Abstract
Purpose :
Astrocyte-neuron interactions are critical to retinal homeostasis. These interactions maintain the survival and normal function of neurons, as well as protect them from various neurochemical insults. In previous work, we have demonstrated that astrocytes protect neurons from metabolic stress through a direct secreted activity, both in vitro and in vivo. The current research aims to extend this work to identify protective signalling mechanisms activated by astrocyte-secreted factors in neurons, along a central PI3K mediated pathway.
Methods :
To produce the astrocyte secretome, astrocyte conditioned media (ACM) was collected from our established primary retinal astrocyte culture model. ACM rescued glutamate-induced cell death in neuronal Ht22 cells, and in primary cortical neurons. Using this in vitro model, a pharmacological screen was carried out to identify pathways, namely the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), which mediated the neuroprotection. Immunoblotting was then carried out to confirm the activation of PI3k in neurons by ACM. Once identified, cross-linked PI3K was immunoprecipitated in neurons treated with ACM and control media to identify its major interactors. ITRAQ Isobaric labeling was carried out on immunoprecipitates, after which samples were analyzed by MS/MS with subsequent bioinformatic mapping.
Results :
The pharmacological screen revealed a number of inhibitors targeting the PI3K pathway. PI3K signaling is associated with neuronal survival, growth, and metabolic activities. In validation studies, the PI3K inhibitor ZSTK474, and downsteam AKT inhibitor GSK690693 each eliminated 90% of ACM-mediated activity in both Ht22 cells and primary neurons. Western blotting showed that ACM treatment was sufficient to induce AKT phosphorylation by 30 minutes. PI3K interactome analysis identified a number of upstream and downstream targets activated in neurons following ACM exposure.
Conclusions :
Our findings demonstrate that ACM neuroprotection is mediated through the PI3K pathway. As several inducers and down-stream targets in the PI3K were identified, these findings will enable additional exploration of mechanisms to promote astrocyte secreted neuroprotective signals.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.