• 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.

Autophagy (2020-03-12)
Mengyu Liu, Huifeng Pi, Yu Xi, Liting Wang, Li Tian, Mengyan Chen, Jia Xie, Ping Deng, Tao Zhang, Chao Zhou, Yidan Liang, Lei Zhang, Mindi He, Yonghui Lu, Chunhai Chen, Zhengping Yu, Zhou Zhou

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.

Product Number
Product Description

Chloroquine diphosphate salt, powder or crystals, 98.5-101.0% (EP)
3-Methyladenine, autophagy inhibitor
Poly-L-lysine solution, mol wt 150,000-300,000, 0.01%, sterile-filtered, BioReagent, suitable for cell culture
Toluidine Blue O, Technical grade
Penicillin-Streptomycin, Solution stabilized, with 10,000 units penicillin and 10 mg streptomycin/mL, 0.1 μm filtered, BioReagent, suitable for cell culture
Alconox® detergent, 0.5 oz packs
Anti-LC3B antibody produced in rabbit, ~1 mg/mL, affinity isolated antibody, buffered aqueous solution
cOmplete, Mini Protease Inhibitor Cocktail, Tablets provided in EASYpacks
Triton X-100, for molecular biology
Anti-β-Actin antibody, Mouse monoclonal, clone AC-15, purified from hybridoma cell culture