Myosotys Rodriguez

HIV-1 and Morphine Regulation of Autophagy in Microglia: Limited Interactions in the Context of HIV-1 Infection and Opioid Abuse

ABSTRACT Microglia are the predominant resident central nervous system (CNS) cell type productively infected by HIV-1, and play a key role in the progression of HIV-associated dementia (HAD). Moreover, neural dysfunction and progression to HAD are accelerated in opiate drug abusers. In the present study, we examined the role of the autophagy pathway in the neuropathogenesis of HIV-1 using primary human microglial cells and determined whether opiates converge at this point. Infection of microglia with the HIV-1SF162 macrophage-tropic strain resulted in increased Beclin1 expression, accompanied by an increase of LC3 protein levels and accumulation of LC3 reporter RFP+ GFP+ (yellow) puncta, suggesting that HIV-1 infection triggers autophagosome formation without promoting protein degradation by the lysosome. Conversely, coexposure with HIV-1 and morphine significantly decreased virus-induced Beclin1 expression and autophagosome formation. Exploration of the possible mechanism(s) used by morphine to disrupt the autophagic process unveiled a significant increase in intracellular pH, which coincided with a reduction in the formation of acidic vesicular organelles and in autophagolysosome formation. Small interfering RNA targeting BECN1, a gene critical for autophagosome formation, significantly reduced viral replication and the virus-induced inflammatory responses. Conversely, morphine-enhanced viral replication and inflammatory responses were not affected by gene silencing with siBeclin1, suggesting that the interactive effect of morphine in HIV-1 pathogenesis is mediated through a Beclin1-independent mechanism. These novel findings may have important implications on the connections between autophagy and HIV-1 pathogenesis mediated by microglial cells in opioid-abusing individuals. IMPORTANCE About 50% of individuals infected with HIV-1 will develop some sort of neurocognitive impairment that cannot be prevented nor eradicated by antiretroviral therapy. The neuropathogenesis is mostly due to inflammatory responses by infected microglia, the resident immune cells of the brain. Cognitive disorders may also be associated with drugs of abuse. In fact, opioid drug users have an increased risk of developing neurocognitive disorders with increased progression to dementia. Although the mechanism(s) by which opioids exacerbate the neuropathogenesis of HIV-1 are not entirely known, it is well accepted that glia are critical to opiate responses. This study gives us new insight into possible autophagic mechanism(s) in microglia that control HIV-1 replication and virus-induced inflammation in the context of opioid abuse and should greatly improve our knowledge in the pathogenesis of HIV-1 resulting from substance abuse to provide a better understanding for the design of candidate antiviral therapies targeting drug-abusing individuals.

Intranasal drug delivery of small interfering RNA targeting Beclin1 encapsulated with polyethylenimine (PEI) in mouse brain to achieve HIV attenuation

We previously reported that activation of the host autophagic protein, Beclin1, by HIV-1 infection represents an essential mechanism in controlling HIV replication and viral-induced inflammatory responses in microglial cells. Existing antiretroviral therapeutic approaches have been limited in their ability to cross the blood-brain barrier effectively and recognize and selectively eliminate persistent HIV-infected brain reservoirs. In the present study and for the first time, the bio-distribution and efficacy of noninvasive intranasal delivery of small interferingxa0RNA (siRNA) against the Beclin1 gene using the cationic linear polyethylenimines (PEI) as a gene carrier was investigated in adult mouse brain. Fluorescein isothiocyanate (FITC)-labeled control siRNA delivered intranasally was found in the cytoplasm of neurons and glial cells of the prefrontal cortex at 4 and 24u2009hours post-delivery, with no major adverse immune reaction encountered. Intranasal delivery of the siRNA targeting Beclin1 significantly depleted the target protein expression levels in brain tissues with no evidence of toxicity. Binding of siRNA to PEI-polymer was characterized and confirmed by Raman spectroscopy. These results indicate that the intranasal drug delivery allows for the direct delivery of the PEI-siRNA nano-complex to the central nervous system, which could potentially offer an efficient means of gene silencing-mediated therapy in the HIV-infected brain.

Electro-Magnetic Nano-Particle Bound Beclin1 siRNA Crosses the Blood–Brain Barrier to Attenuate the Inflammatory Effects of HIV-1 Infection in Vitro

The purpose of this study was to evaluate a novel drug delivery system comprised of ferric-cobalt electro-magnetic nano-material (CoFe2O4@ BaTiO3; MENP) bound to siRNA targeting Beclin1 (MENP-siBeclin1) to cross the blood–brain barrier (BBB) and attenuate the neurotoxic effects of HIV-1 infection in the central nervous system following on-demand release of siRNA using an in vitro primary human BBB model. Beclin1 is a key protein in the regulation of the autophagy pathway and we have recently demonstrated the importance of Beclin1 in regulating viral replication and viral-induced inflammation in HIV-1-infected microglia. The MENP-siBeclin1 nano-formulation did not compromise the physiological function or integrity of the BBB model. Furthermore, the in vitro BBB data revealed that MENP-siBeclin1 could efficiently attenuate viral replication and viral-induced inflammation, likely due to STAT1/ NF-κB signaling pathways. MENP-siBeclin1 also silenced Beclin1 protein expression in HIV-1-infected microglial cells within the model system. In addition, the cytotoxic effects of direct treatment with siBeclin1 and MENP alone or in nano-formulation on primary human neuronal cells showed a minimal amount of cell death. Overall, the data shows that the nano-formulation can silence the BECN1 gene as an effective mechanism to attenuate HIV-1 replication and viral-induced inflammation in the context of the BBB.

Ibudilast (AV411), and its AV1013 analog, reduce HIV-1 replication and neuronal death induced by HIV-1 and morphine

Objective:We explored the antiviral therapeutic potential of ibudilast (AV411, MN-166) and its amino analog, AV1013. Methods:We analyzed whether Ibudilast, a nonselective cyclic AMP phosphodiesterase inhibitor that has been used clinically in Asia for bronchial asthma, poststroke dizziness, and ocular allergies, and AV1013, attenuate HIV-1 replication and the synergistic interactions seen with opiate abuse–HIV-1 comorbidity in neuronal death and inflammation. Results:AV411 and AV1013 inhibited replication by HIV-1 in microglia and significantly suppressed Tat ± morphine-induced tumor necrosis factor-&agr; and MIF production, the activation of the nuclear factor-kappa B subunit p65, and neuronal death. AV411 and AV1013 prevented HIV-1 replication, and attenuated tumor necrosis factor-&agr; and MIF release at concentrations of 100u200anmol/l and 1u200a&mgr;mol/l, which are likely achievable at clinical doses. More importantly, co-exposure with morphine did not negate the inhibitory actions of AV411. Conclusion:Collectively, our data suggest that AV411 and its amino analog, AV1013, may be useful neuroprotective agents counteracting neurotoxicity caused by infected and activated glia, and implicate them as potential therapies for the management of HIV-associated neurocognitive disorders in an opioid-abusing population.

Mammalian microRNA: an important modulator of host-pathogen interactions in human viral infections

MicroRNAs (miRNAs), which are small non-coding RNAs expressed by almost all metazoans, have key roles in the regulation of cell differentiation, organism development and gene expression. Thousands of miRNAs regulating approximately 60xa0% of the total human genome have been identified. They regulate genetic expression either by direct cleavage or by translational repression of the target mRNAs recognized through partial complementary base pairing. The active and functional unit of miRNA is its complex with Argonaute proteins known as the microRNA-induced silencing complex (miRISC). De-regulated miRNA expression in the human cell may contribute to a diverse group of disorders including cancer, cardiovascular dysfunctions, liver damage, immunological dysfunction, metabolic syndromes and pathogenic infections. Current day studies have revealed that miRNAs are indeed a pivotal component of host-pathogen interactions and host immune responses toward microorganisms. miRNA is emerging as a tool for genetic study, therapeutic development and diagnosis for human pathogenic infections caused by viruses, bacteria, parasites and fungi. Many pathogens can exploit the host miRNA system for their own benefit such as surviving inside the host cell, replication, pathogenesis and bypassing some host immune barriers, while some express pathogen-encoded miRNA inside the host contributing to their replication, survival and/or latency. In this review, we discuss the role and significance of miRNA in relation to some pathogenic viruses.

Differing roles of autophagy in HIV-associated neurocognitive impairment and encephalitis with implications for morphine co-exposure.

We investigated the role of autophagy in HIV-infected subjects with neurocognitive impairment (NCI) ± HIV encephalitis (HIVE), many of which had a history of polysubstance abuse/dependence, using post-mortem brain tissues to determine whether differences in autophagy related factors may be more associated with NCI or NCI-encephalitis. Using qRT-PCR, we detected significant differences in gene expression levels with SQSTM1, LAMP1 higher in HIV-infected subjects without NCI while ATG5, SQSTM1 were then lower in HIV infection/NCI and ATG7, SQSTM1 being higher in NCI-HIVE. Immunohistochemical labeling of these autophagy associated proteins (also including Beclin 1 and LC3B) in Iba1-positive microglial cells showed generally higher immunoreactivity in the NCI and NCI-HIVE groups with more focal vs. diffuse patterns of expression in the NCI-HIVE group. Furthermore, analysis of microarray data from these same subjects found significantly higher levels of LAMP1 in NCI-HIVE compared to uninfected subjects in the basal ganglia. Finally, we tested the effect of supernatant from HIV-1-infected microglia and HIV-1 Tat protein in combination with morphine on neurons in vitro and found opposing events with both significant inhibition of autophagic flux and reduced dendrite length for morphine and supernatant treatment while Tat and morphine exposure resulted in lower autophagic activity at an earlier time point and higher levels in the later. These results suggest autophagy genes and their corresponding proteins may be differentially regulated at the transcriptional, translational, and post-translational levels in the brain during various stages of the HIV disease and that infected individuals exposed to morphine can experience mixed signaling of autophagic activity which could lead to more severe NCI than those without opioid use.

Opiate Addiction Therapies and HIV-1 Tat: Interactive Effects on Glial [Ca 2+ ] i , Oxyradical and Neuroinflammatory Chemokine Production and Correlative Neurotoxicity

Few preclinical studies have compared the relative therapeutic efficacy of medications used to treat opiate addiction in relation to neuroAIDS. Here we compare the ability of methadone and buprenorphine, and the prototypic opiate morphine, to potentiate the neurotoxic and proinflammatory ([Ca2+]i, ROS, H2O2, chemokines) effects of HIV-1 Tat in neuronal and/or mixed-glial co-cultures. Repeated observations of neurons during 48 h exposure to combinations of Tat, equimolar concentrations (500 nM) of morphine, methadone, or buprenorphine exacerbated neurotoxicity significantly above levels seen with Tat alone. Buprenorphine alone displayed marked neurotoxicity at 500 nM, prompting additional studies of its neurotoxic effects at 5 nM and 50 nM concentrations ± Tat. In combination with Tat, buprenorphine displayed paradoxical, concentration-dependent, neurotoxic and neuroprotective actions. Buprenorphine neurotoxicity coincided with marked elevations in [Ca2+]i, but not increases in glial ROS or chemokine release. Tat by itself elevated the production of CCL5/RANTES, CCL4/MIP-1β, and CCL2/MCP-1. Methadone and buprenorphine alone had no effect, but methadone interacted with Tat to further increase production of CCL5/RANTES. In combination with Tat, all drugs significantly increased glial [Ca2+]i, but ROS was only significantly increased by co-exposure with morphine. Taken together, the increases in glial [Ca2+]i, ROS, and neuroinflammatory chemokines were not especially accurate predictors of neurotoxicity. Despite similarities, opiates displayed differences in their neurotoxic and neuroinflammatory interactions with Tat. Buprenorphine, in particular, was partially neuroprotective at a low concentration, which may result from its unique pharmacological profile at multiple opioid receptors. Overall, the results reveal differences among addiction medications that may impact neuroAIDS.

Interplay between Autophagy, Exosomes and HIV-1 Associated Neurological Disorders: New Insights for Diagnosis and Therapeutic Applications

The autophagy–lysosomal pathway mediates a degradative process critical in the maintenance of cellular homeostasis as well as the preservation of proper organelle function by selective removal of damaged proteins and organelles. In some situations, cells remove unwanted or damaged proteins and RNAs through the release to the extracellular environment of exosomes. Since exosomes can be transferred from one cell to another, secretion of unwanted material to the extracellular environment in exosomes may have an impact, which can be beneficial or detrimental, in neighboring cells. Exosome secretion is under the influence of the autophagic system, and stimulation of autophagy can inhibit exosomal release and vice versa. Neurons are particularly vulnerable to degeneration, especially as the brain ages, and studies indicate that imbalances in genes regulating autophagy are a common feature of many neurodegenerative diseases. Cognitive and motor disease associated with severe dementia and neuronal damage is well-documented in the brains of HIV-infected individuals. Neurodegeneration seen in the brain in HIV-1 infection is associated with dysregulation of neuronal autophagy. In this paradigm, we herein provide an overview on the role of autophagy in HIV-associated neurodegenerative disease, focusing particularly on the effect of autophagy modulation on exosomal release of HIV particles and how this interplay impacts HIV infection in the brain. Specific autophagy–regulating agents are being considered for therapeutic treatment and prevention of a broad range of human diseases. Various therapeutic strategies for modulating specific stages of autophagy and the current state of drug development for this purpose are also evaluated.

Importance of Autophagy in Mediating Human Immunodeficiency Virus (HIV) and Morphine-Induced Metabolic Dysfunction and Inflammation in Human Astrocytes

Under physiological conditions, the function of astrocytes in providing brain metabolic support is compromised under pathophysiological conditions caused by human immunodeficiency virus (HIV) and opioids. Herein, we examined the role of autophagy, a lysosomal degradation pathway important for cellular homeostasis and survival, as a potential regulatory mechanism during pathophysiological conditions in primary human astrocytes. Blocking autophagy with small interfering RNA (siRNA) targeting BECN1, but not the Autophagy-related 5 (ATG5) gene, caused a significant decrease in HIV and morphine-induced intracellular calcium release. On the contrary, inducing autophagy pharmacologically with rapamycin further enhanced calcium release and significantly reverted HIV and morphine-decreased glutamate uptake. Furthermore, siBeclin1 caused an increase in HIV-induced nitric oxide (NO) release, while viral-induced NO in astrocytes exposed to rapamycin was decreased. HIV replication was significantly attenuated in astrocytes transfected with siRNA while significantly induced in astrocytes exposed to rapamycin. Silencing with siBeclin1, but not siATG5, caused a significant decrease in HIV and morphine-induced interleukin (IL)-8 and tumor necrosis factor alpha (TNF-α) release, while secretion of IL-8 was significantly induced with rapamycin. Mechanistically, the effects of siBeclin1 in decreasing HIV-induced calcium release, viral replication, and viral-induced cytokine secretion were associated with a decrease in activation of the nuclear factor kappa B (NF-κB) pathway.

β-Adrenergic receptor gene expression in HIV-associated neurocognitive impairment and encephalitis: implications for MOR-1K subcellular localization

We previously reported that mRNA expression of the unique alternatively spliced OPRM1 isoform μ-opioid receptor-1K (MOR-1K), which exhibits excitatory cellular signaling, is elevated in HIV-infected individuals with combined neurocognitive impairment (NCI) and HIV encephalitis (HIVE). It has recently been shown that the β2-adrenergic receptor (β2-AR) chaperones MOR-1K, normally localized intracellularly, to the cell surface. Here, we found mRNA expression of the adrenoceptor beta 2 (ADRB2) gene is also elevated in NCI-HIVE individuals, as well as that β2-AR protein expression is elevated in HIV-1-infected primary human astrocytes treated with morphine, and discuss the implications for MOR-1K subcellular localization in this condition.