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Endothelin-Converting Enzyme-1 Is Expressed in Human Cerebral Cortex and Protects Against Alzheimer's Disease

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Endothelin-Converting Enzyme-1 Is Expressed in Human Cerebral Cortex and Protects Against Alzheimer's Disease Molecular Psychiatry (2004) 9, 1122–1128 & 2004 Nature Publishing Group All rights reserved 1359-4184/04 $30.00 www.nature.com/mp ORIGINAL RESEARCH ARTICLE Endothelin-converting enzyme-1 is expressed in human cerebral cortex and protects against Alzheimer’s disease B Funalot1, T Ouimet1, A Claperon1, C Fallet2, A Delacourte3, J Epelbaum4, T Subkowski5,NLe´onard2, V Codron6, J-P David3,7, P Amouyel6, J-C Schwartz1 and N Helbecque6 1Institut National de la Sante´ et de la Recherche Me´dicale (INSERM) Unit 573, Paris, France; 2Department of Neuropathology, Hoˆpital Sainte-Anne, Paris, France; 3INSERM Unit 422, Lille, France; 4INSERM Unit 549, Paris, France; 5BASF Main Laboratories, Ludwigshafen, Germany; 6INSERM Unit 508, Institut Pasteur, Lille, France; 7Department of Gerontology, Hoˆpital Emile-Roux, Limeil-Bre´vannes, France Cerebral accumulation of b-amyloid peptide (Ab) is a central event in the pathogenesis of Alzheimer’s disease (AD). Endothelin-converting enzyme-1 (ECE-1) is a candidate Ab-degrading enzyme in brain, but its involvement in AD pathogenesis was never assessed. We first performed brain immunocytochemistry, using a monoclonal anti-ECE-1 antibody, and observed neuronal ECE-1 expression in various cortical regions of nondemented subjects. In the hippocampus, ECE- 1 immunoreactivity showed a stereotypical pattern inversely correlated with susceptibility to Ab deposition, further suggesting a physiological role in Ab clearance. In order to undertake a genetic association study, we identified a functional genetic variant (ECE1B C-338A) located in a regulatory region of the ECE1 gene. We showed that the A allele is associated with increased transcriptional activity in promoter–reporter gene assays and with increased ECE-1 mRNA expression in human neocortex. In a case–control study involving 401 patients with late-onset AD and 461 aged controls, we found that homozygous carriers of the A allele had a reduced risk of AD (OR ¼ 0.47, 95% CI 0.25–0.88). This finding was strengthened by the analysis of two other genetic variants of the ECE1 gene, which showed that the genetic association is extended over at least 13 kilobases of the gene sequence. Our results suggest that ECE-1 expression in brain may be critical for cortical Ab clearance and offer new potential targets for therapeutic interventions in AD. Molecular Psychiatry (2004) 9, 1122–1128. doi:10.1038/sj.mp.4001584 Published online 31 August 2004 Keywords: endothelin-converting enzyme; Alzheimer’s disease; late-onset Alzheimer’s disease; b-amyloid peptide; clearance; zinc metalloprotease; immunohistochemistry; genetic polymorph- ism; genetic association study Several lines of evidence support an early pathogenic 3.4.24.11), endothelin-converting enzyme-1 (ECE-1, role for the b-amyloid peptide (Ab) in Alzheimer’s EC 3.4.24.71), endothelin-converting enzyme-2 (ECE- disease (AD).1–3 Ab is a physiological peptide con- 2) and insulysin (EC 3.4.24.56), have been shown to tinuously synthesized in brain, produced by cleavage influence endogenous Ab levels in the brains of their of the amyloid precursor protein (APP). In familial respective knockout mice.11–14 Neprilysin, ECE-1 and cases of early-onset AD, various mutations of the ECE-2 are type II integral membrane peptidases, genes encoding APP and presenilins have been classified as members of the M13 family,15 whereas shown to increase Ab production.3,4 By contrast, the insulysin is a cytosolic enzyme belonging to the M16 mechanisms of cortical Ab accumulation in late-onset family.16 The four enzymes are suspected to have a AD (LOAD) patients appear to be more complex and physiological role in Ab clearance in human brain, may involve increased expression and activity of the and therefore to protect against development of AD, b-site APP-cleaving enzyme (BACE-1),5–7 increased but direct evidence of their involvement in AD aggregation of Ab fibrils or decreased Ab clearance.8 pathogenesis is lacking. Neprilysin immunoreactivity Several proteases were shown to hydrolyze Ab in appears to be weak or undetectable in human cerebral vitro or in cell-based assays, and are likely candidates cortex.17,18 By contrast, insulysin immunoreactivity is for a role in Ab clearance in brain.9,10 Among these, marked in hippocampal and neocortical neurons.19,20 only four zinc metalloproteases: neprilysin (EC Several genetic association studies tested for an association between neprilysin or insulysin gene variants and AD, but gave controversial results.21–25 Correspondence: Dr B Funalot, MD, INSERM Unit 573, Centre To date, the expression of ECE-1 in human brain had Paul-Broca, 2ter rue d’Ale´sia, 75014 Paris, France. never been studied, and its potential role in the E-mail: [email protected] Received 16 September 2003; revised 05 May 2004; accepted 19 pathogenesis of AD had never been assessed. We May 2004 have addressed this question by a combination of ECE-1 and Alzheimer’s disease B Funalot et al 1123 expression and genetic studies, and provide evidence gene constructs were transiently transfected in the for a protective role of ECE-1 against AD in the aging ECV304 cell line. The pcDNA3.1/HisB/LacZ plasmid brain. (Invitrogen), containing the b-galactosidase gene under the control of a CMV promoter, was used as an internal standard. SEAP activity was measured in a Materials and methods plate fluorometer (Dynatech) using 4-methylumbelli- Brain immunocytochemistry feryl phosphate as a substrate, and normalized with Brains from eight nondemented subjects were stu- respect to b-galactosidase activity. Results represent died. Ages at death were 38, 56, 82, 84, 85, 86, 92 and the mean of at least six transfection experiments for 99 years. Brain specimens were fixed in 10% each construct. formaldehyde and embedded in paraffin. ECE-1 immunocytochemistry (ICC) was performed using Relative quantitative RT-PCR mouse monoclonal antibodies directed against ECE- We could obtain neocortical samples from seven aged 1 common C-terminal sequence (B61/104, diluted nondemented subjects. All of them were hospitalized 1 : 50, and E15/6, diluted 1 : 20). It was previously for various disorders and died in public hospitals. shown that both antibodies are specific of ECE-1.26 Samples of Brodmann area 9 (prefrontal cortex) were However, we did not test these antibodies against the deep-frozen at autopsy (excluding patients with post- ECE-2 protein, which has B60% identity with ECE-1. mortem delay superior to 24 h) and subsequently Sections were microwaved in citrate buffer, followed stored at À801C. Total RNA was extracted from frozen by endogenous peroxydases inactivation (3% H2O2) samples using the TRIzol method (Invitrogen), fol- and treatment with the blocking agent from the lowing the manufacturer’s instructions. In all, 1 mgof universal HRP immunostaining kit (Immunotech). total RNA was reverse-transcripted using random Primary antibody was identified with a biotinylated hexamers and AMV-reverse transcriptase (Finn- secondary antibody and the avidin–biotin complex, zymes), in a volume of 25 ml, following the manufac- and visualized with 3,3-diaminobenzidine. Immunos- turer’s protocol. A volume of 1 ml of this reaction was tained paraffin sections were counterstained with used in all subsequent PCRs. Amplification of ECE-1 hemalun. The level of ECE-1 staining in the various cDNA was performed using the following primers: regions was assessed as low ( þ ), intermediate ( þþ) ECE-1F: 50-AGT ATG ACA AGG ACG GGA ACC-30 or high ( þþþ). and ECE-1R: 50-CTT ACC AGA CTT CGC ACT TGT G- 30 (resulting in a 459 bp product). Relative quantita- Polymorphism screening tive duplex PCRs were performed in a volume of Genomic DNA was extracted from peripheral whole 25 ml, using 18S RNA as an internal standard.29 Each blood using standard techniques. For each of the three reaction contained ECE-1-specific primers and Quan- promoter regions of the ECE1 gene, B1000 base pairs tumRNA Universal 18S primers (Ambion, resulting in (bp) of genomic DNA were amplified by polymerase a 315 bp product) in the same tube, following the chain reaction (PCR) in 30 healthy individuals, using manufacturer’s instructions. We tested different 18S AmpliTaq Gold (Perkin-Elmer), as described pre- primer-to-competimer ratios and finally used ratios of viously (ECE-1a, ECE-1c),27 or GC-RICH PCR System 2 : 8 for ECE-1 relative quantification. PCR was (Roche Diagnostics), following manufacturer’s in- performed using AmpliTaq Gold (Perkin-Elmer), with structions (ECE-1b-ECE-1d). PCR products were 9 min of denaturation at 951C, followed by 28 cycles screened for sequence variations by direct sequencing comprising 30 s at 941C, 30 s at 621C, and 30 s at 721C on a Li-Cor 4000L automated DNA sequencer, as (the optimal number of cycles had been determined as described previously.27 Wild-type and variant se- recommended). PCR products were loaded onto a 2% quences were analyzed for cis-elements using the agarose gel, stained with ethidium bromide, and computer program MatInspector (http://transfac. visualized by UV transillumination, using a CCD gbf.de/TRANSFAC).28 video camera. Raw images were directly saved as .tif files, and densitometric analysis was performed for Reporter gene assays each sample using the NIH Image software. For each All nucleotide positions (np) are given with respect to subject, relative quantitative RT-PCR were performed the ECE-1b translation start site. Genomic DNAs from and analyzed in triplicate, and the mean of the three two subjects (one homozygous for the A allele and the experiments was used in subsequent tests. other for the C allele of the ECE1B C-338A poly- morphism) were used for promoter activity assays. Genetic association study Genomic DNA regions between np À823 and À2, and French Caucasian patients affected by LOAD were between À823 and þ 258 (encompassing the entire recruited in hospitals.
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