Kim et al. Journal of Neuroinflammation(2024) 21:180
https://doi.org/10.1186/s12974-024-03179-4

Journal of Neuroinflammation

Open Access

RESEARCH

Chronic rapid eye movement sleep
deprivation aggravates the pathogenesisof Alzheimer’s disease by decreasing brain OGlcNAc cycling in mice
Dong Yeol Kim1†, Sang-Min Kim1† and Inn-Oc Han1*
Abstract
This study investigated the role of O-GlcNAc cycling in Alzheimer’s disease-related changes in brain
pathophysiology induced by chronic REM sleep deprivation (CSD) in mice. CSD increased amyloid beta (Aβ)
and p-Tau accumulation and impaired learning and memory (L/M) function. CSD decreased dendritic lengthand spine density. CSD also increased the intensity of postsynaptic density protein-95 (PSD-95) staining. Allof these Alzheimer’s disease (AD) pathogenic changes were effectively reversed through glucosamine (GlcN)
treatment by enhancing O-GlcNAcylation. Interestingly, the lelvel of O-GlcNAcylated-Tau (O-Tau) exhibited an
opposite trend compared to p-Tau, as it was elevated by CSD and suppressed by GlcN treatment. CSD increased
neuroinflammation, as indicated by elevated levels of glial fibrillary acidic protein and IBA-1-positive glial cells inthe brain, which were suppressed by GlcN treatment. CSD promoted the phosphorylation of GSK3β and led to an
upregulation in the expression of endoplasmic reticulum (ER) stress regulatory proteins and genes. These alterations
were effectively suppressed by GlcN treatment. Minocycline not only suppressed neuroinflammation induced by
CSD, but it also rescued the decrease in O-GlcNAc levels caused by CSD. Minocycline also reduced AD neuropathy
without affecting CSD-induced ER stress. Notably, overexpressing O-GlcNAc transferase in the dentate gyrus regionof the mouse brain rescued CSD-induced cognitive dysfunction, neuropathy, neuroinflammation, and ER stressresponses. Collectively, our findings reveal that dysregulation of O-GlcNAc cycling underlies CSD-induced AD
pathology and demonstrate that restoration of OGlcNAcylation protects against CSD-induced neurodegeneration.
Keywords REM sleep, Hexosamine, Cognition, Alzheimer’s disease, O-GlcNAc transferase†

Dong Yeol Kim and Sang-Min Kim contributed equally to this work.

*Correspondence:
Inn-Oc Han
iohan@inha.ac.kr
1
Department of Biomedical Science, Program in Biomedical Scienceand Engineering, Department of Physiology and Biophysics, College ofMedicine, Inha University, Incheon, Korea© The Author(s) 2024. 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) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed 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.

Kim et al. Journal of Neuroinflammation(2024) 21:180

Introduction
Sleep can be categorized into two distinct stages—REM(rapid eye movement) sleep and non-REM sleep—with
the former characterized, in part, by its shorter duration.
Sleep deprivation (SD), particularly deprivation of REM
sleep (REMSD), can have a significant impact on mood,learning and memory (L/M) function, and may even contribute to the development of neuropsychiatric disorders,
including Alzheimer’s disease (AD) [1–3], Parkinson’s
disease [4], and depression [5]. SD can be further classified based on its duration. Acute SD refers to continuous
SD lasting from several hours to a week, whereas chronic
SD involves SD for several hours that persists for weeks
or months. Both acute and chronic SD are associatedwith cognitive impairments and neurodegenerative disorders, but chronic SD in particular has been specifically
implicated in the pathogenesis of AD. Indeed, chronic
insomnia is a known risk factor for accelerated cognitive decline with aging [6] and older adults experiencing
chronic sleep disorders are more likely to exhibit rapid
cognitive decline and have an increased risk of developing AD [7].
Prolonged SD has been reported to decrease glucose
uptake by 10–20% in the brains of humans and rodents[8, 9]. Multiple lines of evidence suggest that disrupted
sleep is likely to impair neurometabolism, particularly inthe hippocampus—a brain region known to be involved
in L/M processes [10–12]. Furthermore, alterations inbrain glucose metabolism are frequently observed before
the manifestation of clinical symptoms of dementia, andseveral studies have demonstrated a reduction in glucose
uptake in the brains of patients with AD [13, 14]. Therefore, it can be hypothesized that prolonged disruption in
glucose metabolism during chronic SD may be a significant contributing factor to the development of cognitive
dysfunction and AD. However, the mechanisms by which
abnormalities in brain glucose metabolism contribute tocognitive impairment or AD are still largely unknown. In
our previous research, we proposed a significant association between dysfunction in the hexosamine biosynthetic
pathway (HBP), which accounts for 2–5% of glucose
metabolism [15], and cognitive impairment induced byacute SD in both mouse and zebrafish [16, 17]. The HBP
produces UDP-N-acetylglucosamine (UDP-GlcNAc),
which serves as a substrate for O-linked-GlcNAc modification (O-GlcNAcylation). O-GlcNAc transferase (OGT)
catalyzes the transfer of a single moiety of GlcNAc from
UDP-GlcNAc to the hydroxyl side chains of serine andthreonine residues on target proteins [18], whereas
O-GlcNAcase (OGA) reverses this process by catalyzing
the removal of O-GlcNAc from proteins [18]. The activity of OGT is responsive to the levels of its substrate,
UDP-GlcNAc, which in turn are influenced by changes inintracellular glucose metabolism. O-GlcNAcylation has

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recently drawn the attention of researchers in the neuroscience community, reflecting the fact that multiple
neuronal proteins undergo O-GlcNAcylation at various
sites—a process that has the potential to contribute tovarious neurological disorders [19]. Notably, the presenceof O-GlcNAc modifications on Tau and amyloid precursor protein (APP), both of which play crucial roles in thedevelopment of AD, has sparked interest in exploring the
involvement of O-GlcNAc in the pathogenesis of AD.
Although researchers are only beginning to investigate
the role and mechanisms of HBP/O-GlcNAc cycling insleep-related brain dysfunction and AD, emerging evidence suggests that dysregulation of O-GlcNAc cycling
is critically involved in the development and progressionof various neurodegenerative brain disorders induced bysleep disturbance.
In this study, we established a chronic murine SDmodel in which mice were subjected to REMSD followed
by a recovery period under normal light-dark conditions.
This cycle of SD and recovery was repeated over a periodof 28 days. CSD model mice exhibited cognitive impairments together with pathological features associatedwith AD. Using this model, we analyzed the reversible
and cyclical alterations in OGT, OGA, and O-GlcNAcylation in the hippocampus in response to SD cycles.
Our results provide compelling evidence that O-GlcNAc
cycling plays a pivotal role in the development of cognitive impairment, neurodegeneration, neuroinflammation, and AD pathogenesis. These novel findings offer
strong evidence supporting the previously hypothesized
relationships between sleep, cognitive impairment, andO-GlcNAc cycling.

Materials and methods
Cell culture

Neuro2a mouse neuroblastoma cell line (Korean Cell
Bank, Seoul, Korea), obtained from the Korean Cell Bankin Seoul, Korea, was used at passage numbers 2 to 6. Neuro2a cells were maintained at 37℃ at 5% CO2 in DMEM
supplemented with 10% fetal bovine serum (Hyclone,
UT, USA), 1% (v/v) streptomycin, and penicillin.
Chronic REM sleep deprivation (CSD) mouse model

Male C57BL/6 N mice, aged 7 to 8 weeks and weighing
between 24 and 26 g, were obtained from SAMTACO(Osan, South Korea). The mice were housed individually in cages (15 cm x 30 cm), and they were maintained
under a 12-hrs light-dark cycle at a temperature of
22 ± 2℃. All animal experiments conducted in this study
were approved by the Institutional Animal Care and Use
Committee of Inha University in Incheon, Korea (INHA
190920-665).
REMSD was created using a modified multiple platform
method, as described in previous studies [17]. Chronic

Kim et al. Journal of Neuroinflammation(2024) 21:180

REMSD (CSD) was induced by transferring mice to a
separate holding area filled to a height of 1 cm with water(maintained at a temperature of 23 ± 2 °C). The watertank used in the study had dimensions of 15 × 30 cm andwas equipped with 12 cylindrical platforms, each with adiameter of 3.5 cm and a height of 3 cm, on which mice
were placed. In this setup, mice were motivated to avoid
the water, leading to disrupted sleep and an inability tomaintain a stable resting position. Before the experiments, mice were positioned on the platforms andlearned to stay on the platform for 30–60 min. Mice inthe sleep-deprived group were placed on the platforms
for three consecutive days, followed by a 4-day period
in their normal cages during which sleep was restored.
This cycle of SD and restoration was repeated a total of
4 times over a period of 28 days. GlcN (200 mg/kg) andminocycline (10 mg/kg) were administered intraperitoneally three times a week during sleep deprivation cycle.
One day after the final sleep-deprivation cycle, passive
avoidance tests and novel object recognition tests (NOR)
were administered. All mice were provided ad libitum
access to water and food throughout the study.
Passive avoidance test

The passive avoidance or step-through test was conducted using a system (Jeungdo Bio&Plant Co, Ltd.,
Seoul, Korea) consisting of light and dark compartments
separated by a door [17]. During the habituation stage,
the mice were placed in a box and allowed to explore thetwo compartments for a period of 10 min. For the test
phase, each mouse was initially placed in the light chamber of the system, and then the door separating the lightand dark chambers was opened, allowing the mouse step
through to the dark chamber. After the mice entered
the dark chamber, the door was promptly closed, anda foot shock of 0.7 mA for 1 s was administered to themice. Learning sessions consisted of 3 trials, with eachtrial being separated by 1-hr intervals. A memory retention test was conducted on the day following the second,
third, and fourth weeks using the same parameters, but
without administering a foot shock. Memory retention
was assessed by measuring the step-through latency, witha maximum measurement time of 300 s.
Novel object recognition test (NOR)

The novel object recognition (NOR) test was performed
following the previously described protocol [20]. NOR
test involved a familiarization session and a test session
in an open-field arena made of acrylic with dimensions of
30 × 30 × 30 cm, containing 2 objects. The mice were given
10 min to freely explore two identical objects, after which
they were returned to their home cages. After 24 h, theanimals were reintroduced to the apparatus, where theyencountered one of the objects from the familiarization

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session and a novel object. The difference in the amountof time spent exploring the new object compared to thefamiliar object indicates the animal’s memory of the previous experience and their learning abilities. The discrimination index (DI), which represents the preference forthe new object over the familiar object, can be calculatedusing the formula: DI = (time of novel one - time of familiar one) / (time of novel one + time of familiar one).
RNA extraction and real-time polymerase chain reaction(RT-PCR)

Total RNA was extracted from cells using TRIzol™ (Invitrogen, CA, USA) following the manufacturer’s protocol.
Complementary DNA (cDNA) was synthesized from 1 µg
of RNA using GoScript Reverse Transcriptase (Promega,
WI, USA) according to the manufacturer’s instructions.
PCR was performed using mouse-specific primers foreach target (GRP78, F: ​G​G​T​G​C​A​G​C​A​G​G​A​C​A​T​C​A​A​G​
T​T, R: ​C​C​C​A​C​C​T​C​C​A​A​T​A​T​C​A​A​C​T​T​G​A; GRP94, F: ​A​
A​T​A​G​A​A​A​G​A​A​T​G​C​T​T​C​G​C​C, R: ​T​C​T​T​C​A​G​G​C​T​C​T​T​
C​T​T​C​T​G​G; CHOP, F: ​C​T​G​C​C​T​T​T​C​A​C​C​T​T​G​G​A​G​A​C,
R: ​C​G​T​T​T​C​C​T​G​G​G​G​A​T​G​A​G​A​T​A , GAPDH, F: ​T​C​A​T​T​
G​A​C​C​T​C​A​A​C​T​A​C​A​G​G​T, R: ​C​T​A​A​G​C​A​G​T​T​G​G​T​G​G​
T​G​C​A​G). The cDNAs were amplified using SYBR Green
Real-time PCR Master Mix (TOYOBO, Osaka, Japan).
The expression levels were normalized to the expressionof GAPDH.
Mouse brain tissue preparation and immunofluorescence

The isolated brains were processed for immunohistochemistry following previously described methods [17].
In brief, the brains were fixed in 4% paraformaldehyde
in 0.1 M PBS (pH 7.4) for 20 min. Subsequently, thebrains were transferred to a fresh solution of 4% paraformaldehyde and kept in this solution for 7 days. The
brains were then embedded in paraffin and sectioned
using a microtome. Serial coronal sections of the mousebrain were made at -1.06, -1.76, and − 2.06 mm relative
to bregma using the microtome (HISTOCUT 820, Leica,
Wetzlar, Germany) [21]. For immunofluorescent staining, paraffin sections of the brain were deparaffinized.
The sections were then washed with 0.1% PBS-T (2.7 mM
KCl, 10 mM Na2HPO4, 137 mM NaCl, 1.8 mM KH2PO4
and 0.1% Tween 20; pH 7.6) and blocked in 10% normal goat serum (Jackson ImmunoResearch, PA, USA)in 0.1% PBS-T. Subsequently, the sections were incubated in 1% PBS-T containing the following antibodies: anti-RL-2 (MA1-072, 1:100, Invitrogen), anti-OGT(1:100, sc-74,546, Santa Cruz Biotechnology, CA, USA),anti-MAP-2 (sc-74,421, 1:00, Santa Cruz Biotechnology,
CA, USA), anti-OGA (14711-1-AP, 1:100, Proteintech,
IL, USA), anti-p-Tau (S231, ab194815, 1:100, Abcam,
Cambridge, UK), anti-PSD-95 (ab18258, 1:100, Abcam,
Cambridge, UK), anti-GlcNAc-Tau (AS55945, 1:100,

Kim et al. Journal of Neuroinflammation(2024) 21:180

Anaspec, TX, USA), anti-Amyloid β (1:100, NBP2-13075,
Novos Biologicals), anti-IBA-1 (ab178846, 1:100, Abcam,
Cambridge, UK), and GFAP (G3893, 1:100, Sigma), andincubated overnight at 4 °C. After washing, the slides
were incubated with a mixture of secondary antibodies, including goat anti-mouse or rabbit IgG conjugatedto Alexa 488 or Alexa 564 (Invitrogen, CA, USA). Cellcounterstaining was performed using DAPI (Sigma) tovisualize the nuclei. The slides were then examined undera confocal LSM 510 META microscope (Carl Zeiss,
Jena, Germany) and analyzed using ZEN Light Edition
software.
Golgi-cox staining

Golgi-Cox staining was performed following the previously described method [17]. Mice were euthanized, andisolated brains were immersed in Golgi-Cox staining
solution (100 ml D.W, 5% potassium dichromate, 50 ml
5% mercuric chloride, and 40 ml 5% potassium chromate) for 8 days at RT in the dark. After washing with
D.W., samples were transferred to tissue protectant buffer and maintained at 4 ℃ in the dark for 8 days. Coronal Sect. (100 μm) were then obtained using a vibratome(VT1000S, Leica, Germany) to generate slides. The slides
were subjected to a series of developing steps involving
gradient ethanol dehydration with 5% sodium thiosulfate.
Subsequently, images were captured using an Olympus
IX 83 light microscope (Olympus, Tokyo, Japan) and analyzed using Cellsens Dimension software (Olympus) andImageJ software (NIH) with a 60x objective oil immersion lens. Neurons were randomly selected for examination of dendrite length and spine density. The lengthof each dendrite and the spine density were measured
using Fiji ImageJ software, following previously describedmethods [22].
Western blotting

The whole hippocampus was homogenized in RIPA buffer (1% Na-deoxycholate, 1% NP-40, 150 mM NaCl, 1
mM EDTA, 10 mM Tris pH 7.4) supplemented with protease and phosphatase inhibitors, 20 mM streptozotocin,
and incubated for 2 h on ice. After incubation, the samples were centrifuged for 15 min at 13,200 rpm. Protein
samples (20 µg) were separated by SDS-PAGE and transferred onto Amersham Biosciences™ Protan™ nitrocellulose membranes (GE Healthcare Life Sciences, Germany).
The membranes were incubated with 5% BSA or 5% skim
milk in TBS-T for 1 h at RT, followed by incubation withprimary antibodies against RL-2 (MA1-072, 1:100, Invitrogen), anti-OGT (1:1000, sc-74546, Santa Cruz Biotechnology, CA, USA), anti-GRP78 (1:1000, sc-13968, Santa
Cruz Biotechnology, CA, USA), anti-GSK3β (1:1000,
sc-377213, Santa Cruz Biotechnology, CA, USA), antip-tau (T231, 1:1000, ab194815), anti-Amyloid β (1:1000,

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NBP2-13075, Novos Biologicals), anti-CHOP (1:1000,
sc-7351, Santa Cruz Biotechnology, CA, USA), antiGRP94 (1:1000, sc-32249, Santa Cruz Biotechnology, CA,USA), anti-ATF-4 (1:1000, Cell Signaling Technology),
anti-p-GSK3β (1:1000, 9336S, Cell Signaling Technology), anti-IBA-1 (1:1000, ab178846, Abcam, Cambridge,UK), GFAP (Sigma), anti-CD68 (1:1000, ab125212,
Abcam, Cambridge, UK), IL-1β (1:1000, 12242 S, Cell
Signaling Technology), TNF-a (1:1000, sc-52746, Santa
Cruz Biotechnology), iNOS (1:1000, 610431, BD bioscience) and anti-β-actin (1:1000, sc-47778, Santa Cruz Biotechnology) overnight at 4 °C. The membranes were thenincubated with horseradish peroxidase (HRP)-conjugatedsecondary antibodies and detected using an Enhanced
Chemiluminescence (ECL) detection system (Promega).
Densitometric quantification of protein bands was performed using the Bio-Rad Image Lab program (Bio-Rad).
Thioflavin S staining

For thioflavin S staining, deparaffinized paraffin sections
were incubated in a filtered 1% aqueous thioflavin S solution for 8 min at room temperature. Subsequently, theslides were washed in a series of ethanol (Duksan, Ansan,
Korea) solutions in the order of 80%, 95%, and distilledwater, with each wash step lasting 3 min. The slides werethen examined under a confocal LSM 510 META microscope (Carl Zeiss) and analyzed using ImageJ software.
Sholl analysis of microglia

Sholl analysis was performed to investigates the branching patterns of microglial processes, according to thepreviously described method [23]. In brief, the sectionsof the hippocampus were stained for IBA-1 and DAPI
and captured as 63X z-stack images at 1 μm intervals
using confocal LSM 510 META microscopy. A maximum
intensity projection image of IBA-1 was created using
ZEN Light software. To analyze intersections at eachradius, the Sholl analysis plugin was employed, startingfrom the first shell at 50 μm and subsequent shells set at
2 μm step sizes. For quantification, intersections were
evaluated to measure microglial ramification from thecenter of the cell soma to the furthest point. The “Analyze
Skeletonize” plugin in ImageJ software was utilized tocount the number of branches and branch lengths fromeach cell.
Stereotaxic intracranial injection of AAV-OGT

To achieve the overexpression of mouse OGT, we utilized the pAAV-CMV-Myc-Flag vector for cloning. The
viral vectors were pseudotyped, with the transgene ofinterest flanked by inverted terminal repeats of AAV8
and packaged in an AAV8 capsid. The mouse OGT vectors were purified, and virus production was carried out
at the Korea Institute of Science and Technology (KIST)

Kim et al. Journal of Neuroinflammation(2024) 21:180

Virus Facility (KIST, Seoul, Korea). The mice were randomly assigned to one of three groups: vector-only (control), vector-only with CSD, and AAV-OGT with CSD.
For the injection, mice were administered 4 µl of either
the pAAV-CMV-Myc-Flag control virus or AAV8-OGTMyc-Flag virus. Prior to the injection, the mice wereanesthetized with isoflurane (Baxter, IL, USA). The
viruses were then injected into the dentate gyrus (DG) of
the hippocampus at specific coordinates (–2.1 mm posterior to bregma, ± 1.2 mm lateral to midline, and − 2.0 mm
ventral to the brain surface) using a 26-gauge stainlesssteel injection needle connected to a 10 µl microsyringe(Hamilton) at a flow rate of 0.3 µl/min. After injection,
the needle was kept in place for 10 min before being
slowly withdrawn.
Serum cortisol and corticosterone levels

Fresh blood samples were obtained from heart and thencentrifuged at 2000 g at 4 °C for 20 min. The serum ofcortisol (Mybiosource) and corticosterone (Abcam)
were determined using enzyme linked immunosorbent assay (ELISA) kits, according to the manufacturer’s
instructions.
Statistical analysis

The data are presented as means ± SEM (standard errorof the mean). Statistical analysis was conducted using
GraphPad Prism 8 software. Data comparisons were performed using one-way analysis of variance (ANOVA),
two-way ANOVA, or unpaired Student’s t-test, as appropriate. Differences were considered statistically significant at p < 0.05. Graphs were generated using GraphPad
Prism 8.

Results
Cognitive dysfunction induced by CSD is improved by GlcNIn this study, we sought to examine the impact of
repeated and sustained chronic SD on the developmentof AD and explore the underlying mechanisms involved.
We validated the SD model by confirming the increasein cortisol and corticosterone levels due to SD in themulti-platform SD model (supplementary Fig. 1). To
this end, we generated a mouse model of chronic REM
sleep deprivation (CSD) in which mice were subjected to
SD for three consecutive days, followed by a 4-day rest
period, a cycle that was repeated for a total of 4 weeks(28 days) (Fig. 1A). We investigated the alterations inbrain O-GlcNAc cycling in response to CSD, and foundthat O-GlcNAcylation level was significantly reduced in
the dentate gyrus (DG) and CA1 of hippocampus andcortex regions of the brains of CSD mice compared with
control group mice (Supplementary Fig. 2A). Interestingly, the levels of OGA and OGT—enzymes involvedin O-GlcNAcylation regulation—remained unchanged

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in the brains of CSD mice compared with mice in control groups (Supplementary Fig. 2B, 2C, 2D and 2E). To
investigate the role of decreased O-GlcNAcylation in
the development of CSD-induced brain dysfunction,
we examined the effects of increasing O-GlcNAcylation
through glucosamine (GlcN) supplementation. Specifically, we investigated the effects of daily GlcN administered throughout the duration of CSD on cognitive
dysfunction, with a particular focus on L/M impairment
induced by CSD. We performed two types of L/M tests.
In the passive avoidance test, which measures fear memory, mice in the control group as well as GlcN-treatedmice successfully learned to associate an electric shockwith the dark room environment and retained this fear
memory after 2, 3, and 4 weeks. However, CSD mice
exhibited reduced learning ability and a lack of memoryin this test (Supplementary Fig. 3, Fig. 1B and C). Importantly, intraperitoneal administration of GlcN to mice in
the CSD group resulted in a notable restoration of L/M
capability. In the NOR test, CSD mice exhibited a decline
in their ability to recognize novel objects compared with
control and GlcN-treated mice. However, mice in the
GlcN-treated CSD group showed improvement in theirability to remember and recognize novel objects (Fig. 1D
and E). Collectively, these findings indicate that GlcN
effectively mitigates the long-term memory and fear
memory dysfunction induced by CSD.
Administration of GlcN rescues CSD-induced decreases in
O-GlcNAcylation levels and dendritic spine density

A subsequent investigation of the effects of GlcN on
CSD-induced O-GlcNAcylation levels showed thatchronic GlcN treatment during CSD effectively restored
O-GlcNAcylation to a level comparable to that in thecontrol group in DG and CA1 regions of the mouse brain(Fig. 2A). We next assessed dendrite morphology anddendritic spine density, observing a reduction in dendrite length and dendritic spine density in the brains of
CSD mice compared with control mice. Injection of GlcN
reversed these reductions, indicating a beneficial effectof GlcN on dendrite morphology (Fig. 2B). In contrast,
no changes in the number of dendrites were observed.
(Fig. 2B). We further found that the levels of postsynaptic
density protein-95 (PSD-95), as well as the ratio of PSD95 to MAP-2 (microtubule-associated protein 2), wereincreased in the brains of CSD mice compared with thoseof control mice (Fig. 2C), suggesting that CSD alters synaptic organization. Interestingly, GlcN treatment effectively attenuated the CSD-induced increase in PSD-95
levels, suggesting its potential to mitigate the synaptic
changes associated with CSD. However, the effect of
GlcN on the PSD-95/MAP2 ratio was not as significant.

Kim et al. Journal of Neuroinflammation(2024) 21:180

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Fig. 1 The effect of GlcN on L/M dysfunction in CSD mice. (A) Schematic experimental procedure of chronic REM sleep deprivation (CSD) timeline diagram. Mice were subjected to REM sleep deprivation for 3 consecutive days (red) using the modified multiple platform method, followed by a normal
sleep-wake cycle for 4 days (blue). This pattern was repeated 4 times over a span of 28 days. (B and C) Mice were subjected to CSD, with or without GlcN.
GlcN (200 mg/kg) was administered intraperitoneally three times a week during sleep deprivation cycle. (B) The bar graph represents the escape latency
during learning trials of the first (T1), second (T2), and third (T3) passive avoidance tests at one-hour intervals following the first cycle of SD. (C) The bar
graph represents escape latency in memory test performed after the second (M1), third (M2), and fourth (M3) cycle during CSD (n = 12/group). (E and F)
The graphs represent the percentage of preference for novel and familiar objects in the Novel Object Recognition (NOR) test. In the familiar test session(D), mice were placed into a box containing two identical objects and allowed to explore the box for 10 min. The time spent exploring each object wasrecorded. In the novel test session (E), mice were placed into the box with one familiar object and one novel object, and the exploration time for eachobject was recorded during a 10-min period. The discrimination index (DI) was calculated using the formula DI = (exploration time of the novel object- exploration time of the familiar object) / (exploration time of the novel object + exploration time of the familiar object). The graph represents the DI
in the control, GlcN, CSD, and CSD + GlcN groups (n = 12/group). Values are presented as mean ± SEM; ***p < 0.001 vs. control group; #p < 0.05, ##p < 0.01,###
p < 0.001 vs. CSD group. Statistical analysis was performed using one-way ANOVA and two-way ANOVA with Tukey’s comparison; CSD, chronic sleep
deprivation; Con, control; GlcN, glucosamine; T, learning trial; M, memory test

GlcN attenuates the accumulation of Aβ and p-Tau inducedby CSD while mitigating the CSD-induced decrease in
O-GlcNAc-Tau levels

Assessments of AD pathology in the brains of mice
injected daily with GlcN during CSD revealed that GlcN
effectively decreased Aβ accumulation induced by CSD(Fig. 3A and D). Neurofilamentary tangles in AD also
contain abnormal p-Tau. In addition, Tau is subjected to
O-GlcNAcylation, which inversely modulates Tau phosphorylation [24]. Therefore, we compared the levels of
Tau phosphorylation and O-GlcNAcylation in response

to CSD, with or without GlcN treatment. This analysis showed that CSD significantly increased the level of
p-Tau in the brain, and that this increase was suppressed
by GlcN (Fig. 3B and D). Conversely, O-GlcNAcylatedTau (O-Tau) was decreased by CSD. Again, this effect was
reverse by treatment with GlcN, which restored O-Tau tolevels similar to those in the control group (Fig. 3C and
D). Thioflavin S staining further demonstrated that CSD
increased protein aggregates containing Aβ and showedthat this increase was inhibited by GlcN treatment(Fig. 3E).

Kim et al. Journal of Neuroinflammation(2024) 21:180

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Fig. 2 Effects of GlcN on O-GlcNAcylation and synaptic density in the brain of CSD Mice. (A) Representative confocal images (40X) of O-GlcNAc, DAPI,
and merged images of the brain from control mice, and those subjected to CSD with or without GlcN. DAPI staining was performed for nuclei visualization. The scale bar represents 50 μm. The accompanying graph represents the quantification of O-GlcNAc levels in DA, CA1 and cortex of the brain (n = 6/
group). (B) Mouse brains were stained using the Golgi-cox method. Slides were prepared from coronal sections with a thickness of 100 μm. Golgi-cox
staining images of dendritic spines in the hippocampal CA1 region were captured at 40X, 63X, and 120X magnifications from control and CSD mice withor without GlcN. The length of each dendrite and the spine density were measured using Fiji Image J software. The scale bar represents 50 μm, and thescale bar for the enlarged images is 5 μm. The graphs present the quantification of dendrite length, number of dendrites, and spine density per µm (n = 6).
(C) Staining images of PSD-95 (green), MAP-2 (red) and DAPI (blue) co-staining of DG, CA1 and cortex brain of control and CSD with or without GlcN mice(n = 6/group). The graphs represent the quantification of PSD-95, MAP2, and PSD-95/MAP2 ratio. The scale bar represents 5 μm. Values are presented as
the mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, vs. control group; #p < 0.05, ###p < 0.001 vs. CSD group. Statistical analysis was performed using one-way
ANOVA with Tukey’s comparison. CSD, chronic sleep deprivation; Con, control; O-Glc, O-GlcNAc; DG, dentate gyrus; PSD-95, postsynaptic density-95;
MAP2, microtubule-associated protein 2

GlcN treatment effectively inhibits CSD-induced glial
activation

We next investigated the effects of CSD on glial activation in the brain and further compared the effects of
GlcN in modulating these responses. We found that
CSD led to an increase in astrocyte activation, as indicated by elevated levels of glial fibrillary acidic protein(GFAP) (Fig. 4A). Additionally, the number of IBA1-positive (activated) microglia in the brains of CSD mice wasincreased (Fig. 4B). Both the increase in GFAP and thepresence of IBA1-positive cells were significantly reduced

by GlcN treatment. Furthermore, a Sholl analysis of
IBA1-positive cells revealed a decrease in the number of
Sholl intersections in microglia from CSD mouse brains
compared with microglia from control mouse brains.
CSD mice treated with GlcN exhibited an increase in thenumber of Sholl intersections compared with mice in
the CSD group (Fig. 4C). A subsequent examination ofchanges in O-GlcNAcylation in microglia in response to
CSD showed that CSD significantly reduced O-GlcNAc
levels in activated microglia (Fig. 4D), an effect thatwas reversed by administration of GlcN (Fig. 4D).

Kim et al. Journal of Neuroinflammation(2024) 21:180

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Fig. 3 The effect of GlcN on CSD-induced Aβ, p-Tau andO-Tau accumulation in the brain of mice. (A-C) Representative confocal images of Aβ (20X) (A),p-Tau (20X) (B), or O-Tau (20X) (C), as well as DAPI and merged images of the brain from control mice and mice subjected to CSD with or without GlcN. The
graphs represent the quantification of Aβ (n = 6/group), p-Tau and O-Tau in the mouse brain (n = 6/group). Values are presented as the mean ± SEM. (D)Western blot analysis using antibodies specific for O-tau, p-tau, Aβ and β-actin was performed on the hippocampus of control and CSD mice with or without GlcN. The graphs represent the densitometric quantification of O-tau, p-tau, and Aβ normalized by β-actin (n = 6/group). (E) Representative imagesof thioflavin S staining are shown, and the graph presents the quantification of thioflavin S staining (n = 6/group). The scale bar represents 50 μm. Values
are measured as the mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc comparison; **p < 0.01, ***p < 0.001, vs.
control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. CSD group; CSD, chronic sleep deprivation; Con, control; DG, dentate gyrus; O-Tau, GlcNAc-Tau; Thio,
thioflavin S; Aβ, amyloid beta

Immunoblot analyses, performed to further investigate
glial activation and inflammatory cytokines, revealed anincrease in GFAP, IBA-1, CD68, iNOS, TNF-α, and IL-1β
levels in the CSD group, and further showed that treatment with GlcN significantly diminished the expressionof these proteins (Fig. 4E and F).
GlcN inhibits CSD-induced stimulation of ER stress andactivation of GSK3β in the brain

Several previous studies have demonstrated an association between ER stress and neurodegenerative diseases,
including AD. To investigate whether CSD induces
ER stress, we examined the expression levels of ER
stress-related proteins in the hippocampus. This analysis revealed a significant increase in the expression of
GRP78, GRP94, CHOP, and ATF-4 in the brains of CSD
mice; it also showed that this increase was effectively
suppressed by GlcN treatment (Fig. 5A). CSD was further
found to activate eukaryotic translation initiation factor 2 (eIF2α), an effect that was also inhibited by GlcN
treatment (Fig. 5B). Next, we investigated CSD-inducedchanges in GSK3β signaling. This pathway was of interest

because it has been proposed that GSK3β is not only
a downstream target of ER stress, but also a kinase
involved in Tau phosphorylation and Aβ processing [25,26]. The brains of mice from the CSD group exhibited a
reduction in GSK3β phosphorylation at serine 9 indicative of activation (Fig. 5C). Moreover, GlcN treatment
effectively suppressed this CSD-induced effect on GSK3β
activation. To investigate the potential involvement of
O-GlcNAc cycling in ER stress and GSK3β activation,
we examined the effect of the OGT inhibitor, OSMI-1, in
N2a cells. These experiments demonstrated that OSMI-1
treatment led to a decrease in O-GlcNAcylation, indicating inhibition of OGT activity. OSMI-1 treatment
resulted in increased expression of the ER stress markers,
CHOP, GRP78, and ATF-4; it also increased phosphorylation of GSK3β (Fig. 5D). Consistent with this, mRNAlevels of CHOP, GRP78, and ATF-4 were increased in adose-dependent manner by OSMI-1, providing further
evidence that OSMI-1 induces ER stress (Fig. 5E).

Kim et al. Journal of Neuroinflammation(2024) 21:180

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Fig. 4 Neuroinflammation induced by CSD with or without GlcN in the mouse brain. (A) Representative immunofluorescent staining of GFAP (an activated astrocyte marker), DAPI, and merged images of the brain from control mice, and mice subjected to CSD with or without GlcN, are shown. The
scale bar represents 100 μm. The graph represents the densitometric quantification of GFAP (n = 6 per group). (B) Representative confocal images (40X
and 240X) of IBA-1, DAPI, and merged images, as well as binary images for Sholl analysis, from the mice brain samples of control and CSD groups, with orwithout GlcN (n = 6/group). Enlarged images are presented in the white boxes. The scale bar is set at 50 μm for the main images and 5 μm for the enlarged
images. (C) The graph represents the intersections from the center of the cell soma to the furthest point of the microglial Sholl image in the CA1 regionof the mice brain, using ZEN Light software (n = 9 ~ 20 cells/group). The Sholl analysis plugin was utilized, with the first shell set at 50 μm and subsequentshells set at 2 μm step sizes. (D) Z-stack staining images of IBA-1 (red), DAPI (blue), and O-GlcNAc (green), along with merged images from the brains ofcontrol and CSD mice with or without GlcN. The scale bar is set at 5 μm. The graph represents the quantification of O-GlcNAc levels within IBA-1 positive
cells (n = 6/group). (E) Western blot analysis using antibodies specific for GFAP, IBA-1, CD68, and β-actin was performed on the hippocampus of control
and CSD mice with or without GlcN. The graphs represent the densitometric quantification of GFAP, IBA-1, and CD68 normalized by β-actin (n = 6/group).
(F) Western blot analysis using antibodies specific for iNOS, TNF-α, IL-1β, and β-actin was performed on the hippocampus of control and CSD mice with orwithout GlcN. The graphs represent the densitometric quantification of iNOS, TNF-α, and IL-1β normalized by β-actin (n = 6/group). Values are presented
as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. CSD group; (A), (B), (D), (E), and (F) were analyzed
using one-way ANOVA with Tukey’s post-hoc comparison. (C) was analyzed using two-way ANOVA with Tukey’s post-hoc comparison. CSD, chronic sleep
deprivation; Con, control; DG, dentate gyrus; GFAP, glial fibrillary acidic protein; IBA-1, ionized calcium-binding adapter molecule 1; TNF-α, tumor necrosis
factor-α; IL-1β, interleukin-1β; iNOS, inducible nitric oxide synthase

Minocycline suppresses CSD-induced glial activation and
AD pathogenesis and rescues the CSD-induced decrease in
O-GlcNAcylation in the brain

We further investigated the impact of minocycline, awell-known anti-inflammatory drug, on CSD-induced
brain pathogenesis and O-GlcNAc cycling. We observedthat daily administration of minocycline during CSD
effectively suppressed CSD-induced activation of astrocytes and microglia, as evidenced by decreased levels of
GFAP and IBA-1, respectively (Fig. 6A and B). A subsequent examination of the effects of minocycline on CSDinduced O-GlcNAc levels and the expression of OGT and
OGA revealed a significant reversal of the CSD-induced
O-GlcNAcylation reduction in the mouse brain (Fig. 6C).

The expression of OGT and OGA did not change significantly following CSD induction and was unaffected byminocycline (Fig. 6C). We further observed that minocycline mitigated the CSD-induced increase in p-Tau
levels and reversed the CSD-induced decrease in O-Tau
levels (Fig. 6D and E). Moreover, minocycline effectively
reduced the CSD-induced accumulation of Aβ (Fig. 6F).
Experiments testing the influence of minocycline on
CSD-induced ER stress and GSK3β activation showed nosignificant effects (supplementary Fig. 4).

Kim et al. Journal of Neuroinflammation(2024) 21:180

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Fig. 5 The effect of CSD or OSMI-1 on ER Stress and GSK3β activation in the mouse brain and N2a cells. (A) Representative Western blot images of GRP78,
GRP94, CHOP, ATF-4, and β-actin from the hippocampus of mice brains. The graphs represent the densitometric quantification normalized by β-actin asa control (n = 6/group). (B) Representative Western blot images of p-eIF2α, eIF2α, and β-actin from the hippocampus of mice brains. The graph illustrates
the densitometric quantification of p-eIF2α normalized to eIF2α as a control (n = 6/group). (C) Representative Western blot images of O-GlcNAc (RL-2),
p-GSK3β, GSK3β, and β-actin from the hippocampus of mice brains. The graphs represent the densitometric quantification of O-GlcNAc normalized toβ-actin and p-GSK3β normalized to GSK3β (n = 6/group). (D) Neuro2a cells were treated with 30 µM OSMI-1 for 24 h. Whole-cell lysates were subjected
to immunoblotting with antibodies against RL-2 (O-GlcNAc), CHOP, GRP78, ATF-4, p-GSK3β, GSK3β, and β-actin. The relative densitometric intensitiesof O-GlcNAc, CHOP, GRP78, and ATF-4 were quantified by normalizing to β-actin. The intensity of p-GSK3β was measured by normalizing to GSK3β. (E)Neuro2a cells were treated with either 15 µM or 30 µM OSMI-1 for 24 h. Quantification of GRP78, GRP94, and CHOP mRNA levels was performed using
quantitative real-time PCR. The values are presented as mean ± SEM.; *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group; ##p < 0.01, ###p < 0.001 vs. CSD
group. Statistical analysis for (A), (B), and (C) was performed using One-way ANOVA with Tukey’s post-hoc comparison. Statistical analysis for (D) and (E)was conducted using Student’s t-test. CSD, chronic sleep deprivation; Con, control; GSK3 β, glycogen synthase kinase 3 beta; Akt, protein kinase B; GRP,
glucose-regulated protein; CHOP, C/EBP homologous protein; eIF2α, eukaryotic translation initiation factor 2 A; ATF-4, activating transcription factor 4;
GRP94, glucose-regulated protein 94

Hippocampal overexpression of OGT restores CSD-induced
L/M dysfunction

To further elucidate the role of alterations in O-GlcNAc
cycling in CSD-induced brain pathogenesis, we utilized aviral delivery system, employing an AAV vector to overexpress OGT in the hippocampus. Injection of OGT
viruses into the hippocampal DG of CSD mice induced
a significant increase in OGT expression (Fig. 7A).
Consistent with this, O-GlcNAc levels were increased
specifically within the DG of CSD mice administered
OGT viruses compared with uninjected CSD mice. No

significant differences in O-GlcNAc levels were observein the CA1 region or cortex, confirming the selective targeting of the DG region (Fig. 7B). We then assessed theimpact of OGT overexpression on L/M functions. In thepassive avoidance test, which measures fear memory,
OGT overexpression in CSD mice significant restored
L/M functions compared with CSD mice that did notreceive viral injections (Fig. 7C and D). In the NOR test,
mice with DG-specific OGT overexpression exhibited anenhanced ability to remember and willingness to explore
novel objects (Fig. 7E).

Kim et al. Journal of Neuroinflammation

Fig. 6 (See legend on next page.)(2024) 21:180

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Kim et al. Journal of Neuroinflammation(2024) 21:180

Page 12 of 17(See figure on previous page.)
Fig. 6 Effects of minocycline on neuroinflammation, O-GlcNAcylation, and degenerative changes in the brain induced by CSD. During CSD, the mice
were administered minocycline (10 mg/kg) three times per week. (A) Representative immunofluorescent staining images (20X) of GFAP, DAPI, andmerged images of the brain from the control, CSD, and CSD plus minocycline groups. The scale bar represents 100 μm. The graph represents the densitometric quantification of GFAP levels (n = 6/groups). (B) Representative immunofluorescent staining images (40X) of IBA-1, DAPI, and merged images forthe control, CSD, and CSD plus minocycline groups (n = 6 per group). The graph illustrates the densitometric quantification of IBA-1 levels. A scale bar of
50 μm is included in the images. (C) Representative confocal staining images (40X) of O-GlcNAc, OGA, OGT, DAPI, and merged images of the mouse brain
from the control, CSD, and CSD plus minocycline group. The scale bar represents 50 μm. Quantification of O-GlcNAc, OGA, and OGT levels in the brain was
conducted (n = 6/group). (D ~ F) Representative confocal images (40X) of O-Tau (D), p-Tau (E), Aβ (F), along with DAPI and merged images of mouse brainsfrom the control, CSD, and CSD plus minocycline groups. A scale bar of 50 μm is provided. The graphs depict the quantification of O-Tau (D), p-Tau (E), andAβ (F) levels (n = 6/group). The values are expressed as the mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group; #p < 0.05, ##p < 0.01, ###p < 0.001
vs. CSD group. Statistical analysis was performed using One-way ANOVA with Tukey’s comparison. CSD, chronic sleep deprivation; Con, control; mino,
minocycline; O-Glc, O-GlcNAc; OGA, O-GlcNAcase; OGT, O-GlcNAc transferase; GFAP, glial fibrillary acidic protein; IBA-1, ionized calcium-binding adapter
molecule 1; O-Tau, O-GlcNAc-Tau; p-Tau, phosphorylated-Tau; Aβ, amyloid beta

OGT overexpression alleviates CSD-induced ER stress andAD pathology

CSD mice that received OGT virus injections exhibited increased levels of O-GlcNAc and p-GSK3β compared with uninjected CSD mice (Fig. 8A). They also
showed significant elevations in protein levels of the ER
stress markers, GRP94, ATF4, and p-eIF2α. Notably, thisincrease was reversed by OGT overexpression in theDG, which restored these proteins to levels comparable
to those in normal controls (Fig. 8B). We further foundthat the decrease in O-Tau levels observed in CSD mice
was specifically reversed by overexpression of OGT in
the DG region of the brain (Fig. 8C). Conversely, p-Tau
levels, which were increased in response to CSD, werereduced by OGT overexpression in the DG region of the
CSD mouse brain (Fig. 8D). OGT overexpression also
suppressed the CSD-induced accumulation of Aβ in the
DG region. Importantly, the CSD-induced changes in
O-Tau, p-Tau, and Aβ levels were not affected by OGT
overexpression in the CA1 region or cortex of CSD mice
(Fig. 8D and E).

Discussion
Growing evidence has demonstrated that CSD inducescognitive impairment, which is also a clinical characteristic of AD, and worsens the pathological changes
associated with AD [27, 28]. The impact of SD on neurocognitive functions may be attributable, at least in part,to disruptions in cerebral glucose metabolism. Deep
sleep is characterized by minimal glucose demand in thebrain, whereas during REM sleep, the level of metabolism is comparable to that in wakefulness [29]. However,
the importance of cerebral glucose metabolism pathways during REMSD and the mechanisms that regulatethem have historically been relatively understudied. In
our previous research, we reported compelling evidence
suggesting that dynamic changes in the cerebral glucose
metabolism of the HBP in the brain may play a criticalrole in the cognitive impairment associated with SD [16,17]. Building upon previous research, the present study
aimed to investigate the hypothesis that repetitive andprolonged REMSD would lead to recurrent disturbancesin cerebral HBP flux and subsequently affect O-GlcNAc
cycling, thereby contributing to the pathogenesis of AD.
Our study revealed a significant decrease in O-GlcNAcylation in the brains of CSD mice compared with normal
control mice. Interestingly, however, unlike the case ofa single episode of SD, where OGT decreases and OGA
increases [17], CSD had no effect on OGA or OGT levels.
This indicates that repetitive SD and subsequent recovery
may contribute to dynamic reciprocal regulation of OGT
and OGA expression, potentially leading to compensatory mechanisms between them. As a result, although theoverall levels of O-GlcNAc may decrease, the intricate
balance between OGT and OGA expression is preserved.
Similarly, a previous report demonstrated that OGA and
OGT levels remain unchanged while O-GlcNAcylationlevels are decreased in the brains of 5xFAD and 3xTg AD
model mice [30].
Sleep plays a role in dendritic spine formation andelimination [31, 32]. Specially, REM sleep is required forstrengthening the new spine for long-term memory formation [33]. In our previous work, we found that a single
episode of REMSD induces a decrease in dendrite lengthand number and reduces spine density [17]. Similarly,
we found that CSD reduces dendrite length and spine
density, both of which are reversed by GlcN treatment.
In contrast, there was no change in the number of dendrites. Typically, SD has been reported to decrease boththe number and density of dendritic spines, but it also
has reported that, during REM sleep, the eliminationof dendritic spines to strengthen new spines facilitates
long-term potentiation [34]. Therefore, it is likely that theextent and type of SD can have diverse effects on dendritic spine number. Nonetheless, it is evident that thedecrease in dendritic spine density in CSD mice points
to diminished synaptic density, implying weakened neural connectivity and reduced information transmission,
with consequent impacts on the development of cognitive disabilities.
The accumulation of extracellular Aβ plaques andincreased intraneuronal Tau expression are significant
pathological events in AD [35, 36]. Studies in transgenic
mouse models of AD have also demonstrated an increase

Kim et al. Journal of Neuroinflammation(2024) 21:180

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Fig. 7 The Effect of OGT overexpression on CSD-induced L/M dysfunction. AAV-OGT or control viruses were injected into the DG of mice brains, andthen the mice were subjected to SD for four cycles to induce CSD. (A) Representative western blot analysis and quantification of OGT and OGA expression levels in the hippocampus from the control, CSD, and CSD plus OGT group. (B) Representative confocal image (20X) of O-GlcNAc, DAPI and merged
immunofluorescence staining in the DG, CA1, and cortex of the mouse brain. Graphs represent densitometric quantification of O-GlcNAc (n = 3/group).
The scale bar represents 100 μm. (C) The graph displays the escape latency during the learning trials of the first (T1), second (T2), and third (T3) passive
avoidance tests at one-hour intervals following the first cycle of SD (n = 6/group). (D) The graph represents the escape latency in the memory test conducted after the second (M1), third (M2), and fourth (M3) cycle during CSD (n = 6/group). (E) The NOR test was conducted to measure the percentage
of preference for familiar and novel objects. In the familiar test session (left), mice were placed into a box containing two identical objects and the timespent exploring each object was recorded. In the novel test session (right), mice were placed into the box with one familiar object and one novel object,
and the exploration time for each object was recorded. The discrimination index (DI) was calculated using the formula DI = (exploration time of the novel
object - exploration time of the familiar object) / (exploration time of the novel object + exploration time of the familiar object). The graph represents the
DI in the control, CSD, and CSD + OGT groups (n = 6/group). The values are expressed as the mean ± SEM; **p < 0.01, *p < 0.01, ***p < 0.001 vs. control group;#p < 0.05, ##p < 0.01, ###p < 0.001 vs. CSD group. Statistical analysis was performed using One-way ANOVA with Tukey’s comparison. CSD, chronic sleep
deprivation; O-Glc, O-GlcNAc; OGA, O-GlcNAcase; OGT, O-GlcNAc transferase; DG, dentate gyrus; T, learning trial; M, memory testin Aβ plaques in the hippocampus following CSD [37]. In
this study, we employed a 28-day repeated SD, demonstrating that this CSD paradigm led to increased deposition of Aβ and p-Tau, accompanied by reduced levels of
OGlcNAcylation in the hippocampus and cortex regionsof wild-type C57 BL/6 mice. To investigate the potential significance of decreased brain O-GlcNAc in CSDinduced AD pathology, we examined the effects of GlcN,
which can serve as a direct substrate of O-GlcNAc, on
these processes. Our findings strongly indicate that therestoration of O-GlcNAc by GlcN significantly contributes to the overall recovery from CSD-induced impairments in L/M, synaptic plasticity, and AD pathology. By
overexpressing OGT in the mouse brain using the AAV
system, we obtained additional strong confirmation that
increasing O-GlcNAc levels can restore various neuropathogenic processes impaired by CSD. These findings
confirm the crucial role of O-GlcNAc cycling in CSDinduced AD pathogenesis. Given that multiple lines ofevidence demonstrate that O-GlcNAcylation limits Tau
phosphorylation and aggregation [38–42], it is plausiblethat CSD might increase Tau phosphorylation throughdecreased O-GlcNAc cycling. Consistent with this, ourresults indicate that CSD leads to a decrease in brain
expression levels of OGlcNAcylated-Tau (O-Tau) but
conversely causes an increase in p-Tau levels. Moreover, treatment with GlcN reversed both of these effects,
reducing p-Tau levels while simultaneously promoting an
increase in O-Tau levels.
There is ongoing controversy regarding the role of
O-GlcNAc cycling in regulating cognitive function andthe development of AD. This controversy arises from
reports of both increased and decreased O-GlcNAc levels in the AD brain [40, 43–47]. The divergent roles of
O-GlcNAc in AD can be attributed to several factors,
including variations in animal models, types of stimuli,
and fluctuations in O-GlcNAc regulatory elements.
However, we propose that maintaining the dynamic
equilibrium of normal O-GlcNAc cycling in the brain
is paramount to ensuring optimal brain function. For
instance, we found that, whereas GlcN was capable ofreversing the AD pathogenesis induced by CSD, we have

Kim et al. Journal of Neuroinflammation(2024) 21:180

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Fig. 8 Effects of OGT Overexpression on ER Stress induction and accumulation of p-Tau, O-Tau, and Aβ induced by CSD. AAV-OGT or control viruses
were injected into the DG of mice brains, and then the mice were subjected to SD for four cycles to induce RSD. (A) Representative western blot analysisof O-GlcNAc and p-GSK3β, in hippocampus from the control, CSD, and CSD plus OGT group. The graphs represent the densitometric quantificationof O-GlcNAc levels normalized to β-actin and p-GSK3β levels normalized to GSK3β (n = 3/group). (B) Representative western blot analysis for GRP78,
GRP94, CHOP, ATF-4, p-eIF2α and eIF2α. The graphs represent densitometric quantification of Western blots, normalized to β-actin (n = 3/group). (C-E)
Representative confocal images (20X) of O-Tau (C), p-Tau (D), Aβ (E) and merged immunofluorescence staining in the DG, CA1, and cortex of mice brain.
Nuclei were counterstained with DAPI. The scale bar represents 100 μm. The values are expressed as the mean ± SEM; **p < 0.01, ***p < 0.001 vs. control
group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. CSD group. Statistical analysis was performed using One-way ANOVA with Tukey’s comparison. CSD, chronic
sleep deprivation; Con, control; DG, dentate gyrus; O-Tau, O-GlcNAc-Tau; p-Tau, phosphorylated-Tau; Aβ, amyloid beta; GSK3β, glycogen synthase kinase
3 beta; GRP78, glucose-regulated protein78; CHOP, C/EBP homologous protein; ATF-4, activating transcription factor 4; GRP94, glucose-regulated protein
94; eIF2α, eukaryotic translation initiation factor 2 A

also discovered that higher (10-fold) concentrations of
GlcN alone lead to notable cognitive impairments inzebrafish and mouse models (unpublished observation),
highlighting the importance of maintaining OGlcNAcylation homeostasis. Additionally, studies have demonstrated a robust 24-hour rhythm of O-GlcNAcylation in
Drosophila, regulated by the expression and activity of
OGT and OGA [48]. This rhythm is influenced by the
integration of circadian and metabolic signals, further
emphasizing the significance of sustaining the dynamic
equilibrium of O-GlcNAc cycling. However, one of theimportant issues regarding the CSD-induced O-GlcNAcylation changes and subsequent cognitive impairment
is the potential involvement of the stress response. Theinterplay between sleep deprivation and stress has beenwell-documented, with sleep deprivation being known
to exacerbate stress [49]. Stress can increase insulin andglucose levels, leading to metabolic changes that are also
observed under CSD conditions. In our study, it remains

challenging to clearly distinguish whether the changes
in O-GlcNAc are solely due to CSD or if they are significantly influenced by the stress response triggered by
CSD.
Emerging evidence indicates that SD leads to cognitive impairment, primarily by inducing neuroinflammation, which disrupts hippocampal neuronal plasticity
and impairs memory processes [50]. Furthermore, neuroinflammation plays a pivotal role in the developmentof SD-associated AD, because it initiates and exacerbates the accumulation of Aβ and p-Tau proteins [2, 51,52]. Glial cells, including microglia and astrocytes, aredensely located in the hippocampus and constitute themajor components of the neuroimmune system [53]. In
this study, we observed that CSD-induced activation ofastrocytes and microglia was effectively suppressed by
GlcN administration or OGT overexpression, indicating
the potential importance of O-GlcNAc cycling in regulating neuroinflammation. We further observed a reduction

Kim et al. Journal of Neuroinflammation(2024) 21:180

in O-GlcNAcylation in microglia from CSD mice, a result
consistent with previous findings showing decreased
O-GlcNAcylation in activated microglia following ischemic brain injury or LPS stimulation [54, 55]. We previously demonstrated that activation of glial cells bysimilar insults (i.e., brain ischemia/reperfusion injury andhypoxic brain damage) is effectively inhibited by GlcNmediated regulation of O-GlcNAc [55, 56]. Therefore,
the demonstration here that GlcN or OGT overexpression suppresses neuroinflammation holds promise for
mitigating the cognitive impairment and AD pathology
induced by CSD. However, whether O-GlcNAc cycling
directly inhibits glial activation or regulates inflammation through the modulation of other neural cell activities remains uncertain. As part of our exploration of therelationship between neuroinflammation and O-GlcNAc
cycling, we assessed the effects of minocycline—an
antibacterial agent known for its anti-inflammatory
properties in the central nervous system [57, 58]—on
CSD-induced brain dysfunction and O-GlcNAcylation.
We found that minocycline-induced suppression of glial
activation not only led to a decrease in CSD-induced
p-Tau and Aβ accumulation, it also rescued the CSDinduced decrease in O-GlcNAcylation levels in the brain,
restoring them to a level similar to that in the normal
state. These results suggest the possibility that activatedglial cells or inflammatory factors directly contribute to
the regulation of O-GlcNAc levels in the brain. However,
it is also plausible that the inflammatory response affects
overall brain metabolism and neuronal function, thereby
modulating O-GlcNAc.
Research from a different perspective has suggested
that ER stress plays a critical role in the onset and progression of AD [59]; consistent with this, ER stress features prominently in the brains of AD patients [60].
One emerging link between ER stress and AD is glycogen synthase kinase-3 (GSK‐3) signaling. Studies haveshown that increased GSK3 activity induces memory
deficits [61, 62] and is a prominent feature of the AD
brain [63]. The identified functions of GSK3 in the brain
center on effects on Tau phosphorylation at most AD
sites [64, 65], and Aβ overproduction [66]. In the current study, we demonstrated that the ER stress markers
were increased in the brains of CSD mice, whereas phosphorylation of GSK3β was decreased, indicating its activation. Given that GlcN and OGT overexpression shared
the property of reversing these molecular changes, wehypothesized that O-GlcNAc cycling might regulate ER
stress and/or GSK3 signaling. Although previous studies have demonstrated activation of GSK-3β during ER
stress [67, 68], the nature of the interplay between ER
stress and GSK-3β signaling in AD has not yet been fully
elucidated. In this study, the OGT inhibitor, OSMI-1,
increased ER stress and GSK‐3β activation in N2a cells,

Page 15 of 17

indicating that O-GlcNAc cycling plays an important rolein the regulation of both ER stress and GSK‐3β signaling.
Consistent with this, it has been reported that increasing
O-GlcNAcylation through adenoviral-mediated overexpression of OGT significantly suppresses ER stress andsubsequent cell death in myocytes [69]. Similar results
have been demonstrated in a rabbit model of renal I/R
injury, where CHOP levels were suppressed upon administration of GlcN [70]. Although it is not yet clear how
O-GlcNAc regulates ER stress, several mechanisms havebeen proposed. First, O-GlcNAcylation of eIF2α caninhibit phosphorylation of eIF2α, which is necessary for
CHOP activation [71]. Second, OGT can suppress ER
stress by stabilizing hypoxia-inducible factor 1α (HIF1α)[72]. Third, it has been proposed that O-GlcNAc plays arole in mitigating ER stress through formation of stressgranules and processing bodies, which regulate mRNAtranslation and degradation, respectively [73]. While thefunctional contributions of CSD-induced ER stress and
GSK-3β activation to CSD-induced AD pathogenesis, aswell as the underlying mechanisms, remain to be elucidated, we tentatively suggest that induction of GSK‐3β
activation or ER stress by CSD may not be the critical,
sole effector of AD pathogenesis or a direct regulatory
target of HBP/O-GlcNAc cycling under CSD conditions.
As evidence for this, inhibition of neuroinflammation by
minocycline did not affect either GSK‐3β activation or
ER stress induced by CSD, despite the fact that it effectively rescued CSD-induced decreases in O-GlcNAc andAD pathogenesis as well as glial activation. Our study
exclusively utilized male mice to minimize experimental
variations caused by hormonal effects, aiming to achieve
clearer results. However, this approach limits our understanding of potential sex differences in neurobiology anddisease mechanisms, as well as the clinical applicabilityof our findings. Future studies should include validation
using female mice to ensure the robustness and broader
applicability of our research.
In the current study, pathogenic changes in the brain
resulting from repeated CSD exposure are evident in our
mice model, and the injury pattern shares some overlapping features with those of AD. The primary significanceof our study lies in its pioneering proposal of a direct
association between O-GlcNAc cycling in the brain
during CSD and the functional and molecular neuronalchanges induced by CSD. Future studies investigating theregulatory mechanisms governing HBP flux during sleep
cycles, as well as the cyclic patterns of OGA and OGT
expression, will be crucial in unraveling the association
between sleep disorders and the development of AD.

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03179-4.

Kim et al. Journal of Neuroinflammation(2024) 21:180

Supplementary Material 1
Supplementary Material 2
Author contributions
DY Kim prepared Figs. (1, 3, 5, 7 and 8), analyzed the data and wrote themain manuscript text. S Kim prepared Figs. (1, 2, 4, and 6), analyzed the data
and wrote the main manuscript text. IO Han designed the experiments,
analyzed the data, and wrote the main manuscript. All authors reviewed themanuscript.
Funding
This work was supported by National Research Foundation (NRF) of Korea
Grants (RS-2024-00346770) and research grant of Inha University.
Data availability
No datasets were generated or analysed during the current study.

Declarations
Ethics declaration
Not applicable.
Conflict of interest
The authors declare that they have no conflicts of interest with the contentsof this article.
Competing interests
The authors declare no competing interests.
Received: 2 March 2024 / Accepted: 17 July 2024

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