HIV Neuropathology Research Articles

HIV-1 and opiates modulate miRNA profiles in extracellular vesicles.

Opiate abuse increases the risk of HIV transmission and exacerbates HIV neuropathology by increasing inflammation and modulating immune cell function. Exosomal EVs(xEV) contain miRNAs that may be differentially expressed due to HIV infection or opiate abuse. Here we develop a preliminary exosomal-miRNA biomarker profile of HIV-infected PBMCs in the context of opiate use. PBMCs infected with HIV were treated with increasing dosages of morphine for 72 hours, the culture supernatants were collected, and the exosomes isolated using differential centrifugation. Exosomal miRNAs were extracted, expression levels determined via Nanostring multiplexed microRNA arrays, and analyzed with Webgestalt. The effect of the exosomes on neuronal function was determined by measuring calcium. Preliminary findings show that HIV-1 infection altered the miRNA profile of PBMC-derived EVs concurrently with opiate exposure. MicroRNA, hsa-miR-1246 was up-regulated 12-fold in the presence of morphine, relative to uninfected control. PBMCs infected with HIV-1 MN, an X4-tropic HIV-1 strain and exposed to morphine, displayed a trend which suggests potential synergistic effects between HIV-1 infection and morphine exposure promoting an increase in viral replication. Dose-dependent differences were observed in miRNA expression as a result of opiate exposure. The xEVs derived from PBMCs exposed to morphine or HIV modulated neuronal cell function. SH-SY5Y cells, treated with xEVs derived from ART-treated PBMCs, exhibited increased viability while for SH-SY5Ys exposed to xEVs derived from HIV-1 infected PBMCs viability was decreased compared to the untreated control. Exposing SH-SY5Y to xEVs derived from HIV-infected PBMCs resulted in significant decrease in calcium signaling, relative to treatment with xEVs derived from uninfected PBMCs. Overall, HIV-1 and morphine induced differential miRNA expression in PBMC-derived exosomes, potentially identifying mechanisms of action or novel therapeutic targets involved in opiate use disorder, HIV neuropathology, TNF signaling pathway, NF-κB signaling pathway, autophagy, and apoptosis in context of HIV infection.

Elevated Galectin-9 across the human brain correlates with HIV neuropathology and detrimental cognitive states.

HIV persistence and neuroinflammation are known to contribute to HIV-associated neuropathology. However, the multifaceted pathways driving impairment remain poorly understood. Galectin-glycan interactions have emerged as significant contributors to neuroinflammatory processes and may play a role in neuroHIV. Here, we quantified Galectin-9 (Gal-9), a pleiotropic immunomodulatory protein, in post-mortem brain tissue across multiple regions from HIV-infected and HIV-uninfected donors to determine causal associations with HIV brain injury. We demonstrate that the staining intensity, total staining area, and cell-associated frequency of Gal-9 were elevated, principally in the frontal lobe and basal ganglia. Higher frontal lobe Gal-9 levels correlated with lower pre-mortem neuropsychological performance test scores in areas of attention and motor skills. Our results suggest that Gal-9 activity across the brain plays a role in neuroHIV pathogenesis and constitutes a promising disease-modifying target.

Neuroinflammatory Changes in Relation to Cerebrospinal Fluid Viral Load in Simian Immunodeficiency Virus Encephalitis.

The exact cause of neurocognitive dysfunction in HIV-positive patients despite successful control of the infection in the periphery is not completely understood. One suggested mechanism is a vicious cycle of microglial activation and release of proinflammatory chemokines/cytokines that eventually leads to neuronal loss and dysfunction. However, the exact role of microglial activation in the earliest stages of the infection with high cerebrospinal fluid (CSF) viral loads (VL) is unclear. In this study, we imaged the translocator protein (TSPO), a mitochondrial membrane receptor known to be upregulated in activated microglia and macrophages, in rhesus macaques before and multiple times after inoculation with a neurotropic simian immunodeficiency virus (SIV) strain (SIVsm804E), using 18F-DPA714 positron emission tomography (PET). The whole-brain standardized uptake values of TSPO at equilibrium reflecting total binding (SUVT) and binding potentials (BPND) were calculated and correlated with CSF and serum markers of disease, and a corresponding postmortem immunostaining analysis was also performed. SUVT was found to be inversely correlated with both CSF VL and monocyte chemoattractant protein 1 (MCP-1) levels. In SIV-infected macaques with very high CSF VL at necropsy (>106 copies/ml), we found decreased TSPO binding by PET, and this was supported by immunostaining which showed glial and neuronal apoptosis rather than microglial activation. On the other hand, with only moderately elevated CSF VL (∼104 copies/ml), we found increased TSPO binding as well as focal and diffuse microglial activation on immunostaining. Our results in the SIV-infected macaque model provide insights into the relationship between HIV neuropathology and CSF VL at various stages of the disease.IMPORTANCE Neurological and cognitive problems are a common complication of HIV infection and are prevalent even in treated individuals. Although the molecular processes underlying brain involvement with HIV are not completely understood, inflammation is suspected to play a significant role. Our work presents an in vivo assessment of neuroinflammation in an animal model of HIV, the simian immunodeficiency virus (SIV)-infected rhesus macaque. Using positron emission tomography (PET) imaging, we identified changes in brain inflammation after inoculation with SIV over time. Interestingly, we found decreased binding of the PET ligand in the presence of very high cerebrospinal fluid (CSF) viral loads. These findings were supported by immunostaining which showed marked glial loss instead of inflammation. This study provides insight into glial and neuronal changes associated with very high CSF viral load and could reflect similar changes occurring in HIV-infected patients.

Decreased glial and synaptic glutamate uptake in the striatum of HIV-1 gp120 transgenic mice

The mechanisms leading to the neurocognitive deficits in humans with immunodeficiency virus type 1 (HIV-1) are not well resolved. A number of cell culture models have demonstrated that the HIV-envelope glycoprotein 120 (gp120) decreases the reuptake of glutamate, which is necessary for learning, memory, and synaptic plasticity. However, the impact of brain HIV-1 gp120 on glutamate uptake systems in vivo remains unknown. Notably, alterations in brain glutamate uptake systems are implicated in a number of neurodegenerative and neurocognitive disorders. We characterized the kinetic properties of system XAG (sodium-dependent) and systems xc- (sodium-independent) [3H]-L-glutamate uptake in the striatum and hippocampus of HIV-1 gp120 transgenic mice, an established model of HIV neuropathology. We determined the kinetic constant Vmax (maximal velocity) and Km (affinity) of both systems XAG and xc- using subcellular preparations derived from neurons and glial cells. We show significant (30-35%) reductions in the Vmax of systems XAG and xc- in both neuronal and glial preparations derived from the striatum, but not from the hippocampus of gp120 mice relative to wild-type (WT) controls. Moreover, immunoblot analysis showed that the protein expression of glutamate transporter subtype-1 (GLT-1), the predominant brain glutamate transporter, was significantly reduced in the striatum but not in the hippocampus of gp120 mice. These extensive and region-specific deficits of glutamate uptake likely contribute to the development and/or severity of HIV-associated neurocognitive disorders. Understanding the role of striatal glutamate uptake systems in HIV-1 gp120 may advance the development of new therapeutic strategies to prevent neuronal damage and improve cognitive function in HIV patients.

Increased in vitro glial fibrillary acidic protein expression, telomerase activity, and telomere length after productive human immunodeficiency virus‐1 infection in murine astrocytes

Although HIV-associated neurocognitive disorders (HAND) result from injury and loss of neurons, productive infection routinely takes place in cells of macrophage lineage. In such a complex context, astrocytosis induced by local chemokines/cytokines is one of the hallmarks of HIV neuropathology. Whether this sustained astrocyte activation is able to alter telomere-aging process is unknown. We hypothesized that interaction of HIV with astrocytes may impact astrocyte telomerase activity (TA) and telomere length in a scenario of astrocytic activation measured by expression of glial fibrillary acidic protein (GFAP). To test this hypothesis, cultured murine astrocytes were challenged with pseudotyped HIV/vesicular stomatitis virus (HIV/VSV) to circumvent the absence of viral receptors; and GFAP, telomerase activity, and telomere length were quantified. As an early and transient event after HIV infection, both TA activity and telomere length were significantly augmented (P < 0.001). Later, a strong negative correlation (-0.8616, P < 0.0001) between virus production and telomerase activity was demonstrated. Once HIV production had reached a peak (7 dpi), the TA decreased, showing levels similar to those of noninfected cells. In contrast, the astrocyte became activated, exhibiting significantly increased levels of GFAP expression directly related to the level of HIV/VSV replication (P < 0.0001). Our results suggest that HIV-infected astrocytes exhibit early disturbance in their cellular functions, such as telomerase activity and telomere length, that may attenuate cell proliferation and enhance the astrocyte dysregulation, contributing to HIV neuropathogenesis. Understanding the mechanisms involved in HIV-mediated persistence by altering the telomere-related aging processes could aid in the development of therapeutic modalities for neurological complications of HIV infection.

Human immunodeficiency virus-associated depression: contributions of immuno-inflammatory, monoaminergic, neurodegenerative, and neurotrophic pathways

In the era of greatly improved pharmacological treatment of HIV infection through highly active antiretroviral therapy (HAART), HIV patients experience reduced viral loads, reduced opportunistic infections, increased CD4+ T cell count, and greater life expectancy. Although life expectancy is increased, patients often develop neurological disturbances that may persist for long periods, seriously jeopardizing quality of life and adherence to the medication protocols of HAART. For these reasons, HIV-associated neurological disorders have gained importance in both clinical and basic investigations of HIV infection. Depression is the most prevalent neuropsychiatric disorder among people living with HIV. In this review, we discuss how HIV can predispose infected individuals to depression by several interrelated mechanisms. These include inducing chronic elevation of cytokines through activation of microglia and astrocytes; decreasing monoaminergic function; inducing neurotoxicity, especially in dopaminergic neurons; and reducing brain-derived neurotrophic factor. These viral pathways interact with psychosocial factors to create the depressive state. HIV depression has a great impact on quality of life and implementation of antiretroviral therapy, and thus, recognition of these modes of action is significant for understanding HIV neuropathology and for selecting modalities for pharmacologic treatment.

Antiretroviral Monocyte Efficacy Score Linked to Cognitive Impairment in Hiv

Monocytes transmigrating to the brain play a central role in HIV neuropathology. We hypothesized that the continued existence of neurocognitive impairment (NCI) despite potent antiretroviral (ARV) therapy is mediated by the inability of such therapy to control this monocyte/macrophage reservoir. Cross-sectional and longitudinal analyses were conducted within a prospectively enrolled cohort. We devised a monocyte efficacy (ME) score based on the anticipated effectiveness of ARV medications against monocytes/macrophages using published macrophage in vitro drug efficacy data. We examined, within an HIV neurocognitive database, its association with composite neuropsychological test scores (NPZ8) and clinical cognitive diagnoses among subjects on stable ARV medications unchanged for >6 months prior to assessment. Among 139 subjects on ARV therapy, higher ME score correlated with better NPZ8 performance (r=0.23, P<0.01), whereas a score devised to quantify expected penetration effectiveness of ARVs into the brain (CPE score) did not (r=0.12, P=0.15). In an adjusted model (adjusted r(2)=0.12), ME score (β=0.003, P=0.02), CD4(+) T-cell nadir (β=0.001, P<0.01) and gender (β=-0.456, P=0.02) were associated with NPZ8, whereas CPE score was not (β=0.003, P=0.94). A higher ME score was associated with better clinical cognitive status (P<0.01). With a range of 12.5-433.0 units, a 100-unit increase in ME score resulted in a 10.6-fold decrease in the odds of a dementia diagnosis compared with normal cognition (P=0.01). ARV efficacy against monocytes/macrophages correlates with cognitive function in HIV-infected individuals on ARV therapy within this cohort. If validated, efficacy against monocytes/macrophages may provide a new target to improve HIV NCI.

Lentiviral vector expressing Nef as a tool to study HIV neuropathology

The goal of this work is to use a lentivirus to create a simplified endogeonous expression system producing only Nef to study the unique contributions that this viral neurotoxin makes to HIV‐related neurological impairment. We used PCR to generate the Nef gene flanked by unique restriction enzymes sites(BamH1 and Sal1). Then we digested a lentiviral packaging vector (pLenti) and this PCR product with the same restriction enzymes to generate DNA fragments with complementary ends. Cloning was performed by incubating with DNA ligase and ATP to create recombinant pLenti‐Nef plasmids. Colonies were screened by PCR to identify those containing plasmids with HIV Nef. These pLenti‐Nef plasmids were recovered by plasmid prep and confirmed by sequencing. The pLenti‐Nef plasmid can be used to generate lentiviral particles capable of delivering the HIV‐Nef gene to target cells in a single round of infection a single round of infection. We will infect rat astrocytes in vitro with these viruses followed by implantation into the hippocampus of Sprague Dawley rats in vivo to mimic the levels of Nef that have been reported in HIV‐infected patients. This investigation will help us understand the HIV disease better at a molecular level by developing a system that allows us to study the role of Nef in driving immune cell chemotaxis into the brain and the loss of neuron function that accompanies HIV neuropathology in parallel. Grant support by RR003050 and RR016470.

Regional Heterogeneity and Diversity in Cytokine and Chemokine Production by Astroglia: Differential Responses to HIV-1 Tat, gp120, and Morphine Revealed by Multiplex Analysis

HIV-infected individuals who abuse opiates show a faster progression to AIDS and higher incidence of encephalitis. The HIV-1 proteins Tat and gp120 have been shown to cause neurodegenerative changes either in vitro or when injected or expressed in the CNS, and we have shown that opiate drugs can exacerbate neurotoxic effects in the striatum through direct actions on pharmacologically discrete subpopulations of mu-opioid receptor-expressing astroglia. Opiate coexposure also significantly enhances release of specific inflammatory mediators by astroglia from the striatum, and we theorize that astroglial reactivity may underlie aspects of HIV neuropathology. To determine whether astroglia from different regions of the central nervous system have distinct, intrinsic responses to HIV-1 proteins and opiates, we used multiplex suspension array analyses to define and compare the inflammatory signature of cytokines released by murine astrocytes grown from cerebral cortex, cerebellum, and spinal cord. Results demonstrate significant regional differences in baseline secretion patterns, and in responses to viral proteins. Of importance for the disease process, astrocytes from all regions have very limited inflammatory response to gp120 protein, as compared to Tat protein, either in the presence or absence of morphine. Overall, the chemokine/cytokine release is higher from spinal cord and cortical astroglia than from cerebellar astroglia, paralleling the relatively low incidence of HIV-related neuropathology in the cerebellum.

Affinity (tropism) of caprine arthritis encephalitis virus for brain cells

One of the constraints in unraveling the mysteries blurring the advancement of research in the quest to totally put HIV problems under control is getting the appropriate animal model that would truly simulate human cases. This problem is more apparent in studies involving the central nervous system. Consequently, a viable animal model to generate information for the production of drugs and vaccines for the prevention and or control of lentiviral induced dementia in affected host animals is pertinent and vital. In this study, explant cultures prepared from the brain of new-born goat-kid were infected with Caprine Arthritis Encephalitis (CAE) virus- a retrovirus affecting goats. The specific brain cell types infected by the (CAE) virus were determined using reverse-transcription polymerase chain reaction (RT-PCR) and transmission electron microscopy (TEM techniques). TEM showed that in 85 – 90% cases, microglia were the cells specifically infected by the virus. Amplification of the genomic sequence of the envelope and the gag genes by RT-PCR confirmed the presence of CAEV proviral DNA in the brain cells of affected animals. No productive infection of the astrocytes was observed. The results of this study showed a lot of similarities in the tropism of CAE virus infection of goat brain cells to that of HIV infection in humans thus suggesting the potential usefulness of the caprine model for the study of HIV neuropathology. The goat model system as a non-primate model therefore could be more adaptable as a simple animal model than primate models with their complexity of anthropological, environmental and safety problems. Key words: CAEV, HIV, affinity, brain cells, RT-PCR, TEM.

Affinity (tropism) of caprine arthritis encephalitis virus for brain cells

One of the constraints in unraveling the mysteries blurring the advancement of research in the quest to totally put HIV problems under control is getting the appropriate animal model that would truly simulate human cases. This problem is more apparent in studies involving the central nervous system. Consequently, a viable animal model to generate information for the production of drugs and vaccines for the prevention and or control of lentiviral induced dementia in affected host animals is pertinent and vital. In this study, explant cultures prepared from the brain of new-born goat-kid were infected with Caprine Arthritis Encephalitis (CAE) virus- a retrovirus affecting goats. The specific brain cell types infected by the (CAE) virus were determined using reverse-transcription polymerase chain reaction (RTPCR) and transmission electron microscopy (TEM techniques). TEM showed that in 85 – 90% cases, microglia were the cells specifically infected by the virus. Amplification of the genomic sequence of the envelope and the gag genes by RT-PCR confirmed the presence of CAEV proviral DNA in the brain cells of affected animals. No productive infection of the astrocytes was observed. The results of this study showed a lot of similarities in the tropism of CAE virus infection of goat brain cells to that of HIV infection in humans thus suggesting the potential usefulness of the caprine model for the study of HIV neuropathology. The goat model system as a non-primate model therefore could be more adaptable as a simple animal model than primate models with their complexity of anthropological, environmental and safety problems.

Role of the transcription factor E2F1 in CXCR4-mediated neurotoxicity and HIV neuropathology

This study sought to determine the role of the transcription factor E2F1 in CXCR4-mediated neurotoxicity and HIV neuropathology. We studied the effect of the HIV envelope protein gp120 on the expression of E2F1-dependent apoptotic proteins in human and rodent neurons and examined the expression pattern of E2F1 in the brain of HIV-infected individuals. Our findings suggest that in cultured neurons gp120 increased E2F1 levels in the nucleus, stimulated its transcriptional activity and enhanced the expression of the E2F1 target proteins Cdc2 and Puma. Studies with neuronal cultures from E2F1 deficient mice demonstrated that the transcription factor is required for gp120-induced neurotoxicity and up-regulation of Cdc2 and Puma. Levels of E2F1 protein were greater in the nucleus of neurons in brains of HIV-infected patients exhibiting dementia when compared to HIV-negative subjects or HIV-positive neurologically normal patients. Overall, these studies indicate that E2F1 is primarily involved in CXCR4-mediated neurotoxicity and HIV neuropathogenesis.

Molecular mimicry between HIV-1 gp41 and an astrocyte isoform of alpha-actinin.

A 100-kDa astrocyte antigen previously shown to cross-react with a monoclonal antibody (MAb) generated against amino acids (aa) 598 to 609 of the transmembrane protein gp41 of human immunodeficiency virus type 1 [HIV-1] has now been molecularly characterized and found to be an alpha-actinin (alpha-actinin) related protein. Western blot analyses of human astrocytoma cells fractionated by differential centrifugation and detergent phase separation showed that the antigen was membrane associated. The astrocyte protein was purified to apparent homogeneity by immunoaffinity chromatography. Amino acid analysis of three peptide fragments obtained by cleavage of the purified 100-kDa protein revealed sequence identities of 77, 83 and 100% to a non-muscle isoform of human alpha-actinin. In addition, the aa 598-609 sequence of gp41 recognized by MAb 781.4, and the aa 581-597 sequence recognized by another cross-reactive MAb 781.3, were 73% and 53% similar to regions of alpha-actinin. This molecular mimicry between gp41 and alpha-actinin was supported by antibody cross-reactivity in Western immunoblot and ELISA analyses. Both anti-gp41 and anti-alpha-actinin MAbs bind to the surface of the human astrocytoma cells as detected by a cell surface binding assay and immunofluorescence. Antibodies made against this immunodominant region of gp41 in the serum and CSF of HIV-infected individuals have access to astrocytes within the CNS. The identification of the astrocyte antigen as an alpha-actinin related protein will allow further work to determine how this immunological cross-reactivity could perturb astrocyte function and contribute to HIV neuropathology.