Viral-mediated knockdown of Atxn2 attenuates TDP-43 pathology and muscle dysfunction in the PFN1C71G ALS mouse model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive motor neuron loss and muscle atrophy. Hyperphosphorylated aggregation of the RNA-binding protein, TDP-43, in the motor cortex and spinal cord are defining molecular features of ALS, suggesting TDP-43 dysfunction underlies disease pathogenesis. This phenomenon, however, has been difficult to recapitulate endogenously in animal models, impeding characterization of TDP-43 pathobiology in neurodegeneration. In this study, we report age-dependent accumulation of TDP-43 pathology in the spinal cord and progressive muscle-related deficits in transgenic mice expressing the ALS-associated PFN1C71G mutant protein. We show that transgenic neuronal expression of PFN1C71G induces early hyperphosphorylation of endogenous TDP-43 in the spinal cord that augments over time, preceding accumulation of insoluble non-phosphorylated TDP-43 and the manifestation of muscle denervation and motor dysfunction. Sustained knockdown of Atxn2 in the central nervous system (CNS) in pre-symptomatic PFN1C71G mice by AAV-driven expression of an artificial microRNA (AAV-amiR-Atxn2) reduces aberrant TDP-43 in the spinal cord, while delaying neurodegeneration and improving muscle and motor function. RNA-sequencing analysis of spinal cord samples from PFN1C71G mice and ALS donors show shared patterns of transcriptional perturbation, including a pro-inflammatory gene signature that is attenuated by AAV-amiR-Atxn2. Notably, impaired regulation of the PFN1C71G skeletal muscle transcriptome exceeds that of the spinal cord and also improved by Atxn2 reduction in the CNS. Lastly, we find significant gene co-expression network homology between PFN1C71G mice and human ALS, with shared dysregulation of modules related to neuroinflammation and neuronal function and uncover novel hub genes that provide biological insight into ALS and potential drug targets that can be further investigated in this mouse model. RNA-seq profiling of C71G mutant PFN1 transgenic mice and age-matched wildtype FVB/NJ controls. Treated with AAV-amiR-Atxn2.

肌萎缩侧索硬化症（Amyotrophic Lateral Sclerosis, ALS）是一种致命的神经退行性疾病，以进行性运动神经元丢失和肌肉萎缩为典型特征。运动皮层与脊髓内RNA结合蛋白（RNA-binding protein）TDP-43的过度磷酸化聚集，是ALS的标志性分子特征，提示TDP-43功能异常是疾病发病机制的核心驱动因素。然而，这一现象难以在动物模型中实现内源性重现，极大阻碍了神经退行性疾病领域中TDP-43病理生物学的研究进展。
本研究报道了表达ALS相关PFN1C71G突变蛋白的转基因小鼠模型：其脊髓内可观察到年龄依赖性的TDP-43病理积累，并伴随进行性肌肉相关功能缺陷。研究证实，PFN1C71G的神经元转基因表达可诱导脊髓内源性TDP-43的早期过度磷酸化，且该修饰随时间逐渐增强，早于不溶性非磷酸化TDP-43的聚集，以及肌肉去神经支配和运动功能障碍的临床表型出现。
通过腺相关病毒（adeno-associated virus, AAV）介导的人工微小RNA（artificial microRNA, amiR）表达系统，在症状前PFN1C71G小鼠的中枢神经系统（central nervous system, CNS）中持续敲低共济失调蛋白2（Ataxin-2, Atxn2），可显著减少脊髓内异常积累的TDP-43，同时延缓神经退行性病变进程，并改善肌肉与运动功能。对PFN1C71G小鼠及ALS患者的脊髓样本进行RNA测序（RNA-sequencing）分析，结果显示二者存在共通的转录扰动模式，其中促炎基因特征可被AAV-amiR-Atxn2所显著减弱。
值得注意的是，PFN1C71G小鼠骨骼肌转录组的调控异常程度高于脊髓样本，且该异常同样可通过中枢神经系统内Atxn2的敲低得到有效改善。最后，本研究发现PFN1C71G小鼠与人类ALS之间存在显著的基因共表达网络同源性，二者在神经炎症与神经元功能相关模块上存在共同的表达失调；同时还发掘出全新的枢纽基因，可为ALS的生物学机制研究提供重要见解，并为潜在药物靶点提供可在该小鼠模型中进一步验证的候选对象。本研究对C71G突变PFN1转基因小鼠及年龄匹配的野生型FVB/NJ对照小鼠开展了RNA测序分析，其中部分小鼠经AAV-amiR-Atxn2处理。