title: Cocaine-mediated microglial activation involves the ER stress-autophagy axis.
abstract: Cocaine abuse leads to neuroinflammation, which, in turn, contributes to the pathogenesis of neurodegeneration associated with advanced HIV-1 infection. Autophagy plays important roles in both innate and adaptive immune responses. However, the possible functional link between cocaine and autophagy has not been explored before. Herein, we demonstrate that cocaine exposure induced autophagy in both BV-2 and primary rat microglial cells as demonstrated by a dose- and time-dependent induction of autophagy-signature proteins such as BECN1/Beclin 1, ATG5, and MAP1LC3B. These findings were validated wherein cocaine treatment of BV-2 cells resulted in increased formation of puncta in cells expressing either endogenous MAP1LC3B or overexpressing GFP-MAP1LC3B. Specificity of cocaine-induced autophagy was confirmed by treating cells with inhibitors of autophagy (3-MA and wortmannin). Intriguingly, cocaine-mediated induction of autophagy involved upstream activation of 2 ER stress pathways (EIF2AK3- and ERN1-dependent), as evidenced by the ability of the ER stress inhibitor salubrinal to ameliorate cocaine-induced autophagy. In vivo validation of these findings demonstrated increased expression of BECN1, ATG5, and MAP1LC3B-II proteins in cocaine-treated mouse brains compared to untreated animals. Increased autophagy contributes to cocaine-mediated activation of microglia since pretreatment of cells with wortmannin resulted in decreased expression and release of inflammatory factors (TNF, IL1B, IL6, and CCL2) in microglial cells. Taken together, our findings suggest that cocaine exposure results in induction of autophagy that is closely linked with neuroinflammation. Targeting autophagic proteins could thus be considered as a therapeutic strategy for the treatment of cocaine-related neuroinflammation diseases.
authors.affiliation: a Department of Pharmacology and Experimental Neuroscience; Nebraska Medical Center; University of Nebraska Medical Center ; Omaha , NE , USA.
authors.forename: Ming-Lei
authors.initials: ML
authors.lastname: Guo
authors.forename: Ke
authors.initials: K
authors.lastname: Liao
authors.forename: Palsamy
authors.initials: P
authors.lastname: Periyasamy
authors.forename: Lu
authors.initials: L
authors.lastname: Yang
authors.forename: Yu
authors.initials: Y
authors.lastname: Cai
authors.forename: Shannon E
authors.initials: SE
authors.lastname: Callen
authors.forename: Shilpa
authors.initials: S
authors.lastname: Buch
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D000071187:Autophagy-Related Protein 5; D000071186:Beclin-1; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003042:Cocaine / Q000494:pharmacology*; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008954:Models, Biological; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013997:Time Factors; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D051017:Apoptosis Regulatory Proteins; C545865:Atg5 protein, rat; D000071187:Autophagy-Related Protein 5; D000071186:Beclin-1; C512829:Becn1 protein, rat; D015415:Biomarkers; D047428:Protein Kinase Inhibitors; D011506:Proteins; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C025946:3-methyladenine; D019869:Phosphatidylinositol 3-Kinases; D003042:Cocaine; D000225:Adenine; D000077191:Wortmannin
keywords: 3-MA, 3-methyladenine; ATF6, activating transcription factor 6; ATG5, autophagy-related 5; BCL2, B-cell CLL/lymphoma 2; BECN1; BECN1, Beclin 1, autophagy related; Baf1, bafilomycin A1; CCL2, chemokine (C-C motif) ligand 2; DAPI: 4, 6-diamidino-2-phenylindole, dihydrochloride; DDIT3, DNA-damage-inducible transcript 3; EGFP, enhanced green fluorescent protein; EIF2AK3, eukaryotic translation initiation factor 2-α kinase 3; EIF2S1, eukaryotic translation initiation factor 2, subunit 1 α, 35kDa; ER stress; ER, endoplasmic reticulum; ERN1, endoplasmic reticulum to nucleus signaling 1; HIV, human immunodeficiency virus; IL1B, interleukin 1, β; IL6, interleukin 6; MAP1LC3B; MAP1LC3B, microtubule-associated protein 1 light chain 3; METH, methamphetamine; MTOR, mechanistic target of rapamycin; NFKB1, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1; PBN, N-tert-butyl-α-phenylnitrone; PPP1R3A, protein phosphatase 1, regulatory subunit 3A; PtdIns3K, class III phosphatidylinositol 3-kinase; ROS, reactive oxygen species; RPS6, ribosomal protein S6; TLR4, toll-like receptor 4; TNF, tumor necrosis factor; autophagy; cocaine; microglial cells; neuroinflammation; rPMCs, rat primary microglial cells; wort, wortmannin