Prevention of methamphetamine-induced microglial cell death by TNF-α and IL-6 through activation of the JAK-STAT pathway

Abstract

BackgroundIt is well known that methamphetamine (METH) is neurotoxic and recent studies have suggested the involvement of neuroinflammatory processes in brain dysfunction induced by misuse of this drug. Indeed, glial cells seem to be activated in response to METH, but its effects on microglial cells are not fully understood. Moreover, it has been shown that cytokines, which are normally released by activated microglia, may have a dual role in response to brain injury. This led us to study the toxic effect of METH on microglial cells by looking to cell death and alterations of tumor necrosis factor-alpha (TNF-α) and interleukine-6 (IL-6) systems, as well as the role played by these cytokines.MethodsWe used the N9 microglial cell line, and cell death and proliferation were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay and incorporation of bromodeoxyuridine, respectively. The TNF-α and IL-6 content was quantified by enzyme-linked immunosorbent assay, and changes in TNF receptor 1, IL-6 receptor-alpha, Bax and Bcl-2 protein levels by western blotting. Immunocytochemistry analysis was also performed to evaluate alterations in microglial morphology and in the protein expression of phospho-signal transducer and activator of transcription 3 (pSTAT3).ResultsMETH induced microglial cell death in a concentration-dependent manner (EC50 = 1 mM), and also led to significant morphological changes and decreased cell proliferation. Additionally, this drug increased TNF-α extracellular and intracellular levels, as well as its receptor protein levels at 1 h, whereas IL-6 and its receptor levels were increased at 24 h post-exposure. However, the endogenous proinflammatory cytokines did not contribute to METH-induced microglial cell death. On the other hand, exogenous low concentrations of TNF-α or IL-6 had a protective effect. Interestingly, we also verified that the anti-apoptotic role of TNF-α was mediated by activation of IL-6 signaling, specifically the janus kinase (JAK)-STAT3 pathway, which in turn induced down-regulation of the Bax/Bcl-2 ratio.ConclusionsThese findings show that TNF-α and IL-6 have a protective role against METH-induced microglial cell death via the IL-6 receptor, specifically through activation of the JAK-STAT3 pathway, with consequent changes in pro- and anti-apoptotic proteins.

The TNF-α/Nf-κB Signaling Pathway has a Key Role in Methamphetamine–Induced Blood–Brain Barrier Dysfunction

“…Considering the differences between rodents and humans, we further used human ECs 20 to reproduce the results obtained with rat primary cultures. As it is also known that METH can induce death of various cell types 4,12 including ECs, 21 we also considered if METH-increased endothelial permeability was due to cell death. Importantly, the concentrations used in the present study did not cause EC death or decreased cell viability.…”

“…11,12 Moreover, TNF-α has been proposed as an important mediator of BBB breakdown. 17 However, nothing is known regarding the role of TNF-α on METH-induced BBB dysfunction.…”

“…12 Primary antibodies used were as follows: mouse anti-claudin-5 (1:200, Invitrogen), rabbit antioccludin (1:150, Invitrogen) and rabbit anti-ICAM-1 (1:200, Santa Cruz). Secondary antibodies were as follows: alkaline phosphatase-conjugated secondary antibody anti-mouse (1:10,000) and anti-rabbit (1:20,000) (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA).…”

“…11,12 Accordingly, Lee et al 13 showed that METH upregulates TNF-α gene and activates nuclear factor kappa B (NF-κB) in human brain ECs. Moreover, astrocytes modulate endothelial responses under pathologic conditions by secreting factors that cause BBB leakage.…”

Pleiotrophin overexpression regulates amphetamine-induced reward and striatal dopaminergic denervation without changing the expression of dopamine D1 and D2 receptors: Implications for neuroinflammation

“…7. It is important to note that different drugs of abuse, including amphetamine and its derivatives, induce neuroinflammation (Coelho-Santos et al, 2012) and that exacerbated neuroinflammatory responses, including astrocytosis, have been linked to neurodegenerative processes and CNS injury in different models (Qin et al, 2007;Sanchez-Guajardo et al, 2013). Our current findings, together with the fact that amphetamine administration increases the levels of expression of PTN in the brain (Le Greves, 2005), suggest the possibility that PTN may be one of the mediators facilitating amphetamine-induced neuroinflammation and, as a result, neurotoxicity (Fig.…”