Sanjay B. Maggirwar, MBA., Ph.D. - US grants

DESCRIPTION (Adapted from applicant's abstract): Programmed cell death (PCD) is a physiological process required for the development of the nervous system. Deregulation of this process may contribute to the neuronal injury and cell death associated with neurodegenerative disorders. Recent data strongly suggest that overexpression of the cRel subunit of NF-kB, alone, is sufficient to protect sympathetic neurons from apoptosis caused by nerve-growth factor (NGF)-deprivation. Therefore, experiments are proposed to investigate the role of NFkB /Rel in mediating the survival of neurons. The present study will utilize sympathetic neurons as an in vitro model for PCD, based on their death after NGF withdrawal. The specific aims are designed to test the hypothesis that NF-kB regulates neuronal death in part by through its ability to compete with the pro-apoptotic factor c-Jun for binding to the co-activator protein p300/CBP, thereby inhibiting the activity of c-Jun. This hypothesis is based on recent evidence showing that NGF deprivation-induced apoptosis of sympathetic neurons requires c-Jun activity. Experiments are proposed to (1) define the specific subdomain(s) within cRel that is necessary for protection of sympathetic neurons from apoptosis due to NGF-withdrawal, (2) examine whether NF-kB can also protect primary neurons derived from the central nervous system (CNS) from a well-defined pro-apoptotic stimulus, and (3) investigate the effect of overexpression of the transcriptional co-activator p300 on NF-kB-mediated survival of neurons. It is expected that these studies will provide new insights into the pathogenesis of neurodegenerative diseases, and that they may ultimately contribute to the identification of pharmacological inhibitors of neuronal cell death.

DESCRIPTION: (Provided by applicant): Severe neurologic complications, including HIV-associated dementia (HAD), occur in a significant proportion of HIV- 1 infected individuals. HAD is marked by cognitive and motor deficits, and pathologic correlates of this syndrome include neuronal apoptosis. Neuronal cell death and damage in HIV- 1 infected individuals are thought to be induced by a number of soluble neurotoxic factors, of both viral and cellular origin. Molecular pathways which may protect neurons from the deleterious effects of these candidate HIV-1 neurotoxins remain poorly understood. Preliminary data presented in this proposal show that the cellular transcription factor NFKB may protect neurons from the pro-apoptotic effects of candidate HIV- 1 neurotoxins. Our data further suggest that this may occur as a result of NFKB-mediated induction of genes, which negatively regulate programmed cell death. The experiments outlined in this proposal are intended to investigate the molecular mechanisms by which NFKB/Rel proteins exert their neuroprotective effects, and to determine the specific contribution of individual NFKB/Rel family members to this process. These studies will be conducted using well-characterized and appropriate neuronal model systems, and a representative array of candidate HIV-1 neurotoxins. Endogenous pathways of NFKB activation in neurons will also be examined, by using specific neurotrophins to activate this transcription factor, and a systematic investigation of NFKB-induced target genes will be performed, with an emphasis on genes involved in the regulation of apoptosis. Finally, the effects of NFKB activation on the caspase cascade will be examined, in neurons exposed to candidate HIV-1 neurotoxins, and in control cells. Taken together, these studies are expected to enhance our understanding of NFKB-mediated neuroprotection, and provide insights that may lead to the development of new therapeutic strategies for HIV-1 associated neurological disease.

DESCRIPTION (provided by applicant): The progressive loss of cognitive abilities in HIV-1-infected individuals, which is currently referred to as HIV-1-associated neurocognitive disorders (HAND; also popularly known as neuroAIDS), remains a substantial clinical concern. Recent studies have shown that the disruption of the blood-brain barrier (BBB) plays a major role in the progression of HAND. However, the underlying mechanisms that regulate the BBB during HIV-1 infection remain poorly defined. In this competitive renewal, we propose to test the hypothesis that BBB permeability is the result of deregulated trans-cellular communication between brain endothelial cells and astrocytes, following enduring effects of HIV-1 proteins on CD40/CD40L signaling. Our project seeks to shift current research and clinical paradigms by defining novel intracellular signaling targets, such as the CD40/CD40L axis and the Sonic hedgehog (Shh) pathway, that play potentially crucial roles in regulating BBB permeability in response to HIV-1 infection, and through functional validation of these targets by using novel anti-Shh drugs as novel therapeutics for HAND. Here we intend to integrate a comprehensive analysis of cellular responses due to HIV-1 with novel methodologies such as intravital multiphoton microscopy in a humanized mouse model. In Aim 1, we will test the hypothesis that CD40/CD40L signaling plays an important role in HIV-1-induced BBB permeability in vivo. Aim 2 will examine whether CD40/CD40L signaling disrupts trans-cellular communication between astrocytes and brain endothelial cells. Finally, in Aim 3 we will investigate whether targeting the Shh pathway renders mice resistant to BBB permeability induced by HIV-1 Tat in a CD40L-dependent manner. The results obtained in these studies are expected to exert a high impact on the neuroAIDS field on three counts: (1) by advancing scientific knowledge and revealing how HIV-1 induces CNS inflammation in infected patients, (2) by promoting novel methodologies and animal models, and (3) by validating novel therapeutic targets for HAND, which are also viable in other neurodegenerative disorders that are secondary to BBB disruption.

DESCRIPTION (provided by applicant): After almost three decades of AIDS epidemic, we are still challenged with the fact that more than one out of three people with productive infection of HIV-1 develops some form of HIV-1 associated neurocognitive disorder (HAND), which remains refractory to the conventional antiviral therapeutics. In addition, the clinical symptoms of HAND are found to coexist with those of other systemic inflammatory diseases in a significant proportion of patients, some of which are associated with a higher number of activated platelets in the circulation. This may suggest that a common underlying mechanism is at work in these infected individuals. In this respect, HIV-infected patients exhibit increased numbers of activated platelets in the plasma in a manner that relates to the severity of the immunologic deficit, however, it is unknown whether platelets play a pivotal role in the pathogenesis of HAND. Here we demonstrate that the systemic administration of physiologically relevant levels of Tat produces evidence of platelet activation in the plasma, followed by enhanced permeability of the BBB and inflammation in the CNS. These effects were abolished when Tat was administered to animals that had first been depleted of platelets, supporting the notion that platelets might play a crucial role in HAND. Based on these findings, we hypothesize that the activation of peripheral platelets by effector molecules released by HIV-1-infected cells elicits abnormal effects on brain microvascular endothelial cells (BMVEC), thereby altering blood-brain barrier (BBB) integrity and exacerbating inflammation in the CNS. Three fundamentally critical questions are addressed in this proposal. The first is whether inflammatory mediators released by HIV-infected cells also target other immune effector cells, such as platelets (Aim 1). Second, if activated platelets in turn activate BMVEC to stimulate inflammatory vascular changes that are connected with BBB permeability (Aim 2);and last whether the effects caused by platelets facilitate frequent passage of peripheral blood monocytes through an otherwise normal BBB (Aim 3). The obvious corollary to this is whether therapeutic manipulation of platelet activity provides protection from neurologic damages induced by HIV. Thus, the experiments proposed herein have great translational potential for the development of new therapeutic strategies for neuroAIDS. PUBLIC HEALTH RELEVANCE: It is speculated that a common mechanism is engaged by HIV-1 to stimulate inflammatory disorders in various body compartments. This notion will be tested by investigating whether the abnormal activation of blood platelets accounts for the observed neurologic deficits in HIV-infected individuals. The outcome of these studies is expected to further our understanding of disease, which ultimately will lead to new therapeutic strategies.

DESCRIPTION (provided by applicant): HIV-1 remains one of the most important infectious causes of global morbidity and mortality. Recent research successes make this an exciting time in HIV/AIDS research - but continued progress will require interdisciplinary, collaborative efforts by highly trained, expert scientists. The goal of this proposal is to prepare young scientists for that role, and to train the next generation of HIV/AIDS researchers. The trainees will be equipped with a broad range of skills and knowledge including virology, molecular biology and immunology as well as training in mentoring, scientific writing, cutting edge research techniques, and other issues important to their career development. Since the previous competitive renewal of this application, we have broadened the research opportunities available, integrated this training program with the University of Rochester (UR)'s new Developmental Center for AIDS Research (DCFAR) and its Clinical & Translational Science Institute (CTSI), added new HIV/AIDS-specific courses, and designed an innovative peer/near-peer evaluation process to enhance trainee career development. We have also added an External Advisory Board to assist with oversight of this program. The program has 4 specific objectives. First: To develop an interactive, productive and exciting interdisciplinary environment in which predoctoral trainees obtain an outstanding education in HIV/AIDS research. This will be achieved by integrating faculty from multiple departments and disciplines. Second: To leverage resources and opportunities created by the UR's new Developmental Center for AIDS Research (DCFAR) and its Clinical & Translational Science Institute (CTSI). This will be achieved by accessing new courses, seminars and cutting-edge scientific core facilities. Third: To provide an enriched training and learning environment for students, with structured seminar programs, distinguished extramural speakers, career development activities, HIV/AIDS-specific courses, an innovative peer/near-peer evaluation process to support trainee career development, enhanced training in the responsible conduct of research, and opportunities for training in mentoring. Students will also be trained in scientific writing, with emphasis on manuscripts and grant applications. Fourth: To foster communication among trainees and faculty through the programs outlined above. This will exploit the intimate environment provided at the UR School of Medicine and Dentistry, where all the participating faculty and trainees work together in the same physical space, under a single roof. Overall, the program will provide outstanding training opportunities in three major emphasis areas: (i) viral replication, RNA biology and reverse transcription, (ii) viral immunology and vaccine biology, and (iii) viral pathogenesis (with emphasis on neuroAIDS).

? DESCRIPTION: Several reports suggest that HIV infected individuals present signs of accelerated vascular aging including atherosclerosis (AS). Available data suggest that the immune dysregualtion associated with HIV infection, not completely restored by the use of combination antiretroviral therapy (cART), plays a role in AS. However, cART directly or indirectly may also play an important role in AS. It has been suggested that the following four events during the aging process are key to evoke AS; 1) Telomere (TL) shortening-mediated DNA damage and apoptosis, 2) impairment of vascular reactivity, 3) inflammation, and 4) impairment of efferocytosis (phagocytosis). In this regard, our group has reported that p90RSK-ERK5 module plays a crucial role in endothelial (EC) dysfunction. Our preliminary data show that cART-induced p90RSK activation inhibited ERK5 transcriptional activity and cART also increased sCD40L plasma levels. Interestingly, sCD40L also increase the activation of p90RSK. Furthermore cART-induced p90RSK activation inhibited ERK5 transcriptional activity thus decreased M? efferocytosis. Lastly, we found that cART caused TL shortening via p90RSK activation, leading to DNA damage and apoptosis. Both increased apoptosis and impairment of efferocytosis evoke secondary stress responses via releasing cellular components including cell-free DNA (cfDNA) and high mobility group box 1 (HMGB1). Based on the rationale above, we propose a novel hypothesis centered on p90RSK activation which provides a link, not previously described, to several observations suggesting an association between cART and accelerated AS in HIV infected individuals. We will test this hypothesis in a 3- year longitudinal study of 180 HIV+ individuals (viral load d50 c/ml) aged e 50 years who are therefore at increased risk of developing measurable changes in markers of AS such as carotid intima-media thickness (CIMT) and carotid artery stiffness (CAS). 90 HIV- controls will be matched for age, gender and Reynolds CVD risk score. We will assess the effect of cART and sCD40L on p90RSK in participants-derived monocytes and secondary stress responses both in monocytes (HMGB1) and plasma (HMGB1, 8-OHdG). We will further define the causal effect of cART and sCD40L on p90RSK-induced accelerated aging of EC and M? in animal studies. Specific Aims: AIM 1. To determine whether chronic exposure to cART and elevated plasma levels of sCD40L in older HIV infected individuals are associated with p90RSK activation leading to enhanced aging of monocytes (reduce telomere length, reduced efferocytosis) and whether these measures of monocyte aging are associated with markers of atherosclerosis (CIMT and CAS). AIM 2. To determine whether the markers of secondary stress response (monocytic and plasma levels of HMGB1, plasma levels of 8-hydroxy-2'- deoxyguanosine (8-OHdG)) in cART-treated older HIV infected individuals are associated with measures of CIMT and CAS. AIM 3. To determine in vitro and in small animal models, the causal effect of cART and sCD40L on p90RSK activation leading to cellular aging of EC and M? which underlie AS.

? DESCRIPTION (provided by applicant): Widespread use of combination antiretroviral therapy (cART) has led to increased survival of Human Immunodeficiency Virus (HIV)-infected patients. As a result, chronic diseases are increasingly replacing acute infections as important causes of morbidity and mortality. One of these chronic diseases is atherothrombosis, the underlying mechanisms of which are not fully elucidated. Here, we demonstrate that monocytes and platelets from HIV-infected, cART-treated individuals exhibit reduced levels of the host restriction factor Tetherin BST-2/CD317), and that Tetherin loss is induced by exposure of healthy cells to the viral protein Tat, and to other inflammatory mediators of cellular origin, via proteasome-dependent mechanisms. We also show that over-expression of degradation-resistant Tetherin, which contains a S3T4S5 substitution mutation, in monocytes causes sequestration of fully formed microparticles (MPs) on the cell surface. These findings will be leveraged to test the overall hypothesis that HIV promotes thrombosis via Tetherin-dependent release of cellular MPs. Briefly, we will examine the loss of Tetherin and associated release of MPs by employing in vitro models of HIV infection (Aim 1), novel mouse models of HIV-associated thrombosis in vivo (Aim 2), and prospectively determine how these events are linked in HIV patients receiving cART (Aim 3). These studies will bring together four established investigators and two emerging investigators with proven expertise in virology, platelet/monocyte biology, MPs, thrombosis, and HIV clinical research. Importantly, proposed studies will leverage research infrastructure offered by the University of Rochester Center for AIDS Research (UR-CFAR). Our studies contain great potential for clinical translation. Changes in Tetherin expression will be directly tested in human subjects and linked with atherothrombosis. As such, revealing the mechanisms through which HIV and cART regulate the production of microparticles in monocytes and platelets will provide a critical basic science foundation so as to understand how these molecular pathways function in mammalian cells, while providing insight into how HIV triggers atherothrombotic events in patients who are or are not receiving cART. In summary, our proposal is discovery-oriented, translational, and is responsive to the goals of RFA (RFA-HL- 14-024).

HIV infected individuals, especially those older, are at increased risk of developing cerebrovascular disease (CBVD). While most of the recent investigations have focused on large vessel disease, cerebral small vessel disease (CSVD) has received much less attention despite its known role and long-term effect on cognitive performance and potentially more direct link to HIV-associated immune dysregulation. CSVD is diagnosed via neuroimaging. Typical findings include small subcortical infarcts, lacunes, white matter hyperintensity, enlarged perivascular spaces, cerebral microbleeds and brain atrophy. Quantitative MR techniques assess indirectly the altered microcirculation and blood brain barrier (BBB) by measuring vascular reactivity, cerebral blood flow, white matter microstructure and tissue susceptibility. Importantly, these quantitative imaging modalities can measure even subtle brain abnormalities that escape the evaluation by standard clinical imaging techniques. CSVD has been associated with markers secreted by myeloid cells. This is particularly relevant to HIV infected individuals. We and others have shown that the aberrant platelet activation during HIV/SIV infection causes an increase in platelet-monocyte complexes (PMCs) that drives monocyte maturation from CD14+/CD16- to the pro-inflammatory CD14(low)/CD16+ phenotype and that the reduced numbers of CD14+/CD16- monocytes are associated with pro-AS changes, BBB permeability and aging. Thus, PMCs could serve as markers and a therapeutic target of CSVD. Based on these observations, we hypothesize that PMCs, by affecting vascular permeability and reactivity, play a crucial role in mediating CSVD, especially in older HIV infected individuals. In a well characterize cohort for CBVD risk factors that includes an older-enriched HIV population (age?50) and age matched uninfected individuals, we propose to address the following Specific Aims pertinent to CSVD in a 3-year longitudinal study. In Aim 1 we will assess whether changes in vascular reactivity (measured via rs-fMRI) and white matter microstructural integrity (measured via DTI) are associated with levels of PMCs. In Sub-Aim 1 we will determine whether areas with increased tissue susceptibility and decreased vascular reactivity are associated with decreased cerebral blood flow (CBF). In Aim 2 we will determine whether changes vascular reactivity and white matter microstructural integrity are associated with soluble products of pro-inflammatory monocytes (plasma levels of sCD163, neopterin, and HMGB1), platelet activation (platelet factor 4 [PF-4]) and with plasma markers of endothelial dysfunction (intercellular adhesion molecule 1 [sICAM- 1], vascular cellular adhesion molecule-1 [sVCAM-1], osteoprotegerin and lipoprotein-associated phospholipase A2 [Lp-PLA2] mass). In Sub-Aim 2 we will determine whether changes in brain iron deposition are associated with levels of PMCs, PF-4, plasma monocyte and endothelial soluble products. In Aim 3 we will model data generated from the previous aims in the context of cognitive performance.

BASIC SCIENCES CORE (CORE C) PROJECT SUMMARY Leveraging the collective scientific expertise and resources across the DC CFAR partner institutions, the Basic Sciences Core (BSC) will provide an array of high impact laboratory services and training to HIV investigators in Washington, DC. Under the leadership of NIH-funded senior scientists and with a cadre of DC CFAR- supported Core service providers, the BSC will provide essential virology, immunology, proteomics, imaging and next-generation sequencing services including single cell analysis that foster scientific discovery in HIV research. These services are aimed at assisting investigators to acquire preliminary data for grant applications; conduct funded research protocols; and foster linkages with the SWGs and clinical and translational investigators. For Specific Aim 1, the Core will provide high impact laboratory services to CFAR investigators which will be evaluated routinely and updated or augmented based on user needs, advances in scientific research or the development of new technologies. For Specific Aim 2, the Core will provide training through individual-level technical support and larger CFAR-wide events. These include the hands-on Core service laboratory ?open house? series and the annual Core Service Providers Research Day. The Core leadership will also collaborate with the Developmental Core to provide mentoring to basic science investigators; encourage submissions for pilot and transitioning investigator awards and microgrants; and strengthen multi-disciplinary research efforts. Lastly, consistent with the overall mission of the DC CFAR, the Core will focus on outreach to women and underrepresented minority investigators to assist their development as independent HIV laboratory scientists.

Summary Despite the development of potent anti-retroviral therapy (ART) that successfully suppresses virus replication in the majority of people living with HIV (PLWH), there is no treatment that can cure this infection entirely. The major obstacle in eradicating HIV is the persistence of various anatomical viral reservoirs (VRs), including the central nervous system (CNS), that have the capacity to produce infectious virus and systemically spread within a short period upon cessation of ART in all, with few exceptional cases. Therefore, developing novel interventions aimed at reducing or eliminating the VRs is one of the key priorities for HIV research. In response to RFA-MH- 20-701, our application proposes basic science and preclinical research in SIV-infected rhesus macaques (RMs) to model aspects of VR in the CNS-resident myeloid cells of PLWH, and to investigate the efficacy of the novel pharmacologic strategy to prevent establishment of HIV persistence in the CNS. Thus, based on the observations outlined in this application we hypothesize that the disruption of PMC formation during acute phase of infection will limit the seeding and maintenance of VR and, as a consequence, the extent of viral rebound in the CNS following analytical therapy interruption (ATI). Three aims are proposed: (1) To investigate whether the systemic disruption of PMC formation during acute phase of infection, regulates viral persistence in the CNS; (2) To investigate whether the systemic disruption of PMC formation during acute phase of infection, regulates the kinetics and extent of viral rebound after ATI; and (3) To investigate whether the systemic disruption of PMC formation during acute phase of infection, regulates the neuroinflammation and synaptodendritic damages associated with long-term ART and ATI. These aims will be achieved by (i) using a well-established model of SIV-infected RMs treated with suppressive ART, and (ii) performing in vivo Ab-mediated disruption of PMC formation during acute phase of untreated infection. Revealing the mechanisms through which platelets regulate the persistence of HIV in myeloid cells will provide a critical understanding of how these cellular interactions function in mammalian cells, and an insight into how a potential HIV cure can be achieved in PLWH.

HIV/AIDS once constituted a lethal diagnosis that ravaged many communities and has now become for some a chronic disease with associated co-morbidities. Likewise, our historic research successes in deciphering the mechanisms of fundamental HIV pathogenesis must now be directed to new questions about HIV elimination, vaccines and therapeutic development, and efforts to treat and prevent subsequent disorders among people surviving the disease. Researchers focused on these questions, with specific skillsets and knowledge, attractive to multiple career paths, are needed to address these emerging challenges in HIV/AIDS disease. The overall goal of this T32 is to prepare doctoral students with the knowledge, methodologic, analytic and leadership skills to become successful future HIV research investigators. To achieve these goals, we will (A) Provide intensive didactic education in HIV/AIDS research. A new required course in HIV Persistence will be offered to complement a core didactic research curriculum. Research in progress will be shared among trainees and experts at an annual retreat, and a catalog of elective courses will round out opportunities to build HIV knowledge in three major emphasis areas: (i) cure research, including T-cell therapy and the reversal of viral latency; (ii) co- morbidities, including malignancies and CNS disease; and (iii) prevention research, including vaccines and novel therapeutics; (B) Recruit two outstanding doctoral students per year for 2 years of support with activities to accelerate their progression to independence. Each trainee will have an individualized project characterized by a) multiple-mentor teams to aid in the design and implementation of hypothesis-driven research projects, b) a curriculum to build HIV specific knowledge and research skill sets, c) professional development including presentation skills and grant-writing d) strong exposure including participation in group-based externship opportunities in multiple career paths for HIV scientists, and strong programmatic oversight; and (C) Cultivate a local infrastructure and multi-departmental culture of collaboration to support HIV research training. This application joins faculty from multiple GW departments and schools, as well as the District of Columbia Center for AIDS Research (DC CFAR) to introduce new perspectives and fresh ideas that will collectively offer outstanding collaborative training in cross-cutting high priority areas of HIV research. Enhanced cross-school and regional investigator orientation and networks, and research progress reports and meetings are intrinsic to this plan. New approaches to enhance mentor development will be expanded and institutionalized to strengthen faculty mentoring practices in effective autonomy-supportive mentoring.