Study design and subjects
Data from 215 individuals with EOAD from Xuanwu Hospital were evaluated in this observational study and compared with healthy cognitively normal controls (Additional file 1: Table S1). Each individual underwent a neuropsychological examination, magnetic resonance imaging (MRI); cerebrospinal fluid analysis was performed for a subset of individuals (n=115) in which levels of Aβ42, Aβ40, Tau and p‐Tau181 were assessed. The criteria for the recruited EOAD patients were set as follows: ①met the National Institute of Neurological and Communicative Disorders and the Stroke and the Alzheimer Disease and Related Disorders Association (NINCDS/ADRA)[12] or the National Institute on Aging-Alzheimer’s Association (NIA-AA) diagnostic criteria [13]; ②the onset age of affected individuals was below 55 years; ③no known pathogenic mutations in PSEN1, PSEN2, APP, MAPT or GRN genes. Healthy controls were cognitively normal; amnesia was not present, Mini-Mental State Examination (MMSE) scores were higher than the appropriate cutoff for dementia, Clinical Memory Scale scores ≧ 90, and global Clinical Dementia Rating scores were equal to 0. Additional 513 DNA samples from patients with EOAD and 4449 healthy controls were collected at the Xuanwu Hospital from 2015 to 2020 and used for Sanger sequencing. Signed informed consent was provided by all the patients and control subjects. The study protocol was approved and monitored by the Ethics Committee of Xuanwu Hospital.
Relative PDE11A protein levels were quantified using immunoblot in samples from fresh frozen postmortem parietal lobe tissue of six AD patients and six cognitively healthy controls, recruited at Washington University in St. Louis Charles F. and Joanne Knight Alzheimer’s Disease Research Center Brain Bank (Knight ADRC) and approved by the institutional review board of Washington University. Written informed consent for brain autopsy was obtained from all participants or their legal representatives.
WES and data analysis
Whole-genomic DNA was obtained from the peripheral blood of all participants. Briefly, the exome was enriched using Agilent SureSelect Human All Exon V5 Kit (Agilent Technologies, Santa Clara, CA, USA) and analyzed using an Illumina HiSeq 2500 (150-bp paired-end, Illumina, San Diego, CA, USA). After conducting quality control, high-quality paired-end reads were mapped to the human genome build GRCh37 using Burrows-Wheeler Aligner software. Verita Trekker was employed to identify variants. Enliven® and ANNOVAR were utilized to perform annotation for Variant Call Format, and variants were selected according to an autosomal dominant model. Nonsynonymous exonic or close-to splice-site variants with minor allele frequency <0.01% were prioritized for analysis. Variant pathogenicity was evaluated using SIFT, Polyphen-2, MutationTaster, M-CAP, CADD, LRT, PROVEAN, DANN, VEST3, fathmm-MKL, GERP, SiPhy, phastCons, and phyloP tools. Candidate variants were prioritized based on OMIM, MGI, GO, KEGG, ACGM, UKBiobank PheWeb web-based tool and the published literature.
Annotation of PDE11A variants
InterPro was used to predict domain maps of the PDE11A protein [14]. DNAMAN software (Lynnon Corporation, Quebec, Canada) was applied for multiple sequence alignment. A homology model of the wildtype PDE11A structure was built in MODELLER software using the crystal structure of the phosphodiesterase template (PDB: 3IBJ) [15]. Based on the homology modeling structure of wildtype PDE11A, Arg202His- and Leu756Gln-mutated proteins were generated with Pymol software [16]. AMBER software was used for molecular dynamics simulations for wildtype PDE11A and the Arg202His- and Leu756Gln variants [17]. To investigate in which major cell type the PDE11A gene is expressed, we queried the gene in a publicly available single-cell/nuclei RNA-seq dataset of AD cohorts of human brain tissues (http://adsn.ddnetbio.com/) [18].
Generation of PDE11A variants
Human wild-type PDE11A cDNA (NM_016953) was cloned into the pCDNA3.1-EGFP vector. Site-directed PDE11A mutagenesis (p.Arg202His and p.Leu756Gln) was performed using a KOD-Plus-Mutagenesis kit (Stratagene, La Jolla, CA, USA). For short hairpin RNA (shRNA), 3 optimal targeting human PDE11A and 1 scrambled control were designed (Additional file 1: Table S2). Lentiviral particles were generated by co-transfecting the transfer vector (psh2-u6-egfp-puro containing shRNA or scramble, pCDH-CMV-MCS-EF1-GFP-T2A-Puro containing WT PDE11A, PDE11A p.Arg202His or PDE11A p.Leu756Gln) and the packaging vectors into HEK293T cells.
Cell culture and transfection
HEK293 cells stably transfected with human WT APP695 (cells termed HEK293-APP695) were propagated in DMEM with 10% fetal bovine serum (FBS) and 300 µg/mL G418 (Thermo Fisher Scientific, Waltham, MA, USA). HEK293 cells were cultured in DMEM with 10% FBS. Primary cortical neurons were isolated from E17-E18 C57BL/6J mouse embryos. Tissues were cut into tiny pieces and digested in 0.125% trypsin (Thermo Fisher Scientific) supplemented with DNase I (100 mg/mL; Sigma-Aldrich, Saint Louis, MO, USA) for 15 minutes at 37℃. The cells were then dissociated into a single-cell suspension and filtered through cell strainers. The cell pellet was resuspended, seeded onto 6-well plates-coated with poly-D-lysine (Sigma-Aldrich) and cultured in neurobasal medium supplemented with 2% B27, L-glutamine (2 mM), and 1% penicillin/streptomycin (Thermo Fisher Scientific). Transfection was performed using Lipofectamine 3000 Reagent (Thermo Fisher Scientific).
Real-time quantitative PCR
Total RNA was purified from freshly harvested cells using an RNeasy Mini Kit (Thermo Fisher Scientific) and TRIzol (Thermo Fisher Scientific). Reverse transcription was carried out using a SuperScript First-Strand Synthesis Kit (Thermo Fisher Scientific), and total mRNA was measured by real-time quantitative polymerase chain reaction (PCR). Diluted cDNA templates, upstream and downstream primers and SYBR Green I Master mix (Thermo Fisher Scientific) were mixed in a 20-μL volume, and reactions were performed using the StepOne Plus real-time PCR platform. Human GAPDH (forward, 5’- ACAGCCTCAAGATCATCAGCAAT-3’; reverse, 5’- GATGGCATGGACTGTGGTCAT-3’) was used as the internal reference gene, and relative expression levels of the PDE11A gene were calculated using the 2-ΔΔCt method. PDE11A q-PCR primers were as follows: forward, 5’- CTGGAGTGGATTGATAGCATCTG-3’; reverse, 5’- CAGTCGTTTTTGGTGTAGCTCTT-3’.
Western blotting
Briefly, cells were lysed in RIPA buffer containing protease inhibitor cocktails and phosphatase inhibitors. The cell lysate was pelleted by centrifugation, and the protein concentration was measured by the BCA assay. Samples were separated by 10% Tris-glycine SDS-PAGE and then electroblotted onto a PVDF membrane. After blocking in 5% nonfat dry milk, the membrane was probed with appropriate primary antibodies (listed in Additional file 1: Table S3) overnight at 4°C. The next day, the membrane was washed with Tris-buffered saline containing Tween-20 (TBS-T) and incubated with horseradish peroxidase-linked secondary antibodies (1:5000; Santa Cruz Biotechnology) for 1 h at room temperature. After washing, the proteins were visualized by chemiluminescence.
ELISA
The concentrations of secreted Aβ40 and Aβ42 in conditioned medium were quantified using commercial enzyme-linked immunosorbent assay kits (IBL International, Hamburg, Germany). Levels of cAMP in cell lysates were determined using a commercially available assay kit (R&D Systems, Minneapolis, MN, USA) according to the protocol provided by the manufacturer.
Statistical analysis
Quantification data were obtained from three independent repeats. Two-tailed unpaired Student’s t test or one-way analysis of variance was performed using the SPSS 21.0 software package (SPSS, Chicago, IL, USA). To determine if the candidate risk gene is enriched in AD, a Fisher’s exact test on the mutation count data was performed. A p value less than 0.05 was used to indicate a statistically significant difference.