Mechanism Of Orai1on Differentiation Of PC12Cells And Rat Cortical Neurons

In most excitable cells, depletion of ER stores elicits sustained Ca²⁺influx bystore-operated Ca²⁺（SOC） entry, defining the important Ca²⁺influx pathway.Store-operated Ca²⁺influx is essential for maintaining ER Ca²⁺content at a preciselevel and functions in various physiological processes such as neuronal differentiationand apoptosis.Orai1, the pore subunit of the Ca²⁺release-activated Ca²⁺（CRAC）channel, and resulting in refilling of ER stores is prolifically expressed in the brain.Previous studies demonstrated that an increase in [Ca²⁺]ininvolving store-operatedCa²⁺entry （SOCE） channels induced neuronal differentiation, but the mechanismsthat initiate its actions are poorly understood. Orai1is prolifically expressed in thebrain and has variously physiological roles such as the composition of store-operatedchannel （SOC）, a scaffold or a receptor for various ligands. In present study, theeffects of Orai1proteins were investigated on neurite outgrowth of PC12cells andinvolvement NGF-induced Rap1-dependent signal pathway. Knockdown of Orai1viashRNA inhibited PC12cells differentiation as well as primary culture of corticalneurons, whereas over expression Orai1had no effect. Interestingly, PKC inhibitorGF109203X reduced Tg-stimulated Ca²⁺influx. Furthermore, GF109203X inhibited outgrowth turning and decreased the numbers of dendritic spines in primary corticalneurons as well as knockdown of Orai1. Based on these findings, Orai1can beinvolved in the physiological roles of Orai1may not be restricted to its participationin SOCE. We conclude that Orai1may acts as a scaffold at the cell surface toassemble a signaling complex to regulate neurite outgrowth for NGF-inducedRap1-dependent signal pathway.1. Effect of Orai1knock-down on Ca²⁺Influx，augmented by treatment ofGF103203xThe level of Ca²⁺influx was examined under with or without NGF-inducedconditions. Knockdown of endogenous Orai1using shOrai1decreased Tg-stimulatedCa²⁺influx as compared with the control. Notably, knockdown Orai1or treatmentwith GFx significantly decreased Tg-stimulated Ca²⁺influx （Fig.1） as well asknockdown Orai1.2. Involvement of Orai1knock-down in the NGF-induced neurite outgrowth,GF103203x augmented the effects of downregulated Orai1which decreased thelevels of Rap1-GTP and pMAPK is also involved in neurite outgrowth of PC12cells induced by NGFTo test the role of Ca²⁺influx in NGF-induced differentiation, we manipulatedthe levels of the SOCE regulatory protein Orai1in PC12cells. Cells were seededsparsely and the full length of the neurites was observed by YFP fuorescence. Fig.2shows representative fuorescence images of each condition. Orai1knock-down ortreatment with GFx reduced neurite outgrowth, which showed shorter neurites incomparison with mock-transfected cells. Here, we evaluated GTP-Rap1and pMAPKlevels up to48h in PC12cells in response to NGF. GTP-Rap1and pMAPK levelswere augmented by stimulated with50ng/ml NGF in PC12cells after48h （Figure3upper panel）. While Orai1knock-down and treatment with GFx decreased the levelsof GTP-Rap1and pMAPK in response to NGF.（Figure3lower panel） This suggests that Rap1is essential for neurite outgrowth.3. Knockdown of Orai1inhibits growth cone turning of neurons, Decreasedendogenous Orai1reduce cortical neuron spine densityTo test the chemotropic responses, we exposed growth cones of isolated corticalneurons in14-20hr cultures to gradients of NGF or GF103203x as they turned inresponse to the guidance cues（Fig.4）. We compared growth cone extensions.ShOrai1growth cones exhibited no apparent bias in the direction of extention,butshcontrolshowed a marked chemotropic turning response toword the source of NGF.To examine the effects of decreased Orai1levels on dendritic spine structure, weimaged the morphology in the scanning electron microscope. When compared withcontrol cultures, knockdown Orai1cultures often displayed swelling of dendrites,axons and cell bodies. The number of labeled spines varied considerably in sectionsfrom control cells and from cells in knockdown Orai1cultures, depending on theplane of the section through the labeled dendrites. As demonstrated in Fig.5, thenumber of spines per one cell was significantly decreased in the cells fromknockdown Orai1（Fig.5A） via shRNA, compared withcontrol cells （Fig.5B）.