Early membrane estrogenic effects required for full expression of slower genomic actions in a nerve cell line.

Interpretations of steroid hormone actions as slow, nuclear, transcriptional events have frequently been seen as competing against inferences of rapid membrane actions. We have discovered conditions where membrane-limited effects potentiate later transcriptional actions in a nerve cell line. Making use of a two-pulse hormonal schedule in a transfection system, early and brief administration of conjugated, membrane-limited estradiol was necessary but not sufficient for full transcriptional potency of the second estrogen pulse. Efficacy of the first pulse depended on intact signal transduction pathways. Surprisingly, the actions of both pulses were blocked by a classical estrogen receptor (ER) antagonist. Thus, two different modes of steroid hormone action can synergize.

Phosphatidylcholine-specific phospholipase C regulates glutamate-induced nerve cell death

Phosphatidylcholine-specific phospholipase C (PC-PLC) is a necessary intermediate in transducing apoptotic signals for tumor necrosis factor and Fas/Apo-1 ligands in nonneuronal cells. The data presented here show that PC-PLC also is required in oxidative glutamate-induced programmed cell death of both immature cortical neurons and a hippocampal nerve cell line, HT22. In oxidative glutamate toxicity, which is distinct from excitotoxicity, glutamate interferes with cystine uptake by blocking the cystine/glutamate antiporter, indirectly causing a depletion of intracellular glutathione. A PC-PLC inhibitor blocks oxidative glutamate toxicity, and exogenous PC-PLC potentiates glutamate toxicity. The inhibition of PC-PLC uncouples the cystine uptake from glutamate inhibition, allowing the maintenance of glutathione synthesis and cell viability. These data suggest that PC-PLC modulates neuronal cell death through a mechanism that is distinct from that involved in nonneuronal apoptosis.

On the actions that one nerve cell can have on another: Distinguishing “drivers” from “modulators”

When one nerve cell acts on another, its postsynaptic effect can vary greatly. In sensory systems, inputs from “drivers” can be differentiated from those of “modulators.” The driver can be identified as the transmitter of receptive field properties; the modulator can be identified as altering the probability of certain aspects of that transmission. Where receptive fields are not available, the distinction is more difficult and currently is undefined. We use the visual pathways, particularly the thalamic geniculate relay for which much relevant evidence is available, to explore ways in which drivers can be distinguished from modulators. The extent to which the distinction may apply first to other parts of the thalamus and then, possibly, to other parts of the brain is considered. We suggest the following distinctions: Cross-correlograms from driver inputs have sharper peaks than those from modulators; there are likely to be few drivers but many modulators for any one cell; and drivers are likely to act only through ionotropic receptors having a fast postsynaptic effect whereas modulators also are likely to activate metabotropic receptors having a slow and prolonged postsynaptic effect.

Transcriptional Regulation of Fibroblast Growth Factor-2 Expression in Human Astrocytes: Implications for Cell Plasticity

Induction of the fibroblast growth factor-2 (FGF-2) gene and the consequent accumulation of FGF-2 in the nucleus are operative events in mitotic activation and hypertrophy of human astrocytes. In the brain, these events are associated with cellular degeneration and may reflect release of the FGF-2 gene from cell contact inhibition. We used cultures of human astrocytes to examine whether expression of FGF-2 is also controlled by soluble growth factors. Treatment of subconfluent astrocytes with interleukin-1β, epidermal or platelet-derived growth factors, 18-kDa FGF-2, or serum or direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or adenylate cyclase with forskolin increased the levels of 18-, 22-, and 24-kDa FGF-2 isoforms and FGF-2 mRNA. Transfection of FGF-2 promoter–luciferase constructs identified a unique −555/−513 bp growth factor-responsive element (GFRE) that confers high basal promoter activity and activation by growth factors to a downstream promoter region. It also identified a separate region (−624/−556 bp) essential for PKC and cAMP stimulation. DNA–protein binding assays indicated that novel cis-acting elements and trans-acting factors mediate activation of the FGF-2 gene. Southwestern analysis identified 40-, 50-, 60-, and 100-kDa GFRE-binding proteins and 165-, 112-, and 90-kDa proteins that interacted with the PKC/cAMP-responsive region. The GFRE and the element essential for PKC and cAMP stimulation overlap with the region that mediates cell contact inhibition of the FGF-2 promoter. The results show a two-stage regulation of the FGF-2 gene: 1) an initial induction by reduced cell contact, and 2) further activation by growth factors or the PKC-signaling pathway. The hierarchic regulation of the FGF-2 gene promoter by cell density and growth factors or PKC reflects a two-stage activation of protein binding to the GFRE and to the PKC/cAMP-responsive region, respectively.

Genetic programs of epithelial cell plasticity directed by transforming growth factor-β

Epithelial–mesenchymal transitions (EMTs) are an essential manifestation of epithelial cell plasticity during morphogenesis, wound healing, and tumor progression. Transforming growth factor-β (TGF-β) modulates epithelial plasticity in these physiological contexts by inducing EMT. Here we report a transcriptome screen of genetic programs of TGF-β-induced EMT in human keratinocytes and propose functional roles for extracellular response kinase (ERK) mitogen-activated protein kinase signaling in cell motility and disruption of adherens junctions. We used DNA arrays of 16,580 human cDNAs to identify 728 known genes regulated by TGF-β within 4 hours after treatment. TGF-β-stimulated ERK signaling mediated regulation of 80 target genes not previously associated with this pathway. This subset is enriched for genes with defined roles in cell–matrix interactions, cell motility, and endocytosis. ERK-independent genetic programs underlying the onset of EMT involve key pathways and regulators of epithelial dedifferentiation, undifferentiated transitional and mesenchymal progenitor phenotypes, and mediators of cytoskeletal reorganization. The gene expression profiling approach delineates complex context-dependent signaling pathways and transcriptional events that determine epithelial cell plasticity controlled by TGF-β. Investigation of the identified pathways and genes will advance the understanding of molecular mechanisms that underlie tumor invasiveness and metastasis.

The nerve cell as a unit.

From Journal of Comparative Neurology.

Mnitoramento do desenvolvimento infantil realizado no Brasil

Objective: To review scientific literature in order to check how infant development surveillance is being carried out in Brazil. Data sources: Search on databases (PubMed, Medline, SciELO and CAPES Database Thesis) for studies on medical practices related to surveillance and monitoring of child development in Brazil from 2000 to 2011. The terms used for research were: child development surveillance, early intervention, developmental screening, and developmental screening tests. There were ten texts on the subject under study. Original articles, reviews, and thesis were analyzed, as well as the reference lists of publications on the topic. Data synthesis: Studies on monitoring of child development in Brazil showed major failures from pediatrician formation to clinical practice. Conclusions: It is urgent to offer continued medical education to pediatricians in order to update their knowledge about child development monitoring, especially due to the increasing numbers of preterm infants.

Motor Recovery and Synaptic Preservation after Ventral Root Avulsion and Repair with a Fibrin Sealant Derived from Snake Venom

Background:Ventral root avulsion is an experimental model of proximal axonal injury at the central/peripheral nervous system interface that results in paralysis and poor clinical outcome after restorative surgery. Root reimplantation may decrease neuronal degeneration in such cases. We describe the use of a snake venom-derived fibrin sealant during surgical reconnection of avulsed roots at the spinal cord surface. The present work investigates the effects of this fibrin sealant on functional recovery, neuronal survival, synaptic plasticity, and glial reaction in the spinal motoneuron microenvironment after ventral root reimplantation.Methodology/Principal Findings:Female Lewis rats (7 weeks old) were subjected to VRA and root replantation. The animals were divided into two groups: 1) avulsion only and 2) replanted roots with fibrin sealant derived from snake venom. Post-surgical motor performance was evaluated using the CatWalk system twice a week for 12 weeks. The rats were sacrificed 12 weeks after surgery, and their lumbar intumescences were processed for motoneuron counting and immunohistochemistry (GFAP, Iba-1 and synaptophysin antisera). Array based qRT-PCR was used to evaluate gene regulation of several neurotrophic factors and receptors as well as inflammatory related molecules. The results indicated that the root reimplantation with fibrin sealant enhanced motor recovery, preserved the synaptic covering of the motoneurons and improved neuronal survival. The replanted group did not show significant changes in microglial response compared to VRA-only. However, the astroglial reaction was significantly reduced in this group.Conclusions/Significance:In conclusion, the present data suggest that the repair of avulsed roots with snake venom fibrin glue at the exact point of detachment results in neuroprotection and preservation of the synaptic network at the microenvironment of the lesioned motoneurons. Also such procedure reduced the astroglial reaction and increased mRNA levels to neurotrophins and anti-inflammatory cytokines that may in turn, contribute to improving recovery of motor function. © 2013 Barbizan et al.

Exploring the multifactorial nature of autism through computational systems biology: Calcium and the Rho GTPase RAC1 under the spotlight

Autism is a neurodevelopmental disorder characterized by impaired social interaction and communication accompanied with repetitive behavioral patterns and unusual stereotyped interests. Autism is considered a highly heterogeneous disorder with diverse putative causes and associated factors giving rise to variable ranges of symptomatology. Incidence seems to be increasing with time, while the underlying pathophysiological mechanisms remain virtually uncharacterized (or unknown). By systematic review of the literature and a systems biology approach, our aims were to examine the multifactorial nature of autism with its broad range of severity, to ascertain the predominant biological processes, cellular components, and molecular functions integral to the disorder, and finally, to elucidate the most central contributions (genetic and/or environmental) in silico. With this goal, we developed an integrative network model for gene-environment interactions (GENVI model) where calcium (Ca2+) was shown to be its most relevant node. Moreover, considering the present data from our systems biology approach together with the results from the differential gene expression analysis of cerebellar samples from autistic patients, we believe that RAC1, in particular, and the RHO family of GTPases, in general, could play a critical role in the neuropathological events associated with autism. © 2013 Springer Science+Business Media New York.

Autocrine secretion of IGF2 regulates adult ß cell functional plasticity

In the pathogenesis of type 2 diabetes, hyperglycemia appears when ß cell mass and insulin secretory capacity are no longer sufficient to compensate for insulin resistance. The reduction in ß cell mass results from increased apoptosis. Therefore, finding strategies to preserve ß cell mass and function may be useful for the treatment or prevention of diabetes. Glucagon-like peptide-1 (GLP-1) protects ß cells against apoptosis, increases their glucose competence, and induces their proliferation. Previous studies in the lab of Prof. Bernard Thorens showed that the GLP-1 anti- apoptotic effect was mediated by robust up-regulation of IGF-1R expression, and this was paralleled with an increase in Akt phosphorylation. This effect was dependent not only on increased IGF-1R expression but also on the autocrine secretion of insulin-like growth factor 2 (IGF2). They also demonstrated that GLP-1 up-regulated IGF-1R expression by a protein a kinase A-dependent translational control mechanism. The main aim of this PhD work has been to further investigate the role of the IGF2/IGF-1 Receptor autocrine loop in ß cell function and to determine the physiological role of IGF2 in ß cell plasticity and its regulation by nutrients. This PhD thesis is divided in 3 chapters. The first chapter describes the role of IGF2/IGF-1R autocrine loop in ß cell glucose competence and proliferation. Here using MIN6 cells and primary mouse islets as an experimental model we demonstrated that the glucose competence of these cells was dependent on the level of IGF-1R expression and on IGF2 secretion. Furthermore, we showed that GLP-1-induced primary ß cell proliferation was significantly reduced by Igf-lr gene inactivation and by IGF2 immunoneutralization or knockdown. In the second chapter we examined the role of this IGF2/IGF-1R autocrine loop on the ß cell functional plasticity during ageing, pregnancy, and in response to acute induction of insulin resistance using mice with ß cell-specific inactivation of ig/2. Here we showed a gender-dependent role of ß cell IGF2 in ageing and high fat diet-induced metabolic stress; we demonstrated that the autocrine secretion of IGF2 is essential for ß cell mass adaptation during pregnancy. Further we also showed that this autocrine loop plays an important role in ß cell expansion in response to acute induction of insulin resistance. The aim of the third chapter was to investigate whether we can modulate the expression and secretion of IGF2 by nutrients in order to increase the activity of autocrine loop. Here we showed that glutamine induces IGF2 biosynthesis and its fast secretion through the regulated pathway, a mechanism enhanced in the presence of glucose. Furthermore, we demonstrated that glutamine-mediated Akt phosphorylation is dependent on IGF2 secretion, indicating that glutamine controls the activity of the IGF2/IGF1R autocrine loop through IGF2 up-regulation. In summary, this PhD work highlights that autocrine secretion of IGF2 is required for compensatory ß cell adaptation to ageing, pregnancy, and insulin resistance. Moreover IGF2/IGF1R autocrine loop is regulated by two feeding-related cues, GLP-1 to increase IGF-1R expression and glutamine to control IGF2 biosynthesis and secretion. -- Dans le diabète de type 2, lorsque la sécrétion d'insuline des cellules Beta du pancréas n'est plus suffisante pour compenser la résistance à l'insuline, une hyperglycémie est observée. Cette baisse de sécrétion d'insuline est Causée par la diminution de la masse de cellules Beta suite à l'augmentation du phénomène de mort cellulaire ou « apoptose ». En diabétologie, une des stratégies médicales concerne la préservation des cellules Beta du pancréas. Une des protéines intervenant dans cette fonction est GLP-1 (Glucagon-like peptide-1). GLP-1 est capable de protéger les cellules Beta contre la mort cellulaire et d'induire leur prolifération. Des études précédemment menées dans le laboratoire du Professeur Bernard Thorens ont montrées que l'activité « anti-apoptotique » de GLP-1 est le résultat l'une augmentation de l'expression du gène IGF-1R sous la dépendance de la sécrétion autocrine d'IGF2 (Insulin-Like Growth Factor). Le but de mon travail de thèse aura été d'étudier le mécanisme de la régulation de GLP-1 par IGF2 et plus précisément de déterminer le rôle physiologique d'IGF2 dans la plasticité des cellules ß ainsi que sa régulation par les nutriments. Ce manuscrit est ainsi divisé en trois chapitres : Le premier chapitre décrit la fonction d'IGF2/IGF- R1 dans la réponse des cellules Beta au glucose ainsi que dans leur capacité à proliférer. Dans ce chapitre nous avons montré l'importance du niveau d'expression d'IGFR-1 et de la sécrétion d'IGF2 dans la régulation du métabolisme du glucose. Dans un deuxième chapitre, nous étudions la boucle de régulation IGF2/IGF-R1 sur la plasticité des cellules Beta lors du vieillissement, de la grossesse ainsi que dans un modèle de souris résistantes à l'insuline. Cette étude met en évidence un dimorphisme sexuel dans le rôle d'IGF2 lors du vieillissement et lors d'un stress métabolique. Nous montrons également l'importance d'IGF2 pour l'adaptation des cellules Beta tout au long de la grossesse ou lors du phénomène de résistance à l'insuline. Dans un troisième chapitre, nous mettons en évidence la possibilité de moduler l'expression et la sécrétion d'IGF2 par les nutriments. En conclusion, ce travail de thèse aura permis de mettre en évidence l'importance d'IGF2 dans la plasticité des cellules ß, une plasticité indispensable lors du vieillissement, de la grossesse ou encore dans le cas d'une résistance à l'insuline.

Adipose-derived stem cells enhance peripheral nerve regeneration.

Traumatic injuries resulting in peripheral nerve lesions often require a graft to bridge the gap. Although autologous nerve auto-graft is still the first-choice strategy in reconstructions, it has the severe disadvantage of the sacrifice of a functional nerve. Cell transplantation in a bioartificial conduit is an alternative strategy to create a favourable environment for nerve regeneration. We decided to test new fibrin nerve conduits seeded with various cell types (primary Schwann cells and adult stem cells differentiated to a Schwann cell-like phenotype) for repair of sciatic nerve injury. Two weeks after implantation, the conduits were removed and examined by immunohistochemistry for axonal regeneration (evaluated by PGP 9.5 expression) and Schwann cell presence (detected by S100 expression). The results show a significant increase in axonal regeneration in the group of fibrin seeded with Schwann cells compared with the empty fibrin conduit. Differentiated adipose-derived stem cells also enhanced regeneration distance in a similar manner to differentiated bone marrow mesenchymal stem cells. These observations suggest that adipose-derived stem cells may provide an effective cell population, without the limitations of the donor-site morbidity associated with isolation of Schwann cells, and could be a clinically translatable route towards new methods to enhance peripheral nerve repair.

Mutant p53 promotes epithelial-mesenchymal plasticity and enhances metastasis in mammary carcinomas of WAP-T mice.

To study the postulated mutant p53 (mutp53) "gain of function" effects in mammary tumor development, progression and metastasis, we crossed SV40 transgenic WAP-T mice with mutant p53 transgenic WAP-mutp53 mice. Compared to tumors in monotransgenic WAP-T mice, tumors in bitransgenic WAP-T x WAP-mutp53 mice showed higher tumor grading, enhanced vascularization, and significantly increased metastasis. Bitransgenic tumors revealed a gene signature associated with the oncogenic epithelial-mesenchymal transition pathway (EMT gene signature). In cultures of WAP-T tumor-derived G-2 cancer cells, which are comprised of subpopulations displaying "mesenchymal" and "epithelial" phenotypes, this EMT gene signature was associated with the "mesenchymal" compartment. Furthermore, ectopic expression of mutp53 in G-2 cells sufficed to induce a strong EMT phenotype. In contrast to these in vitro effects, monotransgenic and bitransgenic tumors were phenotypically similar suggesting that in vivo the tumor cell phenotype might be under control of the tumor microenvironment. In support, orthotopic transplantation of G-2 cells as well as of G-2 cells expressing ectopic mutp53 into syngeneic mice resulted in tumors with a predominantly epithelial phenotype, closely similar to that of endogenous primary tumors. We conclude that induction of an EMT gene signature by mutp53 in bitransgenic tumors primarily promotes tumor cell plasticity, that is, the probability of tumor cells to undergo EMT processes under appropriate stimuli, thereby possibly increasing their potential to disseminate and metastasize.

The required beta cell research for improving treatment of type 2 diabetes.

In healthy individuals, insulin resistance is associated with physiological conditions such as pregnancy or body weight gain and triggers an increase in beta cell number and insulin secretion capacity to preserve normoglycaemia. Failure of this beta cell compensation capacity is a fundamental cause of diabetic hyperglycaemia. Incomplete understanding of the molecular mechanisms controlling the plasticity of adult beta cells mechanisms and how these cells fail during the pathogenesis of diabetes strongly limits the ability to develop new beta cell-specific therapies. Here, current knowledge of the signalling pathways controlling beta cell plasticity is reviewed, and possible directions for future research are discussed.

NADPH-diaphorase-positive ganglion cells of the rat adrenal gland: age- and sex-related changes in their number, size, and distribution.

The rat adrenal gland contains ganglion cells able to synthesize nitric oxide (NO). This messenger molecule controls and modulates adrenal secretory activity and blood flow. The present study analyzed the number, size, and distribution of NO-producing adrenal neurons in adulthood and during postnatal development by means of beta-nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. This method reliably visualizes the enzyme responsible for NO generation. The reactive neurons per adrenal gland were 350-400 in both male and female adult rats. The positive nerve cell bodies were mostly located in the medulla, few being detected within the cortex and the subcapsular region. Dual labeling with anti-microtubule-associated protein 2 antibody, specific for neuronal elements, confirmed this distribution. Anti-microtubule-associated protein 1b antibody identified a subset of NADPH-d-positive neurons, displaying different degrees of maturation according to their position within the adrenal gland. At birth, there were about 220 NADPH-d-labeled neurons per adrenal gland in both sexes. As confirmed by dual immunocytochemical labeling, their great majority was evenly distributed between the cortex and the subcapsular region, the medulla being practically devoid of stained neurons. After birth, the number of adrenal NADPH-d-positive ganglion cells displayed a strong postnatal increase and reached the adult-like distribution after 1-2 months. During the period of increase, there was a transient difference in the numbers of these cells in the two sexes. Thus we present here evidence of plasticity in the number, size, and distribution of NADPH-d-positive adrenal neurons between birth and adulthood; in addition, we describe transient sex-related differences in their number and distribution during the 2nd postnatal week, which are possibly related to the epigenetic action of gonadal hormones during this period.