Expression analysis of pre-phenotype dt27J mouse dorsal root ganglia

Loss of function of dystonin cytoskeletal linker proteins causes neurodegeneration in the sensory ataxia, dystonia musculorum (dt). While much investigation has focused on understanding dt pathology, divergent functions of dystonin isoforms are still unknown. Here, we highlight a novel function of the dystonin-a2 isoform in mediating microtubule (MT) stability, golgi organization and flux through the secretory pathway. Using dystonin-null mice combined with isoform-specific loss of function analysis, we find dystonin-a2 is bound to MAP1B in the area surrounding the centrosome, where it maintains MT acetylation. In dt, the absence of the MAP1B-dystonin-A2 interaction results in a loss of MAP1B perinuclear localization, leading to MT deacetylation and instability. Deacetylated MTs lead to golgi fragmentation and prevent anterograde trafficking of motor proteins. Maintenance of MT acetylation through TSA administration or MAP1B overexperssion in vitro, mitigates the observed defect. These aberrations are apparent in pre-phenotype dorsal root ganglia (DRG) and primary sensory neurons, suggesting they are causal in the dt disorder. P4 dorsal root ganglia (DRG) tissue from 3 WT and 3 dt27J animals from 2 separate litters was subjected to RNA extraction and analyzed.细胞骨架连接蛋白网蛋白（dystonin）功能丧失可引发感觉共济失调、肌张力障碍（dt）表型中的神经退行性病变。尽管已有大量研究聚焦于阐明dt的致病机制，但网蛋白各蛋白亚型（isoform）的多样功能仍未明确。本研究揭示了网蛋白-a2亚型（dystonin-a2 isoform）的全新功能：其可介导微管（microtubule, MT）稳定性、维持高尔基体结构，并调控分泌通路中的物质流。本研究通过构建网蛋白全敲除小鼠，并结合亚型特异性功能缺失分析，发现网蛋白-a2可在中心体（centrosome）周围区域与微管相关蛋白1B（MAP1B）结合，进而维持微管的乙酰化水平。在dt表型小鼠中，MAP1B与网蛋白-A2的相互作用缺失会导致MAP1B的核周定位丧失，进而引发微管去乙酰化及稳定性下降。去乙酰化的微管会引发高尔基体碎片化，并阻碍运动蛋白的顺向运输。在体外实验中，通过曲古抑菌素A（TSA）处理维持微管乙酰化水平，或过表达MAP1B，均可缓解上述观察到的缺陷。这些异常在表型前期的背根神经节（dorsal root ganglia, DRG）及原代感觉神经元中即可观测到，表明它们是dt疾病的致病诱因。本研究对来自2窝幼崽的3只野生型（wild type, WT）及3只dt27J突变体小鼠的P4龄背根神经节（DRG）组织进行了RNA提取与分析。