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With the rapid development of CRISPR-Cas9 technology, gene editing has become a powerful tool for studying gene function. Specifically, in the study of the mechanisms by which natural immune responses combat viral infections, gene knockout mouse models have provided an indispensable platform. This article describes a detailed protocol for constructing gene knockout mice using the CRISPR-Cas9 system. This field focuses on the design of single-guide RNAs (sgRNAs) targeting the antiviral immune gene cGAS, embryo microinjection, and screening and verification of gene editing outcomes. Furthermore, this study provides methods for using cGAS gene knockout mice to analyze the role of specific genes in natural immune responses. Through this protocol, researchers can efficiently generate specific gene knockout mouse models, which not only helps in understanding the functions of the immune system but also offers a powerful experimental tool for exploring the mechanisms of antiviral innate immunity.
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Sistemas CRISPR-Cas , Edición Génica , Inmunidad Innata , Ratones Noqueados , ARN Guía de Sistemas CRISPR-Cas , Animales , Inmunidad Innata/genética , Ratones , ARN Guía de Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Técnicas de Inactivación de Genes/métodos , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Virosis/inmunología , Virosis/genéticaRESUMEN
Introduction: Bruton's tyrosine kinase (BTK) and interleukin (IL)-2 Inducible T-cell Kinase (ITK) inhibitors have anti-inflammatory properties. We investigated the therapeutic effect of ibrutinib, an orally bioavailable BTK/ITK inhibitor, in a mouse model of Graves' orbitopathy (GO). Methods: Genetic immunization was performed through intramuscular administration of the recombinant plasmid, pCMV6-hTSHR cDNA, to 8-week-old female BALB/c mice. Serum levels of T3, T4, and thyroid-stimulating hormone receptor (TSHR) antibodies (TRAbs) were quantified using enzyme-linked immunosorbent assay. Histopathological changes in orbital tissues were examined using immunohistochemistry (IHC) staining for TSHR and various inflammatory markers. Following successful genetic immunization, ibrutinib was orally administered daily for 2 weeks in the GO model mice. After treatment, the mRNA and protein expression levels of BTK, ITK, IL-1ß, and IL-6 in orbital tissues were evaluated using real-time PCR and Western blotting. Results: In total, 20 mice were sacrificed to confirm successful genetic immunization. The GO mouse group exhibited significantly increased serum T3, T4, and TRAb levels. IHC revealed increased expression of TSHR, IL-1ß, IL-6, transforming growth factor-ß1, interferon-γ, CD40, CD4, BTK, and ITK in the GO mouse model. The orbital inflammation was significantly attenuated in ibrutinib-treated mice. The mRNA and protein expression levels of BTK, ITK, IL-1ß, and IL-6 in orbital tissue were lower in ibrutinib-treated GO mouse group compared to the phosphate-buffered saline-treated GO mouse group. Conclusion: The GO mouse model demonstrated enhanced BTK and ITK expression. Ibrutinib, a BTK/ITK inhibitor, suppressed the inflammatory cytokine production. These findings highlight the potential involvement of BTK/ITK in the inflammatory pathogenesis of GO, suggesting its role as a novel therapeutic target.
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Adenina , Agammaglobulinemia Tirosina Quinasa , Modelos Animales de Enfermedad , Oftalmopatía de Graves , Inflamación , Ratones Endogámicos BALB C , Piperidinas , Pirimidinas , Animales , Oftalmopatía de Graves/tratamiento farmacológico , Oftalmopatía de Graves/metabolismo , Oftalmopatía de Graves/patología , Adenina/análogos & derivados , Piperidinas/uso terapéutico , Ratones , Femenino , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/patología , Pirimidinas/uso terapéutico , Pirazoles/uso terapéutico , Pirazoles/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Receptores de Tirotropina/metabolismo , Receptores de Tirotropina/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Melanoma is the most aggressive and deadly form of skin cancer that arises from the transformation of melanocytes, the pigment producing cells of the skin. In the year 2024 there will be approximately 10,000 new cases of melanoma diagnosed and approximately 8,000 deaths attributed to melanoma in the United States. In this study we treated a group of male and female transgenic mice that spontaneously develop metastatic melanoma, TGS, with a G-protein-coupled estrogen receptor agonist LNS8801 to assess the efficacy on disease progression. A second group of male and female TGS mice was also exposed to UVB irradiation to mimic exposure to sunlight. Over the course of the 32-week experiment, visible images were taken by the small animal imaging IVIS system to track tumor progression, and blood and tissue samples were collected for molecular analyses. Results showed that sex-biased effects were observed in the efficacy of LNS8801 and that LNS8801 shows a UV-protective influence in both male and female TGS mice.
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Over the past several decades, a trend toward delayed childbirth has led to increases in parental age at the time of conception. Sperm epigenome undergoes age-dependent changes increasing risks of adverse conditions in offspring conceived by fathers of advanced age. The mechanism(s) linking paternal age with epigenetic changes in sperm remain unknown. The sperm epigenome is shaped in a compartment protected by the blood-testes barrier (BTB) known to deteriorate with age. Permeability of the BTB is regulated by the balance of two mTOR complexes in Sertoli cells where mTOR complex 1 (mTORC1) promotes the opening of the BTB and mTOR complex 2 (mTORC2) promotes its integrity. We hypothesized that this balance is also responsible for age-dependent changes in the sperm epigenome. To test this hypothesis, we analyzed reproductive outcomes, including sperm DNA methylation in transgenic mice with Sertoli cell-specific suppression of mTORC1 (Rptor KO) or mTORC2 (Rictor KO). mTORC2 suppression accelerated aging of the sperm DNA methylome and resulted in a reproductive phenotype concordant with older age, including decreased testes weight and sperm counts, and increased percent of morphologically abnormal spermatozoa and mitochondrial DNA copy number. Suppression of mTORC1 resulted in the shift of DNA methylome in sperm opposite to the shift associated with physiological aging - sperm DNA methylome rejuvenation and mild changes in sperm parameters. These results demonstrate for the first time that the balance of mTOR complexes in Sertoli cells regulates the rate of sperm epigenetic aging. Thus, mTOR pathway in Sertoli cells may be used as a novel target of therapeutic interventions to rejuvenate the sperm epigenome in advanced-age fathers.
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Metilación de ADN , Células de Sertoli , Espermatozoides , Masculino , Animales , Células de Sertoli/metabolismo , Ratones , Espermatozoides/metabolismo , Espermatozoides/fisiología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Serina-Treonina Quinasas TOR/metabolismo , Ratones Noqueados , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/genética , Proteína Reguladora Asociada a mTOR/metabolismo , Proteína Reguladora Asociada a mTOR/genética , Ratones Transgénicos , Envejecimiento/fisiología , Envejecimiento/genética , Transducción de Señal , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Epigénesis GenéticaRESUMEN
The evidence of the correlation between cellular senescence and aging has increased in research with animal models. These models have been intentionally generated to target and regulate cellular senescent cells with the promoter activity of p16Ink4a or p19Arf, genes that are highly expressed in aging cells. However, the senolytic efficiency in various organs and cells from these models represents unexpected variation and diversity in some cases. We have generated a novel knock-in model, p16tdT-hDTR mice, which possess tdTomato and human diphtheria toxin receptor (hDTR) downstream of Cdkn2a, an endogenous p16Ink4a gene. We successfully demonstrated that p16-derived tdTomato and hDTR expressions are observed in these mouse embryo fibroblasts and following treatment with diphtheria toxin (DT) eliminates those cells. Furthermore, we demonstrated the efficacy of eliminating p16-positive cells in vivo, and also observed a tendency to decrease their cutaneous SA-ß-gal activity after subcutaneous DT injection into p16tdT-hDTR mice. In particular, comprehensive gene expression analysis in skin revealed that upregulated genes related to lipid metabolisms with aging exhibited remarkable expressions under the senolysis. These results clearly unveiled p16-positive senescent cells contribute to age-related changes in skin.
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Following bowel surgery, infectious complications, including anastomotic leak (AL), remain major sources of morbidity and mortality. Bowel preparation is often administered with the assumption that gut decontamination reduces post-surgical complications. In this study, we tested this hypothesis using a murine model of colon surgery. The mice were fed either regular chow or a high-fat, high-sugar Western diet. The day before surgery, the mice received one of four interventions: water (control), mechanical bowel preparation (MBP), oral antibiotics (OA), or both MBP and OA. We found no differences in the rates of AL among the experimental groups, and diet did not appear to affect the outcomes. Exploratory analyses showed changes in the gut microbiome consistent with the different treatments, but investigations of fecal short-chain fatty acids and RNA sequencing of colonic tissue did not reveal specific effects of the treatments or the presence of AL. However, we did identify bacterial genera that may be causally associated with AL and developed a predictive index from stool samples as a marker for the presence of AL. Future research is needed to identify and validate a microbial predictive tool and to uncover the microbial-driven mechanisms that lead to AL.
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Fuga Anastomótica , Microbioma Gastrointestinal , Animales , Fuga Anastomótica/etiología , Fuga Anastomótica/microbiología , Fuga Anastomótica/prevención & control , Microbioma Gastrointestinal/efectos de los fármacos , Ratones , Heces/microbiología , Colon/microbiología , Colon/cirugía , Masculino , Ratones Endogámicos C57BL , Antibacterianos/farmacología , Ácidos Grasos Volátiles/metabolismo , Ácidos Grasos Volátiles/análisis , Modelos Animales de EnfermedadRESUMEN
The significance of STING1 gene in tissue inflammation and cancer immunotherapy has been increasingly recognized. Intriguingly, common human STING1 alleles R71H-G230A-R293Q (HAQ) and G230A-R293Q (AQ) are carried by ~60% of East Asians and ~40% of Africans, respectively. Here, we examine the modulatory effects of HAQ, AQ alleles on STING-associated vasculopathy with onset in infancy (SAVI), an autosomal dominant, fatal inflammatory disease caused by gain-of-function human STING1 mutations. CD4 T cellpenia is evident in SAVI patients and mouse models. Using Sting1 knock-in mice expressing common human STING1 alleles HAQ, AQ, and Q293, we found that HAQ, AQ, and Q293 splenocytes resist STING1-mediated cell death ex vivo, establishing a critical role of STING1 residue 293 in cell death. The HAQ/SAVI(N153S) and AQ/SAVI(N153S) mice did not have CD4 T cellpenia. The HAQ/SAVI(N153S), AQ/SAVI(N153S) mice have more (~10-fold, ~20-fold, respectively) T-regs than WT/SAVI(N153S) mice. Remarkably, while they have comparable TBK1, IRF3, and NFκB activation as the WT/SAVI, the AQ/SAVI mice have no tissue inflammation, regular body weight, and normal lifespan. We propose that STING1 activation promotes tissue inflammation by depleting T-regs cells in vivo. Billions of modern humans have the dominant HAQ, AQ alleles. STING1 research and STING1-targeting immunotherapy should consider STING1 heterogeneity in humans.
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Alelos , Linfocitos T CD4-Positivos , Proteínas de la Membrana , Animales , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Linfocitos T CD4-Positivos/inmunología , Humanos , Inflamación/genética , Linfocitos T Reguladores/inmunología , Modelos Animales de EnfermedadRESUMEN
On September 27, 2023, the CureSHANK nonprofit foundation sponsored a conference in Boston, Massachusetts, to identify gaps in knowledge surrounding SHANK3-related epilepsy with the goal of determining future research priorities and recommendations. In addition to patient families and members of the CureSHANK community, participants in the conference included a broad cross-section of preclinical and clinical researchers and clinicians with expertise in SHANK3-related epilepsy as well as representatives from the pharmaceutical industry. Here we summarize the outcomes from comprehensive premeeting deliberations and the final conference recommendations, including (1) gaps in knowledge related to clinical science, (2) gaps in knowledge related to preclinical science, and (3) research priorities moving forward.
A roadmap for SHANK3-related Epilepsy Research: recommendations from the 2023 strategic planning workshop Phelan-McDermid Syndrome, a rare genetic disorder linked to the SHANK3 gene, manifests in a spectrum of clinical phenotypes including intellectual disability, autism spectrum disorder, and epilepsy. Epilepsy has been particularly under-investigated in this syndrome, and most of the animal models studied to date do not display seizures. On September 27, 2023, the CureSHANK nonprofit foundation sponsored a conference in Boston, Massachusetts, to identity gaps in knowledge surrounding SHANK3-related epilepsy. Conference attendees included patient families, basic scientists, clinical researchers, clinicians and representatives from the pharmaceutical industry with interest in SHANK3-related epilepsy. This review summarizes the outcome of this conference, including a summary of current state of knowledge and resources available, gaps in our understanding, priorities for future research in this important manifestation of PMS.
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Arpin was discovered as an inhibitor of the Arp2/3 complex localized at the lamellipodial tip of fibroblasts, where it regulated migration steering. Recently, we showed that arpin stabilizes the epithelial barrier in an Arp2/3-dependent manner. However, the expression and functions of arpin in endothelial cells (EC) have not yet been described. Arpin mRNA and protein are expressed in EC and downregulated by pro-inflammatory cytokines. Arpin depletion in Human Umbilical Vein Endothelial Cells causes the formation of actomyosin stress fibers leading to increased permeability in an Arp2/3-independent manner. Instead, inhibitors of ROCK1 and ZIPK, kinases involved in the generation of stress fibers, normalize the loss-of-arpin effects on actin filaments and permeability. Arpin-deficient mice are viable but show a characteristic vascular phenotype in the lung including edema, microhemorrhage, and vascular congestion, increased F-actin levels, and vascular permeability. Our data show that, apart from being an Arp2/3 inhibitor, arpin is also a regulator of actomyosin contractility and endothelial barrier integrity.
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Actomiosina , Permeabilidad Capilar , Células Endoteliales de la Vena Umbilical Humana , Animales , Humanos , Actomiosina/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Ratones , Serpinas/metabolismo , Serpinas/genética , Ratones Noqueados , Quinasas Asociadas a rho/metabolismo , Quinasas Asociadas a rho/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Fibras de Estrés/metabolismo , Células Endoteliales/metabolismo , Proteínas PortadorasRESUMEN
Tumor cell-derived prostaglandin E2 (PGE2) is a tumor cell-intrinsic factor that supports immunosuppression in the tumor microenvironment (TME) by acting on the immune cells, but the impact of PGE2 signaling in tumor cells on immunosuppressive TME is unclear. We demonstrate that deleting the PGE2 synthesis enzyme or disrupting autocrine PGE2 signaling through EP4 receptors on tumor cells reverses the T cell-low, myeloid cell-rich TME, activates T cells, and suppresses tumor growth. Knockout (KO) of Ptges (the gene encoding PGE2 synthesis enzyme mPGES-1) or the EP4 receptor gene (Ptger4) in KPCY (KrasG12D/P53R172H/Yfp/CrePdx) pancreatic tumor cells abolished growth of implanted tumors in a T cell-dependent manner. Blockade of the EP4 receptor in combination with immunotherapy, but not immunotherapy alone, induced complete tumor regressions and immunological memory. Mechanistically, Ptges and Ptger4 KO tumor cells exhibited altered T and myeloid cell attractant chemokines, became more susceptible to TNF-α killing, and exhibited reduced adenosine synthesis. In hosts treated with an adenosine deaminase inhibitor, Ptger4 KO tumor cells accumulated adenosine and gave rise to tumors. These studies reveal an unexpected finding - a non-redundant role for the autocrine mPGES1-PGE2-EP4 signaling axis in pancreatic cancer cells - further nominating mPGES-1 inhibition and EP4 blockade as immune-sensitizing therapy in cancer.
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The gut microbiota influences physiological functions of the host, ranging from the maintenance of local gut homeostasis to systemic immunity and metabolism. Secreted phospholipase A2 group X (sPLA2-X) is abundantly expressed in colonic epithelial cells but is barely detectable in metabolic and immune tissues. Despite this distribution, sPLA2-X-deficient (Pla2g10-/-) mice displayed variable obesity-related phenotypes that were abrogated after treatment with antibiotics or cohousing with Pla2g10+/+ mice, suggesting the involvement of the gut microbiota. Under housing conditions where Pla2g10-/- mice showed aggravation of diet-induced obesity and insulin resistance, they displayed increased colonic inflammation and epithelial damage, reduced production of polyunsaturated fatty acids (PUFAs) and lysophospholipids, decreased abundance of several Clostridium species, and reduced levels of short-chain fatty acids (SCFAs). These obesity-related phenotypes in Pla2g10-/- mice were reversed by dietary supplementation with ω3 PUFAs or SCFAs. Thus, colonic sPLA2-X orchestrates ω3 PUFA-SCFA interplay via modulation of the gut microbiota, thereby secondarily affecting systemic metabolism.
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The Trp53 gene encodes several isoforms of elusive biological significance. Here, we show that mice lacking the Trp53 alternatively spliced (AS) exon, thereby expressing the canonical p53 protein but not isoforms with the AS C-terminus, have unexpectedly lost a male-specific protection against Myc-induced B-cell lymphomas. Lymphomagenesis was delayed in Trp53+/+Eµ-Myc males compared to Trp53ΔAS/ΔAS Eµ-Myc males, but also compared to Trp53+/+Eµ-Myc and Trp53ΔAS/ΔAS Eµ-Myc females. Pre-tumoral splenic cells from Trp53+/+Eµ-Myc males exhibited a higher expression of Ackr4, encoding an atypical chemokine receptor with tumor suppressive effects. We identified Ackr4 as a p53 target gene whose p53-mediated transactivation is inhibited by estrogens, and as a male-specific factor of good prognosis relevant for murine Eµ-Myc-induced and human Burkitt lymphomas. Furthermore, the knockout of ACKR4 increased the chemokine-guided migration of Burkitt lymphoma cells. These data demonstrate the functional relevance of alternatively spliced p53 isoforms and reveal sex disparities in Myc-driven lymphomagenesis.
Human cells divide many times during a lifetime, a process that requires careful regulation to avoid uncontrolled cell division, which can lead to various disorders, including cancer. For example, TP53, which encodes multiple proteins, is the most commonly mutated gene in cancers. TP53 carries the instructions to make a tumor suppressor protein, known as p53, which can stop cancers from forming and spreading. In humans and mice, TP53 (and the mouse analogue Trp53) can also be read by the cell to make several slightly different versions of the p53 protein, known as isoforms. The p53 isoforms are much less studied and their role in an organism is still unclear. To address this, Fajac et al. used genome editing to make mouse strains that were still able to express p53, but were only able to create a specific subset of p53 isoforms. In these mice, part of the Trp53 gene had been mutated to remove the cell's ability to make isoforms with an alternative C-terminal end. Fajac et al. then allowed these mice to breed with mice that were model organisms for a cancer called B-cell lymphoma. This revealed that male offspring that lacked alternative p53 isoforms were more susceptible to B-cell lymphoma and that they had decreased levels of the protein ACKR4, a receptor for signaling proteins that regulate cellular movement. Human datasets showed that having higher levels of ACKR4 could be linked to a better disease prognosis in male patients with Burkitt lymphoma, a rare but aggressive form of B-cell lymphoma. The same effect was not observed in females, suggesting that measuring ACKR4 gene expression in male patients with Burkitt lymphoma might be useful to identify the patients at higher risk. The study from Fajac et al. provides a new perspective on p53 one of the most studied proteins. It highlights specific p53 isoforms and the ACKR4 protein as a potential way to identify male patients at higher risk from a type of B-cell lymphoma.
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Empalme Alternativo , Isoformas de Proteínas , Proteína p53 Supresora de Tumor , Animales , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ratones , Masculino , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Femenino , Linfoma de Células B/genética , Pronóstico , Humanos , Factores Sexuales , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
SNCA/PARK1 encodes α-synuclein, which is associated with familial Parkinson's disease. Despite its abundance in presynaptic terminals, the aggregation mechanism of α-synuclein and its relationship with Parkinson's disease have not yet been elucidated. Moreover, the ultrastructures of α-synuclein localization sites in neuronal presynaptic terminals remain unclear. Therefore, we herein generated transgenic mice expressing human α-synuclein tagged with mKate2 (hSNCA-mKate2 mice). These mice exhibited normal growth and fertility and had no motor dysfunction relative to their wild-type littermates, even at one year old. α-Synuclein-mKate2 accumulated in presynaptic terminals, particularly between Purkinje cells in the cerebellum and neurons in cerebellar nuclei. α-Synuclein-mKate2 was associated with the presynaptic marker, synaptophysin. In-resin CLEM and immunoelectron or electron microscopy revealed that α-synuclein-mKate2 localized on the surface of synaptic vesicles that were tightly arranged and assembled to form large synaptic pools in the cerebellum with negligible effects on the active zone. These results suggest that α-synuclein-associated ultrastructures in the presynaptic terminals of hSNCA-mKate2 mice reflect the structures of α-synuclein-assembled synaptic vesicle pools, and the size of vesicle pools increased. This transgenic mouse model will be a valuable tool for studying α-synuclein-associated synaptic vesicle pools.
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Ratones Transgénicos , Terminales Presinápticos , Vesículas Sinápticas , alfa-Sinucleína , Animales , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructura , Ratones , Humanos , Células de Purkinje/metabolismo , Células de Purkinje/ultraestructura , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Cerebelo/metabolismo , Cerebelo/ultraestructura , Sinaptofisina/metabolismo , Sinaptofisina/genética , MasculinoRESUMEN
There is an association between noise exposure and cognitive impairment, and noise may have a more severe impact on patients with Alzheimer's disease (AD) and mild cognitive impairment; however, the mechanisms need further investigation. This study used the classic AD animal model APP/PS1 mice to simulate the AD population, and C57BL/6J mice to simulate the normal population. We compared their cognitive abilities after noise exposure, analyzed changes in Cluster of Differentiation (CD) between the two types of mice using transcriptomics, identified the differential CD molecule: CD36 in APP/PS1 after noise exposure, and used its pharmacological inhibitor to intervene to explore the mechanism by which CD36 affects APP/PS1 cognitive abilities. Our study shows that noise exposure has a more severe impact on the cognitive abilities of APP/PS1 mice, and that the expression trends of differentiation cluster molecules differ significantly between C57BL/6J and APP/PS1 mice. Transcriptomic analysis showed that the expression of CD36 in the hippocampus of APP/PS1 mice increased by 2.45-fold after noise exposure (p < 0.001). Meanwhile, Western Blot results from the hippocampus and entorhinal cortex indicated that CD36 protein levels increased by approximately 1.5-fold (p < 0.001) and 1.3-fold (p < 0.05) respectively, after noise exposure in APP/PS1 mice. The changes in CD36 expression elevated oxidative stress levels in the hippocampus and entorhinal cortex, leading to a decrease in PI3K/AKT phosphorylation, which in turn increased M1-type microglia and A1-type astrocytes while reducing the numbers of M2-type microglia and A2-type astrocytes. This increased neuroinflammation in the hippocampus and entorhinal cortex, causing synaptic and neuronal damage in APP/PS1 mice, ultimately exacerbating cognitive impairment. These findings may provide new insights into the relationship between noise exposure and cognitive impairment, especially given the different expression trends of CD molecules in the two types of mice, which warrants further research.
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Antígenos CD36 , Disfunción Cognitiva , Ratones Endogámicos C57BL , Ruido , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Animales , Ratones , Antígenos CD36/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ruido/efectos adversos , Fosfatidilinositol 3-Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Enfermedad de Alzheimer/metabolismo , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Ratones TransgénicosRESUMEN
Cue-potentiated feeding (CPF) describes instances where food intake is increased by exposure to conditioned cues associated with food, often in the absence of hunger. CPF effects have been reported in a range of experimental protocols developed by researchers working across diverse fields spanning behavioural neuroscience, social psychology and ecology. Here we review the evolution of research on cue-potentiated feeding in animal models to identify important behavioural parameters and key neural circuits and pharmacological systems underlying the effect. Overall, evidence indicates that social, discrete and contextual stimuli can be used to elicit CPF effects across multiple species, though effects are often subtle and sensitive to procedural variables. While regular exposure to food cues is thought to be a key risk factor for overeating in so-called 'obesogenic' environments, further work is needed to identify whether CPF promotes positive energy balance and weight gain over the longer term. We suggest several methodological and conceptual areas for inquiry to elucidate the contribution of CPF to the regulation of food choice and energy intake.
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AMP-activated protein kinase (AMPK) is an αßγ heterotrimer protein kinase that functions as a molecular sensor to maintain energy homeostasis. Accumulating evidence suggests a role of AMPK signaling in the regulation of synaptic plasticity and cognitive function; however, isoform-specific roles of AMPK in the central nervous system (CNS) remain elusive. Regulation of the AMPK activities has focused on the manipulation of the α or γ subunit. Meanwhile, accumulating evidence indicates that the ß subunit is critical for sensing nutrients such as fatty acids and glycogen to control AMPK activity. Here, we generated transgenic mice with conditional suppression of either AMPKß1 or ß2 in neurons and characterized potential isoform-specific roles of AMPKß in cognitive function and underlying mechanisms. We found that AMPKß2 (but not ß1) suppression resulted in impaired recognition memory, reduced hippocampal synaptic plasticity, and altered structure of hippocampal postsynaptic densities and dendritic spines. Our study implicates a role for the AMPKß2 isoform in the regulation of synaptic and cognitive function.
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Ischemic stroke is followed by an increased susceptibility to bacterial infections, which exacerbate histological stroke outcome, neurological deficits and memory impairment due to increased neuroinflammation and neurotransmitter dysfunction. Pharmacological activation of nicotinic acetylcholine receptors was suggested to mitigate brain inflammatory responses in ischemic stroke. The functional responses associated with nicotinic acetylcholine receptor activation were unknown. In this study, male NMRI mice subjected to transient intraluminal middle cerebral artery occlusion (MCAO) were intraperitoneally exposed to vehicle treatment or Escherichia coli lipopolysaccharide (LPS; 4 mg/kg)-induced sepsis-like state 24 h post-MCAO, followed by intraperitoneal administration of vehicle or nicotine (0.5 mg/kg) 30 min later. Over 96 h, rectal temperature, neurological deficits, spontaneous locomotor activity, working memory, ischemic injury, synaptic plasticity, and brain inflammatory responses were evaluated by temperature measurement, behavioral analysis, infarct volumetry, electrophysiological recordings, and polymerase-chain reaction analysis. LPS-induced sepsis induced hypothermia, increased general and focal neurological deficits, reduced spontaneous exploration behavior, reduced working memory, and increased infarct volume post-MCAO. Additional treatment with nicotine attenuated LPS-induced hypothermia, reduced neurological deficits, restored exploration behavior, restored working memory, and reduced infarct volume. Local field potential recordings revealed that LPS-induced sepsis decreased long-term potentiation (LTP) in the dentate gyrus post-MCAO, whereas concomitant nicotine exposure restored LTP in the contralateral dentate gyrus. LPS-induced sepsis increased microglial/ macrophage Iba-1 mRNA and astrocytic GFAP mRNA levels post-MCAO, whereas add-on nicotine treatment reduced astrocytic GFAP mRNA. Taken together, these findings indicate that acute nicotine exposure enhances functional stroke recovery. Future studies will have to evaluate the effects of (1) chronic nicotine exposure, a clinically relevant vascular risk factor, and (2) the cessation of nicotine exposure, which is widely recommended post-stroke, but might have detrimental effects in the early stroke recovery phase.
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BACKGROUND: Intracranial aneurysm (IA) is a severe cerebrovascular disease, and effective gene therapy and drug interventions for its treatment are still lacking. Oxidative stress (OS) is closely associated with the IA, but the key regulatory genes involved are still unclear. Through multiomics analysis and experimental validation, we identified two diagnostic markers for IA associated with OS. METHODS: In this study, we first analyzed the IA dataset GSE75436 and conducted a joint analysis of oxidative stress-related genes (ORGs). Differential analysis, functional enrichment analysis, immune infiltration, WGCNA, PPI, LASSO, and other methods were used to identify IA diagnostic markers related to OS. Next, the functions of TLR4 and ALOX5 expression in IA and their potential targeted therapeutic drugs were analyzed. We also performed single-cell sequencing of patient IA and control (superficial temporal artery, STA) tissues. 23,342 cells were captured from 2 IA and 3 STA samples obtained from our center. Cell clustering and annotation were conducted using R software to observe the distribution of TLR4 and ALOX5 expression in IAs. Finally, the expression of TLR4 and ALOX5 were validated in IA patients and in an elastase-induced mouse IA model using experiments such as WB and immunofluorescence. RESULTS: Through bioinformatics analysis, we identified 16 key ORGs associated with IA pathogenesis. Further screening revealed that ALOX5 and TLR4 were highly expressed to activate a series of inflammatory responses and reduce the production of myocytes. Methotrexate (MTX) may be a potential targeted drug. Single-cell analysis revealed a notable increase in immune cells in the IA group, with ALOX5 and TLR4 primarily localized to monocytes/macrophages. Validation through patient samples and mouse models confirmed high expression of ALOX5 and TLR4 in IAs. CONCLUSIONS: Bioinformatics analysis indicated that ALOX5 and TLR4 are the most significant ORGs associated with the pathogenesis of IA. Single-cell sequencing and experiments revealed that the high expression of ALOX5 and TLR4 are closely related to IA. These two genes are promising new targets for IA therapy.
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Araquidonato 5-Lipooxigenasa , Biomarcadores , Aneurisma Intracraneal , Estrés Oxidativo , Receptor Toll-Like 4 , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/genética , Aneurisma Intracraneal/metabolismo , Aneurisma Intracraneal/genética , Animales , Ratones , Humanos , Estrés Oxidativo/fisiología , Araquidonato 5-Lipooxigenasa/metabolismo , Araquidonato 5-Lipooxigenasa/genética , Araquidonato 5-Lipooxigenasa/biosíntesis , Biomarcadores/metabolismo , Masculino , Ratones Endogámicos C57BL , Femenino , MultiómicaRESUMEN
In the ongoing battle against coronavirus disease 2019 (COVID-19), understanding its pathogenesis and developing effective treatments remain critical challenges. The creation of animal models that closely replicate human infection stands as a critical step forward in this research. Here, we present a genetically engineered mouse model with specifically-humanized knock-in ACE2 (hiACE2) receptors. This model, featuring nine specific amino acid substitutions for enhanced interaction with the viral spike protein, enables efficient severe acute respiratory syndrome coronavirus 2 replication in respiratory organs without detectable infection in the central nervous system. Moreover, it mirrors the age- and sex-specific patterns of morbidity and mortality, as well as the immunopathological features observed in human COVID-19 cases. Our findings further demonstrate that the depletion of eosinophils significantly reduces morbidity and mortality, depending on the infecting viral dose and the sex of the host. This reduction is potentially achieved by decreasing the pathogenic contribution of eosinophil-mediated inflammation, which is strongly correlated with neutrophil activity in human patients. This underscores the model's utility in studying the immunopathological aspects of COVID-19 and represents a significant advancement in COVID-19 modeling. It offers a valuable tool for testing vaccines and therapeutics, enhancing our understanding of the disease mechanisms and potentially guiding more targeted and effective treatments.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , COVID-19 , Modelos Animales de Enfermedad , Eosinófilos , SARS-CoV-2 , Animales , COVID-19/inmunología , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Ratones , Humanos , Femenino , Masculino , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Eosinófilos/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Factores Sexuales , Factores de Edad , Replicación Viral , Técnicas de Sustitución del GenRESUMEN
The genes Ocm (encoding oncomodulin) and Slc26a5 (encoding prestin) are expressed strongly in outer hair cells and both are involved in deafness in mice. However, it is not clear if they influence the expression of each other. In this study, we characterise the auditory phenotype resulting from two new mouse alleles, Ocmtm1e and Slc26a5tm1Cre. Each mutation leads to absence of detectable mRNA transcribed from the mutant allele, but there was no evidence that oncomodulin regulates expression of prestin or vice versa. The two mutants show distinctive patterns of auditory dysfunction. Ocmtm1e homozygotes have normal auditory brainstem response thresholds at 4 weeks old followed by progressive hearing loss starting at high frequencies, while heterozygotes show largely normal thresholds until 6 months of age, when signs of worse thresholds are detected. In contrast, Slc26a5tm1Cre homozygotes have stable but raised thresholds across all frequencies tested, 3 to 42 kHz, at least from 4 to 8 weeks old, while heterozygotes have raised thresholds at high frequencies. Distortion product otoacoustic emissions and cochlear microphonics show deficits similar to auditory brainstem responses in both mutants, suggesting that the origin of hearing impairment is in the outer hair cells. Endocochlear potentials are normal in the two mutants. Scanning electron microscopy revealed normal development of hair cells in Ocmtm1e homozygotes but scattered outer hair cell loss even at 4 weeks old when thresholds appeared normal, indicating that there is not a direct relationship between numbers of outer hair cells present and auditory thresholds.