PTB联合小分子诱导星形胶质细胞重编程运动神经元在脊髓损伤后修复中的作用

多聚嘧啶序列结合蛋白（PTB）是一种RNA结合蛋白且在神经元的诱导和分化中扮演重要角色，目前有研究发现通过沉默PTB可以将星形胶质细胞重编程为有功能的神经元，并促进相应疾病模型小鼠的功能恢复。脊髓损伤是一种常见且治疗困难的疾病。脊髓损伤后星形胶质细胞发生反应性增生并在损伤处形成胶质疤痕，进而抑制神经元和轴突的再生；同时运动神经元大量丢失，受损神经元再生能力有限；这些给修复脊髓损伤带来了巨大难度。本项目预实验发现：体外通过病毒沉默PTB可使小鼠脊髓反应性星形胶质细胞重编程为运动神经元，同时联合添加与运动神经元分化相关的小分子维甲酸和嘌吗啡胺可显著提高运动神经元的转分化率；体内沉默PTB联合饲喂维甲酸和嘌吗啡胺可有效促进脊髓损伤小鼠的功能恢复。因此，本项目拟进一步通过各种实验技术手段研究PTB联合维甲酸和嘌吗啡胺的重编程策略在脊髓损伤后修复中的作用，进而实现更好的脊髓损伤修复和功能重建效果。

Polypyrimidine-tract-binding protein(PTB) is a RNA-binding Protein which plays key roles in neurogenesis. Xiang-Dong Fu group reported a one-step strategy to convert brain astrocytes to functional neurons by depleting PTB. They efficiently converted midbrain astrocytes into functional DA neurons that integrate into the nigrostriatal dopamine pathway. Applying this approach to a chemically induced model of Parkinson’s disease, they demonstrated partial replenishment of lost DA neurons and the restoration of striatal dopamine, leading to reversal of motor deficits. Hui Yang group reported that downregulation of PTB resulted in the conversion of Müller glia into retinal ganglion cells (RGCs) with a high effificiency, leading to the alleviation of disease symptoms associated with RGC loss. Furthermore, this approach also induced neurons with dopaminergic features in the striatum and alleviated motor defects in a Parkinson’s disease mouse model. Spinal cord injury (SCI) remains an unsolved problem that causes many years of disability and suffering for millions of patients. After SCI, astrocytes proliferate to form a scar that preserves the integrity of surrounding cells. However, the persistence of a glial scar is detrimental to functional recovery of a damaged spinal cord, largely because this scar not only forms a physical barrier but also secretes inhibitors of axonal growth. Injury to the spinal cord leads to irreversible loss of neurons and the regenerative capacity of the damaged neurons is limited. Our pre-experimental results shows that, mouse primary spinal astrocytes in vitro can be directly transformed into motor neurons by downregulating PTB, and in combination with the small molecules retinoic acid (RA) and purmorphamine (PMA) can significantly enhance the conversion efficioncy of motor neuron. We found that PTB silencing and in combination with RA and PMA in SCI not only improves motor function recovery, but also enhances thermal pain. In this project, we want to explore an efficient conversion of isolated mouse spinal cord astrocytes to motor neurons by PTB knockdown and in combination with small molecules. Applying this approach to SCI, in situ reprogramming of endogenous astrocytes to motor neurons and improving the microenvironment of axon regeneration. We also want to explore the functions of PTB antisense oligonucleotides (PTB ASO) in SCI. Our results may achieve a better SCI repair and functional reconstruction effect.