cells

Review
The Impact of Mitochondrial Dysfunction in Amyotrophic
Lateral Sclerosis
Jiantao Zhao 1,† , Xuemei Wang 1,† , Zijun Huo 1 , Yanchun Chen 1 , Jinmeng Liu 2 , Zhenhan Zhao 1 ,
Fandi Meng 1 , Qi Su 1 , Weiwei Bao 1 , Lingyun Zhang 2 , Shuang Wen 3 , Xin Wang 4 , Huancai Liu 3, *
and Shuanhu Zhou 5, *

1 Department of Histology and Embryology, School of Basic Medical Sciences, Weifang Medical University,
Weifang 261053, China; [email protected] (J.Z.); [email protected] (X.W.);
[email protected] (Z.H.); [email protected] (Y.C.); [email protected] (Z.Z.);
[email protected] (F.M.); [email protected] (Q.S.); [email protected] (W.B.)
2 Neurologic Disorders and Regenerative Repair Laboratory, Weifang Medical University,
Weifang 261053, China; [email protected] (J.L.); [email protected] (L.Z.)
3 Department of Joint Surgery, Affiliated Hospital of Weifang Medical University, School of Clinical Medicine,
Weifang Medical University, Weifang 261061, China; [email protected]
4 Department of Neurosurgery, Brigham and Women’s Hospital, Harvard Medical School,
Boston, MA 02115, USA; [email protected]
5 Department of Orthopedic Surgery, Brigham and Women’s Hospital, Harvard Medical School,
Boston, MA 02115, USA
* Correspondence: [email protected] (H.L.); [email protected] or [email protected] (S.Z.)
† These authors contributed equally to this work.

Abstract: Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and highly fatal neurodegen-
erative disease. Although the pathogenesis of ALS remains unclear, increasing evidence suggests
that a key contributing factor is mitochondrial dysfunction. Mitochondria are organelles in eukary-
otic cells responsible for bioenergy production, cellular metabolism, signal transduction, calcium
Citation: Zhao, J.; Wang, X.; Huo, Z.;
homeostasis, and immune responses and the stability of their function plays a crucial role in neurons.
Chen, Y.; Liu, J.; Zhao, Z.; Meng, F.;
Su, Q.; Bao, W.; Zhang, L.; et al. The
A single disorder or defect in mitochondrial function can lead to pathological changes in cells, such
Impact of Mitochondrial Dysfunction as an impaired calcium buffer period, excessive generation of free radicals, increased mitochondrial
in Amyotrophic Lateral Sclerosis. membrane permeability, and oxidative stress (OS). Recent research has also shown that these mito-
Cells 2022, 11, 2049. https://doi.org/ chondrial dysfunctions are also associated with pathological changes in ALS and are believed to be
10.3390/cells11132049 commonly involved in the pathogenesis of the disease. This article reviews the latest research on
mitochondrial dysfunction and its impact on the progression of ALS, with specific attention to the
Academic Editors: Marie-Christine
Chartier-Harlin and Ritva Tikkanen
potential of novel therapeutic strategies targeting mitochondrial dysfunction.

Received: 13 May 2022 Keywords: amyotrophic lateral sclerosis; mitochondrial dysfunction; neurodegenerative diseases
Accepted: 24 June 2022
Published: 28 June 2022

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in 1. Introduction
published maps and institutional affil- Amyotrophic lateral sclerosis is a complex and common multipathogenic neurodegen-
iations. erative disease, characterized by the progressive loss of upper and lower motor neurons
(MNs). Progressive degeneration of the limbs leads to muscle atrophy and paralysis, and
finally, death (mainly due to respiratory failure) 3 to 5 years after onset. The incidence of
ALS is 0.6 to 3.8 per 100,000 people worldwide, with notable differences between ethnic
Copyright: © 2022 by the authors.
groups [1]. From a genetic point of view, there are two main types of ALS: familial ALS
Licensee MDPI, Basel, Switzerland.
(fALS) and sporadic ALS (sALS). Roughly 90% of cases are sporadic without an associated
This article is an open access article
distributed under the terms and
genetic cause, and 10% are related to family history and dominant inheritance [2]. The
conditions of the Creative Commons
age of onset of ALS is between 55 and 65 years, but the onset of fALS is earlier [3]. In
Attribution (CC BY) license (https:// familial cases, 25% of cases survive on average 2 to 3 years, and though some can survive
creativecommons.org/licenses/by/ up to 5 years, only 5–10% survive to 10 years [4]. There is currently no cure for ALS, and
4.0/).

Cells 2022, 11, 2049. https://doi.org/10.3390/cells11132049 https://www.mdpi.com/journal/cells

Cells 2022, 11, 2049 2 of 21

no drugs have been shown to be effective against the disease. Although two drugs, rilu-
zole and edaravone, have been approved for marketing [5–7], they only delay functional
loss and prolong survival by several months [8]. Based on the current understanding
of the pathogenesis of the disease and the effects of clinical treatments, by the point of
diagnosis, the pathogenic cascade of ALS may have matured, and the degeneration of
neurons has already occurred [9]. Since the discovery of superoxide dismutase 1 (SOD1),
the first gene associated with ALS in 1993, more than 20 other genes have been found to be
causally or highly correlated with its pathogenesis, including transactive response DNA
binding protein 43 kDa (TDP-43) [10], fused in sarcoma/transfer in liposarcoma (FUS),
matrin 3 (MATR3), coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10),
tank-binding kinase 1 (TBK1), tubulin, alpha 4A (TUBA4A), chromosome 21 open reading
frame 2 (C21orf2), chromosome 9 open reading frame 72 (C9orf72), and cyclin F (CCNF),
among others. These genes encode one or more molecular pathways, and their proteins
were identified by genome-wide association studies, genome-wide studies, or exome se-
quencing methods [11]. Even so, our understanding of the pathogenesis of ALS is still in
its infancy, and current research focuses on protein aggregation and misfolding, OS [12],
neuroinflammation, epigenetics, and mitochondrial dysfunction. It is precisely because of
the critical role of mitochondria in maintaining cellular homeostasis that current research
is focused largely on mitochondria, and more and more findings support the idea that
mitochondrial dysfunction plays an active role in ALS pathogenesis [13].
Better research and understanding of disease pathogenesis (which may be multifacto-
rial) and the identification of novel biomarkers and phenotypic modifications will lead to
a better subclinical classification of disease and the development of targeted drugs. Simi-
larly, it will facilitate the development of better prognostic criteria, and interventions in the
disease process will be more effective. In general, factors such as genetic mutations [14]
and weakened autoimmunity are among the many factors that contribute to the death of
MNs. Current evidence suggests that the innate immune system plays a role in ALS. The
release of pro-inflammatory cytokines has been shown to lead to motor neuron damage [15].
Some pathological changes may have already begun before there is any obvious movement
disorder, and many factors leading to the death of MNs are directly or indirectly aggregated
in mitochondria. In this review, we investigate the impact of mitochondrial dysfunction in
ALS and how current research could yield potentially effective interventions and treatments
during disease progression.

2. The Main Function of Mitochondria

Mitochondria, as organelles of almost all eukaryotes, are composed of four-part
structures and are involved in the regulation of many cellular functions, such as ATP
production, Ca2+ signaling, maintenance of cellular homeostasis, and apoptosis. The
dysregulation of these mitochondrial functions can lead to the development of disease [16].
Once the cells are stimulated by the external environment, energy needs can be matched
by increasing mitochondrial mass. Energy supply can be increased by processes that
increase the mass of individual mitochondria, as stimulated by exercise training, electrical
stimulation, hormones, etc. [17].
In addition, mitochondria also maintain, repair, and reorganize themselves through
fusion and fission. At the same time, in order to meet the energy needs of the body,
mitochondria will also increase energy output through fusion and other methods, increasing
the level of oxidative phosphorylation (OXPHOS).
Mitochondria are the main site of OXPHOS and metabolism. Their main function is
to generate ATP for cells, which is essential for the function of energy-demanding organs
such as the brain and heart. Therefore, these organs are most vulnerable to changes in
mitochondrial energy supply [18]. In addition, mitochondria produce reactive oxygen
species (ROS) even under basal energy demands, and approximately 90% are produced
during the process of OXPHOS [19]. Excessive ROS production causes abnormal OS
in vivo, which plays a significant role in the onset and development of ALS. In this process,
Cells 2022, 11, 2049 3 of 21

reducing molecules such as nicotinamide adenine dinucleotide (NADH) and reduced

flavin adenine dinucleotide (FADH2) (reduced analogs of the cytoplasmic substrate) can
enter the electron transport chain (ETC) from the malate-aspartate transport system or the
phosphoglycerol transport system. After several steps in the ETC, oxygen is finally reduced
and energy is released; some is used to generate ATP, and the rest dissipates as heat. The
mitochondrial complex consists of five complexes on the IMM, which undergo a series of
redox processes and finally form ATP. Similarly, the mitochondrial complex is also one of the
main energy supply methods of the body. Enzyme complexes on the inner mitochondrial
membrane (IMM), including NADH-ubiquinone reductase, ubiquinone-cytochrome C
reductase, and cytochrome C oxidase, use the energy released to pump protons into
the mitochondrial intermembrane space against the concentration gradient. Although
the process is efficient, a small number of electrons prematurely reduce oxygen to form
ROS such as superoxide, which can degrade mitochondrial performance. When protons
are pumped into the mitochondrial intermembrane space, an electrochemical gradient is
established on both sides of the IMM, and protons tend to diffuse along the concentration
gradient. ATP synthase (respiratory chain complex V) has a proton channel and an ATP
synthesis site. Driven by the concentration gradient, the protons on both sides of the inner
membrane twitch the rotation of the ATP synthesis site through the proton channel to
synthesize ATP from ADP and Pi. This process is performed by the other four complexes
and OXPHOS. The inhibition or deficiency of mitochondrial respiratory chain complex
activity can lead to severe mitochondrial dysfunction, and long-term inhibition or deficiency
can lead to neurological diseases such as ALS, Parkinson’s disease, Down’s syndrome, and
Leigh’s syndrome, etc. Therefore, mitochondrial respiratory chain complex activity is often
used as an indicator of neurodeficient diseases caused by mitochondrial dysfunction.
In addition to calcium storage, mitochondria also coordinate the functions of structures
such as the endoplasmic reticulum and the extracellular matrix to control the homeostasis
of intracellular calcium concentrations. Not only are there many Ca2+ targets on the mito-
chondrial surface, but the mitochondrial transport of Ca2+ also regulates many aspects of
mitochondrial function [20]. Ca2+ uptake is an electrogenic process mediated by the selec-
tive Ca2+ ion channel, the mitochondrial Ca2+ monotransporter (MCU) [21,22]. However,
Ca2+ uptake results in only a low affinity for the entire complex because of a threshold in
the presence of low cytoplasmic Ca2+ concentrations [23]. Even so, the cytoplasmic Ca2+
signal is rapidly switched into the mitochondrial matrix. The high efficiency of this process
can be attributed to the tight binding between mitochondria and Ca2+ hotspots, and the
ability of mitochondria to rapidly absorb calcium ions makes them a buffer for calcium
ions in cells [24].
Mitochondrial dynamics are regulated by fusion and fission and are involved in cel-
lular stress responses and energy production. It is also a key component in maintaining
mitochondrial homeostasis and regulating mitochondrial function. Mitochondrial fusion
proteins include Mfn1 and Opa1, and fission proteins include Fis1 and Drp1. The extent
of mitochondrial networking is the result of a dynamic balance between fusion and fis-
sion promoted by mitochondrial movement within the cell. In general, more networked
mitochondria appear to be more efficient at producing ATP, especially through aerobic
metabolism [25].

3. Mitochondrial Dysfunction and ALS

Mitochondria are composed of the OMM (outer mitochondrial membrane), the inter
membrane space (IMS), the IMM, and the mitochondrial matrix, which perform multiple
functions in cells, including energy production, material metabolism, the synthesis of iron-
sulfur clusters, and programmed cell death. Mitochondrial homeostasis is dynamically
maintained through processes such as mitochondrial fusion/fission, mitochondrial phago-
cytosis, and apoptosis. Research on mitochondrial dysfunction in various diseases has
revealed that mitochondria are essential for cell development and survival. The activities of
the human body, including normal neuronal function, require a large amount of ATP, and
Cells 2022, 11, x FOR PEER REVIEW 4 of 22

Cells 2022, 11, 2049 4 of 21

has revealed that mitochondria are essential for cell development and survival. The activ-
ities of the human body, including normal neuronal function, require a large amount of
ATP, and mitochondria are the main energy production sites [26]. Furthermore, neuronal
mitochondria
mitochondria are are the
keymain energy
regulators ofproduction
intracellularsites
Ca2+[26]. Furthermore,
homeostasis. neuronal mitochon-
Mitochondria, together
dria
witharethekey regulators reticulum,
endoplasmic of intracellular Ca2+
control homeostasis.
dendritic Mitochondria,
Ca2+ levels and buffertogether
Ca2+ in with the
presyn-
endoplasmic reticulum, control dendritic Ca 2+ levels and buffer Ca2+ in presynaptic termi-
aptic terminals and axonal bundles to regulate neurotransmission [27–31]. Ca transients 2+

nals 2+ transients also control

also and axonal
control thebundles
level of to regulate neurotransmission
mitochondrial matrix Ca2+ to [27–31].
stimulateCamitochondrial ATP pro-
the level[32].
of mitochondrial 2+
matrixmitochondrial
Ca to stimulate mitochondrial ATP production
duction Typically, impaired function is largely caused by damage[32]. to
Typically, impaired
the ETC, which mitochondrial
affects OXPHOS and function is largely caused
ATP production. by damage
Interestingly, to the ETC,
respiratory which
chain de-
affects
ficiencyOXPHOS
has indeed andbeen
ATPfound
production. Interestingly,
in patients with ALSrespiratory
[33]. Damage chainto deficiency
the ETC may haslead
indeedto
been found
changes in patients
in oxygen with ALS [33].
consumption, Damage
a decreased to the ETC maypotential
transmembrane lead to changes
(Δψm), and in oxygen
an in-
consumption,
creased productiona decreased
of ROS, transmembrane
which directlypotential (∆ψm), and
lead to oxidative an increased
damage, including production
reduced
of
ATP synthesis [34]. In addition, inefficient OXPHOS can also generate ROS, causing [34].
ROS, which directly lead to oxidative damage, including reduced ATP synthesis mi-
In addition, inefficient
tochondrial dysfunction OXPHOS can alsomitochondria
[35]. Therefore, generate ROS, causing
are the main mitochondrial
source of both dysfunc-
ATP
tion
and [35].
ROS.Therefore,
Once ROSmitochondria
is overproduced,are the mainlead
it may source of both
to OS, ATPmitochondrial
impair and ROS. Once ROS is
function
overproduced, it may lead to OS, impair mitochondrial function (production
(production of misfolded proteins, oligomers, fibrils, and protein aggregates, etc.), and of misfolded
proteins,
dysregulate oligomers, fibrils, leading
mitochondria, and protein
first aggregates,
to mitophagy etc.),
andand dysregulate
apoptosis, and mitochondria,
eventually to
leading first to mitophagy
cell death [36] (Figure 1). and apoptosis, and eventually to cell death [36] (Figure 1).

Figure 1. Mitochondrial genes such as FUS, SOD1, and CHCHD10 are mutated in ALS patients,
Figure 1. Mitochondrial genes such as FUS, SOD1, and CHCHD10 are mutated in ALS patients,
leading to mitochondrial damage. During mitochondrial fission, OXPHOS decreases due to im-
leading to mitochondrial
paired mitochondria, whichdamage.
results inDuring mitochondrial
decreased fission,
ATP production, OXPHOS
decreased decreases duepoten-
transmembrane to im-
paired mitochondria, which results in decreased ATP production, decreased transmembrane
tial (Δψm), and increased ROS production, while damaged mitochondrial components are elimi- potential
(∆ψm),
nated byand increasedFurthermore,
autophagy. ROS production,
higherwhile damaged mitochondrial
mitochondrial componentsleads
membrane permeability are eliminated
to increasedby
release of cytochrome
autophagy. Furthermore,C, higher
promoting the production
mitochondrial of more
membrane pro-apoptotic
permeability leadsproteins. Mutations
to increased releasein
of
mitochondrial
cytochrome C, DNA (mtDNA)
promoting and excessive
the production of mitochondrial shedding
more pro-apoptotic lead to
proteins. synaptic dysfunction,
Mutations in mitochon-
axonal
drial degeneration,
DNA (mtDNA) andand ultimately neuronal degeneration
excessive mitochondrial sheddingand death.
lead to synaptic dysfunction, axonal
degeneration, and ultimately neuronal degeneration and death.
Numerous potential pathogenic or disease-modifying genes have now been identi-
fied, Numerous
and SOD1potential
plays anpathogenic
important or role in maintaining mitochondrial
disease-modifying genes have now homeostasis and
been identified,
preventing
and apoptosis
SOD1 plays [37]. Therole
an important SOD1-G93A mutant
in maintaining gene binds with
mitochondrial Bcl-2 to form
homeostasis a com-
and prevent-
plexapoptosis
ing and localizes
[37]. in
Themitochondria.
SOD1-G93A Bcl-2
mutant inhibits apoptosis
gene binds withby regulating
Bcl-2 to form acytochrome
complex andC
release and
localizes mitochondrial-initiated
in mitochondria. caspase
Bcl-2 inhibits activation
apoptosis [38,39]. Incytochrome
by regulating addition, glutaredoxins
C release and
mitochondrial-initiated caspase activation [38,39]. In addition, glutaredoxins (Grxs) can
also reduce disulfides to protein thiols that prevent the aggregation of mutant SOD1, pre-
serving mitochondrial function and protecting neuronal cells from apoptosis. However, the
Cells 2022, 11, 2049 5 of 21

overexpression of Grxs1 in IMS may accelerate mitochondrial fragmentation [40]. C9orf72

is a mitochondrial inner membrane-associated protein in which the GGGGCC sequence
repeat expansion is a common genetic cause of ALS [41], characterized by the accumulation
of dipeptide repeat (DPR)-containing proteins in mitochondria and the formation of aggre-
gates in neurons [42]. Translocase of inner mitochondrial membrane domain containing
1 (TIMMDC1) is an essential component in mitochondrial complex I assembly. C9orf72
regulates oxidative phosphorylation by stabilizing TIMMDC1, but C9orf72 haploinsuffi-
ciency and a loss of function lead to the reduced activity of mitochondrial complex I in
C9orf72-ALS patient-derived neurons, directly or indirectly causing or exacerbating the
disease [43]. The activity of mitochondrial complex I decreases, which directly or indirectly
causes or aggravates the disease [44]. Elisa et al. initially found that mitochondrial function
in TDP-43 and C9orf72 fibroblasts were affected under oxidative conditions and impaired
mitochondrial activity in ALS neurons [45]. Second, Tania et al. demonstrated that the
expression of TDP-43 resulted in a dose-dependent decrease in TDP-43 RNA and protein
in SOD1-G93A mice, along with increased levels of Fis1 and Drp1, which play important
roles in the mitochondrial fission machinery. In contrast, the expression of Mfn1, which
plays an important role in mitochondrial fusion, was significantly reduced [46]. Toru et al.
found that the expression of the fission proteins Drp1 and Fis1 was high in Tg mice during
the first 10 weeks of symptoms, while the expression of fusion proteins Mfn1 and Opa1
was gradually decreased [47]. In summary, their findings suggest that fission proteins
such as Fis1 and Drp1 and fusion proteins such as Mfn1 and Opa1 play significant roles in
influencing mitochondrial dynamics. When mitochondrial fusion or fission processes are
overactive, it leads to excessive mitochondrial fragmentation, which activates apoptosis
in cells. It also affects various aspects of mitochondrial function, including ETC, Ca2+
homeostasis, ATP, and ROS production, which directly affect the progression of ALS [48].
Mitophagy can timely remove aging and damaged mitochondria, which plays an
important role in cell growth. The process of mitophagy is to promote the rupture of the
tubular network and the separation of damaged mitochondria after receiving intracellular
and extracellular signals, and subsequently recruit receptors on the mitochondrial surface,
while generating an isolation membrane around mitochondria, targeting mitochondria to
be wrapped by autophagosomes. Then, autophagosomes are transported and fused with
the lysis chamber, lysosomes flow into autophagosomes to degrade mitochondria, and
finally the degraded contents are recycled. Optineurin (OPTN) [49] has been identified
as the primary receptor for PINK1/Parkin-mediated filamentous phagocytosis, which
binds ubiquitin chains of damaged mitochondria to induce phagocytosis [50]. While au-
tophagosome phagocytosis of impaired mitochondria is dependent on OPTN and its kinase
TBK1, the loss of OPTN or TBK1 function results in impaired mitochondrial phagocytosis
and the accumulation of damaged mitochondria [51]. The autophagy-encoding receptor
SQSTM1 can interact with LC3 through its LC3-interacting region (LIR) to help degrade
ubiquitinated molecules. The SQSTM1/p62 ALS-linked L341V mutation is defective in
LC3B recognition, which makes SQSTM1 less susceptible to incorporation into autophagic
vesicles [52]. The loss of SQSTM1 deregulates the autophagy-lysosomal degradation sys-
tem, while mutation of the ALS-linked L341V LIR also reduces the affinity of SQSTM1 for
LC3 and delays SQSTM1 degradation in cells [53].
The dysfunction of mitochondria may stem from morphological defects. Studies have
shown that C9orf72 regulates energy homeostasis by stabilizing mitochondrial complex I.
C9orf72 haploinsufficiency destabilizes mitochondrial complex I and drives motor neuron
degeneration. At the same time, studies have also found that when the morphology of
mitochondria remains intact, C9orf72 can be protected from damage [44]. In cells extracted
from ALS patients, mitochondrial membrane potential and respiratory chain complex
activity were both decreased following changes in mitochondrial morphology. In the SOD1-
G93A transgenic mouse model and other cell models, when mitochondrial morphology is
defective, decreased ATP levels, Ca2+ disturbance, and increased ROS generation can be
observed [54]. These studies on mitochondrial morphological defects directly or indirectly
Cells 2022, 11, x FOR PEER REVIEW 6 of 22

Cells 2022, 11, 2049 SOD1-G93A transgenic mouse model and other cell models, when mitochondrial 6mor- of 21
phology is defective, decreased ATP levels, Ca2+ disturbance, and increased ROS genera-
tion can be observed [54]. These studies on mitochondrial morphological defects directly
or indirectly
affect affect mitochondrial
mitochondrial function andfunction
become and become
direct direct
evidence evidence of mitochondrial
of mitochondrial dysfunction
dysfunction
in ALS. in ALS.
However,
However, there are many
there are many other
other causes
causes ofof mitochondrial
mitochondrial dysfunction.
dysfunction. In
In addition
addition to
to
factors
factors such
such as
as the
the excessive
excessive production
production of of ROS
ROS and
and the
the disruption
disruption of
of calcium
calcium buffering,
buffering,
factors
factors such
suchas asthe
thedisruption
disruptionofof
axonal transport,
axonal mitochondrial
transport, structure,
mitochondrial dynamics,
structure, mi-
dynamics,
tosis, andand
mitosis, apoptotic signaling
apoptotic are also
signaling are disrupted. These These
also disrupted. factorsfactors
are the are
main factors
the mainleading
factors
to the onset
leading of onset
to the the disease,
of the which inwhich
disease, turn affects
in turnthe growth
affects theand development
growth of neurons
and development of
[55].
neurons [55].

4. Mitochondrial
Mitochondrial Dysfunction and Oxidative Stress in ALS
There is is increasing
increasingevidence
evidencethat
thatOSOS is is inextricably
inextricably linked
linked to the
to the pathogenesis
pathogenesis of mi-of
mitochondrial dysfunction and motor neuron degeneration. When normal
tochondrial dysfunction and motor neuron degeneration. When normal cells are attacked cells are attacked
by abnormal substances
substances such
such as hydrogen sulfide (H22S), S), SOD1,
SOD1, andand catalase
catalase (CAT),
(CAT), the
activity of
activity of the
theenzyme
enzymeisisaltered, causing
altered, causingnormal
normal cells to trigger
cells OS. Whereas
to trigger OS. WhereasOS is OS
the is
result
the
of increased
result ROS generation,
of increased often often
ROS generation, accompanied
accompaniedby decreased
by decreasedantioxidant defense
antioxidant [56].
defense
Although
[56]. ROS produced
Although in normal
ROS produced cells iscells
in normal not is
thought to cause
not thought to ALS,
causeexcessive production
ALS, excessive pro-
duction may contribute to the development of the disease. Damage caused by stress
may contribute to the development of the disease. Damage caused by oxidative oxidativecan
also trigger chain reactions such as abnormal protein aggregation, mtDNA
stress can also trigger chain reactions such as abnormal protein aggregation, mtDNA mu- mutations, and
ETC mutations.
tations, and ETCThe mutationThe
mutations. of the ETC notofonly
mutation the leads
ETC not to increased
only leadsROSto production,
increased ROS but
also exacerbates the degree of mutation of the ETC, and eventually leads
production, but also exacerbates the degree of mutation of the ETC, and eventually leads to motor neuron
degeneration
to motor neuron [55]degeneration
(Figure 2). [55] (Figure 2).

Figure 2. OS triggers mitochondrial dysfunction and leads to MN degeneration. The invasion of

Figure 2. OS triggers mitochondrial dysfunction and leads to MN degeneration. The invasion of ab-
abnormal substances such as H2S, SOD, Catalase (CAT), and ROS causes cells to generate OS, re-
normal substances such as H2S, SOD, Catalase (CAT), and ROS causes cells to generate OS, resulting
sulting in the enhancement of PINK1-Parkin-mediated mitophagy and the accumulation of mito-
in the enhancement
chondrial of PINK1-Parkin-mediated
damage. Simultaneously, mitochondrialmitophagy
damage inand thecauses
turn accumulation of mitochondrial
thiol oxidation, triggering
damage.
the Simultaneously,
cascading effects of Camitochondrial damage
2+ imbalance and in turn causes
mitochondrial thiol oxidation,
membrane triggering
rupture, which the cas-
activates cell
cadingIn effects 2+ imbalance and mitochondrial membrane rupture, which activates cell death.
of Cathe
death. addition, impairment of ETC due to mitochondrial damage reduces the production of
NAD the impairment of ETC due to mitochondrial damage reduces the production of NAD+
+ and ATP, and increases the production of ROS, resulting in abnormal protein aggregation,
In addition,
mtDNA
and ATP,mutation, and structural
and increases deformity,
the production andresulting
of ROS, eventually leads to motor
in abnormal neuron
protein degeneration.
aggregation, mtDNA
mutation, and structural deformity, and eventually leads to motor neuron degeneration.
Cells 2022, 11, 2049 7 of 21

4.1. Mitochondria as the Main Source of Reactive Oxygen Species

ROS are one-electron reduction products of a class of oxygen that are mainly produced
in the mitochondria, peroxisomes, and endoplasmic reticulum of cells through the ETC,
cytochrome p-450 activity, prostaglandin synthesis, and phagocytosis [57,58]. Among
them, mitochondrial activity and the metabolism of cytochrome p-450 is the main source
of ROS in mammalian cells [59]. Mitochondria are one of the most important intracellular
sources of ROS. Mitochondria play an important role in the production of oxidative ATP,
and molecular oxygen is reduced to water in the process of the ETC [60]. However,
mitochondrial dysfunction can lead to damage to the ETC, producing an over reduced
state [61,62]. At the same time, damage to the respiratory chain leads to the redundancy
and leakage of electrons, which react with oxygen, further increasing the amount of ROS
generated [63,64]. Under both normal physiological and pathological conditions, ROS
production in mitochondria may occur in the outer membrane, the inner membrane, or
the mitochondrial matrix. The activation of immune cells, inflammatory responses, and
increased mental stress are all causes of endogenous ROS generation. The generation of
exogenous ROS may be due to ingestion or exposure to environmental pollutants, heavy
metals, drugs, chemical solvents, second-hand smoke, alcohol, and radiation, among
other causes [65]. These exogenous substances are degraded or metabolized after entering
the human body, activating the ROS production mechanism, resulting in an increase in
ROS production.

4.2. Oxidative Stress in ALS Induced by ROS

ROS are generated during normal cellular metabolism and are extremely important
for maintaining cellular homeostasis. OS is a state of redox imbalance in which the body’s
antioxidant defense capacity is reduced due to increased ROS production [66,67]. How-
ever, excess ROS can be detrimental, producing unfavorable oxidative modifications to
cellular components, including to the mitochondrial structure, the primary target of ROS-
induced damage [68]. At the same time, excessive ROS can also cause different degrees
of oxidative damage to intracellular proteins, lipids, DNA, and other components [69].
Pathological mechanisms induced directly or indirectly by ROS can cause neuronal damage
and degeneration, especially in the brain [70,71]. The brain is particularly susceptible to
OS [72] because of its high oxygen consumption and low antioxidant defenses. In addition,
the brain is composed mainly of polyunsaturated fatty acids (PUFAs), which are highly
sensitive to lipid peroxide composition [73] and are easily oxidized [74]. Mitochondrial
dysfunction impairs the ATP energy supply to neurons and calcium homeostasis, leading
to high levels of ROS, accelerated mtDNA mutation rates and the lipid peroxidation of
neuronal membranes [56]. In addition, MNs are very sensitive to OS [75], and the central
nervous system has a poor antioxidant capacity and a low activity of protective enzymes
such as CAT and SOD1, resulting in a low cell regeneration capacity. Thus, ALS progresses
irreversibly [76]. Furthermore, the accumulation of mtDNA mutations leads to increased
oxidative damage, a decreased energy production, and increased ROS. Thus, mitochondrial
dysfunction creates a vicious cycle of neuronal damage, genetic mutation, and metabolic
stress, which may lead to apoptosis, which can lead to disease onset or accelerate disease
progression [77].
In many neurodegenerative diseases, ROS can lead to mtDNA mutations, a calcium
homeostasis imbalance, and an increased membrane permeability [78,79]. Because OS is
considered a key pathogenic factor in ALS, hundreds of antioxidant stressors have now
been tested for their therapeutic potential. Riluzole, an anti-glutamatergic drug approved
by the U.S. Food and Drug Administration in 1995, can delay the progression of the disease
and the lifespan of patients to a certain extent, but its efficacy is limited [80]. Studies have
reported that riluzole can inhibit glutamate release and glutamatergic transmission in
the brain and can attenuate oxidative damage in neuronal cells caused by ALS in SOD1-
G93A mice [81,82]. In addition, riluzole has also been reported to increase survival in ALS
patients who have undergone a tracheostomy [83]. Increased survival and improved motor
Cells 2022, 11, 2049 8 of 21

function have been reported in patients receiving riluzole [84,85]. Recently, the antioxidant
drug edaravone, developed by Mitsubishi Tanabe Pharma, was found to be effective in
preventing motor function deterioration in early ALS. The newly approved drug edaravone
is a force multiplier for ALS treatment [86]. Mitochondrial abnormalities have been found
in spinal cord and muscle anatomical samples from ALS patients, along with defects in
mitochondrial respiratory chain complexes and elevated oxidative stress. Meanwhile,
a vicious cycle of abnormal ROS signaling and excessive ROS production was also found
to significantly promote muscle atrophy in mouse models during ALS progression [87].

4.3. Oxidative Stress-Mediated Intracellular Nrf2/Keap1 Signaling Pathway in ALS

ROS can regulate the nuclear factor erythroid 2-related factor 2 (Nrf2)/Keap1 antiox-
idant response element (ARE) intracellular signaling pathway at the molecular level to
promote disease progression [88]. This pathway plays an important role in regulating
ROS-induced cellular OS and has a protective effect against neurodegenerative diseases,
including ALS [89,90]. It has also been reported that the expression of Nrf2 is significantly
reduced in the MNs of SOD1-G93A mice [91]. Another study also reported reduced Nrf2
levels in the motor cortex, the spinal cord, and lower extremity muscle MNs [92]. On the
other hand, the deletion of Nrf2 in SOD1-G93A mice accelerated motor neuron death and
astrocyte activation, resulting in the early onset of the disease [93]. Such findings show that
SOD1 gene mutation leads to the reduction of antioxidant response protein, considered
one of the main cytokines that cause ALS pathogenesis.

4.4. The Role of Mitochondrial Protein Homeostasis in ALS

The maintenance of mitochondrial proteostasis prevents mitochondrial damage. This
protective mechanism ensures the proper hydrolysis and degradation of misfolded, oxida-
tively damaged, and damaged proteins by proteases within the mitochondria. In addition,
proper mitochondrial function can also be maintained by eliminating damaged organelles
that cannot be repaired. Mitochondria have their own unfolded protein response (UPR),
which is activated when too much misfolded protein accumulates. In ALS, the excessive
accumulation of misfolded and oxidatively damaged proteins may slow or even prevent
the progression of the UPR [94]. A study of postmortem spinal cords of sporadic and famil-
ial ALS patients have found that UPR triggers a reduction in general translation and an
increase in the expression of genes encoding chaperones, foldases, and ERAD proteins, but
does not restore mitochondrial homeostasis. Furthermore, the cytoplasmic accumulation
of TDP-43 may be driven by the activation of motor neuron ER stress. ALS mutations in
TDP-43 lead to UPR upregulation in neural 2A cell models. The accumulation of TDP-43
can promote the increase of endoplasmic reticulum stress levels and the subsequent ac-
tivation of apoptosis [95]. Mitochondria contain numerous proteolytic enzymes to build
the mitochondrial proteome, maintain and control its function, or degrade mitochondrial
proteins and peptides. OMA1 is a metalloprotease located on the inner membrane, which
is dormant under normal conditions and rapidly activates under stressful conditions, such
as the loss of membrane potential, heat, or oxidative stress. Studies have shown that under
physiological conditions, CHCHD2 and CHCHD10 interact with OMA1 to inhibit OMA1
activity, the mitochondrial integrated response stress (mtISR), and mitochondrial fusion
processing of OPA1. The double knockout of CHCHD2 and CHCHD10 triggers mtISR,
whereas the single knockdown of CHCHD2 promotes ISR [96]. In addition, missense
mutations of OMA1 have been found in sporadic ALS patients, but more ALS patients
need to be sequenced to determine whether OMA1 plays a role in ALS pathogenesis [97].

4.5. The Role of mtDAMPS in the ALS Inflammation

Neuroinflammation is a common feature of ALS. When tissues are damaged and
invaded by infectious agents, the immune defense system responds rapidly, but may even-
tually lead to neuronal damage due to the persistent production of toxic inflammatory
mediators such as cytokines, ROS, and reactive nitrogen species. Mitochondrial Dam-
Cells 2022, 11, 2049 9 of 21

age Associated Molecular Patterns (mtDAMPS) are endogenous molecules that are often
sequestered by the body’s cells as risk factors. Microglia are the main mediators of neuroin-
flammation, responsible for phagocytosing and removing dead cells, protein aggregates,
other particles, and soluble antigens that threaten the central nervous system. mtDAMPS
will be recognized by the immune receptors of microglia after activation. In addition,
the activated mtDAMPS mediates the expression of inflammatory mediators and also
trigger an intracellular cascade to regulate mitochondrial metabolism and function [98].
TNF is a pleiotropic cytokine involved in many chronic inflammations. Recent studies
have provided compelling evidence that TNF and its downstream signaling pathways
impair OXPHOS, and that the activation of TNFR1 can lead to the expression of many
different cytokines, ultimately leading to apoptosis or programmed cell death. In addi-
tion, the expression of mitochondrial fission protein FIS1 was found to be increased in
the mitochondria of 3T3-L1 adipocytes differentiated by TNF treatment, but the expres-
sion of the fusion protein OPA1 was decreased [99]. These results directly or indirectly
indicate that pro-inflammatory cytokines such as TNF can affect mitochondrial dynamics
in ALS. mtDNA is a key signaling molecule that triggers inflammatory response signals.
A growing number of studies have found that immune-activated mtDNA and mtRNA
can induce an aberrant production of pro-inflammatory cytokines and interferon effectors.
The integrity of mitochondrial membranes is compromised following cellular stress or
mitochondrial damage, when mtDNA is released from mitochondria into the cytoplasm,
triggering aberrant pro-inflammatory and type I interferon (IFN) responses. However, it is
worth noting that the use of VDAC1 oligomerization inhibitor VBIT-4 reduces mtDNA re-
lease and the inflammatory response, which may provide a potential therapeutic approach
for ALS [100].

5. Ca2+ Dysregulation in ALS

Mitochondria are regulators of intracellular Ca2+ movement. When mitochondria
take up Ca2+ , it can promote mitochondrial metabolism and OXPHOS, thereby adjusting
mitochondrial performance [101–103]. Mitochondria further influence electrophysiologi-
cal activity through somatic dendritic formation as well as axonal and presynaptic Ca2+
oscillations [30]. Under basal conditions, the concentration of Ca2+ in mitochondria and
cytoplasm is the same, approximately 100–200 nM. Mitochondria regulate Ca2+ concentra-
tion through voltage-dependent anion-selective channel proteins (VDACs), which promote
Ca2+ from the matrix into the mitochondrial intermembrane space, and through the Ca2+
uniporter complex into the matrix [104].
Therefore, one of the features of impaired MNs in ALS patients is that Ca2+ home-
ostasis is disturbed [105], which has been simulated using in vitro or in vivo models with
mutated genes such as SOD1 [106], TDP-43 [107], FUS, etc. In mitochondria, one of the main
functions of the endoplasmic reticulum (ER) is to regulate the uptake of Ca2+ , especially
to promote Ca2+ exchange with mitochondria after the release of Ca2+ stored in ER, but
TDP-43 disrupts the ER/mitochondria association, reducing mitochondrial Ca levels [108].
Interestingly, upon the activation of α-amino-5-methyl-3-hydroxyisoxazolone-4-propionic
acid (AMPA) receptors, the recovery of physiological Ca2+ concentrations in MNs is de-
layed [109]. Because the high expression of AMPA receptors at postsynaptic terminals
greatly reduces their intrinsic Ca2+ buffering capacity, MNs in ALS are thought to die due
to Ca2+ -induced excitotoxicity [110]. Consequently, MNs are particularly dependent on
proper Ca2+ buffering by mitochondria [111].
Ca2+ exchange is regulated through the interaction of vesicle-associated membrane
protein-associated proteins B and C (VAPB) with the mitochondrial protein, protein tyrosine
phosphatase-interacting protein 51 (PTPIP51). Evidence suggests that VAPB disrupts Ca2+
homeostasis and disrupts mitochondrial anterograde axonal transport, which is critical
for neuronal health and survival [112]. Mutations in VAPB suggest that disturbances in
Ca2+ homeostasis are associated with familial ALS [113]. In addition, TDP-43 has been
shown to disrupt the VAPB-PTPIP52 pathway; a similar mechanism is thought to exist
Cells 2022, 11, 2049 10 of 21

in cases of idiopathic ALS, where pathogenic TDP-43 accumulates. At the same time,
the decreased expression of VAPB in the spinal cord of ALS patients also supports this
contention. A recent study showed that increased calcium permeability of AMPA and
N-methyl-D-aspartic acid receptor (NMDA) is associated with insufficient mitochondrial
Ca2+ uptake and reduced calcium buffering capacity due to an imbalance between MCU1
and MCU2 [114]. Thus, glutamate excitotoxicity in ALS is usually due to an imbalance
in the MCU complex, resulting in defective mitochondrial Ca2+ buffering. However,
another recent study showed specific alterations in the Ca kinetics of MNs in ALS patients,
calling for different treatment strategies [115]. Table 1 summarizes proteins associated with
impaired mitochondrial dynamics in ALS.

Table 1. Proteins associated with impaired mitochondrial dynamics in ALS.

Protein Change References

Mitochondrial fission
SOD1 Drp1 and Fis1 protein levels ↑
[40,45–47]
TDP-43 (mitochondrial fission ↑)
Mitochondrial fusion
SOD1 Mfn1 and Opa1 protein levels ↓
[40,46,47]
TDP-43 (mitochondrial fusion ↓)
Mitochondrial degradation
Accumulation of damaged
OPTN [49–51]
mitochondria
Impaired LC3 recognition
P62 [52,53]
(Autophagy ↓)
Proteins related to disturbed mitochondrial Ca2+ handling
Decreased contacts between
TDP-43 mitochondria and ER [109]
(mitochondrial Ca2+ uptake ↓)
VAPB Disturbed Ca2+ homeostasis [112]

6. Biomarkers Associated with Mitochondrial Dysfunction in ALS

Mutations in genes can lead to mitochondrial fragmentation, repair defects, and
morphological abnormalities in ALS patients, but the lack of precise gene targets hinders
ALS treatment. Identifying signaling pathways and corresponding biomarkers is critical
for discovering new treatments. Findings have identified a number of potential genetic
biomarkers for clinical diagnosis, as shown in Figure 3.
The discovery of these markers provides a basis for in-depth research on the pathogen-
esis, targeted diagnosis, and treatment of ALS. Over the years, scientists have carried out
mechanistic studies to clarify the impact of major markers on the organism after damage,
as shown in Table 2.
Cells 2022, 11, x FOR PEER REVIEW 11 of 22
Cells 2022, 11, 2049 11 of 21

Figure
Figure 3. ALS-related
3. ALS-related biomarkers.
biomarkers. The main
The main biomarkers
biomarkers associated
associated withare
with ALS ALS are shown,
shown, and bi-and
biomarkers associated with mitochondrial dysfunction in ALS are indicated by dark blue
omarkers associated with mitochondrial dysfunction in ALS are indicated by dark blue boxes. boxes.

The2.discovery
Table of these
Genetic markers markerswith
associated provides a basis dysfunction
mitochondrial for in-depthinresearch
ALS. on the patho-
genesis, targeted diagnosis, and treatment of ALS. Over the years, scientists have carried
Protein Location/Coding Sequence
out mechanistic Resultmarkers
studies to clarify the impact of major of Malfunction
on the organism after dam-
SOD1 [116] age, as
IMSshown in Table 2.Mutated SOD1 induces ALS mitochondrial toxicity.
Non-coding region Poly(GR) in C9ORF72-related ALS impairs mitochondrial function and
C9orf72 [117] Table 2. Genetic markers associated
(GGGGCC) increases with mitochondrial
oxidative stress anddysfunction
DNA damage in in
ALS.
iPSC-derived MNs.
Protein Location/Coding Sequence Mutant TDP-43 disrupts
Result of mitochondrial
Malfunction dynamics, and overexpression of
TARDBP
TDP-43
SOD1 [118] TDP-43 results in abnormal mitochondrial aggregation and loss of normal
(chromosome Ip36.2)
IMS Mutated SOD1 induces
function, ALS mitochondrial
resulting toxicity.loss.
in progressive neuronal
[116]
FUS plays a role in a cascade of nuclear loss of function and increased
C9orf72 Non-coding region Poly(GR) in C9ORF72-related ALS impairs mitochondrial function and in-
cytoplasmic functional toxicity in ALS. Furthermore, FUS mutation and
[117]FUS [119] (GGGGCC) Nucleus creases oxidative stressmislocalization
subsequent and DNA damage to the in iPSC-derived
cytoplasm MNs.
sequester additional nuclear
Mutant TDP-43 disrupts
proteins mitochondrial
critical dynamics,
for RNA metabolism, such and overexpression
as motor of
neuron proteins
TDP-43 TARDBP (SMN), blunting the nuclear activity of these proteins.
TDP-43 results in abnormal mitochondrial aggregation and loss of normal
[118] (chromosome Ip36.2)
function, resulting in progressive
VAPB depletion inducesneuronal
increased loss.
autophagic flux and decreased ATP
VAPB [120] ER
production, thereby disrupting neuronal
FUS plays a role in a cascade of nuclear loss of function ion homeostasis and function.
and increased cyto-
MANs plasmic functional toxicity in ALS. Furthermore, FUS mutation and subse-
FUS (mitochondria-associated Regulates Ca 2+ signaling between ER and mitochondria and maintains
SigMar-1 [121] Nucleus quent mislocalization to the cytoplasm sequester additional nuclear proteins
[119] endoplasmic reticulum MAMs structural integrity.
critical for RNA metabolism, such as motor neuron proteins (SMN), blunt-
membranes, MAMs)
ing the nuclear activity of these proteins.
Leucine zipper Dysfunctions in the Nrf2 result in a loss of redox homeostasis, leading to
VAPBNrf2 [122] VAPB depletion induces
ER transcription factor overload with increased autophagic flux
reactive oxygen/nitrogen and decreased ATP pro-
species.
[120] duction, thereby disrupting neuronal ion homeostasis and function.
TRPM7 isoforms cause oxidative stress by inducing hypoxia-activated
TRPM7 [123] MANs Plasma cation currents that increase ROS production.
SigMar-1 (mitochondria-associated en- Regulates Ca2+ signaling between ER and mitochondria and maintains
Deletion of hIGF-1 induces mitochondrial apoptosis, inhibits normal
hIGF-1 [124]
[121] doplasmic reticulum mem- MAMs structural integrity.
mitochondrial mitotic phagocytosis, and promotes motor neuron apoptosis.
branes, MAMs)
Nrf2 Leucine zipper transcription Dysfunctions in the Nrf2 result in a loss of redox homeostasis, leading to
[122] factor 7. Research Progress
overload in reactive
with ALS Treatment
oxygen/nitrogen species.
TRPM7 Antioxidant
TRPM7 isoformsfor
therapy mitochondria
cause may be
oxidative stress byainducing
major direction for future research
hypoxia-activated cat- in-
Plasma volving targeted therapy drugs. Peroxisome-promoting life-activating receptor γ (PPARγ)
[123] ion currents that increase ROS production.
is a ligand-activated transcription factor that can regulate mitochondrial function, main-
hIGF-1 Deletion of hIGF-1 induces mitochondrial apoptosis, inhibits normal mito-
tain normal mitochondrial turnover, stabilize the redox balance, antioxidant response,
[124] chondrial mitotic phagocytosis, and promotes motor neuron apoptosis.
immunity reaction, and fatty acid oxidation, etc. [125]. In SOD1-G93A mice, treatment
with pioglitazone, an agonist of the PPARγ receptor, prolongs the survival of diseased
Cells 2022, 11, 2049 12 of 21

mice, reduces gliosis [126], and protects MNs from p38-mediated neuronal death [127].
Tetramethylpyrazine nitric acid (TMP), a potent free radical-scavenging nitroso moiety,
was found to reduce spinal motor neuron loss and glial cell responses after intraperitoneal
injection in already diseased SOD1-G93A mice. This suggests that the antioxidant activity
of mitochondria is activated by treatment with TBN, a potential targeted drug [128].
Furthermore, in a Drosophila ALS model based on the binding protein TDP-43, piogli-
tazone resolved TDP-43-dependent synergistic motor dysfunction in the MNs and glia but
not in muscle [129]. Because pioglitazone may cause certain features of rhabdomyolysis,
such as muscle pain, elevated phosphocreatine kinase, and weakness, which can worsen
the symptoms of ALS. Hydroxocobalamin attenuated TDP-43 toxicity, reduced OS and
mitochondrial dysfunction, and a combined treatment with a low-sugar diet significantly
attenuated lifespan shortening and motor deficits in flies expressing TDP-43, suggesting
that oral hydroxocobalamin may be a TDP-43-based therapeutic intervention for ALS [130].
Secondly, studies have shown that after mitoquinolmesylate (MitoQ) treatment of SOD1-
G93A mice, the decline in mitochondrial function in the spinal cord and quadriceps muscle
was slowed, and spinal cord nitrification markers and pathological symptoms were signifi-
cantly reduced. Muscle junctions were restored, and lifespan was significantly extended in
mice, suggesting that antioxidant targeting of mitochondria may play a pharmacologic role
in the treatment of ALS, and clinical trials are underway [131].
The magnitude of mitochondrial membrane permeability is also critical for the reg-
ulation of Ca2+ buffering capacity. GNX4728, a regulator of mitochondrial membrane
permeability, increases mitochondrial calcium retention by mitochondrial permeability
transition pore (mPTP) [132]. Olesoxime, such as GNX4728, also works by regulating mPTP.
Studies have demonstrated that olesoxime binds to two outer membrane proteins, voltage-
dependent anion-selective channels, and translocator protein (TSPO), to alter the pore
size of mitochondrial membranes, thereby reducing neuronal cell viability due to damage
impact [133]. In animal experiments, it was found that olesoxime significantly reduces the
death rate of MNs in ALS mice and improves the activation ability of microglia [134]. Since
olesoxime belongs to the family of cholesterol oxime compounds, its toxicity was tolerated
in the first two phases of clinical trials, but in the third phase clinical trial due to its too
high toxicity, the experiment was declared to fail [135].
SigMar-1 is a highly conserved transmembrane protein that is selectively and highly
expressed in MNs of the spinal cord and usually aggregates specifically on the endo-
plasmic reticulum membrane. As an agonist of the SigMar-1 receptor, keratin stabilizes
mitochondria-associated membrane domains by regulating calcium flux, while reducing
ROS generation by affecting PI3K-AKT signaling, thereby ensuring stability in neurodegen-
erative disease states such as ALS Supplies mitochondrial bioenergy [136].
We found that stem cells from human exfoliated deciduous teeth-conditioned medium
(SHED-CM) significantly inhibited the intracellular aggregation and neurotoxicity induced
by mutant SOD1 in ALS. Further, while it is protective against induced iPSC-derived
MNs, the most important finding was that it is effective against both familial and sporadic
ALS. These results suggest that SHED-CM is a potential therapeutic approach to slow the
progression of ALS [137]. HEXA-018, a novel autophagy inducer, can increase the LC3-I/II
ratio and increase the number of autophagic lysosomes. It was found that treatment with
HEXA-018 significantly reduced damage to the ubiquitin–proteasome system and oxidative
stress-induced neurotoxicity. This also suggests HEXA-018 as a new therapeutic candidate
for related neurodegenerative diseases such as ALS [138]. Stem cell transplantation also
provides an opportunity for neurotrophic factors to enter the nervous system and remodel
areas affected by neurodegenerative diseases. However, issues such as the type and number
of cells transplanted remain to be optimized [139].
Table 3 summarizes potential therapeutic targets for improving mitochondrial function
in ALS and drugs effective for mitochondrial function therapy.
Cells 2022, 11, 2049 13 of 21

Table 3. Treatments based on improving mitochondrial function in ALS.

Targets\Drugs Result of Malfunction Therapeutic Directions\Therapeutic Efficacy References

The overexpression of Grx2 interferes with
The verexpression of Grxs1
mitochondrial fragmentation, preserves
Grxs in IMS may accelerate [40]
mitochondrial function, and protects neuronal cells
mitochondrial fragmentation.
from apoptosis.
The binding of OPTN to the ALS-associated E478G
The loss of OPTN or TBK1 function ubiquitin prevented stable binding of the mutant to
results in impaired mitochondrial the mitochondrial surface. Furthermore, the
OPTN and TBK1 [50]
phagocytosis and the accumulation of recruitment of OPTN and LC3B to damaged
damaged mitochondria. mitochondria was significantly reduced using
ALS-associated TBK1 mutants.
C9orf72 haploinsufficiency
Maintaining the integrity of mitochondrial
destabilizes mitochondrial
C9orf72 morphology protects C9orf72 from damage, which [53]
complex I and drives motor
in turn reduces neuronal degeneration.
neuron degeneration.
The accumulation of mtDNA Controlling the amount of ROS production and
mutations leads to increased improving the correctness of replication and repair
mtDNA [77]
oxidative damage, decreased energy mechanisms may be a potential therapeutic
production, and increased ROS. mechanism.
The loss of Nrf2 accelerates motor Mutations in the SOD1 gene lead to reduce Nrf2,
Nrf2 neuron death and astrocyte activation, and finding ways to reduce or inhibit SOD1 gene [93]
leading to early onset of the disease. mutations may improve these problems.
Calcium ions interrupt fine-tuned signaling between
The accumulation of TDP-43 can the ER and mitochondria and initiate apoptotic
promote the increase in endoplasmic signaling cascades, thus serving as a convergence
TDP-43 [95]
reticulum stress level, which in turn point for multiple upstream perturbations of cellular
promotes the activation of apoptosis. homeostasis and constituting a potentially
important therapeutic target.
Reduced mtDNA release and inflammatory
mtDNA is a key signaling molecule
VBIT-4 response with VDAC1 oligomerization inhibitor [100]
that triggers inflammatory responses.
VBIT-4 may offer a potential treatment for ALS.
Hydroxocobalamin attenuated TDP-43 toxicity,
decreased OS and mitochondrial dysfunction, and
TDP-43 toxicity impairs combined treatment with a low-sugar diet
Hydroxocobalamin [130]
mitochondrial function. significantly improved motor deficits, suggesting
that oral hydroxocobalamin may be a TDP-43-based
therapeutic intervention for ALS method.
In SOD1-G93A mice, mitochondrial
MitoQ treatment of SOD1-G93A mice slowed the
function was significantly decreased
rate of decline in mitochondrial function in the
in spinal cord and muscle, and spinal
MitoQ spinal cord and quadriceps, restored muscle [131]
cord nitrification markers and
connectivity and significantly increased
pathological symptoms were
lifespan in mice.
significantly increased.
Changes in mitochondrial membrane
permeability affect Ca2+ buffering GNX4728 is a regulator of mitochondrial membrane
GNX4728 capacity, which in turn affects permeability and increases mitochondrial calcium [132]
mitochondrial metabolism and retention via mPTP.
OXPHOS.
Stem cells from SHED-CM can significantly inhibit
the intracellular aggregation and neurotoxicity
Mutations in the SOD1 gene are
induced by mutant SOD1, have a protective effect on
SHED-CM neurotoxic and induce [137]
MN, and can be considered as a potential
intracellular aggregation.
therapeutic approach to slow down the
progression of ALS.
Cells 2022, 11, 2049 14 of 21

Table 3. Cont.

Targets\Drugs Result of Malfunction Therapeutic Directions\Therapeutic Efficacy References

HEXA-018 increased the LC3-I/II ratio and
increased the number of autophagolysosomes, while
also significantly reducing damage to the
HEXA-018 OS induces neurotoxicity. [138]
ubiquitin-proteasome system and oxidative
stress-induced neurotoxicity. This suggests that
HEXA-018 could be a candidate for ALS treatment.
The inhibition of respiratory chain
Respiratory chain complex activity results in increased Cysteine peptide rT1 can promote ATP synthase and
[140]
complex ROS production and decreased cell survival by targeting ETC.
ATP production.
The mutation of mtDNA causes
Double-stranded DddA potentially corrects highly pure and specific
normal mitochondria to gradually
DNA deaminase pathogenic mutations in mtDNA, a highly [141]
die, resulting in abnormal
toxin A (DddA) innovative therapeutic approach.
mitochondrial function.
Oxidative stress increases ROS TPP can effectively scavenge ROS and reduce
triphenylphosphine
production, leading to cellular oxidative stress, while also transporting functional [142]
cation (TPP)
damage and decreased ETC activity. proteins into mitochondria.

8. Conclusions and Outlook

ALS is the third most common adult-onset neurodegenerative disease [143], for which
there is no cure or treatment. It destroys MNs and skeletal muscles, eventually paralyzing
the patient and causing death [144]. While numerous experimental studies on ALS have
been conducted worldwide, the exact mechanisms of disease onset and progression remain
unclear. There are many reasons for the slow progress of research, including an unclear
pathogenesis, a rapid disease progression, and a lack of suitable animal models [145]. The
crucial role of mitochondria in meeting the physiological requirements of eukaryotes has
attracted a great deal of attention to the chain reaction caused by mitochondrial dysfunction.
Several mechanisms and genes have been firmly associated with mitochondrial dysfunction.
Therefore, a better understanding of the pathogenic mechanism and the interlinkage
between the various factors leading to mitochondrial dysfunction are needed to ensure
more effective treatments [146].
Mitochondria are highly dynamic double-membrane subcellular organelles whose
OXPHOS generates ATP and helps control metabolism [96]. The regulatory mechanism of
mitochondria is extremely important in the process of maintaining cellular homeostasis.
Factors such as genetic mutations or altered mitochondrial dynamics are triggers or facilita-
tors of neurodegeneration. Therefore, the selective vulnerability of MNs in ALS may be
related to mitochondrial numbers, mutated genes, and external influences.
As previously shown, the treatment of mitochondrial dysfunction has mostly focused
on antioxidant, mitochondrial membrane pore regulation and signaling pathway interven-
tion, and has made significant progress. However, clinical trials based on such research still
face many challenges, including the identification of specific mutational loci, the regulation
of signaling pathways, drug-induced neurotoxicity, and suboptimal therapeutic effects. To
date, various targeted drugs have entered clinical trials, such as intravenous injection of the
free radical scavenger edaravone 60 mg/day to ALS patients. After 6 months of treatment,
the ALSFRS-R score of the patients decreased significantly. Edaravone has been approved
for the treatment of ALS in the United States, Japan, Canada, Switzerland, and South
Korea, but has not been approved in the European Union [147]. Masitinib is an inhibitor of
tyrosine kinases. With concomitant riluzole treatment at a dose of 4.5 mg/day as an add-on,
masitinib was also found to reduce ALSFRS-R score [148].
In general, after decades of research and clinical trials, the research on ALS and
mitochondrial dysfunction has grown in depth and thoroughness, but there is an urgent
Cells 2022, 11, 2049 15 of 21

need to find new specific biomarkers and achieve clinical translation of research results.
Mechanism-specific drug development and combination therapies may become the main
new avenues for treatment.

Author Contributions: Y.C., H.L. and S.Z. contributed to the conception of the study. J.Z. and X.W.
(Xuemei Wang) drafted and revised the manuscript. Y.C., H.L., X.W. (Xin Wang) and S.Z. critically
reviewed the manuscript. J.Z., Z.H., J.L. and L.Z. designed the figures and helped with revisions.
Z.Z., F.M., Q.S., W.B. and S.W. made contributions to editing the text and managing references. All
authors have read and agreed to the published version of the manuscript.
Funding: This study was supported by the Shandong Province Natural Science Foundation of
China (Grant No. ZR2020MH150, ZR2020MH149, and ZR2019BH060), Support Program for Youth
Innovation Technology in Colleges and Universities of Shandong Province of China (Grant No.
2019KJK004), Key Project of Shandong Province Higher Educational Science and Technology Program
of China (Grant No. J18KZ013), Shandong Medical and Health Science and Technology Development
Plan Project (Grant No. 2019WS606) and Grants from the Brigham and Women’s Hospital BRI Fund
to Sustain Research Excellence (to X.W.), Gillian Reny Stepping Strong Center for Trauma Innovation,
Osteobiology Research Fund, and Osteobiology Training Fund (to S.Z.).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
ALS amyotrophic lateral sclerosis
OS oxidative stress
MNs motor neurons
SOD1 superoxide dismutase 1
TDP-43 transgenic binding protein 43
FUS fused in sarcoma
MATR 3 matrin 3
CHCHD10 coiled-coil-helix-coiled-coil-helix domain containing 10
TBK1 tank-binding kinase 1
TUBA4A tubulin, alpha 4A
C21orf2 chromosome 21 open reading frame 2
C9orf72 chromosome 9 open reading frame 72
CCNF cyclin F
OXPHOS oxidative phosphorylation
ROS reactive oxygen species
NADH nicotinamide adenine dinucleotide
FADH2 reduced flavin adenine dinucleotide
ETC electron transport chain
IMM inner mitochondrial membrane
MCU mitochondrial calcium uniporter
OMM outer mitochondrial membrane
IMS inter membrane space
Grxs glutaredoxins
DPR dipeptide repeat
TIMMDC1 translocase of inner mitochondrial membrane domain containing 1
Fis1 mitochondrial fission protein 1
Mfn1 mitofusin 1
Drp1 dynamin-related protein 1
Opa1 optic atrophy 1
OPTN optineurin
LIR LC3-interacting region
Cells 2022, 11, 2049 16 of 21

SQSTM1 Sequestosome 1
H2 S hydrogen sulfide
CAT catalase
PUFAs polyunsaturated fatty acids
mtDNA mitochondrial DNA
Nrf2 nuclear factor erythroid 2-related factor 2
ARE antioxidant response element
VDACs voltage-dependent anion-selective channel proteins
ER endoplasmic reticulum
AMPA α-amino-5-methyl-3-hydroxyisoxazolone-4-propionic acid
VAPB vesicle-associated membrane protein-associated proteins B and C
PTPIP51 protein tyrosine phosphatase-interacting protein 51
NMDA N-methyl-D-aspartic acid receptor
MANs mitochondria-associated endoplasmic reticulum membranes
TRPM7 transient receptor potential cation channel subfamily M member 7
hIGF-1 human insulin-like growth factor 1
PPARγ peroxisome-promoting life-activating receptor γ
TMP Tetramethylpyrazine nitric acid
MitoQ Mitoquinolmesylate
mPTP mitochondrial permeability transition pore
TSPO translocator protein
SHED-CM stem cells from human exfoliated deciduous teeth-conditioned medium

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