Mutant NOTCH3ECD Triggers Defects in Mitochondrial Function and Mitophagy in CADASIL Cell Models.

Background: Cerebral autosomal-dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL) is an inherited small-vessel disease that affects the white matter of the brain. Recent studies have confirmed that the deposition of NOTCH3ECD is the main pathological basis of CADASIL; however, whether different mutations present the same pathological characteristics remains to be further studied. Some studies have found that mitochondrial dysfunction is related to CADASIL; however, the specific effects of NOTCH3ECD on mitochondrial remain to be determined. Objective: We aimed to explore the role of mitochondrial dysfunction in CADASIL. Methods: We established transgenic human embryonic kidney-293T cell models (involving alterations in cysteine and non-cysteine residues) via lentiviral transfection. Mitochondrial function and structure were assessed using flow cytometry and transmission electron microscopy, respectively. Mitophagy was assessed using western blotting and immunofluorescence. Results: We demonstrated that NOTCH3ECD deposition affects mitochondrial morphology and function, and that its protein levels are significantly correlated with mitochondrial quality and can directly bind to mitochondria. Moreover, NOTCH3ECD deposition promoted the induction of autophagy and mitophagy. However, these processes were impaired, leading to abnormal mitochondrial accumulation. Conclusions: This study revealed a common pathological feature of NOTCH3ECD deposition caused by different NOTCH3 mutations and provided new insights into the role of NOTCH3ECD in mitochondrial dysfunction and mitophagy.

SNP and Structural Study of the Notch Superfamily Provides Insights and Novel Pharmacological Targets against the CADASIL Syndrome and Neurodegenerative Diseases.

The evolutionary conserved Notch signaling pathway functions as a mediator of direct cell-cell communication between neighboring cells during development. Notch plays a crucial role in various fundamental biological processes in a wide range of tissues. Accordingly, the aberrant signaling of this pathway underlies multiple genetic pathologies such as developmental syndromes, congenital disorders, neurodegenerative diseases, and cancer. Over the last two decades, significant data have shown that the Notch signaling pathway displays a significant function in the mature brains of vertebrates and invertebrates beyond neuronal development and specification during embryonic development. Neuronal connection, synaptic plasticity, learning, and memory appear to be regulated by this pathway. Specific mutations in human Notch family proteins have been linked to several neurodegenerative diseases including Alzheimer's disease, CADASIL, and ischemic injury. Neurodegenerative diseases are incurable disorders of the central nervous system that cause the progressive degeneration and/or death of brain nerve cells, affecting both mental function and movement (ataxia). There is currently a lot of study being conducted to better understand the molecular mechanisms by which Notch plays an essential role in the mature brain. In this study, an in silico analysis of polymorphisms and mutations in human Notch family members that lead to neurodegenerative diseases was performed in order to investigate the correlations among Notch family proteins and neurodegenerative diseases. Particular emphasis was placed on the study of mutations in the Notch3 protein and the structure analysis of the mutant Notch3 protein that leads to the manifestation of the CADASIL syndrome in order to spot possible conserved mutations and interpret the effect of these mutations in the Notch3 protein structure. Conserved mutations of cysteine residues may be candidate pharmacological targets for the potential therapy of CADASIL syndrome.

Blood vessel organoids generated by base editing and harboring single nucleotide variation in Notch3 effectively recapitulate CADASIL-related pathogenesis.

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.

Protein aggregates containing wild-type and mutant NOTCH3 are major drivers of arterial pathology in CADASIL.

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.

Effect of corticosubcortical iron deposition on dysfunction in CADASIL is mediated by white matter microstructural damage.

Iron dysregulation may attenuate cognitive performance in patients with CADASIL. However, the underlying pathophysiological mechanisms remain incompletely understood. Whether white matter microstructural changes mediate these processes is largely unclear. In the present study, 30 cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) patients were confirmed via genetic analysis and 30 sex- and age-matched healthy controls underwent multimodal MRI examinations and neuropsychological assessments. Quantitative susceptibility mapping and peak width of skeletonized mean diffusivity (PSMD) were analyzed. Mediation effect analysis was performed to explore the interrelationship between iron deposition, white matter microstructural changes and cognitive deficits in CADASIL. Cognitive deterioration was most affected in memory and executive function, followed by attention and working memory in CADASIL. Excessive iron in the temporal-precuneus pathway and deep gray matter specific to CADASIL were identified. Mediation analysis further revealed that PSMD mediated the relationship between iron concentration and cognitive profile in CADASIL. The present findings provide a new perspective on iron deposition in the corticosubcortical circuit and its contribution to disease-related selective cognitive decline, in which iron concentration may affect cognition by white matter microstructural changes in CADASIL.

Do Regions of Increased Inflammation Progress to New White Matter Hyperintensities?: A Longitudinal Positron Emission Tomography-Magnetic Resonance Imaging Study.

BACKGROUND: Recent studies have demonstrated increased microglial activation using 11C-PK11195 positron emission tomography imaging, indicating central nervous system inflammation, in cerebral small vessel disease. However, whether such areas of neuroinflammation progress to tissue damage is uncertain. We determined whether white matter destined to become white matter hyperintensities (WMH) at 1 year had evidence of altered inflammation at baseline. METHODS: Forty subjects with small vessel disease (20 sporadic and 20 cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) and 20 controls were recruited to this case-control observational study from in- and out-patient clinics at Addenbrooke's Hospital, Cambridge, UK and imaged at baseline with both 11C-PK11195 positron emission tomography and magnetic resonance imaging; and magnetic resonance imaging including diffusion tensor imaging was repeated at 1 year. WMH were segmented at baseline and 1 year, and areas of new lesion identified. Baseline 11C-PK11195 binding potential and diffusion tensor imaging parameters in these voxels, and normal appearing white matter, was measured. RESULTS: Complete positron emission tomography-magnetic resonance imaging data was available for 17 controls, 16 sporadic small vessel disease, and 14 cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy participants. 11C-PK11195 binding in voxels destined to become new WMH was lower than in normal appearing white matter, which did not progress to WMH (-0.133[±0.081] versus -0.045 [±0.044]; P<0.001). Mean diffusivity was higher and mean fractional anisotropy lower in new WMH voxels than in normal appearing white matter (900 [±80]×10-6 versus 1045 [±149]×10-6 mm2/s and 0.37±0.05 versus 0.29±0.06, both P<0.001) consistent with new WMH showing tissue damage on diffusion tensor imaging a year prior to developing into new WMH; similar results were seen across the 3 groups. CONCLUSIONS: White matter tissue destined to develop into new WMH over the subsequent year is associated with both lower neuroinflammation, and white matter ultrastructural damage at baseline. Our results suggest that this tissue is already damaged 1 year prior to lesion formation. This may reflect that the role of neuroinflammation in the lesion development process occurs at an early stage, although more studies over a longer period would be needed to investigate this further.

Evidence of beta amyloid independent small vessel disease in familial Alzheimer's disease.

We studied small vessel disease (SVD) pathology in Familial Alzheimer's disease (FAD) subjects carrying the presenilin 1 (PSEN1) p.Glu280Ala mutation in comparison to those with sporadic Alzheimer's disease (SAD) as a positive control for Alzheimer's pathology and Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) bearing different NOTCH3 mutations, as positive controls for SVD pathology. Upon magnetic resonance imaging (MRI) in life, some FAD showed mild white matter hyperintensities and no further radiologic evidence of SVD. In post-mortem studies, total SVD pathology in cortical areas and basal ganglia was similar in PSEN1 FAD and CADASIL subjects, except for the feature of arteriosclerosis which was higher in CADASIL subjects than in PSEN1 FAD subjects. Further only a few SAD subjects showed a similar degree of SVD pathology as observed in CADASIL. Furthermore, we found significantly enlarged perivascular spaces in vessels devoid of cerebral amyloid angiopathy in FAD compared with SAD and CADASIL subjects. As expected, there was greater fibrinogen-positive perivascular reactivity in CADASIL but similar reactivity in PSEN1 FAD and SAD groups. Fibrinogen immunoreactivity correlated with onset age in the PSEN1 FAD cases, suggesting increased vascular permeability may contribute to cognitive decline. Additionally, we found reduced perivascular expression of PDGFRß AQP4 in microvessels with enlarged PVS in PSEN1 FAD cases. We demonstrate that there is Aß-independent SVD pathology in PSEN1 FAD, that was marginally lower than that in CADASIL subjects although not evident by MRI. These observations suggest presence of covert SVD even in PSEN1, contributing to disease progression. As is the case in SAD, these consequences may be preventable by early recognition and actively controlling vascular disease risk, even in familial forms of dementia.

Oligomerization, trans-reduction, and instability of mutant NOTCH3 in inherited vascular dementia.

Cerebral small vessel disease (SVD) is a prevalent disease of aging and a major contributor to stroke and dementia. The most commonly inherited SVD, CADASIL, is caused by dominantly acting cysteine-altering mutations in NOTCH3. These mutations change the number of cysteines from an even to an odd number, but the impact of these alterations on NOTCH3 protein structure remain unclear. Here, we prepared wildtype and four mutant recombinant NOTCH3 protein fragments to analyze the impact of CADASIL mutations on oligomerization, thiol status, and protein stability. Using gel electrophoresis, tandem MS/MS, and collision-induced unfolding, we find that NOTCH3 mutant proteins feature increased amounts of inappropriate disulfide bridges, reduced cysteines, and structural instability. Presence of a second protein factor, an N-terminal fragment of NOTCH3 (NTF), is capable of further altering disulfide statuses of both wildtype and mutant proteins, leading to increased numbers of reduced cysteines and further destabilization of NOTCH3 structure. In sum, these studies identify specific cysteine residues alterations and quaternary structure induced by CADASIL mutations in NOTCH3; further, we validate that reductive factors alter the structure and stability of this small vessel disease protein.

Trans-Reduction of Cerebral Small Vessel Disease Proteins by Notch-Derived EGF-like Sequences.

Cysteine oxidation states of extracellular proteins participate in functional regulation and in disease pathophysiology. In the most common inherited dementia, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), mutations in NOTCH3 that alter extracellular cysteine number have implicated NOTCH3 cysteine states as potential triggers of cerebral vascular smooth muscle cytopathology. In this report, we describe a novel property of the second EGF-like domain of NOTCH3: its capacity to alter the cysteine redox state of the NOTCH3 ectodomain. Synthetic peptides corresponding to this sequence (NOTCH3 N-terminal fragment 2, NTF2) readily reduce NOTCH3 N-terminal ectodomain polypeptides in a dose- and time-dependent fashion. Furthermore, NTF2 preferentially reduces regional domains of NOTCH3 with the highest intensity against EGF-like domains 12-15. This process requires cysteine residues of NTF2 and is also capable of targeting selected extracellular proteins that include TSP2 and CTSH. CADASIL mutations in NOTCH3 increase susceptibility to NTF2-facilitated reduction and to trans-reduction by NOTCH3 produced in cells. Moreover, NTF2 forms complexes with the NOTCH3 ectodomain, and cleaved NOTCH3 co-localizes with the NOTCH3 ectodomain in cerebral arteries of CADASIL patients. The potential for NTF2 to reduce vascular proteins and the enhanced preference for it to trans-reduce mutant NOTCH3 implicate a role for protein trans-reduction in cerebrovascular pathological states such as CADASIL.

De novo Mutation Enables NOTCH3ECD Aggregation and Mitochondrial Dysfunction via Interactions with BAX and BCL-2.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) caused by NOTCH3 mutations is the most common monogenic hereditary pattern of cerebral small vessel disease. The aggregation of the mutant NOTCH3 may play a cytotoxic role in CADASIL. However, the main mechanism of this process remains unclear. OBJECTIVE: We aimed to investigate the possible pathogenesis of the mutant NOTCH3 in CADASIL. METHODS: The clinical information of two pedigrees were collected and analyzed. Furthermore, we constructed cell lines corresponding to this mutation in vitro. The degradation of the extracellular domain of NOTCH3 (NOTCH3ECD) was analyzed by Cycloheximide Pulse-Chase Experiment. Flow cytometry and cell counting kit-8 assay were performed to observe the effects of the NOTCH3 mutation on mitochondrial function and apoptosis. RESULTS: We confirmed a de novo heterozygous missense NOTCH3 mutation (c.1690G > A, p. A564T) in two pedigrees. In vitro, the NOTCH3ECD aggregation of A564T mutant may be related to their more difficult to degrade. The mitochondrial membrane potential was attenuated, and cell viability was significant decreased in NOTCH3ECD A564T group. Interestingly, BAX and cytochrome c were significantly increased, which are closely related to the mitochondrial-mediated pathway to apoptosis. CONCLUSION: In our study, the aggregation of NOTCH3ECD A564T mutation may be associated with more difficult degradation of the mutant, and the aggregation may produce toxic effects to induce apoptosis through the mitochondrial-mediated pathway. Therefore, we speculated that mitochondrial dysfunction may hopefully become a new breakthrough point to explain the pathogenesis of cysteine-sparing NOTCH3 mutations.

Proteomic profiling in cerebral amyloid angiopathy reveals an overlap with CADASIL highlighting accumulation of HTRA1 and its substrates.

Cerebral amyloid angiopathy (CAA) is an age-related condition and a major cause of intracerebral hemorrhage and cognitive decline that shows close links with Alzheimer's disease (AD). CAA is characterized by the aggregation of amyloid-ß (Aß) peptides and formation of Aß deposits in the brain vasculature resulting in a disruption of the angioarchitecture. Capillaries are a critical site of Aß pathology in CAA type 1 and become dysfunctional during disease progression. Here, applying an advanced protocol for the isolation of parenchymal microvessels from post-mortem brain tissue combined with liquid chromatography tandem mass spectrometry (LC-MS/MS), we determined the proteomes of CAA type 1 cases (n = 12) including a patient with hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), and of AD cases without microvascular amyloid pathology (n = 13) in comparison to neurologically healthy controls (n = 12). ELISA measurements revealed microvascular Aß1-40 levels to be exclusively enriched in CAA samples (mean: > 3000-fold compared to controls). The proteomic profile of CAA type 1 was characterized by massive enrichment of multiple predominantly secreted proteins and showed significant overlap with the recently reported brain microvascular proteome of patients with cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a hereditary cerebral small vessel disease (SVD) characterized by the aggregation of the Notch3 extracellular domain. We found this overlap to be largely attributable to the accumulation of high-temperature requirement protein A1 (HTRA1), a serine protease with an established role in the brain vasculature, and several of its substrates. Notably, this signature was not present in AD cases. We further show that HTRA1 co-localizes with Aß deposits in brain capillaries from CAA type 1 patients indicating a pathologic recruitment process. Together, these findings suggest a central role of HTRA1-dependent protein homeostasis in the CAA microvasculature and a molecular connection between multiple types of brain microvascular disease.

NOTCH3 variant position is associated with NOTCH3 aggregation load in CADASIL vasculature.

AIMS: CADASIL, the most prevalent hereditary cerebral small vessel disease, is caused by cysteine-altering NOTCH3 variants (NOTCH3cys ) leading to vascular NOTCH3 protein aggregation. It has recently been shown that variants located in one of NOTCH3 protein epidermal growth-factor like repeat (EGFr) domains 1-6, are associated with a more severe phenotype than variants located in one of the EGFr domains 7-34. The underlying mechanism for this genotype-phenotype correlation is unknown. The aim of this study was to analyse whether NOTCH3cys variant position is associated with NOTCH3 protein aggregation load. METHODS: We quantified vascular NOTCH3 aggregation in skin biopsies (n = 25) and brain tissue (n = 7) of CADASIL patients with a NOTCH3cys EGFr 1-6 variant or a EGFr 7-34 variant, using NOTCH3 immunohistochemistry (NOTCH3 score) and ultrastructural analysis of granular osmiophilic material (GOM count). Disease severity was assessed by neuroimaging (lacune count and white matter hyperintensity volume) and disability (modified Rankin scale). RESULTS: Patients with NOTCH3cys EGFr 7-34 variants had lower NOTCH3 scores (P = 1.3·10-5 ) and lower GOM counts (P = 8.2·10-5 ) than patients with NOTCH3cys EGFr 1-6 variants in skin vessels. A similar trend was observed in brain vasculature. In the EGFr 7-34 group, NOTCH3 aggregation levels were associated with lacune count (P = 0.03) and white matter hyperintensity volume (P = 0.02), but not with disability. CONCLUSIONS: CADASIL patients with an EGFr 7-34 variant have significantly less vascular NOTCH3 aggregation than patients with an EGFr 1-6 variant. This may be one of the factors underlying the difference in disease severity between NOTCH3cys EGFr 7-34 and EGFr 1-6 variants.

Hydrolysis of a second Asp-Pro site at the N-terminus of NOTCH3 in inherited vascular dementia.

Cerebrovascular pathology at the biochemical level has been informed by the study of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a vascular disorder caused by NOTCH3 mutations. Previous work in CADASIL described N-terminal proteolysis of NOTCH3 generated by specific non-enzymatic cleavage of the first Asp-Pro sequence of the protein. Here, we investigated whether the second Asp-Pro peptide bond (residues 121-122) of NOTCH3 is cleaved in CADASIL. Monospecific antibodies were generated that recognize the neo-epitope predicted to be generated by cleavage after Asp121. These antibodies were used to localize cleavage events at Asp121 in post-mortem CADASIL and control brain tissue and to investigate factors that regulate cleavage at Asp121. We report that cleavage at Asp121 occurs at a high level in the arterial media of CADASIL cerebral arteries. Leptomeningeal arteries demonstrated substantially more cleavage product than penetrating arteries in the white matter, and control vessels harbored only a small amount of cleaved NOTCH3. Proteolysis at Asp121 occurred in purified preparations of NOTCH3 ectodomain, was increased by acidic pH and reductive conditions, and required native protein conformation for cleavage. Increasing the concentration of NOTCH3 EGF-like domain protein elevated the level of proteolysis. On the other hand, several polyanionic chemicals potently blocked cleavage at Asp121. These studies demonstrate that the NOTCH3 protein in CADASIL is cleaved in multiple locations at labile Asp-Pro peptide bonds. As such, chronic brain vascular disease, like other neurodegenerative conditions, features proteolysis of pathological proteins at multiple sites which may generate small pathological peptides.

Increased extracellular fluid is associated with white matter fiber degeneration in CADASIL: in vivo evidence from diffusion magnetic resonance imaging.

BACKGROUND: White matter hyperintensities (WMHs) are one of the hallmarks of cerebral small vessel disease (CSVD), but the pathological mechanisms underlying WMHs remain unclear. Recent studies suggest that extracellular fluid (ECF) is increased in brain regions with WMHs. It has been hypothesized that ECF accumulation may have detrimental effects on white matter microstructure. To test this hypothesis, we used cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) as a unique CSVD model to investigate the relationships between ECF and fiber microstructural changes in WMHs. METHODS: Thirty-eight CADASIL patients underwent 3.0 T MRI with multi-model sequences. Parameters of free water (FW) and apparent fiber density (AFD) obtained from diffusion-weighted imaging (b = 0 and 1000 s/mm2) were respectively used to quantify the ECF and fiber density. WMHs were split into four subregions with four levels of FW using quartiles (FWq1 to FWq4) for each participant. We analyzed the relationships between FW and AFD in each subregion of WMHs. Additionally, we tested whether FW of WMHs were associated with other accompanied CSVD imaging markers including lacunes and microbleeds. RESULTS: We found an inverse correlation between FW and AFD in WMHs. Subregions of WMHs with high-level of FW (FWq3 and FWq4) were accompanied with decreased AFD and with changes in FW-corrected diffusion tensor imaging parameters. Furthermore, FW was also independently associated with lacunes and microbleeds. CONCLUSIONS: Our study demonstrated that increased ECF was associated with WM degeneration and the occurrence of lacunes and microbleeds, providing important new insights into the role of ECF in CADASIL pathology. Improving ECF drainage might become a therapeutic strategy in future.

Contribution of "Omic" Studies to the Understanding of Cadasil. A Systematic Review.

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 ß (TGFß) 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.

Lunatic fringe promotes the aggregation of CADASIL NOTCH3 mutant proteins.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a genetic small vessel disease characterized by NOTCH3 mutation and abnormal aggregation of NOTCH3 mutant proteins around vessel walls. NOTCH3 is a transmembrane receptor that is degraded by JAGGED1 (JAG1) through a process called trans-endocytosis. There are two types of CADASIL-associated NOTCH3 mutations: signal-active (SA) and signal-deficient (SD) mutations. However, the conditions that lead to abnormal aggregation of NOTCH3 mutant proteins remain poorly understood. Performing a coculture assay, we found that the SA NOTCH3 mutants (C49Y, R90C, R141C, and C185R) were degraded and trans-endocytosed by JAG1 similar to wild-type (WT) NOTCH3, but the SD NOTCH3 mutant (C428S) was not degraded or endocytosed by JAG1, suggesting that other environmental factors may be necessary for the aggregation of SA NOTCH3 mutants. Lunatic fringe (LFNG) is a glycosyltransferase of NOTCH3, but whether LFNG affects the aggregation of NOTCH3 mutants remains unknown. Performing a sucrose gradient ultracentrifugation assay, we found that LFNG might decrease the aggregation propensity of WT NOTCH3 but increase that of C185R NOTCH3. In conclusion, the SD NOTCH3 mutant may be more likely to accumulate than the SA NOTCH3 mutants upon interaction with JAG1. Moreover, LFNG may play an important role in promoting the aggregation of SA NOTCH3 mutants.

Notch3 Signaling and Aggregation as Targets for the Treatment of CADASIL and Other NOTCH3-Associated Small-Vessel Diseases.

Mutations in the NOTCH3 gene can lead to small-vessel disease in humans, including the well-characterized cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a condition caused by NOTCH3 mutations altering the number of cysteine residues in the extracellular domain of Notch3. Growing evidence indicates that other types of mutations in NOTCH3, including cysteine-sparing missense mutations or frameshift and premature stop codons, can lead to small-vessel disease phenotypes of variable severity or penetrance. There are currently no disease-modifying therapies for small-vessel disease, including those associated with NOTCH3 mutations. A deeper understanding of underlying molecular mechanisms and clearly defined targets are needed to promote the development of therapies. This review discusses two key pathophysiological mechanisms believed to contribute to the emergence and progression of small-vessel disease associated with NOTCH3 mutations: abnormal Notch3 aggregation and aberrant Notch3 signaling. This review offers a summary of the literature supporting and challenging the relevance of these mechanisms, together with an overview of available preclinical experiments derived from these mechanisms. It highlights knowledge gaps and future research directions. In view of recent evidence demonstrating the relatively high frequency of NOTCH3 mutations in the population, and their potential role in promoting small-vessel disease, progress in the development of therapies for NOTCH3-associated small-vessel disease is urgently needed.

Genome-wide transcriptome study in skin biopsies reveals an association of E2F4 with cadasil and cognitive impairment.

CADASIL is a small vessel disease caused by mutations in NOTCH3 that lead to an odd number of cysteines in the EGF-like repeat domain, causing protein misfolding and aggregation. The main symptoms are migraine, psychiatric disturbances, recurrent strokes and dementia, being executive function characteristically impaired. The molecular pathways altered by this receptor aggregation need to be studied further. A genome-wide transcriptome study (four cases paired with three healthy siblings) was carried out, in addition to a qRT-PCR for validation purposes (ten new cases and eight new controls). To study the expression profile by cell type of the significant mRNAs found, we performed an in situ hybridization (ISH) (nine cases and eight controls) and a research in the Single-nuclei Brain RNA-seq expression browser (SNBREB). Pathway analysis enrichment was carried out with Gene Ontology and Reactome. Neuropsychological tests were performed in five of the qRT-PCR cases. The two most significant differentially expressed mRNAs (BANP, p-value = 7.23 × 10-4 and PDCD6IP, p-value = 8.36 × 10-4) were selected for the validation study by qRT-PCR. Additionally, we selected two more mRNAs (CAMK2G, p-value = 4.52 × 10-3 and E2F4, p-value = 4.77 × 10-3) due to their association with ischemic neuronal death. E2F4 showed differential expression in the genome-wide transcriptome study and in the qRT-PCR (p = 1.23 × 10-3), and it was upregulated in CADASIL cases. Furthermore, higher E2F4 expression was associated with worse executive function (p = 2.04 × 10-2) and attention and information processing speed (IPS) (p = 8.73 × 10-2). In situ hibridization showed E2F4 expression in endothelial and vascular smooth vessel cells. In silico studies indicated that E2F4 is also expressed in brain endothelial cells. Among the most significant pathways analyzed, there was an enrichment of vascular development, cell adhesion and vesicular machinery terms and autophagy process. E2F4 is more highly expressed in the skin biopsy of CADASIL patients compared to controls, and its expression is present in endothelial cells and VSMCs. Further studies are needed to understand whether E2F4 could be useful as a biomarker, to monitor the disease or be used as a therapeutic target.

Changes in the Morphology, Number, and Protein Levels of Plasma Exosomes in CADASIL Patients.

BACKGROUND: Exosomes are nano-sized extracellular vesicles which are secreted by cells and usually found in body fluids. Previous research has shown that exosomal secretion and autophagy-lysosomal pathway synergistically participates in intracellular abnormal protein elimination. The main pathological manifestations of Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is abnormal accumulation of mutant NOTCH3, and CADASIL vascular smooth muscle cells have been found with autophagy-lysosomal dysfunction. However, whether plasma exosomes change in CADASIL patients is still unclear. OBJECTIVE: We are aimed to investigate the differences of plasma exosomes between CADASIL patients and healthy controls. METHODS: The subjects included 30 CADASIL patients and 30 healthy controls without NOTCH3 mutation. The severity of white matter lesions (WMLs) of CADASIL patients was quantified by Fazekas score. Transmission electron microscopy and nanoparticle tracking analysis were performed to characterize plasma exosomes. In addition, NOTCH3, Neurofilament light and Aß42 levels in plasma exosomes were quantified by enzyme-linked immunosorbent assays. RESULTS: We found that exosomes from CADASIL patients were lower in quantity. In addition, CADASIL plasma exosomes had significantly lower levels of NOTCH3 and significantly increased levels of NFL than those of matched healthy subjects. Interestingly, plasma exosome NOTCH3 levels of CADASIL patients significantly correlated with severity of WMLs. CONCLUSION: The exosome NOTCH3 may be related to the pathological changes of CADASIL, which provides a basis for the pathogenesis research of CADASIL. In addition, plasma exosome NOTCH3 and NFL levels may act as biomarkers to monitor and predict disease progression and measure therapeutic effectiveness in the future clinical trials.

Peripheral arteriopathy caused by Notch3 gain-of-function mutation involves ER and oxidative stress and blunting of NO/sGC/cGMP pathway.

Notch3 mutations cause Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), which predisposes to stroke and dementia. CADASIL is characterised by vascular dysfunction and granular osmiophilic material (GOM) accumulation in cerebral small vessels. Systemic vessels may also be impacted by Notch3 mutations. However vascular characteristics and pathophysiological processes remain elusive. We investigated mechanisms underlying the peripheral vasculopathy mediated by CADASIL-causing Notch3 gain-of-function mutation. We studied: (i) small arteries and vascular smooth muscle cells (VSMCs) from TgNotch3R169C mice (CADASIL model), (ii) VSMCs from peripheral arteries from CADASIL patients, and (iii) post-mortem brains from CADASIL individuals. TgNotch3R169C vessels exhibited GOM deposits, increased vasoreactivity and impaired vasorelaxation. Hypercontractile responses were normalised by fasudil (Rho kinase inhibitor) and 4-phenylbutyrate (4-PBA; endoplasmic-reticulum (ER) stress inhibitor). Ca2+ transients and Ca2+ channel expression were increased in CADASIL VSMCs, with increased expression of Rho guanine nucleotide-exchange factors (GEFs) and ER stress proteins. Vasorelaxation mechanisms were impaired in CADASIL, evidenced by decreased endothelial nitric oxide synthase (eNOS) phosphorylation and reduced cyclic guanosine 3',5'-monophosphate (cGMP) levels, with associated increased soluble guanylate cyclase (sGC) oxidation, decreased sGC activity and reduced levels of the vasodilator hydrogen peroxide (H2O2). In VSMCs from CADASIL patients, sGC oxidation was increased and cGMP levels decreased, effects normalised by fasudil and 4-PBA. Cerebral vessels in CADASIL patients exhibited significant oxidative damage. In conclusion, peripheral vascular dysfunction in CADASIL is associated with altered Ca2+ homoeostasis, oxidative stress and blunted eNOS/sGC/cGMP signaling, processes involving Rho kinase and ER stress. We identify novel pathways underlying the peripheral arteriopathy induced by Notch3 gain-of-function mutation, phenomena that may also be important in cerebral vessels.