Characterization of mice carrying a neurodevelopmental disease-associated GluN2B(L825V) variant.

N-methyl-D-aspartate receptors (NMDARs), encoded by GRIN genes, are ionotropic glutamate receptors playing a critical role in synaptic transmission, plasticity, and synapse development. Genome sequence analyses have identified variants in GRIN genes in patients with neurodevelopmental disorders, but the underlying disease mechanisms are not well understood. Here, we have created and evaluated a transgenic mouse line carrying a missense variant Grin2bL825V , corresponding to a de-novo GRIN2B variant encoding GluN2B(L825V) found in a patient with intellectual disability (ID) and autism spectrum disorder (ASD). We used HEK293T cells expressing recombinant receptors and primary hippocampal neurons prepared from heterozygous Grin2bL825V/+ (L825V/+) and wild-type Grin2b+/+ (+/+) male and female mice to assess the functional impact of the variant. Whole-cell NMDAR currents were reduced in neurons prepared from L825V/+ compared to +/+ mice. Peak amplitude of NMDAR-mediated evoked excitatory postsynaptic currents (NMDAR-eEPSC) was not changed, but NMDAR-eEPSCs in L825V/+ neurons had faster deactivation compared to +/+ neurons and were less sensitive to a GluN2B-selective antagonist ifenprodil. Together, these results suggest a decreased functional contribution of GluN2B subunits to synaptic NMDAR currents in hippocampal neurons from L825V/+ mice. The analysis of the GluN2B(L825V) subunit surface expression and synaptic localization revealed no differences compared to wild-type GluN2B. Behavioral testing of mice of both sexes demonstrated hypoactivity, anxiety, and impaired sensorimotor gating in the L825V/+ strain, particularly affecting males, as well as cognitive symptoms. The heterozygous L825V/+ mouse offers a clinically relevant model of GRIN2B-related ID/ASD and our results suggest synaptic-level functional changes that may contribute to neurodevelopmental pathology.Significance statement Variants in genes for subunits of N-methyl-D-aspartate receptors (NMDARs), a subtype of ionotropic glutamate receptors, are associated with neurodevelopmental disorders. Here we have generated a transgenic mouse model of a de-novo missense GRIN2B gene variant, identified in a patient with intellectual disability and autism, that introduces a single amino acid substitution (L825V) in the NMDAR GluN2B subunit. Di- and triheteromeric NMDARs containing the GluN2B(L825V) subunit have a reduced channel open probability. Synaptic NMDAR currents in neurons from heterozygous L825V/+ mice have accelerated deactivation and reduced ifenprodil sensitivity, suggesting synaptic loss of GluN2B function. L825V/+ mice show increased anxiety, impaired sensorimotor gating, and cognitive deficits, consistent with patient symptoms. Our study describes a clinically relevant mouse model of GRIN2B-related neurodevelopmental pathology.

ABIN1 is a negative regulator of effector functions in cytotoxic T cells.

T cells are pivotal in the adaptive immune defense, necessitating a delicate balance between robust response against infections and self-tolerance. Their activation involves intricate cross-talk among signaling pathways triggered by the T-cell antigen receptors (TCR) and co-stimulatory or inhibitory receptors. The molecular regulation of these complex signaling networks is still incompletely understood. Here, we identify the adaptor protein ABIN1 as a component of the signaling complexes of GITR and OX40 co-stimulation receptors. T cells lacking ABIN1 are hyper-responsive ex vivo, exhibit enhanced responses to cognate infections, and superior ability to induce experimental autoimmune diabetes in mice. ABIN1 negatively regulates p38 kinase activation and late NF-κB target genes. P38 is at least partially responsible for the upregulation of the key effector proteins IFNG and GZMB in ABIN1-deficient T cells after TCR stimulation. Our findings reveal the intricate role of ABIN1 in T-cell regulation.

Towards a Cure for Diamond-Blackfan Anemia: Views on Gene Therapy.

Diamond-Blackfan anemia (DBA) is a rare genetic disorder affecting the bone marrow's ability to produce red blood cells, leading to severe anemia and various physical abnormalities. Approximately 75% of DBA cases involve heterozygous mutations in ribosomal protein (RP) genes, classifying it as a ribosomopathy, with RPS19 being the most frequently mutated gene. Non-RP mutations, such as in GATA1, have also been identified. Current treatments include glucocorticosteroids, blood transfusions, and hematopoietic stem cell transplantation (HSCT), with HSCT being the only curative option, albeit with challenges like donor availability and immunological complications. Gene therapy, particularly using lentiviral vectors and CRISPR/Cas9 technology, emerges as a promising alternative. This review explores the potential of gene therapy, focusing on lentiviral vectors and CRISPR/Cas9 technology in combination with non-integrating lentiviral vectors, as a curative solution for DBA. It highlights the transformative advancements in the treatment landscape of DBA, offering hope for individuals affected by this condition.

Functional canonical RNAi in mice expressing a truncated Dicer isoform and long dsRNA.

Canonical RNA interference (RNAi) is sequence-specific mRNA degradation guided by small interfering RNAs (siRNAs) made by RNase III Dicer from long double-stranded RNA (dsRNA). RNAi roles include gene regulation, antiviral immunity or defense against transposable elements. In mammals, RNAi is constrained by Dicer's adaptation to produce another small RNA class-microRNAs. However, a truncated Dicer isoform (ΔHEL1) supporting RNAi exists in mouse oocytes. A homozygous mutation to express only the truncated ΔHEL1 variant causes dysregulation of microRNAs and perinatal lethality in mice. Here, we report the phenotype and canonical RNAi activity in DicerΔHEL1/wt mice, which are viable, show minimal miRNome changes, but their endogenous siRNA levels are an order of magnitude higher. We show that siRNA production in vivo is limited by available dsRNA, but not by Protein kinase R, a dsRNA sensor of innate immunity. dsRNA expression from a transgene yields sufficient siRNA levels to induce efficient RNAi in heart and muscle. DicerΔHEL1/wt mice with enhanced canonical RNAi offer a platform for examining potential and limits of mammalian RNAi in vivo.

Early embryogenesis in CHDFIDD mouse model reveals facial clefts and altered cranial neurogenesis.

CDK13-related disorder, also known as congenital heart defects, dysmorphic facial features and intellectual developmental disorder (CHDFIDD) is associated with mutations in the CDK13 gene encoding transcription-regulating cyclin-dependent kinase 13 (CDK13). Here, we focused on the development of craniofacial structures and analyzed early embryonic stages in CHDFIDD mouse models, with one model comprising a hypomorphic mutation in Cdk13 and exhibiting cleft lip/palate, and another model comprising knockout of Cdk13, featuring a stronger phenotype including midfacial cleft. Cdk13 was found to be physiologically expressed at high levels in the mouse embryonic craniofacial structures, namely in the forebrain, nasal epithelium and maxillary mesenchyme. We also uncovered that Cdk13 deficiency leads to development of hypoplastic branches of the trigeminal nerve including the maxillary branch. Additionally, we detected significant changes in the expression levels of genes involved in neurogenesis (Ache, Dcx, Mef2c, Neurog1, Ntn1, Pou4f1) within the developing palatal shelves. These results, together with changes in the expression pattern of other key face-specific genes (Fgf8, Foxd1, Msx1, Meis2 and Shh) at early stages in Cdk13 mutant embryos, demonstrate a key role of CDK13 in the regulation of craniofacial morphogenesis.

Tertiary lymphoid structures and B cells determine clinically relevant T cell phenotypes in ovarian cancer.

Intratumoral tertiary lymphoid structures (TLSs) have been associated with improved outcome in various cohorts of patients with cancer, reflecting their contribution to the development of tumor-targeting immunity. Here, we demonstrate that high-grade serous ovarian carcinoma (HGSOC) contains distinct immune aggregates with varying degrees of organization and maturation. Specifically, mature TLSs (mTLS) as forming only in 16% of HGSOCs with relatively elevated tumor mutational burden (TMB) are associated with an increased intratumoral density of CD8+ effector T (TEFF) cells and TIM3+PD1+, hence poorly immune checkpoint inhibitor (ICI)-sensitive, CD8+ T cells. Conversely, CD8+ T cells from immunologically hot tumors like non-small cell lung carcinoma (NSCLC) are enriched in ICI-responsive TCF1+ PD1+ T cells. Spatial B-cell profiling identifies patterns of in situ maturation and differentiation associated with mTLSs. Moreover, B-cell depletion promotes signs of a dysfunctional CD8+ T cell compartment among tumor-infiltrating lymphocytes from freshly isolated HGSOC and NSCLC biopsies. Taken together, our data demonstrate that - at odds with NSCLC - HGSOC is associated with a low density of follicular helper T cells and thus develops a limited number of mTLS that might be insufficient to preserve a ICI-sensitive TCF1+PD1+ CD8+ T cell phenotype. These findings point to key quantitative and qualitative differences between mTLSs in ICI-responsive vs ICI-irresponsive neoplasms that may guide the development of alternative immunotherapies for patients with HGSOC.

Semi-Automated MicroCT Analysis of Bone Anatomy and Mineralization in Mouse Models.

The skeletal system mirrors several processes in the vertebrate body that impact developmental malfunctions, hormonal disbalance, malfunction of calcium metabolism and turn over, and inflammation processes such as arthrosis. X-ray micro computed tomography is a useful tool for 3D in situ evaluation of the skeletal system in a time-related manner, but results depend highly on resolution. Here, we provide the methodological background for a graduated evaluation from whole-body analysis of skeletal morphology and mineralization to high-resolution analysis of femoral and vertebral microstructure. We combine an expert-based evaluation with a machine-learning-based computational approach, including pre-setup analytical task lists. © 2024 Wiley Periodicals LLC. Basic Protocol 1: In vivo microCT scanning and skeletal analysis in mice Basic Protocol 2: Ex vivo high-resolution microCT scanning and microstructural analysis of the femur and L4 vertebra.

Proteolytic profiles of two isoforms of human AMBN expressed in E. coli by MMP-20 and KLK-4 proteases.

Ameloblastin is a protein in biomineralization of tooth enamel. However recent results indicate that this is probably not its only role in an organism. Enamel matrix formation represents a complex process enabled via specific crosslinking of two proteins - the most abundant amelogenin and the ameloblastin (AMBN). The human AMBN (hAMBN) gene possesses 13 protein coding exons with alternatively spliced transcripts and the longest isoform about 447 amino acid residues. It has been described that AMBN molecules in vitro assemble into oligomers via a sequence encoded by exon 5. Enamel is formed by the processing of enamel proteins by two specific proteases - enamelysin (MMP-20) and kallikrein 4 (KLK-4). The scaffold made of AMEL and non-amelogenin proteins is cleaved and removed from the developed tooth enamel. The hAMBN is expressed in two isoforms (ISO I and II), which could lead to their different utilization determined by distinct proteolytic profiles. In this study, we compared proteolytic profiles of both isoforms of hAMBN expressed in E. coli after proteolysis by MMP-20, KLK-4, and their 1:2 mixture. Proteolysis products were analysed and cleavage sites were identified by mass spectrometry. The proteolytic profiles of two AMBN isoforms showed different results, although we have to determine that the analysed AMBN was not post-translationally modified as expressed in prokaryotic cells. These results may lead to the suggestion of potentially divergent roles of AMBN isoforms cleavage products in various cell signalling pathways such as calcium buffering or signalling cascades.

Mitochondrial HER2 stimulates respiration and promotes tumorigenicity.

BACKGROUND: Amplification of HER2, a receptor tyrosine kinase and a breast cancer-linked oncogene, is associated with aggressive disease. HER2 protein is localised mostly at the cell membrane, but a fraction translocates to mitochondria. Whether and how mitochondrial HER2 contributes to tumorigenicity is currently unknown. METHODS: We enriched the mitochondrial (mt-)HER2 fraction in breast cancer cells using an N-terminal mitochondrial targeting sequence and analysed how this manipulation impacts bioenergetics and tumorigenic properties. The role of the tyrosine kinase activity of mt-HER2 was assessed in wild type, kinase-dead (K753M) and kinase-enhanced (V659E) mtHER2 constructs. RESULTS: We document that mt-HER2 associates with the oxidative phosphorylation system, stimulates bioenergetics and promotes larger respiratory supercomplexes. mt-HER2 enhances proliferation and invasiveness in vitro and tumour growth and metastatic potential in vivo, in a kinase activity-dependent manner. On the other hand, constitutively active mt-HER2 provokes excessive mitochondria ROS generation, sensitises to cell death, and restricts growth of primary tumours, suggesting that regulation of HER2 activity in mitochondria is required for the maximal pro-tumorigenic effect. CONCLUSIONS: mt-HER2 promotes tumorigenicity by supporting bioenergetics and optimal redox balance.

Ovol2 promoter mutations in mice and human illuminate species-specific phenotypic divergence.

Pathogenic variants in the highly conserved OVOL2 promoter region cause posterior polymorphous corneal dystrophy (PPCD) 1 by inducing an ectopic expression of the endothelial OVOL2 mRNA. Here we produced an allelic series of Ovol2 promoter mutations in the mouse model including the heterozygous c.-307T>C variant (RefSeq NM_021220.4) causing PPCD1 in humans. Despite the high evolutionary conservation of the Ovol2 promoter, only some alterations of its sequence had phenotypic consequences in mice. Four independent sequence variants in the distal part of the Ovol2 promoter had no significant effect on endothelial Ovol2 mRNA level or caused any ocular phenotype. In contrast, the mutation c.-307T>C resulted in increased Ovol2 expression in the corneal endothelium. However, only a small fraction of adult mice c.-307T>C heterozygotes developed ocular phenotypes such as irido-corneal adhesions, and corneal opacity. Interestingly, phenotypic penetrance was increased at embryonic stages. Notably, c.-307T>C mutation is located next to the Ovol1/Ovol2 transcription factor binding site. Mice carrying an allele with a deletion encompassing the Ovol2 binding site c.-307_-320del showed significant Ovol2 gene upregulation in the cornea endothelium and exhibited phenotypes similar to the c.-307T>C mutation. In conclusion, although the mutations c.-307T>C and -307_-320del lead to a comparably strong increase in endothelial Ovol2 expression as seen in PPCD1 patients, endothelial dystrophy was not observed in the mouse model, implicating species-specific differences in endothelial cell biology. Nonetheless, the emergence of dominant ocular phenotypes associated with Ovol2 promoter variants in mice implies a potential role of this gene in eye development and disease.

Behavioral Characterization of an Angelman Syndrome Mouse Model.

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of AS. We use the rotarod learning paradigm, detailed gait analysis, and nest building test to detect and characterize animal motor impairments. We test animal emotionality in the open field and elevated plus maze tests, as well as the affect in the tail suspension test. When AS mice are tested in the open field test, the results should be interpreted with care, since motor dysfunctions influence mouse behavior in the maze and alter activity scores. The reproducibility and effectiveness of the presented behavioral tests has already been validated in several independent Uba3a mouse lines with different knockout variants, establishing this set of tests as an excellent validation tool in AS research. Models with the relevant construct and face validity will warrant further investigations to elucidate the pathophysiology of the disease and grant the development of causal treatments.

Autoimmune amelogenesis imperfecta in patients with APS-1 and coeliac disease.

Ameloblasts are specialized epithelial cells in the jaw that have an indispensable role in tooth enamel formation-amelogenesis1. Amelogenesis depends on multiple ameloblast-derived proteins that function as a scaffold for hydroxyapatite crystals. The loss of function of ameloblast-derived proteins results in a group of rare congenital disorders called amelogenesis imperfecta2. Defects in enamel formation are also found in patients with autoimmune polyglandular syndrome type-1 (APS-1), caused by AIRE deficiency3,4, and in patients diagnosed with coeliac disease5-7. However, the underlying mechanisms remain unclear. Here we show that the vast majority of patients with APS-1 and coeliac disease develop autoantibodies (mostly of the IgA isotype) against ameloblast-specific proteins, the expression of which is induced by AIRE in the thymus. This in turn results in a breakdown of central tolerance, and subsequent generation of corresponding autoantibodies that interfere with enamel formation. However, in coeliac disease, the generation of such autoantibodies seems to be driven by a breakdown of peripheral tolerance to intestinal antigens that are also expressed in enamel tissue. Both conditions are examples of a previously unidentified type of IgA-dependent autoimmune disorder that we collectively name autoimmune amelogenesis imperfecta.

Omega-3 PUFAs prevent bone impairment and bone marrow adiposity in mouse model of obesity.

Obesity adversely affects bone and fat metabolism in mice and humans. Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) have been shown to improve glucose metabolism and bone homeostasis in obesity. However, the impact of omega-3 PUFAs on bone marrow adipose tissue (BMAT) and bone marrow stromal cell (BMSC) metabolism has not been intensively studied yet. In the present study we demonstrated that omega-3 PUFA supplementation in high fat diet (HFD + F) improved bone parameters, mechanical properties along with decreased BMAT in obese mice when compared to the HFD group. Primary BMSCs isolated from HFD + F mice showed decreased adipocyte and higher osteoblast differentiation with lower senescent phenotype along with decreased osteoclast formation suggesting improved bone marrow microenvironment promoting bone formation in mice. Thus, our study highlights the beneficial effects of omega-3 PUFA-enriched diet on bone and cellular metabolism and its potential use in the treatment of metabolic bone diseases.

Metabolic switch from fatty acid oxidation to glycolysis in knock-in mouse model of Barth syndrome.

Mitochondria are central for cellular metabolism and energy supply. Barth syndrome (BTHS) is a severe disorder, due to dysfunction of the mitochondrial cardiolipin acyl transferase tafazzin. Altered cardiolipin remodeling affects mitochondrial inner membrane organization and function of membrane proteins such as transporters and the oxidative phosphorylation (OXPHOS) system. Here, we describe a mouse model that carries a G197V exchange in tafazzin, corresponding to BTHS patients. TAZG197V mice recapitulate disease-specific pathology including cardiac dysfunction and reduced oxidative phosphorylation. We show that mutant mitochondria display defective fatty acid-driven oxidative phosphorylation due to reduced levels of carnitine palmitoyl transferases. A metabolic switch in ATP production from OXPHOS to glycolysis is apparent in mouse heart and patient iPSC cell-derived cardiomyocytes. An increase in glycolytic ATP production inactivates AMPK causing altered metabolic signaling in TAZG197V . Treatment of mutant cells with AMPK activator reestablishes fatty acid-driven OXPHOS and protects mice against cardiac dysfunction.

A review of standardized high-throughput cardiovascular phenotyping with a link to metabolism in mice.

Cardiovascular diseases cause a high mortality rate worldwide and represent a major burden for health care systems. Experimental rodent models play a central role in cardiovascular disease research by effectively simulating human cardiovascular diseases. Using mice, the International Mouse Phenotyping Consortium (IMPC) aims to target each protein-coding gene and phenotype multiple organ systems in single-gene knockout models by a global network of mouse clinics. In this review, we summarize the current advances of the IMPC in cardiac research and describe in detail the diagnostic requirements of high-throughput electrocardiography and transthoracic echocardiography capable of detecting cardiac arrhythmias and cardiomyopathies in mice. Beyond that, we are linking metabolism to the heart and describing phenotypes that emerge in a set of known genes, when knocked out in mice, such as the leptin receptor (Lepr), leptin (Lep), and Bardet-Biedl syndrome 5 (Bbs5). Furthermore, we are presenting not yet associated loss-of-function genes affecting both, metabolism and the cardiovascular system, such as the RING finger protein 10 (Rfn10), F-box protein 38 (Fbxo38), and Dipeptidyl peptidase 8 (Dpp8). These extensive high-throughput data from IMPC mice provide a promising opportunity to explore genetics causing metabolic heart disease with an important translational approach.

Comprehensive ECG reference intervals in C57BL/6N substrains provide a generalizable guide for cardiac electrophysiology studies in mice.

Reference ranges provide a powerful tool for diagnostic decision-making in clinical medicine and are enormously valuable for understanding normality in pre-clinical scientific research that uses in vivo models. As yet, there are no published reference ranges for electrocardiography (ECG) in the laboratory mouse. The first mouse-specific reference ranges for the assessment of electrical conduction are reported herein generated from an ECG dataset of unprecedented scale. International Mouse Phenotyping Consortium data from over 26,000 conscious or anesthetized C57BL/6N wildtype control mice were stratified by sex and age to develop robust ECG reference ranges. Interesting findings include that heart rate and key elements from the ECG waveform (RR-, PR-, ST-, QT-interval, QT corrected, and QRS complex) demonstrate minimal sexual dimorphism. As expected, anesthesia induces a decrease in heart rate and was shown for both inhalation (isoflurane) and injectable (tribromoethanol) anesthesia. In the absence of pharmacological, environmental, or genetic challenges, we did not observe major age-related ECG changes in C57BL/6N-inbred mice as the differences in the reference ranges of 12-week-old compared to 62-week-old mice were negligible. The generalizability of the C57BL/6N substrain reference ranges was demonstrated by comparison with ECG data from a wide range of non-IMPC studies. The close overlap in data from a wide range of mouse strains suggests that the C57BL/6N-based reference ranges can be used as a robust and comprehensive indicator of normality. We report a unique ECG reference resource of fundamental importance for any experimental study of cardiac function in mice.

Spatial Analysis of Nucleotide Metabolism: From CRISPR Knockout Cancer Cells to MALDI Imaging of Tumors.

Cancer cells depend on nucleotides for proliferation. Inhibition of nucleotide metabolism by antimetabolites is a well-established anticancer therapy. However, resistance and toxicity to antimetabolite treatments reduce their effectiveness. Here, we focus on the pyrimidine de novo synthesis pathway, which is crucial for cancer cell proliferation, yet its pharmacological targeting in cancer has been without much clinical success so far. Hence, it is important to understand how cancer cells cope with the insufficiency of this pathway. Here, we describe a procedure to prepare subcutaneous tumor model deficient in de novo pyrimidine synthesis. For examination of metabolic responses to de novo synthesis blockade in tumors, we propose application of MALDI imaging that allows spatially resolved examination of metabolic responses to de novo synthesis blockade in tumors.

A previously uncharacterized Factor Associated with Metabolism and Energy (FAME/C14orf105/CCDC198/1700011H14Rik) is related to evolutionary adaptation, energy balance, and kidney physiology.

In this study we use comparative genomics to uncover a gene with uncharacterized function (1700011H14Rik/C14orf105/CCDC198), which we hereby name FAME (Factor Associated with Metabolism and Energy). We observe that FAME shows an unusually high evolutionary divergence in birds and mammals. Through the comparison of single nucleotide polymorphisms, we identify gene flow of FAME from Neandertals into modern humans. We conduct knockout experiments on animals and observe altered body weight and decreased energy expenditure in Fame knockout animals, corresponding to genome-wide association studies linking FAME with higher body mass index in humans. Gene expression and subcellular localization analyses reveal that FAME is a membrane-bound protein enriched in the kidneys. Although the gene knockout results in structurally normal kidneys, we detect higher albumin in urine and lowered ferritin in the blood. Through experimental validation, we confirm interactions between FAME and ferritin and show co-localization in vesicular and plasma membranes.

Retinitis pigmentosa-associated mutations in mouse Prpf8 cause misexpression of circRNAs and degeneration of cerebellar granule cells.

A subset of patients with retinitis pigmentosa (RP) carry mutations in several spliceosomal components including the PRPF8 protein. Here, we established two alleles of murine Prpf8 that genocopy or mimic aberrant PRPF8 found in RP patients-the substitution p.Tyr2334Asn and an extended protein variant p.Glu2331ValfsX15. Homozygous mice expressing the aberrant Prpf8 variants developed within the first 2 mo progressive atrophy of the cerebellum because of extensive granule cell loss, whereas other cerebellar cells remained unaffected. We further show that a subset of circRNAs were deregulated in the cerebellum of both Prpf8-RP mouse strains. To identify potential risk factors that sensitize the cerebellum for Prpf8 mutations, we monitored the expression of several splicing proteins during the first 8 wk. We observed down-regulation of all selected splicing proteins in the WT cerebellum, which coincided with neurodegeneration onset. The decrease in splicing protein expression was further pronounced in mouse strains expressing mutated Prpf8. Collectively, we propose a model where physiological reduction in spliceosomal components during postnatal tissue maturation sensitizes cells to the expression of aberrant Prpf8 and the subsequent deregulation of circRNAs triggers neuronal death.