The p53-inducible gene PC3 (TIS21, BTG2) is endowed with antiproliferative activity. Here we report that expression of PC3 in cycling cells induced accumulation of hypophosphorylated, growth-inhibitory forms of pRb and led to G 1 arrest. This latter was not observed in cells with genetic disruption of the Rb gene, indicating that the PC3-mediated G 1 arrest was Rb dependent. Furthermore, (i) the arrest of G 1 -S transition exerted by PC3 was completely rescued by coexpression of cyclin D1 but not by that of cyclin A or E; (ii) expression of PC3 caused a significant down-regulation of cyclin D1 protein levels, also in Rb-defective cells, accompanied by inhibition of CDK4 activity in vivo; and (iii) the removal from the PC3 molecule of residues 50 to 68, a conserved domain of the PC3/BTG/Tob gene family, which we term GR, led to a loss of the inhibition of proliferation as well as of the down-regulation of cyclin D1 levels. These data point to cyclin D1 downregulation as the main factor responsible for the growth inhibition by PC3. Such an effect was associated with a decrease of cyclin D1 transcript and of cyclin D1 promoter activity, whereas no effect of PC3 was observed on cyclin D1 protein stability. Taken together, these findings indicate that PC3 impairs G 1 -S transition by inhibiting pRb function in consequence of a reduction of cyclin D1 levels and that PC3 acts, either directly or indirectly, as a transcriptional regulator of cyclin D1.The control of the cell cycle plays an essential role in cell growth and in the activation of important cellular processes such as differentiation and apoptosis. pRb (retinoblastoma protein) and p53 are two molecules identified as key regulators of the cell cycle.pRb is a nuclear phosphoprotein whose phosphorylation state oscillates regularly during the cell cycle. Its underphosphorylated forms predominate in G 0 and G 1 , while highly phosphorylated forms exist in S, G 2 , and M phases (13,16,21). The primary biological function of underphosphorylated pRb is to inhibit progression toward S phase by controlling a checkpoint in late G 1 (for reviews, see references 8, 22, and 51). In fact, underphosphorylated pRb associates with members of the E2F family of transcription factors, impairing their activity and leading to a cell cycle block in G 1 . Conversely, the phosphorylation of pRb inactivates its growth suppression activity by freeing E2F molecules, thus enabling them to transactivate genes required for the progression of the cell into S phase and the remainder of the cell cycle (52, 97, 114).Cyclin-dependent kinases (CDKs) are the molecules responsible for pRb phosphorylation and its consequent inactivation (reviewed in references 70 and 102). Each CDK has its own functional specificity, based on the period of its activity during the cell cycle and on the specific cyclin partner. CDK4, CDK5, and CDK6 form complexes with D-type cyclins during the G 1 phase (65,69,116). CDK2, when bound to cyclin A or E, is instead essential for G 1 -to-S transition (28, 78), while the c...

Adult neurogenesis in the dentate gyrus plays a critical role in hippocampus-dependent spatial learning. It remains unknown, however, how new neurons become functionally integrated into spatial circuits and contribute to hippocampus-mediated forms of learning and memory. To investigate these issues, we used a mouse model in which the differentiation of adult-generated dentate gyrus neurons can be anticipated by conditionally expressing the pro-differentiative gene PC3 (Tis21/BTG2) in nestin-positive progenitor cells. In contrast to previous studies that affected the number of newly generated neurons, this strategy selectively changes their timing of differentiation. New, adult-generated dentate gyrus progenitors, in which the PC3 transgene was expressed, showed accelerated differentiation and significantly reduced dendritic arborization and spine density. Functionally, this genetic manipulation specifically affected different hippocampus-dependent learning and memory tasks, including contextual fear conditioning, and selectively reduced synaptic plasticity in the dentate gyrus. Morphological and functional analyses of hippocampal neurons at different stages of differentiation, following transgene activation within defined time-windows, revealed that the new, adult-generated neurons up to 3–4 weeks of age are required not only to acquire new spatial information but also to use previously consolidated memories. Thus, the correct unwinding of these key memory functions, which can be an expression of the ability of adult-generated neurons to link subsequent events in memory circuits, is critically dependent on the correct timing of the initial stages of neuron maturation and connection to existing circuits.

PC3 TIS21/BTG2 is the founding member of a family of genes endowed with antiproliferative properties, namely BTG1, ANA/BTG3, PC3B, TOB, and TOB2. PC3 was originally isolated as a gene induced by nerve growth factor during neuronal differentiation of rat PC12 cells, or by TPA in NIH3T3 cells (named TIS21), and is a marker for neuronal birth in vivo. This and other ®ndings suggested its implication in the process of neurogenesis as mediator of the growth arrest before differentiation. Remarkably, its human homolog, named BTG2, was shown to be p53-inducible, in conditions of genotoxic damage. PC3 TIS21/BTG2 impairs G 1 ±S progression, either by a Rb-dependent pathway through inhibition of cyclin D1 transcription, or in a Rb-independent fashion by cyclin E downregulation. PC3 TIS21/BTG2 might also control the G 2 checkpoint. Furthermore, PC3 TIS21/BTG2 interacts with carbon catabolite repressor protein-associated factor 1 (CAF-1), a molecule that associates to the yeast transcriptional complex CCR4 and might in¯uence cell cycle, with the transcription factor Hoxb9, and with the proteinarginine methyltransferase 1, that might control transcription through histone methylation. Current evidence suggests a physiological role of PC3 TIS21/BTG2 in the control of cell cycle arrest following DNA damage and other types of cellular stress, or before differentiation of the neuron and other cell types. The molecular function of PC3 TIS21/BTG2 is still unknown, but its ability to modulate cyclin D1 transcription, or to synergize with the transcription factor Hoxb9, suggests that it behaves as a transcriptional co-regulator.

PC3 is an immediate early gene induced by nerve growth factor in PC12 cells, a cell line derived from a tumor of the adrenal medulla that undergoes neuronal differentiation in the presence of nerve growth factor. This induction is independent of new protein synthesis as it can occur in the presence of cycloheximide. PC3 is also induced with similar kinetics, but at lower levels, by membrane depolarization (both in vivo and in vitro) and epidermal growth factor. It is induced at much lower levels by fibroblast growth factor and interleukin 6. In vivo it is found expressed in tissues, such as brain at embryonic day 13.5, placenta, amnion, and spleen, which are proliferating and/or differentiating. The deduced protein sequence from the cDNA indicates the presence of a signal peptide, suggesting that PC3 is secreted.

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.

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 2 months 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 5 days. 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.

Neurogenesis in the dentate gyrus of the adult hippocampus has been implicated in neural plasticity and memory, but the molecular mechanisms controlling the proliferation and differentiation of newborn neurons and their integration into the synaptic circuitry are still largely unknown. To investigate this issue, we have analyzed the adult hippocampal neurogenesis in a PC3/Tis21-null mouse model. PC3/Tis21 is a transcriptional co-factor endowed with antiproliferative and prodifferentiative properties; indeed, its upregulation in neural progenitors has been shown to induce exit from cell cycle and differentiation. We demonstrate here that the deletion of PC3/Tis21 causes an increased proliferation of progenitor cells in the adult dentate gyrus and an arrest of their terminal differentiation. In fact, in the PC3/Tis21-null hippocampus postmitotic undifferentiated neurons accumulated, while the number of terminally differentiated neurons decreased of 40%. As a result, PC3/Tis21-null mice displayed a deficit of contextual memory. Notably, we observed that PC3/Tis21 can associate to the promoter of Id3, an inhibitor of proneural gene activity, and negatively regulates its expression, indicating that PC3/Tis21 acts upstream of Id3. Our results identify PC3/Tis21 as a gene required in the control of proliferation and terminal differentiation of newborn neurons during adult hippocampal neurogenesis and suggest its involvement in the formation of contextual memories.

Growing evidence indicates that cell cycle arrest and neurogenesis are highly coordinated and interactive processes, governed by cell cycle genes and neural transcription factors. The gene PC3 (Tis21/BTG2) is expressed in the neuroblast throughout the neural tube and inhibits cell cycle progression at the G 1 checkpoint by repressing cyclin D1 transcription. We generated inducible mouse models in which the expression of PC3 was upregulated in neuronal precursors of the neural tube and of the cerebellum. These mice exhibited a marked increase in the production of postmitotic neurons and impairment of cerebellar development. Cerebellar granule precursors of PC3 transgenic mice displayed inhibition of cyclin D1 expression and a strong increase in the expression of Math1, a transcription factor required for their differentiation. Furthermore, PC3, encoded by a recombinant adenovirus, also induced Math1 in postmitotic granule cells in vitro and stimulated the Math1 promoter activity. In contrast, PC3 expression was unaffected in the cerebellar primordium of Math1 null mice, suggesting that PC3 acts upstream to Math1. As a whole, our data suggest that cell cycle exit of cerebellar granule cell precursors and the onset of cerebellar neurogenesis are coordinated by PC3 through transcriptional control of cyclin D1 and Math1, respectively.