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

다국어 초록 (Multilingual 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.

목차 (Table of Contents)

• Contents ⅰ
• List of Figures ⅵ
• List of Tables ⅸ
• Chapter 1. MicroRNA-24-3p regulates neuronal differentiation by controlling hippocalcin expression 1
• Preface 2
• Abstract 3
• I. Introduction 5
• II. Materials and methods 8
• 1. Materials 8
• 2. Cell culture and differentiation conditions 8
• 3. RNA interference 9
• 4. Retrovirus construction and transduction 9
• 5. Transfection of miRNA mimic and inhibitor 10
• 6. RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) 10
• 7. Western blotting assays 11
• 8. Immunofluorescence staining 12
• 9. Measurement of neurite outgrowth 12
• 10. Plasmid construction and dual-luciferase reporter assays 13
• 11. TaqMan RT-qPCR of miRNA 14
• 12. Construction of small hairpin RNA (shRNA)-expressing vectors and lentivirus production 14
• 13. Mice 15
• 14. Stereotaxic surgery 16
• 15. Behavioral studies 16
• 16. Locomotor activity test (LMA) 17
• 17. Contextual fear memory 17
• 18. Novelty suppressed feeding test (NSFT) 18
• 19. Forced swim test (FST) 18
• 20. Dissection of hippocampal dentate gyrus 18
• 21. Immunohistochemistry 19
• 22. Statistical analysis 19
• III. Results 20
• 1. HPCA was up-regulated during the neuronal differentiation of SH-SY5Y cells 20
• 2. HPCA regulated the differentiation of SH-SY5Y cells 20
• 3. Knockdown of HPCA in the hippocampal DG inhibited hippocampal neurogenesis 21
• 4. HPCA deficiency in the hippocampal DG induced manic-like behaviors in mice 22
• 5. miRNA-24-3p targeted endogenous HPCA expression 22
• 6. miR-24-3p modulated neuronal differentiation by inhibiting HPCA in SH-SY5Y cells 24
• IV. Discussion 55
• V. References 58
• VI. Abstract in Korean 65
• Chapter 2. Phospholipase D1 promotes astrocytic differentiation through FAK/AURKA/STAT3 signaling pathway in hippocampal neural stem/progenitor cells 67
• Preface 68
• Abstract 69
• I. Introduction 70
• II. Materials and methods 73
• 1. Materials 73
• 2. Primary culture of hippocampal neural precursor cells 74
• 3. Construction of small hairpin RNA (shRNA)-expressing vectors and lentivirus production 75
• 4. Transient transfection of hippocampal NSPCs 75
• 5. RNA interference 76
• 6. Real-time PCR and RT (reverse transcription)-PCR 77
• 7. Western blot analysis 77
• 8. Immunofluorescence staining 78
• 9. Cell counting and analysis 79
• 10. Statistical analysis 80
• III. Results 81
• 1. PLD1 increases expression of GFAP in hippocampal NPCs 81
• 2. PLD1 deficiency suppresses the astrocytic differentiation of hippocampal NSPCs 82
• 3. PA, a PLD1 product, itself promotes astrocytic differentiation 82
• 4. STAT3 activation is involved in PA-mediated expression of GFAP in hippocampal NPCs 83
• 5. AURKA is required for PA-induced STAT3 activation and astrocytic differentiation 84
• 6. Effects of FAK on PA-induced AURKA/STAT3 activity and astrocytic differentiation 85
• IV. Discussion 109
• V. References 112
• VI. Abstract in Korean 118