PDF(Pubmed)
Abstract：
BACKGROUND: Autophagic defects are involved in Methamphetamine (Meth)-induced neurotoxicity. Syntaxin 17 (Stx17), a member of the SNARE protein family, participating in several stages of autophagy, including autophagosome-late endosome/lysosome fusion. However, the role of Stx17 and potential mechanisms in autophagic defects induced by Meth remain poorly understood.
METHODS: To address the mechanism of Meth-induced cognitive impairment, the adenovirus (AV) and adeno-associated virus (AAV) were injected into the hippocampus for stereotaxis to overexpress Stx17 in vivo to examine the cognitive ability via morris water maze and novel object recognition. In molecular level, the synaptic injury and autophagic defects were evaluated. To address the Meth induced neuronal damage, the epidermal growth factor receptor (EGFR) degradation assay was performed to evaluate the degradability of the \"cargos\" mediated by Meth, and mechanistically, the maturation of the vesicles, including autophagosomes and endosomes, were validated by the Co-IP and the GTP-agarose affinity isolation assays.
RESULTS: Overexpression of Stx17 in the hippocampus markedly rescued the Meth-induced cognitive impairment and synaptic loss. For endosomes, Meth exposure upregulated Rab5 expression and its guanine-nucleotide exchange factor (GEF) (immature endosome), with a commensurate decreased active form of Rab7 (Rab7-GTP) and impeded the binding of Rab7 to CCZ1 (mature endosome); for autophagosomes, Meth treatment elicited a dramatic reduction in the overlap between Stx17 and autophagosomes but increased the colocalization of ATG5 and autophagosomes (immature autophagosomes). After Stx17 overexpression, the Rab7-GTP levels in purified late endosomes were substantially increased in parallel with the elevated mature autophagosomes, facilitating cargo (Aβ42, p-tau, and EGFR) degradation in the vesicles, which finally ameliorated Meth-induced synaptic loss and memory deficits in mice.
CONCLUSIONS: Stx17 decrease mediated by Meth contributes to vesicle fusion defects which may ascribe to the immature autophagosomes and endosomes, leading to autophagic dysfunction and finalizes neuronal damage and cognitive impairments. Therefore, targeting Stx17 may be a novel therapeutic strategy for Meth-induced neuronal injury.
METHODS: To address the mechanism of Meth-induced cognitive impairment, the adenovirus (AV) and adeno-associated virus (AAV) were injected into the hippocampus for stereotaxis to overexpress Stx17 in vivo to examine the cognitive ability via morris water maze and novel object recognition. In molecular level, the synaptic injury and autophagic defects were evaluated. To address the Meth induced neuronal damage, the epidermal growth factor receptor (EGFR) degradation assay was performed to evaluate the degradability of the \"cargos\" mediated by Meth, and mechanistically, the maturation of the vesicles, including autophagosomes and endosomes, were validated by the Co-IP and the GTP-agarose affinity isolation assays.
RESULTS: Overexpression of Stx17 in the hippocampus markedly rescued the Meth-induced cognitive impairment and synaptic loss. For endosomes, Meth exposure upregulated Rab5 expression and its guanine-nucleotide exchange factor (GEF) (immature endosome), with a commensurate decreased active form of Rab7 (Rab7-GTP) and impeded the binding of Rab7 to CCZ1 (mature endosome); for autophagosomes, Meth treatment elicited a dramatic reduction in the overlap between Stx17 and autophagosomes but increased the colocalization of ATG5 and autophagosomes (immature autophagosomes). After Stx17 overexpression, the Rab7-GTP levels in purified late endosomes were substantially increased in parallel with the elevated mature autophagosomes, facilitating cargo (Aβ42, p-tau, and EGFR) degradation in the vesicles, which finally ameliorated Meth-induced synaptic loss and memory deficits in mice.
CONCLUSIONS: Stx17 decrease mediated by Meth contributes to vesicle fusion defects which may ascribe to the immature autophagosomes and endosomes, leading to autophagic dysfunction and finalizes neuronal damage and cognitive impairments. Therefore, targeting Stx17 may be a novel therapeutic strategy for Meth-induced neuronal injury.
摘要：
背景：自噬缺陷与甲基苯丙胺（Meth）诱导的神经毒性有关。语法17(Stx17),SNARE蛋白家族的一员，参与自噬的几个阶段，包括自噬-晚期内体/溶酶体融合。然而,Stx17在Meth诱导的自噬缺陷中的作用和潜在机制仍然知之甚少。
方法：为了解决Meth引起的认知障碍的机制，将腺病毒(AV)和腺相关病毒(AAV)注射到海马体以进行立体定位,从而在体内过表达Stx17,通过Morris水迷宫和新物体识别来检查认知能力.在分子水平上,评估了突触损伤和自噬缺陷。为了解决Meth引起的神经元损伤，进行表皮生长因子受体（EGFR）降解试验以评估由Meth介导的“货物”的降解性，在机械上，囊泡的成熟，包括自噬体和内体，通过Co-IP和GTP-琼脂糖亲和分离试验验证。
结果：在海马中Stx17的过表达明显地挽救了Meth诱导的认知障碍和突触丢失。对于内体，甲基暴露上调Rab5表达及其鸟嘌呤-核苷酸交换因子（GEF）（未成熟核内体），与相应的Rab7活性形式（Rab7-GTP）减少，并阻碍Rab7与CCZ1（成熟内体）的结合；对于自噬体，Meth治疗导致Stx17和自噬体之间的重叠显着减少，但增加了ATG5和自噬体（未成熟的自噬体）的共定位。Stx17过表达后，纯化的晚期内体中的Rab7-GTP水平与成熟自噬体升高平行大幅增加，便利货物(Aβ42，p-tau，和EGFR）在囊泡中降解，最终改善了Meth诱导的小鼠突触丧失和记忆障碍。
结论：Meth介导的Stx17降低导致囊泡融合缺陷，这可能归因于未成熟的自噬体和内体，导致自噬功能障碍，并最终导致神经元损伤和认知障碍。因此,靶向Stx17可能是Meth诱导的神经元损伤的新治疗策略。
方法：为了解决Meth引起的认知障碍的机制，将腺病毒(AV)和腺相关病毒(AAV)注射到海马体以进行立体定位,从而在体内过表达Stx17,通过Morris水迷宫和新物体识别来检查认知能力.在分子水平上,评估了突触损伤和自噬缺陷。为了解决Meth引起的神经元损伤，进行表皮生长因子受体（EGFR）降解试验以评估由Meth介导的“货物”的降解性，在机械上，囊泡的成熟，包括自噬体和内体，通过Co-IP和GTP-琼脂糖亲和分离试验验证。
结果：在海马中Stx17的过表达明显地挽救了Meth诱导的认知障碍和突触丢失。对于内体，甲基暴露上调Rab5表达及其鸟嘌呤-核苷酸交换因子（GEF）（未成熟核内体），与相应的Rab7活性形式（Rab7-GTP）减少，并阻碍Rab7与CCZ1（成熟内体）的结合；对于自噬体，Meth治疗导致Stx17和自噬体之间的重叠显着减少，但增加了ATG5和自噬体（未成熟的自噬体）的共定位。Stx17过表达后，纯化的晚期内体中的Rab7-GTP水平与成熟自噬体升高平行大幅增加，便利货物(Aβ42，p-tau，和EGFR）在囊泡中降解，最终改善了Meth诱导的小鼠突触丧失和记忆障碍。
结论：Meth介导的Stx17降低导致囊泡融合缺陷，这可能归因于未成熟的自噬体和内体，导致自噬功能障碍，并最终导致神经元损伤和认知障碍。因此,靶向Stx17可能是Meth诱导的神经元损伤的新治疗策略。
