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Multiple system atrophy (MSA) is a severe α-synucleinopathy facilitated by glial reactions; the cerebellar variant (MSA-C) preferentially involves olivopontocerebellar fibres with conspicuous demyelination. A lack of aggressive models that preferentially involve olivopontocerebellar tracts in adulthood has hindered our understanding of the mechanisms of demyelination and neuroaxonal loss, and thus the development of effective treatments for MSA. We therefore aimed to develop a rapidly progressive mouse model that recaptures MSA-C pathology. We crossed Plp1-tTA and tetO-SNCA*A53T mice to generate Plp1-tTA::tetO-SNCA*A53T bi-transgenic mice, in which human A53T α-synuclein-a mutant protein with enhanced aggregability-was specifically produced in the oligodendrocytes of adult mice using Tet-Off regulation. These bi-transgenic mice expressed mutant α-synuclein from 8â¯weeks of age, when doxycycline was removed from the diet. All bi-transgenic mice presented rapidly progressive motor deterioration, with wide-based ataxic gait around 22â¯weeks of age and death around 30â¯weeks of age. They also had prominent demyelination in the brainstem/cerebellum. Double immunostaining demonstrated that myelin basic protein was markedly decreased in areas in which SM132, an axonal marker, was relatively preserved. Demyelinating lesions exhibited marked ionised calcium-binding adaptor molecule 1-, arginase-1-, and toll-like receptor 2-positive microglial reactivity and glial fibrillary acidic protein-positive astrocytic reactivity. Microarray analysis revealed a strong inflammatory response and cytokine/chemokine production in bi-transgenic mice. Neuronal nuclei-positive neuronal loss and patchy microtubule-associated protein 2-positive dendritic loss became prominent at 30â¯weeks of age. However, a perceived decrease in tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta in bi-transgenic mice compared with wild-type mice was not significant, even at 30â¯weeks of age. Wild-type, Plp1-tTA, and tetO-SNCA*A53T mice developed neither motor deficits nor demyelination. In bi-transgenic mice, double immunostaining revealed human α-synuclein accumulation in neurite outgrowth inhibitor A (Nogo-A)-positive oligodendrocytes beginning at 9â¯weeks of age; its expression was further increased at 10 to 12â¯weeks, and these increased levels were maintained at 12, 24, and 30â¯weeks. In an α-synuclein-proximity ligation assay, α-synuclein oligomers first appeared in brainstem oligodendrocytes as early as 9â¯weeks of age; they then spread to astrocytes, neuropil, and neurons at 12 and 16â¯weeks of age. α-Synuclein oligomers in the brainstem neuropil were most abundant at 16â¯weeks of age and decreased thereafter; however, those in Purkinje cells successively increased until 30â¯weeks of age. Double immunostaining revealed the presence of phosphorylated α-synuclein in Nogo-A-positive oligodendrocytes in the brainstem/cerebellum as early as 9â¯weeks of age. In quantitative assessments, phosphorylated α-synuclein gradually and successively accumulated at 12, 24, and 30â¯weeks in bi-transgenic mice. By contrast, no phosphorylated α-synuclein was detected in wild-type, tetO-SNCA*A53T, or Plp1-tTA mice at any age examined. Pronounced demyelination and tubulin polymerisation, promoting protein-positive oligodendrocytic loss, was closely associated with phosphorylated α-synuclein aggregates at 24 and 30â¯weeks of age. Early inhibition of mutant α-synuclein expression by doxycycline diet at 23â¯weeks led to fully recovered demyelination; inhibition at 27â¯weeks led to persistent demyelination with glial reactions, despite resolving phosphorylated α-synuclein aggregates. In conclusion, our bi-transgenic mice exhibited progressively increasing demyelination and neuroaxonal loss in the brainstem/cerebellum, with rapidly progressive motor deterioration in adulthood. These mice showed marked microglial and astrocytic reactions with inflammation that was closely associated with phosphorylated α-synuclein aggregates. These features closely mimic human MSA-C pathology. Notably, our model is the first to suggest that α-synuclein oligomers may spread from oligodendrocytes to neurons in transgenic mice with human α-synuclein expression in oligodendrocytes. This model of MSA is therefore particularly useful for elucidating the in vivo mechanisms of α-synuclein spreading from glia to neurons, and for developing therapies that target glial reactions and/or α-synuclein oligomer spreading and aggregate formation in MSA.
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STAT3 gain-of-function (GOF) variants results in a heterogeneous clinical syndrome characterized by early onset immunodeficiency, multi-organ autoimmunity, and lymphoproliferation. While 191 documented cases with STAT3 GOF variants have been reported, the impact of individual variants on immune regulation and the broad clinical spectrum remains unclear. We developed a Stat3p.L387R mouse model, mirroring a variant identified in a family exhibiting common STAT3 GOF symptoms, and rare phenotypes including pulmonary hypertension and retinal vasculitis. In vitro experiments revealed increased STAT3 phosphorylation, nuclear migration, and DNA binding of the variant. Our Stat3p.L387R model displayed similar traits from previous Stat3GOF strains, such as splenomegaly and lymphadenopathy. Notably, Stat3p.L387R/+ mice exhibited heightened embryonic lethality compared to prior Stat3GOF/+ models and ocular abnormalities were observed. This research underscores the variant-specific pathology in Stat3p.L387R/+ mice, highlighting the ability to recapitulate human STAT3 GOF syndrome in patient-specific transgenic murine models. Additionally, such models could facilitate tailored treatment development.
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Modelos Animales de Enfermedad , Mutación con Ganancia de Función , Factor de Transcripción STAT3 , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Animales , Ratones , Humanos , Mutación con Ganancia de Función/genética , Femenino , Masculino , Ratones Transgénicos , Fenotipo , Fosforilación , Ratones Endogámicos C57BLRESUMEN
Idiosyncratic drug toxicities (IDTs) pose a significant challenge; they are marked by life-threatening adverse reactions that emerge aftermarket release and are influenced by intricate genetic and environmental variations. Recent genome-wide association studies have highlighted a strong correlation between specific human leukocyte antigen (HLA) polymorphisms and IDT onset. This review provides an overview of current research on HLA-mediated drug toxicities. In the last six years, HLA-transgenic (Tg) mice have been instrumental in advancing our understanding of these underlying mechanisms, uncovering systemic immune reactions that replicate human drug-induced immune stimulation. Additionally, the potential role of immune tolerance in shaping individual differences in adverse effects highlights its relevance to the interplay between HLA polymorphisms and IDTs. Although HLA-Tg mice offer valuable insights into systemic immune reactions, further exploration is essential to decipher the intricate interactions that lead to organ-specific adverse effects, especially in organs such as the skin or liver. Navigating the intricate interplay of HLA, which may potentially trigger intracellular immune responses, this review emphasizes the need for a holistic approach that integrates findings from both animal models and molecular/cellular investigations. The overarching goal is to enhance our comprehensive understanding of HLA-mediated IDTs and identify factors shaping individual variations in drug reactions. This review aims to facilitate the development of strategies to prevent severe adverse effects, address existing knowledge gaps, and provide guidance for future research initiatives in the field of HLA-mediated IDTs.
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Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Antígenos HLA , Animales , Humanos , Antígenos HLA/genética , Antígenos HLA/inmunología , Ratones Transgénicos , Polimorfismo Genético , RatonesRESUMEN
Plasmalogens (vinyl-ether phospholipids) are an emergent class of lipid drugs against various diseases involving neuro-inflammation, oxidative stress, mitochondrial dysfunction, and altered lipid metabolism. They can activate neurotrophic and neuroprotective signaling pathways but low bioavailabilities limit their efficiency in curing neurodegeneration. Here, liquid crystalline lipid nanoparticles (LNPs) are created for the protection and non-invasive intranasal delivery of purified scallop-derived plasmalogens. The in vivo results with a transgenic mouse Parkinson's disease (PD) model (characterized by motor impairments and α-synuclein deposition) demonstrate the crucial importance of LNP composition, which determines the self-assembled nanostructure type. Vesicle and hexosome nanostructures (characterized by small-angle X-ray scattering) display different efficacy of the nanomedicine-mediated recovery of motor function, lipid balance, and transcriptional regulation (e.g., reduced neuro-inflammation and PD pathogenic gene expression). Intranasal vesicular and hexosomal plasmalogen-based LNP treatment leads to improvement of the behavioral PD symptoms and downregulation of the Il6, Il33, and Tnfa genes. Moreover, RNA-sequencing and lipidomic analyses establish a dramatic effect of hexosomal nanomedicines on PD amelioration, lipid metabolism, and the type and number of responsive transcripts that may be implicated in neuroregeneration.
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Administración Intranasal , Modelos Animales de Enfermedad , Nanomedicina , Nanopartículas , Enfermedad de Parkinson , Plasmalógenos , Animales , Plasmalógenos/química , Plasmalógenos/farmacología , Ratones , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , Nanopartículas/química , Nanomedicina/métodos , Ratones Transgénicos , Metabolismo de los Lípidos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , LiposomasRESUMEN
Metastasis remains a major cause of morbidity and mortality in men with prostate cancer, and the functional impact of the genetic alterations, alone or in combination, driving metastatic disease remains incompletely understood. The proto-oncogene c-MYC, commonly deregulated in prostate cancer. Transgenic expression of c-MYC is sufficient to drive the progression to prostatic intraepithelial neoplasia and ultimately to moderately differentiated localized primary tumors, however, c-MYC-driven tumors are unable to progress through the metastatic cascade, suggesting that a "second-hit" is necessary in the milieu of aberrant c-MYC-driven signaling. Here, we identified cooperativity between c-MYC and KLF6-SV1, an oncogenic splice variant of the KLF6 gene. Transgenic mice that co-expressed KLF6-SV1 and c-MYC developed progressive and metastatic prostate cancer with a histological and molecular phenotype like human prostate cancer. Silencing c-MYC expression significantly reduced tumor burden in these mice supporting the necessity for c-MYC in tumor maintenance. Unbiased global proteomic analysis of tumors from these mice revealed significantly enriched vimentin, a dedifferentiation and pro-metastatic marker, induced by KLF6-SV1. c-MYC-positive tumors were also significantly enriched for KLF6-SV1 in human prostate cancer specimens. Our findings provide evidence that KLF6-SV1 is an enhancer of c-MYC-driven prostate cancer progression and metastasis, and a correlated genetic event in human prostate cancer with potential translational significance.
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Preeclampsia (PE) is characterized by maternal hypertension and placental dysfunction, often leading to fetal growth restriction (FGR). It is associated with an overexpression of the anti-angiogenic sFLT1 protein, which originates from the placenta and serves as a clinical biomarker to predict PE. To analyze the impact of sFLT1 on placental function and fetal growth, we generated transgenic mice with placenta-specific human sFLT1 (hsFLT1) overexpression. Immunohistochemical, morphometrical, and molecular analyses of the placentas on 14.5 dpc and 18.5 dpc were performed with a focus on angiogenesis, nutrient transport, and inflammation. Additionally, fetal development upon placental hsFLT1 overexpression was investigated. Dams exhibited a mild increase in serum hsFLT1 levels upon placental hsFLT1 expression and revealed growth restriction of the fetuses in a sex-specific manner. Male FGR fetuses expressed higher amounts of placental hsFLT1 mRNA compared to females. FGR placentas displayed an altered morphology, hallmarked by an increase in the spongiotrophoblast layer and changes in labyrinthine vascularization. Further, FGR placentas showed a significant reduction in placental glycogen storage and nutrient transporter expression. Moreover, signs of hypoxia and inflammation were observed in FGR placentas. The transgenic spongiotrophoblast-specific hsFLT1 mouse line demonstrates that low hsFLT1 serum levels are sufficient to induce significant alterations in fetal and placental development in a sex-specific manner.
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Retardo del Crecimiento Fetal , Preeclampsia , Ratones , Animales , Embarazo , Humanos , Masculino , Femenino , Ratones Transgénicos , Retardo del Crecimiento Fetal/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Placenta/metabolismo , Preeclampsia/genética , Inflamación/genéticaRESUMEN
One of the distinctive pathological features of Alzheimer's disease (AD) is the deposition of amyloid plaques within the brain of affected individuals. These plaques have traditionally been investigated using labeling techniques such as immunohistochemical imaging. However, the use of labeling can disrupt the structural integrity of the molecules being analyzed. Hence, it is imperative to employ label-free imaging methods for noninvasive examination of amyloid deposits in their native form, thereby providing more relevant information pertaining to AD. This study presents compelling evidence that label-free and nondestructive confocal Raman imaging is a highly effective approach for the identification and chemical characterization of amyloid plaques within cortical regions of an arcAß mouse model of AD. Furthermore, this investigation elucidates how the spatial correlation of Raman signals can be exploited to identify robust Raman marker bands and discern proteins and lipids from amyloid plaques. Finally, this study uncovers the existence of distinct types of amyloid plaques in the arcAß mouse brain, exhibiting significant disparities in terms of not only shape and size but also molecular composition.
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Enfermedad de Alzheimer , Ratones , Animales , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Placa Amiloide/metabolismo , Ratones Transgénicos , Amiloide/metabolismo , Modelos Animales de Enfermedad , Encéfalo/metabolismoRESUMEN
ObjectiveThe human angiotensin converting enzyme2 (hACE2) transgenic mouse model was used to clarify the antiviral efficacy of BD-77 against a novel coronavirus SARS-CoV-2 and explore the action mechanism of BD-77 against SARS-CoV-2. MethodSARS-CoV-2 Omicron and Delta variant strains-infected VeroE6 cell models were established and administered with BD-77 to observe the antiviral effect of BD-77 in vitro. A kit was used to detect the effect of BD-77 in vitro on the binding of spike S protein of SARS-CoV-2 virus (Delta/Omicron) to angiotensin converting enzyme2 (ACE2). Chromatography was adopted to detect the binding of BD-77 to the S protein and N protein of the novel coronavirus. hACE2 transgenic C57BL/6 mice were divided into a blank control group, SARS-CoV-2 infection group, BD-77 administration groups of 37.5 mg·kg-1 and 75 mg·kg-1, with eight mice in each group. The pneumonia model of SARS-CoV-2-infected hACE2 transgenic mice was built to observe the survival of the mice, detect the virus titer of the lung tissue of the mice, and observe the lesions in the lung tissue. ResultBD-77 had a certain inhibitory effect on Omicron and Delta variant strains in vitro, with median inhibitory concentration (IC50) of 526.3 mg·L-1 and 653.0 mg·L-1, respectively. BD-77 had no significant inhibitory effect on the binding of the S protein of WT, Omicron, and Delta variant strains of SARS-CoV-2 to ACE2 and had no binding effect with the S protein and N protein of the novel coronavirus. No mice in the blank group died, while the mortality rate of SARS-CoV-2-infected mice was 75%. There was a large amount of virus replication in the lung tissue of the mice and large areas of inflammatory infiltration in the lung tissue and interstitium. Compared with the model group, BD-77 administration groups of 37.5 mg·kg-1 and 75 mg·kg-1 could reduce the mortality of mice, significantly lower the virus titer in the lung tissue of mice (P<0.05), and improve lung lesions. ConclusionBD-77 demonstrated significant inhibitory effects against SARS-CoV-2 virus in vitro and in vivo. However, its mechanism of action did not involve direct inhibition of the virus itself or intervention in the virus-host binding process. This finding suggests that the mechanism of action of BD-77 needs to be thoroughly investigated and elucidated by further experiments.
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Background: Pancreatic adenocarcinoma (PDAC) is a devastating disease with an urgent need for therapeutic innovation. Immune checkpoint inhibition has shown promise in a variety of solid tumors, but most clinical trials have failed to demonstrate clinical efficacy in PDAC. This low efficacy is partly explained by a highly immunosuppressive microenvironment, which dampens anti-tumor immunity through the recruitment or induction of immunosuppressive cells, particularly regulatory T cells (Tregs). In this context, our laboratory has developed a novel immunotherapeutic strategy aimed at inhibiting the suppressive activity of Tregs, based on a patented (EP3152234B1) monoclonal antibody (mAb) targeting galectin-9 (LGALS9). Materials and methods: CD4+ conventional T cells (TCD4 or Tconv), Treg ratio, and LGALS9 expression were analyzed by immunohistochemistry (IHC) and cytometry in blood and pancreas of K-rasLSL.G12D/+;Pdx-1-Cre (KC) and K-rasWildType (WT);Pdx1-Cre (WT) mice aged 4-13 months. Pancreatic intraepithelial neoplasm (PanIN) progression and grade were quantified using FIJI software and validated by pathologists. The anti-galectin-9 mAb was validated for its use in mice on isolated murine C57BL/6 Treg by immunofluorescence staining and cytometry. Its specificity and functionality were validated in proliferation assays on rLGALS9-immunosuppressed murine Tconv and in suppression assays between murine Treg and Tconv. Finally, 2-month-old KC mice were treated with anti-LGALS9 and compared to WT mice for peripheral and infiltrating TCD4, Treg, and PanIN progression. Results: IHC and cytometry revealed a significant increase in LGALS9 expression and Treg levels in the blood and pancreas of KC mice proportional to the stages of precancerous lesions. Although present in WT mice, LGALS9 is expressed at a basal level with low and restricted expression that increases slightly over time, while Treg cells are few in number in their circulation and even absent from the pancreas over time. Using our anti-LGALS9 mAb in mice, it is shown that (i) murine Treg express LGALS9, (ii) the mAb could target and inhibit recombinant murine LGALS9, and (iii) neutralize murine Treg suppressive activity. Finally, the anti-LGALS9 mAb in KC mice reduced (i) LGALS9 expression in pancreatic cancer cells, (ii) the Treg ratio, and (iii) the total surface area and grade of PanIN. Conclusion: We demonstrate for the first time that an anti-LGALS9 antibody, by specifically targeting endogenous LGALS9 tumor and exogenous LGALS9 produced by Treg, was able to limit the progression of pancreatic neoplastic lesions in mice, opening up new prospects for its use as an immunotherapeutic tool in PDAC.
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Adenocarcinoma , Carcinoma in Situ , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Ratones , Animales , Ratones Transgénicos , Ratones Endogámicos C57BL , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Galectinas , Inmunoterapia , Microambiente TumoralRESUMEN
Numerous Aß proteoforms, identified in the human brain, possess differential neurotoxic and aggregation propensities. These proteoforms contribute in unknown ways to the conformations and resultant pathogenicity of oligomers, protofibrils, and fibrils in Alzheimer's disease (AD) manifestation owing to the lack of molecular-level specificity to the exact chemical composition of underlying protein products with widespread interrogating techniques, like immunoassays. We evaluated Aß proteoform flux using quantitative top-down mass spectrometry (TDMS) in a well-studied 5xFAD mouse model of age-dependent Aß-amyloidosis. Though the brain-derived Aß proteoform landscape is largely occupied by Aß1-42, 25 different forms of Aß with differential solubility were identified. These proteoforms fall into three natural groups defined by hierarchical clustering of expression levels in the context of mouse age and proteoform solubility, with each group sharing physiochemical properties associated with either N/C-terminal truncations or both. Overall, the TDMS workflow outlined may hold tremendous potential for investigating proteoform-level relationships between insoluble fibrils and soluble Aß, including low-molecular-weight oligomers hypothesized to serve as the key drivers of neurotoxicity. Similarly, the workflow may also help to validate the utility of AD-relevant animal models to recapitulate amyloidosis mechanisms or possibly explain disconnects observed in therapeutic efficacy in animal models vs humans.
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Enfermedad de Alzheimer , Amiloidosis , Ratones , Humanos , Animales , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/metabolismo , Ratones Transgénicos , Modelos Animales de Enfermedad , Espectrometría de MasasRESUMEN
Human adenovirus (HAdV) is a highly virulent respiratory pathogen that poses clinical challenges in terms of diagnostics and treatment. Currently, no effective therapeutic drugs or prophylactic vaccines are available for HAdV infections. One factor contributing to this deficiency is that existing animal models, including wild-type and single-receptor transgenic mice, are unsuitable for HAdV proliferation and pathology testing. In this study, a tri-receptor transgenic mouse model expressing the three best-characterized human cellular receptors for HAdV (hCAR, hCD46, and hDSG2) was generated and validated via analysis of transgene insertion, receptor mRNA expression, and protein abundance distribution. Following HAdV-7 infection, the tri-receptor mice exhibited high transcription levels at the early and late stages of the HAdV gene, as well as viral protein expression. Furthermore, the tri-receptor mice infected with HAdV exhibited dysregulated cytokine responses and multiple tissue lesions. This transgenic mouse model represents human HAdV infection and pathogenesis with more accuracy than any other reported animal model. As such, this model facilitates the comprehensive investigation of HAdV pathogenesis as well as the evaluation of potential vaccines and therapeutic modalities for HAdV.
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Infecciones por Adenoviridae , Infecciones por Adenovirus Humanos , Adenovirus Humanos , Ratones , Animales , Humanos , Ratones Transgénicos , Procesamiento Proteico-Postraduccional , Expresión Génica , Modelos Animales de Enfermedad , Adenovirus Humanos/fisiologíaRESUMEN
Transgenic models are useful tools for studying the pathogenesis of and drug development for Alzheimer's Disease (AD). AD models are constructed usually using overexpression or knock-in of multiple pathogenic gene mutations from familial AD. Each transgenic model has its unique behavioral and pathological features. This review summarizes the research progress of transgenic mouse models, and their progress in the unique mechanism of amyloid-ß oligomers, including the first transgenic mouse model built in China based on a single gene mutation (PSEN1 V97L) found in Chinese familial AD. We further summarized the preclinical findings of drugs using the models, and their future application in exploring the upstream mechanisms and multitarget drug development in AD.
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Enfermedad de Alzheimer , Ratones , Animales , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Ratones Transgénicos , Precursor de Proteína beta-Amiloide/genética , Modelos Animales de Enfermedad , Pensamiento , Péptidos beta-Amiloides/genéticaRESUMEN
Antibody inhibitors that block PD-1/PD-L1 interaction have been approved for oncological clinics, yielding impressive treatment effects. Small molecules inhibiting PD-1 signalling are at various stages of development, given that small molecular drugs are expected to outperform protein drugs in several ways. Currently, a significant portion of these small molecular inhibitors achieve this purpose by binding to a limited region of the PD-L1 protein, thereby limiting the choice of chemical structures. Alternative strategies for developing small-molecular PD-1 inhibitors are urgently needed to broaden the choice of chemical structures. Here, we report that 6-mercaptopurine (6-MP) inhibits PD-1 signalling, activates T cell function in vitro and in vivo and shrinks tumours by activating cytotoxic T cells. Mechanistically, 6-MP potently inhibited PD-1 signalling by blocking the recruitment of SHP2 by PD-1. Considering that 6-MP is a chemotherapeutic agent already approved by the FDA for childhood leukaemia, our work revealed a novel anti-tumour mechanism for this drug and suggests that 6-MP warrants further clinical evaluation for other tumour types.
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Mercaptopurina , Neoplasias , Humanos , Niño , Mercaptopurina/farmacología , Mercaptopurina/uso terapéutico , Receptor de Muerte Celular Programada 1 , Neoplasias/tratamiento farmacológico , Transducción de Señal , Linfocitos T/metabolismo , Antígeno B7-H1 , InmunoterapiaRESUMEN
BACKGROUND: Neuroblastoma is the most common solid tumor in infants accounting for approximately 15% of all cancer-related deaths. Over 50% of high-risk neuroblastoma relapse, emphasizing the need of novel drug targets and therapeutic strategies. In neuroblastoma, chromosomal gains at chromosome 17q, including IGF2BP1, and MYCN amplification at chromosome 2p are associated with adverse outcome. Recent, pre-clinical evidence indicates the feasibility of direct and indirect targeting of IGF2BP1 and MYCN in cancer treatment. METHODS: Candidate oncogenes on 17q were identified by profiling the transcriptomic/genomic landscape of 100 human neuroblastoma samples and public gene essentiality data. Molecular mechanisms and gene expression profiles underlying the oncogenic and therapeutic target potential of the 17q oncogene IGF2BP1 and its cross-talk with MYCN were characterized and validated in human neuroblastoma cells, xenografts and PDX as well as novel IGF2BP1/MYCN transgene mouse models. RESULTS: We reveal a novel, druggable feedforward loop of IGF2BP1 (17q) and MYCN (2p) in high-risk neuroblastoma. This promotes 2p/17q chromosomal gains and unleashes an oncogene storm resulting in fostered expression of 17q oncogenes like BIRC5 (survivin). Conditional, sympatho-adrenal transgene expression of IGF2BP1 induces neuroblastoma at a 100% incidence. IGF2BP1-driven malignancies are reminiscent to human high-risk neuroblastoma, including 2p/17q-syntenic chromosomal gains and upregulation of Mycn, Birc5, as well as key neuroblastoma circuit factors like Phox2b. Co-expression of IGF2BP1/MYCN reduces disease latency and survival probability by fostering oncogene expression. Combined inhibition of IGF2BP1 by BTYNB, MYCN by BRD inhibitors or BIRC5 by YM-155 is beneficial in vitro and, for BTYNB, also. CONCLUSION: We reveal a novel, druggable neuroblastoma oncogene circuit settling on strong, transcriptional/post-transcriptional synergy of MYCN and IGF2BP1. MYCN/IGF2BP1 feedforward regulation promotes an oncogene storm harboring high therapeutic potential for combined, targeted inhibition of IGF2BP1, MYCN expression and MYCN/IGF2BP1-effectors like BIRC5.
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Neuroblastoma , Animales , Humanos , Lactante , Ratones , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes myc , Proteína Proto-Oncogénica N-Myc/genética , Proteína Proto-Oncogénica N-Myc/metabolismo , Recurrencia Local de Neoplasia/genética , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismoRESUMEN
AIM: This study mapped the spatiotemporal positions and connectivity of Onecut3+ neuronal populations in the developing and adult mouse brain. METHODS: We generated fluorescent reporter mice to chart Onecut3+ neurons for brain-wide analysis. Moreover, we crossed Onecut3-iCre and Mapt-mGFP (Tau-mGFP) mice to visualize axonal projections. A dual Cre/Flp-dependent AAV construct in Onecut3-iCre cross-bred with Slc17a6-FLPo mice was used in an intersectional strategy to map the connectivity of glutamatergic lateral hypothalamic neurons in the adult mouse. RESULTS: We first found that Onecut3 marks a hitherto undescribed Slc17a6+ /Vglut2+ neuronal cohort in the lateral hypothalamus, with the majority expressing thyrotropin-releasing hormone. In the adult, Onecut3+ /Vglut2+ neurons of the lateral hypothalamus had both intra- and extrahypothalamic efferents, particularly to the septal complex and habenula, where they targeted other cohorts of Onecut3+ neurons and additionally to the neocortex and hippocampus. This arrangement suggests that intrinsic reinforcement loops could exist for Onecut3+ neurons to coordinate their activity along the brain's midline axis. CONCLUSION: We present both a toolbox to manipulate novel subtypes of hypothalamic neurons and an anatomical arrangement by which extrahypothalamic targets can be simultaneously entrained.
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Área Hipotalámica Lateral , Neuronas , Ratones , Animales , Ratones Transgénicos , Neuronas/fisiología , Hipotálamo , EncéfaloRESUMEN
Alzheimer's disease (AD), as the most common type of dementia, has two pathological hallmarks, extracellular senile plaques composed of ß-amyloid peptides and intracellular neurofibrillary tangles containing phosphorylated-tau protein. Amyloid precursor protein (APP) and tau each play central roles in AD, although how APP and tau interact and synergize in the disease process is largely unknown. Here, we showed that soluble tau interacts with the N-terminal of APP in vitro in cell-free and cell culture systems, which can be further confirmed in vivo in the brain of 3XTg-AD mouse. In addition, APP is involved in the cellular uptake of tau through endocytosis. APP knockdown or N-terminal APP-specific antagonist 6KApoEp can prevent tau uptake in vitro, resulting in an extracellular tau accumulation in cultured neuronal cells. Interestingly, in APP/PS1 transgenic mouse brain, the overexpression of APP exacerbated tau propagation. Moreover, in the human tau transgenic mouse brain, overexpression of APP promotes tau phosphorylation, which is significantly remediated by 6KapoEp. All these results demonstrate the important role of APP in the tauopathy of AD. Targeting the pathological interaction of N-terminal APP with tau may provide an important therapeutic strategy for AD.
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Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide , Ratones , Humanos , Animales , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo , Modelos Animales de Enfermedad , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Ratones TransgénicosRESUMEN
In vitro experiments have shown that the E2 protein of human papillomaviruses (HPV) binds to the upstream regulatory region (URR) of the viral genome and modulates transcription. Additionally, it seems to be a necessary component for viral DNA replication together with E1. We have developed a transgenic mouse model containing the URR region of the low-risk virus HPV11 that regulates the expression of the lacZ reporter gene. Most interestingly, in these mice, the transgene was exclusively expressed in the bulge region of the hair follicle but not in any other tissues. Further experimental data indicate that in double transgenic mice that also express the HPV11-E2 protein under the control of the Ubiquitin C-promoter, the transcription of the reporter gene is modulated. When E2 is present, the expression of the reporter gene also occurs exclusively in the bulge region of the hair follicles as it does in the single transgenic mice, but the expression of the lacZ driven by the URR is increased and the statistical spread is greater. Even if the expression of the reporter gene occurs in the hair follicles of the dorsal skin of an animal uniform, E2 obviously has the capacity for both to induce and to repress the URR activity in vivo.
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Replicación del ADN , Replicación Viral , Ratones , Animales , Humanos , ADN Viral/metabolismo , Regiones Promotoras Genéticas , Ratones TransgénicosRESUMEN
Hippo signaling plays an essential role in the development of numerous tissues. Although it was previously shown that the transcriptional effectors of Hippo signaling Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) can fine-tune the regulation of sex differentiation genes in the testes, the role of Hippo signaling in testis development remains largely unknown. To further explore the role of Hippo signaling in the testes, we conditionally deleted the key Hippo kinases large tumor suppressor homolog kinases 1 and -2 (Lats1 and Lats2, two kinases that antagonize YAP and TAZ transcriptional co-regulatory activity) in the somatic cells of the testes using an Nr5a1-cre strain (Lats1flox/flox;Lats2flox/flox;Nr5a1-cre). We report here that early stages of testis somatic cell differentiation were not affected in this model but progressive testis cord dysgenesis was observed starting at gestational day e14.5. Testis cord dysgenesis was further associated with the loss of polarity of the Sertoli cells and the loss of SOX9 expression but not WT1. In parallel with testis cord dysgenesis, a loss of steroidogenic gene expression associated with the appearance of myofibroblast-like cells in the interstitial space was also observed in mutant animals. Furthermore, the loss of YAP phosphorylation, the accumulation of nuclear TAZ (and YAP) in both the Sertoli and interstitial cell populations, and an increase in their transcriptional co-regulatory activity in the testes suggest that the observed phenotype could be attributed at least in part to YAP and TAZ. Taken together, our results suggest that Hippo signaling is required to maintain proper differentiation of testis somatic cells.
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Proteínas Adaptadoras Transductoras de Señales , Diferenciación Sexual , Animales , Masculino , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Testículo/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Alzheimer's disease (AD) patients present with symptoms such as impairment of insulin signaling, chronic inflammation, and oxidative stress. Furthermore, there are comorbidities associated with AD progression. For example, osteoporosis is common with AD wherein patients exhibit reduced mineralization and a risk for fragility fractures. However, there is a lack of understanding on the effects of AD on bone beyond loss of bone density. To this end, we investigated the effects of AD on bone quality using the 5XFAD transgenic mouse model in which 12-month-old 5XFAD mice showed accumulation of amyloid-beta (Aß42) compared with wild-type (WT) littermates (n = 10/group; 50% female, 50% male). Here, we observed changes in cortical bone but not in cancellous bone quality. Both bone mass and bone quality, measured in femoral samples using imaging (micro-CT, confocal Raman spectroscopy, X-ray diffraction [XRD]), mechanical (fracture tests), and chemical analyses (biochemical assays), were altered in the 5XFAD mice compared with WT. Micro-CT results showed 5XFAD mice had lower volumetric bone mineral density (BMD) and increased endocortical bone loss. XRD results showed decreased mineralization with smaller mineral crystals. Bone matrix compositional properties, from Raman, showed decreased crystallinity along with higher accumulation of glycoxidation products and glycation products, measured biochemically. 5XFAD mice also demonstrated loss of initiation and maximum toughness. We observed that carboxymethyl-lysine (CML) and mineralization correlated with initiation toughness, whereas crystal size and pentosidine (PEN) correlated with maximum toughness, suggesting bone matrix changes predominated by advanced glycation end products (AGEs) and altered/poor mineral quality explained loss of fracture toughness. Our findings highlight two pathways to skeletal fragility in AD through alteration of bone quality: (i) accumulation of AGEs; and (ii) loss of crystallinity, decreased crystal size, and loss of mineralization. We observed that the accumulation of amyloidosis in brain correlated with an increase in several AGEs, consistent with a mechanistic link between elevated Aß42 levels in the brain and AGE accumulation in bone. © 2022 American Society for Bone and Mineral Research (ASBMR).
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Enfermedad de Alzheimer , Modelos Animales de Enfermedad , Fracturas Óseas , Ratones , Osteoporosis , Animales , Femenino , Masculino , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Huesos/diagnóstico por imagen , Fracturas Óseas/complicaciones , Fracturas Óseas/diagnóstico por imagen , Productos Finales de Glicación Avanzada/metabolismo , Ratones Transgénicos , Osteoporosis/complicaciones , Osteoporosis/diagnóstico por imagen , Densidad ÓseaRESUMEN
The rapid aging of the population makes the detection and prevention of frailty increasingly important. Oral frailty has been proposed as a novel frailty phenotype and is defined as a decrease in oral function coexisting with a decline in cognitive and physical functions. Oral frailty has received particular attention in relation to Alzheimer's disease (AD). However, the pathomechanisms of oral frailty related to AD remain unknown. It is assumed that the mesencephalic trigeminal nucleus (Vmes), which controls mastication, is affected by AD pathology, and as a result, masticatory function may be impaired. To investigate this possibility, we included male 3 × Tg-AD mice and their non-transgenic counterpart (NonTg) of 3-4 months of age in the present study. Immunohistochemistry revealed amyloid-ß deposition and excessive tau phosphorylation in the Vmes of 3 × Tg-AD mice. Furthermore, vesicular glutamate transporter 1-immunopositive axon varicosities, which are derived from Vmes neurons, were significantly reduced in the trigeminal motor nucleus of 3 × Tg-AD mice. To investigate whether the AD pathology observed in the Vmes affects masticatory function, we analyzed electromyography of the masseter muscle during feeding. The 3 × Tg-AD mice showed a significant delay in masticatory rhythm compared to NonTg mice. Furthermore, we developed a system to simultaneously record bite force and electromyography of masseter, and devised a new method to estimate bite force during food chewing in mice. Since the muscle activity of the masseter showed a high correlation with bite force, it could be accurately estimated from the muscle activity. The estimated bite force of 3 × Tg-AD mice eating sunflower seeds was predominantly smaller than that of NonTg mice. However, there was no difference in masseter weight or muscle fiber cross-sectional area between the two groups, suggesting that the decreased bite force and delayed mastication rhythm observed in 3 × Tg-AD mice were not due to abnormality of the masseter. In conclusion, the decreased masticatory function observed in 3 × Tg-AD mice was most likely caused by AD pathology in the Vmes. Thus, novel quantitative analyses of masticatory function using the mouse model of AD enabled a comprehensive understanding of oral frailty pathogenesis.