探討neuregulin-1與serotonin system在神經及神經膠細胞之相互調節作用

• Main Authors: 曾姿婷, Tseng, Zih-Ting
• Other Authors: 臺灣大學: 藥理學研究所, 符文美
• Format: Thesis
• Language: English
• Published: 2012
• Subjects: 精神分裂症;NRG1;ErbB4;血清素;血清素轉運蛋白; schizophrenia;neuregulin-1;serotonin system;serotonin transporter;erbB4; Mek; Iceland
• Online Access: http://ntur.lib.ntu.edu.tw/handle/246246/251998; http://ntur.lib.ntu.edu.tw/bitstream/246246/251998/1/ntu-101-R99443005-1.pdf

精神分裂症是一種很嚴重的衰退性精神疾病，其病因可能和許多的基因以及環境因素有相關性。其中由Stanfansson等人對於冰島地區的精神分裂症家族做大規模的篩檢發現到Neurgegulin-1 (NRG1)基因是造成精神分裂症的致病基因之一，然而，NRG1基因對於精神分裂症的影響及其詳細機制還不是非常的了解，除此之外，造成精神分裂症當中的其中一種假說為其突觸間血清素的調控異常，而其調控異常的詳細機制仍不清楚，因此本篇論文的目的在於探討血清素及NRG1在神經以及神經膠細胞的相互調控機制。 藉由西方墨點法分析發現，在NRG1+/-小鼠的不同腦部核區出現了血清素轉運蛋白表現量增加的現象，表示其腦部的血清素回收速率和正常小鼠相比較之下高出許多，而另一方面，在體外探討血清素與NRG1調控關係的實驗中，我們使用了人類神經細胞瘤之細胞株SH-SY5Y及人類神經膠瘤之細胞株A172，發現外給NRG1會抑制其血清素轉運蛋白(SERT)的表現量，而其結果也在大鼠皮質初代神經細胞中被證實。而正腎上腺素轉運蛋白(NET)及多巴胺轉運蛋白(DAT)的表現量也同樣受到NRG1影響而產生了抑制效果。另一方面，外給NRG1會在神經細胞瘤細胞中減少SERT, NET 及DAT mRNA的表現量。我們進一步探討外給NRG1所產生的血清素轉運蛋白表現量的減少是透過何種機制來調控，發現此現象在神經細胞瘤中會被PD98059 (MEK抑制劑)及SB203580 (p38抑制劑) 所拮抗，而我們也觀察到外給NRG1會增加這些細胞中ERK 及p38的磷酸化，因此推論此現象可能是經由ERK及p38這兩條訊息傳遞機制所調控。 另一方面，在人類神經細胞瘤細胞株SH-SY5Y外加血清素則可增加NRG1及其受體erbB4的表現量，而在SH-SY5Y、A172及大腦皮質細胞外加血清素受體1A部分促進劑也可增加NRG1及其受體erbB4的表現量，顯示此結果可能是經由血清素受體1A亞型所調控。此外，給予血清素受體1A部分促進劑在神經細胞瘤中會增加erbB4及NRG1 mRNA的表現量。而在人類神經瘤細胞株SH-SY5Y外給正腎上腺素也可增加NRG1及其受體erbB4的表現量。我們進一步探討外給血清素受體1A部分促進劑所產生的erbB4表現量增加是透過何種機制來調控，發現此現象在神經細胞瘤中會被LY294002 (PI3K的抑制劑) 所拮抗，而我們也觀察到外給血清素受體1A促進劑會增加這些細胞中Akt的磷酸化，因此推論此現象可能是經由PI3K/Akt這條訊息傳遞機制所調控。 為了模擬人體長期服用抗憂鬱藥物之療法，我們於正常小鼠長期注射desipramine及duloxetine之後，將小鼠犧牲以觀察其腦部受體之變化。結果發現在不同的腦核區其erbB4的表現量增加，表示抗憂鬱藥物之血清素及正腎上腺素會促進erbB4的表現量。除此之外，藉由高效能液相層析法，我們發現突觸間隙的血清素含量在NRG1+/-小鼠當中的皮質,額狀皮質及紋狀體明顯下降許多，但正腎上腺素及多巴胺的含量和正常小鼠比較差異不大。我們的實驗顯示在NRG1和血清素,正腎上腺素之間存在著正向的調控關係，表示NRG1基因異常所引發的精神分裂症之可能與血清素的功能不足有關。 總結本篇的研究結果，血清素、NRG1和erbB4的失衡可能是造成精神分裂症的原因之一。 Schizophrenia is a severe, debilitating psychiatric disease and multiple genes and environmental insults participate in the pathogenesis of schizophrenia. Neuregulin-1 (NRG1) is one of susceptibility genes which originated from the genome-wide scan of schizophrenia families in Iceland in 2002. However, the relationship between the NRG1 and schizophrenia is still unclear. In addition, the serotonin dysfunction involved in schizophrenia is also still poorly understood. The aim of this study is to investigate the mutual regulation of NRG1 and serotonin system in neuronal cells and astrocytes. It was found that protein expression of serotonin transporter was up-regulated in the various brain regions in NRG+/- mice, indicating that serotonin level was reduced in synaptic cleft because the higher uptake of serotonin in NRG1+/- mice. To evaluate the interaction between NRG1 and serotonin system in vitro, it was found that exogenous treatment of NRG1β decreased the expression of SERT in human neuroblastoma cell lines, SH-SY5Y, primary neurons and human glioblastoma cell lines, A172. The norepinephrine transporter (NET) was also down-regulated in SH-SY5Y cells and A172 cells. In addition, the dopamine transporter (DAT) was also reduced by NRG1β in SH-SY5Y cells. Moreover, the SERT, NET and DAT mRNA expression was reduced in SH-SY5Y cells due to NRG1β application. Furthermore, application of NRG1β reduced the SERT expression, which was antagonized by the treatment of PD98059 (MEK inhibitor) and SB203580 (p38 inhibitor). Moreover, exogenous NRG1β also increased the phosphorylation levels of ERK and P38. Therefore, the down-regulation of SERT by NRG1β treatment may signal through ERK and p38 pathways. On the other hand, application of serotonin and 5-HT1A partial agonist increased the expression of erbB4 and NRG1 in SH-SY5Y cells, A172 cells and primary neurons, indicating that 5-HT1A receptor activation my up-regulate erbB4 and NRG1. Furthermore, the mRNA expression of erbB4 and NRG1 was also up-regulated in SH-SY5Y cells by 5-HT1A partial agonist. Moreover, application of norepinephrine also increased the erbB4 and NRG1 expression. Furthermore, application of 5-HT1A partial agonist increased the erbB4 expression, which was antagonized by the treatment of LY294002 (a PI3K/Akt inhibitor). Moreover, exogenous 5-HT1A partial agonist also increased the phosphorylation level of Akt, indicating that the up-regulation of erbB4 by 5-HT1A partial agonist may be associated with PI3K/Akt pathway. In addition, chronic treatment of desipramine and duloxetine in WT mice increased the erbB4 expression in different regions of brain, indicating that increase of serotonin and norepinephrine by antidepressants may have influence on the expression of erbB4 protein. In addition, serotonin levels but not norepinephrine and dopamine in various brain regions were reduced in NRG1+/- mice. These results indicated that there is positive regulation between NRG1, serotonin and norepinephrine and the serotonin dysregulation maybe implicated in the pathogenesis in schizophrenia accompanied by NRG1 defect. In conclusion, dysregulation between NRG1, serotonin system and erbB4 may be a possible mechanism underlying the etiologies of schizophrenia.