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Loss-of-function mutations in the homotrimeric serine protease HTRA1 cause cerebral vasculopathy. Here, we establish independent approaches to achieve the functional correction of trimer assembly defects. Focusing on the prototypical R274Q mutation, we identify an HTRA1 variant that promotes trimer formation thus restoring enzymatic activity in vitro. Genetic experiments in Htra1 R274Q mice further demonstrate that expression of this protein-based corrector in trans is sufficient to stabilize HtrA1-R274Q and restore the proteomic signature of the brain vasculature. An alternative approach employs supramolecular chemical ligands that shift the monomer-trimer equilibrium towards proteolytically active trimers. Moreover, we identify a peptidic ligand that activates HTRA1 monomers. Our findings open perspectives for tailored protein repair strategies., (© 2024. The Author(s).)

Genetic variants in HTRA1 are associated with stroke risk. However, the mechanisms mediating this remain largely unknown, as does the full spectrum of phenotypes associated with genetic variation in HTRA1. Here we show that rare HTRA1 variants are linked to ischemic stroke in the UK Biobank and BioBank Japan. Integrating data from biochemical experiments, we next show that variants causing loss of protease function associated with ischemic stroke, coronary artery disease and skeletal traits in the UK Biobank and MyCode cohorts. Moreover, a common variant modulating circulating HTRA1 mRNA and protein levels enhances the risk of ischemic stroke and coronary artery disease while lowering the risk of migraine and macular dystrophy in genome-wide association study, UK Biobank, MyCode and BioBank Japan data. We found no interaction between proxied HTRA1 activity and levels. Our findings demonstrate the role of HTRA1 for cardiovascular diseases and identify two mechanisms as potential targets for therapeutic interventions., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Age-related decline in brain endothelial cell (BEC) function contributes critically to neurological disease. Comprehensive atlases of the BEC transcriptome have become available, but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting-based mouse BEC enrichment protocol compatible with proteomics and resolved the profiles of protein abundance changes during aging. Unsupervised cluster analysis revealed a segregation of age-related protein dynamics with biological functions, including a downregulation of vesicle-mediated transport. We found a dysregulation of key regulators of endocytosis and receptor recycling (most prominently Arf6), macropinocytosis and lysosomal degradation. In gene deletion and overexpression experiments, Arf6 affected endocytosis pathways in endothelial cells. Our approach uncovered changes not picked up by transcriptomic studies, such as accumulation of vesicle cargo and receptor ligands, including Apoe. Proteomic analysis of BECs from Apoe-deficient mice revealed a signature of accelerated aging. Our findings provide a resource for analysing BEC function during aging., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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 Aβ 1-40 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., (© 2022. The Author(s).)

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., (© 2021. The Author(s).)

White matter hyperintensities (WMH) are among the most common radiological abnormalities in the ageing population and an established risk factor for stroke and dementia. While common variant association studies have revealed multiple genetic loci with an influence on their volume, the contribution of rare variants to the WMH burden in the general population remains largely unexplored. We conducted a comprehensive analysis of this burden in the UK Biobank using publicly available whole-exome sequencing data (n up to 17 830) and found a splice-site variant in GBE1, encoding 1,4-alpha-glucan branching enzyme 1, to be associated with lower white matter burden on an exome-wide level [c.691+2T>C, β = -0.74, standard error (SE) = 0.13, P = 9.7 × 10-9]. Applying whole-exome gene-based burden tests, we found damaging missense and loss-of-function variants in HTRA1 (frequency of 1 in 275 in the UK Biobank population) to associate with an increased WMH volume (P = 5.5 × 10-6, false discovery rate = 0.04). HTRA1 encodes a secreted serine protease implicated in familial forms of small vessel disease. Domain-specific burden tests revealed that the association with WMH volume was restricted to rare variants in the protease domain (amino acids 204-364; β = 0.79, SE = 0.14, P = 9.4 × 10-8). The frequency of such variants in the UK Biobank population was 1 in 450. The WMH volume was brought forward by ∼11 years in carriers of a rare protease domain variant. A comparison with the effect size of established risk factors for WMH burden revealed that the presence of a rare variant in the HTRA1 protease domain corresponded to a larger effect than meeting the criteria for hypertension (β = 0.26, SE = 0.02, P = 2.9 × 10-59) or being in the upper 99.8% percentile of the distribution of a polygenic risk score based on common genetic variants (β = 0.44, SE = 0.14, P = 0.002). In biochemical experiments, most (6/9) of the identified protease domain variants resulted in markedly reduced protease activity. We further found EGFL8, which showed suggestive evidence for association with WMH volume (P = 1.5 × 10-4, false discovery rate = 0.22) in gene burden tests, to be a direct substrate of HTRA1 and to be preferentially expressed in cerebral arterioles and arteries. In a phenome-wide association study mapping ICD-10 diagnoses to 741 standardized Phecodes, rare variants in the HTRA1 protease domain were associated with multiple neurological and non-neurological conditions including migraine with aura (odds ratio = 12.24, 95%CI: 2.54-35.25; P = 8.3 × 10-5]. Collectively, these findings highlight an important role of rare genetic variation and the HTRA1 protease in determining WMH burden in the general population., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.

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.

Cerebral small vessel disease represents a heterogeneous group of disorders leading to stroke and cognitive impairment. While most small vessel diseases appear sporadic and related to age and hypertension, several early-onset monogenic forms have also been reported. However, only a minority of patients with familial small vessel disease carry mutations in one of known small vessel disease genes. We used whole exome sequencing to identify candidate genes in an autosomal dominant small vessel disease family in which known small vessel disease genes had been excluded, and subsequently screened all candidate genes in 201 unrelated probands with a familial small vessel disease of unknown aetiology, using high throughput multiplex polymerase chain reaction and next generation sequencing. A heterozygous HTRA1 variant (R166L), absent from 1000 Genomes and Exome Variant Server databases and predicted to be deleterious by in silico tools, was identified in all affected members of the index family. Ten probands of 201 additional unrelated and affected probands (4.97%) harboured a heterozygous HTRA1 mutation predicted to be damaging. There was a highly significant difference in the number of likely deleterious variants in cases compared to controls (P = 4.2 × 10(-6); odds ratio = 15.4; 95% confidence interval = 4.9-45.5), strongly suggesting causality. Seven of these variants were located within or close to the HTRA1 protease domain, three were in the N-terminal domain of unknown function and one in the C-terminal PDZ domain. In vitro activity analysis of HTRA1 mutants demonstrated a loss of function effect. Clinical features of this autosomal dominant small vessel disease differ from those of CARASIL and CADASIL by a later age of onset and the absence of the typical extraneurological features of CARASIL. They are similar to those of sporadic small vessel disease, except for their familial nature. Our data demonstrate that heterozygous HTRA1 mutations are an important cause of familial small vessel disease, and that screening of HTRA1 should be considered in all patients with a hereditary small vessel disease of unknown aetiology., (© The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)