Study On Small Moleculars Inducing In Vivo Spinal Cord Neuronal Reprogramming And Involving Mechanism

The central nervous system(CNS)of adult mammals has little or non-regenerative ability.Therefore,the death and hereby loss of neurons induced by CNS diseases,injuries or other medical conditions remains one of the main obstacles to the functional recovery of CNS.As a result,how to replenish and replace these irreversible lost neurons became a major problem regarding the restoration of CNS functions.One of the potential methods to tackle this problem is inducing newborn neurons in vivo.A series of research results in recent years have shown that mature somatic cells could be reprogrammed into mature neurons by forcedexpression of neuron-specific transcription factors.However,the effectiveness and safety of such method is still under consideration,which hinders its possibility of clinical application.As an alternative method to TFs,manipulating cells fate with small molecules which target specific signaling pathways,epigenetic landscapes or other cellular processes offers us a brand-new way of solution regarding this problem.It has been confirmed that various small molecular compounds possess the power to maintain the self-renewal state of stem cells,promote precursor cells specification,improve TFs-induced reprogramming efficiency and /or replace TFs to achiever neuronal reprogramming.The focus of this study is to find one or several small molecular compounds that is/are capable of induce neuronal reprograming in vivo.Astrocytes are the most abundant glial cells in CNS.They play a variety of functions,such as maintenance of internal environmental stability,neuronal energy supply,synaptic pruning and so on.After CNS injuries,astrocytes are activated to exhibit morphological and functional changes,which are called Reactive Astrocytes.Under in vitro conditions,RAs could spontaneously form neuropheres,which could be passaged over multiple generations and differentiate into neurons,astrocytes and oligo-lineage cells,indicating that they have limited stem/precursor-cell characteristics.The results suggested that under induction with appropriate and adequate manipulation with small molecular compounds in vitro,astrocytes could be trans-differentiated into neurons and displayed mature neuronal function.However,it hasn’t been reported whether astrocytes can be reprogrammed into neurons by small molecular compounds in vivo.In this study,we used a variety of experimental methods to find effective small molecular compounds that can induce post-injury reactive astrocytes reprogramming into neurons,with a hope to shed lights on finding and designing clinically reliable methods of neuron regeneration.The main results of this study are as follows:Ⅰ.CHIR99021,LDN193189 CAN SUCCESSFULLY INDUCE NEURONAL REPROGRAMMING OF ASTROCYTES IN VIVO1.Under in vitro conditions,the use of CHIR99021,LDN193189 could induce astrocytes to express DCX,and display a significant morphological change,which ensemble the morphological characteristics of na?ve neurons.2.At about 2 weeks-post-induction,the newly-born neurons gradually matured,expressing neuron-specific markers such as TUBB3,MAP2,Neu N and so on.3.During the reprogramming process,the expression of classical neuron-related transcription factor,such as Neurogenin2,Neuro D1,Myt1 l were continuous increasing,which was accompanied by the up-regulation of neuronal specific genes such as DCX,TUBB3 and so on.4.Within the spinal cords of adult mice,when administrated though intraperitoneal injection,CHIR99021 and LDN193189 could reprogram reactive astrocytes around the lesion into neurons,while normal astrocytes failed to achieve such results.5.Newborn neurons located inside spinal cords could survive for more than 1 year,and such method could be successfully applied in elderly mice.Ⅱ.NOTCH1 SIGNALING PATHWAY PLAYS AN IMPORTANT ROLE IN REPROGRAMMING ASTROCYTES INTO NEURONS.1.Notch1 signaling pathway was involved in the process of neuronal reprogramming of astrocytes using forced expression of Ascl1 or Ngn2.2.Under in vitro culture conditions,Notch1-knockdown lentivirus or Notch1-specific inhibitor DAPT could reprogram astrocytes into neurons,which express neuron-specific marker such as TUBB3,MAP2,NEUN,etc.3.Notch1 is expressed in normal and reactive astrocytes located in spinal cords,and the degree of changes in expression coincides with the degree of astrocytes reactive activity.4.Using Notch1-kowndown lentivirus or intraperitoneal injection of DAPT,reactive astrocytes around the spinal cord lesions could be induced into mature neurons.In summary,this study found that reactive astrocytes were able to be reprogrammed into mature neurons.By combined intraperitoneal administration of CHIR99021 and LDN93189,reactive astrocytes could be successfully reprogrammed into mature neurons.In addition,we found that the Notch1 signaling pathway was involved during the classic astrocyteneuron reprogramming process induced by ASCL1 or NGN2,and knock-down of Notch1 signaling activity could also reprogram spinal cord reactive astrocytes into mature neurons.These results will provide a new idea for the clinical treatment of irreversible neuronal loss caused by CNS lesions or injuries.