The pattern of c-Fos expression and its refractory period in the brain of rats and monkeys

Intense activation of neurons triggers the appearance of immediate expression genes, including c-Fos. This gene is related to various signal cascades involved in biochemical processes such as neuronal plasticity, cell growth and mitosis. Here we investigate the expression pattern and the refractory period of c-Fos in rats and monkey's brains after stimulation with pentylenetetrazol. Rats and monkeys were sacrificed at various times after PTZ-induced seizure. Here we show that rats and monkeys already showed c-Fos expression at 0.5 h after seizure. Yet, the pattern of protein expression was longer in monkeys than rats, and also was not uniform (relative intensity) across different brain regions in monkeys as opposed to rats. in addition monkeys had a regional brain variation with regard to the temporal profile of c-Fos expression, which was not seen in rats. the refractory period after a second PTZ stimulation was also markedly different between rats and monkeys with the latter even showing a summatory effect on c-Fos expression after a second stimulation. However, assessment of c-Fos mRNA in rats indicated a post-transcriptional control mechanism underlying the duration of the refractory period. the difference in the protein expression pattern in rodents and primates characterizes a functional aspect of brain biochemistry that differs between these mammalian orders and may contribute for the more developed primate cognitive complexity as compared to rodents given c-Fos involvement in cognitive and learning tasks.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Physiol, BR-04039032 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Biochem, BR-04039032 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Physiol, BR-04039032 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Biochem, BR-04039032 São Paulo, SP, BrazilWeb of Scienc

CXCL12 N-terminal end is sufficient to induce chemotaxis and proliferation of neural stem/progenitor cells

Neural stem/progenitor cells (NSC) respond to injury after brain injuries secreting IL-1, IL-6, TNF-alpha, IL-4 and IL-10, as well as chemokine members of the CC and CXC ligand families. CXCL12 is one of the chemokines secreted at an injury site and is known to attract NSC-derived neuroblasts, cells that express CXCL12 receptor, CXCR4. Activation of CXCR4 by CXCL12 depends on two domains located at the N-terminal of the chemokine. in the present work we aimed to investigate if the N-terminal end of CXCL12, where CXCR4 binding and activation domains are located, was sufficient to induce NSC-derived neuroblast chemotaxis. Our data show that a synthetic peptide analogous to the first 21 amino acids of the N-terminal end of CXCL12, named PepC-C (KPVSLSYRCPCRFFESHIARA), is able to promote chemotaxis of neuroblasts in vivo, and stimulate chemotaxis and proliferation of CXCR4+ cells in vitro, without affecting NSC fate. We also show that PepC-C upregulates CXCL12 expression in vivo and in vitro. We suggest the N-terminal end of CXCL12 is responsible for a positive feedback loop to maintain a gradient of CXCL12 that attracts neuroblasts from the subventricular zone into an injury site. (C) 2013 the Authors. Published by Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Escola Paulista Med, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Neurobiol Lab, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Physiol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Neurobiol Lab, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Physiol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, São Paulo, BrazilFAPESP: 2005/04061-8FAPESP: 2012/00652-5CNPq: 573909/2008-3]Web of Scienc

Chondroitin Sulfate Impairs Neural Stem Cell Migration Through ROCK Activation

Brain injuries such as trauma and stroke lead to glial scar formation by reactive astrocytes which produce and secret axonal outgrowth inhibitors. Chondroitin sulfate proteoglycans (CSPG) constitute a well-known class of extracellular matrix molecules produced at the glial scar and cause growth cone collapse. The CSPG glycosaminoglycan side chains composed of chondroitin sulfate (CS) are responsible for its inhibitory activity on neurite outgrowth and are dependent on RhoA activation. Here, we hypothesize that CSPG also impairs neural stem cell migration inhibiting their penetration into an injury site. We show that DCX+ neuroblasts do not penetrate a CSPG-rich injured area probably due to Nogo receptor activation and RhoA/ROCK signaling pathway as we demonstrate in vitro with neural stem cells cultured as neurospheres and pull-down for RhoA. Furthermore, CS-impaired cell migration in vitro induced the formation of large mature adhesions and altered cell protrusion dynamics. ROCK inhibition restored migration in vitro as well as decreased adhesion size

Bloqueio da migração de células-tronco neurais por condroitim sulfato via ativação de rock

is characterized by the generation of newborn neurons from neural stem cell (NSC) localized mainly in the subventricular zone in the lateral ventricule and in the hippocampus. Injury alters normal neurogenesis and chemoattractive signals from the injury stimulate neuroblasts migration from the neurogenic niche to the injury site, where the formation of a glial scar takes place, with the production of axonal outgrowth inhibitory molecules, such as chondroitin sulfate proteoglycans. A convergent issue in signaling triggered by soluble factors and extracellular matrix (ECM) components is the activation of RhoGTPase family members, which perform essential roles in cellular migration process. Our hypothesis is that ECM components differently regulate neuroblasts migration through changes in adhesion dynamics and RhoGTPases activity. First of all, we performed a traumatic brain injury model in adult mice motor cortex and observed that neuroblasts migrated toward the injury site but were incapable to enter the injured area, due the presence of chondroitin sulfate proteoglycans. Next, we evaluated NSC migration in vitro using NSC cultured as neurospheres. We observed that chondroitin sulfate (CS) inhibited NSC migration; decreased initial migration speed and altered NSC protrusion dynamics. Besides, CS increased cellular adhesion size during protrusion and decreased NSC area. However, ROCK inhibition stimulated NSC migration both on laminin-coated or CS-coated surfaces and reversed some CS inhibitory effects, increasing cell spreading area and decreasing adhesion size. Finally, the inhibition of Nogo receptor (NgR1), reverted the inhibitory effect of CS on NSC migration in vitro. We conclude that CS exerts its inhibitory effect through the activation of NgR1 and ROCK.A neurogênese é um evento importante no sistema nervoso central em desenvolvimento e no adulto, e é caracterizada pela geração de novos neurônios a partir de células-tronco neurais (CTN) localizadas pricipalmente na zona subventricular e no hipocampo. Uma lesão altera o padrão normal da neurogênese e sinais quimioatrativos estimulam a migração de neuroblastos dos nichos neurogênicos para o local lesado, onde há a formação da cicatriz glial e produção de moléculas inibitórias do crescimento axonal, entre elas, proteoglicanos de condroitim sulfato. A sinalização desencadeada por fatores solúveis e por componentes da matriz ativa membros da família das RhoGTPases, que desempenham funções essenciais no processo de migração celular. Nossa hipótese é que componentes da matriz regulam diferentemente a migração de neuroblastos por alterações na dinâmica de adesão celular e na atividade de RhoGTPases. Utilizando modelo de lesão traumática no córtex motor do camundongo adulto observamos que os neuroblastos que migraram para o local lesado não foram capazes de penetrar na região, rica em proteoglicanos de condroitim sulfato. Em seguida, avaliamos a migração in vitro utilizando CTN cultivadas como neuroesferas. Observamos que o condroitim sulfato (CS) exerceu uma ação inibitória sobre a migração das CTN, diminuindo a velocidade inicial de migração e alterando a dinâmica de protrusão. Além disso, CS promoveu aumento no tamanho das adesões formadas durante a protrusão e dificultando o espraiamento das CTN. Porém, a inibição de ROCK estimulou a migração das CTN tanto em superfícies recobertas por laminina quanto por CS e reverteu alguns de seus efeitos inibitórios, como a retomada da capacidade de espraiamento celular, e a diminuição do tamanho das adesões. Finalmente, a inibição do receptor de Nogo (NgR1), reverteu o efeito inibitório de CS sobre a migração das CTN in vitro. Concluímos, assim, que a inibição da migração de CTN provocada por CS se dá pela ativação de ROCK via ativação de NgR1.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Culture, characterization and differentiation of neural precursors from the central nervous system of guinea pigs (Cavia porcellus Linnaeus, 1758)

Abstract: Potentially neurogenic areas were initially identified by incorporation of bromodeoxyuridine (BrdU) in cells underlying the subventricular zone (SVZ) of the lateral ventricles wall, hippocampus and olfactory bulbs of newborn guinea pigs. Neural precursors from the SVZ were cultured in suspension, generating neurospheres (NSFs), which, upon dissociation were able to generate new NSFs. Upon culture in the absence of growth factors, cells dissociated from NSFs displayed evidence for neural differentiation, giving rise to cells from neural lineage. Flow cytometry analysis for of NSFs-derived cells after differentiation revealed approximately 13.3% nestin positive, 5.5% Beta-III-tubulin positive, 9% GFAP positive and 7.8% mGalC positive. Functional assays by measurement of calcium influx upon gamma butiric amino acid (GABA) and glutamate stimuli, revealed stimulation in differentiated cells, an indicator of neuronal differentiation. The ability of guinea pig SVZ cells to originate functional neurons in vitro is promising for research and towards a future use of neural stem cells in the therapy of neurological disorders

The long non-coding RNA ANRASSF1 in the regulation of alternative protein-coding transcripts RASSF1A and RASSF1C in human breast cancer cells: implications to epigenetic therapy

Alternative protein-coding transcripts of the RASSF1 gene have been associated with dual functions in human cancer: while RASSF1C isoform has oncogenic properties, RASSF1A is a tumour suppressor frequently silenced by hypermethylation. Recently, the antisense long non-coding RNA RASSF1 (ANRASSF1) was implicated in a locus-specific mechanism for the RASSF1A epigenetic repression mediated by PRC2 (Polycomb Repressive Complex 2). Here, we evaluated the methylation patterns of the promoter regions of RASSF1A and RASSF1C and the expression levels of these RASSF1 transcripts in breast cancer and breast cancer cell lines. As expected, RASSF1C remained unmethylated and RASSF1A was hypermethylated at high frequencies in 75 primary breast cancers, and also in a panel of three mammary epithelial cells (MEC) and 10 breast cancer cell lines (BCC). Although RASSF1C was expressed in all cell lines, only two of them expressed the transcript RASSF1A. ANRASSF1 expression levels were increased in six BCCs. In vitro induced demethylation with 5-Aza-2ʹ-deoxicytydine (5-Aza-dC) resulted in up-regulation of RASSF1A and an inverse correlation with ANRASSF1 relative abundance in BCCs. However, increased levels of both transcripts were observed in two MECs (184A1 and MCF10A) after treatment with 5-Aza-dC. Overall, these findings indicate that ANRASSF1 is differentially expressed in MECs and BCCs. The lncRNA ANRASSF1 provides new perspectives as a therapeutic target for locus-specific regulation of RASSF1A