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Mechanism of Enhancement of Memory by YC-1
No full text學習與記憶是目前在神經科學界熱烈被探討的兩個熱門研究，科學家們嘗試用許多方法來理解學習記憶的分子機制。目前已知一氧化氮 (Nitric oxide ； NO)，cGMP，PKG在許多不同的腦部區域都有參與神經可塑性的調節，並扮演相當重要的角色。 YC -1, 它是 benzylindazole 衍生物，可以大大增加soluble guanylate cyclase (sGC) 對於NO的反應力，高達數百倍乃至千倍之多。 我們發現在海馬迴以及杏仁核體腦薄片上，YC-1可以藉由活化NO-cGMP-PKG這條訊息傳遞路徑，顯著的促進長期增益效應 (long-term potentiation ; LTP)之表現。 現在更進一步利用不同的學習記憶模式，探討YC-1對於學習記憶的促進作用；所使用的動物行為模式有：莫氏水迷津試驗、被動性或主動性逃避學習試驗等。實驗結果顯示YC-1在莫氏水迷津試驗，可以縮短逃避時間；在被動性逃避學習試驗，可以增加停留在暗箱的時間，明顯促進大白鼠在學習記憶試驗上的表現。在給予大白鼠足部電擊後30分鐘腹腔注射YC-1，發現無法延長測試中大白鼠在亮區的滯留時間，顯示YC-1可以影響新形成的記憶，使其強化。而過去許多研究報告指出，大腦杏仁核體對於帕伐洛夫恐懼制約 (Pavlovian fear conditioning)的表現與誘發扮演著重要的角色，但是對於主動迴避試驗的訊息傳遞路徑則著墨較少。因此在本論文我們進一步利用YC-1來探討NO在杏仁核所產生的訊息傳遞路徑以及如何促進大白鼠主動迴避試驗之學習與記憶。我們發現YC-1可以藉由 NO-cGMP-PKG-ERK訊息傳遞路徑來促進在杏仁核所產生的長期增益現象，並且進一步增加神經滋養因子 (Brain- derived neurotrophic factor；BDNF) 的表現。YC-1對於杏仁核LTP的促進作用，可以藉由一氧化氮合成酵素 (NO synthase；NOS) 抑制劑 L-NAME，sGC抑制劑 ODQ， PKG抑制劑 Rp-8-Br-PET-cGMPS，ERK抑制劑PD98059以及BDNF receptor TrkB抑制劑K252a所拮抗，顯示YC-1增加神經可塑性的作用是藉由NO-cGMP-PKG-ERK以及促進BDNF合成訊息傳遞路徑而來。YC-1不僅可以促進大白鼠主動迴避試驗之學習，更可以延長記憶之儲存長達一個星期以上。YC-1對於主動迴避試驗之促進作用可以被側腦室給予NOS抑制劑 L-NAME ; PKG抑制劑Rp-8-Br-PET-cGMPS與ERK抑制劑PD98059所抑制，顯示在YC-1主動迴避試驗之促進作用中， NO-cGMP-PKG-ERK的訊息傳遞路徑參與重要的角色。透過西方點墨法得知 YC-1在第一天主動迴避試驗學習30分鐘後，促進杏仁核體中ERK與Akt的活化。此外，YC-1也增加BDNF與CREB mRNA的表現。由以上得知，YC-1可以藉由 NO-cGMP-PKG-ERK 這條訊息傳遞路徑來增加記憶的學習與儲存，並且在許多不同學習記憶模式上，都具有促進記憶鞏固的作用。總結可知：YC-1對新形成的記憶具有促進的作用，但是對於已形成的記憶則不具有修復作用。NO-cGMP-PKG在學習記憶與神經突觸可塑性上扮演重要的角色，因此NO-sGC促進劑具有良好的發展潛力來促進人類的學習與記憶。此外，給予scopolamine，muscaric receptor antagonist，明顯地削弱了水迷津試驗的記憶獲得；事先給予YC-1，阻止了scopolamine所引起的學習缺陷；在卵巢切除的老鼠上給予YC-1也可以改善有卵巢切除所引起的學習記憶缺失，顯示YC-1在治療阿滋海默症與更年期後記憶缺失都有良好的發展潛力。胎神經的生長對於神經細胞發育、形成與突觸新生都有關鍵性的影響，我們在此也進一步探討YC-1在胚胎神經生長與突觸形成所扮演的角色。我們利用形態學分析的方法來探討YC-1在神經瘤細胞與初代培養大腦皮質神經細胞上促進神經生長與突觸新生的作用，我們發現YC-1可以促進神經滋養因子BDNF的合成，而BDNF在促進神經生長與突觸新生均很重要。YC-1的作用可以被NOS抑制劑 L-NAME ; PKG抑制劑KT5823與MEK酵素抑制劑PD98059所拮抗，顯示YC-1促進神經滋養因子BDNF的作用是經過NO-cGMP-PKG-ERK的訊息傳遞路徑所產生，並且NO-cGMP-PKG-ERK訊息傳遞路徑在YC-1促進神經分化扮演重要的角色。我們在初代大腦皮質神經細胞上給予YC-1可以促進神經突觸相關的分子形成，例如：synapsin和synaptophysin。在神經瘤細胞中，我們給予 BDNF antisense oligonucleotide，也可以抑制YC-1所促進的神經生長和神經突觸新生的作用，顯示YC-1促進BDNF合成，並且促進神經分化。由以上實驗結果可知YC-1可以刺激神經滋養因子的合成，促進神經的生長，因此YC-1可以做為一個良好的發展藥物，在治療神經退化疾病所引起的神經萎縮與突觸功能異常。amp;#64029;神分裂症是一種嚴重的慢性精神疾病，其終身盛行率大約為1%，在不同族群與性別之間並無明顯的差異。過去家族、雙胞胎和領養的研究，指出精神分裂症與基因變異有關，但由於遺傳機制複雜，導致精神分裂症發病的相關基因仍不是很清楚。在2000年 Millar 等人利用細胞遺傳篩檢，在一特定蘇格蘭精神病家族中，觀察到患病的成員，有1和11對染色體片段發生平衡式位移 (balanced translocation)，而座落在這斷點上的基因則是 disrupted-in schizophrenia 1 (DISC1)，而有越來越多基因和臨床的證據顯示DISC1與精神分裂症有關。神經滋養因子可以促進神經細胞的反應，調控神經傳遞物質的釋放，並且影響神經細胞的生長與發育。有報告指出當神經滋養因子發生不正常的訊息時會和精神分裂症的形成與進展有關，在精神分裂症病人的腦中與血清中的神經滋養因子有較低的表現。因此，我們想要探討DISC1基因和神經滋養因子之間的關係，我們初步發現在神經膠細胞瘤上給予shRNA降低內生性DISC1的表現量，會抑制細胞受到TNF alpha或血清素刺激時所產生的神經滋養因子合成與釋放，而這個現象有可能是透過影響TNF alpha或血清素刺激時磷酸化ERK所造成的。在內生性DISC1表現降低的神經膠細胞上，其ERK訊息傳遞的生理活性較低，因而降低神經滋養因子的合成和表現，有可能是增加精神分裂症的危險因子。YC-1可以增加BDNF的合成是否可以改善精神分裂症則值得進一步探討。Memory is one of the most fundamental mental processes, and various approaches have been used to understand the mechanisms underlying this process. Nitric oxide (NO), cGMP and protein kinase G (PKG) are involved in the modulation of synaptic plasticity in various brain regions. YC-1, which is a benzylindazole derivative, greatly potentiated the response of soluble guanylate cyclase to NO (up to several hundreds fold). We have previously shown that YC-1 markedly enhances long-term potentiation in hippocampal and amygdala slices via NO-cGMP-PKG-dependent pathway. We here further investigated whether YC-1 promotes learning behavior in Morris water maze and avoidance tests. It was found that YC-1 shortened the escape latency in the task of water maze, increased the retention scores in passive avoidance task. Administration of YC-1 30 min after footshock stimulation did not significantly affect retention scores in response to passive avoidance test. Although much has been learned about the role of the amygdala in Pavlovian fear conditioning, relatively little is known about the signaling pathway involved in the acquisition of an active avoidance reaction. The aim of this study is to investigate the potentiating effects of the NO-guanylate cyclase activator YC-1 on learning and memory of shuttle avoidance test in rats. The potentiation of LTP in amygdala by YC-1 was greatly reduced by NOS inhibitor L-NAME, sGC inhibitor ODQ, PKG inhibitor Rp-8-Br-PET-cGMPS, MEK inhibitor PD98059 and BDNF receptor TrkB antagonist K252a markedly inhibited LTP potentiating action of YC-1. Thus, YC-1 enhanced the induction of LTP in amygdala through NO-cGMP-PKG-ERK pathway and the increase of BDNF expression. The Western blot and PCR methods were used to examine the signaling pathways involved in fear memory. It was found that YC-1 increased the avoidance responses during learning period and the memory retention lasted longer than one week. The enhancement of learning behavior by YC-1 was antagonized by intracerebroventricular injection of NOS inhibitor L-NAME, PKG inhibitor Rp-8-Br-PET-cGMPS and MEK inhibitor PD98059, indicating that NO-cGMP-PKG and ERK pathways are involved in the learning potentiating action of YC-1. In addition, YC-1 increased the activation of ERK and Akt 30 min after Day-1 training in amygdala. YC-1 also potentiated the expression of BDNF and CREB in response to fear memory test. Taken together, these findings suggest that NO-cGMP-PKG-ERK signaling pathway is involved in the action of YC-1 in enhancing the fear memory. YC-1 is thus a good drug candidate for the improvement of learning and memory. In addition, administration of scopolamine, a muscarinic antagonist, markedly impaired the memory acquisition. Pretreatment of YC-1 inhibited the scopolamine-induced learning deficit. Ovariectomized (OVX) rats showed poor memory performance of active shuttle avoidance, but YC-1 ameliorated the OVX-induced memory impairment. These results suggested that YC-1 could improve cognitive ability of Alzheimer’s disease and menopause-induced learning and memory impairment.eurite outgrowth is a critical event in neuronal development, formation, and remodeling of synapses. We here examined the effects of YC-1, a NO-cGMP-PKG activator, on neurite outgrowth and synaptogenesis. Morphometric analysis of N2A cell and rat embryonic cortical neuronal cultures showed that neurite outgrowth and neuronal differentiation was enhanced by YC-1. It was also found that YC-1 enhanced the production of BDNF, which was related to the increase of neurite outgrowth and synaptogenesis. The molecules related to synaptogenesis, such as synapsin and synaptophysin also increased in response to YC-1 treatment in N2A cells and primary cortex cultures. The enhancement of BDNF production was antagonized by concomitant treatment of NOS inhibitor L-NAME, sGC inhibitor ODQ, PKG inhibitor Rp-8-Br-PET-cGMPS and MEK inhibitor PD98059, indicating that NO-cGMP-PKG-ERK pathway is involved in the differentiation potentiating action of YC-1. The enhancement of synaptogenesis can be antagonized by the antisense oligonucleotide of BDNF, suggesting that BDNF is also involved in neuritogenesis and neuronal differentiation promoted by YC-1. These findings indicate that YC-1 promotes neuritogenesis, neuronal differentiation and BDNF production and may be a useful lead compound for the treatment of neurite dystrophy and synaptic dysfunction in neurodegenerative diseases.rowing genetic and clinical evidence has shown that Disrupted in Schizophrenia 1 (DISC1), identified in a pedigree with a familial psychosis with the chromosome translocation (1:11), is one of the most compelling risk genes for schizophrenia and other major mental disorders such as bipolar disorder and major depression. Neurotrophic factors can promote and modulate the neuronal responses across neurotransmitter system in the brain. Abnormal neurotrophic factors signaling may be associated with the pathophysiology of schizophrenia. Decreased neurotrophic factor levels in the brain and the serum of patients with psychotic disorders have been reported. Therefore, we here investigated the regulation of neurotrophic factor expression by DISC1. It was found that knockdown of endogenous DISC1 protein by small hairpin RNA resulted in the suppression of production of BDNF and GDNF induced by TNF alpha and serotonin in A172 glia cells. The phosphorylation of ERK and p38 induced by TNF alpha and serotonin were also inhibited by DISC1 knockdown. YC-1 can increase BDNF expression and ERK phosphorylation. Whether YC-1 is beneficial to Schizophrenia patients needs further investigation.口試委員會審定書 i謝 ii文摘要 iii文摘要 vii目錄 1目錄 4寫表 8一章 緒論 11.1 記憶形成的細胞機轉之探討 12.2 YC-1的藥理作用機轉 21.3 神經分化與神經突觸形成 26.4 神經退化疾病與精神疾病之保護作用 29.5 研究動機 33二章 實驗材料與方法 35.1 實驗動物 36.2 實驗藥品 36.3 電生理活性記錄與測定 36.3.1 海馬迴區域 37.3.2 杏仁核體 37.4 腦部手術與顱內藥物注射 38.5 動物行為試驗 39.5.1 莫氏水迷津（Morris Water maze） 39.5.2 被動性逃避學習 (passive avoidance test) 40.5.3 主動迴避試驗 (Active shuttle avoidance test) 40.5.4 自發性活動量測試 (Locomotor activity) 41.5.5 電擊敏感度測試 (Shock sensitivity test) 42.6 全細胞萃取液 (whole cell lysate) 萃取方法 43.7 西方點墨法 (Western blotting) 43.8 RNA之抽取 43.9 RT-PCR (Reverse transcription-polymerase chain reaction) 44.10 免疫組織染色法 45.11 細胞培養 46.11.1 初級大腦皮質神經細胞 (primary cortical neuron) 46.11.2 神經細胞瘤株N2a cell line 46.11.3 神經膠細胞瘤株A172 cell line 46.11.4 細胞的冷凍保存 47.12 免疫螢光染色法 47.13 細胞存活率測定 (MTT assay) 47.14 細胞轉殖 47.14.1 神經細胞瘤株N2a cell line 48.14.2 神經膠細胞瘤株A172 cell line 48.15 GDNF enzyme-linked immunosorbent assay 48.16 統計方法 49三章 YC-1促進學習與記憶之作用探討 50.1 YC-1促進海馬迴LTP 與莫氏水迷津試驗 51.2 YC-1促進杏仁核LTP之機轉探討 59.3 YC-1促進恐懼記憶之機轉探討 68.4 YC-1改善記憶障礙作用之研究 90四章 YC-1在神經培養細胞促進神經生長、突觸新生之作用探討 101五章 DISC1基因調控神經滋養因子之研究 124六章 結論與未來展望 145考文獻 148作發表 16
Enhancement of learning behaviour by a potent nitric oxide-guanylate cyclase activator YC-1
No full textEnhancement of Long-Term Potentiation by a Potent Nitric Oxide-Guanylyl Cyclase Activator, 3-(5-Hydroxymethyl-2-furyl)-1-benzyl-indazole
No full text
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