Wnt, glucocorticoid and cellular prion protein cooperate to drive a mesenchymal phenotype with poor prognosis in colon cancer.

BACKGROUND: The mesenchymal subtype of colorectal cancer (CRC), associated with poor prognosis, is characterized by abundant expression of the cellular prion protein PrPC, which represents a candidate therapeutic target. How PrPC is induced in CRC remains elusive. This study aims to elucidate the signaling pathways governing PrPC expression and to shed light on the gene regulatory networks linked to PrPC. METHODS: We performed in silico analyses on diverse datasets of in vitro, ex vivo and in vivo models of mouse CRC and patient cohorts. We mined ChIPseq studies and performed promoter analysis. CRC cell lines were manipulated through genetic and pharmacological approaches. We created mice combining conditional inactivation of Apc in intestinal epithelial cells and overexpression of the human prion protein gene PRNP. Bio-informatic analyses were carried out in two randomized control trials totalizing over 3000 CRC patients. RESULTS: In silico analyses combined with cell-based assays identified the Wnt-ß-catenin and glucocorticoid pathways as upstream regulators of PRNP expression, with subtle differences between mouse and human. We uncover multiple feedback loops between PrPC and these two pathways, which translate into an aggravation of CRC pathogenesis in mouse. In stage III CRC patients, the signature defined by PRNP-CTNNB1-NR3C1, encoding PrPC, ß-catenin and the glucocorticoid receptor respectively, is overrepresented in the poor-prognosis, mesenchymal subtype and associates with reduced time to recurrence. CONCLUSIONS: An unleashed PrPC-dependent vicious circle is pathognomonic of poor prognosis, mesenchymal CRC. Patients from this aggressive subtype of CRC may benefit from therapies targeting the PRNP-CTNNB1-NR3C1 axis.

Assessment of the zoonotic potential of atypical scrapie prions in humanized mice reveals rare phenotypic convergence but not identity with sporadic CJD prions.

BACKGROUND: Atypical/Nor98 scrapie (AS) is an idiopathic infectious prion disease affecting sheep and goats. Recent findings suggest that zoonotic prions from bovine spongiform encephalopathy (C-BSE) may co-propagate with atypical/Nor98 prions in AS sheep brains. Investigating the risk AS poses to humans is crucial. METHODS: To assess the risk of sheep/goat-to-human transmission of AS, we serially inoculated brain tissue from field and laboratory isolates into transgenic mice overexpressing human prion protein (Met129 allele). We studied clinical outcomes as well as presence of prions in brains and spleens. RESULTS: No transmission occurred on the primary passage, with no clinical disease or pathological prion protein in brains and spleens. On subsequent passages, one isolate gradually adapted, manifesting as prions with a phenotype resembling those causing MM1-type sporadic Creutzfeldt-Jakob disease in humans. However, further characterization using in vivo and in vitro techniques confirmed both prion agents as different strains, revealing a case of phenotypic convergence. Importantly, no C-BSE prions emerged in these mice, especially in the spleen, which is more permissive than the brain for C-BSE cross-species transmission. CONCLUSIONS: The results obtained suggest a low the zoonotic for AS. Rare adaptation may allow the emergence of prions phenotypically resembling those spontaneously forming in humans.

Targeted mutation and inactivation of the kinesin light chain 3 protein-encoding gene have no impact on mouse fertility†.

The kinesin light chain 3 protein (KLC3) is the only member of the kinesin light chain protein family that was identified in post-meiotic mouse male germ cells. It plays a role in the formation of the sperm midpiece through its association with both spermatid mitochondria and outer dense fibers (ODF). Previous studies showed a significant correlation between its expression level and sperm motility and quantitative semen parameters in humans, while the overexpression of a KLC3-mutant protein unable to bind ODF also affected the same traits in mice. To further assess the role of KLC3 in fertility, we used CRISPR/Cas9 genome editing in mice and investigated the phenotypes induced by the invalidation of the gene or of a functional domain of the protein. Both approaches gave similar results, i.e. no detectable change in male or female fertility. Testis histology, litter size and sperm count were not altered. Apart from the line-dependent alterations of Klc3 mRNA levels, testicular transcriptome analysis did not reveal any other changes in the genes tested. Western analysis supported the absence of KLC3 in the gonads of males homozygous for the inactivating mutation and a strong decrease in expression in males homozygous for the allele lacking one out of the five tetratricopeptide repeats. Overall, these observations raise questions about the supposedly critical role of this kinesin in reproduction, at least in mice where its gene mutation or inactivation did not translate into fertility impairment.

The paralogs' enigma of germ-cell specific genes dispensable for fertility: the case of 19 oogenesin genes†.

Gene knockout experiments have shown that many genes are dispensable for a given biological function. In this review, we make an assessment of male and female germ cell-specific genes dispensable for the function of reproduction in mice, the inactivation of which does not affect fertility. In particular, we describe the deletion of a 1 Mb block containing nineteen paralogous genes of the oogenesin/Pramel family specifically expressed in female and/or male germ cells, which has no consequences in both sexes. We discuss this notion of dispensability and the experiments that need to be carried out to definitively conclude that a gene is dispensable for a function.

Mutation of the MYH3 gene causes recessive cleft palate in Limousine cattle.

BACKGROUND: The palate is a structure separating the oral and nasal cavities and its integrity is essential for feeding and breathing. The total or partial opening of the palate is called a cleft palate and is a common malformation in mammals with environmental or hereditary aetiologies. Generally, it compromises life expectancy in the absence of surgical repair. A new form of non-syndromic cleft palate arose recently in Limousine cattle, with animals referred to the French National Observatory of Bovine Abnormalities since 2012. Since the number of affected animals has increased steadily, this study was undertaken to identify the cause of this disease. RESULTS: Based on pedigree analysis, occurrence of cleft palate in Limousine cattle was concordant with an autosomal recessive mode of inheritance. Genotyping of 16 affected animals and homozygosity mapping led to the identification of a single disease-associated haplotype on Bos taurus chromosome (BTA)19. The genome of two affected animals was sequenced, and their sequences were compared to the ARS-UCD1.2 reference genome to identify variants. The likely causal variants were compared to the variant database of the 1000 bull genome project and two fully linked mutations in exon 24 of the MYH3 (myosin heavy chain) gene were detected: a 1-bp non-synonymous substitution (BTA19:g.29609623A>G) and a 11-bp frameshift deletion (BTA19:g.29609605-29609615del). These two mutations were specific to the Limousine breed, with an estimated allele frequency of 2.4% and are predicted to be deleterious. The frameshift leads to a premature termination codon. Accordingly, mRNA and protein analyses in muscles from wild-type and affected animals revealed a decrease in MYH3 expression in affected animals, probably due to mRNA decay, as well as an absence of the MYH3 protein in these animals. MYH3 is mostly expressed in muscles, including craniofacial muscles, during embryogenesis, and its absence may impair palate formation. CONCLUSIONS: We describe a new form of hereditary cleft palate in Limousine cattle. We identified two fully linked and deleterious mutations, ultimately leading to the loss-of-function of the MYH3 protein. The mutations were included on the Illumina EuroG10k v8 and EuroGMD v1 SNP chips and are used to set up a reliable eradication strategy in the French Limousine breed.

Potential genetic robustness of Prnp and Sprn double knockout mouse embryos towards ShRNA-lentiviral inoculation.

The Shadoo and PrP prion protein family members are thought to be functionally related, but previous knockdown/knockout experiments in early mouse embryogenesis have provided seemingly contradictory results. In particular, Shadoo was found to be indispensable in the absence of PrP in knockdown analyses, but a double-knockout of the two had little phenotypic impact. We investigated this apparent discrepancy by comparing transcriptomes of WT, Prnp0/0 and Prnp0/0Sprn0/0 E6.5 mouse embryos following inoculation by Sprn- or Prnp-ShRNA lentiviral vectors. Our results suggest the possibility of genetic adaptation in Prnp0/0Sprn0/0 mice, thus providing a potential explanation for their previously observed resilience.

Review on PRNP genetics and susceptibility to chronic wasting disease of Cervidae.

To date, chronic wasting disease (CWD) is the most infectious form of prion disease affecting several captive, free ranging and wild cervid species. Responsible for marked population declines in North America, its geographical spread is now becoming a major concern in Europe. Polymorphisms in the prion protein gene (PRNP) are an important factor influencing the susceptibility to prions and their rate of propagation. All reported cervid PRNP genotypes are affected by CWD. However, in each species, some polymorphisms are associated with lower attack rates and slower progression of the disease. This has potential consequences in terms of genetic selection, CWD diffusion and strain evolution. CWD also presents a zoonotic risk due to prions capacity to cross species barriers. This review summarizes our current understanding of CWD control, focusing on PRNP genetic, strain diversity and capacity to infect other animal species, including humans.

Prion strains associated with iatrogenic CJD in French and UK human growth hormone recipients.

Treatment with human pituitary-derived growth hormone (hGH) was responsible for a significant proportion of iatrogenic Creutzfeldt-Jakob disease (iCJD) cases. France and the UK experienced the largest case numbers of hGH-iCJD, with 122 and 81 cases respectively. Differences in the frequency of the three PRNP codon 129 polymorphisms (MM, MV and VV) and the estimated incubation periods associated with each of these genotypes in the French and the UK hGH-iCJD cohorts led to the suggestion that the prion strains responsible for these two hGH-iCJD cohorts were different. In this study, we characterized the prion strains responsible for hGH-iCJD cases originating from UK (n = 11) and France (n = 11) using human PrP expressing mouse models. The cases included PRNP MM, MV and VV genotypes from both countries. UK and French sporadic CJD (sCJD) cases were included as controls. The prion strains identified following inoculation with hGH-iCJD homogenates corresponded to the two most frequently observed sCJD prion strains (M1CJD and V2CJD). However, in clear contradiction to the initial hypothesis, the prion strains that were identified in the UK and the French hGH-iCJD cases were not radically different. In the vast majority of the cases originating from both countries, the V2CJD strain or a mixture of M1CJD + V2CJD strains were identified. These data strongly support the contention that the differences in the epidemiological and genetic profiles observed in the UK and France hGH-iCJD cohorts cannot be attributed only to the transmission of different prion strains.

Co-invalidation of Prnp and Sprn in FVB/N mice affects reproductive performances and highlight complex biological relationship between PrP and Shadoo.

Shadoo and PrP belongs to the same protein family, whose biological function remains poorly understood. Previous experiments reported potential functional redundancies or antagonisms between these two proteins, depending on the tissue analysed. While knockdown experiments suggested the requirement of Shadoo in the absence of PrP during early mouse embryogenesis, knockout ones, on the contrary, highlighted little impact, if any, of the double-knockout of these two loci. In the present study, we reinvestigated the phenotype associated with the concomitant knockout of these two genes using newly produced FVB/N Sprn knockout mice. In this genetic background, the combined two genes' knockout induces intra-uterine growth retardations, likely resulting from placental failures highlighted by transcriptomic analyses that revealed potential redundant or antagonist roles of these two proteins in different developmental-related pathways. It also induced an increased perinatal-lethality and ascertained the role of these two loci in the lactation process.

Cellular prion protein dysfunction in a prototypical inherited metabolic myopathy.

Inherited fatty acid oxidation diseases in their mild forms often present as metabolic myopathies. Carnitine Palmitoyl Transferase 2 (CPT2) deficiency, one such prototypical disorder is associated with compromised myotube differentiation. Here, we show that CPT2-deficient myotubes exhibit defects in focal adhesions and redox balance, exemplified by increased SOD2 expression. We document unprecedented alterations in the cellular prion protein PrPC, which directly arise from the failure in CPT2 enzymatic activity. We also demonstrate that the loss of PrPC function in normal myotubes recapitulates the defects in focal adhesion, redox balance and differentiation hallmarks monitored in CPT2-deficient cells. These results are further corroborated by studies performed in muscles from Prnp-/- mice. Altogether, our results unveil a molecular scenario, whereby PrPC dysfunction governed by faulty CPT2 activity may drive aberrant focal adhesion turnover and hinder proper myotube differentiation. Our study adds a novel facet to the involvement of PrPC in diverse physiopathological situations.

CEP250 is Required for Maintaining Centrosome Cohesion in the Germline and Fertility in Male Mice.

Male gametogenesis involves both mitotic divisions to amplify germ cell progenitors that gradually differentiate and meiotic divisions. Centrosomal regulation is essential for both types of divisions, with centrioles remaining tightly paired during the interphase. Here, we generated and characterized the phenotype of mutant mice devoid of Cep250/C-Nap1, a gene encoding for a docking protein for fibers linking centrioles, and characterized their phenotype. The Cep250 -/- mice presented with no major defects, apart from male infertility due to a reduction in the spermatogonial pool and the meiotic blockade. Spermatogonial stem cells expressing Zbtb16 were not affected, whereas the differentiating spermatogonia were vastly lost. These cells displayed abnormal γH2AX-staining, accompanied by an increase in the apoptotic rate. The few germ cells that survived at this stage, entered the meiotic prophase I and were arrested at a pachytene-like stage, likely due to synapsis defects and the unrepaired DNA double-strand breaks. In these cells, centrosomes split up precociously, with γ-tubulin foci being separated whereas these were closely associated in wild-type cells. Interestingly, this lack of cohesion was also observed in wild-type female meiocytes, likely explaining the normal fertility of Cep250 -/- female mice. Taken together, this study proposes a specific requirement of centrosome cohesion in the male germline, with a crucial role of CEP250 in both differentiating spermatogonia and meiotic spermatocytes.

Improving the Predictive Value of Prion Inactivation Validation Methods to Minimize the Risks of Iatrogenic Transmission With Medical Instruments.

Prions are pathogenic infectious agents responsible for fatal, incurable neurodegenerative diseases in animals and humans. Prions are composed exclusively of an aggregated and misfolded form (PrP Sc ) of the cellular prion protein (PrPC). During the propagation of the disease, PrPSc recruits and misfolds PrPC into further PrPSc. In human, iatrogenic prion transmission has occurred with incompletely sterilized medical material because of the unusual resistance of prions to inactivation. Most commercial prion disinfectants validated against the historical, well-characterized laboratory strain of 263K hamster prions were recently shown to be ineffective against variant Creutzfeldt-Jakob disease human prions. These observations and previous reports support the view that any inactivation method must be validated against the prions for which they are intended to be used. Strain-specific variations in PrPSc physico-chemical properties and conformation are likely to explain the strain-specific efficacy of inactivation methods. Animal bioassays have long been used as gold standards to validate prion inactivation methods, by measuring reduction of prion infectivity. Cell-free assays such as the real-time quaking-induced conversion (RT-QuIC) assay and the protein misfolding cyclic amplification (PMCA) assay have emerged as attractive alternatives. They exploit the seeding capacities of PrPSc to exponentially amplify minute amounts of prions in biospecimens. European and certain national medicine agencies recently implemented their guidelines for prion inactivation of non-disposable medical material; they encourage or request the use of human prions and cell-free assays to improve the predictive value of the validation methods. In this review, we discuss the methodological and technical issues regarding the choice of (i) the cell-free assay, (ii) the human prion strain type, (iii) the prion-containing biological material. We also introduce a new optimized substrate for high-throughput PMCA amplification of human prions bound on steel wires, as translational model for prion-contaminated instruments.

Host prion protein expression levels impact prion tropism for the spleen.

Prions are pathogens formed from abnormal conformers (PrPSc) of the host-encoded cellular prion protein (PrPC). PrPSc conformation to disease phenotype relationships extensively vary among prion strains. In particular, prions exhibit a strain-dependent tropism for lymphoid tissues. Prions can be composed of several substrain components. There is evidence that these substrains can propagate in distinct tissues (e.g. brain and spleen) of a single individual, providing an experimental paradigm to study the cause of prion tissue selectivity. Previously, we showed that PrPC expression levels feature in prion substrain selection in the brain. Transmission of sheep scrapie isolates (termed LAN) to multiple lines of transgenic mice expressing varying levels of ovine PrPC in their brains resulted in the phenotypic expression of the dominant sheep substrain in mice expressing near physiological PrPC levels, whereas a minor substrain replicated preferentially on high expresser mice. Considering that PrPC expression levels are markedly decreased in the spleen compared to the brain, we interrogate whether spleen PrPC dosage could drive prion selectivity. The outcome of the transmission of a large cohort of LAN isolates in the spleen from high expresser mice correlated with the replication rate dependency on PrPC amount. There was a prominent spleen colonization by the substrain preferentially replicating on low expresser mice and a relative incapacity of the substrain with higher-PrPC level need to propagate in the spleen. Early colonization of the spleen after intraperitoneal inoculation allowed neuropathological expression of the lymphoid substrain. In addition, a pair of substrain variants resulting from the adaptation of human prions to ovine high expresser mice, and exhibiting differing brain versus spleen tropism, showed different tropism on transmission to low expresser mice, with the lymphoid substrain colonizing the brain. Overall, these data suggest that PrPC expression levels are instrumental in prion lymphotropism.

Molecular Mechanisms of Trophoblast Dysfunction Mediated by Imbalance between STOX1 Isoforms.

STOX1 is a transcription factor involved in preeclampsia and Alzheimer disease. We show that the knock-down of the gene induces rather mild effect on gene expression in trophoblast cell lines (BeWo). We identified binding sites of STOX1 shared by the two major isoforms, STOX1A and STOX1B. Profiling gene expression of cells overexpressing either STOX1A or STOX1B, we identified genes downregulated by both isoforms, with a STOX1 binding site in their promoters. Among those, STOX1-induced Annexin A1 downregulation led to abolished membrane repair in BeWo cells. By contrast, overexpression of STOX1A or B has opposite effects on trophoblast fusion (acceleration and inhibition, respectively) accompanied by syncytin genes deregulation. Also, STOX1A overexpression led to abnormal regulation of oxidative and nitrosative stress. In sum, our work shows that STOX1 isoform imbalance is a cause of gene expression deregulation in the trophoblast, possibly leading to placental dysfunction and preeclampsia.

The Prion-like protein Shadoo is involved in mouse embryonic and mammary development and differentiation.

Shadoo belongs to the prion protein family, an evolutionary conserved and extensively studied family due to the implication of PrP in Transmissible Spongiform Encephalopathies. However, the biological function of these genes remains poorly understood. While Sprn-knockdown experiments suggested an involvement of Shadoo during mouse embryonic development, Sprn-knockout experiments in 129Pas/C57BL/6J or 129Pas/FVB/NCr mice did not confirm it. In the present study, we analyzed the impact of Sprn gene invalidation in a pure FVB/NJ genetic background, using a zinc finger nuclease approach. The in-depth analysis of the derived knockout transgenic mice revealed a significant increase in embryonic lethality at early post-implantation stages, a growth retardation of young Sprn-knockout pups fed by wild type mice and a lactation defect of Sprn-knockout females. Histological and transcriptional analyses of knockout E7.5 embryos, E14.5 placentas and G7.5 mammary glands revealed specific roles of the Shadoo protein in mouse early embryogenesis, tissue development and differentiation with a potential antagonist action between PrP and Shadoo. This study thus highlights the entanglement between the proteins of the prion family.

The cellular prion protein is a stress protein secreted by renal tubular cells and a urinary marker of kidney injury.

Endoplasmic Reticulum (ER) stress underlies the pathogenesis of numerous kidney diseases. A better care of patients with kidney disease involves the identification and validation of ER stress biomarkers in the early stages of kidney disease. For the first time to our knowledge, we demonstrate that the prion protein PrPC is secreted in a conventional manner by ER-stressed renal epithelial cell under the control of the transcription factor x-box binding protein 1 (XBP1) and can serve as a sensitive urinary biomarker for detecting tubular ER stress. Urinary PrPC elevation occurs in patients with chronic kidney disease. In addition, in patients undergoing cardiac surgery, detectable urine levels of PrPC significantly increase after cardiopulmonary bypass, a condition associated with activation of the IRE1-XBP1 pathway in the kidney. In conclusion, our study has identified PrPC as a novel urinary ER stress biomarker with potential utility in early diagnosis of ongoing acute or chronic kidney injury.

DNAJC2 is required for mouse early embryonic development.

DNAJC2 protein, also known as ZRF1 or MPP11, acts both as chaperone and as chromatin regulator. It is involved in stem cell differentiation and its expression is associated with various cancer malignancies. However, the role of Dnajc2 gene during mouse embryogenesis has not been assessed so far. To this aim, we invalidated Dnajc2 gene in FVB/Nj mice using the CrispR/Cas9 approach. We showed that this invalidation leads to the early post-implantation lethality of the nullizygous embryos. Furthermore, using siRNAs against Dnajc2 in mouse 1-cell embryos, we showed that maternal Dnajc2 mRNAs may allow for the early preimplantation development of these embryos. Altogether, these data demonstrate for the first time the requirement of DNAJC2 for early mouse embryogenesis.

Alpha-1 microglobulin as a potential therapeutic candidate for treatment of hypertension and oxidative stress in the STOX1 preeclampsia mouse model.

Preeclampsia is a human placental disorder affecting 2-8% of pregnancies worldwide annually, with hypertension and proteinuria appearing after 20 weeks of gestation. The underlying cause is believed to be incomplete trophoblast invasion of the maternal spiral arteries during placentation in the first trimester, resulting in oxidative and nitrative stress as well as maternal inflammation and organ alterations. In the Storkhead box 1 (STOX1) preeclampsia mouse model, pregnant females develop severe and early onset manifestations as seen in human preeclampsia e.g. gestational hypertension, proteinuria, and organ alterations. Here we aimed to evaluate the therapeutic potential of human recombinant alpha-1 microglobulin (rA1M) to alleviate the manifestations observed. Human rA1M significantly reduced the hypertension during gestation and significantly reduced the level of hypoxia and nitrative stress in the placenta. In addition, rA1M treatment reduced cellular damage in both placenta and kidneys, thereby protecting the tissue and improving their function. This study confirms that rA1M has the potential as a therapeutic drug in preeclampsia, and likely also in other pathological conditions associated with oxidative stress, by preserving normal organ function.

Low-dose aspirin protective effects are correlated with deregulation of HNF factor expression in the preeclamptic placentas from mice and humans.

Aspirin (acetyl-salicylic acid) is one of the most ancient drugs of the human pharmacopeia. Nonetheless, its action at low doses is not well understood at the molecular level. One of the applications of low-dose aspirin treatment is the prevention of preeclampsia (PE) in patients at risk. Foeto-placental overexpression of the STOX1A transcription factor in mice triggers PE symptoms. Transcriptomic analysis of the placentas, showed that aspirin massively down-regulates genes of the coagulation and complement cascade, as well as genes involved in lipid transport. The genes modified by aspirin treatment are not the ones that are modified by STOX1 overexpression, suggesting that aspirin could act downstream, symptomatically on the preeclamptic disease. Bioinformatics analysis of the promoters of the deregulated genes showed that they are strongly enriched in HNF transcription factors-binding sites, in accordance with existing literature showing their roles as regulators of coagulation. Two of these transcription factors, Hnf1ß and Hnf4α are found down-regulated by aspirin treatment. In parallel, we show that in human patient placentas, aspirin-induced deregulations of genes of the coagulation cascade are also observed. Finally, the expression of Hnf1ß target sequences (Kif12, F2, Hnf4α promoters and a synthetic concatemer of the Hnf1ß-binding site) were investigated by transfection in trophoblast cell models, with or without aspirin treatment and with or without STOX1A overexpression. In this model we observed that STOX1A and aspirin tended to synergize in the down-regulation of Hnf1ß target genes in trophoblasts.

Epigenetic Control of the Notch and Eph Signaling Pathways by the Prion Protein: Implications for Prion Diseases.

Among the ever-growing number of self-replicating proteins involved in neurodegenerative diseases, the prion protein PrP remains the most infamous for its central role in transmissible spongiform encephalopathies (TSEs). In these diseases, pathogenic prions propagate through a seeding mechanism, where normal PrPC molecules are converted into abnormally folded scrapie isoforms termed PrPSc. Since its discovery over 30 years ago, much advance has contributed to define the host-encoded cellular prion protein PrPC as a critical relay of prion-induced neuronal cell demise. A current consensual view is that the conversion of PrPC into PrPSc in neuronal cells diverts the former from its normal function with subsequent molecular alterations affecting synaptic plasticity. Here, we report that prion infection is associated with reduced expression of key effectors of the Notch pathway in vitro and in vivo, recapitulating changes fostered by the absence of PrPC. We further show that both prion infection and PrPC depletion promote drastic alterations in the expression of a defined set of Eph receptors and their ephrin ligands, which represent important players in synaptic function. Our data indicate that defects in the Notch and Eph axes can be mitigated in response to histone deacetylase inhibition in PrPC-depleted as well as prion-infected cells. We thus conclude that infectious prions cause a loss-of-function phenotype with respect to Notch and Eph signaling and that these alterations are sustained by epigenetic mechanisms.