title: Reduced secreted clusterin as a mechanism for Alzheimer-associated CLU mutations.
abstract: BACKGROUND
  The clusterin (CLU) gene has been identified as an important risk locus for Alzheimer's disease (AD). Although the actual risk-increasing polymorphisms at this locus remain to be identified, we previously observed an increased frequency of rare non-synonymous mutations and small insertion-deletions of CLU in AD patients, which specifically clustered in the β-chain domain of CLU. Nonetheless the pathogenic nature of these variants remained unclear. Here we report a novel non-synonymous CLU mutation (p.I360N) in a Belgian Alzheimer patient and have explored the pathogenic nature of this and 10 additional CLU mutations on protein localization and secretion in vitro using immunocytochemistry, immunodetection and ELISAs.
  
  
  RESULTS
  Three patient-specific CLU mutations in the β-chain (p.I303NfsX13, p.R338W and p.I360N) caused an alteration of the subcellular CLU localization and diminished CLU transport through the secretory pathway, indicative of possible degradation mechanisms. For these mutations, significantly reduced CLU intensity was observed in the Golgi while almost all CLU protein was exclusively present in the endoplasmic reticulum. This was further confirmed by diminished CLU secretion in HEK293T and HEK293 FLp-In cell lines.
  
  
  CONCLUSIONS
  Our data lend further support to the contribution of rare coding CLU mutations in the pathogenesis of neurodegenerative diseases. Functional analyses suggest reduced secretion of the CLU protein as the mode of action for three of the examined CLU mutations. One of those is a frameshift mutation leading to a loss of secreted protein, and the other two mutations are amino acid substitutions in the disulfide bridge region, possibly interfering with heterodimerization of the α- and β-chain of CLU.
authors.affiliation: VIB Department of Molecular Genetics, University of Antwerp - CDE, Building V Universiteitsplein 1, B-2610, Antwerpen, Belgium. karolien.bettens@molgen.vib-ua.be.|Institute Born-Bunge, Laboratory of Neurochemistry and Behavior, University of Antwerp, Antwerp, Belgium. karolien.bettens@molgen.vib-ua.be.
authors.forename: Karolien
authors.initials: K
authors.lastname: Bettens
authors.affiliation: VIB Department of Molecular Genetics, University of Antwerp - CDE, Building V Universiteitsplein 1, B-2610, Antwerpen, Belgium. steven.vermeulen@molgen.vib-ua.be.|Institute Born-Bunge, Laboratory of Neurochemistry and Behavior, University of Antwerp, Antwerp, Belgium. steven.vermeulen@molgen.vib-ua.be.
authors.forename: Steven
authors.initials: S
authors.lastname: Vermeulen
authors.affiliation: VIB Department of Molecular Genetics, University of Antwerp - CDE, Building V Universiteitsplein 1, B-2610, Antwerpen, Belgium. caroline.vancauwenberghe@molgen.vib-ua.be.|Institute Born-Bunge, Laboratory of Neurochemistry and Behavior, University of Antwerp, Antwerp, Belgium. caroline.vancauwenberghe@molgen.vib-ua.be.
authors.forename: Caroline
authors.initials: C
authors.lastname: Van Cauwenberghe
authors.affiliation: VIB Department of Molecular Genetics, University of Antwerp - CDE, Building V Universiteitsplein 1, B-2610, Antwerpen, Belgium. bavo.heeman@molgen.vib-ua.be.|Institute Born-Bunge, Laboratory of Neurochemistry and Behavior, University of Antwerp, Antwerp, Belgium. bavo.heeman@molgen.vib-ua.be.
authors.forename: Bavo
authors.initials: B
authors.lastname: Heeman
authors.affiliation: VIB Department of Molecular Genetics, University of Antwerp - CDE, Building V Universiteitsplein 1, B-2610, Antwerpen, Belgium. bob.asselbergh@molgen.vib-ua.be.|Institute Born-Bunge, Laboratory of Neurochemistry and Behavior, University of Antwerp, Antwerp, Belgium. bob.asselbergh@molgen.vib-ua.be.
authors.forename: Bob
authors.initials: B
authors.lastname: Asselbergh
authors.affiliation: VIB Department of Molecular Genetics, University of Antwerp - CDE, Building V Universiteitsplein 1, B-2610, Antwerpen, Belgium. caroline.robberecht@molgen.vib-ua.be.|Institute Born-Bunge, Laboratory of Neurochemistry and Behavior, University of Antwerp, Antwerp, Belgium. caroline.robberecht@molgen.vib-ua.be.
authors.forename: Caroline
authors.initials: C
authors.lastname: Robberecht
authors.affiliation: Institute Born-Bunge, Laboratory of Neurochemistry and Behavior, University of Antwerp, Antwerp, Belgium. sebastiaan.engelborghs@uantwerpen.be.|Department of Neurology and Memory Clinic, Hospital Network Antwerp Middelheim and Hoge Beuken, Antwerp, Belgium. sebastiaan.engelborghs@uantwerpen.be.
authors.forename: Sebastiaan
authors.initials: S
authors.lastname: Engelborghs
authors.affiliation: Department of Psychiatry and Memory Clinic, University of Leuven and University Hospitals Leuven Gasthuisberg, Leuven, Belgium. mathieu.vandenbulcke@med.kuleuven.be.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Vandenbulcke
authors.affiliation: Laboratory for Cognitive Neurology, Department of Neurology, University of Leuven and University Hospitals Leuven Gasthuisberg, Leuven, Belgium. rik.vandenberghe@uzleuven.be.
authors.forename: Rik
authors.initials: R
authors.lastname: Vandenberghe
authors.affiliation: Institute Born-Bunge, Laboratory of Neurochemistry and Behavior, University of Antwerp, Antwerp, Belgium. dedeyn@skynet.be.|Department of Neurology and Memory Clinic, Hospital Network Antwerp Middelheim and Hoge Beuken, Antwerp, Belgium. dedeyn@skynet.be.|Department of Neurology and Alzheimer Research Center, University of Groningen and University Medical Center Groningen, Groningen, The Netherlands. dedeyn@skynet.be.
authors.forename: Peter Paul
authors.initials: PP
authors.lastname: De Deyn
authors.affiliation: VIB Department of Molecular Genetics, University of Antwerp - CDE, Building V Universiteitsplein 1, B-2610, Antwerpen, Belgium. marc.cruts@molgen.vib-ua.be.|Institute Born-Bunge, Laboratory of Neurochemistry and Behavior, University of Antwerp, Antwerp, Belgium. marc.cruts@molgen.vib-ua.be.
authors.forename: Marc
authors.initials: M
authors.lastname: Cruts
authors.affiliation: VIB Department of Molecular Genetics, University of Antwerp - CDE, Building V Universiteitsplein 1, B-2610, Antwerpen, Belgium. christine.vanbroeckhoven@molgen.vib-ua.be.|Institute Born-Bunge, Laboratory of Neurochemistry and Behavior, University of Antwerp, Antwerp, Belgium. christine.vanbroeckhoven@molgen.vib-ua.be.
authors.forename: Christine
authors.initials: C
authors.lastname: Van Broeckhoven
authors.affiliation: VIB Department of Molecular Genetics, University of Antwerp - CDE, Building V Universiteitsplein 1, B-2610, Antwerpen, Belgium. kristel.sleegers@molgen.vib-ua.be.|Institute Born-Bunge, Laboratory of Neurochemistry and Behavior, University of Antwerp, Antwerp, Belgium. kristel.sleegers@molgen.vib-ua.be.
authors.forename: Kristel
authors.initials: K
authors.lastname: Sleegers
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000544:Alzheimer Disease / Q000453:epidemiology / Q000235:genetics* / Q000503:physiopathology; D019943:Amino Acid Substitution; D001530:Belgium / Q000453:epidemiology; D001692:Biological Transport; D051152:Clusterin / Q000235:genetics / Q000378:metabolism*; D003553:Cystine / Q000737:chemistry; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005091:Exons / Q000235:genetics; D005260:Female; D016368:Frameshift Mutation; D006056:Golgi Apparatus / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008297:Male; D020125:Mutation, Missense; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014161:Transduction, Genetic; D014162:Transfection
chemical_list: C495242:CLU protein, human; D051152:Clusterin; D011993:Recombinant Fusion Proteins; D003553:Cystine