Analysis of the pathogenesis of neurodegenerative disorders using animal models with lysosomal lipid storage diseases.

神经节苷脂沉积症是一种由于溶酶体β-半乳糖苷酶(β-Gal)缺乏导致GM1神经节苷脂及其衍生物进行性神经积聚所致的进行性神经退行性疾病。人们对这种疾病的神经退行性变的机制知之甚少。在本研究中，我们建立了几只转基因(TG)/基因敲除(KO)小鼠，包括GM1合成酶TG/β-Gal KO小鼠，并试图利用这些动物模型来分析疾病的发病机制。我们建立了4个GM1合成酶TG小鼠品系，并成功培育了3个GM1合成酶TG/β-Gal KO小鼠品系。对其中一个品系的TG/KO小鼠进行的脂质分析表明，它们脑中GM1的存储水平与KO小鼠几乎相同，尽管TG/KO小鼠的GM1/Asial GM1存储比KO小鼠略有增加。这3个品系的TG/KO小鼠的症状严重程度和平均寿命与KO小鼠相似。为了分析发病机制，我们使用了出生后第8天的β-GalKO小鼠的小脑颗粒细胞。细胞质膜上GM1神经节苷脂分布异常，并伴有溶酶体GM1积聚。由于GM1位于脂筏中，并且已知它调节Trk神经营养因子受体介导的信号转导，我们研究了它在β-GalKO小鼠大脑中的行为。免疫沉淀分析表明，与野生型相比，KO小鼠大脑皮层、中脑和小脑中Trk A蛋白的磷酸化水平显著降低，GM1与Trk的结合存在缺陷。与此一致的是，Trk A的下游信号，即通过PLC Gamma的Ca++信号也受到了损害。Trk相关信号的损伤可能参与了GM1神经节苷脂沉着症小鼠脑内神经元功能的丧失和随后的神经变性。

GM1-gangliosidosis is a progressive neurodegenerative disease caused by a deficiency of lysosomal beta-galactosidase (beta-Gal) resulting in progressive neural accumulation of GM1 ganglioside and its derivatives. Little is known about the mechanisms of neurodegeneration in this disease. In this study, we generated several transgenic(TG)/knockout (KO) mice including the GM1 synthase TG/beta-Gal KO mice and also we tried to analyze the pathogenesis of the disorder using these animal models. We established 4 lines of GM1 synthase TG mice and then successfully generated 3 lines of GM1 synthase TG/beta-Gal KO mice. Lipid analysis of one line of TG/KO mice revealed that the level of GM1 storage in their brains was almost same as the KO mice, although the ratio of GM1/asial GM1 storage was slightly increased in the TG/KO mice than that of the KO mice. The severity of the symptoms and the average life spans of these 3 lines of TG/KO mice were similar to those of the KO mice. For analysis of the pathogenesis, we used cerebellar granule cells from postnatal day 8 beta-Gal KO mice. These cells showed abnormal distribution of GM1 ganglioside in plasma membrane, which was accompanied by lysosomal GM1 accumulation. Since GM1 locates in lipid rafts and it is known to regulate Trk neurotrophin receptor-mediated signal transduction, we investigated its behavior in the brain of beta-Gal KO mice. By immunoprecipitation assay, the association of GM1 with Trk was proved to be defective and the phosphorylation of Trk A protein was significantly decreased in the cerebral cortex, midbrain and cerebellum of KO mice compared to wild type littermates. Consistent with this, downstream signaling from Trk A, namely Ca++ signaling via PLCgamma was also impaired. The impairment of Trk-related signaling may contribute to the loss of neuronal function and following neurodegeneration in GM1-gangliosidosis mouse brain.