Seth W. Perry - US grants

[unreadable] DESCRIPTION (provided by applicant): HIV-associated neurologic disease (HAND) remains a source of significant morbidity, and is increasing in prevalence, despite success reducing viral load in HIV-infected individuals with highly active antiretroviral therapy (HAART). While HAART can frequently delay the onset or progression of HAND, and may in part reverse the course of HAND with timely initiation, it cannot always prevent HAND, and it cannot forever halt the course of HAND as HIV-infected individuals age, thus accounting for HAND's increased prevalence. Therefore exceptional need remains for adjunctive therapies that can directly address the neurologic deficits of the increasing and aging population of HIV-infected individuals. A large body of evidence suggests HAND pathogenesis results from a toxic milieu of secretory neurotoxins secreted from HIV-1 infected, brain-resident mononuclear phagocytes and glia, which act in concert to impart a range of toxic effects neuronal function, particularly synaptic function. Neuropathologic studies demonstrating neuronal apoptosis in brain tissue of patients that had neurologic deficits indicate that the degree of frank neuronal loss does not correlate well with pre-mortem neuropsychologic deficits. Rather, alterations in dendritic architecture and synaptic structure correlate far better with these deficits, and our laboratory and others have shown that the HIV neurotoxins Tat and platelet activating factor (PAF) adversely affect synaptic function, and may even render "normal" physiologic synaptic activity harmful in their presence. From this convergent evidence, we believe HAND arises from a reversible metabolic synaptic dysfunction, and is amenable to direct therapeutic intervention. The dopamine system appears particularly vulnerable in HAND, and there is evidence that a "reversible" synaptic dysfunction applies to dopaminergic synapses as well. Supported by others' complementary findings, we present a breadth of preliminary evidence here that dopamine transporter (DAT) activity and function is disrupted by the HIV neurotoxin Tat, resulting in hyperactive DAT activity and dopamine uptake at the synapse. We hypothesize this hyperactive DAT activity is: 1. Sufficient in itself to cause HAND deficits, and 2. May ultimately result in permanent nigral or striatal neuronal loss and neurologic deficit, consequent to unsustainable metabolic demands and/or enhanced auto-oxidative dopamine toxicity pre-synaptically, or over/under-stimulation of post-synaptic striatal connections. Mechanistically, binding of alpha-synuclein to DAT has been shown to increase membrane DAT in models of Parkinson's disease (PD). Separately, we have found significant roles for glycogen kinase three-beta (GSK-3beta) in models of HAND, and others' have implicated GSK-3beta in controlling alpha-synuclein activity. No direct links have yet been established between GSK and synuclein as mediators of DAT dysfunction in HAND (or PD), despite preliminary success of GSK- blockade as a potential therapeutic approach for HAND. The studies herein propose to explore these interactions as a possible root cause of dopaminergic dysfunction in neuroAIDS. PUBLIC HEALTH RELEVANCE This project proposes to study the mechanisms by which HIV-neurotoxins in the brain alter dopaminergic synapses, particularly activity of the dopamine transporter, to impart neurologic deficits in HIV Dementia, with the goal of developing adjunctive therapeutics for this disease. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Worldwide 33.4 million people lived with HIV/AIDS in 2008, with 2.7 million new infections, and 2 million related deaths. In the US, 1.4 million people had HIV/AIDS in 2008, with 55,000 new infections, and 25,000 related deaths. Certain disadvantaged or vulnerable populations bear a disparate share of this burden, including women and children globally, and Black American (BA) and Hispanic minorities in USA. BAs acquired 46% of new infections in 2006, despite comprising only 13% of the population. Among females, BAs share an even more disproportionate burden, acquiring 61% of all new female HIV infections in 2006. For 2006 male infections, 76% were MSM. In these vulnerable groups, drug use including amphetamines, opiates, alcohol, and cocaine, is associated with high-risk sexual behavior, and accelerates progression of and mortality from HIV. Drug use is also an independent risk factor for seroconversion, and predictive of disease progression, development of AIDS, and AIDS mortality. Thus it is increasingly important to understand impacts of comorbid drug abuse on HIV infection, particularly as it affects vulnerable populations and therapy development [1-10]. Some evidence implicates glycogen synthase kinase 3 (GSK3) as a possible regulator of peripheral HIV infection, and GSK3 also may mediate effects of some abused drugs, such as cocaine and methamphetamine. However there has been little investigation of exactly how or whether GSK3 directly affects HIV infection with and without comorbid drug abuse, particularly as regards key HIV tropic peripheral CD4+ T cells and macrophages. Hence, this proposal will investigate the unifying hypothesis that GSK3 activation increases endocytosis to enhance coreceptor mediated HIV infection of peripherally derived human CD4+ T cells and macrophages, and that this is the mechanism by which cocaine and amphetamines enhance HIV infection of these cell types. First we will determine if GSK3 transgene overexpression or knockdown directly affects HIV infection of CD4+ T cells and macrophages. Next, using fluorescent, immunocytochemical, and western blot assays for endocytosis, and siRNA knockdown of Rab5, we will determine whether GSK3 increases in HIV infection in CD4+ T cells and macrophages by enhancing Rab5-mediated endocytosis. In Aim 2, using similar approaches, we will first determine whether Meth or cocaine treatment increases GSK3 expression and activity, and endocytosis, in these cell types. We will further determine whether Meth or cocaine increase HIV infection of these cells, and whether these effects can be abrogated by manipulating GSK3 and/or Rab5 expression. In all of these studies, with and without cocaine and Meth, we will determine whether GSK3's effects on HIV infection of CD4+ T cells and macrophages can be ameliorated by the GSK3-inhibiting compounds lithium and AR-A014418, to determine whether GSK3 inhibition is a potentially viable therapeutic strategy for reducing viral load in HIV disease. Thus these studies will provide new insights into the molecular mechanisms regulating HIV infection of highly HIV-tropic peripheral immune cells, to identify new therapies for treating HIV disease with comorbid drug abuse. PUBLIC HEALTH RELEVANCE: This project explores new mechanistic theories that may help explain how HIV enters human immune cells to cause HIV disease. Better understanding of these disease mechanisms concerning how HIV causes infection of principal cell types involved in HIV, will help identify additional therapeutic targets for preventative intervention to treat HIV and AIDS.

? DESCRIPTION (provided by applicant): The vast majority of breast cancer deaths arise from metastatic disease. Altered deposition or organization of fibrillar collagen types I, III, and V in breast tissue or tumor stroma have been linked to increased breast cancer risk and metastasis, but precisely how these changes in collagen homeostasis promote breast cancer pathology is not well understood. However increased collagen type I fiber density and alignment have been shown to promote metastasis in breast cancer, and tumor cells may preferentially migrate along aligned collagen I fibers. Moreover, integrins appear to be key regulators of breast tumor metastasis, and integrins interact with collagen fibers. Yet surprisingly, despite these connections, exactly how fibrillar collagens and integrins interact to regulate breast cancer metastasis remains largely unknown. Supported by preliminary data linking changes in Col1 fibril structure with metastatic potential and clinical BC progression, this proposal will provide insight into these key questions by testing a novel unifying hypothesis for how altered collagen expression in BC may dynamically regulate Col1's fibrillar structure, which may in turn dictate its biologic function in breast tumor metastasis. In Aim 1, we will use a 3D organotypic collagen gel model, multiphoton second harmonic generation (SHG) microscopy, and electron microscopy to test whether collagen III/I and V/I ratios alter heterotypic collagen fibril composition and structre to affect a2ß1 binding site availability on Col1 fibers. In Aim 2, we will then test the functiona consequences of altered collagen III/I and V/I ratios and a2ß1-Col1 interactions on breast cancer metastasis using complementary in vitro and in vivo model systems. In vitro, we will use 3D collagen gels and real-time total internal reflection fluorescence microscopy (TIRFM) to determine how changing collagen III/I and V/I ratios affects breast tumor cell's ability to migrate along collagen fibers via a2ß1-engagement of Col1. To further validate our findings in vivo, we will use an orthotopic mammary tumor model, and human breast cancer biopsies, to assess how changes in collagen III/I and V/I ratios, collagen structure as measured by SHG, and integrin binding site availability on Col1, affect metastatic outcome in these in vivo model systems. These studies will provide important new linkages between existing evidence for the roles of fibrillar collagens and integrins in breast cancer. [These studies also highlight the possibility of using targeted modulation of individual collagen subtypes, or targeted manipulation of integrin binding sites on specific collagen subtypes, as a novel therapeutic approach for preventing breast cancer metastasis].