Human brain samples
We conducted an observational, case-control study comparing postmortem hippocampal samples from AD patients and control hippocampus. Frozen postmortem hippocampal samples from 30 Alzheimer’s disease (AD) cases and 12 controls were provided by the Navarrabiomed Brain Bank. After death, half brain specimens from donors were cryopreserved at −80 °C.
Formalin-fixed (4 %), paraffin-embedded tissue from mid-hippocampus region was cut (5-mm sections) and placed on StarFrost Microscope Slides. After deparaffinizing, endogenous peroxidase activity was quenched by incubation with 0.3 % (vol/vol) H2O2 in methanol for 30 minutes at room temperature. Antigen retrieval was performed by soaking the sections in 10-mM citrate buffer pH 6.0, heated and boiled for 10 minutes in a microwave oven.
Assessment of β-amyloid deposition was carried out with a mouse monoclonal (S6F/3D) anti β-amyloid antibody (Leica Biosystems Newcastle Ltd, Newcastle upon Tyne, UK) which identified the presence of amyloid pathology.
Evaluation of neurofibrillary pathology was performed with a mouse monoclonal antibody anti-human PHF-TAU, clone AT-8, (Tau AT8) (Innogenetics, Ghent, Belgium), which identified hyperphosphorylated tau (p-tau)  in the shape of dystrophic neurites and neurofibrillary tangles. The reaction product was visualized using an automated slide immunostainer (Leica Bond Max) with Bond Polymer Refine Detection (Leica Biosystems, Newcastle Ltd). Omission of the primary antibody was used as a negative control for all stainings.
Neuropathological examination was completed following the usual recommendations  and assessment of AD was performed according to the updated National Institute on Aging-Alzheimer's Association guidelines . Samples were further classified, based on the ABC score, into four groups: control (n = 12), initial AD (including A1B1C1, A1B2C1, A1B2C2, and A1B2C3 scores) (n = 9), intermediate AD (including A2B2C2, A2B2C3, and A2B3C3 scores) (n = 7) and advanced AD (including A3B2C1, A3B2C3, A3B3C2, and A3B3C3 scores) (n = 14) (Additional file 1: Figure S1). Importantly, to avoid spurious associations, those individuals showing coexisting protein deposits different from p-tau or β-amyloid were not eligible for the study. This approach maximizes chances of finding true associations with AD, even though reducing the final sample size. Neuropathological and demographic features of subjects, including age, gender, ABC score and postmortem interval are listed in Additional file 2: Table S1.
Quantitative assessment of β-amyloid and p-tau deposits in brain tissues
In order to quantitatively assess the β-amyloid and p-tau burden for further statistical analysis we applied a method to quantify protein deposits. This method generates a numeric measurement that reflects the extent of β-amyloid and p-tau deposition. Sections of the mid hippocampus were examined after performing immunostaining with anti β-amyloid and anti p-tau antibodies as described above. Three pictures were obtained for each immunostained section using an Olympus BX51 microscope at 10x magnification power. Morphological deposits of β-amyloid, as described by Braak and Braak (neuritic, immature and compact plaque), were manually determined and those areas were further edited and analyzed with ImageJ software  (Additional file 1: Figure S2). Next, β-amyloid plaque count referred to as amyloid plaque score (APS) and total area of β-amyloid deposition were automatically measured by ImageJ and averaged for each section. Regarding p-tau deposit, pictures were also analyzed with ImageJ software, by adjusting in the threshold color hue (maximum range: 45–197), saturation (maximum range: 7–243) and brightness (maximum range: 50–195), in order to obtain an averaged quantitative measure of the global p-tau deposit for each section (Additional file 1: Figure S2).
CRTC1 methylation profiling by bisulfite cloning sequencing
Genomic DNA was isolated from hippocampal tissue by standard methods . Next, 500 ng of genomic DNA was bisulfite converted using the EpiTect Bisulfite Kit (QIAGEN, Redwood City, CA, USA) according to the manufacturer’s instructions. Two promoter regions (Prom1 and Prom2) within the CRTC1 gene were amplified by PCR (Additional file 1: Figure S3). Genomic coordinates were obtained from GRCh37/Hg19 assembly. Primer pair sequences were designed by MethPrimer  and are listed in Additional file 2: Table S2. PCR products were cloned using the TopoTA Cloning System (Invitrogen, Carlsbad, CA, USA) and a minimum of 12 independent clones were sequenced for each examined subject and region. Methylation graphs were obtained with the QUMA software .
CRTC1 mRNA expression analysis
Total RNA was isolated from the 42 hippocampus 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 a 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 an Applied 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Sequences of primer pair were designed using the Real Time PCR tool (IDT, Coralville, IA, USA) and are listed in Additional file 2: Table S2. Relative expression level of CRTC1 mRNA in a particular sample was calculated as previously described  and the geometric mean of GAPDH and ACTB genes were used to normalize expression values.
Statistical data analysis
Statistical analysis was performed with SPSS 21.0 (IBM, Inc., USA). Data represent the mean ± SEM or median (range), depending on the type of variable. Differences with p-value < 0.05 were considered significant. Statistical significance for expression and bisulfite intergroup differences were assessed by the Mann-Whitney U test and the Kruskal–Wallis test. Spearman’s rank correlation coefficient was used to determine correlation between AD-related pathology and methylation levels. When multiple comparisons were performed, as was the case of correlation between methylation levels and AD-related pathological burden, a Bonferroni correction was applied and the significance threshold was set at p-corrected value = 0.008. GraphPad Prism version 6.00 for Windows (GraphPad Software, La Jolla, CA, USA) was used to draw the graphs except for methylation figures that were obtained by QUMA software.
Ethics, consent and permissions
Our study was carried out in accordance with the Declaration of Helsinki and handling of human brain samples was performed according to the current Spanish national legislation (Law 14/2007 and Royal Decree RD1716/2011). The Ethics Committee of the “Complejo Hospitalario de Navarra” approved the use of human subjects for this study (90/2014). Written informed consent was obtained from all subjects or next of kin, previous to brain donation, to perform research projects related to neurodegenerative conditions. The consent form is held by the authors’ institution and is available for review by the Editor-in-Chief.