Eric de Waal

Detection of protein carbonyls in aging liver tissue: A fluorescence-based proteomic approach.

Protein carbonyls are commonly used as a marker of protein oxidation in cells and tissues. Currently, 2,4-dinitrophenyl hydrazine (DNPH) is widely used (spectrophotometrically or immunologically) to quantify the global carbonyl levels in proteins and identify the specific proteins that are carbonylated. We have adapted a fluorescence-based approach using fluorescein-5-thiosemicarbazide (FTC), to quantify the global protein carbonyls as well as the carbonyl levels on individual proteins in the proteome. Protein carbonyls generated in vitro were quantified by labeling the oxidized proteins with FTC followed by separating the FTC-labeled protein from free probe by gel electrophoresis. The reaction of FTC with protein carbonyls was found to be specific for carbonyl groups. We measured protein carbonyl levels in the livers of young and old mice, and found a significant increase (two-fold) in the global protein carbonyl levels with age. Using 2-D gel electrophoresis, we used this assay to directly measure the changes in protein carbonyl levels in specific proteins. We identified 12 proteins showing a greater than two-fold increase in carbonyl content (pmoles of carbonyls/microg of protein) with age. Most of the 12 proteins contained transition metal binding sites, with Cu/Zn superoxide dismutase containing the highest molar ratio of carbonyls in old mice. Thus, the fluorescence-based assay gives investigators the ability to identify potential target proteins that become oxidized under different pathological and physiological conditions.

Caspase-2 Deficiency Enhances Aging-Related Traits in Mice

Alteration of apoptotic activity has been observed in a number of tissues in aging mammals, but it remains unclear whether and/or how apoptosis may affect aging. Caspase-2 is a member of the cysteine protease family that plays a critical role in apoptosis. To understand the impact of compromised apoptosis function on mammalian aging, we conducted a comparative study on caspase-2 deficient mice and their wild-type littermates with a specific focus on the aging-related traits at advanced ages. We found that caspase-2 deficiency enhanced a number of traits commonly seen in premature aging animals. Loss of caspase-2 was associated with shortened maximum lifespan, impaired hair growth, increased bone loss, and reduced body fat content. In addition, we found that the livers of caspase-2 deficient mice had higher levels of oxidized proteins than those of age-matched wild-type mice, suggesting that caspase-2 deficiency compromised the animals ability to clear oxidatively damaged cells. Collectively, these results suggest that caspase-2 deficiency affects aging in the mice. This study thus demonstrates for the first time that disruption of a key apoptotic gene has a significant impact on aging.

In Vitro Culture Increases the Frequency of Stochastic Epigenetic Errors at Imprinted Genes in Placental Tissues from Mouse Concepti Produced Through Assisted Reproductive Technologies

ABSTRACT Assisted reproductive technologies (ART) have enabled millions of couples with compromised fertility to conceive children. Nevertheless, there is a growing concern regarding the safety of these procedures due to an increased incidence of imprinting disorders, premature birth, and low birth weight in ART-conceived offspring. An integral aspect of ART is the oxygen concentration used during in vitro development of mammalian embryos, which is typically either atmospheric (∼20%) or reduced (5%). Both oxygen tension levels have been widely used, but 5% oxygen improves preimplantation development in several mammalian species, including that of humans. To determine whether a high oxygen tension increases the frequency of epigenetic abnormalities in mouse embryos subjected to ART, we measured DNA methylation and expression of several imprinted genes in both embryonic and placental tissues from concepti generated by in vitro fertilization (IVF) and exposed to 5% or 20% oxygen during culture. We found that placentae from IVF embryos exhibit an increased frequency of abnormal methylation and expression profiles of several imprinted genes, compared to embryonic tissues. Moreover, IVF-derived placentae exhibit a variety of epigenetic profiles at the assayed imprinted genes, suggesting that these epigenetic defects arise by a stochastic process. Although culturing embryos in both of the oxygen concentrations resulted in a significant increase of epigenetic defects in placental tissues compared to naturally conceived controls, we did not detect significant differences between embryos cultured in 5% and those cultured in 20% oxygen. Thus, further optimization of ART should be considered to minimize the occurrence of epigenetic errors in the placenta.

Primary epimutations introduced during intracytoplasmic sperm injection (ICSI) are corrected by germline-specific epigenetic reprogramming

The use of assisted reproductive technologies (ART) has become increasingly common worldwide and is now responsible for 2–3% of children born in developed countries. Multiple reports have suggested that ART-conceived children are more likely to develop rare epigenetic disorders such as Beckwith-Wiedemann Syndrome or Angelman Syndrome, both of which involve dysregulation of imprinted genes. Anecdotal reports suggest that animals produced with ART that manifest apparent epigenetic defects typically do not transmit these epimutations to subsequent generations when allowed to breed naturally, but this hypothesis has not been directly studied. We analyzed allele-specific DNA methylation and expression at three imprinted genes, H19, Snrpn, and Peg3, in somatic cells from adult mice generated with the use of intracytoplasmic sperm injection (ICSI), a type of ART. Epimutations were detected in most of the ICSI-derived mice, but not in somatic cells of their offspring produced by natural mating. We examined germ cells from the ICSI mice that exhibited epimutations in their somatic cells and confirmed normal epigenetic reprogramming of the three imprinted genes analyzed. Collectively, these results confirm that ART procedures can lead to the formation of primary epimutations, but while such epimutations are likely to be maintained indefinitely in somatic cells of the ART-derived individuals, they are normally corrected in the germ line by epigenetic reprogramming and thus, not propagated to subsequent generations.

The cumulative effect of assisted reproduction procedures on placental development and epigenetic perturbations in a mouse model

Assisted reproductive technologies (ART) are associated with several complications including low birth weight, abnormal placentation and increased risk for rare imprinting disorders. Indeed, experimental studies demonstrate ART procedures independent of existing infertility induce epigenetic perturbations in the embryo and extraembryonic tissues. To test the hypothesis that these epigenetic perturbations persist and result in adverse outcomes at term, we assessed placental morphology and methylation profiles in E18.5 mouse concepti generated by in vitro fertilization (IVF) in two different genetic backgrounds. We also examined embryo transfer (ET) and superovulation procedures to ascertain if they contribute to developmental and epigenetic effects. Increased placental weight and reduced fetal-to-placental weight ratio were observed in all ART groups when compared with naturally conceived controls, demonstrating that non-surgical embryo transfer alone can impact placental development. Furthermore, superovulation further induced overgrowth of the placental junctional zone. Embryo transfer and superovulation defects were limited to these morphological changes, as we did not observe any differences in epigenetic profiles. IVF placentae, however, displayed hypomethylation of imprinting control regions of select imprinted genes and a global reduction in DNA methylation levels. Although we did not detect significant differences in DNA methylation in fetal brain or liver samples, rare IVF concepti displayed very low methylation and abnormal gene expression from the normally repressed allele. Our findings suggest that individual ART procedures cumulatively increase placental morphological abnormalities and epigenetic perturbations, potentially causing adverse neonatal and long-term health outcomes in offspring.

A DNMT3A2-HDAC2 Complex Is Essential for Genomic Imprinting and Genome Integrity in Mouse Oocytes

Maternal genomic imprints are established during oogenesis. Histone deacetylases (HDACs) 1 and 2 are required for oocyte development in mouse, but their role in genomic imprinting is unknown. We find that Hdac1:Hdac2(-/-) double-mutant growing oocytes exhibit global DNA hypomethylation and fail to establish imprinting marks for Igf2r, Peg3, and Srnpn. Global hypomethylation correlates with increased retrotransposon expression and double-strand DNA breaks. Nuclear-associated DNMT3A2 is reduced in double-mutant oocytes, and injecting these oocytes with Hdac2 partially restores DNMT3A2 nuclear staining. DNMT3A2 co-immunoprecipitates with HDAC2 in mouse embryonic stem cells. Partial loss of nuclear DNMT3A2 and HDAC2 occurs in Sin3a(-/-) oocytes, which exhibit decreased DNA methylation of imprinting control regions for Igf2r and Srnpn, but not Peg3. These results suggest seminal roles of HDAC1/2 in establishing maternal genomic imprints and maintaining genomic integrity in oocytes mediated in part through a SIN3A complex that interacts with DNMT3A2.

Detection and quantification of protein disulfides in biological tissues a fluorescence-based proteomic approach.

While most of the amino acids in proteins are potential targets for oxidation, the thiol group in cysteine is one of the most reactive amino acid side chains. The thiol group can be oxidized to several states, including the disulfide bond. Despite the known sensitivity of cysteine to oxidation and the physiological importance of the thiol group to protein structure and function, little information is available on the oxidative modification of cysteine residues in proteins because of the lack of reproducible and sensitive assays to measure cysteine oxidation in the proteome. We have developed a fluorescence-based assay that allows one to quantify both the global level of protein disulfides in the cellular proteome as well as the disulfide content of individual proteins. This fluorescence-based assay is able to detect an increase in global protein disulfide levels after oxidative stress in vitro or in vivo. Using this assay, we show that the global protein disulfide levels increase significantly with age in liver cytosolic proteins, and we identified 11 proteins that show a more than twofold increase in disulfide content with age. Thus, the fluorescence-based assay we have developed allows one to quantify changes in the oxidation of cysteine residues to disulfides in the proteome of a cell or tissue.