Browsing by Subject "Glutamate"
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Item CELL SURFACE TRANSLOCATION OF ANNEXIN A2 FACILITATES GLUTAMATE-INDUCED EXTRACELLULAR PROTEOLYSIS(2014-03) Maji, Sayantan; Vishwanatha, Jamboor K.; Valapala, MallikaNeurodegenerative diseases like age related macular degeneration (AMD) and Retinitis Pigmentosa (RP) are major causes of blindness affecting millions of people around the world. One of the major reasons of cell death observed in these diseases is the increased accumulation of glutamate, an excitatory amino acid. Unfortunately, the mechanisms behind glutamate induced toxicity are not yet known. Here we are investigating a possible role of a protein Annexin A2 (AnxA2) in glutamate induced toxicity. We found that glutamate causes increased membrane translocation of AnxA2. Increased membrane localization of AnxA2 and thereby its function can lead to the death of the eye cells leading to degenerative diseases like AMD and RP. The present study shows one of the possible mechanisms that can lead to glutamate induced cell death of the eye. Thus, using AnxA2 targeted therapy as an adjunctive therapy can lead to better and more efficient outcomes. Purpose (a): Glutamate-induced intracellular increase in Ca2+ levels leads to the hyper-activation of several normal Ca2+-mediated physiological processes including the activation of intracellular kinases, phosphatases, phospholipases and proteases which contribute to the degeneration of the retinal neurons as seen in many diseases including age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Despite intensive research, the mechanisms that contribute to glutamate-induced cellular loss are yet to be elucidated. AnxA2, a Ca2+-dependent phospholipid binding protein serves as an extracellular proteolytic center by recruiting tissue plasminogen activator and plasminogen, and mediating localized generation of plasmin. We investigated whether AnxA2 plays a major role in glutamate induced neuronal excitotoxicity in a cone-photoreceptor cell line, 661W. Understanding the molecular mechanisms of glutamate-induced retinal degeneration can lead to the development of better therapeutic approaches for neurodegenerative diseases including AMD and RP. Our study provides new insights into one of the mechanisms that might contribute to glutamate-induced loss of photoreceptors in the retina. Methods (b): Ratiometric Ca2+ imaging and time lapse confocal microscopy were used to study glutamate-induced Ca2+ influx. EDTA eluates of 661W cells were immunoblotted to study the membrane translocation of endogenous as well as AnxA2-GFP in the presence or absence of different treatments. To determine whether glutamate induced membrane translocation of AnxA2 is dependent on the phosphorylation of the 23rd tyrosine residue or not, phosphomimetic and non-phosphomimetic variants were studied. Results (c): Glutamate translocated both endogenous and AnxA2-GFP to the cell surface in a process dependent on the activity of the NMDA receptor. Glutamate-induced translocation of AnxA2 is dependent on the phosphorylation of tyrosine 23 at the N-terminus and mutation of tyrosine 23 to a non-phosphomimetic variant inhibits the translocation process. The cell surface translocated AnxA2 forms an active plasmin-generating complex and this activity can be neutralized by a hexapeptide directed against the N-terminus. Conclusions (d): These results suggest an involvement of AnxA2 in potentiating glutamate-induced cell death processes. Thereby, targeting AnxA2 can be used as an adjunctive therapy in neurodegenerative diseases like AMD and RP.Item HIV-1 TAT EXPRESSION ALTERS HUMAN ASTROCYTE INFLAMMATORY BIOMARKER PROFILES AND GLUTAMATE METABOLISM(2014-03) Joshi, Chaitanya R.; Ghorpade, AnujaPurpose (a): More than 50% of the human immunodeficiency virus type 1 (HIV-1) infected individuals exhibit some form of HIV-associated neurocognitive disorders (HAND). Several studies reported that HIV-1 transactivator of transcription (Tat) protein was associated with HAND pathophysiology. HIV-1 Tat induces apoptosis and dysregulates cytokine/chemokine profiles leading to neurotoxicity. Previous studies have studied the in vivo HIV-1 Tat regulation using transgenic animal models. Although animal models have helped determine the in vivo disease pathology, application of in vitro neural cell models will be critical to decipher cellular and molecular mechanisms associated with HAND. Here, we report an in vitro model system developed by transfecting human astrocytes with a full-length (101 AA) HIV-1 Tat protein expressing plasmid (pTat). HIV-1 Tat expressing in vitro system was used to evaluate HIV-1 Tat regulation of astrocyte inflammatory responses and altered neuroprotective function i.e. glutamate uptake from synapse. Methods (b): Primary human astrocytes were transfected with pTat by nucleofection and HIV-1 Tat expression was evaluated by immunocytochemistry. Effects of HIV-1 Tat on cell viability and replication were determined with metabolic activity and cell proliferation assays. Proinflammatory cytokines and chemokines were assayed using ELISAs. HIV-1 Tat regulation of glutamate clearing ability of astrocytes was determined using a modified amplex red glutamic acid/glutamate oxidase assay. Additionally, mRNA and protein expression of excitatory amino acid transporter-2 (EAAT-2), the major glutamate transporter in astrocytes, was measured by RT-PCR and western blot analysis respectively. Results (c): The immunostaining confirmed HIV-1 Tat expression in transfected astrocytes, while glial fibrillary acidic protein (GFAP) staining indicated morphological alterations. The pTat transfection did not significantly change cell metabolism as compared to controls. However, HIV-1 Tat expression altered chemokine and cytokine levels; specifically HIV-1 Tat increased CCL2 levels significantly (P. Conclusions (d): HIV-1 Tat expression upregulated inflammatory biomarkers and altered glutamate clearing ability of astrocytes, implicating a direct role for astrocyte-expressed HIV-1 Tat in HAND neuropathogenesis.