Akhirin regulates the proliferation and differentiation of neural stem cells/progenitor cells at neurogenic niches in mouse brain

Specialized microenvironment, or neurogenic niche, in embryonic and postnatal mouse brain plays critical roles during neurogenesis throughout adulthood. The subventricular zone (SVZ) and the dentate gyrus (DG) of hippocampus in the mouse brain are two major neurogenic niches where neurogenesis is directed by numerous regulatory factors. Now, we report Akhirin (AKH), a stem cell maintenance factor in mouse spinal cord, plays a pivotal regulatory role in the SVZ and in the DG. AKH showed specific distribution during development in embryonic and postnatal neurogenic niches. Loss of AKH led to abnormal development of the ventricular zone and the DG along with reduction of cellular proliferation in both regions. In AKH knockout mice (AKH^−/−), quiescent neural stem cells (NSCs) increased, while proliferative NSCs or neural progenitor cells decreased at both neurogenic niches. In vitro NSC culture assay showed increased number of neurospheres and reduced neurogenesis in AKH^−/−. These results indicate that AKH, at the neurogenic niche, exerts dynamic regulatory role on NSC self-renewal, proliferation and differentiation during SVZ and hippocampal neurogenesis.     

Spinal cord injury reveals multilineage differentiation of ependymal cells

Spinal cord injury often results in permanent functional impairment. Neural stem cells present in the adult spinal cord can be expanded in vitro and improve recovery when transplanted to the injured spinal cord, demonstrating the presence of cells that can promote regeneration but that normally fail to do so efficiently. Using genetic fate mapping, we show that close to all in vitro neural stem cell potential in the adult spinal cord resides within the population of ependymal cells lining the central canal. These cells are recruited by spinal cord injury and produce not only scar-forming glial cells, but also, to a lesser degree, oligodendrocytes. Modulating the fate of ependymal progeny after spinal cord injury may offer an alternative to cell transplantation for cell replacement therapies in spinal cord injury.     

Origin of new glial cells in intact and injured adult spinal cord

Several distinct cell types in the adult central nervous system have been suggested to act as stem or progenitor cells generating new cells under physiological or pathological conditions. We have assessed the origin of new cells in the adult mouse spinal cord by genetic fate mapping. Oligodendrocyte progenitors self-renew, give rise to new mature oligodendrocytes, and constitute the dominating proliferating cell population in the intact adult spinal cord. In contrast, astrocytes and ependymal cells, which are restricted to limited self-duplication in the intact spinal cord, generate the largest number of cells after spinal cord injury. Only ependymal cells generate progeny of multiple fates, and neural stem cell activity in the intact and injured adult spinal cord is confined to this cell population. We provide an integrated view of how several distinct cell types contribute in complementary ways to cell maintenance and the reaction to injury.     

Sox11 promotes endogenous neurogenesis and locomotor recovery in mice spinal cord injury

We introduced a lentiviral vector containing the Sox11 gene into injured spinal cords of mice to evaluate the therapeutic potential of Sox11 in spinal cord injury. Sox11 markedly improved locomotor recovery after spinal cord injury and this recovery was accompanied by an up-regulation of Nestin/Doublecortin expression in the injured spinal cord. Sox11 was mainly located in endogenous neural stem cells lining the central canal and in newly-generated neurons in the spinal cord. In addition, Sox 11 significantly induced expressions of BDNF in the spinal cords of LV-Sox11-treated mice. We concluded that Sox11 induced activation of endogenous neural stem cells into neuronal determination and migration within the injured spinal cord. The resultant increase of BDNF at the injured site might form a distinct neurogenic niche which induces a final neuronal differentiation of these neural stem cells. Enhancing Sox11 expression to induce neurogenic differentiation of endogenous neural stem cells after injury may be a promising strategy in restorative therapy after SCI in mammals.     

LPA receptor expression in the central nervous system in health and following injury

Lysophosphatidic acid (LPA) is released from platelets following injury and also plays a role in neural development but little is known about its effects in the adult central nervous system (CNS). We have examined the expression of LPA receptors 1-3 (LPA_1–3) in intact mouse spinal cord and cortical tissues and following injury. In intact and injured tissues, LPA₁ was expressed by ependymal cells in the central canal of the spinal cord and was upregulated in reactive astrocytes following spinal cord injury. LPA₂ showed low expression in intact CNS tissue, on grey matter astrocytes in spinal cord and in ependymal cells lining the lateral ventricle. Following injury, its expression was upregulated on astrocytes in both cortex and spinal cord. LPA₃ showed low expression in intact CNS tissue, viz. on cortical neurons and motor neurons in the spinal cord, and was upregulated on neurons in both regions after injury. Therefore, LPA_1–3 are differentially expressed in the CNS and their expression is upregulated in response to injury. LPA release following CNS injury may have different consequences for each cell type because of this differential expression in the adult nervous system.     

Interleukin-17A regulates ependymal cell proliferation and functional recovery after spinal cord injury in mice

Ependymal cells have been suggested to act as neural stem cells and exert beneficial effects after spinal cord injury (SCI). However, the molecular mechanism underlying ependymal cell regulation after SCI remains unknown. To examine the possible effect of IL-17A on ependymal cell proliferation after SCI, we locally administrated IL-17A neutralizing antibody to the injured spinal cord of a contusion SCI mouse model, and revealed that IL-17A neutralization promoted ependymal cell proliferation, which was paralleled by functional recovery and axonal reorganization of both the corticospinal tract and the raphespinal tract. Further, to test whether ependymal cell-specific manipulation of IL-17A signaling is enough to affect the outcomes of SCI, we generated ependymal cell-specific conditional IL-17RA-knockout mice and analyzed their anatomical and functional response to SCI. As a result, conditional knockout of IL-17RA in ependymal cells enhanced both axonal growth and functional recovery, accompanied by an increase in mRNA expression of neurotrophic factors. Thus, Ependymal cells may enhance the regenerative process partially by secreting neurotrophic factors, and IL-17A stimulation negatively regulates this beneficial effect. Molecular manipulation of ependymal cells might be a viable strategy for improving functional recovery.Subject terms: Neuroimmunology, Spinal cord injury     

Early neurogenesis during caudal spinal cord regeneration in adult Gekko japonicus

Gekko japonicus undergoes dramatic changes in the caudal spinal cord after tail amputation. The amputation induces cell proliferation in the caudal ependymal tube. We performed hematoxylin and eosin staining at different time points in the regeneration process to investigate the morphological characterization of the regenerated appendages. The central canal extended to the blastema post-amputation and the cartilage and muscle tissue appeared 3 weeks after injury. We performed the bromodeoxyuridine (BrdU) incorporation assay to detect proliferating cells during the regeneration process. BrdU positive cells were detected in the peri-central canal. Furthermore, nestin and neuron-specific enolase (NSE) immunocytochemistry were applied to detect neural stem/progenitor cells and neurons. Two weeks after injury, nestin-positive cells undergoing proliferation were located outside of the ependymal tube, and NSE positive cells appeared after 3 weeks of amputation. These data suggest that neurogenesis is an early event during caudal spinal cord regeneration in gecko.     

Identity of tendon stem cells – how much do we know?

Tendon stem cells are multi‐potent adult stem cells with broad differentiation plasticity that render them of great importance in cell‐based therapies for the repair of tendons. We called them tendon‐derived stem cells (TDSCs) to indicate the tissue origin from which the stem cells were isolated in vitro. Based on the work of other sources of MSCs and specific work on TDSCs, some properties of TDSCs have been characterized / implicated in vitro. Despite these findings, tendon stem cells remained controversial cells. This was because MSCs residing in different organs, although very similar, were not identical cells. There is evidence of differences in stem cell‐related properties and functions related to tissue origins. Similar to other stem cells, tendon stem cells were identified and characterized in vitro. Their in vivo identities, niche (both anatomical locations and regulators) and roles in tendons were less understood. This review aims to summarize the current evidence of the possible anatomical locations and niche signals regulating the functions of tendon stem cells in vivo. The possible roles of tendon stem cells in tendon healing and non‐healing are presented. Finally, the potential strategies for understanding the in vivo identity of tendon stem cells are discussed.     

Human embryonic stem cell-derived neural precursor transplants in collagen scaffolds promote recovery in injured rat spinal cord

Background aimsSeveral studies have reported functional improvement after transplantation of in vivo-derived neural progenitor cells (NPC) into injured spinal cord. However, the potential of human embryonic stem cell-derived NPC (hESC-NPC) as a tool for cell replacement of spinal cord injury (SCI) should be considered.MethodsWe report on the generation of NPC as neural-like tubes in adherent and feeder-free hESC using a defined media supplemented with growth factors, and their transplantation in collagen scaffolds in adult rats subjected to midline lateral hemisection SCI.ResultshESC-NPC were highly expressed molecular features of NPC such as Nestin, Sox1 and Pax6. Furthermore, these cells exhibited the multipotential characteristic of differentiating into neurons and glials in vitro. Implantation of xenografted hESC-NPC into the spinal cord with collagen scaffold improved the recovery of hindlimb locomotor function and sensory responses in an adult rat model of SCI. Analysis of transplanted cells showed migration toward the spinal cord and both neural and glial differentiation in vivo.ConclusionsThese findings show that transplantation of hESC-NPC in collagen scaffolds into an injured spinal cord may provide a new approach to SCI.     

Central Canal Ependymal Cells Proliferate Extensively in Response to Traumatic Spinal Cord Injury but Not Demyelinating Lesions

The adult mammalian spinal cord has limited regenerative capacity in settings such as spinal cord injury (SCI) and multiple sclerosis (MS). Recent studies have revealed that ependymal cells lining the central canal possess latent neural stem cell potential, undergoing proliferation and multi-lineage differentiation following experimental SCI. To determine whether reactive ependymal cells are a realistic endogenous cell population to target in order to promote spinal cord repair, we assessed the spatiotemporal dynamics of ependymal cell proliferation for up to 35 days in three models of spinal pathologies: contusion SCI using the Infinite Horizon impactor, focal demyelination by intraspinal injection of lysophosphatidylcholine (LPC), and autoimmune-mediated multi-focal demyelination using the active experimental autoimmune encephalomyelitis (EAE) model of MS. Contusion SCI at the T9–10 thoracic level stimulated a robust, long-lasting and long-distance wave of ependymal proliferation that peaked at 3 days in the lesion segment, 14 days in the rostral segment, and was still detectable at the cervical level, where it peaked at 21 days. This proliferative wave was suppressed distal to the contusion. Unlike SCI, neither chemical- nor autoimmune-mediated demyelination triggered ependymal cell proliferation at any time point, despite the occurrence of demyelination (LPC and EAE), remyelination (LPC) and significant locomotor defects (EAE). Thus, traumatic SCI induces widespread and enduring activation of reactive ependymal cells, identifying them as a robust cell population to target for therapeutic manipulation after contusion; conversely, neither demyelination, remyelination nor autoimmunity appears sufficient to trigger proliferation of quiescent ependymal cells in models of MS-like demyelinating diseases.     

TGF‐beta signalling in the adult neurogenic niche promotes stem cell quiescence as well as generation of new neurons

Members of the transforming growth factor (TGF)‐β family govern a wide range of mechanisms in brain development and in the adult, in particular neuronal/glial differentiation and survival, but also cell cycle regulation and neural stem cell maintenance. This clearly created some discrepancies in the field with some studies favouring neuronal differentiation/survival of progenitors and others favouring cell cycle exit and neural stem cell quiescence/maintenance. Here, we provide a unifying hypothesis claiming that through its regulation of neural progenitor cell (NPC) proliferation, TGF‐β signalling might be responsible for (i) maintaining stem cells in a quiescent stage, and (ii) promoting survival of newly generated neurons and their functional differentiation. Therefore, we performed a detailed histological analysis of TGF‐β1 signalling in the hippocampal neural stem cell niche of a transgenic mouse that was previously generated to express TGF‐β1 under a tetracycline regulatable Ca‐Calmodulin kinase promoter. We also analysed NPC proliferation, quiescence, neuronal survival and differentiation in relation to elevated levels of TGF‐β1 in vitro and in vivo conditions. Finally, we performed a gene expression profiling to identify the targets of TGF‐β1 signalling in adult NPCs. The results demonstrate that TGF‐β1 promotes stem cell quiescence on one side, but also neuronal survival on the other side. Thus, considering the elevated levels of TGF‐β1 in ageing and neurodegenerative diseases, TGF‐β1 signalling presents a molecular target for future interventions in such conditions.     

新生鼠中枢神经系统内Nestin蛋白免疫阳性的组织分布

为了观察Nestin在新生SD大鼠中枢神经系统中的分布,探讨神经干细胞在新生鼠的分布.采用免疫荧光法,显示含神经干细胞特征性的标志物Nestin的阳性结构在新生SD大鼠中枢神经系统中的分布.结果表明在新生SD大鼠中枢神经系统中,Nestin在前脑、脑干和小脑的各个部位均有表达,阳性结构多为纤细的纤维状突起,分布密集,标记强度多为中等强度,分布相对比较均匀.在脊髓实质的Nestin免疫阳性产物明显减少,分布稀疏,染色也较浅,中央管Nestin免疫染色阳性的室管膜细胞很少,但在脊髓中央管的背侧(延髓见于腹侧和背侧)可见到“喷泉”状免疫强阳性纤维束垂直伸展,直达软膜.由此可得出结论:新生SD大鼠中枢神经系统的广泛脑区均存在大量的神经干细胞,而脊髓的神经干细胞数目较少,提示神经干细胞在生后从神经系统的尾端开始逐渐减少.     

Salidroside attenuates neuroinflammation and improves functional recovery after spinal cord injury through microglia polarization regulation

Spinal cord injury (SCI) is a severe neurological disease; however, few drugs have been proved to treat SCI effectively. Neuroinflammation is the major pathogenesis of SCI secondary injury and considered to be the therapeutic target of SCI. Salidroside (Sal) has been reported to exert anti‐inflammatory effects in airway, adipose and myocardial tissue; however, the role of Sal in SCI therapeutics has not been clarified. In this study, we showed that Sal could improve the functional recovery of spinal cord in rats as revealed by increased BBB locomotor rating scale, angle of incline, and decreased cavity of spinal cord injury and apoptosis of neurons in vivo. Immunofluorescence double staining of microglia marker and M1/M2 marker demonstrated that Sal could suppress M1 microglia polarization and activate M2 microglia polarization in vivo. To verify how Sal exerts its effects on microglia polarization and neuron protection, we performed the mechanism study in vitro in microglia cell line BV‐2 and neuron cell line PC12. The results showed that Sal prevents apoptosis of PC12 cells in coculture with LPS‐induced M1 BV‐2 microglia, also the inflammatory secretion phenotype of M1 BV‐2 microglia was suppressed by Sal, and further studies demonstrated that autophagic flux regulation through AMPK/mTOR pathway was involved in Sal regulated microglia polarization after SCI. Overall, our study illustrated that Sal could promote spinal cord injury functional recovery in rats, and the mechanism may relate to its microglia polarization modulation through AMPK‐/mTOR‐mediated autophagic flux stimulation.     

Regional differences of proliferation activity in the spinal cord ependyma of adult rats

Increased proliferation activity in the central canal ependyma of adult rodent spinal cord was described after injury and is thought to participate in recovery processes. Proliferation activity is scarce under physiological conditions, but still could be of importance, as in vitro studies showed that the spinal cord ependyma is an internal source of neural stem cells. Data from these studies indicate that there are regional differences in the distribution of proliferation activity along the rostro-caudal axis. We analyzed the proliferation activities in the ependyma within the entire extent of intact adult rat spinal cord. To identify proliferating cells we performed immunohistochemistry either for cell cycle S-phase marker BrdU or for the nuclear protein Ki-67. BrdU and Ki-67 positive cells were counted on sections selected from four spinal cord regions — cervical, thoracic, lumbar and sacral/coccygeal. Analysis showed that the number of BrdU positive cells within the ependyma was very low in all subdivisions of the spinal cord. Both BrdU and Ki-67 labeling revealed a significantly higher number of proliferating cells in the ependyma of sacrococcygeal part in comparison to all other spinal cord regions, suggesting that the caudal spinal cord might have potentially higher regeneration capacity compared to more rostral parts.     

Purinergic signalling in a latent stem cell niche of the rat spinal cord

The ependyma of the spinal cord harbours stem cells which are activated by traumatic spinal cord injury. Progenitor-like cells in the central canal (CC) are organized in spatial domains. The cells lining the lateral aspects combine characteristics of ependymocytes and radial glia (RG) whereas in the dorsal and ventral poles, CC-contacting cells have the morphological phenotype of RG and display complex electrophysiological phenotypes. The signals that may affect these progenitors are little understood. Because ATP is massively released after spinal cord injury, we hypothesized that purinergic signalling plays a part in this spinal stem cell niche. We combined immunohistochemistry, in vitro patch-clamp whole-cell recordings and Ca²⁺ imaging to explore the effects of purinergic agonists on ependymal progenitor-like cells in the neonatal (P1–P6) rat spinal cord. Prolonged focal application of a high concentration of ATP (1 mM) induced a slow inward current. Equimolar concentrations of BzATP generated larger currents that reversed close to 0 mV, had a linear current–voltage relationship and were blocked by Brilliant Blue G, suggesting the presence of functional P2X7 receptors. Immunohistochemistry showed that P2X7 receptors were expressed around the CC and the processes of RG. BzATP also generated Ca²⁺ waves in RG that were triggered by Ca²⁺ influx and propagated via Ca²⁺ release from internal stores through activation of ryanodine receptors. We speculate that the intracellular Ca²⁺ signalling triggered by P2X7 receptor activation may be an epigenetic mechanism to modulate the behaviour of progenitors in response to ATP released after injury.     

Role of PTHrP nuclear localization and carboxyl terminus sequences in postnatal spinal cord development

Parathyroid hormone‐related peptide (PTHrP) acts under physiological conditions to regulate normal development of several tissues and organs. The role of PTHrP in spinal cord development has not been characterized. Pthrp knock in (Pthrp KI) mice were genetically modified to produce PTHrP in which there is a deficiency of the nuclear localization sequence (NLS) and C‐terminus. Using this genetically modified mouse model, we have characterized its effect on spinal cord development early postnatally. The spinal cords from Pthrp KI mice displayed a significant reduction in its length, weight, and cross‐sectional area compared to wild‐type controls. Histologically, there was a decreased development of neurons and glial cells that caused decreased cell proliferation and increased apoptosis. The neural stem cells (NSCs) cultures also revealed decreased cell proliferation and differentiation and increased apoptosis. The proposed mechanism of delayed spinal cord development in Pthrp KI mice may be due to alteration in associated pathways in regulation of cell‐division cycles and apoptosis. There was significant downregulation of Bmi‐1 and upregulation of cyclin‐dependent kinase inhibitors p27, p21, and p16 in Pthrp KI animals. We conclude that NLS and C‐terminus peptide segments of PTHrP play an important role in inhibiting cell apoptosis and stimulation of cellular proliferation necessary for normal spinal cord development.     

神经干细胞巢成分及调节机制的研究进展

神经干细胞（NSCs）是一类具有自我更新和多向分化潜能的细胞。在特定的条件下能够分化成神经元、星形胶质细胞和少突胶质细胞,从而参与神经发生和损伤修复。调节NSCs的特定微环境,通常称为神经干细胞巢,包括多个细胞群,其贡献目前正在积极探索。了解NSCs及其微环境成分之间的相互作用,对于开发治疗神经退行性疾病及脊髓损伤的疗法至关重要。本篇综述描述并讨论了最新的研究,确定了新的成分在神经干细胞巢中的作用。这些发现给这个领域带来了新的概念。本综述评估这些最新进展,提高对NSCs微环境及其对NSCs功能的影响的认识。     

Adipokinetic hormone counteracts oxidative stress elicited in insects by hydrogen peroxide: in vivo and in vitro study

The role of adipokinetic hormone (AKH) in counteracting oxidative stress elicited in the insect body is studied in response to exogenously applied hydrogen peroxide, an important metabolite of oxidative processes. In vivo experiments reveal that the injection of hydrogen peroxide (8 µmol) into the haemocoel of the firebug, Pyrrhocoris apterus L. (Heteroptera: Pyrrhocoridae) increases the level of AKH by 2.8‐fold in the central nervous system (CNS) and by 3.8‐fold in the haemolymph. The injection of hydrogen peroxide also increases the mortality of experimental insects, whereas co‐injection of hydrogen peroxide with Pyrap‐AKH (40 pmol) reduces mortality to almost control levels. Importantly, an increase in haemolymph protein carbonyl levels (i.e. an oxidative stress biomarker) elicited by hydrogen peroxide is decreased by 3.6‐fold to control levels when hydrogen peroxide is co‐injected with Pyrap‐AKH. Similar results are obtained using in vitro experiments. Oxidative stress biomarkers such as malondialdehyde and protein carbonyls are significantly enhanced upon exposure of the isolated CNS to hydrogen peroxide in vitro, whereas co‐treatment of the CNS with hydrogen peroxide and Pyrap‐AKH reduces levels significantly. Moreover, a marked decrease in catalase activity compared with controls is recorded when the CNS is incubated with hydrogen peroxide. Incubation of the CNS with hydrogen peroxide and Pyrap‐AKH together curbs the negative effect on catalase activity. Taken together, the results of the present study provide strong support for the recently published data on the feedback regulation between oxidative stressors and AKH action, and implicate AKH in counteracting oxidative stress. The in vitro experiments should facilitate research on the mode of action of AKH in relation to oxidative stress, and could help clarify the key pathways involved in this process.