Cyp11a1基因在嗅球表現功能之探討

Abstract

P450膽固醇側鏈截切酶 (cytochrome P450 side chain cleavage, P450scc) 為CYP11A1基因的產物，參與類固醇生成的關鍵步驟，能將膽固醇轉換為Pregnenolone，主要表現在腎上腺及性腺。CYP11A1也表現在腦中，控制神經類固醇的生成，並對腦的生理功能有所影響。由於腦中CYP11A1表現量很低，因此目前對於CYP11A1表現分布及轉錄調控尚未明瞭。本實驗室以CYP11A1啟動子驅動Cre重組酶，建立SCCmtp-Cre基因轉殖鼠，以Cre的表現探討CYP11A1啟動子在類固醇生成組織的轉錄活性。本論文利用SCCmtp-Cre與ROSAR-Td-tomato小鼠交配，分析螢光蛋白在腦的表現，以了解內生性CYP11A1可能的表現分布。我觀察到Td-tomato在腦中許多區域都有表現，包括間腦、下視丘、杏仁核以及嗅覺系統的嗅球與嗅覺上皮。我們發現Td-tomato會專一地表現在嗅球的granule cell layer (GCL)，且較集中於內層。這些有螢光表現之細胞，其神經纖維會延伸至mitral cell layer (MCL) 與external plexiform layer (EPL)，符合granule cells的特性。免疫螢光染色分析顯示，Td-tomato螢光細胞可表現神經元的標誌蛋白NeuN，但沒有星狀細胞的標誌蛋白GFAP之表現，說明其為分化的神經元。GCL由granule cells組成，會分泌γ-aminobutyric acid (GABA) 為神經傳導物。以GABA的生成酵素glutamic acid decarboxylase 67 (GAD67)、glutamic acid decarboxylase 65 (GAD65) 進行染色，結果發現Td-tomato螢光細胞幾乎都有這兩者的表現。有些granule cells會表現鈣離子結合蛋白calretinin或calbindin，但這兩者都沒有表現在Td-tomato螢光細胞。 我們利用Cyp11a1基因剔除鼠 (KO) 進一步探討Cyp11a1在嗅球的生理功能。Western blot分析顯示，KO中GAD65蛋白質量顯著下降，而GAD67量沒有統計差異。相反地，calretinin量在KO顯著上升，而calbindin量則沒有差異。存在於glomerular layer的蛋白olfactory marker protein (OMP)，在KO的表現量也顯著上升。利用TUNEL assay觀察Cyp11a1基因剔除對細胞凋亡的影響，結果發現KO在GCL的細胞凋亡數目顯著多於野生型。這些結果說明，Cyp11a1基因在嗅球可能扮演重要功能。

CYP11A1 encodes cholesterol side-chain cleavage enzyme (P450scc). P450scc converts cholesterone into pregnenolone, which is the key step of steroid biosynthesis. CYP11A1 is abundantly expressed in the adrenal glands and gonads. In addition, it is also expressed in the brain to control the synthesis of neurosteroids that are involved in many neural functions. Because the amount of endogenous mRNA of Cyp11a1 is very low, the expression of CYP11A1 gene in the brain has not been well characterized. We previously generated a SCCmtp-Cre transgenic mouse line in which the expression of Cre recombinase gene is under the control of human CYP11A1 promoter. To study the expression of SCC promoter activity in the brain, SCCmtp-Cre was crossed to ROSA26R Cre-dependent Td-Tomato reporter mice. Td-tomato signal was found in several brain areas including the olfactory system (olfactory bulb and olfactory epithelium), diencephalon, hypothalamus, and amygdala. In the olfactory bulb, Td-tomato signal is specifically present in the granule cell layer. We can see the process of Td-tomato extend into mitral cell layer and external plexiform layer. Immunofluoresence analysis showed that Td-tomato are NeuN-positive (neuron marker) and GFAP-negative (astrocyte marker), indicating that these cells are differentiated neurons. Granule cells release γ-aminobutyric acid (GABA) which is synthesized by glutamic acid decarboxylase 65 (GAD65) or 67 (GAD67). Our data showed that most of Td-tomato cells co-localized with these two enzymes. Some granule cells express calcium binding protein calretinin or calbindin. However, these two markers were not co-localized with Td-tomato signal. We used Cyp11a1 knockout mice (KO) to explore the function of Cyp11a1 in the olfactory bulb. Western blot analysis showed that the protein levels of GAD 65, but not GAD67 are significantly decreased in KO mice. In contrast, the protein levels of calretinin, but not calbindin are significantly increased in KO mice. Olfactory marker protein in glomerulus is also significantly increased in KO mice. In addition, TUNEL assays showed that KO mice have more apoptotic cells in GCL compared to wild-type mice. These results suggested that Cyp11a1 may play a critical role in the olfactory bulb.