Search

The pseudoautosomal region (PAR) of mammalian sex chromosomes is a small region of sequence identity that allows pairing, crossover, recombination, and proper chromosome segregation during male meiosis. The structure of the mouse PAR is largely unknown. Here, we developed a new assembly method to robustly resolve repetitive sequences and employed highly accurate long-read sequencing data to reveal the entire PAR sequence. The PAR of the widely-used inbred strain C57BL/6J is ∼700 kb, comprising 10 protein- coding genes in a mass of complex repetitive sequences. A large segmental duplication exhibiting copy-number polymorphisms even among C57BL/6J littermates is present. High GC-content exons and short introns are common properties of PAR genes and are the consequence of maintaining gene function, while PAR is rapidly evolving. Elucidating the mouse PAR sequence completes the mouse euchromatic genome sequencing and enables the exploration of the function and evolution of the PAR using modern molecular genetic approaches.

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.

Additional file 2: Supplementary tables 1-3. Table S1: Table of primers used for qPCR and RT-qPCR to assess the levels of mtDNA, nDNA and mRNA targets. Table S2: Table of primers used for PCR to assess structural variants of mtDNA. Table S3: Table of primers used for qPCR to assess the expression of endogenous and transgenic PolgD181A.

Additional file 1: Supplementary figures 1-5. Fig. S1: Additional analysis of mtDNA single nucleotide variants. Fig. S2: Additional analysis of mtDNA deletions. Fig. S3: Additional analysis of mtDNA multimers. Fig. S4: Additional analysis of mtDNA multimers and inversions. Fig. S5: Polg expression analysis.

Background Although inflammatory cytokines are established biomarkers of mood disorders, their molecular mechanism is not known. We hypothesized that circulating mitochondrial DNA (mtDNA) contributes to inflammation and could be used as biomarkers. We investigated if circulating mtDNA level is associated with inflammatory cytokines and can be used as a biomarker of mood disorders. Methods Plasma mtDNA concentration was measured with real-time quantitative PCR targeting two regions of the mtDNA and plasma levels of four cytokines (GM-CSF, IL-2, IL-4, and IL-6) were measured with a multiplex immunoassay method in 109 patients with major depressive disorder (MDD). The most significantly correlated cytokine was verified with an enzyme-linked immunosorbent assay (ELISA). The data from 28 patients with bipolar disorder (BD), 17 patients with schizophrenia (SZ), and 29 healthy controls were compared. Results MtDNA levels showed a nominal positive correlation with GM-CSF, IL-2 and IL-4 in patients with MDD. The most significant correlation with IL-4 ( ρ = 0.38, P ρ = 0.19, P = 0.049). Unexpectedly, patients with MDD and BD showed significantly lower plasma mtDNA levels than controls. MtDNA levels were lower in the depressive state than in the euthymic state in patients with MDD. Patients with depression, bipolar disorder, and schizophrenia did not show significantly higher levels of these four cytokines than controls. Limitations There is a possibility that the patients in this study are different from previous studies in which increased cytokine levels were reported. MtDNA levels should be measured in patients showing elevated plasma cytokine levels. A larger sample is required to generalize the results. Conclusions The present findings coincide with our hypothesis that circulating mtDNA contributes to the inflammation in MDD. Further studies are needed to conclude whether plasma mtDNA would be a biomarker of mood disorders.

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 (N1000), 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.

Aim Rare missense variants, which likely account for a substantial portion of the genetic ‘dark matter’ for a common complex disease, are challenging because the impacts of variants on disease development are difficult to substantiate. This study aimed to examine the impacts of amino acid substitution variants in the POLG1 found in bipolar disorder, as an example and proof of concept, in three different modalities of assessment: in silico predictions, in vitro biochemical assays, and clinical evaluation. We then tested whether deleterious variants in POLG1 contributed to the genetics of bipolar disorder. Methods We searched for variants in the POLG1 gene in 796 Japanese patients with bipolar disorder and 767 controls and comprehensively investigated all 23 identified variants in the three modalities of assessment. POLG1 encodes mitochondrial DNA polymerase and is one of the causative genes for a Mendelian-inheritance mitochondrial disease, which is occasionally accompanied by mood disorders. The healthy control data from the Tohoku Medical Megabank Organization were also employed. Results Although the frequency of carriers of deleterious variants varied from one method to another, every assessment achieved the same conclusion that deleterious POLG1 variants were significantly enriched in the variants identified in patients with bipolar disorder compared to those in controls. Conclusion Together with mitochondrial dysfunction in bipolar disorder, the present results suggested deleterious POLG1 variants as a credible risk for the multifactorial disease.

Depression is a common debilitating human disease whose etiology has defied decades of research. A critical bottleneck is the difficulty in modeling depressive episodes in animals. Here, we show that a transgenic mouse with chronic forebrain expression of a dominant negative mutant of Polg1, a mitochondrial DNA (mtDNA) polymerase, exhibits lethargic behavioral changes, which are associated with emotional, vegetative and psychomotor disturbances, and response to antidepression drug treatment. The results suggested a symptomatic similarity between the lethargic behavioral change that was recurrently and spontaneously experienced by the mutant mice and major depressive episode as defined by DSM-5. A comprehensive screen of mutant brain revealed a hotspot for mtDNA deletions and mitochondrial dysfunction in the paraventricular thalamic nucleus (PVT) with similar defects observed in postmortem brains of patients with mitochondrial disease with mood symptoms. Remarkably, the genetic inhibition of PVT synaptic output by Cre-loxP-dependent expression of tetanus toxin triggered de novo depression-like episodes. These findings identify a novel preclinical mouse model and brain area for major depressive episodes with mitochondrial dysfunction as its cellular mechanism.

Objective Mutations in nuclear-encoded mitochondrial DNA (mtDNA) polymerase (POLG) are known to cause autosomal dominant chronic progressive external ophthalmoplegia (adCPEO) with accumulation of multiple mtDNA deletions in muscles. However, no animal model with a heterozygous Polg mutation representing mtDNA impairment and symptoms of CPEO has been established. To understand the pathogenic mechanism of CPEO, it is important to determine the age dependency and tissue specificity of mtDNA impairment resulting from a heterozygous mutation in the Polg gene in an animal model. Methods We assessed behavioral phenotypes, tissue-specific accumulation of mtDNA deletions, and its age dependency in heterozygous PolgD257A knock-in mice carrying a proofreading-deficient mutation in the Polg. Results Heterozygous PolgD257A knock-in mice exhibited motor dysfunction in a rotarod test. Polg+/D257A mice had significant accumulation of multiple mtDNA deletions, but did not show significant accumulation of point mutations or mtDNA depletion in the brain. While mtDNA deletions increased in an age-dependent manner regardless of the tissue even in Polg+/+ mice, the age-dependent accumulation of mtDNA deletions was enhanced in muscles and in the brain of Polg+/D257A mice. Interpretation Heterozygous PolgD257A knock-in mice showed tissue-specific, age-dependent accumulation of multiple mtDNA deletions in muscles and the brain which was likely to result in neuromuscular symptoms. Polg+/D257A mice may be used as an animal model of adCPEO associated with impaired mtDNA maintenance.

Background Diagnostic biomarkers of major depressive disorder, bipolar disorder, and schizophrenia are urgently needed, because none are currently available. Methods We performed a comprehensive metabolome analysis of plasma samples from drug-free patients with major depressive disorder (n=9), bipolar disorder (n=6), schizophrenia (n=17), and matched healthy controls (n=19) (cohort 1) using liquid chromatography time-of-flight mass spectrometry. A significant effect of diagnosis was found for 2 metabolites: nervonic acid and cortisone, with nervonic acid being the most significantly altered. The reproducibility of the results and effects of psychotropic medication on nervonic acid were verified in cohort 2, an independent sample set of medicated patients [major depressive disorder (n=45), bipolar disorder (n=71), schizophrenia (n=115)], and controls (n=90) using gas chromatography time-of-flight mass spectrometry. Results The increased levels of nervonic acid in patients with major depressive disorder compared with controls and patients with bipolar disorder in cohort 1 were replicated in the independent sample set (cohort 2). In cohort 2, plasma nervonic acid levels were also increased in the patients with major depressive disorder compared with the patients with schizophrenia. In cohort 2, nervonic acid levels were increased in the depressive state in patients with major depressive disorder compared with the levels in the remission state in patients with major depressive disorder and the depressive state in patients with bipolar disorder. Conclusion These results suggested that plasma nervonic acid is a good candidate biomarker for the depressive state of major depressive disorder.

There is an urgent need for diagnostic biomarkers of bipolar disorder (BD) and schizophrenia (SZ); however, confounding effects of medication hamper biomarker discovery. In this study, we conducted metabolome analyses to identify novel plasma biomarkers in drug-free patients with BD and SZ.We comprehensively analyzed plasma metabolites using capillary electrophoresis time-of-flight mass spectrometry in patients with SZ (n = 17), BD (n = 6), and major depressive disorder (n = 9) who had not received psychotropics for at least 2 weeks, and in matched healthy controls (n = 19). The results were compared with previous reports, or verified in an independent sample set using an alternative analytical approach.Lower creatine level and higher 2-hydroxybutyric acid level were observed in SZ than in controls (uncorrected P = 0.016 and 0.043, respectively), whereas they were unaltered in a previously reported dataset. Citrulline was nominally significantly decreased in BD compared to controls (uncorrected P = 0.043); however, this finding was not replicated in an independent sample set of medicated patients with BD. N-methyl-norsalsolinol, a metabolite of dopamine, was suggested as a candidate biomarker of BD; however, it was not detected by the other analytical method. Levels of betaine, a previously reported candidate biomarker of schizophrenia, were unchanged in the current dataset.Our preliminary findings suggest that the effect of confounding factors, such as duration of illness and medication, should be carefully controlled when searching for plasma biomarkers. Further studies are required to establish robust biomarkers for these disorders.

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.

The chicken pineal gland is a photosensitive neuroendocrine organ producing melatonin in circadian clock-regulated and light-sensitive manners. To understand the relationship between the photoreceptive molecule pinopsin and the light-dependent melatonin suppression that is sensitive to pertussis toxin treatment, we have searched for pertussis toxin-sensitive G protein alpha-subunits expressed in the chicken pineal gland. Here we report the cDNA cloning of the pineal transducin alpha-subunit (Gtalpha), which is highly homologous to human retinal rod cell-specific Gt(1)alpha. Concurrent cDNA cloning of chicken retinal Gt(1)alpha and Gt(2)alpha (rod and cone cell-specific alpha-subunits of transducin, respectively) revealed that the chicken pineal Gtalpha is identical to the retinal Gt(1)alpha. Double-immunostaining analysis of the chicken pineal sections localized Gt(1)alpha-immunoreactivity in the rudimentary outer segments of both follicular and parafollicular pinealocytes that were immunopositive to anti-pinopsin antibody. To examine whether pineal Gt(1)alpha is involved in the pineal phototransduction pathway, trypsin protection assay was applied for detecting the conversion of GDP-bound Gt(1)alpha into the guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-bound form in the pineal membrane homogenate. It was clearly demonstrated that the pineal Gt(1)alpha is activated in a light-dependent manner in the presence of GTPgammaS. These data together suggest strongly that pineal Gt(1)alpha mediates the phototransduction pathway triggered by pinopsin in the chicken pinealocytes.

Melatonin is secreted from the pineal gland in a circadian manner. It is well established that the synthesis of melatonin shows a diurnal rhythm reflecting a daily change in serotonin N-acetyltransferase (NAT) activity, and the overall secretion of melatonin requires a cellular release process, which is poorly understood. To investigate the possible involvement of Golgi-derived vesicles in the release, we examined the effect of brefeldin A (BFA), a reversible inhibitor of Golgi-mediated secretion, on melatonin secretion of cultured chick pineal cells. We show here that treatment with BFA completely disassembles the Golgi apparatus and reduces melatonin secretion. In more detailed time course experiments, however, the inhibition of melatonin secretion is only observed after the removal of BFA in parallel with the reassembly of the Golgi apparatus. This inhibition of melatonin secretion is not accompanied by accumulation of melatonin in the cells. These observations indicate that chick pineal melatonin is released independently of the Golgi-derived vesicles, and suggest inhibition of melatonin synthesis after the removal of BFA. By measuring the activities and mRNA levels of melatonin-synthesizing enzymes, we found that the removal of BFA specifically inhibits NAT activity at the protein level. On the other hand, BFA causes no detectable phase-shift of the chick pineal oscillator regulating the circadian rhythm of melatonin secretion. The results presented here suggest that the Golgi-mediated vesicular transport is involved in neither the melatonin release nor the time-keeping mechanism of the circadian oscillator, but rather contributes to the regulation of NAT activity.

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.

The pathophysiology of bipolar disorder is still unclear, although family, twin and linkage studies implicate genetic factors. Here we identified XBP1, a pivotal gene in the endoplasmic reticulum (ER) stress response, as contributing to the genetic risk factor for bipolar disorder. Using DNA microarray analysis of lymphoblastoid cells derived from two pairs of twins discordant with respect to the illness, we found downregulated expression of genes related to ER stress response in both affected twins. A polymorphism (-116C-->G) in the promoter region of XBP1, affecting the putative binding site of XBP1, was significantly more common in Japanese patients (odds ratio = 4.6) and overtransmitted to affected offspring in trio samples of the NIMH Bipolar Disorder Genetics Initiative. XBP1-dependent transcription activity of the -116G allele was lower than that of the -116C allele, and in the cells with the G allele, induction of XBP1 expression after ER stress was markedly reduced. Valproate, one of three mood stabilizers, rescued the impaired response by inducing ATF6, the gene upstream of XBP1. These results indicate that the -116C-->G polymorphism in XBP1 causes an impairment of its positive feedback system and increases the risk of bipolar disorder.

Light is a major environmental signal for entrainment of the circadian clock, but little is known about the intracellular phototransduction pathway triggered by light activation of the photoreceptive molecule(s) responsible for the phase shift of the clock in vertebrates. The chicken pineal gland and retina contain the autonomous circadian oscillators together with the photic entrainment pathway, and hence they represent useful experimental models for the clock system. Here we show the expression of G11alpha, an alpha subunit of heterotrimeric G-protein, in both tissues by cDNA cloning, Northern blot, and Western blot analyses. G11alpha immunoreactivity was colocalized with pinopsin in the chicken pineal cells and also with rhodopsin in the outer segments of retinal photoreceptor cells, suggesting functional coupling of G11alpha with opsins in the clock-containing photosensitive tissues. The physical interaction was examined by coimmunoprecipitation experiments, the results of which provided evidence for light- and GTP-dependent coupling between rhodopsin and G11alpha. To examine whether activation of endogenous G11 leads to a phase shift of the oscillator, Gq/11-coupled m1-type muscarinic acetylcholine receptor (mAChR) was ectopically expressed in the cultured pineal cells. Subsequent treatment of the cells with carbamylcholine (CCh), an agonist of mAChR, induced phase-dependent phase shifts of the melatonin rhythm in a manner very similar to the effect of light. In contrast, CCh treatment induced no measurable effect on the rhythm of nontransfected (control) cells or cells expressing G(i/o)-coupled m2-type mAChR, indicating selectivity of the G-protein activation. Together, our results demonstrate the existence of a G11-mediated opsin-signaling pathway contributing to the photic entrainment of the circadian clock.

Pinopsin is a photoreceptive molecule present in the outer segments of chicken pinealocytes. In this paper, the localization of alpha-subunits of G-proteins, rod transducin (Gt1) and Gq/11, was examined by immunoelectron microscopy to investigate whether these G-proteins colocalize with pinopsin in the outer segments. Ultrathin sections of the chicken pineal gland were double-immunolabeled with antibodies to pinopsin and either Gt1alpha or Gq/11alpha. As shown previously, the outer segments around the follicular lumen exhibited divergent morphology with ciliary, bulbous, or lamellate shapes, and most of them displayed pinopsin immunoreactivity. The majority (90%) of pinopsin-immunopositive outer segments were labeled by anti-Gt1alpha and/or anti-Gq/11alpha antibodies. Application of double-immunolabeling to serial sections demonstrated that a large number of the pinopsin-immunopositive outer segments contained both Gt1alpha and Gq/11alpha immunoreactivities. These results suggest that Gt1alpha and Gq/11alpha are functionally coupled with light-activated pinopsin within a single outer segment.

The avian pinealocytes have an intrinsic circadian clock function that controls rhythmic synthesis of melatonin, and an environmental light signal can reset the phase of the clock. In addition to the photoendocrine function, the melatonin synthesis of the pinealocytes is regulated by neural signals from sympathetic nerves. Thus the avian pinealocytes show diagnostic characters which seem to represent an evolutionary transition from photosensory cells of lower vertebrates to the neuroendocrinal cells of mammals. To understand the evolutionary background of the regulatory mechanism for the melatonin synthesis in this organ, we screened the chicken pineal cDNA library to find alpha-subunits of heterotrimeric G-proteins involved in the photic and neural regulations. In addition to the transducin-like alpha-subunit (Gt alpha) supposed to mediate the photic pathway, we isolated cDNA clones encoding Gi2 alpha, Gi3 alpha, and Go1 alpha and its splicing variant Go2 alpha. The deduced amino acid sequence of each G alpha had a potential site for pertussis toxin-catalyzed ADP-ribosylation. As it is known that adrenergic receptor-mediated inhibition of melatonin synthesis is blocked by pertussis toxin, the G-proteins identified in the present study are likely to contribute to this neuroendocrine function of the chicken pineal cells.

Kasahara and Kato reply - Our announcement1 that the redox cofactor pyrroloquinoline quinone (PQQ) is a new vitamin has been challenged as being premature by Felton and Anthony 2 and by Rucker .3.

Impaired endoplasmic reticulum (ER) stress response has been suggested as a possible pathophysiological mechanism of bipolar disorder (BD). The expression of ER stress-related genes, spliced form or unspliced form of XBP1, GRP78 (HSPA5), GRP94 (HSP90B1), CHOP (DDIT3), and calreticulin (CALR), were examined in lymphoblastoid cells derived from 59 patients with BD and 59 age- and sex-matched control subjects. Basal mRNA levels and induction by 4 h or 12 h of treatment with two ER stressors, thapsigargin or tunicamycin, were examined using real-time quantitative reverse transcription-polymerase chain reaction. Induction of the spliced form of XBP1 as well as total XBP1 by thapsigargin was significantly attenuated in patients with BD. Induction of GRP94 by thapsigargin was also decreased in the BD group. A haplotype of GRP94, protective against BD, exhibited significantly higher GRP94 expression upon ER stress. This report confirms and extends earlier observations of impaired ER stress response in larger samples of lymphoblastoid cell lines derived from BD patients. Altered ER stress response may play a role in the pathophysiology of BD by altering neural development and plasticity.

Genetically modified mice harboring mitochondrial DNA defects show aberrant cyclic change in wheel-running activity, which is improved by lithium

Nicotinamides and flavins are essential cofactors in enzyme-catalysed reduction–oxidation (redox) reactions and are classified as vitamins because they must be supplied in the diet. Another redox cofactor, pyrroloquinoline quinone (PQQ), was first discovered in bacteria1 and is also likely to be important in mammals2,3, but the biochemical pathways in which it participates are unknown. Here we identify a PQQ-dependent dehydrogenase enzyme that is crucial for the degradation of the amino acid lysine in mice. PQQ is acting as a mammalian redox cofactor in this reaction, and therefore qualifies as a newcomer to the B group of vitamins.