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-β (Aβ) peptides and formation of Aβ deposits in the brain vasculature resulting in a disruption of the angioarchitecture. Capillaries are a critical site of Aβ 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 Aβ1-40 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 Aβ 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

Common coding variant in SERPINA1 increases the risk for large artery stroke

Large artery atherosclerotic stroke (LAS) shows substantial heritability not explained by previous genome-wide association studies. Here, we explore the role of coding variation in LAS by analyzing variants on the HumanExome BeadChip in a total of 3,127 cases and 9,778 controls from Europe, Australia, and South Asia. We report on a nonsynonymous single-nucleotide variant in serpin family A member 1 (SERPINA1) encoding alpha-1 antitrypsin [AAT; p.V213A; P = 5.99E-9, odds ratio (OR) = 1.22] and confirm histone deacetylase 9 (HDAC9) as a major risk gene for LAS with an association in the 3?-UTR (rs2023938; P = 7.76E-7, OR = 1.28). Using quantitative microscale thermophoresis, we show that M1 (A213) exhibits an almost twofold lower dissociation constant with its primary target human neutrophil elastase (NE) in lipoprotein-containing plasma, but not in lipid-free plasma. Hydrogen/deuterium exchange combined with mass spectrometry further revealed a significant difference in the global flexibility of the two variants. The observed stronger interaction with lipoproteins in plasma and reduced global flexibility of the Val-213 variant most likely improve its local availability and reduce the extent of proteolytic inactivation by other proteases in atherosclerotic plaques. Our results indicate that the interplay between AAT, NE, and lipoprotein particles is modulated by the gate region around position 213 in AAT, far away from the unaltered reactive center loop (357-360). Collectively, our findings point to a functionally relevant balance between lipoproteins, proteases, and AAT in atherosclerosis

Régulation protéolytique du récepteur de l'urokinase et inflammation pulmonaire

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Stroke Genetics: Turning Discoveries into Clinical Applications

The field of medical and population genetics in stroke is moving at a rapid pace and has led to unanticipated opportunities for discovery and clinical applications. Genome-wide association studies have highlighted the role of specific pathways relevant to etiologically defined subtypes of stroke and to stroke as a whole. They have further offered starting points for the exploration of novel pathways and pharmacological strategies in experimental systems. Mendelian randomization studies continue to provide insights in the causal relationships between exposures and outcomes and have become a useful tool for predicting the efficacy and side effects of drugs. Additional applications that have emerged from recent discoveries include risk prediction based on polygenic risk scores and pharmacogenomics. Among the topics currently moving into focus is the genetics of stroke outcome. While still at its infancy, this field is expected to boost the development of neuroprotective agents. We provide a brief overview on recent progress in these areas

Disruption of the endothelial barrier by proteases from the bacterial pathogen Pseudomonas aeruginosa: implication of matrilysis and receptor cleavage

International audienceWithin the vasculature, uncontrolled pericellular proteolysis can lead to disruption of cell-to-cell and cell-to-matrix interactions and subsequent detachment-induced cell apoptosis, or anoikis, contributing to inflammatory vascular diseases, with the endothelium as the major target. Most studies so far have focused on endogenous proteinases. However, during bloodstream infections, bacterial proteinases may also trigger endothelial anoikis. We thus investigated the potential apoptotic activity of the proteinases secreted by the haematotropic opportunistic pathogen, Pseudomonas aeruginosa, and particularly its predominant metalloproteinase, LasB. For this, we used the secretome of the LasB-expressing pseudomonal strain, PAO1, and compared it with that from the isogenic, LasB-deficient strain (PAO1?lasB), as well as with purified LasB. Secretomes were tested for apoptotic activity on cultured human endothelial cells derived from the umbilical vein or from the cerebral microvasculature. We found that the PAO1 secretome readily induced endothelial cell anoikis, as did secretomes of LasB-positive clinical pseudomonal isolates, while the PAO1?lasB secretome had only a limited impact on endothelial adherence and viability. Notably, purified LasB reproduced most of the effects of the LasB-containing secretomes, and these were drastically reduced in the presence of the LasB-selective inhibitor, phosphoramidon. A precocious and extensive LasB-dependent degradation of several proteins associated with the endothelial extracellular matrix, fibronectin and von Willebrand factor, was observed by immunofluorescence and/or immunoblotting analysis of cell cultures. Moreover, the PAO1 secretome, but not that from PAO1?lasB, specifically induced rapid endoproteolysis of two major interendothelial junction components, VE-cadherin and occludin, as well as of the anti-anoikis, integrin-associated urokinase receptor, uPAR. Taken as a prototype for exogenous haemorrhagic proteinases, pseudomonal LasB thus appears to induce endothelial anoikis not only via matrilysis, as observed for many pro-apoptotic proteinases, but also via cleavage of some essential cell-to-cell and cell-to-matrix adhesion receptors implicated in the maintenance of the endothelial barrier

Reply to Liu et al.: Loss of TGF-β signaling in CARASIL pathogenesis

We thank Liu et al. (1) for their comments on our work on the link between the cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL)-related protease high temperature requirement protein A1 (HtrA1) and the TGF-β signaling pathway. To advance our understanding of the mechanisms underlying HtrA1 deficiency, we used dermal fibroblasts from CARASIL patients and Htra1 knockout mice as experimental systems (2). We agree with Liu et al. that findings from fibroblasts might or might not fully reflect the physiological processes occurring in a vessel wall and that alterations in other cell types

Reply to Liu et al.: Loss of TGF-β signaling in CARASIL pathogenesis

We thank Liu et al. (1) for their comments on our work on the link between the cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL)-related protease high temperature requirement protein A1 (HtrA1) and the TGF-β signaling pathway. To advance our understanding of the mechanisms underlying HtrA1 deficiency, we used dermal fibroblasts from CARASIL patients and Htra1 knockout mice as experimental systems (2). We agree with Liu et al. that findings from fibroblasts might or might not fully reflect the physiological processes occurring in a vessel wall and that alterations in other cell types

Rab31-dependent regulation of transforming growth factor ß expression in breast cancer cells

Background!#!The small GTP-binding protein Rab31 plays an important role in the modulation of tumor biological-relevant processes, including cell proliferation, adhesion, and invasion. As an underlying mechanism, Rab31 is presumed to act as a molecular switch between a more proliferative and an invasive phenotype. This prompted us to analyze whether Rab31 overexpression in breast cancer cells affects expression of genes involved in epithelial-to-mesenchymal transition (EMT)-like processes when compared to Rab31 low-expressing cells.!##!Methods!#!Commercially available profiler PCR arrays were applied to search for differentially expressed genes in Rab31 high- and low-expressing CAMA-1 breast cancer cells. Differential expression of selected candidate genes in response to Rab31 overexpression in CAMA-1 cells was validated by independent qPCR and protein assays.!##!Results!#!Gene expression profiling of key genes involved in EMT, or its reciprocal process MET, identified 9 genes being significantly up- or down-regulated in Rab31 overexpressing CAMA-1 cells, with the strongest effects seen for TGFB1, encoding TGF-ß1 (> 25-fold down-regulation in Rab31 overexpressing cells). Subsequent validation analyses by qPCR revealed a strong down-regulation of TGFB1 mRNA levels in response to increased Rab31 expression not only in CAMA-1 cells, but also in another breast cancer cell line, MDA-MB-231. Using ELISA and Western blot analysis, a considerable reduction of both intracellular and secreted TGF-ß1 antigen levels was determined in Rab31 overexpressing cells compared to vector control cells. Furthermore, reduced TGF-ß activity was observed upon Rab31 overexpression in CAMA-1 cells using a sensitive TGF-ß bioassay. Finally, the relationship between Rab31 expression and the TGF-ß axis was analyzed by another profiler PCR array focusing on genes involved in TGF-ß signaling. We found 12 out of 84 mRNAs significantly reduced and 7 mRNAs significantly increased upon Rab31 overexpression.!##!Conclusions!#!Our results demonstrate that Rab31 is a potent modulator of the expression of TGF-ß and other components of the TGF-ß signaling pathway in breast cancer cells

Sequestration of latent TGF-β binding protein 1 into CADASIL-related Notch3-ECD deposits.

International audienceIntroduction:Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy(CADASIL) represents the most common hereditary form of cerebral small vessel disease characterized by early-onsetstroke and premature dementia. It is caused by mutations in the transmembrane receptor Notch3, which promote theaggregation and accumulation of the Notch3 extracellular domain (Notch3-ECD) within blood vessel walls. This processis believed to mediate the abnormal recruitment and dysregulation of additional factors including extracellular matrix(ECM) proteins resulting in brain vessel dysfunction. Based on recent evidence indicating a role for the transforminggrowth factor-β(TGF-β) pathway in sporadic and familial small vessel disease we studied fibronectin, fibrillin-1 andlatent TGF-βbinding protein 1 (LTBP-1), three ECM constituents involved in the regulation of TGF-βbioavailability, inpost-mortembrain tissue from CADASIL patients and control subjects.Results:Fibronectin and fibrillin-1 were found to be enriched in CADASIL vessels without co-localizing with Notch3-ECDdeposits, likely as a result of fibrotic processes secondary to aggregate formation. In contrast, LTBP-1 showed both anaccumulation and a striking co-localization with Notch3-ECD deposits suggesting specific recruitment into aggregates.We also detected increased levels of the TGF-βprodomain (also known as latency-associated peptide, LAP) indicatingdysregulation of the TGF-βpathway in CADASIL development.In vitroanalyses revealed a direct interaction betweenLTBP-1 and Notch3-ECD and demonstrated a specific co-aggregation of LTBP-1 with mutant Notch3.Conclusion:We propose LTBP-1 as a novel component of Notch3-ECD deposits and suggest its involvement inpathological processes triggered by Notch3-ECD aggregation