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Review
2016:
A ‘Mitochondria
’
Odyssey Catherine Cherry,1,2 Brian Thompson,1,2 Neil Saptarshi,1,2
Jianyu Wu,1 and Josephine Hoh1,*
The integration of the many roles of mitochondria in cellular function and the
contributionof mitochondrial dysfunction to disease are major areas of research.
Within this realm, the roles of mitochondria in immune defense, epigenetics, and
stem cell
(SC)
development
have
recently
come
into
the
spotlight.
With
new
understanding, mitochondria
may
bring
together
these
seemingly
unrelated
elds, a crucial process in treatment and prevention for various diseases. In this
review we describe novel ndings in these three arenas, discussing the signi-
cance of the interplay between mitochondria and the cell nucleus in response to
environmental cues. While we optimistically anticipate that further research in
these areas can have a profound impact on disease management, we also bring
forth some
of
the
key
questions
and
challenges
that
remain.
‘Thus Spoke Mitochondria’Mitochondria
are
cellular
organelles
with
important
roles
in
signaling
and
bioenergetics.
They
are
surrounded
by
two
membranes,
the
inner
mitochondrial
membrane
(IMM)
and
the
outer
mitochondrial
membrane
(OMM).
In
most
cell
types,
mitochondria
are
not
isolated
organelles;
they
radiate
from
the
cell
nucleus
in
a
reticular
network,
displaying
high
levels
of
interconnectivity
and
plasticity
facilitating
their
functional
roles
within
the
cell
[1].
The
eld
of
biology
has
come
a
long
way
in
understanding
mitochondria
since
their
discovery
over a century ago [2]. In 2015, the UK made changes to legislation allowing the use of
mitochondrial
replacement
therapies
to
help
prevent
the
development
of
mitochondrial
diseases
[3].
Despite
rapid
progress
in
mitochondrial
biology,
little
emphasis
has
been
placed
on
mitochondrial involvement in epigenetics, SC biology, or immune defense. These three areas
are
intricately
linked
by
the
functional
roles
of
mitochondria.
Consequently,
by
appreciating
this
link we may also improve our understanding of the environmental signals that control gene
function
and
inuence
mitochondrial
dysfunction
and
disease.
This
review
aims
to
tie
together
the
recent
steps
forward
in
these
three
underrepresented
elds
of
mitochondrial
biology.
In
addition,
to
facilitate
the
development
of
strategic
approaches
toanswer complex questions in these elds, we discuss rapidly evolving technologies and
experimental
tools
to
study
mitochondria
in
great
detail.
Of
clinical
relevance,
we
provide
examples of treatments using mitochondria that are either licensed or currently in development
aiming
to
treat
various
pathologies.
Immunity, SC Biology, and Epigenetics
Mitochondria
in
Immunity
Mitochondria
play
a
signicant
role
in
the
human
immune
system.
Pattern
recognition
receptors
(PRRs)
recognize
pathogen-associated
molecular
pathogens
(PAMPs)
and
activate
signaling
cascades
that
promote
inammatory
responses
[4].
On
viral
infection,
these
inammatory
Trends
Mitochondria play a pivotal role in the
immune system by detecting foreign
invaders through signaling pathways
(e.g., inammasomes) and generating
immune responses. Modulation of thisrole might open up new therapeutic
potential.
Methylat ion by DNA methyltrans-
ferases contributes to the epigenetic
modicat ion of mitochondrial DNA.
Dysregulation of the mitochondrial epi-
genome within cel ls has been impli-
cated in various diseases.
Mi tochondria contr ibute to t issue
regeneration and integrity, which are
maintained by stem cell renewal and
differentiation.Stemcellspresent excit-
ing medical possibilities in regenerative
medicine.Understanding specic
mito-chondrial biology in stem cells is vital.
Novel techniques are al lowing the
study of mitochondria in much greater
detail than before.
Possible new therapeutic avenues are
emerging with increased scientic
knowledge l inking mitochondria to
immunity, epigenetics, and stem cell
biology.
1School of Medicine, Departments of
Environmental Health Science and
Ophthalmology, Yale University, New
Haven, CT, USA 2These authors contributed equally.
*Correspondence:
[email protected] (J. Hoh).
Trendsin MolecularMedicine, May2016,Vol. 22,No. 5 http://dx.doi.org/10.1016/j.molmed.2016.03.009 391© 2016 Publishedby Elsevier Ltd.
mailto:[email protected]://dx.doi.org/10.1016/j.molmed.2016.03.009http://dx.doi.org/10.1016/j.molmed.2016.03.009http://crossmark.crossref.org/dialog/?doi=10.1016/j.molmed.2016.03.009&domain=pdfmailto:[email protected]
8/17/2019 A Mitochondria Odyssey
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responses
are
triggered
and
virally
infected
cells
can
be
eliminated
by
mitochondria-driven
apoptosis.
In
these
molecular
events,
protein-signaling
complexes
that
drive
the
production
of
interferons
(IFNs)
form
active
complexes
on
mitochondria
[5].
When
present,
viral
RNA
forms
a
complex
with
Rig-1-like
receptors
(see
Glossary )
and
translocates
to
the
mitochondrialantiviral
signaling
protein
(MAVS)
in
the
OMM.
MAVS
forms
aggregates
in
the
OMM
that
can
subsequently
activate
the
key
signaling
mediators
IFN
regulatory
factor
3
(IRF3)
and
the
transcription
factor
nuclear
factor
kappa
B
(NF-k B)
pathway
in
the
cytoplasm
(Figure
1 A)
[6].
It is increasingly recognized that mitochondrial DNA (mtDNA) and mitochondrial reactive
oxygen
species
(mtROS)
play
signicant
roles
in
the
cellular
immune
response.
mtDNA
released
during Bcl-2-mediated apoptosis can bind to cGMP– AMP synthase (cGAS) causing the
generation
of
cGAMP,
which
in
turn
activates
stimulator
of
IFN
genes
(STING).
This
results
in
the
production
of
IFN
(Figure
1B)
[5].
Caspase-3,
-9,
and
-7
of
the
apoptotic
caspase
cascade
Glossary
Acetyl-CoA: metabolic intermediate
produced during fatty acid
metabolism.
Age-related macular
degeneration (AMD): leading cause
of vision loss in elderly populations. In
the dry form, debris or ‘drusen’
accumulates. In the wet form, blood
vessels grow from the choroid.
Diabetic retinopathy: complication
of
diabetes affecting the eyes and
leading to vision loss.
Genome-scale analysis: analysis of
genomic features such as DNA
sequence and gene expression over
the whole genome. The genome is
searched for small variations called
SNPs that occur more frequently in
people with a particular disease.
Heteroplasmy: the mix of non-
mutated and mutated mtDNA that
can exist in a cel l. The level of
heteroplasmy can differ between
cells, tissues, and individuals.
Mammosphere: a clump of human
mammary gland cells.
Mitochondrial DNA (mtDNA):
circular genome inside nucleoids in
the inner mitochondrial membrane
that encodes for 13 proteins and 24
RNA molecules.
Mitochondrial ssion: the process
of two mitochondria separating.
Mitochondrial fusion: joining of two
more mitochondria to form a
network.
MT-RNR1: the mitochondrial gene
that encodes 12s RNA.
Nucleoid architecture: pattern by
which DNA is compacted, folded, or
wrapped.
Oxidative phosphorylation
(OXPHOS): metabolic pathway in
which mitochondria produce ATP.
Rig-1-like receptor: a PRR in the
cytoplasm.
Stemness: common molecular
processes underlying the core SC
properties of self-renewal and the
generation of differentiated progeny.
Superoxide: a compound containingthe anion O2
.
MAVS
Viral RNA
RIG1
NF-κ BIRF3/7
mtDNA
CRIF1
LEM
ROS
Acvaon of NLRP3 inflammasome
Producon of
IFNs and
cytokines
RAGE
TLR9TFAM
mtDNA
(D)
(A)
(B)
(C)
pDC
Mature IL-1β
producon
cGAS
cGAMP
STING
ATP + GTP
IRF3
mtDNA
Cell
damage/necrosis
Figure 1. Mitochondria and the Immune System. (A)ViralRNA formsa complex with RIG1 andbindsto mitochondrial
antiviral signaling protein (MAVS) on the outer mitochondrial membrane (OMM). This then stimulates the nuclear factor
kappa
B (NF-k B) and interferon (IFN) regulatory factor 3/7 (IRF3/7) pathways resulting in the production of IFNs and
cytokines.(B) Mitochondrial DNA (mtDNA) released from themitochondria is a stress signal andcan activate the stimulator
of IFNgenes (STING) pathway. mtDNAbinds to cGMP– AMP synthase (cGAS) generating cGAMP, which activates STING.
IRF3 can then induce expression of IFN and other IFN-stimulated genes (ISGs). (C) Transcriptional factor A, mitochondrial
(TFAM) is a
mtDNA-binding protein. After cell damage/necrosis TFAM acts as a danger signal and enhances the
plasmacytoid dendrit ic cell (pDC) response by binding to the receptor for advanced glycation end products (RAGE)
and toll-like receptor 9 (TLR9). (D) CR6-interacting factor (CRIF1) generates reactive oxygen species (ROS) through an
interaction with the lymphocyte expansion molecule (LEM). Mitochondrial ROS (mtROS) stimulate the immune system by
activating the Nod-like receptor family, pyrin domain containing 3 (NLRP3) inammasome pathway, which generates
downstream mature IL-1b.
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can
silence
immune
activation
[7,8],
which
may
prevent
dying
cells
from
producing
IFN.
Of
note,
loss
of
the
caspase
cascade
in
vivo
and
in
vitro
leads
to
elevated
IFN-b levels.
With
the
goal
of
silencing
immune
activation,
caspase
inhibitors
have
been
included
in
animal
preclinical
trials
yielding
promising
results.
For
example,
VX-765,
a
selective
inhibitor
of
caspase-1
[9,10],
iscurrently
being
investigated.
However,
none
of
these
caspase
inhibitors
is
currently
licensed.
Of
note,
caspase
inhibition
can
amplify
IFN
production,
which
is
an
interesting
concept
from
a
pharmacological
standpoint.
The Nod-like receptor family, pyrin domain containing 3 (NLRP3) inammasome can be
activated
by
a
wide
range
of
ligands
including
bacterial
toxins
and
PAMPs
[11].
NLRP3
enables
the activation of caspase-1 which cleaves IL-1b into its mature form. Experiments in murine bone
marrow-derived
macrophages
(BMDMs)
showed
that
oxidized
mtDNA
released
into
the
cyto-
plasm
could
bind
and
activate
the
NLRP3
inammasome
during
programmed
cell
death
(Figure 1D) [12]. Another study using murine BMDMs reported that mtDNA release depended
on
the
NLRP3
inammasome
and
mtROS
and
that
mtDNA
could
further
amplify
inammasome
signaling (caspase-1 activation) [13]. Of note, the autophagy proteins microtubule-associated
protein-1 light chain 3B (LC3B) and Beclin-1 were required to maintain mitochondrial integrity[13]. mtDNA is a ligand of the NLRP3 inammasome and this system provides a positive
feedback
loop
to
prolong
the
activation
of
the
NLRP3
inammasome.
Notably,
dysregulation
of
the NLRP3 inammasome has been associated with many diseases, including Alzheimer's
disease and type 2 diabetes [14]. Hence, besides immune responses and inammation, it is
conceivable
that
the
mechanisms
and
regulation
of
mtDNA
in
the
context
of
inammasome
activation could be applicable to many other diseases.
The mtDNA-binding protein transcriptional factor A, mitochondrial (TFAM) regulates nucleoid
architecture, abundance, and segregation [15]. A TFAM heterozygous (TFAM+/ ) knockout
mouse line has been shown to display 40–60% mtDNA depletion and mild mtDNA repair
defects,
which
can
cause
an
increase
in
mtDNA
mutations
[5].
In
TFAM+/ mouse
embryonic
broblasts
(MEFs),
mtDNA
stress
was
induced
with
lack
of
TFAM
and
in
the
absence
of
major
oxidative phosphorylation (OXPHOS) defects [5]. This resulted in decreased total mtDNA,
creating
larger
nucleoids
and
instigating
mitochondrial
hyperfusion.
mtDNA
instability
and
mitochondria dysregulation have been observed in many human diseases; hence, cells isolated
from
this
mouse
model
can
be
used
to
study
cellular
responses
to
mtDNA
stress
in
vitro.
Specically, challenging TFAM+/ MEFs with herpes simplex virus 1 or vesicular stomatitis virus
demonstrated
that
the
mice
were
resistant
to
infection
compared
with
wild-type
(WT)
MEFs
[5].
Depletion
of
mtDNA
in
WT
MEFs
however,
reduced
the
resistance
to
viral
infection,
suggesting
that virally induced mtDNA stress boosted the host's antiviral responses, as evidenced by
induced
type
1
IFN
and
IFN-stimulated
gene
(ISG)
responses
[5].
Julian
and
colleagues
[16,17]
built
on
recent
evidence
suggesting
that
mtDNA
is
the
principal
regulator
of
systemic
inammation
in
the
immune
response
[18].
The
damage-associated
molecular
pattern
(DAMP)
nuclear
DNA-binding
high-mobility
group
box
protein1
(HMGB1)can
be
secreted
by
immune
cells
and
act
as
a
mediator
of
inammation
[19].
HMGB1
has
been
shown
to
engage
the
receptor
for
advanced
glycation
end
products
(RAGE),
which
in
turn
induces
cytokine
secretion
through
activation
of
the
transcription
factor
NF-k B
and
enhance
responses
to
CpG
DNA
in
murine
plasmacytoid
dendritic
cells
(pDCs)
[20].
HMGB1
has
also
been
shown
to
direct
cell
migration
of
murine
mesoangioblasts
(mesoderm
SCs)
in
a
NF-k B-
dependent
manner
[21].
TFAM
is
a
HMGB1
structural
homolog.
Consequently,
it
has
been
postulated
that
TFAM
engages
the
RAGE
to
enhance
pDC
activation
via
toll-like
receptor
9
(TLR9),
as
shown
in
Figure
1C
[16,20].
pDCs
are
antigen-presenting
cells
that
promote
immune
responses to self-antigens and to self-DNA released from necrotic cells. In these studies,
exposure
to
TFAM
alone
did
not
activate
pDCs
but
did,
however,
amplify
type
1
IFN
and
tumor
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necrosis
factor
alpha
(TNF/ ) responses to CpG-DNA in cultured splenocytes [16,17]. Conse-
quently,
TFAM-mediated
stimulation
of
pDCs
enhanced
their
cytokine
responses
to
CpG
DNA,
suggesting
a
strong
link
between
mitochondria
and
immune
activation
stemming
from
necro-
tized
cells.
These
ndings
may
have
strong
implications
in
pathological
conditions
wherenecrotic
or
apoptotic
cells
are
present
and
can
trigger
an
immune
response.
From
another
angle,
T
lymphocyte
proliferation
can
increase
with
changes
in
metabolic
respira-
tion
and
ROS
production,
which
are
critical
processes
in
mitochondrial
function
[22].
Compared
with resting T cells, activated T cells have a different metabolic program that includes the ability to
adapt
to
changing
environments,
as
has
been
shown
in
numerous
studies.
For
instance,
one
report identied AMP-activated protein kinase (AMPK) as a metabolic checkpoint that regulates
T
cell
adaptation
and
maintains
cell
viability
[23].
In
addition,
a
recently
identied mutation
in
the
lymphocyte
expansion
molecule
(LEM)
has
been
shown
to
impact
T
cell
immunity
and
to
modulate mitochondrial function. In one study, LEM mutations were identied via high-through-
put
exome
sequencing
[24]
in
lymphocytic
choriomeningitis
virus
clone13
(LCMV
Cl13)-infected
mice (‘Retro’ strain). Specic mutations enhanced the production of LCMV-specic cytotoxic
CD8+ T cells (CTLs) as well as long-lived memory T cell numbers [25]. Moreover, LEM in CTLswas shown to interact with CR6-interacting factor (CRIF1), a protein needed for the translation
and
insertion
of
OXPHOS
peptides
into
the
IMM
[25,26].
Presumably,
LEM
interacts
with
the
OXPHOS protein CRIF1 to increase the levels of mtROS (Figure 1D). The discovery of LEM and
the insights into its role further implicate mitochondria in immunity, in both effector and memory
T
cell
responses.
Whether
upregulation
of
LEM
in
the
mouse
Retro
strain
(where
the
phenotype
was bred to heterozygosity) can restore CTL immunity and enhance memory in different
contexts
may
prove
to
be
an
exciting
opportunity
to
explore
future
therapeutic
avenues.
Recently,
another
interesting
link
between
mitochondria
and
immunity
has
been
reported.
Shahni and colleagues identied signal transducer and activator of transcription 2 (STAT2)
as
an
activator
of mitochondrial ssion [27]. A mutation in STAT2 resulted in complete loss of
expression,
leading
to
severe
multiorgan
dysfunction
with
impaired
mitochondrial
ssion
in
three human patients who had received the live-attenuated mumps–measels–rubella (MMR)
vaccination:
two
were
siblings
presenting
with
neurological
deterioration
and
one
was
a
STAT2-
decient patient. The STAT2 deciency had not caused symptoms until exposure to viral
challenge
in
this
patient
(the
MMR
vaccination).
Furthermore,
in
patient
broblasts
there
was
decreased expression of activated dynamin-related protein 1 (DRP1 ) and increased expression
of
inactive
DRP1, which
the
authors
deemed
responsible
for
the
observed
hyperfused,
elon-
gated
mitochondria
[27].
The
authors
recapitulated
this
effect
by
silencing
STAT2
in
SHSY5Y
neuroblastoma cells, while STAT2 overexpression rescued the phenotype [27]. The link between
mitochondrial
dynamics
and
immunization
(memory
responses)
described
here
suggests
that
disruption
of
the
JAK –STAT signaling pathway may impair mitochondrial dynamics and function
and
could
potentially
provide
clues
to
why
patients
with
mitochondrial
diseases
are
susceptible
to
viral
infections.
Together
these
examples
further
illustrate
the
interconnected
nature
of
mitochondrial
biology
in
various
cellular
processes
and
host
responses.
Mitochondrial
Epigenetics
Epigenetics – the study of heritable changes in gene expression that do not alter DNA sequences
– is a major eld of investigation in mitochondrial biology. Epigenetics can determine the
expression
of
nucleus-encoded
genes
in
accordance
with
environmental
cues.
Of
relevance,
an
increasing
number
of
disorders
and
complex
phenomena
such
as
aging
have
been
associ-
ated
with
mitochondrial
dysfunction
and
epigenetics.
Cytosine
methylation
is
an
epigenetic
modication
of
DNA
catalyzed
by
DNA
methyltransferases
(DNMTs)
leading,
in
principle,
to
transcriptional silencing. Epigenetic studies in mitochondrial biology have been mostly focused
on
the
transcriptional
control
or
modication
of
nuclear
DNA
(nDNA),
since
DNMTs
were
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originally thought to be unable to access mitochondria in vertebrates. Also, since mtDNA does not
contain
histones,
it
was
thought
that
mtDNA
could
not
be
epigenetically
modied, as
depicted
in
Figure 2 [28]. Moreover, early attempts to utilize mtDNA methylation as a biomarker for cancer
detection
across
15
cancer
cell
lines
from
patients
with
gastric
and
colorectal
cancer
were
hindered
by the lack of DNA methylation reported across all samples tested. Direct sequencing conrmed an
absence
of
methylated
mtDNA,
further
describing
mtDNA
methylation
as
a
rare
event
[29].
However,
nDNA
modications
could
not
accurately
represent
the
whole
picture
when
considering
overall cellular gene regulation. Thus, several epigenomic hypotheses have been formed that take
into
account
the
regulation
and
crosstalk
of
both
nDNA
and
mtDNA
in
modulating
cell
function.
The
rst
breakthrough
suggesting
a
role
for
epigenetics
in
mitochondria
came
from
the
identication and characterization of DNMT1 in mitochondria from MEFs and HCT116 human
colon
carcinoma
cells
[30],
where
immunoprecipitation
against
5-methylcytosine
(5-mc)
or
5-hydroxymethylcytosine (5-hmc) demonstrated signicant enrichment compared with IgG
controls [30]. Mitochondrial DNMT1 identication subsequently gave way to the identication
of
DNMT3a,
which
also
localized
inside
mouse
and
human
central
nervous
system
mitochondrialfractions [31].
Using bisulte genomic sequencing and next-generation sequencing on mtDNA regions from
human
HEK293
and
HCT116
cell
lines,
a
study
reported
that
the
overall
CpG
island
methylation
frequency was less than 0.1% [32]. This called into question both the methods involved in the
detection
of
CpG
methylation
and
the
overarching
physiological
signicance
of
mtDNA
meth-
ylation
in
epigenetic
regulation.
However,
the
pathophysiological
relevance
of
methylation
levels
in disease etiology cannot be ignored. Recent evidence demonstrated a signicant increase in
5-mc
and
decrease
in
DNMT3a
levels
in
spinal
cord
neurons
and
skeletal
muscle
myobrils
from
transgenic murine amyotrophic lateral sclerosis (ALS) models [33]. The salient message from this
mtDNA
CpG
CpG CpG
CpGCpG
DNMTs
(A)
(B)
mtDNA
CpG
Figure 2. Epigenetic Modications of Mitochondrial DNA (mtDNA). (A) Two examples of mtDNA CpG islands in hypermethylated states are shown. DNA
methyltransferases (DNMTs) such as DNMT3a or DNMT1 methylate the 440 identied mtDNA islands. Methyl groups are indicated by a closed circle whereas open
circles represent unmethylatedmtDNA.Hypermethylation has been implicated in cancer, amyotrophic lateral sclerosis (ALS), diabetic retinopathy, and the response to
environmental toxicant exposure. (B) Twoexamplesof mtDNA CpGislands in hypomethylated statesare shown.Methyl groupsare indicatedby a closedcirclewhereas
open circles represent unmethylated mtDNA. Hypomethylation of mtDNA has been detected in patients with, for example, Down's syndrome.
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study
was
that
mtDNA
methylation
was
tissue
specic and
could
contribute
to
the
degree
of
tissue
inammation
seen
in
ALS
pathology
in
mice
[33].
Mitochondrial genome-scale analysis
has
provided
a
platform
where
large-scale
bisulte
sequencing
can
be
mapped
to
the
human
mitochondrial
genome
and
methylation
patterns
ascertained
with
an
expected
degree
of methylation
heterogeneity
across
39
cell
line
publicly
available
datasets
[34]. Such
studies
suggest
that
epigenetic
modication
of
mtDNA
is
more
prevalent
than
previously
thought.
When
more
specically
considering
disease,
DNMT1
hypermethylation
might
play
a
role
in
the
pathogenesis of diabetic retinopathy [35]. In patients with diabetes, nucleus-encoded DNMT1
is
translocated
into
retinal
mitochondria,
hypermethylating
the
mtDNA
control
(D-loop)
region
where transcription and replication elements are located [35]. Hypermethylation of this region
causes
aberrant
transcription
of
mitochondrial
genes
crucial
to
the
regulation
of
the
electron
transport
chain,
thus
leading
to
the
generation
of superoxide radicals promoting a hypergly-
cemic superoxide radical milieu [35]. This nding underscores the importance of mtDNA
methylation
outside
classical
CpG
sites
and
highlights
the
regulatory
role
of
epigenetic
mod-
ications in mitochondria that can contribute to disease [35,36].
Anecdotally, environmental exposure to toxicants has been shown to have a major impact not
only
on
nDNA
but
potentially
on
mtDNA
as
well.
For
example,
workers
highly
exposed
to
airborne pollutants (e.g., metals, traf c-derived particles, benzene) have been reported to exhibit
increased mtDNA methylation in the 12S rRNA region (MT-RNR1) compared with workers
exposed
to
low
levels
of
airborne
pollutants
[37].
Of
signicance,
aberrant
methylation
of
MT-RNR1 could lead to aberrant mitochondrial ribosome function and protein production
[37].
Although
further
validation
is
required
in
these
studies,
impaired
mitochondrial
protein
production stemming from epigenetic changes in MT-RNR1 presumably might lead to envi-
ronment-associated
pathologies
(e.g.,
cancer,
lung
disease).
With
continued
advances
in
the
understanding
of
the
regulatory
role
of
the
mitochondrial
epigenome
in
mitochondrial
function,
a
novel
layer
of
regulatory
crosstalk
between
the
nucleus
and the mitochondrion is emerging. A mitochondria-to-nucleus pathway, shown in Figure 3, can
transmit
signals
from
mtROS
to
the
nucleus
and
modulate
gene
expression.
An
example
of
this
process has been reported with the inactivation of the histone demethylase Rph1p at sub-
telomeric
heterochromatin
[38].
In
this
study,
mtROS
signaled
through
Tel1p
and
Rad53p
(homologs of the mammalian DNA damage response kinases ATM and Chk2) to ensure yeast
longevity.
This
pathway
subsequently
inactivated
Rph1p
leading
to
transcriptional
silencing
of
telomeric
genes
[38].
Moreover,
another
study
has
provided
evidence
linking
mtDNA
to
mito-
chondrial metabolites to regulate nuclear gene expression in skeletal muscle SCs (SMSCs) [39].
This
work
demonstrated
that
SMSCs
undergo
a
metabolic
switch
from
fatty
acid
oxidation
to
glycolysis
(transpiring
in
mitochondria)
when
transitioning
from
quiescence
to
proliferation.
This
led
to
decreases
in
both
intracellular
NAD+ levels
and
the
activity
of
the
histone
deacetylase
sirtuin
1
(SIRT1),
resulting
in
elevated
histone
H4K16
acetylation
and
activation
of
muscle
gene
transcription
[39].
Therefore,
such
changes
in
metabolic
state
can
inuence
metabolitesand
the
epigenetic
regulation
of
gene
expression
[39].
Bidirectional
regulation
of
gene
expression
between
nDNA
and
mtDNA
presents
an
additional
layer
of
complexity
with
the
presence
of
miRNA.
miRNAs
that
reside
on
both
the
OMM
and
IMM
can
affect
epigenetic
regulation
and
in
turn
be
themselves
epigenetically
regulated.
Early
studies
revealed
the
presence
of
15
nucleus-encoded
miRNAs
present
in
mitochondria
of
murine
liver
tissues
[40].
This
initially
raised
the
question
of
the
function
of
miRNAs
in
mitochondria,
bringing
forth
the
possibility
of
an
alternative
mechanism
of
nuclear
control
of
mtDNA
function.
The
miRNA miR-1 has been found to enter mitochondria and stimulate the translation of mtDNA in
muscle
cells
[41]. In
addition,
miRNA –mtDNA crosstalk was also suggested in a rat model of
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traumatic
brain
injury
(TBI)
[42]. Animals
with
TBI – a leading cause of cognitive defects in
humans – exhibited mitochondrial dysfunction and dysregulation of a set of miRNAs expressed
in the hippocampus region of the brain [42]. Collectively, these studies are timely, reinforcing an
epigenetic
coregulatory
role
of
nDNA,
mtDNA,
and,
presumably,
miRNA.
However,
whether
miRNA dysregulation is a cause or a result of mitochondrial dysfunction in these contexts
remains
to
be
determined.
Filling
this
gap
in
understanding
should
be
a
major
focus
of
future
research.
The
epigenetics
of
both
nDNA
and
mtDNA
are
clearly
important
regulatory
processes
for
proper
cell function. Studies currently positioned to obtain a better understanding of mtDNA epigenetic
modications
and
the
crosstalk
between
the
two
epigenomes
are
now
at
the
forefront
of
biomedical
research.
Mitochondria
in
SC
Biology
SCs
have
become one of
the most potentially promising
therapeutic avenues for regenerativemedicine. Recent studies in
SC
research
have demonstrated
that SCs isolated from human
blastocysts
not only show conventional hallmark
characteristicsof
naive pluripotency
but
also
exhibit additional
functional
features
such
as mitochondrial respiration [43]. SCs have
two
dening
qualities:
self-renewal by
the production of
identical
daughter
cells
and the
ability
to
produce
independent
daughter
cells
that
can differentiate into
many
different
cells. Research-
ers
have
faced multiple challenges
associated with
studying SCs, some of
which
have
been
overcome with
the
development
of
iPSCs.
This methodology
is
progressively allowing
the
scientic
community
to
understand various
complex
factors
involved
in
regulating the main-
tenance of SCs, with the role of mitochondria just beginning to be woven into this complex
picture.
Rph1p
Sirt1
mtROS
NAD+
Nucleus
Cytosol
miRNA
Figure
3.
Mitochondria–Nucleus
Epigenetic
Bidirectional
Pathways.
Mitochondrial reactive oxygen species
(mtROS) and mitochondrial metabolites such as NAD+ can contribute to nuclear epigenetic regulation by inhibition of
histone
demethylase (Rph1p) and regulation of histone deacetylase SIRT1.miRNAsmove to themitochondria where they
can
modulate epigenetic regulation and mitochondrial gene expression.
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Asymmetric
division
allows
SCs
to
generate
daughter
cells
with
differing
fates
[44].
There
is
evidence
suggesting
that
damaged
proteins
are
inherited
asymmetrically
[45]
and
this
trend
has
also
been
observed
in
Saccharomyces
cerevisiae
[46].
However,
there
is
limited
supportive
evidence
to
indicate
that
mitochondria
asymmetrically
divide
in
mammalian
systems.
To
addressthis,
recent
work
was
conducted
in
SCs
using
tag
techniques.
Photoactivatable
GFP
was
tagged
to
MOM
protein
25
(paGFP-Omp25)
and
Snap-tag
was
used
to
track
apportioned
mitochondria
in
daughter
cells
originating
from
stem-like
cells
[44]
recently
identied
from
immortalized
human
mammary
cells
[47].
The
data
indicated
that
mitochondria
were
not
evenly
distributed among daughter cells. Interestingly, through uorescence-activated cell sorting
(FACS)
and
replating
of
daughter
cell
populations
it
was
observed
that
daughter
cell
populations
that received ‘younger’ mitochondria displayed stronger stem-like characteristics such as a
mammosphere-forming ability (Figure 4 ). This study illustrates the power of controlling the
apportioning
of
mitochondria
into
daughter
cells
[44].
SC
differentiation
is
a
tightly
regulated
process
that
is
crucial
for
both
animal
development
and
tissue homeostasis [48]. However, little is known about the intrinsic cellular mechanisms
governing this process. One recent study using in vivo RNAi in Drosophila melanogaster discovered that mitochondrial ATP synthase, a protein that chemically synthesizes ATP from
ADP
and
Pi [49], plays an important role in regulating germ cell differentiation and ensuring
germline development [48]. Furthermore, knockdown studies of other members of the OXPHOS
system demonstrated that ATP synthase acts during differentiation through a mechanism that is
separate
from
its
role
in
OXPHOS.
ATP
synthase
expression
was
observed
to
be
specically
regulated during differentiation [48]. With the use of electron micrographs, native polyacrylamide
Young
Old
Division 0
Division 1
Division 2
Division 3
Mitochondria
Figure 4. Mitochondria in Stem Cell Differentiation. Asymmetrically dividing stem cells acquire young (yellow) and old
(blue)mitochondria.Stem cells that acquire more youngmitochondria have an enhanced proliferation advantageover stem
cells that acquire a greater number of old mitochondria. This has important implications in senescence.
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gel
electrophoresis,
and
in-gel
ATPase
assays,
it
was
demonstrated
that
ATP
synthase
dimer-
ization
is
required
for
mitochondrial
crista
formation
during
differentiation
[48].
Combining
these
ndings
with
previous
results
demonstrating
differences
between
SCs
and
differentiated
cells
(e.g.,
cardiomyocytes,
follicle
cells)
in
mitochondrial
fusion
and
ssion
as
well
as
IMMstructure,
[50,51],
it
is
becoming
increasingly
apparent
that
mitochondria
could
play
important
roles
in
the
regulation
of
SC
differentiation
and
function.
Reinforcing
these ndings, recent research has
suggested
that
mitochondrial proteins such
as mitofusion-1 and -2 (Mfn1/2), which are known to be intimately involved in controlling
mitochondrial dynamics
and
energy
production,
play
an
extensive role in
regulating cell fate
transition [52]. It was observed that during the early stages of reprogramming, around day 7,
mitochondrial function
was
downregulated
with
an
associated
decrease in
Mfn1 / 2
levels.
Mfn1 / 2
genetic
ablation or
pharmacological
inhibition of
mitochondrial fusion in
both human
ESCs andmouse MEFs resulted in reprogramming changes and, furthermore, demonstrated
a
glycolytic
bioenergetic transition
to
meet
the energy demands
of
proliferating
pluripotent
cells [52]. Other research has shown that mitochondrial uncoupling protein 2 (UCP2), a
protein that regulates mitochondrial respiration by controllingmetabolite transportation out of mitochondria [53], appears to play an important role in the regulation of SC differentiation by
blocking the shift from glycolysis to
cellular respiration [54]. Furthermore,
recent work
has
shown that in both human andmouse ESCs, bioenergetics processes, namely glycolysis, are
crucial in the maintenance of the pluripotent state [55]. This study employed high-resolution
NMR
and 13C
glucose-tracing using
mass
spectrometry in
pluripotent SCs to
document that
glycolysis-mediated changes in acetyl-CoA occurred with differentiation (decreased glycol-
ysis) and also led to
H3K9/K27 histone acetylation
[55]. In
addition,
increases in
ROS
production have been associated with SC ‘aging’ or loss of regenerative capacity [56],
suggesting
that
ROS
production
may
play
a
regulatory
role in stemness and SC proliferation.
Taken together, these results demonstrate that through the control of mitochondrial dynamics
and
bioenergetics, novel approaches
to
promoting
somatic cel l reprogramming may be
obtained.
iPSCs
can be
created from somatic
cells through
forced expression
of
reprogramming
factors. iPSCs have different gene expression patterns [57], differentiation potentiality [58],
and
DNA
methylation
patterns
[59]
compared with ESCs. These dif ferences
have
led
researchers to develop a technique known as somatic cell nuclear transfer (SCNT). SCNT
allows
the transfer of
a
somatic cell nucleus into
an oocyte
with
subsequent
reprogramming
to
convert it into
a
pluripotent
cell [60]. Recent evidence hasemerged
showing
thatmouse
ESCs
with different mtDNA haplotypes display differential expression of genes associated with DNA
methyltransferases
and processes of
energy metabolism and pluripotency [61].
However,
a
problem
surfaces:
mismatching mitochondria between donor and recipient during ESC
nuclear
transfer (NT-ESC)
might
lead to
immunoreactivity [62]. For example,
mouse
NT-ESCs
were
generated
withmismatched
C57BL/6J
mitochondria andBALB/c nuclei. On
injection of
mismatched
NT-ESCs into
BALB/c
mice, there
was
an increase in
helper
T
cell activation
inaddition
to
NT-ESC-directed antibody production [62].
This result poses
a
challenge
to
the
development
of
SCNT
therapy as
theheterogeneity level in
human
mitochondria is
higher
than
that
in
mice.
Recent Advances in Mitochondrial Therapeutics
The past few years have shown a glimpse of rapidly evolving techniques that allow mitochondria
to
be
studied
in
greater
detail.
Novel
sequencing
methods
and
immunoassays
coupled
with
intricate cellular approaches are just some of the ways in which the study of mitochondria has
improved.
Mitoash
and
MitoParaquat
for
mtROS
measurements
and
Seahorse
for
bioener-
getics are summarized in Table 1. Gradient centrifugation ( Table 1 ) allows the isolation of
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mitochondria
for
the
quantication
of
mtDNA
methylation
and
mapping
of
5-mC
and
5-hmC.
Moreover, existing nDNA tools are providing exciting prospects that can be applied to mtDNA.
Bisulte sequencing and liquid chromatography–electrospray ionization tandem mass spec-
trometry
(LC–ESI-MS/MS) ( Table 1 ) as well as af nitymethods and restriction methods are being
used, but the best way seems to involve a combination of several methods. For instance, the
combination
of
bisulte
sequencing
and
methylated
mtDNA
immunoprecipitation
assays
has
accurately shown methylated and hydroxymethylated cytosines in mtDNA [34,36].
This progression is leading towards novel therapeutics and improvements of existing treatments
that
may
achieve
what
was
previously
inconceivable.
Summarized
in
Table
2
are
examples
of
promising
new
treatments
that
utilize
our
knowledge
of
mitochondria:
MitoC,
phenformin,
mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs), and SBI-
0206965.
In
particular,
compelling
research
is
being
conducted
into
developing
mitochondrial
replacement therapies (MRTs). It is hoped that with the combined utilization of in vitro fertilization
and
MRTs,
mutated
maternal
mtDNA
and
unhealthy
mitochondria
can
be
replaced
with
unmutated donor mtDNA and healthy mitochondria. Macaque- and human-based studies have
demonstrated
that
MRT
may
be
a
viable
mitochondrial
disease
prevention
strategy
[63,64].
A
study
with
human
oocytes
has
shown
that
following
nuclear
genome
exchange,
the
swapped
pluripotent cells and derived broblasts exhibited normal metabolic proles and respiratory chain
enzyme
activity
compared
with
ESCs
and
ESC-derived
broblast
controls
[63].
Another
study
showed
that
replacing
mutant
mtDNA
with
healthy
mtDNA
using
spindle–chromosomal com-
plex
transfer
(ST)
gave
rise
to
healthy
rhesus
macaque
monkeys
[64].
To
improve
fertility
potential,
AUGMENT SM treatment
( Table
2 )
has
been
marketed.
This
involves
the
transfer
of
healthy
energy-producing
mitochondria
(AUGMENT SM
processed)
from
a
woman's
own
pro-genitor
egg
cells
taken
from
the
ovarian
lining
(EggPCs)
into
her
mature
egg
cells
in
combination
with
sperm
during
in
vitro
fertilization
procedures;
it
is
licensed
in
only
some
countries
[65].
Despite
these
promising
results,
there
has
been
much
resistance
towards
using
MRTs.
Many
opponents
of
MRT
cite
that
studies
performed
on
invertebrates
and
mice
have
demonstrated
altered
parameters
of
health
such
as
energy
production,
fertility,
and
learning
[66–68]. After
taking
into
consideration
the
risks
of
MRT,
in
early
2015
the
UK
was
the
rst
country
to
change
legislation
allowing
the
use
of
MRT
on
parents
who
want
to
conceive
a
child
but
are
at
risk
for
having
a
child
with
a
mitochondrial
disease
[3].
The
lessons
learned
from
these
earlier
pioneering
studies will be pivotal in setting the course for MRT-granting legislation to be passed on to other
nations.
Table 1. Mitochondrial Technique Advances
Technique Application Principle Refs
Mitoash Measurement of superoxide
production
An optical readout is produced at the
single-mitochondrion level
[79–81]
MitoParaquat Measurement of superoxides A triphenylphosphonium (TPP) lipophilic
cation conjugated to redox cycler
paraquat; accumulation on matrix increases
superoxides at the avin site of complex I
[82]
Seahorse Bioscience Measures extracellular ux in
living cells
Fluorescent oxygen sensors are used in a
microplate assay format
[83]
Gradient centrifugation Mitochondrial purication Sucrose stop density gradient centrifugation
analysis
[84–87]
LC–ESI-MS/MS mtDNA methylation quantication Separation and measurement of specic
bases in DNA with subsequent analysis
of
any modications
[88,89]
400 Trends in MolecularMedicine, May2016,Vol. 22,No. 5
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Concluding Remarks: ‘Eyes Wide Shut’We came to the eld of mitochondrial biology through genetic studies of one of the most
prevalent
eye
diseases
in
aging
populations, age-related macular degeneration (AMD)
[69–72]. Mitochondrial dysfunction had already been documented in age-related diseases
including
AMD
[72–74]. Further studies on the same AMD-associated gene family implicated
the mitochondrial protein high temperature-dependent serine peptidase 2 (HtrA2) in AMD
disease
[75–78]. As mentioned above, MRT is currently being used to eliminate dysfunctional
mitochondria
in
rare
inherited
diseases.
For
common
disorders,
a
new
theme
has
emerged
from
recent work: mitochondria can play a pivotal role in immunity, epigenetic regulation, and SC
development.
Deciphering
the
interplay
between
mitochondria
and
nuclear
processes
will
be
critical in understanding the mitochondrial role in cellular function in these three areas in health
and
disease.
From
a
public
health
point
of
view,
it
will
be
signicant
to
follow
these
lines
of
investigation, potentially providing further clues to the participation of mitochondria in mediating
responses
to
environmental
cues,
infection,
tissue
transplantation,
aging,
and
cellular
dysfunc-
tion
as
in
the
case
of
autoimmune
disorders
and
neurodegenerative
diseases,
among
others.
Answers to some of these queries (see Outstanding Questions) may yield novel approaches to
better
manage
or
prevent
severe
disease
and/or
to
facilitate
tissue
regeneration
strategies.
When
contemplating
the
treatment
of
mitochondria-related
disorders,
the
most
important
task
is
to
understand
how
to
better
translate
our
experimental
knowledge
to
patients
and
human
populations
while
accounting
for heteroplasmy within individuals. Despite recent signicant
progress,
scientists
need
to
continue
to
focus
on
the
multiple
hurdles
and
challenges
that
remainahead
and
that
need
to
be
overcome.
Acknowledgments
The authors are grateful to Professor Steve Waxman for the opportunity and for advice in writing this review. They are also
grateful to the anonymous reviewers for their input andcomments. This work is fundedby theSackler Foundation and the
Rosebay Medical Foundation.
References1. Burte, F. et al. (2015) Disturbed mitochondrial dynamics and
neurodegenerative disorders. Nat. Rev. Neurol. 11, 11–24
2. Ernster, L. andSchatz, G. (1981) Mitochondria: a historicalreview.
J. Cell Biol. 91, 227s–255s
Table 2. Mitochondrial Therapeutic Advances
Name Type Mechanism Application Refs
MitoC Antioxidant
(ascorbate)
conjugated
to TPP
The antioxidant is targeted
to mitochondria by TPP
and can be taken up by
mitochondria
Mitochondria-targeted
antioxidant and tool to
explore the role of
ascorbate in mitochondria
[90]
Phenformin Mitochondria l
inhibitor
Induces apoptosis in LKB1-
decient non-small cell lung
cancer (NSCLC) cells
Metabolism-based
therapeutic for
LKB1-decient tumors
[91]
SBI-0206965 ULK1 kinase
inhibitor
Inhibitor of autophagy and
mitophagy
Combined use with
rapamycin to kill tumor
cells
[92,93]
AUGMENT SM
treatment
Mitochondrial
transfer
Transfer of mitochondria
from a woman's own
immature EggPCs to
supplement the existing
mitochondria in her
mature eggs
Improving infertility and
in vitro fertilization (IVF)
procedures
http://www.
augmenttreatment.com
[65]
mitoTALENs Nuclease Targeted to mtDNA
mutation
Keeping heteroplasmy
below threshold levels
[94,95]
Outstanding Questions
Canwe link knowledgeof mutantmito-
chondria to the prediction of disease
recurrence risk?
Howdo wedealwith mitochondrial het-
eroplasmy for
various diseases and
account for
this in therapeutic design?
What steps can we take to ef ciently
and effectively collect human cohorts
to study mitochondrial d isorders in
both rare and chronic diseases?
How do mitochondr ia adapt to the
changing environment we experience?
How are the well -known functions
of the mitochondrion such as energy
production linked to its emerging roles
in epigenetic regulation, stem cell-
induced tissue regeneration, and
immune defense?
Do we know whether mitochondrial
dysfunction is a primary cause of a
disease or a
secondary effect resulting
froma given disorder?Is causality con-
text dependent?
Can we rout inely replace damaged
mitochondria within somatic stem cells
with funct ioning mitochondria and
inject them into t issues? If so, is the
existing epigenetic program faithfully
carried over into the grafted cells?
Within the eld of mitochondrial trans-
fer, how can we accurately analyze
cellular subsets andMHCs in respond-
ing (recipient) cells?
Would mitochondrial progeny from tis-
sue regeneration processes act in the
same manner as parental mitochon-
dria? If so, under what circumstances?
How can scientists integrate the fast-
growing eld of mitochondrial research
with that of stem cells, epigenetics and
immunobiology to treat diseases not
previously thoughtto beassociatedwith
mitochondrial dysfunction? Can these
new elds be used to improve diagnos-
tic
and therapeutic procedures?
Froman evolutionarypointof view, since
mitochondria were thought to be incor-
porated into multicellular organisms
from single-cell bacteria, are we sti ll
acquiring new mitochondria from our
own microbiome? If so, are they trans-
mittable
and functional in mitochondrial
next-generation progeny?
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