Abstract：
Multiple system atrophy (MSA) is a severe α-synucleinopathy facilitated by glial reactions; the cerebellar variant (MSA-C) preferentially involves olivopontocerebellar fibres with conspicuous demyelination. A lack of aggressive models that preferentially involve olivopontocerebellar tracts in adulthood has hindered our understanding of the mechanisms of demyelination and neuroaxonal loss, and thus the development of effective treatments for MSA. We therefore aimed to develop a rapidly progressive mouse model that recaptures MSA-C pathology. We crossed Plp1-tTA and tetO-SNCA*A53T mice to generate Plp1-tTA::tetO-SNCA*A53T bi-transgenic mice, in which human A53T α-synuclein-a mutant protein with enhanced aggregability-was specifically produced in the oligodendrocytes of adult mice using Tet-Off regulation. These bi-transgenic mice expressed mutant α-synuclein from 8 weeks of age, when doxycycline was removed from the diet. All bi-transgenic mice presented rapidly progressive motor deterioration, with wide-based ataxic gait around 22 weeks of age and death around 30 weeks of age. They also had prominent demyelination in the brainstem/cerebellum. Double immunostaining demonstrated that myelin basic protein was markedly decreased in areas in which SM132, an axonal marker, was relatively preserved. Demyelinating lesions exhibited marked ionised calcium-binding adaptor molecule 1-, arginase-1-, and toll-like receptor 2-positive microglial reactivity and glial fibrillary acidic protein-positive astrocytic reactivity. Microarray analysis revealed a strong inflammatory response and cytokine/chemokine production in bi-transgenic mice. Neuronal nuclei-positive neuronal loss and patchy microtubule-associated protein 2-positive dendritic loss became prominent at 30 weeks of age. However, a perceived decrease in tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta in bi-transgenic mice compared with wild-type mice was not significant, even at 30 weeks of age. Wild-type, Plp1-tTA, and tetO-SNCA*A53T mice developed neither motor deficits nor demyelination. In bi-transgenic mice, double immunostaining revealed human α-synuclein accumulation in neurite outgrowth inhibitor A (Nogo-A)-positive oligodendrocytes beginning at 9 weeks of age; its expression was further increased at 10 to 12 weeks, and these increased levels were maintained at 12, 24, and 30 weeks. In an α-synuclein-proximity ligation assay, α-synuclein oligomers first appeared in brainstem oligodendrocytes as early as 9 weeks of age; they then spread to astrocytes, neuropil, and neurons at 12 and 16 weeks of age. α-Synuclein oligomers in the brainstem neuropil were most abundant at 16 weeks of age and decreased thereafter; however, those in Purkinje cells successively increased until 30 weeks of age. Double immunostaining revealed the presence of phosphorylated α-synuclein in Nogo-A-positive oligodendrocytes in the brainstem/cerebellum as early as 9 weeks of age. In quantitative assessments, phosphorylated α-synuclein gradually and successively accumulated at 12, 24, and 30 weeks in bi-transgenic mice. By contrast, no phosphorylated α-synuclein was detected in wild-type, tetO-SNCA*A53T, or Plp1-tTA mice at any age examined. Pronounced demyelination and tubulin polymerisation, promoting protein-positive oligodendrocytic loss, was closely associated with phosphorylated α-synuclein aggregates at 24 and 30 weeks of age. Early inhibition of mutant α-synuclein expression by doxycycline diet at 23 weeks led to fully recovered demyelination; inhibition at 27 weeks led to persistent demyelination with glial reactions, despite resolving phosphorylated α-synuclein aggregates. In conclusion, our bi-transgenic mice exhibited progressively increasing demyelination and neuroaxonal loss in the brainstem/cerebellum, with rapidly progressive motor deterioration in adulthood. These mice showed marked microglial and astrocytic reactions with inflammation that was closely associated with phosphorylated α-synuclein aggregates. These features closely mimic human MSA-C pathology. Notably, our model is the first to suggest that α-synuclein oligomers may spread from oligodendrocytes to neurons in transgenic mice with human α-synuclein expression in oligodendrocytes. This model of MSA is therefore particularly useful for elucidating the in vivo mechanisms of α-synuclein spreading from glia to neurons, and for developing therapies that target glial reactions and/or α-synuclein oligomer spreading and aggregate formation in MSA.
摘要：
多系统萎缩（MSA）是由神经胶质反应促进的严重α-突触核蛋白病；小脑变体（MSA-C）优先涉及橄榄桥脑小脑纤维，并伴有明显的脱髓鞘。缺乏在成年期优先涉及橄榄桥脑小脑束的侵袭性模型阻碍了我们对脱髓鞘和神经轴突丢失机制的理解。从而开发了MSA的有效治疗方法。因此,我们旨在开发一种快速进展的小鼠模型,其重新捕获MSA-C病理学。我们将Plp1-tTA和tetO-SNCA*A53T小鼠杂交以产生Plp1-tTA::tetO-SNCA*A53T双转基因小鼠，其中使用Tet-Off调节在成年小鼠的少突胶质细胞中特异性产生人A53Tα-突触核蛋白-一种具有增强的聚集性的突变蛋白。这些双转基因小鼠从8周龄开始表达突变型α-突触核蛋白，当从饮食中去除多西环素时。所有双转基因小鼠都表现出快速进行性运动恶化，22周龄左右有广泛的共济失调步态，30周龄左右死亡。他们在脑干/小脑也有明显的脱髓鞘。双重免疫染色表明，髓鞘碱性蛋白在轴突标记物SM132的区域明显减少，相对保存。脱髓鞘病变表现出明显的电离钙结合衔接分子1-，精氨酸酶-1-,Toll样受体2阳性小胶质细胞反应性和胶质纤维酸性蛋白阳性星形细胞反应性。微阵列分析揭示了双转基因小鼠中强烈的炎症反应和细胞因子/趋化因子的产生。在30周龄时，神经元核阳性神经元丢失和斑片状微管相关蛋白2阳性树突状丢失变得突出。然而,与野生型小鼠相比，双转基因小鼠黑质致密质中酪氨酸羟化酶阳性神经元的减少并不显著，甚至在30周大的时候。野生型,Plp1-tTA,tetO-SNCA*A53T小鼠既没有运动障碍也没有脱髓鞘。在双转基因小鼠中，双重免疫染色显示人α-突触核蛋白在9周龄时开始在神经突生长抑制剂A（Nogo-A）阳性少突胶质细胞中积累；其表达在10至12周时进一步增加，这些增加的水平在12周,24周和30周保持。在α-突触核蛋白邻近连接测定中，α-突触核蛋白寡聚物早在9周龄时就首次出现在脑干少突胶质细胞中，然后扩散到星形胶质细胞，Neuropil,和12周龄和16周龄的神经元。脑干神经纤维中的α-突触核蛋白寡聚体在16周龄时最丰富，此后减少；然而，Purkinje细胞中的细胞逐渐增加，直到30周龄。双重免疫染色显示，早在9周龄时，脑干/小脑的Nogo-A阳性少突胶质细胞中就存在磷酸化的α-突触核蛋白。在定量评估中，在双转基因小鼠中，磷酸化的α-突触核蛋白在12、24和30周逐渐积累。相比之下,在野生型中未检测到磷酸化α-突触核蛋白，tetO-SNCA*A53T,或检查任何年龄的Plp1-tTA小鼠。发音脱髓鞘和微管蛋白聚合，促进蛋白阳性少突胶质细胞丢失，在24和30周龄时与磷酸化的α-突触核蛋白聚集体密切相关。多西环素饮食在23周时对突变型α-突触核蛋白表达的早期抑制导致完全恢复脱髓鞘；在27周时抑制导致持续的脱髓鞘并伴有神经胶质反应，尽管解决磷酸化α-突触核蛋白聚集体。总之,我们的双转基因小鼠表现出逐渐增加的脱髓鞘和脑干/小脑神经轴突丢失，在成年期迅速进行性运动恶化。这些小鼠表现出明显的小胶质细胞和星形细胞反应，炎症与磷酸化的α-突触核蛋白聚集体密切相关。这些特征紧密地模拟人MSA-C病理学。值得注意的是,我们的模型首次表明α-突触核蛋白寡聚体可能从少突胶质细胞扩散到转基因小鼠的神经元，在少突胶质细胞中表达人α-突触核蛋白。因此，这种MSA模型对于阐明α-突触核蛋白从神经胶质扩散到神经元的体内机制特别有用，以及用于开发针对神经胶质反应和/或α-突触核蛋白寡聚体扩散和MSA中聚集体形成的疗法。
