Manipulating NeuroD1 expression by MicroRNAs to Optimize Neuronal Conversion for Spinal Cord Injury Repair

项目摘要： 脊髓损伤（SCI）是一种破坏性的中枢神经系统（CNS）损伤， 患者的感觉功能。治疗SCI的一个重大挑战是补充在治疗过程中失去的神经元。 病理过程。体内重编程是最近开发的技术，并且代表了一个主要的 再生医学的重大突破这项创新技术将内源性神经胶质细胞转化为 功能性神经元的修复。在体内将反应性星形胶质细胞重编程为功能性神经元， 已在多份报告中成功证明，其中包括PI实验室通过使用单一转录的报告 在损伤的大脑中，神经因子NeuroD 1（Guo等人，2014，Cell Stem Cell）。PI正在进行的研究计划是 确定这种体内重编程技术是否可以在受损的脊髓中再生功能性神经元。 他们的初步结果已经表明，NeuroD 1可以有效地将反应性星形胶质细胞转化为新的 脊髓神经元然而，由高度和持续表达的NeuroD 1转化的神经元是 几乎完全是兴奋性（即兴奋性）神经元亚型，这与以下事实一致： NeuroD 1是一种发育过程中的神经元谱系决定因子。实际上， 并且需要抑制性神经元来重建用于功能修复的最佳神经元回路。PI认为， 在新转化的神经元中，高水平的NeuroD 1驱使它们进入多巴胺能亚型， 他们可以通过调节NeuroD 1表达水平来产生抑制性神经元。 神经元转化过程为此，他们设计了一种新的NeuroD 1表达病毒结构， 包含一个microRNA（miRNA）响应元件。特别是，他们插入了一个miR-16的串联重复序列， 124（一种神经元miRNA）靶序列，从而 NeuroD 1的表达可通过miR-124的抑制机制来调节。因此，我们可以实现高 星形胶质细胞中NeuroD 1的表达水平（miR-124低）使神经元重编程发生， 转化神经元中NeuroD 1水平降低（miR-124高），从而允许产生抑制性 神经元亚型在该提议中，PI将确定在小鼠中通过ND 1 - 124 T的神经元转化的效率。 受伤的脊髓更重要的是，它们将确定转化神经元的特定亚型， 这种转化是否改善SCI后的功能恢复。他们的核心假设是， 神经元转化期间的NeuroD 1表达有益于产生多样化的神经元亚型， 改善SCI后动物的行为结果。PI提出了两个具体目标：1）确定神经元 ND 1 - 124 T的转化效率和损伤脊髓中转化神经元的神经元亚型; 2） 确定ND 1 - 124 T转化神经元的功能整合及其对动物行为的影响， 挫伤性脊髓损伤PI认为，完成该提案将优化治疗干预 脊髓损伤和其他神经系统疾病。

Project Summary: Spinal cord injury (SCI) represents a devastating central nervous system (CNS) injury that impairs mobility and sensory function of afflicted patients. A significant challenge in treating SCI is to replenish the neurons lost during the pathological process. In vivo reprogramming is a recently developed technology and represents a major breakthrough in regenerative medicine. This innovative technology literally converts endogenous glial cells into functional neurons for repair purposes. In vivo reprogramming reactive astrocytes into functional neurons has been successfully demonstrated in several reports including one from the PI’s lab by using a single transcription factor, NeuroD1, in the injured brain (Guo et al, 2014, Cell Stem Cell). The PI’s ongoing research programs is to determine if this in vivo reprogramming technology can regenerate functional neurons in the injured spinal cord. Their preliminary results have already shown that NeuroD1 can efficiently convert reactive astrocytes into new neurons in the spinal cord. However, the neurons converted by highly and continuously expressed NeuroD1 are almost exclusively of the glutamatergic (i.e. excitatory) neuronal subtype, which is consistent with the fact that NeuroD1 is a glutamatergic neuron-lineage determination factor during development. In reality, both excitatory and inhibitory neurons would be needed to rebuild optimal neuronal circuitry for functional repair. The PI believes that high level of NeuroD1 in newly converted neurons drive them into the glutamatergic subtype, and reason that they can generate inhibitory neurons using NeuroD1 by modulating NeuroD1 expression level during the neuronal conversion process. Toward that, they have engineered a new NeuroD1-expression viral construct that contains a microRNA (miRNA)-responsive element. In particular, they have inserted a tandem repeat of miR- 124 (a neuronal miRNA) target sequence at the 3’-end of the NeuroD1-coding sequence (ND1-124T), so that NeuroD1 expression can be regulated by the inhibitory mechanism of miR-124. Thus, we can achieve a high level of NeuroD1 expression in astrocytes (low in miR-124) for neuronal reprogramming to occur, and a much reduced level of NeuroD1 in converted neurons (high in miR-124) thereby allowing generation of inhibitory neuronal subtypes. In this proposal, the PI will determine the efficiency of neuronal conversion by ND1-124T in the injured spinal cord. More importantly, they will determine the specific subtypes of the converted neurons and whether such conversion improves functional recovery after SCI. Their central hypothesis is that controlled NeuroD1 expression during neuronal conversion is beneficial in generating diversified neuronal subtypes and improving animal’s behavioral outcomes after SCI. The PI proposes two specific aims: 1) To determine neuronal conversion efficiency of ND1-124T and neuronal subtypes of converted neurons in the injured spinal cord; 2) To determine functional integration of ND1-124T-converted neurons and their effects on animal’s behavior after contusive SCI. The PI believes that completion of the proposal will lead to optimized intervention for the treatment of SCI as well as other neurological disorders.