Browsing by Subject "excitotoxicity"
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Item Humanin Attenuates NMDA-Induced Excitotoxicity by Inhibiting ROS-dependent JNK/p38 MAPK Pathway(MDPI, 2018-09-29) Yang, Xiaorong; Zhang, Hongmei; Wu, Jinzi; Yin, Litian; Yan, Liang-Jun; Zhang, CeHumanin (HN) is a novel 24-amino acid peptide that protects neurons against N-methyl-d-aspartate (NMDA)-induced toxicity. However, the contribution of the different mitogen-activated protein kinases (MAPKs) signals to HN neuroprotection against NMDA neurotoxicity remains unclear. The present study was therefore aimed to investigate neuroprotective mechanisms of HN. We analyzed intracellular Ca(2+) levels, reactive oxygen species (ROS) production, and the MAPKs signal transduction cascade using an in vitro NMDA-mediated excitotoxicity of cortical neurons model. Results showed that: (1) HN attenuated NMDA-induced neuronal insults by increasing cell viability, decreasing lactate dehydrogenase (LDH) release, and increasing cell survival; (2) HN reversed NMDA-induced increase in intracellular calcium; (3) pretreatment by HN or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), an intracellular calcium chelator, decreased ROS generation after NMDA exposure; (4) administration of HN or N-Acetyl-l-cysteine (NAC), a ROS scavenger, inhibited NMDA-induced JNK and p38 MAPK activation. These results indicated that HN reduced intracellular elevation of Ca(2+) levels, which, in turn, inhibited ROS generation and subsequent JNK and p38 MAPK activation that are involved in promoting cell survival in NMDA-induced excitotoxicity. Therefore, the present study suggests that inhibition of ROS-dependent JNK/p38 MAPK signaling pathway serves an effective strategy for HN neuroprotection against certain neurological diseases.Item Methamphetamine Activates Trace Amine Associated Receptor 1 to Regulate Astrocyte Excitatory Amino Acid Transporter-2 via Differential CREB Phosphorylation During HIV-Associated Neurocognitive Disorders(Frontiers Media S.A., 2020-11-25) Cisneros, Irma E.; Ghorpade, Anuja; Borgmann, KathleenMethamphetamine (METH) use, referred to as methamphetamine use disorder (MUD), results in neurocognitive decline, a characteristic shared with HIV-associated neurocognitive disorders (HAND). MUD exacerbates HAND partly through glutamate dysregulation. Astrocyte excitatory amino acid transporter (EAAT)-2 is responsible for >90% of glutamate uptake from the synaptic environment and is significantly decreased with METH and HIV-1. Our previous work demonstrated astrocyte trace amine associated receptor (TAAR) 1 to be involved in EAAT-2 regulation. Astrocyte EAAT-2 is regulated at the transcriptional level by cAMP responsive element binding (CREB) protein and NF-kappaB, transcription factors activated by cAMP, calcium and IL-1beta. Second messengers, cAMP and calcium, are triggered by TAAR1 activation, which is upregulated by IL-1beta METH-mediated increases in these second messengers and signal transduction pathways have not been shown to directly decrease astrocyte EAAT-2. We propose CREB activation serves as a master regulator of EAAT-2 transcription, downstream of METH-induced TAAR1 activation. To investigate the temporal order of events culminating in CREB activation, genetically encoded calcium indicators, GCaMP6s, were used to visualize METH-induced calcium signaling in primary human astrocytes. RNA interference and pharmacological inhibitors targeting or blocking cAMP-dependent protein kinase A and calcium/calmodulin kinase II confirmed METH-induced regulation of EAAT-2 and resultant glutamate clearance. Furthermore, we investigated METH-mediated CREB phosphorylation at both serine 133 and 142, the co-activator and co-repressor forms, respectively. Overall, this work revealed METH-induced differential CREB phosphorylation is a critical regulator for EAAT-2 function and may thus serve as a mechanistic target for the attenuation of METH-induced excitotoxicity in the context of HAND.Item PROLONGED NMDA STIMULATION INDUCES NEUROPROTECTIVE PATHWAYS AND ENHANCES SURVIVABILITY OF PRIMARY RETINAL GANGLION CELLS(2014-03) Mueller, Brett H.; Park, Yong; Ma, Hai-Ying; Yorio, ThomasPurpose (a): Calcium influx through postsynaptic NMDA receptors has been shown to stimulate a number of key pro-survival genes; however, prolonged stimulation has been shown to have excitotoxic effects leading to apoptosis in neurons. Previous studies have shown a rapid dephosphorylation of CREB in primary hippocampal neurons treated for 1-2 h with100µM NMDA . It is hypothesized that the activation of CREB-specific phosphatases is one of the main pathways that cause apoptosis during NMDA excitotoxicity. The current study investigated the role of NMDA stimulation on the phosphorylation of CREB in primary RGCs, and assessed if NMDA overstimulation caused excitotoxic changes similar to those seen in primary hippocampal neurons. In addition, the occurrence of NMDA excitotoxicity in bipolar and photoreceptor cells was also investigated. Methods (b): Purification and culture of RGCs were performed by sequential immunopanning using Thy 1 antibody from P3-P7 Sprague-Dawley rats. Mixed retinal cultures that remained following isolation of RGCs from the retina were plated once the RGCs were separated and purified. Calcium imaging was used to measure the intracellular changes in calcium following treatment of cells with 100µM NMDA. Western blots were performed to determine signaling pathways linked to NMDA induced cell survival or excitotoxicity. Calcein AM and ethidium homodimer were used to quantify cell survival and cell death. Cells were also subjected to a trophic factor deprivation insult for 6 hours and 24 hours. Results (c): Treatment of primary RGCs with NMDA (100 µM) for 6h caused a greater than 2-3 fold induction of the transcription factor pCREB. MK801 (NMDA antagonist) completely abolished endogenous levels of pCREB and blocked NMDA induction of pCREB. NMDA (100 µM) treatment for 6 and 24 hrs under trophic factor deprivation, protected RGCs from trophic factor deprivation induced cellular death. The mixed retinal cultures (retinal cells without RGCs) had an opposite effect, where the levels of pCREB were diminished and the neurons died when treated with 100 µM of NMDA. Conclusions (d): The data suggests that NMDA signaling is essential for RGC survivability and blocking calcium ion influx through this receptor by the NMDA blocker, MK801 can be detrimental to RGC function and survival. These results also demonstrate that primary RGCs behave differently than other neurons in the retina, and are not susceptible to NMDA excitotoxicity.Item The Flip-Flop of AMPA Receptors in Retinal Ganglion Cell Survival(2015-12-01) Park, Yong H.; Yorio, Thomas; Clark, Abbot F.; Singh, MeharvanElevated glutamate produces a cascade of events inducing neuronal death (known as “excitotoxicity”) in which the AMPA receptor (AMPAR), one of the ionotropic glutamate receptors (iGluRs), is hypothesized to play a role in neurodegenerative diseases such as glaucoma. Overstimulation of iGluRs increases intracellular calcium, leading to cellular dysfunction, and eventually apoptotic death of neurons. The activation of AMPARs has been well demonstrated to induce cell death in vivo; however, the mechanism of AMPAR mediated excitotoxicity is not fully understood. We hypothesized that AMPAR desensitization is the determinant of excitotoxicity in retinal ganglion cells (RGCs) in vitro. In this study, we evaluated AMPARs’ dual role in RGCs in mediating both neuroprotection and excitotoxicity following AMPAR stimulation. Overstimulation of AMPARs (100μM s-AMPA) in purified RGCs was not able to induce the apoptotic pathway or produce RGC death. s-AMPA (desensitizing agonist) was able to increase RGC survival and increase the phosphorylation of cAMP response element-binding protein through the influx of Ca2+. However, RGC survival decreased when RGC cultures were stimulated with kainic acid (non-desensitizing AMPAR agonist) or when co-treated with s-AMPA and cyclothiazide (inhibits desensitization). Following an ischemic-like insult, AMPAR’s alternative spliced flip (decrease desensitization) and flop (increase desensitization) isoforms in purified RGCs were characterized, for the first time, to determine if the flip and flop isoforms play role in RGC excitotoxicity. A decrease in the mRNA expression of GLUA2 and 3 flop isoforms was observed. In conclusion, we found a dual role for AMPARs in RGCs, where these receptors can mediate both cell survival and cell death. Additionally, for the first time, the decrease in AMPAR desensitization was associated with AMPAR-mediated excitotoxicity, through the changes in the post-transcriptional modifications (alternative splicing and RNA editing of the R/G site) of the AMPAR. This new mechanism of RGC death through AMPAR desensitization gives us a better insight into the pathogenesis and new targets to combat diseases, such as glaucoma, in which neurodegeneration is a hallmark.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 HAND