The Effect Of Astrocytic Heterogeneity On Neuronal Reprogramming

Astrocytes,the most abundant cell type in mammalian central nervous system(CNS),widely play roles in CNS physiological and pathological processes,such as regulating the cerebral blood flow,modulation of synaptic transmission,participation in blood–brain barrier,transmitter uptake and release.Under pathology conditions,astrocytes wrap around and limit the lesion through their activation and proliferation,which would be critical for neural recovery following CNS diseases and injuries.When astrocytes were first found,they were thought as a homologous cell type,which were just distinguished as fibrous and protoplasmic astrocytes by their morphologies.With the deepening research in astrocytes,now we have known that they could display cellular heterogeneous characteristic in many aspects including development,gene expression and biological function.Under various pathology conditions astrocytes also exhibit different reactive response,and play heterogeneous roles during pathological process.Because the neurons couldn't be regenerated or proliferated,and overactive astrocytes around the lesion would be harmful to neuronal axon regeneration and reconnection,they both become major obstacle for the treatment of CNS diseases and injuries.In recent years,with the development of cellular reprogramming and regenerative medicine,directly converting astrocytes,especially the overactive astrocytes to neurons gradually became a new strategy for treating CNS diseases or injuries.Currently,people have successfully induced astrocytes directly to different types of mature neurons through utilizing various transcription factors and small molecule compounds in vitro and vivo experiments.Nevertheless,reprogramming efficiency of induced neurons derived from astrocytes in mammalian CNS was still very low,furthermore,reprogramming efficiency of regional astrocytes such as cortex,spinal cord,was distinct from each other region.Besides various microenvironment which astrocytes situated in,it was still not clear that whether astroglial heterogeneity could influence neuronal reprogramming process.In our study,we cultured the cortex,cerebellum and spinal cord astrocytes from postnatal or adult mouse in vitro,then directly reprogrammed them to neurons with transcription factor NGN2 or ASCL1.The results showed that there was a significant difference in reprogramming efficiency of induced-neurons between different cultured astrocytes,and the choice of transcription factors didn't affect the trend of difference in reprogramming efficiency.Through analyzing a series of key molecules which closely related to neuronal specialization during embryonic development and neuronal transdifferentiation during genetic reprogramming process,we found that the various expression of Notch1 signaling molecules in different astrocytes might play an important role in neuronal reprogramming.Furthermore,we inhibited Notch1 signaling by ?-secretase inhibitor DAPT or lentiviral vector harboring RNAi sequence.Compared with control group,the neuronal reprogramming efficiency of interference group was obviously improved.These results demonstrated that the reprogramming efficiency was influenced by astroglial heterogeneity,which was likely mediated by Notch1 signaling in astrocytes.The study would be helpful for us utilize genetic reprogramming better to directly convert astrocytes to neurons and treat CNS diseases and injuries.