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Hypermethylation of Mest Promoter Causes Aberrant Wnt Signaling in the Patients of Alzheimer’s Disease

Renuka Prasad University of Seoul Hwajin Jung University of Seoul College of Natural Sciences Anderson Tan University of Seoul College of Natural Sciences Yonghee Song University of Seoul College of Natural Sciences Sungho Moon University of Seoul College of Natural Sciences Mohammed R. Shaker Korea University College of Medicine and School of Medicine Woong Sun Korea University College of Medicine and School of Medicine Junghee Lee Boston University School of Medicine Hoon Ryu Korea Institute of Science and Technology Hyun Kook Lim Catholic University of Korea Eek-hoon Jho (  ej70@uos.ac.kr ) University of Seoul https://orcid.org/0000-0003-2414-6234


Background
Mis-regulation of Wnt/β-catenin signaling is one of the major causes of diseases due to its participation in cell proliferation, self-renewal, and cell fate determination during embryonic development and tissue homeostasis in adult [1]. Consistently, mutations in Wnt signaling components is tightly linked with age-related neurodegenerative conditions, including Alzheimer's disease (AD) [2]. Previous studies have reported the association between LRP6, which is a co-receptor of Wnt ligand, single nucleotide polymorphisms Val 1062 allele and late-onset AD. LRP6 Val 1062 allele shows reduced β-catenin signaling [3].
Mest is a paternally expressed gene and expressed abundantly in the mesodermal tissue and adult brain [4,5]. Imprinting phenomenon refers to the mono-allelic expression of a specific gene due to the only one parent specific methylation of the gene promoter [6]. Mest is expressed only from paternal genome since the maternal gene promoter is methylated.
Loss of imprinting (LOI) of Mest is often correlated with altered growth [7]. Mest is a candidate gene for Silver-Russell Syndrome [8], and hypermethylation of Mest promoter is associated with oligozoospermia [9]. In addition, LOI of Mest is also involved in breast, colorectal and lung cancer [10][11][12]. Genomic imprinting is loosely controlled during aging. Since aberrant Mest hypermethylation and LOI are often associated with various diseases, it is vital to study the functional role and methylation status of Mest promoter in AD patients.
The epigenome-wide association study of AD patients suggests that Mest promoter is potentially hypermethylated in the cortex of AD patients [13]. However, the underlying mechanism on how Mest promoter hypermethylation associated with AD pathology still remain elusive. Herein, we investigated the methylation status of Mest promoter and how it affects Wnt signaling using the brain samples of AD patients and neurons. In the current study, we showed that Mest promoter is hypermethylated in AD patients and the mRNA levels of Mest were reduced while the Wnt target genes were elevated. Mest depletion resulted in ４ upregulation of Wnt signaling and tau phosphorylation causing neurodegeneration.

Postmortem brain samples
Frozen postmortem human cortex samples from 9 AD patients and their 9 age-matched controls were used in this study. Neuropathological examination of normal subject and AD human brain samples was determined using procedures previously established by the Boston University Alzheimer's Disease Center (BUADC). Next of kin provided informed consent for participation and brain donation. Institutional review board approval for ethical permission was obtained through the BUADC center. This study was reviewed by the Institutional Review Board of the Boston University School of Medicine (Protocol H-28974) and was approved for exemption because it only included tissues collected from post-mortem subjects not classified as human subjects. The study was performed in accordance with institutional regulatory guidelines and principles of human subject protection in the Declaration of Helsinki. The sample information is listed in Supplementary Table 1 and 2.

DNA extraction and bisulfite conversion
Total genomic DNA was isolated from postmortem frontal cortex of AD patients using a AccuPrep® Genomic DNA Extraction Kit (BIONEER, K-3032) according to the manufacturer's instructions. Each 500ng of genomic DNA was subjected to bisulfite conversion by EZ DNA methylation kit (Zymo Research, D5001) according to the manufacturer's instructions.

Mest promoter amplification and bisulfite sequencing
The individual genomic DNA was amplified by PCR using the primers indicated in ５   Supplementary Table 4. The amplification was done using the following conditions: by 95 °C   for 10 min followed by 40cycles (95 °C for 30 s, 55 °C for 35 s, and 72 °C for 40 s and a final   extension at 72 °C for 7 min). The PCR products were run on 1% agarose gels. The PCR products were isolated from the gel slice by Gel extraction kit (ELPIS BIOTECH, EBD-1005).
The PCR products were subcloned into pGEM-T easy vector system (Promega, A1360) according to the manufacturer's instructions. The ligation products were transformed into E.coli (DH5α), then spread into LB / ampicillin / IPTG / X-Gal plates. Next day, 12 colonies were picked and amplified / collected plasmid DNA (LaboPass, CMP0112) according to the manufacturer's instructions. The plasmid DNA was sent to DNA sequencing (Bionics).
Mouse embryonic stem cell E14 cell lines were used for the experiments. mESCs were cultured on a tissue culture dish pre-coated with 0.2% gelatin (Sigma) at 37 °C in a 5% CO2 incubator. Cell culture media consists of 15% fetal bovine serum (Bio West), Tylosine (Sigma), 0.1 mM β-mercaptoethanol (GIBCO), 100X nonessential amino acid (Corning), 1 mM sodium pyruvate (GIBCO), 100X GlutaMAX (GIBCO). LIF-conditioned media was generated by transfection of LIF cDNA to Cos7 cells using Lipofectamine 2000. For subculture, cells were treated with 0.05% trypsin for 5 min at 37 °C. After trypsinization, neutralization was done by treatment of FBS containing complete media and centrifuged. Subsequently, mESCs were ６ resuspended in culture media and plated on a pre-coated culture dish.

Hippocampal primary culture
Mouse primary hippocampal neuronal cell cultures were prepared from ICR (Young bio, Korea) mice as described (Kim et al., 2011). Experiments were carried as per the standard ７ ethical guidelines and were approved by the University of Seoul Institutional Animal Care and Use Committee (UOS IACUC). Briefly, hippocampus was isolated from embryonic day 18.5 mouse embryos using Hank's balanced salts solution. Isolated hippocampus was trypsinized and triturated with fire-polished Pasteur pipettes, then strained through 40-μm nylon mesh.
These cells were cultured in Neurobasal medium with 2% B27 supplement, 25 μM L-Glutamate, 0.5 mM L-glutamine, and 1% antibiotics mixture. The same growth media without Glutamate and antibiotics was used for media changes after 4 d. One half of the medium was changed once per week.

Gene knockout using CRISPR-Cas9
pLKO.1 -TRC was digested with AgeI and EcoRI and gel purified to extract the vector fragment. The Mest gRNA cassette was PCR amplified the from template plasmid (PLKO-gRNA-Gsk3a) using the primer sequences listed in Supplementary Table 3. The insert (~200bp) was then ligated to vector using NEB Gibson assembly kit (Catlog # E5510).  Supplementary Table 3.

Immunofluorescence analysis
The P19 and primary hippocampal neurons were seeded onto poly-D-lysine and Laminin coated glass coverslips in 12-well plates, fixed for 10 min in 4% paraformaldehyde in PBS, and then permeabilized for 20 min with 0.1% Triton X-100 in PBS at room temperature.

Isolation of mRNA and qPCR analysis
For quantitative real-time PCR, total RNA was isolated using TRIZOL reagent (Invitrogen) and cDNA synthesis was performed using ImProm-II™ Reverse Transcriptase (Promega) according to the manufacturer's protocol. Quantitative PCR (qPCR) was carried out by using CFX connect real-time PCR (BIO-RAD) with SYBR Green PCR Master Mix (TOYOBO). The primer sequences used for qPCR are listed in Supplementary Table 4.

Statistical analysis
All statistical data are expressed as the mean ± SD and number of sample size (n=3) were indicated in each figure legends. Most of the experiments were repeated three times. The statistical significance of differences between different groups was analyzed using the Student's t-test. P-values were calculated using Student's t-test and the values of *, p <0.05; **, p <0.01; ***, p <0.001 ****, p <0.0001 were considered significant. Representative example of methylation pattern of Mest promoter region in DNA extracted from the brain of normal aged subjects showed equal number of clones consisting of methylated and unmethylated alleles (Fig. 1a). Interestingly, 44.4% of the AD cases showed hypermethylation in Mest promoter in comparison to 11.1% observed in normal aged (NA) controls ( Fig. 1b and c). To show the observed Mest promoter hypermethylation was not a PCR-generated artifact, we examined H19, another paternally imprinted gene, as a control.

Hypermethylation of Mest promoter and upregulation of
We found that H19 promoter exhibited hypermethylation in 22.2% of AD cases, indicating that the Mest promoter hypermethylation seen in AD patients is specific to Mest ( Fig. 1c and S1A).
Another interesting finding is that Mest promoter methylation is tightly controlled in which the clones showed all or none methylation pattern (Fig. 1b), while H19 promoter methylation pattern is loosely controlled in both normal aged and AD cases (Fig. S1A). Our results are consistent with reports that cortex-specific Mest promoter hypermethylation is associated with AD neuropathology [13].
Consistently, the mRNA levels of Mest are downregulated in the temporal cortex of sporadic AD postmortem brains in comparison to normal subjects (n=7) (Fig. 1d). Canonical Wnt target genes (Axin2, c-Myc) were also upregulated in AD patients, which is consistent １２ with the previously reported role of Mest as a negative regulator of Wnt signaling (Fig. 1e and S1B) [15]. These results suggest a potential link between hypermethylation of Mest promoter and elevation of Wnt signaling in AD.

Mest KO in embryonic carcinoma and embryonic stem cells lead to neuron differentiation blockade
To assess the functional significance of Mest in neurons, P19 embryonic carcinoma cells were differentiated to neuronal lineage [16,17] and the levels of its mRNA and protein were examined ( Fig. S2A-B). The levels of both Mest mRNA and protein were increased during neuronal differentiation ( Fig. 2a-b). Next, we generated P19 Mest KO cells using CRISPR/Cas9 system. The presence of indel mutations was confirmed by T7 endonuclease 1 (T7E1) assay and genomic DNA sequencing (Fig. 2c-d). Mest mRNA and protein levels were reduced significantly in KO clone (Fig. 2e-f). P19 Mest KO cells exhibited neuron differentiation arrest with the increased expression of cleaved caspase-3 (Fig. 2g).
We also investigated the role of Mest in the early differentiation of mouse embryonic stem cells (mESCs) by generating Mest KO mESCs. Mest protein and mRNA levels were significantly reduced in Mest KO cells compared to control (Fig. S3A-B). Indel mutations were also observed in Mest KO clones (Fig. S3C). Mest levels were increased during neuronal differentiation, whereas neuronal differentiation marker, Nestin levels were significantly decreased in Mest KO cells (Fig. S3D-E). By using embryonic body (EB) differentiation method, we checked the levels of Mest during differentiation. We found that Mest expression is low in mESCs and increased with differentiation (Fig. S3F). Various lineage-specific markers (Nestin, GSC, GATA4) were significantly impaired during differentiation of Mest KO cells, suggesting that the depletion of Mest affects differentiation potential of mESCs ( Fig.   S3G-I).

Mest depletion results in elevation of Wnt signaling and Tau phosphorylation causing neurodegeneration.
To examine the effect of abnormal Wnt/β-catenin signaling levels in neurodegeneration, primary hippocampal neurons of DIV7 were treated with small molecules, which regulate Wnt signaling. XAV939 antagonizes Wnt signaling via stimulation of β-catenin degradation and stabilization of Axin [18]. Hippocampal neurons treated with XAV939 showed increased apoptosis and neurodegeneration ( Fig. S4A-B, A'). These results are consistent with our previous reports that reduced Wnt signaling leads to neurodegeneration by small molecule ICG-001 [19]. The GSK-3 inhibitor, LiCl was used as Wnt signaling activator [20]. Treatment of LiCl also showed increase of apoptotic cells and neurodegeneration ( Fig. S4C-D, B').
Similarly, treatment of either XAV939 or LiCl showed significant reduction of neuronal markers in P19 day 8 neurons (Fig. S4E-F). Consistently, Wnt target genes were downregulated and upregulated in XAV939 and LiCl treated groups respectively. Overall, either activation or inhibition of Wnt signaling leads to neurodegeneration indicating that Wnt signaling must be in controlled levels for the maintenance of neuronal survival.
Next, in order to mimic the low level of Mest in AD patients, we employed inducible split-Cas9 system [21] to suppress endogenous Mest expression. LSC-5 Mest gRNA was constructed and transduced into P19 cells (Fig. S5A-B). Treatment of P19 LSC-5 Mest gRNA stable cells with rapamycin suppressed Mest mRNA expression and increased Wnt target gene levels (Fig. 3a). Mest inducible KO (MiKO) cells also showed reduction of neuronal markers levels. MiKO cells formed large aggregates while control cells exhibited a proper neurite formation (Fig. 3b). Immunofluorescent examination showed that control cells exhibited intact neuron morphology with decent neuronal differentiation upon staining with Tuj1 whereas MiKO cells displayed fragmented neurites with few surviving neurons and with positive signals for cleaved caspase 3 (Fig. 3b). Wnt signaling components such as p-LRP6, １４ β-catenin, ABC were also increased in MiKO cells (Fig. 3c). These findings propose that Mest suppression forced the activation of Wnt signaling resulting in neurodegeneration.
To corroborate our findings, primary hippocampal neurons were transduced with shMest lentivirus. The mRNA expression of neuronal markers in Mest knockdown cells were significantly reduced and the expression levels of Wnt target genes were increased (Fig. 3d).
Mest knockdown neurons also stained positive for cleaved caspase 3 (CC3) suggesting occurrence of apoptosis, while shGFP treated cells remained intact with the expression of Tuj1 (Fig. 3e). Tau phosphorylation causes formation of neurofibrillary tangles. A gradual increase in Tau phosphorylation at Tyr18, Thr231 and Ser199 sites correlate with AD severity [22]. Interestingly, we found that Mest knockdown in rat cortical neurons increased tau phosphorylation at Ser199 and Thr231 residues (Fig. 3f). Overall, these results demonstrate that Mest suppression results in increased Wnt signaling and tau phosphorylation therefore possibly causing neurodegeneration. . Notably, we observed Mest promoter hypermethylation in the patients of AD brain. The current study revealed that the Mest promoter hypermethylation maybe the epigenetic mechanism responsible for the reduction of Mest mRNA levels in AD patients (Fig.1). Our results also corroborates previous reports that Mest promoter is hypermethylated and its mRNA level is decreased significantly in AD brain [13,36]. The reasons for the enrichment of hypermethylated alleles in Mest promoter in AD patients is unclear and requires further study. It would be interesting to elucidate the upstream mechanisms which causes the hypermethylation of Mest promoter in the patients of AD brain.

Discussion
Tau protein is predominantly phosphorylated at Ser/Thr-Pro sites causing formation of neurofibrillary tangles [37]. Tau phosphorylation is highest at Tyr18, Thr231 and Ser199 residues in the cortex and it is correlated with the progression of AD [22]. In the present study, we showed that Mest depletion caused tau hyperphosphorylation at Ser199 and Thr231 sites in rat cortical neurons (Fig. 3f). The microarray studies on AD cortex with neurofibrillary tangles also showed diminished levels of Mest [38]. Finding the tau kinase responsible for tau phosphorylation upon Mest reduction requires further investigation.
The current results should be interpreted with caution, as larger number of AD patient samples should be analyzed. The role of Mest in other signaling pathways is still not clear.

１６
Within the broader framework of AD, our results hint us to speculate that increased Wnt signaling upon reduced expression of Mest due to loss of imprinting may cause neurodegeneration.

Conclusion
In conclusion, we found that Mest promoter hypermethylation causes the reduction of Mest A. Methylation profile of H19 promoter in normal aged and AD brain sample. DNA from brain samples from at least 9 subjects from normal aged (NA) and AD subjects were analyzed.
There are 23 CpG sites in each row. Each row of circles represents a single cloned allele with at least 12 clones sequenced per subject. The % methylation is summarized in Fig. 2b. B. DMSO, XAV939 (5 μM); 10 mM Nacl, 10 mM LiCl. mRNA levels were normalized to β-actin.