Musashi1‐CreERT2: A new cre line for conditional mutagenesis in neural stem cells

The RNA‐binding protein Musashi1 (Msi1) is one of two mammalian homologues of DrosophilaMusashi, which is required for the asymmetric cell division of sensory organ precursor cells. In the mouse central nervous system (CNS), Msi1 is preferentially expressed in mitotically active progenitor cells in the ventricular zone (VZ) of the neural tube during embryonic development and in the subventricular zone (SVZ) of the postnatal brain. Previous studies showed that cells in the SVZ can contribute to long‐term neurogenesis in the olfactory bulb (OB), but it remains unclear whether Msi1‐expressing cells have self‐renewing potential and can contribute to neurogenesis in the adult. Here, we describe the generation of Msi1‐CreER^T2 knock‐in mice and show by cell lineage tracing that Msi1‐CreER^T2‐expressing cells mark neural stem cells (NSCs) in both the embryonic and adult brain. Msi1‐CreER^T2 mice thus represent a new tool in our arsenal for genetically manipulating NSCs, which will be essential for understanding the molecular mechanisms underlying neural development. genesis, 51:128–134, 2013. © 2012 Wiley Periodicals, Inc.     

Age-dependent changes in the subcallosal zone neurogenesis of mice

There are several known neurogenic areas including subventricular zone and subgranular layer in the dentate gyrus of the hippocampus. Both germinal centers exhibit an age-dependent decline in cell proliferation and neurogenesis, which may be associated with age-related decline in brain function. We recently identified the subcallosal zone (SCZ) as a novel neural stem cell niche with a potential to spontaneously produce new neuroblasts. We examined whether SCZ neurogenesis is also regulated by the age of mice. The number of newly generated neuroblasts was reduced in the SCZ with age, and only marginal number of DCX-labeled neuroblasts was found in 6-month-old SCZ, which is most likely due to reduced proliferation of progenitor cells and loss of neural stem cells (NSCs). This age-dependent changes in the SCZ occurred earlier than that of other neurogenic brain regions. The neurosphere assay in vitro confirmed the depletion of NSCs within the SCZ of young adults. However, marked induction of neuroblast production in the SCZ was seen in 6-month-old mice after traumatic brain injury. Taken together, these results indicate that a rapid decline in SCZ neurogenesis in mice is due to depletion of NSCs and reduced capacity to produce neuroblasts.     

In vitro characterization of subventricular zone isolated neural stem cells,from adult monkey and rat brain

Neural stem cells (NSCs) are multipotent, self-renewable cells who are capable of differentiating into neurons, astrocytes, and oligodendrocytes. NSCs reside at the subventricular zone (SVZ) of the adult brain permanently to guarantee a lifelong neurogenesis during neural network plasticity or undesirable injuries. Although the specious inaccessibility of adult NSCs niche hampers their in vivo identification, researchers have been seeking ways to optimize adult NSCs isolation, expansion, and differentiation, in vitro. NSCs were isolated from rhesus monkey SVZ, expanded in vitro and then characterized for NSCs-specific markers expression by immunostaining, real-time PCR, flow cytometry, and cell differentiation assessments. Moreover, cell survival as well as self-renewal capacity were evaluated by TUNEL, Live/Dead and colony assays, respectively. In the next step, to validate SVZ-NSCs identity in other species, a similar protocol was applied to isolate NSCs from adult rat’s SVZ as well. Our findings revealed that isolated SVZ-NSCs from both monkey and rat preserve proliferation capacity in at least nine passages as confirmed by Ki67 expression. Additionally, both SVZ-NSCs sources are capable of self-renewal in addition to NESTIN, SOX2, and GFAP expression. The mortality was measured meager with over 95% viability according to TUNEL and Live/Dead assay results. Eventually, the multipotency of SVZ-NSCs appraised authentic after their differentiation into neurons, astrocytes, and oligodendrocytes. In this study, we proposed a reliable method for SVZ-NSCs in vitro maintenance and identification, which, we believe is a promising cell source for therapeutic approach to recover neurological disorders and injuries condition.

     

Identification,characterization, and functional investigation of circular RNAs in subventricular zone of adult rat brain

Adult neural stem cells (NSCs) are able to self-renew and generate new neural cells. Identifying regulators of NSCs is significant for the development of NSC-based therapies for neurodegenerative diseases and brain injuries. Recently, circular RNAs (circRNAs) have been characterized in various cell lines and brain tissues, and found to participate in multiple biological processes. However, the expression pattern of circRNAs in adult NSCs is still unknown. Here, the subventricular zone (SVZ) of the lateral ventricle was isolated as the niche of NSCs in adult rat brain for RNA sequencing and the characteristics of circRNAs profiling in both SVZ and cerebral cortex were also investigated. As a result, 29 049 and 31 975 circRNAs were identified in SVZ and cortex, respectively. Among them, 41 were SVZ-specific and 48 were cortex-specific. 467 circRNAs were also found to express predominately in SVZ, while the cortex had other 423 circRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the SVZ-specific circRNAs have close relationship with the regulation of NSC expansion and NSC-niche interaction, while the other differentially expressed circRNAs might be involved in neural cellular construction and nerve system function. Furthermore, the interactions between circRNAs and microRNAs were also explored, and the result showed that one SVZ-specific circRNA was capable to competitively bind miR-138-5p as a potential derepressive regulator in NSCs proliferation. Hence, our work has laid the foundations to decipher regulation mechanisms of circRNAs in adult NSCs and to develop circRNAs as novel biomarkers for adult NSCs.     

Reduced Cerebral Oxygen Content in the DG and SVZ In Situ Promotes Neurogenesis in the Adult Rat Brain In Vivo

Neurogenesis in the adult brain occurs mainly within two neurogenic structures, the dentate gyrus (DG) of the hippocampus and the sub-ventricular zone (SVZ) of the forebrain. It has been reported that mild hypoxia promoted the proliferation of Neural Stem Cells (NSCs)in vitro. Our previous study further demonstrated that an external hypoxic environment stimulated neurogenesis in the adult rat brain in vivo. However, it remains unknown how external hypoxic environments affect the oxygen content in the brain and result in neurogenesis. Here we use an optical fiber luminescent oxygen sensor to detect the oxygen content in the adult rat brain in situ under normoxia and hypoxia. We found that the distribution of oxygen in cerebral regions is spatiotemporally heterogeneous. The Po₂ values in the ventricles (45∼50 Torr) and DG (approximately 10 Torr) were much higher than those of other parts of the brain, such as the cortex and thalamus (approximately 2 Torr). Interestingly, our in vivo studies showed that an external hypoxic environment could change the intrinsic oxygen content in brain tissues, notably reducing oxygen levels in both the DG and SVZ, the major sites of adult neurogenesis. Furthermore, the hypoxic environment also increased the expression of HIF-1α and VEGF, two factors that have been reported to regulate neurogenesis, within the DG and SVZ. Thus, we have demonstrated that reducing the oxygen content of the external environment decreased Po₂ levels in the DG and SVZ. This reduced oxygen level in the DG and SVZ might be the main mechanism triggering neurogenesis in the adult brain. More importantly, we speculate that varying oxygen levels may be the physiological basis of the regionally restricted neurogenesis in the adult brain.     

Repetitive Transcranial Magnetic Stimulation Promotes Neural Stem Cell Proliferation via the Regulation of MiR-25 in a Rat Model of Focal Cerebral Ischemia

Repetitive transcranial magnetic stimulation (rTMS) has increasingly been studied over the past decade to determine whether it has a therapeutic benefit on focal cerebral ischemia. However, the underlying mechanism of rTMS in this process remains unclear. In the current study, we investigated the effects of rTMS on the proliferation of adult neural stem cells (NSCs) and explored microRNAs (miRNAs) that were affected by rTMS. Our data showed that 10 Hz rTMS significantly increased the proliferation of adult NSCs after focal cerebral ischemia in the subventricular zone (SVZ), and the expression of miR-25 was obviously up-regulated in the ischemic cortex after rTMS. p57, an identified miR-25 target gene that regulates factors linked to NSC proliferation, was also evaluated, and it exhibited down-regulation. To further verify the role of miR-25, rats were injected with a single dose of antagomir-25 and were subjected to focal cerebral ischemia followed by rTMS treatment. The results confirmed that miR-25 could be repressed specifically and could drive the up-regulation of its target gene (p57), which resulted in the inhibition of adult NSC proliferation in the SVZ after rTMS. Thus, our studies strongly indicated that 10 Hz rTMS can promote the proliferation of adult NSCs in the SVZ after focal cerebral ischemia by regulating the miR-25/p57 pathway.     

Role of the nuclear receptor Tailless in adult neural stem cells

Tailless (Tlx) is an orphan nuclear receptor which is specifically expressed in the neural stem cells of the two largest germinal neurogenesis zones in the adult mouse brain, the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus. By interacting with its cofactors, Tlx represses its target genes and plays an important role in the maintenance of adult NSCs. This review provides a snapshot of current knowledge about Tlx function in adult NSCs.     

Immunohistochemical Detection of Neural Stem Cells and Glioblastoma Stem Cells in the Subventricular Zone of Glioblastoma Patients

Glioblastoma usually recurs after therapy consisting of surgery, radiotherapy, and chemotherapy. Recurrence is at least partly caused by glioblastoma stem cells (GSCs) that are maintained in intratumoral hypoxic peri-arteriolar microenvironments, or niches, in a slowly dividing state that renders GSCs resistant to radiotherapy and chemotherapy. Because the subventricular zone (SVZ) is a major niche for neural stem cells (NSCs) in the brain, we investigated whether GSCs are present in the SVZ at distance from the glioblastoma tumor. We characterized the SVZ of brains of seven glioblastoma patients using fluorescence immunohistochemistry and image analysis. NSCs were identified by CD133 and SOX2 but not CD9 expression, whereas GSCs were positive for all three biomarkers. NSCs were present in all seven samples and GSCs in six out of seven samples. The SVZ in all samples were hypoxic and expressed the same relevant chemokines and their receptors as GSC niches in glioblastoma tumors: stromal-derived factor-1α (SDF-1α), C-X-C receptor type 4 (CXCR4), osteopontin, and CD44. In conclusion, in glioblastoma patients, GSCs are present at distance from the glioblastoma tumor in the SVZ. These findings suggest that GSCs in the SVZ niche are protected against radiotherapy and chemotherapy and protected against surgical resection due to their distant localization and thus may contribute to tumor recurrence after therapy.     

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.     

Tauroursodeoxycholic Acid Enhances Mitochondrial Biogenesis,Neural Stem Cell Pool,and Early Neurogenesis in Adult Rats

Although neurogenesis occurs in restricted regions of the adult mammalian brain, neural stem cells (NSCs) produce very few neurons during ageing or after injury. We have recently discovered that the endogenous bile acid tauroursodeoxycholic acid (TUDCA), a strong inhibitor of mitochondrial apoptosis and a neuroprotective in animal models of neurodegenerative disorders, also enhances NSC proliferation, self-renewal, and neuronal conversion by improving mitochondrial integrity and function of NSCs. In the present study, we explore the effect of TUDCA on regulation of NSC fate in neurogenic niches, the subventricular zone (SVZ) of the lateral ventricles and the hippocampal dentate gyrus (DG), using rat postnatal neurospheres and adult rats exposed to the bile acid. TUDCA significantly induced NSC proliferation, self-renewal, and neural differentiation in the SVZ, without affecting DG-derived NSCs. More importantly, expression levels of mitochondrial biogenesis-related proteins and mitochondrial antioxidant responses were significantly increased by TUDCA in SVZ-derived NSCs. Finally, intracerebroventricular administration of TUDCA in adult rats markedly enhanced both NSC proliferation and early differentiation in SVZ regions, corroborating in vitro data. Collectively, our results highlight a potential novel role for TUDCA in neurologic disorders associated with SVZ niche deterioration and impaired neurogenesis.     

Effect of conditioned medium from neural stem cells on glioma progression and its protein expression profile analysis

BACKGROUNDEmerging evidence suggests that the spread of glioma to the subventricular zone (SVZ) is closely related to glioma recurrence and patient survival. Neural stem cells (NSCs) are the main cell type in the SVZ region and exhibit tumor-homing ability.AIMTo evaluate the effects of conditioned medium (CM) derived from SVZ NSCs on the cancer-related behaviors of glioma cells.METHODSThe characteristics of SVZ hNSCs were identified by immunofluorescence. The normoxic-hNSC-CM and hypoxic-hNSC-CM (3% O₂, oxygen-glucose deprived [OGD] culturing) were collected from 80%-90% confluent SVZ NSCs in sterile conditions. The CCK8 and Transwell assays were used to compare and evaluate the effects of normoxic-CM and hypoxic-CM on glioma proliferation and invasion. Then proteins secreted from SVZ NSCs into the CM were investigated by mass spectrometry, and the potential effects of candidate protein NCAN in the regulation of glioma progression were examined by CCK8 and Transwell assays.RESULTSThe CM from SVZ NSCs significantly increased the proliferation and invasion of glioma cells, particularly the CM from OGD NSCs induced under hypoxic conditions. Furthermore, the secreted protein neurocan (NCAN) in CM from OGD NSCs was identified by proteomic analysis. NCAN was expressed in glioma cells and played regulatory roles in mediating the progression of glioma cells mainly via the Rho/Rho-associated protein kinase pathway.CONCLUSIONOur study identified a potential interactive mechanism between SVZ NSCs and glioma cells, in which SVZ NSCs promote glioma progression via the secreted protein NCAN. These findings suggested that exploring the CM derived from cells could be a novel strategy for optimizing treatments and that NCAN derived from SVZ NSCs may be a potential new target in glioma progression.     

Neural stem cells confer unique pinwheel architecture to the ventricular surface in neurogenic regions of the adult brain

Neural stem cells (NSCs, B1 cells) are retained in the walls of the adult lateral ventricles but, unlike embryonic NSCs, are displaced from the ventricular zone (VZ) into the subventricular zone (SVZ) by ependymal cells. Apical and basal compartments, which in embryonic NSCs play essential roles in self-renewal and differentiation, are not evident in adult NSCs. Here we show that SVZ B1 cells in adult mice extend a minute apical ending to directly contact the ventricle and a long basal process ending on blood vessels. A closer look at the ventricular surface reveals a striking pinwheel organization specific to regions of adult neurogenesis. The pinwheel's core contains the apical endings of B1 cells and in its periphery two types of ependymal cells: multiciliated (E1) and a type (E2) characterized by only two cilia and extraordinarily complex basal bodies. These results reveal that adult NSCs retain fundamental epithelial properties, including apical and basal compartmentalization, significantly reshaping our understanding of this adult neurogenic niche.     

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G₁ phase due to a G₁ specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G₁ phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.     

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells¹. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level². Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development^3,4 . More recently this was applied to study the postnatal rodent forebrain^5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.     

The Molecular Profiles of Neural Stem Cell Niche in the Adult Subventricular Zone

Neural stem cells (NSCs) reside in a unique microenvironment called the neurogenic niche and generate functional new neurons. The neurogenic niche contains several distinct types of cells and interacts with the NSCs in the subventricular zone (SVZ) of the lateral ventricle. While several molecules produced by the niche cells have been identified to regulate adult neurogenesis, a systematic profiling of autocrine/paracrine signaling molecules in the neurogenic regions involved in maintenance, self-renewal, proliferation, and differentiation of NSCs has not been done. We took advantage of the genetic inducible fate mapping system (GIFM) and transgenic mice to isolate the SVZ niche cells including NSCs, transit-amplifying progenitors (TAPs), astrocytes, ependymal cells, and vascular endothelial cells. From the isolated cells and microdissected choroid plexus, we obtained the secretory molecule expression profiling (SMEP) of each cell type using the Signal Sequence Trap method. We identified a total of 151 genes encoding secretory or membrane proteins. In addition, we obtained the potential SMEP of NSCs using cDNA microarray technology. Through the combination of multiple screening approaches, we identified a number of candidate genes with a potential relevance for regulating the NSC behaviors, which provide new insight into the nature of neurogenic niche signals.     

Jagged1 signals in the postnatal subventricular zone are required for neural stem cell self-renewal

Neural stem cells (NSCs) in the postnatal mammalian brain self-renew and are a source of neurons and glia. To date, little is known about the molecular and cellular mechanisms regulating the maintenance and differentiation of these multipotent progenitors. We show that Jagged1 is required by mitotic cells in the subventricular zone (SVZ) and stimulates self-renewal of multipotent epidermal growth factor-dependent NSCs. Jagged1-expressing cells line the adult SVZ and are juxtaposed to Notch1-expressing cells, some of which are putative NSCs. In vitro, endogenous Jagged1 acts through Notch1 to promote NSC maintenance and multipotency. In vivo, reducing Jagged1/Notch1 signaling decreases the number of proliferating cells in the SVZ. In addition, soluble Jagged1 promotes self-renewal and neurogenic potential of multipotent neural progenitors in vitro. Our findings suggest a central role for Jagged1 in the NSC niche in the SVZ for maintaining a population of NSCs in the postnatal brain.     

GalR3 activation promotes adult neural stem cell survival in response to a diabetic milieu

Type 2 diabetes impairs adult neurogenesis which could play a role in the CNS complications of this serious disease. The goal of this study was to determine the potential role of galanin in protecting adult neural stem cells (NSCs) from glucolipotoxicity and to analyze whether apoptosis and the unfolded protein response were involved in the galanin‐mediated effect. We also studied the regulation of galanin and its receptor subtypes under diabetes in NSCs in vitro and in the subventricular zone (SVZ) in vivo. The viability of mouse SVZ‐derived NSCs and the involvement of apoptosis (Bcl‐2, cleaved caspase‐3) and unfolded protein response [C/EBP homologous protein (CHOP) Glucose‐regulated protein 78/immunoglobulin heavy‐chain binding protein (GRP78/BiP), spliced X‐box binding protein 1 (XBP1), c‐Jun N‐terminal kinases (JNK) phosphorylation] were assessed in the presence of glucolipotoxic conditions after 24 h. The effect of diabetes on the regulation of galanin and its receptor subtypes was assessed on NSCs in vitro and in SVZ tissues isolated from normal and type 2 diabetes ob/ob mice. We show increased NSC viability following galanin receptor (GalR)3 activation. This protective effect correlated with decreased apoptosis and CHOP levels. We also report how galanin and its receptors are regulated by diabetes in vitro and in vivo. This study shows GalR3‐mediated neuroprotection, supporting a potential future therapeutic development, based on GalR3 activation, for the treatment of brain disorders.