RESUMO
Myotonic dystrophy type 1 (DM1) is a complex neuromuscular disorder caused by expansion of a CTG repeat in the 3'-untranslated region (UTR) of the DMPK gene. Mutant DMPK transcripts form aberrant structures and anomalously associate with RNA-binding proteins (RBPs). As a first step toward better understanding of the involvement of abnormal DMPK mRNA folding in DM1 manifestation, we used SHAPE, DMS, CMCT, and RNase T1 structure probing in vitro for modeling of the topology of the DMPK 3'-UTR with normal and pathogenic repeat lengths of up to 197 CUG triplets. The resulting structural information was validated by disruption of base-pairing with LNA antisense oligonucleotides (AONs) and used for prediction of therapeutic AON accessibility and verification of DMPK knockdown efficacy in cells. Our model for DMPK RNA structure demonstrates that the hairpin formed by the CUG repeat has length-dependent conformational plasticity, with a structure that is guided by and embedded in an otherwise rigid architecture of flanking regions in the DMPK 3'-UTR. Evidence is provided that long CUG repeats may form not only single asymmetrical hairpins but also exist as branched structures. These newly identified structures have implications for DM1 pathogenic mechanisms, like sequestration of RBPs and repeat-associated non-AUG (RAN) translation.
Assuntos
Regiões 3' não Traduzidas , Sequências Repetidas Invertidas , Miotonina Proteína Quinase/genética , Repetições de Trinucleotídeos , Pareamento de Bases , Expressão Gênica , Humanos , Modelos Genéticos , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Distrofia Miotônica/patologia , Miotonina Proteína Quinase/metabolismo , Conformação de Ácido Nucleico , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Study motivation and knowledge retention benefit from regular student self-assessments. Inclusion of certainty-based learning (CBL) in computer-assisted formative tests may further enhance this by enabling students to identify whether they are uninformed or misinformed regarding the topics tested, which may trigger future study actions including instructor consultation. METHODS: Using a cross-over study design involving two out of thirteen computer-assisted formative assessments (CAFAs) of a first-year cell biology course, we compared student-instructor interactions, student learning experiences and final exam scores between two (bio)medical science student cohorts who worked with different CBL-containing CAFAs. RESULTS: A total of 389 students participated in the study. After completion 159 (41%) filled in a questionnaire on their experience with CBL during supervised CAFAs. In the control group the median duration of student-instructor interactions was 90 s (range 60-140 s), and this increased with 20 s to 110 s (range 60-150 s) in the group working with a CBL-based CAFA. The number of interactions was similar in both groups (0.22 per student per hour, regardless of CBL inclusion). Forty percent of the students expected that CBL would positively influence their study behavior, and 23% also anticipated a positive effect on examination scores. Student examination scores, however, were not affected by CBL. Almost half of the students (43%) were in favor of CBL inclusion in future computer-assisted learning modules, whereas 33% did not see merit in including CBL in CAFAs. CONCLUSIONS: Incorporation of CBL in a single formative assessment led to a slight increase in student-instructor interaction times, but had effect neither on the number of student-instructor interactions nor on exam scores. CBL inclusion positively influenced student's appreciation of the coursework, presumably by helping students to evaluate their mastery level and identify misconceptions. A more extensive enrollment of CBL beyond an individual formative assessment, throughout a course or a curriculum, may possibly reveal positive effects on study efficacy.
Assuntos
Avaliação Educacional/métodos , Estudantes de Medicina/psicologia , Adolescente , Instrução por Computador/métodos , Estudos Cross-Over , Feminino , Feedback Formativo , Humanos , Masculino , Adulto JovemRESUMO
The congenital form of myotonic dystrophy type 1 (cDM) is caused by the large-scale expansion of a (CTGâ¢CAG)n repeat in DMPK and DM1-AS. The production of toxic transcripts with long trinucleotide tracts from these genes results in impairment of the myogenic differentiation capacity as cDM's most prominent morpho-phenotypic hallmark. In the current in vitro study, we compared the early differentiation programs of isogenic cDM myoblasts with and without a (CTG)2600 repeat obtained by gene editing. We found that excision of the repeat restored the ability of cDM myoblasts to engage in myogenic fusion, preventing the ensuing myotubes from remaining immature. Although the cDM-typical epigenetic status of the DM1 locus and the expression of genes therein were not altered upon removal of the repeat, analyses at the transcriptome and proteome level revealed that early abnormalities in the temporal expression of differentiation regulators, myogenic progression markers, and alternative splicing patterns before and immediately after the onset of differentiation became normalized. Our observation that molecular and cellular features of cDM are reversible in vitro and can be corrected by repeat-directed genome editing in muscle progenitors, when already committed and poised for myogenic differentiation, is important information for the future development of gene therapy for different forms of myotonic dystrophy type 1 (DM1).
Assuntos
Mioblastos/patologia , Distrofia Miotônica/genética , Repetições de Trinucleotídeos , Linhagem Celular , Epigênese Genética , Edição de Genes , Terapia Genética , Humanos , Desenvolvimento Muscular , Mioblastos/citologia , Mioblastos/metabolismo , Distrofia Miotônica/patologia , Distrofia Miotônica/terapia , Miotonina Proteína Quinase/genéticaRESUMO
Muscular manifestation of myotonic dystrophy type 1 (DM1), a common inheritable degenerative multisystem disorder, is mainly caused by expression of RNA from a (CTG·CAG)n-expanded DM1 locus. Here, we report on comparative profiling of expression of normal and expanded endogenous or transgenic transcripts in skeletal muscle cells and biopsies from DM1 mouse models and patients in order to help us in understanding the role of this RNA-mediated toxicity. In tissue of HSA(LR) mice, the most intensely used 'muscle-only' model in the DM1 field, RNA from the α-actin (CTG)250 transgene was at least 1000-fold more abundant than that from the Dmpk gene, or the DMPK gene in humans. Conversely, the DMPK transgene in another line, DM500/DMSXL mice, was expressed â¼10-fold lower than the endogenous gene. Temporal regulation of expanded RNA expression differed between models. Onset of expression occurred remarkably late in HSA(LR) myoblasts during in vitro myogenesis whereas Dmpk or DMPK (trans)genes were expressed throughout proliferation and differentiation phases. Importantly, quantification of absolute transcript numbers revealed that normal and expanded Dmpk/DMPK transcripts in mouse models and DM1 patients are low-abundance RNA species. Northern blotting, reverse transcriptase-quantitative polymerase chain reaction, RNA-sequencing and fluorescent in situ hybridization analyses showed that they occur at an absolute number between one and a few dozen molecules per cell. Our findings refine the current RNA dominance theory for DM1 pathophysiology, as anomalous factor binding to expanded transcripts and formation of soluble or insoluble ribonucleoprotein aggregates must be nucleated by only few expanded DMPK transcripts and therefore be a small numbers game.
Assuntos
Perfilação da Expressão Gênica/métodos , Músculo Esquelético/citologia , Distrofia Miotônica/genética , RNA Mensageiro/genética , Expansão das Repetições de Trinucleotídeos , Actinas/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Desenvolvimento Muscular , Músculo Esquelético/patologia , Distrofia Miotônica/patologia , Miotonina Proteína Quinase/genética , Miotonina Proteína Quinase/metabolismoRESUMO
Myotonic dystrophy type 1 (DM1) is caused by (CTGâ CAG)n-repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5' or 3' unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPK alleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTGâ CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPK gene were normalized. Dual sgRNA-guided excision of the (CTGâ CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Instabilidade Genômica , Distrofia Miotônica/genética , Miotonina Proteína Quinase/genética , Expansão das Repetições de Trinucleotídeos , Repetições de Trinucleotídeos , Animais , Proteínas de Bactérias/genética , Sequência de Bases , Proteína 9 Associada à CRISPR , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Códon , Modelos Animais de Doenças , Endonucleases/genética , Fibroblastos/metabolismo , Expressão Gênica , Ordem dos Genes , Loci Gênicos , Humanos , Camundongos , RNA Guia de Cinetoplastídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de SequênciaRESUMO
The unstable (CTG·CAG)n trinucleotide repeat in the myotonic dystrophy type 1 (DM1) locus is bidirectionally transcribed from genes with terminal overlap. By transcription in the sense direction, the DMPK gene produces various alternatively spliced mRNAs with a (CUG)n repeat in their 3' UTR. Expression in opposite orientation reportedly yields (CAG)n-repeat containing RNA, but both structure and biologic significance of this antisense gene (DM1-AS) are largely unknown. Via a combinatorial approach of computational and experimental analyses of RNA from unaffected individuals and DM1 patients we discovered that DM1-AS spans >6 kb, contains alternative transcription start sites and uses alternative polyadenylation sites up- and downstream of the (CAG)n repeat. Moreover, its primary transcripts undergo alternative splicing, whereby the (CAG)n segment is removed as part of an intron. Thus, in patients a mixture of DM1-AS RNAs with and without expanded (CAG)n repeat are produced. DM1-AS expression appears upregulated in patients, but transcript abundance remains very low in all tissues analyzed. Our data suggest that DM1-AS transcripts belong to the class of long non-coding RNAs. These and other biologically relevant implications for how (CAG)n-expanded transcripts may contribute to DM1 pathology can now be explored experimentally.
Assuntos
Distrofia Miotônica/genética , Miotonina Proteína Quinase/genética , RNA Antissenso/genética , RNA Mensageiro/química , Expansão das Repetições de Trinucleotídeos , Regiões 3' não Traduzidas , Adolescente , Processamento Alternativo , Estudos de Casos e Controles , Linhagem Celular , Biologia Computacional/métodos , Humanos , Masculino , Miotonina Proteína Quinase/química , Fases de Leitura Aberta , Poliadenilação , RNA Antissenso/química , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Sítio de Iniciação de Transcrição , Regulação para CimaRESUMO
Oncogenic transformation involves reprogramming of cell metabolism, whereby steady-state levels of intracellular NAD(+) and NADH can undergo dramatic changes while ATP concentration is generally well maintained. Altered expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of NAD(+)-salvage, accompanies the changes in NAD(H) during tumorigenesis. Here, we show by genetic and pharmacological inhibition of NAMPT in glioma cells that fluctuation in intracellular [NAD(H)] differentially affects cell growth and morphodynamics, with motility/invasion capacity showing the highest sensitivity to [NAD(H)] decrease. Extracellular supplementation of NAD(+) or re-expression of NAMPT abolished the effects. The effects of NAD(H) decrease on cell motility appeared parallel coupled with diminished pyruvate-lactate conversion by lactate dehydrogenase (LDH) and with changes in intracellular and extracellular pH. The addition of lactic acid rescued and knockdown of LDH-A replicated the effects of [NAD(H)] on motility. Combined, our observations demonstrate that [NAD(H)] is an important metabolic component of cancer cell motility. Nutrient or drug-mediated modulation of NAD(H) levels may therefore represent a new option for blocking the invasive behavior of tumors.
Assuntos
Movimento Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Glioma/fisiopatologia , NAD/metabolismo , Invasividade Neoplásica/fisiopatologia , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Northern Blotting , Western Blotting , Glioma/metabolismo , Humanos , Concentração de Íons de Hidrogênio , L-Lactato Desidrogenase/metabolismo , Ácido Láctico/farmacologia , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Imagem com Lapso de Tempo , Células Tumorais CultivadasRESUMO
Human mitochondrial complex I (CI) deficiency is associated with progressive neurological disorders. To better understand the CI pathomechanism, we here studied how deletion of the CI gene NDUFS4 affects cell metabolism. To this end we compared immortalized mouse embryonic fibroblasts (MEFs) derived from wildtype (wt) and whole-body NDUFS4 knockout (KO) mice. Mitochondria from KO cells lacked the NDUFS4 protein and mitoplasts displayed virtually no CI activity, moderately reduced CII, CIII and CIV activities and normal citrate synthase and CV (F(o)F(1)-ATPase) activity. Native electrophoresis of KO cell mitochondrial fractions revealed two distinct CI subcomplexes of ~830kDa (enzymatically inactive) and ~200kDa (active). The level of fully-assembled CII-CV was not affected by NDUFS4 gene deletion. KO cells exhibited a moderately reduced maximal and routine O(2) consumption, which was fully inhibited by acute application of the CI inhibitor rotenone. The aberrant CI assembly and reduced O(2) consumption in KO cells were fully normalized by NDUFS4 gene complementation. Cellular [NAD(+)]/[NADH] ratio, lactate production and mitochondrial tetramethyl rhodamine methyl ester (TMRM) accumulation were slightly increased in KO cells. In contrast, NDUFS4 gene deletion did not detectably alter [NADP(+)]/[NADPH] ratio, cellular glucose consumption, the protein levels of hexokinases (I and II) and phosphorylated pyruvate dehydrogenase (P-PDH), total cellular adenosine triphosphate (ATP) level, free cytosolic [ATP], cell growth rate, and reactive oxygen species (ROS) levels. We conclude that the NDUFS4 subunit is of key importance in CI stabilization and that, due to the metabolic properties of the immortalized MEFs, NDUFS4 gene deletion has only modest effects at the live cell level. This article is part of a special issue entitled: 17th European Bioenergetics Conference (EBEC 2012).
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Embrião de Mamíferos/enzimologia , Fibroblastos/enzimologia , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular Transformada , Complexo I de Transporte de Elétrons/genética , Embrião de Mamíferos/citologia , Estabilidade Enzimática/fisiologia , Fibroblastos/citologia , Deleção de Genes , Humanos , Ácido Láctico/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , NAD/genética , NAD/metabolismo , NADP/genética , NADP/metabolismo , Fosforilação/fisiologia , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Complexo Piruvato Desidrogenase/genética , Complexo Piruvato Desidrogenase/metabolismoRESUMO
Extracellular ATP and ADP trigger inflammatory, vasodilatatory, and prothrombotic signaling events in the vasculature, and their turnover is governed by networks of membrane-associated enzymes. The contribution of soluble activities to intravascular nucleotide homeostasis remains controversial. By using thin-layer chromatographic assays, we revealed transphosphorylation of [γ-(32)P]ATP and AMP by human and murine sera, which was progressively inhibited by specific adenylate kinase (AK) inhibitor Ap(5)A. This phosphotransfer reaction was diminished markedly in serum from knockout mice lacking the major AK isoform, AK1, and in human serum immunodepleted of AK1. We also showed that â¼75% ADP in cell-free serum is metabolized via reversible AK1 reaction 2ADP â ATP + AMP. The generated ATP and AMP are then metabolized through the coupled nucleotide pyrophosphatase/phosphodiesterase and 5'-nucleotidase/CD73 reactions, respectively. Constitutive presence of another nucleotide-converting enzyme, nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, known as CD39), was ascertained by the relative deficiency of serum from CD39-null mice to dephosphorylate [(3)H]ADP and [γ-(32)P]ATP, and also by diminished [(3)H]ADP hydrolysis by human serum pretreated with NTPDase1 inhibitors, POM-1 and ARL-67156. In summary, we have identified hitherto unrecognized soluble forms of AK1 and NTPDase1/CD39 that contribute in the active cycling between the principal platelet-recruiting agent ADP and other circulating nucleotides.
Assuntos
Difosfato de Adenosina/sangue , Adenilato Quinase/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Trifosfato de Adenosina/sangue , Adenilato Quinase/genética , Animais , Humanos , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/enzimologiaRESUMO
DMPK, the product of the mutated gene in myotonic dystrophy type 1, belongs to the subfamily of Rho-associated serine-threonine protein kinases, whose members play a role in actin-based cell morphodynamics. Not much is known about the physiological role of differentially localized individual DMPK splice isoforms. We report here that prominent stellar-shaped stress fibers are formed during early and late steps of differentiation in DMPK-deficient myoblast-myotubes upon complementation with the short cytosolic DMPK E isoform. Expression of DMPK E led to an increased phosphorylation status of MLC2. We found no such effects with vectors that encode a mutant DMPK E which was rendered enzymatically inactive or any of the long C-terminally anchored DMPK isoforms. Presence of stellar structures appears associated with changes in cell shape and motility and a delay in myogenesis. Our data strongly suggest that cytosolic DMPK participates in remodeling of the actomyosin cytoskeleton in developing skeletal muscle cells. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.
Assuntos
Actomiosina/metabolismo , Diferenciação Celular , Citosol/enzimologia , Mioblastos/citologia , Mioblastos/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Actinas/química , Actinas/metabolismo , Animais , Movimento Celular , Polaridade Celular , Proliferação de Células , Forma Celular , Isoenzimas/metabolismo , Camundongos , Desenvolvimento Muscular , Miosina Tipo II/metabolismo , Miotonina Proteína Quinase , Fosforilação , Estrutura Quaternária de Proteína , Transporte Proteico , Fibras de Estresse/metabolismo , Fibras de Estresse/ultraestrutura , Frações Subcelulares/metabolismoRESUMO
Serine/threonine kinases of the AGC group are important regulators of cell growth and motility. To examine the candidate substrate profile for two members of this group, DMPK E and Lats2, we performed in vitro kinase assays on peptide arrays. Substrate peptides for both kinases exhibited a predominance of basic residues surrounding the phosphorylation target site. 3D homology modeling of the kinase domains of DMPK E and Lats2 indicated that presence of two negative pockets in the peptide binding groove provides an explanation for the substrate preference. These findings will aid future research toward signaling functions of Lats2 and DMPK E within cells.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Linhagem Celular , Chlorocebus aethiops , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/genética , Modelos Moleculares , Miotonina Proteína Quinase , Fosforilação , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Transdução de Sinais , Especificidade por Substrato , Proteínas Supressoras de Tumor/genéticaRESUMO
Myotonic dystrophy (DM) is a complex, dominantly inherited, multisystem disorder and the archetypal example of an RNA gain-of-function disease. Unstable expansions of (CTG*CAG)n or (CCTG*CAGG)n repeat tracts in the DMPK and ZNF9 genes cause the two known subtypes of myotonic dystrophy, DM1 and DM2, for which no cure or effective molecular treatment exists. Focus in therapeutic development is currently on toxic, expanded (C/CUG)n RNAs. A series of recent papers provide proof of concept of promising strategies using antisense oligonucleotides or small organic compounds aimed at either complete elimination of expanded (CUG)n RNA transcripts or prevention of detrimental protein binding to thermodynamically stable (C/CUG)n hairpin structures. These developments offer new hope to patients with DM, even though several hurdles still have to be overcome before they can be introduced into clinical practice.
Assuntos
Distrofia Miotônica/tratamento farmacológico , RNA Mensageiro/genética , Humanos , Distrofia Miotônica/genética , Miotonina Proteína Quinase , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Proteínas de Ligação a RNA/genéticaRESUMO
Myotonic dystrophy type 1 (DM1) is caused by toxicity of an expanded, noncoding (CUG)n tract in DM protein kinase (DMPK) transcripts. According to current evidence the long (CUG)n segment is involved in entrapment of muscleblind (Mbnl) proteins in ribonuclear aggregates and stabilized expression of CUG binding protein 1 (CUGBP1), causing aberrant premRNA splicing and associated pathogenesis in DM1 patients. Here, we report on the use of antisense oligonucleotides (AONs) in a therapeutic strategy for reversal of RNA-gain-of-function toxicity. Using a previously undescribed mouse DM1 myoblast-myotube cell model and DM1 patient cells as screening tools, we have identified a fully 2'-O-methyl-phosphorothioate-modified (CAG)7 AON that silences mutant DMPK RNA expression and reduces the number of ribonuclear aggregates in a selective and (CUG)n-length-dependent manner. Direct administration of this AON in muscle of DM1 mouse models in vivo caused a significant reduction in the level of toxic (CUG)n RNA and a normalizing effect on aberrant premRNA splicing. Our data demonstrate proof of principle for therapeutic use of simple sequence AONs in DM1 and potentially other unstable microsatellite diseases.
Assuntos
Distrofia Miotônica/genética , Oligonucleotídeos/genética , RNA/genética , Alelos , Animais , Proteínas CELF1 , Inativação Gênica , Camundongos , Modelos Genéticos , Músculo Esquelético/metabolismo , Mutação , Mioblastos/metabolismo , Distrofia Miotônica/terapia , Oligonucleotídeos/química , Oligonucleotídeos Antissenso/genética , Interferência de RNA , Splicing de RNA , Proteínas de Ligação a RNA/genéticaRESUMO
The kinetics of phosphoryl exchange involving ATP and ADP have been investigated successfully by in vivo (31)P magnetic resonance spectroscopy using magnetization transfer. However, magnetization transfer effects seen on the signals of ATP also could arise from intramolecular cross-relaxation. This relaxation process carries information on the association state of ATP in the cell. To disentangle contributions of chemical exchange and cross-relaxation to magnetization transfer effects seen in (31)P magnetic resonance spectroscopy of skeletal muscle, we performed saturation transfer experiments on wild type and double-mutant mice lacking the cytosolic muscle creatine kinase and adenylate kinase isoforms. We find that cross-relaxation, observed as nuclear Overhauser effects (NOEs), is responsible for magnetization transfer between ATP phosphates both in wild type and in mutant mice. Analysis of (31)P relaxation properties identifies these effects as transferred NOEs, i.e. underlying this process is an exchange between free cellular ATP and ATP bound to slowly rotating macromolecules. This explains the ß-ATP signal decrease upon saturation of the γ-ATP resonance. Although this usually is attributed to ß-ADP â ß-ATP phosphoryl exchange, we did not detect an effect of this exchange on the ß-ATP signal as expected for free [ADP], derived from the creatine kinase equilibrium reaction. This indicates that in resting muscle, conditions prevail that prevent saturation of ß-ADP spins and puts into question the derivation of free [ADP] from the creatine kinase equilibrium. We present a model, matching the experimental result, for ADP â ATP exchange, in which ADP is only transiently present in the cytosol.
Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Adenilil Ciclases/metabolismo , Creatina Quinase/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Difosfato de Adenosina/genética , Trifosfato de Adenosina/genética , Adenilil Ciclases/genética , Animais , Creatina Quinase/genética , Citosol/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Mutantes , Proteínas Musculares/genética , Isótopos de FósforoRESUMO
Phagocytosis requires locally coordinated cytoskeletal rearrangements driven by actin polymerization and myosin motor activity. How this actomyosin dynamics is dependent upon systems that provide access to ATP at phagosome microdomains has not been determined. We analyzed the role of brain-type creatine kinase (CK-B), an enzyme involved in high-energy phosphoryl transfer. We demonstrate that endogenous CK-B in macrophages is mobilized from the cytosolic pool and coaccumulates with F-actin at nascent phagosomes. Live cell imaging with XFP-tagged CK-B and beta-actin revealed the transient and specific nature of this partitioning process. Overexpression of a catalytic dead CK-B or CK-specific cyclocreatine inhibition caused a significant reduction of actin accumulation in the phagocytic cup area, and reduced complement receptor-mediated, but not Fc-gammaR-mediated, ingestion capacity of macrophages. Finally, we found that inhibition of CK-B affected phagocytosis already at the stage of particle adhesion, most likely via effects on actin polymerization behavior. We propose that CK-B activity in macrophages contributes to complement-induced F-actin assembly events in early phagocytosis by providing local ATP supply.
Assuntos
Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/fisiologia , Creatina Quinase Forma BB/metabolismo , Fagocitose , Trifosfato de Adenosina/provisão & distribuição , Animais , Adesão Celular , Proteínas do Sistema Complemento/metabolismo , Creatina Quinase Forma BB/fisiologia , Creatinina/análogos & derivados , Creatinina/farmacologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mutantes/metabolismo , Proteínas Opsonizantes/metabolismo , Fagocitose/fisiologia , Fagossomos/metabolismo , Polímeros/metabolismo , Transporte Proteico/fisiologia , Fatores de Tempo , Zimosan/metabolismoRESUMO
PURPOSE: Brain-type creatine kinase (CK-B) and ubiquitous mitochondrial creatine kinase (UbCKmit) act as components of local phosphocreatine ATP shuttles that help in the compartmentalization and maintenance of pools of high-energy phosphate molecules in both neurons and glial cells. We investigated the role of these brain-type creatine kinases during extreme energy-demanding conditions in vivo (generalized tonic-clonic seizures) and in vitro. METHODS: The physiologic response of wild-types and mice lacking both CK-B and UbCKmit (CK--/--mice) to pentylenetetrazole (PTZ)-induced seizures was measured using electroencephalography (EEG) recordings and behavioral monitoring. In vitro intracellular Ca(2+) kinetics in hippocampal granule neurons were monitored upon single and repetitive depolarizations. RESULTS: PTZ induced in only a few CK--/-- mice PTZ seizure-like behavior, but in all wild-types a full-blown seizure. EEG analysis showed that preseizure jerking was associated with high-amplitude discharges. Wild-type EEG recordings showed continuous runs of rhythmic 4-6 Hz activity, whereas no rhythmic EEG activities were observed in the few CK--/-- mice that developed a behavioral seizure. All other CK--/-- mice displayed a sudden postictal depression without any development of a generalized seizure. Hippocampal granule neurons of CK--/-- mice displayed a higher Ca(2+) removal speed following repetitive KCl-induced depolarizations. DISCUSSION: Deficiency for creatine kinase is affecting brain energy metabolism and will likely contribute to the disturbance of seizure development. Because CK--/-- hippocampal neurons exhibited an increase in Ca(2+) removal rate of elevated intracellular levels, we conclude that altered Ca(2+) clearance in CK--/-- neurons could play a role in the abnormal EEG and seizure activity.
Assuntos
Encéfalo/metabolismo , Cálcio/metabolismo , Creatina Quinase Forma BB/deficiência , Creatina Quinase Forma BB/metabolismo , Neurônios/metabolismo , Convulsões/induzido quimicamente , Convulsões/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/enzimologia , Creatina Quinase/deficiência , Creatina Quinase/efeitos dos fármacos , Creatina Quinase/metabolismo , Creatina Quinase Forma BB/efeitos dos fármacos , Creatina Quinase Mitocondrial/metabolismo , Modelos Animais de Doenças , Eletroencefalografia , Metabolismo Energético , Hipocampo/metabolismo , Técnicas In Vitro , Camundongos , Camundongos Knockout , Neuroglia/metabolismo , Neurônios/enzimologia , Pentilenotetrazol , Cloreto de Potássio/farmacologia , Convulsões/enzimologiaRESUMO
Parkinson's disease (PD) is a highly prevalent neurodegenerative disease for which no disease-modifying treatments are available, mainly because knowledge about its pathogenic mechanism is still incomplete. Recently, a key role for lipids emerged, but lipid profiling of brain samples from human subjects is demanding. Here, we used an unbiased approach, lipidomics, to determine PD-linked changes in the lipid profile of a well-established cell model for PD, the catecholaminergic neuronal cell line SH-SY5Y treated with the neurotoxin 6-hydroxydopamine (6-OHDA). We observed changes in multiple lipid classes, including phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM), and total cholesterol, in 6-OHDA-treated SH-SY5Y cells. Furthermore, we found differences in the length and degree of unsaturation of the fatty acyl chains, indicating changes in their metabolism. Except for the observed decreased PS levels, the alterations in PC, PG, PI, and cholesterol levels are in agreement with the results of previous studies on PD-patient material. Opposite to what has been previously described, the cholesterol-lowering drug statins did not have a protective effect, while low doses of cholesterol supplementation partially protected SH-SY5Y cells from 6-OHDA toxicity. However, cholesterol supplementation triggered neuronal differentiation, which could have confounded the results of cholesterol modulation. Taken together, our results show that 6-OHDA-treated SH-SY5Y cells display many lipid changes also found in PD patient and animal model brains, although the SH-SY5Y cell model seems less suitable to study the involvement of cholesterol in PD initiation and progression.
Assuntos
Lipídeos/análise , Modelos Biológicos , Oxidopamina/toxicidade , Doença de Parkinson/metabolismo , Linhagem Celular Tumoral , Colesterol/metabolismo , Humanos , Lipidômica , Sinvastatina/farmacologiaRESUMO
Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons from the substantia nigra (SN) that project to the dorsal striatum (caudate-putamen). To better understand the molecular mechanisms underlying PD, we performed combined lipid profiling and RNA sequencing of SN and putamen samples from PD patients and age-matched controls. SN lipid analysis pointed to a neuroinflammatory component and included elevated levels of the endosomal lipid Bis (Monoacylglycero)Phosphate 42:8, while two of the three depleted putamen lipids were saturated sphingomyelin species. Remarkably, we observed gender-related differences in the SN and putamen lipid profiles. Transcriptome analysis revealed that the top-enriched pathways among the 354 differentially expressed genes (DEGs) in the SN were "protein folding" and "neurotransmitter transport", and among the 261 DEGs from putamen "synapse organization". Furthermore, we identified pathways, e.g., "glutamate signaling", and genes, encoding, e.g., an angiotensin receptor subtype or a proprotein convertase, that have not been previously linked to PD. The identification of 33 genes that were common among the SN and putamen DEGs, which included the α-synuclein paralog ß-synuclein, may contribute to the understanding of general PD mechanisms. Thus, our proof-of-concept data highlights new genes, pathways and lipids that have not been explored before in the context of PD.
Assuntos
Perfilação da Expressão Gênica/métodos , Lipídeos/sangue , Doença de Parkinson/fisiopatologia , Putamen/fisiopatologia , Substância Negra/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , MasculinoRESUMO
BACKGROUND: The Warburg phenotype in cancer cells has been long recognized, but there is still limited insight in the consecutive metabolic alterations that characterize its establishment. We obtained better understanding of the coupling between metabolism and malignant transformation by studying mouse embryonic fibroblast-derived cells with loss-of-senescence or H-RasV12/E1A-transformed phenotypes at different stages of oncogenic progression. RESULTS: Spontaneous immortalization or induction of senescence-bypass had only marginal effects on metabolic profiles and viability. In contrast, H-RasV12/E1A transformation initially caused a steep increase in oxygen consumption and superoxide production, accompanied by massive cell death. During prolonged culture in vitro, cell growth rate increased gradually, along with tumor forming potential in in vitro anchorage-independent growth assays and in vivo tumor formation assays in immuno-deficient mice. Notably, glucose-to-lactic acid flux increased with passage number, while cellular oxygen consumption decreased. This conversion in metabolic properties was associated with a change in mitochondrial NAD+/NADH redox, indicative of decreased mitochondrial tricarboxic acid cycle and OXPHOS activity. CONCLUSION: The high rate of oxidative metabolism in newly transformed cells is in marked contrast with the high glycolytic rate in cells in the later tumor stage. In our experimental system, with cells growing under ambient oxygen conditions in nutrient-rich media, the shift towards this Warburg phenotype occurred as a step-wise adaptation process associated with augmented tumorigenic capacity and improved survival characteristics of the transformed cells. We hypothesize that early-transformed cells, which potentially serve as founders for new tumor masses may escape therapies aimed at metabolic inhibition of tumors with a fully developed Warburg phenotype.
Assuntos
Transformação Celular Neoplásica , Fibroblastos/metabolismo , Glicólise , Fosforilação Oxidativa , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/fisiologia , Animais , Linhagem Celular Transformada , Proliferação de Células , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/ultraestrutura , Ácido Láctico/metabolismo , Masculino , Metaboloma , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Eletrônica de Varredura , Mitocôndrias/metabolismo , NAD/metabolismo , Transplante de Neoplasias , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Consumo de Oxigênio , Retroviridae/genética , Superóxidos/metabolismo , Proteínas ras/genética , Proteínas ras/fisiologiaRESUMO
We developed a new dedicated measurement protocol for dynamic (31)P MRS analysis in contracting calf muscles of the mouse, using minimally invasive assessment of the contractile force combined with the acquisition of spectroscopic data gated to muscle contraction and determination of phosphocreatine (PCr) recovery rate and ATP contractile cost. This protocol was applied in a comparative study of six wild type (WT) mice and six mice deficient in cytosolic creatine kinase and adenylate kinase isoform 1 (MAK(-/-) mice) using 70 repeated tetanic contractions at two contractions per minute. Force levels during single contractions, and metabolite levels and tissue pH during resting conditions were similar in muscles of MAK(-/-) and WT mice. Strikingly, muscle relaxation after contraction was significantly delayed in MAK(-/-) mice, but during repeated contractions, the decrease in the force was similar in both mouse types. Gated data acquisition showed a negligible PCr breakdown in MAK(-/-) immediately after contraction, without a concomitant decrease in ATP or tissue pH. This protocol enabled the determination of rapid PCr changes that would otherwise go unnoticed due to intrinsic low signal-to-noise ratio (SNR) in mouse skeletal muscles combined with an assessment of the PCr recovery rate. Our results suggest that MAK(-/-) mice use alternative energy sources to maintain force during repeated contractions when PCr breakdown is reduced. Furthermore, the absence of large increases in adenosine diphosphate (ADP) or differences in force compared to WT mice in our low-intensity protocol indicate that creatine kinase (CK) and adenylate kinase (AK) are especially important in facilitating energy metabolism during very high energy demands.