Dennis L. Kolson - US grants

The proposal describes a research program which will examine the biological effects of human immunodeficiency virus type 1 (HIV-1) on neuronal cells, while providing fundamental training in virology and neurobiology which will lead to an independent research career in neurovirology. The AIDS Dementia Complex (ADC) is the most common CNS manifestation of HIV-1 infection. Neuropathological studies indicate that macrophages, rather than neurons, are the major site of productive infection in the CNS, suggesting indirect effects of infection on neuronal cells. Potential mechanisms include HIV-1 proteins produced and released by infected cells, as well as macrophage secretion products, that could effect neuronal cell function. A likely candidate mediator is the HIV-1 regulatory protein, tat (trans-activator or transcription) which is known to be released by some infected cells and can effect cell function when applied to uninfected lymphocytes. In preliminary studies we have shown that tat also induces morphological changes in primary brain cell cultures, and may provide a link between HIV-1 infection and neuronal cell dysfunction. Our hypothesis is that HIV-1 gene products and cellular products produced by infected macrophages within the CNS can affect neural cell function, and play a role in the pathogenesis of ADC. During Phase I of this program we will examine the effect of tat on primary rat brain cultures and transformed human neuronal cell lines. We will investigate the ability of tat to affect neural cell gene expression using transformed human neural cell lines. In addition, we will utilize a mixed cell co- culture system of primary rat brain cells and human monocyte-derived macrophages to study directly the interaction of products of HIV-infected macrophages with neuronal cells. In Phase II, we will employ the techniques and models developed in Phase II to examine the effects of other accessory HIV-1 gene products on neural cell functions. We will utilize primary rat brain cells and transformed neural cell lines to examine the effects of these gene products on our neural cell growth, morphology, and neural specific gene expression, as well as electrical activity. These studies utilizing our primary brain cell/macrophage co-cultivation system and transformed cell lines should aid in further understanding HIV-1/neural cell interactions, and help clarify the mechanism(s) of HIV-1 induction of CNS dysfunction.

The proposal describes a research program which will examine the biological effects of human immunodeficiency virus type 1 (HIV-1) on neuronal cells, while providing fundamental training in virology and neurobiology which will lead to an independent research career in neurovirology. The AIDS Dementia Complex (ADC) is the most common CNS manifestation of HIV-1 infection. Neuropathological studies indicate that macrophages, rather than neurons, are the major site of productive infection in the CNS, suggesting indirect effects of infection on neuronal cells. Potential mechanisms include HIV-1 proteins produced and released by infected cells, as well as macrophage secretion products, that could effect neuronal cell function. A likely candidate mediator is the HIV-1 regulatory protein, tat (trans-activator or transcription) which is known to be released by some infected cells and can effect cell function when applied to uninfected lymphocytes. In preliminary studies we have shown that tat also induces morphological changes in primary brain cell cultures, and may provide a link between HIV-1 infection and neuronal cell dysfunction. Our hypothesis is that HIV-1 gene products and cellular products produced by infected macrophages within the CNS can affect neural cell function, and play a role in the pathogenesis of ADC. During Phase I of this program we will examine the effect of tat on primary rat brain cultures and transformed human neuronal cell lines. We will investigate the ability of tat to affect neural cell gene expression using transformed human neural cell lines. In addition, we will utilize a mixed cell co- culture system of primary rat brain cells and human monocyte-derived macrophages to study directly the interaction of products of HIV-infected macrophages with neuronal cells. In Phase II, we will employ the techniques and models developed in Phase II to examine the effects of other accessory HIV-1 gene products on neural cell functions. We will utilize primary rat brain cells and transformed neural cell lines to examine the effects of these gene products on our neural cell growth, morphology, and neural specific gene expression, as well as electrical activity. These studies utilizing our primary brain cell/macrophage co-cultivation system and transformed cell lines should aid in further understanding HIV-1/neural cell interactions, and help clarify the mechanism(s) of HIV-1 induction of CNS dysfunction.

HIV-associated dementia complex (HADC) is characterized by profound cognitive impairment and variable pathological changes. While one mechanism of neurologic dysfunction is believed to be cell loss resulting from release of toxins from HIV-infected macrophages and microglia, clinical signs are often not correlated with the degree of neuronal loss. Moreover, since marked neurologic improvement occurs with antiretroviral therapy, functional abnormalities in neurons may be important in addition to cell death. In addition to macrophage infection, recent in situ PCR studies have demonstrated restricted infection in neurons and astrocytes. Our hypothesis is that alterations in neural cell functions, as well as cell death, result from productive HIV-1 infection of macrophages/microglia and restricted infection in astrocytes and neurons, and that both neuronal dysfunction and cell death contribute to neurologic dysfunction in HADC. To study functional responses of human neurons relevant to the pathogenesis of HADC, we have developed a unique human neuronal cell culture system and panel of assays for specialized neuronal cell functions which can be applied in mixed co-cultures of neurons, primary macrophages, and astrocytes. Our goal is to better understand the functional and toxic responses of neurons to HIV-1 infection in the brain and how these responses are modulated through glial elements. The First Award proposal builds upon my background in neurotransmitter metabolism, HIV virology, and development of this unique system for functional analysis of differentiated human neurons. First, we will identify alterations of neuronal functions that result from HIV-1 infection in macrophages, and determine how such infection may ultimately kill neurons. This involves a detailed analysis of neurotransmitter enzyme expression and high-affinity neurotransmitter uptake in neurons, utilizing our recently developed glial/neuronal culture system. Second, we will define the role(s) of astrocytes in modulating neuronal functions. To do this, we will analyze effects of viral gene expression within astrocytes on astrocytic and neuronal functions, utilizing viral vectors and co-cultivation with HIV-1 infected macrophages. Third, we will determine the functional consequences of direct HIV-1 infection of neurons. We will utilize chronically-infected clonal neuronal cell derivatives to analyze effects of infection on cell functions in differentiating neurons and their modulation through glial contact. Because neurological dysfunction may occur independently of cell death it is critical to examine neuronal cell functional responses to HIV-1 infection in the brain. It is our expectation that these studies will provide a foundation for, ultimately, the rational development of novel therapeutic approaches to preservation of neurological function in HADC.

This study (ACTG 242) was a multicenter trial sponsored by the National Institute of Allergy and Infectious Diseases and was designed to determine the safety and efficacy of amitriptyline versus mexiletine in the treatment of painful peripheral neuropathy in AIDS. The two primary endpoints were 1) change of mean pain intensity score between baseline and eight weeks of treatment; and 2) assessment of drug-related toxicity. Patients (240 anticipated from all participating centers) were randomized in a double-blinded manner to one of three treatment arms: placebo, amitriptyline, or mexiletine and treated for ten weeks. Response to treatment was assessed by neurological examination and patient responses to standardized self-assessment pain questionnaires. However, interim analysis (137 patients) conducted during enrollment of our ACTG site (University of Pennsylvania) indicated no benefit above that seen in placebo-treated patients and the study was terminated on 1/3/97.

neurotransmitter antagonist; human therapy evaluation; NMDA receptors; AIDS dementia complex; drug screening /evaluation; brain disorder chemotherapy; AIDS therapy; clinical research; human subject;

DESCRIPTION (adapted from the Abstract): HIV-associated dementia complex develops in 20% of AIDS patients and has been linked to release of neurotoxic metabolites and cytokines by infected macrophages and microglia in the brain. Recently, macrophage infection has also been associated with release of certain chemokines, both in vitro and in the brain of ADC. Little is known about the role of chemokines and chemokine receptor interactions in the pathogenesis of ADC. The Principal Investigator and his associates have demonstrated functional chemokine receptors on human neurons in vitro and chemokine receptors on neurons and microglia in vivo. Further, they found that chemokines modulated certain neuronal glial function in vitro and that CNS HIV strains differ in their abilities to induce chemokines in macrophages. To test the hypothesis that chemokines induced by HIV-1 within the CNS contribute to the pathogenesis of ADC through chemokine receptor/ligand interaction, the Investigator proposes three specific aims: (1) to determine the distribution of selected chemokine receptors in CNS cell type, and to identify specific chemokine production within the CNS, by analysis of primary glial cells cultured from human brain, cerebrospinal fluid from HIV-1 infected patients, and autopsied brain specimens; (2) to define the effects of specific chemokines on functional neurotransmitter pathways and other relevant functions in neurons and glia, determining the effects of chemokines on neurotransmitter uptake and enzyme expression, glial cell chemotaxis, and neuronal calcium fluxes; (3) to analyze differences between primary CNS isolates for patients with and without ADC in inducing chemokine production.

HIV-1 infection of CD4-expressing cells requires interaction between envelope, CD4 and one or more chemokine receptors (seven transmembrane-spanning G protein-coupled receptors). Infection requires envelope binding to both CD4 and chemokine receptor, followed by fusion of virus and cell membrane. In the brain, infection occurs in macrophages and microglia, which express CCR5, CXCR4, and CCR3chemokine receptors. We have demonstrated that effective interaction of envelope with such receptors occurs when CD4 and chemokine receptor are expressed on the virion and envelop is expressed on the target cell. We showed that such CD4/chemokine receptor pseudovirions specifically target HIV-1 infected cells, as predicted by envelope utilization patterns by prototypic HIV-1 strains. In addition, we have used HIV-based vector pseudotypes to express transgenes in human astrocytes and neurons. Our hypothesis is that HIV-1 based vectors utilized as chemokine receptor/CD4 pseudovirus can target HIV-1- infected cells to deliver antiviral and toxicity genes to limit infection and potentially eliminate infected cellular reservoirs in the central nervous system (CNS), and that HIV-1 based vectors also allow introduction of neuroprotective genes to limit HIV-1- induced neurotoxicity. Our goal is to develop HIV-1-based pseudotype strategies to eliminate CNS reservoirs for HIV-1 and to protect neurons form effects of HIV-1 infection. To do this we will: 1) Determine the efficiency of chemokine receptor/CD4 pseudotypes (reverse pseudotypes) in transducing and killing HIV-1 infected macrophages. We will utilize reverse pseudotypes expressing a pro-apoptosis genes construct to induce apoptosis gene construct to induce apoptosis in macrophages infected with primary CNS HIV-1 isolates with different chemokine receptor utilization patterns; 2) Define the ability of HIV-1- based pseudotypes to transduce human neurons, astrocytes and macrophages to establish neuroprotective strategies against HIV-1. We will utilize pseudotype transduction of primary CNS cells to test anti-apoptosis genes as neuroprotectants; and 3) Determine the ability of pseudotypes selected from specific aims 1 & 2 to transduce primary CNS cells in vivo and to target HIV-1 infected macrophages in vivo. In collaboration with our Neuropathology core and Dr. Kerr, we will utilize a SCID-hu mouse to establish a model for HIV-1 infection of the brain to study vector transduction in vivo. The ability of HIV-based vectors to target and express in post-mitotic cells suggests new approaches for the treatment and prevention of AIDS dementia.

DESCRIPTION (provided by applicant): Neuronal apoptosis is a hallmark of AIDS dementia complex (ADC), and may represent a final common pathway of cell death in response to neurotoxins released from HIV-infected macrophages(HIV/MDM). Some forms of neuronal apoptosis are blocked by certain anti-apoptosis members of the bct-2gone family (Bcl-2 & Bcl-xL), which block the intrinsic (mitochondrial-associated) pathway. Neuronal apoptosis may also be blocked by activation of AKT/protein kinase B signaling (AKT/PKB), which acts at points involving the intrinsic and extrinsic (death receptor-associated) apoptosis pathways. How the intrinsic and/or extrinsic apoptosis pathways are involved in neuronal apoptosis in ADC is unknown, and this represents an important gap in our understanding of HIV neuropathogenesis and neuroprotective strategies. We have developed a novel in vitro model for HIV-induced neuronal apoptosis based on a unique human neuronal cell line (NT2.N). We found that Bcl-2 & Bcl-xL as well as activators of neuronal AKT/PKB block HIV/MDM-induced neuronal apoptosis. We have also demonstrated expression of the newly described seven-transmembrane HIV co-receptor APJ in neurons, and found that its native ligand, apelin, activates AKT/PKB and blocks such apoptosis. Finally, we have used a novel single-cell mRNA amplification technique to analyze gene expression in apoptotic neurons in vivo. Our hypothesis is that HIV induced neuronal apoptosis is mediated through specific pathways involving both intrinsic & extrinsic apoptosis mechanisms that may be differentially induced by HIV/MDM and HIV proteins, and that APJ activated signaling may modulate these pathways and mediate neuroprotective responses against effectors of HIV-associated apoptosis. Our aim is to better understand the pathways of HIV-induced neuronal apoptosis and the mechanisms of apelin/APJ neuroprotection, through our in vitro model and analysis of gene expression in apoptotic neurons in rive. We will: 1) Determine the pathways of neuronal apoptosis induced by HIV/MDM &: associated neurotoxins; 2) Define the neuroprotective signaling pathways for APJ/apelin against HIV/MDM; and 3) Determine the expression of genes modulating intrinsic & extrinsic apoptosis pathways and neuroprotective pathways in defined subclasses of apoptotic neurons in HIV-infected brain.

The cardinal pathological features of HIV-associated dementia (HAD) include astrogliosis/activation, macrophage/microglia (M/M) activation, synaptic damage, and apoptosis of neurons and astrocytes. The mechanisms of M/M activation in HAD remain uncertain, but factors released by HIV-activated M/M (glutamate, Fas-L, TNF-alpha, viral proteins, others) are likely responsible for astrocytic & neuronal injury through multiple mechanisms. Moreover, astrocyte activation and injury are critical in amplifying neuronal damage, most likely through impairment of astrocyte high-affinity glutamate scavenging and related neuroprotective functions. The mechanisms by which HIV/MM injure astrocvtes have received relatively little attention despite the evidence that this is critical in HAD pathogenesis. Our hypothesis is that HIV-activated M/M induce both astrocyte and neuronal injury and apoptosis as well as astrocyte activation and associated dysfunction; and that this exacerbates neuronal injury through loss of neuroprotective functions such as glutamate scavenging. We have developed in vitro models for neuronal and astrocytic apoptosis and dysfunction and have shown that mitochondrial-mediated (intrinsic) apoptosis triggered by HIV-M/M excitotoxins defines a major pathway by which HIV-M/M injure neurons. We also established a unique human astrocyte cell model with inducible trans-gene expression, and have modulated mitochondrial-mediated astrocyte apoptosis through Bcl-2 expression. Furthermore, we found that Fas-L, which is unregulated in HAD brain, suppresses glutamate transport in astrocytes. and that TNF-alpha suppresses glutamate transport in astrocytes and enhances transport in M/M. Finally, we have carried out in vivo studies using a novel single cell mRNA amplification/gene profiling approach to begin to define gene expression patterns of individual brain cells in HAD. Our goal is to utilize our in vitro models to define the pathways of astrocyte activation and associated dysfunction & apoptosis elicited by HIV/MM, and exploit our in vivo gene expression analysis approach to define the pathways activated in vivo in HAD. We will: 1) Identify the mechanisms and pathways of astrocyte activation and apoptosis induced by HIV-M/M and the role of Fas death receptors; 2) Determine the mechanisms of death receptor (Fas, TNF) modulation of high-affinity glutamate transport in astrocytes and M/M and the effects on neuronal survival; and 3) Define the in vivo pathways of astrocyte apoptosis and the patterns of activation regulating high-affinity glutamate transporter expression in HAD using our single-cell mRNA analysis to validate and extend in vitro findings. Understanding the mechanisms of astrocyte injury in HAD may identify additional targets for comprehensive neuroprotection in HAD.

DESCRIPTION (provided by applicant): Neuronal damage in HIV-infected brain resulting in HIV-associated dementia (HAD) is thought to occur in part through oxidative stress-associated N-methyl-D-aspartate receptor (NMDAR) excitation initiated by HIV infection of brain macrophages. Brain regions enriched in NMDAR expressing the NR2A and NR2B subunits, such as the hippocampus, are particularly vulnerable to HIV. HIV proteins as well as glutamate, quinolinic acid (QUIN), platelet activating factor (PAF) and reactive oxygen species (ROS) released from activated glia can act as direct or indirect triggers for HIV-induced NMDAR-mediated excitotoxicity. In our initial funding period, we used our NT2.N neurotoxicity model to show that HIV-1-infected monocyte-derived macrophages (HIV/MDM) trigger the NMDAR-activated intrinsic (mitochondrial) apoptosis pathway. Furthermore, our recent study using our primary rodent hippocampal culture model demonstrates that HIV/MDM release excitotoxins that kill neurons by NMDAR-mediated calpain activation, and that hippocampal vulnerability is determined by NR2A and NR2B subunit expression. Our hypothesis is that HIV/MDM-induced NMDAR activation, altered NR subunit phosphorylation &localization are associated with macrophage stress responses and determine hippocampal neuronal vulnerability to HIV induced damage. Our studies of HIV/MDM neurotoxicity and the roles of NMDAR and the macrophage integrated stress response (ISR) have shown that i) hippocampal neuronal vulnerability is age-dependent and is determined by NR2A and NR2B expression patterns;ii) HIV/MDM rapidly induce neuronal calpain and cdk5 activation, altered NR2B phosphorylatioh &cleavage, and calpain- and cdk5-dependent death;iii) HIV replication in MDM induces the macrophage integrated stress response (ISR);and iv) the ISR is activated in macaque brain and human brain in individuals infected with immunodeficiency virus. To better define how HIV infection of macrophages triggers neurodegeneration cascades and to identify new targets for neuroprotection we will: 1) Define the pathways of calpain-induced NMDAR-mediated neurotoxicity induced by HIV/MDM and the functional roles for NR2 subunits;2) Determine whether specific phosphorylation and synaptic localization of NMDAR modulate HIV/MDM neurotoxicity and whether similar NR phosphorylation patterns are expressed in HAD;and 3) Determine the role of the macrophage Integrated Stress Response (ISR) in HIV/MDM-induced neurotoxicity. These studies offer appealing targets at multiple steps in the neurodegeneration cascade initiated by HIV infection that can potentially be exploited for identfication of currently available drugs and for the development of new neuroprotective strategies that can be effective against HIV-induced neurodegeneration. Project Narrative: This project investigates the mechanisms by which HIV-1 infection of macrophages induces neurodegeneration, using an in vitro infection model and pathological analyses of HIV-infected human brain and simian immunodeficiency virus (SIV)-infected macaque/monkey brain tissues. Our hypothesis, based on our preliminary studies, is that such infection alters the macrophage integrated stress response (ISR), leading to excitotoxin production and neuronal NMDA receptor activation by mechanisms that determine why selective neuronal populations are damaged in HIV-1 infection. We are attempting to validate our in vitro observations in human brain tissue and in the SIV macaque model so that neuroprotection studies can eventually be rationally designed for testing in the macaque model.

DESCRIPTION (provided by applicant): HIV-associated neurocognitive disorders (HAND) remain prevalent despite the use of antiretroviral therapy (ART), and CNS inflammation & neurodegeneration associated with HIV replication in macrophages/microglia remain as neuropathological features. Persistent systemic inflammation & monocyte activation, CNS inflammation, macrophage activation, correlate with HAND in patients on ART. Thus, although ART is the backbone of HIV therapy, there is a critical need for adjunctive therapies to suppress persistent inflammation and virus replication, and to decrease the high burden of HAND- associated disability. Accordingly, drugs that suppress inflammation and HIV replication systemically and within the CNS are especially attractive as adjunctive neuroprotectants. We are proposing a dual-investigator MPI study (Kolson, Aldvoni) to investigate a novel drug, dimethyl fumarate, (DMF, Fumaderm(R)), now in a phase III clinical trial for multiple sclerosis) as a candidate neuroprotectant for HAND. Using our HIV neurotoxicity model we found that DMF and its primary in vivo metabolite, MMF, suppress a) HIV replication, b) associated inflammatory responses, and c) neurotoxin production in monocyte- derived macrophages (MDM). DMF/MMF also d) induces monocyte antioxidant responses and e) suppresses chemotaxis. In addition, using transcriptome analyses of HIV-infected T lymphocytes and MDM, we also demonstrated that HIV reprograms host gene expression in a cell-dependent manner to modulate pathways of virus spread, inflammatory mediators, and apoptosis, which can intersect pathways of HIV/MDM neurotoxin production & neurodegeneration. Because DMF is orally-deliverable, CNS- penetrating, and minimally toxic, we hypothesize that DMF can be an effective neuroprotectant in HAND and we further hypothesize that transcriptome analyses can identify host pathways modified by DMF/MMF that underlie its neuroprotection. We will: 1) Define mechanisms of DMF/MMF suppression of HIV replication and MDM neurotoxin production by HIV replication and Tat expression~ 2) Define DMF/MMF effects on suppression of monocyte & macrophage activation through anti-oxidant responses & other pathways~ 3) Determine mechanisms of DMF/MMF modulation of monocyte chemotaxis & transendothelial migration~ and 4) Determine the ability of DMF/MMF to suppress monocyte activation and induce antioxidant responses in SIV-infected rhesus macaques. This should provide a rationale for a future clinical trial in HIV patients.

DESCRIPTION: HIV associated neurocognitive disorders (HAND) remain prevalent (~50%) despite remarkably improved patient survival due to improved antiretroviral therapy (ART) regimens. In HAND, CNS neuroinflammation & oxidative stress persist despite ART and continue to contribute to neuropathogenesis, which emphasizes the critical need for identifying host targets for adjunctive therapy for HAND prevention. We recently identified the cellular detoxifying/antioxidant enzyme, heme oxygenase-1 (HO-1), as such a target and we have demonstrated a significant deficiency of HO-1 expression in brain tissue from individuals with HAND. We also identified the recently FDA-approved CNS-penetrating multiple sclerosis drug (dimethyl fumarate/DMF/Tecfidera(R)), which stimulates HO-1 expression, as a unique candidate neuroprotectant for prevention of HAND. We propose to define the association between brain HIV-1 infection, HO-1 expression and HAND, and to establish a proof-of-concept therapeutic link to HO-1 induction as an adjunctive approach for HAND neuroprotection in a pilot dimethyl fumarate/DMF-treatment study of SIV-infected macques. Using our in vitro HIV neurodegeneration model we showed that HO-1 acts as a specific suppressor of HIV- induced neurodegeneration and that dimethyl fumarate/DMF induction of HO-1 is highly neuroprotective. Through analysis of autopsied brain tissue specimens (dorsolateral frontal cortex) from more than 150 HIV+ individuals, we found a significant deficiency of HO-1 expression and showed that this HO-1 deficiency correlates with neurocognitive (executive) dysfunction. Thus, HO-1 brain deficiency is directly linked with neurocognitive dysfunction in HIV-infected individuals, and a therapeutic approach that potentially can correct this deficiency and improve neurocognitive outcomes is at hand. We hypothesize that HIV-mediated suppression of brain HO-1 expression contributes to neuropathogenesis of HAND and that induction of HO-1 expression with dimethyl fumarate/DMF in ART-treated HIV+ individuals can limit the neuroinflammation, neurodegeneration and neurocognitive decline associated with HAND. We will determine: 1) correlations between regional brain HO-1 expression, immune activation, viral load, and neurocognitive performance in HIV+ individuals~ 2) mechanisms of HO-1 dysregulation by HIV~ and 3) whether DMF treatment alters HO-1 expression, neuroimmune activation, and neuropathogenesis of brain SIV infection in macaques.

Kolson, Dennis L. Project Summary HIV associated neurocognitive disorders (HAND) persist (~30% prevalence) worldwide in antiretroviral therapy (ART)-treated individuals despite a profound reduction in severe HAND (HIV-associated dementia/HAD). Biomarkers of oxidative stress in both systemic and CNS body compartments are strongly correlated with HAND, even in ART-treated individuals. Recently, we analyzed autopsied brains (>150 NNTC donors) and found a deficiency of a critical enzyme modulator of oxidative stress, heme oxygenase-1 (HO-1,) in those with HAND. This effect was independent of ART use and it correlated with brain macrophage activation and type I interferon responses. We further defined a link between brain HO-1 deficiency and HIV neuropathogenesis by showing that: i) HIV infection of monocyte-derived macrophages (MDM) consistently and selectively reduces HO-1 expression and increases neurotoxin (glutamate) release; ii) ART treatment of established HIV infection in MDM (HIV/MDM) does not prevent neurotoxin release, while iii) restoration of HO-1 expression in HIV/MDM does prevent neurotoxin release independent of ART and HIV replication. Our new preliminary studies also implicate dysregulation of brain HO-1 expression through a common HO-1 gene promoter sequence variation (GTn dinucleotide repeat length) and through regional variation in brain HO-1 expression in HAND pathogenesis. This HO-1 GTn dinucleotide repeat variation has previously been correlated with plasma markers of HIV disease progression (sCD14, HIV load) and our brain analyses demonstrate a strong correlation between HO-1 promoter GTn repeat length and the presence of HIV encephalitis. Additionally, our preliminary studies of autopsied rhesus macaque brains (n=18) demonstrated consistent regional (9 regions) brain differences in HO-1 expression levels, with lowest levels in deep brain structures where, in humans, HIV effects are particularly profound. Thus, our studies identify HIV-driven brain HO-1 deficiency as a major contributor to HAND pathogenesis, and they suggest that HO-1 promoter GTn repeat variation and brain regional HO-1 variation could be risk factors for HAND despite the use of ART. We hypothesize that HIV- induced brain HO-1 loss is a risk for HAND and that HO-1 promoter GTn repeat variation influences not only systemic HIV disease progression but also CNS disease progression and HAND. We further hypothesize that regional brain HO-1 levels contribute to selective regional vulnerability to HIV injury. We will: 1) Determine the correlation between HO-1 promoter GTn repeat variation and neurocognition in HIV+ subjects (CHARTER patient cohort); 2) Identify HO-1 variation associations with compartmental pathology and HIV disease markers (brain, spleen/NNTC autopsy cohort); and 3) Define the neuropathological in vitro responses of macrophages, astrocytes, endothelial cells relevant for blood-brain barrier function to HO-1 modulation and effects of the HO- 1 promoter GTn repeat variation on these responses. These studies can provide critical information for assessing cellular and clinical responses to HO-1-targeted therapies.