興奮性胺基酸受體在發育期大腦皮質神經元之神經營養功能研究

1. 興奮性胺基酸受體在發育期大腦皮質神經元之神經營養功能研究興奮性胺基酸受體在發育期大腦皮質神經元之神經營養功能研究 • 本論文主旨在研究興奮性胺基酸受體在腦神經發育過程中之神經營養功能。我們以懷孕十六至十八天大白鼠胚胎的大腦皮質 (cerebral cortex) 神經元，經體外培養四至六天(4-6 days in vitro)，投予紅藻胺酸 (Kainic acid，簡稱KA)，一種作用於興奮性胺基酸受體中 AMPA (alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid) /KA受體亞型的催動劑，也是一種造成癲癇的神經毒素，以探討發育期大腦皮質神經元在神經保護功能 (neuroprotection) 與神經毒性 (neurotoxicity)上的調節與平衡。在利用乳酸脫氫脢釋放 (lactate dehydrogenase release assay)測定細胞死亡程度的實驗中發現，紅藻胺酸的神經毒性在發育期是偏低的。以500uM 紅藻胺酸作5?15分鐘短暫刺激後16到24小時所造成之神經細胞死亡並不顯著，但若預先投予酪胺酸磷酸化脢抑制劑 (tyrosine kinaseinhibitor) 10 uM genistein，100 nM K-252a 與 10 uM AG-879，在對細胞不具毒性的濃度下，可以相當顯著的提高紅藻胺酸對神經元所造成的細胞毒性。由此可見，紅藻胺酸藉由受體的活化可以引發酪胺酸磷酸化 (tyrosine phosphorylation)，並與細胞存活有關。此外，當以抗神經生長因子之抗體 (anti-NGF) (4 ug/ml)中和其活性後，可顯著增加紅藻胺酸對細胞的神經毒性，此證據顯示 AMPA/KA 受體的活化可能造成神經生長因子的釋放而行其神經保護的作用。再者，紅藻胺酸可在發育期神經元細胞膜上分子量 140 kD 與 75 kD的蛋白質產生酪胺酸磷酸化，而此磷酸化蛋白並進一步以免疫沉澱法證實為NGF之受體TrkA。TrkA之酪胺酸磷酸化，亦即其活性受活化的程度可被鈣離子螯化劑 BAPTA， anti-NGF與磷脂脢 C 抑制劑 U73122所阻斷。因此，紅藻胺酸引發 NGF 的釋放，可能透過增加細胞內鈣濃度及活化磷脂脢 C。長時間紅藻胺酸刺激可引發一較顯著毒性，並可被 U73122與 GDPBS起細胞內鈣上升，除了會被 AMPA/KA 受體之拮抗劑 CNQX，NMDA 受體拮抗劑 AP-5 與MK-801，以及 L- 型鈣離子通道抑制劑 nifedipine 抑制以外，也可被 genistein，AG-879 及神經生長因子抗體所降低。顯示紅藻胺酸所引起細胞內鈣離子增加是經由 AMPA/KA 受體本身，NMDA 受體活化，L- 型鈣離子通道開啟，及磷脂脢C活化等多重路徑所造成。此細胞內鈣增加可能造成 NGF 的釋放與 TrkA 的活化。另一方面，我們利用測量多磷酸纖維糖代謝探討紅藻胺酸在發育期神經元引發之磷脂脢 C 活化其神經營養功能的相關性，結果顯示需要鈣離子之參與。而其不受 MK-801，AP-5或 MCPG 抑制，但是會被 CNQX 完全阻斷。證實其為 AMPA/KA 受體活化的作用。此外，鈉離子通道阻斷劑 TTX 與神經生長因子抗體對此無作用。顯示NGF 釋放及TrkA 活化發生於磷脂脢C活化之後。此一磷脂脢之活化更經證實為透過 G-protein，因為其可被 GDPBS 抑制。GDPBS 亦可顯著降低紅藻胺酸引起的細胞內鈣上升及細胞毒性。反之，發育期神經元若長時間受紅藻胺酸刺激，仍會顯著地造成細胞死亡亡，而此現象可被 U73122 及 GDPBS緩解，表示 AMPA/KA 受體透過 G-protein 活化的磷脂脢 C 會導致神經細胞死亡，而且可能透過增加細胞自殺死亡促進蛋白Bax或減少抑制蛋白Bcl-2 的表現來影響其平衡機轉。綜觀以上，紅藻胺酸對於發育期神經元同時具有神經毒性與神經保護作用，其神經保護作用與神經毒性皆透過細胞內鈣增加而啟動，並與神經生長因子釋放及磷脂脢 C 所引發的訊息傳遞有關。而且其平衡機制可能受到神經生長因子受體 TrkA 的活化及細胞內鈣濃度變化來參與調節，以共同完成大腦皮質發育成熟的過程。

2. Excitatory Amino Acid Receptors-Mediated Neurotrophic Functions in Developing Cortical Neurons • The aim of this thesis is to elucidate the neurotrophic function ofexcitatory amino acid (EAA) receptors, with special focus on the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) / kainate (KA) in the developingnervous system. We used 4 to 6 days in vitro (DIV) culturedcortical neurons from embryonic rat brains to investigate the balance betweenneuroprotection andneurotoxicity of KA in brain development. In the lactate dehydrogenase (LDH)release assay, KA showed low toxicity in developing cortical neurons.Insignificant cell death was detected by the brief (5-15min) KA (500 uM) stimulation,whereas neurons pretreated with tyrosine kinaseinhibitors genistein (10 uM)and AG-879 (10 uM) at a non-toxic concentration,potentiated KA-induced cell death. It is conceivable that the KA receptor maymediate tyrosine phosphorylation responsible for neuronal survival. Both briefand prolong KA stimulation showed an enhanced neurotoxicity by nerve growthfactor (NGF) deprivation, and this evidence suggests the activation of AMPA/KAreceptors triggers a NGF-mediated neuroprotection mechanism to protect neuronsfrom KA neurotoxicity. KA induced tyrosine phosphorylations on 140 kD and 75 kD membrane proteins in the Western blot analysis, which were proved to be TrkA and p75NGFR by the TrkA phosphorylation analysis. The KA-induced TrkA activation requires intracellular calciumand phospholipase C (PLC) activation since it can be abolished by the addition of BAPTA and U73122. Conspicuous cell death in the developing cortical neurons was observed by prolonged (30min) administration of KA, and this KA neurotoxicity was attenuated by U73122 and GDPBS. When using fluorescence spectrophotometry to determine the intracellular calcium changes, KA- increased fura-2 labeling in the developing neurons was reduced by CNQX,AP-5, MK-801, U73122 and nifedipine, but not intracellular calcium release inhibitor dantrolene. On theother hand, the KA-induced intracellular calcium increase was reduced by bothAG-879 and anti-NGF in the initial phase, and it can be potentiated by AG-879after 5 min KA stimulation. These resultssuggest that KA-mediated intracellular calcium homeostasis during neuronal development is closely correlated with the NMDA receptor activity, PLC activity, and NGF-mediated neuroprotection. TheKA - induced PLC activation results inincrease of poly-phosphoinositides (poly-PI) turnover, which was BAPTA-, GDPBS-and U73122-sensitive, fully blocked byCNQX, but not by TTX, AP-5, MK-801, MCPG,anti-NGF and AG-879.These datasupport that KA directly activate AMPA/KA receptor to trigger a calcium-dependent and G-protein-linked PLC signaling indeveloping cortical neurons. GDPBS also reduced the KA neurotoxicity and KA-increased intracellular calcium. Finally, KA may mediate apoptosis, such as increase apoptosis promoting protein Baxand decrease antagonist Bcl-2 expressionfor neuronal development. This function was reduced by U73122 and genistein. In conclusion, KA initiates both the neuroprotective and neurotoxicevents in developing neurons. The neuroprotectionis mediated by the release of NGF and the neurotoxicity is via the PLC - mediated signal transduction. Both events were calcium-dependent, balanced for proper cell fate determination.