a mitochondria odyssey

8/17/2019 A Mitochondria Odyssey

1/13

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.

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2/13

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.

392 Trends in MolecularMedicine, May2016,Vol. 22,No. 5


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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

Trends inMolecularMedicine, May2016, Vol. 22, No. 5 393


4/13

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.



6/13

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



7/13

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.



8/13

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.



9/13

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



10/13

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]



11/13

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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