Roles of p53 and p27(Kip1) in the regulation of neurogenesis in the murine adult subventricular zone.

The tumor suppressor protein p53 (Trp53) and the cell cycle inhibitor p27(Kip1) (Cdknb1) have both been implicated in regulating proliferation of adult subventricular zone (aSVZ) cells. We previously reported that genetic ablation of Trp53 (Trp53-/-) or Cdknb1 (p27(Kip1-/-) ) increased proliferation of cells in the aSVZ, but differentially affected the number of adult born neuroblasts. We therefore hypothesized that these molecules might play non-redundant roles. To test this hypothesis we generated mice lacking both genes (Trp53-/- ;p27(Kip1-/-) ) and analysed the consequences on aSVZ cells and adult neuroblasts. Proliferation and self-renewal of cultured aSVZ cells were increased in the double mutants compared with control, but the mice did not develop spontaneous brain tumors. In contrast, the number of adult-born neuroblasts in the double mutants was similar to wild-type animals and suggested a complementation of the p27(Kip1-/-) phenotype due to loss of Trp53. Cellular differences detected in the aSVZ correlated with cellular changes in the olfactory bulb and behavioral data on novel odor recognition. The exploration time for new odors was reduced in p27(Kip1-/-) mice, increased in Trp53-/- mice and normalized in the double Trp53-/- ;p27(Kip1-/-) mutants. At the molecular level, Trp53-/- aSVZ cells were characterized by higher levels of NeuroD and Math3 and by the ability to generate neurons more readily. In contrast, p27(Kip1-/-) cells generated fewer neurons, due to enhanced proteasomal degradation of pro-neural transcription factors. Together, these results suggest that p27(Kip1) and p53 function non-redundantly to modulate proliferation and self-renewal of aSVZ cells and antagonistically in regulating adult neurogenesis.

Loss of p53 induces changes in the behavior of subventricular zone cells: implication for the genesis of glial tumors.

The role of multipotential progenitors and neural stem cells in the adult subventricular zone (SVZ) as cell-of-origin of glioblastoma has been suggested by studies on human tumors and transgenic mice. However, it is still unknown whether glial tumors are generated by all of the heterogeneous SVZ cell types or only by specific subpopulations of cells. It has been proposed that transformation could result from lack of apoptosis and increased self-renewal, but the definition of the properties leading to neoplastic transformation of SVZ cells are still elusive. This study addresses these questions in mice carrying the deletion of p53, a tumor-suppressor gene expressed in the SVZ. We show here that, although loss of p53 by itself is not sufficient for tumor formation, it provides a proliferative advantage to the slow- and fast-proliferating subventricular zone (SVZ) populations associated with their rapid differentiation. This results in areas of increased cell density that are distributed along the walls of the lateral ventricles and often associated with increased p53-independent apoptosis. Transformation occurs when loss of p53 is associated with a mutagenic stimulus and is characterized by dramatic changes in the properties of the quiescent adult SVZ cells, including enhanced self-renewal, recruitment to the fast-proliferating compartment, and impaired differentiation. Together, these findings provide a cellular mechanism for how the slow-proliferating SVZ cells can give rise to glial tumors in the adult brain.

Combinatorial profiles of oligodendrocyte-selective classes of transcriptional regulators differentially modulate myelin basic protein gene expression.

Recent studies suggest that specific neural basic helix-loop-helix (HLH; i.e., Olig1 and Olig2, Mash1), associated inhibitory HLH (i.e., Id2 and Id4), high-mobility group domain (i.e., Sox10), and homeodomain (i.e., Nkx2.2) transcription factors are involved in oligodendrocyte (OL) lineage specification and progressive stages of maturation including myelination. However, the developmental interplay among these lineage-selective determinants, in a cell- and maturational stage-specific context, has not yet been defined. We show here in vivo and in vitro developmental expression profiles for these distinct classes of transcriptional regulators of OLs. We show that progressive stages of OL lineage maturation are characterized by dynamic changes in the subcellular distribution of these transcription factors and by different permutations of combinatorial transcriptional codes. Transient transfections of these precise combinatorial codes with a luciferase reporter gene driven by the myelin basic protein promoter define how changes in the molecular composition of these transcriptional complexes modulate myelin gene expression. Our overall findings suggest that the dynamic interplay between developmental stage-specific classes of transcriptional activators and associated inhibitory factors orchestrate myelin gene expression during terminal maturation of the mammalian CNS.

Histone deacetylase activity is necessary for oligodendrocyte lineage progression.

Gene expression can be modulated by chromatin changes induced by histone acetylation and deacetylation. Acetylation of histone lysine residues by acetyltransferases is associated with transcriptionally active chromatin, whereas the removal of acetyl groups by histone deacetylases (HDACs) correlates with repressed chromatin. Recent evidence has shown that histone deacetylation is responsible for restricting neuronal gene expression, whereas histone acetylation is necessary for astrocytic differentiation We now asked whether histone acetylation or deacetylation was necessary for oligodendrocyte differentiation. Neonatal rat cortical progenitors were kept proliferating and undifferentiated in the presence of mitogens and induced to stop proliferating and differentiate into oligodendrocytes by mitogen removal. Histone deacetylation was observed during the temporal window between exit from the cell cycle and onset of differentiation, which was characterized by acquisition of branched morphology and myelin gene expression. Blocking HDAC activity during this critical window using the inhibitor trichostatin A (TSA) prevented the progression of progenitors into mature oligodendrocytes. TSA-treated progenitors were able to exit from the cell cycle but did not progress to oligodendrocytes. Their development was arrested at the progenitor stage, characterized by simple morphology and lack of myelin gene expression. The effect of TSA on progenitor differentiation was lineage specific, because TSA did not affect the ability of these cells to differentiate into type II astrocytes when cultured in the presence of serum. From these data, we conclude that histone deacetylation is a necessary component of the oligodendrocyte differentiation program.

Multiple roles of Id4 in developmental myelination: predicted outcomes and unexpected findings.

Myelination in the central nervous system is a complex process requiring the integration of oligodendrocyte progenitor differentiation and the coordinate expression of myelin genes. This study addresses the role of the helix-loop-helix protein Id4 in these two events. Overexpression of Id4 in oligodendrocyte progenitors prevents differentiation and consequently decreases the endogenous expression of all myelin genes. Conversely, progenitors lacking Id4 display precocious differentiation both in vitro and in vivo, and this phenotype is partially compensated by increased apoptosis. Besides this role, Id4 also has the ability to decrease the activity of specific myelin promoters, since Id4 overexpression decreases the activity of luciferase reporter genes driven by the ceramide galactosyltransferase (CGT) or myelin basic protein (MBP) promoter, but not by a myelin proteolipid protein (PLP) promoter. Consistent with these results, the expression levels of MBP and CGT are greater in neonatal Id4 null mice when compared with wild-type siblings and correlate with the early detection of MBP immunoreactive myelinated fibers. In contrast, the levels of other myelin proteins, such as PLP and myelin associated glycoprotein (MAG) are decreased in the Id4 null mice. MAG expression is localized to the soma rather than the fibers of immunoreactive cells in the neonatal brain and compensated at later developmental stages. These data support the role of Id4 as oligodendrocyte differentiation inhibitor with the ability to differentially regulate the expression and subcellular distribution of myelin gene products.

A molecular insight of Hes5-dependent inhibition of myelin gene expression: old partners and new players.

This study identifies novel mechanisms of Hes5 function in developmental myelination. We report here upregulation of myelin gene expression in Hes5-/- mice compared to wild-type siblings and downregulation in overexpressing progenitors. This effect was only partially explained by the ability to regulate the levels of Mash1 and bind to N boxes in myelin promoters, as deletion of the DNA-binding domain of Hes5 did not suppress its inhibitory role on myelin gene expression. Novel mechanisms of Hes5 function in the oligodendrocyte lineage include the regulation of feedback loops with the cell-specific transcriptional activator Sox10. In progenitors with low levels of Sox10, Hes5 further decreases the bioavailability of this protein by transcriptional inhibition and direct sequestration of this activator. Increasing levels of Sox10 in progenitors, in turn, bind to Hes5 and titrate out its inhibitory effect by sequestration and displacement of the repressive complexes from myelin promoters. Thus, Hes5-dependent modulation of myelin gene expression involves old players (i.e. Mash1) and novel mechanisms of transcriptional regulation that include cell-specific regulatory loops with transcriptional activators (i.e. Sox10).

S-phase entry of oligodendrocyte lineage cells is associated with increased levels of p21Cip1.

The mechanisms regulating the number of myelinating cells in the central nervous system are crucial for both normal development and repair in pathological conditions. Among relevant growth factors involved in this process, fibroblast growth factor-2 (FGF2) induces oligodendrocyte progenitors (OLPs) to proliferate and stimulates mature oligodendrocytes (OLs) to reenter the S-phase of the cell cycle. S-phase entry is modulated by the formation of complexes between cyclins and cyclin-dependent kinases (CDKs), on one hand, and by their interactions with cell cycle inhibitors (e.g., p18INK, p27Kip1, p21Cip1), on the other. Although the roles of cyclin E/CDK2 complexes and the inhibitor p27Kip1 have been extensively investigated relative to proliferation and differentiation in the OL lineage, less is known about the regulation of the formation of cyclin D1/CDK4 complexes and the role of p21Cip1 in these events. In this study, we show that the FGF2-mediated increase in bromodeoxyuridine (BrdU) incorporation into OL progenitors and mature OLs occurs concomitantly with increase in the levels of p21Cip1 and the formation of p21Cip1/cyclin D1/CDK4 ternary complexes. These complexes are functionally active is indicated by the ensuing FGF2-dependent hyperphosphorylation of the downstream target Rb. In untreated mature OLs that do not incorporate BrdU, the levels of p21Cip1 are low, and the level of the inhibitor p18INK is high. Furthermore, p18INK sequesters CDK2 into binary complexes, precluding the formation of p21Cip1/cyclin D1/CDK4 ternary complexes in these cells. Therefore, we propose that p21Cip1 is acting as a positive regulator, rather than an inhibitor, of cell cycle entry by favoring the assembly of active cyclin D1/CDK4 complexes.

Oligodendrocyte progenitor proliferation and maturation is differentially regulated by male and female sex steroid hormones.

Using primary cultures of oligodendrocyte progenitors isolated from male and female neonatal rodent brains, we observed more oligodendrocytes in female-derived compared to male-derived cultures. To determine whether the observed differences were due to a differential effect of sex hormones on proliferation, we treated cultures with increasing doses of 17beta-estradiol, testosterone or progesterone and labeled cells with bromodeoxyuridine to identify cells in S phase. Treatment with 17beta-estradiol, but not progesterone or testosterone, delayed the exit of oligodendrocyte progenitor cells from the cell cycle. In addition, 17beta-estradiol treatment enhanced membrane sheet formation, while progesterone increased cellular branching. Interestingly, the estrogen modulator tamoxifen mimicked the effect of 17beta-estradiol on cell cycle exit, but not on membrane formation. Immunocytochemical localization of estrogen receptors (ERs) showed ERbeta mainly localized to the cytoplasm of oligodendrocytes, suggesting that the effect of 17beta-estradiol on membrane formation could be mediated by interaction with this receptor. We conclude that sex steroids differentially regulate oligodendrocyte progenitor number and myelin formation, possibly contributing to gender-specific differences in repair.

Oligodendrocyte process outgrowth in vitro is modulated by epigenetic regulation of cytoskeletal severing proteins.

Process outgrowth is crucial in oligodendrocyte (OL) development and myelination. It is well accepted that increased levels of proteins affecting the polymerization of cytoskeletal components promote branching. Interestingly, we have suggested that other mechanisms may contribute to oligodendrocyte process outgrowth. We have previously shown that pharmacological inhibitors of histone deacetylation prevent oligodendrocyte branching and we now seek to explore in detail the relationship between these two events. The results presented here indicate that pharmacological inhibitors of histone deacetylation prevent branching, similar to the effect of low doses of cytoskeletal depolymerizing agents. The lack of process outgrowth does not correlate with changes in the levels of tubulin or actin, but correlates with increased levels of microtubule (i.e., stathmin) and microfilaments (i.e., gelsolin) depolymerizing proteins. These data suggest that in OL progenitors, the high levels of depolymerizing proteins maintain a simple morphology, while branching is favored by reduced levels of these cytoskeletal components, consequent to the effect of histone deacetylation on gene expression. We therefore hypothesize that epigenetic regulation of stathmin and gelsolin is a novel regulatory mechanism contributing to OL process outgrowth. In conclusion, our results suggest that process outgrowth in vitro is regulated not only by increased levels of proteins affecting polymerization, but also by decreased levels of proteins affecting depolymerization. The levels of these severing proteins are regulated by chromatin modifiers and therefore suggest that their expression in developing OL is decreased by an epigenetic mechanism.

p27(Kip1) enhances myelin basic protein gene promoter activity.

The process of oligodendrocyte differentiation is a complex event that requires cell cycle withdrawal, followed by the activation of a specific transcriptional program responsible for the synthesis of myelin genes. Because growth arrest precedes differentiation, we sought to investigate the role of cell cycle molecules in the activation of myelin gene promoters. We hypothesized that the cell cycle inhibitor p27(Kip1), which is primarily responsible for arresting proliferating oligodendrocyte progenitors, may be involved in the transcriptional regulation of myelin genes. In agreement with this hypothesis, overexpression of p27(Kip1) in the CG4 cell line, but not in 3T3 fibroblasts, enhances the expression of luciferase driven by the myelin basic protein (MBP) promoter. Interestingly, this effect is specific for p27(Kip1); overexpression of other cell cycle inhibitors had no effect. Additionally, this effect is independent of halting the cell cycle; treatment with the cell cycle blocker roscovitine did not affect MBP promoter usage. We conclude that p27(Kip1) contributes to oligodendrocyte differentiation by regulating transcription of the MBP gene.