2012-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/693882摘要：LMBRD1 蛋白質在2005 年首先被我們發現其功能。LMBRD1 至少有2 種isoforms，NESI及LMBR1。NESI 對具有proline-rich 之核輸出訊息的蛋白質之核輸出重要，而LMBR1可當為lysosomal 之維生素B12 之輸出者。最近我們證明一帶有單一lmbrd1 對偶基因刪除(LUHKO)老鼠有心室肥大的現象，可能導因於持續活化胰島素受體之訊息傳導路徑所致。LUHKO 老鼠表現有心律及心肌收縮增加的現象，導致代償病理性肥大並伴隨有纖維化。更進一步的機制分析，暗示LMBRD1 蛋白質可能參與胰島素受體之內吞作用及調節其代謝之訊息傳遞。另外，我們利用迴避試驗證明LUHKO 老鼠有記憶及學習的障礙。同時，證明LUHKO 老鼠比正常老鼠在腦部紋狀體有較多的18FDOPA 吸收。另外，利用18F-fallypride 吸收實驗證明LUHKO 老鼠比正常老鼠在腦部紋狀體有較多的多巴胺D2/D3 受體的堆積。同時，利用shRNA 實驗證明在神經瘤N2A 細胞，LMBRD1 有減弱retinoic acid 所誘導的神經細胞分化。有趣的發現是在lmbrd1 knockdown 的海馬迴初代培養細胞顯示有明顯的neurite outgrowth 現象，但卻減少spine 的形成。更進一步的實驗證明在N2A 細胞lmbrd1 knockdown 會抑制FAK-SHP2-β-catenin 複合體的形成，而增加磷酸化FAK 的量。這些結果暗示LMBRD1 在動作協調、學習及記憶在神經分化的過程之FAK-SHP2-β-catenin 路徑中佔有重要的角色。本計畫主要擬探討在於胚胎發育及出生後LMBRD1 在心臟及腦部組織的分布情形以瞭解疾病發生的原因。另將以shRNA 的方法進行LMBRD1 在調節心臟及腦部發育及相關疾病之機制分析。初代培養細胞將用以研究LMBRD1 如何參與心臟胰島素受體在細胞膜上之再利用及神經分化研究之平台。接著將心肌細胞之lmbrd1 基因knockdown 再表現LMBRD1 蛋白質，以進行表現特徵之回復實驗。利用已建立之LUHKO 老鼠具有心肌病變及心智障礙的表現型，可用以進一步瞭解LMBRD1 參與組織/器官發育、致病、病程及在細胞和分子層次上之機制。以上這些訊息可用於與LMBRD1 相關疾病之新藥發展、早期診斷及治療效率、副作用及預後之評估。<br> Abstract: LMBR1 domain containing protein 1 (LMBRD1) was first functionally identified in2005. LMBRD1 contains at least two major isoforms, NESI and LMBR1. NESI is critical forthe nuclear export of proteins with Pro-rich NES (nuclear export signal), whereas LMBR1serves as a putative lysosomal cobalamin transporter. Recently, we demonstrated that thelmbrd1 ubiquitous heterozygous knockout (LUHKO) mice with cardiac hypertrophy mayresult from constitutively activation of insulin receptor signaling pathway. In addition,LUHKO mice exhibit an increase in heart rate and cardiac muscle contractility, leading tocompensated pathological hypertrophy with the development of fibrosis. Further mechanisticstudies suggest that LMBRD1 functions as an adaptor specific for the clathrin mediatedendocytosis of insulin receptor and regulates insulin receptor metabolic signaling pathways.On the other hand, we demonstrated that LUHKO mice associate with memory and learningimpairments by avoidance tests. In addition, 18FDOPA uptake in LUHKO mice was increasedin striatum compared to that of wild-type littermates. Results from 18F-fallypride uptakeindicated that the dopamine D2/D3 receptor was accumulated in striatum region in LUHKOmice. In addition, LMBRD1 was demonstrated to attenuate the retinoic acid-mediated thedifferentiation of neuroblastoma N2A cells by shRNA knockdown experiments. Interestingly,lmbrd1 knockdown hippocampal primary culture cells showed the significant neuriteoutgrowth with less spine formation. Furthermore, the lmbrd1 knockdown inhibited theFAK-SHP2-β-catenin complex formation and increased the phospho-FAK level in N2A cells.These results indicate that LMBRD1 may play significant roles in motor coordination,learning, and memory mediated by the FAK-SHP2-β-catanin pathway during neuronaldifferentiation. In this study, the distribution of LMBRD1 expression in heart and brain tissuesduring embryogenesis and after birth will be further investigated to understand the cause ofdiseases. Mechanistic studies of LMBRD1 involved in regulating cardiac and braindevelopment and diseases will be analyzed by shRNA method. Primary culture cells will beused as the platform to elucidate the roles of LMBRD1 in the recycling of insulin receptor onthe plasma membrane and neuronal differentiation. Subsequently, in validating the molecularpathway, additional LMBRD1 will be re-introduced into the knockdown cells for phenotypicrescue. Moreover, the LUHKO mice with the cardiomyopathy and mental illness can be usedto understand the tissue/organ development, pathogenesis, progression, and underlyingmechanisms at the cellular and molecular levels. The information can be used to develop newtherapeutics for LMBRD1-associated diseases and early diagnosis, and to evaluate theefficacy of treatments, side effects, and prognosis.