REVIEW
Neuronal Death Mechanisms and Therapeutic Strategy
in Ischemic Stroke
Rui Mao1 • Ningning Zong1 • Yujie Hu1 • Ying Chen1 • Yun Xu1,2,3,4,5
Received: 1 December 2021 / Accepted: 18 January 2022
Ó The Author(s) 2022
Abstract Ischemic stroke caused by intracranial vascular
occlusion has become increasingly prevalent with considerable
mortality and disability, which gravely burdens the
global economy. Current relatively effective clinical treatments
are limited to intravenous alteplase and thrombectomy.
Even so, patients still benefit little due to the short
therapeutic window and the risk of ischemia/reperfusion
injury. It is therefore urgent to figure out the neuronal death
mechanisms following ischemic stroke in order to develop
new neuroprotective strategies. Regarding the pathogenesis,
multiple pathological events trigger the activation of
cell death pathways. Particular attention should be devoted
to excitotoxicity, oxidative stress, and inflammatory
responses. Thus, in this article, we first review the principal
mechanisms underlying neuronal death mediated by these
significant events, such as intrinsic and extrinsic apoptosis,
ferroptosis, parthanatos, pyroptosis, necroptosis, and
autophagic cell death. Then, we further discuss the
possibility of interventions targeting these pathological
events and summarize the present pharmacological
achievements.
Keywords Ischemic stroke Á Neuronal death Á Mechanisms
Á Therapeutic strategy
Introduction
Globally, stroke is among the primary causes of mortality
and disability [1]. Among strokes, ischemic stroke (IS)
accounts for [70%. Neuronal death caused by cerebral
ischemia determines the mortality and disability rate of
stroke. In general, reversing neuronal death in the ischemic
infarct core is very challenging, and for many years,
strategies that have focused on preventing neuronal death
in the penumbra (the region surrounding the infarct core)
and secondary neuronal loss have failed because of unclear
mechanisms.
Multiple paradigms of cell death have been progressively
defined since the last century. Aside from the
commonly-known forms of cell death, such as apoptosis,
neurons can undergo several rarely known modes, such as
ferroptosis, pyroptosis, and parthanatos [2]. Any kind of
cell death is inseparable from a cascade of detrimental
events that occur in IS, especially in the pathological
processes of excitotoxicity, oxidative stress, and inflammatory
responses. These deleterious pathological events
are mutually independent and extensively intersect in
promoting cell death.
Here, we review the mechanisms of the significant
events following IS and the different forms of neuronal
death secondary to these pathological factors. We also
Rui Mao, Ningning Zong, Yujie Hu and Ying Chen contributed
equally to this work.
& Yun Xu
xuyun20042001@aliyun.com
1
Department of Neurology, Affiliated Drum Tower Hospital,
Medical School of Nanjing University, Nanjing 210008,
China
2
The State Key Laboratory of Pharmaceutical Biotechnology,
Institute of Brain Science, Nanjing University, Nanjing
210008, China
3
Jiangsu Key Laboratory for Molecular Medicine, Medical
School of Nanjing University, Nanjing 210008, China
4
Jiangsu Province Stroke Center for Diagnosis and Therapy,
Nanjing 210008, China
5
Nanjing Neurology Clinic Medical Center, Nanjing 210008,
China
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https://doi.org/10.1007/s12264-022-00859-0 www.springer.com/12264
illustrate the therapeutics of excitotoxicity, oxidative stress,
and inflammatory responses, in order to enrich the
currently limited clinical treatment. Considering the particularity
of autophagy, we list this separately for discussion
(Fig. 1).
Excitotoxicity and Neuronal Death
As the primary neurotransmitter in the central nervous
system (CNS), glutamate is responsible for rapid synaptic
transmission so that communication among neurons can be
realized. But it has neurotoxic effects under pathological
conditions. This special type of neurotoxicity mediated by
excitatory amino-acids is called excitotoxicity, which is the
key link between ischemia and neuronal death in strokes.
Fig. 1 A The main structure of this review. The content in the dashed frame represents the relevant therapeutic targets. B A simple schematic of
several death mechanisms in ischemic stroke.
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Among the currently-known ionotropic and metabotropic
glutamate receptors, N-methyl-d-aspartate receptors
(NMDARs) play an important role in permitting excessive
Ca2?
influx, which in turn leads to ischemic cell death.
Growing evidence has demonstrated the dual effects of
NMDARs on neuronal outcomes [3], which can be boiled
down to several hypotheses, such as the ‘NMDAR subtype’
hypothesis and the ‘NMDAR location’ hypothesis.
Overactivated NMDARs during cerebral ischemia are
heterotetrameric complexes involving two essential NR1
(GluN1) subunits and two NR2 (GluN2) subunits [4]. The
‘NMDAR subtype’ hypothesis emphasizes that GluN2AR
conduces to neuronal survival while GluN2BR induces
neuronal death [5]. In addition, as the second hypothesis
suggests, synaptic NMDARs contribute to neuronal survival
while extrasynaptic receptors activate distinct downstream
death signaling proteins [6]. But multiple studies
have demonstrated that both synaptic and extrasynaptic
NMDARs participate in triggering the cell death signaling
pathway [3].
The pro-survival effects of NMDARs are attributable to
activation of the PI3K (phosphoinositide-3-kinase)–Akt
(protein kinase B) pathway, activation of the ERK
(extracellular signal regulated kinase) pathway, and expression
of the CREB (cAMP-response element binding
protein)-related gene in an activity-dependent manner.
It is worth noting that NMDARs mediate neuronal death
or survival according to their activity. Hyper-activity or
hypo-activity is often detrimental, whereas normal
NMDAR activity promotes cell survival. Both high concentrations
of NMDA and strong pharmacological inhibition
induce neuronal death. Research has revealed the
dynamic changes of NMDAR activity in the acute neurodestructive
phase and the subsequent recovery phase of
IS [6, 7]. A late-onset persistent decline of NMDAR
activity has been reported after overactivation. This can
prevent secondary neuronal loss in the penumbra and
increase neurogenesis in the dentate gyrus [8].
Excitotoxicity and Apoptosis by the Intrinsic/Mitochondrial
Pathway
In 1972, Kerr et al. first described the morphological
features of apoptosis, including nuclear and cytoplasmic
condensation, apoptotic body formation, and cell fragmentation
[9]. As is widely known, apoptotic cell death is
programmed and can be initiated by the intrinsic (mitochondrial)
or the extrinsic (death receptor) pathway. Ca2?
overload mediated by excitotoxicity during cerebral
ischemia leads to apoptosis principally through the former
pathway.
Increased intracellular Ca2?
mainly depends on rapid
Ca2?
influx via NMDARs and other non-excitotoxic
means, including acid-sensing ion channels [10], transient
receptor potential channels [11], and Na?
/Ca2?
exchangers
[12]. The dysfunction of these homeostasis regulators
gradually causes Ca2?
overload, hence triggering calpain
activation [13]. Calpain can cleave Bcl-2 interacting
domain (BID) to produce tBID, which interacts with Bax
(Bcl-2-associated X protein). Bax forms homo-oligomers
and then inserts them into the mitochondrial outer membrane.
As a result, mitochondrial permeability transition
pores take shape [2, 14, 15] and thus allow the release of
apoptogens.
Cytochrome C (Cytc) is one of the most important proapoptotic
factors. Upon entering the cytosol, Cytc combines
with apoptotic protein-activating factor-1 (Apaf-1) and
procaspase-9 to form the apoptosome. Procaspase-9
depends on autocleavage to become mature, thereby
progressively activating the executor caspase-3 to mediate
intrinsic apoptosis [16, 17] (Fig. 2).
In addition, when neurons undergo ischemic insults,
NMDARs can activate death-associated protein kinase 1
Fig. 2 Apoptosis by the intrinsic/mitochondrial pathway during
cerebral ischemia. Increased extracellular glutamate activates
NMDARs, causing excessive Ca2?
influx. Activated calpain cleaves
BID to tBID, which interacts with Bax and helps to form the mPTP.
Cytc is released from these pores and combines with Apaf-1 and procaspase-9.
The apoptosome stepwise activates the executioner
caspase-3, and consequently induces apoptosis. Apaf-1, apoptotic
protein-activating factor-1; Bcl-2, B-cell leukemia/lymphoma 2; BID,
Bcl-2 interacting domain; Cytc, cytochrome C; mPTP, mitochondrial
permeability transition pore; NMDA, N-methyl-d-aspartate; tBID,
truncated Bid.
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(DAPK1) and promote DAPK1-p53-dependent apoptotic
cell death [18]. Phosphatase and tensin homolog deleted on
chromosome TEN (PTEN) dephosphorylates PIP3 into
PIP2, inhibiting the PI3K–Akt survival signaling pathway
[19]. GluN2BR-mediated PTEN nuclear translocation has
also been shown to induce apoptosis [20]. C-Jun N-terminal
kinase (JNK) is also involved in caspase-dependent
apoptosis by inducing the phosphorylation of Bcl-2-associated
death promoter [21].
Excitotoxicity and Ferroptosis
Ferroptosis, a lately discovered form of non-apoptotic
death, was first defined in 2012. It is iron-dependent and is
fundamentally attributable to the overwhelming lipid
peroxidation. From a morphological point of view, ferroptosis
with mitochondrial shrinkage can be distinguished
from other forms of cell death [22, 23]. There is evidence
that ferroptosis may be triggered by excitotoxic stress when
neurons are exposed to hypoxia.
The overactivation of NMDARs following stroke can
induce iron uptake through the NMDAR-Dexras1-PAP7divalent
metal transporter 1 (DMT1) signaling cascade,
leading to increased redox-active iron in the neuronal
cytosol [24, 25]. Apart from intracellular iron deposition,
the reduction of the cysteine-GSH-GPX4 axis activity
promotes ferroptosis as well [26]. During excitotoxic
stress, the extravagant accumulation of glutamate inhibits
the cystine/glutamate antiporter, System Xc,
which
reduces the influx of cystine and limits the biosynthesis
of GSH. GPX4, the key negative regulator of ferroptosis,
uses GSH to catalyze lipid hydroperoxides into alcohols
[27]. Hence, low levels of GSH can weaken the activity of
GPX4 and promote overwhelming peroxidation.
Ca2?
overload can activate cytosolic phospholipase A2a
(cPLA2a), which mediates the release of arachidonic acid
and lysophospholipid to provide substrates for lipid
peroxidation [28]. All the mechanisms described above
can work together to eventually trigger ferroptotic neuronal
death in IS [14]. Many studies have reported that ferroptosis
inhibitors including Ferrostatin-1 and Liproxstatin-1
prevent glutamate-induced neurotoxicity and have neuroprotection
effects [26, 29] (Fig. 3).
Excitotoxicity and Parthanatos
Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear
protein that limits genomic instability and facilitates DNA
base repair as well as the regulation of inflammatory
processes [30]. Nevertheless, in many neurodegenerative
diseases, rapid PARP-1 activation can result in a unique
cell death mode called parthanatos [31, 32].
During cerebral ischemia, the C-terminal domains of
GluN2B binding to postsynaptic density protein-95 (PSD-
95) [33] link NMDARs to downstream neurotoxic
molecules such as nitric oxide synthase (nNOS) [34]. In
addition to PSD-95, PSD-93 is another molecular adaptive
protein that glues NMDAR and nNOS together [35]. The
GluN2B-PSD95/93-nNOS complex combines excitotoxicity
with neuronal DNA injury that is caused by oxidative or
nitrosative stress. When DNA damage occurs, PARP-1 can
use NAD?
as a substrate to synthesize PAR, thereby
inducing the nuclear translocation of apoptosis-inducing
factor (AIF) [36–38].
The formation of mPTPs allows the release of AIF into
the neuronal cytosol during excitotoxic stress. During this
process, AIF recruits migration inhibitory factor (MIF) and
they both rapidly translocate to the nucleus, which
eventually mediates DNA fragmentation and chromatin
condensation [39]. Besides, PAR promotes bioenergetic
collapse and causes neuronal death by inhibiting hexokinase,
one of the key rate-limiting enzymes of glycolysis
[40]. At the same time, NAD?
depletion impairs metabolic
processes, causes mitochondrial dysfunction, and aggravates
cellular damage [36] (Fig. 4).
Excitotoxicity also induces autophagic cell death in IS.
Fig. 3 Ferroptosis during cerebral ischemia. Normally, GPX4 uses
GSH to catalyze lipid hydroperoxides into alcohols. Increased
extracellular glutamate inhibits the activity of System Xcas
well
as the cysteine-GSH-GPX4 axis. Meanwhile, Ca2?
overload activates
cPLA2a to provide substrates for lipid peroxidation. Fe2?
-activated
lipoxygenase (LOX) also participates in this process. Increased redoxactive
iron and overwhelming lipid peroxidation contribute to
ferroptosis. cPLA2a, cytosolic phospholipase A2a; GPX4, c-Lglutamyl-L-cysteinylglycine
peroxidase 4; GSH, glutathione; GSSG,
oxidized glutathione; LOX, lipoxygenase; PUFA, polyunsaturated
fatty acid; TRF, transferrin.
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Therapeutics of Excitotoxicity in Ischemic Stroke
Targeting Glutamate
Previous preclinical research has shown that decreasing the
release or enhancing the reuptake of glutamate are effective
therapeutic candidates against ischemic injury. However,
the majority of these established agents are inefficient in
reducing morbidity or mortality in stroke clinical trials, and
have more or less neurological side-effects. Currently,
there are several potential therapeutic targets worth exploration.
For instance, the latest research found that the
subunit Swell1 of the volume-regulated anion channel
activated by cell swelling is responsible for the release of
glutamate [41]. N-myc downstream regulated gene 2
interacts with Na?
/K?
-ATPase b1 to facilitate glutamate
uptake in astrocytes [42].
Compared with drug treatment, blood glutamate scavenging
does not interfere with normal brain neurophysiology
[43]. Peritoneal dialysis has been found effective in
reducing blood glutamate in rats during cerebral ischemia
[44]. This finding needs to be further verified in clinical
trials.
Targeting NMDARs
As early as the late 1980s, researchers discovered that the
NMDAR antagonist dizocilpine (MK-801) significantly
protects the brain against focal cerebral ischemia in the rat
[45]. With an in-depth understanding of their
pharmacological effects, NMDAR antagonists can be
divided into competitive antagonists at the recognition
site, uncompetitive ion channel blockers, and subunitspecific
antagonists [46]. A series of drugs have been
reported to be effective against ischemic insults both
in vitro and in vivo, such as uncompetitive NMDAR
antagonists (ketamine, phencyclidine, dextromethorphan,
and dizocilpine) and glycine-binding site antagonists
(gavestinel and licostine). Nevertheless, there is a lack of
clinical success regarding the fact that these agents may
induce pathomorphological changes and neurological dysfunction
[5].
In recent years, based on the ‘NMDAR subtype’
hypothesis, more and more targeted therapies and interventions
have entered the preclinical stage. GluN2BRselective
NMDA antagonists, like CP-101 606 and Ro
25-6981 [47, 48], failed in clinical trials due to safety
reasons and limited efficacy, even though they were
effective against neuronal damage. Similar drugs still face
the same problems, including a short therapeutic window
and serious side-effects [3]. Along with progress in the
reduction of adverse effects, the pH-dependent GluN2BRselective
antagonists, the 93-series, may be more clinically
promising because they have less impact on the healthy
brain at normal pH [49]. A novel synthesized brainpenetrant
GluN2BR antagonist called compound 45e also
exhibits superior neuroprotective activity [50]. By contrast,
some current studies prefer to exploit GluN2AR-positive
allosteric modulators to enhance neuronal survival signaling
[51, 52].
Although the validity of the ‘NMDAR location’ hypothesis
is still under discussion, interventions based on it have
already attained certain achievements. Memantine, which
is used to treat Alzheimer’s disease, has been found to
preferentially block the open channels of extrasynaptic
NMDARs at therapeutic concentrations. Its pharmacological
effects on neuroprotection and neuroplasticity have
been confirmed in preclinical strokes [53, 54].
Targeting Downstream Death Signaling Proteins
DAPK1 activation is inseparable from calcineurin activation.
It is through reducing calcineurin activity that
Protopanaxadiol ginsenoside-Rd alleviates the DAPK1mediated
phosphorylation of GluN2B [55]. Besides, Caytaxin
also inhibits DAPK1 catalytic activity. It combines
with DAPK1 at the presynaptic site and kicks in 2 h after
middle cerebral artery occlusion (MCAO) [56]. Likewise,
the interference peptide Tat-NR2B-CT blocks the effects of
DAPK1 on GluN2B [57].
The PTEN inhibitor bpv, which is a bisperoxovanadium
compound, decreases MCAO-induced neuronal apoptosis
by preventing the downregulation of phospho-mTOR.
Fig. 4 Parthanatos during cerebral ischemia. PSD-95 links NMDARs
to nNOS. Neuronal DNA injury that is caused by oxidative or
nitrosative stress activates PARP-1, which uses NAD?
to synthesize
PAR. PAR inhibits HK and promotes the nuclear translocation of
AIF. The mPTP allows the release of AIF into the cytoplasm where it
binds to MIF. The AIF–MIF complex enters the nucleus and mediates
DNA fragmentation. HK, hexokinase; MIF, migration inhibitory
factor; NAD?
, nicotinamide adenine dinucleotide; nNOS, nitric oxide
synthase; PAR, poly (ADP-ribose); PARP-1, poly (ADP-ribose)
polymerase-1; PSD-95, postsynaptic density protein-95.
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Therefore, it can be concluded that PTEN deletion protects
the ischemic brain by activating the mTOR survival
signaling pathway [58]. Moreover, a considerable number
of microRNAs prevent cerebral ischemic damage through
the PTEN-PI3K-Akt pathway, including miR-130a [59],
miR-532-5p [60], miR-188-5p [61], and miR-217-5p [62].
JNK-IN-8, a highly specific JNK inhibitor, suppresses
the JNK/NF-jB pathway and the activation of microglia. It
decreases the expression of several pro-inflammatory
factors and further controls neuroinflammation as well as
ischemic injury [63]. Roflumilast, approved for the treatment
of chronic obstructive pulmonary disease, has been
found to prevent neurons from ischemia/reperfusion injury
by attenuating the phosphorylation of JNK [64]. Regrettably,
none of the above agents have achieved clinical
application for the treatment of IS.
Tat-NR2B-9c (NA-1) is an interference peptide that
binds to PSD-95. The neuroprotective effect of NA-1
against ischemic insults has been demonstrated in rodent
models and non-human primate models [65]. It is encouraging
that the phase III clinical trial, ESCAPE-NA1,
achieved significant results. This trial enrolled 1,105
patients who suffered acute IS within 12 h to measure
the efficacy and safety of NA-1 [66]. According to the
results, there are still problems to be solved with respect to
the timing and strategy of NA-1 though its value cannot be
denied. TP95 is a cell-penetrating peptide containing PSD-
95 cleavage sites. When delivered to the cortex, it
downregulates PSD-95 and improves the neurological
outcome during excitotoxic stress [67].
Oxidative Stress and Neuronal Death
When an IS occurs, neurons generate excessive amounts of
superoxide compounds, including hydroxyl radical and
nitric oxide, due to hypoxia and subsequent reperfusion
stimulation. The generation of these molecules is related to
nicotinamide adenine dinucleotide phosphate (NADPH)
oxidase, cyclooxygenases, and xanthine oxidase [68, 69],
among which, NADPH oxidase is considered to be the
major source of reactive oxygen species (ROS) in the brain.
As an electron donor, NADPH produces O2
-
in
response to NADPH oxidase (NOX) catalysis. NOX is a
membrane-bound enzyme complex consisting of seven
subtypes that have been detected in neuronal cells,
microglia, fibroblasts, and endothelial cells [70]. After
the occurrence of IS, NOX2, NOX1, NOX4, and NOX5 in
neuronal cells are activated to produce peroxides such as
H2O2 and O2- [68]. NOX has been studied from the
perspective of the nervous system and it was found that the
expression of NOX2 and NOX4 are increased in neuronal
cells in IS. Besides, through negatively regulating NOX2,
miR-320 reduces the generation of ROS in IS so as to
protect neurons [71].
The continuous accumulation of oxidative molecules in
cells leads to the peroxidation of lipids and the crosslinking
of macromolecules such as DNA and proteins.
These oxidative molecules also mediate signaling pathways
which induce different types of cell death [72], such
as ferroptosis, apoptosis, and autophagy.
Oxidative Stress and Apoptosis
After a stroke, the depolarization of the cell membrane
caused by ATP depletion is accompanied by the massive
production of ROS. ROS can directly damage the integrity
of plasma membrane and can lead to DNA strand breaks as
well. Besides, it seems to participate in the release of Cytc
from mitochondria into the cytoplasm of neurons. A
research team also found that ultraviolet irradiation or
anticancer drugs generate ROS, which then mediate
apoptosis by activating apoptosis signal-regulating kinase
1 [73]. In brief, the damage caused by ROS promotes
apoptosis in many ways [14].
Oxidative stress primarily damages mitochondria,
including mitochondrial DNA, Ca2?
ion balance, and the
mitochondrial membrane, and induces apoptosis by the
intrinsic pathway. The key element of apoptosis in the
mitochondrial pathway is mitochondrial outer membrane
permeabilization, which can be regulated by affecting the
Bcl-2 protein family or downstream caspase [73, 74].
Under oxidative stress, as a nuclear transcription factor,
p53 is activated and then regulates pro-apoptotic genes
such as Bax and Bak (Bcl-2 antagonist or killer). It also
directly acts on mitochondria, resulting in the release of
Cytc and the activation of caspase, and finally induces
apoptosis [75]. It has been shown that oxidative stress
responsive apoptosis inducing protein acts on the MAPK
signaling pathway and the JAK/STAT signaling pathway to
activate caspase, causing caspase-dependent apoptosis
[76].
In addition, studies have shown that TRPM2, which
belongs to the transient receptor potential channel superfamily,
is associated with the normal transport of Ca2?
and
is highly sensitive to oxidative stress. When stimulated by
oxidative stress, TRPM2 channels cause the destruction of
intracellular Ca2?
and Zn2?
homeostasis. The increasing
intracellular Ca2?
concentration plays an important role in
initiating apoptosis [14, 77].
Oxidative Stress and Ferroptosis
Furthermore, iron overload and excessive lipid peroxidation
are closely linked to oxidative stress and contribute to
ferroptosis.
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Normally, under the gradual action of transferrin and
DMT1, the iron in a cell is continuously transformed in the
state of Fe2?
and Fe3?
, and circulates throughout the body,
and is eventually transported to the labile iron pool (LIP) in
the cytoplasm in the form of Fe2?
[78]. The iron level in
the LIP is transferred by ferritin (FT), and regulated by
both FT and iron response protein (IRP), to keep the Fe2?
concentration in the LIP in a narrow range to achieve
dynamic balance [79]. When cells are under oxidative
stress, ROS and NOS act on IRP1, IRP2, and other
proteins, leading to intracellular iron overload by regulating
the IRP/iron response element system [80, 81]. Consequently,
excessive iron damages cells through the Fenton
reaction and lipid peroxidation. During cerebral ischemia,
the accumulating iron in cells enters the brain parenchyma
through the damaged blood-brain barrier [78]. Excessive
Fe2?
combines with a large number of superoxides in the
brain to produce Fe3?
and hydroxyl radical. At the same
time, Fe2?
is an active molecule in the catalytic subunit of
LOX, which catalyzes lipid peroxidation. The overproduced
hydroxyl radical and lipid peroxide induce oxidative
stress and cell death [82].
Lipid peroxidation also provides abundant ROS for the
oxidative stress of neuronal cells. Fatty acids in the brain
stem account for about half of the mass, of which
polyunsaturated fatty acid (PUFA) accounts for *40%.
PUFA often contains multiple double bonds, among which
arachidonic acid and adrenergic acid are particularly
susceptible to oxidization to form lipid peroxidation
products [83, 84]. The production of lipid peroxides results
in a massive accumulation of ROS and lipid hydrogen
peroxide. And the amount and location of PUFA are related
to the degree of lipid peroxidation, which indirectly
determines the degree of the ferroptosis effect [85].
GSH and oxidized glutathione (GSSG) constitute the
antioxidant system in cells. This system eliminates oxidative
species to protect cells from oxidative damage.
Previous studies have shown that there is a certain
relationship between cell oxidative death and the significant
depletion of GSH/GSSG, and have also shown that the
inducer used in the test consumes GSH by inhibiting
system Xc[86].
At the same time, GSH combines with
Fe2?
in the LIP, so GSH directly prevents Fe2?
from
oxidizing to generate hydroxyl radical and reduce the
damage of cells caused by oxidative stress [85]. Sesamin
reduces the ischemic brain injury in mice by reducing the
levels of lipid peroxidation and superoxide anion and
restoring the level of GSH [87].
Oxidative stress also participates in facilitating
autophagy.
Therapeutics of Oxidative Stress in Ischemic Stroke
Here is a brief list of some novel drugs with therapeutic
potential. (1) Prussian blue (PB) has been used clinically as
an antidote to metal poisoning such as by cesium, and as a
complex contrast agent for ultrasound. New studies have
found that PB has the same internal enzyme activity as
Fe3O4 to protect cells from oxidative stress and plays an
anti-apoptotic role by eliminating ROS and adjusting the
expression of p53 and Bcl-2 in cells [88]. (2) Calycosin-7O-b-D-glucoside
is the main component of Astragalus
isoflavones. It plays a protective role in neurons after IS
through decreasing SOD activity, apoptosis rate, and Bax
protein expression, and activating the STRTI/FoxO1/PGC-
1a signaling pathway [89]. (3) Melatonin, which is already
used to regulate sleep and delays aging, has antioxidation
and anti-autophagy effects by blocking NF-jB signal
transduction and activating the mTOR signaling pathway
in experimental stroke [90]. (4) Chlorogenic acid is a
polyphenol. It protects the brain against stroke by activating
the Nrf2 signaling pathway, which enhances the
activity of SOD and GSH, and reduces the content of
intracellular ROS and the rate of apoptosis [91]. (5)
Carvacrol, a phenolic monoterpenoid that targets ferroptosis,
protects hippocampal neurons after ischemia/reperfusion
in gerbils by reducing the levels of ROS, iron, and
TfR1 protein and increasing the expression levels of GPX4
and Fpn1 protein [92]. (6) Edaravone Dexbomeol is a
compound of edaravone and dexcamphenol. It has been
demonstrated that this novel neuroprotective drug
improves the prognosis of acute IS patients by inhibiting
the expression of iROS and TNF-a. In a phase II clinical
trial, NCT01929096, its safety and tolerability are better
than edaravone used alone [93].
Inflammatory Responses and Neuronal Death
Inflammatory responses cover the whole process from the
acute stage to convalescence in IS. On the one hand, the
inflammatory response puts pressure on brain cells and
causes neuron death, and on the other hand, contributes to
tissue repair as one part of the innate defense system. In the
acute phase of IS, the inflammatory response causes
irreversible brain damage.
The inflammatory response starts as early as the onset of
ischemia. As noted above, oxidative stress and excitotoxicity
occur immediately when the blood vessels in the brain
are blocked, with the activation of microglia proceeding
within minutes. In parallel, damage-associated molecular
patterns (DAMPs) released by dead cells, such as purines,
also activate microglia [94]. Subsequently, activated
microglia release numerous pro-inflammatory factors
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which recruit peripheral immune cells and activate other
brain cells such as astrocytes. Pro-inflammatory factors
promote ICAM-1 and selectin expression on endothelial
cells, making it easier for peripheral immune cells to reach
the ischemic area. Recruited peripheral immune cells also
secrete pro-inflammatory factors [95].
Astrocytes also contribute to the inflammatory response
and exacerbate damage to neurons after IS. Cytokines
released by activated microglia like IL-1a, TNF-a, and
C1q induce the activation of reactive astrocytes and the
subsequent production of pro-inflammatory mediators
including IL-1, TNF-a, IL-6, interferon c (IFN-c), MMP-
9, radicals, and chemokines like chemokine (C-C motif)
ligand 2 [96, 97]. MMP-9 not only damages brain tissue
but also disrupts the blood-brain barrier, leading to
peripheral immune cell infiltration [98]. ATP released by
dead cells activates the inflammasome via astrocytic
pannexin 1 channels [99]. In addition to pattern recognition
receptors, astrocytes also express the class II major
histocompatibility complex (MHC II) which is the initiator
of the T and B lymphocyte-mediated adaptive immune
response. The antigen in reactive astrocytes presented by
MHC II is induced by the pro-inflammatory cytokine IFN-c
and associated with increased lysosomal exocytosis [100].
In general, pro-inflammatory factors, glial cells, and
immune cells form a positive feedback loop to further
exacerbate brain damage.
Inflammatory Responses and Phagocytosis
In addition to inducing inflammation, microglia are also
capable of consuming dead and dying neurons [101].
Neurons that suffer from hypoxia and stress expose
phosphatidylserine, which is recognized as an ‘‘eat-me’’
signal by microglia [102]. Phagocytosis seems to be
beneficial. However, it has been found that living neurons
in the ischemic penumbra are also engulfed by microglia,
leading to delayed neuronal death at least 24 h after
cerebral ischemia. Blocking this phagocytosis strongly
reduces neuronal loss after IS [103]. Phosphatidylserine
blockade rescues up to 90% of neurons in primary rat
cultures stimulated by lipoteichoic acid or lipopolysaccharide
(LPS) [104]. Activated microglia also display sialidase
activity, leading to the surface deacetylation of microglia
and the complement receptor 3-mediated phagocytosis of
neurons [105].
Inflammatory Responses and Apoptosis
by the Extrinsic/Death Receptor Pathway
Exogenous apoptosis is triggered by TNF-a, FASL and
TRAIL. These ligands bind to death receptors with death
domains including TNF-R1, Fas, and TRAIL-R. Take
TNF-a as an example. The death domain is a critical
structure connecting downstream complex I [TRADD
(tumor necrosis factor receptor type 1-associated death
domain), TRAF2, cIAP1/2, and RIPK1] after ligation of
TNF-R1. CYLD de-ubiquitylates RIPK1 and the rest of
complex I recruits FADD (Fas-Associated protein with
Death Domain) via homotypic death domain interactions
[106]. FADD recruits and then homodimerizes caspase-8,
triggers the caspase cascade, and results in mitochondrial
membrane leakage, DNA cleavage, and apoptosis in
neuronal cells [107]. TNF-R1 also triggers NF-jB signaling
via RIPK1 ubiquitylation by cIAP1/2. The limited
amount of RIPK1 might explain the crosstalk of NF-jB
signaling and the apoptosis pathway emanating from TNFR1.
The sensitivity of cells to TNF-induced apoptosis
increases when NF-jB activity is absent, while artificial
activation of NF-jB prevents apoptosis (Fig. 5).
Inflammatory Responses and Necroptosis
Necroptosis is one type of lytic-programmed cell death. In
addition to apoptosis, TNF-a also triggers neuronal
necroptosis after IS. Necroptosis signal conduction is
triggered by death receptors such as TNFR1, TRAILR,
Fas, IFNR, and TLR3/4 [108]. The activation of death
receptors results in the de-ubiquitylation of receptorinteracting
protein kinase 1 (RIPK1) by the de-ubiquitinating
enzyme CYLD [109]. After RIPK1 activation, the
preserved complex I recruits RIPK3 via homotypic RHIM
domain interactions, consequently forming the RIPK1RIPK3-MLKL
(mixed lineage kinase domain-like) signaling
complex. MLKL forms a homotrimer and subsequently
locates to the cell plasma membrane. RIPK3-mediated
phosphorylation of MLKL induces necroptosis [110].
Recently, growing numbers of molecules have been
discovered to modulate TNF-induced neuronal necroptosis.
Jinho et al. revealed that CHIP (carboxyl terminus of
Hsp70-interacting protein) negatively regulates RIPK3 and
RIPK1 via E3 ligase-mediated ubiquitylation. CHIP-depleted
cells express higher levels of RIPK3 and exhibit
enhanced sensitivity to necroptosis induced by TNF-a
[111]. Accumulating evidence has demonstrated that CHIP
is involved in the pathological progression of IS through
different mechanisms [112]. CHIP overexpression prevents
neuronal degeneration in vitro and in vivo [113]. These
findings suggest that targeting key molecules in necroptosis
signaling may be effective for treating neuronal injury after
IS.
Inflammatory Responses and Pyroptosis
Pyroptosis is a gasdermin D (GSDMD)-mediated form of
regulated cell death featuring continuous cell expansion
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Neurosci. Bull.
until the cell membrane ruptures, resulting in the release of
cell contents and subsequent strong inflammatory
responses. Pyroptosis is classified into the caspase-1dependent
classical pathway and caspase-4/5/11-dependent
pathways. Pathogen-associated pattern molecules and
DAMPs are recognized by pattern recognition receptors,
which subsequently assemble into inflammasomes that
recruit caspase-1 for self-splicing. Activated caspase-1
cleaves GSDMD to form the GSDMD nitrogen terminus
and the carbon terminus, which binds to the phospholipid
proteins on the cell membrane to form pores, release the
contents, and induce pyroptosis. Activated caspase-1 also
cleaves pro-IL-1b and pro-IL-18 to form active IL-1b and
IL-18, and releases them into the extracellular space,
causing an inflammatory response. Intracellular caspase-4/
5/11 directly binds to LPS to undergo auto-oligomeric
activation. They cleave GSDMD and cause pyroptosis
[114].
Pyroptosis actively participates in the inflammatory
response and contributes to neuronal death during IS. An
elevated level of pyroptosis has been reported around the
infarcted area in the early stage of ischemia-reperfusion.
The ablation of GSDMD significantly reduces pyroptosis
and the infraction volume after ischemia/reperfusion by
inhibiting the secretion of mature IL-18 and IL-1b from
microglia [115]. Wang et al. revealed that melatonintreated
exosomes effectively reduce the activation of
caspase-1 and inflammasome-mediated neuronal pyroptosis
through the TLR4/NF-jB signaling pathway, therefore
decreasing the infarct volume and improving recovery
from the functional deficit [116]. Lammert et al. reported
that pyroptosis induced by the AIM2 inflammasome results
in the elimination of genetically compromised neuronal
cells during neurodevelopment [117] (Fig. 6).
Therapeutics of Inflammatory Responses
in Ischemic Stroke
Targeting Microglia
Emerging evidence points out that target microglia are
effective in alleviating the inflammatory response and
neuronal injury after IS. Zhang et al. reported that miR-
146a-5p attenuates microglia-mediated neuroinflammation,
leading to a reduction in infarct volume and neural deficits
through the IRAK1/TRAF6 pathway [118]. Gentianine, an
alkaloid obtained from Gentiana scabra Bunge, inhibits the
activation of microglia and ameliorates inflammatory
responses via the TLR4/NF-jB signaling pathway in a
transient MCAO model [119]. Growing numbers of studies
have shown that lncRNAs regulate the inflammation and
polarization of microglia during cerebral ischemia-reperfusion
injury [120]. In vitro, SNHG4 up-regulates STAT6
and represses inflammation by adsorbing miR-449c-5p in
microglia [121]. Knockdown of the LncRNA MEG3
inhibits M1 polarization and inflammation and promotes
M2 polarization through Kru¨ppel-like factor 4 in vitro and
in vivo [122]. Jin et al. revealed that lncRNA NEAT1 is
strongly correlated with activated-microglia mRNAs via
the transcriptome-wide analysis of mice with focal
ischemia. Knockdown of Neat1 significantly represses the
activation of microglia and subsequent brain damage
caused by pro-inflammatory cytokines released by microglia
[123]. Knockdown of the lncRNA NEAT1 also
alleviates the apoptosis of N2a cells and increases neuronal
viability [124].
Targeting Immune Cells
Several genes in immune cells have been shown to play
crucial roles in modulating the inflammatory response in
the ischemic brain. Genetic deletion of PKM2 in myeloid
cells reduces peripheral neutrophil infiltration and inflammatory
cytokines following cerebral ischemia-reperfusion.
Inhibition of the nuclear translocation of PKM2 significantly
reduces neutrophil hyperactivation and improves
functional deficit recovery following stroke [125]. Meng
et al. reported that infiltrating CD3?
CD4-
CD8T
cells
enhance immune and inflammatory responses, resulting in
destructive ischemic brain injury via modulating the FasL/
Fig. 5 Death ligands induce apoptosis and necroptosis. Death ligands
(TNF-a, FASL, and TRAIL) bind to death receptors. CYLD deubiquitylates
RIPK1 and the rest of complex I recruits FADD. FADD
homodimerizes caspase-8, resulting in apoptosis. RIPK1 activation
contributes to the formation of the RIPK1–RIPK3–MLKL complex.
RIPK3-mediated phosphorylation of MLKL induces necroptosis.
Death receptors also triggers NF-jB signaling via RIPK1 ubiquitylation
by cIAP1/2. CYLD inhibits this process. FADD, Fas-associated
death domain; cIAP1/2, cellular inhibitor of apoptosis 1/2; MLKL,
mixed lineage kinase domain-like; RIPK, receptor interacting serine/
threonine kinases; TRADD, tumor necrosis factor receptor type
1-associated death domain; TRAF, TNFR-associated factor.
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R. Mao et al.: Neuronal Death Mechanisms and Therapeutic Strategy in Ischemic Stroke
PTPN2/TNF-a signaling pathway [126]. Weitbrecht
revealed that CD4?
T cells facilitate delayed B cell
infiltration into the brain and CD4 depletion attenuates
post-stroke cognitive impairment [127]. Nakajima et al.
reported that mucosa-associated invariant T cell deficiency
suppresses the activation of microglia and attenuates
cytokine production, including IL-1b and IL-6 in a
transient MCAO model [128]. Small extracellular vesicles
derived from embryonic stem cells trigger the TGF-b/
Smad pathway in CD4?
T cells, and thereby decrease the
inflammatory response and neuronal death after IS [129].
Targeting Pro-inflammatory Mediators
Growing evidence suggests that therapy against proinflammatory
mediators is potent in alleviating inflammation
during IS. The confinement of MMP-9 expression via
lentivirus injection of hypoxia response element on day 7
after transient MCAO contributes to improved neurological
outcomes, increased peri-infarct microvessels, and alleviation
of ischemia-induced brain atrophy [130]. Nanoparticles
of the pharmacologically active oligosaccharide
material TPCD reduces the expression of pro-inflammatory
cytokines (TNF-a, IL-1b, and IL-6) and inhibits neuronal
apoptosis [131]. The TNF-a receptor inhibitor R-7050
reverses the activation of TNF receptor-I, NF-jB, and IL-6,
and reduces the metabolic alterations in a rat model of
permanent cerebral ischemia [132]. Silencing of lncRNA
SNHG15 decreases the levels of pro-inflammatory cytokines
(TNF-a and IL-1b) and apoptosis of N2a cells via
sequestering miR-18a and subsequently activating the ERK
signaling pathway [133]. Several immunomodulatory drugs
are in clinical trials. A recent randomized controlled phase
II trial has shown that an IL-1 receptor antagonist reduced
inflammation and improved the clinical outcome in 80
patients within 5 h of IS onset, indicating the potential
clinical applications of targeted inflammatory drugs [134].
Autophagy and Autophagy-Dependent Cell Death
(ADCD)
Autophagy, a term first coined by Christian de Duve in the
1960s [135], refers to a conserved intracellular catabolic
degradation pathway that is activated in response to
starvation and stress [136]. By delivering cytoplasmic
constituents to lysosomes and digesting them, cells can
maintain homeostasis by recycling degraded metabolic
elements. The process is vital to the survival of cells
[137, 138]. However, it is well-known that a dual effect is
manifested in autophagy. In detail, excessive activation of
autophagy has a detrimental impact on cellular functions
and then causes cell injury or even death [139]. Similarly,
the role of autophagy underlying IS remains unclear since
it serves as a double-edged sword that can either protect or
damage neurons upon ischemic insult [140]. In fact, either
impaired or excessive induction of autophagy leads to
neuronal cell injury, and the boundary between lethal and
non-lethal basal autophagy remains vague.
Recently, the concept of ADCD has been put forward
and sparked a heated debate. The Nomenclature Committee
of Cell Death defines ADCD as ‘a form of regulated
cell death that mechanistically depends on the autophagic
machinery (or components thereof)’ [141]. This concept
describes cell death exclusively caused by autophagy,
without the involvement of other death mechanisms like
apoptosis or necroptosis. However, the concept is severely
challenged given the widespread interplay between
Fig. 6 Pyroptosis during ischemic stroke. DAMPs bind to PRRs,
resulting in the formation of inflammasomes and the production of
caspase-1. On the one hand, caspase-1 cleaves GSDMD to form pores
on the cell membrane, resulting in content release and pyroptosis. On
the other hand, caspase-1 cleaves pro-IL-1b and pro-IL-18 to form
active IL-1b and IL-18, causing inflammatory responses. GSDMD,
gasdermin D; DAMPs, damage-associated molecular patterns; PRR,
pattern recognition receptor.
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Neurosci. Bull.
autophagy and apoptosis or necrosis [142]. Moreover,
historically, morphological criteria are solely applied to
distinguish ‘autophagic cell death’, but they have been
confirmed to be misleading [143, 144]. According to the
Nomenclature Committee, only when at least two molecular
components associated with autophagy are genetically
silenced to block cell death is this phenomenon defined as
ADCD. The most well-accepted form of ADCD is
‘autosis’, which was named by Levine and his colleagues
in 2015 and is dependent on Na?
/K?
-ATPase [145].
Role of Autophagy in Ischemic Stroke
In effect, the role of autophagy in IS has never been clearly
defined and described: some studies have shown autophagy
to be deleterious in IS while others consider it to be
neuroprotective. Here, we review some of these findings
and focus on the negative effects of autophagy [146].
The neuronal deletion of Atg7 in mice prevents hypoxiainduced
neuronal autophagy and reduces neuronal death in
multiple brain regions, and thereby results in a 42%
decrease of tissue loss compared to wild-type mice.
Increased numbers of microtubule-associated protein 1
light chain 3-, lysosomal-associated membrane protein 1-,
and cathepsin D-positive cells have also been found,
indicating upregulated autophagy after severe ischemic
insult [147]. Overexpression of TP53-induced glycolysis
and apoptosis regulator (TIGAR) significantly reduces the
activation of ischemia/reperfusion-induced autophagy,
alleviates brain damage, and protects against neuronal
injury by suppressing autophagy through upregulating
phosphorylated mTOR and S6KP70 [148]. Moreover,
dysfunctional lysosomal storage is associated with the
early burst of autophagy, then inducing synaptic impairment
in neurons [149]. Besides, autophagy is considered to
be involved in blood-brain barrier disruption [150].
Enhanced autophagic activity and loss of occludin have
been reported in brain endothelial cells in a mouse model
of MCAO; this ultimately resulted in permeability changes
and contributed to an amplification of ischemic brain
damage [150]. The activation of autophagy can also
activate the cathepsin-tBid-mitochondrial apoptotic signaling
pathway by losing stabilization of the lysosomal
membrane in ischemic cells [151].
Autophagy may induce apoptosis and eventually result
in neuronal cell death through these pathways: Primarily,
p53 upregulates transactivating damage-regulated autophagy
modulator 1 (DRAM), triggers mitochondrial outer
membrane permeabilization and upregulates pro-apoptotic
genes such as Bax and Bak [152, 153]. In addition, BH3only
proteins and BH3 mimics competitively bind to Bcl-2
and induce autophagy. This then elicits the inhibition of
anti-apoptotic proteins like Bcl-2 and Bcl-XL and
activation of pro-apoptotic members such as Bax and
Bak [154, 155]. Another category is Ser/Thr kinases which
includes DAPK, JNK, and protein kinase B (PKB/AKT).
Among them, DAPK and AKT play similar roles as BH3only
proteins. Activation of p19ARF along with p53 and
protein phosphatase 2A is also involved in DAPK pathways
[155]. Besides, oncogenes like MYC and RAS, as
well as ROS, ceramide, and Ca2?
overload are somewhat
considered to regulate both autophagy and apoptosis
[155, 156].
Induction of Autophagy in Ischemic Stroke
As discussed above, a variety of stress factors occur in IS.
Pathological changes like excitotoxicity, oxidative stress,
and inflammatory responses all trigger multiple signaling
cascades of autophagy. Then, excessive initiation of
autophagy and subsequent cellular death occur on account
of these stimulants [146, 157].
Overactivated NMDARs can bring about increased
intracellular Ca2?
as well as redox imbalance [3]. Such
alterations are able to cause unfolded or misfolded proteins
to accumulate in the endoplasmic reticulum (ER) lumen,
leading to ER stress [158]. Afterwards, maladaptive
autophagy is initiated via three major transmembrane
sensors, among which, PERK and its downstream mediators
like ATF4 and DDIT3 promote autophagosome
formation by upregulating the transcription of microtubule
associated protein 1 light chain 3 beta (MAP1LC3) and
ATG5 [159]. IRE1 has been reported to cause upregulation
of BECN1 through X box-binding protein 1 (XBP1) and
mitogen-activated protein kinase 8 (MAPK8) [160]. This
leads to the induction of excessive autophagy. Furthermore,
ATF6 participates in autophagic initiation through
upregulating the DAPK1-mediated phosphorylation of
BECN1 [160, 161]. It also has an effect on the activation
of CCAAT/enhancer binding protein homologous protein
(CHOP) and XBP1, which are able to stimulate overactive
autophagy [160] (Fig. 7).
Oxidative stress is triggered by accumulation of ROS.
Increased ROS upregulates nuclear p53, and then facilitates
autophagy by transactivating damage-regulated autophagy
modulator1, inhibiting mechanistic target of rapamycin
complex 1, and activating hypoxia inducible factor1 (HIF-
1) [162, 163]. In addition, activation of HIF-1 triggers
autophagy by enhancing the transcription of BCL2/adenovirus
E1B 19 kDa interacting protein 3 (BNIP3) and
BNIP3-like (BNIP3L/NIX) [164]. BNIP3 also induces
autophagy by directly inhibiting Ras homolog protein
enriched in brain [165]. Moreover, the forkhead box O
(FOXO) family of transcription factors, especially FOXO1
and FOXO3, play a role in autophagic induction by
upregulating the expression of various ATGs including
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ULK1/2, BECN1, and PIK3C as well as BNIP3 [164, 166].
Besides, ROS leads to the upregulation of NF-E2-related
factor 2, which enhances the expression of p62 and induces
excessive autophagy [167]. Furthermore, ROS accumulation
induces the transcription of PERK, whose effects on
autophagy are described in detail above [168] (Fig. 8).
It is well accepted that autophagy plays a positive role in
suppressing the inflammatory response. However, autophagy
can also upregulate inflammation in an unconventional
way [169]. And in turn, inflammasomes in neuronal
cells can trigger the formation of autophagosomes and
activate autophagy [170]. Since exogenous apoptosis is
triggered by inflammation, autophagy is induced subsequently
through apoptosis-dependent pathways like p53,
BH3-only proteins, DAPK, JNK, and PKB. Besides,
persistent or excessive activation of the inflammatory
response leads to the pathological stimulation of autophagy
[171].
Therapeutics of Autophagy in Ischemic Stroke
Targeting Excessive Autophagy
As noted above, neuronal deletion of Atg7, as well as
TIGAR, schizandrin, and dexmedetomidine (DEX) is
thought to be neuroprotective in IS via suppressing
autophagy. Apart from these, the most acknowledged
inhibitor is 3-methyladenine, which inhibits PI3K. However,
it only experimental now and its effect in vivo
remains doubtful [172]. The ability of ULK1/2 inhibitors
such as SBI-0206965 and ULK101 to down-regulate
autophagy is promising [172, 173]. Moreover, chloroquine
and hydroxychloroquine, which have been widely demonstrated
to be safe in cancer therapy, are considered to act on
the inhibition of autophagy [174]. Propofol at a dose of
100 lmol/L is reported to significantly reduce BECN1 and
ULK1, as well as inhibit the Ca2?
/CaMKKb/AMPK/
mTOR axis [175, 176]. Besides, VSP34 inhibitors include
SAR405, compound 13, and SB02024. Among them,
SB02024 is highly potent and is a promising candidate
for clinical therapeutics. In addition, DEX is thought to
inhibit autophagy in IS by upregulating CCND1, SQSTM1,
and HIF1A, and downregulating LC3A, BECN1, BAX,
Fig. 7 Autophagy induced by excitotoxicity. NMDAR leads to Ca2?
influx, causing unfolded protein and ER stress. The three transmembrane
sensors IRE1, ATF6, and PERK activate a complex cascade
with autophagic induction. IRE1 upregulates BECN1 via MAPK8 and
XBP1. XBP1 also transactivates FOXO1 and TFEB, bypassing
BECN1, and induces autophagy. PERK upregulates ATF4 and CHOP
via the eIF2a axis, then enhances the transcription of MAP1LC3,
ATG5, and ATG12. ATF6 upregulates the DAPK1-mediated phosphorylation
of BECN1 and is believed to activate both XBP1 and
CHOP. IRE1, inositol-requiring enzyme 1; ATF, activating transcription
factor; PERK, PKR-like endoplasmic reticulum kinase; MAPK8,
mitogen-activated protein kinase 8; XBP1, box-binding protein 1;
FOXO1, forkhead box O1; TFEB, transcription factor EB; CHOP,
CCAAT/enhancer binding protein homologous protein; eIF2a,
eukaryotic initiation factor 2a; MAP1LC3, microtubule associated
protein 1 light chain 3 beta; DAPK1, death-associated protein kinase
1.
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Neurosci. Bull.
and DRAM [176]. Intriguingly, lithium, initially a drug for
bipolar disorder, has been reported to suppress autophagy
by inhibiting PI3K and upregulating Bcl-2 [177]. Melatonin
is also thought to effectively inhibit autophagy via
modulating a decrease in ER stress [14].
Targeting Adaptive Autophagy
As the first drug identified in autophagy induction,
rapamycin has been widely examined in the context of
IS, along with its analogues such as temsirolimus. Unfortunately,
it has multiple adverse effects, given the
autophagy-independent functions of mTOR [14, 178]. In
addition, metformin, a drug used for diabetes, has been
reported to activate AMPK through mitochondrial depletion
of ATP. Aspirin also induces autophagy via upregulating
transcription factor EB in brain cells [179]. Other
compounds like rilmenidine, eugenol, progesterone, and
resveratrol have been reported to induce adaptive autophagy
in IS [176]. Besides, knockdown of circular RNA
Hectd1 expression significantly decreases the infarct area,
attenuates neuronal deficits, and ameliorates astrocyte
activation by activating autophagy in transient MCAO
mice [180]. Moreover, overexpression of poly (ADPribose)
polymerase family, member 14 (PARP14) alleviates
microglial activation by modulating microglial
autophagy and thus promotes post-stroke functional recovery
[181].
Conclusions and Future Directions
In this review, we summarize the pathological role of
excitotoxicity, oxidative stress, and inflammatory
responses and the involvement of multiple death modes
mediated by these deleterious events in IS. Despite all of
the data, the exact sequence of these pathological events
has not yet been completely clarified according to current
studies. It should also be pointed out that this review does
not cover some forms of neuronal death that are rarely
found and that could not be directly summarized above,
such as paraptosis.
The deep understanding of the intricate mechanisms
based on research over the past decades provides latent
treatment options. We point out some possible future
directions, or problems that need to be solved urgently.
Future studies can pay more attention to combining
NMDAR-targeted agents with stem cell therapy. Stem cell
therapy following ischemic insults has proved to be a novel
and promising therapeutic strategy in recent years. It
promotes CNS plasticity and regeneration, angiogenesis,
and immunomodulation [182]. The efficacy and safety of
cell-based therapy have been confirmed in animal models.
However, evidence of its neuroprotective effect is yet
insufficient in humans. Several drugs that are able to
enhance neurogenesis may assist stem cell therapy, such as
above-mentioned Tat-NR2B-9c [183].
Oxidative stress causes damage to neurons during the
occurrence of IS and in subsequent treatment, blood
reperfusion also produces large amounts of ROS leading
to neuronal damage. Therefore, how to reduce ROS
generation or how to block the ROS cascade will be
closely related to the prognosis.
It is worth noting that the inflammatory response is a
double-edged sword. Microglia are able to release proinflammatory
or anti-inflammatory cytokines at different
times and in distinct states, resulting in detrimental or
beneficial effects [184]. Proliferating astrocytes also form a
physical barrier against inflammation via upregulating
intermediate filament proteins and secreting extracellular
matrix components [97]. The physical barrier limits
inflammation in the early stage of ischemia. However, it
has been shown that the glial scar around the ischemic
region prevents axonal outgrowth in the late period.
Astrocytes also contribute to long-term recovery after IS
by secreting neurotrophic factors [185]. How to modulate
the participation of astrocytes in the inflammatory response
to minimize neuronal injury is a challenging question.
Fig. 8 Autophagy induced by oxidative stress. Increased ROS
upregulates nuclear p53, transactivates DRAM1, inhibits mTOR1,
and activates HIF-1. Besides, HIF-1 due to oxygen deprivation
triggers the transcription of BNIP3 and NIX. BNIP3 also inhibits
mTOR1 via inhibiting Rheb. The FOXO family, especially FOXO1
and FOXO3, upregulates ATGs including ULK1/2, BECN1, and
PIK3C, as well as BNIP3. ROS also upregulates NRF2 to enhance the
levels of p62 and upregulates PERK as shown in Fig. 6. DRAM1,
damage-regulated autophagy modulator1; mTOR1, mechanistic target
of rapamycin complex 1; HIF-1, hypoxia inducible factor 1; BNIP3,
BCL2/adenovirus E1B 19 kDa interacting protein 3; NIX, BNIP3like;
Rheb, Ras homolog protein enriched in brain; FOXO, forkhead
box O; ULK1/2, Unc-51 like autophagy activating kinase; NRF2, NFE2-related
factor 2.
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Hence, where and when to intervene in the inflammatory
process remains to be studied.
Multiple therapeutics against autophagy have been
reported recently. However, given the presence of both
pro-survival and pro-death autophagy in IS, the management
targeting proper sites needs to be demonstrated. Some
studies have shown that in the early stage of ischemia,
well-functioning basal autophagy dominates, and that
lethal autophagy develops with prolonged initiation, especially
after reperfusion [186]. But the cut-off point remains
vague and further exploration is required due to the paucity
of available evidence. Besides, inhibition of one specific
regulatory molecule is never sufficient to suppress the
whole process of autophagy. This fact therefore lowers the
reliability of many current studies. Another puzzle
involved in autophagy then emerges: intricate cross-links
between autophagy and other mechanisms. This complexity
makes it challenging to regulate autophagy properly,
and enormous blanks in ADCD remain to be explored
In addition, future investigations may focus on the
exploration of multi-target brain protectants and the
feasibility of their clinical application.
Acknowledgements This review was supported by the National
Natural Science Foundation of China (81920108017, 82130036, and
81630028), the Key Research and Development Program of Jiangsu
Province of China (BE2020620), and Jiangsu Province Key Medical
Discipline (ZDXKA2016020).
Conflict of interest All authors claim that there are no conflicts of
interest.
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as
long as you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons licence, and indicate
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indicated otherwise in a credit line to the material. If material is not
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use is not permitted by statutory regulation or exceeds the permitted
use, you will need to obtain permission directly from the copyright
holder. To view a copy of this licence, visit http://creativecommons.
org/licenses/by/4.0/.
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