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1.
Development ; 150(22)2023 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-37905445

RESUMO

Failures in growth and differentiation of the early human placenta are associated with severe pregnancy disorders such as pre-eclampsia and fetal growth restriction. However, regulatory mechanisms controlling development of placental epithelial cells, the trophoblasts, remain poorly elucidated. Using trophoblast stem cells (TSCs), trophoblast organoids (TB-ORGs) and primary cytotrophoblasts (CTBs) of early pregnancy, we herein show that autocrine NOTCH3 signalling controls human placental expansion and differentiation. The NOTCH3 receptor was specifically expressed in proliferative CTB progenitors and its active form, the nuclear NOTCH3 intracellular domain (NOTCH3-ICD), interacted with the transcriptional co-activator mastermind-like 1 (MAML1). Doxycycline-inducible expression of dominant-negative MAML1 in TSC lines provoked cell fusion and upregulation of genes specific for multinucleated syncytiotrophoblasts, which are the differentiated hormone-producing cells of the placenta. However, progenitor expansion and markers of trophoblast stemness and proliferation were suppressed. Accordingly, inhibition of NOTCH3 signalling diminished growth of TB-ORGs, whereas overexpression of NOTCH3-ICD in primary CTBs and TSCs showed opposite effects. In conclusion, the data suggest that canonical NOTCH3 signalling plays a key role in human placental development by promoting self-renewal of CTB progenitors.


Assuntos
Placenta , Trofoblastos , Humanos , Gravidez , Feminino , Placenta/metabolismo , Receptor Notch3/genética , Receptor Notch3/metabolismo , Diferenciação Celular/genética , Proliferação de Células/genética , Células-Tronco , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo
2.
Nature ; 582(7811): 259-264, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32499639

RESUMO

The synovium is a mesenchymal tissue composed mainly of fibroblasts, with a lining and sublining that surround the joints. In rheumatoid arthritis the synovial tissue undergoes marked hyperplasia, becomes inflamed and invasive, and destroys the joint1,2. It has recently been shown that a subset of fibroblasts in the sublining undergoes a major expansion in rheumatoid arthritis that is linked to disease activity3-5; however, the molecular mechanism by which these fibroblasts differentiate and expand is unknown. Here we identify a critical role for NOTCH3 signalling in the differentiation of perivascular and sublining fibroblasts that express CD90 (encoded by THY1). Using single-cell RNA sequencing and synovial tissue organoids, we found that NOTCH3 signalling drives both transcriptional and spatial gradients-emanating from vascular endothelial cells outwards-in fibroblasts. In active rheumatoid arthritis, NOTCH3 and Notch target genes are markedly upregulated in synovial fibroblasts. In mice, the genetic deletion of Notch3 or the blockade of NOTCH3 signalling attenuates inflammation and prevents joint damage in inflammatory arthritis. Our results indicate that synovial fibroblasts exhibit a positional identity that is regulated by endothelium-derived Notch signalling, and that this stromal crosstalk pathway underlies inflammation and pathology in inflammatory arthritis.


Assuntos
Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Receptor Notch3/metabolismo , Transdução de Sinais , Membrana Sinovial/patologia , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Células Endoteliais/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Receptor Notch3/antagonistas & inibidores , Receptor Notch3/deficiência , Receptor Notch3/genética , Antígenos Thy-1/metabolismo
3.
J Biol Chem ; 300(10): 107787, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39303912

RESUMO

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a genetic vascular dementia characterized by age-related degeneration of vascular mural cells and accumulation of a NOTCH3 mutant protein. NOTCH3 functions as a signaling receptor, activating downstream gene expression in response to ligands like JAG1 and DLL4, which regulate the development and survival of mural cells. This signal transduction process is thought to be connected with NOTCH3 endocytic degradation. However, the specific cellular circumstances that modulate turnover and signaling efficacy of NOTCH3 mutant protein remain largely unknown. Here, we found elevated NOTCH3 and Radical fringe (RFNG) expression in senescent human pericyte cells. We then investigated impacts of RFNG on glycosylation, degradation, and signal activity of three NOTCH3 CADASIL mutants (R90C, R141C, and C185R) in EGF-like repeat-2, 3, and 4, respectively. Liquid chromatography with tandem mass spectrometry analysis showed that RFNG modified NOTCH3 WT and C185R to different degrees. Additionally, coculture experiments demonstrated that RFNG significantly promoted JAG1-dependent degradation of NOTCH3 WT but not that of R141C and C185R mutants. Furthermore, RFNG exhibited a greater inhibitory effect on JAG1-mediated activity of NOTCH3 R141C and C185R compared to that of NOTCH3 WT and R90C. In summary, our findings suggest that NOTCH3 R141C and C185R mutant proteins are relatively susceptible to accumulation and signaling impairment under cellular conditions of RFNG and JAG1 coexistence.


Assuntos
CADASIL , Proteína Jagged-1 , N-Acetilglucosaminiltransferases , Receptor Notch3 , Transdução de Sinais , Receptor Notch3/metabolismo , Receptor Notch3/genética , Humanos , Proteína Jagged-1/metabolismo , Proteína Jagged-1/genética , CADASIL/metabolismo , CADASIL/genética , CADASIL/patologia , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genética , Pericitos/metabolismo , Pericitos/patologia , Senescência Celular , Mutação , Glicosilação , Proteólise , Mutação de Sentido Incorreto , Glicosiltransferases
4.
Ann Neurol ; 95(6): 1040-1054, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38520151

RESUMO

OBJECTIVES: Intracerebral hemorrhage (ICH) and cerebral microbleeds (CMB) in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy are more common in East Asian populations than in people of white European ancestry. We hypothesized that the ethnic difference is explained by the East Asian-specific NOTCH3 p.R75P mutation. METHODS: This retrospective observational study included 118 patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy in Japanese and Korean cohorts. We investigated whether the p.R75P mutation is associated with symptomatic ICH and multiple CMB (>5) using quasi-Poisson regression models. We predicted the NOTCH3 extracellular domain protein structures in silico and graded NOTCH3 extracellular domain immunostaining in skin vessels of some patients, with subsequent comparisons between p.R75P and other conventional mutations. RESULTS: Among 63 Japanese patients (median age 55 years; 56% men), 15 had a p.R75P mutation, significantly associated with symptomatic ICH (adjusted relative risk 9.56, 95% CI 2.45-37.31), multiple CMB (3.00, 1.34-6.71), and absence of temporopolar lesions (4.91, 2.29-10.52) after adjustment for age, sex, hypertension, and antithrombotics. In the Korean cohort (n = 55; median age 55 years; 51% men), the p.R75P mutation (n = 13) was also associated with symptomatic ICH (8.11, 1.83-35.89), multiple CMB (1.90, 1.01-3.56), and absence of temporopolar lesions (2.32, 1.08-4.97). Structural analysis revealed solvent-exposed free cysteine thiols in conventional mutations, directly causing aggregation, whereas a stereochemically incompatible proline residue structure in p.R75P lowers correct disulfide bond formation probability, indirectly causing aggregation. Pathologically, the p.R75P mutation resulted in less vascular NOTCH3 extracellular domain accumulation than the other conventional mutations. INTERPRETATION: NOTCH3 p.R75P mutation is associated with hemorrhagic presentations, milder temporopolar lesions, and distinct mutant protein structure properties. ANN NEUROL 2024;95:1040-1054.


Assuntos
CADASIL , Hemorragia Cerebral , Mutação , Receptor Notch3 , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Povo Asiático/genética , CADASIL/genética , Hemorragia Cerebral/genética , População do Leste Asiático/genética , Japão , Mutação/genética , Receptor Notch3/genética , República da Coreia , Estudos Retrospectivos
5.
J Biol Chem ; 299(6): 104838, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37209821

RESUMO

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a cerebral small vessel disease that results from mutations in NOTCH3. How mutations in NOTCH3 ultimately result in disease is not clear, although there is a predilection for mutations to alter the number of cysteines of the gene product, supporting a model in which alterations of conserved disulfide bonds of NOTCH3 drives the disease process. We have found that recombinant proteins with CADASIL NOTCH3 EGF domains 1 to 3 fused to the C terminus of Fc are distinguished from wildtype proteins by slowed mobility in nonreducing gels. We use this gel mobility shift assay to define the effects of mutations in the first three EGF-like domains of NOTCH3 in 167 unique recombinant protein constructs. This assay permits a readout on NOTCH3 protein mobility that indicates that (1) any loss of cysteine mutation in the first three EGF motifs results in structural abnormalities; (2) for loss of cysteine mutants, the mutant amino acid residue plays a minimal role; (3) the majority of changes that result in a new cysteine are poorly tolerated; (4) at residue 75, only cysteine, proline, and glycine induce structural shifts; (5) specific second mutations in conserved cysteines suppress the impact of loss of cysteine CADASIL mutations. These studies support the importance of NOTCH3 cysteines and disulfide bonds in maintaining normal protein structure. Double mutant analysis suggests that suppression of protein abnormalities can be achieved through modification of cysteine reactivity, a potential therapeutic strategy.


Assuntos
CADASIL , Receptor Notch3 , Humanos , CADASIL/genética , Cisteína/genética , Cisteína/metabolismo , Dissulfetos , Fator de Crescimento Epidérmico/genética , Mutação , Receptor Notch3/genética
6.
J Biol Chem ; 299(1): 102772, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36470429

RESUMO

Mutations in NOTCH3 underlie cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common inherited cerebral small vessel disease. Two cleavages of NOTCH3 protein, at Asp80 and Asp121, were previously described in CADASIL pathological samples. Using monoclonal antibodies developed against a NOTCH3 neoepitope, we identified a third cleavage at Asp964 between an Asp-Pro sequence. We characterized the structural requirements for proteolysis at Asp964 and the vascular distribution of the cleavage event. A proteome-wide analysis was performed to find proteins that interact with the cleavage product. Finally, we investigated the biochemical determinants of this third cleavage event. Cleavage at Asp964 was critically dependent on the proline adjacent to the aspartate residue. In addition, the cleavage product was highly enriched in CADASIL brain tissue and localized to the media of degenerating arteries, where it deposited with the two additional NOTCH3 cleavage products. Recombinant NOTCH3 terminating at Asp964 was used to probe protein microarrays. We identified multiple molecules that bound to the cleaved NOTCH3 more than to uncleaved protein, suggesting that cleavage may alter the local protein interactome within disease-affected blood vessels. The cleavage of purified NOTCH3 protein at Asp964 in vitro was activated by reducing agents and NOTCH3 protein; cleavage was inhibited by specific dicarboxylic acids, as seen with cleavage at Asp80 and Asp121. Overall, we propose homologous redox-driven Asp-Pro cleavages and alterations in protein interactions as potential mechanisms in inherited small vessel disease; similarities in protein cleavage characteristics may indicate common biochemical modulators of pathological NOTCH3 processing.


Assuntos
CADASIL , Receptor Notch3 , Humanos , Encéfalo/metabolismo , CADASIL/genética , CADASIL/patologia , Doenças de Pequenos Vasos Cerebrais/genética , Doenças de Pequenos Vasos Cerebrais/patologia , Mutação , Receptor Notch3/genética , Receptor Notch3/metabolismo , Ligação Proteica , Análise Serial de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
7.
Cancer Sci ; 115(2): 412-426, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38115797

RESUMO

Docetaxel is the preferred chemotherapeutic agent in patients with castrate-resistant prostate cancer (CRPC). However, patients eventually develop docetaxel resistance and in the absence of effective treatment options. Consequently, it is essential to investigate the mechanisms generating docetaxel resistance and develop novel alternative therapeutic targets. RNA sequencing was undertaken on docetaxel-sensitive and docetaxel-resistant prostate cancer (PCa) cells. Subsequently, chemoresistance, cancer stemness, and lipid metabolism were investigated. To obtain insight into the precise activities and action mechanisms of NOTCH3 in docetaxel-resistant PCa, immunoprecipitation, mass spectrometry, ChIP, luciferase reporter assay, cell metabolism, and animal experiments were performed. Through RNA sequencing analysis, we found that NOTCH3 expression was markedly higher in docetaxel-resistant cells relative to parental cells, and that this trend was continued in docetaxel-resistant PCa tissues. Experiments in vitro and in vivo revealed that NOTCH3 enhanced stemness, lipid metabolism, and docetaxel resistance in PCa. Mechanistically, NOTCH3 is bound to TUBB3 and activates the MAPK signaling pathway. Moreover, NOTCH3 was directly regulated by MEF2A in docetaxel-resistant cells. Notably, targeting NOTCH3 and the MEF2A/TUBB3 signaling axis was related to docetaxel chemoresistance in PCa. Overall, these results demonstrated that NOTCH3 fostered stemness, lipid metabolism, and docetaxel resistance in PCa via the TUBB3 and MAPK signaling pathways. Therefore, NOTCH3 may be employed as a prognostic biomarker in PCa patients. NOTCH3 could be a therapeutic target for PCa patients, particularly those who have developed docetaxel resistance.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias da Próstata , Masculino , Animais , Humanos , Docetaxel/farmacologia , Docetaxel/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Transdução de Sinais/genética , Tubulina (Proteína)/metabolismo , Receptor Notch3/genética
8.
Development ; 148(7)2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33722902

RESUMO

Niemann-Pick disease type C (NPC) is a rare, fatal, neurodegenerative lysosomal disease caused by mutations of either NPC1 or NPC2. NPC2 is a soluble lysosomal protein that functions in coordination with NPC1 to efflux cholesterol from the lysosomal compartment. Mutations of either gene result in the accumulation of unesterified cholesterol and other lipids in the late endosome/lysosome, and reduction of cellular cholesterol bioavailability. Zygotic null npc2m/m zebrafish showed significant unesterified cholesterol accumulation at larval stages, a reduction in body size, and motor and balance defects in adulthood. However, the phenotype at embryonic stages was milder than expected, suggesting a possible role of maternal Npc2 in embryonic development. Maternal-zygotic npc2m/m zebrafish exhibited significant developmental defects, including defective otic vesicle development/absent otoliths, abnormal head/brain development, curved/twisted body axes and no circulating blood cells, and died by 72 hpf. RNA-seq analysis conducted on 30 hpf npc2+/m and MZnpc2m/m embryos revealed a significant reduction in the expression of notch3 and other downstream genes in the Notch signaling pathway, suggesting that impaired Notch3 signaling underlies aspects of the developmental defects observed in MZnpc2m/m zebrafish.


Assuntos
Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Transporte Biológico , Colesterol/metabolismo , Desenvolvimento Embrionário , Endossomos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Larva/anatomia & histologia , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Receptor Notch3/genética , Receptor Notch3/metabolismo , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética
9.
Haematologica ; 109(8): 2606-2618, 2024 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-38385272

RESUMO

Multiple myeloma (MM) remains incurable due to disease relapse and drug resistance. Notch signals from the tumor microenvironment (TME) confer chemoresistance, but the cellular and molecular mechanisms are not entirely understood. Using clinical and transcriptomic datasets, we found that NOTCH3 is upregulated in CD138+ cells from newly diagnosed MM (NDMM) patients compared to healthy individuals and increased in progression/relapsed MM (PRMM) patients. Further, NDMM patients with high NOTCH3 expression exhibited worse responses to bortezomib (BOR)-based therapies. Cells of the TME, including osteocytes, upregulated NOTCH3 in MM cells and protected them from apoptosis induced by BOR. NOTCH3 activation (NOTCH3OE) in MM cells decreased BOR anti-MM efficacy and its ability to improve survival in in vivo myeloma models. Molecular analyses revealed that NDMM and PRMM patients with high NOTCH3 exhibit CXCL12 upregulation. TME cells upregulated CXCL12 and activated the CXCR4 pathway in MM cells in a NOTCH3-dependent manner. Moreover, genetic or pharmacologic inhibition of CXCL12 in NOTCH3OE MM cells restored sensitivity to BOR regimes in vitro and in human bones bearing NOTCH3OE MM tumors cultured ex vivo. Our clinical and preclinical data unravel a novel NOTCH3-CXCL12 pro-survival signaling axis in the TME and suggest that osteocytes transmit chemoresistance signals to MM cells.


Assuntos
Quimiocina CXCL12 , Resistencia a Medicamentos Antineoplásicos , Mieloma Múltiplo , Receptor Notch3 , Transdução de Sinais , Microambiente Tumoral , Animais , Humanos , Camundongos , Bortezomib/farmacologia , Bortezomib/uso terapêutico , Linhagem Celular Tumoral , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Mieloma Múltiplo/genética , Receptor Notch3/metabolismo , Receptor Notch3/genética , Transdução de Sinais/efeitos dos fármacos
10.
Cell Commun Signal ; 22(1): 256, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38705997

RESUMO

BACKGROUND: Melanoma is a highly heterogeneous cancer, in which frequent changes in activation of signaling pathways lead to a high adaptability to ever changing tumor microenvironments. The elucidation of cancer specific signaling pathways is of great importance, as demonstrated by the inhibitor of the common BrafV600E mutation PLX4032 in melanoma treatment. We therefore investigated signaling pathways that were influenced by neurotrophin NRN1, which has been shown to be upregulated in melanoma. METHODS: Using a cell culture model system with an NRN1 overexpression, we investigated the influence of NRN1 on melanoma cells' functionality and signaling. We employed real time cell analysis and spheroid formation assays, while for investigation of molecular mechanisms we used a kinase phosphorylation kit as well as promotor activity analysis followed by mRNA and protein analysis. RESULTS: We revealed that NRN1 interacts directly with the cleaved intracellular domain (NICD) of Notch1 and Notch3, causing a potential retention of NICD in the cytoplasm and thereby reducing the expression of its direct downstream target Hes1. This leads to decreased sequestration of JAK and STAT3 in a Hes1-driven phosphorylation complex. Consequently, our data shows less phosphorylation of STAT3 while presenting an accumulation of total protein levels of STAT3 in association with NRN1 overexpression. The potential of the STAT3 signaling pathway to act in both a tumor suppressive and oncogenic manner led us to investigate specific downstream targets - namely Vegf A, Mdr1, cMet - which were found to be upregulated under oncogenic levels of NRN1. CONCLUSIONS: In summary, we were able to show that NRN1 links oncogenic signaling events between Notch and STAT3 in melanoma. We also suggest that in future research more attention should be payed to cellular regulation of signaling molecules outside of the classically known phosphorylation events.


Assuntos
Melanoma , Neuropeptídeos , Fator de Transcrição STAT3 , Transdução de Sinais , Humanos , Carcinogênese/genética , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Melanoma/metabolismo , Melanoma/genética , Melanoma/patologia , Fosforilação , Ligação Proteica , Receptor Notch1/metabolismo , Receptor Notch1/genética , Receptor Notch3/metabolismo , Receptor Notch3/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética
11.
Arterioscler Thromb Vasc Biol ; 43(12): 2301-2311, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37855127

RESUMO

BACKGROUND: The regional heterogeneity of vascular components and transcriptomes is an important determinant of aortic biology. This notion has been explored in multiple mouse studies. In the present study, we examined the regional heterogeneity of aortas in nonhuman primates. METHODS: Aortic samples were harvested from the ascending, descending thoracic, suprarenal, and infrarenal regions of young control monkeys and adult monkeys with high fructose consumption for 3 years. The regional heterogeneity of aortic structure and transcriptomes was examined by histological and bulk RNA sequencing analyses, respectively. RESULTS: Immunostaining of CD31 and αSMA (alpha-smooth muscle actin) revealed that endothelial and smooth muscle cells were distributed homogeneously across the aortic regions. In contrast, elastic fibers were less abundant and dispersed in the infrarenal aorta compared with other regions and associated with collagen deposition. Bulk RNA sequencing identified a distinct transcriptome related to the Notch signaling pathway in the infrarenal aorta with significantly increased NOTCH3 mRNA compared with other regions. Immunostaining revealed that NOTCH3 protein was increased in the media of the infrarenal aorta. The abundance of medial NOTCH3 was positively correlated with the dispersion of elastic fibers. Adult cynomolgus monkeys with high fructose consumption displayed vascular wall remodeling, such as smooth muscle cell loss and elastic fiber disruption, predominantly in the infrarenal region. The correlation between NOTCH3 and elastic fiber dispersion was enhanced in these monkeys. CONCLUSIONS: Aortas of young cynomolgus monkeys display regional heterogeneity of their transcriptome and the structure of elastin and collagens. Elastic fibers in the infrarenal aorta are dispersed along with upregulation of medial NOTCH3.


Assuntos
Aorta Abdominal , Tecido Elástico , Animais , Camundongos , Aorta Abdominal/metabolismo , Macaca fascicularis/metabolismo , Tecido Elástico/metabolismo , Receptor Notch3/genética , Receptor Notch3/metabolismo , Elastina/metabolismo , Colágeno/metabolismo , Frutose
12.
BMC Neurol ; 24(1): 77, 2024 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-38408980

RESUMO

BACKGROUND: CADASIL(Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy)is an inherited small vessel disease caused by mutations in NOTCH3 gene. Although NOTCH3 has numerous hotspots of gene mutations, mutations in exons 9 are rare. The p.C484T gene mutation type associated with it has not been reported in any relevant cases yet. Furthermore, CADASIL patients rarely present with acute bilateral multiple subcortical infarcts. CASE PRESENTATION: We report the case of a Chinese female patient with CADASIL who experienced "an acute bilateral subcortical infarction" because of"hemodynamic changes and hypercoagulability". In genetic testing, we discovered a new Cys484Tyr mutation in exon 9, which has also been found in the patient's two daughters. CONCLUSIONS: It is important to note that this discovery not only expands the mutation spectrum of Notch3 mutations in CADASIL patients, but also examines the mechanism behind acute bilateral subcortical infarction in CADASIL patients via case reviews and literature reviews, in order to provide some clinical recommendations for early intervention, diagnosis, and treatment in similar cases in the future.


Assuntos
CADASIL , Humanos , Feminino , CADASIL/complicações , CADASIL/diagnóstico por imagem , CADASIL/genética , Imageamento por Ressonância Magnética , Mutação/genética , Receptor Notch3/genética , Testes Genéticos , Éxons
13.
Mol Biol Rep ; 51(1): 714, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38824264

RESUMO

BACKGROUND: NOTCH3 variants are known to be linked to cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). However, some null NOTCH3 variants with homozygous inheritance cause neurological symptoms distinct from CADASIL. The aim of this study was to expand the clinical spectrum of this distinct condition and provide further evidence of its autosomal recessive inheritance. METHODS AND RESULTS: Whole exome sequencing (WES) was performed on a proband who exhibited livedo racemosa, ataxia, cognitive decline, seizures, and MRI white matter abnormalities without anterior temporal pole lesions. Segregation analysis was conducted with Sanger sequencing. WES of the proband identified a novel homozygous NOTCH3 null variant (c.2984delC). The consanguineous parents were confirmed as heterozygous variant carriers. In addition, three heterozygous NOTCH3 null variants were reported as incidental findings in three unrelated cases analyzed in our center. CONCLUSION: The findings of this study suggest an autosomal recessive inheritance pattern in this early-onset leukoencephalopathy, in contrast to CADASIL's dominant gain-of-function mechanism; which is a clear example of genotype-phenotype correlation. Comprehensive genetic analysis provides valuable insights into disease mechanisms and facilitates diagnosis and family planning for NOTCH3-associated neurological disorders.


Assuntos
Sequenciamento do Exoma , Genes Recessivos , Linhagem , Fenótipo , Receptor Notch3 , Humanos , Receptor Notch3/genética , Masculino , Feminino , Sequenciamento do Exoma/métodos , Genes Recessivos/genética , Adulto , Estudos de Associação Genética , CADASIL/genética , Imageamento por Ressonância Magnética/métodos , Alelos , Homozigoto , Consanguinidade , Mutação com Perda de Função/genética , Mutação/genética , Heterozigoto
14.
Brain ; 146(6): 2364-2376, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-36380532

RESUMO

Genetic leukoencephalopathies (gLEs) are a highly heterogeneous group of rare genetic disorders. The spectrum of gLEs varies among patients of different ages. Distinct from the relatively more abundant studies of gLEs in children, only a few studies that explore the spectrum of adult gLEs have been published, and it should be noted that the majority of these excluded certain gLEs. Thus, to date, no large study has been designed and conducted to characterize the genetic and phenotypic spectra of gLEs in adult patients. We recruited a consecutive series of 309 adult patients clinically suspected of gLEs from Beijing Tiantan Hospital between January 2014 and December 2021. Whole-exome sequencing, mitochondrial DNA sequencing and repeat analysis of NOTCH2NLC, FMR1, DMPK and ZNF9 were performed for patients. We describe the genetic and phenotypic spectra of the set of patients with a genetically confirmed diagnosis and summarize their clinical and radiological characteristics. A total of 201 patients (65%) were genetically diagnosed, while 108 patients (35%) remained undiagnosed. The most frequent diseases were leukoencephalopathies related to NOTCH3 (25%), NOTCH2NLC (19%), ABCD1 (9%), CSF1R (7%) and HTRA1 (5%). Based on a previously proposed pathological classification, the gLEs in our cohort were divided into leukovasculopathies (35%), leuko-axonopathies (31%), myelin disorders (21%), microgliopathies (7%) and astrocytopathies (6%). Patients with NOTCH3 mutations accounted for 70% of the leukovasculopathies, followed by HTRA1 (13%) and COL4A1/2 (9%). The leuko-axonopathies contained the richest variety of associated genes, of which NOTCH2NLC comprised 62%. Among myelin disorders, demyelinating leukoencephalopathies (61%)-mainly adrenoleukodystrophy and Krabbe disease-accounted for the majority, while hypomyelinating leukoencephalopathies (2%) were rare. CSF1R was the only mutated gene detected in microgliopathy patients. Leukoencephalopathy with vanishing white matter disease due to mutations in EIF2B2-5 accounted for half of the astrocytopathies. We characterized the genetic and phenotypic spectra of adult gLEs in a large Chinese cohort. The most frequently mutated genes were NOTCH3, NOTCH2NLC, ABCD1, CSF1R and HTRA1.


Assuntos
Leucoencefalopatias , Criança , Humanos , Adulto , Leucoencefalopatias/genética , Leucoencefalopatias/patologia , Mutação/genética , Bainha de Mielina/patologia , Análise de Sequência de DNA , Receptor Notch3/genética , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Proteína do X Frágil da Deficiência Intelectual
15.
Brain ; 146(7): 2913-2927, 2023 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-36535904

RESUMO

Cysteine-altering missense variants (NOTCH3cys) in one of the 34 epidermal growth-factor-like repeat (EGFr) domains of the NOTCH3 protein are the cause of NOTCH3-associated small vessel disease (NOTCH3-SVD). NOTCH3-SVD is highly variable, ranging from cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) at the severe end of the spectrum to non-penetrance. The strongest known NOTCH3-SVD modifier is NOTCH3cys variant position: NOTCH3cys variants located in EGFr domains 1-6 are associated with a more severe phenotype than NOTCH3cys variants located in EGFr domains 7-34. The objective of this study was to further improve NOTCH3-SVD genotype-based risk prediction by using relative differences in NOTCH3cys variant frequencies between large CADASIL and population cohorts as a starting point. Scientific CADASIL literature, cohorts and population databases were queried for NOTCH3cys variants. For each EGFr domain, the relative difference in NOTCH3cys variant frequency (NVFOR) was calculated using genotypes of 2574 CADASIL patients and 1647 individuals from population databases. Based on NVFOR cut-off values, EGFr domains were classified as either low (LR-EGFr), medium (MR-EGFr) or high risk (HR-EGFr). The clinical relevance of this new three-tiered EGFr risk classification was cross-sectionally validated by comparing SVD imaging markers and clinical outcomes between EGFr risk categories using a genotype-phenotype data set of 434 CADASIL patients and 1003 NOTCH3cys positive community-dwelling individuals. CADASIL patients and community-dwelling individuals harboured 379 unique NOTCH3cys variants. Nine EGFr domains were classified as an HR-EGFr, which included EGFr domains 1-6, but additionally also EGFr domains 8, 11 and 26. Ten EGFr domains were classified as MR-EGFr and 11 as LR-EGFr. In the population genotype-phenotype data set, HR-EGFr individuals had the highest risk of stroke [odds ratio (OR) = 10.81, 95% confidence interval (CI): 5.46-21.37], followed by MR-EGFr individuals (OR = 1.81, 95% CI: 0.84-3.88) and LR-EGFr individuals (OR = 1 [reference]). MR-EGFr individuals had a significantly higher normalized white matter hyperintensity volume (nWMHv; P = 0.005) and peak width of skeletonized mean diffusivity (PSMD; P = 0.035) than LR-EGFr individuals. In the CADASIL genotype-phenotype data set, HR-EGFr domains 8, 11 and 26 patients had a significantly higher risk of stroke (P = 0.002), disability (P = 0.041), nWMHv (P = 1.8 × 10-8), PSMD (P = 2.6 × 10-8) and lacune volume (P = 0.006) than MR-EGFr patients. SVD imaging marker load and clinical outcomes were similar between HR-EGFr 1-6 patients and HR-EGFr 8, 11 and 26 patients. NVFOR was significantly associated with vascular NOTCH3 aggregation load (P = 0.006), but not with NOTCH3 signalling activity (P = 0.88). In conclusion, we identified three clinically distinct NOTCH3-SVD EGFr risk categories based on NFVOR cut-off values, and identified three additional HR-EGFr domains located outside of EGFr domains 1-6. This EGFr risk classification will provide an important key to individualized NOTCH3-SVD disease prediction.


Assuntos
CADASIL , Acidente Vascular Cerebral , Humanos , Receptor Notch3/genética , CADASIL/diagnóstico por imagem , CADASIL/genética , Fator de Crescimento Epidérmico/genética , Imageamento por Ressonância Magnética , Acidente Vascular Cerebral/genética , Medição de Risco , Receptores Notch/genética , Receptores Notch/metabolismo , Mutação/genética
16.
PLoS Genet ; 17(5): e1009553, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33945523

RESUMO

The CBFB gene is frequently mutated in several types of solid tumors. Emerging evidence suggests that CBFB is a tumor suppressor in breast cancer. However, our understanding of the tumor suppressive function of CBFB remains incomplete. Here, we analyze genetic interactions between mutations of CBFB and other highly mutated genes in human breast cancer datasets and find that CBFB and TP53 mutations are mutually exclusive, suggesting a functional association between CBFB and p53. Integrated genomic studies reveal that TAp73 is a common transcriptional target of CBFB and p53. CBFB cooperates with p53 to maintain TAp73 expression, as either CBFB or p53 loss leads to TAp73 depletion. TAp73 re-expression abrogates the tumorigenic effect of CBFB deletion. Although TAp73 loss alone is insufficient for tumorigenesis, it enhances the tumorigenic effect of NOTCH3 overexpression, a downstream event of CBFB loss. Immunohistochemistry shows that p73 loss is coupled with higher proliferation in xenografts. Moreover, TAp73 loss-of-expression is a frequent event in human breast cancer tumors and cell lines. Together, our results significantly advance our understanding of the tumor suppressive functions of CBFB and reveal a mechanism underlying the communication between the two tumor suppressors CBFB and p53.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Subunidade beta de Fator de Ligação ao Core/genética , Regulação Neoplásica da Expressão Gênica , Proteína Tumoral p73/genética , Proteína Supressora de Tumor p53/genética , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade beta de Fator de Ligação ao Core/deficiência , Subunidade beta de Fator de Ligação ao Core/metabolismo , Feminino , Genes Supressores de Tumor , Humanos , Imuno-Histoquímica , Camundongos , Mutação , Receptor Notch3/genética , Receptor Notch3/metabolismo , Transcrição Gênica , Proteína Tumoral p73/deficiência , Proteína Tumoral p73/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Int J Mol Sci ; 25(16)2024 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-39201482

RESUMO

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is caused by NOTCH3 mutations affecting the number of cysteines. The pathogenic role of cysteine-sparing NOTCH3 mutations with typical clinical CADASIL syndrome is still debated. This review aimed to characterize NOTCH3 cysteine-sparing mutations in patients with clinical suspicion of CADASIL. Articles on NOTCH3 cysteine-sparing mutations with clinical suspicion of CADASIL were reviewed. Clinical and radiological cerebral phenotypes data were extracted and characterized across regions and compared with phenotypes of typical CADASIL patients. We screened 298 NOTCH3 cysteine-sparing mutation individuals from 20 publications, and mutations in exon 3 were the most frequently reported (21.46%). Gait impairment (76.47%), cognitive impairment (67.47%), and stroke (62.37%) were the three most common clinical phenotypes; the most frequent radiological cerebral phenotypes were lacunes (74.29%) and cerebral microbleeds (72.73%). Compared with CADASIL patients, cognitive impairment and cerebral microbleed frequencies were significantly higher in patients with NOTCH3 cysteine-sparing mutations, while the white matter hyperintensities in anterior temporal polar and external capsule were rarely observed. Compared with Western patients, radiological phenotypes were more common than clinical phenotypes in cysteine-sparing Asian patients. More than half of cysteine-sparing patients had positive granular osmiophilic material deposits. NOTCH3 cysteine-sparing mutations in patients with clinical suspicion of CADASIL mainly manifested with gait and cognitive impairment but rare white matter hyperintensities in anterior temporal pole and external capsule. Further studies are warranted to pay attention to atypical NOTCH3 variants, which could guide specific diagnosis and help unravel underlying mechanisms.


Assuntos
CADASIL , Cisteína , Mutação , Fenótipo , Receptor Notch3 , Humanos , CADASIL/genética , CADASIL/diagnóstico por imagem , CADASIL/patologia , Receptor Notch3/genética , Cisteína/genética , Disfunção Cognitiva/genética
18.
Int J Mol Sci ; 25(11)2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38892440

RESUMO

NOTCH3 receptor signaling has been linked to the regulation of smooth muscle cell proliferation and the maintenance of smooth muscle cells in an undifferentiated state. Pulmonary arterial hypertension (World Health Organization Group 1 idiopathic disease: PAH) is a fatal disease characterized clinically by elevated pulmonary vascular resistance caused by extensive vascular smooth muscle cell proliferation, perivascular inflammation, and asymmetric neointimal hyperplasia in precapillary pulmonary arteries. In this review, a detailed overview of the specific role of NOTCH3 signaling in PAH, including its mechanisms of activation by a select ligand, downstream signaling effectors, and physiologic effects within the pulmonary vascular tree, is provided. Animal models showing the importance of the NOTCH3 pathway in clinical PAH will be discussed. New drugs and biologics that inhibit NOTCH3 signaling and reverse this deadly disease are highlighted.


Assuntos
Hipertensão Arterial Pulmonar , Receptor Notch3 , Transdução de Sinais , Humanos , Receptor Notch3/metabolismo , Receptor Notch3/genética , Animais , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/patologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia
19.
Laeknabladid ; 110(7): 360-364, 2024 Jul.
Artigo em Is | MEDLINE | ID: mdl-38934718

RESUMO

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is a hereditary small vessel disease of the brain characterized by progressive white matter lesions, subcortical infarcts, and cognitive decline. This autosomal dominant disorder is caused by mutations in the NOTCH3 gene located on chromosome 19, resulting in the accumulation of granular osmiophilic material within the walls of small arteries and arterioles. Clinically, CADASIL typically manifests in mid-adulthood with recurrent ischemic events, migraine with aura, mood disturbances, and cognitive impairment. Neuroimaging plays a crucial role in the diagnosis of CADASIL, with characteristic findings including white matter hyperintensities particularly in the anterior temporal lobe and external capsule.


Assuntos
CADASIL , Predisposição Genética para Doença , Mutação , Fenótipo , Receptor Notch3 , Humanos , CADASIL/genética , CADASIL/diagnóstico , Receptor Notch3/genética , Valor Preditivo dos Testes , Fatores de Risco , Prognóstico , Hereditariedade , Imageamento por Ressonância Magnética , Cognição , Encéfalo/patologia , Encéfalo/diagnóstico por imagem
20.
Stroke ; 54(10): e452-e464, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37602377

RESUMO

Lacunar infarcts and vascular dementia are important phenotypic characteristics of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, the most common inherited cerebral small vessel disease. Individuals with the disease show variability in the nature and onset of symptoms and rates of progression, which are only partially explained by differences in pathogenic mutations in the NOTCH3 gene. Recognizing the disease early in its course and securing a molecular diagnosis are important clinical goals, despite the lack of proven disease-modifying treatments. The purposes of this scientific statement are to review the clinical, genetic, and imaging aspects of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, contrasting it with other inherited small vessel diseases, and to provide key prevention, management, and therapeutic considerations with the intent of reducing practice variability and encouraging production of high-quality evidence to support future treatment recommendations.


Assuntos
CADASIL , Demência Vascular , Humanos , CADASIL/diagnóstico , CADASIL/genética , CADASIL/terapia , Receptor Notch3/genética , American Heart Association , Demência Vascular/genética , Demência Vascular/terapia , Infarto Cerebral , Mutação/genética , Receptores Notch/genética , Imageamento por Ressonância Magnética
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