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Monoamine oxidase (MAO) is a key enzyme responsible for the degradation of neurotransmitters and trace amines. MAO has two subtypes (MAO-A and MAO-B) that are encoded by different genes. In the brain, MAO-B is highly expressed in the paraventricular thalamic nucleus (PVT); however, its substrate in PVT remains unclear. To identify the MAO-B substrate in PVT, we generated Maob knockout (KO) mice and measured five candidate substrates (i.e., noradrenaline, dopamine, 3-methoxytyramine, serotonin, and phenethylamine [PEA]) by liquid chromatography tandem mass spectrometry. We showed that only PEA levels were markedly elevated in the PVT of Maob KO mice. To exclude the influence of peripheral MAO-B deficiency, we developed brain-specific Maob KO mice, finding that PEA in the PVT was increased in brain-specific Maob KO mice, whereas the extent of PEA increase was less than that in global Maob KO mice. Given that plasma PEA levels were elevated in global KO mice, but not in brain–specific KO mice, and that PEA passes across the blood–brain barrier, the substantial accumulation of PEA in the PVT of Maob KO mice was likely due to the increase in plasma PEA. These data suggest that PEA is a substrate of MAO-B in the PVT as well as other tissues.

Bipolar disorder (BD) is a severe psychiatric disorder characterized by repeated conflicting manic and depressive states. In addition to genetic factors, complex gene–environment interactions, which alter the epigenetic status in the brain, contribute to the etiology and pathophysiology of BD. Here, we performed a promoter-wide DNA methylation analysis of neurons and nonneurons derived from the frontal cortices of mutant Polg1 transgenic (n = 6) and wild-type mice (n = 6). The mutant mice expressed a proofreading-deficient mitochondrial DNA (mtDNA) polymerase under the neuron-specific CamK2a promoter and showed BD-like behavioral abnormalities, such as activity changes and altered circadian rhythms. We identified a total of 469 differentially methylated regions (DMRs), consisting of 267 neuronal and 202 nonneuronal DMRs. Gene ontology analysis of DMR-associated genes showed that cell cycle-, cell division-, and inhibition of peptide activity-related genes were enriched in neurons, whereas synapse- and GABA-related genes were enriched in nonneurons. Among the DMR-associated genes, Trim2 and Lrpprc showed an inverse relationship between DNA methylation and gene expression status. In addition, we observed that mutant Polg1 transgenic mice shared several features of DNA methylation changes in postmortem brains of patients with BD, such as dominant hypomethylation changes in neurons, which include hypomethylation of the molecular motor gene and altered DNA methylation of synapse-related genes in nonneurons. Taken together, the DMRs identified in this study will contribute to understanding the pathophysiology of BD from an epigenetic perspective.

Cardiolipin (CL) is a hallmark phospholipid of mitochondria and plays a significant role in maintaining the mitochondrial structure and functions. Despite the physiological importance of CL, mutant organisms, yeast, Arabidopsis, C elegans, and Drosophila, which lack CL synthase (Crls1) gene and consequently are deprived of CL, are viable. Here we report conditional Crls1-deficient mice using targeted insertion of loxP sequences flanking the functional domain of CRLS1 enzyme. Homozygous null mutant mice exhibited early embryonic lethality at the peri-implantation stage. We generated neuron-specific Crls1 knockout (cKO) mice by crossing with Camk2α-Cre mice. Neuronal loss and gliosis were gradually manifested in the forebrains, where CL levels were significantly decreased. In the surviving neurons, malformed mitochondria with bubble-like or onion-like inner membrane structures were observed. We showed decreased supercomplex assembly and reduced enzymatic activities of electron transport chain complexes in the forebrain of cKO mice, resulting in affected mitochondrial calcium dynamics, a slower rate of Ca2+ uptake and a smaller calcium retention capacity. These observations clearly demonstrate indispensable roles of CL as well as of Crls1 gene in mammals.

Background Patients with bipolar disorder (BD) have increased plasma IL-6 levels, which are higher in depressed BD (dBD) than remitted BD (rBD). However, the mechanism that differentiates the cytokine levels between dBD and rBD is not understood. First, we determined whether brain-derived mtDNA can be detected in plasma using neuron-specific mutant Polg1 transgenic (Tg) mice. Second, we investigated whether the brain-derived plasma circulating cell-free mitochondrial DNA (ccf-mtDNA) differentiate the cytokine levels between dBD and rBD. Methods Mouse plasma ccf-mtDNA levels were measured using real-time PCR targeting two regions of the mtDNA (CO1 and D-loop) in Tg mice and non-Tg littermates. Human plasma ccf-mtDNA levels were measured using real-time PCR targeting two regions of the mtDNA (ND1 and ND4) and IL-6 levels were evaluated in 10 patients in different states (depressed and remitted) of BD in a longitudinal manner and 10 healthy controls. Results The mouse plasma CO1/D-loop ratio was significantly lower in Tg than non-Tg mice (P = 0.0029). Human plasma ccf-mtDNA copy number, ND4/ND1 ratio, and IL-6 levels were not significantly different between dBD and rBD. Human plasma ccf-mtDNA levels showed a nominal significant correlation with delusional symptoms (P = 0.033, ρ = 0.68). Limitations A larger sample size is required to generalize the results and to determine whether plasma ccf-mtDNA is associated with systemic inflammation. Conclusions Tg mice revealed that brain-derived mtDNA could be present in peripheral blood. The present findings did not coincide with our hypothesis that plasma ccf-mtDNA differentiates the cytokine levels between dBD and rBD.

The hormone melatonin is synthesized from serotonin by two enzymatic reactions (AANAT and ASMT/HIOMT) in the pineal gland following a circadian rhythm with low levels during the day and high levels at night. The robust nightly peak of melatonin secretion is an output signal of the circadian clock to the whole organism. However, so far the regulatory roles of endogenous melatonin in mammalian biological rhythms and physiology processes are poorly understood. Here, we establish congenic mouse lines (>N10 generations) that are proficient or deficient in melatonin synthesis (AH+/+ or AH-/- mice, respectively) on the C57BL/6J genetic background by crossing melatonin-proficient MSM/Ms with C57BL/6J. AH+/+ mice displayed robust nightly peak of melatonin secretion and had significantly higher levels of pineal and plasma melatonin vs AH-/- mice. Using this mice model, we investigated the role of endogenous melatonin in regulating multiple biological rhythms, physiological processes, and rhythmic behaviors. In the melatonin-proficient (AH+/+) mice, the rate of re-entrainment of wheel-running activity was accelerated following a 6-hour phase advance of dark onset when comparted with AH-/- mice, suggesting a role of endogenous melatonin in facilitating clock adjustment. Further in the AH+/+ mice, there was a significant decrease in body weight, gonadal weight and reproductive performance, and a significant increase in daily torpor (a hypothermic and hypometabolic state lasting only hours during adverse conditions). Endogenous melatonin, however, had no effect in the modulation of the diurnal rhythm of 2-[125 I]-iodomelatonin receptor expression in the SCN, free-running wheel behavior in constant darkness, life span, spontaneous homecage behaviors, and various types of social-emotional behaviors. The findings also shed light on the role of endogenous melatonin in mice domestication and provide new insights into melatonin's action in reducing energy expenditure during a food shortage. In summary, the congenic mice model generated in this study offers a significant advantage toward understanding of the role of endogenous melatonin in regulating melatonin receptor-mediated rhythm behaviors and physiological functions.

Bipolar disorder is a common mental illness occurring in approximately 1% of individuals and requires lifelong treatment. Although genetic factors are known to contribute to this disorder, the genetic architecture has not yet been completely clarified. Our initial trio-based exome sequencing study of bipolar disorder showed enrichment of de novo, loss-of-function (LOF) or protein-altering mutations in a combined group with bipolar I and schizoaffective disorders, and the identified de novo mutations were enriched in calcium-related genes. These findings suggested a role for de novo mutations in bipolar disorder. The validity of these statistical associations will be strengthened if the functional impact of the mutations on cellular function and behavior are identified. In this study, we focused on two de novo LOF mutations in calcium-related genes, EHD1 and MACF1, found in patients with bipolar disorder. We first showed that the EHD1 mutation resulted in a truncated protein with diminished effect on neurite outgrowth and inhibited endocytosis. Next, we used CRISPR/Cas9 to establish two knock-in mouse lines to model the in vivo effects of these mutations. We performed behavioral screening using IntelliCage and long-term wheel running analysis. Ehd1 mutant mice showed higher activity in the light phase. Macf1 mutant mice showed diminished attention and persistence to rewards. These behavioral alterations were similar to the phenotypes in previously proposed animal models of bipolar disorder. These findings endorse the possible role of de novo mutations as a component of the genetic architecture of bipolar disorder, which was suggested by the statistical evidence.

XBP1 is a transcription factor induced by unconventional splicing associated with endoplasmic reticulum stress and plays a role in development. Brain-derived neurotrophic factor (BDNF) causes splicing of Xbp1 mRNA in neurites, and Xbp1 is required for BDNF-induced neurite extension and branching. To search for the molecular mechanisms of how Xbp1 plays a role in neural development, comprehensive gene expression analysis was performed in primary telencephalic neurons obtained from Xbp1 knockout mice at embryonic day 12.5. By searching for the genes induced by BDNF in wild type neurons but not in Xbp1 knockout mice, we found that upregulation of three GABAergic markers, somatostatin (Sst), neuropeptide Y (Npy), and calbindin (Calb1), were compromised in Xbp1 knockout neurons. Attenuated upregulation of Npy and Calb1 in Xbp1 knockout neurons was confirmed by quantitative RT-PCR. This finding may be relevant to impaired BDNF-induced neurite extension in Xbp1 knockout neurons.

Wolfram disease is a rare genetic disorder frequently accompanying depression and psychosis. Non-symptomatic mutation carriers also have higher rates of depression and suicide. Because WfS1, the causative gene of Wolfram disease, is located at 4p16, a linkage locus for bipolar disorder, mutations of WfS1 were suggested to be involved in the pathophysiology of bipolar disorder. In this study, we performed behavioral and gene expression analyses of Wfs1 knockout mice to assess the validity as an animal model of mood disorder. In addition, the distribution of Wfs1 protein was examined in mouse brain. Wfs1 knockout mice did not show abnormalities in circadian rhythm and periodic fluctuation of wheel-running activity. Behavioral analysis showed that Wfs1 knockout mice had retardation in emotionally triggered behavior, decreased social interaction, and altered behavioral despair depending on experimental conditions. Wfs1-like immunoreactivity in mouse brain showed a similar distribution pattern to that in rats, including several nuclei potentially relevant to the symptoms of mood disorders. Gene expression analysis showed down-regulation of Cdc42ep5 and Rnd1, both of which are related to Rho GTPase, which plays a role in dendrite development. These findings may be relevant to the mood disorder observed in patients with Wolfram disease.

Animal models of human diseases should meet three sets of criteria: construct validity, face validity, and predictive validity. To date, several putative animal models of bipolar disorder have been reported. They are classified into various categories: pharmacological models, nutritional models, environmental models, and genetic models. None of them, however, totally fulfills the three validity criteria, and thus may not be useful for drug development. Mounting evidence suggests that mitochondrial dysfunction has a role in bipolar disorder. To test whether accumulation of mtDNA deletions in the brain can cause bipolar disorder, we generated transgenic mice with neuron-specific expression of mutant Polg (D181A). These mice showed altered diurnal activity rhythm and periodic activity change associated with the estrous cycle. These phenotypes were worsened by administration of a tricyclic antidepressant, but improved after lithium treatment. This mouse model of bipolar disorder potentially fulfills the three validity criteria, and therefore might be used for future drug development studies.

We previously reported that neuron-specific mutant Polg1 (mitochondrial DNA polymerase) transgenic (Tg) mice exhibited bipolar disorder (BD)-like phenotypes such as periodic activity change and altered circadian rhythm. In this study, we re-evaluated two datasets resulting from DNA microarray analysis to estimate a biological pathway associated with the disorder. The gene lists were derived from the comparison between post-mortem brains of BD patients and control subjects, and from the comparison between the brains of Tg and wild-type mice. Gene ontology analysis showed that 16 categories overlapped in the altered gene expression profiles of BD patients and the mouse model. In the brains of Tg mice, 33 genes showed similar changes in the frontal cortex and hippocampus compared to wild-type mice. Among the 33 genes, SFPQ and PPIF were differentially expressed in post-mortem brains of BD patients compared to control subjects. The only gene consistently down-regulated in both patients and the mouse model was PPIF, which encodes cyclophilin D (CypD), a component of the mitochondrial permeability transition pore. A blood-brain barrier-permeable CypD inhibitor significantly improved the abnormal behaviour of Tg mice at 40 mg/kg.d. These findings collectively suggest that CypD is a promising target for a new drug for BD.

Melatonin is a pineal hormone produced at night; however, many strains of laboratory mice are deficient in melatonin. Strangely enough, the gene encoding HIOMT enzyme (also known as ASMT) that catalyzes the last step of melatonin synthesis is still unidentified in the house mouse ( Mus musculus ) despite the completion of the genome sequence. Here we report the identification of the mouse Hiomt gene, which was mapped to the pseudoautosomal region (PAR) of sex chromosomes. The gene was highly polymorphic, and nonsynonymous SNPs were found in melatonin-deficient strains. In C57BL/6 strain, there are two mutations, both of which markedly reduce protein expression. Mutability of the Hiomt likely due to a high recombination rate in the PAR could be the genomic basis for the high prevalence of melatonin deficiency. To understand the physiologic basis, we examined a wild-derived strain, MSM/Ms, which produced melatonin more under a short-day condition than a long-day condition, accompanied by increased Hiomt expression. We generated F2 intercrosses between MSM/Ms and C57BL/6 strains and N2 backcrosses to investigate the role of melatonin productivity on the physiology of mice. Although there was no apparent effect of melatonin productivity on the circadian behaviors, testis development was significantly promoted in melatonin-deficient mice. Exogenous melatonin also had the antigonadal action in mice of a melatonin-deficient strain. These findings suggest a favorable impact of melatonin deficiency due to Hiomt mutations on domestic mice in breeding colonies.

We developed transgenic (Tg) mice modeling an autosomally inherited mitochondrial disease, chronic progressive external ophthalmoplegia, patients with which sometimes have comorbid mood disorders. The mutant animals exhibited bipolar disorder-like phenotypes, such as a distorted day-night rhythm and a robust activity change with a period of 4-5 days, and the behavioral abnormalities were improved by lithium. In this study, we tested the effect of electroconvulsive stimulation (ECS) on the behavioral abnormalities of the model. Electroconvulsive therapy, which has long been used in clinical practice, provides fast-acting relief to depressive patients and drug-resistant patients. We performed long-term recordings of wheel-running activity of Tg and non-Tg mice. While recording, we administrated a train of ECS to mice, six times over two weeks or three times over a week. The treatment ameliorated the distorted day-night rhythm within three times of ECS, but it had no effect on the activity change with a period of 4-5 days in the female mice. To study the mechanism of the action, we investigated whether ECS could alter the circadian phase but found no influence on the circadian clock system. The potent and fast-acting efficacy of ECS in the mutant mice supports the predictive validity of the mice as a model of bipolar disorder. This model will be useful in developing a safe and effective alternative to lithium or electroconvulsive therapy.

Accumulation of unfolded proteins in the endoplasmic reticulum initiates intracellular signaling termed the unfolded protein response (UPR). Although Xbp1 serves as a pivotal transcription factor for the UPR, the physiological role of UPR signaling and Xbp1 in the central nervous system remains to be elucidated. Here, we show that Xbp1 mRNA was highly expressed during neurodevelopment and activated Xbp1 protein was distributed throughout developing neurons, including neurites. The isolated neurite culture system and time-lapse imaging demonstrated that Xbp1 was activated in neurites in response to brain-derived neurotrophic factor (BDNF), followed by subsequent translocation of the active Xbp1 into the nucleus. BDNF-dependent neurite outgrowth was significantly attenuated in Xbp1(-/-) neurons. These findings suggest that BDNF initiates UPR signaling in neurites and that Xbp1, which is activated as part of the UPR, conveys the local information from neurites to the nucleus, contributing the neurite outgrowth.

Maintenance of mitochondrial DNA (mtDNA) depends on nuclear-encoded proteins such as mtDNA polymerase (POLG), whose mutations are involved in the diseases caused by mtDNA defects including mutation and deletion. The defects in mtDNA and in intracellular Ca2+([Ca2+]i) homeostasis have been reported in bipolar disorder (BD). To understand the relevance of the mtDNA defects to BD, we studied transgenic (Tg) mice in which mutant POLG (mutPOLG) was expressed specifically in neurons. mtDNA defects were accumulated in the brains of mutPOLG Tg mice in an age-dependent manner and the mutant mice showed BD-like behavior. However, the molecular and cellular basis for the abnormalities has not been clarified. In this study, we investigated Ca2+regulation by isolated mitochondria and [Ca2+]idynamics in the neurons of mutPOLG Tg mice. Mitochondria from the mutant mice sequestered Ca2+more rapidly, whereas Ca2+retention capacity and membrane potential, a driving force of Ca2+uptake, of mitochondria were unaffected. To elucidate the molecular mechanism of the altered Ca2+uptake, we performed DNA microarray analysis and found that the expression of cyclophilin D (CyP-D), a component of the permeability transition pore, was downregulated in the brains of mutPOLG Tg mice. Cyclosporin A, an inhibitor of CyP-D, mimicked the enhanced Ca2+uptake in mutant mice. Furthermore, G-protein-coupled receptor-mediated [Ca2+]iincrease was attenuated in hippocampal neurons of the mutant mice. These findings suggest that mtDNA defects lead to enhancement of Ca2+uptake rate via CyP-D downregulation and alter [Ca2+]idynamics, which may be involved in the pathogenesis of BD.

There is no established genetic model of bipolar disorder or major depression, which hampers research of these mood disorders. Although mood disorders are multifactorial diseases, they are sometimes manifested by one of pleiotropic effects of a single major gene defect. We focused on chronic progressive external ophthalmoplegia (CPEO), patients with which sometimes have comorbid mood disorders. Chronic progressive external ophthalmoplegia is a mitochondrial disease, which is accompanied by accumulation of mitochondrial DNA (mtDNA) deletions caused by mutations in nuclear-encoded genes such as POLG (mtDNA polymerase). We generated transgenic mice, in which mutant POLG was expressed in a neuron-specific manner. The mice showed forebrain-specific defects of mtDNA and had altered monoaminergic functions in the brain. The mutant mice exhibited characteristic behavioral phenotypes, a distorted day-night rhythm and a robust periodic activity pattern associated with estrous cycle. These abnormal behaviors resembling mood disorder were worsened by tricyclic antidepressant treatment and improved by lithium, a mood stabilizer. We also observed antidepressant-induced mania-like behavior and long-lasting irregularity of activity in some mutant animals. Our data suggest that accumulation of mtDNA defects in brain caused mood disorder-like mental symptoms with similar treatment responses to bipolar disorder. These findings are compatible with mitochondrial dysfunction hypothesis of bipolar disorder.

Animal models of mental disorders should ideally have construct, face, and predictive validity, but current animal models do not always satisfy these validity criteria. Additionally, animal models of depression rely mainly on stress-induced behavioral changes. These stress-induced models have limited validity, because stress is not a risk factor specific to depression, and the models do not recapitulate the recurrent and spontaneous nature of depressive episodes. Although animal models exhibiting recurrent depressive episodes or bipolar depression have not yet been established, several researchers are trying to generate such animals by modeling clinical risk factors as well as by manipulating a specific neural circuit using emerging techniques.

Mitochondria have their own DNA (mitochondrial DNA [mtDNA]). Although mtDNA copy number is dependent on tissues and its decrease is associated with various neuromuscular diseases, detailed distribution of mtDNA copies in the brain remains uncertain. Using real-time quantitative PCR assay, we examined regional variation in mtDNA copy number in 39 brain regions of male mice. A significant regional difference in mtDNA copy number was observed (P