三甲基氯化锡(TMT)作为杀菌剂和塑料稳定剂的成分在工业和农业领域广泛使用,并且通常被认为具有强大的神经毒性,尤其是在海马体;然而,TMT诱导神经毒性的机制仍然难以捉摸。在这里,我们将Neuro-2a细胞暴露于不同浓度的TMT(2、4和8μM)24小时。结合生物信息学分析,揭示了巨自噬/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显著损害自噬通量,例如通过抑制溶酶体蛋白水解和改变溶酶体pH,从而导致自噬清除缺陷并随后导致神经细胞死亡。机械上,独创性通路分析的分子相互作用网络确定了一个下调的分子,KIF5A(驱动蛋白家族成员5A),作为TMT受损自噬通量的关键靶标。TMT降低KIF5A蛋白表达,破坏了KIF5A和溶酶体之间的相互作用,溶酶体轴突运输受损。此外,Kif5a过表达恢复轴突运输,溶酶体功能障碍增加,并在体外拮抗TMT诱导的神经毒性。重要的是,在服用TMT的小鼠中,癫痫发作症状和海马组织形态学损伤,TMT抑制海马中KIF5A的表达。Kif5a的基因转移增强了海马中的自噬清除并减轻了TMT诱导的体内神经毒性。我们的结果首次证明了KIF5A依赖性轴突运输缺陷在TMT诱导的神经毒性中通过溶酶体功能紊乱引起自噬通量受损;操作KIF5A可能是拮抗TMT诱导的神经毒性的治疗方法。PBS;缩写:3-MA:3-甲基腺嘌呤;AAV:ABA-ABA相关的β1酶;ABV:ABA-ABA相关的β酶1;ABB-ABA-ABA-ABA-β酶1;ABB类ABA-ABA-ABA-ABA-ββ;ABB类ABA-ABA-β-β-Ac-βββββββ酶:
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.