1994 — 1997 |
Meeker, Rick B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cellular Mechanisms of Fiv Neurotoxicity @ University of North Carolina Chapel Hill
Feline immunodeficiency virus (FIV) is a neurotropic lentivirus homologous to human immunodeficiency virus type-1 (HIV-1) with similar cellular targets, disease progression and clinical outcome. Growing evidence supports the presence of neurological disease accompanying FIV infection indicating that FIV may provide a useful animal model for AIDS dementia (HIV-1- associated cognitive-motor complex). Current models of HIV- related neurotoxicity suggest that excitotoxic mechanisms may be the final common pathway for neurodegeneration. We hypothesize that similar mechanisms of neurodegeneration will be seen in cats infected with FIV. If similar mechanisms are found, the cat would provide a versatile and convenient animal model inn which both in vitro and in vivo studies could be combined in the same species for analysis of CNS infectivity and neurotoxicity and the development of neuroprotective therapeutic strategies. We propose to develop an in vitro CNS model for the analysis of FIV neurotropism neurotoxicity. To accomplish this, we will establish primary cultures of embryonic (E40-E58) feline neural tissue including neurons, astrocytes and microglia. Virus infection and replication in these cultures will be assessed by inoculating cultures with FIV and evaluating proviral content by PCR-Southern blot analysis. The cellular target(s) of FIV will be determined by evaluating infectivity of purified cultures of astrocytes and microglia as well as by immunohistochemistry or in situ hybridization analyses in mixed cultures. Conditioned medium from various combinations of purified and co-cultured cells inoculated with FIV will be tested on mixed neuronal cultures to evaluate the secretion of toxic factors as well as the role of cell-cell interactions in the production of the toxic substances. To compare the FIV model to HIV, a strong focus will be placed on the role of excitatory amino acid and VIP receptors, calcium channels and cytokines will be used to characterize the pharmacology of the neurotoxicity. Results from the in vitro studies will be correlated with in vivo receptor studies (membrane binding, autoradiography) and neuropathology of late-stage FIV infected cats to relate the in vitro mechanisms of neurotoxicity to patterns of neurodegenerative changes in feline AIDS. Together, we believe these studies will establish FIV infections in cats as a valuable animal model for the investigation of the neurodegenerative changes associated with AIDS dementia in humans.
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1 |
1995 — 2000 |
Meeker, Rick B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Excitatory Amino Acid Regulation of Vasopressin Cells @ University of North Carolina Chapel Hill
Our studies will focus on the chemical and physiological factors which control the secretion of vasopressin by magnocellular neurons in the hypothalamic supraoptic nucleus. These factors will be identified, characterized and an attempt will be made to relate these to the osmotic regulation of vasopressin secretion. The use of an in vitro approach with the hypothalamo-neurohypophysial complex (HNC) will give the degree of control necessary to achieve these goals. The HNC contains the entire supraoptic-neuroendocrine final common pathway including the two intact supraoptic nuclei (NSO), the supraoptico-neurohypophysial tract (SOHT) and the neurosecretory terminals of the neural lobe (NL). Every major site suspected of being involved in the osmotic regulation of vasopressin secretion is present in the HNC. The HNC explant retains osmosensitivity in vitro and can approach in vivo responsiveness. Thus, the entire functional pathway is present for the exploration of the functional integration of osmotic stimuli and the transduction of this information at the level of the supraoptic nucleus. We will characterize the release of vasopressin and establish the conditions which allow precise temporal and semi-quantitative analys of responsiveness via the SOHT. Effects of chemical and osmotic stimuli and their interactions will be characterized and will include screening for the effects of various putative neurotransmitters on the release of vasopressin (cholinergic, angiotensin II, norepinephrine, enkephalin, dopamine). In particular, we will focus on the specific contribution of the NSO with particular emphasis on the nicotinic cholinergic system which has been strongly implicated in the control of vasopressin secretion. The localization of putative nicotinic receptors on vasopressin-containing magnocellular neurons in NSO will be examined. Parallel experiments will be run to assess specific properties of these receptors which may contribute to osmotic sensitivity of the NSO. Investigation of the electrophysiological properties of identified cells in the NSO during chemical and osmotic challenges will provide detailed temporal comparison of the physiological responses with the release of vasopressin. Concomitant double- and triple-labeling procedures on dye-marked neurons will be used for cytoarchitectonic reconstruction and electron microscopic analysis of synaptic inputs on immunocytochemically-identified neurons in the NSO. Thus, the HNC explant represents a simple, viable, highly controlled mammalian neural system in which unknown physiological processes express their effects on a well defined final pathway. Ideal neuroendocrine control system.
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1 |
2000 — 2001 |
Meeker, Rick B |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Fiv Penetration and Persistence in Neonatal Cat Brain @ University of North Carolina Chapel Hill
Description (Adapted from Applicant's abstract): Human immunodeficiency virus (HIV) penetrates into the central nervous system (CNS) and leads to particularly rapid disease progression in children suggesting heightened viral penetration and pathogenic interactions in the immature brain. While antiretroviral therapies have been effective in reducing HIV infection in newborns, there are many important unresolved issues. Can exposure of the brain prior to antiretrovirals establish a latent reservoir in the brain? What is the relative penetration and distribution of virus in the neonatal brain? What is the cytokine and chemokine response of neonatal brain to HIV? Is chronic exposure to antiretroviral drugs toxic to the developing brain? Because early interactions of the virus with brain probably set the stage for subsequent disease progression, early direct intervention in conjunction with antiretrovirals is likely to be important for effective treatment of the neurological and behavioral complications of HIV infection. However, the development of effective therapeutic strategies requires more knowledge regarding the dissemination of virus in the brain, establishment and control of local viral reservoirs, clearance of virus from the brain and the mechanisms of pathogenesis. We will use feline immunodeficiency virus (FIV) as an animal model of HIV-induced CNS disease to examine virus penetration and cytokine and chemokine expression in the immature cat brain by quantitative RT-PCR. Cytotoxicity assays of CSF will be used to evaluate the production of toxic factors in cats infected with FIV and cats treated systemically with zidovudine. After systemic virus is suppressed in vivo with antiretroviral treatment, we will: 1). Evaluate virus transfer from the brain to the systemic circulation upon removal of anti retrovirrals, 2). Measure the stability of the brain viral reservoir and 3). Determine if neurological disease progresses in the absence of a systemic viremia. It is anticipated that these studies will clarify some of the early interactions of lentiviruses with neonatal brain and reveal possible therapeutic strategies to reduce CNS infection and pathogenesis.
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1 |
2002 — 2005 |
Meeker, Rick B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Microglia and Choroid Plexus Macrophages in Neuro-Aids @ University of North Carolina Chapel Hill
Human immunodeficiency virus (HIV) rapidly penetrates the central nervous system (CNS) and can lead to varying levels of neuronal loss and neurological impairments including AIDS dementia. Because interactions of the virus with the CNS prior to the development of AIDS probably set the stage for subsequent disease progression, early intervention is likely to be important for effective treatment of the neurological and behavioral complications of HIV infection. Microglia and macrophages are thought to play a key role in the penetration of the virus into the nervous system and subsequent pathogenesis. However, our understanding of the interactions of the virus with brain microglia and macrophages is still quite limited. Specifically, little is know about the relative role of microglia and macrophages in pathogenesis, the stimuli that provoke the activation of these cells and release of putative toxins, and the mechanisms that lead to neuronal dysfunction. Using feline immunodeficiency virus (FIV) we have developed a model of viral infection and pathogenesis that may help to explain how each cell might contribute to evolving pathogenesis. This model suggests that the macrophage population within the choroid plexus could maintain a virus reservoir capable of infecting T-cells locally and perhaps contribute to the trafficking of macrophages and secretion of neurotoxins. Using primary cultures of brain microglia, choroid plexus macrophages and neurons, we will determine the viral and non-viral stimuli that induce microglial and macrophage activation and secretion of toxins. Microglial/macrophage activation will be assessed by evaluating increases in intracellular calcium in vitro, production of cytokine and chemokine mRNA and the accumulation of substances toxic to cortical cat neurons in primary culture. In addition, we will pharmacologically characterize the actions of the secreted toxins on neurons that lead to the destabilization of intracellular calcium. Microglia, macrophages or T-cells infected in vitro will then be infused into the brain ventricles to generate an in vivo model of central nervous system pathogenesis. These studies will clarify the role of microglia, macrophages and T-cells in the inflammatory response that develops in the brain following lentivirus infection and help to generate an animal model of macrophage-induced toxicity that can be used for identification and evaluation of improved strategies for early therapeutic intervention.
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1 |
2006 — 2009 |
Meeker, Rick B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Choroid Plexus Macrophages in Fiv Neuropathogenesis @ Univ of North Carolina Chapel Hill
DESCRIPTION (provided by applicant): Human immunodeficiency virus (HIV) rapidly penetrates into and infects the central nervous system (CNS). Inflammatory activity resulting from the interaction of HIV with macrophages and microglia in the nervous system leads to varying levels of neuronal loss and neurological impairments. While disease severity has been reduced with the advent of highly active antiretroviral therapy, CNS disease persists. The progression of CNS disease, although slowed, is expected to exert an increasingly heavy toll as patients with HIV live longer. The development of therapeutic strategies designed to protect against neuronal damage have been hindered by a lack of animal models that recapitulate the conditions that lead to neuropathogenesis in HIV infection. While simian immunodeficiency virus (SIV) models have been quite valuable, animals are costly and in short supply. Feline immunodeficiency virus (FIV) offers an alternative model of lentiviral neuropathogenesis which recapitulates all essential aspects of HIV infection in humans. However, a rapid model of FIV-induced CNS disease has not yet been developed. Our recent data have indicated that FIV introduced directly into the cerebral ventricles can produce greater CNS infection and increased viral RNA in the cerebrospinal fluid (CSF). In these studies a unique variant of FIV was identified in CSF which correlated with the onset of neurological disease. The proposed studies will grow this neurovirulent virus from CSF and introduce it into the brain of naive cats to encourage the development of a neurotropic strain which can be used to more effectively study CNS disease progression. Viral titers will be closely tracked in the plasma and CSF in conjunction with the analysis of viral genotype diversity using the heteroduplex tracking assay. This data should provide significant insights into the processes that control the development and evolution of viral reservoirs within the CNS. In addition, choroid plexus macrophages infected with FIV were found to encourage the trafficking of monocytes into the brain. A paradigm has been developed to assess the contribution of these cells to immune cell trafficking across the blood-brain barrier. This new in vivo model offers the potential to define the mechanisms that control the early penetration of monocytes into the brain. This infiltration is thought to trigger the inflammatory processes that lead to neuronal dysfunction and death. Thus, the model will be ideally suited for the development and testing of therapeutic strategies designed to control monocyte invasion and inflammation in the brain. Together, these studies will provide a a much needed model of HIV infection that will foster the development of therapies that restrict entry of the virus into the brain and minimize the brain damage associated with HIV infection.
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1 |
2007 — 2008 |
Meeker, Rick B |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Neuroprotective and Anti-Inflammatory Actions of Novel Growth Factor Mimetics @ University of North Carolina Chapel Hill
[unreadable] DESCRIPTION (provided by applicant): Human immunodeficiency virus (HIV) rapidly penetrates into and infects the central nervous system (CMS). Inflammatory activity resulting from the interaction of HIV with macrophages and microglia in the nervous system leads to varying levels of neurological impairment and neuronal loss. While disease severity has been reduced with the advent of highly active antiretroviral therapy, CMS disease persists and is expected to exert an increasingly heavy toll as patients with HIV live longer. Currently there are no therapeutic treatments that effectively control the inflammatory interactions that disable and destroy neurons. Several studies have indicated that neurotrophin receptor activation of protein kinase B (commonly known as Akt) has substantial therapeutic potential for the treatment of HIV-associated CMS disease. However, it has been difficult to exploit this potential due to the poor penetration of peptides into the brain and difficulties in controlling the balance between neuroprotective and pro-apoptotic neurotrophin signaling. The recent identification of small, non-peptide molecules that cross the blood-brain barrier and mimic the actions of neurotrophins at the p75 neurotrophin receptor (p75NTR) or receptor tyrosine kinase B (TrkB) offers an important opportunity to develop the therapeutic potential of the neurotrophin-Akt signaling pathway. These neurotrophin mimetics represent a new class of compounds that appear to have excellent neuroprotective and anti-inflammatory properties, are non-toxic and have good oral availability. The proposed studies will use primary neural cultures to evaluate the therapeutic potential of three of these compounds. Protection against inflammation and damage generated by exposing rat neural cultures to HIV-1 envelope proteins or feline neural cultures to feline immunodeficiency virus (FIV) will be assessed using conventional measures of cell death as well as newly developed techniques to assess alterations in calcium homeostasis. In addition, the anti-inflammatory properties of each compound will be assessed by measuring cytokine, chemokine and growth factor secretion in both mixed and purified microglial cultures. The proposed studies will determine the basic pharmacology and therapeutic potential of the non-peptide neurotrophin mimetics in assays specifically designed to maximize the efficiency of screening and allow rapid translation to in vivo models. [unreadable] [unreadable] [unreadable]
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1 |
2009 — 2013 |
Meeker, Rick B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Neurotrophin Mimetic Therapeutics For Hiv-Associated Neural Dysfunction @ Univ of North Carolina Chapel Hill
DESCRIPTION (provided by applicant): Human immunodeficiency virus (HIV) rapidly penetrates into and infects the central nervous system (CNS). Inflammatory activity resulting from the interaction of HIV with macrophages and microglia in the nervous system leads to varying levels of neurological impairment and neuronal loss. While disease severity has been reduced with the advent of highly active antiretroviral therapy, CNS disease persists and is expected to exert an increasingly heavy toll as patients with HIV live longer. Currently there are no therapeutic treatments that effectively control the inflammatory interactions that disable and destroy neurons. Several studies have indicated that neurotrophin receptor activation and subsequent signaling through protein kinase B (commonly known as Akt) has substantial therapeutic potential for the treatment of HIV-associated CNS disease. However, it has been difficult to exploit this potential due to the poor penetration of peptides into the brain and difficulties in controlling the balance between neuroprotective and pro-apoptotic neurotrophin signaling. The recent identification of small, non-peptide molecules that cross the blood-brain barrier and mimic the actions of neurotrophins offers an important opportunity to develop the therapeutic potential of the neurotrophin-Akt signaling pathway. These compounds are targeted to specific epitopes of the p75 neurotrophin receptor (p75NTR) or receptor tyrosine kinase B (TrkB), thereby allowing a greater degree of control over signaling than seen with the endogenous ligands. Early work has shown that these compounds have potent neuroprotective properties. The proposed studies will use primary neural cultures to evaluate the therapeutic potential of three of these compounds. Protection against early neuronal damage will be tested by exposing rat neural cultures to conditioned medium from macrophages exposed to the HIV-1 envelope protein, gp120. Alterations in calcium homeostasis and microtubule-associated protein immunoreactivity will be used as sensitive indices of neuronal dysfunction. In addition, the direct anti- inflammatory properties of each compound will be assessed by measuring cytokine, chemokine and growth factor secretion in microglial and macrophage cultures. Assays are designed to generate basic pharmacological data needed to progress from culture experiments to pre-clinical whole animal studies. The proposed studies will establish dosing guidelines, mechanistic information and cellular targeting profiles necessary for therapeutic development of the neurotrophin mimetics.
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0.988 |
2011 |
Kay, Heidi A Meeker, Rick B Sacktor, Ned C |
R43Activity Code Description: To support projects, limited in time and amount, to establish the technical merit and feasibility of R&D ideas which may ultimately lead to a commercial product(s) or service(s). |
A Novel Cns-Penetrable Antiretroviral
DESCRIPTION (provided by applicant): Since the introduction of highly active antiretroviral therapy (HAART) in the mid-1990's for treatment of human immunodeficiency virus type 1 (HIV-1) infection, overall incidence rates of associated neurological maladies have declined. Unfortunately, the HIV-associated prevalence of both central nervous system (CNS) and peripheral nervous system (PNS) disorders is on the rise, suggesting a failure of antiretroviral therapies to protect neurological tissues despite reducing viral replication. Novel antiretroviral therapeutic approaches are urgently needed for individuals suffering from NeuroAIDS. Most antiretrovirals cannot penetrate into the CNS where both direct and indirect viral burden lead to neuronal damage. Although neurons do not directly support HIV infection, soluble factors and neurotoxins produced by CNS-resident infected cells - such as microglia and astrocytes - are known to lead to neuronal death. Additionally, viral and bacterial proteins can lead to neuronal death through inflammatory responses of CNS- resident cells, even in the absence of direct viral infection. This proposal aims to develop a novel therapeutic suitable for such CNS complications of HIV infection. FX101 is a novel potential antiretroviral therapeutic especially suited for NeuroAIDS, as evidenced by evaluation of virological, immunological and soluble factors in vitro and in vivo, together with its capacity to penetrate the CNS compartment. Specific aims of this project are: 1) demonstrate preclinical safety outcomes and central nervous system uptake of FX101 in vivo; 2) examine in vitro responses of microglial and macrophage cells to FX101 both directly and in concert with viral protein or lipopolysaccharide activation;and 3) evaluate translational therapeutic efficacy of FX101 using human HIV-infected ex vivo whole blood cultures The proposed work will develop along two years and will be continuously evaluated toward the goal of filing an IND application and commercialization of the therapeutic. PUBLIC HEALTH RELEVANCE: This research proposal examines preclinical toxicological studies and human translational efficacy of a novel blood-brain-barrier penetrable therapeutic for the treatment of HIV/AIDS, with or without drug abuse. It is expected that this work will reduce the burden of neurological diseases impacting the central nervous system as a consequence of lentiviral infections and associated pathologies, leading to better treatments for HIV/AIDS-Associated Neuropsychological Disorders.
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0.906 |
2012 — 2013 |
Hudson, Lola C (co-PI) [⬀] Kay, Heidi A Meeker, Rick B |
R43Activity Code Description: To support projects, limited in time and amount, to establish the technical merit and feasibility of R&D ideas which may ultimately lead to a commercial product(s) or service(s). |
Fiv Testing of a Cns-Relevant Antiretroviral Compound
DESCRIPTION (provided by applicant): Specific Aims Neurological complications of HIV-1/AIDS persist despite HAART therapies. Current knowledge identifies several sources of neuronal aggravation: (1) direct virus infection of astrocytes and microglial cells; (2) infiltratin of virus-infected monocyte/macrophages; (3) cytokine dysregulation indirectly affecting nerve cells; (4) breakdown of the blood-brain-barrier; (5) abnormal antigen-presentation leading to autoimmunity; (6) apoptotic suicide of neuronal cells and (7) toxins associated with HAART therapies. Novel antiretroviral therapeutic approaches are urgently needed for individuals suffering from NeuroAIDS. This goal is to investigate and develop a novel therapeutic for the treatment of NeuroAIDS. Based upon preliminary studies, this small molecule exhibits a unique molecular action resulting in reductions to the proviral reservoir together with direct inhibition f virus productions. Since the compound is blood-brain barrier penetrable, it potentially affords adjunctive therapeutic value specifically appropriate for central nervous system (CNS) lentivirus burdens. The working hypothesis is that FX101 is a novel antiretroviral compound especially suited for NeuroAIDS, uniquely targeting lentiviral nucleocapsid proteins. Using the FIV model, we seek to investigate antiviral efficacies with emphasis to the CNS compartment and to relate these findings to the proposed mechanism of action. The proposed studies are designed to accomplish four goals: 1) Establish safety and pharmacokinetics of FX101 in cats, including penetration into the CNS. 2) Measure effects of FX101 on FIV burden in plasma, CSF, T-cell subsets and brain tissue. 3) Evaluate the uptake and distribution of FX101 in macrophages and its ability to suppress virus production. 4) Examine and compare virion progeny and nucleocapsid proteins produced during exposure to FX101. We propose to examine pharmacokinetic, safety and antiretroviral efficacy of this novel compound towards application of a CNS-relevant antiretroviral compound for HIV.
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0.906 |
2013 — 2016 |
Meeker, Rick B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Neurotrophin Protection in Hiv and Aging @ Univ of North Carolina Chapel Hill
DESCRIPTION (provided by applicant): The introduction of antiretroviral therapies that suppress the systemic HIV burden has greatly extended the life span of infected individuals. However, because these compounds have poor penetration into the central nervous system (CNS), a persistent, active viral reservoir remains that supports the gradual progression and increasing prevalence of CNS disease. As the HIV- infected population ages, chronic inflammation is expected to interact adversely with a variety of age-related disease processes that give rise to cognitive and psychiatric disorders. Our ability to predict and prevent adverse interactions is currently limited by an incomplete understanding of how chronic HIV-associated immune activation causes neurotoxicity and how it might synergize with other diseases. Dendritic beading, pruning and synapse loss are common pathological features of HIV infection, aging and Alzheimer disease. Evidence supports an association between this damage and a loss of neurotrophic support as well as a calcium- dependent disruption of actin regulation and intracellular transport. In independent studies of HIV neuropathogenesis and aging we have demonstrated that the novel p75 neurotrophin ligand, LM11A-31, prevents neural dysfunction and damage, in part, by restoring calcium homeostasis. Recent studies of early Alzheimer pathogenesis suggest that alteration in neurotrophin signaling through the p75 neurotrophin receptor may underlie the development of pathology. Based on these observations, we hypothesize that neurotrophin and calcium signaling converge on mechanisms common to HIV and Alzheimer pathogenesis that regulate the actin cytoskeleton and microtubule-associated proteins. The proposed studies will use in vitro and in vivo models of HIV-associated neuropathogenesis in combination with natural neurotrophins (NGF, proNGF), pathogenic molecules associated with aging (Abeta oligomers) and LM11A-31 to define both deleterious and neuroprotective interactions. Immunostaining and western blot analyses of changes in actin polymerization, tau phosphorylation, microtubule associated protein phosphorylation and functional assessments of mitochondrial transport will be used to determine how each challenge contributes to the development of pathology and/or protection. Normal aging in gp120 transgenic mice will be examined to determine the impact of chronic inflammation model on natural aging using standard measures of cholinergic degeneration as well as more global assessments of microtubule-associated proteins and synapses. The contribution of the p75 neurotrophin receptor to pathogenesis will be evaluated in p75 Exon III knockout mice. The efficacy of treatment with LM11A-31 will then be tested in the aging gp120 mice at a time coincident with the most active development of pathology. These studies will provide insights into the cellular dysfunction associated with age- and HIV- associated loss of neurotrophin support and will determine the therapeutic potential of the novel p75 ligand, LM11A-31.
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0.988 |
2014 — 2015 |
Meeker, Rick B |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Lm11a-31 Neuroprotective Efficacy in An Animal Model of Hiv @ Univ of North Carolina Chapel Hill
DESCRIPTION (provided by applicant): Even after the introduction of combination antiretroviral therapies, HIV infection persists in the central nervous system (CNS). The chronic presence of virus and the associated inflammation supports an increasing prevalence of CNS disease as the HIV-infected population ages. In vitro studies have shown that neuronal dysfunction is triggered by a destabilization of neuronal calcium followed by the appearance of damage in the form of dendritic beading, pruning of processes and synapse loss. We have demonstrated that the novel p75 neurotrophin receptor ligand, LM11A-31, prevents the neural dysfunction and damage, in feline neural cultures infected with feline immunodeficiency virus (FIV) or rat neural cultures exposed to HIV virions, gp120 or toxic macrophage conditioned medium. As seen in many disease models, neuronal p75 increases in response to HIV, thereby providing a therapeutic target that is upregulated by the disease process. The protective effect was seen at nanomolar concentrations and was due, in part, to a restoration of calcium homeostasis, thereby preventing the initial dysfunction that leads to neuronal damage. Separate studies demonstrated that the compound crossed the blood-brain barrier, accumulated in the brain compartment and was free of adverse effects at high concentrations in vitro and in vivo in both mice and cats. Tests for adverse effects included physiological (body weight, CBC, urinalysis, blood chemistry, organ weights and histology) and behavioral measures (thermal sensitivity, gait analysis, veterinary examination). Neuroprotective efficacy has also been reported in animal models of aging, Alzheimer disease, Huntington disease, spinal cord injury and traumatic brain injury. Based in part on the above studies, LM11A-31 has been approved for Phase I studies for development as a treatment for Alzheimer disease. Our in vitro results and preliminary in vivo studies indicate that LM11A-31 will also be an effective intervention to protect the nervous system against HIV-associated damage. The proposed studies will test this assumption using the FIV model of HIV-associated neuropathogenesis while also addressing issues important for the proposed therapeutic use of the compound in the context of HIV infection. This natural infectious model has been optimized for therapeutic testing and recapitulates features of infection and CNS disease important for translation to humans. A major endpoint will be the reversal of FIV-induced cognitive deficits. Additional endpoints will include demonstration of reduced inflammation in the brain, assessment of toxicity/adverse effects with long-term treatment and evaluation of effects on virus titers and disease progression. These studies will establish initial in vivo efficacy and safety of LM11A-31 in preparation for subsequent studies designed to establish LM11A-31 as a treatment for HIV-associated neural dysfunction.
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0.988 |
2017 — 2018 |
Meeker, Rick B |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
A Degradomics Strategy For the Analysis of Inflammation-Associated Neuronal Vulnerability @ Univ of North Carolina Chapel Hill
Inflammation is thought to play a pivotal role in the progression of neurodegenerative diseases by establishing conditions that increase the vulnerability of neurons to toxic insults. The activation of microglia and macrophages and perhaps their long-term sensitization are associated with the increased vulnerability. The inflammatory events that sensitize neurons very likely precede the development of severe pathology and cognitive decline. Although it is widely acknowledged that substances secreted from macrophages and microglia induce neuronal damage the precise mechanisms of pathogenesis are not well understood. In animal models of Alzheimer Disease (AD) the first signs of pathology in neurons include the dysregulation of calcium, development of focal swellings (beading) within axons and dendrites, actin aggregation, loss of transport systems and loss of trophic support. No current treatments are available that effectively reduce the inflammatory response and neural damage. We have shown that these early pathogenic changes can be can duplicated by exposing primary neurons to microglial conditioned medium (MCM) collected after treatment with amyloid beta oligomers (A?o). Accumulation of excess intracellular calcium though NMDA receptors appears to be a triggering event although the medium does not contain toxic levels of glutamate or other excitatory compounds. Proteomic profiling of the medium revealed that MMP-9 was robustly secreted and correlated with the neurotoxic effects. We were able to show that exogenous MMP-9 added to neural cultures enhanced the sensitivity of the NMDA receptor. This sensitization may represent an initial trigger that increases neruonal vulnerability. A growing body of data supporting the essential role of MMP-9 in normal synaptic plasticity emphasizes the need to better understand the actions of MMP-9 under inflammatory conditions. However, many protein targets have been identified that could impact plasticity and neural function. In addition, the presence of endogenous inhibitors and the need for activation of secreted proMMP-9 introduces complexities that have made it difficult to evaluate the exact role of MMP- 9. Recent developments in the ability to assess degradomic profiles using Terminal Amine Isotopic Labeling of Substrates (TAILS) provides the opportunity to precisely define the activity of MMP-9 under different conditions. By quantifying proteins cleaved by MMP-9 in wild type and MMP-9 KO neurons challenged with MCM or the purified enzyme, we can generate a clear picture of the targets specific to each experimental condition as well as the contributions of intracellular versus exogenous MMP-9. The specific role of each cleavage event can then be tested in the culture model and subsequently validated in a mouse model of AD. Results from these studies will provide a new tool to characterize the functional activity of proteolytic enzymes in the context of normal and disease states. We further expect that the results will identify cleavage fragments that might be used as markers of early pathogenesis and for the screening of precisely targeted, protective small interfering molecules.
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0.988 |
2018 — 2021 |
Meeker, Rick B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Neural Network Dysfunction in Early Hiv Neuropathogenesis @ Univ of North Carolina Chapel Hill
HIV infection of the nervous system results in chronic infection, inflammation and cognitive decline in many patients with no effective treatments. Inflammation appears early in the disease process and causes progressive neural damage due, in part, to factors released by activated microglia and macrophages. In cultured neurons these factors induce intracellular calcium accumulation, cytoskeletal damage and focal swelling, much like the early Alzheimer disease (AD)pathology, suggesting a common substrate for disease progression. In gp120 transgenic mice and AD mouse models we recently identified a unique form of Tau that accumulated in the neuritic swellings. The same Tau accumulated naturally in the hippocampus of aging and gp120 Tg mice in parallel with p75NTR expression and Iba-1 immunoreactive microglia suggesting an important link between inflammation, aging and neurodegeneration. In preliminary multielectrode array (MEA) studies, these early indices of neural damage and Tau accumulation correlated with the appearance of burst-like activity patterns, increased spike frequencies and a decreased density of neuron interconnections, all signs of network dysfunction. At the cellular level, hyperresponsiveness contrasted with the restricted network activity highlighting the need to better understand how changes in neuronal function translate to network function. Treatment strategies targeted to these early, reversible manifestations of the disease process have the potential to stabilize cognitive function and perhaps suppress pathogenesis. We propose a series of experiments that will provide complementary in vitro and in vivo analyses of the temporal development of network dysfunction in mouse models of HIV-associated inflammation. In vitro studies of mixed neural cultures from gp120 Tg, Tau overexpressing, p75 neurotrophin receptor deficient and wild type mice will utilize high content recording of neural activity on 4096 electrode culture grids, calcium imaging of primary neurons, morphometry and immunocytochemistry to examine the contribution of HIV pathology to the development of neural and network dysfunction under both normal and disease prone conditions. Parallel studies will examine the relative contribution of microglia as well as HIV Tat protein. Subsequent microwire array recording in vivo to examine hippocampal mesoscale neural network activity, communication and function in gp120 Tg mice crossed to Tau overexpressing and p75 deficient mice will begin to reveal how network behavior is modified as pathology progresses. Mice with confirmed network dysfunction will be evaluated for cognitive function which will be correlated with MRI/PET studies of synaptic loss and microglial activation with the SV2A synaptic vesicle protein probe 11C-UCB-J and the mitochondrial TSPO probe 18F-PBR111, respectively. We believe this integrated approach will identify and characterize HIV-associated network dysfunction and open new avenues for disease modifying therapeutic intervention.
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0.988 |