Reprogramming Astrocytes To Motor Neurons By Activation Of Endogenous Ngn2 And Isl1

Objective: The occurrence of the acute central nervous system(CNS)injury and chronic neurodegenerative diseases is related to irreversible neuronal loss,which eventually leads to permanent neurological dysfunction.Spinal cord injury(SCI)is a common CNS injury,which is the most serious complication of spinal cord injury.SCI leads to irreversible neuron loss and the interruption of the ascending and descending spinal tract,leading to sensory and motor dysfunction below the injury plane,even severe disability.So far,there is no effective method to promote the recovery of neural function in the clinic,so the regeneration and functional reconstruction of neurons is one of the most challenging medical problems.In recent years,cell reprogramming has been widely used in cell replacement therapy,disease modeling,drug screening,and so on.Direct neuron reprogramming has also attracted extensive attention,and will become a new strategy for the treatment of neurodegenerative diseases and CNS injury.The purpose of this study is to reprogram astrocytes into motor neurons(MNs)directly by gene editing and to explore the possibility of reprogramming endogenous non-neural cells into spinal MNs.As a proof of concept study,we used the CRISPRa system to activate endogenous transcription factors Ngn2 and Isl1,and converted mouse spinal cord astrocytes into functional MNs directly and efficiently in vitro and in vivo,which provided a theoretical and experimental basis for reprogramming technology applied in clinical cell therapy of SCI.Methods: The primary astrocytes were isolated and cultured from the spinal cord of neonatal mice,and the purity of third-generation astrocytes was identified by immunofluorescence staining;Lentiviral vectors of sg RNA-Ngn2,sg RNA-Isl1 were constructed;The activation efficiency of sg RNA was detected by q RT-PCR and western blot;sg RNA-Ngn2,sg RNA-Isl1,and d Cas9-VP64 were then transfected into astrocytes and reprogrammed into neurons;q RT-PCR,immunofluorescence staining and whole-cell patch-clamp were used to analyze the characteristics of the induced neurons.The mouse embryonic fibroblasts(MEFs)were converted into neurons by similar methods.The subtypes and functions of the induced neurons were analyzed by immunofluorescence staining and whole-cell patch-clamp;After that,sg RNA-Ngn2,sg RNA-Isl1,and d Cas9-VP64 were injected into the spinal cord of adult h GFAP-Cre ERT2;Ai14 mice by AAV vector,and the neurogenesis was analyzed by immunofluorescence staining at different time point.To further investigate the progress of axonal projections of the induced neurons in the h GFAP-Cre ERT2;Ai14mice spinal cord,a retrograde tracer,cholera toxin subunit B(CTB)was injected into the sciatic nerve at 42 dpi.The mice were sacrificed 7 days after CTB injection for analysis of the CTB-labeled neurons in the lumbar segment of the spinal cord.Results:1)Activation of endogenous Ngn2 and Isl1 with d Cas9-VP642)Activation of Ngn2 and Isl1 enabled mouse spinal cord astrocytes to acquire motor neuronal properties.3)MEFs could be efficiently converted into i MNs by activation of Ngn2 and Isl1.4)In vivo activation of endogenous Ngn2 and Isl1 could efficiently convert astrocytes into motor neurons in the adult spinal cord.5)Astrocytes in the white matter of the spinal cord fail to reprogram to neurons.6)The in vivo converted i MNs in the spinal cord could extend their axonal projections into the sciatic nerve.Conclusion: Two transcription factors Ngn2 and Isl1 targeted by the CRISPRa system could convert astrocytes from the mouse spinal cord into functional motor neurons efficiently in vitro and in vivo.