Mitochondrial CHCHD2 and CHCHD10: Roles in Neurological Diseases and Therapeutic Implications

Abstract

CHCHD2 mutations have been identified in various neurological diseases such as Parkinson’s disease (PD), frontotemporal dementia (FTD), and Alzheimer’s disease (AD). It is also the first mitochondrial gene whose mutations lead to PD. CHCHD10 is a homolog of CHCHD2; similar to CHCHD2, various mutations of CHCHD10 have been identified in a broad spectrum of neurological disorders, including FTD and AD, with a high frequency of CHCHD10 mutations found in motor neuron diseases. Functionally, CHCHD2 and CHCHD10 have been demonstrated to interact with each other in mitochondria. Recent studies link the biological functions of CHCHD2 to the MICOS complex (mitochondrial inner membrane organizing system). Multiple experimental models suggest that CHCHD2 maintains mitochondrial cristae and disease-associated CHCHD2 mutations function in a loss-of-function manner. However, both CHCHD2 and CHCHD10 knockout mouse models appear phenotypically normal, with no obvious mitochondrial defects. Strategies to maintain or enhance mitochondria cristae could provide opportunities to correct the associated cellular defects in disease state and unravel potential novel targets for CHCHD2-linked neurological conditions.

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https://doi.org/10.1177/1073858419871214
The Neuroscientist
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Review
Neurodegenerative disorders, comprising of a broad spec-
trum of diseases including Alzheimer’s disease (AD),
Parkinson’s disease (PD), amyotrophic lateral sclerosis
(ALS), and frontotemporal dementia (FTD), present a
major challenge in the aging population. Diverse neurode-
generative disorders display selective dysfunction and
neuron death in different brain regions. One common
pathological hallmark shared by all those disorders is
mitochondrial dysfunction. Whether mitochondrial dys-
function is the cause or the consequence of pathogenic
process has been often debated. Current evidence suggests
that mitochondrial dysfunction contributes to multiple
pathological process of human diseases in that mitochon-
dria participate in almost all aspects of cellular function,
whereas mitochondrial dysfunction also directly causes
multiple inherited diseases and is heavily implicated in
age-related pathology including neurodegeneration.
Mitochondria and Mitochondrial
Genome
Mitochondria are distinct and powerful intracellular
organelles, playing a vital role in generating cellular
energy via oxidative phosphorylation. Mitochondria are
particularly important for energy-intensive, postmitotic
cells such as neurons, cardiac cells, and muscle cells. A
continual supply of energy from mitochondria is critical
to neuron function in that human brain consumes 20% of
the body’s total energy with only ~2% of total body mass.
Apart from energy generation, mitochondria perform
critical metabolic functions, harbor proapoptotic factors,
and are the major sources of damaging reactive oxygen
species.
Mitochondrion is unique in that it is the only cell organ-
elle under the control of both nuclear DNA (nDNA) and
its own mitochondrial DNA (mtDNA). mtDNA replicates
independent of cell cycle, and even replicates in postmi-
totic cells such as neurons. Such feature makes mtDNA
existing in multiple numbers of copies, varying from hun-
dreds to thousands, in different populations of cells.
871214NROXXX10.1177/1073858419871214The NeuroscientistZhou et al.
review-article2019
1Neuroscience Research laboratory, National Neuroscience Institute,
Duke NUS Medical School, Singapore
2Department of Neurology, Singapore General Hospital, Singapore
Corresponding Author:
Eng-King Tan, National Neuroscience Institute, Duke-NUS Medical
School, Singapore.
Email: gnrtek@sgh.com.sg
Mitochondrial CHCHD2 and CHCHD10:
Roles in Neurological Diseases and
Therapeutic Implications
Wei Zhou1, Dongrui Ma2, and Eng-King Tan1,2
Abstract
CHCHD2 mutations have been identified in various neurological diseases such as Parkinson’s disease (PD),
frontotemporal dementia (FTD), and Alzheimer’s disease (AD). It is also the first mitochondrial gene whose mutations
lead to PD. CHCHD10 is a homolog of CHCHD2; similar to CHCHD2, various mutations of CHCHD10 have been
identified in a broad spectrum of neurological disorders, including FTD and AD, with a high frequency of CHCHD10
mutations found in motor neuron diseases. Functionally, CHCHD2 and CHCHD10 have been demonstrated to
interact with each other in mitochondria. Recent studies link the biological functions of CHCHD2 to the MICOS
complex (mitochondrial inner membrane organizing system). Multiple experimental models suggest that CHCHD2
maintains mitochondrial cristae and disease-associated CHCHD2 mutations function in a loss-of-function manner.
However, both CHCHD2 and CHCHD10 knockout mouse models appear phenotypically normal, with no obvious
mitochondrial defects. Strategies to maintain or enhance mitochondria cristae could provide opportunities to correct
the associated cellular defects in disease state and unravel potential novel targets for CHCHD2-linked neurological
conditions.
Keywords
CHCHD2, CHCHD10, neurological disorders, iPSC, animal models

2 The Neuroscientist 00(0)
Human nDNA is 3.3 billion bp, while mtDNA is small and
circular DNA including 16,569 nucleotides. mtDNA con-
tains 37 genes, including 22 tRNAs, 2 rRNAs, and 13
mRNAs encoding 13 electron transport chain proteins.
Due to its small size, mtDNA is unable to produce all
mitochondrial proteins, which is over 1000, needed for its
functionality; thus, mitochondria rely heavily on imported
nuclear gene products. Mitochondrial genome compasses
additional over 1000 nDNA mitochondrial genes. Since
mitochondria involve both nDNA and mtDNA-encoded
proteins, mutations on either nDNA mitochondrial genes
or mtDNA genes that disrupt mitochondrial function lead
to a broad spectrum of clinically and genetically diverse
and chronic human diseases, which can affect any parts of
the body including neurons, muscles, eyes, and livers
(Fig. 1).
Next-generation sequencing enables the discovery of
novel mutations of mitochondrial genes. However, many
pathogenic candidate genes remain uncharacterized,
leaving unmet diagnostic and treatment need for patients
whose defective gene has not yet been established. In
2014, mutations of mitochondrial gene CHCHD10,
encoded by nDNA, were first reported in human patients
with FTD and ALS. In 2015, mutations of mitochondrial
Figure 1. Genetic mutations in mitochondrial genome cause mitochondrial dysfunction, leading to cellular dysfunction
manifested in different organs of patients with diverse pathology. Mitochondria are unique cellular organelles that are responsible
for cellular respiration, apoptosis, ROS generation, and so on. Mitochondria play a vital role in generating cellular energy,
and therefore are particularly important for cells that are highly energy demanding. Mitochondrial function is maintained by
mitochondrial genes that are encoded by either nuclear DNA or mitochondrial DNA. Genetic mutations in mitochondrial
genome lead to mitochondrial dysfunction in different types of cells, tissues, and organs, especially in neurons and neurological
system. In human patients, such manifested defects could be highly variable and presented in various organs or multiple organs
that may not be apparently linked.

Zhou et al. 3
gene CHCHD2, a homologue of CHCHD10 and encoded
by nDNA, were identified in PD patients. Subsequently,
CHCHD2 or CHCHD10 mutations have been reported in
different population of patients with PD, ALS, FTD, and
AD. In particular, CHCHD2 is the first mitochondrial
gene whose mutations lead to PD and this could provide
an opportunity to unravel novel therapeutic targets. Here
we provide a concise review of the clinical associations
between mutations of mitochondrial genes CHCHD2 and
CHCHD10 and a broad spectrum of neurological disor-
ders, discuss the pathophysiological roles of CHCHD2/
CHCHD10 based on various cellular and animal models,
and highlight the potential therapeutic targets for these
disorders.
CHCHD2 and Human Neurological
Disorders
CHCHD2 is a member of a family of proteins containing
CHCH (coiled-coil-helix-coiled-coil-helix) domain (Fig.
2A). CHCH domain is characterized by a pair of cyste-
ines separated by nine amino acids, named CX9C motifs
(Fig. 2B). CHCH domain adopts a CHCH fold stabilized
by two disulfide bonds that are formed by the four cyste-
ines in the twin CX9C motifs (i.e., two pairs of cysteines
each separated by nine residues) (Fig. 2C). The majority
of CHCH domain-containing proteins, such as CHCHD2,
have a single CHCH domain, while the rest, such as
CHCHD5, have two. The family of CHCH domain-con-
taining proteins is evolutionarily conserved with diverse
physiological functions. Many CHCH domain-contain-
ing proteins are localized in the mitochondria (Modjtahedi
and others 2016).
In addition to a C-terminal CHCH domain, CHCHD2
has an N-terminal mitochondrial targeting sequence.
Thirteen disease-related amino acid residues span the
whole CHCHD2 protein, 151 amino acids in length
(illustrated in Fig. 2A; details listed in Table 1). Most of
those residues are conserved in evolution, while some are
changed in invertebrates such as Drosophila (Fig. 2B).
The missense mutations Thr61Ile and Arg145Gln of
CHCHD2 were reported in inherited late-onset autoso-
mal dominant PD cases in a large Japanese family
(Funayama and others 2015). Thr61Ile was later reported
in a Chinese autosomal dominant PD family (Shi and oth-
ers 2016). Ala32Thr, Pro34Leu, and Ile80Val were identi-
fied in PD patients with European ancestry (Jansen and
others 2015). A nonsense heterozygous variant of
CHCHD2 (Gln126X), leading to a truncated protein, was
identified in a German PD patient (age at onset >40
years) (Koschmidder and others 2016). CHCHD2
Arg8His (Ikeda and others 2017) and Ala79Ser (Yang
and others 2019) were identified in sporadic PD cases.
CHCHD2 Val66Met was reported in an Italian patient
with multiple system atrophy (Nicoletti and others 2017).
The variant Pro2Leu was identified in PD, essential
tremor, AD, and FTD (Foo and others 2015; Funayama
and others 2015; Shi and others 2016; Wu and others
2016). The rare missense variants Ser5Arg, Ala32Thr,
and Ser85Arg of CHCHD2 were identified in AD and
FTD (Che and others 2018). Most disease-related
CHCHD2 mutations are heterozygous, with the excep-
tion of Ala71Pro. Homozygous CHCHD2 Ala71Pro,
along with a rare homozygous Asp211Asn mutation of
TOP1MT (mitochondrial topoisomerase I), was reported
in a 30-year-old Caucasian patient with early-onset PD
(Lee and others 2018b).
Pathophysiological Roles of
CHCHD2/CHCHD10 in Cellular
Models
CHCHD2 is located in both mitochondria and nuclei
(Aras and others 2015; Liu and others 2015; Zhou and
others 2018a). Both the N-terminal mitochondria target-
ing sequence and the C-terminal CHCH domain contrib-
ute to CHCHD2’s mitochondrial location. A truncation
mutation, CHCHD2 Q126X, which loses its CHCH
domain, is located in the cytosol (Zhou and others 2018a).
Endogenous CHCHD2 is primarily located in mitochon-
dria (Liu and others 2015; Zhou and others 2018a). Upon
apoptotic stimuli, CHCHD2 loses its mitochondrial local-
ization and seems to be translocated to the nucleus (Liu
and others 2015). In the nucleus, CHCHD2 functions as a
transcription factor. CHCHD2 transcriptionally regulates
the expression of COX (cytochrome c oxidase) subunit 4
isoform 2 (COX4I2) and CHCHD2 itself upon oxidative
or hypoxic stress (Aras and others 2013; Aras and others
2015). CHCHD2 responds to different stressors.
CHCHD2 was upregulated at both the transcript and pro-
tein levels by mitochondrial unfolded protein stress
(mtUPR) caused by the overexpression of truncated mito-
chondrial matrix protein ornithine transcarbamylase
(ΔOTC) in Drosophila (Meng and others 2017).
Since mitochondria play a significant role in neuron
system, there is much interest in understanding
CHCHD2’s mitochondrial role(s). CHCHD2 promotes
mitochondrial function. Knockdown of CHCHD2
reduced the activity of mitochondrial respiratory com-
plex IV and, to some extent, the activity of complex I
(Baughman and others 2009). Knockdown of CHCHD2
reduced mitochondrial oxygen consumption in various
cellular models (Baughman and others 2009; Zhou and
others 2018a). We and others also found that overexpres-
sion of CHCHD2 WT in cell lines did not enhance or
reduce the mitochondrial oxygen consumption rate. We
generated isogenic human embryonic stem cells (hESCs)
harboring heterozygous PD-associated CHCHD2 mutants

4 The Neuroscientist 00(0)
Figure 2. The schematic view of disease-related CHCHD2 mutations in human CHCHD2 protein. (A) Disease-associated
CHCHD2 mutations were labeled on the functional domains of human CHCHD2. There is an N-terminal mitochondrial targeting
sequence and C-terminal CHCH (coiled-coil-helix-coiled-coil-helix) domain in the human CHCHD2 protein. T61I (Thr61Ile),
Q126X (Gln126X), and R145Q (Arg145Gln), whose functions have been experimentally proven, are labeled red. Common risk
variants such as P2L (Pro2Leu), S5R (Ser5Arg), A32T (Ala32Thr), P34L (Pro34Leu), V66M (Val66Met), I80L (Ile80Val), and S85R
(Ser85Arg) are labeled yellow. A71P (Ala71Pro), labeled with *, was reported as a homozygous mutation in an early-onset PD
(continued)

Zhou et al. 5
Q126X or R145Q and highlighted that PD-associated
CHCHD2 Q126X and R145Q led to mitochondrial dys-
function with a reduction in both mitochondrial oxygen
consumption and the abundancy of components of the
mitochondrial respiratory complex (Zhou and others
2018a). A human induced pluripotent stem cell (hiPSC)
line derived from fibroblasts of a patient carrying hetero-
zygous CHCHD2 T61I was reported (Wang and others
2018), although mitochondrial oxygen consumption rates
from either patient-derived fibroblasts or hiPSCs are yet
to be shown. The generation of patient-derived fibro-
blasts or hiPSCs carrying CHCHD2 mutations with
proper control and careful characterization of those
patient-derived lines will provide more evidence.
CHCHD2 inhibits mitochondrial apoptosis.
Downregulation of CHCHD2 significantly increased cel-
lular ROS (Aras and others 2015). Transient knockdown
of CHCHD2 sensitized cells to multiple apoptotic stim-
uli. CHCHD2 interacts with Bcl-xL to regulate Bax local-
ization, activation, and oligomerization. When cells were
challenged by apoptotic stressors, CHCHD2 decreased in
mitochondria, thus losing its binding to Bcl-xL and allow-
ing apoptosis to proceed (Liu and others 2015). CHCHD2
was also shown to bind to cytochrome c along with
MICS1, a member of the Bax inhibitor-1 superfamily
(Meng and others 2017).
CHCHD2 and Mitophagy
Mitochondrial integrity and homeostasis are vital to main-
taining proper mitochondrial function. When terminally
damaged, mitochondria are degraded through the process
of targeted mitochondrial autophagy (mitophagy) to pre-
vent potentially catastrophic consequences. PINK1/Parkin
signaling is responsible for mitochondrial quality control
via mitophagy in the pathogenesis of PD. It has been spec-
ulated that mitochondrial CHCHD2 may be involved in
the process of PINK/Parkin-mediated mitophagy; never-
theless, evidence supporting a direct role of CHCHD2 in
PINK1/Parkin-mediated mitophagy is lacking. In
Drosophila model, loss of CHCHD2 did not further
enhance the mitochondrial defects caused by loss of
PINK1 or Parkin, suggesting that CHCHD2 may play a
role independent of Parkin/PINK1 (Meng and others
2017). On the other hand, PINK1 or Parkin
overexpression decreased the mitochondrial integrity of
dCHCHD2-deficient flies (Meng and others 2017). As
PINK1 or Parkin overexpression could activate mitoph-
agy, these results suggest that PINK1/Parkin recognized
and cleared the dysfunctional mitochondria caused by loss
of CHCHD2 in flies. Furthermore, in vivo evidence of
mitophagy mediated by PINK1/Parkin seems inconsistent
even though PINK1 and Parkin’s role in mitophagy is well
established. A recent study reported no substantial impact
on basal mitophagy in PINK1 or Parkin null flies, even
though these flies exhibit locomotor defects and dopami-
nergic neuron loss (Lee and others 2018a). Studies using
mice deficient in PINK1 also showed that basal mitoph-
agy is unaffected by loss of PINK1 (McWilliams and oth-
ers 2018). However, other investigators reported that
physiological mitophagy increased with age in Drosophila
(Cornelissen and others 2018), and mitophagy was abro-
gated by PINK1 or Parkin deficiency in vivo. Further
studies in mammalian systems may provide further in-
depth insights into the in vivo or in vitro role of mitophagy
as well as CHCHD2’s relationship with mitophagy.
CHCHD2, CHCHD10, and MICOS
Structurally, mitochondria are composed of a matrix sur-
rounded by two layers of lipid-based membranes, the
mitochondrial outer membrane (OM) and the mitochon-
drial inner membrane (IM) (Fig. 3). The matrix contains
mtDNA and enzymes for various metabolic pathways,
including the TCA cycle and fatty acid oxidation. OM,
which separates the mitochondria from the cytosol, is
responsible for communication between mitochondria
and other membranous organelles. IM shows a complex
ultrastructure with multiple cristae protruding into the
matrix (Fig. 3). The cristae provide an extended mem-
brane surface for multiple enzymes and cofactors com-
posing the mitochondrial respiratory chain, named
complexes I to V (complex I, NADH ubiquinone reduc-
tase; complex II, succinate ubiquinone reductase; com-
plex III, ubiquinol cytochrome c reductase; complex IV,
cytochrome c oxidase; complex V, F1FO-ATP synthase).
Therefore, the cristae are essential for OXPHOS enzymes
to produce ATP.
Such delicate mitochondrial cristae are maintained by
protein modulators and specific phospholipids in IM,
case, along with a homozygous TOP1MT Asp211Asn mutation. The remaining mutations were heterozygous in various human
neurodegenerative disorders. (B) Multiple sequence alignment of the CHCHD2 protein from H. sapiens (human), M. musculus
(mouse), R. norvegicus (rat), C. griseus (hamster), D. rerio (zebrafish), and D. melanogaster (fly, CG5010). Cysteines in the CHCH
domain are highlighted in blue; T61, Q126, and R145 residues are highlighted in red; the remaining disease-linked residues are
highlighted in yellow. Two CX9C motifs are labeled. (C) A typical coiled coil-helix-coiled coil helix (CHCH) fold formed by twin
CX9C motifs, in which two disulfide bonds (labeled blue) are formed by the four cysteines. The representative illustration was
adapted from the solution NMR (nuclear magnetic resonance) structure of human CHCHD4/Mia40, a membrane of the CHCH
domain-containing protein family. Protein Data Bank ID: 2L0Y.
Figure 2. (continued)

6 The Neuroscientist 00(0)
Table 1. Summary of Amino Acid Mutations of CHCHD2/CHCHD10 with the Associated Human Neurological Disorders.
Protein Mutation Genotype Associated Diseases Ethnicity Population Reference
CHCHD2 Pro2Leu +/− Sporadic PD Japanese (Funayama and others 2015)
Early-onset and sporadic PD Chinese (Foo and others 2015)
Sporadic PD Chinese (Shi and others 2016)
ET Chinese (Wu and others 2016)
AD, FTD Chinese (Che and others 2018)
Ser5Arg +/− AD, FTD Chinese (Che and others 2018)
Arg8His +/− Sporadic PD Japanese (Ikeda and others 2017)
Ala32Thr +/− PD Western European
ancestry
(Jansen and others 2015)
AD, FTD Chinese (Che and others 2018)
Pro34Leu +/− PD Western European
ancestry
(Jansen and others 2015)
Thr61Ile +/− Late-onset autosomal dominant PD Japanese (Funayama and others 2015)
Autosomal dominant PD Chinese (Shi and others 2016)
Val66Met +/− MSA Italian (Nicoletti and others 2017)
Ala71Pro −/− Early-onset PD, along with a homozygous TOP1MT
Asp211Asn mutation
Caucasian (Lee and others 2018b)
Ala79Ser +/− Sporadic PD Chinese (Yang and others 2019)
Ile80Val +/− PD Western European
ancestry
(Jansen and others 2015)
Ser85Arg +/− AD, FTD Chinese (Che and others 2018)
Gln126X +/− Early-onset PD German (Koschmidder and others 2016)
Arg145Gln +/− Late-onset autosomal dominant PD Japanese (Funayama and others 2015)
CHCHD10 Pro12Ser +/− ALS Spanish (Dols-Icardo and others 2015)
Arg15Leu/Ser +/− R15L-familial ALS German (Müller and others 2014)
R15S with G58R in cis-autosomal dominant
mitochondrial myopathy
Hispanic (Ajroud-Driss and others 2015)
R15L-familial ALS USA (Johnson and others 2014)
R15L-family with a history suggestive of autosomal-
dominant motor neuron disorder
German (Kurzwelly and others 2015)
R15L-sporadic ALS Caucasian (Zhang and others 2015)
His22Tyr +/− Sporadic FTD Chinese (Jiao and others 2016)
Pro23Thr/Ser/
Leu
+/− P23T-FTLD Italian (Zhang and others 2015)
−/− P23S-Sporadic FTD Chinese (Jiao and others 2016)
+/− P23L-Familial ALS Chinese (Shen and others 2017)
Pro24Leu +/− FTD Chinese (Che and others 2017)
Ser30Leu +/− Sporadic PD Chinese (Zhou and others 2018b)
Ala32Asp +/− Sporadic FTD Chinese (Jiao and others 2016)
Pro34Ser +/− FTD-ALS French (Chaussenot and others 2014)
Sporadic ALS Italian (Chio and others 2015)
PD Caucasian (Zhang and others 2015)
AD
Ala35Asp +/− FTLD Italian (Zhang and others 2015)
Sporadic late-onset AD Chinese (Xiao and others 2016)
Val57Glu +/− Sporadic FTD Chinese (Jiao and others 2016)
Gly58Arg +/− G58R with R15S in cis-autosomal dominant
mitochondrial myopathy
Hispanic (Ajroud-Driss and others 2015)
Ser59Leu +/− Late-onset FTD-ALS with cerebellar ataxia and
mitochondrial myopathy
French (Bannwarth and others 2014)
FTD and FTD-ALS Spanish (Chaussenot and others 2014)
Gly66Val +/− Familial ALS Finnish (Müller and others 2014)
Late-onset SMAJ Caucasian (Penttila and others 2015)
Axonal CMT2 European (Auranen and others 2015)
Pro80Leu +/− Sporadic early-onset ALS and muscle mitochondrial
pathology
Italian (Ronchi and others 2015)
ALS Caucasian (Zhang and others 2015)
Gln82X +/− FTD Spanish (Dols-Icardo and others 2015)
Tyr92Cys +/− Sporadic ALS Chinese (Zhou and others 2017)
Gln102His +/− Sporadic ALS Chinese (Zhou and others 2017)
Q108P/X +/− FTD Belgium (Perrone and others 2017)
PD = Parkinson’s disease; ET = essential tremor; AD = Alzheimer’s disease; FTD = frontotemporal dementia; MSA = multiple system atrophy; ALS = amyotrophic
lateral sclerosis; SMAJ = late-onset spinal motor neuropathy.

Zhou et al. 7
Figure 3. The pathophysiological roles of CHCHD2/CHCHD10 and potential intervening strategies for CHCHD2-related
diseases. Various in vivo, such as flies and mice, and in vitro, such as cell lines and hiPSC models, have been used to investigate
the pathophysiological role of CHCHD2 and its homologue CHCHD10. Both proteins are located in mitochondria. CHCHD2
and CHCHD10 form homodimer along with CHCHD2-CHCHD10 heterodimer to maintain MICOS. MICOS is a protein
complex located in the mitochondrial inner membrane (IM) that determines the number and shape of crista junctions with
IM-specific lipids such as cardiolipin. The mammalian MICOS are composed of key components Mitofilin/Mic60 and MINOS1/
Mic10, with other components including CHCHD3 and CHCHD6. CHCHD10 was previously believed to associate with MICOS.
CHCHD2 mutation causes the impairment of CHCHD2-CHCHD10 complex, leading to destabilized MICOS and eventually
mitochondrial dysfunction including reduced mitochondrial oxygen consumption rate and OXPHOS complexes. Strategies to
enhance or stabilize MICOS complex or CHCHD2 could ameliorate or repair the mitochondrial defects caused by CHCHD2
mutations. In line with that, we found the FDA-approved peptidyl drug Elamipretide (MTP-131), which specifically targets the
IM lipid cardiolipin, can ameliorate the cristae defects and mitochondrial dysfunction caused by CHCHD2 mutation. Several
questions remain on the pathophysiological roles of CHCHD2/CHCHD10, for example: (1) Is there any mouse models on
CHCHD2? (2) How do CHCHD2/CHCHD10 work with MICOS to maintain mitochondrial cristae? See the text for detailed
description. OM = mitochondrial outer membrane; IMS = mitochondrial internal membrane space; IM = mitochondrial
inner membrane; mtDNA = mitochondrial DNA; TFAM = transcriptional factor A, mitochondrial, which is a key activator of
mitochondrial transcription as well as a participant in mtDNA replication.

8 The Neuroscientist 00(0)
such as cardiolipin (Ikon and Ryan 2017). A large protein
complex named MICOS (the mitochondrial contact site
and cristae organizing system) is essential for mitochon-
drial membrane architecture and the formation of cristae
junctions (Fig. 3). The MICOS complex is highly con-
served from yeast to human and comprises at least 7 com-
ponents in mammals: Mitofilin/Mic60, CHCHD3/Mic19,
CHCHD6/Mic25, APOO/Mic26, APOOL/Mic27, QIL1/
Mic13, and MINOS1/Mic10. The list of the components
of MICOS complex may grow, as multiple pieces of evi-
dence suggest the presence of unidentified components of
MICOS. These components form two distinct MICOS
subcomplexes marked by the core components Mitofilin/
Mic60 and MINOS1/Mic10. The Mitofilin/Mic60 sub-
complex and MINOS1/Mic10 subcomplex play different
roles in maintaining contact sites between IM and OM
and in the formation of stable cristae junctions, respec-
tively (Wollweber and others 2017). Previous studies
revealed that complete absence or impairment of any
MICOS components caused drastic alterations of mito-
chondrial cristae and ultimately mitochondrial dysfunc-
tion, and the loss of one subunit of MICOS also prevented
the stable accumulation of other MICOS components in
mitochondria (van der Laan and others 2016; Wollweber
and others 2017). Disruption of the components of
MICOS has been associated with numbers of neurologi-
cal disorders (van der Laan and others 2016).
Recent studies link the biological functions of
CHCHD2 to MICOS complex. Such linkage starts from
CHCHD10, a homolog of CHCHD2. CHCHD10, with a
C-terminal CHCH domain, shares 53% protein sequence
identity with CHCHD2 (Fig. 4). It is speculated that both
proteins play similar and/or redundant biological roles;
furthermore, fast-moving research reveals rather contro-
versial conclusions on their roles based on different cel-
lular or animal models.
CHCHD10 mutations on 16 amino acid residues,
across its whole protein sequence, 142 amino acid in
length (illustrated in Fig. 4; details listed in Table 1), have
been identified in a broad spectrum of complicated neu-
rological disorders, including the motor neuron disease
ALS (Bannwarth and others 2014; Chaussenot and others
2014; Chio and others 2015; Dols-Icardo and others
2015; Johnson and others 2014; Kurzwelly and others
2015; Marroquin and others 2016; Müller and others
2014; Perrone and others 2017; Ronchi and others 2015;
Shen and others 2017; Teyssou and others 2016; Zhang
and others 2015; Zhou and others 2017), mitochondrial
myopathy (Ajroud-Driss and others 2015), late-onset spi-
nal motor neuronopathy (SMAJ) (Penttila and others
2015), Charcot-Marie-Tooth disease type 2 (CMT2)
(Auranen and others 2015), cognitive decline similar to
FTD (Bannwarth and others 2014; Chaussenot and others
2014; Che and others 2017; Dols-Icardo and others 2015;
Jiao and others 2016; Perrone and others 2017), PD
(Zhang and others 2015; Zhou and others 2018b), AD
(Xiao and others 2016; Zhang and others 2015), and cer-
ebellar ataxia (Bannwarth and others 2014). With a 53%
sequence identity and 64% sequence similarity (by Blast)
between CHCHD2 and CHCHD10, we found that 11 out
of 13 disease-associated CHCHD2 residues are con-
served in CHCHD10, and 12 out of 16 disease-associated
CHCHD10 residues are conserved in CHCHD2 (Fig.
4C), hinting a comparable pathological role of CHCHD2
and CHCHD10.
Similar to other CHCH domain-containing proteins,
CHCHD10 is a mitochondrial protein. Efforts were made
to investigate the mechanisms of CHCHD10’s patho-
physiological role. In 2016, Genin and colleagues, using
patient-derived fibroblasts and cell lines, reported that
disease-associated CHCHD10 S59L mutations caused
the loss of mitochondrial cristae and disassembly of
MICOS complex. They found that CHCHD10 bound to
Mitofilin/Mic60 in MICOS in HeLa cells and that
CHCHD10 comigrated with Mitofilin/Mic60, CHCHD3/
Mic19, and CHCHD6/Mic25 in human fibroblast lysates
(Genin and others 2016). The authors did not address
whether CHCHD10 S59L has impaired binding to
Mitofilin/Mic60 compared with CHCHD10 wildtype and
how CHCHD10 S59L causes the loss of mitochondrial
cristae.
In 2017, Meng and colleagues reported that CHCHD2
loss in flies caused mildly distorted mitochondrial cristae
in indirect flight muscles (Meng and others 2017). They
found that CHCHD2 bound to itself to form a homodi-
mer. In early 2018, two back-to-back papers reported a
CHCHD2-CHCHD10 interaction underlying CHCHD10-
associated ALS pathogenesis (Burstein and others 2018;
Straub and others 2018). However, authors of both papers
reported the failure to identify a CHCHD10-Mitofilin/
Mic60 interaction either in human fibroblasts (Straub and
others 2018) or in mouse heart mitochondria (Burstein
and others 2018). Similarly, we reported an endogenous
CHCHD2-CHCHD10 interaction but no endogenous
CHCHD10-Mitofilin/Mic60 bindings in dopaminergic
SK-N-SH cells and human brain lysates (Zhou and others
2018a). While the CHCHD2-CHCHD10 interaction has
been corroborated by many studies (Burstein and others
2018; Huang and others 2018; Purandare and others
2018; Straub and others 2018; Zhou and others 2018a),
many investigators were unable to validate the
CHCHD10-Mitofilin/Mic60 interaction. Moving ahead,
several interesting questions remain to be addressed: (1)
Is MICOS the primary target through which CHCHD10
fulfills its biological function? (2) Is MICOS the primary
target through which CHCHD2 fulfills its biological
function? (3) Does the CHCHD2-CHCHD10 complex
play any role in MICOS complex? (4) If the answer to

Zhou et al. 9
Figure 4. The schematic view of disease-related CHCHD10 mutations in human CHCHD10 protein. (A) Disease-associated
CHCHD10 mutants were labeled on the functional domains of the human CHCHD10 protein. There is an N-terminal mitochondrial
targeting sequence and a C-terminal CHCH (coiled-coil-helix-coiled-coil-helix) domain in the human CHCHD10 protein. Mutations/
variants identified in various human neurological disorders are labeled orange. Y92C and Q102C, labeled with *, were found in a
(continued)

10 The Neuroscientist 00(0)
Question 3 is yes, how does the CHCHD2-CHCHD10
complex work with MICOS components? If the answer is
no, how is the CHCHD2-CHCHD10 complex implicated
in the pathogenesis of various neurological disorders?
Regarding CHCHD10 and CHCHD2’s role in MICOS,
Straub and colleagues found that neither CHCHD10 nor
CHCHD2 comigrated with key components of the
MICOS complex, Mitofilin/Mic60 or CHCHD3/Mic19,
by using cell lysates from human fibroblasts (Burstein
and others 2018), similar to Genin and colleagues (Genin
and others 2016); however, these observations were
sharply opposite to those from Genin and colleagues in
2016. Straub and colleagues, therefore, concluded that
CHCHD10 did not associate with MICOS. Separately,
Huang and colleagues generated stable HeLa and
HEK293 cell lines deficient in CHCHD2, CHCHD10, or
both and observed very subtle abnormal mitochondrial
ultrastructure in CHCHD2/CHCHD10 double knockout
cell lines and no impairment of MICOS components in
either CHCHD2 or CHCHD10 knockout cell lines
(Huang and others 2018). Therefore, it was concluded
that both CHCHD2 and CHCHD10 were dispensable for
the MICOS complex. Genin and colleagues further
reported that disease-associated CHCHD10 G66V did
not impair the MICOS complex by using patient-derived
fibroblasts (Genin and others 2018) after they reported
MICOS defects in patient cells carrying CHCHD10 S59L
(Genin and others 2016). This observation complicated
CHCHD10’s role in MICOS as G66 is in proximity with
S59 in CHCHD10 (Fig. 4A). Taken together, multiple
lines of evidence seems to point to a non-MICOS-related
role of CHCHD10, and possibly CHCHD2.
We investigated the mechanism(s) of CHCHD2 muta-
tions associated with PD (Zhou and others 2018a). We
chose dopaminergic SK-N-SH cell line, which resembles
some physiological features of dopaminergic neurons.
We engaged super-resolution microscopy (STED), which
provides a resolution smaller than the wavelength of
light, and found that CHCHD2 showed a unique distribu-
tion pattern along mitochondria that was very similar to
the distribution pattern of Mitofilin/Mic60 revealed pre-
viously by STED. High-resolution imaging showed some
overlapping signals for CHCHD2 and Mitofilin/Mic60,
suggesting that they might not bind to each other. We
generated isogenic lines harboring PD-related CHCHD2
mutants R145Q or Q126X on pluripotent hESCs line H9.
hESCs can be differentiated into neural progenitor cells
(NPCs) and dopaminergic neurons under proper culture
conditions.
By using SK-N-SH, hESCs and NPCs, we found that
CHCHD2 knockdown caused distorted mitochondrial
cristae, destabilized MICOS and resulted in mitochon-
drial dysfunction. The same observations, as well as
reduced CHCHD2 itself, were made in isogenic hESCs
and derived NPCs carrying CHCHD2 R145Q or Q126X.
With these results, we found a CHCHD2-CHCHD10
interaction but not a CHCHD10-Mitofilin/Mic60 interac-
tion. PD-associated CHCHD2 mutants impaired their
binding to CHCHD10. To investigate whether the defects
caused by CHCHD2 mutants were indeed mediated by
CHCHD10, we examined SK-N-SH cells with CHCHD10
transient knockdown. We found that transient knockdown
of CHCHD10 in SK-N-SH caused a destabilized MICOS
complex and distorted mitochondrial ultrastructure.
CHCHD10 knockdown caused the reduction of CHCHD2
and vice versa. We showed colocalization of CHCHD2
and CHCHD10 in mouse brain sections, and both
CHCHD2 and CHCHD10 appeared as “rail-like” clusters
at the rim of mitochondria by super resolution micros-
copy, suggesting that both proteins were in proximity to
MICOS. To identify how CHCHD2 was involved in
MICOS, we tried to determine the endogenous protein-
protein interaction without any crosslinker, but we failed
to find any interaction between CHCHD2 and Mitofilin/
Mic60, MINOS1/Mic10, CHCHD3/Mic19, or CHCHD6/
Mic25. As the current understanding of MICOS compo-
nents is still limited and additional unknown subunits of
the mammalian MICOS have been suggested (van der
Laan and others 2016), we speculate that unidentified
MICOS components may bridge the CHCHD2-
CHCHD10 complex to known MICOS components. For
example, CHCHD2 binds to MICS1, a protein maintain-
ing cristae structure (Meng and others 2017). MICS1 is a
protein yet to be understood in terms of whether MICS1
maintains cristae structure through MICOS and whether
CHCHD10 associates with the MICS1-CHCHD2 com-
plex. Further study is needed to extend the understanding
of MICOS.
Previous studies on CHCHD10 knockdown or knock-
out showed little or mild defects (Burstein and others
longer transcript of human CHCHD10. (B) Multiple sequence alignment of the CHCHD10 protein from H. sapiens (human), M.
musculus (mouse), R. norvegicus (rat), and D. rerio (zebrafish). A potential fly ortholog of human CHCHD10, CG31007, was included
in the alignment and labeled D. melanogaster (fly). Cysteines within the CHCH domain are highlighted in blue; disease-linked residues
are highlighted in red. Two CX9C motifs are labeled. (C) Sequence alignment of the CHCHD2 protein and the CHCHD10
protein from H. sapiens (human). Cysteines within the CHCH domain are highlighted in blue; disease-linked CHCHD2 residues are
highlighted in green; disease-linked CHCHD10 residues are highlighted in red. Conserved residues in CHCHD2 or CHCHD10,
which were reported in neurological disorders, are highlighted in yellow with rectangles.
Figure 4. (continued)

Zhou et al. 11
2018; Huang and others 2018) in MICOS, while ours
showed significant defects in MICOS. One reason could
be cell type-specific effects. For example, dopaminergic
cells could be a more relevant model than HEK293/HeLa
cells. Another reason could be the different gene manipu-
lation strategy. During gene silencing, especially when
the target gene is indispensable, loss of one gene could be
compensated by another gene with overlapping function
to maintain the fitness of living organisms (El-Brolosy
and Stainier 2017). When generating stable lines or
knockout animals, chronic cultured cells could gradually
activate cellular mechanisms to compensate for the lack
of the target gene, while transient knockdown of the tar-
geted gene shows instant cellular responses upon losing
the target gene; therefore, the results of stable gene silenc-
ing may be compromised compared with those of tran-
sient genetic manipulation. With such a scenario, chronic
cellular or animal models harboring knock-in mutants
may be a better choice for phenocopy genetic mutation-
related human diseases.
hiPSC technology allows the isolation of patient-
derived cells that carry all genetic alterations that cause
disease. Those patient-origin cells have a unique advan-
tage to recapitulate disease-relevant phenotypes, provid-
ing an experimental system to study the pathogenesis of
the disease in vitro. However, one major challenging
issue of patient-derived hiPSCs is the variability between
hiPSC lines of a given lineage (Khurana and others 2015).
Such variation between lines is unpredictable and is
believed to be mostly caused by the differences in genetic
backgrounds as well as the reprogramming strategy. In
addition, the process of deriving fibroblast subclones
from parental fibroblasts may also introduce variations,
as both fibroblast subclones and derived clonal hiPSCs
were reported to contain similar numbers of de novo vari-
ants compared with their parental fibroblasts (Kwon and
others 2017). Therefore, to eliminate variations between
lines, it would be better to generate isogenic pairs of dis-
ease-specific and control hiPSCs that differ exclusively at
the disease-causing mutation to define subtle disease-
relevant differences. CRISPR/Cas9 allows relatively easy
genome editing, whereas the specificity of Cas9 and its
off-target effects need to be carefully monitored and ade-
quately addressed in isogenic pairs of hiPSCs.
Animal Models of CHCHD2/
CHCHD10
The discovery of inherited forms of PD led to the genera-
tion of novel genetic animal models of PD. Although flies
are distantly related to humans in evolutionary terms,
many neuronal circuits in the vertebrates and flies appear
to be analogous, with both vertebrates and flies having
dopaminergic neurons and circuits. It is therefore
possible that PD-associated pathogenic mutations may
have similar effects in both flies and humans. Fly models
with manipulated PD genes, such as PINK1, Parkin,
Fbxo7, TRAP1, LRRK2, and α-synuclein, have success-
fully phenocopied many characteristics of PD, including
reduced locomotion, the reduction of dopaminergic neu-
rons in the brain, and mitochondrial and synaptic pheno-
types, suggesting that fly nervous system could provide
unique opportunities to elucidate neurophysiological per-
turbations caused by genetic mutations (West and others
2015).
Tio and colleagues reported transgenic fly models with
overexpressed human CHCHD2 WT, PD-related CHCHD2
mutants T61I, R145Q or risk-variant P2L (Tio and others
2017). They reported that all transgenic fly lines exhibited
PD-related phenotypes such as locomotor dysfunction,
dopaminergic neuron degeneration, lifespan reduction,
mitochondrial dysfunction, and impairment in synaptic
transmission. Among all transgenic lines, CHCHD2 T61I
and R145Q overexpression showed more severe phenotypes
than others. As CHCHD2 generally promotes mitochondrial
function, the toxicity observed in flies with CHCHD2 WT
overexpression may not be caused by CHCHD2 per se.
Meng and colleagues carried out a series of experi-
ments on flies by manipulating Drosophila CHCHD2,
CG5010 (Meng and others 2017). They reported that flies
without dCHCHD2 were largely normal at a younger
stage but had a shorter life span compared to the control
group. The loss of dCHCHD2 led to oxidative stress,
dopaminergic neuron loss, and motor dysfunction with
age. CHCHD2 loss in flies caused distorted mitochon-
drial cristae in the indirect flight muscles and impaired
oxygen respiration in the mitochondria. The authors
found that these PD-associated phenotypes can be res-
cued by the overexpression of the translation inhibitor
4E-BP and by the introduction of human CHCHD2 but
not its PD-associated T61I or R145Q mutants. Their
study on flies provides evidence on CHCHD2’s role in
PD pathogenesis in vivo, and rescue experiments showed
that CHCHD2 T61I and R145Q acted as loss-of-function
mutants. There is currently no fly study on CHCHD10.
An uncharacterized protein CG31007 could be a potential
fly ortholog of CHCHD10. CG31007 shares 43% amino
acid sequence identity and 64% sequence similarity (by
Blast) with human CHCHD10 (Fig. 4B). On the other
hand, Woo and others, showed CHCHD10 exerts a pro-
tective role in mitochondria and synaptic integrity by
using C. elegans model, and they reported FTD/ALS-
related CHCHD10 R15L and S59L exhibit loss-of-func-
tion phenotypes (Woo and others 2017).
Similar to many other mouse models of genetic PD,
which have had limited success in showing disease-
related phenotypes, CHCHD2 knockout mouse models
appear normal. CHCHD2-null mice developed normally

12 The Neuroscientist 00(0)
with no distorted mitochondrial ultrastructure observed
in CHCHD2 null MEFs (mouse embryonic fibroblasts)
(Meng and others 2017). CHCHD10 knockout mice were
also phenotypically normal, with no mitochondrial
defects in the brain, heart, or skeletal muscles (Burstein
and others 2018). Such normal phenotypes could be due
to, at least partially, genetic compensation as discussed
above. In line with that, knock-in mouse models with
disease-associated mutations may recapitulate some
pathological features of human diseases. Recent studies
on transgenic mice harboring CHCHD10 S59L demon-
strated features of motor neuron disease with mitochon-
drial defects and motor-neuron death (Anderson and
others 2019; Genin and others 2019). The in vivo studies
on CHCHD2/CHCHD10 are summarized in Table 2.
Drugs Targeting CHCHD2
Multiple experimental models suggest that CHCHD2
maintains mitochondrial cristae and disease-associated
CHCHD2 mutations function in a loss-of-function man-
ner. Therefore, strategies to maintain or enhance mito-
chondrial cristae as well as CHCHD2 could provide
opportunities to correct CHCHD2 mutations-caused cel-
lular defects (Fig. 3). We sought a potential strategy to
correct defects caused by CHCHD2 mutations using iso-
genic NPCs carrying heterozygous PD-related CHCHD2
R145Q as an in vitro model for PD. By testing various
candidate compounds, we reported that mitochondrial
defects caused by CHCHD2 R145Q can be ameliorated
by Elamipretide/MTP-131 (Zhou and others 2018a).
Elamipretide/MTP-131 (Bendavia) is a cell-permeable
and mitochondria-targeting peptide that protects mito-
chondrial cristae by crossing the mitochondrial outer
membrane and interacting with phospholipid cardiolipin
in the IM. Cardiolipin plays a crucial role in cristae for-
mation, mitochondrial fusion, mtDNA integrity, and oxi-
dative phosphorylation (Ikon and Ryan 2017).
Elamipretide/MTP-131 is currently being tested in clini-
cal trials for ischemia reperfusion injury, mitochondrial
myopathies, and heart failure. The US Food and Drug
Administration has granted fast track designation for
Elamipretide/MTP-131 as a treatment for people with
rare mitochondrial diseases (2017–2018). Considering
the good safety profile and known pharmacokinetics and
pharmacodynamics of Elamipretide/MTP-131, it could
be used as a potential treatment for CHCHD2 mutation-
related neuronal disorders. Further vigorous testing of
Elamipretide/MTP-131 in a proper in vivo model of
CHCHD2 will provide stronger evidence to use this drug
for patients with neurological disorders associated with
CHCHD2 mutations. Moreover, Elamipretide/MTP-131
directly targets cardiolipin, but not CHCHD2. Further
investigation on alternative strategies to enhance/stabi-
lize CHCHD2 directly, thereby to stabilize MICOS, could
be more efficient to correct the defects and benefit rele-
vant patients.
Current Limitations and Challenges
CHCHD2/CHCHD10 are important mitochondrial pro-
teins whose mutations have been associated with various
neurological diseases among different populations.
Several important questions regarding their mechanisms
and relevant models are yet to be answered: (1) Does
CHCHD2/CHCHD10 play any role in mitophagy? (2)
How do CHCHD2/CHCHD10 work with MICOS to
maintain mitochondrial cristae? (3) Are there any dopa-
minergic neurons carrying CHCHD2/CHCHD10 muta-
tions? As mitochondrial function is vital for hiPSCs to
Table 2. Summary of Animal Models on CHCHD2/CHCHD10 Studies.
Animal Models CHCHD2 CHCHD10
Drosophila dCHCHD2 null flies show mitochondrial
defects in muscle, with neuron defects
(Meng and others 2017)
Not availanle
Overexpression of human WT, T61I, R145Q,
P2L in flies all show neuron toxicity (Tio
and others 2017)
C. elegans Not available Loss of C. elegans har-1, the closest orthologue to
mammalian CHCHD10, impairs mitochondria
and movement (Woo and others 2017)
Mouse Knockout Normal development; no mitochondrial
defects in MEFs (Meng and others 2017)
Normal without disease pathology (Burstein and
others 2018)
Knockin Not available CHCHD10S59L/+ mimic the mitochondrial
myopathy with mtDNA instability displayed by
the patients (Anderson and others 2019; Genin
and others 2019)

Zhou et al. 13
differentiate into functional neurons, it could be challeng-
ing to derive dopaminergic neurons from hiPSC or hESC
carrying CHCHD2/CHCHD10 mutations. (4) Are there
any hiPSC lines derived from human patients carrying
CHCHD2/CHCHD10 mutations with corrected
CHCHD2/CHCHD10 mutations as controls? (5) Are
there any CHCHD2 mouse models phenocopying PD or
related neurological disorders? As discussed before, a
knockin mouse model harboring diseases-related
CHCHD2 mutations may work similarly as knockin
mouse model harboring diseases-related CHCHD10
mutations. (6) Even with the similarity between CHCHD2
and CHCHD10, they obviously perform differential cel-
lular functions and are under possible varied regulatory
modes. Further characterization of functions and regula-
tion of CHCHD2 and CHCHD10 will shed new light on
the pathology associated with both proteins. We antici-
pate the answers to the above-mentioned questions will
facilitate the discovery of novel targeted therapy for neu-
rological patients who carry mutations of CHCHD2, and
possibly of CHCHD10.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial sup-
port for the research, authorship, and/or publication of this
article: The authors’ work is supported by NMRC (National
Medical Research Council) Parkinson’s Disease Trans-
lational Clinical Program Grant, Parkinson’s Disease Large
Collaborative Grant (SPARK II) and STaR Award (EK-T).
ORCID iD
Wei Zhou https://orcid.org/0000-0002-0872-6223
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