RESUMO

Dendritic spines are small, actin-rich protrusions that act as the receiving sites of most excitatory inputs in the central nervous system. The remodeling of the synapse architecture is mediated by actin cytoskeleton dynamics, a process precisely regulated by the small Rho GTPase family. Wnt ligands exert their presynaptic and postsynaptic effects during formation and consolidation of the synaptic structure. Specifically, Wnt5a has been identified as an indispensable synaptogenic factor for the regulation and organization of the postsynaptic side; however, the molecular mechanisms through which Wnt5a induces morphological changes resulting from actin cytoskeleton dynamics within dendritic spines remain unclear. In this work, we employ primary rat hippocampal cultures and HT22 murine hippocampal neuronal cell models, molecular and pharmacological tools, and fluorescence microscopy (laser confocal and epifluorescence) to define the Wnt5a-induced molecular signaling involved in postsynaptic remodeling mediated via the regulation of the small Rho GTPase family. We report that Wnt5a differentially regulates the phosphorylation of Cofilin in neurons through both Ras-related C3 botulinum toxin substrate 1 and cell division cycle 42 depending on the subcellular compartment and the extracellular calcium levels. Additionally, we demonstrate that Wnt5a increases the density of dendritic spines and promotes their maturation via Ras-related C3 botulinum toxin substrate 1. Accordingly, we find that Wnt5a requires the combined activation of small Rho GTPases to increase the levels of filamentous actin, thus promoting the stability of actin filaments. Altogether, these results provide evidence for a new mechanism by which Wnt5a may target actin dynamics, thereby regulating the subsequent morphological changes in dendritic spine architecture.

Assuntos

RESUMO

BACKGROUND TcP21 is a ubiquitous secreted protein of Trypanosoma cruzi and its recombinant form (rP21) promotes parasite cell invasion and acts as a phagocytosis inducer by activating actin polymerisation in the host cell. OBJECTIVE Our goal was to evaluate if the additional supplementation of rP21 during a prime/boost/challenge scheme with T. cruzi TCC attenuated parasites could modify the well-known protective behavior conferred by these parasites. METHODS The humoral immune response was evaluated through the assessment of total anti-T. cruzi antibodies as well as IgG subtypes. IFN-γ, TNF-α and IL-10 were measured in supernatants of splenic cells stimulated with total parasite homogenate or rP21. FINDINGS Our results demonstrated that, when comparing TCC+rP21 vs. TCC vaccinated animals, the levels of IFN-γ were significantly higher in the former group, while the levels of IL-10 and TNF-α were significantly lower. Further, the measurement of parasite load after lethal challenge showed an exacerbated infection and parasite load in heart and skeletal muscle after pre-treatment with rP21, suggesting the important role of this protein during parasite natural invasion process. MAIN CONCLUSION Our results demonstrated that rP21 may have adjuvant capacity able to modify the cytokine immune profile elicited by attenuated parasites.

Assuntos

RESUMO

RhoGDI proteins have been implicated in several human cancers; changes in their expression levels have shown pro- or anti-tumorigenic effects. Pancreatic Ductal Adenocarcinoma (PDAC) is a complex pathology, with poor prognosis, and most patients die shortly after diagnosis. Efforts have been focused on understanding the role of RhoGDI's in PDAC, specially, RhoGDI1 and RhoGDI2. However, the role of RhoGDI3 has not been studied in relation to cancer or to PDAC. Here, we characterized the expression and functionality of RhoGDI3 and its target GTPases, RhoG and RhoB in pancreatic cell lines from both normal pancreatic tissue and tissue in late stages of PDAC, and compared them to human biopsies. Through immunofluorescences, pulldown assays and subcellular fractionation, we found a reduction in RhoGDI3 expression in the late stages of PDAC, and this reduction correlates with tumor progression and aggressiveness. Despite the reduction in the expression of RhoGDI3 in PDAC, we found that RhoB was underexpressed while RhoG was overexpressed, suggesting that cancerous cells preserve their capacity to activate this pathway, thus these cells may be more eager to response to the stimuli needed to proliferate and become invasive unlike normal cells. Surprisingly, we found nuclear localization of RhoGDI3 in non-cancerous pancreatic cell line and normal pancreatic tissue biopsies, which could open the possibility of novel nuclear functions for this protein, impacting gene expression regulation and cellular homeostasis.

Assuntos

RESUMO

Introdução: O carcinoma de vulva é um tumor de baixa ocorrência, sendo responsável por menos de 3% de todos os tumores malignos que acometem mulheres. Em mulheres jovens a ocorrência da doença está atrelada a fatores de risco como tabagismo e infecção por HPV, entretanto em mulheres acima dos 50 anos ocorre por mecanismos genéticos ainda pouco elucidados. Nos últimos anos poucos autores estudaram alterações genômicas em carcinomas vulvares, contudo nenhum deles determinou um fator prognóstico definitivo, nem tampouco correlacionou esses achados com a expressão gênica, mesmo representando pontos-chave na compreensão do processo de carcinogênese. A partir da análise de arranjos de hibridação genômica comparativa (CGH-array) realizada previamente em nosso laboratório, dois genes candidatos, ROCK1 e RhoD, localizados em regiões com alta frequência de ganhos em nossas amostras de neoplasias vulvares, foram selecionados para este estudo. Objetivo: Validar a expressão dos genes candidatos, ROCK1 e RhoD, que foram identificados em regiões com ganho de cópias nas amostras de carcinoma vulvar pelo método de CGHarray, a fim de determinar melhores e mais acurados valores prognósticos no carcinoma vulvar...

Introduction: The vulvar carcinoma is a low occurrence tumor, accounting for less than 3% of all malignant tumors that affect women. In young women the occurrence of the disease is linked to risk factors such as smoking and HPV infection, but the majority of cases of vulvar cancer occurs in women over 50 years by genetic mechanisms still poorly understood. Recently, few authors studied genomic changes in vulvar carcinomas, however none of them determined on prognostic factor, nor correlate these findings with gene expression, even representing key points in understanding the carcinogenesis process. From the analysis of comparative genomic hybridization arrays (array CGH) previously performed in our laboratory, two candidate genes, ROCK1 and Rhod, located in regions with high frequency gains in our samples of vulvar cancer were selected for this study. Objective: To validate the expression of the candidate genes, RhoD and ROCK1, which have been identified in regions with a gain of copies in vulvar carcinoma samples by CGH-array method, in order to determine the best and most accurate prognostic values in vulvar carcinoma. Methods: 16 cases of vulvar cancer were rescued from AC Camargo Cancer Center’s Biobank...

Assuntos

RESUMO

Evidence has been obtained that indicates the presence of small 22 kDa GTP-binding Rho proteins through ADP-ribosylation by Clostridium botulinum C3 exotoxin in Mucor circinelloides. Rho protein was detected at all stages of growth studied. During polarized growth, both under aerobic conditions and during the yeast-mycelia transition, the radiolabeling of the [32P]ADP-ribosylated protein increased when tube formation occurred and decreased as the hyphae branched. However, when Mucor grew isotropically, the Rho protein band was thick and its intensity did not vary significantly even after bud formation and separation of daughter cells. Crude extracts of yeast and mycelial cells exhibited a broad 22 kDa band of the [32P]ADP-ribosylated Rho protein that was resolved into a protein with a pI of 6.0, after two-dimensional electrophoresis, corresponding to the Rho1p homolog. Furthermore, [32P]ADP-ribosylated Rho protein from soluble and particulate extracts of multipolarized mycelial cells obtained from the yeast-mycelia transition was separated into two proteins with pI of 6.0 and 6.4, respectively, after two-dimensional electrophoresis. These correspond to the Rho1p and Rho3p homologs, respectively. Therefore, our results show that an increase in Rho accumulation is associated with polarized growth.

Assuntos