Eph/ephrin signaling: networks

Bidirectional signaling has emerged as an important signature by which Ephs and ephrins control biological functions. Eph/ephrin signaling participates in a wide spectrum of developmental processes, and cross-regulation with other communication pathways lies at the heart of the complexity underlying their function in vivo. Here, we review in vitro and in vivo data describing molecular, functional, and genetic interactions between Eph/ephrin and other cell surface signaling pathways. The complexity of Eph/ephrin function is discussed in terms of the pathways that regulate Eph/ephrin signaling and also the pathways that are regulated by Eph/ephrin signaling.     

Eph/ephrin molecules--a hub for signaling and endocytosis

The development, homeostasis, and regeneration of complex organ systems require extensive cell-cell communication to ensure that different cells proliferate, migrate, differentiate, assemble, and function in a coordinated and timely fashion. Eph receptor tyrosine kinases and their ephrin ligands are critical regulators of cell contact-dependent signaling and patterning. Eph/ephrin binding can lead to very diverse biological readouts such as adhesion versus repulsion, or increased versus decreased motility. Accordingly, depending on cell type and context, a limited and conserved set of receptor-ligand interactions is translated into a large variety of downstream signaling processes. Recent evidence indicates that the endocytosis of Eph/ephrin molecules, together with the internalization of various associated tissue-specific effectors, might be one of the key principles responsible for such highly diverse and adaptable biological roles. Here, we summarize recent insights into Eph/ephrin signaling and endocytosis in three biological systems; i.e., the brain, intestine, and vasculature.     

Beta-catenin signaling promotes proliferation of progenitor cells in the adult mouse subventricular zone

The subventricular zone (SVZ) is the largest germinal zone in the mature rodent brain, and it continuously produces young neurons that migrate to the olfactory bulb. Neural stem cells in this region generate migratory neuroblasts via highly proliferative transit-amplifying cells. The Wnt/beta-catenin signaling pathway partially regulates the proliferation and neuronal differentiation of neural progenitor cells in the embryonic brain. Here, we studied the role of beta-catenin signaling in the adult mouse SVZ. beta-Catenin-dependent expression of a destabilized form of green fluorescent protein was detected in progenitor cells in the adult SVZ of Axin2-d2EGFP reporter mice. Retrovirus-mediated expression of a stabilized beta-catenin promoted the proliferation of Mash1+ cells and inhibited their differentiation into neuroblasts. Conversely, the expression of Dkk1, an inhibitor of Wnt signaling, reduced the proliferation of Mash1+ cells. In addition, an inhibitor of GSK3 beta promoted the proliferation of Mash1+ cells and increased the number of new neurons in the olfactory bulb 14 days later. These results suggest that beta-catenin signaling plays a role in the proliferation of progenitor cells in the SVZ of the adult mouse brain.     

Eph and ephrin signaling in the development of the central auditory system

Acoustic communication relies crucially on accurate interpretation of information about the intensity, frequency, timing, and location of diverse sound stimuli in the environment. To meet this demand, neurons along different levels of the auditory system form precisely organized neural circuits. The assembly of these precise circuits requires tight regulation and coordination of multiple developmental processes. Several groups of axon guidance molecules have proven critical in controlling these processes. Among them, the family of Eph receptors and their ephrin ligands emerge as one group of key players. They mediate diverse functions at multiple levels of the auditory pathway, including axon guidance and targeting, topographic map formation, as well as cell migration and tissue pattern formation. Here, we review our current knowledge of how Eph and ephrin molecules regulate different processes in the development and maturation of central auditory circuits.     

Nonsynaptic GABA signaling in postnatal subventricular zone controls proliferation of GFAP-expressing progenitors

In the postnatal subventricular zone (SVZ), local cues or signaling molecules released from neuroblasts limit the proliferation of glial fibrillary acidic protein (GFAP)-expressing progenitors thought to be stem cells. However, signals between SVZ cells have not been identified. We show that depolarization of neuroblasts induces nonsynaptic SNARE-independent GABA(A) receptor currents in GFAP-expressing cells, the time course of which depends on GABA uptake in acute mouse slices. We found that GABA(A) receptors are tonically activated in GFAP-expressing cells, consistent with the presence of spontaneous depolarizations in neuroblasts that are sufficient to induce GABA release. These data demonstrate the existence of nonsynaptic GABAergic signaling between neuroblasts and GFAP-expressing cells. Furthermore, we show that GABA(A) receptor activation in GFAP-expressing cells limits their progression through the cell cycle. Thus, as GFAP-expressing cells generate neuroblasts, GABA released from neuroblasts provides a feedback mechanism to control the proliferation of GFAP-expressing progenitors by activating GABA(A) receptors.     

Eph receptors and ephrin signaling pathways: a role in bone homeostasis

The maintenance of bone homeostasis is tightly controlled, and largely dependent upon cellular communication between osteoclasts and osteoblasts, and the coupling of bone resorption to bone formation. This tight coupling is essential for the correct function and maintenance of the skeletal system, repairing microscopic skeletal damage and replacing aged bone. A range of pathologic diseases, including osteoporosis and cancer-induced bone disease, disrupt this coupling and cause subsequent alterations in bone homeostasis. Eph receptors and their associated ligands, ephrins, play critical roles in a number of cellular processes including immune regulation, neuronal development and cancer metastasis. Eph receptors are also expressed by cells found within the bone marrow microenvironment, including osteoclasts and osteoblasts, and there is increasing evidence to implicate this family of receptors in the control of normal and pathological bone remodeling.     

Fractone-heparan sulfates mediate BMP-7 inhibition of cell proliferation in the adult subventricular zone

Bone morphogenetic protein-7 (BMP-7) is a heparin-binding growth factor that inhibits cell proliferation in the subventricular zone (SVZ) of the lateral ventricle, the primary neurogenic niche in the adult brain. However, the physiological mechanisms regulating the activity of BMP-7 in the SVZ are unknown. Here, we report the inhibitory effect of BMP-7 on cell proliferation through the anterior SVZ after intracerebroventricular injection in the adult mouse. To determine whether the inhibition of cell proliferation induced by BMP-7 is dependant on heparin-binding, heparitinase-1 was intracerebroventricularly injected to N-desulfate heparan sulfate proteoglycans before BMP-7 was injected. Heparatinase-1 drastically reduced the inhibitory effect of BMP-7 on cell proliferation in the SVZ. To determine where BMP-7 binds within the niche, we visualized biotinylated-BMP-7 after intracerebroventricular injection, using streptavidin Texas red on frozen brain sections. BMP-7 binding was seen as puncta in the SVZ at the location of fractones, the particulate specialized extracellular matrix of the SVZ, which have been identified primarily by N-sulfated heparan sulfate immunoreactivity (NS-HS+). BMP binding was also seen in NS-HS+ blood vessels of the SVZ. Injection of heparitinase-1 prior to biotinylated BMP-7 resulted in the absence of signal for biotinylated-BMP-7 in the fractones and blood vessels, indicating that the binding is heparan sulfate dependant. These results indicate that BMP-7 requires heparan sulfates to bind and inhibit cell proliferation in the SVZ neurogenic niche. Heparan sulfates concentrated in fractones and SVZ blood vessels emerge as a functional stem cell niche component involved in growth factor activity.     

Anosmin-1 over-expression increases adult neurogenesis in the subventricular zone and neuroblast migration to the olfactory bulb

New subventricular zone (SVZ)-derived neuroblasts that migrate via the rostral migratory stream are continuously added to the olfactory bulb (OB) of the adult rodent brain. Anosmin-1 (A1) is an extracellular matrix protein that binds to FGF receptor 1 (FGFR1) to exert its biological effects. When mutated as in Kallmann syndrome patients, A1 is associated with severe OB morphogenesis defects leading to anosmia and hypogonadotropic hypogonadism. Here, we show that A1 over-expression in adult mice strongly increases proliferation in the SVZ, mainly with symmetrical divisions, and produces substantial morphological changes in the normal SVZ architecture, where we also report the presence of FGFR1 in almost all SVZ cells. Interestingly, for the first time we show FGFR1 expression in the basal body of primary cilia in neural progenitor cells. Additionally, we have found that A1 over-expression also enhances neuroblast motility, mainly through FGFR1 activity. Together, these changes lead to a selective increase in several GABAergic interneuron populations in different OB layers. These specific alterations in the OB would be sufficient to disrupt the normal processing of sensory information and consequently alter olfactory memory. In summary, this work shows that FGFR1-mediated A1 activity plays a crucial role in the continuous remodelling of the adult OB     

Wnt expression in the adult rat subventricular zone after stroke

Neurogenesis occurs in adult brain neural progenitor cells (NPCs) located in the subventricular zone (SVZ) of the lateral ventricle and the subgrandular zone of the hippocampal dentate gyrus. After ischemic stroke, NPCs in the SVZ proliferate and migrate towards the ischemic boundary region to replenish damaged neurons. During development, the Wnt pathways contribute to stem cell maintenance and promote neurogenesis. We hypothesized that stroke up regulates Wnt family genes in SVZ cells. Non-ischemic and ischemic cultured SVZ cells and a single population of non-ischemic and ischemic SVZ cells isolated by laser capture microdissection (LCM) were analyzed for Wnt pathway expression using real-time RT-PCR and immunostaining. The number of neurospheres increased significantly (p<0.05) in SVZ cells derived from ischemic (32+/-4.7/rat) compared with the number in non-ischemic SVZ cells (18+/-3/rat). Wnt family gene mRNA levels were detected in SVZ cells isolated from both cultured and LCM SVZ cells, however there was no up regulation between non-ischemic and ischemic SVZ cells. Immunostaining on brain sections also demonstrated no up regulation of Wnt pathway protein between ischemic and non-ischemic SVZ cells. Expression of the Wnt family genes in SVZ cells support the hypothesis that the Wnt pathway may be involved in neurogenesis in the adult brain. However, ischemia does not up regulate Wnt family gene expression.     

小鼠脑室下区细胞增殖能力随年龄增加逐渐下降与β-catenin的相关研究

目的:脑内成体干细胞状态和年龄的关系非常密切,本研究探讨小鼠老化过程中脑室下区(subven-tricular zone,SVZ)内成体干细胞随年龄增加增殖活性的变化和脑内β-catenin信号的相关性。方法:C57B/L小鼠分别饲养至2,12,18月时处死,各组动物处死前腹腔注射BrdU(60 mg/kg,处死前3 d开始注射,连续注射3 d,1次/d)。部分动物断头取脑,冰台上游离前额皮层,冷冻备用,部分动物灌注固定,连续冠状冰冻切片备用。利用免疫组织化学方法检测SVZ区BrdU和Ki67表达。利用Western blot技术检测前额皮层内β-catenin的表达水平。结果:12,18月时小鼠SVZ区BrdU和Ki67阳性细胞的数量与2月时比较明显有所减少(P<0.05)。Western blot结果可见前额皮层内β-catenin的表达水平在2月时达高峰,12月和18月时逐渐减少。结论:SVZ区干细胞的增殖能力随年龄增加逐渐下降可能与脑内β-catenin信号的减弱有关。     

Deprivation of sensory inputs to the olfactory bulb up-regulates cell death and proliferation in the subventricular zone of adult mice

The main olfactory bulb (MOB) is the first relay on the olfactory sensory pathway and the target of the neural progenitor cells generated in the subventricular zone (SVZ) lining the lateral ventricles and which migrate along the rostral extension of the SVZ, also called the rostral migratory stream (RMS). Within the MOB, the neuroblasts differentiate into granular and periglomerular interneurons. A reduction in the number of granule cells during sensory deprivation suggests that neurogenesis may be influenced by afferent activity. Here, we show that unilateral sensory deafferentation of the MOB by axotomy of the olfactory receptor neurons increases apoptotic cell death in the SVZ and along the rostro-caudal extent of the RMS. The vast majority of dying cells in the RMS are migrating neuroblasts as indicated by double Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling/PSA-NCAM labeling. Counting bromodeoxyuridine-labeled cells in animals killed immediately or 4 days after tracer administration showed a bilateral increase in proliferation in the SVZ and RMS which was balanced by cell death on the operated side. These data suggest that olfactory inputs are required for the survival of newborn neural progenitors. The greatest enhancement in proliferation occurred in the extension of the RMS located in the MOB, revealing a population of local precursors mitotically stimulated following axotomy. Together, these findings indicate that olfactory inputs may strongly modulate the balance between neurogenesis and apoptosis in the SVZ and RMS and provide a model for further investigation of the underlying molecular mechanisms of this activity-dependent neuronal plasticity.     

Characterization of epidermal growth factor-induced dysplasia in the adult rat subventricular zone

Epidermal growth factor (EGF) is a mitogen widely used when culturing adult neural stem cells in vitro. Although proliferative effects can also be observed in vivo, intracerebroventricular infusion of EGF has been found to counteract neuronal determination and promote glial differentiation instead. However, EGF receptor activation has different effects on the subventricular zone (SVZ) in mice and rats, possibly because of species differences in SVZ cell composition. Specifically in the rat, EGF stimulation of the SVZ induces the formation of hyperplastic polyps. The present study aims at molecular and morphological characterization of these subventricular polyps. Using immunohistochemistry, electron microscopy, and gene expression analysis, we demonstrate in hyperplastic EGF-induced polyps an upregulation in protein expression of Sox2, Olig2, GFAP, nestin, and vimentin. We found polyp-specific dysplastic changes in the form of coexpression of Sox2 and Olig2. This highly proliferative, Sox2/Olig2 coexpressing dysplastic cell type is >10-fold enriched in the hyperplastic polyps compared with control SVZ and most likely causes the polyp formation. Unique ultrastructural features of the polyps include a lack of ependymal cell lining as well as a large number of cells with large, light, ovoid nuclei and a cytoplasm with abundant ribosomes, whereas other polyp cells contain invaginated nuclei but fewer ribosomes. EGF also induced changes in the expression of Id genes Id1, Id2, and Id4 in the SVZ. Taken together, we here demonstrate dysplastic, structural, and phenotypical changes in the rat SVZ following EGF stimulation, which are specific to hyperplastic polyps.     

成年哺乳动物脑内具有神经生发功能的脑室下区

成年哺乳动物脑侧脑室外侧壁的脑室下区 (SVZ)具有持续存在神经生发现象 ,表明此区存在着神经干细胞。成年神经干细胞 (adult neural stem cells)主要是指存在于成年神经系统的多能干细胞 (m ultipotent stem cells) [1 ] ,它们具有自我更新潜能 (self- renewal potential)和多向分化潜能 (m ul-tipotentiality) ,它们的子代细胞中既有与自身完全相同的细胞还有可通过非对称性分裂分化为神经元或胶质细胞的细胞。发现和分离培养以及鉴定出成年神经干细胞是近年来神经科学领域中的重大成果之一 ,它为探讨神经组织发育机制和神经组织损伤修…     

Radial glial origin of the adult neural stem cells in the subventricular zone

Adult neurogenesis persists within restricted areas of the mammalian brain, giving rise prevalently to neuronal precursors that integrate inside the hippocampus and olfactory bulb. The source of this continuous cell production consists of neural stem cells which have been identified as elements of the astroglial lineage. This counterintuitive finding overlaps with the recent discovery that embryonic radial glia can themselves act as stem cells, capable of producing both neurons and glia during development. Although radial glia was thought to disappear early postnatally at the end of neurogenesis by transformation into parenchymal astrocytes, it has recently been demonstrated that some radial glial cells somehow persist within the adult forebrain subventricular zone, hidden among astrocytes of the glial tubes. This transformation occurs in parallel with overall morphological and molecular changes within the neurogenic site, whose specific steps, mechanisms, and outcomes are not yet fully understood. The modified radial glia appear to be neural progenitor cells belonging to the astroglial lineage (type B cells) assuring both stem cell self-renewal and production of a differentiated progeny in the adult subventricular zone, and also playing regulatory roles in stem cell niche maintenance.     

Glutamate enhances survival and proliferation of neural progenitors derived from the subventricular zone

Extracellular glutamate levels increase as a consequence of perinatal hypoxia/ischemia, causing the death of neurons and oligodendrocytes. Precursors in the subventricular zone (SVZ) also die following perinatal hypoxia/ischemia; therefore we hypothesized that glutamate would stimulate the death of neural precursors. Here we demonstrate using calcium imaging that SVZ derived neural stem/progenitor cells respond to both ionotropic and metabotropic excitatory amino acids. Therefore, we tested the effects of high levels of glutamate receptor agonists on the proliferation, survival, and differentiation of SVZ derived neural stem/progenitor cells in vitro. We show that high levels of glutamate, up to 1 mM, are not toxic to neural precursor cultures. In fact, stimulation of either the kainate receptor or group 2 metabotropic glutamate receptors (group 2 mGluR) reduces basal levels of apoptosis and increases neural precursor proliferation. Furthermore, group 2 mGluR activation expands the number of multipotent progenitor cells present in these cultures while maintaining equivalent mature cell production. We conclude that the glutamate released following perinatal hypoxia/ischemia may act to acutely promote the proliferation of multipotent precursors in the subventricular zone.     

Increased adult neurogenesis in the subventricular zone in a rat model of sepsis

Neurogenesis occurs in the adult brain throughout the lives of all mammals. The dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ) of the lateral ventricles have been established as the primary sites of adult neurogenesis, and recent studies have shown that inflammation has a modulating effect on adult neurogenesis. However, only limited studies have investigated how neurogenesis is affected during sepsis and sepsis-associated encephalopathy. Therefore, we investigated adult neurogenesis in the cecal ligation and puncture (CLP) model of sepsis using a cell proliferation marker, 5-bromo-2′-deoxyuridine (BrdU). Twenty-four rats were placed into the following three groups: an un-operated control group, a sham-operated group that underwent exactly the same procedures except for CLP, and a CLP group that survived surgical procedures and developed signs of sepsis. Rats were monitored for twenty-four hours before they were euthanized and their brains were harvested. Significantly higher numbers of BrdU-immunoreactive cells were observed in the SVZ of the lateral ventricles in the CLP group as compared with both control groups, while no significant difference was found in the number of DG granule cells between the three groups. The majority of BrdU-positive cells in the SVZ co-expressed the neuronal marker doublecortin but not the astrocytic marker glial fibrillary acidic protein. Taken together, our results suggest that sepsis induced by CLP in rats increases region-specific cellular regeneration, in a possible attempt to compensate for the devastating effect of sepsis and sepsis-associated encephalopathy on the brain.     

Increased adult neurogenesis in the subventricular zone in a rat model of sepsis

Neural mechanisms underlying word processing have been extensively studied. It has been revealed that when individuals are engaged in active word processing, a complex network of cortical regions is activated. However, it is entirely unknown whether the word-processing regions are intrinsically organized without any explicit processing tasks during the resting state. The present study investigated the intrinsic functional connectivity between word-processing regions during the resting state with the use of fMRI methodology. The low-frequency fluctuations were observed between the left middle fusiform gyrus and a number of cortical regions. They included the left angular gyrus, left supramarginal gyrus, bilateral pars opercularis, and left pars triangularis of the inferior frontal gyrus, which have been implicated in phonological and semantic processing. Additionally, the activations were also observed in the bilateral superior parietal lobule and dorsal lateral prefrontal cortex, which have been suggested to provide top-down monitoring on the visual-spatial processing of words. The findings of our study indicate an intrinsically organized network during the resting state that likely prepares the visual system to anticipate the highly probable word input for ready and effective processing.     

Migration and differentiation of adult rat subventricular zone progenitor cells transplanted into the adult rat striatum

Adult brain subventricular zone progenitor cells undergo neurogenesis in the olfactory bulb. We tested the hypothesis that cultured adult subventricular zone progenitor cells migrate and differentiate into neurons when transplanted into the adult striatum. Cells in the adult rat subventricular zone were isolated and cultured for 8 days in medium containing basic fibroblast growth factor. These cells proliferated as assayed by bromodeoxyuridine immunostaining, and the majority of them were neuron-specific class III beta-tubulin (TuJ1) immunoreactive at 8 days of culture. These cultured cells were labeled in vitro with bromodeoxyuridine or with lipophilic dye-coated particles and were transplanted into the adult rat striatum. Twenty-eight days after transplantation, the cells migrated 0.5-1.5 mm from the midline of the graft to the surrounding host striatum. Migration of grafted cells in the host striatum was also detected on magnetic resonance imaging in living rats. Morphological analysis revealed that many of these migrated cells exhibited multibranched processes from the cell soma resembling host medium-size striatal projection neurons. Only a few astrocyte-like cells were detected. Double immunostaining showed that many bromodeoxyuridine immunoreactive cells were microtubule-associated protein 2 or immunoreactive with a mouse monoclonal antibody against neuronal nuclear protein, whereas only a few bromodeoxyuridine immunoreactive cells had glial fibrillary acidic protein immunoreactivity. Morphology of bromodeoxyuridine and microtubule-associated protein 2 immunoreactive cells was similar to those of host microtubule-associated protein 2 immunoreactive cells. These results suggest that transplanted cultured adult subventricular zone progenitor cells can migrate and differentiate in response to guidance cues within the adult striatum.     

Peripherally injected lipopolysaccharide induces apoptosis in the subventricular zone of young adult mice

Because the subventricular zone (SVZ) constantly supplies newly generated neurons to the olfactory bulb (OB) along the rostral migratory stream (RMS) in adult brain, SVZ-RMS-OB axis has been thought to work as a unit. We previously reported that peripherally injected lipopolysaccharide (LPS) induces apoptosis in the OB in young adult mice. Therefore, this study was undertaken to examine whether peripherally injected LPS induces apoptotic cell death also in the SVZ. Two mouse strains were used: C3H/HeN and Toll-like receptor 4-mutated C3H/HeJ, and wild-type C57BL/6 and TNFR1^−/−-2^−/−, in which the genes tumor necrosis factor receptor (TNFR)1 and TNFR2 are knocked out. Immunohistochemical study and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay done on the SVZ-RMS pathway of young adult male mice showed that peripherally injected LPS switches on the apoptotic signal by cleaving pro-caspase-3, thus possibly increasing the number of cells dying from apoptosis in these areas in adult mice. Activation of the tumor necrosis factor (TNF)-α-TNFR system played a critical role in fully inducing apoptosis in this pathway. We suggest that TNF-α was probably released not from microglia but from astrocytes in the SVZ and RMS.