Dynamics of N6-methyladenosine modification during Alzheimer's disease development

doi:10.21203/rs.3.rs-2861775/v1

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Dynamics of N6-methyladenosine modification during Alzheimer's disease development

https://doi.org/10.21203/rs.3.rs-2861775/v1

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N6-methyladenosine (m6A) modification is a common RNA modification in the central nervous system and has been linked to various neurological disorders, including Alzheimer's disease (AD). However, little is known about the dynamic of mRNA m6A modification and m6A enzymes during the development of AD.Therefore, this study examined the expression profiles of m6A and its enzymes in the development of AD. The results showed that changes in the expression levels of m6A regulatory factors occurred in the early stages of AD, indicating the potential involvement of m6A modification in disease onset. Moreover, the analysis of mRNA m6A expression profiles using m6A-seq revealed significant differences in m6A modification between AD and control brains.The differentially methylated genes were enriched in GO and KEGG terms related to processes such as inflammation response, immune system processes. And the differently expressed genes (DEGs) are negative associated with microglia homeostasis genes and but positive for “disease-associated microglia” (DAM) associated genes, suggesting that dysregulation of mRNA m6A modification may contribute to the development of AD by affecting the function and gene expression of microglia.

Alzheimer’s disease

N6-methyladenosine

ALKBH5

METTL3

microglia

WTAP

Alzheimer's disease (AD) is a neurodegenerative disorder in the central nervous system (CNS) that lead to dementia, being the most common cause of dementia in the elderly¹. The pathological changes of AD mainly involve the deposition of β-amyloid (Aβ) to form plaques and the formation of neurofibrillary tangles (NFTs), in which the microtubule-associated protein tau plays a major role and eventually leads to neuronal damage². Environmental exposure and genetic susceptibility are risk factors for AD³. However, the heterogeneity pathologies of AD among individuals with similar or identical susceptibility genes suggests the involvement of epigenetics in the disease.

Epigenetics refers to the heritable phenotype changes that occur without changes in the DNA sequence, which is different from genetics, which is caused by changes in the nucleotide sequence. Epigenetics includes DNA methylation, histone modification, chromatin remodeling, and non-coding RNA regulation. N6-methyladenosine (m6A) is a widely occurring methylation modification on mRNA, which occurs at the sixth nitrogen atom of adenine⁴. This modification is dynamically reversible, and the regulatory factors involved in m6A methylation modification include "Writers" (methyltransferases), "Erasers" (demethylases), and "Readers" (binding proteins) of m6A⁵. The main components of methyltransferase complex (MACOM) are methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and Wilms tumor 1-associated protein (WTAP), which cooperate with each other to perform catalytic functions^{6, 7}. Demethylases include fat and obesity-associated protein (FTO) and ALKB homolog 5 (ALKBH5)^{8, 9}, while m6A methylation binding proteins include various binding proteins containing YTH domains¹⁰, heterogeneous nuclear ribonucleoproteins (hnRNPs)¹¹, and eukaryotic initiation factor 3 (eIF3)¹². The study by Miao et al¹³ has found that m6A modification is not random, and its content is related to the length and number of exons and the distance from neighboring genes. Recent studies have revealed that epigenetic modifications can affect the development of the nervous system, such as neurogenesis, axon growth, synaptic plasticity, circadian rhythm, cognitive function, and stress response^14–16. When the expression patterns of genes controlled by epigenetics are dysregulated, it may lead to autoimmune diseases, cancer, and various other diseases. m6A-related proteins are widely expressed in neurons and regulate neurogenesis, memory formation and consolidation, cerebellar development, and axon regeneration^17–19. Moreover, m6A modification abnormalities have been found in various neurodegenerative diseases, including AD^{20, 21}.

It is worth noting that although many studies have reported changes in m6A in AD, including alterations in METTL3, METTL14, and FTO, the dynamics of m6A and m6A enzymes during AD development remain unclear^{22, 23}. To this end, we analyzed m6A methylation levels in hippocampal brain tissues from 5xFAD transgenic mice at different ages, and used real-time quantitative PCR (RT-qPCR) to investigate the expression of m6A regulatory factors during different stages of AD progression. We also employed immunofluorescence staining to investigate the association of methyltransferases METTL3 and WTAP with Aβ, and applied mRNA m6A methylation sequencing (MeRIP-seq or m6A-seq) technology to investigate the changes in m6A modification at the genome-wide level during the AD process. These findings may offer a new perspective for understanding the potential role of m6A modification in AD pathogenesis and identifying new therapeutic targets for AD treatment.

Animals

5xFAD(B6.Cg-Tg(APPSwFlLon,PSEN1M146LL286V)6799Vas/Mmjax, Stock No: 008730) mice was purchased from The Jackson laboratory. They were used in this study alongside their wild-type littermates at 3, 6, and 9 months of age. The mice were housed in a 12-hour light/dark cycle and kept at a constant temperature of 22–24℃. Food and water were provided ad libitum.

Tissue preparation for RNA extraction and immunohistochemistry

To obtain brain tissue for analysis, mice were anesthetized and perfused with either 50 ml of 0.01M cold PBS or 0.9% Nacl. The right hemisphere was used to collect hippocampal tissue, which was flash-frozen in dry ice and stored at -80°C prior to cryosection. Tissue RNA samples were extracted using the manufacturer's protocol (Takara, Cat# 9767). The left hemisphere was postfixed in 4% paraformaldehyde (PFA) overnight and then dehydrated with 30% sucrose in 0.01 M PBS at 4°C for 2–3 days. Finally, the tissues were embedded in optimal cutting temperature compound (OCT) and stored at -80°C before cryosection.

Quantification of the m6A Modification

In this study, we used the m6A RNA Methylation Quantification Kit (Epigentek, Cat# P-9005)to measure the m6A content in the total RNAs. In this assay, total RNA is bound to strip wells using a RNA high binding solution. m6A is detected using specific capture and detection antibodies. The detected signal is enhanced and then quantified colorimetrically by reading the absorbance in a microplate spectrophotometer at a wavelength of 450 nm. The total amount of RNA in each reaction was 300 ng. RNA m6A relative quantification assay was performed following the manufacturer's protocol.

Quantitative Real-Time PCR

Genomic DNA was removed from total RNA and cDNA was rapidly synthesized through PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Cat# RR047A). TB Green® Premix Ex Taq™ (Tli RNaseH Plus) (Takara, Cat# RR420A) and a LightCycler480 System (Roche Diagnostics) were used for qRT-PCR. Gapdh was used as internal control, relative expression of the following genes, Ythdf1, Ythdf2, Ythdf3, Ythdc1, Mettl3, Mettl14, Wtap, Fto and Alkbh5, were calculated using the 2-ΔΔCt method. Details of primer sequences used in the study are shown in supplementary table 6.

Immunohistochemistry

All tissues were frozen in the freezing microtome for 15 minutes before cryosection. Coronal sections of the brain were made at a thickness of 30 µm by Leica CM1950 cryostat and rinsed by 0.01M PBS for 3 times. Brain were blocked and permeabilized with 0.01M PBS containing 0.3% Triton X-100 and 4% normal donkey serum (NDS) for 2 hours at room temperature (RT). Next, the samples were incubated in primary antibody diluted 1%NDS in PBST at 4 ° C ON. After rinsed by 0.01M PBS 10 minutes for 3 times, the secondary antibody was incubated for 2 hours at RT, together with 4’,6-diamidino-2-phenylindole (DAPI, 1:1000). Next, samples are rinsed by 0.01M PBS for 3 times and then mounted anti-fade mounting medium. Then sealed the edge of the cover glass with nail enamel.

Primary antibodies included the following: Rabbit anti-METTL14 (sigma, Cat# HPA038002 1:500); Rabbit anti-YTHDF3 (Proteintech, Cat# 25537-1-AP 1:500); Rabbit anti-ALKBH5 (Millipore, Cat# ABE547 1:500); Goat anti-IBA1 (Abcam, Cat# ab5076 1:500); Goat anti-Olig2 (Biotechne, Cat# AF2418-SP 1:500); Rabbit anti-YTHDF1 (Proteintech, Cat# 17479-1-AP 1:500); Rabbit anti-YTHDF2 (Proteintech, Cat# 24744-1-AP 1:500); Rabbit anti-WTAP (Proteintech, Cat# 10200-1-AP 1:500); Rabbit anti-m6A (synaptic systems, Cat# 202003 1:500); Rabbit anti-METTL3 (Abcam, Cat# ab195352 1:500); Mouse anti-β-Amyloid (Biolegend, Cat# 803014 1:2000); Mouse anti-NeuN (Abcam, Cat# ab104224 1:1000); Mouse anti-GFAP (Sigma, Cat# G3893 1:500). Secondary antibodies included the following: AlexaFluor-488-, AlexaFluor-555-, or AlexaFluor-647-conjugated secondary antibodies against goat, rabbit, mouse (Invitrogen, 1:1000) antibodies.

MeRIP-seq

Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA), and the RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA). Poly (A) RNA was purified from 50µg total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) by two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 86℃ 7min. The cleaved RNA fragments were incubated for 2h at 4℃ with m6A-specific antibody (No. 202003, Synaptic Systems, Germany) in IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630). The IP RNA was reverse-transcribed to cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which was next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA). An A-base was added to the blunt ends of each strand, preparing for ligation to the indexed adapters. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. At last, we performed paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 platform(LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.

MeRIP-seq data analysis

The quality of IP and Input sample sequences was assessed using FastQC and RseQC. Low-quality reads and those containing adaptors were removed using fastp (v0.19.4) with default settings. Reads were mapped to the reference genome (mm10) using HISAT2 (v2.0.4). Peak calling and differential peak analysis were conducted in R (v4.2.2) using the exomePeak package (v2.16.0). Peak annotations were performed with ANNOVAR. De novo and known motif discovery, as well as motif localization relative to the peak summit, were carried out using MEME and HOMER (v4.10). Transcript and gene expression levels were determined from input libraries using StringTie (v2.1.2) by calculating FPKM (total exon fragments/mapped reads (millions) x exon length (kB). Differential analysis was based on the FPKM matrix and performed using edgeR (v3.40.2) with quasi-likelihood (QL) F-tests. Genes with an absolute log2(fold change) greater than log2 (1.5) and a p-value less than 0.05 were considered as significant differentially expressed genes (DEGs). Volcano plots were created using the EnhancedVolcano package (v1.16.0). Gene Ontology (GO) and KEGG pathway analyses were conducted using the enrichGO and enrichKEGG functions of the clusterProfiler package (v4.6.2). Categories with a false discovery rate (FDR) of 0.05 or lower were deemed significantly enriched, and these categories were visualized using ggplot2 (v3.4.2). Detailed information can be found in the accompanying R script.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism (GraphPad Software, Version 8.0, La Jolla, USA). Each data point represented the average statistical result of three sections in different regions of the brain. Results were determined independently at double-blind manner. All data were shown as mean ± standard deviation (SD) (bar plot). Data from multiple groups were evaluated statistically by one-way analysis of variance (ANOVA) with Holm-Sidak’s multiple comparisons test (post hoc). Student’s t test was used for comparison between the two groups. Statistical significance was set as P < 0.05.

Aβ plaque accumulation is associated with mRNA m6A modification

To investigate the changes in mRNA m6A levels during the progression of AD, we performed m6A detection in the hippocampal from 5xFAD and age-matched wild-type (WT) mice. The 5xFAD mouse model begins to express a significant amount of Aβ42 at 2 months of age, leading to the development of pathological features such as amyloid-like protein accumulation and glial hyperplasia²⁴. These mice exhibit a deterioration in learning and memory at the age of 6 months old and neuronal apoptosis at the age of 9 months old²⁴. Therefore, we analyzed the m6A level at four time points, including 1-month (1 mo), 3-month (3 mo), 6-month (6 mo), and 9-month (9 mo). We observed a gradual decrease in m6A levels in both 5xFAD and WT mice as they aged. However, no significant difference was detected in the total m6A levels between 5xFAD and WT mice at the various timepoints (Fig. 1A). We then investigated the expression profiles of m6A in neuron, microglia, oligodendrocytes, and astrocytes during AD progression by co-staining cell-specific markers with m6A (Fig. 1B and sFig.1). The m6A antibody was used to identify and quantify the presence of m6A modification in RNA molecules. We found that m6A was highly expressed in neurons than glial cells in the adult mouse brain (Fig. 1B). However, no significant differences in total m6A expression were observed between AD and control groups across aging (Fig. 1C).

The accumulation of Aβ plays a central role in the pathogenesis of AD, which contributes to neuronal dysfunction and neurodegeneration. Several mechanisms have been proposed to explain the impact of Aβ on neuronal function, including synaptic dysfunction, oxidative stress and inflammation, mitochondrial dysfunction, neuronal death and tau hyper phosphorylation ^{25, 26}. But, the impact of Aβ aggregation on mRNA m6A modification remains illusive. To this end, we examine the distribution of Aβ plaques and m6A profiles using immunofluorescence (Fig. 1D-1F). Next, we quantified the fluorescence intensity of m6A around Aβ and non-Aβ regions. The results showed that the levels of m6A surrounding Aβ plaques were significantly lower compared to the non-Aβ region and control brain, as early as 3 months of age (Fig. 1D).

Profiles the m6A enzymes expression in the adult brain.

The observed reduction in m6A levels surrounding Aβ plaques suggested a potential link between Aβ plaques formation and alterations in mRNA m6A modification during AD development. To further explore this relationship, We examined the features of YTH domain family proteins, including YTHDF1, YTHDF2, YTHDF3, and YTHDC1, which are involved in reading methylated RNA; methyltransferases METTL3, METTL14, and WTAP; and demethylases ALKBH5 and FTO in normal and AD brain. We firstly assessed the expression of these m6A enzymes in neurons and glial cells using whole brain scRNA-seq data from adult mouse brain (YX Li, unpublished data). We observed that Ythdf1 ~ 3, Ythdc1, Igf2bp3, Mettl3, Mettl14, Wtap, Alkbh5, and Fto were abundantly expressed in 16 populations identified, except for hemoglobin-expressing vascular cells (Hb-VC) (sFig2). All regulatory factors were highly enriched in neurons, including mature neuron (mNEUNR), immature neuron (ImmN) and neuroendocrine cell (NendC) (Fig. 2A, 2E, 2I and sFig.2). Additionally, Ythdf2, Ythdf3, Wtap, Alkbh5 and Fto were highly expressed in glial cells, such as astrocytes, oligodendrocytes, and microglia (Fig. 2A, 2E, 2I and sFig.2).

Next, we further investigated the cellular localization of these regulatory factors in the adult mouse brain using immunofluorescence. The m6A regulatory factors, including writers (METTL3, METTL14 and WTAP), erasers (FTO and ALKBH5), and readers (YTHDF1, YTHDF2 and YTHDF3), were co-stained with the neuronal and glia cell markers. We found that these m6A regulatory factors were widely and strongly expressed in neurons within the hippocampus and cortex of the adult mouse brain in the steady state (Fig. 2). In contrast to observation from scRNA-seq, these proteins were hardly detectable in glia cells under the steady-state condition (sFig.3, sFig4 and sFig5).

Dynamic changes of m6A-related regulatory factors in the progression of Alzheimer's disease

Next, we investigated the alterations in m6A-related regulatory factors at the RNA level during different stages of AD progression. Firstly, we analyzed published mouse bulk RNA-seq data, focusing on the dynamic changes of m6A enzymes during the development of AD ²⁷. We found that Wtap exhibited significant downregulation in expression during the early stage of AD (4-month-old), whereas Ythdc1, Ythdf3, and Fto showed significant changes in expression in the later stage of AD (18-month-old) compared to control samples (sFig. 6).

In this study, we conducted RT-qPCR experiments on total RNA extracted from the hippocampus of adult mice at 1, 3, 6, and 9 months of age (Fig. 3 and supplementary table 1). In the early stages of AD, we have observed a marked alteration in a number of m6A-related enzymes, including Mettl3, Wtap, Ythdf1, Ythdc1, and Alkbh5 (Fig. 3 and supplementary table 1). Previous studies have demonstrated that YTHDF2 involved in regulating hippocampal dentate gyrus-dependent learning and memory ²⁸. YTHDF3 has to be shown to be correlated with cognitive recovery²⁹. At 6 months, Ythdf2 and Ythdf3 expression were significantly downregulated in the 5xFAD group compared to the WT group (P < 0.05), Ythdc1 expression was also downregulated in the 5xFAD group (Fig. 3C). Unexpectedly, there were no significant differences in the expression levels of the proteins involved in mRNA m6A methylation modification between the 5xFAD and WT control groups (P > 0.05) at 9 months of age (Fig. 3). The changes in WTAP that we observed at 3 months of age were consistent with the previous sequencing data, indicating that alterations in m6A enzymes indeed occur early on during AD development (Fig. 3E).

The expression profile of METTL3 and WTAP is closely associated with the deposition of Aβ

m6A modification is catalyzed by the m6A methyltransferase complex, which consists of METTL3, METTL14, and WTAP. Among them, METTL3 is the core component of the m6A methyltransferase complex and plays a catalytic role, which is responsible for the catalytic activity of the complex, transferring the methyl group from S-adenosylmethionine (SAM) to the target RNA molecule ³⁰. Specifically, WTAP functions as a regulatory factor in the complex, modulating the localization and activity of the complex to promote efficient m6A modification ⁷. Previous studies have demonstrated that the expression of WTAP and METTL3 is dysregulated in the brains of AD patients and in animal models of AD ²². In the aforementioned RT-qPCR experiment, we observed a significant downregulation of Mettl3 and Wtap in the hippocampal region of 5xFAD mice as early as 3-month-old. This suggested the potential involvement of m6A regulatory factors in the onset of AD. To further validate this findings, we examined the expression profile of these proteins in the hippocampus during AD progression. The analysis of the fluorescence intensity showed no significant difference in the expression of METTL3 and WTAP in the AD group compared to the WT group at 1-month age (sFig. 7). The expression of WTAP and METTL3 surrounding the Aβ exhibited a decrease in abundance only after the appearance of significant Aβ accumulation (Fig. 4). The reduction of WTAP began as early as 3 months old, whereas the decrease in METTL3 expression started close to 6 months old. This implies that the decline in METTL3 expression occurs earlier than WTAP during the development of AD (Fig. 4E and 4K).

MeRIP-seq analysis of AD brain

To gain a deeper understanding of RNA m6A modification changes in AD, Methylated RNA Immunoprecipitation Sequencing (MeRIP-seq) was performed on the whole brain of 9-month-old 5xFAD and age-matched C57BL/6 control mice, with four mice in each group. MeRIP-seq results were analyzed, and m6A peaks with |fold change|>1.5 and P < 0.05 were selected for both the WT and 5xFAD groups, peaks represented regions in the genome that are modified with m6A. The results showed that there were 11,664 and 11,573 m6A modification sites in the WT and 5xFAD groups, respectively, with 10,884 overlapping m6A modification sites between the two groups (Fig. 5C, supplementary table2). The exomePeak package was used for genome-wide peak scanning, and it was found that m6A peaks were mainly distributed in the coding region (CDS), 3' untranslated region (3'UTR), and 5' untranslated region (5'UTR) in both the AD and WT control groups (sFig. 5B). The differential peaks between the two groups were then analyzed, and it was found that, compared to the WT group, the 5xFAD group had 607 upregulated and 645 downregulated m6A peaks (Fig. 5D). The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations suggested that differentially expressed m6A-modified genes found in 5xFAD were involved in DNA damage, cellular senescence, apoptosis, and N-glycan biosynthesis (Fig. 5E and 5F, and supplementary table 3).

To investigate the relationship between m6A methylation and gene expression in the AD process, we performed RNA sequencing (RNA-seq) on brain tissues from 5xFAD and WT mice. The analysis of differentially expressed genes (DEGs) showed that, compared to the WT group, 77 genes were significantly upregulated and 31 genes were significantly downregulated in the 5xFAD group (Fig. 6A). Subsequently, based on the results of differential genes and differential peaks, a correlation analysis was performed to integrate transcription with m6A methylation levels (Fig. 6B). A four-quadrant plot was generated to observe changes in gene expression as m6A methylation levels changed. The Hyper-up quadrant represents upregulated m6A peaks and upregulated gene expression; the Hyper-down quadrant represents upregulated m6A peaks and downregulated gene expression; the Hypo-up quadrant represents downregulated m6A peaks and upregulated gene expression; and the Hypo-down quadrant represents downregulated m6A peaks and downregulated gene expression. We identified 25 genes in the hyper-up quadrant (like Clec7a, Mpeg1, H2q7, H2k1), 14 genes in the hypo-down quadrant (Pigh, Tomt, Zbed5), 33 genes in the hyper-down quadrant (Cap6, Cstad, Lsm2, Cradd) and 31 genes in the hypo-up quadrant (Ilr1, Gvin1, Ch25h, Ifit3) (Fig. 6B and supplementary table 4).

Next, we performed GO term enrichment and KEGG analysis on the genes identified in the four quadrants. The results showed that the genes with differential m6A peaks identified in the AD were enriched in processes such as regulation of IFNγ production, VEGF production, and regulation of Th1 immune response (Fig. 6C). These are crucial for immune system function ^31–33.The KEGG analysis revealed that the genes with expression related to m6A peak changes in the AD group were mainly enriched in several pathways, including neutrophil extracellular trap formation, cytokine-cytokine receptor interaction, steroid hormone biosynthesis, platelet activation, primary bile acid biosynthesis, and glycosaminoglycan degradation (Fig. 6D and supplementary table 5). These pathways have been implicated in the clearance of Aβ plaques, regulation of innate immune response, regulation of neuroprotective, regulation of cerebral blood flow and lipid metabolism in the brain, respectively ^34–37.

mRNA m6A methylation and the correlation with microglia transcription

Based on the annotation in the differential peaks identified in m6A-seq data, there may be a connection between mRNA m6A methylation and the regulation of microglial gene expression, which is relevant to the pathogenesis of AD. Indeed, microglia are crucial for AD development, and disease-associated microglia (DAM) are a specific subset of microglia that have been identified in the context of neurodegenerative diseases such as AD. We observed 25 DAM genes were highly expressed in 9-month-old AD brains. (Fig. 7Aand 7B). In these 25 genes, most of them exhibit increased m6A modifications, such as Csf1, Ccl6, Mpge1, and Cd68 (Fig. 7D). These studies propose a link between m6A modifications and the function of microglia in AD. Thus, we employed the MACS technique to isolate microglia from 9-month-old AD mice and examined the expression of m6A and m6A enzymes (Fig. 7E). Our findings revealed an upward trend in m6A levels within microglia (Fig. 7F). Notably, m6A writers Ythdf1, Ythdf2, Ythdf3, and the eraser Alkbh5 exhibited a significant upregulation (Fig. 7G). We further observed upregulated expression of ALKBH5 but not m6A writers in microglia surrounding Aβ plaques within the hippocampal region (Fig. 7H), suggesting the implication of ALKBH5 in microglial response to Aβ plaques accumulation.

mRNA m6A methylation has been found to be associated with the occurrence of cancer, such as breast cancer and lung cancer ³⁸, but the relationship between RNA m6A methylation and the central nervous system is largely unknown. Although the level of mRNA m6A modification is relatively high in the brain, research on m6A-modified RNA in the brain is still a nascent field, and the significance of this epigenetic marker in the CNS has only recently been recognized ^{23, 39}. In this study, we focused on investigating the dynamic changes in mRNA m6A and m6A enzymes during the development of AD. Immunofluorescence staining revealed that RNA m6A modification is relatively high in neurons, but low in glial cells. But the precise reason for the low level of m6A expression in glial cells compared to neurons is not yet fully understood. The overall level of m6A modification gradually decreases with age, but there was no significant difference detected in total m6A levels between AD and normal controls at the same age. However, after detecting Aβ accumulation at 3 months of age, we observed a decrease in m6A levels surrounding Aβ plaques. Furthermore, we observed changes in the expression of m6A enzymes during AD development, with downregulation of METTL3 and WTAP occurring as early as 3 months of age, suggesting a link between dysregulated m6A RNA modification and the onset of AD.

Similarly, a previous study analyzed a public RNA-seq dataset and found that the RNA methyltransferase METTL3 and the component of the m6A methyltransferase complex RBM15B were downregulated and upregulated, respectively, in the hippocampus ⁴⁰. Additionally, our finding is consistent with other studies that demonstrated decreased levels of METTL3 and METTL14 in the brain tissue from patients with mild cognitive impairment, a precursor stage of AD, suggesting a potential role for mRNA m6A modification in the early stages of the disease ²². In adult mouse hippocampus, the reduction of m6A modification caused by Mettl3 gene knockout resulted in cognitive and memory impairment, which are characteristic manifestations of AD. This study also demonstrated that METTL3 is necessary for the maintenance of dendritic spine and synaptic integrity, as well as neuronal survival. Another study showed that suppression of FTO demethylase to restore m6A modification levels in an AD model rescued pathological changes in the brain, such as neuronal death. However, in contrast to our finding, another study found that the level of mRNA m6A methylation is increased in the cortex and hippocampus of an AD mouse model (APP/PS1 transgenic mice) compared to control, and the expression level of METTL3 is upregulated while FTO is downregulated in AD mice ⁴¹. The reason for this discrepancy may be attributed to the different AD mouse models used.

Lastly, analysis of m6A-seq in the brains of AD and WT mice, we found that the differentially methylated peaks were enriched in GO and KEGG terms related to processes such as inflammation response, immune system processes, signal transduction, positive regulation of lipid metabolism, and cytokine production. These findings suggest a link between mRNA m6A methylation and the regulation of microglia gene expression. Microglia are immune cells that play a critical role in the central nervous system, and dysregulation of microglial activation has been implicated in the pathogenesis of neurodegenerative diseases, including AD ⁴². One study found that silencing of the m6A methyltransferase METTL14 resulted in decreased expression of pro-inflammatory genes and increased expression of anti-inflammatory genes in microglia, suggesting that m6A methylation regulates microglial polarization and activation ⁴³. Another study found that the m6A methyltransferase METTL3 was upregulated in activated microglia in response to lipopolysaccharide (LPS) stimulation. Inhibition of METTL3 resulted in decreased expression of pro-inflammatory cytokines and chemokines in microglia, indicating that m6A methylation is involved in the regulation of microglial inflammatory responses ⁴⁴. In addition, a recent study found that m6A methylation plays a critical role in the regulation of microglial phagocytosis ⁴⁵. Our observation suggested ALKBH5 is close like to microglia response to Aβ accumulation. Taken together, these findings suggest that mRNA m6A methylation plays a crucial role in the regulation of microglia function and gene expression, and dysregulation of this process may contribute to the pathogenesis of neurodegenerative diseases such as AD.

Ethics approval and consent to participate

All animal procedures adhered to the guidelines set by the Institutional Animal Care and Use Committee and were conducted in the Animal Facility of the Department of Laboratory Animal Science at Fudan University.

Consent for publication

Not applicable.

Availability of data and materials

MeRIP-seq data are available in the Genome Sequence Archive (GSA) with accession code CRA010646.

Competing interests

The authors have no conflicts of interest to disclose. All authors grant permission to publish the submitted content.

Funding

This work was supported by grants from the National Natural Science Foundation of China (32170958).

Author contributions

Y.R. and B.P. conceived and designed this study. Y.R. and B.P supervised and conceptualized this study. Y.R., and Y.W. wrote the manuscript. Y.W. performed most experiments. X.L. analyzed the sequencing results, J.W.,T.-F.Y. provided necessary study support. All authors discussed the results and commented on this manuscript.

Acknowledgements

The authors thank Yuxiao Jin, Shuai Gao (Fudan University) for donating 5xFAD mice. Fang Lei and Jiachen Zhu (Fudan University) for the excellent laboratory management. Yanxia Rao would like to express her gratitude to Pai Peng for his patience and support during the drafting of this manuscript. In addition, the authors express their gratitude and respect to all animals sacrificed in this study. In memory of Prof. Huaxi Xu. This study was supported by STI2030-Major Projects (2022ZD0207200) (Y.R.) and (2022ZD0204700) (B.P.), National Natural Science Foundation of China (32170958) (B.P.), Program of Shanghai Academic/Technology Research Leader (21XD1420400) (B.P.), Shanghai Pilot Program for Basic Research (21TQ014) (B.P.), The Innovative Research Team of High-Level Local University in Shanghai (B.P.).

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No competing interests reported.

sf1.tif
Supplementary Figure 1. m6A was not expressed in glial cells in adult mouse brain. (A) Double labeling stain with m6A and microglia marker (Iba1), astrocyte marker (GFAP) and oligodendrocyte (Olig2) antibody in the hippocampus of 3, 6 and 9-mouth-old WT and 5xFAD mice. (B-D) Quantification of the expression of m6A in microglia, astrocyte and oligodendrocyte in the hippocampus of 3, 6 and 9-mouth-old WT and 5xFAD mice. Scale bar is 20 µm.
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Supplementary Figure 2. Using scRNA-seq data from the whole brain to evaluate the expression of m6A-related genes in neurons and glial cells of adult normal mice.
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Supplementary Figure 3. m6A readers (YTHDF1-3) was co-stained with IBA1, Olig2 and GFAP in 3-month-old normal adult mice, respectively. Scale bar are provided in the images.
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Supplementary Figure 4. m6A writers (METTL3/14 and WTAP) was co-stained with IBA1, Olig2 and GFAP in 3-month-old normal adult mice, respectively. Scale bar are provided in the images.
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Supplementary Figure 5. m6A erasers (ALKBH5 and FTO) was co-stained with IBA1, Olig2 and GFAP in 3-month-old normal adult mice, respectively. Scale bar are provided in the images.
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Supplementary Figure 6. Analysis of the public database of 5xFAD mouse brain transcriptome (GSE168137) at 4, 8, 12 and 18-months of age href="#_ENREF_27" title="Forner, 2021 #59">²⁷
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Supplementary Figure 7. (A) Representative confocal images of co-staining of WTAP and 6E10 (Aβ). (B) Statistics of the fluorescence intensity of WTAP in 1-month-old WT and 5xFAD mice brains. N=4 or 5 mice for each group. Two-tailed independent t test. (C) Representative confocal images of co-staining of WTAP and 6E10 (Aβ). (D) Statistics of the fluorescence intensity of METTL3 in 1-month-old WT and 5xFAD mice brains. N=4 or 5 mice for each group. Two-tailed independent t test. Scale bar is 50 µm.
supplementarytable1qPCRrawdata.xlsx
supplementarytable25FADvsCtrldiffPeakexpression.xlsx
supplementarytable3diffpeakKEGGandGOBPresults.xlsx
supplementarytable4m6Adownupandgenedownuplists.xlsx
supplementarytable54quartantKEGGandGOBPresults.xlsx
supplementarytable6primerantibodyusedinthisstudy.xlsx

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Dynamics of N6-methyladenosine modification during Alzheimer's disease development

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Abstract

Figures

Introduction

Materials and methods

Animals

Tissue preparation for RNA extraction and immunohistochemistry

Quantification of the m6A Modification

Quantitative Real-Time PCR

Immunohistochemistry

MeRIP-seq

MeRIP-seq data analysis

Statistical Analysis

Results

Aβ plaque accumulation is associated with mRNA m6A modification

Profiles the m6A enzymes expression in the adult brain.

Dynamic changes of m6A-related regulatory factors in the progression of Alzheimer's disease

The expression profile of METTL3 and WTAP is closely associated with the deposition of Aβ

MeRIP-seq analysis of AD brain

mRNA m6A methylation and the correlation with microglia transcription

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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