Identification of PDE11A variants
We sequenced and analyzed the whole exome of 270 individuals (the pipeline is shown in Fig. 1). The cohort consisted of 215 EOAD cases and 55 unrelated control samples. About 80,000 variants per sample passed our quality control filters. For investigation of novel genes, patients carrying known EOAD risk genes (TREM2, VPS35, SORL1, MARK4, RUFY1 and TCIRG1) or genes associated with late-onset AD (ABCA7, ADAM17, IGHG3, PLD3, UNC5C, BIN1, CD2AP, CLU, CR1, EPHA1, MS4A4A, PLCG2, ABI3, AKAP9 and ZNF655) were excluded (n=13). We then selected only mutations rare in the population (< 0.01% MAF) and coding mutations, lowering the count to 317. Next, we excluded synonymous variants and used in silico analysis to restrict our findings to those predicted as damaging for the protein, revealing 32 variants. To further narrow the search for variants of interest, we used data from OMIM, MGI, GO, KEGG, ACGM and UKBiobank PheWeb to perform a systems-level analysis of the 32 mutated genes (Additional file 1: Table S4).
Among them, PDE11A met all criteria. We identified two variants in the PDE11A gene (NM_016953: rs752822096: c.605G>A: p.Arg202His and NM_016953: rs201572288: c.2267T>A: p.Leu756Gln). PDE11A is a dominant gene located on an autosome. A strong association was shown (p-value = 1.0 × 10−13) between PDE11A and AD using the UKBiobank PheWeb tool. Based on genetic databases, these two variants are rare in the East Asian population (p.Arg202His, gnomAD exomes_EAS: 0.0000; p.Leu756Gln, gnomAD exomes_EAS: 0.00431). Confirmation by Sanger sequencing is shown in Figure 2a. The p.Arg202His and p.Leu756Gln variants are predicted by eleven bioinformatics tools, including Polyphen2 HDIV, Polyphen2 HVAR, SIFT, LRT, PROVEAN, MutationTaster, DANN, VEST3, fathmm-MKL, CADD and M-CAP, to be damaging to the protein and by three algorithms (GERP, phastCons, phyloP) to be conserved (Table 1). The p.Arg202His and p.Leu756Gln variants are likely pathogenic and of uncertain significance, respectively, according to the American College of Medical Genetics and Genomics guidelines [19]. Subsequent Sanger sequencing analysis in an expanded cohort of individuals (N=620: 310 EOAD, 310 controls) identified two p.Leu756Gln carriers among EOAD patients but none in normal controls.
PDE11A variants and clinical features
The PDE11A p.Arg202His variant was detected in a 54-year-old female patient who had visited our hospital complaining of progressive memory decline over the past 4 years. She presented with amnesia as well as executive function and orientation deficits. She scored 9/30 on the MMSE and 8/30 on the Montreal Cognitive Assessment (MoCA), which was below the recommended cutoff values of 22 and 24, respectively. She had a CDR score of 3 and only remembered one word from the WHO-UCLA Delayed Recall Memory Test. MRI revealed moderate cerebral atrophy, especially in the hippocampus (MTA= 4). Her APOE genotype was ε3/ε3. Her parents and siblings were cognitively normal without complaints. Both her parents were deceased, and no DNA was available.
PDE11A p.Leu756Gln was found in a male patient who presented episodic memory decline at the age of 52 years. He had progressive difficulties in understanding and orientation, and he developed motor aphasia and personality changes in subsequent years. MRI showed atrophy of the temporoparietal lobe. The patient’s APOE genotype was ε4/ε3. He denied a family history of dementia.
Functional annotation of rare PDE11A variants
PDE11A p.Arg202His is uniquely present in the PDE11A4 isoform. The PDE11A protein sequence in which the two rare variants are located is highly conserved amino acids across different species (Fig. 2b), and their GERP scores are 4.34 and 5.57 respectively, implicating potential interference of important protein biological functions by the mutations. PDE11A p.Arg202His and p.Leu756Gln are predicted by Poly-Phen2 and SIFT to be damaging or possibly damaging (Table 1). As depicted in the schematic diagram of full-length PDE11A in Figure 2C, the two variants are located near or in functional domains, including the cGMP-specific phosphodiesterase, adenylyl cyclase and FhlA (GAF) and catalytic domains, suggesting a potential functional impact of these variants on the PDE11A protein.
Global conformations of the p.Arg202His and p.Leu756Gln variants changed significantly from wild-type human PDE11A in three-dimensional (3D) homology models (Fig. 2d-f). Specifically, the 3D model predicts that p.Arg202His abolishes critical hydrogen bonds with surrounding amino acids; in contrast, p.Leu756Gln leads to a new hydrogen bonded network, which affects the helical structure. Taken together, the model predicts that both p.Arg202His and p.Leu756Gln variants identified in patients with AD may impair PDE11A function.
PDE11A expression in AD brain tissues
The PDE11A gene is expressed in several regions of the mouse hippocampus, including the CA1, the subiculum, and the amygdalohippocampal area [20]. Moreover, Pde11a-knockout mice exhibit enlarged lateral ventricles and abnormal social investigation [20]. PDE11A is also reported to be involved in the regulation of cGMP-mediated signaling. These results suggest that PDE11A has an important role in the brain, with a possible role in central nervous system disorders.
We assessed PDE11A expression in single-nuclei RNA sequencing data from AD cases and control brain samples[18] and found the PDE11A gene to be expressed in almost all types of cells, including neurons, astrocytes, and microglia. (Fig. 3a-b).
To characterize the involvement of PDE11A in AD, we analyzed the PDE11A protein in fresh frozen postmortem brain tissues from cognitively normal healthy controls (n = 6) and patients with AD (n = 6). All cases were matched for age and sex. This analysis revealed significantly decreased levels of PDE11A in those with AD relative to healthy controls (Fig. 3c-d).
Effects of PDE11A variants on Aβ homeostasis
To further confirm the pathogenesis of PDE11A variants, we performed in vitro studies to test the effect on Aβ homeostasis. Lower levels of the PDE11A protein was observed in AD patients. Therefore, we used PDE11A shRNA to knockdown (KD) PDE11A levels in cell models. A markedly lower PDE11A mRNA and protein levels were obtained in HEK cells (Additional file 2: Figure S1A-C).
HEK293-APP695 cells were transfected with the PDE11A plasmid (PDE11A WT, p.Arg202His, p.Leu756Gln, scramble or shRNA) showed no significant differences in secreted Aβ40 or Aβ42 levels or the Aβ42/Aβ40 ratio (Additional file 2: Figure S2A-C). PDE11A variants or KD did not change the levels of APP or BACE-1(Additional file 2: Figure S3A-C). These results suggest that PDE11A may not affect Aβ homeostasis.
PDE11A variants affect Tau phosphorylation
To understand the influence of PDE11A variants on Tau phosphorylation, lentiviruses with human MAPT and PDE11A (WT PDE11A, PDE11A p.Arg202His, PDE11A p.Leu756Gln, shRNA or scramble) were used to transduce primary neurons simultaneously. Effects on Tau phosphorylation levels in transduced primary neurons were assessed by immunoblotting. A significant reduction in Tau phosphorylation was detected at multiple sites, including T181, S404, S202/T205, S416, S214, and S396, in WT PDE11A-expressing neurons compared to neurons infected with mock lentivirus (Fig. 4). Moreover, both variants notably increased Tau phosphorylation at multiple sites compared with the WT (Fig. 4a-b). PDE11A shRNA treatment reduced PDE11A mRNA and protein levels by 60% and significantly increased Tau phosphorylation at multiple sites compared with scramble shRNA treatment (Fig. 4a-c). These results suggest that both mutations could be a loss-of-function.
The protein level of GSK-3β, a major kinase involved in Tau phosphorylation, and its inactive form p-Ser9-GSK3β were not significantly altered in any of the groups (Additional file 2: Figure S4A-C). These data suggest that PDE11A variants likely affect Tau phosphorylation independent of GSK-3β signaling.
PDE11A variants exhibit alterations in cAMP/PKA signaling
The cAMP/PKA/CREB signaling plays an important role in AD. However, the link with PDE11A is unknown. To further clarify the underlying mechanisms, we tested the effects of PDE11A on cAMP/PKA/CREB signaling. Transduction of WT PDE11A (with MAPT) in primary neurons decreased cAMP levels compared to the mock group (with MAPT). The p.Arg202His and p.Leu756Gln variants increased cAMP levels relative to WT PDE11A (Fig. 5a-b). These results suggest that the p.Arg202His and p.Leu756Gln variants reduced the ability of PDE11A to degrade cAMP. Total PKA and p(Thr197)-PKA levels, as well as the ratio of phosphorylated CREB (p-CREB) to CREB, were also increased in variants PDE11A-expressing neurons compared to WT PDE11A-expressing neurons (Fig. 5c-d). Similar results were obtained in PDE11A shRNA-treated neurons compared to scramble shRNA-treated neurons (Fig. 5c and 5e).
In addition, pretreating the neurons with the PKA inhibitor (H89) reduced PDE11A p.Arg202His and p.Leu756Gln variant-induced Tau phosphorylation (Fig. 6a-c). H89 pretreatment also decrease p-PKA and p-CREB/CREB levels (Fig. 6d-h). Furthermore, these results were recapitulated using PDE11A shRNA. Therefore, these data demonstrate that the PDE11A variants affect the cAMP/PKA pathway, which is associated with increased Tau phosphorylation through a loss-of-function mechanism.