Retrograde transmission of pathogenic TDP-43 to the CNS

项目摘要/摘要 TDP-43是一种常见的蛋白质，聚集在多种神经退行性疾病和肌肉疾病中。TDP- 43是一种普遍表达的RNA结合蛋白，主要定位于细胞核，但重新定位到 细胞应激条件下的细胞质，在神经元上形成应力颗粒，在骨骼中形成肌颗粒 肌肉。正常情况下，TDP-43在压力消除后返回到细胞核，但通过未知的疾病 机制错误折叠的TDP-43仍然以磷酸化的不溶性聚集体的形式留在细胞质中。研究 已经证明TDP-43形成淀粉样寡聚体，能够自我模板化转化，其中 错误折叠的蛋白质会导致其天然对应蛋白错误折叠，从而导致 聚合病理学。错误折叠的容易聚集的蛋白质，被称为蛋白质病态种子，是 能够通过相连的神经元网络进行细胞间的传输。在某些疾病中，如 多系统蛋白病，患者骨骼肌和神经元均有TDP-43聚集。在这些 在一些病例中，肌病症状在痴呆症发作前几年就开始了。因此，中央 这一提议背后的假设是TDP-43能够在骨骼中自我模板化转换 肌肉和传导到中枢神经系统(CNS)导致神经退化。为了 为了测试这一点，将首先测试从人类骨骼肌肌颗粒中分离出的TDP-43的能力 使用硫代黄素试验和TDP-43 FRET传感器线在体外进行自我模板转化。那么， 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，并 表达TDP-43的小鼠具有突变的核定位信号，导致细胞质TDP-43增加- 43表达。将监测小鼠的运动或认知变化，并在特定时间点处死小鼠 在蛋白质疗法种子注射之后。将检查骨骼肌是否有局部传播的迹象，以及 病理学，以确认种子是否可以在肌纤维的环境中繁殖。此外，脊椎 将检查脊髓和大脑是否有TDP-43的病理迹象，以确定是否肌肉注射 TDP-43蛋白病变种子可导致TDP-43蛋白病变传递到中枢神经系统。为了确认肌肉- TDP-43种子在较短的时间线上向神经元传递，原代小鼠成肌细胞表达 荧光标记的TDP-43将被蛋白质病种子处理，然后与原代小鼠共培养 神经元。这些神经元将被监测荧光TDP-43，这表明存在种子。如果成功，建议的 实验将显著提高对TDP-43蛋白病变及其是否可以传播的理解 从外围到中枢神经系统。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。圆满完成这些目标和培训计划 申请人将准备成为蛋白质聚集领域的独立研究员 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.