Browsing by Subject "astrocytes"
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Item Astrocyte-mediated HIV-1 Tat neurotoxicity and its molecular mechanisms: astrocyte activation and impaired neurogenesis(2016-08-01) Fan, Yan; He, Johnny J.; Ghorpade, Anuja; Barber, Robert C.Human immunodeficiency virus type 1 (HIV-1) invasion of the central nervous system (CNS) often causes motor and cognitive dysfunction, which is termed HIV-associated neurocognitive disorders (HAND). Although the introduction of combination antiretroviral therapy (cART) has effectively suppressed viral replication, improved immune function and increased life expectancy among HIV-infected individuals, it has failed to provide complete protection from HAND or to reverse the disease. HIV-1 Tat protein is a major pathogenic factor in HAND. Studies including ours have demonstrated that Tat is taken up by HIV-1 uninfected brain cells and alters the function of these cells, especially astrocytes, neurons and neural progenitor cells. However, the underlying mechanisms are still unclear. There are two parts to my dissertation research. In the first part, we determined the roles of signal transducer and activator of transcription 3 (STAT3) in Tat-induced glial fibrillary acidic protein (GFAP) transactivation. Astrocytes, the most abundant cells in the brain, not only provide the scaffold support in the brain, but also are essential for maintaining the homeostasis of the brain. GFAP is the specific molecular marker for astrocytes, but it also modulates astrocyte function both physiologically and pathologically. In this study, we first showed that STAT3 expression and phosphorylation led to significant increases in GFAP transcription and protein expression. Then we determined that Tat expression was associated with increased STAT3 expression and phosphorylation in Tat-expressing astrocytes and HIV-infected astrocytes. In addition, we showed that GFAP, Egr-1 and p300 transcription all showed positive response to STAT3 and its phosphorylation. Moreover, knockdown of STAT3 resulted in significant decreases in Tat-induced GFAP and Egr-1 transcription and protein expression. Taken together, these findings show that STAT3 is involved in and acts upstream of Egr-1 and p300 in the Tat-induced GFAP transactivation cascade and suggest important roles of STAT3 in controlling astrocyte proliferation and activation in the HIV-infected CNS. In the second part of the dissertation research, we took advantage of the doxycyclineinducible and astrocyte-specific HIV-1 Tat transgenic mice (iTat) and determined the relationship between Tat expression and neurogenesis. Tat expression in astrocytes was associated with detection of fewer neuron progenitor cells (NPC), fewer immature neurons and fewer mature neurons in the dentate gyrus of the hippocampus of the mouse brain. In vitro NPC-derived neurosphere assays showed that Tat-containing conditioned media from astrocytes and recombinant Tat protein inhibited NPC proliferation and migration and altered NPC differentiation, while immunodepletion of Tat from Tat-containing conditioned media or heat inactivation of recombinant Tat abrogated those effects. Notch signaling downstream gene Hes1 promoter-driven luciferase reporter gene assay and Western blotting showed that recombinant Tat and Tat-containing conditioned media activated Hes1 transcription and protein expression, which were abrogated by Tat heat inactivation, immunodepletion, and cysteine mutation at position 30. Moreover, Notch signaling inhibitor DAPT significantly rescued Tat-impaired NPC differentiation in vitro and neurogenesis in vivo. Taken together, these results show that Tat adversely affects NPC and neurogenesis through Notch signaling and point to the potential of developing Notch signaling inhibitors as HAND therapeutics.Item Astrocytes & Ischemic stroke(2014-08-01) Roy Choudhury, Gourav; Yang, Shaohua; Singh, Meharvan; Schreihofer, DerekAlthough less appreciated, recent findings introduced critical contributions of astrocytes to numerous CNS functions, like neurogenesis, synaptogenesis, ion homeostasis, neurotransmission, and blood brain barrier formation. Their active participation in the progression of specific CNS pathologies has garnered major attention and culminated in thorough investigation of astrocyte function in brain. Reactive astrogliosis, characterized by increases in glial fibrillary acidic protein (GFAP) and cellular hypertrophy, describes the extensive structural and functional changes that astrocytes undergo in response to tissue injury. Despite of extensive investigation, the molecular mechanism of reactive astrogliosis in ischemic stroke still remains elusive. p38 MAPK is a well studied signal transducing pathway known to be involved in modulating cell type specific responses to ischemic injury. The first study presented in the dissertation delineates the involvement p38 MAPK signaling pathway in reactive astrogliosis after ischemic stroke. Results showed that astrocyte specific deletion of p38 MAPK attenuated oxygen-glucose deprivation (OGD)-induced increase in GFAP expression in primary astrocytes in vitro. Additionally, inhibition of p38 MAPK (SB239063/genetic deletion) slowed astrocyte migration without affecting astrocyte proliferation. In vivo deletion of p38 MAPK from astrocytes attenuated reactive astrogliosis after permanent middle cerebral artery occlusion in mice. These findings strongly indicated that p38 MAPK plays a critical role in reactive astrogliosis after ischemic stroke. During ischemic stroke, astrocyte dysfunction causes extensive cell death through excitotoxicity, disruption of ion and water homeostasis. Restoration of astrocyte function thus may be beneficial to ischemic tissue in the long term. Methylene blue (MB), a metabolic enhancer, has been well studied and known to improve cellular respiration, glucose metabolism and attenuate superoxide production by efficient electron transport in mitochondria. In the second part of this dissertation we determined the effect of MB in astrocytes under oxygen glucose deprivation (OGD) and reoxygenation stress and the underlying protective mechanisms. Our studies demonstrated that MB improved astrocyte bioenergetics and promoted astrocyte survival following OGD and reoxygenation. In conclusion both the studies presented, provide a unique perspective of the importance of astroglial response in ischemic injury and how its modulation can benefit the healing and recovery of the brain following ischemic injury.Item Cal'MAM'ity at the Endoplasmic Reticulum-Mitochondrial Interface: A Potential Therapeutic Target for Neurodegeneration and Human Immunodeficiency Virus-Associated Neurocognitive Disorders(Frontiers Media S.A., 2021-10-21) Proulx, Jessica; Park, InWoo; Borgmann, KathleenThe endoplasmic reticulum (ER) is a multifunctional organelle and serves as the primary site for intracellular calcium storage, lipid biogenesis, protein synthesis, and quality control. Mitochondria are responsible for producing the majority of cellular energy required for cell survival and function and are integral for many metabolic and signaling processes. Mitochondria-associated ER membranes (MAMs) are direct contact sites between the ER and mitochondria that serve as platforms to coordinate fundamental cellular processes such as mitochondrial dynamics and bioenergetics, calcium and lipid homeostasis, autophagy, apoptosis, inflammation, and intracellular stress responses. Given the importance of MAM-mediated mechanisms in regulating cellular fate and function, MAMs are now known as key molecular and cellular hubs underlying disease pathology. Notably, neurons are uniquely susceptible to mitochondrial dysfunction and intracellular stress, which highlights the importance of MAMs as potential targets to manipulate MAM-associated mechanisms. However, whether altered MAM communication and connectivity are causative agents or compensatory mechanisms in disease development and progression remains elusive. Regardless, exploration is warranted to determine if MAMs are therapeutically targetable to combat neurodegeneration. Here, we review key MAM interactions and proteins both in vitro and in vivo models of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. We further discuss implications of MAMs in HIV-associated neurocognitive disorders (HAND), as MAMs have not yet been explored in this neuropathology. These perspectives specifically focus on mitochondrial dysfunction, calcium dysregulation and ER stress as notable MAM-mediated mechanisms underlying HAND pathology. Finally, we discuss potential targets to manipulate MAM function as a therapeutic intervention against neurodegeneration. Future investigations are warranted to better understand the interplay and therapeutic application of MAMs in glial dysfunction and neurotoxicity.Item Evaluation of NK Cell – Astrocyte Interactions: Potential Role in HIV-Associated Neurocognitive Disorders and HIV- Associated Dementia(2015-05-01) Bowen, Kelly E.; Mathew, Porunelloor A.; Mathew, Stephen O.; Hodge, Lisa M.NK cells play important roles in immunity against pathogens and cancer. NK cell functions are regulated by inhibitory and activating receptors binding corresponding ligands on the surface of target cells. During pathological conditions, NK cells were shown to be recruited to the CNS and could impact CNS physiology by killing glial cells and by secreting IFN-g. Astrocytes are intimately involved in immunological and inflammatory events occurring in the CNS and reactive astrogliosis is a key feature in HIV-associated neurocognitive disorders (HAND). There is little data on NK cell-astrocyte interactions and ligands expressed on astrocytes that could impact NK cell function. This study aimed to identify NK-associated ligands expressed by human astrocytes that confer this NK-directed cytotoxicity of astrocytes and assay the cytotoxicity differences in presence and absence of HIV 3S peptide. Using a fusion protein consisting of the extracellular domain of NKp44 fused to Fc portion of human IgG, we determined the expression of a novel ligand for NKp44 (NKp44L) on astrocytes. Incubation of astrocytes with 3S peptide downregulated NKp44L expression on astrocytes implicating protection from NK mediated killing. Thus, our study demonstrated that NKp44 has a protective effect on astrocytes from NK cell mediated killing during HIV infection. Astrocytes could also secrete cytokines that affect the expression of NK receptors on NK cells. We evaluated the expression of receptors on NK cells after co-culture with astrocytes. CD38 expression was increased on primary NK cells after incubation with astrocytes. CD38 is expressed on both NK cells and astrocytes and has an important implication in HIV-1 infection. Blocking CD38 signaling in our studies decreased astrocyte lysis, suggesting CD38 signaling has important implications in NK-astrocyte interactions. Future studies providing novel insights into the role of NK cells in the pathogenesis of HAND and other brain disorders might result in the development of NK cell based therapies for brain pathologies.Item Impact Of Culture Conditions on Primary Astrocyte Phenotype(2019-05) Prah, Jude K.; Yang, Shaohua; Singh, Meharvan; Forster, Michael J.; Yan, Liang-Jun; Fudala, RafalAlthough previously thought to be passive support cells in the central nervous system (CNS), recent findings introduced critical contributions of astrocytes to numerous CNS functions like energy metabolism, ion and water homeostasis, blood brain barrier formation and neurotransmission. Their dysfunction has been implicated in the initiation and progression of specific CNS pathologies with astrocyte now given serious attention as cellular target for neuroprotection and treatment of neurological disorders. In spite of the aforementioned advances, our understanding of the mechanisms and pathways regulating astrocytic function, dysfunction and astrogliosis is still rudimentary. This is as a result of the complex interwoven nature of different cells in the CNS. Because of the complexities of the brain structure and function in vivo, methods of in vitro primary culture that overcome the influence of complex brain environment provide critical tools for understanding brain cell function at the cellular and molecular levels. The current primary astrocytes cultures are mostly maintained in serum-containing hyperglycemic medium which is non-physiological and produces astrocyte with a reactive, morphological and functional phenotype different from in vivo quiescent astrocytes. The first study presented in the dissertation delineates a serum free astrocyte culture condition that maintains primary astrocytes in a quiescent state. Results showed that primary astrocytes isolated from the cerebral cortex of postnatal day 1 C57BL6 mice and cultured in an astrocyte base medium supplemented with fibroblast growth factor (FGF2) and epidermal growth factor (EGF) (ABM- FGF2-EGF) have higher process bearing morphologies similar to in vivo astrocytes and different from the flat polygonal fibroblast like morphologies exhibited by astrocytes cultured under the traditional FBS condition developed by McCarthy and de Vellis (1980) (MD-10% FBS). Additionally astrocytes cultured in ABM-FGF2-EGF had enhanced glycolytic metabolism, higher glycogen content, lower GFAP and vimentin, increased glutamine synthase and glutamate transporter mRNA levels compared to astrocytes in the MD-10% FBS condition. These findings strongly indicates that astrocytes cultured in ABM-FGF2-EGF medium compared to the usual FBS medium promote quiescent and biosynthetic phenotype similar to in vivo astrocytes. This media provides a novel method for studying astrocytes function in vitro under physiological and pathological condition. Hyperglycemia could increase neuronal glucose level which leads to neuronal damage in a phenomenal referred to as glucose neurotoxicity. On the other hand the impact of hyperglycemia on astrocytes has been less explored although astrocytes are critical for glucose uptake and metabolism and many primary astrocyte cultures are maintained in high glucose conditions. In the second part of this dissertation we investigated the impact of hyperglycemia on astrocyte phenotype and function. Our studies demonstrated that hyperglycemic levels (25 mM) induce cell cycle arrest, ROS production, cytokine expression and inhibited astrocyte proliferation. High glucose enhanced glycolysis and increased metabolic potentials of astrocyte. In addition high glucose activated AMP-activated kinase (AMPK) signaling pathways and induces reactive astrocyte phenotype. In conclusion both studies presented a unique perspective of how culture conditions influence astrocyte phenotype and experimental outcome. Our study also provided a mechanism which may underline the role of astrocytes in hyperglycemia induced neurological complications.Item Intercellular Nef transfer and HIV-1 infection of astrocytes(2015-05-01) Luo, Xiaoyu; He, Johnny J.; Ghorpade, Anuja; Wordinger, Robert J.Acquired immune deficiency syndrome (AIDS) is a pandemic caused by human immunodeficiency virus type 1 (HIV-1). It is a major health issue in many parts of the world ever since its discovery in 1981. The most devastating effect of HIV-1 infection is the graduate loss of CD4+ T cells, which eventually leads to the dysfunction of the immune system, susceptibility to opportunistic infections and cancer. HIV-1 Nef protein is long known as an essential pathogenic factor for HIV-1/AIDS pathogenesis. A few recent studies including ours have demonstrated that Nef can be transferred to neighboring cells and alters the function of these cells. However, the underlying mechanism of intercellular Nef transfer is in dispute. In the first part of our study, we characterized two potential underlying mechanisms for intercellular Nef transfer: direct cell-cell contact and exosomes using several complementary strategies and a panel of exosomal markers. First, we showed that Nef was transferred from Nef-expressing or HIV-infected CD4+ T lymphocytes to CD4+ T lymphocytes and astrocytes, and that the transfer was mainly associated with tunneling nanotube formation. Then we determined that Nef enhanced virological synapse formation and induced cytoskeleton re-arrangement and cell surface protrusions, suggesting that Nef promotes the establishment of intercellular connection and communication between infected cells and uninfected cells. Thirdly, we examined the possibility of Nef transfer through exosomes. In the exosome uptake assay, Nef transfer was undetectable while exosome marker CD81 transferred rapidly. In contrast, Nef was detected in crude exosomes collected from Nef-transfected 293T. In addition, two different populations of exosomes were successfully separated by OptiPrep gradient fractionation and determined as AChE+/CD81low/TSG101low exosomes and AChE- /CD81high/TSG101high exosomes. We determined that Nef was selectively secreted into the AChE+/CD81low/TSG101low population. Lastly, microscopic imaging showed no significant Nef detection in exosomal vesicle-like structures in and out the cell. Taken together, this study shows that Nef transfer requires direct cell-cell contact such as tunneling nanotubes, not cell-free exosomes. In addition, this study reveals existence of two types of exosomes: AChE+/CD81low/TSG101low exosomes and AChE/CD81high/TSG101high exosomes. In the second part, we characterized HIV-1 infection of astrocytes. Astrocytes are the most abundant cells in the central nervous system (CNS) and play important roles in HIV-1/neuroAIDS. Detection of HIV-1 proviral DNA, RNA and early gene products but not late structural gene products in astrocytes in vivo and in vitro indicates that astrocytes are susceptible to HIV-1 infection albeit in a restricted manner. We, as well as others have shown that cell-free HIV-1 is capable of entering CD4- astrocytes through human mannose receptor-mediated endocytosis. In this study, we took advantage of several newly developed fluorescence protein-based HIV-1 reporter viruses and further characterized HIV-1 interaction with astrocytes. First, we found that HIV-1 was successfully transferred to astrocytes from HIV-infected CD4+ T cells in a cell-cell contact- and gp120-dependent manner. In addition, we demonstrated that compared to endocytosis-mediated cell-free HIV-1 entry and subsequent degradation of endocytosed virions, cell-cell contact between astrocytes and HIV-infected CD4+ T cells led to robust HIV-1 infection of astrocytes but retained the restricted nature of viral gene expression. Furthermore, we showed that HIV-1 latency was established in astrocytes. Lastly, we demonstrated that infectious progeny HIV-1 was readily recovered from latently infected astrocytes in a cell-cell contact-mediated manner. Taken together, our studies point to the importance of the cell-cell contact-mediated HIV-1 interaction with astrocytes and provide direct evidence to support the notion that astrocytes are HIV-1 latent reservoirs in the CNS.Item Metabolic Heterogeneity of Cerebral Cortical and Cerebellar Astrocytes(MDPI, 2023-01-22) Sun, Yuanhong; Winters, Ali; Wang, Linshu; Chaudhari, Kiran; Berry, Raymond; Tang, Christina; Liu, Ran; Yang, ShaohuaAstrocytes play critical roles in regulating neuronal synaptogenesis, maintaining blood-brain barrier integrity, and recycling neurotransmitters. Increasing numbers of studies have suggested astrocyte heterogeneity in morphology, gene profile, and function. However, metabolic phenotype of astrocytes in different brain regions have not been explored. In this paper, we investigated the metabolic signature of cortical and cerebellar astrocytes using primary astrocyte cultures. We observed that cortical astrocytes were larger than cerebellar astrocytes, whereas cerebellar astrocytes had more and longer processes than cortical astrocytes. Using a Seahorse extracellular flux analyzer, we demonstrated that cortical astrocytes had higher mitochondrial respiration and glycolysis than cerebellar astrocytes. Cerebellar astrocytes have lower spare capacity of mitochondrial respiration and glycolysis as compared with cortical astrocytes. Consistently, cortical astrocytes have higher mitochondrial oxidation and glycolysis-derived ATP content than cerebellar astrocytes. In addition, cerebellar astrocytes have a fuel preference for glutamine and fatty acid, whereas cortical astrocytes were more dependent on glucose to meet energy demands. Our study indicated that cortical and cerebellar astrocytes display distinct metabolic phenotypes. Future studies on astrocyte metabolic heterogeneity and brain function in aging and neurodegeneration may lead to better understanding of the role of astrocyte in brain aging and neurodegenerative disorders.Item Modulation of Astrocyte Phenotype in Response to T-cell Interaction(2021-05) Hersh, Jessica M.; Yang, Shaohua; Smith, Michael L.; Jin, Kunlin; Hodge, Lisa M.We determined that T-cell astrocyte interaction modulates interleukin-10 (IL-10) production from both cell types. The impact of IL-10 on astrocytes was compared to IL-10 generated from T-cell-astrocyte interactions in vitro. We demonstrated that T-cells directly interact with astrocytes to upregulate gene expression and secretion of IL-10, confirmed by elevated STAT3p/STAT3 expression in astrocytes. IL-10 increased astrocytes proliferation. In addition, IL-10 treatment and CD4+ co-culture shifts primary astrocytes toward a more energetic phenotype. These findings indicate that direct interaction of CD4+ T-cells with astrocytes, activated the IL-10 anti-inflammatory pathway, altering astrocyte phenotype, metabolism, and proliferation.Item Ocular Hypertension Results in Hypoxia within Glia and Neurons throughout the Visual Projection(MDPI, 2022-04-29) Jassim, Assraa Hassan; Nsiah, Nana Yaa; Inman, Denise M.The magnitude and duration of hypoxia after ocular hypertension (OHT) has been a matter of debate due to the lack of tools to accurately report hypoxia. In this study, we established a topography of hypoxia in the visual pathway by inducing OHT in mice that express a fusion protein comprised of the oxygen-dependent degradation (ODD) domain of HIF-1alpha and a tamoxifen-inducible Cre recombinase (CreERT2) driven by a ubiquitous CAG promoter. After tamoxifen administration, tdTomato expression would be driven in cells that contain stabilized HIF-1alpha. Intraocular pressure (IOP) and visual evoked potential (VEP) were measured after OHT at 3, 14, and 28 days (d) to evaluate hypoxia induction. Immunolabeling of hypoxic cell types in the retina and optic nerve (ON) was performed, as well as retinal ganglion cell (RGC) and axon number quantification at each time point (6 h, 3 d, 14 d, 28 d). IOP elevation and VEP decrease were detected 3 d after OHT, which preceded RGC soma and axon loss at 14 and 28 d after OHT. Hypoxia was detected primarily in Muller glia in the retina, and microglia and astrocytes in the ON and optic nerve head (ONH). Hypoxia-induced factor (HIF-alpha) regulates the expression of glucose transporters 1 and 3 (GLUT1, 3) to support neuronal metabolic demand. Significant increases in GLUT1 and 3 proteins were observed in the retina and ON after OHT. Interestingly, neurons and endothelial cells within the superior colliculus in the brain also experienced hypoxia after OHT as determined by tdTomato expression. The highest intensity labeling for hypoxia was detected in the ONH. Initiation of OHT resulted in significant hypoxia that did not immediately resolve, with low-level hypoxia apparent out to 14 and 28 d, suggesting that continued hypoxia contributes to glaucoma progression. Restricted hypoxia in retinal neurons after OHT suggests a hypoxia management role for glia.Item The Role of a Membrane Androgen Receptron in the Brain(2007-02-01) Gatson, John Wayne; Simpkins, James; Koulen, Peter; Basu, AlakanandaGatson, Joshua Wayne, The Role of a Membrane Androgen Receptor in the Brain. Doctor of Philosophy (Biomedical Sciences), February 2007, pp187, 34 illustrations. In the brain, depending on the insult type, androgens have been shown to protect from or exacerbate the levels of cell death. This discrepancy is partly due to the array of receptors that androgens may activate during injury. For example, activation of intracellular androgen receptors (AR) leads to the activation of pro-survival pathways and protects from various toxins such beta-amyloid. In contrast, previous studies have demonstrated that testosterone causes an increase in lesion size following stroke. The damaging effects of androgens in the brain may be mediated by a membrane-associated AR (mAR), since activation of mAR in peripheral tissue results in a decrease in cell growth and an increase in apoptotic cell death during serum deprivation. Here, I hypothesize that activation of a mAR in cortical astrocytes, suppresses the ERK and Akt signaling pathways and increases cell death in the presence of a metabolic and oxidative stressor. In this study, we found that glia express both isoforms of the AR (AR-B and AR-A) and that dihydrotestosterone (DHT) elicits ERK and Akt phosphorylation in rat glioma (C6) cells. The effect of DHT on the activation of these signaling pathways is AR dependent, since flutamide blocked this effect. In contrast to the intracellular receptor, we concluded that DHT-BSA (membrane impermeant form of DHT) binds to DHT displaceable sites on the plasma membrane. Also, treatment with DHT-BSA in the C6 cells resulted in a significant decrease in phosphor-ERK and Akt levels, suggesting the existence of two different pathways through which DHT can influence the activity of these signaling pathways. With respect to cell survival, the C6 cells and primary cortical astrocytes were treated with the metabolic and oxidative insult, iodoacetic acid (IAA), in the presence or absence of DHT, DHT-BSA, or estradiol. Following treatment, DHT and estradiol protected the glia from IAA-induced toxicity, whereas DHT-BSA caused a significant increase in cell death in the presence of a sublethal concentration of IAA. These results indicate that activation of the intracellular pathway is protective and activation of a membrane pathway is damage-inducing during injury, further supporting our results from the ERK and Akt signaling studies. To further characterize this mAR in the brain, we decided to look for indices of apoptosis such as caspase activation and TUNEL staining. It was found that DHT-BSA treatment in the presence of IAA, resulted in an increase in caspase-3/7 activation and increased TUNEL staining. In addition, PKC-delta mediated DHT-BSA-induced cell death, since antagonism of PKC-delta with rottlerin afforded protection. In conclusion, we have partially characterized a novel mAR in astrocytes during injury. Here, the damaging effects of androgens, at least in astrocytes, may in fact be mediated by a mAR, which may be a therapeutic target stroke or reperfusion injury.Item The role of exosomes and microRNA in Astrocyte-mediated HIV-1 Tat neurotoxicity(2016-08-01) Rahimian, Pejman; Johnny J. HeHuman immunodeficiency virus invades the central nervous system (CNS) soon after the initial infection, often leading to neurological complications including cognitive and motor dysfunction, which have been collectively termed HIV/neuroAIDS. The introduction of combination antiretroviral therapy in the mid-1990’s led to reduced viral replication, improved immune function and increased life expectancy among HIV-infected individuals. As a result, the incidence of the most severe form of cognitive impairment due to HIV, so called HIV-associated dementia, reduced dramatically. However, the treatment regimen was not successful in protecting the patients from neuroAIDS as more discrete forms of CNS dysfunction, so-called minor cognitive motor disorders, have become more common. HIV-1 Tat protein is an indispensable factor for successful transcription and replication of the viral genome. Aside from nucleus-bound functions, Tat is diffusely and unconventionally secreted outside of infected cells and contributes immensely to the pathology of neuroAIDS as a potent neurotoxin. The presence of Tat in the CNS despite the implementation of combination anti-retroviral therapy and the strong correlation of pathological hallmarks of neuroAIDS with continued Tat expression in CNS cells warrant a thorough understanding of the partially explained unconventional secretion mechanism(s) by Tat. Exosomal secretion of cargo has been established as an extremely efficient pathway of glia-neuron communications and astrocytes have been shown to utilize this delivery mechanism for the provision of neurotrophic factors and danger-associated molecular patterns to neurons. My dissertation research consisted of two parts. In the first part, we investigated the possibility of exosomal association and distribution of Tat protein from astrocytes and its delivery to neurons. We demonstrated significant presence of HIV-1 Tat in exosomes derived from Tat-expressing primary astrocytes, astrocytoma cell lines, and HIV-infected T cells. We further showed that exosome-associated Tat from Tat-expressing astrocytes was capable of causing neurite shortening and neuron death, further supporting that this new form of extracellular Tat is biologically active. Lastly, we constructed a Tat mutant deleted of its basic domain and determined the role of the basic domain in Tat trafficking into exosomes. Basic domain-deleted Tat exhibited no apparent effects on Tat trafficking into exosomes, while maintained its dominant negative function in Tat-mediated LTR transactivation. Taken together, these results show a significant fraction of Tat is secreted and present in the form of exosomes and may contribute to the stability of extracellular Tat and broaden the spectrum of its target cells. In the second part, we investigated the mechanism of neurite shortening by Tat. Dendritic pruning and synaptic loss of neurons are the most prominent pathological hallmarks of neuroAIDS in the cART era. Although Tat has been implicated in the synaptodendritic damage to neurons, the exact mechanisms of this injury by Tat have not yet been elucidated. Several important controllers of dendritic plasticity have been shown to be post-transcriptionally regulated by a brain-enriched microRNA, miR-132, which is abundantly expressed in the brains of the HIV-infected individuals with cognitive impairment. We found significant induction of miR-132 in both astrocytic and neuronal cell lines following Tat transfection. Tat expression in primary astrocytes from our doxycycline-inducible Tat transgenic mice (iTat) and HIV-infected primary human astrocytes also led to significant upregulation of this microRNA. We confirmed the repression of miR-132 target genes involved in the regulation of dendritic length following Tat expression. Using a basic-domain-deletion mutant of Tat we further demonstrated that Tat-induced miR-132 expression involved CREB phosphorylation. Lastly, we showed that following Tat expression in astrocytes, exosome-associated miR-132 was significantly increased and caused neurite shortening in primary mouse cortical neurons. Taken together, these results demonstrate for the first time the role of miR-132 in Tat-induced damage of the dendritic arbor.Item Trace amine associated receptor 1 (TAAR1), a novel astrocyte receptor for METH-mediated neurotoxicity in HIV-1-associated neurocognitive disorders (HAND)(2015-05-01) Cisneros, Irma E.; Ghorpade, Anuja; Wordinger, Robert J.; Forster, Michael J.This dissertation explores the role of astrocyte trace amine associated receptor 1 (TAAR1), a novel G-protein coupled receptor (GPCR), in modulating the effects of methamphetamine (METH) on astrocyte-mediated excitotoxicity, thereby exacerbating HIV-associated neurocognitive disorders (HAND). The rising pandemic of methamphetamine (METH) abuse has multiple effects and interactions with HIV-1 in infected individuals, affecting both the periphery and the central nervous system (CNS). Moreover, there is a high prevalence of HIV-1 infection among METH users. Underlying evidence provides insight into the cellular mechanisms associated with METH and HIV-1 neurodegeneration, including the effects and byproducts of glial cells, specifically astrocytes. While indirect effects of METH and HIV-1 have been proposed in astrocytes the direct mechanisms by which they contribute to neurodegeneration and continue to evolve. Particularly, imbalance in glutamate homeostasis plays a vital role in METH- & HIV-1-mediated neurodegeneration. We propose METH activates a novel GPCR, trace amine associated receptor 1 (TAAR1), thereby regulating astrocyte-mediated glutamate uptake via excitatory amino acid transporter-2 (EAAT-2), exacerbating HIV-1-induced excitotoxicity. Importantly, our data demonstrate astrocyte functions leading to neurotoxic outcomes like excitotoxicity can be directly exacerbated through TAAR1 regulation. Additionally, extrinsic regulation of TAAR1 signaling, including cAMP, calcium, PKA and PKC, not only reduce activation of subsequent signaling factors, but also reduce or eliminate METH- and IL-1β-mediated alterations in astrocytes glutamate clearance abilities. Finally, preliminary studies indicate that astrocyte-TAAR1 may be a novel therapeutic target for the common morbidity of METH abuse in HANDItem TRANSIENT METHAMPHETAMINE-ASSOCIATED HYPERTHERMIA MODULATES ASTROCYTE TRACE AMINE ASSOCIATED RECEPTOR-1 ACTIVATION AND EXACERBATES HIV-1-INDUCED NEURODEGENERATION(2013-04-12) Cisneros, IrmaPurpose: Methamphetamine (METH) is a highly abused and addictive psychostimulant. METH heightens sexual arousal and decreases inhibition increasing the probability for acquiring human immunodeficiency virus-1 (HIV-1). HIV-1 results in cognitive effects, such as HIV-associated dementia (HAD) characterized by similar neurotoxic mechanisms as METH. Astrogliosis and hyperthermia are key pathological features of METH exposure and HAD. In context of our studies, METH abuse increases brain temperature by approximately 2° C, mimicking a fever common during early HIV-1 infection. A moderate increase in brain temperature exacerbates neuroinflammatory processes synergistically effecting METH/HIV-1-associated neurodegeneration. Methods: Astrocytes sensitivity to METH led to the investigation of astrocyte TAAR1 as a receptor mechanism for METH-induced effects in astrocytes. Previously we showed localization and function of astrocyte TAAR1. Documented TAAR1 thermoregulatory responses led the expansion of our studies to investigate METH-associated hyperthermia in METH/HIV-1-induced astrocyte activation. Results: Furthermore, preliminary data suggest TAAR1 regulation in the presence of thermal stress. Elevated temperatures increased GFAP expression and cytokine secretion. We propose activation of astrocyte TAAR1 mediates METH/HIV-1-induced neurodegeneration, further modulated by METH-associated hyperthermia. Conclusions: The results will lead to understanding of the mechanisms and pathological features associated with METH and HIV-1 neurodegeneration and potential therapeutic targets in the CNS.