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Andrea C. Bohrer (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Immunology and Microbiology. Inflammation and Innate Immunity Unit. Bethesda, MD, United States of America.); Ehydel Castro (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Immunology and Microbiology. Inflammation and Innate Immunity Unit. Bethesda, MD, United States of America.); Zhidong Hu (Fudan University. Shanghai Public Health Clinical Center. Department of Scientific Research. Shanghai, China / Fudan University. Shanghai Emerging and Re-emerging Infectious Disease Institute. Tuberculosis Center. Shanghai, China.); Artur T. L. Queiroz (The KAB Group / Multinational Organization Network Sponsoring Translational and Epidemiological Research. Salvador, BA, Brasil / Fundacao Oswaldo Cruz. Instituto Goncalo Moniz. Salvador, BA, Brasil.); Claire E. Tocheny (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Immunology and Microbiology. Inflammation and Innate Immunity Unit. Bethesda, MD, United States of America.); Maike Assmann (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Immunology and Microbiology. Inflammation and Innate Immunity Unit. Bethesda, MD, United States of America.); Shunsuke Sakai (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Parasitic Diseases. T Lymphocyte Biology Section. Bethesda, MD, United States of America.); Christine Nelson (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Parasitic Diseases. T Lymphocyte Biology Section. Bethesda, MD, United States of America.); Paul J. Baker (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Immunology and Microbiology. Inflammation and Innate Immunity Unit. Bethesda, MD, United States of America.); Hui Ma (Fudan University. Shanghai Public Health Clinical Center. Department of Scientific Research. Shanghai, China / Fudan University. Shanghai Emerging and Re-emerging Infectious Disease Institute. Tuberculosis Center. Shanghai, China.); Lin Wang (Fudan University. Shanghai Emerging and Re-emerging Infectious Disease Institute. Tuberculosis Center. Shanghai, China / Fudan University. Shanghai Public Health Clinical Center. Department of Thoracic Surgery. Shanghai, China.); Wen Zilu (Fudan University. Shanghai Emerging and Re-emerging Infectious Disease Institute. Tuberculosis Center. Shanghai, China / Fudan University. Shanghai Public Health Clinical Center. Department of Thoracic Surgery. Shanghai, China.); Elsa du Bruyn (University of Cape Town. Institute of Infectious Disease and Molecular Medicine. Wellcome Centre for Infectious Diseases Research in Africa. Cape Town, South Africa.); Catherine Riou (University of Cape Town. Institute of Infectious Disease and Molecular Medicine. Wellcome Centre for Infectious Diseases Research in Africa. Cape Town, South Africa.); Keith D. Kauffman (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Parasitic Diseases. T Lymphocyte Biology Section. Bethesda, MD, United States of America.); Tuberculosis Imaging Program (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Division of Intramural Research. Tuberculosis Imaging Program. Bethesda, MD, United States of America.); Ian N. Moore (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Comparative Medicine Branch. Infectious Disease Pathogenesis Section. Bethesda, MD, United States of America.); Franca Del Nonno (Istituto Di Ricovero e Cura a Carattere Scientifico. National Institute for Infectious Diseases "L. Spallanzani". Pathology Unit. Rome, Italy.); Linda Petrone (Istituto Di Ricovero e Cura a Carattere Scientifico. Department of Epidemiology and Preclinical Research National Institute for Infectious Diseases. Translational Research Unit. Rome, Italy.); Delia Goletti (Istituto Di Ricovero e Cura a Carattere Scientifico. Department of Epidemiology and Preclinical Research National Institute for Infectious Diseases. Translational Research Unit. Rome, Italy.); Adrian R. Martineau (University College London. Institute of Immunity and Transplantation. London, United Kingdom.); David M. Lowe (University College London. Institute of Immunity and Transplantation. London, United Kingdom.); Mark R. Cronan (Max Planck Institute for Infection Biology. In Vivo Cell Biology of Infection Unit. Berlin, Germany / Duke University School of Medicine. Department of Molecular Genetics and Microbiology. Durham, NC, United States of America.); Robert J. Wilkinson (University of Cape Town. Institute of Infectious Disease and Molecular Medicine. Wellcome Centre for Infectious Diseases Research in Africa. Cape Town, South Africa / Imperial College London. Department of Infectious Diseases. London, United Kingdom.); Clifton E. Barry, III (University of Cape Town. Institute of Infectious Disease and Molecular Medicine. Wellcome Centre for Infectious Diseases Research in Africa. Cape Town, South Africa / National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Immunology and Microbiology. Tuberculosis Research Section. Bethesda, MD, United States of America.); Laura E. Via (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Division of Intramural Research. Tuberculosis Imaging Program. Bethesda, MD, United States of America / National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Immunology and Microbiology. Tuberculosis Research Section. Bethesda, MD, United States of America.); Daniel L. Barber (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Parasitic Diseases. T Lymphocyte Biology Section. Bethesda, MD, United States of America.); Amy D. Klion (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Parasitic Diseases. Human Eosinophil Section. Bethesda, MD, United States of America.); Bruno B. Andrade (The KAB Group / Multinational Organization Network Sponsoring Translational and Epidemiological Research. Salvador, BA, Brasil / Fundacao Oswaldo Cruz. Instituto Goncalo Moniz. Salvador, BA, Brasil.); Yanzheng Song (Fudan University. Shanghai Emerging and Re-emerging Infectious Disease Institute. Tuberculosis Center. Shanghai, China / Fudan University. Shanghai Public Health Clinical Center. Department of Thoracic Surgery. Shanghai, China.); Ka-Wing Wong (Fudan University. Shanghai Public Health Clinical Center. Department of Scientific Research. Shanghai, China / Fudan University. Shanghai Emerging and Re-emerging Infectious Disease Institute. Tuberculosis Center. Shanghai, China.); Katrin D. Mayer-Barber (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Immunology and Microbiology. Inflammation and Innate Immunity Unit. Bethesda, MD, United States of America.).

The function of the Agtpbp1 gene has mainly been delineated by studying Agtpbp1pcd (pcd) mutant mice, characterized by losses in cerebellar Purkinje and granule cells along with degeneration of retinal photoreceptors, mitral cells of the olfactory bulb, thalamic neurons, and alpha-motoneurons. As a result of cerebellar degeneration, cerebellar GABA and glutamate concentrations in Agtpbp1pcd mutants decreased while monoamine concentrations increased. The salient behavioral phenotypes include cerebellar ataxia, a loss in motor coordination, and cognitive deficits. Similar neuropathogical and behavioral profiles have been described in childhood-onset human subjects with biallelic variants of AGTPBP1, including cerebellar ataxia and hypotonia.

Dendritic cell immunoreceptor (DCIR) is a C-type lectin receptor with a carbohydrate recognition domain and an immunoreceptor tyrosine-based inhibitory motif. Previously, we showed that Dcir−/− mice spontaneously develop autoimmune enthesitis and sialadenitis, and also develop metabolic bone abnormalities. However, the ligands for DCIR functionality remain to be elucidated. Here we showed that DCIR is expressed on osteoclasts and DCs and binds to an asialo-biantennary N-glycan(s) (NA2) on bone cells and myeloid cells. Osteoclastogenesis was enhanced in Dcir−/− cells, and NA2 inhibited osteoclastogenesis. Neuraminidase treatment, which exposes excess NA2 by removing the terminal sialic acid of N-glycans, suppressed osteoclastogenesis and DC function. Neuraminidase treatment of mice ameliorated collagen-induced arthritis and experimental autoimmune encephalomyelitis in a DCIR-dependent manner, due to suppression of antigen presentation by DCs. These results suggest that DCIR activity is regulated by the modification of the terminal sialylation of biantennary N-glycans, and this interaction is important for the control of both autoimmune and bone metabolic diseases.

Reactive oxygen species (ROS) metabolism is regulated by the oxygen-mediated enzyme reaction and antioxidant mechanism within cells under physiological conditions. Xanthine oxidoreductase (XOR) exhibits two inter-convertible forms (xanthine oxidase (XO) and xanthine dehydrogenase (XDH)), depending on the substrates. XO uses oxygen as a substrate and generates superoxide (O2•−) in the catalytic pathway of hypoxanthine. We previously showed that superoxide dismutase 1 (SOD1) loss induced various aging-like pathologies via oxidative damage due to the accumulation of O2•− in mice. However, the pathological contribution of XO-derived O2•− production to aging-like tissue damage induced by SOD1 loss remains unclear. To investigate the pathological significance of O2•− derived from XOR in Sod1−/− mice, we generated Sod1-null and XO-type- or XDH-type-knock-in (KI) double-mutant mice. Neither XO-type- nor XDH-type KI mutants altered aging-like phenotypes, such as anemia, fatty liver, muscle atrophy, and bone loss, in Sod1−/− mice. Furthermore, allopurinol, an XO inhibitor, or apocynin, a nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor, failed to improve aging-like tissue degeneration and ROS accumulation in Sod1−/− mice. These results showed that XOR-mediated O2•− production is relatively uninvolved in the age-related pathologies in Sod1−/− mice.

GM1 gangliosidosis is a rare lysosomal storage disease caused by a deficiency of β-galactosidase due to mutations in the GLB1 gene. We established a C57BL/6 mouse model with Glb1G455R mutation using CRISPR/Cas9 genome editing. The β-galactosidase enzyme activity of Glb1G455R mice measured by fluorometric assay was negligible throughout the whole body. Mutant mice displayed no marked phenotype at eight weeks. After 16 weeks, GM1 ganglioside accumulation in the brain of mutant mice was observed by immunohistochemical staining. Meanwhile, a declining performance in behavioral tests was observed among mutant mice from 16 to 32 weeks. As the disease progressed, the neurological symptoms of mutant mice worsened, and they then succumbed to the disease by 47 weeks of age. We also observed microglia activation and proliferation in the cerebral cortex of mutant mice at 16 and 32 weeks. In these activated microglia, the level of autophagy regulator LC3 was up-regulated but the mRNA level of LC3 was normal. In conclusion, we developed a novel murine model that mimicked the chronic phenotype of human GM1. This Glb1G455R murine model is a practical in vivo model for studying the pathogenesis of GM1 gangliosidosis and exploring potential therapies.

Mutations in the IKBKB gene cause severe immunodeficiency, characterized clinically by persistent respiratory or gastrointestinal infections. Targeted gene panel sequencing revealed a novel homozygous missense mutation in the IKBKB gene of a patient with immune dysregulation and combined T and B cell functional defects. PBMCs from the patient, Ikbkb Y397H mice, and transfected cells were used to elucidate how the Y395H mutation triggers IKKβ deficiency and impairs immune function. Here, we found that cells from both the patient and Ikbkb Y397H mice lacked or showed decreased levels of IKKβ protein, along with impaired lymphocyte function. IKKα and IKKγ protein expression by human PBMCs harboring the Y395H mutation was normal, but degradation of IKKβ protein was accelerated. Binding of human NF-κB to DNA in patient PBMCs fell upon stimulation with TNF-α or LPS. Additionally, a structural model of Y395H revealed loss of the hydrogen bond with D389. These data suggest that IKBKB deficiency induces abnormal IKKβ protein degradation, leading to impaired NF-κB signaling and immune function. We postulate that the Y395H variant in the IKKβ protein lost the hydrogen bond with D389, thereby affecting interaction between Y395 and D389 and increasing protein instability.

Animal models that recapitulate human diseases and disorders are widely used to investigate etiology, diagnosis, and treatment of those conditions in people. Disorders during pregnancy are particularly difficult to explore as interventions in pregnant women are not easily performed. Therefore, models that allow for pre-conception investigations are advantageous for elucidating the mechanisms involved in adverse pregnancy outcomes that are responsible for both maternal and fetal morbidity, such as preeclampsia. The Blood Pressure High (BPH)/5 mouse model has been used extensively to study the pathogenesis of preeclampsia. The female BPH/5 mouse is obese with increased adiposity and borderline hypertension, both of which are exacerbated with pregnancy making it a model of superimposed preeclampsia. Thus, the BPH/5 model shares traits with a large majority of women with pre-existing conditions that predisposes them to preeclampsia. We sought to explore the genome of the BPH/5 female mouse and determine the genetic underpinnings that may contribute to preeclampsia-associated phenotypes in this model. Using a whole genome sequencing approach, we are the first to characterize the genetic mutations in BPH/5 female mice that make it unique from the closely related BPH/2 model and the normotensive background strain, C57Bl/6. We found the BPH/5 female mouse to be uniquely different from BPH/2 and C57Bl/6 mice with a genetically complex landscape. The majority of non-synonymous consequences within the coding region of BPH/5 females were missense mutations found most abundant on chromosome X when comparing BPH/5 and BPH/2, and on chromosome 8 when comparing BPH/5 to C57Bl/6. Genetic mutations in BPH/5 females largely belong to immune system-related processes, with overlap between BPH/5 and BPH/2 models. Further studies examining each gene mutation during pregnancy are warranted to determine key contributors to the BPH/5 preeclamptic-like phenotype and to identify genetic similarities to women that develop preeclampsia.

Norrie disease is caused by mutation of the NDP gene, presenting as congenital blindness followed by later onset of hearing loss. Protecting patients from hearing loss is critical for maintaining their quality of life. This study aimed to understand the onset of pathology in cochlear structure and function. By investigating patients and juvenile Ndp-mutant mice, we elucidated the sequence of onset of physiological changes (in auditory brainstem responses, distortion product otoacoustic emissions, endocochlear potential, blood-labyrinth barrier integrity) and determined the cellular, histological, and ultrastructural events leading to hearing loss. We found that cochlear vascular pathology occurs earlier than previously reported and precedes sensorineural hearing loss. The work defines a disease mechanism whereby early malformation of the cochlear microvasculature precedes loss of vessel integrity and decline of endocochlear potential, leading to hearing loss and hair cell death while sparing spiral ganglion cells. This provides essential information on events defining the optimal therapeutic window and indicates that early intervention is needed. In an era of advancing gene therapy and small-molecule technologies, this study establishes Ndp-mutant mice as a platform to test such interventions and has important implications for understanding the progression of hearing loss in Norrie disease., JCI Insight, 7 (3), ISSN:2379-3708

Spinocerebellar ataxia type 1 (SCA1) is an adult-onset neurodegenerative disorder characterized by motor incoordination, mild cognitive decline, respiratory dysfunction, and early lethality. It is caused by the expansion of the polyglutamine (polyQ) tract in Ataxin-1 (ATXN1), which stabilizes the protein, leading to its toxic accumulation in neurons. Previously, we showed that serine 776 (S776) phosphorylation is critical for ATXN1 stability and contributes to its toxicity in cerebellar Purkinje cells. Still, the therapeutic potential of disrupting S776 phosphorylation on noncerebellar SCA1 phenotypes remains unstudied. Here, we report that abolishing S776 phosphorylation specifically on the polyQ-expanded ATXN1 of SCA1-knockin mice reduces ATXN1 throughout the brain and not only rescues the cerebellar motor incoordination but also improves respiratory function and extends survival while not affecting the hippocampal learning and memory deficits. As therapeutic approaches are likely to decrease S776 phosphorylation on polyQ-expanded and WT ATXN1, we further disrupted S776 phosphorylation on both alleles and observed an attenuated rescue, demonstrating a potential protective role of WT allele. This study not only highlights the role of S776 phosphorylation to regulate ATXN1 levels throughout the brain but also suggests distinct brain region-specific disease mechanisms and demonstrates the importance of developing allele-specific therapies for maximal benefits in SCA1.

Objective Statins pleiotropically provide additional benefits in reducing atherosclerosis, but their effects on intraplaque angiogenesis (IPA) and hemorrhage (IPH) remain unclear. Therefore, we discriminated statin’s lipid-lowering dependent and independent effects on IPA and IPH. Approach and results ApoE3*Leiden mice are statin-responsive due to ApoE and LDLR presence, but also allow to titrate plasma cholesterol levels by diet. Therefore, ApoE3*Leiden mice were fed a high-cholesterol-inducing-diet (HCD) with or without atorvastatin (A) or a moderate-cholesterol-inducing-diet (MCD). Mice underwent vein graft surgery to induce lesions with IPA and IPH. Cholesterol levels were significantly reduced in MCD (56%) and HCD + A (39%) compared to HCD with no significant differences between MCD and HCD + A. Both MCD and HCD + A have a similar reduction in vessel remodeling and inflammation comparing to HCD. IPA was significantly decreased by 30% in HCD + A compared to HCD or MCD. Atorvastatin treatment reduced the presence of immature vessels by 34% vs. HCD and by 25% vs. MCD, resulting in a significant reduction of IPH. Atorvastatin’s anti-angiogenic capacity was further illustrated by a dose-dependent reduction of ECs proliferation and migration. Cultured mouse aortic-segments lost sprouting capacity upon atorvastatin treatment and became 30% richer in VE-Cadherin expression and pericyte coverage. Moreover, Atorvastatin inhibited ANGPT2 release and decreased VE-Cadherin(Y685)-phosphorylation in ECs. Conclusions Atorvastatin has beneficial effects on vessel remodeling due to its lipid-lowering capacity. Atorvastatin has strong pleiotropic effects on IPA by decreasing the number of neovessels and on IPH by increasing vessel maturation. Atorvastatin improves vessel maturation by inhibiting ANGPT2 release and phospho(Y658)-mediated VE-Cadherin internalization.

Titin truncating variants are a well-established cause of cardiomyopathy; however, the role of titin missense variants is less well understood. Here we describe the generation of a mouse model to investigate the underlying disease mechanism of a previously reported titin A178D missense variant identified in a family with non-compaction and dilated cardiomyopathy. Heterozygous and homozygous mice carrying the titin A178D missense variant were characterised in vivo by echocardiography. Heterozygous mice had no detectable phenotype at any time point investigated (up to 1 year). By contrast, homozygous mice developed dilated cardiomyopathy from 3 months. Chronic adrenergic stimulation aggravated the phenotype. Targeted transcript profiling revealed induction of the foetal gene programme and hypertrophic signalling pathways in homozygous mice, and these were confirmed at the protein level. Unsupervised proteomics identified downregulation of telethonin and four-and-a-half LIM domain 2, as well as the upregulation of heat shock proteins and myeloid leukaemia factor 1. Loss of telethonin from the cardiac Z-disc was accompanied by proteasomal degradation; however, unfolded telethonin accumulated in the cytoplasm, leading to a proteo-toxic response in the mice.We show that the titin A178D missense variant is pathogenic in homozygous mice, resulting in cardiomyopathy. We also provide evidence of the disease mechanism: because the titin A178D variant abolishes binding of telethonin, this leads to its abnormal cytoplasmic accumulation. Subsequent degradation of telethonin by the proteasome results in proteasomal overload, and activation of a proteo-toxic response. The latter appears to be a driving factor for the cardiomyopathy observed in the mouse model. Supplementary Information The online version contains supplementary material available at 10.1007/s00395-021-00853-z.

Homozygous Dab1 yotari mutant mice, Dab1yot (yot/yot) mice, have an autosomal recessive mutation of Dab1 and show reeler-like phenotype including histological abnormality of the cerebellum, hippocampus, and cerebral cortex. We here show abnormal hippocampal development of yot/yot mice where granule cells and pyramidal cells fail to form orderly rows but are dispersed diffusely in vague multiplicative layers. Possibly due to the positioning failure of granule cells and pyramidal cells and insufficient synaptogenesis, axons of the granule cells did not extend purposefully to connect with neighboring regions in yot/yot mice. We found that both hippocampal granule cells and pyramidal cells of yot/yot mice expressed proteins reactive with the anti-Dab1 antibody. We found that Y198- phosphorylated Dab1 of yot/yot mice was greatly decreased. Accordingly the downstream molecule, Akt was hardly phosphorylated. Especially, synapse formation was defective and the distribution of neurons was scattered in hippocampus of yot/yot mice. Some of neural cell adhesion molecules and hippocampus associated transcription factors of the neurons were expressed aberrantly, suggesting that the Reelin-Dab1 signaling pathway seemed to be importantly involved in not only neural migration as having been shown previously but also neural maturation and/or synaptogenesis of the mice. It is interesting to clarify whether the defective neural maturation is a direct consequence of the dysfunctional Dab1, or alternatively secondarily due to the Reelin-Dab1 intracellular signaling pathways.

Although mindfulness-based stress reduction (MBSR) improves cognitive function, the mechanism is not clear. In this study, people aged 65 years and older were recruited from elderly communities in Chitose City, Japan, and assigned to a non-MBSR group or a MBSR group. Before and after the intervention, the Japanese version of the Montreal Cognitive Assessment (MoCA-J) was administered, and blood samples were collected. Then, neuron-derived extracellular vesicles (NDEVs) were isolated from blood samples, and microRNAs, as well as the target mRNAs, were evaluated in NDEVs. A linear mixed model analysis showed significant effects of the MBSR x time interaction on the MoCA-J scores, the expression of miRNA(miR)-29c, DNA methyltransferase 3 alpha (DNMT3A), and DNMT3B in NDEVs. These results indicate that MBSR can improve cognitive function by increasing the expression of miR-29c and decreasing the expression of DNMT3A, as well as DNMT3B, in neurons. It was also found that intracerebroventricular injection of miR-29c mimic into 5xFAD mice prevented cognitive decline, as well as neuronal loss in the subiculum area, by down-regulating Dnmt3a and Dnmt3b in the hippocampus. The present study suggests that MBSR can prevent neuronal loss and cognitive impairment by increasing the neuronal expression of miR-29c.

The kinase PDK1 is a crucial regulator for immune cell development by connecting PI3K to downstream AKT signaling. However, the roles of PDK1 in CD4+ T cell differentiation, especially in T follicular helper (Tfh) cell, remain obscure. Here we reported PDK1 intrinsically promotes the Tfh cell differentiation and germinal center responses upon acute infection by using conditional knockout mice. PDK1 deficiency in T cells caused severe defects in both early differentiation and late maintenance of Tfh cells. The expression of key Tfh regulators was remarkably downregulated in PDK1-deficient Tfh cells, including Tcf7, Bcl6, Icos, and Cxcr5. Mechanistically, ablation of PDK1 led to impaired phosphorylation of AKT and defective activation of mTORC1, resulting in substantially reduced expression of Hif1α and p-STAT3. Meanwhile, decreased p-AKT also suppresses mTORC2-associated GSK3β activity in PDK1-deficient Tfh cells. These integrated effects contributed to the dramatical reduced expression of TCF1 and ultimately impaired the Tfh cell differentiation.

The cohesin complex plays an essential role in chromosome maintenance and transcriptional regulation. Recurrent somatic mutations in the cohesin complex are frequent genetic drivers in cancer, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Here, using genetic dependency screens of stromal antigen 2–mutant (STAG2-mutant) AML, we identified DNA damage repair and replication as genetic dependencies in cohesin-mutant cells. We demonstrated increased levels of DNA damage and sensitivity of cohesin-mutant cells to poly(ADP-ribose) polymerase (PARP) inhibition. We developed a mouse model of MDS in which Stag2 mutations arose as clonal secondary lesions in the background of clonal hematopoiesis driven by tet methylcytosine dioxygenase 2 (Tet2) mutations and demonstrated selective depletion of cohesin-mutant cells with PARP inhibition in vivo. Finally, we demonstrated a shift from STAG2- to STAG1-containing cohesin complexes in cohesin-mutant cells, which was associated with longer DNA loop extrusion, more intermixing of chromatin compartments, and increased interaction with PARP and replication protein A complex. Our findings inform the biology and therapeutic opportunities for cohesin-mutant malignancies., We developed models of cohesin-mutant myelodysplastic syndromes and acute myeloid leukemia and demonstrated a shift from STAG2- to STAG1-cohesin complexes, increased DNA damage, and sensitivity to PARP inhibition.

The coat protein I (COPI) complex mediates retrograde trafficking from the Golgi to the endoplasmic reticulum (ER). Five siblings with persistent bacterial and viral infections and defective humoral and cellular immunity had a homozygous p.K652E mutation in the γ1 subunit of COPI (γ1-COP). The mutation disrupts COPI binding to the KDEL receptor and impairs the retrieval of KDEL-bearing chaperones from the Golgi to the ER. Homozygous Copg1(K652E) mice had increased ER stress in activated T and B cells, poor antibody responses, and normal numbers of T cells that proliferated normally, but underwent increased apoptosis upon activation. Exposure of the mutants to pet store mice caused weight loss, lymphopenia, and defective T cell proliferation that recapitulated the findings in the patients. The ER stress-relieving agent tauroursodeoxycholic acid corrected the immune defects of the mutants and reversed the phenotype they acquired following exposure to pet store mice. This study establishes the role of γ1-COP in the ER retrieval of KDEL-bearing chaperones and thereby the importance of ER homeostasis in adaptive immunity.

Immunoglobulin and T cell receptor gene assembly depends on V(D)J recombination initiated by the RAG1-RAG2 recombinase. The RAG1 N-terminal region (NTR; aa 1–383) has been implicated in regulatory functions whose influence on V(D)J recombination and lymphocyte development in vivo is poorly understood. We generated mice in which RAG1 lacks ubiquitin ligase activity (P326G), the major site of autoubiquitination (K233R), or its first 215 residues (Δ215). While few abnormalities were detected in R1.K233R mice, R1.P326G mice exhibit multiple features indicative of reduced recombination efficiency, including an increased Igκ+:Igλ+ B cell ratio and decreased recombination of Igh, Igκ, Igλ, and Tcrb loci. Previous studies indicate that synapsis of recombining partners during Igh recombination occurs through two pathways: long-range scanning and short-range collision. We find that R1Δ215 mice exhibit reduced short-range Igh and Tcrb D-to-J recombination. Our findings indicate that the RAG1 NTR regulates V(D)J recombination and lymphocyte development by multiple pathways, including control of the balance between short- and long-range recombination.

ATP11A translocates phosphatidylserine (PtdSer), but not phosphatidylcholine (PtdCho), from the outer to the inner leaflet of plasma membranes, thereby maintaining the asymmetric distribution of PtdSer. Here, we detected a de novo heterozygous point mutation of ATP11A in a patient with developmental delays and neurological deterioration. Mice carrying the corresponding mutation died perinatally of neurological disorders. This mutation caused an amino acid substitution (Q84E) in the first transmembrane segment of ATP11A, and mutant ATP11A flipped PtdCho. Molecular dynamics simulations revealed that the mutation allowed PtdCho binding at the substrate entry site. Aberrant PtdCho flipping markedly decreased the concentration of PtdCho in the outer leaflet of plasma membranes, whereas sphingomyelin (SM) concentrations in the outer leaflet increased. This change in the distribution of phospholipids altered cell characteristics, including cell growth, cholesterol homeostasis, and sensitivity to sphingomyelinase. Matrix-assisted laser desorption ionization-imaging mass spectrometry (MALDI-IMS) showed a marked increase of SM levels in the brains of Q84E-knockin mouse embryos. These results provide insights into the physiological importance of the substrate specificity of plasma membrane flippases for the proper distribution of PtdCho and SM.

Group 2 innate lymphoid cells (ILC2s) are unique in their ability to produce low levels of type 2 cytokines at steady state, and their production capacity is dramatically increased upon stimulation with IL-33. However, it is unknown how constitutive cytokine production is regulated in the steady state. Here, we found that tristetraprolin (TTP/Zfp36), an RNA-binding protein that induces mRNA degradation, was highly expressed in naive ILC2s and was downregulated following IL-33 stimulation. In ILC2s from Zfp36−/− mice, constitutive IL-5 production was elevated owing to the stabilization of its mRNA and resulted in an increased number of eosinophils in the intestine. Luciferase assay demonstrated that TTP directly regulates Il5 mRNA stability, and overexpression of TTP markedly suppressed IL-5 production by ILC2s, even under IL-33 stimulation. Collectively, TTP-mediated posttranscriptional regulation acts as a deterrent of excessive cytokine production in steady-state ILC2s to maintain body homeostasis, and downregulation of TTP may contribute to massive cytokine production under IL-33 stimulation.

Alzheimer's disease (AD) is characterized by progressive neurodegeneration leading to loss of cognitive abilities and ultimately to death. Postmortem investigations revealed decreased expression of cerebral insulin-like growth factor (IGF)-1 receptor (IGF-1R) and insulin receptor substrate (IRS) proteins in patients with AD. To elucidate the role of insulin/IGF-1 signaling in AD, we crossed mice expressing the Swedish mutation of amyloid precursor protein (APP(SW), Tg2576 mice) as a model for AD with mice deficient for either IRS-2, neuronal IGF-1R (nIGF-1R(-/-)), or neuronal insulin receptor (nIR(-/-)), and analyzed survival, glucose, and APP metabolism. In the present study, we show that IRS-2 deficiency in Tg2576 mice completely reverses premature mortality in Tg2576 females and delays beta-amyloid (Abeta) accumulation. Analysis of APP metabolism suggested that delayed Abeta accumulation resulted from decreased APP processing. To delineate the upstream signal responsible for IRS-2-mediated disease protection, we analyzed mice with nIGF-1R or nIR deficiency predominantly in the hippocampus. Interestingly, both male and female nIGF-1R(-/-)Tg2576 mice were protected from premature death in the presence of decreased Abeta accumulation specifically in the hippocampus formation. However, neuronal IR deletion had no influence on lethality of Tg2576 mice. Thus, impaired IGF-1/IRS-2 signaling prevents premature death and delays amyloid accumulation in a model of AD.

International audience; LMNA mutations in patients are responsible for a dilated cardiomyopathy. Molecular mechanisms underlying the origin and development of the pathology are unknown. Herein, using mouse pluripotent embryonic stem cells (ESCs) and a mouse model both harboring the p.H222P Lmna mutation, we found early defects in cardiac differentiation of mutated ESCs and dilatation of mutated embryonic hearts at E13.5, pointing to a developmental origin of the disease. Using mouse ESCs, we demonstrated that cardiac differentiation of Lmna(H222P/+) was impaired at the mesodermal stage. Expression of Mesp1, a mesodermal cardiogenic gene involved in epithelial-to-mesenchymal transition of epiblast cells, as well as Snail and Twist expression, was decreased in Lmna(H222P/+) cells compared with WT cells in the course of differentiation. In turn, cardiomyocyte differentiation was impaired. ChIP assay of H3K4me1 in differentiating cells revealed a specific decrease of this histone mark on regulatory regions of Mespl and Twist in Lmna(H222P/+) cells. Downregulation or inhibition of LSD1 that specifically demethylated H3K4me1 rescued the epigenetic landscape of mesodermal Lmna(H222P/+) cells and in turn contraction of cardiomyocytes. Inhibition of LSD1 in pregnant mice or neonatal mice prevented cardiomyopathy in E13.5 Lmna(H222P/H222P) offspring and adults, respectively. Thus, LSD1 appeared to be a therapeutic target to prevent or cure dilated cardiomyopathy associated with a laminopathy.

Prion diseases are progressive, neurodegenerative diseases affecting humans and animals. Also known as the transmissible spongiform encephalopathies, for the hallmark spongiform change seen in the brain, these diseases manifest increased oxidative damage early in disease and changes in antioxidant enzymes in terminal brain tissue. Superoxide dismutase 2 (SOD2) is an antioxidant enzyme that is critical for life. SOD2 knock-out mice can only be kept alive for several weeks post-birth and only with antioxidant therapy. However, this results in the development of a spongiform encephalopathy. Consequently, we hypothesized that reduced levels of SOD2 may accelerate prion disease progression and play a critical role in the formation of spongiform change. Using SOD2 heterozygous knock-out and litter mate wild-type controls, we examined neuronal long-term potentiation, disease duration, pathology, and degree of spongiform change in mice infected with three strains of mouse adapted scrapie. No influence of the reduced SOD2 expression was observed in any parameter measured for any strain. We conclude that changes relating to SOD2 during prion disease are most likely secondary to the disease processes causing toxicity and do not influence the development of spongiform pathology.

A high-fat diet (HFD) can induce hyperglycemia and metabolic syndromes that, in turn, can trigger visual impairment. To evaluate the acute effects of HFD feeding on retinal degeneration, we assessed retinal function and morphology, inflammatory state, oxidative stress, and gut microbiome in dystrophic retinal degeneration 10 (rd10) mice, a model of retinitis pigmentosa, fed an HFD for 2 to 3 wk. Short-term HFD feeding impaired retinal responsiveness and visual acuity and enhanced photoreceptor degeneration, microglial cell activation, and Müller cell gliosis. HFD consumption also triggered the expression of inflammatory and oxidative markers in rd10 retinas. Finally, an HFD caused gut microbiome dysbiosis, increasing the abundance of potentially proinflammatory bacteria. Thus, HFD feeding drives the pathological processes of retinal degeneration by promoting oxidative stress and activating inflammatory-related pathways. Our findings suggest that consumption of an HFD could accelerate the progression of the disease in patients with retinal degenerative disorders. This work was supported by grants from the Spanish Ministry of the Economy and Competitiveness (RTI2018-094248-B-I00), Spanish Ministry of Science and Innovation cofinanced by European Regional Development Fund (MICINN-FEDER PID2019-106230RB-I00), Instituto de Salud Carlos III co-financed by European Regional Development Fund (RETICS-FEDER-RD16/0008/0016), Asociación Retina Asturias (ASOCIACIONRETINA1-20I), Federación de Asociaciones de Retinosis Pigmentaria de España and Fundación Lucha Contra la Ceguera (FUNDALUCE18-01), and Generalitat Valenciana (PROMETEO/2021/024, IDIFEDER/2017/064).

Nrf2, a transcription factor that regulates the response to oxidative stress, has been shown to rescue cone photoreceptors and slow vision loss in mouse models of retinal degeneration (rd). The retinal pigment epithelium (RPE) is damaged in these models, but whether it also could be rescued by Nrf2 has not been previously examined. We used an adeno-associated virus (AAV) with an RPE-specific (Best1) promoter to overexpress Nrf2 in the RPE of rd mice. Control rd mice showed disruption of the regular array of the RPE, as well as loss of RPE cells. Cones were lost in circumscribed regions within the cone photoreceptor layer. Overexpression of Nrf2 specifically in the RPE was sufficient to rescue the RPE, as well as the disruptions in the cone photoreceptor layer. Electron microscopy showed compromised apical microvilli in control rd mice but showed preserved microvilli in Best1-Nrf2–treated mice. The rd mice treated with Best1-Nrf2 had slightly better visual acuity. Transcriptome profiling showed that Nrf2 upregulates multiple oxidative defense pathways, reversing declines seen in the glutathione pathway in control rd mice. In summary, Nrf2 overexpression in the RPE preserves RPE morphology and survival in rd mice, and it is a potential therapeutic for diseases involving RPE degeneration, including age-related macular degeneration (AMD).

Brain homeostasis is regulated by the viability and functionality of neurons. HAT (histone acetyltransferase) and HDAC (histone deacetylase) inhibitors have been applied to treat neurological deficits in humans; yet, the epigenetic regulation in neurodegeneration remains elusive. Mutations of HAT cofactor TRRAP (transformation/transcription domain-associated protein) cause human neuropathies, including psychosis, intellectual disability, autism, and epilepsy, with unknown mechanism. Here we show that Trrap deletion in Purkinje neurons results in neurodegeneration of old mice. Integrated transcriptomics, epigenomics, and proteomics reveal that TRRAP via SP1 conducts a conserved transcriptomic program. TRRAP is required for SP1 binding at the promoter proximity of target genes, especially microtubule dynamics. The ectopic expression of Stathmin3/4 ameliorates defects of TRRAP-deficient neurons, indicating that the microtubule dynamics is particularly vulnerable to the action of SP1 activity. This study unravels a network linking three well-known, but up-to-date unconnected, signaling pathways, namely TRRAP, HAT, and SP1 with microtubule dynamics, in neuroprotection.

Connexin 30 (Cx30; also known as Gjb6 when referring to the mouse gene) is expressed in ependymal cells of the brain ventricles, in leptomeningeal cells and in astrocytes rich in connexin 43 (Cx43), leading us to question whether patients harboring GJB6 mutations exhibit any brain anomalies. Here, we used mice harboring the human disease-associated A88V Cx30 mutation to address this gap in knowledge. Brain Cx30 levels were lower in male and female Cx30A88V/A88V mice compared with Cx30A88V/+ and Cx30+/+ mice, whereas Cx43 levels were lower only in female Cx30 mutant mice. Characterization of brain morphology revealed a disrupted ependymal cell layer, significant hydrocephalus and enlarged ventricles in 3- to 6-month-old adult male and female Cx30A88V/A88V mice compared with Cx30A88V/+ or Cx30+/+ sex-matched littermate mice. To determine the functional significance of these molecular and morphological changes, we investigated a number of behavioral activities in these mice. Interestingly, only female Cx30A88V/A88V mice exhibited abnormal behavior compared with all other groups. Cx30A88V/A88V female mice demonstrated increased locomotor and exploratory activity in both the open field and the elevated plus maze. They also exhibited dramatically reduced ability to learn the location of the escape platform during Morris water maze training, although they were able to swim as well as other genotypes. Our findings suggest that the homozygous A88V mutation in Cx30 causes major morphological changes in the brain of aging mice, possibly attributable to an abnormal ependymal cell layer. Remarkably, these changes had a more pronounced consequence for cognitive function in female mice, which is likely to be linked to the dysregulation of both Cx30 and Cx43 levels in the brain., Summary: This study reveals that adult mice harboring a disease-linked Cx30 mutant exhibit morphological changes in the brain that result in behavioral abnormalities that are more pronounced in female mice.

Haematopoietic stem cells (HSCs) are characterized by their self-renewal potential associated to dormancy. Here we identify the cell surface receptor neogenin-1 as specifically expressed in dormant HSCs. Loss of neogenin-1 initially leads to increased HSC expansion but subsequently to loss of self-renewal and premature exhaustion in vivo. Its ligand netrin-1 induces Egr1 expression and maintains quiescence and function of cultured HSCs in a Neo1 dependent manner. Produced by arteriolar endothelial and periarteriolar stromal cells, conditional netrin-1 deletion in the bone marrow niche reduces HSC numbers, quiescence and self-renewal, while overexpression increases quiescence in vivo. Ageing associated bone marrow remodelling leads to the decline of netrin-1 expression in niches and a compensatory but reversible upregulation of neogenin-1 on HSCs. Our study suggests that niche produced netrin-1 preserves HSC quiescence and self-renewal via neogenin-1 function. Decline of netrin-1 production during ageing leads to the gradual decrease of Neo1 mediated HSC self-renewal., Haematopoietic stem cells (HSCs) are characterized by their self-renewal potential and associated dormancy. Here the authors show that niche produced netrin-1 preserves HSC quiescence and self-renewal via neogenin-1, and that decline of netrin-1 production during ageing leads to decreased Neo1 mediated HSC self-renewal.

Summary Mammalian genomes contain long domains with distinct average compositions of A/T versus G/C base pairs. In a screen for proteins that might interpret base composition by binding to AT-rich motifs, we identified the stem cell factor SALL4, which contains multiple zinc fingers. Mutation of the domain responsible for AT binding drastically reduced SALL4 genome occupancy and prematurely upregulated genes in proportion to their AT content. Inactivation of this single AT-binding zinc-finger cluster mimicked defects seen in Sall4 null cells, including precocious differentiation of embryonic stem cells (ESCs) and embryonic lethality in mice. In contrast, deletion of two other zinc-finger clusters was phenotypically neutral. Our data indicate that loss of pluripotency is triggered by downregulation of SALL4, leading to de-repression of a set of AT-rich genes that promotes neuronal differentiation. We conclude that base composition is not merely a passive byproduct of genome evolution and constitutes a signal that aids control of cell fate., Graphical Abstract, Highlights • SALL4 binds to short AT-rich motifs via its C-terminal zinc-finger cluster, ZFC4 • SALL4 ZFC4 represses early differentiation genes in proportion to their AT content • Mutating ZFC4 leads to abnormal neuronal differentiation and embryonic lethality • The other zinc-finger clusters ZFC1 and ZFC2 seem dispensable in embryonic stem cells, The mammalian genome comprises domains with varying ratios of A/T to G/C base pairs. The significance of mosaic base composition was unknown, but we discovered that the stem cell protein SALL4 reads A/T content to inhibit the expression of many genes, thereby stabilizing the pluripotent state.

Omega-3 fatty acids from fish oil reduce triglyceride levels in mammals, yet the mechanisms underlying this effect have not been fully clarified, despite the clinical use of omega-3 ethyl esters to treat severe hypertriglyceridemia and reduce cardiovascular disease risk in humans. Here, we identified in bile a class of hypotriglyceridemic omega-3 fatty acid–derived N-acyl taurines (NATs) that, after dietary omega-3 fatty acid supplementation, increased to concentrations similar to those of steroidal bile acids. The biliary docosahexaenoic acid–containing (DHA-containing) NAT C22:6 NAT was increased in human and mouse plasma after dietary omega-3 fatty acid supplementation and potently inhibited intestinal triacylglycerol hydrolysis and lipid absorption. Supporting this observation, genetic elevation of endogenous NAT levels in mice impaired lipid absorption, whereas selective augmentation of C22:6 NAT levels protected against hypertriglyceridemia and fatty liver. When administered pharmacologically, C22:6 NAT accumulated in bile and reduced high-fat diet–induced, but not sucrose-induced, hepatic lipid accumulation in mice, suggesting that C22:6 NAT is a negative feedback mediator that limits excess intestinal lipid absorption. Thus, biliary omega-3 NATs may contribute to the hypotriglyceridemic mechanism of action of fish oil and could influence the design of more potent omega-3 fatty acid–based therapeutics.

Degenerative diseases of the retina are responsible for the death of photoreceptors and subsequent loss of vision in patients. Nevertheless, the inner retinal layers remain intact over an extended period of time, enabling the restoration of light sensitivity in blind retinas via the expression of optogenetic tools in the remaining retinal cells. The chimeric Opto-mGluR6 protein represents such a tool. With exclusive ON-bipolar cell expression, it combines the light-sensitive domains of melanopsin and the intracellular domains of the metabotropic glutamate receptor 6 (mGluR6), which naturally mediates light responses in these cells. Albeit vision restoration in blind mice by Opto-mGluR6 delivery was previously shown, much is left to be explored in regard to the effects of the timing of the treatment in the degenerated retina. We performed a functional evaluation of Opto-mGluR6-treated murine blind retinas using multi-electrode arrays (MEAs) and observed long-term functional preservation in the treated retinas, as well as successful therapeutical intervention in later stages of degeneration. Moreover, the treatment decreased the inherent retinal hyperactivity of the degenerated retinas to levels undistinguishable from healthy controls. Finally, we observed for the first time micro electroretinograms (mERGs) in optogenetically treated animals, corroborating the origin of Opto-mGluR6 signalling at the level of mGluR6 of ON-bipolar cells.

Chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory bone disease that presents with bone destruction and pain. Although genetic studies have identified signalling pathways involving CRMO, molecularly targeted drugs remain unavailable. We used an animal model of CRMO as an in vivo screening system for candidate therapeutic agents. A gain-of-function mutation in Fgr, a member of Src family kinases (SFKs), causes peripheral paw inflammation and reduced bone mineral density (BMD) in Ali18 mice. The SFK inhibitor dasatinib was selected for administration to Ali18 mice daily for 2 weeks. Local inflammation and BMD were assessed by clinical scoring and computed tomography, respectively. Pilot studies in a small number of animals showed that dasatinib administration effectively suppressed the early phase of autoinflammation in Ali18 mice. Serial oral gavage of dasatinib to a group of Ali18 mice confirmed significant suppression of paw swelling with no side effects. Histological analysis revealed that abnormal proliferative bone marrow cells and inflammatory infiltration into the skin in the affected area were clearly reduced in the animals with dasatinib administration. Further, trabecular BMD in Ali18 long bones was restored to levels similar to that found in wild type mice. Our results indicate that autoinflammation and related-bone phenotypes were completely suppressed by the dasatinib kinase inhibitor in CRMO model animals. Thus, it is strongly suggested that dasatinib can be used for clinical treatments of CRMO with the combination of molecular diagnosis of the FGR locus. SIGNIFICANCE OF THE STUDY: Autoinflammation and related-bone phenotypes were effectively suppressed by the kinase inhibitor dasatinib in CRMO model animals. In combination with molecular analysis of the FGR locus, dasatinib is a strong candidate for the clinical treatments of CRMO. We propose that the animal model employed in this study can be used to screen this and other potential drugs for CRMO.

RNA polymerase (Pol) III synthesizes abundant short non-coding RNAs that have essential functions in protein synthesis, secretion and other processes. Despite the ubiquitous functions of these RNAs, mutations in Pol III subunits cause Pol III-related leukodystrophy, an early-onset neurodegenerative disease. The basis of this neural sensitivity and the mechanisms of disease pathogenesis are unknown. Here we show that mice expressing pathogenic mutations in the largest Pol III subunit, Polr3a, specifically in Olig2-expressing cells, have impaired growth and developmental delay, deficits in cognitive, sensory and fine sensorimotor function, and hypomyelination in multiple regions of the cerebrum and spinal cord. In contrast, the gross motor defects and cerebellar hypomyelination that are common features of severely affected patients are absent in the mice, suggesting a relatively mild form of the disease in this conditional model. Our results show that disease pathogenesis in the mice involves defects that reduce both the number of mature myelinating oligodendrocytes and the ability of these cells to produce a myelin sheath of normal thickness. Thus, the findings suggest cell-specific roles for Pol III in the development and/or survival of oligodendrocytes as well as their function in myelination.Significance StatementPathogenic mutations in subunits of RNA polymerase (Pol) III cause a prevalent autosomal recessive form of leukodystrophy. However, understanding of the mechanisms of pathogenesis, including how ubiquitously-expressed Pol III mutations affect primarily the central nervous system, has been limited by the absence of an animal model of the disease. We show that conditional knock-in of pathogenic Polr3a mutations in the Olig2 lineage in mice results in growth, neurobehavioral and hypomyelination phenotypes reflecting a subset of clinical features of Pol III-related leukodystrophy patients. Myelination defects in the mice identify neural-specific roles for Pol III transcription. The phenotypes of Pol III-related leukodystrophic mice enable genetic and pharmacological approaches aimed at mitigating the consequences of this disease in humans.

Gabapentinoids such as gabapentin and pregabalin, which bind specifically to the α

International audience; Background: Takayasu arteritis (TAK) is a large vessel vasculitis resulting in artery wall remodeling with segmental stenosis and/or aneurysm formation. Mast cells (MCs) are instrumental in bridging cell injury and inflammatory response.Objectives: This study sought to investigate the contribution of MCs on vessel permeability, angiogenesis, and fibrosis in patients with TAK. Methods: MC activation and their tissue expression were assessed in sera and in aorta from patients with TAK and from healthy donors (HDs). In vivo permeability was assessed using a modified Miles assay. Subconfluent cultured human umbilic vein endothelial cells and fibroblasts were used in vitro to investigate the effects of MC mediators on angiogenesis and fibrogenesis.Results: This study found increased levels of MC activation markers (histamine and indoleamine 2,3-dioxygenase) in sera of patients with TAK compared with in sera of HDs. Marked expression of MCs was shown in aortic lesions of patients with TAK compared with in those of noninflammatory aorta controls. Using Miles assay, this study showed that sera of patients with TAK significantly increased vascular permeability in vivo as compared with that of HDs. Vessel permeability was abrogated in MC-deficient mice. MCs stimulated by sera of patients with TAK supported neoangiogenesis (increased human umbilic vein endothelial cell proliferation and branches) and fibrosis by inducing increased production of fibronectin, type 1 collagen, and a-smooth muscle actin by fibroblasts as compared to MCs stimulated by sera of HD.Conclusions: MCs are a key regulator of vascular lesions in patients with TAK and may represent a new therapeutic target in large vessel vasculitis.

International audience; Beiging is an attractive therapeutic strategy to fight against obesity and its side metabolic complications. The loss of function of the nuclear transcription factor RORa has been related to a lean phenotype with higher thermogenesis in sg/sg mice lacking this protein. Here we show that pharmacological modulation of RORa activity exerts reciprocal and cell-autonomous effect on UCP1 expression ex vivo, in cellulo, and in vivo. The RORa inverse-agonist SR3335 upregulated UCP1 expression in brown and subcutaneous white adipose tissue (scWAT) explants of wild-type (WT) mice, whereas the RORa agonist SR1078 had the opposite effect. We confirmed the reciprocal action of these synthetic RORa ligands on gene expression, mitochondrial mass, and uncoupled oxygen consumption rate in cultured murine and human adipocytes. Time course analysis revealed stepwise variation in gene expression, first of TLE3, an inhibitor of the thermogenic program, followed by a reciprocal effect on PRDM16 and UCP1. Finally, RORa ligands were shown to be useful tools to modulate in vivo UCP1 expression in scWAT with associated changes in this fat depot mass. SR3335 and SR1078 provoked the opposite effects on the WT mice body weight, but without any effect on sg/sg mice. This slimming effect of SR3335 was related to an increased adaptive thermogenesis of the mice, as assessed by the rectal temperature of cold-stressed mice and induction of UCP1 in scWAT, as well as by indirect calorimetry in presence or not of a b3-adrenoceptor agonist. These data confirmed that RORa ligands could be useful tools to modulate thermogenesis and energy homeostasis. NEW & NOTEWORTHY The regulation of adipose tissue browning was not fully deciphered and required further studies explaining how the regulation of this process may be of interest for tackling obesity and related metabolic disorders. Our data confirmed the involvement of the transcription factor RORa in the regulation of nonshivering thermogenesis, and importantly, revealed the possibility to in vivo modulate its activity by synthetic ligands with beneficial consequences on fat mass and body weight of the mice.

mTOR activation is essential and sufficient to cause polycystic kidneys in Tuberous Sclerosis Complex (TSC) and other genetic disorders. In disease models, a sharp increase of proliferation and cyst formation correlates with a dramatic loss of oriented cell division (OCD). We find that OCD distortion is intrinsically due to S6 kinase 1 (S6K1) activation. The concomitant loss of S6K1 in Tsc1-mutant mice restores OCD but does not decrease hyperproliferation, leading to non-cystic harmonious hyper growth of kidneys. Mass spectrometry-based phosphoproteomics for S6K1 substrates revealed Afadin, a known component of cell-cell junctions required to couple intercellular adhesions and cortical cues to spindle orientation. Afadin is directly phosphorylated by S6K1 and abnormally decorates the apical surface of Tsc1-mutant cells with E-cadherin and α-catenin. Our data reveal that S6K1 hyperactivity alters centrosome positioning in mitotic cells, affecting oriented cell division and promoting kidney cysts in conditions of mTOR hyperactivity., mTOR activation is known to generate polycystic kidneys, which show both increased proliferation and loss of oriented cell division (OCD). Here, Bonucci et al. show that loss of OCD is linked to S6K1 activation through its direct target Afadin and is separable from hyperproliferation.

Goal-directed behaviors are essential for normal function and significantly impaired in neuropsychiatric disorders. Despite extensive associations between genetic mutations and these disorders, the molecular contributions to goal-directed dysfunction remain unclear. We examined mice with constitutive and brain region-specific mutations in Neurexin1α, a neuropsychiatric disease-associated synaptic molecule, in value-based choice paradigms. We found Neurexin1α knockouts exhibited reduced selection of beneficial outcomes and impaired avoidance of costlier options. Reinforcement modeling suggested that this was driven by deficits in updating and representation of value. Disruption of Neurexin1α within telencephalic excitatory projection neurons, but not thalamic neurons, recapitulated choice abnormalities of global Neurexin1α knockouts. Furthermore, this selective forebrain excitatory knockout of Neurexin1α perturbed value-modulated neural signals within striatum, a central node in feedback-based reinforcement learning. By relating deficits in value-based decision-making to region-specific Nrxn1α disruption and changes in value-modulated neural activity, we reveal potential neural substrates for the pathophysiology of neuropsychiatric disease-associated cognitive dysfunction.

Congenital aniridia is caused by heterozygous mutations in the PAX6 gene. In this disease, congenital iris and foveal hypoplasia is associated with juvenile onset cataract, glaucoma, and corneal keratopathy. In rodents, Pax6 mutations result in a congenital reduction in ocular size that is not typically described in human aniridia. Here, the ocular morphometry of aniridia patients is compared with the lens phenotype of Pax6(+/tm1/Pgr) mice to reveal whether there are species differences in Pax6 regulation of lens development and homeostasis. Ultrasound biometry (UBM) revealed that eleven percent of aniridia patients exhibited mild microphthalmia while the anterior chamber depth of aniridic eyes was significantly reduced from 6 months of age onward. Although aniridic lens thickness was normal from birth, it was significantly decreased in aniridic lenses older than 30. Notably, 86% of aniridic lenses exhibited cataractous changes in this cohort. In addition, a significant proportion of aniridia patients develop lens subluxation as they age associated with reduced lens diameter as measured by anterior segment optical coherence tomography (AS-OCT). Analysis of young adult Pax6(+/tm1/Pgr) mouse lenses by micro-computed tomography (microCT), bright field and dark field imaging revealed that they are reduced in size but did not exhibit overt cataracts at this age. Overall, this study reveals that congenital microphthalmia as assessed by axial length, or microphakia, as assessed by lens thickness, are not typical in human aniridia, although these are primary manifestations of Pax6 mutations in mice, suggesting that PAX6 regulates some aspects of lens development differently between these species.

Mutations that increase the protein kinase activity of LRRK2 are one of the most common causes of familial Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif, impacting interaction with effectors. We describe and validate a new, multiplexed targeted mass spectrometry assay to quantify endogenous levels of LRRK2-phosphorylated Rab substrates (Rab1, Rab3, Rab8, Rab10, Rab35 and Rab43) as well as total levels of Rabs, LRRK2 and LRRK2-phosphorylated at the Ser910 and Ser935 biomarker sites. Exploiting this assay, we quantify for the first time the relative levels of each of the pRab proteins in different cells (mouse embryonic fibroblasts, human neutrophils) and mouse tissues (brain, kidney, lung and spleen). We define how these components are impacted by Parkinson’s pathogenic mutations (LRRK2[R1441C] and VPS35[D620N]) and LRRK2 inhibitors. We find that the VPS35[D620N], but not LRRK2[R1441C] mutation, enhances Rab1 phosphorylation in a manner blocked by administration of an LRRK2 inhibitor, providing the first evidence that endogenous Rab1 is a physiological substrate for LRRK2. We exploit this assay to demonstrate that in Parkinson’s patients with VPS35[D620N] mutations, phosphorylation of multiple Rab proteins (Rab1, Rab3, Rab8, Rab10 and Rab43) is elevated. We highlight the benefits of this assay over immunoblotting approaches currently deployed to assess LRRK2 Rab signalling pathway.

In the United States, 5–12% of adults have at least one symptom of temporomandibular joint (TMJ) disorders, including TMJ osteoarthritis (TMJ-OA). However, there is no chondroprotective agent that is approved for clinical application. We showed that LOXL2 is elevated in the regenerative response during fracture healing in mice and has a critical role in chondrogenic differentiation. Indeed, LOXL2 is an anabolic effector that attenuates pro-inflammatory signaling in OA cartilage of the TMJ and knee joint, induces chondroprotective and regenerative responses, and attenuates NF-kB signaling. The specific goal of the study was to evaluate if adenoviral delivery of LOXL2 is anabolic to human and mouse TMJ condylar cartilage in vivo and evaluate the protective and anabolic effect on cartilage-specific factors. We employed two different models to assess TMJ-OA. In one model, clinical TMJ-OA cartilage from 5 different samples in TMJ-OA cartilage plugs were implanted subcutaneously in nude mice. Adenovirus LOXL2 -treated implants showed higher mRNA levels of LOXL2, ACAN, and other anabolic genes compared to the adenovirus-Empty-treated implants. Further characterization by RNA-seq analysis showed LOXL2 promotes proteoglycan networks and extracellular matrix in human TMJ-OA cartilage implants in vivo. In order to evaluate if LOXL2-induced functional and sex-linked differences, both male and female four-month-old chondrodysplasia (Cho/+) mice, which develop progressive TMJ-OA due to a point mutation in the Col11a1 gene, were subjected to intraperitoneal injection with Adv-RFP-LOXL2 every 2 weeks for 12 weeks. The data showed that adenovirus delivery of LOXL2 upregulated LOXL2 and aggrecan (Acan), whereas MMP13 expression was slightly downregulated. The fold change expression of Acan and Runx2 induced by Adv-RFP-LOXL2 was higher in females compared to males. Interestingly, Adv-RFP-LOXL2 injection significantly increased Rankl expression in male but there was no change in females, whereas VegfB gene expression was increased in females, but not in males, as compared to those injected with Adv-RFP-Empty in respective groups. Our findings indicate that LOXL2 can induce specifically the expression of Acan and other anabolic genes in two preclinical models in vivo. Further, LOXL2 has beneficial functions in human TMJ-OA cartilage implants and promotes gender-specific anabolic responses in Cho/+ mice with progressive TMJ-OA, suggesting its merit for further study as an anabolic therapy for TMJ-OA.

Many studies of the autoimmune disease Sjogren's syndrome have been performed using spontaneous mouse models. In the present study, we describe the characteristics of McH/lpr-RA1 mice and propose their use as a novel murine model of autoimmune sialadenitis. The McH/lpr-RA1 mouse is a recombinant congenic strain derived from generation F54 or more of MRL-Faslpr x (MRL- Faslpr x C3H- Faslpr) F1. We show for the first time that this mouse spontaneously develops autoimmune sialadenitis and vasculitis in submandibular gland tissues. Sialadenitis was accompanied by extensive inflammatory cell infiltration and tissue destruction. Immunohistochemical studies revealed that the salivary gland lesions strongly expressed four sialadenitis-related molecules: SSA and SSB (autoantigens of Sjogren's syndrome), gp91phox (an accelerator of reactive oxygen species production) and single strand DNA (a marker of apoptotic cells). In contrast, expression of aquaporin-5 (AQP5), which stimulates salivary secretion was weak or negligible. Statistical correlation analyses indicated that the apoptosis of salivary gland cells provoked by oxidative stress contributed to the severe sialadenitis and reduced expression of AQP5. Our study has demonstrated that McH/lpr-RA1 mice spontaneously develop the pathognomonic features of autoimmune sialadenitis and thus could be used as a new animal model of Sjogren's syndrome.

Epithelial cells organize an ordered array of non-centrosomal microtubules, the minus ends of which are regulated by CAMSAP3. The role of these microtubules in epithelial functions, however, is poorly understood. Here, we show that the kidneys of mice in which Camsap3 is mutated develop cysts at the proximal convoluted tubules (PCTs). PCTs were severely dilated in the mutant kidneys, and they also exhibited enhanced cell proliferation. In these PCTs, epithelial cells became flattened along with perturbation of microtubule arrays as well as of certain subcellular structures such as interdigitating basal processes. Furthermore, YAP and PIEZO1, which are known as mechanosensitive regulators for cell shaping and proliferation, were activated in these mutant PCT cells. These observations suggest that CAMSAP3-mediated microtubule networks are important for maintaining the proper mechanical properties of PCT cells, and its loss triggers cell deformation and proliferation via activation of mechanosensors, resulting in the dilation of PCTs.

Salivary gland dysfunction is a common complication of diabetes mellitus (DM). Long non-coding RNA (lncRNA) is evidenced to involve in the functional regulation of salivary gland, however, its role in DM-impaired gland is unknown. Therefore, this study aimed to investigate the expression profiles and functional networks of lncRNA in the parotid glands (PGs) of DM mice.Microarray was used to detect lncRNA and messenger RNA (mRNA) expression profiles in the PGs from db/db and db/m mice. Eleven differently expressed (DE) lncRNAs validated by qRT-PCR were selected for coding-non-coding gene co-expression (CNC) and competing endogenous RNA (ceRNA) network analysis, as well as the following Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Pearson's coefficient correlation analysis was used to analyze the correlations between DE lncRNAs expression and DM pathology.By using a 2-fold change and P0.05 as the cutoff criteria, 1650 DE lncRNAs (758 upregulated and 892 downregulated) and 1073 mRNAs (563 upregulated and 510 downregulated) were identified in the PGs of db/db mice compared to db/m mice. GO and KEGG analysis of DE mRNA suggested that activated inflammation response and downregulated ion transport might count for the dysfunction of diabetic PG. CNC and ceRNA networks analysis of 11 DE lncRNAs showed that the inflammation process and its related signaling pathways including advanced glycation end product (AGE)-receptor for AGE (RAGE) signaling pathway in diabetic complications, cytokine-cytokine receptor interaction, chemokine signaling pathway, apoptosis, and cell adhesion molecules were significantly enriched. The alterations of lncRNAs were closely correlated with higher blood glucose and serum insulin levels in mice.We identified multiple lncRNAs/mRNAs and several signaling pathways that may involve in the pathogenesis of diabetic salivary injury, providing new insight into potential target of diabetic hyposalivation.

Ataxia and Male Sterility (AMS) is a mutant mouse strain that contains a missense mutation in the coding region of Nna1, a gene that encodes a deglutamylase. AMS mice exhibit early cerebellar Purkinje cell degeneration and an ataxic phenotype in an autosomal recessive manner. To understand the underlying mechanism, we generated neuronal stem cell (NSC) lines from wild-type (NMW7), Nna1 mutation heterozygous (NME), and Nna1 mutation homozygous (NMO1) mouse brains. The NNA1 levels were decreased, and the glutamylated tubulin levels were increased in NMO1 cultures as well as in the cerebellum of AMS mice at both 15 and 30 days of age. However, total &beta, tubulin protein levels were not altered in the AMS cerebellum. In NMO1 neurosphere cultures, &beta, tubulin protein levels were increased without changes at the transcriptional level. NMO1 grew faster than other NSC lines, and some of the neurospheres were attached to the plate after 3 days. Immunostaining revealed that SOX2 and nestin levels were decreased in NMO1 neurospheres and that the neuronal differentiation potentials were reduced in NMO1 cells compared to NME or NMW7 cells. These results demonstrate that the AMS mutation decreased the NNA1 levels and increased glutamylation in the cerebellum of AMS mice. The observed changes in glutamylation might alter NSC properties and the neuron maturation process, leading to Purkinje cell death in AMS mice.

Purpose The purpose of this study was to test the hypothesis that anti-oxidant and / or anti-inflammation drugs that suppress rod death in cyclic light-reared Pde6brd10 mice are also effective in dark-reared Pde6brd10 mice. Methods In untreated dark-reared Pde6brd10 mice at post-natal (P) days 23 to 24, we measured the outer nuclear layer (ONL) thickness (histology) and dark-light thickness difference in external limiting membrane-retinal pigment epithelium (ELM-RPE) (optical coherence tomography [OCT]), retina layer oxidative stress (QUEnch-assiSTed [QUEST] magnetic resonance imaging [MRI]); and microglia/macrophage-driven inflammation (immunohistology). In dark-reared P50 Pde6brd10 mice, ONL thickness was measured (OCT) in groups given normal chow or chow admixed with methylene blue (MB) + Norgestrel (anti-oxidant, anti-inflammatory), or MB or Norgestrel separately. Results P24 Pde6brd10 mice showed no significant dark-light ELM-RPE response in superior and inferior retina consistent with high cGMP levels. Norgestrel did not significantly suppress the oxidative stress of Pde6brd10 mice that is only found in superior central outer retina of males at P23. Overt rod degeneration with microglia/macrophage activation was observed but only in the far peripheral superior retina in male and female P23 Pde6brd10 mice. Significant rod protection was measured in female P50 Pde6brd10 mice given 5 mg/kg/day MB + Norgestrel diet; no significant benefit was seen with MB chow or Norgestrel chow alone, nor in similarly treated male mice. Conclusions In early rod degeneration in dark-reared Pde6brd10 mice, little evidence is found in central retina for spatial associations among biomarkers of the PDE6B mutation, oxidative stress, and rod death; neuroprotection at P50 was limited to a combination of anti-oxidant/anti-inflammation treatment in a sex-specific manner.

CHD8 encodes a chromatin-remodeling factor and is one of the most recurrently mutated genes in individuals with autism spectrum disorder (ASD). Although we have recently shown that mice heterozygous for Chd8 mutation manifest myelination defects and ASD-like behaviors, the detailed mechanisms underlying ASD pathogenesis have remained unclear. Here we performed diffusion tensor imaging (DTI) and resting-state functional magnetic resonance imaging (rsfMRI) in oligodendrocyte lineage-specific Chd8 heterozygous mutant mice. DTI revealed that ablation of Chd8 specifically in oligodendrocytes of mice was associated with microstructural changes of specific brain regions including the cortex and striatum. The extent of these changes in white matter including the corpus callosum and fornix was correlated with total contact time in the reciprocal social interaction test. Analysis with rsfMRI revealed changes in functional brain connectivity in the mutant mice, and the extent of such changes in the cortex, hippocampus, and amygdala was also correlated with the change in social interaction. Our results thus suggest that changes in brain microstructure and functional connectivity induced by oligodendrocyte dysfunction might underlie altered social interaction in mice with oligodendrocyte-specific CHD8 haploinsufficiency.

Interleukin-10 (IL-10) plays a central role in regulation of intestinal mucosal homeostasis and prevention of inflammatory bowel disease (IBD). We previously reported that CD11b(hi) regulatory dendritic cells (DCs) can produce more IL-10, and CD11b can negatively regulate Toll-like receptors (TLRs)-induced inflammatory responses in macrophages. However whether CD11b and its signaling can control autoimmunity via IL-10 production remains unclear. Here we found that CD11b deficient (Itgam(-/-)) mice were more susceptible to dextran sulfate sodium (DSS)-induced colitis, with more tumor necrosis factor α (TNF-α) while less IL-10 production. CD11b inhibited nuclear factor-kappa B (NF-κB) while promoted activator protein 1 (AP-1) activation through activating sarcoma oncogene (Src), leading to decreased TNF-α while increased IL-10 production. Src interacted with and promoted c-casitas B lineage lymphoma proto-oncogene (c-Cbl)-mediated degradation of the inhibitory subunit p85 of phosphatidylinositol 3-kinase (PI3K). Importantly, Src inhibitor dasatinib aggravated DSS-induced colitis by decreasing IL-10 while increasing TNF-α in vivo. Therefore, CD11b promotes IL-10 production by activating Src-Akt signal pathway. An axis of CD11b-Src pathway is important in balancing homeostasis of TLR-induced pro-inflammatory and anti-inflammatory responses.

The transcription factor PAX6 is involved in the development of the eye and pancreatic islets, besides being associated with sleep–wake cycles. Here, we investigated a point mutation in the RED subdomain of PAX6, previously described in a human patient, to present a comprehensive study of a homozygous Pax6 mutation in the context of adult mammalian metabolism and circadian rhythm. Pax6Leca2 mice lack appropriate retinal structures for light perception and do not display normal daily rhythmic changes in energy metabolism. Despite β cell dysfunction and decreased insulin secretion, mutant mice have normal glucose tolerance. This is associated with reduced hepatic glucose production possibly due to altered circadian variation in expression of clock and metabolic genes, thereby evading hyperglycemia. Hence, our findings show that while the RED subdomain is important for β cell functional maturity, the Leca2 mutation impacts peripheral metabolism via loss of circadian rhythm, thus revealing pleiotropic effects of PAX6., Nirav Chhabra et al. characterize adult mice carrying a homozygous mutation in Pax6 that was identified in a patient with foveal hypoplasia. They find that the Pax6 point mutation has pleiotropic effects, including defects in the mouse retinal structures, loss of the optic nerve, changes in energy metabolism and circadian rhythms, and dysregulation of genes expressed in the pancreas.