Double Mutant Animals Research Articles

Activated SKN-1 alters the aging trajectories of long-lived C. elegans mutants.

In the presence of stressful environments, the SKN-1 cytoprotective transcription factor is activated to induce the expression of gene targets that can restore homeostasis. However, chronic activation of SKN-1 results in diminished health and a reduction of lifespan. Here we demonstrate the necessity of modulating SKN-1 activity to maintain the longevity-promoting effects associated with genetic mutations that impair daf-2/insulin receptor signaling, the eat-2 model of caloric restriction, and glp-1-dependent loss of germ cell proliferation. A hallmark of animals with constitutive SKN-1 activation is the age-dependent loss of somatic lipids and this phenotype is linked to a general reduction in survival in animals harboring the skn-1gf allele, but surprisingly, daf-2lf; skn-1gf double mutant animals do not redistribute somatic lipids which suggests the insulin signaling pathway functions downstream of SKN-1 in the maintenance of lipid distribution. As expected, the eat-2lf allele, which independently activates SKN-1, continues to display somatic lipid depletion in older ages with and without the skn-1gf activating mutation. In contrast, the presence of the skn-1gf allele does not lead to somatic lipid redistribution in glp-1lf animals that lack a proliferating germline. Taken together, these studies support a genetic model where SKN-1 activity is an important regulator of lipid mobilization in response to nutrient availability that fuels the developing germline by engaging the daf-2/insulin receptor pathway.

Febuxostat ameliorates muscle degeneration and movement disorder of the dystrophin mutant model in Caenorhabditis elegans

Duchenne muscular dystrophy (DMD) is an inherited disorder with mutations in the dystrophin gene characterized by progressive muscle degeneration and weakness. Therapy such as administration of glucocorticoids, exon skipping of mutant genes and introduction of dystrophin mini-genes have been tried, but there is no radical therapy for DMD. In this study, we used C. elegans carrying mutations in the dys-1 gene as a model of DMD to examine the effects of febuxostat (FBX). We applied FBX to dys-1 mutant animals harboring a marker for muscle nuclei and mitochondria, and found that FBX ameliorates the muscle loss. We next used a severer model dys-1; unc-22 double mutant and found the dys-1 mutation causes a weakened muscle contraction. We applied FBX and other compounds to the double mutant animals and assayed the movement. We found that the administration of FBX in combination of uric acid has the best effects on the DMD model.

Mitophagy in Astrocytes Is Required for the Health of Optic Nerve.

Mitochondrial dysfunction in astrocytes has been implicated in the development of various neurological disorders. Mitophagy, mitochondrial autophagy, is required for proper mitochondrial function by preventing the accumulation of damaged mitochondria. The importance of mitophagy, specifically in the astrocytes of the optic nerve (ON), has been little studied. We introduce an animal model in which two separate mutations act synergistically to produce severe ON degeneration. The first mutation is in Cryba1, which encodes βA3/A1-crystallin, a lens protein also expressed in astrocytes, where it regulates lysosomal pH. The second mutation is in Bckdk, which encodes branched-chain ketoacid dehydrogenase kinase, which is ubiquitously expressed in the mitochondrial matrix and involved in the catabolism of the branched-chain amino acids. BCKDK is essential for mitochondrial function and the amelioration of oxidative stress. Neither of the mutations in isolation has a significant effect on the ON, but animals homozygous for both mutations (DM) exhibit very serious ON degeneration. ON astrocytes from these double-mutant (DM) animals have lysosomal defects, including impaired mitophagy, and dysfunctional mitochondria. Urolithin A can rescue the mitophagy impairment in DM astrocytes and reduce ON degeneration. These data demonstrate that efficient mitophagy in astrocytes is required for ON health and functional integrity.

TGFβ-2 haploinsufficiency causes early death in mice with Marfan syndrome

To assess the contribution of individual TGF-β isoforms to aortopathy in Marfan syndrome (MFS), we quantified the survival and phenotypes of mice with a combined fibrillin1 (the gene defective in MFS) hypomorphic mutation and a TGF-β1, 2, or 3 heterozygous null mutation. The loss of TGF-β2, and only TGF-β2, resulted in 80% of the double mutant animals dying earlier, by postnatal day 20, than MFS only mice. Death was not from thoracic aortic rupture, as observed in MFS mice, but was associated with hyperplastic aortic valve leaflets, aortic regurgitation, enlarged aortic root, increased heart weight, and impaired lung alveolar septation. Thus, there appears to be a relationship between loss of fibrillin1 and TGF-β2 in the postnatal development of the heart, aorta and lungs.

PTRN-1 (CAMSAP) and NOCA-2 (NINEIN) are required for microtubule polarity in Caenorhabditis elegans dendrites.

The neuronal microtubule cytoskeleton is key to establish axon-dendrite polarity. Dendrites are characterized by the presence of minus-end out microtubules. However, the mechanisms that organize these microtubules with the correct orientation are still poorly understood. Using Caenorhabditis elegans as a model system for microtubule organization, we characterized the role of 2 microtubule minus-end related proteins in this process, the microtubule minus-end stabilizing protein calmodulin-regulated spectrin-associated protein (CAMSAP/PTRN-1), and the NINEIN homologue, NOCA-2 (noncentrosomal microtubule array). We found that CAMSAP and NINEIN function in parallel to mediate microtubule organization in dendrites. During dendrite outgrowth, RAB-11-positive vesicles localized to the dendrite tip to nucleate microtubules and function as a microtubule organizing center (MTOC). In the absence of either CAMSAP or NINEIN, we observed a low penetrance MTOC vesicles mislocalization to the cell body, and a nearly fully penetrant phenotype in double mutant animals. This suggests that both proteins are important for localizing the MTOC vesicles to the growing dendrite tip to organize microtubules minus-end out. Whereas NINEIN localizes to the MTOC vesicles where it is important for the recruitment of the microtubule nucleator γ-tubulin, CAMSAP localizes around the MTOC vesicles and is cotranslocated forward with the MTOC vesicles upon dendritic growth. Together, these results indicate that microtubule nucleation from the MTOC vesicles and microtubule stabilization are both important to localize the MTOC vesicles distally to organize dendritic microtubules minus-end out.

Nuclear receptors NHR-49 and NHR-79 promote peroxisome proliferation to compensate for aldehyde dehydrogenase deficiency in C. elegans.

The intracellular level of fatty aldehydes is tightly regulated by aldehyde dehydrogenases to minimize the formation of toxic lipid and protein adducts. Importantly, the dysregulation of aldehyde dehydrogenases has been implicated in neurologic disorder and cancer in humans. However, cellular responses to unresolved, elevated fatty aldehyde levels are poorly understood. Here, we report that ALH-4 is a C. elegans aldehyde dehydrogenase that specifically associates with the endoplasmic reticulum, mitochondria and peroxisomes. Based on lipidomic and imaging analysis, we show that the loss of ALH-4 increases fatty aldehyde levels and reduces fat storage. ALH-4 deficiency in the intestine, cell-nonautonomously induces NHR-49/NHR-79-dependent hypodermal peroxisome proliferation. This is accompanied by the upregulation of catalases and fatty acid catabolic enzymes, as indicated by RNA sequencing. Such a response is required to counteract ALH-4 deficiency since alh-4; nhr-49 double mutant animals are sterile. Our work reveals unexpected inter-tissue communication of fatty aldehyde levels and suggests pharmacological modulation of peroxisome proliferation as a therapeutic strategy to tackle pathology related to excess fatty aldehydes.

The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

Telomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans.

Global loss of Tfr2 with concomitant induced iron deficiency greatly ameliorates the phenotype of a murine thalassemia intermedia model.

β-thalassemias result from mutations in β-globin, causing ineffective erythropoiesis and secondary iron overload due to inappropriately low levels of the iron regulatory hormone hepcidin. Mutations in transferrin receptor 2 (TFR2) lead to hereditary hemochromatosis (HH) as a result of inappropriately increased iron uptake from the diet, also due to improperly regulated hepcidin. TFR2 is also thought to be required for efficient erythropoiesis through its interaction with the erythropoietin receptor in erythroid progenitors. Transmembrane serine protease 6 (TMPRSS6), a membrane serine protease expressed selectively in the liver, participates in regulating hepcidin production in response to iron stores by cleaving hemojuvelin (HJV). We have previously demonstrated that inhibiting TMPRSS6 expression with a hepatocyte-specific siRNA formulation, induces hepcidin, mitigates anemia, and reduces iron overload in murine models of β-thalassemia intermedia and HH. Here, we demonstrate that Tmprss6 siRNA treatment of double mutant Tfr2Y245X/Y245X HH Hbbth3/+ thalassemic mice induces hepcidin and diminishes tissue and serum iron levels. Importantly, treated double mutant animals produce more mature red blood cells and have a nearly 50% increase in hemoglobin compared to untreated β-thalassemic mice. Furthermore, we also show that treatment of Tfr2Y245X/Y245X HH mice leads to increased hepcidin expression and reduced total body iron burden. These data indicate that siRNA suppression of Tmprss6, in conjunction with the targeting of TFR2, may be superior to inhibiting Tmprss6 alone in the treatment of the anemia and secondary iron loading in β-thalassemia intermedia and may be useful as a method of suppressing the primary iron overload in TFR2-related (type 3) hereditary hemochromatosis.

P3828Zebrafish models for arrhythmogenic cardiomyopathy type 8: a starting platform for exercise stress test and drug treatment

Abstract Background Arrhythmogenic Cardiomyopathy (AC) is an inherited heart disease characterized by progressive substitution of the myocardium with fibro-fatty tissue, leading to electrical instability and high risk of sudden death, particularly in young subjects and athletes. In recent years, our laboratory has produced zebrafish (zf) mutant lines modelling AC type 8, an AC form linked to mutations in the junctional protein Desmoplakin (Dsp). Mutations in the DSP gene have been identified in both dominant and recessive AC cases, characterized by left-dominant and biventricular forms of the disease. Sports medicine has highlighted that they are the most dangerous forms, being less easily identifiable by ECG. Purpose Taking advantage of our zf Dsp mutant lines, we aim to fully characterize the pathological phenotype, analyze the perturbation of cell communication pathways, evaluate the role of the physical exercise, and test the efficacy of candidate drugs. Methods Among our zf lines we have identified double mutant animals, bearing both zf dspa and dspb mutations in heterozygous condition, as the best model able to recapitulate the human AC phenotype. This model underwent physical stress tests in the presence/absence of candidate drug treatment. Phenotyping included heart rhythm measurement, gene expression analysis using Real Time PCR and signaling pathway transgenes, immune-histochemistry, whole-mount in situ hybridization, standard histology and ultrastructural TEM analysis. Results Preliminary results from mutant phenotyping indicate alterations in heart rate, sudden cardiac death, structural alterations of the myocardium associated with junctional disorganization and, in parallel, dysregulation of Wnt, Hippo and TGFbeta pathways. Specifically, Dsp mutant animals can range from an 8% decrease to a 14% increase of heart rhythm compared to the physiological range (120–140 beats per minute in zf larvae). At the adult stage, about 1% of the fish mutant population dies suddenly. The histological examination shows a 50% reduction of the myocardial cell mass, in parallel with a 50% decrease of Dsp signal, detected by TEM, associated with the so-called “pale desmosome” phenotype. Signaling dysregulation includes an 80% loss of Wnt/Beta-catenin, a 300% increase of TGFbeta and a 500% increase of Hippo/YAP-TAZ signaling in the cardiac tissue. Physical stress tests and pathway-directed drug treatment have clarified that these factors can modulate the pathological phenotype, as preliminarily evidenced by the rescue of Wnt signal decrease to normal levels through SB216763 treatment of Dsp-deficient individuals at rest. Conclusion Preliminary evidences corroborate the zf organism as a suitable model for AC cellular and molecular phenotyping, exploitable for the dissection of the genetic events leading to the onset and progression of the disease, and applicable to the analysis of chemical and mechanical modulators of AC-associated features. Acknowledgement/Funding Cariparo 2017 SHoCD; TRANSAC; CPDA133979/13; RP-2014-ehz745.06700394

Abstract 4654: Hotspot mutations in the core spliceosomal protein SF3B1 promote breast tumorigenesis

Abstract The core RNA splicing factor SF3B1 is targeted by recurrent hotspot mutations in leukemias and solid tumors with the K700E substitution observed in 2% of unselected breast cancer patients. These mutations promote usage of aberrant intron proximal 3’ splice sites. In breast cancer, the links between these aberrant splicing events and the transformed phenotype are not well understood. Here, we integrated findings from multiple genetically modified model systems and human tumor specimens to understand the contributions of mutant SF3B1 to breast cancer pathogenesis. To identify the aberrant splicing events in breast cancers specifically, we first performed RNA-seq analyses of MCF10A breast epithelial cells with CRISPR knock-in of the K700E mutation. Dysregulation of splicing at over 1,000 splice junctions was discovered, most of which involve the use of alternative 3’ splice sites. Gene expression analysis revealed upregulation of expression of NFκB pathway genes in the SF3B1 mutant cells. SF3B1 mutant cells showed higher phosphorylation of RelA (p65) and, accordingly, greater migration in wound-healing assays. Mis-splicing of exon 5 in MAP3K7, a gene encoding a central regulator of NFκB pathway, significantly reduced MAP3K7 protein expression in SF3B1 K700E mutant cells. Consistent with these data, pharmacologic MAP3K7 inhibition in SF3B1 wild-type breast cells increased phosphorylation of RelA. To further understand the effects of the K700E mutation on breast cancer development and progression, we generated a conditional knock-in mouse model, in which Sf3b1 K700E mutant allele was activated through MMTV promoter-controlled expression of Cre recombinase in mammary epithelium. Accelerated tumor formation was not identified in the mammary gland of these mice. However, RNA-seq analysis of mammary epithelial cells from the Sf3b1 K700E mutant mice revealed dysregulation of alternative splicing including Map3k7. As we found the majority of SF3B1 k700E mutations to co-occur with activating mutations in the PI3K pathway, we additionally introduced the Pik3ca H1047R mutant allele, by mating the MMTV-Cre Sf3b1 K700E mice with Rosa26-Pik3ca H1047R knock-in line. Markedly decreased survival was observed in double mutant animals, which developed mammary tumors faster, compared with single mutant controls. Overall, these data reveal that breast cancer-associated Sf3b1 mutations induce the NFkB pathway to promote invasion and cooperate with the PI3K pathway to accelerate mammary tumor development. Citation Format: Bo Liu, Michelle Ki, Omar Abdel-Wahab, Sarat Chandarlapaty. Hotspot mutations in the core spliceosomal protein SF3B1 promote breast tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4654.

Exploring the Mechanisms of Thalassemic Erythropoiesis Improvement Caused By Bone Marrow Tfr2 Deletion

Transferrin receptor 2 (TFR2), the type 3 hemochromatosis gene, is an activator of the iron hormone hepcidin in the liver and a partner of erythropoietin (EPO) receptor in erythroid cells. The loss of bone marrow (BM) Tfr2 increases erythroblast EPO sensitivity inducing erythrocytosis in mice (Nai et al, Blood 2015). We explored whether deletion of BM Tfr2 improves anemia and ineffective erythropoiesis in β-thalassemias, iron-loading anemias due to recessive β-globin gene mutations. We generated thalassemic mice (Hbbth3/+) with selective BM inactivation of Tfr2 (Tfr2BMKO/Hbbth3/+) through BM transplantation (BMT). Deletion of BM Tfr2 ameliorates RBC morphology with consistent and persistent increase of RBC count and Hb levels in thalassemic mice, accompanied by reduced iron accumulation. Around 22 weeks after BMT the improvement fades in double mutantanimals: Hb levels return comparable to those of Hbbth3/+mice, while RBC count persists higher. Anemia improvement in double mutant mice reduces serum EPO levels and improves erythropoiesis, in particular 22 weeks after BMT. Overall these data prove that the loss of the beneficial effect of deleting Tfr2 is not accounted for by erythropoiesis failure, but likely by exhaustion of splenic iron consumed by the enhanced erythropoiesis.In order to elucidate the molecular mechanisms of the phenotype improvement, we investigated whether the EPO-EPOR signaling pathway is overactive, as occurs in Tfr2 null erythroid cells (Nai et al, Blood 2015). Taking into account that Tfr2BMKO/Hbbth3/+ mice have lower serum EPO than Hbbth3/+, the expression levels of target genes of the EPOR-JAK2-STAT5 (Erfe and Bcl-xl) and of EPOR-PI3K-AKT pathway (Fasl, Epor and Ccng2) is consistent with the signaling being inappropriately active in double mutant mice.To start unraveling the global molecular/cellular processes underlying the remarkable phenotype amelioration, we performed RNAseq analysis on spleen samples from double mutant and Hbbth3/+ control mice at the time point of maximal erythropoiesis improvement (22 weeks post BMT). Spleens are enlarged in both genotypes with about 80% Ter119+ (erythroid) cells in both. In total we identified 2796 genes (1997 protein coding) differentially regulated between the two genotypes. The analysis of iron-related genes reveals a strong reduction of the expression of the iron exporter Fpn, Hmox1 and Alas2, suggestive of decreased hemolysis and/or of free heme accumulation in double mutants. Gene ontology analysis reveals enrichment of genes involved in cell cycle and proliferation, mitochondrial function, as well as proteasome activity and of most of the antioxidant targets (Sod1, Sod2, Fth1, Txn1, Txn2, Gstpi) of the canonical NF-kB pathway. Underrepresented genes are those involved in lipid handling, leukocyte/lymphocyte differentiation and coagulation.In summary, the RNAseq patterns indicate an increased spleen erythroid commitment and a mitochondrial metabolic shift, similar to the shift occurring during hematopoietic development to sustain erythroid proliferation and differentiation. We speculate that this effect is mediated by the enhanced EPO sensitivity. Interestingly EPO directly stimulates mitochondrial genes expression in adipocytes. Also the increased proteasome activity may significantly contribute to the improved erythropoiesis, since proteasomal degradation is required in the process of erythroblast enucleation. Finally, the activation of the NF-kB antioxidant response may contrast ROS increase and limit ineffective erythropoiesis.In conclusion, targeting erythroid TFR2 might become a novel erythropoiesis stimulating approach, worth to be tested in other forms of anemia. DisclosuresMuckenthaler:Novartis: Research Funding. Camaschella:vifor Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Ablation of cytoskeletal scaffolding proteins, Band 4.1B and Whirlin, leads to cerebellar purkinje axon pathology and motor dysfunction.

The cerebellar cortex receives neural information from other brain regions to allow fine motor coordination and motor learning. The primary output neurons from the cerebellum are the Purkinje neurons that transmit inhibitory responses to deep cerebellar nuclei through their myelinated axons. Altered morphological organization and electrical properties of the Purkinje axons lead to detrimental changes in locomotor activity often leading to cerebellar ataxias. Two cytoskeletal scaffolding proteins Band 4.1B (4.1B) and Whirlin (Whrn) have been previously shown to play independent roles in axonal domain organization and maintenance in myelinated axons in the spinal cord and sciatic nerves. Immunoblot analysis had indicated cerebellar expression for both 4.1B and Whrn; however, their subcellular localization and cerebellum-specific functions have not been characterized. Using 4.1B and Whrn single and double mutant animals, we show that both proteins are expressed in common cellular compartments of the cerebellum and play cooperative roles in preservation of the integrity of Purkinje neuron myelinated axons. We demonstrate that both 4.1B and Whrn are required for the maintenance of axonal ultrastructure and health. Loss of 4.1B and Whrn leads to axonal transport defects manifested by formation of swellings containing cytoskeletal components, membranous organelles, and vesicles. Moreover, ablation of both proteins progressively affects cerebellar function with impairment in locomotor performance detected by altered gait parameters. Together, our data indicate that 4.1B and Whrn are required for maintaining proper axonal cytoskeletal organization and axonal domains, which is necessary for cerebellum-controlled fine motor coordination.

Functional Interactions Between rsks-1/S6K, glp-1/Notch, and Regulators of Caenorhabditis elegans Fertility and Germline Stem Cell Maintenance.

The proper accumulation and maintenance of stem cells is critical for organ development and homeostasis. The Notch signaling pathway maintains stem cells in diverse organisms and organ systems. In Caenorhabditis elegans, GLP-1/Notch activity prevents germline stem cell (GSC) differentiation. Other signaling mechanisms also influence the maintenance of GSCs, including the highly-conserved TOR substrate ribosomal protein S6 kinase (S6K). Although C. elegans bearing either a null mutation in rsks-1/S6K or a reduction-of-function (rf) mutation in glp-1/Notch produce half the normal number of adult germline progenitors, virtually all these single mutant animals are fertile. However, glp-1(rf) rsks-1(null) double mutant animals are all sterile, and in about half of their gonads, all GSCs differentiate, a distinctive phenotype associated with a significant reduction or loss of GLP-1 signaling. How rsks-1/S6K promotes GSC fate is unknown. Here, we determine that rsks-1/S6K acts germline-autonomously to maintain GSCs, and that it does not act through Cyclin-E or MAP kinase in this role. We found that interfering with translation also enhances glp-1(rf), but that regulation through rsks-1 cannot fully account for this effect. In a genome-scale RNAi screen for genes that act similarly to rsks-1/S6K, we identified 56 RNAi enhancers of glp-1(rf) sterility, many of which were previously not known to interact functionally with Notch. Further investigation revealed at least six candidates that, by genetic criteria, act linearly with rsks-1/S6K. These include genes encoding translation-related proteins, cacn-1/Cactin, an RNA exosome component, and a Hedgehog-related ligand. We found that additional Hedgehog-related ligands may share functional relationships with glp-1/Notch and rsks-1/S6K in maintaining germline progenitors.

Deletion of MCP-1 Impedes Pathogenesis of Acid Ceramidase Deficiency

Farber Disease (FD) is an ultra-rare Lysosomal Storage Disorder caused by deficient acid ceramidase (ACDase) activity. Patients with ACDase deficiency manifest a spectrum of symptoms including formation of nodules, painful joints, and a hoarse voice. Classic FD patients will develop histiocytes in organs and die in childhood. Monocyte chemotactic protein (MCP-1; CCL2) is significantly elevated in both FD patients and a mouse model we previously generated. Here, to further study MCP-1 in FD, we created an ACDase;MCP-1 double mutant mouse. We show that deletion of MCP-1 reduced leukocytosis, delayed weight loss, and improved lifespan. Reduced inflammation and fibrosis were observed in livers from double mutant animals. Bronchial alveolar lavage fluid analyses revealed a reduction in cellular infiltrates and protein accumulation. Furthermore, reduced sphingolipid accumulation was observed in the lung and liver but not in the brain. The neurological and hematopoietic defects observed in FD mice were maintained. A compensatory cytokine response was found in the double mutants, however, that may contribute to continued signs of inflammation and injury. Taken together, targeting a reduction of MCP-1 opens the door to a better understanding of the mechanistic consequences of ceramide accumulation and may even delay the progression of FD in some organ systems.

Deletion of Slc26a1 and Slc26a7 Delays Enamel Mineralization in Mice.

Amelogenesis features two major developmental stages—secretory and maturation. During maturation stage, hydroxyapatite deposition and matrix turnover require delicate pH regulatory mechanisms mediated by multiple ion transporters. Several members of the Slc26 gene family (Slc26a1, Slc26a3, Slc26a4, Slc26a6, and Slc26a7), which exhibit bicarbonate transport activities, have been suggested by previous studies to be involved in maturation-stage amelogenesis, especially the key process of pH regulation. However, details regarding the functional role of these genes in enamel formation are yet to be clarified, as none of the separate mutant animal lines demonstrates any discernible enamel defects. Continuing with our previous investigation of Slc26a1−/− and Slc26a7−/− animal models, we generated a double-mutant animal line with the absence of both Slc26a1 and Slc26a7. We showed in the present study that the double-mutant enamel density was significantly lower in the regions that represent late maturation-, maturation- and secretory-stage enamel development in wild-type mandibular incisors. However, the “maturation” and “secretory” enamel microstructures in double-mutant animals resembled those observed in wild-type secretory and/or pre-secretory stages. Elemental composition analysis revealed a lack of mineral deposition and an accumulation of carbon and chloride in double-mutant enamel. Deletion of Slc26a1 and Slc26a7 did not affect the stage-specific morphology of the enamel organ. Finally, compensatory expression of pH regulator genes and ion transporters was detected in maturation-stage enamel organs of double-mutant animals when compared to wild-type. Combined with the findings from our previous study, these data indicate the involvement of SLC26A1and SLC26A7 as key ion transporters in the pH regulatory network during enamel maturation.

Caenorhabditis elegans RIG-I Homolog Mediates Antiviral RNA Interference Downstream of Dicer-Dependent Biogenesis of Viral Small Interfering RNAs.

ABSTRACTDicer enzymes process virus-specific double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) to initiate specific antiviral defense by related RNA interference (RNAi) pathways in plants, insects, nematodes, and mammals. Antiviral RNAi in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1), not found in plants and insects but highly homologous to mammalian retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), intracellular viral RNA sensors that trigger innate immunity against RNA virus infection. However, it remains unclear if DRH-1 acts analogously to initiate antiviral RNAi in C. elegans. Here, we performed a forward genetic screen to characterize antiviral RNAi in C. elegans. Using a mapping-by-sequencing strategy, we uncovered four loss-of-function alleles of drh-1, three of which caused mutations in the helicase and C-terminal domains conserved in RLRs. Deep sequencing of small RNAs revealed an abundant population of Dicer-dependent virus-derived small interfering RNAs (vsiRNAs) in drh-1 single and double mutant animals after infection with Orsay virus, a positive-strand RNA virus. These findings provide further genetic evidence for the antiviral function of DRH-1 and illustrate that DRH-1 is not essential for the sensing and Dicer-mediated processing of the viral dsRNA replicative intermediates. Interestingly, vsiRNAs produced by drh-1 mutants were mapped overwhelmingly to the terminal regions of the viral genomic RNAs, in contrast to random distribution of vsiRNA hot spots when DRH-1 is functional. As RIG-I translocates on long dsRNA and DRH-1 exists in a complex with Dicer, we propose that DRH-1 facilitates the biogenesis of vsiRNAs in nematodes by catalyzing translocation of the Dicer complex on the viral long dsRNA precursors.

A novel immunodeficient NOD.SCID-rd1 mouse model of retinitis pigmentosa to investigate potential therapeutics and pathogenesis of retinal degeneration.

ABSTRACTRetinitis pigmentosa (RP) is a common retinal degeneration disease caused by mutation in any gene of the photo transduction cascade and results in photoreceptor dystrophy. Over decades, several animal models have been used to address the need for the elucidation of effective therapeutics and factors regulating retinal degeneration to prohibit or renew the damaged retina. However, controversies over the immune privilege of retina during cell transplantation and the role of immune modulation during RP still remain largely uninvestigated because of the lack of suitable animal models. Here, we have developed an immunocompromised mouse model, NOD.SCID-rd1, for retinitis pigmentosa (RP) by crossing CBA/J and NOD SCID mice and selecting homozygous double mutant animals for further breeding. Characterization of the newly developed RP model indicates a similar retinal degeneration pattern as CBA/J, with a decreased apoptosis rate and rhodopsin loss. It also exhibits loss of T cells, B cells and NK cells. The NOD.SCID-rd1 model is extremely useful for allogenic and xenogenic cell-based therapeutics, as indicated by the higher cell integration capacity post transplantation. We dissect the underlying role of the immune system in the progression of RP and the effect of immune deficiency on immune privilege of the eye using comparative qPCR studies of this model and the immune-competent RP model.

Induction of endoplasmic reticulum stress by deletion of Grp78 depletes Apc mutant intestinal epithelial stem cells.

Intestinal epithelial stem cells are highly sensitive to differentiation induced by endoplasmic reticulum (ER) stress. Colorectal cancer develops from mutated intestinal epithelial stem cells. The most frequent initiating mutation occurs in Apc, which results in hyperactivated Wnt signalling. This causes hyperproliferation and reduced sensitivity to chemotherapy, but whether these mutated stem cells are sensitive to ER stress induced differentiation remains unknown. Here we examined this by generating mice in which both Apc and ER stress repressor chaperone Grp78 can be conditionally deleted from the intestinal epithelium. For molecular studies, we used intestinal organoids derived from these mice. Homozygous loss of Apc alone resulted in crypt elongation, activation of the Wnt signature and accumulation of intestinal epithelial stem cells, as expected. This phenotype was however completely rescued on activation of ER stress by additional deletion of Grp78. In these Apc-Grp78 double mutant animals, stem cells were rapidly lost and repopulation occurred by non-mutant cells that had escaped recombination, suggesting that Apc-Grp78 double mutant stem cells had lost self-renewal capacity. Although in Apc-Grp78 double mutant mice the Wnt signature was lost, these intestines exhibited ubiquitous epithelial presence of nuclear β-catenin. This suggests that ER stress interferes with Wnt signalling downstream of nuclear β-catenin. In conclusion, our findings indicate that ER stress signalling results in loss of Apc mutated intestinal epithelial stem cells by interference with the Wnt signature. In contrast to many known inhibitors of Wnt signalling, ER stress acts downstream of β-catenin. Therefore, ER stress poses a promising target in colorectal cancers, which develop as a result of Wnt activating mutations.

Absence of CCL2 and CCL3 Ameliorates Central Nervous System Grey Matter But Not White Matter Demyelination in the Presence of an Intact Blood-Brain Barrier.

A broad spectrum of diseases is characterized by myelin abnormalities, oligodendrocyte pathology, and concomitant glia activation, among multiple sclerosis (MS). Our knowledge regarding the factors triggering gliosis and demyelination is scanty. Chemokines are pivotal for microglia and astrocyte activation and orchestrate critical steps during the formation of central nervous system (CNS) demyelinating lesions. Redundant functions of chemokines complicate, however, the study of their functional relevance. We used the cuprizone model to study redundant functions of two chemokines, CCL2/MCP1 and CCL3/MIP1α, which are critically involved in the pathological process of cuprizone-induced demyelination. First, we generated a mutant mouse strain lacking functional genes of both chemokines and demonstrated that double-mutant animals are viable, fertile, and do not present with gross abnormalities. Astrocytes and peritoneal macrophages, cultured form tissues of these animals did neither express CCL2 nor CCL3. Exposure to cuprizone resulted in increased CCL2 and CCL3 brain levels in wild-type but not mutant animals. Cuprizone-induced demyelination, oligodendrocyte loss, and astrogliosis were significantly ameliorated in the cortex but not corpus callosum of chemokine-deficient animals. In summary, we provide a novel powerful model to study the redundant function of two important chemokines. Our study reveals that chemokine function in the CNS redounds to region-specific pathophysiological events.

The class-specific BCR tonic signal modulates lymphomagenesis in a c-myc deregulation transgenic model.

Deregulation of c-myc by translocation onto immunoglobulin (Ig) loci can promote B cell malignant proliferations with phenotypes as diverse as acute lymphoid leukemia, Burkitt lymphoma, diffuse large B cell lymphoma, myeloma... The B cell receptor (BCR) normally providing tonic signals for cell survival and mitogenic responses to antigens, can also contribute to lymphomagenesis upon sustained ligand binding or activating mutations. BCR signaling varies among cell compartments and BCR classes. For unknown reasons, some malignancies associate with expression of either IgM or class-switched Ig. We explored whether an IgA BCR, with strong tonic signaling, would affect lymphomagenesis in c-myc IgH 3'RR transgenic mice prone to lymphoproliferations. Breeding c-myc transgenics in a background where IgM expression was replaced with IgA delayed lymphomagenesis. By comparison to single c-myc transgenics, lymphomas from double mutant animals were more differentiated and less aggressive, with an altered transcriptional program. Larger tumor cells more often expressed CD43 and CD138, which culminated in a plasma cell phenotype in 10% of cases. BCR class-specific signals thus appear to modulate lymphomagenesis and may partly explain the observed association of specific Ig classes with human B cell malignancies of differential phenotype, progression and prognosis.