TFEB drives mTORC1 hyperactivation and kidney disease in Tuberous Sclerosis Complex.

Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, leading to hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) and lesions  in multiple organs including lung (lymphangioleiomyomatosis) and kidney (angiomyolipoma and renal cell carcinoma). Previously, we found that TFEB is constitutively active in TSC. Here, we generated two mouse models of TSC in which kidney pathology is the primary phenotype. Knockout of TFEB rescues kidney pathology and overall survival, indicating that TFEB is the primary driver of renal disease in TSC. Importantly, increased mTORC1 activity in the TSC2 knockout kidneys is normalized by TFEB knockout. In TSC2-deficient cells, Rheb knockdown or Rapamycin treatment paradoxically increases TFEB phosphorylation at the mTORC1-sites and relocalizes TFEB from nucleus to cytoplasm. In mice, Rapamycin treatment normalizes lysosomal gene expression, similar to TFEB knockout, suggesting that Rapamycin's benefit in TSC is TFEB-dependent. These results change the view of the mechanisms of mTORC1 hyperactivation in TSC and may lead to therapeutic avenues.

Autophagy-related proteins: Potential diagnostic and prognostic biomarkers of aging-related diseases.

Autophagy plays a key role in cellular, tissue and organismal homeostasis and in the production of the energy load needed at critical times during development and in response to nutrient shortage. Autophagy is generally considered as a pro-survival mechanism, although its deregulation has been linked to non-apoptotic cell death. Autophagy efficiency declines with age, thus contributing to many different pathophysiological conditions, such as cancer, cardiomyopathy, diabetes, liver disease, autoimmune diseases, infections, and neurodegeneration. Accordingly, it has been proposed that the maintenance of a proper autophagic activity contributes to the extension of the lifespan in different organisms. A better understanding of the interplay between autophagy and risk of age-related pathologies is important to propose nutritional and life-style habits favouring disease prevention as well as possible clinical applications aimed at promoting long-term health.

Fluid shear stress triggers cholesterol biosynthesis and uptake in inner medullary collecting duct cells, independently of nephrocystin-1 and nephrocystin-4.

Renal epithelial cells are subjected to fluid shear stress of urine flow. Several cellular structures act as mechanosensors-the primary cilium, microvilli and cell adhesion complexes-that directly relay signals to the cytoskeleton to regulate various processes including cell differentiation and renal cell functions. Nephronophthisis (NPH) is an autosomal recessive tubulointerstitial nephropathy leading to end-stage kidney failure before adulthood. NPHP1 and NPHP4 are the major genes which code for proteins that form a complex at the transition zone of the primary cilium, a crucial region required for the maintenance of the ciliary composition integrity. These two proteins also interact with signaling components and proteins associated with the actin cytoskeleton at cell junctions. Due to their specific subcellular localization, we wondered whether NPHP1 and NPHP4 could ensure mechanosensory functions. Using a microfluidic set up, we showed that murine inner medullary collecting ductal cells invalidated for Nphp1 or Nphp4 are more responsive to immediate shear exposure with a fast calcium influx, and upon a prolonged shear condition, an inability to properly regulate cilium length and actin cytoskeleton remodeling. Following a transcriptomic study highlighting shear stress-induced gene expression changes, we showed that prolonged shear triggers both cholesterol biosynthesis pathway and uptake, processes that do not seem to involve neither NPHP1 nor NPHP4. To conclude, our study allowed us to determine a moderate role of NPHP1 and NPHP4 in flow sensation, and to highlight a new signaling pathway induced by shear stress, the cholesterol biosynthesis and uptake pathways, which would allow cells to cope with mechanical stress by strengthening their plasma membrane through the supply of cholesterol.

Fluid flow-induced shear stress controls the metabolism of proximal tubule kidney epithelial cells through primary cilium-dependent lipophagy and mitochondria biogenesis.

The kidney, similar to many other organs, has to face shear stress induced by biological fluids. How epithelial kidney cells respond to shear stress is poorly understood. Recently we showed in vitro and in vivo that proximal tubule epithelial cells use lipophagy to fuel mitochondria with fatty acids. Lipophagy is stimulated by a primary cilium-dependent signaling that converges at AMP kinase. AMP kinase is a central signaling hub to trigger lipophagy and also to stimulate mitochondrial biogenesis. These two pathways contribute to generate ATP needed to support energy-consuming cellular processes such as glucose reabsorption, gluconeogenesis. These findings demonstrate the role of the primary cilium and selective macroautophagy/autophagy to integrate shear stress and to sustain the execution of a specific cellular program.

The primary cilium and lipophagy translate mechanical forces to direct metabolic adaptation of kidney epithelial cells.

Organs and cells must adapt to shear stress induced by biological fluids, but how fluid flow contributes to the execution of specific cell programs is poorly understood. Here we show that shear stress favours mitochondrial biogenesis and metabolic reprogramming to ensure energy production and cellular adaptation in kidney epithelial cells. Shear stress stimulates lipophagy, contributing to the production of fatty acids that provide mitochondrial substrates to generate ATP through ß-oxidation. This flow-induced process is dependent on the primary cilia located on the apical side of epithelial cells. The interplay between fluid flow and lipid metabolism was confirmed in vivo using a unilateral ureteral obstruction mouse model. Finally, primary cilium-dependent lipophagy and mitochondrial biogenesis are required to support energy-consuming cellular processes such as glucose reabsorption, gluconeogenesis and cytoskeletal remodelling. Our findings demonstrate how primary cilia and autophagy are involved in the translation of mechanical forces into metabolic adaptation.

Interplay between primary cilia, ubiquitin-proteasome system and autophagy.

Cilia are microtubule-based organelles located at the cell surface of many eukaryotic cell types. Cilia control different cellular functions ranging from motility (for motile cilia) to signal transduction pathways (for primary cilia). A variety of signaling pathways are coordinated by this organelle during development, cell migration and cell differentiation. Interestingly, aberrant ciliogenesis or altered cilium signaling has been associated with human diseases, notably in cancer. Disruption of cilia through mutation of genes encoding cilia proteins has been also linked to multiple human disorders referred as ciliopathies. Recent studies highlight the interplay between cilia and proteostasis. Here we review findings regarding the crosstalk between cilia and two proteolytic systems, the ubiquitin proteasome system and the autophagy-lysosomal system and discuss the potential implications in human disease including ciliopathies.

Oleuropein Aglycone Protects against MAO-A-Induced Autophagy Impairment and Cardiomyocyte Death through Activation of TFEB.

Age-associated diseases such as neurodegenerative and cardiovascular disorders are characterized by increased oxidative stress associated with autophagy dysfunction. Oleuropein aglycone (OA), the main polyphenol found in olive oil, was recently characterized as an autophagy inducer and a promising agent against neurodegeneration. It is presently unknown whether OA can have beneficial effects in a model of cardiac stress characterized by autophagy dysfunction. Here, we explored the effects of OA in cardiomyocytes with overexpression of monoamine oxidase-A (MAO-A). This enzyme, by degrading catecholamine and serotonin, produces hydrogen peroxide (H2O2), which causes oxidative stress, autophagic flux blockade, and cell necrosis. We observed that OA treatment counteracted the cytotoxic effects of MAO-A through autophagy activation, as displayed by the increase of autophagic vacuoles and autophagy-specific markers (Beclin1 and LC3-II). Moreover, the decrease in autophagosomes and the increase in autolysosomes, indicative of autophagosome-lysosome fusion, suggested a restoration of the defective autophagic flux. Most interestingly, we found that the ability of OA to confer cardioprotection through autophagy induction involved nuclear translocation and activation of the transcriptional factor EB (TFEB). Our data provide strong evidence of the beneficial effects of OA, suggesting its potential use as a nutraceutical agent against age-related pathologies involving autophagy dysfunction, including cardiovascular diseases.

Monoamine Oxidase Is Overactivated in Left and Right Ventricles from Ischemic Hearts: An Intriguing Therapeutic Target.

Growing evidence indicates that reactive oxygen species (ROS) may play a key role in human heart failure (HF). Monoamine oxidase (MAO) is emerging as a major ROS source in several cardiomyopathies. However, little is known about MAO activity in human failing heart and its relationship with redox imbalance. Therefore, we measured MAO activity in the left (LV) and in the right (RV) ventricle of human nonfailing (NF) and in end-stage ischemic (IHD) and nonischemic failing hearts. We found that both MAO isoforms (MAO-A/B) significantly increased in terms of activity and expression levels only in IHD ventricles. Catalase and aldehyde dehydrogenase-2 activities (ALDH-2), both implicated in MAO-catalyzed catecholamine catabolism, were significantly elevated in the failing LV, whereas, in the RV, statistical significance was observed only for ALDH-2. Oxidative stress markers levels were significantly increased only in the failing RV. Actin oxidation was significantly elevated in both failing ventricles and related to MAO-A activity and to functional parameters. These data suggest a close association between MAO-A-dependent ROS generation, actin oxidation, and ventricular dysfunction. This latter finding points to a possible pathogenic role of MAO-A in human myocardial failure supporting the idea that MAO-A could be a new therapeutic target in HF.

The Polyphenol Oleuropein Aglycone Modulates the PARP1-SIRT1 Interplay: An In Vitro and In Vivo Study.

Poly(ADP-ribose) polymerase-1 (PARP1) activation contributes to the cascade of events initiated by amyloid-ß (Aß) peptide eventually leading to cell death in Alzheimer's disease brain. A significant accumulation of PAR polymers and increase of PARP1 expression were detected in the cortex at the early (3.5 months) and intermediate (6 months) stage of Aß deposition in the TgCRND8 mouse model. Our previous data highlighted the beneficial effects of oleuropein aglycone (OLE), the main polyphenol found in the olive oil, against neurodegeneration both in cultured cells and in model organisms. Here we found that 8-week OLE treatment (50 mg/kg of diet) to 6-month-old TgCRND8 mice rescued to control values PARP1 activation and the levels of its product, PAR. In N2a neuroblastoma cells, PARP1 activation and PAR formation upon exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were abolished by pretreatment for 24 h with either OLE (100µM) or PARP inhibitors. A significant reduction of the NAD+ content, compared to controls, was found in N2a cells exposed to MNNG (100µM) for 90 min; the latter was slightly attenuated by cell treatment for 24 h with PJ-34 or with OLE. In vitro and in vivo, the OLE-induced reduction of PARP1 activation was paralleled by the overexpression of Sirtuin1 (SIRT1), and, in vivo, by a decrease of NF-κB and the pro-apoptotic marker p53. In N2a cells, we also found that OLE potentiates the MNNG-induced increase of Beclin1 levels. In conclusion, our data show that OLE treatment counteracts neuronal damage through modulation of the PARP1-SIRT1 interplay.

Oleuropein aglycone induces autophagy via the AMPK/mTOR signalling pathway: a mechanistic insight.

The healthy effects of plant polyphenols, some of which characterize the so-called Mediterranean diet, have been shown to arise from epigenetic and biological modifications resulting, among others, in autophagy stimulation. Our previous work highlighted the beneficial effects of oleuropein aglycone (OLE), the main polyphenol found in the extra virgin olive oil, against neurodegeneration both in cultured cells and in model organisms, focusing, in particular, autophagy activation. In this study we investigated more in depth the molecular and cellular mechanisms of autophagy induction by OLE using cultured neuroblastoma cells and an OLE-fed mouse model of amylod beta (Aß) deposition. We found that OLE triggers autophagy in cultured cells through the Ca2+-CAMKKß-AMPK axis. In particular, in these cells OLE induces a rapid release of Ca2+ from the SR stores which, in turn, activates CAMKKß, with subsequent phosphorylation and activation of AMPK. The link between AMPK activation and mTOR inhibition was shown in the OLE-fed animal model in which we found that decreased phospho-mTOR immunoreactivity and phosphorylated mTOR substrate p70 S6K levels match enhanced phospho-AMPK levels, supporting the idea that autophagy activation by OLE proceeds through mTOR inhibition. Our results agree with those reported for other plant polyphenols, suggesting a shared molecular mechanism underlying the healthy effects of these substances against ageing, neurodegeneration, cancer, diabetes and other diseases implying autophagy dysfunction.