Neural stem cells: a pharmacological tool for brain diseases?

Pharmacological Research 47 (2003) 289–297

Neural stem cells: a pharmacological tool for brain diseases? Luciano Conti∗ , Tiziana Cataudella, Elena Cattaneo Department of Pharmacological Sciences, Center of Excellence on Neurodegenerative Diseases, University of Milano, Via Balzaretti 9, 20133 Milano, Italy Accepted 11 December 2002

Abstract Stem cells are believed to provide a tool by which new cells and tissues can be made and by which damaged ones can be replaced or repaired. Over the past few years, the existence of a subset of stem cells has been documented in the fetal brain, therefore named neural stem cells (NSCs). To this regard, the more recent demonstration that similar cells are present in the adult mammalian brain and retain the capability to produce new neurons, has undermined the dogma that neurons are only generated during the fetal life and has stimulated investigations into the regulation and role of adult neurogenesis. Here, we will review the recent advancements on the biology of brain stem cells and discuss the mechanisms and drugs regulating adult neurogenesis, aiming at better estimating the possible future applications of NSCs for brain repair. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Neural stem cells; Brain diseases; Pharmacological control; Adult neurogenesis

1. Introduction In the last few years, stem cell technologies have been frequently in the popular news producing high expectations in the public opinion about their therapeutic potential. These expectations arise from reports showing that stem cell differentiation and their functional integration into a transplanted tissue can be controlled by the host environment in which they are placed. This is particularly important for the central nervous system (CNS) because the brain exhibits a poor functional capability to replace cells lost in the course of an injury or disease. Beside the promises offered by the embryonic stem cells, tissue-specific fetal stem cells, and stem cells residing in various adult organs have stimulated research into the nature of these cells and their functional roles and therapeutic potential. Cells with stem-like properties, initially identified in fetal, and more recently in the adult mammalian brain, can grow in culture, displaying the potential to self-renew and to generate all the different cell types of the nervous system: neurons, astrocytes and oligodendrocytes. The present challenge is to determine the physiological relevance of adult NSCs and to increase our knowledge on ∗ Corresponding author. Tel.: +39-02-5031-8333; fax: +39-02-5031-8284. E-mail address: [email protected] (L. Conti).

the factors and mechanisms controlling their proliferation and differentiation. The final purpose is the development of strategies and drugs for an optimal pharmacological control of NSCs for therapeutical applications.

2. Definition and location of neural stem cells (NSCs) NSCs are generally considered to be proliferating cells that can produce identical cells as well as “committed” progenitor cells and their differentiated counterparts, i.e. neurons and glia [1]. Although we have no direct means of distinguishing NSCs from brain progenitors, the latter are generally considered to be more limited in their potential and can produce only restricted phenotypes [2]. In the developing brain, the NSCs are found in a precise location, i.e. the ventricular zone (VZ) and subventricular zone (SVZ). Indeed, at early stages, the cavity of the neural tube gives rise to the ventricular system, while the epithelial layer surrounding the cavity contains the stem cells that will give rise to the neuronal and glial cells of the future brain [1]. Early retroviral labeling studies demonstrated the presence of dividing cells in the mammalian VZ, with about 50% of the labeled cells that remained in colonies within the VZ, suggesting the occurrence of self-renewal at this site [3]. Subsequent studies demonstrated that this high rate of proliferation is balanced by apoptosis in order

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to maintain a steady population and to allow for the correct brain modelling [4,5]. As development continues, neurons migrate away, mainly guided by radially oriented glial processes, and the ventricular zone diminishes in size [6]. At all stages, the NSCs remain attached to the basal lamina. At this site, the cells divide asymmetrically while a more “committed” progeny (i.e. progenitors, neuroblasts) migrate away from the ventricular zone towards the overlying cortex. Such cells can further divide and can be distinguished from immature NSCs because of the acquisition of specific phenotypic markers [7]. These events occur in an orderly and coordinated fashion, so that the right numbers and classes of cells are produced in a precise sequence [1]. The final fate decision of a given stem/progenitor cell depends on extrinsic signals and specific patterns of transcriptional activation, generally resulting in the generation of neuronal cell types prior to glia, as shown in several brain regions [8]. The first evidence that similar cells exist also in the adult brain is from Altman and Das [9]. These authors found that some regions of the adult brain exhibit ongoing neurogenesis as judged by incorporation of 3 H-thymidine. In the early 90s, it was also shown that cells could be isolated from the adult mouse brain and expanded in vitro in the presence of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2) [10]. These cells fulfilled two criteria thought to identify cells with “stem-like” properties, i.e. they are able to autoreplicate (although in bulk cultures) and to give rise to neurons and astrocytes. Subsequent studies indicated that the adult NSCs originate mostly from two regions of the adult CNS, i.e. the hippocampus and the SVZ of the lateral ventricle [11]. Noteworthy, although both fetal and adult NSCs appear to share commonalties, their existence may underlay very different meanings. Indeed, while the fetal NSCs actively divide for a restricted interval of the fetal life and then progressively disappear when their progeny has reached the proper size, the adult NSCs may define a long-term subset of stem cells that are capable to show perpetual self-renewal. Furthermore, one might speculate that the adult NSCs, rather than following an orderly sequence of events to produce specific products at specific times, respond instead to local environmental influences to produce a cellular output proper to a given condition [12]. To this regard, it is not yet well established whether the adult NSCs are a particular subset of the fetal NSCs persisting into adult life or, rather, a new and unrelated cell type. This is an important point to address since the adult NSCs may display different functional and integrative capacity following intracerebral transplantation [1]. In the last few years, some groups have also claimed that the differentiative potential of fetal and adult NSCs may be closer to that of the ES cells than expected [13–16]. Indeed, stem cells committed to a particular lineage seem to be able to acquire mature phenotypes of a different germinal layer. Other reports, however, discuss this extensive plasticity as the result of their fusion with cells of the host [17,18], or as

a consequence of transformation events [19], or artifacts due to the in vitro expansion [20]. Overall, the available evidence indicates that the number of cells that are able to transdifferentiate, if any, is very low and, at the moment, of little clinical relevance [19,21]. Future research should establish the actual impact and occurrence of these phenomena. It will also have to be clarified whether somatic stem cells exist outside the CNS that can consistently produce neurons able to functionally integrate into the host CNS, thus opening to allografts strategies.

3. NSCs: isolation and expansion NSCs have been identified in many areas of the developing brain and also in specific regions of the adult CNS of most mammals, including human. The results obtained, however, indicated that cells from different brain areas are not identical, displaying different growth characteristics, trophic factor requirements and specific patterns of differentiation [1]. This suggests that more restricted progenitors arise during brain development. Undoubtedly, in order to reach an understanding of NSCs, it is necessary to identify them in vivo and also to define their real potential in their original environment. Indeed, it may well be that their isolation and in vitro propagation will affect their proliferative and differentiative properties, resulting in the propagation of cells whose characteristics are different from those present in the original cells in vivo [19]. Nonetheless, in vitro studies may greatly impact cell therapy approaches to brain diseases if NSCs are shown to consistently produce neurons of a specific type either in vitro or, more importantly, after their transplantation. In vitro studies can also importantly assist in the analyses of the biology of NSCs, given the more controlled environment and the opportunities for multiple observations of the same cells throughout time. In vitro experiments also reveal that NSCs differ in their potential according to the developmental stage at which they are isolated and according to the site from where they were obtained [1]. At least two classes of putative stem cell seem to exist in the CNS, which have different growth factor requirements. One group of cells is EGF-responsive and can be expanded as floating cell aggregates, called neurospheres, for many passages [22]. According to the literature, these EGF-dependent cells cannot be isolated before E14 in the murine brain, and some groups reported a change in growth factor requirements, the cells becoming FGF-responsive with the in vitro passages [23]. These neurospheres are not, however, pure collections of actively dividing stem cells but rather they are a heterogeneous pool of cells that can be propagated on uncoated dishes following mechanical dissociation. In one experiment, Gritti et al. [24] showed that single cells isolated from passaged neurospheres can be grown in isolation to form secondary neurospheres containing cells able to give rise to neurons and glia. Whether cells

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freshly isolated from the fetal neural tube (or adult rodent brain) can also do the same is unknown, as well as it is not yet fully demonstrated if one single cell isolated from fetal or adult brain can generate millions of secondary cells or neurospheres having the same biochemical and functional properties. In addition, each neurosphere is highly heterogeneous since it contains cells that are at different stages of their differentiation. The presence of NSCs within these neurospheres is inferred by the ability to expand them for several passages in vitro and by the demonstration that neurons and glia are generated in differentiating conditions. However, neurosphere propagation is also compatible with the activity of dividing glioblasts or glial cells endowed with stem-like properties. Such putative multipotent cells present in a neurosphere are immunoreactive for nestin and readily incorporate BrdU. Noteworthy, according to literature data, only a minority of the cells in a given neurosphere (less than 1%) is truly multipotent and can generate secondary neurospheres [19]. The second group of precursor cells isolated from the CNS have been shown to be FGF-dependent. These cells can be propagated both as adherent cultures and as floating neurospheres [25]. FGF-dependent stem cells are not EGF-responsive as demonstrated by the lack of EGF receptor expression. Ciccolini and Svendsen [26] suggested that EGF-dependent cells might arise from FGF-dependent precursors. Both types of neural precursors have been isolated in multiple brain regions and appear to coexist. Furthermore, both can self-renew and can differentiate in vitro into the three neural cell types and have also been successfully intracerebrally transplanted into rodent brain and shown to generate the expected neuronal and glial phenotype [27]. Nevertheless, in these experiments, the efficiency of neuronal differentiation was very poor. Despite the results highlighting the presence of a stem-like activity in in vitro expanded neurospheres, the field suffers from a major lack of prospective methods to identify NSCs. Indeed, all data and knowledge on NSCs, so far, have been inferred from retrospective analyses of the behaviour of the cultures. In other words, the identification of a cell as an NSC is generally achieved retrospectively through the examination of its progeny, using clonal and differentiation assays. Recently, isolation of NSCs from the embryonic or adult forebrain has been successfully achieved by the sorting of cells infected with viral particles or obtained from transgenic mice bearing vital fluorescent proteins under the nestin or musashi promoters [28–30]. Nevertheless, there are only a few markers of putative NSCs, and the markers used with some degree of specificity for the NSC (e.g. nestin and musashi) or glial lineages (GFAP for astrocytes) are also sometimes expressed by other cell types [31]. For example, NSCs, neuronal progenitors, and muscle progenitors all express nestin [32]; GFAP positive astrocytes can be induced to exhibit characteristics of stem cells, indicating that also the GFAP per se does not unequivocally identify a particular cell type [33,34]. In another promising attempt at isolating stem cells from the brain, it was found that a fraction of cells

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with stem-like properties (i.e. able to generate spheres from a single cell as well as to produce differentiated progeny) can be isolated by combining sorting for cell size and antigenic properties [35]. The fraction of cells having a diameter above 12 ␮m, and low expression of peanut agglutinin antigen and heat stable antigen was indeed retaining the vast majority of the stem-like activity.

4. NSCs in the adult brain In spite of the evidence of 3 H-thymidine incorporating cells in the adult rodent brain back to 1960s [9], for several years there has been no direct demonstration that the newly generated cells were effectively neurons. The evidence of the last 10 years, however, suggests that NSCs able to generate new neurons populate the adult brain. Indeed, as for the fetal brain, cells with stem-like properties can be isolated from diverse regions of the adult rodent brain and can be induced to proliferate in vitro by addition of various epigenetic factors as EGF and FGF2 [10,36]. Use of BrdU incorporation coupled to immunohistochemistry techniques has provided evidence that new neurons are indeed formed throughout the life of adult mammals indicating that cells with stem-like properties may be located in restricted brain areas or distributed intraparenchymally. Two brain regions are widely accepted as examples of continued cell proliferation throughout adulthood [11,37]. 4.1. NSCs in the subependymal layer Dividing cells persisting throughout life have been identified in the anterior part of the subependymal layer (SEL), a remnant of the ventricular zones of the fetal brain, and in the rostral migratory stream moving toward the olfactory bulbs [38,39]. These NSCs give rise to migrating cells that eventually differentiate into interneurons within the olfactory bulb where neurogenesis seems to occur at a considerably high rate, with approximately 80,000 new granule neurons per bulb (about 1% of total olfactory granule neurons) per day [40]. These migrating neuronal cells form long chains and migrate without the guidance of axons or radial glia [41]. GFAP positive astrocytes unsheathe these chains. Two contrasting hypotheses indicate different cells as the multipotent stem cells that give rise to these migrating neurons. The first derives from the study of Johansson et al. [42] that indicated that the NSCs are the ependymal cells, i.e. ciliated cells organized into a monolayer which face the ventricles. In their studies, the authors demonstrated that ependymal cells, which were marked by an intraventricular injection of a liposoluble dye, exhibit proliferative capability in vivo (and in vitro) and appear as neurons in the olfactory bulb. Other groups have challenged this hypothesis. A recent study by Capela and Temple [43] has identified Lewis X (LeX), a membrane carbohydrate, as a novel marker for adult NSCs and found that the ependymal cells are LeX negative and are

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not able to generate neurospheres in vitro. Noteworthy, studies by Alvarez-Buylla and coworkers [33,44] indicate that astrocytes present in the subependymal layer may generate intermediate progenitor cells that in turn differentiate into the migrating neural cells. This was further demonstrated using retroviral labelling techniques to specifically label astrocytes in the subependymal layer and tracking their migration to the olfactory bulb. They also showed that when cultured in vitro in the presence of FGF2 and EGF, purified astrocytes promptly generate neurospheres capable to differentiate into neuronal phenotypes. 4.2. NSCs in the hippocampus Hippocampus is the second well understood example of adult neurogenesis in mammals, where a proliferative zone in the hilus of the dentate gyrus produces neurons and glia. An approximate estimation of the stem cell population in the adult hippocampus indicates the generation of 100–150 new granule cells (about 0.03% of total granule neurons) per day in young, adult rodents [45]. Another study indicated that more than twice these many cells are effectively generated but not all survive and differentiate into neurons. These dividing cells arise in the subgranular zone and migrate into the granule cell layer differentiating into granule neurons [46]. The newly generated neurons establish axonal projections to area CA3 along the mossy fiber tracts, as do all other granule cells in the granule cell layer, becoming indistinguishable from the surrounding local cells [37]. Although data are quantitatively more variable, there appears to be a similar rate of neurogenesis in the human dentate gyrus. Indeed, Gage and coworkers have identified a similar type of cell in post mortem studies performed on a group of oncology patients who had BrdU injection [47]. In all treated patients, BrdU-labeled cells were present in the granule layer and coexpressed neuronal markers. It has also been recently claimed that while neurogenesis occurs in both the dentate gyrus and the subependymal zone, NSCs reside only in the latter region [48]. Microdissection and culture of cells from both brain regions indicated that only committed neuronal and glial progenitors arise in the dentate gyrus, while “true” NSCs were present in the subependymal zone. On the other hand, studies performed by Gage and coworkers [49–51] employing retroviral labeling of NSCs from the adult rodent hippocampus showed that these cells are able to generate either neurons or astrocytes. Noteworthy, postmitotic neurons generated from those cells displayed action potentials and functional synaptic connections. However, given the possible heterogeneity of the culture, these data are also consistent with the presence of committed neural progenitors for each lineage in the starting material. 4.3. NSCs in other brain regions New data seem to indicate that new neurons are generated under basal conditions also in regions like the cere-

bral cortex and the substantia nigra [52,53]. Indeed, a third widespread population of stem cells has been suggested to reside in the brain parenchyma in the form of cells with astrocyte-like properties. Recent evidence indicates the possibility that de-differentiating glial cells may exhibit stem cell-like properties, suggesting neurogenic potential also in other brain areas [44]. The cells of the subependymal layer and hippocampus could be involved in steady-state neurogenesis for the replacement of cells in the constantly remodelling olfactory bulb and hippocampus [54]. In the hippocampus, the functional implications of new granule cells in the dentate gyrus have not been clearly elucidated, although recent reports indicate that loss of these cells in animal experiments affects short-term memory [55]. Clearly, the rate of adult neurogenesis is affected by intrinsic and extrinsic factors. Generation and survival of new neurons in the hippocampus diminishes with age and increased concentrations of corticosteroids and can be substantially increased by provision of an enriched environment or by increased physical exercise [40]. New studies also indicate that, in the brain, endogenous NSCs respond to signals coming from degenerating cells or drugs, migrate to the region of cell loss, and differentiate properly in the attempt to restore neural circuit integrity [56]. This ability opens to the potential use of drugs to fully exploit the potential of NSCs.

5. Physiology and pharmacological control of adult neurogenesis New neurons are daily generated in the adult brain; however, the rate of neurogenesis is limited and certainly not enough to allow the replacement of neuronal cells lost during neurodegenerative processes [57]. Is adult neurogenesis an inefficient attempt to replace lost neurons, or is it an unrelated process? Literature data indicate that in physiological conditions the total number of neurons in the brain regions remains constant. This implies that the newly generated neurons are able to functionally integrate into the mature brain replacing the lost “aged/damaged” cells or that most of the cells generated through division of stem or progenitor cells have a short life. Both situations have been observed indicating that the rate of proliferation, apoptosis and survival in the adult brain is strictly regulated. 5.1. Social and physical activities increase neurogenesis The issue about the possible environmental influence on neurogenesis in the adult brain has been particularly investigated in hippocampus because of its involvement in learning and memory processes. For example, hippocampal-dependent learning has been shown to promote neurogenesis in dentate gyrus, where the generation of new neurons is important to encode memory formation. To this regard, Kempermann et al. [45] demonstrated that

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adult hippocampal neurogenesis is subjected to regulation by the environment. Indeed, an increase in the number of hippocampal neurons in the dentate gyrus and in the size of the granule layer was shown in adult mice placed in an enriched environment including enhanced social interactions and tools to play compared with littermate controls. Similarly, other studies described an increased neurogenesis in the dentate gyrus in mice given access to a running wheel, thus suggesting that, in healthy animals, ongoing neurogenesis in the adult hippocampus may contribute to behaviour, learning and memory [58]. 5.2. Trophic factors as mediators of neurogenesis in the adulthood Although the exact mechanisms underlying the increase of neurogenesis have not been fully addressed, both learning and exercise are reported to increase the expression of specific trophic factors. These factors include brain-derived neurotrophic factor (BDNF) and FGF2 [59,60]. The possibility of a direct relationship between these factors and increased neurogenesis in response to exercise, learning and memory has been the subject of numerous studies. For example, in avian brain, where BDNF is involved in the formation of new neurons, singing activity upregulates BDNF expression [61]. Consistently, intracerebroventricular administration of FGF2 and BDNF leads to an increase in neurogenesis in the subependymal layer and olfactory bulb [60]. Similarly, neurogenesis in both hippocampus and olfactory bulb is enhanced following peripheral administration of FGF2 and insulin-like growth factor 1 (IGF-1) [62,63]. Yoshimura et al. [64] demonstrated that FGF2 overexpression may stimulate proliferation of NSCs even if it is required to maintain the normal rate of neurogenesis. This result was inferred by analysis of hippocampal neurogenesis in FGF2 knockout mice, which exhibited the same neurogenetic capability as the wild-type littermates. This basal level of neurogenesis greatly increased following FGF2 delivery both in null mutants and wild-type mice. Physical exercise is thought to induce an increase in neurogenesis by a mechanism that may involve growth factor production as well [46]. Indeed, physical activity increases IGF-1 levels in blood leading to an increase in the hippocampal production of new granule neurons [65]. Although these studies indicate a role of specific factors in the modulation of neurogenesis, cell proliferation, migration and differentiation are likely regulated by a complex interaction between multiple factors requiring appropriate spatial and temporal presentation of adequate levels of given molecules. Grafting of purified or enriched populations of NSCs into different regions of the postnatal or adult brain showed that optimal integration of the donor NSCs is achieved exclusively when cells are grafted in neurogenic areas [1]. Adult hippocampal NSCs grafted back into the hippocampus have the ability to adopt the appropriate phenotype of mature granule neurons of the hippocampus. Grafting

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of these same cells into cerebellum, a non-neurogenic region, leads to a limited survival and integration with prominent glial differentiation and poor neuronal differentiation. On the contrary, these cells when grafted into the olfactory bulb produce differentiated cells exhibiting olfactory neuron phenotype and not hippocampal phenotypes. Similar results were obtained with NSCs derived from different embryonic and adult murine brain areas as well as human NSCs. These studies clearly indicate that NSCs are competent to respond to various environmental factors [27]. Thus, although neurogenic regions of the adult brain express signals appropriate to direct a wide variety of stem cells to adopt a neuronal fate, these signals are either lacking or insufficient in non neurogenic regions of the adult brain under normal conditions. 5.3. Neurogenesis in the lesioned brain Elucidation of the neurogenic potential and neurogenic signals present in injured brain is of primary importance in CNS repair issues employing endogenous or grafted NSCs. Nevertheless, several lines of evidence now indicate that, despite of the limited neurogenic potential of the intact adult CNS, injury may activate a latent self-repair program and that neurogenic signals may be reactivated to compensate for neuronal loss. In fact, different insults, including prolonged seizures and ischemia or neurodegenerative disorders, may induce the proliferation of NSCs and the generation of new neurons in different brain areas [40,56,66]. Depending on the nature of the injury, proliferation of the endogenous NSCs has been demonstrated to occur both in well characterized neurogenic regions [56,67] and in areas where the generation of new neurons is not normally detected [52,68,69]. However, the endogenous regenerative potentials is very limited, leading to the replacement of a number of neurons not sufficient to produce a functional recovery [68,69]. Several studies now indicate that the injury-induced neurogenic potential may be significantly enhanced by growth factors infusion. To this regard, Nakatomi et al. [56] showed that following global ischemia, adult neural progenitors can be stimulated in situ by intraventricular infusion of EGF and FGF2 to replace CA1 pyramidal neurons leading to anatomical and functional reconstruction of CA1. This study emphasizes two important issues: (i) adult progenitors can regenerate specific neuronal subtypes appropriate for the site of damage. Indeed, the authors demonstrated that hippocampal CA1 pyramidal neurons can be efficiently regenerated after ischemia. (ii) Short-term growth factors infusion stimulates neural progenitors from areas close to the site of injury to contribute to the regeneration in the hippocampus. This represents a feature proper to the injured brain. Indeed, in the intact adult brain, growth factors treatments stimulate proliferation of progenitors located in normally neurogenic areas. In contrast, the combination of ischemic signals and growth factor stimulation is able to transiently instruct normally resting progenitors. This suggests that the regenerative potential of the adult brain is wider than expected and

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the poor regenerative capability normally detected may be the result of a limited availability of appropriate signaling factors or due to the presence of inhibitory signals. Taken together, all these evidence indicate that adult NSCs may provide a novel target for drug therapies for neurological diseases. Drugs stimulating the production of specific growth factors capable to positively regulate adult NSCs proliferation and differentiation will represent a future challenge. Indeed, there is the suggestion that a number of pharmacological-based therapies already in use in clinical trials for neurological disorders probably work, at least in part, through a similar mechanism. For example, riluzole, a drug recently employed for treatment of the Amyotrophic Lateral Sclerosis [70,71] and Huntington’s Disease [72–74] has been recently demonstrated to enhance glial cell line-derived neurotrophic factor (GDNF) and BDNF production [75,76]. Alternative strategies aimed to implement the number of newly generated and fully functional neurons from adult NSCs may come from studies of signaling mechanisms downstream of growth factors receptors. An example is represented by the cell-autonomous developmental switch in expression from the ShcA to the ShcC adapter protein that may instruct NSCs to withdraw from the cell cycle and to initiate a program of neuronal differentiation [77,78]. 5.4. Effects of stress and pharmacotherapy on adult neurogenesis Evidences in the last few years indicate that hippocampal neurogenesis may be significantly inhibited in response to stress, depression and aging, possibly further emphasizing the dementia and other memory problems observed in depressed patients. Abnormal adult neurogenesis is now considered one possible mechanism underlying neurological and mood disorders [79]. Major depression, for example, may have its biological origins from a disturbance in neuronal plasticity. One working hypothesis considers that a failure of neuronal plasticity and adult neurogenesis may represent one of the correlated effects or the driving cause of depression [80,81]. Stress may be one of the key factors that initiate the cellular pathogenesis of depression through interference with adult hippocampal neurogenesis. In fact, it is now accepted that hippocampal neurogenesis is reduced as a plastic response to stress [82]. Sustained psychosocial and physiological stress has been demonstrated to produce a hippocampal volume loss reported also with high resolution MRI [83]. Noteworthy, the observed volume loss is not only due to a decrease in cell number but also to atrophy in the neuropil and synaptic bulk, indicating a more general effect of stress. Several studies have linked the stress-mediated decrease in hippocampal neurogenesis to the stress-induced activation of the hypothalamic–pituitary–adrenal (HPA) axis resulting in elevation of glucocorticoids levels [84]. To this regard, it has been shown that administration of glucocorticoids de-

creases the proliferation of granule cell precursors in adult rat hippocampus and during development [85]. The negative effect exerted by glucocorticoids on hippocampal neurogenesis accounts also for the decreased neurogenesis in aged animals, where an increase of such hormones is observed. Indeed, experimental removal of adrenal steroids restores neurogenesis to the levels observed in young adult rats, thus demonstrating that aged animals retain the capacity for a rate of neurogenesis similar to that is observed in young animals [86]. The production of growth factors may provide the link explaining the influence of stress on adult neurogenesis. In fact, BDNF and FGF2 levels, molecules known to increase neurogenesis, are negatively modulated by glucocorticoids [87,88]. The link between stress, mood disorders and hippocampal neurogenesis is also demonstrated by the observation that administration of antidepressant therapies, including drugs, electroconvulsive therapy and physical exercise, all influence adult hippocampal neurogenesis [89,90]. Several studies indicate that serotonin (5-HT), whose role in mood disorders has been deeply investigated, may play a role also on the regulation of neurogenesis [91]. Indeed, drugs able to increase serotonin levels in the brain (such as fluoxetine and Prozac) have a powerful antidepressant action. Increasing evidence indicates that 5-HT stimulates the generation of new neurons in the dentate gyrus of mammalian brain [91]. Increase of 5-HT release and 5-HT1A -receptor density following adrenalectomy [92] or administration of NMDA antagonists [93] induces neurogenesis. On the other hand, administration of serotonin reuptake inhibitors affects normal adult neurogenesis [89]. In particular, serotoninergic deafferentation of the hippocampus and inhibition of serotonin synthesis lead to reduced cell division in hippocampus [94]. This situation was rescued after grafting serotonin-producing cells in the same area. Another drug used in the pharmacotherapy of mood disorders, lithium, has been also shown to stimulate neurogenesis in the hippocampus possibly via an increased expression of Bcl2, a molecule exerting antiapoptotic functions [95]. These results point out the possibility that increased apoptosis and decreased cell proliferation may act synergistically to reduce adult neurogenesis. Taken together, these data raise the possibility that positive outcomes of drug administration may result, in part, from their influence on neurogenesis also indicating that these observations should be taken into consideration when designing novel treatments for mood disorders.

6. Conclusions All together, the discussed items clearly indicate that NSC research is one of the most exciting fields of neuroscience. However, there is still a very long way to go before these cells can be used safely and efficaciously for the cure of neurological disorders.

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