Granular Osmiophilic Material Research Articles

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.

Abstract 3116: Elucidating The Molecular Progression Of Hereditary Vascular Dementia In A Novel Mouse Model

Aging is an immutable risk factor for the development of numerous cardiovascular diseases, including vascular dementia. Vascular dementia is the second most common cause of dementia with up to 20% of dementia cases in the elderly attributed to dysfunctional vasculature. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), is a mendelian vascular dementia characterized by progressive dysfunction of vascular smooth muscle cells (vSMCs) due to gene variants in NOTCH3 . With over 250 known disease - causing variants in NOTCH3 identified in the past 24 years, the allelic heterogeneity observed in CADASIL has long contributed to our impaired understanding of the molecular progression from NOTCH3 variation to clinical presentation. Recent work published by our group has shown that Notch3 is lost from in the cerebral vasculature of mouse ( Notch3 -/- ) and humans during aging. With time, the progressive loss of Notch3 in the aging mouse brain vasculature drives the formation of cerebral micro-aneurysms as well as impairs vascular reactivity and cerebral blood flow. These vascular consequences also hinder glymphatic flow which promotes an accelerated aggregation of glycosaminoglycans and the development of neuronal gene signature analogous to those observed in other neurodegenerative diseases such as Alzheimer’s disease. To assess if CADASIL represents a loss of NOTCH3 function and thus an accelerated cerebral aging or novel gain of function in NOTCH3, we used CRISPR-Cas9 genome editing to create a novel CADASIL mouse model: Notch3C175Y (N3Y). This alteration faithfully recapitulates the human variant within the same locus and shows the pathological hallmark of CADASIL: GOMs (granular osmiophilic material). Histopathological assessment of the N3Y mouse shows dysregulation of the vSMC organization as early as 8 weeks of age along with novel structural abnormalities as compared to the Notch3 -/- . Using single-cell RNA sequencing we also performed in-depth molecular characterization of the vascular changes in N3Y. Analysis at 2 months and at 24 months allowed us to identify temporal progression of the disease and provided novel molecular insights into the mechanisms of this mendelian form of vascular dementia.

Disturbances of Ocular Circulation in Color Doppler Imaging, Retinal Changes and Electrophysiological Tests with Neuro-Ophthalmological Clinical Symptoms in the Course of CADASIL Syndrome—A Case Report

The authors present a new paper examining the disturbances in ocular circulation and electrophysiological changes in the presence of neuro-ophthalmic manifestations in a patient with cerebral autosomal dominant arteriopathy with subcortical infracts and leucoencephalopathy (CADASIL). Symptoms reported by the patient included: transient vision loss (TVL), migraines, diplopia, bilateral peripheral visual field loss and convergence insufficiency. CADASIL was confirmed by the presence of NOTCH3 gene mutation (p.Cys212Gly), the presence of granular osmiophilic material (GOM) in cutaneous vessels in an immunohistochemistry test (IHC) and bilateral focal vasogenic lesions in the white matter of the cerebral hemisphere, with micro-focal infarct in the left external capsule on a magnetic resonance imaging test (MRI). Color Doppler imaging (CDI) confirmed decreased blood flow and increased vascular resistance in the retinal and posterior ciliary arteries, with reduced P50 wave amplitude on a pattern electroretinogram (PERG). An eye fundus examination and fluorescein angiography (FA) revealed the constriction of retinal vessels and a peripheral retinal pigment epithelium (RPE) atrophy with focal drusen. The authors suggest that the cause of TVL may be changes in the hemodynamics of the retinochoroid vessels associated with the narrowing of small vessels and the presence of druses in the retina-which is supported by a decrease in the amplitude of the P50 wave in PERG, changes in OCT correlating simultaneously with changes in MRI imaging and other neurological symptoms.

A clinical case of anosognosia in a CADASIL disease.

IntroductionCADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) is a cerebrovascular disease, tht appears in 1.98/100,000. It´s caused by a mutation of the Notch3 gene and is characterized by accumulation of granular osmiophilic material in the middle layer of the small and median sized cerebral arteries.Sypmtoms are migraine, recurrent cerebral ischemic episodes, dementia, neuropsychiatric disorders (anosognosia, character disorders, apathy and cognitive impairment). It usually appears between 30-60 years, although there is an important variability. There is no curative treatment, only palliative.ObjectivesClinical review of anosognosia and its presence in CADASIL disease.MethodsClinical case and literatura review.ResultsWe presented the clinical case of a 68-year-old man, who was diagnosed with CADASIL after a stroke 3 years earlier. In his family, his brother was diagnosed also with CADASIL. The patient had previously presented disturbances in impulse control (hyperorality) and important executive failures. He currently presented anosognosia, deficits in verbal memory, spatial perception and executive functions, in addition to behavioral alterations and apathy. Due to these deficits he was prohibited from certain activities (driving, hunting).The patient was not aware of these deficits and becouse of his “no knowledge of his illness”, he disagreed with these prohibitions, so he showed rage and anger at the impotence of not understanding why certain actions are prohibited.In the consultation, mnesic errors and in naming objects were also objectified, for which it was recommended to carry out cognitive stimulation on a daily basis. In addition, he presented failures of sphincter incontinence, especially of urine and occasionally also of the anal sphincter. He had previously had episodes of myoclonus or fasciculations.A genetic study by massive sequencing confirmed the heterozygous presence of the pathogenic variant c.1819C>T p.(Arg607Cys) in the NOTCH3 gene, a CADASIL disease.ConclusionsThe anosognosia that many patients with CADASIL disease present constitutes a problem because it contributes to the delay in consultation and, therefore, the delay in the adequate diagnostic approach, therapeutic possibilities and family genetic counseling. Due in part to anosognosia, CADASIL is considered an underdiagnosed entity. Due to the lack of awareness and the consequent lack of recognition of the deficit, these people are often seen as stubborn and difficult to deal with by people in their immediate environment.In addition, there is general difficulty in the rehabilitation process, since patients do not think the neccesity to be treated. This can generate frustration and despair both in their relatives and in the health personnel.For all these reasons, both in anosognosia and in CADASIL disease, adequate psychological support is needed for both those affected and their families.Disclosure of InterestNone Declared

Update on the Epidemiology, Pathogenesis, and Biomarkers of Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leukoencephalopathy

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic disorder of the cerebral small blood vessels. It is caused by mutations in the NOTCH3 gene on chromosome 19, and more than 280 distinct pathogenic mutations have been reported to date. CADASIL was once considered a very rare disease with an estimated prevalence of 1.3-4.1 per 100,000 adults. However, recent large-scale genomic studies have revealed a high prevalence of pathogenic NOTCH3 variants among the general population, with the highest risk being among Asians. The disease severity and age at onset vary significantly even among individuals who carry the same NOTCH3 mutations. It is still unclear whether a significant genotype-phenotype correlation is present in CADASIL. The accumulation of granular osmiophilic material in the vasculature is a characteristic feature of CADASIL. However, the exact pathogenesis of CADASIL remains largely unclear despite various laboratory and clinical observations being made. Major hypotheses proposed so far have included aberrant NOTCH3 signaling, toxic aggregation, and abnormal matrisomes. Several characteristic features have been observed in the brain magnetic resonance images of patients with CADASIL, including subcortical lacunar lesions and white matter hyperintensities in the anterior temporal lobe or external capsule, which were useful in differentiating CADASIL from sporadic stroke in patients. The number of lacunes and the degree of brain atrophy were useful in predicting the clinical outcomes of patients with CADASIL. Several promising blood biomarkers have also recently been discovered for CADASIL, which require further research for validation.

Detection of Vascular Notch3 Deposits in Unfixed Frozen Skin Biopsy Sample in CADASIL

This study aimed to evaluate the utility of immunohistochemical staining of vascular Notch3 deposits in biopsied unfixed frozen skin samples from patients with suspected cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). We analyzed vascular Notch3 deposits in unfixed frozen skin biopsy samples obtained from 43 patients with suspected CADASIL by immunohistochemistry using antibodies against the extracellular domain (ECD) of Notch3. We also sequenced the NOTCH3 gene in all patients, as well as evaluated their symptoms and neuroimages. We found granular Notch3 ECD deposits in the vessel walls of unfixed frozen skin biopsy samples in 10 of the 43 suspected patients with CADASIL. All 10 cases with skin Notch3 ECD deposits also carried reported pathogenic variants in the NOTCH3 gene associated with CADASIL. NOTCH3 variants of unknown significance were found in the other four patients without vascular Notch3 ECD or granular osmiophilic material deposits in biopsied skin samples. The remaining 29 cases without vascular Notch3 ECD deposits did not have variants in the NOTCH3 gene. Immunohistochemical evaluation of vascular Notch3 ECD deposits in unfixed frozen biopsied skin samples may be useful for detecting Notch3 deposits in CADASIL.

Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leukoencephalopathy Family Members With a Pathogenic NOTCH3 Variant Can Have a Normal Brain Magnetic Resonance Imaging and Skin Biopsy Beyond Age 50 Years.

Background:To determine whether extremely mild small vessel disease (SVD) phenotypes can occur in NOTCH3 variant carriers from Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) pedigrees using clinical, genetic, neuroimaging, and skin biopsy findings.Methods:Individuals from CADASIL pedigrees fulfilling criteria for extremely mild NOTCH3-associated SVD (mSVDNOTCH3) were selected from the cross-sectional Dutch CADASIL cohort (n=200), enrolled between 2017 and 2020. Brain magnetic resonance imaging were quantitatively assessed for SVD imaging markers. Immunohistochemistry and electron microscopy was used to quantitatively assess and compare NOTCH3 ectodomain (NOTCH3ECD) aggregation and granular osmiophilic material deposits in the skin vasculature of mSVDNOTCH3 cases and symptomatic CADASIL patients.Results:Seven cases were identified that fulfilled the mSVDNOTCH3 criteria, with a mean age of 56.6 years (range, 50–72). All of these individuals harbored a NOTCH3 variant located in one of EGFr domains 7-34 and had a normal brain magnetic resonance imaging, except the oldest individual, aged 72, who had beginning confluence of WMH (Fazekas score 2) and 1 cerebral microbleed. mSVDNOTCH3 cases had very low levels of NOTCH3ECD aggregation in skin vasculature, which was significantly less than in symptomatic EGFr 7-34 CADASIL patients (P=0.01). Six mSVDNOTCH3 cases had absence of granular osmiophilic material deposits.Conclusions:Our findings demonstrate that extremely mild SVD phenotypes can occur in individuals from CADASIL pedigrees harboring NOTCH3 EGFr 7-34 variants with normal brain magnetic resonance imaging up to age 58 years. Our study has important implications for CADASIL diagnosis, disease prediction, and the counseling of individuals from EGFr 7-34 CADASIL pedigrees.

Molecular Chaperone BRICHOS Inhibits CADASIL-Mutated NOTCH3 Aggregation In Vitro.

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is the most common familial form of stroke, which is caused by mutations located in the epidermal growth factor (EGF)-like repeats of the NOTCH3 gene. Mutations cause the NOTCH3 (N3) protein to misfold and aggregate. These aggregates will be a component of granular osmiophilic material, which when accumulated around the arteries and arterioles is believed to cause the degradation of vascular smooth muscle cells (VSMC). VSMC degradation affects blood flow regulation and leads to white matter and neuronal death. Currently, there is no treatment for CADASIL. The dementia-relevant BRICHOS domain is a small multitalented protein with functions that include ATP-independent chaperone-like properties. BRICHOS has been shown to prevent the aggregation of both fibrillar and non-fibrillar structures. Therefore, the objective of this study is to investigate whether BRICHOS exhibits anti-aggregating properties on a recombinant CADASIL-mutated N3 protein consisting of the first five repeats of EGF (EGF1–5), harboring a cysteine instead of an arginine in the position 133, (R133C). We found that the N3 EGF1–5 R133C mutant is more prone to aggregate, while the wildtype is more stable. Recombinant human Bri2 BRICHOS is able to interact and stabilize the R133C-mutated N3 protein in a dose-dependent manner. These results suggest an anti-aggregating impact of BRICHOS on the N3 EGF1–5 R133C protein, which could be a potential treatment for CADASIL.

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

AimsCADASIL, the most prevalent hereditary cerebral small vessel disease, is caused by cysteine‐altering NOTCH3 variants (NOTCH3 cys ) 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 NOTCH3 cys variant position is associated with NOTCH3 protein aggregation load.MethodsWe quantified vascular NOTCH3 aggregation in skin biopsies (n = 25) and brain tissue (n = 7) of CADASIL patients with a NOTCH3 cys 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).ResultsPatients with NOTCH3 cys 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 NOTCH3 cys 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.ConclusionsCADASIL 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 NOTCH3 cys EGFr 7–34 and EGFr 1–6 variants.

Notch3-Dependent Effects on Adult Neurogenesis and Hippocampus-Dependent Learning in a Modified Transgenic Model of CADASIL.

We and others have reported that Notch3 is a regulator of adult hippocampal neurogenesis. Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), the most common genetic form of vascular dementia, is caused by mutations in Notch3. The present study intended to investigate whether there is a correlation between altered adult hippocampal neurogenesis and spatial memory performance in CADASIL transgenic mice. To overcome visual disabilities that hampered behavioral testing of the original mice (on an FVB background) we back-crossed the existing TgN3R169C CADASIL mouse model onto the C57BL/6J background. These animals showed an age-dependent increase in the pathognomonic granular osmiophilic material (GOM) deposition in the hippocampus. Analysis in the Morris water maze task at an age of 6 and 12 months revealed deficits in re-learning and perseverance in the CADASIL transgenic mice. Overexpression of Notch3 alone resulted in deficits in the use of spatial strategies and diminished adult neurogenesis in both age groups. The additional CADASIL mutation compensated the effect on strategy usage but not on adult neurogenesis. In brain bank tissue samples from deceased CADASIL patients we found signs of new neurons, as assessed by calretinin immunohistochemistry, but no conclusive quantification was possible. In summary, while our study confirmed the role of Notch3 in adult neurogenesis, we found a specific effect of the CADASIL mutation only on the reversion of the Notch3 effect on behavior, particularly visible at 6 months of age, consistent with a loss of function. The mutation did not revert the Notch3-dependent changes in adult neurogenesis or otherwise affected adult neurogenesis in this model.

IMPAIRED NO SIGNALLING, ER STRESS AND RHO KINASE MEDIATE PERIPHERAL VASCULAR DYSFUNCTION IN A MODEL OF SMALL VESSEL DISEASE OF THE BRAIN (CADASIL)

Objective: CADASIL, a monogenic condition due to Notch3 mutations, is a very aggressive small vessel disease of the brain resulting in premature vascular dementia and stroke. Changes in cerebral vessels include vascular dysfunction and narrowing, and accumulation of granular osmiophilic material (GOM). It is not clear whether small peripheral arteries undergo similar damage. Thus our aim is to assess vascular dysfunction and associated mechanisms in mesenteric resistance arteries from CADASIL mice. Design and method: Mesenteric arteries (MA) from male CADASIL-causing Notch3 mutation (TgNotch3R169C) and wildtype (TgNotch3WT) mice (6 months old) were investigated. Blood pressure (BP) was assessed by tail-cuff and vascular function by myography. Results: BP and cardiac function were unchanged in CADASIL mice. By electron microscopy GOM deposits were identified in MA from CADASIL mice. Notch3 (WT: 2.0 ± 0.5 vs. 6.0 ± 1.3) and HeyL (WT: 1.1 ± 0.4 vs. 2.9 ± 0.6) mRNA were augmented in CADASIL mice (p < 0.01). CADASIL mice exhibited endothelial (Emax 109.9 ± 7.4 vs. 81.3 ± 5.4) and VSMC-dysfunction (pD2 7.8 ± 0.1 vs 6.8 ± 0.3); effects associated with increased eNOS inhibition (p-Thr495) (1.8-fold increase) and decreased cGMP levels (1.2 ± 0.2 vs 0.59 ± 0.2) (p < 0.05). Contraction to Ang II (Emax 33.7 ± 6.8 vs. 72.8 ± 4.4), phenylephrine (Emax: 70.6 ± 7.2 vs. 92.1 ± 4.2) and U46619 (Emax: 123.4 ± 4.4 vs. 215.1 ± 24.4) (p < 0.05) was increased in CADASIL; effects attenuated by fasudil (Rho-kinase inhibitor) and 4-PBA (ER stress inhibitor) (p < 0.05). MA structure and mechanics from CADASIL mice remain unchanged at this stage. U46619-induced calcium influx was increased in TgNotch3R169C VSMCs (AUC: 1.3-fold increase) as well as SERCA (1.5 ± 0.7 vs. 8.1 ± 1.4) and TRPM2 (0.7 ± 0.3 vs. 4.5 ± 1.6) mRNA levels (p < 0.05). BiP protein expression (ER stress marker) (1.7-fold increase) and gene expression of Rho GEFs (LARG: 1.1 ± 0.1 vs. 2.1 ± 0.3; PDZ: 0.9 ± 0.1 vs. 2.9 ± 0.4) were increased in MA from TgNotch3R169C vs. TgNotch3WT mice (p < 0.05). These effects may be associated with the increase in oxidative stress observed in VSMCs from peripheral arteries and brain vessels from CADASIL patients (p < 0.05). Conclusions: Our findings demonstrate that the cerebral vasculopathy associated with Notch3 mutations is also present in peripheral small vessels and identify ER stress and Rho kinase as potential new therapeutic targets in CADASIL, for which there are no disease-specific treatments.

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.

Ultrastructure of mitochondria and damage to small blood vessels in siblings with the same mutation in the NOTCH 3 and coexisting diseases.

We performed ultrastructural studies ofmitochondria and evaluated theappearance ofsmall blood vessels ofthree middle-aged siblings affected by thesame mutation in theNOTCH3 gene, causing CADASIL. CADASIL pathognomonic features include granular osmiophilic material (GOM), which we observed. GOMs were located in damaged and thickened basement membranes (BM) ofcapillaries and arterioles. Our patients were also burdened by type II diabetes (first patient), impaired glucose metabolism (second patient), and hypertension (third patient). Theultrastructure ofthecapillaries in thefirst and second patients differed from thethird patient. In diabetes/impaired glucose metabolism patients (first and second patients), we observed: pathologies ofmitochondria in damaged endothelium and pericytes ofcapillaries; extremely thickened (BM) with visible remains ofvascular cells; well-preserved GOMs anchored in therebuilt capillary extracellular matrix. We identified degenerated or vestigial small blood vessels ofskeletal muscles in thefirst patient. Thecapillary damage in thethird patient (with hypertension) was milder compared to thediabetes/impaired glucose metabolism patients. We conclude that in patients with amutation in theNOTCH3 gene, theco-occurrence ofdiseases such as type II diabetes/impaired glucose metabolism can cause amultiplication thedamages to small blood vessels by modifying/masking thepathogenesis ofCADASIL.

Abstract MP31: Peripheral Vascular Dysfunction In A Model Of Small Vessel Disease Of The Brain (CADASIL) Involves Impaired Redox-sensitive Cyclic GMP/PKG Signaling

CADASIL, a monogenic condition due to Notch3 mutations, is a very aggressive small vessel disease of the brain resulting in premature vascular dementia and stroke. Changes in cerebral vessels include vascular dysfunction and narrowing, and accumulation of granular osmiophilic material (GOM). It is not clear whether small peripheral arteries undergo similar damage. Therefore, our aim is to assess vascular dysfunction and associated mechanisms in mesenteric resistance arteries from CADASIL mice. Mesenteric arteries (MA) from male CADASIL-causing Notch3 mutation (TgNotch3 R169C ) and wildtype (TgNotch3 WT ) mice (6 months old) were investigated. GOM deposits in MA from CADASIL mice were identified by electron microscopy. mRNA expression of Notch3 (WT: 2.0±0.5 vs. 6.0±1.3) and its downstream target HeyL (WT: 1.1±0.4 vs. 2.9±0.6) was augmented in CADASIL mice (p<0.01), suggesting increased Notch3 activation. CADASIL mice exhibited endothelial-dependent (Emax 109.9±7.4 vs. 81.3±5.4) and -independent dysfunction (pD 2 7.8±0.1 vs. 6.8±0.3); effects associated with increased eNOS inhibition (p-Thr 495 ) (1.8-fold increase) and decreased cGMP levels (1.2±0.2 vs. 0.59±0.2) (p<0.05). Plasma lipid peroxidation (0.8±0.1 vs. 2.0±0.3; p<0.05) and vascular reactive oxygen species (ROS) production (7.2±1.9 vs. 75.4±35.0; p<0.05) were increased in TgNotch3 R169C mice; processes associated with upregulation of soluble guanylate cyclase (sGC) oxidation and decreased sGC activity. H 2 O 2 levels were decreased in TgNotch3 R169C mice (1.9±0.2 vs. 1.1±1.9; p<0.05), which was associated with reduced activation of protein kinase G (PKG). Observations in TgNotch3 R169C mice were recapitulated in human CADASIL, where ROS levels (0.8±0.1 vs. 4.1±2.7; p<0.05) and sGC oxidation were also increased. Our findings demonstrate that the vasculopathy associated with a CADASIL Notch3 gain-of-function mutation in peripheral small vessels involves reduction in eNOS activation and redox-sensitive processes leading to impaired sGC/cGMP signalling pathway. We identify a potential new therapeutic target in CADASIL, for which there are no disease-specific treatments.

Human iPS cell-derived mural cells as an in vitro model of hereditary cerebral small vessel disease

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is one of the most common forms of hereditary cerebral small vessel diseases and is caused by mutations in NOTCH3. Our group has previously reported incorporation of NOTCH3 extracellular domain (N3ECD) in the CADASIL-specific granular osmiophilic materials and increase of PDGFRβ immunoreactivity in CADASIL postmortem brains. Here, we aimed to establish an in vitro model of CADASIL, which can recapitulate those CADASIL phenotypes, using induced pluripotent stem cells (iPSCs). We have refined a differentiation protocol of endothelial cells to obtain mature mural cells (MCs) with their characteristic properties. iPSCs from three CADASIL patients with p.Arg182Cys, p.Arg141Cys and p.Cys106Arg mutations were differentiated into MCs and their functional and molecular profiles were compared. The differentiated CADASIL MCs recapitulated pathogenic changes reported previously: increased PDGFRβ and abnormal structure/distribution of filamentous actin network, as well as N3ECD/LTBP-1/HtrA1-immunopositive deposits. Migration rate of CADASIL MCs was enhanced but suppressed by knockdown of NOTCH3 or PDGFRB. CADASIL MCs showed altered reactivity to PDGF-BB. Patient-derived MCs can recapitulate CADASIL pathology and are therefore useful in understanding the pathogenesis and developing potential treatment strategies.

Homozygous NOTCH3 p.R587C mutation in Chinese patients with CADASIL: a case report

BackgroundCerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited small vessel disease caused by mutations in NOTCH3 gene with remarkable phenotypic heterogeneity. Cases of CADASIL associated with homozygous NOTCH3 mutations are rare and subsequently understudied. In this study, we investigate the genetic and phenotypic features within patients of CADASIL with homozygous NOTCH3 mutations.Case presentationWe recruited two affected individuals with CADASIL from a mainland Chinese family. The proband (Patient 1), a 60-year-old male, presented with slow progressive gait instability, severe cognitive impairment, and emotional disorder for more than 2 years with a history of ischemic stroke and hypertension. His younger brother (Patient 2) presented with apparent gait difficulties, dysarthria as well as cognitive decline at 59 years old. Brain magnetic resonance imaging (MRI) showed diffused white matter lesions involving bilateral periventricular white matter, semioval center region, and anterior temporal lobes. Molecular genetic testing identified a homozygous variant, c.1759C > T (p.R587C), in NOTCH3 gene in both patients. Pathological analysis revealed granular osmiophilic material (GOM) deposits in small arterial walls of skin from the proband. The diagnosis of CADASIL was confirmed.ConclusionsOur cases of CADASIL with homozygous mutation c.1759C > T (p.R587C) in NOTCH3 share similar manifestation to the patients with heterozygous same mutation reported previously. Other than genetic factors, vascular risk factors or environmental factors might contribute to the phenotypic variation of CADASIL.

NOTCH3 is non-enzymatically fragmented in inherited cerebral small-vessel disease

The small-vessel disorder cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) arises from mutations in the human gene encoding NOTCH3 and results in vascular smooth muscle cell degeneration, stroke, and dementia. However, the structural changes in NOTCH3 involved in CADASIL etiology are unclear. Here, we discovered site-specific fragmentation of NOTCH3 protein in pathologically affected vessels of human CADASIL-affected brains. EM-based experiments to pinpoint NOTCH3 localization in these brains indicated accumulation of NOTCH3 fragmentation products in the basement membrane, collagen fibers, and granular osmiophilic material within the cerebrovasculature. Using antibodies generated against a disease-linked neo-epitope found in degenerating vascular medium of CADASIL brains, we mapped the site of fragmentation to the NOTCH3 N terminus at the peptide bond joining Asp80 and Pro81 Cleavage at this site was predicted to separate the first epidermal growth factor (EGF)-like domain from the remainder of the protein. We found that the cleavage product from this fragmentation event is released into the conditioned medium of cells expressing recombinant NOTCH3 fragments. Mutagenesis of Pro81 abolished the fragmentation, and low pH and reducing conditions enhanced NOTCH3 proteolysis. Furthermore, substitution of multiple cysteine residues of the NOTCH3 N terminus activated proteolytic release of the first EGF-like repeat, suggesting that the elimination of multiple disulfide bonds in NOTCH3 accelerates its fragmentation. These characteristics link the signature molecular genetic alterations present in individuals with CADASIL to a post-translational protein alteration in degenerating brain arteries. The cellular consequences of these pathological NOTCH3 fragments are an important area for future investigation.

Naturally occurring NOTCH3 exon skipping attenuates NOTCH3 protein aggregation and disease severity in CADASIL patients.

CADASIL is a vascular protein aggregation disorder caused by cysteine-altering NOTCH3 variants, leading to mid-adult-onset stroke and dementia. Here, we report individuals with a cysteine-altering NOTCH3 variant that induces exon 9 skipping, mimicking therapeutic NOTCH3 cysteine correction. The index came to our attention after a coincidental finding on a commercial screening MRI, revealing white matter hyperintensities. A heterozygous NOTCH3 c.1492G>T, p.Gly498Cys variant, was identified using a gene panel, which was also present in four first- and second-degree relatives. Although some degree of white matter hyperintensities was present on MRI in all family members with the NOTCH3 variant, the CADASIL phenotype was mild, as none had lacunes on MRI and there was no disability or cognitive impairment above the age of 60 years. RT-PCR and Sanger sequencing analysis on patient fibroblast RNA revealed that exon 9 was absent from the majority of NOTCH3 transcripts of the mutant allele, effectively excluding the mutation. NOTCH3 aggregation was assessed in skin biopsies using electron microscopy and immunohistochemistry and did not show granular osmiophilic material and only very mild NOTCH3 staining. For purposes of therapeutic translatability, we show that, in cell models, exon 9 exclusion can be obtained using antisense-mediated exon skipping and CRISPR/Cas9-mediated genome editing. In conclusion, this study provides the first in-human evidence that cysteine corrective NOTCH3 exon skipping is associated with less NOTCH3 aggregation and an attenuated phenotype, justifying further therapeutic development of NOTCH3 cysteine correction for CADASIL.

Progression and Classification of Granular Osmiophilic Material (GOM) Deposits in Functionally Characterized Human NOTCH3 Transgenic Mice

CADASIL is a NOTCH3-associated cerebral small vessel disease. A pathological ultrastructural disease hallmark is the presence of NOTCH3-protein containing deposits called granular osmiophilic material (GOM), in small arteries. How these GOM deposits develop over time and what their role is in disease progression is largely unknown. Here, we studied the progression of GOM deposits in humanized transgenic NOTCH3Arg182Cys mice, compared them to GOM deposits in patient material, and determined whether GOM deposits in mice are associated with a functional CADASIL phenotype. We found that GOM deposits are not static, but rather progress in ageing mice, both in terms of size and aspect. We devised a GOM classification system, reflecting size, morphology and electron density. Six-month-old mice showed mostly early stage GOM, whereas older mice and patient vessels showed predominantly advanced stage GOM, but also early stage GOM. Mutant mice did not develop the most severe GOM stage seen in patient material. This absence of end-stage GOM in mice was associated with an overall lack of histological vascular pathology, which may explain why the mice did not reveal functional deficits in cerebral blood flow, cognition and motor function. Taken together, our data indicate that GOM progress over time, and that new GOM deposits are continuously being formed. The GOM staging system we introduce here allows for uniform GOM deposit classification in future mouse and human studies, which may lead to more insight into a potential association between GOM stage and CADASIL disease severity, and the role of GOM in disease progression.