Browsing by Subject "Excitotoxicity"
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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 ISOLATION OF PRIMARY ASTROCYTES FROM HUMAN BRAIN TISSUE AND ASSESSMENT OF PROTOTYPICAL INFLAMMATORY RESPONSES FOR NEURODEGENERATIVE RESEARCH(2014-03) Borgmann, Kathleen R.; Tang, Lin; Ghorpade, AnujaPurpose (a): A common link in CNS disease is inflammation and the contribution of astrocyte inflammatory responses to neurodegeneration remains a focus of investigation. Non-human glial models may be limited in providing data that extrapolate directly to human neurodegenerative diseases, thus much remains to be learned in the genetically relevant context of primary human astroglial cultures. Methods (b): Here we describe the isolation and purification of primary human astrocytes from fetal brain in detail. We expand this protocol to include the assessment of astrocyte responses to inflammation through changes in cell morphology and expression of astrocyte specific markers, mitochondrial pore opening and activity, proinflammatory chemokine secretion and glutamate uptake. Results (c): Pure cultures were uniform in size and shape, and at least 95% positive for astrocyte markers. Mitochondrial pore staining revealed punctate calcein staining, which was decreased during inflammation. Upon treatment with a prototypical mediator of astrocyte inflammatory responses, interleukin (IL)-1beta, astrocyte processes became constricted; indicating a reactive astrocytic state, chemokine secretion increased significantly and the ability of astrocytes to clear glutamate was significantly impaired. Untreated cultures that demonstrated reactive phenotypes or those that failed to attain reactive states upon IL-1beta-treatment were excluded. Conclusions (d): These parameters established a framework to assess the overall purity, health, responsiveness to inflammation and thus the suitability of the culture for experimental use of primary human astrocyte cultures for neurodegenerative research.