Kelly L. Jordan-Sciutto, PhD - US grants

The proposed study will define the functional role of a noel protein, FAC1, during brain development and Alzheimer's disease (AD). The FAC1 gene product has been shown to be expressed at high levels during human brain development, low levels in adult control brain, and re-expressed during early stages of AD. Functional analysis of specific domains within FAC1 is required to understand the role of FAC1in these processes. We have identified a zinc finger motif and acidic activation domain in the N-terminus of FAC1, features common to transcription factors. The first Specific Aim will test the hypothesis that FAC1 interacts with other proteins and assay their ability to regulate FAC1 function. Characterization of FAC1 interacting proteins during brain development and various neurodegeneratie discases will further define molecular mechanisms guiding transcriptional and translational events during these processes.

DESCRIPTION: HIV encephalitis is remarkable for neuronal damage and loss in the absence of significant HIV infection of neuroglial cells. Neuronal cell death is believed to be mediated by activated, HIV-infected macrophagesecreted products including chemokines, cytokines, neurotrophic factors (NTF), and quinolinic acid. Several of these factors including NTF have the ability to induce changes in the cell cycle regulatory machinery. We have observed changes in expression and subcellular localization of three key regulators of the cell cycle, pRb. E2F1, and p53, in models for HIV encephalitis (HIVE). This has led us to propose the hypothesis that neuronal degeneration observed in HIVE is caused by changes in activity of cell cycle machinery in terminally differentiated neurons. Activation of cytokine, chemokine, and neurotrophic factor receptors in non-neuronal systems results in second messenger cascade, which result in phosphorylation of pocket proteins like the retinoblastoma susceptibility gene product, pRb. Phosphorylation of these proteins leads to deregulation of proteins controlling gene expression, most notably E2F1. Since pRb, E2F1 and p53 control the two key pathways determining cell survival, we hypothesize that changes in activity of these proteins will alter neuronal viability. Using both retrospective autopsy studies and an in vitro model of HIVE, we will study the activity and expression of members of these two pathways and their effects on survival of neuronal and glial elements. Our first aim focuses on determining the regulation of pRb and its family members by phosphorylation in response to macrophage secreted factors. Specific Aim 2 will determine if E2F1 activity is altered by macrophage secreted factor signaling and study the impact of activated E2F1 on neuronal and glial survival. Finally specific aim 3 will address the contribution of p53 to neuronal and glial survival in response to macrophage secreted factors. These specific aims will help define the role of cell cycle regulators in neuronal and glial survival during HIVE and define therapeutic targets to disrupt the molecular cascade leading to the associated neurodegenerative disease.

[unreadable] DESCRIPTION (provided by applicant): including chemokines, cytokines, neurotrophic factors (NTF), reactive oxygen species, and viral proteins. Most of these factors stimulate changes in cell cycle regulatory machinery which determines cellular outcomes in non-neuronal systems. This has led us to propose that cell cycle proteins exhibit altered activity in neurons of patients with HIVE and this activity determines neuronal survival in response to the onslaught of macrophage secreted factors present in the extracellular milieu. We have observed increased inactivation of pRb by phosphorylation (ppRb) and increased cytoplasmic E2F1 in HIVE and SIVE. Using in vitro cultures, NTF and chemokines stimulate increased ppRb and cytoplasmic E2F1, but hydrogen peroxide does not. Because the changes in E2F1 distribution and pRb phosphorylation occur in cells responding to neurotrophic/survival signals, but not in cells responding to oxidative stress, we propose that neurons in the disease with increased ppRb and cytoplasmic E2F1 are "surviving" neurons. This has led us to hypothesize that E2F1 and ppRb determine neuronal viability dependent on their subcellular distribution and interaction partners which is determined by the prevailing signaling cues in the extracellular milieu. The following aims are proposed: 1) To determine whether cytoplasmic E2F1 provides neuroprotection from HIVE-associated toxins, 2) To determine the role of MDMx in regulating cell survival and E2F1 subcellular localization in neurons responding to neuroprotective versus neurotoxic factors, and 3) To determine if post-translational modification of pRb in response to trophic factors occurs on different amino acids as compared to those residues modified in response to toxic factors. These studies will elucidate the role of cell cycle proteins in determining neuronal survival in HIVE and other neurodegenerative diseases with inflammatory components. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Despite the introduction of highly active antiretroviral therapy (HAART), the prevalence of human immunodeficiency virus associated dementia (HAD) has been steadily increasing. Neuronal injury and loss in HAD is initiated by HIV-infected, activated macrophage/microglia via soluble neurotoxic mediators including reactive oxygen species (ROS), excitotoxins, and other metabolites as well as viral proteins. These mediators, which induce oxidative stress, are known to injure neurons directly and alter astrocytic homeostatic functions such as glutamate homeostasis which can lead to excitotoxic neuronal injury. Importantly, several studies in vitro and in vivo have shown that oxidative stress and excitotoxicity are likely critical mechanisms of pathogenesis in HAD. How neurons and astrocytes respond to oxidative stress in HIV infection of the central nervous system (CNS) is largely undefined and underexplored. A major downstream response to oxidative stress is induction of the recently described integrated stress response (ISR) which may be the critical link between oxidative stress and excitotoxicity. Activation of the ISR results in translational attenuation, transactivation of ER chaperone gene promoters, activation of protein degradation pathways and activation of the endogenous antioxidant response. Induction of these responses is regulated by one or more of three distinct signaling pathways: pancreatic endoplasmic reticulum kinase (PERK), IRE1 alpha, and ATF6. Whether one or all of these pathways are induced in neuronal or astrocytic cells during HIV induced CNS injury is not known;however, we have demonstrated that supernatants from HIV infected monocyte derived macrophages (HIV-M/M) induce several components of the ISR including PERK in primary astrocytes in vitro. In addition, we have found that several antiretroviral drugs used in HAART also induce ISR in primary astrocytes. Thus, we hypothesize that that activation of the ISR in astrocytes by HIV M/M and/or HAART exposure is a major determinant of neuronal injury/survival in HAD. Our goal is to determine the role of ISR activation in astrocytes by HIV M/M and HAART therapeutics in mediating neuronal damage in vitro and in vivo in HAD. To do this we will: 1) Determine the mechanism by which ISR activation triggered by HIV-M/M supernatants impacts astrocyte neuroprotective functions and survival, 2) Define the role of HAART drugs in ISR activation in astrocytes, and 3) Define and validate the contribution of chronic activation of the ISR pathways in HAD. By investigating the role of ISR, we expect to uncover a novel mechanism for neuronal dysfunction and loss in HIVE and elucidate the impact of current HAART therapeutics on astrocytic mediated neuronal survival. Patients with HIV associated dementia have increased cellular stress in their brain due to inflammatory infiltration and oxidative stress. These stressors are known activate a protective pathway called the integrated stress response in non-neural cells. This proposal aims to assess the state of activation of the integrated stress response in astrocytes exposed to HIV-infected macrophage products and highly active antiretroviral therapeutic agents and determine the impact of such activation on neuronal survival.

Despite a reduction in the incidence of HIV-associated dementia (HAD) in the era of Highly Active Anti-Retroviral Therapy (HAART), the prevalence of HAD is increasing, as is the frequency of minor cognitive diagnoses in HIV-positive patients. Although HAART is neurotoxic in both the peripheral nervous system and cultured neurons, the contribution of HAART to HIV-associated neurocognitive disorders is unknown. The toxicity of HAART compounds is due to distinct antiretroviral class-dependent mechanisms, such as alteration of protein metabolism and mitochondrial damage-induced generation of ROS (ROS), both of which have been implicated in other neurodegenerative conditions. These mechanisms, as well as HAART drugs themselves, can initiate the endoplasmic reticulum (ER) stress response, a multi-pronged pro-survival signaling pathway that may become deleterious if chronically activated, and autophagy, which has been implicated in neurodegeneration. An understanding of the cellular mechanisms triggered by the different classes of HAART drugs, in both neurons and macrophages, is crucial for guiding the design of new drugs and the assembly of combinational anti-retroviral therapies that minimize neurological impact. We hypothesize that HAART compounds induce neuronal toxicity both directly and indirectly (e.g. alteration of macrophage function), and that these mechanisms are mediated by cell-specific activation of stress responses that differ among classes of HAART compounds. Supporting this hypothesis, we have observed that individual antiretroviral drugs are neurotoxic in CNS cultures and that nontoxic drugs, when applied in recommended HAART combinations, become neurotoxic. We have also demonstrated an increase of downstream indicators of ER stress response activation in neurons and microglia/macrophages of patients with HIV-associated neurocognitive disorders, and in cultures of neurons and monocyte derived macrophages (MDM) following treatment with HAART compounds. Based on these findings, we propose to: 1) Determine the mechanisms of direct neuronal toxicity of HAART drug classes alone and in recommended, therapeutic combinations;2) Determine the mechanisms of direct toxicity of HAART drug classes (alone and in combination) in monocyte derived macrophages, 3) Determine the contributions of HAART-exposed MDM to neurotoxicity. By investigating the cell-specific mechanisms of HAART toxicity in the central nervous system we will uncover an unexplored source for cognitive impairment in HIV-positive patients, identify targets to mitigate side-effects of HAART compounds that effectively control viral replilcation, and create a model to detect neurotoxicity of current and future HAART compounds alone and in combination.

Abstract Neuronal damage and loss in patients with HIV-associated dementia (HAD) is most closely associated with macrophage infiltration and neuroinflammation of the CNS. Most of these factors stimulate changes in cell cycle regulatory machinery which determine cellular outcomes even in the absence of cell division. This has led us to propose the following overarching hypothesis: neurons in HIV-positive patients with HAD exhibit altered cell cycle protein activity and this activity determines neuronal survival in response to factors released by HIV infected macrophages. Induction of cell cycle machinery classically results in increased transcriptional activity of the E2F family of transcription factors leading to the expression of gene products necessary for DNA synthesis and progression to S-phase. Activity of the E2F family is repressed by direct interaction with the Retinoblastoma tumor suppressor protein, pRb, and its family members, an interaction that is disrupted when pRb is hyperphosphorylated. In support of our hypothesis, we have observed increased E2F1 and hyper-phosphorylated pRb in the CNS of patients with HIV encephalitis, in a simian model of disease, and in neurons in our in vitro HIV neurodegeneration model. Surprisingly, E2F1 is localized to the cytoplasm, a site inconsistent with its known transcriptional roles. Altered E2F function is further supported by altered E2F DNA binding activity in SIVE. The consistent and predominant cytoplasmic localization of E2F1 in neurons suggests a novel role for this protein. Interestingly, E2F1 has been reported to bind to a unique RNA hairpin structure and mediate stabilization of at least one mRNA species, Axin 2; however, little is known about this aspect of E2F1 function. We thus hypothesize that E2F1, acting independently of pRb, plays a novel role in modulating neurodegeneration in HIV infection via altered nucleic acid binding activity, subcellular distribution, and calpain cleavage. To test this hypothesis, we propose to: 1) determine the potential cytoplasmic role for the E2F1 nucleic acid binding domain as a mechanism modulating neurodegeneration. 2) determine the ability of calpain-cleaved E2F1 to modulate E2F1 functions such as nucleic acid binding and neuronal survival. 3) determine the role for E2F1 localization in HIV-induced neurotoxicity. Given its potentially unique function and regulation in post-mitotic neurons, we believe E2F1 may serve as a target to prevent neuronal loss in HIV associated dementia.

DESCRIPTION (provided by applicant): Antiretroviral therapy (ART) has led to significant decrease in incidence of the most severe forms of HIV associated neurocognitive disorder (HAND); however, the level of less severe forms of cognitive, behavioral and motor dysfunction have been reported to persist in 30-50% of patients. HIV-associated neuropathology has shifted from a rapidly progressing encephalitic condition to a prolonged neurodegenerative disease with pathologic features including astrogliosis, microgliosis and dendritic damage. However, white matter changes remain a common feature of HAND in the pre- and post-ART era. Intriguingly, a recent transcriptome analysis has shown that genes associated with oligodendrocyte differentiation and myelin production are down regulated in untreated patients with HAND as well as in patients with HAND treated with ART. These findings indicate that HIV and ART may disrupt myelin development and maintenance. However, the effects of ART compounds alone or in combination with HIV-infected cells on the oligodendrocytes and their precursor cells have not been studied. We hypothesize, that ART compounds alter oligodendrocyte differentiation, function, and survival, contributing to the persistence of HAND in the post-ART era. To this end, we have demonstrated that 2 antiretroviral compounds (ARV), one nucleoside reverse transcriptase inhibitor (NRTI), AZT, and one protease inhibitor (PI), ritonavir, alter oligodendrocyte morphology and decrease oligodendrocyte survival in a dose dependent manner in vitro. Further, at subtoxic concentrations, both AZT and ritonavir disrupt maturation of oligodendrocyte precursors (OPCs) in vitro induce oxidative damage and induce the endogenous antioxidant response as indicated by upregulation of heme oxygenase 1 (HO1). Oxidative stress has been shown to alter oligodendrocyte survival and differentiation in both perinatal white matter injury and Multiple Sclerosis models. A recent study has shown that a fumaric acid ester (FAE) with antioxidant properties is efficacious in MS clinical trials. Given th presence of oxidative stress in the CNS of patients with HAND, including those on ART we propose to test the hypothesis that HIV-infected macrophages (HIVMDM) and ARV compounds induce oxidative stress altering oligodendrocyte differentiation, function, and survival in vitro ad in vivo. To test this we will: a) determine the contribution of HIVMDM and ARVs to the development and maintenance of mature oligodendrocytes, b) determine the role of HIVMDM- and ARV-induced oxidative stress in blocking oligodendrocyte differentiation and myelination, c) determine the effect of ARV-induced oxidative stress on oligodendrocytes in vivo, and d) determine the state of oligodendrocyte damage and stress in the context of lentiviral-infection and ART in primates.

? DESCRIPTION (provided by applicant): HIV-Associated Neurocognitive Disorders (HAND) continue to affect ~50% of HIV(+) patients1-4, despite suppression of HIV replication with antiretroviral compounds1-4. With antiretroviral therapy, HAND is now a chronic, progressing disease leading to a spectrum of neurologic disorders. In more severe forms of disease, such as HIV-Associated Dementia (HAD), there is neuronal loss, but in milder forms of disease, synaptic damage is the more salient pathologic change8-14. Both neuronal loss and synaptic simplification are associated with macrophage infiltration and neuroinflammation 15-18, which are believed to be mediated by release of excitotoxins, reactive oxygen species (ROS), cytokines and viral proteins10,15,19-42. Transcriptomic, Proteomic, and Metabolomic approaches have identified several classes of genes and proteins that are dysregulated in HAND, including those regulating innate immunity, inflammasome formation, cell cycle regulation, myelination and synaptic development and function43-51. As synaptic damage correlates best with HAND progression, we are most interested in understanding these changes; however, the mechanisms underlying synaptodendritic damage remain elusive. A particular hurdle to understanding the contribution of gene expression in neurons is the presence of polyribosome tracts in the distal axons and dendrites of neurons where translation is regulated locally to provide rapid changes in gene expression resulting in synaptic plasticity. Local translation, splicing, and stability of RNA as wll as delivery of RNA to these sites require RNA binding proteins (RBPs). It is now clear that RNA regulation by RBPs contributes significantly to regulation of synapse formation, maintenance, and turnover as evidenced by the growing number of genetic disorders of the nervous system with defects in RBPs including Fragile X Syndrome, Frontotemporal Dementia, Spinocerabellar Ataxia, Spinomuscular Atrophy, and Parkinson disease53-55. In order to gain insight into the synaptic changes observed in neurons responding to HIV-associated neurotoxins in HAND, we propose to use a novel, high yield approach, protein interaction profile-sequencing (PIP-Seq), to identify global changes in RNA sequences bound by RBPs in neurons exposed to HIV-associated neurotoxins in an in vitro model of disease, and in the CNS of primates in an in vivo model of lentiviral encephalitis. PIP-Seq employs a novel RNase-mediated protein footprinting strategy in which all unbound RNA is digested by RNAses and RNA sequences protected by RBPs are subjected to deep sequencing. Combining information gained from PIP-seq with bioinformatics approaches, this high risk, high yield approach will provide insights into: the RBP-bound RNA sequences in neurons at baseline, the changes in RBP-bound RNA sequences in neurons responding to HIV-associated neurotoxins, the region of RNA molecules bound by RBP, the conservation of the bound sites across vertebrate species, the overrepresented biologic processes in which differentially RBP-bound RNAs function, the ability to distinguish putative SNPs linked to changes in RBP activity, and the means to identify known and novel RBPs that regulate RNAs linked to HIV-associated changes. Finally, identified RBP-bound RNA sequences will be compared with previous transcriptomic and proteomic results to gain insight into the mechanisms underlying changes in observed gene expression in HAND. This novel, high risk, high yield approach stands to provide valuable insight into changes in neuronal responses in HAND and in diseases beyond, and will provide the underpinnings for studies to increase both our understanding of basic neuronal function and how disease processes impact that function.

? DESCRIPTION (provided by applicant): Clinical intervention over the past two decades has changed HIV infection from a death sentence to a chronic disease. Despite pharmacological management of HIV-infection, approximately 50% of patients continue to present with neurologic impairment termed HIV-associated neurocognitive disorders (HAND), with persistent neuroinflammation and oxidative tissue damage. Current approaches to mitigate HAND have focused on agents targeting a single cellular function, which may limit their efficacy in a complex multisystem disease. Therefore, novel, pluripotent agents capable of acting simultaneously on diverse pathways and cellular processes may present an opportunity in the short and long term management of HIV. Thus, brain penetrant agents capable of controlling oxidative and inflammatory processes with minimal toxicity over prolonged administration to humans are attractive candidates in mitigating or preventing such effects associated with HAND. Our group has been investigating protective properties of secoisolariciresinol diglucoside (SDG), the main bioactive lignan phenolic in wholegrain flaxseed. Dietary SDG boosts endogenous antioxidant defenses in murine tissues, and exhibits potent anti-inflammatory and antioxidant activity in several tissues including the CNS. SDG and its metabolites can cross the blood-brain barrier (BBB) and enter brain tissues, making it an attractive candidate for mitigation of HIV-induced neurotoxicity. We therefore hypothesize that SDG, as a pluripotent agent, will mitigate HIV-associated neurotoxicity by reducing BBB damage, neuroinflammation, and oxidative stress. SDG is safe and well tolerated in humans and findings from numerous recent clinical trials enhance the translational aspect of our proposed study, which aims to provide novel insights into the usefulness of SDG, and to elucidate its mechanisms of action. Using in vitro modeling, Aims 1-3 will determine the effects of SDG on BBB damage, leukocyte trafficking across the BBB, microglial activation and neurotoxicity induced by HIV infection, and will elucidate mechanisms that underlie the efficacy of SDG as an agent that reduces neuroinflammation and oxidative stress. Aim 4 will investigate the ability of SDG to mitigate BBB damage, neuroinflammation, oxidative stress and neurotoxicity in the humanized mouse model of HIV infection, the huNSG-HIV mouse. Together, these studies will provide evidence to support SDG as adjunctive treatment for management of HAND.

HIV-Associated Neurocognitive Disorder (HAND) continues to affect ~50% of HIV(+) patients despite widespread implementation of antiretroviral therapy (ART) and successful viral suppression. Several risk factors including older age, low nadir CD4+ cell count, metabolic syndrome, depression and hepatitis C co-infection associate with HAND. Early interventions and complete viral suppression are most likely to improve neurocognitive (NC) prognosis; the Mind Exchange Program recommends consideration of risk factors for diagnosis and treatment within the first six months. Many of the mechanisms implicated in HAND persistence, including unremitting CNS viral replication, age-related pathologies, comorbidities (e.g., drug abuse, ART-associated toxicities) can induce the unfolded protein response (UPR), which results in activation of one or more of the 3 initiators, PERK, ATF6, and IRE1?, when cellular stressors disrupt their binding to the chaperone, binding protein (BiP), with wide ranging consequences. Most pertinent here is PERK-mediated phosphorylation of eukaryotic translation initiation factor 2? (eIF2?) slows global translation, while selectively enhancing translation of a subset of genes including the beta-site amyloid precursor protein cleaving enzyme 1 (BACE1). Chronic and/or sustained UPR activation may have detrimental outcomes, supported by multiple studies. Consistently, UPR is implicated in several neurodegenerative diseases, including Alzheimer and Parkinson. We previously showed UPR activation in HAND prefrontal cortex. Based on multiple lines of evidence from in vitro and in vivo models of HIV-induced neurodegeneration, suggesting that the dysregulation of the PERK arm of the UPR pathway may be contributing to HIV- as well as antiretroviral-mediated neurodegenerative processes, we propose a role for PERK dysregulation as a mechanism for the continuing neuronal perturbations still observed in HAND patients despite ART?s success. We propose that PERK activation contributes to HAND development, and a genetic variant of PERK with increased activity is a predictive risk factor for HAND. In this application, we will examine whether a protein-coding PERK haplotype resulting from three single nucleotide polymorphisms (SNPs), which may confer increased kinase activity, may underlie the alteration/s of PERK?s protein function and/or changes in its amount. We will determine: 1) the mechanisms of PERK-mediated neurotoxicity in vitro, 2) the mechanisms of PERK-mediated neuronal injury in a preclinical rodent model of HIV-induced synaptic damage, gliosis and behavioral/cognitive deficits, and 3) the associations between PERK genetic haplotype and/or expression with HAND risk in human adults.

Major neuropsychiatric disorders, including Aids Dementia, Alzheimer's Disease, depression and schizophrenia, may involve aberrant phagocytosis of cerebral cortical synapses by monocytes and/or microglia. However, efforts to target this process for therapeutic intervention have been limited. One reason for the limited success in the development of therapeutic interventions for CNS microglia/macrophage activation is that species differences between the immune systems of humans and model organisms render the co-culture of human immune cells with non-human neurons less informative. Fortunately, recent progress in generating cortical cells, including neurons, astrocytes and microglia, from human stem cells provides a new opportunity to address this challenge. This proposal is a collaborative effort between the labs of Kelly Jordan-Sciutto, who studies neuro-AIDS and monocyte activity in vitro, and Stewart Anderson, who studies forebrain development in relation to neuropsychiatric disorders using mouse and human stem cells. Differentiation of iPSCs into forebrain-like excitatory neurons (iNrns), and their co-culture with astrocyte-like cells (iAstrs), results in a synaptically dense network. By plating human monocytes over those neurons we observe IBA1+ cells that appear to have ingested synaptic markers. Moreover, we are able to generate human microglia like cells (iMgl), infect them with HIV, and find that they react to this infection by cytokine release and the formation of multinucleated cells. Importantly, exposure to an antiretroviral agent reduces their reverse transcriptase activity. We also find that we can co-culture iMgl together with i-Nrns and i-Astrs. In this exploratory R21 we will fine-tune this human iMgl, iAstr, iNrn culture system to test the hypothesis that HIV infected microglia decrease excitatory synapse density in vitro. If so, we will determine whether the effect is mimicked by conditioned media, is associated with increased compliment deposition, and is affected by ART. We will also test whether viral suppression occurs in the iMgl within the triculture context. Success would lead to new approaches for mechanistic studies and therapeutics development for the multiple neuropsychiatric disorders, such as HIV Associated Dementia, in which monocyte/microglial activation may play a crucial role in their pathogenesis.

Despite effective viral control by Antiretroviral therapy (ART), HIV associated neurocognitive disorder (HAND) persists in 30-50% of patients. In the ART era, neuropathology has shifted from a rapidly progressing encephalitic condition to a prolonged neurodegenerative disease with pathologic features including astrogliosis, microgliosis, dendritic damage, and especially white matter deficits. White matter alterations include decreased myelin sheath thickness, myelin lesions, and abnormal myelin protein expression. The severity of white matter damage correlates with the amount of time on ART therapy. Transcriptome analysis of HIV patients on ART revealed decreases in genes associated with oligodendrocyte maturation and myelination. These myelin deficits are especially critical in the pediatric population in which myelination is in progress and is necessary to establish critical neural connections that affect motor control, cognition and behavior. Children with vertically transmitted HIV on ART therapy still exhibit memory loss, lack of motor control and executive function and demonstrate white matter deficits and ventricular enlargement by imaging. However, the actual cell and molecular effects on myelin generation caused by products of HIV or the antiretroviral drugs used to treat it have not been studied. The findings in our preliminary studies are consistent with the data from children. Using supernatants from HIV infected macrophages, we show inhibition of the differentiation of oligodendrocytes and myelin protein synthesis in a well-characterized purified tissue culture system. Additionally, we tested select drugs from three classes of antiretrovirals, the protease inhibitor class, the nucleoside reverse transcriptase class, and the integrase inhibitor class and found that select agents inhibit differentiation of oligodendrocyte precursors to mature oligodendrocytes in culture in a dose-dependent manner, independent of cell death. Intravenous administration of ritonavir to mice for only two weeks significantly decreased the expression of several myelin proteins. Further, myelin basic protein (MBP) was significantly decreased in the cortex of adult HIV patients who were on ART and exhibited HAND. We hypothesize that the products of the HIV virus as well as a subset of ART compounds inhibit developmental myelination resulting in motor, behavioral and cognitive deficits. To test this hypothesis, in the first aim we will use the HIV transgenic rat. This animal model of HIV displays deficits in learning and memory and a transcriptome analysis found decreases in key myelin proteins however the actual effects on myelination have not been studied. We will assess developmental myelination by histology, immunochemistry, myelin expression and electron microscopy of myelin structure. In Aim #2, we will determine if the antiretrovirals inhibit or retard developmental myelination in both normal rats and HIV rats during the period of greatest myelin development. Results from these aims should help devise more rational drug therapies without myelin deficits to reduce HAND.

PROJECT SUMMARY HIV-Associated Neurocognitive Disorder (HAND) persists in up to 50% of the HIV-positive population, often in spite of low or undetectable viral loads due to effective antiretroviral therapy (ART). Microglia are the resident macrophages in the central nervous system (CNS) and are thought to be a persistent reservoir of HIV in ART treated patients. Microglia secrete a panoply of factors that impact neuronal function and these secreted factors are altered during HIV infection of microglia. In addition to HIV virus-host interactions mediated by microglia, many exogenous factors can modify these interactions or directly impact the CNS itself, making it difficult to dissect the underlying causes of neural pathology leading to HAND. To establish a physiologically- relevant platform to investigate the dynamics of HIV infection in the context of ART and other co-factors, we will use human induced pluripotent stem cells to generate human microglia that we can co-culture with our well-characterized 3D model of the developing cerebral cortex. This integrated 3D cerebral organoid model will recapitulate many of the critical cell populations thought to contribute to HAND-related pathology including glutamatergic neurons, astrocytes, and microglia. We will evaluate the efficacy of the cerebral organoids to model HIV infections by measuring HIV replication kinetics and suppression, as well as HIV protein expression in different cell types (Aim 1). We will then measure the impact of HIV on neuronal development and function at several time points following acute exposure (Aim 2). We will perform morphological and electrophysiological analyses and RNA sequencing at both the single cell and population level to determine the cell type-specific transcriptional responses to HIV exposure. Finally, we will evaluate the impact of exogenous drugs on these measures of viral replication and neuronal function (Aim 3). As a proof-of-concept, we will test a preferred regimen of ART, as well as cannabinoids, both with and without HIV exposure to begin to dissect the impact of these common co-factors on cellular properties that may contribute to pathology underlying HAND. Successful completion of these experiments will result in a validated platform to model the dynamics of neuroinflammatory disorders using brain region-specific cerebral organoids integrated with human microglia.

Project Summary The objective of the University of Pennsylvania (Penn) Post-baccalaureate Research and Education Program (PennPREP) is to guide recent college graduates from underrepresented (UR) groups into graduate school to pursue biomedical research. At least 9 UR scholars are identified each year who are interested in biomedical research but lack the experience or expertise necessary for graduate school training. Each scholar is matched with a research mentor among the graduate training faculty and provided with a significant one year (or possibly longer) research project. An Individual Student Development Plan (IDP) is developed for each scholar to ensure that the scholar undertakes training appropriate to his or her own scientific needs and interests, including graduate or advanced undergraduate coursework. Scholars also develop the skills necessary for success in graduate school by completing workshops in biostatistics, grant writing, the responsible conduct of research, critical analysis of scientific literature, and oral and written presentation skills. Scholars also participate in a one- on-one writing workshop with a professional writing instructor. Scholars meet weekly as a group to discuss scientific journal articles and their own research, in order to further develop their skills in the critical evaluation of research and the delivery of scientific presentations, as well as to increase their exposure to research outside their own laboratory. In addition, scholars receive advising for the graduate school application process, including selecting programs, writing application essays, and practicing interviews. If necessary, scholars will take a GRE or MCAT preparation course. Scholars also participate in various lunches and seminars with Penn faculty, postdoctoral fellows and graduate students who share their training experiences, on-going research, and academic paths. As of 2012, scholars meet annually with scholars, faculty, and advisors of PREP programs at other institutions in order to network and gain additional perspectives. In addition, all PREP scholars are expected to attend a national conference each year, such as ABRCMS or a discipline-specific meeting as an additional means of broadening exposure to research and establishing networks. Several opportunities are in place for PREP alumni to remain engaged with the program including: (1) PREPConnect, via which PREP alumni connect virtually (e.g. Skype), once/month with current scholars to offer mentoring and guidance, and (2) the PREP Alumni Advisory Board, which will meet twice/year to evaluate current programming initiatives, events and meetings. Mechanisms are in place for measuring the effectiveness of the program's various components and improving the training experience over time. The ultimate measure of the program's success is the percentage of scholars who are admitted to PhD or MD-PhD training programs and pursue a research career. Over the past 13 years, 84% (62/74) of PennPREP scholars who have successfully completed the program have been admitted to PhD or MD-PhD programs. Nineteen of these PennPREP alumni have received the PhD and moved on to post-doctoral training and/or research careers.

Abstract: Antiretroviral therapy (ART) has dramatically extended the lives of people living with HIV (PLWH); however, they continue to experience a plethora of co-morbid conditions including neuronal disorders and pain. Between 40 and 72% of PLWH use cannabis to mitigate anxiety, stress, ART side effects, pain, and/or for pleasure with over 55% of patients using cannabis at least daily. Interestingly, a recent study found that people who use cannabis heavily had reduced inflammatory signatures in PLWH on ART. These and a numerous other studies support the anti-inflammatory and immunomodulator effects of phytocannabinoids in a number of organ systems including heart, colon, kidney, liver and the gut; however, their medicinal use is confounded by the psychotropic activities. Efforts to separate the anti-inflammatory effects from the psychotropic effects have revealed differential activities of 3 endogenous receptors including cannabis receptor 1 (CB2) CB2 which exhibit differential tissue expression and agonism with endo- and phyto-cannabinoids. Several reports have shown that cannabinoids attenuate HIV infection and/or replication in T-cells, macrophages, dendritic cells and human fetal microglia cultured ex vivo. However, the effect of cannabinoids on HIV infection of microglia in the context of ART and the normal cellular environment of neighboring neurons and astrocytes in the CNS has not been examined. Several studies specifically implicate CB2 agonism which has been shown to have anti- inflammatory properties in the heart, gut, experimental autoimmune encephalitis and neuropathic pain via inflammasome activation. This has led us to hypothesize that Cannabinoid signaling influences HIV infection and chronic inflammation in the presence of ARV in the central nervous system by attenuating the inflammasome. In order to examine HIV infection in the context of cells of the CNS, we have developed a human induced pluripotent stem cell tri-culture model composed of iNeurons, iAstrocytes, and iMicroglia. This model recapitulates several key aspects of HIV infection in the CNS including increased cytokine production, oxidative stress response, inflammatory signaling, and integrated stress response. ARV treatment reduces HIV infection and inflammatory signaling pathways; however, a subset of pathways remain elevated despite viral suppression. We propose to further develop this model to determine the ability of cannabinoids to modulate HIV-induced inflammation and subsequent neuronal dysfunction via reducing inflammasome activation by: 1) Determining the effect of cannabinoids on chronic HIV infection and ART in the context of iMgl/iNrn/iAstr triculture. 2) Determining the effect of cannabinoids on cytokine levels, inflammatory gene expression profile, and microglial activation in iMgl/iNrn/iAstr triculture. 3) Determining the effect of cannabinoids on neurons and astrocytes in HIV infection and ART in iMgl/iNrn/iAstr triculture.