Effect of the Ketone Body, D-ß-Hydroxybutyrate, on Sirtuin2-Mediated Regulation of Mitochondrial Quality Control and the Autophagy-Lysosomal Pathway.

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.

Nucleophagy contributes to genome stability through degradation of type II topoisomerases A and B and nucleolar components.

The nuclear architecture of mammalian cells can be altered as a consequence of anomalous accumulation of nuclear proteins or genomic alterations. Most of the knowledge about nuclear dynamics comes from studies on cancerous cells. How normal healthy cells maintain genome stability, avoiding accumulation of nuclear damaged material, is less understood. Here, we describe that primary mouse embryonic fibroblasts develop a basal level of nuclear buds and micronuclei, which increase after etoposide-induced DNA double-stranded breaks. Both basal and induced nuclear buds and micronuclei colocalize with the autophagic proteins BECN1 and LC3B (also known as MAP1LC3B) and with acidic vesicles, suggesting their clearance by nucleophagy. Some of the nuclear alterations also contain autophagic proteins and type II DNA topoisomerases (TOP2A and TOP2B), or the nucleolar protein fibrillarin, implying they are also targets of nucleophagy. We propose that basal nucleophagy contributes to genome and nuclear stability, as well as in response to DNA damage.

Chromatin Structure from Development to Ageing.

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.

Rats exhibit age-related mosaic loss of chromosome Y.

Mosaic loss of the Y chromosome (LOY) is the most frequent chromosomal aberration in aging men and is strongly correlated with mortality and disease. To date, studies of LOY have only been performed in humans, and so it is unclear whether LOY is a natural consequence of our relatively long lifespan or due to exposure to human-specific external stressors. Here, we explored whether LOY could be detected in rats. We applied a locus-specific PCR and target sequencing approach that we used as a proxy to estimate LOY in 339 samples covering eleven tissues from young and old individuals. We detected LOY in four tissues of older rats. To confirm the results from the PCR screening, we re-sequenced 60 full genomes from old rats, which revealed that the Y chromosome is the sole chromosome with low copy numbers. Finally, our results suggest that LOY is associated with other structural aberrations on the Y chromosome and possibly linked to the mosaic loss of the X chromosome. This is the first report, to our knowledge, demonstrating that the patterns of LOY observed in aging men are also present in a rodent, and conclude that LOY may be a natural process in placental mammals.

Selective Moonlighting Cell-Penetrating Peptides.

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.

Enhanced Activity of Exportin-1/CRM1 in Neurons Contributes to Autophagy Dysfunction and Senescent Features in Old Mouse Brain.

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.

Programmed Cell Senescence in the Mouse Developing Spinal Cord and Notochord.

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.

Connecting Mind-Body Therapy-Mediated Effects to Pathological Features of Alzheimer's Disease.

Alzheimer's disease (AD) is a complex, multifactorial neurodegenerative disorder that represents a major and increasing global health challenge. In most cases, the first clinical symptoms of AD are preceded by neuropathological changes in the brain that develop years to decades before their onset. Therefore, research in the last years has focused on this preclinical stage of AD trying to discover intervention strategies that might, if implemented effectively, delay or prevent disease progression. Among those strategies, mind-body therapies such as yoga and meditation have gained increasing interest as complementary alternative interventions. Several studies have reported a positive impact of yoga and meditation on brain health in both healthy older adults and dementia patients. However, the underlying neurobiological mechanisms contributing to these effects are currently not known in detail. More specifically, it is not known whether yogic interventions, directly or indirectly, can modulate risk factors or pathological mechanisms involved in the development of dementia. In this article, we first review the literature on the effects of yogic practices on outcomes such as cognitive functioning and neuropsychiatric symptoms in patients with mild cognitive impairment and dementia. Then, we analyze how yogic interventions affect different risk factors as well as aspects of AD pathophysiology based on observations of studies in healthy individuals or subjects with other conditions than dementia. Finally, we integrate this evidence and propose possible mechanisms that might explain the positive effects of yogic interventions in cognitively impaired individuals.

Antimicrobial Peptide against Mycobacterium Tuberculosis That Activates Autophagy Is an Effective Treatment for Tuberculosis.

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.

Role of autophagy in the chemopreventive effect of the IFC-305 compound in the sequential model of cirrhosis-hepatocellular carcinoma in the rat and in vitro.

Hepatocellular carcinoma (HCC) can be originated from various etiologies and is preceded mostly by cirrhosis. Unfortunately, there is no effective treatment due to its late prognosis. Alterations in autophagy have been reported during the development and progression of HCC. Autophagy allows for the maintenance of a positive energy balance and the proper functioning of organelles through the selective degradation of cellular components. It has been demonstrated that autophagy suppresses spontaneous tumorigenesis in the liver. Therefore, autophagy has become a therapeutic target for effective HCC therapies. We have previously demonstrated that the adenosine-derived compound, IFC-305, has a chemopreventive effect on HCC, in addition to maintaining mitochondrial function in a sequential model of cirrhosis-HCC. Thus, the aim of this work was to determine if IFC-305 has an effect on autophagy in the sequential model of cirrhosis-HCC induced by diethylnitrosamine or in vitro in the HCC cell line HepG2 and mouse embryonic fibroblasts. The results of this work showed that IFC-305 modifies the levels of the BECN1, p62/SQSTM1 and LC3-II proteins that play an important role in the autophagic process. In vivo, IFC-305 regulates the levels of the PINK1 and PARKIN proteins that specifically mark mitochondria for repair or degradation. In the HepG2 cell line, its effect was accompanied by a decrease in cell viability. Interestingly, in nontumoral cells the time to autophagy induction was different compared to the HepG2 cells. This study suggests that autophagy induction may be part of the mechanism by which IFC-305 maintains mitochondrial function, thereby facilitating the prevention and reversal of HCC.

Lamin B receptor: role on chromatin structure, cellular senescence and possibly aging.

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.

Autophagy Regulation by the Translation Machinery and Its Implications in Cancer.

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.

The nuclear receptor NR4A1 is regulated by SUMO modification to induce autophagic cell death.

NR4A is a nuclear receptor protein family whose members act as sensors of cellular environment and regulate multiple processes such as metabolism, proliferation, migration, apoptosis, and autophagy. Since the ligand binding domains of these receptors have no cavity for ligand interaction, their function is most likely regulated by protein abundance and post-translational modifications. In particular, NR4A1 is regulated by protein abundance, phosphorylation, and subcellular distribution (nuclear-cytoplasmic translocation), and acts both as a transcription factor and as a regulator of other interacting proteins. SUMOylation is a post-translational modification that can affect protein stability, transcriptional activity, alter protein-protein interactions and modify intracellular localization of target proteins. In the present study we evaluated the role of SUMOylation as a posttranslational modification that can regulate the activity of NR4A1 to induce autophagy-dependent cell death. We focused on a model potentially relevant for neuronal cell death and demonstrated that NR4A1 needs to be SUMOylated to induce autophagic cell death. We observed that a triple mutant in SUMOylation sites has reduced SUMOylation, increased transcriptional activity, altered intracellular distribution, and more importantly, its ability to induce autophagic cell death is impaired.

Cortical neurons develop a senescence-like phenotype promoted by dysfunctional autophagy.

Senescent cells accumulate in various tissues and organs with aging altering surrounding tissue due to an active secretome, and at least in mice their elimination extends healthy lifespan and ameliorates several chronic diseases. Whether all cell types senesce, including post-mitotic cells, has been poorly described mainly because cellular senescence was defined as a permanent cell cycle arrest. Nevertheless, neurons with features of senescence have been described in old rodent and human brains. In this study we characterized an in vitro model useful to study the molecular basis of senescence of primary rat cortical cells that recapitulates senescent features described in brain aging. We found that in long-term cultures, rat primary cortical neurons displayed features of cellular senescence before glial cells did, and developed a functional senescence-associated secretory phenotype able to induce paracrine premature senescence of mouse embryonic fibroblasts but proliferation of rat glial cells. Functional autophagy seems to prevent neuronal senescence, as we observed an autophagic flux reduction in senescent neurons both in vitro and in vivo, and autophagy impairment induced cortical cell senescence while autophagy stimulation inhibited it. Our findings suggest that aging-associated dysfunctional autophagy contributes to senescence transition also in neuronal cells.

Re-thinking the Etiological Framework of Neurodegeneration.

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.

Reduced lifespan of mice lacking catalase correlates with altered lipid metabolism without oxidative damage or premature aging.

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.

Cellular Senescence, Neurological Function, and Redox State.

SIGNIFICANCE: Cellular senescence, characterized by permanent cell cycle arrest, has been extensively studied in mitotic cells such as fibroblasts. However, senescent cells have also been observed in the brain. Even though it is recognized that cellular energetic metabolism and redox homeostasis are perturbed in the aged brain and neurodegenerative diseases (NDDs), it is still unknown which alterations in the overall physiology can stimulate cellular senescence induction and their relationship with the former events. Recent Advances: Recent findings have shown that during prolonged inflammatory and pathologic events, the blood-brain barrier could be compromised and immune cells might enter the brain; this fact along with the brain's high oxygen dependence might result in oxidative damage to macromolecules and therefore senescence induction. Thus, cellular senescence in different brain cell types is revised here. CRITICAL ISSUES: Most information related to cellular senescence in the brain has been obtained from research in glial cells since it has been assumed that the senescent phenotype is a feature exclusive to mitotic cells. Nevertheless, neurons with senescence hallmarks have been observed in old mouse brains. Therefore, although this is a controversial topic in the field, here we summarize and integrate the observations from several studies and propose that neurons indeed senesce. FUTURE DIRECTIONS: It is still unknown which alterations in the overall metabolism can stimulate senescence induction in the aged brain, what are the mechanisms and signaling pathways, and what is their relationship to NDD development. The understanding of these processes will expose new targets to intervene age-associated pathologies.-Antioxid. Redox Signal. 28, 1704-1723.

Connecting chaperone-mediated autophagy dysfunction to cellular senescence.

Chaperone-mediated autophagy (CMA) is one of the main pathways of the lysosome-autophagy proteolytic system. It regulates different cellular process through the selective degradation of cytosolic proteins. In ageing, the function of CMA is impaired causing an inefficient stress response and the accumulation of damaged, oxidized or misfolded proteins, which is associated with numerous age-related diseases. Deficient protein degradation alters cellular proteostasis and activates signaling pathways that culminate in the induction of cellular senescence, whose accumulation is a typical feature of ageing. However, the relationship between CMA activity and cellular senescence has been poorly studied. Here, we review and integrate evidence showing that CMA dysfunction correlates with the acquisition of many hallmarks of cellular senescence and propose that loss of CMA function during aging promotes cellular senescence.

Autophagy fails to prevent glucose deprivation/glucose reintroduction-induced neuronal death due to calpain-mediated lysosomal dysfunction in cortical neurons.

Autophagy is triggered during nutrient and energy deprivation in a variety of cells as a homeostatic response to metabolic stress. In the CNS, deficient autophagy has been implicated in neurodegenerative diseases and ischemic brain injury. However, its role in hypoglycemic damage is poorly understood and the dynamics of autophagy during the hypoglycemic and the glucose reperfusion periods, has not been fully described. In the present study, we analyzed the changes in the content of the autophagy proteins BECN1, LC3-II and p62/SQSTM1 by western blot, and autophagosome formation was followed through time-lapse experiments, during glucose deprivation (GD) and glucose reintroduction (GR) in cortical cultures. According to the results, autophagosome formation rapidly increased during GD, and was followed by an active autophagic flux early after glucose replenishment. However, cells progressively died during GR and autophagy inhibition reduced neuronal death. Neurons undergoing apoptosis during GR did not form autophagosomes, while those surviving up to late GR showed autophagosomes. Calpain activity strongly increased during GR and remained elevated during progressive neuronal death. Its activation led to the cleavage of LAMP2 resulting in lysosome membrane permeabilization (LMP) and release of cathepsin B to the cytosol. Calpain inhibition prevented LMP and increased the number of neurons containing lysosomes and autophagosomes increasing cell viability. Taken together, the present results suggest that calpain-mediated lysosome dysfunction during GR turns an adaptive autophagy response to energy stress into a defective autophagy pathway, which contributes to neuronal death. In these conditions, autophagy inhibition results in the improvement of cell survival.