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Samih Alqawlaq, Izzy Livne-Bar, Darren Chan, Jeremy M Sivak; Retinal astrocytes protect neurons against metabolic stress by inducing the PI3K pathway. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4213.
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© ARVO (1962-2015); The Authors (2016-present)
Maintenance of retinal function heavily relies on the support roles of astrocytes, which carry out major homeostatic roles to protect neurons from oxidative and metabolic stresses. The purpose of the current research is to identify candidate astrocyte-secreted factors involved in regulating metabolic stress in relevant neuronal cell models.
Astrocyte conditioned media (ACM) was collected from cultures of primary retinal astrocytes, previously described and characterized by our group. ACM significantly reduces metabolic stress induced cell death in the immortalized neural Ht22 cell line, and in primary cortical neurons. Using this model as a screening platform, two directions were pursued: 1) A quantitative antibody (Ab) array to compare enrichment of known neuroprotective factors in ACM, compared to control media. 2) A kinase inhibitor (KI) library screen of over 400 tool compounds, using the Ht22 cell model as a platform for ACM neuroprotection assessment. This screen was followed by pathway validation via western blotting and validation of select KIs in primary cortical neurons.
The Ab array showed enrichment of several growth factors in ACM, several of which are known to bind to receptor tyrosine kinases (RTKs), including basic FGF (3-fold enrichment), and NGF (2-fold enrichment). Notably, ACM was not significantly enriched for BDNF or CNTF. In complementary studies the KI screen prominently identified several inhibitors targeting the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) pathway. Each inhibitor hit reduced ACM-mediated neuroprotection, without causing any direct neurotoxicity. For example; the PI3K inhibitor ZSTK474, and the AKT inhibitor GSK690693 each eliminated approximately 90% of ACM-mediated effect in both Ht22 cells and primary neurons. Western blotting of Ht22 cells showed that ACM treatment induced AKT phosphorylation by 30 mins, indicating PI3K activation.
Neuroprotection of ACM against metabolic stress was established as a screening platform in HT22 cells and primary neurons, allowing high throughput screening of pathway targets. Observed neuroprotection is mediated through the PI3K pathway, and several potential corresponding pathway inducers were identified in ACM. These findings will enable additional testing and validation of candidate pathways and targets to mediate astrocyte secreted neuroprotective signals.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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