Maji, Sayantan
Vishwanatha, Jamboor K.
Valapala, Mallika


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Neurodegenerative 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.