摘要:脊髓肌肉萎縮症(Spinal Muscular Atrophy, 簡稱SMA)為一遺傳性運動神經元疾病,目前臨床上尚未有治療的方法。SMA的疾病遺傳模式為體染色體隱性遺傳,其發生率約為6,000 至10,000 分之一,而大多數SMA患者的兩股運動神經元存活基因 (Survivalmotor neuron gene, 簡稱SMN1)皆發生了基因突變。SMA模式小鼠已由小鼠Smn基因剔除合併人類SMN2 基因轉殖的方法所建立,我們已詳細的分析了SMA小鼠表現型的特色,因此這個SMA模式小鼠對後續SMA致病機制及治療性試驗的相關研究可帶來極大的幫助。RAGE接受器(receptor for advanced glycation end products, RAGE)屬於免疫球蛋白家族的膜蛋白,其ligand包括advanced glycation end products (AGE), S100, HMGB1 和β-amyloid等。傳統上認為RAGE的活化會引起發炎反應,進而造成組織的傷害。然而近年來研究發現,RAGE在體內亦參與了許多重要的細胞基本反應,如細胞存活、移動、分化及修復等,其作用路徑和RAGE所表現的細胞種類、外在環境及ligand的種類和濃度習習相關。RAGE亦表現於神經元,但其於神經系統的角色和利弊,需取決於個別組織和病態生理上的條件。SMA的致病機轉,目前認為可能和SMN表現不足所引起的neurite異常和神經元的細胞凋零有關;S100 和HMGB1 對RAGE的活化不但可增進神經元的neurite生長,亦可抑制細胞的凋零。此外有研究證實RAGE的表現可促進周邊神經和肌肉組織的修復,此現象可能有機會對減緩SMA的疾病進程有所助益。我們最近亦首度發現,SMA小鼠脊髓內RAGE的mRNA及蛋白質表現皆明顯下降,且和疾病的嚴重度呈現正相關,因此,RAGE可能在SMA的病理機制上扮演重要的角色。然而目前為止,RAGE於SMA的關係性仍尚待研究,而以基因轉殖或基因治療的方式提昇RAGE的表現,在SMA的反應及療效仍然未知。本研究計畫的第一個目的為:了解RAGE於運動神經元所扮演的角色。我們將嘗試驗證是否抑制運動神經元細胞內SMN的含量(SMA-like運動神經元),會影響細胞內RAGE及RAGE相關因子的表現,而若進而增加RAGE的表現,是否可改進SMA-like運動神經元的異常細胞形態及細胞凋亡現象。第二個目的為:了解RAGE於SMA模式小鼠中所扮演的角色。我們將製備傳統SMA小鼠和具RAGE基因轉殖的SMA小鼠,並分析經RAGE基因轉殖的SMA小鼠之表現型。第三個目的為:了解經腦室注射之RAGE基因治療,是否對SMA小鼠有所療效。我們將以腺相關病毒對SMA小鼠進行RAGE基因治療,並比較不同劑量、不同疾病時期、和不同疾病嚴重度下之RAGE基因治療的療效。
Abstract: Spinal muscular atrophy (SMA) is a hereditary progressive motor neuron disease withoutavailable curative treatment currently. SMA exhibits an autosomal recessive pattern of inheritancewith an incidence of 1 in 6,000-10,000 newborns. Most SMA patients have homozygous genemutation on the survival of motor neuron 1 gene (SMN1). The mice model of SMA has been generatedby the mouse Smn knockout-human SMN2 transgenic method. Following our characterization of thephenotype for SMA mice, this rodent model is suitable for use in investigating disease mechanismsand testing therapies for SMA.Receptor for advanced glycation end products (RAGE) is a multi-ligand transmembrane proteinbelonging to immunoglobulin superfamily. The ligands include advanced glycation end products(AGE), S100, HMGB1, and beta-amyloid, etc. RAGE expresses on neuronal cell membrane, too. Intraditional view, activation of RAGE primarily induces inflammatory reactions, leading to tissue injury.However, recent studies have demonstrated that RAGE is also important for multiple essential cellularprocesses, such as cell survival, migration, differentiation, and cell repair. The individual down-streampathway following RAGE activation depends on the cell type, external environment, and ligand typeand concentration.SMN protein deficiency in SMA causes neurite dysfunction; the degeneration of spinal motorneurons in SMA is mediated via apoptosis. RAGE activation by S100 and/or HMGB1 stimulation notonly enhanced neurite outgrowth, but also inhibited cell apoptosis. In addition, the RAGE expressionpromoted regeneration of peripheral nerves and muscles in the situation of acute injury. RAGE mightthus be a potential therapeutic target for SMA treatment. Notably, we recently found that the levels ofRAGE transcript and protein were decreased in spinal cord of SMA mice as compared to controllittermates. The RAGE expression was correlated with the SMA disease severity. Therefore, RAGEmay contribute to pathogenesis of SMA. However, the roles of RAGE in SMA are not fullyunderstood and effects of RAGE-related treatment are still unclear.The first aim of this project is to investigate the role of RAGE in cultured motor neurons. We willtry to understand whether SMN knock-down in motor neurons will influence the expression of RAGEand RAGE-associated factors and whether up-regulation of RAGE will rescue potential abnormalphenotypes after SMN knock-down in motor neurons. The second aim of this project is to investigatethe role of RAGE in SMA using an SMA mouse model. We will investigate the effects of RAGEup-regulation in SMA mice by means of generating RAGE transgenic SMA mice. The third aim of thisproject is to investigate the therapeutic responses of intracerebroventricular delivery ofadenoassociated viral vectors carrying RAGE gene (AAV-RAGE) in SMA mice. We intend to test theeffects of AAV-RAGE therapy with different treatment time and dosage in SMA mice with differentdisease severity.