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Mitochondrial activity and quality control are essential for neuronal homeostasis as neurons rely on glucose oxidative metabolism. The ketone body, D-ß-hydroxybutyrate (D-BHB), is metabolized to acetyl-CoA in brain mitochondria and used as an energy fuel alternative to glucose. We have previously reported that D-BHB sustains ATP production and stimulates the autophagic flux under glucose deprivation in neurons; however, the effects of D-BHB on mitochondrial turnover under physiological conditions are still unknown. Sirtuins (SIRTs) are NAD+-activated protein deacetylases involved in the regulation of mitochondrial biogenesis and mitophagy through the activation of transcription factors FOXO1, FOXO3a, TFEB and PGC1α coactivator. Here, we aimed to investigate the effect of D-BHB on mitochondrial turnover in cultured neurons and the mechanisms involved. Results show that D-BHB increased mitochondrial membrane potential and regulated the NAD+/NADH ratio. D-BHB enhanced FOXO1, FOXO3a and PGC1α nuclear levels in an SIRT2-dependent manner and stimulated autophagy, mitophagy and mitochondrial biogenesis. These effects increased neuronal resistance to energy stress. D-BHB also stimulated the autophagic-lysosomal pathway through AMPK activation and TFEB-mediated lysosomal biogenesis. Upregulation of SIRT2, FOXOs, PGC1α and TFEB was confirmed in the brain of ketogenic diet (KD)-treated mice. Altogether, the results identify SIRT2, for the first time, as a target of D-BHB in neurons, which is involved in the regulation of autophagy/mitophagy and mitochondrial quality control.
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NAD , Sirtuína 2 , Animais , Camundongos , Ácido 3-Hidroxibutírico/farmacologia , Ácido 3-Hidroxibutírico/metabolismo , Autofagia , Glucose/metabolismo , Corpos Cetônicos/metabolismo , Corpos Cetônicos/farmacologia , Lisossomos/metabolismo , Mitocôndrias/metabolismo , NAD/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Sirtuína 2/metabolismoRESUMO
Nuclear structure influences genome architecture, which contributes to determine patterns of gene expression. Global changes in chromatin dynamics are essential during development and differentiation, and are one of the hallmarks of ageing. This chapter describes the molecular dynamics of chromatin structure that occur during development and ageing. In the first part, we introduce general information about the nuclear lamina, the chromatin structure, and the 3D organization of the genome. Next, we detail the molecular hallmarks found during development and ageing, including the role of DNA and histone modifications, 3D genome dynamics, and changes in the nuclear lamina. Within the chapter we discuss the implications that genome structure has on the mechanisms that drive development and ageing, and the physiological consequences when these mechanisms fail.
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Cromatina , Lâmina Nuclear , Cromatina/genética , Cromatina/metabolismo , Lâmina Nuclear/genética , Lâmina Nuclear/metabolismo , Genoma , Simulação de Dinâmica MolecularRESUMO
Cell penetrating peptides (CPPs) are molecules capable of passing through biological membranes. This capacity has been used to deliver impermeable molecules into cells, such as drugs and DNA probes, among others. However, the internalization of these peptides lacks specificity: CPPs internalize indistinctly on different cell types. Two major approaches have been described to address this problem: (i) targeting, in which a receptor-recognizing sequence is added to a CPP, and (ii) activation, where a non-active form of the CPP is activated once it interacts with cell target components. These strategies result in multifunctional peptides (i.e., penetrate and target recognition) that increase the CPP's length, the cost of synthesis and the likelihood to be degraded or become antigenic. In this work we describe the use of machine-learning methods to design short selective CPP; the reduction in size is accomplished by embedding two or more activities within a single CPP domain, hence we referred to these as moonlighting CPPs. We provide experimental evidence that these designed moonlighting peptides penetrate selectively in targeted cells and discuss areas of opportunity to improve in the design of these peptides.
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Brain aging is characterized by dysfunctional autophagy and cellular senescence, among other features. While autophagy can either promote or suppress cellular senescence in proliferating cells, in postmitotic cells, such as neurons, autophagy impairment promotes cellular senescence. CRM1 (exportin-1/XPO1) exports hundreds of nuclear proteins into the cytoplasm, including the transcription factors TFEB (the main inducer of autophagy and lysosomal biogenesis genes) and STAT3, another autophagy modulator. It appears that CRM1 is a modulator of aging-associated senescence and autophagy, because pharmacological inhibition of CRM1 improved autophagic degradation in flies, by increasing nuclear TFEB levels, and because enhanced CRM1 activity is mechanistically linked to senescence in fibroblasts from Hutchinson-Gilford progeria syndrome patients and old healthy individuals; furthermore, the exogenous overexpression of CRM1 induced senescence in normal fibroblasts. In this work, we tested the hypothesis that impaired autophagic flux during brain aging occurs due to CRM1 accumulation in the brain. We found that CRM1 levels and activity increased in the hippocampus and cortex during physiological aging, which resulted in a decrease of nuclear TFEB and STAT3. Consistent with an autophagic flux impairment, we observed accumulation of the autophagic receptor p62/SQSTM1 in neurons of old mice, which correlated with increased neuronal senescence. Using an in vitro model of neuronal senescence, we demonstrate that CRM1 inhibition improved autophagy flux and reduced SA-ß-gal activity by restoring TFEB nuclear localization. Collectively, our data suggest that enhanced CRM1-mediated export of proteins during brain aging perturbs neuronal homeostasis, contributing to autophagy impairment, and neuronal senescence.
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Envelhecimento/metabolismo , Autofagia , Encéfalo/metabolismo , Senescência Celular , Carioferinas/metabolismo , Neurônios/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Envelhecimento/patologia , Animais , Encéfalo/patologia , Camundongos , Camundongos Transgênicos , Neurônios/patologia , Ratos , Ratos Wistar , Proteína Exportina 1RESUMO
Programmed cell senescence is a cellular process that seems to contribute to embryo development, in addition to cell proliferation, migration, differentiation and programmed cell death, and has been observed in evolutionary distant organisms such as mammals, amphibians, birds and fish. Programmed cell senescence is a phenotype similar to stress-induced cellular senescence, characterized by the expression of the cell cycle inhibitors p21CIP1/WAF and p16INK4A, increased activity of a lysosomal enzyme with beta-galactosidase activity (coined senescence-associated beta-galactosidase) and secretion of growth factors, interleukins, chemokines, metalloproteases, etc., collectively known as a senescent-associated secretory phenotype that instructs surrounding tissue. How wide is the distribution of programmed cell senescence during mouse development and its specific mechanisms to shape the embryo are still poorly understood. Here, we investigated whether markers of programmed cell senescence are found in the developing mouse spinal cord and notochord. We found discrete areas and developmental windows with high senescence-associated beta galactosidase in both spinal cord and notochord, which was reduced in mice embryos developed ex-utero in the presence of the senolytic ABT-263. Expression of p21CIP1/WAF was documented in epithelial cells of the spinal cord and the notochord, while p16INK4A was observed in motoneurons. Treatment with the senolytic ABT-263 decreased the number of motoneurons, supporting their senescent phenotype. Our data suggest that a subpopulation of motoneurons in the developing spinal cord, as well as some notochord cells undergo programmed cell senescence.
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Mycobacterium tuberculosis (MTB) is the principal cause of human tuberculosis (TB), which is a serious health problem worldwide. The development of innovative therapeutic modalities to treat TB is mainly due to the emergence of multi drug resistant (MDR) TB. Autophagy is a cell-host defense process. Previous studies have reported that autophagy-activating agents eliminate intracellular MDR MTB. Thus, combining a direct antibiotic activity against circulating bacteria with autophagy activation to eliminate bacteria residing inside cells could treat MDR TB. We show that the synthetic peptide, IP-1 (KFLNRFWHWLQLKPGQPMY), induced autophagy in HEK293T cells and macrophages at a low dose (10 µM), while increasing the dose (50 µM) induced cell death; IP-1 induced the secretion of TNFα in macrophages and killed Mtb at a dose where macrophages are not killed by IP-1. Moreover, IP-1 showed significant therapeutic activity in a mice model of progressive pulmonary TB. In terms of the mechanism of action, IP-1 sequesters ATP in vitro and inside living cells. Thus, IP-1 is the first antimicrobial peptide that eliminates MDR MTB infection by combining four activities: reducing ATP levels, bactericidal activity, autophagy activation, and TNFα secretion.
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The nuclear envelope is composed by an outer nuclear membrane and an inner nuclear membrane, which is underlain by the nuclear lamina that provides the nucleus with mechanical strength for maintaining structure and regulates chromatin organization for modulating gene expression and silencing. A layer of heterochromatin is beneath the nuclear lamina, attached by inner nuclear membrane integral proteins such as Lamin B receptor (LBR). LBR is a chimeric protein, having also a sterol reductase activity with which it contributes to cholesterol synthesis. Lukasova et al. showed that when DNA is damaged by ɣ-radiation in cancer cells, LBR is lost causing chromatin structure changes and promoting cellular senescence. Cellular senescence is characterized by terminal cell cycle arrest and the expression and secretion of various growth factors, cytokines, metalloproteinases, etc., collectively known as senescence-associated secretory phenotype (SASP) that cause chronic inflammation and tumor progression when they persist in the tissue. Therefore, it is fundamental to understand the molecular basis for senescence establishment, maintenance and the regulation of SASP. The work of Lukasova et al. contributed to our understanding of cellular senescence establishment and provided the basis that lead to the further discovery that chromatin changes caused by LBR reduction induce an up-regulated expression of SASP factors. LBR dysfunction has relevance in several diseases and possibly in physiological aging. The potential bifunctional role of LBR on cellular senescence establishment, namely its role in chromatin structure together with its enzymatic activity contributing to cholesterol synthesis, provide a new target to develop potential anti-aging therapies.
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Senescência Celular , Receptores Citoplasmáticos e Nucleares , Cromatina , Membrana Nuclear , Receptor de Lamina BRESUMO
Various metabolic pathways and molecular processes in the cell act intertwined, and dysregulating the interplay between some of them may lead to cancer. It is only recently that defects in the translation process, i.e., the synthesis of proteins by the ribosome using a messenger (m)RNA as a template and translation factors, have begun to gain strong attention as a cause of autophagy dysregulation with effects in different maladies, including cancer. Autophagy is an evolutionarily conserved catabolic process that degrades cytoplasmic elements in lysosomes. It maintains cellular homeostasis and preserves cell viability under various stress conditions, which is crucial for all eukaryotic cells. In this review, we discuss recent advances shedding light on the crosstalk between the translation and the autophagy machineries and its impact on tumorigenesis. We also summarize how this interaction is being the target for novel therapies to treat cancer.
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Neurodegenerative diseases are among the leading causes of disability and death worldwide. The disease-related socioeconomic burden is expected to increase with the steadily increasing life expectancy. In spite of decades of clinical and basic research, most strategies designed to manage degenerative brain diseases are palliative. This is not surprising as neurodegeneration progresses "silently" for decades before symptoms are noticed. Importantly, conceptual models with heuristic value used to study neurodegeneration have been constructed retrospectively, based on signs and symptoms already present in affected patients; a circumstance that may confound causes and consequences. Hence, innovative, paradigm-shifting views of the etiology of these diseases are necessary to enable their timely prevention and treatment. Here, we outline four alternative views, not mutually exclusive, on different etiological paths toward neurodegeneration. First, we propose neurodegeneration as being a secondary outcome of a primary cardiovascular cause with vascular pathology disrupting the vital homeostatic interactions between the vasculature and the brain, resulting in cognitive impairment, dementia, and cerebrovascular events such as stroke. Second, we suggest that the persistence of senescent cells in neuronal circuits may favor, together with systemic metabolic diseases, neurodegeneration to occur. Third, we argue that neurodegeneration may start in response to altered body and brain trophic interactions established via the hardwire that connects peripheral targets with central neuronal structures or by means of extracellular vesicle (EV)-mediated communication. Lastly, we elaborate on how lifespan body dysbiosis may be linked to the origin of neurodegeneration. We highlight the existence of bacterial products that modulate the gut-brain axis causing neuroinflammation and neuronal dysfunction. As a concluding section, we end by recommending research avenues to investigate these etiological paths in the future. We think that this requires an integrated, interdisciplinary conceptual research approach based on the investigation of the multimodal aspects of physiology and pathophysiology. It involves utilizing proper conceptual models, experimental animal units, and identifying currently unused opportunities derived from human data. Overall, the proposed etiological paths and experimental recommendations will be important guidelines for future cross-discipline research to overcome the translational roadblock and to develop causative treatments for neurodegenerative diseases.
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The relationship between the mechanisms that underlie longevity and aging and the metabolic alterations due to feeding conditions has not been completely defined. In the present work, through the deletion of the gene encoding catalase, hydrogen peroxide (H2O2) was uncovered as a relevant regulator of longevity and of liver metabolism. Mice lacking catalase (Cat-/-) fed ad libitum with a regular diet showed a shorter lifespan than wild type mice, which correlated with reduced body weight, blood glucose levels and liver fat accumulation, but not with increased oxidative damage or consistent premature aging. High fat diet (HFD) and fasting increased oxidative damage in the liver of wild type animals but, unexpectedly, this was not the case for that of Cat-/- mice. Interestingly, although HFD feeding similarly increased the body weight of Cat-/- and wild-type mice, hyperglycemia and liver steatosis did not develop in the former. Fat accumulation due to fasting, on the other hand, was diminished in mice lacking catalase, which correlated with increased risk of death and low ketone body blood levels. Alteration in expression of some metabolic genes in livers of catalase deficient mice was consistent with reduced lipogenesis. Specifically, Pparγ2 expression up-regulation in response to a HFD and down-regulation upon fasting was lower and higher, respectively, in livers of Cat-/- than of wild type mice, and a marked decay was observed during Cat-/- mice aging. We propose that catalase regulates lipid metabolism in the liver by an evolutionary conserved mechanism that is determinant of lifespan without affecting general oxidative damage.
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Catalase/genética , Metabolismo dos Lipídeos/genética , Longevidade/genética , PPAR gama/genética , Acatalasia/genética , Acatalasia/metabolismo , Senilidade Prematura/genética , Senilidade Prematura/patologia , Animais , Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Humanos , Peróxido de Hidrogênio/metabolismo , Resistência à Insulina/genética , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Knockout , Obesidade/genética , Obesidade/patologia , Estresse Oxidativo/genéticaRESUMO
Multifunctionality is a common trait of many natural proteins and peptides, yet the rules to generate such multifunctionality remain unclear. We propose that the rules defining some protein/peptide functions are compatible. To explore this hypothesis, we trained a computational method to predict cell-penetrating peptides at the sequence level and learned that antimicrobial peptides and DNA-binding proteins are compatible with the rules of our predictor. Based on this finding, we expected that designing peptides for CPP activity may render AMP and DNA-binding activities. To test this prediction, we designed peptides that embedded two independent functional domains (nuclear localization and yeast pheromone activity), linked by optimizing their composition to fit the rules characterizing cell-penetrating peptides. These peptides presented effective cell penetration, DNA-binding, pheromone and antimicrobial activities, thus confirming the effectiveness of our computational approach to design multifunctional peptides with potential therapeutic uses. Our computational implementation is available at http://bis.ifc.unam.mx/en/software/dcf.