| Ca2+ fluxes across the plasma membrane and between intracellular compartments play crical roles in fundamental functions of neurons. The endoplasmic reticulum (ER) represents a dynamic Ca2+ storage in various tissues. Regulated ER Ca2+ release regulates a wide varity of neuronal functions such as protein synthesis and signal transduction. In the present study, we determine the effects of Ca2+ handing proteins on endoplasmic reticulum Ca2+ content and neuronal differentiation in mouse embryonic stem (ES) cells.The ER Ca2+ homeostasis and Ca2+ release are regulated by ryanodine receptors (RyRs), inositol 1,4,5-triphosphate receptors (IP3Rs) and sarco (endo) plasmic reticulum Ca2+-ATPase (SERCA). Inhibition of ER Ca2+ uptake by blockade of SERCA would alter this balance. In the present study, we determine the frequency and duration of spontaneous ER Ca+ transients during neuronal differentiation from mouse ES cells. And we investigated the role of SERCA activity in ER Ca2+ homeostasis and neurogenesis. Junctophilin (Jp) subtypes Jp-3 and Jp-4 are expressed specifically in brain. They are members of membrane protein family and exclusively present in excitable cells. In general, Jps consist of a C-terminal domain spanning the membrane of endoplasmatic reticulum (ER), which serves as an intracellular Ca2+ store in excitable cells, and as a long cytoplasmatic domain interacting with plasma membrane. Jps take part in enabling communication between plasma membrane of an excitable cell and intracellular ion channels.Chapter one:Role of SERCA in endoplasmic reticulum Ca2+ content and neuronal neurite development in mouse embryonic stem cellsObjective:To evaluate the ER Ca+ mobilization on the directed differentiation of mouse ES cells into neuronal cells in vitro. And evaluate whether SERCA activity is required in neurogenesis. Methods:The hanging drop method was employed for embryonic body formation to mimic embryo development in vivo.10-7mol/L retinoic acid (RA) was taken as the positive control and 0.1% DMSO as the solvent control. At the final of differentiation, morphological evaluation and immunocytochemistry staining were conduced to indentify the neurocyte derivatives. The expression of RyRs, IP3Rs and SERCA were detected by immunocytochemistry staining. Samples of ES, EB, d 8+0, d 8+5, and d 8+10 were collected. Western blot and RT-PCR were performed to investigate the expression of RyR, IP3R1 and SERCA2b during neurogenesis. The amplitudes of the intracellular Ca2+ transients were tested by Fluo 4-AM staining both in neural progenitor cells (d 8+3) and in differentiated neuronal cells (d 8+10). SERCA inhibitor CPA (cyclopiazonic acid, CPA) was used to evaluate the functional important of SERCA in neurogenesis in mouse ES cells. Western blot was performed to investigate the expression of the neurocyte-specific proteins includingβ-tubulinⅢ, nestin, NEFM and MAP2. FM1-43 staining was performed to investigate the synaptic vesicle recycling. Western blot was performed to investigate protein expression of ERK/JNK/p38 MAPK, in order to study the mechanism between intracellular Ca2+ mobilization and activity-dependent neurogenesis. Results:ER Ca2+ handing proteins (RyR, IP3R1 and SERCA2b) expressed at the early differentiation stage, and undergo developmental changes. The intracellular Ca2+ transients were detected in neural progenitor cells. The developmental regulation of the ER Ca+ transient is detected during neurogenesis. The cell treated with SERCA2b inhibitor CPA showed inhibited neuronal differentiation, and decrease in expression of neuron maker proteins. FM1-43 staining and Western blot showed long lasting loss of function of the ER Ca2+ storage could inhibit neurite outgrowth and synaptic vesicle in neurons derived from mouse ES cells. Treated with CPA, the phosphorylation of ERK1/2, JNK and p38 were both decreased. Conclusion:The developmental regulation of the ER Ca2+ transient is detected during neurogenesis. Impaired SERCA activity diminished ER Ca2+ content. Besides, SERCA acts as a powerful regulator in neurogenesis, including neurite outgrowth and synaptic vesicle. SERCA activity regulates the activity-dependent neurogenesis via activation of mitogen-activated protein kinase.Chapter two:Junctophilin-3, Junctophilin-4 regulate neuron differentiation of mouse embryonic stem cell in vitroObjective:To evaluate the inductive effects of Jp-3, Jp-4 on the directed differentiation of mouse ES cells into neurons. Methods:Knockdown Jp-3, Jp-4 by siRNA in ES cells, samples of d 8+0, d 8+2, d 8+6 and d 8+8 were collected. Expression of the pluripotency marker gene Oct-4, epiblast gene Fgf-5, mesoderm gene Brachyury, endoderm gene Afp and neuron progenitor cell gene nestin during early neuron differentiation were analyzed by semiquantitative RT-PCR. Samples of d 8+0, d 8+5, and d 8+10 were collected, expression of mitochondrial related genes (Mfh1, Mfn2, PGC1-α, NRF-1) were analyzed by semiquantitative RT-PCR. MitoTracker*Red staining was performed to detect mitochondria injury. RT-PCR was performed to evaluate secretion of neurotransmitters (VGluT1, GAD65, AchE) on ES cell derived neurons (d 8+10). Results:Differentiation towards ectoderm and neurogenesis was determined by expression of Fgf-5 and nestin. siJp-3, siJp-4 selectively decreased Fgf-5, and nestin expression during the early stage. Jp-3, Jp-4 necessary sensitive time window during neuron differentiation was demonstrated. Jp-3, Jp-4 control the differentiation of ES cells into neuron, as well as ectoderm at early stage. siJp-3, Jp-4 cells reduced the expression of peroxisome proliferator-activated receptorγcoactivator-la gene Pgc-1α, transcription factor regulating oxidative phosphorylation enzyme gene Nrf-1 and ATP-dependent mitochondrial fusion gene Mfn-1, Mfn-2. As showed by MitoTracker*Red staining, si Jp-3, siJp-4 caused mitochondrial fragmentation and reduced mitochondrial density in neuronal processes. The expression of neurotransmitters (VGluTl, GAD65, AchE) were inhibited by siJp-3, siJp-4. Conclusion:Jp-3, Jp-4 takes fundamental part in neurogenesis, mitochondrial fusion and neurotransmitter secretion. |
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