Genome-wide association studies (GWAS) have identified a region upstream the BIN1 gene as the most important genetic susceptibility locus in Alzheimer’s disease (AD) after APOE. We report that BIN1 transcript levels were increased in AD brains and identified a novel 3 bp insertion allele ∼28 kb upstream of BIN1, which increased (i) transcriptional activity in vitro, (ii) BIN1 expression levels in human brain and (iii) AD risk in three independent case-control cohorts (Meta-analysed Odds ratio of 1.20 (1.14–1.26) (P=3.8 × 10−11)). Interestingly, decreased expression of the Drosophila BIN1 ortholog Amph suppressed Tau-mediated neurotoxicity in three different assays. Accordingly, Tau and BIN1 colocalized and interacted in human neuroblastoma cells and in mouse brain. Finally, the 3 bp insertion was associated with Tau but not Amyloid loads in AD brains. We propose that BIN1 mediates AD risk by modulating Tau pathology.

The only recognized genetic determinant of the common forms of Alzheimer's disease (AD) is the ɛ4 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 ɛ4 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 ɛ4 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 Aβ40 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.

To more rapidly identify candidate genes located within chromosomal regions of interest defined by genome scan studies in Alzheimer's disease (AD), we have developed a customized microarray containing all the ORFs (n=2741) located within nine of these regions. Levels of gene expression were assessed in total RNA from brain tissue of 12 controls and 12 AD patients. Of all genes showing differential expression, we focused on the ornithine transcarbamylase (OTC) gene on Xp21.1., a key enzyme of the urea cycle which we found to be expressed in AD brains but not in controls, as confirmed by RT–PCR. We also detected mRNA expression of all the other urea cycle enzymes in AD brains. Immunochemistry experiments revealed that the OTC expression was strictly restricted to vascular endothelial cells in brain. Furthermore, OTC activity was 880% increased in the CSF of probable AD cases compared with controls. We analysed the association of the OTC −389 G/A and −241 A/G promoter polymorphisms with the risk of developing AD. We observed that rare haplotypes may be associated with the risk of AD through a possible modulation of the methylation of the OTC promoter. In conclusion, our results suggest the involvement of a new pathway in AD brains involving the urea cycle.

Background: It has been proposed that, independent of the ε4 allele, APOE promoter polymorphisms (−491 A/T and −219 G/T) may be risks factor for Alzheimer’s disease by modulating APOE expression. Objective: To measure the level of APOE expression in Alzheimer’s disease. Methods: Brains were obtained at necropsy from 114 patients with early and late onset sporadic Alzheimer’s disease in Greater Manchester (UK) during years 1986 to 2001. Total RNA was extracted from 84 brains. Purified lymphocytes were obtained from fresh blood from 16 probable Alzheimer cases from Lille (France). APOE and β-actin gene expression was determined by reverse transcriptase polymerase chain reaction in brain and lymphocytes. Results: An inverse correlation between APOE expression level and Aβ loads was observed. As previously described and extended to 114 cases here, an association between the −219 TT genotype and a higher level of parenchymal Aβ deposition was found, irrespective of APOE ε4 allele status. This effect was more pronounced in older individuals, whereas higher Aβ load appeared more closely related to ε4 in the younger age group (cut off point at the median age at death (72.5 years)). The −219 TT genotype was associated with a decrease in APOE expression. There was a 60% decrease in APOE expression in lymphocytes from probable Alzheimer cases v controls (p = 0.01). Conclusions: In the oldest individuals, reduced APOE expression, modulated in part by −219 G/T polymorphism, may influence risk and constitute a determinant Aβ load in Alzheimer’s disease.

Although possession of the ε4 allele of the apolipoprotein E gene appears to be an important biological marker for Alzheimer’s disease (AD) susceptibility, strong evidence indicates that at least one additional risk gene exists on chromosome 12. Here, we describe an association of the 3′-UTR +1073 C/T polymorphism of the OLR1 (oxidised LDL receptor 1) on chromosome 12 with AD in French sporadic (589 cases and 663 controls) and American familial (230 affected sibs and 143 unaffected sibs) populations. The age and sex adjusted odds ratio between the CC+CT genotypes versus the TT genotypes was 1.56 (p=0.001) in the French sample and 1.92 (p=0.02) in the American sample. Furthermore, we have discovered a new T/A polymorphism two bases upstream of the +1073 C/T polymorphism. This +1071 T/A polymorphism was not associated with the disease, although it may weakly modulate the impact of the +1073 C/T polymorphism. Using 3′-UTR sequence probes, we have observed specific DNA protein binding with nuclear proteins from lymphocyte, astrocytoma, and neuroblastoma cell lines, but not from the microglia cell line. This binding was modified by both the +1071 T/A and +1073 C/T polymorphisms. In addition, a trend was observed between the presence or absence of the +1073 C allele and the level of astrocytic activation in the brain of AD cases. However, Aβ40, Aβ42, Aβ total, and Tau loads or the level of microglial cell activation were not modulated by the 3′-UTR OLR1 polymorphisms. Finally, we assessed the impact of these polymorphisms on the level of OLR1 expression in lymphocytes from AD cases compared with controls. The OLR1 expression was significantly lower in AD cases bearing the CC and CT genotypes compared with controls with the same genotypes. In conclusion, our data suggest that genetic variation in the OLR1 gene may modify the risk of AD.

Background: There is evidence that inflammatory processes may contribute to the development of Alzheimer's disease through production of cytokines and free radicals that damage neurones. A recent study has shown that transforming growth factor beta1 (TGF-beta1) signalling in astrocytes promotes Abeta production and could play a critical role in the formation of amyloid plaques in the brain. Objectives: To explore the impact of the -800 and -509 TGF-beta1 promoter polymorphisms and the +25 polymorphism on the risk of occurrence of Alzheimer's disease in a large population of sporadic cases and controls, and on the amyloid beta (Abeta) load in the brains of Alzheimer patients. Methods: The TGF-beta1 genotypes of the three polymorphisms were determined in 678 sporadic Alzheimer's disease patients and 667 controls. They were also characterised, along with Abeta load, in the brains of 81 necropsy confirmed Alzheimer patients. Results: No significant variations in the distribution of the genotypes and haplotypes were observed between Alzheimer patients and controls, or in the amount of Abeta deposition. Conclusions: These results do not suggest an influence of genetic variability at the TGF-beta1 gene locus on the occurrence of Alzheimer's disease.

Background: There is evidence that inflammatory processes may contribute to the development of Alzheimer's disease through production of cytokines and free radicals that damage neurones. A recent study has shown that transforming growth factor β1 (TGF-β1) signalling in astrocytes promotes Aβ production and could play a critical role in the formation of amyloid plaques in the brain. Objectives: To explore the impact of the −800 and −509 TGF-β1 promoter polymorphisms and the +25 polymorphism on the risk of occurrence of Alzheimer's disease in a large population of sporadic cases and controls, and on the amyloid β (Aβ) load in the brains of Alzheimer patients. Methods: The TGF-β1 genotypes of the three polymorphisms were determined in 678 sporadic Alzheimer's disease patients and 667 controls. They were also characterised, along with Aβ load, in the brains of 81 necropsy confirmed Alzheimer patients. Results: No significant variations in the distribution of the genotypes and haplotypes were observed between Alzheimer patients and controls, or in the amount of Aβ deposition. Conclusions: These results do not suggest an influence of genetic variability at the TGF-β1 gene locus on the occurrence of Alzheimer's disease.

Background: The NOTCH4 gene is located at 6p21.3, a site shown in several studies to have significant linkage with Alzheimer's disease.Objective: To investigate the potential impact of two polymorphisms within this gene on the risk of developing Alzheimer's disease.Methods: Genotyping of promoter and 5'-UTR polymorphisms was done in Scottish, English, and French populations. The potential functionality of the 5'-UTR polymorphism was assessed by testing its impact on Ab load in Alzheimer brains and also by undertaking electrophoretic mobility shift assays and transfection experiments.Results: No association of the Notch4 polymorphisms alone with the disease was observed in any of the populations. However, an interaction of the 5'-UTR C/T polymorphism with the epsilon4 allele of the APOE gene was detected in United Kingdom populations but not in the French. No relation between the 5'-UTR polymorphism and Ab loads was detected overall or in the presence or absence of the e4 allele. No DNA protein specific binding was found with proteins from neuroblastoma, glioma, or astrocytoma cells, and no allele dependent transcriptional activity was detected.Conclusions No association between two NOTCH4 polymorphisms alone and Alzheimer's disease was observed in the three populations, but there was evidence of an increased risk associated with the 5'-UTR CC genotype in e4 bearers in the United Kingdom. As no functionality for this polymorphism could be determined, it is likely that the interaction is spurious or results from a linkage disequilibrium of this 5'-UTR polymorphism with another marker elsewhere in the 6p21.3 locus.