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1.
Biochem Biophys Res Commun ; 691: 149253, 2024 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-38043196

RESUMO

Mitochondrial dysfunction is implicated in neuropsychiatric disorders. Inhibition of mitochondrial permeability transition pore (mPTP) and thereby enhancement of mitochondrial Ca2+ retention capacity (CRC) is a promising treatment strategy. Here, we screened 1718 compounds to search for drug candidates inhibiting mPTP by measuring their effects on CRC in mitochondria isolated from mouse brains. We identified seco-cycline D (SCD) as an active compound. SCD and its derivative were more potent than a known mPTP inhibitor, cyclosporine A (CsA). The mechanism of action of SCD was suggested likely to be different from CsA that acts on cyclophilin D. Repeated administration of SCD decreased ischemic area in a middle cerebral artery occlusion model in mice. These results suggest that SCD is a useful probe to explore mPTP function.


Assuntos
Proteínas de Transporte da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , Camundongos , Animais , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mitocôndrias/metabolismo , Ciclofilinas/metabolismo , Ciclosporina/farmacologia , Cálcio/farmacologia , Encéfalo/metabolismo
2.
FASEB J ; 34(1): 1465-1480, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914590

RESUMO

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.


Assuntos
Sinalização do Cálcio , Cardiolipinas/metabolismo , Embrião de Mamíferos/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Prosencéfalo/embriologia , Animais , Cálcio/metabolismo , Cardiolipinas/genética , Embrião de Mamíferos/patologia , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/patologia , Neurônios/patologia , Prosencéfalo/patologia , Transferases (Outros Grupos de Fosfato Substituídos)/deficiência , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
3.
BMC Biol ; 18(1): 150, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097039

RESUMO

BACKGROUND: The accumulation of mtDNA mutations in different tissues from various mouse models has been widely studied especially in the context of mtDNA mutation-driven ageing but has been confounded by the inherent limitations of the most widely used approaches. By implementing a method to sequence mtDNA without PCR amplification prior to library preparation, we map the full unbiased mtDNA mutation spectrum across six distinct brain regions from mice. RESULTS: We demonstrate that ageing-induced levels of mtDNA mutations (single nucleotide variants and deletions) reach stable levels at 50 weeks of age but can be further elevated specifically in the cortex, nucleus accumbens (NAc), and paraventricular thalamic nucleus (PVT) by expression of a proof-reading-deficient mitochondrial DNA polymerase, PolgD181A. The increase in single nucleotide variants increases the fraction of shared SNVs as well as their frequency, while characteristics of deletions remain largely unaffected. In addition, PolgD181A also induces an ageing-dependent accumulation of non-coding control-region multimers in NAc and PVT, a feature that appears almost non-existent in wild-type mice. CONCLUSIONS: Our data provide a novel view of the spatio-temporal accumulation of mtDNA mutations using very limited tissue input. The differential response of brain regions to a state of replication instability provides insight into a possible heterogenic mitochondrial landscape across the brain that may be involved in the ageing phenotype and mitochondria-associated disorders.


Assuntos
Encéfalo/metabolismo , Mapeamento Cromossômico , Replicação do DNA , DNA Mitocondrial/química , Mutação , Animais , DNA Mitocondrial/análise , Feminino , Camundongos
4.
Mol Psychiatry ; 23(10): 2039-2049, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29892051

RESUMO

Although mitochondrial and serotonergic dysfunctions have been implicated in the etiology of bipolar disorder (BD), the relationship between these unrelated pathways has not been elucidated. A family of BD and chronic progressive external ophthalmoplegia (CPEO) caused by a mutation of the mitochondrial adenine nucleotide translocator 1 (ANT1, SLC25A4) implicated that ANT1 mutations confer a risk of BD. Here, we sequenced ANT1 in 324 probands of NIMH bipolar disorder pedigrees and identified two BD patients carrying heterozygous loss-of-function mutations. Behavioral analysis of brain specific Ant1 heterozygous conditional knockout (cKO) mice using lntelliCage showed a selective diminution in delay discounting. Delay discounting is the choice of smaller but immediate reward than larger but delayed reward and an index of impulsivity. Diminution of delay discounting suggests an increase in serotonergic activity. This finding was replicated by a 5-choice serial reaction time test. An anatomical screen showed accumulation of COX (cytochrome c oxidase) negative cells in dorsal raphe. Dorsal raphe neurons in the heterozygous cKO showed hyperexcitability, along with enhanced serotonin turnover in the nucleus accumbens and upregulation of Maob in dorsal raphe. These findings altogether suggest that mitochondrial dysfunction as the genetic risk of BD may cause vulnerability to BD by altering serotonergic neurotransmission.


Assuntos
Translocador 1 do Nucleotídeo Adenina/genética , Translocador 1 do Nucleotídeo Adenina/metabolismo , Transtorno Bipolar/genética , Animais , Transtorno Bipolar/metabolismo , Desvalorização pelo Atraso/fisiologia , Núcleo Dorsal da Rafe/metabolismo , Feminino , Humanos , Comportamento Impulsivo , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Oftalmoplegia Externa Progressiva Crônica/metabolismo , Recompensa , Neurônios Serotoninérgicos/metabolismo , Neurônios Serotoninérgicos/fisiologia
6.
Neurosci Lett ; 814: 137467, 2023 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-37652351

RESUMO

Maternal immune activation is one of the environmental risk factors for offspring to develop psychiatric disorders. A synthetic viral mimetic immunogen, polyinosinic-polycytidylic acid (poly(I:C)), is used to induce maternal immune activation in animal models of psychiatric disorders. In the mouse poly(I:C) model, the existence of segment filamentous bacteria (SFB) in the maternal intestine has been reported to be important for the induction of ASD-related behavioral alterations as well as atypical cortical development called cortical patches. This study aimed to elucidate the effect of a single poly(I:C) injection during embryonic day (E) 9 to E16 on offspring's behavior in the ensured absence of maternal SFB by vancomycin drinking in C57BL/6N mice. The cortical patches were not found at either injection timings with poly(I:C) or PBS vehicle, tested in male or female offspring at postnatal day 0 or 1. Prepulse inhibition was decreased in male adult offspring most strongly at poly(I:C) injection timings later than E11, whereas a modest but significant decrease was observed in female offspring with an injection during E12 to E15. The decrease in social interaction was observed in female offspring most conspicuously at injection timings later than E11, whereas a significant decrease was observed in male offspring with an injection during E12 to E15. In conclusion, this study indicated that behavioral alterations could be induced without maternal SFB. The effect on behavior was substantially different between males and females.


Assuntos
Bactérias , Poli I-C , Humanos , Camundongos , Adulto , Animais , Feminino , Masculino , Camundongos Endogâmicos C57BL , Poli I-C/farmacologia , Modelos Animais de Doenças , Citoesqueleto
7.
Sci Transl Med ; 15(700): eabq7721, 2023 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-37315111

RESUMO

Intracranial aneurysms (IAs) are a high-risk factor for life-threatening subarachnoid hemorrhage. Their etiology, however, remains mostly unknown at present. We conducted screening for sporadic somatic mutations in 65 IA tissues (54 saccular and 11 fusiform aneurysms) and paired blood samples by whole-exome and targeted deep sequencing. We identified sporadic mutations in multiple signaling genes and examined their impact on downstream signaling pathways and gene expression in vitro and an arterial dilatation model in mice in vivo. We identified 16 genes that were mutated in at least one IA case and found that these mutations were highly prevalent (92%: 60 of 65 IAs) among all IA cases examined. In particular, mutations in six genes (PDGFRB, AHNAK, OBSCN, RBM10, CACNA1E, and OR5P3), many of which are linked to NF-κB signaling, were found in both fusiform and saccular IAs at a high prevalence (43% of all IA cases examined). We found that mutant PDGFRBs constitutively activated ERK and NF-κB signaling, enhanced cell motility, and induced inflammation-related gene expression in vitro. Spatial transcriptomics also detected similar changes in vessels from patients with IA. Furthermore, virus-mediated overexpression of a mutant PDGFRB induced a fusiform-like dilatation of the basilar artery in mice, which was blocked by systemic administration of the tyrosine kinase inhibitor sunitinib. Collectively, this study reveals a high prevalence of somatic mutations in NF-κB signaling pathway-related genes in both fusiform and saccular IAs and opens a new avenue of research for developing pharmacological interventions.


Assuntos
Aneurisma Intracraniano , NF-kappa B , Animais , Camundongos , Aneurisma Intracraniano/genética , Mutação/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais/genética , Humanos
8.
Biochim Biophys Acta ; 1807(3): 270-4, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21145305

RESUMO

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<4.8×10(-35)). High levels of mtDNA copies were found in the ventral tegmental area and substantia nigra, two major nuclei containing dopaminergic neurons. In contrast, cerebellar vermis and lobes had significantly lower copy numbers than other regions. Hippocampal dentate gyrus also had a relatively low mtDNA copy number. This study is the first quantitative analysis of regional variation in mtDNA copy number in mouse brain. Our findings are important for the physiological and pathophysiological studies of mtDNA in the brain.


Assuntos
Encéfalo/fisiologia , Variações do Número de Cópias de DNA/genética , DNA Mitocondrial/genética , Dosagem de Genes , Mitocôndrias/genética , Animais , Núcleo Celular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase
9.
Sci Rep ; 12(1): 17, 2022 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-34996979

RESUMO

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.


Assuntos
Núcleos da Linha Média do Tálamo/enzimologia , Monoaminoxidase/metabolismo , Fenetilaminas/metabolismo , Animais , Dopamina/análogos & derivados , Dopamina/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Núcleos da Linha Média do Tálamo/química , Núcleos da Linha Média do Tálamo/metabolismo , Monoaminoxidase/química , Monoaminoxidase/genética , Neurotransmissores/metabolismo , Norepinefrina/metabolismo , Serotonina/metabolismo , Especificidade por Substrato
10.
Mol Brain ; 15(1): 9, 2022 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-34991677

RESUMO

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.


Assuntos
Metilação de DNA , DNA Mitocondrial , Animais , Metilação de DNA/genética , Epigênese Genética , Lobo Frontal , Humanos , Camundongos , Camundongos Transgênicos , Neurônios
11.
Sci Rep ; 7(1): 13624, 2017 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-29051587

RESUMO

Retrotransposon long interspersed nuclear element-1 (LINE-1) occupies a large proportion of the mammalian genome, comprising approximately 100,000 genomic copies in mice. Epigenetic status of the 5' untranslated region (5'-UTR) of LINE-1 is critical for its promoter activity. DNA methylation levels in the 5'-UTR of human active LINE-1 subfamily can be measured by well-established methods, such as a pyrosequencing-based assay. However, because of the considerable sequence and structural diversity in LINE-1 among species, methods for such assays should be adapted for the species of interest. Here we developed pyrosequencing-based assays to examine methylcytosine (mC) and hydroxymethylcytosine (hmC) levels of the three active LINE-1 subfamilies in mice (TfI, A, and GfII). Using these assays, we quantified mC and hmC levels in four brain regions and four nonbrain tissues including tail, heart, testis, and ovary. We observed tissue- and subfamily-specific mC and hmC differences. We also found that mC levels were strongly correlated among different brain regions, but mC levels of the testis showed a poor correlation with those of other tissues. Interestingly, mC levels in the A and GfII subfamilies were highly correlated, possibly reflecting their close evolutionary relationship. Our assays will be useful for exploring the epigenetic regulation of the active LINE-1 subfamilies in mice.


Assuntos
Metilação de DNA , Elementos Nucleotídeos Longos e Dispersos/genética , Regiões 5' não Traduzidas , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/análise , Animais , Sequência de Bases , Encéfalo/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Ovário/metabolismo , Análise de Sequência de DNA , Testículo/metabolismo
12.
Mol Brain ; 7: 5, 2014 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-24443933

RESUMO

BACKGROUND: Pre-mRNAs of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)-propanoic acid (AMPA)/kainate glutamate receptors undergo post-transcriptional modification known as RNA editing that is mediated by adenosine deaminase acting on RNA type 2 (ADAR2). This modification alters the amino acid sequence and function of the receptor. Glutamatergic signaling has been suggested to have a role in mood disorders and schizophrenia, but it is unknown whether altered RNA editing of AMPA/kainate receptors has pathophysiological significance in these mental disorders. In this study, we found that ADAR2 expression tended to be decreased in the postmortem brains of patients with schizophrenia and bipolar disorder. RESULTS: Decreased ADAR2 expression was significantly correlated with decreased editing of the R/G sites of AMPA receptors. In heterozygous Adar2 knockout mice (Adar2+/- mice), editing of the R/G sites of AMPA receptors was decreased. Adar2+/- mice showed a tendency of increased activity in the open-field test and a tendency of resistance to immobility in the forced swimming test. They also showed enhanced amphetamine-induced hyperactivity. There was no significant difference in amphetamine-induced hyperactivity between Adar2+/- and wild type mice after the treatment with an AMPA/kainate receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline. CONCLUSIONS: These findings collectively suggest that altered RNA editing efficiency of AMPA receptors due to down-regulation of ADAR2 has a possible role in the pathophysiology of mental disorders.


Assuntos
Adenosina Desaminase/metabolismo , Transtornos do Humor/enzimologia , Transtornos do Humor/genética , Edição de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores de Glutamato/genética , Esquizofrenia/genética , Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Mudanças Depois da Morte , Quinoxalinas/farmacologia , Edição de RNA/efeitos dos fármacos , Receptores de AMPA/metabolismo , Receptores de Glutamato/metabolismo , Esquizofrenia/enzimologia
13.
Ann Clin Transl Neurol ; 1(11): 909-20, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25540805

RESUMO

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 Polg (D257A) knock-in mice carrying a proofreading-deficient mutation in the Polg. RESULTS: Heterozygous Polg (D257A) 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 Polg (D257A) 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.

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