DKO Mice Research Articles

Micro-dystrophin gene therapy demonstrates long-term cardiac efficacy in a severe Duchenne muscular dystrophy model

Micro-dystrophin gene replacement therapies for Duchenne muscular dystrophy (DMD) are currently in clinical trials, but have not been thoroughly investigated for their efficacy on cardiomyopathy progression to heart failure. We previously validated Fiona/dystrophin-utrophin-deficient (dko) mice as a DMD cardiomyopathy model that progresses to reduced ejection fraction indicative of heart failure. Adeno-associated viral (AAV) vector delivery of an early generation micro-dystrophin prevented cardiac pathology and functional decline through 1 year of age in this new model. We now show that gene therapy using a micro-dystrophin optimized for skeletal muscle efficacy (AAV-μDys5), and which is currently in a clinical trial, is able to fully prevent cardiac pathology and cardiac strain abnormalities and maintain normal (>45%) ejection fraction through 18months of age in Fiona/dko mice. Early treatment with AAV-μDys5 prevents inflammation and fibrosis in Fiona/dko hearts. Collagen in cardiac fibrotic scars becomes more tightly packed from 12 to 18months in Fiona/dko mice, but the area of fibrosis containing tenascin C does not change. Increased tight collagen correlates with unexpected improvements in Fiona/dko whole-heart function that maintain impaired cardiac strain and strain rate. This study supports micro-dystrophin gene therapy as a promising intervention for preventing DMD cardiomyopathy progression.

β-Cryptoxanthin Attenuates Cigarette-Smoke-Induced Lung Lesions in the Absence of Carotenoid Cleavage Enzymes (BCO1/BCO2) in Mice.

High dietary intake of β-cryptoxanthin (BCX, an oxygenated provitamin A carotenoid) is associated with a lower risk of lung disease in smokers. BCX can be cleaved by β-carotene-15,15'-oxygenase (BCO1) and β-carotene-9',10'-oxygenase (BCO2) to produce retinol and apo-10'-carotenoids. We investigated whether BCX has protective effects against cigarette smoke (CS)-induced lung injury, dependent or independent of BCO1/BCO2 and their metabolites. Both BCO1-/-/BCO2-/- double knockout mice (DKO) and wild type (WT) littermates were supplemented with BCX 14 days and then exposed to CS for an additional 14 days. CS exposure significantly induced macrophage and neutrophil infiltration in the lung tissues of mice, regardless of genotypes, compared to the non-exposed littermates. BCX treatment significantly inhibited CS-induced inflammatory cell infiltration, hyperplasia in the bronchial epithelium, and enlarged alveolar airspaces in both WT and DKO mice, regardless of sex. The protective effects of BCX were associated with lower expression of IL-6, TNF-α, and matrix metalloproteinases-2 and -9. BCX treatment led to a significant increase in hepatic BCX levels in DKO mice, but not in WT mice, which had significant increase in hepatic retinol concentration. No apo-10'-carotenoids were detected in any of the groups. In vitro BCX, at comparable doses of 3-OH-β-apo-10'-carotenal, was effective at inhibiting the lipopolysaccharide-induced inflammatory response in a human bronchial epithelial cell line. These data indicate that BCX can serve as an effective protective agent against CS-induced lung lesions in the absence of carotenoid cleavage enzymes.

GM-CSF-producing CCR2+ CCR6+ Th17 cells are pathogenic in dextran sodium sulfate-induced colitis model in mice.

Although excessive immune responses by Th17 cells, a helper T cell subset, are implicated in the pathogenesis of inflammatory bowel disease (IBD), the mechanism by which its localization in an inflamed colon is regulated remains unclear. Chemokines and their receptors are involved in the pathogenesis of IBD, however, the relative significance of each receptor on Th17 cells remains unknown. We generated C-C motif chemokine receptor 2 (CCR2) knockout (KO) and CCR6 KO mice in the syngeneic background using the CRISPR/Cas9 system and found that the phenotypes of experimental colitis worsened in both mutant mice. Surprisingly, the phenotype of colitis in CCR2/CCR6-double knockout (CCR2/6 DKO) mice was opposite to that of the single-deficient mice, with significantly milder experimental colitis (p < .05). The same was true for the symptoms in CCR6 KO mice, but not in wild type mice treated with a CCR2 inhibitor, propagermanium. Colonic CCR2+ CCR6+ Th17 cells produced a potentially pathogenic cytokine GM-CSF whose levels in the gut were significantly reduced in CCR2/6 DKO mice (p < .05). These results suggest that GM-CSF-producing CCR2+ CCR6+ Th17 cells are pathogenic and are attracted to the inflamed colon by either CCR2 or CCR6 gradient, which subsequently exacerbates experimental colitis in mice.

PS-BPB05-1: DUAL DISRUPTION OF ENDOTHELIAL NITRIC OXIDE SYNTHASE AND APOE GENE ACCELERATES KIDNEY FIBROSIS AND AGING AFTER INJURY

Objective: Advances in medical science have made it possible to control diseases such as cancer, autoimmune diseases, and infectious diseases, extending the lifespan of affected people around the world. On the other hand, the number of patients with Chronic Kidney Disease (CKD) is increasing worldwide, and hypertension is a key factor. Suppression of CKD is important for extending healthy life expectancy. In this study, we will clarify the effects of the interaction between hypertension and atherosclerosis on renal fibrosis and aging. Design and method: Wild type (WT) mouse, apolipoprotein E-/- (ApoE KO) mouse, and endothelial nitric oxide synthase (eNOS)-/- ; ApoE-/- (DKO) mouse were obtained by crossing eNOS+/- mouse and ApoE+/- mouse. Unilateral ureteral obstruction (UUO) was performed on 8–10 weeks old male mice after blood pressure and lipid profile were measured. Mice were sacrificed 10 days after UUO. The degree of renal tubular injury, fibrosis and kidney senescence were evaluated. Results: DKO mice exhibited the most pronounced blood pressure and lipid abnormalities among the three groups, with a significant elevation of blood pressure, total cholesterol, and low HDL cholesterol compared to WT mice. DKO mice manifested elevated blood pressure, higher total cholesterol and lower HDL than WT mice. Compared with WT mice, ApoE KO and DKO mice showed sustained kidney injury molecule-1 expression and kidney fibrosis was significantly higher in ApoEKO and DKO mice. mRNA expression of genes related to kidney fibrosis was the highest in DKO mice. The mRNA expression of Zinc alpha2 Glycoprotein and heme oxygenase-1 were significantly decreased in DKO mice. Furthermore, mRNA expression of p53, p21 and p16 were increased both in ApoEKO and DKO mice, with DKO mice being the highest. Senescence associated beta-gal positive tubule area was significantly increased in DKO. Increased DNA damage and target of rapamycin autophagy spatial coupling compartments (TASCCs, Sci Transl Med. 2019;11(476):eaav4754. J Clin Invest. 2019;129(11):4797- 4816.) formation was found in DKO mice. Conclusion: Mice at high risk for cardiovascular disease developed kidney fibrosis and aging even in the young mice after injury. Vulnerability to oxidative stress due to endothelial cell dysfunction promotes fibrosis and aging. Managing lifestyle-related diseases including hypertension from a young age is important for CKD prevention.

Upregulated FOXM1 stimulates chondrocyte senescence in Acot12-/-Nudt7-/- double knockout mice.

Rationale: One of the hallmarks of osteoarthritis (OA), the most common degenerative joint disease, is increased numbers of senescent chondrocytes. Targeting senescent chondrocytes or signaling mechanisms leading to senescence could be a promising new therapeutic approach for OA treatment. However, understanding the key targets and links between chondrocyte senescence and OA remains unclear. Methods: Senescent chondrocytes were identified from Nudt7-/-, Acot12-/-, double-knockout mice lacking Acot12 and Nudt7 (dKO) and applied to microarray. The presence of forkhead transcription factor M1 (FOXM1) was detected in aged, dKO, and destabilization of the medial meniscus (DMM) cartilages and articular chondrocytes, and the effect of FoxM1 overexpression and acetyl-CoA treatment on cartilage homeostasis was examined using immunohistochemistry, quantitative real-time PCR (qRT-PCR), cell apoptosis and proliferation assay, and safranin O staining. Delivery of Rho@PAA-MnO2 (MnO2 nanosheet) or heparin-ACBP/COS-GA-siFoxM1 (ACBP-siFoxM1) nanoparticles into DMM cartilage was performed. Results: Here, we propose the specific capture of acetyl-CoA with the delivery of (FoxM1 siRNA (siFoxM1) to prevent cartilage degradation by inhibiting the axis of chondrocyte senescence. dKO stimulate chondrocyte senescence via the upregulation of FoxM1 and contribute to severe cartilage breakdown. We found that the accumulation of acetyl-CoA in the dKO mice may be responsible for the upregulation of FoxM1 during OA pathogenesis. Moreover, scavenging reactive oxygen species (ROS) induced by chondrocyte senescence via the implantation of MnO2 nanosheets or delivery of siFoxM1 functionalized with acetyl-CoA binding protein (ACBP) to capture acetyl-CoA using an injectable bioactive nanoparticle (siFoxM1-ACBP-NP) significantly suppressed DMM-induced cartilage destruction. Conclusion: We found that the loss of Acot12 and Nudt7 stimulates chondrocyte senescence via the upregulation of FoxM1 and accumulation of acetyl-CoA, and the application of siFoxM1-ACBP-NP is a potential therapeutic strategy for OA treatment.

Innately expressed estrogen-related receptors in the skeletal muscle are indispensable for exercise fitness.

Transcriptional determinants in the skeletal muscle that govern exercise capacity, while poorly defined, could provide molecular insights into how exercise improves fitness. Here, we have elucidated the role of nuclear receptors, estrogen-related receptor alpha and gamma (ERRα/γ) in regulating myofibrillar composition, contractility, and exercise capacity in skeletal muscle. We used muscle-specific single or double (DKO) ERRα/γ knockout mice to investigate the effect of ERRα/γ deletion on muscle and exercise parameters. Individual knockout of ERRα/γ did not have a significant impact on the skeletal muscle. On the other hand, DKO mice exhibit pale muscles compared to wild-type (WT) littermates. RNA-seq analysis revealed a predominant decrease in expression of genes linked to mitochondrial and oxidative metabolism in DKO versus WT muscles. DKO muscles exhibit marked repression of oxidative enzymatic capacity, as well as mitochondrial number and size compared to WT muscles. Mitochondrial function is also impaired in single myofibers isolated from DKO versus WT muscles. In addition, mutant muscles exhibit reduced angiogenic gene expression and decreased capillarity. Consequently, DKO mice have a significantly reduced exercise capacity, further reflected in poor fatigue resistance of DKO mice in in vivo contraction assays. These results show that ERRα and ERRγ together are a critical link between muscle aerobic capacity and exercise tolerance. The ERRα/γ mutant mice could be valuable for understanding the long-term impact of impaired mitochondria and vascular supply on the pathogenesis of muscle-linked disorders.

Simultaneous loss of skeletal muscle myosin heavy chain IIx and IIb causes severe skeletal muscle hypoplasia in postnatal mice.

The skeletal muscle myosin heavy chain (MyHC) is a fundamental component of the sarcomere structure and muscle contraction. Two of the three adult fast MyHCs, MyHC-IIx and MyHC-IIb, are encoded by Myh1 and Myh4, respectively. However, skeletal muscle disorders have not yet been linked to these genes in humans. MyHC-IIb is barely detectable in human skeletal muscles. Thus, to characterize the molecular function of skeletal muscle MyHCs in humans, investigation of the effect of simultaneous loss of MyHC-IIb and other MyHCs on skeletal muscle in mice is essential. Here, we generated double knockout (dKO) mice with simultaneous loss of adult fast MyHCs by introducing nonsense frameshift mutations into the Myh1 and Myh4 genes. The dKO mice appeared normal after birth and until 2 weeks of age but showed severe skeletal muscle hypoplasia after 2 weeks. In 3-week-old dKO mice, increased expression of other skeletal muscle MyHCs, such as MyHC-I, MyHC-IIa, MyHC-neo, and MyHC-emb, was observed. However, these expressions were not sufficient to compensate for the loss of MyHC-IIb and MyHC-IIx. Moreover, the aberrant sarcomere structure with altered expression of sarcomere components was observed in dKO mice. Our findings imply that the simultaneous loss of MyHC-IIb and MyHC-IIx is substantially detrimental to postnatal skeletal muscle function and will contribute to elucidating the molecular mechanisms of skeletal muscle wasting disorders caused by the loss of skeletal muscle MyHCs.

Mir204 and Mir211 suppress synovial inflammation and proliferation in rheumatoid arthritis by targeting Ssrp1.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease characterized by synovial hyperplasia. Mir204 and Mir211 are homologous miRNAs with the same gene targeting spectrum. It is known that Mir204/211 play an important role in protecting osteoarthritis development; however, the roles of Mir204/211 in RA disease have not been determined. In the present study, we investigated the effects and molecular mechanisms of Mir204/211 on synovial inflammation and hyperproliferation in RA. The effects of Mir204/211 on the inflammation and abnormal proliferation in primary fibroblast-like synoviocytes (FLSs) were examined by Mir204/211 gain-of-function and loss-of-function approaches in vitro and in vivo. We identified the structure-specific recognition protein 1 (Ssrp1) as a downstream target gene of Mir204/211 based on the bioinformatics analysis. We overexpressed Ssrp1and Mir204/211 in FLS to determine the relationship between Ssrp1 and Mir204/211 and their effects on synovial hyperplasia. We created a collagen-induced arthritis (CIA) model in wild-type as well as Mir204/211 double knockout (dKO) mice to induce RA phenotype and administered adeno-associated virus (AAV)-mediated Ssrp1-shRNA (AAV-shSsrp1) by intra-articular injection into Mir204/211 dKO mice. We found that Mir204/211 attenuated excessive cell proliferation and synovial inflammation in RA. Ssrp1 was the downstream target gene of Mir204/211. Mir204/211 affected synovial proliferation and decelerated RA progression by targeting Ssrp1. CIA mice with Mir204/211 deficiency displayed enhanced synovial hyperplasia and inflammation. RA phenotypes observed in Mir204/211 deficient mice were significantly ameliorated by intra-articular delivery of AAV-shSsrp1, confirming the involvement of Mir204/211-Ssrp1signaling during RA development. In this study, we demonstrated that Mir204/211 antagonize synovial hyperplasia and inflammation in RA by regulation of Ssrp1. Mir204/211 may serve as novel agents to treat RA disease.

Minocycline Inhibits Microglial Activation and Improves Visual Function in a Chronic Model of Age-Related Retinal Degeneration.

Age-related macular degeneration (AMD) is a chronic disease, which progresses slowly from early to late stages over many years. Inflammation critically contributes to the pathogenesis of AMD. Here, we investigated the therapeutic potential of minocycline in a chronic model of AMD (i.e., the LysMCre-Socs3fl/flCx3cr1gfp/gfp double knockout [DKO] mice). Five-month-old DKO and wild type (WT) (Socs3fl/fl) mice were gavage fed with minocycline (25 mg/kg daily) or vehicle (distilled water) for 3 months. At the end of the treatment, visual function and retinal changes were examined clinically (using electroretinography, fundus photograph and optic coherence tomography) and immunohistologically. Three months of minocycline treatment did not affect the body weight, behaviour and general health of WT and DKO mice. Minocycline treatment enhanced the a-/b-wave aptitudes and increased retinal thickness in both WT and DKO. DKO mouse retina expressed higher levels of Il1b, CD68 and CD86 and had mild microglial activation, and decreased numbers of arrestin+ photoreceptors, PKCα+ and secretagogin+ bipolar cells compared to WT mouse retina. Minocycline treatment reduced microglial activation and rescued retinal neuronal loss in DKO mice. Our results suggest that long-term minocycline treatment is safe and effective in controlling microglial activation and preserving visual function in chronic models of AMD.

TRIAC Treatment Improves Impaired Brain Network Function and White Matter Loss in Thyroid Hormone Transporter Mct8/Oatp1c1 Deficient Mice.

Dysfunctions of the thyroid hormone (TH) transporting monocarboxylate transporter MCT8 lead to a complex X-linked syndrome with abnormal serum TH concentrations and prominent neuropsychiatric symptoms (Allan-Herndon-Dudley syndrome, AHDS). The key features of AHDS are replicated in double knockout mice lacking MCT8 and organic anion transporting protein OATP1C1 (Mct8/Oatp1c1 DKO). In this study, we characterize impairments of brain structure and function in Mct8/Oatp1c1 DKO mice using multimodal magnetic resonance imaging (MRI) and assess the potential of the TH analogue 3,3',5-triiodothyroacetic acid (TRIAC) to rescue this phenotype. Structural and functional MRI were performed in 11-weeks-old male Mct8/Oatp1c1 DKO mice (N = 10), wild type controls (N = 7) and Mct8/Oatp1c1 DKO mice (N = 13) that were injected with TRIAC (400 ng/g bw s.c.) daily during the first three postnatal weeks. Grey and white matter volume were broadly reduced in Mct8/Oatp1c1 DKO mice. TRIAC treatment could significantly improve white matter thinning but did not affect grey matter loss. Network-based statistic showed a wide-spread increase of functional connectivity, while graph analysis revealed an impairment of small-worldness and whole-brain segregation in Mct8/Oatp1c1 DKO mice. Both functional deficits could be substantially ameliorated by TRIAC treatment. Our study demonstrates prominent structural and functional brain alterations in Mct8/Oatp1c1 DKO mice that may underlie the psychomotor deficiencies in AHDS. Additionally, we provide preclinical evidence that early-life TRIAC treatment improves white matter loss and brain network dysfunctions associated with TH transporter deficiency.

Epigenetic regulation of white adipose tissue plasticity and energy metabolism by nucleosome binding HMGN proteins

White adipose tissue browning is a key metabolic process controlled by epigenetic factors that facilitate changes in gene expression leading to altered cell identity. We find that male mice lacking the nucleosome binding proteins HMGN1 and HMGN2 (DKO mice), show decreased body weight and inguinal WAT mass, but elevated food intake, WAT browning and energy expenditure. DKO white preadipocytes show reduced chromatin accessibility and lower FRA2 and JUN binding at Pparγ and Pparα promoters. White preadipocytes and mouse embryonic fibroblasts from DKO mice show enhanced rate of differentiation into brown-like adipocytes. Differentiating DKO adipocytes show reduced H3K27ac levels at white adipocyte-specific enhancers but elevated H3K27ac levels at brown adipocyte-specific enhancers, suggesting a faster rate of change in cell identity, from white to brown-like adipocytes. Thus, HMGN proteins function as epigenetic factors that stabilize white adipocyte cell identity, thereby modulating the rate of white adipose tissue browning and affecting energy metabolism in mice.

IMMU-07. HUMANIZED BI-SPECIFIC ANTIBODY SHOWS POTENT ACTIVITY IN PATIENT-DERIVED XENOGRAFT MODELS OF GLIOBLASTOMA

Abstract BACKGROUND Advancements in bioengineering have allowed bispecific Abs to develop with therapeutic applications in immuno-oncology. Here we describe a fully humanized bispecific t cell engager (BiTE) that binds the GBM-associated antigen interleukin 13 receptor alpha 2 (IL13Rα2) and the T Cell receptor CD3 epsilon (CD3ε) to elicit an anti-tumor immune response in patient-derived models of GBM. METHODS In vitro, BiTE-mediated activation of T Cells was assessed via flow cytometry as measurements of T cell activation markers CD25 and CD69 and cytokines IFNγ and TNFα. BiTE-directed T cell-mediated cytotoxicity was assessed in GBM 6 and GBM12patient-derived xenograft lines. In vivo, GBM6 patient-derived xenograft cells were intracranially implanted in NSG-MHC I/II DKO mice, reconstituted with HLA-matched donor peripheral blood lymphocytes (PBMC), and subsequently treated with 2.5 mg/kg BiTE via i.p. injection x 4 days/week of therapeutic BiTE for three weeks. Animals were followed for survival, and the brains of long-term survivors were analyzed for residual disease and T cell infiltration. RESULTS In vitro, BiTE potently activated T cells as determined by a high expression of CD25 and CD69 and the production of TNFα and IFNγ. BiTE also directed T cell-mediated cytotoxicity against GBM cells in a dose-dependent manner, with significant activity still seen in the low nanogram range. In vivo, BiTE significantly extended the survival of tumor-bearing animals in GBM6patient-derived xenograft models. Histological analysis of long-term surviving mice shows complete absence of tumor and robust presence of CD8+ T cells localized to the original site of tumor implantation. CONCLUSIONS Our data demonstrate the efficacy of humanized BiTE in patient-derived GBM models, and our results support the investigation of this novel therapeutic in a clinical trial setting.FUNDING: R01 NS122395-01 and R01 NS095642-01

BMP10 functions independently from BMP9 for the development of a proper arteriovenous network

Hereditary hemorrhagic telangiectasia (HHT) is a genetic vascular disorder characterized by the presence of arteriovenous malformation (AVM) in multiple organs. HHT is caused by mutations in genes encoding major constituents for transforming growth factor-β (TGF-β) family signaling: endoglin (ENG), activin receptor-like kinase 1 (ALK1), and SMAD4. The identity of physiological ligands for this ENG-ALK1 signaling pertinent to AVM formation has yet to be clearly determined. To investigate whether bone morphogenetic protein 9 (BMP9), BMP10, or both are physiological ligands of ENG-ALK1 signaling involved in arteriovenous network formation, we generated a novel Bmp10 conditional knockout mouse strain. We examined whether global Bmp10-inducible knockout (iKO) mice develop AVMs at neonatal and adult stages in comparison with control, Bmp9-KO, and Bmp9/10-double KO (dKO) mice. Bmp10-iKO and Bmp9/10-dKO mice showed AVMs in developing retina, postnatal brain, and adult wounded skin, while Bmp9-KO did not display any noticeable vascular defects. Bmp10 deficiency resulted in increased proliferation and size of endothelial cells in AVM vessels. The impaired neurovascular integrity in the brain and retina of Bmp10-iKO and Bmp9/10-dKO mice was detected. Bmp9/10-dKO mice exhibited the lethality and vascular malformation similar to Bmp10-iKO mice, but their phenotypes were more pronounced. Administration of BMP10 protein, but not BMP9 protein, prevented retinal AVM in Bmp9/10-dKO and endothelial-specific Eng-iKO mice. These data indicate that BMP10 is indispensable for the development of a proper arteriovenous network, whereas BMP9 has limited compensatory functions for the loss of BMP10. We suggest that BMP10 is the most relevant physiological ligand of the ENG-ALK1 signaling pathway pertinent to HHT pathogenesis.

Non-uniform dystrophin re-expression after CRISPR-mediated exon excision in the dystrophin/utrophin double-knockout mouse model of DMD

Duchenne muscular dystrophy (DMD) is the most prevalent inherited myopathy affecting children, caused by genetic loss of the gene encoding the dystrophin protein. Here we have investigated the use of the Staphylococcus aureus CRISPR-Cas9 system and a double-cut strategy, delivered using a pair of adeno-associated virus serotype 9 (AAV9) vectors, for dystrophin restoration in the severely affected dystrophin/utrophin double-knockout (dKO) mouse. Single guide RNAs were designed to excise Dmd exon 23, with flanking intronic regions repaired by non-homologous end joining. Exon 23 deletion was confirmed at the DNA level by PCR and Sanger sequencing, and at the RNA level by RT-qPCR. Restoration of dystrophin protein expression was demonstrated by western blot and immunofluorescence staining in mice treated via either intraperitoneal or intravenous routes of delivery. Dystrophin restoration was most effective in the diaphragm, where a maximum of 5.7% of wild-type dystrophin expression was observed. CRISPR treatment was insufficient to extend lifespan in the dKO mouse, and dystrophin was expressed in a within-fiber patchy manner in skeletal muscle tissues. Further analysis revealed a plethora of non-productive DNA repair events, including AAV genome integration at the CRISPR cut sites. This study highlights potential challenges for the successful development of CRISPR therapies in the context of DMD.

Validation of Mct8/Oatp1c1 dKO mice as a model organism for the Allan-Herndon-Dudley Syndrome

ObjectiveThe Allan-Herndon-Dudley syndrome (AHDS) is a severe disease caused by dysfunctional central thyroid hormone transport due to functional loss of the monocarboxylate transporter 8 (MCT8). In this study, we assessed whether mice with concomitant deletion of the thyroid hormone transporters Mct8 and the organic anion transporting polypeptide (Oatp1c1) represent a valid preclinical model organism for the AHDS. MethodsWe generated and metabolically characterized a new CRISPR/Cas9 generated Mct8/Oatp1c1 double-knockout (dKO) mouse line for the clinical features observed in patients with AHDS. ResultsWe show that Mct8/Oatp1c1 dKO mice mimic key hallmarks of the AHDS, including decreased life expectancy, central hypothyroidism, peripheral hyperthyroidism, impaired neuronal myelination, impaired motor abilities and enhanced peripheral thyroid hormone action in the liver, adipose tissue, skeletal muscle and bone. ConclusionsWe conclude that Mct8/Oatp1c1 dKO mice are a valuable model organism for the preclinical evaluation of drugs designed to treat the AHDS.

The gelatinases, matrix metalloproteinases 2 and 9, play individual roles in skeleton development

The gelatinases, a subgroup of the matrix metalloproteinases (MMPs) superfamily are composed of two members; MMP2 and MMP9. They are known to degrade gelatin among other components of the extracellular matrix. Recently, the two gelatinases were found to be necessary for neural crest cell migration and to compensate for each other loss in these cells. To characterize their involvement in the skeletal system, and to better reveal their individual or common roles, we have generated double knockout (dKO) mice, lacking both MMP2 and MMP9. Comprehensive analysis of the skeleton morphological and mechanical parameters at postnatal day (P) 0, P21, 3 months (M) and 8M of age, revealed an unexpected distinct role for each gelatinase; MMP2 was found to be involved merely in intramembranous ossification which led to a smaller skull and inferior cortical parameters upon its loss, while MMP9 was found to affect only the endochondral ossification process, which led to shorter long-bones in its absence. Importantly, the dKO mice demonstrated a combination of both the skull and long bone phenotypes as found in the single-KOs, and not a severer additive phenotype. Transcriptome analysis on the cortical bone, the growth plate and the skull frontal bone, found many genes that were differentially expressed as a direct or indirect result of MMP-loss, and reinforced the specific and distinct role of each gelatinase in each bone type. Altogether, these results suggest that although both gelatinases share the same substrates and are highly expressed in flat and long bones, they are indispensable and control separately the development of different bones.

Favine/CCDC3 deficiency accelerated atherosclerosis and thrombus formation is associated with decreased MEF2C-KLF2 pathway

SummaryCurrently, no mouse models manifest calcification and thrombus formation, which is frequently associated with human atherosclerosis. We demonstrated that lack of Favine/CCDC3 in apoE knockout mice accelerated atherosclerosis accompanied by large cholesterol crystals and calcification, and also promoted thrombus formation in the left ventricle and arteries. Circulating Favine was detectable in WT mouse plasma. RNA-sequencing analysis of aortae in DKO mice showed similar gene expression patterns of human atherosclerosis with unstable and vulnerable plaques. Importantly, human FAVINE mRNA expressions were lower in atheroma plaque than in adjacent intact aortic tissue and decreased with the progression of atherosclerosis. Pathway analysis of aortae in DKO mice suggested the decrease of the MEF2C-KLF2-mediated transcriptional pathway. Favine insufficiency and its attenuated downstream pathways may increase atherosclerosis progression with calcification and thrombus, which have not previously been fully modeled in experimental animals. Favine and its downstream pathways may have therapeutic potential for atherosclerosis.

Hepatic Deletion of X-Box Binding Protein 1 in FXR Null Mice Leads to Enhanced Liver Injury

FXR regulates bile acid metabolism, and FXR null (Fxr−/−) mice have elevated bile acid levels and progressive liver injury. The inositol-requiring enzyme 1α/X-box binding protein 1 (XBP1) pathway is a protective unfolded protein response pathway activated in response to endoplasmic reticulum stress. Here, we sought to determine the role of the inositol-requiring enzyme 1α/XBP1 pathway in hepatic bile acid toxicity using the Fxr−/− mouse model. Western blotting and quantitative PCR analysis demonstrated that hepatic XBP1 and other unfolded protein response pathways were activated in 24-week-old Fxr−/− compared with 10-week-old Fxr−/− mice but not in WT mice. To further determine the role of the liver XBP1 activation in older Fxr−/− mice, we generated mice with whole-body FXR and liver-specific XBP1 double KO (DKO, Fxr−/−Xbp1LKO) and Fxr−/−Xbp1fl/fl single KO (SKO) mice and characterized the role of hepatic XBP1 in cholestatic liver injury. Histologic staining demonstrated increased liver injury and fibrosis in DKO compared with SKO mice. RNA sequencing revealed increased gene expression in apoptosis, inflammation, and cell proliferation pathways in DKO mice. The proapoptotic C/EBP-homologous protein pathway and cell cycle marker cyclin D1 were also activated in DKO mice. Furthermore, we found that total hepatic bile acid levels were similar between the two genotypes. At age 60 weeks, all DKO mice and no SKO mice spontaneously developed liver tumors. In conclusion, the hepatic XBP1 pathway is activated in older Fxr−/− mice and has a protective role. The potential interaction between XBP1 and FXR signaling may be important in modulating the hepatocellular cholestatic stress responses.

The intramembrane proteases SPPL2a and SPPL2b regulate the homeostasis of selected SNARE proteins.

Signal peptide peptidase (SPP) and SPP-like (SPPL) aspartyl intramembrane proteases are known to contribute to sequential processing of type II-oriented membrane proteins referred to as regulated intramembrane proteolysis. The ER-resident family members SPP and SPPL2c were shown to also cleave tail-anchored proteins, including selected SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins facilitating membrane fusion events. Here, we analysed whether the related SPPL2a and SPPL2b proteases, which localise to the endocytic or late secretory pathway, are also able to process SNARE proteins. Therefore, we screened 18 SNARE proteins for cleavage by SPPL2a and SPPL2b based on cellular co-expression assays, of which the proteins VAMP1, VAMP2, VAMP3 and VAMP4 were processed by SPPL2a/b demonstrating the capability of these two proteases to proteolyse tail-anchored proteins. Cleavage of the four SNARE proteins was scrutinised at the endogenous level upon SPPL2a/b inhibition in different cell lines as well as by analysing VAMP1-4 levels in tissues and primary cells of SPPL2a/b double-deficient (dKO) mice. Loss of SPPL2a/b activity resulted in an accumulation of VAMP1-4 in a cell type- and tissue-dependent manner, identifying these proteins as SPPL2a/b substrates validated in vivo. Therefore, we propose that SPPL2a/b control cellular levels of VAMP1-4 by initiating the degradation of these proteins, which might impact cellular trafficking.

Development and validation of an LC-MS/MS methodology for the quantification of thyroid hormones in dko MCT8/OATP1C1 mouse brain

The Allan-Herndon Dudley Syndrome (AHDS) is a rare disease caused by the progressive loss of monocarboxylate transporter 8 (MCT8). In patients with AHDS, the absence of MCT8 impairs transport of thyroid hormones (TH) through the blood brain barrier, leading to a central state of TH deficiency. In mice, the AHDS is mimicked by simultaneous deletion of the TH transporters MCT8 and the solute carrier organic anion transporter family member 1c1 (OATP1C1). To support preclinical mouse studies, an analytical methodology was developed and successfully applied for quantifying selected thyroid hormones in mouse whole brain and in specific regions using liquid chromatography tandem mass-spectrometry (LC-MS/MS). An important requirement for the methodology was its high sensitivity since a very low concentration of THs was expected in MCT8/OATP1C1 double-knockout (dko) mouse brain. Seven THs were targeted: L-thyroxine (T4), 3,3´,5‐triiodo‐L‐thyronine-thyronine (T3), 3,3´,5´‐triiodo‐L‐thyronine-thyronine (rT3), 3,3´‐diiodo‐L‐thyronine (3,3´‐T2, T2), 3,5-diiodo-L-thyronine (rT2, 3,5-T2), 3-iodo-L-thyronine (T1), 3-iodothyronamine (T1AM). Isotope dilution liquid chromatography triple-quadrupole mass spectrometry methodology was applied for detection and quantification. The method was validated in wild-type animals for mouse whole brain and for five different brain regions (hypothalamus, hippocampus, prefrontal cortex, brainstem and cortex). Instrumental calibration curves ranged from 0.35 to 150 pg/µL with good linearity (r2 >0.996). The limit of quantification was from 0.08 to 0.6 pg/mg, with an intra- and inter-day precision of 4.2–14.02% and 0.4–17.9% respectively, and accuracies between 84.9% and 114.8% when the methodology was validated for the whole brain. In smaller, distinct brain regions, intra- and inter-day precision were 0.6–20.7% and 2.5–15.6% respectively, and accuracies were 80.2–128.6%. The new methodology was highly sensitive and allowed for the following quantification in wild-type mice: (i) for the first time, four distinct thyroid hormones (T4, T3, rT3 and 3,3´‐T2) in only approximately 100 mg of mouse brain were detected; (ii) the quantification of T4 and T3 for the first time in distinct mouse brain regions were reported. Further, application of our method to MCT8/OATP1C1 dko mice revealed the expected, relative lack of T3 and T4 uptake into the brain, and confirmed the utility of our analytical method to study TH transport across the blood brain barrier in a preclinical model of central TH deficiency.