Mitochondrial Electron Transport Chain Complex II Dysfunction Causes Premature Aging of Hematopoietic Stem Cells.

Mitochondria are indispensable in maintaining hematopoietic stem cells (HSCs), and mitochondrial complex II (MCII) has been recognized as a key component of HSCs. However, the physiological role of MCII on long-term hematopoiesis and hematopoietic reconstitution capacity remains unknown. Hence, this study evaluated the impact of MCII dysfunctions on long-term HSC maintenance and hematopoietic homeostasis among conditional transgenic mice with a missense mutation in the succinate dehydrogenase complex subunit C gene (SdhcV69E). HSCs collected from SdhcV69E mice had a higher reactive oxygen species (ROS) accumulation and DNA damage in response to mitochondrial activation. Via the aging stress response, MCII dysfunctions caused decreased white blood cell count with myeloid-skewing property, macrocytic anemia, and thrombocytosis. Moreover, the HSCs of aged SdhcV69E mice exhibited greater ROS accumulation and lower membrane potential. Transplantation-induced replicative stress also caused premature senescent hematopoiesis. Furthermore, accelerated ROS accumulation and profound DNA damage in HSCs were observed in the SdhcV69E-derived cell recipients. The long-term hematopoietic reconstitution capacity was remarkably impaired in HSCs from the SdhcV69E-derived cell recipients. Taken together, MCII plays an essential role in long-term hematopoiesis, and MCII dysfunctions with aging or replicative stresses caused excessive ROS accumulation and DNA damage in HSCs, leading to premature senescence.

DETECTION OF TRANSGENERATIONAL GENETIC EFFECTS BASED ON WHOLE-GENOME SEQUENCING IN THE MOUSE MODEL.

It has become feasible to detect de novo mutations in mammalian genomes by using whole-genome sequencing. The power to detect numbers of de novo mutations should provide a useful tool to assess the transgenerational genetic effects of radiations on living organisms. By reviewing the spontaneous mutations in the mouse as a model, an action plan is proposed to detect the induced mutations after accumulating mutations for several generations with continuous exposure to low-dose radiations. Some susceptibility differences against radiations between humans and model animals for the transgenerational effect have been suggested. The applicability of the mouse model for the assessment of low-dose radiation is also discussed.

Establishment of mouse line showing inducible priapism-like phenotypes.

Purpose: Penile research is expected to reveal new targets for treatment and prevention of the complex mechanisms of its disorder including erectile dysfunction (ED). Thus, analyses of the molecular processes of penile ED and continuous erection as priapism are essential issues of reproductive medicine. Methods: By performing mouse N-ethyl-N-nitrosourea mutagenesis and exome sequencing, we established a novel mouse line displaying protruded genitalia phenotype (PGP; priapism-like phenotype) and identified a novel Pitpna gene mutation for PGP. Extensive histological analyses on the Pitpna mutant and intracavernous pressure measurement (ICP) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS)/MS analyses were performed. Results: We evaluated the role of phospholipids during erection for the first time and showed the mutants of inducible phenotypes of priapism. Moreover, quantitative analysis using LC-ESI/MS/MS revealed that the level of phosphatidylinositol (PI) was significantly lower in the mutant penile samples. These results imply that PI may contribute to penile erection by PITPα. Conclusions: Our findings suggest that the current mutant is a mouse model for priapism and abnormalities in PI signaling pathways through PITPα may lead to priapism providing an attractive novel therapeutic target in its treatment.

Early embryonic mutations reveal dynamics of somatic and germ cell lineages in mice.

De novo mutations accumulate with zygotic cell divisions. However, the occurrence of these mutations and the way they are inherited by somatic cells and germ cells remain unclear. Here, we present a novel method to reconstruct cell lineages. We identified mosaic mutations in mice using deep whole-genome sequencing and reconstructed embryonic cell lineages based on the variant allele frequencies of the mutations. The reconstructed trees were confirmed using nuclear transfer experiments and the genotyping of approximately 50 offspring of each tree. The most detailed tree had 32 terminal nodes and showed cell divisions from the fertilized egg to germ cell- and somatic cell-specific lineages, indicating at least five independent cell lineages that would be selected as founders of the primordial germ cells. The contributions of each lineage to germ cells and offspring varied widely. At the emergence of the germ cell-specific lineages, 10-15 embryonic mutations had accumulated, suggesting that the pregastrulation mutation rate is 1.0 mutation per mitosis. Subsequent mutation rates were 0.7 for germ cells and 13.2 for tail fibroblasts. Our results show a new framework to assess embryonic lineages; further, we suggest an evolutionary strategy for preserving heterogeneity owing to postzygotic mutations in offspring.

Assessing reproducibility of inherited variants detected with short-read whole genome sequencing.

BACKGROUND: Reproducible detection of inherited variants with whole genome sequencing (WGS) is vital for the implementation of precision medicine and is a complicated process in which each step affects variant call quality. Systematically assessing reproducibility of inherited variants with WGS and impact of each step in the process is needed for understanding and improving quality of inherited variants from WGS. RESULTS: To dissect the impact of factors involved in detection of inherited variants with WGS, we sequence triplicates of eight DNA samples representing two populations on three short-read sequencing platforms using three library kits in six labs and call variants with 56 combinations of aligners and callers. We find that bioinformatics pipelines (callers and aligners) have a larger impact on variant reproducibility than WGS platform or library preparation. Single-nucleotide variants (SNVs), particularly outside difficult-to-map regions, are more reproducible than small insertions and deletions (indels), which are least reproducible when > 5 bp. Increasing sequencing coverage improves indel reproducibility but has limited impact on SNVs above 30×. CONCLUSIONS: Our findings highlight sources of variability in variant detection and the need for improvement of bioinformatics pipelines in the era of precision medicine with WGS.

Depression-like behaviors induced by defective PTPRT activity through dysregulated synaptic functions and neurogenesis.

PTPRT has been known to regulate synaptic formation and dendritic arborization of hippocampal neurons. PTPRT-/- null and PTPRT-D401A mutant mice displayed enhanced depression-like behaviors compared with wild-type mice. Transient knockdown of PTPRT in the dentate gyrus enhanced the depression-like behaviors of wild-type mice, whereas rescued expression of PTPRT ameliorated the behaviors of PTPRT-null mice. Chronic stress exposure reduced expression of PTPRT in the hippocampus of mice. In PTPRT-deficient mice the expression of GluR2 (also known as GRIA2) was attenuated as a consequence of dysregulated tyrosine phosphorylation, and the long-term potentiation at perforant-dentate gyrus synapses was augmented. The inhibitory synaptic transmission of the dentate gyrus and hippocampal GABA concentration were reduced in PTPRT-deficient mice. In addition, the hippocampal expression of GABA transporter GAT3 (also known as SLC6A11) was decreased, and its tyrosine phosphorylation was increased in PTPRT-deficient mice. PTPRT-deficient mice displayed reduced numbers and neurite length of newborn granule cells in the dentate gyrus and had attenuated neurogenic ability of embryonic hippocampal neural stem cells. In conclusion, our findings show that the physiological roles of PTPRT in hippocampal neurogenesis, as well as synaptic functions, are involved in the pathogenesis of depressive disorder.

Involvement of Rev1 in alkylating agent-induced loss of heterozygosity in Oryzias latipes.

Translesion synthesis (TLS) polymerases mediate DNA damage bypass during replication. The TLS polymerase Rev1 has two important functions in the TLS pathway, including dCMP transferase activity and acting as a scaffolding protein for other TLS polymerases at the C-terminus. Because of the former activity, Rev1 bypasses apurinic/apyrimidinic sites by incorporating dCMP, whereas the latter activity mediates assembly of multipolymerase complexes at the DNA lesions. We generated rev1 mutants lacking each of these two activities in Oryzias latipes (medaka) fish and analyzed cytotoxicity and mutagenicity in response to the alkylating agent diethylnitrosamine (DENA). Mutant lacking the C-terminus was highly sensitive to DENA cytotoxicity, whereas mutant with reduced dCMP transferase activity was slightly sensitive to DENA cytotoxicity, but exhibited a higher tumorigenic rate than wild-type fish. There was no significant difference in the frequency of DENA-induced mutations between mutant with reduced dCMP transferase activity and wild-type cultured cell. However, loss of heterozygosity (LOH) occurred frequently in cells with reduced dCMP transferase activity. LOH is a common genetic event in many cancer types and plays an important role on carcinogenesis. To our knowledge, this is the first report to identify the involvement of the catalytic activity of Rev1 in suppression of LOH.

Crim1C140S mutant mice reveal the importance of cysteine 140 in the internal region 1 of CRIM1 for its physiological functions.

Cysteine-rich transmembrane bone morphogenetic protein regulator 1 (CRIM1) is a type I transmembrane protein involved in the organogenesis of many tissues via its interactions with growth factors including BMP, TGF-ß, and VEGF. In this study, we used whole-exome sequencing and linkage analysis to identify a novel Crim1 mutant allele generated by ENU mutagenesis in mice. This allele is a missense mutation that causes a cysteine-to-serine substitution at position 140, and is referred to as Crim1C140S. In addition to the previously reported phenotypes in Crim1 mutants, Crim1C140S homozygous mice exhibited several novel phenotypes, including dwarfism, enlarged seminal vesicles, and rectal prolapse. In vitro analyses showed that Crim1C140S mutation affected the formation of CRIM1 complexes and decreased the amount of the overexpressed CRIM1 proteins in the cell culture supernatants. Cys140 is located in the internal region 1 (IR1) of the N-terminal extracellular region of CRIM1 and resides outside any identified functional domains. Inference of the domain architecture suggested that the Crim1C140S mutation disturbs an intramolecular disulfide bond in IR1, leading to the protein instability and the functional defects of CRIM1. Crim1C140S highlights the functional importance of the IR1, and Crim1C140S mice should serve as a valuable model for investigating the functions of CRIM1 that are unidentified as yet.

Mice with endogenous TDP-43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis.

TDP-43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP-43 function at physiological levels both in vitro and in vivo Interestingly, we find that mutations within the C-terminal domain of TDP-43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP-43 loss- and gain-of-function effects. TDP-43 gain-of-function effects in these mice reveal a novel category of splicing events controlled by TDP-43, referred to as "skiptic" exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain-of-function mutation in endogenous Tardbp causes an adult-onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain-of-function and skiptic exons in ALS patient-derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP-43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.

A Comprehensive Mouse Transcriptomic BodyMap across 17 Tissues by RNA-seq.

The mouse has been widely used as a model organism for studying human diseases and for evaluating drug safety and efficacy. Many diseases and drug effects exhibit tissue specificity that may be reflected by tissue-specific gene-expression profiles. Here we construct a comprehensive mouse transcriptomic BodyMap across 17 tissues of six-weeks old C57BL/6JJcl mice using RNA-seq. We find different expression patterns between protein-coding and non-coding genes. Liver expressed the least complex transcriptomes, that is, the smallest number of genes detected in liver across all 17 tissues, whereas testis and ovary harbor more complex transcriptomes than other tissues. We report a comprehensive list of tissue-specific genes across 17 tissues, along with a list of 4,781 housekeeping genes in mouse. In addition, we propose a list of 27 consistently and highly expressed genes that can be used as reference controls in expression-profiling analysis. Our study provides a unique resource of mouse gene-expression profiles, which is helpful for further biomedical research.

An ENU-induced p.C225S missense mutation in the mouse Tgfb1 gene does not cause Camurati-Engelmann disease-like skeletal phenotypes.

Camurati-Engelmann disease (CED) is a rare sclerosing bone disorder in humans with autosomal dominant inheritance. Mutations in the gene (TGFB1) that encodes transforming growth factor-ß1 (TGF-ß1) are causative for CED. TGF-ß1 signaling is enhanced by the CED-causing mutations. In this study, we performed Tgfb1 mutation screening in an ENU-mutagenized mouse genomic DNA library. We identified a missense mutation in which cysteine was substituted by serine at position 225 (p.C225S), that corresponded to the CED-causing mutation (p.C225R). TGF-ß1 mutant protein carrying p.C225S was secreted normally into the extracellular space. Reporter gene assays showed that the p.C225S mutants enhanced TGF-ß signaling at the same level as p.C225R mutants. We generated p.C225S homozygous mice and confirmed that the mature TGF-ß1 levels in the culture supernatants of the calvarial cells from the homozygotes were significantly higher than those from wild-type mice. Although the skull and femur are sclerotic in CED, these phenotypes were not observed in p.C225S homozygous mice. These results suggest that human and mouse bone tissue react differently to TGF-ß1. These findings are useful to pharmacological studies using mouse models in developing drugs that will target TGF-ß signaling.

Illegitimate translation causes unexpected gene expression from on-target out-of-frame alleles created by CRISPR-Cas9.

CRISPR-Cas9 is efficient enough to knock out both alleles directly by introducing out-of-frame mutations. We succeeded in making biallelic on-target frameshift mutations of the endogenous Gli3 gene; however, the GLI3 protein was expressed in all six of the established cell lines carrying homozygous out-of-frame mutations. We developed a dual-tagged expression vector and proved that illegitimate translation (ITL) was the cause of the unexpected Gli3 expression. Thus, gene expression must be examined even if designed on-target out-of-frame sequences are introduced by genome editing. In addition, it is highly recommended to pre-examine the occurrence of ITL in vitro prior to the design and construction of any genome-editing vectors. In vitro assay systems such as the dual-tagged ITL assay system developed in this study should aid the identification and elucidation of ITL-based human diseases and gene expression.

Dose-dependent de novo germline mutations detected by whole-exome sequencing in progeny of ENU-treated male gpt delta mice.

Germline mutations are an important component of genetic toxicology; however, mutagenicity tests of germline cells are limited. Recent advances in sequencing technology can be used to detect mutations by direct sequencing of genomic DNA (gDNA). We previously reported induced de novo mutations detected using whole-exome sequencing in the offspring of N-ethyl-N-nitrosourea (ENU)-treated mice in a single-dose experiment (85mg/kg, i.p., weekly on two occasions). In this study, two lower doses (10 and 30mg/kg) were added, and dose-response of inherited germline mutations was analyzed. Male gpt delta transgenic mice treated with ENU in three dose groups were mated with untreated females 10 weeks after the last treatment, and offspring were obtained. The ENU-treated male mice showed dose-dependent increases in gpt mutant frequencies in their sperm, testis, and liver. gDNA of one family (parents and four offspring) from each dose group was used for whole-exome sequencing, and unique de novo mutations in the offspring were detected. Frequencies of inherited mutations increased with dosage more than 25-fold in the highest dose group. The mutation spectrum of the inherited mutations showed characteristics of ENU-induced mutations, such as A:T base substitutions. No confirmed mutations were observed in the control group. Filtering using the alternate reads ratio resulted in the mutation frequencies and spectra similar to those obtained by the Sanger sequencing confirmation. These results suggest that direct sequencing analysis may be a useful tool to investigate inherited germline mutations induced by environmental mutagens.

Estimation of the frequency of inherited germline mutations by whole exome sequencing in ethyl nitrosourea-treated and untreated gpt delta mice.

BACKGROUND: Germline mutations are heritable and may cause health disadvantages in the next generation. To investigate trans-generational mutations, we treated male gpt delta mice with N-ethyl-N-nitrosourea (ENU) (85 mg/kg intraperitoneally, weekly on two occasions). The mice were mated with untreated female mice and offspring were obtained. Whole exome sequencing analyses were performed to identify de novo mutations in the offspring. RESULTS: At 20 weeks after the treatment, the gpt mutant frequencies in the sperm of ENU-treated mice were 21-fold higher than those in the untreated control. Liver DNA was extracted from six mice, including the father, mother, and four offspring from each family of the ENU-treated or untreated mice. In total, 12 DNA samples were subjected to whole exome sequencing analyses. We identified de novo mutations in the offspring by comparing single nucleotide variations in the parents and offspring. In the ENU-treated group, we detected 148 mutation candidates in four offspring and 123 (82 %) were confirmed as true mutations by Sanger sequencing. In the control group, we detected 12 candidate mutations, of which, three (25 %) were confirmed. The frequency of inherited mutations in the offspring from the ENU-treated family was 184 × 10(-8) per base, which was 17-fold higher than that in the control family (11 × 10(-8) per base). The de novo mutation spectrum in the next generation exhibited characteristic ENU-induced somatic mutations, such as base substitutions at A:T bp. CONCLUSIONS: These results suggest that direct sequencing analyses can be a useful tool for investigating inherited germline mutations and that the germ cells could be a good endpoint for evaluating germline mutations, which are transmitted to offspring as inherited mutations.

Novel allelic mutations in murine Serca2 induce differential development of squamous cell tumors.

Dominant mutations in the Serca2 gene, which encodes sarco(endo)plasmic reticulum calcium-ATPase, predispose mice to gastrointestinal epithelial carcinoma [1-4] and humans to Darier disease (DD) [14-17]. In this study, we generated mice harboring N-ethyl-N-nitrosourea (ENU)-induced allelic mutations in Serca2: three missense mutations and one nonsense mutation. Mice harboring these Serca2 mutations developed tumors that were categorized as either early onset squamous cell tumors (SCT), with development similar to null-type knockout mice [2,4] (aggressive form; M682, M814), or late onset tumors (mild form; M1049, M1162). Molecular analysis showed no aberration in Serca2 mRNA or protein expression levels in normal esophageal cells of any of the four mutant heterozygotes. There was no loss of heterozygosity at the Serca2 locus in the squamous cell carcinomas in any of the four lines. The effect of each mutation on Ca(2+)-ATPase activity was predicted using atomic-structure models and accumulated mutated protein studies, suggesting that putative complete loss of Serca2 enzymatic activity may lead to early tumor onset, whereas mutations in which Serca2 retains residual enzymatic activity result in late onset. We propose that impaired Serca2 gene product activity has a long-term effect on squamous cell carcinogenesis from onset to the final carcinoma stage through an as-yet unrecognized but common regulatory pathway.

Mouse models for ROS1-fusion-positive lung cancers and their application to the analysis of multikinase inhibitor efficiency.

ROS1-fusion genes, resulting from chromosomal rearrangement, have been reported in 1-2% of human non-small cell lung cancer cases. More than 10 distinct ROS1-fusion genes, including break-point variants, have been identified to date. In this study, to investigate the in vivo oncogenic activities of one of the most frequently detected fusions, CD74-ROS1, as well as another SDC4-ROS1 fusion that has also been reported in several studies, we generated transgenic (TG) mouse strains that express either of the two ROS1-fusion genes specifically in lung alveolar type II cells. Mice in all TG lines developed tumorigenic nodules in the lung, and a few strains of both TG mouse lines demonstrated early-onset nodule development (multiple tumor lesions present in the lung at 2-4 weeks after birth); therefore, these two strains were selected for further investigation. Tumors developed progressively in the untreated TG mice of both lines, whereas those receiving oral administration of an ALK/MET/ROS1 inhibitor, crizotinib, and an ALK/ROS1 inhibitor, ASP3026, showed marked reduction in the tumor burden. Collectively, these data suggest that each of these two ROS1-fusion genes acts as a driver for the pathogenesis of lung adenocarcinoma in vivo The TG mice developed in this study are expected to serve as valuable tools for exploring novel therapeutic agents against ROS1-fusion-positive lung cancer.

STEP signaling pathway mediates psychomotor stimulation and morphine withdrawal symptoms, but not for reward, analgesia and tolerance.

Striatal-enriched protein tyrosine phosphatase (STEP) is abundantly expressed in the striatum, which strongly expresses dopamine and opioid receptors and mediates the effects of many drugs of abuse. However, little is known about the role of STEP in opioid receptor function. In the present study, we generated STEP-targeted mice carrying a nonsense mutation (C230X) in the kinase interaction domain of STEP by screening the N-ethyl-N-nitrosourea (ENU)-driven mutant mouse genomic DNA library and subsequent in vitro fertilization. It was confirmed that the C230X nonsense mutation completely abolished functional STEP protein expression in the brain. STEP(C230X-/-) mice showed attenuated acute morphine-induced psychomotor activity and withdrawal symptoms, whereas morphine-induced analgesia, tolerance and reward behaviors were unaffected. STEP(C230X-/-) mice displayed reduced hyperlocomotion in response to intrastriatal injection of the µ-opioid receptor agonist DAMGO, but the behavioral responses to δ- and κ-opioid receptor agonists remained intact. These results suggest that STEP has a key role in the regulation of psychomotor action and physical dependency to morphine. These data suggest that STEP inhibition may be a critical target for the treatment of withdrawal symptoms associated with morphine.

Self-directed exploration provides a Ncs1-dependent learning bonus.

Understanding the mechanisms of memory formation is fundamental to establishing optimal educational practices and restoring cognitive function in brain disease. Here, we show for the first time in a non-primate species, that spatial learning receives a special bonus from self-directed exploration. In contrast, when exploration is escape-oriented, or when the full repertoire of exploratory behaviors is reduced, no learning bonus occurs. These findings permitted the first molecular and cellular examinations into the coupling of exploration to learning. We found elevated expression of neuronal calcium sensor 1 (Ncs1) and dopamine type-2 receptors upon self-directed exploration, in concert with increased neuronal activity in the hippocampal dentate gyrus and area CA3, as well as the nucleus accumbens. We probed further into the learning bonus by developing a point mutant mouse (Ncs1(P144S/P144S)) harboring a destabilized NCS-1 protein, and found this line lacked the equivalent self-directed exploration learning bonus. Acute knock-down of Ncs1 in the hippocampus also decoupled exploration from efficient learning. These results are potentially relevant for augmenting learning and memory in health and disease, and provide the basis for further molecular and circuit analyses in this direction.

Degradation of Stop Codon Read-through Mutant Proteins via the Ubiquitin-Proteasome System Causes Hereditary Disorders.

During translation, stop codon read-through occasionally happens when the stop codon is misread, skipped, or mutated, resulting in the production of aberrant proteins with C-terminal extension. These extended proteins are potentially deleterious, but their regulation is poorly understood. Here we show in vitro and in vivo evidence that mouse cFLIP-L with a 46-amino acid extension encoded by a read-through mutant gene is rapidly degraded by the ubiquitin-proteasome system, causing hepatocyte apoptosis during embryogenesis. The extended peptide interacts with an E3 ubiquitin ligase, TRIM21, to induce ubiquitylation of the mutant protein. In humans, 20 read-through mutations are related to hereditary disorders, and extended peptides found in human PNPO and HSD3B2 similarly destabilize these proteins, involving TRIM21 for PNPO degradation. Our findings indicate that degradation of aberrant proteins with C-terminal extension encoded by read-through mutant genes is a mechanism for loss of function resulting in hereditary disorders.

Bcl11b SWI/SNF-complex subunit modulates intestinal adenoma and regeneration after γ-irradiation through Wnt/ß-catenin pathway.

SWI/SNF chromatin remodeling complexes constitute a highly related family of multi-subunit complexes to modulate transcription, and SWI/SNF subunit genes are collectively mutated in 20% of all human cancers. Bcl11b is a SWI/SNF subunit and acts as a haploinsufficient tumor suppressor in leukemia/lymphomas. Here, we show expression of Bcl11b in intestinal crypt cells and promotion of intestinal tumorigenesis by Bcl11b attenuation in Apc (min/+) mice. Of importance, mutations or allelic loss of BCL11B was detected in one-third of human colon cancers. We also show that attenuated Bcl11b activity in the crypt base columnar (CBC) cells expressing the Lgr5 stem cell marker enhanced regeneration of intestinal epithelial cells after the radiation-induced injury. Interestingly, BCL11B introduction in human cell lines downregulated transcription of ß-catenin target genes, whereas Bcl11b attenuation in Lgr5(+) CBCs increased expression of ß-catenin targets including c-Myc and cyclin D1. Together, our results argue that Bcl11b impairment promotes tumor development in mouse and human intestine at least in part through deregulation of ß-catenin pathway.