Human hippocampal samples and neuropathological examination
Brain hippocampal samples from 30 AD patients and 12 controls were provided by Navarrabiomed Brain Bank. After death, half brain specimens from donors were cryopreserved at − 80 °C. Neuropathological examination was completed following the usual recommendations  and according to the updated National Institute on Aging-Alzheimer’s Association guidelines . Assessment of β-amyloid deposition was carried out by immunohistochemical staining of paraffin-embedded sections (3–5 μm thick) with a mouse monoclonal (S6F/3D) anti β-amyloid antibody (Leica Biosystems Newcastle Ltd., Newcastle upon Tyne, UK). Evaluation of neurofibrillary pathology was performed with a mouse monoclonal antibody anti-human PHF-TAU, clone AT-8, (Tau AT8) (Innogenetics, Gent, Belgium), which identifies hyperphosphorylated tau (p-tau) . The reaction product was visualized using an automated slide immunostainer (Leica Bond Max) with Bond Polymer Refine Detection (Leica Biosystems, Newcastle Ltd.).
To avoid spurious findings related to multiprotein deposits, “pure” AD cases with deposits of only p-tau and β-amyloid were eligible for the study and controls were free of any pathological protein aggregate. This approach maximizes chances of finding true associations with AD, even though reducing the final sample size. A summary of characteristics of subjects included in this study is shown in Additional file 1: Table S1. AD subjects were older than controls (82.3 ± 11.3 versus 50.7 ± 21.5; p value < 0.01), and no differences were found regarding gender (p value = 0.087). The postmortem interval (PMI) were not significantly different between groups (8.2 ± 4.4 h in the control group versus 7.9 ± 7.1 h in the AD group; p value = 0.91).
PLD3 mRNA expression analysis by RT-qPCR
Total RNA was isolated from hippocampal homogenates using RNeasy Lipid Tissue Mini kit (QIAGEN, Redwood City, CA, USA), following the manufacturer’s instructions. Genomic DNA was removed with recombinant DNase (TURBO DNA-free™ Kit, Ambion, Inc., Austin, TX, USA). RNA integrity was checked by 1.25% agarose gel electrophoresis under denaturing conditions. Concentration and purity of RNA were both evaluated with NanoDrop spectrophotometer. Only RNA samples showing a minimum quality index (260 nm/280 nm absorbance ratios between 1.8 and 2.2 and 260 nm/230 nm absorbance ratios higher than 1.8) were included in the study. Complementary DNA (cDNA) was reverse transcribed from 1500 ng total RNA with SuperScript® III First-Strand Synthesis Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) after priming with oligo-d (T) and random primers. RT-qPCR reactions were performed in triplicate with Power SYBR Green PCR Master Mix (Invitrogen, Carlsbad, CA, USA) in a QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and repeated twice within independent cDNA sets. Sequences of primer pair were designed using Real Time PCR tool (IDT, Coralville, IA, USA) and are listed in Additional file 1: Table S2. Relative expression level of PLD3 mRNA in a particular sample was calculated as previously described  and ACTB gene was used as the reference gene to normalize expression values.
PLD3 protein expression analysis by Western blot
Human hippocampus tissue from patients and control samples was lysed with 100 μL lysis buffer containing urea, thiourea, and DTT. After centrifugation at 35.000 rpm for 1 h at 15 °C, extracted proteins were quantified following the Bradford-Protein Assay (Bio-Rad, Hercules, CA, USA) by using a spectrophotometer.
Next, 5 μg of protein per sample were resolved in 4–20% Criterion TGX stain-free gels (Bio-Rad) and electrophoretically transferred onto nitrocellulose membranes using a Trans-blot Turbo transfer system (25 V, 7 min) (Bio-Rad). Equal loading of the gel was assessed by stain-free digitalization and by Ponceau staining. Membranes were probed with rabbit anti-human PLD3 primary antibody (Sigma-Aldrich; 1:250) in 5% nonfat milk and incubated with peroxidase-conjugated anti-rabbit secondary antibody (Cell Signaling; 1:2000). Immunoblots were then visualized by exposure to an enhanced chemiluminescence Clarity Western ECL Substrate (Bio-Rad) using a ChemidocMP Imaging System (Bio-Rad). Expression levels of PLD3 were standardized by the corresponding band intensity of GAPDH (Calbiochem; 1:10000).
Quantitative assessment of β-amyloid and p-tau deposits in hippocampal samples
In order to quantitatively assess the β-amyloid and p-tau burden for further statistical analysis, we applied a method to quantify protein deposits, as described in detail elsewhere . In brief, hippocampal sections were examined after performing immunostaining with anti β-amyloid and anti p-tau antibodies. Focal deposit of β-amyloid, including neuritic, immature, and compact plaques , was analyzed with the ImageJ software. Moreover, β-amyloid plaque count, referred to as amyloid plaque score (APS), was measured. Finally, p-tau deposit was also analyzed with ImageJ software in order to obtain an average quantitative measure of the global p-tau deposit for each section.
PLD3 methylation measurement by pyrosequencing
Genomic DNA was isolated from frozen hippocampal tissue by phenol-chloroform method . Next, 500 ng of genomic DNA was bisulfite converted using the EpiTect Bisulfite Kit (Qiagen, Redwood City, CA, USA) according to the manufacturer’s protocol. Primers to amplify and sequence two promoter regions of PLD3 were designed with PyroMark Assay Design version 184.108.40.206 (Qiagen) (Additional file 1: Table S2), and PCR reactions were carried out on a VeritiTM Thermal Cycler (Applied Biosystems, Foster City, CA, USA). Next, 20 μl of biotinylated PCR product was immobilized using streptavidin-coated sepharose beads (GE Healthcare Life Sciences, Piscataway, NJ, USA) and 0.3 μM sequencing primer was annealed to purified DNA strands. Pyrosequencing was performed using the PyroMark Gold Q96 reagents (Qiagen) on a PyroMark™ Q96 ID System (Qiagen). For each particular cytosine-phosphate-guanine dinucleotide (CpG), methylation levels were expressed as percentage of methylated cytosines over the sum of total cytosines. Unmethylated and methylated DNA samples (EpiTect PCR Control DNA Set, Qiagen) were used as controls for the pyrosequencing reaction.
PLD3 methylation validations by bisulfite cloning sequencing
Bisulfite-converted genomic DNA was used to validate pyrosequencing results. Primer pair sequences were designed by MethPrimer  and are listed in Additional file 1: Table S2. PCR products were cloned using the TopoTA Cloning System (Invitrogen, Carlsbad, CA, USA), and a minimum of 10–12 independent clones were sequenced for each examined subject and region. Methylation graphs were obtained with QUMA software .
Statistical data analysis
Statistical analysis was performed with SPSS 21.0 (IBM, Inc., USA). Before performing differential analysis, we checked that all continuous variables showed a normal distribution, as per one-sample Kolgomorov-Smirnov test and the normal quantil-quantil (QQ) plots. Data represents the mean ± standard deviation (SD). Significance level was set at p value < 0.05. Statistical significance for PLD3 mRNA levels and pyrosequecing intergroup differences was assessed by T test. One-way analysis of variance (ANOVA) followed by Games-Howell post hoc analysis was used to analyze differences in the expression levels of PLD3 mRNA between Braak and Braak stage groups. A logistic regression model (ENTER method) was fit to assess the independent association of PLD3 mRNA levels with AD status, using gender and age as covariates. Kendall’s tau-b correlation coefficient was used to determine correlation between AD-related pathology and PLD3 mRNA expression levels. Difference between two bisulfite cloning sequencing groups was evaluated with Mann-Whitney U test. GraphPad Prism version 6.00 for Windows (GraphPad Software, La Jolla, CA, USA) was used to draw graphs except for methylation figures that were obtained by QUMA software.