Two Novel Pde11a Genetic Variants Increase Tau Phosphorylations in Early-onset Alzheimer's Disease

Background: Alzheimer’s disease (AD) is a leading cause of dementia in the elderly and has become a major health issue. However, a large number of genetic risk factors remain undiscovered. Methods: To identify novel risk genes and better understand the molecular pathway underlying AD, whole-exome sequencing (WES) was performed in 215 early-onset AD (EOAD) patients and 55 unrelated healthy controls of Han Chinese ethnicity. Subsequent direct sequencing was performed in 620 individuals to validate the selected rare mutations. Computational annotation and in vitro functional studies were performed to evaluate the role of candidate mutations in EOAD and the underlying mechanisms. Results: We identied two rare missense mutations in the phosphodiesterase 11A (PDE11A) gene, resulting in p.Arg202His, and p.Leu756Gln, in 4 individuals with EOAD. Both mutations are located in evolutionarily highly conserved amino acids, are predicted to alter the protein conformation, and classied as pathogenic. Furthermore, we found signicantly decreased protein levels of PDE11A in brain samples of AD patients. Expression of PDE11A variants and knockdown experiments with specic short hairpin RNA (shRNA) for PDE11A both resulted in an increase of AD-associated Tau hyperphosphorylation at T181, S404, S202, S416, S214, S396 and AT8 epitopes in vitro. PDE11A variants or PDE11A shRNA also caused increased cAMP levels, protein kinase A (PKA) activation, and cAMP response element-binding protein (CREB) phosphorylation. Additionally, pretreatment with a PKA inhibitor (H89) suppressed PDE11A mutation-induced p-Tau formation. Conclusions: Our results demonstrate that both PDE11A mutations and PDE11A knockdown increase Tau phosphorylation through the cAMP/PKA pathway, suggesting that PDE11A is a novel risk gene for AD. This study provides insight into the involvement of Tau phosphorylation via the cAMP/PKA pathway in EOAD pathogenesis and provides a potential new target for intervention.

EOAD is an almost entirely genetically determined disease with a heritability ranging 92% to 100% [4].
High-penetrant mutations in amyloid precursor protein (APP) and the presenilins 1 and 2 (PSEN1 and PSEN2) are the main genetic risk factors underlying EOAD but are only found in approximately 11% of all EOAD patients [6]. Whole-exome sequencing (WES) has recently identi ed rare missense variants in TREM2, ABCA7, CASP7, ADAM10 and other genes that increase the risk for developing AD [7][8][9][10][11]. However, a large number of EOAD patients remain genetically unexplained. EOAD patients are more likely to carry pathogenic variants [4], and suitable for identifying novel AD risk genes. The number of patients with dementia in China accounts for approximately 25% of the entire population with dementia worldwide [1]. However, very few studies on risk genes in EOAD cohorts have been carried out in China.
In this study, WES was performed in 215 EOAD patients and 55 unrelated healthy controls of Han Chinese ethnicity to identify novel AD risk genes. We identi ed two rare nonsynonymous variants (p.Arg202His and p.Leu756Gln) in PDE11A in four individuals with EOAD. Functional analyses in vitro revealed that both variants enhanced Tau phosphorylation. We also investigated the mechanisms through which PDE11A may be relevant in AD.

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 le 1: Table S1). Each individual underwent a neuropsychological examination, magnetic resonance imaging (MRI); cerebrospinal uid 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: met 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]; the onset age of affected individuals was below 55 years; no known pathogenic mutations in PSEN1, PSEN2, APP, MAPT or GRN genes; none of the patients were homozygous for Apolipoprotein E ε4. 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 310 DNA samples from patients with EOAD and 310 healthy controls were collected at the Xuanwu Hospital from 2017 to 2019 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 quanti ed 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. Brie y, 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

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].

Real-time quantitative PCR
Total RNA was puri ed from freshly harvested cells using an RNeasy Mini Kit (Thermo Fisher Scienti c) and TRIzol (Thermo Fisher Scienti c). Reverse transcription was carried out using a SuperScript First-Strand Synthesis Kit (Thermo Fisher Scienti c), 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 Scienti c) 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
Brie y, 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 le 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 quanti ed 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
Quanti cation 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). A p value less than 0.05 was used to indicate a statistically signi cant difference.

Identi cation 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 lters. 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 ndings 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 le 1: Table S4).
Among them, PDE11A met all criteria. We identi ed 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). Con rmation 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 signi cance, 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) identi ed 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 de cits. 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 di culties 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-speci c 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 signi cantly from wild-type human PDE11A in three-dimensional (3D) homology models (Fig. 2d-f). Speci cally, 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 identi ed 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 signi cantly decreased levels of PDE11A in those with AD relative to healthy controls (Fig. 3c-d).

Effects of PDE11A variants on Aβ homeostasis
To further con rm 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 le 2: Figure S1A-C).

PDE11A variants affect Tau phosphorylation
To understand the in uence 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 signi cant 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 signi cantly 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 signi cantly altered in any of the groups (Additional le 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.

Discussion
In the present study, we report PDE11A as a novel candidate risk gene for EOAD. Our results showed that PDE11A variants did not affect amyloid production but cause dysregulation of cAMP/PKA signaling and increase Tau phosphorylation. These ndings potentially improve our understanding of the molecular mechanisms of EOAD and reveal new AD target pathways.
We selected 215 cases of very early-onset AD (EOAD, with age of onset before 55 years) to detect new AD risk genes. EOAD has higher heritability than late-onset AD [21] and absence of known causal or risk AD gene mutations, sequencing EOAD increases the chances of discovering new AD genes. Using a systematic analysis of WES data, we identi ed PDE11A as a novel candidate risk gene for EOAD. Subsequent direct genotyping of two variants in 310 controls and 310 EOAD patients revealed two additional unrelated p.Leu756Gln carriers among AD patients but none in healthy controls. The human PDE11A gene is located at 2q31.2 and consists of 23 exons [22]. Four transcript isoforms designated PDE11A1-4, which result from different transcription initiation sites and alternative splicing, encode different PDE11A isoforms with unique N-terminal domains. Germline mutations, expression changes, and functional alterations in PDE11A have been linked to brain function, tumorigenesis, and in ammation [20,23,24]. For example, PDE11A is required for intact brain function across the lifespan [25], and studies have suggested that variations in PDE11A may be associated with bipolar disorder, depression and other psychiatric diseases [26,27]. Moreover, PDE11A negatively regulates the e cacy of lithium in treating bipolar disorder [28]. Expression of PDE11A4 mRNA in the brain tends to occur in CA1 neurons and in the subiculum in ventral hippocampal (VHIPP) formation. Atrophy of VHIPP has been observed in patients with AD or mild cognitive impairment [29,30]. Importantly, the extent of anatomical and functional de cits in the VHIPP appears to correlate with the severity of clinical impairment in patients with AD [30,31]. In addition, PDE11A4 can directly in uence synaptic plasticity [32]. PDE11A4 also plays a role in systems consolidation, which has relevance for cognitive de cits [33]. Phosphodiesterases (PDEs) have been linked to mental processes of emotions, learning and memory, with implications in mood and cognitive disorders, including Huntington disease, Parkinson's disease and AD [34][35][36][37]. This evidence suggests that PDE11A plays a role in brain function and may also be involved in AD.
In our study, we identi ed two rare nonsynonymous variants p.Arg202His and p.Leu756Gln in PDE11A in EOAD patients. The p.Arg202His variant is only present in the PDE11A4 isoform, whereas the p.Leu756Gln variant is present in all PDE11A isoforms. PDE11A isoforms are expressed in a tissuespeci c manner [24]. In a previous study, Kelly et al. found that PDE11A4 is highly expressed in hippocampal areas but not in any of the other tissues examined. PDE11A4 is the only PDE with restricted expression in hippocampal areas, which are affected in AD [38]. Both variants identi ed in this study affect PDE11A4 isoforms though a loss of function mechanism. Both variants are highly conserved evolutionarily and located near or in an important PDE11A4 domain. In addition, both mutations disrupt hydrogen bonding and dramatically alter protein coformation. Furthermore, both variants are predicted to be damaging by eleven in silico tools and are considered conserved by all four algorithms applied. Taken together, these ndings suggest that important biological functions can be attributed to these variants. Moreover, using bulk RNA-Seq data derived from 1536 individuals [39], we found that the PDE11A gene is signi cantly associated with neuronal proportion in cortical tissues. Neuronal loss in the cerebral cortex is one of the main characteristic pathological changes of AD. We also detected signi cantly decreased protein levels of PDE11A in brain tissues from AD patients. It appears that these variants may affect PDE11A function in the brain and facilitate disease onset.
We conducted cellular and biochemical studies and found that the PDE11A variants increase Tau phosphorylation at multiple sites. PDE11A shRNA also induces signi cant Tau hyperphosphorylation, whereas overexpression of PDE11A markedly decreases Tau phosphorylation. These ndings suggest a putative role for PDE11A and variants in tau phosphorylation. In contrast, the conditioned medium of cells transduced with lentivirus carrying the p.Arg202His or p.Leu756Gln variant or PDE11A shRNA showed no signi cant differences in Aβ 42 or Aβ 40 levels or Aβ 42 to Aβ 40 ratio, indicating that the variants may not affect Aβ peptides homeostasis. GSK-3β signaling affects Tau hyperphosphorylation. However, PDE11A variants or shRNA groups did not affect GSK-3β levels, indicating that the GSK-3β pathway is unaffected by the PDE11A mutations. It is likely that other signaling pathways contribute to Tau hyperphosphorylation. The underlying mechanisms connecting PDE11A and Tau needs to be further evaluated.
Our data demonstrate that PDE11A affects Tau phosphorylation through the cAMP/PKA pathway. PDEs hydrolyze cAMP and are key enzymes regulating intracellular cAMP levels. cAMP is known as a crucial second messenger that participates in transducing signals in many types of cells. cAMP activates PKA, which phosphorylates and activates CREB. Activated CREB binds to CREs in target genes to regulate their expression. cAMP/PKA signaling pathway dysregulation has been reported in AD patients, and the pathway plays a crucial role in AD pathogenesis [40]. Therefore, we reasoned that PDE11A variants might affect AD-relevant phentypes through cAMP/PKA signaling. Our results showed higher cAMP levels in lysates from cells transduced with PDE11A p.Arg202His, p.Leu756Gln or shRNA compared with those expressing WT PDE11A or scramble shRNA. We also found that PDE11A p.Arg202His or p.Leu756Gln variant led to elevated PKA, p-PKA and p-CREB levels. Increases in cAMP levels and PKA activity have been observed in the cerebral microvessels of AD patients [41], and elevated levels of cAMP in the CSF of AD patients have been reported [42]. Van et al. [43] documented an increase p-PKA levels in the temporal cortex of early-stage AD patients. Expression of PKA and p-CREB is decreased in AD patients and in vitro models [44,45], suggesting that PKA and p-CREB levels may vary on different tissues at different stages of disease progression.
PKA phosphorylates Tau, and it is important for the formation of NFTs [46,47]. Martinez et al. [42] showed that cAMP levels correlate with levels of Tau protein in the CSF. In the present study, H89 (a speci c PKA inhibitor) pretreatment suppressed PDE11A mutation-associated p-Tau formation, which further con rmed the role of the cAMP/PKA/CREB signaling pathway in PDE11A-mediated regulation of Tau phosphorylation. Carlyle et al. [48] reported an age-related increase in cAMP-dependent PKA-mediated phosphorylation of Tau as a risk factor for aged cortex degeneration. PKA activation induces Tau hyperphosphorylation and spatial memory de cits, which are reversed by the PKA inhibitor H89 inhibits [47]. Ikezu et al. [49] identi ed two rare variants in AKAP9 in African American AD patients. AKAP9 mutations had no effect on Aβ but signi cantly increased Tau phosphorylation in cells treated with a PDE4 inhibitor [50]. PKA enhances Tau phosphorylation by other Tau kinases [51]. Thus, the effect of PKA on AD-associated Tau phosphorylation may be either direct or indirect, and the exact mechanism has yet to be elucidated.

Limitations
First, the individuals carrying the PDE11A variants did not report AD family history. Due to absence of DNA samples from relatives, we could not determine whether these PDE11A mutations are de novo variants. Second, a replication case-control study in a large independent Chinese cohort and an independent replication of the association of PDE11A variants in different populations would have been con rmatory of our genetic ndings. Third, our results highlight the need to investigate the role of PDE11A in AD using in vivo models in future research. Such in vivo studies will provide evidence of PDE11A effects on cognition and con rm the mechanism through which PDE11A is involved in AD.

Conclusion
We report for the rst time two novel rare PDE11A variants in four individuals with EOAD. These PDE11A variants enhance both cAMP/PKA signaling and Tau phosphorylation. PDE11A could be a novel candidate genetic predisposing factor in AD.

Availability of data and materials
The data generated and analyzed in this study are available from the corresponding author on reasonable request.

Ethics approval
The study protocol was approved and monitored by the Ethics Committee of Xuanwu Hospital. Signed informed consent was provided by all the patients and control subjects. The recruited fresh frozen postmortem parietal lobe tissues were approved by the institutional review board of Washington    The PDE11A variants led to signi cantly high Tau phosphorylation levels. The primary neurons were coinfected with MAPT and PDE11A lentivirus (PDE11A WT, mutants, scramble or shRNA). Seventy-two hours 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. Western blot (A) and quantitative analysis (B, C) of phosphorylated Tau levels. The data are represented as the mean ± SEM, based on three unrelated measurements. *p < 0.05, **p < 0.01 by one-way ANOVA or Student's t-test.

Figure 4
The PDE11A expression in brain tissues of AD. (A, B) Based on single-nucleus RNA sequencing data, PDE11A gene was expressed almost in all kinds of cells. Western blot (C) and quantitative analysis (D) of PDE11A protein levels in post-mortem brain tissues. The data are represented as the mean ± SEM, based on three unrelated measurements. **p < 0.01 by Student's t-test.  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.