Retrograde transmission of pathogenic TDP-43 to the CNS

项目总结/摘要 TDP-43是在多种神经退行性疾病和肌病中发现聚集的常见蛋白质。TDP- 43是一种广泛表达的RNA结合蛋白，主要定位于细胞核，但重新定位于细胞核。 在细胞应激条件下，在细胞质中形成应激颗粒，在神经元中形成应激颗粒，在骨骼肌中形成肌颗粒。 肌肉.正常情况下，当压力消除时，TDP-43返回到细胞核，但通过未知疾病 错误折叠的TDP-43作为磷酸化的不溶性聚集体保留在细胞质中。研究 已经显示TDP-43形成能够自模板转化的淀粉样低聚物，其中 错误折叠的蛋白质导致其天然对应物变得错误折叠，从而导致 综合病理学这种错误折叠的易聚集蛋白质，被称为蛋白质种子， 能够通过连接的神经网络进行细胞间的传递。在某些疾病中，如 在多系统蛋白质病中，患者在骨骼肌和神经元中具有TDP-43聚集。在这些 在一些病例中，肌病症状在痴呆发作前开始数年就开始了。因此中央 这一提议背后的假设是TDP-43能够在骨骼中自模板化转化， 肌肉和传递到中枢神经系统（CNS）引起神经变性。为了 为了测试这一点，首先将测试从人骨骼肌肌颗粒中分离的TDP-43的能力， 使用硫磺素测定和TDP-43 FRET传感器线进行体外自模板转化。然后 将分离的TDP-43或重组TDP-43种子注射到野生型小鼠的后肢肌肉中， 表达TDP-43的小鼠，其具有突变的核定位信号，其导致细胞质TDP-43增加。 43表情。将监测小鼠的运动或认知变化，并在特定时间点处死 蛋白质疗法种子注射后将检查骨骼肌是否有局部传播的迹象， 病理学以确认种子是否可以在肌纤维的环境中繁殖。此外，脊柱 将检查脊髓和脑的TDP-43病理学迹象，以确定是否肌内注射 TDP-43蛋白质病种子可导致TDP-43蛋白质病传播至CNS。为了确认肌肉- TDP-43种子在较短的时间轴上向神经元传递，原代小鼠成肌细胞表达 荧光标记的TDP-43用蛋白质疗法种子处理，然后与原代小鼠共培养 神经元将监测神经元的荧光TDP-43，指示接种。如果成功，建议 实验将显着提高对TDP-43蛋白质病的理解，以及它是否可以传播 从外周到中枢神经系统这有可能极大地影响诊断方法和治疗 神经退行性疾病的选择。成功完成这些目标以及培训计划 提供的将准备申请人成为一个独立的研究者在蛋白质聚集领域 和神经肌肉疾病

Project Summary/Abstract TDP-43 is a common protein found aggregated in multiple neurodegenerative diseases and myopathies. TDP- 43 is a ubiquitously expressed RNA-binding protein that is primarily localized in the nucleus but relocates to the cytoplasm under conditions of cellular stress, forming stress granules in neurons and myo-granules in skeletal muscle. Normally TDP-43 returns to the nucleus when the stress is resolved, but through unknown disease mechanisms misfolded TDP-43 remains in the cytoplasm as phosphorylated insoluble aggregates. Studies have shown that TDP-43 forms amyloid-like oligomers which are capable of self-templated conversion in which the misfolded protein causes its native counterpart to become misfolded, leading to propagation of the aggregate pathology. The misfolded aggregation-prone proteins, referred to as proteopathic seeds, are capable of cell-to-cell transmission through connected neuronal networks. In some diseases such as multisystem proteinopathy, patients have TDP-43 aggregation in both skeletal muscle and neurons. In these cases, myopathy symptoms begin several years prior to the onset of dementia. Therefore, the central hypothesis behind this proposal is that TDP-43 is capable of self-templated conversion in skeletal muscle and transmission to the central nervous system (CNS) to cause neurodegeneration. In order to test this, TDP-43 isolated from human skeletal muscle myo-granules will first be tested for the ability to undergo self-templated conversion in vitro using a thioflavin assay and a TDP-43 FRET sensor line. Then, the isolated TDP-43 or recombinant TDP-43 seeds will be injected into the hindlimb muscles of wild type mice and mice expressing TDP-43 with a mutated nuclear localization signal which causes increased cytoplasmic TDP- 43 expression. Mice will be monitored for motor or cognitive changes and sacrificed at specific time points following proteopathic seed injection. Skeletal muscle will be examined for signs of local transmission and pathology to confirm whether seeds can propagate in the environment of a myofiber. Additionally, the spinal cord and brain will be examined for signs of TDP-43 pathology to determine whether intramuscular injected TDP-43 proteopathic seeds can lead to transmission of TDP-43 proteinopathy to the CNS. To confirm muscle- to-neuron transmission of TDP-43 seeds on a shorter timeline, primary mouse myoblasts expressing fluorescently-tagged TDP-43 will be treated with proteopathic seeds and then co-cultured with primary mouse neurons. The neurons will be monitored for fluorescent TDP-43, indicating seeding. If successful, the proposed experiments will significantly improve the understanding of TDP-43 proteinopathy and whether it can spread from the periphery to the CNS. This has the potential to greatly impact the diagnosis methods and treatment options for neurodegenerative diseases. The successful completion of these aims as well as the training plan provided will prepare the applicant to become an independent investigator in the field of protein aggregation and neuromuscular disease.