2013 — 2017 |
Fitting, Sylvia |
K99Activity Code Description: To support the initial phase of a Career/Research Transition award program that provides 1-2 years of mentored support for highly motivated, advanced postdoctoral research scientists. R00Activity Code Description: To support the second phase of a Career/Research Transition award program that provides 1 -3 years of independent research support (R00) contingent on securing an independent research position. Award recipients will be expected to compete successfully for independent R01 support from the NIH during the R00 research transition award period. |
Opiate Drug Abuse & Hiv-Induced Excitotoxicity in Striatal Neurons @ Virginia Commonwealth University
DESCRIPTION (provided by applicant): HIV-1 infected individuals who are injecting opioid drugs show increased cognitive defects and undergo an accelerated rate of progression to AIDS. Accumulating evidence suggests that opioid drug abuse intrinsically exacerbates the pathogenesis of HIV-1. We have found that neuronal death is preceded by a prolonged period of synaptic culling, functional losses, and dendritic pathology that are presumed reversible. Importantly, opioid abuse potentiates the neuropathogenesis of HIV-1 by synergistically increasing dendritic pathology (varicosity formation, beading, fragmentation, pruning), while promoting additive dendritic spine losses (plasticity). This has been verified in medium spiny neurons (MSN) of the striatum and synaptic pruning has been confirmed electron microscopically. Moreover, behavioral defects in locomotor activity are accompanied by synaptic losses and dendritic pathology in the absence of demonstrable neuron death, suggesting that sublethal neuronal injury and reduced synaptic connectivity underlie the ability of opioids to aggravate HIV-1-associated neurological disorders (HAND). While death per se is significant, the interruption of events preceding neuron death may be more strategic therapeutically. This grant will focus on the functional level of MSN by investigating the underlying physiological mechanisms of opioid +/- HIV-induced excitotoxicity. It is hypothesized that Tat induces changes in the cellular homeostasis and excitability of MSN that are exacerbated by opioid drugs through a complex sequence of events involving OR-mediated pathways. In vitro approaches are being proposed by assessing the effects of opioid drug and HIV-1 Tat-induced neurotoxicity in dissociated cortical-striatal cell cultures. Whole-cell patch-clamp recordings wil be conducted in voltage- and current-clamp mode by assessing action potentials as well as sodium, potassium, AMPA, NMDA, and calcium (Ca2+) currents. The role of OR will be elucidated by applying pharmacological (OR antagonists), genetic (OR knockout mice) and silencing (silencing NMDAR) strategies to identify mechanisms underlying opioid + HIV protein interaction. To sort out whether opioids exacerbate the excitotoxic effects of Tat in the striatum via OR on MSN we will conduct experiments in vivo using two types of Cre-lox mice. Conditionally deleting OR at key sites will define the targets and associated mechanisms by which opioids exacerbate neuronal excitability (action potentials, ion channel activity, ion imaging, mitochondrial membrane potential), injury (including dendritic pathology and spine density), and behavioral defects (locomotor activity) in the striatum.
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0.988 |
2016 — 2017 |
Fitting, Sylvia |
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.) |
Investigation of Endocannabinoid-Mediated Neuroprotection in Models of Neuroaids @ Univ of North Carolina Chapel Hill
? DESCRIPTION (provided by applicant): The endocannabinoid system has an important role in many neurodegenerative and neuroinflammatory processes/diseases including Parkinson's disease, Alzheimer's disease, and neuropathic pain. In the era of combined antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) is now considered a chronic disease that specifically targets the brain and causes a high prevalence of mild forms of neurocognitive impairments, also referred to as HIV-associated neurocognitive disorders (HAND) [1, 2]. The best correlate of HAND has been shown to be synaptodendritic damage with injury to synapses and dendrites underlying the neurocognitive impairments seen in HIV-1 positive individuals [3]. It is proposed that increasing endocannabinoid signaling will be protective in models of such neuronal damage. Preliminary studies from our laboratory have shown that anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) used as direct ligands are neuroprotective against HIV-1 Tat-induced dysfunction and injury. However, use of endocannabinoids in vivo is not optimal/feasible because of their rapid degradation by catabolic enzymes: the main enzyme responsible for degradation of AEA, fatty acid amide hydrolase (FAAH), and the main enzyme responsible for degradation of 2-AG, monoacylglycerol lipase (MAGL). Recently, a new class of selective inhibitors of those enzymes have been developed that show neuroprotective, anti-inflammatory, antinociceptive and anxiolytic effects [4-6]. Thus, the focus of this proposal is to determine 1) the neuroprotective effects of endocannabinoids in HIV-1 Tat-induced injury using endocannabinoid catabolic enzyme inhibitors as a tool, and to define 2) the receptor- mediated mechanisms by which endocannabinoids are naturally protective in HIV-1 Tat neurotoxicity. We hypothesize that endocannabinoids will be protective in neuro-acquired immunodeficiency syndrome (neuroAIDS) via a presynaptic CB1 receptor-dependent mechanism. In Specific Aim 1, we will determine the mechanisms by which FAAH inhibitor PF3845 and MAGL inhibitor MJN110 are neuroprotective against HIV-1 Tat toxicity. Cell imaging and electrophysiology studies on cultured prefrontal cortex neurons will assess the effects of the enzyme inhibitors on HIV-1 Tat-induced disturbances of ion homeostasis (i.e. increases in [Ca2+]I and [Na+]i), synaptic neurotransmission, synaptodendritic injury and cell death. In Specific Aim 2, we will determine if endocannabinoids are naturally neuroprotective in HIV-1 Tat-induced toxicity via a presynaptic CB1 receptor-mediated pathway. Whole cell-patch clamp recordings will be conducted ex vivo on prefrontal cortex slices of CB1 and CB2 knockout mice, including paired-pulse stimulation, dual whole patch recordings, and extracellular cell-attached recordings. New knowledge will be gained about how the endocannabinoid system protects neurons from HIV-1 Tat-induced toxicity by focusing on changes in synaptic plasticity of excitation and inhibition. Understanding the effects of enzyme inhibitors in the context of neuroAIDS may uncover novel therapeutic targets for HAND and other diseases in which cognitive deficits occur.
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0.988 |
2018 — 2021 |
Fitting, Sylvia |
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. |
Endocannabinoid-Mediated Neuroprotection in Models of Neuroaids in Vivo @ Univ of North Carolina Chapel Hill
Project Summary/Abstract Several neurodegenerative disorders, including Parkinson?s and Alzheimer?s diseases, display alterations in the function of the endocannabinoid (eCB) system. In the era of combined antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) is now considered a chronic disease with an inflammatory component that specifically targets the brain and causes a high prevalence of HIV-associated neurocognitive disorders (HAND). The eCB system has attracted interest as a target for treatment of neurodegenerative disorders, due to the potential neuroprotective, anti-inflammatory, & neurotrophic properties of CBs. However, therapeutic use of eCBs in vivo is unlikely due to their rapid degradation by catabolic enzymes. The main enzymes responsible for degradation of two major eCBs, anandamide (AEA) and 2-arochinonoylglycerol (2- AG), are respectively: fatty acid amide hydrolase (FAAH), and monoacylglycerol lipase (MAGL). A new class of selective inhibitors of those enzymes has been developed that show neuroprotective and anti-inflammatory effects in preclinical animal models of neurodegenerative diseases. These new pharmacological tools allow for selective elevation of eCB signaling, which enables investigation of physiological actions of particular eCBs as well as reveal therapeutic potential of such precise modulation. The aim of the present study is to unravel the role of the eCB signaling in neuroprotection against HIV-1 protein toxicity that results in behavioral changes as a consequence of HAND. To achieve this goal, we plan to use Tat and gp120 transgenic (tg) mice, as well- established models of neuroAIDS, that will allow us to determine: 1) the neuroprotective effects of eCBs on prefrontal cortex (PFC)-dependent behavior in vivo, with use of catabolic enzyme inhibition; 2) the mechanisms underlying protective effects of eCBs in PFC ex vivo; and 3) the effects of chronic eCB catabolic enzyme inhibitors on neuroinflammation using positron emission tomography (PET) imaging in vivo. We hypothesize that in the HIV-1 protein tg mouse models, eCB catabolic enzyme inhibitors will show protective effects on behavior, function, and structure via a CB1R/CB2R-mediated mechanism. In Specific Aim 1, Tat tg mice will be trained on the operant conditioning Go/No-Go task and eCB enzyme inhibitors will be tested for protective effects against Tat-induced interference in cognitive function, including a decrease of behavioral inhibition and increased impulsivity. Moreover, the same animals will undergo electrophysiology studies on PFC pyramidal neurons ex vivo to establish the effects of eCB enzyme inhibitors on synaptic currents. In Specific Aim 2, we will conduct behavioral, functional, and morphological imaging studies to determine if eCBs are neuroprotective in Tat toxicity via a CB1R/CB2R-mediated mechanism ex vivo. In Specific Aim 3, non-invasive longitudinal PET imaging studies in Tat and gp120 tg mice will investigate the effects of eCB enzyme inhibitors on active inflammatory processes using the tracer [18F]-PBR111. Understanding the role of the eCB system in neuro- AIDS may uncover novel therapeutic targets for HAND and other diseases in which cognitive deficits occur.
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0.988 |
2018 — 2019 |
Fitting, Sylvia Kourkoutis, Lena F |
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.) |
Mapping Hiv-Tat +/- Endocannabinoid Induced Synaptic Changes At the Macromolecular Level Via Cryo-Electron Tomography @ Univ of North Carolina Chapel Hill
Project Summary/Abstract The human brain has about 100 billion neurons and each neuron is connected to up to 10,000 other neurons, passing information to each other through as many as 1,000 trillion synaptic connections, equivalent to a computer with a 1 trillion bit per second processor. Normal brain function critically depends on the proper function of these neural networks, with synapses being crucial components of neurons to communicate and transmit information through neuronal networks. In the era of combined antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) is considered a chronic disease that specifically targets the brain and is associated with high prevalence of mild forms of neurocognitive impairments, also referred to as HIV- associated neurocognitive disorders (HAND). Strong indicators of HAND are dendritic injury and disruption of the synaptic machinery. While the HIV-1 protein transactivator of transcription (Tat) is known to induce synaptic dysfunction and impairments in neurocognition, the endocannabinoid (eCBs) system has been shown to play a protective role in neurodegenerative diseases, such as multiple sclerosis, Parkinson?s disease, and Alzheimer?s disease. Thus, the focus of this proposal is to determine 1) HIV-1 Tat-induced synaptic injury at the macromolecular level via cryo-electron microscopy (cryo-EM), and to define 2) the neuroprotective functional and structural effects of eCBs in Tat-induced synaptic injury using eCB catabolic enzyme inhibitors as a tool. We hypothesize that eCBs will affect Tat-induced functional and structural changes at the synapse level. In Specific Aim 1, we will visualize the macromolecular architecture of control versus Tat-treated primary hippocampal neurons preserved in their close-to-physiological state using cryo-EM. Our recent advances in accessing the internal structure of frozen-hydrated biological material using cryo-focused ion beam milling (cryo-FIB) will enable us to assess structural changes of neuronal networks at and below the synaptic level via 3D cryo-electron tomography (CET). The microstructure of control and Tat-treated hippocampal neurons will be compared with focus on synaptic vesicles on the presynaptic side and the postsynaptic density (PSD) on the postsynaptic side. In Specific Aim 2, we will determine if eCB enzyme inhibitors are naturally neuroprotective in the presence of Tat by focusing on functional and structural changes on the synapse level. Functional information, in particular on communication between neurons by tracking firing patterns of neurons on a millisecond time scale, will be obtained by electrophysiology. At the same time microscopy techniques will be used to reveal changes in the molecular structure of synapses. Fluorescence light microscopy provides functional readout, and the opportunity to visualize and monitor subcellular functional changes in ion homeostasis at the synapse level in real time. Combining CET, electrophysiology and fluorescence techniques will allow us to examine how microscopic changes in the structure of synapses affect synaptic function in the context of neuroAIDS and to assess the role of eCB enzyme inhibitors.
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0.988 |