Hedong Li - US grants

Project Summary: Spinal cord injury (SCI) is a devastating neurological disorder that often impairs the daily function of patients for their entire life. One of the major obstacles in treating SCI is how to restore the lost neuronal functions. Despite decades of research efforts, current strategies including stem cell transplantation have not resulted in a successful clinical therapy. Therefore, there is an urgent need to develop novel technology to treat SCI. This research team has recently developed an innovative approach to reprogram reactive astrocytes into functional neurons in situ for brain repair by injecting viral particles expressing a single neural transcription factor NeuroD1 into the adult mouse cortex (Guo et al., Cell Stem Cell, BEST of 2014 article). This proposal will introduce this cutting-edge in vivo reprogramming technology into SCI. In particular, the PI proposes to convert endogenous reactive astrocytes into functional neurons by forced expression of a neuronal specific microRNA (miRNA) miR-124. Most of current in vivo reprogramming studies including the one from this team have been done by using viral vectors expressing neurogenic transcription factors; and in vivo neuronal conversion by miRNAs has not been reported. MiRNAs are small non-coding RNAs that play pivotal roles during neural development and diseases. The miRNA function could be potent in that one miRNA may regulate many target genes through the unique imperfect base-pairing mechanism. Furthermore, their small size (~22 nucleotides) makes them attractive for therapeutic application since they may easily penetrate tissues and be taken up by target cells. MiR-124 plays critical roles in neurogenesis, neuronal differentiation and maturation, which makes it an ideal candidate for neuronal reprogramming. Therefore, in this proposal, the PI will test the hypothesis that forced expression of miR-124 can convert reactive astrocytes into functional neurons in the injured spinal cord, and that converted neurons can integrate into the local neuronal circuitry and promote functional recovery after SCI. The PI proposes two specific aims: 1) To determine conversion of reactive astrocytes into neurons by miR-124 after SCI; 2) To determine functional integration of miR-124-converted neurons and their effects on animal's behavior after contusive SCI. Completion of the proposed study here will show feasibility of miR-124-mediated glia-neuron conversion in vivo and lay out foundation for therapeutic application of this small RNA molecules as a synthetic drug in the future. In addition, the success of this proposal will potentially lead to a novel therapeutic treatment for SCI as well as other neurological diseases such as traumatic brain injury (TBI) and amyotrophic lateral sclerosis (ALS).

Project Summary: One major obstacle in treating spinal cord injury (SCI) is to replenish neurons that are lost during the course of injury and to restore local neuronal circuitry for functional repair. Stem cell transplantation therapy once showed great promise. However, its efficacy has not been satisfactory in clinical trials; immunorejection and ethical issues remain problematic. In vivo reprogramming is emerging as a potentially new breakthrough in regenerative medicine. This innovative technology literally converts endogenous glial cells into functional neurons for repair purposes, bypassing the challenging questions that stem cell replacement therapies are facing. Previous research from the PI?s lab has demonstrated that reactive astrocytes can be successfully converted in situ into functional neurons in both injured brain and brain of a model for Alzheimer?s disease by overexpression of a single transcription factor NeuroD1 (Guo et al, 2014; BEST of 2014 Article in Cell Stem Cell). However, molecular mechanisms of the reprogramming process remain elusive. MicroRNAs (miRNAs) are small non-coding RNAs that play pivotal roles during neural development and diseases. The function of miRNAs could be extensive given that one miRNA may regulate many target genes through the unique imperfect base-pairing mechanism exerting a global impact on the gene expression profile in a cell. In this proposal, the PI will combine his expertise on SCI and miRNA, and examine miRNA function during NeuroD1-mediated neuronal conversion in a mouse model of SCI. The central hypothesis of this proposal is that miRNAs play essential roles during the neuronal reprogramming process, and that forced expression of miRNAs, as well as NeuroD1, can reprogram reactive astrocytes into neurons for functional repair after SCI. The PI proposes three specific aims: 1) To determine miRNA function during NeuroD1-mediated neuronal conversion in the injured spinal cord; 2) To determine conversion of reactive astrocytes into neurons by miRNAs after SCI; 3) To determine functional integration of miRNA-converted neurons and their effects on animal?s behavior after contusive SCI. Completion of this proposal will show feasibility of miRNA-mediated glia-to-neuron conversion and lay out foundation for therapeutic application of this small RNA molecule as a synthetic drug in the future. The PI believes that this proposal will lead to a novel therapeutic treatment for SCI as well as other neurological disorders.

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.