Erythropoietin up-regulates SOCS2 in neuronal progenitor cells derived from SVZ of adult rat

We examined the effects of EPO on expression of suppressor of cytokine signaling 2 (SOCS2) and found that treatment of neural progenitor cells derived from the adult subventricular zone (SVZ) with recombinant human EPO (rhEPO) stimulated progenitor cell differentiation into neurons, but not astrocytes. Quantitative RT-PCR revealed that SOCS2 mRNA levels were increased in the progenitor cells treated with rhEPO. Immunostaining showed that neurons but not astrocytes were SOCS2 immunoreactive. Incubation of the progenitor cells with rhEPO in the presence of a neutralizing antibody against EPO abolished the effects of EPO on neuronal differentiation and expression of SOCS2. Our data suggest that up-regulation of SOCS2 in neuronal progenitor cells derived from the adult SVZ may regulate EPO enhanced neuronal differentiation.     

Suppressor of cytokine signalling-2 and epidermal growth factor regulate neurite outgrowth of cortical neurons

Factors that regulate neurite outgrowth are important in determining the wiring of the central nervous system. Here we describe that the intracellular regulator of cytokine signalling, suppressor of cytokine signalling-2 (SOCS2) and epidermal growth factor (EGF), both of which are expressed in the cortical plate during neural development, promote neurite outgrowth of cortical neurons. Cortical neurons derived from transgenic mice that over-express SOCS2 had an increased rate of neurite outgrowth and an increased length and number of primary neurites compared with wild-type neurons. EGF produced a similar effect in wild-type cortical neurons and further enhanced the SOCS2-induced neurite outgrowth. The mechanism of neurite outgrowth induction by SOCS2 and EGF at least partially overlapped as phosphorylation of the EGF receptor in SOCS2 over-expressing or EGF-stimulated neurons was increased on Tyrosine845, the Src binding site and neurite outgrowth in both protocols was blocked by inhibitors of the EGF receptor kinase and Src kinase.     

Galanin and galanin-like peptide modulate neurite outgrowth via protein kinase C-mediated activation of extracellular signal-related kinase

The neuropeptide galanin is widely distributed in the central nervous system and plays a role in a number of processes in the adult brain. Galanin also has neurotrophic effects in the developing nervous system and after nerve injury. The current study investigated the mechanism by which galanin promotes neurite outgrowth in the neuronal cell line PC12 and in neurospheres derived from adult hippocampal progenitor cells. We demonstrated that galanin can induce extracellular signal-related kinase (ERK) phosphorylation transiently in a concentration-dependent manner in neurons. Galanin-like peptide, which is thought to signal primarily through the GalR2 receptor subtype, induced ERK phosphorylation with similar kinetics to galanin. In functional studies, the ability of galanin and galanin-like peptide to induce neurite outgrowth was dependent on activation of both protein kinase C and ERK. This study identified a novel physiological role for galanin-induced ERK phosphorylation and identified ERK and protein kinase C as important signaling components in the galanin-mediated modulation of neurite outgrowth.     

Comparative analysis of CNS populations in knockout mice with altered growth hormone responsiveness

Recently we have shown that growth hormone (GH) inhibits neuronal differentiation and that this process is blocked by suppressor of cytokine signalling-2 (SOCS2). Here we examine several cortical and subcortical neuronal populations in GH hyper-responsive SOCS2 null (-/-) mice and GH non-responsive GH receptor null (GHR-/-) mice. While SOCS2-/- mice showed a 30% decrease in density of NeuN positive neurons in cortex compared to wildtype, GHR-/- mice showed a 25% increase even though brain size was decreased. Interneuron sub-populations were variably affected, with a slight decrease in cortical parvalbumin expressing interneurons in SOCS2-/- mice and an increase in cortical calbindin and calretinin and striatal cholinergic neuron density in GHR-/- mice. Analysis of glial cell numbers in cresyl violet or glial fibrillary acidic protein (GFAP) stained sections of cortex showed that the neuron : glia ratio was increased in GHR-/- mice and decreased in SOCS2-/- mice. The astrocytes in GHR-/- mice appeared smaller, while they were larger in SOCS2-/- mice. Neuronal soma size also varied in the different genotypes, with smaller striatal cholinergic neurons in GHR-/- mice. While the size of layer 5 pyramidal neurons was not significantly different from wildtype, SOCS2-/- neurons were larger than GHR-/- neurons. In addition, primary dendritic length was similar in all genotypes but dendritic branching of pyramidal neurons in the cortex appeared sparser in GHR-/- and SOCS2-/- mice. These results suggest that GH, possibly regulated by SOCS2, has multiple effects on central nervous system (CNS) development and maturation, regulating the number and size of multiple neuronal and glial cell types.     

Expression of α5 integrin rescues fibronectin responsiveness in NT2N CNS neuronal cells

The extracellular matrix protein fibronectin is implicated in neuronal regeneration in the peripheral nervous system. In the central nervous system (CNS), fibronectin is up‐regulated at sites of penetrating injuries and stroke; however, CNS neurons down‐regulate the fibronectin receptor α5β1 integrin during differentiation and generally respond poorly to fibronectin. NT2N CNS neuron‐like cells (derived from NT2 precursor cells) have been used in preclinical and clinical studies for treatment of stroke and a variety of CNS injury and disease models. Here we show that, like primary CNS neurons, NT2N cells down‐regulate α5β1 integrin during differentiation and respond poorly to fibronectin. The poor neurite outgrowth by NT2N cells on fibronectin can be rescued by transducing NT2 precursors with a retroviral vector expressing α5 integrin under the control of the murine stem cell virus 5′ long terminal repeat. Sustained α5 integrin expression is compatible with the CNS‐like neuronal differentiation of NT2N cells and does not prevent robust neurite outgrowth on other integrin ligands. Thus, α5 integrin expression in CNS neuronal precursor cells may provide a strategy for enhancing the outgrowth and survival of implanted cells in cell‐replacement therapies for CNS injury and disease. © 2009 Wiley‐Liss, Inc.     

Hepatocyte growth factor promotes proliferation and neuronal differentiation of neural stem cells from mouse embryos

Hepatocyte growth factor (HGF), originally cloned as a hepatocyte mitogen, has recently been reported to exhibit neurotrophic activity in addition to being expressed in different parts of the nervous system. At present, the effects of HGF on neural stem cells (NSCs) are not known. In this study, we first report the promoting effect of HGF on the proliferation of neurospheres and neuronal differentiation of NSCs. Medium containing only HGF was capable of inducing neurosphere formation. Addition of HGF to medium containing fibroblast growth factor 2 or epidermal growth factor increased both the size and number of newly formed neurospheres. More neurons were also obtained when HGF was added in differentiation medium. In contrast, neurosphere numbers were reduced after repeated subculture by mechanical dissociation, suggesting that HGF-formed neurospheres comprised predominantly progenitor cells committed to neuronal or glial lines. Together, these results suggest that HGF promotes proliferation of neurospheres and neuronal differentiation of NSCs derived from mouse embyos.     

ROCK inhibition enhances neurite outgrowth in neural stem cells by upregulating YAP expressionin vitro

Spontaneous axonal regeneration of neurons does not occur after spinal cord injury because of inhibition by myelin and other inhibitory factors. Studies have demonstrated that blocking the Rho/Rho-kinase (ROCK) pathway can promote neurite outgrowth in spinal cord injury models. In the present study, we investigated neurite outgrowth and neuronal differentiation in neural stem cells from the mouse subventricular zone after inhibition of ROCK in vitro. Inhibition of ROCK with Y-27632 increased neurite length, enhanced neuronal differentiation, and upregulated the expression of two major signaling pathway effectors, phospho-Akt and phospho-mitogen-activated protein kinase, and the Hippo pathway effector YAP. These results suggest that inhibition of ROCK mediates neurite outgrowth in neural stem cells by activating the Hippo signaling pathway.     

Enhanced neurogenesis from neural progenitor cells with G1/S-phase cell cycle arrest is mediated by transforming growth factor beta1

We have previously demonstrated that a G1/S-phase cell cycle blocker, deferoxamine (DFO), increased the number of new neurons from rat neurosphere cultures, which correlated with prolonged expression of cyclin-dependent kinase (cdk) inhibitor p27(kip1) [H. J. Kim et al. (2006)Brain Research, 1092, 1-15]. The present study focuses on neuronal differentiation mechanisms following treatment of neural stem/progenitor cells (NPCs) with a G1/S-phase cell cycle blocker. The addition of DFO (0.5 mm) or aphidicolin (Aph) (1.5 microm) to neurospheres for 8 h, followed by 3 days of differentiation, resulted in an increased number of neurons and neurite outgrowth. DFO induced enhanced expression of transforming growth factor (TGF)-beta1 and cdk5 at 24 h after differentiation, whereas Aph only increased TGF-beta1 expression. DFO-induced neurogenesis and neurite outgrowth were attenuated by administration of a cdk5 inhibitor, roscovitine, suggesting that the neurogenic mechanisms differ between DFO and Aph. TGF-beta1 (10 ng/mL) did not increase neurite outgrowth but rather the number of beta-tubulin III-positive cells, which was accompanied by enhanced p27(kip1) mRNA expression. In addition, TGF-beta receptor type II expression was observed in nestin-positive NPCs. Results indicated that DFO-induced TGF-beta1 signaling activated smad3 translocation from the cytoplasm to the nucleus. In contrast, TGF-beta1 signaling inhibition, via a TGF-beta receptor type I inhibitor (SB-505124), resulted in decreased DFO-induced neurogenesis, in conjunction with decreased p27(kip1) protein expression and smad3 translocation to the nucleus. These results suggest that cell cycle arrest during G1/S-phase induces TGF-beta1 expression. This, in turn, prompts enhanced neuronal differentiation via smad3 translocation to the nucleus and subsequent p27(kip1) activation in NPCs.     

Age-dependent effects of TWEAK/Fn14 receptor activation on neural progenitor cells

TWEAK/Fn14 signaling regulates progenitor cell proliferation, differentiation, and survival in multiple organ systems. This study examined the effects of TWEAK (tumor necrosis factor-like weak inducer of apoptosis) treatment on cultured mouse neural progenitor cells. The receptor for TWEAK is expressed by neural progenitor cells from the early embryonic stages through postnatal development. Although embryonic day 12 (E12) and postnatal day 1 (PN1) neural progenitor cells both express the receptor for TWEAK, TWEAK treatment of cultured E12 and PN1 progenitor cells resulted in age-dependent effects on proliferation and on neurite extension by neuronal progeny. TWEAK treatment did not alter proliferation of E12 neural progenitor cells but shifted PN1 progenitor cells toward cell-cycle phases G0 and G1 and reduced the rate at which they incorporated CldU. Conversely, the effects of TWEAK on axon elongation were more prominent in the earlier developmental stage. TWEAK induced extensive neurite outgrowth by the neuronal progeny of E12 but not PN1 progenitors. Treatment of the E12 progenitor cells with a TWEAK-neutralizing antibody repressed neurite extension, indicating that endogenous activation of this pathway may be required for neurite extension by the embryonic neuronal progeny. These studies indicate that TWEAK/Fn14 receptor activation exerts different effects on neural progenitor cells and their progeny depending on the developmental stage of the cells.     

Behavior of hippocampal stem/progenitor cells following grafting into the injured aged hippocampus

Multipotent neural stem/progenitor cells (NSCs) from the embryonic hippocampus are potentially useful as donor cells to repopulate the degenerated regions of the aged hippocampus after stroke, epilepsy, or Alzheimer's disease. However, the efficacy of the NSC grafting strategy for repairing the injured aged hippocampus is unknown. To address this issue, we expanded FGF-2-responsive NSCs from the hippocampus of embryonic day 14 green fluorescent protein-expressing transgenic mice as neurospheres in vitro and grafted them into the hippocampus of 24-month-old F344 rats 4 days after CA3 region injury. Engraftment, migration, and neuronal/glial differentiation of cells derived from NSCs were analyzed 1 month after grafting. Differentiation of neurospheres in culture dishes or after placement on organotypic hippocampal slice cultures demonstrated that these cells had the ability to generate considerable numbers of neurons, astrocytes, and oligodendrocytes. Following grafting into the injured aged hippocampus, cells derived from neurospheres survived and dispersed, but exhibited no directed migration into degenerated or intact hippocampal cell layers. Phenotypic analyses of graft-derived cells revealed neuronal differentiation in 3%-5% of cells, astrocytic differentiation in 28% of cells, and oligodendrocytic differentiation in 6%-10% cells. The results demonstrate for the first time that NSCs derived from the fetal hippocampus survive and give rise to all three CNS phenotypes following transplantation into the injured aged hippocampus. However, grafted NSCs do not exhibit directed migration into lesioned areas or widespread neuronal differentiation, suggesting that direct grafting of primitive NSCs is not adequate for repair of the injured aged brain without priming the microenvironment.     

Rhotekin modulates differentiation of cultured neural stem cells to neurons

Rhotekin is a downstream signal of Rho and is expressed in the central nervous system. However, the physiological role of rhotekin in the development of neural stem cells (NSCs) into neurons is unknown. In this study, we knocked down the expression of rhotekin protein with small interfering RNA (siRNA) in the NSCs and in neural differentiated cells and measured cell proliferation, differentiation, neurite length, and survival. By using immunocytochemistry and Western blot, the production of rhotekin was observed in NSCs and neuronal cells. Furthermore, rhotekin production was increased in accordance with neural differentiation. Rhotekin knock-down reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) activity and increased the cell death 72 hr after transfection in neurons. On the other hand, in NSCs, rhotekin knock-down increased MTT activity and the number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells. In the present study, we demonstrated that rhotekin is required for maintenance and survival of neurons and positively regulates differentiation and neurite outgrowth. Moreover, we found that rhotekin is produced in NSCs and that the role of rhotekin is to regulate cell proliferation negatively. In conclusion, these results suggest that rhotekin is one of the key molecules in the differentiation of NSCs into neurons.     

Activation of protein kinase A induces neuronal differentiation of HiB5 hippocampal progenitor cells

Cyclic AMP-dependent protein kinase (PKA) signaling has been shown to be a critical regulator for neuronal or glial differentiation in the developing brain and several neuronal cell lines. However, the involvement of the PKA signaling cascade in hippocampal neuronal development and differentiation is poorly understood. The present study was performed to investigate whether activation of the PKA pathway directly regulates differentiation of hippocampal progenitor cell line, HiB5. Treatment of hippocampal HiB5 cells with 0.5 mM dibutyryl-cyclic AMP (dbcAMP) at 39 degrees C in N2 medium caused dramatic morphological changes including neurite outgrowth within 24 h and an inhibition of proliferation. During these processes, PKA activity as well as phosphorylation of the cAMP responsive element binding protein (CREB) were augmented. To characterize dbcAMP-induced differentiation of HiB5 cells, the expressions of several neuronal marker genes were investigated. After 24 h of dbcAMP treatment, the expression of NF-H and NF-M neuronal makers increased with a concomitant decrease in nestin (a marker for neural precursor cells) and GFAP an astrocyte marker expression, suggesting that HiB5 cells can develop a neuronal phenotype. Using the doxycycline-inducible, enhanced GFP-fused PKA catalytic subunit alpha (PKAcalpha-EGFP) overexpression system, we found that overexpressed PKAcalpha-EGFP induces neurite outgrowth in HiB5 cells. Taken together, these pharmacological and genetic transfection studies provide compelling evidence for the role of PKA activation on neuronal differentiation in HiB5 hippocampal progenitor cells.     

DIRECTIONAL NEURITE OUTGROWTH AND AXONAL DIFFERENTIATION OF EMBRYONIC HIPPOCAMPAL NEURONS ARE PROMOTED BY A NEURITE OUTGROWTH DOMAIN OF THE B2-CHAIN OF LAMININ

Molecular cues involved in directional neurite outgrowth and axonal differentiation of embryonic hippocampal neurons were studied on substrates coated in a striped 5 μm pattern with synthetic peptides from a neurite outgrowth (RDIAEIIKDI, P1543) and cell attachment (CDPGYIGSR, P364) domain of the B2- and B1-chains of laminin, respectively. Both peptides supported neuronal attachment, but only the B2-chain-derived P1543 promoted expression of a mature neuronal phenotype. Directional neurite outgrowth and axonal differentiation of embryonic hippocampal neurons were selectively induced by striped substrates of the B2-chain-derived P1543. Axonal differentiation was determined by expression of a phosphorylated epitope of the 200 kDa neurofilament protein in the longer “axonal” neurite of the bipolar embryonic hippocampal neurons. Ethanol (100 mM), a neuroactive compound known to delay neuronal development, impaired both directional neurite outgrowth and expression of a phosphorylated epitope of the 200 kDa neurofilament protein on a patterned P1543 substratum. The present results provide direct evidence that a 10 amino acid peptide (P1543), derived from a neurite outgrowth domain of the B2-chain of laminin, may be an axonal guidance and differentiation factor for embryonic hippocampal neurons in vitro. Published by Elsevier Science Ltd.     

Role of brominated diphenly ether-209 in the differentiation of neural stem cells in vitro

Brominated diphenly ether-209 (BDE-209, decaBDE) is among the most common flame retardants. In a previous study, it was confirmed that exposure to BDE-209 can decrease learning and memory in mice. However, it is still unknown whether BDE-209 has an effect on cultured neural stem cells (NSCs). To analyse the role of BDE-209 in the differentiation of neural stem cells, NSCs obtained from neonatal rats were cultured as neurospheres in DMEM/F12 medium that contained different concentrations of BDE-209 for 7 days. BDE-209 was found to inhibit neurite outgrowth and the differentiation of NSCs into neurons in a concentration-dependent manner. BDE-209 also enhanced the ratio of differentiation of NSCs into glial cells.     

Impaired neurogenesis is an early event in the etiology of familial Alzheimer's disease in transgenic mice

Formation of new neurons in the adult brain takes place in the subventricular zone and in the subgranule layer of the dentate gyrus throughout life. Neurogenesis is thought to play a role in hippocampus‐ and olfaction‐dependent learning and memory. However, whether impairments in neurogenesis take place in learning and memory disorders, such as Alzheimer's disease, is yet to be established. Importantly, it remains to be elucidated whether neurogenic impairments play a role in the course of the disease or are the result of extensive neuropathology. We now report that transgenic mice harboring familial Alzheimer's disease‐linked mutant APPswe/PS1ΔE9 exhibit severe impairments in neurogenesis that are evident as early as 2 months of age. These mice exhibit a significant reduction in the proliferation of neural progenitor cells and their neuronal differentiation. Interestingly, levels of hyperphosphorylated tau, the cytotoxic precursor of the Alzheimer's disease hallmark neurofibrillary tangles, are particularly high in the neurogenic niches. Isolation of neural progenitor cells in culture reveals that APPswe/PS1ΔE9‐expressing neurospheres exhibit impaired proliferation and tau hyperphosphorylation compared with wildtype neurospheres isolated from nontransgenic littermates. This study suggests that impaired neurogenesis is an early critical event in the course of Alzheimer's disease that may underlie memory impairments, at least in part, and exacerbate neuronal vulnerability in the hippocampal formation and olfaction circuits. Furthermore, impaired neurogenesis is the result of both intrinsic pathology in neural progenitor cells and extrinsic neuropathology in the neurogenic niches. Finally, hyperphosphorylation of the microtubule‐associated protein tau, a critical player in cell proliferation, neuronal maturation, and axonal transport, is a major contributor to impaired neurogenesis in Alzheimer's disease. © 2010 Wiley‐Liss, Inc.