25: synaptosomal-associated protein 25; SQSTM1/p62: sequestosome 1; SYN1: synapsin I; SYT1: synaptotagmin I; TBST: tris-buffered saline Tween-20; TEM: transmission electron microscopy; TFEB: transcription factor EB; tMCAO: transient middle cerebral artery occlusion; TTC: 2,3,5-triphenyltetrazolium chloride; TUBB3: tubulin, beta 3 class III.

摘要

巨自噬/自噬对神经元稳态和功能至关重要。越来越多的证据表明，自噬在脑缺血期间受损，导致神经元功能障碍和神经变性。然而，自噬机制瞬时改变后的结果尚不完全清楚。本研究使用海马神经元氧-葡萄糖剥夺 (OGD) 大鼠模型和大脑中动脉闭塞 (MCAO) 小鼠模型研究缺血应激对自噬和突触结构的影响。急性缺血后，初始自噬修饰以上调方式发生。随后，溶酶体数量增加，溶酶体体积增加，表明溶酶体储存功能失调。这些变化是通过 3-甲基腺嘌呤 (3-MA) 处理或 ATG7（自噬相关 7）敲低抑制自噬来防止的，或者被雷帕霉素 (RAPA)（一种已知的自噬激活剂）模拟。这表明功能失调的溶酶体储存与自噬的早期爆发有关。功能失调的溶酶体储存导致自噬功能障碍，因为再灌注中 MTOR 依赖性溶酶体生物发生的基础水平不足以清除瞬时自噬上调后未降解的货物。进一步的研究表明，突触超微结构的损伤，伴随着溶酶体储存功能障碍，可能是由于突触蛋白的动态更新失败造成的。这表明自噬-溶酶体机制在维持突触结构中的重要作用。该研究支持先前的证据，即在神经元自噬上调后可能会发生功能失调的溶酶体储存。适当的自噬体-溶酶体功能对于维持神经元突触功能至关重要，并且比少数已知的突触蛋白影响更大。

缩写：3-MA：3-甲基腺嘌呤；ACTB：肌动蛋白β；AD：阿尔茨海默病；ALR：自噬溶酶体重组；ATG7：自噬相关7；CTSB：组织蛋白酶 B；CTSD：组织蛋白酶 D；DAPI：4ʹ,6-二脒基-2-苯基吲哚；DEGs：差异表达基因；DMEM：Dulbecco 改良的 Eagle 培养基；DMSO：二甲亚砜；GO：基因本体论；HBSS：汉克斯平衡盐溶液；HPCA：海马钙素；icv：脑室内；KEGG：京都基因和基因组百科全书；LAMP1：溶酶体相关膜蛋白 1；MAP1LC3B/LC3：微管相关蛋白 1 轻链 3 β；LSD：溶酶体贮积症；MAP2：微管相关蛋白2；MCAO：大脑中动脉闭塞；mCTSB：成熟的CTSB；mCTSD：成熟的CTSD；MOI：感染复数；MTOR：雷帕霉素激酶的机制靶点；OGD/R：氧-葡萄糖剥夺/复氧；PBS：磷酸盐缓冲盐水；PRKAA/AMPKα：蛋白激酶 AMP 激活的催化亚基 α；proCTSD：原组织蛋白酶 D；RAPA：雷帕霉素；RNA-seq：RNA测序；RPS6KB/p70S6K：核糖体蛋白S6激酶；SDS-PAGE：十二烷基硫酸钠-聚丙烯酰胺凝胶电泳；SIM：结构照明显微镜；SNAP25：突触体相关蛋白 25；SQSTM1/p62：sequestosome 1；SYN1：突触素 I；SYT1：突触结合蛋白 I；TBST：tris 缓冲盐水 Tween-20；TEM：透射电子显微镜；TFEB：转录因子EB；tMCAO：暂时性大脑中动脉闭塞；TTC：氯化2,3,5-三苯基四唑；TUBB3：微管蛋白，β 3 III 类。RNA-seq：RNA测序；RPS6KB/p70S6K：核糖体蛋白S6激酶；SDS-PAGE：十二烷基硫酸钠-聚丙烯酰胺凝胶电泳；SIM：结构照明显微镜；SNAP25：突触体相关蛋白 25；SQSTM1/p62：sequestosome 1；SYN1：突触素 I；SYT1：突触结合蛋白 I；TBST：tris 缓冲盐水 Tween-20；TEM：透射电子显微镜；TFEB：转录因子EB；tMCAO：暂时性大脑中动脉闭塞；TTC：氯化2,3,5-三苯基四唑；TUBB3：微管蛋白，β 3 III 类。RNA-seq：RNA测序；RPS6KB/p70S6K：核糖体蛋白S6激酶；SDS-PAGE：十二烷基硫酸钠-聚丙烯酰胺凝胶电泳；SIM：结构照明显微镜；SNAP25：突触体相关蛋白 25；SQSTM1/p62：sequestosome 1；SYN1：突触素 I；SYT1：突触结合蛋白 I；TBST：tris 缓冲盐水 Tween-20；TEM：透射电子显微镜；TFEB：转录因子EB；tMCAO：暂时性大脑中动脉闭塞；TTC：氯化2,3,5-三苯基四唑；TUBB3：微管蛋白，β 3 III 类。透射电子显微镜；TFEB：转录因子EB；tMCAO：暂时性大脑中动脉闭塞；TTC：氯化2,3,5-三苯基四唑；TUBB3：微管蛋白，β 3 III 类。透射电子显微镜；TFEB：转录因子EB；tMCAO：暂时性大脑中动脉闭塞；TTC：氯化2,3,5-三苯基四唑；TUBB3：微管蛋白，β 3 III 类。