Adult oligodendrocytes can arise from either white matter OPC or neural progenitor cells from the subventricular zone (SVZ). Therefore a study investigated the function of Cdk2 in the adult subventricular zone and more precisely in chondroitin sulfate proteoglycan NG2-expressing progenitors taking advantage of a viable Cdk2−/− mouse mutant 3738. NG2⁺ cells from the SVZ display cellular properties of transient-amplifying (type C) cells and are able to generate oligodendrocytes in the adult 622. Proliferation of NG2⁺ cells was demonstrated to be selectively impaired in the adult SVZ of mice lacking Cdk2 (Table 1). Importantly, loss of Cdk2 did not affect proliferation of slowly dividing GFAP⁺-nestin⁺ stem cells or PSA-NCAM⁺ neuroblasts 3039 indicating that intrinsic cell cycle regulators play distinct roles in different cell populations of the adult SVZ. Analysis in cultures obtained from adult SVZ NG2⁺ cells is consistent with the in vivo findings, as a significant decrease in the formation of neurospheres was also observed in Cdk2−/− cells 39. Moreover, it appears that Cdk2 might play a specific role in adult neural progenitors because loss of this kinase did not affect proliferation of embryonic fibroblasts or human colon cancer cell lines in culture 373840.

The pathological condition concerns corpus callosum LPC-induced demyelination. SVZ subventricular zone; CC corpus callosum; NO not observed; NC not concerned

The decrease in NG2⁺ cell proliferation observed in the adult SVZ may result from a shift in balance between cell proliferation and differentiation and/or cell death. Caspase-3⁺ apoptotic cells were quantified and no differences were observed in cell death in the absence of Cdk2 at P8 or P90. These results were confirmed by quantification of TUNEL⁺ cells 39. However, cellular and molecular analyses in vivo and in vitro demonstrate that the loss of Cdk2 promotes NG2⁺ cell lineage commitment and differentiation in oligodendrocytes of adult SVZ cells 39 (Table 1). Regarding these results, Cdk2 appears to contribute not only to cell cycle regulation but also to the decision to differentiate. At variance with findings in the adult SVZ, in vivo and in vitro analysis demonstrated that both NG2⁺ cell proliferation and self-renewal capacities were not affected by the loss of the Cdk2 gene up to postnatal day 15 implying during perinatal period compensatory activity of other Cdks which is a well known phenomenon 41. Cdk1 could play this role as in the absence of interphase Cdks (Cdk4, Cdk6 and Cdk2), it can execute all the events that are required to drive mammalian cell division 42. More precisely, in p27−/−; Cdk2−/− double KO mice, Cdk1 compensates the loss of Cdk2 function, binding to cyclin E and regulating G1/S transition 3743. However, probably due to the importance of the genetic locus for Cdk function 44 in specific cell types, Cdk1 is unable to compensate for the loss of Cdk2 in germinal cells as Cdk2−/− mice are sterile 3738. In SVZ protein extracts from Cdk2−/− P8 and P90 mice, Cdk1 expression was evaluated and difference with wild-type mice could not be found. Actually, compensatory mechanisms in perinatal Cdk2−/− SVZ cells, which persist until postnatal day 15, involve increased Cdk4 expression that results in retinoblastoma protein inactivation 39. A subsequent decline in Cdk4 activity to wild-type levels in postnatal day 28 Cdk2−/− cells coincides with lower NG2⁺ proliferation and self-renewal capacity similar to adult levels. Cdk4 compensation was confirmed by silencing experiments in perinatal Cdk2−/− SVZ cells that abolishes Cdk4 up-regulation and reduces cell proliferation and self-renewal to adult levels. Conversely, Cdk4 overexpression in adult SVZ cells restores proliferative capacity to wild-type levels 39. Thus, although Cdk2 is functionally redundant in perinatal SVZ, it is important for adult progenitor cell proliferation and self-renewal, through age-dependent regulation of Cdk4.

The subventricular zone does not constitute the only persistant germinative zone in the adult as granule neurons undergo continuous renewal throughout life in the dentate gyrus (DG) of the hippocampus. Thus, the requirement of Cdk2 has also been investigated in this region using Cdk2 deficient mice 45. Surprisingly, the quantification of cell cycle markers first revealed that the lack of Cdk2 activity does not influence spontaneous or seizure-induced proliferation of neural progenitor cells in the adult DG. Using bromodeoxyuridine incorporation assays, it was shown that the number of mature newborn granule neurons generated de novo was similar in both wild-type and Cdk2-deficient adult mice. Moreover, the apparent lack of cell output reduction in Cdk2−/− mice DG did not result from a reduction in apoptosis of newborn granule cells as analyzed by TUNEL assays 45. So, contrary to its role in NPC proliferation in the adult SVZ, Cdk2 seems to be dispensable for NPC proliferation, differentiation and survival of adult-born DG granule neurons in vivo. This discrepancy could be explained by compensatory mechanisms, which may be region-specific and may reflect the low proliferative potential of hippocampal progenitors, as compared to their SVZ counterpart. Alternatively, these differences may have resulted from a non essential role played by Cdk2 in neurogenic regions of the brain, such as the adult hippocampus, as compared to the SVZ, which is directly involved in oligodendrogenesis, a process that more closely depends on the control of OPC proliferation by Cdk2.

The data described previously indicate that Cdk2 loss does affect neither the number of white matter oligodendrocytes and OPCs, nor their proliferation rate. However, since oligodendrocytes are responsible for myelin sheath synthesis with each cell myelinating up to 40 independent axonal segments 60, loss of Cdk2 could impact CNS myelin synthesis. To explore this possibility, electron microscopy was used but did not reveal any changes in axonal preservation, in the number of myelinated axons, or differences in myelin structure, after Cdk2 loss in the postnatal (P15) or adult (P90) corpus callosum, since myelin compaction and g ratio did not significantly differ in Cdk2−/− and WT mice 30. In addition, Western blot quantification of MBP, one of the most abundant CNS myelin protein 61, showed no difference in its levels between the two genotypes. Taken together, these data suggest that myelination during early postnatal development is either Cdk2-independent or is efficiently compensated by other Cdks (Table 1).

Although some OPCs are still produced in the adult from SVZ cells 102262, decreased proliferation of these cells under physiological conditions in the absence of Cdk2 39 seems sufficient to maintain a normal density of oligodendroglial cells in the corpus callosum. However, when challenged with demyelination induced by LPC injection, a process known to enhance proliferation of resident adult OPCs and SVZ precursors 1821, cell proliferation was found to be reduced not only in the SVZ, but also in the lesion of Cdk2−/− mice 30 (Table 1). These alterations were not an indirect consequence of axonal loss, as no obvious alteration in axons and/or in axonal density, were observed by electron microscopy. Since decreased proliferation specifically affected cells giving rise to oligodendrocytes, including OPCs and type C cells of the SVZ, rather than microglia and astrocytes, the consequences of altered proliferation on OPC differentiation into oligodendrocytes were investigated. Reduced proliferation was shown to be correlated with enhanced cell cycle exit in adult OPCs of Cdk2 −/− mice, as compared to wild-type, as well as with enhanced differentiation of adult OPCs into differentiated CC1⁺ oligodendrocytes 30. Interestingly, while NG2⁺ OPC densities were identical in the corpus callosum of wild-type and Cdk2−/− non-injured mice, their reduced proliferation at 7 days post injection (dpi) coincided with a reduction in OPC density in Cdk2−/− mice, as compared to wild-types. In contrast, CC1⁺ oligodendrocyte density increased in Cdk2−/− mice 1 and 2 weeks later, a delay corresponding to the onset of remyelination and the oligodendrocyte/myelin regeneration period in LPC lesions, respectively. However, differences between Cdk2−/− mice and wild-types disappeared by 21 days, when the process of repair was nearly accomplished 30. Moreover, increased cell cycle exit and oligodendrocyte differentiation in Cdk2−/− mice had a direct impact on the number of remyelinated axons, and on the extent of axonal remyelination, as assessed by g ratio measurements in the lesion (Table 1). The fact that remyelination in wild-type mice levelled with that of Cdk2−/− mice at 21 dpi further underlines that Cdk2 acts as a modulator rather than as an on-off switch of the differentiation/remyelination process. These results are consistent with previous in vitro findings showing that Cdk2 loss decreased OPC proliferation 29 and promoted lineage commitment of NG2-expressing progenitors in the adult SVZ, and differentiation of adult neural progenitor cells 39. In view of these studies, Cdk2 appears as an intrinsic molecular mechanism that controls the timing of oligodendrocyte differentiation in vivo. This conclusion is supported by the fact that accelerated differentiation of oligodendroglial cells in the absence of Cdk2 occurred also in vitro30.

Requirement for Cdk2 in adult OPCs could contribute to a better understanding of remyelination failure in multiple sclerosis. In this inflammatory disease, endogenous remyelination exists, but is not efficient in all lesions. Among several possible hypotheses, dysregulation of OPC proliferation has been proposed to play a role in remyelination failure, as demonstrated by reduced OPC proliferation in some lesions, although in others OPCs are numerous, but appear unable to fully differentiate 63. A previous study also demonstrated that persistent CNS inflammation significantly altered cell kinetics of precursors and stem cells in experimental autoimmune encephalomyelitis, a model of multiple sclerosis 64. In view of the above mentioned data 3039, it would be of interest to investigate whether Cdk2 expression is altered in MS and could explain some of the default of remyelination observed in MS lesions.

Differential requirement of another Cdk, Cdk4, for myelination/remyelination processes, was also described in the PNS. Cell cycle function of Cdk4 is closely related to Cdk2, as both Cdk2 and Cdk4 are activated by their regulatory proteins cyclin E and D, respectively, to sequentially phosphorylate Rb. Although Cdk2−/− or Cdk4−/− mice are viable, Cdk2−/−; Cdk4−/− double KO mice die in utero around E15 65. Peripheral myelin formation depends on axonal signals that tightly control proliferation and differentiation of the associated Schwann cells 66. Requirement for three different Cdks was analyzed in Schwann cells using Cdk-deficient mice 67. Mice lacking Cdk4 showed a drastic decrease in the proliferation rate of Schwann cells at postnatal days 2 and 5. However, proliferation was unaffected at embryonic day 18 which could be due to compensatory mechanisms that are effective during early development. In contrast, ablation of Cdk2 and Cdk6 had no significant influence on postnatal Schwann cell proliferation. Taken together, these findings indicate that postnatal Schwann cell proliferation is uniquely controlled by Cdk4. Despite the lack of the postnatal wave of Schwann cell proliferation, axons were normally myelinated in adult Cdk4-deficient sciatic nerves. Following nerve injury, Schwann cells lacking Cdk4 were unable to re-enter the cell cycle, while Schwann cells deficient in Cdk2 or Cdk6 displayed proliferation rates comparable to controls 67. No compensatory effects such as elevated Cdk4 levels were observed in uninjured or injured nerves of Cdk2- or Cdk6-deficient mice. So requirement for Cdk4 in Schwann cells, as requirement for Cdk2 in OPCs, seems to be different during developmental myelination compared to pathological event.

Periventricular white matter injury (PWMI) is a common form of brain injury affecting preterm infants. A major factor that predisposes to PWMI is hypoxia. Influences of hypoxia on oligodendrocytes development were investigated in vitro by maintaining purified cultures of OPCs at 21% O₂ or hypoxia (1% or 4% O₂) for up to 7 days 68. 1% O₂ led to an increase in the proportion of myelin basic protein (MBP)-positive oligodendrocytes after 1 week in culture, and a decrease in the proportion of PDGFRα+ cells suggesting premature oligodendrocyte maturation. Increased expression of the cell cycle regulatory proteins p27^Kip1 and phospho-Cdk1 was seen as well. Hypoxia appears to interfere with the normal process of oligodendrocyte differentiation by inducing premature OPC maturation that may contribute to hypomyelination in the developing hypoxic brain. The specific role of Cdk2 was not investigated in these studies but giving the role of this kinase in oligodendrocytes, we can suppose that the premature OPC maturation observed in response to increased expression of p27^Kip1 involves Cdk2 increased inhibition and premature cell cycle exit. The role of Cdk2 in response to hypoxia has been studied in other cell types and it appears that Cdk2 when associated with cyclin A, is involved in hypoxia-induced apoptosis in cardiomyocytes 69. Indeed, some investigators have interpreted the rapid induction of Cdk2 activity in cardiomyocytes as evidence of post-mitotic cells reentering the cell cycle and undergoing proliferative growth. However, an increasing amount of data indicates that proteins classically thought to be involved in cell cycle regulation also play a critical role in the control of cell death, particularly through apoptotic pathways. Numerous stimuli unrelated to cell cycle progression induce Cdk2 activity and subsequently apoptosis such as irradiation 70, Tumor necrosis factor-α 71, Fas 72, staurosporine 71, hypoxia 6973, and ischemia 74. Although Cdk2 activity is elevated during apoptosis, activation of the mitotic processes does not occur even in cells capable of proliferating 72. Likewise, perturbations of cell cycle progression, without affecting Cdk2 activity, fail to block apoptosis 75 supporting the notion that Cdk2 activation is not simply indicative of aberrant mitosis but, instead, that Cdk2 plays a direct role in cell death.

After transient focal cerebral ischemia, cyclin Dl and Cdk4 were selectively expressed in morphologically intact or altered oligodendrocytes and neurons localized to the ischemic tissue. Apoptotic cells were not immunoreactive to these proteins at 46 hours of reperfusion after 2 hours of middle cerebral artery occlusion (I/R injury). However, cyclin D1 and Cdk4 induction is not involved in the onset of astrocyte and microglial cell proliferation in response to ischemia 76. p21 protein levels in oligodendrocytes and neurons were also found to gradually increase to 250% and 140% of contralateral levels in areas bordering the infarct core up to 6 h following middle cerebral artery occlusion whereas a low level of constitutively p21 was found in nuclei of these cells. In contrast, p53, p27 and cyclin E levels decrease in the infarct core and border areas with time after middle cerebral artery occlusion. The selective up-regulation of p21 in the border zone of a focal ischemic infarct indicates its involvement in an adaptive response to ischemic injury 77. The potential contribution of Cdk2 to oligodendrocyte survival in response to cerebral ischemia was not investigated in these studies but was observed in other cell type. Indeed, myocardial ischemia activates Cdk2, resulting in the phosphorylation and inactivation of Rb. Blocking Cdk2 activity reduces apoptosis in cultured cardiac myocytes. Genetic or pharmacological inhibition of Cdk2 activity in vivo during I/R injury led to a 36% reduction in infarct size, when compared to control mice, associated with a reduction in apoptotic myocytes 78. These data suggest that Cdk2 signaling pathways are critical regulators of cardiac I/R injury in vivo whereas the loss of Cdk2 was shown to have no effect on apoptosis consecutive to focal demyelination in the adult brain 30 confirming the cell specific role of Cdk2.

Traumatic spinal cord injury (SCI) evokes a complex cascade of events with initial mechanical damage leading to secondary injury processes that contribute to further tissue loss and functional impairment. Growing evidence suggests that the cell cycle is activated following SCI 798081. Expression of key cell cycle activator genes, such as cyclin D, Cdk4 and Rb were up-regulated at the early time points (i.e., 4 h and 24 h) after SCI 82. Upregulation of cell cycle proteins after injury appears to contribute not only to apoptotic cell death of postmitotic cells, including neurons and oligodendrocytes, but also to posttraumatic gliosis and microglial activation. Inhibition of key cell cycle regulatory pathways reduces injury-induced cell death, as well as microglial and astroglial proliferation both in vitro and in vivo. Treatment with cell cycle pharmacological or endogenous inhibitors like p21 in rodent SCI models prevents neuronal cell death and reduces inflammation, as well as the surrounding glial scar, resulting in markedly reduced lesion volumes and improved motor recovery 82. In a recent study, Cdk2 and cyclin H protein expression and their association were demonstrated to be increased in proliferating microglia and astrocytes in a rat spinal cord contusion model 83 whereas loss of Cdk2 did not affect proliferation of these cells in response to focal demyelination of the adult brain 30. Cyclin H regulates cell cycle transitions by forming trimeric Cdk-activating kinase (CAK) complex with Cdk7 and MAT1 that phosphorylates and activates Cdk2. So Cdk2 and cyclin H may play a proliferative role in spinal cord injury.