Mutant Mouse Lines Research Articles

Integrin \u03b111\u03b21 is a receptor for collagen XIII

Collagen XIII is a conserved transmembrane collagen mainly expressed in mesenchymal tissues. Previously, we have shown that collagen XIII modulates tissue development and homeostasis. Integrins are a family of receptors that mediate signals from the environment into the cells and vice versa. Integrin α11β1 is a collagen receptor known to recognize the GFOGER (O=hydroxyproline) sequence in collagens. Interestingly, collagen XIII and integrin α11β1 both have a role in the regulation of bone homeostasis. To study whether α11β1 is a receptor for collagen XIII, we utilized C2C12 cells transfected to express α11β1 as their only collagen receptor. The interaction between collagen XIII and integrin α11β1 was also confirmed by surface plasmon resonance and pull-down assays. We discovered that integrin α11β1 mediates cell adhesion to two collagenous motifs, namely GPKGER and GF(S)QGEK, that were shown to act as the recognition sites for the integrin α11-I domain. Furthermore, we studied the in vivo significance of the α11β1-collagen XIII interaction by crossbreeding α11 null mice (Itga11−/−) with mice overexpressing Col13a1 (Col13a1oe). When we evaluated the bone morphology by microcomputed tomography, Col13a1oe mice had a drastic bone overgrowth followed by severe osteoporosis, whereas the double mutant mouse line showed a much milder bone phenotype. To conclude, our data identifies integrin α11β1 as a new collagen XIII receptor and demonstrates that this ligand-receptor pair has a role in the maintenance of bone homeostasis.

Abstract 16286: Absence of Fxyd1 is Associated With a Female-specific Pro-inflammatory and Hypercholesterolemic Environment: Implications for Atherosclerosis

Introduction: Bioavailability of nitric oxide (NO) is often compromised in early atherosclerosis, driving disease onset. This is usually due to dysfunctional endothelial nitric oxide synthase (eNOS). We have found that the transmembrane protein, FXYD1, is highly concentrated in the caveolae sub-cellular region and is closely associated with eNOS. Our preliminary data suggest that FXYD1 protects eNOS from redox-dependent inactivation. So, we hypothesise that FXYD1 may protect against atherosclerosis. Hypothesis: We aimed to test the role of FXYD1 in a murine model of atherosclerosis Methods: We produced a novel mutant mouse line by cross-breeding FXYD1 heterozygote (FXYD1 +/- ) and apolipoprotein E knock out (ApoE -/- ) mice. FXYD1 -/- /ApoE -/- and FXYD1 +/+ /ApoE -/- littermates were fed a high fat/high cholesterol diet from 6-8 weeks of age for 16 weeks. At sacrifice, blood was taken, and plasma was separated for analysis of cholesterol and pro-inflammatory cytokines using ELISA. The thoracic aorta was isolated and formalin-fixed to assess atherosclerotic plaque development using oil red O stain. Immunoblotting for eNOS expression was performed in heart lysates. Results: Plasma total cholesterol was significantly increased in FXYD1-/-/ApoE-/- female mice (n = 14 compared to FXYD1+/+/ApoE-/- mice (n = 21; p<0.05)). Circulating IL-1β levels were also significantly increased in female FXYD1-/-/ApoE-/- vs. FXYD1+/+/ApoE-/- mice. Despite this, there was no effect on plaque size as determined by oil red O stain of fixed aortae. eNOS protein expression was decreased in heart tissue from FXYD1-/-/ApoE-/- vs. FXYD1+/+/ApoE-/- female mice which may lead to lower NO generation. All of the FXYD1-mediated changes were specific to female mice as we detected no differences in these parameters in littermate males. Conclusions: FXYD1-/- atherosclerotic female mice have higher circulating lipids and pro-inflammatory cytokines, but this does not appear to alter plaque development in the short term. Ongoing studies will determine the effect on plaque stability and role of FXYD1 in lipidomics. FXYD1 appears to have an anti-inflammatory role and may also be involved in cholesterol metabolism or handling. This could provide a novel therapeutic angle for females.

TMOD-24. GENERATION AND CHARACTERIZATION OF A NOVEL TRANSGENIC IDO REPORTER MOUSE FOR IDO POSTTRANSLATIONAL MODIFICATION ANALYSIS IN SITU

Abstract Glioblastoma (GBM) is the most common and aggressive primary central nervous system tumor in adults with a median survival of 14.6 months. GBM is a potently immunosuppressive cancer due in-part to the prolific expression of immunosuppressive indoleamine 2,3 dioxygenase 1 (IDO). Tumor cell IDO facilitates the intratumoral accumulation of regulatory T cells (Tregs; CD4+CD25+FoxP3+). Although immunosuppressive IDO activity is canonically characterized by the conversion of tryptophan into kynurenine, we have utilized transgenic and syngeneic mouse models and mutant glioma lines to demonstrate that tumor cell IDO increases Treg accumulation independent of tryptophan metabolism. Here, we address the gap in our understanding of IDO signaling activity in vivo. Subcutaneously-engrafted human GBM expressing human IDO-GFP cDNA was isolated from immunodeficient humanized NSG-SGM3 mice. The tumor was immunoprecipitated for the GFP tag using GFP-TRAP followed by mass spectrometry which revealed a novel methylation site on a lysine residue at amino acid 373 in the IDO C-terminus region. Western blot analysis of IDO protein also revealed the presence of tyrosine phosphorylation. Additionally, we recently created a new transgenic IDO reporter mouse model whereby endogenous IDO is fused to GFP via a T2A linker (IDO→GFP). This model allows for the isolation of IDO+ cells in real-time and without causing cell death, thereby creating the opportunity for downstream molecular analysis of in situ-isolated GFP+ cells. Collectively, our work suggests that IDO non-enzyme activity may involve the post-translational modifications we recently identified. As IDO activity may differ between in vitro and in vivo modeling systems, we will use the new IDO→GFP reporter mouse model for an improved mechanistic understanding of how immunosuppressive IDO facilitates Treg accumulation in vivo.

Chd8 haploinsufficiency impairs early brain development and protein homeostasis later in life

BackgroundChromodomain helicase DNA-binding protein 8 (Chd8) is a high-confidence risk gene for autism spectrum disorder (ASD). However, how Chd8 haploinsufficiency impairs gene expression in the brain and impacts behavior at different stages of life is unknown.MethodsWe generated a mutant mouse line with an ASD-linked loss-of-function mutation in Chd8 (V986*; stop codon mutation). We examined the behavior of Chd8 mutant mice along with transcriptional changes in the cerebral cortex as a function of age, with a focus on one embryonic (E14.5) and three postnatal ages (1, 6, and 12 months).ResultsChd8V986*/+ mutant mice displayed macrocephaly, reduced rearing responses and reduced center time in the open field, and enhanced social novelty preference. Behavioral phenotypes were more evident in Chd8V986*/+ mutant mice at 1 year of age. Pup survival was reduced in wild-type x Chd8V986*/+ crosses when the mutant parent was female. Transcriptomic analyses indicated that pathways associated with synaptic and neuronal projections and sodium channel activity were reduced in the cortex of embryonic Chd8V986*/+ mice and then equalized relative to wild-type mice in the postnatal period. At 12 months of age, expression of genes associated with endoplasmic reticulum (ER) stress, chaperone-mediated protein folding, and the unfolded protein response (UPR) were reduced in Chd8V986*/+ mice, whereas genes associated with the c-MET signaling pathway were increased in expression.LimitationsIt is unclear whether the transcriptional changes observed with age in Chd8V986*/+ mice reflect a direct effect of CHD8-regulated gene expression, or if CHD8 indirectly affects the expression of UPR/ER stress genes in adult mice as a consequence of neurodevelopmental abnormalities.ConclusionsCollectively, these data suggest that UPR/ER stress pathways are reduced in the cerebral cortex of aged Chd8V986*/+ mice. Our study uncovers neurodevelopmental and age-related phenotypes in Chd8V986*/+ mice and highlights the importance of controlling for age when studying Chd8 haploinsufficient mice.

Introduction of a de novo Creb-binding protein gene mutation in sperm to produce a Rubinstein-Taybi syndrome model using inbred C57BL/6 mice

Neurodevelopmental disorders, including intellectual disability and autism spectrum disorder, are often caused by de novo autosomal dominant mutations. While mouse models are frequently used to investigate these disorders, the genetic background sometimes affects the appearance or severity of mutant phenotypes. In a previous report, we developed a system to produce de novo heterozygous mutant mice using the Cre-LoxP system without the need to maintain the heterozygous mutant line itself (Takagi et al. 2015). To further verify the applicability of the de novo mutation system in sperm, we used this system to produce a mouse model for Rubinstein-Taybi syndrome, using a Cbp heterozygous mutant, which has been reported to be difficult to maintain on a C57BL/6 background. Here, we show that de novo Cbp- loss-of-function heterozygous mutant mice with a C57BL/6 background, present with a clear craniofacial phenotype and reduced locomotor activity in the open field test, which was not observed in the loss-of-function of Cbp heterozygous mutant line mice with a mixed genetic background, but was observed in the dominant negative Cbp heterozygous mutant line with a mixed genetic background. Meanwhile, the de novo heterozygous Cbp mutant mice still showed great variability in survival rates despite their inbred background. These results further confirmed that the de novo mutation system used in germ cells is effective for stable production and analysis of an autosomal dominant disorder mouse model, which is often difficult to maintain as a mutant mouse line.

Male-biased aganglionic megacolon in the TashT mouse model of Hirschsprung disease involves upregulation of p53 protein activity and Ddx3y gene expression.

Hirschsprung disease (HSCR) is a complex genetic disorder of neural crest development resulting in incomplete formation of the enteric nervous system (ENS). This life-threatening neurocristopathy affects 1/5000 live births, with a currently unexplained male-biased ratio. To address this lack of knowledge, we took advantage of the TashT mutant mouse line, which is the only HSCR model to display a robust male bias. Our prior work revealed that the TashT insertional mutation perturbs a Chr.10 silencer-enriched non-coding region, leading to transcriptional dysregulation of hundreds of genes in neural crest-derived ENS progenitors of both sexes. Here, through sex-stratified transcriptome analyses and targeted overexpression in ENS progenitors, we show that male-biased ENS malformation in TashT embryos is not due to upregulation of Sry-the murine ortholog of a candidate gene for the HSCR male bias in humans-but instead involves upregulation of another Y-linked gene, Ddx3y. This discovery might be clinically relevant since we further found that the DDX3Y protein is also expressed in the ENS of a subset of male HSCR patients. Mechanistically, other data including chromosome conformation captured-based assays and CRISPR/Cas9-mediated deletions suggest that Ddx3y upregulation in male TashT ENS progenitors is due to increased transactivation by p53, which appears especially active in these cells yet without triggering apoptosis. Accordingly, in utero treatment of TashT embryos with the p53 inhibitor pifithrin-α decreased Ddx3y expression and abolished the otherwise more severe ENS defect in TashT males. Our data thus highlight novel pathogenic roles for p53 and DDX3Y during ENS formation in mice, a finding that might help to explain the intriguing male bias of HSCR in humans.

A tailored Cln3Q352X mouse model for testing therapeutic interventions in CLN3 Batten disease

CLN3 Batten disease (CLN3 disease) is a pediatric lysosomal storage disorder that presents with progressive blindness, motor and cognitive decline, seizures, and premature death. CLN3 disease results from mutations in CLN3 with the most prevalent mutation, a 966 bp deletion spanning exons 7–8, affecting ~ 75% of patients. Mouse models with complete Cln3 deletion or Cln3Δex7/8 mutation have been invaluable for learning about both the basic biology of CLN3 and the underlying pathological changes associated with CLN3 disease. These models, however, vary in their disease presentation and are limited in their utility for studying the role of nonsense mediated decay, and as a consequence, in testing nonsense suppression therapies and read-through compounds. In order to develop a model containing a disease-causing nonsense point mutation, here we describe a first-of-its-kind Cln3Q352X mouse model containing a c.1054C > T (p.Gln352Ter) point mutation. Similar to previously characterized Cln3 mutant mouse lines, this novel model shows pathological deficits throughout the CNS including accumulation of lysosomal storage material and glial activation, and has limited perturbation in behavioral measures. Thus, at the molecular and cellular level, this mouse line provides a valuable tool for testing nonsense suppression therapies or read through compounds in CLN3 disease in the future.

A murine model of the exercise pressor reflex.

The decerebrate mouse provides a novel working model of the exercise pressor reflex (EPR). The decerebrate mouse model of the EPR is similar to the previously described decerebrate rat model. Studying the EPR in transgenic mouse models can define exact mechanisms of the EPR in health and disease. The exercise pressor reflex (EPR) is defined by a rise in mean arterial pressure (MAP) and heart rate (HR) in response to exercise and is necessary to match metabolic demand and prevent premature fatigue. While this reflex is readily tested in humans, mechanistic studies are largely infeasible. Here, we have developed a novel murine model of the EPR to allow for mechanistic studies in various mouse models. We observed that ventral root stimulation (VRS) in an anaesthetized mouse causes a depressor response and a reduction in HR. In contrast, the same stimulation in a decerebrate mouse causes a rise in MAP and HR which is abolished by dorsal rhizotomy or by neuromuscular blockade. Moreover, we demonstrate a reduced MAP response to VRS using TRPV1 antagonism or in Trpv1 null mice while the response to passive stretch remains intact. Additionally, we demonstrate that intra-arterial infusion of capsaicin results in a dose-related rise in MAP and HR that is significantly reduced by a selective and potent TRPV1 antagonist or is completely abolished in Trpv1 null mice. These data serve to validate the development of a decerebrate mouse model for the study of cardiovascular responses to exercise and further define the role of the TRPV1 receptor in mediating the EPR. This novel model will allow for extensive study of the EPR in unlimited transgenic and mutant mouse lines, and for an unprecedented exploration of the molecular mechanisms that control cardiovascular responses to exercise in health and disease.

Glucocorticoids limit lipopolysaccharide-induced lethal inflammation by a double control system.

Lipopolysaccharides (LPS) can lead to a lethal endotoxemia, which is a systemic inflammatory response syndrome (SIRS) characterized by a systemic release of cytokines, such as TNF. Endotoxemia is studied intensely, as a model system of Gram-negative infections. LPS- and TNF-induced SIRS involve a strong induction of interferon-stimulated genes (ISGs), some of which cause cell death in the intestinal epithelium cells (IECs). It is well known that glucocorticoids (GCs) protect against endotoxemia. By applying numerous mutant mouse lines, our data support a model whereby GCs, via their glucocorticoid receptor (GR), apply two key mechanisms to control endotoxemia, (i) at the level of suppression of TNF production in a GR monomer-dependent way in macrophages and (ii) at the level of inhibition of TNFR1-induced ISG gene expression and necroptotic cell death mediators in IECs in a GR dimer-dependent way. Our data add new important insights to the understanding of the role of TNF in endotoxemia and the two separate key roles of GCs in suppressing TNF production and activity.

Ribosome binding protein GCN1regulates the cell cycle and cell proliferation and is essential for the embryonic development of mice.

Amino acids exert many biological functions, serving as allosteric regulators and neurotransmitters, as constituents in proteins and as nutrients. GCN2-mediated phosphorylation of eukaryotic initiation factor 2 alpha (elF2α) restores homeostasis in response to amino acid starvation (AAS) through the inhibition of the general translation and upregulation of amino acid biosynthetic enzymes and transporters by activating the translation of Gcn4 and ATF4 in yeast and mammals, respectively. GCN1 is a GCN2-binding protein that possesses an RWD binding domain (RWDBD) in its C-terminus. In yeast, Gcn1 is essential for Gcn2 activation by AAS; however, the roles of GCN1 in mammals need to be established. Here, we revealed a novel role of GCN1 that does not depend on AAS by generating two Gcn1 mutant mouse lines: Gcn1-knockout mice (Gcn1 KO mice (Gcn1-/-)) and RWDBD-deleted mutant mice (Gcn1ΔRWDBD mice). Both mutant mice showed growth retardation, which was not observed in the Gcn2 KO mice, such that the Gcn1 KO mice died at the intermediate stage of embryonic development because of severe growth retardation, while the Gcn1ΔRWDBD embryos showed mild growth retardation and died soon after birth, most likely due to respiratory failure. Extension of pregnancy by 24 h through the administration of progesterone to the pregnant mothers rescued the expression of differentiation markers in the lungs and prevented lethality of the Gcn1ΔRWDBD pups, indicating that perinatal lethality of the Gcn1ΔRWDBD embryos was due to simple growth retardation. Similar to the yeast Gcn2/Gcn1 system, AAS- or UV irradiation-induced elF2α phosphorylation was diminished in the Gcn1ΔRWDBD mouse embryonic fibroblasts (MEFs), suggesting that GCN1 RWDBD is responsible for GCN2 activity. In addition, we found reduced cell proliferation and G2/M arrest accompanying a decrease in Cdk1 and Cyclin B1 in the Gcn1ΔRWDBD MEFs. Our results demonstrated, for the first time, that GCN1 is essential for both GCN2-dependent stress response and GCN2-independent cell cycle regulation.

Loss of mural cell-derived laminin aggravates hemorrhagic brain injury

BackgroundMural cells synthesize and deposit laminin to the basement membrane. To investigate the function of mural cell-derived laminin, we generated a mutant mouse line lacking mural cell-derived laminin (termed PKO). In a previous study, we showed that the PKO mice were grossly normal under homeostatic condition, but developed blood-brain barrier (BBB) breakdown with advanced age (> 8 months), suggesting that these mutants are intrinsically weak. Based on these findings, we hypothesized that PKO mice have exacerbated injuries in pathological conditions.MethodsUsing collagenase-induced intracerebral hemorrhage (ICH) as an injury model, we examined various stroke outcomes, including hematoma volume, neurological function, neuronal death, BBB integrity, paracellular/transcellular transport, inflammatory cell infiltration, and brain water content, in PKO mice and their wildtype littermates at young age (6–8 weeks). In addition, transmission electron microscopy (TEM) analysis and an in vitro ICH model were used to investigate the underlying molecular mechanisms.ResultsCompared to age-matched wildtype littermates, PKO mice display aggravated stroke outcomes, including larger hematoma size, worse neurological function, increased neuronal cell death, enhanced BBB permeability, increased transcytosis, and elevated inflammatory cell infiltration. These mutants also exhibit high baseline brain water content independent of aquaporin-4 (AQP4). In addition, mural cell-derived laminin significantly reduced caveolin-1 without affecting tight junction proteins in the in vitro ICH model.ConclusionsThese results suggest that mural cell-derived laminin attenuates BBB damage in ICH via decreasing caveolin-1 and thus transcytosis, regulates brain water homeostasis, and plays a beneficial role in ICH.

A Murine Model of the Exercise Pressor Reflex

The exercise pressor reflex (EPR) is an involuntary response to exercise. It is defined by a rise in mean arterial pressure (MAP) and heart rate (HR) in response to muscle contraction and is necessary for providing adequate blood flow to the exercising muscle to match metabolic demand and prevent premature fatigue. Proper function of this reflexive response is essential to healthy cardiovascular responses to exercise. Animal models available to test the EPR such as cat, dog, and rabbit have been utilized extensively to gain physiological information across species but generating disease or translating observations to humans has been challenging in these models. While this reflex can be tested rapidly and non‐invasively in humans, an elucidation of the mechanisms that regulate this reflex at the cellular and molecular level limit the utility of such studies. To test the EPR in the setting of heart failure, we previously developed a reliable and reproducible decerebrate rat model that allowed us to study the EPR in cardiomyopathic animals. We observed that this model yielded similar responses to exercise to those observed in humans. Here, we have developed a novel murine model of the EPR to allow for unlimited exploration of physiological and pathological cellular and molecular mechanisms in a variety of transgenic and genetically modified mouse lines. Like the rat model, we observed that ventral root stimulation (VRS; to evoke contraction of skeletal muscle) in an anesthetized mouse causes a depressor response and a reduction in HR. In contrast, the same stimulation in a decerebrate mouse causes a rise in MAP and HR. The responses observed in the decerebrate murine preparation are abolished by disruption of afferent innervation of the muscle via dorsal rhizotomy or by neuromuscular blockade. Moreover, we demonstrate that TRPV1 antagonism in wild type (WT) mice and Trpv1 null mice display a reduced MAP response to VRS while the response to mechanosensitive stimulation (passive stretch) remains intact. Additionally, we demonstrate that intra‐arterial infusion of capsaicin (a TRPV1 agonist) results in a dose related rise in MAP and HR that is significantly reduced by a selective and potent TRPV1 antagonist and, further, that these responses are completely abolished in Trpv1 null mice. Lastly, we observe no role of the Piezo2 receptor in mediating the EPR under physiological conditions. These data serve to validate the development of a decerebrate mouse model for the study of cardiovascular responses to exercise and further define the role of the TRPV1 receptor in mediating the EPR. This novel model will allow for extensive study of the EPR in unlimited transgenic and mutant mouse lines, thus allowing for an unprecedented exploration of the molecular mechanisms that control cardiovascular responses to exercise in health and disease.

A Mouse Model for Cerebro‐Costo‐Mandibular Syndrome (CCMS) with an Intronic Deletion in SnrpB

SNRPB, an essential effector of the spliceosomal complex, is implicated in the splicing of mature mRNA and is mutated in a rare craniofacial disorder known as Cerebro‐costo‐mandibular syndrome (CCMS). Most CCMS patients carry point mutations in the highly conserved alternative exon 2 (AE2) of SNRPB, leading to increased inclusion of a pre‐termination codon (PTC) and reduced levels of the protein‐coding transcript. Given that SNRPB is implicated in all steps of splicing, it was thought that its requirement would be equivalent in all cells and tissues. However, CCMS patients predominantly present with micrognathia and rib defects with variable penetrance. We hypothesize that a subset of transcripts is highly sensitive to the levels of SNRPB during development and is tissue specific. Here, we are generating a mouse model for CCMS to uncover those transcripts that play an essential role in craniofacial development. Using the CRISPR/Cas9 gene‐editing system, we developed a mutant mouse line with a 61 base‐pair (bp) intronic deletion upstream of the AE2. Interestingly, a subset of the heterozygous and homozygous embryos and pups for this deletion show morphological abnormalities in the face, head, ribs, and limbs. Moreover, a significant percentage of the mutants with the 61‐bp deletion die in their first year of life. A subgroup of the heterozygous and homozygous mutant embryos show increased inclusion of the PTC containing AE2 and reduced SNRPB protein levels. We presume that this predominant inclusion of the AE2 induces non‐sense mediated decay resulting in decreased levels of SNRPB. As our data suggest that the 61 bp intronic sequence plays a critical role in regulating SNRPB levels in mice, we are currently investigating if similar intronic sequences in intron 2 of human SNRPB are involved in the preferential splicing of AE2. In addition, we generated a SnrpB conditional knockout mouse model, which will be used in parallel with the deletion model to investigate the cell‐type‐specific roles of SNRPB during craniofacial development. Our novel model for CCMS will further our understanding of the role of SNRPB in craniofacial development.Support or Funding InformationCanadian Institutes of Health Research (CIHR)

Evidence for significance of serine 1487 in β-adrenergic regulation of Cav1.2 channel protein function in genetically engineered mice

Stress responses are mediated through activation of the β-adrenergic signaling pathway. Chronic sympathetic stimulation can result in the development of hypertrophy, arrhythmia and sudden cardiac death but the molecular mechanisms are still not understood. We identified a single serine, following phosphorylation by cAMP-dependent protein kinase A, responsible for the increase in function of the human cardiac Cav1.2 channel protein in an artificial cell system. To confirm whether the serine is involved in in vivo channel stimulation by β-adrenergic signaling, we created CRISPR/Cas9 mutant mouse lines with glutamate and alanine-substitution of the serine1487 on Cav1.2 protein to mimic the phosphorylated and the non-phosphorylated protein. The average litter size (6.7 ± 0.7; N = 16 and 6.1 ± 0.4; N = 24 respectively) and survival rate (48.6% vs. 42.5%) were similar in the two lines, but homozygous mutation of Cav1.2 S1487E caused 100% mortality with a median survival of ~1 day. The relative size of the hearts of homozygous S1487A mice were smaller than S1487E (heart weight/tibial length: 7.0 ± 0.1; N = 3 vs 7.8 ± 0.3; N = 3). Administration of intraperitoneal injection of 20 mg/kg isoproterenol increased the heart rate up to 30% in both glutamate and alanine-substituted heterozygous mutants in a similar manner to the negatively genotyped littermates, but did not alter the heart rate of the homozygous S1487A mutants. Chronic isoproterenol treatment (20 mg/kg isoproterenol daily injection for 2 weeks) induced hypertrophy in the wild type and heterozygous S1497A mutant mice, but there were no significant changes in the posterior wall thickness of the heart of homozygous S1487A mutants.

IL-7R-Dependent Phosphatidylinositol 3-Kinase Competes with the STAT5 Signal to Modulate T Cell Development and Homeostasis.

T cell development and homeostasis requires IL-7R α-chain (IL-7Rα) signaling. Tyrosine Y449 of the IL-7Rα is essential to activate STAT5 and PI3K, whereas PI3K recruitment requires IL-7Rα methionine M452. How IL-7Rα activates and regulates both signaling pathways differentially remains unclear. To characterize differential signaling, we established two lines of IL-7Rα mutant mice: IL-7R-Y449F mice and IL-7R-M452L mice. IL-7R-Y449F mice showed decreased PI3K and STAT5 signals, whereas IL-7R-M452L mice showed decreased PI3K but significantly increased STAT5 signaling, owing to a competition between PI3K and STAT5 signaling through Y449 of IL-7Rα. The number of T, B, and mature innate lymphoid cells were markedly reduced in IL-7R-Y449F mice, whereas IL-7R-M452L mice showed impaired early T cell development and memory precursor effector T cell maintenance with the downregulation of transcription factor T cell factor-1. Peripheral T cell numbers increased in IL-7R-M452L mice with enhanced survival and homeostatic proliferation. Furthermore, although wild type and IL-7R-Y449F mice showed comparable Th1/Th2 differentiation, IL-7R-M452L mice exhibited impaired Th17 differentiation. We conclude that PI3K competes with STAT5 under IL-7Rα and maintains an appropriate signal balance for modulating T cell development and homeostasis. To our knowledge, this study provides a new insight into complex regulation of IL-7Rα signaling, which supports immune development and responses.

High-throughput discovery of genetic determinants of circadian misalignment.

Circadian systems provide a fitness advantage to organisms by allowing them to adapt to daily changes of environmental cues, such as light/dark cycles. The molecular mechanism underlying the circadian clock has been well characterized. However, how internal circadian clocks are entrained with regular daily light/dark cycles remains unclear. By collecting and analyzing indirect calorimetry (IC) data from more than 2000 wild-type mice available from the International Mouse Phenotyping Consortium (IMPC), we show that the onset time and peak phase of activity and food intake rhythms are reliable parameters for screening defects of circadian misalignment. We developed a machine learning algorithm to quantify these two parameters in our misalignment screen (SyncScreener) with existing datasets and used it to screen 750 mutant mouse lines from five IMPC phenotyping centres. Mutants of five genes (Slc7a11, Rhbdl1, Spop, Ctc1 and Oxtr) were found to be associated with altered patterns of activity or food intake. By further studying the Slc7a11tm1a/tm1a mice, we confirmed its advanced activity phase phenotype in response to a simulated jetlag and skeleton photoperiod stimuli. Disruption of Slc7a11 affected the intercellular communication in the suprachiasmatic nucleus, suggesting a defect in synchronization of clock neurons. Our study has established a systematic phenotype analysis approach that can be used to uncover the mechanism of circadian entrainment in mice.

Loss of Chondroitin Sulfate Modification Causes Inflammation and Neurodegeneration in skt Mice.

One major aspect of the aging process is the onset of chronic, low-grade inflammation that is highly associated with age-related diseases. The molecular mechanisms that regulate these processes have not been fully elucidated. We have identified a spontaneous mutant mouse line, small with kinky tail (skt), that exhibits accelerated aging and age-related disease phenotypes including increased inflammation in the brain and retina, enhanced age-dependent retinal abnormalities including photoreceptor cell degeneration, neurodegeneration in the hippocampus, and reduced lifespan. By positional cloning, we identified a deletion in chondroitin sulfate synthase 1 (Chsy1) that is responsible for these phenotypes in skt mice. CHSY1 is a member of the chondroitin N-acetylgalactosaminyltransferase family that plays critical roles in the biosynthesis of chondroitin sulfate, a glycosaminoglycan (GAG) that is attached to the core protein to form the chondroitin sulfate proteoglycan (CSPG). Consistent with this function, the Chsy1 mutation dramatically decreases chondroitin sulfate GAGs in the retina and hippocampus. In addition, macrophage and neutrophil populations appear significantly altered in the bone marrow and spleen of skt mice, suggesting an important role for CHSY1 in the functioning of these immune cell types. Thus, our study reveals a previously unidentified impact of CHSY1 in the retina and hippocampus. Specifically, chondroitin sulfate (CS) modification of proteins by CHSY1 appears critical for proper regulation of immune cells of the myeloid lineage and for maintaining the integrity of neuronal tissues, since a defect in this gene results in increased inflammation and abnormal phenotypes associated with age-related diseases.

A Common Outer Retinal Change in Retinal Degeneration by Optical Coherence Tomography Can Be Used to Assess Outcomes of Gene Therapy.

Identifying early disease hallmarks in animal models with slow disease progression may expedite disease detection and assessment of treatment outcomes. Using optical coherence tomography, a widely applied noninvasive method for monitoring retinal structure changes, we analyzed retinal optical sections from six mouse lines with retinal degeneration caused by mutations in different disease-causing genes. While images from wild-type mice revealed four well-separated hyper-reflective bands in the outer retina (designated as outer retina reflective bands, ORRBs) at all ages, the second band (ORRB2) and the third band (ORRB3) were merged in retinas of five mutant mouse lines at early ages, suggesting the pathological nature of this alteration. This ORRB change appeared to be degenerating photoreceptor related, and occurred before obvious morphological changes that can be identified on both hematoxylin and eosin-stained sections and electron microscopic sections. Importantly, the merging of ORRB2 and ORRB3 was reversed by treatment with adeno-associated viral vector-mediated gene replacement therapies, and this restoration occurred much earlier than measurable functional or structural improvement. Our data suggest that the ORRB change could be a common hallmark of early retinal degeneration and its restoration could be used for rapid and noninvasive assessment of therapeutic effects following gene therapy or other treatment interventions.

A subpopulation of Bdnf-e1–expressing glutamatergic neurons in the lateral hypothalamus critical for thermogenesis control

ObjectiveBrown adipose tissue (BAT)–mediated thermogenesis plays a key role in energy homeostasis and the maintenance of body temperature. Previous work suggests that brain-derived neurotrophic factor (BDNF) is involved in BAT thermogenesis, but the underlying neural circuits and molecular mechanism remain largely unknown. This is in part due to the difficulties in manipulating BDNF expression in different brain regions through different promoters and the lack of tools to identify neurons in the brain specifically involved in BAT thermogenesis. MethodsWe have created several lines of mutant mice in which BDNF transcription from a specific promoter was selectively disrupted by replacing Bdnf with green fluorescent protein (GFP; Bdnf-e1, -e4, and -e6−/− mice). As such, cells expressing Bdnf-e1, -e4, or -e6 were labeled with GFP. To identify BAT-connected thermogenesis neurons in brain, we applied the retrograde pseudorabies virus labeling method from BAT. We also used chemogenetic tools to manipulate specific neurons coupled with BAT temperature recording. Moreover, we developed a new TrkB agonist antibody to rescue the BAT thermogenesis deficits. ResultsWe show that selective disruption of Bdnf expression from promoter 1 (Bdnf-e1) resulted in severe obesity and deficits of BAT-mediated thermogenesis. Body temperature response to cold was impaired in Bdnf-e1−/− mice. BAT expression of Ucp1 and Pcg1a, genes known to regulate thermogenesis, was also reduced, accompanying a decrease in the sympathetic activity of BAT. Staining of cells expressing Bdnf-e1 transcript, combined with transsynaptic, retrograde-tracing labeling of BAT-connected neurons, identified a group of excitatory neurons in lateral hypothalamus (LH) critical for thermogenesis regulation. Moreover, an adaptive thermogenesis defect in Bdnf-e1−/− mice was rescued by injecting an agonistic antibody for TrkB, the BDNF receptor, into LH. Remarkably, activation of the excitatory neurons (VGLUT2+) in LH through chemogenetic tools resulted in a rise of BAT temperature. ConclusionsThese results reveal a specific role of BDNF promoter I in thermogenesis regulation and define a small subset of neurons in LH that contribute to such regulation.

Beyond detoxification: Pleiotropic functions of multiple glutathione S-transferase isoforms protect mice against a toxic electrophile.

Environmental and endogenous electrophiles cause tissue damage through their high reactivity with endogenous nucleophiles such as DNA, proteins, and lipids. Protection against damage is mediated by glutathione (GSH) conjugation, which can occur spontaneously or be facilitated by the glutathione S-transferase (GST) enzymes. To determine the role of GST enzymes in protection against electrophiles as well as the role of specific GST families in mediating this protection, we exposed mutant mouse lines lacking the GSTP, GSTM, and/or GSTT enzyme families to the model electrophile acrylamide, a ubiquitous dietary contaminant known to cause adverse effects in humans. An analysis of urinary metabolites after acute acrylamide exposure identified the GSTM family as the primary mediator of GSH conjugation to acrylamide. However, surprisingly, mice lacking only this enzyme family did not show increased toxicity after an acute acrylamide exposure. Therefore, GSH conjugation is not the sole mechanism by which GSTs protect against the toxicity of this substrate. Given the prevalence of null GST polymorphisms in the human population (approximately 50% for GSTM1 and 20–50% for GSTT1), a substantial portion of the population may also have impaired acrylamide metabolism. However, our study also defines a role for GSTP and/or GSTT in protection against acrylamide mediated toxicity. Thus, while the canonical detoxification function of GSTs may be impaired in GSTM null individuals, disease risk secondary to acrylamide exposure may be mitigated through non-canonical pathways involving members of the GSTP and/or GSTT families.