NOTCH3 signalling controls human trophoblast stem cell expansion and differentiation.

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.

The N-acetylglucosaminyltransferase Radical Fringe contributes to defects in JAG1-dependent turnover and signaling of NOTCH3 CADASIL mutants.

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. LC-MS/MS 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.

A tumor-suppressive function for Notch3 in the parous mammary gland.

Notch3 promotes mammary luminal cell specification and forced Notch3 activation can induce mammary tumor formation. However, recent studies suggest a tumor-suppressive role for Notch3. Here, we report on Notch3 expression and functional analysis in the mouse mammary gland. Notch3 is expressed in the luminal compartment throughout mammary gland development, but switches to basal cells with initiation of post-lactational involution. Deletion of Notch3 caused a decrease of Notch activation in luminal cells and diminished luminal progenitors at puberty, as well as reduced alveolar progenitors during pregnancy. Parous Notch3-/- mammary glands developed hyperplasia with accumulation of CD24hiCD49flo cells, some of which progressed to invasive tumors with luminal features. Notch3 deletion abolished Notch activation in basal cells during involution, accompanied by altered apoptosis and reduced brown adipocytes, leading to expansion of parity-identified mammary epithelial cells (PI-MECs). Interestingly, the postpartum microenvironment is required for the stem cell activity of Notch3-/- PI-MECs. Finally, high expression of NOTCH3 is associated with prolonged survival in patients with luminal breast cancer. These results highlight an unexpected tumor-suppressive function for Notch3 in the parous mammary gland through restriction of PI-MEC expansion.

Structural changes in NOTCH3 induced by CADASIL mutations: Role of cysteine and non-cysteine alterations.

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.

A midposition NOTCH3 truncation in inherited cerebral small vessel disease may affect the protein interactome.

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.

NOTCH3 promotes docetaxel resistance of prostate cancer cells through regulating TUBB3 and MAPK signaling pathway.

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.

The role of Niemann-Pick type C2 in zebrafish embryonic development.

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.

Inhibition of Notch3/Hey1 ameliorates peribiliary hypoxia by preventing hypertrophic hepatic arteriopathy in biliary atresia progression.

Emerging evidence indicates the presence of vascular abnormalities and ischemia in biliary atresia (BA), although specific mechanisms remain undefined. This study examined both human and experimental BA. Structural and hemodynamic features of hepatic arteries were investigated by Doppler ultrasound, indocyanine green angiography, microscopic histology, and invasive arterial pressure measurement. Opal multiplex immunohistochemistry, western blot, and RT-PCR were applied to assess Notch3 expression and the phenotype of hepatic arterial smooth muscle cells (HASMCs). We established animal models of Notch3 inhibition, overexpression, and knockout to evaluate the differences in overall survival, hepatic artery morphology, peribiliary hypoxia, and HASMC phenotype. Hypertrophic hepatic arteriopathy was evidenced by an increased wall-to-lumen ratio and clinically manifested as hepatic arterial hypertension, decreased hepatic artery perfusion, and formation of hepatic subcapsular vascular plexuses (HSVPs). We observed a correlation between overactivation of Notch3 and phenotypic disruption of HASMCs with the exacerbation of peribiliary hypoxia. Notch3 signaling mediated the phenotype alteration of HASMCs, resulting in arterial wall thickening and impaired oxygen supply in the portal microenvironment. Inhibition of Notch3/Hey1 ameliorates portal hypoxia by restoring the balance of contractile/synthetic HASMCs, thereby preventing hypertrophic arteriopathy in BA.

Deficiency of thrombospondin-2 alleviates intimal hyperplasia in mice by modulating vascular smooth muscle cell proliferation and migration.

Thrombospondin-2 (Tsp2), a glycoprotein in the extracellular matrix, plays a critical role in the maintenance of vascular homeostasis. However, its role in the pathogenesis of cardiovascular disorders such as intimal hyperplasia is not fully elucidated. This study, therefore, aims to explore the effect of Tsp2 on intimal hyperplasia and its associated underlying mechanisms. Intimal hyperplasia (IH) was established using a modified wire-mediated femoral artery injury model. Immunofluorescence and qPCR identified upregulated Tsp2 expression in the injured femoral artery compared with the uninjured femoral artery. Similarly, TSP2 expression was also increased in human samples from the atherosclerotic femoral artery and colocalized with vascular smooth muscle cells (VSMCs). Compared with the wild-type littermates, Tsp2 knockout mice displayed a mitigated IH in the injured femoral artery, as demonstrated by a decreased neointimal area and intimal/median ratio. Primary mouse VSMCs were cultured to explore the mechanism by which Tsp2 influenced IH in vitro. PDGF-stimulated VSMCs presented an elevated Tsp2 expression and enhanced migration and proliferation. However, Tsp2 knockdown by siRNA blocked the increased migration and proliferation of VSMCs. Further analysis identified an association between Notch3 and IH when the intracellular domain of Notch3 (Nicd3) was upregulated in PDGF-stimulated VSMCs and femoral arteries with IH in human tissues. Along with the overexpression and downregulation of Tsp2, the Nicd3 expression was also up and downregulated accordingly. Tsp2 was associated with IH and may serve as a therapeutic target for IH. Downregulation of Tsp2 could mitigate the progression of IH by modulating the proliferation and migration of VSMCs.

Characteristics and temporal evolution of asymptomatic diffusion-weighted imaging lesions in patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL).

BACKGROUND AND PURPOSE: The role of asymptomatic diffusion-weighted imaging-positive (aDWI+) lesions in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) patients remains unclear, and their radiographic features may differ from those of symptomatic diffusion-weighted imaging-positive (sDWI+) lesions. We aimed to investigate the clinicoradiographic characteristics of aDWI+ lesions in CADASIL patients. METHODS: We conducted a retrospective analysis using data from the Taiwan CADASIL Registry. aDWI+ lesions were defined as incidentally detected DWI+ lesions without corresponding acute neurological deficits. We compared the baseline clinical characteristics of patients with and without aDWI+ lesions and analyzed their radiological features and evolution in relation to sDWI+ lesions. RESULTS: Among 154 enrolled patients (mean age 62 ± 10 years), 17 (11%) had aDWI+ lesions. Baseline clinical characteristics were similar in the two groups, but those with aDWI+ lesions had more lacunes (median 8 vs. 2), multiple cerebral microbleeds (CMBs; 85% vs. 40%), and anterior temporal white matter hyperintensity (WMH; 47% vs. 14%). Multivariable analysis showed that aDWI+ lesions were associated with anterior temporal WMH (odds ratio 5.7, 95% confidence interval 1.5-21.0) after adjusting for multiple lacunes, multiple CMBs, and total WMH score. Compared to sDWI+ lesions, aDWI+ lesions were more often small infarcts (<1 cm; 89% vs. 23%) and less likely to involve the corticospinal tract (11% vs. 96%). Among the 11 aDWI+ lesions with follow-up magnetic resonance imaging, seven became microinfarcts, three became lacunes, and one disappeared. CONCLUSIONS: aDWI+ lesions in CADASIL are not uncommon and are associated with higher burdens of small vessel disease and anterior temporal WMH. Further research is needed to assess their long-term impact on CADASIL.

First report of a p.Cys484Tyr Notch3 mutation in a CADASIL patient with acute bilateral multiple subcortical infarcts-case report and brief review.

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.

Distinct neurological phenotypes associated with biallelic loss of NOTCH3 function: evidence for recessive inheritance.

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.

Three-tiered EGFr domain risk stratification for individualized NOTCH3-small vessel disease prediction.

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.

Disabled-2: a protein up-regulated by high molecular weight hyaluronan has both tumor promoting and tumor suppressor roles in ovarian cancer.

Although the pro-tumorigenic functions of hyaluronan (HA) are well documented there is limited information on the effects and targets of different molecular weight HA. Here, we investigated the effects of 27 kDa, 183 kDa and 1000 kDa HA on ES-2 ovarian cancer cells overexpressing the stem cell associated protein, Notch3. 1000 kDA HA promoted spheroid formation in ES-2 cells mixed with ES-2 overexpressing Notch3 (1:3). We report disabled-2 (DAB2) as a novel protein regulated by 1000 kDa HA and further investigated its role in ovarian cancer. DAB2 was downregulated in ovarian cancer compared to normal tissues but increased in metastatic ovarian tumors compared to primary tumors. High DAB2 expression was associated with poor patient outcome and positively correlated with HA synthesis enzyme HAS2, HA receptor CD44 and EMT and macrophage markers. Stromal DAB2 immunostaining was significantly increased in matched ovarian cancer tissues at relapse compared to diagnosis and associated with reduced survival. The proportion of DAB2 positive macrophages was significantly increased in metastatic ovarian cancer tissues compared to primary cancers. However, DAB2 overexpression significantly reduced invasion by both A2780 and OVCAR3 cells in vivo. Our research identifies a novel relationship between HA signalling, Notch3 and DAB2. We highlight a complex relationship of both pro-tumorigenic and tumor suppressive functions of DAB2 in ovarian cancer. Our findings highlight that DAB2 has a direct tumor suppressive role on ovarian cancer cells. The pro-tumorigenic role of DAB2 may be mediated by tumour associated macrophages and requires further investigation.

LncRNA WWTR1-AS1 upregulates Notch3 through miR-136 to increase cancer cell stemness in cervical squamous cell carcinoma.

BACKGROUND: This Study investigated the role of WWTR1-AS1 in cervical squamous cell carcinoma (CSCC). RESULTS: WWTR1-AS1 expression was upregulated in CSCC tissues. WWTR1-AS1 was predicted to interact with miR-136, whereas correlation analysis revealed that there was no close correlation between WWTR1-AS1 and miR-136 across CSCC samples. Moreover, WWTR1-AS1 and miR-136 did not regulate the expression of each other. In addition, overexpression of WWTR1-AS1 increased the expression levels of Notch3, which could be targeted by miR-136. Cell stemness analysis indicated that the overexpression of WWTR1-AS1 and Notch3 increased CSCC cell stemness and the capacity of CSCC cell to grow as spheroids. Overexpression of miR-136 decreased CSCC cell stemness and reversed the effects of overexpression of WWTR1-AS1 on Notch3 in CSCC cells. CONCLUSION: Therefore, WWTR1-AS1 may upregulate Notch3 through miR-136 to increase cancer cell stemness in CSCC.

NOTCH3 and Pulmonary Arterial Hypertension.

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.

Phenotypes Associated with NOTCH3 Cysteine-Sparing Mutations in Patients with Clinical Suspicion of CADASIL: A Systematic Review.

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.

[The hereditary vessel disease CADASIL].

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.

Hypoxia-inducible factor-1α and poly [ADP ribose] polymerase 1 cooperatively regulate Notch3 expression under hypoxia via a noncanonical mechanism.

Upregulation of Notch3 expression has been reported in many cancers and is considered a marker for poor prognosis. Hypoxia is a driving factor of the Notch3 signaling pathway; however, the induction mechanism and role of hypoxia-inducible factor-1α (HIF-1α) in the Notch3 response are still unclear. In this study, we found that HIF-1α and poly [ADP-ribose] polymerase 1 (PARP-1) regulate Notch3 induction under hypoxia via a noncanonical mechanism. In the analyzed cancer cell lines, Notch3 expression was increased during hypoxia at both the mRNA and protein levels. HIF-1α knockdown and Notch3 promoter reporter analyses indicated that the induction of Notch3 by hypoxia requires HIF-1α and also another molecule that binds the Notch3 promoter's guanine-rich region, which lacks the canonical hypoxia response element. Therefore, using mass spectrometry analysis to identify the binding proteins of the Notch3 promoter, we found that PARP-1 specifically binds to the Notch3 promoter. Interestingly, analyses of the Notch3 promoter reporter and knockdown of PARP-1 revealed that PARP-1 plays an important role in Notch3 regulation. Furthermore, we demonstrate that PARP inhibitors, including an inhibitor specific for PARP-1, attenuated the induction of Notch3 by hypoxia. These results uncover a novel mechanism in which HIF-1α associates with PARP-1 on the Notch3 promoter in a hypoxia response element-independent manner, thereby inducing Notch3 expression during hypoxia. Further studies on this mechanism could facilitate a better understanding of the broader functions of HIF-1α, the roles of Notch3 in cancer formation, and the insights into novel therapeutic strategies.

Influence of different spectra of NOTCH3 variants on the clinical phenotype of CADASIL - experience from Slovakia.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common hereditary vascular disorder causing ischaemic attacks and strokes in middle-aged adults. Though the clinical spectrum includes some typical symptoms, recognition of the disease, especially at an earlier stage, is very difficult because of the highly variable manifestation and incomplete clinical picture. Characteristic brain MRI findings and the presence of pathogenic variants in the NOTCH3 gene are fundamental for CADASIL diagnosis. In this paper, we provide the first comprehensive report on CADASIL patients from Slovakia. Altogether, we identified 23 different pathogenic variants in 35 unrelated families. In our cohort of patients with clinical suspicion of CADASIL, we found a causal genetic defect and confirmed the diagnosis in 10.2% of cases. We present the case reports with up-to-date unpublished NOTCH3 variants and describe their phenotype-genotype correlation: p.(Cys65Phe), p.(Pro86Leu/Ser502Phe), p.(Arg156*), p.(Cys408Arg), p.(Tyr423Cys), p.(Asp1720His), and p.(Asp1893Thrfs*13). The most frequently described location for pathogenic variants was in exon 4, whereas the most common single variant was p.Arg1076Cys in exon 20. Based on the results of our study, we propose a re-evaluation of the criteria for the selection of patients suitable for NOTCH3 gene analysis. We hereby state that the currently used protocol of a high score requirement is not ideal for assessing molecular analysis, and it will be desirable to be less strict in criteria for genetic testing.