Norman Haughey - US grants

DESCRIPTION (provided by applicant): The success of Highly Active Antiretroviral Treatment (HAART) in combating HIV infection has resulted in a dramatic increase in the expected lifespan of HIV infected patients. Neurological deficits associated with HIV infection (collectively termed HIV associated dementia; HAD) also declined with the advent of HAART therapy, although in some cohorts, it is the incidence rather than the prevalence (which continues to increase) that has declined. This suggests that neurological dysfunction is increasing as HIV infected individuals' age. Neurons in the "aged" brain are more vulnerable to toxic insult. Age-related increases in the lipid ceramide and cholesterol are thought to play key roles in increasing neuronal sensitivity to insult by priming apoptotic pathways. Ceramide signaling directly inhibits the anti-apoptotic protein Bcl-2 and decreases mitochondrial function by disrupting electron transport and promotes the generation of free radical species. Increases in cholesterol disrupt cellular signaling by decreasing the fluidity of the plasma membrane and can increase ceramide levels by slowing the conversion of ceramide to sphingomyelin. In our preliminary studies we found dramatic increases in ceramide and cholesterol in brains of HIV-1 infected patients with dementia compared to HIV-1 seropositive or seronegative controls. Because some HIV-1 proteins are thought to play key roles in neuronal dysfunction and death, we exposed human neuroglial cultures gp120 and Tat and found that these proteins increased ceramide levels. When we exposed cultures to several different insults, including Tat, cell death was dramatically reduced by inhibition of de novo ceramide synthesis, suggesting a central role for ceramide in some forms of neuronal cell death. Our findings suggest that HIV-1 infection could compound increases in ceramide and cholesterol that occur with age resulting in ceramide overload and neuronal cell death. Drugs that lower cholesterol levels (and indirectly ceramide) have therapeutic benefit in some neurodegenerative diseases such as Alzheimer's and ischemic stroke. We propose to determine the mechanisms of interaction of age, ceramides, cholesterols, gp120 and Tat that contribute to neuronal dysfunction and death in HAD. In vivo, we will test the protective effects of cholesterol reduction, inhibition of ceramide synthesis and reduction of oxidative stress in models of HIV protein-induced toxicity.

DESCRIPTION (provided by applicant): The introduction of highly active antiretroviral therapy (HAART) regimes in the mid-1990's has resulted in a 40-50% decrease in the incidence of HIV-associated dementia (HIVD). Nonetheless, AIDS-associated neurological diseases continue to be major causes of morbidity and mortality, and in some cohorts, the prevalence of HIVD is on the increase, despite effective peripheral control of viral replication. We hypothesize that a dysfunction of neural progenitor cells in the hippocampus contributes to the neurological manifestations of HIVD. The formation of new neurons (neurogenesis) occurs through adult life and is thought to contribute to the processes of learning and memory. Neurodegenerative and neuroinflammatory conditions interfere with neurogenesis and thus disrupt memory formation by preventing the functional incorporation of new neurons into existing hippocampal circuitry. HIV and HIV-derived proteins are known to promote neuroinflammation and can induce the dysfunction and death of neural cells though direct actions at chemokine receptors and by indirect effects that are mediated by macrophages and glia. It is thus possible that HIV and HIV proteins contribute to learning and memory deficits in HIV-infected patients by disrupting neural progenitor cell function. In preliminary experiments we have characterized the expression of chemokine receptors in two populations of neural progenitor cells and have evidence that the HIV-1 protein Tat disrupts neurogenesis in vitro and decreases the proliferation rate of neural progenitor cells in the molecular layer of the dentate gyrus of mice transgenic for Tat. Using a multilineage human neurosphere model and purified populations of lineage-restricted progenitor cells, we propose to determine the effects of HIV-1 and the HIV-1 proteins gp120 and Tat on neural progenitor cell proliferation, survival and cell fate decisions. Findings from our in vitro studies will be confirmed in mice transgenic for gp120 or Tat by quantification of differences in neural progenitor cell proliferation, survival and function. The object of these studies is to identify and characterize mechanisms of HIV, gp120 and Tat-mediated dysfunctions in neural progenitor cells. Findings from these studies could identify novel therapeutic targets for the treatment of HIV-related neurological disease.

[unreadable] DESCRIPTION (provided by applicant): Human immunodeficiency virus type -1 (HIV-1) infection is the commonest cause of dementia in adults less than 40 years of age. Alcohol is a common drug of abuse in HIV-infected patients that can worsen the decline of frontal lobe function that is associated with HIV encephalitis. Our goals are to determine the mechanisms of neuronal dysfunction and death associated with alcohol abuse in the setting of HIV-dementia that are dependent on the pathological modification of ceramide and sterol content in neuronal membranes. Although a number of channelopathies have been identified in HIV-dementia, we have deliberately focused our attention on alterations in N-methyl-d-aspartate (NMDA) receptor function because of the importance of this excitatory receptor in neuronal dysfunction and death. Our preliminary findings suggest that the alcohol can rapidly alter the cholesterol and ceramide content of neuronal membranes, while the HIV-1 proteins gp120 and Tat alter the sphingomyelin and ceramide composition, and promote the trafficking of NMDA receptors to rafts. Based on these observations we hypothesize that alcohol may promote neuronal dysfunction in HIV-dementia by disorganizing the structure of lipid rafts and perturbing the function of raft-located NMDA receptors. Accordingly, pharmacological agents that stabilize ceramide and cholesterol metabolism may be neuroprotective by preventing the disorganization of lipid rafts. Using in vitro and in vivo models of HIV-dementia, we propose to determine the mechanisms of how alcohol dysregulates neuronal function in acute, chronic and withdrawal conditions. [unreadable] [unreadable] Public Health Relevance: Alcohol is a common drug of abuse in HIV-infected patients that can hasten the onset of dementia and worsen the severity of cognitive decline. Determination of how alcohol interacts with viral products to damage brain cells is critical for the rational design of therapeutics designed to protect brain functions. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): HIV-dementia is the most common form of dementia in persons under 40 years of age. Therapeutic interventions designed to improve cognitive function or to prevent further cognitive decline in patients with HIV-dementia have focused on drugs that interact with receptors such as excitatory amino acid receptors and voltage operated calcium channels. Unfortunately, clinical trials with these agents have had limited success. In this proposal we look at a common pathway that is deregulated by HIV infection. We present the first evidence that begins to identify a mechanism whereby increased levels of ceramide and cholesterol in the brains and CSF of HIV infected patients with dementia can lead to neuronal dysfunction and death. We further identify and test potential neuroprotective therapeutics that stabilize sphingolipid metabolism. The long-term goals of this research are to discover and test therapeutic agents that protect neuronal function by stabilizing sphingolipid biochemistry. Sphingomyelin, ceramide, cholesterol and ganglosides are the primary constituents of specialized membrane domains called 'lipid rafts". These specialized regions of cellular membranes are thought be important to coordinate cellular signaling by localizing functional groups of proteins in the plasma membrane and by coupling transmembrane receptors with signal transduction machinery. Lipid rafts can be modified by ceramide to form larger domains, which serve to cluster receptor molecules. The generation of a high receptor density might be required for initiation of receptor-specific signaling and has been implicated as pivotal step in the formation of "death domains". We have identified a ceramide-dependent mechanism that promotes the clustering of N-methyl-D- aspartate (NMDA) receptors into lipid rafts in response to the neurotoxic HIV-1 proteins gp120 and Tat. NMDA-evoked calcium bursts in these microdomains are sufficiently elevated to activate calcium dependent death effectors. Using biochemical, biophysical, molecular, electrophysiological and imaging techniques we propose to determine the mechanisms that direct the neurotoxic effects of the HIV-1 proteins gp120 and Tat by disrupting sphingolipid metabolism. These findings may lead to the rational design of pharmaceutical agents that are neuroprotective by mechanisms that stabilize sphingolipid metabolism and prevent the abnormal clustering of dysfunctional NMDA receptors.

DESCRIPTION (provided by applicant): The success of antiretroviral therapy (ART) in combating HIV infection has resulted in a dramatic increase in the expected lifespan of HIV-infected patients. An aging population infected with HIV has raised the concern that viral- or viral-associated factors could interact with age-related neuropathologies. Indeed, there is increasing evidence that Alzheimer's disease (AD)-like pathology may be present in some individuals infected with HIV. In particular, there have been repeated observations that pathogenic forms of amyloid (A) accumulate in brains of patients infected with HIV at much higher frequency than is seen in HIV seronegative patients. These observations suggest that there may be some viral- or viral-associated factors that promote abnormal amyloid processing. In preliminary studies we have found that the viral coat protein gp120 can induce activity of secretases involved in the pathogenic processing of amyloid. The experiments outlined in this application are designed to eludicate potential mechanisms by which gp120 may perturb amyloid processing to promote the generation of pathogenic [unreadable]-amyloid peptides. PUBLIC HEALTH RELEVANCE: The success of drug therapies used to combat infection with HIV (the causative virus of Acquired Immunodeficiency Syndrome;AIDS) has dramatically increased the expected lifespan of people infected with HIV. Although neurological complications associated with HIV infection also declined with the advent of drug therapies, an aging population of people infected with HIV has raised the concern that virus- or virus-associated factors could contribute to an increase of Alzheimer's in this population. Indeed, several studies have found evidence for Alzheimer's-like pathology in the brains of people infected with HIV. The studies outlined in this application are designed to determine the mechanisms by which infection with HIV could promotes Alzheimer's-like neuropathology.

SURROGATE MARKERS CORE PROJECT SUMMARY (See instructions): Neurocognitive impairment in HIV-infected individuals remains a significant problem despite antiretroviral therapy. There is a critical need for surrogate markers that can be used to predict cognitive impairment and to monitor the effects of antiretroviral, or neuroprotective therapy in HIV-infected individuals. The primary goals of the Surrogate Marker / Pharmacokinetic Core will be to: 1) Assist in the development and monitoring of surrogate markers for HIV-associated neurocognitive disorders (HAND). 2) To provide mentorship and consultation for Neuro-AIDS researchers in the development of clinically useful surrogate markers for HAND and to validate these as predictive and associative markers for cognitive impairment and as surrogate markers for therapeutic effectiveness. An additional task ofthe core will be to assist in the development of small molecule therapeufics by providing consultation on pharamcokinetic/pharmacodynamic considerations for drug development, and conducting pharmacokinetic and analysis of potential drugs and drug metabolites.

DESCRIPTION (provided by applicant): Despite the effectiveness of combined antiretroviral therapy (CART) to suppress HIV replication, HIV- associated Neurocognitive Disorders (HAND) continues to be major causes of morbidity and mortality. The management of HAND in this growing population will require new therapeutics to protect the CNS. Prominent neuroimmune activation is evident during the first months following HIV transmission, and brain inflammation persists in many patients despite CART. Sustained neuroinflammation in HIV-infected subjects is thought to contribute to neurological damage, in part by promoting the transmigration of leukocytes from the periphery into the CNS. It is not currently understood how CNS inflammation is communicated to the peripheral immune system. Our data suggests that brain neutral sphingomyelinase-2 (nSMase2) is a key regulator of this neuroimmune axis. nSMase2 activity is up-regulated by inflammatory cytokines, HIV-proteins and calcium to induce the release of exosomes from stimulated astrocytes. Exosomes are lipoprotein complexes that appear to carry inflammatory signals to periphery, and modulate the activation of leukocytes. The findings from this project will lead to a better molecular understanding of neuroimmunity that will aid on the development of a new class of neuroprotective drugs that reduce neuroinflammation by inhibition of nSMase2.

DESCRIPTION (provided by applicant): HIV infection in United States and several other developed countries is now largely driven by drug abusing populations. Since both HIV and drugs of abuse independently cause injury to the brain, researchers face multiple challenges in the design of studies to address the pathogenesis or develop effective therapies. This is further complicated by the fact that these patients often abuse multiple drugs and have other co-morbidities. Nonetheless, it is clear that the use of antiretroviral drugs alone is not sufficient and neuroprotective strategies need to be used in these patient populations. Yet no clinical trials have been conducted to date in this patient population. On the contrary, drug abusers have been excluded from most clinical trials on HIV dementia. While the research community recognizes the importance of studying the combined effects of these substances challenges faced include the large number of variables that need to be considered and the lack of ideal small animal models to study these combined effects. Despite these drawbacks, we rationalize that information available to date clearly suggests that there are common subcellular mechanisms involved in neural injury by HIV proteins and drugs of abuse, which can be exploited to develop novel therapeutic approaches for this patient population. HIV proteins and select drugs of abuse may contribute to the pathogenesis of HIVD through non-mutually exclusive mechanisms including 1) direct neurotoxicity, 2) glial activation, and 3) increased viral replication. We recently screened over 2000 compounds that have been approved by the Federal Drug Administration for human use and found that two compounds within the class of the selective serotinergic release inhibitors (SSRI), fluoxetine and paroxetine, have potent neuroprotective effects against the combined effects of drugs of abuse and HIV proteins. Interestingly, in a recent retrospective clinical study it was found that HIV patients on antidepressants had better neurocognitive function and lower CSF viral loads (Letendre et al, 2007). In another study with magnetic resonance spectroscopy, HIV infected patients on SSRIs had higher levels of N-acetyl aspartate suggestive of neuroprotection (Linda Chang, University of Hawaii, personal communication). For these reasons, we have designed an experimental study to determine if fluoxetine and paroxetine can impact the combined effects of HIV and drugs of abuse in various cell types in the brain and determine the mechanisms by which such neuroprotection may occur. Another novel aspect of our study is that we will study the combined effects of opiates and cocaine, since they are commonly used together. Thus this study will provide critical information needed to design future clinical trials with these agents for HIV infected drug abusers. We propose three specific aims. (1). Determine if SSRI can modulate the effect of HIV proteins and drugs of abuse on neuronal function (2) Determine if SSRI can modulate effects of HIV proteins and drugs of abuse on glial cell function (3) Determine if SSRI can modulate the effect of HIV proteins and drugs of abuse on neural progenitor cell function. PUBLIC HEALTH RELEVANCE Currently, there is no available neuroprotective therapy for HIV infected drug abusers who may be afflicted by a dementing illness. We have screened over 2,00 compounds and identified a class of antidepressant compounds that have novel brain protective properties. We will explore their mechanism of action and conduct other preclinical studies that are needed to take them to clinical trials.

DESCRIPTION (provided by applicant): Neurocognitive impairments in HIV-infected individuals, collectively known as HIV-Associated Neurocognitive Impairments (or HAND) remains a significant problem in the era of Combined Antiretroviral Therapy (CART). In many HIV-infected individuals there is evidence of accelerated aging, including aberrant processing of amyloid precursor protein (APP). These disruptions seem to result in accumulations of pathogenic forms of amyloid-ß (ß) in brain and are thus likely to also decrease the formation of soluble APP? (sAPP?), an important neurotrophic peptide. Our preliminary data suggest that accumulations of sphingolipids and complex glycolipids in intracellular compartments accelerates Aß formation by enhancing the activity of ß- and ?- secretases (that process APP to Aß), and by perturbing the intracellular trafficking / clearance of Aß. Previously we have documented accumulations of multiple sphingolipid species in HIV-infected individuals. These combined findings prompted us to determine if the accumulations of sphingolipid products in endosomes, lysosomes and/or autophagosomes are associated with aberrant APP processing, increased Aß deposition and decreased sAPP? in the setting of HIV-infection. In this application we propose a comprehensive approach to address this question, using human brain tissues, cellular/molecular approaches, and transgenic model systems to determine if increased brain levels of these lipid metabolites shifts APP processing to a more amyloidogenic (Aß) and less trophic (sAPP?) pheotype and if interventions that target sphingolipid metabolism can reverse these effects.

DESCRIPTION (provided by applicant): As people are now aging with HIV-infection, there is evidence that the cumulative incidence of HAND is continuing to increase 8-12. It is not currently understood why some HIV-infected individuals develop cognitive impairments while others do not. Likewise, there are no biological measures that can identify individuals with asymptomatic neurocognitive impairments (ANI) who are likely to progress to more severe forms of cognitive impairments including mild neurocognitive disorder (MND) or frank dementia (HIV associated dementia; HAD), or who is likely to spontaneously improve. The objective of this proposal is to use combinational informatics approaches to identify neural systems that are perturbed early in the course of HAND, and to determine how longitudinal changes in neural systems are related to changes in cognitive function. These combined informatics approaches will break new ground in the molecular understanding of HAND that will aid in the development new classes of neuroprotective drugs. These approaches are also likely to pinpoint a unique set of surrogate markers that may identify HIV infected individuals at a prodromal phase of HAND, when neuroprotective therapeutic intervention would have the greatest benefit. To accomplish these goals we propose to use a combinational informatics systems approach to interrogate a unique set of clinical samples that have undergone extensive analysis at multiple performance sites.

ABSTRACT (Project 1. Mechanistic studies) Despite continued advances in combination antiretroviral therapy (cART) to treat infection by the Human Immunodeficiency Virus (HIV), a considerable number of people infected with HIV develop neurocognitive impairments that are collectively known as HIV-Associated Neurocognitive Disorders (HAND). Although the severity of cognitive impairment in the post-cART era is generally milder than was observed pre-cART, neurocognitive impairment eventually occurs in approximately 30-50% of people infected with HIV, suggesting that there is ongoing cerebral injury despite reasonable control of viral replication. This treatment-gap highlights the importance of discovering an adjunctive therapy that could protect the CNS. Although the precise mechanisms for these residual cognitive impairments are not understood, they are thought to involve a loss of central bioenergetic homeostasis, and persistent low-level inflammation that contributes to dendritic and synaptic damage. In this complex environment, an ideal therapeutic agent for HAND would have multiple effects that regulate neuroprotection, neurotrophic and anti-inflammatory responses. Insulin has numerous actions in brain that regulate many of the same neural pathways perturbed by HIV infection including energy metabolism, lipid metabolism, neurotransmitter channel activity, neurite outgrowth, synaptic strength, and inflammatory signaling, suggesting that insulin might protect the CNS in the setting of HIV-infection. Our preliminary data supports this hypothesis showing that insulin protects cultured neurons from inflammatory, excitotoxic, and HIV- protein induced toxicity as well as prevents cognitive deficits in a murine Eco HIV infection model of HAND. The mechanistic studies proposed in Project 1 will focus on elucidating the mechanism of this therapeutic effect including evaluating the interactions of insulin signaling with ceramide and cellular bioenergetics, and the effects of these interactions on dendritic structure, synaptic functions and microglial activation using in vitro model systems that recapitulate key aspects of CNS HIV infection. The findings from these studies will provide new mechanistic insights into the therapeutic effects of insulin and may help to provide information on biomarkers to be utilized in translational studies.

? DESCRIPTION (provided by applicant): In the era of Antiretroviral Therapy (ART), reductions in neuronal connectivity, synaptic simplification and reductions in dendritic complexity are correlated with the severity of cognitive impairments in HIV-infected individuals. There is also considerable evidence that synaptic damage, and cognitive impairments are worse in HIV-infected people who use opiate drugs. Although a number of studies have shown that morphine interacts with HIV and components of HIV to dysregulate astrocyte and neuronal biology, the mechanisms for these interactions are not understood. Bi-directional communication between astrocytes and neurons regulates synaptic formation, synaptic strength, and participates in the regulation of neural circuitry by coordinating activity among groups of neurons. Recent advancements in the biology of extracellular vesicles have begun to implicate glial released microparticles as mediators of glia to neuron communication. In preliminary experiments we found that astrocyte exosomes (released in response to a endogenous stimuli) regulated a neurotrophic response when applied onto neurons. We determined that the molecular cargo of exosomes is complex and contains ~280 distinct proteins, 114 miRNA, and hundreds of lipid species. Moreover, the molecular composition of astrocyte exosomes was modified by the stimulus used to induce release. Based on these preliminary findings we reasoned that a scientific focus on any one protein, lipid or miRNA would be unlikely to produce a true representation of function for these important signaling complexes. Therefore, we used bioinformatics approaches to understand how the entire composition of exosomes interacts with neuronal signaling pathways, and focused our efforts on a small number of the identified pathways. In particular we concentrated on neural pathways associated with synapse formation, spine formation, and neurite outgrowth, as these pathways were consistent with the neurotrophic response to astrocyte exosomes released in response to an endogenous stimuli. The goals of this application are to understand how HIV and morphine modulate the cargo of astrocyte exosomes and how these differences in cargo regulate dendritic and synaptic functions in neurons.

? DESCRIPTION (provided by applicant): HIV-associated neurocognitive disorders (HAND) continue to be a remarkably prevalent condition in HIV- infected individuals despite the potent effects of combination antiretroviral therapy (cART). The development of HAND represents an important treatment issue for HIV patients that impacts quality of life, mortality, and everyday functioning. Currently, despite 25 years of research, no specific treatments have entered clinical practice for HAND. The overarching aim of this proposal is the development of a novel therapy, intranasal insulin, for HIV-associated neurocognitive disorders (HAND). We have identified this as an innovative, and high potential target based on our preliminary research. HIV-infection and cART are well known to cause alterations in lipid distribution, glucose homeostasis, and energy metabolism that are associated with alterations in insulin signaling. Several decades of research have shown that insulin has multiple actions in brain that regulate many of the same neural pathways perturbed by HIV infection including energy metabolism, lipid metabolism, neurotransmitter channel activity, neurite outgrowth, synaptic strength, and inflammatory signaling suggesting that insulin might protect the CNS in the setting of HIV-infection. In preliminary experiments we found that insulin protected neurons from a broad variety of insults including toxic HIV proteins, as well as ischemic, oxidative, inflammatory, and excitotoxic challenges, in addition to dampening the inflammatory response of microglia. In a novel animal model of HAND produced by infection of conventional mice with a chimeric HIV (EcoHIV) we found that intranasal insulin treatment for 9 days completely reversed cognitive impairment in infected animals. These data are consistent with human studies in healthy volunteers, Alzheimer's and type 2 diabetes patients showing that intranasal insulin improves cognitive function. These preliminary findings strongly suggest that insulin delivered directly to brain may preserve or restore neuronal function in HIV-infected individuals.

? DESCRIPTION (provided by applicant): Bi-directional communication between astrocytes and neurons regulates synaptic formation, synaptic strength, and participates in the regulation of neural circuitry by coordinating activity among groups of neurons. Astrocyte dysfunction in the settings of HIV-infection, and other neurodegenerative conditions has been postulated to disrupt the activity of neural networks involved in memory and executive functions. Although HIV-associated perturbations in the composition and quantity of various cytokine, chemokine and growth factors released from astrocytes have been demonstrated, these observations have thus far been insufficient to explain how astrocyte stress contributes to neuronal dysfunction. These questions are particularly important in the era of Antiretroviral Therapy (ART), where reductions in neuronal connectivity, synaptic simplification and reductions in dendritic complexity are thought to be primary contributors to cognitive impairments in HIV- infected individuals. Advancements in our understanding of the biology of extracellular vesicles have begun to implicate glial released microvesicles as primary mediators of glia to neuron communication. In preliminary experiments we provide evidence that a variety of stimuli can induce astrocytes to shed microvesicles. The molecular cargo of astrocyte-shed microvesicles was complex and contained more than 200 distinct proteins, 100 miRNA, and hundreds of bioreactive lipid species. Moreover, the protein, miRNA and lipid composition of astrocyte exosomes was modified by the stimulus used to induce release. These astrocyte-shed exosomes directly interacted with neurons to modify neuronal structure and function. Based on these preliminary findings we reasoned that a scientific focus on any one protein, lipid or miRNA would be unlikely to produce a true representation of the functions regulated by this complex signaling vesicles. Therefore, we used bioinformatic and systems biology approaches to understand how the protein, miRNA and lipid composition of exosomes interacts to regulate neuronal signaling pathways identified by whole genome sequencing of target neurons. In this application we focused our efforts on a small number of the identified pathways. In particular we concentrated on neural pathways associated with synapse formation, spine formation, and neurite outgrowth, as these neuronal structures appear to be damaged in many HIV infected individuals. The goals of this application are to understand how endogenous excitatory stimuli and inflammatory stimuli associated with HIV-infection modulate the cargo of astrocyte-shed exosomes and how these exosomes regulate/dysregulate the structure and function of their target neurons.

ABSTRACT Bi-directional communication between astrocytes and neurons regulates synaptic formation, synaptic strength, and participates in the regulation of neural circuitry by coordinating activity among groups of neurons. Astrocyte dysfunction in Alzheimer?s (AD), and other neurodegenerative conditions has been postulated to contribute to perturbations in activity of neural networks involved in memory and executive functions. Although AD associated modifications in the composition and quantity of various cytokine, chemokine and growth factors released from astrocytes have been demonstrated, these observations have thus far been insufficient to explain how astrocyte stress contributes to neuronal dysfunction. Advancements in our understanding of the biology of extracellular vesicles have begun to implicate glial released microvesicles as primary mediators of glia to neuron communication. In preliminary experiments we provide evidence that a variety of stimuli can induce astrocytes to shed microvesicles. The molecular cargo of these astrocyte-shed microvesicles was complex, and contained more than 200 distinct proteins, 100 miRNA, and hundreds of bioreactive lipid species. Moreover, the protein, miRNA and lipid composition of astrocyte exosomes was modified by the stimulus used to induce release and could be further modified by pre-treatment with oligomeric A? peptides. These astrocyte- shed exosomes directly interacted with neurons to modify neuronal structure and function. Based on these preliminary findings we reasoned that a scientific focus on any one protein, lipid or miRNA would be unlikely to produce a true representation of the functions regulated by this complex signaling vesicles. Therefore, we used bioinformatic and systems biology approaches to understand how the protein, miRNA and lipid composition of exosomes interacts to regulate neuronal signaling pathways identified by whole genome sequencing of target neurons. In this application we focused our efforts on a small number of the identified pathways. In particular we concentrated on neural pathways associated with synapse formation, spine formation, and neurite outgrowth, as these neuronal structures are damaged in AD. The goals of this application are to understand how endogenous excitatory stimuli and inflammatory stimuli associated with AD modulate the cargo of astrocyte-shed exosomes and how these exosomes regulate/dysregulate the structure and function of target neurons.

PROJECT SUMMARY ? BIOMARKER CORE The Johns Hopkins University NIMH Center for Novel Therapeutics for HIV-associated Cognitive Disorders Biomarker Core will provide support for pre-clinical development and clinical testing associated with therapeutics to treat cognitive disorders in HIV-infected patients. This support will include a series of measures of neurobiological functions including cell stress, neuronal injury/protection, oxidative injury, energy metabolism, immune activation and insulin signaling. These measures will be used to support experimental outcome measures in pre-clinical projects, and will aid in determining the biological response to clinical stage therapeutics.

PROJECT SUMMARY ? OVERALL HIV-associated neurocognitive disorders (HAND) remain very prevalent, even among aviremic HIV+ individuals treated with CART. In the era of CART, the prevalence of HAND in HIV+ individuals with advanced infection remains around 40-50% [1, 2], and HAND may now be the most common form of young-age neurocognitive impairment globally [1]. Currently there are no uniformly accepted clinical, neuroimaging, or laboratory outcome measures for clinical trials for the treatment of HAND. HIV-related neuroscience research at Johns Hopkins University has focused on this challenging problem, exploring critical pathogenic mechanisms for neurological damage. Despite tremendous efforts to understand the mechanisms underlying the persistence of HAND, no definitive adjunctive therapeutics have yet entered clinical practice. There is also an unfilled need to develop surrogate markers and more robust and simpler screening instruments for HAND, to allow for earlier detection, for tracking of the course of HAND, and improving the efficiency of clinical trials. Until the NIMH Center was established at JHU collaborations had been limited by the lack of a central organizing structure for this type of research, and limited resources to facilitate cross-disciplinary and translational research. The JHU NIMH Center has addressed these needs over the past 11 years and has provided a resource to catalyze interdisciplinary research in HIV neuroscience, with the aim of leading to new therapies. Accomplishments of the Center are highlighted in the Overall Strategy section and in each individual core, and the key accomplishment is our proven ability to move HAND therapeutics from the discovery phase, through animal models, and on to clinical trials, with the ultimate goal of shifting clinical practice.

ABSTRACT People living with HIV (PLWH) have higher rates of drug addiction compared with uninfected populations, and often show faster rates of disease progression, including early and rapidly progressing cognitive impairments. We have previously demonstrated that synaptic damage resulting from acute morphine administration self- repairs during drug withdrawal. These repair mechanisms were not active in gp120 transgenic mice, and synaptic repair failed during drug withdrawal. Although these observations in addition to a number of other studies that have demonstrated interactions of morphine with HIV that ultimately reduce dendritic spine density, the precise mechanisms for these interactions are not understood. Our preliminary findings suggests that the initial dendritic damage induced by morphine involves activation of a non-canonical Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing (NLRP) inflammasome pathway in astrocytes that facilitates the release of EVs carrying complement C3. This complement protein is opsonized in dendritic spines and targets them for elimination by phagocytosis. The removal of morphine stops the shedding of complement C3 from astrocytes, and dendritic spines can self-repair. However, in the setting of viral infection there is a sustained activation of pattern recognition receptors on microglia with chronic activation of the classical NLRP inflammasome pathway that maintains a state of persistent inflammation, This chronic inflammatory state disallows dendritic spines to self-repair. Here we propose to use primary cell culture, EcoHIV infected mice, and conditional transgenic systems to test the hypothesis that interactions between TLRs, the inflammasome, and the complement system contribute to neuronal damage in PLHW who abuse opiates. .

PROJECT SUMMARY ? ADMINISTRATIVE CORE The rationale for a centralized Administrative Core is to have streamlined management and administration in order to maximize efficiency of personnel, resources, and provide overall scientific and programmatic leadership, fiscal oversight and support, and to integrate communications and information resources to maximize the JHU NIMH Center utility, value, and cohesiveness in support of neuroAIDS research at Johns Hopkins. The Administrative Core consists of the Executive Committee (EC), an Operations Office (OO), and an External Advisory Committee (EAC) to provide 'high-altitude' guidance and oversight. The Executive Committee consists of the Director, Co-Directors, and a Program Manager for the Center. The objectives of the Administrative Core are to facilitate the development of novel therapeutics for HIV-associated neurocognitive disorders by facilitating collaborative research in HIV-related neuroscience among the widest possible range of the JHU academic community; increasing resources for HIV-related neuroscience; enhancing the productivity of HIV-related neuroscience research locally, nationally and internationally; encouraging high- risk, innovative 'developmental' research; encouraging and provide resources for new investigators; and providing educational resources for non-neurologists involved in HIV care for HIV-related neurological complications, and to facilitate participation in research.