The PROK2/PROKR2 signaling pathway is required for the migration of most olfactory bulb interneurons

Neural stem cells in the subventricular zone (SVZ) of the lateral ventricle generate new interneurons, which migrate tangentially through the rostral migratory stream (RMS) to the olfactory bulb (OB). The PROK2 (prokineticin 2) and PROKR2 (prokineticin receptor 2) signaling pathway has been identified to cause human Kallmann syndrome, a developmental disease that associates hypogonadism with anosmia (OB developmental defects). However, the identities and properties of PROK2⁺ and PROKR2⁺ cells in the SVZ-RMS-OB remain largely unknown. Here we examine the expression patterns of Prok2 and Prokr2 in the SVZ-RMS-OB using Prok2^EGFP transgenic and Prokr2^LacZ/+ knockin mice. Our results show that Prokr2 is expressed in postmitotic immature interneurons in the SVZ-RMS-OB. Prok2 is not expressed in the SVZ, but a few PROK2⁺ cells are found in the medial part of the RMS; they are not neural progenitors or migrating neuroblasts. In the OB, Prok2 is expressed in a subset of granule cells and tufted cells, but no coexpression of Prok2 and Prokr2 in the OB cells is observed. In Prok2 and Prokr2 mutant mice, severe tangential and radial migration defects of neuroblasts in the SVZ-RMS-OB result in loss of ~75% of GABAergic interneurons in the OB. These analyses demonstrate that PROK2/PROKR2 signaling is crucial for the tangential and radial migration of OB interneurons.     

γ‐Aminobutyric acid‐mediated regulation of the activity‐dependent olfactory bulb dopaminergic phenotype

γ‐Aminobutyric acid (GABA) regulates the proliferation and migration of olfactory bulb (OB) interneuron progenitors derived from the subventricular zone (SVZ), but the role of GABA in the differentiation of these progenitors has been largely unexplored. This study examines the role of GABA in the differentiation of OB dopaminergic interneurons using neonatal forebrain organotypic slice cultures prepared from transgenic mice expressing green fluorescent protein (GFP) under the control of the tyrosine hydroxylase (Th) gene promoter (ThGFP). KCl‐mediated depolarization of the slices induced ThGFP expression. The addition of GABA to the depolarized slices further increased GFP fluorescence by inducing ThGFP expression in an additional set of periglomerular cells. These findings show that GABA promoted differentiation of SVZ‐derived OB dopaminergic interneurons and suggest that GABA indirectly regulated Th expression and OB dopaminergic neuron differentiation through an acceleration of the maturation rate for the dopaminergic progenitors. Additional studies revealed that the effect of GABA on ThGFP expression required activation of L‐ and P/Q‐type Ca²⁺ channels as well as GABA_A and GABA_B receptors. These voltage‐gated Ca²⁺ channels and GABA receptors have previously been shown to be required for the coexpressed GABAergic phenotype in the OB interneurons. Together, these findings suggest that Th expression and the differentiation of OB dopaminergic interneurons are coupled to the coexpressed GABAergic phenotype and demonstrate a novel role for GABA in neurogenesis. © 2009 Wiley‐Liss, Inc.     

Lineage analysis of quiescent regenerative stem cells in the adult brain by genetic labelling reveals spatially restricted neurogenic niches in the olfactory bulb

The subventricular zone (SVZ) of the lateral ventricles is the major neurogenic region in the adult mammalian brain, harbouring neural stem cells within defined niches. The identity of these stem cells and the factors regulating their fate are poorly understood. We have genetically mapped a population of Nestin-expressing cells during postnatal development to study their potential and fate in vivo . Taking advantage of the recombination characteristics of a nestin::CreER ^T2 allele, we followed a subpopulation of neural stem cells and traced their fate in a largely unrecombined neurogenic niche. Perinatal nestin::CreER ^T2 -expressing cells give rise to multiple glial cell types and neurons, as well as to stem cells of the adult SVZ. In the adult SVZ nestin::CreER ^T2 -expressing neural stem cells give rise to several neuronal subtypes in the olfactory bulb (OB). We addressed whether the same population of neural stem cells play a role in SVZ regeneration. Following anti-mitotic treatment to eliminate rapidly dividing progenitors, relatively quiescent nestin::CreER ^T2 -targeted cells are spared and contribute to SVZ regeneration, generating new proliferating precursors and neuroblasts. Finally, we have identified neurogenic progenitors clustered in ependymal-like niches within the rostral migratory stream (RMS) of the OB. These OB-RMS progenitors generate neuroblasts that, upon transplantation, graft, migrate and differentiate into granule and glomerular neurons. In summary, using conditional lineage tracing we have identified neonatal cells that are the source of neurogenic and regenerative neural stem cells in the adult SVZ and occupy a novel neurogenic niche in the OB.     

Purines regulate adult brain subventricular zone cell functions: Contribution of reactive astrocytes

Brain injuries modulate activation of neural stem cells (NSCs) in the adult brain. In pathological conditions, the concentrations of extracellular nucleotides (eNTs) raise several folds, contribute to reactive gliosis, and possibly directly affect subventricular zone (SVZ) cell functioning. Among eNTs and derived metabolites, the P2Y₁ receptor agonist ADP strongly promotes astrogliosis and might also influence SVZ progenitor activity. Here, we tested the ability of the stable P2Y₁ agonist adenosine 5′‐O‐(2‐thiodiphosphate) (ADPβS) to control adult NSC functions both in vitro and in vivo, with a focus on the possible effects exerted by reactive astrocytes. In the absence of growth factors, ADPβS promoted proliferation and differentiation of SVZ progenitors. Moreover, ADPβS‐activated astrocytes markedly changed the pattern of released cytokines and chemokines, and strongly modulated neurosphere‐forming capacity of SVZ progenitors. Notably, a significant enhancement in proliferation was observed when SVZ cells, initially grown in the supernatant of astrocytes exposed to ADPβS, were shifted to normal medium. In vivo, ADPβS administration in the lateral ventricle of adult mice by osmotic minipumps caused diffused reactive astrogliosis, and a strong response of SVZ progenitors. Indeed, proliferation of glial fibrillary acidic protein‐positive NSCs increased and led to a significant expansion of SVZ transit‐amplifying progenitors and neuroblasts. Lineage tracing experiments performed in the GLAST::CreERT2;Rosa‐YFP transgenic mice further demonstrated that ADPβS promoted proliferation of glutamate/aspartate transporter‐positive progenitors and sustained their progression toward the generation of rapidly dividing progenitors. Altogether, our results show that the purinergic system crucially affects SVZ progenitor activities both directly and through the involvement of reactive astrocytes. GLIA 2014;62:428–439     

Calorie restriction activates new adult born olfactory‐bulb neurones in a ghrelin‐dependent manner but acyl‐ghrelin does not enhance subventricular zone neurogenesis

The ageing and degenerating brain show deficits in neural stem/progenitor cell (NSPC) plasticity that are accompanied by impairments in olfactory discrimination. Emerging evidence suggests that the gut hormone ghrelin plays an important role in protecting neurones, promoting synaptic plasticity and increasing hippocampal neurogenesis in the adult brain. In the present study, we investigated the role of ghrelin with respect to modulating adult subventricular zone (SVZ) NSPCs that give rise to new olfactory bulb (OB) neurones. We characterised the expression of the ghrelin receptor, growth hormone secretagogue receptor (GHSR), using an immunohistochemical approach in GHSR‐eGFP reporter mice to show that GHSR is expressed in several regions, including the OB but not in the SVZ of the lateral ventricle. These data suggest that acyl‐ghrelin does not mediate a direct effect on NSPC in the SVZ. Consistent with these findings, treatment with acyl‐ghrelin or genetic silencing of GHSR did not alter NSPC proliferation within the SVZ. Similarly, using a bromodeoxyuridine pulse‐chase approach, we show that peripheral treatment of adult rats with acyl‐ghrelin did not increase the number of new adult‐born neurones in the granule cell layer of the OB. These data demonstrate that acyl‐ghrelin does not increase adult OB neurogenesis. Finally, we investigated whether elevating ghrelin indirectly, via calorie restriction (CR), regulated the activity of new adult‐born cells in the OB. Overnight CR induced c‐Fos expression in new adult‐born OB cells but not in developmentally born cells, whereas neuronal activity was absent following re‐feeding. These effects were not present in ghrelin^?/? mice, suggesting that adult‐born cells are uniquely sensitive to changes in ghrelin mediated by fasting and re‐feeding. In summary, ghrelin does not promote neurogenesis in the SVZ and OB; however, new adult‐born OB cells are activated by CR in a ghrelin‐dependent manner.     

Developmental characterization of Zswim5 expression in the progenitor domains and tangential migration pathways of cortical interneurons in the mouse forebrain

GABAergic interneurons play an essential role in modulating cortical networks. The progenitor domains of cortical interneurons are localized in developing ventral forebrain, including the medial ganglionic eminence (MGE), caudal ganglionic eminence (CGE), preoptic area (POA), and preoptic hypothalamic border domain (POH). Here, we characterized the expression pattern of Zswim5, an MGE-enriched gene in the mouse forebrain. At E11.5–E13.5, prominent Zswim5 expression was detected in the subventricular zone (SVZ) of MGE, POA, and POH, but not CGE of ventral telencephalon where progenitors of cortical interneurons resided. At E15.5 and E17.5, Zswim5 expression remained in the MGE/pallidum primordium and ventral germinal zone. Zswim5 mRNA was markedly decreased after birth and was absent in the adult forebrain. Interestingly, the Zswim5 expression pattern resembled the tangential migration pathways of cortical interneurons. Zswim5-positive cells in the MGE appeared to migrate from the MGE through the SVZ of LGE to overlying neocortex. Indeed, Zswim5 was co-localized with Nkx2.1 and Lhx6, markers of progenitors and migratory cortical interneurons. Double labeling showed that Ascl1/Mash1-positive cells co-expressed Zswim5. Zswim5 expressing cells contained none or at most low levels of Ki67 but co-expressed Tuj1 in the SVZ of MGE. These results suggest that Zswim5 is immediately upregulated as progenitors exiting cell cycle become postmitotic. Given that recent studies have elucidated that the cell fate of cortical interneurons is determined shortly after becoming postmitotic, the timing of Zswim5 expression in early postmitotic interneurons suggests a potential role of Zswim5 in regulation of neurogenesis and tangential migration of cortical interneurons.     

Transiently impaired neurogenesis in MPTP mouse model of Parkinson's disease

It is still being debated whether neurogenesis in the subventricular zone (SVZ) is enhanced in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injury in the adult mouse brain. Our previous studies provided evidence that MPTP induces apoptosis of migrating neuroblasts (neural progenitor cells, A cells) in the SVZ and rostral migratory stream (RMS). We investigated cellular kinetics in the adult SVZ and olfactory bulb (OB) after MPTP damage. Cells were labeled with bromodeoxyuridine (BrdU), and the effects of MPTP on the survival and fate of migrating and residing neuroblasts were evaluated. Two days after BrdU labeling and MPTP treatment, the number of BrdU-positive cells in the SVZ and OB of MPTP-treated mice was significantly lower than in the SVZ and OB of saline controls. Additionally, fewer BrdU-positive cells migrated to the OB of treated mice than to that of saline controls, and the cells that did migrate diffused radially into the granule cell layer (GCL) when observed at 7, 14, and 28 days. In the OB GCL, the differentiation of BrdU-positive cells into mature neurons significantly attenuated 14 and 28 days after MPTP injury. Moreover, the impaired neurogenesis was followed by a recovery of A cells in the SVZ and OB, suggesting activation of the self-repair process as a result of MPTP-induced depletion of BrdU-positive cells. Our findings clarify the kinetics underlying neurogenesis in MPTP-treated mice and may contribute to the development of an animal model of Parkinson's disease, and the demonstration of cellular kinetics in SVZ may also provide a new insight into assessing neurogenesis in MPTP-treated mouse.     

The adult spinal cord harbors a population of GFAP‐positive progenitors with limited self‐renewal potential

Adult neural stem cells (aNSCs) of the forebrain are GFAP‐expressing cells that are intercalated within ependymal cells of the subventricular zone (SVZ). Cells showing NSCs characteristics in vitro can also be isolated from the periaqueductal region in the adult spinal cord (SC), but contradicting results exist concerning their glial versus ependymal identity. We used an inducible transgenic mouse line (hGFAP‐CreERT2) to conditionally label GFAP‐expressing cells in the adult SVZ and SC periaqueduct, and directly and systematically compared their self‐renewal and multipotential properties in vitro. We demonstrate that a population of GFAP⁺ cells that share the morphology and the antigenic properties of SVZ‐NSCs mostly reside in the dorsal aspect of the central canal (CC) throughout the spinal cord. These cells are non‐proliferative in the intact spinal cord, but incorporate the S‐phase marker EdU following spinal cord injury. Multipotent, clonal YFP‐expressing neurospheres (i.e., deriving from recombined GFAP‐expressing cells) were successfully obtained from both the intact and injured spinal cord. These spheres however showed limited self‐renewal properties when compared with SVZ‐neurospheres, even after spinal cord injury. Altogether, these results demonstrate that significant differences exist in NSCs lineages between neurogenic and non‐neurogenic regions of the adult CNS. Thus, although we confirm that a population of multipotent GFAP⁺ cells co‐exists alongside with multipotent ependymal cells within the adult SC, we identify these cells as multipotent progenitors showing limited self‐renewal properties. GLIA 2013;61:2100–2113     

Ultrastructure of the subventricular zone in Macaca fascicularis and evidence of a mouse‐like migratory stream

Recent publications have shown that the lateral wall of the lateral ventricles in the Macaca fascicularis brain, in particular the subventricular zone (SVZ), contains neural stem cells throughout adulthood that migrate through a migratory pathway (RMS) to the olfactory bulb (OB). To date, a detailed and systematic cytoarchitectural and ultrastructural study of the monkey SVZ and RMS has not been done. We found that the organization of the SVZ was similar to that of humans, with the ependymal layer surrounding the lateral ventricles, a hypocellular GAP layer formed by astrocytic and ependymal expansions, and the astrocyte ribbon, composed of astrocytic bodies. We found no cells corresponding to the type C proliferating precursor of the rodent brain. Instead, proliferating cells, expressed as Ki‐67 immunoreactivity, were predominantly young neurons concentrated in the anterior regions, and occasional astrocytes of the ribbon. We observed displaced ependymal cells of still unknown significance. New neurons tended to organize in chain‐like structures, which were surrounded by astrocytes. This pattern was highly reminiscent of that observed in rodent RMS, but not in humans. These chains spread from the frontal SVZ along a GAP‐like layer, uniquely composed of astrocytic expansions, to the olfactory bulb (OB). The number of neuronal chains and the number of chain‐forming cells decreased gradually upon reaching the OB. The purpose of this work is to provide a reference for future studies in the field of adult neurogenesis that may lead to an understanding of the fate and functionality of newborn neurons in primates, and ultimately in humans. J. Comp. Neurol. 514:533–554, 2009. © 2009 Wiley‐Liss, Inc.     

Neurogenesis in the circumventricular organs of adult mouse brains

The circumventricular organs (CVOs), including the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), median eminence (ME), and area postrema (AP), allow parenchyma cells to sense a variety of blood‐derived substances and/or secreted peptides into blood circulation. In the present study, we examined continuous neurogenesis in the CVOs of adult mice. The immunohistochemistry of neural progenitor cell (NPC) marker proteins revealed that Math1‐ and Mash1‐positive cells were observed in the discrete regions of CVOs, including the capillary plexus in the OVLT, the internal zone of the ME, and the lateral zone in the AP. A few Mash1‐ and Math1‐positive cells were seen throughout the SFO, and many Math1‐ but not Mash1‐positive cells were observed at the arcuate nucleus. Math‐positive cells were often seen to localize in close proximity to the vasculature. Bromodeoxyuridine (BrdU) immunohistochemistry revealed the incorporation of BrdU in a subpopulation of Mash1‐, Math1‐, HuC/D‐, and microtubule‐associated protein 2 (MAP2)‐positive cells. Mash1‐ and Math1‐positive cells expressed exclusively high level of plasminogen, whereas a subpopulation of HuC/D‐ and MAP2‐positive neurons expressed low or undetectable level of plasminogen. Thus, the present study demonstrates that newborn cells express NPC marker proteins and plasminogen to localize closely at vascular matrix and moreover differentiate into neurons expressing mature neuron marker proteins, indicating that new neurons are possibly generated to integrate into new neural circuits. © 2013 Wiley Periodicals, Inc.     

NG2 cells are distinct from neurogenic cells in the postnatal mouse subventricular zone

NG2 cells express the chondroitin sulfate proteoglycan NG2 and are a fourth type of glia distinct from astrocytes, oligodendrocytes, and microglia. NG2 cells generate oligodendrocytes but have also been reported to represent neuronal progenitor cells in the postnatal mouse subventricular zone (SVZ). We performed a detailed immunohistochemical analysis of NG2 cells in the mouse SVZ, rostral migratory stream (RMS), and olfactory bulb granule cell layer (OB GCL), which constitute a neurogenic niche in the postnatal forebrain. NG2 cells in the SVZ and RMS expressed the oligodendrocyte precursor cell antigen platelet‐derived growth factor receptor‐α but did not express antigens known to be expressed by neuronogenic cells in the SVZ, such as doublecortin, PSA‐NCAM, beta‐tubulin, Dlx2, or GFAP. More than 99.5% of the proliferating cells in the SVZ were NG2 negative. In the olfactory bulb, NG2 cells were found to generate primarily oligodendrocytes and a small number of astrocytes but not neurons. In the SVZ and RMS, NG2 cells were sparse and made up a much smaller fraction of the cells compared with the surrounding nonneurogenic parenchyma. Parenchymal NG2 cells were often located along the border of the SVZ and RMS. The abundance of NG2 cells increased in the distal parts of the RMS and especially in the OB GCL, where NG2 cell processes were seen in close proximity to many maturing interneurons. Our findings indicate that NG2 cells do not represent neuronal progenitor cells in the postnatal SVZ but are likely to be oligodendrocyte precursor cells. J. Comp. Neurol. 512:702–716, 2009. © 2008 Wiley‐Liss, Inc.     

Loss of galectin‐3 decreases the number of immune cells in the subventricular zone and restores proliferation in a viral model of multiple sclerosis

Multiple sclerosis (MS) frequently starts near the lateral ventricles, which are lined by subventricular zone (SVZ) progenitor cells that can migrate to lesions and contribute to repair. Because MS‐induced inflammation may decrease SVZ proliferation and thus limit repair, we studied the role of galectin‐3 (Gal‐3), a proinflammatory protein. Gal‐3 expression was increased in periventricular regions of human MS in post‐mortem brain samples and was also upregulated in periventricular regions in a murine MS model, Theiler's murine encephalomyelitis virus (TMEV) infection. Whereas TMEV increased SVZ chemokine (CCL2, CCL5, CCL, and CXCL10) expression in wild type (WT) mice, this was inhibited in Gal‐3^?/? mice. Though numerous CD45+ immune cells entered the SVZ of WT mice after TMEV infection, their numbers were significantly diminished in Gal‐3^?/? mice. TMEV also reduced neuroblast and proliferative SVZ cell numbers in WT mice but this was restored in Gal‐3^?/? mice and was correlated with increased numbers of doublecortin+ neuroblasts in the corpus callosum. In summary, our data showed that loss of Gal‐3 blocked chemokine increases after TMEV, reduced immune cell migration into the SVZ, reestablished SVZ proliferation and increased the number of progenitors in the corpus callosum. These results suggest Gal‐3 plays a central role in modulating the SVZ neurogenic niche's response to this model of MS. GLIA 2016;64:105–121     

CXCR4 prevents dispersion of granule neuron precursors in the adult dentate gyrus

Neurogenesis in the adult dentate gyrus (DG) generates new granule neurons that differentiate in the inner one‐third of the granule cell layer (GCL). The migrating precursors of these neurons arise from neural stem cells (NSCs) in the subgranular zone (SGZ). Although it is established that pathological conditions, including epilepsy and stroke, cause dispersion of granule neuron precursors, little is known about the factors that regulate their normal placement. Based on the high expression of the chemokine CXCL12 in the adult GCL and its role in guiding neuronal migration in development, we addressed the function of the CXCL12 receptor CXCR4 in adult neurogenesis. Using transgenic reporter mice, we detected Cxcr4‐GFP expression in NSCs, neuronal‐committed progenitors, and immature neurons of adult and aged mice. Analyses of hippocampal NSC cultures and hippocampal tissue by immunoblot and immunohistochemistry provided evidence for CXCL12‐promoted phosphorylation/activation of CXCR4 receptors in NSCs in vivo and in vitro. Cxcr4 deletion in NSCs of the postnatal or mature DG using Cre technology reduced neurogenesis. Fifty days after Cxcr4 ablation in the mature DG, the SGZ showed a severe reduction of Sox2‐positive neural stem/early progenitor cells, NeuroD‐positive neuronal‐committed progenitors, and DCX‐positive immature neurons. Many immature neurons were ectopically placed in the hilus and inner molecular layer, and some developed an aberrant dendritic morphology. Only few misplaced cells survived permanently as ectopic neurons. Thus, CXCR4 signaling maintains the NSC pool in the DG and specifies the inner one‐third of the GCL as differentiation area for immature granule neurons. © 2013 Wiley Periodicals, Inc.     

Characterization of TROY/TNFRSF19/TAJ-expressing cells in the adult mouse forebrain

A member of the tumor necrosis factor receptor superfamily (TNFRSF), TROY/TNFRSF19/TAJ, is highly expressed in the brain of adult mice. Northern blot analysis using mRNA taken from regions of the adult CNS showed the expression of TROY in all regions examined, including the olfactory bulb, cerebral cortex, striatum, and hippocampus. In situ hybridization and immunohistochemistry revealed that TROY mRNA and protein were strongly expressed in the rostral migratory stream (RMS) and subventricular zone (SVZ) of adult mice. In the adult SVZ, some glial fibrillary acidic protein (GFAP)-positive cells (type B cells) are thought to be multipotent neural stem cells. These type B cells divide slowly and generate epidermal growth factor receptor (EGFR)-positive transit-amplifying precursor cells (type C cells) in the presence of epidermal growth factor (EGF). Type C cells give rise to neuron-specific class III beta-tubulin (TuJ1)-positive neuroblasts (type A cells) that migrate to the olfactory bulb along the RMS. TROY-expressing cells were GFAP-positive, EGFR-positive, and TuJ1-negative in the adult SVZ. From these findings, TROY appears to be expressed in type B and type C cells, but not in type A cells, which was supported by immunoelectron microscopy. In addition, TROY was expressed in GFAP-positive astrocytes of the various regions, such as the cerebral cortex, striatum, and hippocampus. Thus, TROY was expressed in uncommitted precursor cells and astroglial lineage cells, suggesting that TROY plays some roles in the regulation of gliogenesis in the adult CNS.     

Adult subventricular zone neuronal precursors continue to proliferate and migrate in the absence of the olfactory bulb.

Neurons continue to be born in the subventricular zone (SVZ) of the lateral ventricles of adult mice. These cells migrate as a network of chains through the SVZ and the rostral migratory stream (RMS) into the olfactory bulb (OB), where they differentiate into mature neurons. The OB is the only known target for these neuronal precursors. Here, we show that, after elimination of the OB, the SVZ and RMS persist and become dramatically larger. The proportion of dividing [bromodeoxyuridine (BrdU)-labeled] or dying (pyknotic or terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeled) cells in the RMS was not significantly affected at 3 d or 3 weeks after bulbectomy (OBX). However, by 3 months after OBX, the percentage of BrdU-labeled cells in the RMS decreased by half and that of dying cells doubled. Surprisingly, the rostral migration of precursors continued along the RMS after OBX. This was demonstrated by focal microinjections of BrdU and grafts of SVZ cells carrying LacZ under the control of a neuron-specific promoter gene. Results indicate that the OB is not essential for proliferation and the directional migration of SVZ precursors.