James Haorah, Ph.D - US grants

[unreadable] DESCRIPTION (provided by applicant): Alcohol is the most commonly used and abused drug in the United States. Deleterious alcohol-related health effects, attributed to internal organ toxicity, include irreversible brain tissue injury. Brain tissue of chronic alcoholics features neurodegeneration paralleling neuro-cognitive deficits. The causes of HIV-1-associated neurotoxicity, clinically manifesting as HIV-1 associated dementia (HAD), include excitotoxic effects of glutamate, secretory products of chronically activated glial cells, and oxidative stress, similar culprits to ones mediating alcohol-induced neuronal injury. Alcohol abuse and HIV-1 infection of central nervous system (CNS) could result in combined toxic effects leading to neuronal demise and cognitive dysfunction. The current grant application is focused on putative mechanisms of enhanced neurotoxicity in the setting of alcohol abuse and HIV-1 CNS infection. Specifically, we will study unique aspects of astrocyte dysfunction caused by HIV-1 CNS infection and alcohol abuse. We hypothesize that astrocyte dysfunction caused by alcohol metabolites and oxidative stress results in (1) increased glutamate levels via down regulation of excitatory amino acid transporter (EAAT-2, the primary astrocyte glutamate scavenger) causing neuronal injury; (2) production of pro-inflammatory factors (via activation of Src kinases and phospholipase A2); and (3) enhanced activity of metalloproteases (MMPs) resulting in loss of blood brain barrier (BBB) integrity. We mechanistically address the role of oxidative stress in astrocytes leading to production of pro-inflammatory molecules and impairment of glutamate uptake by astrocytes. Using a combination of in vitro systems, an HIVE animal model chronically exposed to alcohol, and in vivo magnetic resonance spectroscopy, we will investigate the astrocyte dysfunction as a focal point of combined effects of HIV-1 and alcohol abuse in the CNS. We will address the following questions: (1) What are the underlying mechanisms causing astrocyte pro-inflammatory phenotype in the setting of alcohol abuse and HIV-1 CNS infection that cause activation of MMPs and BBB dysfunction? (Aim 1); (2) What is the role/contribution of enhanced production of reactive oxygen species, Rho GTPases, and NF-?B signaling in diminished expression and function of EAAT-2 in astrocytes? (Aim 2); (3) How effective are the therapeutics that target oxidative stress and EAAT-2 dysregulation in ameliorating neurotoxicity and BBB impairment in an animal model for HIV-1 encephalitis and alcohol abuse? (Aim 3). Antioxidants and specific signaling inhibitors will be utilized to delineate pathways involved in these effects. We believe that the proposed works are highly significant as they will uncover novel mechanisms involved in the combined effects of HIV-1 and alcohol abuse in the CNS and propose therapeutic approaches based on these investigations. [unreadable] [unreadable] Public Health Relevance: Alcohol is abused by millions of Americans has long lasting toxic effects on the central nervous system. Clinical studies indicated that alcohol dependence has an additive effect on cognitive deficits associated with HIV-1 infection. Current proposal aims to understand mechanisms of combined effects of HIV-1 and alcohol abuse in the brain and to propose neuroprotective therapies. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Alcohol is the most commonly used and abused drug in the United States. Deleterious health effects of alcohol attribute to toxicity of internal organs including irreversible brain tissue injury. Brain tissue from chronic alcoholics exhibits neuronal cell death and degeneration paralleling neuro-cognitive deficits. Alcohol abuse leads to white matter abnormalities that could be associated with blood brain barrier (BBB) dysfunction detected in alcohol abusers or seen in animal model. The BBB formed by brain microvascular endothelial cells (BMVEC), pericytes and astrocytes connect the BMVEC assuring BBB structural tightness. The actin cytoskeleton is linked to tight junctions (TJ) proteins through intracellular zonula occludens (ZO-1-3) and this dynamic complex readily adapts to a variety of physiologic or pathologic conditions. The intracellular signaling processes that regulate phosphorylation of TJ proteins control TJ assembly and BBB integrity. The exact mechanism of BBB dysfunction seen in alcohol abusers remains elusive. We demonstrated the previously unrecognized effects of ethanol (EtOH) on BMVEC, underlying the cause of BBB impairment. We showed that EtOH increases the activity and content of EtOH-metabolizing enzymes, cytochrome P450-2E1 (CYP2E1) and alcohol dehydrogenase in human BMVEC. EtOH metabolism by CYP2E1/ADH in BMVEC enhances production of acetaldehyde (AA) and reactive oxygen species (ROS). We propose that these reactive EtOH metabolites (AA and ROS) activate myosin light chain kinase (MLCK) via IP3R-gated intracellular calcium signaling pathway resulting in phosphorylation of cytoskeletal and TJ proteins as acute effect. Further, the reactive EtOH metabolites activate matrix metalloproteinases -2 and -9 (MMP-2 and -9) via protein tyrosine kinase (PTK) signaling pathway leading to degradation of endothelial specific basement membrane (BM) or extracellular matrix (ECM) proteins. Changes in cytoskeletal and TJ assembly, or degradation of BM/ECM proteins increased BBB permeability and augmented leukocyte migration across the BBB, suggesting break down of BBB function. BBB dysfunction due to alcohol abuse could be associated with in neuro-inflammatory disorders and neurological diseases. Thus, we propose to study the mechanism of alcohol abuse disruption of blood-brain barrier due to oxidative stress stemming from alcohol metabolism in brain endothelial cells, which activates myosin light chain kinase through intracellular calcium release (acute effects) and activation of matrix metalloproteinases via PTK signaling pathway (delayed effects). [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This proposal advocates the function of glial cell astrocytic acetaldehyde dehydrogenase 2 (ALDH2) as the first line neuroprotective defense mechanisms in the brain from alcohol abuse. Neutralization of reactive acetaldehyde by ALDH2 promotes the key initial protective biochemical mechanisms in alcohol abuse. The statement is supported by our recent findings that activation of NADPH oxidase and inducible nitric oxide synthase by acetaldehyde increases the levels of reactive oxygen species and nitric oxide in primary human neurons. To prevent the action of acetaldehyde as transducer for oxidative stress, a therapeutic stabilization of astrocytic ALDHs by acetyl-L-carnitine (ALC) is an innovative approach, which has not been studied so far. This protective mechanism of astrocytes is arisen from the fact that ALDHs are genuine antioxidants that can balance oxidant and antioxidant levels in alcohol stress. Our proposal is that stabilization of the inherent astrocytic ALDH2 protein in the brain shields the neurons from adverse effects of alcohol and acetaldehyde toxicity. Therefore, astrocytes are active defenders of neurons from the deleterious effects of alcohol. The central hypothesis of the proposal is that effective detoxification of acetaldehyde by ALC-stabilized astrocytic ALDH2 can prevent the oxidative damage and neurotoxicity. We propose to investigate the protective mechanisms by using primary mice and human astrocytes and neuronal co-cultures as in vitro system and chronic ethanol liquid diets feeding as our animal model. Success of the project will impact knowledge gap in unraveling the cellular and biochemical mechanisms of alcohol effects in neurobiology and health care. PUBLIC HEALTH RELEVANCE: The relevance of the proposal addresses the novelty that astrocytes are the active defenders of neurons from the deleterious effects of alcohol by virtue of their inherent ALDH2 function. Stabilization of astrocytic ALDH2 protein in brain shields the sensitive neurons from the adverse effects of alcohol toxicity. Therefore, astrocytes are the active defenders of the brain and the success of the proposal has a huge potential impact for prevention of many alcohol-related neurological disorders in health care.

DESCRIPTION (provided by applicant): Stroke is the third leading cause of death and the most prevalent cause of permanent disability. This application evolves from the findings that chronic alcohol use is strongly associated with atherosclerosis and stroke with not well define underlying molecular/cellular mechanisms. Here, we propose to examine the hypothesis that immune cell adhesion and cholesterol deposit at the site of oxidative injury in capillary walls causes formation cholesterol crystals (CC) to activate NLRP3 inflammasome for induction of atherosclerosis in heavy chronic alcohol intake. The clinical relevant of this application is that Joint therapy of acetyl-L-carnitine (ALC) and Lipitor can prevent this alcohol-elicited CC formation and atherosclerotic lesions. We will address this innovative idea in our recently developed unique animal model of human disease with two objectives. Our first objective is to examine if immune cell adhesion and cholesterol deposit at the vasculature enhances CC formation, NLRP3 activation and atherosclerotic lesions in response to dose-time-/and temperature-dependent effects of alcohol. After establishing this kinetic profile, we will then address the molecular mechanisms of CC-induced activation of NLRP3 and downstream caspase 1 signaling pathways in primary human brain endothelial cell culture. Our second objective is to evaluate if a joint therapy of ALC/Lipitor can reverse the early development of atherosclerosis in chronic alcohol intake. This will assess the protective effect of ALC/Lipitor from alcohol-induced atherosclerotic bulging, brain infarct volume, intracranial blood pressure and in vivo imaging of CC using cyclodextrin nanoparticles specific to CC by MR brain imaging.

Abstract: Application is submitted here in response to PA-17-296. The efficient clearance of waste metabolites is compromised in the brain due to lack of lymphatic system and cerebrospinal fluid (CSF) is not able to clear large size waste metabolites. Recent discovery of glymphatic system is able to transport small size water-soluble waste metabolites directly to perivenous space by aquaporin-4 water channels of the astrocyte end-feet. Thus, unravelling the mechanisms of clearing large size waste metabolites in the CNS become significant for therapeutic prevention of neurological diseases associated with entangled proteins. Here, we propose to address the clearance mechanisms of fluorescent labeled ?-amyloid protein from interstitial fluid to perivascular space or from CSF subarachnoid into perivenous drainage through perivascular clearance path. We propose that this dynamic perivascular-perivenous drainage path is significantly promoted by low dose alcohol (ethanol), and not by high dose or chronic alcohol use. Our working hypothesis is that activation of cerebral arterial endothelial specific nitric oxide synthase (eNOS) by alcohol (5 mM) led to generation of a potent vasodilator nitric oxide, which mediates the interactive reactivity of endothelial-smooth muscle cells and subsequent inter-convective movement of waste metabolites towards perivascular space. We will address the hypothesis, first, by establishing the existence of perivascular clearance path and how alcohol promotes this drainage path, and second by evaluating the underlying molecular mechanisms of perivascular clearance by alcohol-elicited endothelial NO that promotes endothelial- smooth muscle cells dilative reactivity and subsequent waste metabolites clearance. Knowledge gained from this project is expected to bear a significant impact for possible prevention of neurological disorders such as cerebral amyloid angiopathy and Alzheimer's disease.