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During aging, brain performances decline. Cellular senescence is one of the aging drivers and a key feature of a variety of human age‐related disorders. The transcriptional repressor RE1‐silencing transcription factor (REST) has been associated with aging and higher risk of neurodegenerative disorders. However, how REST contributes to the senescence program and functional impairment remains largely unknown. Here, we report that REST is essential to prevent the senescence phenotype in primary mouse neurons. REST deficiency causes failure of autophagy and loss of proteostasis, increased oxidative stress, and higher rate of cell death. Re‐establishment of autophagy reverses the main hallmarks of senescence. Our data indicate that REST has a protective role in physiological aging by regulating the autophagic flux and the senescence program in neurons, with implications for neurological disorders associated with aging., Cellular senescence is one of the aging drivers and a key feature of a variety of human age‐related disorders. The transcriptional repressor REST deficiency leads to cellular senescence in primary neurons. Rest depletion causes DNA damage, impairment of autophagic flux, mitochondrial dysfunction, and alteration of SASP genes expression. We describe a novel transcriptional regulator of the senescence program.

Neuroinflammation is associated with synapse dysfunction and cognitive decline in patients and animal models. One candidate for translating the inflammatory stress into structural and functional changes in neural networks is the transcriptional repressor RE1-silencing transcription factor (REST) that regulates the expression of a wide cluster of neuron-specific genes during neurogenesis and in mature neurons. To study the cellular and molecular pathways activated under inflammatory conditions mimicking the experimental autoimmune encephalomyelitis (EAE) environment, we analyzed REST activity in neuroblastoma cells and mouse cortical neurons treated with activated T cell or microglia supernatant and distinct pro-inflammatory cytokines. We found that REST is activated by a variety of neuroinflammatory stimuli in both neuroblastoma cells and primary neurons, indicating that a vast transcriptional change is triggered during neuroinflammation. While a dual activation of REST and its dominant-negative splicing isoform REST4 was observed in N2a neuroblastoma cells, primary neurons responded with a pure full-length REST upregulation in the absence of changes in REST4 expression. In both cases, REST upregulation was associated with activation of Wnt signaling and increased nuclear translocation of β-catenin, a well-known intracellular transduction pathway in neuroinflammation. Among single cytokines, IL-1β caused a potent and prompt increase in REST transcription and translation in neurons, which promoted a delayed and strong synaptic downscaling specific for excitatory synapses, with decreased frequency and amplitude of spontaneous synaptic currents, decreased density of excitatory synaptic connections, and decreased frequency of action potential-evoked Ca2+ transients. Most important, the IL-1β effects on excitatory transmission were strictly REST dependent, as conditional deletion of REST completely occluded the effects of IL-1β activation on synaptic transmission and network excitability. Our results demonstrate that REST upregulation represents a new pathogenic mechanism for the synaptic dysfunctions observed under neuroinflammatory conditions and identify the REST pathway as therapeutic target for EAE and, potentially, for multiple sclerosis.

// Evangelia Vartholomaiou 1,* , Marta Madon-Simon 1,* , Stephane Hagmann 1 , Guillaume Muhlebach 1 , Wolfgang Wurst 2,3,4,5 , Thomas Floss 2 and Didier Picard 1 1 Departement de Biologie Cellulaire, Universite de Geneve, Sciences III, Geneve, Switzerland 2 Helmholtz Zentrum Munchen, Deutsches Forschungszentrum fur Gesundheit und Umwelt, Neuherberg, Germany 3 Deutsches Zentrum fur Neurodegenerative Erkrankungen e. V., Munchen, Germany 4 Munich Cluster for Systems Neurology, Munchen, Germany 5 Technische Universitat Munchen-Weihenstephan, Neuherberg, Germany * These authors have contributed equally to this work Correspondence: Didier Picard, email: // Keywords : Hsp90; Trap1; breast cancer; metastasis; mouse model Received : January 12, 2017 Accepted : February 15, 2017 Published : February 23, 2017 Abstract The Hsp90 family of molecular chaperones includes the cytosolic isoforms Hsp90α and Hsp90β, and the mitochondrial isoform Trap1. Hsp90α/β support a large number of client proteins in the cytoplasm and the nucleus whereas Trap1 regulates oxidative phosphorylation in mitochondria. Many of the associated proteins and cellular processes are relevant to cancer, and there is ample pharmacological and genetic evidence to support the idea that Hsp90α/β and Trap1 are required for tumorigenesis. However, a direct and comparative genetic test in a mouse cancer model has not been done. Here we report the effects of deleting the Hsp90α or Trap1 genes in a mouse model of breast cancer. Neither Hsp90α nor Trap1 are absolutely required for mammary tumor initiation, growth and metastasis induced by the polyoma middle T-antigen as oncogene. However, they do modulate growth and lung metastasis in vivo and cell proliferation, migration and invasion of isolated primary carcinoma cells in vitro . Without Hsp90α, tumor burden and metastasis are reduced, correlating with impaired proliferation, migration and invasion of cells in culture. Without Trap1, the appearance of tumors is initially delayed, and isolated cells are affected similarly to those without Hsp90α. Analysis of expression data of human breast cancers supports the conclusion that this is a valid mouse model highlighting the importance of these molecular chaperones.

The dynamic interaction of DNA methylation and transcription factor binding in regulating spatiotemporal gene expression is essential for embryogenesis, but the underlying mechanisms remain understudied. In this study, using mouse models and integration of in vitro and in vivo genetic and epigenetic analyses, we show that the binding of REST (repressor element 1 (RE1) silencing transcription factor; also known as NRSF) to its cognate RE1 sequences is temporally regulated by non-CpG methylation. This process is dependent on DNA methyltransferase 3B (DNMT3B) and leads to suppression of adult cardiac genes in developing hearts. We demonstrate that DNMT3B preferentially mediates non-CpG methylation of REST-targeted genes in the developing heart. Downregulation of DNMT3B results in decreased non-CpG methylation of RE1 sequences, reduced REST occupancy, and consequently release of the transcription suppression during later cardiac development. Together, these findings reveal a critical gene silencing mechanism in developing mammalian hearts that is regulated by the dynamic interaction of DNMT3B-mediated non-CpG methylation and REST binding.

TAR DNA-binding protein 43 (TDP-43) is a key player in neurodegenerative diseases including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Accumulation of TDP-43 is associated with neuronal death in the brain. How increased and disease-causing mutant forms of TDP-43 induce cell death remains unclear. Here we addressed the role of TDP-43 during neural development and show that reduced TDP-43 causes defects in neural stem/progenitor cell proliferation but not cell death. However, overexpression of wild type and TDP-43A315T proteins induce p53-dependent apoptosis of neural stem/progenitors and human induced pluripotent cell (iPS)-derived immature cortical neurons. We show that TDP-43 induces expression of the proapoptotic BH3-only genes Bbc3 and Bax, and that p53 inhibition rescues TDP-43 induced cell death of embryonic mouse, and human cortical neurons, including those derived from TDP-43G298S ALS patient iPS cells. Hence, an increase in wild type and mutant TDP-43 induces p53-dependent cell death in neural progenitors developing neurons and this can be rescued. These findings may have important implications for accumulated or mutant TDP-43 induced neurodegenerative diseases., Scientific Reports, 8, ISSN:2045-2322

Pf1, also known as Phf12 (plant homeodomain [PHD] zinc finger protein 12), is a member of the PHD zinc finger family of proteins. Pf1 associates with a chromatin-interacting protein complex comprised of MRG15, Sin3B, and histone deacetylase 1 (HDAC1) that functions as a transcriptional modulator. The biological function of Pf1 remains largely elusive. We undertook the generation of Pf1 knockout mice to elucidate its physiological role. We demonstrate that Pf1 is required for mid- to late gestation viability. Pf1 inactivation impairs the proliferative potential of mouse embryonic fibroblasts (MEFs) and is associated with a significant decrease in bromodeoxyuridine incorporation; an increase in senescence-associated β-galactosidase (SA-β-Gal) activity, a marker of cellular senescence; and elevated levels of phosphorylated H2AX (γ-H2A.X), a marker associated with DNA double-strand breaks. Analysis of transcripts differentially expressed in wild-type and Pf1-deficient cells revealed the impact of Pf1 in multiple regulatory arms of the ribosome biogenesis pathways. Strikingly, assessment of the morphology of the nucleoli exposed an abnormal nucleolar structure in Pf1-deficient cells. Finally, proteomic analysis of the Pf1-interacting complexes highlighted proteins involved in ribosome biogenesis. Taken together, our data reveal an unsuspected function for the Pf1-associated chromatin complex in the ribosomal biogenesis and senescence pathways.

Rationale : 22q11 deletion syndrome arises from recombination between low-copy repeats on chromosome 22. Typical deletions result in hemizygosity for TBX1 associated with congenital cardiovascular disease. Deletions distal to the typically deleted region result in a similar cardiac phenotype but lack in extracardiac features of the syndrome, suggesting that a second haploinsufficient gene maps to this interval. Objective : The transcription factor HIC2 is lost in most distal deletions, as well as in a minority of typical deletions. We used mouse models to test the hypothesis that HIC2 hemizygosity causes congenital heart disease. Methods and Results : We created a genetrap mouse allele of Hic2. The genetrap reporter was expressed in the heart throughout the key stages of cardiac morphogenesis. Homozygosity for the genetrap allele was embryonic lethal before embryonic day E10.5, whereas the heterozygous condition exhibited a partially penetrant late lethality. One third of heterozygous embryos had a cardiac phenotype. MRI demonstrated a ventricular septal defect with over-riding aorta. Conditional targeting indicated a requirement for Hic2 within the Nkx2.5+ and Mesp1 + cardiovascular progenitor lineages. Microarray analysis revealed increased expression of Bmp10 . Conclusions : Our results demonstrate a novel role for Hic2 in cardiac development. Hic2 is the first gene within the distal 22q11 interval to have a demonstrated haploinsufficient cardiac phenotype in mice. Together our data suggest that HIC2 haploinsufficiency likely contributes to the cardiac defects seen in distal 22q11 deletion syndrome.

Sphingolipids and the derived gangliosides have critical functions in spermatogenesis, thus mutations in genes involved in sphingolipid biogenesis are often associated with male infertility. We have generated a transgenic mouse line carrying an insertion in the sphingomyelin synthase gene Sms1, the enzyme which generates sphingomyelin species in the Golgi apparatus. We describe the spermatogenesis defect of Sms1-/- mice, which is characterized by sloughing of spermatocytes and spermatids, causing progressive infertility of male homozygotes. Lipid profiling revealed a reduction in several long chain unsaturated phosphatidylcholins, lysophosphatidylcholins and sphingolipids in the testes of mutants. Multi-Spectral Optoacoustic Tomography indicated blood-testis barrier dysfunction. A supplementary diet of the essential omega-3 docosahexaenoic acid and eicosapentaenoic acid diminished germ cell sloughing from the seminiferous epithelium and restored spermatogenesis and fertility in 50% of previously infertile mutants. Our findings indicate that SMS1 has a wider than anticipated role in testis polyunsaturated fatty acid homeostasis and for male fertility.

Mitochondrial diseases often exhibit tissue-specific pathologies, but this phenomenon is poorly understood. Here we present regulation of mitochondrial translation by the Mitochondrial Translation Optimization Factor 1, MTO1, as a novel player in this scenario. We demonstrate that MTO1 mediates tRNA modification and controls mitochondrial translation rate in a highly tissue-specific manner associated with tissue-specific OXPHOS defects. Activation of mitochondrial proteases, aberrant translation products, as well as defects in OXPHOS complex assembly observed in MTO1 deficient mice further imply that MTO1 impacts translation fidelity. In our mouse model, MTO1-related OXPHOS deficiency can be bypassed by feeding a ketogenic diet. This therapeutic intervention is independent of the MTO1-mediated tRNA modification and involves balancing of mitochondrial and cellular secondary stress responses. Our results thereby establish mammalian MTO1 as a novel factor in the tissue-specific regulation of OXPHOS and fine tuning of mitochondrial translation accuracy.

The maintenance and control of pluripotency is of great interest in stem cell biology. The dual specificity T-box/basic-helix-loop-helix-zipper transcription factor Mga is expressed in the pluripotent cells of the inner cell mass (ICM) and epiblast of the peri-implantation mouse embryo, but its function has not been investigated previously. Here, we use a loss-of-function allele and RNA knockdown to demonstrate that Mga depletion leads to the death of proliferating pluripotent ICM cells in vivo and in vitro, and the death of embryonic stem cells (ESCs) in vitro. Additionally, quiescent pluripotent cells lacking Mga are lost during embryonic diapause. Expression of Odc1, the rate-limiting enzyme in the conversion of ornithine into putrescine in the synthesis of polyamines, is reduced in Mga mutant cells, and the survival of mutant ICM cells as well as ESCs is rescued in culture by the addition of exogenous putrescine. These results suggest a mechanism whereby Mga influences pluripotent cell survival through regulation of the polyamine pool in pluripotent cells of the embryo, whether they are in a proliferative or quiescent state.

Recently, mutations in the mitochondrial translation optimization factor 1 gene (MTO1) were identified as causative in children with hypertrophic cardiomyopathy, lactic acidosis and respiratory chain defect. Here, we describe an MTO1-deficient mouse model generated by gene trap mutagenesis that mirrors the human phenotype remarkably well. As in patients, the most prominent signs and symptoms were cardiovascular and included bradycardia and cardiomyopathy. In addition, the mutant mice showed a marked worsening of arrhythmias during induction and reversal of anaesthesia. The detailed morphological and biochemical workup of murine hearts indicated that the myocardial damage was due to complex I deficiency and mitochondrial dysfunction. In contrast, neurological examination was largely normal in Mto1-deficient mice. A translational consequence of this mouse model may be to caution against anaesthesia-related cardiac arrhythmias which may be fatal in patients.

Targeted mouse mutants are instrumental for the analysis of gene function in health and disease. We recently provided proof-of-principle for the fast-track mutagenesis of the mouse genome, using transcription activator-like effector nucleases (TALENs) in one-cell embryos. Here we report a routine procedure for the efficient production of disease-related knockin and knockout mutants, using improved TALEN mRNAs that include a plasmid-coded poly(A) tail (TALEN-95A), circumventing the problematic in vitro polyadenylation step. To knock out the C9orf72 gene as a model of frontotemporal lobar degeneration, TALEN-95A mutagenesis induced sequence deletions in 41% of pups derived from microinjected embryos. Using TALENs together with mutagenic oligodeoxynucleotides, we introduced amyotrophic lateral sclerosis patient-derived missense mutations in the fused in sarcoma (Fus) gene at a rate of 6.8%. For the simple identification of TALEN-induced mutants and their progeny we validate high-resolution melt analysis (HRMA) of PCR products as a sensitive and universal genotyping tool. Furthermore, HRMA of off-target sites in mutant founder mice revealed no evidence for undesired TALEN-mediated processing of related genomic sequences. The combination of TALEN-95A mRNAs for enhanced mutagenesis and of HRMA for simplified genotyping enables the accelerated, routine production of new mouse models for the study of genetic disease mechanisms.

Alternative mRNA splicing provides transcript diversity and may contribute to human disease. We demonstrate that expression of several genes regulating RNA processing is decreased in both liver and skeletal muscle of obese humans. We evaluated a representative splicing factor, SFRS10, downregulated in both obese human liver and muscle and in high-fat-fed mice, and determined metabolic impact of reduced expression. SFRS10-specific siRNA induces lipogenesis and lipid accumulation in hepatocytes. Moreover, Sfrs10 heterozygous mice have increased hepatic lipogenic gene expression, VLDL secretion, and plasma triglycerides. We demonstrate that LPIN1, a key regulator of lipid metabolism, is a splicing target of SFRS10; reduced SFRS10 favors the lipogenic β isoform of LPIN1. Importantly, LPIN1β-specific siRNA abolished lipogenic effects of decreased SFRS10 expression. Together, our results indicate that reduced expression of SFRS10, as observed in tissues from obese humans, alters LPIN1 splicing, induces lipogenesis, and therefore contributes to metabolic phenotypes associated with obesity.

Loss of function of DJ-1 (PARK7) is associated with autosomal recessive early-onset Parkinson's disease (PD), one of the major age-related neurological diseases. In this study, we extended former studies on DJ-1 knockout mice by identifying subtle morphological and behavioural phenotypes. The DJ-1 gene trap-induced null mutants exhibit less dopamine-producing neurons in the ventral tegmental area (VTA). They also exhibit slight changes in behaviour, i.e. diminished rearing behaviour and impairments in object recognition. Furthermore, we detected subtle phenotypes, which suggest that these animals compensate for the loss of DJ-1. First, we found a significant upregulation of mitochondrial respiratory enzyme activities, a mechanism known to protect against oxidative stress. Second, a close to significant increase in c-Jun N-terminal kinase 1 phosphorylation in old DJ-1-deficient mice hints at a differential activation of neuronal cell survival pathways. Third, as no change in the density of tyrosine hydroxylase (TH)-positive terminals in the striatum was observed, the remaining dopamine-producing neurons likely compensate by increasing axonal sprouting. In summary, the present data suggest that DJ-1 is implicated in major non-motor symptoms of PD appearing in the early phases of the disease-such as subtle impairments in motivated behaviour and cognition-and that under basal conditions the loss of DJ-1 is compensated.

Recombinase-mediated cassette exchange (RMCE) exploits the possibility to unidirectionally exchange any genetic material flanked by heterotypic recombinase recognition sites (RRS) with target sites in the genome. Due to a limited number of available pre-fabricated target sites, RMCE in mouse embryonic stem (ES) cells has not been tapped to its full potential tRecombinase-mediated cassette exchange (RMCE) exploits the possibility to unidirectionally exchange any genetic material flanked by heterotypic recombinase recognition sites (RRS) with target sites in the genome. Due to a limited number of available pre-fabricated target sites, RMCE in mouse embryonic stem (ES) cells has not been tapped to its full potential to date. Here, we introduce a universal system, which allows the targeted insertion of any given transcriptional unit into 85 742 previously annotated retroviral conditional gene trap insertions, representing 7013 independent genes in mouse ES cells, by RMCE. This system can be used to express any given cDNA under the control of endogenous trapped promoters in vivo, as well as for the generation of transposon 'launch pads' for chromosomal region-specific 'Sleeping Beauty' insertional mutagenesis. Moreover, transcription of the gene-of-interest is only activated upon Cre-recombinase activity, a feature that adds conditionality to this expression system, which is demonstrated in vivo. The use of the RMCE system presented in this work requires one single-cloning step followed by one overnight gateway clonase reaction and subsequent cassette exchange in ES cells with efficiencies of 40% in average.o date. Here, we introduce a universal system, which allows the targeted insertion of any given transcriptional unit into 85 742 previously annotated retroviral conditional gene trap insertions, representing 7013 independent genes in mouse ES cells, by RMCE. This system can be used to express any given cDNA under the control of endogenous trapped promoters in vivo, as well as for the generation of transposon 'launch pads' for chromosomal region-specific 'Sleeping Beauty' insertional mutagenesis. Moreover, transcription of the gene-of-interest is only activated upon Cre-recombinase activity, a feature that adds conditionality to this expression system, which is demonstrated in vivo. The use of the RMCE system presented in this work requires one single-cloning step followed by one overnight gateway clonase reaction and subsequent cassette exchange in ES cells with efficiencies of 40% in average.

Gene trapping is used to introduce insertional mutations into genes of mouse embryonic stem cells (ESCs). It is performed with gene trap vectors that simultaneously mutate and report the expression of the endogenous gene at the site of insertion and provide a DNA tag for rapid identification of the disrupted gene. Gene traps have been employed worldwide to assemble libraries of mouse ESC lines harboring mutations in single genes, which can be used to make mutant mice. However, most of the employed gene trap vectors require gene expression for reporting a gene trap event and therefore genes that are poorly expressed may be under-represented in the existing libraries. To address this problem, we have developed a novel class of gene trap vectors that can induce gene expression at insertion sites, thereby bypassing the problem of intrinsic poor expression. We show here that the insertion of the osteopontin enhancer into several conventional gene trap vectors significantly increases the gene trapping efficiency in high-throughput screens and facilitates the recovery of poorly expressed genes.

Nucleostemin (NS) is a putative GTPase expressed preferentially in the nucleoli of neuronal and embryonic stem cells and several cancer cell lines. Transfection and knockdown studies indicated that NS controls the proliferation of these cells by interacting with the p53 tumor suppressor protein and regulating its activity. To assess the physiological role of NS in vivo, we generated a mutant mouse line with a specific gene trap event that inactivates the NS allele. The corresponding NS(-/-) embryos died around embryonic day 4. Analyses of NS mutant blastocysts indicated that NS is not required to maintain pluripotency, nucleolar integrity, or survival of the embryonic stem cells. However, the homozygous mutant blastocysts failed to enter S phase even in the absence of functional p53. Haploid insufficiency of NS in mouse embryonic fibroblasts leads to decreased cell proliferation. NS also functions in early amphibian development to control cell proliferation of neural progenitor cells. Our results show that NS has a unique ability, derived from an ancestral function, to control the proliferation rate of stem/progenitor cells in vivo independently of p53.

International audience; A type of retroviral gene trap vectors has been developed that can induce conditional mutations in most genes expressed in mouse embryonic stem (ES) cells. The vectors rely on directional site-specific recombination systems that can repair and re-induce gene trap mutations when activated in succession. After the gene traps are inserted into the mouse genome, genetic mutations can be produced at a particular time and place in somatic cells. In addition to their conditional features, the vectors create multipurpose alleles amenable to a wide range of post-insertional modifications. Here we have used these directional recombination vectors to assemble the largest library of ES cell lines with conditional mutations in single genes yet assembled, presently totaling 1,000 unique genes. The trapped ES cell lines, which can be ordered from the German Gene Trap Consortium, are freely available to the scientific community.

A major task in the second phase of the genome sequencing projects is the identification of coding sequence within the three billion base pairs of each the mouse and human genomes. At present, in addition to computer-aided programs, several high-throughput mutagenesis programs are being undertaken worldwide in order to achieve this goal (Ref. 1). Gene-trap mutagenesis screens take advantage of random insertions into transcription units to drive a selection–reporter cassette and simultaneously to mutate the tagged genes. One of the advantages of gene-trap mutagenesis is that no a priori knowledge is needed about the structure of the tagged gene. However, because most gene-trap vectors contain splice-acceptor or splice-donor sites to capture translated gene sequence, the precise location of vector integration within introns is not known. Therefore, it is often difficult to generate external probes for Southern blots or external primers for PCR analysis in order to distinguish between homozygous and heterozygous mutants. To overcome this serious limitation in high-throughput mutagenesis screens, we developed a real-time PCR strategy that allows us to discriminate between mutants with either one or two copies of the gene-trap vector inside their genomes. Real-time quantitative PCR is based on the quantification of a fluorescent dye [5′-6-carboxyfluorescein (5′-FAM)] that is quenched by 3′-6-carboxy-tetramethylrhodamine (3′ TAMRA) when attached to a probe located between two PCR primers but is activated by the 5′ exonuclease activity of the Taq DNA polymerase (Ref. 2,3 ). Here, we describe a rapid method based on the quantification of a gene-trap vector relative to a standard locus within the mouse genome. This method allows the rapid genotyping of any gene-trap animal without prior knowledge of the mutated genes. Here, as an example, we describe the genotyping of a PT1βgeo insertion (Ref. 4) into the mouse Neurochondrin gene (Ref. 5) by comparing a multiplex PCR assay and a novel real-time-PCR-based method.

We would like to respond to Brosch et al. regarding our manuscript "Expression of the Splicing Factor Gene SFRS10 Is Reduced in Human Obesity and Contributes to Enhanced Lipogenesis" (Pihlajamaki et al., 2011b). Brosch performed RT-PCR in liver samples from 13 lean and 34 obese individuals, finding no differences in SFRS10 or LPIN1 expression. We wish to address points raised by Brosch, including experimental strategy and analysis of human SFRS10 expression.