Mutant Mouse Models Research Articles

Effect of oxidative stress on telomere maintenance in aortic smooth muscle cells

Reactive oxygen species (ROS) and telomere dysfunction are both associated with aging and the development of age-related diseases. Although there is evidence for a direct relationship between ROS and telomere dysfunction as well as an independent association of oxidative stress and telomere attrition with age-related disorders, there has not been sufficient exploration of how the interaction between oxidative stress and telomere function may contribute to the pathophysiology of cardiovascular diseases (CVD). To better understand the complex relationships between oxidative stress, telomerase biology and pathophysiology, we examined the telomere biology of aortic smooth muscle cells (ASMCs) isolated from mutant mouse models of oxidative stress. We discovered that telomere lengths were significantly shorter in ASMCs isolated from superoxide dismutase 2 heterozygous (Sod2+/−) mice, which exhibit increased arterial stiffness with aging, and the observed telomere attrition occurred over time. Furthermore, the telomere erosion occurred even though telomerase activity increased. In contrast, telomeres remained stable in wild-type and superoxide dismutase 1 heterozygous (Sod1+/−) mice, which do not exhibit CVD phenotypes. The data indicate that mitochondrial oxidative stress, in particular elevated superoxide levels and decreased hydrogen peroxide levels, induces telomere erosion in the ASMCs of the Sod2+/− mice. This reduction in telomere length occurs despite an increase in telomerase activity and correlates with the onset of disease phenotype. Our results suggest that the oxidative stress caused by imbalance in mitochondrial ROS, from deficient SOD2 activity as a model for mitochondrial dysfunction results in telomere dysfunction, which may contribute to pathogenesis of CVD.

Cav3 T-Type Voltage-Gated Ca2+ Channels and the Amyloidogenic Environment: Pathophysiology and Implications on Pharmacotherapy and Pharmacovigilance.

Voltage-gated Ca2+ channels (VGCCs) were reported to play a crucial role in neurotransmitter release, dendritic resonance phenomena and integration, and the regulation of gene expression. In the septohippocampal system, high- and low-voltage-activated (HVA, LVA) Ca2+ channels were shown to be involved in theta genesis, learning, and memory processes. In particular, HVA Cav2.3 R-type and LVA Cav3 T-type Ca2+ channels are expressed in the medial septum-diagonal band of Broca (MS-DBB), hippocampal interneurons, and pyramidal cells, and ablation of both channels was proven to severely modulate theta activity. Importantly, Cav3 Ca2+ channels contribute to rebound burst firing in septal interneurons. Consequently, functional impairment of T-type Ca2+ channels, e.g., in null mutant mouse models, caused tonic disinhibition of the septohippocampal pathway and subsequent enhancement of hippocampal theta activity. In addition, impairment of GABA A/B receptor transcription, trafficking, and membrane translocation was observed within the septohippocampal system. Given the recent findings that amyloid precursor protein (APP) forms complexes with GABA B receptors (GBRs), it is hypothesized that T-type Ca2+ current reduction, decrease in GABA receptors, and APP destabilization generate complex functional interdependence that can constitute a sophisticated proamyloidogenic environment, which could be of potential relevance in the etiopathogenesis of Alzheimer’s disease (AD). The age-related downregulation of T-type Ca2+ channels in humans goes together with increased Aβ levels that could further inhibit T-type channels and aggravate the proamyloidogenic environment. The mechanistic model presented here sheds new light on recent reports about the potential risks of T-type Ca2+ channel blockers (CCBs) in dementia, as observed upon antiepileptic drug application in the elderly.

S100A9-CXCL12 activation in BRCA1-mutant breast cancer promotes an immunosuppressive microenvironment associated with resistance to immunotherapy

Immune checkpoint blockade (ICB) is a powerful approach for cancer therapy although good responses are only observed in a fraction of cancer patients. Breast cancers caused by deficiency of breast cancer-associated gene 1 (BRCA1) do not have an improved response to the treatment. To investigate this, here we analyze BRCA1 mutant mammary tissues and tumors derived from both BRCA1 mutant mouse models and human xenograft models to identify intrinsic determinants governing tumor progression and ICB responses. We show that BRCA1 deficiency activates S100A9-CXCL12 signaling for cancer progression and triggers the expansion and accumulation of myeloid-derived suppressor cells (MDSCs), creating a tumor-permissive microenvironment and rendering cancers insensitive to ICB. These oncogenic actions can be effectively suppressed by the combinatory treatment of inhibitors for S100A9-CXCL12 signaling with αPD-1 antibody. This study provides a selective strategy for effective immunotherapy in patients with elevated S100A9 and/or CXCL12 protein levels.

Deep phenotyping reveals movement phenotypes in mouse neurodevelopmental models

BackgroundRepetitive action, resistance to environmental change and fine motor disruptions are hallmarks of autism spectrum disorder (ASD) and other neurodevelopmental disorders, and vary considerably from individual to individual. In animal models, conventional behavioral phenotyping captures such fine-scale variations incompletely. Here we observed male and female C57BL/6J mice to methodically catalog adaptive movement over multiple days and examined two rodent models of developmental disorders against this dynamic baseline. We then investigated the behavioral consequences of a cerebellum-specific deletion in Tsc1 protein and a whole-brain knockout in Cntnap2 protein in mice. Both of these mutations are found in clinical conditions and have been associated with ASD.MethodsWe used advances in computer vision and deep learning, namely a generalized form of high-dimensional statistical analysis, to develop a framework for characterizing mouse movement on multiple timescales using a single popular behavioral assay, the open-field test. The pipeline takes virtual markers from pose estimation to find behavior clusters and generate wavelet signatures of behavior classes. We measured spatial and temporal habituation to a new environment across minutes and days, different types of self-grooming, locomotion and gait.ResultsBoth Cntnap2 knockouts and L7-Tsc1 mutants showed forelimb lag during gait. L7-Tsc1 mutants and Cntnap2 knockouts showed complex defects in multi-day adaptation, lacking the tendency of wild-type mice to spend progressively more time in corners of the arena. In L7-Tsc1 mutant mice, failure to adapt took the form of maintained ambling, turning and locomotion, and an overall decrease in grooming. However, adaptation in these traits was similar between wild-type mice and Cntnap2 knockouts. L7-Tsc1 mutant and Cntnap2 knockout mouse models showed different patterns of behavioral state occupancy.LimitationsGenetic risk factors for autism are numerous, and we tested only two. Our pipeline was only done under conditions of free behavior. Testing under task or social conditions would reveal more information about behavioral dynamics and variability.ConclusionsOur automated pipeline for deep phenotyping successfully captures model-specific deviations in adaptation and movement as well as differences in the detailed structure of behavioral dynamics. The reported deficits indicate that deep phenotyping constitutes a robust set of ASD symptoms that may be considered for implementation in clinical settings as quantitative diagnosis criteria.

Pregnancy Complications and Neonatal Mortality in a Serotonin Transporter Null Mouse Model: Insight Into the Use of Selective Serotonin Reuptake Inhibitor During Pregnancy.

Selective serotonin reuptake inhibitors (SSRI) are widely prescribed to pregnant woman. Although some SSRI compounds are known to cause pregnancy loss and fetal malformations, other SSRI continue to be used by pregnant women. However, several studies have associated the use of SSRI with adverse pregnancy outcomes: intrauterine growth restriction, preterm birth, and neonatal morbidity. Nonetheless, interpretation of studies in humans are typically complicated by the adverse pregnancy outcomes caused by depression itself. Therefore, we used a mutant mouse model with genetic ablation of the serotonin transporter, the target site for SSRI, to unravel the role of the serotonin transporter on pregnancy outcomes. The serotonin transporter null mice had increased pregnancy loss (17.5 vs. 0%), decreased number of pups born (6.6 ± 0.2 vs. 7.5 ± 0.2), and increased neonatal mortality (2.3-fold). Furthermore, preterm birth, dystocia, and fetal malformations were only observed in serotonin transporter null mice. This genetically ablated serotonin transporter mouse recapitulates several adverse pregnancy outcomes similar to those in women undergoing SSRI treatment during gestation. Additionally, neonatal loss in the present study reproduced a sudden infant death phenotype as in humans and mice with altered serotonergic signaling. In conclusion, findings from this study demonstrate a role for serotonin transporter in pregnancy maintenance and neonatal health. Additionally, it suggests that the adverse pregnancy outcomes in women taking SSRI during gestation might be due to altered serotonin transporter function caused by SSRI independent of underlying depression. This is a critical finding, given the number of women prescribed SSRI during pregnancy, and provides the framework for critical research in this area.

Tick Intrastadial Feeding and Its Role on IgE Production in the Murine Model of Alpha-gal Syndrome: The Tick "Transmission" Hypothesis.

Recent studies have provided strong evidence indicating that lone star tick bites are a cause of AGS (alpha-gal syndrome, also known as red meat allergy RMA) in humans. AGS is characterized by an increase in IgE antibody production against galactose-alpha-1,3-galactose (aGal), which is a common glycan found in mammalian tissue, except in Old World monkeys and humans. The main causative factor of AGS, the lone star tick (Amblyomma americanum), is broadly distributed throughout the east and midwest of the United States and is a vector of a wide range of human and animal pathogens. Our earlier glycomics study of the salivary glands of partially fed male and female ticks revealed relatively high levels of aGal epitopes. In this study, we found that partially fed males of A. americanum on bovine blood, which engage in multiple intrastadial feedings, carry a large amount of aGal in the salivary glands. In our current work, we aimed to test whether ticks mediate the transmission of the aGal sensitizer acquired from nonhuman blood to humans in the intrastadial host switch (referred to as the “transmission” hypothesis). To test this hypothesis, we used an alpha-galactosyltransferase knockout mutant mouse (aGT-KO) model system infested with ticks that were unfed or partially fed on bovine blood. Based on the levels of total IgE and specific IgG and IgE antibodies against aGal after tick feedings, aGT-KO mice significantly responded to tick feeding and injection of aGal (Galα1-3Galβ1-4GlcNAc) conjugated to human serum albumin or mouse serum albumin (aGal-HSA or aGal-MSA) by increasing total IgE and aGal-specific IgE levels compared to those in C57BL/6 control mice. All of the treatments of aGT-KO mice involving the feeding of partially fed and unfed ticks functioned as sensitizers that increased the levels of specific IgE against aGal, with large individual variations. The data in this study do not support the “transmission” component of AGS, although they confirmed that aGT-KO mice can be used as a model for RMA studies.

Dendritic cell functions in vivo: A user's guide to current and next- generation mutant mouse models.

DCs do not just excel in antigen presentation. They orchestrate information transfer from innate to adaptive immunity by sensing and integrating a variety of danger signals, and translating them to naïve T cells, to mount specifically tailored immune responses. This is accomplished by distinct DC types specialized in different functions and because each DC is functionally plastic, assuming different activation states depending on the input signals received. Mouse models hold the key to untangle this complexity and determine which DC types and activation states contribute to which functions. Here, we aim to provide comprehensive information for selecting the most appropriate mutant mouse strains to address specific research questions on DCs, considering three in vivo experimental approaches: (i) interrogating the roles of DC types through their depletion; (ii) determining the underlying mechanisms by specific genetic manipulations; (iii) deciphering the spatiotemporal dynamics of DC responses. We summarize the advantages, caveats, suggested use, and perspectives for a variety of mutant mouse strains, discussing in more detail the most widely used or accurate models. Finally, we discuss innovative strategies to improve targeting specificity and next-generation mutant mouse models, and briefly address how humanized mouse models can accelerate translation into the clinic.

Molecular basis of force-pCa relation in MYL2 cardiomyopathy mice: Role of the super-relaxed state of myosin

In this study, we investigated the role of the super-relaxed (SRX) state of myosin in the structure-function relationship of sarcomeres in the hearts of mouse models of cardiomyopathy-bearing mutations in the human ventricular regulatory light chain (RLC, MYL2 gene). Skinned papillary muscles from hypertrophic (HCM-D166V) and dilated (DCM-D94A) cardiomyopathy models were subjected to small-angle X-ray diffraction simultaneously with isometric force measurements to obtain the interfilament lattice spacing and equatorial intensity ratios (I11/I10) together with the force-pCa relationship over a full range of [Ca2+] and at a sarcomere length of 2.1 μm. In parallel, we studied the effect of mutations on the ATP-dependent myosin energetic states. Compared with wild-type (WT) and DCM-D94A mice, HCM-D166V significantly increased the Ca2+ sensitivity of force and left shifted the I11/I10-pCa relationship, indicating an apparent movement of HCM-D166V cross-bridges closer to actin-containing thin filaments, thereby allowing for their premature Ca2+ activation. The HCM-D166V model also disrupted the SRX state and promoted an SRX-to-DRX (super-relaxed to disordered relaxed) transition that correlated with an HCM-linked phenotype of hypercontractility. While this dysregulation of SRX ↔ DRX equilibrium was consistent with repositioning of myosin motors closer to the thin filaments and with increased force-pCa dependence for HCM-D166V, the DCM-D94A model favored the energy-conserving SRX state, but the structure/function-pCa data were similar to WT. Our results suggest that the mutation-induced redistribution of myosin energetic states is one of the key mechanisms contributing to the development of complex clinical phenotypes associated with human HCM-D166V and DCM-D94A mutations.

Matrix Metalloproteinase 7 Expression and Apical Epithelial Defects in Atp8b1 Mutant Mouse Model of Pulmonary Fibrosis.

Abnormalities in airway epithelia and lung parenchyma are found in Atp8b1 mutant mice, which develop pulmonary fibrosis after hyperoxic insult. Microarray and ingenuity pathway analysis (IPA) show numerous transcripts involved in ciliogenesis are downregulated in 14-month (14 M) -old Atp8b1 mouse lung compared with wild-type C57BL/6. Lung epithelium of Atp8b1 mice demonstrate apical abnormalities of ciliated and club cells in the bronchial epithelium on transmission electron microscopy (TEM). Matrix metalloproteinase 7 (MMP7) regulates of ciliogenesis and is a biomarker for idiopathic pulmonary fibrosis (IPF) in humans. Mmp7 transcript and protein expression are significantly upregulated in 14 M Atp8b1 mutant mouse lung. MMP7 expression is also increased in bronchoalveolar lavage fluid (BAL). Immunohistochemistry is localized MMP7 to bronchial epithelial cells in the Atp8b1 mutant. In conclusion, MMP7 is upregulated in the aged Atp8b1 mouse model, which displays abnormal ciliated cell and club cell morphology. This mouse model can facilitate the exploration of the role of MMP7 in epithelial integrity and ciliogenesis in IPF. The Atp8b1 mutant mouse is proposed as a model for IPF.

Primitive Erythropoiesis in the Mouse is Independent of DOT1L Methyltransferase Activity.

DOT1-like (DOT1L) histone methyltransferase is essential for mammalian erythropoiesis. Loss of DOT1L in knockout (Dot1l-KO) mouse embryos resulted in lethal anemia at midgestational age. The only recognized molecular function of DOT1L is its methylation of histone H3 lysine 79 (H3K79). We generated a Dot1l methyltransferase mutant (Dot1l-MM) mouse model to determine the role of DOT1L methyltransferase activity in early embryonic hematopoiesis. Dot1l-MM embryos failed to survive beyond embryonic day 13.5 (E13.5), similarly to Dot1l-KO mice. However, when examined at E10.5, Dot1l-MM embryos did not exhibit overt anemia like the Dot1l-KO. Vascularity and the presence of red blood cells in the Dot1l-MM yolk sacs as well as in the AGM region of Dot1l-MM embryos appeared to be similar to that of wildtype. In ex vivo cultures of yolk sac cells, Dot1l-MM primitive erythroblasts formed colonies comparable to those of the wildtype. Although ex vivo cultures of Dot1l-MM definitive erythroblasts formed relatively smaller colonies, inhibition of DOT1L methyltransferase activity in vivo by administration of EPZ-5676 minimally affected the erythropoiesis. Our results indicate that early embryonic erythropoiesis in mammals requires a DOT1L function that is independent of its intrinsic methyltransferase activity.

Loss-of-function mutation in Pcsk1 increases serum APOA1 level and LCAT activity in mice

BackgroundThe convertase subtilisin/kexin family 1 gene (PCSK1) has been associated in various human genetics studies with a wide spectrum of metabolic phenotypes, including early-onset obesity, hyperphagia, diabetes insipidus, and others. Despite the evident influence of PCSK1 on obesity and the known functions of other PCSKs in lipid metabolism, the role of PCSK1 specifically in lipid and cholesterol metabolism remains unclear. This study evaluated the effect of loss of PCSK1 function on high-density lipoprotein (HDL) metabolism in mice.ResultsHDL cholesterol, apolipoprotein A1 (APOA1) levels in serum and liver, and the activities of two enzymes (lecithin-cholesterol acyltransferase, LCAT and phospholipid transfer protein, PLTP) were evaluated in 8-week-old mice with a non-synonymous single nucleotide mutation leading to an amino acid substitution in PCSK1, which results in a loss of protein’s function. Mutant mice had similar serum HDL cholesterol concentration but increased levels of serum total and mature APOA1, and LCAT activity in comparison to controls.ConclusionsThis study presents the first evaluation of the role of PCSK1 in HDL metabolism using a loss-of-function mutant mouse model. Further investigations will be needed to determine the underlying molecular mechanism.

Increased RyR2 open probability induces neuronal hyperactivity and memory loss with or without Alzheimer's disease-causing gene mutations.

Neuronal hyperactivity is an early neuronal defect commonly observed in familial and sporadic Alzheimer's disease (AD), but the underlying mechanisms are unclear. We employed a ryanodine receptor 2 (RyR2) mutant mouse model harboring the R4496C+/- mutation that markedly increases the channel's open probability (Po) to determine the impact of increased RyR2 activity in neuronal function without AD gene mutations. Genetically increasing RyR2 Po induced neuronal hyperactivity in vivo in anesthetized and awake mice. Increased RyR2 Po induced hyperactive behaviors, impaired learning and memory, defective dendritic spines, and neuronal cell death. Increased RyR2 Po exacerbated the onset of neuronal hyperexcitability and learning and memory impairments in 5xFAD mice. Increased RyR2 Po exacerbates the onset of familial AD-associated neuronal dysfunction, and induces AD-like defects in the absence of AD-causing gene mutations, suggesting that RyR2-associated neuronal hyperactivity represents a common target for combating AD with or without AD gene mutations.

The Two β-Arrestins Regulate Distinct Metabolic Processes: Studies with Novel Mutant Mouse Models.

The two β-arrestins (β-arrestin-1 and -2; alternative names: arrestin-2 and -3, respectively) are well known for their ability to inhibit signaling via G protein-coupled receptors. However, β-arrestins can also act as signaling molecules in their own right. Although the two proteins share a high degree of sequence and structural homology, early studies with cultured cells indicated that β-arrestin-1 and -2 are not functionally redundant. Recently, the in vivo metabolic roles of the two β-arrestins have been studied using mutant mice selectively lacking either β-arrestin-1 or -2 in cell types that are of particular relevance for regulating glucose and energy homeostasis. These studies demonstrated that the β-arrestin-1 and -2 mutant mice displayed distinct metabolic phenotypes in vivo, providing further evidence for the functional heterogeneity of these two highly versatile signaling proteins.

2008 – INTEGRATIVE SINGLE-CELL ANALYSIS OF PRELEUKAEMIC MUTANT MOUSE MODELS ILLUSTRATES MUTATION-SPECIFIC HAEMATOPOIETIC PERTURBATIONS

Acute myeloid leukaemia can develop from antecedent conditions such as myeloproliferative neoplasms as well as clonal haematopoiesis through stepwise acquisition of multiple mutations. However, the perturbing effects of individual mutations on the entire haematopoietic system remain elusive. To better characterise the mutation-specific haematopoietic disruption, we conducted single-cell RNA sequencing of eight different mutant mouse models with the following mutations: Flt3 internal tandem duplication (n=3), Npm1c (n=2), Idh1 R132H (n=3), Dnmt3a R882H (n=2), Ezh2 knock-out (n=2), Utx knock-out (n=2), Jak2 V617F (n=2) and Calr 52-bp deletion (n=2). Compared with the wild-type samples, each mutant model showed significant accumulation of distinct subsets of progenitors: granulomonocytic progenitors in the Flt3 mutant, stem/immature cells in the Idh1 mutant, lymphoid and erythroid progenitors in the Ezh2 knock-out, granulocyte progenitors and megakaryocytes in the Utx mutant, late erythroid progenitors in the Jak2 mutant, and stem cells and megakaryocytes in the Calr mutant. Npm1 and Dnmt3a mutants showed smaller effects on the global cell type distribution. Transcriptome-based inference of cellular metabolism further revealed that energy generating glycolysis and TCA-cycle reactions were differentially associated with the cell type abundance, where Jak2 and Calr mutations activated these metabolic processes in the accumulated stages of progenitors, while Idh1, Ezh2 and Utx mutations downregulated these energy generation processes in the abundant cell types. I will present an integrated analysis of eight preleukemic mouse models, and the different methods which I have utilised to investigate differential abundance of cell populations, metabolic analysis, differential gene expression and differentiation biases of the progenitor cell populations. Acute myeloid leukaemia can develop from antecedent conditions such as myeloproliferative neoplasms as well as clonal haematopoiesis through stepwise acquisition of multiple mutations. However, the perturbing effects of individual mutations on the entire haematopoietic system remain elusive. To better characterise the mutation-specific haematopoietic disruption, we conducted single-cell RNA sequencing of eight different mutant mouse models with the following mutations: Flt3 internal tandem duplication (n=3), Npm1c (n=2), Idh1 R132H (n=3), Dnmt3a R882H (n=2), Ezh2 knock-out (n=2), Utx knock-out (n=2), Jak2 V617F (n=2) and Calr 52-bp deletion (n=2). Compared with the wild-type samples, each mutant model showed significant accumulation of distinct subsets of progenitors: granulomonocytic progenitors in the Flt3 mutant, stem/immature cells in the Idh1 mutant, lymphoid and erythroid progenitors in the Ezh2 knock-out, granulocyte progenitors and megakaryocytes in the Utx mutant, late erythroid progenitors in the Jak2 mutant, and stem cells and megakaryocytes in the Calr mutant. Npm1 and Dnmt3a mutants showed smaller effects on the global cell type distribution. Transcriptome-based inference of cellular metabolism further revealed that energy generating glycolysis and TCA-cycle reactions were differentially associated with the cell type abundance, where Jak2 and Calr mutations activated these metabolic processes in the accumulated stages of progenitors, while Idh1, Ezh2 and Utx mutations downregulated these energy generation processes in the abundant cell types. I will present an integrated analysis of eight preleukemic mouse models, and the different methods which I have utilised to investigate differential abundance of cell populations, metabolic analysis, differential gene expression and differentiation biases of the progenitor cell populations.

3044 – IP-10 LOSS MODULATES ERYTHROID CELL PRODUCTION IN MOUSE MODELS OF MYELOPROLIFERATIVE NEOPLASMS

The malignant transformation of hematopoietic stem cells (HSCs) is a multi-step process. In myeloproliferative neoplasms (MPNs), the JAK2 V617F missense mutation is found in most patients and loss of function TET2 mutations co-occur in approximately 10% of patients. Despite comprehensive genomic profiling, disease heterogeneity exists amongst individuals which cannot be solely resolved by genetic approaches. The microenvironment surrounding HSCs can be diverse and dynamic and deregulated cytokine levels have been implicated in the development of myeloid malignancies. One molecule in particular, IP-10/CXCL10, correlated strongly with disease severity and with JAK2 / TET2 mutational status with highest levels present in the serum of double-mutant patients and in JAK2/TET2 double-mutant mouse models compared to wild-type controls and to other MPN mouse models (e.g., CALRdel52) which recapitulate the MPN phenotype. To test the potential functional role of IP-10 in disease development and maintenance, we generated an allelic series of mutant mouse models carrying mutations in the JAK2, TET2 and IP10 genes. In transplantation experiments, JAK2/TET2 double mutant cells were slowed in their ability to drive an MPN in IP-10-/- animals, but eventually developed a strong disease phenotype, suggesting that the mutant clone itself eventually begins to produce IP-10. In double-mutant JAK2V617F/ V617F IP-10-/- models and triple mutant JAK2V617F/ V617F TET2-/- IP-10-/-, partial amelioration of disease phenotype in some animals as measured by serial monitoring of peripheral blood hemoglobin and hematocrit levels as well as erythroid differentiation assays. Together, these studies suggest that IP-10 may play a role in disease modulation at the red cell progenitor stage and set the stage for future studies exploring its reduction as a potential therapeutic modality in MPNs. The malignant transformation of hematopoietic stem cells (HSCs) is a multi-step process. In myeloproliferative neoplasms (MPNs), the JAK2 V617F missense mutation is found in most patients and loss of function TET2 mutations co-occur in approximately 10% of patients. Despite comprehensive genomic profiling, disease heterogeneity exists amongst individuals which cannot be solely resolved by genetic approaches. The microenvironment surrounding HSCs can be diverse and dynamic and deregulated cytokine levels have been implicated in the development of myeloid malignancies. One molecule in particular, IP-10/CXCL10, correlated strongly with disease severity and with JAK2 / TET2 mutational status with highest levels present in the serum of double-mutant patients and in JAK2/TET2 double-mutant mouse models compared to wild-type controls and to other MPN mouse models (e.g., CALRdel52) which recapitulate the MPN phenotype. To test the potential functional role of IP-10 in disease development and maintenance, we generated an allelic series of mutant mouse models carrying mutations in the JAK2, TET2 and IP10 genes. In transplantation experiments, JAK2/TET2 double mutant cells were slowed in their ability to drive an MPN in IP-10-/- animals, but eventually developed a strong disease phenotype, suggesting that the mutant clone itself eventually begins to produce IP-10. In double-mutant JAK2V617F/ V617F IP-10-/- models and triple mutant JAK2V617F/ V617F TET2-/- IP-10-/-, partial amelioration of disease phenotype in some animals as measured by serial monitoring of peripheral blood hemoglobin and hematocrit levels as well as erythroid differentiation assays. Together, these studies suggest that IP-10 may play a role in disease modulation at the red cell progenitor stage and set the stage for future studies exploring its reduction as a potential therapeutic modality in MPNs.

Progressive Degeneration of the Retina in Loxl3 Mutant Mouse Model of Stickler Syndrome

Progressive Degeneration of the Retina in Loxl3 Mutant Mouse Model of Stickler Syndrome

Effects of TYROBP Deficiency on Neuroinflammation of a Alzheimer’s Disease Mouse Model Carrying a PSEN1 p.G378E Mutation

ObjectiveTo study the effects of TYRO protein kinase-binding protein (TYROBP) deficiency on learning behavior, glia activation and pro-inflammatory cycokines, andTau phosphorylation of a new Alzheimer’s disease (AD) mouse model carrying a PSEN1 p.G378E mutation. MethodsA new AD mouse model carrying PSEN1 p.G378E mutation was built based on our previously found AD family which might be ascribed to the PSEN1 mutation, and then crossed with TYROBP deficient mice to produce the heterozygous hybrid mice (PSEN1G378E/WT; Tyrobp+/–) and the homozygous hybrid mice (PSEN1G378E/G378E; Tyrobp–/–). Water maze test was used to detect spatial learning and memory ability of mice. After the mice were sacrificed, the hippocampus was excised for further analysis. Immunofluorescence was used to identify the cell that expresses TYROBP and the number of microglia and astrocyte. Western blot was used to detect the expression levels of Tau and phosphorylatedTau (p-Tau), and ELISA to measure the levels of pro-inflammatory cytokines. ResultsOur results showed that TYROBP specifically expressed in the microglia of mouse hippocampus. Absence of TYROBP in PSEN1G378E mutation mouse model prevented the deterioration of learning behavior, decreased the numbers of microglia and astrocytes, and the levels of interleukin-6, interleukin-1β and tumor necrosis factor-α in the hippocampus (all P < 0.05). The ratios of AT8/Tau5, PHF1/Tau5, pT181/Tau5, pT231/ Tau5 and p-ERK/ERK were all higher in homozygous hybrid mice (PSEN1G378E/G378E; Tyrobp–/– mice) compared with PSEN1G378E/G378E mice (all P < 0.05). ConclusionsTYROBP deficiency might play a protective role in the modulation of neuroinflammation of AD. However, the relationship between neuroinflammation processes involving microglia and astrocyte activation, and release of pro-inflammatory cytokines, and p-Tau pathology needs further study.

Cdx regulates gene expression through PRC2-mediated epigenetic mechanisms

The extra-embryonic yolk sac contains adjacent layers of mesoderm and visceral endoderm. The mesodermal layer serves as the first site of embryonic hematopoiesis, while the visceral endoderm provides a means of exchanging nutrients and waste until the development of the chorioallantoic placenta. While defects in chorioallantoic fusion and yolk sac hematopoiesis have been described in Cdx mutant mouse models, little is known about the gene targets and molecular mechanisms through which Cdx members regulate these processes. To this end, we used RNA-seq to examine Cdx-dependent gene expression changes in the yolk sac. We find that loss of Cdx function impacts the expression of genes involved in yolk sac hematopoiesis, as previously described, as well as novel Cdx2 target genes. In addition, we observed Cdx-dependent changes in PRC2 subunit expression accompanied by altered H3K27me3 deposition at a subset of Cdx target genes as early as E7.5 in the embryo proper. This study identifies additional Cdx target genes and provides further evidence for Cdx-dependent epigenetic regulation of gene expression in the early embryo, and that this regulation is required to maintain gene expression programs in the extra-embryonic yolk sac at later developmental stages.

Involvement of increased endoplasmic reticulum stress in the development of cataracts in BALB.NCT-Cpoxnct mice

The BALB.NCT-Cpoxnct is a mutant mouse model for hereditary cataracts. We previously uncovered that the primary cause of the cataracts of BALB.NCT-Cpoxnct is a mutation in the coproporphyrinogen oxidase (Cpox) gene. Because of the mutation, excessive coproporphyrin is accumulated in the BALB.NCT-Cpoxnct lens. In this study, we analyzed the changes in transcriptome and proteins in the lenses of 4- and 12-week-old BALB.NCT-Cpoxnct to further elucidate the molecular etiology of cataracts in this mouse strain. Transcriptome analysis revealed that endoplasmic reticulum (ER) stress was increased in the BALB.NCT-Cpoxnct lens that induced persistent activation of the PERK signaling pathway of the ER stress response. Also, levels of crystallin transcripts and proteins were reduced in the BALB.NCT-Cpoxnct lens. Analysis of proteins disclosed aggregation of crystallins and keratins prior to the manifestation of cataracts in 4-week-old BALB.NCT-Cpoxnct mice. At 12 weeks of age, insoluble crystallins were accumulated in the cataractous BALB.NCT-Cpoxnct lens. Overall, our data suggest the following sequence of events in the BALB.NCT-Cpoxnct lens: accumulated coproporphyrin induces the aggregation of proteins including crystallins. Aggregated proteins increase ER stress that, in turn, leads to the repression of global translation of proteins including crystallins. The decline in the molecular chaperone crystallin aggravates aggregation and insolubilization of proteins. This vicious cycle would eventually lead to cataracts in BALB.NCT-Cpoxnct.

Does Chronic Sleep Fragmentation Lead to Alzheimer's Disease in Young Wild-Type Mice?

Chronic sleep insufficiency is becoming a common issue in the young population nowadays, mostly due to life habits and work stress. Studies in animal models of neurological diseases reported that it would accelerate neurodegeneration progression and exacerbate interstitial metabolic waste accumulation in the brain. In this paper, we study whether chronic sleep insufficiency leads to neurodegenerative diseases in young wild-type animals without a genetic pre-disposition. To this aim, we modeled chronic sleep fragmentation (SF) in young wild-type mice. We detected pathological hyperphosphorylated-tau (Ser396/Tau5) and gliosis in the SF hippocampus. 18F-labeled fluorodeoxyglucose positron emission tomography scan (18F-FDG-PET) further revealed a significant increase in brain glucose metabolism, especially in the hypothalamus, hippocampus and amygdala. Hippocampal RNAseq indicated that immunological and inflammatory pathways were significantly altered in 1.5-month SF mice. More interestingly, differential expression gene lists from stress mouse models showed differential expression patterns between 1.5-month SF and control mice, while Alzheimer's disease, normal aging, and APOEε4 mutation mouse models did not exhibit any significant pattern. In summary, 1.5-month sleep fragmentation could generate AD-like pathological changes including tauopathy and gliosis, mainly linked to stress, as the incremented glucose metabolism observed with PET imaging suggested. Further investigation will show whether SF could eventually lead to chronic neurodegeneration if the stress condition is prolonged in time.