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

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

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

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

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

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

CADA-PRO: A Patient Questionnaire Measuring Key Cognitive, Motor, Emotional, and Behavioral Outcomes in CADASIL.

BACKGROUND: Cerebral small vessel disease (cSVD) of ischemic type, either sporadic or genetic, as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), can impact the quality of daily life on various cognitive, motor, emotional, or behavioral aspects. No instrument has been developed to measure these outcomes from the patient's perspective. We thus aimed to develop and validate a patient-reported questionnaire. METHODS: In a development study, 79 items were generated by consensus between patients, family representatives, and cSVD experts. A first sample of patients allowed assessing the feasibility (missing data, floor and ceiling effect, and acceptability), internal consistency, and dimensionality of a first set of items. Thereafter, in a validation study, we tested a reduced version of the item set in a larger sample to assess the feasibility, internal consistency, dimensionality, test-retest reliability, concurrent validity, and sensitivity to change. RESULTS: The scale was developed in 44 patients with cSVD and validated in a second sample of 89 individuals (including 43 patients with CADASIL and 46 with another cSVD). The final CADASIL Patient-Reported Outcome scale comprised 18 items covering 4 categories of consequences (depression/anxiety, attention/executive functions, motor, and daily activities) of the disease. The proportion of missing data was low, and no item displayed a major floor or ceiling effect. Both the internal consistency and test-retest reliability were good (Cronbach alpha=0.95, intraclass correlation coefficient=0.88). In patients with CADASIL, CADASIL Patient-Reported Outcome scores correlated with the modified Rankin Scale, Starkstein Apathy Scale, Hospital Anxiety and Depression scale, Working Memory Index, and trail making test times. In patients with other cSVDs, CADASIL Patient-Reported Outcome correlated only with Hospital Anxiety and Depression scale and Starkstein Apathy Scale. CONCLUSIONS: The CADASIL Patient-Reported Outcome may be an innovative instrument for measuring patient-reported outcomes in future cSVD trials. Full validation was obtained for its use in patients with CADASIL, but further improvement is needed for its application in other cSVDs.

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

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

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

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

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

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

Disentangling the effects of Alzheimer's and small vessel disease on white matter fibre tracts.

Alzheimer's disease and cerebral small vessel disease are the two leading causes of cognitive decline and dementia and coexist in most memory clinic patients. White matter damage as assessed by diffusion MRI is a key feature in both Alzheimer's and cerebral small vessel disease. However, disease-specific biomarkers of white matter alterations are missing. Recent advances in diffusion MRI operating on the fixel level (fibre population within a voxel) promise to advance our understanding of disease-related white matter alterations. Fixel-based analysis allows derivation of measures of both white matter microstructure, measured by fibre density, and macrostructure, measured by fibre-bundle cross-section. Here, we evaluated the capacity of these state-of-the-art fixel metrics to disentangle the effects of cerebral small vessel disease and Alzheimer's disease on white matter integrity. We included three independent samples (total n = 387) covering genetically defined cerebral small vessel disease and age-matched controls, the full spectrum of biomarker-confirmed Alzheimer's disease including amyloid- and tau-PET negative controls and a validation sample with presumed mixed pathology. In this cross-sectional analysis, we performed group comparisons between patients and controls and assessed associations between fixel metrics within main white matter tracts and imaging hallmarks of cerebral small vessel disease (white matter hyperintensity volume, lacune and cerebral microbleed count) and Alzheimer's disease (amyloid- and tau-PET), age and a measure of neurodegeneration (brain volume). Our results showed that (i) fibre density was reduced in genetically defined cerebral small vessel disease and strongly associated with cerebral small vessel disease imaging hallmarks; (ii) fibre-bundle cross-section was mainly associated with brain volume; and (iii) both fibre density and fibre-bundle cross-section were reduced in the presence of amyloid, but not further exacerbated by abnormal tau deposition. Fixel metrics were only weakly associated with amyloid- and tau-PET. Taken together, our results in three independent samples suggest that fibre density captures the effect of cerebral small vessel disease, while fibre-bundle cross-section is largely determined by neurodegeneration. The ability of fixel-based imaging markers to capture distinct effects on white matter integrity can propel future applications in the context of precision medicine.

Decreased water exchange rate across blood-brain barrier in hereditary cerebral small vessel disease.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and heterozygous HTRA1 mutation-related cerebral small vessel disease (CSVD) are the two types of dominant hereditary CSVD. Blood-brain barrier (BBB) failure has been hypothesized in the pathophysiology of CSVD. However, it is unclear whether there is BBB damage in the two types of hereditary CSVD, especially in heterozygous HTRA1 mutation-related CSVD. In this study, a case-control design was used with two disease groups including CADASIL (n = 24), heterozygous HTRA1 mutation-related CSVD (n = 9) and healthy controls (n = 24). All participants underwent clinical cognitive assessments and brain MRI. Diffusion-prepared pseudo-continuous arterial spin labelling was used to estimate the water exchange rate across the BBB (kw). Correlation and multiple linear regression analyses were used to examine the association between kw and disease burden and neuropsychological performance, respectively. Compared with the healthy controls, kw in the whole brain and multiple brain regions was decreased in both CADASIL and heterozygous HTRA1 mutation-related CSVD patients (Bonferroni-corrected P < 0.007). In the CADASIL group, decreased kw in the whole brain (ß = -0.634, P = 0.001), normal-appearing white matter (ß = -0.599, P = 0.002) and temporal lobe (ß = -0.654, P = 0.001) was significantly associated with higher CSVD score after adjusting for age and sex. Reduced kw in the whole brain was significantly associated with poorer neuropsychological performance after adjusting for age, sex and education in both CADASIL and heterozygous HTRA1 mutation-related CSVD groups (ß = 0.458, P = 0.001; ß = 0.884, P = 0.008). This study showed that there was decreased water exchange rate across the BBB in both CADASIL and heterozygous HTRA1 mutation-related CSVD patients, suggesting a common pathophysiological mechanism underlying the two types of hereditary CSVD. These results highlight the potential use of kw for monitoring the course of CADASIL and heterozygous HTRA1 mutation-related CSVD, a possibility which should be tested in future research.

PIP2 corrects cerebral blood flow deficits in small vessel disease by rescuing capillary Kir2.1 activity.

Cerebral small vessel diseases (SVDs) are a central link between stroke and dementia-two comorbidities without specific treatments. Despite the emerging consensus that SVDs are initiated in the endothelium, the early mechanisms remain largely unknown. Deficits in on-demand delivery of blood to active brain regions (functional hyperemia) are early manifestations of the underlying pathogenesis. The capillary endothelial cell strong inward-rectifier K+ channel Kir2.1, which senses neuronal activity and initiates a propagating electrical signal that dilates upstream arterioles, is a cornerstone of functional hyperemia. Here, using a genetic SVD mouse model, we show that impaired functional hyperemia is caused by diminished Kir2.1 channel activity. We link Kir2.1 deactivation to depletion of phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane phospholipid essential for Kir2.1 activity. Systemic injection of soluble PIP2 rapidly restored functional hyperemia in SVD mice, suggesting a possible strategy for rescuing functional hyperemia in brain disorders in which blood flow is disturbed.

Heterogeneous blood-brain barrier dysfunction in cerebral small vessel diseases.

INTRODUCTION: We explored how blood-brain barrier (BBB) leakage rate of gadolinium chelates (Ktrans) and BBB water exchange rate (kw) varied in cerebral small vessel disease (cSVD) subtypes. METHODS: Thirty sporadic cSVD, 40 cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), and 13 high-temperature requirement factor A serine peptidase 1 (HTRA) -related cSVD subjects were investigated parallel to 40 healthy individuals. Subjects underwent clinical, cognitive, and MRI assessment. RESULTS: In CADASIL, no difference in Ktrans, but lower kw was observed in multiple brain regions. In sporadic cSVD, no difference in kw, but higher Ktrans was found in the whole brain and normal-appearing white matter. In HTRA1-related cSVD, both higher Ktrans in the whole brain and lower kw in multiple brain regions were observed. In each patient group, the altered BBB measures were correlated with lesion burden or clinical severity. DISCUSSION: In cSVD subtypes, distinct alterations of kw and Ktrans were observed. The combination of Ktrans and kw can depict the heterogeneous BBB dysfunction. HIGHLIGHTS: We measured BBB leakage to gadolinium-based contrast agent (Ktrans) and water exchange rate (kw) across BBB in three subtypes of cSVD. CADASIL is characterized by lower kw, HTRA1-related cSVD exhibits both higher Ktrans and lower kw, while sporadic cSVD is distinguished by higher Ktrans. There are distinct alterations in kw and Ktrans among subtypes of cSVD, indicating the heterogeneous nature of BBB dysfunction.

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.

White Matter Hyperintensities of the Corpus Callosum Are Associated With Clinical Severity in CADASIL.

BACKGROUND: In CADASIL (Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leukoencephalopathy), clinical severity is not related to the total burden of white matter hyperintensities (WMHs), presumably because of heterogeneous underlying tissue alterations. We aimed to investigate whether WMHs in the corpus callosum (WMHCC) are due to secondary degeneration and related to clinical severity. METHODS: We evaluated data from 228 CADASIL patients included in an ongoing prospective cohort with available 3-dimensional fluid-attenuated inversion recovery magnetic resonance imaging sequences. We analyzed in a blind manner WMHCC and lacunes in presumably connected areas to determine whether WMHCC are related to secondary degeneration. We evaluated the links between WMHCC and the Mattis dementia rating scale and the modified Rankin Scale-widely used measures of global cognitive performances and disability, respectively. Linear regression models were adjusted for age, sex, level of education, brain volume, number of lacunes, and volume of WMH. RESULTS: Among 228 patients, only 105 (46%) had WMHCC while all had WMH in the rest of the white matter. In 74% of cases, WMHCC crossed a presumably connected nearby lacune, which was significantly higher than the expected value if the spatial distributions of WMHCC and nearby lacunes were unrelated (11%; P<0.001). Patients with WMHCC had worse Mattis dementia rating scale (median [P25-P75], 138 [122-142] versus 143 [140-143]; P<0.001) and worse modified Rankin Scale (2 [1-3] versus 1 [0-1]; P<0.001). In adjusted models, Mattis dementia rating scale was significantly associated with WMHCC (estimate, -6.2 [95% CI, -11.8 to -0.1]). CONCLUSIONS: In CADASIL, WMHCC are likely related to secondary degeneration and are independently related to clinical severity, in contrast to the total burden of WMH.

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.

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

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

Prospects for Diminishing the Impact of Nonamyloid Small-Vessel Diseases of the Brain.

Small-vessel diseases (SVDs) of the brain are involved in about one-fourth of ischemic strokes and a vast majority of intracerebral hemorrhages and are responsible for nearly half of dementia cases in the elderly. SVDs are a heavy burden for society, a burden that is expected to increase further in the absence of significant therapeutic advances, given the aging population. Here, we provide a critical appraisal of currently available therapeutic approaches for nonamyloid sporadic SVDs that are largely based on targeting modifiable risk factors. We review what is known about the pathogenic mechanisms of vascular risk factor-related SVDs and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most frequent hereditary SVD, and elaborate on two mechanism-based therapeutic approaches worth exploring in sporadic SVD and CADASIL. We conclude by discussing opportunities and challenges that need to be tackled if efforts to achieve significant therapeutic advances for these diseases are to be successful.

White Matter Tract Injury by MRI in CADASIL Patients is Associated With Iron Accumulation.

BACKGROUND: Widespread white matter (WM) injury is a hallmark feature of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). However, controversies about the mechanism of WM tract injury exist persistently. Excessive iron accumulation, frequently reported in CADASIL patients, might cause WM tract injury. PURPOSE: To test the association between iron accumulation and WM tract injury in CADASIL patients. STUDY TYPE: Retrospective. POPULATION: A total of 35 CADASIL patients (age = 50.4 ± 6.4, 62.9% female) and 48 healthy controls (age = 55.7 ± 8.0, 68.8% female). FIELD STRENGTH/SEQUENCE: Diffusion-weighted spin-echo echo-planar sequence; enhanced susceptibility-weighted angiography (ESWAN) gradient echo sequence on a 3 T scanner. ASSESSMENT: The phase images acquired by ESWAN were used to calculate quantitative susceptibility mapping (QSM). Iron accumulation was evaluated in deep gray matters using QSM. WM tract injury was quantified by diffusion metrics based on WM major tracts skeleton. We compared iron deposition between groups and analyzed the correlation between WM tract injury and iron deposition in regions showing significant differences from healthy controls. Exploratory analysis was carried out to investigate whether WM tract injury mediated the relationship between iron deposition and cognitive impairment evaluated by Mini-Mental State Examination (MMSE). STATISTICAL TESTS: General linear model (GLM), partial correlation, stepwise linear regression and mediation analysis were used. The threshold of statistical significance was set as p < 0.05. RESULTS: Compared with healthy controls, CADASIL patients had significantly increased iron deposition in the caudate and putamen. Aberrant iron deposition in these two regions was significantly associated with decreased WM fractional anisotropy (FA) (caudate, r = -0.373； putamen, r = - 0.421), and increased radial diffusivity (RD) (caudate, r = 0.372; putamen, r = 0.386). Furthermore, WM tract injury mediated the relationship between iron deposition and cognitive impairment. DATA CONCLUSION: Patients with CADASIL show increased iron deposition in the caudate and putamen that is correlated to WM tract injury, which may in turn mediate the association with cognitive impairment. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 2.

Diffusion prepared pseudo-continuous arterial spin labeling reveals blood-brain barrier dysfunction in patients with CADASIL.

OBJECTIVES: Diffusion prepared pseudo-continuous arterial spin labeling (DP-pCASL) is a newly proposed MRI method to noninvasively measure the function of the blood-brain barrier (BBB). We aim to investigate whether the water exchange rate across the BBB, estimated with DP-pCASL, is changed in patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), and to analyze the association between the BBB water exchange rate and MRI/clinical features of these patients. METHODS: Forty-one patients with CADASIL and thirty-six age- and sex-matched controls were scanned with DP-pCASL MRI to estimate the BBB water exchange rate (kw). The MRI lesion burden, the modified Rankin scale (mRS), and the neuropsychological scales were also examined. The association between kw and MRI/clinical features was analyzed. RESULTS: Compared with that in the controls, kw in patients with CADASIL was decreased at normal-appearing white matter (NAWM) (t = - 4.742, p < 0.001), cortical gray matter (t = - 5.137, p < 0.001), and deep gray matter (t = - 3.552, p = 0.001). After adjustment for age, gender, and arterial transit time, kw at NAWM was negatively associated with the volume of white matter hyperintensities (ß = - 0.754, p = 0.001), whereas decreased kw at NAWM was independently associated with an increased risk of abnormal mRS scale (OR = 1.058, 95% CI: 1.013-1.106, p = 0.011) in these patients. CONCLUSIONS: This study found that the BBB water exchange rate was decreased in patients with CADASIL. The decreased BBB water exchange rate was associated with an increased MRI lesion burden and functional dependence of the patients, suggesting the involvement of BBB dysfunction in the pathogenesis of CADASIL. CLINICAL RELEVANCE STATEMENT: DP-pCASL reveals BBB dysfunction in patients with CADASIL. The decreased BBB water exchange rate is associated with MRI lesion burden and functional dependence, indicating the potential of DP-pCASL as an evaluation method for disease severity. KEY POINTS: • DP-pCASL reveals blood-brain barrier dysfunction in patients with CADASIL. • Decreased BBB water exchange rate, an indicator of BBB dysfunction detected by DP-pCASL, was associated with MRI/clinical features of patients with CADASIL. • DP-pCASL can be used as an evaluation method to assess the severity of disease in patients with CADASIL.

Presumed periventricular venous infarction on magnetic resonance imaging and its association with increased white matter edema in CADASIL.

OBJECTIVES: Venous pathology could contribute to the development of parenchymal lesions in cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). We aim to identify presumed periventricular venous infarction (PPVI) in CADASIL and analyze the associations between PPVI, white matter edema, and microstructural integrity within white matter hyperintensities (WMHs) regions. METHODS: We included forty-nine patients with CADASIL from a prospectively enrolled cohort. PPVI was identified according to previously established MRI criteria. White matter edema was evaluated using the free water (FW) index derived from diffusion tensor imaging (DTI), and microstructural integrity was evaluated using FW-corrected DTI parameters. We compared the mean FW values and regional volumes with different levels of FW (ranging from 0.3 to 0.8) in WMHs regions between the PPVI and non-PPVI groups. We used intracranial volume to normalize each volume. We also analyzed the association between FW and microstructural integrity in fiber tracts connected with PPVI. RESULTS: We found 16 PPVIs in 10 of 49 CADASIL patients (20.4%). The PPVI group had larger WMHs volume (0.068 versus 0.046, p = 0.036) and higher FW in WMHs (0.55 versus 0.52, p = 0.032) than the non-PPVI group. Larger areas with high FW content were also found in the PPVI group (threshold: 0.7, 0.47 versus 0.37, p = 0.015; threshold: 0.8, 0.33 versus 0.25, p = 0.003). Furthermore, higher FW correlated with decreased microstructural integrity (p = 0.009) in fiber tracts connected with PPVI. CONCLUSIONS: PPVI was associated with increased FW content and white matter degeneration in CADASIL patients. CLINICAL RELEVANCE STATEMENT: PPVI is an important factor related with WMHs, and therefore, preventing the occurrence of PPVI would be beneficial for patients with CADASIL. KEY POINTS: •Presumed periventricular venous infarction is important and occurs in about 20% of patients with CADASIL. •Presumed periventricular venous infarction was associated with increased free water content in the regions of white matter hyperintensities. •Free water correlated with microstructural degenerations in white matter tracts connected with the presumed periventricular venous infarction.