Jerel Adam Fields - US grants

DESCRIPTION (provided by applicant): The goal of this proposal is to characterize regulation of primary human astrocyte tissue inhibitors of metalloproteases (TIMP) expression during Human Immunodeficiency Virus (HIV)-associated dementia (HAD). HIV-associated neurocognitive disorders are a group of disorders of which HAD is the most severe. HAD affects 10-27% of HIV-infected individuals in the United States. The disease is characterized by an accumulation of activated and infected macrophages/microglia in the central nervous system, which secrete cytokines, infectious virions and viral proteins that activate surrounding astrocytes leading to astrogliosis and altered gene expression. Neural dysfunction is believed to be the key component causing dementia. Astrocytes are essential in maintaining neural homeostasis in the CNS during injury and repair, in part through the production of neuroprotective TIMPs. An imbalance between matrix metalloproteases and TIMPs is thought to contribute to several neurodegenerative disorders, including HAD. TIMP-1, the inducible form of a family of 4 TIMPs, is a multifunctional protein that displays neuroprotective properties via homeostatic maintenance and possibly by inhibiting apoptosis. TIMP-1 levels are decreased in HAD patients compared to HIV-1 seronegative controls and primary human astrocytes acutely stimulated with HAD-relevant stimuli increase TIMP-1 expression, but levels fall after 3 days of exposure. Additionally, deleting the CAAT site at -310 in the TIMP-1 promoter increases transcription. Thus, we propose that down regulation of astrocyte TIMP-1 during HAD is mediated through differential transcriptional regulation via a CAAT site in the TIMP-1 promoter leading to reduced astrocyte TIMP-1 expression and ultimately exacerbating neurodegeneration. Two specific aims have been designed to address regulation of astrocyte TIMP-1 expression during HAD: Aim 1 determine the expression profile of CAAT enhancer binding protein 2 in activated primary human astrocytes and their influence on TIMP-1 transcription and Aim 2 identify signal transduction pathways leading to TIMP-1 down regulation in primary human astrocytes. To complete Aim 1 basic cell culture of primary human astrocytes and HAD-relevant stimuli like cytokines and viral proteins will be used along with analysis tools such as western blot, real-time polymerase chain reaction, immunocytochemistry and enzyme linked immunosorbent assay. Aim 2 will be completed by combining pathway-specific inhibitors with a panel of luciferase expression plasmids, CAAT-binding factor overexpression plasmids and pathway-indicating plasmids with and without HAD-relevant stimuli. Completion of these studies will shed light on the regulation of human astrocyte TIMP-1 expression in HAD. PUBLIC HEALTH RELEVANCE: HIV-1-associated dementia (HAD), the most severe manifestation of HIV-1-associated neurocognitive disorders, is an important neurological complication of HIV-1 infection and is characterized by cognitive, behavioral and motor dysfunction. An estimated 10-27% of HIV-seropositive patients progress to develop HAD in developed worlds such as the United States, despite the availability of highly active antiretroviral therapy. Our studies will provide a better understanding of the specific mechanistic contributions of activated astrocytes to HIV-1-neuropathogensis and neuroinflammation.

DESCRIPTION: The goal of this proposal is to characterize neuronal mitochondria function during HIV-associated neurocognitive disorders (HAND) and the mechanisms by which HIV proteins disrupt homeostatic neuronal mitochondria fission/fusion processes. Despite 30 years of research and the advent of highly active antiretroviral therapy (HAART), HAND persist. Moreover, HAART increases life expectancy and an increasingly large pool of HIV patients are managing viral infection confounded by processes of aging. Accordingly, there is great need for therapies capable ameliorating the devastating effects of HIV infiltrating the central nervous system (CNS). HIV enter the CNS in monocytes and subsequently reproduce, infect CNS cells and initiate a battery of inflammatory cascades that ultimately result in synaptic degradation, neurodegeneration and HAND. Progress has been made, but effective treatment for HAND remains elusive. Recent studies show that HIV proteins may interfere with normal function of mitochondrial fission/fusion proteins DRP1 and Mfn2, which may affect fission/fusion and mitophagy. Our preliminary data show DRP1 is increased and Mfn1 and Mfn 2 are decreased in postmortem brain tissue of HIV infected patients, suggesting a pro-fission environment. We found that recombinant HIV negative regulatory factor (Nef) and glycoprotein (gp)120 similarly increase neuronal DRP1 and decrease neuronal Mfn1 and Mfn2. These and other studies suggest mitochondria fission/fusion may be impaired in the CNS during HIV infection; a potential contributor to HAND. Therefore, we hypothesize that HIV proteins, Nef and/or gp120 bind DRP1, Mfn1 and/or Mfn2 and disrupt homeostatic mitochondria fission/fusion processes in neurons. To explore this possibility we propose the following aims: Aim 1: To characterize DRP1, Mfn1 and Mfn2 expression patterns and their interactions with HIV proteins in postmortem brain tissues from HIV infected donors. Aim 2: To investigate the role of HIV proteins in the cellular mechanisms of mitochondrial fission/fusion dysfunction and resulting neurotoxicity. To complete Aim 1 tissue from the California NeuroAIDS consortium will be analyzed for DRP1, Mfn1 and Mfn2 expression and their interactions with HIV proteins by co-immunoprecipitation, immunoblot, real-time polymerase chain reaction (RT2PCR), enzyme-linked immunosorbent assay (ELISA) and quantitative immunohistochemistry. Aim 2 will be completed using lentiviral (LV) vectors expressing Nef, gp120, dominant negative (DN) DRP1, Mfn1 and Mfn2 in neuronal and microglial cell lines. DRP1, Mfn1 and Mfn 2 expression levels and function will be assessed via coimmunoprecipitation, immunoblot, RT2PCR, and quantitative immunocytochemistry. Completion of these studies will shed light on mitochondrial function during HIV infection. Scheme A [is that] HIV-infected microglia secrete progeny virus, cytokines and toxic viral proteins that lead to neurodegeneration and HAND. [Scheme] B [is that] proper functioning mitochondria fission/fusion processes are necessary for maintaining and constructing new neural pathways. Interference may lead to neurodegeneration via toxic viral proteins: 1. Binding neuron receptor and affecting DRP1 expression and/or 2. Binding fission/fusion machinery directly (DRP1, Mfn1, Mfn2) and altering function or half-life. These studies will provide valuable insight into neuronal mitochondria dynamics during HAND and other CNS disease, and possibly provide novel therapeutic targets.

PROJECT SUMMARY Over 36.7 million people worldwide, including 1.2 million people in the USA are HIV positive. HIV-associated sensory neuropathy (HIV-SN) is the most frequent neurological manifestation of HIV and is characterized as a distal symmetrical, predominantly sensory, polyneuropathy. HIV-SN may represent 2 clinically indistinguishable neuropathies with distinct pathogenesis: a distal axonal degeneration caused by interaction of sensory neurons with HIV-associated proteins such as gp120 and Tat and also an anti-retroviral therapy (ART)-induced toxic neuropathy, ART may also potentiate the HIV-SN induced by HIV proteins. Recent studies have implicated mitochondrial dysfunction in the pathogenesis of HIV-SN and disruption of neuronal mitochondrial biogenesis and quality control (biogenesis-mitophagy axis) occurs in HIV-SN patients. There is no current treatment that targets a pathological process underlying HIV-SN, but the emerging appreciation of the role of mitochondrial dysfunction in the underlying pathogenesis provides a potential therapeutic approach. The academic founders of WinSanTor recently reported that neurite outgrowth from peripheral sensory neurons is under a cholinergic constraint mechanism mediated by type 1 muscarinic receptors (M1R). Stimulation of the M1R restrains mitochondrial function, thereby limiting neuronal energy supply and neurite growth. Conversely, inhibition of M1R activates AMP-activated protein kinase (AMPK) with subsequent enhancement of mitochondrial bioenergetic function and neurite regeneration. M1R inhibition also prevents and reverses indices of distal degenerative neuropathy in animal models of diabetic and chemotherapy-induced neuropathy. These promising findings suggest that the therapeutic efficacy of M1R antagonists has the potential to extend across diverse peripheral neuropathies in which mitochondrial function is compromised. We have recently found that mouse models that overexpress HIV-associated proteins exhibit mitochondrial dysfunction and develop symptoms of neuropathy. Further, reduced neurite outgrowth from sensory neurons exposed to the HIV protein gp120 in vitro was prevented by treatment with the M1R antagonist pirenzepine, while loss of corneal nerves induced by delivery of gp120 to the eye of normal mice was both prevented and reversed by concurrent topical application of a M1R antagonist. Based on these results, the goal of this Phase I STTR project is to 1) evaluate efficacy of pirenzepine against neuropathy in mice expressing HIV-Tat protein; 2) evaluate efficacy of pirenzepine against neuropathy in mice expressing HIV-gp120 protein with concurrent ART therapy. Successful completion of this Phase I project will support further pre-clinical development of pirenzepine as a novel therapeutic for treatment of HIV- SN. In Phase II, we will further define the safety/toxicology profiles of pirenzepine to support filing of an IND application with the FDA.

SUMMARY/ABSTRACT Over 1.3 million people in the USA are infected with HIV and almost half suffer from HIV-associated neurocognitive disorders (HAND), despite having access to antiretroviral therapy (ART). Our recent findings show mitochondrial dysfunction may play a crucial role in the neurodegenerative process involved in HAND. The research goals of this K01 proposal are to determine how dysfunctional mitochondria contribute to the progression of HAND in the era of ART. The training goals are for the applicant to attain expertise in transcriptomics and mitochondrial bioenergetics research. Mitochondria power the body by performing the final steps of converting the food we eat into the molecular units of energy our cells can use. Maintaining a healthy pool of mitochondria requires generation of new mitochondria and recycling of old mitochondria. We've recently discovered that in brains of HAND decedents, there is blockage in both the generation of new mitochondria and recycling of damaged mitochondria. The mechanisms of neurodegeneration causing HAND are not completely understood, however our previous findings show that HIV proteins can alter the splitting and conjoining of mitochondria, and this results in mitochondrial enlargement and damage. New data suggest that in HAND brains, cells attempt generate new mitochondria, but the process is blocked prematurely. To extend these studies, we hypothesize that disruption in mitochondrial biogenesis and downstream effects on bioenergetics are key neurodegenerative mechanisms driving HAND. We will test this hypothesis in two Specific Aims: AIM 1: Investigate mitochondrial biogenesis-related gene expression networks in brains of HIV+ decedents who were on ART, and in a mouse model expressing HIV proteins in the brain. In Aim 1, mitochondrial biogenesis will be investigated using brain tissues from a cohort of HAND decedents, and from mice expressing HIV proteins in the brain. Mitochondrial biogenesis gene expression in brains will be determined by RNA sequencing (seq) and transcriptomic analyses. Findings will then be validated using biochemical and microscopy methods. We will further investigate the how HIV proteins or ART affect mitochondria in AIM 2: Determine the underlying mechanisms of disrupted mitochondrial biogenesis and bioenergetics in neurons exposed to gp120, Tat and/or ART, in vitro. For Aim 2, mitochondrial activity of neurons will be measured after knockdown and overexpression of mitochondrial biogenesis pathway proteins in human primary neuroglial cultures treated with HIV proteins or ART. Using these experiments, we will identify important interactions between HIV proteins or ART and mitochondrial biogenesis, and the effects on neuronal bioenergetic capacity. The information gathered from these studies may lead to novel therapeutic targets in ART-era HAND patients and novel mechanisms of dysfunctional mitochondrial biogenesis and bioenergetics that could be relevant to other neurodegenerative diseases.