Regeneration of dorsal spinal cord neurons after injury viain situNeuroD1-mediated astrocyte-to-neuron conversion

AbstractSpinal cord injury (SCI) often leads to impaired motor and sensory functions, partially because the injury-induced neuronal loss cannot be easily replenished through endogenous mechanisms.In vivoneuronal reprogramming has emerged as a novel technology to regenerate neurons from endogenous glial cells by forced expression of neurogenic transcription factors. We have previously demonstrated successful astrocyte-to-neuron conversion in mouse brains with injury or Alzheimer’s disease by overexpressing a single neural transcription factorNeuroD1via retroviruses. Here we demonstrate regeneration of dorsal spinal cord neurons from reactive astrocytes after SCI via adeno-associated virus (AAV), a more clinically relevant gene delivery system. We find thatNeuroD1converts reactive astrocytes into neurons in the dorsal horn of stab-injured spinal cord with high efficiency (∼95%). Interestingly,NeuroD1-converted neurons in the dorsal horn mostly acquire glutamatergic neuronal subtype, expressing spinal cord-specific markers such as Tlx3 but not brain-specific markers such as Tbr1, suggesting that the astrocytic lineage and local microenvironment affect the cell fate of conversion. Electrophysiological recordings show that theNeuroD1-converted neurons can functionally mature and integrate into local spinal cord circuitry by displaying repetitive action potentials and spontaneous synaptic responses. We further show thatNeuroD1-mediated neuronal conversion can occur in the contusive SCI model, allowing future studies of evaluating this reprogramming technology for functional recovery after SCI. In conclusion, this study may suggest a paradigm shift for spinal cord repair usingin vivoastrocyte-to-neuron conversion technology to generate functional neurons in the grey matter.