Citation: Transl Psychiatry (2013) 3, e272; doi:10.1038/tp.2013.49 & 2013 Macmillan Publishers Limited All rights reserved 2158-3188/13 www.nature.com/tp Common exonic missense variants in the C2 domain of the human KIBRA protein modify lipid binding and cognitive performance K Duning1,10, DO Wennmann1,10, A Bokemeyer1,10, C Reissner2, H Wersching3, C Thomas1, J Buschert1, K Guske1, V Franzke1, A Flo¨el4,11, H Lohmann4, S Knecht4, S-M Brand5,MPo¨ter6, U Rescher6, M Missler2, P Seelheim7, C Pro¨pper8, TM Boeckers8, L Makuch9, R Huganir9, T Weide1, E Brand1, H Pavensta¨dt1,10 and J Kremerskothen1,10 The human KIBRA gene has been linked to human cognition through a lead intronic single-nucleotide polymorphism (SNP; rs17070145) that is associated with episodic memory performance and the risk to develop Alzheimer’s disease. However, it remains unknown how this relates to the function of the KIBRA protein. Here, we identified two common missense SNPs (rs3822660G/T [M734I], rs3822659T/G [S735A]) in exon 15 of the human KIBRA gene to affect cognitive performance, and to be in almost complete linkage disequilibrium with rs17070145. The identified SNPs encode variants of the KIBRA C2 domain with distinct Ca2 þ dependent binding preferences for monophosphorylated phosphatidylinositols likely due to differences in the dynamics and folding of the lipid-binding pocket. Our results further implicate the KIBRA protein in higher brain function and provide direction to the cellular pathways involved. Translational Psychiatry (2013) 3, e272; doi:10.1038/tp.2013.49; published online 18 June 2013 Introduction modules.1,4,11 C2 domains were initially identified as Ca2 þ - KIBRA (also called WWC1 for WW-and-C2-domain–contain- sensitive lipid-binding motifs in PKC, but are also found in various other proteins including the family of synaptotagmins ing-protein-1) represents a cytoplasmic protein highly 12 expressed in kidney and brain that was recently linked to and phosphoinositide-specific phospholipase C. In contrast higher brain functions,1–5 presumably based on differences in to other lipid-binding motifs (for example, Pleckstrin homology hippocampal processing.6,7 Intriguingly, a genome-wide domains, Bin–Amphiphysin–Rvs domains, Phox-homology screen for candidate genes affecting human cognitive domains, Fab-YOTB-Vac1-EEA1 domains), C2 domains functions and memory formation revealed an intronic single- display a unique structure that lacks a defined lipid-binding 13 nucleotide polymorphism (SNP; rs17070145, residing in the pocket and a conserved cationic patch. Whereas the 2 þ ninth intron of the human KIBRA gene) that was associated majority of known C2 domains have a role in Ca -dependent with superior performance in episodic memory tasks.5 vesicle membrane association, these protein modules are 2 þ Although a number of subsequent studies reproduced and also involved in Ca -insensitive membrane targeting as well 13 confirmed the association between rs17070145 SNP and as in intracellular protein–protein interactions. Although the cognitive performance in different cohorts,4–6,8 including a function of the KIBRA C2 domain in membrane association meta analysis9 and a putative protective association against has not been analyzed yet, a recent study in null-mutant mice Alzheimer’s disease,10 the impact of the intronic variant on demonstrated that KIBRA is involved in the regulation of the KIBRA protein function remains elusive. vesicle-based turnover of postsynaptic a-amino-3-hydroxy-5- KIBRA is a member of the WWC family of proteins that was methyl-4-isoxazolepropionic acid (AMPA) receptors in the initially identified to interact with the actin-binding protein hippocampus.7 Additional studies suggested that KIBRA may dendrin1 as well as with the atypical protein kinase C z also function in other cellular processes including regulation of (PKCz).2 KIBRA contains two amino terminal WW domains endosomal vesicle sorting,14 directed cell migration11,15 and that are known to bind to PPxY motifs in target molecules (for establishment of cell polarity.16 example, dendrin and synaptopodin), a C2 domain and a In this study, we describe the identification of two novel carboxyterminal-binding motif for PSD-95/Discs-large/ZO-1 adjacent SNPs in the human KIBRA gene that results in 1Internal Medicine D, Department of Nephrology, Hypertension and Rheumatology, University Hospital Mu¨nster, Mu¨nster, Germany; 2Department of Anatomy and Molecular Neurobiology, University Mu¨nster, Mu¨nster, Germany; 3Institute of Epidemiology and Social Medicine, University of Mu¨nster, Mu¨nster, Germany; 4Department of Neurology, University Hospital Mu¨nster, Mu¨nster, Germany; 5Institute of Sports Medicine, University of Mu¨nster, Mu¨nster, Germany; 6Institute of Medical Biochemistry, Center for Molecular Biology of Inflammation, Mu¨nster, Germany; 7Institute of Biochemistry, University of Mu¨nster, Mu¨nster, Germany; 8Institute of Anatomy and Cell Biology, University Ulm, Ulm, Germany and 9Howard Hughes Medical Center, John Hopkins University, Baltimore, MD, USA Correspondence: Dr J Kremerskothen, Internal Medicine D, Department of Nephrology, Hypertension and Rheumatology, University Hospital Mu¨nster, Albert- Schweitzer-Campus 1, 48149 Mu¨nster, Germany. E-mail: [email protected] 10These authors contributed equally to this study. 11Current address: Department of Neurology, Center for Stroke Research Berlin, Cluster of Excellence NeuroCure, Charite Universita¨tsmedizin, Berlin, Germany. Keywords: C2 domain; human cognition; KIBRA; membrane binding; phosphatidylinositols Received 3 January 2013; revised 22 April 2013; accepted 23 April 2013 Human exonic KIBRA variants affect lipid binding and cognitive performance K Duning et al 2 structural changes within the C2 domain of the encoded genotype. Effects of the KIBRA rs3822660 on the cognitive protein. Genetic analysis demonstrated a striking linkage domains were tested with multivariable analyses of variance between the exonic SNPs affecting the C2 domain and the using the general linear model procedure in SAS 9.2 well-characterized intronic SNP known to be associated with (www.sas.com/software/sas9/ ). The model included the human cognitive performance. A biochemical characteriza- following covariates: age, gender, education, history of arterial tion revealed a Ca2 þ -dependent-binding specificity of the hypertension, intake of antihypertensive medication, history KIBRA C2 domain for monophosphorylated phosphatidylino- of diabetes, lifestyle index, history of depression and intake sitols. Finally, the identified exonic SNPs were shown to affect of antidepressive medication. Allelic associations of KIBRA the lipid-binding specificities of the C2 domain accompanied rs3822660 and rs17070145 were tested using a w2 test. by changes in the structural dynamics of the protein motif. In conclusion, our data elucidate the function of the KIBRA C2 Reagents. Standard highest-grade reagents were from domain in membrane association and present for the first time Sigma-Aldrich (Deisenhofen, Germany). a linkage between natural KIBRA protein variants and cognitive performance in humans. Antibodies. Monoclonal antibodies directed against glutathion-S-transferase (GST) were purchased from Materials and methods Sigma-Aldrich. Horseradish peroxidase-conjugated second- ary antibodies were purchased from Dianova (Hamburg, Population-based cohort study and neuropsychological Germany). testing. We used clinical, neuropsychological and genetic data of 545 participants aged above 50 years from the Preparation of GST fusion proteins. Human KIBRA population-based SEARCH Health study (Systematic eva- cDNA1 (Acc. No. AF506799) fragments were cloned into a luation and alteration of risk factors for cognitive health). modified pGEX-KG vector. The constructs pGEX-KG, pGEX- Details of the study protocol were described elsewhere.8,17 KG-human KIBRA C2 wild-type (WT; aa 661–776), pGEX- Sixteen single-neuropsychological tests were condensed into KG-KIBRAC2 var. (I734/A735) were used for the isopropyl four composite scores using principal component analysis b-D-1-thiogalactopyranoside-induced expression of GST (varimax rotation and including coefficients with absolute fusion proteins in Escherichia coli BL21 as previously values above 0.4), reflecting the cognitive domains of verbal described.11 GST and GST-C2 fusion proteins were purified memory, word fluency, working memory and executive from bacterial lysates using glutathione sepharose beads functions/psychomotor speed.8,17 (Amersham Bioscience, Freiburg, Germany) according to the manufacturer’s instructions. Identification and genotyping of KIBRA polymorphisms. Genomic DNA was extracted from white blood cells using Western blotting. Western blotting experiments were per- QIAamp DNA Blood Kit (Qiagen, Hilden, Germany). We formed using standard techniques.11 Antibodies were diluted screened 8773 bp for genetic variants using single-strand in blocking buffer containing 5% skim milk powder in conformation polymorphism analysis and subsequent direct phosphate-buffered saline (PBS)/Tween. After washing, the sequencing (ABI 3730). As both exon 15 SNP variants membranes were incubated with respective secondary (rs3822660 and rs3822659) were in complete linkage antibodies coupled to horseradish peroxidase. Finally, the disequilibrium with one another (tested by direct sequencing membranes were washed and developed using the in 100 individuals), only the frequency of exonic SNP variant Lumi-Light chemiluminescence detection kit