Skip to main content
Download PDF

Long-term exercise training down-regulates m6A RNA demethylase FTO expression in the hippocampus and hypothalamus: an effective intervention for epigenetic modification

BMC Neuroscience volume 23, Article number: 54 (2022) Cite this article

Abstract

Background

Exercise boosts the health of some brain parts, such as the hippocampus and hypothalamus. Several studies show that long-term exercise improves spatial learning and memory, enhances hypothalamic leptin sensitivity, and regulates energy balance. However, the effect of exercise on the hippocampus and hypothalamus is not fully understood. The study aimed to find epigenetic modifications or changes in gene expression of the hippocampus and hypothalamus due to exercise.

Methods

Male C57BL/6 mice were randomly divided into sedentary and exercise groups. All mice in the exercise group were subjected to treadmill exercise 5 days per week for 1 h each day. After the 12-week exercise intervention, the hippocampus and hypothalamus tissue were used for RNA-sequencing or molecular biology experiments.

Results

In both groups, numerous differentially expressed genes of the hippocampus (up-regulated: 53, down-regulated: 49) and hypothalamus (up-regulated: 24, down-regulated: 40) were observed. In the exercise group, increased level of N6-methyladenosine (m6A) was observed in the hippocampus and hypothalamus (p < 0.05). Furthermore, the fat mass and obesity-associated gene (FTO) of the hippocampus and hypothalamus were down-regulated in the exercise group (p < 0.001). In addition, the Fto co-expression genes of the mouse brain were studied and analyzed using database to determine the potential roles of exercise-downregulated FTO in the brain.

Conclusion

The findings demonstrate that long-term exercise might elevates the levels of m6A-tagged transcripts in the hippocampus and hypothalamus via down-regulation of FTO. Hence, exercise might be an effective intervention for epigenetic modification.

Peer Review reports

Introduction

The brain is the master organ of the central nervous system that modulates body organ functioning. The hippocampus and hypothalamus are parts of the brain crucial for the body's physiological functions. The hippocampus is a highly plastic region associated with stress response, learning, and memory [1, 2], and the hypothalamus is a critical central regulatory center for blood sugar, energy balance, and water balance [3]. Dysfunction of these parts can lead to adverse effects.

Exercise targets various aspects of brain function and broadly influences brain health. Studies on humans and animals suggested that physical exercise improves spatial learning and memory [4, 5]. Some studies reported that exercise controls obesity by enhancing hypothalamic leptin sensitivity [6, 7]. However, the cellular and molecular effects of exercise on the hippocampus and hypothalamus remain unknown. Therefore, it is important to study the effect of exercise on gene expression in the brain, and find a non-drug method to maintain brain health.

Recently, epigenetic regulation in various biological functions and pathogenesis of diseases has gained attention. m6A is one of the most common post-transcriptional RNA modifications in mRNA, represents another novel epigenetic marker, play critical roles in the regulation of gene expression. Through mutual interplay with methyltransferases, demethylases and m6A binding proteins to balance the m6A level, and to insure the mRNA transcripts can be properly spliced, transported, transcripts, and degraded [8]. Exercise as a lifestyle intervention can fine-tune gene expressions and biological processes via epigenetic modifications [9]. In the present study, a transcriptome profiling technology RNA-sequencing was used to identify differentially expressed genes of the hippocampus and hypothalamus in exercise training models. In the exercise group, an increased level of m6A was observed in the hippocampus and hypothalamus. Furthermore, the m6A RNA methylation regulator expression was assessed. Bioinformatic analysis showed that the Fto gene was down-regulated in the hippocampus and hypothalamus, which responds to exercise. Based on previous bioinformatics analyses, we further confirm Fto expression and other m6A RNA methylation regulators in the hippocampus and hypothalamus of exercise mice using qPCR and western blot analysis.

FTO is associated with an increased risk of diabetes and obesity [10]. Recently, the FTO gene and its expression product have attracted widespread interest due to its identification as an m6A RNA demethylase [11,12,13]. FTO is highly expressed in the brain and likely involved in many nuclear RNA processing events, such as mRNA translation, splicing, and metabolism [11, 13]. Previous study showed that highly intensive exercise decreases the skeletal muscle FTO mRNA [14]. However, the evidence on the effects of long-term exercise on FTO expression is scarce. Hence, evidence on the molecular biological mechanisms of exercise-induced changes of FTO-m6A expression on brain function and the biological process has been provides in this study.

Experimental procedure

Animals and diet administration

The C57BL/6 mice were provided by the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). Eight-week-old male C57BL/6 J mice were randomly divided into sedentary (n = 24) and exercise (n = 24) groups. The mice in the exercise group were trained on a treadmill 5 days per week for 12 weeks (Fig. 1). The schedule was as follows: 5 min of warm-up at 0–12 m/min, 50 min of the main exercise at 12 m/min (moderate-intensity exercise with 75% maximum oxygen consumption), and 5 min of cool down at 12–0 m/min [15]. The mice in the sedentary group were controls. And the control mice were exposed to treadmill noise and vibration without runing. The mice were fed a standard diet and water ad libitum in a 12 h-light/12 h-dark cycle at the Guangzhou Sport University. Five days after the final exercise training, mice were assessed for body composition and metabolic status. The mice were euthanized under anesthesia (sodium pentobarbital 50 μg/g) for collection of the hippocampus and hypothalamus tissues. This study was approved by the Institutional Animal Care and Use Committee of Guangzhou Sport University (2021DWLL-05).

Fig. 1
figure 1

Construction of 12-week exercise training model in exercise mice and exercise protocol

Full size image

Metabolic assessments

Mice were placed in an Oxymax Comprehensive Lab Animal Monitoring System (Columbus Instruments, USA) to detect the oxygen consumption (VO2), the carbon dioxide production (VCO2), respiratory exchange ratio (RER), energy intake, and energy expenditure. The body composition was assessed using the EchoMRI quantitative magnetic resonance (QMR) method (EchoMRI-500H, USA).

Library construction for RNA-sequencing

The quantity and purity of the total RNA were analyzed using the Bioanalyzer 2100 and RNA 1000 Nano LabChip Kit (Agilent, USA) with RIN number > 7.0. Poly(A) RNA was purified from the total RNA (5 μg) using poly-T oligo-attached magnetic beads with two rounds of purification. Subsequently, the mRNA was fragmented into small pieces using divalent cations under elevated temperatures. The cleaved RNA fragments were then reverse transcribed using the mRNA Seq sample preparation kit (Illumina, USA) to create the final cDNA library [the average insert size for the libraries was 300 bp (± 50 bp)]. In addition, the paired-end sequencing was performed on an Illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.

RNA extraction and quantitative real-time PCR (qPCR)

Total RNA from the mouse hippocampus and hypothalamus tissues were extracted using HiPure Universal RNA Kit (Magen, China). The cDNA was synthesized from 1 μg of total RNA using PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Japan). qPCR analysis was conducted using TB Green® Premix Ex Taq™ (TaKaRa, Japan) with Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific, USA). The relative cycle threshold (CT) values were normalized using β-actin. All primers used for qPCR are listed in Table 1.

Table 1 Mouse specific primer sequences used for qPCR (β-actin for the housekeeping as an internal control)
Full size table

Protein extraction and western blot

The mouse hippocampus and hypothalamus tissues were lysed on ice using RIPA lysis buffer (100 mm NaCl, 20 mm Tris, pH8.0, 1 mm EDTA, pH8.0, 0.5% Triton X-100, and 0.5% Nonidet P-40) containing a protease and phosphatase inhibitor cocktail (Beyotime Biotechnology, China). These tissues were quantified using the BCA Protein Assay Kit (Pierce, Germany). The same amount of protein (15 μg) was resolved on a 12% SDS-PAGE under a denaturing condition, transferred onto a PVDF membrane, and blocked in 5% non-fat milk. The blots were cut prior to hybridisation with antibodies. After the tissues were incubated with FTO antibody (Cat#: 98768, Santa Cruz Biotech, USA) or β-actin antibody (Cat#: 60008-1-Ig, ProteinTech Group, USA) overnight at 4 °C and secondary antibody (Peroxidase-conjugated Affinipure Goat Anti Mouse/Rabbit IgG, ProteinTech Group, USA) for 2 h at room temperature, the bands were exposed using enhanced chemiluminescence (Pierce, USA) and X-ray film. Quantitative data were obtained using ImageJ software.

m6A level

The m6A RNA methylation status of the mouse hippocampus and hypothalamus were detected using enzyme-linked immunoassay (ELISA) with an EpiQuik™ m6A RNA Methylation Quantification Kit (Epigebtek, China) following the manufacturer's protocol. The detected signal was quantified by reading the absorbance in a microplate spectrophotometer. The amount of m6A is proportional to the OD intensity measured.

Bioinformatic analysis of Fto gene co-expression network

The transcriptome and expression profiles of Fto and other m6A RNA methylation regulators were analyzed using the R package “Limma” (R software version R3.6.3). The Fto gene co-expression network in the mouse brain were analyzed using the coexpedia database. To explore the functional annotation and pathway enrichment of the co-expression network in the brain, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 online analysis tool.

Statistical analyses

All experimental data were analyzed using the SPSS 20.0 software, and the results were expressed as mean ± SEM. Kolmogorov–Smirnov and Shapiro–Wilk normality tests were performed and homogeneity of variance was tested with the Levene. Statistical significance of differences between the two groups were calculated using Student's t-test. When variables did not fulfill assumptions of normality, the Kruskal–Wallis test was applied. p < 0.05 were considered significant and marked with an asterisk (*).

Results

Characterization of mice with 12-week exercise training

A long-term aerobic exercise training mouse model with moderate-intensity was used to analysis the effect of exercise on weight, body compose, and metabolic parameters. Compared with the mice in the sedentary group, those in the exercise group showed lower body weight (p < 0.05) and fat percentages (p < 0.05) after 12-week exercise training (Fig. 2a). In addition, the VCO2 (p < 0.01) and RER (p < 0.001) decreased in the exercise group compared to that in the sedentary group (Fig. 2e and f). But there is no statistical difference in energy intake, energy expenditure, and VO2 between the exercise group and the sedentary group (Fig. 2b–d).

Fig. 2
figure 2

Metabolic parameters of the exercise group compared to the sedentary group. a Body composition, b energy intake, c energy expenditure, d average VO2, e average VCO2, and f average 24 h RER in the exercise group compared to the sedentary group. n = 16 per group, *p < 0.05

Full size image

Alteration of hippocampal and hypothalamic RNA-sequencing in mice with 12-week exercise training

RNA-sequencing determined the transcriptome and expression profiles in the hippocampus and hypothalamus of 5 mice with 12-week exercise training (exercise group) and 5 control mice (sedentary group). Heatmaps and volcano maps showed significant differentially expressed genes in the hippocampus and hypothalamus between the two groups (Fig. 3). As shown in Fig. 3c, 102 differentially expressed genes were observed in the hippocampus: 53 and 49 genes were up-regulated and down-regulated, respectively, in the exercise group compared to that in the sedentary group. However, 64 differentially expressed genes were observed in the hypothalamus: 24 and 40 genes were up-regulated and down-regulated, respectively, in the exercise group compared to that in the sedentary group (Fig. 3d).

Fig. 3
figure 3

Visualization analysis of differentially expressed genes in hippocampus and hypothalamus. a, b Heatmap based on RNA-sequencing data of the exercise group compared to the sedentary group in hippocampus and hypothalamus, respectively. c, d Volcano map based on RNA-sequencing data of the exercise group compared to the sedentary group in hippocampus (up-regulated: 53, down-regulated: 49) and hypothalamus (up-regulated: 24, down-regulated: 40), respectively. Red, blue, and white colors respectively represent the relatively high, low, and equal expression in heatmaps and volcano maps. n = 5 per group, p < 0.05

Full size image

Genes with significant changes (|log2-fold-change| > 1 and normalized p < 0.05) were identified in the exercise and sedentary group, and their functions were annotated using GO and KEGG pathway analyses [16]. The results reported that differentially expressed genes in the hippocampus majorly enriched in biological process (BP), including “cell adhesion,” “biological process” and “ventricular cardiac muscle tissue morphogenesis”; cellular component (CC), including “membrane,” “integral component of membrane” and “cytoplasm”; and molecular function (MF), including “protein binding,” “metal ion binding,” and “calcium ion binding” (Fig. 4a). In addition, differentially expressed genes in the hypothalamus majorly enriched in BP, including “biological process,” “regulation of transcription, DNA-templated,” and “positive regulation of transcription by RNA polymerase II”; CC, including “nucleus,” “cytoplasm,” and “membrane”; and MF, including “protein binding,” “metal ion binding,” and “molecular function” (Fig. 4b). Furthermore, genes involved in the KEGG pathways of the hippocampus enriched “hypertrophic cardiomyopathy signaling pathway,” “dilated cardiomyopathy signaling pathway,” “cardiac muscle contraction signaling pathway,” “adrenergic signaling in cardiomyocytes signaling pathway,” “viral myocarditis signaling pathway,” and “cell adhesion molecules signaling pathway” (Fig. 4c). Similarly, genes involved in the KEGG pathway of the hypothalamus enriched “phosphonate and phosphinate metabolism signaling pathway,” “choline metabolism in cancer signaling pathway,” and “hepatitis B signaling pathway” (Fig. 4d).

Fig. 4
figure 4

Visualization analysis of GO and KEGG of the exercise group compared to the sedentary group in hippocampus and hypothalamus. a, b GO analysis classified regulators into BP, CC, and MF groups. c, d KEGG pathway enrichment. n = 5 per group, p < 0.05

Full size image

Increased m6A level and down-regulated FTO expression in the hippocampus and hypothalamus of mice with 12-week exercise training

Exercise as a positive lifestyle intervention may regulate the downstream genes and various biological processes by changing the RNA methylation. Considering this, the level of m6A was detected using ELISA. A high level of m6A was observed in hippocampus (p < 0.05) and hypothalamus (p < 0.05) of mice in the exercise group (Fig. 5a), indicating that exercise increased the level of m6A in the hippocampus and hypothalamus.

Fig. 5
figure 5

Exercise increased the level of m6A and down-regulated FTO expression in hippocampus and hypothalamus. a The level of m6A was detected using the ELISA method. n = 6 per group, *p < 0.05. b The heatmaps showed 13 m6A RNA methylation regulators in hippocampus and hypothalamus of the exercise group compared to the sedentary group. hip.E and hyp.E were marked with blue, hip.S and hyp.S were marked with red, position of white spots on the way represented the median value of expression, n = 5 per group, p < 0.05. c qPCR showed 13 m6A RNA methylation regulators mRNA expression. The relative levels of these genes were normalized to β-actin, and the relative mRNA levels in the sedentary group were normalized as “1”. n = 6 per group, ***p < 0.01, ***p < 0.001. d Western blot analysis of FTO protein levels. The relative levels of FTO were normalized to β-actin, and the relative protein levels in the sedentary group were normalized as “1”. n = 6 per group, ***p < 0.001

Full size image

To determine the reason of these changes, the transcriptome and expression profiles of 13 m6A RNA methylation regulators in hippocampus and hypothalamus of mice were compared between both the groups. Rank sum test was used to analyze the statistically significant differences, and the results were shown using heatmaps (Fig. 5b). Comparative analyses and qPCR (Fig. 5b and c) showed low Fto (p < 0.001) and Ythdc1 (p < 0.05) mRNA expressions in the hippocampus and Fto (p < 0.01) mRNA expression in the hypothalamus of mice in the exercise group, respectively. Further, the level of FTO were detected using western blot, revealing significant downregulation of FTO expressions in the hippocampus (p < 0.001) and hypothalamus (p < 0.001) of mice in the exercise group (Fig. 5d).

Potential roles of exercise-downregulated FTO in the brain

Several studies have suggested the importance of FTO in modulating brain functions. However, the genes interacting with FTO were unknown. Hence, the Fto gene co-expression network of the mouse brain was analyzed using the coexpedia database to explore the potential role of FTO in the brain. The results showed 54 co-expression genes of Fto (Fig. 6a and Table 2). The Fto/co-expression genes majorly enriched BP, including “in utero embryonic development,” “protein stabilization,” and “protein autophosphorylation”; CC, including “membrane,” “cytoplasm,” and “nucleoplasm”; and MF, including “protein binding,” “nucleotide binding,” and “protein kinase binding” (Fig. 6b). Furthermore, Fto/co-expression genes involved in the KEGG pathway of the hypothalamus enriched “vasopressin regulated water reabsorption signaling pathway,” “synaptic vesicle cycle signaling pathway,” “protein processing in endoplasmic reticulum signaling pathway,” and “CAMP signaling pathway” (Fig. 6c). Therefore, FTO plays diverse physiological and pathological functions in the brain tissues. Further, exercise may play a role in brain functions and biological processes by regulating the FTO expression.

Fig. 6
figure 6

Visualization analysis of Fto interaction network, GO and KEGG in the mouse brain. a The red ellipse represents Fto gene, and the green ellipses represent the Fto gene co-expression network. b GO analysis classified the co-expression network into BF, CC, and MF terms. c KEGG pathway enrichment

Full size image
Table 2 Fto co-expression genes in the mouse brain
Full size table

Discussion

Physical exercise has substantial beneficial effects not only on physical health but also on brain function. Most studies suggested the importance of exercise on the brain, particularly the hippocampus and hypothalamus. For example, long-term exercise can prevent cognitive dysfunction induced by obesity [17] or aging [18] and improve spatial learning and memory ability. Endurance exercise can alter the gene expression status of the hippocampus, thereby affecting human cognitive function [19]. In addition, exercise ameliorates the hypothalamic leptin resistance [20] and insulin resistance [21] to affect the energy balance. However, the molecular mechanisms through which exercise affects brain function are unclear. The development of high-throughput sequencing provides a beneficial tool to study the role of exercise in regulating the biological processes in the brain by altering the gene expressions.

The molecular mechanisms of exercise that regulates brain function were investigated. We used an exercise mouse model to observe the effects of exercise on gene expression in the hippocampus and hypothalamus. Using high-throughput sequencing technology, differential genes were found to be involved in many important cellular functions and signaling pathways. For example, some enriched functions of the differentially expressed genes in the hippocampus were associated with the synaptic transmission process (GO: 0099025, GO: 0099029, GO: 0099576, GO: 0060080, GO: 0099151, and GO: 0051932), indicating that exercise may regulate synaptic activity. In addition, some enriched functions of the differentially expressed genes in the hypothalamus were associated with the neural function (GO: 0032809 and GO: 0043005), neurogenesis (GO: 0021626 and GO: 0014037), and glucagon secretion regulation (GO: 0070029), suggesting that exercise promotes hypothalamic health and its function. Regarding the KEGG pathway, the “cell adhesion molecules signaling pathway” plays a crucial role in the hippocampal neuronal survival, differentiation, axonal growth, and synaptic development [22, 23].

Recently, the significance of epigenetic regulation in various biological functions and disease pathogenesis has increased. As an epigenetic marker, the reversible m6A is the most prevalent post-transcriptional regulation of mammalian gene expression. m6A is abundant in the nervous system, and the cellular dynamics of m6A are associated with neural function, neurogenesis, and neuronal survival [24,25,26]. The dysregulation of m6A is related to many biological processes, including neurodevelopment and neurodegenerative diseases. Reportedly, the upregulation of m6A occurs with brain maturation [27], behavioral experience [28], and memory formation [29]. In this study, a high level of m6A was observed in the hippocampus and hypothalamus of mice in the exercise group (Fig. 5a). Since the dynamic equilibrium of m6A is governed by m6A-related components, such as methylesterases, demethylases, and reading proteins, the expression of 13 m6A RNA methylation regulator genes, including METTL3, METTL14, WTAP, RBM15, ZC3H13, FTO, ALKBH5, YTHDF1, YTHDF3, YTHDC2, YTHDF2, YTHDC1, and HNRNPC were analyzed in the hippocampus and hypothalamus of mice in the exercise group. The result showed that only Fto was down-regulated in the hippocampus and hypothalamus of the mice in exercise group (Fig. 5b and c). In addtion, western blot experiment was performed, confirming the finding (Fig. 5d).

FTO as an m6A demethylase is a crucial component of m6A modification [30, 31]. Several studies suggested that FTO knockdown with siRNA increased the amount of m(6)A in mRNA, and FTO overexpression decreased the amount of m(6)A in human cells [12]. The above evidence proves that FTO expression may contribute to m6A levels. Hence, presumably, elevated levels of m6A in the hippocampus and hypothalamus after exercise are due to the downregulation of FTO. Although polymorphisms within the intron 1 of the FTO gene were first reported to be associated with obesity [10, 32, 33], the physiological role of the FTO gene remains unclear. FTO is widely found in central and peripheral tissues of mammals [34]. In peripheral tissues, FTO is related to energy metabolism [35, 36] and cancer progression [37,38,39]. In central tissues, FTO is highly expressed in the brain and essential for development of the central nervous system in humans [40, 41]. Numerous preclinical evidence reported that altered FTO expression is partially responsible for energy balance, epilepsy, neurodevelopment, and neurodegenerative diseases. In animal studies, FTO can activate the phosphorylation of Tau, which is one of the markers of Alzheimer's disease (AD) [42]. In human studies, the genetic variation in the introns of the FTO gene possibly contributes to the risk of AD [43, 44]. However, specific mechanism of the FTO gene variants that contribute to the risks of AD is still unclear and requires further research. Moreover, the FTO inhibitor can regulate the neuronal excitability with anticonvulsant activity [45],and is responsible for glioblastoma progression [46]. Axonal FTO is reportedly involved in neuronal development by regulating the m6A modification of axonal mRNA [47]. Decreasing FTO in the dorsal hippocampus aids in memory formation [29]. However, the loss of FTO leads to impairment of neuronal differentiation and a processing defect of brain-derived neurotrophic factor (BDNF) within the hippocampus, which increasing anxiety and impairing the working memory [48]. In addition, the complete or neural-specific Fto gene deletion results in postnatal growth retardation of mice [34]. The m6A RNA demethylase FTO alleviates the deficits in dopaminergic neurotransmission in response to arsenite exposure [49]. FTO is related to appetite and food intake in the hypothalamus [50]. Further research found that mice with low expression of FTO remain sensitive to the anorexigenic effects of leptin [51]. All these studies strongly suggest that FTO plays vital roles in the physiological and pathological functions of the brain.

Although most studies have focused on the impact of FTO overexpression or knockdown in the brain, the genes that related to FTO are still important as they perform many subsequent molecular functions and biological processes. It has been reported that FTO as a transcriptional coactivator promotes gene transcription, ultimately affecting adipose tissue development [36]. However, the mechanism of FTO interaction with downstream genes to further regulate nerve function remain largely unknown.

FTO could be regulated not only by nutrition but also by exercise. Previous studies found that physical activity might weaken the effect of the FTO variant on BMI [52,53,54,55,56]. In addition, gender also influences the FTO genotype on exercise for weight loss. It is observed that males carrying the FTO risk allele lose more weight after a 12-week regular exercise [57]. An acute decreased skeletal muscle FTO mRNA expression was observed after high-intensity exercise by Danaher et al. [14]. Most researchers focus on the reducing obesity risk caused by FTO gene polymorphisms under exercise, while there are few on its function. In the present study, we indicated that exercise attenuates FTO expression. FTO, a demethylase, plays an important role in energy metabolism. The abnormal FTO expression modifies the level of m6A of target genes and is involved in many physiological and pathological processes. Overall, the study reported for the first time that long-term exercise can down-regulated the FTO expression in the hippocampus and hypothalamus, indicating that FTO may be a promising key player between exercise and the brain.

However, it is unclear whether exercise-induced FTO downregulation can regulate downstream target genes and the biological processes. Hence, the Fto/co-expression genes were downloaded from the database for GO enrichment and KEGG signal pathway analyses. Based on the results of bioinformation analyses, the significant enrichment pathway primarily correlated with vasopressin-regulated signaling pathway, water reabsorption signaling pathway, synaptic vesicle cycle signaling pathway, endocrine signaling pathway, calcium reabsorption signaling pathway, protein processing in endoplasmic reticulum signaling pathway, salivary secretion signaling pathway, cAMP signaling, insulin secretion signaling pathway, and morphine addiction signaling pathway (Fig. 6c). The result suggests that FTO and its co-expression genes are involved in many important biological processes in the brain. In addtion, the known and unknown proteins co-expressed with FTO may be regulated by FTO-m6A to alter their expression and function. Thus, exercise may regulate the expression and function of the related genes via FTO-dependent demethylation of mRNA m6A.

We found that the Vamp2 gene expression in mouse brain is involved in two KEGG signaling pathways, include insulin secretion and synaptic vesicle cycle. Vesicle-associated membrane protein 2 (VAMP2) has been implicated in the insulin-regulated trafficking of GLUT4 in insulin-sensitive cells. VAMP2 inhibited insulin-stimulated GLUT4 translocation and decreased insulin sensitivity [58, 59]. Insulin-sensitive tissue or cells include liver, skeletal muscle, adipocytes, and hypothalamus. In addition, VAMP2 may have important roles in synaptic trafficking in the hippocampus [60]. The epileptogenesis is dramatically attenuated in hippocampus of Vamp+/− mice [61]. Hence, presumably, long-term exercise may regulate the expression and function of VAMP2 in hypothalamus and hippocampus via FTO-dependent demethylation of mRNA m6A, but further research is needed to confirm that VAMP2 is a target of FTO. Hence, FTO could be a valuable therapeutic target for brain diseases in the future.

Conclusion

The gene changes after exercise training were confirmed using RNA-sequencing analysis. Long-term exercise training showed increased level of m6A and down-regulated FTO expressions in the hippocampus and hypothalamus. Lifestyle intervention such as exercise might be an effective intervention for epigenetic modification. In addition, reviewed of studies on the role and co-expression genes of Fto in mice brain revealed that the relationship between FTO and downstream genes is not completely reported, requiring additional research to elucidate their roles in the brain in response to exercise. Nevertheless, further research is warranted to understand the signaling pathways of FTO involved in and their impacts on brain health.

Availability of data and materials

The RNA-sequencing datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The data used to bioinformatic analysis of Fto gene co-expression network can be accessed at coexpedia database: https://www.coexpedia.org/mm_single.php?gene=FTO.

Abbreviations

m6A:

N6-methyladenosine

FTO:

Fat mass and obesity-associated gene

VO2 :

Oxygen consumption

VCO2 :

Carbon dioxide production

RER:

Respiratory exchange ratio

GO:

Gene ontology

KEGG:

Kyoto encyclopedia of genes and genomes

mRNA:

Messenger RNA

METTL3:

Methyltransferase-like 3

METTL14:

Methyltransferase-like 14

WTAP:

Wilms' tumor 1 associated protein

RBM15:

RNA binding motif protein 15

ZC3H13:

Zinc finger CCCH domain-containing protein 13

ALKBH5:

Alkylation repair homolog protein 5

YTHDF1:

YTH domain-containing family protein 1

YTHDF3:

YTH domain-containing family protein 3

YTHDC2:

YTH domain-containing protein1

YTHDF2:

YTH domain-containing family protein 2

YTHDC1:

YTH domain-containing protein1

HNRNPC:

Heterogeneous nuclear ribonucleoprotein C

AD:

Alzheimer’s disease

BDNF:

Brain-derived neurotrophic factor

References

  1. Snyder JS, Soumier A, Brewer M, Pickel J, Cameron HA. Adult hippocampal neurogenesis buffers stress responses and depressive behaviour. Nature. 2011;476(7361):458–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Leuner B, Gould E. Structural plasticity and hippocampal function. Annu Rev Psychol. 2010;61(111–40):c1-3.

    Google Scholar 

  3. Williams G, Bing C, Cai XJ, Harrold JA, King PJ, Liu XH. The hypothalamus and the control of energy homeostasis: different circuits, different purposes. Physiol Behav. 2001;74(4–5):683–701.

    Article  CAS  PubMed  Google Scholar 

  4. Cassilhas RC, Lee KS, Fernandes J, Oliveira MG, Tufik S, Meeusen R, et al. Spatial memory is improved by aerobic and resistance exercise through divergent molecular mechanisms. Neuroscience. 2012;202:309–17.

    Article  CAS  PubMed  Google Scholar 

  5. Cassilhas RC, Viana VA, Grassmann V, Santos RT, Santos RF, Tufik S, et al. The impact of resistance exercise on the cognitive function of the elderly. Med Sci Sports Exerc. 2007;39(8):1401–7.

    Article  PubMed  Google Scholar 

  6. Kang S, Kim KB, Shin KO. Exercise training improves leptin sensitivity in peripheral tissue of obese rats. Biochem Biophys Res Commun. 2013;435(3):454–9.

    Article  CAS  PubMed  Google Scholar 

  7. Silva VRR, Micheletti TO, Katashima CK, Lenhare L, Morari J, Moura-Assis A, et al. Exercise activates the hypothalamic S1PR1-STAT3 axis through the central action of interleukin 6 in mice. J Cell Physiol. 2018;233(12):9426–36.

    Article  CAS  PubMed  Google Scholar 

  8. Niu Y, Zhao X, Wu YS, Li MM, Wang XJ, Yang YG. N6-methyl-adenosine (m6A) in RNA: an old modification with a novel epigenetic function. Genom Proteom Bioinform. 2013;11(1):8–17.

    Article  CAS  Google Scholar 

  9. Denham J. Exercise and epigenetic inheritance of disease risk. Acta Physiol. 2018;222(1).

    Article  Google Scholar 

  10. Frayling TM, Timpson NJ, Weedon MN, Zeggini E, Freathy RM, Lindgren CM, et al. A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity. Science. 2007;316(5826):889–94.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Fu Y, Jia G, Pang X, Wang RN, Wang X, Li CJ, et al. FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA. Nat Commun. 2013;4:1798.

    Article  PubMed  Google Scholar 

  12. Berulava T, Ziehe M, Klein-Hitpass L, Mladenov E, Thomale J, Rüther U, et al. FTO levels affect RNA modification and the transcriptome. Eur J Hum Genet. 2013;21(3):317–23.

    Article  CAS  PubMed  Google Scholar 

  13. Zhao X, Yang Y, Sun BF, Shi Y, Yang X, Xiao W, et al. FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis. Cell Res. 2014;24(12):1403–19.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Danaher J, Stathis CG, Wilson RA, Moreno-Asso A, Wellard RM, Cooke MB. High intensity exercise downregulates FTO mRNA expression during the early stages of recovery in young males and females. Nutr Metab. 2020;17:68.

    Article  CAS  Google Scholar 

  15. Fernando P, Bonen A, Hoffman-Goetz L. Predicting submaximal oxygen consumption during treadmill running in mice. Can J Physiol Pharmacol. 1993;71(10–11):854–7.

    Article  CAS  PubMed  Google Scholar 

  16. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28(1):27–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Graham LC, Grabowska WA, Chun Y, Risacher SL, Philip VM, Saykin AJ, et al. Exercise prevents obesity-induced cognitive decline and white matter damage in mice. Neurobiol Aging. 2019;80:154–72.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Marosi K, Bori Z, Hart N, Sárga L, Koltai E, Radák Z, et al. Long-term exercise treatment reduces oxidative stress in the hippocampus of aging rats. Neuroscience. 2012;226:21–8.

    Article  CAS  PubMed  Google Scholar 

  19. Wrann CD, White JP, Salogiannnis J, Laznik-Bogoslavski D, Wu J, Ma D, et al. Exercise induces hippocampal BDNF through a PGC-1α/FNDC5 pathway. Cell Metab. 2013;18(5):649–59.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Flores MB, Fernandes MF, Ropelle ER, Faria MC, Ueno M, Velloso LA, et al. Exercise improves insulin and leptin sensitivity in hypothalamus of Wistar rats. Diabetes. 2006;55(9):2554–61.

    Article  CAS  PubMed  Google Scholar 

  21. da Luz G, Frederico MJ, da Silva S, Vitto MF, Cesconetto PA, de Pinho RA, et al. Endurance exercise training ameliorates insulin resistance and reticulum stress in adipose and hepatic tissue in obese rats. Eur J Appl Physiol. 2011;111(9):2015–23.

    Article  PubMed  Google Scholar 

  22. Zhang B, Wei K, Li X, Hu R, Qiu J, Zhang Y, et al. Upregulation of Cdh1 signaling in the hippocampus attenuates brain damage after transient global cerebral ischemia in rats. Neurochem Int. 2018;112:166–78.

    Article  CAS  PubMed  Google Scholar 

  23. Zhang Y, Yao W, Qiu J, Qian W, Zhu C, Zhang C. The involvement of down-regulation of Cdh1-APC in hippocampal neuronal apoptosis after global cerebral ischemia in rat. Neurosci Lett. 2011;505(2):71–5.

    Article  CAS  PubMed  Google Scholar 

  24. Yoon KJ, Ringeling FR, Vissers C, Jacob F, Pokrass M, Jimenez-Cyrus D, et al. Temporal control of mammalian cortical neurogenesis by m(6)A methylation. Cell. 2017;171(4):877-89.e17.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Chen J, Zhang YC, Huang C, Shen H, Sun B, Cheng X, et al. m(6)A regulates neurogenesis and neuronal development by modulating histone methyltransferase Ezh2. Genom Proteom Bioinform. 2019;17(2):154–68.

    Article  Google Scholar 

  26. Wang L, Liang Y, Lin R, Xiong Q, Yu P, Ma J, et al. Mettl5 mediated 18S rRNA N6-methyladenosine (m(6)A) modification controls stem cell fate determination and neural function. Genes Dis. 2022;9(1):268–74.

    Article  CAS  PubMed  Google Scholar 

  27. Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, Jaffrey SR. Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons. Cell. 2012;149(7):1635–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Widagdo J, Zhao QY, Kempen MJ, Tan MC, Ratnu VS, Wei W, et al. Experience-dependent accumulation of N6-methyladenosine in the prefrontal cortex is associated with memory processes in mice. J Neurosci. 2016;36(25):6771–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Walters BJ, Mercaldo V, Gillon CJ, Yip M, Neve RL, Boyce FM, et al. The role of the RNA demethylase FTO (fat mass and obesity-associated) and mRNA methylation in hippocampal memory formation. Neuropsychopharmacology. 2017;42(7):1502–10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Gerken T, Girard CA, Tung YC, Webby CJ, Saudek V, Hewitson KS, et al. The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase. Science. 2007;318(5855):1469–72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Jia G, Fu Y, Zhao X, Dai Q, Zheng G, Yang Y, et al. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol. 2011;7(12):885–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Berulava T, Horsthemke B. The obesity-associated SNPs in intron 1 of the FTO gene affect primary transcript levels. Eur J Hum Genet. 2010;18(9):1054–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Church C, Moir L, McMurray F, Girard C, Banks GT, Teboul L, et al. Overexpression of Fto leads to increased food intake and results in obesity. Nat Genet. 2010;42(12):1086–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Gao X, Shin YH, Li M, Wang F, Tong Q, Zhang P. The fat mass and obesity associated gene FTO functions in the brain to regulate postnatal growth in mice. PLoS ONE. 2010;5(11): e14005.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Claussnitzer M, Dankel SN, Kim KH, Quon G, Meuleman W, Haugen C, et al. FTO obesity variant circuitry and adipocyte browning in humans. N Engl J Med. 2015;373(10):895–907.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Wu Q, Saunders RA, Szkudlarek-Mikho M, Serna Ide L, Chin KV. The obesity-associated Fto gene is a transcriptional coactivator. Biochem Biophys Res Commun. 2010;401(3):390–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Li Z, Weng H, Su R, Weng X, Zuo Z, Li C, et al. FTO plays an oncogenic role in acute myeloid leukemia as a N(6)-methyladenosine RNA demethylase. Cancer Cell. 2017;31(1):127–41.

    Article  PubMed  Google Scholar 

  38. Tao L, Mu X, Chen H, Jin D, Zhang R, Zhao Y, et al. FTO modifies the m6A level of MALAT and promotes bladder cancer progression. Clin Transl Med. 2021;11(2): e310.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Su R, Dong L, Li Y, Gao M, Han L, Wunderlich M, et al. Targeting FTO suppresses cancer stem cell maintenance and immune evasion. Cancer Cell. 2020;38(1):79-96.e11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Boissel S, Reish O, Proulx K, Kawagoe-Takaki H, Sedgwick B, Yeo GS, et al. Loss-of-function mutation in the dioxygenase-encoding FTO gene causes severe growth retardation and multiple malformations. Am J Hum Genet. 2009;85(1):106–11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Yeo GS. FTO and obesity: a problem for a billion people. J Neuroendocrinol. 2012;24(2):393–4.

    Article  PubMed  Google Scholar 

  42. Li H, Ren Y, Mao K, Hua F, Yang Y, Wei N, et al. FTO is involved in Alzheimer’s disease by targeting TSC1-mTOR-Tau signaling. Biochem Biophys Res Commun. 2018;498(1):234–9.

    Article  CAS  PubMed  Google Scholar 

  43. Reitz C, Tosto G, Mayeux R, Luchsinger JA. Genetic variants in the fat and obesity associated (FTO) gene and risk of Alzheimer’s disease. PLoS ONE. 2012;7(12): e50354.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Keller L, Xu W, Wang HX, Winblad B, Fratiglioni L, Graff C. The obesity related gene, FTO, interacts with APOE, and is associated with Alzheimer’s disease risk: a prospective cohort study. J Alzheimer’s Dis. 2011;23(3):461–9.

    Article  CAS  Google Scholar 

  45. Zheng G, Cox T, Tribbey L, Wang GZ, Iacoban P, Booher ME, et al. Synthesis of a FTO inhibitor with anticonvulsant activity. ACS Chem Neurosci. 2014;5(8):658–65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Cui Q, Shi H, Ye P, Li L, Qu Q, Sun G, et al. m(6)A RNA methylation regulates the self-renewal and tumorigenesis of glioblastoma stem cells. Cell Rep. 2017;18(11):2622–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Yu J, Chen M, Huang H, Zhu J, Song H, Zhu J, et al. Dynamic m6A modification regulates local translation of mRNA in axons. Nucleic Acids Res. 2018;46(3):1412–23.

    Article  CAS  PubMed  Google Scholar 

  48. Spychala A, Rüther U. FTO affects hippocampal function by regulation of BDNF processing. PLoS ONE. 2019;14(2): e0211937.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Bai L, Tang Q, Zou Z, Meng P, Tu B, Xia Y, et al. m6A demethylase FTO regulates dopaminergic neurotransmission deficits caused by arsenite. Toxicol Sci. 2018;165(2):431–46.

    Article  CAS  PubMed  Google Scholar 

  50. Tung YC, Ayuso E, Shan X, Bosch F, O’Rahilly S, Coll AP, et al. Hypothalamic-specific manipulation of Fto, the ortholog of the human obesity gene FTO, affects food intake in rats. PLoS ONE. 2010;5(1): e8771.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Tung YC, Gulati P, Liu CH, Rimmington D, Dennis R, Ma M, et al. FTO is necessary for the induction of leptin resistance by high-fat feeding. Mol Metab. 2015;4(4):287–98.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Liaw YC, Liaw YP. Physical activity might reduce the adverse impacts of the FTO gene variant rs3751812 on the body mass index of adults in Taiwan. Genes. 2019;10(5).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Mitchell JA, Church TS, Rankinen T, Earnest CP, Sui X, Blair SN. FTO genotype and the weight loss benefits of moderate intensity exercise. Obesity. 2010;18(3):641–3.

    Article  PubMed  Google Scholar 

  54. Cho HW, Jin HS, Eom YB. The interaction between FTO rs9939609 and physical activity is associated with a 2-fold reduction in the risk of obesity in Korean population. Am J Hum Biol. 2021;33(3): e23489.

    Article  PubMed  Google Scholar 

  55. Hiraike Y, Yang CT, Liu WJ, Yamada T. FTO obesity variant-exercise interaction on changes in body weight and BMI: The Taiwan Biobank Study. J Clin Endocrinol Metab. 2021;106(9):e3673–81.

    Article  PubMed  Google Scholar 

  56. Shinozaki K, Okuda M. Physical activity modifies the FTO effect on body mass index change in Japanese adolescents. Pediatrics Int. 2018;60(7):656–61.

    Article  Google Scholar 

  57. Wang W, Yang K, Wang S, Zhang J, Shi Y, Zhang H, et al. The sex-specific influence of FTO genotype on exercise intervention for weight loss in adult with obesity. Eur J Sport Sci. 2021:1–6.

    Article  PubMed  Google Scholar 

  58. Martin S, Tellam J, Livingstone C, Slot JW, Gould GW, James DE. The glucose transporter (GLUT-4) and vesicle-associated membrane protein-2 (VAMP-2) are segregated from recycling endosomes in insulin-sensitive cells. J Cell Biol. 1996;134(3):625–35.

    Article  CAS  PubMed  Google Scholar 

  59. Martin LB, Shewan A, Millar CA, Gould GW, James DE. Vesicle-associated membrane protein 2 plays a specific role in the insulin-dependent trafficking of the facilitative glucose transporter GLUT4 in 3T3-L1 adipocytes. J Biol Chem. 1998;273(3):1444–52.

    Article  CAS  PubMed  Google Scholar 

  60. Fukuda M. Vesicle-associated membrane protein-2/synaptobrevin binding to synaptotagmin I promotes O-glycosylation of synaptotagmin I. J Biol Chem. 2002;277(33):30351–8.

    Article  CAS  PubMed  Google Scholar 

  61. Matveeva EA, Price DA, Whiteheart SW, Vanaman TC, Gerhardt GA, Slevin JT. Reduction of vesicle-associated membrane protein 2 expression leads to a kindling-resistant phenotype in a murine model of epilepsy. Neuroscience. 2012;202:77–86.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank the author in the study.

Funding

This study was funded by the National Natural Science Foundation of China (32100920), the Innovative Talents Project of Colleges and Universities of Guangdong Province, China (2019KQNVX063), the Featured Innovation Projects of Colleges and Universities of Guangdong Province, China (2021KTSCX060), Natural Science Foundation of Guangdong Province, China (2019A1515012204), Special Projects in Key Fields of Colleges and Universities of Guangdong Province, China (2021ZDZX2054), and Science and Technology Program of Guangzhou, China (202102080026).

Author information

Author notes

  1. Shu-Jing Liu, Tong-Hui Cai and Chun-Lu Fang contributed equally to this work

Authors and Affiliations

  1. Center for Scientific Research and Institute of Exercise and Health, Guangzhou Sport University, Guangzhou, China

    Shu-Jing Liu, Chun-Lu Fang, Wen-Qi Yang, Yuan Wei, Fu Zhou, Ling Liu, Yuan Luo, Zi-Yi Guo, Ge Zhao, Ya-Ping Li & Liang-Ming Li

  2. The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510260, China

    Tong-Hui Cai & Shao-Zhang Lin

Authors
  1. Shu-Jing Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tong-Hui Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chun-Lu Fang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shao-Zhang Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wen-Qi Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yuan Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Fu Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ling Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yuan Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Zi-Yi Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Ge Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Ya-Ping Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Liang-Ming Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

LML designed the research; SJL, CLF, LL, YL, ZYG, GZ, YPL, and SZL performed the research; THC, CLF, WQY, YW, and FZ analyzed data; LML, SJL, and THC wrote the main manuscript text and prepared Figs. 1, 2, 3, 4, 5, 6. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Liang-Ming Li.

Ethics declarations

Ethics approval and consent to participate

This study is reported in accordance with the ARRIVE guidelines (https://arriveguidelines.org). All animal experiments in this study were in strict accordance with relevant guidelines and regulations in the Guide for the Care and Use of Laboratory Animals and approval by the Ethics Committee of Guangzhou Sport University, and the permit number of ethics approval was 2021DWLL-05.

Consent for publication

Not applicable.

Competing interests

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Liu, SJ., Cai, TH., Fang, CL. et al. Long-term exercise training down-regulates m6A RNA demethylase FTO expression in the hippocampus and hypothalamus: an effective intervention for epigenetic modification. BMC Neurosci 23, 54 (2022). https://doi.org/10.1186/s12868-022-00742-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12868-022-00742-8

Keywords

BMC Neuroscience

ISSN: 1471-2202

Contact us