Susan Morgello, MD - US grants

Neurologic abnormalities are common in AIDS. While it is clear that CNS infection with HIV may manifest as a subcortical encephalitis, unfortunately, not all patients with AIDS dementia complex reveal this histopathology. This has led to a search for alternative explanations for dementia. In particular, recent emphasis has been placed upon the possible role of neuronal damage in the genesis of AIDS dementia complex. This neuropathologic investigation will examine the extent and nature of grey matter abnormalities in the brains of patients dying with AIDS, and will correlate these with clinical neurologic features. Specifically, this project will utilize autopsy brains from AIDS patients that are histopathologically sorted into 3 groups: HIV encephalitis, aseptic leptomeningitis, and no infectious abnormalities, as well as brains from HIV-negative controls to determine: 1. if HIV infection results in decreased neuronal counts or neuronal atrophy; 2. if there is a pattern of selective vulnerability in neurons undergoing pathologic changes; 3. if abnormal glial responses occur in grey matter and can be correlated with neuronal changes; 4. if CNS glucose transporters show any decrement with HIV infection; and 5. if any detected neuronal abnormalities can be correlated with clinical data, specifically the presence of AIDS dementia complex. The methods entailed in this study are computer-assisted morphometric analysis in concert with histochemistry and immunohistochemistry; immuno-blotting; and polymerase chain reaction.

Well characterized brains have great utility in studies of HIV neuropathogenesis and studies relating to adequate treatment of viral reservoirs. The Manhattan HIV Brain Bank will utilize pre-extant bank infrastructure located at the Mount Sinai School of Medicine (MSSM), and coordinate patient resources at MSSM, Beth Israel Medical Center (BIMC), and St. Luke's-Roosevelt Hospital Center (SLRHC), three of New York City's largest medical complexes, to: 1. Identify and follow a well- characterized cohort with advanced HIV disease, using neurologic, neuropsychologic, medical, and laboratory examinations; 2. Establish a multi-institutional database in which clinical data are recorded; 3. Obtain autopsy-derived central nervous system (CNS), peripheral nervous system (PNS) and systemic tissues from these patients within 24 hours of their demise; 4. Provide complete pathologic diagnoses of these tissues that will be maintained in a separate but interactive pathology database; 4. Dissect, store, catalogue and distribute these tissues to extramural investigators; 5. Evaluate the appropriateness of tissue requests with a bank review committee that will function under the guidelines of the Network Steering Committee; and 6. Utilize the pathology and clinical databases in correlational studies to elucidate the pathogenesis and natural history of HIV-related neurologic diseases. Within the context of the Manhattan HIV Brain Bank, we will establish a nationwide resource for the provision of numerous well-characterized brains and tissues for study, and additionally, establish a multi- institutional longitudinal clinical/neurologic and neuropsychiatric database that will serve as a template for following a significant proportion of the medically-serviced, HIV-infected population of New York City, and assist in future studies of HIV neuropathogenesis.

This work was developed based on the notion that direct examination of postmortem human brain by extremely sensitive techniques would be important to determine persistent reservoirs of active/restricted infection HIV-1. In this way the true potential of highly active anti- retroviral therapies (HAART) can be determined. The hope is that these therapies will, inevitably, completely eliminate HIV from infected patients. As correctly stated by the investigators there are currently no reliable, consistent, and sensitive techniques for in situ identification of HIV in postmortem brain. The technique being developed in the application is the ramification amplification assay (RAM). This is a newly invented, solution-phase, isothermal procedure that utilizes a unique DNA polymerize to generate large, multimeric molecules from circular probes that are covalently locked on to their RNA targets. In solution phase, the technique appears to be more sensitive than single round (35 cycle) polymerase chain reaction (PCR). Dr. Morgello's proposes that this technique would be ideally suited for the sensitive in situ detection of active and restricted HIV infection in human postmortem brain. The questions being answered are : (1) What is the cellular reservoir/distribution of HIV in human brain? (2) Does this change with histologic evidence of inflammatory disease? (3) What is the proportion of productive, restricted and/or latent HIV infection in the brain?

PROJECT SUMMARY The Manhattan HIV Brain Bank (MHBB), member of the National NeuroAIDS Tissue Consortium (NNTC), is a research resource committed to providing scientists with densely annotated, high quality biospecimens in support of neuroAIDS and AIDS research. Located in New York City, it conducts a longitudinal, observational neurologic, neuropsychologic, and psychiatric study with individuals who agree to be organ donors upon demise. While harmonizing its assessments and activities with other members of the NNTC, MHBB retains important and unique characteristics. MHBB is a predominantly minority cohort with excellent representation of underserved populations, including African Americans and Latinos of Caribbean origin; approximately half of MHBB participants are women; and the population overall displays a high frequency of medical and psychosocial co-morbidities that contribute to nervous system dysfunction. The cohort also has excellent representation of individuals with long-term viral suppression, suited to the support of research into viral reservoirs. In providing its biospecimen and data resources, MHBB implements standard NNTC protocols to synchronize the type and quality of materials provided to researchers with other NNTC clinical sites. However, MHBB has also created a variety of customized resources to better serve its clients, inclusive of providing digital slides and viewing software to enhance case selection and analysis; obtaining fresh blood samples from the cohort for functional assays, which when coupled with MHBB's extensive nervous system characterization, provide a cost efficient mechanism to accomplish translational neuroAIDS research; and performing specialized tissue dissections to create unique collections like the Brain Arterial Remodeling Study. MHBB is committed to fostering young scientists embarking on careers in neuroAIDS research, with past support of an R25 mechanism, and plans to promote F- and K- type awards utilizing its resources in the next funding period. MHBB maintains high quality standards for the information it collects, with mature, databank-assisted protocols for quality assurance on the local level, use of monthly data reports and annual audits provided by the NNTC coordinating center, and participation in all NNTC subcommittees to assure compliance with standardized NNTC assessments. With a relatively small and dedicated staff, MHBB is able to rapidly adapt to NNTC initiatives, as its organizational structure and standard operating procedures result in frequent communication between all levels and domains of expertise. In addition, MHBB utilizes the talents and expanded expertise of its scientific advisor and multiple collaborators, who provide a robust translational science environment to enhance the study's operations.

Well characterized brains have great utility in studies of HIV neuropathogenesis and studies relating to adequate treatment of viral reservoirs. The Manhattan HIV Brain Bank will utilize pre-extant bank infrastructure located at the Mount Sinai School of Medicine (MSSM), and coordinate patient resources at MSSM, Beth Israel Medical Center (BIMC), and St. Luke's-Roosevelt Hospital Center (SLRHC), three of New York City's largest medical complexes, to: 1. Identify and follow a well- characterized cohort with advanced HIV disease, using neurologic, neuropsychologic, medical, and laboratory examinations; 2. Establish a multi-institutional database in which clinical data are recorded; 3. Obtain autopsy-derived central nervous system (CNS), peripheral nervous system (PNS) and systemic tissues from these patients within 24 hours of their demise; 4. Provide complete pathologic diagnoses of these tissues that will be maintained in a separate but interactive pathology database; 4. Dissect, store, catalogue and distribute these tissues to extramural investigators; 5. Evaluate the appropriateness of tissue requests with a bank review committee that will function under the guidelines of the Network Steering Committee; and 6. Utilize the pathology and clinical databases in correlational studies to elucidate the pathogenesis and natural history of HIV-related neurologic diseases. Within the context of the Manhattan HIV Brain Bank, we will establish a nationwide resource for the provision of numerous well-characterized brains and tissues for study, and additionally, establish a multi- institutional longitudinal clinical/neurologic and neuropsychiatric database that will serve as a template for following a significant proportion of the medically-serviced, HIV-infected population of New York City, and assist in future studies of HIV neuropathogenesis.

DESCRIPTION (provided by the applicant): The Manhattan HIV Brain Bank, a member of the National NeuroAIDS Tissue Consortium, is a resource for the provision of well-characterized tissues and fluids from HIV-infected patients to NeuroAIDS investigators. In the previous funding period, infrastructure to facilitate the collection and distribution of tissues, fluids and information was built. We have thus established a successful HIV organ donation program in a seriously ill, largely minority and disadvantaged inner city population. In the next funding period, we propose: to increase the provision of information, tissues, and fluids from HIV-infected patients to qualified researchers investigating HIV pathogenesis; to establish a web site from which qualified investigators can learn about our program and manage transactions regarding specimen withdrawals, as well as download modular portions of a malleable database to facilitate recruitment, neurologic analysis, and neuropsychologic analysis of diverse patient populations; to continue development of collaborations with other large studies interested in HIV pathogenesis; for purposes of standardization, to participate in NNTC quality assurance programs; to continue to recruit HIV-infected patients into an organ donation program preceded by a longitudinal neurologic, neuropsychologic, and psychiatric study; to examine traditional clinico-pathologic correlations of premortem function with postmortem pathology; to focus on co-morbidities and health disparities in HIV-related CNS and PNS disorders; to define contributions of liver disease, substance disorders, and HIV on cognitive and neurologic dysfunction; and to create an organ donation program in an HIV-negative control cohort, that will serve as a source of virologic control materials and also aid in the dissection of hepatic influences on NeuroAIDS disorders.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. With the availability of highly active antiretroviral therapy (HAART), shifts in the spectrum of HIV-related nervous system diseases are being observed. However, there has been no large-scale, comprehensive study of these manifestations and their potential pathogenesis. The CHARTER protocol was designed as an observational, non-interventional study of the nervous system complications of HIV and HAART therapy, designed to encompass a representative sample of HIV-infected patients at 6 subsites across the United States. It will have both cross-sectional and longitudinal components, and will recruit patients in all stages of HIV disease. Hypothesis: HAART will have variable effects on CNS and PNS function, dependent upon the type of regimen and patient-specific factors. Viral replication and drug resistance are important determinants in the longitudinal manifestations of disease. This study will: 1 determine if patients taking HAART have reduced risk of CNS and PNS complications of HIV infection compared to those who are na[unreadable]ve to or have stopped prior ARV. 2 determine if the CNS penetration profile of HAART is related to antiviral effects within the CNS (as measured by CSF HIV RNA levels) and to risk of HIV-associated neurological disease (as measured by cognitive function). 3 determine if the mechanism of neurocognitive damage in late stage HIV disease differs from that in earlier stages of HIV disease. 4 measure the relationship of HIV replication within the CNS (as measured by CSF HIV RNA) and of immune competence (as measured by CD4 levels) to the prevalence and incidence of syndromic HIV-related neurocognitive impairment. 5 determine the prevalence of discordant ARV resistance in HIV plasma and CSF. 6 determine if the predictors and risk factors for peripheral neuropathy from HIV differ from that of d-drug ARV neurotoxicity.

DESCRIPTION (provided by applicant): With the over-representation of racial and ethnic minority (REM) populations in HIV/AIDS, there is a need to recruit and mentor REM scientists involved in neuroAIDS investigations. The Mount Sinai NeuroAIDS Disparities Institute (MSINAD) will stimulate entry and retention of REM and non-REM scientists working in the field of neuroAIDS disparities. A summer institute will be created with a multi-disciplinary curriculum spanning the fields of neurology, neuropsychology, psychiatry, neuropathology, and neuroscience. Scholars accepted into the program will be given both didactic seminars and a research practicum;didactics will cover both topics specific to neuroAIDS disorders, and general tools for academic success, including application development. Scholars will be matched with both a scientific and a development mentor, and have access to a multi-disciplinary panel of senior scientists as well as junior faculty in AIDS disparities research as role models. By summer's end, scholars will have a strong grounding in topics necessary to conduct high quality, multi-disciplinary translational neuroAIDS research in REM communities. At the midpoint of the program, a 2 day symposium will be held to bring together a group of experts in neuroAIDS disorders, and to allow current and past MSINAD scholars to present their research concepts and progress. The MSINAD will build upon the infrastructures of two programs. The Manhattan HIV Brain Bank is a multi-disciplinary research resource that maintains an HIV-positive REM cohort that undergoes longitudinal neurologic, neuropsychologic, and psychiatric assessments. It routinely interacts with and supports a variety of neuroscientists investigating HIV manifestations and neuropathogenesis, has conducted pilot analyses of neuroAIDS disparities, and has successfully recruited and trained minority investigators who are entering the neuroAIDS research work force. The Mount Sinai Center for Multicultural and Community Affairs is a Center of Excellence for Minority Health, focused on reducing REM health outcome disparities by developing increased health care work force diversity, and providing support services at a variety of educational training and practice levels. Its Faculty Development Programs support minorities in a variety of research and clinical career pathways. By combining the strengths of these two programs, the MSINAD will help create high quality, REM researchers whose long term careers will make a lasting contribution to the area of neuroAIDS disparities.

HIV entry into the CNS occurs early after peripheral infection and is mediated by the transmigration of infected monocytes across the BBB, establishing CNS viral reservoirs, neuronal damage, and low level inflammation, despite antiretroviral therapy (ART), that mediate HIV-associated neurocognitive disorders, HAND, in >50% of infected people even in those with undetectable virus. The mechanisms of HIV infected monocyte transmigration across the BBB have only been minimally characterized. A mature CD14+CD16+ monocyte subset is key to HIV CNS pathogenesis. We showed that these cells selectively transmigrate across our model of the human BBB in response to the chemokine CCL2, and that when HIV infected, they transmigrate in even greater numbers. This is due, in part, to their increased junctional proteins JAM-A and ALCAM, and increased CCR2, the receptor for CCL2. CD14+CD16+ monocytes in HIV-infected individuals are heterogeneous, consisting of cells that are infected with HIV (HIV+), and cells exposed to viral and host factors, but not infected with the virus (HIVexp). It is not known whether the transmigration of HIV+ and HIVexp CD14+CD16+ monocytes across the BBB differs, and how this affects CNS neuropathogenesis. HIV+ monocytes that cross the BBB may differentiate into long-lived CNS macrophages and establish and maintain CNS viral reservoirs contributing to CNS damage. With ART, productively infected CD14+CD16+ monocytes in the peripheral blood are significantly reduced, with a small number of these cells still having detectable viral DNA. We propose that the neuronal damage and chronic inflammation that mediate cognitive impairment, even in the presence of ART, is dependent on continued reseeding of the brain with HIV+CD14+CD16+ monocytes, maintaining CNS viral reservoirs and continuing the influx of these infected as well as uninfected monocytes into the brain. Mechanisms that ensure that these HIV+CD14+CD16+ monocytes replenish CNS viral reservoirs over extended periods are not known. We hypothesize that CCR2, and junctional proteins are higher on HIV+ monocytes compared to HIVexp monocytes, resulting in their preferential transmigration across the BBB, and that this is associated with the establishment and maintenance of CNS viral reservoirs, and with neuronal and structural damage, low level inflammation, and cognitive impairment. We will characterize HIV+ and HIVexp monocytes using primary human mature CD14+CD16+ monocytes infected in vitro. We will also characterize the phenotype and transmigration of HIV+ and HIVexp mature monocytes from a longitudinal cohort of HIV-infected people stably suppressed on ART, and determine whether these circulating monocyte characteristics correlate with cognitive impairment and neurobiologic abnormalities using neuroimaging. We will determine whether CCR2, JAM-A, or ALCAM are biomarkers of HAND. We will use blocking antibodies and cenicriviroc in transmigration assays to assess JAMA, ALCAM, and CCR2 as potential therapeutic targets to limit CNS entry of peripheral blood HIV+ monocytes and potentially reduce reservoirs and HAND.

Summary Motor dysfunction is prevalent in combination antiretroviral therapy (cART) era HIV+ populations, however, its genesis is unclear, as cerebrovascular disease is likely to contribute to its etiology. We propose to study the spectrum, underlying neural circuitry, and cell type-specific molecular signatures of HIV-associated motor dysfunction, with the following aims and hypotheses: Aim 1. Identify neural regions associated with motor impairment in HIV+ patients with and without cerebrovascular disease. 160 cART-treated subjects will be recruited to a structural and functional magnetic resonance imaging study, to test the hypothesis that the neuroanatomical basis of motor task performance will vary by motor status (normal/abnormal) and presence or absence of cerebrovascular comorbidity. For this 2x2 analysis, participants in the Manhattan HIV Brain Bank (MHBB) will form a nidus for recruitment, with multimodal assessment of motor function. These analyses will be conducted in years 1 and 2. Then, having identified regions most strongly implicated in motor dysfunction, in years 3 through 5 we will examine autopsy brains from the MHBB cohort for: Aim 2. Cell-type specific transcriptome and epigenome mapping in dorsolateral striatum, ventral midbrain, and selected gray and white matter regions of interest (ROI) as defined in aim 1 to identify molecular signatures of motor dysfunction. Our hypothesis is that the molecular genesis of motor dysfunction can be elucidated through regional and cell-type specific analysis of transcriptome and open chromatin-associated histone acetylation and methylation landscape. Specifically, that neuronal, astrocyte, and oligodendrocyte signatures in HIV+ brain regions implicated in motor function will be affected by HIV-inflammatory burden and regional cerebrovascular disease; and that motor function will be predicted by these regional changes. Fluorescence-activated nuclei sorting in neuroanatomic regions implicated in motor dysfunction will be used for cell-type specific fractions of input material for genome-scale RNA-Seq and histone ChIP-seq. In contiguous tissue, assays of HIV DNA by nested PCR, monocyte/microglial cell activation by CD68 and CD163 immunohistochemistry, and cerebral small vessel disease (CSVD) by morphometric analysis of arteriolar wall thickening, will be done. 100 MHBB HIV+ brains will be selected on the basis of cognitive profiles and vascular risk to best approximate living subjects in aim #1, along with brains accrued from imaged individuals through subsequent organ donation. 50 demographically similar HIV- brains will also be studied. Candidate genes in molecular signatures of motor dysfunction with and without vascular disease will be tested via qPCR. With these aims, we will elucidate the neural circuitry of motor dysfunction in cART-treated HIV, cerebrovascular contributions to its genesis, and characterize its regional neurogenomics. Insight into molecular alterations and targets for amelioration will be relevant to a wider spectrum of neurodegenerative disorders with vascular contributions in HIV- populations.

HIV-associated neurocognitive disorders (HAND) persist in the era of combination antiretroviral therapy (cART). Proof of HIV latency in human CNS is currently lacking, despite continued high prevalence of HIV- associated neurologic disease and increasing recognition of CNS viral escape in people stably suppressed with cART. One of the major issues regarding CNS HIV in need for study is HIV integration. With other words, whether CNS HIV integration has biologically significant impact, contributing to pathogenesis? Issues of CNS functional deficit are further complicated by the co-registered epidemic of opiate and other substance use disorders (SUD) in people living with HIV/AIDS (PLWHA), as SUD also have profound impact on CNS function, and potentially on HIV latency. Nowhere in the CNS is this more evident than in the neuroanatomic overlap of HIV and SUD in striatonigral dopaminergic circuitry and frontostriatal projections, sites of predilection for functional and neurobiologic disease as well as for increased burden of HIV infection. Accordingly, directly utilizing brain tissues in these regions, from neurologically well-characterized HIV-infected individuals with and without SUD, the goal of this application will be: (i) to replicate for brain some of the emerging genomic mechanisms recently discovered in peripheral cells, linking HIV host genome integration and virus latency to nuclear topography and open chromatin; (ii) to explore whether HIV signatures in transcriptomes and epigenomes in dopaminergic circuitry is associated with prospectively monitored neurological status in the years before death and exposure to drug of abuse; (iii) explore HIV expression and integration in potential reservoir cells of the brain, including microglia and astrocytes derived from HIV+ brain at autopsy and (iv) explore the impact of substance history on HIV integration and activity in primary microglial cultures derived from HIV- brains at autopsy and (vi) expose primary microglia in culture to drugs of abuse and HIV. The innovative experiments proposed here are expected to offer novel insights into epigenomic landscapes in specific brain cells and explore potential links between neurogenomic status of the infected brain and neurological and cognitive symptoms and substance abuse. While recognizing the high-risk aspects, these analyses will nevertheless have predictable, high gain benefits in understanding the complex neurobiology underlying HIV-associated CNS disease in PLWHA and SUD.