Enhanced neurogenesis in the ischemic striatum following EGF-induced expansion of transit-amplifying cells in the subventricular zone

In the subventricular zone (SVZ) of the adult mammalian brain, neural stem cells continually produce transit-amplifying precursors, which generate neuroblasts migrating into the olfactory bulb. Previous studies have suggested that SVZ cells also have the capacity to generate some striatal neurons after cerebral ischemia. The infusion of epidermal growth factor (EGF) has been demonstrated to increase the number of these regenerated neurons. However, which cell types in the SVZ are stimulated to proliferate or differentiate after EGF infusion remains unknown. In this paper, we demonstrated that cerebral ischemia results in an increase in the number of EGF receptor (EGFR)-positive transit-amplifying cells in the SVZ. EGF infusion into the ischemic brain caused the number of transit-amplifying cells to increase and the number of neuroblasts to decrease. On the other hand, after an interval of 6 days after the discontinuation of EGF infusion, a significant increase in the number of neuroblasts was found, both in the striatum and the SVZ. These results suggest that the replacement of neurons in injured striatum can be enhanced by an EGF-induced expansion of transit-amplifying cells in the SVZ.     

Controlled epi-cortical delivery of epidermal growth factor for the stimulation of endogenous neural stem cell proliferation in stroke-injured brain

One of the challenges in treating central nervous system (CNS) disorders with biomolecules is achieving local delivery while minimizing invasiveness. For the treatment of stroke, stimulation of endogenous neural stem/progenitor cells (NSPCs) by growth factors is a promising strategy for tissue regeneration. Epidermal growth factor (EGF) enhances proliferation of endogenous NSPCs in the subventricular zone (SVZ) when delivered directly to the ventricles of the brain; however, this strategy is highly invasive. We designed a biomaterials-based strategy to deliver molecules directly to the brain without tissue damage. EGF or poly(ethylene glycol)-modified EGF (PEG-EGF) was dispersed in a hyaluronan and methylcellulose (HAMC) hydrogel and placed epi-cortically on both uninjured and stroke-injured mouse brains. PEG-modification decreased the rate of EGF degradation by proteases, leading to a significant increase in protein accumulation at greater tissue depths than previously shown. Consequently, EGF and PEG-EGF increased NSPC proliferation in uninjured and stroke-injured brains; and in stroke-injured brains, PEG-EGF significantly increased NSPC stimulation. Our epi-cortical delivery system is a minimally-invasive method for local delivery to the brain, providing a new paradigm for local delivery to the brain.     

Characterization of adult neural stem cells and their relation to brain tumors

The adult mammalian brain contains neural stem cells that are capable of generating new neurons and glia over the course of a lifetime. Neural stem cells reside in 2 germinal niches, the subventricular zone (SVZ) and the dentate gyrus subgranular zone. These primary progenitors have been identified in their niche in vivo; these cells have characteristics of astrocytes. Recent studies have shown that adult SVZ stem cells are derived from radial glia, the stem cells in the developing brain, which in turn are derived from the neuroepithelum, the earliest brain progenitors. Thus, SVZ stem cells are a continuum from neuroepithelium to radial glia to astrocytes, and are contained within what has been considered the lineage for astrocytes. However, it seems that only a small subset of the astrocytes present in the adult brain have stem cell properties. Recent findings have shown that SVZ stem cell astrocytes express a receptor for platelet-derived growth factor (PDGF), suggesting that the ability to respond to specific growth factor stimuli, such as PDGF, epidermal growth factor and others, may be unique to these stem cell astrocytes. Intriguingly, activation of these same signaling pathways is widely implicated in brain tumor formation. Since the adult brain has very few proliferating cells capable of accumulating the numerous mutations required for transformation, the adult neural stem and/or progenitor cells may be likely candidates for the brain tumor cell of origin. Indeed, activation of the PDGF or epidermal growth factor pathways in adult neural stem or progenitor cells confers tumor-like properties on these cells, lending support to this hypothesis.     

Changes in cognition induced by social isolation in the mouse are restored by electro-acupuncture

Social isolation by individual housing the mice eliminates social interaction and induces pathophysiological changes including decreased learning capabilities. Acupuncture with the manipulation of the needles according to traditional Chinese medicine or with the electrical stimulation applied on needles inserted into the acupoints (referred as electro-acupuncture, EA) may modulate stress response, mood, learning and memory process. We investigated whether or not EA could reverse memory impairments induced in the male mouse by social isolation. We also studied any changes due to EA or social isolation in neurotrophic factors as nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) known to have a role in regulating memory and learning processes. We found that 30 min daily session of EA for a period of 4 days at the acupoint Zusanli (low burst frequency of 2 Hz, each pulse was a square electric wave and had a duration of 180, length of 0.1, and internal burst frequency of 80 Hz; intensity 1.0-1.5 mA) reverses the changes in the passive avoidance responses of isolated mice. These findings were associated with decreased NGF and BDNF in the hippocampus and decreased NGF in the striatum of isolated mice exposed to EA. In conclusion the present data in the mouse show that EA may modulate cognition in mice subjected to social isolation.     

Cell division and apoptosis in the adult neural stem cell niche are differentially affected in transthyretin null mice

Thyroid hormones (THs) are fundamental in regulation of growth and development, particularly of the brain. THs are required for full proliferative activity of neural stem cells in the subventricular zone (SVZ) of adult mouse brains, and also affect the normal fate of progenitor cells: apoptosis. Transthyretin (TTR) is a TH distributor protein in the blood and cerebrospinal fluid. TTR secretion by the choroid plexus is involved in transport of THs from blood into cerebrospinal fluid. We investigated the regulation of neural stem cell cycle in the SVZ of adult TTR null mice. Markers for neural stem cell mitosis that are reduced during hypothyroidism, did not differ between genotypes. However, in TTR null mice the level of apoptosis, the fate of most progenitor cells, was as low as that in brains of hypothyroid wildtype mice. Thus, lack of TTR results in reduced availability of TH to progenitor cells in the SVZ. We show that proliferation and apoptosis in the SVZ neural stem cell niche are differentially affected by the lack of TTR synthesis.     

An enriched environment mitigates the brain-disruptive effects of prenatal diethylstilbestrol exposure in mice

An enriched environment is known to promote structural changes in the brain and to enhance learning and memory performance in rodents. We previously reported that prenatal exposure to diethylstilbestrol (DES) impaired passive avoidance responses and increased levels of phosphorylated Ca²⁺/calmodulin-dependent protein kinase II (pCaMKII) in the hippocampus of mice. In this study, we examined whether an enriched environment affects the behavioral and neurochemical changes induced in mice prenatally exposed to DES. Male DES-exposed mice were placed in a standard or enriched environment at 3 weeks of age and subjected to behavioral testing after 3 weeks of exposure to these environments. Immunoblot analysis and 5-bromodeoxyuridine (BrdU) immunohistochemistry were then performed. In DES-exposed mice reared in an enriched environment, passive avoidance responses were significantly improved compared to those in mice reared in a standard environment. Moreover, the increase in level of pCaMKII in the hippocampus of DES-exposed mice was reversed by rearing in an enriched environment. Numbers of BrdU-positive cells in the dentate gyrus were significantly increased in normal and DES-exposed mice reared in the enriched environment compared to those in mice reared in the standard environment. These findings suggest that rearing in an enriched environment may mitigate the defects in brain function induced by prenatal exposure to endocrine disrupters such as DES.     

On the functional role of polypeptide growth factors in rodent neurobehavioral development

A number of polypeptide growth factor molecules have been discovered and characterized in the recent years. Most of them appear to play important roles in the ontogeny of both vertebrates and invertebrates, and some growth factors are specifically involved in brain development of altricial mammals such as rats and mice. In particular, Nerve Growth Factor (NGF) seems to be involved in degenerative processes of the aged CNS and to control brain development, particularly cholinergic systems. Data concerning NGF effects on neurobehavioral development of neonatal mice are reported, and compared with the specific alterations caused by neonatal Epidermal Growth Factor (EGF) exposure on a number of physical and behavioral scores. While NGF mainly anticipated behavioral development, EGF exerted both growth-promoting and growth-inhibiting effects.     

Induction of striatal neurogenesis enhances functional recovery in an adult animal model of neonatal hypoxic-ischemic brain injury

While intraventricular administration of epidermal growth factor (EGF) expands the proliferation of neural stem/progenitor cells in the subventricular zone (SVZ), overexpression of brain-derived neurotrophic factor (BDNF) is particularly effective in enhancing striatal neurogenesis. We assessed the induction of striatal neurogenesis and consequent functional recovery after chronic infusion of BDNF and EGF in an adult animal model of neonatal hypoxic-ischemic (HI) brain injury. Permanent brain damage was induced in CD-1® (ICR) mice (P7) by applying the ligation of unilateral carotid artery and hypoxic condition. At 6 weeks of age, the mice were randomly assigned to groups receiving a continuous 2-week infusion of one of the following treatments into the ventricle: BDNF, EGF, BDNF/EGF, or phosphate buffered saline (PBS). Two weeks after treatment, immunohistochemical analysis revealed an increase in the number of BrdU⁺ cells in the SVZ and striata of BDNF/EGF-treated mice. The number of new neurons co-stained with BrdU and βIII-tubulin was also significantly increased in the neostriata of BDNF/EGF-treated mice, compared with PBS group. In addition, the newly generated cells were expressed as migrating neuroblasts labeled with PSA-NCAM or doublecortin in the SVZ and the ventricular side of neostriata. The new striatal neurons were also differentiated as mature neurons co-labeled with BrdU⁺/NeuN⁺. When evaluated post-surgical 8 weeks, BDNF/EGF-treated mice exhibited significantly longer rotarod latencies at constant speed (48 rpm) and under accelerating condition (4–80 rpm), relative to PBS and untreated controls. In the forelimb-use asymmetry test, BDNF/EGF-treated mice showed significant improvement in the use of the contralateral forelimb. In contrast, this BDNF/EGF-associated functional recovery was abolished in mice receiving a co-infusion of 2% cytosine-b-d-arabinofuranoside (Ara-C), a mitotic inhibitor. Induction of striatal neurogenesis by the intraventricular administration of BDNF and EGF promoted functional recovery in an adult animal model of neonatal HI brain injury. The effect of Ara-C to completely block functional recovery indicates that the effect may be the result of newly generated neurons. Therefore, this treatment may offer a promising strategy for the restoration of motor function for adults with cerebral palsy (CP).     

Tumor necrosis factor-alpha modulates survival, proliferation, and neuronal differentiation in neonatal subventricular zone cell cultures

Tumor necrosis factor (TNF)-alpha has been reported to modulate brain injury, but remarkably, little is known about its effects on neurogenesis. We report that TNF-alpha strongly influences survival, proliferation, and neuronal differentiation in cultured subventricular zone (SVZ) neural stem/progenitor cells derived from the neonatal P1-3 C57BL/6 mice. By using single-cell calcium imaging, we developed a method, based on cellular response to KCl and/or histamine, that allows the functional evaluation of neuronal differentiation. Exposure of SVZ cultures to 1 and 10 ng/ml mouse or 1 ng/ml human recombinant TNF-alpha resulted in increased differentiation of cells displaying a neuronal-like profile of [Ca2+](i) responses, compared with the predominant profile of immature cells observed in control, nontreated cultures. Moreover, by using neutralizing antibodies for each TNF-alpha receptor, we found that the proneurogenic effect of 1 ng/ml TNF-alpha is mediated via tumor necrosis factor receptor 1 activation. Accordingly, the percentage of neuronal nuclear protein-positive neurons was increased following exposure to mouse TNF-alpha. Interestingly, exposure of SVZ cultures to 1 ng/ml TNF-alpha induced cell proliferation, whereas 10 and 100 ng/ml TNF-alpha induced apoptotic cell death. Moreover, we found that exposure of SVZ cells to TNF-alpha for 15 minutes or 6 hours caused an increase in the phospho-stress-activated protein kinase/c-Jun N-terminal kinase immunoreactivity initially in the nucleus and then in growing axons, colocalizing with tau, consistent with axonogenesis. Taken together, these results show that TNF-alpha induces neurogenesis in neonatal SVZ cell cultures of mice. TNF-alpha, a proinflammatory cytokine and a proneurogenic factor, may play a central role in promoting neurogenesis and brain repair in response to brain injury and infection.     

Immunocytochemical localization of nerve growth factor,epidermal growth factor,renin and protease A in the submandibular glands of Tfm/Y mice

The submandibular glands of mice with testicular feminization (Tfm/Y) and their normal adult male littermates (Ta/Y) were studied by immunocytochemical techniques for the demonstration of epidermal growth factor (EGF), nerve growth factor (NGF), renin and protease A. In the glands of both the affected and normal males, these polypeptides were restricted to cells of the granular convoluted tubules (GCT), with the exception of protease A, which was also found in small amounts in striated duct cells. Compared to those of Ta/Y males, GCTs were narrower in the glands of Tfm/Y mice and contained a markedly reduced number of cells immunoreactive for EGF, NGF and renin. However, the number of GCT cells that stained for protease A in the glands of Tfm/Y males was not as drastically decreased.     

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.     

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.     

Selective upregulation of 3-phosphoglycerate dehydrogenase (Phgdh) expression in adult subventricular zone neurogenic niche

In the adult rodent brain, constitutive neurogenesis occurs in two restricted regions, the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus, where multipotent neural stem/progenitor cells generate new neurons. Using Western blotting and immunohistochemistry for established markers, we demonstrated that the expression of 3-phosphoglycerate dehydrogenase (Phgdh), an enzyme involved in de novo synthesis of l-serine, was upregulated in the SVZ. The expression was selective to cells having morphological features and expressing markers of astrocyte-like primary neural stem cells (type B cells) and their progeny, actively proliferating progenitors (type C cells). By contrast, Phgdh protein expression was virtually absent in committed neuronal precursors (type A cells) derived from type C cells. High levels of Phgdh were also expressed by glial tube cells located in the rostral migratory stream (RMS). Interestingly, ensheathment of type A cells by these Phgdh-expressing cells was persistent in the SVZ and RMS, suggesting that l-serine mediates trophic support for type A cells via these glial cells. In vitro neurosphere assays confirmed that growth-factor-responsive, transient amplifying neural progenitors in the SVZ, but not differentiated neurons, expressed Phgdh. In the aged brain, a decline in Phgdh expression was evident in type B and C cells of the SVZ. These observations support the notion that availability of l-serine within neural stem/progenitor cells may be a critical factor for neurogenesis in developing and adult brain.     

Simultaneous visualization of the binding of nerve growth factor and epidermal growth factor to single rat pheochromocytoma (PC12) cells through indirect immunohistofluorescence

The rat pheochromocytoma cell line, PC12, which has receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF), was used to develop a technique for the simultaneous visualization of separate growth factor receptors by indirect immunohistofluorescence. The cells were incubated with saturating concentrations of nerve growth factor and epidermal growth factor. After fixation, the cells were treated with anti-NGF sheep antiserum and then with antisheep rabbit IgG conjugated with fluorescein; they also were treated with anti-EGF rabbit antiserum and then with anti-rabbit sheep IgG conjugated with rhodamine. Fluorescence microscopy showed that a single PC12 cell bound both NGF and EGF. The fluorescence due to EGF binding was reduced when the cells were grown in the presence of NGF. A similar reduction of fluorescence was observed after addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Both manipulations are known to reduce the specific binding of 125I-EGF to these cells. Subclones of PC12 cells, NR11 and NR20, reported not to have NGF receptors, did not demonstrate NGF binding when tested with this indirect immunohistofluorescence method. Thus, the binding of growth factors which is demonstrable by indirect immunohistofluorescence method seems to reflect the presence of the specific cell surface receptors for both peptides on individual PC12 cells.     

Induction of neurogenesis in nonconventional neurogenic regions of the adult central nervous system by niche astrocyte-produced signals

The central nervous system (CNS) of adult mammals regenerates poorly; in vivo, neurogenesis occurs only in two restricted areas, the hippocampal subgranular zone (SGZ) and the subventricular zone (SVZ). Neurogenic potential depends on both the intrinsic properties of neural progenitors and the environment, or niche, in which progenitor cells reside. Isolation of multipotent progenitor cells from broad CNS regions suggests that the neurogenic potential of the adult CNS is dictated by local environmental cues. Here, we report that astrocytes in the neurogenic brain regions, the SGZ and SVZ, of adult mice release molecular signals, such as sonic hedgehog (Shh), that stimulate adult neural progenitors to reenter the cell cycle and generate new neurons in vitro and in vivo. Transplantation of SGZ astrocytes or application of Shh caused de novo neurogenesis from the non-neurogenic neocortex of adult mice. These findings identify a molecular target that can activate the dormant neurogenic potential from nonconventional neurogenic regions of the adult CNS and suggest a novel mechanism of neural replacement therapy for treating neurodegenerative disease and injury without transplanting exogenous cells.     

Cilostazol attenuates ischemic brain injury and enhances neurogenesis in the subventricular zone of adult mice after transient focal cerebral ischemia

Evidence suggests that neurogenesis occurs in the adult mammalian brain, and that various stimuli, for example, ischemia/hypoxia, enhance the generation of neural progenitor cells in the subventricular zone (SVZ) and their migration into the olfactory bulb. In a mouse stroke model, focal ischemia results in activation of neural progenitor cells followed by their migration into the ischemic lesion. The present study assessed the in vivo effects of cilostazol, a type 3 phosphodiesterase inhibitor known to activate the cAMP-responsive element binding protein (CREB) signaling, on neurogenesis in the ipsilateral SVZ and peri-infarct area in a mouse model of transient middle cerebral artery occlusion. Mice were divided into sham operated (n=12), vehicle- (n=18) and cilostazol-treated (n=18) groups. Sections stained for 5-bromodeoxyuridine (BrdU) and several neuronal and a glial markers were analyzed at post-ischemia days 1, 3 and 7. Cilostazol reduced brain ischemic volume (P<0.05) and induced earlier recovery of neurologic deficit (P<0.05). Cilostazol significantly increased the density of BrdU-positive newly-formed cells in the SVZ compared with the vehicle group without ischemia. Increased density of doublecortin (DCX)-positive and BrdU/DCX-double positive neural progenitor cells was noted in the ipsilateral SVZ and peri-infarct area at 3 and 7 days after focal ischemia compared with the vehicle group (P<0.05). Cilostazol increased DCX-positive phosphorylated CREB (pCREB)-expressing neural progenitor cells, and increased brain derived neurotrophic factor (BDNF)-expressing astrocytes in the ipsilateral SVZ and peri-infarct area. The results indicated that cilostazol enhanced neural progenitor cell generation in both ipsilateral SVZ and peri-infarct area through CREB-mediated signaling pathway after focal ischemia.     

Nasal administration of cholera toxin B subunit-nerve growth factor improves the space learning and memory abilities in beta-amyloid protein(25-35)-induced amnesic mice

Nerve growth factor (NGF) is a potential drug for Alzheimer's disease treatment, but delivering NGF to the brain is difficult. To increase the content of NGF in brain, we prepared cholera toxin B subunit (CB) -NGF by the improved sodium metaperiodate method and compared its pharmacodynamics with NGF. In vitro, CB-NGF, as well as NGF, could promote neurite outgrowth and increase choline acetyltransferase activities. But the time window of TrkA phosphorylation induced by CB-NGF and NGF was different. In vivo, nasal administration of CB-NGF could increase the stay time and partially improve abilities of space learning and memory in amnesic mice, and protected the cholinergic neurons in basal forebrain against Abeta(25-35). CB-NGF treatment has better curative effects than NGF in Alzheimer's disease model mice.