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Meiotic spindles are critical to ensure chromosome segregation during gamete formation. Oocytes lack centrosomes and use alternative microtubule-nucleation mechanisms for spindle building. How these mechanisms are regulated is still unknown. Aurora kinase A (AURKA) is essential for mouse oocyte meiosis because in pro-metaphase I it triggers microtubule organizing-center fragmentation and its expression compensates for the loss of the two other Aurora kinases (AURKB/AURKC). Although knockout mouse models were useful for foundational studies, AURK spatial and temporal functions are not yet resolved. We provide high-resolution analyses of AURKA/AURKC requirements during meiotic spindle-building and identify the subcellular populations that carry out these functions: 1) AURKA is required in early spindle assembly and later for spindle stability, whereas 2) AURKC is required in late pro-metaphase, and 3) Targeted AURKA constructs expressed in triple AURK knockout oocytes reveal that spindle pole-localized AURKA is the most important population controlling spindle building and stability mechanisms.
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After fertilization, remodeling of the oocyte and sperm genomes is essential to convert these highly differentiated and transcriptionally quiescent cells into early cleavage-stage blastomeres that are transcriptionally active and totipotent. This developmental transition is accompanied by cell cycle adaptation, such as lengthening or shortening of the gap phases G1 and G2. However, regulation of these cell cycle changes is poorly understood, especially in mammals. Checkpoint kinase 1 (CHK1) is a protein kinase that regulates cell cycle progression in somatic cells. Here, we show that CHK1 regulates cell cycle progression in early mouse embryos by restraining CDK1 kinase activity due to CDC25A phosphatase degradation. CHK1 kinase also ensures the long G2 phase needed for genome activation and reprogramming gene expression in two-cell stage mouse embryos. Finally, Chk1 depletion leads to DNA damage and chromosome segregation errors that result in aneuploidy and infertility.
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Mammalian oocytes are arrested at meiotic prophase I. The dual-specificity phosphatase CDC25B is essential for cyclin-dependent kinase 1 (CDK1) activation that drives resumption of meiosis. CDC25B reverses the inhibitory effect of the protein kinases WEE1 and MYT1 on CDK1 activation. Cdc25b-/- female mice are infertile because oocytes cannot activate CDK1. To identify a role for CDC25B following resumption of meiosis, we restored CDK1 activation in Cdc25b-/- oocytes by inhibiting WEE1 and MYT1, or expressing EGFP-CDC25A or constitutively active EGFP-CDK1 from microinjected complementary RNAs. Forced CDK1 activation in Cdc25b-/- oocytes allowed resumption of meiosis, but oocytes mostly arrested at metaphase I (MI) with intact spindles. Similarly, approximately a third of Cdc25b+/- oocytes with a reduced amount of CDC25B arrested in MI. MI-arrested Cdc25b-/- oocytes also displayed a transient decrease in CDK1 activity similar to Cdc25b+/+ oocytes during the MI-MII transition, whereas Cdc25b+/- oocytes exhibited only a partial anaphase-promoting complex/cyclosome activation and anaphase I entry. Thus, CDC25B is necessary for the resumption of meiosis and the MI-MII transition.
Assuntos
Meiose , Oócitos , Anáfase , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Animais , Feminino , Mamíferos , Metáfase , Camundongos , Oócitos/metabolismo , Fosfatases cdc25RESUMO
OBJECTIVE: The MATRICS consensus cognitive battery (MCCB) is a widely used neuropsychological battery for the assessment of cognitive dysfunction in schizophrenia. However, the accuracy of measurement is dependent on suitable normative data which are in the Czech Republic lacking. METHOD: The Czech academic research translation of the MCCB battery was administered to a sample of healthy volunteers aged 17 to 62 years (N = 573) and the effects of age, education and sex were examined. In addition, a comparison was made to examine the differences between the US and current normative data. RESULTS: Consistent with previous studies, significant effects of age, sex and education were found, however, in sex and education in distinct MCCB-domains. By comparing the original and current normative data, significant differences with small to large effect sizes were revealed in all domains except for Verbal Learning. CONCLUSIONS: We present nationally specific MCCB regression-based and tabular normative data applicable in research and clinical settings.
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Cognição , Psicologia do Esquizofrênico , Consenso , República Tcheca , Humanos , Testes NeuropsicológicosRESUMO
The Aurora protein kinases are well-established regulators of spindle building and chromosome segregation in mitotic and meiotic cells. In mouse oocytes, there is significant Aurora kinase A (AURKA) compensatory abilities when the other Aurora kinase homologs are deleted. Whether the other homologs, AURKB or AURKC can compensate for loss of AURKA is not known. Using a conditional mouse oocyte knockout model, we demonstrate that this compensation is not reciprocal because female oocyte-specific knockout mice are sterile, and their oocytes fail to complete meiosis I. In determining AURKA-specific functions, we demonstrate that its first meiotic requirement is to activate Polo-like kinase 1 at acentriolar microtubule organizing centers (aMTOCs; meiotic spindle poles). This activation induces fragmentation of the aMTOCs, a step essential for building a bipolar spindle. We also show that AURKA is required for regulating localization of TACC3, another protein required for spindle building. We conclude that AURKA has multiple functions essential to completing MI that are distinct from AURKB and AURKC.
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Aurora Quinase A/genética , Proteínas de Ciclo Celular/genética , Proteínas Fetais/genética , Meiose/genética , Proteínas Associadas aos Microtúbulos/genética , Oócitos/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Animais , Aurora Quinase B/genética , Aurora Quinase C/genética , Divisão do Núcleo Celular/genética , Segregação de Cromossomos/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Centro Organizador dos Microtúbulos/metabolismo , Oócitos/metabolismo , Fuso Acromático/genética , Polos do Fuso/genética , Quinase 1 Polo-LikeRESUMO
Cell therapies represent a promising approach to slow down the progression of currently untreatable neurodegenerative diseases (e.g., Alzheimer's and Parkinson's disease or amyotrophic lateral sclerosis), as well as to support the reconstruction of functional neural circuits after spinal cord injuries. In such therapies, the grafted cells could either functionally integrate into the damaged tissue, partially replacing dead or damaged cells, modulate inflammatory reaction, reduce tissue damage, or support neuronal survival by secretion of cytokines, growth, and trophic factors. Comprehensive characterization of cells and their proliferative potential, differentiation status, and population purity before transplantation is crucial to preventing safety risks, e.g., a tumorous growth due to the proliferation of undifferentiated stem cells. We characterized changes in the proteome and secretome of human neural stem cells (NSCs) during their spontaneous (EGF/FGF2 withdrawal) differentiation and differentiation with trophic support by BDNF/GDNF supplementation. We used LC-MS/MS in SWATH-MS mode for global cellular proteome profiling and quantified almost three thousand cellular proteins. Our analysis identified substantial protein differences in the early stages of NSC differentiation with more than a third of all the proteins regulated (including known neuronal and NSC multipotency markers) and revealed that the BDNF/GDNF support affected more the later stages of the NSC differentiation. Among the pathways identified as activated during both spontaneous and BDNF/GDNF differentiation were the HIF-1 signaling pathway, Wnt signaling pathway, and VEGF signaling pathway. Our follow-up secretome analysis using Luminex multiplex immunoassay revealed significant changes in the secretion of VEGF and IL-6 during NSC differentiation. Our results further demonstrated an increased expression of neuropilin-1 as well as catenin ß-1, both known to participate in the regulation of VEGF signaling, and showed that VEGF-A isoform 121 (VEGF121), in particular, induces proliferation and supports survival of differentiating cells.
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BACKGROUND: Huntington's disease (HD) is a devastating neurodegenerative disorder caused by CAG triplet expansions in the huntingtin gene. Oxidative stress is linked to HD pathology, although it is not clear whether this is an effect or a mediator of disease. The transgenic (TgHD) minipig expresses the N-terminal part of human-mutated huntingtin and represents a unique model to investigate therapeutic strategies towards HD. A more detailed characterization of this model is needed to fully utilize its potential. METHODS: In this study, we focused on the molecular and cellular features of fibroblasts isolated from TgHD minipigs and the wild-type (WT) siblings at different ages, pre-symptomatic at the age of 24-36 months and with the onset of behavioural symptoms at the age of 48 months. We measured oxidative stress, the expression of oxidative stress-related genes, proliferation capacity along with the expression of cyclin B1 and D1 proteins, cellular permeability, and the integrity of the nuclear DNA (nDNA) and mitochondrial DNA in these cells. RESULTS: TgHD fibroblasts isolated from 48-month-old animals showed increased oxidative stress, which correlated with the overexpression of SOD2 encoding mitochondrial superoxide dismutase 2, and the NEIL3 gene encoding DNA glycosylase involved in replication-associated repair of oxidized DNA. TgHD cells displayed an abnormal proliferation capacity and permeability. We further demonstrated increased nDNA damage in pre-symptomatic TgHD fibroblasts (isolated from animals aged 24-36 months). CONCLUSIONS: Our results unravel phenotypic alterations in primary fibroblasts isolated from the TgHD minipig model at the age of 48 months. Importantly, nDNA damage appears to precede these phenotypic alterations. Our results highlight the impact of fibroblasts from TgHD minipigs in studying the molecular mechanisms of HD pathophysiology that gradually occur with age.
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Envelhecimento/metabolismo , Fibroblastos/metabolismo , Proteína Huntingtina/metabolismo , Animais , Animais Geneticamente Modificados , Divisão Celular , Dano ao DNA , DNA Mitocondrial/genética , Regulação da Expressão Gênica , Humanos , Proteína Huntingtina/genética , Peroxidação de Lipídeos , N-Glicosil Hidrolases/biossíntese , N-Glicosil Hidrolases/genética , Estresse Oxidativo , Fenótipo , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genética , Suínos , Porco MiniaturaRESUMO
RNAi is the sequence-specific mRNA degradation guided by siRNAs produced from long dsRNA by RNase Dicer. Proteins executing RNAi are present in mammalian cells but rather sustain the microRNA pathway. Aiming for a systematic analysis of mammalian RNAi, we report here that the main bottleneck for RNAi efficiency is the production of functional siRNAs, which integrates Dicer activity, dsRNA structure, and siRNA targeting efficiency. Unexpectedly, increased expression of Dicer cofactors TARBP2 or PACT reduces RNAi but not microRNA function. Elimination of protein kinase R, a key dsRNA sensor in the interferon response, had minimal positive effects on RNAi activity in fibroblasts. Without high Dicer activity, RNAi can still occur when the initial Dicer cleavage of the substrate yields an efficient siRNA. Efficient mammalian RNAi may use substrates with some features of microRNA precursors, merging both pathways even more than previously suggested. Although optimized endogenous Dicer substrates mimicking miRNA features could evolve for endogenous regulations, the same principles would make antiviral RNAi inefficient as viruses would adapt to avoid efficacy.