博碩士論文 109821004 詳細資訊




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姓名 林詠瑞(Yung-Jui Lin)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 KLHL17在癲癇與自閉症中之角色
(The role of KLHL17 in epilepsy and autism spectrum disorder)
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摘要(中) 嬰兒點頭痙攣症後群 (Infantile spasm, IS),為一種罕見的癲癇性痙攣,好發於一歲以前幼童,亦常共發其他神經疾病,例如:自閉症與智能遲緩。人類基因體學研究發現KLHL17為高機率IS致病基因。KLHL17/Actinfillin (Kelch-like 17,又被稱為Actinfillin),為腦專一纖維性肌動蛋白 (Filamentous-actin, F-actin)結合蛋白。KLHL17亦能作為Cul3的受質結合蛋白,協助降解GRIK2/GluR6 (Kainite 受體次單元)。本實驗室之前的研究發現,KLHL17調控海馬迴神經細胞 (in vitro)的樹突棘擴大作用與突觸活性,然而對於KLHL17在小鼠腦內 (in vivo)的調控機制及生理功能還未釐清。因此本論文分為兩部分,第一部分使用Klhl17基因缺失小鼠研究KLHL17在體內的功能,而第二部份為協助實驗室其他KLHL17相關計畫進行。在第一部分的實驗,首先進行形態之特徵分析,實驗結果顯示,不論公母,Klhl17+/-小鼠不會影響腦部巨觀的解剖學變化,然而我發現Klhl17缺失小鼠的海馬迴與體覺皮質區投射神經元樹突棘形態發育異常。有趣的是,在海馬迴樹突棘寬度缺陷方面,Klhl17+/-母鼠呈現較為嚴重的缺陷,顯示性別及腦區的差異性。接著將公母小鼠分成四組,進行四組行為測試,並進一步分析其中異常分子機制。第一組行為測試為癲癇測試,Klhl17+/-公鼠較母鼠有較高的癲癇易受性。第二~四組則是系列的類自閉症相關行為。Klhl17+/-突變鼠在幼時有些許叫聲溝通異常。Klhl17+/-公母鼠均有社交行為的缺陷,但是在特定測試中,母鼠社交缺陷較不明顯。然而Klhl17+/-公鼠並無行為固化之特徵,反而有更好的變通性;在母鼠則不明顯。另外,我發現早期Ultrasonic vocalization test的經歷會差異性的影響Klhl17+/-公母鼠的活動力、空間記憶、及社交行為。總之,性别的差異會影響Klhl17缺失的性狀。為了解其中可能的分子機制,我也分析海馬迴總蛋白質體以及突觸蛋白質體,發現一些受KLHL17和性别影響的蛋白質分子,然而這些異常分子機制與上述神經樹突棘發育形態的異常或行為異常的關聯,還有待後續實驗釐清。在第二部分,主要發現KLHL17參與神經细胞活性調控内質網的突觸分佈。總結,本論文從不同的角度研究KLHL17,期待藉此了解KLHL17之生理功能和神經系统疾病的關係。
摘要(英) Infantile spasm (IS) is a severe epileptic seizure that usually occurs before one year of age. The human genetic study suggested that KLHL17 is a high-risk gene of IS. In mice, Klhl17 encodes a brain-specific actin-binding protein. It also acts as a substrate receptor of CUL3 to degrade GRIK2/GLUR6 (a subunit of the kainite receptor). We have previously reported that KLHL17 regulates dendritic spine enlargement and synaptic activity. However, the in vivo function of KLHL17 remains unknown. This thesis is divided into two parts to address this question. In the first part, I use Klhl17 deletion mice to characterize the in vivo function of KLHL17. In the second part, the main goal is to assist other KLHL17-related projects in the laboratory. In the first part, although Klhl17 deletion does not affect overall brain anatomy in both male and female mice, we found that the morphology of dendritic spines exhibits sex- and region-biased differences in the hippocampal CA1 region and somatosensory cortex of Klhl17 deletion mice. I further analyzed the behavioral deficits of Klhl17 deficient mice. Consistent with the association of KLHL17 and IS, I found higher seizure susceptibility in male but not female Klhl17 deficient mice. Klhl17+/- mice also showed slightly social communication deficits in early life. Both male and female Klhl17+/- mice all exhibited social deficits, though in a specific test, female Klhl17+/- mice social deficits were not evident. However, male and female Klhl17+/- mice did not have the trait of cognitive inflexibility, and precisely, male Klhl17+/- mice showed improved results. Moreover, I found that early ultrasonic vocalization experience would alter locomotivity, spatial memory, and social behavior in male and female Klhl17+/- mice. Further, I analyzed proteomics results in the total hippocampal lysate and synaptosomal fractions to understand the possible molecular mechanism. I found some misregulated proteins affected by KLHL17 sex-differentially. However, it is still unclear whether dysregulation of those synaptic proteins is linked to dendritic spine deficits or abnormal behaviors. In the second part, majorly, I showed that KLHL17 regulates the distribution of ER in the dendritic spine upon neuronal activation. To sum up, I use different aspects and approaches to study the function of KLHL17, and much more understand the relationship between KLHL17 and neurologic disorders.
關鍵字(中) ★ 癲癇
★ 自閉症
★ 神經元
★ 小鼠
★ KLHL17
關鍵字(英) ★ Epilepsy
★ Autism spectrum disorder
★ Neuron
★ mouse
★ KLHL17
論文目次 目錄
中文摘要……………………………………………………………………………………….Ⅰ
英文摘要…………………………………………………………………………………..…..Ⅱ
致謝…………………………………………………………………………………………..Ⅳ
目錄……………………………………………………………………………………….…...Ⅴ
縮寫說明…………………………………………………………………………………..…Ⅸ
第一章、緒論
1. KLHL17/ AF的基本介紹………………………………………………………………..1
2.嬰兒點頭痙攣(Infantile spasm, IS)…………………………………………………….…2
3.自閉症
3-1.自閉症的基本介紹……………………………………………………………………3
3-2.自閉症的成因與機制探討………………………………………………………..…..4
3-2-1.自閉症與肌動骨架蛋白的調控…………………………………………….……5
3-2-2.自閉症與蛋白質的恆定性…………………………………………………...…..6
4.研究動機………………………………………………………………………………....10
第二章、材料與方法………………………………………………………………….....11~30
第三章、結果
1. Klhl17基因缺失不會影響小鼠外觀、體重、腦部結構……………………………...31
2. Klhl17基因缺失對神經形態生成的影響具有性別差異性
2-1.海馬迴CA1投射神經元…………………………………………………….….….32
2-2.體覺皮質區投射神經元………………………………………………………...….33
3. Klhl17基因缺失小鼠分子機制探討
3-1. Klhl17+/-小鼠海馬迴總蛋白體及突觸體差異性表現的蛋白質分子數量…...….34
3-2.受Klhl17缺失影響的蛋白質分子分析…………………………………………....34
3-3.西方墨點法分析………………………………………………………………...….36
3-4.自閉症相關的差異性表現蛋白質分子………………………………………….....37
4. Klhl17基因缺失小鼠行為分析
4-1.第一組行為實驗: Klhl17基因缺失小鼠的癲癇易受性…………………..………37
4-1-1.公母Klhl17+/-小鼠展現不同癲癇易受性……………………………….…….38
4-2.第二組行為實驗分析
4-2-1.第二組行為實驗分析- Open field…………………………………………...….39
4-2-2.第二組行為實驗分析- Cued Y maze……………………………………….…..39
4-2-3.第二組行為實驗分析- Elevated plus maze……………………………………..39
4-2-4.第二組行為實驗分析- Reciprocal social interaction…………………………...40
4-2-5.第二組行為實驗分析- Three chamber social interaction………………………40
4-2-6.第二組行為實驗分析- T-maze reversal learning…………………………….....41
4-3.第三組行為實驗分析
4-3-1.第三組行為實驗分析- Novel object recognition……………………………….41
4-3-2.第三組行為實驗分析- Food preference and STFP…………...………………...42
4-4.第四組行為實驗分析
4-4-1.第四組行為實驗分析- Ultrasonic vocalization……………………………..….43
4-4-2.第四組行為實驗分析- Open field……………………………………………...43
4-4-3.第四組行為實驗分析- Cued Y maze………………………………………...…43
4-4-4.第四組行為實驗分析- Elevated plus maze……………………………………..44
4-4-5.第四組行為實驗分析- Reciprocal social interaction…………………………...44
4-4-6.第四組行為實驗分析- Contextual fear memory………………………………..44
4-5.早期特殊經驗對於Klhl17+/-小鼠行為的影響…………………………………....45
4-6.第二、三組社交行為實驗分析………………………………………………...….45
5. 第二部分–KLHL17的其他功能
5-1.活化神經細胞造成 KLHL17與內質網於樹突棘分布改變……………………...46
5-2. Klhl17基因缺失影響Synaptopodin表現量、樹突棘聚集與分布改變………...48
第四章、討論
1. Klhl17基因缺失對於異常樹突棘形態與異常行為的相關性………………………..50
2. Klhl17+/-公母鼠展現不同癲癇易受性………………………………………………..51
3. Klhl17+/-小鼠展現類似於自閉症患者的症狀……………………………………..…52
4.早期特殊經驗對於Klhl17+/-小鼠展現不同行為特徵………………………………..53
5.Klhl17基因缺失對於海馬迴蛋白質分子表現的改變………………………………...54
6.突觸體蛋白質組學分析討論………………………………………………………..….55
第五章、圖表
圖一: Klhl17/Actinfilin基因缺失小鼠建立與鑑定……………………………………...56
圖二: Klhl17基因缺失小鼠和鼠腦外觀,以及體重分析…………………………...…57
圖三: Klhl17/Actinfilin缺失小鼠前腦切片比較……………………………………...…58
圖四: Klhl17基因缺失影響海馬迴CA1投射神經元的樹突棘形態發生…………….60
圖五: Klhl17基因缺失影響體覺皮質區投射神經元樹突棘形態發生……………...…61
圖六: 海馬迴總蛋白體及突觸體蛋白萃取流程…………………………...…………….62
圖七~九:海馬迴總蛋白體的分析比較……………………………………………….63~74
圖十~十二:海馬迴突觸體蛋白的分析比較……………………………….............…75~86
圖十三:海馬迴總蛋白體或突觸體蛋白重疊蛋白質分析………………………........….87
圖十四:海馬迴總蛋白體和突觸蛋白體GO比較分析……………………………….…89
圖十五:海馬迴總蛋白質體和突觸體蛋白西方墨點法證明………………………........90
圖十六:海馬迴總蛋白體和突觸蛋白體差異性表現的蛋白質與自閉症之相關性……92
圖十七~十八: 第一組行為實驗(癲癇相關)……………………………………………..93
圖十九: Klhl17基因缺失小鼠行為分析流程圖(第二~四組)………………………..….95
圖二十~二十六:第二組行為實驗結果……………………………………..……….96~102
圖二十七~二十九:第三組行為實驗結果………………………………………….103~105
圖三十~三十五:第四組行為實驗結果…………………………………………….106~111
圖三十六:第二組與第四組行為實驗分析比較(經歷早期特殊經驗與否)…………….112
圖三十七: KLHL17/AF在海馬迴初代神經細胞内的分布情形……………………..…113
圖三十八:活化海馬迴初代神經細胞引發樹突棘形態變化…………………………....114
圖三十九:海馬迴初代神經細胞活性影響KLHL17和内質網在樹突之分布…………116
圖四十: Klhl17缺失小鼠影響Synaptopodin (SYNPO)在海馬迴CA1區域的表現量..117
圖四十一: Klhl17缺失小鼠影響Synaptopodin (SYNPO)的聚集(Clustering)…………118
第六章、附圖……………………………………………………………………………….119
第七章、參考文獻……………………………………………………………………120~127
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指導教授 薛一蘋 陳盛良(Yi-Ping Hsueh Shen-Liang Chen) 審核日期 2022-8-23
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