Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : July 7, 2022
Accepted : August 29, 2022
Published online : September 30, 2022
doi:10.24659/gsr.9.3_135

Original article

Amyloid beta clearance and microglia: Effects of glycative stress and melatonin
Yoshikazu Yonei 1), Toshio Taira 2), Shino Otaka 2), Shizuko Sekiguchi 2), ANM Mamun-or-Rashid 1)
Masayuki Yagi 1), Hiroaki Masuzaki 3)1) Anti-Aging Medical Research Center/Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan2) Sapporo Division, Cosmo Bio Co., Ltd., Otaru, Hokkaido, Japan
3) Division of Endocrinology, Diabetes and Metabolism, Hematology and Rheumatology,(Second Department of Internal Medicine), Graduate School of Medicine,
University of the Ryukyus, Nishihara, Okinawa, Japan

Abstract

The physiological role of amyloid-β (Aβ) is unknown, while it plays an important role in the onset and progression of
Alzheimer's disease (AD). Aβ polymerization leads to enhanced neurotoxicity, persistent degradation, and deposition in
the brain, resulting in decreased Aβ clearance. Diabetes mellitus and poor sleep quality are representative risk factors forthe development of AD. Methylglyoxal (MGO) and acrolein are increased in diabetic patients, a representative disease withhigh glycative stress, and melatonin secretion is decreased during poor sleep quality. In this study, we focused on the Aβphagocytosis of microglia, which plays a role in Aβ clearance, and examined the effects of Aβ glycation and melatonin.
Glycated Aβ was prepared by MGO or acrolein treatment. Fluorescently labeled TAMRA-Aβ and primary rat microglial
cells (Cosmo Bio) were used in the experiments. Several new findings were obtained from this experiment. First, microglia
phagocytose Aβ, while their phagocytic capacity for glycated Aβ was markedly reduced. Second, Aβ phagocytosis wasenhanced by melatonin. Concurrently, spontaneous death of cultured microglia was greater when Aβ was not added than whenAβ was. These findings suggest that prevention of Aβ glycation by countermeasures against glycative stress and preventionof AD progression by lifestyle, i.e., improvement of sleep quality, are important, rather than elimination of Aβ as has been
conventionally practiced.

KEY WORDS: Alzheimer's disease, microglia, amyloid beta, methylglyoxal, acrolein, melatonin, sleep quality

Introduction
Amyloid beta (Aβ) is a type of protein produced in thebrain. It is also present in the brain of healthy individuals,
but its physiological role is not well understood. Aβ is
normally degraded and eliminated in a short period of time
as a relic in the brain (Aβ clearance). However, oxidative
stress or glycation stress modifies the structure of Aβ,
resulting in the formation of abnormal Aβ 1, 2). In particular,when proteins form cross-links due to glycation stress,
Aβ binds to each other and forms abnormal Aβ polymers.
The formation of abnormal Aβ polymers causes persistentdegradation, increased neurotoxicity, and decreased Aβ

clearance. When the resulting Aβ is not excreted and isdeposited in the brain, it forms Aβ senile plaques (amyloid
pathology). Neurotoxic abnormal Aβ can also affect neurons
when it clings to the periphery of previously healthy neurons.
This triggers the formation of neurofibrillary tangles, inwhich tau protein, a microtubule-binding protein, fibrillates
and aggregates in the cytoplasm. This results in gradualbrain atrophy and progression of Alzheimer's disease. Thisis the concept of the "amyloid cascade hypothesis" with theaddition of glycation stress 3-5).
To date, there have been no reports on the effects
on microglial phagocytosis when Aβ is subjected totranslational modifications. In this study, we focused on

Correspondence to: Professor Yoshikazu Yonei, MD, PhD
Anti-Aging Medical Research Center,
Graduate School of Life and Medical Sciences, Doshisha University
1-3, Tatara Miyakodani, Kyotanabe, Kyoto, 610-0394 Japan
TEL & FAX: +81-774-65-6394 e-mail: yyonei@mail.doshisha.ac.jp
Co-authors: Taira T, primarycelltaira@gmail.com;
Otaka S, shino-otaka@cosmobio.co.jp; Sekiguchi S, shizuko-sekiguchi@cosmobio.co.jp;
Mamun-or-Rashid ANM, mamunbtgeiu@gmail.com; Yagi M, myagi@mail.doshisha.ac.jp;
Masuzaki H, hiroaki@med.u-ryukyu.ac.jp_ 135 _

Glycative Stress Research 2022; 9 (3) : 135-145(c) Society for Glycative Stress ResearchMicroglial Aβ Phagocytosis, Glycative Stress, and Melatonin

microglia (MG), which play an important role in the Aβ
clearance, and examined the uptake of Aβ into cells and thechange in uptake capacity when Aβ is subjected to glycative
modification. Concurrently, we examined the effect of
melatonin (Mel), which is involved in sleep quality.

Methods

Preparation of test reagents

Fluorescently labeled TAMRA-Aβ (Cosmo Bio, Tokyo,
Japan) was used as the experimental reagent (hereinafterreferred to as T-Aβ). This reagent consists of Aβ(1-42)
labeled with red fluorescent carboxytetramethylrhodamine( TAMRA) via a PEG spacer 6).

Preparation of glycated T-Aβ withmethylglyoxal (MGO)

0.5 mg of T-Aβ was dissolved in 50 µL of trifluoroacetic
acid (TFA) (Nacalai Tesque, Kyoto, Japan) and then 450 µL
of ultrapure water was added. 100 µL each was dispensed
into 5 microtubes and concentrated and dried overnightusing a centrifugal evaporator. After drying, 200 µL/tube
of 0.2 M phosphate buffer (pH 6.0) was added to two of thefive tubes to dissolve them, and they were combined in one
tube to make 400 µL of T-Aβ solution. 80 µL/well of T-Aβ
solution was dispensed into four wells in 96 well black plates
and MGO ( Fujifilm Wako Pure Chemicals, Osaka, Japan)
was added to final concentrations of 0 mM (Blank), 0.4 mM,
2 mM, and 10 mM. After addition, the plates were placed in
an incubator at 37 °C for glycation. Fluorescence values at
the wavelengths of fluorescent end glycation end products
AGEs (advanced glycation end products), (excitation: 34 0
nm, fluorescence: 430 nm), were measured by a microplate
reader before and 1 hour, 17 hours, and 65 hours after the
addition, as an index of glycation.
Since there was a possibility that the toxicity of residual
MGO could adversely affect cells when glycated T-Aβ was
added to cells, we performed two processes to remove MGO
from the glycated T-Aβ solution: 1) crude purification by
ether precipitation and 2) simple purification by reverse
phase solid phase extraction.

1) Crude purification by ether precipitation
The 0.5 mg/mL T-Aβ solution glycated with 10 mM
MGO was divided into microtubes in 30 µL portions, and270 µL of TFA was added and stirred and mixed well. In
addition, 1. 5 mL of t - butyl methyl ether ( Merck KGaA,
Darmstadt, Germany) was added and the glycated T-Aβ was
precipitated by standing at −80 °C for 3 hours and then at 4 °C
overnight. The T-Aβ solution was centrifuged at 12,000 × g
for 15 min to remove the supernatant containing residual
MGO, 1 mL of t-butyl methyl ether was added again andstirred, then centrifuged at 12,000 × g for 15 min to remove
the supernatant. After drying by nitrogen blowing, 500 µL
of ACN was added, sonicated for 30 seconds, and 500 µL
of 0.1% TFA was added to dissolve the glycated T-Aβ. The
absorbance was measured at 556 nm using an ultra trace
spectrophotometer. After measurement, the samples werelyophilized and stored under refrigeration.

2) Simple purification by reversed-phasesolid-phase extraction
The SPE column (HyperSep™ C18 cartridge; Thermo
Fisher Scientific, Tokyo, Japan) was conditioned by washingwith 0.75 mL of 95 % ACN/ 0.1% TFA and equilibrated
three times with 2.25 mL of 0.1% TFA. ACN was obtained
from Merck KgaA. 40 µL of 0.5 mg/mL T-Aβ solution
glycated with 2 mM MGO and 100 µL of 0.1% TFA wereapplied to the SPE column, washed 5 times with 2.25 mL of
0.1% TFA and once more with 0.35 mL of 95 % ACN/0.1%
TFA. After washing, glycated T-Aβ was eluted with 0.6 mLof 95 % ACN/ 0.1% TFA, and the eluate was collected. To
the collected eluate, 0.6 mL of 0.1% TFA was added, andthe absorbance at 556 nm was measured using an ultra-trace
spectrophotometer. Then, the samples were lyophilized andstored under refrigeration.

Preparation of glycated T-Aβ with acrolein
0.5 mg of T-Aβ was dissolved in 50 µL of TFA andthen 450 µL of ultrapure water was added; 100 µL each wasdispensed into 5 microtubes and concentrated and driedovernight using a centrifugal evaporator. After drying, 200µL/tube of 0.2 M phosphate buffer (pH 6.0) was added
to two of the five tubes to dissolve and combined in one
tube to make 400 µL of T-Aβ solution. T-Aβ solution wasdispensed in 4 wells at 80 µL/well into 96 well black plates,
and acrolein (Fujifilm Wako Pure Chemicals) was added
to final concentrations of 0 mM (Blank), 0.4 mM, 2 mM
and 10 mM. The plates were placed in an incubator at 37 °C
for glycation. Fluorescence values at the wavelengths offluorescent AGEs (excitation: 340 nm, fluorescence: 430nm) were measured in a microplate reader before addition,
14 hours after addition, and 62 hours after addition, and wereused as indicators of glycation.

MG Primary Cultured Cells
Cultured microglial cell strains that have been used forexperiments generally exhibit macrophage-like properties,
and their functions, i.e., response to inflammation andneuroprotection, are distinct from those of microglia in vivo.
Whereas, primary microglia are prepared from the neonatal
brain, and are grown in a mixed culture system that includes
microglia and other cells, mainly astrocytes and neurocytes,
thereby maintaining the original functions of microglia inpart 7). In this study, we used a primary microglia culturekit (Cosmo Bio) to verify the function of microglia inrecognizing and eliminating Aβ 8).

Effect of T-Aβ and glycated T-Aβ on primary
microglial cells

Floating MG were collected from one 75 cm2 flask of
mixed culture of primary MG prepared from SD rat brain
according to protocol 8) using the medium supplied withthe kit and seeded onto 8 wells of 96 well black plates (thenumber of seedings was unknown because the number ofseedings was not countable). Since MG cell numbers are
reduced by adhesion to centrifuge tubes, measures weretaken to prevent excessive adhesion by using instrumentation_ 13 6 _Glycative Stress Researchcoated with a macrophage anti-adhesion coating agent. Nonglycated T-Aβ and MGO-glycated T-Aβ were added tothe medium at a final concentration of 1 µM the day after
seeding, and the cells were exposed for 4 days to observe
T-Aβ uptake into the cells by fluorescence microscopy.
After observation, the cells were washed twice withmedium to remove T-Aβ and glycated T-Aβ. Changes in T-Aβ
uptake into cells were observed on day one (culture day 5)
and day 4 (culture day 8) after removal, and on day 8 (cultureday 12), 1/10 of the medium was added with a cell proliferation/
cytotoxicity assay reagent according to protocol 9), and a color
reaction was performed in a 37 °C incubator for 2 hours.
Cytotoxicity was evaluated by measuring absorbance at 450
nm (reference: 650 nm) in a microplate reader.

Composition of the experimental group
The composition of the experimental group is shown
below.
		
T-Aβ Mel Glycationa) Non-T-Aβ group (no treatment) −−−b) T-Aβ group			+−−c) T-Aβ + Mel group		++−d) Gly-T-Aβ group		+−+e) Gly-T-Aβ + Mel group		
+++Note that there are two types of glycated T-Aβ:
MGO-treated and acrolein-treated. The final concentrationof Mel (melatonin) in the culture medium was 100 µM. In
this study, two MG seeding experiments were performed.
The first experiment was conducted as a, b, c, d, and e
above, with MGO-treated Gly-T-Aβ used for d and e. In thesecond experiment, a, b, and d were used, and MGO-treated
and acrolein-treated Gly-T-Aβ were used in d. Since the
MG culture conditions in the two experiments were different,

the analyses were performed separately, and no integrated
analysis of T-Aβ phagocytosis was performed.

Statistical analysis
MG cell counts were measured as the number of nuclei
per unit area using an inverted phase-contrast fluorescencemicroscope (BZ-X710, Keyence, Tokyo, Japan) with an image
analyzer (BZ-H3CM). Analysis of T-Aβ phagocytosis by MG
was performed on the area of T-Aβ-derived fluorescence(area of fluorescence intensity above the reference value)
divided by the total cell area (μm 2 ). The figure showed
the value adjusted for the area of nonspecific background
fluorescence (nonspecific high intensity area in the non-TAβ group). Statistical analysis was performed using Excel
statistics (Bell Curve, Tokyo, Japan) and evaluated by analysis
of variance (ANOVA) followed by Fisher's post hoc test.

Results

Generation of glycated AβDuring MGO treatment of Aβ, fluorescent AGEs
tended to increase with increasing MGO concentration andglycation time (Fig. 1). 65 h after addition of 10 mM MGO,
the fluorescence value decreased from 17 h. Therefore, thereaction time of one day and night at 10 mM was adopted forglycation conditions.
Similarly, during acrolein treatment, fluorescent AGEs
tended to increase with increasing acrolein concentration andglycation time (Fig. 2). Since fluorescence values were lower
at 62 h after acrolein addition than at 14 h, a reaction timeof one day and night at 10 mM was adopted for glycation
conditions.

AGE fluorescence intensity

2,000
1,600
1,200
800
400
0

MGO

17 hr

1 hr

0 hr
0 mM

0.4 mM

Fig. 1. Glycation of T-Aβ by MGO treatment.

2.0 mM

65 hr
10 mM

Results are expressed as mean of duplicate values. Fluorescent intensity derived from AGEs were measured by Ex 340
nm/Em 430 nm. MGO concentration; 0 mM, 0. 4 mM, 2.0 mM, 10 mM. T-Aβ, TAMRA-β-amyloid(1- 42); MGO,
methylglyoxal; AGEs, advanced glycation end products.

Fig. 1_ 137 _

Microglial Aβ Phagocytosis, Glycative Stress, and Melatonin

AGE fluorescence intensity

350
300
250
200
150
100
50
0

Acrolein

0 hr

14 hr

0 mM

0.4 mM

62 hr
2.0 mM

10 mM

Fig. 2. Glycation of T-Aβ by acrolein treatment.

Results are expressed as mean of duplicate values. Fluorescent intensity derived from AGEs were measured by Ex 340
nm/Em 430 nm. Acrolein concentration; 0 mM, 0. 4 mM, 2.0 mM, 10 mM. T-Aβ, TAMRA-β-amyloid(1- 42); AGEs,
advanced glycation end products.

Fig. 2First culture experiment

MG cell count and Aβ phagocytosis
Cultures were fluorescently labeled T-Aβ added at day
2 after MG seeding and observed on day 10.
In the non-T-Aβ group, MG cell counts showed adecrease in the number of viable cells due to cell death on
day 10 compared to day 2, experimental start date (Fig. 3-a,no photo on experimental start date). The cause of cell death
was determined to be spontaneous death.
In the T-Aβ group, MG cell counts showed no decrease
on day 10 compared to day 2 (Fig. 3-b); MG cell counts on
day 10 were higher and spontaneous death was lower thanin the non-T-Aβ group. T-Aβ labeled fluorescence was seen
to be incorporated into the MG cytoplasm, confirming that
Aβ is phagocytosed (taken into the cell) by MG. No specificmorphological changes were observed.
In the T-Aβ + Mel group, the number of MG cells
increased on day 10 compared to day 2 (Fig. 3-c) MG cellnumbers were higher than in the T-Aβ group without Mel.
It was not possible to determine whether there was lessspontaneous death or enhanced MG proliferation. In the
T-Aβ + Mel group, fluorescent uptake of MG cells wasprominent and Aβ phagocytosis by MG was more active. No
specific morphological changes were observed.
In contrast, in the MGO-treated Gly-T-Aβ group, thenumber of MG cells increased on day 10 compared to day 2(Fig. 3-d ). Little fluorescent uptake of MG cells was observed.
No specific morphological changes were observed.
In the Gly-T-Aβ + Mel group, MG cell counts weresimilar on day 10 compared to day 2 (Fig. 3-e), and MG cellcounts on day 10 were lower than those in the Mel-free GlyT-Aβ group. The fluorescent uptake of MG cells was slight.
The image data of T-Aβ phagocytosis by MG wasquantitatively evaluated by image analysis ( Fig. 4 ), and theimage data of the T-Aβ + Mel group showed a markedly
higher trend intensity compared to the T-Aβ group, indicatingthat T-Aβ phagocytosis was increased by Mel. However, twoof the three T-Aβ + Mel group data values were excluded fromthe analysis because of their abnormally high uptake
fluorescence brightness; values that were not excluded areshown in the figure. Compared to the T-Aβ group, thefluorescent brightness in MG of the Gly- T-Aβ group was
significantly lower (p = 0.03), indicating reduced phagocytosis.

Second culture experimentComparison of Aβ phagocytosis with MGO and acrolein
Fluorescently labeled T-Aβ was added at 2 days after
MG seeding, and fluorescence microscopy images at 10 days
are shown in Fig. 5.
In the T-Aβ group, labeled fluorescence was seen in the
MG cytoplasm, reconfirming that Aβ is phagocytosed by
MG (Fig. 5-b). Whereas, in the MGO- and acrolein-treated
Gly-T-Aβ groups, little labeled fluorescence was seen in theMG, and glycosylated Aβ phagocytosis by MG was virtuallyabsent (Fig. 5-c, d ). There was no difference between the
MGO - and acrolein-treated groups.
Cell counts on day 10 of culture are shown in Fig. 6;
there were no significant differences in cell counts among
the Non-T-Aβ, MGO-treated Gly-T-Aβ, acrolein-treated
Gly-T-Aβ, and T-Aβ groups.
The results of Aβ phagocytosis by MG are summarized
in Fig. 7. When MG phagocytosis of T-Aβ was compared
between the T-Aβ and Gly- T-Aβ groups, phagocytosis ofglycated T-Aβ was markedly reduced (p < 0.001), with nodifference between MGO and acrolein treatments.

_ 13 8 _Glycative Stress Research

aa

cc

bb

Fig. 3.

T-Aβ phagocytosis by microglia:
Fluorescence microscopy imagea) Non-T-Aβ group (no treatment), b) T-Aβgroup, c) T-Aβ + Mel group, d) Gly-T-Aβgroup, e) Gly-T-Aβ + Mel group. Images (xeedd100) of cultured cells 10 days after seeding(8 days after addition of T-Aβ). Red color
portions indicate T-Aβ - derived fluorescence
uptaken by microglia. Note the enhanced
fluorescence by Mel ( c ) compared with
the T-Aβ group (b) . Gly-T-Aβ is preparedby MGO treatment (10 m M, one day).
T-Aβ, TAMRA- β -amyloid (1- 42); MGO,
methylglyoxal; Mel, melatonin; Gly, glycated.

T-Aß fluorescence/Cell area (µm 2)

ig. 3

*

2.50

**

2.00
1.50
1.00
0.50
0.00

T-Aß
Melatonin
Glycation
by MGO

Group

−
−
−

＋
−
−

＋
＋
−

＋
−
＋

＋
＋
＋

Non-T-Aß

T-Aß

T-Aß + Mel

Gly-T-A ß

Gly-T-Aß + Mel

Fig. 4. Comparison of T-Aβ phagocytosis by MG: The first experiment

Y axis shows the values: T-Aβ fluorescence intensity / cell area (μm 2 ) followed by adjustment for the area of nonspecific background
fluorescence (nonspecific high intensity area in the non-T-Aβ group). Results are expressed as mean ± SD of triplet values (n = 4 except
Non-T-Aβ group and T-Aβ + Mel group, n = 2), * p < 0.05, ** p < 0. 01 by Fisher's post hoc test ( T-Aβ + Mel group excluded). T-Aβphagocytosis is inhibited by MGO-glycation while enhanced by Mel (100 μM). Gly-T-Aβ is prepared by MGO treatment (10 mM, oneday). MG, microglia; T-Aβ, TAMRA-β-amyloid (1- 42); MGO, methylglyoxal; Mel, melatonin; Gly, glycated; SD, standard deviation.

Fig. 4_ 13 9 _Microglial Aβ Phagocytosis, Glycative Stress, and Melatonin

a aa

bbb

c cc

ddd

Fig.
55
Fig.
Fig. 5. T-Aβphagocytosis by MG: Comparison between MGO and acrolein treatment.

a) Non-T-Aβ group (no treatment), b) T-Aβ group, c) MGO-Gly-T-Aβ group, d) Acrolein-Gly-T-Aβ group. Images(x 4 00) of cultured cells 10 days after seeding (8 days after addition of T-Aβ). Red color portions indicate T-Aβderived fluorescence uptaken by MG. No difference between MGO (c) and acrolein (d) treatment. Note the much lessfluorescence in c) and d) than that in b). Gly-T-Aβ is prepared by MGO or acrolein treatment (10 mM, one day). MG,
microglia; T-Aβ, TAMRA-β-amyloid(1- 42); MGO, methylglyoxal; Gly, glycated.

700

Number of microglia

600
500
400
300
200
100
0

Gly-T-Aß group

Non-T-Aß group(MGO)

Fig. 6. Live cell counts of MG 10 days after seeding.

T-Aß group(Acrolein)

Results are expressed as mean ± SD of triplet values, counted the number of nuclei by image analyzer (n = 4, except
Non -T-Aβ group, n = 2). Gly-T-Aβ is prepared by MGO or acrolein treatment (10 mM, one day). MG, microglia;
T-Aβ, TAMRA- β -amyloid (1- 42); MGO, methylglyoxal; Gly, glycated; SD, standard deviation.

Fig. 6_ 14 0 _Glycative Stress Research

T-Aß fluorescence/Cell area (µm 2 )

450

***

400
350
300
250
200
150
100
50
0

Gly-T-Aß group

Non-T-Aß group(MGO)

T-Aß group(Acrolein)

Fig. 7. Comparison of T-Aβ phagocytosis by MG: The second experiment

Y axis shows the values: T-Aβ fluorescence intensity/cell area (μm 2 ). Results are expressed as mean ± SD, n = 3, *** p < 0.001
vs other groups, by Fisher's post hoc test. T-Aβ phagocytosis is inhibited by MGO - and acrolein-treatment (10 mM, one day).
MG, microglia; T-Aβ, TAMRA-β - amyloid (1- 42); MGO, methylglyoxal; Gly, glycated; SD, standard deviation.

Fig. 7

Discussion
Alzheimer's disease (AD) is a leading cause of
progressive dementia; autopsy brains of AD patients havebeen found to have an accumulation of activated MG aroundsenile plaques, suggesting the involvement of impaired Aβ
clearance. However, the reason for the presence of Aβ in thehealthy state and its physiological role are not clear.
Aβ is produced in vivo under physiological conditions.
γ-secretase is a membrane protein complex composed ofpresenilin, nicastrin, Aph-1, and Pen-2 with an active center.
The substrate (APP or Notch) is cleaved by γ-secretase
after cleavage at the lumenal side, and the resulting Aβ
is secreted extracellularly 3, 4). γ-secretase is involved incarboxy-terminal cleavage in the Aβ production pathway anddetermines the amount of Aβ and its aggregation. Therefore,
it is considered an important AD drug target molecule, andvarious small molecule compounds have been developed asγ-secretase inhibitors. However, therapies that inhibit Aβ
production have repeatedly failed in clinical trials, suggestingthat Aβ has some physiological significance and that lowering
Aβ levels in the brain may not lead to AD treatment 5). Aβ
production occurs regardless of age or presence of AD, andin the normal brain, Aβ is properly removed and does notoften accumulate in the brain 10, 11).
These findings indicate that homeostasis of Aβ
clearance is important for controlling AD progression. Aβ
deposition, which is pathognomonic at autopsy, reflects thebalance of Aβ production, deposition, and removal at thattime. Activated MG accumulate around senile plaques andplay an important role in Aβ clearance. Phagocytosis of Aβ

by cultured rat MG is regulated by the low molecular weight
G protein Rac1 and the Wiskott-Aldrich syndrome protein
family verprolinhomologous protein (WAVE) that acts
downstream actin fiber reorganization 12).
When Aβ is taken up by MG, it is processed intracellularly
and the N-terminal fragment is lost 11). Activated MG in
advanced AD model mice express the nicotinic acetylcholine
receptor α7 nAChR, and CD14/TLR complexes are expressedto perform clearance functions 13). Regarding the morphology
of MG, it has been reported that the morphology of MG
prepared from rat neonates before Aβ42 treatment was rodtype, but immediately after treatment, they changed theirmorphology to ameboid-type and began phagocytosis 14). The
morphology of the cultured MG used in this experiment was
ameboid type from the second day of culture.

Factors affecting Aβ clearance
There are few reports on factors affecting Aβ clearance
in MG. Galantamine, an acetylcholinesterase (AChE)
inhibitor, binds to microglial nAChR as a ligand, promoting
the expression of the CD14/ TLR complex and facilitating Aβclearance 15).
Microglial Aβ phagocytosis is enhanced by heat shockproteins (Hsp) and, conversely, inhibited by high mobility
groupbox protein-1 (HMGB 1), a known nuclear protein 16).
Rutin (quercetin-3-rutinoside) is a natural flavonoid withantioxidant and AGE formation inhibitory properties. It has
been suggested that sodium rutin (NaR) may increase MG
and promote Aβ clearance by increasing the expression levelof phagocytosis-related receptors in MG 17). With the results_ 141 _

Microglial Aβ Phagocytosis, Glycative Stress, and Melatoninof this experiment, Mel was newly added as a factor that
promotes microglial Aβ phagocytosis. Since Mel secretion
increases as "sleep quality" improves, it is important to aim
for good sleep to maintain Aβ clearance homeostasis in orderto prevent the progression of AD.
In an experiment comparing mouse brain MG divided
into two groups, a subset that produces high levels ofreactive oxygen species (ROS) and a subset that does notproduce ROS, the former showed neurotoxicity and the latterneuroprotection when co-cultured with neurons 18). In culture
experiments in which HIV-1 Nef protein was introduced toincrease NADPH oxidase activity, neuroprotective microglial
clones were transformed into toxic microglial clones. Thesefindings suggest that MG may change from a neuroprotective
subset to a neurotoxic subset in response to the environment.
It is a subject for future study how MG in contact with Aβ
and glycated Aβ undergo changes and whether there aredifferences between the two types of Aβ.

Involvement of Aldehydes in AD
Diabetes mellitus, a typical disease with high glycationstress, is said to be the source of all diseases and increases
the risk of developing various age-related diseases. The
fundamental factor is the tendency to produce aldehydes(compounds with -CHO groups), open-ring glucose andopen-ring fructose, as well as lipid peroxidation products
such as 4-hydroxynonenal (HNE), acrolein, MGO, glyoxal(GO), glyceraldehyde, 3 deoxyglucosone (3DG) are increased.
As a carbonyl stress, these aldehydes induce protein
carbonylation and further produce AGEs, which contribute toneurodegeneration in AD 19-22). Aβ amyloid protein glycationby aldehydes is a potentially toxic morphological changewith enhanced toxicity 23).
AGE formation by Aβ glycation alters Aβ aggregation
capacity 24). Aβ glycation meaningfully delays β conversion
to mature fibers, and toxic forms. i.e., oligomers, are more
likely to remain 25, 26). In the central nervous system, glycationof Aβ as well as tau protein and α-synuclein is thought
to be involved in the onset and progression of cognitive
impairment.
MGO increases the size of Aβ aggregates 26); MGO
concentrations in cerebrospinal fluid (CSF ) of AD patientsare higher than in healthy subjects 27) and MGO-derived
hydroimidazolone compounds are more abundant 28). Regarding
the development of cognitive impairment, it has beenreported that higher serum MGO levels are associated withgreater cognitive decline 29).
Acrolein, on the other hand, is produced by lipid
peroxidation and polyamine metabolism and is highly
nucleophilic and reactive. Acrolein undergoes Michael
addition reactions with nucleophilic amino acids, i.e., cysteine,lysine, and histidine, to induce protein carbonylation. Oraladministration of acrolein to rats (2.5 mg/kg/day, for 8 weeks)
resulted in cognitive decline and hippocampal atrophy 30).
Clinically, acrolein is increased in the hippocampus andtemporal lobe of AD patients 31) and acrolein-conjugatedprotein in plasma and spinal fluid is higher than in normal
subjects 32, 33). It is assumed that acrolein is involved in theonset and progression of AD 34).
There are only a limited number of studies on theeffects of Aβ morphological changes; glycated Aβ formed
by MGO has been reported to be highly toxic, inducing
increased RAGE expression and glycogen synthase kinase-3(GSK-3) activation 35). Therefore, in this study, we preparedglycated Aβ by MGO and acrolein and verified the effect on
microglial phagocytosis, and found that phagocytosis wasdecreased for glycated Aβ.

Involvement of Mel
Mel is a hormone secreted by the pineal gland thathas effects of antioxidation 36) and glucose metabolism
homeostasis 37). Concurrently, poor sleep quality and circadianrhythm disturbances are known to increase the risk ofdeveloping AD 38), and Mel has attracted attention as a factor
linking these events 39). Mel acts in an inhibitory manner
on amyloid precursor protein (APP) processing and Aβ
generation by modulating the regulatory network of secretase
expression 40).
Mel inhibits Aβ aggregation 41- 44), ameliorates
neurotoxicity 45, 46), improves lymphatic drainage throughlymphatic vessels 47), and promotes Aβ clearance throughBBB transport and degradation pathways.
Since Aβ4 0 is highly aggregative and is easily
deposited in the brain, and Aβ42 is easily excreted, theplasma Aβ40/42 ratio is significant as an indicator of
impaired Aβ clearance, and a high ratio is associated witha high risk of developing AD 48). We have reported that
Aβ40/42 increases with age 49) and that Aβ40/42 is higher in
populations with poor sleep quality 50). This finding suggeststhat Aβ clearance may be decreased in populations with low
Mel secretion, which is compatible with the results of thepresent study.
Mel also has a positive effect on memory 51, 52). In OXYS
rats, which exhibit impaired Mel secretion, Mel administration
suppresses increased anxiety and deterioration of reference
memory 53). Mel has also been shown to improve memory
function in D-galactose-treated mice by suppressing
the elevation of ROS and RAGE (receptor for advanced
glycation end products) 51). It was reported that Mel crosses
the blood-brain barrier and is converted to N1- acetyl-5methoxykynuramine (AMK) in brain tissue, which promotes
long-term object memory in mice 54).
When Aβ is taken up by neurons, the ionic gradientin the inner mitochondrial membrane is altered, inducing
mitochondrial damage and reducing respiratory control ratio
and ATPase hydrolytic activity 55). Furthermore, mitophagy
is impaired, resulting in mitochondrial repair 56). In AD,
mitochondrial aldehyde dehydrogenase (ALDH2) 57) and
RAGE 51) have been implicated in mitochondrial andmitophagic disturbances.
We have studied Mel from the viewpoint of underlying
glycation stress, and reported that Mel suppresses postprandial
hyperglycemia (blood glucose spike) 58) and promotes AGEs
degradation 59). Furthermore, in the present study, we foundthat Mel promotes microglial Aβ phagocytosis. These findings
indicate that improving sleep quality and enhancing one's
own Mel secretion are important for primary prevention inorder to delay the progression of AD as much as possible,
which may be called a paradigm shift in AD prevention
measures.

_ 14 2 _Glycative Stress Research

Conclusion

MG have previously been shown to play an importantrole in brain homeostasis as cells involved in the regulationof synaptic function, removal of foreign substances fromthe brain, or immunity. The present experiments provide
several new findings. First, MG not only phagocytose anddegrade Aβ, but also act more protectively toward MG
than without Aβ, suggesting that Aβ may contribute to
microglial homeostasis. Second, Aβ phagocytosis by MG isfacilitated by Mel. This is consistent with epidemiologicalfindings that AD incidence is higher in individuals withpoor "sleep quality" who have decreased Mel secretion.
Third, MG may have difficulty phagocytosing glycated Aβ,
resulting in decreased Aβ clearance. This is consistent withthe epidemiological finding of a higher incidence of AD indiabetic patients with high glycation stress and increased
glycated Aβ. Based on the above findings, it is considered
important to prevent the progression of AD by preventing
Aβ glycation through measures against glycative stress andlifestyle habits, i.e., improving sleep quality, rather thanReference
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Conflict of Interest Statement
The authors claim no conflict of interest in this study.

Acknowledgments
This work was supported by Grants-in-Aid for Scientific
Research (JSPS KAKENHI) (20K11593). This study waspresented at the 25th Meeting of the Society for Glycation
Stress, September 10 th, 2022, Kyoto, Japan.

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