Cell Cycle Regulation During Neurogenesis in the Embryonic and ...

Stem Cell Rev and Rep (2013) 9:794–805 DOI 10.1007/s12015-013-9460-5

Cell Cycle Regulation During Neurogenesis in the Embryonic and Adult Brain Arquimedes Cheffer & Attila Tárnok & Henning Ulrich

Published online: 31 July 2013 # Springer Science+Business Media New York 2013

Abstract Throughout the development of the central nervous system, neural crest cells and the primary neural stem cells originate several non-neuronal and neuronal cell types. Undifferentiated stem cells exist in the adult brain, mainly in the dentate gyrus of the hippocampus and in the subventricular zone of the lateral ventricles, and can produce new neurons, participating in brain plasticity and tissue regeneration. Neurogenesis in the embryonic and adult brain occurs under the control of intrinsic and extrinsic factors. However, the mechanisms, by which cell cycle components control neural stem cell proliferation and consequently neurogenesis, still lack further investigation. We discuss here recent knowledge obtained on cell cycle components as key regulators of neural stem and progenitor cell proliferation and differentiation in the embryonic and adult brain. Keywords Cell cycle . Neural stem cells . Proliferation . Neurogenesis . Embryonic and adult brain

Introduction The central nervous system (CNS) develops from a small number of cells proliferating and interacting in a very particular manner in order to form a functional neural network with regional identities containing over hundreds of billions of cells in human beings. In this dynamic process, neural A. Cheffer : H. Ulrich (*) Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil e-mail: [email protected] A. Tárnok Department of Pediatric Cardiology, Heart Centre, and Translational Center of Regenerative, Medicine Leipzig, TRM, Medical Faculty, University Leipzig, Leipzig, Germany

crest (neuroepithelial) cells—or the primary neural stem cells (NSCs)—give rise to several cell types, including nonneuronal and neuronal phenotypes. Among the neuronal cell types, i.e. peripheral nervous system neurons can be cited [1, 2]. This process involves initial proliferation, followed by migration resulting in differentiation of neurons and glial cells under the control of intrinsic (transcription factors and cell cycle constituents) and extrinsic factors (hormones, growth factors, neurotransmitters and their receptors) [3, 4]. The previous view of the non-neurogenic nature of the adult CNS has been shown to be not valid based on the discovery of stem and progenitor cells, capable of proliferating and participating in brain tissue regeneration. The incorporation of [3H]-thymidine into DNA of dividing neural stem and progenitor cells provided a clear evidence for the generation of new neurons in postnatal mouse brains, including the hippocampus, neocortex and olfactory bulb (OB), as detailed in a recent review [1]. Moreover, some reports even suggest that in adulthood other stem cells remain, such as very small embryonic like (VSEL) and pericytederived stem cells with differentiation potential into neural phenotypes [5–8]. Moreover, neurodegenerative and neuropsychiatric diseases have been connected with deficient neuroregenerative capacities. Since proliferation and differentiation of neural stem and progenitor cells can be an endogenous response to brain injury, its further stimulation can present a treatment strategy as alternative to stem cell transfer [3]. On the other site, neoplasia, diagnosed as brain tumors, is initiated by proliferation of stem cells which have lost control of cell cycle regulation [9]. In eukaryotes, the cell cycle corresponds to a sequence of events, occurring during the lifetime of a eukaryotic cell, and is divided into four distinct phases: mitosis and cell division occur during the relatively brief M phase, which is followed by the G1 phase. This is the main period of cell growth and it covers the longest part of the cell cycle. G1 phase gives way

Stem Cell Rev and Rep (2013) 9:794–805

to the S phase, period of the cell cycle, when DNA is synthesized. Following S phase, in the relatively short G2 phase, the cell prepares for dividing, followed by entering the M phase again and beginning a new round of the cell cycle. G1, S and G2 phases collectively are called interphase (Fig. 1). Terminal-differentiated cells, such as neurons, never divide and therefore they enter a quiescent phase (G0) rather than continuing in the cycle [10]. The progression through the cell cycle is driven by the concerted action of proteins known as cyclins and cyclin-dependent kinases (Cdks) [11, 12]. During G1 phase, Cdk4/6-cyclin D complexes continuously phosphorylate the retinoblastoma-associated protein (pRb), an important regulator of the G1/S cell cycle transition. The phosporylated pRb is then not anymore capable of interacting with E2f transcription factors and consequently fails to inhibit promoter binding of those. As result, transcription of genes necessary for cell cycle progression, including cyclin E, occur. Cdk2-cyclin E complexes further phosphorylate pRb, inactivating it completely and leading to a wave of transcriptional activity required for DNA duplication phase (S phase) [13, 14]. In the S phase, Cdk2 then complexes with cyclin A, phosphorylates pRb and drives the cell through S phase, at the end of which, cyclin A binds to Cdk1. The resulting complex is important for the completion of the G2 phase. Finally, Cdk1-cyclin B complexes facilitate the G2/M transition. At the cell cycle’s G2/M boundary, Cdk1 is activated, which in turn triggers mitosis (M phase) [15]. Beyond their control by binding to the adequate cyclin, Cdk activities are also regulated by cyclin-dependent kinase inhibitors (CKIs), which induce cell cycle arrest in response to antiproliferative signals including contact with other cells, DNA damage, terminal differentiation, and senescence. The CKIs have been classified into two families, according to their sequence and functional similarities: the Kip/Cip family (kinase interacting protein/cytokine-inducible protein), which inhibits most Cdkcyclin complexes, except Cdk4/6-cyclin D; the Ink4 family inhibiting specifically Cdk4 and Cdk6 and their complexes with cyclin D, which mediate the progression through the G1 phase [13, 14, 16]. Altogether, these elements control the cell cycle, which finally results in mitotic separation of the two daughter cells [11]. Several studies have shed light on the role and importance of the cell cycle components in controlling the proliferation and differentiation of NSCs during the embryonic development of the nervous system. However, more recently, efforts have been made to increase our understanding about how the NSC proliferation and neurodifferentiation in adulthood are regulated by cell cycle constituents. We show here achievements in the field of basic research allowing the study of cellular processes occurring during brain development and the investigation of the cell cycle components as key regulators of these processes. We believe that such studies are crucial to develop subsequent therapeutic strategies.

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Fig. 1 Cell cycle regulation. Cell cycle components discussed in this review and involved in regulating NSC proliferation and differentiation throughout CNS development are shown at their approximate positions

Cell Cycle Components as Key Regulators of Neural Stem Cell Proliferation and Neurogenesis in the Embryonic and Adult Nervous System Although in mammalian cells the progression through the cell cycle involves the participation of at least five different Cdks (Cdk1, Cdk2, Cdk3, Cdk4, and Cdk6), recent work has demonstrated that mouse embryos, lacking all so called interphase Cdks (Cdk2, Cdk3, Cdk4 and Cdk6), are able to undergo organogenesis and develop to midgestation. This is possible due to Cdk1 ability in binding to all cyclins, leading to the phosphorylation of pRb, and subsequently, to the expression of genes, whose regulation depends on E2f transcription factors. On the other hand, mouse embryos lacking Cdk1 fail to reach the morula and blastocyst stages, suggesting that Cdk1 is the only one required for embryonic development [17]. This hinders any study of the role and requirement of Cdk1 during the embryonic development or even for the proliferation/differentiation of a determined cell type during adulthood based on gene expression or activity silencing. Therefore, we rely on data on other Cdks, on which our review is focused. Cdk2 Even though Cdk2 seems to be dispensable for NSC proliferation, differentiation and survival of adult-born dentate gyrus (DG) granule neurons in vivo, experimental evidence suggests that this Cdk plays an important role during the embryonic development of the nervous system [18]. Lim and Kaldis observed a striking ablation of the intermediate zone and cortical plate in Cdk2 and Cdk4 double-knockout (KO) mouse embryonic brain. NSC analysis revealed that they were able to proliferate, but they displayed an altered cell cycle profile and differentiated more easily into neurons,

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as indicated by a notable reduction in S phase duration and an increased tendency towards neurogenic divisions [19]. Moreover, it had already been demonstrated that even dominant-negative Cdk2 pRb-deficient mouse embryos show partially decreased cell growth, indicating that Cdk2 might have other targets [20]. Indeed, the Cdk2-cyclin E complex also functions by promoting G1 phase progression in oligodendrocyte precursor cells (OPCs), which could be related to the importance of Cdk2 for adult OPC renewal and present one of the underlying mechanisms that drive adult progenitors to differentiate and thus regenerate myelin, although Cdk2 loss does not affect myelination [21, 22]. It is worthwhile to mention that Cdk2 is required for proliferation and selfrenewal of NSCs derived from the adult subventricular zone (SVZ) [23]. Cdk4/6 Cdk4 and Cdk6 are discussed together, since, given their homology sequence, they are thought to play similar roles. In 2003, Calegari and Huttner proposed, based on their studies with mouse embryo culture, a model called the cell cycle length hypothesis. According to this model, time could be a key limiting factor for a cell fate change to occur; consequently, a relatively long G1 phase would allow the switch from proliferation to neurogenesis while a short G1 phase would promote cell-cycle progression [24]. Following this study, many investigations were performed to test such hypothesis. For instance, shortening the cell cycle by overexpressing Cdk4-cyclin D, a complex involved in promoting the G1/S transition, inhibited neurogenesis while augmented generation and expansion of basal progenitors, resulting in a thicker SVZ and larger surface area of the postnatal cortex, as observed by Lange and co-workers [20]. The opposite effect was obtained, when expression of this complex was blocked by RNA interference and cell cycle duration increased [25]. The cell cycle length hypothesis is also supported by the afore-mentioned work showing that NSCs derived from Cdk2 and Cdk4 double-KO mice are more likely to differentiate into neurons, preventing proper proliferation of the progenitor pool [19]. Although Cdk4 and Cdk6 are believed to play similar roles, a study with P19 embryonal carcinoma cells revealed that while Cdk4-cyclin D1 complexes are associated with exponentially growing cells, these complexes are replaced by Cdk4/6-cyclin D2/D3 complexes after density arrest. Neuronal differentiation is accompanied by cytoplasmic accumulation of D-type cyclins and Cdk4/6, related to neuritogenesis [26]. Research has also been carried out to unveil the role of Cdk4/6 in the regulation of NSC proliferation in the adult brain and similar results compared to those observed during embryogenesis were obtained. During brain development, overexpression of the Cdk4-cyclin D1 complex in the adult murine hippocampus induced expansion of NSCs, and neurogenesis was inhibited at the same time [27]. Similarly,


increased proliferation was also observed in adult mouse SVZ cells overexpressing Cdk4 [23]. However, the most surprising results have been those showing that specifically Cdk6 is essential for cell proliferation within the DG of the hippocampus and the SVZ of the lateral ventricles. While the numbers of cells in the DG and SVZ of Cdk4(−/−) and their respective wild-type (WT) littermates were similar, a twofold reduction in the numbers of cells in the DG and SVZ of Cdk6(−/−) animals were found. Furthermore, Cdk6(−/−) precursor cells, committed to a neuronal fate, prematurely exited the cell cycle and exhibited a lengthened G1 phase, which reduced the production of newborn neurons [28]. Cdk5 Different from Cdk1 and other interphase Cdks, Cdk5 does not play any apparent roles in regulating the cell cycle. It is mainly expressed in postmitotic neurons, being implicated in their maturation, migration, and survival during embryonic development, but also playing distinct functions in adult neurogenesis [29]. Several evidences support these CdK5 roles, such as the detection of Cdk5 mRNA and their regulators p35 and p39 in specific regions of the developing brain and adult rat nervous system by in situ hybridization [30]. It is known that Cdk5 is crucial for the proper migration of cortical neurons in the developing cerebral cortex [31]. Cdk5 is mainly involved in the transitional phase of neurons from multipolar into bipolar morphology during radial neuronal migration, and a deficiency of Cdk5 results in abnormal morphology of pyramidal neurons [32]. It was also demonstrated that Cdk5 plays an important role in neuroblast migration from the SVZ toward the OB in the adult rodent brain [33]. Similarly, Jessberger and colleagues overexpressed Cdk5 in NSCs from rat and mouse hippocampus and did not observe any significant effects on the proliferation of adult rat or mouse NPCs, as measured by the number of bromo-deoxy-uridine (BrdU)incorporating cells. Moreover, the number of anti-MAP2 antibody-labeled neuronal cells was not significantly different between control cells, cells overexpressing Cdk5 and cells induced to neuronal differentiation. The same results were obtained when Cdk5 expression was inhibited. Together, these observations indicate that Cdk5 activity is not required for proliferation or differentiation of NSCs. Furthermore, the authors of this work demonstrated, by expressing a dominant negative form of Cdk5 or a short-hairpin RNA (shRNA) against Cdk5 mRNA that Cdk5 is crucial for migration, development of proper dendritic architecture, and pathfinding of newborn granule cells in the course of neurogenesis during adulthood [34]. Strikingly, the genetic deletion of Cdk5 in adult hippocampal precursor cells reduces dramatically the number of mature neurons, without affecting the number of immature neurons, suggesting that such decrease in the number of mature neurons in Cdk5-KO mice results from enhanced cell death. Indeed, the number of activated caspase 3-immunoreactive cells was greater in Cdk5-KO mice compared to WT controls.


These data suggest that Cdk5 expression in immature neurons is required for survival of adult-generated dentate granule neurons [35]. Although little is known about the mechanisms underlying Cdk5 functions, a recent work showed that Cdk5 phosphorylation of p27Kip1 is crucial for neurite outgrowth and neural differentiation. This idea is supported by the fact that (i) neurogenesis is specifically suppressed by transfection of p27Kip1 siRNA into Cdk5(+/+) NSCs; and (ii) Cdk5(+/+) NSCs, whose differentiation is inhibited by a mutant p27/Thr187A, which cannot be phosphorylated, are rescued by co-transfection of the phosphorylation-mimicking mutant p27/Thr187D [36]. Besides p27Kip1, another Cdk5 target is Pctaire1, a Cdk-related protein kinase, whose phosphorylation increases neuronal differentiation together with elevating Cdk5 activity. It was also demonstrated that knockdown of Pctaire1 expression impairs dendrite development, and more surprisingly that expression of a mutant of Pctaire1, which cannot be phosphorylated, also decreases dendrite complexity [37]. Different targets have also been found, by which Cdk5 is implicated in cell survival. For instance, it was suggested that Cdk5 regulates neuronal survival by precise epigenetic control through modulation of histone acetylation. Suppression of Cdk5 activity by the specific inhibitor roscovitine effectively attenuated both histone H3 acetylation (via phosphorylation of a component of the histone deacetylase/HDAC complex) and reexpression of cyclin proteins in granule neurons under conditions of potassium ion deprivation, resulting in protection of neurons against apoptotic cell death [38]. This finding is consistent with experimental evidence that terminal-differentiated neurons reenter the cell cycle before undergoing programmed cell death, and obviously such reentry requires reexpression of cell cycle proteins [39–41]. Overall, these findings suggest that CdK5 have different roles in normal developing neurons and during apoptosis, mediating in the earlier case pro-survival functions. Such difference results from altered regulation of the HDAC complex during neuronal apoptosis by Cdk5. It is also possible that Cdk5 has other targets. Actually, the antiapoptotic protein Bcl-2 (B-cell lymphoma protein 2) was identified as a novel substrate of Cdk5 in developing neurons. Interestingly, it was found that Cdk5-mediated phosphorylation of Bcl-2 at Ser70 is required for the neuroprotective effect of Bcl-2. For instance, overexpression of Bcl-2 in a mutant lacking the Cdk5 phosphorylation site abolished the protective effect of Cdk5 re-expression in Cdk5(−/−) neurons [42]. The protective effect exerted by Cdk5 during brain development is still reinforced by the observation that Cdk5 conditional-KO mice exhibit, besides complex neurological deficits, neuronal loss and microglial activation, which is correlated with a massive inflammatory reaction accompanying neurodegenerative disorders [43]. D-type cyclins D-type cyclins are regulatory proteins of the cell cycle, whose binding is required to activate Cdk4/6, that

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is responsible for the regulation of the progression through G1 phase [11]. Three D-type cyclins (D1, D2, and D3) have been identified in mammals, whereas D1 and D2 types are mostly involved in proliferation and differentiation of embryonic and adult NSCs. A body of evidence suggests that these two D-type cyclins are required along developmental stages. Glickstein and colleagues evaluated cyclin D1 and D2 expression in mice from embryonic day 12.5 until postnatal day 60 and observed transition from cyclin D1 to D2 expression in neuroprogenitors as the brain developed [44]. On one hand, cyclin D1 expressed at early stages of life is capable of compensating for missing cyclin-D2 expression agreeing with the observation that wild type and cyclin D2KO mice have surprisingly similar rates of proliferation and neurogenesis. On the other hand, cyclin D2 is essential for adult proliferation and neurogenesis in the DG and SVZ as detailed in Fig. 2 [45]. In this same work, the authors, based on results of a BrdU incorporation assay, suggested that adult neurogenesis does not occur in mice lacking cyclin D2, while genetic ablation of cyclin D1 does not affect adult neurogenesis [45]. However, the presence of cyclin D1 in the region of adult neurogenesis in the DG was detected [44]. This indicates that cyclin D1 is not capable of compensating for for missing cyclin D2 during adult neurogenesis in the DG. On the other hand, cyclin D1, but not cyclin D2, seems to be essential for neurogenesis in developing mouse spinal cord, as indicated by cyclin D1-selective staining of neuronal precursors. Noteworthy, cyclin D1 regulates neurogenesis in a cell cycle-independent manner, since cyclin-D1 downregulation does not have any effects on cell survival and proliferation [46]. Cyclin D1 is also suggested to be involved in the mechanism, by which insulin-like growth factor (IGF)I mediates proliferation and survival in oligodendrocyte lineage cells [47]. Concurrently, a study using mouse embryonic stem cells induced to neural differentiation, showed that Notch-induced proliferative response is, at least partially, mediated by cyclin D1 [48]. Finally, the supposed role played by cyclin D1 during brain development is again strengthened by the observation that overexpression of Cdk4-cyclin D1 prevents G1 phase lengthening, inhibits neurogenesis and increases NSC proliferation during embryonic mouse development and in the adult mouse brain [25, 27]. It is worth noting a recent work demonstrating that there was a significant reduction of BrdU+ cells in the subgranular zone of the DG and SVZ of cyclin D1-KO mice compared with cyclin D1(+/+) and cyclin D1(+/−) mice, indicative of decreased proliferation of NSCs in the absence of cyclin D1 expression. The authors of this study also reported that cyclin-D1 KO led to apoptosis of NSCs and inhibited differentiation into astrocytes without affecting the differentiation into neurons [49]. As plausible explanation for this observation, NSCs committed to a neural fate mostly express cyclin D2. Indeed, it has already been observed that those few cells

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Retinoblastoma-associated protein (pRb) and E2f transcription factors The ability of pRb to regulate transcription, and consequently cell proliferation and differentiation, is controlled by the activity of Cdk-cyclin complexes. As previously mentioned phosphorylation of pRB by Cdk-cyclin complexes prevents it from binding to E2f transcription factors and from inhibiting the E2f-mediated expression of genes required for S-phase entry and for DNA synthesis, resulting in cell growth [13, 14]. In mammalian cells, pRb belongs to a family of three proteins that also includes p107 and p130, which are structurally and functionally related to pRb and belong to the same cellular pathway, but display distinct functions from pRb in specific contexts [50]. It is known that pRb is expressed in the

developing nervous system from E8.5 to E17.5 throughout the ventricular and intermediate zones of the cortex. Expression of p107 overlaps with that of pRb in the ventricular zone, while expression of p130 is more diffuse [51]. While mice deficient in pRb die between days 13 and 15 of gestation, making impossible any study on the role of pRb in adult neurogenesis, p107(−/−) or p130(−/−) mice 3 develop normally and exhibit no obvious adult phenotype, suggesting the existence of compensatory mechanisms among Rb-family members during development [52]. Despite the importance of Rb proteins in brain development, evidences point to them as regulators rather than essential components of the differentiation program, since neural lineage cells derived from triple KO-mouse embryos are capable of exiting the cell cycle in G0/G1 and differentiating into neurons, as indicated by the expression of markers of postmitotic neuronal cells such as MAP2, Tuj1, and NF200

Fig. 2 Schematic presentation of cell cycle control and NSC proliferation and differentiation in adulthood. a Two neurogenic regions exist in the adult brain: the DG of the hippocampus and the SVZ of the lateral ventricles. b As discussed in the text, deficiency of Cdk6, cyclins D1 and D2, and E2f1 decreases NSC proliferation in the adult DG. On the other hand, deficiency of cyclin D1 and D2 expression impairs NSC differentiation into astrocytes and neurons, respectively. Deficiency of p21Cip1 and p27Kip1 increases NSC proliferation and simultaneously differentiation into neurons. Cdk2-deficient animals show impaired differentiation into oligodendrocytes. c In the SVZ, deficiency of Cdk2/6, cyclin D1/D2, and E2f1 impairs NSC proliferation, similarly to what occurs in the DG, except by the fact that Cdk2 is also important in the SVZ. Deficient p21Cip1 and p107 expression, on the other hand,

increases NSC proliferation. Deficiency of p16Ink4a results in similar effects on NSC proliferation, agreeing with the observation that p16lnk4a expression augments with age in several tissues, including the brain, which might be associated with lower regenerative capacity. Interestingly, deficiency of p27Kip1 expression increases type-C NSC proliferation and consequently reduces the number of neuroblasts. The effects induced by insufficient cyclin D1 and D2 and Cdk2 expression on differentiation into astrocytes, neurons, and oligodendrocytes, respectively, are similar to those ones observed in the DG. NPC, neural progenitor cell; GPC, glial progenitor cell; N, neuron; A, astrocyte, O, oligodendrocyte. Arrows with solid or dashed liner indicate proliferation or differentiation, respectively (+) indicates an increase in proliferation or differentiation, while (-) indicates the opposite effect

generated within the adult DG of mice lacking cyclin D2 belong to the astroglial lineage [45].


[53]. Such regulatory role is sustained by a body of evidences (see also Fig. 3 for a summary of these results). For instance, p107 is highly expressed in NSCs of the embryonic ventricular zone in adult mice and p107(−/−) mice had 50% more constitutively proliferating cells than littermate controls as determined by BrdU labeling [54]. It was also found that p107-mediated regulation of neural precursor number occurs through repression of Hes1 transcription, a downstream mediator of the Notch-signaling pathway, and strikingly, p107 deficiency led to a decreased number of matures neurons, as demonstrated by results of immunohistochemistry studies with NeuN, a marker for mature CNS neurons, and a reduction in the overall size/thickness of the cortex. It was later suggested that this decrease is not caused by increased cell death of committed neurons, but by apoptosis elevated in uncommitted progenitor cells, which is in accordance with previous work [55]. Moreover, short- and long-term BrdUlabeling studies revealed a striking defect in the rate at which p107(−/−) progenitors commit to a neuronal fate, indicating that p107 regulates the transition from proliferating neural progenitor cells to committed neuroblast [56]. Although little is known about the role played by p130 in adult neurogenesis, this protein is thought to promote differentiation of NSCs and maintain neurons in a differentiated state [57]. In addition to controlling cell cycle-related processes, Rb proteins are also involved in regulating other events, which are equally important for adequate nervous system development. Rb is indicated, for instance, to mediate neuronal migration throughout cortex development in a cell cycle-independent mechanism, controlling the expression of genes such as neogenin, which encodes a receptor involved in cell migration and axon guidance [58]. Finally, a work by Mclear and colleagues is noteworthy to mention, in which the authors used a different strategy to inactivate all three Rb family proteins, allowing assessing the role of these proteins in NSCs during early phases of CNS development. Basically, transgenic mice were generated, in which T121, the N-terminal fragment of SV40 large T antigen binding to and inactivating Rb-family proteins, was expressed under the nestin gene control after exposure to the cyclic-AMP response element in GFAPpositive neuroprogenitors. Since nestin is expressed only in NSCs, inactivation of pRb, p107 and p130 is restricted to these precursors, being not any more present following differentiation into neural and glial lineages. Consequently, mice are viable, in some cases until adulthood, permitting the assessment of both developmental defects and subsequent morphological and behavioral consequences. As results, an increase in proliferation and apoptosis was observed in the telencephalon, as well as cellular disorganization of the cerebral cortex and cerebellum and behavioral abnormalities [59]. In mammalians, there are eight E2f members divided into four groups. The first one is composed by E2f1-3

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functioning as transcriptional activators. The second one includes E2f4 and E2f5 and is often referred to as the suppressive E2fs. E2f6-8 represent the two remaining groups and also function as transcriptional repressors; however, they are considered as atypical E2fs, as they are not regulated by the Rb family of proteins [50]. The analysis of transcriptional factor expression during mouse brain development, from the gestational day 10 until adulthood, revealed that several expression of specific isoforms of E2f1, E2f3 and E2f5 was increased while expression of most species of E2f factors were down-regulated, pointing at a role of these factors in differentiation and maturation of the nervous system [60]. In agreement, E2f1 is the most expressed E2f factor in the developing nervous system [61]. Even though mice lacking the E2f1 factor have revealed normal brain development, suggesting the existence of compensatory mechanisms among E2f factors, these animals showed significantly decreased stem cell and progenitor division in the proliferative zones of the lateral ventricle wall and the hippocampus in adulthood, which impaired the production of newborn neurons in the adult OB and DG [62]. Moreover, there is evidence that Rb proteins regulate E2f1 and E2f3 factors during NSC differentiation, as indicated by the fact that Rb-deficient mice exhibit a significant enhancement of E2f1 and E2f3 activities throughout differentiation concomitant with the aberrant expression of E2Finducible genes [63]. As stated earlier, mice lacking the E2f1 factor show normal brain development, indicating that compensatory mechanisms among E2f factors exist. In this sense, E2f3a and b are reported to support E2f target-gene expression and cell proliferation in the absence of other E2F activators, such as E2f1 and E2f2 [64]. Not only Rb proteins, but also many mitogenic and trophic factors are involved in neurogenesis and supposedly regulate E2f factor activities. For instance, BrdU incorporation into NSCs in vitro and in the adult rat brain in vivo, as an index of cell proliferation, demonstrated that vascular endothelial cell growth factor (VEGF) exerts its proliferative effect through up-regulation of E2f family transcription factors, in part by increasing nuclear expression of E2f1, E2f2, and E2f3, being consistent with regulation of the G1/S phase transition [65]. At the same time, the E2f factor could bind to the trophic factor promoter and regulate its expression and consequently the growth factor responsiveness. Indeed, the p107/E2f3 pathway has already been reported to control the pool of FGF-responsive NSCs by directly acting on FGF2 gene expression in vivo [66]. The proliferative effect promoted by E2f3 is such pronounced that it has to be degraded by the anaphase-promoting complex/ cyclosome and its activator Cdh1 (APC/C(Cdh1)), as cells withdraw from the cell cycle [67]. It has already been mentioned that Rb exhibits roles beyond cell cycle regulation during nervous system development; it also mediates migration in vivo, a role that is specifically dependent on E2f3 activity [58, 68].

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Fig. 3 Cell cycle component expression and requirement during embryonic brain development. Cdk1 is the only Cdk required for adequate embryonic development, since suppression of its expression causes mice to fail in reaching morula and blastocyst stages, while mouse embryos lacking all the interphase Cdks only develop to midgestation. Cyclin D1 is expressed mainly at early stages of the brain development, and its expression decreases progressively till adulthood, being replaced by cyclin D2, whose expression is essential for adult NSC proliferation and neurogenesis in the DG and SVZ. pRb family proteins are also differently expressed throughout SNC development. While pRb is

expressed from E8.5 to E17.5 and crucial for embryo viability, p107 expression deceases till adulthood, while p130 reaches maximal expression at around E14 and maintains at constant expression levels. The pRb family regulates the transition from proliferation into differentiation, and protects NSCs from apoptosis. E2f members show a similar expression pattern, with some of them (E2f2/4/6) decreasing and others (E2f1/3/5) increasing along brain development. While p21Cip1 expression augments from E12.5 to 20.5, p57Kip2 plays a role in early and middle phase of corticogenesis, and p27Kip1 participates in middle and late corticogenesis

Cdk inhibitors (CKIs) The four members of the Ink4 family (p16lnk4a, p15lnk4b, p18lnk4c and p19lnk4d) bind to the isolated Cdk and prevent its association with the cyclin and thus its activation. However, they can also bind to and inhibit the Cdk-cyclin complex without dissociating from the cyclin, suggesting that they may have multiple mechanisms of action. The lnk4 inhibitors inhibit specifically G1-phase Cdks [16]. The expression of lnk4 members varies during mouse development and aging, with p16lnk4a expression increasing with age in several tissues, including brain [69, 70]. Indeed, it is well known that aging is related to reduced regenerative capacity in tissues containing stem cells, which could be explained by increased expression of cell cycle inhibitors [71]. For instance, reduced NSC proliferation in the SVZ and neurogenesis in the OB in the forebrain of ageing mouse

correlates with increased expression of p16Ink4a. Inversely, ageing mice lacking p16lnk4a show a significantly smaller decline in SVZ proliferation and OB neurogenesis. On the other hand, proliferation and neurogenesis in the DG is not affected by such deficiency, suggesting regional differences in the cell cycle machinery controlling proliferation and neurogenesis [70]. It should be stressed that some transcriptional factors associated with the maintenance of the NSC self-renewal capacity decrease p16Ink4a expression [72]. The three members of the Cip/Kip family (p21cip1, p27Kip1 and p57Kip2) bind and inhibit the active Cdk-cyclin complex and have a broader Cdk preference when compared to Ink4 inhibitors [16]. Using human neuroblastoma cells, Liu and coworkers demonstrated that the retinoic acid-induced differentiation into neurons caused a tenfold increase in p21cip1 protein


levels [73]. It has also been reported that the exogenously expressed homeodomain transcription factor Pitx2 in NSCs isolated from embryonic cortex causes a rapid decrease in proliferation associated with a rapid and dramatic 20-fold increase in expression p21Cip1 and accumulation of NSCs in G1 phase. It was found that Pitx2 increases p21Cip1 expression by binding directly to its promoter, as investigated by chromatin immunoprecipitation (ChIP) [74]. Interestingly, the mood stabilizer, valproic acid, inhibited proliferation and induced differentiation of NSCs derived from cerebral cortex of developing rat embryos into neurons, as assessed by BrdU incorporation and Tuj1 expression, a specific marker of immature neurons, respectively. These effects occurred together with increased expression of p21Cip1 [75]. The Cdk inhibitor p21Cip1 is expressed by NSCs in the subgranular zone of the DG of the hippocampus in early neuronal progenitors and in immature neurons, but not in mature neurons or astroglia, in mice. More specifically, confocal microscopy and flow cytometry analysis revealed that p21Cip1 is abundantly expressed in the nuclei of cells of the DG. There it is co-localized with NeuN and among doublecortin+ (DCX+) cells, 42.8% were positive for p21Cip1, indicating that p21Cip1 is expressed in neuroblasts and in newly developing neurons. p21Cip1 is also expressed in hippocampal-derived NSCs in vitro. NSCs derived from p21Cip1-deficient mice proliferate to a higher extent than those derived from wild-type mice. In agreement with these results, the transient suppression of p21Cip1 expression by transfecting NSCs with a shRNA against p21Cip1 leads to increased proliferation. Furthermore, similar to those results obtained with valproic acid, the treatment of mice with some antidepressants, such as fluoxetine, imipramine and desipramine, decreases p21Cip1 expression, and consequently increases hippocampal neurogenesis [76, 77]. It was also observed that the loss of p21Cip1 induces initially the post-natal proliferation of NSCs derived from the mammalian forebrain. This increase leads, in turn, to exhaustion of NSCs in aging mice, impairing their self-renewal capacity. Therefore, p21Cip1 supposedly contributes to the relative quiescence of adult NSCs and, consequently, to the maintenance of NCS selfrenewal and their population [78]. The expression of p27Kip1 increases throughout the differentiation of mouse embryonic stem cells. It was also found that the absence of p27Kip1 augments the neural pool, without affecting its differentiation, as indicated by results of western-blot analysis of neural markers N-CAM (neuron/ glia-specific), GAP-43 (neuron/glia-specific), and GFAP (astrocyte-specific) [79]. The Phox2a-induced transcription of p27Kip1 is related to the coordination of cell cycle exit and differentiation. Phox2a is a homeodomain transcription factor required for CNS- and neural crest-derived noradrenergic neuron differentiation. Small interfering RNA-mediated silencing of p27Kip1 suppresses p27Kip1 transcription and neuronal differentiation, suggesting a link between p27Kip1

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expression and differentiation [80]. We have already mentioned a recent work showing that Cdk5 phosphorylation of p27Kip1 is crucial for neurite outgrowth and neural differentiation [36]. Altogether, these works demonstrate the importance of p27Kip1 for the developing nervous system, even though in most of the studies the role of this CKI during postnatal and adult life brain development has been investigated. Post-natal analysis of mice lacking p19Inkd and p27Kip1 revealed that subpopulations of CNS neurons proliferated in all parts of the brain, including normally dormant cells of the hippocampus, cortex, hypothalamus, pons, and brainstem, indicating that p27Kip1 cooperates with p19Inkd to maintain differentiated neurons in a quiescent state [81]. Curiously, p27Kip1 is the only member of the Kip family expressed in the SVZ and its absence has been reported to increase BrdU incorporation compared with WT mice [82, 83]. It should be said that the loss of p27Kip1 resulted in selective increase of transit-amplifying precursors (type-C cells), concomitant with a decrease in the number of neuroblasts (type-A cells). The number of more quiescent precursors (type-B cells) was not affected. This signifies that loss of p27Kip1 enhances proliferation of type C cells and not of B cells or A neuroblasts, as shown by the increased number of [3H]thymidine- or BrdU-positive C cells and by the increase in the total number of type-C cells [83]. As consequence, different SVZ cell types rely on the action of distinct molecules regulating G1/S transition, representing for the majority of eukaryotic cells, the decisional point between exit or re-entry into the cell cycle. Similar results were obtained with NSCs from the adult hippocampal subgranular zone, since the absence of p27Kip1 also increases proliferation in the DG as it does in the SVZ. Neuronalcommitted NSCs seem to be more affected by the lack of p27Kip1. Although deeper investigations are needed for elucidation of underlying mechanisms, it is clear that p27Kip1— deficient mice have more newly born neurons in the DG when compared with their WT littermates [84]. Strikingly, p27Kip1 has been found to promote neurogenesis and radial migration of cortical projection neurons in the mouse cerebral cortex by cell-cycle independent mechanisms; the former, by stabilizing the neurogenin2 protein, and the latter, by blocking RhoA signaling [85]. Finally, p27Kip1 has also been shown to regulate proliferation of oligodendroglial precursors. An increased number of proliferating embryonic glial and neonatal oligodendrocyte progenitor cells in p27Kip1(−/−) mice was observed at the peak of gliogenesis [86]. CKI p57Kip2 is abundantly expressed in the developing embryonic cortex at early and middle stages of corticogenesis, when it causes cell cycle exit, transition from proliferation to neuronal differentiation, and increases neurite outgrowth [87]. This transition from proliferation to differentiation is consistent with its nuclear expression, as precursors exit the cell cycle and begin expressing neuronal characteristics [88]. On

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the other hand, p57Kip2 overexpression at later stages promotes precocious glial differentiation. CKI p57Kip2 is also two-fold more effective than p27Kip1 in inducing neuronal differentiation and it is not responsive to astrogliogenic effects of ciliary neurotrophic factor, indicating that CKIs differentially modulate cell fate decisions [87]. Published data regarding the role of p57Kip2 in regulating glial fate decision are quite conflicting. It has been recently reported that shRNAmediated suppression of p57Kip2 expression in cultured adult neural stem cells strongly reduces astroglial characteristics, while oligodendroglial precursor features increase. This discrepancy is likely to result from the fact that even a lower expression of p57Kip2 is capable of inducing gliogenesis, at the expense of lineage restriction in direction of astrocytes and neurons, as it was speculated for the absence of p27Kip1 [83]. The effects of suppression of expression of a specific cell cycle component on proliferation and differentiation of NSCs are summarized in Fig. 2. How one can observe, requirements for a cell cycle component and correspondent effects are cell-type and region-(DG or SVZ) specific. Differential expression patterns for cell cycle components during brain development are schematized in Fig. 3.

Conclusions In this review, we have highlighted some of the mechanisms linking cell cycle components to cellular processes occurring during brain development and discussed how they can work as key regulators of these processes. In the last few years, our understanding how the cell cycle machinery is used to regulate NSC proliferation and differentiation during brain development and adulthood, has increased. However, several questions still lack answers. For instance, we do not yet know whether Cdk1, the only Cdk required for embryonic development, is crucial for NSC proliferation in the adult brain. Furthermore, the role of pRb in the adult neurogenesis could be investigated by using a strategy similar to that employed by Mclear and colleagues to inactivate all three Rb family proteins, and to assess the role of these proteins in NSCs during early phases of CNS development [59]. However, an interesting approach would be evaluating the effects of silencing of gene expression of pRb in the grown animal for studying its effect in adult neurogenesis. A significant number of cell cycle components seem to be not essential for the organism survival in most conditions due to compensatory mechanisms existing among them. However, the requirement of such factors exists in certain conditions depending on age and the respective cell type. Understanding how NSC differentiation is linked to proliferation and consequently to the cell cycle may be relevant for the treatment of brain disorders associated with abnormal proliferation or failing neuroregenerative capacities,


such as tumors and neurodegenerative diseases, respectively. For example, Cdk6 inhibition by small molecules might be beneficial in the treatment of medulloblastoma [89]. Cell cycle inhibitors function as tumor suppressors, and consequently abnormalities in these proteins can give rise to developmental defects and cancer affecting the CNS [90]. Cell cycle control is also implicated in neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease, and amyotrophic lateral sclerosis [39, 91]. Silencing Cdk5 has drawn attention as a strategy for the treatment of the afore-mentioned diseases [92]. In summary, although many mechanisms still remain to be elucidated concerning the roles of cell cycle components in controlling NSC proliferation and differentiation throughout brain development, much knowledge has already been obtained by identifying more targets for novel treatments of brain diseases. On the other hand, adult NSCs also represent a novel treatment strategy for neurodegenerative diseases in view of cellular therapy. We believe that, in the near future, further studies will allow a more efficient way of manipulating the cell cycle of transplanted and endogenous NSCs for subsequent therapeutic applications. Acknowledgments This work was supported by research grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico (CNPq), Brazil (awarded to H.U.) and the Bundesministerium für Bildung und Forschung, (BMBF), Germany (awarded to A.T); A.C. acknowledges a postdoctoral fellowship from FAPESP. Conflict of Interest The authors indicate no potential conflicts of interest.

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