Alberto Miranda

Prion Protein Expression Regulates Embryonic Stem Cell Pluripotency and Differentiation

Cellular prion protein (PRNP) is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB) differentiation in mouse Prnp-null (KO) and WT embryonic stem cell (ESC) lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC) markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5) in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel) and SPRN (Shadoo), whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

Role of the Goat K222-PrPC Polymorphic Variant in Prion Infection Resistance

ABSTRACT The prion protein-encoding gene (prnp) strongly influences the susceptibility of small ruminants to transmissible spongiform encephalopathies (TSEs). Hence, selective breeding programs have been implemented to increase sheep resistance to scrapie. For goats, epidemiological and experimental studies have provided some association between certain polymorphisms of the cellular prion protein (PrPC) and resistance to TSEs. Among them, the Q/K polymorphism at PrPC codon 222 (Q/K222) yielded the most promising results. In this work, we investigated the individual effects of the K222-PrPC variant on the resistance/susceptibility of goats to TSEs. For that purpose, we generated two transgenic mouse lines, expressing either the Q222 (wild type) or K222 variant of goat PrPC. Both mouse lines were challenged intracerebrally with a panel of TSE isolates. Transgenic mice expressing the wild-type (Q222) allele were fully susceptible to infection with all tested isolates, whereas transgenic mice expressing similar levels of the K222 allele were resistant to all goat scrapie and cattle BSE isolates but not to goat BSE isolates. Finally, heterozygous K/Q222 mice displayed a reduced susceptibility to the tested panel of scrapie isolates. These results demonstrate a highly protective effect of the K222 variant against a broad panel of different prion isolates and further reinforce the argument supporting the use of this variant in breeding programs to control TSEs in goat herds. IMPORTANCE The objective of this study was to determine the role of the K222 variant of the prion protein (PrP) in the susceptibility/resistance of goats to transmissible spongiform encephalopathies (TSEs). Results showed that transgenic mice expressing the goat K222-PrP polymorphic variant are resistant to scrapie and bovine spongiform encephalopathy (BSE) agents. This protective effect was also observed in heterozygous Q/K222 animals. Therefore, the single amino acid exchange from Q to K at codon 222 of the cellular prion protein provides resistance against TSEs. All the results presented here support the view that the K222 polymorphic variant is a good candidate for selective breeding programs to control and eradicate scrapie in goat herds.

Male Mice Produced by In Vitro Culture Have Reduced Fertility and Transmit Organomegaly and Glucose Intolerance to Their Male Offspring

ABSTRACT It has been reported that suboptimal in vitro culture (IVC) of mouse embryos can affect the postnatal expression of epigenetically sensitive alleles, resulting in altered postnatal growth, organ dimensions, health, and behavior in the offspring. Although these detrimental impacts on the offspring are well described, the relative contribution of the IVC-produced fathers is unclear. In this work, we have analyzed if suboptimal IVC (achieved by altering the culture medium by the addition of FCS) can affect male fertility and if organ size and glucose clearance, two of the adverse effects produced by suboptimal IVC conditions, were transmitted to the next two generations. IVC-produced males had lower sperm concentrations (5.8 × 106 spermatozoa in IVC vs. 14.5 × 106 spermatozoa in control), and these sperm exhibited decreased overall motility (49.6% vs. 72.8% in control) and progressive motility (22.6% vs. 32.2% in control). Fertility tests demonstrated that the percentage of pregnancies was reduced for IVC males (35% for IVC-produced males vs. 86% for in vivo controls). These features were related to a modified gene expression pattern in adult male testes, showing an altered gene expression in genes involved in DNA repair and apoptosis that was confirmed by TUNEL assay. Regarding the IVC related adverse phenotype transmitted to offspring, male glucose intolerance was shown only in F1 and F2 male but not female offspring. The same occurred with male abnormalities in the organ size of the liver, which were transmitted to F1 and F2 males but not to F1 females; moreover, analysis of the F0, F1, and F2 males revealed greater coefficients of variance in body weight and glucose intolerance than the control group. Finally, we analyzed, through gene silencing, the effect of IVC on the mRNA expression at the blastocyst stage for 11 known gene expression modifiers of epigenetic reprogramming. Suboptimal IVC reduced the expression of Kap1, Sox2, Hdac1, Dnmt1, and Dnmt3a, suggesting a molecular epigenetic role for gene expression modifiers in the origin and transmission of these abnormal phenotypes.

The Mkk2 MAPKK Regulates Cell Wall Biogenesis in Cooperation with the Cek1-Pathway in Candida albicans.

The cell wall integrity pathway (CWI) plays an important role in the biogenesis of the cell wall in Candida albicans and other fungi. In the present work, the C. albicans MKK2 gene that encodes the putative MAPKK of this pathway was deleted in different backgrounds and the phenotypes of the resultant mutants were characterised. We show here that Mkk2 mediates the phosphorylation of the Mkc1 MAPK in response to cell wall assembly interfering agents such as zymolyase or tunicamycin and also to oxidative stress. Remarkably, mkk2 and mkc1 mutants display related but distinguishable- cell wall associated phenotypes and differ in the pattern of MAPK phosphorylation under different stress conditions. mkk2 and mkc1 mutants display an altered expression of GSC1, CEK1 and CRH11 genes at different temperatures. Combined deletion of MKK2 with HST7 supports a cooperative role for the Cek1-mediated and CWI pathways in regulating cell wall architecture under vegetative growth. However, and in contrast to Mkc1, Mkk2 does not seem to play a role in the virulence of C. albicans in the mouse systemic model or the Galleria mellonella model of infection.

The role of prion protein in stem cell regulation.

Cellular prion protein (PrP(C)) has been well described as an essential partner of prion diseases due to the existence of a pathological conformation (PrP(Sc)). Recently, it has also been demonstrated that PrP(C) is an important element of the pluripotency and self-renewal matrix, with an increasing amount of evidence pointing in this direction. Here, we review the data that demonstrate its role in the transcriptional regulation of pluripotency, in the differentiation of stem cells into different lineages (e.g. muscle and neurons), in embryonic development, and its involvement in reproductive cells. Also highlighted are recent results from our laboratory that describe an important regulation by PrP(C) of the major pluripotency gene Nanog. Together, these data support the appearance of new strategies to control stemness, which could represent an important advance in the field of regenerative medicine.

New challenges in the analysis of gene transcription in bovine blastocysts.

In the last years, enormous progress has been made in the analysis of gene transcription at the blastocyst stage. The study of gene expression at this early stage of development is challenging because of the very small amount of starting material, which limits the use of traditional mRNA analysis approaches such as Northern blot. Another problem is the difficulty for data normalization, particularly the identification of the best housekeeping gene with the lowest fluctuation under different developmental conditions. Moreover, the transcriptional analysis of embryo biopsies or individual embryos needs to take into consideration that the blastocyst is a transitional stage of development, which is composed of three different types of cells (trophoblast, epiblast and primitive ectoderm) with different patterns of gene expression, and that there are large differences between male and female blastocysts. In this review, we analyse the different specific and sensitive tools available to compare mRNA expression levels of specific genes at the blastocyst stage, and how the protocol and the analytical method used can influence the results dramatically. Finally, we describe future research challenges to identify candidate genes related to developmental competence of bovine blastocysts, not only in terms of pregnancy rates but also in relation to adverse long-term consequences in the adult animal.

Transcriptome profiling of liver of non-genetic low birth weight and long term health consequences

BackgroundIt is believed that the main factors of low prenatal growth in mammals are genetic and environmental. We used isogenic mice maintained in standard conditions to analyze how natural non-genetic microsomia (low birth weight) is produced in inbred mice and its long term effect on health. To better understand the molecular basis of non-genetic microsomia, we undertook transcriptome profiling of both male and female livers from small and normal size mice at birth.ResultsNaturally occurring neonatal microsomia was defined as a gender-specific weanling weight under the 10th percentile of the colony. Birth weight variation was similar in inbred and outbred lines. Mice were phenotyped by weight, size, blood pressure, organ size, their response to a glucose challenge, and survival rates. Regardless of diet, adult mice born with microsomia showed a significantly lower body weight and size, and differences in the weight of several organs of microsomic adult mice compared to normal birth weight adults were found. After a high-fat diet, microsomic mice were less prone to obesity, showing a better glucose tolerance and lower blood pressure. Through a transcriptome analysis, we detected a different pattern of mRNA transcription in the liver at birth comparing male vs female and microsomic vs normal mice, noting some modifications in epigenetic regulatory genes in females and modifications in some growth factor genes in males. Finally, using embryo transfer of embryos of different quality and age, we identified a putative preimplantation origin of this non-genetic microsomia.Conclusions(1) neonatal microsomia is not always a risk factor for adult metabolic syndrome, (2) neonatal non-genetic microsomia displays changes in the expression of important epigenetic genes and changes in liver mRNA transcription profile at birth, exaggerating sexual dimorphism, and (3) random preimplantation phenotypic variability could partially explain body birth weight variation in isogenic lines.

Maintenance of Pluripotency in Mouse Stem Cells: Use of Hyaluronan in the Long-Term Culture

The use of embryonic stem (ES) cells in regenerative medicine is one of the most important promises of this decade. The knowledge about culture medium, isolation protocols and pluripotency related genes is growing but their therapeutic use is still restricted by a risk of teratoma formation. To test the hypothesis that some cells remain undifferentiated after a differentiation culture process, we have electroporated mouse ES cells with a construct comprising the mTert promoter coupled to green fluorescent protein (GFP). When ES cells colonies are in culture, they express the green fluorescent protein as a consequence of their telomerase activity. After LIF removal a natural differentiation process into embryoid bodies (EB) occurs, such that the GFP signal gradually diminishes due to this differentiation. However, after 2 weeks of incubation, we could observe small groups of fluorescent cells remaining in the differentiated monolayer. After isolation and expansion of these groups under ES cell culture conditions, ES cell morphology was recovered. Molecular analysis showed that after several passages in ES medium, pluripotency quality characteristics of these cells are completely recovered. According to our results, a specific screening of differentiated cells should be performed before their therapeutic use to avoid the risk of teratoma formation stimulated by an adequate environment. Moreover, to maintain undifferentiated stem cells in defined conditions is of critical importance to improve their in vitro culture. We evaluated the effects of the replacement of fibroblast feeder layer (mEF) by gelatin or the glycosaminoglycan hyaluronan (HA). Long-term culture onto HA was more effective in maintaining the pluripotent state of mES cells when compared to gelatin. The rate of terminal differentiation was the highest onto mEF, intermediate onto HA and reduced onto gelatin. In addition, our findings strongly suggest that the use of hyaluronan, without feeder cells, may offer a valuable alternative for unifying and standardizing the conditions for long-term defined culture conditions of undifferentiated mES cells.

Sperm-Mediated Gene Transfer: Concepts and Controversies

Description: Sperm-mediated gene transfer (SMGT) represents a novel set of technologies for animal (or in the future, human) genetic modification using the sperm as a vector, as opposed to more traditional established routes such as fertilized eggs or embryonic stem cells. Studies of sperm-mediated gene transfer (SMGT) indicate that sperm cells possess the ability to be utilized as carriers of exogenous genetic sequences, offering the potential of a novel cost-effective route for germline genetic modification. The fate of transgenes borne by sperm cells has been inconsistent, and analysis of offspring from SMGT experiments has shown a mixed picture in terms of genomic integration of the transgene, suggesting an episomal mode of inheritance. Various distinct steps in transgene uptake by the sperm cell have been described or proposed, including a model based upon endogenous reverse transcriptase activity. Although mature sperm cells are naturally protected against uptake of foreign nucleic acid molecules, certain environmental conditions, for example at key times within the reproductive tract, may reduce this protection, suggesting that SMGT may occasionally take place in nature. If correct, this carries profound implications for evolution and human genetic health. This e-book brings together theoretical and empirical reviews from experts in SMGT, providing comprehensive coverage of the major trends, developments and controversies in this novel field. This e-book is intended as a reference for professional researchers in the field of animal genetic modification (transgenesis) as well as teachers, scientists and physicians interested in medical genetics in general and gene therapy in particular.