RESUMO
Human AUTS2 mutations are linked to a syndrome of intellectual disability, autistic features, epilepsy, and other neurological and somatic disorders. Although it is known that this unique gene is highly expressed in developing cerebral cortex, the molecular and developmental functions of AUTS2 protein remain unclear. Using proteomics methods to identify AUTS2 binding partners in neonatal mouse cerebral cortex, we found that AUTS2 associates with multiple proteins that regulate RNA transcription, splicing, localization, and stability. Furthermore, AUTS2-containing protein complexes isolated from cortical tissue bound specific RNA transcripts in RNA immunoprecipitation and sequencing assays. Deletion of all major functional isoforms of AUTS2 (full-length and C-terminal) by conditional excision of exon 15 caused breathing abnormalities and neonatal lethality when Auts2 was inactivated throughout the developing brain. Mice with limited inactivation of Auts2 in cerebral cortex survived but displayed abnormalities of cerebral cortex structure and function, including dentate gyrus hypoplasia with agenesis of hilar mossy neurons, and abnormal spiking activity on EEG. Also, RNA transcripts that normally associate with AUTS2 were dysregulated in mutant mice. Together, these findings indicate that AUTS2 regulates RNA metabolism and is essential for development of cerebral cortex, as well as subcortical breathing centers.
Assuntos
Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Giro Denteado/crescimento & desenvolvimento , Giro Denteado/metabolismo , RNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Animais Recém-Nascidos , Córtex Cerebral/anormalidades , Córtex Cerebral/metabolismo , Eletroencefalografia , Éxons/genética , Deleção de Genes , Regulação da Expressão Gênica , Deficiência Intelectual/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA-Seq , RespiraçãoRESUMO
There is growing evidence that stochastic events play an important role in determining individual longevity. Studies in model organisms have demonstrated that genetically identical populations maintained under apparently equivalent environmental conditions display individual variation in life span that can be modeled by the Gompertz-Makeham law of mortality. Here, we report that within genetically identical haploid and diploid wild-type populations, shorter-lived cells tend to arrest in a budded state, while cells that arrest in an unbudded state are significantly longer-lived. This relationship is particularly notable in diploid BY4743 cells, where mother cells that arrest in a budded state have a shorter mean life span (25.6 vs. 35.6) and larger coefficient of variance with respect to individual life span (0.42 vs. 0.32) than cells that arrest in an unbudded state. Mutations that cause genomic instability tend to shorten life span and increase the proportion of the population that arrest in a budded state. These observations suggest that randomly occurring damage may contribute to stochasticity during replicative aging by causing a subset of the population to terminally arrest prematurely in the S or G2 phase of the cell cycle.
Assuntos
Pontos de Checagem do Ciclo Celular , Viabilidade Microbiana , Leveduras/fisiologia , Processos EstocásticosRESUMO
AUTS2 syndrome is a genetic disorder that causes intellectual disability, microcephaly, and other phenotypes. Syndrome severity is worse when mutations involve 3' regions (exons 9-19) of the AUTS2 gene. Human AUTS2 protein has two major isoforms, full-length (1259 aa) and C-terminal (711 aa), the latter produced from an alternative transcription start site in exon 9. Structurally, AUTS2 contains the putative "AUTS2 domain" (â¼200 aa) conserved among AUTS2 and its ohnologs, fibrosin, and fibrosin-like-1. Also, AUTS2 contains extensive low-complexity sequences and intrinsically disordered regions, features typical of RNA-binding proteins. During development, AUTS2 is expressed by specific progenitor cell and neuron types, including pyramidal neurons and Purkinje cells. AUTS2 localizes mainly in cell nuclei, where it regulates transcription and RNA metabolism. Some studies have detected AUTS2 in neurites, where it may regulate cytoskeletal dynamics. Neurodevelopmental functions of AUTS2 have been studied in diverse model systems. In zebrafish, auts2a morphants displayed microcephaly. In mice, excision of different Auts2 exons (7, 8, or 15) caused distinct phenotypes, variously including neonatal breathing abnormalities, cerebellar hypoplasia, dentate gyrus hypoplasia, EEG abnormalities, and behavioral changes. In mouse embryonic stem cells, AUTS2 could promote or delay neuronal differentiation. Cerebral organoids, derived from an AUTS2 syndrome patient containing a pathogenic missense variant in exon 9, exhibited neocortical growth defects. Emerging technologies for analysis of human cerebral organoids will be increasingly useful for understanding mechanisms underlying AUTS2 syndrome. Questions for future research include whether AUTS2 binds RNA directly, how AUTS2 regulates neurogenesis, and how AUTS2 modulates neural circuit formation.
RESUMO
BACKGROUND: Glioblastoma (GBM) is the most common primary brain tumor in adults with a median survival of approximately 15 months; therefore, more effective treatment options for GBM are required. To identify new drugs targeting GBMs, we performed a high-throughput drug screen using patient-derived neurospheres cultured to preferentially retain their glioblastoma stem cell (GSC) phenotype. METHODS: High-throughput drug screening was performed on GSCs followed by a dose-response assay of the 5 identified original "hits." A PI3K/mTOR dependency to a proteasome inhibitor (carfilzomib), was confirmed by genetic and pharmacologic experiments. Proteasome Inhibition Response Signatures were derived from proteomic and bioinformatic analysis. Molecular mechanism of action was determined using three-dimensional (3D) GBM-organoids and preclinical orthotopic models. RESULTS: We found that GSCs were highly sensitive to proteasome inhibition due to an underlying dependency on an increased protein synthesis rate, and loss of autophagy, associated with PTEN loss and activation of the PI3K/mTOR pathway. In contrast, combinatory inhibition of autophagy and the proteasome resulted in enhanced cytotoxicity specifically in GSCs that did express PTEN. Finally, proteasome inhibition specifically increased cell death markers in 3D GBM-organoids, suppressed tumor growth, and increased survival of mice orthotopically engrafted with GSCs. As perturbations of the PI3K/mTOR pathway occur in nearly 50% of GBMs, these findings suggest that a significant fraction of these tumors could be vulnerable to proteasome inhibition. CONCLUSIONS: Proteasome inhibition is a potential synthetic lethal therapeutic strategy for GBM with proteasome addiction due to a high protein synthesis rate and autophagy deficiency.
Assuntos
Antineoplásicos , Neoplasias Encefálicas , Glioblastoma , Animais , Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Humanos , Camundongos , Células-Tronco Neoplásicas , PTEN Fosfo-Hidrolase/genética , Complexo de Endopeptidases do Proteassoma , ProteômicaRESUMO
Rapamycin extends lifespan and attenuates age-related pathologies in mice when administered through diet at 14 parts per million (PPM). Recently, we reported that daily intraperitoneal injection of rapamycin at 8 mg/kg attenuates mitochondrial disease symptoms and progression in the Ndufs4 knockout mouse model of Leigh Syndrome. Although rapamycin is a widely used pharmaceutical agent dosage has not been rigorously examined and no dose-response profile has been established. Given these observations we sought to determine if increased doses of oral rapamycin would result in more robust impact on mTOR driven parameters. To test this hypothesis, we compared the effects of dietary rapamycin at doses ranging from 14 to 378 PPM on developmental weight in control and Ndufs4 knockout mice and on health and survival in the Ndufs4 knockout model. High dose rapamycin was well tolerated, dramatically reduced weight gain during development, and overcame gender differences. The highest oral dose, approximately 27-times the dose shown to extend murine lifespan, increased survival in Ndufs4 knockout mice similarly to daily rapamycin injection without observable adverse effects. These findings have broad implications for the effective use of rapamycin in murine studies and for the translational potential of rapamycin in the treatment of mitochondrial disease. This data, further supported by a comparison of available literature, suggests that 14 PPM dietary rapamycin is a sub-optimal dose for targeting mTOR systemically in mice. Our findings suggest that the role of mTOR in mammalian biology may be broadly underestimated when determined through treatment with rapamycin at commonly used doses.
RESUMO
Many genes that affect replicative lifespan (RLS) in the budding yeast Saccharomyces cerevisiae also affect aging in other organisms such as C. elegans and M. musculus. We performed a systematic analysis of yeast RLS in a set of 4,698 viable single-gene deletion strains. Multiple functional gene clusters were identified, and full genome-to-genome comparison demonstrated a significant conservation in longevity pathways between yeast and C. elegans. Among the mechanisms of aging identified, deletion of tRNA exporter LOS1 robustly extended lifespan. Dietary restriction (DR) and inhibition of mechanistic Target of Rapamycin (mTOR) exclude Los1 from the nucleus in a Rad53-dependent manner. Moreover, lifespan extension from deletion of LOS1 is nonadditive with DR or mTOR inhibition, and results in Gcn4 transcription factor activation. Thus, the DNA damage response and mTOR converge on Los1-mediated nuclear tRNA export to regulate Gcn4 activity and aging.
Assuntos
Envelhecimento/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Longevidade/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Caenorhabditis elegans/genética , Restrição Calórica , Dano ao DNA/genética , Deleção de Genes , Regulação da Expressão Gênica/genética , Genoma , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genéticaRESUMO
Mitochondrial dysfunction contributes to numerous health problems, including neurological and muscular degeneration, cardiomyopathies, cancer, diabetes, and pathologies of aging. Severe mitochondrial defects can result in childhood disorders such as Leigh syndrome, for which there are no effective therapies. We found that rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, robustly enhances survival and attenuates disease progression in a mouse model of Leigh syndrome. Administration of rapamycin to these mice, which are deficient in the mitochondrial respiratory chain subunit Ndufs4 [NADH dehydrogenase (ubiquinone) Fe-S protein 4], delays onset of neurological symptoms, reduces neuroinflammation, and prevents brain lesions. Although the precise mechanism of rescue remains to be determined, rapamycin induces a metabolic shift toward amino acid catabolism and away from glycolysis, alleviating the buildup of glycolytic intermediates. This therapeutic strategy may prove relevant for a broad range of mitochondrial diseases.
Assuntos
Doença de Leigh/tratamento farmacológico , Doenças Mitocondriais/tratamento farmacológico , Terapia de Alvo Molecular , Complexos Multiproteicos/antagonistas & inibidores , Fármacos Neuroprotetores/uso terapêutico , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/patologia , Modelos Animais de Doenças , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Glicólise/efeitos dos fármacos , Doença de Leigh/genética , Doença de Leigh/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Camundongos Mutantes , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologiaRESUMO
Although environmental stress likely plays a significant role in promoting aging, the relationship remains poorly understood. To characterize this interaction in a more comprehensive manner, we examined the stress response profiles for 46 long-lived yeast mutant strains across four different stress conditions (oxidative, ER, DNA damage, and thermal), grouping genes based on their associated stress response profiles. Unexpectedly, cells lacking the mitochondrial AAA protease gene AFG3 clustered strongly with long-lived strains lacking cytosolic ribosomal proteins of the large subunit. Similar to these ribosomal protein mutants, afg3Δ cells show reduced cytoplasmic mRNA translation, enhanced resistance to tunicamycin that is independent of the ER unfolded protein response, and Sir2-independent but Gcn4-dependent lifespan extension. These data demonstrate an unexpected link between a mitochondrial protease, cytoplasmic mRNA translation, and aging.
Assuntos
Adenosina Trifosfatases/genética , Citosol/metabolismo , Mitocôndrias/genética , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/metabolismo , Fatores Etários , Longevidade , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
Chronological aging of budding yeast cells results in a reduction in subsequent replicative life span through unknown mechanisms. Here we show that dietary restriction during chronological aging delays the reduction in subsequent replicative life span up to at least 23days of chronological age. We further show that among the viable portion of the control population aged 26days, individual cells with the lowest mitochondrial membrane potential have the longest subsequent replicative lifespan. These observations demonstrate that dietary restriction modulates a common molecular mechanism linking chronological and replicative aging in yeast and indicate a critical role for mitochondrial function in this process.
Assuntos
Restrição Calórica , Mitocôndrias/fisiologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Animais , Divisão Celular/fisiologia , Técnicas de Cultura/métodos , Citometria de Fluxo , Glucose/metabolismo , Potencial da Membrana Mitocondrial/fisiologia , Reprodução/fisiologia , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Fatores de TempoRESUMO
Dietary restriction (DR) increases lifespan and attenuates age-related phenotypes in many organisms; however, the effect of DR on longevity of individuals in genetically heterogeneous populations is not well characterized. Here, we describe a large-scale effort to define molecular mechanisms that underlie genotype-specific responses to DR. The effect of DR on lifespan was determined for 166 single gene deletion strains in Saccharomyces cerevisiae. Resulting changes in mean lifespan ranged from a reduction of 79% to an increase of 103%. Vacuolar pH homeostasis, superoxide dismutase activity, and mitochondrial proteostasis were found to be strong determinants of the response to DR. Proteomic analysis of cells deficient in prohibitins revealed induction of a mitochondrial unfolded protein response (mtUPR), which has not previously been described in yeast. Mitochondrial proteotoxic stress in prohibitin mutants was suppressed by DR via reduced cytoplasmic mRNA translation. A similar relationship between prohibitins, the mtUPR, and longevity was also observed in Caenorhabditis elegans. These observations define conserved molecular processes that underlie genotype-dependent effects of DR that may be important modulators of DR in higher organisms.
Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Restrição Calórica , Dieta , Saccharomyces cerevisiae/genética , Aerobiose , Animais , Autofagia , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/metabolismo , Genótipo , Proibitinas , Saccharomyces cerevisiae/citologia , Resposta a Proteínas não Dobradas/genéticaRESUMO
Chronological and replicative aging have been studied in yeast as alternative paradigms for post-mitotic and mitotic aging, respectively. It has been known for more than a decade that cells of the S288C background aged chronologically in rich medium have reduced replicative lifespan relative to chronologically young cells. Here we report replication of this observation in the diploid BY4743 strain background. We further show that the reduction in replicative lifespan from chronological aging is accelerated when cells are chronologically aged under standard conditions in synthetic complete medium rather than rich medium. The loss of replicative potential with chronological age is attenuated by buffering the pH of the chronological aging medium to 6.0, an intervention that we have previously shown can extend chronological lifespan. These data demonstrate that extracellular acidification of the culture medium can cause intracellular damage in the chronologically aging population that is asymmetrically segregated by the mother cell to limit subsequent replicative lifespan.