Elucidating the molecular mechanisms of acute reelin signaling in regulating the proteome and actin cytoskeleton during neurodevelopment
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Abstract
Proper neurodevelopment requires that neurons attain both the correct position in the brain and appropriate morphology to facilitate neuronal communication and information transmission. Dysfunction of these developmental events often leads to severe neurodevelopmental and neuropsychiatric disorders. Reelin signaling is required for numerous aspects of neurodevelopment. Reelin is a secreted glycoprotein that acts at both acute and chronic time scales to regulate proper lamination of the neocortex, neurite outgrowth and arborization, and synaptic plasticity. Canonically, Reelin signaling is initiated by Reelin binding to two lipoprotein receptors, apolipoprotein E receptor 2 (APOER2) and very low-density lipoprotein receptor (VLDLR), which results in tyrosine phosphorylation of the intracellular adaptor protein, disabled 1 (Dab1) by Src family kinases (SFK). Additionally, Reelin signaling can occur through the non-canonical signaling pathway which utilizes non-lipoprotein receptors. Due to the multi-faceted nature of Reelin signaling, many of the downstream events and molecular mechanisms underlying Reelin-mediated neurite outgrowth have yet to be elucidated. Additionally, the differential downstream effects of Reelin signaling via canonical and non-canonical receptors are not yet well understood. Therefore, understanding the molecular mechanisms underpinning Reelin regulated neurite outgrowth and arborization, and the differential function of Reelin signaling through canonical and non-canonical signaling in development will provide valuable information into proper neurodevelopment. We conducted an unbiased proteomics screen of primary murine neurons at 7 days in vitro (DIV7) after acute Reelin stimulation using tandem mass tag (TMT) LC-MS/MS and observed distinct changes in the neuronal proteome and phospho-proteome downstream of the canonical and non-canonical Reelin signaling pathways. Canonical Reelin signaling had a major impact on the regulation of the neuronal cytoskeleton, with an emphasis on actin-filament based processes, whereas non-canonical signaling regulated protein translation and mRNA metabolism in addition to cytoskeletal dynamics. Additionally, we identified a novel downstream effector in the Reelin signaling pathway: the glycolytic enzyme and actin binding protein, aldolase A. Dynamic regulation of the actin cytoskeleton is an essential component of cell motility and growth. We demonstrate that acute Reelin signaling leads to depolymerization of F-actin in immature neurites and increased activity of the actin severing protein, cofilin, through reduced phosphorylation at serine 3. In concordance with our proteomics data, we show that Reelin signaling led to increased de novo translation of aldolase A as well as the dissociation of existing aldolase A from the actin cytoskeleton, suggesting aldolase A functions to stabilize the F-actin cytoskeleton. In addition, shRNA knockdown of aldolase A ablated Reelin-mediated neurite growth in vitro and led to reduced neuronal arborization and perturbed neuronal positioning in the mouse neocortex. Lentiviral rescue in primary murine neurons with actin binding mutant aldolase A (R42A) and catalytically dead mutant aldolase A (D33S) demonstrated these effects are dependent on actin binding and independent of aldolase A’s glycolytic function. Overall, our data provides detailed information into important proteomic changes that occur downstream of canonical and non-canonical Reelin signaling and how these pathways differentially contribute to neurodevelopment. We also provide strong evidence demonstrating the importance of aldolase A in regulating Reelin-mediated neurite arborization.
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2024