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.     

Region-specific expression of cell cycle inhibitors in the adult brain

In the adult brain, neural proliferation is almost absent and neurons are generally not renewed. By contrast, in the olfactory organ, olfactory neurons are produced continuously throughout life. To investigate whether specific cell cycle inhibitors are involved in the control of neural quiescence in adulthood, we compared their expression either in different regions of the adult brain weakly or non neurogenic or, for comparison, in the olfactory mucosa. We show that numerous cell cycle inhibitors are expressed in the adult brain either in an ubiquitous fashion (as p19Ink4d) or in specific brain regions (p15Ink4b in the forebrain, p27Kip1 and p21Cip1 in the cerebellum). By contrast p18Ink4c was expressed detectably only in the highly neurogenic olfactory epithelium. The present data suggest that various CDK inhibitors may be involved in a region-specific fashion in the maintenance of nerve cell quiescence in adults.     

Neuronal expression of cycline dependent kinase inhibitors of the INK4 family in Alzheimer's disease

Summary. Neurodegeneration and cell death in Alzheimer's disease might be associated with aberrant proliferative mechanisms and activation of cell-cycle related events. We reported previously on the elevated expression of the cyclin dependent kinase inhibitor p16^INK4a in Alzheimer's disease closely associated with neurofibrillary degeneration. In the present study, we demonstrate that other members of the INK4-family of cyclin dependent kinase inhibitors such as p15^INK4b, p18^INK4c and p19^INK4d that bind directly to cdk4/6 or to complexes of cdk4/6 with D-type cyclins are all elevated. In contrast, no indication of altered expression of the cyclin dependent kinase inhibitors p21^Cip1 and p27^Kip1 were observed. Inhibitors of the INK4-family were strongly expressed in tangle-bearing neurones and neuritic components of plaques. A much lower expression was also seen in astrocytes. These findings add further evidence to the suggestion that a dysfunction of cell cycle regulation is of critical importance in the pathomechanism of Alzheimer's disease. Accepted January 28, 1998; received January 13, 1998     

Effects of prenatal exposure to ethanol on the cyclin-dependent kinase system in the developing rat cerebellum

Prenatal exposure to ethanol inhibits neurogenesis in the developing cerebellum. Cyclin-dependent kinases (CDKs) are a family of protein kinases that play multiple roles in the regulation of cell proliferation, differentiation and survival. The activity of CDKs is positively regulated by CDK activators, cyclins, and negatively regulated by CDK inhibitors (CDKIs). We hypothesize that impaired cerebellar development induced by gestational ethanol exposure is mediated by disruption of the CDK system. Pregnant rats were fed ad libitum with an ethanol-containing liquid diet (Et) or pair-fed an isocaloric control diet (Ct). Cerebella were collected from pups (postnatal day (P) 0 through P21) and examined for CDK, cyclin, or CDKI expression using a quantitative immunoblotting procedure. In Ct-treated rats, the expression of CDK2 and its activator, cyclin A, paralleled the pattern of granule cell proliferation. Prenatal ethanol exposure produced a significant down-regulation of CDK2/cyclin A expression. Although the amounts of CDK4/CDK6 and their activator, cyclin D2, did not oscillate during postnatal development, their expression in Et-treated pups was significantly (P<0.05) higher than in controls. The expression of a CDK inhibitor, p27(Kip), was inversely correlated to proliferation of cerebellar granule progenitors. Prenatal ethanol exposure caused the down-regulation of p27(Kip) between P0 and P21. Thus, prenatal exposure to ethanol disturbed the expression of cell cycle machineries in the postnatal cerebellum. This may account for the teratogenic effects of ethanol on the developing cerebellum.     

The cyclin-dependent kinase inhibitors p19(Ink4d) and p27(Kip1) are coexpressed in select retinal cells and act cooperatively to control cell cycle exit

Cyclin-dependent kinase inhibitors (cdki's), including p19(Ink4d) and p27(Kip1), mediate exit from the cell cycle. To determine the function of these cdki's in regulating neurogenesis, we examined retina from wild-type, Ink4d-null, and Ink4d/Kip1-double null animals. Ink4d was expressed in progenitors and select neurons in the mature retina. Ink4d-null retina showed an extended period of proliferation, followed by apoptosis. Colabeling for p19(Ink4d) and p27(Kip1) revealed that a subpopulation of cells expressed both inhibitors. Deletion of Ink4d and Kip1 resulted in continued proliferation that was synergistic. This hyperproliferation led to an increase in number of horizontal cells and differentiated neurons reentering the cell cycle. Deletion of Ink4d and Kip1 also exacerbated the retinal dysplasia observed in Kip1-null mice, which was shown to be partly dependent on p53. These data indicate that select retinal cells express both p19(Ink4d) and p27(Kip1) and that they act cooperatively to ensure cell cycle exit.     

Voltage-activated K+ channels and membrane depolarization regulate accumulation of the cyclin-dependent kinase inhibitors p27(Kip1) and p21(CIP1) in glial progenitor cells.

Neural cell development is regulated by membrane ion channel activity. We have previously demonstrated that cell membrane depolarization with veratridine or blockage of K+ channels with tetraethylammonium (TEA) inhibit oligodendrocyte progenitor (OP) proliferation and differentiation (); however the molecular events involved are largely unknown. Here we show that forskolin (FSK) and its derivative dideoxyforskolin (DFSK) block K+ channels in OPs and inhibit cell proliferation. The antiproliferative effects of TEA, FSK, DFSK, and veratridine were attributable to OP cell cycle arrest in G1 phase. In fact, (1) cyclin D accumulation in synchronized OP cells was not affected by K+ channel blockers or veratridine; (2) these agents prevented OP cell proliferation only if present during G1 phase; and (3) G1 blockers, such as rapamycin and deferoxamine, mimicked the anti-proliferative effects of K+ channel blockers. DFSK also prevented OP differentiation, whereas FSK had no effect. Blockage of K+ channels and membrane depolarization also caused accumulation of the cyclin-dependent kinase inhibitors p27(Kip1) and p21(CIP1) in OP cells. The antiproliferative effects of K+ channel blockers and veratridine were still present in OP cells isolated from INK4a-/- mice, lacking the cyclin-dependent kinase inhibitors p16(INK4a) and p19(ARF). Our results demonstrate that blockage of K+ channels and cell depolarization induce G1 arrest in the OP cell cycle through a mechanism that may involve p27(Kip1) and p21(CIP1) and further support the conclusion that OP cell cycle arrest and differentiation are two uncoupled events.     

Microglial cell cycle-associated proteins control microglial proliferation in vivo and in vitro and are regulated by GM-CSF and density-dependent inhibition

Hyperplasia of microglia is one characteristic of reactive gliosis, which is observed in various pathologic conditions in the central nervous system (CNS). To investigate the regulation mechanisms of microglial proliferation, the effect of granulocyte macrophage colony-stimulating factor (GM-CSF) on the expression of cell cycle-associated proteins was examined in the microglial cell line GMI-M6-3. After GM-CSF administration, cyclins D1, E, and A and cyclin-dependent kinase inhibitor p21Cip1 were increased, and another cyclin-dependent kinase inhibitor, p27Kip1, was decreased with morphologic transformation into ameboid form. By contrast, downregulation of these cyclins and p21Cip1, and strong upregulation of p27Kip1 accompanied by ramification were observed with GM-CSF deprivation. We also found that GMI-M6-3 exhibited homotypic contact inhibition of proliferation without any morphologic transformation. The increase of p27Kip1 and the decrease of cyclin A were suggested to play an important role in microglial contact inhibition. In addition, the direct effect of p27Kip1 to inhibit microglial proliferation was demonstrated both in vitro and in vivo by overexpression of p27Kip1.     

Upregulation of p21Cip1 in activated glial cells

The cdk inhibitor p21(Cip1), also named p21(Cip1/Waf1), is intimately involved in coupling growth arrest to cellular differentiation in several cell types. p21(Cip1) is a multifunctional protein that might regulate cell-cycle progression at different levels. In a recent study, we found no differences in the rate of proliferation between glial cells from wild-type and p21(Cip1-/-) mice. In the present study, we examined differences in glial activation between glial cells from wild-type and p21(Cip1-/-) mice, using mixed glial cultures, microglia-enriched cultures, and astrocyte-enriched cultures. We compared the effect of lipopolysaccharide and two forms (oligomeric and fibrillar) of the 1-42 beta-amyloid peptide on glial activation. We observed an attenuation of nuclear translocation of the nuclear factor kappa-B in p21(Cip1-/-) glial cells, when compared with glial cells from wild-type mice. In contrast, tumor necrosis factor-alpha release was enhanced in p21(Cip1-/-)microglial cells. In addition glial activation induced by lipopolysaccharide and the fibrillar form of the 1-42 beta-amyloid peptide upregulated p21(Cip1). Our results support a role for p21(Cip1) in the activation of glial cells, particularly in microglia.     

Increased nitric oxide levels and nitric oxide synthase isoform expression in the cerebellum of the taiep rat during its severe demyelination stage

We have previously reported progressive reactive astrocytes in the cerebellum of taiep rats, one of the most regions affected by demyelination, and activation of cerebellar glial cells in vitro. Based on the hypothesis that activated glial cells produce high levels of reactive nitrogen intermediates, we assessed the production of nitric oxide (NO) and the expression of the three NO synthases (NOS) in the cerebellum of 6-month-old taiep rats. A significant 40% increase of NO levels was measured in taiep rats when compared with controls. The protein and mRNA levels of the three NOS isoforms were also significantly increased. In contrast to controls, immunostaining assays against nNOS or iNOS showed an increased number of immunoreactive glial cells in the granular layer (nNOS) and Purkinje layer (iNOS) of cerebellum of taiep rats. Microglia-macrophages and both CD4- and CD8-immunoreactive cells were observed in cerebellar white matter of taiep rats only, thus suggesting other possible cell sources of those NOSs. Differences in the cellular location for eNOS immunoreactivity were not observed. The enhanced levels of NO, NOS proteins, mRNAs, and NOS immunoreactivities in glial cells and microglia strongly suggest glial activation together with the professional immune cells can aggravate the demyelination of aged taiep rats.     

Potassium-induced apoptosis in rat cerebellar granule cells involves cell-cycle blockade at the G1/S transition

The role of regulators controlling the G1/S transition of the cell cycle was analyzed during neuronal apoptosis in post-mitotic cerebellar granule cells in an attempt to identify common mechanisms of control with transformed cells. Cyclin D1 and its associated kinase activity CDK4 (cyclin-dependent kinase 4) are major regulators of the G1/S transition. Whereas cyclin D1 is the regulatory subunit of the complex, CDK4 represents the catalytic domain that, once activated, will phosphorylate downstream targets such as the retinoblastoma protein, allowing cell-cycle progression. Apoptosis was induced in rat cerebellar granule cells by depleting potassium in presence of serum. Western-blot analyses were performed and protein kinase activities were measured. As apoptosis proceeded, loss in cell viability was coincident with a significant increase in cyclin D1 protein levels, whereas CDK4 expression remained essentially constant. Synchronized to cyclin D1 accumulation, cyclin-dependent kinase inhibitor p27Kip1 drastically dropped to 20% normal values. Cyclin D1/CDK4-dependent kinase activity increased early during apoptosis, reaching a maximum at 9-12 h and decreasing to very low levels by 48 h. Cyclin E, a major downstream target of cyclin D1, decreased concomitantly to the reduction in cyclin D1/CDK4-dependent kinase activity. We suggest that neuronal apoptosis takes place through functional alteration of proteins involved in the control of the G1/S transition of the cell cycle. Thus, apoptosis in post-mitotic neurons could result from a failed attempt to re-enter cell cycle in response to extracellular conditions affecting cell viability and it could involve mechanisms similar to those that promote proliferation in transformed cells.     

Dynamic changes of PIRH2 and p27kip1 expression in injured rat sciatic nerve

p53-induced ring-H2 protein (PIRH2), a newly identified E3 ubiquitin ligase, has been reported to be interacted with p27Kip1 and promote ubiquitination of p27Kip1 independently of p53. p27kip1, a member of the Cip/Kip family of cyclin-dependent kinases inhibitors (CKIs), was shown to control cell cycle progression and promote cell proliferation. While the distribution and function of PIRH2 and p27kip1 in nervous system lesion and regeneration remains unclear. Here, we performed a sciatic nerve injury model in adult rats and studied the dynamic changes of PIRH2 and p27kip1 expression by western blot and RT-PCR in injured rat sciatic nerve. Sciatic nerve crush resulted in a significant up-regulation of PIRH2 and a down-regulation of p27kip1. Besides, we observed that they were expressed widely in both Schwann cells and axons in adult rat sciatic nerve by double immunofluorescence staining. Results obtained by coimmunoprecipitation and double labeling further showed their interaction in the regenerating process. Thus, these results indicate that PIRH2 and p27kip1 likely play an important role in peripheral nerve injury and regeneration.     

Developmental regulation of tyrosine kinase substrate p36 (calpactin heavy chain) in rat cerebellum

The tyrosine kinase substrate p36 (calpactin heavy chain) is a calcium-dependent membrane- and cytoskeletal-binding protein. Using an affinity-purified antiserum raised against the p36 heavy chain from bovine adrenal medulla, we have examined the cellular distribution of p36 in developing and adult cerebellum. From immunoblotting, the level of p36 in cerebellum was found to decline during development. In dissociated cell cultures of P4 cerebellum, all cell types were labeled by anti-p36. In vibratome sections from cerebella of P10 rats, anti-p36 stained Purkinje cell bodies strongly and all other cell types less strongly, with the exception of cells in the external germinal layer, which were unstained. By 18 days postnatally p36 was present at higher levels in Bergmann glia and astrocytes of the white matter. In sections of adult cerebella, anti-p36 staining was restricted to Bergmann glial processes and to the processes of a subclass of astrocytes in the granular layer and the white matter. At no developmental stage was anti-p36 staining detectable in axons or dendrites in vibratome sections. These results suggest that p36 plays a role in some aspect of cellular differentiation common to all cerebellar cell types and may have additional functions in astrocytes of the adult cerebellum. This work was supported by project grants to R.D.B. from the Medical Research Council of the United Kingdom.     

Maternal choline availability alters the localization of p15Ink4B and p27Kip1 cyclin-dependent kinase inhibitors in the developing fetal rat brain hippocampus

Previously we have shown that changes in maternal dietary choline are associated with permanent behavioral changes in offspring. Importantly, in adult male rats, feeding a choline-deficient diet increases the localization of cyclin-dependent kinase inhibitors (CDKIs) in the liver, whereas young adult CDKI knockout mice (p15Ink4B or p27Kip1) exhibit behavioral abnormalities. Thus, maternal dietary choline-CDKI interactions could underlie the changes we observe in fetal hippocampal development and cognitive function in offspring. Here, timed-pregnant rats on embryonic day E12 were fed the AIN-76 diet with varying levels of dietary choline for 6 days, and, on E18, fetal brain sections were collected, and the localization of CDKI proteins was studied using immunohistochemistry and an unbiased image analysis method. In choline-supplemented animals compared to controls, the number of cells with nuclear immunoreactivity for p15Ink4b CDKI protein was decreased 2- to 3-fold in neuroepithelial ventricular zones and adjacent subventricular zones corresponding to the fimbria, primordial dentate gyrus and Ammon's horn regions in the fetal hippocampus. In contrast, maternal dietary choline deficiency significantly decreased nuclear p15Ink4b immunoreactivity in the neuroepithelial layer of the dentate gyrus. Unlike p15Ink4b, the CDKI protein p27Kip1 was observed almost exclusively in the cytoplasm, though the protein was distributed throughout the proliferating and postmitotic zones in the E18 fetal hippocampus. Maternal dietary choline supplementation decreased the cytoplasmic staining intensity for p27Kip1 throughout the fetal hippocampus compared to control animals. Choline deficiency increased the staining intensity of p27Kip1 throughout the hippocampus in association with increased expression of MAP-1 and vimentin proteins. These results link maternal dietary choline availability to CDKI protein immunoreactivity and commitment to differentiation during fetal hippocampal development.     

Spatial, temporal, and cellular distribution of the activated extracellular signal regulated kinases 1 and 2 in the developing and mature rat cerebellum

The extracellular signal regulated kinases 1 and 2 (ERK1/2) are important members of an intracellular signaling cascade that is involved in many aspects of the cellular physiology and development of neurons and glia. ERK1/2 are expressed in many brain regions including the cerebellum; however, their role during cerebellar development is poorly understood. Immunohistochemical approaches using phosphorylation-state specific antiserum that recognizes only the activated-ERK1/2 (pERK) were used to characterize the spatial and temporal patterns of activated-ERK in the developing and adult rat cerebellum. The distribution and cell type-specificity of pERK-immunoreactivity (IR) followed an age-related pattern, with the density of pERK-IR Purkinje cells decreasing between P6 and P15 and increasing at later times. Immunopositive granule cell neurons increased from P6 to P12, became decreased during much of late postnatal cerebellar development, and absent in adults. Co-localization of pERK with glial fibrillary acidic protein or the neuronal marker beta-tubulin revealed that activated ERK is present in maturing Purkinje and granule cells, and the soma of Bergmann glia on P4, P10 and P15; pERK was detected in astrocytes on P10 and P15. Associated with weaning, there was a general increase in activated-ERK in all cell types on P22. In adults, pERK-IR was confined to the Purkinje cell layer and scattered cells in the corpus medullare. In summary, a high degree of developmental plasticity was observed in the spatiotemporal distribution of cerebellar pERK-IR suggesting that the ERK-pathway plays a dynamic role in regulating neuronal and glial migration, proliferation and differentiation in the developing cerebellum. In the mature cerebellum, ERK signaling may also mediate postsynaptic information processing.     

Developmental regulation of tyrosine kinase substrate p36 (calpactin heavy chain) in rat cerebellum

The tyrosine kinase substrate p36 (calpactin heavy chain) is a calcium-dependent membrane- and cytoskeletal-binding protein. Using an affinity-purified antiserum raised against the p36 heavy chain from bovine adrenal medulla, we have examined the cellular distribution of p36 in developing and adult cerebellum. From immunoblotting, the level of p36 in cerebellum was found to decline during development. In dissociated cell cultures of P4 cerebellum, all cell types were labeled by anti-p36. In vibratome sections from cerebella of P10 rats, anti-p36 stained Purkinje cell bodies strongly and all other cell types less strongly, with the exception of cells in the external germinal layer, which were unstained. By 18 days postnatally p36 was present at higher levels in Bergmann glia and astrocytes of the white matter. In sections of adult cerebella, anti-p36 staining was restricted to Bergmann glial processes and to the processes of a subclass of astrocytes in the granular layer and the white matter. At no developmental stage was anti-p36 staining detectable in axons or dendrites in vibratome sections. These results suggest that p36 plays a role in some aspect of cellular differentiation common to all cerebellar cell types and may have additional functions in astrocytes of the adult cerebellum.     

Density dependent modulation of cell cycle protein expression in astrocytes.

The proliferation of type-1 astrocytes is strongly inhibited by homotypic cell-contact. To examine the mechanisms mediating this inhibition of proliferation, the expression of cell cycle related proteins was compared between exponential growth-phase and contact-inhibited astrocytes. Expression of the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 was upregulated 10-fold in confluent compared with growth-phase cultures. Density-induced expression of p27Kip1 was reversible. When confluent cultures of astrocytes expressing high levels of p27Kip1 were replated at low density, the expression of p27Kip1 decreased rapidly. In contrast to p27Kip1, the expression levels of the cell cycle protein, cyclin A was decreased ten-fold in confluent cultures compared with those in growth phase. In addition, the ratio of hyperphosphorylated to hypophosphorylated retinoblastoma protein (pRb) decreased concomitantly with the increase of p27Kip1 and the decrease of cyclin A levels. These results suggest that increased expression of p27Kip1 and decreased expression of cyclin A underlie the reduction in proliferation of contact inhibited astrocytes. High levels of mitogenic stimulation could transiently override contact-dependent inhibition of astrocyte proliferation. Addition of exogenous epidermal growth factor (EGF) resulted in elevated proliferation at high density and formation of multiple cell layers. Addition of EGF did not substantially alter levels of p27Kip1 or cyclin A, but did elevate the levels of cyclin D1. Such changes in cell cycle protein expression may contribute to elevated cell proliferation seen in reactive gliosis after injury to the adult CNS.     

p16INK4A and p14ARF tumor suppressor pathways are deregulated in malignant rhabdoid tumors

Malignant rhabdoid tumors (MRTs) are aggressive tumors associated with mutations in the SMARCB1 gene. In experimental systems, the loss of SMARCB1 is hypothesized to alter p16(INK4A) pathways resulting in the repression of tumor suppressors. To determine whether these pathways are deregulated in human MRT, we used immunohistochemistry on tissue microarrays to evaluate p16(INK4A)/E2F1/RB and p14(ARF)/MDM2/p53 pathways in 25 atypical teratoid/rhabdoid tumors (AT/RT) and 11 non-CNS MRT. p16(INK4A) was negative or showed focal weak expression. p16(INK4A) downstream targets CDK4/cyclin D1/ppRB were variably expressed at moderate to low levels; E2F1 was negative. Unexpectedly, p14(ARF) expression was seen in many cases, which correlated positively with p53 and inversely with MDM2 immunostaining in AT/RT. TP53 mutational analysis in 19 of 25 AT/RT and in 8 of 11 non-CNS MRT cases showed point mutations in only 3 AT/RT cases, suggesting that p53 expression was driven mainly by p14(ARF). Finally, nucleophosmin, a protein that stabilizes p53, was positive in most cases and colocalized with p53. Together, these data suggest that, in MRT, there is deregulation not only of p16(INK4A) but also of the p14(ARF) pathway. These results provide insights into cell cycle deregulation in the pathogenesis of human MRT and may aid in the design and evaluation of potential therapies for these tumors.     

Abundant expression of zinc transporters in Bergman glia of mouse cerebellum

Zinc transporters (ZnTs) are membrane proteins involved in zinc ion transportation in mammalian cells. Seven members of ZnT family, ZnT1-7, have been cloned and characterized. These transporter proteins have different cellular and sub-cellular locations, suggesting that they may play different roles in zinc homeostasis in normal and pathological conditions in different tissues. Cerebellum is one of the most zinc-enriched regions in the central nervous system, but little is known about zinc metabolism in the cerebellum. In the present study, we investigated the detailed distributions of four members (ZnT1, ZnT3, ZnT4 and ZnT6) of the ZnT family, in the mouse cerebellum. Immunostaining and confocal microscopic observations revealed a similar staining pattern of ZnTs in the molecular layer and the Purkinje cell layer. Double labeling with anti-S-100beta or anti-MAP2 and anti-ZnTs clearly showed that the Bergman glial cell bodies in the Purkinje cell layer and their radial processes in the molecular layer exhibited strong immunofluorescence of all the tested ZnTs. However, the somata of the Purkinje cells contained a moderate immunostaining for ZnT1, but virtually lack of other three ZnTs. In the granular layer, ZnTs appeared with different immunostaining patterns. ZnT1 was expressed in a small number of neuronal cell bodies and their primary dendrites, whereas ZnT3 and ZnT4 were present in nerve terminals but not in the neuronal somata. ZnT6 was undetectable in either the cell bodies or processes in the granular layer. The present results indicate that the Bergman glial cells may play an important role in zinc metabolism in the mouse cerebellar cortex.     

Alterations in cyclin A,B, and D1 in mouse dentate gyrus following TMT-induced hippocampal damage

The interactions of glia and neurons during injury and subsequent neurodegeneration are a subject of interest both in disease and chemical-induced brain injury. One such model is the prototypical hippocampal toxicant trimethyltin (TMT). An acute injection of TMT (2.0 mg/kg, i.p.) to postnatal day 21 CD-1 male mice produced neuronal necrosis and loss of dentate granule cells, astrocyte hypertrophy, and microglia activation in the hippocampus within 24 h. Neuronal necrosis and microglia differentiation to a phagocytic phenotype is temporally correlated with peak elevations in TNF-α, cyclin A2, cyclin B1 and cyclin D1 at 72 h post-TMT. TNF-α mRNA levels were significantly elevated in the hippocampus by 12 h and remained elevated for 72 h. mRNA levels for cyclin A2 and cyclin B1 were elevated by approximately 2-fold at 72 h. Immunohistochemistry suggested a cellular localization of cyclin A to microglia in the region of neuronal necrosis in the dentate, cyclin B in glial cells in juxtaposition to neurons in the hilus of the hippocampus and cyclin D1 to non-glial cells in the dentate. mRNA levels for cyclin D1 were elevated approximately 1.5-fold by 72 h as determined by RNase protection assay. No changes were seen in mRNA levels for cyclins E, F, G1, G2, H or I nor cyclin dependent kinases. These elevations are not associated with proliferation of microglia as determined by BrdU incorporation and Ki-67 immunohistochemistry. Upregulation of cell cycle genes was associated with cellular processes other than proliferation and may contribute to the differentiation of microglia to a phagocytic phenotype. These data suggest an integrated role for cell cycle regulation of neural cells in the manifestation of hippocampal pathophysiology.     

Cyclin D1 is required for proliferation of olig2‐expressing progenitor cells in the injured cerebral cortex

Little is known about the molecular mechanisms driving proliferation of glial cells after an insult to the central nervous system (CNS). To test the hypothesis that the G1 regulator cyclin D1 is critical for injury‐induced cell division of glial cells, we applied an injury model that causes brain damage within a well‐defined region. For this, we injected the neurotoxin ibotenic acid into the prefrontal cortex of adult mice, which leads to a local nerve cell loss but does not affect the survival of glial cells. Here, we show that cyclin D1 immunoreativity increases drastically after neurotoxin injection. We find that the cyclin D1‐immunopositive (cyclin D1+) cell population within the lesioned area consists to a large extent of Olig2+ oligodendrocyte progenitor cells. Analysis of cyclin D1‐deficient mice demonstrates that the proliferation rate of Olig2+ cells diminishes upon loss of cyclin D1. Further, we show that cyclin‐dependent kinase (cdk) 4, but not cdk6 or cdk2, is essential for driving cell division of Olig2‐expressing cells in our injury model. These data suggest that distinct cell cycle proteins regulate proliferation of Olig2+ progenitor cells following a CNS insult.