Role of Dysregulated Autophagy in HIV Tat, Cocaine, and cART Mediated NLRP3 Activation in Microglia.

Despite the ability of combination antiretroviral therapy (cART) to suppress viremia, there is persistence low levels of HIV proteins such as Transactivator of transcription (Tat) in the central nervous system (CNS), contributing to glial activation and neuroinflammation. Accumulating evidence also implicates the role of drugs of abuse in exacerbating neurological complications associated with HIV-1. The combined effects of HIV Tat, drugs of abuse, and cART can thus create a toxic milieu in the CNS. The present study investigated the combinatorial effects of HIV-Tat, cocaine, and cART on autophagy and NLRP3 inflammasome activation. We selected a combination of three commonly used cART regimens: tenofovir, emtricitabine, and dolutegravir. Our results demonstrated that exposure of mouse primary microglia (MPMs) to these agents-HIV Tat (25 ng/ml), cocaine (1 µM), and cART (1 µM each) resulted in upregulation of autophagy markers: Beclin1, LC3B-II, and SQSTM1 with impaired lysosomal functioning involving increased lysosomal pH, decreased LAMP2 and cathepsin D, ultimately leading to dysregulated autophagy. Our findings also demonstrated activation of the NLRP3 signaling in microglia exposed to these agents. We further demonstrated that gene silencing of key autophagy protein BECN1 significantly blocked NLRP3-mediated activation of microglia. Silencing of NLRP3, however, failed to block HIV Tat, cocaine, and cART-mediated dysregulation of the autophagy-lysosomal axis; these in vitro phenomena were also validated in vivo using iTat mice administered cocaine and cART. This study thus underscores the cooperative effects of HIV Tat, cocaine, and cART in exacerbating microglial activation involving dysregulated autophagy and activation of the NLRP3 inflammasome signaling.

Involvement of lncRNA TUG1 in HIV-1 Tat-Induced Astrocyte Senescence.

HIV-1 infection in the era of combined antiretroviral therapy has been associated with premature aging. Among the various features of HIV-1 associated neurocognitive disorders, astrocyte senescence has been surmised as a potential cause contributing to HIV-1-induced brain aging and neurocognitive impairments. Recently, lncRNAs have also been implicated to play essential roles in the onset of cellular senescence. Herein, using human primary astrocytes (HPAs), we investigated the role of lncRNA TUG1 in HIV-1 Tat-mediated onset of astrocyte senescence. We found that HPAs exposed to HIV-1 Tat resulted in significant upregulation of lncRNA TUG1 expression that was accompanied by elevated expression of p16 and p21, respectively. Additionally, HIV-1 Tat-exposed HPAs demonstrated increased expression of senescence-associated (SA) markers-SA-ß-galactosidase (SA-ß-gal) activity and SA-heterochromatin foci-cell-cycle arrest, and increased production of reactive oxygen species and proinflammatory cytokines. Intriguingly, gene silencing of lncRNA TUG1 in HPAs also reversed HIV-1 Tat-induced upregulation of p21, p16, SA-ß gal activity, cellular activation, and proinflammatory cytokines. Furthermore, increased expression of astrocytic p16 and p21, lncRNA TUG1, and proinflammatory cytokines were observed in the prefrontal cortices of HIV-1 transgenic rats, thereby suggesting the occurrence of senescence activation in vivo. Overall, our data indicate that HIV-1 Tat-induced astrocyte senescence involves the lncRNA TUG1 and could serve as a potential therapeutic target for dampening accelerated aging associated with HIV-1/HIV-1 proteins.

Substance use, microbiome and psychiatric disorders.

Accumulating evidence from several studies has shown association between substance use, dysregulation of the microbiome and psychiatric disorders such as depression, anxiety, and psychosis. Many of the abused substances such as cocaine and alcohol have been shown to alter immune signaling pathways and cause inflammation in both the periphery and the central nervous system (CNS). In addition, these substances of abuse also alter the composition and function of the gut microbiome which is known to play important roles such as the synthesis of neurotransmitters and metabolites, that affect the CNS homeostasis and consequent behavioral outcomes. The emerging interactions between substance use, microbiome and CNS neurochemical alterations could contribute to the development of psychiatric disorders. This review provides an overview of the associative effects of substance use such as alcohol, cocaine, methamphetamine, nicotine and opioids on the gut microbiome and psychiatric disorders involving anxiety, depression and psychosis. Understanding the relationship between substance use, microbiome and psychiatric disorders will provide insights for potential therapeutic targets, aimed at mitigating these adverse outcomes.

Protective Role of Lactobacillus rhamnosus Probiotic in Reversing Cocaine-Induced Oxidative Stress, Glial Activation and Locomotion in Mice.

Cocaine abuse is known to cause inflammation, oxidative injury and alterations in the gut microbiota. Although emerging studies have demonstrated the role of gut microbiota in modulating neurological complications and behavior, the mechanism(s) underlying these processes remain unclear. In the present study, we investigated the protective effect of Lactobacillus rhamnosus probiotic on cocaine-induced oxidative stress, glial activation, and locomotion in mice. In this study, groups of male C56BL6 mice were administered gut-resident commensal bacteria L. rhamnosus probiotic (oral gavage) concurrently with cocaine (20 mg/kg, i.p.) or saline for 28 days and assessed for oxidative stress and cellular activation in both the gut and brain as well as alterations in locomotion behavior. Cocaine-induced gut dysregulation was associated with increased formation of 4-hydroxynonenal (4-HNE) adducts, increased expression of pERK-1/2, pNF-kB-p65 and antioxidant mediators (SOD1, GPx1). In cocaine administered mice, there was increased activation of both microglia and astrocytes in the striatum and cortex of the brain as shown by enhanced expression of CD11b and GFAP, respectively. Cocaine administration also resulted in increased locomotor activity in the open field test in these mice. Administration of L. rhamnosus attenuated cocaine-induced gut oxidative stress and inflammation as well as glial activation and locomotion. These results suggest the potential of microbial-based interventions to attenuate cocaine-mediated behavioral responses and neuroinflammation, in addition to systemic inflammation and oxidative damage.

NLRP3 Inflammasome Is Involved in Cocaine-Mediated Potentiation on Behavioral Changes in CX3CR1-Deficient Mice.

Microglia, the primary immunocompetent cells of the brain, are suggested to play a role in the development of drug addiction. Previous studies have identified the microglia-derived pro-inflammatory factor IL1ß can promote the progression of cocaine addiction. Additionally, the activation status of microglia and "two-hit hypothesis" have been proposed in the field of drug addiction to explain how early life stress (ELS) could significantly increase the incidence of drug addiction in later life. However, the mechanisms underlying microglia prime and full activation and their roles in drug addiction remain greatly unexplored. Here, we employed CX3CR1-GFP mice (CX3CR1 functional deficiency, CX3CR1-/-) to explore whether primed microglia could potentiate cocaine-mediated behavioral changes and the possible underlying mechanisms. CX3CR1-/- mice revealed higher hyperlocomotion activity and conditional place preference than wild-type (WT) mice did under cocaine administration. In parallel, CX3CR1-/- mice showed higher activity of NLR family pyrin domain-containing 3 (NLRP3) inflammasome than WT mice. Interestingly, CX3CR1 deficiency itself could prime NLRP3 signaling by increasing the expression of NLPR3 and affect lysosome biogenesis under basal conditions. Taken together, our findings demonstrated that the functional status of microglia could have an impact on cocaine-mediated reward effects, and NLRP3 inflammasome activity was associated with this phenomenon. This study was consistent with the two-hit hypothesis and provided solid evidence to support the involvement of microglia in drug addiction. Targeting the NLRP3 inflammasome may represent a novel therapeutic approach for ameliorating or blocking the development of drug addiction.

Astrocytes & Astrocyte derived Extracellular Vesicles in Morphine Induced Amyloidopathy: Implications for Cognitive Deficits in Opiate Abusers.

While opiates like morphine play a major role in the pharmacotherapy for the control of pain associated with various diseases, paradoxically, their long-term use is associated with cognitive impairments. Furthermore, morphine administration has been shown to result in neuronal synaptodendritic injury in rodent brains, leading to neurodegeneration. We recently reported the role of astrocytes as contributors of amyloidosis associated with HIV-associated neurological disorders. Herein we hypothesize that morphine could induce astrocytic amyloidosis, which, in turn, could be disseminated to various regions in the brain by astrocyte-derived EVs (ADEVs). In this study we demonstrate brain region-specific up-regulation of astrocytic amyloids in morphine dependendent rhesus macaques. In addition, herein we also demonstrate increased expression of ß-site cleaving enzyme (BACE1), APP, and Aß in human primary astrocytes (HPAs) exposed to morphine. Mechanisms involved in this process included the up-regulation of hypoxia-inducible factor (HIF-1α), its translocation and binding to the promoter of BACE1, leading to increased BACE1 activity and, generation of Aß 1-42. Gene silencing approaches confirmed the regulatory role of HIF-1α in BACE1 mediated amyloidosis leading to astrocyte activation and neuroinflammation. We next sought to assess whether morphine-stimulated ADEVs could carry amyloid cargoes. Results showed that morphine exposure induced the release of morphine-ADEVs, carrying amyloids. Interestingly, silencing HIF-1α in astrocytes not only reduced the numbers of ADEV released, but also the packaging of amyloid cargos in the ADEVs. These findings were further validated in brain derived EVs (BEVs) isolated from macaques, wherein it was shown that BEVs from morphine-dependent macaques, carried varieties of amyloid cargoes including the cytokine IL-1ß. This is the first report implicating the role of HIF-1α-BACE1 axis in morphine-mediated induction of astrocytic amyloidosis, leading, in turn, to neuroinflammation and release of the amyloid cargoes via ADEVs. These findings set the groundwork for the future development of therapeutic strategies for targeting cognitive deficits in chronic opiate users.

HIV-1 and drug abuse comorbidity: Lessons learned from the animal models of NeuroHIV.

Various research studies that have investigated the association between HIV infection and addiction underpin the role of various drugs of abuse in impairing immunological and non-immunological pathways of the host system, ultimately leading to augmentation of HIV infection and disease progression. These studies have included both in vitro and in vivo animal models wherein investigators have assessed the effects of various drugs on several disease parameters to decipher the impact of drugs on both HIV infection and progression of HIV-associated neurocognitive disorders (HAND). However, given the inherent limitations in the existing animal models of HAND, these investigations only recapitulated specific aspects of the disease but not the complex human syndrome. Despite the inability of HIV to infect rodents over the last 30 years, multiple strategies have been employed to develop several rodent models of HAND. While none of these models can accurately mimic the overall pathophysiology of HAND, they serve the purpose of modeling some unique aspects of HAND. This review provides an overview of various animal models used in the field and a careful evaluation of methodological strengths and limitations inherent in both the model systems and study designs to understand better how the various animal models complement one another.

Biogenesis, physiological functions and potential applications of extracellular vesicles in substance use disorders.

Substance use disorder (SUD) is a growing health problem that affects several millions of people worldwide, resulting in negative socioeconomic impacts and increased health care costs. Emerging evidence suggests that extracellular vesicles (EVs) play a crucial role in SUD pathogenesis. EVs, including exosomes and microvesicles, are membrane-encapsulated particles that are released into the extracellular space by most types of cells. EVs are important players in mediating cell-to-cell communication through transfer of cargo such as proteins, lipids and nucleic acids. The EV cargo can alter the status of recipient cells, thereby contributing to both physiological and pathological processes; some of these play critical roles in SUD. Although the functions of EVs under several pathological conditions have been extensively reviewed, EV functions and potential applications in SUD remain less studied. In this review, we provide an overview of the current knowledge of the role of EVs in SUD, including alcohol, cocaine, heroin, marijuana, nicotine and opiate abuse. The review will focus on the biogenesis and cargo composition of EVs as well as the potential use of EVs as biomarkers of SUD or therapeutic targets in SUD.

NLRP3 Inflammasome Blockade Reduces Cocaine-Induced Microglial Activation and Neuroinflammation.

Cocaine use disorder is a major health crisis that is associated with increased oxidative stress and neuroinflammation. While the role of NLRP3 inflammasome in mediating neuroinflammation is well-recognized, whether cocaine induces this response remains unexplored. Based on the premise that cocaine induces both reactive oxygen species (ROS) as well as microglial activation, we hypothesized that cocaine-mediated microglial activation involves both ROS and NLRP3 signaling pathways. We examined activation of the NLRP3 pathway in microglia exposed to cocaine, followed by validation in mice administered either cocaine or saline for 7 days, with or without pretreatment with the NLRP3 inhibitor, MCC950, and in postmortem cortical brain tissues of chronic cocaine-dependent humans. We found that microglia exposed to cocaine exhibited significant induction of NLRP3 and mature IL-1ß expression. Intriguingly, blockade of ROS (Tempol) attenuated cocaine-mediated priming of NLRP3 and microglial activation (CD11b). Blockade of NLRP3 by both pharmacological (MCC950) as well as gene silencing (siNLRP3) approaches underpinned the critical role of NLRP3 in cocaine-mediated activation of inflammasome and microglial activation. Pretreatment of mice with MCC950 followed by cocaine administration for 7 days mitigated cocaine-mediated upregulation of mature IL-1ß and CD11b, in both the striatum and the cortical regions. Furthermore, cortical brain tissues of chronic cocaine-dependent humans also exhibited upregulated expression of the NLRP3 pathway mediators compared with non-cocaine dependent controls. Collectively, these findings suggest that cocaine activates microglia involving the NLRP3 inflammasome pathway, thereby contributing to neuroinflammation. NLRP3 can thus be considered as a potential therapeutic target for alleviating cocaine-mediated neuroinflammation.

HIV TAT-mediated microglial senescence: Role of SIRT3-dependent mitochondrial oxidative stress.

The advent of combined antiretroviral treatment (cART) as a treatment for HIV-1 infection has not only resulted in a dramatic decrease in the peripheral viral load but has also led to increased life expectancy of the infected individuals. Paradoxically, increased lifespan is accompanied with higher prevalence of age-related comorbidities, including HIV-associated neurocognitive disorders (HAND). Present study was aimed at exploring the role of HIV TAT protein in mediating microglial mitochondrial oxidative stress, ultimately resulting in neuroinflammation and microglial senescence. Our findings demonstrated that exposure of mouse primary microglial cells (mPMs) to HIV TAT protein resulted in a senescence-like phenotype, that was characterized by elevated expression of both p16 and p21 proteins, increased numbers of senescence-associated-ß-galactosidase positive cells, augmented cell-cycle arrest, increased release of proinflammatory cytokines and decreased telomerase activity. Additionally, exposure of mPMs to HIV TAT also resulted downregulation of SIRT3 with a concomitant increase in mitochondrial oxidative stress. Dual luciferase reporter assay identified miR-505 as a novel target of SIRT3, which was upregulated in mPMs exposed to HIV TAT. Furthermore, transient transfection of mPMs with either the SIRT3 plasmid or miRNA-505 inhibitor upregulated the expression of SIRT3 and mitochondrial antioxidant enzymes, with a concomitant decrease in microglial senescence. These in vitro findings were also validated in the prefrontal cortices and striatum of HIV transgenic rats as well as cART-treated HIV-infected individuals. In summary, this study underscores a yet undiscovered novel mechanism(s) underlying HIV TAT-mediated induction of senescence phenotype in microglia, involving the miR-505-SIRT3 axis-mediated induction of mitochondrial oxidative stress.

N-Acetylcysteine Reverses Antiretroviral-Mediated Microglial Activation by Attenuating Autophagy-Lysosomal Dysfunction.

Successful suppression of viral replication by combined antiretroviral therapy (cART) in HIV-1 infected individuals is paradoxically also accompanied by an increased prevalence of HIV-associated neurocognitive disorders (HAND) in these individuals. HAND is characterized by a state of chronic oxidative stress and inflammation. Microglia are extremely sensitive to a plethora of stimuli, including viral proteins and cART. The current study aimed to assess the effects of cART-mediated oxidative stress on the induction of inflammatory responses in microglia. In the present study, we chose a combination of three commonly used antiretroviral drugs-tenofovir disoproxil fumarate, emtricitabine, and dolutegravir. We demonstrated that exposure of microglia to the chosen cART cocktail induced generation of reactive oxygen species, subsequently leading to lysosomal dysfunction and dysregulated autophagy, ultimately resulting in the activation of microglia. Intriguingly, the potent antioxidant, N-acetylcysteine, reversed the damaging effects of cART. These in vitro findings were further corroborated in vivo wherein cART-treated HIV transgenic (Tg) rats demonstrated increased microglial activation, exaggerated lysosome impairment, and dysregulated autophagy in the prefrontal cortices compared with HIV Tg rats not exposed to cART. Similar to in vitro findings, the treatment of HIV Tg rats with N-acetylcysteine also mitigated the deleterious effects of cART. Taken together, our findings suggest that oxidative stress-mediated lysosomal dysfunction plays a critical role in the pathogenesis of HAND in drug-treated HIV-infected individuals and that antioxidant-mediated mitigation of oxidative stress could thus be considered as an adjunctive therapeutic strategy for ameliorating/dampening some of the neurological complications of HAND.

Opioid-Mediated HIV-1 Immunopathogenesis.

Despite the ability of combination antiretroviral therapy to dramatically suppress viremia, the brain continues to be a reservoir of HIV-1 low-level replication. Adding further complexity to this is the comorbidity of drug abuse with HIV-1 associated neurocognitive disorders and neuroHIV. Among several abused drugs, the use of opiates is highly prevalent in HIV-1 infected individuals, both as an abused drug as well as for pain management. Opioids and their receptors have attained notable attention owing to their ability to modulate immune functions, in turn, impacting disease progression. Various cell culture, animal and human studies have implicated the role of opioids and their receptors in modulating viral replication and virus-mediated pathology both positively and negatively. Further, the combinatorial effects of HIV-1/HIV-1 proteins and morphine have demonstrated activation of inflammatory signaling in the host system. Herein, we summarized the current knowledge on the role of opioids on peripheral immunopathogenesis, viral immunopathogenesis, epigenetic profiles of the host and viral genome, neuropathogenesis of SIV/SHIV-infected non-human primates, blood-brain-barrier, HIV-1 viral latency, and viral rebound. Overall, this review provides recent insights into the role of opioids in HIV-1 immunopathogenesis. Graphical abstract.

Engineered Extracellular Vesicles Loaded With miR-124 Attenuate Cocaine-Mediated Activation of Microglia.

MicroRNA-124 (miR-124), a brain-enriched microRNA, is known to regulate microglial quiescence. Psychostimulants such as cocaine have been shown to activate microglia by downregulating miR-124, leading, in turn, to neuroinflammation. We thus rationalized that restoring the levels of miR-124 could function as a potential therapeutic approach for cocaine-mediated neuroinflammation. Delivering miRNA based drugs in the brain that are effective and less invasive, however, remains a major challenge in the field. Herein we engineered extracellular vesicles (EVs) and loaded them with miR-124 for delivery in the brain. Approach involved co-transfection of mouse dendritic cells with Dicer siRNA and RVG-Lamp2b plasmid to deplete endogenous miRNAs and for targeting the CNS, respectively. Mouse primary microglia (mPm) were treated with purified engineered EVs loaded with either Cy5-miR-124 or Cy5-scrambled miRNA oligos in the presence or absence of cocaine followed by assessing EV uptake and microglial activation. In vivo studies involved pretreating mice intranasally with engineered EVs followed by cocaine injection (20 mg/kg, i.p.). mPm exposed to EV-miR-124 exhibited reduced expression of miR-124 targets - TLR4 and STAT3 as well as ERK-1/2 and Iba1. In cocaine administered mice, EV-Cy5-miR-124 delivered intranasally were detected in the CNS and significantly reduced the expression of inflammatory markers TLR4, MYD88, STAT3 and NF-kB p65 while also downregulating the microglial activation marker, Iba1. Collectively, these findings suggest that engineered EVs can deliver miR-124 into the CNS, thereby alleviating cocaine-mediated microglial activation. Manipulating EV miRNAs can thus be envisioned as an efficient means for delivery of RNA-based therapeutics to target organs.

Role of Inflammasomes in HIV-1 and Drug Abuse Mediated Neuroinflammaging.

Despite the effectiveness of combined antiretroviral therapy (cART) in suppressing virus replication, chronic inflammation remains one of the cardinal features intersecting HIV-1, cART, drug abuse, and likely contributes to the accelerated neurocognitive decline and aging in people living with HIV-1 (PLWH) that abuse drugs. It is also estimated that ~30-60% of PLWH on cART develop cognitive deficits associated with HIV-1-associated neurocognitive disorders (HAND), with symptomatology ranging from asymptomatic to mild, neurocognitive impairments. Adding further complexity to HAND is the comorbidity of drug abuse in PLWH involving activated immune responses and the release of neurotoxins, which, in turn, mediate neuroinflammation. Premature or accelerated aging is another feature of drug abusing PLWH on cART regimes. Emerging studies implicate the role of HIV-1/HIV-1 proteins, cART, and abused drugs in altering the inflammasome signaling in the central nervous system (CNS) cells. It is thus likely that exposure of these cells to HIV-1/HIV-1 proteins, cART, and/or abused drugs could have synergistic/additive effects on the activation of inflammasomes, in turn, leading to exacerbated neuroinflammation, ultimately resulting in premature aging referred to as "inflammaging" In this review, we summarize the current knowledge of inflammasome activation, neuroinflammation, and aging in central nervous system (CNS) cells such as microglia, astrocytes, and neurons in the context of HIV-1 and drug abuse.

Extracellular Vesicles in Viral Infections of the Nervous System.

Almost all types of cells release extracellular vesicles (EVs) into the extracellular space. EVs such as exosomes and microvesicles are membrane-bound vesicles ranging in size from 30 to 1000 nm in diameter. Under normal conditions, EVs mediate cell to cell as well as inter-organ communication via the shuttling of their cargoes which include RNA, DNA and proteins. Under pathological conditions, however, the number, size and content of EVs are found to be altered and have been shown to play crucial roles in disease progression. Emerging studies have demonstrated that EVs are involved in many aspects of viral infection-mediated neurodegenerative diseases. In the current review, we will describe the interactions between EV biogenesis and the release of virus particles while also reviewing the role of EVs in various viral infections, such as HIV-1, HTLV, Zika, CMV, EBV, Hepatitis B and C, JCV, and HSV-1. We will also discuss the potential uses of EVs and their cargoes as biomarkers and therapeutic vehicles for viral infections.

Targeting endoplasmic reticulum stress and autophagy as therapeutic approaches for neurological diseases.

Neurological diseases are multifactorial, devastating diseases that are causative for various neurodegenerative disorders. Emerging evidence points that accumulation of unfolded, misfolded, insoluble, and damaged proteins inside the CNS cells such as microglia, astrocytes, neurons, oligodendrocytes, pericytes, and endothelial cells, leads to endoplasmic reticulum (ER) stress and dysregulated autophagy, which, in turn, sets the stage for ensuing neuropathogenesis. Studies have also demonstrated that chronic ER stress/unfolded protein response (UPR) activates autophagy, and conversely, that blockade of autophagy aggravates ER stress with ensuing cell death, in turn, leading to the development and progression of neurodegeneration. ER stress and autophagy signaling pathways are thus of particular interest as target(s) for pharmacological intervention for the development of therapeutic strategies for various neurological diseases. Herein, we summarized the current knowledge of chronic ER stress/UPR and autophagy signaling pathways and their regulation in CNS cells such as microglia, astrocytes, neurons, oligodendrocytes, pericytes, and endothelial cells. We also reviewed various neurological diseases wherein ER stress/UPR, and autophagy play key roles and also discussed possible pharmacological interventions involving these processes.

Mitigation of cocaine-mediated mitochondrial damage, defective mitophagy and microglial activation by superoxide dismutase mimetics.

Although cocaine exposure has been shown to potentiate neuroinflammation by upregulating glial activation in the brain, the role of mitophagy in this process remains an enigma. In the present study, we sought to examine the role of impaired mitophagy in cocaine-mediated activation of microglia and to determine the ameliorative potential of superoxide dismutase mimetics in this context. Our findings demonstrated that exposure of mouse primary microglial cells (mPMs) to cocaine resulted in decreased mitochondrial membrane potential, that was accompanied by increased expression of mitophagy markers, PINK1 and PRKN. Exposure of microglia to cocaine also resulted in increased expression of DNM1L and OPTN with a concomitant decrease in the rate of mitochondrial oxygen consumption as well as impaired mitochondrial functioning. Additionally, in the presence of cocaine, microglia also exhibited upregulated expression of autophagosome markers, BECN1, MAP1LC3B-II, and SQSTM1. Taken together, these findings suggested diminished mitophagy flux and accumulation of mitophagosomes in the presence of cocaine. These findings were further confirmed by imaging techniques such as transmission electron microscopy and confocal microscopy. Cocaine-mediated activation of microglia was further monitored by assessing the expression of the microglial marker (ITGAM) and the inflammatory cytokine (Tnf, Il1b, and Il6) mRNAs. Pharmacological, as well as gene-silencing approaches aimed at blocking both the autophagy/mitophagy and SIGMAR1 expression, underscored the role of impaired mitophagy in cocaine-mediated activation of microglia. Furthermore, superoxide dismutase mimetics such as TEMPOL and MitoTEMPO were shown to alleviate cocaine-mediated impaired mitophagy as well as microglial activation.Abbreviations: 3-MA: 3-methyladenine; Δψm: mitochondrial membrane potential; ACTB: actin, beta; AIF1: allograft inflammatory factor 1; ATP: adenosine triphosphate; BAF: bafilomycin A1; BECN1: beclin 1, autophagy related; CNS: central nervous system; DNM1L: dynamin 1 like; DMEM: Dulbecco modified Eagle medium; DAPI: 4,6-Diamidino-2-phenylindole; DRD2: dopamine receptor D2; ECAR: extracellular acidification rate; FBS: fetal bovine serum; FCCP: Trifluoromethoxy carbonylcyanide phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IL1B: interleukin 1, beta; IL6: interleukin 6; ITGAM: integrin subunit alpha M; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; mPMs: mouse primary microglial cells; MRC: maximal respiratory capacity; NFKB: nuclear factor kappa B; NLRP3: NLR family pyrin domain containing 3; NTRK2: neurotrophic receptor tyrosine kinase 2; OCR: oxygen consumption rate; OPTN: optineurin; PBS: phosphate buffered saline; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; siRNA: small interfering RNA; SQSTM1: sequestosome 1; TNF: tumor necrosis factor.

Antiretroviral-Mediated Microglial Activation Involves Dysregulated Autophagy and Lysosomal Dysfunction.

In the era of combined antiretroviral therapy (cART), as infected individuals continue to have longer lifespans, there is also an increased prevalence of HIV-associated neurocognitive disorders (HAND). Inflammation is one of the underlying features of HAND, with the role of viral proteins and antiretroviral drugs implicated in this process. Microglia are extremely sensitive to a plethora of stimuli, including viral products and cART. The current study was undertaken to understand the molecular mechanism(s) underlying cART-mediated activation of microglia. Herein we chose a combination of three commonly used drugs, tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and dolutegravir (DTG). We demonstrated that exposure of microglia to this cART cocktail induced lysosomal membrane permeabilization (LMP), which subsequently resulted in impaired lysosomal functioning involving elevated pH and decreased cathepsin D (CTSD) activity. cART exposure of microglia resulted in increased formation of autophagosomes as demonstrated by a time-dependent increase of autophagy markers, with a concomitant defect in the fusion of the lysosomes with the autophagosome. Taken together, our findings suggest a novel mechanism by which cART impairs lysosomal functioning, resulting in dysregulated autophagy and increased neuroinflammation. Interventions aimed at lysosome protection could likely be envisioned as promising therapeutic targets for abrogating cART-mediated microglia activation, which in turn, could thus be considered as adjunctive therapeutics for the treatment of HAND pathogenesis.

Cocaine Induces Inflammatory Gut Milieu by Compromising the Mucosal Barrier Integrity and Altering the Gut Microbiota Colonization.

Cocaine use disorder (CUD), a major health crisis, has traditionally been considered a complication of the CNS; however, it is also closely associated with malnourishment and deteriorating gut health. In light of emerging studies on the potential role of gut microbiota in neurological disorders, we sought to understand the causal association between CUD and gut dysbiosis. Using a comprehensive approach, we confirmed that cocaine administration in mice resulted in alterations of the gut microbiota. Furthermore, cocaine-mediated gut dysbiosis was associated with upregulation of proinflammatory mediators including NF-κB and IL-1ß. In vivo and in vitro analyses confirmed that cocaine altered gut-barrier composition of the tight junction proteins while also impairing epithelial permeability by potentially involving the MAPK/ERK1/2 signaling. Taken together, our findings unravel a causal link between CUD, gut-barrier dysfunction and dysbiosis and set a stage for future development of supplemental strategies for the management of CUD-associated gut complications.