antioxidants

Review
Role of Oxidative Damage in Alzheimer’s Disease and
Neurodegeneration: From Pathogenic Mechanisms to
Biomarker Discovery
Francesca Romana Buccellato 1, *, Marianna D’Anca 2 , Chiara Fenoglio 3 , Elio Scarpini 1,2 and
Daniela Galimberti 1,2

1 Department of Biomedical, Surgical and Dental Sciences, University of Milan, 20122 Milan, Italy;
[email protected] (E.S.); [email protected] (D.G.)
2 Fondazione IRCSS ca’ Granda, Ospedale Policlinico, 20122 Milano, Italy; [email protected]
3 Department of Pathophysiology and Transplantation, University of Milan, 20122 Milan, Italy;
[email protected]
* Correspondence: [email protected]; Tel.: +39-02 55033814

Abstract: Alzheimer’s disease (AD) is a neurodegenerative disorder accounting for over 50% of
all dementia patients and representing a leading cause of death worldwide for the global ageing
population. The lack of effective treatments for overt AD urges the discovery of biomarkers for
early diagnosis, i.e., in subjects with mild cognitive impairment (MCI) or prodromal AD. The brain



is exposed to oxidative stress as levels of reactive oxygen species (ROS) are increased, whereas


cellular antioxidant defenses are decreased. Increased ROS levels can damage cellular structures
Citation: Buccellato, F.R.; D’Anca, or molecules, leading to protein, lipid, DNA, or RNA oxidation. Oxidative damage is involved in
M.; Fenoglio, C.; Scarpini, E.; the molecular mechanisms which link the accumulation of amyloid-β and neurofibrillary tangles,
Galimberti, D. Role of Oxidative containing hyperphosphorylated tau, to microglia response. In this scenario, microglia are thought
Damage in Alzheimer’s Disease and to play a crucial role not only in the early events of AD pathogenesis but also in the progression of
Neurodegeneration: From Pathogenic
the disease. This review will focus on oxidative damage products as possible peripheral biomarkers
Mechanisms to Biomarker Discovery.
in AD and in the preclinical phases of the disease. Particular attention will be paid to biological
Antioxidants 2021, 10, 1353.
fluids such as blood, CSF, urine, and saliva, and potential future use of molecules contained in
https://doi.org/10.3390/antiox1009
such body fluids for early differential diagnosis and monitoring the disease course. We will also
1353
review the role of oxidative damage and microglia in the pathogenesis of AD and, more broadly, in
Academic Editors: Stanley Omaye neurodegeneration.
and Eugenio Barone
Keywords: Alzheimer’s disease; oxidative damage; neurodegeneration; biomarker
Received: 16 July 2021
Accepted: 17 August 2021
Published: 26 August 2021
1. Introduction
Publisher’s Note: MDPI stays neutral The longer life expectancy will lead to an increase in ageing-related neurodegenerative
with regard to jurisdictional claims in disease in the next future [1,2]. A rising challenge in the ageing population is the treatment
published maps and institutional affil-
and management of dementia patients, as public health care worldwide will face an increase
iations.
in cases and the global cost of dementia patient treatment [3,4].
The most common cause of dementia is Alzheimer’s disease (AD), a progressive
neurodegenerative disorder, affecting over 50 million people worldwide [4]. The disease is
irreversible and presents with neurodegeneration caused by toxic aggregation of extracel-
Copyright: © 2021 by the authors. lular amyloid plaques, and intracellular neurofibrillary tangles of hyperphosphorylated
Licensee MDPI, Basel, Switzerland. tau protein [5]. Individuals with AD show memory loss, intellectual disabilities, changes
This article is an open access article in personality, and behavior. At present, acetylcholinesterase (AChE) inhibitors donepezil,
distributed under the terms and
galantamine, rivastigmine, and the N-Methyl-D-aspartate (NMDA) antagonist memantine
conditions of the Creative Commons
are the only symptomatic medications approved by American and European regulatory
Attribution (CC BY) license (https://
agencies to treat AD [6]. In June 2021, the FDA gave a conditional approval of the first
creativecommons.org/licenses/by/
disease-modifying drug for the treatment of AD, aducanumab, a monoclonal antibody
4.0/).

Antioxidants 2021, 10, 1353. https://doi.org/10.3390/antiox10091353 https://www.mdpi.com/journal/antioxidants

Antioxidants 2021, 10, 1353 2 of 22

designed against the amyloid build-up, even if its efficacy has to be confirmed in large
phase III–IV studies. In the hope that this treatment will be effective, the search for new
potential treatments has never stopped and there is increasing interest in healthy food
habits and alternative treatments which could prevent the cognitive decline or alleviate the
memory loss in AD.
Although the aetiology of AD is still debated, the most studied pathogenic mechanism
has been the “amyloid cascade hypothesis” proposed in 1991 by Hardy and Allsop, who
suggested that inappropriate processing of amyloid-β (Aβ) precursor leads to the build-up
of amyloid plaques, the formation of tau tangles, and ultimately to neuronal death [7].
About 2–3% of AD cases are due to autosomal dominant mutations. The rest are
sporadic cases and are prevalent in the aging population [8]. Independently of genetic
aspects, it is well known that AD pathogenesis starts decades before the symptoms appear.
Following the new research framework (2018-NIA-AA-RF), AD is not defined by its clinical
consequences but by its underlying pathology measured by lifetime biomarkers. Regardless
of the presence of clinical symptoms, both Aβ and phosphorylated tau pathology are
required for classification as AD, whereas Aβ deposition alone is an early sign, labelling
the pathologic change towards AD [9]. In 2021, new recommendations for the clinical
diagnosis of AD were proposed by the International working group, highlighting that the
diagnosis of AD should be restricted to people who have positive biomarkers together
with specific AD phenotypes. Cognitively unimpaired individuals who are only positive
to biomarkers should be considered at-risk for progression to AD [10].
There are different pathogenic hypotheses to understanding AD aetiology and the
early stages of AD [11]. The study of AD pathogenesis is significant not only for discovering
new therapeutic targets and set up new clinical trials, but also to highlight potential
biomarkers for the diagnosis of early stages of AD, in individuals with Mild Cognitive
Impairment (MCI), or prodromal AD.
In AD, reactive microglia and astrocytes are associated with the amyloid plaques
and respond to Aβ with the expression of pro-inflammatory cytokines [12–14]. Activated
microglia are also observed close to tau protein in neurofibrillary tangles [12]. Further,
activated microglia can respond to injured neurons in a neuroprotective manner and,
afterwards, become toxic, contributing to neuroinflammation and neurodegeneration [15].
Microglia can respond to Aβ, or to other danger-associated molecules released by damaged
cells, by producing reactive oxygen species (ROS) [16]. Thus, the overproduction of ROS
and a decrease in antioxidant defenses can lead to oxidative stress (OS) [17]. The role of
microglia in contributing to OS induced by ROS production and some of the molecular
mechanisms implicated in ROS production will be analysed in this review.
Past and recent studies have demonstrated that oxidative damage is not only an early
event in the pathogenesis of AD, occurring before the onset of clinical symptoms but also a
factor contributing to the disease progression [17–21]. The role of oxidative damage in the
pathogenesis of AD and more broadly in neurodegeneration will be discussed.
Oxidative damage products, as the ones deriving from oxidation of lipid, DNA, RNA,
or glycation ends products, are extensively studied, and are suggested as biomarkers
in neurodegeneration and AD [22–24]. They are detected in body fluids such as blood,
plasma, serum, cerebrospinal fluid (CSF), urine, and saliva [25–27]. The availability of
such molecules in peripheral circulation and their use as potential biomarkers of AD is of
paramount importance, overtaking the invasiveness and cost of the usual diagnostic and
imaging tools. See Figure 1 for summary of the interplay between ROS, oxidative damage
products, and cellular components in AD.
 In addition, we will analyze the potential use of this body of knowledge to identify
sensitive and reliable biomarkers in AD and in the early stages of the disease as MCI and
prodromal AD. Recent data from metabolomic and proteomic studies can help to identify
the potential biomarker [22,27]. The ideal biomarker should permit a diagnosis before the
Antioxidants 2021, 10, 1353
clinical onset of the disease and correlate with neuropathological findings and/or the 3 of 22

progression of the disease.

Figure 1. The neurodegeneration

Figure 1. The neurodegeneration occurring in AD occurring in AD can
can be related be related
to ROS to ROS overproduction
overproduction and/or a decrease and/or a
in antioxidant
decrease in antioxidant defenses. Aβ deposition can also be directly responsible for ROS increase
defenses. Aβ deposition can also be directly responsible for ROS increase when it interacts with microglial surface receptors
when
or mitochondria. it interacts with
Mitochondria microglial
are the primary surface
source ofreceptors
ROS butor mitochondria.
also Mitochondria
a target of ROS are in
and alteration theenergy
primary
metabolism
source of ROS but also a target of ROS and alteration in energy metabolism have been described in
have been described in AD patients. ROS may also be considered a second messenger and induce different signaling
AD patients. ROS may also be considered a second messenger and induce different signaling
pathways or the synthesis of antioxidant enzymes (Nrf2). Oxidative damage to mitochondrial DNA and RNA, proteins,
pathways or the synthesis of antioxidant enzymes (Nrf2). Oxidative damage to mitochondrial
and lipids areDNA
detected in ADproteins,
and RNA, brain. Increased
and lipidslevels of iron in
are detected andAD other metals
brain. can be
Increased responsible
levels of iron andof ROS
otherproduction
and iron has been ++ influx alterations and the consequent mitochondrial dysfunction
metals can be responsible of ROS production and iron has been related to ferroptosis. Ca influx are also
related to ferroptosis. Ca ++

crucial in oxidative damage.

alterations andAβ thecan trigger NO
consequent production, an
mitochondrial RNS. Oxidative
dysfunction damage
are also crucialaffects synapses,
in oxidative causing
damage. Aβcognitive
impairment incanAD.trigger NO production,
Decreased an RNS.
levels of different Oxidative
lipids damage
have been affectswith
correlated synapses, causing
cognitive cognitive
deficit in AD, suggesting their
impairment
use as biomarkers in AD. clinical
or in potential Decreased levels of different lipids have been correlated with cognitive deficit
intervention.
in AD, suggesting their use as biomarkers or in potential clinical intervention.
In this review, we will discuss the current knowledge on oxidative damage products.
2. Products of
In Oxidative
addition, weDamage in ADthe potential use of this body of knowledge to identify sensitive
will analyze
and reliable
OS is defined biomarkers
by the disruptionin AD andequilibrium
of the in the early stages
between of the
ROSdisease
and/oras reactive
MCI and prodromal
AD. Recent
nitrogen species (RNS) data from
levels metabolomic
and and
antioxidants proteomic
defenses studies
[18]. can help
Increased to identify
levels of ROSthe potential
biomarker [22,27]. The ideal biomarker should permit a diagnosis
and/or RNS together with decreased levels of antioxidants can lead to oxidative damage before the clinical onset
of the disease and correlate with neuropathological findings and/or the
to cells, molecules, and biological systems [18]. In the brains of individuals with AD and progression of the
disease.
preclinical AD (i.e., evidence of Aβ and tau deposition in the brain without any clinical
manifestation of the disease), as well as patients with MCI (a mild clinical impairment
2. Products of Oxidative Damage in AD
with no proof of underlying pathology with biomarkers), many oxidative damage
OS is defined
products are measured, by the disruption
and numerous of the
investigations equilibrium
have pointed tobetween
OS as aROSkey and/or
and reactive
nitrogen species (RNS) levels and antioxidants defenses
early player in the pathogenesis of neurodegeneration and AD [18,19]. [18]. Increased levels of ROS
and/or RNS together with decreased levels of antioxidants can lead to
The brain is very susceptible to OS because it is rich in polyunsaturated fatty acids,oxidative damage
to cells,consumption
has a high oxygen molecules, and
andbiological systems
a high content [18]. Inin
of metals the brains of iron,
particular, individuals
that canwith AD and
preclinical AD (i.e., evidence of Aβ and tau deposition in the brain without any clinical
catalyze oxidative reactions, in contrast, it has less antioxidant enzymes in comparison
manifestation of the disease), as well as patients with MCI (a mild clinical impairment with
with other organs [28]. The result of the reaction of ROS with different substrates can be
no proof of underlying pathology with biomarkers), many oxidative damage products are
protein, nucleic acid oxidation or lipid peroxidation. All of these markers of OS have been
measured, and numerous investigations have pointed to OS as a key and early player in
described as an early alteration in the AD brain, and this concept has been used in the past
the pathogenesis of neurodegeneration and AD [18,19].
to support the ‘oxidative stress hypothesis’ in the pathogenesis of AD [20,28–30]. The
The brain is very susceptible to OS because it is rich in polyunsaturated fatty acids,
has a high oxygen consumption and a high content of metals in particular, iron, that can
catalyze oxidative reactions, in contrast, it has less antioxidant enzymes in comparison
with other organs [28]. The result of the reaction of ROS with different substrates can
be protein, nucleic acid oxidation or lipid peroxidation. All of these markers of OS have
been described as an early alteration in the AD brain, and this concept has been used in
the past to support the ‘oxidative stress hypothesis’ in the pathogenesis of AD [20,28–30].
The oxidative stress hypothesis can be applied to other neurodegenerative diseases, such as
Parkinson’s Disease (PD) and Amyotrophic Lateral Sclerosis (ALS) where the accumulation
Antioxidants 2021, 10, 1353 4 of 22

of oxidative damage over time leads to the late life onset and to the progressive nature of
these diseases [29]. It has been hypothesized that tissue injury, aging, ischemia, increased
metal levels or energy metabolism deficit can induce ROS generation independently from
Aβ deposition [29]. Even if ROS generation is secondary to other initiating causes, it plays
an important role in the events that lead to neuron death in AD [29].
3-Nitrotyrosine is a product of oxidative damage on proteins generated when a
superoxide radical reacts with nitric oxide (NO) to form peroxynitrite, which cause nitration
of proteins on tyrosine residues. 3-nitrotyrosine and protein carbonyls, these last produced
mainly by the action of free radicals on the peptide chain or by oxidation of amino acids,
are used as biomarkers of oxidative damage in the brain of AD and MCI [18,24,31–33]. NO,
also classified as an RNS, is synthesized from L-arginine by three different isoforms of
the enzyme NO synthase (NOS), i.e., endothelial (eNOS), neuronal (nNOS), and inducible
(iNOS) which catalyze a two-step oxidation of L-arginine to NO and L-citrulline [34,35].
NO is a gaseous molecule, with a lone pair of electrons which can easily diffuse across
membranes acting as a vasodilator, neuromodulator, and inflammatory mediator [34].
At high concentration, NO reacts with superoxide radical and generates peroxynitrite,
contributing to OS. iNOS is implicated in the pathophysiology of neurodegenerative
diseases including AD, as Aβ can trigger NO production with an unknown mechanism,
which leads to increased levels of free radicals, mitochondrial damage, and consequently
neuronal function alterations [34]. Due to its gaseous nature and its short half-life, it is
difficult to measure NO as a marker of OS, but its metabolites in the arginine/NO pathway
such as L-arginine, asymmetric and symmetric dimethylarginine, and dimethylamine have
been studied in metabolomic analysis. Untargeted and targeted metabolomics studies have
indicated that L-arginine/nitric oxide (NO) pathway is altered in AD [35,36].
Membrane phospholipids contain arachidonic and docosahexaenoic (DHA) acids,
which are abundant in the brain and vulnerable to ROS attack. Decreased levels of phos-
phatidylcholine, phosphatidylethanolamine and the phospholipid precursors, choline and
ethanolamine, have been described in AD and are considered specific for the pathogenic
mechanism of AD. The depletion of phospholipids in AD has been referred to an increase
of ROS-mediated lipid peroxidation [29]. 4-hydroxy-2-trans-nonenal (HNE) is produced in
the brain via lipid peroxidation of arachidonic acid present in neuronal membranes [37].
HNE could be considered an apoptotic inducer because of its ability to spread OS and form
protein adducts involved in neurodegenerative diseases [38]. This highly reactive aldehyde
and also hexenal and 2-propene-1-al (acrolein) can react with specific amino acid residues
on proteins altering the conformation and functions of such proteins. Increased levels of
HNE are observed in AD and MCI brains [24,38]. Neurofilaments are a primary target of
HNE as HNE-adducts to neurofilaments have been found [38]. Further, HNE have been
detected in Aβ plaques and in the cerebrospinal fluid in AD patients [39,40]. By altering
membranes, HNE could impair ionic and energetic metabolism and cause neuronal cell
death [37].
Redox proteomic studies confirm previous results that carbonylated proteins are
present in CSF of amnestic MCI patients compared with controls, and these proteins remain
oxidized in the progression towards AD [40]. F2-, F3- F4-isoprostanes are prostaglandin
isomers also produced by ROS attack on arachidonic acid in membrane phospholipids.
Analysis of specific isoprostane may reflect increased OS in AD [41].
Advanced glycation end products (AGE) are irreversible products of the glycosylation
of lysine or arginine residues of proteins and are considered biomarkers of oxidative
damage. The impact of AGE adducts on various aging related diseases including AD, the
role of increased oxidative stress in promoting the production of AGE-related adducts
and the consequences in aging-related diseases are well documented in the review of
Rungratanawanich et al. (2021) [42]. In the brain, OS can cause the production of AGEs
and OS can also be generated due to AGEs formation in this organ. This vicious cycle may
increase oxidative damage, leading to the initiation and progression of neurodegenerative
diseases. AGEs and the receptor for advanced glycation end-products (RAGE) can play
Antioxidants 2021, 10, 1353 5 of 22

an important role in AD pathogenesis as higher levels of RAGE expression in neuronal,

microglial, and endothelial cells in patients with AD compared with age-matched, non-
demented controls are found [43–46]. The levels of expression of RAGE are correlated to
the severity of the disease indicated by clinical score of the amyloid plaque or tangle [45,47].
Therefore, one of the most abundant AGE-protein products in the brain is the AGE-albumin
adduct, which causes RAGE overexpression in primary neurons from human AD brains.
OS and Aβ aggregation increase the formation of AGE-albumin adduct [48]. Aβ can
also bind RAGEs to activate the NF-κB, and other signal transducers and activators of
transcription pathways, leading to neuronal cell death and neurodegeneration [49].
Oxidation of DNA and RNA produced by ROS also occurs in AD [50–53]. 8-Hydroxy-
deoxyguanosine (8-OHdG) is the product of oxidative DNA damage while 8-oxo-guanine
(8-oxoGua), and 8-oxo-guanosine (8-oxoGuo) are markers of oxidative RNA damage. Both
these oxidative damage products are the most commonly measured biomarkers of OS.
Oxidative RNA damage can affect not only mRNAs but also non-coding RNA species [54].
Dysregulation of microRNAs (miRNAs) has been implicated in neurodegenerative disor-
ders such as AD and this matter is reviewed by Nunomura and Perry (2020). Further, an
in vitro study has demonstrated that miRNAs can be directly oxidized and can misrecog-
nize mRNAs that are not their original targets [55]. OS can also affect the expression of
multiple miRNAs, which regulate genes involved in the OS response in neurodegeneration
in AD, PD, ALS, and Huntington’s disease (HD) [56,57].
The brain iron is stored within the ferritin protein complex, preventing iron from
converting hydrogen peroxide into a highly toxic hydroxyl free radical. Through the
production of these ROS, iron can induce severe brain damage [28,58]. Iron and ferritin are
described to be associated with amyloid deposition and microglia [59,60]. Their levels are
elevated in neuronal tissue and in the amyloid plaques [61]. Alongside the capacity of iron
to induce OS alone catalyzing the formation of ROS, iron can bind histidine residues of Aβ
and in this way trigger the redox activity of the iron-Aβ aggregate [62].

3. Mitochondria as Sources of ROS and Target of Oxidative Damage

In the AD brain, mitochondria can be sources of ROS and also the target of OS, es-
pecially mitochondrial DNA (mtDNA) is susceptible to oxidative damage [19,53,63]. It is
demonstrated that there is a progressive age-related accumulation of oxidative damage
to DNA in the human brain and that mtDNA is preferentially affected. The increased
amount of 8-OHdG in mtDNA and nuclear DNA is demonstrated in the aging human
brain and in AD [50,51,64]. Hirai et al. have demonstrated that oxidative damage marked
by 8-OHdG and nitrotyrosine occurs in the neurons of AD patients. Morphometric anal-
ysis showed a reduction of mitochondria in these same neurons and an accumulation
of mtDNA and mitochondrial proteins [53]. These abnormalities are found in neurons
lacking neurofibrillary tangles, suggesting that mitochondrial abnormalities are one of
the earliest pathological changes in AD. These results confirmed that mtDNA is a target
of oxidative damage and that the increased damage to DNA contributes to AD and the
neurodegenerative process [51,53].
The studies on oxidative-associated damage on mitochondria in AD have sustained the
“mitochondrial cascade hypothesis” of AD pathogenesis, alongside the classical amyloid
cascade hypothesis [65]. In this explanation, the mitochondrial function declines with age
and leads to increased production of ROS [65,66]. Besides, amyloid oligomers can access
mitochondria, contributing to compromise their function and triggering a vicious circle
in which it is not defined if the accumulation of Aβ came before mitochondrial declined
function or after [67].
Dysregulation of Ca2+ homeostasis has also been reported as cause of mitochondrial-
induced production of ROS in AD [68,69]. Aβ can be toxic on neuronal mitochondria
and causes Ca2+ elevation, changes in mitochondrial size and shape in APP/PS1 AD
mouse model [70]. Excessive Ca2+ accumulation into mitochondria leads to decreased
mitochondrial membrane potential and increased ROS production in the same AD mouse
Antioxidants 2021, 10, 1353 6 of 22

model [67,68,70]. In this way the toxic action exerted by Aβ causes mitochondrial frag-
mentation that has been reported to precede neuronal cell death [71,72]. The maintenance
of a healthy mitochondrial pool is essential for neuronal fitness, indeed dysfunctional
mitochondria are degraded through selective autophagy, a mechanism called mitophagy.
Dysregulated mitophagy has also been implicated in neurodegenerative diseases, as PD
and AD, as well as aging [73]. Excessive calcium influx induced by overstimulation of the
NMDA receptor is the mechanism implicated in the neuroprotective effect of Memantine.
Memantine is a drug, approved in US and in EU, for the treatment of the progressive
symptomatic decline in patients with moderate to severe AD [74]. The drug exerts its
neuroprotective effect blocking NMDA Receptor (NMDAR). Aβ oligomers can target and
activate NMDAR leading to a rapid increase in neuronal calcium levels and ROS produc-
tion in mature hippocampal neurons in culture [75]. During pathological activation of the
NMDAR, memantine blocks excessive calcium entry through the channel and Aβ-induced
OS, providing a specific biological mechanism for the therapeutic action of memantine.
Recently, sirtuin 3 (SIRT3) defects have been considered to contribute to OS in AD
mitochondria. Indeed, SIRT3 has been linked to dysregulation of mitochondrial DNA
expression, ROS accumulation and neuronal damage in AD [76,77]. The importance of
SIRT3 is highlighted in this report as SIRT3 has been found exclusively in mitochondria
and its function is to eliminate reactive oxygen species and inhibit apoptosis [76,77].
Glucose and energy metabolism can be affected by the oxidative damage to the
mitochondria. Glucose metabolism is dysregulated in AD and MCI and the metabolic
rate of glucose is decreased in the brain of patients with senile dementia [18]. De Leon
in a longitudinal FDG-PET study prognosticated the future cognitive decline and MCI
among normal elderly individuals using decreased glucose metabolism as a predictor of
the clinical change [78]. Using redox proteomics to analyze the protein profile in brain
tissue samples of patients with AD, Di Domenico and Butterfield suggest that in affected
brain areas, oxidative modification of the glycolytic enzyme aldolase, triosephosphate
isomerase, glyceraldehyde- 3-phosphate dehydrogenase, phosphoglycerate mutase 1, and
α-enolase occurs. In addition, oxidative modifications to enzymes involved in TCA cycle,
ATP production in brain mitochondria are described in the brains of individuals with MCI
and AD [24]. Large scale analysis of proteomic in AD brains and CSF demonstrated that
activation of microglia and astrocytes are associated with energy metabolism [27]. This acti-
vation may be neuroprotective and anti-inflammatory in preventing the progression to AD.
Increased levels of glucose, carbohydrate, and protein metabolism in CSF of patients with
AD were also observed [27]. Taken together, these results suggest the link between energy
metabolism and OS in AD. Glucose metabolism, ATP production, and OS are deeply linked
in the energy metabolism of the cell, while mitochondria are the cellular organelles deputed
to control the energy metabolism. Deficiency in energy metabolism deriving from one or
more levels can represent a key role in the pathogenesis of AD.

4. Neurodegeneration and OS
AD neuropathology is characterized, macroscopically, by cerebral cortical thinning
and atrophy where synapse and neuronal loss contribute to the atrophy. From a microscopic
point of view, the hallmarks of the pathology of AD are neuritic amyloid plaques and
neurofibrillary tangles. The alternative “Aβ oligomer hypothesis”, supported by in vitro,
in vivo, and ex vivo models, points to the toxic Aβ oligomers (AβO)s rather than amyloid
plaques as key players in AD pathogenesis [79–81]. (AβO)s are considered the most toxic
and pathogenic form of Aβ [79,82]. In this hypothesis, after cleavage from the membrane,
Aβ peptides aggregate to form AβOs. Part of the oligomers which are not going to form
fibrils may induce the neuron damage responsible for cognitive decline and dementia.
The collective body of evidence, reviewed by Cline et al., supports a pathogenic mechanism
in which AD neuropathology and cognitive loss are the consequences of the AβOs toxicity
on neurons. Blocking AβOs or AβO receptor has been considered a disease-modifying
treatment in AD. Indeed, Aducanumab has been designed to target AβOs and fibrils to
Antioxidants 2021, 10, 1353 7 of 22

slow the cognitive decline. Additionally, RAGE has been identified as an AβO targeted
receptor and Azeliragon, an inhibitor of RAGE, is in phase 3 clinical trial as the therapy
shows significant delay in AD cognitive decline. The mechanism of toxicity of AβOs can
be exerted by binding specific receptors and activate receptor transduction mechanism, by
a direct interaction with different cells as neurons, microglia, astrocytes, or organelles such
as mitochondria [83,84]. As discussed before, mitochondria respond to the toxic action of
AβOs producing ROS, which can mediate apoptosis of the neuronal cells [69,82].
Cognitive deficits in MCI patients and decreased executive and reasoning abilities
in AD are caused by the oxidative damage of small Aβ oligomers to the synaptic mem-
branes [83–85]. In vivo and in vitro studies support a direct relationship between OS and
synaptic dysfunction in AD [18,86–90].
The Aβ oligomer hypothesis sustains another emerging knowledge: the cellular and
molecular basis for neuroanatomic selectivity seen in the AD, and the neuron to neuron
spread of the toxic Aβ protein [79,91,92]. Indeed, the progression of AD pathology corre-
lates to the diffusion of amyloid deposition to specific neurological structures, following
this regional and temporal pattern is possible to identify specific stages in the progression
of the disease. The staging of the progression was postulated by Braak [93]. In this re-
gard, it is possible to hypothesize regional and temporal differences between the different
neuronal population responses to OS. In this view, entorhinal cortex (EC) neurons are
particularly prone to damage by OS and mitochondria dysfunction [94,95]. The EC is a
vital component of the medial temporal lobe, contributing, during pathological condition,
to downstream changes in its afferent region, the hippocampus. Neurodegeneration in the
EC and hippocampus has been clearly linked to impairments in memory and cognitive
function in the early phases of AD [91,96]. Oxidative damage to RNA in EC, hippocampus,
subiculum, and temporal neocortex of subjects with AD has been described. The levels
of 8-OHdG increase early before pathological changes occur, and this can explain that the
selective vulnerability of neurons in the EC during AD may be related to the vulnerability
of these particular neurons to oxidative damage [19].
Iron levels in the hippocampus of AD patients, are significantly elevated [97]. Abnor-
malities in iron homeostasis in brain tissue can increase ROS production, causing noxious
oxidative damage to sensitive cellular structures, and trigger a new cell death process called
ferroptosis [98,99]. There is still not clear evidence to relate ferroptosis to neurodegenera-
tion occurring in AD, but several studies have considered targeting iron metabolism, using
iron chelators, as a potential drug in the treatment of AD [100] (Deferiprone clinical trial,
3D Study NCT03234686). Ferroptosis-dependent cytotoxicity induced by the activation of
OS has also been described in human astrocytes from AD patients [101].

5. Microglia and Astrocytes as Sources of ROS in AD

Colocalization of reactive microglia with Aβ deposits is a hallmark of AD pathology
and has been reported also in brains from AD mouse models [102,103]. Microglia express
pattern recognition receptors (PRRs), innate immune cell receptors that respond to danger-
(DAMP) or pathogen-associated molecular patterns [102,103]. The nucleotide-binding-like
receptor (NLR) belongs to PRRs and can trigger the formation of multiprotein complexes,
called inflammasome [104]. NLRP3 inflammasome is expressed exclusively by microglia in
the CNS and may detect noxious agents or disturbances in the cellular environment [104].
Various studies have described a role of NLRP3 signaling in different neurologic disorders,
including multiple sclerosis (MS), ALS, prion diseases, and AD [105,106]. In AD, Aβ can act
as DAMP and its interaction with microglia creates an oxidative and neuroinflammatory
environment through an increased production of ROS and release of proinflammatory
cytokines [107]. Further, NLRP3 activity is associated with impaired clearance of Aβ by
microglia [104].
NADPH oxidase (NOX) belongs to a group of ROS generating enzymes that can
be activated by different stimuli in response to neuronal injury, inflammation, or OS.
NOX activation is evident in the AD brain, in addition NOX2 is highly expressed in
Antioxidants 2021, 10, 1353 8 of 22

microglia [108,109]. In this scenario, activated microglia might contribute, through the
activation of NOX2, to trigger or maintain the OS in AD. NO X4 is the astrocytes isoform,
and its levels are reported significantly elevated in astrocytes of patients with AD and
APP/PS1 double-transgenic mouse model of AD [101]. The levels of two products of
oxidative damage, 4-HNE and MDA, were also significantly elevated, suggesting that the
elevation of NOX4 promotes the impairment of mitochondrial metabolism, mitochondrial
ROS production and fragmentation in human astrocytes [101]. NOX4-mediated ferroptosis
is also described in human astrocytes in this experimental setting [101].
ROS produced by activated microglia can also act as a second messenger, inducing
different signaling pathways. Zhang and colleagues (2016) describe several cellular sig-
naling pathways that ROS can induce [110]. In particular, the transcription factor nuclear
factor-erythroid 2 p45-related factor 2 (Nrf2) is involved in the cellular redox metabolism,
protecting cells against OS by binding to antioxidant response elements (ARE) in the pro-
moters of antioxidant genes. The trigger of the Keap1-Nrf2-ARE signaling pathway is
responsible for inducing a protective mechanism against OS in many diseases including
AD and PD [111,112]. The increased level of intracellular ROS promotes the dissociation of
Nrf2 and Keap1. Then, dissociated Nrf2 is transferred to the nucleus where it targets genes
encoding for detoxification enzymes such as glutathione synthetase (GSS), glutathione
reductase (GR), thioredoxin (TRX), thioredoxin reductase (TRR), and peroxiredoxin (PRX)
to prevent the OS [110]. Downregulation of Nrf2-target genes have been reported in AD,
and other neurodegenerative conditions [112]. Nrf-2 activation can occur not only in
response to OS but can be induced by plant extracts or synthetic compounds with antioxi-
dant properties [113]. The Nrf2 activating compound dimethyl fumarate is an approved
FDA-therapy for MS and clinical trial is underway for its use in Friedrich’s ataxia. Another
Nrf2 activating compound is Centella asiatica extracts, which can activate Nrf2 in neurob-
lastoma cells, isolated primary neurons and in the brains of AD and aging mouse models.
This activation improved mitochondrial and cognitive function, and enhanced synaptic
density [113–115].
Proliferation and activation of microglia in the brain, around amyloid plaques, is a
prominent characteristic of AD [102]. In the attempt of digesting Aβ, microglia can release
not only ROS but also pro-inflammatory cytokines or chemokines to recruit other cells.
Anti-inflammatory cytokines, then, modulate phagocytosis to limit the process [116]. In
this way, activated microglia can contribute to neuroinflammation and neuronal cell death.
Indeed, chemokines and cytokines increased levels in plasma and CSF are a very early
event in AD pathogenesis, even preceding the clinical onset of the disease, as demonstrated
by subjects with MCI who developed AD over time, representing a crucial step in the
progression of the disease [117–119].
Recent findings highlight the potential involvement of the triggering receptor ex-
pressed on myeloid cells 2 (TREM2) in AD pathology, neurodegeneration, and neuroin-
flammation. In the brain, TREM2 is exclusively expressed by microglia and its function
is to maintain microglial health during stress events and to enable the progression of mi-
croglia towards the profile of disease-associated microglia (DAM) [120]. TREM2 facilitate
microglial phagocytosis and clearance of Aβ and apoptotic neurons [121]. The mutation
in TREM2, has been associated with a three-fold higher risk to develop AD [122]. Re-
garding the relation of TREM2 with OS, it has been supposed that a down-regulation
of microglial TREM2 expression and signaling might be one of the major pathogenetic
mechanisms in sporadic cases of AD in which advanced age, OS, neuroinflammation
contribute to the suppression of wild-type TREM2, considering that TREM2 mutation is
rare in humans [123].

6. Focus on Experimental Models of AD and Potential Antioxidant Therapeutic

Targets
The pioneering studies, conducted to validate the early changes of products of oxida-
tive damage, found in specimens of AD patients, in a transgenic mouse model (Tg2576) of
AD amyloidosis, dated back more than 20 years [20,124]. In the last 20 years several other
Antioxidants 2021, 10, 1353 9 of 22

experimental models are designed to reproduce the hallmarks of the pathology of AD and
to study products of oxidative damage in animal models.
Some of the molecular mechanisms of the pathogenesis we have revised in the present
review are studied in these models. To counterbalance the increase of OS that is char-
acteristic in age-related neurodegenerative diseases antioxidant treatment are studied in
experimental models and in human trials.
While some of the studies of early biomarkers focused on wide-proteomic analysis
to find a reliable and specific marker of OS in human fluids, the molecular mechanisms
to justify the involvement of mitochondria, microglia and the use of antioxidant pathway
as therapeutic target in AD are better analyzed in animal experimental models or in-vitro
models. In the recent literature we can find significant trends in the new approaches
to study the role of oxidative damage in AD. Indirect proofs of the role of oxidative
damage derive from the studies of biochemical/molecular mechanisms, which minimize
the accumulation of such deleterious products in experimental models of AD.
To maintain genome integrity, the misincorporation of oxidized nucleotides is pre-
vented by various repair enzymes, such as human MutT homolog 1 (MTH1). MTH1
hydrolyzes 8-oxo-dGTP to its corresponding monophosphates, 8-oxo-dGMP, avoiding
8-oxo-dG incorporation into DNA. Meanwhile, 8-OxoG DNA glycosylase-1 (OGG1) excises
8-oxoG paired with cytosine in DNA, minimizing 8-oxoG accumulation in DNA [125].
Oka et al., studied the role of 8-oxoG accumulation in the pathogenesis of AD, utilizing
a knockout of Mth1 and Ogg1 genes in a 3xTg-AD background. They demonstrated that
MTH1 and OGG1 deficiency increased 8-oxoG accumulation in nuclear and mitochondrial
genomes, causing microglial activation and neuronal loss with impaired cognitive function
at 4–5 months of age. The use of minocycline, an inhibitor of microglial activation and neu-
roinflammation, in this model decreased the nuclear accumulation of 8-oxoG in microglial
cells, and inhibited microgliosis and neuronal loss. Further, gene expression profiling
revealed that MTH1 and OGG1 efficiently suppress progression of AD by inducing various
protective genes in 3xTg-AD brain. These results highlighted that the potential suppression
of 8-oxoG accumulation in brain genomes could be a new approach for the prevention and
treatment of AD [125].
On the same tune, the report of Pao et al. 2021 shows that histone deacetylase (HDAC)1
modulates OGG1-initated 8-oxoguanine (8-oxoG) repair in the brain [126]. HDACs en-
zymes remove acetyl groups from lysine residues of histones and non-histone proteins
modulating transcription, chromatin remodeling, and DNA repair. Deregulation of HDAC1
induces alteration in cell cycle activity and DNA damage leading to neuronal death in
cultured neurons and mouse brain [127]. Further, sirtuin (SIRT) 1 can also bind the class I
histone deacetylase HDAC1, deacetylate HDAC1 and stimulate its enzymatic activity [128].
HDAC1-deficient mice show DNA damage accumulation and cognitive impairment dur-
ing aging [126]. Further, this work showed elevated 8-oxoG along with reduced HDAC1
activity and downregulation of a gene set critical for brain function in the 5XFAD mouse
model of AD, uncovering important roles for HDAC1 in 8-oxoG repair and highlights the
therapeutic potential of HDAC1 activation to counteract the functional decline in brain
aging and neurodegeneration [126].
AD pathogenesis has been linked to mitochondria as their health is essential in main-
taining energy homeostasis and in preventing neuronal dysfunction. Stojakovic et al.
observed that mitochondrial respiratory chain complex I might be a therapeutic target in
AD [129]. Chronic treatment with complex I inhibitor, the tricyclic pyrone (CP2), which
can penetrate the blood brain barrier (BBB) and accumulate in mitochondria, improves
cognitive and motor function in transgenic mice, expressing a form of the amyloid pre-
cursor protein (APP) and presenilin 1 that leads to early onset AD (APP/PS1). Further,
this treatment can reduce total Aβ levels triggering autophagy, one of the neuroprotective
pathways essential for Aβ clearance. Taken together, the data suggest that CP2 treatment
induces multiple protective mechanisms including autophagy, anti-inflammatory, and
antioxidant responses, which contribute to reduce Aβ pathology [129].
Antioxidants 2021, 10, 1353 10 of 22

Mitochondrial Tu translation elongation factor (TUFM or EF-Tu) can maintain mi-

tochondrial respiratory chain activity and protect cell against OS. TUFM protein level
is decreased in the hippocampus and cortex in the aged APP/PS1 mice [130]. Further,
cellular ROS levels were affected by TUFM expression, and TUFM-mediated regulation
of apoptosis and Tau phosphorylation is counteracted by the treatment with TEMPO, a
potent antioxidant drug. Collectively, TUFM protein levels were decreased in APP/PS1
mice. Taken together these results pointed at TUFM involvement in AD pathology by
regulating BACE1 translation, apoptosis, and Tau phosphorylation, in which ROS plays an
important role [130].
Regarding the role of mitochondria and Aβ oligomers in AD pathology, Takeda and
collaborators have studied the role of mitochondrial ubiquitin ligase, as MITOL is an
integral mitochondrial outer membrane protein which regulates mitochondrial functions
and morphology [131]. It is supposed to be downregulated in patients with AD [131,132].
MITOL-deleted APP/PS1 mice show severe cognitive impairment, synapse alteration,
and neuroinflammation mediated by excessive generation of toxic and dispersible Aβ
oligomers. It is possible to affirm that MITOL deletion triggers mitochondrial impairments
and exacerbates cognitive decline in APP/PS1 mouse model with AD [131].
Glutathione (GSH) is a cellular antioxidant, and its depletion has been observed in
AD [133]. The effect of dietary supplementation with γ-glutamylcysteine (γ-GC), the
immediate precursor of GSH biosynthesis has been studied in the brains of APP/PS1
mice [134]. The authors report that the diet with γ-GC lowered the levels of brain lipid
peroxidation, protein carbonyls and apoptosis, and maintain antioxidant status in APP/PS1
mice. Aβ pathology was reduced, and AChE activity was improved in APP/S1 mice on
the γ-GC diet compared to APP/PS1 mice fed a standard chow diet. γ-GC may also lower
inflammation and enhance Aβ plaque clearance in vivo as suggested by cytokines and
matrix metalloproteinase levels in the brain. Supplementation with γ-GC improve learning
and memory in this AD animal model as determined by their performance in Morris water
maze [134].
The intranasal administration of a nano-formulation of graphene oxide loaded with
dauricine has been studied in a mouse model of AD for its capability of protecting against
oxidative damage induced by Aβ. This report highlights also the potential of non-invasive
drug delivery system based on nanoparticles that can pass the BBB and reach the brain [135].

7. Products of Oxidative Damage in Blood, CSF and Other Body Fluids of AD Patients
The amyloid/tau/neurodegeneration (AT(N)) classification is used to divide biomark-
ers into those measuring β-amyloid (Aβ) deposition (A) (CSF Aβ levels or Aβ-positron
emission tomography (PET)), pathologic phosphorylated tau (T) (CSF phospho-tau (p-tau)
levels or tau-PET), and neurodegeneration (N) (18F-fluorodeoxyglucose-PET (FDG-PET),
magnetic resonance imaging (MRI), or CSF total tau (t- tau) levels) [10,136]. These biomark-
ers are required for the biological diagnosis of AD. Blood-based biomarkers are not avail-
able for AD and the potential ones are derived from Aβ and Tau [137]. A non-invasive,
peripheral approach of screening would be useful and the potential use of products of
oxidative damage as markers in the early stages of the disease has been extensively studied.
Although the brain is the most affected in AD the search for markers of OS on peripheral
fluids has been always active (Table 1).
Few studies have analyzed the potential of urine, the most peripheral and available
human fluid as a source of biomarkers in AD. The largest metabolic analysis performed on
urine of AD and MCI patients have permitted to shape a urinary metabolic phenotyping.
Urine contains metabolites that reflect a response to injury, including OS, occurring at
high distance or carry information originated from the gut microbiome, a novel area of
research in neurodegeneration and AD [138]. The strength of this report, even if not
specific on oxidative markers, lies in the potential to shape a metabolic profile associated
with AD and to correlate such metabolites to genetic variants [138]. Peña-Bautista and
colleagues have recently developed some analytical methods to determine a panel of lipid
Antioxidants 2021, 10, 1353 11 of 22

peroxidation biomarkers in urine, plasma, and saliva samples [25,138–144]. Saliva is also
considered a promising source of biomarkers which measure products of oxidative damage.
These potential neurodegenerative biomarkers could represent a promising screening test
for minimally invasive AD diagnosis and for monitoring the progression of the diseases.
A very recent study of this group has evaluated lipid peroxidation compounds in preclinical
AD patients and healthy elderly individuals [142].
Recently, the plasma lipidome signature has been proposed as a potential biomarker
of AD. Liu and colleagues (2021) have examined the differences in plasma lipidome
between AD patients and healthy age-matched controls and compared the lipid profiles to
identify specific alterations in lipid metabolism [145]. Decreased levels of ethanolamine
plasmalogens (PlsEtns) have been found to correlate with the severity of AD. However, the
potential use of signature species of PlsEtns as biomarkers in AD as well as their potential
in therapy have yet to be explored [146].
OS and inflammation can perturbate the high-density lipoprotein (HDL) proteome,
possibly playing a role in AD pathogenesis. Loss of HDL-associated proteins with an-
tioxidant action such apolipoprotein phospholipase A2, glutathione peroxidase-3, and
paraoxonase (PON)-1 and -3 are supposed to be related to the early stage of AD. PON-3
modulating factors should be considered of interest in the study of potential AD treat-
ments [147]. Additionally, Apolipoprotein A-I levels were decreased in serum of AD
patients [148]
Salivary levels of AGE have been described to correlate with the deterioration in mini
mental state examination (MMSE) score and have been suggested as potentially useful in
the diagnosis of dementia [26].
AD can modify a number of metabolites which, even if not considered a product
of oxidative damage, can be related to OS pathways. Serum metabolome analysis can
dissect the metabolic heterogeneity in AD pathology and can associate specific metabolic
alterations to AD phenotypes, permitting a more precise and personalized medicine.
Regarding this experimental approach, several studies have been done. Arnold et al. (2020)
highlighted the sex-specific dysregulations of energy metabolism, energy homeostasis,
and (metabolic/nutrient) stress response, presenting the first evidence and molecular
readouts for sex-related metabolic differences in AD [149]. The metabolic alterations
described in this work suggest that females experience greater impairment of mitochondrial
energy production than males [149]. A metabolomic study using high-resolution mass
spectrometry with liquid chromatography conducted on CSF from normal controls and
MCI revealed a metabolic signature characterized by impairments in sugars, methionine,
and tyrosine regulation in MCI [150]. Blood metabolic signature revealed decreased levels
of specific amino acids and lipids levels in AD, and three markers of oxidative potentially
predictive in demarcating AD patients from control subjects: the isoprostane-pathway
derivatives 8,12-iPF-2a IV, and PGD2, and the nitro-fatty acid NO2-aLA (C18:3) [22].
As previously discussed, NO is an RNS, and is implicated in OS in various neurode-
generative diseases. Untargeted and targeted metabolomics studies have indicated that
L-arginine/NO pathway is altered in AD [35,36]. Further, using targeted metabolomics,
Fleszar and colleagues have assessed L-arginine, L-citrulline, dimethylamine, asymmetric
dimethyl arginine, and symmetric dimethylarginine in blood samples from AD, mixed-type
dementia, and non-demented individuals and they demonstrated that L-Arginine/NO
pathway is altered in neurodegenerative and vascular dementia in association with neu-
rodegenerative and vascular markers of brain damage and severity of cognitive impair-
ment [35].
Protein profiling approach is considered a promising strategy to assess pathological
changes in both asymptomatic AD and later stages of the disease [151]. In this approach,
proteomic data collected from postmortem brain tissue of control, asymptomatic AD,
and AD cases have been organized in groups or modules with biological significance in
underlying the disease and correlated with clinical features of AD [147]. Alterations in
these disease-associated modules are strongly conserved across AD cohorts [151]. The
Antioxidants 2021, 10, 1353 12 of 22

largest proteomic study allowed to identify a protein network module linked to sugar
metabolism as one of the modules most significantly associated with AD pathology and
cognitive impairment [27]. Proteins from this module were elevated in CSF in early stages
of the disease [27]. In this case, no specific protein has been linked to OS, but a group
of proteins involved in one of the molecular mechanisms were potentially dysregulated
during OS in AD. Obviously, the potential availability of blood-based biomarkers would
overtake the invasiveness and cost of the usual diagnostic tools. Recently, a large-scale
proteomic profile of human serum specimens has revealed consistent mitochondrial protein
changes between control and AD samples [152]. Several redox markers were found altered
in whole blood cells from AD patients, some of them (GR, GSH, GSSG, and GSSG/GSH)
are already altered in MCI patients, suggesting their potential use as markers of prodromal
AD. Further, they are associated with MMSE scores and seem to be useful to monitor
cognitive decline in AD progression [153].

Table 1. Summary of recent studies on OS-related biomarkers for diagnosis and progression evaluation in AD.

Body Fluid Biomarkers Disease Stage Method Reference

AGE AD, VD, MD Fluorescence [26]
Saliva AOPP, 8-isop Colorimetric [26]
Lipid peroxidation compounds AD UPLC-MS/MS [140]
AGE AD, VD, MD Fluorescence [26]
AOPP, 8-isop, 8-OH-dG Colorimetric [26]
Plasma
Lipid peroxidation compounds MCI [139]
Isoprostanes AD/AD progression UPLC-MS/MS [141,143]
Lipid peroxidation compounds AD UPLC-MS/MS [25]
8-OHdG/2-dG MCI/AD UPLC-MS/MS [144]
Urine
3-NO2 -Tyr/p-Tyr [144]
m-Tyr/Phe, o-Tyr/Phe [144]
Energy metabolism AD Metabolomic [149]
Serum
Mitochondrial function AD Proteomic [152]
Energy metabolism AD Proteomic [27]
CSF
Sugars, Met/Tyr regulation MCI Metabolomic [150]
Blood Isoprostanes, PDG2 AD Metabolomic [22]
Fatty acid NO2-aLA (C18:3) [22]
L-arginine/NO pathway AD, VD, MD Metabolomic [35]
Blood cells GR, GSH, GSSG, GSSG/GSH AD/MCI Fluorometric assay [153]
Frontal cortex 8-oxo-dG, 4-HNE AD Immunofluorescence [154]
Abbreviations: 3-NO2 -Tyr/p-Tyr, 3-nitrotyrosine; 4-HNE, 4-hydroxy-2-trans-nonenal 8-isop, 8-isoprostanes; 8-OH-dG, 8-hydroxy-2-
deoxyguanosine; AD, Alzheimer’s diseases; AGE, advanced glycation end products; AOPP, advanced 8-isoprostanes oxidation protein
products; MD, mixed dementia; m-Tyr/Phe, meta-tyrosine; o-Tyr/Phe, orto-tyrosine; PGD2, prostaglandin2; VD, vascular dementia;
UPLC/MS, liquid chromatography coupled to tandem mass spectrometry.

8. Antioxidants and Other OS Counteracting Drugs as Potential Therapy in AD

According to the definition, not only an increase in ROS levels but also a decrease in
antioxidants levels can lead to OS. In response to OS in the brain, a number of protective
mechanisms are up regulated to maintain the oxidative balance, in particular compensatory
induction of antioxidant enzymes such as superoxide dismutase, glutathione reductase,
catalase have been found in vulnerable neurons in AD [31]. Total antioxidant capacity
(TAC) (including glutathione, ascorbic acid, uric acid, and bilirubin) is measured in plasma
samples from AD and MCI patients and has been found depleted [117]. In addition, several
nonenzymatic antioxidants such as vitamin C, vitamin A, vitamin E, and carotenoids were
lower in the plasma of patients with AD compared with controls and are associated to
increased levels of oxidative damage markers [155]. Reduction of antioxidants such as
glutathione, glutathione peroxidase, glutathione-S-transferase, and superoxide dismutase
Antioxidants 2021, 10, 1353 13 of 22

are demonstrated post-mortem in the mitochondrial and synaptosomal fractions in the

frontal cortex of MCI and AD patients [31].
Oxidative damage and antioxidant response were assessed in neurons and astrocytes
of post-mortem frontal cortices of AD and non-demented individuals with Alzheimer’s
Neuropathology (NDAN). NDAN patients display amyloid and tau pathology, but they
resist to dementia [154]. The levels of 8-oxo-dG and 4-HNE in relation to the accumula-
tion of neurotoxic Aβ peptide were increased in cortices of AD patients in comparison
to NDAN. Further, NDAN patients show cell-specific mechanisms to counteract redox
imbalance [154]. These individuals display low oxidative damage, associated with high
levels of antioxidant response (SOD2, PGC1α, PPARα, CAT). PGC1α expression, as a
key regulator of antioxidant response, was lower in AD and similar to control levels in
NDAN individuals, while expression of miRNA-485, a PGC1α upstream regulator, was
significantly increased in AD cortex as compared to the control and NDAN subjects [154].
Activation of PGC1α-mediated antioxidant pathway may prevent OS in AD [154].
Antioxidant dietary supplements are seen as a strategy to improve well-being and
prevent disease. Synaptic dysfunction and synapse loss are key hallmarks of AD, present
in the early stages of the disease before clinical symptoms appear [79,86,156]. Synaptic
dysfunction as a therapeutic target has been studied in animal models and in clinical
trials [157–159]. To this aim, dietary supplements containing antioxidants and phospho-
lipids are designed to improve the synaptic and subsequently the cognitive function [160].
Dietary enrichment of nutrients that are precursors of synaptic membrane phospholipids
can enhance phosphatide synthesis, the number of dendritic spines and the levels of pre-
or post-synaptic proteins, which are all needed for the formation of new synapse. Pre-
clinical studies showed that administration of specific combinations of the phospholipid
synthesis-promoting nutrients can enhance neurotransmission and cognitive function [159].
This background plus the notion that circulating levels of these nutrients are lower
in AD compared with healthy controls led to one of the first multicenter clinical trial to
test the hypothesis that a diet supplement containing antioxidants and a combination of
phospholipid synthesis-promoting nutrients could improve the synaptic function and the
cognitive symptoms [160]. Souvenaid, a medical food which contains Fortasin Connect,
was developed and administered to mild AD patients [160]. The study showed prelimi-
nary suggestion of continued improvement in memory performance. Recently, Tadokoro
et al. introduced a novel antioxidative supplement, Twendee X (TwX). The multicenter
interventional study conducted on MCI patients indicates that the antioxidant supplement
TwX is beneficial for cognitive function in MCI [161]. Controlling NO overproduction
with phytochemicals and nutraceuticals, and selective iNOS inhibition have been stud-
ied as potential disease-modifying-treatments in PD, AD, MS, and other inflammatory
disorders [34,37,162].
Considering that a potential drug treatment for AD can counteract the biological
mechanism that trigger OS, we have analyzed the current knowledge on clinical trials
on AD treatment. Cummings and colleagues have reviewed www.clinicaltrials.gov (ac-
cessed on 11 August 2021) and described the pipeline of drugs in development for AD
for the sixth year in a row [163]. The potential drugs have been classified based on the
phase of the clinical trial, common Alzheimer’s and related dementias research ontology
(CADRO) targets and mechanism of action of the drug. Looking at the clinical trials
that have reached phase 3, beside the very recently approved Aducanumab, there is a
progressive interest in the non-amyloid targets [163]. Indeed, some of the treatments are de-
signed to modify the biology of the disease acting on synaptic dysfunction, mitochondrial
dysfunction, microglial modulation, and reducing OS [163]. Of the 18 agents in phase 3
classified as disease-modifying treatments, three have OS as mechanism of action, two have
metabolism/bioenergetic, two have antioxidant properties, and among the three agents
acting on synaptic plasticity/neuroprotection, Azeliragon is also meant to ameliorate OS
(Table 2). Interestingly, Blarcamesine or ANAVEX2-73 (NTC03790709) is a chemical com-
pound known as an agonist of the sigma 1 receptor [164]. The therapeutic purpose of the
Antioxidants 2021, 10, 1353 14 of 22

treatment with this compound is its potential to correct a number of metabolic and synaptic
abnormalities, as this receptor has been shown to restore cellular homeostasis and synaptic
plasticity when activated by targeting mitochondrial dysfunction and associated OS, pro-
tein misfolding, proteostasis, and associated autophagy, and neuroinflammation [164]. The
multinational trial using biomarker and precision medicine to monitor patients with mild
AD or MCI is still ongoing. The same compound is also in development for Parkinson’s
disease dementia.

Table 2. Summary table of phase 3 clinical trials with agents related to OS mechanism of action or antioxidant properties in
patients with AD.

Agent Mechanism of Action N. ClinicalTrial.gov Estimated End Date

Azeliragon RAGE antagonist NCT03980730 July 2023
Sigma 1 receptor antagonist, ameliorate OS
Blarcamesine (ANAVEX2-73) NCT03790709 December 2021
and mitochondrial disfunction
Ginko biloba Plant extracts with antioxidants properties NCT03090516 March 2020
Icosapent ethyl Decrease OS, improve synaptic function NCT02719327 November 2021
Metformin Improve CNS glucose metabolism NCT04098666 April 2024
Omega 3 (DHA + EPA) Antioxidant, ameliorate OS NCT03691519 December 2023
Energy metabolism
Tricaprilin Improve mitochondrial and neuronal NCT04187547 February 2023
function inducing ketosis

Another molecule in Phase 3 is Azeliragon (NTC03980730), an inhibitor of RAGE

that can be administered orally [163]. RAGE binds AGEs, products of the glycosylation
of proteins. AGEs are adducts produced and slowly accumulated in the body during
aging and under OS. Hyperglicemic conditions as the ones observed in diabetes can also
cause the nonenzymatic glycation of proteins and the formation of AGEs. As previously
discussed in this review, AGEs through the binding to the receptor, can produce ROS and
be involved in neurodegeneration [42]. RAGE can also bind amyloid and is upregulated in
astrocytes and microglia of AD patients [47]. The clinical trial will end in 2023.
Icosapent ethyl (NCT02719327) is a purified form of the omega-3 fatty acid, eicos-
apentaenoid acid, administered as a dietary supplement. It is an approved drug for the
treatment of severe hypertriglyceridemia, in the current trial it is supposed to counteract
OS, reducing inflammation and improving synaptic function. The same agent omega 3
(NCT03691519) is currently in another phase 3 trial, classified in OS for CADRO mechanism
and for its antioxidant action [163]. DHA exerts its indirect antioxidant action modulat-
ing thioredoxin and glutathione pathways, and DHA supplementation is beneficial in
improving cognitive function in mild AD [165]. However, omega 3 fatty acids are prone to
peroxidation and an altered balance between cholesterol and oxysterols can reverse the
beneficial effects. Thus, diet supplements with appropriate omega 3/omega 6 and low
content of cholesterol can be considered a strategy in AD prevention [166].
Energy metabolism is also implicated in AD pathogenesis, and its dysregulation can
be linked to OS, as discussed previously. Regarding the role of glucose metabolism in AD
and the association of Type 2 Diabetes with AD, the medicament metformin (NTC04098666)
is in phase 3 [163]. Metformin is an insulin sensitizing medicine, a first-line treatment and
a widely prescribed oral treatment for type 2 diabetes. Glucose metabolism amelioration in
CNS is supposed to improve the cognitive decline of aMCI patients or prevent the cognitive
decline in obese people [163,167]. The trial will end in 2024. Tricaprilin (NCT04187547) is
an oral formulation of the caprylic triglyceride, designed to improve mitochondrial and
neuronal function by inducing mild ketosis.
Plants extracts with antioxidant properties as Ginkgo Biloba extracts are also extensively
studied as dietary supplements for AD patients. Ginkgo Biloba (NCT03090516) extracts are
supposed to improve mitochondrial function and brain blood flow, acting as a cognitive
enhancer [163]. As reported, the trial is in phase 3. Regardless, discordant results, from
Antioxidants 2021, 10, 1353 15 of 22

testing this extract on the cognitive function of AD patients and healthy people, were found
and reviewed by Liu et al., 2020 [168].

9. Conclusions and Future Perspectives

The study of accumulation of the products of oxidative damage in the brain and
specimens has surely shed light on the early events of AD pathology and has contributed
to the knowledge of AD pathogenesis. Aβ oligomers can induce oxidative damage to
membranes of neuron synapses, mitochondria, and other brain cells, such as astrocytes
and microglia. On the other hand, OS is a state which can be determined by aging and
other comorbidities, representing a risk factor for AD in health people or a progression
factor for the MCI patients. In this case, a systemic OS environment could be one of the
components leading to neuronal damage in AD the brain.
Despite the failure of studies on oxidative damage products in AD to reveal, till now,
one or more reliable marker, oxidative damage can be localized to mitochondria in AD
brain and ROS may act as second messenger, initiating a downstream signaling pathway to
face up the alteration in the redox balance [169]. Besides, oxidative damage may be linked
to the genetic risk of AD, and in this view more relevant in patients carrying the APOE ε4
allele [169].
At the time being, metabolomic and proteomic blood-signature seems to be the more
efficient approach to provide a complete view of the OS-mediated pathological changes
caused by AD and together with the clinical biomarkers and neuropathology the more
appropriate guide towards a personalized medicine in AD patients.
Regarding a potential use of metabolites related to oxidative damage as biomarkers,
it has to be acknowledged that it is unlikely to find a peripheral specific marker, as many
pathways related to oxidative damage occur not only in AD but in other neurodegenerative
conditions. Nevertheless, the identification of a biomarker with a high negative predictive
value may be useful in the context of primary care setting, i.e., a screening tool not to be
intended as a diagnostic but as a gatekeeper to further confirm the diagnostic procedures
(CSF biomarkers, PET with amyloid tracer) [170].
Concerning the treatment of patients with antioxidative compounds, clearly any
supplementation can be considered curative, but, in the context of a multifactorial disease,
it may help to reduce or limit pathogenic events eventually leading to neurodegeneration.
Furthermore, the progressive interest in non-amyloid treatments could be an alternative
potential approach to diseases-modifying treatments and the possibility that non-invasive
and new drug delivery system could pass the BBB a future perspective in AD study.

Author Contributions: F.R.B.; M.D.; C.F.; writing—original draft preparation, F.R.B., D.G.; E.S.;
writing—review and editing, M.D.; figure design and preparation, D.G.; E.S.; supervision. All authors
have read and agreed to the published version of the manuscript.
Funding: The APC was funded by Italian Ministry of Health (Ricerca Corrente). This work was
supported by grants from Fondazione Gigi and Pupa Ferrari Onlus. M.D. is supported by the Italian
Ministry of Health, grant RF-2018-12365333.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Hou, Y.; Dan, X.; Babbar, M.; Wei, Y.; Hasselbalch, S.G.; Croteau, D.L.; Bohr, V.A. Ageing as a risk factor for neurodegenerative
disease. Nat. Rev. Neurol. 2019, 15, 565–581. [CrossRef]
2. Partridge, L.; Deelen, J.; Slagboom, P.E. Facing up to the global challenges of ageing. Nature 2018, 561, 45–56. [CrossRef]
3. Wimo, A.; Jönsson, L.; Bond, J.; Prince, M.; Winblad, B. Alzheimer Disease International The worldwide economic impact of
dementia 2010. Alzheimer’s Dement. 2013, 9, 1–11. [CrossRef] [PubMed]
4. Prince, M.; Ali, G.C.; Guerchet, M.; Prina, A.M.; Albanese, E.; Wu, Y.T. Recent global trends in the prevalence and incidence of
dementia, and survival with dementia. Alzheimer’s Res. Ther. 2016, 8, 23. [CrossRef] [PubMed]
5. Stelzmann, R.A.; Norman Schnitzlein, H.; Reed Murtagh, F. An english translation of alzheimer’s 1907 paper, “über eine
eigenartige erkankung der hirnrinde”. Clin. Anat. 1995, 8, 429–431. [CrossRef] [PubMed]
Antioxidants 2021, 10, 1353 16 of 22

6. Yiannopoulou, K.G.; Papageorgiou, S.G. Current and Future Treatments in Alzheimer Disease: An Update. J. Central Nerv. Syst.
Dis. 2020, 12, 1–12. [CrossRef]
7. Hardy, J.; Allsop, D. Amyloid deposition as the central event in the aetiology of Alzheimer’s disease. Trends Pharmacol. Sci. 1991,
12, 383–388. [CrossRef]
8. Galimberti, D.; Scarpini, E. Treatment of Alzheimers Disease: Symptomatic and Disease-Modifying Approaches. Curr. Aging Sci.
2010, 3, 46–56. [CrossRef]
9. Carandini, T.; Arighi, A.; Sacchi, L.; Fumagalli, G.G.; Pietroboni, A.M.; Ghezzi, L.; Colombi, A.; Scarioni, M.; Fenoglio, C.;
De Riz, M.A.; et al. Testing the 2018 NIA-AA research framework in a retrospective large cohort of patients with cognitive
impairment: From biological biomarkers to clinical syndromes. Alzheimer’s Res. Ther. 2019, 11, 84. [CrossRef]
10. Dubois, B.; Villain, N.; Frisoni, G.B.; Rabinovici, G.D.; Sabbagh, M.; Cappa, S.; Bejanin, A.; Bombois, S.; Epelbaum, S.;
Teichmann, M.; et al. Clinical diagnosis of Alzheimer’s disease: Recommendations of the International Working Group. Lancet
Neurol. 2021, 20, 484–496. [CrossRef]
11. Liu, P.-P.; Xie, Y.; Meng, X.-Y.; Kang, J.-S. History and progress of hypotheses and clinical trials for Alzheimer’s disease. Signal
Transduct. Target. Ther. 2019, 4, 1–22. [CrossRef]
12. Serrano-Pozo, A.; Mielke, M.L.; Gómez-Isla, T.; Betensky, R.A.; Growdon, J.H.; Frosch, M.P.; Hyman, B.T. Reactive Glia not only
Associates with Plaques but also Parallels Tangles in Alzheimer’s Disease. Am. J. Pathol. 2011, 179, 1373–1384. [CrossRef]
13. Hanisch, U.-K.; Kettenmann, H. Microglia: Active sensor and versatile effector cells in the normal and pathologic brain. Nat.
Neurosci. 2007, 10, 1387–1394. [CrossRef]
14. Galimberti, D.; Baron, P.; Medaa, L.; Prata, E.; Scarpini, E.; Delgado, R.; Catania, A.; Lipton, J.M.; Scarlatoa, G. α-MSH Peptides
Inhibit Production of Nitric Oxide and Tumor Necrosis Factor-α by Microglial Cells Activated with β-Amyloid and Interferon γ.
Biochem. Biophys. Res. Commun. 1999, 263, 251–256. [CrossRef]
15. Clark, I.A.; Vissel, B. Amyloid β: One of three danger-associated molecules that are secondary inducers of the proinflammatory
cytokines that mediate A lzheimer’s disease. Br. J. Pharmacol. 2015, 172, 3714–3727. [CrossRef]
16. Schilling, T.; Eder, C. Amyloid-β-induced reactive oxygen species production and priming are differentially regulated by ion
channels in microglia. J. Cell. Physiol. 2011, 226, 3295–3302. [CrossRef]
17. Butterfield, D.A. β-Amyloid-Associated Free Radical Oxidative Stress and Neurotoxicity: Implications for Alzheimer’s Disease.
Chem. Res. Toxicol. 1997, 10, 495–506. [CrossRef]
18. Butterfield, D.A.; Halliwell, B. Oxidative stress, dysfunctional glucose metabolism and Alzheimer disease. Nat. Rev. Neurosci.
2019, 20, 148–160. [CrossRef] [PubMed]
19. Nunomura, A.; Perry, G.; Aliev, G.; Hirai, K.; Takeda, A.; Balraj, E.K.; Jones, P.K.; Ghanbari, H.; Wataya, T.; Shimohama, S.; et al.
Oxidative Damage Is the Earliest Event in Alzheimer Disease. J. Neuropathol. Exp. Neurol. 2001, 60, 759–767. [CrossRef] [PubMed]
20. Praticò, D.; Uryu, K.; Leight, S.; Trojanoswki, J.Q.; Lee, V.M.-Y. Increased Lipid Peroxidation Precedes Amyloid Plaque Formation
in an Animal Model of Alzheimer Amyloidosis. J. Neurosci. 2001, 21, 4183–4187. [CrossRef] [PubMed]
21. Ansari, M.A.; Scheff, S.W. NADPH-oxidase activation and cognition in Alzheimer disease progression. Free. Radic. Biol. Med.
2011, 51, 171–178. [CrossRef] [PubMed]
22. de Leeuw, F.A.; Peeters, C.F.; Kester, M.I.; Harms, A.C.; Struys, E.A.; Hankemeier, T.; van Vlijmen, H.W.; van der Lee, S.J.;
van Duijn, C.M.; Scheltens, P.; et al. Blood-based metabolic signatures in Alzheimer’s disease. Alzheimer’s Dementia: Diagn. Assess.
Dis. Monit. 2017, 8, 196–207. [CrossRef] [PubMed]
23. Praticò, D. Oxidative stress hypothesis in Alzheimer’s disease: A reappraisal. Trends Pharmacol. Sci. 2008, 29, 609–615. [CrossRef]
[PubMed]
24. Di Domenico, F.; Barone, E.; Perluigi, M.; Butterfield, D.A. The Triangle of Death in Alzheimer’s Disease Brain: The Aberrant
Cross-Talk Among Energy Metabolism, Mammalian Target of Rapamycin Signaling, and Protein Homeostasis Revealed by Redox
Proteomics. Antioxid. Redox Signal. 2017, 26, 364–387. [CrossRef]
25. Peña-Bautista, C.; Vigor, C.; Galano, J.-M.; Oger, C.; Durand, T.; Ferrer, I.; Cuevas, A.; López-Cuevas, R.; Baquero, M.;
López-Nogueroles, M.; et al. New screening approach for Alzheimer’s disease risk assessment from urine lipid peroxidation
compounds. Sci. Rep. 2019, 9, 14244. [CrossRef]
26. Choromańska, M.; Klimiuk, A.; Kostecka-Sochoń, P.; Wilczyńska, K.; Kwiatkowski, M.; Okuniewska, N.; Waszkiewicz, N.;
Zalewska, A.; Maciejczyk, M. Antioxidant Defence, Oxidative Stress and Oxidative Damage in Saliva, Plasma and Erythrocytes
of Dementia Patients. Can Salivary AGE be a Marker of Dementia? Int. J. Mol. Sci. 2017, 18, 2205. [CrossRef] [PubMed]
27. Johnson, E.C.B.; Dammer, E.B.; Duong, D.; Ping, L.; Zhou, M.; Yin, L.; Higginbotham, L.A.; Guajardo, A.; White, B.; Troncoso,
J.C.; et al. Large-scale proteomic analysis of Alzheimer’s disease brain and cerebrospinal fluid reveals early changes in energy
metabolism associated with microglia and astrocyte activation. Nat. Med. 2020, 26, 769–780. [CrossRef]
28. Jellinger, K.; Paulus, W.; Grundke-Iqbal, I.; Riederer, P.; Youdim, M.B.H. Brain iron and ferritin in Parkinson’s and Alzheimer’s
diseases. J. Neural Transm. 1990, 2, 327–340. [CrossRef] [PubMed]
29. Markesbery, W.R. Oxidative Stress Hypothesis in Alzheimer’s Disease. Free. Radic. Biol. Med. 1997, 23, 134–147. [CrossRef]
30. Smith, M.A.; Rottkamp, C.A.; Nunomura, A.; Raina, A.K.; Perry, G. Oxidative stress in Alzheimer’s disease. Biochim. et Biophys.
Acta (BBA) Mol. Basis Dis. 2000, 1502, 139–144. [CrossRef]
31. Ansari, M.A.; Scheff, S.W. Oxidative Stress in the Progression of Alzheimer Disease in the Frontal Cortex. J. Neuropathol. Exp.
Neurol. 2010, 69, 155–167. [CrossRef] [PubMed]
Antioxidants 2021, 10, 1353 17 of 22

32. Scheff, S.W.; Ansari, M.A.; Mufson, E.J. Oxidative stress and hippocampal synaptic protein levels in elderly cognitively intact
individuals with Alzheimer’s disease pathology. Neurobiol. Aging 2016, 42, 1–12. [CrossRef] [PubMed]
33. Sultana, R.; Perluigi, M.; Butterfield, D.A. Lipid peroxidation triggers neurodegeneration: A redox proteomics view into the
Alzheimer disease brain. Free. Radic. Biol. Med. 2012, 62, 157–169. [CrossRef] [PubMed]
34. Subedi, L.; Gaire, B.P.; Parveen, A.; Kim, S.Y. Nitric Oxide as a Target for Phytochemicals in Anti-Neuroinflammatory Prevention
Therapy. Int. J. Mol. Sci. 2021, 22, 4771. [CrossRef] [PubMed]
35. Fleszar, M.G.; Wiśniewski, J.; Zboch, M.; Diakowska, D.; Gamian, A.; Krzystek-Korpacka, M. Targeted metabolomic analysis of
nitric oxide/L-arginine pathway metabolites in dementia: Association with pathology, severity, and structural brain changes. Sci.
Rep. 2019, 9, 13764. [CrossRef]
36. Hurtado, M.O.; Kohler, I.; De Lange, E.C. Next-generation biomarker discovery in Alzheimer’s disease using metabolomics–from
animal to human studies. Bioanalysis 2018, 10, 1525–1546. [CrossRef]
37. Dalleau, S.; Baradat, M.; Guéraud, F.; Huc, L. Cell death and diseases related to oxidative stress:4-hydroxynonenal (HNE) in the
balance. Cell Death Differ. 2013, 20, 1615–1630. [CrossRef]
38. Perry, E.A.; Castellani, R.J.; Moreira, P.I.; Nunomura, A.; Harris, P.L.R.; Sayre, L.M.; Szweda, P.A.; Szweda, L.I.; Zhu, X.;
Smith, M.A.; et al. Neurofilaments are the major neuronal target of hydroxynonenal-mediated protein cross-links. Free. Radic.
Res. 2013, 47, 507–510. [CrossRef]
39. Butterfield, D.A.; Swomley, A.M.; Sultana, R. Amyloid beta-peptide (1-42)-induced oxidative stress in Alzheimer disease:
Importance in disease pathogenesis and progression. Antioxid. Redox Signal 2013, 19, 823–835. [CrossRef]
40. Di Domenico, F.; Pupo, G.; Giraldo, E.; Badìa, M.C.; Monllor, P.; Lloret, A.; Schininà, M.E.; Giorgi, A.; Cini, C.; Tramutola, A.; et al.
Oxidative signature of cerebrospinal fluid from mild cognitive impairment and Alzheimer disease patients. Free. Radic. Biol. Med.
2016, 91, 1–9. [CrossRef]
41. Praticò, D. The neurobiology of isoprostanes and Alzheimer’s disease. Biochim. Biophys. Acta (BBA) Mol. Cell Biol. Lipids 2010,
1801, 930–933. [CrossRef]
42. Rungratanawanich, W.; Qu, Y.; Wang, X.; Essa, M.M.; Song, B.-J. Advanced glycation end products (AGEs) and other adducts in
aging-related diseases and alcohol-mediated tissue injury. Exp. Mol. Med. 2021, 53, 168–188. [CrossRef]
43. Fang, F.; Yu, Q.; Arancio, O.; Chen, D.; Gore, S.; Yan, S.S.; Yan, S.F. RAGE mediates Aβ accumulation in a mouse model of
Alzheimer’s disease via modulation of β- and γ-secretase activity. Hum. Mol. Genet. 2018, 27, 1002–1014. [CrossRef] [PubMed]
44. Origlia, N.; Bonadonna, C.; Rosellini, A.; Leznik, E.; Arancio, O.; Yan, S.S.; Domenici, L. Microglial Receptor for Advanced
Glycation End Product-Dependent Signal Pathway Drives-Amyloid-Induced Synaptic Depression and Long-Term Depression
Impairment in Entorhinal Cortex. J. Neurosci. 2010, 30, 11414–11425. [CrossRef] [PubMed]
45. Miller, M.C.; Tavares, R.; Johanson, C.; Hovanesian, V.; Donahue, J.E.; Gonzalez, L.; Silverberg, G.D.; Stopa, E.G. Hippocampal
RAGE immunoreactivity in early and advanced Alzheimer’s disease. Brain Res. 2008, 1230, 273–280. [CrossRef] [PubMed]
46. Sasaki, N.; Toki, S.; Chowei, H.; Saito, T.; Nakano, N.; Hayashi, Y.; Takeuchi, M.; Makita, Z. Immunohistochemical distribution of
the receptor for advanced glycation end products in neurons and astrocytes in Alzheimer’s disease. Brain Res. 2001, 888, 256–262.
[CrossRef]
47. Lue, L.-F.; Walker, D.G.; Brachova, L.; Beach, T.G.; Rogersa, J.; Schmidt, A.M.; Stern, D.M.; Du Yan, S. Involvement of Microglial
Receptor for Advanced Glycation Endproducts (RAGE) in Alzheimer’s Disease: Identification of a Cellular Activation Mechanism.
Exp. Neurol. 2001, 171, 29–45. [CrossRef] [PubMed]
48. Byun, K.; Bayarsaikhan, E.; Kim, D.; Kim, C.Y.; Mook-Jung, I.; Paek, S.H.; Kim, S.U.; Yamamoto, T.; Won, M.-H.; Song, B.-J.; et al.
Induction of Neuronal Death by Microglial AGE-Albumin: Implications for Alzheimer’s Disease. PLoS ONE 2012, 7, e37917.
[CrossRef]
49. Bortolotto, V.; Grilli, M. Every Cloud Has a Silver Lining: Proneurogenic Effects of Aβ Oligomers and HMGB-1 via Activation of
the RAGE-NF-κB Axis. CNS Neurol. Disord. Drug Targets 2018, 16, 1066–1079. [CrossRef]
50. Mecocci, P.; MacGarvey, U.; Kaufman, A.E.; Koontz, D.; Shoffner, J.M.; Wallace, D.C.; Beal, M.F. Oxidative damage to mitochon-
drial DNA shows marked age-dependent increases in human brain. Ann. Neurol. 1993, 34, 609–616. [CrossRef]
51. Mecocci, P.; MacGarvey, U.; Beal, M.F. Oxidative damage to mitochondrial DNA is increased in Alzheimer’s disease. Ann. Neurol.
1994, 36, 747–751. [CrossRef]
52. Abe, T.; Tohgi, H.; Isobe, C.; Murata, T.; Sato, C. Remarkable increase in the concentration of 8-hydroxyguanosine in cerebrospinal
fluid from patients with Alzheimer’s disease. J. Neurosci. Res. 2002, 70, 447–450. [CrossRef]
53. Hirai, K.; Aliev, G.; Nunomura, A.; Fujioka, H.; Russell, R.L.; Atwood, C.S.; Johnson, A.B.; Kress, Y.; Vinters, H.V.;
Tabaton, M.; et al. Mitochondrial Abnormalities in Alzheimer’s Disease. J. Neurosci. 2001, 21, 3017–3023. [CrossRef]
54. Nunomura, A.; Perry, G. RNA and Oxidative Stress in Alzheimer’s Disease: Focus on microRNAs. Oxidative Med. Cell. Longev.
2020, 2020, 2638130. [CrossRef] [PubMed]
55. Wang, J.; Gao, J.; Ding, S.-L.; Wang, K.; Jiao, J.-Q.; Wang, Y.; Sun, T.; Zhou, L.-Y.; Long, B.; Zhang, X.-J.; et al. Oxidative Modification
of miR-184 Enables It to Target Bcl-xL and Bcl-w. Mol. Cell 2015, 59, 50–61. [CrossRef] [PubMed]
56. Amakiri, N.; Kubosumi, A.; Tran, J.; Reddy, P.H. Amyloid Beta and MicroRNAs in Alzheimer’s Disease. Front. Neurosci. 2019, 13,
430. [CrossRef] [PubMed]
57. Konovalova, J.; Gerasymchuk, D.; Parkkinen, I.; Chmielarz, P.; Domanskyi, A. Interplay between MicroRNAs and Oxidative
Stress in Neurodegenerative Diseases. Int. J. Mol. Sci. 2019, 20, 6055. [CrossRef] [PubMed]
Antioxidants 2021, 10, 1353 18 of 22

58. Everett, J.; Brooks, J.; Lermyte, F.; O’Connor, P.B.; Sadler, P.J.; Dobson, J.; Collingwood, J.; Telling, N.D. Iron stored in ferritin is
chemically reduced in the presence of aggregating Aβ(1-42). Sci. Rep. 2020, 10, 10332. [CrossRef] [PubMed]
59. Bulk, M.; Van Der Weerd, L.; Breimer, W.; Lebedev, N.; Webb, A.; Goeman, J.; Ward, R.J.; Huber, M.; Oosterkamp, T.H.; Bossoni, L.
Quantitative comparison of different iron forms in the temporal cortex of Alzheimer patients and control subjects. Sci. Rep. 2018,
8, 6898. [CrossRef]
60. Kenkhuis, B.; Somarakis, A.; de Haan, L.; Dzyubachyk, O.; IJsselsteijn, M.E.; de Miranda, N.F.; Lelieveldt, B.P.; Dijkstra, J.; van
Roon-Mom, W.M.; Höllt, T.; et al. Iron loading is a prominent feature of activated microglia in Alzheimer’s disease patients. Acta
Neuropathol. Commun. 2021, 9, 27. [CrossRef] [PubMed]
61. Meadowcroft, M.D.; Peters, D.G.; Dewal, R.P.; Connor, J.R.; Yang, Q.X. The effect of iron in MRI and transverse relaxation of
amyloid-beta plaques in Alzheimer’s disease. NMR Biomed. 2014, 28, 297–305. [CrossRef]
62. Nakamura, M.; Shishido, N.; Nunomura, A.; Smith, M.A.; Perry, G.; Hayashi, Y.; Nakayama, K.; Hayashi, T. Three Histidine
Residues of Amyloid-β Peptide Control the Redox Activity of Copper and Iron. Biochemistry 2007, 46, 12737–12743. [CrossRef]
[PubMed]
63. Lin, M.T.; Beal, M.F. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nat. Cell Biol. 2006, 443,
787–795. [CrossRef] [PubMed]
64. Mecocci, P.; Beal, M.F.; Cecchetti, R.; Polidori, M.C.; Cherubini, A.; Chionne, F.; Avellini, L.; Romano, G.; Senin, U. Mitochondrial
membrane fluidity and oxidative damage to mitochondrial DNA in aged and AD human brain. Mol. Chem. Neuropathol. 1997, 31,
53–64. [CrossRef]
65. Swerdlow, R.H.; Khan, S.M. A “mitochondrial cascade hypothesis” for sporadic Alzheimer’s disease. Med. Hypotheses 2004, 63,
8–20. [CrossRef]
66. Swerdlow, R.H.; Burns, J.M.; Khan, S.M. The Alzheimer’s Disease Mitochondrial Cascade Hypothesis. J. Alzheimer’s Dis. 2010, 20,
S265–S279. [CrossRef]
67. Oka, S.; Leon, J.; Sakumi, K.; Ide, T.; Kang, N.; LaFerla, F.M.; Nakabeppu, Y. Human mitochondrial transcriptional factor A breaks
the mitochondria-mediated vicious cycle in Alzheimer’s disease. Sci. Rep. 2016, 6, 37889. [CrossRef] [PubMed]
68. Mattson, M.P. Calcium and neurodegeneration. Aging Cell 2007, 6, 337–350. [CrossRef] [PubMed]
69. Bezprozvanny, I.; Mattson, M.P. Neuronal calcium mishandling and the pathogenesis of Alzheimer’s disease. Trends Neurosci.
2008, 31, 454–463. [CrossRef]
70. Xie, H.; Guan, J.-S.; Borrelli, L.A.; Xu, J.; Serrano-Pozo, A.; Bacskai, B.J. Mitochondrial alterations near amyloid plaques in an
Alzheimer’s disease mouse model. J. Neurosci. 2013, 33, 17042–17051. [CrossRef]
71. Xie, H.; Hou, S.; Jiang, J.; Sekutowicz, M.; Kelly, J.; Bacskai, B.J. Rapid cell death is preceded by amyloid plaque-mediated
oxidative stress. Proc. Natl. Acad. Sci. USA 2013, 110, 7904–7909. [CrossRef]
72. Wang, X.; Su, B.; Siedlak, S.L.; Moreira, P.; Fujioka, H.; Wang, Y.; Casadesus, G.; Zhu, X. Amyloid-β overproduction causes
abnormal mitochondrial dynamics via differential modulation of mitochondrial fission/fusion proteins. Proc. Natl. Acad. Sci.
USA 2008, 105, 19318–19323. [CrossRef] [PubMed]
73. Kerr, J.S.; Adriaanse, B.A.; Greig, N.H.; Mattson, M.P.; Cader, Z.; Bohr, V.A.; Fang, E.F. Mitophagy and Alzheimer’s Disease:
Cellular and Molecular Mechanisms. Trends Neurosci. 2017, 40, 151–166. [CrossRef]
74. Robinson, D.M.; Keating, G.M. Memantine: A Review of Its Use in Alzheimer’s Disease. Drugs 2006, 66, 1515–1534. [CrossRef]
[PubMed]
75. De Felice, F.G.; Velasco, P.T.; Lambert, M.P.; Viola, K.; Fernandez, S.J.; Ferreira, S.T.; Klein, W.L. Aβ Oligomers Induce Neuronal
Oxidative Stress through an N-Methyl-D-aspartate Receptor-dependent Mechanism That Is Blocked by the Alzheimer Drug
Memantine. J. Biol. Chem. 2007, 282, 11590–11601. [CrossRef] [PubMed]
76. Lee, J.; Kim, Y.; Liu, T.; Hwang, Y.J.; Hyeon, S.J.; Im, H.; Lee, K.; Alvarez, V.E.; McKee, A.C.; Um, S.-J.; et al. SIRT3 deregulation is
linked to mitochondrial dysfunction in Alzheimer’s disease. Aging Cell 2017, 17, e12679. [CrossRef] [PubMed]
77. Zhang, J.; Xiang, H.; Liu, J.; Chen, Y.; He, R.-R.; Liu, B. Mitochondrial Sirtuin 3: New emerging biological function and therapeutic
target. Theranostics 2020, 10, 8315–8342. [CrossRef]
78. de Leon, M.J.; Convit, A.; Wolf, O.T.; Tarshish, C.Y.; DeSanti, S.; Rusinek, H.; Tsui, W.; Kandil, E.; Scherer, A.J.; Roche, A.; et al.
Prediction of cognitive decline in normal elderly subjects with 2-[18F]fluoro-2-deoxy-D-glucose/positron-emission tomography
(FDG/PET). Proc. Natl. Acad. Sci. USA 2001, 98, 10966–10971. [CrossRef]
79. Selkoe, D.J.; Hardy, J. The Amyloid Hypothesis of Alzheimer’s Disease at 25 Years. EMBO Mol. Med. 2016, 8, 595–608. [CrossRef]
[PubMed]
80. Cline, E.N.; Bicca, M.A.; Viola, K.L.; Klein, W.L. The Amyloid-β Oligomer Hypothesis: Beginning of the Third Decade. J.
Alzheimer’s Dis. 2018, 64, S567–S610. [CrossRef]
81. Fontana, I.C.; Zimmer, A.R.; Rocha, A.S.; Gosmann, G.; Souza, D.O.; Lourenco, M.V.; Ferreira, S.T.; Zimmer, E.R. Amyloid-β
oligomers in cellular models of Alzheimer’s disease. J. Neurochem. 2020, 155, 348–369. [CrossRef]
82. Hayden, E.Y.; Teplow, D.B. Amyloid β-protein oligomers and Alzheimer’s disease. Alzheimer’s Res. Ther. 2013, 5, 60. [CrossRef]
83. Mroczko, B.; Groblewska, M.; Litman-Zawadzka, A.; Kornhuber, J.; Lewczuk, P. Cellular Receptors of Amyloid β Oligomers
(AβOs) in Alzheimer’s Disease. Int. J. Mol. Sci. 2018, 19, 1884. [CrossRef]
84. Jarosz-Griffiths, H.; Noble, E.; Rushworth, J.V.; Hooper, N.M. Amyloid-β Receptors: The Good, the Bad, and the Prion Protein. J.
Biol. Chem. 2016, 291, 3174–3183. [CrossRef] [PubMed]
Antioxidants 2021, 10, 1353 19 of 22

85. Manczak, M.; Anekonda, T.S.; Henson, E.; Park, B.S.; Quinn, J.; Reddy, P.H. Mitochondria are a direct site of Aβ accumulation in
Alzheimer’s disease neurons: Implications for free radical generation and oxidative damage in disease progression. Hum. Mol.
Genet. 2006, 15, 1437–1449. [CrossRef] [PubMed]
86. Terry, R.D.; Masliah, E.; Salmon, D.P.; Butters, N.; Bs, R.D.; Hill, R.; Hansen, L.A.; Katzman, R. Physical basis of cognitive
alterations in alzheimer’s disease: Synapse loss is the major correlate of cognitive impairment. Ann. Neurol. 1991, 30, 572–580.
[CrossRef] [PubMed]
87. Selkoe, D.J. Alzheimer’s Disease Is a Synaptic Failure. Science 2002, 298, 789–791. [CrossRef]
88. Ferreira, S.T.; Klein, W.L. The Aβ oligomer hypothesis for synapse failure and memory loss in Alzheimer’s disease. Neurobiol.
Learn. Mem. 2011, 96, 529–543. [CrossRef] [PubMed]
89. Robinson, J.L.; Molina-Porcel, L.; Corrada, M.; Raible, K.; Lee, E.B.; Lee, V.M.-Y.; Kawas, C.H.; Trojanowski, J.Q. Perforant
path synaptic loss correlates with cognitive impairment and Alzheimer’s disease in the oldest-old. Brain 2014, 137, 2578–2587.
[CrossRef]
90. Butterfield, D.A.; Boyd-Kimball, D. Redox proteomics and amyloid β-peptide: Insights into Alzheimer disease. J. Neurochem.
2018, 151, 459–487. [CrossRef]
91. Stranahan, A.M.; Mattson, M.P. Selective Vulnerability of Neurons in Layer II of the Entorhinal Cortex during Aging and
Alzheimer’s Disease. Neural Plast. 2010, 2010, 108190. [CrossRef]
92. Mrdjen, D.; Fox, E.J.; Bukhari, S.A.; Montine, K.S.; Bendall, S.C.; Montine, T.J. The basis of cellular and regional vulnerability in
Alzheimer’s disease. Acta Neuropathol. 2019, 138, 729–749. [CrossRef]
93. Braak, H.; Braak, E. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol. 1991, 82, 239–259. [CrossRef]
[PubMed]
94. Gómez-Isla, T.; Price, J.L.; McKeel, D.W., Jr.; Morris, J.C.; Growdon, J.H.; Hyman, B.T. Profound Loss of Layer II Entorhinal Cortex
Neurons Occurs in Very Mild Alzheimer’s Disease. J. Neurosci. 1996, 16, 4491–4500. [CrossRef] [PubMed]
95. Terni, B.; Boada, J.; Portero-Otin, M.; Pamplona, R.; Ferrer, I. Mitochondrial ATP-Synthase in the Entorhinal Cortex Is a Target of
Oxidative Stress at Stages I/II of Alzheimer’s Disease Pathology. Brain Pathol. 2010, 20, 222–233. [CrossRef]
96. Wang, X. Selective neuronal vulnerability to oxidative stress in the brain. Front. Aging Neurosci. 2010, 2, 12. [CrossRef] [PubMed]
97. Raven, E.P.; Lu, P.H.; Tishler, T.A.; Heydari, P.; Bartzokis, G. Increased Iron Levels and Decreased Tissue Integrity in Hippocampus
of Alzheimer’s Disease Detected in vivo with Magnetic Resonance Imaging. J. Alzheimer’s Dis. 2013, 37, 127–136. [CrossRef]
98. Li, J.; Cao, F.; Yin, H.-L.; Huang, Z.-J.; Lin, Z.-T.; Mao, N.; Sun, B.; Wang, G. Ferroptosis: Past, present and future. Cell Death Dis.
2020, 11, 88. [CrossRef]
99. Yan, H.-F.; Zou, T.; Tuo, Q.-Z.; Xu, S.; Li, H.; Belaidi, A.A.; Lei, P. Ferroptosis: Mechanisms and links with diseases. Signal
Transduct. Target. Ther. 2021, 6, 49. [CrossRef] [PubMed]
100. Nikseresht, S.; Bush, A.I.; Ayton, S. Treating Alzheimer’s disease by targeting iron. Br. J. Pharmacol. 2019, 176, 3622–3635.
[CrossRef] [PubMed]
101. Park, M.W.; Cha, H.W.; Kim, J.; Kim, J.H.; Yang, H.; Yoon, S.; Boonpraman, N.; Yi, S.S.; Yoo, I.D.; Moon, J.-S. NOX4 promotes
ferroptosis of astrocytes by oxidative stress-induced lipid peroxidation via the impairment of mitochondrial metabolism in
Alzheimer’s diseases. Redox Biol. 2021, 41, 101947. [CrossRef] [PubMed]
102. Sarlus, H.; Heneka, M.T. Microglia in Alzheimer’s disease. J. Clin. Investig. 2017, 127, 3240–3249. [CrossRef] [PubMed]
103. Schwabe, T.; Srinivasan, K.; Rhinn, H. Shifting paradigms: The central role of microglia in Alzheimer’s disease. Neurobiol. Dis.
2020, 143, 104962. [CrossRef] [PubMed]
104. Venegas, C.; Heneka, M.T. Inflammasome-mediated innate immunity in Alzheimer’s disease. FASEB J. 2019, 33, 13075–13084.
[CrossRef] [PubMed]
105. Deora, V.; Lee, J.D.; Albornoz, E.A.; McAlary, L.; Jagaraj, C.J.; Robertson, A.A.B.; Atkin, J.; Cooper, M.A.; Schroder, K.;
Yerbury, J.; et al. The microglial NLRP3 inflammasome is activated by amyotrophic lateral sclerosis proteins. Glia 2019, 68,
407–421. [CrossRef] [PubMed]
106. Heneka, M.T.; McManus, R.; Latz, E. Inflammasome signalling in brain function and neurodegenerative disease. Nat. Rev.
Neurosci. 2018, 19, 610–621. [CrossRef]
107. Venegas, C.; Heneka, M.T. Danger-associated molecular patterns in Alzheimer’s disease. J. Leukoc. Biol. 2016, 101, 87–98.
[CrossRef]
108. Shimohamaa, S.; Taninob, H.; Kawakamib, N.; Okamurac, N.; Kodamac, H.; Yamaguchid, T.; Hayakawad, T.; Nunomurae, A.;
Chibae, S.; Perry, G.; et al. Activation of NADPH Oxidase in Alzheimer’s Disease Brains. Biochem. Biophys. Res. Commun. 2000,
273, 5–9. [CrossRef]
109. Geng, L.; Fan, L.M.; Liu, F.; Smith, C.; Li, J.-M. Nox2 dependent redox-regulation of microglial response to amyloid-β stimulation
and microgliosis in aging. Sci. Rep. 2020, 10, 1582. [CrossRef]
110. Zhang, J.; Wang, X.; Vikash, V.; Ye, Q.; Wu, D.; Liu, Y.; Dong, W. ROS and ROS-Mediated Cellular Signaling. Oxidative Med. Cell.
Longev. 2016, 2016, 4350965. [CrossRef]
111. Dodson, M.; Castro-Portuguez, R.; Zhang, D.D. NRF2 plays a critical role in mitigating lipid peroxidation and ferroptosis. Redox
Biol. 2019, 23, 101107. [CrossRef]
Antioxidants 2021, 10, 1353 20 of 22

112. Wang, Q.; Li, W.-X.; Dai, S.-X.; Guo, Y.-C.; Han, F.-F.; Zheng, J.-J.; Li, G.-H.; Huang, J.-F. Meta-Analysis of Parkinson’s Disease and
Alzheimer’s Disease Revealed Commonly Impaired Pathways and Dysregulation of NRF2-Dependent Genes. J. Alzheimer’s Dis.
2017, 56, 1525–1539. [CrossRef] [PubMed]
113. Brandes, M.S.; Gray, N.E. NRF2 as a Therapeutic Target in Neurodegenerative Diseases. ASN Neuro 2020, 12. [CrossRef] [PubMed]
114. Gray, N.E.; Zweig, J.A.; Caruso, M.; Zhu, J.Y.; Wright, K.M.; Quinn, J.F.; Soumyanath, A. Centella asiatica attenuates hippocampal
mitochondrial dysfunction and improves memory and executive function in β-amyloid overexpressing mice. Mol. Cell. Neurosci.
2018, 93, 1–9. [CrossRef] [PubMed]
115. Matthews, D.G.; Caruso, M.; Murchison, C.F.; Zhu, J.Y.; Wright, K.M.; Harris, C.J.; Gray, N.E.; Quinn, J.F.; Soumyanath, A. Centella
Asiatica Improves Memory and Promotes Antioxidative Signaling in 5XFAD Mice. Antioxidants 2019, 8, 630. [CrossRef] [PubMed]
116. Plaza-Zabala, A.; Sierra-Torre, V.; Sierra, A. Autophagy and Microglia: Novel Partners in Neurodegeneration and Aging. Int. J.
Mol. Sci. 2017, 18, 598. [CrossRef]
117. Guidi, I.; Galimberti, D.; Lonati, S.; Novembrino, C.; Bamonti, F.; Tiriticco, M.; Fenoglio, C.; Venturelli, E.; Baron, P.;
Bresolin, N.; et al. Oxidative imbalance in patients with mild cognitive impairment and Alzheimer’s disease. Neurobiol. Aging
2006, 27, 262–269. [CrossRef]
118. Galimberti, D.; Fenoglio, C.; Lovati, C.; Venturelli, E.; Guidi, I.; Corrà, B.; Scalabrini, D.; Clerici, F.; Mariani, C.; Bresolin, N.; et al.
Serum MCP-1 levels are increased in mild cognitive impairment and mild Alzheimer’s disease. Neurobiol. Aging 2006, 27,
1763–1768. [CrossRef]
119. Galimberti, D.; Schoonenboom, N.; Scheltens, P.; Fenoglio, C.; Bouwman, F.; Venturelli, E.; Guidi, I.; Blankenstein, M.; Bresolin,
N.; Scarpini, E. Intrathecal Chemokine Synthesis in Mild Cognitive Impairment and Alzheimer Disease. Arch. Neurol. 2006, 63,
538–543. [CrossRef]
120. Ulland, T.K.; Colonna, M. TREM2—A key player in microglial biology and Alzheimer disease. Nat. Rev. Neurol. 2018, 14, 667–675.
[CrossRef]
121. Takahashi, K.; Rochford, C.D.; Neumann, H. Clearance of apoptotic neurons without inflammation by microglial triggering
receptor expressed on myeloid cells-2. J. Exp. Med. 2005, 201, 647–657. [CrossRef] [PubMed]
122. Guerreiro, R.; Wojtas, A.; Bras, J.; Carrasquillo, M.; Rogaeva, E.; Majounie, E.; Cruchaga, C.; Sassi, C.; Kauwe, J.S.; Younkin, S.;
et al. TREM2 Variants in Alzheimer’s Disease. N. Engl. J. Med. 2013, 368, 117–127. [CrossRef] [PubMed]
123. Liu, W.; Taso, O.; Wang, R.; Bayram, S.; Graham, A.C.; Garcia-Reitboeck, P.; Mallach, A.; Andrews, W.D.; Piers, T.M.; Botia, J.A.;
et al. Trem2 promotes anti-inflammatory responses in microglia and is suppressed under pro-inflammatory conditions. Hum.
Mol. Genet. 2020, 29, 3224–3248. [CrossRef] [PubMed]
124. Pratico, D.; Lee, V.M.-Y.; Trojanowski, J.Q.; Rokach, J.; Fitzgerald, G.A. Increased F 2 -isoprostanes in Alzheimer’s disease:
Evidence for enhanced lipid peroxidation in vivo. FASEB J. 1998, 12, 1777–1783. [CrossRef] [PubMed]
125. Oka, S.; Leon, J.; Sakumi, K.; Abolhassani, N.; Sheng, Z.; Tsuchimoto, D.; LaFerla, F.M.; Nakabeppu, Y. MTH1 and OGG1 maintain
a low level of 8-oxoguanine in Alzheimer’s brain, and prevent the progression of Alzheimer’s pathogenesis. Sci. Rep. 2021, 11,
5819. [CrossRef]
126. Pao, P.-C.; Patnaik, D.; Watson, L.A.; Gao, F.; Pan, L.; Wang, J.; Adaikkan, C.; Penney, J.; Cam, H.P.; Huang, W.-C.; et al. HDAC1
modulates OGG1-initiated oxidative DNA damage repair in the aging brain and Alzheimer’s disease. Nat. Commun. 2020, 11,
2484. [CrossRef]
127. Kim, D.; Frank, C.L.; Dobbin, M.M.; Tsunemoto, R.K.; Tu, W.; Peng, P.L.; Guan, J.-S.; Lee, B.-H.; Moy, L.Y.; Giusti-Rodríguez, P.;
et al. Deregulation of HDAC1 by p25/Cdk5 in Neurotoxicity. Neuron 2008, 60, 803–817. [CrossRef]
128. Dobbin, M.M.; Madabhushi, R.; Pan, L.; Chen, Y.; Kim, D.; Gao, J.; Ahanonu, B.; Pao, P.-C.; Qiu, Y.; Zhao, Y.; et al. SIRT1
collaborates with ATM and HDAC1 to maintain genomic stability in neurons. Nat. Neurosci. 2013, 16, 1008–1015. [CrossRef]
129. Stojakovic, A.; Trushin, S.; Sheu, A.; Khalili, L.; Chang, S.-Y.; Li, X.; Christensen, T.; Salisbury, J.L.; Geroux, R.E.; Gateno, B.; et al.
Partial inhibition of mitochondrial complex I ameliorates Alzheimer’s disease pathology and cognition in APP/PS1 female mice.
Commun. Biol. 2021, 4, 61. [CrossRef]
130. Zhong, B.; Zhou, G.; Song, L.; Wen, Q.; Deng, X.; Ma, Y.; Hu, L.; Chen, G. TUFM is involved in Alzheimer’s disease-like
pathologies that are associated with ROS. FASEB J. 2021, 35, e21445. [CrossRef]
131. Takeda, K.; Uda, A.; Mitsubori, M.; Nagashima, S.; Iwasaki, H.; Ito, N.; Shiiba, I.; Ishido, S.; Matsuoka, M.; Inatome, R.; et al.
Mitochondrial ubiquitin ligase alleviates Alzheimer’s disease pathology via blocking the toxic amyloid-β oligomer generation.
Commun. Biol. 2021, 4, 192. [CrossRef]
132. Bai, Z.; Han, G.; Xie, B.; Wang, J.; Song, F.; Peng, X.; Lei, H. AlzBase: An Integrative Database for Gene Dysregulation in
Alzheimer’s Disease. Mol. Neurobiol. 2014, 53, 310–319. [CrossRef]
133. Haddad, M.; Hervé, V.; Ben Khedher, M.R.; Rabanel, J.-M.; Ramassamy, C. Glutathione: An Old and Small Molecule with Great
Functions and New Applications in the Brain and in Alzheimer’s Disease. Antioxid. Redox Signal. 2021, 35. [CrossRef] [PubMed]
134. Liu, Y.; Chen, Z.; Li, B.; Yao, H.; Zarka, M.; Welch, J.; Sachdev, P.; Bridge, W.; Braidy, N. Supplementation with γ-glutamylcysteine
(γ-GC) lessens oxidative stress, brain inflammation and amyloid pathology and improves spatial memory in a murine model of
AD. Neurochem. Int. 2020, 144, 104931. [CrossRef] [PubMed]
135. Wang, K.; Wang, L.; Chen, L.; Peng, C.; Luo, B.; Mo, J.; Chen, W. Intranasal administration of dauricine loaded on graphene oxide:
Multi-target therapy for Alzheimer’s disease. Drug Deliv. 2021, 28, 580–593. [CrossRef]
Antioxidants 2021, 10, 1353 21 of 22

136. Jack, C.R.; Bennett, D.A.; Blennow, K.; Carrillo, M.C.; Dunn, B.; Haeberlein, S.B.; Holtzman, D.M.; Jagust, W.; Jessen, F.; Karlawish,
J.; et al. NIA-AA Research Framework: Toward a biological definition of Alzheimer’s disease. Alzheimer’s Dement. 2018, 14,
535–562. [CrossRef] [PubMed]
137. Nakamura, A.; Kaneko, N.; Villemagne, V.L.; Kato, T.; Doecke, J.; Dore, V.; Fowler, C.; Li, Q.-X.; Martins, R.; Rowe, C.; et al. High
performance plasma amyloid-β biomarkers for Alzheimer’s disease. Nat. Cell Biol. 2018, 554, 249–254. [CrossRef]
138. Kurbatova, N.; Garg, M.; Whiley, L.; Chekmeneva, E.; Jiménez, B.; Gómez-Romero, M.; Pearce, J.; Kimhofer, T.; D’Hondt, E.;
Soininen, H.; et al. Urinary metabolic phenotyping for Alzheimer’s disease. Sci. Rep. 2020, 10, 21745. [CrossRef]
139. Peña-Bautista, C.; Vigor, C.; Galano, J.-M.; Oger, C.; Durand, T.; Ferrer, I.; Cuevas, A.; López-Cuevas, R.; Baquero, M.; López-
Nogueroles, M.; et al. Plasma lipid peroxidation biomarkers for early and non-invasive Alzheimer Disease detection. Free. Radic.
Biol. Med. 2018, 124, 388–394. [CrossRef]
140. Peña-Bautista, C.; Carrascosa-Marco, P.; Oger, C.; Vigor, C.; Galano, J.-M.; Durand, T.; Baquero, M.; López-Nogueroles, M.; Vento,
M.; García-Blanco, A.; et al. Validated analytical method to determine new salivary lipid peroxidation compounds as potential
neurodegenerative biomarkers. J. Pharm. Biomed. Anal. 2018, 164, 742–749. [CrossRef]
141. Peña-Bautista, C.; Álvarez, L.; Durand, T.; Vigor, C.; Cuevas, A.; Baquero, M.; Vento, M.; Hervás, D.; Cháfer-Pericás, C. Clinical
Utility of Plasma Lipid Peroxidation Biomarkers in Alzheimer’s Disease Differential Diagnosis. Antioxidants 2020, 9, 649.
[CrossRef] [PubMed]
142. Peña-Bautista, C.; Álvarez-Sánchez, L.; Ferrer, I.; López-Nogueroles, M.; Cañada-Martínez, A.; Oger, C.; Galano, J.-M.; Durand, T.;
Baquero, M.; Cháfer-Pericás, C. Lipid Peroxidation Assessment in Preclinical Alzheimer Disease Diagnosis. Antioxidants 2021, 10,
1043. [CrossRef] [PubMed]
143. Peña-Bautista, C.; Álvarez, L.; Baquero, M.; Ferrer, I.; García, L.; Hervás-Marín, D.; Cháfer-Pericás, C. Plasma isoprostanoids
assessment as Alzheimer’s disease progression biomarkers. J. Neurochem. 2020, 157, 2187–2194. [CrossRef]
144. Peña-Bautista, C.; Tirle, T.; López-Nogueroles, M.; Vento, M.; Baquero, M.; Cháfer-Pericás, C. Oxidative Damage of DNA as Early
Marker of Alzheimer’s Disease. Int. J. Mol. Sci. 2019, 20, 6136. [CrossRef]
145. Liu, Y.; Thalamuthu, A.; Mather, K.A.; Crawford, J.; Ulanova, M.; Wong, M.W.K.; Pickford, R.; Sachdev, P.S.; Braidy, N. Plasma
lipidome is dysregulated in Alzheimer’s disease and is associated with disease risk genes. Transl. Psychiatry 2021, 11, 344.
[CrossRef]
146. Su, X.Q.; Wang, J.; Sinclair, A.J. Plasmalogens and Alzheimer’s disease: A review. Lipids Heal. Dis. 2019, 18, 100. [CrossRef]
147. Zimetti, F.; Adorni, M.P.; Marsillach, J.; Marchi, C.; Trentini, A.; Valacchi, G.; Cervellati, C. Connection between the Altered HDL
Antioxidant and Anti-Inflammatory Properties and the Risk to Develop Alzheimer’s Disease: A Narrative Review. Oxidative Med.
Cell. Longev. 2021, 2021, 6695796. [CrossRef] [PubMed]
148. Zuin, M.; Cervellati, C.; Trentini, A.; Passaro, A.; Rosta, V.; Zimetti, F.; Zuliani, G. Association between Serum Concentrations
of Apolipoprotein A-I (ApoA-I) and Alzheimer’s Disease: Systematic Review and Meta-Analysis. Diagnostics 2021, 11, 984.
[CrossRef] [PubMed]
149. Arnold, M.; Nho, K.; Kueider-Paisley, A.; Massaro, T.; Huynh, K.; Brauner, B.; MahmoudianDehkordi, S.; Louie, G.; Moseley,
M.A.; Thompson, J.W.; et al. Sex and APOE ε4 genotype modify the Alzheimer’s disease serum metabolome. Nat. Commun. 2020,
11, 1148. [CrossRef]
150. Hajjar, I.; Liu, C.; Jones, D.P.; Uppal, K. Untargeted metabolomics reveal dysregulations in sugar, methionine, and tyrosine
pathways in the prodromal state of AD. Alzheimer’s Dement. Diagn. Assess. Dis. Monit. 2020, 12, e12064. [CrossRef]
151. Rayaprolu, S.; Higginbotham, L.; Bagchi, P.; Watson, C.M.; Zhang, T.; Levey, A.I.; Rangaraju, S.; Seyfried, N.T. Systems-based
proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau. Neuropsychopharmacology 2020, 46, 98–115.
[CrossRef] [PubMed]
152. Dey, K.K.; Wang, H.; Niu, M.; Bai, B.; Wang, X.; Li, Y.; Cho, J.-H.; Tan, H.; Mishra, A.; High, A.A.; et al. Deep undepleted human
serum proteome profiling toward biomarker discovery for Alzheimer’s disease. Clin. Proteom. 2019, 16, 16. [CrossRef]
153. de Toda, I.M.; Miguélez, L.; Vida, C.; Carro, E.; De la Fuente, M. Altered Redox State in Whole Blood Cells from Patients with
Mild Cognitive Impairment and Alzheimer’s Disease. J. Alzheimer’s Dis. 2019, 71, 153–163. [CrossRef] [PubMed]
154. Fracassi, A.; Marcatti, M.; Zolochevska, O.; Tabor, N.; Woltjer, R.; Moreno, S.; Taglialatela, G. Oxidative Damage and Antioxidant
Response in Frontal Cortex of Demented and Nondemented Individuals with Alzheimer’s Neuropathology. J. Neurosci. 2020, 41,
538–554. [CrossRef] [PubMed]
155. Mecocci, P.; Polidori, M.C.; Cherubini, A.; Ingegni, T.; Mattioli, P.; Catani, M.; Rinaldi, P.; Cecchetti, R.; Stahl, W.; Senin, U.; et al.
Lymphocyte oxidative DNA damage and plasma antioxidants in Alzheimer disease. Arch. Neurol. 2002, 59, 794–798. [CrossRef]
[PubMed]
156. Scheff, S.W.; Neltner, J.H.; Nelson, P.T. Is synaptic loss a unique hallmark of Alzheimer’s disease? Biochem. Pharmacol. 2014, 88,
517–528. [CrossRef]
157. Sakamoto, T.; Cansev, M.; Wurtman, R.J. Oral supplementation with docosahexaenoic acid and uridine-50 -monophosphate
increases dendritic spine density in adult gerbil hippocampus. Brain Res. 2007, 1182, 50–59. [CrossRef]
158. De Bruin, N.; Kiliaan, A.; De Wilde, M.; Broersen, L. Combined uridine and choline administration improves cognitive deficits in
spontaneously hypertensive rats. Neurobiol. Learn. Mem. 2003, 80, 63–79. [CrossRef]
Antioxidants 2021, 10, 1353 22 of 22

159. van Wijk, N.; Broersen, L.M.; de Wilde, M.C.; Hageman, R.J.; Groenendijk, M.; Sijben, J.W.; Kamphuis, P.J. Targeting Synaptic
Dysfunction in Alzheimer’s Disease by Administering a Specific Nutrient Combination. J. Alzheimer’s Dis. 2013, 38, 459–479.
[CrossRef]
160. Rikkert, M.G.O.; Verhey, F.R.; Blesa, R.; von Arnim, C.A.; Bongers, A.; Harrison, J.; Sijben, J.; Scarpini, E.; Vandewoude, M.F.;
Vellas, B.; et al. Tolerability and Safety of Souvenaid in Patients with Mild Alzheimer’s Disease: Results of Multi-Center, 24-Week,
Open-Label Extension Study. J. Alzheimer’s Dis. 2015, 44, 471–480. [CrossRef]
161. Tadokoro, K.; Morihara, R.; Ohta, Y.; Hishikawa, N.; Kawano, S.; Sasaki, R.; Matsumoto, N.; Nomura, E.; Nakano, Y.; Takahashi,
Y.; et al. Clinical Benefits of Antioxidative Supplement Twendee X for Mild Cognitive Impairment: A Multicenter, Randomized,
Double-Blind, and Placebo-Controlled Prospective Interventional Study. J. Alzheimer’s Dis. 2019, 71, 1063–1069. [CrossRef]
[PubMed]
162. Minhas, R.; Bansal, Y.; Bansal, G. Inducible nitric oxide synthase inhibitors: A comprehensive update. Med. Res. Rev. 2019, 40,
823–855. [CrossRef]
163. Cummings, J.; Lee, G.; Zhong, K.; Fonseca, J.; Taghva, K. Alzheimer’s disease drug development pipeline: 2021. Alzheimer’s
Dement Transl. Res. Clin. Interv. 2021, 7, e12179. [CrossRef]
164. Kaufmann, W.E.; Sprouse, J.; Rebowe, N.; Hanania, T.; Klamer, D.; Missling, C.U. ANAVEX®2-73 (blarcamesine), a Sigma-1
receptor agonist, ameliorates neurologic impairments in a mouse model of Rett syndrome. Pharmacol. Biochem. Behav. 2019, 187,
172796. [CrossRef]
165. Díaz, M.; Mesa-Herrera, F.; Marín, R. DHA and Its Elaborated Modulation of Antioxidant Defenses of the Brain: Implications in
Aging and AD Neurodegeneration. Antioxidants 2021, 10, 907. [CrossRef] [PubMed]
166. Corsinovi, L.; Biasi, F.; Poli, G.; Leonarduzzi, G.M.; Isaia, G. Dietary lipids and their oxidized products in Alzheimer’s disease.
Mol. Nutr. Food Res. 2011, 55, S161–SS172. [CrossRef]
167. Correia, S.; dos Santos, R.X.C.; Perry, G.; Zhu, X.; Moreira, P.I.; Smith, M.A. Insulin-resistant brain state: The culprit in sporadic
Alzheimer’s disease? Ageing Res. Rev. 2011, 10, 264–273. [CrossRef] [PubMed]
168. Liu, H.; Ye, M.; Guo, H. An Updated Review of Randomized Clinical Trials Testing the Improvement of Cognitive Function of
Ginkgo biloba Extract in Healthy People and Alzheimer’s Patients. Front. Pharmacol. 2020, 10, 1688. [CrossRef] [PubMed]
169. Zabel, M.; Nackenoff, A.; Kirsch, W.M.; Harrison, F.E.; Perry, G.; Schrag, M. Markers of oxidative damage to lipids, nucleic acids
and proteins and antioxidant enzymes activities in Alzheimer’s disease brain: A meta-analysis in human pathological specimens.
Free. Radic. Biol. Med. 2017, 115, 351–360. [CrossRef]
170. Hampel, H.; O’Bryant, S.E.; Molinuevo, J.L.; Zetterberg, H.; Masters, C.; Lista, S.; Kiddle, S.; Batrla, R.; Blennow, K. Blood-based
biomarkers for Alzheimer disease: Mapping the road to the clinic. Nat. Rev. Neurol. 2018, 14, 639–652. [CrossRef]