HIV‐1, Drug Addiction, and Autophagy

Despite the dramatic success of combined antiretroviral therapies (cART) in controlling peripheral virus replication, the prevalence of HIV‐1‐associated neurocognitive disorders (HAND) is on a rise as infected individuals continue to live longer. Almost half of the infected individuals on ART develop HAND, out of which at least 30% suffer from a comorbid condition of substance abuse. Involvement of autophagy has been implicated not only in HIV‐1 infection of the CNS but also in CNS cells exposed to drugs such as amphetamine, opiates, and cocaine, contributing in turn, to cellular dysfunction. HIV‐1 is known to interfere with the autophagy pathway, resulting in turn to upregulation of HIV‐1 replication. Specifically, different HIV‐1 proteins such as TAT, gp120, and Nef have been shown to act on various stages of autophagy such as initiation and maturation and to affect overall autophagy levels. Whether or not abused drugs and HIV‐1 can cooperate to dysregulate autophagy, however, remains unclear. This chapter is focused on identifying the molecular mechanism(s) underlying HIV‐1 (proteins) and cocaine, opiate, methamphetamine‐mediated impairment of autophagy. Such effects could underlie the synergistic effects of HIV‐1 and abused drugs in exacerbating symptoms of HAND

Alcohol in dementia: Implications of Alzheimer’s like pathology in alcohol abuse

Alcohol use is widespread, with 85.6 percent of people ages 18 and older reported drinking alcohol in their lifetime in 2019. Mild to heavy alcohol use is associated with multi-organ dysfunction at the cellular and genetic levels, as well as epigenetic modifications, leading to liver and brain damage and dementia. The role of astrocytes as contributors to Alzheimer’s like pathology associated with cognitive decline in opiate abusers and people with HIV-associated neurological disorders (HAND) has been recently reported from our group. We hypothesize that alcohol could also induce astrocytic amyloidosis. In this study we demonstrated that exposure of human primary astrocytes (HPA) to ethanol resulted in a dose dependent (6.25-200 mM) increase in AD markers-amyloid precursor protein (APP), Aβ 1-42, β-site cleaving enzyme (BACE1), as well as the inflammatory marker IL1βand lncRNA BACE1AS. Next, we demonstrated a time-dependent (0-96h, 12.5 mM) upregulation of AD markers, oxidative stress (4-HNE), alcohol metabolizing enzymes alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH2), and cytochrome P450 2E1 (CYP2E1), as well as proinflammatory cytokines (TNF-α, IL1β, IL6) in HPAs exposed to alcohol. Gene silencing approaches confirmed the regulatory role of lncRNA BACE1-AS in amyloidosis and its interaction with alcohol metabolic pathways leading to neuroinflammation and oxidative stress. Further, in vivo study validated our in vitro findings, demonstrating up-regulation of APP, Aβ1-42, 4-HNE and IL1β in the cortices of ethanol-fed mice (4- weeks, ad libidum) compared to saline controls. This is the first report implicating the role of lncRNA BACE-AS in alcohol-mediated induction of astrocytic amyloidosis, leading to neuroinflammation and oxidative stress, which, in turn, could contribute to cognitive impairments. These findings set the stage for future development of therapeutic strategies aimed at targeting cognitive deficits in alcohol users and abusers.https://digitalcommons.unmc.edu/surp2021/1047/thumbnail.jp

Cationic Surface Modification of PLG Nanoparticles Offers Sustained Gene Delivery to Pulmonary Epithelial Cells

Biodegradable polymeric nanoparticles are currently being explored as a nonviral gene delivery system; however, many obstacles impede the translation of these nanomaterials. For example, nanoparticles delivered systemically are inherently prone to adsorbing serum proteins and agglomerating as a result of their large surface/volume ratio. What is desired is a simple procedure to prepare nanoparticles that may be delivered locally and exhibit minimal toxicity while improving entry into cells for effectively delivering DNA. The objective of this study was to optimize the formulation of poly(D,L-lactide-co-glycolide) (PLG) nanoparticles for gene delivery performance to a model of the pulmonary epithelium. Using a simple solvent diffusion technique, the chemistry of the particle surface was varied by using different coating materials that adsorb to the particle surface during formation. A variety of cationic coating materials were studied and compared to more conventional surfactants used for PLG nanoparticle fabrication. Nanoparticles (~200 nm) efficiently encapsulated plasmids encoding for luciferase (80–90%) and slowly released the same for 2 weeks. In A549 alveolar lung epithelial cells, high levels of gene expression appeared at day 5 for certain positively charged PLG particles and gene expression was maintained for at least 2 weeks. In contrast, PEI gene expression ended at day 5. PLG particles were also significantly less cytotoxic than PEI suggesting the use of these vehicles for localized, sustained gene delivery to the pulmonary epithelium

Cocaine-mediated induction of microglial activation involves the ER stress-TLR2 axis

Effects of PBN (ROS scavenger) and APO (NADPH oxidase inhibitor) on cocaine-induced ROS formation in BV2 cells. (A) Green fluorescence was observed using a Zeiss Observer Z1 inverted microscope (Carl Zeiss MicroImaging). It revealed that PBN and APO resulted in significant inhibition of cocaine-mediated induction of ROS production. (JPG 7600 kb

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

Hypoxia-inducible factor-1 α/platelet derived growth factor axis in HIV-associated pulmonary vascular remodeling

<p>Abstract</p> <p>Background</p> <p>Human immunodeficiency virus (HIV) infected patients are at increased risk for the development of pulmonary arterial hypertension (PAH). Recent reports have demonstrated that HIV associated viral proteins induce reactive oxygen species (ROS) with resultant endothelial cell dysfunction and related vascular injury. In this study, we explored the impact of HIV protein induced oxidative stress on production of hypoxia inducible factor (HIF)-1α and platelet-derived growth factor (PDGF), critical mediators implicated in the pathogenesis of HIV-PAH.</p> <p>Methods</p> <p>The lungs from 4-5 months old HIV-1 transgenic (Tg) rats were assessed for the presence of pulmonary vascular remodeling and HIF-1α/PDGF-BB expression in comparison with wild type controls. Human primary pulmonary arterial endothelial cells (HPAEC) were treated with HIV-associated proteins in the presence or absence of pretreatment with antioxidants, for 24 hrs followed by estimation of ROS levels and western blot analysis of HIF-1α or PDGF-BB.</p> <p>Results</p> <p>HIV-Tg rats, a model with marked viral protein induced vascular oxidative stress in the absence of active HIV-1 replication demonstrated significant medial thickening of pulmonary vessels and increased right ventricular mass compared to wild-type controls, with increased expression of HIF-1α and PDGF-BB in HIV-Tg rats. The up-regulation of both HIF-1α and PDGF-B chain mRNA in each HIV-Tg rat was directly correlated with an increase in right ventricular/left ventricular+septum ratio. Supporting our <it>in-viv</it>o findings, HPAECs treated with HIV-proteins: Tat and gp120, demonstrated increased ROS and parallel increase of PDGF-BB expression with the maximum induction observed on treatment with R5 type gp-120_CM. Pre-treatment of endothelial cells with antioxidants or transfection of cells with HIF-1α small interfering RNA resulted in abrogation of gp-120_CMmediated induction of PDGF-BB, therefore, confirming that ROS generation and activation of HIF-1α plays critical role in gp120 mediated up-regulation of PDGF-BB.</p> <p>Conclusion</p> <p>In summary, these findings indicate that viral protein induced oxidative stress results in HIF-1α dependent up-regulation of PDGF-BB and suggests the possible involvement of this pathway in the development of HIV-PAH.</p

Immunoprophylaxis against AIDS in macaques with a lentiviral DNA vaccine

AbstractWe earlier reported that immunization of macaques with a reverse transcriptase-deleted SHIVKU2 (ΔrtSHIVKU2) plasmid that contained HIV-1(HXB2) env and SIV gag–nef induced protection against AIDS caused by challenge virus SHIV89.6P with a heterologous env. We further deleted vif and integrase from ΔrtSHIVKU2 and substituted the 3′LTR with SV40 poly A sequences, creating Δ4SHIVKU2 (M) and a parallel construct containing gag–nef of HIV-1SF2, Δ4SHIVKU2 (H). Six macaques received two intramuscular injections of the (M) DNA, and another six received three injections of the (H) DNA. Three of the latter group received two post-challenge boosts with (M) DNA vaccine. Seven virus control macaques were inoculated with SHIV89.6P. All twelve immunized macaques were challenged with SHIV89.6P virus, and CMI responses were measured by ELISPOT assays.Virus control animals all developed progressive infection, whereas vaccinated macaques from both groups controlled virus replication, with plasma viral loads dropping to undetectable levels between weeks 6 and 126 p.i. This DNA vaccine was efficacious even though it encoded Env, Gag, and Nef that were genetically distinct from the proteins in the challenge virus. The DNA vaccine induced broad-based protection without using viral proteins to boost the immunity

Neuronal-Derived Extracellular Vesicles are Enriched in the Brain and Serum of HIV-1 Transgenic Rats

Despite the efficacy of combination antiretroviral therapy (ART) in controlling human immunodeficiency virus (HIV-1) replication, cytotoxic viral proteins such as HIV-1 transactivator of transcription (Tat) persist in tissues such as the brain. Although HIV-1 does not infect neuronal cells, it is susceptible to viral Tat protein-mediated toxicity, leading to neuroinflammation that underlies HIV-associated neurocognitive disorders (HAND). Given the role of extracellular vesicles (EVs) in both cellular homoeostasis and under pathological conditions, we sought to investigate the alterations in the quantity of neuronal-derived EVs in the brain–as defined by the presence of cell adhesion molecule L1 (L1CAM) and to evaluate the presence of L1CAM+ EVs in the peripheral circulation of HIV-1 transgenic (HIV-1 Tg) rats. The primary goal of this study was to investigate the effect of long-term exposure of HIV-1 viral proteins on the release of neuronal EVs in the brain and their transfer in the systemic compartment. Brain and serum EVs were isolated from both wild type and HIV-1 Tg rats using differential ultracentrifugation with further purification using the Optiprep gradient method. The subpopulation of neuronal EVs was further enriched using immunoprecipitation. The current findings demonstrated increased presence of L1CAM+ neuronal-derived EVs both in the brain and serum of HIV-1 Tg rats. © 2019, © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles

MiR-9 promotes microglial activation by targeting MCPIP1

Microglia participate in innate inflammatory responses within the central nervous system. The highly conserved microRNA-9 (miR-9) plays critical roles in neurogenesis as well as axonal extension. Its role in microglial inflammatory responses, however, remains poorly understood. Here we identify a unique role of miR-9 in mediating the microglial inflammatory response via distinct signalling pathways. MiR-9-mediated regulation of cellular activation involved downregulated expression of the target protein, monocyte chemotactic protein-induced protein 1 (MCPIP1) that is crucial for controlling inflammation. Results indicate that miR-9-mediated cellular activation involved signalling via the NF-kappa B pathway, but not the beta-catenin pathway

Astrocyte EV-Induced lincRNA-Cox2 Regulates Microglial Phagocytosis: Implications for Morphine-Mediated Neurodegeneration

Impairment of microglial functions, such as phagocytosis and/ or dysregulation of immune responses, has been implicated as an underlying factor involved in the pathogenesis of various neurodegenerative disorders. Our previous studies have demonstrated that long intergenic noncoding RNA (lincRNA)-Cox2 expression is influenced by nuclear factor kB (NF-kB) signaling and serves as a coactivator of transcriptional factors to regulate the expression of a vast array of immune- related genes in microglia. Extracellular vesicles (EVs) have been recognized as primary facilitators of cell-to-cell communication and cellular regulation. Herein, we show that EVs derived from astrocytes exposed to morphine can be taken up by microglial endosomes, leading, in turn, to activation of Toll-like receptor 7 (TLR7) with a subsequent upregulation of lincRNA-Cox2 expression, ultimately resulting in impaired microglial phagocytosis. This was further validated in vivo, wherein inhibition of microglial phagocytic activity was also observed in brain slices isolated from morphine-administrated mice compared with control mice. Additionally, we also showed that intranasal delivery of EVs containing lincRNA-Cox2 siRNA (small interfering RNA) was able to restore microglial phagocytic activity in mice administered morphine. These findings have ramifications for the development of EV-loaded RNA-based therapeutics for the treatment of various disorders involving functional impairment of microglia