Proteomics analysis identifies new markers associated with capillary cerebral amyloid angiopathy in Alzheimer's disease

Abstract

Alzheimer's disease (AD) is characterized by amyloid beta (Aβ) deposits as plaques in the parenchyma and in the walls of cortical and leptomeningeal blood vessels of the brain called cerebral amyloid angiopathy (CAA). It is suggested that CAA type-1, which refers to amyloid deposition in both capillaries and larger vessels, adds to the symptomatic manifestation of AD and correlates with disease severity. Currently, CAA cannot be diagnosed pre-mortem and disease mechanisms involved in CAA are elusive. To obtain insight in the disease mechanism of CAA and to identify marker proteins specifically associated with CAA we performed a laser dissection microscopy assisted mass spectrometry analysis of post-mortem human brain tissue of (I) AD cases with only amyloid deposits in the brain parenchyma and no vascular related amyloid, (II) AD cases with severe CAA type-1 and no or low numbers of parenchymal amyloid deposits and (III) cognitively healthy controls without amyloid deposits. By contrasting the quantitative proteomics data between the three groups, 29 potential CAA-selective proteins were identified. A selection of these proteins was analysed by immunoblotting and immunohistochemistry to confirm regulation and to determine protein localization and their relation to brain pathology. In addition, specificity of these markers in relation to other small vessel diseases including prion CAA, CADASIL, CARASAL and hypertension related small vessel disease was assessed using immunohistochemistry.Increased levels of clusterin (CLU), apolipoprotein E (APOE) and serum amyloid P-component (APCS) were observed in AD cases with CAA. In addition, we identified norrin (NDP) and collagen alpha-2(VI) (COL6A2) as highly selective markers that are clearly present in CAA yet virtually absent in relation to parenchymal amyloid plaque pathology. NDP showed the highest specificity to CAA when compared to other small vessel diseases. The specific changes in the proteome of CAA provide new insight in the pathogenesis and yields valuable selective biomarkers for the diagnosis of CAA.

Keywords: Alzheimer’s disease; Amyloid beta; Biomarker; Cerebral amyloid angiopathy; Human brain; Laser microdissection; Post-mortem tissue; Proteomics.

Fig. 1

Workflow used in this study. Amyloid Beta pathology was visualized in human postmortem occipital lobe tissue. Unaffected grey matter was isolated from healthy control cases. Grey matter with high burden of Aβ pathology was isolated from the AD and CAA cases thereby isolating tissue with high plaque load or high CAA type-1 burden, respectively Tissue was lysed and the proteins were separated using SDS-PAGE and subjected to in-gel trypsin digestion. Peptides were analysed using LC-MS-MS. A database search for protein identification and protein quantification was performed using MaxQuant software. ANOVA (Kruskall Wallis) and t-tests were performed to identify significantly regulated proteins

Fig. 2

Three strategies used to select proteins that are differentially expressed in CAA type-1 compared to control and AD brains. Criteria of each of the selection strategies are specified, numbers of resulted proteins indicated, and selected proteins are listed in tables and figures as indicated

Fig. 3

Relative abundance of proteins with altered expression in CAA type-1 compared to Alzheimer’s disease cases and controls as determined by MS. Three groups of selected proteins (panels a-c), with altered levels (MS-derived, log2 LFQ intensity values) in CAA type-1 compared to the control groups and the AD groups. Selection criteria are specified in Fig. 2. Gene names are indicated

Fig. 4

Immunoblotting analysis of proteins with altered expression in CAA type-1. a Immunoblotting was performed for, NDP, COL6A2, APCS and APOE on occipital lobe tissue lysates of non-demented controls (N) AD cases (A) and CAA cases (C). Immunoreactivity was observed at the correct molecular weight for each protein. b A significant difference (p < 0.05) was found in the expression of NDP in the CAA group when compared to control but not when compared to the AD group. For COL6A2, APCS and APOE significance was not reached between any of the groups using this technique. Data are expressed as mean ± SEM

Fig. 5

Immunohistochemical analysis of selected proteins. Representative images were taken. Aβ pathology was visualized. a The control case does not have any Aβ pathology. b Plaque pathology is confirmed in the AD case and (c) CAA type-1 pathology is confirmed in the CAA type-1 case. d-f Extensive NDP immunoreactivity is observed in the CAA type-1 cases whereas absent in both control and AD cases without CAA. g-i COL6A2 immunoreactivity is hardly observed in the control and AD cases, however, extensive immunoreactivity is observed in the CAA type cases and includes both capillaries and large vessels. j-l Immunoreactivity for HTRA1 is absent in control tissue, however, is observed both related to plaque pathology and CAA at comparable intensity. m-o Immunoreactivity for APCS is absent in control cases but is observed both related to plaque and CAA type-1 pathology. However, the intensity of the staining observed in the AD cases is considerably less. p-r Also, APOE immunoreactivity is observed related to both plaque and CAA type-1 pathology, yet its intensity in CAA type-1 is far greater. Scale bar, 100 μm in images A to R. Scale bar in image (C`) 10 μm and in all zoomed images, which are marked with a grave accent (`)

Fig. 6

Semi-quantitative analysis of immunohistochemical data of proteins with altered expression in CAA type-1. Immunohistochemical stainings were quantified by measuring the percentage of pixels that showed positive immunoreactivity. Significance was calculated using a one-way ANOVA (Kruskal-Wallis test) and posthoc Dunn’s Multiple Comparison Test. A significant increase (p < 0.05) in immunoreactivity in the CAA group compared to both control and AD groups was observed for NDP and COL6A2. For APOE, APCS and HTRA1 significant differences were only found when comparing CAA with control, but not with the AD group. Data are expressed as mean ± SEM

Fig. 7

Expression of CAA type-1 markers in other small vessel diseases. IHC for Aβ, NDP, COL6A2, APCS and APOE was performed. In a CAA type-1 case immunoreactivity for all marker proteins is confirmed (a-e). Also immunoreactivity is seen for all markers in the cotton wool case and Aβ pathology was confirmed (f). For NDP, APOE and APCS immunoreactivity is also seen localizing to severe dyshoric angiopathy (g, i and j). COL6A2 immunoreactivity is restricted to the vessel wall (h). In de Prp-CAA case Aβ pathology was absent (k). Extensive immunoreactivity was observed for NDP, COL6A2, APOE and APCS (l-o). No immunoreactivity was observed for Aβ, NDP, COL6A2, APOE and APCS in the white matter of control tissue (p-t). In the CADASIL case no Aβ pathology was present (u). Mild immunoreactivity for NDP (v) COL62A staining was most pronounced (w). APOE and APCS also displayed mild immunoreactivity related to the affected vessels (x, y). In hypertension related small vessel disease no Aβ was detected (z) immunoreactivity of COLA6A2 was moderate (ab) while immunoreactivity for NDP, APOE and APCS were low but present (aa, ac and ad). In the CARASAL case only prominent immunoreactivity of COL6A2 was seen in affected vessels (ag) while NDP, APOE and APCS were absent (af, ah and ai). Scale bar in (a) indicates 100 μm