Molecular mechanisms of miR-24-3p-mediated neuronal differentiation and PLD1-induced astrocytic differentiation

Abstract

Chapter 1. Hippocalcin (HPCA) is a neuron-specific calcium-binding protein predominantly expressed in the nervous system. In the present study, we demonstrate that HPCA regulates neuronal differentiation in SH-SY5Y cells. We observed that the expression level of HPCA was increased during neuronal differentiation. Depletion of HPCA inhibited both neurite outgrowth and synaptophysin (SYP) expression, whereas overexpression of HPCA induced neuronal differentiation. Hippocampal deficiency of HPCA exhibited manic-like behavior, including hyperactivity, decreased anxiety-like behavior, reduced depressive-related behavior, and impaired learning and memory. Furthermore, HPCA depletion reduced the levels of synaptic plasticity-related proteins. Thus, HPCA regulates neuronal differentiation both in vitro and in vivo. Interestingly, we also found that the expression of HPCA mRNA was modulated by miR-24-3p. Using a dual-luciferase assay, we showed that co-transfection of a plasmid containing the miR-24-3p binding site from the 3'-untranslated region (3'UTR) of the HPCA gene and an miR-24-3p mimic effectively reduced luminescence activity. The effect was abolished when miR-24-3p seed sequences in the 3'UTR of the HPCA gene were mutated. miR-24-3p expression was decreased during neuronal differentiation of SH-SY5Y cells, suggesting that the decreased expression level of miR-24-3p might have upregulated mRNA expression of HPCA. In line with this, upregulation of miR-24-3p by an miRNA mimic led to a decrease in HPCA expression, accompanied by diminished neuronal differentiation. In contrast, downregulation of miR-24-3p by an antisense inhibitor promoted neurite outgrowth as well as levels of SYP expression. Taken together, these results suggest that miR-24-3p is an important miRNA that regulates neuronal differentiation by controlling HPCA expression.

Chapter 2. Phospholipase D1 (PLD1) plays a crucial role in cell differentiation of different cell types. However, the involvement of PLD1 in astrocytic differentiation remains unknown. In the present study, we investigated the possible role of PLD1 and its product phosphatidic acid (PA) in astrocytic differentiation of hippocampal neural stem/progenitor cells (NSPCs) from hippocampi of E16.5 rat embryos. We showed that overexpression of PLD1 increased expression level of glial fibrillary acidic protein (GFAP), an astrocyte marker. Depletion of PLD1 by transfection with Pld1 shRNA inhibited expression of GFAP. Moreover, PA itself was sufficient to promote astrocytic differentiation. These results suggest that PLD1 plays a critical role in regulating astrocytic differentiation of hippocampal NSPCs. Treatment with PA increased the phosphorylation of Signal transducer and activator of transcription 3 (STAT3) at tyrosine 705, suggesting that STAT3 activation is involved in PA-mediated astrocytic differentiation. When STAT3 activity was blocked by Stat3 siRNA, PA-induced GFAP expression was decreased. PA-induced STAT3 activation was regulated by Focal Adhesion Kinase (FAK)/Aurora Kinase A (AURKA) pathway. As expected, treatment with a specific FAK inhibitor (PF-573228) or an AURKA inhibitor (AurAi) suppressed the PA-induced STAT3 activation. Furthermore, PA-induced astrocytic differentiation was blocked by PF-573228 as well as AurAi. Taken together, these results suggest that PLD1 is an important regulator that regulates astrocytic differentiation through the FAK/AURKA/STAT3 signaling pathway in hippocampal NSPCs.