Andrew Levine - US grants

[unreadable] DESCRIPTION (provided by applicant): In the proposed study we will examine how illicit stimulant use affects the longitudinal course of neurocognitive functioning in individuals infected with human immunodeficiency virus (HIV), and how host-genetic variation modulates this. While it is has been established that both HIV infection and stimulant use result in neurocognitive impairment, and that these effects are additive or even synergistic, the interactive relationship between these two factors as it evolves over time has not yet been examined. Further, while the role of host- genetics in the progression of HIV/AIDS has received growing attention during the past few years, its contribution to the progression and characteristics of neurocognitive deficits over time remains to be determined. To address this, we will employ two genetic association methods. First, we will utilize a candidate gene approach with a large sample (N = 1000) of HIV+ and HIV- individuals that will focus on polymorphisms that affect the metabolism and activity of dopamine (DA). This neurotransmitter is implicated in the neuropathogenesis of HIV- associated neurocognitive disorders (HAND) and cognitive sequelae of chronic stimulant abuse. Further, research has shown that functional polymorphisms in DA-related genes can affect neuropsychological functioning via alterations in underlying neurophysiology, and that this occurs in brain regions similar to those affected in neuroAIDS. Therefore, examination of DA-related polymorphisms may add a critical tier of knowledge for explaining inter-individual variation in neuropsychological outcomes and progression among HIV+ drug users. In this exploratory study, we will also examine variants of other genes associated with neurobiological and immune functioning, including ApoE, brain-derived neurotrophic factor, and tumor necrosis factor-alpha. In the second approach, we will examine existing data from a genome-wide association (GWA) scan of 496 HIV+ individuals. We will be able to examine over 555,000 single nucleotide polymorphisms (SNPs) in an effort to detect novel genes associated with neurocognitive phenotypes, such as progression and diagnosis of HAND. Such a large scale genetic analysis of HAND neuropathogenesis has yet to be conducted. The proposed study will use the Multicenter AIDS Cohort Study (MACS), a longitudinal study of the natural history and clinical outcomes in HIV+ individuals. Neuropsychological, psychiatric, and virologic data on cohort of largely Caucasian, gay males are collected at regular intervals. Data exist for both HIV+ and HIV- individuals, as well as individuals with varying stimulant use patterns. Fluids are available for DNA extraction and genotyping. The proposed research team consists of experts in psychiatric genetics, neuropsychology, NeuroAIDS, and statistics with proven ability by successfully carrying out similar studies and to work with MACS data. The results of these investigations will lead to greater understanding of the neuropathogenesis of HAND, identification of pharmaceutical targets, and ultimately greater quality of life for those infected with HIV. The results of these investigations will be immediately translatable to public health efforts. Specifically, they will further illuminate understanding of the neuropathogenesis of HAND. Further, the identification of host- genetic factors in the neuropathogenesis of HAND will enable development of specific pharmaceutical treatments, ultimately leading to greater quality of life for those infected with HIV. [unreadable] [unreadable] [unreadable]

The prevalence of HIV-associated neurocognitive disorders (HAND) remains high despite widespread use of intensive antiretroviral medicines. Up to 50% of HIV-infected individuals will develop some degree of HIV-related neurocognitive impairment during their lifetimes, ranging from mild deficits that do not significantly impact day-to-day functioning to debilitating dementia. HAND is likely the result of chronic neuroinflammation, mediated in large part by the infiltration of monocytes into the brain. This neuroinflammation, and subsequently the severity of HAND, are enhanced by use of stimulant drugs, including cocaine and methamphetamine. The cellular cascade of events associated with HIV-associated dementia has been well-described, including alterations in the phenotype of blood monocytes, and changes in gene expression and protein expression within brain tissue. However, the vast majority of HAND involves insidious, often chronic mild neurocognitive deficits that do not evolve into dementia. Despite this, the cellular changes associated with mild HAND have not yet been well-delineated. In the proposed study, we will apply innovative systems biology approaches to the investigation of mild HAND pathogenesis. We focus our investigation on circulating blood monocytes, as these cells are an early and key component of this pathogenesis. We will use integrated weighted gene co-expression network analysis (IWGCNA), developed by the co-PI/PD, to develop meaningful biological pathways derived from monocyte-specific gene expression microarrays, HAND-associated genetic markers, and clinical diagnosis of HAND. Using structural equation modeling, we will then determine how stimulant use and virologic biomarkers (e.g. viral load) modify these pathways. This analytic strategy will be first be implemented on a large cohort of HIV-infected individuals from the Multicenter AIDS Cohort Study (MACS). We will then validate our findings on a separate cohort from the National Neurological AIDS Bank (NNAB). To accomplish this ambitious study, we have assembled a collaborative team that includes leaders in immunology, systems biology, neuroAIDS, neuropsychology, and genetics. Further, because we will be relying largely on existing resources and data, the monetary cost and patient burden necessary for the study are extraordinarily low. The results of the study will lead to greater understanding of the mechanisms involved in HAND, provide potential biomarkers of HAND, and identify targets for pharmaceutical prophylactics and therapeutics.

DESCRIPTION (provided by applicant): The prevalence of HIV-associated neurocognitive disorders (HAND) is near 50% despite the widespread use of combined antiretroviral therapy (cART). HAND is a clinical diagnosis based on neurocognitive impairments ranging in severity from mild neurocognitive deficits to a debilitating dementia. HAND is the result of a chronic neuroinflammatory state fueled by host immune activation; however, the key pathophysiological pathways involved remain unclear. In recent years, a small number of host genetic susceptibility loci have been identified that increase risk for HAND or influence its course, suggesting that perturbation of the physiological pathways their gene products are involved in modifies HAND pathogenesis. However, all studies of HAND-related genetic susceptibility loci have relied solely on clinical outcomes; nothing is known of what pathophysiological changes these genetic variants cause. An innovative approach for determining the pathophysiological mechanisms through which genetic susceptibility loci influence HAND is to examine their influence on neuropathological intermediate phenotypes, or NIPs. At its most basic level, HAND is believed to be the end result of a sequence of physiological events that commences with HIV-induced cellular changes that are modified by genetic factors. In the context of the present study, we deem quantifiable neuropathological changes that occur proximal to the beginning of this causal chain of HAND pathogenesis as the NIPs necessary for the behavioral impairments characteristic of HAND. There exist a number of candidate NIPs that are associated with HAND, including synaptodendritic simplification; macrophage infiltration; microglial and astrocyte activation, and beta-amyloid deposition. Through examining the relationship between genetic susceptibility loci and NIPs in brain tissue derived from a clinically well-characterized cohort, it will be possible t determine 1) which genetic variants are biologically relevant to HAND, 2) the pathophysiological mechanisms through which they exert their effect on the neurobehavioral phenotypes, and 3) the relative importance of NIPs as underlying causative factors of HAND. This study is highly relevant to the mission of NIH to elucidate the physiological mechanisms leading to HAND. To accomplish our aims, we will use immunohistochemistry and high throughput quantification procedures to characterize NIPs across multiple brain regions. Our aim is to determine the extent to which genotype and important co-factors (e.g., age, substance use, cART use) modify these NIPs. Our second aim will determine the linear causative relationship between genetic susceptibility loci, NIPs, and neurocognitive impairment. Towards this end we will use Network Edge Orienting to determine the causative relationship between genotype and NIPs. This will be integrated with structural equation modeling, thus allowing creation of a comprehensive pathway model of HAND that includes genotype, NIPs, neurocognitive functioning, and important co-factors.

? DESCRIPTION (provided by applicant): Highly Active Antiretroviral Therapy (HAART) greatly reduces quantity of active virus and has resulted in a marked increase in life expectancy of HIV-infected (HIV+) individuals. While HAART has also led to a reduction in the incidence of AIDS-defining illnesses, a variety of HIV-Associated Non-AIDS (HANA) conditions more commonly associated with older age are increasingly commonplace in HIV+ individuals. Aging and HIV appear to share common features of immune dysfunction, and the two may act synergistically in the aging population with long term HIV infection. A burgeoning literature suggests that HIV infection accelerates aging; however the underlying molecular mechanisms remain poorly understood. In particular, it not known how measures of disease severity, such as HIV viral load, affect dynamic epigenetic processes such as DNA methylation (DNAm). It is also not known how long term use of HAART impacts DNAm. The PI recently developed a novel biomarker of aging (referred to as epigenetic clock), which is the first age prediction method based on DNAm levels that accurately predicts age in more than one human tissue or fluid. As a matter of fact, it works in the vast majority of tissues/fluids/organs. It is arguably the first accurate measure of age that allows one to compare the ages of different parts of the human body. Using two DNAm data sets from the peripheral blood mononuclear cells of HIV+ individuals, we have demonstrated that even low levels of HIV replication significantly accelerates age according to the epigenetic clock. This R21 proposal will generate crucial data for testing the plausible hypothesis that HIV viral load also accelerates aging in a wide variety of affected human tissues, thus helping to explain the higher incidence of HANA conditions affecting a variety of organ systems. The genome wide DNAm profiles will not only allow us to test this hypothesis but more broadly allow us to identify additional DNAm markers that correlate with viral load in different tissues. To study the clinical ramifications, we will correlate the methylation data with various markers of tissue pathology typically associated with aging (e.g., fibrosis), and pre-mortem diagnoses of HANA conditions. To study the effect of antiretroviral medication (ARV), we will correlate the methylation data with ARV usage data tracked up to 15 years pre-mortem while participants were enrolled in a longitudinal cohort study. This proposal leverages the tissue samples from deceased HIV+ and HIV- subjects and the latest version of the well-validated Ilumina Infinium 450K array, allowing high-resolution genome wide DNAm profiles. Our overarching goal is to show that accelerated cellular aging, as measured by DNAm, has clinical relevance in the context of HIV/HANA. Working from that foundation, we will further investigate the molecular mechanisms in in vitro systems. This research will lead to development of epigenetic biomarkers of HANA conditions, produce useful surrogate measures of clinical outcomes, and open up a new area for therapeutics.

? DESCRIPTION (provided by applicant): DNA methylation (DNAm), an epigenetic process, is critical for neurodevelopment and for neurologic functioning throughout the lifespan. Aberrations in DNAm result in developmental, neurodegenerative, and psychiatric disorders. We have discovered that HIV+ adults have altered DNAm in both brain and peripheral blood mononuclear cells, resulting in accelerated biological aging. Perinatally HIV-infected (PHIV+) children are at increased risk of neurodevelopmental delay and lasting neurocognitive dysfunction. In this study, we examine the role of DNAm in the neurocognitive development of PHIV+ adolescents, while also considering important psychosocial, medical, virologic, and other factors. The immediate goal of this study is to delineate the complex relationship between these various factors in the pathogenesis of neurocognitive deficits in PHIV+ adolescents. The intermediate goal is to utilize the information gained to develop intervention strategies aimed at improving the lives of PHIV+ children as they progress into adulthood. The long term aim of this study is to develop a biomarker of currently concealed neuropathological processes that result from HIV, so that appropriate interventions can be provided to at-risk youths. This study will consider a host of environmental factors known to impact neurodevelopment, including stress, antiretroviral (ARV) medication use, economic adversity, medical comorbidities, and home/family environment. It will leverage the extant resource of the Cape Town Adolescent Antiretroviral Cohort (CTAAC), thereby maximizing efficiency and cost effectiveness, while minimizing human subjects' burden. The combination of cross sectional and longitudinal aims will allow fast translation of the findings and true characterization of neurodevelopmental and cognitive divergences resulting from epigenetic dysregulation due to HIV. In addition, the genome-wide methylation data generated can be used to enhance other research arms of the CTAAC, maximizing the potential return of investment. We also include a capacity building element by funding a postdoctoral fellow from UCT to be trained in biostatistics by UCLA researchers. This study is possible through the collaboration of two teams: The University of Cape Town team, which is currently carrying out the CTAAC neuro sub-study involving 200 HIV+ and 50 HIV- adolescents with diffusor tensor imaging, comprehensive neuropsychological testing, and assessment of childhood psychosocial factors, will provide all data and biological samples, as well as expertise in neuroimaging and pediatric neuroHIV. The University of California - Los Angeles team, which has extensive expertise in epigenetic biostatistical methods and genomic investigations of neuroHIV will process DNA samples and conduct the statistical analyses.

Project Summary HIV infection frequently results in varying degrees of neurocognitive and functional impairment, collectively termed HIV-associated neurocognitive disorders (HAND). Pharmaceutical interventions for HAND have had very limited success. Based on results of large-scale transcriptomic analyses in human samples and in vitro studies of HIV neuropathogenesis, antioxidant inflammation modulators that modify the nrf2/KEAP1 axis may be effective for the prevention and/or treatment of HAND. Our preliminary data show one such compound, Bardoxolone, is effective in reducing HIV replication in human monocytes and macrophages, as well as increasing neuroprotective factors in neurons and macrophages. However, before moving forward with a costly and labor intensive clinical trial, it is important to further characterize the safety and efficacy of Bardoxolone for treating and/or preventing HAND. We propose three studies. First, we will examine the ability of Bardoxolone to mitigate HIV-related cognitive-behavioral deficits in HIV-infected humanized NOG mice. Second, we assess the efficacy of Bardoxolone in reducing viral replication and HIV-induced inflammatory factors in plasma of the humanized mice. Finally, we examine brain tissue of the same humanized mice to determine if Bardoxolone mitigates HIV-induced neural injury and increases expression of neuroprotective factors. Results of these experiments will provide a strong foundation to proceed with an Investigational New Drug application to the FDA and ultimately clinical trials.

Project Summary Based on estimates derived from large consortium studies, HIV-associated neurocognitive disorders (HAND) affects between 22-84% of infected individuals at any one time. The vast majority of HAND diagnoses are mild, and are termed Asymptomatic Neurocognitive Impairment (ANI) in the most recent research diagnostic criteria. As many as 70% of those diagnosed with HAND have ANI, according to data from the CHARTER study. Yet some argue that ANI is over diagnosed, citing the fact that when the diagnostic criteria are applied to otherwise healthy, uninfected individuals, there is a very high rate of false-positive diagnoses. Another likely reason for the overestimated prevalence of ANI is suboptimal effort. That is, individuals enrolled in research studies from which prevalence rates are derived may not put forth sufficient effort on neurocognitive testing, thereby inflating estimates of mild HAND prevalence. Preliminary data from the Multicenter AIDS Cohort Study (MACS), the largest HIV study incorporating neurocognitive measures, indicates that over 50% of MACS participants who recently completed comprehensive neurocognitive testing reported suboptimal effort. The most common reasons being fatigued/tired, having poor concentration/distraction, and ?other?. Furthermore, there was an ordinal relationship between effort level and HAND severity, indicating that those with the poorest effort have the highest rates of ANI diagnosis. The preliminary data also allowed for identification of predictors of suboptimal effort, including low education and unemployment. These preliminary findings are extremely important for the future study of HAND. They suggest not only that rates of HAND are inflated due to poor effort among research participants, but also that there are specific causes for, and predictors of, suboptimal effort that can be addressed. In the proposed study, we will further explore suboptimal effort in the MACS. The results may lead to adjustments in HAND prevalence estimates and establishing methods for optimizing effort in research studies.