| 摘要: | 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。;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. |
