Manuela Ceccarelli

Running Rescues Defective Adult Neurogenesis by Shortening the Length of the Cell Cycle of Neural Stem and Progenitor Cells

Physical exercise increases the generation of new neurons in adult neurogenesis. However, only few studies have investigated the beneficial effects of physical exercise in paradigms of impaired neurogenesis. Here, we demonstrate that running fully reverses the deficient adult neurogenesis within the hippocampus and subventricular zone of the lateral ventricle, observed in mice lacking the antiproliferative gene Btg1. We also evaluated for the first time how running influences the cell cycle kinetics of stem and precursor subpopulations of wild‐type and Btg1‐null mice, using a new method to determine the cell cycle length. Our data show that in wild‐type mice running leads to a cell cycle shortening only of NeuroD1‐positive progenitor cells. In contrast, in Btg1‐null mice, physical exercise fully reactivates the defective hippocampal neurogenesis, by shortening the S‐phase length and the overall cell cycle duration of both neural stem (glial fibrillary acidic protein+ and Sox2+) and progenitor (NeuroD1+) cells. These events are sufficient and necessary to reactivate the hyperproliferation observed in Btg1‐null early‐postnatal mice and to expand the pool of adult neural stem and progenitor cells. Such a sustained increase of cell proliferation in Btg1‐null mice after running provides a long‐lasting increment of proliferation, differentiation, and production of newborn neurons, which rescues the impaired pattern separation previously identified in Btg1‐null mice. This study shows that running positively affects the cell cycle kinetics of specific subpopulations of newly generated neurons and suggests that the plasticity of neural stem cells without cell cycle inhibitory control is reactivated by running, with implications for the long‐term modulation of neurogenesis. Stem Cells 2014;32:1968–1982

Btg1 is Required to Maintain the Pool of Stem and Progenitor Cells of the Dentate Gyrus and Subventricular Zone

Btg1 belongs to a family of cell cycle inhibitory genes. We observed that Btg1 is highly expressed in adult neurogenic niches, i.e., the dentate gyrus and subventricular zone (SVZ). Thus, we generated Btg1 knockout mice to analyze the role of Btg1 in the process of generation of adult new neurons. Ablation of Btg1 causes a transient increase of the proliferating dentate gyrus stem and progenitor cells at post-natal day 7; however, at 2u2009months of age the number of these proliferating cells, as well as of mature neurons, greatly decreases compared to wild-type controls. Remarkably, adult dentate gyrus stem and progenitor cells of Btg1-null mice exit the cell cycle after completing the S phase, express p53 and p21 at high levels and undergo apoptosis within 5u2009days. In the SVZ of adult (two-month-old) Btg1-null mice we observed an equivalent decrease, associated to apoptosis, of stem cells, neuroblasts, and neurons; furthermore, neurospheres derived from SVZ stem cells showed an age-dependent decrease of the self-renewal and expansion capacity. We conclude that ablation of Btg1 reduces the pool of dividing adult stem and progenitor cells in the dentate gyrus and SVZ by decreasing their proliferative capacity and inducing apoptosis, probably reflecting impairment of the control of the cell cycle transition from G1 to S phase. As a result, the ability of Btg1-null mice to discriminate among overlapping contextual memories was affected. Btg1 appears, therefore, to be required for maintaining adult stem and progenitor cells quiescence and self-renewal.

Tis21 Knock-Out Enhances the Frequency of Medulloblastoma in Patched1 Heterozygous Mice by Inhibiting the Cxcl3-Dependent Migration of Cerebellar Neurons

A failure in the control of proliferation of cerebellar granule neuron precursor cells (GCPs), located in the external granular layer (EGL) of the cerebellum, gives rise to medulloblastoma. To investigate the process of neoplastic transformation of GCPs, we generated a new medulloblastoma model by crossing Patched1 heterozygous mice, which develop medulloblastomas with low frequency, with mice lacking the Tis21 gene. Overexpression of Tis21 is known to inhibit proliferation and trigger differentiation of GCPs; its expression decreases in human medulloblastomas. Double-knock-out mice show a striking increase in the frequency of medulloblastomas and hyperplastic EGL lesions, formed by preneoplastic GCPs. Tis21 deletion does not affect the proliferation of GCPs but inhibits their differentiation and, chiefly, their intrinsic ability to migrate outside the EGL. This defect of migration may represent an important step in medulloblastoma formation, as GCPs, remaining longer in the EGL proliferative niche, may become more prone to transformation. By genome-wide analysis, we identified the chemokine Cxcl3 as a target of Tis21. Cxcl3 is downregulated in Tis21-null GCPs of EGL and lesions; addition of Cxcl3 to cerebellar slices rescues the defective migration of Tis21-null GCPs and, remarkably, reduces the area of hyperplastic lesions. As Tis21 activates Cxcl3 transcription, our results suggest that Tis21 induces migration of GCPs through Cxcl3, which may represent a novel target for medulloblastoma therapy.

Genetic control of adult neurogenesis: interplay of differentiation, proliferation and survival modulates new neurons function, and memory circuits

Within the hippocampal circuitry, the basic function of the dentate gyrus is to transform the memory input coming from the enthorinal cortex into sparse and categorized outputs to CA3, in this way separating related memory information. New neurons generated in the dentate gyrus during adulthood appear to facilitate this process, allowing a better separation between closely spaced memories (pattern separation). The evidence underlying this model has been gathered essentially by ablating the newly adult-generated neurons. This approach, however, does not allow monitoring of the integration of new neurons into memory circuits and is likely to set in motion compensatory circuits, possibly leading to an underestimation of the role of new neurons. Here we review the background of the basic function of the hippocampus and of the known properties of new adult-generated neurons. In this context, we analyze the cognitive performance in mouse models generated by us and others, with modified expression of the genes Btg2 (PC3/Tis21), Btg1, Pten, BMP4, etc., where new neurons underwent a change in their differentiation rate or a partial decrease of their proliferation or survival rate rather than ablation. The effects of these modifications are equal or greater than full ablation, suggesting that the architecture of circuits, as it unfolds from the interaction between existing and new neurons, can have a greater functional impact than the sheer number of new neurons. We propose a model which attempts to measure and correlate the set of cellular changes in the process of neurogenesis with the memory function.

Altered cerebellum development and impaired motor coordination in mice lacking the Btg1 gene: Involvement of cyclin D1.

Cerebellar granule neurons develop postnatally from cerebellar granule precursors (GCPs), which are located in the external granule layer (EGL) where they massively proliferate. Thereafter, GCPs become postmitotic, migrate inward to form the internal granule layer (IGL), further differentiate and form synapses with Purkinje cell dendrites. We previously showed that the Btg family gene, Tis21/Btg2, is required for normal GCP migration. Here we investigated the role in cerebellar development of the related gene, Btg1, which regulates stem cell quiescence in adult neurogenic niches, and is expressed in the cerebellum. Knockout of Btg1 in mice caused a major increase of the proliferation of the GCPs in the EGL, whose thickness increased, remaining hyperplastic even after postnatal day 14, when the EGL is normally reduced to a few GCP layers. This was accompanied by a slight decrease of differentiation and migration of the GCPs and increase of apoptosis. The GCPs of double Btg1/Tis21-null mice presented combined major defects of proliferation and migration outside the EGL, indicating that each gene plays unique and crucial roles in cerebellar development. Remarkably, these developmental defects lead to a permanent increase of the adult cerebellar volume in Btg1-null and double mutant mice, and to impairment in all mutants, including Tis21-null, of the cerebellum-dependent motor coordination. Gain- and loss-of-function strategies in a GCP cell line revealed that Btg1 regulates the proliferation of GCPs selectively through cyclin D1. Thus, Btg1 plays a critical role for cerebellar maturation and function.

Tis21 is required for adult neurogenesis in the subventricular zone and for olfactory behavior regulating cyclins, BMP4, Hes1/5 and Ids.

Bone morphogenic proteins (BMPs) and the Notch pathway regulate quiescence and self-renewal of stem cells of the subventricular zone (SVZ), an adult neurogenic niche. Here we analyze the role at the intersection of these pathways of Tis21 (Btg2/PC3), a gene regulating proliferation and differentiation of adult SVZ stem and progenitor cells. In Tis21-null SVZ and cultured neurospheres, we observed a strong decrease in the expression of BMP4 and its effectors Smad1/8, while the Notch anti-neural mediators Hes1/5 and the basic helix-loop-helix (bHLH) inhibitors Id1-3 increased. Consistently, expression of the proneural bHLH gene NeuroD1 decreased. Moreover, cyclins D1/2, A2, and E were strongly up-regulated. Thus, in the SVZ Tis21 activates the BMP pathway and inhibits the Notch pathway and the cell cycle. Correspondingly, the Tis21-null SVZ stem cells greatly increased; nonetheless, the proliferating neuroblasts diminished, whereas the post-mitotic neuroblasts paradoxically accumulated in SVZ, failing to migrate along the rostral migratory stream to the olfactory bulb. The ability, however, of neuroblasts to migrate from SVZ explants was not affected, suggesting that Tis21-null neuroblasts do not migrate to the olfactory bulb because of a defect in terminal differentiation. Notably, BMP4 addition or Id3 silencing rescued the defective differentiation observed in Tis21-null neurospheres, indicating that they mediate the Tis21 pro-differentiative action. The reduced number of granule neurons in the Tis21-null olfactory bulb led to a defect in olfactory detection threshold, without effect on olfactory memory, also suggesting that within olfactory circuits new granule neurons play a primary role in odor sensitivity rather than in memory.

Control of the Normal and Pathological Development of Neural Stem and Progenitor Cells by the PC3/Tis21/Btg2 and Btg1 Genes

The PC3/Tis21/Btg2 and Btg1 genes are transcriptional cofactors belonging to the Btg/Tob family, which regulate the development of several cell types, including neural precursors. We summarize here the actions of these genes on neural precursors in the adult neurogenic niches and the cognitive defects associated when their expression is altered. We consider also recent findings implicating them in neural and non‐neural tumors, since common developmental mechanisms are involved. PC3/Tis21 is required for the regulation of the maturation of stem and progenitor cells in the adult dentate gyrus and subventricular zone (SVZ), by controlling both their exit from the cell cycle and the ensuing terminal differentiation. Such actions are effected by regulating the expression of several genes, including cyclin D1, BMP4, Id3. In cerebellar precursors, however, PC3/Tis21 regulates chiefly their migration rather than proliferation or differentiation, with important implications for the onset of medulloblastoma, the cerebellar tumor. In fact PC3/Tis21 is a medulloblastoma‐suppressor, as its overexpression in cerebellar precursors inhibits this tumor; PC3/Tis21 shows anti‐tumor activity also in non‐neural tumors. Btg1 presents a different functional profile, as it controls proliferation in adult stem/progenitor cells of dentate gyrus and SVZ, where is required to maintain their self‐renewal and quiescence, but is apparently devoid of a direct control of their terminal differentiation or migration. Notably, physical exercise in Btg1‐null mice rescues the loss of proliferative capability occurring in older stem cells. Both genes could be further investigated as therapeutical targets, namely, Btg1 in the process of aging and PC3/Tis21 as a tumor‐suppressor. J. Cell. Physiol. 230: 2881–2890, 2015.

Medulloblastoma or not? Crucial role in tumorigenesis of the timing of migration of cerebellar granule precursor cells, regulated by Nos2 and Tis21.

A commentary on n nNos2 inactivation promotes the development of medulloblastoma in Ptch1(+/−) mice by deregulation of Gap43-dependent granule cell precursor migration n nby Haag, D., Zipper, P., Westrich, V., Karra, D., Pfleger, K., Toedt, G., Blond, F., Delhomme, N., Hahn, M., Reifenberger, J., Reifenberger, G., and Lichter, P. (2012) PLoS Genet. 8:e1002572. doi: 10.1371/journal.pgen.1002572 n nTis21 knock-out enhances the frequency of medulloblastoma in Patched1 heterozygous mice by inhibiting the Cxcl3-dependent migration of cerebellar neurons n nby Farioli-Vecchioli, S., Cina, I., Ceccarelli, M., Micheli, L., Leonardi, L., Ciotti, M. T., De Bardi, M., Di Rocco, C., Pallini, R., Cavallaro, S., and Tirone, F. (2012) J. Neurosci. 32, 15547–15564. n nMedulloblastoma is a very aggressive tumor of the cerebellum and one of the most common malignant pediatric brain tumors. Medulloblastoma comprises four tumor subtypes; about one fourth of medulloblastomas originate from precursor cells of granule neurons (GCPs), carrying an aberrant activation of the Sonic Hedgehog proliferative signaling (Shh; Yang et al., 2008; Gibson et al., 2010). n nIt is in fact accepted that prolonged mitotic activity of GCPs at the surface of the cerebellum during its postnatal morphogenesis makes the cells potential targets of transforming insults (Wang and Zoghbi, 2001). n nTwo recent studies (Farioli-Vecchioli et al., 2012a; Haag et al., 2012) have highlighted that the localization of preneoplastic GCPs (pGCPs) during cerebellar development plays a crucial role for their malignant progression. These studies show that ablation of Nos2 (nitric oxide synthase) or of Tis21 (also known as Btg2 or PC3) leads to impairment of the migration of GCPs from the surface of the cerebellum toward the internal layers. This occurs in consequence of the decrease of expression of two genes regulated by Nos2 and Tis21, i.e., Gap43 and the chemokine Cxcl3, respectively, which specifically induce GCPs migration. Ablation of either Nos2 or Tis21 in Shh-activated mice leads to a large increase in the frequency of medulloblastoma. The explanation for such an increase, supported by data, is that the prolongation of the permanence in the external proliferative cerebellar region under control of Shh exponentially increases the possibility of neoplastic transformation. In the study of Farioli-Vecchioli et al. (2012a) the specificity of the effect of Cxcl3 on the migration of GCPs is guaranteed by the observations that Cxcl3 cell-autonomously regulates their migration without affecting either proliferation or differentiation. Furthermore, ablation of Tis21 does not influence the proliferation of GCPs [also suggesting that other genes of the same family expressed in the cerebellum, such as Btg1 (Farioli-Vecchioli et al., 2012b), may vicariate the known antiproliferative action of Tis21]. Although the ablation of Tis21 reduces the differentiation of GCPs (Farioli-Vecchioli et al., 2012a), it is known that GCPs exit the cell cycle and start differentiating after migrating away from the surface of cerebellum (Choi et al., 2005). n nSome additional considerations arise from the Farioli-Vecchioli et al. (2012a) study. First, Canzoniere et al. (2004) have previously proposed that Tis21 overexpression induces differentiation of GCPs by inducing Math1, a gene known to support the differentiation of GCPs (Gazit et al., 2004). Consistently, the ablation of Tis21 causes the down-regulation of Math1 in cerebellar precursors (Figure A1A). This, however, appears to conflict with recent reports indicating that Math1 is required for the formation of medulloblastomas induced by constitutive activation of the Shh pathway (Zhao et al., 2008; Flora et al., 2009). Moreover, Math1 behaves as a tumor suppressor in colorectal cancer (Bossuyt et al., 2009). An interesting possibility reconciling these observations was proposed by Flora et al. (2009). When cerebellar precursors are in a proliferative environment Math1 makes the cells competent to transduce the proliferative signal of Shh. In contrast, when the cells are exposed to a differentiative signal, Math1 has a prodifferentiative action. In keeping with this idea, it is possible that the ablation of the prodifferentiative gene Tis21 in Shh-activated mice, depriving the GCPs of a differentiative stimulus, would favor the pro-Shh action of Math1. Consequently, the action of activated Shh on GCPs at the surface of the cerebellum would became more penetrant. A putative model of the Math1 pathway in GCPs (relative to Tis21) is illustrated in Figure u200bFigure1A1A (based on the above references and on: Hammerle and Tejedor, 2007; Farioli-Vecchioli et al., 2009); Figure A1A shows the expression values in Tis21-knockout GCPs of Math1 and of Id3, an inhibitor of proneural genes which is a direct target of Tis21 in the dentate gyrus and in GCPs (Farioli-Vecchioli et al., 2009 and data not shown). n n n nFigure 1 n nProposed models of plasticity and transformation of cerebellar granule precursor cells (GCPs) in response to Math1, Tis21, Nos2, Cxcl3, or Gap43. (A) Hypothetical model for the ambivalent role of Math1 (Atoh1) in tumorigenesis and differentiation; in ... n n n n n nFigure A1 n n(A) Math1 and Id3 mRNA expression values by real time PCR analysis performed in GCPs from 7-days-old mice of the four genotypes indicated. Math1 mRNA levels are reduced in GCPs of Tis21 knockout mice, either in a Patched1 wild-type background (Tis21KO ... n n n nSecondly, Tis21 is a transcriptional cofactor, known to be recruited as part of protein complexes containing histone modifying factors to which Tis21 is known to bind, namely, the protein-arginine N-methyltransferase PRMT1, and the histone deacetylases HDAC1 and HDAC4 (Lin et al., 1996; Passeri et al., 2006; Farioli-Vecchioli et al., 2007). These complexes may control the activity of multiple transcription factors by epigenetic mechanisms and account for at least part of the changes in gene expression observed. Such a possibility was tested by interrogating the array of 344 Tis21-regulated genes, either Tis21-knockout-specific or medulloblastoma-specific (Farioli-Vecchioli et al., 2012a), in a search for Tis21-dependent genes targets of epigenetic modifiers. Indeed, a significant number of genes turn out to be epigenetically modifiable, either being responsive to histone deacetylase inhibitors, or because their products bind histone deacetylase proteins. Some of these genes are involved in cell migration, contraction or motility, or in tumorigenesis (see Figures u200bFigures1B,1B, A1B). n nThis suggests that the transcriptional control exerted by Tis21 on cohorts of genes involved in the neoplastic transformation of GCPs may occur at least in part epigenetically. It would be interesting to verify whether the same occurs for Nos2. n nA more general and important question raised by these studies concerns the possibility of controlling the development of medulloblastoma by regulating the migration of GCPs. Farioli-Vecchioli et al. (2012a) show that exogenous Cxcl3 can reduce the area of medulloblastoma lesions in cerebellar slices. It is known that pGCPs can still differentiate and migrate like normal GCPs, although they are able to generate a tumor when transplanted (Kessler et al., 2009). Thus, it is possible to speculate that the migration-promoting action of Cxcl3 or Gap43 may induce pGCPs to differentiate and exit the neoplastic program. Nonetheless, it is plausible that, after a yet undefined period of time, pGCPs may become irreversibly transformed and lose the ability to differentiate. If so, it is necessary that the treatment with proteins regulating GCPs migration such as Cxcl3 or Gap43 takes place at the very initial stages of the tumor (see model in Figure u200bFigure1C).1C). Otherwise, the induction of migration at later stages may contribute to cancer cell spreading, in which case it would be more appropriate to inhibit migration, using for instance the chemokine Cxcl12, which keeps GCPs at the surface of cerebellum, and is upregulated in Tis21-null GCPs (Tiveron and Cremer, 2008; Farioli-Vecchioli et al., 2012a). These considerations highlight the need for routine checks, with more powerful techniques to diagnose brain tumors at a very early stage.

Terminal Differentiation of Adult Hippocampal Progenitor Cells Is a Step Functionally Dissociable from Proliferation and Is Controlled by Tis21, Id3 and NeuroD2

Cell proliferation and differentiation are interdependent processes. Here, we have asked to what extent the two processes of neural progenitor cell amplification and differentiation are functionally separated. Thus, we analyzed whether it is possible to rescue a defect of terminal differentiation in progenitor cells of the dentate gyrus, where new neurons are generated throughout life, by inducing their proliferation and/or their differentiation with different stimuli appropriately timed. As a model we used the Tis21 knockout mouse, whose dentate gyrus neurons, as demonstrated by us and others, have an intrinsic defect of terminal differentiation. We first tested the effect of two proliferative as well as differentiative neurogenic stimuli, one pharmacological (fluoxetine), the other cognitive (the Morris water maze (MWM) training). Both effectively enhanced the number of new dentate gyrus neurons produced, and fluoxetine also reduced the S-phase length of Tis21 knockout dentate gyrus progenitor cells and increased the rate of differentiation of control cells, but neither factor enhanced the defective rate of differentiation. In contrast, the defect of terminal differentiation was fully rescued by in vivo infection of proliferating dentate gyrus progenitor cells with retroviruses either silencing Id3, an inhibitor of neural differentiation, or expressing NeuroD2, a proneural gene expressed in terminally differentiated dentate gyrus neurons. This is the first demonstration that NeuroD2 or the silencing of Id3 can activate the differentiation of dentate gyrus neurons, complementing a defect of differentiation. It also highlights how the rate of differentiation of dentate gyrus neurons is regulated genetically at several levels and that a neurogenic stimulus for amplification of neural stem/progenitor cells may not be sufficient in itself to modify this rate.

Suppression of Medulloblastoma Lesions by Forced Migration of Preneoplastic Precursor Cells with Intracerebellar Administration of the Chemokine Cxcl3

Medulloblastoma (MB), tumor of the cerebellum, remains a leading cause of cancer-related mortality in childhood. We previously showed, in a mouse model of spontaneous MB (Ptch1+/-/Tis21-/-), that a defect of the migration of cerebellar granule neuron precursor cells (GCPs) correlates with an increased frequency of MB. This occurs because GCPs, rather than migrating internally and differentiating, remain longer in the proliferative area at the cerebellar surface, becoming targets of transforming insults. Furthermore, we identified the chemokine Cxcl3 as responsible for the inward migration of GCPs. As it is known that preneoplastic GCPs (pGCPs) can still migrate and differentiate like normal GCPs, thus exiting the neoplastic program, in this study we tested the hypothesis that pGCPs within a MB lesion could be induced by Cxcl3 to migrate and differentiate. We observed that the administration of Cxcl3 for 28 days within the cerebellum of 1-month-old Ptch1+/-/Tis21-/- mice, i.e., when MB lesions are already formed, leads to complete disappearance of the lesions. However, a shorter treatment with Cxcl3 (2 weeks) was ineffective, suggesting that the suppression of MB lesions is dependent on the duration of Cxcl3 application. We verified that the treatment with Cxcl3 causes a massive migration of pGCPs from the lesion to the internal granular layer, where they differentiate. Thus, the induction of migration of pGCPs in MB lesions may open new ways to treat MB that exploit the plasticity of the pGCPs, forcing their differentiation. It remains to be tested whether this plasticity continues at advanced stages of MB. If so, these findings would set a potential use of the chemokine Cxcl3 as therapeutic agent against MB development in human preclinical studies.