P2‐038: ASTROCYTES PROTECT NEURONS FROM Aβ TOXICITY BY SECRETING TIMP‐1

Astrocyte activation is one of the major hallmarks of neurodegenerative diseases including Alzheimer's disease (AD). Astrocytes mediate Aβ induced neurotoxicity by secreting a set of harmful cytokines. However, it is believed that controlled astrocyte activation is rather beneficial to neurons and inhibition of astrocyte activation delays neuron's recovery. Tissue inhibitor of matrix metalloproteinase (TIMP-1) has been found to have neuroprotective potential. It is secreted from astrocyte and found to be increased in AD brain. But the role of TIMP-1 protein in AD is not clearly understood. So, our aim was to investigate whether TIMP-1 acts as a neuroprotective factor in AD condition. Cultured astrocyte (14DIV) were treated with Aβ for 6h,16h and 24h and activation was confirmed by immunocytochemistry using glial Fibrillary acidic protein(GFAP) and ki67 antibody respectively. Cortical neuron cultures (6DIV) were treated with astrocyte conditioned medium( ACM) exposed to Aβ for different time points. Neuronal viability and morphology were checked after 24h by direct nuclei counting and immunocytochemistry using β3-tubulin antibody respectively. Then the ACM was subjected to cytokine array profiling and TIMP-1 secretion was quantitatively measured by ELISA. Intracellular TIMP-1 level was checked by western blot, immunocytochemistry and qRT-PCR. TIMP-1 cytokine was neutralized by neutralizing antibody from 6h ACM and transferred to cortical neuron culture. Finally, cortical neuron culture was treated with rat recombinant TIMP-1 protein in presence and absence of Aβ. The neuron viability and morphology were checked by the aforementioned methods. Characteristic morphological changes and significant increase in cell proliferation indicate that astrocyte activation starts at 6h of Aβ exposure and continues up to 24h in cultured condition. The 6h ACM offered a significant protection to neurons and TIMP-1 was found to be significantly secreted at this time point. Interestingly TIMP-1 protein level was found to be increased but no significant change in mRNA transcript levels was found. The neuron health was significantly compromised when TIMP-1 was neutralized from 6h Aβ treated ACM and recombinant TIMP-1 protein showed significant neuronal survival even in presence of Aβ. Taken together, our study shows that astrocytes treated with Aβ for 6h provides protection to neurons by secreting TIMP-1 protein, at least in part.