以第十七型脊髓小腦運動失調症小鼠模式探討ERK活化與Purkinje神經細胞退化之相關性

Abstract

脊髓小腦共濟失調第十七型（SCA17）是常染色體顯性遺傳小腦共濟失調的一種亞型，是由TATA結合蛋白（TBP）基因中CAG / CAA重複序列的異常擴展所引起。SCA17的臨床特徵是漸進性共濟失調、痙攣、舞蹈症、類帕金森症和認知障礙。而於神經病理上，以小腦萎縮、Purkinje細胞丟失和神經膠質增生為SCA17疾病常見之特徵，並且最終導致共濟失調和行為障礙。實驗室先前已經建立了SCA17轉基因小鼠，以研究異常的TBP聚集體如何誘導SCA17病理作用。在這項研究中，我們主要分析了4-8周大的小鼠，即疾病發作的起始年齡。 我們發現在4週齡大SCA17轉殖基因小鼠中，出現了Purkinje細胞減少，Purkinje細胞核中出現TBP蛋白的累積和小膠質細胞活化。研究尚發現自6週齡大SCA17轉殖基因小鼠，開始發生Purkinje細胞退化情況，亦同時出現星形膠質和Bergmann膠質細胞的增生，並且觀察到在增生的星形膠質細胞和Bergmann膠質細胞中， ERK有被顯著活化之現象。而6週齡大的SCA17轉基因小鼠，也表現出步態紊亂與運動不協調的情況。進一步確認Purkinje細胞死亡的分子機制，6週和8週齡大的SCA17轉基因小鼠中，Bax / Bcl2比例，活化態的caspase-3和89 kD片段的PARP都有顯著增加。綜和上述，SCA17小鼠自6週齡開始，步態異常及運動不協調的表現與神經病理發病的機制在時序上是相呼應的。我們的研究表明活化的pERK是存在星形膠質細胞和Bergmann膠質細胞中，並且可能導致SCA17小鼠小腦神經發炎現象及神經元凋亡。 此外，我們篩選出了一種GSK3β潛力抑制劑PHA-767491，也已知是一種Cdc7 / CDK抑制劑。我們發現PHA-767491透過抑制神經炎症機制對蛋白質聚集相關疾病，如AD、SCA17各種模式均產生了神經保護作用。在SCA17小腦初代細胞培養和器官切片培養中，給予PHA-767491能降低神經膠質細胞的增生。在SCA17小鼠的動物模型中，給予PHA-767491亦可改善步態異常和降低了神經炎症情況，根據以上結果，我們認為聚焦在抑制神經炎症的途徑，可能是一種治療SCA17疾病的潛在策略。

Spinocerebellar ataxia type 17 (SCA17) is one subtype of autosomal dominant cerebellar ataxia group caused by the CAG/CAA expansion in the TATA-binding protein (TBP) gene. The clinical features are progressive ataxia, spasticity, chorea, Parkinsonism and cognitive impairment. Cerebellar atrophy, Purkinje cell loss and gliosis are the neurological hallmarks of SCA17 disease, which ultimately lead to ataxia and behavioral disorders. We have established the SCA17 transgenic mice to study how abnormal TBP aggregates could induce the SCA17 pathology.

In this study, we focused the analysis of mice at 4-8 week-old, the disease onset initiation age. We found that a reduction of Purkinje cell count, mutant TBP aggregation in the Purkinje cell nuclei and the activation ofmicroglia appeared in the 4 week-old SCA17 transgenic mice. The degeneration of Purkinje cells occurs since 6 weeks old. The presence of astrogliosis and Bergmann’s gliosis was also observed since then. The activated ERK was identified in the reactive astrocytes and Bergmann’s glial cells. Simultaneously, SCA17 transgenic mice showed abnormal motor coordination. Furthermore, the expression of Bax/Bcl2 ratio, active caspase-3 and cleaved PARP were significantly increased in the 6- and 8- week-old SCA17 transgenic mice. The gait abnormalities and motor incoordination in the 6 week-old SCA17 mice are corresponded to the neurological pathogenesis. Our study suggests that the activation of ERK in astrocytes and Bergmann glia may contribute to the elevated gliosis and the neuronal apoptosis in the SCA17 mouse cerebellum.

Further, we identified a potential GSK3 inhibitor PHA-767491, also known as Cdc7/CDK inhibitor, which has a neuroprotective effect by inhibiting the neuroinflammation in the protein aggregation diseases, AD and SCA17 models. PHA-767491 reduced the active glial cells in both the SCA17 primary and organotypic slice cultures. In addition, PHA-767491 improved the gait abnormalities and reduced inflammation in the SCA17 transgenic mice. From the above data, we suggest that inhibition of the neuroinflammatory pathways might be a potential therapeutic strategy for the SCA17 disease.