Dynamic regulation of NeuroD1 expression level by a novel viral construct during astrocyte-to-neuron reprogramming

Astrocyte-to-neuron reprogramming presents a viable approach for regenerative medicine. The reprogramming factor NeuroD1 has demonstrated the capability of neuronal reprogramming with high efficiency and yet primarily glutamatergic neuronal subtype. However, diversified neuronal subtypes are needed to establish appropriate neuronal connectivity in disease/injury conditions. We reason that continuously high level of NeuroD1 expression forces the reprogrammed neurons into glutamatergic subtype and that reducing NeuroD1 level after reprogramming may allow the generation of neurons with diversified subtypes. For this purpose, we engineered a novel viral expression vector by which NeuroD1 expression can be dynamically regulated during the reprogramming process. Specifically, the target site of a neuron-specific microRNA (miR-124) is incorporated in the expression system. Therefore, this novel construct would still achieve a high NeuroD1 expression level in astrocytes for reprogramming to occur and yet reduce its level in the reprogrammed neurons by suppression of endogenous miR-124. In this study, we demonstrated that this construct elicits a dynamic gene expression pattern with much reduced level of NeuroD1 at later stages of neuronal reprogramming. We also showed that this construct still retains relatively high reprogramming efficiency and can generate mature neurons with an enhanced GABAergic neuronal phenotype.