Small molecules reprogram reactive astrocytes into neuronal cells in the injured adult spinal cord.

INTRODUCTION: Ectopic expression of transcription factor-mediated in vivo neuronal reprogramming provides promising strategy to compensate for neuronal loss, while its further clinical application may be hindered by delivery and safety concerns. As a novel and attractive alternative, small molecules may offer a non-viral and non-integrative chemical approach for reprogramming cell fates. Recent definitive evidences have shown that small molecules can convert non-neuronal cells into neurons in vitro. However, whether small molecules alone can induce neuronal reprogramming in vivo remains largely unknown. OBJECTIVES: To identify chemical compounds that can induce in vivo neuronal reprogramming in the adult spinal cord. METHODS: Immunocytochemistry, immunohistochemistry, qRT-PCR and fate-mapping are performed to analyze the role of small molecules in reprogramming astrocytes into neuronal cells in vitro and in vivo. RESULTS: By screening, we identify a chemical cocktail with only two chemical compounds that can directly and rapidly reprogram cultured astrocytes into neuronal cells. Importantly, this chemical cocktail can also successfully trigger neuronal reprogramming in the injured adult spinal cord without introducing exogenous genetic factors. These chemically induced cells showed typical neuronal morphologies and neuron-specific marker expression and could become mature and survive for more than 12 months. Lineage tracing indicated that the chemical compound-converted neuronal cells mainly originated from post-injury spinal reactive astrocytes. CONCLUSION: Our proof-of-principle study demonstrates that in vivo glia-to-neuron conversion can be manipulated in a chemical compound-based manner. Albeit our current chemical cocktail has a lowreprogramming efficiency, it will bring in vivo cell fate reprogramming closer to clinical application in brain and spinal cord repair. Future studies should focus on further refining our chemical cocktail and reprogramming approach to boost the reprogramming efficiency.

Identification and characterization of sex-dependent gene expression profile in glioblastoma.

Glioblastoma (GBM) is the most lethal primary tumor in the human brain and lacks favorable treatment options. Sex differences in the outcome of GBM are broadly acknowledged, but the underlying molecular mechanisms remain largely unknown. To identify the sex-dependent critical genes in the progression of GBM, raw data from several microarray datasets with the same array platform were downloaded from the Gene Expression Omnibus (GEO) database. These datasets included tumorous and normal tissue from patients with GBM and crucial sex features. Then, the differentially expressed genes (DEGs) in female and male tumors were identified via bioinformatics analysis, respectively. Functional signatures of the identified DEGs were further annotated by Gene Ontology (GO) and pathway enrichment analyses. Venn diagram and functional protein-protein interaction (PPI) network analyses were performed to screen out the sex-specific DEGs. Survival analysis of patients with differences in the expression level of selected genes was then carried out using the data from The Cancer Genome Atlas (TCGA). Here, we showed that ECT2, AURKA, TYMS, CDK1, NCAPH, CENPU, OIP5, KIF14, ASPM, FBXO5, SGOL2, CASC5, SHCBP1, FN1, LOX, IGFBP3, CSPG4, and CD44 were enriched in female tumor samples, whereas TNFSF13B, CXCL10, CXCL8, CXCR4, TLR2, CCL2, and FCGR2A were enriched in male tumor samples. Among these key genes, interestingly, ECT2 was associated with increased an survival rate for female patients, whileTNFSF13B could be regarded as a potential marker of poor prognosis in male patients. These results suggested that sex differences in patients may be attributed to the heterogeneous gene activity, which might influence the oncogenesis and the outcomes of GBM.

Topoisomerase IIA in adult NSCs regulates SVZ neurogenesis by transcriptional activation of Usp37.

Topoisomerase IIA (TOP2a) has traditionally been known as an important nuclear enzyme that resolves entanglements and relieves torsional stress of DNA double strands. However, its function in genomic transcriptional regulation remains largely unknown, especially during adult neurogenesis. Here, we show that TOP2a is preferentially expressed in neurogenic niches in the brain of adult mice, such as the subventricular zone (SVZ). Conditional knockout of Top2a in adult neural stem cells (NSCs) of the SVZ significantly inhibits their self-renewal and proliferation, and ultimately reduces neurogenesis. To gain insight into the molecular mechanisms by which TOP2a regulates adult NSCs, we perform RNA-sequencing (RNA-Seq) plus chromatin immunoprecipitation sequencing (ChIP-Seq) and identify ubiquitin-specific protease 37 (Usp37) as a direct TOP2a target gene. Importantly, overexpression of Usp37 is sufficient to rescue the impaired self-renewal ability of adult NSCs caused by Top2a knockdown. Taken together, this proof-of-principle study illustrates a TOP2a/Usp37-mediated novel molecular mechanism in adult neurogenesis, which will significantly expand our understanding of the function of topoisomerase in the adult brain.

NOTCH1 signaling regulates the latent neurogenic program in adult reactive astrocytes after spinal cord injury.

Background: Direct reprogramming of astrocytes into neurons opens up a new avenue for neuroregenerative medicine. However, the poor understanding of the molecular mechanisms underpinning the latent neurogenic program in astrocytes has largely restricted this strategy towards safe and effective clinical therapies. Methods: Immunocytochemistry, immunohistochemistry, western blotting, qRT-PCR, gene knockdown and fate-mapping are performed to analyze the role of NOTCH1 signaling in regulation of the latent neurogenic program in reactive astrocytes after spinal cord injury. Results: Western blotting analysis highlights that NOTCH1 is a key signaling mediating Ascl1- and Neurog2-driven astrocyte-to-neuron conversion. Inhibition of NOTCH1 signaling in cultured astrocytes by shRNA or DAPT (a NOTCH1 inhibitor) is sufficient to reprogram them into neurons by upregulating the expression of pro-neural transcription factors, including NeuroD1, NeuroD2, Pax6, Lmx1a and Lhx6. In the spinal cord of adult mouse, the expression of Notch1 is detected in resident astrocytes, which was significantly increased after spinal cord injury (SCI). Genetical knockdown of NOTCH1 signaling alone successfully triggers endogenous reactive astrocytes reprogramming into neurons in the injured adult spinal cord. Importantly, pharmacologically blocking NOTCH1 signaling with small molecule DAPT alone can also induce in situ astrocyte-to-neuron conversion after SCI. Conclusions: We identify NOTCH1 as a key common signaling pathway in reactive astrocyte that provides a barrier for cell fate conversion. This proof-of-principle study will significantly expand our molecular understanding of astroglial-lineage reprogramming and overcoming the NOTCH1 gatekeeper with small molecules may provide a transgene-free approach for in vivo chemical neuronal reprogramming with potential clinical application in neuroregeneration.

[Biological characteristics and functions of NG2-glia].

NG2-glia are a major type of glial cells that are widely distributed in the central nervous system (CNS). Under physiological conditions, they mainly differentiate into oligodendrocytes and contribute to the myelination of axons, so they are generally called oligodendrocyte progenitor cells. Emerging evidence suggests that NG2-glia not only act as the precursors of oligodendrocytes but also possess many other biological properties and functions. For example, NG2-glia can form synapse with neurons and participate in energy metabolism and immune regulation. Under pathological conditions, NG2-glia can also differentiate into astrocytes, Schwann cells and even neurons, which are involved in CNS injury and repair. Therefore, a deeper understanding of the biological characteristics and functions of NG2-glia under physiological and pathological conditions will be helpful for the treatment of CNS injury and disease. This article reviews the recent advances in the biological characteristics and functions of NG2-glia.

Direct neuronal reprogramming of olfactory ensheathing cells for CNS repair.

Direct conversion of readily available non-neural cells from patients into induced neurons holds great promise for neurological disease modeling and cell-based therapy. Olfactory ensheathing cells (OECs) is a unique population of glia in olfactory nervous system. Based on the regeneration-promoting properties and the relative clinical accessibility, OECs are attracting increasing attention from neuroscientists as potential therapeutic agents for use in neural repair. Here, we report that OECs can be directly, rapidly and efficiently reprogrammed into neuronal cells by the single transcription factor Neurogenin 2 (NGN2). These induced cells exhibit typical neuronal morphologies, express multiple neuron-specific markers, produce action potentials, and form functional synapses. Genome-wide RNA-sequencing analysis shows that the transcriptome profile of OECs is effectively reprogrammed towards that of neuronal lineage. Importantly, these OEC-derived induced neurons survive and mature after transplantation into adult mouse spinal cords. Taken together, our study provides a direct and efficient strategy to quickly obtain neuronal cells from adult OECs, suggestive of promising potential for personalized disease modeling and cell replacement-mediated therapeutic approaches to neurological disorders.

Inhibition of Glioma Development by ASCL1-Mediated Direct Neuronal Reprogramming.

Direct conversion of non-neural cells into induced neurons holds great promise for brain repair. As the most common malignant tumor in the central nervous system, glioma is currently incurable due to its exponential growth and invasive behavior. Given that neurons are irreversible postmitotic cells, reprogramming glioma cells into terminally differentiated neuron-like cells represents a potential approach to inhibit brain tumor development. We here show that human glioma cells can be directly, rapidly and efficiently reprogrammed into terminally differentiated neuron-like cells by the single transcription factor ASCL1 (Achaete-scute complex-like 1, also known as MASH1). These induced cells exhibit typical neuron-like morphology and express multiple neuron-specific markers. Importantly, ASCL1-mediated neuronal reprogramming drives human glioma cells to exit the cell cycle and results in dramatic inhibition of proliferation, both in vitro and in vivo. Taken together, this proof-of-principle study demonstrates a potential strategy for impeding brain tumor development by ASCL1-induced direct neuronal reprogramming.

Conditional ablation of reactive astrocytes to dissect their roles in spinal cord injury and repair.

Astrocytes become reactive in response to spinal cord injury (SCI) and ultimately form a histologically apparent glial scar at the lesion site. It is controversial whether astrocytic scar is detrimental or beneficial to the axonal regeneration and SCI repair. Therefore, much effort has focused on understanding the functions of reactive astrocytes. Here, we used a lentivirus-mediated herpes simplex thymidine kinase/ganciclovir (HSVtk/GCV) system to selectively kill scar-forming reactive proliferating astrocytes. The suicide gene expression was regulated by human glial fibrillary acidic protein (hGFAP) promoter, which is active primarily in astrocytes. Conditional ablation of reactive astrocytes in a mouse SCI model with crush injury impeded glial scar formation and resulted in widespread infiltration of inflammatory cells, increased neuronal loss, and severe tissue degeneration, which ultimately led to the failure of spontaneous functional recovery. These results suggest that reactive proliferating astrocytes play key roles in the healing process after SCI, shedding light on the potential benefit for the repair after central nervous system (CNS) injury.

Region-Restrict Astrocytes Exhibit Heterogeneous Susceptibility to Neuronal Reprogramming.

The adult CNS has poor ability to replace degenerated neurons following injury or disease. Recently, direct reprogramming of astrocytes into induced neurons has been proposed as an innovative strategy toward CNS repair. As a cell population that shows high diversity on physiological properties and functions depending on their spatiotemporal distribution, however, whether the astrocyte heterogeneity affect neuronal reprogramming is not clear. Here, we show that astrocytes derived from cortex, cerebellum, and spinal cord exhibit biological heterogeneity and possess distinct susceptibility to transcription factor-induced neuronal reprogramming. The heterogeneous expression level of NOTCH1 signaling in the different CNS regions-derived astrocytes is shown to be responsible for the neuronal reprogramming diversity. Taken together, our findings demonstrate that region-restricted astrocytes reveal different intrinsic limitation of the response to neuronal reprogramming.

Identification of characteristic genes distinguishing neural stem cells from astrocytes.

BACKGROUND: Neural stem cells (NSCs) have unique biological characteristics such as continuous proliferation and multipotential differentiation, providing a possible method for restoration of central nervous system (CNS) function after injury or disease. NSCs and astrocytes share many similar biological properties including cell morphology and molecular expression and can trans-differentiate into each other under certain conditions. However, characteristic genes specifically expressed by NSCs have not been well described. METHODS: To provide insights into the characteristic expression of NSCs, bioinformatics analysis of two microarrays of mouse NSCs and astrocytes was performed. Compared to astrocytes, the differentially expressed genes (DEGs) in NSCs were identified and annotated by GO, KEGG and GSEA analysis, respectively. Then key genes were screened by protein-protein interaction (PPI) network and modules analysis, and were verified using multiple high-throughput sequencing resources. Finally, the expression difference between the two cell types was confirmed by Real-time Quantitative PCR (qPCR), western blotting and immunochemical analysis. RESULTS: In the present study, 282 and 250 NSC-enriched genes from two microarrays were identified and annotated respectively, and the 77 overlapping DEGs were then selected. From the PPI network 24 key genes in three modules were screened out. Importantly, sequencing data of tissues showed that these 24 key genes tended to be highly expressed in NSCs compared with astrocytes. Furthermore, qPCR and western blot analysis of cultured NSCs and astrocytes showed two genes (KIF2C and TOP2A) were not only differentially expressed in RNA level but also at the protein level. Importantly, the NSC-specific genes KIF2C and TOP2A were validated by immunohistochemistry in vivo. CONCLUSION: In present study, we identified 2 hub genes (KIF2C and TOP2A) that might serve as potential biomarkers for distinguishing NSCs from astrocytes, contributing to our comprehensive understanding of the biological properties and functions of NSCs.

TROY interacts with RKIP to promote glioma development.

TROY is a component of the Nogo receptor complex and plays the key role in neuronal survival, migration, and differentiation. Here, we show the up-regulation of TROY in human glioma tissues and cells. Inhibition of TROY expression slowed glioma development in vivo and in vitro. Raf kinase inhibitor (RKIP) was found to interact with TROY. The physical interaction of TROY/RKIP was confirmed via co-immunoprecipitation (co-IP) assays. Furthermore, we found that the TROY/RKIP interaction was enhanced by fetal bovine serum (FBS) exposure, and TROY knockdown also led to down-regulation of NF-κB. Finally, disruption of the TROY/RKIP interaction using the TAT-TROY (234-371 aa) protein reduced the glioma development in xenografted mice. This suggests the TROY/RKIP interaction is a potential target for therapy of gliomas.

Hemopexin is required for adult neurogenesis in the subventricular zone/olfactory bulb pathway.

The neural stem cells (NSCs) of the subventricular zone (SVZ) reside within a specialized niche critical for neurogenesis. Hemopexin, a plasma glycoprotein, has been extensively studied as a heme scavenger at the systemic level. However, little is known about its function in the central nervous system, especially in neurogenesis. In the present study, we demonstrate that deletion of hemopexin leads to neurogenic abnormalities in the SVZ/olfactory bulb (OB) pathway. The lateral ventricle is enlarged in hemopexin-deficient mice, and more apoptosis was observed in Dcx+ cells. Lineage differentiation of NSCs was also inhibited in the SVZ of hemopexin-deficient mice, with more stem cells stayed in an undifferentiated, GFAP+ radial glia-like cell stage. Moreover, hemopexin deletion resulted in impaired neuroblast migration in the rostral migratory stream. Furthermore, exogenous hemopexin protein inhibited apoptosis and promoted the migration and differentiation of cultured NSCs. Finally, immunohistochemical analysis demonstrated that deletion of hemopexin reduced the number of interneurons in the OB. Together, these results suggest a new molecular mechanism for the NSC niche that regulates adult neurogenesis in the SVZ/OB pathway. Our findings may benefit the understanding for olfactory system development.

[Astrocytes as therapeutic targets after spinal cord injury].

Spinal cord injury (SCI) is a challenging medical problem in the field of neurology, showing high incidence rate, disability rate, treatment cost and low-aged trend. Despite the clinical application of drug intervention, surgical treatment and modern rehabilitation training, no ideal curative effect has been achieved. Therefore, future study is necessary to clarify detailed pathological mechanism of SCI and identify the potential target cells for therapeutic intervention. In the central nervous system (CNS), astrocytes are the most abundant and widely distributed glial cells which play multiple key roles in maintaining homeostasis of the CNS in physiological and pathological conditions. Increasing evidence indicates that astrocytes are ideal therapeutic target cells for SCI. Here, we review current knowledge of the roles of astrocytes in the pathological reaction after SCI, astroglial transplantation and astrocyte reprogramming.

Establishment and characterization of primary astrocyte culture from adult mouse brain.

As a major class of glial cells, astrocytes have been indicated to play multi-roles in physiological and pathological brain. Astrocyte cultures derived from postnatal mouse brains have been extensively used to characterize their biological properties. However, the inability to culture adult mouse primary astrocytes has long stymied studies of function in adult brain. Here, we developed a protocol to successfully establish highly enriched astrocyte cultures from the brains of adult mouse. Cortical tissues were collected to prepare cell suspension by enzymatic digestion and mechanical dissociation, and then plated onto vessels pre-coated with gelatin and matrigel and cultured in DMEM medium containing 20% fetal bovine serum (FBS). Forskolin (FSK) and glial-derived neurotrophic factor (GDNF) were use to promote astrocyte proliferation and survival respectively. These adult astrocyte cultures were identified by immunocytochemical, immunobloting and PCR analysis. Furthermore, biological and functional analysis indicated that they possess the biochemical and physiological properties of astrocytes, suggestive of a useful cell model for astroglial studies in adult brain.

[Glial cells function as neural stem cells and progenitor cells].

Glial cells, including astrocytes, oligodendrocyte progenitor cells (OPCs), NG2-glia, etc, are broadly distributed throughout the central nervous system (CNS). Also, it has been well known that glial cells play multi-roles in physiological and pathological processes in the CNS, such as maintaining homeostasis, providing neurotrophins for neurons and regulating neural signal transmission. Recently, increasing evidence showed that glial cells may also function as neural stem/progenitor cells and contribute to adult neurogenesis or neuroregeneration. In pathological conditions, for instance, astrocytes and OPCs could be activated to proliferate and differentiate. When cultured in vitro, they could form neurospheres which possess the ability to differentiate into astrocytes, oligodendrocytes and neurons. Additionally, forced expression of exogenous genes in astrocytes and NG2-glia can successfully reprogram them into neurons, which may also be suggestive of their stem/progenitor cell features. Here, we review current knowledge of the stem cell-like properties of glial cells, including what types of glial cells can function as stem/progenitor cells, how they can acquire the stem/progenitor potential and what progenies can be produced. These insights may foster a better understanding of glial cell biology and function in physiological or pathological processes in the CNS and lead to the idea of using the stem/progenitor-like glial cells as endogenous cell source for neural repair.

The p53 Pathway Controls SOX2-Mediated Reprogramming in the Adult Mouse Spinal Cord.

Although the adult mammalian spinal cord lacks intrinsic neurogenic capacity, glial cells can be reprogrammed in vivo to generate neurons after spinal cord injury (SCI). How this reprogramming process is molecularly regulated, however, is not clear. Through a series of in vivo screens, we show here that the p53-dependent pathway constitutes a critical checkpoint for SOX2-mediated reprogramming of resident glial cells in the adult mouse spinal cord. While it has no effect on the reprogramming efficiency, the p53 pathway promotes cell-cycle exit of SOX2-induced adult neuroblasts (iANBs). As such, silencing of either p53 or p21 markedly boosts the overall production of iANBs. A neurotrophic milieu supported by BDNF and NOG can robustly enhance maturation of these iANBs into diverse but predominantly glutamatergic neurons. Together, these findings have uncovered critical molecular and cellular checkpoints that may be manipulated to boost neuron regeneration after SCI.

Heterogeneous astrocytes: Active players in CNS.

Astrocytes, the predominant cell type that are broadly distributed in the brain and spinal cord, play key roles in maintaining homeostasis of the central nerve system (CNS) in physiological and pathological conditions. Increasing evidence indicates that astrocytes are a complex colony with heterogeneity on morphology, gene expression, function and many other aspects depending on their spatio-temporal distribution and activation level. In pathological conditions, astrocytes differentially respond to all kinds of insults, including injury and disease, and participate in the neuropathological process. Based on current studies, we here give an overview of the roles of heterogeneous astrocytes in CNS, especially in neuropathologies, which focuses on biological and functional diversity of astrocytes. We propose that a precise understanding of the heterogeneous astrocytes is critical to unlocking the secrets about pathogenesis and treatment of the mazy CNS.

[Structural, mechanistic and functional insights into topoisomerase II].

Topoisomerases are nuclear enzymes that regulate the overwinding or underwinding of DNA helix during replication, transcription, recombination, repair, and chromatin remodeling. These enzymes perform topological transformations by providing a transient DNA break, through which the unique problems of DNA entanglement that occur owing to unwinding and rewinding of the DNA helix can be resolved. In mammals, topoisomerases are classified into two types, type I topoisomerase (Top1) and type II topoisomerase (Top2), depending on the number of strands cut in one round of action. Top1 induces single-strand breaks in DNA, and Top2 induces double-strand breaks. In cells from vertebrate species, there are two forms of Top2, designated alpha and beta. Top2α is involved in the cellular proliferation and pluripotency, while Top2ß plays key roles in neurodevelopment. In this review, we cover recent advances in structural, mechanistic and functional insights into Top2.

Neuroprotective effects of nitidine against traumatic CNS injury via inhibiting microglia activation.

Glial cell response to injury has been well documented in the pathogenesis after traumatic brain injury (TBI) and spinal cord injury (SCI). Although microglia, the resident macrophages in the central nervous system (CNS), are responsible for clearing debris and toxic substances, excessive activation of these cells will lead to exacerbated secondary damage by releasing a variety of inflammatory and cytotoxic mediators and ultimately influence the subsequent repair after CNS injury. In fact, inhibition of microgliosis represents a therapeutic strategy for CNS trauma. We here showed that nitidine, a benzophenanthridine alkaloid, restricted reactive microgliosis and promoted CNS repair after traumatic injury. Nitidine was shown to prevent cultured microglia from LPS-induced reactive activation by regulation of ERK and NF-κB signaling pathway. Furthermore, the nitidine-mediated inhibition of microgliosis was also shown in injured brain and spinal cord, which significantly increased neuronal survival and decreased neural tissue damage after injury. Importantly, behavioral analysis revealed that nitidine-treated mice with SCI had improved functional recovery as assessed by Basso Mouse Scale and swimming test. Together, these findings indicated that nitidine increased CNS tissue sparing and improved functional recovery by attenuating reactive microgliosis, suggestive of the potential therapeutic benefit for CNS injury.

In vivo conversion of astrocytes to neurons in the injured adult spinal cord.

Spinal cord injury (SCI) leads to irreversible neuronal loss and glial scar formation, which ultimately result in persistent neurological dysfunction. Cellular regeneration could be an ideal approach to replenish the lost cells and repair the damage. However, the adult spinal cord has limited ability to produce new neurons. Here we show that resident astrocytes can be converted to doublecortin (DCX)-positive neuroblasts by a single transcription factor, SOX2, in the injured adult spinal cord. Importantly, these induced neuroblasts can mature into synapse-forming neurons in vivo. Neuronal maturation is further promoted by treatment with a histone deacetylase inhibitor, valproic acid (VPA). The results of this study indicate that in situ reprogramming of endogenous astrocytes to neurons might be a potential strategy for cellular regeneration after SCI.