An endogenous PI3K interactome promoting astrocyte-mediated neuroprotection identifies a novel association with RNA-binding protein ZC3H14

Abstract

Astrocytes can support neuronal survival through a range of secreted signals that protect against neurotoxicity, oxidative stress, and apoptotic cascades. Thus, analyzing the effects of the astrocyte secretome may provide valuable insight into these neuroprotective mechanisms. Previously, we characterized a potent neuroprotective activity mediated by retinal astrocyte conditioned media (ACM) on retinal and cortical neurons in metabolic stress models. However, the molecular mechanism underlying this complex activity in neuronal cells has remained unclear. Here, a chemical genetics screen of kinase inhibitors revealed phosphoinositide 3-kinase (PI3K) as a central player transducing ACM-mediated neuroprotection. To identify additional proteins contributing to the protective cascade, endogenous PI3K was immunoprecipitated from neuronal cells exposed to ACM or control media, followed by MS/MS proteomic analyses. These data pointed toward a relatively small number of proteins that co-immunoprecipitated with PI3K, and surprisingly only five were regulated by the ACM signal. These hits included expected PI3K interactors, such as the platelet-derived growth factor receptor A (PDGFRA), as well as novel RNA binding protein (RBP) interactors ZC3H14 (zinc finger CCCH-type containing 14) and THOC1 (THO complex protein 1). In particular, ZC3H14 has recently emerged as an important RBP with multiple roles in post-transcriptional regulation. In validation studies, we show that PI3K recruitment of ZC3H14 is necessary for PDGF-induced neuroprotection, and that this interaction is present in primary retinal ganglion cells. Thus, we identified a novel non-cell autonomous neuroprotective signaling cascade mediated through PI3K that requires recruitment of ZC3H14 and may present a promising strategy to promote astrocyte-secreted pro-survival signals.

A Stereocontrolled Total Synthesis of Lipoxin B4 and its Biological Activity as a Pro‐Resolving Lipid Mediator of Neuroinflammation

“…We previously reported that in addition to their known pro‐resolution functions, lipoxins demonstrate an exciting novel protective bioactivity directly on neurons [14] . In particular, LXB 4 was surprisingly 20‐fold more potent than LXA 4 in an established neuronal injury model, [14,39] and also active in a variety of primary neurons, including cortical and hippocampal cells, and retinal ganglion cells. These broad neuroprotective effects were subsequently correlated with demonstrated LXB 4 efficacy in vivo using both acute and chronic models of retinal ganglion cell death associated with the common neurodegenerative disease glaucoma, a leading cause of vision loss and blindness worldwide [40–42] .…”

Evidence of an Annexin A4 mediated plasma membrane repair response to biomechanical strain associated with glaucoma pathogenesis

“…For western blot analysis, cell and tissue protein was collected in RIPA lysis buffer containing protease and phosphatase inhibitors, homogenized, and run on a 12% sodium dodecyl sulfate polyacrylamide gel according to standard practices. The protein was then transferred onto a polyvinylidene difluoride membrane and blocked with 5% bovine serum albumin as previously described (Alqawlaq et al, 2020). Membranes were incubated with primary antibodies raised against ANXA4 (1:2000 dilution; Sigma) and glyceraldehyde 3‐phosphate dehydrogenase (1:10,000 dilution; Calbiochem).…”

“…Yet, the cellular injury and mechanosensing mechanisms linking this strain to cellular damage are critically unclear, and remain a key research question. The resulting chronic strain injury induces a cascade of pathological effects that prominently include cytoskeletal remodeling and para‐inflammatory activation of ONH astrocytes (Almasieh et al, 2012; Alqawlaq et al, 2019, 2020; Livne‐Bar et al, 2016), which are crucial for providing structural and homeostatic support to retinal ganglion cell (RGC) axons as they exit the eye to form the optic nerve (Alqawlaq et al, 2020; Guo et al, 2018; Livne‐Bar et al, 2017).…”

Protective Effects of Lipoxin A4and B4Signaling on the Inner Retina in a Mouse Model of Experimental Glaucoma

“…[7][8][9][10] We and others have uncovered increasing evidence that astrocytes can also play essential roles in maintaining and promoting neuronal homeostasis. [11][12][13][14][15][16] A recent study by our groups indicated that the levels of astrocyte-derived lipoxins A 4 (LXA 4 ) and B 4 (LXB 4 ) in the retina and optic nerve decreased following acute excitotoxic injury. 17 Lipoxins are members of the specialized pro-resolving mediator (SPM) family of polyunsaturated fatty acid metabolites, derived from arachidonic acid.…”

“…7–10 We and others have uncovered increasing evidence that astrocytes can also play essential roles in maintaining and promoting neuronal homeostasis. 11–16…”