Population descriptives
Since publication of our previous data [3], 74 additional Alzheimer patients (mean age of onset 82 ± 11.3 years, %female 56.3) were screened for CLU coding exons. Patients were ascertained at the Memory Clinic of the ZNA Middelheim and Hoge Beuken, Antwerp, Belgium (S.E.,P.P.D.D.) in the frame of a prospective study of neurodegenerative and vascular dementia in Flanders, the Dutch-speaking region of Belgium [20, 21], and at the Memory Clinic of the University Hospitals of Leuven (UHL), Gasthuisberg, Leuven, Belgium (M.V.,R.V.) as part of a prospective study on the molecular genetics of cognitive impairment using the same clinical assessments and biosampling schemes. Each patient underwent a neuropsychological examination and structural and/or functional neuroimaging [22].
Genetic screening
We designed a PCR-based target enrichment assay using MASTR™ technology (Multiplicom, Niel, Belgium) including fourteen CLU fragments (3.3 kb) covering the 9 CLU coding exons, flanking intron-exon boundaries and UTR regions (NM_001831.2 and NM_20339.1). Primers for multiplex PCR were designed using mPCR. Multiplex PCR was performed for amplification of the target region, followed by purification of the pooled amplicon library using Agencourt AMPureXP beads (Beckman Coulter, CA, USA). Patient-specific barcodes (Multiplicom, Niel, Belgium) were incorporated during a universal PCR step. Barcoded samples were pooled prior to bridge amplification and sequencing on MiSeq, using the reagent kit v2, generating 250 bp paired-end reads (Illumina, San Diego, CA, USA). Data was processed with the Illumina pipeline and Genomecomb’s [23] data annotation and filtering package, including read alignment against build GRCh37/hg19 and mapping steps with Burrows-Wheeler Aligner (BWA) [24] and variant calling with Genome Analysis Toolkit (GATK) [25]. Rare CLU variants were filtered on impact (frameshift, nonsense, non-synonymous and splice variants), low-frequency in 1000 Genomes Project [26] (frequency <0.01) and European American samples of the NHLBI GO Exome Sequencing Project (ESP6500 data set) (frequency <0.01). Filtered variants were visually inspected using interactive genomics viewer (IGV). In total, 78.5 % of the target region had a coverage ≥ 50x in all samples. Rare CLU variants were validated using Sanger sequencing as described before [3]. The effects of non-synonymous substitutions on protein function were evaluated with PolyPhen2 [5], Pmut [27] and SIFT [4] programs.
cDNA constructs
The effect of CLU mutations on CLU secretion was modeled using a synthetic cDNA construct of NM001831.3 (GeneCopoeia), starting from the second ATG codon for exclusive expression of the short secreted isoform (449 AA) [10]. Using attB sites containing primers, CLU ORF was amplified and subsequently recombined into the pDONR221 vector using the Gateway Cloning System (Life Technologies). In total, 11 CLU missense and small insertion-deletion mutations were introduced in this pDONR construct by site-directed mutagenesis using Kapa High Fidelity Polymerase. All sequences were verified using Sanger sequencing and subsequently transferred to pEGFPC-GW, an in-house designed gateway-compatible destination vector with C-terminal EGFP tag (without stopcodon), to allow the expression of CLU-EGFP fusion proteins.
In addition, the ORFs from wild-type and CLU mutants (with stop codon) were cloned into the pEF/FRT/V5 Gateway vector (Life Technologies) and the pLENTI6/V5 vector (Life Technologies) to generate stable CLU expressing cell lines by Flp recombinase-mediated integration, or by lentiviral vector transduction, respectively.
Cell culture
HeLa cells (Henrietta Lack cells, cervical cancer cells; ATCC) were maintained in Minimal Essential Medium (MEM) supplemented with 10 % (v/v) fetal bovine serum, 100 U/ml penicillin/streptomycin at 37 °C in a humidified 5 % CO2 atmosphere.
Flp-In HEK293 cells (Life technologies), stable Flp-In cell lines and transduced HEK293T cells expressing the CLU gene were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with Zeocin (400 μg/ml, Life Technologies, HygromycinB (200 μg/ml, Ducheva Biochemie) or Blasticidin (4 μg/ml, Invivogen) respectively.
Lentiviral vector production
Human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors were produced by a standardized three plasmid transient transfection protocol [28]. Briefly, HEK293T cells were transfected with a second-generation packaging plasmid pCMV dR8.91, an envelope plasmid encoding the VSV-G protein, and the pLenti6/V5 transfer plasmid carrying the CLU CDS with mutations of interest using calcium phosphate as transfection reagent. The cell supernatant containing the lentiviral particles was harvested 48 h post-transfection, filtered using a 0.45 μm PVDF membrane (Millex), aliquoted and stored at −20 °C. The total amount of LV was determined by measuring the lentivirus associated p24 antigen content using a commercial enzyme linked immunosorbent assay (HIV-1 p24 ELISA, Cell Biolabs).
Transfections and lentiviral transductions
Nearly confluent HeLa cells were transfected in 35 mm dishes with plasmids encoding the wild-type and mutant CLU-EGFP fusion protein using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s recommendations.
Secondly, to generate stable CLU overexpressing HEK Flp-In cell lines, 1.2×106 cells per 100 mm plate were co-transfected with a total of 10 μg DNA of the pEF/FRT/V5 and pOG44 plasmids at a 1/9 ratio, using Lipofectamine 2000 (Life technologies). Cells were selected with HygromycinB antibiotic after 48 h. Integration of the CLU wild-type and mutant cDNA was sequence-verified on gDNA isolated from these cells (DNA blood minikit, Qiagen).
Thirdly, HEK293T cells (HCL4517, Fermentas) were transduced for 24 h with various LV vectors normalized to P24 antigen (infectious titer of 2.107 TU). Selection in DMEM media containing Blasticidin resulted in stable polyclonal CLU expressing HEK293T cell lines.
Live cell imaging and immunocytochemistry on HeLa cells
Examination of living (unfixed) HeLa cells was performed 48 h after transfection with CLU-EGFP expressing constructs. Cells grown on glass bottom imaging dishes (MatTek Corporation) were imaged on a Zeiss LSM 700 confocal microscope equipped with a stage top incubator (Pecon Gmbh), maintaining conditions of 37 °C and 5 % CO2.
For immunocytochemical staining, HeLa cells grown on glass coverslips were fixed in 3 % paraformaldehyde (PFA) at 48 h after transfection with the CLU-EGFP constructs. After washing with PBS and blocking for 1 h with Donkey Serum (1:500, Abcam, Cambridge) at 37 °C in PBT (PBS + 0.02 % Triton-X-100, 0.5 % BSA), cells were incubated for 1 h at room temperature with goat anti-EGFP antibody (1:2000, Abcam, Cambridge) together with a marker for either Golgi (rabbit anti-giantin, 1:1000, Covance, Rotterdam, The Netherlands) or ER (mouse anti-PDI, 1:100, Abcam, Cambridge). Subsequently, secondary antibody incubation for 1 h at room temperature (Donkey anti-Rabbit Alexa Fluor 594 and Donkey anti-Goat, both at 1:500, Life Technologies), F-actin cytoskeleton staining (Phalloidin conjugated to Alexa Fluor 647 (25 min, 1:30, Life Technologies) and nuclear DAPI staining (4’, 6-diamidino-2-phenylindol, 100 ng/ml, 10 min, Bio-Rad, Nazareth Eke, Belgium) was performed with intermittent washes in PBT. After a final wash step with PBS, sections were mounted using DAKO fluorescent mounting medium and stored at 4 °C in the dark before being subjected to fluorescence microscopy.
Image acquisition and quantification
Confocal images used for quantification (1892×1892, 106×106 nm2 pixels) were acquired on a Zeiss LSM 700 microscope with Zen 2009 software (Zeiss, Zaventem, Belgium), using a EC Plan-Neofluar 40x /1.30 Oil objective, while keeping identical acquisition settings (laser intensities, detector gain, channel settings). Images of EGFP-CLU and the organelle markers (ER or Golgi) were acquired in different tracks (serial frame scanning) to avoid any possible crosstalk between the channels. Images were acquired and quantified with genotype labels blinded for the investigators.
ImageJ [29] was used to measure both the relative EGFP intensity in the Golgi and the colocalization with the ER. In both cases the F-actin cytoskeleton staining was first used to manually delineate the regions of the cells, which were stored in ImageJ ROI manager files. For quantification of the relative EGFP signal in the Golgi, the Golgi region was segmented by using an automatic threshold followed by filtering of the binary mask. The ratio of mean EGFP intensity in and outside the Golgi was calculated, corrected for the background signal, and based upon the mean intensity in the Golgi and the mean intensity outside Golgi in the remaining cell region. A fixed cutoff threshold of EGFP mean intensity outside the Golgi region was used to exclude cells with low EGFP signal. A minimum of 20 cells per genotype were used to perform quantification. To quantify EGFP colocalization with PDI, the ImageJ Coloc2 plugin was used to measure the Pearson’s correlation coefficient between the two fluorescent channels in individual cells. For both types of measurements (ER and Golgi), ImageJ scripts (macro) were written and used to process and analyze all images (and all genotypes) in batch.
Protein extraction
Both HEK Flp-In and HEK293T cells were grown to confluence in a 100 mm dish format. After washing with Dulbecco’s Phosphate-buffered saline (PBS), cell medium was replaced by serum-free OPTIMEM I medium (Life Technologies). Cell media and cell lysates were harvested 24 h later. One ml of cell medium, supplemented with the protease inhibitor AEBSF (1 mM final, Calbiochem), was centrifugated (15.000 g for 5 min, 4 °C). Supernatants were aliquoted and immediately stored at −80 °C. After an initial wash with PBS, cell lysates were prepared by collecting the cells with a cell scraper followed by lysis with 2 ml NP40 buffer (Life Technologies) supplemented with the protease inhibitor AEBSF. The cell lysates were incubated on ice for 10 min, centrifugated (15.000 g for 5 min, 4 °C) and immediately stored at −80 °C.
CLU ELISA
Secreted CLU from conditioned medium and CLU from cell lysates of HEK293T cells was quantified by a commercial CLU ELISA (Human Clusterin Elisa RD194034200R, Biovendor, Germany). All undiluted samples were run in duplicate. Besides the quality control samples provided by the manufacturer, inter-assay samples were run in each experiment. Three independent experiments were performed per mutation. Normalization was performed using TGF-ß1. Because of a slight immunoreactive response of the sample matrix, as observed in measurement of nontransfected cells, the average of the immunoreactive response was computed across three experiments and subtracted from the observed data for each mutation.
Western blot and immunodetection
Protein concentrations were determined with a bicinchoninic acid colorimetric assay (BCA) (ThermoScientific). Equal amounts of protein (20 μg) were loaded and separated on NuPAGE® 10 % Bis-Tris gels (Life Technologies). Proteins were transferred onto polyvinylidene difluoride membranes (Hybond P, Amersham Biosciences). After blotting, membranes were blocked in 5 % skimmed milk in PBS containing 0.1 % Tween® 20 (Merck) (PBT) for 2 h at room temperature and probed overnight with a mouse anti-clusterin antibody (1:200, B-5 Santa Cruz recognizing the CLU β–chain) at 4 °C. Equal loading was controlled using a mouse α-tubulin antibody (1:10000, GeneTex). Immunodetection was achieved with horseradish peroxidase (HRP)-conjugated sheep anti-mouse antibody (1:10000, BioSciences) and an ECL Plus™ chemiluminescent detection system (GE Healthcare). Bands were visualized and quantified using ImageQuant LAS 4000 equipment (GE Healthcare Life Sciences).
Statistical analysis
For quantification of the colocalization experiments, Pearson’s R correlation value was determined between ER and CLU reactivity (using ImageJ Coloc2 plugin). For Golgi measurements, ratios of mean intensity inside Golgi versus outside Golgi apparatus were determined. Means of the independent measurements were compared to wt using ANOVA (Tukey HSD test). Extreme outliers (n = 23 from 568 measurements), defined as values with more than 3 standard deviations from the mean were removed from the analysis.
For quantification of western blot data, CLU protein levels in cell media and lysates were evaluated through quantification of band intensities with ImageQuant software (GE Healthcare Life Sciences). Independent measures of CLU intensity ratios between cell lysates and cell media were used for further analysis. Cochran-Mantel-Haenszel test was used to compare ratios of CLU in cell medium versus lysate between CLU wt and mutant genotypes. Bonferroni correction for 11 different constructs is applied. P-values <0.0045 are considered significant. Results are represented as mean ratios of CLU in cell medium and lysate (Fig. 4, Additional file 4: Figure S4).
Consent
All participants and/or legal guardians gave written informed consent for participation in genetic studies, and for publication of study results, in an anonymized manner. Clinical study protocol and informed consent forms for ascertainment were approved by the Ethics Committee of the respective hospitals at the sampling sites. Genetic study protocols and informed consent forms were approved by the Ethics Committees of the University of Antwerp and the University Hospital of Antwerp, Belgium.