Liliana Matos

Superoxide dismutase expression in human cumulus oophorus cells

Success in assisted reproductive techniques (ART) is influenced by gamete and embryo quality but the assessment of these parameters has been thwarted by the lack of reliable biomarkers. Follicular fluid and cumulus oophorus cells may provide biomarkers due to their close relationship to the oocyte. These cells produce antioxidants and thus protect the oocyte from oxidative damage exerted by reactive oxygen species (ROS). ROS and antioxidants are known to intervene in reproductive physiology and pathology, but their roles are unclear. It is hypothesized that superoxide dismutase (SOD), a first line antioxidant enzyme, is associated with oocyte quality. Cells obtained in the course of ART for the treatment of infertility due to male factor or female pathology were processed for SOD intracellular isoforms (CuZnSOD and MnSOD) immunodetection, total SOD activity and isoforms content. Cells presented strong positive staining for CuZnSOD and MnSOD. SOD activity decreased with increasing female age but was increased in endometriosis and in ovulatory dysfunction. When male factor was the cause for infertility, successful ART was associated with higher SOD activity. Variations in SOD emphasize the relevance of oxidative stress in the oocyte maturation process. These variations also suggest that SOD is a potential biomarker for ART success.

Copper ability to induce premature senescence in human fibroblasts.

Human diploid fibroblasts (HDFs) exposed to subcytotoxic concentrations of oxidative or stressful agents, such as hydrogen peroxide, tert-butylhydroperoxide, or ethanol, undergo stress-induced premature senescence (SIPS). This condition is characterized by the appearance of replicative senescence biomarkers such as irreversible growth arrest, increase in senescence-associated β-galactosidase (SA β-gal) activity, altered cell morphology, and overexpression of several senescence-associated genes. Copper is an essential trace element known to accumulate with ageing and to be involved in the pathogenesis of some age-related disorders. Past studies using either yeast or human cellular models of ageing provided evidence in favor of the role of intracellular copper as a longevity modulator. In the present study, copper ability to cause the appearance of senescent features in HDFs was assessed. WI-38 fibroblasts exposed to a subcytotoxic concentration of copper sulfate presented inhibition of cell proliferation, cell enlargement, increased SA β-gal activity, and mRNA overexpression of several senescence-associated genes such as p21, apolipoprotein J (ApoJ), fibronectin, transforming growth factor β-1 (TGF β1), insulin growth factor binding protein 3, and heme oxygenase 1. Western blotting results confirmed enhanced intracellular p21, ApoJ, and TGF β1 in copper-treated cells. Thus, similar to other SIPS-inducing agents, HDF exposure to subcytotoxic concentration of copper results in premature senescence. Further studies will unravel molecular mechanisms and the biological meaning of copper-associated senescence and lead to a better understanding of copper-related disorder establishment and progression.

ER Stress Response in Human Cellular Models of Senescence

The aging process is characterized by progressive accumulation of damaged biomolecules in the endoplasmic reticulum, as result of increased oxidative stress accompanying cellular senescence. In agreement, we hypothesized that WI-38 human cellular models of replicative senescence and stress-induced premature senescence (SIPS) induced by hydrogen peroxide (H2O2-SIPS) or copper sulfate (CuSO4-SIPS) would present endoplasmic reticulum chaperoning mechanisms impairment and unfolded protein response activation. Results show that in replicative senescence and CuSO4-SIPS, immunoglobulin binding protein, calnexin, protein disulfide isomerase, and ER oxireductin-1 levels adjust to restore proteostasis and inositol-requiring enzyme-1 (IRE1)-, activating transcription factor 6 (ATF6)-, and pancreatic ER kinase (PERK)-mediated unfolded protein response are activated. However, H2O2-SIPS does not exhibit IRE1 and ATF6 pathways activation but a PERK-mediated upregulation of CCAAT/enhancer-binding protein homologous protein, showing that CuSO4-SIPS mimics better the endoplasmic reticulum molecular events of replicative senescence than H2O2-SIPS. Moreover, unfolded protein response activation is required for both SIPS models induction, because PERK and IRE1 inhibitors decreased senescence-associated beta-galactosidase appearance. In CuSO4-SIPS, the decrease in senescence levels is associated with PERK-driven, but IRE1 independent, cell cycle arrest while in H2O2-SIPS cell proliferation is PERK independent. These results add a step further on the molecular mechanisms that regulate senescence induction; moreover, they validate CuSO4-SIPS model as a useful tool to study cellular stress responses during aging, hoping to postpone age-related health decline.

Therapeutic strategies based on modified U1 snRNAs and chaperones for Sanfilippo C splicing mutations

BackgroundMutations affecting RNA splicing represent more than 20% of the mutant alleles in Sanfilippo syndrome type C, a rare lysosomal storage disorder that causes severe neurodegeneration. Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides.MethodsIn this study we tested several therapeutic approaches specifically designed for different splicing mutations depending on how the mutations affect mRNA processing. For three mutations that affect the donor site (c.234 + 1G > A, c.633 + 1G > A and c.1542 + 4dupA), different modified U1 snRNAs recognizing the mutated donor sites, have been developed in an attempt to rescue the normal splicing process. For another mutation that affects an acceptor splice site (c.372-2A > G) and gives rise to a protein lacking four amino acids, a competitive inhibitor of the HGSNAT protein, glucosamine, was tested as a pharmacological chaperone to correct the aberrant folding and to restore the normal trafficking of the protein to the lysosome.ResultsPartial correction of c.234 + 1G > A mutation was achieved with a modified U1 snRNA that completely matches the splice donor site suggesting that these molecules may have a therapeutic potential for some splicing mutations. Furthermore, the importance of the splice site sequence context is highlighted as a key factor in the success of this type of therapy. Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding.ConclusionsWe have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications.

AGEs, contributors to placental bed vascular changes leading to preeclampsia

Abstract Glycation of proteins or other biomolecules and their further long-term degradation result in the formation of advanced glycation end products, AGEs. AGEs and other ligands interact with their receptors, RAGEs, localized to a variety of tissues, but mainly in endothelium and vascular wall cells. This interaction triggers diverse signaling pathways that converge on the activation of NF-κB and the initiation of a local inflammatory reaction that, when prolonged, results in dysfunctional features. Preeclampsia is a serious vascular disorder centred at the placenta–uterine interface, the placental bed, but the condition extends to the mother´s circulation. RAGEs have notorious expression in the placental bed tissues along pregnancy but, in addition, RAGEs and their ligands are expressed in the fetal membranes and are found in the amniotic fluid and the mother´s serum. Disorders complicating pregnancies and having an important vascular involvement, as preeclampsia and diabetes mellitus, have additional enhanced AGE/RAGE expression variation. This indicates that for their assessment, the assay of RAGEs or their ligands may become useful diagnostic or prognostic procedures.

From bedside to cell biology: A century of history on lysosomal dysfunction

Lysosomal storage disorders (LSDs) are a group of rare genetic diseases, generally caused by a deficiency of specific lysosomal enzymes, which results in abnormal accumulation of undegraded substrates. The first clinical reports describing what were later shown to be LSDs were published more than a hundred years ago. In general, the history and pathophysiology of LSDs has impacted on our current knowledge of lysosomal biology. Classically, depending on the nature of the substrates, LSDs can be divided into different subgroups. The mucopolysaccharidoses (MPSs) are those caused by impaired degradation of glycosaminoglycans (GAGs). Amongst LSDs, the MPSs are a major group of pathologies with crucial historical relevance, since their study has revealed important biological pathways and highlighted interconnecting pathological cascades which are still being unveiled nowadays. Here we review the major historical discoveries in the field of LSDs and their impact on basic cellular knowledge and practical applications. Attention will be focused on the MPSs, with occasional references to other LSDs. We will show as studies on the metabolic basis of this group of diseases have increased our knowledge of the complex degradative pathways associated with the lysosome and established the basis to the development of specific therapeutic approaches aiming at correcting or, at least ameliorating their associated phenotypes.

Age-Related Effects of Dexamethasone Administration in Adrenal Zona Reticularis

Abstract:  Suppression of adrenocorticotropic hormone results in reduced adrenal steroid output, adrenocortical cell atrophy, and apoptosis in young rats. To verify such effects during aging, dexamethasone was injected into rats for 3 days at five different ages; at day 4, adrenals and blood were collected for morphologic and corticosterone assay. Adrenal structure was similar at all ages, but in dexamethasone‐injected animals there were ultrastructural features of apoptosis and a higher percentage of TUNEL and caspase‐3‐labeled nuclei and cytoplasm; their corticosterone decreased significantly. In both groups, there was age‐related decrease in the percentage of apoptotic cells, significant only in dexamethasone‐injected rats. The data suggest that aged adrenocortical cells are less susceptible to the lack of adrenocorticotropic hormone (ACTH), possibly as a result of their decreased functional ability.

Resveratrol Attenuates Copper-Induced Senescence by Improving Cellular Proteostasis

Copper sulfate-induced premature senescence (CuSO4-SIPS) consistently mimetized molecular mechanisms of replicative senescence, particularly at the endoplasmic reticulum proteostasis level. In fact, disruption of protein homeostasis has been associated to age-related cell/tissue dysfunction and human disorders susceptibility. Resveratrol is a polyphenolic compound with proved antiaging properties under particular conditions. In this setting, we aimed to evaluate resveratrol ability to attenuate cellular senescence induction and to unravel related molecular mechanisms. Using CuSO4-SIPS WI-38 fibroblasts, resveratrol is shown to attenuate typical senescence alterations on cell morphology, senescence-associated beta-galactosidase activity, and cell proliferation. The mechanisms implicated in this antisenescence effect seem to be independent of senescence-associated genes and proteins regulation but are reliant on cellular proteostasis improvement. In fact, resveratrol supplementation restores copper-induced increased protein content, attenuates BiP level, and reduces carbonylated and polyubiquitinated proteins by autophagy induction. Our data provide compelling evidence for the beneficial effects of resveratrol by mitigating CuSO4-SIPS stressful consequences by the modulation of protein quality control systems. These findings highlight the importance of a balanced cellular proteostasis and add further knowledge on molecular mechanisms mediating resveratrol antisenescence effects. Moreover, they contribute to identifying specific molecular targets whose modulation will prevent age-associated cell dysfunction and improve human healthspan.

Antioxidant Supplementation Modulates Age-Related Placental Bed Morphology and Reproductive Outcome in Mice

ABSTRACT The number of women who delay their first childbirth is increasing. This demographic shift is an important health issue because advanced maternal age is a risk factor for reproductive capacity loss and the occurrence of placental bed disorders that may lead to placenta abruption, preeclampsia, and placenta insufficiency. A redox imbalance status, resulting from the enhanced production of reactive oxygen species or their deficient neutralization, is proposed to occur in this setting. Thus, uterine redox status was evaluated in young (8- to 12-wk-old) and reproductively aged (38- to 42-wk-old) mice. In addition, it was hypothesized that specific dietary antioxidant supplementation would restore the balance and improve the reproductive outcome of aging female mice. To test this hypothesis, two different antioxidants, the nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor apocynin and the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL), were added to the drinking water of female mice prior to and during pregnancy. Compared to younger females, uteri from reproductively aged nonpregnant mice exhibited areas of endometrial cystic dilation, increased level of NOX1 expression, and enhanced protein carbonylation, especially in the apical surface of the luminal epithelium. Both antioxidants decreased protein carbonylation level in the uterus of reproductively aged mice. When reproductively aged females became pregnant, the litter size was smaller and fetuses were heavier. The change was accompanied by a significant decrease in decidua thickness. Provision of apocynin significantly increased litter size and restored decidua thickness. Reproductively aged mice provided with TEMPOL did not evidence such benefits, but whereas apocynin normalized fetal birth weight, TEMPOL further increased it. These findings emphasize that uterine redox balance is important for reproductive success and suggest that age-related redox imbalance might be compensated by specific antioxidant supplementation.

Follicular Fluid redox involvement for ovarian follicle growth

As the human ovarian follicle enlarges in the course of a regular cycle or following controlled ovarian stimulation, the changes in its structure reveal the oocyte environment composed of cumulus oophorus cells and the follicular fluid (FF).In contrast to the dynamic nature of cells, the fluid compartment appears as a reservoir rich in biomolecules. In some aspects, it is similar to the plasma, but it also exhibits differences that likely relate to its specific localization around the oocyte. The chemical composition indicates that the follicular fluid is able to detect and buffer excessive amounts of reactive oxygen species, employing a variety of antioxidants, some of them components of the intracellular milieu.An important part is played by albumin through specific cysteine residues. But the fluid contains other molecules whose cysteine residues may be involved in sensing and buffering the local oxidative conditions. How these molecules are recruited and regulated to intervene such process is unknown but it is a critical issue in reproduction.In fact, important proteins in the FF, that regulate follicle growth and oocyte quality, exhibit cysteine residues at specific points, whose untoward oxidation would result in functional loss. Therefore, preservation of controlled oxidative conditions in the FF is a requirement for the fine-tuned oocyte maturation process. In contrast, its disturbance enhances the susceptibility to the establishment of reproductive disorders that would require the intervention of reproductive medicine technology.