Exome sequencing revealed PDE11A as a novel candidate gene for early-onset Alzheimer's disease

Abstract

To identify novel risk genes and better understand the molecular pathway underlying Alzheimer's disease (AD), whole-exome sequencing was performed in 215 early-onset AD (EOAD) patients and 255 unrelated healthy controls of Han Chinese ethnicity. Subsequent validation, computational annotation and in vitro functional studies were performed to evaluate the role of candidate variants in EOAD. We identified two rare missense variants in the phosphodiesterase 11A (PDE11A) gene in individuals with EOAD. Both variants are located in evolutionarily highly conserved amino acids, are predicted to alter the protein conformation and are classified as pathogenic. Furthermore, we found significantly decreased protein levels of PDE11A in brain samples of AD patients. Expression of PDE11A variants and knockdown experiments with specific short hairpin RNA (shRNA) for PDE11A both resulted in an increase of AD-associated Tau hyperphosphorylation at multiple epitopes in vitro. PDE11A variants or PDE11A shRNA also caused increased cyclic adenosine monophosphate (cAMP) levels, protein kinase A (PKA) activation and cAMP response element-binding protein phosphorylation. In addition, pretreatment with a PKA inhibitor (H89) suppressed PDE11A variant-induced Tau phosphorylation formation. This study offers insight into the involvement of Tau phosphorylation via the cAMP/PKA pathway in EOAD pathogenesis and provides a potential new target for intervention.

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Figures

Figure 1

Workflow of the current study. Exome sequencing was performed in 215 Chinese individuals with EOAD and 255 controls. Subsequent direct sequencing was performed in an independent cohort to validate the selected rare variants. Then in vitro functional studies were performed to evaluate the role of candidate variants in EOAD and the underlying mechanisms.

Figure 2

Functional annotation of PDE11A variants. (A) Sanger sequencing confirmation of PDE11A variants. (B) Protein homologs in different species were aligned. The p.Arg202His and p.Leu756Gln are conserved in all PDE11A orthologs. The variants are highlighted in the red boxes. (C) Predicted domain maps of PDE11A protein with the identified missense variants marked: G: GAF domain (amino acids 217–380 and amino acids 402–568), P: HD/PDEase domain (amino acids 663–839), C: 3′5′-cyclic nucleotide phosphodiesterase, catalytic domain (amino acids 588–912). The p.Arg202His variant is located near the GAF domain, and p.Leu756Gln variant in the 3′5′-cyclic nucleotide phosphodiesterase, catalytic domain. (D) Predicted three-dimensional structure of wide type (WT) and mutant PDE11A protein. (E) Structure comparison between wild-type PDE11A and R202H variant. Critical hydrogen bonds with surrounding amino acids were predicted to be eliminated. Dashed lines indicate hydrogen bonds. (F) Structure comparison between wild-type PDE11A and L756Q variant. Q756 mutant was predicted to induce more hydrogen bonds and then affect helix structure.

Figure 3

The PDE11A expression in brain tissues of AD. (A and B) Based on single-nucleus RNA sequencing data, PDE11A gene was expressed almost in all kinds of cells. (C) Representative western blots illustrate the expression of PDE11A in postmortem brain tissues. (D) The histogram shows the quantification of PDE11A detected by immunoblot relative to control levels. The data are represented as the mean ± SEM, based on three unrelated measurements. **P < 0.01 by Student’s t-test.

Figure 4

The PDE11A variants led to significantly high Tau phosphorylation levels. The cells were co-infected with MAPT and PDE11A lentivirus (PDE11A WT, mutants, scramble or shRNA). A total of 72 h after infection, cell lysates were used to detect levels of p-Tau(T181), p-Tau(S404), p-Tau(S202), p-Tau(S416), p-Tau(S214), p-Tau(S396), p-Tau(AT8) and Tau. (A) Representative western blots illustrate the expressions of phosphorylated Tau. (B and C) The histograms show the quantification of phosphorylated Tau levels detected by immunoblot relative to control levels. The data are represented as the mean ± SEM, based on three unrelated measurements. *P < 0.05, **P < 0.01 by one-way ANOVA and Dunnett test or Student’s t-test.

Figure 5

The PDE11A variants affect cAMP/PKA signaling. ELISA analysis showed cAMP levels in cells after infection with WT PDE11A (with MAPT), decreased as expected; they increased following infection with PDE11A p.Arg202His, PDE11A p.Leu756Gln (A) or shRNA (with MAPT) (B). (C) Western blot of PKA signaling. The histograms show the quantification of p-PKA, PKA, p-CREB/CREB levels detected by immunoblot relative to control levels in cells infected with PDE11A mutants (D) or shRNA (with MAPT) (E). The data are represented as the mean ± SEM. *P < 0.05, **P < 0.01 by one-way ANOVA and Dunnett test or Student’s t-test.

Figure 6

The effects of PDE11A mutants on phosphorylation of Tau after treatment with PKA inhibitor. SH-SY5Y cells were treated with or without the PKA inhibitor H89 (10M). Then, cells were co-transfected with MAPT and PDE11A mutant or shRNA plasmids. (A) Cell lysates were collected to examine Tau phosphorylation levels using western blot. (B and C) The histograms show the quantification of phosphorylated Tau levels detected by immunoblot relative to control levels. The data are represented as the mean ± SEM. *P < 0.05, **P < 0.01 by one-way ANOVA and Dunnett test.