Regeneration of Functional Neurons After Spinal Cord Injury via in situ NeuroD1-Mediated Astrocyte-to-Neuron Conversion.

Brendan Puls,Gong Chen,Kevin He,Lei Zhang,Yan Ding,Austin Redilla,Cody Forsyth,Xiaoyun Ding,Alice Cai,Zifei Pei,Mei Jiang,Zhuofan Lei,Tanvi Rana,Fengyu Zhang,Hedong Li,Mengjie Pan,Morgan Metzger,Matthew G Keefe ,Y Bai ,Michael M Lai

doi:10.3389/fcell.2020.591883

Abstract

Spinal 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 vivo neuronal 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 factor NeuroD1. Here we demonstrate regeneration of spinal cord neurons from reactive astrocytes after SCI through AAV NeuroD1-based gene therapy. We find that NeuroD1 converts 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 after conversion. Electrophysiological recordings show that the NeuroD1-converted neurons can functionally mature and integrate into local spinal cord circuitry by displaying repetitive action potentials and spontaneous synaptic responses. We further show that NeuroD1-mediated neuronal conversion can occur in the contusive SCI model with a long delay after injury, allowing future studies to further evaluate this in vivo reprogramming technology for functional recovery after SCI. In conclusion, this study may suggest a paradigm shift from classical axonal regeneration to neuronal regeneration for spinal cord repair, using in vivo astrocyte-to-neuron conversion technology to regenerate functional new neurons in the gray matter.

Highlights

Spinal cord injury (SCI) is a devastating central nervous system (CNS) disorder and often leads to loss of motor and sensory functions below the injury site, even paralysis depending on the severity of the injury (Adams and Hicks, 2005)
We previously demonstrated that expressing NeuroD1 in reactive astrocytes after brain injury can directly convert astrocytes into neurons (Guo et al, 2014; Chen et al, 2020; Zhang et al, 2020)
At 1 wpi, we found that the control GFP retroviruses infected a mixture of glial cell types including reactive astrocytes (GFAP+ and some GFAP+/Olig2+), oligodendrocyte progenitor cells (OPCs) (Olig2+), and microglia (Iba1+) (Figures 1C,D), but not NeuN+ neurons (Figure 1D)

Summary

Introduction

Spinal cord injury (SCI) is a devastating central nervous system (CNS) disorder and often leads to loss of motor and sensory functions below the injury site, even paralysis depending on the severity of the injury (Adams and Hicks, 2005). Resident astrocytes react to injuryinduced cytokines and dramatically upregulate the expression of a number of proteins such as the astrocytic marker GFAP and the neural progenitor markers Nestin and Vimentin (Sofroniew, 2009) These reactive astrocytes become proliferative and hypertrophic in cell morphology. The spinal neurons that are lost during and after the injury need to be replaced in order to rebuild the local neuronal circuits. In this regard, stem cell transplantation has been reported to achieve certain success (Tuszynski et al, 2014; Lu et al, 2017), the identity of transplanted cells and restoration of functional circuitry in the injury site still require further investigation (Goldman, 2016)

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