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

doi:10.21203/rs.3.rs-474622/v1

Download PDF

Research article

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

https://doi.org/10.21203/rs.3.rs-474622/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background: The expression of mesoderm-specific transcript (Mest) is regulated by genomic imprinting where only the paternal allele is active for transcription. Mest is a candidate gene for Silver-Russell Syndrome, and hypermethylation of Mest promoter is associated with oligozoospermia. In addition, Loss of imprinting (LOI) is often associated with various neurodegenerative diseases and cancers. Since aberrant Mest hypermethylation and LOI are implicated in various diseases, it is vital to study Mest promoter hypermethylation and its functional role in AD patients.

Methods: To assess the Mest promoter methylation, bisulfite sequencing technique was used. CRIPSR/Cas9 system was used to generate the Mest knockout (KO) in both embryonic carcinoma and mouse embryonic stem cells. Lentiviral Split-Cas9 system (LSC-5) was employed to generate inducible Mest KO in neurons.

Results: We found that Mest promoter is hypermethylated, which led to the reduction of Mest mRNA levels and activation of Wnt signaling in the brain tissues of AD patients. Mest KO in both embryonic carcinoma and mouse embryonic stem cells leads to neuronal differentiation arrest. Depletion of Mest in primary hippocampal neurons via lentivirus expressing sh-Mest or inducible KO system caused neurodegeneration. Notably, depletion of Mest in rat primary cortical neurons leads to tau phosphorylation at S199 and T231 sites.

Conclusion: Our study has unfolded the epigenetic modification; Mest promoter hypermethylation in AD. Biochemically, we have linked this with Wnt signaling activation and Tau phosphorylation in neurons.

Cellular & Molecular Neuroscience

Neurobiology of Disease

Mest

Hypermethylation

Wnt signaling

Neurodegeneration

Alzheimer’s disease.

Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF.

figS1.jpg
H19 Promoter methylation and Wnt target gene expression in AD brain samples. 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. Figure represents mRNA expression of Wnt target genes of individual normal aged vs AD subjects. Target mRNA levels were normalized to β-actin. Data shown as mean ± SD; *, p < .05; **, p < .01; ***, p < .001; ****, p < .0001 compared with the corresponding control group. Student’s t test was used for statistical analysis.
figS2.jpg
Increased expression of Mest during neuronal differentiation of P19 cells. A. Scheme of P19 neuronal differentiation. UD; undifferentiated, EB; Embryonic body, 8dN; 8 day neuron, RA; Retinoic acid. B. qPCR analyses for Tuj1, MAP2 and Neun mRNA expression during neuronal differentiation. mRNA levels were normalized to β-actin. Data shown as mean ± SD; *, p < .05; **, p < .01; ***, p < .001; ****, p < .0001 compared with the corresponding control group. Student’s t test was used for statistical analysis.
figS3.jpg
Mest KO in E14 mESCs leads to delayed differentiation. A. Mest mRNA expression analysis by RT-PCR in E14 mESC cells transduced with Mest gRNA. The clone 2 showed the absence of Mest transcripts. B. Protein levels of Mest in clone 2. The protein lysates were extracted from clone 2 and Cas 9 (control). Mest expression levels were analyzed by western blot. C. Representative Sanger sequencing trace of one Mest targeted clone showing deletion within the Mest coding exon 2 in E14 mESCs. D. Mest mRNA levels were increased in control cells but not in Mest KO cells. The qPCR analysis for Mest mRNA expression during mESC differentiation in N2B27 media. E. Nestin mRNA expression levels were reduced in Mest KO cells upon neuron differentiation in N2B27 media. F. Mest mRNA expression was absent in Mest KO cells while the control cells showed increased mRNA expression of Mest in embryonic body differentiation. G-I. The mRNA expression of ectodermal (Nestin), mesodermal (GSC), and endodermal (GATA4) markers were reduced in Mest KO cells. mRNA levels were normalized to β-actin. Data shown as mean ± SD; *, p < .05; **, p < .01; ***, p < .001; ****, p < .0001 compared with the corresponding control group. Student’s t test was used for statistical analysis.
figS4.jpg
Either extremes of Wnt signaling causes neurodegeneration in neurons. A-D; Mouse primary hippocampal neurons cultured in vitro for 7 days were incubated with the small molecules indicated in the figure for 48 hours and then immunostained with anti-Tuj1 (green) and Cleaved Caspase-3 ((CC-3), (red)) antibodies. DMSO, 5 μM XAV939; 10 mM Nacl,10 mM LiCl. A’-B’; To determine the percentage of Tuj1+ and cleaved caspase-3(CC-3)+ cells, the number of Tuj1+ & CC-3+cells was divided by the number of hoechst+ cells in four randomly selected areas for each sample. Scale bars, 25 μm. For each sample, more than 150 DAPI+ cells were counted. Data are expressed as the means ± SEM from the indicated number of samples. E-F; The P19 8 day differentiated neurons were treated with the small molecules indicated in the figure for 48 hours and harvested for qPCR analysis. DMSO, XAV939 (5 μM); 10 mM Nacl, 10 mM LiCl. mRNA levels were normalized to β-actin. Data shown as mean ± SD; *, p < .05; **, p < .01; ***, p < .001; ****, p < .0001 compared with the corresponding control group. Student’s t test was used for statistical analysis.
figS5.jpg
P19 LSC-5 stable cell generation and validation A. Linear map of lentiviral split-Cas9 plasmid containing U6 promoter–driven Mest gRNA. B. LSC-5 control and Mest-gRNA lentivirus was transduced into P19 cells and selected with puromycin. LSC-5 control and Mest gRNA P19 stable cells were validated for the expression of LSC-5 vector component FRB by qPCR. P19 LSC-5 control and Mest gRNA cells were selected for rapamycin inducible Mest KO.
TableS1.jpg
TableS2.jpg
TableS3.jpg
TableS4.jpg

Download PDF

Version 1

posted

You are reading this latest preprint version

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

Status:

Version 1

Abstract

Figures

Full Text

Supplementary Files

Status:

Version 1