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

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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.