Kong et al. Journal of Neuroinflammation(2024) 21:208
https://doi.org/10.1186/s12974-024-03189-2

Journal of Neuroinflammation

Open Access

RESEARCH

Tet1-mediated 5hmC regulates hippocampal
neuroinflammation via wnt signaling as
a novel mechanism in obstructive sleep
apnoea leads to cognitive deficit
Yaru Kong1,2,3†, Jie Ji4†, Xiaojun Zhan3†, Weiheng Yan1,2,3, Fan Liu1,2,5, Pengfei Ye3, Shan Wang5* and Jun Tai1,3*
Abstract
Background Obstructive sleep apnoea (OSA) is a sleep-disordered breathing characterized by intermittent hypoxia(IH) that may cause cognitive dysfunction. However, the impact of IH on molecular processes involved in cognitive
function remains unclear.
Methods C57BL / 6 J mice were exposed to either normoxia (control) or IH for 6 weeks. DNA hydroxymethylation
was quantified by hydroxymethylated DNA immunoprecipitation (hMeDIP) sequencing. ten-eleven translocation
1 (Tet1) was knocked down by lentivirus. Specifically, cognitive function was assessed by behavioral experiments,
pathological features were assessed by HE staining, the hippocampal DNA hydroxymethylation was examined by
DNA dot blot and immunohistochemical staining, while the Wnt signaling pathway and its downstream effects werestudied using qRT-PCR, immunofluorescence staining, and Luminex liquid suspension chip analysis.
Results IH mice showed pathological changes and cognitive dysfunction in the hippocampus. Compared with thecontrol group, IH mice exhibited global DNA hydroxylmethylation in the hippocampus, and the expression of threehydroxylmethylases increased significantly. The Wnt signaling pathway was activated, and the mRNA and 5hmC levelsof Wnt3a, Ccnd2, and Prickle2 were significantly up-regulated. Further caused downstream neurogenesis abnormalities
and neuroinflammatory activation, manifested as increased expression of IBA1 (a marker of microglia), GFAP (a marker
of astrocytes), and DCX (a marker of immature neurons), as well as a range of inflammatory cytokines (e.g. TNFa, IL3,
IL9, and IL17A). After Tet1 knocked down, the above indicators return to normal.
Conclusion Activation of Wnt signaling pathway by hippocampal Tet1 is associated with cognitive dysfunction
induced by IH.
Keywords 5hmC1, IH2, Tet13, Wnt pathway4, hippocampus5†

Yaru Kong, Jie Ji and Xiaojun Zhan contributed equally to this work.

*Correspondence:
Shan Wangwsaquarius@sina.com
Jun Taitrenttj@163.com
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, 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 you modified thelicensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it.The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to thematerial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation
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Kong et al. Journal of Neuroinflammation(2024) 21:208

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Graphical Abstract

Introduction
Obstructive sleep apnoea (OSA) is the most common
type of sleep-disordered breathing, affecting approximately 1 billion adults worldwide; the largest numberof OSA in China [1]. OSA is characterized by recurrent
upper airway obstruction during sleep, resulting in intermittent hypoxia (IH), sleep fragmentation, further systemic inflammation and oxidative stress [2]. Untreated
OSA is widely recognized to be associated with anincreased risk of hypertension [3], diabetes [4], and cognitive dysfunction [5], leading to substantial health andsocial burdens. In particular, the consequences of cognitive dysfunction, which includes impaired neurodevelopment in children [6, 7] and neurodegenerative diseases inelderly individuals [8], have received extensive attention
in recent years. Cognitive dysfunction in OSA patients
may be related to regional hippocampal volume changes;
an increase in the volume suggests inflammation andglial activation and a decrease in volume may be a resultof long-term neuronal damage [9]. IH may play a central
role in causing attention, memory and executive function impairment in OSA patients [10]. In vivo experiments have demonstrated that IH can drive cognitive
dysfunction through microglia-mediated hippocampal

inflammatory injury in mice [11]. However, the specificmolecular mechanism involved is unclear.
DNA methylation, one of the most studied epigenetic
modifications, occurs at the 5-carbon position of cytosine residues to generate 5-methylcytosine (5mC), which
was once considered to be stable [12]. The ten-eleventranslocation (TET) enzymes TET1-3 are members ofthe 2-oxoglutarate-dependent dioxygenase (2-OGDD)
family and oxidize DNA 5mC to 5-hydroxymethylcytosine (5hmC), suggesting a novel mechanism for DNA
demethylation [13, 14]. While the levels of 5mC are comparable across organs throughout the body, the distribution of 5hmC varies depending on tissue or cell type, withparticularly high levels in mature neurons in the central
nervous system (CNS) [15]. Given that 5hmC levels were
found to increase progressively from early postnatal toadulthood in the cerebellum and hippocampus of mice,
it is suggested that 5hmC may play a directly involvedin neurodevelopment and aging [16]. More importantly,
5hmC levels were significantly increased in the brain of
Alzheimer’s disease patients, indicating that the changesof 5hmC are not only age-dependent, but may be suggestive of neurodegeneration and disease [17]. Furthermore, TETs mediated DNA hydroxymethylation may be

Kong et al. Journal of Neuroinflammation(2024) 21:208

involved in regulating neuronal activity and cognitive
processes [18–21]. TET1 has been found to be the mostcharacterized member of the TET family involved inlearning and memory; however, this phenomenon is also
controversial [22]. Both the loss and overexpression of
Tet1 have been reported to enhance or impair memory,which may be related to the cell type specificity of TET1
[18, 23–27]. For instance, knockdown of Tet1 specificallyin hippocampal astrocytes not only significantly reduced
global 5hmC levels, but also resulted in abnormal neuronal development and impaired cognitive function [24].
However, Tet1 overexpression in hippocampal CA1 pyramidal cells contributed to long-term memory impairment after episodic fear conditioning, through strong
raised 5hmC levels [25]. Although it is well establishedthat TETs requires oxygen for catalytic activity, the datareported on the response of TETs to hypoxia are conflicting. In a variety of tumour cells, hypoxia can directlyreduce the oxidative activity of TETs to promote DNA
demethylation, while total TETs expression is unchanged[28, 29]. However, hypoxia increases TET1 expression
and 5hmC levels in certain types of cells, such as neuroblastoma [28, 30] and glioblastoma cells [31], probably
because Tet1 coactivates with hypoxia-inducible factor
independently of enzymatic activity [30, 32, 33]. In addition, TET1 also links cellular redox status and epigenome
maintenance, which mediates the increased 5hmC formation under oxidative stress [34]. Therefore, TETs and5hmC changes in hippocampal cognitive dysfunction
induced by IH, a specific chronic mild hypoxia, deserve
to be investigated.
The Wnt signaling pathway is the most important cascade in the nervous system, which is divided into thecanonical β-catenin pathway, as well as the atypical Ca+
and planar cell polarity (PCP) pathways, coordinating
synaptic function, adult neurogenesis, and neuroimmune
interactions in the brain [35–37]. Its dysregulation participates in neuroinflammation and neurogenesis, causing
cognitive dysfunction in a variety of neurodegenerativeand neuropsychiatric diseases [38, 39]. In ApoE-KO AD
organoids, aberrant Wnt pathway activation leads toabnormal neurogenesis, including decreased excitatoryneurons and increased glial and inhibitory neurons [40].
Abnormal activation of Wnt pathway was also found in
the hippocampus of LPS-induced depression mice, withdecreased mature neurons and activation of glial cells,
leading to cognitive decline [41]. In addition, activationof the Wnt pathway was found in OSA patients withhypertension and IH-treated cardiomyocytes [42, 43].
Therefore, we hypothesized that the Wnt pathway plays
an important role in IH-mediated hippocampal cognitive
dysfunction in mice.
Here, we assessed the role of DNA demethylationin cognitive dysfunction in IH mice. In addition, thePage 3 of 21

therapeutic effect of hippocampal-specific knockdownof Tet1 was investigated. This study aims to reveal theneuroinflammation -related molecular regulatory mechanism of OSA with cognitive dysfunction from the perspective of Wnt signaling activation in the hippocampus.

Materials and methods
Animals

8-week-old male BALB/c mice, weighing 18–20 g, werepurchased from Beijing Shenghe Experimental Animal Technology Co., Ltd. The mice were kept at a specific pathogen-free condition of 18–26 ℃ and 40–70%
humidity, accompanied by a standard light-dark cycle.
Experiments were performed in strict accordance withinstitutional ethical codes and were approved by the Animal Care and Use Ethics Committee of the Capital Institute of Pediatrics (DWLL2021016).
Cell culture and transfection

Human embryonic kidney cell line 293T and mouse brainneuroma cell line N2A were obtained from the Capital
Institute of Pediatrics Cell Bank. The cells were cultured
in Dulbecco’s modified Eagle’s medium (DMEM; Gibco,USA) with 10% fetal bovine serum (FBS; Gibco), 100 µg/
mL penicillin and 100 µg/mL streptomycin (Gibco) at
37 °C and 5% CO2. To interfere the expression of Tet1, ashRNA interference sequence targeting Tet1 (5’-3’: ​G​C​
A​A​T​C​A​G​T​T​A​G​C​A​G​A​C​T​T​G​A) was designed and synthesized and named shTet1. The negative control virus
was named shNC. These are purchased from Genechem(Shanghai, China). 293Ts were used for infections with a
MOI of 1. N2As were used for infections with a MOI of
20. 72 h after transfection, the relative amount of green
fluorescence in 293Ts was observed under a high-content
microscope (Theo Fisher, USA), and N2As were collected
for PCR identification.
Group, modeling and treatment
Group

Acclimatization to the new environment for 3 days. Mice
were randomly divided into 4 groups: control group, IH
group, IH + shNC group and IH + shTet1 group. In orderto prevent any interference between experiments, certain
mice were exclusively monitored for hippocampal oxygen
partial pressure (PO2), functional magnetic resonanceimaging (fMRI) parameters, or behavioral tests. The
remaining mice had their hippocampal tissues utilized
for molecular biology experiments.
Modeling

IH group, IH + shNC group, and IH + shTet1 groupwere exposed to IH for 6 weeks using a hyperbaric oxygen chamber (Beijing Zhongshi Dichuang Technology
Development Co., Ltd., China). The IH mode was 1 min

Kong et al. Journal of Neuroinflammation(2024) 21:208

alternating cycle (oxygen concentration 5-21%) for 8 h /d (8:00 am to 4:00 pm) [44]. The control group was maintained in room air. At the end of the 4th week of IH, bodyweight (n = 20/ group) was measured in the control and
IH groups, and a part of mice were euthanized after hippocampal PO2 (n = 6/ group) and fMRI parameters (n = 6/
group) were measured.
Treatment

In order to interfere the expression of Tet1, lentiviral shRNA interference sequences targeting Tet1 were
designed, synthesized and named shTet1. The negative
control viruses were named shNC. After 4 weeks of IH
treatment, IH + shNC and IH + shTet1 mice were injectedwith shNC and shTet1 in the hippocampus, respectively.
In brief, mice were anesthetized with isoflurane and fixedin the stereotaxy (Beijing Zhongshi Dichuang Technology Development Co., Ltd.). Artificial tear ointment wasapplied to the eyes of the mice. Hair was cut off from the
top of the skull and a longitudinal incision was made in
the skin. Blunt dissection was used to remove connective tissue overlying the skull. Two holes were made in
the skull using a small dental drill, with coordinates of± 1.9 mm inter-aural, − 1.4 mm relative to the bregma,
and + 1.8 mm from the dural surface [45]. The injection
infusion rate was set at 0.8 µL/min, and the total infusion
volume was 1µL/hemisphere. After infusion, the needle
was left in place for 1 min to minimize diffusion and thenslowly withdrawn. Finally, the incision was sutured andthe mice were returned to their cages.
At the end of the 6th week of IH, a part of mice in eachgroup were assessed for behavior and euthanized, andthe brains of the remaining mice were quickly removed
and processed for study after euthanasia.
Hippocampal PO2

The PO2 value in the hippocampus of mice was detectedby tissue oxygen partial pressure detection device (Beijing Zhongshi Dichuang Technology Development Co.,
Ltd.). In brief, a stereotaxy was used to fix isofluraneanesthetized mice and guide the probe into the hippocampus. PO2 was detected in real time. It was recorded
until PO2 values were in the 10% range for 3 min. The left
and right hippocampi were averaged.
fMRI data acquisition and analysis

The MRI scanning was performed by the BRUKER 7.0-T
MRI (Bruker Pharmasca 7.0/16us, Germany) scanner.
Anatomical images were acquired using rapid acquisition
with relaxation enhancement (RARE) sequence with thefollowing parameters: matrix size 256 × 256, field of view(FOV) = 2 cm × 2 cm, repetition time (TR)/ echo time(TE) = 4500/45 ms, and number of excitation (NEX) = 4.
Functional scans were acquired using gradient-echo

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planar imaging (EPI): slice thickness = 1 mm, matrix
size = 80 × 64, FOV = 2.43 cm x 2.1 cm, TR/TE = 2000/15
ms, and NEX = 1. Statistical parametric mapping software
was used for image pre-processing and statistical analysis. The blood oxygenation level dependent (BOLD) values were analyzed and compared between the the control
and IH groups. All functional image post-processing wasdone by an experienced observer who was blinded to thescanned objects. The voxel-level height threshold was
P < 0.05, and the clusterextent threshold was 20 voxels.
RNA-sequencing and transcriptomics analysis

Total RNA was extracted by Trizol (Invitrogen, USA).
Ribo-Zero rRNA Removal kit (Illumina, USA) was used
to remove rRNAs from the total RNA. High-quality RNA
was preprocessed by TruSeq Stranded Total RNA Library
Prep kit (Illumina) to construct cDNA libraries. The
libraries were qualified and quantified with BioAnalyzer
2100 system (Agilent Technologies, USA). High-quality
libraries were denatured into single-stranded DNA, captured on Illumina flowcell and amplified into clusters in
situ, and then 150 cycle sequencing was performed on
NovaSeq 6000 sequencer (Illumina). The sequencing data
were imported into Gene set enrichment analysis (GSEA)
software (v.4.3.3) to enrich signaling pathways. The
Canonical pathways set was obtained from the MSigDB
database (https://www.gsea-msigdb.org/gsea/msigdb/),
and NOM P value < 0.05 was considered significantly
enriched [46]. The sequencing data were imported into
the R platform 4.2.0, and the “DESeq2” package [47] wasused to analyze the differentially expressed genes (DEGs)of the two groups. Genes with adjust P values < 0.05 and|log2 (Fold change)| ≥ 1 were considered to have differential expression. DEGs were used for Gene ontology(GO) analysis, Kyoto encyclopedia of genes and genomes(KEGG) pathway analysis and protein-protein interaction(PPI) network construction. Protein-protein interaction(PPI) of DEGs was performed using STRING (https://cn.string-db.org), and PPIs with scores > 0.4 were visualized as networks using Cytoscape (v3.7.1) [48]. The top
20 target proteins were scored by the maximum cliqness
centrality (MCC) algorithm using plug-in CytoHubba ofCytoscape. Gene ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis were performed, and significantly enriched GO terms
and pathways were screened according to the adjusted
P < 0.05.
Hydroxymethylated/methylated DNA immunoprecipitation(hMeDIP / MeDIP) sequencing

Genomic DNA was extracted from the hippocampus ofmice in the control and IH groups using the DNeasy kit(QIAGEN, Germany) and sonicated to fragments ranging from 100 to 300 bp. Subsequently, the fragmented

Kong et al. Journal of Neuroinflammation(2024) 21:208

DNA was end repaired with the Universal DNA Library
Prep kit (GenSeq, China), adding dA tails and adaptor
sequences. A portion of the DNA labeled input (about10%) after the adaptor was added was retained, and theremaining was subjected to immunoprecipitation reaction with the 5mC MeDIP kit and 5hmC MeDIP kit(GenSeq), respectively. The Illumina PCR Primers and
2×HiFi PCR Mix (GenSeq) were used for library amplification and purification of immunoprecipitated DNA as
well as Input DNA that had not been immunoprecipitated. Purified DNA libraries were quantified by Qubit(ThermoFisher) fluorescence assay and sequenced with
NovaSeq 6000 sequencer.

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Hematoxylin-eosin (HE) staining

Brain tissues were fixed in 4% paraformaldehyde for 48 h.
The tissues were washed with phosphate buffered saline(PBS), dehydrated with ethanol, embedded in paraffin,
and sliced. Sections were stained with H&E and examined under a light microscope (Nikon, 80i, Japan). Representative images were obtained at high magnificationsof 400×.
Immunohistochemistry (IHC) staining

DNA samples were fragmented to 100 to 300 bp, andeach sample was divided into purified immunoprecipitated DNA and input DNA. The purified DNA was used
to verify the target gene. Primer sequences are shown in
Supplementary Table S1.

The tissue antigen was repaired. Sections were blockedwith 5% goat serum protein and then incubated with primary antibodies against TET1 (dilution 1:500; GeneTex,USA), TET2 (dilution 1:200; Cell Signaling Technology),
and TET3 (dilution 1:500; GeneTex) antibodies overnightat 4 °C. After washing with PBS, the sections were incubated with peroxidase-coupled secondary antibodies for
30 min at 37 °C. Representative images were obtained athigh magnifications of 400×. Image J software was used
to analyze the images.

Quantitative real-time (qRT)-PCR

Immunofluorescence (IF) staining

Total RNA was extracted by Trizol. Reverse transcriptionof cDNA using PrimeScriptTM RT kit (ABM, Canada).
qRT-PCR with the LightCycler 480 system (Roche, Switzerland) was performed using Maxima SYBR Green/
ROX qPCR Master Mix (ABM). The relative expression
level was calculated by 2 − ΔΔCt. Primer sequences areshown in Supplementary Table S1.

Sections were incubated with primary antibodies against
TET1 (dilution 1:100; GeneTex), DCX (dilution 1:100;
Cell Signaling Technology), Iba-1 (dilution 1:100; Cell
Signaling Technology), GFAP (dilution 1:100; Cell Signaling Technology) antibodies overnight at 4 °C and subsequently incubated with Cy5-labeled secondary antibodies
for 30 min at 37 °C. Nuclei were stained with DAPI. Representative images were observed under a fluorescencemicroscope (Leica, Wetzlar, Germany). Representative
images were obtained at high magnifications of 400×.

hMeDIP-PCR

Cell-type annotation

According to the existing annotations of the brain in the
CellMarker database (http://biocc.hrbmu.edu.cn/CellMarker/) [49], DEGs, differentially methylated/demethylated genes (DMG/DDG)-enriched cell types weremarked.
DNA extraction and dot blot

The TIANamp Genomic DNA Kit (Tiangen, Beijing,
China) was used to extract genomic DNA from mice hippocampus. Genomic DNA was denatured and dot onto
a nitrocellulose filter membrane (Whatman, UK) andviolet cross-linked. The membrane was blocked with 5%
milk and then incubated with primary antibodies against
5hmC (dilution 1: 1000; Cell Signaling Technology) and
5mC (dilution 1:1000; Cell Signaling Technology) antibodies overnight at 4 °C. After washing with TBST, membrane was incubated with peroxidase-coupled secondary
antibodies for 1 h at 37 °C and then developed using
enhanced chemiluminescence (7sea biotech, Shanghai,
China). The signals were quantified using ImageJ software. In addition, 0.02% methylene blue was stained in
0.3 M sodium acetate (pH 5.2) to visualize DNA as a total
genomic DNA loading control.

Luminex liquid suspension chip analysis

Luminex liquid suspension chip analysis was performed
using the Bio-Plex Pro Mouse Cytokine Grp I Panel
23-plex Assay (Bio-Rad, USA). In brief, mouse hippocampi were incubated for 30 min in 96-well plates
embedded with microspheres, followed by 30 min incubation with detection antibodies. Subsequently, Streptavidin-PE was added for 10 min and values were read using
the Bio-Plex 200 system (Luminex Corporation, USA).
Behavior assessment
Barnes maze

Mice were assessed for behavior 2 weeks after stereotaxic injection. Short-term and long-term spatial learning and memory were assessed using the Barnes maze[50]. The maze consisted of a circular platform with 20
holes arranged equally spaced at the edges, one of which
was connected to a dark chamber, the target hole. The
stimulus of light (a 200 W white light lamp) forced mice
to search for the target hole. Each mouse was allowed toadapt for 2 min the day before testing. During the training

Kong et al. Journal of Neuroinflammation(2024) 21:208

session, each mouse underwent a 4-day spatial acquisition phase in which two 3-min trials spaced 15 min apartwere performed each day. Each mouse was placed in thecenter of the platform and covered with an opaque chamber for 5 s. The chamber was subsequently removed andthe mice allowed to explore freely for 3 min. Aversive
light was turned on to encourage the mouse to run andescape into the target hole. When the target hole failed to
be entered within 3 min, the mice were gently guided into
the target hole using a transparent chamber. Mice wereallowed to explore the target hole for 1 min before being
returned to their main cage and the platform cleaned
with 70% ethanol. Mice were tested for short-term memory on day 5 and long-term memory on day 12. The time
to find the target hole was recorded for each trial, and the
trajectory was depicted using the Tracking Master system v4.0 (Beijing Zhongshi Technology Co., Ltd.).
Y-maze maze

Y-maze maze consisted of three equiangularly arranged
arms, labeled A, B, and C, each 40 cm long, 5 cm wide,
and 13 cm high [51]. Each mouse was placed distal toarm A and moved freely through the maze over an 8-min
period, and the order and number of its arm entries wererecorded. Consecutive entry into all three arms (i.e.,
ABC, BAC, CBA) was considered a single spontaneousalternating behavior. Percentage alternation was calculated as the ratio of actual and possible alternation (thetotal number of entries into the arm minus 2), multipliedby 100. The number of entering arms was used as an indicator of motor function.
Novel object recognition (NOR)

NOR was performed in an open field 0.3 m long, 0.3 mwide, and 0.45 m high [52]. Mice were trained for 5 min,during which time they were placed in the center of thearena in the presence of two identical objects. After 1 h oftraining, mice were replaced into the open field for testing in which one of the objects was replaced by a novel
object. Time spent exploring old and new objects wasquantified using the Tracking Master system v4.0. The
discrimination index was calculated as (Tnovel - TFamiliar)/ (TNovel + TFamiliar).
In addition, Pearson correlation analysis was performed between behavioral data and global 5hmC levels,
Wnt pathway gene expression levels and their 5hmC levels in the hippocampus, and P < 0.05 represented a significant correlation.
Statistical analysis

All data are in average ± standard deviation (SD) andrepeated at least 3 times. Pearson correlation analysis was
performed using SPSS software (v.25). Other data wereanalyzed using GraphPad Prism v. 9.4. The differences

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between the groups were determined by the student
T-test (two groups) or one-way analysis of variance(ANOVA). P values < 0.05 for difference was statisticallysignificant.

Results
IH exposure induced the upregulation of TETs and total
5hmC in the hippocampus of mice

The IH mouse model was established as described inprevious studies (Fig. 1A). At the end of the 4th week
of IH, weight, PO2 and fMRI were measured in the control and IH groups. The body weight and hippocampal
PO2 in the IH group were significantly lower than thosein the control group (Fig. 1B). fMRI analysis of the IH
mice revealed significantly lower BOLD signal activationin the hippocampus, especially on the left side, than in
the control mice (Fig. 1C). Previous studies have shownthat TETs mediated 5hmC modification plays an important role in regulating cognitive function. At the end of
the 6th week of IH, the hippocampal tissues of the control and IH groups were collected for DNA dot blot, IHC
staining and qPCR. The total amount of 5hmC in the hippocampus of control and IH mice was quantified using
DNA dot blot analysis. As shown in Figs. 1D and 5hmC
levels were significantly increased by IH compared withthose in the controls. We determined the expressionof TET1, TET2 and TET3 enzymes in the hippocampus by IHC and qPCR. As shown in Fig. 1E, comparedwith those in the controls, the mRNA expression levelsof Tet1, Tet2, and Tet3 in the IH group increased significantly. Interestingly, Tet1 level increased approximately
2-fold. Meanwhile, the protein expression levels of TET1,
TET2 and TET3 were also increased in the hippocampal
dentate gyrus of IH mice (Fig. 1E). Overall, IH induced
the expression of DNA hydroxymethyltransferase TET
and modification of global 5hmC.
IH exposure caused DEGs in the hippocampus of miceAt the end of the 6th week of IH, the hippocampal tissuesof the control and IH groups were collected for RNA-seq
and hMeDIP/ MeDIP-seq. The hMeDIP-seq process isshown in Fig. 2A. To evaluate the effect of IH on the hippocampus, the hippocampi of the control and IH mice
were subjected to transcriptome sequencing. A total of
253 signaling pathways were enriched by GSEA (NOM
P value < 0.05), including those related to cell cycle (e.g.
cellcycle pathway and cyclin A B1 B2 associated events
during G2 transition) and neuroregulation (e.g. TYROBP
causal network in microglia and dopaminergic neurogenesis) (Supplementary Figure S1, Supplementary
Table S2). In addition, MAPK pathway and hypoxia via
HIF1A dn were also enriched. A total of 3263 DEGs
were identified, including 1 309 upregulated DEGs and1 954 downregulated DEGs (Supplementary Table S3).

Kong et al. Journal of Neuroinflammation(2024) 21:208

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Fig. 1 IH exposure induced the upregulation of TETs and total 5hmC in the hippocampus of mice. (A) The illustration of IH modeling. (B) Body weightand hippocampal PO2. (C) BOLD-fMRI analysis of the hippocampus. (D) Total amount of 5hmC in the hippocampus using standard DNA dot blot. (E) The
expression of TET1, TET2, and TET3 in the hippocampus using IHC staining and qPCR. Analysis was performed by Student’s t test. All data are presented
as the mean ± standard deviation

Hierarchical clustering and volcano plot were used toanalyse the DEGs and visualize the genomic data (Fig. 2B,
Supplementary Figure S2A). STING and Cytoscape wereused to construct the PPI network and visualization, andthe CytoHubba plugin with MCC algorithm was used toscore and rank the target proteins. The top 20 target proteins are mostly related to cell cycle (e.g., Cdk1, Ccnb1,
Bub1) or Wnt (e.g., Wnt3a, Wnt2) (Supplementary Figure S2B). GO and KEGG pathway enrichment analyses
were subsequently performed using the DEGs. A total of
1 703 GO terms were obtained, including 3 676 BP, 303
CC, and 573 MF (adjusted P value < 0.05) (Supplementary
Table S4). As shown in Fig. 2C, compared with those in
the controls, the regulated transcripts in the hippocampiof IH mice were associated with the BP terms axonogenesis, axon guidance, and the pattern specification process,MF terms, such as ion and gated channel activity, andin CC terms, such as the synaptic membrane and ionchannel complex. A total of 67 Signaling pathways wereenriched according to KEGG pathway analysis (adjusted
P value < 0.05) (Supplementary Table S5). Twenty pathways, including neuroactive ligand‒receptor interaction,
the cAMP Signaling pathway, the Wnt Signaling pathway, and the MAPK Signaling pathway, were screened forvisualization based on the P value (Fig. 2D). These results
showed that IH altered the expression of inflammatoryand neuroactive-related genes in the hippocampus.
IH exposure caused DMG/DDG in the hippocampus of mice

MeDIP-seq and hMeDIP-seq were performed on 5mC
and 5hmC, respectively, to map the genome differences
between the hippocampi of control and IH mice. As

Kong et al. Journal of Neuroinflammation(2024) 21:208

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Fig. 2 IH exposure caused DEGs in the hippocampus of mice. (A) The illustration of hMeDIP-sequencing. (B) DEGs were displayed by hierarchical clustering. (C) GO enrichment. (D) KEGG pathway enrichment

shown in Fig. 3A, there were significant differences in themean normalized counts of 5mC- and 5hmC-enriched
regions in the hippocampus between control and IH
mice. Heatmap clustering was used to visualize the 5 kb
distribution of 5mC and 5hmC peaks upstream anddownstream of the transcription start site (TSS) (Fig. 3B).
Compared with control mice, IH mice presented
decreased enrichment of 5mC in this genomic region,
whereas the corresponding level of 5hmC increased(Fig. 3C). Genes containing altered 5hmC peaks wereanalysed, and 12 606 genes were found to be differentially demethylated in the hippocampus between control
and IH mice; 10 994 of these genes were demethylated,and 1 612 were not demethylated (Supplementary Table
S6). GO analysis of the DDGs revealed a total of 5 824
enriched GO items, including 4 723 BP, 392 CC, and 709

MF terms (Supplementary Table S7). According to theadjusted P value, 30 GO terms were screened for visual
analysis (Fig. 3D). The results of the BP analysis indicatedthat DDGs were mainly enriched in terms related to axonogenesis, synapse organization, and cell junction assembly terms. In the MF, the most enriched terms included
GTPase regulator activity, nucleotide-triphosphatase regulator activity, and metal ion transmembrane transporter
activity. The three most enriched CC terms were glutamatergic synapse, neuron to neuron, and synapse asymmetric synapse. Genes containing altered 5mC peaks
were analysed, and 16 033 genes were found to be differentially methylated in the hippocampus between control and IH mice, 15 620 of which were methylated and413 of which were unmethylated (Supplementary Table
S8). GO analysis of the DMGs revealed a total of 4 732

Kong et al. Journal of Neuroinflammation(2024) 21:208

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Fig. 3 IH exposure caused DMG / DDG in the hippocampus of mice. (A) Mean normalized counts of 5mC- and 5hmC-enriched regions in the hippocampus. (B) (C) 5 kb distribution of 5mC and 5hmC peaks upstream and downstream of the TSS. (D) GO analysis of the DDGs. (E) GO analysis of the DMGs. (F)KEGG pathway analysis. (G) Cell types enrichment

enriched GO items, including 3 977 BP, 288 CC, and 467
MF terms (Supplementary Table S9). According to theadjusted P value, 30 GO terms were screened for visual
analysis (Fig. 3E). The BP results indicated that DMGs

were mainly enriched in ameboidal-type cell migration,
cell junction assembly and axonogenesis terms. In theMF, the most enriched terms included metal ion transmembrane transporter activity, gated channel activity,

Kong et al. Journal of Neuroinflammation(2024) 21:208

and passive transmembrane transporter activity. The
three most enriched CC terms were collagen-containing extracellular matrix, receptor complex, and synaptic
membrane. Moreover, they were enriched in CCs suchas receptor complexes and synaptic membranes. KEGG
analysis of the DDGs and DMGs revealed 121 and 123
enriched Signaling pathways, respectively (Supplementary Table S10, Supplementary Table S11). According to
the adjusted P value, 10 pathways were screened separately for visualization; among these pathways, the DDGenriched pathways included axon guidance, the cAMP
Signaling pathway, and the Wnt Signaling pathway, andthe corresponding DMGs included the calcium Signaling pathway, the PI3K-Akt Signaling pathway, and axon
guidance (Fig. 3F). The cell types enriched in the upregulated and downregulated DEGs, upregulated DDGs, andupregulated DMGs are marked. As shown in Fig. 3G, theupregulated DEGs were enriched mainly in neurons andbasket cells, and the downregulated DEGs were enrichedmainly in ependymal cells and the other 4 cell types. The
upregulated DMGs were enriched mainly in ependymal cells and the other 7 cell types, and the upregulated
DDGs were enriched mainly in 10 cell types, including smooth muscle cells, Cajal–Retzius cells, olfactoryensheathing glia, oligodendrocytes, endothelial cells
and Bergmann glial cells. These data indicated that IH
induced global enrichment of 5hmC markers, especiallyin genes related to inflammation and neural activity.
IH exposure promoted the binding of 5hmC to wnt
pathway genes in the hippocampi of mice

Given the critical function of 5hmC alterations in controlling gene expression, we investigated the relationshipbetween 5hmC and mRNA levels. Supplementary Figure
S3A shows the proportion of 5mC and 5hmC peaks ineach genomic region. Significant differences were foundby comparing the enrichment of 5mC and 5hmC in promoter regions in the hippocampus of control and IH
mice (Supplementary Figure S3B). The RNA-sequencing
data was combined with the methylation, demethylationand gene expression in promoter regions data (Fig. 4A,
Supplementary Table S12, S13). Compared with thosein the control group, the expression and demethylationlevels of genes involved in the Wnt (Wnt3a, Ccnd2 andPrickle2), synaptic vesicle cycle (Snap25, Syt1), MAPK
(Il1b, Fgf13) and cAMP (Htr4, Adcy1) Signaling pathways were increased. The expression of the above genes
in the hippocampi of control and IH mice was examined
by qPCR. The results showed that the above genes wereupregulated after IH stimulation (Fig. 4B). 5hmC profiles
revealed high 5hmC levels during IH treatment (Fig. 4C,
Supplementary Figure S4). The demethylation levelsof specific regions in Wnt3a, Ccnd2 and Prickle2 werevalidated by hMeDIP-qPCR. The results showed that

Page 10 of 21

IH stimulation increased the binding of the above genes
to 5hmC (Fig. 4D). These findings suggested that 5hmC
modification may play an epigenetic regulatory role in
the expression of Wnt3a, Ccnd2 and Prickle2 in the Wnt
pathway in the hippocampi of IH-treated mice.
Tet1 intervention improved hippocampal cognitive
dysfunction in IH mice

Taken together, our data demonstrate that the expressionof DNA hydroxymethyltransferases, especially TET1, issignificantly altered in the hippocampus of IH mice, suggesting a critical role for DNA hydroxymethylation. To
investigate IH further, we altered the DNA hydroxymethylation levels in the hippocampus by knocking down Tet1
using a lentivirus plasmid (Fig. 5A). IH mice treated with
shNC or shTet1 are shown in Fig. 5A. Figure 5B illustrates
the design of the lentivirus carrying shTet1. 293T cells
were transfected with shNC or shTet1 for 72 h, and green
fluorescence was observed under a high-content microscope, confirming successful transfection (Supplementary Figure S5). The qPCR results showed that the Tet1
mRNA level in N2A cells transfected with shTet1 was significantly lower than that in N2A cells transfected with
shNC, while there was no significant difference between
the shNC-transfected cells and the control cells (Fig. 5C).
At the end of the 6th week of IH, hippocampal tissues
were collected for IHC staining, qPCR and IF staining.
IHC and qPCR showed that both the staining and relative mRNA expression of total TET1 in the hippocampus of shTet1-treated mice were significantly lower thanthose in the IH group (Fig. 5D). IF staining demonstrated
the subcellular localization of TET1 in the hippocampus, and the localization changes after IH treatment andTet1 knockdown (Fig. 5E). Hippocampus is an important
brain region responsible for encoding and storing memories. Thus, behavioral assessments, including Barnes
maze, Y maze, and NOR tests, were performed during
weeks 6 to 8 of IH. The exploratory trajectories of theBarnes maze-treated mice on Days 5 and 12 showed thatthe short-term and long-term memory deficits, respectively, were impaired after IH exposure but recovered
after Tet1 knockdown (Fig. 6A). The latency to identify target box was significantly greater in the IH groupthan in the control group, while it was significantly lower
in the IH + shTet1 group than in the IH group (Fig. 6B).
Figure 6C shows the changes in latency to reach the target box during the 4-day training period in each groupof mice. The Y-maze test showed that spontaneous alternation behaviour was significantly lower in the IH groupthan in the control group but was significantly greaterin the IH + shTet1 group than in the IH group (Fig. 6D).
There was no significant difference in the total numberof arm entries between the groups, suggesting that thechanges in spontaneous alternation behaviour were notKong et al. Journal of Neuroinflammation(2024) 21:208

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Fig. 4 IH exposure promoted the binding of 5hmC to Wnt pathway genes in the hippocampi of mice. (A) The connection by assimilating RNA-sequencing data into methylation, demethylation and gene expression in promoter regions. (B) The expression of Wnt3a, Ccnd2, Prickle2, Il1b, Fgf13, Htr4, Adcy1,
Snap25, and Syt1 in the hippocampus using qPCR. (C) 5hmC profiles of Wnt3a, Ccnd2, and Prickle2. (D) The demethylation levels of specific regions in
Wnt3a, Ccnd2 and Prickle2 using hMeDIP-qPCR. Analysis was performed by Student’s t test. All data are presented as the mean ± standard deviation

due to motor deficits (Fig. 6E). The NOR test showed thatthe preference of mice for new objects was significantly
lower in the IH group than in the control group but wassignificantly greater in the IH + shTet1 group than in the
IH group (Fig. 6F). There was no significant differencein the total interaction time with old or new objects
between the groups, suggesting that the changes in thediscrimination index were not due to lack of interest(Fig. 6G). These findings suggested that Tet1 knockdown
can ameliorate the short-term and long-term memory
impairment caused by IH. Besides, Pearson correlation
analysis was performed between cognitive function andglobal 5hmC levels, Wnt pathway gene expression levels
and their 5hmC levels. As shown in Table 1, latency toKong et al. Journal of Neuroinflammation(2024) 21:208

Page 12 of 21

Fig. 5 Tet1 knockdown was effective in vitro and in vivo. (A) The illustration of Tet1 intervention. (B) The design of the lentivirus carrying shTet1. (C) The
expression of Tet1 in N2A cells using qPCR. (D) The expression of TET1 in the hippocampus using IHC staining and qPCR. (E) The subcellular localization of
TET1 in the hippocampus using IF staining. Analysis was performed by Student’s t test. All data are presented as the mean ± standard deviation

identify target box at day 5 in the Barnes maze was significantly positively correlated with global 5hmC levels
and Prickle2 mRNA expression. The proportion of alternating behaviors in the Y-maze was negatively correlatedwith the mRNA levels of Wnt3a, Ccnd2, and Prickle2, as
well as the 5hmC levels of Wnt3a and Prickle2. The discrimination index in the NOR was significantly positively
correlated with the 5hmC level of Wnt3a.

Kong et al. Journal of Neuroinflammation(2024) 21:208

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Fig. 6 Tet1 intervention improved hippocampal cognitive dysfunction in IH mice. Measured in the Barnes maze: (A) Exploratory trajectories on days 5
and 12. (B) Latency to identify target box on days 5 and 12. (C) Latency to identify target box during the 4-day training period. Measured in the Y-maze:(D) Alternation behavior. (E) Number of arm entries. Measured in the NOR: (F) Discrimination index. (G) Total interaction time. Analysis was performed byStudent’s t test. All data are presented as the mean ± standard deviation

Kong et al. Journal of Neuroinflammation(2024) 21:208

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Table 1 Correlation analysis of the cognition function and 5hmC demethylation

global 5hmC level
5hmC level of Wnt3amRNA level of Wnt3a5hmC level of Ccnd3
mRNA level of Ccnd2
5hmC level of Prickle2
mRNA level of Prickle2

Latency to identify target box
on the 5th day of the Barns
maze

Latency to identify target box
on the 12th day of the Barns
maze

Alternation behaviors of
the Y-maze

0.816*
0.766
0.782
0.799
0.37
0.809
0.898*

0.219
0.7
0.773
0.695
0.747
0.715
0.28

-0.52
-0.850*-0.919**-0.852*-0.77-0.854*-0.838*Discriminationindex ofthe NOR-0.397-0.754-0.824*-0.762-0.735-0.762-0.736*p<0.05 ** p<0.01

Tet1 knockdown limited excessive wnt signaling activationand hippocampal neurogenesis in mice

Tet1 knockdown alleviated IH-induced hippocampal
neuroinflammation in mice

Wnt pathway plays a crucial role in regulating neurogenesis and neuroinflammation, and its dysregulation canlead to various neuropathologies. While moderate neurogenesis and neuroinflammation have positive effects
on the brain; however, their excess is often catastrophic.
In this study, brains were collected at the end of the 6th
week of IH for HE staining of hippocampal CA1, CA3
and DG regions. As illustrated in Fig. 7A, the neurons
in the hippocampi of control mice were well-organized,
clearly stained, and exhibited a normal microstructure
under normal oxygen conditions. In contrast, the IH
group showed evident pathological changes; the neurons appeared loosely arranged with light staining, andthere was observed neuron loss and nuclear pyknosis.
However, following treatment with shTet1, the morphology of all tissues tended to normalize. Then, we utilized
DNA dot blot and qPCR to assess the global 5hmC levels and Wnt pathway gene expression. Our findings
revealed a decrease in the 5hmC level in the hippocampiof shTet1 mice compared to IH mice, as determined bystandard dot blot (Fig. 7B). The mRNA levels of Wnt3a,
Ccnd2, and Prickle2 were significantly lower in the hippocampi of shTet1 mice compared to IH mice, as determined by qPCR (Fig. 7C). Additionally, shTet1 injection
significantly reduced IH-induced demethylation levels of
specific regions in Wnt3a, Ccnd2, and Prickle2, as determined by hMeDIP-qPCR (Fig. 7D). Finally, IF staining
was used to examine the expression of DCX (a marker
of immature neurons) in the hippocampal DG area
(Fig. 7E). Quantitative analysis revealed that IH treatment significantly increased DCX expression, while it
was significantly reduced after shTet1 injection. These
results indicated that Tet1-targeted therapy may effectively counteract IH-induced abnormal hippocampal
neurogenesis by potentially regulating excessive levels of
5hmC and Wnt pathway genes.

We then investigated n hippocampal neuroinflammatory changes in the hippocampus following IH, as well as
improvements observed after shTet1 injection. IF staining
was utilized to assess the expression of IBA1 (a marker
of microglia) and GFAP (a marker of astrocytes) in the
hippocampal DG area (Fig. 8A-B). Quantitative analysis revealed that IH treatment significantly elevated theexpression of IBA1, and GFAP. Following shTet1 injection, there was a significant decrease in GFAP expression, while IBA1 showed a tendency towards reduction,although not statistically significant. Luminex liquid suspension chip analysis was used to compare the differential expression of 23 cytokines in the hippocampus of thecontrol group, IH group and IH + shTet1 group (Fig. 8C).
Thirteen cytokines were significantly upregulated after
IH, and the five most upregulated proteins were MIP1a,
IL17A, KC, IL3 and IL12 (p40). The expression of eightcytokines, such as TNFa, IL3, IL9 and IL17A, decreased
significantly after shTet1 injection (Fig. 8C, Supplementary Table S14). Taken together, these data indicatethat Tet1-targeted therapy may be effective against IHinduced hippocampal inflammation, possibly also bylimiting the excessive levels of 5hmC and Wnt pathway
genes.

Discussion
IH is an important feature of OSA and plays a majorrole in hippocampal oxidative stress injury and cognitive dysfunction [53]; however, the exact mechanism
involved remains poorly understood. In this study, forthe first time, we revealed the effects of IH on the DNA
demethylation process in the mouse hippocampus andlinked these effects with changes in hippocampus-related
cognitive functions. In addition, we found that IH regulated neuroinflammation and neurogenesis through thedemethylation of Wnt Signaling pathway genes, which
was reversed by Tet1 knockdown.
The enzymatic activity of TETs, members of the
2-OGDD superfamily, may be regulated by oxygen

Kong et al. Journal of Neuroinflammation(2024) 21:208

Fig. 7 Tet1 knockdown increased hippocampal microglia. (A) Hippocampal morphology using HE staining. (B)(C)(D) The expression of DCX, GFAP
and IBA1 in the hippocampus using IF staining. Analysis was performed byStudent’s t test. All data are presented as the mean ± standard deviation

availability and redox-based mechanisms, thereby catalysing the progressive oxidation of 5mC from DNA [54,55]. Unlike those of Tet2 and Tet3, the enzymatic activityof Tet1 is more sensitive to changes in oxygen concentration within the physiological hypoxic range. Specifically,
Oxygen-dependent Tet1 activity mediates the phasic
burst of 5hmC levels during early differentiation in vitro[56]. In various tumor cell lines, hypoxia leads to reduced
5hmC levels by decreasing the activity of TET enzymes
without concomitant TET expression [28]. It should be
noted that the inhibition of the DNA hydroxymethylation
process was observed only at oxygen concentrations

Page 15 of 21

below about 2%. The IH used in our study is a specificchronic mild hypoxia with alternating oxygen concentrations from 5 to 21%, most likely far from sufficient toinhibit TET enzyme activity. On the other hand, hypoxia
induces transcriptional activation of TET1 but not TET2or TET3 in a variety of neuroblastoma cell lines, resulting
in elevated global 5hmC levels and enrichment of 5hmC
at canonical hypoxia response genes. This reflected theoxygen-dependent non-enzymatic function of TET1 [28,30]. 5hmC is a stable epigenetic marker that generally
promotes gene expression [57, 58]. 5hmC is most abundant in the central nervous system, and its continuous
increase in the hippocampus and other brain regions ofmice from early postnatal to adulthood and possibly into
old age indicates its close association with brain function
and disease [58, 59]. In our study, TETs expression andtotal 5hmC levels were significantly higher in the hippocampi of IH mice than in those of control mice. These
results suggested that TETs play, at least in part, a direct
nonenzymatic role under IH, that is, by upregulating
their expression to raise 5hmC levels.
Among TET enzymes, TET1 has been the earliest andmost studied in the field of neuroscience, and its influence on neurogenesis extends throughout prenatal, juvenile and adult life. First, both catalytic and non-catalytic
functions of TET1 drive the differentiation of mouseembryonic stem cells into neural precursors [60]. Then,
TET1 can accelerate neurogenesis during mouse embryonic brain development [61]. Besides, 5hmC specificallyaccumulates in the Tet1-binding region during abnormal
cortical development in mice at young ages, which may
further affect neurodevelopment and brain function [62].
Tet1 can even control adult hippocampal neurogenesis
in mice, further regulating psychological behavior andcognitive function [26, 63]. However, the effect of TET1
on cognitive function is quite controversial, and both its
loss and overexpression have been reported to enhanceor impair memory by mediating DNA hydroxymethylation, which may be partially attributed to the differential
regulation of its two isoforms in neurons and neurogliocytes, respectively [18, 23–27]. Tet1S is highly enrichedin neurons, and its repression enhances hippocampusdependent memory in mice, whereas Tet1FL is moreabundant in glial cells, and its repression impairs memory [27]. However, overexpression of Tet1 specificallyin hippocampal CA1 pyramidal cells caused memory
impairment [25]. In addition, Tet2 also has cell type specificity in regulating cognitive processes, as its loss in adultneural progenitor cells impairs neurogenesis and cognitive function, whereas its loss in neurons enhances hippocampus-dependent memory by modulating synaptic
plasticity [64, 65]. Loss of Tet3 in brain neurons impairs
hippocampal spatial orientation and short-term memory[19, 66]. Therefore, we hypothesized that IH-induced

Kong et al. Journal of Neuroinflammation(2024) 21:208

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Fig. 8 Tet1 knockdown alleviated IH-induced hippocampal neuroinflammation in mice. (A) The expression of Wnt3a, Ccnd2, and Prickle2 in the hippocampus using qPCR. Analysis was performed by Student’s t test. All experiments were repeated at least three times. All data are presented as themean ± standard deviation. (B) Total amount of 5hmC in the hippocampus using standard DNA dot blot. (C) Differential expression of 23 cytokines was
displayed by hierarchical clustering, and 13 of them were significantly upregulated after IH. Analysis was performed by Student’s t test. All data are presented as the mean ± standard deviation

cognitive dysfunction may be associated with enhanced
global DNA hydroxymethylation in the hippocampus.
We found that IH mice exhibited significant decreases inspatial learning and short-term and long-term memory.
Pathological changes were found in the hippocampus by

HE staining, and a decreased BOLD signal was found by
fMRI, especially on the left side of the hippocampus. We
will further investigate the reasons for this bilateral asymmetry in the future. Furthermore, we conducted cell type
annotation of DDGs and found that IH-induced 5hmC

Kong et al. Journal of Neuroinflammation(2024) 21:208

was extensively enriched in neurons, a variety of glial
cells, smooth muscle cells, and endothelial cells. Interestingly, IF staining revealed that TET1 was not expressed
throughout the DG. Even if its expression was significantly upregulated after IH, it remained relatively weak in
specific cells. This finding contradicts previous literature
which reported high levels of TET1 expression in neurons throughout the hippocampus. In the future, it will
be necessary to investigate colocalization of TET1 withdifferent cell types in the hippocampus in the future.
Studies have shown that IH can cause hippocampal
inflammatory damage and subsequent cognitive dysfunction, which may be mediated mainly by microglia [11,67, 68]. We found several signaling pathways that wereenriched in the DDGs and DEGs. These genes were foundto be involved in neuroregulation -related pathways,
including axon guidance, neuroactive ligand‒receptor
interaction, and Calcium signaling pathways, as well as
inflammation -related pathways, such as MAPK signaling
pathways, NF-κB signaling pathways and Th17 cell differentiation. Strikingly, the Wnt signaling pathway, which isconsidered to regulate neurogenesis, neuroinflammation[37], was also enriched. In addition, the top 20 key target
proteins obtained by PPI analysis were mostly related tocell cycle and Wnt. The Wnt signaling pathway is divided
into the canonical Wnt/β-catenin pathway and the noncanonical Wnt signaling pathway, which include the Ca+
pathway and PCP pathway [69]. Neurogenesis, a highly
dynamic process that continues throughout the lifetimein rodents and humans, plays an important role in hippocampus-dependent learning and memory [70]. Lossof normal neurogenesis or the generation of abnormal
neurogenesis can disrupt hippocampal neural circuits,
leading to cognitive dysfunction [71]. The Wnt signaling
pathway is the main factor controlling adult hippocampal
neurogenesis, and proper Wnt signaling pathway activation is required to regulate the proliferation, differentiation and maturation of neurons [36, 72]. During the earlystages of neurogenesis, canonical Wnt Signaling determines cell proliferation and maturation and subsequently,
together with noncanonical Wnt signaling, regulates differentiation and morphological development [73–75]. In
the canonical Wnt pathway, Wnt3a, an upstream gene,promotes neurogenesis by shortening the cell cycle ofhippocampal neural progenitor cells [76]. Its downstream gene, Ccnd2, whose knockdown caused a defectin progenitor cell proliferation almost completely abolished adult neurogenesis [77]. Prickle2, a member of the
non-canonical Wnt pathway, was found to be deficient in
dendritic branching, synapse number and postsynaptic
density [78]. However, excessive activation of this pathway may impair the maturation of neurons, causing dendrite, spine and synapse dysplasia [75, 79, 80]. The effectof the Wnt signaling pathway on neuroinflammation isPage 17 of 21

mainly achieved by regulating microglial activation. In avariety of CNS diseases, microglial activation mediatedby canonical Wnt signaling is generally characterized byan anti-inflammatory protective effect, while noncanonical Wnt signaling -mediated microglial activation is proinflammatory and promotes disease progression [81–88].
Even if, for instance, Wnt/β-catenin activity coincides
with early neuronal damage and microglial inflammationin the hippocampi of mice with experimental autoimmune encephalomyelitis, this change can be interpreted
as active inflammation activating Wnt signaling, triggering hippocampal neurogenesis, and replenishing damaged neurons [89]. However, Wnt3a plays dual regulatory
roles in microglial and may be related to the activationof other inflammatory pathways. Intraventrically injected
GSK3β inhibitors to activate canonical signaling pathways in premature rabbits with intraventricular haemorrhage inhibited microglial inflammation, while rt-Wnt3a
promoted inflammation [90]. In vitro, Wnt3a can convert microglia to a proinflammatory phenotype, probably
through its activation of the ERK1/2 pathway in addition to the canonical Wnt pathway [91, 92]. Activationof the NF-κB pathway can be observed when a Wnt/βcatenin pathway agonist is added to microglia, and viceversa; this bidirectional positive feedback effect may
cause sustained activation of both pathways and exacerbate inflammatory damage [93]. In addition, activatedmicroglia can also enhance the neurotoxicity of astrocytes by secreting a variety of inflammatory mediators tojointly drive inflammatory damage in the CNS [11, 94].
Therefore, we identified Wnt3a, Prickle2 and Ccnd2 in
the Wnt signaling pathway as key demethylation regulators and demonstrated increased demethylation and gene
expression in the hippocampi of IH mice. The level of
DCX (a marker of immature neurons) in the DG regionof the hippocampus were significantly increased after IH,
indicating abnormal hippocampal neurogenesis. Besides,
the levels of IBA1 (a marker of microglia) and GFAP (amarker of astrocytes) in the DG region of the hippocampus were significantly increased after IH. Additionally,a variety of inflammatory cytokines were significantly
upregulated, indicating a neuroinflammatory state in the
hippocampus.
To summarize, we heve decided to validate the induced
phenotypic changes by eliminating demethylation via
hippocampus-specific knockdown of Tet1 for the following reasons: (1) TET1 has been identified as a hypoxiasensitive sensor in specific cell types and induces high5hmC levels in response to hypoxia. We suggested thatit may play an important role in IH. (2) TET1 is essential for neurogenesis, and since the hippocampal DG
is one of the major regions of adult neurogenesis, we
proposed that it is more likely to regulate hippocampal
function in the IH context. (3) Studies on the effects of

Kong et al. Journal of Neuroinflammation(2024) 21:208

cognitive function have focused more on TET1 but arehighly contradictory, and we aimed to determine the roleof TET1 in IH-induced cognitive dysfunction. IF staining
showed that TET1 expression was significantly reducedin the nucleus of the DG region. As we predicted, shTet1
treatment resulted in significant improvements in spatial
learning and memory, and significant reductions in Wnt
Signaling pathway gene demethylation and gene expression. The number of DCX, GFAP and IBA1 positive cells
and the levels of multiple inflammatory cytokines tended
to be normal, indicating that abnormal neurogenesis andneuroinflammation were limited. In future studies, theeffectiveness of TET1 knockdown will be investigatedin different subtypes and cell type distributions. In addition, recent studies have shown that intrathecal injectionof TET1 specific inhibitor Bobcat 339 can achieve similar therapeutic effects as Tet1 siRNA on inflammatory
pain in mice [95]. It prompted us to consider utilizing
this inhibitor in the future to further explore the mechanism of DNA hydroxymethylation in IH-induced cognitive dysfunction. Finally, 5hmC point mutations in Wnt
pathway genes were essential to verify that TET1 affects
cognitive function by specifically regulating 5hmC.

Conclusions
Our results demonstrated that Wnt pathway genedemethylation was involved in the development of IHinduced cognitive dysfunction in mice by regulating hippocampal neuroinflammation and neurogenesis, which
could be reversed by specific knockdown of hippocampal
Tet1. We hope that our study contributes to the understanding of the underlying mechanisms of cognitive
impairment in OSA patients and suggests a potentiallyeffective target for intervention.
Abbreviations
2-OGDD	2-oxoglutarate-dependent dioxygenase
5hmC	5-hydroxymethylcytosine
5mC	5-methylcytosine
BOLD	Blood oxygenation level dependent
CNS	Central nervous system
DDGs	Differentially demethylated genes
DEGs	Differentially expressed genes
DMGs	Differentially methylated genes
fMRI	Functional magnetic resonance imaging
HE	Hematoxylin-eosin
hMeDIP	Hydroxymethylated DNA immunoprecipitation
IF	Immunofluorescence
IHC	Immunohistochemistry
IH	Intermittent hypoxia
MeDIP	Methylated DNA immunoprecipitation
NOR	Novel object recognition
OSA	Obstructive sleep apnoea
PO2	Partial pressure of oxygen
qRT-PCR	Quantitative real-time qRT-PCR
TET	Ten-eleven translocation

Page 18 of 21

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03189-2.
Supplementary Material 1
Supplementary Material 2
Supplementary Material 3
Supplementary Material 4
Supplementary Material 5
Supplementary Material 6
Supplementary Material 7
Supplementary Material 8
Supplementary Material 9
Supplementary Material 10
Supplementary Material 11
Supplementary Material 12
Supplementary Material 13
Supplementary Material 14
Supplementary Material 15
Supplementary Material 16
Supplementary Material 17
Supplementary Material 18
Supplementary Material 19
Supplementary Material 20
Acknowledgements
Not applicable.
Author contributions
All authors contributed to the conception and design of the study. KY
and JJ conducted most of the experiments, prepared the manuscript andcontributed equally. ZX, LF, and YP help analyze data and comment on
previous manuscripts. All the authors approved the final draft. Dr. Fei Yang
and Dr. Shan wang are the guarantor of this work, and as such, they has fullaccess to all the data in the study and is responsible for the integrity of thedata and the accuracy of the data analysis. All authors read and approved thefinal manuscript.
Funding
This work was supported by grants from National natural science foundationof China (82271193), Public service development and reform pilot projectof Beijing Medical Research Institute (BMR2021-3), Capital’s Funds for Health
Improvement and Research (2022-2-1132), Beijing Hospitals Authority’s Ascent
Plan (DFL20221102), and Research Foundation of Capital Institute of Pediatrics(LCY-2023-23).
Data availability
Data is provided within the manuscript or supplementary information files.

Declarations
Ethics approval and consent to participate
All experiments were approved by the Animal Care and Use Ethics Committeeof the Capital Institute of Pediatrics (DWLL2021016). Anesthesia andeuthanasia of animals were consulted with the American Veterinary Medical
Association (AVMA) Guidelines for the Euthanasia of Animals (2020).

Kong et al. Journal of Neuroinflammation(2024) 21:208

Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1Children’s Hospital Capital Institute of Pediatrics, Chinese Academy ofMedical Sciences & Peking Union Medical College, Beijing 100020, China
2
Graduate School of Peking Union Medical College, Beijing 100730, China
3
Department of Otolaryngology, Head and Neck Surgery, Children’s
Hospital Capital Institute of Paediatrics, Beijing 100020, China
4
Department of Otolaryngology, Head and Neck Surgery, BeijingChildren’s Hospital, Capital Medical University, National Center forChildren’s Health, Beijing 100045, China
5
Beijing Municipal Key Laboratory of Child Development and Nutriomics,
Capital Institute of Pediatrics, Beijing 100020, China
Received: 11 April 2024 / Accepted: 29 July 2024

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