Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : February 25, 2023
Accepted : May 20, 2023
Published online : June 30, 2023
doi:10.24659/gsr.10.2_79

Review article

Effects of DNA methylation and demethylation on LRP1 and RAGE genes as
crucial factors in Alzheimer's disease pathophysiology
Nikola Barić
Private General Practice Office, Labin, Croatia

Abstract

Presently there is a growing number of scientific papers that indicate a strong connection between epigenetics and the
Alzheimer's disease (AD) pathophysiology. This study also concentrates on some events related to this serious, chronic,
and lethal neurodegenerative disease. Actually, this disease, with a still incomplete knowledge of its etiology and closely
connected to the ageing process and old age, in conditions of intensive ageing of the world population (so called senectual
explosion), is increasingly becoming a burden for contemporary society. The most recent scientific papers related to AD
pathophysiology indicate the complex interaction of a number of genes and oxidative and glycative stress, as well as epigenetic
phenomena. The two mentioned types of stress have been under intensive research for a longer period of time; however,
epigenetics is a new challenging field that penetrates into the essence of this disease with promising results. Epigenetics,
as opposed to genetics that is concerned with gene mutations, investigates special events that are not directly connected togenes, but events that affect genes not changing their sequential structure. This study does not analyze histone acetylation(adding the acetyl group CH 3CO- to histones) and deacetylation, nor histone methylation (adding the methyl group -CH 3 tohistones) and demethylation; it is primarily concerned with DNA methylation as the crucial epigenetic event essential for theprocess of the transcription of genes and their adequate expression. Special emphasis is directed to the analysis of the lowdensity lipoprotein receptor-related protein (LRP1) receptor, the extremely important factor in the amyloid beta (Aβ) drainage
from the brain, and to the decline of the LRP1 expression on the membrane abluminal side of the blood brain barrier (BBB)
endothelial cells. This paper does not analyze oxidative and glycative damage of the LRP1 receptor, and it does not focus on
AD therapy; it is primarily concerned with epigenetics.

KEY WORDS: Alzheimer's disease (AD), LRP1 receptor, RAGE receptor, disorder of Aβ drainage from the brain,		epigenetics

Introduction

A short review on genes etiologically linked toAlzheimer's disease

Before analyzing the essential features of the DNA
methylation process, it is necessary to emphasize that theaim of the presented study is to explain the role of the lowdensity lipoprotein1 (LRP1, LRP1 gene location 12q13.3)
receptor in the Aβ peptide drainage from the brain, andits decline in Alzheimer's disease (AD). This study does
not analyze other mechanisms of the mentioned clearance
that primarily include effects of hydroxyl radicals (*OH),
belong to a great group of reactive oxygen species; ROS,
and oxidative and glycative stress; it is exclusively involvedin epigenetics, with reference to the DNA methylation. Itshould also be stressed that the study does not analyze AD
etiologically important genes (APP, amyloid precursor
gene, chr. 21.q21; PSEN1 gene, chr. 14q24.2; PSEN2
gene, chr.1q42.13; BACE1 gene, chr. 11q23.3; γ -secretase
complex-CTFβ chr3p22.1; PSEN1 gene chr, 14q24.2;
Pen2 gene chr.19q13.12; ApH1 gene chr. 1q21.2; ADAM10
gene, chr. 15q21.3; APOE gene, chr.19q13.32, ApoEε4
allele, the strongest genetic risk factor for LOAD); however,
it is necessary to mention them. The last two mentioned
genes are linked to LOAD ( late AD onset, 95 % of allcases), which develops after the age of 65, and the former
mentioned genes to EOAD (early AD onset, 5 % of allcases) which develop before the age of 65 1).

Correspondence to: Nikola Barić MD, PhD
Presika 153, 52220 Labin, Istria, Croatia
TEL : +385- 052-852- 008
e-mail: nikolabaric100@gmail.com

Glycative Stress Research 2023; 10 (2): 79-88(c) Society for Glycative Stress Research_ 79 _

DNA methylation and demethylation in Alzheimer's disease

Amyloid beta efflux and influx transport receptor
proteins critically involved in Aβ drainage fromthe brain

The authors of three research articles 2- 4) emphasize thegenerally accepted view that amyloid beta (Aβ) accumulation
and aggregation is the trigger for the development ofprogressive neurodegeneration and dementia associatedwith AD. They also point out that the sporadic late AD form
(LOAD, onset over 65 years, 95 % of all cases), is primarily
caused by the decreased Aβ clearance from the brain, andnot by its stronger production. In the mentioned drainage
process, the low-density lipoprotein receptor-related
protein 1 ( LRP1), the member of the large LDL receptor
group, has the crucial role. LRP1 is dominantly located on
the abluminal side of the BBB capillary endothelial cell
membrane, and by the endocytosis mechanism, it ejects Aβfrom the brain tissues into the drainage capillary blood. It
should be noted that in the other form of AD, onset (EOAD,
5 % of all cases) develops before the age of 65. These studies
continually point out the imbalance of Aβ homeostasis which
results in the pathological accumulation of cerebral Aβ, themain characteristic of AD. Special attention is given to theresults after measuring the mRNA efflux genes ( LRP1;
P-gp, P-glycoprotein, ABCB1 gene) and the influx gene
(RAGE, receptor for advanced glycation end products, genelocation 6p21.32), where in the efflux group, in the capillary
blood and in the endothelial cells, a significant decline ofmRNA LRP1 has been found, and in the influx group, anincrease of mRNA RAGE has been found. The first groupis accompanied by a significant gene transcription drop, andthe second group by its increase 2- 4).

Essential features of DNA methylation

Yang J et al. 5) emphasize that the DNA methylation
process occurs in all cells in the body and it is evolutionarily
conserved among insects and mammalian species. The
main characteristic of this process is the transfer of themethyl group (- CH 3) from the methyl donor, S-adenosylmethionine (SAM) onto the active site of the DNA cytosine
methyltransferase enzyme and C5 everted cytosine fromone chain of the corresponding DNA molecule. The
mentioned process can be seen during the DNA basecytosine methylation by the effect of the enzyme human
DNA (cytosine-5) methyltransferase M.Hhal (Dnmt M.Hhal).
Dnmt M.Hhal is a 5-methylcytosine methyltransferase(m5C-MTase) that forms part of a type II restrictionmodification system from Haemophilus haemolyticus. The
enzyme recognizes the specific tetranucleotide sequence,
5-GCGC-3. The methyltransferase binding to DNA begins
with three H-bonds breaking between cytosine (target base)and its pair guanine. Immediately after this event occurs thecytosine eversion, so that it projects out of the double helix,
then the nucleophilic attack (Nfa) of Cys81 nucleophile(from the Dnmt) on cytosine C6, and C6 attack (Nfa) on C5
with its activation and rise in nucleophilicity. The activated
C5 attacks (Nfa) the SAM methyl group and induces its
abstraction. At the same time, three bases (Glu119, Arg163,
Arg165) on the other Dnmt side, form H-bonds between
NH 2, N 3 and O 2. The nucleophilic attack of Cys81 resultsin the formation of the covalent adduct, Michael adducts(creation of carbon-carbon bond at the acceptor's β-Carbon).
Three amino acids on the other Dnmt side are essential for
maintaining the target cytosine in the flipped-out position.
Before the C5 nucleophilic attack on target - CH 3, C5
deprotonation occurs. The Β-elimination of the C5 proton
utilizes as base an OH- derived from conserved crystal waterthat is part of a proton wire water channel. The methylation
results in transcriptional blockade (epigenetic phenomenon)(Fig. 1).
In the following analysis of the DNA methylation
process, the investigation results and views of some eminent
researchers are presented.
Liu Y et al. 6) emphasize the importance of the ADassociated promoter DNA hypermethylation, which
establishes an epigenetic barrier for transcriptional
activation. They point out functional activities of selected
neurons, microglia, and astrocyte-enriched genes (AGAP2,
DUSP6 and GPR37L1), which are DNA hypermethylated
at promoters in AD. According to the authors, in mammals,
DNA methylation primarily occurs at the 5-carbon of thecytosine base in the form of 5-methylcytosine (5mC).
Cheng X et al. 7) describe the approach and binding ofDnmt to DNA. This induces the eversion (base flipping) of
the target nucleotide with its projection out of the double
helix and entrance into the Dnmt pocket. The catalytic
process induces the nucleophilic attack of Cys81 on cytosine
C6 with the formation of the covalent Michael adduct.
Cytosine C6 induces a nucleophilic attack on C5, which
momentarily induces the same attack on the methyl group of
S-adenosyl-L-methionine (AdoMet), which is absorbed andtransferred to C5.
Lin CC et al. 8) emphasize that DNA methyltransferases
are primary enzymes for cytosine methylation at CpG
sites of epigenetic gene regulation in mammals. DNMT3B
methyltransferase uses two flexible loops to enclose DNA
and employs its catalytic loop to evert the target nucleotide
to project out of the double helix. DNMT3B recognizes
DNA with CpGpG sites via residues Asn779 (asparagine,
it contains an α-amino acid which is in the protonated state-NH 3+ ) and Lys777 in its target recognition domain loop.
This facilitates processive methylation of CpG sites repeatedin tandem. The authors also emphasize the importance of the
proton wire water channel essential for the C5 deprotonation.
According to Xing P et al. 9), the promoter region of
LRP1 gene is enriched with CpG islands that govern thesensitivity of the LRP1 gene to DNA methylation. WhenCpG are completely methylated, the transcriptional activity
disappears completely and the expression of LRP1 issilenced. They think that the epigenetic mechanism might beinvolved as hypermethylation of the LRP1 gene promoter,especially as the promoter of this gene is rich in CpG
islands.
Tohgi H et al. 10) have investigated the methylation statusof cytosines in the promoter region of RAGE in the humancortex autopsy using the bisulfite method, polymerase chain
reaction (PCR) and the direct sequencing of PCR products.
They have found that the total number of methylcytosines in
the mentioned region is significantly reduced with age. This
reduction of methylcytosines at transcription factor bindingsites can increase the expression of RAGE.
Silverberg GD et al. 11) emphasize the decreased
methylation in the RAGE promoter region. During the_ 80 _Glycative Stress Research

Aging-related increase in SAM-synthetase

SAM, remarkably increase during aging
RNA-polymerase can not
approach the DNA
Xing P et al. (Ref 37)
emphasize that thepromotor region of LRP1
is enriched with CpG
islands that govern
sensitivity of the LRP1
gene to DNA
methylation.
Deoxyribose

One DNA strand

Gene promoter
CpG island

phosphate

Nucleotide

Flipping of cytosine
into a pocket of the
enzyme;Nucleophilic attack

Cytosine eversionof -CH3-CH32β-elimination of the C5 proton
utilizes as base an OH- derived
from a conserved crystal waterthat is part of a proton wire water
channelProton-abstracting base+2

Β-eliminationof the C5 proton

Arg163H-bondGlu119....... -2

Amino acids essential
for maintaining thetarget cytosine in the
flipped-out position

Proton donator to cytosine N3
Proton transfer
Active site
Pocket of the enzyme
Active site

Dnmt (M.Hhal)enzymeL-methionine

The methylation results intranscriptional blockade;2

H-bonds

base

Everted cytosine(reactant)
Nfa.Cytosine
cofactor
Two concerted processes(target)
AdoMet deprotonation
Donator of a pair of electron
SAM binding site SAM Nfa.
transfer
S+
S-adenosyl-L -methionine

Bulk water

H-bonds breaking

Arg165Nfa.

(AdoMet) methyl group

Guanine=Deoxyribose

Formationof covalent
Cys 81 adduct
Michael adduct

Adenosyl cation

phosphateUpregulation of the methylation of
DNA contributes to the pathogenesisof AD

CytosineDNA

Deprotonated Cys81 thiolate
Donator of a pair of electrons
Potent nucleophile
S-adenosyl-L -methionine(AdoMet) methyl group

Deoxyribose (sugar)

The level of SAM
remarkably increased in theaged tissues;
Hayashi Y et al. (Ref 13)

RNA polymerase can not approach to cytosine;(Epigenetic phenomenon)

Fig. 1. Schematic presentation of DNA methylation by human DNA cytosine methyltransferase M.Hhal (epigenetic
event).

The enzyme Dnmt M.Hhal catalyzes the transfer of a methyl group from S-adenosyl- L-methionine (AdoMet) to the C5 position of cytosine.
Between cytosine and guanine, there are three H-bonds, according to Yang J et al. (Ref 5) and Cheng X et al. (Ref 7), and the methyltransferase
binding to DNA begins with three H-bonds breaking between cytosine (target base) and its pair guanine, cytosine eversion ("base flipping"),
so that it projects out of the double helix, nucleophilic attack (Nfa) of Cys81 nucleophile on cytosine C6, and C6 attack on C5 with its
activation and rise in nucleophilicity. C5 attacks the SAM methyl group and induces its abstraction. Concurrently, three bases (Glu119, Arg163,
Arg165) on the other Dnmt side, form the H-bonds between NH2, N3 and O2. SAM remarkably increases in the brain tissues (cerebellum,
cerebrum, hippocampus) during aging. AD, Alzheimer's disease; Dnmt M.Hhal, DNA methyltransferase M.Hhal; Arg, arginine; Cys, cysteine;Glu, glutamine; SAM, S-adenosyl-L-methionine (AdoMet); Nfa, nucleophilic attack; LRP1, low density lipoprotein receptor-related protein;
CpG islands, the CpG sites are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases
along its 5' → 3' direction and CpG sites occur with high frequency in genomic regions called CpG islands.

_ 81 _

DNA methylation and demethylation in Alzheimer's disease

ageing process and in AD, there is an increased accumulationof AGEs, as well as increased values of Aβ, cytokines( interleukin 1, tumor necrosis factor), and of nuclear factor
κB (NFκB), of which, all can decrease the promoter
methylation and elevate the RAGE gene transcription.
It is evident that changes in LRP1 and RAGE genes
expression have a crucial role in the Aβ peptide drainage
drop from the brain and in the Aβ accumulation andaggregation in the brain tissues, which is the reason forbrain neurodegeneration and AD.

SAM cofactor biosynthesis

Following is the explanation of the origin of thisimportant cofactor, the methyl donor required for almost all
methyltransferase (MTase) activities. SAM originates by
the effect of evolutionally conserved enzyme S-adenosylmethionine-synthase (SAM-S) that links amino acidmethionine and adenosine triphosphate (ATP). Actually,
SAM is a complex compound of methionine and adenosyl
cation; the latter is the compound of ribose sugar and the
adenine purine base.
Experimenting on worms (Caenorhabditis elegans),a group of researchers have found that methionine dietary
restriction extends their longevity. The same experimentswith mammals accelerate their pathology, but accelerating
the SAM catabolism by glycine N-methyltransferase (Gnmt),
the researchers have suppressed the pathology and extended
their longevity. The downstream metabolites of SAM arepolyamines and cysteine. They are inductors of autophagy
and elevators of oxidative stress resistance (Fig. 2) 12).
Here, it is important to point out that the SAM and
Sam-S level rises in old-age tissues and in AD. Analyzing
age-related changes in SAM biosynthesis in brain tissues(cerebellum, cerebrum, hippocampus) of young and old mice,the authors of a study have found that the elevation of DNA
methylation and of histones accelerates the pathogenesis of
AD. They conclude that the increase in SAM and Sam-S
levels is an age-related phenomenon common across the
species and tissues. They emphasize that SAM is the
major cellular donor of the methyl group for methylation
modifications. However, the factors that induce the agingrelated increase in Sam-S levels are not known 13).

The process of DNA methylation in thecourse of ageing

Xiao FH et al. 14) emphasize that DNA methylation
represents a link between genetic and environmental signals
via the regulation of gene transcription. DNA methylation
presents globally the decreasing and site-specific increasing
in aging. It is the most common epigenetic modification andit plays the crucial role in many biological processes. It isevident that it is closely associated with aging, age-related
and longevity diseases.
In another study, Xiao FH et al. 15) present the dynamics
of DNA methylation during the human life cycle indetail. The dynamics shows a strong correlation of DNA
methylation events with old age and the consequences ofageing. The telomere shortening that occurs during cellular
replication has until recently been considered a goodpredictor for ageing and its consequences. However, exact

investigations have shown that the insufficient detectionof telomers and their weak correlation with age-related
outcomes is a limiting factor in their role in the prediction
system. The authors determine the chronological age
and predict biological age, especially emphasizing age
methylated changes in certain CpG loci. They point out that
DNA methylation based on epigenetic clocks serves as a
new standard to determine chronological age. The increase
in DNA methylation level, associated with age, on selected
CpG sites typically located near transcription start sites, hasgreat value in analyzing the character of DNA methylation.
In order to further analyze the DNA methylation
process, it is necessary to explain the meaning of the CpG
site and CpG island.
A promoter, as related to genomics, is a region of
DNA upstream of a gene where relevant proteins (such as
RNA polymerase and transcription factors) bind to initiate
the transcription of that gene. The resulting transcription
produces an RNA molecule (such as mRNA). The
transcription start site is the location where transcription
starts at the 5'-end of a gene sequence. Transcription factorsthat include a wide number of proteins, excluding RNA
polymerase, initiate and regulate the transcription of genes.
Previously in the text, it has been stressed that thepromoter region of LRP1 gene is enriched with CPG
islands that govern the sensitivity of the LRP1 gene to
DNA methylation. When CpG is completely methylated,
the transcriptional activity completely disappears and theexpression of LRP1 is silenced 9).
CpG islands (CGIs) are regions of the genome that
contain a large number of CpG dinucleotide repeats. In
mammalian genomes, CpG islands usually extend by 300 –3000 base pairs. They are located within and close to sites ofabout 40 % of mammalian gene promoters.
The CpG sites or CG sites are regions of DNA wherea cytosine nucleotide is followed by a guanine nucleotide in
the linear sequence of bases along its 5' → 3' direction. CpG
sites occur with high frequency in genomic regions called
CpG islands (or CG islands).
Slieker RC et al. 16) investigate the tissue-specificcharacter of age-related DNA methylation changes at thelevel of the CpG functional genomic region and the nearest
gene. They have found that the age-related DNA methylation
changes are highly tissue-specific. Linear regression analysis
between ELOVL2 CpG (cg16867657) with ELOVL2 gene
( Fatty acid elongase 2) and age (years) has shown a strong
statistically significant linear positive correlation.
Hernandez DG et al. 17) have examined DNA methylation
at more than 27,000 CpG sites throughout the human genome
in the brain structures from 387 human donors between theage of 1 and 102 years. They have identified CpG loci thatshow a highly significant positive correlation between DNA
methylation and chronological age. The major part of theseloci is within CpG islands, and there is a positive correlation
between age and DNA methylation level. They have also
discovered that the CpG sites, where the DNA methylationlevel is significantly positively associated with age, arelocated close to genes involved in the DNA binding and in
the regulation of transcription.
A growing number of recent studies have shown that
DNA methylation plays an important role during mammalianembryogenesis 18-21). Researchers in these investigations have

_ 82 _Glycative Stress Research!"#$%#!

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Fig. 2. Schematic presentation of SAM synthesis

SAM (AdoMet) acts as a methyl donor necessary for almost all methyltransferase activity. In humans, it is mainly produced
from L-methionine and ATP by methionine transadenylase in the liver. ATP, the source of energy for use and storage at thecellular level, is complex, composed from a nitrogenous base adenine, a sugar ribose, and three serially bounded phosphategroups. The three MATs (SAMs, AdoMet synthetases, and methionine adenosyltransferases) convert methionine into SAM.
During aging, the systemic SAM levels in the body are increased. SAM acts as a cofactor, a substance whose presence isessential for the activity of an enzyme. SAM consists of adenosyl cation attached to the sulfur (S) of methionine-the linkage
is a sulfonium functional group. MAT1A, MAT2A, and MAT2B are three genes which encode a catalytic regulatory subunit
used in formation of the MATs isozymes. MAT1A gene is expressed only in the adult liver. MAT2A and MAT2B are expressedin fetal liver and nonhepatic tissues. ATP, adenosine triphosphate; SAM, AdoMet, S-adenosyl-methionine; MAT, S-adenosylmethionine synthase.

_ 83 _

DNA methylation and demethylation in Alzheimer's disease

found modifications associated with childhood adversity andepigenetic modifications. They point out DNA methylation
modifications in genes that regulate the hypothalamus
pituitary adrenal axis as well as the immune system.
The analysis of the mentioned studies, especially of thepresented graphs showing the positive linear regression line
of DNA methylation and the age of the investigated subjects(Smith ZD et al. 18), Hahn MA et al. 19), Greenberg MVC et al. 20),
Hao G et al. 21), Hernandez DG et al. 17), Slieker RC et al. 16),shows the lifelong DNA methylation of a number of genes.
These authors point out changes in brain tissues in particular.
Considering the increase of the SAM and Sam-S levels related
to ageing, it is obvious that there is a program determinedby evolution that dictates the scope and speed of the DNA
methylation in the genome. The theory of programmed
ageing, practically applicable to all living beings, actually
determines the duration of certain life cycle phases with the
maximally possible lifetime limit of each species. All thesephases, as well as the maximally possible duration of life, arecrucial conditions for the species to survive in the conditionsof severe struggle for survival (Fig. 3). It seems that the
DNA methylation program represents the crucial link inthese events. At many CpG loci, there is a standard average
methylation level. However, CpG sites of LRP1 genes have amuch more sheer methylation regression line direction, whichindicates the accelerated rate of methylation, and increased
local ageing with decreased LRP1 gene transcription.
It has been mentioned that SAM occurs through thebiosynthesis of methionine and ATP. The biosynthesis occurs
by effect of the evolutionally conserved enzyme methionine
adenosyltransferase (Sam-S, MAT, SAM-synthetase). It has
been found that with ageing there is an aging-related increase
in SAM levels.
Hoffert KM et al. 22) present human genes which encode
methionine adenosyl transferases (MATs), also known as
AdoMet synthetases. Three genes, MAT1A (10q22.3), MAT2A(2p11.2), and MAT2B (5q34), each encode a catalytic or
regulatory subunit used in formation MATI (homotetramer),
MAT II (heterotrimer), and MATIII (homodimer) isozymes.
MAT1A is expressed only in the adult liver, while MAT2A
and MAT2B are expressed in the fetal liver and non-hepatic
tissues. The mechanism of their action is not yet wellunderstood. This action is connected with the upregulation or
downregulation of some cancers (Fig. 2).
Ciccarone F et al. 23) have found that the level of DNMT1(19p13.2) gradually dropped with aging, but this was only
observed up to the age of 64 years. The expression of
DNMT3B (20q11.21) decreased linearly with increasing age
and this association was particularly evident in females. The
authors emphasize that age affects the expression of both
mentioned DNA methyltransferases as an almost independent
variable in respect to all other variables evaluated.
It is necessary to further emphasize that with ageing,
and in old age, remarkably elevated levels of SAM and agingrelated increases in Sam-S are increasingly manifested. Is
it the question of the effect of certain transcription factorsthat intensify the transcription, or is it the question of theblockade of transcription blockers, or of the positive or
negative biofeedback mechanism? In these events, theprogrammed ageing theory probably has the crucial role.
DNA methylation is closely linked to this program. The
DNA methylation programmed levels during the individual

lifetime cycle are correlated with the tendency for chronic
diseases and longevity. The individual terminal old age andthe final inevitable death are the constituent parts of thelife cycle. DNA methylation is the crucial regulator of thisinevitability. The above-mentioned questions require special
attention 13, 15, 24-29).
Obviously, the LRP1 gene is subjected to all thementioned effects. He Y et al. 30), analyzing the critical side
effects of cisplatin, the cancer chemotherapeutic, have foundthat in the background of the mentioned events lies the strong
LRP1 gene promoter methylation, and the strong decline ofthis gene expression. If the LRP1 gene expression declines,
this also happens to the Sha1/PI3K/AKT signaling pathway
important for M2 microglia polarization. M2 microglia
phenotype has a strong protective function. Unfortunately, thenecessary further investigations of LRP1 gene methylationand transcription suppression are extremely complicated, sothat in the available literature there are few facts covering
this subject. However, the author of this study believes inthe validity of the thesis related to the great importance of
the LRP1 gene methylation in AD pathophysiology, and the
crucial impact of epigenetics in these events.
Following the profound analysis of the complex DNA
methylation process, it is necessary to elaborate on theessential characteristics of the active DNA demethylation
process that originates through the interplay of DNA
oxidative reactions and DNA repair mechanisms (Fig. 4 ).
In their mini-review, Bayraktar G et al. 31) analyze thecurrent knowledge on the regulatory mechanisms controllingthe active DNA demethylation. The first step of this process
is 5mC oxidation induced by the ten-eleven translocation(TET) family of dioxygenases with the generation of
5-hydroxymethylcytosine (5hmC). The second step involves
further hydroxylation of 5hmC by TET enzymes with thegeneration of 5-formylcytosine (5fC) and 5-carboxylcytosine(5caC). Thymine DNA glycosylase (TDG), after recognizing
DNA, induces the 5fC and 5caC excision of the glycosidic
bond, resulting in an apyrimidinic (AP) site. AP site can be
efficiently repaired by Base Excision Repair (BER) enzymes
with the generation of mark-free cytosine. At the end of
their report the authors emphasize that the contribution of
methylation and active DNA demethylation in AD remains to
be determined.
The analysis of the presented graphs in the studies by
Slieker RC et al. 16) and Hernandez DG et al. 17) shows thatthe methylation level of a series of CpG sites related to oldage, practically starts to grow from the moment of birth (ongraphs, separately marked for different brain regions). Guo H
et al. 32) emphasize that during the embryonal period (period
from fertilization until the end of 8th week) and the fetal
period (from 9th/10th week until birth) of life, there has
been an intensive demethylation process of the methylated
paternal and maternal genes, and at the same time the major
wave of genome-wide demethylation is completed at the
2-cell stage (zygotic stage). Demethylation of the paternal
genome is much faster than of the maternal genome. The
inverse correlation between promoter methylation and gene
expression gradually strengthens during early embryonicdevelopment reaching its peak at the post-implantation stage.
They conclude that the DNA methylation and demethylation
are the crucial elements in the epigenetic regulation of
mammalian embryonic development.

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Fig. 3. Schematic presentation of age-related methylation changes in brain regions.

The presented graph clearly shows the relation of the level of DNA methylation in some brain regions (y axis) and age(x axis). This hypothetical relation, according to Slieker RC et al. (Ref 16) and Hernandez DG et al. (Ref 17), presents
the methylation of individual CpG loci on the DNA of several genes. Here, the presented graph does not represent thereal situation, its purpose is only educational. It is important to emphasize that on Figure 2 in Ref 17, the authors havemeasured the DNA methylation level at 10 CpG sites. For example, the code for one of these sites is cg06993413. On theirgraph, the CpG sites in the frontal cortex are green, and on the temporal cortex red. For all 10 sites the methylation levels
increase significantly with age in all investigated brain regions (four). On this graph all circles present the same CpG
site, only its location is different (different colors). CpG sites, regions of DNA where a cytosine nucleotide is followed bya guanine nucleotide in the linear sequence of bases along its 5' → 3' direction; LRP1, low density lipoprotein receptorrelated protein; RAGE, receptor for advanced glycation endproducts.

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DNA methylation and demethylation in Alzheimer's disease!

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"'%!' #%! +'#&"Fig. 4. Schematic presentation of active DNA demethylation processes.

The figure from Bayraktar G et al. (Ref 31) shows the two active DNA demethylation pathways. Both pathways involve a number ofenzymes and compounds. In the first step, 5mC is oxidized by the TET family to produce 5hmC. In the other pathway, TDG induces
glycosidic bond cleavage of 5fC and 5caC to generate AP sites, which are repaired by BER to produce cytosine. The active DNA
demethylation in AD yet remains to be determined. 5mC, 5-methylcyutosine; 5hmC, 5-hydroxymethylcytosine; 5caC, 5-carboxylcytosine;
TET, ten-eleven translocation; TDG, thymine DNA glycosylase; BER, base excision repair enzymes; AP, apyrimidinic; CpG, where acytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' → 3' direction; AID/APOBEC, activityinduced cytidinedeaminase/apolipoprotein B mRNA editing complex deaminase.

Gkountela S et al. 33), using laboratory mice in theirinvestigations, answer the question of the purposefulness ofthe global DNA demethylation in the preimplantation phaseof the mammal embryonal phase, including humans. The
mentioned purposefulness lies in the strong prevention of theinherited transmission of abnormal cytosine methylations
from parents to children. DNA demethylation in the preimplant phase of the embryonal development removes thecytosine methylation acquired in the parents' gametes before
fertilization. Their findings give the explanation of theregression lines in diagrams by Slieker RC et al. 16) and
Hernandez DG et al. 17).
Bayraktar G et al. 31) and Kang J et al. 34) intensively
investigated the process of active DNA demethylation.
They are concerned with this problem in the early phase of
mammal embryogenesis, including in humans. Their special
interest is directed to the function of the dioxygenases

TET family. Three mammalian TET proteins, TET1
(gene,10q21.3), TET2 (gene4q24) i TET3 (gene2p13)
successively oxidize 5mC to 5hmC. The embryos without
Tet1 i Tet3 show a heavy loss of 5-hydroxymethylcytosine(5hmC), and an increase of 5-methylcytosine (5mC) ateight cell stages. TET proteins maintain the consistency ofgene transcription. According to Bernstein C et al. 35), theactive demethylation reactions depend on the family of
5-methylcytosine oxidases (TET) and on DNA base excision
repair enzymes (BER). They have found that in mice,
within 6 hours of fertilization, the paternal chromosomes
are close to 100% actively demethylated through TET3 andrepair activity. Methyl groups on maternal DNA passively
become highly diluted over the next four days. Qi Q et al. 36)
also emphasizes the great importance of dioxygenases
as inductors of DNA demethylation during mammaliandevelopment (Fig. 4 ).

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Conclusion
The analysis of the presented studies undoubtedly shows
the crucial role of DNA methylation and demethylationin human embryogenesis. Early zygote demethylation
significantly removes possible harmful inherited genomes.
Consequently, the transfer of damaging epigenetic featuresfrom parents to children is prevented, and there is strong
support for the optimal embryonal and fetal development. All
this also has a strong effect on the overall further lifetimeof the individual. The optimization of epigenetic effects on
receptor LRP1 genes and RAGE most probably slows down
the AD pathophysiology.

Conflicts of Interest
The authors declare that they have no conflict of interest.

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