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What Can Mitochondrial DNA Analysis Tell Us About Mood Disorders?

  • Takaoki Kasahara
    Affiliations
    Laboratory for Molecular Dynamics of Mental Disorders, RIKEN Brain Science Institute, Wako-shi, Saitama, Japan
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  • Tadafumi Kato
    Correspondence
    Address correspondence to Tadafumi Kato, M.D., Ph.D., RIKEN Brain Science Institute, Laboratory for Molecular Dynamics of Mental Disorders, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
    Affiliations
    Laboratory for Molecular Dynamics of Mental Disorders, RIKEN Brain Science Institute, Wako-shi, Saitama, Japan
    Search for articles by this author
Open AccessPublished:September 21, 2017DOI:https://doi.org/10.1016/j.biopsych.2017.09.010

      Abstract

      Variants in mitochondrial DNA (mtDNA) and nuclear genes encoding mitochondrial proteins in bipolar disorder, depression, or other psychiatric disorders have been studied for decades, since mitochondrial dysfunction was first suggested in the brains of patients with these diseases. Candidate gene association studies initially resulted in findings compatible with the mitochondrial dysfunction hypothesis. Many of those studies, however, were conducted with modest sample sizes (N < 1000), which could cause false positive findings. Furthermore, the DNA samples examined in these studies, including genome-wide association studies, were generally derived from peripheral tissues. One key unanswered question is whether there is an association between mood disorders and somatic mtDNA mutations (deletions and point mutations) in brain regions that accumulate a high amount of mtDNA mutations and/or are involved in the regulation of mood. Two lines of robust evidence supporting the importance of mtDNA mutations in brain tissues for mood disorders have come from clinical observation of mitochondrial disease patients who carry primary mtDNA mutations or accumulate secondary mtDNA mutations due to nuclear mutations and an animal model study. More than half of mitochondrial disease patients have comorbid mood disorders, and mice with neuron-specific accumulation of mtDNA mutations show spontaneous depression-like episodes. In this review, we first summarize the current knowledge of mtDNA and its genetics and discuss what mtDNA analysis tells us about neuropsychiatric disorders based on an example of Parkinson’s disease. We also discuss challenges and future directions beyond mtDNA analysis toward an understanding of the pathophysiology of “idiopathic” mood disorders.

      Keywords

      Mitochondrial DNA (mtDNA) is the genetic material of mitochondria, the intracellular organelles that are present in almost all cells of the body. There are vast differences in patterns of genetic inheritance and variations between mtDNA and the nuclear genome. mtDNA was a target of genetic analysis before the nuclear genome, because mtDNA is overwhelmingly smaller (1.7 × 104 base pairs [bp] vs. 3.2 × 109 bp) (Figure 1), and there are hundreds or thousands of copies of mtDNA in each cell (
      • Larsson N.G.
      • Gustafsson C.M.
      DNA replication and transcription in mammalian mitochondria.
      ,
      • D’Erchia A.M.
      • Atlante A.
      • Gadaleta G.
      • Pavesi G.
      • Chiara M.
      • De Virgilio C.
      • et al.
      Tissue-specific mtDNA abundance from exome data and its correlation with mitochondrial transcription, mass and respiratory activity.
      ). The complete sequence of human mtDNA was determined more than 20 years before completion of the Human Genome Project (
      • Anderson S.
      • Bankier A.T.
      • Barrell B.G.
      • de Bruijn M.H.
      • Coulson A.R.
      • Drouin J.
      • et al.
      Sequence and organization of the human mitochondrial genome.
      ).
      Figure thumbnail gr1
      Figure 1Human mitochondrial DNA (mtDNA) and complexes for respiratory chain/oxidative phosphorylation system. The 37 mtDNA genes include two ribosomal RNA genes (12S and 16S), 22 transfer RNA genes (shown by one-letter codes), and 13 genes encoding structural subunits of the complexes for respiratory chain/oxidative phosphorylation (7 subunits of complex I [white], one of complex III [light gray], three of complex IV [polka dot], and two of complex V [dark gray]). Complexes I, III, IV, and V contain both mtDNA-encoded subunits (represented by hexagonal shapes) and nuclear gene–encoded subunits (ovoid shapes), whereas complex II is comprised of only nuclear gene–encoded subunits. The standard gene symbols of mtDNA genes are italicized. Replication and transcription are usually initiated within the displacement loop (D-loop) region, where no genes are located. The most common pathogenic mutation in MELAS, m.3243A>G, is shown in the direction of 10 o’clock. Since an identical sequence stretch of 13 base pairs (bp) (ACCTCCCTCACCA) is located in MT-ATP8 and MT-ND5, the region of 4799 bp between the two sites is prone to be deleted in human cells. The human mtDNA genome comprises about 16.6 kb. The first published sequence of human mtDNA
      (
      • Anderson S.
      • Bankier A.T.
      • Barrell B.G.
      • de Bruijn M.H.
      • Coulson A.R.
      • Drouin J.
      • et al.
      Sequence and organization of the human mitochondrial genome.
      )
      , referred to as the Cambridge reference sequence, was 16,569 bp. Resequencing of the mtDNA belonging to a European haplogroup confirms that there are 11 incorrect nucleotides in the Cambridge Reference Sequence. One of them is the CC doublet at positions 3106 and 3107, which is actually a single cytosine residue. In the revised Cambridge Reference Sequence (GenBank NC_012920.1)
      (
      • Andrews R.M.
      • Kubacka I.
      • Chinnery P.F.
      • Lightowlers R.N.
      • Turnbull D.M.
      • Howell N.
      Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA.
      )
      , nucleotide numbers are maintained by insertion of ‘N’ at position 3107. The length of human mtDNA varies generally depending on haplogroup background.
      The more we understand mtDNA and mitochondria, however, the more difficult a target mtDNA turns out to be in genetics. mtDNA is maternally transmitted and does not appear to undergo recombination, and therefore conventional, family-based association studies are not applicable, and mtDNA polymorphisms cannot be treated as independent polymorphisms. Thus, the entire mtDNA sequence should be analyzed as if it is one genotype, which is called a haplogroup. Because haplogroups vary greatly across human populations (
      • Kivisild T.
      Maternal ancestry and population history from whole mitochondrial genomes.
      ), genetic association studies of mtDNA are subject to a larger degree of population stratification than those on the nuclear genome. A simulation test predicted that approximately 6000 cases and a similar number of control subjects would be required to achieve 90% power to detect a 10% change in frequency, even when the most common haplogroup is associated (
      • Samuels D.C.
      • Carothers A.D.
      • Horton R.
      • Chinnery P.F.
      The power to detect disease associations with mitochondrial DNA haplogroups.
      ). A case-control association study of mtDNA polymorphisms in approximately 50,000 individuals from 11 common diseases and control subjects identified several polymorphisms associated with eight diseases, including schizophrenia, after multiple testing correction (
      • Hudson G.
      • Gomez-Duran A.
      • Wilson I.J.
      • Chinnery P.F.
      Recent mitochondrial DNA mutations increase the risk of developing common late-onset human diseases.
      ). However, the analysis of functional effect of each mtDNA polymorphism would be hampered by other polymorphisms within the same haplogroup. For example, we showed that mitochondrial calcium levels were lower in cybrids (transmitochondrial hybrid cells) with m.10398A>G (
      • Kazuno A.A.
      • Munakata K.
      • Nagai T.
      • Shimozono S.
      • Tanaka M.
      • Yoneda M.
      • et al.
      Identification of mitochondrial DNA polymorphisms that alter mitochondrial matrix pH and intracellular calcium dynamics.
      ) that is protective for multiple diseases (
      • Hudson G.
      • Gomez-Duran A.
      • Wilson I.J.
      • Chinnery P.F.
      Recent mitochondrial DNA mutations increase the risk of developing common late-onset human diseases.
      ). However, m.10398A>G was closely linked to another polymorphism, m.8701A>G, and it was difficult to assess the functional effects of the two polymorphisms separately (
      • Kazuno A.A.
      • Munakata K.
      • Nagai T.
      • Shimozono S.
      • Tanaka M.
      • Yoneda M.
      • et al.
      Identification of mitochondrial DNA polymorphisms that alter mitochondrial matrix pH and intracellular calcium dynamics.
      ). Rare variants, which are present sporadically across haplotypes, also interfere with association studies and functional analyses. Another complicating factor is heteroplasmy (
      • Stewart J.B.
      • Chinnery P.F.
      The dynamics of mitochondrial DNA heteroplasmy: Implications for human health and disease.
      ), which refers to the presence of two (or more) different sequences of mtDNA in a single individual cell. In patients with a mitochondrial disease caused by a maternally inherited heteroplasmic mtDNA mutation, such as m.3243A>G in individuals with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (Online Mendelian Inheritance in Man #540000), the ratio of mutant mtDNA compared with the wild-type varies among cells and tissues, and the clinical manifestations vary among individuals (
      • Stewart J.B.
      • Chinnery P.F.
      The dynamics of mitochondrial DNA heteroplasmy: Implications for human health and disease.
      ,
      • DiMauro S.
      • Hirano M.
      MELAS, GeneReviews at Gene Tests: Medical Genetics Information Resource.
      ,
      • Hämäläinen R.H.
      • Manninen T.
      • Koivumäki H.
      • Kislin M.
      • Otonkoski T.
      • Suomalainen A.
      Tissue- and cell-type-specific manifestations of heteroplasmic mtDNA 3243A>G mutation in human induced pluripotent stem cell-derived disease model.
      ). Modifier genes in the nuclear genome have been suggested to account for the individual difference (
      • Chen C.
      • Chen Y.
      • Guan M.X.
      A peep into mitochondrial disorder: Multifaceted from mitochondrial DNA mutations to nuclear gene modulation.
      ). Further complicating matters is the fact that the somatic mutation rate of mtDNA is much higher than that of the nuclear genome (
      • Schneider S.
      • Excoffier L.
      Estimation of past demographic parameters from the distribution of pairwise differences when the mutation rates vary among sites: Application to human mitochondrial DNA.
      ,
      • Greaves L.C.
      • Beadle N.E.
      • Taylor G.A.
      • Commane D.
      • Mathers J.C.
      • Khrapko K.
      • et al.
      Quantification of mitochondrial DNA mutation load.
      ,
      • Ju Y.S.
      • Alexandrov L.B.
      • Gerstung M.
      • Martincorena I.
      • Nik-Zainal S.
      • Ramakrishna M.
      • et al.
      Origins and functional consequences of somatic mitochondrial DNA mutations in human cancer.
      ). An inability to detect mutations in blood cells cannot exclude the possibility that the brain (or other organs) of a patient carries somatic mutations. To clarify the relationship between mtDNA mutations and mood disorders and to test the mitochondrial dysfunction hypothesis of the diseases (
      • Kato T.
      • Kato N.
      Mitochondrial dysfunction in bipolar disorder.
      ,
      • Manji H.
      • Kato T.
      • Di Prospero N.A.
      • Ness S.
      • Beal M.F.
      • Krams M.
      • et al.
      Impaired mitochondrial function in psychiatric disorders.
      ,
      • Kato T.
      Neurobiological basis of bipolar disorder: Mitochondrial dysfunction hypothesis and beyond.
      ), it is of great significance to study mental symptoms in mitochondrial disease patients and in animal models with mtDNA mutations (
      • Manji H.
      • Kato T.
      • Di Prospero N.A.
      • Ness S.
      • Beal M.F.
      • Krams M.
      • et al.
      Impaired mitochondrial function in psychiatric disorders.
      ).
      We review the latest knowledge on the unique characteristics of mtDNA and the relationship between mtDNA mutations and Parkinson’s disease (PD) as a model case of mtDNA mutation–associated nervous system disease. We then briefly summarize knowledge from research on polymorphisms and deletions of mtDNA in patients with mood disorders and review the research on psychiatric symptoms of mitochondrial disease patients and those of animal models. The challenges and future directions after mtDNA analysis will also be discussed.

      The Current Understanding of mtDNA

      Over a period of a billion years since commencement of symbiosis, the origin of mitochondria, most of the symbiotic genome has been transferred to the host genome. SAR11, the Rickettsiales (Alphaproteobacteria) that are closest to the inferred symbiotic bacteria (
      • Thrash J.C.
      • Boyd A.
      • Huggett M.J.
      • Grote J.
      • Carini P.
      • Yoder R.J.
      • et al.
      Phylogenomic evidence for a common ancestor of mitochondria and the SAR11 clade.
      ,
      • Rodríguez-Ezpeleta N.
      • Embley T.M.
      The SAR11 group of alpha-proteobacteria is not related to the origin of mitochondria.
      ), have a genome of about 1000 kb, whereas the human mtDNA is no longer than 16.6 kb (Figure 1). It carries 13 protein-coding genes, two mitochondrial ribosomal RNAs, and 22 mitochondrial transfer RNAs. All 13 proteins are involved in mitochondrial oxidative phosphorylation (Figure 1). The other genes essential for mitochondrial biology are encoded by the nuclear genome; of the approximately 20,000 genes in the human nuclear genome, at least 1100 genes are estimated to encode proteins that are transported to and function in the mitochondria (
      • Calvo S.E.
      • Clauser K.R.
      • Mootha V.K.
      MitoCarta2.0: An updated inventory of mammalian mitochondrial proteins.
      ). According to the endosymbiotic theory, transferring of mtDNA genes to the nuclear genome has been beneficial for mtDNA (the endosymbiont genome) because the mutation rate of mtDNA is much higher than that of the nuclear DNA (
      • Brown W.M.
      • George Jr., M.
      • Wilson A.C.
      Rapid evolution of animal mitochondrial DNA.
      ,
      • Ku C.
      • Nelson-Sathi S.
      • Roettger M.
      • Sousa F.L.
      • Lockhart P.J.
      • Bryant D.
      • et al.
      Endosymbiotic origin and differential loss of eukaryotic genes.
      ). Mitochondria generate a lot of reactive oxygen species (ROS) as byproducts of the mitochondrial respiration process, and the repair system of mtDNA is less efficient than that of nuclear DNA (
      • Alexeyev M.
      • Shokolenko I.
      • Wilson G.
      • LeDoux S.
      The maintenance of mitochondrial DNA integrity—critical analysis and update.
      ). In addition, humans and other eukaryotes have a diploid genome and the sex system offering large gene pools, which robustly protect endosymbiont-derived genes in the nuclear genome. The main mutagenic product of base damage in mtDNA is 8-oxoguanine, causing G→T transversion upon replication (
      • Croteau D.L.
      • Bohr V.A.
      Repair of oxidative damage to nuclear and mitochondrial DNA in mammalian cells.
      ). This is the first step of a vicious cycle of ROS generation and mtDNA mutations. ROS produced by respiration cause mtDNA mutations, which lead to dysfunction in the electron transport chain and to a further increase in ROS generation due to this dysfunction (
      • Trifunovic A.
      Mitochondrial DNA and ageing.
      ,
      • Schon E.A.
      • DiMauro S.
      • Hirano M.
      Human mitochondrial DNA: Roles of inherited and somatic mutations.
      ).
      Multiple deletions or a single deletion in mtDNA can also occur as somatic mutations. Small truncated mtDNAs with large deletions (for example, 30% of the total length) can be maintained in mitochondria if the region involved in replication (mtDNA control region or the D-loop region) is intact. Replication of deleted mtDNAs can be completed in a shorter time and they may increase in abundancy preferentially (
      • Nekhaeva E.
      • Bodyak N.D.
      • Kraytsberg Y.
      • McGrath S.B.
      • Van Orsouw N.J.
      • Pluzhnikov A.
      • et al.
      Clonally expanded mtDNA point mutations are abundant in individual cells of human tissues.
      ,
      • Fuke S.
      • Kametani M.
      • Yamada K.
      • Kasahara T.
      • Kubota-Sakashita M.
      • Kujoth G.C.
      • et al.
      Heterozygous Polg mutation causes motor dysfunction due to mtDNA deletions.
      ). Probably by chance, human mtDNA has two identical 13–bp sequences separated by 4977 bp (Figure 1), and the approximately 5-kb DNA stretch between the two sites is likely to be lost (called “common deletion”). This suggests that a repair system working on mtDNA is similar to microhomology-mediated end joining, one of the nuclear DNA repair systems (
      • Tadi S.K.
      • Sebastian R.
      • Dahal S.
      • Babu R.K.
      • Choudhary B.
      • Raghavan S.C.
      Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions.
      ), and a mtDNA replication-dependent repair pathway (
      • Phillips A.F.
      • Millet A.R.
      • Tigano M.
      • Dubois S.M.
      • Crimmins H.
      • Babin L.
      • et al.
      Single-molecule analysis of mtDNA replication uncovers the basis of the common deletion.
      ), although mtDNA was previously thought to lack efficient DNA repair mechanisms.
      Our sensitive quantitative polymerase chain reaction method for assaying deleted mtDNA in mutant mtDNA polymerase (Polg) knock-in mice showed that multiple deleted mtDNAs were more abundantly accumulated in the neural tissues (
      • Fuke S.
      • Kametani M.
      • Yamada K.
      • Kasahara T.
      • Kubota-Sakashita M.
      • Kujoth G.C.
      • et al.
      Heterozygous Polg mutation causes motor dysfunction due to mtDNA deletions.
      ). Although neurons are in a postmitotic state, mtDNAs in the cells are actively metabolized (i.e., replicated and degraded). Pulse labeling of mtDNA using 5-bromo-2-deoxyuridie incorporation revealed that the time to reach the half maximum levels of 5-bromo-2-deoxyuridie incorporation was shortened in the brain (approximately 4 hours) compared to the liver (
      • Fuke S.
      • Kametani M.
      • Yamada K.
      • Kasahara T.
      • Kubota-Sakashita M.
      • Kujoth G.C.
      • et al.
      Heterozygous Polg mutation causes motor dysfunction due to mtDNA deletions.
      ). The small truncated mtDNAs are able to be replicated and transcribed, and thus produce mutant mitochondrial proteins, which accelerate the vicious cycle of ROS in mitochondria (
      • Manji H.
      • Kato T.
      • Di Prospero N.A.
      • Ness S.
      • Beal M.F.
      • Krams M.
      • et al.
      Impaired mitochondrial function in psychiatric disorders.
      ).
      New problems have emerged in the era of next-generation sequencing: next-generation sequencing helps us to examine mtDNA, but it is difficult to distinguish between heteroplasmy and sequence errors. The presence of mtDNA-like sequences in the nuclear genome (nuclear mitochondrial sequences) is also a major obstacle (
      • Hazkani-Covo E.
      • Zeller R.M.
      • Martin W.
      Molecular poltergeists: Mitochondrial DNA copies (numts) in sequenced nuclear genomes.
      ). Human mtDNA genes are still being transferred into the nuclear DNA, and thereby a number of nuclear mitochondrial sequences exist across the nuclear genome. During the human lineage of evolution, mutations have occurred in the nuclear mitochondrial sequences, which are likely to produce spurious variant calls. To reduce the risk, a bioinformatics pipeline, MToolBox, will be helpful, which filters out nuclear mitochondrial sequences and also reconstructs the mtDNA sequence from next-generation sequencing data (
      • Calabrese C.
      • Simone D.
      • Diroma M.A.
      • Santorsola M.
      • Gutta C.
      • Gasparre G.
      • et al.
      MToolBox: A highly automated pipeline for heteroplasmy annotation and prioritization analysis of human mitochondrial variants in high-throughput sequencing.
      ).
      In addition to point mutations and deletions, depletion of mtDNA should also be considered a predisposing factor for mitochondrial diseases and related disorders (
      • El-Hattab A.W.
      • Scaglia F.
      Mitochondrial DNA depletion syndromes: Review and updates of genetic basis, manifestations, and therapeutic options.
      ). In most cases of mtDNA depletions, nuclear mutations in the enzymes for mtDNA replication are involved (
      • El-Hattab A.W.
      • Scaglia F.
      Mitochondrial DNA depletion syndromes: Review and updates of genetic basis, manifestations, and therapeutic options.
      ). To detect depletion of mtDNA or to measure mtDNA copy number, quantitative polymerase chain reaction can be performed using primers targeting specific regions of mtDNA (for example, the D-loop region, MT-CO1 genes) (Figure 1). Primers and probes should be carefully designed and validated because many nuclear mitochondrial sequences are almost identical to the mtDNA sequences.

      The Lesson from PD

      There are many diseases in which mitochondrial dysfunction is involved (
      • Lin M.T.
      • Beal M.F.
      Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases.
      ,
      • Nunnari J.
      • Suomalainen A.
      Mitochondria: In sickness and in health.
      ,
      • Chow J.
      • Rahman J.
      • Achermann J.C.
      • Dattani M.T.
      • Rahman S.
      Mitochondrial disease and endocrine dysfunction.
      ), such as diabetes mellitus and PD, because of the presence of mitochondria in almost all cells in humans. Before we discuss psychiatric disorders and mtDNA mutations, we review the mtDNA genetics in PD as a good precedent of neuronal diseases with the potential involvement of mtDNA mutations. The link between PD and mitochondrial dysfunction (
      • Perier C.
      • Vila M.
      Mitochondrial biology and Parkinson’s disease.
      ,
      • Hu Q.
      • Wang G.
      Mitochondrial dysfunction in Parkinson’s disease.
      ,
      • Bose A.
      • Beal M.F.
      Mitochondrial dysfunction in Parkinson’s disease.
      ) first came from the observation that accidental exposure to 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine resulted in parkinsonism. In the brain, 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine is converted into 1-methyl-4-phenylpyridinium, an inhibitor of mitochondrial complex I. 1-Methyl-4-phenylpyridinium is selectively taken up by dopaminergic neurons in the substantia nigra (SN), resulting in their degeneration. Genetics of familial PD has also suggested mitochondrial dysfunction to play a role in PD (
      • Nuytemans K.
      • Theuns J.
      • Cruts M.
      • Van Broeckhoven C.
      Genetic etiology of Parkinson disease associated with mutations in the SNCA, PARK2, PINK1, PARK7, and LRRK2 genes: A mutation update.
      ,
      • Lin M.K.
      • Farrer M.J.
      Genetics and genomics of Parkinson’s disease.
      ). Briefly, PINK1 (encoded by PARK6) detects damaged mitochondria with reduced membrane potential, and PINK1 kinase activity initiates Parkin (PARK2) translocation to mitochondria and mitophagy. In addition, DJ-1 (PARK7) is a mitochondrial peroxiredoxin-like peroxidase scavenging mitochondrial ROS.
      Despite the solid link between PD and mitochondrial dysfunction, association with mtDNA polymorphisms in case-control association studies was not reproducible (
      • Kirches E.
      Do mtDNA mutations participate in the pathogenesis of sporadic Parkinson’s disease?.
      ). This was due to the small number of subjects, the lack of identification of haplogroups, a failure to control population stratification, and other factors. An increase in sample size may resolve this problem in part (
      • Hudson G.
      • Gomez-Duran A.
      • Wilson I.J.
      • Chinnery P.F.
      Recent mitochondrial DNA mutations increase the risk of developing common late-onset human diseases.
      ). In contrast, the role of somatic mtDNA mutations has been strongly suggested in PD. Patients with chronic progressive external ophthalmoplegia (CPEO) frequently have comorbid Parkinsonism (
      • Bose A.
      • Beal M.F.
      Mitochondrial dysfunction in Parkinson’s disease.
      ,
      • Luoma P.
      • Melberg A.
      • Rinne J.O.
      • Kaukonen J.A.
      • Nupponen N.N.
      • Chalmers R.M.
      • et al.
      Parkinsonism, premature menopause, and mitochondrial DNA polymerase γ mutations: Clinical and molecular genetic study.
      ,
      • Davidzon G.
      • Greene P.
      • Mancuso M.
      • Klos K.J.
      • Ahlskog J.E.
      • Hirano M.
      • et al.
      Early-onset familial parkinsonism due to POLG mutations.
      ). CPEO is a Mendelian inheritance mitochondrial disease (Online Mendelian Inheritance in Man #157640) in which mutations in the POLG gene and others cause an increase in multiple deletions in mtDNA. Importantly, mtDNA deletions are accumulated in neurons microdissected from the SN of postmortem brains from patients with idiopathic PD compared to age-matched control subjects (
      • Bender A.
      • Krishnan K.J.
      • Morris C.M.
      • Taylor G.A.
      • Reeve A.K.
      • Perry R.H.
      • et al.
      High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease.
      ).
      What the lesson from PD tells is to focus not on mtDNA polymorphisms in peripheral tissues but on somatic mtDNA mutations in tissues responsible for the disease. For brain disorders, studying patients with mitochondrial diseases and comorbid mood disorders will ultimately be informative and lead to drug targets and understanding the mental illnesses (
      • Manji H.
      • Kato T.
      • Di Prospero N.A.
      • Ness S.
      • Beal M.F.
      • Krams M.
      • et al.
      Impaired mitochondrial function in psychiatric disorders.
      ).

      mtDNA Variants in Mood Disorders

      In the past 20 years, a number of studies have explored mtDNA variants in patients with psychiatric diseases (
      • Baron M.
      Novel strategies in molecular genetics of mental illness.
      ,
      • Kato T.
      The other, forgotten genome: Mitochondrial DNA and mental disorders.
      ). The mtDNA variants explored included polymorphisms, rare variants, heteroplasmic mutations causative for mitochondrial diseases, such as the mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes mutation m.3243A>G (
      • Munakata K.
      • Iwamoto K.
      • Bundo M.
      • Kato T.
      Mitochondrial DNA 3243A>G mutation and increased expression of LARS2 gene in the brains of patients with bipolar disorder and schizophrenia.
      ), and other somatic mutations, including the common deletion (
      • Stine O.C.
      • Luu S.U.
      • Zito M.
      • Casanova M.
      The possible association between affective disorder and partially deleted mitochondrial DNA.
      ,
      • Kato T.
      • Stine O.C.
      • McMahon F.J.
      • Crowe R.R.
      Increased levels of a mitochondrial DNA deletion in the brain of patients with bipolar disorder.
      ,
      • Shao L.
      • Martin M.V.
      • Watson S.J.
      • Schatzberg A.
      • Akil H.
      • Myers R.M.
      • et al.
      Mitochondrial involvement in psychiatric disorders.
      ,
      • Sequeira A.
      • Martin M.V.
      • Rollins B.
      • Moon E.A.
      • Bunney W.E.
      • Macciardi F.
      • et al.
      Mitochondrial mutations and polymorphisms in psychiatric disorders.
      ,
      • Mamdani F.
      • Rollins B.
      • Morgan L.
      • Sequeira P.A.
      • Vawter M.P.
      The somatic common deletion in mitochondrial DNA is decreased in schizophrenia.
      ).
      Most of the reported genetic association studies of mtDNA polymorphisms and mood disorders were performed in small samples and cannot be considered robust findings at the current standard. Hundreds of mtDNA polymorphisms can be tested using recent single nucleotide polymorphism arrays that are used for genome-wide association study analyses. A modest association of mtDNA polymorphisms m.3666G>A and m.15784T>C with bipolar disorder (BD) was detected in genome-wide association study (965 cases and 3938 control subjects) (
      • Sequeira A.
      • Martin M.V.
      • Rollins B.
      • Moon E.A.
      • Bunney W.E.
      • Macciardi F.
      • et al.
      Mitochondrial mutations and polymorphisms in psychiatric disorders.
      ). Even if in a larger-scale association study some mtDNA polymorphism associations will be found to be significant after correcting for multiple testing, they are not likely to bring us closer to connecting the polymorphisms and neuroscience because the odds ratios are expected to be low. Contrary to polymorphisms (common variants), rare variants of mtDNA, which are detected using whole-genome sequencing or mtDNA resequencing array, could provide explicit cues to the involvement of aberrant mtDNA or mitochondrial dysfunction in the disease. Vawter et al. (
      • Rollins B.
      • Martin M.V.
      • Sequeira P.A.
      • Moon E.A.
      • Morgan L.Z.
      • Watson S.J.
      • et al.
      Mitochondrial variants in schizophrenia, bipolar disorder, and major depressive disorder.
      ) sequenced entire mtDNAs (14 BD cases, 15 major depressive disorder cases, and 20 control subjects) and identified two rare variants, m.114C>T and m.16300A>G, in patients with BD (the worldwide frequencies of these variants were <.004) (
      • Rollins B.
      • Martin M.V.
      • Sequeira P.A.
      • Moon E.A.
      • Morgan L.Z.
      • Watson S.J.
      • et al.
      Mitochondrial variants in schizophrenia, bipolar disorder, and major depressive disorder.
      ). “Private mutations” that had not been found in controls so far were not identified in the study. The same is equally true of other mtDNA variants, which were once identified as private mutations in patients with mood disorders (
      • Sequeira A.
      • Martin M.V.
      • Rollins B.
      • Moon E.A.
      • Bunney W.E.
      • Macciardi F.
      • et al.
      Mitochondrial mutations and polymorphisms in psychiatric disorders.
      ,
      • Kirk R.
      • Furlong R.A.
      • Amos W.
      • Cooper G.
      • Rubinsztein J.S.
      • Walsh C.
      • et al.
      Mitochondrial genetic analyses suggest selection against maternal lineages in bipolar affective disorder.
      ,
      • Munakata K.
      • Tanaka M.
      • Mori K.
      • Washizuka S.
      • Yoneda M.
      • Tajima O.
      • et al.
      Mitochondrial DNA 3644T>C mutation associated with bipolar disorder.
      ,
      • Sequeira A.
      • Rollins B.
      • Magnan C.
      • van Oven M.
      • Baldi P.
      • Myers R.M.
      • et al.
      Mitochondrial mutations in subjects with psychiatric disorders.
      ), but almost all of them now turn out to be polymorphisms or rare variants with one exception: m.4564G>A found in a patient with BD (
      • Kirk R.
      • Furlong R.A.
      • Amos W.
      • Cooper G.
      • Rubinsztein J.S.
      • Walsh C.
      • et al.
      Mitochondrial genetic analyses suggest selection against maternal lineages in bipolar affective disorder.
      ).
      Another line of study is the measurement of mtDNA copy number in the brain (
      • Vawter M.P.
      • Tomita H.
      • Meng F.
      • Bolstad B.
      • Li J.
      • Evans S.
      • et al.
      Mitochondrial-related gene expression changes are sensitive to agonal-pH state: Implications for brain disorders.
      ,
      • Kakiuchi C.
      • Ishiwata M.
      • Kametani M.
      • Nelson C.
      • Iwamoto K.
      • Kato T.
      Quantitative analysis of mitochondrial DNA deletions in the brains of patients with bipolar disorder and schizophrenia.
      ), peripheral cells (
      • Cai N.
      • Li Y.
      • Chang S.
      • Liang J.
      • Lin C.
      • Zhang X.
      • et al.
      Genetic control over mtDNA and its relationship to major depressive disorder.
      ,
      • Tyrka A.R.
      • Parade S.H.
      • Price L.H.
      • Kao H.T.
      • Porton B.
      • Philip N.S.
      • et al.
      Alterations of mitochondrial DNA copy number and telomere length with early adversity and psychopathology.
      ), and plasma (
      • Kageyama Y.
      • Kasahara K.
      • Kato M.
      • Sakai S.
      • Deguchi Y.
      • Tani M.
      • et al.
      The relationship between circulating mitochondrial DNA and inflammatory cytokines in patients with major depression.
      ). Quantification of the mtDNA copy number in the brain is particularly important in the study of mood disorders, as the copy number has implications for biogenesis and the turnover of mitochondria (
      • Clay Montier L.L.
      • Deng J.J.
      • Bai Y.
      Number matters: Control of mammalian mitochondrial DNA copy number.
      ), and the activity and the structural change of mitochondrial in neurons are crucial for proper synaptic functioning (
      • Li Z.
      • Okamoto K.
      • Hayashi Y.
      • Sheng M.
      The importance of dendritic mitochondria in the morphogenesis and plasticity of spines and synapses.
      ,
      • Fu Z.X.
      • Tan X.
      • Fang H.
      • Lau P.M.
      • Wang X.
      • Cheng H.
      • Bi G.Q.
      Dendritic mitoflash as a putative signal for stabilizing long-term synaptic plasticity.
      ). Although the regulation of mtDNA copy number is complex and the sampling methods affect the apparent mtDNA copy number, it should be investigated in a large number of samples in a refined way.
      In order to investigate the link between mtDNA variants and psychiatric diseases, it is undoubtedly reasonable to examine somatic mtDNA mutations in the tissues responsible for the diseases, just as mtDNA mutations in neurons of the SN should be examined in PD (
      • Bender A.
      • Krishnan K.J.
      • Morris C.M.
      • Taylor G.A.
      • Reeve A.K.
      • Perry R.H.
      • et al.
      High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease.
      ). The common deletion was quantified in candidate brain regions, such as the prefrontal cortex, anterior cingulate cortex, amygdala, caudate nucleus, dorsolateral prefrontal cortex, hippocampus, nucleus accumbens, and others, which have been implicated by magnetic resonance imaging and other methods (
      • Birur B.
      • Kraguljac N.V.
      • Shelton R.C.
      • Lahti A.C.
      Brain structure, function, and neurochemistry in schizophrenia and bipolar disorder—A systematic review of the magnetic resonance neuroimaging literature.
      ), and found an increase in the level of the common deletion in BD in brain (
      • Stine O.C.
      • Luu S.U.
      • Zito M.
      • Casanova M.
      The possible association between affective disorder and partially deleted mitochondrial DNA.
      ,
      • Kato T.
      • Stine O.C.
      • McMahon F.J.
      • Crowe R.R.
      Increased levels of a mitochondrial DNA deletion in the brain of patients with bipolar disorder.
      ,
      • Shao L.
      • Martin M.V.
      • Watson S.J.
      • Schatzberg A.
      • Akil H.
      • Myers R.M.
      • et al.
      Mitochondrial involvement in psychiatric disorders.
      ,
      • Sequeira A.
      • Martin M.V.
      • Rollins B.
      • Moon E.A.
      • Bunney W.E.
      • Macciardi F.
      • et al.
      Mitochondrial mutations and polymorphisms in psychiatric disorders.
      ,
      • Mamdani F.
      • Rollins B.
      • Morgan L.
      • Sequeira P.A.
      • Vawter M.P.
      The somatic common deletion in mitochondrial DNA is decreased in schizophrenia.
      ,
      • Fuke S.
      • Kametani M.
      • Kato T.
      Quantitative analysis of the 4977-bp common deletion of mitochondrial DNA in postmortem frontal cortex from patients with bipolar disorder and schizophrenia.
      ). To focus on somatic mtDNA mutations, however, brings another question: Are the somatic mutations observed an effect of medication (
      • Ponchaut S.
      • Veitch K.
      Valproate and mitochondria.
      ,
      • Modica-Napolitano J.S.
      • Lagace C.J.
      • Brennan W.A.
      • Aprille J.R.
      Differential effects of typical and atypical neuroleptics on mitochondrial function in vitro.
      ,
      • Moren C.
      • Juarez-Flores D.L.
      • Cardellach F.
      • Garrabou G.
      The role of therapeutic drugs on acquired mitochondrial toxicity.
      ) or the cause or the consequence of the disease (
      • Manji H.
      • Kato T.
      • Di Prospero N.A.
      • Ness S.
      • Beal M.F.
      • Krams M.
      • et al.
      Impaired mitochondrial function in psychiatric disorders.
      ,
      • Kato T.
      Molecular neurobiology of bipolar disorder: A disease of ‘mood-stabilizing neurons’?.
      )? To investigate such questions, an alternative approach based on animal models would be useful.
      There has been an alternative approach that focuses on the mental condition of mitochondrial disease patients since the publication by Fattal et al. (
      • Fattal O.
      • Link J.
      • Quinn K.
      • Cohen B.H.
      • Franco K.
      Psychiatric comorbidity in 36 adults with mitochondrial cytopathies.
      ). Although sample sizes were small, they gave a substantially consistent result—namely, that major depressive disorder is present in about half of mitochondrial disease patients and BD is present in 7% to 21% (Table 1) (
      • Fattal O.
      • Link J.
      • Quinn K.
      • Cohen B.H.
      • Franco K.
      Psychiatric comorbidity in 36 adults with mitochondrial cytopathies.
      ,
      • Koene S.
      • Kozicz T.L.
      • Rodenburg R.J.
      • Verhaak C.M.
      • de Vries M.C.
      • Wortmann S.
      • et al.
      Major depression in adolescent children consecutively diagnosed with mitochondrial disorder.
      ,
      • Smits B.W.
      • Fermont J.
      • Delnooz C.C.
      • Kalkman J.S.
      • Bleijenberg G.
      • van Engelen B.G.
      Disease impact in chronic progressive external ophthalmoplegia: More than meets the eye.
      ,
      • Anglin R.E.
      • Rosebush P.I.
      • Noseworthy M.D.
      • Tarnopolsky M.
      • Mazurek M.F.
      Psychiatric symptoms correlate with metabolic indices in the hippocampus and cingulate in patients with mitochondrial disorders.
      ,
      • Mancuso M.
      • Orsucci D.
      • Ienco E.C.
      • Pini E.
      • Choub A.
      • Siciliano G.
      Psychiatric involvement in adult patients with mitochondrial disease.
      ,
      • Inczedy-Farkas G.
      • Remenyi V.
      • Gal A.
      • Varga Z.
      • Balla P.
      • Udvardy-Meszaros A.
      • et al.
      Psychiatric symptoms of patients with primary mitochondrial DNA disorders.
      ). The comorbidity with mood disorders has been reported in patients with primary mtDNA mutations, such as m.3243A>G, m.3271T>C, m.3460G>A, m.8344A>G, m.9035C>T, and the common deletion, as well as in patients with CPEO harboring somatic mtDNA deletions due to mutations in nuclear genes encoding mitochondrial proteins involved in mtDNA replication, such as mtDNA polymerase (POLG), mtDNA helicase, and adenine nucleotide transporter (
      • Koene S.
      • Kozicz T.L.
      • Rodenburg R.J.
      • Verhaak C.M.
      • de Vries M.C.
      • Wortmann S.
      • et al.
      Major depression in adolescent children consecutively diagnosed with mitochondrial disorder.
      ,
      • Smits B.W.
      • Fermont J.
      • Delnooz C.C.
      • Kalkman J.S.
      • Bleijenberg G.
      • van Engelen B.G.
      Disease impact in chronic progressive external ophthalmoplegia: More than meets the eye.
      ,
      • Anglin R.E.
      • Rosebush P.I.
      • Noseworthy M.D.
      • Tarnopolsky M.
      • Mazurek M.F.
      Psychiatric symptoms correlate with metabolic indices in the hippocampus and cingulate in patients with mitochondrial disorders.
      ,
      • Mancuso M.
      • Orsucci D.
      • Ienco E.C.
      • Pini E.
      • Choub A.
      • Siciliano G.
      Psychiatric involvement in adult patients with mitochondrial disease.
      ,
      • Inczedy-Farkas G.
      • Remenyi V.
      • Gal A.
      • Varga Z.
      • Balla P.
      • Udvardy-Meszaros A.
      • et al.
      Psychiatric symptoms of patients with primary mitochondrial DNA disorders.
      ,
      • Kasahara T.
      • Kubota M.
      • Miyauchi T.
      • Noda Y.
      • Mouri A.
      • Nabeshima T.
      • et al.
      Mice with neuron-specific accumulation of mitochondrial DNA mutations show mood disorder-like phenotypes.
      ,
      • Copeland W.C.
      Inherited mitochondrial diseases of DNA replication.
      ). Importantly, some literature has described that at least some of the mitochondrial disease patients suffered from mood disorders before the onset or diagnosis of mitochondrial disease (
      • Suomalainen A.
      • Majander A.
      • Haltia M.
      • Somer H.
      • Lönnqvist J.
      • Savontaus M.L.
      • et al.
      Multiple deletions of mitochondrial DNA in several tissues of a patient with severe retarded depression and familial progressive external ophthalmoplegia.
      ,
      • Siciliano G.
      • Tessa A.
      • Petrini S.
      • Mancuso M.
      • Bruno C.
      • Grieco G.S.
      • et al.
      Autosomal dominant external ophthalmoplegia and bipolar affective disorder associated with a mutation in the ANT1 gene.
      ,
      • Mancuso M.
      • Ricci G.
      • Choub A.
      • Filosto M.
      • DiMauro S.
      • Davidzon G.
      • et al.
      Autosomal dominant psychiatric disorders and mitochondrial DNA multiple deletions: Report of a family.
      ).
      Table 1Comorbid Mood Disorders in Mitochondrial Disease Patients
      Medical ConditionSubjects, nDiagnosis MethodPrevalence of Comorbid Psychiatric DisordersReference
      Mitochondrial diseases36MINIMDD 54%, BD 17%Fattal et al.
      • Fattal O.
      • Link J.
      • Quinn K.
      • Cohen B.H.
      • Franco K.
      Psychiatric comorbidity in 36 adults with mitochondrial cytopathies.
      Mitochondrial diseases (adolescents)35DSM-IVMDD 14%, suspected depression 9%Koene et al.
      • Koene S.
      • Kozicz T.L.
      • Rodenburg R.J.
      • Verhaak C.M.
      • de Vries M.C.
      • Wortmann S.
      • et al.
      Major depression in adolescent children consecutively diagnosed with mitochondrial disorder.
      CPEO19SCIDDepressive state 32%Smits et al.
      • Smits B.W.
      • Fermont J.
      • Delnooz C.C.
      • Kalkman J.S.
      • Bleijenberg G.
      • van Engelen B.G.
      Disease impact in chronic progressive external ophthalmoplegia: More than meets the eye.
      Mitochondrial diseases15CIDIMDD 67%, BD 7%Anglin et al.
      • Anglin R.E.
      • Rosebush P.I.
      • Noseworthy M.D.
      • Tarnopolsky M.
      • Mazurek M.F.
      Psychiatric symptoms correlate with metabolic indices in the hippocampus and cingulate in patients with mitochondrial disorders.
      Mitochondrial diseases24MINIMDD 37%, BD 21%
      Calculated from the results of MINI.
      Mancuso et al.
      • Mancuso M.
      • Orsucci D.
      • Ienco E.C.
      • Pini E.
      • Choub A.
      • Siciliano G.
      Psychiatric involvement in adult patients with mitochondrial disease.
      Primary mtDNA mutation disease19SCIDBD 16%, other mood disorders 32%
      Other mood disorders include MDD, dysthymia, and adjustment disorder with depressed mood.
      Inczedy-Farkas et al.
      • Inczedy-Farkas G.
      • Remenyi V.
      • Gal A.
      • Varga Z.
      • Balla P.
      • Udvardy-Meszaros A.
      • et al.
      Psychiatric symptoms of patients with primary mitochondrial DNA disorders.
      BD, bipolar disorder; CIDI, Composite International Diagnostic Interview; CPEO, chronic progressive external ophthalmoplegia; DSM-IV, Diagnostic and Statistical Manual of Mental Disorders, 4th edition; MDD, major depressive disorder; MINI, Mini-International Neuropsychiatric Interview; SCID, Structured Clinical Interview for DSM.
      a Calculated from the results of MINI.
      b Other mood disorders include MDD, dysthymia, and adjustment disorder with depressed mood.
      In addition to clinical observation of mitochondrial disease patients, another important strategy is animal model research, in which behaviors of mice with mtDNA abnormalities are examined. There have been reported “mito-mice” carrying a heteroplasmic single mtDNA deletion (
      • Inoue K.
      • Nakada K.
      • Ogura A.
      • Isobe K.
      • Goto Y.
      • Nonaka I.
      • et al.
      Generation of mice with mitochondrial dysfunction by introducing mouse mtDNA carrying a deletion into zygotes.
      ) or a heteroplasmic pathogenic mutation (
      • Sato A.
      • Nakada K.
      • Akimoto M.
      • Ishikawa K.
      • Ono T.
      • Shitara H.
      • et al.
      Rare creation of recombinant mtDNA haplotypes in mammalian tissues.
      ) and knock-in mice carrying the proofreading-deficient mutation in the Polg gene (
      • Trifunovic A.
      • Wredenberg A.
      • Falkenberg M.
      • Spelbrink J.N.
      • Rovio A.T.
      • Bruder C.E.
      • et al.
      Premature ageing in mice expressing defective mitochondrial DNA polymerase.
      ,
      • Kujoth G.C.
      • Hiona A.
      • Pugh T.D.
      • Someya S.
      • Panzer K.
      • Wohlgemuth S.E.
      • et al.
      Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging.
      ). These mutant mice modeled mitochondrial diseases with primary mtDNA mutations or secondary mtDNA mutations led by nuclear genome mutations, respectively. In these mutant mice, mitochondrial dysfunction occurred in the whole body, which would impede behavioral analyses because of weakness of skeletal muscles, cardiomyopathy, motor abnormalities, and/or premature aging (
      • Inoue K.
      • Nakada K.
      • Ogura A.
      • Isobe K.
      • Goto Y.
      • Nonaka I.
      • et al.
      Generation of mice with mitochondrial dysfunction by introducing mouse mtDNA carrying a deletion into zygotes.
      ,
      • Sato A.
      • Nakada K.
      • Akimoto M.
      • Ishikawa K.
      • Ono T.
      • Shitara H.
      • et al.
      Rare creation of recombinant mtDNA haplotypes in mammalian tissues.
      ,
      • Trifunovic A.
      • Wredenberg A.
      • Falkenberg M.
      • Spelbrink J.N.
      • Rovio A.T.
      • Bruder C.E.
      • et al.
      Premature ageing in mice expressing defective mitochondrial DNA polymerase.
      ,
      • Kujoth G.C.
      • Hiona A.
      • Pugh T.D.
      • Someya S.
      • Panzer K.
      • Wohlgemuth S.E.
      • et al.
      Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging.
      ). Thus, we generated transgenic (Tg) mice expressing proofreading-deficient POLG in a neuron-specific manner; in other words, the mutant mice are a model of central nervous system manifestation of CPEO (
      • Kasahara T.
      • Kubota M.
      • Miyauchi T.
      • Noda Y.
      • Mouri A.
      • Nabeshima T.
      • et al.
      Mice with neuron-specific accumulation of mitochondrial DNA mutations show mood disorder-like phenotypes.
      ). mtDNAs in the skeletal muscles and the heart remained intact in the aged Tg mice, and accumulation of point mutations and multiple deletions in mtDNA was profound only in the forebrain (
      • Kasahara T.
      • Kubota M.
      • Miyauchi T.
      • Noda Y.
      • Mouri A.
      • Nabeshima T.
      • et al.
      Mice with neuron-specific accumulation of mitochondrial DNA mutations show mood disorder-like phenotypes.
      ).
      The greatest problem of animal models for psychiatric disorders is how to evaluate the psychiatric symptoms of the models. Since diagnosis based on the DSM-5 by an interview is currently used both in clinical setting and in research fields, we considered that behaviors of the mutant Polg Tg mice should be assessed based on the DSM-5 (
      American Psychiatric Association
      Diagnostic and Statistical Manual of Mental Disorders, 5th ed.
      ). The inner experience/feelings (such as depressed mood and feeling of worthlessness) assessed by verbal communication that were part of the diagnostic criteria are unable to be evaluated in mice. However, other items that define a major depressive episode, such as changes in appetite, sleep disturbance, fatigability, loss of interest, retardation, and concentration, could be operationally defined, even in mice. The mutant Polg Tg mice showed hypoactivity episodes lasting 2 to 3 weeks at a frequency of about once half a year, and the behavioral and physiological phenotypes during the episodes satisfied the diagnostic criteria of major depressive episode of the DSM-5 (
      • Kasahara T.
      • Takata A.
      • Kato T.M.
      • Kubota-Sakashita M.
      • Sawada T.
      • Kakita A.
      • et al.
      Depression-like episodes in mice harboring mtDNA deletions in paraventricular thalamus.
      ). The episodes were also accompanied with biological changes, including elevated levels of corticosteroid and altered body temperature (
      • Kasahara T.
      • Takata A.
      • Kato T.M.
      • Kubota-Sakashita M.
      • Sawada T.
      • Kakita A.
      • et al.
      Depression-like episodes in mice harboring mtDNA deletions in paraventricular thalamus.
      ). Furthermore, the episodes responded to treatment by a selective serotonin reuptake inhibitor (escitalopram) and a mood stabilizer (lithium) (
      • Kasahara T.
      • Takata A.
      • Kato T.M.
      • Kubota-Sakashita M.
      • Sawada T.
      • Kakita A.
      • et al.
      Depression-like episodes in mice harboring mtDNA deletions in paraventricular thalamus.
      ). The mutant Polg Tg mice clearly demonstrated that accumulation of mtDNA mutations (multiple deletions and point mutations) in the brain triggered mood disorder–like phenotypes (
      • Kasahara T.
      • Takata A.
      • Kato T.M.
      • Kubota-Sakashita M.
      • Sawada T.
      • Kakita A.
      • et al.
      Depression-like episodes in mice harboring mtDNA deletions in paraventricular thalamus.
      ).

      Beyond the mtDNA Variants

      To proceed to the next stage, at least two major directions are possible. One is to clarify what happens in the neurons with accumulation of mtDNA mutations. It will likely lead to drug development. With mtDNA defect, the respiratory chain activity declines and the mitochondrial membrane potential decreases. Since the mitochondrial membrane potential is important for translocation of mitochondrial proteins encoded by the nuclear genome (
      • Bohnert M.
      • Pfanner N.
      • van der Laan M.
      A dynamic machinery for import of mitochondrial precursor proteins.
      ), almost all functions of mitochondria (for example, adenosine triphosphate synthesis, Ca2+ regulation, and metabolic processes, such as beta oxidation) will be impaired. If dysfunction of a particular mitochondrial pathway is linked to the onset of a psychiatric symptom, drugs that improve the dysfunction or bypass the pathway will be potent against mood symptoms. Mitochondria in the neurons could possibly have neuron-specific functions, as several genes encoding mitochondrial proteins are specifically expressed in neurons (Figure 2). Another possibility is that when the mtDNA defect causes mitochondrial dysfunction and cell damage, the contents of mitochondria may leak out of the cells and act as damage-associated molecular patterns, which initiate the innate immune response (
      • Goulopoulou S.
      • Matsumoto T.
      • Bomfim G.F.
      • Webb R.C.
      Toll-like receptor 9 activation: A novel mechanism linking placenta-derived mitochondrial DNA and vascular dysfunction in pre-eclampsia.
      ,
      • Wenceslau C.F.
      • McCarthy C.G.
      • Szasz T.
      • Spitler K.
      • Goulopoulou S.
      • Webb R.C.
      Mitochondrial damage-associated molecular patterns and vascular function.
      ). This may explain the enigmatic inflammation often seen in psychiatric disorders (
      • Kageyama Y.
      • Kasahara K.
      • Kato M.
      • Sakai S.
      • Deguchi Y.
      • Tani M.
      • et al.
      The relationship between circulating mitochondrial DNA and inflammatory cytokines in patients with major depression.
      ,
      • Goldsmith D.R.
      • Rapaport M.H.
      • Miller B.J.
      A meta-analysis of blood cytokine network alterations in psychiatric patients: Comparisons between schizophrenia, bipolar disorder and depression.
      ,
      • Köhler C.A.
      • Freitas T.H.
      • Maes M.
      • de Andrade N.Q.
      • Liu C.S.
      • Fernandes B.S.
      • et al.
      Peripheral cytokine and chemokine alterations in depression: A meta-analysis of 82 studies.
      ). Alternatively, if it is the same as in the case of PD, neurons in a particular nucleus or a brain region may be degenerated owing to mtDNA mutations and consequent mitochondrial dysfunction. Such a neuronal loss, however, in a specific brain region, which corresponds to the SN in the case of PD, has not been found yet in postmortem brains of patients with mood disorders (
      • Manji H.
      • Kato T.
      • Di Prospero N.A.
      • Ness S.
      • Beal M.F.
      • Krams M.
      • et al.
      Impaired mitochondrial function in psychiatric disorders.
      ).
      Figure thumbnail gr2
      Figure 2Genes encoding brain-specific mitochondrial proteins in the human genome. This Venn diagram shows an overlap of two human gene sets identified by the brain-specific transcriptome
      (
      • Uhlén M.
      • Fagerberg L.
      • Hallstrom B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Proteomics. Tissue-based map of the human proteome.
      )
      and Human MitoCarta2.0
      (
      • Calvo S.E.
      • Clauser K.R.
      • Mootha V.K.
      MitoCarta2.0: An updated inventory of mammalian mitochondrial proteins.
      )
      , which provides genes expressed highly in the brain and genes encoding mitochondrial proteins, respectively. The numbers in parentheses indicate numbers of genes in each set: approximately 19,500, all genes in the genome; 1437, genes showing a more than fivefold increase in messenger RNA levels in the brain compared with average levels in all tissues; 415, genes showing a more than fivefold increase in messenger RNA levels in the brain compared with all other tissues tested
      (
      • Uhlén M.
      • Fagerberg L.
      • Hallstrom B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Proteomics. Tissue-based map of the human proteome.
      )
      . The numbers that are not denoted by parentheses indicate numbers of genes in each region of the Venn diagram. Genes encoding brain-specific mitochondrial proteins are PAK7, SLC25A22, and TUBB3, all of which are expressed in neurons (
      • Pandey A.
      • Dan I.
      • Kristiansen T.Z.
      • Watanabe N.M.
      • Voldby J.
      • Kajikawa E.
      • et al.
      Cloning and characterization of PAK5, a novel member of mammalian p21-activated kinase-II subfamily that is predominantly expressed in brain.
      ,
      • Molinari F.
      • Raas-Rothschild A.
      • Rio M.
      • Fiermonte G.
      • Encha-Razavi F.
      • Palmieri L.
      • et al.
      Impaired mitochondrial glutamate transport in autosomal recessive neonatal myoclonic epilepsy.
      ,
      • Caccamo D.V.
      • Herman M.M.
      • Frankfurter A.
      • Katsetos C.D.
      • Collins V.P.
      • Rubinstein L.J.
      An immunohistochemical study of neuropeptides and neuronal cytoskeletal proteins in the neuroepithelial component of a spontaneous murine ovarian teratoma. Primitive neuroepithelium displays immunoreactivity for neuropeptides and neuron-associated beta-tubulin isotype.
      ).
      To identify the unknown brain region(s) with neurons with severe mitochondrial dysfunction is of paramount importance and a possible future step toward the understanding of mood disorders themselves. A comprehensive search for brain region(s) with higher levels of somatic mtDNA mutations can identify the pathological or responsible site(s) of mood disorders. It is also possible to investigate mitochondrial respiratory chain activity using activity staining methods (
      • Ross J.M.
      Visualization of mitochondrial respiratory function using cytochrome c oxidase/succinate dehydrogenase (COX/SDH) double-labeling histochemistry.
      ,
      • Spinazzi M.
      • Casarin A.
      • Pertegato V.
      • Salviati L.
      • Angelini C.
      Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells.
      ). Although the human brain is too huge to search comprehensively, such studies become feasible with the rapid development of digital microscope slide scanners, brain clearing technology, machine learning for image analysis, and other technologies. We searched for regions with a higher level of multiple deletions in the brains of mutant Polg mice. The mouse brain slices were divided into small pieces of 200 × 200 μm2 by laser microdissection, and quantitative polymerase chain reaction was performed to specifically amplify mtDNA deletions on extracted DNA from each piece. We found that mtDNA deletions were accumulated most in the paraventricular nucleus of the thalamus (
      • Kasahara T.
      • Takata A.
      • Kato T.M.
      • Kubota-Sakashita M.
      • Sawada T.
      • Kakita A.
      • et al.
      Depression-like episodes in mice harboring mtDNA deletions in paraventricular thalamus.
      ). Activity staining of mitochondrial complexes revealed that there were a number of neurons deficient in the activity of complex IV (cytochrome c oxidase; COX), which includes three mtDNA-encoded subunits, but the neurons retained the activity of complex II, which consists of only nuclear genome–encoded subunits. We found a higher level of mtDNA deletions in the COX-negative neurons in the paraventricular nucleus of the thalamus (
      • Kasahara T.
      • Takata A.
      • Kato T.M.
      • Kubota-Sakashita M.
      • Sawada T.
      • Kakita A.
      • et al.
      Depression-like episodes in mice harboring mtDNA deletions in paraventricular thalamus.
      ), just like COX-deficient neurons in the SN of PD patients (
      • Bender A.
      • Krishnan K.J.
      • Morris C.M.
      • Taylor G.A.
      • Reeve A.K.
      • Perry R.H.
      • et al.
      High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease.
      ). In addition, we used anti-MTCO1 (a mtDNA-encoded subunit of COX) and anti-SDHA (a subunit of complex II) antibodies, performed immunofluorescent staining on fixed brain sections, and obtained a similar result: significantly increased number of COX-negative (MTCO1-negative, SDHA-positive) neurons in the paraventricular nucleus of the thalamus of mutant Polg mice than in control mice (
      • Kasahara T.
      • Takata A.
      • Kato T.M.
      • Kubota-Sakashita M.
      • Sawada T.
      • Kakita A.
      • et al.
      Depression-like episodes in mice harboring mtDNA deletions in paraventricular thalamus.
      ). Using the immunofluorescent staining method, we examined postmortem brains of two CPEO patients with comorbid mood symptoms, and both contained high levels of COX-negative cells in paraventricular thalamus compared with age-matched controls (
      • Kasahara T.
      • Takata A.
      • Kato T.M.
      • Kubota-Sakashita M.
      • Sawada T.
      • Kakita A.
      • et al.
      Depression-like episodes in mice harboring mtDNA deletions in paraventricular thalamus.
      ).
      However, not all patients with mtDNA mutations have mood disorders or other psychiatric diseases (
      • Fattal O.
      • Link J.
      • Quinn K.
      • Cohen B.H.
      • Franco K.
      Psychiatric comorbidity in 36 adults with mitochondrial cytopathies.
      ,
      • Koene S.
      • Kozicz T.L.
      • Rodenburg R.J.
      • Verhaak C.M.
      • de Vries M.C.
      • Wortmann S.
      • et al.
      Major depression in adolescent children consecutively diagnosed with mitochondrial disorder.
      ,
      • Smits B.W.
      • Fermont J.
      • Delnooz C.C.
      • Kalkman J.S.
      • Bleijenberg G.
      • van Engelen B.G.
      Disease impact in chronic progressive external ophthalmoplegia: More than meets the eye.
      ,
      • Anglin R.E.
      • Rosebush P.I.
      • Noseworthy M.D.
      • Tarnopolsky M.
      • Mazurek M.F.
      Psychiatric symptoms correlate with metabolic indices in the hippocampus and cingulate in patients with mitochondrial disorders.
      ,
      • Mancuso M.
      • Orsucci D.
      • Ienco E.C.
      • Pini E.
      • Choub A.
      • Siciliano G.
      Psychiatric involvement in adult patients with mitochondrial disease.
      ,
      • Inczedy-Farkas G.
      • Remenyi V.
      • Gal A.
      • Varga Z.
      • Balla P.
      • Udvardy-Meszaros A.
      • et al.
      Psychiatric symptoms of patients with primary mitochondrial DNA disorders.
      ). In addition, mtDNA mutations or mitochondrial dysfunction do not explain the etiology of all patients with mood disorders. We searched for variants in POLG gene in patients with “idiopathic” BD, who were not diagnosed with mitochondrial disease, and comprehensively investigated the deleterious effects of identified nonsynonymous variants (
      • Kasahara T.
      • Ishiwata M.
      • Kakiuchi C.
      • Fuke S.
      • Iwata N.
      • Ozaki N.
      • et al.
      Enrichment of deleterious variants of mitochondrial DNA polymerase gene (POLG1) in bipolar disorder.
      ). As a result, deleterious POLG variants were significantly enriched in patients; however, the frequency of carriers of the deleterious variants was low in patients with BD: variants predicted as deleterious, 2.4%; deleterious variants biochemically defined, 2.3%; and CPEO-related mutations, 0.38%.
      Genetic and clinical studies point out that idiopathic mood disorders are heterogeneous (
      American Psychiatric Association
      Diagnostic and Statistical Manual of Mental Disorders, 5th ed.
      ,
      • Nestler E.J.
      • Hyman S.E.
      Animal models of neuropsychiatric disorders.
      ,
      • Mühleisen T.W.
      • Leber M.
      • Schulze T.G.
      • Strohmaier J.
      • Degenhardt F.
      • Treutlein J.
      • et al.
      Genome-wide association study reveals two new risk loci for bipolar disorder.
      ,
      • Ikeda M.
      • Takahashi A.
      • Kamatani Y.
      • Okahisa Y.
      • Kunugi H.
      • Mori N.
      • et al.
      A genome-wide association study identifies two novel susceptibility loci and trans population polygenicity associated with bipolar disorder.
      ), in which causative genetic factors, endophenotypes, and psychiatric manifestations vary (but the core symptoms of mood disorders are present). This suggests that multiple brain regions, circuits, or neurons are affected in individual patients and also in the patient population. We are starting on the road to identify each of the affected sites as genetic risk factors have been emerging (
      • Mühleisen T.W.
      • Leber M.
      • Schulze T.G.
      • Strohmaier J.
      • Degenhardt F.
      • Treutlein J.
      • et al.
      Genome-wide association study reveals two new risk loci for bipolar disorder.
      ,
      • Ikeda M.
      • Takahashi A.
      • Kamatani Y.
      • Okahisa Y.
      • Kunugi H.
      • Mori N.
      • et al.
      A genome-wide association study identifies two novel susceptibility loci and trans population polygenicity associated with bipolar disorder.
      ,
      • Kataoka M.
      • Matoba N.
      • Sawada T.
      • Kazuno A.A.
      • Ishiwata M.
      • Fujii K.
      • et al.
      Exome sequencing for bipolar disorder points to roles of de novo loss-of-function and protein-altering mutations.
      ). In such a situation, to study mitochondrial disease patients and of animal models with mtDNA mutations will provide a powerful clue to identify the affected sites in brains of patients with idiopathic mood disorders because they undergo a variety of symptoms, including the core symptoms of mood disorders, and they have damaged neurons in the brain (
      • Hämäläinen R.H.
      • Manninen T.
      • Koivumäki H.
      • Kislin M.
      • Otonkoski T.
      • Suomalainen A.
      Tissue- and cell-type-specific manifestations of heteroplasmic mtDNA 3243A>G mutation in human induced pluripotent stem cell-derived disease model.
      ,
      • Kasahara T.
      • Takata A.
      • Kato T.M.
      • Kubota-Sakashita M.
      • Sawada T.
      • Kakita A.
      • et al.
      Depression-like episodes in mice harboring mtDNA deletions in paraventricular thalamus.
      ,
      • Carelli V.
      • Chan D.C.
      Mitochondrial DNA: Impacting central and peripheral nervous systems.
      ,
      • Lax N.Z.
      • Gorman G.S.
      • Turnbull D.M.
      Review: Central nervous system involvement in mitochondrial disease.
      ).

      Acknowledgments and Disclosures

      This work was supported by the Advanced Genome Research and Bioinformatics Study to Facilitate Medical Innovation from the Japan Agency for Medical Research and Development (Grant No. 16815678 to T. Kato) and MEXT Grants-in-Aid for Scientific Research (Grant No. 17H01573 to T. Kato).
      We thank Dr. Emilie Kristine Bagge for comments and careful reading of the manuscript.
      T. Kato received a grant from Takeda Pharmaceutical outside of this work. T. Kasahara reports no biomedical financial interests or potential conflicts of interest.

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