Bioscience Reports, Vol. 23, Nos. 5 and 6, October and December 2003 (# 2004)
Polymorphism, Heteroplasmy, Mitochondrial Fusion
and Diabetes
Aya Sato,1,2 Hitoshi Endo,2 Kazuo Umetsu,3 Hideyuki Sone,1 Yoshiko Yanagisawa,1
Azusa Saigusa,1 Sayuri Aita,1 and Yasuo Kagawa1,2,4
Received August 20, 2003
Mitochondrial DNA (mtDNA) is highly susceptible to mutations that result in
polymorphisms and diseases including diabetes. We analyzed heteroplasmy,
polymorphisms related to diabetes, and complementation by fusogenic proteins.
Cytoplast fusion and microinjection allow, defects in mutated mtDNA inside a
heteroplasmic cell to be complemented by fusing two mitochondria via human fusogenic
proteins. We characterized three hfzos as well as two OPA1s that prevent apoptosis. Two
coiled coil domains and GTPase domains in these fusogenic proteins regulate membrane
fusion. The hfzo genes were expressed mainly in the brain and in muscle that are
postmitotic, but not in the pancreas. Under the influence of polymorphisms of mtDNA and
nDNA, the vicious circle of reactive oxygen species and mutations in cell can be alleviated
by mitochondrial fusion.
KEY WORDS: Polymorphism, mtDNA, fusogenic protein, cytoplasts, cybrids, ooplasmic
transfer, diabetes.
ABBREVIATIONS: BMI, body mass index; CM, cardiomyopathy; CPEO, chronic progressive external ophthalmoplegia; mtDNA; mtDNA with deletion; Hþ ; electrochemical potential difference of a proton across the inner mitochondrial membrane; DM2, type 2
diabetes mellitus; hfzo, human fusogenic protein; KSS, Kearn–Sayre syndrome; LHON,
Leber’s hereditary optic neuropathy; MELAS, mitchondrial encephalomyopathy, lactic acidosis, and strokelike symptoms; MERRF, myoclonic epilepsy and ragged-red fiber disease; mt, mitochondria; Mgm1, mitochondria genome maintenace gene; mtDNA,
mitochondrial DNA; NARP, neurogenic muscle weakness, ataxia, and retinitis pigmentosa;
nDNA, nuclear DNA; OPA1, optic atrophy type 1; PGC, PPAR gamma coactivator; PPAR, peroxisome proliferator activated receptor; ROS, reactive oxygen species; 0 cell, cell
devoid of mtDNA; SNP(s), single nucleotide polymorphism(s); syn mutation, defect in
tRNA gene of mtDNA resulting in decreased protein synthesis; UCP, uncoupling protein;
YBP, years before the present.
1
High Technology Research Center, Kagawa Nutrition University, Chiyoda Sakado, Saitama 350–0288,
Japan.
2
Department of Biochemistry, Jichi Medical School, Minamikawachi, Tochigi, 329–0498, Japan.
3
Department of Forensic Medicine, Yamagata University, Yamagata 990–2331, Japan.
4
To whom correspondence should be addressed. Department of Medical Chemistry, Kagawa Nutrition
University, 3–9–21 Chiyoda, Sakado, Saitama, 350–0288, Japan. Fax: +81-492-82-3618; Tel: +81-49282-3618; E-mail: [email protected]
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0144-8463/03/1000–0313/0 # 2004 Plenum Publishing Corporation
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INTRODUCTION
Mitochondria are endosymbiotic organelles that produce energy in eukaryotic cells.
Several thousand copies of self-proliferating circular mitochondrial DNA (mtDNA)
are found in a cell [1–3] (Fig. 1). The mitochondrial membrane generates Hþ
(electrochemical potential difference of a proton across the inner mitochondrial
membrane) by complexes I, III and IV of the electron transport machinery. This
difference aross the membrane drives protons through a complex called ATP
synthase [3], and ATP synthesis by this process is called oxidative phosphorylation.
Among the mammalian enzymes, only the four enzyme complexes described above
are encoded by both mtDNA and nuclear DNA (nDNA). Since nDNA and mtDNA
are separated by the mitochondrial membranes, mitochondria have an independent
translation system composed of mitochondrion-specific ribosomal RNAs (rRNAs),
transfer RNAs (tRNA) and messenger RNAs (mRNAs).
Human mtDNA is a 16,569 nucleotide pair (np) molecule that encodes genes for
13 mRNAs involved in oxidative phosphorylation plus the two rRNAs and 22
Fig. 1. The human mtDNA genome. Mitochondrial DNA (mtDNA) consists of
16,569 bps numbered according to Andrews et al. (2). Abbreviations: rRNA (yellow),
Ribosomal RNA; ND1 to ND6 and ND4L (light blue), subunits of NADH
dehydrogenase complex (Complex I); COI to COIII (violet), subunits of cytochrome c
oxidase (Complex IV); ATP6 and ATP8 (orange), subunits of ATP synthase; Cyt b
(orange), cytochrome b of CoQ-cytochrome c reductase (Complex III), and Ile, Met
etc. (red), tRNA genes are indicated by three letters of their cognate amino acids
(CUN, AGY and UUR are codons). Point mutations corresponding to mitochondrial
diseases, such as MELAS (A3243G) and MERRF (A8344G) are also indicated (see
footnotes for abbreviations). Red letters, novel mutations [22]; green letters, 6
polymorphisms determined in this study; blue letters, mutations reported in relation
to DM2.
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tRNAs necessary for the expression of these genes (Fig. 1) [2]. The C-rich light (L)
chain is a sense chain encoding small and large rRNAs, 12 of the peptides, and 14 of
the tRNAs. The G-rich heavy (H) chain is the other sense chain encoding one
polypeptide (ND6, NADH dehydrogenase subunit 6) and 8 tRNAs (Fig. 1).
MtDNA replication uses one origin for each chain. The H-chain origin ðOH Þ is
located in the major control region called the D-loop and the complementary Hchain is replicated from the other origin ðOL Þ: The D-loop contains the two mtDNA
promoters, one for the H-chain and the other for the L-chain. Both the replication
and transcription of mtDNA are controlled by a transcription factor called mtTFA
[3].
MtDNA is highly sensitive to mutation because of an inaccurate DNA repair
system, damage by reactive oxygen species (ROS) in mitochondria, and the absence
of protective histone [1, 3]. Mutations in mtDNA cause the decline of energy
metabolism in many diseases and during the aging process [1]. Many tRNA
mutations or deletions in mtDNA inactivate one or more tRNA gene essential for
protein synthesis (syn mutation, shown as x in Fig. 2) [3]. The major concept of this
article especially regarding the restoration of mitochondrial functions in hybrid
mitochondria is summarized in Fig. 2. Mitochondrial Diseases A and B including
diabetes (DM) corresponds to syn mutations A and B, respectively. If Cell A with
syn mutation A and Cell B with syn mutation B are fused, the resulting Cell A–B
Fig. 2. Mutations, heteroplasmy, mitochondrial fusion and complementation. Left-upper:
destruction of tRNA genes A and B (marked x) results in syn mutations A and B, respectively.
Left-bottom: fusion of Cell A with syn mutation A and Cell B with syn mutation B. Resulting
Cell A-B contains different mtDNAs (heteroplasmy) and nDNAs (heterokaryon). Right bottom:
fusogenic protein fuses mitochondria A and B and form mitochondria A-B. Right upper:
complementation of two different syn mutations by exchange of remaining wild type tRNAs A and
B. Cell functions are restored. For more specific example of diseases, see Ref. 4.
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(Fig. 2) contains different mtDNAs (heteroplasmy) and nDNAs (heterokaryon).
Owing to compartmentalization by the mitochondrial membrane, tRNAs are
confined within mitochondria. When cell A–B contains a fusogenic protein,
mitochondria A and B are fused to form mitochondria A–B. In the resulting
mitochondria A-B, two different syn mutations can be complemented by an
exchange of the remaining wild type tRNAs A and B, which restores both
translation and cellular functions (Fig. 2 right upper) [4]. Such inter-mitochondrial
complementation (trans- complementation) in cell hybrids between two types of
recessive mutation remains controversial [4, 5] as will be discussed under
‘‘transcomplementation of defective mtDNA’’. The most crucial evidence for the
complementation of deleted mtDNA ðmtDNAÞ in which some tRNAs are lost, is
the formation of a fusion protein (not the fusogenic proteins described later) that
tandemly connects two reading frames on both sides of the deletion gap [6]. When
the eliminated tRNAs of mtDNA are supplied from the coexisting wild type
mtDNA encoding the missing tRNAs in heteroplasmic cells, fusion protein is
translated from the fusion mRNA transcribed from mtDNA [6].
Enucleated cells that contain mitochondria are called cytoplasts (Fig. 3).
Cytoplast fusion [7] and mitochondrial microinjection [8] are essential to transfer the
complementation of defective mtDNA by forming heteroplasmic cells. Cells fused
Fig. 3. Mitochondrial transfer by cytoplast fusion into a cell to form
a cybrid. Red and green, young and aged mitochondria, respectively.
Cybrid of a 0 cell is homoplasmic (bottom), while that of a cell with
different mtDNA is heteroplasmic (upper). Heteroplasmic oocyte is
also formed by mitochondrial transfer (right).
Mitochondrial Genetics, Fusion and Diabetes
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with cytoplasts are called cybrids (Fig. 3). The electrofusion of cytoplasts containing
mtDNA with a zygote results in transmitochondrial mice composed of
heteroplasmic cells [9–11]. On the other hand, microinjection of a somatic cell
nucleus into an enucleated oocyte results in a cloned animal that is composed of
nuclear DNA (nDNA) of the somatic cell and mtDNA of an oocyte [12] (Fig. 4).
Microinjecting ooplasma containing mitochondria into another oocyte is called
ooplasmic transfer [13] (Fig. 4, center). Although transgeneration gene therapy is
prohibited in humans, ooplasmic transfer has resulted in many healthy transmitochondrial offsprings carrying heteroplamic mtDNA [13] (Fig. 4, right bottom).
The present review aims to clarify the relationships between mtDNA mutations,
heteroplasmy, polymorphisms, fusogenic proteins and DM2.
MUTATION OF MITOCHONDRIAL DNA
MtDNA is 20-fold more susceptible to mutation than nDNA [1, 14, 15]. The
higher levels of oxidative damage and mutation detected in mtDNA have been
ascribed to (a) the compartmentalization of mtDNA within the inner mitochondrial
membrane where reactive oxygen species (ROS) are formed, (b) the lack of
protective histones in mtDNA, (c) the limited repertoire of DNA repair mechanisms
in mitochondria, and (d) high information density because of the lack of exons in
mtDNA. Mitochondria only express uracil glycosylase and can therefore only
correct cytosine deamination. Mitochondria lack the repair system for pyrimidine
dimers and the mitochondrial DNA polymerase has no proofreading ability. In
addition to being the principal source of ATP, mitochondrial metabolism is also a
source of the ROS that destroy mtDNA [1, 14, 15]. These ROS include superoxide,
hydrogen peroxide, hydroxy radicals and singlet oxygen atoms that are produced
continuously at a high rate as by-products of electron transport. Moreover, defective
mitochondria play an important role in apoptosis [1].
Oxidative lesions in mtDNA accumulate as a function of age in tissues, and
hence an aging theory of mtDNA has been proposed [16]. The amount of 8hydroxydeoxyguanosine, a biomarker of oxidative DNA damage, increases during
aging and causes somatic mutations in mtDNA. ROS have been implicated in cell
dysfunction and apoptosis associated with type 1 and 2 diabetes mellitus [14–15, 17].
The superoxide content of isolated pancreatic islet increased in response to glucose
stimulation [17].
Figure 1 shows some of the loci of the mutations in mtDNA that cause disease.
The mutation points in mtDNA of mitchondrial encephalomyopathy, lactic acidosis,
and strokelike symptoms (MELAS) [18, 19] and myoclonic epilepsy and ragged-red
fiber disease (MERRF) [20] have already been identified [21]. Mutations causing
mtDNA disease fall into four groups that are briefly described below. These are: (a)
missense mutations in a reading frame, (b) protein synthesis mutations (syn
mutations) that is, base substitution or modification defects of a tRNA gene, (c)
insertion–deletion mutations and (d) copy number mutations [3, 21].
(a) Examples of missence mutation are those in Leber’s hereditary optic
neuropathy (LHON) [22, 23] and neurogenic muscle weakness, ataxia, and retinitis
pigmentosa (NARP) [24]. Over 95% of LHON cases are primarily the result of one
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of three mtDNA point mutations, G3460A, G11778A, and T14484C, which all
involve genes for complex I subunits [23]. An intriguing feature of LHON is that
only approximately 50% of males and approximately 10% of females who harbour a
pathogenic mtDNA mutation actually develop the optic neuropathy [23]. A marked
incomplete penetrance and gender bias imply that additional mutations of mtDNA
and/or nDNA must be modulating the phenotypic expression of LHON. In this
connection, the first author and Goto et al. recently described a patient with LHONCM who carried double mutation (G11778A in ND4) and (G12192A in tRNAHis )
(Fig. 1, red letters) [22]. NARP is the result of a T-to-G transversion at np 8993 (T to
G) that changes amino acid 156 of subunit 6 (Fo portion) of ATP synthase (ATP6)
[24]. Because of the decreased respiration associated with NARP, large amounts of
ROS are released from the NARP fibroblasts that cause apoptosis [25]. Mutations
and polymorphisms of mtDNA mutations that cause DM2 have been found at genes
for ATP synthase (ATP6 T8993G/C), complex I (ND1, G3316A; ND2, A4917G;
ND3, C10398T and ND4, T11778C), complex IV (COXII, A8245G, etc.), tRNAs
(C15904T, etc.) and D-loop (T16189C) [data on the internet address described in the
reference section] (Fig. 1). The distinction between SNPs (prevalence > 1%; by
definition) and genetic disease mutation is not clear, because the sample was small
ðn < 100Þ: To identify other SNPs responsible for DM2, complete sequencing of 5
mtDNAs from DM2 patients and 6 mtDNAs from controls at our Nutrition Clinic
revealed 58 novel SNPs including those in the significant coding region, 9 novel
SNPs in the diabetics, and a different set of 9 novel SNPs in the controls (Sato, A. et
al. Japanese Biochemical Society Meeting Abstract 75(8) 1024 (2003)). However,
according to our meta-analysis and a wide survey in Asian populations, the
reproducibility of DM2-related SNPs in other ethnic groups is poor, if we exclude
the effects of nDNA SNPs for UCP etc. Not all point mutations are neutral or
pathological. For example, the C150T SNP of mtDNA confers a survival advantage
upon centenarians and twins, perhaps because of de novo replication origin [1].
(b) The known synthetic mutations ðsyn Þ are of tRNA and are associated with
mitochondrial myopathy and other systemic phenotypes. They consist of MERRF
[20], MELAS [18, 19], and maternally inherited cardiomyopathy (CM) [26]. The
major mutations found in MERRF, MELAS and CM are localized at np 8344(A to
G; tRNALys ) [20], np 3243 (A to G; tRNALeuðUURÞ ) [18, 19] and np 4269 (A to G;
tRNAIle ) [26], respectively (Fig. 1, black letters). In addition to the most frequent
SNP (A3243G of tRNA-Leu-UUR), many other SNPs are diabetogenic [3, 14, 15,
17]. We identified novel pathogenic mutations of tRNAs (G5773A, G5827A,
A14692G) by sequencing mtDNA (Fig. 1, red letters) [22]. A new type of syn
mutant, modification defect at an anticodon wobble nucleotide of tRNA, was
reported [27]. Modified uridines that contain taurine are lacking in mutant
mitochondrial tRNAs for Leu (UUR) and Lys from pathogenic cells of the
MELAS and MERRF, respectively [28].
(c) Deletions in mtDNA cause much of the chronic progressive external
ophthalmoplegia (CPEO) and Kearn–Sayre syndrome (KSS) [29]. Ocular myopathy
is sometimes associated with autosomal dominant mutations [30]. Affected
individuals with these pedigrees harbor multiple mtDNA deletions, all of which
are flanked by direct repeats, suggesting an increased frequency of slip-replication
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[30]. In fact, all 12 deletions are flanked by short direct repeats (4–12 bp) [30]. Some
deletions such as the polymorphic ‘‘9 bp deletion’’ (Fig. 1, green letter, bottom) are
not pathogenic, and are used as markers of human evolution.
Many other mutations are included in this category, including those that cause
diabetes mellitus [3, 15]. The incidence of the so called ‘‘common deletion’’ of
4977 bp from mtDNA increases during aging in human skin fibroblasts from fetal,
young and old donors (the highest level is 0.3%) [31]. Common deletions of
heterogenous mtDNA between 8468 and 13446 bp have also been found in the
striated muscle of old diabetic patients because of hyperglycemia and ROS [32]. A
deletion is generally much easier to detect than a mutation, so many point mutations
caused by aging or ROS damage could remain unreported [1, 14, 16].
(d) A significant decrease in the number of mtDNA molecules is associated with
familial mitochondrial myopathy [3]. In addition, one tumor called oncocytoma
over-produces mitochondria, resulting in both the copy number of mtDNA and
resultant transcripts [3]. A mechanism of significantly increasing the amount of
mtDNA in oncocytoma may prove useful for future mitochondrial gene therapy [7].
The copy number of mtDNA is regulated by by a factor called PGC together with
nDNA for energy metabolism including UCP genes [14].
The pathological mechanism by which these diseases with mtDNA mutation
arise remains unknown, but apoptosis might play an important role [1, 25, 33]. In
fact, caspase-3 is activated in the ragged red fibers of MELAS, MERRF and CPEO,
together with cytochrome c release from mitochondria [34].
Many missense, syn mutations and deletions in mtDNA cause diabetes
mellitus [14, 35–37]. About 2% of diabetics harbor the MELAS (A3243G) mutation,
for example, that is accompanied by maternal inheritance (100%), hearing loss
(100%), myopathy (43%) and neuropsychiatric symptoms. This type is referred to as
mitochondrial diabetes [35–37] and mutated mtDNA is shown as an example
(T14577C, Fig. 1, blue letter) [36].
HETEROPLASMY AND HOMOPLASMY
Heteroplasmy means that cells contain a mixture of mtDNAs with different
sequences (Fig. 2, left bottom and Fig 3, left middle), whereas homoplasmy means
that they contain 100% of the mtDNAs have an identical sequence (Fig. 2, left upper
and Fig 3, left upper) [3, 7, 14]. Cytoplasts are enucleated cells that contain
mitochondria (Fig. 3, upper). The mitochondria of other cytoplasts such as
enucleated oocytes, synaptosomes and platelets (Fig. 3, right upper) can also be
transferred. Heteroplasmy can be induced by cytoplast fusion (Fig. 3, upper middle)
to Cell A to form cybrids (Fig. 3, left middle). If Cell A is homoplasmic, Cytoplast A
fused with Cell B which is 0 cell devoid of mitochondrial DNA, the resulting cybrid
is homoplasmic but the nucleus is delived from Cell B. If aged mtDNAs are mutated,
mitochondrial tranfer from a young homoplasmic cytoplast will rejuvenate the aged
cell (Fig. 3). In a mutation of nDNA two copies of the allele thus allow only three
combinations: two homozygotes and one heterozygote. However, individual human
cells have hundreds of mitochondria and thousands of mtDNA molecules, so the
potential combinations of two different mtDNAs in a heteroplasmic cell are
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numerous. The majority of pathogenic mutations are heteroplasmic, with mutated
and wild-type mtDNA coexisting with the same cell. For example, cells with the
MELAS mutation are heteroplasmic [18], because they needs wild-type mtDNA to
support the oxidative phospholyration that is completely lost in MELAS
mitochondria. However, the cells with weakly pathogenic mutations of tRNA are
homoplasmic [22]. The ratios of mtDNAs differ and consequently, so do the energy
states of each cell [3, 6, 18, 38, 39]. Exceeding a specific threshold ratio (%) of mutant
mitochondria impairs cellular functions (threshold expression) [38]. The maximal
oxygen uptake in patients with mitochondrial myopathy correlated with the ratio
(%) of heteroplasmy ðr > 0:82 : p < 0:005Þ [38]. The ratio affects electron transport
dependent proton electrochemical potential gradient ðHþ Þ [3] that drives ATP
synthase [3]. The formation of Hþ is dependent on mitochondrial oxidative
phosphorylation, hence there is the association between the level of MELAS
heteroplasmy and the time of DM2 onset (r ¼ 0:67; p < 0:01) [39].
During the mitotic or meiotic cytokinesis of heteroplasmic cell, both mutated
and normal mtDNA unevenly divide into daughter cells. Thus, cells will eventually
segregate into two different homoplasmic lineages. This phenomenon of replicative
segregation is called stochastic segregation [7, 14]. The accumulation of mutated
mtDNA, especially in postmitotic cells during differentiation and aging, is clonal
[40]. Even cells or individuals with identical nuclear genotypes can have different
phenotypes owing to heteroplasmy and stochastic segregation. In a MELAS patient,
for example, the ratios of mutant mtDNA differ among organs [37], and if the
patient’s hypophysis contains the highest ratio of mutant mtDNA, symptoms of
endocrine diseases will become manifest [37]. The same MELAS mutation in
mtDNA may cause encephalomyopathy [18, 19], diabetes mellitus [35], myopathy
[38] or even hypopituitarism [37]. This is attributed to the stochastic segregation of
the mutant mtDNA that is unevenly distributed among the various tissues of the
patient [3, 7, 37].
Heteroplasmic cells can be formed artificially either by cell fusion or by
mitochondrial microinjection [7, 11, 13], but somatically aquired mutations during
aging also generate heteroplasmic cells that are normally present in healthy
individuals [31, 40, 41]. The frequency of age-dependent heteroplasmy at
hypervariable region II was found in 11.6% of individuals, but its distribution
differed across tissue types, being higher in muscle [41].
The age-dependent accumulation of somatic mutation of mtDNA is rarely
inherited by progeny, because of the bottleneck effect [1, 42], that is, of the
thousands of mtDNA, very few mitochondria located near the nucleus of oocyte can
proliferate. By this mechanism oocytes with normal mtDNA are selected. Aged
oocytes rich in mutated mtDNA (4977 bp deletion between ATPase 8 and ND5) [43]
may be removed because they are dysfunctional. When the defective mtDNA is
concentrated in an embryo by the bottleneck effect, the embryo is removed by
abortion (Fig. 4, bottom). Owing to selection by the bottleneck effect, a
heteroplasmic individual will becomes homoplasmic of wild type mtDNA after
three generations. Heteroplasmy, threshold expression and stochastic segregation are
characteristic of maternal, non-Mendelian genetics of mtDNA.
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Fig. 4. Ooplasmic transfer: mitochondrial renewal by microinjection of young mitochondria into an oocyte. Red and green, young
and aged mitochondria, respectively. ES cells (embryonic stem
cells) with renewed mitochondria can be differentiated into an
organ suitable for transplantation.
Since several mtDNAs exist within a mitochondrion, heteroplasmy should be
divided into intermitochondrial and intramitochondrial groups [5, 11]. Intramitochondrial heteroplasmy is stable, whereas the mutant genome is amplified in
intermitochondrial heteroplasmy, perhaps by the more rapid replication of short
deleted mtDNA or by stimulation of replication of mutant mtDNA due to
inadequate function [3].
The risk of disease developing in an individual is dependent not only on the dose
of mutated mtDNA in the zygote but also on subsequent changes in distribution,
which might be at random or influenced by various selective factors throughout life
[37, 41]. Defects in cellular oxidative phosphorylation are roughly proportional to
the ratios of mutant mitochondria [18, 38], and thus compensatory dependence on
glycolysis (or lactic acidosis in an individual) is characteristic of cells with mutant
mtDNA [18].
The developmental competence of early embryos appears to be directly related
to the metabolic capacity of a finite complement of maternally inherited
mitochondria that begin to replicate after implantation. Mitochondiral dysfunctions
resulting from a variety of influences, such as genetic abnormalities and oxidative
stress, can influence ATP level in embryos, which in turn may result in
developmental arrest and abortion (Fig. 4) [43, 44]. Deletions and mutations in
the mtDNA of fertilized heteroplasmic oocyte might be concentrated in some cell
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lineages or organs such as muscle [40, 41] by stochastic segregation during embryonic
development and may result in infertility. Ooplasmic transfer of active young
mitochondria (mitochondrial donation, and perhaps with other factors) by
microinjection [7, 8] is a practical means of renewing aged mitochondria [13, 44]
(Fig. 4, left upper). This procedure will create heteroplasmic embryos and in fact,
many healthy offspring with transferred young mtDNA have already been delivered
(Fig. 4, right bottom) [13, 44]. A cloned animal contains oocyte mtDNA [12]. The
rejuvenation seen in cloned sheep is partly caused by refreshing mutated mtDNA
with the wild-type mtDNA in the oocyte [12] (Fig. 4). If ooplasmic transfer is applied
to cloned human oocyte and embryonic stem cells (ES cells in Fig. 4) isolated from
the blastula, transplantation organs might be improved.
However, nDNA-mtDNA interactions constrain function of heteroplasmic cells
[45]. Owing to the co-evolution of nDNA and mtDNA, many specific interactions
that are crucial for optimized ATP synthesis with four complexes are encoded by
both nDNA and mtDNA [1, 45]. These interactions have been tested by introducing
cytoplast containing rat mtDNA into mouse 0 cells [45]. The xenomitochondrial
cybrid thus formed respired approximately 50% less than the parental mouse cells.
The assembly of Complexes I and IV in the mitochondrial membrane was defective
[45]. However, 10% mouse mtDNA co-existing with the rat mtDNA (mouse-rat
mtDNA heteroplasmy) was sufficient to restore respiration to normal levels [45].
This nDNA-mtDNA interaction was also found in the pathogenesis of LHON [23].
Thus, heteroplasmy might play important role both in the mammalian evolution and
prevention of mitochondrial diseases caused by rapid mtDNA mutation.
POLYMORPHISMS AMONG ETHNIC GROUPS
To differentiate general genetic variations from pathogenic (MELAS etc.) or
disease-sensitive mutations (C5178A etc.), analyzes of the polymorphic background
in mtDNA of various ethnic groups are needed. By culturing heteroplasmic cells of
patients with MELAS, we could separate homoplasmic cell lineages with MELAS
from those with polymorphic wild-type by stochastic segregation, and identify
pathogenic mutation [18]. Homoplasmic mutations present a more difficult challenge
in terms of diagnosis and assigning pathogenicity. The simplest type of polymorphism is a single nucleotide polymorphism (SNP) that results from a single base
mutation substituting one nucleotide for another [46, 47]. Some SNPs are
advantageous for survival [1, 46] and others are harmful like the sensitivity genes
for the diabetes [46, 48]. However, most sequence variations, such as synonymous
codons, do not directly impact phenotypic variation and thus are not directly subject
to the force of selection. In the polymorphism of mtDNA, the frequency of mutation
is high in both D-loop region [41, 47] and at the third letter of the codons
(synonymous codons), which are ‘‘silent’’ for the phenotype. According to the
neutral theory of molecular evolution, most mutations with rare allele advantage
might be balanced by random genetic drift. By definition the rarer mutant must arise
at a frequency of over 1% in the healthy population. On the other hand, a mutation
at tRNA (syn mutation) such as MELAS [18] is rare (less than 1% of the total
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population) because of natural selection of fatal diabetes or encephalomyopathy,
and it is not referred to as a polymorphism.
The polymorphism of mtDNA reflects the course of human evolution [47, 49–
51]. Because of its high mutation rate and maternal inheritance, mtDNA diversity
has proven useful in studies of human evolution and of the genetic affinities of
human groups from different geographic regions [47–51]. Haplotypic and
phylogenetic analyzes of neutral SNPs in mtDNA have clarified much of the
controversy concerning the time and pattern of colonization in various geographic
regions [47, 49]. The most recent common mtDNA ancestor of modern Homo
sapiens is estimated to have occurred at 143 18 kYBP; and the divergence of
Caucasoid and Mongoloid at 70 13 kYBP [46, 47, 49]. Nucleotide sequences of the
D-loop region of human mtDNA from the East Asian population have revealed the
paleomongoloid (the anthropological prototype of Mongoloid) origin of the Jomon
and Ainu peoples of Japan (10 kYBP) and showed that approximately 65% of the
gene pool in mainland Japanese was derived from continental gene flow after the
Yayoi Age (2 kYBP) [50, 51]. Paleomongoloid peoples might have arrived in the
Americas and in the Pacific islands around 15 kYBP [49]. The conclusion that
Mongoloids and Caucasoids diverged early in human history has important
implications for the use of mtDNA in more detailed phylogenetic studies [49]. The
origin of the Japanese people and their relationship with the peoples in Southeast
Asia has been analyzed using mtDNA [50–52], and HLA (human leukocyte antigen)
and other genetic markers. Genetic variation of mtDNAs from Southeast Asian
population with a 9bp deletion is wide spread, and some aboriginal populations
exhibited several unique mtDNA clusters that have not been identified in other
populations [53].
We have been studying polymorphism in the Asia/Pacific regions [50, 52]. The
simultaneous determination of six SNPs (nt3010, nt4386, nt5178, nt8272–8289,
nt8794, nt10398) is referred to as‘‘multiplex amplified product-length polymorphism
analysis (APLP)’’ [54] (Fig. 1, green letters and Table 1). Two of these haplotypes, B1
(estimated ancestral haplotype) and C1, were distributed among all populations
tested. However, the haplotypes A1 (D5), A2 (D4), B2 (M7a), B3 (B4) and C2 (A)
were mostly restricted to Mongoloid populations (names in parenthesis are used in
other groups). The map in Fig. 5 shows that haplogroup A2 is dominant in Japanese,
whereas haplogroup B2 is specific among Japanese, and B2 is a marker of the Jomon
people (former inhabitants of Japan found in Okinawa and in Northern Japan).
More detailed studies by Umetsu et al. have revealed a genetic gradient from Japan
to Korea (Seoul), Taiwan and many areas of China (Shenyang etc.). We found some
mtDNAs characteristic of Caucasoid among Mongols.
For example, the longevity-associated SNP of C5178A of mtDNA [48, 55]
forms haplogroups A2 and A3 among Nothern Mongoloids (G3010A þ A10398G)
which is rare in Southeast Asia and absent among Caucasoids (Fig. 5). The
longevity-associated SNP of the C150T of mtDNA was found among Caucasoid (52
centenarians, p ¼ 0:0035) [1]. However, the complete sequencing of Japanese
mtDNA did not reveal any difference in the prevalence of C150T between
centenarians (n ¼ 96; 19.79%) and control groups (n ¼ 576; 19.44%) (Tanaka,
M., data can be found on http://www.giib.or.jp/mtsnp/index_e. html).
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Table 1. Haplotype-specific Polymorphic Sites in Human mtDNAs
Polymorphic site
Haplogroup and haplotype
A:
B:
C:
A1
A2
A3
B1
B2
B3
B4
B5
B6
B7
B8
C1
C2
C3
C4
C5
C6
C7
3010
4386
5178
8794
9bp
10398
G
A
A
G
G
G
A
A
A
G
G
G
G
G
G
A
G
G
T
T
T
T
C
T
C
T
T
T
T
T
T
T
C
T
T
T
A
A
A
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
T
C
C
C
T
C
2
2
1
2
2
1
2
2
3
2a
2b
2
2
1
2
2
3
3
G
G
G
G
G
G
G
G
G
G
G
A
A
A
A
A
A
A
Designations in this column indicate the following: 1, deletion of the 9-bp repeat; 2, 2-copies of the 9-bp
repeat; 3, 3 copies of 9-bp repeat; 2a, 4-bp insertion (heteroplasmic); 2b, 3-bp deletion (heteroplasmic).
Owing to the modernization accompanied by imigration in Asia, there was sex
bias in frequencies of polymorphism (examples: male vs. female): in Mongol
(p < 0:001), B1 (27.8% vs. 40.2%) and in Palau (p < 0:001), C1 (59.3% vs. 35.4%).
The sex bias in mtDNA polymorphism is expected in Spanish conquest of Amerind
in 16th> centry: for example, in Metizo family (Indio vs. Conquistador) [56]. The
analysis of the gene pool of Antioquia revealed that approximately 90% of the
mtDNAs are native Amerind, while approximately 94% of Y chromosomes
(paternal inheritance) are European [56]. According to surveys of DM2, the
frequency of maternal inheritance is significantly higher than that of paternal
inheritance (maternal vs. paternal percentage: Europeans: 21.7% vs. 9.9%, Pacific
islanders: 15.7% vs. 5.3%; p < 0:001) [57]. An example of the sex bias is
mitochondrial diabetes accompanied by MELAS and other mitochondrial encephalomyopathies with mtDNA mutations [35–37]. However, the frequency of MELAS
among diabetics is only 2%, which cannot explain the large sex bias. Some
polymorphisms of mtDNA may contribute to the sex bias of DM2 frequency [48].
Some SNPs were associated with DM2, because any dysfunction of mtDNA will
retard insulin secretion [14, 35]. Mitochondrial diabetes is a consequence of pancreatic
-cell dysfunction caused by mutations in mtDNA [14]. The -cell mitochondria serve
as glucose sensors, generating ATP that couples glucose concentration to the
exocytosis of vesicles containing insulin [14]. The longevity-associated A-type SNP of
C5178A of mtDNA [55] prevents DM2 [48], but our survey [46] showed that, its
frequency is very different among human populations: 40, 33, 30, 4 and 0% in
Mitochondrial Genetics, Fusion and Diabetes
Fig. 5. Polymorphism of mitochondrial DNA in Asia/Pacific region. A1-C4: haplogroups determined by restriction fragments of
mitochondrial DNA (see Table 1). Frequencies of haplogroups are summarized as circular graphs.
325
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Aya Sato et al.
mainland Japan, Okinawa, China, Thailand and Caucasoids, respectively (Fig. 5).
Thus, this polymorphism is related to DM2, but is not essential to prevent it. We
sequenced totally the mtDNAs of 6 diabetic patients and 6 control groups from our
clinic, and discovered 58 novel SNPs. Detailed case-control analyzes of those SNPs
revealed that most do not directly affect the pathogenesis of DM2 (Sato, A. et al., The
abstract of Japanese Biochemical Society 75(8), 1024 (2003)).
The clients of our clinic who were not treated with drugs were classified
according to mtDNA C5178A SNPs. The fasting blood glucose level of the clients
with the A type (n ¼ 39; 86:9 9:7 mg=dl) was significantly lower than that of those
with the C type (n ¼ 65; 95:1 10:0 mg=dl) (p < 0:0001) before applying our regime.
After receiving dietary and exercise advice, clients with the C type
(90:4 10:8 mg=dl) responded better than those with the A type
(84:4 12:6 mg=dl) (p < 0:012). The tendency in BMI and its response between
clients with A and C types was similar. Thus, diet and exercise might prevent
diabetes even among those with C type SNPs.
Since the interactions between mtDNA and nDNA in mitochondrial
metabolism are close [23, 45, 58], polymorphisms caused by mutation, might be
adjusted by selection during evolution. The selection occurs not only in the struggle
for survival, but also in the abortion of misfit embryo during the gestation (Fig. 4).
In fact, there is a concentration of remodeled replication origin in mtDNA in twins
[1]. The incidence of C150T is much higher in twins (monozygotic 30% and dizygotic
22%) than that in control Italians (3.4%). The formation of a new replication origin
at position 149, substituting for that at 151, may be advantageous for twin survival
[1]. The nDNA-mtDNA interaction during the pathogenic expression of mtDNA
mutation is not only limited to heteroplasmic cells (ragged red fiber formation in
MELAS etc.) but also found in homoplasmic cells (tissue specific expression in
LHON etc.) [58]. The nuclear modifier does not induce any pathology per se in
LHON, but it contributes to the pathogenic effect of mtDNA mutation [58]. The
adverse effects of heteroplasmy caused by aging process in some postmitotic cells [41]
can be corrected by complementation.
MITOCHONDRIAL TRANSFER AND COMPLEMENTATION
Recent developments in human genomics and physics have enabled mtDNA
transfer, of which cytoplast fusion (Fig. 3) and microinjection (Fig. 4) are the key
methods. In transgenic mouse, DNA for green fluorescent protein and subunits of
ATP synthase of mitochondria were useful in the future developments [59].
Microinjection of mitochondria into oocyte [7, 8] is applied to in vitro human
embryonic transplantation (Fig. 4) [13].
Microinjection: A gene for green fluorescence protein or mitochondrial ATP
synthase [60] microinjected into an oocyte was expressed in vivo in all organs of
tested transgenic mice (Fig. 4) [59]. We thus microinjected mouse liver mitochondria
labeled with a fluorescent marker (PKH26) into a fertilized oocyte [8]. C3H/HeJ
female mice were treated with Serotropin and hCG at 5–6 weeks of age, and fertilized
[8]. Mitochondria were introduced through borosilicate glass capillary micropippettes (diameter about 7 m) attached to a micromanipulator (Leitz) equipped with a
Mitochondrial Genetics, Fusion and Diabetes
327
Piezomicropipette driving unit (PMM-01). Fluorescence was observed by confocal
microscopy. In contrast to the loss of sperm mitochondria after fertilization, the
exogenous mitochondria were retained near nuclei at 1-cell stage and then
distributed throughout the cytoplasm at 2-cell stage. When all mitochondria were
stained with Rhodamine 123, both exogenous and endogenous mitochondria were
co-localized at 4- and 8-cell stages. The microinjected control liposomes were
dispersed rapidly at 1-cell stage and then appeared as a speckled pattern [8]. We then
microinjected submitochondrial particles and liposomes containing submitochondria
particles [8]. All particles were distributed in cytoplasm at the 2-cell stage, and
colocalized with endogenous mitochondria at the 4-cell stage. These results suggested
that a factor fuses submitochondrial fraction or liposome to mitochondria, which is
described under ‘‘Fusogenic proteins’’.
Cytoplast fusion: Cytoplast fusion is another useful method of transferring
mitochondria into cells (Fig 3). Cytoplasts are enucleated by centrifugation (23,000g,
348C for 10 min) in the presence of cytochalasin B (10 g=ml) (Fig. 3, upper-middle
and bottom middle) [61, 62]. The mtDNA in postmortem human brain synaptosomes and in platelets were fused into a 0 cell by the cell fusion method [62].
Platelets, synaptosomes and enucleated oocytes are equivalent to cytoplasts that
have mitochondria and no nucleus (Fig. 3, right upper). Recipient of cytoplast
mtDNA in the presence of PEG (polyethylene glycol) can be 0 cells (Fig. 3, bottom
left) [7, 61, 63]. Owing to the absence of histones, mtDNA can be removed without
damaging nDNA by adding ethidium bromide (or ditercalinium for mouse mtDNA)
to the culture medium [61, 63]. Theoretically, 0 cells lack oxidative phosphorylation
and can proliferate in glucose medium for glycolysis. However, in contrast to yeast
cells, adding pyruvate to glucose medium is essential to support human 0 cells,
because most NAD is reduced in the absence of electron transport. The oxidant
regenerate NAD from NADH is required, so we use glucose-rich media
supplemented with pyruvate (0.1 mg/ml) as the oxidant, to prevent an extreme
reductive state [7,14]. We selected cells with mutant mtDNA as well as human 0
cells from wild type cells using DM170 (galactose-pyruvate medium) and DM201
(glucose-pyruvate medium) [14]. Human 0 cells obtained from HeLa cells [7, 61] or
cells with mutant mtDNA lacking oxidative phosphorylation cannot survive in
DM170 [7], because conversion of galactose into glucose is very slow in mammalian
cells. Cells with wild type mtDNA capable of oxidative phosphorylation obtain
energy from pyruvate. Galactose in the medium is mainly needed for polysaccharide
synthesis [14].
Cytoplasts fused with nucleated cells or 0 cells transfer their mitochondria to
the other type of cells [7, 61]. The resulting hybrid cells are called cybrids (Fig. 3,
right bottom). When two cells are fused, the resulting cells are called heterokaryon
hybrids (Fig. 2, right bottom). These hybrids contain two different nuclei
(heterokaryon), and different mitochondria. Cybrids and hybrids containing wild
type mtDNA can grow on galactose medium, and are easily separated from 0 cells
or cells with defective oxidative phosphorylation [7, 61, 63].
We have restored the cytochrome oxidase activity of HeLa 0 cells by
introducing wild-type mtDNA from original HeLa cells [61] or synaptosomes [62].
Other activities dependent on mtDNA, such as NADH dehydrogenase, were also
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Aya Sato et al.
restored only when the wild-type mtDNA was introduced. In fact, the translation of
all 13 subunits of oxidative phosphorylation which is dependent on normal mtDNA,
was equally restored by introducing normal mtDNA into the 0 HeLa cells [7, 61].
However, when mutant mtDNAs from the patients were introduced into the HeLa
0 cells, cytochrome oxidase activity was not restored. Most mtDNA in cells from
aged individuals was wild type, and the activity of cybrids was almost normal [61],
despite some accumulation of mutated mtDNA [15, 16]. During the aging process,
the major involvement of nDNA was established by the cybrid method [61, 62],
perhaps caused by telomere shortening [14]. Thus, replacement of defective mtDNA
with wild-type mtDNA by cytoplast (or platelet etc.) fusion is theoretically possible,
especially if there are mitochondrial fusogenic factors in the cell to transcomplement
defects in the aged mtDNA (Fig. 3).
In fact transmitochondrial mice (mito-mice) can be produced by electrofusing
cytoplasts containing mtDNA4696 (4696 bp deletion from tRNA(Lys) to ND5)
with zygotes [9, 10]. Embryo development does not screen out the defective mtDNA
after this procedure [9], and exogenous mtDNA is not removed after oocyte
microinjection [8]. These findings have positively impacted gene therapy for
mitochondrial diseases [7].
The cybrid method will be applied to true in vivo human gene therapy. For
example, liver-directed gene therapy can treat homozygous familial hypercholesterolemia by cell replacement [64], and diabetes mellitus by pancreatic islet
transplantaion [65]. Such patients underwent cell transfusion through a portal
venous catheter [64, 65]. When mtDNA is mutated, cells removed from patients can
proliferate in the glucose medium, and those cells rich in wild-type mtDNA can then
be selected in the galactose-pyruvate medium by stochastic segregation. Similar to
the growth factors used in transduced cell proliferation in human gene therapy [7],
wild-type mtDNA proliferates when a specific mitochondrial translation factor [3] is
induced by PCG [14]. Cells thus enriched in wild-type mtDNA can be converted into
cytoplasts and infused. Proteins that assist cell-fusion (Sendai virus protein) or
polyethylene glycol can be used during cytoplast-mutant cell fusion.
Transcomplementation of defective mtDNA: Most point mutations (MELAS
etc.) in mtDNA occur in tRNA, and virtually any deletion of at least 1–2 kb (CPEO
etc.) eliminates a tRNA gene essential for translation (Fig. 1). Mitochondrial fusion
complements the defective mtDNA (Fig. 2, mitochondria A–B) [7, 11]. If wild-type
mtDNA molecules introduced into a cell were in fact separated by mitochondrial
membrane, complementation of the defective mtDNA in other mitochondria would
be impossible. However, the formation of fusion proteins of deleted mtRNA
transcribed from deleted mtDNA in heteroplasmic cells indicates intermitochondrial
communication, because such deletions result in the absence of several tRNAs [6].
These fusion mtRNAs are translated into fusion proteins using tRNAs transcribed
from normal mtDNA coexisting in the cells [6] (Fig. 2). Thus, the amount of
introduced normal mtDNA needed to complement the defective mtDNA is only
about 20–60% of the total mtDNA in a cell [6, 11].
The controversy over the intermitochondrial mtDNA complementation
(transcomplementation) [4, 5, 11] is based on an argument on mitochondrial fusion
and mixing mtDNA and/or mtDNA products in vivo. The issues are the generality
Mitochondrial Genetics, Fusion and Diabetes
329
and frequency of this phenomenon and the importance of its control by the nucleus
[5]. Mitochondria carrying either one or two recessive tRNA mutations have been
brought together in the same cell, but even 42–99 days after cell fusion under
conditions that do not select for respiration, the rate of mitochondrial protein
synthesis by the clones was marginal and respiration capacity did not recover [5, 66].
A large-scale investigation revealed that less than 1% of the 0 cells consisted of
transcomplementing clones [66], and this ratio did not recover over six days [66].
However, transmitochondrial mice carrying exogenous mtDNA4696 [9,10]
constitute the evidence of extensive in vivo transcomplementation in all tissues
tested. If transcomplementation has not occurred, then both cytochrome oxidase
positive and negative mitochondria should coexist in a cell, because each cell
contains 20–85% mtDNA [4,11]. The absence of cytochrome oxidase negative
mitochondria supports both the generality and importance of transcomplementation. Ttranscomplementation has also been established by detecting of fusion
proteins as discussed earlier [6].
FUSOGENIC AND DYNAMIN-RELATED PROTEINS
A dynamic balance between fission and fusion determines the morphology of
proliferating mitochondria. Several factors including fusogenic and dynamin-related
proteins are involved in these processes. Dynamin is a microtubule-associated GTPbinding protein that is required for endocytosis. Since mtDNA is enclosed in the
outer and inner mitochondrial membranes, fusogenic proteins are also needed to
introduce wild type mtDNA into mitochondria containing mutant mtDNA from
heteroplasmic cells (Fig. 1). The first known protein mediator of mitochondrial
fusion is fzo (fuzzy oinions), which was discovered in Drosophila melanogaster [67].
During Drosophila spermatogenesis, mitochondria of the early post meiotic
spermatide aggregate, fuse, and elongate beside the growing flagellar axoneme.
Mutant fzo males are sterile, because mitochondrial fusion is blocked. A large
predicted transmembrane GTPase that appears in spermatide mitochondria late in
meiosis II is encoded by fzo [67]. Homologs of fzo have been identified in mammals,
nematodes and yeast [67]. Detailed studies on fzo has been performed in yeast,
because genetic manipulation is easy [68]. The point mutations that alter the GTPase
domain of yeast fzo (Fzo1p) do not affect Fzo1p localization but disrupt
mitochondrial fusion. Sub-organellar fractionation has suggested that Fzo1p spans
the outer mitochondrial membrane and is tightly associated with the inner
mitochondrial membrane.
The authors cloned and characterized three human orthologs (human fzos,
hfzos), namely hfzo1(741 aa), hfzo2 (769 aa) and hfzo3 (757 aa) (Fig. 6). The genes
are expressed mainly in the brain, as well as skeletal and heart muscles, which are
postmitotic and need mitochondrial fusion to complement mutations caused by ROS
etc. All other mitotic cells can remove defective cells that are heavily loaded with
mutant mtDNA, but may require hfzos for mitochondrial fusion to complement
mutant mtDNA if mitosis is terminated by telomeric loss after aging.
We found 17 exons in hfzos 1 and 2, and 19 exons in hfzo3 (Fig. 6). These
proteins contain a GTPase domain, two coiled coil domains and two tandem
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Fig. 6. Gene and protein structure of human fusogenic proteins. TM, transmembrane domain;
CC, coiled coil domain; AA, amino acid residues.
transmembrane domains for the inner and outer membranes (Fig. 6). Green
fluorescent protein (GFP) was genetically attached to hfzo to locate them (Fig. 7,
GFP). The fragments of hfzo DNA were subcloned into the pCMV-GFP plasmid
[58], which was constructed from the GFP gene and the mammalian expression
vector pCMV-SPORT to produce GFP-hfzo-fragment fusion proteins (hfzo-gfp).
Transient transfection of each hfzo gene into HeLa cells aggregated the elongated
mitochondria (blue bars in Fig. 7, right panel) into a large mass (yellow bars in Fig.
7) or a perinuclear cluster (brown bars in Fig. 7). The presequence at the N-terminal
(mitochondrial target signal) that guide hfzo into mitochondria is missing from
hfzo1. However, Fig. 7 (hfzo1-gfp) shows that the C-terminal can also attach to
mitochondria (like the C-terminal of fzo2-gfp), and that about 60% of mitochondria
were aggregated as a large mass. High magnification fluorescence micrscopy of the
aggregated mitochondria found that hfzo was located at the contact sites. Electron
microscopy of the aggregates revealed small holes between two mitochondria. We
created mutant genes with a single amino acid substitution (K to T) in the GTPase
region by PCR mutagenesis. The aggregation capacity was decreased in the mutated
fzo-gfps (Fig. 7). Truancation of hfzo from the GTPase region to the C-terminal
removed the fusion capacity, whereas truncation between N-terminal and GTPase
region did not affect aggregation activity.
Gel filtration analysis of hfzo revealed a 440 kDa complex on the mitochondrial
membrane. Figure 8 shows our proposed hypothetical structures for the active hfzoGTP tetramer and the inactive hfzo-GDP dimer. The hfzo tetramer spans both the
outer and inner membranes of mitochondria and regulates mitochondrial membrane
docking and fusion (Fig 8). In other laboratory, hfzo is called mitofusin (Mfn1 and
Mitochondrial Genetics, Fusion and Diabetes
331
Fig. 7. Human fusogenic proteins (hfzos). Mitochondrial clustering activity of truncated and mutated hfzos revealed by labelling with green fluorescent protein.
Left upper panel: protein structures of human fusogenic proteins (hfzo1, 2 and 3) marked with green fluorescent protein (GFP). Hfzos treated by site-directed
mutagenesis from K (Lysine) to T (Threonine) of GTPase region, and truncated hfzos at N-terminus (head), C-terminus (CTC), and ATP synthase subunit (F1)
are compared. Left bottom panel: Microscopy of HeLa cells. Mitochondria are elongated shape (blue), aggregated around perinuclear region (brown), and fused in
large mass (yellow).
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Aya Sato et al.
Fig. 8. Model of function of human fusogenic proteins on mitochondria: Active GTP-form tetramer facilitates membrane fusion. OM, mitochondrial outer
membrane; IM, mitochondrial inner membrane; N, N-terminus; C, C-terminus; green, coiled coil region; red, GDP bound GTPase region; yellow, GTP bound
GTPase region.
Mitochondrial Genetics, Fusion and Diabetes
333
Mfn2), and high levels of Mfn1 are expressed in the heart, wheras Mfn2 is expressed
in both heart and muscle [69].
In addition to Fzo1p, other factors for mitochondrial fusion have recently been
identified in the mitochondria of Saccharomyces cervisiae [70]. They provided direct
evidence that the dynamin-related Mgm1 (mitochondrial genome maintenance gene
1) protein is also requied to regulate mitochondrial fusion, because Mgm1 mutants
cannot fuse the inner and outer mitochondrial membranes [70]. Mgm1 contains a
GTPase domain, which is essential for mitochondrial fusion. We also cloned and
characterized the human ortholog of Mgm1, dynamin-related protein OPA1 (optic
atrophy type 1)[71]. OPA1 is a causal gene for autosomal dominant optic atrophy in
which the GTPase domain is usually mutated. Skipping exons 4, 4b and 5b generates
eight alternative splicing variants of OPA1. The OPA1 gene product consists of
mitochondrial target signal, an N-terminal coiled-coil domain, a GTPase domain, a
dynamin central region, and a C-terminal coiled-coil domain [71]. These domains are
highly conserved from yeasts to humans. Both isoforms 1 (960 aa, mature 88 kDa,
exons 4b and 5b skipped) and 7 (997 aa, mature 93 kDa, exon 4 skipped) have been
expressed in HeLa cells [71]. Green fluorescent protein (GFP) was genetically
attached to the isoforms, and localized by fluorescence microscopy. Isoform 1 was
located in the outer membrane and isoform 7 with a long N-terminal coiled coil
domain was bound to the inner membrane. Both isoforms faced the mitochondrial
intermembrane space [67]. OPA1 protein induced mitochondrial aggregation in
cultured cells, and like hfzo, OPA1 isoforms can form different molecular mass
complexes. OPA1-isoform 1(Q297V)-GFP is constitutively active for GTP binding,
and aggregated mitochondria arounde the nucleus, whereas OPA1-isoform
1(K301A)-GFP stabilizes the GDP-bound form, and mitochondria do not
aggregate. The sturucture of mitochondria depends on the balance of fusion and
fission processes. We have already observed the complete morphological change in
heteroplasmic cells containing both wild type and MELAS mitochondria showing
the dissipation of Hþ and intermitochondrial fusion [72]. Down regulation of
OPA1 in HeLa cells using specific small interfering RNA leads to to a drastic
disorganization of the cristae and to the dissipation of Hþ [73]. These events are
followed by cytochrome c release and caspase-dependent apoptotic nuclear events
[73]. Loss of OPA1 commits cells to apoptosis without any other stimulus [73]. Thus,
OPA-1 regulates GTP dependent mitochondrial morphology [73, 74].
In conclusion, mitochondrial fusion machinery must sequentially fuse the outer
and inner mitochondrial membranes [74]. These coordinated fusion events rely on a
transmembrane GTPase that is known as hfzo, OPA-1 [71, 73] and many related
proteins [74]. Since hfzo is not expressed in proliferating cells, including the pancreas,
an extended human lifespan will need hfzo to complement mutated mtDNA, after
proliferation is stopped by telomeric shortening.
CONCLUSIONS
Mutations in mtDNA (Fig. 1) caused by factors such as aging [40], ROS stress
[14, 31] and evolution [47, 49] result in heteroplasmy that can be compensated by
mitochondrial fusion (Fig. 2) [4, 7,11]. Mitochondrial transfer by cytoplast fusion
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Aya Sato et al.
(Fig. 3) [7, 61, 62] or by microinjection (Fig. 4) [7, 8] will rescue defects of mtDNA
[11, 72]. In fact, human ooplasmic transfer is a proven success [13]. Polymorphisms
of mtDNA (Fig. 5, Table 1) may be related to ethnic differences in functions [48].
The C150T SNP found in Italian leucocytes is frequent among centernarians and
twins [1, 75], but not among Japanese. While C5178A SNP in haplogroup A2 was
frequent among Japanese centenarians [55], but not among Caucasians. SNPs
usually disturb normal subunit-subunit interactions in an enzyme complex that is
encoded by both mtDNA and nDNA. The cytb gene of mtDNA among centenarians
contains fewer SNPs than those of control groups. Thus, Tanaka proposed the
‘‘golden mean’’ hypothesis [76] that the accumulation of mutations in mtDNA (cytb
gene) causes disturbances, and only centenarians with a few deleterious mutations in
their mtDNA can escape fatal diseases until the age of 100 years. Because of
mtDNA-nDNA interactions, transcomplementation with fusogenic proteins (Figs.
6–7) is important in postmitotic (brain and muscles) and aged cells. The fusogenic
proteins hfzo and OPA1 [71] are GTPases with coiled-coil structures (Figs. 8).
Decrease in OPA-1 results in caspase-dependent apoptosis [73].
DM2 is caused by both environmetal and genetic factors, via glucose sensing
function of mitochondria [14, 77]. Except for the monogenic subtype of DM2
including MELAS, most DM2 results from polygenic heredity due to many SNPs
being susceptible to deleterious environmental influences [78]. These SNPs include
both mtDNA (5178C, 15497A etc.) and nDNA (UCP-3–55t etc.) [46]. The etiology
of DM2 has been analyzed with respect to the mitochondria-dependent apoptosis [1,
25, 34] of -cell. A vicious circle of mutation, decreased mitochondrial function,
changed Hþ , ROS production [17], and enhanced mutation [15] might be
initiated by environmental factors and terminated by the apoptosis of -cells under
the influence of susceptible SNPs. There are several defense mechanisms against
apoptosis including fusogenic proteins [71, 73] and ROS scavengers [14]. Further
experiments of mitochondrial diseases are needed to elucidate mitochondrial diseases
[79].
ACKNOWLEDGMENTS
We thank the Asian peoples and clients at our Clinic for co-operation with our
SNP studies. This study was supported by Grants in Aid for the High Technology
Research Center from the Ministry of Education, Science and Culture of Japan.
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