Transcriptome analysis reveals genes associated with stem cell activation by physical exercise in the dentate gyrus of aged p16Ink4a knockout mice.

Throughout adulthood neural stem cells divide in neurogenic niches-the dentate gyrus of the hippocampus and the subventricular zone-producing progenitor cells and new neurons. Stem cells self-renew, thus preserving their pool. Furthermore, the number of stem/progenitor cells in the neurogenic niches decreases with age. We have previously demonstrated that the cyclin-dependent kinase inhibitor p16Ink4a maintains, in aged mice, the pool of dentate gyrus stem cells by preventing their activation after a neurogenic stimulus such as exercise (running). We showed that, although p16Ink4a ablation by itself does not activate stem/progenitor cells, exercise strongly induced stem cell proliferation in p16Ink4a knockout dentate gyrus, but not in wild-type. As p16Ink4a regulates stem cell self-renewal during aging, we sought to profile the dentate gyrus transcriptome from p16Ink4a wild-type and knockout aged mice, either sedentary or running for 12 days. By pairwise comparisons of differentially expressed genes and by correlative analyses through the DESeq2 software, we identified genes regulated by p16Ink4a deletion, either without stimulus (running) added, or following running. The p16Ink4a knockout basic gene signature, i.e., in sedentary mice, involves upregulation of apoptotic, neuroinflammation- and synaptic activity-associated genes, suggesting a reactive cellular state. Conversely, another set of 106 genes we identified, whose differential expression specifically reflects the pattern of proliferative response of p16 knockout stem cells to running, are involved in processes that regulate stem cell activation, such as synaptic function, neurotransmitter metabolism, stem cell proliferation control, and reactive oxygen species level regulation. Moreover, we analyzed the regulation of these stem cell-specific genes after a second running stimulus. Surprisingly, the second running neither activated stem cell proliferation in the p16Ink4a knockout dentate gyrus nor changed the expression of these genes, confirming that they are correlated to the stem cell reactivity to stimulus, a process where they may play a role regulating stem cell activation.

Cyclin D3 deficiency promotes a slower, more oxidative skeletal muscle phenotype and ameliorates pathophysiology in the mdx mouse model of Duchenne muscular dystrophy.

We previously reported that cyclin D3-null mice display a shift toward the slow, oxidative phenotype in skeletal muscle, improved exercise endurance, and increased energy expenditure. Here, we explored the role of cyclin D3 in the physiologic response of skeletal muscle to external stimuli and in a model of muscle degenerative disease. We show that cyclin D3-null mice exhibit a further transition from glycolytic to oxidative muscle fiber type in response to voluntary exercise and an improved response to fasting. Since fast glycolytic fibers are known to be more susceptible to degeneration in Duchenne muscular dystrophy (DMD), we examined the effects of cyclin D3 inactivation on skeletal muscle phenotype in the mdx mouse model of DMD. Compared with control mdx mice, cyclin D3-deficient mdx mice display a higher proportion of slower and more oxidative myofibers, reduced muscle degenerative/regenerative processes, and reduced myofiber size variability, indicating an attenuation of dystrophic histopathology. Furthermore, mdx muscles lacking cyclin D3 exhibit reduced fatigability during repeated electrical stimulations. Notably, cyclin D3-null mdx mice show enhanced performance during recurrent trials of endurance treadmill exercise, and post-exercise muscle damage results decreased while the regenerative capacity is boosted. In addition, muscles from exercised cyclin D3-deficient mdx mice display increased oxidative capacity and increased mRNA expression of genes involved in the regulation of oxidative metabolism and the response to oxidative stress. Altogether, our findings indicate that depletion of cyclin D3 confers benefits to dystrophic muscle, suggesting that cyclin D3 inhibition may represent a promising therapeutic strategy against DMD.

Role of Hydroxytyrosol and Oleuropein in the Prevention of Aging and Related Disorders: Focus on Neurodegeneration, Skeletal Muscle Dysfunction and Gut Microbiota.

Aging is a multi-faceted process caused by the accumulation of cellular damage over time, associated with a gradual reduction of physiological activities in cells and organs. This degeneration results in a reduced ability to adapt to homeostasis perturbations and an increased incidence of illnesses such as cognitive decline, neurodegenerative and cardiovascular diseases, cancer, diabetes, and skeletal muscle pathologies. Key features of aging include a chronic low-grade inflammation state and a decrease of the autophagic process. The Mediterranean diet has been associated with longevity and ability to counteract the onset of age-related disorders. Extra virgin olive oil, a fundamental component of this diet, contains bioactive polyphenolic compounds as hydroxytyrosol (HTyr) and oleuropein (OLE), known for their antioxidant, anti-inflammatory, and neuroprotective properties. This review is focused on brain, skeletal muscle, and gut microbiota, as these systems are known to interact at several levels. After the description of the chemistry and pharmacokinetics of HTyr and OLE, we summarize studies reporting their effects in in vivo and in vitro models of neurodegenerative diseases of the central/peripheral nervous system, adult neurogenesis and depression, senescence and lifespan, and age-related skeletal muscle disorders, as well as their impact on the composition of the gut microbiota.

Adult hippocampal neurogenesis and social behavioural deficits in the R451C Neuroligin3 mouse model of autism are reverted by the antidepressant fluoxetine.

Neuron generation persists throughout life in the hippocampus but is altered in animal models of neurological and neuropsychiatric diseases, suggesting that disease-associated decline in cognitive and emotional hippocampal-dependent behaviours might be functionally linked with dysregulation of postnatal neurogenesis. Depletion of the adult neural stem/progenitor cell (NSPCs) pool and neurogenic decline have been recently described in mice expressing synaptic susceptibility genes associated with autism spectrum disorder (ASDs). To gain further insight into mechanisms regulating neurogenesis in mice carrying mutations in synaptic genes related to monogenic ASDs, we used the R451C Neuroligin3 knock-in (Nlgn3 KI) mouse, which is characterized by structural brain abnormalities, deficits in synaptic functions and reduced sociability. We show that the number of adult-born neurons, but not the size of the NSPC pool, was reduced in the ventral dentate gyrus in knock-in mice. Notably, this neurogenic decline was rescued by daily injecting mice with 10 mg/Kg of the antidepressant fluoxetine for 20 consecutive days. Sustained treatment also improved KI mice's sociability and increased the number of c-Fos active adult-born neurons, compared with vehicle-injected KI mice. Our study uncovers neurogenesis-mediated alterations in the brain of R451C KI mouse, showing that the R451C Nlgn3 mutation leads to lasting, albeit pharmacologically reversible, changes in the brain, affecting neuron formation in the adult hippocampus. Our results suggest that fluoxetine can ameliorate social behaviour in KI mice, at least in part, by rescuing adult hippocampal neurogenesis, which may be relevant for the pharmacological treatment of ASDs.

Transcriptome Analysis in a Mouse Model of Premature Aging of Dentate Gyrus: Rescue of Alpha-Synuclein Deficit by Virus-Driven Expression or by Running Restores the Defective Neurogenesis.

The dentate gyrus of the hippocampus and the subventricular zone are neurogenic niches where neural stem and progenitor cells replicate throughout life to generate new neurons. The Btg1 gene maintains the stem cells of the neurogenic niches in quiescence. The deletion of Btg1 leads to an early transient increase of stem/progenitor cells division, followed, however, by a decrease during adulthood of their proliferative capability, accompanied by apoptosis. Since a physiological decrease of neurogenesis occurs during aging, the Btg1 knockout mouse may represent a model of neural aging. We have previously observed that the defective neurogenesis of the Btg1 knockout model is rescued by the powerful neurogenic stimulus of physical exercise (running). To identify genes responsible for stem and progenitor cells maintenance, we sought here to find genes underlying this premature neural aging, and whose deregulated expression could be rescued by running. Through RNA sequencing we analyzed the transcriptomic profiles of the dentate gyrus isolated from Btg1 wild-type or Btg1 knockout adult (2-month-old) mice submitted to physical exercise or sedentary. In Btg1 knockout mice, 545 genes were deregulated, relative to wild-type, while 2081 genes were deregulated by running. We identified 42 genes whose expression was not only down-regulated in the dentate gyrus of Btg1 knockout, but was also counter-regulated to control levels by running in Btg1 knockout mice, vs. sedentary. Among these 42 counter-regulated genes, alpha-synuclein (Snca), Fos, Arc and Npas4 showed significantly greater differential regulation. These genes control neural proliferation, apoptosis, plasticity and memory and are involved in aging. In particular, Snca expression decreases during aging. We tested, therefore, whether an Snca-expressing lentivirus, by rescuing the defective Snca levels in the dentate gyrus of Btg1 knockout mice, could also reverse the aging phenotype, in particular the defective neurogenesis. We found that the exogenous expression of Snca reversed the Btg1 knockout-dependent decrease of stem cell proliferation as well as the increase of progenitor cell apoptosis. This indicates that Snca has a functional role in the process of neural aging observed in this model, and also suggests that Snca acts as a positive regulator of stem cell maintenance.

Tumor Growth in the High Frequency Medulloblastoma Mouse Model Ptch1+/-/Tis21KO Has a Specific Activation Signature of the PI3K/AKT/mTOR Pathway and Is Counteracted by the PI3K Inhibitor MEN1611.

We have previously generated a mouse model (Ptch1+/-/Tis21KO ), which displays high frequency spontaneous medulloblastoma, a pediatric tumor of the cerebellum. Early postnatal cerebellar granule cell precursors (GCPs) of this model show, in consequence of the deletion of Tis21, a defect of the Cxcl3-dependent migration. We asked whether this migration defect, which forces GCPs to remain in the proliferative area at the cerebellar surface, would be the only inducer of their high frequency transformation. In this report we show, by further bioinformatic analysis of our microarray data of Ptch1+/-/Tis21KO GCPs, that, in addition to the migration defect, they show activation of the PI3K/AKT/mTOR pathway, as the mRNA levels of several activators of this pathway (e.g., Lars, Rraga, Dgkq, Pdgfd) are up-regulated, while some inhibitors (e.g. Smg1) are down-regulated. No such change is observed in the Ptch1+/- or Tis21KO background alone, indicating a peculiar synergy between these two genotypes. Thus we investigated, by mRNA and protein analysis, the role of PI3K/AKT/mTOR signaling in MBs and in nodules from primary Ptch1+/-/Tis21KO MB allografted in the flanks of immunosuppressed mice. Activation of the PI3K/AKT/mTOR pathway is seen in full-blown Ptch1+/-/Tis21KO MBs, relative to Ptch1+/-/Tis21WT MBs. In Ptch1+/-/Tis21KO MBs we observe that the proliferation of neoplastic GCPs increases while apoptosis decreases, in parallel with hyper-phosphorylation of the mTOR target S6, and, to a lower extent, of AKT. In nodules derived from primary Ptch1+/-/Tis21KO MBs, treatment with MEN1611, a novel PI3K inhibitor, causes a dramatic reduction of tumor growth, inhibiting proliferation and, conversely, increasing apoptosis, also of tumor CD15+ stem cells, responsible for long-term relapses. Additionally, the phosphorylation of AKT, S6 and 4EBP1 was significantly inhibited, indicating inactivation of the PI3K/AKT/mTOR pathway. Thus, PI3K/AKT/mTOR pathway activation contributes to Ptch1+/-/Tis21KO MB development and to high frequency tumorigenesis, observed when the Tis21 gene is down-regulated. MEN1611 could provide a promising therapy for MB, especially for patient with down-regulation of Btg2 (human ortholog of the murine Tis21 gene), which is frequently deregulated in Shh-type MBs.

Interaction Between Neurogenic Stimuli and the Gene Network Controlling the Activation of Stem Cells of the Adult Neurogenic Niches, in Physiological and Pathological Conditions.

In the adult mammalian brain new neurons are continuously generated throughout life in two niches, the dentate gyrus of the hippocampus and the subventricular zone. This process, called adult neurogenesis, starts from stem cells, which are activated and enter the cell cycle. The proliferative capability of stem cells progressively decreases during aging. The population of stem cells is generally quiescent, and it is not clear whether the potential for stem cells to expand is limited, or whether they can expand and then return to quiescence, remaining available for further activation. Certain conditions may deregulate stem cells quiescence and self-renewal. In fact we discuss the possibility of activation of stem cells by neurogenic stimuli as a function of the intensity of the stimulus (i.e., whether this is physiological or pathological), and of the deregulation of the system (i.e., whether the model is aged or carrying genetic mutations in the gene network controlling quiescence). It appears that when the system is aged and/or carrying mutations of quiescence-maintaining genes, preservation of the quiescent state of stem cells is more critical and stem cells can be activated by a neurogenic stimulus which is ineffective in normal conditions. Moreover, when a neurogenic stimulus is in itself a cause of brain damage (e.g., kainic acid treatment) the activation of stem cells occurs bypassing any inhibitory control. Plausibly, with strong neurogenic stimuli, such as kainic acid injected into the dentate gyrus, the self-renewal capacity of stem cells may undergo rapid exhaustion. However, the self-renewal capability of stem cells persists when normal stimuli are elicited in the presence of a mutation of one of the quiescence-maintaining genes, such as p16Ink4a, p21Cip1 or Btg1. In this case, stem cells become promptly activated by a neurogenic stimulus even during aging. This indicates that stem cells retain a high proliferative capability and plasticity, and suggests that stem cells are protected against the response to stimulus and are resilient to exhaustion. It will be interesting to assess at which functional degree of deregulation of the quiescence-maintaining system, stem cells will remain responsive to repeated neurogenic stimuli without undergoing exhaustion of their pool.

Deletion of Btg1 Induces Prmt1-Dependent Apoptosis and Increased Stemness in Shh-Type Medulloblastoma Cells Without Affecting Tumor Frequency.

About 30% of medulloblastomas (MBs), a tumor of the cerebellum, arise from cerebellar granule cell precursors (GCPs) undergoing transformation following activation of the Sonic hedgehog (Shh) pathway. To study this process, we generated a new MB model by crossing Patched1 heterozygous (Ptch1 +/-) mice, which develop spontaneous Shh-type MBs, with mice lacking B-cell translocation gene 1 (Btg1), a regulator of cerebellar development. In MBs developing in Ptch1 +/- mice, deletion of Btg1 does not alter tumor and lesion frequencies, nor affect the proliferation of neoplastic precursor cells. However, in both tumors and lesions arising in Ptch1 +/- mice, ablation of Btg1 increases by about 25% the apoptotic neoplastic precursor cells, as judged by positivity to activated caspase-3. Moreover, although Btg1 ablation in early postnatal GCPs, developing in the external granule cell layer, leads to a significant increase of proliferation, and decrease of differentiation, relative to wild-type, no synergy occurs with the Ptch1 +/- mutation. However, Btg1 deletion greatly increases apoptosis in postnatal GCPs, with strong synergy between Btg1-null and Ptch1 +/- mutations. That pronounced increase of apoptosis observed in Ptch1 +/- /Btg1 knockout young or neoplastic GCPs may be responsible for the lack of effect of Btg1 ablation on tumorigenesis. This increased apoptosis may be a consequence of increased expression of protein arginine methyltransferase 1 (Prmt1) protein that we observe in Btg1 knockout/Ptch1 +/- MBs. In fact, apoptotic genes, such as BAD, are targets of Prmt1. Moreover, in Btg1-null MBs, we observed a two-fold increase of cells positive to CD15, which labels tumor stem cells, raising the possibility of activation of quiescent tumor cells, known for their role in long-term resistance to treatment and relapses. Thus, Btg1 appears to play a role in cerebellar tumorigenesis by regulating the balance between apoptosis and proliferation during MB development, also influencing the number of tumor stem cells.

Hydroxytyrosol stimulates neurogenesis in aged dentate gyrus by enhancing stem and progenitor cell proliferation and neuron survival.

The dentate gyrus of the hippocampus is one of two brain areas generating throughout life new neurons, which contribute to the formation of episodic/associative memories. During aging, the production of new neurons decreases and a cognitive decline occurs. Dietary factors influence neuronal function and synaptic plasticity; among them the phenolic compound hydroxytyrosol (HTyr), present in olive oil, displays neuroprotective effects. As age impacts primarily on the hippocampus-dependent cognitive processes, we wondered whether HTyr could stimulate hippocampal neurogenesis in vivo in adult and aged wild-type mice as well as in the B-cell translocation 1 gene (Btg1) knockout mouse model of accelerated neural aging. We found that treatment with HTyr activates neurogenesis in the dentate gyrus of adult, aged, and Btg1-null mice, by increasing survival of new neurons and decreasing apoptosis. Notably, however, in the aged and Btg1-null dentate gyrus, HTyr treatment also stimulates the proliferation of stem and progenitor cells, whereas in the adult dentate gyrus HTyr lacks any proliferative effect. Moreover, the new neurons generated in aged mice after HTyr treatment are recruited to existing circuits, as shown by the increase of BrdU+ /c-fos+ neurons. Finally, HTyr treatment also reduces the markers of aging lipofuscin and Iba1. Overall, our findings indicate that HTyr treatment counteracts neurogenesis decline during aging.

Running-Activated Neural Stem Cells Enhance Subventricular Neurogenesis and Improve Olfactory Behavior in p21 Knockout Mice.

In the subventricular zone (SVZ) of the adult brain, the neural stem cells (NSCs) ensure a continuous supply of new neurons to the olfactory bulb (OB), playing a key role in its plasticity and olfactory-related behavior. The activation and expansion of NSCs within the SVZ are finely regulated by environmental and intrinsic factors. Running represents one of the most powerful neurogenic stimuli, although is ineffective in enhancing SVZ neurogenesis. The cell cycle inhibitor p21 is an intrinsic inhibitor of NSCs' expansion through the maintenance of their quiescence and the restrain of neural progenitor proliferation. In this work, we decided to test whether running unveils the intrinsic neurogenic potential of p21-lacking NSCs. To test this hypothesis, we examined the effect of three different paradigms of voluntary running (5, 12, and 21 days) on SVZ neurogenesis of p21 knockout (KO) male mice at two different stages of development, 2 and 12 months of age. In vivo and in vitro data clearly demonstrate that physical activity is consistent with the activation and expansion of NSCs and with the enhancement of SVZ neurogenesis in p21 KO mice. We also found that 12 days of running contribute to the increase in the number of new neurons functionally active within the OB, which associates with an improvement in olfactory performance strictly dependent on adult SVZ neurogenesis, i.e., the odor detection threshold and short-term olfactory memory. These data suggest that in the adult SVZ of p21 KO mice, NSCs retain a high neurogenic potential, triggered by physical activity, with long-term consequences in olfactory-related behavior.

p16Ink4a Prevents the Activation of Aged Quiescent Dentate Gyrus Stem Cells by Physical Exercise.

In the neurogenic niches-the dentate gyrus of the hippocampus and the subventricular zone (SVZ) adjacent to lateral ventricles-stem cells continue to divide during adulthood, generating progenitor cells and new neurons, and to self-renew, thus maintaining the stem cell pool. During aging, the numbers of stem/progenitor cells in the neurogenic niches are reduced. The preservation of the neurogenic pool is committed to a number of antiproliferative genes, with the role of maintaining the quiescence of neural cells. The cyclin-dependent kinase inhibitor p16Ink4a, whose expression increases with age, controls the expansion of SVZ aging stem cells, since in mice its deficiency prevents the decline of neurogenesis in SVZ. No change of neurogenesis is however observed in the p16Ink4a-null dentate gyrus. Here, we hypothesized that p16Ink4a plays a role as a regulator of the self-renewal of the stem cell pool also in the dentate gyrus, and to test this possibility we stimulated the dentate gyrus neural cells of p16Ink4a-null aging mice with physical exercise, a powerful neurogenic activator. We observed that running highly induced the generation of new stem cells in the p16Ink4a-null dentate gyrus, forcing them to exit from quiescence. Stem cells, notably, are not induced to proliferate by running in wild-type (WT) mice. Moreover, p16Ink4a-null progenitor cells were increased by running significantly above the number observed in WT mice. The new stem and progenitor cells generated new neurons, and continued to actively proliferate in p16Ink4a-null mice longer than in the WT after cessation of exercise. Thus, p16Ink4a prevents aging dentate gyrus stem cells from being activated by exercise. Therefore, p16Ink4a may play a role in the maintenance of dentate gyrus stem cells after stimulus, by keeping a reserve of their self-renewal capacity during aging.

Depression and adult neurogenesis: Positive effects of the antidepressant fluoxetine and of physical exercise.

Of wide interest for health is the relation existing between depression, a very common psychological illness, accompanied by anxiety and reduced ability to concentrate, and adult neurogenesis. We will focus on two neurogenic stimuli, fluoxetine and physical exercise, both endowed with the ability to activate adult neurogenesis in the dentate gyrus of the hippocampus, known to be required for learning and memory, and both able to counteract depression. Fluoxetine belongs to the class of selective serotonin reuptake inhibitor (SSRI) antidepressants, which represent the most used pharmacological therapy; physical exercise has also been shown to effectively counteract depression symptoms in rodents as well as in humans. While there is evidence that the antidepressant effect of fluoxetine requires its pro-neurogenic action, exerted by promoting proliferation, differentiation and survival of progenitor cells of the hippocampus, on the other hand fluoxetine exerts also neurogenesis-independent antidepressant effects by influencing the plasticity of the new neurons generated. Similarly, the antidepressant action of running also correlates with an increase of hippocampal neurogenesis and plasticity, although the gene pathways involved are only partially coincident with those of fluoxetine, such as those involved in serotonin metabolism and synapse formation. We further discuss how extra-neurogenic actions are also suggested by the fact that, unlike running, fluoxetine is unable to stimulate neurogenesis during aging, but still displays antidepressant effects. Moreover, in specific conditions, fluoxetine or running activate not only progenitor but also stem cells, which normally are not stimulated; this fact reveals how stem cells have a long-term, hidden ability to self-renew and, more generally, that neurogenesis is subject to complex controls that may play a role in depression, such as the type of neurogenic stimulus or the state of the local niche. Finally, we discuss how fluoxetine or running are effective in counteracting depression originated from stress or neurodegenerative diseases.

Fluoxetine or Sox2 reactivate proliferation-defective stem and progenitor cells of the adult and aged dentate gyrus.

The dentate gyrus of the hippocampus and the subventricular zone are neurogenic niches where the production of new neurons from glia-like stem cells continues throughout adult life. It is not clear whether the pool of stem cells is fated to be exhausted or is conserved until old age. We observed that the antiproliferative gene Btg1 maintains the quiescence of stem cells, and its ablation causes an increase of stem/progenitor cells proliferation in neonatal mice followed by progressive loss of proliferation during adulthood. Fluoxetine is an antidepressant, which exerts a powerful neurogenic effect on dentate gyrus progenitor cells, but is ineffective on stem cells. Here we show that adult dentate gyrus stem cells in the Btg1 knockout mice, with reduced self-renewal and proliferative capability, can be reactivated by fluoxetine, which increases their number greatly above the level of control or fluoxetine-treated wild-type mice. The increase of mitotic index above wild-type in Btg1 knockout fluoxetine-treated stem cells indicates that fluoxetine forces quiescent stem cells to enter the cycle. Stem cell proliferation undergoes continuous reactivation until fluoxetine is administered. Remarkably, fluoxetine reactivates proliferation-defective stem cells also in aged Btg1 knockout mice (15-month-old), an effect absent in wild-type aged mice. Moreover, overexpression of Sox2 retrovirally transduced in Btg1 knockout dentate gyrus cells significantly increases the number of neuroblasts, indicating that Sox2 is able to promote the self-renewal of proliferation-defective stem cells. Overall, the deletion of an antiproliferative gene, such as Btg1, reveals that dentate gyrus stem cells retain a hidden plasticity for self-renewal also in old age, in agreement with a model of permanent self-renewal.

Tis21-gene therapy inhibits medulloblastoma growth in a murine allograft model.

Medulloblastoma (MB), the tumor of the cerebellum, is the most frequent brain cancer in childhood and a major cause of pediatric mortality. Based on gene profiling, four MB subgroups have been identified, i.e., Wnt or Sonic Hedgehog (Shh) types, and subgroup 3 or 4. The Shh-type MB has been shown to arise from the cerebellar precursors of granule neurons (GCPs), where a hyperactivation of the Shh pathway leads to their neoplastic transformation. We have previously shown that the gene Tis21 (PC3/Btg2) inhibits the proliferation and promotes the differentiation and migration of GCPs. Moreover, the overexpression or the deletion of Tis21 in Patched1 heterozygous mice, a model of spontaneous Shh-type MB, highly reduces or increases, respectively, the frequency of MB. Here we tested whether Tis21 can inhibit MB allografts. Athymic nude mice were subcutaneously grafted with MB cells explanted from Patched1 heterozygous mice. MB allografts were then injected with adeno-associated viruses either carrying Tis21 (AAV-Tis21) or empty (AAV-CBA). We observed that the treatment with AAV-Tis21 significantly inhibited the growth of tumor nodules, as judged by their volume, and reduced the number of proliferating tumor cells (labeled with Ki67 or BrdU), relative to AAV-CBA-treated control mice. In parallel, AAV-Tis21 increased significantly tumor cells labeled with early and late neural differentiation markers. Overall the results suggest that Tis21-gene therapy slows down MB tumor growth through inhibition of proliferation and enhancement of neural differentiation. These results validate Tis21 as a relevant target for MB therapy.

Metastatic group 3 medulloblastoma is driven by PRUNE1 targeting NME1-TGF-ß-OTX2-SNAIL via PTEN inhibition.

Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-ß signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-ß activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-ß/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3.10.1093/brain/awy039_video1awy039media15742053534001.

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.

Physical exercise rescues defective neural stem cells and neurogenesis in the adult subventricular zone of Btg1 knockout mice.

Adult neurogenesis occurs throughout life in the dentate gyrus (DG) and the subventricular zone (SVZ), where glia-like stem cells generate new neurons. Voluntary running is a powerful neurogenic stimulus triggering the proliferation of progenitor cells in the DG but, apparently, not in the SVZ. The antiproliferative gene Btg1 maintains the quiescence of DG and SVZ stem cells. Its ablation causes intense proliferation of DG and SVZ stem/progenitor cells in young mice, followed, during adulthood, by progressive decrease of the proliferative capacity. We have previously observed that running can rescue the deficit of DG Btg1-null neurogenesis. Here, we show that in adult Btg1-null SVZ stem and neuroblast cells, the reduction of proliferation is associated with a longer cell cycle and a more frequent entry into quiescence. Notably, running increases proliferation in Btg1-null SVZ stem cells highly above the levels of sedentary wild-type mice and restores normal values of cell cycle length and quiescence in stem and neuroblast cells, without affecting wild-type cells. Btg1-null SVZ neuroblasts show also increased migration throughout the rostral migratory stream and a deficiency of differentiated neurons in the olfactory bulb, possibly a consequence of premature exit from the cycle; running, however, normalizes migration and differentiation, increasing newborn neurons recruited to the olfactory circuitry. Furthermore, running increases the self-renewal of Btg1-null SVZ-derived neurospheres and, remarkably, in aged Btg1-null mice almost doubles the proliferating SVZ stem cells. Altogether, this reveals that SVZ stem cells are endowed with a hidden supply of self-renewal capacity, coupled to cell cycle acceleration and emerging after ablation of the quiescence-maintaining Btg1 gene and following exercise.

HDAC1, HDAC4, and HDAC9 Bind to PC3/Tis21/Btg2 and Are Required for Its Inhibition of Cell Cycle Progression and Cyclin D1 Expression.

PC3/Tis21 is a transcriptional cofactor that inhibits proliferation in several cell types, including neural progenitors. Here, we report that PC3/Tis21 associates with HDAC1, HDAC4, and HDAC9 in vivo, in fibroblast cells. Furthermore, when HDAC1, HDAC4, or HDAC9 are silenced in fibroblasts or in a line of cerebellar progenitor cells, the ability of PC3/Tis21 to inhibit proliferation is significantly reduced. Overexpression of HDAC1, HDAC4, or HDAC9 in fibroblasts and in cerebellar precursor cells synergizes with PC3/Tis21 in inhibiting the expression of cyclin D1, a cyclin selectively inhibited by PC3/Tis21. Conversely, the depletion of HDAC1 or HDAC4 (but not HDAC9) in fibroblasts and in cerebellar precursor cells significantly impairs the ability of PC3/Tis21 to inhibit cyclin D1 expression. An analysis of HDAC4 deletion mutants shows that both the amino-terminal moiety and the catalytic domain of HDAC4 associate to PC3/Tis21, but neither alone is sufficient to potentiate the inhibition of cyclin D1 by PC3/Tis21. As a whole, our findings indicate that PC3/Tis21 inhibits cell proliferation in a way dependent on the presence of HDACs, in fibroblasts as well as in neural cells. Considering that several reports have demonstrated that HDACs can act as transcriptional corepressors on the cyclin D1 promoter, our data suggest that the association of PC3/Tis21 to HDACs is functional to recruit them to target genes, such as cyclin D1, for repression of their expression. J. Cell. Physiol. 232: 1696-1707, 2017. © 2016 Wiley Periodicals, Inc.

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.

Functional Genomics Identifies Tis21-Dependent Mechanisms and Putative Cancer Drug Targets Underlying Medulloblastoma Shh-Type Development.

We have recently generated a novel medulloblastoma (MB) mouse model with activation of the Shh pathway and lacking the MB suppressor Tis21 (Patched1+/-/Tis21KO ). Its main phenotype is a defect of migration of the cerebellar granule precursor cells (GCPs). By genomic analysis of GCPs in vivo, we identified as drug target and major responsible of this defect the down-regulation of the promigratory chemokine Cxcl3. Consequently, the GCPs remain longer in the cerebellum proliferative area, and the MB frequency is enhanced. Here, we further analyzed the genes deregulated in a Tis21-dependent manner (Patched1+/-/Tis21 wild-type vs. Ptch1+/-/Tis21 knockout), among which are a number of down-regulated tumor inhibitors and up-regulated tumor facilitators, focusing on pathways potentially involved in the tumorigenesis and on putative new drug targets. The data analysis using bioinformatic tools revealed: (i) a link between the Shh signaling and the Tis21-dependent impairment of the GCPs migration, through a Shh-dependent deregulation of the clathrin-mediated chemotaxis operating in the primary cilium through the Cxcl3-Cxcr2 axis; (ii) a possible lineage shift of Shh-type GCPs toward retinal precursor phenotype, i.e., the neural cell type involved in group 3 MB; (iii) the identification of a subset of putative drug targets for MB, involved, among the others, in the regulation of Hippo signaling and centrosome assembly. Finally, our findings define also the role of Tis21 in the regulation of gene expression, through epigenetic and RNA processing mechanisms, influencing the fate of the GCPs.