Transcriptomic and genetic studies identify IL-33 as a candidate gene for Alzheimer's disease

Abstract

The only recognized genetic determinant of the common forms of Alzheimer's disease (AD) is the epsilon 4 allele of the apolipoprotein E gene (APOE). To identify new candidate genes, we recently performed transcriptomic analysis of 2741 genes in chromosomal regions of interest using brain tissue of AD cases and controls. From 82 differentially expressed genes, 1156 polymorphisms were genotyped in two independent discovery subsamples (n=945). Seventeen genes exhibited at least one polymorphism associated with AD risk, and following correction for multiple testing, we retained the interleukin (IL)-33 gene. We first confirmed that the IL-33 expression was decreased in the brain of AD cases compared with that of controls. Further genetic analysis led us to select three polymorphisms within this gene, which we analyzed in three independent case-control studies. These polymorphisms and a resulting protective haplotype were systematically associated with AD risk in non-APOE epsilon 4 carriers. Using a large prospective study, these associations were also detected when analyzing the prevalent and incident AD cases together or the incident AD cases alone. These polymorphisms were also associated with less cerebral amyloid angiopathy (CAA) in the brain of non-APOE epsilon 4 AD cases. Immunohistochemistry experiments finally indicated that the IL-33 expression was consistently restricted to vascular capillaries in the brain. Moreover, IL-33 overexpression in cellular models led to a specific decrease in secretion of the A beta(40) peptides, the main CAA component. In conclusion, our data suggest that genetic variants in IL-33 gene may be associated with a decrease in AD risk potentially in modulating CAA formation.

Figure 1

(A) Expression level of IL-33 in the brain of AD cases and controls. All values of IL-33 mRNA were reported as arbitrary units following normalisation by β-actin mRNA quantification. mRNA quantifications (IL-33 and β-actin) were carried out in triplicate in all individuals (n = 45 controls and n=43 cases). Cross: mean of IL-33 expression in cases and controls; Middle line: Median; Upper horizontal line: inclusion of 75% of the individuals; lower horizontal line: inclusion of 25% of the individuals. *: individuals exhibiting extreme values (out of the global distribution). (B) Representative IL-33 immunolabelling in the colon mucosa of a patient with Crohn’s diseas as a positive control; (C) Representative IL-33 immunolabelling in AD brains. In the superficial cortex from an Alzheimer patient, the small meningeal and superficial cortical small vessels show the same pattern of labelling than in the one observed in controls. (D,E) Representative IL-33 immunolabelling in the control brains. This labelling is mostly located in endothelial cells and vascular smooth muscle cells of small vessels, in the nucleus and/or the cytoplasm

Figure 2

Impact of the rs1157505, rs11792633 and rs7044343 SNPs on the risk of developing AD in the American, English, French, prospective-3C study and combined populations according to the APOE status. (A), (B) and (C) Allelic OR for the rs1157505, rs11792633 and rs7044343 SNPs respectively. (D) OR for the haplotype 222 (including the rare alleles of the rs1157505, rs11792633 and rs704343 SNPs).

Figure 3

Overexpression of IL-33 in the neuroblastoma SKNSH-SY5Y cell line stably transfected with APP^695wt. (A) Representative experiment of APP metabolism variations following transfection of the IL-33 expression vector (APPs, APP soluble; APP-CTF, APP carboxy terminal fragments); (B) Representative experiment of Aβ₄₀ and Aβ₄₂ secretions following transfection of the IL-33 expression vector; (C) Mean variations of the Aβ₄₀ and Aβ₄₂ secretions from three independent experimentations in duplicates; (D) IL-33 accumulates in nucleus (colocalisation with dense regions of DAPI staining, indicating association with heterochromatin). No signal was detected using an empty vector (Mock) (Data not shown); (E) detection of the ST2 receptor expression in the SY5Y-AP^P695wt cell line by immunofluorescence experiments and western blot; (F) Representative experiment of APP metabolism variations in response to human recombinant IL-33 protein; (G) Representative experiment of Aβ₄₀ and Aβ₄₂ secretions in response to human recombinant IL-33 protein; (H) Mean variations of the Aβ₄₀ and Aβ₄₂ secretions from three independent experimentations in duplicates.