Anuja Ghorpade, Ph.D - US grants

DESCRIPTION (provided by the applicant): Astrocytes participate in maintenance of brain homeostasis and disease pathogenesis. Reactive astrogliosis is associated with neural injury in a wide range of neurodegenerative diseases, including HIV-1 -associated dementia (HAD). We hypothesize that activation of astrocytes may transform these normally supportive cells into neurotoxic immune effectors. Interleukin-I (IL-1) beta is elevated in HAD and is known to activate astrocytes, thus, we have chosen it as a prototypical immune activator. RT-PCR, ELISA and Immunocytochemistry analyses of IL-1 beta-activated astrocytes showed an upregulation of Fas ligand (FasL), a death protein. Elevated levels of FasL were observed in cerebrospinal fluid of HAD patients. Preliminary data showed that culture supernatants from IL-1 beta-activated astrocytes induced injury in rat and human neurons in cell-free and co-culture systems. Activated astrocytes led to caspase activation in human neurons. These findings lead us to believe that expression of FasL by activated astrocytes represents a novel pathway for neuronal injury in HAD. In order to explore this hypothesis, a series of cellular, molecular, pharmacological and electrophysiological techniques will be utilized to address the following questions: 1) What role does FasL play in astrocyte-mediated neuronal injury? 2) What is the mechanism for FasL-induced neurotoxicity? And 3) How is FasL expression regulated in activated astrocytes? We believe that answers to these questions will be important in understanding the mechanism of HAD and other neurodegenerative diseases and may provide novel approaches for therapeutic intervention.

tissue resource /registry; cell bank /registry; biomedical facility; glia; monocyte; lymphocyte; tissue /cell culture; centrifugation; laboratory rat; human tissue;

DESCRIPTION: HIV-1-associated neurocognitive disorders (HAND) remain a significant societal burden in the era of post-antiretroviral therapy. In the brain, regulation of matrix metalloproteinases (MMPs), via their tissue inhibitors (TIMPs), remains an important homeostatic function of astrocytes. Over the past few years of funding we demonstrated that astrocyte-TIMP-1, the multifunctional inducible member of the TIMP family plays key roles in neuroinflammation and HAND neuropathogenesis. We identified critical signaling pathways in regulation of TIMP-1 production by astrocytes and described novel TIMP-1 neuroprotective effects through activation of neuronal pro-survival mechanisms. As a developmental component, we tested multiple nanoformulations and standardized A5P50 as a successful candidate for brain delivery studies. In this competing renewal, we propose to extend our works in novel directions for advancing basic insights into the mechanisms with clinical relevance. Specifically, in this proposal, we will test our overarching hypothesis that astrocyte-TIMP-1 is regulated by upstream promoter elements via C/EBP¿-MAPK/p38 signaling (Aim 1) and mediates neuroprotection via a TIMP-1/tetraspanin/integrin tripartite complex (Aim 2). Investigations will include promoter-reporter and overexpression constructs along with the knockdown or inhibition of key signaling elements to decipher these mechanisms. Furthermore, we previously optimized nanoformulations that will be used for successful GFAP-restrictive gene delivery to the brain. In Aim 3 we propose to test the A5P50 nanoformulations of GFAP-driven astrocyte TIMP-1 expression in the setting of HAND. The GFAP/Tat-transgenic animal model will be used and A5P50-conjugated GFAP driven-TIMP-1 will be administered via tail-vein injection. Changes will be evaluated at the tissue level by analysis of RNA, protein and histopathology for reversal of neural damage and deleterious side effects on extracellular matrix. Behavior studies will be performed for proof-of-concept of clinical outcomes. Combined molecular, cellular, in vitro, in vivo and translational approaches will be employed. Taken together, studies proposed in this renewal will not only unravel novel mechanisms underlying astrocyte responses during HAND-associated neuroinflammation, they will: provide innovative insight into the tetraspanin/integrin neuronal signaling, have broader implications for inflammatory diseases that involve MMP/TIMP imbalance and obtain critical proof-of-concept data for future therapeutic strategies relevant to HAND.

Currently, almost 35 million people live with HIV-1 infection worldwide. HIV-1-associated dementia (HAD) along with mild neurocognitive disorder and asymptomatic neurocognitive impairment comprises the HIV- associated neurocognitive disorder (HAND). Inflammation associated with milder forms of HIV encephalitis shows presence of activated microglia, reactive astrogliosis and neuronal injury in areas of inflammation. A few years ago, we began novel investigations into the potential mechanisms associated with glial activation & their contribution in neuro-AIDS. Primary human glial cells were used in preliminary experiments and CXCL8 was among the key molecules upregulated in activated astrocytes and other neural cells. Importantly, CXCL8 was neuroprotective in cultured human neurons exposed to HIV-1-related neurotoxins. Biological relevance of this observation was further confirmed as HIV-1-infected brain tissues demonstrated greater CXCL8 levels as compared to age-matched controls. We propose that CXCL8 plays a key regulatory role in the intercellular interactions in HIV-1 CNS infection. Microglial infection and activation leads to upregulation of IL-1¿. IL-1¿, a prototypical inflammatory stimulus for astrocytes, enhances CXCL8 production by astrocytes in CNS. CXCL8 further recruits microglia and regulates microglial activation and HIV-1 infection. Taken together, ultimately these events may lead to CXCL8-mediated direct or indirect neuroprotection. To these ends, we will address the following specific questions: How is CXCL8 regulated in activated astrocytes in HAD and what mechanisms are involved? (Aim 1) How does glial CXCL8 regulate microglial recruitment, activation and infection? (Aim 2) How does CXCL8 regulate neuronal survival and/or function during the process of HAD? (Aim 3) In Aim 1, human brain tissue specimens and primary human neural cells will be utilized to delineate CXCL8 profiles and to identify the cellular sources for CXCL8 in HAD. Primary human neural cells will be exposed to HAD-specific stimuli and CXCL8 regulation will be evaluated. The intracellular signaling pathways involved in IL-8 regulation, specifically, NF-¿B, p38MAPK and/or SHP2 will be studied in astrocytes. The role of intercellular interactions between activated astrocytes and microglia via CXCL8-mediated in regulation of microglial activation, recruitment and HIV-1 infection will be evaluated in Aim 2. The mechanisms of CXCL8 neuroprotection and the ensuing signal transduction specifically through Akt/PKB, ERK1/2, Bcl-2 and Bax and the role of TNF receptors in these neuroprotective events will be investigated using primary human neurons in Aim 3. Taken together, the studies proposed in this application will provide novel data about CXCL8-mediated glial cross-talk and neuropathogenesis and lead to novel insights into regulation of glial inflammatory responses that have both basic and clinical significance.

DESCRIPTION (provided by applicant): Methamphetamine (METH), an addictive stimulant has long lasting toxic effects on the central nervous system (CNS). Clinical studies indicated that METH dependence has an additive effect on neuropsychological deficits associated with HIV-1 infection. Oxidative stress, excitotoxicity, BBB impairment and glial cell activation, all have been independently implicated in the mechanisms of METH- and HIV-1-associated neurotoxicity. This proposal will investigate specific mechanisms operative in HIV-1 CNS infection and METH abuse that lead to an overall increase in oxidative stress and NF-kB signaling resulting in impairment of astrocytes and endothelial cell function. We propose that METH-mediated oxidative stress in astrocytes leads to a down regulation excitotoxic amino acid transporter (EAAT) -2, the primary astrocyte glutamate scavenger, while in endothelial cells, such an increase in oxidative stress results in loss of BBB integrity. Indeed, our preliminary data suggest that METH exposure caused ROS generation in astrocytes and human primary BMVEC, diminished EAAT-2 expression, in astrocytes, decreased BBB integrity in vitro that was restored by antioxidant treatment, caused decreased expression/re-distribution of tight junction proteins, enhanced adhesion and migration of monocytes across endothelial monolayers and activated small GTPases in BMVEC that were previously implicated in the BBB injury during HIV-1 encephalitis (HIVE). New preliminary data demonstrated increased permeability of BBB in vivo in animals exposed to METH that was prevented by antioxidant treatment. This proposal will investigate a novel concept of mechanistic commonality, i.e. METH-mediated oxidative stress exerts its cell-specific effects in astrocytes and endothelial cells aggravating injury caused by HIV-1 CNS infection and delineate therapeutic options for these targets using in vivo studies. Using a combination of in vitro assays and METH/HIVE animal model, we will address the following questions: What is the role(s) of enhanced reactive oxygen species, ROS production and NF-kB signaling in diminished expression/function of EAAT-2 in astrocytes? (Aim 1) What are underlying mechanisms of BBB dysfunction and enhanced monocyte adhesion/migration across brain endothelium (via oxidative stress, interference with NF-kB and GTPase signaling)? (Aim 2);and Can therapeutics decreasing oxidative stress and normalizing EAAT-2 function ameliorate BBB dysfunction and neurotoxicity in an animal model for HIVE and METH abuse? (Aim 3) We will address these questions utilizing primary human astrocytes and brain microvascular cells, in vitro BBB models and evaluate combined effects of METH and HIV-1 relevant stimuli on EAAT-2 and BBB function. Antioxidants and specific signaling inhibitors will be utilized to delineate pathways involved in these effects. Our HIVE animal model will be employed to investigate the biological outcomes of these cell-specific mechanisms in cognitive function, BBB damage and neurotoxicity. We believe that the proposed works are highly significant, as they will uncover novel mechanisms involved in the combined effects of HIV-1 and METH in the CNS and propose therapeutic approaches based on these investigations. PUBLIC HEALTH RELEVANCE: Methamphetamine, a highly addictive stimulant abused by millions of Americans has long lasting toxic effects on the central nervous system. Clinical studies indicated that methamphetamine dependence has an addictive effect on cognitive deficits associated with HIV-1 infection. Current proposal aims to understand mechanisms of combined effects of HIV-1 and methamphetamine in the brain and to propose neuroprotective therapies.

DESCRIPTION (provided by applicant): The pathogenesis of neurodegenerative disorders, including Human Immunodeficiency Virus (HIV)-1 associated dementia (HAD-now called HAND: HIV-1-associated neurocognitive disorder), is exacerbated by an imbalance between metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). As the TIMPs exhibit diverse non-classical functions including anti-apoptotic effects, the induction of TIMP-1 in neuroinflammatory conditions likely serves multiple roles in addition to modulating MMP activity. Our work in the previous funding cycle demonstrated that differential TIMP-1 expression in acute versus chronic activation of astrocytes and HAD brain tissues. We showed that in acute activation IL-12 upregulates TIMP-1 through multiple mechanisms, while chronic activation leads to TIMP-1 downregulation primarily through transcriptional control. We identified a novel CCAAT displacement protein (CDP) binding repressor element involved in astrocyte TIMP-1 regulation. Further, neurotoxicity assays were performed using staurosporine, macrophage-tropic HIV-1 virus and glutamate to induce cell death in cultured human neurons. In these assays, TIMP-1 had equivalent neuroprotective effects as brain derived neurotrophic factor (BDNF). These neuroprotective effects were independent of MMP binding as shown with the TIMP-1 T2G mutant. Neurotrophic signaling through Bcl-2/Bcl-xL is a possible pathway for TIMP-1 neuroprotection and recently a tetraspanin has been identified as a potential TIMP-1 binding partner. Thus, the overarching hypothesis for this proposal is that astrocyte-TIMP-1 is differentially regulated in disease through CDP repression and contributes to loss of TIMP-1-mediated neuroprotection rendered via tetraspanin receptors on neurons. Furthermore, TIMP-1 CNS delivery strategies will be explored using novel nanomedicine approaches. Combined molecular, cellular, in vitro, in vivo and translational approaches will be used. In this competing renewal, we continue an in depth investigation into the mechanisms of astrocyte-TIMP-1 regulation via CDP, neuroprotective effects of TIMP-1 and explore a potential therapeutic nanomedicine strategy, based on our progress in the previous round of funding. We will first investigate the role of CDP in the regulation of astrocyte-TIMP-1 in HAD (Aim 1). Next, we will study the mechanisms of TIMP-1-mediated neuroprotection via tetraspanin signaling (Aim 2). Specifically, we will delineate the TIMP-1 neuronal tetraspanin receptors and related signal transduction mechanism leading to neuroprotection. Finally, we will explore the therapeutic application of GFAP-driven TIMP-1 expression using CNS-targeted nanoparticles (Aim 3). GFAP-TIMP- 1/luciferase constructs in Tat-conjugated PLGA nanoparticles will be intravenously administered to mice and CNS-specific TIMP-1 and luciferase expression will be evaluated. Thus, studies proposed in this renewal will unravel novel mechanisms underlying astrocyte responses during chronic neuroinflammation in HAD, have broader implications in other inflammatory diseases that involve MMP/TIMP imbalance and provide critical proof-of-concept data for future therapeutic strategies. PUBLIC HEALTH RELEVANCE: HIV-1-associated dementia (HAD), now referred to as HIV-1-associated neurocognitive disorder, is an important neurological complication of HIV-1 infection and is characterized by cognitive, behavioral and motor dysfunction. An estimated 10-15% of HIV-seropositive (HIV+) patients progress to develop HAD in developed worlds such as the United States, despite the availability of highly active antiretroviral therapy. Reactive astrogliosis, recruitment to and proliferation of astrocytes at the site of injury, is the pathological hallmark of all neuroinflammatory conditions and is observed in areas of inflammation in HAD. How neuroprotective responses of astrocytes are altered in neuroinflammation and contribute to disease is intriguing and is the focus of our and many other recent investigations. Our studies will provide a better understanding of the specific mechanistic contributions of activated astrocytes to HIV-1-neuropathogensis and neuroinflammation.

Woridwide, -40 million people live with Human Immunodeficiency Virus (HIV)-I disease and associated Acquired Immune Deficiency Syndrome (AIDS), half are women. HIV/AIDS has affected more women than any other disease over the past two decades. Women of color, particularly, African American and Hispanic American women carry the most significant burden of this disease. Clearly, HIV/AIDS in minorities and women will continue to be a significant burden and a critical area of Health Disparities research. Importantly, almost 50% of HIV/AIDS patients suffer from some neurocognitive impairment. Thus, studies that pertain to identification of novel biomarkers that predict disease progression and neurocognitive impairment are critical. Recent data demonstrate that sCD40L, a novel biomarker is significantly elevated in HIV-infected patients with neurocognitive impairment. However, no information is available regarding patterns of sCD40L changes in context of race and gender. This is important, as expression of disease characteristics in HIV/AIDS can be race- and/or gender-specific. Viral loads, metabolic parameters and body composition, plasma lipid levels etc. differ in the setting of specific race and gender. In this application, we propose to investigate the role of SCD40L as a prognostic marker for disease progression in HIV-1 neurological manifestations in the context of race and gender. Although limited to evaluations of race and gender as statistical risk factors, this work will synergize basic science with issues related to health disparities. Specifically, we will identify and analyze plasma sCD40L levels in a cohort of HIV+/- patients and correlate these to standard disease variables and cognitive function. All parameters will be correlated with each other and against neurocognitive measures to test the hypothesis that sCD40L levels correlate with neurocognitive impairment in the context of race and gender. We will also analyze the immune activation status of diverse patient leukocytes. Levels of sCD40L & other proinflammatory cytokines including TNF-a will be analyzed. Ours will be one ofthe first studies to fully describe the HIV-1-positive population characteristics in relation to specific cognitive outcomes, socioeconomic strata and sCD40L and will likelv vield data that will have direct therapeutic implications.

? DESCRIPTION (provided by applicant): As a popular psychostimulant, methamphetamine (METH) use leads to long-lasting, strong euphoric effects. While METH abuse is common in the general population, approximately 10-15% of human immunodeficiency virus-1 (HIV-1) patients report METH abuse. METH exacerbates the severity and onset of HIV-associated neurocognitive disorders (HAND). Both METH and HAND neuropathogenesis mechanistically concur with neuroinflammation, astrocyte activation, brain hyperthermia, oxidative stress and excitotoxicity. Thus, METH affects a multitude of astrocyte functions, yet the mechanism through which this is attained is unclear. Recently, we reported trace amine associated receptor 1 (TAAR1) as a novel astrocyte receptor for METH signaling. In this proposal, we will investigate METH-mediated regulation and activation of TAAR1 and the downstream effects that lead to exacerbation of HAND in the context of METH comorbidity. Our preliminary data in this proposal suggest a critical role of pCREB in astrocyte-METH signaling. Recently, TAAR1 has also emerged as a promising pharmacotherapeutic target. We propose that METH-abuse in HAND modulates astrocyte-TAAR1 and downstream signaling via cAMP, [Ca2+]i, PKA/ERK, PKC and NF-kB regulating CREB. These pathways play critical mechanistic role(s) in astrocyte-mediated neurotoxic outcomes, including mitochondrial damage, oxidative stress, excitotoxicity and neuroinflammation. Further, we propose astrocyte-TAAR1 as a novel therapeutic target in HAND and METH comorbidity. We will conduct investigations in the molecular regulation of TAAR1 in Aim 1, delineate the complex intracellular signaling pathways altering astrocyte function in Aim 2 and lastly extend the work to potential therapeutic avenues using a HAND relevant animal model in Aim 3. First, we will investigate the regulation of astrocyte-TAAR1 and following cAMP changes leading to gliosis and neuroinflammation (Aim 1: Molecular). Human astrocytes activated with virotoxins, or those expressing viral proteins, with/out METH will be used. TAAR1-GFP overexpression model will be used to identify TAAR1-specific responses. Next, we will delineate astrocyte-TAAR1 intracellular signaling leading to METH comorbidity in HAND (Aim 2: Signaling and function). Lastly, we will evaluate TAAR1 as a potential therapeutic target (Aim 3: Translational). Taken together, we employ a combined cellular and molecular approach with ex vivo, in vitro and in vivo studies that will have broader implication(s) for astrocyte-TAAR1 regulation, its role in substance abuse-based neurological deficits and as a novel therapeutic target for METH comorbidity in HAND.

Project Summary/Abstract Despite the advent of successful antiretroviral therapy (ART), the quest for a human immunodeficiency virus (HIV)-1 cure continues. In order to achieve total eradication, we must successfully resolve persistent viral reservoirs in various tissue compartments. HIV-associated neurocognitive disorders (HAND) remain a significant societal burden in post-ART era. In response to the RFA-MH-17-100, we propose to uncover strategies to address astrocytes as a major CNS reservoir for HIV-1. Our preliminary data show HIV-1 latency can be recapitulated in cultured human astrocytes. The lack of powerful experimental tools to evaluate latency versus reactivation has in part, been responsible for the lack of information in viral latency in the CNS. We propose to employ the powerful dual color HIV-1 reporter virus that harbors CMV-driven mCherry (red, R) and HIV-1 LTR-driven GFP (green, G). Preliminary studies using primary human astrocytes infected with RG-HIV-1 VSV-pseudotyped virions are shown. All astrocytes infected with RG- HIV-1 express the CMV-driven mCherry (red), whereas only those that have an active HIV-1 LTR will express GFP (green) and are positive for HIV-1 p24. Red-alone astrocytes (mCherry+/GFP-) represent latently infected cells amenable to easy monitoring, sorting, manipulation and mechanistic investigations. We have established this model system using primary human astrocytes, which presents a simple, convenient and powerful tool for these works. We propose that latently infected astrocytes express identifiable biomarkers, even in the absence of reactivation. And that guide RNA/Cas9 editing can excise or silence proviral gene expression to prevent reactivation and improve astrocyte physiological function. To mitigate the conundrum of distinguishing latently infected astrocytes from those that have active HIV-1 LTR function, our first goal is to identify targetable biomarkers for HIV-1 latency in human astrocytes (Aim 1). Second, we will target latently infected astrocytes without reactivation using guide RNA and Cas9 gene editing techniques to excise proviral components, rendering it silent and incapable of reactivation (Aim 2). A successful integration of Aims 1 and 2 will lead to novel therapies to cure and eradicate HIV-1 CNS reservoirs. In summary, these investigations responding to RFA-MH-17-100 will provide novel and timely insight into delineating approaches to identify and target astrocyte viral reservoirs, key CNS barriers to HIV-1/HAND cure, and eliminate them without viral reactivation.