Browsing by Subject "HIV-1"
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Item ASTROCYTE-ELEVATED GENE-1 PROTECTS HUMAN ASTROCYTES FROM OXIDATIVE STRESS-INDUCED CELL DEATH: A POTENTIAL SURVIVAL MECHANISM IN HAND AND GLIOMA PATHOGENESIS(2013-04-12) Vartak, NehaPurpose: Astrocyte elevated gene-1 (AEG-1), a novel human immunodeficiency virus (HIV-1)- and tumor necrosis factor-ɑ-inducible oncogene, has generated significant interest in the field of cancer research as a therapeutic target for many metastatic cancers. However, little is known of its role in astrocyte behavior and function during HIV-1 central nervous system (CNS) infection, and whether it contributes towards the development of HIV-1-associated neurocognitive disorders. Based on its putative role in cancer as a chemotherapy resistance marker, here, we investigated whether AEG-1 induction in astrocytes alters their responses to oxidative stress, a hallmark feature of neuroinflammatory disorders. Methods: Oxidative stress induced changes in AEG-1 mRNA and protein were assessed by real-time PCR, immunoblotting and confocal microscopy. Astrocyte responses to oxidative stress were assayed by measuring the changes in catalase activity, protein carbonylation and glutathion production. AEG-1 mediated protection against oxidative damage was assessed by measuring the anti-oxidant mechanisms upon AEG-1 knockdown. Results: Analysis of AEG-1 mRNA levels in an aging cohort of HIV-1 seropositive and seronegative human brain tissues showed a significant positive correlation to aging. A dose-dependent increase in AEG-1 astrocyte nucleolar localization was noted following treatment with oxidative stress stimuli, hydrogen peroxide, as assayed by immunostaining and confocal microscopy. Cell death and cell survival assays to quantify apoptotic nuclei, mitochondrial depolarization and activity, and cell membrane permeability demonstrated a novel role of AEG-1 in protecting astrocytes from oxidative-stress-induced damage. Conclusions: Together, findings from this study suggest that AEG-1 may play a role in protecting astrocytes from oxidative stress-induced DNA damage, a plausible mechanism of astrocyte survival during HIV-1 CNS infection-induced toxicity.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 Chemokine CXCL8 Mediated Intercellular Interactions in HIV-1 associated Dementia(2013-12-01) Mamik, Manmeet K.; Ghorpade, AnujaThis dissertation explores the role of chemokine CXCL8 during human immune deficiency virus (HIV)-1 infection in the brain. Chemokine CXCL8 is an important neutrophil chemoattractant implicated in various neurodegenerative disorders. It is upregulated in the brains and cerebrospinal fluid of HIV-1 infected individuals suggesting its potential role in HIV-1 associated neuroinflammation. Astrocytes are known to be the major contributors to the CXCL8 pool. Interleukin (IL)-1β activated astrocytes exhibit significant upregulation of CXCL8. In order to determine the signaling pathways involved in CXCL8 regulation in astrocytes, we employed pharmacological inhibitors for non-receptor Src homology-2 domain-containing protein tyrosine phosphatase (SHP) 2 and mitogen-activated protein kinases (MAPK) pathway and observed reduced expression of CXCL8 following IL-1β stimulation. Thus, our findings suggest an important role for SHP2 in CXCL8 expression in astrocytes during inflammation, as SHP2, directly or indirectly, modulates p38 and extracellular signal regulated kinase (ERK) MAPK in the signaling cascade leading to CXCL8 production. In the post-antiretroviral therapy (ART) era, low level of productive replication of HIV-1 in brain is a critical component of neuropathogenesis regulation. HIV-1 replication is a complex mechanism involving both host and viral factors. The majority of viral replication in brain occurs in perivascular macrophages and/or 2 microglia. In this study, we investigated the effect of CXCL8 on productive infection of HIV-1 in human monocytes-derived macrophages (MDM) and primary human microglia. The results show that CXCL8 mediates productive infection of HIV-1 in MDM and microglia via receptors CXCR1 and CXCR2 and induces HIV-1 long terminal repeat (LTR) promoter activity. Detailed understanding of astrocyte signaling and HIV-1 replication, as presented in the thesis, will be relevant to glial-neuronal interactions, which are central to neuroinflammation in HIV-1 and many other neurodegenerative conditions. Also, modulation of levels of CXCL8 can be a therapeutic strategy for control of productive HIV-1 replication in the brain.Item HIV-1 TAT INDUCED LYSOSOMAL EXOCYTOSIS IN ASTROCYTES AND ITS CONTRIBUTIONS TO TAT NEUROTOXICITY(2014-03) Fan, Yan; He, JohnnyPurpose (a): HIV-1 Tat protein is considered to be the critical reason in the processing of HIV-associated neuropathogenesis. Our previous studies demonstrates that HIV-1 Tat expression leads to ER stress in astrocytes through GFAP aggregation and suggest that disruption of ER homeostasis, i.e., ER stress may be involved in HIV-associated neuropathogenesis. But what the neurotoxic factor is in this indirect astrocyte-mediated Tat neurotoxicity system is still unknown. In this study, we take advantage of our Tat-inducible transgenic mice, and proteomic analysis was performed to explore the neurotoxic factor in the astrocyte-mediated Tat neurotoxicity system. Methods (b): Brain-targeted inducible Tat transgenic and GFAP knockout mice were used in the study. Primary astrocytes and neurons cultures were prepared from mouse embryos. U373 cells and primary astrocytes and were either transfected with pTat.Myc or treated with doxycycline to induce Tat expression. Cells were prepared and analyzed for β–hexosaminidase activity by NAG assay after ionomycin mediated Ca2+ influx. Culture supernatants were collected for immunodepletion and analyzed for their neurotoxicity toward primary mouse or human neurons using MTT assay. TIRF microscopy is utilized to visualize lysosome exocytosis events. Results (c): Base on the protein sequencing and pathway analysis, we proposed that HIV-1 Tat induces lysosomal exocytosis in astrocytes. Then, NAG assay and TIRF microscopy provide consistent evidences that verified our hypothesis. Moreover, we attested that inhibition of Tat induced lysosomal exocytosis in astrocytes by vaculin-1 can abolish Tat induced neuron death. More interestingly, we observed that Tat induced lysosomal exocytosis and neuron death only appears in astrocytes but not in other cell types, such as 293T and Huh 7.5.1 cells. Further more, we proved that GFAP plays a critical role in Tat induced lysosomal exocytosis and neuron death. Conclusions (d): Taken together, these results demonstrate that HIV-1 Tat induces lysosomal exocytosis and hydrolytic enzymes contained within lysosome are suggested to contribute to neuronal death, which is a novel insight into astrocytes-mediated Tat neurotoxicity.Item HIV-1 TAT INDUCES ER STRESS IN ASTROCYTES AND CAUSES NEUROTOXICITY THROUGH GFAP ACTIVATION AND AGGREGATION(2013-04-12) Fan, YanPurpose: HIV-1 Tat is a major pathogenic factor for HIV-associated neurodegenerative diseases. One of the consistent hallmarks of HIV-1 infection of the central nervous system (CNS) is astrocytosis, which is characterized by increased cytoplasmic accumulation of intermediate filament glial fibrillary acidic protein (GFAP). Our previous studies show that Tat induces GFAP expression in astrocytes and GFAP is a critical regulator of Tat neurotoxicity. However, the molecular mechanisms responsible for GFAP activation-mediated Tat neurotoxicity is not known. Thus, we attempted in this study to determine the underlying unknown molecular mechanisms. Methods: Brain-targeted inducible Tat transgenic and GFAP knockout mice were used in the study. Primary astrocytes were either transfected with pTat.cMyc or treated with doxycycline to induce Tat expression. Cells were harvested to detect GFAP, eIF2a, ATF6, Oasis and Bip expression by Western blotting to , or to detect Tat, GFAP and XBP-1 by RT-PCR. Culture supernatants were collected and analyzed for neurotoxicity toward primary mouse or human neurons using MTT assay. GFAP, MAP-2 and Bip expression in astrocytes, Tat-expressing mouse brain and the brains of HIV-infected individuals were also determined by IF and IHC staining. Flow cytometry was performed to determine proteasomal activity by monitor expression intensity of proteasomal activity reporter pZsProSensor-1. Results: We showed that HIV-1 Tat-induced GFAP up-regulation and aggregation in astrocytes activated endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways, such as PERK, IRE1, ATF6 and OASIS, We further showed that supernatants from Tat-expressing astrocytes were neurotoxic. Importantly, we showed that the neurotoxicity in these culture supernatants were significantly diminished when GFAP was null or when the cultures were treated with 4-sodium phenyl butyrate (4-PBA), a chemical chaperon capable of blocking the ER stress, suggesting that GFAP activation and aggregation-induced ER stress in the presence of Tat expression is at least in part responsible for Tat neurotoxicity. Lastly, we showed that GFAP activation and aggregation resulted in lower proteasome activity and induction of autophagy in astrocytes. Conclusions: Taking together, this study demonstrates that HIV-1 Tat expression leads to ER stress in astrocytes through GFAP aggregation and suggest that disruption of ER homeostasis, i.e., ER stress may be involved in HIV-associated neuropathogenesis.Item IMPLICATIONS OF ASTROCYTIC NRF2-ARE SIGNALING PATHWAY IN METHAMPHETAMINE AND HIV-1gp120 INDUCED OXIDATIVE STRESS(2014-03) Shelake, Sagar; Ghorpade, AnujaDespite the advent of the antiretroviral therapy, HIV-1 associated neurocognitive disorders (HAND) continue to be a significant issue for HIV-1 infected patients. HIV-1 infection of CNS combined with Methamphetamine (METH) abuse causes overall increase in oxidative stress in astrocyte. In HAND patients, oxidative stress induced apoptosis in astrocyte compromises the normal physiology and function of CNS. NF-E2–related factor 2 (Nrf2) transcription factor plays vital role in cellular protective response against oxidative stress due to environmental agents such as electrophiles, drug abuse, smoking, radiation. Although HIV-1 gp120and METH have been implicated in the pathogenesis of HAND, little is known about their combined effect on the regulation of Nrf2 in human astrocytes. In this study, we investigated the combinatorial effect of gp120 and METH on Nrf2-ARE signaling pathway in primary human fetal astrocytes. Astrocytes were treated with METH and gp120 followed by immunocytochemistry analysis. The oxidative stress and apoptosis was detected by protein carbonylation and DNA fragmentation, respectively. The levels of phospho-Nrf2 and Nrf2 were analyzed by western blot. Repeated treatment of METH and gp120 induced the reactive astrocyte phenotype as observed by GFAP immunostaining. METH and gp120 significantly increased oxidative stress and apoptosis. Further investigation revealed that METH and gp120 significantly increased Nrf2 phosphorylation and nuclear translocation in a time-dependent manner. Taken together, these results suggest the involvement of Nrf2-ARE signaling pathway as a protective response to METH- and gp120- induced oxidative stress in human astrocytes. Purpose (a): Despite the advent of the antiretroviral therapy, HIV-1 associated neurocognitive disorders (HAND) continue to be a significant issue for HIV-1 infected patients. HIV-1 infection of CNS combined with Methamphetamine (METH) abuse causes overall increase in oxidative stress in astrocyte. In HAND patients, oxidative stress induced apoptosis in astrocyte compromises the normal physiology and function of CNS. NF-E2–related factor 2 (Nrf2) transcription factor plays vital role in cellular protective response against oxidative stress due to environmental agents such as electrophiles, drug abuse, smoking, radiation. Although HIV-1 gp120and METH have been implicated in the pathogenesis of HAND, little is known about their combined effect on the regulation of Nrf2 in human astrocytes. In this study, we investigated the combinatorial effect of gp120 and METH on Nrf2-ARE signaling pathway in primary human fetal astrocytes. Methods (b): Astrocytes were treated with METH and gp120 followed by immunocytochemistry analysis. The oxidative stress and apoptosis was detected by protein carbonylation and DNA fragmentation, respectively. The levels of phospho-Nrf2 and Nrf2 were analyzed by western blot. Results (c): Repeated treatment of METH and gp120 induced the reactive astrocyte phenotype as observed by GFAP immunostaining. METH and gp120 significantly increased oxidative stress and apoptosis. Further investigation revealed that METH and gp120 significantly increased Nrf2 phosphorylation and nuclear translocation in a time-dependent manner. Conclusions (d): Taken together, these results suggest the involvement of Nrf2-ARE signaling pathway as a protective response to METH- and gp120- induced oxidative stress in human astrocytes.Item TAAR1-dependent astrocyte dysregulation during HAND and METH exposure(2018-12) Mythen, Shannon; Ghorpade, Anuja; Basu, Alakananda; Barber, Robert C.; Gatch, Michael B.; Krishnamoorthy, Raghu R.Excitatory amino-acid transporter (EAAT)-2 is predominantly expressed in astrocytes and clears glutamate from tripartite synapses preventing excitotoxicity. EAAT- 2 dysregulation occurs during human immunodeficiency virus (HIV)-associated neuroinflammation and methamphetamine (METH) abuse, leading to neurotoxic outcomes. Trace amine associated receptor (TAAR) 1, a METH receptor in astrocytes, triggers EAAT-2 dysfunction. Protein kinase C (PKC) signaling promotes ubiquitination of EAAT-2 C-terminal lysine residues, resulting in EAAT-2 internalization. As a G protein coupled receptor, TAAR1's signaling is implicated in PKC activation. In this work, we investigated the role of TAAR1 in PKC-mediated EAAT-2 ubiquitination during HIV- associated neurocognitive disorders (HAND) and METH comorbidities. We evaluated a TAAR1 overexpression model in primary astrocytes to elucidate TAAR1-mediated functional changes. We found that TAAR1-selective inhibitor, EPPTB, reduced EAAT-2 ubiquitination, and a PKC activator decreased glutamate clearance in METH-pretreated human astrocytes. Therapies targeting astrocyte dysfunction may improve outcomes during HAND, METH abuse and other neuroinflammatory disorders.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 Transcriptome and functional profiles of R/G-HIV+ human astrocytes: Implications for shock or lock therapies in the brain(2020-05) Edara, Venkata Viswanadh; Borgmann, Kathleen; Berg, Rance E.; Krishnamoorthy, Raghu R.; Mathew, Porunelloor A.; Yang, Shaohua; Patterson, Rita M.A significant number of people living with human immunodeficiency virus (HIV) suffer from HIV-associated neurocognitive disorders (HAND). Many previous studies investigating HIV in astrocytes as a heterogenous population have established the relevance of astrocytes to HIV-associated neuropathogenesis. However, these studies were unable to differentiate the state of infection, i.e. active or restricted, or to evaluate how this affects astrocyte biology. In this study a pseudotyped doubly labelled fluorescent reporter R/G-HIV-1 was used to identify and enrich restricted and active populations of HIV+ astrocytes based on the viral promoter activity. Here we report, the majority of human astrocytes restricted R/G-HIV-1 gene expression early during infection and were resistant to reactivation by vorinostat and interleukin-1β. However, actively infected astrocytes were inducible, leading to increased expression of viral proteins upon reactivation. R/G-HIV-1 infection also significantly decreased cell proliferation and glutamate clearance ability of astrocytes, which may contribute to excitotoxicity. Moreover, transcriptome analyses to compare gene expression patterns of astrocytes harboring active vs restricted long terminal repeats revealed that the gene expression patterns were similar, and the active population demonstrated more widespread and robust changes. Our data suggest that harboring the HIV genome profoundly alters astrocyte biology and strategies that keep the virus latent (e.g. Block and Lock), or those that reactivate the latent virus (e.g. Shock and Kill) may be detrimental to astrocyte function and possibly augment their deleterious contributions to HAND.Item Ubiquitin-protein ligase E3A (UBE3A) mediation of viral infection and human diseases(Elsevier B.V., 2023-08-05) Chaudhary, Pankaj; Proulx, Jessica; Park, In-WooThe Ubiquitin-protein ligase E3A, UBE3A, also known as E6-associated protein (E6-AP), is known to play an essential role in regulating the degradation of various proteins by transferring Ub from E2 Ub conjugating enzymes to the substrate proteins. Several studies indicate that UBE3A regulates the stabilities of key viral proteins in the virus-infected cells and, thereby, the infected virus-mediated diseases, even if it were reported that UBE3A participates in non-viral-related human diseases. Furthermore, mutations such as deletions and duplications in the maternally inherited gene in the brain cause human neurodevelopmental disorders such as Angelman syndrome (AS) and autism. It is also known that UBE3A functions as a transcriptional coactivator for the expression of steroid hormone receptors. These reports establish that UBE3A is distinguished by its multitudinous functions that are paramount to viral pathology and human diseases. This review is focused on molecular mechanisms for such intensive participation of UBE3A in disease formation and virus regulation.Item Uncovering the Multifaceted HAND of Reactive astrocytes in HIV-Associated Neurocognitive Disorders(2017-08-01) Nooka, Shruthi; Ghorpade, Anuja; Luedtke, Robert; Mathew, Porunelloor A.Despite the advent of antiretroviral therapy (ART), central nervous system (CNS) complications associated with HIV-1 infection, collectively referred to as HIV-associated neurocognitive disorders (HAND), continue to increase. HIV infection promotes cognitive dysfunction and neurodegeneration through persistent inflammation, oxidative stress from infected and/or activated macrophages, astrocytes and neurons. Additionally, recent studies demonstrated that neurotoxic side effects of antiretroviral (ARV) drugs are among several contributing factors to this continued prevalence of HAND. In recent years, a new appreciation of the role of astrocytes in regulating HIV-1 CNS infection has emerged. Thus, investigating the elusive cellular and molecular mechanisms regulated by astrocytes during HIV-1/ART-induced neurotoxicity could provide insight into HAND pathogenesis. The work presented in this thesis contributes to the documentation of astrocyte dysfunction in HIV-1-infection. The accumulated data reveal the multifaceted mechanisms and roles of astrocytes in HIV-1 CNS infection. A neuropathological feature of HIV-1 infection includes reactive astrogliosis, which is a hallmark of many neurodegenerative diseases. Our studies reveal the regulation of β-catenin signaling on major aspects of reactive astrogliosis i.e., proliferation, wound healing and inflammation. HAND-relevant inflammatory stimuli activate β-catenin signaling in astrocytes. Our in vitro studies reveal knockdown of β-catenin impairs astrocyte responses to injury. Further, reduced levels of β-catenin also show less proliferation and inflammatory responses in astrocytes. We also demonstrated that Wnt/ β-catenin and NF-κB crosstalk links with inflammation during HIV-1 CNS infection. We next investigated endoplasmic reticulum (ER) stress associated with HAND-relevant neuroinflammation. Our studies show that ART (abacavir) and interleukin-1β increase cytosolic calcium in astrocytes, which in turn regulates ER stress and mitochondrial depolarization. We also identify astrocyte elevated gene (AEG)-1 as an ER stress inducible gene. In addition, AEG-1 interacts with calnexin, which emphasizes AEG-1 as a scaffolding protein regulating ER calcium signaling. Further, HIV-1-coupled inflammation and oxidative stress significantly increase regulator of ribosome synthesis (RRS1) expression, suggesting inhibition of rRNA transcription in astrocytes. Further, AEG-1 overexpression enhanced oxidative stress-induced RRS1 expression, i.e., nucleolar stress in astrocytes. Taken together, this study identified novel regulatory mechanisms in reactive astrocytes during HIV-1-induced neurodegeneration that might serve as innovative therapeutic targets for HAND.