Cocaine induces astrocytosis through ER stress-mediated activation of autophagy

Cocaine is known to induce inflammation, thereby contributing in part, to the pathogenesis of neurodegeneration. A recent study from our lab has revealed a link between macroautophagy/autophagy and microglial activation. The current study was aimed at investigating whether cocaine could also mediate activation of astrocytes and, whether this process involved induction of autophagy. Our findings demonstrated that cocaine mediated the activation of astrocytes by altering the levels of autophagy markers, such as BECN1, ATG5, MAP1LC3B-II, and SQSTM1 in both human A172 astrocytoma cells and primary human astrocytes. Furthermore, cocaine treatment resulted in increased formation of endogenous MAP1LC3B puncta in human astrocytes. Additionally, astrocytes transfected with the GFP-MAP1LC3B plasmid also demonstrated cocaine-mediated upregulation of the green fluorescent MAP1LC3B puncta. Cocaine-mediated induction of autophagy involved upstream activation of ER stress proteins such as EIF2AK3, ERN1, ATF6 since blockage of autophagy using either pharmacological or gene-silencing approaches, had no effect on cocaine-mediated induction of ER stress. Using both pharmacological and gene-silencing approaches to block either ER stress or autophagy, our findings demonstrated that cocaine-induced activation of astrocytes (measured by increased levels of GFAP) involved sequential activation of ER stress and autophagy. Cocaine-mediated-increased upregulation of GFAP correlated with increased expression of proinflammatory mediators such as TNF, IL1B, and IL6. In conclusion, these findings reveal an association between ER stress-mediated autophagy and astrogliosis in cocaine-treated astrocytes. Intervention of ER stress and/or autophagy signaling would thus be promising therapeutic targets for abrogating cocaine-mediated neuroinflammation.

已知可卡因可诱导炎症反应，从而在一定程度上参与神经退行性疾病的发病机制。本实验室近期的一项研究揭示了巨自噬/自噬（macroautophagy/autophagy）与小胶质细胞激活（microglial activation）之间的关联。本研究旨在探究可卡因是否同样可介导星形胶质细胞（astrocytes）的激活，以及该过程是否涉及自噬的诱导。

我们的研究结果显示，在人A172星形细胞瘤细胞（human A172 astrocytoma cells）与原代人星形胶质细胞（primary human astrocytes）中，可卡因可通过改变自噬标志物（autophagy markers，如BECN1、ATG5、MAP1LC3B-II及SQSTM1）的表达水平，介导星形胶质细胞的激活。此外，可卡因处理可使人星形胶质细胞内源性MAP1LC3B斑点的形成增多。进一步实验发现，转染GFP-MAP1LC3B质粒的星形胶质细胞中，可卡因介导的绿色荧光MAP1LC3B斑点上调现象同样显著。

可卡因介导的自噬诱导涉及内质网应激（ER stress）蛋白（如EIF2AK3、ERN1、ATF6）的上游激活，因为通过药理学或基因沉默（gene-silencing）手段阻断自噬，并不会影响可卡因介导的内质网应激诱导。通过药理学及基因沉默手段分别阻断内质网应激或自噬后，我们的研究结果证实，可卡因诱导的星形胶质细胞激活（以胶质纤维酸性蛋白（GFAP）水平升高为检测指标）依赖于内质网应激与自噬的序贯激活。

可卡因介导的GFAP上调与促炎介质（proinflammatory mediators，如肿瘤坏死因子（TNF）、白细胞介素1β（IL1B）及白细胞介素6（IL6））的表达升高呈显著正相关。综上，本研究结果揭示了可卡因处理的星形胶质细胞中，内质网应激介导的自噬与星形胶质细胞增生（astrogliosis）之间的关联。因此，靶向干预内质网应激和/或自噬信号通路，或可成为阻断可卡因介导的神经炎症（neuroinflammation）的潜在治疗策略。