Survival Of Neuroblasts Research Articles

Interaction between subventricular zone microglia and neural stem cells impacts the neurogenic response in a mouse model of cortical ischemic stroke

After a stroke, the neurogenic response from the subventricular zone (SVZ) to repair the brain is limited. Microglia, as an integral part of the distinctive SVZ microenvironment, control neural stem / precursor cell (NSPC) behavior. Here, we show that discrete stroke-associated SVZ microglial clusters negatively impact the innate neurogenic response, and we propose a repository of relevant microglia–NSPC ligand–receptor pairs. After photothrombosis, a mouse model of ischemic stroke, the altered SVZ niche environment leads to immediate activation of microglia in the niche and an abnormal neurogenic response, with cell-cycle arrest of neural stem cells and neuroblast cell death. Pharmacological restoration of the niche environment increases the SVZ-derived neurogenic repair and microglial depletion increases the formation and survival of newborn neuroblasts in the SVZ. Therefore, we propose that altered cross-communication between microglial subclusters and NSPCs regulates the extent of the innate neurogenic repair response in the SVZ after stroke.

Adult hippocampal neurogenesis in Alzheimer's disease: A roadmap to clinical relevance.

Adult hippocampal neurogenesis (AHN) drops sharply during early stages of Alzheimer's disease (AD), via unknown mechanisms, and correlates with cognitive status in AD patients. Understanding AHN regulation in AD could provide a framework for innovative pharmacological interventions. We here combine molecular, behavioral, and clinical data and critically discuss the multicellular complexity of the AHN niche in relation to AD pathophysiology. We further present a roadmap toward a better understanding of the role of AHN in AD by probing the promises and caveats of the latest technological advancements in the field and addressing the conceptual and methodological challenges ahead.

Engineered antibody-functionalized porous silicon nanoparticles for therapeutic targeting of pro-survival pathway in endogenous neuroblasts after stroke

Generation of new neurons by utilizing the regenerative potential of adult neural stem cells (NSCs) and neuroblasts is an emerging therapeutic strategy to treat various neurodegenerative diseases, including neuronal loss after stroke. Committed to neuronal lineages, neuroblasts are differentiated from NSCs and have a lower proliferation rate. In stroke the proliferation of the neuroblasts in the neurogenic areas is increased, but the limiting factor for regeneration is the poor survival of migrating neuroblasts. Survival of neuroblasts can be promoted by small molecules; however, new drug delivery methods are needed to specifically target these cells. Herein, to achieve specific targeting, we have engineered biofunctionalized porous silicon nanoparticles (PSi NPs) conjugated with a specific antibody against polysialylated neural cell adhesion molecule (PSA-NCAM). The PSi NPs loaded with a small molecule drug, SC-79, were able to increase the activity of the Akt signaling pathway in doublecortin positive neuroblasts both in cultured cells and in vivo in the rat brain. This study opens up new possibilities to target drug effects to migrating neuroblasts and facilitate differentiation, maturation and survival of developing neurons. The conjugated PSi NPs are a novel tool for future studies to develop new therapeutic strategies aiming at regenerating functional neurocircuitry after stoke.

Possible Existence of the Hypothalamic-Pituitary-Hippocampal (HPH) Axis: A Reciprocal Relationship Between Hippocampal Specific Neuroestradiol Synthesis and Neuroblastosis in Ageing Brains with Special Reference to Menopause and Neurocognitive Disorders

The hippocampus-derived neuroestradiol plays a major role in neuroplasticity, independent of circulating estradiol that originates from gonads. The response of hypothalamus-pituitary regions towards the synthesis of neuroestradiol in the hippocampus is an emerging scientific concept in cognitive neuroscience. Hippocampal plasticity has been proposed to be regulated via neuroblasts, a major cellular determinant of functional neurogenesis in the adult brain. Defects in differentiation, integration and survival of neuroblasts in the hippocampus appear to be an underlying cause of neurocognitive disorders. Gonadotropin receptors and steroidogenic enzymes have been found to be expressed in neuroblasts in the hippocampus of the brain. However, the reciprocal relationship between hippocampal-specific neuroestradiol synthesis along neuroblastosis and response of pituitary based feedback regulation towards regulation of estradiol level in the hippocampus have not completely been ascertained. Therefore, this conceptual article revisits (1) the cellular basis of neuroestradiol synthesis (2) a potential relationship between neuroestradiol synthesis and neuroblastosis in the hippocampus (3) the possible involvement of aberrant neuroestradiol production with mitochondrial dysfunctions and dyslipidemia in menopause and adult-onset neurodegenerative disorders and (4) provides a hypothesis for the possible existence of the hypothalamic-pituitary-hippocampal (HPH) axis in the adult brain. Eventually, understanding the regulation of hippocampal neurogenesis by abnormal levels of neuroestradiol concentration in association with the feedback regulation of HPH axis might provide additional cues to establish a neuroregenerative therapeutic management for mood swings, depression and cognitive decline in menopause and neurocognitive disorders.

Genetic reprogramming of somatic cells into neuroblasts through a co-induction of the doublecortin gene along the Yamanaka factors: A promising approach to model neuroregenerative disorders

Neural stem cell (NSC) mediated adult neurogenesis represents the regenerative plasticity of the brain. The functionality of the neurogenic process appears to be operated by neuroblasts, the multipotent immature neuronal population of the adult brain. While neuroblasts have been realized to play a major role in synaptic remodeling and immunogenicity, neurodegenerative disorders have been characterized by failure in the terminal differentiation, maturation, integration and survival of newborn neuroblasts. Advancement in understanding the impaired neuroregenerative process along the neuropathological conditions has currently been limited by lack of an appropriate experimental model of neuroblasts. The genetic reprogramming of somatic cells into pluripotent state offers a potential strategy for the experimental modeling of brain disorders. Thus, the induced pluripotent stem cell (iPSC) based direct reprogramming of somatic cells into neuroblasts would represent a potential tool to understand the regenerative biology of the adult brain. Therefore, this concise article discusses the significance of iPSCs, the functional roles of neuroblasts in the adult brain and provides a research hypothesis for the direct reprogramming of somatic cells into neuroblasts through the co-induction of a potential proneurogenic marker, the doublecortin (DCX) gene along with the Yamanaka factors. The proposed cellular model of adult neurogenesis may provide us with further insights into neuropathogenesis of many neurodegenerative disorders and will provide a potential experimental platform for diagnostic, drug discovery and regenerative therapeutic strategies.

Unique role for dentate gyrus microglia in neuroblast survival and in VEGF-induced activation.

Neurogenic roles of microglia (MG) are thought to include an active role in adult hippocampal neurogenesis in addition to their established roles in pruning surplus dendrites and clearing dead neuroblasts. However, identification of such a role and its delineation in the neurogenic cascade is yet to be established. Using diphtheria toxin-aided MG ablation, we show that MG reduction in the DG-the site where neuronal stem cells (NSCs) reside-is sufficient to impede overall hippocampal neurogenesis due to reduced survival of newly formed neuroblasts. To examine whether MG residing in the hippocampal neurogenic zone are inherently different from MG residing elsewhere in the hippocampus, we compared growth factor responsiveness of DG MG with that of CA1 MG. Strikingly, transgenic induction of the potent neurogenic factor VEGF elicited robust on-site MG expansion and activation exclusively in the DG and despite eliciting a comparable angiogenic response in the CA1 and elsewhere. Temporally, DG-specific MG expansion preceded both angiogenic and neurogenic responses. Remarkably, even partial MG reduction during the process of VEGF-induced neurogenesis led to reducing the number of newly formed neuroblasts to the basal level. Transcriptomic analysis of MG retrieved from the naïve DG and CA1 uncovered a set of genes preferentially expressed in DG MG. Notably the tyrosine kinase Axl is exclusively expressed in naïve and VEGF-induced DG MG and its inhibition prevented neurogenesis augmentation by VEGF. Taken together, findings uncover inherent unique properties of DG MG of supporting both basal- and VEGF-induced adult hippocampal neurogenesis.

Specific inhibition of ADAM17/TACE promotes neurogenesis in the injured motor cortex

Brain injuries in the adult mammalian brain are accompanied by a fast neurogenic response inside neurogenic niches. However, this response does not contribute to the generation of new neurons within damaged tissues like the cerebral cortex, which are essentially non-neurogenic. This occurs because injuries create a hostile environment that favors gliogenesis. Overexpression and sequential activation of the ADAM17/TGFα/EGFR signaling cascade are crucial for the generation of this gliogenic/non-neurogenic environment. Here, we demonstrate that chronic local infusion of a general metalloprotease inhibitor in areas of traumatic cortical injury in adult mice moderately increased the number of neuroblasts around the lesion, by facilitating the survival of neuroblasts and undifferentiated progenitors, which had migrated to the perilesional area from the subventricular zone. Next, we generated a dominant-negative version of ADAM17 metalloprotease, consisting of a truncated protein containing only the pro-domain (ADAM17-Pro). Specific inhibition of ADAM17 activity by ADAM17-Pro overexpression increased the generation of new neurons in vitro. Local overexpression of ADAM17-Pro in injured cortex in vivo, mediated by lentiviral vectors, dramatically increased the number of neuroblasts observed at the lesion 14 days after injury. Those neuroblasts were able to differentiate into cholinergic and GABAergic neurons 28 days after injury. We conclude that ADAM17 is a putative target to develop new therapeutic tools for the treatment of traumatic brain injury.

Draxin regulates hippocampal neurogenesis in the postnatal dentate gyrus by inhibiting DCC-induced apoptosis

Hippocampal neurogenesis in the dentate gyrus (DG) is controlled by diffusible molecules that modulate neurogenic processes, including cell proliferation, differentiation and survival. To elucidate the mechanisms underlying hippocampal neurogenesis, we investigated the function of draxin, originally identified as a neural chemorepellent, in the regulation of neuronal survival in the DG. Draxin was expressed in Tbr2 (+) late progenitors and NeuroD1 (+) neuroblasts in the dentate granule cell lineage, whereas expression of its receptor DCC (deleted in colorectal cancer) was mainly detectable in neuroblasts. Our phenotypic analysis revealed that draxin deficiency led to enhanced apoptosis of DCC-expressing neuroblasts in the neurogenic areas. Furthermore, in vitro assays using a hippocampal neural stem/progenitor cell (HNSPC) line indicated that draxin inhibited apoptosis in differentiating HNSPCs, which express DCC. Taken together, we postulate that draxin plays a pivotal role in postnatal DG neurogenesis as a dependence receptor ligand for DCC to maintain and promote survival of neuroblasts.

Effects of crenolanib, a nonselective inhibitor of PDGFR, in a mouse model of transient middle cerebral artery occlusion

Neurogenesis in the subventricular zone (SVZ) plays a vital role in neurologic recovery after stroke. However, only a small fraction of newly generated neuroblasts from the SVZ will survive long-term. Successful migration and survival of neuroblasts requires angiogenesis, lesion-derived chemo-attractants, and appropriate local microenvironments, which are partly regulated by the platelet-derived growth factor receptor (PDGFR) signaling pathway. In this study, we investigated the effects of PDGFR inhibition in a mouse model of transient middle cerebral artery occlusion (MCAO). We blocked the pathway using a nonselective PDGFR inhibitor, crenolanib, during the acute post-MCAO phase (days 1–3) or during the sub-acute phase (days 7–9). Downregulating the PDGFR signaling pathway with crenolanib from day 1 to day 3 after MCAO significantly decreased the migration of neuroblasts from the SVZ to the peri-infarct region, decreased angiogenesis, and lowered expression of vascular endothelial growth factor, stromal cell-derived factor-1, and monocyte chemotactic protein-1. Downregulation of the PDGFR signaling pathway on days 7–9 with crenolanib significantly increased apoptosis of the neuroblasts that had migrated to the peri-infarct region, increased the number of activated microglia, and decreased the expression of brain-derived neurotrophic factor, neurotrophin-3, and interleukin-10. Crenolanib treatment increased the apoptosis of pericytes and decreased the pericyte/vascular coverage, but had no effects on apoptosis of astrocytes. We conclude that the PDGFR signaling pathway plays a vital role in the SVZ neurogenesis after stroke. It can also affect angiogenesis, lesion-derived chemo-attractants, and the local microenvironment, which are all important to stroke-induced neurogenesis.

Monocyte depletion early after stroke promotes neurogenesis from endogenous neural stem cells in adult brain

Ischemic stroke, caused by middle cerebral artery occlusion, leads to long-lasting formation of new striatal neurons from neural stem/progenitor cells (NSPCs) in the subventricular zone (SVZ) of adult rodents. Concomitantly with this neurogenic response, SVZ exhibits activation of resident microglia and infiltrating monocytes. Here we show that depletion of circulating monocytes, using the anti-CCR2 antibody MC-21 during the first week after stroke, enhances striatal neurogenesis at one week post-insult, most likely by increasing short-term survival of the newly formed neuroblasts in the SVZ and adjacent striatum. Blocking monocyte recruitment did not alter the volume of the ischemic lesion but gave rise to reduced astrocyte activation in SVZ and adjacent striatum, which could contribute to the improved neuroblast survival. A similar decrease of astrocyte activation was found in and around human induced pluripotent stem cell (iPSC)-derived NSPCs transplanted into striatum at one week after stroke in monocyte-depleted mice. However, there was no effect on neurogenesis in the graft as determined 8weeks after implantation. Our findings demonstrate, for the first time, that a specific cellular component of the early inflammatory reaction in SVZ and adjacent striatum following stroke, i.e., infiltrating monocytes, compromises the short-term neurogenic response neurogenesis from endogenous NSPCs.

Stroke alters behavior of human skin-derived neural progenitors after transplantation adjacent to neurogenic area in rat brain

BackgroundIntracerebral transplantation of human induced pluripotent stem cells (iPSCs) can ameliorate behavioral deficits in animal models of stroke. How the ischemic lesion affects the survival of the transplanted cells, their proliferation, migration, differentiation, and function is only partly understood.MethodsHere we have assessed the influence of the stroke-induced injury on grafts of human skin iPSCs-derived long-term neuroepithelial-like stem cells using transplantation into the rostral migratory stream (RMS), adjacent to the neurogenic subventricular zone, in adult rats as a model system.ResultsWe show that the occurrence of an ischemic lesion, induced by middle cerebral artery occlusion, in the striatum close to the transplant does not alter the survival, proliferation, or generation of neuroblasts or mature neurons or astrocytes from the grafted progenitors. In contrast, the migration and axonal projection patterns of the transplanted cells are markedly influenced. In the intact brain, the grafted cells send many fibers to the main olfactory bulb through the RMS and a few of them migrate in the same direction, reaching the first one third of this pathway. In the stroke-injured brain, on the other hand, the grafted cells only migrate toward the ischemic lesion and virtually no axonal outgrowth is observed in the RMS.ConclusionsOur findings indicate that signals released from the stroke-injured area regulate the migration of and fiber outgrowth from grafted human skin-derived neural progenitors and overcome the influence on these cellular properties exerted by the neurogenic area/RMS in the intact brain.

Distinct Effects of miR-210 Reduction on Neurogenesis: Increased Neuronal Survival of Inflammation But Reduced Proliferation Associated with Mitochondrial Enhancement.

Neurogenesis is essential to brain development and plays a central role in the response to brain injury. Stroke and head trauma stimulate proliferation of endogenous neural stem cells (NSCs); however, the survival of young neurons is sharply reduced by postinjury inflammation. Cellular mitochondria are critical to successful neurogenesis and are a major target of inflammatory injury. Mitochondrial protection was shown to improve survival of young neurons. This study tested whether reducing cellular microRNA-210 (miR-210) would enhance mitochondrial function and improve survival of young murine neurons under inflammatory conditions. Several studies have demonstrated the potential of miR-210 inhibition to enhance and protect mitochondrial function through upregulation of mitochondrial proteins. Here, miR-210 inhibition significantly increased neuronal survival and protected the activity of mitochondrial enzymes cytochrome c oxidase and aconitase in differentiating NSC cultures exposed to inflammatory mediators. Unexpectedly, we found that reducing miR-210 significantly attenuated NSC proliferation upon induction of differentiation. Further investigation revealed that increased mitochondrial function suppressed the shift to primarily glycolytic metabolism and reduced mitochondrial length characteristic of dividing cells. Activation of AMP-regulated protein kinase-retinoblastoma signaling is important in NSC proliferation and the reduction of this activation observed by miR-210 inhibition is one mechanism contributing to the reduced proliferation. Postinjury neurogenesis occurs as a burst of proliferation that peaks in days, followed by migration and differentiation over weeks. Our studies suggest that mitochondrial protective miR-210 inhibition should be delayed until after the initial burst of proliferation, but could be beneficial during the prolonged differentiation stage.SIGNIFICANCE STATEMENT Increasing the success of endogenous neurogenesis after brain injury holds therapeutic promise. Postinjury inflammation markedly reduces newborn neuron survival. This study found that enhancement of mitochondrial function by reducing microRNA-210 (miR-210) levels could improve survival of young neurons under inflammatory conditions. miR-210 inhibition protected the activity of mitochondrial enzymes cytochrome c oxidase and aconitase. Conversely, we observed decreased precursor cell proliferation likely due to suppression of the AMP-regulated protein kinase-retinoblastoma axis with miR-210 inhibition. Therefore, mitochondrial protection is a double-edged sword: early inhibition reduces proliferation, but inhibition later significantly increases neuroblast survival. This explains in part the contradictory published reports of the effects of miR-210 on neurogenesis.

Proliferation and Survival of Embryonic Sympathetic Neuroblasts by MYCN and Activated ALK Signaling.

MYCN overexpression combined with activated anaplastic lymphoma kinase (ALK) is sufficient to induce neuroblastoma (NB) in mouse sympathoadrenal cells. To address cellular and molecular effects elicited by MYCN/ALK cooperation, we used cultures of chick sympathetic neuroblasts. We demonstrate that MYCN increases proliferation but not survival, whereas long-term expression of ALKF1174L elicits cell-cycle exit, differentiation, and survival of postmitotic neurons. Combined MYCN/ALKF1174L expression allows long-term proliferation and survival of neuroblasts with differentiated characteristics. In the presence of ALKF1174L signaling, MYCN induces the expression of the ubiquitin ligase SKP2 (S-phase kinase-associated protein 2), which targets p27 for degradation and is also upregulated in high-risk NB. SKP2 inhibition supports a function for SKP2 in the maintained neuroblast proliferation downstream of MYCN/ALK, which may represent an early step toward tumorigenesis.

Cell therapy for Parkinson׳s disease: Functional role of the host immune response on survival and differentiation of dopaminergic neuroblasts

Parkinson׳s disease (PD) is a neurodegenerative disorder, whose cardinal pathology is the loss of dopaminergic neurons in the substantia nigra. Current treatments for PD have side effects in the long term and do not halt disease progression or regenerate dopaminergic cell loss. Attempts to compensate neuronal cell loss by transplantation of dopamine-producing cells started more than 30 years ago, leading to several clinical trials. These trials showed safety and variable efficacy among patients. In addition to variability in efficacy, several patients developed graft-induced dyskinesia. Nevertheless, they have provided a proof of concept that motor symptoms could be improved by cell transplantation.Cell transplantation in the brain presents several immunological challenges. The adaptive immune response should be abolished to avoid graft rejection by the host. In addition, the innate immune response will always be present after transplanting cells into the brain. Remarkably, the innate immune response can have dramatic effects on the survival, differentiation and proliferation of the transplanted cells, but has been hardly investigated.In this review, we analyze data on the functional effects of signals from the innate immune system on dopaminergic differentiation, survival and proliferation. Then, we discussed efforts on cell transplantation in animal models and PD patients, highlighting the immune response and the immunomodulatory treatment strategies performed. The analysis of the available data lead us to conclude that the modulation of the innate immune response after transplantation can increase the success of future clinical trials in PD by enhancing cell differentiation and survival.This article is part of a Special Issue entitled SI: PSC and the brain.

ALCAR Exerts Neuroprotective and Pro-Neurogenic Effects by Inhibition of Glial Activation and Oxidative Stress via Activation of the Wnt/β-Catenin Signaling in Parkinsonian Rats.

Oxidative stress and neuroinflammation are known causative factors in progressive degeneration of dopaminergic (DAergic) neurons in Parkinson's disease (PD). Neural stem cells (NSCs) contribute in maintaining brain plasticity; therefore, survival of NSCs and neuroblasts during neurodegenerative process becomes important in replenishing the pool of mature neuronal population. Acetyl-L-carnitine (ALCAR), present in almost all body cells, increases endogenous antioxidants and regulates bioenergetics. Currently, no information is available about the putative mechanism and neuroprotective effects of ALCAR in 6-hydroxydopamine (6-OHDA)-induced rat model of PD-like phenotypes. Herein, we investigated the effect of ALCAR on death/survival of DAergic neurons, neuroblasts and NSCs and associates mechanism of neuroprotection in 6-OHDA-induced rat model of PD-like phenotypes. ALCAR (100mg/kg/day, intraperitoneal (i.p.)) treatment started 3days prior to 6-OHDA lesioning and continued for another 14day post-lesioning. We found that ALCAR pretreatment in 6-OHDA-lesioned rats increased expression of neurogenic and the Wnt pathway genes in the striatum and substantia nigra pars compacta (SNpc) region. It suppressed the glial cell activation, improved antioxidant status, increased NSC/neuroblast population and rescued the DAergic neurons in nigrostriatal pathway. ALCAR pretreatment in 6-OHDA-lesioned rats decreased GSK-3β activation and increased nuclear translocation of β-catenin. Functional deficits were restored following ALCAR pretreatment in 6-OHDA-lesioned rats as demonstrated by improved motor coordination and rotational behaviour, confirming protection of DAergic innervations in lesioned striatum. These results indicate that ALCAR exerts neuroprotective effects through the activation of Wnt/β-catenin pathway, suggesting its therapeutic use to treat neurodegenerative diseases by enhancing regenerative capacity.

Delayed post-treatment with bone marrow-derived mesenchymal stem cells is neurorestorative of striatal medium-spiny projection neurons and improves motor function after neonatal rat hypoxia-ischemia.

Perinatal hypoxia-ischemia is a major cause of striatal injury and may lead to cerebral palsy. This study investigated whether delayed administration of bone marrow-derived mesenchymal stem cells (MSCs), at one week after neonatal rat hypoxia-ischemia, was neurorestorative of striatal medium-spiny projection neurons and improved motor function. The effect of a subcutaneous injection of a high-dose, or a low-dose, of MSCs was investigated in stereological studies. Postnatal day (PN) 7 pups were subjected to hypoxia-ischemia. At PN14, pups received treatment with either MSCs or diluent. A subset of high-dose pups, and their diluent control pups, were also injected intraperitoneally with bromodeoxyuridine (BrdU), every 24h, on PN15, PN16 and PN17. This permitted tracking of the migration and survival of neuroblasts originating from the subventricular zone into the adjacent injured striatum. Pups were euthanized on PN21 and the absolute number of striatal medium-spiny projection neurons was measured after immunostaining for DARPP-32 (dopamine- and cAMP-regulated phosphoprotein-32), double immunostaining for BrdU and DARPP-32, and after cresyl violet staining alone. The absolute number of striatal immunostained calretinin interneurons was also measured. There was a statistically significant increase in the absolute number of DARPP-32-positive, BrdU/DARPP-32-positive, and cresyl violet-stained striatal medium-spiny projection neurons, and fewer striatal calretinin interneurons, in the high-dose mesenchymal stem cell (MSC) group compared to their diluent counterparts. A high-dose of MSCs restored the absolute number of these neurons to normal uninjured levels, when compared with previous stereological data on the absolute number of cresyl violet-stained striatal medium-spiny projection neurons in the normal uninjured brain. For the low-dose experiment, in which cresyl violet-stained striatal medium-spiny neurons alone were measured, there was a lower statistically significant increase in their absolute number in the MSC group compared to their diluent controls. Investigation of behavior in another cohort of animals showed that delayed administration of a high-dose of bone marrow-derived MSCs, at one week after neonatal rat hypoxia-ischemia, improved motor function on the cylinder test. Thus, delayed therapy with a high- or low-dose of adult MSCs, at one week after injury, is effective in restoring the loss of striatal medium-spiny projection neurons after neonatal rat hypoxia-ischemia and a high-dose of MSCs improved motor function.

Fibroblast growth factor and endothelin-1 receptors mediate the response of human striatal precursor cells to hypoxia

Fetal striatal transplantation has emerged as a new therapeutic strategy in Huntington’s disease (HD). Hypoxia is one of the microenvironmental stress conditions to which fetal tissue is exposed as soon as it is isolated and transplanted into the diseased host brain. Mechanisms that support neuroblast survival and replenishment of damaged cells within the HD brain in the hypoxic condition have yet to be fully elucidated. This study is aimed at investigating the molecular pathways associated with the hypoxic condition in human fetal striatal neuroblasts (human striatal precursor (HSP) cells), using the hypoxia-mimetic agent cobalt chloride (CoCl2). We analyzed the effect of CoCl2 on HSP cell proliferation and on the expression of hypoxia-related proteins, such as hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF). Moreover, we evaluated fibroblast growth factor 2 (FGF2; 50ng/ml) and endothelin-1 (ET-1; 100nM) proliferative/survival effects in HSP cells in normoxic and hypoxic conditions. Dose–response experiments using increasing concentrations of CoCl2 (50–750μM) showed that the HSP cell growth was unaffected after 24h, while it increased at 48h, with the maximal effect observed at 400μM. In contrast, cell survival was impaired at 72h. Hypoxic conditions determined HIF-1α protein accumulation and increased gene and protein expression of VEGF, while FGF2 and ET-1 significantly stimulated HSP cell proliferation both in normoxic and hypoxic conditions, thus counteracting the apoptotic CoCl2 effect at 72h. The incubation with selective receptor (FGFR1, endothelin receptor A (ETA) and endothelin receptor B (ETB)) inhibitors abolished the FGF2 and ET-1 neuroprotective effect. In particular, ET-1 stimulated HSP cell survival through ETA in normoxic conditions and through ETB during hypoxia. Accordingly, ETA expression was down-regulated, while ETB expression was up-regulated by CoCl2 treatment. Overall, our results support the idea that HSP cells possess the machinery for their adaptation to hypoxic conditions and that neurotrophic factors, such as FGF2 and ET-1, may sustain neurogenesis and long-term survival through complex receptor-mediated mechanisms.

TrkB interacts with ErbB4 and regulates NRG1-induced NR2B phosphorylation in cortical neurons before synaptogenesis.

BackgroundNeuregulin 1 (NRG1) and NMDARs play important roles in various neuronal functions including neural development. NMDARs also promote many cellular events such as proliferation and survival of neuroblasts before synapse formation. Although many recent studies have indicated that NRG1 regulates NMDAR function in cortical neurons, the effect of NRG1 on NMDAR activation before synapse formation is not well studied.ResultsNRG1 induces activation of NMDAR subunit NR2B, and tropomyosin-related kinase receptor B (TrkB), the receptor for BDNF via activation of phospholipase C-gamma (PLC-γ) in immature primary cortical neurons. Our data using TrkB inhibitor (K252a), TrkB siRNA and TrkB−/− neurons demonstrated that TrkB inhibition suppresses NRG1-induced NR2B activation in neurons. We found that NRG1 stimulation leads to GABAA receptor-mediated TrkB activation. Co-immunoprecipitation and proximity ligase assay showed that TrkB interacts with ErbB4 (NRG1 receptor) and the TrkB-ErbB4 interaction was increased following NRG1 treatment. A significant reduction in TrkB-ErbB4 interaction was observed in the prefrontal cortex of schizophrenia subjects. We found significant increase in released BDNF levels following NRG1 treatment, which was inhibited by ErbB4 inhibitor, AG1478. In addition, pretreatment with BDNF neutralizing antibody, but not control IgG abolished NRG1-induced increases in phospho-TrkB and phospho-NR2B levels. Moreover, studies using TrkB mutants showed that intercellular domain of TrkB is necessary for TrkB-ErbB4 interaction and NR2B activation.ConclusionsBDNF/TrkB signaling plays an important role in the NRG1-stimulated NR2B regulation. These findings could be of relevance to many neurodevelopmental disorders, as NRG1 and BDNF signaling pathways have been implicated in autism and schizophrenia.

Nanofibrous scaffolds releasing a small molecule BDNF-mimetic for the re-direction of endogenous neuroblast migration in the brain

Brain tissue engineering has the potential to harness existing elements of neurogenesis within the adult brain to overcome a microenvironment that is otherwise inhibitory to regeneration, especially following severe tissue damage. This study investigates the ability of electrospun poly ε-caprolactone (PCL) to re-direct the migratory pathway of endogenous neuroblasts from the disrupted subventricular zone (SVZ). A small molecule non-peptide ligand (BDNF-mimetic) that mimicked the trophic properties of brain-derived neurotrophic factor (BDNF) was incorporated into electrospun PCL scaffolds to improve neuroblast survival and promote neuroblast migration towards the implant. PCL scaffolds were able to support neuroblast infiltration and migration along the implant tract. In the presence of the BDNF-mimetic, neuroblasts were able to migrate towards the implant via the parenchyma, and their persistence within the implants was prolonged. In addition, the BDNF-mimetic improved implant integration and increased local neuronal plasticity by increasing neurite sprouting at the tissue-implant interface. SMI32+ neurites were observed inside scaffolds at 21 days but not 8 days post implantation, indicating that at least some of the infiltrated neuroblasts had differentiated into neurons.

TrkB interacts with ErbB4 and regulates NRG1-induced NR2B phosphorylation in cortical neurons before synaptogenesis

Neuregulin 1 (NRG1) and NMDARs play important roles in various neuronal functions including neural development. NMDARs also promote many cellular events such as proliferation and survival of neuroblasts before synapse formation. Although many recent studies have indicated that NRG1 regulates NMDAR function in cortical neurons, the effect of NRG1 on NMDAR activation before synapse formation is not well studied. NRG1 induces activation of NMDAR subunit NR2B, and tropomyosin-related kinase receptor B (TrkB), the receptor for BDNF via activation of phospholipase C-gamma (PLC-γ) in immature primary cortical neurons. Our data using TrkB inhibitor (K252a), TrkB siRNA and TrkB−/− neurons demonstrated that TrkB inhibition suppresses NRG1-induced NR2B activation in neurons. We found that NRG1 stimulation leads to GABAA receptor-mediated TrkB activation. Co-immunoprecipitation and proximity ligase assay showed that TrkB interacts with ErbB4 (NRG1 receptor) and the TrkB-ErbB4 interaction was increased following NRG1 treatment. A significant reduction in TrkB-ErbB4 interaction was observed in the prefrontal cortex of schizophrenia subjects. We found significant increase in released BDNF levels following NRG1 treatment, which was inhibited by ErbB4 inhibitor, AG1478. In addition, pretreatment with BDNF neutralizing antibody, but not control IgG abolished NRG1-induced increases in phospho-TrkB and phospho-NR2B levels. Moreover, studies using TrkB mutants showed that intercellular domain of TrkB is necessary for TrkB-ErbB4 interaction and NR2B activation. BDNF/TrkB signaling plays an important role in the NRG1-stimulated NR2B regulation. These findings could be of relevance to many neurodevelopmental disorders, as NRG1 and BDNF signaling pathways have been implicated in autism and schizophrenia.