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Emanuel DiCicco-Bloom

Publications and source records attributed to Emanuel DiCicco-Bloom.

10 recordsLinked to original sources
PubMed2006

DNAse I pre-treatment markedly enhances detection of nuclear cyclin-dependent kinase inhibitor p57Kip2 and BrdU double immunostaining in embryonic rat brain.

As a member of the CIP/KIP family of cyclin-dependent kinase inhibitors (CKIs), p57Kip2 binds tightly to G1 cyclin/cyclin-dependent kinase complexes to block cell cycle progression. CKIs play critical roles in regulating the transition from proliferation to differentiation in many tissues, including the nervous system. Conversely, CKI dys-regulation contributes to neoplasia and cancer progression. While the combined detection of CKI immunoreactivity and S phase entry using bromodeoxyuridine (BrdU) incorporation may be particularly informative, successful immunostaining may be limited due to "masked" antigen epitopes and acid-induced signal degradation. We now report an improved double immunofluorescent method for detecting p57Kip2 and BrdU in paraformaldehyde-fixed frozen sections of embryonic rat brain. We substituted deoxyribonuclease I (DNAse I) for HCl pre-treatment to expose antigenic sites in frozen sections, and employed a biotinylated tyramide-based system to enhance p57Kip2 visualization. We identified a time- and dose-dependent relationship between DNAse I treatment and double labeling of p57Kip2 and BrdU, increasing both the numbers and intensities of immunopositive nuclei. With excess DNAse I treatment, however, there was signal degradation for both BrdU and total DNA, as reflected by DAPI staining. The use of DNAse I pre-treatment significantly increases the reliability and sensitivity of immunodetection of CKI nuclear factors, and should be useful for both developmental neurobiology studies as well as cancer diagnostic applications.

Animals↗
PubMed2006

Methylmercury elicits rapid inhibition of cell proliferation in the developing brain and decreases cell cycle regulator, cyclin E.

The developing brain is highly sensitive to methylmercury (MeHg). Still, the initial changes in cell proliferation that may contribute to long-term MeHg effects are largely undefined. Our previous studies with growth factors indicate that acute alterations of the G1/S-phase transition can permanently affect cell numbers and organ size. Therefore, we determined whether an environmental toxicant could also impact brain development with rapid (6-7h) effects on DNA synthesis and cell cycle machinery in neuronal precursors. In vivo studies in newborn rat hippocampus and cerebellum, two regions of postnatal neurogenesis, were followed by in vitro analysis of two precursor models, cortical and cerebellar cells, focusing on the proteins that regulate the G1/S transition. In postnatal day 7 (P7) pups, a single subcutaneous injection of MeHg (3microg/g) acutely (7h) decreased DNA synthesis in the hippocampus by 40% and produced long-term (2 weeks) reductions in total cell number, estimated by DNA quantification. Surprisingly, cerebellar granule cells were resistant to MeHg effects in vivo at comparable tissue concentrations, suggesting region-specific differences in precursor populations. In vitro, MeHg altered proliferation and cell viability, with DNA synthesis selectively inhibited at an early timepoint (6h) corresponding to our in vivo observations. Considering that G1/S regulators are targets of exogenous signals, we used a well-defined cortical cell model to examine MeHg effects on relevant cyclin-dependent kinases (CDK) and CDK inhibitors. At 6h, MeHg decreased by 75% levels of cyclin E, a cell cycle regulator with roles in proliferation and apoptosis, without altering p57, p27, or CDK2 nor levels of activated caspase 3. In aggregate, our observations identify the G1/S transition as an early target of MeHg toxicity and raise the possibility that cyclin E degradation contributes to both decreased proliferation and eventual cell death.

Analysis of Variance↗
PubMed2005

Moderate lead exposure elicits neurotrophic effects in cerebral cortical precursor cells in culture.

Lead (Pb) persists as an environmental toxicant despite aggressive environmental and occupational regulation. Neurotoxicological effects of acute Pb poisoning range from subtle cognitive deficits, to clumsiness and ataxia, to coma and seizures. In adult neurotoxicity, reductions of blood Pb levels are often associated with reversal of clinical signs. In children, however, the effects are more likely to endure, with even low levels of chronic Pb exposure correlating with decreasing IQ. These persistent effects likely result from neurodevelopmental insults, such as altered cell survival or maturation, although the mechanisms have not been fully defined. In the present study we define the effects of moderate-level Pb exposure on mammalian neurogenesis using a well-characterized cortical precursor model. Gestational day 14.5 rat cerebral cortical precursor cells were cultured in defined media and cell number, precursor proliferation, apoptosis, and neuritic process outgrowth were assessed following exposure to a range of Pb acetate concentrations. Surprisingly, whereas a concentration of 30 microg/ml Pb acetate was acutely toxic to neurons, concentrations between 1 and 10 microg/ml Pb acetate (approximately 3 microM and 30 microM Pb, respectively) increased cell number: 10 times as many cells exposed to 10 microg/ml Pb were present on day 4 as compared to control. The increase in cell number was not a result of increased proliferation, however, as DNA synthesis did not increase. Rather, Pb exposure maintained the survival of cortical precursors, as the progressive apoptosis occurring under control conditions was markedly reduced by the metal. Additionally, neuritic process initiation and outgrowth increased in a concentration-dependent manner, with processes four times as abundant on day 1 and twice as long on day 2. These results suggest that brief exposure to lead during neurogenesis directly affects cell survival and process development, potentially altering cortical arrangement. Consequently, alterations in neural circuitry may underlie some of the neurological effects of Pb exposure during brain development.

Animals↗
PubMed2003

Direct cAMP signaling through G-protein-coupled receptors mediates growth cone attraction induced by pituitary adenylate cyclase-activating polypeptide.

Developing axons are guided to their appropriate targets by environmental cues through the activation of specific receptors and intracellular signaling pathways. Here we report that gradients of pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide widely expressed in the developing nervous system, induce marked attraction of Xenopus growth cones in vitro. PACAP exerted its chemoattractive effects through PAC1, a PACAP-selective G-protein-coupled receptor (GPRC) expressed at the growth cone. Furthermore, the attraction depended on localized cAMP signaling because it was completely blocked either by global elevation of intracellular cAMP levels using forskolin or by inhibition of protein kinase A using specific inhibitors. Moreover, local direct elevation of intracellular cAMP by focal photolysis of caged cAMP compounds was sufficient to induce growth cone attraction. On the other hand, blockade of Ca2+, phospholipase C, or phosphatidyl inositol-3 kinase signaling pathways did not affect PACAP-induced growth cone attraction. Finally, PACAP-induced attraction also involved the Rho family of small GTPases and required local protein synthesis. Taken together, our results establish cAMP signaling as an independent pathway capable of mediating growth cone attraction induced by a physiologically relevant peptide acting through GPCRs. Such a direct cAMP pathway could potentially operate in other guidance systems for the accurate wiring of the nervous system.

Animals↗
PubMed2002

Pituitary adenylate cyclase-activating polypeptide and sonic hedgehog interact to control cerebellar granule precursor cell proliferation.

Although positive and negative signals control neurogenesis in the embryo, factors regulating postnatal proliferation are less well characterized. In the vertebrate cerebellum, Sonic Hedgehog (Shh) is an efficacious mitogen for cerebellar granule neuron precursors (GNPs), and mutations activating the Shh pathway are linked to medulloblastoma, a tumor derived from GNPs. Although the mitogenic effects of Shh can be blocked by increasing cAMP or protein kinase A activity, the physiological factors antagonizing this stimulation are undefined. In the embryo, pituitary adenylate cyclase-activating polypeptide (PACAP) receptor 1 (PAC1) signaling regulates neural precursor proliferation. We now show that in the developing cerebellum, PAC1 mRNA colocalizes with gene transcripts for Shh receptor Patched 1 and target gene Gli1 in the external germinal layer. We consequently investigated the interactions of PACAP and Shh in proliferation of purified GNPs in culture. Shh exhibited mitogenic activity in both rat and mouse cultures, stimulating DNA synthesis approximately 10-fold after 48 hr of exposure. PACAP markedly inhibited Shh-induced thymidine incorporation by 50 and 85% in rat and mouse GNPs, respectively, but did not significantly affect the stimulation induced by other mitogens. This selective effect was reproduced by the specific PAC1 agonist maxadilan, as well as by the adenylate cyclase activator forskolin, suggesting that PAC1 provides a potent inhibitory signal for Shh-induced proliferation in developing cerebellum. In contrast, in the absence of Shh, PACAP and maxadilan modestly stimulated DNA synthesis, an effect reproduced by activating protein kinase C. These observations suggest that G-protein-coupled receptors, such as PAC1, serve as sensors of environmental cues, coordinating diverse neurogenetic signals.

Adenylyl Cyclases↗
PubMed2002

Pituitary adenylate cyclase activating polypeptide anti-mitogenic signaling in cerebral cortical progenitors is regulated by p57Kip2-dependent CDK2 activity.

Generation of distinct cell types and numbers in developing cerebral cortex is subject to regulation by extracellular factors that positively or negatively control precursor proliferation. Although signals stimulating proliferation are well described, factors halting cell cycle progression are less well defined. At the molecular level, production and association of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs) regulate cycle progression. We now report that the endogenous peptide, pituitary adenylate cyclase activating polypeptide (PACAP), negatively regulates the cell cycle by inhibiting p57Kip2-dependent CDK2 activity in embryonic cortex. Protein levels of CDK2 and members of the CIP/KIP family of CKIs (p27Kip1, p57Kip2) were detected in developing rat cortex from embryonic day 13.5 through postnatal day 2. With advancing development, CDK2 protein levels decreased, whereas CKI expression increased, suggesting that stimulatory and inhibitory cycle proteins control cell cycle exit. Using a well defined, nonsynchronized, 8 hr precursor culture, PACAP decreased the fraction of cells crossing the G1/S boundary, inhibiting DNA synthesis by 35%. CDK2 kinase activity was inhibited 75% by PACAP, whereas kinase protein and its regulatory cyclin E subunit were unaffected. Moreover, decreased kinase activity was accompanied by a twofold increase in levels of p57Kip2 protein, but not p21Cip1 or p27Kip1, suggesting that p57Kip2 mediates PACAP anti-mitogenic effects. Indeed, immunoprecipitation of CDK2 complex revealed increased p57Kip2 association with the kinase and concomitant reduction in free inhibitor after PACAP exposure, suggesting that p57Kip2 interactions directly regulate CDK2 activity. These observations establish a mechanism whereby anti-mitogenic signals actively induce cell cycle withdrawal in developing cortex.

Animals↗
PubMed2002

Hippocampal granule neuron production and population size are regulated by levels of bFGF.

Numerous studies of the proliferative effects of basic fibroblast growth factor (bFGF) in culture, including neonatal and adult hippocampal precursors, suggest that the factor plays a ubiquitous and life-long role in neurogenesis. In contrast, in vivo, bFGF is devoid of effects on neurons in mature hippocampus, raising the possibility that bFGF exhibits developmental stage-specific activity in the complex animal environment. To define neurogenetic effects in the newborn, a single subcutaneous injection of bFGF (20 ng/gm) was administered to postnatal day 1 (P1) rats, and hippocampal DNA content was quantified: bFGF elicited an increase in total DNA throughout adulthood, by 48% at P4, 25% at P22, and 17% at P180, suggesting that bFGF increases hippocampal cell number. To define mechanisms, bromodeoxyuridine (BrdU) was injected at P1 and mitotically labelled cells were assessed at P22: there was a twofold increase in BrdU-positive cells in the dentate granule cell layer (GCL), indicating that bFGF enhanced the generation of neurons, or neuronogenesis, from a cohort of precursors. Moreover, enhanced mitosis and survival led to a 33% increase in absolute GCL neuron number, suggesting that neuron production depends on environmental levels of bFGF. To evaluate this possibility, bFGF-knockout mice were analyzed: hippocampal DNA content was decreased at all ages examined (P3, -42%; P21, -28%; P360, -18%), and total GCL neuron and glial fibrillary acidic protein (GFAP)-positive cell number were decreased by 30%, indicating that bFGF is necessary for normal hippocampal neurogenesis. We conclude that environmental levels of bFGF regulate neonatal hippocampal neurogenesis. As adult hippocampal neuronogenesis was unresponsive to bFGF manipulation in our previous study [Wagner, J.P., Black, I.B. & DiCicco-Bloom, E. (1999) J. Neurosci., 19, 6006], these observations suggest distinct, stage-specific roles of bFGF in the dentate gyrus granule cell lineage.

Aging↗
PubMedDate not supplied

Basic fibroblast growth factor exhibits dual and rapid regulation of cyclin D1 and p27 to stimulate proliferation of rat cerebral cortical precursors.

While extracellular signals play a major role in brain neurogenesis, little is known about the cell cycle machinery underlying mitogen stimulation of precursor proliferation. Current models suggest that the D cyclins function as primary sensors of extracellular mitogens. Here we define the mechanisms by which basic fibroblast growth factor (bFGF) stimulates cortical precursors, with particular attention to the responses of cell cycle promitogenic and antimitogenic regulators. bFGF produced a 4-fold increase in DNA synthesis and a 3-fold rise in bromodeoxyuridine labeling, suggesting that the factor promotes the G1/S transition. There was also a 3-fold increase in cyclin-dependent kinase 2 (CDK2) kinase activity, which is critical for S phase entry. CDK2 activation was apparently cyclin E dependent, since only its protein and mRNA levels were elevated at 24 h, whereas CDK2, p27KIP1 and p57KIP2 levels were unaltered. Late G1 phase CDK2/cyclin E activity depends on early G1 D cyclin function. Indeed, cyclin D1, but not cyclin D3, was upregulated selectively at 8 h after bFGF treatment, a time when cyclin E was unchanged. The sequential activation of cyclin D1 and cyclin E supports the idea that cyclin E gene transcription is regulated by cyclin-D/CDK4/6-mediated pRb phosphorylation and subsequent E2F transcription factor release. However, in addition to increased D1 cyclin, we unexpectedly detected a 75% reduction in p27KIP1 protein at 8 h, suggesting that both pro- and antimitogenic regulators are targets of extracellular mitogens during brain development.

Animals↗