Misfolded GBA/ß-glucocerebrosidase impairs ER-quality control by chaperone-mediated autophagy in Parkinson disease.

Inhibition of chaperone-mediated autophagy (CMA), a selective type of lysosomal degradation for intracellular proteins, may contribute to pathogenesis in neurodegenerative diseases including Parkinson disease (PD). Pathogenic variants of PD-related proteins that reside in the cytosol, including SNCA/alpha-synuclein, LRRK2 (leucine rich repeat kinase 2), UCHL1 (ubiquitin Cterminal hydrolase 1) and VPS35 (VPS35 retromer complex component), exert inhibitory effects on CMA. Decreased CMA activity has also been reported in sporadic PD patients, consistent with an association between CMA inhibition and PD. We have now reported the first example of CMA dysfunction caused by a non-cytosolic PD-related protein, GBA/ß-glucocerebrosidase, the most common genetic risk factor for PD, which uncovers a new role for CMA in endoplasmic reticulum (ER) quality control.

Protective role of chaperone-mediated autophagy against atherosclerosis.

Chaperone-mediated autophagy (CMA) contributes to regulation of energy homeostasis by timely degradation of enzymes involved in glucose and lipid metabolism. Here, we report reduced CMA activity in vascular smooth muscle cells and macrophages in murine and human arteries in response to atherosclerotic challenges. We show that in vivo genetic blockage of CMA worsens atherosclerotic pathology through both systemic and cell-autonomous changes in vascular smooth muscle cells and macrophages, the two main cell types involved in atherogenesis. CMA deficiency promotes dedifferentiation of vascular smooth muscle cells and a proinflammatory state in macrophages. Conversely, a genetic mouse model with up-regulated CMA shows lower vulnerability to proatherosclerotic challenges. We propose that CMA could be an attractive therapeutic target against cardiovascular diseases.

Mutant glucocerebrosidase impairs α-synuclein degradation by blockade of chaperone-mediated autophagy.

The most common genetic risk factors for Parkinson's disease (PD) are a set of heterozygous mutant (MT) alleles of the GBA1 gene that encodes ß-glucocerebrosidase (GCase), an enzyme normally trafficked through the ER/Golgi apparatus to the lysosomal lumen. We found that half of the GCase in lysosomes from postmortem human GBA-PD brains was present on the lysosomal surface and that this mislocalization depends on a pentapeptide motif in GCase used to target cytosolic protein for degradation by chaperone-mediated autophagy (CMA). MT GCase at the lysosomal surface inhibits CMA, causing accumulation of CMA substrates including α-synuclein. Single-cell transcriptional analysis and proteomics of brains from GBA-PD patients confirmed reduced CMA activity and proteome changes comparable to those in CMA-deficient mouse brain. Loss of the MT GCase CMA motif rescued primary substantia nigra dopaminergic neurons from MT GCase-induced neuronal death. We conclude that MT GBA1 alleles block CMA function and produce α-synuclein accumulation.

Autophagy and the hallmarks of aging.

Autophagy, an essential cellular process that mediates degradation of proteins and organelles in lysosomes, has been tightly linked to cellular quality control for its role as part of the proteostasis network. The current interest in identifying the cellular and molecular determinants of aging, has highlighted the important contribution of malfunctioning of autophagy with age to the loss of proteostasis that characterizes all old organisms. However, the diversity of cellular functions of the different types of autophagy and the often reciprocal interactions of autophagy with other determinants of aging, is placing autophagy at the center of the aging process. In this work, we summarize evidence for the contribution of autophagy to health- and lifespan and provide examples of the bidirectional interplay between autophagic pathways and several of the so-called hallmarks of aging. This central role of autophagy in aging, and the dependence on autophagy of many geroprotective interventions, has motivated a search for direct modulators of autophagy that could be used to slow aging and extend healthspan. Here, we review some of those ongoing therapeutic efforts and comment on the potential of targeting autophagy in aging.

Gαq activation modulates autophagy by promoting mTORC1 signaling.

The mTORC1 node plays a major role in autophagy modulation. We report a role of the ubiquitous Gαq subunit, a known transducer of plasma membrane G protein-coupled receptors signaling, as a core modulator of mTORC1 and autophagy. Cells lacking Gαq/11 display higher basal autophagy, enhanced autophagy induction upon different types of nutrient stress along with a decreased mTORC1 activation status. They are also unable to reactivate mTORC1 and thus inactivate ongoing autophagy upon nutrient recovery. Conversely, stimulation of Gαq/11 promotes sustained mTORC1 pathway activation and reversion of autophagy promoted by serum or amino acids removal. Gαq is present in autophagic compartments and lysosomes and is part of the mTORC1 multi-molecular complex, contributing to its assembly and activation via its nutrient status-sensitive interaction with p62, which displays features of a Gαq effector. Gαq emerges as a central regulator of the autophagy machinery required to maintain cellular homeostasis upon nutrient fluctuations.

Chaperone-mediated autophagy prevents collapse of the neuronal metastable proteome.

Components of the proteostasis network malfunction in aging, and reduced protein quality control in neurons has been proposed to promote neurodegeneration. Here, we investigate the role of chaperone-mediated autophagy (CMA), a selective autophagy shown to degrade neurodegeneration-related proteins, in neuronal proteostasis. Using mouse models with systemic and neuronal-specific CMA blockage, we demonstrate that loss of neuronal CMA leads to altered neuronal function, selective changes in the neuronal metastable proteome, and proteotoxicity, all reminiscent of brain aging. Imposing CMA loss on a mouse model of Alzheimer's disease (AD) has synergistic negative effects on the proteome at risk of aggregation, thus increasing neuronal disease vulnerability and accelerating disease progression. Conversely, chemical enhancement of CMA ameliorates pathology in two different AD experimental mouse models. We conclude that functional CMA is essential for neuronal proteostasis through the maintenance of a subset of the proteome with a higher risk of misfolding than the general proteome.

Chaperone-mediated autophagy sustains haematopoietic stem-cell function.

The activation of mostly quiescent haematopoietic stem cells (HSCs) is a prerequisite for life-long production of blood cells1. This process requires major molecular adaptations to allow HSCs to meet the regulatory and metabolic requirements for cell division2-4. The mechanisms that govern cellular reprograming upon stem-cell activation, and the subsequent return of stem cells to quiescence, have not been fully characterized. Here we show that chaperone-mediated autophagy (CMA)5, a selective form of lysosomal protein degradation, is involved in sustaining HSC function in adult mice. CMA is required for protein quality control in stem cells and for the upregulation of fatty acid metabolism upon HSC activation. We find that CMA activity in HSCs decreases with age and show that genetic or pharmacological activation of CMA can restore the functionality of old mouse and human HSCs. Together, our findings provide mechanistic insights into a role for CMA in sustaining quality control, appropriate energetics and overall long-term HSC function. Our work suggests that CMA may be a promising therapeutic target for enhancing HSC function in conditions such as ageing or stem-cell transplantation.

In Vivo Remodeling of Altered Autophagy-Lysosomal Pathway by a Phosphopeptide in Lupus.

The phosphopeptide P140/Lupuzor, which improves the course of lupus disease in mice and patients, targets chaperone-mediated autophagy (CMA), a selective form of autophagy that is abnormally upregulated in lupus-prone MRL/lpr mice. Administered intravenously to diseased mice, P140 reduces the expression level of two major protein players of CMA, LAMP2A and HSPA8, and inhibits CMA in vitro in a cell line that stably expresses a CMA reporter. Here, we aimed to demonstrate that P140 also affects CMA in vivo and to unravel the precise cellular mechanism of how P140 interacts with the CMA process. MRL/lpr mice and CBA/J mice used as control received P140 or control peptides intravenously. Lysosome-enriched fractions of spleen or liver were prepared to examine lysosomal function. Highly purified lysosomes were further isolated and left to incubate with the CMA substrate to study at which cellular step P140 interacts with the CMA process. The data show that P140 effectively regulates CMA in vivo in MRL/lpr mice at the step of substrate lysosomal uptake and restores some alterations of defective lysosomes. For the first time, it is demonstrated that by occluding the intralysosome uptake of CMA substrates, a therapeutic molecule can attenuate excessive CMA activity in a pathological pro-inflammatory context and protect against hyperinflammation. This recovery effect of P140 on hyperactivated CMA is not only important for lupus therapy but potentially also for treating other (auto)inflammatory diseases, including neurologic and metabolic disorders, where CMA modulation would be highly beneficial.

Humanin is an endogenous activator of chaperone-mediated autophagy.

Chaperone-mediated autophagy (CMA) serves as quality control during stress conditions through selective degradation of cytosolic proteins in lysosomes. Humanin (HN) is a mitochondria-associated peptide that offers cytoprotective, cardioprotective, and neuroprotective effects in vivo and in vitro. In this study, we demonstrate that HN directly activates CMA by increasing substrate binding and translocation into lysosomes. The potent HN analogue HNG protects from stressor-induced cell death in fibroblasts, cardiomyoblasts, neuronal cells, and primary cardiomyocytes. The protective effects are lost in CMA-deficient cells, suggesting that they are mediated through the activation of CMA. We identified that a fraction of endogenous HN is present at the cytosolic side of the lysosomal membrane, where it interacts with heat shock protein 90 (HSP90) and stabilizes binding of this chaperone to CMA substrates as they bind to the membrane. Inhibition of HSP90 blocks the effect of HNG on substrate translocation and abolishes the cytoprotective effects. Our study provides a novel mechanism by which HN exerts its cardioprotective and neuroprotective effects.

Interplay of pathogenic forms of human tau with different autophagic pathways.

Loss of neuronal proteostasis, a common feature of the aging brain, is accelerated in neurodegenerative disorders, including different types of tauopathies. Aberrant turnover of tau, a microtubule-stabilizing protein, contributes to its accumulation and subsequent toxicity in tauopathy patients' brains. A direct toxic effect of pathogenic forms of tau on the proteolytic systems that normally contribute to their turnover has been proposed. In this study, we analyzed the contribution of three different types of autophagy, macroautophagy, chaperone-mediated autophagy, and endosomal microautophagy to the degradation of tau protein variants and tau mutations associated with this age-related disease. We have found that the pathogenic P301L mutation inhibits degradation of tau by any of the three autophagic pathways, whereas the risk-associated tau mutation A152T reroutes tau for degradation through a different autophagy pathway. We also found defective autophagic degradation of tau when using mutations that mimic common posttranslational modifications in tau or known to promote its aggregation. Interestingly, although most mutations markedly reduced degradation of tau through autophagy, the step of this process preferentially affected varies depending on the type of tau mutation. Overall, our studies unveil a complex interplay between the multiple modifications of tau and selective forms of autophagy that may determine its physiological degradation and its faulty clearance in the disease context.

Role of chaperone-mediated autophagy in metabolism.

Different types of autophagy coexist in most mammalian cells, and each of them fulfills very specific tasks in intracellular degradation. Some of these autophagic pathways contribute to cellular metabolism by directly hydrolyzing intracellular lipid stores and glycogen. Chaperone-mediated autophagy (CMA), in contrast, is a selective form of autophagy that can only target proteins for lysosomal degradation. Consequently, it was expected that the only possible contribution of this pathway to cellular metabolism would be by providing free amino acids resulting from protein breakdown. However, recent studies have demonstrated that disturbance in CMA leads to important alterations in glucose and lipid metabolism and in overall organism energetics. Here, we describe the unique mechanisms by which CMA contributes to the regulation of cellular metabolism and discuss the possible implications of these previously unknown functions of CMA for the pathogenesis of common metabolic diseases.

Cancer Prevention and Health Benefices of Traditionally Consumed Borago officinalis Plants.

Nowadays, healthy eating is increasing the demand of functional foods by societies as sources of bioactive products with healthy qualities. For this reason, we tested the safety of the consumption of Borago officinalis L. and its main phenolic components as well as the possibility of its use as a nutraceutical plant to help in cancer prevention. The in vivo Drosophila Somatic Mutation and Recombination Test (SMART) and in vitro HL-60 human cell systems were performed, as well-recognized methods for testing genotoxicity/cytotoxicity of bioactive compounds and plant products. B. officinalis and the tested compounds possess antigenotoxic activity. Moreover, B. officinalis wild type cultivar exerts the most antigenotoxic values. Cytotoxic effect was probed for both cultivars with IC50 values of 0.49 and 0.28 mg · mL(-1) for wild type and cultivated plants respectively, as well as their constituent rosmarinic acid and the assayed phenolic mixture (IC50 = 0.07 and 0.04 mM respectively). B. officinalis exerts DNA protection and anticarcinogenic effects as do its component rosmarinic acid and the mixture of the main phenolics presented in the plant. In conclusion, the results showed that B. officinalis may represent a high value plant for pleiotropic uses and support its consumption as a nutraceutical plant.

Antigenotoxicity and Tumor Growing Inhibition by Leafy Brassica carinata and Sinigrin.

Cruciferous vegetables are well known and worldwide consumed due to their health benefits and cancer prevention properties. As a desirable cruciferous plant, Ethiopian mustard (Brassica carinata A. Braun) and its glucosinolate sinigrin were tested in the in vivo Drosophila melanogaster (SMART) and the in vitro HL60 (human promyelocytic leukaemia cell line) systems. High performance liquid chromatography (HPLC) analysis of plant samples confirmed the presence of sinigrin as principal B. carinata glucosinolate. SMART was performed by feeding D. melanogaster larvae either with different concentrations of plant/compound samples or combining them with hydrogen peroxide (a potent oxidative mutagen) being both antimutagenics. HL60 assays showed the tumoricidal activity of plant samples (IC50 = 0.28 mg·mL(-1)) and the breakdown products of sinigrin hydrolysis (IC50 = 2.71 µM). Our results enhance the potential of B. carinata as health promoter and chemopreventive in both systems and the leading role of sinigrin in these effects.

α-Synuclein-independent histopathological and motor deficits in mice lacking the endolysosomal Parkinsonism protein Atp13a2.

Accumulating evidence from genetic and biochemical studies implicates dysfunction of the autophagic-lysosomal pathway as a key feature in the pathogenesis of Parkinson's disease (PD). Most studies have focused on accumulation of neurotoxic α-synuclein secondary to defects in autophagy as the cause of neurodegeneration, but abnormalities of the autophagic-lysosomal system likely mediate toxicity through multiple mechanisms. To further explore how endolysosomal dysfunction causes PD-related neurodegeneration, we generated a murine model of Kufor-Rakeb syndrome (KRS), characterized by early-onset Parkinsonism with additional neurological features. KRS is caused by recessive loss-of-function mutations in the ATP13A2 gene encoding the endolysosomal ATPase ATP13A2. We show that loss of ATP13A2 causes a specific protein trafficking defect, and that Atp13a2 null mice develop age-related motor dysfunction that is preceded by neuropathological changes, including gliosis, accumulation of ubiquitinated protein aggregates, lipofuscinosis, and endolysosomal abnormalities. Contrary to predictions from in vitro data, in vivo mouse genetic studies demonstrate that these phenotypes are α-synuclein independent. Our findings indicate that endolysosomal dysfunction and abnormalities of α-synuclein homeostasis are not synonymous, even in the context of an endolysosomal genetic defect linked to Parkinsonism, and highlight the presence of α-synuclein-independent neurotoxicity consequent to endolysosomal dysfunction.

Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide.

The P140 peptide, a 21-mer linear peptide (sequence 131-151) generated from the spliceosomal SNRNP70/U1-70K protein, contains a phosphoserine residue at position 140. It significantly ameliorates clinical manifestations in autoimmune patients with systemic lupus erythematosus and enhances survival in MRL/lpr lupus-prone mice. Previous studies showed that after P140 treatment, there is an accumulation of autophagy markers sequestosome 1/p62 and MAP1LC3-II in MRL/lpr B cells, consistent with a downregulation of autophagic flux. We now identify chaperone-mediated autophagy (CMA) as a target of P140 and demonstrate that its inhibitory effect on CMA is likely tied to its ability to alter the composition of HSPA8/HSC70 heterocomplexes. As in the case of HSPA8, expression of the limiting CMA component LAMP2A, which is increased in MRL/lpr B cells, is downregulated after P140 treatment. We also show that P140, but not the unphosphorylated peptide, uses the clathrin-dependent endo-lysosomal pathway to enter into MRL/lpr B lymphocytes and accumulates in the lysosomal lumen where it may directly hamper lysosomal HSPA8 chaperoning functions, and also destabilize LAMP2A in lysosomes as a result of its effect on HSP90AA1. This dual effect may interfere with the endogenous autoantigen processing and loading to major histocompatibility complex class II molecules and as a consequence, lead to lower activation of autoreactive T cells. These results shed light on mechanisms by which P140 can modulate lupus disease and exert its tolerogenic activity in patients. The unique selective inhibitory effect of the P140 peptide on CMA may be harnessed in other pathological conditions in which reduction of CMA activity would be desired.

Evaluation of potential antigenotoxic, cytotoxic and proapoptotic effects of the olive oil by-product "alperujo", hydroxytyrosol, tyrosol and verbascoside.

Olive oil is an integral ingredient of the "Mediterranean diet". The olive oil industry generates large quantities of a by-product called "alperujo" (AL) during the two-phase centrifugation system developed in the early nineties. AL could be a potent exploitable source of natural phenolic antioxidants. Our results showed that AL and its distinctive phenols hydroxytyrosol, tyrosol and verbascoside were not genotoxic in the Somatic Mutation and Recombination Test (SMART) of Drosophila melanogaster and exerted antigenotoxic activity against DNA oxidative damage generated by hydrogen peroxide (H2O2). Alperujo and hydroxytyrosol also exhibited notable antiproliferative and caspase 3-dependent proapoptotic effects toward the human tumoral cell line HL60. AL can provide a cheap and efficient source of chemopreventive phenolic compounds with strong antioxidant properties, becoming a promising and potent therapeutic drug in the future.

Impact of light/dark cycle patterns on oxidative stress in an adriamycin-induced nephropathy model in rats.

The principal goal of this study was to determine the effect of the photoperiod on oxidative damage biomarkers in rats submitted to different light/darkness patterns, in a hyperlipidemic nephropathy model (induced by adriamycin), as well as its possible relationship with melatonin and leptin secretion rhythms. To test this hypothesis, six different groups were used (N = 6 rats per group): control (12 h/12h light:dark); exposure to permanent illumination (24 h light); exposure to darkness (22 h dark); injected with adriamycin, 12h/12h light:dark; injected with adriamycin + exposure to permanent illumination and injected with adriamycin + exposure to darkness (22 h dark). The different photoperiods were begun two weeks prior to medication and were maintained up to the day of the animal's sacrifice, ten days after medication. The following parameters were analysed: i) weight evolution; ii) in plasma: urea, creatinine, uric acid, total proteins, albumen, lactate dehydrogenase, creatinine-quinase, aspartate aminotransferase, alanine aminotransferase and total cholesterol; iii) in urine: urea, creatinine, total proteins and microalbumen; iv) biomarkers of oxidative damage in kidneys, heart, liver and brain: lipoperoxides, total glutathione, reduced glutathione, catalase, glutathione peroxidase, glutathione reductase and glutathione transferase; v) melatonin (pineal gland tissue and plasma) and leptin (plasma). From the results obtained it was concluded that the administration of adriamycin generated oxidative stress in renal, cerebral, hepatic and cardiac tissue. Additionally, in the healthy animal, but of a lesser relevance in the adriamycin animal, permanent light worsened the oxidative stress, whereas darkness improved it. This could be related to the circadian rhythm of the inverse release shown by melatonin and leptin, accentuating the release of melatonin in the darkness phase and that of leptin in the light phase. The correlation between melatonin and leptin in the healthy animal seemed to confirm the relationship between both variables and their influence on oxidative damage biomarkers.

Effect of natalizumab on oxidative damage biomarkers in relapsing-remitting multiple sclerosis.

BACKGROUND: Natalizumab is a monoclonal antibody used to treat multiple sclerosis. This study sought to determine whether the protective action of natalizumab involved a reduction in oxidative damage. METHODS: Twenty-two multiple sclerosis patients fulfilling the revised McDonald criteria were assigned to treatment with 300 mg natalizumab intravenously once monthly (infusion every 4 weeks) in accordance with Spanish guidelines. Carbonylated proteins, 8-hydroxy-2'-deoxyguanosine, total glutathione, reduced glutathione, superoxide dismutase, glutathione peroxidase, and myeloperoxidase levels were measured at baseline and after 14 months' treatment, and the antioxidant gap was calculated. RESULTS: Natalizumab prompted a drop in oxidative-damage biomarker levels, together with a reduction both in myeloperoxidase levels and in the myeloperoxidase/neutrophil granulocyte ratio. Interestingly, natalizumab induced nuclear translocation of Nrf2 and a fall in serum vascular cell adhesion molecule-1 levels. CONCLUSION: These findings suggest that natalizumab has a beneficial effect on oxidative damage found in MS patients.

Profile of oxidant and antioxidant activity in prepubertal children related to age, gender, exercise, and fitness.

Tissue damage resulting from oxidative stress induced by a pathological condition might have more serious consequences in children than in adults. Researchers have not yet identified particular markers - alone or in combination with others - of oxidative stress, or their role in pediatric diseases. The aim of this study was to identify gender-based biomarkers for measuring oxidative stress. Oxidative biomarkers were studied in 138 healthy Spanish children (85 boys, 53 girls) 7 to 12 years of age, at the prepubertal (Tanner I) stage, independent of body mass index (BMI), age, fitness (measured by 20-m shuttle run test), and physical activity (measured by participation in an after-school exercise program). The oxidative biomarkers measured were lipid peroxidation products, total nitrites, protein carbonyls, and oxidized glutathione (GSSG). The antioxidant biomarkers measured were total glutathione (TG), reduced glutathione (GSH), superoxide dismutase activity (SOD), and glutathione peroxidase activity. In the study population, height, weight, waist circumference, and BMI were lower in girls than in boys. For oxidative biomarkers, boys had higher levels of protein carbonyl than girls (p < 0.001). In spite of this, girls had higher levels of GSSG (p < 0.001) and TG (p = 0.001), and a lower GSH/GSSG ratio (p < 0.001) than boys. For the antioxidant response, girls had higher levels of SOD (p = 0.002) than boys. All analyses were adjusted for BMI, age, fitness, and physical activity. In conclusion, prepubertal girls had higher oxidative stress than boys, in addition to higher levels of SOD, independent of age, BMI, fitness, and physical activity.