Browsing by Subject "Tat"
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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 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 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.