Transgenic Mice Research Articles

Cyclin‐dependent kinase 13 is indispensable for normal mouse heart development

AbstractCongenital heart disease (CHD) has an incidence of approximately 1%. Over the last decade, sequencing studies including large cohorts of individuals with CHD have begun to unravel the genetic mechanisms underpinning CHD. This includes the identification of variants in cyclin‐dependent kinase 13 (CDK13), in individuals with syndromic CHD. CDK13 encodes a serine/threonine protein kinase. The cyclin partner of CDK13 is cyclin K; this complex is thought to be important in transcription and RNA processing. Pathogenic variants in CDK13 cause CDK13‐related disorder in humans, characterised by intellectual disability and developmental delay, recognisable facial features, feeding difficulties and structural brain defects, with 35% of individuals having CHD. To obtain a greater understanding for the role that this essential protein kinase plays in embryonic heart development, we have analysed a presumed loss of function Cdk13 transgenic mouse model (Cdk13tm1b). The homozygous mutants were embryonically lethal in most cases by E15.5. X‐gal staining showed Cdk13 expression localised to developing facial regions, heart and surrounding areas at E10.5, whereas at E12.5, it was more widely present. In the E15.5 heart, staining was seen throughout. RT‐qPCR showed significant reduction in Cdk13 transcript expression in homozygous compared with WT and heterozygous hearts at E10.5 and E12.5. Detailed morphological 3D analysis of embryonic and postnatal hearts was performed using high‐resolution episcopic microscopy, which affords a more detailed analysis of structures such as cardiac valve leaflets and endocardial cushions, compared with more traditional histological techniques. We show that both the homozygous and heterozygous Cdk13tm1b mutants exhibit a range of CHD, including ventricular septal defects, bicuspid aortic valve, double outlet right ventricle and atrioventricular septal defects. 100% (n = 4) of homozygous hearts displayed CHD. Differential expression was seen in Cdk13tm1b homozygous mutants for two genes known to be necessary for normal heart development. The types of defects, and the presence of CHD in heterozygous mice (17.02%, n = 8/47), are consistent with the CDK13‐related disorder phenotype in humans. This study provides important insights into the effects of reduced function of CDK13 in the mouse heart and contributes to our understanding of the mechanism behind this disorder as a cause of CHD.

Abstract A015: Aging limits stemness and tumor development in the lung by reprogramming iron homeostasis

Abstract Aging is the most important and well-recognized risk factor for cancer. Aging leads to systemic decline of physical fitness, significantly changing the macroenvironment from the early tumor initiation to the terminal stage of metastasis. However, the underlying mechanisms only start to emerge. Here, using physiologically aged autochthonous genetically engineered mouse models (GEMMs) and primary cells, we demonstrate that aging suppresses lung cancer initiation and progression by degrading stemness of the alveolar cell of origin. Using single-cell transcriptomics, we uncover a distinct compositional landscape of cancer cell subsets in aged lung tumors, revealing a delay in the molecular progression of the lung lesions and an age-specific gene expression signature. Interestingly, key components of this gene signature were traced back to the aged cell of origin. Furthermore, the age-specific signature derived from GEMMs was predictive of patient age in human lung cancer, indicating conservation of the age-associated changes across species. Interestingly, our preliminary data suggested that tumor cells derived from aged tumor-bearing mice displayed higher metastatic potential, indicating that different mechanisms may govern tumor initiation and metastasis. Functional interrogation of the age-specific gene signature revealed that the loss of stemness and tumorigenic capacity is underpinned by aging-associated induction of the transcription factor NUPR1. We find that NUPR1 is induced in the cell of origin in aged mice and humans, which leads to a functional iron insufficiency in the aged cells. Genetic inactivation of the NUPR1 or iron supplementation restores stemness and promotes tumorigenic potential of aged alveolar cells. Conversely, targeting NUPR1 is detrimental to young alveolar cells by promoting ferroptosis. We find that aging-associated DNA hypomethylation at specific enhancer sites results in elevated NUPR1 expression, which is recapitulated in young cells by inhibition of DNA methylation. We uncover that, unlike the young, aged alveolar cells are functionally iron insufficient, which leads to loss of stemness and resistance to ferroptosis. Our results indicate that aging-associated reprogramming of the cell of origin determines tumor initiation potential, alters trajectory of tumor evolution and produces age-dependent vulnerabilities. These findings demonstrate that aging fundamentally changes the biology of lung cancer, shedding light on novel therapeutic modulation of cellular iron homeostasis or ferroptosis in regenerative medicine and in cancer prevention for the elderly population. Citation Format: Xueqian Zhuang, Qing Wang, Simon Joost, Alexander Ferrena, Melissa Blum, Klavdija Krause, Zhuxuan Li, Deyou Zheng, Emily S Wong, Tuomas Tammela. Aging limits stemness and tumor development in the lung by reprogramming iron homeostasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A015.

Overexpression of autophagy enhancer PACER/RUBCNL in neurons accelerates disease in the SOD1G93A ALS mouse model

AbstractAmyotrophic lateral sclerosis (ALS) is a debilitating and fatal paralytic disorder associated with motor neuron death. Mutant superoxide dismutase 1 (SOD1) misfolding and aggregation have been linked to familial ALS, with the accumulation of abnormal wild-type SOD1 species being also observed in postmortem tissue of sporadic ALS cases. Both wild-type and mutated SOD1 are reported to contribute to motoneuron cell death. The autophagic pathway has been shown to be dysregulated in ALS. Recent evidence suggests a dual time-dependent role of autophagy in the progression of the disease. PACER, also called RUBCNL (Rubicon-like), is an enhancer of autophagy and has been found diminished in its levels during ALS pathology in mice and humans. Pacer loss of function disturbs the autophagy process and leads to the accumulation of SOD1 aggregates, as well as sensitizes neurons to death. Therefore, here we investigated if constitutive overexpression of PACER in neurons since early development is beneficial in an in vivo model of ALS. We generated a transgenic mouse model overexpressing human PACER in neurons, which then was crossbred with the mutant SOD1G93A ALS mouse model. Unexpectedly, PACER/SOD1G93A double transgenic mice exhibited an earlier disease onset and shorter lifespan than did littermate SOD1G93A mice. The overexpression of PACER in neurons in vivo and in vitro increased the accumulation of SOD1 aggregates, possibly due to impaired autophagy. These results suggest that similar to Pacer loss-of function, Pacer gain-of function is detrimental to autophagy, increases SOD1 aggregation and worsens ALS pathogenesis. In a wider context, our results indicate the requirement to maintain a fine balance of PACER protein levels to sustain proteostasis.

Abstract C007: Identifying the role of Interleukin-17 signaling in intestinal stem cells during pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC), the most aggressive type of pancreatic cancer, is predicted to become the second leading cause of cancer related deaths by 2030. Interleukin 17 (IL-17) signaling can promote tumor growth. In pancreatic cancer, IL-17 signaling in cancer cells is capable of triggering the formation of neutrophil extracellular traps (NETs) which is detrimental to the anti-tumor effect of immunotherapy. IL-17 signaling has been shown to have critical roles in maintaining gut homeostasis. Intestinal epithelial cells directly interact with the gut microbiome and are actively involved in infection control, injury repair, immune responses, and the production of antimicrobial peptides and cytokines, and need to be constantly replenished by intestinal stem cells (ISCs). Our lab’s study has previously shown that deletion of Interleukin 17 receptor A (IL-17RA) in the entire gut epithelium induces pro-tumorigenic IL-17 signaling due to the disruption of gut homeostasis, suggesting that immune responses induced by gut IL-17/IL-17RA signaling can affect the behavior of distant tumors. However, the mechanism of IL-17 signaling within specific gut epithelial compartments such as ISCs, enterocytes, secretory cells, etc. and their role in maintaining gut homeostasis and cancer risk are not well-studied until now. Thus, we aimed to identify the importance of IL-17 signaling in ISCs during pancreatic cancer. We hypothesized that disruption of IL-17/IL-17RA signaling within ISCs impacts tumor growth. Our preliminary data shows that IL-17 signaling in enterocytes maintains intestinal homeostasis and disruption of this axis triggers systemic increase in B cells that promotes tumor growth by increasing IL-17 levels in circulation. To identify the function of IL-17/IL-17RA signaling in ISCs, we utilized Leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5), a lineage marker for ISCs, to drive Cre enzyme tissue specific expression to specifically knockout Il17ra in gut ISCs - an inducible transgenic mouse model Il17ra fl/fl ;Lgr5- EGFP-CreERT2 (cKO). We used multiplex staining and imaging to validate the cell type specific loss of IL-17RA expression in tamoxifen treated cKO mice. We used implantable pancreatic tumor models and performed immunoprofiling using flow cytometry. We found that the pancreatic tumor growth was altered by the remote signal from gut ISCs in cKO mice. Furthermore, we performed RNAseq in both tumor and gut tissue which identified that the major changes in gene expression were associated with both innate and adaptive immune pathways, especially B cell activation. We conclude that the IL-17/IL-17RA signaling in gut ISCs changes intestinal immune responses locally that can affect distant tumor immunity. Citation Format: Haoyue Liu, Vidhi Chandra, Le Li, Olivereen Le Roux, Virginia Tahan, Florencia McAllister. Identifying the role of Interleukin-17 signaling in intestinal stem cells during pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C007.

A humanized neutralizing antibody protects against human adenovirus type 7 infection in humanized desmoglein-2 and CD46 double-receptor transgenic mice.

Human adenovirus type 7 (HAdV7) has become a major public health threat due to its widespread transmission, severe associated pneumonia, and a lack of effective anti-HAdV7 drugs. The aim of the current study is to design a humanized monoclonal antibody (mAb) demonstrating efficacy against HAdV-7 infections in vitro and in vivo. The humanized neutralizing antibody, 3G5-hu, was derived from the murine mAb 3G5. Antibody activity was evaluated using a flow cytometry-based neutralization (FCN) assay to identify humanized mAbs retaining potent neutralizing activity. Additionally, a humanized hDSG2/hCD46 dual-receptor transgenic mouse model was developed to simulate HAdV-7 infection. Using recombinant HAdV-7 expressing enhanced green fluorescent protein and clinically isolated wild-type HAdV-7, the half-maximal effective concentration of 3G5-hu against HAdV-7 was determined to be < 30 ng/mL. Notably, 3G5-hu exhibits high specificity for the hexon protein of the HAdV-7 capsid (affinity: KD = 9.02 × 10- 11 M). Microneutralization studies with wild-type HAdV-7 and rAd7EGFP confirmed that humanized mAb 3G5-hu neutralizes 10-30 ng/mL HAdV-7 (approximately 67-200 pM). Furthermore, hDSG2/hCD46 double-receptor transgenic mice are more susceptible to HAdV-7 infection than single-receptor transgenic mice. Meanwhile, the humanized mAb 3G5-hu provides good protection against HAdV-7 infection in hDSG2/hCD46 knock-in transgenic mice. The newly designed humanized mAb 3G5-hu specifically neutralizes HAdV-7 in vitro and in vivo. 3G5-hu elicits protection against HAdV-7 infection in hDSG2/hCD46 knock-in transgenic mice. The findings of this study provide insights to guide the future development of preventative and therapeutic treatments for HAdV-7 infection.

Neural Crest-Derived Mesenchymal Cells Support Thymic Reconstitution After Lethal Irradiation.

Reconstitution of the thymus is essential for assessing thymic function following injury. However, the currently employed cytoreductive regimes unvaryingly affect the thymic microenvironment, thereby impeding the recovery of T lymphopoiesis. The thymic stroma is composed of epithelial and mesenchymal cells. Thymic mesenchymal cells originate from the Neural crest (NC) and mesoderm and contribute to thymus organogenesis, yet their role in thymic regeneration is unclear. In this study, using transgenic mice expressing NC-specific Cre and Cre-driven DT receptors, we investigated the role of NC-derived mesenchymal cells in thymic regeneration following total body irradiation. We revealed that NC-derived mesenchymal cells have reduced susceptibility to irradiation and induce the upregulation of hematopoietic factors that promote thymus regeneration after irradiation. Additionally, using adult thymic organ culture and renal capsule transplantation, depletion of NC-derived mesenchymal cells resulted in a reduction of DN1-like early T-cell progenitors (ETP) and impaired thymic regeneration. Furthermore, among the numerous factors upregulated by NC-derived mesenchymal cells, Periostin and Flt3L were markedly increased after irradiation and promoted abundance of DN1-like ETPs during thymic reconstitution. Collectively, these findings highlight the importance of NC-derived mesenchymal cells in thymic regeneration.

Targeting resident astrocytes attenuates neuropathic pain after spinal cord injury.

Astrocytes derive from different lineages and play a critical role in neuropathic pain after spinal cord injury (SCI). Whether selectively eliminating these main origins of astrocytes in lumbar enlargement could attenuate SCI-induced neuropathic pain remains unclear. Through transgenic mice injected with an adeno-associated virus vector and diphtheria toxin, astrocytes in lumbar enlargement were lineage traced, targeted, and selectively eliminated. Pain-related behaviors were measured with an electronic von Frey apparatus and a cold/hot plate after SCI. RNA sequencing, bioinformatics analysis, molecular experiment, and immunohistochemistry were used to explore the potential mechanisms after astrocyte elimination. Lineage tracing revealed that the resident astrocytes but not ependymal cells were the main origins of astrocytes-induced neuropathic pain. SCI-induced mice to obtain significant pain symptoms and astrocyte activation in lumbar enlargement. Selective resident astrocyte elimination in lumbar enlargement could attenuate neuropathic pain and activate microglia. Interestingly, the type I interferons (IFNs) signal was significantly activated after astrocytes elimination, and the most activated Gene Ontology terms and pathways were associated with the type I IFNs signal which was mainly activated in microglia and further verified in vitro and in vivo. Furthermore, different concentrations of interferon and Stimulator of interferon genes (STING) agonist could activate the type I IFNs signal in microglia. These results elucidate that selectively eliminating resident astrocytes attenuated neuropathic pain associated with type I IFNs signal activation in microglia. Targeting type I IFNs signals is proven to be an effective strategy for neuropathic pain treatment after SCI.

Organophosphate Insecticide Malathion Induces Alzheimer's Disease-Like Cognitive Impairment in Mice: Evidence of the Microbiota-Gut-Brain Axis.

Evidence suggests that exposure to organophosphate pesticides increases the risk of neurodegenerative diseases, but the mechanisms remain unclear. This study investigated the effects of malathion on Alzheimer's disease (AD)-like symptoms at environmentally relevant concentrations using wild-type (WT) and APP/PS1 transgenic mouse models. Results showed that malathion exposure induced AD-like cognitive impairment, amyloid-β (Aβ) accumulation, and neuroinflammation in WT mice, with worsened symptoms in APP/PS1 mice. Mechanistic studies revealed that malathion induced AD-like gut microbiota dysbiosis (reduced Lactobacillus and Akkermansia, and increased Dubosiella), causing gut barrier impairment and tryptophan metabolism disruptions. This resulted in a significant increase in indole derivatives and activation of the colonic aryl hydrocarbon receptor (AhR), promoting the kynurenine (KYN) pathway while inhibiting the serotonin (5-HT) pathway. Increased neurotoxic KYN metabolites (3-hydroxykynurenine and quinolinic acid) triggered gut and systemic inflammation, upregulating hippocampal IL-6 and IL-1β mRNA levels and thereby causing neuroinflammation. Gut tryptophan metabolism disruptions caused hippocampal neurotransmitter imbalances, reducing the levels of 5-HT and its derivatives. These effects promoted AD progression in both WT and APP/PS1 mice. This study highlights the crucial role of the microbiota-gut-brain axis in AD-like cognitive impairment induced by malathion exposure, providing insights into the neurodegenerative disease risks posed by organophosphate pesticides.

Targeting resident astrocytes attenuates neuropathic pain after spinal cord injury

Astrocytes derive from different lineages and play a critical role in neuropathic pain after spinal cord injury (SCI). Whether selectively eliminating these main origins of astrocytes in lumbar enlargement could attenuate SCI-induced neuropathic pain remains unclear. Through transgenic mice injected with an adeno-associated virus vector and diphtheria toxin, astrocytes in lumbar enlargement were lineage traced, targeted, and selectively eliminated. Pain-related behaviors were measured with an electronic von Frey apparatus and a cold/hot plate after SCI. RNA sequencing, bioinformatics analysis, molecular experiment, and immunohistochemistry were used to explore the potential mechanisms after astrocyte elimination. Lineage tracing revealed that the resident astrocytes but not ependymal cells were the main origins of astrocytes-induced neuropathic pain. SCI-induced mice to obtain significant pain symptoms and astrocyte activation in lumbar enlargement. Selective resident astrocyte elimination in lumbar enlargement could attenuate neuropathic pain and activate microglia. Interestingly, the type I interferons (IFNs) signal was significantly activated after astrocytes elimination, and the most activated Gene Ontology terms and pathways were associated with the type I IFNs signal which was mainly activated in microglia and further verified in vitro and in vivo. Furthermore, different concentrations of interferon and Stimulator of interferon genes (STING) agonist could activate the type I IFNs signal in microglia. These results elucidate that selectively eliminating resident astrocytes attenuated neuropathic pain associated with type I IFNs signal activation in microglia. Targeting type I IFNs signals is proven to be an effective strategy for neuropathic pain treatment after SCI.

A novel mouse model recapitulating the MMR-defective SCLC subtype uncovers an actionable sensitivity to immune checkpoint blockade.

Small cell lung cancer (SCLC) has an extremely poor prognosis. Despite high initial response rates to chemotherapy and modest survival improvements with the addition of immune checkpoint inhibitors (ICI), almost all patients experience relapse and fatal outcomes. Recent genomic insights uncovered extensive molecular heterogeneity in addition to the almost uniform loss of RB1 and TRP53. Additionally, defective DNA mismatch repair (MMR) has recently been described in some SCLC cases. Here, we generated a novel SCLC mouse model capturing MMR deficiency and assessed immunotherapy responses. We developed an MMR-deficient genetically engineered mouse model (GEMM) of SCLC by introducing a conditional Msh2 gene, crucial for maintaining MMR integrity, into the standard Rb1fl/fl;Trp53fl/fl (RP) model. Genomic characteristics and preclinical therapy responses were evaluated by focusing on overall survival and whole exome sequencing (WES) analyses. MMR-defective SCLC tumors (Rb1fl/fl;Trp53fl/fl;Msh2fl/fl (RPM)) developed later than tumors in MMR-proficient mice. However, the time from tumor manifestation to death of the affected animals was substantially shortened (median survival 55 days in RP vs. 46.5 days in RPM), indicating increased aggressiveness of MMR-defective tumors. RPM tumors exhibited MMR deficiency, high tumor mutational burden (TMB), and an elevated load of candidate neoantigens, compared to RP lesions (p = 0.0106), suggesting increased immunogenicity. Importantly, the overall survival of RPM animals was significantly improved when exposed to ICI. We propose a novel RPM mouse model as a suitable system to mimic MMR-defective SCLC and tumors with high TMB. We provide in vivo evidence that Msh2 deficiency enhances ICI sensitivity. These findings could contribute to stratifying SCLC patients to immunotherapy, thereby improving treatment outcomes.

Astragaloside IV alleviates septic myocardial injury through DUSP1-Prohibitin 2 mediated mitochondrial quality control and ER-autophagy.

Septic cardiomyopathy (SCM) is a complication of myocardial injury in patients with severe sepsis. This study highlights the potential of Astragaloside IV(AS) in the treatment of septic cardiomyopathy and provides a reference for developing cardioprotective drugs targeting DUSP1-PHB2-related mitochondria-ER interaction. Dual specificity phosphatase-1 (DUSP1)/Prohibitin 2 cardiomyocyte-specific knockout mice (DUSP1/PHB2CKO) /DUSP1 transgenic mice (DUSP1/PHB2TG) were used to generate LPS-induced sepsis models. The pathological mechanism by which AS-IV improves heart injury was detected using cardiac ultrasound, fluorescence staining, transmission electron microscopy, and western blotting. After siRNA treatment of cardiomyocytes with DUSP-1/PHB2, changes in mitochondrial function and morphology were determined using qPCR, western blotting, ELISA, and laser confocal microscopy, and the targeted therapeutic effects of AS-IV were further examined. SCM treatment leads to severe mitochondrial dysfunction. However, Astragaloside IV (AS) treatment normalizes mitochondrial homeostasis and ER function. Notably, the protective effect was blocked in DUSP1/Prohibitin 2 cardiomyocyte-specific knockout mice (DUSP1/PHB2CKO) but remained unaffected in DUSP1 transgenic mice (DUSP1/PHB2TG). This study highlights the potential of AS in the treatment of septic cardiomyopathy and provides a reference for developing cardioprotective drugs targeting DUSP1-PHB2 related mitochondria-ER interaction.

Elimination of olfactory sensory neurons by zinc sulfate inoculation prevents SARS-CoV-2 infection of the brain in K18-hACE2 transgenic mice.

Coronavirus disease-2019 (COVID-19), attributed to the severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2), has posed global health challenges since it first emerged in 2019, and its impact continues to persist. The neurotropic nature of SARS-CoV-2 remains undisclosed, though researchers are proposing hypotheses on how the virus is transmitted to the central nervous system. One of the prevailing hypotheses is that SARS-CoV-2 travels through the olfactory nerve system via the olfactory epithelium (OE). Using a K18-human angiotensin converting-enzyme 2 (hACE2) transgenic mouse model with impaired olfactory sensory neurons (OSNs) induced by zinc sulfate, we examined the role of the olfactory nerve in the brain invasion by SARS-CoV-2. Mice lacking OSNs exhibited reduced levels of viral transmission to the brain, leading to significantly improved outcomes following SARS-CoV-2 infection. Moreover, a positive correlation was observed between viral persistence in the OE and brain infection. These results indicate that early inhibition of the olfactory nerve pathway effectively prevents viral invasion of the brain in K18-hACE2 mice. Our study underscores the significance of the olfactory nerve pathway in the transmission of SARS-CoV-2 to the brain.

Semaglutide promotes the transition of microglia from M1 to M2 type to reduce brain inflammation in APP/PS1/tau mice.

A growing number of studies show that the diabetes drug Semaglutide is neuroprotective in Alzheimer's disease (AD) animal models, but its mode of action is not fully understood. In order to explore the mechanism of Semaglutide, 7-month-old APP/PS1/tau transgenic (3xTg) mice and wild-type (WT) mice were randomly divided into four groups: control group (WT + PBS), AD model group (3xTg + PBS), Semaglutide control group (WT + Semaglutide) and Semaglutide treatment group (3xTg + Semaglutide). Semaglutide (25 nmol/kg) or PBS was administered intraperitoneally once every two days for 30 days, followed by behavioral and molecular experiments. The results show that Semaglutide can improve working memory and spatial reference memory of 3xTg-AD mice, promote the release of anti-inflammatory factors and inhibit the production of pro-inflammatory factors in the cortex and hippocampus, and reduce Aβ deposition in the hippocampal CA1 region of 3xTg mice. Semaglutide can inhibit the apoptosis of BV2 cells induced by Aβ1-42 in a dose-dependent manner and promote the transformation of microglia from M1 to M2, thereby exerting anti-inflammatory and neuroprotective effects. Therefore, we speculate that Semaglutide shows an anti-inflammatory effect by promoting the transformation of microglia from M1 to M2 type in the brain of 3xTg mice, and thus exerts a neuroprotective effect.

Conditional Activation of c-MYC in Distinct Catecholaminergic Cells Drives Development of Neuroblastoma or Somatostatinoma.

c-MYC is an important driver of high-risk neuroblastoma. A lack of c-MYC-driven genetically engineered mouse models (GEMM) has hampered the ability to better understand mechanisms of neuroblastoma oncogenesis and to develop effective therapies. Here, we showed that conditional c-MYC induction via Cre recombinase driven by a tyrosine hydroxylase (Th) promoter led to a preponderance of PDX1+ somatostatinoma, a type of pancreatic neuroendocrine tumor (PNET). However, c-MYC activation via an improved Cre recombinase driven by a dopamine β-hydroxylase (Dbh) promoter resulted in neuroblastoma development. The c-MYC murine neuroblastoma tumors recapitulated the pathologic and genetic features of human neuroblastoma and responded to anti-GD2 immunotherapy and DFMO, an FDA-approved inhibitor targeting the MYC transcriptional target ODC1. Thus, c-MYC overexpression results in different but related tumor types depending on the targeted cell. The GEMMs represent valuable tools for testing immunotherapies and targeted therapies for these diseases.

The PI3K-AKT-mTOR axis persists as a therapeutic dependency in KRASG12D-driven non-small cell lung cancer.

KRASG12C and KRASG12D inhibitors represent a major translational breakthrough for non-small cell lung cancer (NSCLC) and cancer in general by directly targeting its most mutated oncoprotein. However, resistance to these small molecules has highlighted the need for rational combination partners necessitating a critical understanding of signaling downstream of KRAS mutant isoforms. We contrasted tumor development between KrasG12C and KrasG12D genetically engineered mouse models (GEMMs). To corroborate findings and determine mutant subtype-specific dependencies, isogenic models of KrasG12C and KrasG12D initiation and adaptation were profiled by RNA sequencing. We also employed cell line models of established KRAS mutant NSCLC and determined therapeutic vulnerabilities through pharmacological inhibition. We analysed differences in survival outcomes for patients affected by advanced KRASG12C or KRASG12D-mutant NSCLC. KRASG12D exhibited higher potency in vivo, manifesting as more rapid lung tumor formation and reduced survival of KRASG12D GEMMs compared to KRASG12C. This increased potency, recapitulated in an isogenic initiation model, was associated with enhanced PI3K-AKT-mTOR signaling. However, KRASG12C oncogenicity and downstream pathway activation were comparable with KRASG12D at later stages of tumorigenesis in vitro and in vivo, consistent with similar clinical outcomes in patients. Despite this, established KRASG12D NSCLC models depended more on the PI3K-AKT-mTOR pathway, while KRASG12C models on the MAPK pathway. Specifically, KRASG12D inhibition was enhanced by AKT inhibition in vitro and in vivo. Our data highlight a unique combination treatment vulnerability and suggest that patient selection strategies for combination approaches using direct KRAS inhibitors should be i) contextualised to individual RAS mutants, and ii) tailored to their downstream signaling.

Spatially integrated cortico-subcortical tracing data for analyses of rodent brain topographical organization.

The cerebral cortex extends axonal projections to several subcortical brain regions, including the striatum, thalamus, superior colliculus, and pontine nuclei. Experimental tract-tracing studies have shown that these subcortical projections are topographically organized, reflecting the spatial organization of sensory surfaces and body parts. Several public collections of mouse- and rat- brain tract-tracing data are available, with the Allen mouse brain connectivity atlas being most prominent. There, a large body of image data can be inspected, but it is difficult to combine data from different experiments and compare spatial distribution patterns. To enable co-visualization and comparison of topographical organization in mouse brain cortico-subcortical projections across experiments, we represent axonal labelling data as point data in a common 3D brain atlas space. We here present a collection of point-cloud data representing spatial distribution of corticostriatal, corticothalamic, corticotectal, and corticopontine projections in mice and exemplify how these spatially integrated point data can be used as references for experimental investigations of topographic organization in transgenic mice, and for cross-species comparison with corticopontine projections in rats.

ER stress-induced YAP upregulation leads to chondrocyte phenotype loss in age-related osteoarthritis

BackgroundOsteoarthritis (OA) is a common degenerative joint disease, leading to pain and restricted mobility. Age-related endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of OA, but the underlying mechanisms remain unclear. This study aims to explore the relationship between age-related ER stress, YAP overexpression, and chondrocyte phenotype loss in the development of OA.MethodsCartilage samples were collected from patients undergoing amputation, and age-related ER stress markers and YAP expression were assessed using immunohistochemical staining and qPCR. Transgenic mice with cartilage-specific YAP overexpression (YAPOE) were created, and Pamrevlumab was administered to evaluate its therapeutic effects.ResultsHigher expression of ER stress markers and YAP were showed in aged tissues compared to younger tissues. YAP overexpression led to decreased levels of cartilage phenotype markers and increased osteogenesis-related proteins. In vivo, YAPOE mice exhibited OA-like cartilage degeneration, which was mitigated by Pamrevlumab treatment.ConclusionAge-related ER stress induces YAP overexpression, contributing to OA pathogenesis. Pamrevlumab effectively prevents this phenotype loss in YAPOE mice, suggesting its potential as a therapeutic agent for OA. These findings provide new insights into the molecular mechanisms of OA and highlight the importance of targeting the ER stress-YAP-CTGF signaling pathway in OA treatment and prevention.

Abstract 4132120: Down syndrome-trisomy-mediated gene, Dscr-1, protects against lifestyle-related vascular diseases

Epidemiological studies indicate that individuals with Down syndrome (DS) have an increased risk of leukemia and neuronal diseases but a significantly reduced incidence of most solid tumors and advanced vascular dysfunction. This suggests that one or a combination of trisomy genes on human chromosome 21 (HSA21) or murine chromosome 16 ( Mmu16 ) may be responsible for protecting against vasculopathy. Our previous research has shown that the Down syndrome critical region (Dscr)-1 gene, located on HSA21 or Mmu16 , encodes a feedback modulator of calcineurin-NFAT signaling in endothelial cells (ECs). Null mutation or overexpression of Dscr-1 has been found to increase or prevent septic mortality, angiogenesis, liver steatosis, and susceptibility of tumor metastasizing to the lung, respectively, demonstrating its significant role in these processes. In a unique approach, we crossed DS model mice with ApoE -null mice and fed them a high-fat diet (HFD) for 12 weeks to survey the effects of DS-related genes on atherosclerosis. The results were striking, with HFD-mediated increased LDL and triglyceride levels in ApoE -null mice significantly reduced in the combined DS plus ApoE -null mice. This innovative method allowed us to observe a remarkable (~35%) reduction in ApoE-null mediated atheroma formation in the DS model. This finding was also replicated in EC-specific DSCR-1 transgenic (DSCR-1 ECTg ) mice. Furthermore, to test the effect of HSA21, we have collected DS patient-derived trisomy and disomy iPS cells and executed them for the chromatin conformation capture analysis and the in vitro ECs differentiation experiments. DS-derived trisomy iPS cells already changed the whole chromatin structure, typically within the X chromosome. Moreover, the trisomy iPS-derived ECs revealed less proliferation compared to the disomy. RNA-seqs indicated trisomy iPS derived ECs dramatically (21-fold) increased DSCR-1 , but some HSA21 genes, Alzheimer’s related App and Bace2 , e.g., were inversely reduced the expression. Our comprehensive studies not only shed new light on the mechanisms of atherosclerosis in vascular pathology but also offer potential therapeutic strategies. The discovery of the Dscr-1 gene on the DS chromosome, which could be a powerful tool in combating lifestyle-related vascular diseases, underscores the significance of our research.

Abstract 4118993: β1 Adrenergic Receptor Autoantibodies Promote Heart Failure Though Activation of Prostaglandin E2 Receptor EP1/Phosphodiesterase 4B Pathway

Background: β1 adrenergic receptor (β1-AR) autoantibodies (β1-AA) can cause heart failure (HF) through activating of β1-AR; However, β-AR antagonists cannot completely reverse the cardiac injury induced by β1-AA and improve the survival rate of patients with β1-AA-positive HF. However, the β1-AR-independent mechanisms of heart failure induced by β1-AA are unclear. Methods: Proteomics of mice hearts was used to identify the specific changed proteins induced by β1-AA. The expression of phosphodiesterase 4B (PDE4B) was detected by western blot in neonatal mice mouse cardiomyocytes (NMCMs) and heart tissues. The binding proteins with β1-AA were enriched by immunoprecipitation (IP) and identified by mass spectrum. The interaction between β1-AA and prostaglandin E2 Receptor EP1 was detected by co-IP and surface plasmon resonance. The cardiac function and cardiac remodeling of PDE4B transgenic (TG) mice and EP1 knockout (KO) mice. Results: Firstly, PDE4B was changed the most proteins induced by β1-AA compared to β1 adrenergic receptor dobutamine. Moreover, the decrease of PDE4B induced by β1-AA cannot be reversed by metoprolol in vivo and in vitro. Secondly, the cAMP/PKA pathway and cardiac injury/dysfunction induced by β1-AA were inhibited after activation of PDE4B or PDE4B-TG in vivo and in vitro. Thirdly, β1-AA can bind with EP1 and activate Gq/IP3 pathway. The activation effect was abolished by EP1 antagonist. Lastly, blocked EP1 or EP1-KO will reverse the decreased PDE4B and cardiac injury/dysfunction induced by β1-AA. Conclusions: Our work reveals that β1-AA can activate of EP1/PDE4B pathway to result in heart failure.

Abstract 4134449: Increasing Histidyl dipeptides in Skeletal Muscle Preserves Muscle Mass and Function During Heart Failure.

Background: In heart failure patients muscle wasting is a serious comorbidity. Oxidative stress and inflammatory cytokines, which cause muscle wasting, are increased in the skeletal muscle of heart failure patients. Oxidative stress leads to oxidation of lipids, generating toxic lipid peroxidation products, such as acrolein, which activates TNFa. Whether lipid peroxidation products accumulate in muscle and their removal from muscle influences muscle atrophy during heart failure has not been studied. In the muscle there are histidyl dipeptides, such as carnosine, ranging between 5-10 mM, synthesized via enzyme carnosine synthase (CARNS) that bind lipid peroxidation products. We examined whether intramuscular histidyl dipeptides influences heart failure-induced muscle wasting. Hypothesis: Histidyl dipeptides, alleviates intramuscular inflammation, preserves muscle mass and function during heart failure. Methods: Wild type (WT) and muscle-specific CARNS transgenic (Tg) mice were subjected to sham and transverse aortic constriction (TAC) surgeries. Cardiac function, muscle strength, atrophic and inflammatory markers, aldehyde modified protein, CARNS, and histidyl dipeptides were measured after 12 weeks. Results: In heart failure mice, weight of gastrocnemius (sham: 18.64 ± 1.73 vs TAC: 14.10 ± 0.90 mg, p=0.005), soleus (sham: 6.06 ± 0.82 vs TAC: 4.14 ± 0.70 mg, p=0.001), and tibialis anterior (sham:10.76 ± 1.62 vs TAC: 6.22 ± 1.83 mg, p=0.003), were decreased. Muscle strength was diminished (sham: 2.81 ± 0.36 vs TAC: 1.52 ± 0.27 N, p=0.004). Atrophic marker, atrogin1, aldehyde protein adducts and TNF-a were increased in the gastrocnemius muscle of TAC mice (~2-3-fold vs sham, p&lt;0.05). CARNS expression and carnosine was decreased in different muscles of TAC mice (gastrocnemius; sham: 43.38 ± 20.46 vs TAC: 24.48 ± 6.49 nmoles/mg protein, p=0.03; soleus; sham: 22.04 ± 11.36 vs TAC: 11.02 ± 4.69 nmoles/mg protein, p=0.03; and tibialis; sham: 27.9 ± 11.70 vs TAC: 15.50 ± 3.48 nmoles/mg protein, p=0.01). Gastrocnemius muscle weight (WT TAC: 14.38 ± 1.08 vs Tg TAC: 17.30 ± 1.32 mg, p=0.004) and muscle strength (WT TAC: 3.06 ± 0.32 vs Tg TAC: 3.98 ± 0.41 N) were preserved, atrogin and TNFa expressions were decreased in gastrocnemius muscle of Tg TAC mice (~2-3-fold, p=0.04). Conclusion: Increasing intramuscular histidyl dipeptides alleviates muscle inflammation, preserves muscle mass, and function during heart failure.