Jerome Ricard

EphrinB3 regulates cell proliferation and survival in adult neurogenesis

Interactions between ephrins and their receptors have been implicated in many processes during central nervous system development. In the adult, ephrins and Eph receptors have been implicated in controlling cell proliferation and neuroblast migration, although there is no direct evidence for the role of ephrinB3 in these functions. In addition, activation of Eph receptors has been shown to regulate transduction pathways important in cell cycle control as well as cell death. We show that ephrinB3 contributes to the control of cell proliferation and survival in the adult subventricular zone (SVZ). EphrinB3(-/-) mice exhibit a significant increase in dividing cells along the lateral ventricle, and altered expression of proteins involved in cell cycle regulation. Gain-of-function approach by infusing soluble ephrinB3-Fc molecules in ephrinB3(-/-) can suppress cell proliferation to wild type levels. At the same time, ephrinB3 also regulates cell survival as greater numbers of cells die in the SVZ of ephrinB3(-/-) mice. Together, our results suggest that ephrinB3 negatively regulates cell cycle progression and cell apoptosis in the adult subventricular zone.

EphrinB3 is an Anti-apoptotic Ligand that Inhibits the Dependence Receptor Functions of EphA4 Receptors during adult neurogenesis

Eph receptors have been implicated in regulating a diverse array of cellular functions in the developing nervous system. Recently, Eph receptors have been shown to promote cell death in adult germinal zones; however, their mechanisms of action remain ill-defined. In this study, we demonstrate that EphA4 is a new member of the dependence receptors family, which can initiate cell death in the absence of its ligand ephrinB3. Upon removal of its ligand, EphA4 triggers cell death that is dependent on caspase activation as caspase inhibitors prevent cell death. EphA4 itself is cleaved by caspase-3-like caspase in the intracellular domain at position D773/774, which is necessary for cell death initiation as mutation of the cleavage site abolishes apoptosis. In the adult subventricular zone, abolishing ephrinB3 results in increased cell death, while the absence of EphA4 results in excessive numbers of neuroblasts. Furthermore, infusion of soluble ephrinB3 into the lateral ventricle reduced cell death, and together these results support a dependence role for EphA4 in adult neurogenesis.

EphB3 limits the expansion of neural progenitor cells in the subventricular zone by regulating p53 during homeostasis and following traumatic brain injury.

Ephrins and Eph receptor(s) have recently been implicated in regulating neurogenesis in the adult subventricular zone (SVZ) and rostral migratory stream. Here, we examined the role of ephrinB3‐EphB3 signaling in mediating the SVZ response to traumatic brain injury (TBI). Analysis of EphB3 expression showed colocalization with glial fibrillary acidic protein‐positive neural stem progenitor cells (NSPCs) and doublecortin‐positive neuroblasts, whereas ephrinB3 was expressed outside the neurogenic region. TBI resulted in a significant reduction in EphB3 expression, which coincided with enhanced NSPC survival and proliferation at 3 and 7 days postinjury. Analysis of mice lacking either ephrinB3 (ephrinB3−/−) or EphB3 (EphB3−/−) showed a significant increase in bromodeoxyuridine (BrdU) incorporation and Ki67 immunoreactivity in the SVZ. Interestingly, cell death was dissimilar between knockout mice, where cell death was reduced in EphB3−/− but increased in ephrinB3−/− mice. Lateral ventricle infusion of soluble preclustered ephrinB3‐Fc reversed the proliferative and cell death defects in ephrinB3−/− but not EphB3−/− mice and prevented TBI‐induced proliferation in wild‐type NSPCs. Coincidently, tumor suppressor p53 expression was increased following EphB3 stimulation and is reduced in the absence of either EphB3 or ephrinB3. Furthermore, pharmacological inhibition and siRNA knockdown of p53‐attenuated ephrinB3‐Fc‐mediated growth suppression while having no effect on cell death in cultured NSPCs. These data demonstrate that EphB3 signaling suppresses NSPC proliferation in a p53‐dependent manner, induces cell death in the absence of ligand stimulation and is transiently reduced in the SVZ to initiate the expansion and survival of endogenous adult NSPCs following TBI. STEM CELLS 2010;28:1231–1242

Central but not systemic administration of XPro1595 is therapeutic following moderate spinal cord injury in mice

BackgroundGlial cell activation and overproduction of inflammatory mediators in the central nervous system (CNS) have been implicated in acute traumatic injuries to the CNS, including spinal cord injury (SCI). Elevated levels of the proinflammatory cytokine tumor necrosis factor (TNF), which exists in both a soluble (sol) and a transmembrane (tm) form, have been found in the lesioned cord early after injury. The contribution of solTNF versus tmTNF to the development of the lesion is, however, still unclear.MethodsWe tested the effect of systemically or centrally blocking solTNF alone, using XPro1595, versus using the drug etanercept to block both solTNF and tmTNF compared to a placebo vehicle following moderate SCI in mice. Functional outcomes were evaluated using the Basso Mouse Scale, rung walk test, and thermal hyperalgesia analysis. The inflammatory response in the lesioned cord was investigated using immunohistochemistry and western blotting analyses.ResultsWe found that peripheral administration of anti-TNF therapies had no discernable effect on locomotor performances after SCI. In contrast, central administration of XPro1595 resulted in improved locomotor function, decreased anxiety-related behavior, and reduced damage to the lesioned spinal cord, whereas central administration of etanercept had no therapeutic effects. Improvements in XPro1595-treated mice were accompanied by increases in Toll-like receptor 4 and TNF receptor 2 (TNFR2) protein levels and changes in Iba1 protein expression in microglia/macrophages 7 and 28 days after SCI.ConclusionsThese studies suggest that, by selectively blocking solTNF, XPro1595 is neuroprotective when applied directly to the lesioned cord. This protection may be mediated via alteration of the inflammatory environment without suppression of the neuroprotective effects of tmTNF signaling through TNFR2.

Inhibition of astroglial NF-kappaB enhances oligodendrogenesis following spinal cord injury

BackgroundAstrocytes are taking the center stage in neurotrauma and neurological diseases as they appear to play a dominant role in the inflammatory processes associated with these conditions. Previously, we reported that inhibiting NF-κB activation in astrocytes, using a transgenic mouse model (GFAP-IκBα-dn mice), results in improved functional recovery, increased white matter preservation and axonal sparing following spinal cord injury (SCI). In the present study, we sought to determine whether this improvement, due to inhibiting NF-κB activation in astrocytes, could be the result of enhanced oligodendrogenesis in our transgenic mice.MethodsTo assess oligodendrogenesis in GFAP-IκBα-dn compared to wild-type (WT) littermate mice following SCI, we used bromodeoxyuridine labeling along with cell-specific immuno-histochemistry, confocal microscopy and quantitative cell counts. To further gain insight into the underlying molecular mechanisms leading to increased white matter, we performed a microarray analysis in naïve and 3 days, 3 and 6 weeks following SCI in GFAP-IκBα-dn and WT littermate mice.ResultsInhibition of astroglial NF-κB in GFAP-IκBα-dn mice resulted in enhanced oligodendrogenesis 6 weeks following SCI and was associated with increased levels of myelin proteolipid protein compared to spinal cord injured WT mice. The microarray data showed a large number of differentially expressed genes involved in inflammatory and immune response between WT and transgenic mice. We did not find any difference in the number of microglia/leukocytes infiltrating the spinal cord but did find differences in their level of expression of toll-like receptor 4. We also found increased expression of the chemokine receptor CXCR4 on oligodendrocyte progenitor cells and mature oligodendrocytes in the transgenic mice. Finally TNF receptor 2 levels were significantly higher in the transgenic mice compared to WT following injury.ConclusionsThese studies suggest that one of the beneficial roles of blocking NF-κB in astrocytes is to promote oligodendrogenesis through alteration of the inflammatory environment.

Neural progenitors proliferation is inhibited by EphB3 in the developing subventricular zone

The subventricular zone (SVZ) of the mammalian forebrain is a major source of multipotent stem cells during development, and contributes to neurogenesis throughout the lifespan of the organism. Several studies described molecules regulating adult neurogenesis, however, few of them have examined neurogenesis in the early postnatal period. Adult neurogenesis is regulated in part by ephrinB3 and its receptors, so we examined the role of EphB3 on neural stem/progenitor cell (NSPC) proliferation in early postnatal development in the SVZ. To examine NSPC proliferation, we used BrdU incorporation in both cultured NSPCs and neonatal gene‐targeted knockout mice, as well as Ki67 immunostaining in EphB3−/− mice. We observed a significant increase in proliferation in cultured NSPCs derived from EphB3−/− mice and in the SVZ of EphB3−/− mice. These studies support an anti‐proliferative role for EphB3 in regulating NSPC numbers in the developing SVZ.

EphrinB3 blocks EphB3 dependence receptor functions to prevent cell death following traumatic brain injury

Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins, have a variety of roles in the developing and adult central nervous system that require direct cell–cell interactions; including regulating axon path finding, cell proliferation, migration and synaptic plasticity. Recently, we identified a novel pro-survival role for ephrins in the adult subventricular zone, where ephrinB3 blocks Eph-mediated cell death during adult neurogenesis. Here, we examined whether EphB3 mediates cell death in the adult forebrain following traumatic brain injury and whether ephrinB3 infusion could limit this effect. We show that EphB3 co-labels with microtubule-associated protein 2-positive neurons in the adult cortex and is closely associated with ephrinB3 ligand, which is reduced following controlled cortical impact (CCI) injury. In the complete absence of EphB3 (EphB3−/−), we observed reduced terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL), and functional improvements in motor deficits after CCI injury as compared with wild-type and ephrinB3−/− mice. We also demonstrated that EphB3 exhibits dependence receptor characteristics as it is cleaved by caspases and induces cell death, which is not observed in the presence of ephrinB3. Following trauma, infusion of pre-clustered ephrinB3-Fc molecules (eB3-Fc) into the contralateral ventricle reduced cortical infarct volume and TUNEL staining in the cortex, dentate gyrus and CA3 hippocampus of wild-type and ephrinB3−/− mice, but not EphB3−/− mice. Similarly, application of eB3-Fc improved motor functions after CCI injury. We conclude that EphB3 mediates cell death in the adult cortex through a novel dependence receptor-mediated cell death mechanism in the injured adult cortex and is attenuated following ephrinB3 stimulation.

High-Content Analysis of Proapoptotic EphA4 Dependence Receptor Functions Using Small-Molecule Libraries

Small-molecule compounds (SMCs) can provide an inexpensive and selective approach to modifying biological responses. High-content analysis (HCA) of SMC libraries can help identify candidate molecules that inhibit or activate cellular responses. In particular, regulation of cell death has important implications for many pathological conditions. Dependence receptors are a new classification of proapoptotic membrane receptors that, unlike classic death receptors, initiate apoptotic signals in the absence of their ligands. EphA4 has recently been identified as a dependence receptor that may have important functions in conditions as disparate as cancer biology and CNS injury and disease. To screen potential candidate SMCs that inhibit or activate EphA4-induced cell death, HCA of an SMC library was performed using stable EphA4-expressing NIH 3T3 cells. Our results describe a high-content method for screening dependence receptor-signaling pathways and demonstrate that several candidate SMCs can inhibit EphA4-mediated cell death.

Reproducible expansion and characterization of mouse neural stem/progenitor cells in adherent cultures derived from the adult subventricular zone

Endogenous neural stem/progenitor cells (NSPCs) residing in the subventricular zone (SVZ) of the adult mouse forebrain have been shown to enhance their neurogenic potential in response to CNS injury. Mechanisms involved in regulating adult neurogenesis under naïve or stressed conditions can be studied using a monolayer cell-culture system of the nestin-expressing NSPC lineage to analyze proliferation, survival, and differentiation. Here, a protocol for the expansion of NSPCs for studies aimed at understanding the functional role of NSPCs in maintaining adult neurogenic processes is described. This unit outlines detailed procedures for: (1) isolation, maintenance, and culture of the NSPC component of the SVZ niche from the lateral wall of the lateral ventricle; (2) characterization of NSPC functions by examining proliferation, survival, and differentiation; and (3) efficient siRNA transfection methods in 96-well format.

Neurogenesis: Is the Adult Stem Cell Young or Old?

Stem cell biology is one of the most exciting, controversial, and debated fields in science today. It has been suggested that neuronal replacement therapy using stem cell transplants may be one possible answer to a host of neuropathological disorders including spinal cord injury, stroke, and neurodegenerative diseases. Important sources for stem cells include the developing embryo and adult central nervous system, but will these populations of cells exhibit similar behavior and responses to stimuli? This review will discuss some important similarities and differences between the embryonic and adult stem cell, as well as the basis for developing therapeutic approaches for stem cell replacement. IUBMB Life, 56: 1‐6, 2004