Crucial Role Of Iunctophilin3 And 4 In Early Neuronal Differentiation Of Embryonic Stem Cells

Junctophilins ?JPs? exist in excitable cells and contribute to forming junctional membrane complexes ?JMC? by interacting with the plasma membrane ?PM? and spanning the endoplasmic/sarcoplasmic reticulum ?ER/SR? membrane. These complexes allow for functional crosstalk between Ca²⁺ channels at the PM and the ER/SR, thus regulating intracellular Ca²⁺homeostasis and functions of excitable cells. JP3 and JP4 are predominantly expressed in brain. Mice lacking both JP3 and JP4 ?JP-DKO mice? demonstrate severe growth retardation and lethality 3-4 weeks after birth under normal housing conditions. These studies implied crucial role of JP3,4 for neuronal development. Moreover, trinucleotide repeat expansion in the JP3 gene has been reported to associate with Huntington disease-like 2 ?HDL2?. Although these findings unveil a novel role of JP3 and JP4 for brain functions, whether JP3,4 involve in the establishment of neural system during neuronal development still remain unclear.Undertaker ?UTA?, as a Drosophila Junctophilin, is reported to be required in phagocytosis of apoptotic cells by keeping Ca²⁺ homeostasis during Drosophila embryogenesis. Large amount of cells undergo apoptosis during neuronal development. Cues including'find me'and'eat me'signals exposed by dying cells trigger phagocytes rapidly clearing corpses to ensure normal development. Non-engulfed apoptotic cells typically undergo secondary necrosis, which results in toxicity and increases risk for autoimmune disease later in life. However, whether JP3 and JP4 play the same roles during mammalian neuronal development has not yet been elucidated. In addition to strict and appropriate micro-environment, neuronal polarization and axon outgrowth also need extracellular molecular signaling. It has been known that cell proliferation, cell growth and death are highly coordinated and tightly controlled during development. Activation of apoptosis can trigger compensatory proliferation in neighboring cells to maintain tissue homeostasis. However, whether apoptotic cells can exert positive influence on neurite outgrowth has been little known before. Besides, the underlying functions of JP3,4 in linking apoptosis to neuronal polarization remain elusive.The initial event in establishing a polarized neuron is the specification of a single axon. Early in neuronal development, competition among undifferentiated neurites results in the growth of one neurite at the expense of the rest. Neuronal polarization and neurite outgrowth require localized calcium signal and cAMP. Studies showing that calcium influx through a transient receptor potential ?TRP? channel initiated axon specification in hippocampal neurons by signaling through calcium-and calmodulin-dependent protein kinase I ?CaMKI?. Moreover, calcium signal could interact with cAMP pathway to form a positive feedback and amplify cAMP signal. Local neurite with high cAMP concentrations reinforced its own cAMP signaling through positive feedback and grew extensively to become the axon, while the other neurites became dendrites. Even though calcium and cAMP widely involve in neuronal polarization and neurite outgrowth, it is still not fully understood that how they are strictly and precisely localized and regulated during early neuronal development. Moreover, wherher the JP3,4 mediated calcium oscillasion involves in neuronal polarization remains unclear.During early phase of development, neurons form excess axon branches, dendritic arbors and synapses. In the later phase, unnecessary neural processed are selectively removed without loss of the other parts of the axons, dendrites and parental neurons to refine functional neural circuit. The selective elimination of synapses and neural processes is also required for remodelling of synaptic connectivity. The engulfment receptor Megf10 was reported to be essential for pruning central nervous system synapses. Developing mice deficient in both Megf10 and MERTK pathways fail to refine the retinogeniculate connections normally and retain excess functional synapses. Drosophila Draper, an orthologue of Megf10, was found to coordinate with UTA to engulf corpses. Thus, whether JP3,4 could involve in axon pruning remain to be elucidated.1. Junctophilin-3,-4 linked phagocytosis played crucial roles in neuronal polarization.To assess the potential role of JP3 and JP4 during neuronal differentiation, a typical 4-/4+protocol was applied to imitate the neuronal development in vivo. In our present studies, we found that both JP3 and JP4 are crucial for neuronal polarization and neurite outgrowth. JP3,4 contributed to maintaining cAMP/Ca²⁺ positive feedback to sustain local neuronal polarization. Early knockdown of JP3,4 severely not only disrupted calcium transient within neural precursors but also decreased intracellular cAMP level and inhibited cAMP-LKB1-SAD/MARK2 pathway. These results implied an important role of JP3,4 in maintaining cAMP/Ca²⁺ positive feedback during neuronal polarization. Further studies revealed that JP3,4 also involved in engulfment pathway to ensure appropriate neurite growth micro-environment. Knockdown of JP3,4 lead to severe inhibition of corpse removal. Moreover, we found essential roles of JP3,4 in connecting apoptosis to neuronal polarization during neuronal development. Apoptotic cells stimulation elevated the expression of JP4 and Megf10. Meanwhile, the interaction between JP3 and RyR2,3 or JP4 and Megf10 were significantly enhanced after apoptotic cell treatment. Notably, we found apoptotic cells existed communication with neural precursors not only by attracting migration and phagocytosis, but also by promoting neuronal polarization and axon growth. Collectively, these results unveil critical roles of JP3,4 in regulating neuronal polarization and neurite outgrowth during neuronal development.2. Involvement of JP3,4 in axon pruning during neuronal differentiation.The role of JP3,4 in axon pruning during neuronal differentiation was investigated in this section. Knockdown of Megf10 by lentivirus transfection lead to downregulation of JP3 and upregulation of JP4. Inhibition of JP3 increased expression of Megf10, while knockdown of JP4 had no obvious influence on Megf10. These results argue that there is molecular regulation between Megf10 and JP3,4. Following studies showed that apoptotic stimulation on d 8+5 could also elevated expression of JP3,4 and Megf10. The results of immunofluorescence and electron micrographs indicated that phagocytes hold more efficient phagocytotic ability with two or more corpses engulfed simultaneously. The ?-tubulin? positive fragment was found engulfed by phagocytes expressed Megf10 on d 8+5. The 3D reconstruction of JP4 and ?-tubulin? couterstain cells showed that ?-tubulin? positive component was enclosed by JP4. Further study indicated that it was Nestin positive cells, not neurons that executed the axon pruning, which prompt that glial precursors may take part in neural process elimination.Conclusion:1. Our studies unveiled crucial role of JP3,4 in neuronal polarization. JP3,4 maintained intracellular calcium homeostasis and involved in phagocytosis to ensure neuronal polarization. Apoptotic cells could not only attract phagocyte, but also promote neuronal polarization. Moreover, JP3,4 could sense the signal released from apoptotic cells, and simultaneously regulate cAMP signals for neuronal polarization. These findings extended the notion that JP3,4 can link phagocytosis to regulate neuronal polarization, suggesting a novel role of JP3,4 in neuronal development.2. JP3,4 also involved in axon pruning during neuronal development. JP3,4 and Megf10 not only interacted with each other, but also had molecular regulation. JP4 and Megf10 took a role in axon pruning. These findings partly revealed that essential role of JP3,4 in precise circuit formation.