脊髓肌肉萎縮症：由動物模式到治療

Abstract

脊髓肌肉萎縮症 (Spinal Muscular Atrophy, 簡稱SMA) 為一遺傳性神經退化性疾病，因脊髓之運動神經元退化，造成患者四肢無力和肌肉萎縮。傳統上，疾病依臨床症狀及發病時間的不同，可分成第一型/嚴重型、第二型/中間型和第三型/輕型，目前尚無有效的根本治療。SMA為一自體隱性遺傳疾病，SMA的相關基因為運動神經元存活基因（SMN），一般正常人在第5條染色體上分別有一個SMN1及SMN2基因，SMN1基因所製造出的SMN蛋白質是正常且功能完整的，但SMN2基因的蛋白質產物大多是不穩定且無功能的SMN蛋白質。SMA疾病的表現導因於SMN蛋白質的不足，而大約有百分之九十六的SMA病患因在兩股染色體上之SMN1基因皆發生突變，其體內只能產生少數由SMN2基因所製造的正常SMN蛋白質，所以病患因缺乏SMN蛋白質而產生SMA的臨床表現。 將小鼠二股的Smn基因剔除，並同時植入人類的SMN2基因，此小鼠(Smn-/-SMN2+/-)表現和人類SMA的病患相似，具有脊髓運動神經元的退化和四肢肌肉的萎縮，可作為SMA的小鼠模式。但目前的SMA小鼠多有存活時間過短的缺點，不利於治療性研究的進行，而第三型(輕型)SMA小鼠雖可存活數月，但尚無明顯的表現型分析，因此本論文首先嘗試進行第三型小鼠的表現型分析，以期此SMA小鼠有機會成為適當且理想的SMA小鼠模式運用於後續治療性研究。 將第三型SMA小鼠和正常小鼠分別以其接受試驗的時間分為4組，包括三、六、九及十二個月大，每隻小鼠皆接受形態學分析、運動功能試驗、電生理檢查及脊髓病理分析。運動功能試驗包括了傾斜試驗、轉輪試驗、RotaRod試驗及下落試驗。在電生理測試中，以電刺激小鼠的坐骨神經，記錄腓腸肌經活化後所產生訊號 (CMAP)的振幅。在脊髓病理分析中，將小鼠腰髓以H&E法染色，觀察並計算脊髓前角區域內的神經元密度。 分析發現，第三型SMA小鼠的脊髓運動神經元於6個月大時開始退化，而在9個月大時，脊髓運動神經元的數目和正常小鼠相比，只剩下71%，同時在小鼠9個月大時，可檢測出SMA小鼠明顯的形態學異常，如尾末端壞死變短、耳殼不規則及後肢缺指的現象，而四個運動功能測試皆顯示有功能的下降，且腓腸肌出現了肌肉萎縮的情形；而在小鼠12個月大時，脊髓運動神經元的數目和正常小鼠相比只剩60%，該時在電生理檢查中，CMAP的振幅較正常小鼠要小。以上結果顯示，本論文所運用的形態學分析、運動功能檢測、脊髓及肌肉分析和電生理檢查，可以用來評估第三型SMA小鼠的表現型，且其在9個月大至12個月大時，是比較治療反應最適合的時間點。 過去在細胞研究上已發現，藥物valproic acid (VPA)的治療可使細胞內的SMN表現量增加，由於體內SMN蛋白質的多寡和疾病嚴重度呈逆相關，因此，VPA具有治療SMA的潛力。此外，以VPA治療SMA的療效機轉尚未非常明瞭，VPA除了可能使神經元內的SMN蛋白質表現量增加外，過去曾有研究發現VPA亦可能增加抗細胞凋亡因子的表現和促進神經元及肌肉細胞的再生。因此本論文接著以VPA治療第三型SMA模式小鼠，嘗試證實VPA於SMA的治療效果，另外並探索VPA對SMA小鼠的療效機制。 將第三型SMA小鼠隨機分為VPA治療組及無治療組，在治療組中，從小鼠6個月大起，取VPA置於飲用水中 (0.2 mg/ml)給小鼠飲用，VPA治療持續至小鼠12個月大，另取同年紀經VPA治療及未治療的正常小鼠作為比較。療效評估法包括了形態學分析、運動功能試驗、電生理檢查及脊髓和肌肉病理分析。此外亦使用西方點墨法、同步定量PCR和免疫螢光染色法來探索VPA於SMA的治療機制。 研究顯示，以VPA長期治療第三型SMA模式小鼠，可以減少SMA小鼠形態學的異常、減緩運動功能的退化、避免電生理檢查中CMAP振幅的下降、減少神經肌肉交界處的去神經化現象、降低肌肉纖維的萎縮及延緩脊髓運動神經元的退化，然而VPA的療效似乎仍不足以根治SMA小鼠的病態表現。而VPA對SMA的治療作用機制是多元的，可能的作用包括了增進SMN2基因promoter之活性及使SMN訊息RNA的alternative splicing趨向於全長SMN的狀態，因而使脊髓運動神經元及肌肉組織內的SMN蛋白質含量增加；此外，VPA提升了小鼠脊髓神經元內Bcl-2和Bcl-xL的表現量，因而可能具有抗細胞凋亡的作用；最後，VPA於SMA小鼠的脊髓內誘發了神經細胞生成及增進了神經膠質細胞的增生，也許此作用可形成神經保護的現象。 SMA患者脊髓內有過度細胞凋亡的現象。而Bcl-xL是一個抗細胞凋亡因子，具有抑制神經細胞死亡的能力，由於SMA病患的病理組織中曾發現有抗細胞凋亡因子Bcl-xL表現下降的情形，因此若能增加SMA模式小鼠體內神經細胞之Bcl-xL表現，則可能可減緩脊髓神經元的細胞凋亡，達到治療的目的。 將SMA小鼠和Bcl-xL基因轉殖小鼠交配，得到含有Bcl-xL轉殖基因的第三型SMA模式小鼠 (SMA/Bcl-xL, Smn -/-SMN2+/-Bcl-xL+/-)，另取原SMA小鼠作為對照。分別進行形態學分析、運動功能試驗、電生理檢查、脊髓病理分析及存活分析。此外亦使用西方點墨法和免疫螢光染色法了解脊髓內Bcl-xL和SMN蛋白質的表現量及細胞凋亡的狀態。 結果顯示， SMA/Bcl-xL小鼠的脊髓內Bcl-xL含量約為SMA小鼠的二倍，但 SMA/Bcl-xL小鼠和SMA小鼠的脊髓內SMN蛋白質含量皆較正常小鼠明顯偏低。而分析SMA/Bcl-xL小鼠的表現型可知，SMA/Bcl-xL小鼠的存活時間較SMA小鼠長約1.5倍、尾巴及耳緣的退化較少、運動功能較佳、肌肉萎縮較輕、電生理分析結果較為正常、脊髓運動神經元退化較少、且脊髓內有較少的細胞凋亡反應。 以上研究發現，在不影響SMN蛋白質表現的情況下，Bcl-xL的基因轉殖對SMA有明顯的治療作用。由於目前全世界SMA的相關研究，幾乎皆是以SMN蛋白質為標的，嘗試尋找可以使SMN蛋白質表現增加的藥物，因此未來Bcl-xL可作為一個新的SMA治療標的，尋找有潛力的治療藥物。

Spinal muscular atrophy (SMA) is characterized by the degeneration of spinal motor neurons, and it is associated with muscle paralysis and atrophy. Childhood SMA exhibits an autosomal recessive pattern of inheritance. Based on age of onset of symptoms and achievement of motor milestones, SMA has been subdivided into three clinical types. No curative treatment is available so far for SMA. Several mouse models for SMA have been generated through different methods. Because of the disadvantage of short survival time, heterogeneous phenotype or the non-homologous genetic situation compared to human SMA in these mouse models, there is still no standard preclinical therapeutic testing system for SMA. Type III (mild-type) SMA mice have a lifespan longer than half a year, offering a chance to become a good therapeutic testing system. This study first characterized the type III SMA mice by a series of examinations. For characterization of SMA mice, type III SMA mice (Smn−/−SMN2+/−) and wild-type mice (Smn+/+) were subdivided into four subgroups, according to their evaluated age: 3, 6, 9 and 12 months old. Each mouse underwent morphological, motor functional, electrophysiological and pathological studies. The results revealed that motor neuron degeneration in type III SMA mice started at 6 months of age, and the number of residual motor neurons reduced to 71% of wild-type mice at the age of 9 months. At the same time, SMA mice exhibited clear morphological changes such as short tails and irregular ears, motor function decline demonstrated by four motor functional tests and muscle atrophy measured by gastrocnemius muscle weight. By 12 months old, the number of residual motor neurons reduced to 60% of wild-type mice. In addition, the amplitudes of compound muscle action potentials (CMAPs) in SMA mice had decreased significantly. Valproic acid (VPA), a histone deacetylase inhibitor, increased SMN protein levels in cells derived from SMA patients. VPA also improved muscle strength in some SMA patients. SMA-related physicians and patients are becoming more interested in VPA; however, the therapeutic effects of VPA in treating SMA are still not fully understood. This study thus further investigated the treatment effects and therapeutic mechanisms of VPA in type III SMA mice. Type III SMA mice were randomly assigned to treated and untreated groups. In treated mice, VPA was added to the daily drinking water at 0.2 mg/ml from 6 to 12 months of age. The therapeutic effects were evaluated using morphological tests, motor functional tests, electrophysiological studies, spinal pathological tests, and muscle pathological studies. For investigation of therapeutic mechanisms of VPA, Western blots, real-time PCR, and immunohistochemistry were used. We found that VPA-treated SMA mice showed considerably reduced degeneration of spinal motor neurons compared to untreated SMA mice. As such, their motor function was better preserved with less muscle denervation and atrophy, greater CMAP amplitudes on sciatic nerves, and less morphological abnormalities than seen in untreated SMA mice. In addition, VPA exhibited multiple therapeutic effects in SMA. VPA elevated SMN protein levels in the spinal motor neurons. VPA also increased the levels of Bcl-2 and Bcl-xL proteins in the spinal neurons, which may reduce motor neuron apoptosis in SMA. Furthermore, VPA probably could induce neurogenesis and promoted astrocyte proliferation in the SMA mouse spinal cord, which might contribute to therapeutic effects by enhancing neuroprotection. Moreover, Bcl-xL is an anti-apoptotic member of the Bcl-2 family and acts by inhibiting proapoptotic members of the Bcl-2 family through heterodimerization. Viral-mediated Bcl-xL expression can protect motor neuron death in primary motor neuron cultures. Since the degeneration of spinal motor neurons in SMA is mediated by apoptosis, over-expression of Bcl-xL may be of benefit in SMA. This study then investigated the benefits of Bcl-xL transgenes in type III SMA mice. Crossing type III SMA mice with Bcl-xL transgenic mice created SMA/Bcl-xL mice. The Bcl-xL transgenic effects were evaluated using morphological tests, motor functional tests, electrophysiological studies, spinal pathological tests, and survival analysis. For investigation of SMN and Bcl-xL expression and degrees of spinal apoptosis, Western blots and immunohistochemistry were used. The levels of Bcl-xL protein in spinal neurons of SMA/Bcl-xL mice were nearly double of that in SMA mice while the levels of SMN protein were similar between these two groups. The SMA/Bcl-xL mice showed preserved motor function, normalized electrophysiological tests, diminished muscle atrophy, and less motor neuron apoptosis and degeneration. Although the SMA/Bcl-xL mice still showed the typical SMA morphological phenotypes throughout their lives, they lived 1.5 times longer than SMA mice. In summary, the above studies established a standard therapeutic testing system for SMA with type III SMA mice. The morphological, motor functional, electrophysiological and spinal pathological tests here could be used to evaluate treatment responses in both treated and non-treated mice between 9 and 12 months of age. In addition, by a combination of effects such as increased SMN protein levels, anti-apoptosis, and probable neuroprotection, VPA showed considerable therapeutic benefit in the mouse model of type III SMA. Over-expression of Bcl-xL has a potential for amelioration of SMA.