Search

Abstract Purpose Corneal dysmorphologies (CDs) are typically classified as either regressive degenerative corneal dystrophies (CDtrs) or defective growth and differentiation-driven corneal dysplasias (CDyps). Both eye disorders have multifactorial etiologies. While previous work has elucidated many aspects of CDs, such as presenting symptoms, epidemiology, and pathophysiology, the genetic mechanisms remain incompletely understood. The purpose of this study was to analyze phenotype data from 8,707 knockout mouse lines to identify new genes associated with the development of CDs in humans. Methods 8,707 knockout mouse lines phenotyped by the International Mouse Phenotyping Consortium were queried for genes associated with statistically significant (P

Angiography by means of micro–computed tomography (m-CT) is extensively used for the diagnosis of vasculature disorders. To establish a connection between m-CT images and genuine histopathology findings, we developed two novel titanium dioxide nanoparticle (TiO2-NP)–based perfusion contrast agents: TiNpCA-1 and TiNpCA-2. Three-dimensionally reconstructed m-CT images in mice perfused with these contrast agents showed high resolution and accuracy in various organs without deformation or dilation of vessels. Vessels

Abstract Because of the advent of genome-editing technology, gene knockout (KO) hamsters have become attractive research models for diverse diseases in humans. This study established a new KO model of diabetes by disrupting the insulin receptor substrate-2 (Irs2) gene in the golden (Syrian) hamster. Homozygous KO animals were born alive but with delayed postnatal growth until adulthood. They showed hyperglycemia, high HbA1c, and impaired glucose tolerance. However, they normally responded to insulin stimulation, unlike Irs2 KO mice, an obese type 2 diabetes (T2D) model. Consistent with this, Irs2 KO hamsters did not increase serum insulin levels upon glucose administration and showed β-cell hypoplasia in their pancreas. Thus, our Irs2 KO hamster provide a unique T2D animal model that is distinct from the obese T2D models. This model may contribute to a better understanding of the pathophysiology of human non-obese T2D with β-cell dysfunction, the most common type of T2D in East Asian countries, including Japan.

Increased levels of lactate, an end-product of glycolysis, have been proposed as a potential surrogate marker for metabolic changes during neuronal excitation. These changes in lactate levels can result in decreased brain pH, which has been implicated in patients with various neuropsychiatric disorders. We previously demonstrated that such alterations are commonly observed in five mouse models of schizophrenia, bipolar disorder, and autism, suggesting a shared endophenotype among these disorders rather than mere artifacts due to medications or agonal state. However, there is still limited research on this phenomenon in animal models, leaving its generality across other disease animal models uncertain. Moreover, the association between changes in brain lactate levels and specific behavioral abnormalities remains unclear. To address these gaps, the International Brain pH Project Consortium investigated brain pH and lactate levels in 109 strains/conditions of 2294 animals with genetic and other experimental manipulations relevant to neuropsychiatric disorders. Systematic analysis revealed that decreased brain pH and increased lactate levels were common features observed in multiple models of depression, epilepsy, Alzheimer’s disease, and some additional schizophrenia models. While certain autism models also exhibited decreased pH and increased lactate levels, others showed the opposite pattern, potentially reflecting subpopulations within the autism spectrum. Furthermore, utilizing large-scale behavioral test battery, a multivariate cross-validated prediction analysis demonstrated that poor working memory performance was predominantly associated with increased brain lactate levels. Importantly, this association was confirmed in an independent cohort of animal models. Collectively, these findings suggest that altered brain pH and lactate levels, which could be attributed to dysregulated excitation/inhibition balance, may serve as transdiagnostic endophenotypes of debilitating neuropsychiatric disorders characterized by cognitive impairment, irrespective of their beneficial or detrimental nature.

Maternally inherited symbionts that kill male insect hosts are well known in bacteria, but are also beginning to be recognised in viruses. In this study, the authors identify a gene from a symbiotic virus genome that is responsible for the male-killing phenotype of this virus in the fly Drosophila biauraria.

Abstract Protocadherin 9 (Pcdh9) is a member of the cadherin superfamily and is uniquely expressed in the vestibular and limbic systems; however, its physiological role remains unclear. Here, we studied the expression of Pcdh9 in the limbic system and phenotypes of Pcdh9-knock-out mice (Pcdh9 KO mice). Pcdh9 mRNA was expressed in the fear extinction neurons that express protein phosphatase 1 regulatory subunit 1 B (Ppp1r1b) in the posterior part of the basolateral amygdala (pBLA), as well as in the Cornu Ammonis (CA) and Dentate Gyrus (DG) neurons of the hippocampus. We show that the Pcdh9 protein was often localised at synapses. Phenotypic analysis of Pcdh9 KO mice revealed no apparent morphological abnormalities in the pBLA but a decrease in the spine number of CA neurons. Further, the Pcdh9 KO mice were related to features such as the abnormal optokinetic response, less approach to novel objects, and reduced fear extinction during recovery from the fear. These results suggest that Pcdh9 is involved in eliciting positive emotional behaviours, possibly via fear extinction neurons in the pBLA and/or synaptic activity in the hippocampal neurons, and normal optokinetic eye movement in brainstem optokinetic system-related neurons.

Abstract Autism spectrum disorder (ASD) is a continuum of neurodevelopmental disorders and needs new therapeutic approaches. Recently, oxytocin (OXT) showed potential as the first anti-ASD drug. Many reports have described the efficacy of intranasal OXT therapy to improve the core symptoms of patients with ASD; however, the underlying neurobiological mechanism remains unknown. The OXT/oxytocin receptor (OXTR) system, through the lateral septum (LS), contributes to social behavior, which is disrupted in ASD. Therefore, we selectively express hM3Dq in OXTR-expressing (OXTR+) neurons in the LS to investigate this effect in ASD mouse models developed by environmental and genetic cues. In mice that received valproic acid (environmental cue), we demonstrated successful recovery of impaired social memory with three-chamber test after OXTR+ neuron activation in the LS. Application of a similar strategy to Nl3 R451C knock-in mice (genetic cue) also caused successful recovery of impaired social memory in single field test. OXTR+ neurons in the LS, which are activated by social stimuli, are projected to the CA1 region of the hippocampus. This study identified a candidate mechanism for improving core symptoms of ASD by artificial activation of DREADDs, as a simulation of OXT administration to activate OXTR+ neurons in the LS.

Zinc plays a critical role in many physiological processes, and disruption of zinc homeostasis induces various disorders, such as growth retardation, osteopenia, immune deficiency, and inflammation. However, how the imbalance in zinc homeostasis leads to heart disease is not yet fully understood. Cardiovascular diseases are a major cause of death worldwide, and the development of novel therapeutic targets to treat it is urgently needed. We report that a zinc transporter, ZIP13, regulates cardiovascular homeostasis. We found that the expression level of Zip13 mRNA was diminished in both primary neonatal cardiomyocytes and mouse heart tissues treated with the cardiotoxic agent doxycycline. Primary neonatal cardiomyocytes from Zip13 gene-knockout (KO) mice exhibited abnormal irregular arrhythmic beating. RNA-seq analysis identified 606 differentially expressed genes in Zip13-KO mouse-derived primary neonatal cardiomyocytes and Gene ontology (GO) analysis revealed that both inflammation- and cell adhesion-related genes were significantly enriched. In addition, telemetry echocardiography analysis suggested that arrhythmias were likely to occur in Zip13-KO mice, in which elevated levels of the cardiac fibrosis marker Col1a1, vascular inflammation-related gene eNOS, and Golgi-related molecule GM130 were observed. These results indicate the physiological importance of ZIP13-it maintains cardiovascular homeostasis by resolving inflammation and stress response. Our findings suggest that optimizing ZIP13 expression and/or function may improve cardiovascular disease management.

Abstract 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.

De novo mutations significantly contribute to autism spectrum disorders (ASD). Here, the authors demonstrate that ASD-associated de novo mutations in the POGZ gene, one of a high-confidence ASD gene, lead to ASD-related impaired neuronal development and disrupted mature cortical network function.

Sirtuins (SIRT1–7 in mammals) are evolutionarily conserved nicotinamide adenine dinucleotide-dependent lysine deacetylases/deacylases that regulate fundamental biological processes including aging. In this study, we reveal that male Sirt7 knockout (KO) mice exhibited an extension of mean and maximum lifespan and a delay in the age-associated mortality rate. In addition, aged male Sirt7 KO mice displayed better glucose tolerance with improved insulin sensitivity compared with wild-type (WT) mice. Fibroblast growth factor 21 (FGF21) enhances insulin sensitivity and extends lifespan when it is overexpressed. Serum levels of FGF21 were markedly decreased with aging in WT mice. In contrast, this decrease was suppressed in Sirt7 KO mice, and the serum FGF21 levels of aged male Sirt7 KO mice were higher than those of WT mice. Activating transcription factor 4 (ATF4) stimulates Fgf21 transcription, and the hepatic levels of Atf4 mRNA were increased in aged male Sirt7 KO mice compared with WT mice. Our findings indicate that the loss of SIRT7 extends lifespan and improves glucose metabolism in male mice. High serum FGF21 levels might be involved in the beneficial effect of SIRT7 deficiency.

Angiogenesis is a process to generate new blood vessels from pre-existing vessels and to maintain vessels, and plays critical roles in normal development and disease. However, the molecular mechanisms underlying angiogenesis are not fully understood. This study examined the roles of exocyst complex component (Exoc) 3-like 2 (Exoc3l2) during development in mice. We found that Exoc3l1, Exoc3l2, Exoc3l3 and Exoc3l4 are expressed abundantly in endothelial cells at embryonic day 8.5. The generation of Exoc3l2 knock-out (KO) mice showed that disruption of Exoc3l2 resulted in lethal in utero. Substantial numbers of Exoc3l2 KO embryos exhibited hemorrhaging. Deletion of Exoc3l2 using Tie2-Cre transgenic mice demonstrated that Exoc3l2 in hematopoietic and endothelial lineages was responsible for the phenotype. Taken together, these findings reveal that Exoc3l2 is essential for cardiovascular and brain development in mice.

Tracheal development arises due to tube morphogenesis but how this is regulated is unclear. Here, the authors identify polarization of smooth muscle progenitors as controlling murine tracheal development, activating noncanonical Wnt signaling followed by subepithelial morphogenesis and ring cartilage development.

Trabecular bone structure has an important influence on bone strength, but little is known about its genetic regulation. To elucidate the genetic factor(s) regulating trabecular bone structure, we compared the trabecular bone structures of two genetically remote mouse strains, C57BL/6J and Japanese wild mouse-derived MSM/Ms. Phenotyping by X-ray micro-CT revealed that MSM/Ms has structurally more fragile trabecular bone than C57BL/6J. Toward identification of genetic determinants for the difference in fragility of trabecular bone between the two mouse strains, we employed phenotype screening of consomic mouse strains in which each C57BL/6J chromosome is substituted by its counterpart from MSM/Ms. The results showed that many chromosomes affect trabecular bone structure, and that the consomic strain B6-Chr15MSM, carrying MSM/Ms-derived chromosome 15 (Chr15), has the lowest values for the parameters BV/TV, Tb.N, and Conn.D, and the highest values for the parameters Tb.Sp and SMI. Subsequent phenotyping of subconsomic strains for Chr15 mapped four novel trabecular bone structure-related QTL (Tbsq1-4) on mouse Chr15. These results collectively indicate that genetic regulation of trabecular bone structure is highly complex, and that even in the single Chr15, the combined action of the four Tbsqs controls the fragility of trabecular bone. Given that Tbsq4 is syntenic to human Chr 12q12-13.3, where several bone-related SNPs are assigned, further study of Tbsq4 should facilitate our understanding of the genetic regulation of bone formation in humans.

Omphalocele is a human congenital anomaly in ventral body wall closure and may be caused by impaired formation of the primary abdominal wall (PAW) and/or defects in abdominal muscle development. Here, we report that mice doubly deficient in homeobox genes Six4 and Six5 showed the same ventral body wall closure defects as those seen in human omphalocele. SIX4 and SIX5 were localized in surface ectodermal cells and somatic mesoderm-derived mesenchymal and coelomic epithelial cells (CECs) in the PAW. Six4−/−;Six5−/− fetuses exhibited a large omphalocele with protrusion of both the liver and intestine, or a small omphalocele with protrusion of the intestine, with complete penetrance. The umbilical ring of Six4−/−;Six5−/− embryos was shifted anteriorly and its lateral size was larger than that of normal embryos at the E11.5 stage, before the onset of myoblast migration into the PAW. The proliferation rates of surface ectodermal cells in the left and right PAW and somatic mesoderm-derived cells in the right PAW were lower in Six4−/−;Six5−/− embryos than those of wild-type embryos at E10.5. The transition from CECs of the PAW to rounded mesothelial progenitor cells was impaired and the inner coelomic surface of the PAW was relatively smooth in Six4−/−;Six5−/− embryos at E11.25. Furthermore, Six4 overexpression in CECs of the PAW promoted ingression of CECs. Taken together, our results suggest that Six4 and Six5 are required for growth and morphological change of the PAW, and the impairment of these processes is linked to the abnormal positioning and expansion of the umbilical ring, which results in omphalocele.

The cytoplasmic peptide:N-glycanase (Ngly1 in mammals) is a de-N-glycosylating enzyme that is highly conserved among eukaryotes. It was recently reported that subjects harboring mutations in the NGLY1 gene exhibited severe systemic symptoms (NGLY1-deficiency). While the enzyme obviously has a critical role in mammals, its precise function remains unclear. In this study, we analyzed Ngly1-deficient mice and found that they are embryonic lethal in C57BL/6 background. Surprisingly, the additional deletion of the gene encoding endo-β-N-acetylglucosaminidase (Engase), which is another de-N-glycosylating enzyme but leaves a single GlcNAc at glycosylated Asn residues, resulted in the partial rescue of the lethality of the Ngly1-deficient mice. Additionally, we also found that a change in the genetic background of C57BL/6 mice, produced by crossing the mice with an outbred mouse strain (ICR) could partially rescue the embryonic lethality of Ngly1-deficient mice. Viable Ngly1-deficient mice in a C57BL/6 and ICR mixed background, however, showed a very severe phenotype reminiscent of the symptoms of NGLY1-deficiency subjects. Again, many of those defects were strongly suppressed by the additional deletion of Engase in the C57BL/6 and ICR mixed background. The defects observed in Ngly1/Engase-deficient mice (C57BL/6 background) and Ngly1-deficient mice (C57BL/6 and ICR mixed background) closely resembled some of the symptoms of patients with an NGLY1-deficiency. These observations strongly suggest that the Ngly1- or Ngly1/Engase-deficient mice could serve as a valuable animal model for studies related to the pathogenesis of the NGLY1-deficiency, and that cytoplasmic ENGase represents one of the potential therapeutic targets for this genetic disorder.

Escherichia coli RNase E (Eco-RNase E), encoded by rne (Eco-rne), is considered the global RNA decay initiator. Although Eco-RNase E is an essential gene product in E. coli, some bacterial species, such as Bacillus subtilis, do not possess Eco-RNase E sequence homologues. B. subtilis instead possesses RNase J1/J2 (Bsu-RNase J1/J2) and RNase Y (Bsu-RNase Y) to execute RNA decay. Here we found that E. coli lacking the Eco-rne gene (Δrne E. coli) was viable conditional on M9 minimal media by introducing Bsu-RNase J1/J2 or Bsu-RNase Y. We also cloned an extremely short Eco-RNase E homologue (Wpi-RNase E) and a canonical sized Bsu-RNase J1/J2 homologue (Wpi-RNase J) from Wolbachia pipientis, an α-proteobacterial endosymbiont of arthropods. We found that Wpi-RNase J restored the colony-forming ability (CFA) of Δrne E. coli, whereas Wpi-RNase E did not. Unexpectedly, Wpi-RNase E restored defective CFA due to lack of Eco-RNase G, a paralogue of Eco-RNase E. Our results indicate that bacterial species that lack Eco-RNase E homologues or bacterial species that possess Eco-RNase E homologues which lack Eco-RNase E-like activities have a modest Eco-RNase E-like function using RNase J and/or RNase Y. These results suggest that Eco-RNase E-like activities might distribute among a wide array of bacteria and that functions of RNases may have changed dynamically during evolutionary divergence of bacterial lineages.

Lysine residues can be post-translationally modified by various acyl modifications in bacteria and eukarya. Here, we showed that two major acyl modifications, acetylation and succinylation, were changed in response to the carbon source in the Gram-positive model bacterium Bacillus subtilis. Acetylation was more common when the cells were grown on glucose, glycerol, or pyruvate, whereas succinylation was upregulated when the cells were grown on citrate, reflecting the metabolic states that preferentially produce acetyl-CoA and succinyl-CoA, respectively. To identify and quantify changes in acetylation and succinylation in response to the carbon source, we performed a stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic analysis of cells grown on glucose or citrate. We identified 629 acetylated proteins with 1355 unique acetylation sites and 204 succinylated proteins with 327 unique succinylation sites. Acetylation targeted different metabolic pathways under the two growth conditions: branched-chain amino acid biosynthesis and purine metabolism in glucose and the citrate cycle in citrate. Succinylation preferentially targeted the citrate cycle in citrate. Acetylation and succinylation mostly targeted different lysine residues and showed a preference for different residues surrounding the modification sites, suggesting that the two modifications may depend on different factors such as characteristics of acyl-group donors, molecular environment of the lysine substrate, and/or the modifying enzymes. Changes in acetylation and succinylation were observed in proteins involved in central carbon metabolism and in components of the transcription and translation machineries, such as RNA polymerase and the ribosome. Mutations that modulate protein acylation affected B. subtilis growth. A mutation in acetate kinase (ackA) increased the global acetylation level, suggesting that acetyl phosphate-dependent acetylation is common in B. subtilis, just as it is in Escherichia coli. Our results suggest that acyl modifications play a role in the physiological adaptations to changes in carbon nutrient availability of B. subtilis.