麩胺酸受體調控大腦皮質神經元發育期間神經營養因子受體表現之分子機轉探討

1. 麩胺酸受體調控大腦皮質神經元發育期間神經營養因子受體表現之分子機轉探討麩胺酸受體調控大腦皮質神經元發育期間神經營養因子受體表現之分子機轉探討 • 神經營養因子在神經發育過程中，對神經的分化、存活等皆扮演著重要的角色。先前研究發現細胞去極化的作用，會促進神經營養因子及其受體的表現。在此，我們欲證實興奮性麩胺酸受體的活化，在神經發育期中，是否會調控神經營養因子受體的表現。大腦皮質神經元初代體外培養第五天時，我們發現麩胺酸受體亞型促效劑AMPA/KA在50μM濃度下，確實會改變細胞膜上神經生長因子受體TrkA的表現；而細胞質中TrkA的改變則無明顯差異。由此可推測，AMPA/KA造成TrkA的增加，並非由細胞質送出至細胞膜上所造成的。而利用第二型鈣離子/鈣制素依存型蛋白質激(CaMKⅡ)的抑制劑KN-93與KA的共同作用下，會降低TrkA的表現，且會造成細胞的死亡，故可知CaMKⅡ的活化可能為KA的神經保護作用所必需。我們更進一步證實，KA的作用會影響存在於細胞核中的CaMKⅡ受質環腺嘌呤核單磷酸反應物質結合蛋白(CREB)的活化。而雖然KN-93本身便會降低CREB的活化，但對KA增加CREB活化並無法抑制之。另一方面，在KA被移除後，活化態的CREB會減少，KN-93的移除則會造成其增加；而兩者合併給予，KN-93並無法改變KA被移除所造成活化態CREB減少的現象。由以上結果推測，KA藉由CaMKⅡ所造成TrkA表現增加的過程，應是透過其他轉錄因子，而非CREB的作用。然而此因子究竟為何，尚待更深入的研究以釐清。最後，神經營養因子受體活化後所啟動的下游訊息傳遞系統與KA對發育期大腦皮質神經元的保護作用之關聯性，亦於此探討之。實驗結果發現KA在PI3 Kinase抑制劑Wortmannin 40nM的作用下，會抑制其原有對神經元的保護作用，造成細胞的死亡。而MAP Kinase抑制劑PD98059在20M的濃度下，並無法影響KA的神經保護作用。總而言之，在發育時期之大腦皮質神經元中，麩胺酸受體KA的活化會藉由引發CaMKⅡ的活化而增加TrkA的表現，進而啟動下游之PI3 Kinase訊息傳遞系統，達到神經保護作用，以維持神經細胞正常的發育。

2. Molecular Mechanism of Glutamate Receptor-Mediated Neurotrophin Receptor Expressions In Developing Cortical Neurons • Neurotrophins play important roles in neuronal differentiation and survival to optimize neuronal development. It has been shown previously that expressions of neurotrophins and their receptors can be facilitated by depolarization. We herein postulated that excitatory glutamate receptors might serve as a physiological trigger to mediate neurotrophin receptor expressions in developing neurons. In the primary cultured cortical neurons at 5 days in vitro, we found that surface expression of neurotrophin receptors TrkA was significantly increased by glutamate receptor subtype agonists, kainate (KA) and α-amino-3-hydroxy-5-methyl-4-isopropionate (AMPA) at 50μM concentration. No significant changes in cytosolic TrkA expression upon each glutamate receptor agonists stimulation suggesting that the increase of surface expression is not due to cytosol-surface translocation. Furthermore, the kainate-increased TrkA expression was significantly reduced by calcium / calmodulin dependent protein kinaseⅡ (CaMKⅡ) inhibitor KN-93. KN-93 also increased neuronal death when added to the kainate-treated neurons, suggesting that neurotrophic activity of kainate is acting upon CaMKⅡ. We further examined if kainate could induce phosphorylation of cAMP response element binding protein (CREB), one of the potential targets activated by CaMKⅡ. CREB phosphorylation in nuclear fraction was significantly increased during kainate stimulation with no change of total level of CREB. However, KN-93, by itself reduced CREB phosphorylation, did not block kainate-induced CREB phosphorylation. Phospho-CREB decreased after kainate stimulation was removed, and increased after KN-93 was removed. However, removal of KN-93 plus kainate still resulted in decrease of CREB phosphorylation. These results suggest that kainate-increased TrkA expression mediated by CaMKⅡ may act upon activation of transcription factors other than CREB. Lastly, application of inhibitors of TrkA downstream mitogen-activated protein (MAP) kinase and phosphoinositol-3 (PI-3) kinase pathways to block kainate-induced neurotrophic activity possibly mediated by TrkA activation was performed. It was shown that 40nM wortmannin, a specific of PI3 kinase inhibitor, but not 20M PD98059, a specific MAP kinase inhibitor, significantly increased neuronal death when applied with kainate. In summary, activation of glutamate receptors, especially the kainate receptor, can induce TrkA expression via CaMK activation in developing cortical neurons. Increased TrkA level leads to neuronal protection via PI3 kinase pathway to survive neurons from various insults to optimize neuronal development.