Functions of neurotrophins and growth factors in neurogenesis and brain repair

Authors


Abstract

The identification and isolation of multipotent neural stem and progenitor cells in the brain, giving rise to neurons, astrocytes, and oligodendrocytes initiated many studies in order to understand basic mechanisms of endogenous neurogenesis and repair mechanisms of the nervous system and to develop novel therapeutic strategies for cellular regeneration therapies in brain disease. A previous review (Trujillo et al., Cytometry A 2009;75:38–53) focused on the importance of extrinsic factors, especially neurotransmitters, for directing migration and neurogenesis in the developing and adult brain. Here, we extend our review discussing the effects of the principal growth and neurotrophic factors as well as their intracellular signal transduction on neurogenesis, fate determination and neuroprotective mechanisms. Many of these mechanisms have been elucidated by in vitro studies for which neural stem cells were isolated, grown as neurospheres, induced to neural differentiation under desired experimental conditions, and analyzed for embryonic, progenitor, and neural marker expression by flow and imaging cytometry techniques. The better understanding of neural stem cells proliferation and differentiation is crucial for any therapeutic intervention aiming at neural stem cell transplantation and recruitment of endogenous repair mechanisms. © 2012 International Society for Advancement of Cytometry

INTRODUCTION

Cells from neural tube and crest form the nervous system during embryo development. In this process neural stem cells (NSC) extensively proliferate and differentiate into oligodendrocytes, astrocytes, and neurons that can be identified by expression of specific marker proteins (1). For a long time scientists believed that no new neurons were born in the adult central nervous system (CNS). This view was changed during the first half of the 20th century, when many research groups identified cell division in the brain of birds and rodents, but only in the 1990's the idea of neurogenesis was accepted (2). In 1992, Reynolds and Weiss isolated NSC from the striatum of adult mice and cultivated them in the presence of epidermal growth factor (EGF) and observed some clusters of dividing neural stem and progenitor cells. After EGF removal, cells were able to differentiate and expressed neural or glial markers, an indicative of stem cells in the adult brain (3). Those findings gave rise to a very useful in vitro model to study cell fate determination, called neurosphere.

Previous published research and review articles of our group focused on functions and signaling mechanisms of the kinin-kalikrein, cholinergic and purinergic systems in triggering neural differentiation and phenotype determination (4–8). However, the mechanisms how neurotrophins and growth factors determine cell fate is still far from being completely understood. This review aims to highlight the new findings about roles of neurotrophins and growth factors on the modulation of NSC proliferation, survival, and differentiation (Fig. 1). Another goal is to provide an update of new markers characterizing neural stem and progenitor cells (Fig. 1, Table 1). Cellular phenotypes are identified, to great extent, through imaging or flow cytometry analysis of neural cell marker expression. Several classical markers are used, such as Nestin for neural progenitor cells, GFAP for glial cells, β3-Tubulin for neuronal cells, microtubule associated protein 2 (MAP2), neuron-specific enolase (NSE), and NeuN for mature neurons, S100β for mature astrocytes, and Gal C for oligodendrocytes (Fig. 1). Recently, novel markers were identified, contributing to the understanding of roles and effects of grow factors and neurotrophins in neural stem cell fate determination.

Figure 1.

Effects of growth factors and neurotrophins on neural stem and progenitor cells from different regions of adult and embryonic (EMB) brain. In contrast to the developing nervous system, the adult CNS maintains NSC only in defined neurogenic areas in the brain, such as the subventricular zone (SVZ), the dentate gyrus (HDG) and the subgranular zone (SZ) of the hippocampus, the olfactory bulb (OB), and the spinal cord (SC). Moreover, NSC also persists in the adult peripheral nervous system (PNS), where they can originate dorsal root ganglion (DRG) cells and other peripheral neural phenotypes. The diagram shows the influence of growth factors and neurotrophins on proliferation, survival and differentiation of NSC into progenitor cells, following differentiation into neurons, astrocytes, or oligodendrocytes. Markers for cell differentiation and apoptosis are also indicated.

Table 1. Markers utilized for flow cytometry and immunohistochemistry to identify neural stem, progenitor, and differentiated cells
Markers profileCell identifiedCell originReference
CD44+Astrocyte progenitor cellsPostnatal mouse cerebellum(195)
CD133+/CD15+Neural stem cellsHuman fetal brain E50-55(196)
E-PHA binding N-glycansNeural stem cellsMice fetal brain E12, E14, E16(197)
GD3Neural stem cellsMouse striata and subventricular zone(198)
HRD1+/nestin+/GFAP+Neural stem cellsMice subventricular zone(199)
HRD1+/nestin+Neural stem cellsMice dentate gyrus(199)
Id1high+/GFAP+Type B1 astrocytesMice subventricular zone(200)
Ki-67Neural progenitor cellsPig subventricular zone(201)
QKFNeural stem cellsMice subventricular zone(202)
CD184+ CD271 CD44 CD24+Neural stem cellsHuman embryonic stem cells(203)
CD184 CD44 CD15Low CD24+NeuronHuman embryonic stem cells(203)
CD184+ CD44+Glial cellsHuman embryonic stem cells(203)
Vimentin+ nestin+ Sox2+Radial glial cellsRat germinal zone E16,5(204)
CD140a+/CD9+Oligodendrocyte progenitor cellsFetal human forebrain(205)
mAb 4860OligodendrocyteMouse telencephalus E13(206)
CD15+ CD29High CD24LowNeural stem cellsHuman embryonic stem cells(207)
CD15 CD29High CD24LowNeural crest-like cellsHuman embryonic stem cells(207)
CD15 CD29Low CD24HighNeuroblast and neuronsHuman embryonic stem cells(207)

Nerve Growth Factor (NGF)

In 1951, Levi-Montalcini and Hamburger first noticed that a mouse sarcoma stimulated the growth of sympathetic and sensory neurons and, together with Cohen, they isolated NGF (9). Pro-NGF, a precursor form, is processed to the mature form by furins, that then activates two types of receptors: the tyrosine kinase receptor TrkA, which binds specifically NGF, and the member of the tumor necrosis factor receptor family, p75NTR, which binds any neurotrophin. (10, 11). The expression pattern of these receptors and the concentration of NGF determines effects exerted by this polypeptide (12, 13). The proliferative effect of NGF on NSC proliferation was reported by Cattaneo and McKay. They found that cells exposed to fibroblast growth factor 2 (FGF2), followed by NGF treatment, displayed an increase in the Nestin+ population (14). Later it was demonstrated that a previous exposition to growth factors is necessary for TrkA expression (15). NGF promotes proliferation through the phosphorylation of ERK1/2 in NSC (16). Zhang et al. demonstrated that lower concentrations of NGF (2–5 ηg/ml) are more effective to promote proliferation (13).

NGF was shown to influence the migration of oligodendrocytes in the CNS (17) and of Schwann cells in the peripheral nervous system (PNS) (18), mediated through the p75NTR (19). Another effect of NGF is the induction of neurite outgrowth. NGF-producing NSC have longer neurites than naïve NSC (20), and this effect is triggered by the down-regulation of ATF5 transcription factor expression (21). Potential clinical applications of this trait are being investigated, for instance, the use of dorsal root ganglion cells together with NGF for regeneration of spinal ganglion neurons (22).

It has been demonstrated that NGF regulates the differentiation of NSC into mature neural phenotypes, a trait inhibited by EGF (15). NGF signaling leads to differentiation into neurons and astrocytes, but not into oligodendrocytes (23-26). NSC isolated from specific regions were able to differentiate into glutamatergic and sensory neurons and also into nociceptors (13, 27, 28). Differentiation is induced by NGF by down-regulation of ATF5 (21) and upregulation of TIMP-2 metalloproteinase inhibitor expression (29).

It has been suggested that NGF also plays a role in apoptosis, mediated by the p75NTR (12, 30). Although NGF has been more associated with TrkA-mediated neuroprotection and cell survival, in both CNS (13) and PNS (27), TrkA is also involved in apoptosis at low concentrations of NGF (100 fg/ml) (31). P3IK/Akt and mitogen-activated protein kinase (MAPK) pathways were shown to be involved in neuronal survival (32).

Brain-Derived Neurotrophic Factor (BDNF)

BDNF is another member of the neurotrophin family essential for developmental events of the nervous system, including proliferation, migration, differentiation, survival, apoptosis, and synaptic plasticity (33). BDNF-mediated effects are controversially discussed (34). It has been suggested that BDNF promotes only survival of neurons from the rat SVZ (subventricular zone) (35). However, it also enhanced both survival and differentiation of postnatal hippocampal stem cells (36). Our group has observed that BDNF alone has no effect on rat telencephalon-derived NSC proliferation and differentiation (Fig. 2).

Figure 2.

BDNF-mediated effects on proliferation and differentiation of rat telencephalon-derived NSC by imaging cytometry technique. (A). Immunostaining for Nestin, MAP-2, β3-Tubulin, and GFAP of neurospheres on day 7 of differentiation in the presence or absence (control) of 20 ηg/ml BDNF. Briefly, cells plated onto coverslips were blocked for 1 h with 3% FBS in PBS/0.1% Triton X-100, followed by a 2 h incubation with primary antibodies against β3-Tubulin (Sigma-Aldrich, 2G10 monoclonal), Nestin (Millipore, rat-401 monoclonal), and GFAP (DAKO, 6F2 monoclonal) at 1:500 dilution. NSC were washed with PBS and Alexa 555 and Alexa 488 (Molecular Probes, clone not informed) at 1:500 dilutions were added for 1 h. Cell nuclei were counterstained with DAPI. Coverslips were mounted and analyzed under a fluorescence microscope (Axiovert 200, Zeiss). Scale bars = 20 μm. (B). Immunodetection of BrdU incorporation after a 12 h pulse of 0.2 mM BrdU (Sigma-Aldrich) in neurospheres on day 7 of differentiation in the presence or absence (control) of 20 ηg/ml BDNF. Cells were fixed with ice-cold methanol for 10 min, washed with PBS and incubated for 30 min in 1.5 M HCl. Following the washing step, cells were incubated for 2 h with rat anti-BrdU (Abcam; 1:200 dilution, ICR1 monoclonal). Cells were again washed with PBS followed by addition of Alexa fluor 488 secondary antibodies (Molecular Probes, clone not informed) at 1:500 dilution. After washing with PBS, DAPI solution (Sigma-Aldrich; 0.3 μg/ml) was used as a nuclear stain. Coverslips were mounted and analyzed under a fluorescence microscope (Axiovert 200, Zeiss). BrdU incorporating nuclei are shown in green. The percentages of BrdU-positive cells were calculated as the ratio of immunolabeled cells over the total number of DAPI-stained cells. Scale bar = 20 μm.

BDNF exerts its effects by TrkB and p75NTR activation, the latter is known to have a role in postmitotic neural survival (37–39). Young et al. have demonstrated p75NTR expression defines a population of cells in the SVZ that persists in adulthood and is able to respond to stimulation by neurotrophins. The results of this work suggest that p75NTR is a specific postnatal marker that can be useful for identification and purification of those cells by flow cytometry. TrkB seems to be involved in proliferative mechanisms, while BDNF-induced neurogenesis occurs via p75NTR activation alone, independently from TrkB (40-42). BDNF has been so far described as the only neurotrophin able to affect dendritic development of SVZ-derived neurons via its high affinity receptor TrkB (43).

It has been proposed that TrkB and p75NTR can affect each other (44). Alternative TrkB mRNA splicing originates eight receptor isoforms, which form heterodimers with full-length receptors or competitively bind to available ligands. Truncated Trk receptors can inhibit full-length Trk receptors either by acting as dominant negative receptors or by forming nonfunctional heterodimers (45, 46).

Delayed differentiation of NSC caused by inhibition of nitric oxide (NO) production was shown to be reversed by BDNF (unpublished data), probably by upregulation of p75NTR expression (47, 48). Moreover, NO inhibits cell proliferation (49), but this effect is also abolished by BDNF (50), indicating that p75NTR is also involved in the regulation of cell proliferation.

Takahashi et al. demonstrated that hippocampus-derived stem cell clones did not reveal any response following stimulation with neurotrophins; however, following cell exposure to retinoic acid (RA) the expression of all neurotrophins receptors became upregulated. Treatment with BDNF or neurotrophin-3 (NT-3) after exposition to RA led to augmented expression of mature neuronal markers (51). In neuroblastoma cell lines and sympathetic neurons from newborn rats, expression of TrkB and subsequent dependence on BDNF, actions were induced by RA (52, 53).

Taken together, these results show that BDNF-induced neurogenesis is determined by the interaction of TrkB isoforms and p75NTR with others factors, such as NO and RA.

Neurotrophin-3 (NT-3)

NT-3 was identified and cloned in 1990. Its primary structure is very similar to NGF and BDNF (54, 55), and it binds to TrkC and p75NTR. Unlike other neurotrophins, NT-3 binds to TrkA and TrkB, although with lower affinity than its original ligands, NGF and BDNF (12, 56). The effect of NT-3 on cell fate determination depends on the expression of these receptors (12). In vitro studies demonstrated that exogenous NT-3 increased proliferation of neural crest and somite derived NSC as well as of cells cultured on NT-3 impregnated scaffolds (26, 57, 58). Cells overexpressing NT-3 were also shown to proliferate faster in vitro (59). Effects of NT-3 on proliferation were dose-dependent. Low doses promoted cell proliferation (1-20 ηg/ml) while higher doses (50 ηg/ml) actually lowered it (60).

NT-3 also affects migration and neurite outgrowth. Cells overexpressing NT-3 migrated more than nontransfected ones in rat injured spinal cord (61). NSC overexpressing NT-3 displayed longer neurites in vitro (62) and promoted neurite outgrowth in vivo (63).

NSC expressing NT-3 revealed a significant increase in the number of MAP2-positive cells after 14 and 56 days in culture (62). Interestingly, NSC expressing TrkC treated with NT-3 showed a higher MAP2-positive population than those treated with NT-3 or TrkC alone (64). Co-transplantation of Schwann cells expressing NT-3 or NSC resulted in an increase in neuronal population in rat injured spinal cord (65). Besides the role in proliferation and differentiation events, NT-3 is strongly involved in neuronal specification (27, 66, 67). Engraftment of NSC expressing NT-3 in infarcted brains increased the number of cholinergic, GABAergic, and glutamatergic neurons (68); moreover, co-transplantation of NSC and Schwan cells expressing NT-3 promoted differentiation into serotoninergic neurons in rat injured spinal cord (63). NT-3 also participates in the differentiation of oligodendrocytes (23, 57, 69, 70) from cortical multipotent cells, but not of primary culture of cortical oligodendrocyte progenitors, which differentiate only in response to stimulation by platelet-derived growth factor (PDGF) (71).

Different mechanisms are involved in NT-3-induced differentiation. NT-3, as well as transforming growth factor (TGF)-β1 and FGF2, upregulates norepinephrine transporter expression, promoting differentiation into noradrenergic neurons (72, 73). MAPK, together with PI3K/Akt pathways, is also activated by NT-3, inducing neuronal differentiation of the NSC (74, 75). Although some evidence points at NT-3 as a promoter of NMDA-induced cell death (76), its survival-promoting effects are more remarkable. NSC expressing NT-3 had a higher viability than the control group (62), and the same effect was observed in vivo, when were rats subjected to axotomization of Clarke's nucleus axons (77).

Epidermal Growth Factor (EGF)

The discovery of EGF by Stanley Cohen initiated a new era in the research field of growth and differentiation (Nobel Prize in Physiology and Medicine in 1986). Cohen isolated and characterized the EGF receptor (78, 79). EGF binds to the EGF receptor (EGFR), which in turn promotes enzymatic activity (79). The EGFR, also named ErbB-1, belongs to a family of four structurally related receptor tyrosine kinases. EGF has several important roles during development of the nervous system, including induction of proliferation and migration of NSC. There is a consensus that EGF is not mitogenic at the beginning of neural development, since its expression becomes detectable only at later stages (80, 81). Therefore, exogenous FGF2, but not EGF, stimulated the proliferation of mouse neuroepithelial cells from embryonic day 10 (E10) as well as of rat cortical cells obtained on E13. On the other hand, EGF is critical for the proliferation of EGF-responsive NSC isolated from the E14.5 subgranular zone (SGZ) (82). In other words, FGF2-responsive NSC divide symmetrically for self-renewal and proliferation, and asymmetrically, yielding EGF-responsive cells (83, 84). EGF-dependent SVZ precursor expansion measured by using the neurosphere assay is lost when the EGFR is inhibited, and the constitutive expression of active receptors is sufficient to rescue the proliferation of NSC induced by hypoxic/ischemic brain injury. These results reveal the EGFR as a key regulator of the expansion of SVZ precursors in response to brain injury (85, 86).

It is well known that the EGF/EGFR promotes phosphoinositide 3-kinase (PI3K) and extracellular-signal-regulated kinase1/2 (ERK1/2) pathway activation, resulting in NSC proliferation (87-89). Recent studies demonstrate that EGF activates adenylate cyclase and inhibits cAMP-specific phosphodiesterase, leading to intracellular cAMP accumulation and subsequent PKA activation which, in turn, stimulates CREB (90, 91). This transcription factor is required for EGF-induced cell proliferation in cultured adult NSC of the SVZ (92). Another recent study showed that EGFR-mediated signaling promotes Sox2 expression, which binds to the EGFR promoter and directly upregulates EGFR expression by a positive feedback loop in NSC of the mouse embryonic cortices (E18.5). Knockdown of Sox2 down-regulates EGFR expression and attenuates colony formation of NSC, whereas overexpression of Sox2 augments EGFR expression and promotes progenitor cells self-renewal (88). Moreover, Pax6 is also induced in regulation of EGF-induced proliferation of SVZ-derived NSC. Expression of EGFR in neurospheres from Pax6 mutant mice (E18.5), was down-regulated in vitro and in vivo, as determined by flow cytometry (93). EGFR has also been associated with the maintenance of multipotency. Flow cytometry analysis revealed that, independently from age or region of the brain, most cells overexpressing EGFR are multipotent precursor cells. However, these cells did not show higher neurogenic capabilities, indicating that EGFR is not directly linked to differentiation (94).

EGFR signaling plays an important role in migration of adult and embryonic neural precursors (94-96). This is associated with increased phosphorylation of Akt and focal adhesion kinase (97). Overexpression of EGF promotes radial migration toward the cortical plate (95), and ventrolateral migration in the lateral cortical stream (96) of fetal telencephalon. Interestingly, EGFR overexpression in nonmigratory cortical nerve/glial antigen 2 (NG2+) cells converts these cells into a migratory phenotype in vitro and in vivo (98). EGF-evoked effects have been associated with the progression from transit-amplifying precursor cells to neuroblasts. EGFR+ cells, purified by flow cytometry, demonstrated functional voltage-dependent Ca2+ channels and later on differentiated into neuroblasts (99). EGFR activity has also been connected with enhanced gliogenesis, increasing the number of newborn glia and decreasing the number of newborn neurons in vivo and in vitro (100). First of all, EGF infusion induces vast proliferation and migration of SVZ progenitors; however, seven days later, most labeled cells derived from SVZ primary precursors (type B1 cells) gave rise to the oligodendrocyte lineage, including NG2+ progenitors, and premyelinating and myelinating oligodendrocytes. SVZ B1 cells also originated a population of S100β+/GFAP+ cells in the striatum and septum, but neuronal differentiation was not observed (101). Reduced EGFR signaling in progenitor cells of the adult SVZ attenuates the production of oligodendrocytes, whereas EGFR overexpression expanded the oligodendrocyte population (102, 103). EGF induced proliferation and migration of isolated SVZ B cells which, in turn, gave rise to migratory cells expressing Olig2/NG2, but not to neuronal phenotypes. Upon EGF removal, Olig2/NG2 migratory cells stopped migrating and originated to cells expressing an oligodendrocyte- specific marker (104).

Fibroblast Growth Factor (FGF)

The nervous system has a limited capacity for self-repair. Thus, efforts have been made to improve the repair process by transplanting exogenous cells into sites of injury. In this context, FGFs can be used to maintain and expand embryonic stem cells (ESC) and NSC and to guide differentiation into specific neuronal cell subtypes in vitro. Therefore FGF plays a major role in such cell replacement therapies (105, 106). The FGF family comprises 22 ligands and 4 receptors, of which FGFR-1, 2 and 3 influence neurogenesis. Co-activation of FGFR-1 and 3 promoted symmetrical divisions of NSC, whereas inactivation of either of them resulted in asymmetrical divisions and neurogenesis. Developmental upregulation of FGFR2 expression correlated with a shift of NSC into a multi-potential state (107).

Signaling pathways involved in the maintenance of human ESC pluripotency are not fully understood. Leukemia inhibitory factor (LIF) signaling, which is essential for mouse ESC self-renewal, is not active in undifferentiated human ESC (108, 109) and activin signaling is not sufficient to sustain long-term growth of them in a chemically defined medium (106). In this context, recent studies have shown that addition of FGF2, in combination with activin, maintains long-term expression of pluripotency markers in human ESC. In addition, inhibition of the FGF signaling pathway causes human ESC differentiation (106).

Exogenous FGF2 alone improved the commitment of mouse and human ESC to a neural fate and generated NSC (105, 110). These cells proliferate in response to FGF2 and can differentiate into neurons, astrocytes, and oligodendrocytes (111, 112). This acquired multipotency results from the induction of multiple genes by FGF2, like Olig2 and EGFR, making the cells responsive to EGF and increasing their proliferative capacity (95, 113). The addition of high concentrations of both FGF2 and EGF is a standard procedure to expand NSC and progenitor cells as floating neurospheres or adherent cultures (105, 114). The determination of EGF- and FGF2-induced proliferation of NSC can be analyzed by imaging and flow cytometry techniques, as shown in Figure 3. NSC proliferation was assessed using BrdU (5-bromo-2'-deoxyuridine, an analogue of thymidine) incorporation. The percentage of BrdU+ cells increased from 8.4% to 35.8% in the presence of EGF and FGF2 (Fig. 3A). EdU (5-ethynyl-2'-deoxyuridine), another thymidine analogue, has advantages over BrdU, since EDU does not result in epitope destruction permitting co-staining by antibodies. It allows the identification of proliferative neural progenitor cells, as shown in Figure 3B.

Figure 3.

Determination of EGF and FGF2-induced proliferation of mouse telencephalon-derived NSC by flow cytometry and immunostaining. (A). BrdU incorporation was used to measure cell proliferation. Undifferentiated cells were stimulated to proliferate with EGF and FGF2 (both 20 ηg/ml) for a 24 h period compared to proliferation rates of unstimulated cells, followed by a 2 h incubation in the presence of BrdU (100 μM). An increase in the percentage of BrdU+ cells (35.8%) was observed in the presence of growth factors when compared with unstimulated cells cultured in the absence of these factors (8.4%). Protocol for BrdU labeling: cells were washed with PBS, fixed with 70% ethanol for 4 h at 4°C, and then incubated with 2 M HCl for 30 min at room temperature. Following another washing step, the preparation was treated with 0.1 M sodium tetraborate (pH 8.5) for 5 min and then washed again. The preparation was then incubated with primary anti-BrdU antibody (Axyll, ICR1 monoclonal) at 1:100 dilution in PBS containing 2% FBS at room temperature for 2 h, followed by washing with PBS; addition of a secondary antibody solution (Alexa Fluor 488, Molecular Probes, clone not informed) at 1:500 dilution in PBS containing 2% FBS at room temperature and incubation for 1 hour protected from light. After a final washing step, cells were resuspended in PBS and analyzed by flow cytometry in agreement with the MiFlowCyt standards (208). Further details are provided in the Supplementary Material. (B). Immunostaining of undifferentiated neurospheres for Nestin and EdU was used to assess cell proliferation. Cells were stimulated to proliferate with EGF and FGF2 (both 20 ηg/ml) over a 24h period followed by a 14h incubation in the presence of EdU (100 μM, Life Technologies) showing several progenitor cells proliferating in the presence of these factors. Proliferation was assessed by Click-iT EdU Imaging Kits (Life Technologies) according to the manufacturer's procedure (209). The preparation was then incubated with primary anti-Nestin antibody (Millipore, rat-401 monoclonal) at 1:500 dilution in PBS containing 2% FBS at room temperature, for 2 h, followed by washing with PBS and incubation for 1 h with of a secondary antibody solution (Alexa Fluor 555, Molecular Probes, clone not informed) at 1:500 dilution in PBS containing 2% FBS at room temperature and protected from the light. After a final washing step, slides were mounted with coverslips and analyzed under a fluorescence microscope (Axiovert 200, Zeiss) (×100 magnification). Scale bar = 20 μm.

FGF2 promotes proliferation of NSC by activation of MAPK/ERK pathway, upregulation of cyclin D2, and down-regulation of the cyclin-dependent kinase inhibitor p27kip1 expression (107, 115). FGF2 signaling is mediated through increased expression of β-catenin, nuclear translocation, and phosphorylation of GSK-3β and tyrosine phosphorylation of β-catenin. Overexpression of β-catenin, in the presence of FGF2, keeps NSC in a proliferative state and, in the absence of FGF2, enhances neuronal differentiation (116). Although FGF2 generally acts as a mitogen for NSC, in granule cell precursors, obtained from the developing cerebellum, FGF2 strongly inhibits the proliferative response to Sonic hedgehog by the activation of ERK and c-Jun N-terminal kinases (JNK). FGF2 also promotes granule cell differentiation in vitro and in vivo (117). Schwindt et al. reported that short-term removal of EGF and FGF2 from the medium promotes neurogenesis and neurite extension in human and rat neural progenitor cells (118, 119).

FGFs are essential for regular neurogenesis in the brain and spinal cord, and the development of multiple neuronal lineages in the embryo. Mice lacking FGF2 have neuronal deficits in the spinal cord and cerebral cortex (50% reduction in the number of cortical neurons at birth), and phenotypically anomalous neurons in the hippocampus (120). In vitro, FGFs have been used to stimulate and guide the differentiation of mouse (FGF2 and FGF4) and human (FGF2, FGF8, and FGF20) stem cells. FGF4 has similar effects of FGF2 in NSC obtained from embryo and adult mouse (121, 122). Furthermore, the addition of FGF4 increased the number of NSC generated from ES cells (122, 123). Finally, FGF-4 has also been suggested to be a potent mitogen for NSC (122). FGF4 is produced in an autocrine fashion by undifferentiated mouse ESC (124). If left unchecked, this factor acts in the block self-renewal and promotes commitment to mesodermal or neural lineages. On the other hand, without FGF4/ERK1/2 input, commitment of ESC to any lineage does not occur and alterations in expression of pluripotency markers Oct4, Rex1, and Nanog are not observed (125). Bone morphogenetic protein (BMP) and BMP signaling inhibitors can act downstream of FGF4 to promote non-neural and neural fates, respectively (125, 126). LIF/Stat3 inhibits lineage commitment and intervenes downstream of Erk 1/2 to override the autoinductive capacity of FGF4 (125). Another important study with mouse NSC was done by Palmer et al. (127), showing that FGF2 is critical for neuronal generation from adult NSC derived from non-neurogenic regions. Several studies also showed that mouse NSC, isolated from neocortex or dorsal spinal cord, which do not generate the oligodendrocyte lineage in vivo, can be isolated by flow cytometry and induced in vitro by FGF2 to express Olig2 and NG2 and then give rise to oligodendrocytes (113, 128, 129).

In human ESC, FGFs can also guide the differentiation in vitro. First, FGF signaling through FGFR1 was demonstrated to be required for olfactory bulb morphogenesis (130). A second study demonstrated that 100% of FGF8-treated aggregates (0% of untreated controls) co-expressed Tbx21/Reelin/Tbr1, which is characteristic of neuronal projections in the olfactory bulb, suggesting that FGF8 is sufficient to induce differentiation of olfactory bulb neurons from telencephalic progenitors (131). However, another study showed that FGF2 provoked human fetal forebrain-derived NSC to express the motor neuron marker Hb9, which is blocked by specific inhibition of FGFR. Thus, treatment with FGF2 within a specific time window generates cholinergic neurons with spinal motor neuron properties (131). The FGFR1c ligand FGF20 has potent effects in generating large numbers of dopaminergic neurons from hESC co-cultured with mouse stromal cells (132). These effects in neural cell type specification make FGFs useful for stem cell-based therapies of neurologic disorders.

Brain morphogenesis comprises essential steps, like migration of newborn neurons, glial cells and NSC, formation of neural circuits, and repair of injuries. In this way, some in vitro studies have been performed demonstrating the effects of FGF2 in these processes. For instance, the reduction in Neurogenin expression in cultured NSC can be partially restored by a brief exposure to FGF2 during the early phase of differentiation, resulting in increased migration and survival of neurons after transplantation (133). Another study revealed similar results demonstrating that FGF2 overexpression significantly enhances the migratory capacity of grafted NSC in complex three-dimensional structures, such as cortical slices (134).

The balance between apoptosis and cell survival is tightly controlled during brain maturation, as well as during neurogenesis in vitro (135). In vitro studies showed that FGF1, FGF2, and FGF4 are survival factors for neuronal cells isolated from distinct regions of the brain in the embryo (122, 136). FGF5 has also survival effects in cultured embryonic spinal motor neurons (137). However, FGF2 induces a switch in death receptor signaling, thereby upregulating TNF-α-mediated death and down-regulating the Fas-death pathway in both progenitor and primary hippocampal cells (138). Lastly, among the molecules implicated in the maintenance of NSC and neural differentiation, FGFs may have the most widespread roles in generating the cellular diversity and morphological complexity of the nervous system (105).

Glial Cell Line-Derived Neurotrophic Factor (GDNF)

Neurotrophic factors are essential for differentiation and neuron survival and maintenance of its phenotype. GDNF was found in culture supernatants of the B49 glial cell line, and it was first related with survival promotion of cultured rat mesencephalic dopaminergic neurons (139, 140). It has been demonstrated that GDNF increases the differentiation of NSC into dopaminergic neurons. The treatment of NSC with 25 ηg/ml of GDNF for 5 days increased the population of dopaminergic neurons from 2.9% to 50%, as demonstrated by flow cytometry (141).

The neurotrophic and neuroprotective effects of GDNF have been broadly described and are mainly exerted through cell survival, involving complex interactions between multiple signaling cascades (142), like the activation of PI3K/Akt and MAPK/ERK pathways (143, 144). The antiapoptotic effect of Akt is triggered by downstream targets, including Bad (Bcl-2-associated death promoter), FKHR-1 (forkhead transcription factor 1), and NF-κB (145). Neuronal survival is also supported by the activation of the MAPK/ERK pathway (146, 147). Nicole et al. (143) demonstrated that, after GDNF treatment, cortical neurons and astrocytes displayed activation of the MAPK pathway, which is responsible for the regulation of cell proliferation and differentiation. Recently, studies of GDNF signaling and function in adult brain were made using genetic animal models with deficiency in the GDNF-dependent pathways. Experiments with a conditional GDNF null mouse model allowed one to demonstrate a major physiological neuroprotective effect of GDNF and its absolute requirement for survival of dopaminergic and noradrenergic neurons in adult brain (148).

Storch et al. reported that dopaminergic neurons can be obtained from long-term cultures of human fetal mesencephalic precursor cells by incubation in differentiation medium containing interleukins, LIF, and GDNF. The resulting neurons were immunoreactive for tyrosine hydroxylase and exhibited morphological and functional properties of dopaminergic neurons in vitro (149). Addition of GDNF to E12 mouse ventral mesencephalon-derived neurospheres resulted in significantly higher cell numbers expressing early dopaminergic markers, Nurr1 and Ptx3 (150). In the presence of NT-3 and GDNF, ESC cultures did not augment proliferation, however, the number of neurons in the cultures was increased 7 days after plating. Pretreatment of ESC with GDNF also reduced the vulnerability of ESC-derived neurons to NMDA-induced death (151). Cell transplantation has been shown to be an effective therapy for CNS disorders in animal models. During the early phases of the implantation process, cells are exposed to an environment that causes hypoxia-ischemia damage, which may induce cell death. Optimization of cell transplantation efficacy depends critically on improving grafted cell survival. Wang et al. investigated the neuroprotective effects of GDNF on NSC survival, both in vivo and in vitro. NSC pretreated with GDNF for 3 days were subjected to oxygen-glucose deprivation (OGD) (152). GDNF was shown to increase NSC survival and also to reduce the number of apoptotic cells significantly, as compared to cells treated with saline. Pretreatment of NSC with GDNF also increased cell survival after transplantation into the striatum of a Parkinson Disease (PD) rat model (152). The results reported by Lei et al. partially elucidated the mechanisms involved in PD, as well as the GDNF protective effects upon ventral midbrain dopaminergic neurons. They showed that Nurr1, a critical transcription factor, is essential for the regulation of expression of a set of genes involved in dopamine metabolism (tyrosine hydroxylase, vesicular monoamine transporter (Vmat2), dopamine transporter, and aromatic L-amino acid decarboxylase). Nurr1 cross-talks with Pitx3, which is involved in the development and maintenance of dopaminergic neurons of the substantia nigra compacta (SNc) and the ventral tegmental area (VTA) (153).

The beneficial effects of GDNF were also demonstrated in a rat model of stroke. The injection of stem cells into the tail vein has been demonstrated to increase the expression of GDNF in the ischemic boundary zone (154). Studies from Lee et al. (155), using GDNF-secreting human NSC, resulted in an improvement of motor performance and an increase in survival of transplanted NSC in a mouse model for intracerebral hemorrhage (ICH). Adult mouse striatum was lesioned with bacterial collagenase to induce the ICH model. In GDNF grafted ICH brain, they found a significant increase in the concentration of antiapoptotic and cell survival-promoting factors (Bcl2, Akt, ERK-MAPK), together with a marked reduction in proapoptotic proteins (p53, Caspase 9 and 3, Bax) when compared with the control group (155). Rats subjected to middle cerebral occlusion and reperfusion and then treated with GDNF-secreting rat NSC revealed improved neurological function and increased expression of synaptophysin and postsynaptic density-95 (PSD-95) proteins. Interestingly, in the GDNF-treated group, the number of NSC was augmented, and cell survival was also positively affected by GDNF, as detected by a decrease in TUNEL labeling and caspase-3 expression. The neurotrophic factors BDNF and NT-3 were also detected in the GDNF-treated group. Transplanted NSC in the control group (naive) also promoted improvements, as GDNF-secreting NSC do, but the neuroprotective effects of GDNF-NSC were more drastic than those observed of control NSC (156).

Genetically modified human NSC secreting GDNF were transplanted unilaterally into the spinal cord of a transgenic mutant superoxide dismutase (SOD1 G93A) rat model for amyotrophic lateral sclerosis (ALS). GDNF promoted a remarkable preservation of motor neurons at early and end stages of the disease, but enhanced neuronal survival did not improve ipsilateral limb use, suggesting that additional strategies should be used for maintenance of neuromuscular connections and functional recovery (157). A transgenic mouse model for Huntington's disease, N171-82Q was transplanted with GDNF-secreting NSC derived from mouse striatum. GDNF expressing NSC transplanted mice were able to maintain motor function and showed increased striatal neuronal survival.

Transplantation studies with GDNF-modified human amniotic fluid-derived mesenchymal stem cells (AFMSC) confirmed the ability of GDNF to promote peripheral nerve regeneration. GDNF-modified AFMSC promoted improvement in muscle action potential ratio and motor function. The administration of AFMSC alone also resulted in the same effect, but it was more moderate. Moreover, early regeneration markers, such as neurofilament, had increased expression. In addition, Schwann cell apoptosis was reduced, supporting a neuroregenerative environment promoted by AFMSC and GDNF (158). GDNF-transfected NSC were able to promote sciatic nerve regeneration in rats when seeded in a poly (D,L-lactide) conduit. Thicker myelin sheaths, a higher number of myelinated axons, and a larger area of nerve regeneration were found after GDNF overexpression. Another important aspect for nerve regeneration is the vascularization rescue. The number of blood vessels was significantly increased in the group receiving GDNF-transfected NSC. The regeneration capacity of rat sciatic nerve in the presence of GDNF-transfected NSC was confirmed by histology, functional gait, and electrophysiology (159).

In summary, GDNF promoted neuronal regeneration and survival in vitro (140, 160) and can be useful for improving clinical outcome in various animal models of neurological disorders, such as Parkinson disease, spinal cord injury, and ischemia. However, underlying mechanisms for induction of neurogenesis and neuroprotection are not yet elucidated.

Platelet-Derived Growth Factor (PDGF)

PDGF was discovered in the early of 1970s by Russel Ross et al., when they investigated the role played by the smooth muscle cells (SMCs) in the formation of atherosclerotic lesions. It was demonstrated that, in the absence of the endothelium, SMCs migrated and proliferated to form an initial atherosclerotic lesion. These results led Ross to believe that, in any way, the removal of the endothelium facilitated the penetration of certain plasma factors, having an effect on SMCs. Further studies with animals indicated platelets as the source of such factors and the putative factor was named PDGF (161). The PDGF ligand family includes four members (PDGF-A–D). PDGFs are disulfide-linked homo- and heterodimers: PDGF-AA, -AB, -BB, -CC, and -DD. PDGF-A and -B are secreted as active ligands, while C and D ligands, produced as latent factors, are activated under enzymatic cleavage of their N-terminal portion. These PDGF ligands exert their cellular effects by binding to structurally related tyrosine kinase receptors, PDGFR-α and PDGFR-β (162).

Although PDGF has been initially discovered in the platelets, nowadays it is well known that a plethora of cells is able to synthesize, store, and release PDGF, and of particular importance are the effects of this growth factor on embryogenesis and normal CNS development. A growing body of evidence suggests that proliferation, migration, differentiation, and survival processes of NSC are regulated by PDGF and their receptors.

Forsberg-Nilsson et al. demonstrated that cultured NSC from the rat embryonic cortex migrate to take their final position when stimulated by PGDF, which is blocked by incubation with PDGF specific antibodies. This finding suggests a role for PDGF in cell migration in the developing cortex (163). Mice neurospheres lacking PDGFRβ show reduced capacity of migration. Moreover, when PDGFR inhibitor STI571 was added to culture medium, the effects of FGF2 on control neurospheres were blocked. FGF2 increases the activity of the PDGFRB promoter as well as the expression and phosphorylation of PDGFRβ. These data indicate the presence of a cross-talking between PDGF and FGF2, in which the effects of FGF2 in migration and neural differentiation of cells is potentiated by activation of the PDGFRβ (164).

It has been reported that NSC from the embryonic rat cortex proliferate even after removal of growth factors, such as FGF2. Taking into account that these progenitors express PDGF receptors, and produce PDGF-BB during early NSC differentiation, and that cell numbers are reduced in cultures treated with PDGF receptor inhibitors, one may conclude that PDGF is important to maintain progenitor cell division (165). Effects of platelet microparticles on mouse neurospheres are suggested to be mediated by ERK and Akt, both involved in cell proliferation. Since such effects are partially abolished when cells are incubated with an antibody against PDGF, we can once more conclude that cell proliferation is to some extent controlled by PDGF and their receptors (166). The key role of PDGF in cell proliferation is also reinforced by the fact that PDGF-B overexpression causes both GFAP-expressing astrocytes and Nestin-expressing CNS progenitors to proliferate in culture. Furthermore, gene transfer of PDGF in neural progenitors and astrocytes induces the formation of oligodendrogliomas and either oligodendrogliomas or mixed oligoastrocytomas, respectively (167). Similar results are obtained following PDGF treatment of NSC from the adult brain SVZ specifically labeled with PDGFRα. Cell proliferation is induced, leading to hyperplasias resembling gliomas (168).

NSC from the embryonic rat cortex pretreated with PDGF do not complete neuronal differentiation, that is, although they are positive for the neuronal marker β3-Tubulin, they are almost completely devoid of neurites. The same pretreatment does not alter the proportion of both GFAP-positive cells (marker for glial cells) and cells expressing neuronal markers. It should be stressed that only a few oligondendrocytes are detected in comparison with astrocytes, and the latter ones show an immature morphology. However, when NSC cultures treated with PDGF were exposed to additional differentiation factors, like B27, CTNF, and NT3, however, the differentiation proceeded into neurons, astrocytes, and oligodendrocytes. As mentioned above, these cells produce PDGF-BB and, when this action was inhibited, neurons and oligodendrocytes differentiate more rapidly. This points PDGF as an inducer of partial differentiation of NSC (165). Neurospheres from PDGFRβ knockout mice are less responsive to PDGF and, consequently, have a lower differentiation into neurons (164). Hayon et al. also observed that platelet microparticle increases the differentiation of NSC to neurons and glia, which is blocked by specific antibody against PDGFR (166). NSC specifically labeled with PDGFα from the adult brain SVZ act as progenitors of neurons and oligodendrocytes, but not neurogenesis. This suggests that PDGF helps to balance differentiation between neurons and oligodendrocytes (168).

Recent studies demonstrate that PDGF does not only act as a mitogen for progenitors but, also, it reduces apoptosis rates. For instance, it has been reported that PDGF treatment of NSC derived from rat, reduced the number of apoptotic nuclei by half when compared with control measurements (169). Kwon also evaluated the antiapoptotic effect of PDGF by analyzing the presence of phosphatidylserine in the outer surface of NSC by staining with annexin V (phosphatidylserine, almost exclusively located on the inner side of the plasma membrane, appears in the outer surface of the cells at the beginning of apoptosis). It was observed that PDGF treatment abolish staining for annexin V, indicating a decrease in the number of apoptotic cells (170). Taken together, all these works suggest that PDGF is important in the early phase of differentiation process, increasing the number of progenitors and immature neurons, functioning as a mitogen and a protective factor against apoptosis.

Insulin-Like Growth Factor (IGF)

IGFs are polypeptide hormones with potent anabolic and mitogenic effects that control cell proliferation, survival, and differentiation. These factors act by binding to cell-surface heterotetrameric tyrosine kinase receptors and activating multiple intracellular signaling cascades. Two subtypes of IGF receptors have been identified: (I) the IGF-1 receptor (IGF1R), to which IGF-1 preferentially binds instead of insulin; (II) the IGF-2 receptor (IGF2F), also called mannose-6-phosphate receptor, is devoid of signal transduction capacity, interacting mainly with IGF2, and preventing this from competing with IGF-1 by IGF1R (171).

Recent studies demonstrate that IGF-1 and its receptor play an important role in growth and differentiation of NSC. This is supported by the fact that IGF-binding protein-3, to which IGF-1 is always bound, inhibits the growth of rat NSC and promotes neurogenesis, as indicated by decreasing Nestin expression (172). Choi et al. also demonstrated, via flow cytometry analysis with antibodies against the neuronal markers β3-Tubulin and NeuN, that IGF-1 alone or in combination with other growth factors is able to stimulate the proliferation and differentiation of rat NSC (26). Surprising effects were also obtained in vivo, when animals had been subjected to a peripheral infusion of IGF-1. Such treatment led NSC derived from hippocampus to proliferate and differentiate selectively into neurons (151). On the other hand, multipotent adult rat hippocampus-derived NSC can be stimulated by IGF-1 to differentiate into oligodendrocytes (173). These works suggest an important role of IGF-1 in cell fate determination. IGF-1 may even play a neuroprotective role, in addition to its role as endogenous diffusible factors that mediate postischemic neural progenitor proliferation (174). The suggested role for IGF-1 as a key element in NSC growth is reinforced by the observation that neither EGF nor FGF2 are able to induce proliferation of mouse striatal NSC in the absence of IGF-1 (175).

Although little is known about the mechanisms by which IGF-1 mediates proliferation of NSC, a body of evidence points at participation of Akt. IGF-1 treatment of NSC increased the phosphorylation of Akt, but not of Erk. Moreover, the addition of U0126, a specific inhibitor of Akt, abolished cell survival induced by IGF-1. The same result is not obtained when an inhibitor of Erk is added, confirming once more that IGF-1 effects are mediated by Akt (176). As already mentioned, IGF-1 is also able to induce survival of NSC. This may, to certain extent, be accounted by a protection against apoptosis, as recently published by Gualco et al. The authors have shown that the expression of Survinin, an antiapoptotic protein, is IGF1R-dependent. In contrast to wild-type animals, the embryos of knock-out IGF1R animals with low Survinin levels, revealed increased numbers of apoptotic neurons in a earlier differentiation phenotype and reduced NSC proliferation rates (177).

Novel Proteins Implicated in Neural Stem and Progenitor Cell Proliferation and Phenotypic Characterization

In addition to many well-characterized antigens expressed by NSC (7), recent studies have identified novel markers specifically expressed by neural stem and progenitor cells (Table 1). The population expressing these markers can be screened by multiplex flow cytometry together with cell cycle analysis and proliferation, using BrdU, EdU, propidium iodide (PI), or 5-fluoruracil as DNA stains (178, 179). As a result of such assays, implications of these novel markers in proliferation and differentiation of NSC have been suggested as follows. CD44 is a membrane glycoprotein expressed by NSC with importance in adhesion and proliferation. Zhang et al. demonstrated that EGF plays a role induces up-regulation of CD44 expression (180) and later Pollard et al. demonstrated that this increase can also occur after treatment with FGF2 (181). The MAPK/ERK pathway is involved in this effect, since its inhibition reverses EGF-induced upregulation of CD44 expression (182). CD44v6, an alternative splicing form of CD44, also participates in the regulation of cell proliferation, acting as a co-receptor for growth factors (183). Like CD44, expression of this isoform is also subject to upregulation by both IGF and PDGF (184). CD133, also known as Prominin1, is expressed in NSC, but is not known to be present in progenitor cells already committed to a defined neural fate (185). Combined treatment of brain tumor stem cells with EGF and FGF2 augments the expression of CD133, forming aggregates of stem and progenitor cells known as gliospheres, with more than 50% of cells expressing CD133 (186). GD3 is a ganglioside present in the membrane of neural cells. Its expression in NSC is not affected by exposure of cells to EGF (187), nor to PDGF (188). C17.2 immortalized murine NSC expressing recombinant GD3 synthase had its EGF-dependent activation of the Ras-MAPK pathway repressed (189). GD3 synthase-transfected PC12 pheochromocytoma cells exhibited constant activation of the TrkA receptor, independently from the presence of NGF. The increased expression of GD1b and GT1b gangliosides results in conformational changes of the receptor, leading to its dimerization and activation (190). Other gangliosides, like GD2, are also expressed by NSC and can be used as a phenotypic marker to identify these cells (191). Id1 is a nuclear factor that acts as inhibitor of cell differentiation. Treatment of rat SVZ causes an up-regulation of Id1 (192). Expression of Id1 is also increased in FGF15 null mice dorsolateral midbrain (193). Neuroblastoma cells treated with FGF2 showed induced expression of both Id1 mRNA and protein (194). Other markers for NSC or differentiated neural progenies have been recently characterized (Table 1). However, the effects of neurotrophins and growth factors on some of these markers have so far, not been studied.

CONCLUSION

The regulation of NSC migration, proliferation, differentiation, and cell death is extremely complex. Among a diversity of agents involved in the modulation of these processes, neurotrophins and growth factors play an important role. These molecules affect not only stem cells, but also committed progenitors, as reviewed in Figure 1. Imaging and flow cytometry analysis, combined with other techniques, have been important to uncover these roles, by allowing the characterization of distinct cell populations, according to the expression of neural phenotype-specific markers (Table 1, Fig. 1). A better understanding of the mechanisms underlying the regulation of proliferation, differentiation, and cell death, brings new advances in the neurogenesis field and cell therapy.

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