The Study On Conversion Of Astrocytes Into Neurons Mediated By Long Noncoding RNA TUNA

Objective The adult cerebral cortex and spinal cord lack the capacity to regenerate neurons following degenerative disease,stroke or traumatic injury.Conversion of nonneuronal cells into neurons has been reported as a novel cell replacement therapy for central nervous system(CNS)repair that would enable direct therapeutic application and avoid immunosuppression.Astrocytes are the most abundant cell type in the brain and develop from the same precursor cells as neurons.In addition,astrocytes are always highly proliferative;therefore,they are considered the perfect starting cells with which to regenerate neurons.Neurogenic transcription factors,micro RNAs,and small-molecule compounds have been found to successfully convert nonneuronal cells into neurons.Recent studies have shown that TUNA(Tcl1Upstream Neuron-Associated long noncoding RNA)forms a complex with three RNA-binding proteins(RBPs),and the TUNA-RBP complex has been detected at the promoters of neurogenic transcription factors,including Sox2,Nanog,and Fgf4.Knockdown of TUNA or the individual RBPs blocks the differentiation of mouse embryonic stem cells(m ESCs)into neurons.However,the neuronal reprogramming ability of long noncoding RNAs(lncRNAs)remains unexplored.Here,we show that overexpression of the lncRNA TUNA alone in mouse astrocytes induces their conversion into neuroblasts in vitro and neural stem cells in vivo.This method of direct reprogramming of astrocytes is promising for the treatment of a wide range of central nervous system(CNS)-associated injuries or diseases.Materials and methods To culture postnatal astroglia from the C57BL/6J mice cerebral cortex,we followed the protocol described previously by Schildge et al.and Heinrich et al.Astrocytes were cultured and purified and identified by immunofluorescence.The TUNA-expressing lentiviruses were injected into the astrocyte cultures and analyzed for their ability to induce neurogenesis.Neurogenesis was initially examined by Immunocytochemistry.After TUNA-induced astrocyte-neuroblast conversion,BDNF was added to the cell culture to further induce the differentiation of neuroblasts into neurons.To determine whether neurogenesis could also be induced under this clinically relevant pathological condition,we injected lentiviruses into the scratch model astrocyte culture 6 h after the scratch was made.Lentivirus expressing TUNA was individually injected into the right striatum of adult mice and analyzed for its ability to induce adult neurogenesis.The status of neurogenesis was screened by staining for the expression of representative markers for neurons at different stages.Results 1.More than 95% of the cells expressed the astrocytic marker GFAP,the viral infection efficiency was determined to be approximately 40%.2.At 1 week post viral infection(wpi),TUBB3+ cells were identified in cell cultures supplemented with lentivirus expressing TUNA but not in those supplemented with the control lentivirus.At 1 wpi,the expression of the lncRNA TUNA in cortical astrocytes infected with the TUNA-expressing virus was1,500–3,000-fold higher than that in astrocytes infected with the control virus.After TUNA-induced astrocyte-neuroblast conversion,BDNF was added to the cell culture to further induce the differentiation of neuroblasts into neurons.By 2 wpi,neurons derived from TUNA-transduced astroglia had acquired MAP2 immunoreactivity,indicative of dendritic maturation.Neu N,an RBP that regulates alternative splicing events that might be expressed in mature neurons,was not detected in TUNA-transduced astroglia.3.By 2 wpi,neurons derived from TUNA-transduced astroglia had acquired MAP2 immunoreactivity,indicative of dendritic maturation.No MAP2+ cells were detected in the scratch model cultures injected with the control virus,h GFAP-GFP,at 2 wpi.In contrast to the MAP2+ cells in the normal primary astrocyte culture,the MAP2+ cells in the scratch model were also positive for the neuronal marker Neu N.4.The NSC marker Nestin was not detected in the intact left striatum.Although stab-injury-induced reactive astrocytes were found to express Nestin,Nestin expression was not detected in the control group,indicating that the viral delivery method was minorly invasive.TUNA-induced Nestin+ cells were mainly identified surrounding the virus-injected region and showed typical immature neuronal morphology with bipolar or multipolar processes.The number of Nestin+ cells surrounding the core injection sites was estimated to be 25(X 1,000)at 1 wpi,and the number of induced Nestin+ cells were dramatically decreased at 2 wpi and 4 wpi.Together.Conclusions Adding to the most recent methods developed to convert nonneuronal cells to neuronal cells,we provide evidence showing that the single lncRNA TUNA is also sufficient to convert endogenous differentiated astrocytes into neurons in vitro and into NSCs in vivo.Our method to generate neuronal cells from astrocytes provides a new strategy in the pursuit of regeneration-based cell therapies for the treatment of CNS-associated injuries or diseases.Further studies that aim to improve neuronal differentiation will be critical to refine this reprogramming technology.