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News & Views
Sea squirt alternative oxidase bypasses
fatal mitochondrial heart disease
Ann Saada
1,2
Mitochondrial diseases are a diverse group
of inborn disorders affecting cellular
energy production by oxidative phospho-
rylation (OXPHOS) via the five (CI-CV)
mitochondrial respiratory chain (MRC)
complexes. The sea squirt alternative
oxidase (AOX) is able to bypass the distal
part of the MRC and was shown to allevi-
ate the consequences of CIII and CIV
defects in several cellular and Drosophila
models. In this issue of EMBO Molecular
Medicine, Rajendran et al (2018) demon-
strate the first proof of concept in
mammals, by showing that AOX is capable
to extend lifespan and prevent heart fail-
ure in a CIII deficient mouse model, raising
the possibility of future human AOX
bypass treatment.
EMBO Mol Med (2018)e9962
See also: J Rajendran et al (2018)
Mitochondria are intracellular orga-
nelles with a separate genome
(mtDNA) and translation system,
present in all enucleated cells. They are
involved in numerous cellular pathways (in-
termediate metabolism, iron and calcium
metabolism, cell death, etc.), but their main
function is to provide cellular energy (ATP)
via the mitochondrial respiratory chain
(MRC), which is composed of ~89 proteins
in five multimeric complexes (CI-CV, with
CI, CIII, CIV as super complexes) embedded
in the mitochondrial inner membrane (IM).
CI and CII transfer electrons from NADH
and FADH
2
originate from the tricarboxylic
acid (TCA) to coenzyme Q (Q, ubiquinone).
Q also subsequently receives electrons from
additional pathways (pyrimidine synthesis,
fatty acid oxidation and glycolysis); elec-
trons from reduced Q (QH
2,
ubiquinol) are
transferred via CIII, cytochrome cand CIV
(cytochrome coxidase) to the final electron
acceptor oxygen, forming water. Simultane-
ously, protons are translocated across the
IM by CI, CIII and CIV, creating an electro-
chemical gradient (proton-motive force),
which is utilized by CV (ATP synthase) to
generate ATP. This oxygen-requiring
process, termed oxidative phosphorylation
(OXPHOS), provides the vast majority of the
cell’s energy requirements. Under normal
conditions, a small fraction of the electrons
escape the MRC and form oxygen free radi-
cals (ROS), which may participate in cell
signalling (El-Khoury et al, 2016; Fig 1A).
Mitochondrial diseases affecting the
OXPHOS are a heterogeneous group of
prevalent genetic disorders transmitted
either by maternal inheritance due to muta-
tions in mtDNA genes (encoding 13 MRC
core subunits, 2 mRNAs, 22 tRNAs) or by
Mendelian inheritance due to mutations in
nuclear genes encoding the remaining MRC
subunits or one of the numerous assembly,
transcription, translation and replication
factors needed for mtDNA and OXPHOS
maintenance. The clinical manifestations are
extremely variable, can occur at any age and
involve several organs, mainly highly
energy-dependent, such as brain, heart,
muscle, liver and kidneys. OXPHOS dysfunc-
tion is also implicated in common neurode-
generative disorders including Parkinson’s
disease (PD) and Alzheimer’s disease (AD).
The cellular consequences of impaired
OXPHOS are complex and include energy-
deficit oxidative stress by overproduction of
ROS, accumulation of toxic metabolites and
metabolic derangement (Suomalainen &
Battersby, 2018; Fig 1B). Therapy is chal-
lenging and is mainly supportive by admin-
istration of Q and vitamins (mostly MRC co-
factors), although numerous pharmacologi-
cal and genetic options are currently under
intensive investigation (Garone & Viscomi,
2018).
An original idea for mitochondrial
disease therapy, elaborated by Pierre Rustin
and Howard Jacobs, was to bypass blockade
in the distal (CIII-CIV) part of the electron
transport by alternative oxidase (AOX)
(Fig 1C). This peculiar enzyme, found in
plants and several lower animals, is capable
of transferring electrons from QH
2
directly to
oxygen without proton translocation and is
unaffected by cyanide, in contrast to the
cyanide-sensitive cytochrome c oxidase
(CIV). Indeed, allotropic expression of the
single peptide AOX from a sea squirt, Ciona
intestinalis, conferred cyanide resistance
and restored electron flow to CIV-deficient
cells in culture (Hakkaart et al, 2006; Dassa
et al, 2009). AOX expression was also able
to complement and alleviate several fruit fly
Drosophila models of COX deficiency, and
interestingly as well as models of PD and
AD. Although some flies with complete loss
of CIV activity or mutated mito-ribosomal
protein were not rescued, the results still
implicated a wide-spectrum therapeutic use
of AOX (Fernandez-Ayala et al, 2009; Kemp-
painen et al, 2014; El-Khoury et al, 2016).
Moreover, AOX expression was well toler-
ated in mice, with only minor phenotypic
1The Monique and Jacques Roboh Department of Genetic Research, Department of Genetics and Metabolic Diseases, Hadassah Medical Center, Jerusalem, Israel.
E-mails: annsr@hadassah.org.il and anns@ekmd.huji.ac.il
2Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel
DOI 10.15252/emmm.201809962
ª2018 The Author. Published under the terms of the CC BY 4.0license EMBO Molecular Medicine e9962 |2018 1of 3
Published online: December 10, 2018
effects on the OXPHOS, but without any
apparent negative effect on physiology (Szi-
bor et al, 2017).
In this issue of EMBO Molecular Medicine,
Rajendran and colleagues provide the first
proof of concept that AOX bypass alleviates
the pathological manifestations and expands
lifespan in a relevant genetic mouse model
of a human mitochondrial disorder (Rajen-
dran et al, 2018). The researchers crossed
AOX into a mouse model of CIII deficiency,
which recapitulates many of the manifesta-
tions related to human GRACILE (foetal
growth, restriction, aminoaciduria, cholesta-
sis, liver iron overload, lactic acidosis and
early death during infancy) syndrome
caused by mutations in BCS1L, encoding a
CIII assembly factor (Fernandez-Vizarra &
Zeviani, 2015). Lifespan of the CIII-deficient
AOX mice was markedly extended from ~7
to ~19 months, and lethal cardiomyopathy
was prevented. Even though the effect
appeared to be tissue-specific, as liver
disease was still present, kidney and brain
manifestations were ameliorated and growth
was restored. Heart mitochondrial ultrastruc-
ture, respiration and normalized metabolic
alterations confirmed that AOX is indeed
capable to bypass the OXPHOS defect.
Although restoration of the specific muta-
tion by viral transfer or gene editing would
theoretically be a more precise approach,
AOX could provide a universal treatment
H+H+
H+
Δψm
IMS
Matrix
IMM
NADH NAD+
FADH2
Succinate
FAD
Fumarate
III
III
ADP+Pi
ATP
IV
H+
½O2+2H+H2O
V
Cyt c
Q
QH2
A Normal respiration
B Blockade of CIII and /or C IV
C Blockade of CIII and /or C IV and alleviation of electron ow by AOX
e–
e–
e–e–
e–
e–
e–
e–
H+H+
H+
Δψm
IMS
Matrix
IMM
III
III IV
H+
1/2O2+2H+H2O
V
Cyt c
Q
QH2
e–e–
e–
e–
e–
e–
H+H+
H+
Δψm
IMS
Matrix
IMM
III
III IV
H+
1/2O2+2H+H2O
V
Cyt c
Q
QH2
e–
e–
e–
e–
e–
e–
AOX
NADH NAD+
FADH2
Succinate
FAD
Fumarate ADP+P
i
ATP
NADH NAD+
FADH2
Succinate
FAD
Fumarate ADP+P
i
ATP
½O2+2H+
H2O
ROS
DHOD
ETF-QO
G3PD
e–
e–
e–
e–
DHOD
ETF-QO
G3PD
DHOD
ETF-QO
G3PD
© EMBO
Figure 1. Simplified scheme of the mitochondrial respiratory chain and bypass CIII and CIV by AOX.
(A) Normal respiration, (B) blockade of CIII and/or CIV, (C) alleviation of electron flow by AOX. G3PG: glycerol-3-phosphate dehydrogenase, DHD: dihydroorotate dehydrogenase,
ETF-QO: electron transfer flavoprotein dehydrogenase, Cyt c: cytochrome C, Q: coenzyme Q ROS: oxygen free radicals, AOX: alternative oxidase, IM: mitochondrial inner
membrane.
2of 3EMBO Molecular Medicine e9962 |2018 ª2018 The Author
EMBO Molecular Medicine AOX bypasses mito disease Ann Saada
Published online: December 10, 2018
strategy for all mutations affecting CIII and
CIV, and possibly other diseases involving
OXPHOS. The precise mechanism of AOX
action has not yet been fully elucidated, but
could include reducing oxidative and cellu-
lar stresses, and preventing NAD
+
deficit by
relieving the accumulation of stalled elec-
trons, albeit at the expense of less efficient
energy production. An additional advantage
of AOX is that the enzyme becomes active
only when needed, i.e., when a significant
amount of QH
2
accumulates, enabling fine-
tuning according to specific tissue demands.
Whether AOX or other alternative enzyme
bypass therapies are feasible in humans is
presently “unchartered territory”, but will
most probably be explored in the near future.
Acknowledgements
Chaya Miller and Liza Douiev are acknowledged for
proofreading and fruitful discussions.
Conflict of interest
The author declares that he has no conflict of
interest.
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License: This is an open access article under the
terms of the Creative Commons Attribution 4.0
License, which permits use, distribution and repro-
duction in any medium, provided the original work
is properly cited.
ª2018 The Author EMBO Molecular Medicine e9962 |2018 3of 3
Ann Saada AOX bypasses mito disease EMBO Molecular Medicine
Published online: December 10, 2018
