Analysis of Cyp11a1 expression and function in olfactory bulb
| Autor: | Szu-Ying Liu, 劉思穎 |
|---|---|
| Rok vydání: | 2014 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 102 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. &;#8195 |
| Databáze: | Networked Digital Library of Theses & Dissertations |
| Externí odkaz: |
|