Heterozygous Controls Research Articles

Sacsin levels in PBMCs: A diagnostic assay for SACS variants in peripheral blood cells - A PROSPAX study.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a common recessive ataxia that is still underdiagnosed worldwide. An easily accessible diagnostic biomarker might help to diagnostically confirm patients presenting SACS variants of unknown significance (VUS) or atypical phenotypes. To detect sacsin in peripheral blood mononuclear cells (PBMCs) and to validate its diagnostic biomarker quality to discriminate biallelic SACS patients (including patients with VUS and/or atypical phenotypes) against healthy controls, non-ARSACS spastic ataxia patients, and heterozygous SACS carriers. Sacsin protein levels in PBMCs were assessed in patients versus controls and validated in skin-derived fibroblasts. Patients with biallelic SACS variants - including patients with VUS and/or atypical phenotypes - showed loss of sacsin in PBMCs, with discriminative performance against healthy, heterozygous, and non-ARSACS controls. This included all investigated SACS missense variants. Also, C-terminal variants escaping nonsense-mediated decay, while not differing from controls in expression level, showed lower molecular weight in this assay. Assessing sacsin levels using PBMCs offers an easy, peripherally accessible diagnostic biomarker for ARSACS, with PBMCs being much less invasive and easier to handle than fibroblasts. Additionally, this might be a potential target-engagement blood biomarker for sacsin-increasing therapies. © 2024 International Parkinson and Movement Disorder Society.

CD4, but not Cxcr6, is necessary for control of Pneumocystis murina infection

CD4+ T cells are critical to control of Pneumocystis infection, and Cxcr6 has been shown to be upregulated in these cells during infection, but the roles of CD4 and Cxcr6 in this setting are undefined. To explore this, mice deficient in CD4 or Cxcr6 expression were utilized in a co-housing mouse model that mimics the natural route of Pneumocystis infection. Organism load and anti-Pneumocystis antibodies were assayed over time, and immunohistochemistry, flow cytometry, and quantitative PCR were used to characterize host immune responses during infection. CD4 was found to be necessary for clearance of Pneumocystis murina, though partial control was seen in it's absence; based on ThPOK expression, double negative T cells with T helper cell characteristics may be contributing to this control. Using a Cxcr6 deficient mouse expressing gfp, control of infection in the absence of Cxcr6 was similar to that in heterozygous control mice. It is noteworthy that gfp + cells were seen in the lungs with similar frequencies between the 2 strains. Interferon-ɣ and chemokine/ligands Cxcr3, Cxcl9, and Cxcl10 increased during P. murina infection in all models. Thus, CD4, but not Cxcr6, is needed for clearance of P. murina infection.

Phenotypic quantification of Nphs1-deficient mice.

Steroid-resistant nephrotic syndrome (SRNS) is the second most frequent cause of chronic kidney disease in children and young adults. The most severe form of steroid-resistant nephrotic syndrome is congenital nephrotic syndrome Finnish type (CNSF), caused by biallelic loss-of-function variants inNPHS1,encoding nephrin. Since each of the 68 monogenic causes of steroid-resistant nephrotic syndrome represents a rare cause of the disease, tailoring therapeutic interventions to multiple molecular targets remains challenging, suggesting gene replacement therapy (GRT) as a viable alternative. To set the ground for a gene replacement study in vivo, we established rigorous, quantifiable, and reproducible phenotypic assessment of a conditional Nphs1 knockout mouse model. By breeding a floxed Nphs1fl/- mouse (Nphs1tm1Afrn/J) previously studied for pancreatic β-cell survival with a podocin promoter-driven Cre recombinase mouse model (Tg(NPHS2-Cre)295Lbh/J), we generated mice with podocyte-specific nephrin deficiency (Nphs1fl/fl NPHS2-Cre +). We observed a median survival to postnatal day P5 in nephrin-deficient mice, whereas heterozygous control mice and wild type (WT) control group showed 90% and 100% survival, respectively (at P50 days). Light microscopy analysis showed a significantly higher number of renal-tubular microcysts per kidney section in nephrin-deficient mice compared to the control groups (P < 0.0022). Transmission electron microscopy demonstrated reduced foot process (FP) density in nephrin-deficient mice compared to controls (P < 0.0001). Additionally, proteinuria quantitation using urine albumin-to-creatinine ratio (UACR) was significantly higher in nephrin-deficient mice compared to controls. This study represents the first comprehensive description of the kidney phenotype in anephrin-deficient mouse model, laying the foundation for future gene replacement therapy endeavors.

Quantifiable and reproducible phenotypic assessment of a constitutive knockout mouse model for congenital nephrotic syndrome of the Finnish type

Steroid-resistant nephrotic syndrome (SRNS) is the second most frequent cause of childhood chronic kidney disease. Congenital nephrotic syndrome of the Finnish type (CNF) (MIM# 256300) is caused by biallelic variants in the gene NPHS1, encoding nephrin, an integral component of the kidney filtration barrier. No causal treatments exist, and children inevitably require kidney replacement therapy. In preparation for gene replacement therapy (GRT) in CNF, we established a quantifiable and reproducible phenotypic assessment of the nephrin-deficient CNF mouse model: 129/Sv-Nphs1tm1Rkl/J. We assessed the phenotypic spectrum of homozygous mice (Nphs1tm1Rkl/Nphs1tm1Rkl) compared to heterozygous controls (Nphs1tm1Rkl/Nphs1WT) by the following parameters: 1. cohort survival, 2. podocyte foot process (FP) density per glomerular basement membrane (GBM) using transmission electron microscopy, 3. tubular microcysts in brightfield microscopy, and 4. urinary albumin/creatinine ratios. Nphs1tm1Rkl/Nphs1tm1Rkl mice exhibited: 1. perinatal lethality with median survival of 1 day, 2. FP effacement with median FP density of 1.00 FP/µm GBM (2.12 FP/µm in controls), 3. tubular dilation with 65 microcysts per section (6.5 in controls), and 4. increased albumin/creatinine ratio of 238 g/g (4.1 g/g in controls). We here established four quantifiable phenotyping features of a CNF mouse model to facilitate future GRT studies by enabling sensitive detection of phenotypic improvements.

Inhibition of somatostatin enhances the long-term metabolic outcomes of sleeve gastrectomy in mice

ObjectiveBariatric surgery is an effective treatment to obesity, leading to weight loss and improvement in glycemia, that is characterized by hypersecretion of gastrointestinal hormones. However, weight regain and relapse of hyperglycemia are not uncommon. We set to identify mechanisms that can enhance gastrointestinal hormonal secretion following surgery to sustain weight loss. MethodsWe investigated the effect of somatostatin (Sst) inhibition on the outcomes of bariatric surgery using a mouse model of sleeve gastrectomy (SG). ResultsSst knockout (sst-ko) mice fed with a calorie-rich diet gained weight normally and had a mild favorable metabolic phenotype compared to heterozygous sibling controls, including elevated plasma levels of GLP-1. Mathematical modeling of the feedback inhibition between Sst and GLP-1 showed that Sst exerts its maximal effect on GLP-1 under conditions of high hormonal stimulation, such as following SG. Obese sst-ko mice that underwent SG had higher levels of GLP-1 compared with heterozygous SG-operated controls. The SG-sst-ko mice regained less weight than controls and maintained lower glycemia months after surgery. Obese wild-type mice that underwent SG and were treated daily with a Sst receptor inhibitor for two months had higher GLP-1 levels, regained less weight, and improved metabolic profile compared to saline-treated SG-operated controls, and compared to inhibitor or saline-treated sham-operated obese mice. ConclusionsOur results suggest that inhibition of Sst signaling enhances the long-term favorable metabolic outcomes of bariatric surgery.

TMIC-12. RAB27 REGULATION OF VASCULAR WALL INTEGRITY MEDIATES T-CELL INFILTERATION IN GLIOBLASTOMA

Abstract Endothelial secretome exerts a potent influence on the phenotype of cancer cells and immune microenvironment. Recently, we have reported that glioblastoma stem cells acquire a more aggressive mesenchymal state under influence of vascular endothelial cell derived extracellular vesicles (EVs). Since Rab27, GTPases involved in multiple aspects of EV (exosome) biogenesis, has been implicated in control of angiogenic pathways through regulation of VEGFR1 turnover and possibly other mechanisms, we interrogated the role of Rab27 in the function of normal and glioblastoma associated brain microvasculature. Using mice with disrupted Rab27a and Rab27b isoforms, we documented that these proteins control development and stability of the brain vasculature including pathological angiogenesis in glioblastoma. Thus, relative to wild-type C57bl/6 (WT) and double heterozygous Rab27a+/-;Rab27b+/- (dHET) mice, animals with Rab27a/b double knock-out (Rab27a/b-dKO) exhibit reduced microvascular density in brain cortices. When neoangiogensis was induced following orthotopic inoculation of mouse glioma cells (GL261), multiple morphological anomalies were observed in the tumour vasculature relative to tumours established in control mice. Moreover, vascular permeability of tumours in Rab27a/b-dKO mice markedly exceeded that of similar tumours in wild type or heterozygous controls resulting in augmented T-cell infiltration into the otherwise ‘cold’ brain tumor microenvironment. Our results suggest that key switches in the cellular secretome, such as Rab27a and Rab27b, may impact endothelial membrane dynamic and intercellular interactions capabilities, whereby they may play a critical role in ensuring the stability and sustained cellular barrier function of the vasculature during brain tumour progression. Therapeutic implications of these findings are being explored.

DNMT3A R882H Exhibits Greater Inflammatory Potential Than R882C in Primary Hematopoietic Stem and Progenitor Cell Knock-in Model and Population Data

Clonal hematopoiesis (CH) is an age-associated phenomenon which is known to increase the risk for hematologic malignancy and cardiovascular disease. The most commonly mutated gene in CH is de novo DNA methyltransferase DNMT3A. R882H and R882C are the most common hotspot mutations in this gene, and these mutations are commonly grouped together in interpretation of clinical data even though in vitro experiments suggest divergence in their biochemical consequences. Here, we used CRISPR knock-in of primary hematopoietic stem and progenitor cells (HSPCs) along with population data from the UK Biobank (UKB) to evaluate the extent to which R882H and R882C differentially predispose to inflammatory phenotypes. Using two homology donor (HD) templates with distinct self-cleaving fluorescent tags, we used FACS to isolate homozygous R882H- or R882C-mutant cells from six unique samples of cord blood HSPCs. We subsequently cultured these isogeneic mutant cells and their mock-electroporated wild-type (WT) controls in monocyte differentiation media and evaluated their transcriptomes via bulk RNA sequencing (RNAseq) and production of secreted cytokines in a Luminex-based assay. Analysis of transcripts using differential expression for repeated measures (Dream) revealed that, when compared to WT, the mutants demonstrated many shared differentially expressed genes (DEGs) but also exhibited many DEGs unique to each variant. Furthermore, gene set enrichment analysis (GSEA) of Hallmark pathways revealed common enrichment of cell cycle related pathways in both variants, but found that R882H displayed greater enrichment of inflammation-related pathways than either WT or R882C (Figure 1A). Rank-order GO and KEGG pathway enrichment comparison of R882H vs R882C showed further evidence of bias in inflammation-related pathways among the most upregulated gene sets (e.g., GO:0019882, Antigen Processing &amp; Presentation and KEGG:hsa04145, Phagosome). IL-6 is significantly regulated by R882 genotype in our model; levels of IL-6 for R882H (13,065 ± 2,446; mean ± s.e. in pg/mL) but not R882C (10,340 ± 2,446) were significantly different than WT (6,281 ± 1,730; p = 0.014). We next sought to better understand if this biased inflammatory priming would also be observed for heterozygous mutations and across different cell types, so using mutant and WT HD templates we performed single cell RNAseq on CRISPR'd heterozygous mutants and knock-in WT controls. Comparing R882H to R882C in this context demonstrated upregulation of numerous inflammation-related genes, including CXCL8 (IL-8), S100A8, and S100A9. GSEA of Hallmark pathways revealed enrichment of inflammatory pathways (e.g., Inflammatory Response &amp; TNFA Signaling via NFKB) in monocytic cells but also in progenitor cells. We then investigated whether these transcriptional differences between R882H and R882C might be due to divergent patterns of DNA (hypo)methylation. Employing 5-letter next-generation sequencing (A, C, G, T, methyl-C), we compared the methylation landscape of three isogeneic donors with homozygous R882H, R882C, or mock-electroporated WT genotypes. As expected, comparison of either mutant to WT showed a global hypomethylation. Comparison of R882H to R882C revealed no systematic bias in methylation of gene promoters but a pervasive hypomethylation of gene bodies. A gene set of the 50 most upregulated genes from our single cell RNAseq experiment was significantly enriched in rank order analysis of R882H vs R882C hypomethylation of gene bodies (NES = 1.47, p = 0.036) but not promoters (NES = -0.86, p = 0.71). Finally, we asked if there were differences in patient phenotypes for those with R882H or R882C clonal hematopoiesis. Here, we used our previously published UKB clonal hematopoiesis cohort (Vlasschaert et al. 2023, Blood). In Cox proportional hazards models controlling for basic demographics (N = 451K) or demographics plus cardiovascular-relevant covariates (N = 390K), we found that R882H but not R882C was associated with significantly greater incidence of heart failure and of a composite measure consisting of death or coronary artery disease (Figure 1B). In conclusion, our study found evidence in experimental models and patient data that DNMT3A R882H may pose a greater risk for inflammatory phenotypes than R882C.

Urinary Metabolic Distinction of Niemann-Pick Class 1 Disease through the Use of Subgroup Discovery.

In this investigation, we outline the applications of a data mining technique known as Subgroup Discovery (SD) to the analysis of a sample size-limited metabolomics-based dataset. The SD technique utilized a supervised learning strategy, which lies midway between classificational and descriptive criteria, in which given the descriptive property of a dataset (i.e., the response target variable of interest), the primary objective was to discover subgroups with behaviours that are distinguishable from those of the complete set (albeit with a differential statistical distribution). These approaches have, for the first time, been successfully employed for the analysis of aromatic metabolite patterns within an NMR-based urinary dataset collected from a small cohort of patients with the lysosomal storage disorder Niemann-Pick class 1 (NPC1) disease (n = 12) and utilized to distinguish these from a larger number of heterozygous (parental) control participants. These subgroup discovery strategies discovered two different NPC1 disease-specific metabolically sequential rules which permitted the reliable identification of NPC1 patients; the first of these involved 'normal' (intermediate) urinary concentrations of xanthurenate, 4-aminobenzoate, hippurate and quinaldate, and disease-downregulated levels of nicotinate and trigonelline, whereas the second comprised 'normal' 4-aminobenzoate, indoxyl sulphate, hippurate, 3-methylhistidine and quinaldate concentrations, and again downregulated nicotinate and trigonelline levels. Correspondingly, a series of five subgroup rules were generated for the heterozygous carrier control group, and 'biomarkers' featured in these included low histidine, 1-methylnicotinamide and 4-aminobenzoate concentrations, together with 'normal' levels of hippurate, hypoxanthine, quinolinate and hypoxanthine. These significant disease group-specific rules were consistent with imbalances in the combined tryptophan-nicotinamide, tryptophan, kynurenine and tyrosine metabolic pathways, along with dysregulations in those featuring histidine, 3-methylhistidine and 4-hydroxybenzoate. In principle, the novel subgroup discovery approach employed here should also be readily applicable to solving metabolomics-type problems of this nature which feature rare disease classification groupings with only limited patient participant and sample sizes available.

Characterisation of the hydrogen sulfide system in early diabetic kidney disease.

A deficiency in hydrogen sulfide has been implicated in the development and progression of diabetic chronic kidney disease. The purpose of this study was to determine the effect of diabetes on the H2S system in early-stage diabetic kidney disease. We characterised gene and protein expression profile of the enzymes that regulate H2S production and degradation, and H2S production capacity, in the kidney from 10-week-old C57BL6J db/db mice (n=6), in age-matched heterozygous controls (n=7), and in primary endothelial cells (HUVECs) exposed to high glucose. In db/db mice renal H2S levels were significantly reduced (p=0.009). Protein expression of the H2S production enzymes was differentially affected by diabetes: Cystathionine β-synthase (CBS) was significantly lower in both db/db mice and high glucose treated HUVECS (p<0.0001; p= 0.0318) whereas 3-Mercatopyruvate sulfurtransferase (3-MST) expression was higher in the db/db kidney (p<0.0001), yet lower in the HUVECs (p=0.0001). Diabetes had no effect on the expression of Cystathionine γ-lyase (CSE) in the db/db kidney (p=ns), but was associated with reduced expression in the HUVECs (p=0.0004). Protein expression of degradation enzyme Sulfide quinone reductase (SQOR) was significantly higher in db/db kidney (p=0.048) and lower in the high glucose treated HUVECs (p= 0.008). Immunofluorescence studies revealed differential localization of the H2S enzymes in the kidney, including both tubular and vascular localisation, suggestive of functionally distinct actions in the kidney. The results of this study provide foundational knowledge for future research looking at the H2S system in both kidney physiology and the aetiology of chronic diabetic kidney disease.

Gender Differences in a Mouse Model of Hepatocellular Carcinoma Revealed Using Multi-Modal Imaging.

The worldwide incidence of hepatocellular carcinoma (HCC) continues to rise, in part due to poor diet, limited exercise, and alcohol abuse. Numerous studies have suggested that the loss or mutation of PTEN plays a critical role in HCC tumorigenesis through the activation of the PI3K/Akt signaling axis. The homozygous knockout of PTEN in the livers of mice results in the accumulation of fat (steatosis), inflammation, fibrosis, and eventually progression to HCC. This phenotype bears a striking similarity to non-alcoholic steatohepatitis (NASH) which is thought to occupy an intermediate stage between non-alcoholic fatty liver disease (NAFLD), fibrosis, and HCC. The molecular and physiological phenotypes that manifest during the transition to HCC suggest that molecular imaging could provide a non-invasive screening platform to identify the hallmarks of HCC initiation prior to the presentation of clinical disease. We have carried out longitudinal imaging studies on the liver-specific PTEN knockout mouse model using CT, MRI, and multi-tracer PET to interrogate liver size, steatosis, inflammation, and apoptosis. In male PTEN knockout mice, significant steatosis was observed as early as 3 months using both magnetic resonance spectroscopy (MRS) and computed tomography (CT). Enhanced uptake of the apoptosis tracer 18F-TBD was also observed in the livers of male PTEN homozygous knockout mice between 3 and 4 months of age relative to heterozygous knockout controls. Liver uptake of the inflammation tracer [18F]4FN remained relatively low and constant over 7 months in male PTEN homozygous knockout mice, suggesting the suppression of high-energy ROS/RNS with PTEN deletion relative to heterozygous males where the [18F]4FN liver uptake was elevated at early and late time points. All male PTEN homozygous mice developed HCC lesions by month 10. In contrast to the male cohort, only 20% (2 out of 10) of female PTEN homozygous knockout mice developed HCC lesions by month 10. Steatosis was significantly less pronounced in the female PTEN homozygous knockout mice relative to males and could not accurately predict the eventual occurrence of HCC. As with the males, the [18F]4FN uptake in female PTEN homozygous knockout mice was low and constant throughout the time course. The liver uptake of 18F-TBD at 3 and 4.5 months was higher in the two female PTEN knockout mice that would eventually develop HCC and was the most predictive imaging biomarker for HCC in the female cohort. These studies demonstrate the diagnostic and prognostic role of multi-modal imaging in HCC mouse models and provide compelling evidence that disease progression in the PTEN knockout model is highly dependent on gender.

Sex Differences in renal ischemia-reperfusion injury following deletion of Netrin-1 in the kidney

Netrin-1, an angiogenic guidance cue, is heavily expressed in the kidney. Our lab has previously reported that deletion of Netrin-1 in Foxd1-positive stromal progenitors ( Foxd1 GC/+ Netrin1 fl/fl ) results in abnormal vascular patterning during kidney development, including a reduction in interlobar branching. It remains unknown how these changes in vascular patterning affect kidney function and physiology. In the current study, we hypothesized that changes in developmental patterning of the kidney vasculature would protect from renal ischemia-reperfusion injury in a sex-specific manner due to ischemic preconditioning to the kidney tubular cells. To test this hypothesis, we performed warm, bilateral ischemia-reperfusion by clamping the renal pedicles for 26-minutes in wild-type ( Netrin1 fl/fl or Netrin1 fl/+ )(3M, 3F), heterozygous ( Foxd1 GC/+ Netrin1 fl/+ ) (2M, 4F), and mutant ( Foxd1 GC/+ Netrin1 fl/fl ) (3M, 1F) male (M) and female (F) mice. Following 24-hours of reperfusion, blood and kidneys were collected for analysis of kidney injury markers. We found that plasma creatinine (p 2-wayANOVA =0.021) and blood urea nitrogen (p 2-wayANOVA =0.009) were decreased in female mice compared to male mice regardless of genotype. In both male and female mutant mice, there was a decrease in these markers of injury compared to heterozygous and wild-type control mice, although this did not reach statistical significance. In agreement, immunofluorescent staining of kidney injury marker 1 (KIM-1) indicated less tubular injury in female mice compared to males. These data suggest that female mice are less susceptible to ischemic injury than male mice despite both sexes having similar alterations in vascular patterning following deletion of Netrin-1. Further, our results indicate a protective effect of these developmental patterning changes due to Netrin-1 deletion. We suspect that changes in the vasculature in our mutant mice provide a preconditioning-like environment to mediate this effect. Future studies will investigate the underlying mechanisms which drive this injury phenotype between sexes. Understanding how renal vascular development impacts kidney injury is critical to the development of new therapeutic strategies to prevent the sustained injury following periods of ischemia. NIH R01DK121014 to LO This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

INNATE TL1A SIGNALING PROMOTES INTESTINAL NEUTROPHIL ACTIVATION AND COLITIS ASSOCIATED CANCER

Intestinal inflammation associated with chronic inflammatory bowel disease (IBD) increases the risk of developing colitis-associated cancer (CAC), but the cellular mechanisms driving CAC are not well defined. Polymorphisms in TNFSF15 (called TNF-like cytokine 1a or TL1A) are highly associated with IBD and the aim of this work is to evaluate a potential mechanistic role of TL1A in regulating CAC. Data from the human atlas protein database revealed that TL1A protein is present in 50% of colorectal cancer tissue samples and that high expression of TNFSF15 correlated with reduced survival. To test the functional role for TL1A in tumorigenesis, we used the well-established AOM/DSS model of CAC in mice deficient for the TL1A receptor (called death receptor 3 or DR3) and mice deficient for myeloid derived TL1A (CD11C cre+TNFSF15fl/fl). We found that both DR3-deficient mice and myeloid derived TL1A deficient mice had a significant reduction in tumor number compared to heterozygous littermate controls. To evaluate the contribution of innate lymphocytes, we used DR3-deficient mice on a RAG-deficient background in our AOM/DSS model. Even in the absence of B and T cells, DR3-deficient mice still showed significant reduction in tumor burden, suggesting innate lymphoid cell (ILC) contribution. DR3-deficient mice also revealed a significant reduction in neutrophil infiltration. Neutrophil depletion with α-Ly6G similarly resulted in a significant reduction of tumor numbers in our CAC model in a DR3 dependent manner. To test the direct regulation of neutrophils by ILCs, we establish a co-culture model. TL1A activated ILCs triggered neutrophil activation (CD177) and maturation (CD11B) which required GM-CSF. In vivo activation with a DR3 agonist confirmed intestinal ILC3 production of GM-CSF but also impacted bone marrow granulopoiesis by expanding both mature neutrophils and granulocyte-macrophage progenitors (GMPs).

Notch1 Is Involved in Physiologic Cardiac Hypertrophy of Mice via the p38 Signaling Pathway after Voluntary Running.

Appropriate exercise such as voluntary wheel-running can induce physiological cardiac hypertrophy. Notch1 plays an important role in cardiac hypertrophy; however, the experimental results are inconsistent. In this experiment, we aimed to explore the role of Notch1 in physiological cardiac hypertrophy. Twenty-nine adult male mice were randomly divided into a Notch1 heterozygous deficient control (Notch1+/- CON) group, a Notch1 heterozygous deficient running (Notch1+/- RUN) group, a wild type control (WT CON) group, and a wild type running (WT RUN) group. Mice in the Notch1+/- RUN and WT RUN groups had access to voluntary wheel-running for two weeks. Next, the cardiac function of all of the mice was examined by echocardiography. The H&E staining, Masson trichrome staining, and a Western blot assay were carried out to analyze cardiac hypertrophy, cardiac fibrosis, and the expression of proteins relating to cardiac hypertrophy. After two-weeks of running, the Notch1 receptor expression was decreased in the hearts of the WT RUN group. The degree of cardiac hypertrophy in the Notch1+/- RUN mice was lower than that of their littermate control. Compared to the Notch1+/- CON group, Notch1 heterozygous deficiency could lead to a decrease in Beclin-1 expression and the ratio of LC3II/LC3I in the Notch1+/- RUN group. The results suggest that Notch1 heterozygous deficiency could partly dampen the induction of autophagy. Moreover, Notch1 deficiency may lead to the inactivation of p38 and the reduction of β-catenin expression in the Notch1+/- RUN group. In conclusion, Notch1 plays a critical role in physiologic cardiac hypertrophy through the p38 signaling pathway. Our results will help to understand the underlying mechanism of Notch1 on physiological cardiac hypertrophy.

Effects of changes in myosin biomechanics on canonical and non-canonical signaling and HCM phenotypes.

Myosin heavy chain 7 (MYH7) mutations are among the most commonly found in patients with hypertrophic cardiomyopathy (HCM). To address cellular responses to biomechanical alternations due to mutations in MYH7, we employed human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as an in vitro model and deeply phenotyped the transcriptional changes in these cells using single cell RNA sequencing (scRNAseq). We profiled hiPSC-CMs carrying heterozygous G265E mutant and control wild type MYH7 alleles at 30 and 60 day after differentiation and found potential disruption in hypertrophic gene expression as well as genes regulating mitochondrial metabolism. Intriguingly, the gene expression phenotype in these mutant lines appear somewhat subtle and vary considerably from line to line and differentiation batch to batch. To assess whether the mutant cells are more susceptible to physiological stress, we treated day 30 hiPSC-CMs with isoproterenol and observed an increased in their metabolic gene defect that was corroborated using a Seahorse metabolic assay. Given the more subtle phenotype of MYH7 G256E heterozygous mutant line, we considered whether a homozygous mutant would provide a more robust phenotype manifestation at the transcriptional level. To address this, we profiled day 30 hiPSC-CM from a MYH R369Q homozygous mutant line and found a significantly greater consistency in transcriptional phenotype among different lines and batches of differentiation. In summary, our work revealed the power of MYH7 mutation-specific hiPSC-CMs as an in vitro model of HCM and the ability of scRNAseq to deeply phenotype these cells to reveal their aberrant cellular signaling and metabolic response. When combined with a comprehensive understanding of MYH7 mutation effects at the single molecular and whole tissue level, we hope to enable future development of novel therapy against HCM.

INNATE TL1A SIGNALING PROMOTES INTESTINAL NEUTROPHIL ACTIVATION AND COLITIS ASSOCIATED CANCER

Abstract Intestinal inflammation associated with chronic inflammatory bowel disease (IBD) increases the risk of developing colitis-associated cancer (CAC), but the cellular mechanisms driving CAC are not well defined. Polymorphisms in TNFSF15 (called TNF-like cytokine 1a or TL1A) are highly associated with IBD and the aim of this work is to evaluate a potential mechanistic role of TL1A in regulating CAC. Data from the human atlas protein database revealed that TL1A protein is present in 50% of colorectal cancer tissue samples and that high expression of TNFSF15 correlated with reduced survival. To test the functional role for TL1A in tumorigenesis, we used the well-established AOM/DSS model of CAC in mice deficient for the TL1A receptor (called death receptor 3 or DR3) and mice deficient for myeloid derived TL1A (CD11C cre+TNFSF15fl/fl). We found that both DR3-deficient mice and myeloid derived TL1A deficient mice had a significant reduction in tumor number compared to heterozygous littermate controls. To evaluate the contribution of innate lymphocytes, we used DR3-deficient mice on a RAG-deficient background in our AOM/DSS model. Even in the absence of B and T cells, DR3-deficient mice still showed significant reduction in tumor burden, suggesting innate lymphoid cell (ILC) contribution. DR3-deficient mice also revealed a significant reduction in neutrophil infiltration. Neutrophil depletion with α-Ly6G similarly resulted in a significant reduction of tumor numbers in our CAC model in a DR3 dependent manner. To test the direct regulation of neutrophils by ILCs, we establish a co-culture model. TL1A activated ILCs triggered neutrophil activation (CD177) and maturation (CD11B) which required GM-CSF. In vivo activation with a DR3 agonist confirmed intestinal ILC3 production of GM-CSF but also impacted bone marrow granulopoiesis by expanding both mature neutrophils and granulocyte-macrophage progenitors (GMPs). CONCLUSION: Our data reveals a new link between intestinal ILC production of GM-CSF and neutrophil activation that promotes CAC and bone marrow granulopoiesis.

Tmprss6 Depletion-Mediated Iron Deficiency Attenuates the Clinical Course of Plasmodium Berghei Infection in Mice

Background Anaemia and malaria cause a great burden of disease and commonly co-exist. Field studies have linked iron deficiency with decreased malaria risk but may be confounded. We aimed to define the association between iron status and malaria risk in an in vivo experimental system. Using the P. berghei mouse malaria model, we assessed the impact of both severe iron overload and iron deficiency using inducible hepcidin (Hamp) knockout (iHamp-KO), Tmprss6 knockout (Tmprss6-KO) and Tmprss6 knockdown (Tmprss6-KD) mouse models. Homozygous mutations of HAMP (encoding hepcidin) cause severe iron overload, while homozygous inactivation of TMPRSS6 leads to hepcidin overexpression and iron deficiency. MethodsP. berghei infection of C57BL/6 mice is an experimental model of cerebral and severe malaria. The majority of mice develop neurological symptoms between day 6-10 post infection, surviving mice usually develop hyperparasitaemia and symptoms of severe anaemia by day 20. Mice were infected with PbmCherryLuci sporozoites by intravenous injection. Luciferase activity was used to assess liver infection and egress. Mice were analysed by IVIS at 24, 36, 52 and 55/56 hours post infection. From day 3 post infection peripheral parasitaemia was assessed daily. Mice were euthanised if they showed signs of cerebral malaria (such as loss of self-righting reflex and hind-limb paresis), severe anaemia, or if parasitaemia reached >20% (iHamp-KO and Tmprss6-KO) or >30% (Tmprss6-KD) in line with animal ethics requirements. iHamp-KO mice received tamoxifen intraperitoneally daily for 5 days 3 months prior to infection to induce Hamp deletion. Controls were iHamp-KO littermates that received vehicle alone. Tmprss6-KO mice were infected at about 24 weeks of age and compared to littermate heterozygote controls which are known to have a negligible phenotype. Tmprss6-KD was achieved through treatment of WT mice with siTMP, a GalNAc siRNA conjugate targeting Tmprss6. Mice received siTMP 5mg/kg s.c. 3 weekly starting 8 weeks prior to P. berghei infection, or a single treatment on day 3 (expected onset of blood stage infection) or day 6 (expected onset of high risk period for cerebral malaria), while controls received PBS. Results iHamp-KO mice had lower peak liver infection (p=0.0097), but egress was not affected, and there was no effect on overall survival or survival from cerebral malaria. Tmprss6-KO mice showed higher liver infection compared with heterozygous littermate controls at all timepoints, but egress was not different. Tmprss6-KO mice had improved overall survival compared with heterozygous controls (p=0.0004) and improved survival from cerebral malaria (p=0.0004). All 7 heterozygous controls succumbed to cerebral malaria by day 10, compared to 1 of 6 Tmprss6-KO animals (Figure 1). We further investigated effects of iron deficiency on malaria severity using Tmprss6 knockdown. Mice treated with siTMP for 8-10 weeks had markedly reduced Tmprss6 and elevated Hamp expression in the liver, and lower haemoglobin, MCV, and plasma iron. Tmprss6-KD mice, treated 8 weeks prior to P. berghei infection exhibited reduced parasitaemia, and took longer to reach 10%, 20% and 30% parasitaemia (Figure 2). There was no change in survival from cerebral malaria. Tmprss6-KD after infection did not change parasitaemia or survival. Conclusion Experimental iron deficiency via Tmprss6 downregulation attenuated malaria disease phenotype in mice. These data provide experimental evidence to support observational data that iron deficiency provides protection against malaria infection. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

ANGI-11. RAB27 IS REQUIRED FOR REGULATING INTACT VASCULATURE IN GLIOBLASTOMA

Abstract Rab27 is a GTPase involved in the biogenesis of extracellular vesicles (EVs) and cellular secretion pathway. Among two Rab27 isoforms (a and b) Rab27a has been identified in regulating angiogenesis during development and wound healing via VEGFR1. Here, we show that Rab27 plays a role in the development and stability of the brain vasculature including pathological angiogenesis in glioblastoma.. Thus, relative to wild-type C57bl/6 (WT) and double heterozygous Rab27a+/-;Rab27b+/- (dHET) mice, animals with Rab27a/b double knock-out (Rab27a/b-dKO) exhibit reduced microvascular density in brain cortices. Additionally, we also observed a reduction in permeability of Evans Blue in the Rab27 dKO cortices relative to controls. When neoangiogensis was induced following orthotopic inoculation of mouse glioma cells (GL261), a similar reduction and morphological anomalies were observed in the tumour vasculature relative to tumours established in control mice. Moreover, vascular permeability of tumours in Rab27a/b-dKO mice markedly exceeded that of similar tumours in wild type or heterozygous controls. Our results suggest Rab27a and Rab27b may play a critical role in ensuring stability of the vasculature under physiological conditions and in glioma.

The genetic risk factor CEL-HYB1 causes proteotoxicity and chronic pancreatitis in mice

Background & aimsThe CEL gene encodes the digestive enzyme carboxyl ester lipase. CEL-HYB1, a hybrid allele of CEL and its adjacent pseudogene CELP, is a genetic variant suggested to increase the risk of chronic pancreatitis (CP). Our aim was to develop a mouse model for CEL-HYB1 that enables studies of pancreatic disease mechanisms. MethodsWe established a knock-in mouse strain where the variable number of tandem repeat (VNTR) region of the endogenous mouse Cel gene was substituted with the mutated VNTR of the human CEL-HYB1 allele. Heterozygous and homozygous Cel-HYB1 mice and littermate wildtype controls were characterized with respect to pancreatic pathology and function. ResultsWe successfully constructed a mouse model with pancreatic expression of a humanized CEL-HYB1 protein. The Cel-HYB1 mice spontaneously developed features of CP including inflammation, acinar atrophy and fatty replacement, and the phenotype became more pronounced as the animals aged. Moreover, Cel-HYB1 mice were normoglycemic at age 6 months, whereas at 12 months they exhibited impaired glucose tolerance. Immunostaining of pancreatic tissue indicated the formation of CEL protein aggregates, and electron microscopy showed dilated endoplasmic reticulum. Upregulation of the stress marker BiP/GRP78 was seen in pancreatic parenchyma obtained both from Cel-HYB1 animals and from a human CEL-HYB1 carrier. ConclusionsWe have developed a new mouse model for CP that confirms the pathogenicity of the human CEL-HYB1 variant. Our findings place CEL-HYB1 in the group of genes that increase CP risk through protein misfolding-dependent pathways.

PMCA1 depletion in mouse eggs amplifies calcium signaling and impacts offspring growth†.

Egg activation in mammals is triggered by oscillations in egg intracellular calcium (Ca2+) level. Ca2+ oscillation patterns can be modified in vitro by changing the ionic composition of culture media or in vivo by conditions affecting mitochondrial function, such as obesity and inflammation. In mice, disruption of Ca2+ oscillations in vitro impacts embryo development and offspring growth. Here we tested the hypothesis that, even without in vitro manipulation, abnormal Ca2+ signaling following fertilization impacts offspring growth. Plasma membrane Ca2+ ATPases (PMCA) extrude cytosolic Ca2+ to restore Ca2+ homeostasis. To disrupt Ca2+ signaling in vivo, we conditionally deleted PMCA1 (cKO) in oocytes. As anticipated, in vitro fertilized cKO eggs had increased Ca2+ exposure relative to controls. To assess the impact on offspring growth, cKO females were mated to wild type males to generate pups that had high Ca2+ exposure at fertilization. Because these offspring would be heterozygous, we also tested the impact of global PMCA1 heterozygosity on offspring growth. Control heterozygous pups that had normal Ca2+ at fertilization were generated by mating wild type females to heterozygous males; these control offspring weighed significantly less than their wild type siblings. However, heterozygous offspring from cKO eggs (and high Ca2+ exposure) were larger than heterozygous controls at 12week-of-age and males had altered body composition. Our results show that global PMCA1 haploinsufficiency impacts growth and support that abnormal Ca2+ signaling after fertilization in vivo has a long-term impact on offspring weight. These findings are relevant for environmental and medical conditions affecting Ca2+ handling and for design of culture conditions and procedures for domestic animal and human assisted reproduction.

Application of data augmentation techniques towards metabolomics

Niemann–Pick Class 1 (NPC1) disease is a rare and debilitating neurodegenerative lysosomal storage disease (LSD). Metabolomics datasets of NPC1 patients available to perform this type of analysis are often limited in the number of samples and severely unbalanced. In order to improve the predictive capability and identify new biomarkers in an NPC1 disease urinary dataset, data augmentation (DA) techniques based on computational intelligence have been employed to create synthetic samples, i.e. the addition of noise, oversampling techniques and conditional generative adversarial networks. These techniques have been used to evaluate their predictive capacities on a set of urine samples donated by 13 untreated NPC1 disease and 47 heterozygous (parental) carrier control participants. Results on the prediction have also been obtained using different machine learning classification models and the partial least squares techniques. These results provide strong evidence for the ability of DA techniques to generate good quality synthetic data. Results acquired show increases in sensitivity of 20%–50%, an F1 score of 6%–30%, and a predictive capacity of 0.3 (out of 1). Additionally, more conventional forms of multivariate data analysis have been employed. These have allowed the detection of unusual urinary metabolite profiles, and the identification of biomarkers through the use of synthetically augmented datasets. Results indicate that urinary branched-chain amino acids such as valine, 3-aminoisobutyrate and quinolinate, may be employable as valuable biomarkers for the diagnosis and prognostic monitoring of NPC1 disease.