Browsing by Subject "Astrocyte"
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Item Endothelin-1-Induced Proliferation of Human Optic Nerve Head Astrocytes Under Hypoxia(2003-11-01) Desai, Devashish; Thomas Yorio; Ganesh Prasanna; Clark, Abbot F.Desai, Devashish, Endothelin-1-Induced Proliferation of Cultured Human Optic Nerve Head Astrocytes under Hypoxia. Master of Science (Biomedical Sciences). Purpose: Optic nerve head astrocytes (ONAs) normally support and protect the axons of retinal ganglion cells exiting the eye. Along with effects related to elevated intraocular pressure (IOP), proliferation and activation of ONAs, known as ‘astrogliosis’, is also thought to contribute to the pathophysiology of glaucoma by distributing axonal transport and preventing axon regeneration. Concentrations of endothelin-1 (ET-1) are elevated in glaucomatous eyes and in animal models for glaucoma. ET-1 injection into the eye causes reduction of ocular blood flow. ET-1 causes a time-dependent proliferation of human ONAs. Tumor necrosis factor-α (TNF-α), a cytokine, which is also elevated in glaucomatous optic nerve head, promotes ET-1 release from ocular cells and could potentially stimulate ET-1 secretion from the ONAs. Hypoxia resulting from ischemia, which is produced by the elevation of IOP or vasospasm in the retinal vasculature, is considered a significant factor contributing to the stress as the glaucomatous optic nerve head. Methods: Concentrations of ET-1 secreted by hONAs into cell culture media after hypoxia and TNF-α treatment was measured using an enzyme-linked immunosorbent assay (ELISA). Proliferation of hONAs was measured using a proliferation assay (formazan assay), performed at the end of various time periods of incubation with TNPα and ET-1 under normoxia or hypoxia. The involvement of mitogen activated protein kinase (MAPK) in hONA proliferation was examined using MAPK inhibitors and Western blot analyses. Results: Cell culture media collected from hONAs after 24-hour hypoxia with concurrent TNF-α treatment showed a 500% increase in the irET-1. Under normoxia, both TNF-α and ET-1 caused moderate proliferation of hONAs. Under hypoxia, TNF-α-induced proliferation was greatly increased. Conclusion: Hypoxia augments TNF-a and ET-1 growth of optic nerve head astrocytes, by way of increasing ET-1 synthesis and release as well as mitogenesis. Therefore reactive ONAs could be the common denominator underlying optic nerve damage in glaucoma since their localization makes them susceptible to mechanistic and ischemic influences in addition to influences of ET-1 and TNF-α. Keywords: astrocyte; endothelin-1; tumor-necrosis factor-α; hypoxia; proliferation; astrogliosis; glaucoma; optic nerveItem 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 TAARgeting Astrogliosis and Mitochondrial Dysfunction during METH Exposure and HIV-relevant Neuroinflammation(2017-12-01) Borgmann, Kathleen; Ghorpade, Anuja; Wordinger, Robert J.; Berg, Rance E.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 determine the outcome of neurological disease, we investigate the mechanisms regulating astrocyte-mediated neurotoxicity in the context of METH and HIV comorbidity. To these ends, we examined the expression, localization and function of the novel METH astrocyte receptor, trace amine associated receptor 1 (TAAR1) in an extended METH in vitro model, which mimics chronic residual METH concentrations between binges, and HIV-associated activation. In our model, TAAR1 levels and localization to the endoplasmic reticulum and plasma membranes increased with METH and HIV-induced astrogliosis. Extended physiological METH exposure led to augmented calcium flux, a mechanism known to mediate ER and mitochondrial, and oxidative stress. METH induced dysregulation of astrocyte mitochondrial morphology by elevating mitofusin expression and inhibitory phosphorylation of dynamin-related protein-1. While METH decreased oxygen consumption and ATP levels during acute exposure, chronic treatment significantly enhanced both. Together, these changes increased expression of antioxidant proteins, augmenting the astrocyte’s oxidative capacity, but also oxidative damage. METH and HIV activation impaired excitatory amino acid transporter 2 (EAAT2) expression and activity, which were recovered by inhibition of TAAR1 with EPPTB, a TAAR1 selective antagonist. 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. We propose that equilibrium between agonism of neuronal TAAR1 and antagonism of astrocyte TAAR1 will need to be further investigated to balance the neuroprotective benefits of TAAR1 targeting drugs in the CNS during HIV and METH comorbidity.