Down-regulation of EPB41L4A-AS1 mediated the brain aging and neurodegenerative diseases via damaging synthesis of NAD+ and ATP

doi:10.21203/rs.3.rs-927236/v1

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Down-regulation of EPB41L4A-AS1 mediated the brain aging and neurodegenerative diseases via damaging synthesis of NAD⁺ and ATP

https://doi.org/10.21203/rs.3.rs-927236/v1

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Background: Long noncoding RNA EPB41L4A-AS1 plays a very important role in metabolism. Aging and neurodegenerative diseases are typical metabolic-related processes. As a metabolism-related lncRNA, EPB41L4A-AS may be involved in the development of brain aging and neurodegenerative diseases. In this study, we aim to reveal the mechanism of EPB41L4A-AS in aging and neurodegenerative diseases.

Methods: Age-related differential expression analysis was applied on the gene expression profile of the hippocampus in the Genotype-Tissue Expression database to obtain age-related differentially expressed genes and the weighted correlation network analysis algorithm was used to construct a gene co-expression network for age-related differentially expressed genes to obtain different gene clustering modules. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, protein-protein interaction network, and correlation analysis were used to reveal the mechanism of EPB41L4A-AS1. The mechanism was verified using Gene Expression Omnibus profile GSE5281 and biology experiments (construction of cell lines, Real-time quantitative PCR, Western blot, measurement of ATP and NAD⁺ levels, nicotinamide riboside treatment, Chromatin Immunoprecipitation) in neurons and glial-derived cells.

Results: EPB41L4A-AS1 is down-regulated in aging and Alzheimer’s disease. EPB41L4A-AS1 related genes are genes of the electron transport chain and NAD⁺ synthesis pathway. Furthermore, these genes are highly related to neurodegenerative diseases and EPB41L4A-AS1 has a positive correlation with them. In addition, biology experiments proved that the down-regulation of EPB41L4A-AS1 can reduce the expression of these genes via modification of the acetylation of lysine 27 on histone 3, resulting in the down-regulation of NAD⁺ and ATP levels, while the overexpression of EPB41L4A-AS1 and nicotinamide riboside treatment can restore the levels of NAD⁺ and ATP.

Conclusions: Down-regulation of EPB41L4A-AS1 not only disturbs NAD⁺ biosynthesis but also affects ATP production. As a result, the high demand of brain for NAD⁺ and ATP can not be met, which promotes the development of brain aging and neurodegenerative diseases. However, overexpression of EPB41L4A-AS1 and nicotinamide riboside, a substrate of NAD⁺ synthesis, can reduce EPB41L4A-AS1 down-regulation mediated decrease of NAD⁺ and ATP synthesis. Our results provide a new perspective on brain aging and neurodegenerative diseases.

General Biochemistry

Molecular Genetics

Molecular Biology

Aging

Neurodegenerative diseases

EPB41L4A- AS1

NAD+

ATP

H3K27Ac

Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs that are usually longer than 200 nucleotides in length [1]. LncRNAs have been regarded as useless components in cells for a long time, but in recent years, more and more studies show that lncRNAs play important regulatory roles in cells with the continuous deepening of research. It has been reported that lncRNAs are involved in many processes, such as the MEK/ERK signaling pathway [2], ferroptosis [3], metastasis of clear cell renal cell carcinoma [4], and lncRNAs are considered to be highly related to the occurrence of cancers [5,6]. Not only that, recent studies show that lncRNAs play an important regulatory role in cell metabolisms, such as glycolysis [7] and Aβ clearance [8].

EPB41L4A-AS1 was first reported as a protein-coding gene, named TIGA1 related to cell proliferation [9], but now it is found that EPB41L4A-AS1 mainly functions as a lncRNA which is associated with cell metabolism and immune response [7,10,11]. In cancer cells, downregulation of EPB41L4A-AS1 induces glutamine dependency and promotes glycolysis [7]. In placental trophoblast cells, the downregulation of EPB41L4A-AS1 inhibits the Warburg effect which is needed for fast growth of placental trophoblast cells and causes recurrent miscarriage [10]. Furthermore, down-regulation of EPB41L4A-AS1 can activate the MYD88-Dependent NF-κB pathway in diabetes-related inflammation [11].

The decline of brain energy metabolism is an important reason for age-associated functional decline and diseases of the brain and it has subtly appeared even before the diagnosis of neurodegenerative diseases [12]. Mitochondrial dysfunction is the main cause of the decline of brain energy metabolism in the aging brain and neurodegenerative diseases. NAD⁺ depletion is a contributing factor for mitochondrial dysfunction, which will reduce the production of ATP, the main currency of brain energy metabolism, leading to brain dysfunction [13~15]. NAD⁺ is not only a key coenzyme in energy metabolism but also a cofactor or substrate of hundreds of enzymes, playing multiple roles for DNA damage repair, gene expression, and stress response [13,16,17]. Therefore, the decline of NAD⁺ during aging influences many key cellular functions and induces many aging-associated diseases, including neurodegenerative diseases [16,18]. However, the underlying mechanism that causes NAD⁺ depletion in the aging brain and neurodegenerative diseases is still unclear, especially the role of lncRNAs.

In the present study, we found that the expression of EPB41L4A-AS1 gradually decreased as people get older. Because EPB41L4A-AS1 plays an important role in metabolism, we speculate that the down-regulation of EPB41L4A-AS1 has an important effect on brain metabolism and may be related to the occurrence of aging and neurodegenerative diseases. By biology experiments, we find that the expressive disorder of EPB41L4A-AS1 induces metabolic damage, manifested as a decrease of NAD⁺ and ATP synthesis. The aging-related down-regulation of EPB41L4A-AS1 mediated the development of brain aging and neurodegenerative diseases and overexpression of EPB41L4A-AS1 can reduce the symptoms. Our results provide new insight into the mechanism of brain aging and neurodegenerative diseases.

Data sets and bioinformatics analysis

Two data sets were used in the bioinformatics analysis. GTEx (Genotype-Tissue Expression) includes the gene expression data of 54 normal tissues of the human body while the age, gender, tissue location of the samples are also collected. In this study, we used the gene expression data of 13 brain regions. GEO data set GSE5281 was used to check whether the theory we proposed is right in Alzheimer’s disease.

Differential gene expression analysis was used to find differentially expressed genes. differentially expressed genes (DEGs) often have important biological functions. R packages edgeR [19] and limma were used in this process. The required input data are the gene expression matrix and sample grouping matrix. In this study, we grouped samples according to their ages to obtain genes with the age difference.

WGCNA (weighted correlation network analysis) was used to perform cluster analysis on genes, and classify genes with similar expression patterns into the same module, thereby obtaining several modules. Besides, through the analysis of sample traits of modules, the most relevant modules for the target trait (age) were selected. R package WGCNA [20] was necessary for the process.

GO (Gene Ontology) [21] and KEGG (Kyoto Encyclopedia of Genes and Genomes) [22] enrichments were used to show the biological functions and pathways of the target genes. The target genes may be involved in many functions or pathways, however, the gene ratios and P-values of each function or pathway can show which function or pathway is the most important.

Correlation analysis can reveal the correlation coefficients between EPB41L4A-AS1 and the target genes, thus revealing an important regulatory role between them. The significance test shows the credibility of this role. The correlation coefficient was calculated using the Pearson method, and the significance test was performed using the Mann-Whitney test method.

Protein-protein interaction (PPI) network can display the interactions between proteins, including interactions from text mining, experiments, databases, co-expression, neighborhood, gene fusion, and co-occurrence [23]. The website STRING (https://www.string-db.org/) was used in this process.

Cell lines and transfection

SH-SY5Y (ATCC, CL-0208) and U251 (ATCC, CL-0237) cells were cultured in DMEM/F 12 medium (Thermo Fisher Scientific) or DMEM medium (Thermo Fisher Scientific) with 10% FBS (Thermo Fisher Scientific) in 5% CO2-humidified incubator at 37 ℃. SiRNAs for EPB41L4A-AS1, negative control siRNA vectors, lentivirus carrying EPB41L4A-AS1 RNAi, negative control shRNA vectors, EPB41L4A-AS1 overexpression plasmids, and empty plasmids used as negative control were purchased from GenePharma (Suzhou, China). For transient transfection, siRNAs for EPB41L4A-AS1 and EPB41L4A-AS1 overexpression plasmids were transfected using Lipofectamine™ 2000 Transfection Reagent (Thermo Fisher Scientific) by the manufacturer protocol. To obtain EPB41L4A-AS1 stable knockdown cell lines, U251 and SH-SY5Y cells were transfected with shEPB41L4A-AS1 lentivirus, followed by 1 µg/mL final concentration of puromycin (Thermo Fisher Scientific) selection.

Real-time quantitative PCR

RNA iso Plus (Takara) was used to extract total RNA. Reverse transcription of the total RNA was performed by One-Step RT-PCR SuperMix (TransGen Biotech). PerfectStartTM Green qPCR SuperMix (TransGen Biotech) was used to perform Real-time qRT-PCR. All mRNA levels were measured from three independent experiments and normalized to ACTB. Primers needed for Real-time quantitative PCR are shown in Table S1.

Western blot

Proteins were extracted from cultured cells by cell extract buffer (50 mM Tris-HCl, pH 8.0, 4 M urea, and 1% Triton X-100) with protease inhibitor mixture (Roche Diagnostics, Cat.No:04693132001). Cell lysates were resolved by SDS-PAGE and then analyzed by western blotting. The following antibodies were used: ACTB (Proteintech, #20536-1-AP, 1:5000), NMNAT2 (Abnova, #H00023057-M04, 1:1000), NDUFS4 (Novus, #NBP1-31465, 1:1000), NDUFS6 (Novus, #NBP1-49831, 1:1000).

Measurement of ATP and NAD⁺ levels

ATP level was measured using Enhanced ATP Assay Kit (Beyotime Biotechnology) and NAD⁺, NADH levels were measured using Amplite^TM Colorimetric NAD/NADH Ratio Assay Kit (AAT Bioquset) according to the manufacturer's protocol.

NR (nicotinamide riboside) treatment for cells

NR was purchased from MCE (#HY-123033). Cells were cultured in the medium containing 100 mM NR after adhesion, and the follow-up experiments were carried out 24 hours later. Cells cultured in the medium without NR were used as controls.

ChIP (Chromatin Immunoprecipitation) assay

ChIP assay was conducted as described previously in [8]. In brief, the cells were fixed with 1% formaldehyde and sonicated to shear the DNA. After centrifugation, the supernatants were incubated with H3K27Ac (Abcam, ab4729). Chromatin DNA was purified with protein G Dynabeads (Invitrogen, 10004D) and subjected to real-time PCR. The region-specific primers used are listed in Table S1.

EPB41L4A-AS1 is down-regulated in aging and neurodegenerative diseases

EPB41L4A-AS1 is a metabolism-related lncRNA. Brain metabolic patterns are altered in aging and neurodegenerative diseases, especially in AD. Therefore, we think the expression of EPB41L4A-AS1 may be dysregulated in these conditions. To address these issues, the Mann-Whitney test method was used to compare the expression of EPB41L4A-AS1 in different age groups, and between normal and AD groups. As shown in Fig. 1A-B, EPB41L4A-AS1 expression gradually decreases with age increasing, and its expression is also down-regulated in the AD group, compared with normal people.

Differences of gene expression in the hippocampus of different age groups

The hippocampus is the main damaged area in aging and neurodegenerative diseases, so gene expression profiles (32972 genes) of 197 samples from the hippocampus (31 samples 20-50 years old, 52 samples 50-60 years old, 114 samples 60-80 years old) were used for gene differential expression analysis (age-related). To get better results, the expression profiles of two sets of samples (20-50 years old, 60-80 years old) were used as an expression matrix.

A total of 6538 DEGs (3246 up-regulated and 3292 down-regulated) between the elderly group (60-80 years) and the young group (20-50 years) were screened out under the threshold of p-Value < 0.05. The down-regulation of EPB41L4A-AS1 is also confirmed in the volcano plot of DEGs (Fig. 1C).

Construction of Co-Expression Modules

To find genes having similar expression patterns with EPB41L4A-AS1, the expression profile of all DEGs in 197 samples from the hippocampus was extracted to construct a weighted co-expression network using the WGCNA algorithm. The appropriate samples were screened out by sample clustering (Fig. 1D), and the appropriate power value was screened out (Fig. 1E-F). Scale-free networks were constructed with proper independence degree and average connectivity when the power value was equal to 14. The DEGs with similar expression patterns were cluster into four distinct gene co-expression modules (Fig. 1G).

Interaction Analysis with Clinical Traits and GO, KEGG Enrichment

The heatmap (Fig. 2A) demonstrates the Topological Overlap Matrix (TOM) among 400 randomly chosen genes in the analysis. The correlation of three modules successfully clustered is shown in the heatmap (Fig. 2B). As a result, each module is independent of the other.

EPB4L4A-AS1 is located in the turquoise module, so we speculate that the protein-coding genes in the turquoise module may be regulated by EPB41L4A-AS1. At the same time, module-trait relationships (Fig. 2C) show that there is a negative correlation (r = -0.35, p < 0.001) between the genes in the turquoise module and age.

To obtain the further function of the turquoise module, GO and KEGG enrichments are applied to all the genes in the turquoise module. The top 10 GO terms and KEGG pathways are visualized in Fig. 2D-E. As showed in the figures, genes in the turquoise module are mainly enriched in nerve excitement transmission and neurodegenerative diseases (such as Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), Parkinson’s disease (PD), and Prion disease) pathway.

Correlation of EPB41L4A-AS1 and Protein-coding Genes (PCGs)

The genes of five neurodegenerative disease-related pathways are processed by the intersection (Fig. 3A). As a result, 55 PCGs are related to all five diseases. A more in-depth GO enrichment analysis of these 55 genes shows that these 55 genes mainly influence oxidative phosphorylation, ATP metabolic process, and electron transport (Fig. 3B). Therefore, we speculated that these 55 PCGs play very important roles in aging and neurodegenerative diseases. It has been proved by cluster analysis that the expression patterns of EPB41L4A-AS1 and these PCGs are similar, thus correlation analysis of EPB41L4A-AS1 and these PCGs is necessary. It shows that EPB41L4A-AS1 is mainly positively correlated with these 55 PCGS in the hippocampus (Fig. 3C) and EPB41L4A-AS1 and these 55 PCGs are mainly down-regulated as brain ages (Fig. 3D). To prove the universality of this relationship, the correlation analysis of 13 brain regions is displayed in the heatmap (Fig. S1A). Furthermore, the fold changes in gene expression of EPB41L4A-AS1 and 55 PCGs in 13 brain regions show that EPB41L4A-AS1 and 55 PCGs are universally down-regulated in the brain with aging (Fig. S1.B). As a result, the relationship is universal.

Gene Function Verification in AD Dataset

To investigate if there are some similarities of the expressive pattern of EPB41L4A-AS1 related genes between the aging group and AD neurodegenerative diseases group, gene expression dataset GSE5281 was used in the next step. For technical reasons, the expression data of 45 PCGs among 55 PCGs were detected in dataset GSE5281. Therefore, we applied the correlation analysis of EPB41L4A-AS1 and 45 PCGs in GSE5281 (Fig. 3E). The result shows that there is a strong positive correlation between EPB41L4A-AS1 and other PCGs. Also, EPB41L4A-AS1 and these PCGs are down-regulated in AD samples (Fig. 3F). The results further prove that the relationship we proposed before is reliable.

Protein-Protein Interaction Network of 55 PCGs

To reveal the protein-protein interaction network of the 55 PCGs, 55 PCGs were uploaded to STRING and the network is shown in Fig. 4A. The 55 PCGs are mainly the components of complex I-V, which mainly affect the third stage of cellular respiration through molecular biological analysis. The function of complex I is to catalyze the production of NAD⁺ from NADH [24,25] while complex I-V can catalyze the production of ATP from ADP by affecting the electron transport chain [26,27].

At this stage, we found that EPB41L4A-AS1 has a strong positive correlation with 55 PCGs, and these 55 genes were involved in the formation of complex I-V on the electron transport chain. By analyzing the reactions related to the electron transport chain, we speculated that the down-regulation of EPB41L4A-AS1 will down-regulate the expression of the components of complex I-V, thus reducing the activity of complex I-V, and ultimately leading to the down-regulation of NAD⁺ and ATP levels.

Previous studies have reported that brain NAD⁺ is associated with ATP energy production and membrane phospholipid turnover in humans [28], and ATP plays a very important role in the normal brain [29]. Furthermore, NAD⁺ has been reported to be strongly related to aging and neurodegenerative diseases [18,30], and NAD⁺ can inhibit Alzheimer’s disease [31]. As a result, when NAD⁺ and ATP levels go down, the body’s energy and anti-aging ability will decrease, which may eventually lead to aging and neurodegenerative diseases [32].

Analysis of NAD⁺ synthesis pathway genes

NAD⁺ level is not only related to electron transport chain but also maintained by three independent biosynthetic pathways, kynurenine pathway (or de novo synthesis pathway), Preiss–Handler pathway, and salvage pathway [16]. Furthermore, it has been reported that the kynurenine pathway is associated with neurodegenerative diseases [33,34], while the salvage pathway protects neurons from chemotherapy-induced degeneration [35]. Inspired by these, we analyzed the expression levels of genes in the NAD⁺ biosynthesis pathways in aging and neurodegenerative diseases. As a result, we found that only NMNAT2 (enriched in the cytoplasm [36]) is a DEG and it mainly expresses in the brain (Fig. S2A). NMNATs catalyze the reaction of NAMN (Preiss–Handler pathway) and NMN (salvage pathway) to form NAD⁺ and NMNAT2 is the main form of NMNATs in the brain (Fig. S2B). In addition, NMNAT2 is located in the same module (turquoise module) as EPB41L4A-AS1 in the WGCNA process, and it’s also a down-regulated gene with aging (Fig. 4B). Correlation analysis of EPB41L4A-AS1 and NMNAT2 also showed that EPB41L4A-AS1 has a strong positive regulation on NMNAT2 in aging (Fig. 4C). For further study, we analyzed the expression level of NMNAT2 in normal and AD samples and its correlation with EPB41L4A-AS1 in dataset GSE5281. As shown in Fig. 4D-E, NMNAT2 is down-regulated in Alzheimer’s disease and has a strong correlation with EPB41L4A-AS1.

In short, EPB41L4A-AS1 may regulate the NAD⁺ and ATP generation via impacting the genes of the electron transport chain and NAD⁺ synthesis pathway (NMNAT2), and thus plays an important role in brain aging and neurodegenerative diseases. The mechanism is shown in Fig. S2C.

EPB41L4A-AS1 has a positive regulation of NAD⁺ and ATP synthesis in SH-SYSY and U251 cells

In this process, we applied experiments on SH-SY5Y and U251 cells to prove the role and mechanism of EPB41L4A-AS1 in brain aging and neurodegenerative diseases. For each complex of the mitochondrial respiratory chain, we selected two PCGs with the highest correlation coefficient with EPB41L4A-AS1 for experiments. As a result, a total of 11 PCGs (10 genes of 5 complexes, NMNAT2) were used to check if EPB41L4A-AS1 plays a regulatory role in them.

Firstly, we used siRNAs to knock down EPB41L4A-AS1 in SH-SY5Y cells to observe the changes of 11 PCGs. Two siRNAs (si-EPB41L4A-AS1-1, si-EPB41L4A-AS1-2) were successfully transiently transfected and the relative mRNA expressions of 11 target genes were down-regulated (Fig. 5A). We also applied sh-EPB41L4A-AS1 lentivirus transfection to obtain EPB41L4A-AS1 stable knockdown cell lines in the next step so it can prove if the changes of 11 target genes are stable when EPB41L4A-AS1 is knocked down. Two sh-EPB41L4A-AS1 cell lines (sh-EPB41L4A-AS1-1, sh-EPB41L4A-AS1-2) were obtained and the results are as same as transient transfection (Fig. 5B). Finally, EPB41L4A-AS1 overexpression plasmids were successfully transfected in the sh-NC cell line and two sh-EPB41L4A-AS1 cell lines. As shown in Fig. 5B, the expression levels of the 11 PCGs rebound when EPB41L4A-AS1 is overexpressed.

To increase the reliability of cell experiments, we carried out the same experiments on U251 cells in the next step. As shown in Fig. S3, the expressions of 11 target genes were down-regulated when siRNAs were transiently transfected (Fig. S3A) and sh-EPB41L4A-AS1 lentivirus transfections were applied (Fig. S3B). Also, the expressions of the 11 target genes were up-regulated as EPB41L4A-AS1 is overexpressed (Fig. S3B).

In the next step, we applied Western blot analysis to reveal the protein levels of the NAD⁺ and ATP synthesis genes in SH-SY5Y and U251 cells. For ATP synthesis genes, NDUFS4 and NDUFS6 were chosen as the representative genes because the proportion of genes constituting complex I is the largest in previous bioinformatics analysis. Therefore, the protein levels of NMNAT2, NDUFS4, and NDUFS6 were measured in this step. As shown in Fig. 6A and Fig. 6B, the protein levels of these genes are down-regulated when EPB41L4A-AS1 is knocked down, and once EPB41L4A-AS1 is overexpressed, their protein levels were up-regulated.

We finally measured NAD⁺, NADH, and ATP levels in SH-SY5Y and U251 cells. As shown in Fig. 6C and Fig. 6D, NADH is not changed significantly while NAD⁺, NAD⁺/NADH ratio, and ATP levels are down-regulated in sh-EPB41L4A-AS1 cells, and overexpression of EPB41L4A-AS1 upregulated them. So far, we have proved the correctness of our proposed regulatory mechanism from the perspectives of bioinformatics, molecular biology, and cell biology.

NR treatment can improve the NAD⁺ and ATP levels in SH-SY5Y and U251 cells

Overexpression of EPB41L4A-AS1 can restore the levels of ATP and NAD⁺. NAD⁺ depletion is a contributing factor for mitochondrial dysfunction, which will also reduce the production of ATP. Therefore, we conducted the NR (nicotinamide riboside, a substrate of NAD⁺ synthesis) treatment experiment to see if NR can rescue EPB41L4A-AS1 down-regulation mediated decrease of NAD⁺ and synthesis, then, furthermore to rescue the decrease of ATP synthesis mediated by the decrease of NAD⁺ synthesis. As shown in Fig. 7A and Fig. 7B, NAD⁺ and ATP levels rebound after NR treatment in SH-SY5Y and U251 cells. The results show that NR and EPB41L4A-AS1 have the same effect in increasing the levels of ATP and NAD⁺.

EPB41L4A-AS1 has a positive regulation of the acetylation of histones

According to the reported mechanism, EPB41L4A-AS1, functioning as a lncRNA, regulated gene expression at the epigenetic level. It mainly affects the acetylation of histones [7]. In this study, we conducted a chromatin immunoprecipitation (ChIP) assay to measure the H3K27Ac levels at NMNAT2 gene promoter in SH-SY5Y and U251 cells. As shown in Fig. 7C-D, EPB41L4A-AS1 inhibition down-regulates H3K27Ac located nearby to the transcription start site (TSS) of NMNAT2. The results explain the regulatory mechanism of EPB41L4A-AS1 at the epigenetic level.

In this study, we performed bioinformatics analysis to reveal the mechanism of lncRNA EPB41L4A-AS1 in brain aging in the first step. Through analyzing the data from GTEx and GEO data sets, we found that EPB41L4A-AS1 expression is down-regulated in the aging brain and AD group, compared with normal people. Then, we compared gene expression in the hippocampus between the elderly and the young group and generated the construction of co-expression modules. The DEGs with similar expression patterns were clustered into four distinct gene co-expression modules. EPB4L4A-AS1 is located in the turquoise module and the genes in the turquoise module have a relationship with aging and neurodegenerative diseases. Previous studies have shown that the occurrence of neurodegenerative diseases is closely related to changes in oxidative phosphorylation [37,38], which further illustrates the importance of the genes in the turquoise module.

In the turquoise module, 55 genes are obtained after the intersection of 5 neurodegenerative diseases. Through the correlation analysis and significance test of EPB41L4A-AS1 and 55 PCGs in 13 brain regions, we found that EPB41L4A-AS1 has a strong positive regulatory effect on these genes. In the next step, we verified the regulation mechanism using the AD dataset GSE5281 and got a very consistent result. Furthermore, through the functional analysis of these 55 genes, we found that these genes are highly related to the electron transport chain and ATP synthesis in the cell respiration process. PPI network also proves that they are mainly involved in the formation of complexes I-V in the process of respiration. All of the five complexes affect the final ATP synthesis and among them, the number of genes related to complex I is the largest, and complex I is mainly involved in the conversion of NADH to NAD⁺. Because of the importance of NAD⁺, we analyzed the NAD⁺ synthesis pathway genes in the following research and found that NMNAT2 is a down-regulated DEG in aging and Alzheimer’s disease. Also, NMNAT2 is highly correlated with EPB41L4A-AS1 both in aging and Alzheimer’s disease. Thus, we verified the mechanism that down-regulation of EPB41L4A-AS1 mediated the brain aging and development of neurodegenerative diseases via damaging synthesis of NAD⁺ and ATP.

Finally, we proved the regulation mechanism by cell experiments on U251 and SH-SY5Y cells. As predicted by bioinformatics, genes regulated by EPB41L4A-AS1 are down-regulated when EPB41L4A-AS1 was knocked down in terms of molecular biology, and NAD⁺ and ATP levels are also down-regulated from a cell biology point of view. Also, these reductions are restored once EPB41L4A-AS1 is overexpressed. Furthermore, the NR treatment experiment shows the positive significance of EPB41L4A-AS1 in treatment, and experiments at the epigenetic level show the regulatory mechanism of EPB41L4A-AS1 is based on adjusting H3K24Ac levels of histones.

As a critical metabolite and coenzyme, NAD⁺ is always in high demand. Sustaining its stable intracellular level is important for the normal function of multiple metabolic pathways and cellular processes. NAD⁺ level is maintained by three independent biosynthetic pathways, including the kynurenine pathway, the Preiss–Handler pathway, and the NAM salvage pathway [16,39,40]. The balance of NAD⁺ between synthesis and degradation can be broken during aging, but its mechanism is still unclear [16]. In this investigation, we found that NMNAT2, a brain-specific nicotinamide mononucleotide adenylyltransferase is down-regulated by EPB41L4A-AS1. NMNATs are involved in the Preiss–Handler pathway and the NAM salvage pathway, and its decrease with age may be the main cause of NAD⁺ level going down in the aging brain. It was reported that NMNAT2 depletion is related to SARM1-dependent axon degeneration in Parkinson's disease and other axonal disorders [41].

Many studies show that brain NAD⁺ plays a central role in maintaining energy homeostasis and ATP production [28]. In this study, we found that the aging-induced down-regulation of EPB41L4A-AS1 not only disturbs NAD⁺ biosynthesis but also affects ATP production. NAD⁺ accepts hydride equivalents, forming NADH, which provides electrons to the electron transport chain to generate ATP [28]. On one side, the decrease of NAD⁺ disrupted the process, affecting the generation of ATP. On the other side, the down-regulation of EPB41L4A-AS1 inhibits the expression of genes related to the electron transport chain, causing the decrease of ATP synthesis. Under this condition, the high demand of the brain for NAD⁺ and ATP can not be met, which promotes the development of brain aging and neurodegenerative diseases.

In conclusion, this study reported that EPB41L4A-AS1 has a strong positive regulation on the genes which form complex I-V on the electron transport chain and genes in NAD⁺ biosynthetic pathways. Down-regulation of EPB41L4A-AS1 mediated by aging will lead to a decrease in the expression of these genes, which will make the NAD⁺ and ATP generation go down. As NAD⁺ and ATP go down, many normal physiological functions will be inhibited, the metabolism will be impeded, and thus aging and symptoms of neurodegenerative diseases will occur. Conversely, these symptoms will be relieved when EPB41L4A-AS1 is overexpressed, which provides a new method for anti-aging and treatment of neurodegenerative diseases.

lncRNA: long noncoding RNA; GTEx: Genotype-Tissue Expression; DEGs: differentially expressed genes; WGCNA: weighted correlation network analysis; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; PPI: protein-protein interaction; GEO: Gene Expression Omnibus; NR: nicotinamide riboside; Chip: Chromatin Immunoprecipitation; H3K27Ac: acetylation of lysine 27 on histone 3; PCG: protein-coding gene; AD: Alzheimer’s disease; ALS: amyotrophic lateral sclerosis; HD: Huntington’s disease; PD: Parkinson’s disease; TSS: transcription start site

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

All data analyzed during this study are included in this article.

Competing interests

The authors have declared they have no conflict of interests.

Funding

This work was supported by the Shenzhen International Cooperation Research Project [GJHZ20180929162002061].

Authors' contributions

T-P.Y. conducted bioinformatics analysis. T-P.Y. and Y-Z.W. cultured cells and processed cells. C.L. and Y-Y.W. participated in molecular biology experiments. W-J.L., S-K.Z., S-M.W., N-H.X. and W-D.X. guided data analysis. Z-Q.W. conducted epigenetic experiments and Y-O.Z. wrote the manuscript. All authors read and approved the final draft.

Acknowledgements

The authors would like to thank both the State Key Laboratory of Chemical Oncogenomics for supporting and all the lab colleagues for assistance.

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Fig.S1.jpg
Fig. S1 (A) Correlation analysis of EPB41L4A-AS1 and 55 PCGs in 13 brains regions. (B) Fold changes in gene expression of EPB41L4A-AS1 and 55 PCGs in 13 brain regions, the fold changes are calculated by Mean(Old group, 60-80 years) / Mean(Young group, 20-50 years).
Fig.S2.jpg
Fig. S2 (A) Gene expression for NMNAT2 in 54 normal tissues. (B) Gene expression of NMNATs in 13 brain regions. (C) The mechanism of lncRNA EPB41L4A-AS1 in regulating brain metabolism, lncRNA EPB41L4A-AS1 regulates NAD+ and ATP level by affecting the genes of complex I-V and NAD+ synthesis pathway gene NMNAT2.
Fig.S3.jpg
Fig. S3 EPB41L4A-AS1 regulates the expression of 11 PCGs in U251 cells. (A) Relative RNA expression of genes in si-EPB41L4A-AS1 and siNC cells. (B) Relative RNA expression of genes in sh-EPB41L4A-AS1 and shNC cells, OENC: cells transfected with empty plasmids, OE: cells transfected with EPB41L4A-AS1 overexpression plasmids. Data are shown as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, student’s t-test.
TableS1.docx
Table S1 List of the primer sequences for real-time quantitative PCR

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Down-regulation of EPB41L4A-AS1 mediated the brain aging and neurodegenerative diseases via damaging synthesis of NAD⁺ and ATP

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