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Loss of arterial smooth muscle cells (SMCs) and abnormal accumulation of the extracellular domain of the NOTCH3 receptor (Notch3ECD) are the 2 core features of CADASIL, a common cerebral small vessel disease caused by highly stereotyped dominant mutations in NOTCH3. Yet the relationship between NOTCH3 receptor activity, Notch3ECD accumulation, and arterial SMC loss has remained elusive, hampering the development of disease-modifying therapies. Using dedicated histopathological and multiscale imaging modalities, we could detect and quantify previously undetectable CADASIL-driven arterial SMC loss in the CNS of mice expressing the archetypal Arg169Cys mutation. We found that arterial pathology was more severe and Notch3ECD accumulation greater in transgenic mice overexpressing the mutation on a wild-type Notch3 background (TgNotch3R169C) than in knockin Notch3R170C/R170C mice expressing this mutation without a wild-type Notch3 copy. Notably, expression of Notch3-regulated genes was essentially unchanged in TgNotch3R169C arteries. We further showed that wild-type Notch3ECD coaggregated with mutant Notch3ECD and that elimination of 1 copy of wild-type Notch3 in TgNotch3R169C was sufficient to attenuate Notch3ECD accumulation and arterial pathology. These findings suggest that Notch3ECD accumulation, involving mutant and wild-type NOTCH3, is a major driver of arterial SMC loss in CADASIL, paving the way for NOTCH3-lowering therapeutic strategies.

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is caused by mutations in the NOTCH3 gene, affecting the number of cysteines in the extracellular domain of the receptor, causing protein misfolding and receptor aggregation. The pathogenic role of cysteine-sparing NOTCH3 missense mutations in patients with typical clinical CADASIL syndrome is unknown. The aim of this article is to describe these mutations to clarify if any could be potentially pathogenic. Articles on cysteine-sparing NOTCH3 missense mutations in patients with clinical suspicion of CADASIL were reviewed. Mutations were considered potentially pathogenic if patients had: (a) typical clinical CADASIL syndrome; (b) diffuse white matter hyperintensities; (c) the 33 NOTCH3 exons analyzed; (d) mutations that were not polymorphisms; and (e) Granular osmiophilic material (GOM) deposits in the skin biopsy. Twenty-five different mutations were listed. Four fulfill the above criteria: p.R61W; p.R75P; p.D80G; and p.R213K. Patients carrying these mutations had typical clinical CADASIL syndrome and diffuse white matter hyperintensities, mostly without anterior temporal pole involvement. Cysteine-sparing NOTCH3 missense mutations are associated with typical clinical CADASIL syndrome and typical magnetic resonance imaging (MRI) findings, although with less involvement of the anterior temporal lobe. Hence, these mutations should be further studied to confirm their pathological role in CADASIL.

Key Points Notch signalling has key roles in the development and homeostasis of most organs, including — notably — the haematopoietic system, skin, vascular system and liver. An increasing number of known human diseases relate to dysregulated Notch signalling, including developmental congenital disorders and cancers with de novo NOTCH mutations. The core pathway is simple but provides ample potential therapeutic possibilities. Viable targets include inhibition of receptor cleavage by γ-secretase inhibitors and blockade of specific receptors or ligands with antibodies. Crosstalk between Notch and other signalling mechanisms may provide possibilities for combinatorial treatments, especially in cancer where targeting several pathways simultaneously may offer considerable benefits. Numerous clinical trials are underway with the aim of modulating Notch signalling. The Notch signalling pathway, which is crucial for the development and homeostasis of most tissues, has been linked to a range of diseases, including cancer. Andersson and Lendahl discuss where and how to intervene therapeutically in the Notch signalling pathway, highlighting current achievements and remaining obstacles. The Notch signalling pathway is evolutionarily conserved and is crucial for the development and homeostasis of most tissues. Deregulated Notch signalling leads to various diseases, such as T cell leukaemia, Alagille syndrome and a stroke and dementia syndrome known as CADASIL, and so strategies to therapeutically modulate Notch signalling are of interest. Clinical trials of Notch pathway inhibitors in patients with solid tumours have been reported, and several approaches are under preclinical evaluation. In this Review, we focus on aspects of the pathway that are amenable to therapeutic intervention, diseases that could be targeted and the various Notch pathway modulation strategies that are currently being explored.

Human blood vessel organoids (hBVOs) offer a promising platform for investigating vascular diseases and identifying therapeutic targets. In this study, we focused on in vitro modeling and therapeutic target finding of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common form of hereditary stroke disorder caused by mutations in the NOTCH3 gene. Despite the identification of these mutations, the underlying pathological mechanism is elusive, and effective therapeutic approaches are lacking. CADASIL primarily affects the blood vessels in the brain, leading to ischemic strokes, migraines, and dementia. By employing CRISPR/Cas9 base-editing technology, we generated human induced pluripotent stem cells (hiPSCs) carrying Notch3 mutations. These mutant hiPSCs were differentiated into hBVOs. The NOTCH3 mutated hBVOs exhibited CADASIL-like pathology, characterized by a reduced vessel diameter and degeneration of mural cells. Furthermore, we observed an accumulation of Notch3 extracellular domain (Notch3ECD), increased apoptosis, and cytoskeletal alterations in the NOTCH3 mutant hBVOs. Notably, treatment with ROCK inhibitors partially restored the disconnection between endothelial cells and mural cells in the mutant hBVOs. These findings shed light on the pathogenesis of CADASIL and highlight the potential of hBVOs for studying and developing therapeutic interventions for this debilitating human vascular disorder.

Notch3 is one of four mammalian Notch proteins, which act as signalling receptors to control cell fate in many developmental and adult tissue contexts. Notch signalling continues to be important in the adult organism for tissue maintenance and renewal and mis-regulation of Notch is involved in many diseases. Genetic studies have shown that Notch3 gene knockouts are viable and have limited developmental defects, focussed mostly on defects in the arterial smooth muscle cell lineage. Additional studies have revealed overlapping roles for Notch3 with other Notch proteins, which widen the range of developmental functions. In the adult, Notch3, in collaboration with other Notch proteins, is involved in stem cell regulation in different tissues in stem cell regulation in different tissues, and it also controls the plasticity of the vascular smooth muscle phenotype involved in arterial vessel remodelling. Overexpression, gene amplification and mis-activation of Notch3 are associated with different cancers, in particular triple negative breast cancer and ovarian cancer. Mutations of Notch3 are associated with a dominantly inherited disease CADASIL (cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy), and there is further evidence linking Notch3 misregulation to hypertensive disease. Here we discuss the distinctive roles of Notch3 in development, health and disease, different views as to the underlying mechanisms of its activation and misregulation in different contexts and potential for therapeutic intervention.

CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) is a small vessel disease caused by mutations in NOTCH3 that lead to an odd number of cysteines in the epidermal growth factor (EGF)-like repeat domain, causing protein misfolding and aggregation. The main symptoms are migraines, psychiatric disorders, recurrent strokes, and dementia. Omic technologies allow the massive study of different molecules for understanding diseases in a non-biased manner or even for discovering targets and their possible treatments. We analyzed the progress in understanding CADASIL that has been made possible by omics sciences. For this purpose, we included studies that focused on CADASIL and used omics techniques, searching bibliographic resources, such as PubMed. We excluded studies with other phenotypes, such as migraine or leukodystrophies. A total of 18 articles were reviewed. Due to the high prevalence of NOTCH3 mutations considered pathogenic to date in genomic repositories, one can ask whether all of them produce CADASIL, different degrees of the disease, or whether they are just a risk factor for small vessel disease. Besides, proteomics and transcriptomics studies found that the molecules that are significantly altered in CADASIL are mainly related to cell adhesion, the cytoskeleton or extracellular matrix components, misfolding control, autophagia, angiogenesis, or the transforming growth factor β (TGFβ) signaling pathway. The omics studies performed on CADASIL have been useful for understanding the biological mechanisms and could be key factors for finding potential drug targets.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary cerebral small vessel disease caused by NOTCH3 mutation. The condition leads to recurrent ischemic strokes, vascular dementia, early-onset and high disability, and its prevalence has long been underestimated. Pathologically, CADASIL involves the degeneration and loss of brain vascular smooth muscle cells (VSMCs), but the mechanisms remain unclear. Using a transgenic mouse model of CADASIL (NOTCH3-R545C) and NOTCH3 mutant (R90C and R544C) cell models, the study identifies impaired NOTCH3 signaling, resulting from reduced SUMOylation, as a pivotal pathogenic mechanism that compromises cell survival and proliferation. We found that the NOTCH3-R545C mice exhibited anxiety-like behaviors, spatial working memory deficits, and reduced mural cell coverage. In primary VSMCs and HEK293 cells, the NOTCH3 mutation diminished cell viability, proliferation and NOTCH3 cleavage. Mechanistically, NOTCH3 mutations reduced NOTCH3 SUMOylation. This reduction diminished the interaction between the NOTCH3 intracellular domain (NOTCH3ICD) and the transcription factor RBPjκ, thereby impairing downstream NOTCH3 signaling. Overexpression of the SUMOylation molecule SUMO1 restored NOTCH3 cleavage, stability, transcriptional activity, target gene expression, and cell survival/proliferation. In contrast, the deSUMOylation enzyme SENP1 and SUMOylation-deficient NOTCH3 mutants exacerbated these impairments. These findings demonstrate that reversible SUMOylation of NOTCH3 serves as a critical regulator of VSMC homeostasis, with SUMO1 and SENP1 functioning as key mediators. This study provides novel insights into CADASIL pathogenesis by linking NOTCH3 SUMOylation to vascular dysfunction and further highlights SUMOylation as a potential target for the therapeutic development of CADASIL.

Aims The aim of this study is to investigate the role of glymphatic function of cerebral autosomal dominant arteriopathy, subcortical infarcts, and leukoencephalopathy (CADASIL), the most common monogenic small vessel disease caused by NOTCH3 mutation, and to explore potential therapeutic strategies to improve glymphatic function. Methods We assessed glymphatic influx and efflux function in CADASIL mouse models (Notch3R170C) and correlated these findings with brain atrophy in CADASIL patients. We also investigated the underlying mechanisms of glymphatic impairment, focusing the expression of AQP4 in astrocytic endfeet. Results CADASIL mouse exhibited both impaired glymphatic influx and efflux, which impedes waste clearance and promotes brain senescence. In accordance, brain atrophy in CADASIL patients is associated with perivascular space enlargement, indicating that glymphatic impairment contributes to advanced brain senescence in CADASIL. The glymphatic malfunction in CADASIL is attributed to diminished AQP4 expression in astrocytic endfeet, which is the core mediator of glymphatic activity. Mechanistically, AQP4 expression is regulated by NOTCH3‐RUNX1‐CMYB signaling. Reinforcing AQP4 expression in astrocytes by AAV‐based therapy resumes the glymphatic functions in CADASIL mice, which further prevents brain senescence. Conclusion We propose that to improve glymphatic function by reinforcing AQP4 expression is a promising therapeutic strategy in CADASIL. Beside mural cells, NOTCH3 is expressed in astrocytes. Expression of Aquaporin 4 (AQP4), the core mediator of glymphatic functions, is decreased in CADASIL brain. Consequently, glymphatic activities are impaired. Diminished AQP4 expression is attributed to the suppression of RUNX1‐CMYB‐AQP4 signaling due to NOTCH3 mutation in astrocytes. To reinforce AQP4 expression by astrocyte‐targeting AAV resumes glymphatic functions in CADASIL mice, which subsequently prevents brain senescence.

Pericytes regulate vessel stability and pericyte dysfunction contributes to retinopathies, stroke and cancer. Here we define Notch as a key regulator of pericyte function during angiogenesis. In Notch1 +/− ; Notch3 −/− mice, combined deficiency of Notch1 and Notch3 altered pericyte interaction with the endothelium and reduced pericyte coverage of the retinal vasculature. Notch1 and Notch3 were shown to cooperate to promote proper vascular basement membrane formation and contribute to endothelial cell quiescence. Accordingly, loss of pericyte function due to Notch deficiency exacerbates endothelial cell activation caused by Notch1 haploinsufficiency. Mice mutant for Notch1 and Notch3 develop arteriovenous malformations and display hallmarks of the ischemic stroke disease CADASIL. Thus, Notch deficiency compromises pericyte function and contributes to vascular pathologies.

Background Mutations in NOTCH3 cause CADASIL, a dominantly inherited condition, linked to recurrent stroke and vascular dementia and associated with accumulation of the ECD of NOTCH3. The latter has a toxic effect on VSMCs. Misregulated signalling may also play a role in disease progression. ECD detachment is an obligatory step in NOTCH3 activation, but some CADASIL mutants prevent ligand-induced activation and so ligand interactions are not a common underlying requirement. Here we investigated whether basal NOTCH3 endocytosis that is associated with ligand-independent activation mechanisms can be source of ECD shedding in CADASIL mutants. Methods We used transient transfection of hTERT-RPE1 cells to express WT, R90C, C212Y and C455R mutant NOTCH3 constructs. Internalisation of NOTCH3 was followed using a pulse-chase endocytic uptake assay after surface NOTCH3 labelling of live cells. Immunolocalisation of NOTCH3 ECD and ICD was used to define the subcellular localisation of expressed NOTCH3 in the secretory and endocytic pathway of transfected cells, and endogenous NOTCH3 in MCF7 cells and VSMCs derived from human ES cells. To investigate NOTCH3 signalling we used a luciferase reporter assay under control of a NOTCH-responsive reporter element. Results Both WT and CADASIL NOTCH3 proteins are endocytosed before ECD shedding and then undergo dissociation and independent trafficking of the ECD and ICD in the endosome. The relative amount of ICD compared to ECD that colocalised with endosomal markers increases as NOTCH3 progresses through the endosomal trafficking pathway from early endosome to lysosome. The R90C mutant showed earlier separation of ECD compared to WT or other CADASIL mutants tested. All WT and mutant constructs activated downstream signalling when expressed in hTERT-RPE1 cells, and these basal signalling levels were not affected by the C455R mutation which removes ligand-activated signalling. R90C showed distinctly different requirements for activation being less sensitive to metalloprotease inhibition and more sensitive to inhibition of the lysosomal protein TRPML. Conclusions Basal NOTCH3 endocytosis and signalling is a potential source of ECD shedding and accumulation in CADASIL. Different mechanisms may apply to different CADASIL mutants and understanding the variety of mechanisms by which NOTCH3 signalling and ECD shedding occur will inform new targeted approaches to treatments of small vessel disease. Tuning NOTCH3 activity through modulation of the endocytic pathway may offer better tolerated approaches than direct targeting of NOTCH3 signalling.