RESUMO
Regulation of mRNA translation in astrocytes gains a growing interest. However, until now, successful ribosome profiling of primary astrocytes has not been reported. Here, we optimized the standard 'polysome profiling' method and generated an effective protocol for polyribosome extraction, which enabled genome-wide assessment of mRNA translation dynamics along the process of astrocyte activation. Transcriptome (RNAseq) and translatome (Riboseq) data generated at 0, 24 and 48 h after cytokines treatment, revealed dynamic genome-wide changes in the expression level of â¼12 000 genes. The data clarify whether a change in protein synthesis rate results from a change in mRNA level or translation efficiency per se. It exhibit different expression strategies, based on changes in mRNA abundance and/or translation efficiency, which are specifically assigned to gene subsets depending on their function. Moreover, the study raises an important take-home message related to the possible presence of 'difficult to extract' polyribosome sub-groups, in all cell types, thus illuminating the impact of ribosomes extraction methodology on experiments addressing translation regulation.
Assuntos
Astrócitos , Biossíntese de Proteínas , Astrócitos/metabolismo , Polirribossomos/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Perfilação da Expressão Gênica/métodosRESUMO
Eukaryotic translation initiation factor 2B (eIF2B) is a master regulator of protein synthesis under normal and stress conditions. Mutations in any of the five genes encoding its subunits lead to vanishing white matter (VWM) disease, a recessive genetic deadly illness caused by progressive loss of white matter in the brain. In this study we used fibroblasts, which are not involved in the disease, to demonstrate the involvement of eIF2B in mitochondrial function and abundance. Mass spectrometry of total proteome of mouse embryonic fibroblasts (MEFs) isolated from Eif2b5R132H/R132H mice revealed unbalanced stoichiometry of proteins involved in oxidative phosphorylation and of mitochondrial translation machinery components, among others. Mutant MEFs exhibit 55% decrease in oxygen consumption rate per mtDNA content and 47% increase in mitochondrial abundance (p < 0.005), reflecting adaptation to energy requirements. A more robust eIF2B-associated oxidative respiration deficiency was found in mutant primary astrocytes, which exhibit > 3-fold lower ATP-linked respiration per cell despite a 2-fold increase in mtDNA content (p < 0.03). The 2-fold increase in basal and stimulated glycolysis in mutant astrocytes (p ≤ 0.03), but not in MEFs, demonstrates their higher energetic needs and further explicates their involvement in the disease. The data demonstrate the critical role of eIF2B in tight coordination of expression from nuclear and mitochondrial genomes and illuminates the importance of mitochondrial function in VWM pathology. Further dissection of the signaling network associated with eIF2B function will help generating therapeutic strategies for VWM disease and possibly other neurodegenerative disorders.
Assuntos
Astrócitos/ultraestrutura , Fator de Iniciação 2B em Eucariotos/genética , Mitocôndrias/genética , Mutação/genética , Fosforilação Oxidativa , Consumo de Oxigênio/genética , Animais , Animais Recém-Nascidos , Antimicina A/farmacologia , Astrócitos/efeitos dos fármacos , Astrócitos/fisiologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Tamanho Celular , Células Cultivadas , Cloranfenicol/farmacologia , Fator de Iniciação 2B em Eucariotos/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/ultraestrutura , Antígenos de Histocompatibilidade/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/fisiologia , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Inibidores da Síntese de Proteínas/farmacologia , Ionóforos de Próton/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Gtf2i encodes the general transcription factor II-I (TFII-I), with peak expression during pre-natal and early post-natal brain development stages. Because these stages are critical for proper brain development, we studied at the single-cell level the consequences of Gtf2i's deletion from excitatory neurons, specifically on mitochondria. Here we show that Gtf2i's deletion resulted in abnormal morphology, disrupted mRNA related to mitochondrial fission and fusion, and altered autophagy/mitophagy protein expression. These changes align with elevated reactive oxygen species levels, illuminating Gtf2i's importance in neurons mitochondrial function. Similar mitochondrial issues were demonstrated by Gtf2i heterozygous model, mirroring the human condition in Williams syndrome (WS), and by hemizygous neuronal Gtf2i deletion model, indicating Gtf2i's dosage-sensitive role in mitochondrial regulation. Clinically relevant, we observed altered transcript levels related to mitochondria, hypoxia, and autophagy in frontal cortex tissue from WS individuals. Our study reveals mitochondrial and autophagy-related deficits shedding light on WS and other Gtf2i-related disorders.
Assuntos
Fatores de Transcrição TFIII , Síndrome de Williams , Humanos , Autofagia/genética , Heterozigoto , Neurônios/metabolismo , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFIII/metabolismo , Síndrome de Williams/genética , Síndrome de Williams/metabolismoRESUMO
Translation initiation factor 2B (eIF2B) is a master regulator of global protein synthesis in all cell types. The mild genetic Eif2b5(R132H) mutation causes a slight reduction in eIF2B enzymatic activity which leads to abnormal composition of mitochondrial electron transfer chain complexes and impaired oxidative phosphorylation. Previous work using primary fibroblasts isolated from Eif2b5(R132H/R132H) mice revealed that owing to increased mitochondrial biogenesis they exhibit normal cellular ATP level. In contrast to fibroblasts, here we show that primary astrocytes isolated from Eif2b5(R132H/R132H) mice are unable to compensate for their metabolic impairment and exhibit chronic state of low ATP level regardless of extensive adaptation efforts. Mutant astrocytes are hypersensitive to oxidative stress and to further energy stress. Moreover, they show migration deficit upon exposure to glucose starvation. The mutation in Eif2b5 prompts reactive oxygen species (ROS)-mediated inferior ability to stimulate the AMP-activated protein kinase (AMPK) axis, due to a requirement to increase the mammalian target of rapamycin complex-1 (mTORC1) signalling in order to enable oxidative glycolysis and generation of specific subclass of ROS-regulating proteins, similar to cancer cells. The data disclose the robust impact of eIF2B on metabolic and redox homeostasis programs in astrocytes and point at their hyper-sensitivity to mutated eIF2B. Thereby, it illuminates the central involvement of astrocytes in Vanishing White Matter Disease (VWMD), a genetic neurodegenerative leukodystrophy caused by homozygous hypomorphic mutations in genes encoding any of the 5 subunits of eIF2B.
Assuntos
Trifosfato de Adenosina/metabolismo , Astrócitos/metabolismo , Encéfalo/metabolismo , Metabolismo Energético , Fator de Iniciação 2B em Eucariotos/metabolismo , Leucoencefalopatias/metabolismo , Mitocôndrias/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose , Astrócitos/patologia , Encéfalo/patologia , Movimento Celular , Células Cultivadas , Modelos Animais de Doenças , Fator de Iniciação 2B em Eucariotos/genética , Leucoencefalopatias/genética , Leucoencefalopatias/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mitocôndrias/genética , Mitocôndrias/patologia , Mutação , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Vanishing white matter (VWM) disease is an autosomal genetic leukodystrophy caused by mutations in subunits of eukaryotic translation initiation factor 2B (eIF2B). The clinical symptoms exhibit progressive loss of white matter in both hemispheres of the brain, accompanied by motor functions deterioration, neurological deficits, and early death. To date there is no treatment for VWM disease. The aim of this work was to expedite rational development of a therapeutic opportunity. Our approach was to design a computer-aided strategy for an efficient and reliable screening of drug-like molecules; and to use primary cultures of fibroblasts isolated from the Eif2b5R132H/R132H VWM mouse model for screening. The abnormal mitochondria content phenotype of the mutant cells was chosen as a read-out for a simple cell-based fluorescent assay to assess the effect of the tested compounds. We obtained a hit rate of 0.04% (20 hits out of 50,000 compounds from the selected library). All primary hits decreased mitochondria content and brought it closer to WT levels. Structural similarities between our primary hits and other compounds with known targets allowed the identification of three putative cellular pathways/targets: 11ß-hydroxysteroid dehydrogenase type 1, Sonic hedgehog (Shh), and Sigma-1-Receptor (S1R). In addition to initial experimental indication of Shh pathway impairment in VWM mouse brains, the current study provides evidence that S1R is a relevant target for pharmaceutical intervention for potential treatment of the disease. Specifically, we found lower expression level of S1R protein in fibroblasts, astrocytes, and whole brains isolated from Eif2b5R132H/R132H compared to WT mice, and confirmed that one of the hits is a direct binder of S1R, acting as agonist. Furthermore, we provide evidence that treatment of mutant mouse fibroblasts and astrocytes with various S1R agonists corrects the functional impairments of their mitochondria and prevents their need to increase their mitochondria content for compensation purposes. Moreover, S1R activation enhances the survival rate of mutant cells under ER stress conditions, bringing it to WT levels. This study marks S1R as a target for drug development toward treatment of VWM disease. Moreover, it further establishes the important connection between white matter well-being and S1R-mediated proper mitochondria/ER function.
RESUMO
Epidemiological evidence suggests that carotenoids prevent several types of cancer, including mammary and endometrial cancers. On the other hand, such studies have also shown that estrogens are the most important risk factors for these cancer types. Genistein, the phytoestrogen mainly found in soy, also shows significant estrogenic activity when tested at concentrations found in human blood. The aim of this study was to determine whether carotenoids inhibit signaling of steroidal estrogen and phytoestrogen which could explain their cancer preventive activity. Similar to the known effect of 17beta-estradiol (E(2)), treatment of breast (T47D and MCF-7) and endometrial (ECC-1) cancer cells with phytoestrogens induced cell proliferation, cell-cycle progression and transactivation of the estrogen response element (ERE). However, each of the tested carotenoids (lycopene, phytoene, phytofluene, and beta-carotene) inhibited cancer cell proliferation induced by either E(2) or genistein. The inhibition of cell growth by lycopene was accompanied by slow down of cell-cycle progression from G1 to S phase. Moreover, the carotenoids inhibited estrogen-induced transactivation of ERE that was mediated by both estrogen receptors (ERs) ERalpha and ERbeta. The possibility that this inhibition results from competition of carotenoid-activated transcription systems on a limited pool of shared coactivators with the ERE transcription system was tested. Although cotransfection of breast and endometrial cancer cells with four different coactivators (SRC-1, SRC-2, SRC-3, and DRIP) strongly stimulated ERE reporter gene activity, it did not oppose the inhibitory effect of carotenoids. These results suggest that dietary carotenoids inhibit estrogen signaling of both 17beta-estradiol and genistein, and attenuate their deleterious effect in hormone-dependent malignancies.