Abstract
Purpose :
Astrocytes can support neuronal homeostasis with a variety of secreted signals. Thus, analysis of the astrocyte secretome provides valuable insight into these protective functions. Previously, we characterized a potent neuroprotective effect mediated by astrocyte conditioned media (ACM) in neurons and the retina. Here, we identify phosphoinositide 3-kinase (PI3k) as a key signaling axis for transducing ACM mediated neuroprotection from metabolic stress. To characterize the associated signaling cascade we performed a PI3k interactome analysis for changes in neuronal protein-protein interactions following ACM exposure.
Methods :
To produce the astrocyte secretome ACM was collected from our established primary retinal culture model. ACM reduced metabolic cell death in neuronal Ht22 cells, and in primary neurons. Using this model, a chemical genetics screen identified the PI3k pathway as a central mediator for ACM neuroprotection. Endogenous PI3k was then cross-linked and immunoprecipitated in neurons treated with ACM vs 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. Resulting hits were were then validated through follow-up biochemical and functional studies.
Results :
The chemical screen revealed that seven of the top ten hits directly targeted the PI3K pathway. In validation studies, selective PI3K inhibitors significantly blocked ACM-mediated activity, while ACM treatment rapidly induced downstream AKT phosphorylation. Remarkably, following careful bioinformatic analyses, only five significantly ACM-induced PI3k interactors were identified, with the top hit being the zinc finger RNA binding protein ZC3H14. Subsequent studies validated the PI3k-dependent recruitment of ZC3H14 biochemically, and ZC3H14 knockdown demonstrated that this interaction is necessary for ACM mediated neuroprotection.
Conclusions :
Our findings demonstrate that ACM neuroprotection is mediated through the PI3K pathway, and identified the novel interactor ZC3H14. These results advance our understanding of the signaling pathways mediating astrocyte neuroprotective activities.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.