Cell Biology

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21650

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    Silencing Astrocyte Elevated Gene-1 during Calcium Signaling and Glutamate Excitotoxicity
    (2018-03-14) Stacey, Satomi; Ghorpade, Anuja Dr; Proulx, Jessica
    Purpose: During central nervous system (CNS) injury or infection, astrocytes undergo inflammatory and functional changes, ultimately regulating cognitive impairment. Among these devastating changes is the downregulation of excitatory amino acid transporter 2 (EAAT2). EAAT2 is critical for glutamate uptake in synaptic clefts, so when downregulated, results in glutamate excitotoxicity. Our previous studies highlighted astrocyte elevated gene-1 (AEG-1) overexpression as a novel modulator of EAAT2 repression during HIV-1 associated neuroinflammation. Additionally, elevated AEG-1 levels are evident in several cancers, neurodegeneration, oxidative stress, and aging. AEG-1 cooperates in several cell signaling pathways mediating cell development, inflammation, proliferation, differentiation, metabolism, apoptosis, and autophagy. While AEG-1 is ubiquitously expressed, levels are higher in muscle-dominated organs and endocrine glands, suggesting a role of AEG-1 in calcium associated signaling. Furthermore, we have shown AEG-1 to have a direct interaction with the calcium-binding chaperone calnexin. Both calcium and glutamate are prominent CNS signaling molecules reported to be dysregulated during conditions AEG-1 is often upregulated. However, the role of AEG-1 in calcium signaling has not yet been explored. Materials & Methods: AEG-1 specific siRNA was used to isolate AEG-1 dependent outcomes. Astrocyte activation was measured via morphological staining and cytokine ELISA. EAAT2 protein and mRNA expression were examined in conjunction with functional glutamate clearance assay to correlate potential glutamate excitotoxicity. Calcium signaling was measured by live cell confocal microscopy using a genetically encoded calcium sensor. Results: AEG-1 siRNA significantly decreased protein and mRNA expression of AEG-1 compared to non-specific siRNA. Preliminary studies for calcium signaling required standardization and suggest a decreased calcium response when AEG-1 is knocked down. Additional investigations are still in progress. Conclusion: The current study focuses on investigating how AEG-1 regulates calcium signaling and astrocyte activation in connection with glutamate excitotoxicity. These findings will help better understand AEG-1 mediated dysregulation of astrocyte function, possibly identifying a novel therapeutic target for cognitive impairment.
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    TAARGETING ASTROGLIOSIS DURING METH AND HIV-1 EXPOSURE
    (2018-03-14) Mythen, Shannon; Swanta, Naomi; Ghorpade, Anuja; Borgmann, Kathleen
    Purpose: As a popular psychostimulant, methamphetamine (METH) use leads to long-lasting, strong euphoric effects. METH exacerbates the severity and onset of HIV-associated neurocognitive disorders (HAND), which affect 30-70% of the 37.6 million people globally infected with HIV. Most neurodegenerative diseases share neuroinflammation as a common pathogenic mechanism. Neuroinflammation, HIV and METH dysregulate a wide range of brain functions including neuronal signaling, glial activation, viral infection, oxidative stress and excitotoxicity. Since neuroglia often determine the outcomes of neurological disease, we investigated the mechanisms regulating astrocyte-mediated neurotoxicity in the context of METH and HIV comorbidity. Methods: To these ends, we examined the expression, localization and function of the novel METH astrocyte receptor, trace amine associated receptor 1 (TAAR1) in an in vitro model of HIV-associated activation wherein extended METH exposure is administered to mimic residual METH concentrations that occur in humans between binges of METH-taking. Results: In this model, TAAR1 levels, and its localization to the endoplasmic reticulum (ER) and plasma membranes, increased with METH and HIV-induced astrogliosis. Calcium flux, which mediates ER, mitochondrial, and oxidative stress, also was increased, corroborating our prior studies on astrocyte mitochondrial dysregulation. The astrocyte responses to METH and HIV-relevant stimuli were blocked with the TAAR1-selective antagonist EPPTB. Extended METH and HIV activation impaired excitatory amino acid transporter 2 (EAAT2) expression and activity, which were recovered by following 24 hour exposure with EPPTB. Conclusion: Together, these data highlight several mechanisms regulating METH/HIV-induced, astroglia-mediated neurotoxicity and the potential for astrocyte targeted intervention via TAAR1 during chronic disease.
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    Targeting Astrocyte HIV-1 Proviral Reservoirs in HAND
    (2018-03-14) Ghorpade, Anuja PhD; Edara, Venkata Viswanadh
    Purpose: Although antiretroviral therapy (ART) has greatly reduced the incidence of HIV-associated dementia (HAD), nearly 50-70% of HIV-1 infected individuals develop HIV-associated neurocognitive disorders (HAND). Between 5-20% of astrocytes harbor HIV-1 provirus, and they do not actively propagate viral infection. However, it is well established that astrocytes produce viral proteins, which cause changes in astrocyte function and aggravate HAND pathogenesis. Moreover, in vivo it is difficult to distinguish latently infected astrocytes from healthy cells. Thus, there is a great need to identify latently infected astrocytes and develop strategies to target this specific population. We hypothesize that HIV-1 proviral reservoirs alter astrocyte function and gene expression patterns, which could serve as biomarkers to facilitate targeted therapy. Methods: The dual-labeled, fluorescent reporter Red/Green-HIV-1 (R/G-HIV-1) was used to visualize viral promoter (LTR) activity in primary human astrocytes. Astrocytes were spinoculated with pseudotyped R/G-HIV-1-WT. Exposed uninfected (R-/G-), astrocytes with active (R+/G+) and silent (R+/G-) LTRs were enriched using fluorescence activated cell sorting (FACS). Subsequently, these cells were used to evaluate viral protein expression, functional studies, and preliminary RNA sequencing. Results: Astrocytes with silent viral promoter are devoid of late viral proteins such as p24, indicating a functionally silent HIV-1 LTR. Vorinostat, an HDAC inhibitor, reactivated silent HIV-1 LTR in R/G-HIV-1-infected astrocytes. Preliminary data indicate that astrocytes with silent (R+/G-) and active (R+/G+) LTRs have significantly impaired glutamate clearance ability and cell proliferation compared to exposed uninfected (R-/G-) cells. Interleukin-1β (IL-1β) a HAND relevant stimuli, further reduced glutamate clearance ability of these independent populations. However, harboring the HIV-1 provirus did not alter inflammatory responses of astrocytes, such as CXCL8 and CCL2 production, either alone or in presence of IL-1β. Conclusions: Our data suggest that harboring HIV-1 provirus with either active or silent viral promoters interfered with astrocyte function and growth. Hence, we propose that identifying biomarkers for astrocytes harboring HIV provirus, and therapeutic gene editing to eliminate proviral gene expression, will improve physiological function compared to HIV-1 infected cells.
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    Polymeric Nanoparticle-Mediated GFAP-TIMP-1 Gene Delivery to Human Astrocytes
    (2018-03-15) Labhasetwar, Vinod; Ghorpade, Anuja; Joshi, Chaitanya
    Purpose: The neuroprotective functions of astrocyte tissue inhibitor of metalloproteinases-1 (TIMP-1) during HIV-1-induced apoptosis are documented. However, astrocyte TIMP-1 levels decrease during chronic inflammation typical of HIV-associated neurocognitive disorders (HAND). We hypothesize that nanoparticle (NPs) mediated, astrocyte-targeted TIMP-1-gene delivery could restore TIMP-1 levels and serve as a HAND therapy. Optimally biocompatible polymeric NPs with validated in vivo reporter gene expression would serve as gene delivery vehicles. TIMP-1 overexpression may be restricted to astrocytes, using a glial fibrillary acidic protein (GFAP) promoter-driven gene expression, without adversely affecting the astrocyte structure and functions. Methods: We tested biocompatibility, dose-time kinetics, and in vivo gene delivery with arginine-modified polyethylenimine (PEI) analogs (AnPn) in vitro (primary human neural cells) and in vivo (mice) using a cytomegalovirus (CMV) promoter-driven luciferase plasmid (pLuc). Truncated and full-length GFAP promoter-driven plasmids encoding luciferase-reporter (gfa-Luc) and TIMP-1 (gfa-TIMP-1) were sub-cloned. Astrocytes transfected with AnPn-delivered gfa-Luc and gfa-TIMP-1 were evaluated for morphological changes, inflammatory biomarker profiles, and glutamate clearance to evaluate if critical astrocyte functions are affected. Results: Select PEI analogs (AnPn) led to robust and sustained reporter gene expression in astrocytes. Successful in vivo reporter gene delivery to the brain was confirmed by luminescence assay and immunohistochemistry. Truncated GFAP promoters led to a detectable gene expression in astrocytes compared to full-length GFAP promoters, which was regulated by inflammatory stimuli. Lastly, gfa-TIMP-1 plasmids were successfully delivered using AnPn to astrocytes in the presence and absence of HIV-relevant stimuli. Immunocytochemistry, and proinflammatory biomarker profiles of transfected astrocytes were analyzed to delineate NP-specific versus TIMP-1-specific changes. Conclusions: These studies confirmed successful polymeric NPs-mediated reporter gene delivery in vitro and in vivo. Subsequently, NPs-mediated GFAP promoter-driven reporter and TIMP-1 gene expression was demonstrated in vitro. These findings serve as proof-of-concept towards testing therapeutic TIMP-1 gene delivery and demand future investigations geared towards clinical translations for HAND treatment.
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    miR-200b-3p and miR-211-5p downregulate the expression of extracellular matrix and associated proteins in human optic nerve head astrocytes
    (2018-03-14) Tovar-Vidales, Tara; Clark, Abbot F.; Lopez, Navita
    PURPOSE: microRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that epigenetically regulate post-transcriptional gene expression. miRNAs are known to modulate cellular functions such as extracellular matrix (ECM) turnover. There is evidence that dysregulation of miRNA expression has a role in the pathogenesis of fibrotic diseases including glaucoma. Glaucoma is a leading cause of irreversible blindness and is associated with fibrotic changes to the optic nerve head (ONH), the initial site of glaucomatous damage to the retina and optic nerve. Our previous study showed that expression of the profibrotic cytokine TGFβ2 is elevated in the ONH of glaucoma eyes compared to age-matched normal eyes. Currently there is a lack of knowledge regarding the roles of miRNAs in the ONH. The purpose of this study was to determine: (a) differences in the expression of profibrotic and anti-fibrotic miRNAs in normal ONH astrocytes treated with or without TGFβ2 and (b) whether candidate miRNAs (miR-200b-3p and miR-211-5p) regulate the expression of ECM and ECM associated proteins in the ONH. METHODS: Primary normal human ONH astrocytes (ONA) were grown to 100% confluency. ONA were treated with 5ng/ml TGFβ2 or with control for 24hrs. miRNA qPCR arrays were performed to compare the expression levels of profibrotic and anti-fibrotic miRNAs in normal ONA treated with or without TGFβ2. ONA were transfected with miR-200b-3p and miR-211-5p mimics at 10nM, 5nM, and 1nM concentrations to confirm target predictions based on the TargetScan database. An all stars negative control siRNA was included which will not recognise any mammalian gene. RESULTS: The miRNA qPCR arrays analysed from normal ONA exposed to TGFβ2 showed that TGFβ2 downregulated the expression of hsa-miR-200b-3p and hsa-miR-211-5p. Transfection of miR-200b-3p mim downregulated fibronectin (FN), gremlin, and tissue transglutaminase II in ONA. Transfection of miR-211-5p downregulated FN and gremlin in ONA . CONCLUSIONS: Our results suggest that TGFβ2, which is elevated in the glaucomatous ONH, modulates the expression of miRNAs in ONA. These miRNAs target FN, TGM2, and gremlin to modify the ECM in the ONH. Downregulation of anti-fibrotic miRNAs may contribute to fibrosis of the ONH.
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    Elucidating molecular mechanisms of TAAR1-dependent astrocyte regulation during HIV-associated neurocognitive disorders and methamphetamine exposure
    (2018-03-14) Ghorpade, Anuja; Mythen, Shannon
    Elucidating molecular mechanisms of TAAR1-dependent astrocyte regulation during HIV-associated neurocognitive disorders and methamphetamine exposure Shannon Mythen and Anuja Ghorpade Department of Microbiology, Immunology and Genetics, UNT Health Science Center, Fort Worth, TX Purpose: A study conducted in 2012 estimated that 1.2 million people in the U.S. reported the use of methamphetamine (METH) in the previous year, with 133,000 new users age 12 or older. METH is a highly addictive substance that leads to an imbalance in dopamine and norepinephrine release causing euphoric effects. Long term METH use has been linked to many central nervous system (CNS) abnormalities including deficits in memory, executive function, anxiety and depression. METH use is associated with risky sexual behavior, lowered inhibitions and increased likelihood for acquiring HIV. METH abuse exacerbates the onset of HIV-associated neurocognitive disorders (HAND) and promotes a neurotoxic environment by increasing oxidative stress and excitotoxicity. Our lab previously identified trace amine associated receptor 1 (TAAR1) as a novel stimulatory G protein coupled receptor in primary human astrocytes. The expression of TAAR1 is modulated by METH and HAND-relevant stimuli. We hypothesize that TAAR1 dysregulates astrocyte intracellular signaling during HAND and METH exposure thus contributing to disease pathogenesis. Methods: To mimic upregulation of TAAR1 during HIV CNS disease, TAAR1 overexpression was studied in human astrocytes. We then used a physiologically relevant model of extended METH exposure and low level HIV-associated activation. First, exogenous TAAR1 expression and intracellular localization were characterized. Next, TAAR1 localization with HAND relevant stimuli and METH exposure were assessed. Finally, TAAR1-dependent signaling and activation of downstream modulators were evaluated. We used a TAAR1 selective antagonist, EPPTB, to determine TAAR1-dependent changes in astrocyte function. Results: The overexpression model was standardized for TAAR1 levels in astrocytes. TAAR1 function mirrored expression levels in transfected astrocytes. Exposure to METH and HAND-relevant stimuli altered astrocyte functional responses, including proliferation and reactive morphological phenotype. Conclusions: Our study aims to delineate therapeutically targetable mechanisms that regulate astrocytes during neuroinflammation in HAND and METH exposure. Therefore, we propose astrocyte TAAR1 as a potential target to combat neurocognitive decline.
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    Canonical Wnt signaling in optic nerve head
    (2018-03-14) Liu, Yang; Clark, Abbot; Allums, Elliott
    Purpose: While canonical Wnt signaling has been explored in areas of the eye like the trabecular meshwork and ciliary body during glaucomatous pathology, it has not been explored in the lamina cribrosa. Complement protein 1, subunit q (C1q) has been shown to be an activator of the canonical Wnt pathway leading to an increase in fibrosis in various tissues, but not explored in the lamina cribrosa (LC) either. As C1q concentration increases in blood serum and central nervous system with an increase in age, C1q activation of canonical Wnt signaling may be an important factor in glaucomatous pathology. Therefore, we aimed to prove that a functional canonical Wnt signaling pathway is expressed in the lamina cribrosa and that C1q is an activator of the pathway. Methods: Primary mouse optic nerve head (ONH) astrocytes from C57BL/6J mice were cultured and characterized. When confluent, cells were serum starved overnight and then treated for 24 hours with 100 nM Wnt3a or left untreated as a control. Following treatment, cells were collected and had cytosolic and nuclear fractions separated. Fractions were then western blotted and probed for β-Catenin. Bands were analyzed via densotometry and fold changes in expression were compared to control cells. Results: In a single primary mouse ONH astrocyte cell strain, β-Catenin expression increased 1.354-fold when treated with 100 nM Wnt3a compared to control in the cytosolic fraction and 1.145-fold when treated with 100 nM Wnt3a compared to control in the nuclear fraction. Without additional cell strains, statistical significance is not able to be determined. Conclusions: Our very preliminary results support our hypothesis that a functional canonical Wnt signaling pathway is expressed in the LC. Additional cell strains will need to be examined to fully determine presence of a functional canonical Wnt signaling pathway as well as determining if C1q activates the pathway in this population.