KIF5A-dependent axonal transport deficiency disrupts autophagic flux in trimethyltin chloride-induced neurotoxicity
KIF5A-dependent axonal transport deficiency disrupts autophagic flux in trimethyltin chloride-induced neurotoxicity
复制标题
KIF5A 依赖性轴突运输缺陷破坏三甲基氯化锡诱导的神经毒性中的自噬通量
DOI：
10.1080/15548627.2020.1739444
复制
发表时间：
2020-03-30
期刊：
影响因子：
13.3
通讯作者：
Zhou, Zhou
中科院分区：
文献类型：
--
作者：
Liu, Mengyu;Pi, Huifeng;Zhou, Zhou
氯化三甲基锡（Trimethyltin chloride，TMT）是一种广泛应用于工业和农业领域的杀菌剂和塑料稳定剂，具有较强的神经毒性，尤其是海马神经毒性，但其神经毒性的诱导机制尚不清楚。在此，我们将Neuro-2a细胞暴露于不同浓度的TMT（2、4和8 μM）24 h。蛋白质组学分析结合生物信息学分析揭示了自噬/自噬-溶酶体机制在TMT诱导的神经毒性中的重要作用。进一步的分析表明，TMT通过抑制溶酶体功能，如通过抑制溶酶体蛋白水解和改变溶酶体pH值，显著损害自噬通量，从而导致自噬清除缺陷，随后导致神经细胞死亡。从机制上讲，免疫途径分析的分子相互作用网络确定了下调的分子KIF 5A（驱动蛋白家族成员5A）作为TMT受损自噬通量的关键靶点。TMT降低KIF 5A蛋白表达，破坏KIF 5A与溶酶体之间的相互作用，并损害溶酶体轴突运输。此外，Kif 5a过表达恢复轴突运输，增加溶酶体功能障碍，并拮抗体外TMT诱导的神经毒性。重要的是，在TMT给药的小鼠癫痫症状和海马组织形态学损伤，TMT抑制海马KIF 5A的表达。Kif 5a的基因转移增强了海马中的自噬清除，并减轻了体内TMT诱导的神经毒性。我们的研究结果是第一个证明KIF 5A依赖性轴突运输缺陷，通过干扰溶酶体功能在TMT诱导的神经毒性引起自噬通量损害;操纵KIF 5A可能是一种治疗方法，拮抗TMT诱导的神经毒性。缩略语：3-甲基丙烯酸：3-甲基腺嘌呤; AAV病毒：腺相关病毒; ACTB：肌动蛋白β; AGC：自动增益控制ATG：自噬相关; ATP6V0D1：ATP酶H+转运溶酶体V0亚基D1; ATP 6 V1 E1：ATP酶H+转运溶酶体V1亚基E1; CA：羊角; CQ：氯喹; CTSB：组织蛋白酶B; CTSD：组织蛋白酶D; DCTN 1：动力蛋白亚基1; DG：齿状回; DYNLL 1：动力蛋白轻链LC 8 - 1型; FBS：胎牛血清; GABARAP：GABA A型受体相关蛋白; GABARAPL 1：GABA A型受体相关蛋白样1; GABARAPL 2：GABA A型受体相关蛋白样2; GAPDH：甘油醛-3-磷酸脱氢酶; IPA：免疫途径分析; KEGG：京都基因和基因组百科全书; KIF 5A：驱动蛋白家族成员5A; LAMP：溶酶体相关膜蛋白; MAP 1 LC 3 B/LC 3 B：微管相关蛋白1轻链3 β; NBR 1：NBR 1自噬货物受体; OPTN：视神经磷酸酶; PBS：磷酸盐缓冲盐水; PFA：多聚甲醛; PIK 3C 3/VPS 34：磷脂酰肌醇3-激酶催化亚基3型; PRM：平行反应监测; siRNA：小干扰RNA; SQSTM 1/p62：多价螯合体1; SYP：突触素; TAX1BP 1：Tax 1结合蛋白1; TMT：三甲基氯化锡;浴缸：微管蛋白
ABSTRACT Trimethyltin chloride (TMT) is widely used as a constituent of fungicides and plastic stabilizers in the industrial and agricultural fields, and is generally acknowledged to have potent neurotoxicity, especially in the hippocampus; however, the mechanism of induction of neurotoxicity by TMT remains elusive. Herein, we exposed Neuro-2a cells to different concentrations of TMT (2, 4, and 8 μM) for 24 h. Proteomic analysis, coupled with bioinformatics analysis, revealed the important role of macroautophagy/autophagy-lysosome machinery in TMT-induced neurotoxicity. Further analysis indicated significant impairment of autophagic flux by TMT via suppressed lysosomal function, such as by inhibiting lysosomal proteolysis and changing the lysosomal pH, thereby contributing to defects in autophagic clearance and subsequently leading to nerve cell death. Mechanistically, molecular interaction networks of Ingenuity Pathway Analysis identified a downregulated molecule, KIF5A (kinesin family member 5A), as a key target in TMT-impaired autophagic flux. TMT decreased KIF5A protein expression, disrupted the interaction between KIF5A and lysosome, and impaired lysosomal axonal transport. Moreover, Kif5a overexpression restored axonal transport, increased lysosomal dysfunction, and antagonized TMT-induced neurotoxicity in vitro. Importantly, in TMT-administered mice with seizure symptoms and histomorphological injury in the hippocampus, TMT inhibited KIF5A expression in the hippocampus. Gene transfer of Kif5a enhanced autophagic clearance in the hippocampus and alleviated TMT-induced neurotoxicity in vivo. Our results are the first to demonstrate KIF5A-dependent axonal transport deficiency to cause autophagic flux impairment via disturbance of lysosomal function in TMT-induced neurotoxicity; manipulation of KIF5A may be a therapeutic approach for antagonizing TMT-induced neurotoxicity. Abbreviations: 3-MA: 3-methyladenine; AAV: adeno-associated virus; ACTB: actin beta; AGC: automatic gain control; ATG: autophagy-related; ATP6V0D1: ATPase H+ transporting lysosomal V0 subunit D1; ATP6V1E1: ATPase H+ transporting lysosomal V1 subunit E1; CA: cornu ammonis; CQ: chloroquine; CTSB: cathepsin B; CTSD: cathepsin D; DCTN1: dynactin subunit 1; DG: dentate gyrus; DYNLL1: dynein light chain LC8-type 1; FBS: fetal bovine serum; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IPA: Ingenuity Pathway Analysis; KEGG: Kyoto Encyclopedia of Genes and Genomes; KIF5A: kinesin family member 5A; LAMP: lysosomal-associated membrane protein; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; PBS: phosphate-buffered saline; PFA: paraformaldehyde; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PRM: parallel reaction monitoring; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; SYP: synaptophysin; TAX1BP1: Tax1 binding protein 1; TMT: trimethyltin chloride; TUB: tubulin.