Cell Cycle Reactivation Research Articles

Origination of asexual plantlets in three species of Crassulaceae.

During asexual plant reproduction, cells from different organs can be reprogrammed to produce new individuals, a process that requires the coordination of cell cycle reactivation with the acquisition of other cellular morphological characteristics. However, the factors that influence the variety of asexual reproduction have not yet been determined. Here, we report on plantlet formation in Kalanchoe daigremontiana, Graptopetalum paraguayense, and Crassula portulacea (Crassulaceae) and analyse the effect of initiating cells on asexual reproduction in these three species. Additionally, the roles of WUSCHEL (WUS) and CUP-SHAPED COTYLEDON 1 (CUC1) in the asexual reproduction of these species were analysed through qRT-PCR. Our results indicated that pre-existing stem cell-like cells at the sites of asexual reproduction were responsible for the formation of plantlets. These cells were arrested in different phases of the cell cycle and showed different cell morphological characteristics and cell counts. The accumulation of auxin and cytokinin at the sites of asexual plantlet formation indicated their important functions, particularly for cell cycle reactivation. These differences may influence the pattern and complexity of asexual reproduction in these Crassulaceae species. Additionally, the dynamic expression levels of CUC1 and WUS may indicate that CUC1 functions in the formation of callus and shoot meristems; whereas, WUS was only associated with shoot induction.

Down-regulation of miRNA-106b inhibits growth of melanoma cells by promoting G1-phase cell cycle arrest and reactivation of p21/WAF1/Cip1 protein.

MiR-106b is overexpressed in various types of cancers and is associated with the regulation of the carcinogenic processes. Using RT-PCR, we have identified overexpression of miRNA-106b in various melanoma cell lines (A375, Hs294t, SK-Mel28, SK-Mel 119, Mel 1241, Mel 1011 and Mel 928) as compared to its expression in normal human epidermal melanocytes (NHEM). The overexpression of miR-106b in melanoma cells (A375, Hs294t) was associated with greater cell proliferation capacity than NHEM. Treatment of A375 and Hs294t cells with anti-miR-106b resulted in inhibition of cell proliferation as well as G1-phase arrest. We determined the effects of grape seed proanthocyanidins (GSPs) on the expression of miRNA-106b and its underlying molecular targets. Treatment of A375 and Hs294t cells with GSPs resulted in suppression of the levels of miRNA-106b, cytotoxicity, G1-phase arrest and reactivation of p21/WAF1/Cip1. Dietary GSPs significantly inhibited growth of A375 melanoma cell tumor xenografts in nude mice, which was associated with reduction in the levels of miRNA-106b, tumor cell proliferation and increases in the levels of p21/WAF1/Cip1 protein. These studies suggest that miRNA-106b plays a crucial role in melanoma growth and that GSPs act as an inhibitor of miR-106b thereby blocking melanoma growth in vitro and in vivo models.

Poly(ADP-ribosyl)ation is required to modulate chromatin changes at c-MYC promoter during emergence from quiescence.

Poly(ADP-ribosyl)ation is a post-translational modification of various proteins and participates in the regulation of chromatin structure and transcription through complex mechanisms not completely understood. We have previously shown that PARP-1, the major family member of poly(ADP-ribose)polymerases, plays an important role in the cell cycle reactivation of resting cells by regulating the expression of Immediate Early Response Genes, such as c-MYC, c-FOS, JUNB and EGR-1. In the present work we have investigated the molecular mechanisms by which the enzyme induces c-MYC transcription upon serum stimulation of quiescent cells. We show that PARP-1 is constitutively associated in vivo to a c-MYC promoter region recognized as biologically relevant for the transcriptional regulation of the gene. Moreover, we report that serum stimulation causes the prompt accumulation of ADP-ribose polymers on the same region and that this modification is required for chromatin decondensation and for the exchange of negative for positive transcriptional regulators. Finally we provide evidence that the inhibition of PARP activity along with serum stimulation impairs c-MYC induction by preventing the proper accumulation of histone H3 phosphoacetylation, a specific chromatin mark for the activation of Immediate Early Response Genes. These findings not only suggest a novel strategy by which PARP-1 regulates the transcriptional activity of promoters but also provide new information about the complex regulation of c-MYC expression, a critical determinant of the transition from quiescence to proliferation.

A GLOBAL (PHOSPHO)PROTEOMIC WORKFLOW SUITABLE FOR ANALYSIS OF FRESH/FROZEN BRAIN TISSUE IN TRANSGENIC MODELS OF NEURODEGENERATION

Many neurodegenerative diseases are characterised by abnormal posttranslational modification (PTM) of one or more proteins leading to their aggregation and, by mechanisms not yet fully understood, ultimately to neuronal cell death. One of the most common forms of pathological PTM is phosphorylation, with phosphorylated forms of tau (AD), TDP-43 (ALS, FTLD) and alpha-synuclein (PD) all associated with disease, whereas decreased phosphorylation on huntingtin (HD) is thought to be protective. The role of cell signalling pathways, mostly regulated by kinase/phosphatase cascades is also increasingly being recognised as modulating key aspects of neurodegeneration such as re-activation of cell cycle in neuronal cells and activation of immune cells such as microglia and astrocytes. Accordingly, there is a strong rationale to develop and apply global phosphorylation profiling to neuronal cell cultures and brain tissues used in neurodegenerative disease research. We have recently reported SysQuant, a global phosphoproteomic workflow that employs isobaric Tandem Mass Tags (TMT) and differential chromatography to measure fractions of both enriched phosphopeptides and the un-enriched nonphosphorylated peptides derived from tryptic digestion of tumour cell lysates of up to 10 samples in a single mass spectrometry experiment(1). We have now applied SysQuant to frozen brain tissue from a mouse model of human tauopathy (TMHT Mouse, QPS Austria) treated with vehicle or vehicle + kinase inhibitors, and we are able to demonstrate significant, potentially protective effects on the phosphorylation of aggregating proteins, as well as regulation of key pathways such as glycolysis/gluconeogenesis, calcium signalling, oxidative phosphorylation, citrate cycle along with all major neurodegenerative KEGG pathways. By using a TMT 10plex labelling strategy we were able to generate a detailed, quantitative phosphoproteomic map for 3 x 3 x 3 animals treated with vehicle or one of two kinase inhibitors from hippocampus, cortex and rest of brain regions in approximately 8 weeks. Data from the hippocampus is presented here.

DNA damage signaling regulates age-dependent proliferative capacity of quiescent inner ear supporting cells.

Supporting cells (SCs) of the cochlear (auditory) and vestibular (balance) organs hold promise as a platform for therapeutic regeneration of the sensory hair cells. Prior data have shown proliferative restrictions of adult SCs forced to re-enter the cell cycle. By comparing juvenile and adult SCs in explant cultures, we have here studied how proliferative restrictions are linked with DNA damage signaling. Cyclin D1 overexpression, used to stimulate cell cycle re-entry, triggered higher proliferative activity of juvenile SCs. Phosphorylated form of histone H2AX (γH2AX) and p53 binding protein 1 (53BP1) were induced in a foci-like pattern in SCs of both ages as an indication of DNA double-strand break formation and activated DNA damage response. Compared to juvenile SCs, γH2AX and the repair protein Rad51 were resolved with slower kinetics in adult SCs, accompanied by increased apoptosis. Consistent with thein vitro data, in a Rb mutant mouse model in vivo, cell cycle re-entry of SCs was associated with γH2AX foci induction. In contrast to cell cycle reactivation, pharmacological stimulation of SC-to-hair-cell transdifferentiation in vitro did not trigger γH2AX. Thus, DNA damage and its prolonged resolution are critical barriers in the efforts to stimulate proliferation of the adult inner ear SCs.

The effect of Platelet Lysate on osteoblast proliferation associated with a transient increase of the inflammatory response in bone regeneration

Platelet Lysate (PL) contains a cocktail of growth factors and cytokines, which actively participates in tissue repair and its clinical application has been broadly described. The aim of this study was to assess the regenerative potential of PL for bone repair. We demonstrated that PL stimulation induces a transient increase of the inflammatory response in quiescent human osteoblasts, via NF-kB activation, COX-2 induction, PGE2 production and secretion of pro-inflammatory cytokines. Furthermore, we showed that long-term PL stimulation enhances proliferation of actively replicating osteoblasts, without affecting their differentiation potential, along with changes of cell morphology, resulting in increased cell density at confluence. In confluent resting osteoblasts, PL treatment induced resumption of proliferation, change in cell morphology and increase of cell density at confluence. A burst of PL treatment (24-h) was sufficient to trigger such processes in both conditions. These results correlated with up-regulation of the proliferative and survival pathways ERKs and Akt and with cell cycle re-activation via induction of CyclinD1 and phosphorylation of Rb, following PL stimulation. Our findings demonstrate that PL treatment results in activation and expansion of resting osteoblasts, without affecting their differentiation potential. Therefore PL represents a good therapeutic candidate in regenerative medicine for bone repair.

Protein kinase C activation causes neurite retraction via cyclinD1 and p60‐katanin increase in rat hippocampal neurons

Neurons are differentiated postmitotic cells residing in G0 phase of the cell cycle and are unable to proceed through G1 phase, in which cyclinD1 needs to be up-regulated for initiation. Yet, a growing body of evidence has shown that cell cycle re-activation via cyclinD1 up-regulation drives neurons into apoptosis. By contrast, there is also evidence demonstrating cell cycle proteins playing roles in neuronal differentiation. cyclinD1 has been shown to be differently regulated by protein kinase C alpha (PKC-α) in various mitotic cells. Based on these different effects, we investigated the role of PKC-α on cyclinD1 regulation in hippocampal neurons. Neurons were treated with PKC activator, PMA, and analysed for subcellular distributions of PKC-α and cyclinD1. Remarkably, PMA treatment increased nuclear PKC-α and cyclinD1, but not PKC-ε in hippocampal neurons. Increases in nuclear PKC-α and cyclinD1 were accompanied by microtubule re-organisation via increases in tau and retinoblastoma protein phosphorylation levels. Increased p60-katanin and p53 changed the neuronal morphology into neurons with shorter, but increased number of side branches. Since up-regulation of cell cycle is associated with apoptosis in neurons, we also analysed changes in Bax, Bcl-2 early and PARP (poly(ADP-ribose)polymerase), caspase3 late apoptotic markers. However, we did not observe any indication of apoptosis. These data suggest that in addition to their previously known roles in mitotic cells on cell cycle regulation, PKC-α and cyclinD1 seem to be important for differentiation, and nuclear PKC-α and cyclinD1 interfere with differentiation by promoting microtubule re-organisation through PKC signaling without triggering apoptosis.

Phosphorylated retinoblastoma protein (p-Rb) is involved in neuronal apoptosis after traumatic brain injury in adult rats

Phosphorylated retinoblastoma protein (p-Rb), a well identified cell cycle related protein, is involved in regulating the biological functions of various cell types including neurons. One attractive biological function of p-Rb is releasing E2F transcription factor to induce S-phase entry and cellular proliferation of mitotic cells. However, some studies point out that the role of p-Rb in post-mitotic cells such as mature neurons is unique; it may induce cellular apoptosis rather than proliferation via regulating cell cycle reactivation. Up to now, the knowledge of p-Rb function in CNS is still limited. To investigate whether p-Rb is involved in CNS injury and repair, we performed a traumatic brain injury model in adult rats. Up-regulation of p-Rb was observed in the injured brain cortex by western blot analysis and immunohistochemistry staining. Terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling (TUNEL) and 4',6-diamidino-2-phenylindole (DAPI) staining suggested that p-Rb was relevant to neuronal apoptosis after brain injury. In addition, glutamate excitotoxic model of primary cortex neurons was introduced to further investigate the role of p-Rb in neuronal apoptosis; the result implied p-Rb was associated with cell cycle activation in the apoptotic neurons. Based on our data, we suggested that p-Rb might play an important role in neuronal apoptosis after traumatic brain injury in rat; which might also provide a basis for the further study on its role in regulating cell cycle re-entry in apoptotic neurons, and might gain a novel strategy for the clinical therapy for traumatic brain injury.

The Drosophila SERTAD protein Taranis determines lineage-specific neural progenitor proliferation patterns

Neural progenitors of the Drosophila larval brain, called neuroblasts, can be divided into distinct populations based on patterns of proliferation and differentiation. Type I neuroblasts produce ganglion mother cells (GMCs) that divide once to produce differentiated progeny, while type II neuroblasts produce self-renewing intermediate neural progenitors (INPs) and thus generate lineages containing many more progeny. We identified Taranis (Tara) as an important determinant of type I lineage-specific neural progenitor proliferation patterns. Tara is an ortholog of mammalian SERTAD proteins that are known to regulate cell cycle progression. Tara is differentially-expressed in neural progenitors, with high levels of expression in proliferating type I neuroblasts but no detectable expression in type II lineage INPs. Tara is necessary for cell cycle reactivation in quiescent neuroblasts and for cell cycle progression in type I lineages. Cell cycle defects in tara mutant neuroblasts are due to decreased activation of the E2F1/Dp transcription factor complex and delayed progression through S-phase. Mis-expression of tara in type II lineages delays INP cell cycle progression and induces premature differentiation of INPs into GMCs. Premature INP differentiation can also be induced by loss of E2F1/Dp function and elevated E2F1/Dp expression suppresses Tara-induced INP differentiation. Our results show that lineage-specific Tara expression is necessary for proper brain development and suggest that distinct cell cycle regulatory mechanisms exist in type I versus type II neural progenitors.

Gene profiling identifies commonalities in neuronal pathways in excitotoxicity: Evidence favouring cell cycle re-activation in concert with oxidative stress

Excitotoxicity, induced by the aberrant rise in cytosolic Ca2+ level, is a major neuropathological process in numerous neurodegenerative disorders. It is triggered when extracellular glutamate (Glu) concentration reaches neuropathological levels resulting in dysregulation and hyper-activation of ionotropic glutamate receptor subtype (iGluRs). Even though all three members of the iGluRs, namely N-methyl-d-aspartate (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPAR) and kainate (KAR) receptors are implicated in excitotoxicity, their individual contributions to downstream signaling transduction have not been explored. In this study, we report a comprehensive description of the recruitment of cellular processes in neurons upon iGluR activation during excitotoxicity through temporal (5h, 15h, and 24h) global gene profiling of AMPA, KA, NMDA, and Glu excitotoxic models. DNA microarray analyses of mouse primary cortical neurons treated with these four pharmacological agonists are further validated via real-time PCR. Bi-model analyses against Glu model demonstrate that NMDARs and KARs play a more pivotal role in Glu-mediated excitotoxicity, with a higher degree of global gene profiling overlaps, as compared to that of AMPARs. Comparison of global transcriptomic profiles reveals aberrant calcium ion binding and homeostasis, organellar (lysosomal and endoplasmic reticulum) stress, oxidative stress, cell cycle re-entry and activation of cell death processes as the main pathways that are significantly modulated across all excitotoxicity models. Singular profile analyses demonstrate substantial transcriptional regulation of numerous cell cycle proteins. For the first time, we show that iGluR activation forms the basis of cell cycle re-activation, and together with oxidative stress fulfill the “two-hit” hypothesis that accelerates neurodegeneration.

ERK activation and expression of neuronal cell cycle markers in the hippocampus after entorhinal cortex lesion

Current findings suggest that neuronal cell death is frequently associated with the aberrant expression of cell cycle-regulatory proteins in postmitotic neurons. Aberrant cell cycle reentry has been implicated in diverse neurodegenerative conditions, including Alzheimer's disease (AD). Previously we reported that the appearance of cell cycle markers in postmitotic neurons of the entorhinal cortex (EC) after excitotoxic hippocampal damage is associated with the expression of phospho-tau and amyloid precursor protein (APP). However, the question of the signaling pathway involved in this cell cycle reentry remains unresolved. Differentiated neurons use the molecular mechanisms initially acquired to direct cell proliferation, such as the Ras-extracellular signal-regulated kinase (ERK1/2) pathway, to regulate synaptic plasticity. In this work we explored whether ERK1/2-related signaling might contribute to the cell cycle reentry in hippocampal neurons after a unilateral EC lesion. We showed that, within the first 24 hr after hippocampal deafferentation, numerous neurons expressed phospho-ERK1/2, concomitantly with the gradual increases in cyclin D1 and cyclin B immunoreactivity in the dentate gyrus and hilus. Several of these immunopositive cells to phospho-ERK1/2 and cyclin B in hippocampus are postmitotic neurons, insofar as they are positive to NeuN. The intracisternal administration of U0126 (an MEK inhibitor), previous to the excitotoxic lesion, decreased the activation of ERK1/2 and the expression of cyclin D1 and cyclin B in the hippocampus. The present findings support the notion that ERK1/2 plays a role in cell cycle reactivation in mature neurons efferently connected to the lesion site.

Sucrose synthase activity and carbohydrates content in relation to phosphorylation status of Vicia faba root meristems during reactivation from sugar depletion

Carbohydrate starvation of Vicia faba root meristems leads to readjustment of carbohydrate metabolism and blocks the cell cycle in two principal control points (PCP1/2). The cell cycle reactivation is possible after sucrose provision, although with a delay of about 12h. During this period, the cells are sensitive to 6-dimethylaminopurine (6-DMAP) and okadaic acid (OA), inhibitors of protein kinases and phosphatases, respectively. The aim of the present study was to investigate whether those inhibitors are involved in inhibition of cell cycle revival through interference with the activities of two sucrose-cleaving enzymes: sucrose synthase (SuSy; EC 2.4.1.13) and invertase (INV; EC 3.2.1.26). In sugar-starved cells, the in situ activity of both enzymes decreased significantly. Following supplementation of root meristems with sugar, INV remained inactive, but SuSy activity increased. Despite the lack of INV activity, glucose was present in meristem cells, but its content was low in cells treated with OA. In the latter case, the size of plastids was reduced, they had less starch, and Golgi structures were affected. In sugar-starved cells, SuSy activity was induced more by exogenous sucrose than by glucose. The sucrose-induced activity was strongly inhibited by OA (less by 6-DMAP) at early stages of regeneration, but not at the stages preceding DNA replication or mitotic activities. The results indicate that prolongation of regeneration and a marked decrease in the number of cells resuming proliferation (observed in previous studies) and resulting from the action of inhibitors, are correlated with the process of SuSy activation at the beginning of regeneration from sugar starvation.

Cdk5 Levels Oscillate during the Neuronal Cell Cycle: Cdh1 UBIQUITINATION TRIGGERS PROTEOSOME-DEPENDENT DEGRADATION DURING S-PHASE

When cell cycle re-activation occurs in post-mitotic neurons it places them at increased risk for death. The cell cycle/cell death association has been reported in many neurodegenerative diseases including Alzheimer disease (AD), yet the mechanisms by which a normal neuron suppresses the cycle remain largely unknown. Recently, our laboratory has shown that Cdk5 (cyclin-dependent kinase 5) is a key player in this protective function. When a neuron is under stress, Cdk5 is transported to the cytoplasm; this eliminates its cell cycle suppression activity and the neuron re-enters S-phase. In the current study we show that a similar principle applies during a normal cell cycle. When a neuronal cell enters S phase, Cdk5 is transported to the cytoplasm where it is ubiquitinated by the E3 ligase APC-Cdh1. Ubiquitinated Cdk5 is then rapidly degraded by the proteasome. The ubiquitination site of Cdk5 appears to be in the p35 binding area; in the presence of high levels of p35, the ubiquitination of Cdk5 was blocked, and the degradation in S phase was attenuated. The data suggest an unsuspected role for Cdk5 during the progression of a normal cell cycle and offer new pharmaceutical targets for regulating neuronal cell cycling and cell death.

Nerve Growth Factor-Induced Cell Cycle Reentry in Newborn Neurons Is Triggered by p38MAPK-Dependent E2F4 Phosphorylation

Cumulative evidence indicates that activation of cyclin D-dependent kinase 4/6 (cdk4/6) represents a major trigger of cell cycle reentry and apoptosis in vertebrate neurons. We show here the existence of another mechanism triggering cell cycle reentry in differentiating chick retinal neurons (DCRNs), based on phosphorylation of E2F4 by p38(MAPK). We demonstrate that the activation of p75(NTR) by nerve growth factor (NGF) induces nuclear p38(MAPK) kinase activity, which leads to Thr phosphorylation and subsequent recruitment of E2F4 to the E2F-responsive cdc2 promoter. Inhibition of p38(MAPK), but not of cdk4/6, specifically prevents NGF-dependent cell cycle reentry and apoptosis in DCRNs. Moreover, a constitutively active form of chick E2F4 (Thr261Glu/Thr263Glu) stimulates G(1)/S transition and apoptosis, even after inhibition of p38(MAPK) activity. In contrast, a dominant-negative E2F4 form (Thr261Ala/Thr263Ala) prevents NGF-induced cell cycle reactivation and cell death in DCRNs. These results indicate that NGF-induced cell cycle reentry in neurons depends on the activation of a novel, cdk4/6-independent pathway that may participate in neurodegeneration.

Transcriptome of Protoplasts Reprogrammed into Stem Cells in Physcomitrella patens

BackgroundDifferentiated plant cells can retain the capacity to be reprogrammed into pluripotent stem cells during regeneration. This capacity is associated with both cell cycle reactivation and acquisition of specific cellular characters. However, the molecular mechanisms underlying the reprogramming of protoplasts into stem cells remain largely unknown. Protoplasts of the moss Physcomitrella patens easily regenerate into protonema and therefore provide an ideal system to explore how differentiated cells can be reprogrammed to produce stem cells.Principal findingsWe obtained genome-wide digital gene expression tag profiles within the first three days of P. patens protoplast reprogramming. At four time-points during protoplast reprogramming, the transcript levels of 4827 genes changed more than four-fold and their expression correlated with the reprogramming phase. Gene ontology (GO) and pathway enrichment analysis of differentially expressed genes (DEGs) identified a set of significantly enriched GO terms and pathways, most of which were associated with photosynthesis, protein synthesis and stress responses. DEGs were grouped into six clusters that showed specific expression patterns using a K-means clustering algorithm. An investigation of function and expression patterns of genes identified a number of key candidate genes and pathways in early stages of protoplast reprogramming, which provided important clues to reveal the molecular mechanisms responsible for protoplast reprogramming.ConclusionsWe identified genes that show highly dynamic changes in expression during protoplast reprogramming into stem cells in P. patens. These genes are potential targets for further functional characterization and should be valuable for exploration of the mechanisms of stem cell reprogramming. In particular, our data provides evidence that protoplasts of P. patens are an ideal model system for elucidation of the molecular mechanisms underlying differentiated plant cell reprogramming.

Deregulated Cdk5 Triggers Aberrant Activation of Cell Cycle Kinases and Phosphatases Inducing Neuronal Death

Aberrant activation of cell cycle proteins is believed to play a critical role in Alzheimer's disease (AD) pathogenesis; although, the molecular mechanisms leading to their activation in diseased neurons remain elusive. The goal of this study was to investigate the mechanistic link between Cdk5 deregulation and cell cycle re-activation in β-amyloid(1-42) (Aβ(1-42))-induced neurotoxicity. Using a chemical genetic approach, we identified Cdc25A, Cdc25B and Cdc25C as direct Cdk5 substrates in mouse brain lysates. We show that deregulated Cdk5 directly phosphorylates Cdc25A, Cdc25B and Cdc25C at multiple sites, which not only increases their phosphatase activities but also facilitates their release from 14-3-3 inhibitory binding. Cdc25A, Cdc25B and Cdc25C in turn activate Cdk1, Cdk2 and Cdk4 kinases causing neuronal death. Selective inhibition of Cdk5 abrogates Cdc25 and Cdk activations in Aβ(1-42)-treated neurons. Similarly, phosphorylation-resistant mutants of Cdc25 isoforms at Cdk5 sites are defective in activating Cdk1, Cdk2 and Cdk4 in Aβ(1-42)-treated primary cortical neurons, emphasizing a major role of Cdk5 in the activation of Cdc25 isoforms and Cdks in AD pathogenesis. These results were further confirmed in human AD clinical samples, which had higher Cdc25A, Cdc25B and Cdc25C activities that were coincident with increased Cdk5 activity, as compared to age-matched controls. Inhibition of Cdk5 confers the highest neuroprotection against Aβ(1-42) toxicity, whereas inhibition of Cdc25 isoforms was partially neuroprotective, further emphasizing a decisive role of Cdk5 deregulation in cell-cycle-driven AD neuronal death.

Restrictions in cell cycle progression of adult vestibular supporting cells in response to ectopic cyclin D1 expression.

Sensory hair cells and supporting cells of the mammalian inner ear are quiescent cells, which do not regenerate. In contrast, non-mammalian supporting cells have the ability to re-enter the cell cycle and produce replacement hair cells. Earlier studies have demonstrated cyclin D1 expression in the developing mouse supporting cells and its downregulation along maturation. In explant cultures of the mouse utricle, we have here focused on the cell cycle control mechanisms and proliferative potential of adult supporting cells. These cells were forced into the cell cycle through adenoviral-mediated cyclin D1 overexpression. Ectopic cyclin D1 triggered robust cell cycle re-entry of supporting cells, accompanied by changes in p27Kip1 and p21Cip1 expressions. Main part of cell cycle reactivated supporting cells were DNA damaged and arrested at the G2/M boundary. Only small numbers of mitotic supporting cells and rare cells with signs of two successive replications were found. Ectopic cyclin D1-triggered cell cycle reactivation did not lead to hyperplasia of the sensory epithelium. In addition, a part of ectopic cyclin D1 was sequestered in the cytoplasm, reflecting its ineffective nuclear import. Combined, our data reveal intrinsic barriers that limit proliferative capacity of utricular supporting cells.

Dysfunction of TGF-β1 signaling in Alzheimer’s disease: perspectives for neuroprotection

Alzheimer's disease (AD) is a neurodegenerative disorder that affects about 35 million people worldwide. Current drugs for AD only treat the symptoms and do not interfere with the underlying pathogenic mechanisms of the disease. AD is characterized by the presence of β-amyloid (Aβ) plaques, neurofibrillary tangles, and neuronal loss. Identification of the molecular determinants underlying Aβ-induced neurodegeneration is an essential step for the development of disease-modifying drugs. Recently, an impairment of the transforming growth factor-β1 (TGF-β1) signaling pathway has been demonstrated to be specific to the AD brain and, particularly, to the early phase of the disease. TGF-β1 is a neurotrophic factor responsible for the initiation and maintenance of neuronal differentiation and synaptic plasticity. The deficiency of TGF-β1 signaling is associated with Aβ pathology and neurofibrillary tangle formation in AD animal models. Reduced TGF-β1 signaling seems to contribute both to microglial activation and to ectopic cell-cycle re-activation in neurons, two events that contribute to neurodegeneration in the AD brain. The neuroprotective features of TGF-β1 indicate the advantage of rescuing TGF-β1 signaling as a means to slow down the neurodegenerative process in AD.

PhyscomitrellaCyclin-Dependent Kinase A Links Cell Cycle Reactivation to Other Cellular Changes during Reprogramming of Leaf Cells

During regeneration, differentiated plant cells can be reprogrammed to produce stem cells, a process that requires coordination of cell cycle reactivation with acquisition of other cellular characteristics. However, the factors that coordinate the two functions during reprogramming have not been determined. Here, we report a link between cell cycle reactivation and the acquisition of new cell-type characteristics through the activity of cyclin-dependent kinase A (CDKA) during reprogramming in the moss Physcomitrella patens. Excised gametophore leaf cells of P. patens are readily reprogrammed, initiate tip growth, and form chloronema apical cells with stem cell characteristics at their first cell division. We found that leaf cells facing the cut undergo CDK activation along with induction of a D-type cyclin, tip growth, and transcriptional activation of protonema-specific genes. A DNA synthesis inhibitor, aphidicolin, inhibited cell cycle progression but prevented neither tip growth nor protonemal gene expression, indicating that cell cycle progression is not required for acquisition of protonema cell-type characteristics. By contrast, treatment with a CDK inhibitor or induction of dominant-negative CDKA;1 protein inhibited not only cell cycle progression but also tip growth and protonemal gene expression. These findings indicate that cell cycle progression is coordinated with other cellular changes by the concomitant regulation through CDKA;1.

Cell-dependent kinase inhibitor 2A and 2B genetic variability in patients with Alzheimer’s disease

The cell cycle is a highly regulated and fundamental cellular process that involves complex feedback regulation of many proteins, and any failure of its integrity could result in important consequences for the cell. The abnormal cell cycle re-entry has been hypothesized to play a role in neurodegenerative disease, primarily Alzheimer’s disease (AD). An association study of cell-dependent kinase inhibitor (CDKN) 2A and 2B, involved in cell cycle G1 phase progression, was carried out in a population of 241 patients with AD and 238 age-matched controls. Three tagging single nucleotide polymorphisms (SNPs), namely rs3731239 T/C, rs2811710 C/T and rs3217992 G/A, spanning CDKN2A and CDKN2B, and covering 100% gene variability, were analyzed by allelic discrimination. Allelic and genotypic frequencies of the three SNPs were similar between patients and controls (P [ 0.05), even after stratification for gender or apolipoprotein E (ApoE) status, as well as the haplotype distribution. Therefore, according to these data, CDKN2A and CDKN2B do not likely act as risk factors for AD. Nevertheless, we cannot exclude that other genes encoding for proteins involved in cell proliferation play a role in AD susceptibility. Alzheimer’s disease is a multifactorial disease, caused by the interaction between genetic and environmental risk factors. Previous data suggest that cell cycle reactivation plays a role in the pathogenesis of AD. In particular, there is evidence of accumulation of cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors in cortical neurons from patients with AD [1, 4, 5], suggesting that neurons, usually in the G0 phase, re-enter the cell cycle during the progression of AD. Two studies [2, 6] identified a linkage region in 9p21.3 in late onset AD; this region contains CDKN2A (cell-dependent kinase inhibitor), which regulates cell cycle G1 phase progression. In this study, we carried out an association study of CDKN2A and the adjacent gene CDKN2B by analyzing three tagging SNPs, namely rs3731239 (T/C), rs2811710 (C/T) and rs3217992 (G/A), which cover 100% gene variability. Two hundred forty-one patients with AD were recruited at the Alzheimer Units of Ospedale Maggiore Policlinico and Ospedale L. Sacco. All patients underwent a standard battery of examinations, including medical history, physical and neurological examination, screening laboratory tests, neurocognitive evaluation, brain magnetic resonance imaging (MRI) or computed tomography (CT), and, if indicated, positron emission computed tomography (PET). Dementia severity was assessed by the Clinical Dementia Rating (CDR) and the Mini Mental Scale Examination (MMSE). Disease duration was defined as the time in years between the first symptoms (by history) and the clinical diagnosis. The presence of significant vascular brain damage was excluded (Hachinski ischemic score \4). The diagnosis of probable AD was made by exclusion according to NINCDS-ADRDA criteria [3]. F. Cortini C. Fenoglio E. Venturelli C. Villa M. Serpente C. Cantoni G. Fumagalli N. Bresolin E. Scarpini D. Galimberti (&) Department of Neurological Sciences, ‘‘Dino Ferrari’’ Center, University of Milan, Fondazione Ca Granda, IRCCS Ospedale Maggiore Policlinico, Milan, Italy e-mail: daniela.galimberti@unimi.it