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

Abstract

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.

Keywords: CADASIL; Cerebral amyloid angiopathy; Cerebral small vessel disease; HTRA1; Proteomics; Proteostasis.

Fig. 1

Study workflow and characterization of microvessel preparations used for the proteomic analysis. a Study workflow (top): Parenchymal microvessels were isolated from cryopreserved post-mortem brain samples of patients with cerebral amyloid angiopathy type 1 (CAA), control subjects (CON) or patients with Alzheimer’s disease (AD) and analyzed by LC–MS/MS. Confocal microscopy images (bottom) of a brain tissue section of a representative CAA, control and AD case immunostained for the amyloid beta (Aβ) peptide (red) and for the basement membrane marker collagen IV (pseudocoloured in white). Prominent vascular Aβ immunoreactivity in arterioles and capillaries (indicated by filled yellow arrows) are only observed in CAA. Parenchymal amyloid plaques, indicated by empty yellow arrows, are observed in CAA and AD. b Confocal microscopy image of a capillary network isolated from a CAA patient with hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) and stained for Aβ (red) and for the basement membrane marker collagen IV (pseudocoloured in white). c, d Level of the amyloid species Aβ_1-40 (c) and Aβ_1-42 (d) in the microvessel extracts determined by ELISA. p-values were calculated using ANOVA with Tukey post hoc analysis, **p = 0.0078, ****p < 0.0001. Of note, the HCHWA-D case showed the highest levels of both Aβ_1-40 (40,929 pg/μg) and Aβ_1-42 (1699 pg/μg) across all samples

Fig. 2

The brain microvascular proteome of patients with cerebral amyloid angiopathy. a Venn diagram demonstrating the overlap of 3752 proteins identified in at least 6 of the 12 samples from patients with cerebral amyloid angiopathy type 1 (CAA) and 6 of the 12 samples from control (CON) subjects. b Volcano plot illustrating log₂ LFQ ratios (CAA vs CON) and -log10 p values of all quantified proteins. Red symbols (n = 35) indicate proteins with a significant change in abundance (p < 0.05, log₂ ratio <  − 1.0/ > 1.0). c Abundance distribution according to the mean iBAQ intensity for each protein. Significantly altered proteins are indicated in red. d Protein localization of significantly altered proteins and of all identified proteins according to UniProt subcellular localization information

Fig. 3

The brain microvascular proteome of patients with Alzheimer’s disease. a Venn diagram demonstrating the overlap of 3780 proteins identified in at least 6 of the 13 samples from patients with Alzheimer’s disease (AD) and 6 of the 12 samples from control (CON) subjects. b Volcano plot illustrating log₂ LFQ ratios (AD vs CON) and − log10 p values of all quantified proteins. Blue symbols (n = 82) indicate proteins with a significant change in abundance (p < 0.05, log₂ ratio <  − 1.0/ > 1.0). c Abundance distribution according to the mean iBAQ intensity for each protein. Significantly altered proteins are indicated in blue. The three most and least abundant proteins are labeled with gene names. d Protein localization of significantly altered proteins and of all identified proteins according to UniProt subcellular localization information

Fig. 4

The CAA brain microvascular proteome displays a distinct profile independent of AD. a Venn diagram demonstrating the overlap between significantly altered proteins in CAA and AD (n = 12; p = 3.13 × 10^–12). The overlapping proteins are displayed in yellow, the 23 and 70 proteins specifically altered in CAA and AD are shown in red and blue respectively. b Scatter plot of the log₂ LFQ ratios in CAA versus CON against AD versus CON. Proteins exclusively altered in CAA are labeled with gene names, as are TNC and VGF. Significantly altered proteins are highlighted according to the colors in a, non-significantly altered proteins are shown in grey. c Circos diagram illustrating the subcellular localization information (UniProt) of the overlapping proteins and of the proteins specifically altered in CAA or AD

Fig. 5

The CAA proteomic profile overlaps with the CADASIL proteomic profile. a Venn diagrams illustrating the overlap between proteins significantly altered in CAA (red) or AD patients (blue) with proteins significantly altered in CADASIL (teal) as reported in our earlier study [72]. A comparison between the CAA and CADASIL proteomic profiles results in an overlap of 12 proteins (p = 1.47 × 10^–13) (orange), whereas no overlap was observed between the AD and CADASIL proteomic profiles. For better comparability, the CADASIL dataset was analyzed using the same significance and fold change thresholds as applied to the CAA and AD samples (p < 0.05 and log₂ fold change <  − 1.0/ > 1.0). b Scatter plot of the log₂ LFQ ratios in CAA versus CON against CADASIL versus CON. Significantly altered proteins are highlighted according to the colors in a, non-significantly altered proteins are shown in grey. Overlapping proteins are labeled with gene names, as is NOTCH3. The serine protease HTRA1 is highlighted in green. c Profile plot of Aβ_1-40 levels (determined by ELISA) (black) and iBAQ intensities for the overlapping proteins (orange) in each individual CAA patient. For formal assessment of statistical correlations see Additional file 1: Figure 1. Case 8 represents a patient with hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D)

Fig. 6

Accumulation of HTRA1 and its substrates in the CAA proteomic profile. a Immunofluorescence staining of an isolated capillary from a CAA patient (visualized by collagen IV staining) demonstrates a near complete co-localization of Aβ and HTRA1 immunoreactivity. b Venn diagram illustrating the overlap between proteins significantly altered in CAA with proteins significantly altered in CADASIL and in HTRA1 deficient mice as reported in our earlier study [72]. Eight of the shared proteins in CAA and CADASIL were also enriched in HTRA1 deficient mice (red filling). c Table with LFQ ratio information in the CAA, CADASIL and HTRA1 deficient proteomic profiles for the overlapping proteins, as for APCS, C3, C1QC and HTRA1. The latter four proteins are shared between CAA and CADASIL but not part of the HTRA1 deficient profile as they were not identified (APCS), not significantly altered (C3 and C1QC) or exclusively detected under HTRA1 wild-type conditions (HTRA1). Similar proteomic changes in HTRA1 deficient mice were recently reported by Kato et. al [34]. For most proteins in vitro cleavage data have been reported earlier. d, e APCS and PRSS23 are HTRA1 substrates. Shown are immunoblots of conditioned supernatants from HEK293 cells expressing PRSS23 (d) (detected via Myc-tag) or APCS (e), co-incubated with supernatants containing wild-type or active-site mutant (S328A) HTRA1 (detected via V5-tag) in the presence or absence of a HTRA1-specific inhibitor (5 μM). Molecular weight marker bands in kDa are indicated. The band above APCS in the S328A condition represent an unspecific cross-reactivity of the antibody. HTRA1 bands of lower molecular weight represent autoproteolysis fragments