Publications by authors named "Carmine Settembre"

59 Publications

Role of FAM134 paralogues in endoplasmic reticulum remodeling, ER-phagy, and Collagen quality control.

EMBO Rep 2021 Sep 2;22(9):e52289. Epub 2021 Aug 2.

Institute of Biochemistry II (IBC2), Faculty of Medicine, Goethe University, Frankfurt am Main, Germany.

Degradation of the endoplasmic reticulum (ER) via selective autophagy (ER-phagy) is vital for cellular homeostasis. We identify FAM134A/RETREG2 and FAM134C/RETREG3 as ER-phagy receptors, which predominantly exist in an inactive state under basal conditions. Upon autophagy induction and ER stress signal, they can induce significant ER fragmentation and subsequent lysosomal degradation. FAM134A, FAM134B/RETREG1, and FAM134C are essential for maintaining ER morphology in a LC3-interacting region (LIR)-dependent manner. Overexpression of any FAM134 paralogue has the capacity to significantly augment the general ER-phagy flux upon starvation or ER-stress. Global proteomic analysis of FAM134 overexpressing and knockout cell lines reveals several protein clusters that are distinctly regulated by each of the FAM134 paralogues as well as a cluster of commonly regulated ER-resident proteins. Utilizing pro-Collagen I, as a shared ER-phagy substrate, we observe that FAM134A acts in a LIR-independent manner and compensates for the loss of FAM134B and FAM134C, respectively. FAM134C instead is unable to compensate for the loss of its paralogues. Taken together, our data show that FAM134 paralogues contribute to common and unique ER-phagy pathways.
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http://dx.doi.org/10.15252/embr.202052289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8447607PMC
September 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Beclin-1-mediated activation of autophagy improves proximal and distal urea cycle disorders.

EMBO Mol Med 2021 Feb 28;13(2):e13158. Epub 2020 Dec 28.

Telethon Institute of Genetics and Medicine, Pozzuoli, Italy.

Urea cycle disorders (UCD) are inherited defects in clearance of waste nitrogen with high morbidity and mortality. Novel and more effective therapies for UCD are needed. Studies in mice with constitutive activation of autophagy unravelled Beclin-1 as druggable candidate for therapy of hyperammonemia. Next, we investigated efficacy of cell-penetrating autophagy-inducing Tat-Beclin-1 (TB-1) peptide for therapy of the two most common UCD, namely ornithine transcarbamylase (OTC) and argininosuccinate lyase (ASL) deficiencies. TB-1 reduced urinary orotic acid and improved survival under protein-rich diet in spf-ash mice, a model of OTC deficiency (proximal UCD). In Asl mice, a model of ASL deficiency (distal UCD), TB-1 increased ureagenesis, reduced argininosuccinate, and improved survival. Moreover, it alleviated hepatocellular injury and decreased both cytoplasmic and nuclear glycogen accumulation in Asl mice. In conclusion, Beclin-1-dependent activation of autophagy improved biochemical and clinical phenotypes of proximal and distal defects of the urea cycle.
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http://dx.doi.org/10.15252/emmm.202013158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7863400PMC
February 2021

Regulation of autophagosome biogenesis by OFD1-mediated selective autophagy.

EMBO J 2021 02 28;40(4):e105120. Epub 2020 Dec 28.

Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy.

Autophagy is a lysosome-dependent degradation pathway essential to maintain cellular homeostasis. Therefore, either defective or excessive autophagy may be detrimental for cells and tissues. The past decade was characterized by significant advances in molecular dissection of stimulatory autophagy inputs; however, our understanding of the mechanisms that restrain autophagy is far from complete. Here, we describe a negative feedback mechanism that limits autophagosome biogenesis based on the selective autophagy-mediated degradation of ATG13, a component of the ULK1 autophagy initiation complex. We demonstrate that the centrosomal protein OFD1 acts as bona fide autophagy receptor for ATG13 via direct interaction with the Atg8/LC3/GABARAP family of proteins. We also show that patients with Oral-Facial-Digital type I syndrome, caused by mutations in the OFD1 gene, display excessive autophagy and that genetic inhibition of autophagy in a mouse model of the disease, significantly ameliorates polycystic kidney, a clinical manifestation of the disorder. Collectively, our data report the discovery of an autophagy self-regulated mechanism and implicate dysregulated autophagy in the pathogenesis of renal cystic disease in mammals.
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http://dx.doi.org/10.15252/embj.2020105120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7883294PMC
February 2021

MLL4-associated condensates counterbalance Polycomb-mediated nuclear mechanical stress in Kabuki syndrome.

Nat Genet 2020 12 9;52(12):1397-1411. Epub 2020 Nov 9.

Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy.

The genetic elements required to tune gene expression are partitioned in active and repressive nuclear condensates. Chromatin compartments include transcriptional clusters whose dynamic establishment and functioning depend on multivalent interactions occurring among transcription factors, cofactors and basal transcriptional machinery. However, how chromatin players contribute to the assembly of transcriptional condensates is poorly understood. By interrogating the effect of KMT2D (also known as MLL4) haploinsufficiency in Kabuki syndrome, we found that mixed lineage leukemia 4 (MLL4) contributes to the assembly of transcriptional condensates through liquid-liquid phase separation. MLL4 loss of function impaired Polycomb-dependent chromatin compartmentalization, altering the nuclear architecture. By releasing the nuclear mechanical stress through inhibition of the mechanosensor ATR, we re-established the mechanosignaling of mesenchymal stem cells and their commitment towards chondrocytes both in vitro and in vivo. This study supports the notion that, in Kabuki syndrome, the haploinsufficiency of MLL4 causes an altered functional partitioning of chromatin, which determines the architecture and mechanical properties of the nucleus.
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http://dx.doi.org/10.1038/s41588-020-00724-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610431PMC
December 2020

Mechanisms by which autophagy regulates memory capacity in ageing.

Aging Cell 2020 09 30;19(9):e13189. Epub 2020 Jul 30.

Telethon Institute of Genetics and Medicine, Telethon Foundation, Pozzuoli, Italy.

Autophagy agonists have been proposed to slow down neurodegeneration. Spermidine, a polyamine that acts as an autophagy agonist, is currently under clinical trial for the treatment of age-related memory decline. How Spermidine and other autophagy agonists regulate memory and synaptic plasticity is under investigation. We set up a novel mouse model of mild cognitive impairment (MCI), in which middle-aged (12-month-old) mice exhibit impaired memory capacity, lysosomes engulfed with amyloid fibrils (β-amyloid and α-synuclein) and impaired task-induced GluA1 hippocampal post-translation modifications. Subchronic treatment with Spermidine as well as the autophagy agonist TAT-Beclin 1 rescued memory capacity and GluA1 post-translational modifications by favouring the autophagy/lysosomal-mediated degradation of amyloid fibrils. These findings provide new mechanistic evidence on the therapeutic relevance of autophagy enhancers which, by improving the degradation of misfolded proteins, slow down age-related memory decline.
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http://dx.doi.org/10.1111/acel.13189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511873PMC
September 2020

MiT/TFE factors control ER-phagy via transcriptional regulation of FAM134B.

EMBO J 2020 09 27;39(17):e105696. Epub 2020 Jul 27.

Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy.

Lysosomal degradation of the endoplasmic reticulum (ER) via autophagy (ER-phagy) is emerging as a critical regulator of cell homeostasis and function. The recent identification of ER-phagy receptors has shed light on the molecular mechanisms underlining this process. However, the signaling pathways regulating ER-phagy in response to cellular needs are still largely unknown. We found that the nutrient responsive transcription factors TFEB and TFE3-master regulators of lysosomal biogenesis and autophagy-control ER-phagy by inducing the expression of the ER-phagy receptor FAM134B. The TFEB/TFE3-FAM134B axis promotes ER-phagy activation upon prolonged starvation. In addition, this pathway is activated in chondrocytes by FGF signaling, a critical regulator of skeletal growth. FGF signaling induces JNK-dependent proteasomal degradation of the insulin receptor substrate 1 (IRS1), which in turn inhibits the PI3K-PKB/Akt-mTORC1 pathway and promotes TFEB/TFE3 nuclear translocation and enhances FAM134B transcription. Notably, FAM134B is required for protein secretion in chondrocytes, and cartilage growth and bone mineralization in medaka fish. This study identifies a new signaling pathway that allows ER-phagy to respond to both metabolic and developmental cues.
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http://dx.doi.org/10.15252/embj.2020105696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459426PMC
September 2020

TFEB regulates murine liver cell fate during development and regeneration.

Nat Commun 2020 05 18;11(1):2461. Epub 2020 May 18.

Jan and Dan Duncan Neurological Research Institute, Texas Children Hospital, Houston, TX, 77030, USA.

It is well established that pluripotent stem cells in fetal and postnatal liver (LPCs) can differentiate into both hepatocytes and cholangiocytes. However, the signaling pathways implicated in the differentiation of LPCs are still incompletely understood. Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, is known to be involved in osteoblast and myeloid differentiation, but its role in lineage commitment in the liver has not been investigated. Here we show that during development and upon regeneration TFEB drives the differentiation status of murine LPCs into the progenitor/cholangiocyte lineage while inhibiting hepatocyte differentiation. Genetic interaction studies show that Sox9, a marker of precursor and biliary cells, is a direct transcriptional target of TFEB and a primary mediator of its effects on liver cell fate. In summary, our findings identify an unexplored pathway that controls liver cell lineage commitment and whose dysregulation may play a role in biliary cancer.
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http://dx.doi.org/10.1038/s41467-020-16300-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7235048PMC
May 2020

Beating the ER: novel insights into FAM134B function and regulation.

EMBO J 2020 03 19;39(5):e104546. Epub 2020 Feb 19.

Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy.

To maintain cellular homeostasis, the endoplasmic reticulum (ER) necessitates a continuous removal of ER fragments via a selective, receptor-mediated, form of autophagy known as ER-phagy. In this issue of The EMBO Journal, Jiang et al (2020) shed light on how the best characterized autophagy receptor FAM134B mediates ER membrane fragmentation, the earliest event during ER-phagy. They propose a dynamic model for FAM134B protein oligomerization and ER membrane scission, which are driven by CAMK2B-mediated phosphorylation of the receptor and are altered in sensory neuropathy.
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http://dx.doi.org/10.15252/embj.2020104546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049802PMC
March 2020

Emerging lysosomal pathways for quality control at the endoplasmic reticulum.

FEBS Lett 2019 09 13;593(17):2319-2329. Epub 2019 Aug 13.

Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy.

Protein misfolding occurring in the endoplasmic reticulum (ER) might eventually lead to aggregation and cellular distress, and is a primary pathogenic mechanism in multiple human disorders. Mammals have developed evolutionary-conserved quality control mechanisms at the level of the ER. The best characterized is the ER-associated degradation (ERAD) pathway, through which misfolded proteins translocate from the ER to the cytosol and are subsequently proteasomally degraded. However, increasing evidence indicates that additional quality control mechanisms apply for misfolded ER clients that are not eligible for ERAD. This review focuses on the alternative, ERAD-independent, mechanisms of clearance of misfolded polypeptides from the ER. These processes, collectively referred to as ER-to-lysosome-associated degradation, involve ER-phagy, microautophagy or vesicular transport. The identification of the underlying molecular mechanisms is particularly important for developing new therapeutic approaches for human diseases associated with protein aggregate formation.
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http://dx.doi.org/10.1002/1873-3468.13571DOI Listing
September 2019

Transcriptional Regulation of Autophagy: Mechanisms and Diseases.

Front Cell Dev Biol 2019 2;7:114. Epub 2019 Jul 2.

Telethon Institute of Genetics and Medicine, Pozzuoli, Italy.

Macro (Autophagy) is a catabolic process that relies on the cooperative function of two organelles: the lysosome and the autophagosome. The recent discovery of a transcriptional gene network that co-regulates the biogenesis and function of these two organelles, and the identification of transcription factors, miRNAs and epigenetic regulators of autophagy, demonstrated that this catabolic process is controlled by both transcriptional and post-transcriptional mechanisms. In this review article, we discuss the nuclear events that control autophagy, focusing particularly on the role of the MiT/TFE transcription factor family. In addition, we will discuss evidence suggesting that the transcriptional regulation of autophagy could be targeted for the treatment of human genetic diseases, such as lysosomal storage disorders (LSDs) and neurodegeneration.
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http://dx.doi.org/10.3389/fcell.2019.00114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614182PMC
July 2019

Autophagy Is Required for Memory Formation and Reverses Age-Related Memory Decline.

Curr Biol 2019 02 17;29(3):435-448.e8. Epub 2019 Jan 17.

INSERM U1151, Institut Necker Enfants-Malades (INEM), Team 14, Université Paris Descartes-Sorbonne-Paris Cité, 75014 Paris, France. Electronic address:

Age-related declines in cognitive fitness are associated with a reduction in autophagy, an intracellular lysosomal catabolic process that regulates protein homeostasis and organelle turnover. However, the functional significance of autophagy in regulating cognitive function and its decline during aging remains largely elusive. Here, we show that stimulating memory upregulates autophagy in the hippocampus. Using hippocampal injections of genetic and pharmacological modulators of autophagy, we find that inducing autophagy in hippocampal neurons is required to form novel memory by promoting activity-dependent structural and functional synaptic plasticity, including dendritic spine formation, neuronal facilitation, and long-term potentiation. We show that hippocampal autophagy activity is reduced during aging and that restoring its levels is sufficient to reverse age-related memory deficits. Moreover, we demonstrate that systemic administration of young plasma into aged mice rejuvenates memory in an autophagy-dependent manner, suggesting a prominent role for autophagy to favor the communication between systemic factors and neurons in fostering cognition. Among these youthful factors, we identify osteocalcin, a bone-derived molecule, as a direct hormonal inducer of hippocampal autophagy. Our results reveal that inducing autophagy in hippocampal neurons is a necessary mechanism to enhance the integration of novel stimulations of memory and to promote the influence of systemic factors on cognitive fitness. We also demonstrate the potential therapeutic benefits of modulating autophagy in the aged brain to counteract age-related cognitive impairments.
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http://dx.doi.org/10.1016/j.cub.2018.12.021DOI Listing
February 2019

TFEB controls vascular development by regulating the proliferation of endothelial cells.

EMBO J 2019 02 27;38(3). Epub 2018 Dec 27.

Department of Oncology, University of Turin, Candiolo, Italy

Transcription factor TFEB is thought to control cellular functions-including in the vascular bed-primarily via regulation of lysosomal biogenesis and autophagic flux. Here, we report that TFEB also orchestrates a non-canonical program that controls the cell cycle/VEGFR2 pathway in the developing vasculature. In endothelial cells, TFEB depletion halts proliferation at the G1-S transition by inhibiting the CDK4/Rb pathway. TFEB-deficient cells attempt to compensate for this limitation by increasing VEGFR2 levels at the plasma membrane via microRNA-mediated mechanisms and controlled membrane trafficking. TFEB stimulates expression of the miR-15a/16-1 cluster, which limits VEGFR2 transcript stability and negatively modulates expression of MYO1C, a regulator of VEGFR2 trafficking to the cell surface. Altered levels of miR-15a/16-1 and MYO1C in TFEB-depleted cells cause increased expression of plasma membrane VEGFR2, but in a manner associated with low signaling strength. An endothelium-specific Tfeb-knockout mouse model displays defects in fetal and newborn mouse vasculature caused by reduced endothelial proliferation and by anomalous function of the VEGFR2 pathway. These previously unrecognized functions of TFEB expand its role beyond regulation of the autophagic pathway in the vascular system.
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http://dx.doi.org/10.15252/embj.201798250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356157PMC
February 2019

A selective ER-phagy exerts procollagen quality control via a Calnexin-FAM134B complex.

EMBO J 2019 01 17;38(2). Epub 2018 Dec 17.

Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy

Autophagy is a cytosolic quality control process that recognizes substrates through receptor-mediated mechanisms. Procollagens, the most abundant gene products in Metazoa, are synthesized in the endoplasmic reticulum (ER), and a fraction that fails to attain the native structure is cleared by autophagy. However, how autophagy selectively recognizes misfolded procollagens in the ER lumen is still unknown. We performed siRNA interference, CRISPR-Cas9 or knockout-mediated gene deletion of candidate autophagy and ER proteins in collagen producing cells. We found that the ER-resident lectin chaperone Calnexin (CANX) and the ER-phagy receptor FAM134B are required for autophagy-mediated quality control of endogenous procollagens. Mechanistically, CANX acts as co-receptor that recognizes ER luminal misfolded procollagens and interacts with the ER-phagy receptor FAM134B. In turn, FAM134B binds the autophagosome membrane-associated protein LC3 and delivers a portion of ER containing both CANX and procollagen to the lysosome for degradation. Thus, a crosstalk between the ER quality control machinery and the autophagy pathway selectively disposes of proteasome-resistant misfolded clients from the ER.
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http://dx.doi.org/10.15252/embj.201899847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331724PMC
January 2019

Defective collagen proteostasis and matrix formation in the pathogenesis of lysosomal storage disorders.

Matrix Biol 2018 10 2;71-72:283-293. Epub 2018 Jun 2.

Telethon Institute of Genetics and Medicine (TIGEM), Via dei Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy.

The lysosome is a catabolic organelle devoted to the degradation of cellular components, such as protein complexes and whole or portion of organelles that reach the lysosomes through (macro)autophagy. The lysosomes also function as signaling organelles by controlling the activity of key metabolic kinases, such as the mechanistic target of Rapamycin complex 1 (mTORC1). Lysosome dysfunction has dramatic consequences on cellular homeostasis and causes lysosomal storage disorders (LSDs). Here we review the recently proposed mechanisms by which impairment of lysosome/autophagy pathway affects extracellular matrix formation and skeletal development and growth. In particular, we will highlight the role of autophagy as a collagen quality control pathway in collagen-producing cells. An impairment of autophagy, such as the one observed in LSDs, leads to a collagen proteostatic defects and can explain, at least in part, the skeletal phenotypes characterizing patients with lysosomal storage disorders.
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http://dx.doi.org/10.1016/j.matbio.2018.06.001DOI Listing
October 2018

mTORC1 hyperactivation arrests bone growth in lysosomal storage disorders by suppressing autophagy.

J Clin Invest 2017 Oct 5;127(10):3717-3729. Epub 2017 Sep 5.

Telethon Institute of Genetics and Medicine (TIGEM), and.

The mammalian target of rapamycin complex 1 (mTORC1) kinase promotes cell growth by activating biosynthetic pathways and suppressing catabolic pathways, particularly that of macroautophagy. A prerequisite for mTORC1 activation is its translocation to the lysosomal surface. Deregulation of mTORC1 has been associated with the pathogenesis of several diseases, but its role in skeletal disorders is largely unknown. Here, we show that enhanced mTORC1 signaling arrests bone growth in lysosomal storage disorders (LSDs). We found that lysosomal dysfunction induces a constitutive lysosomal association and consequent activation of mTORC1 in chondrocytes, the cells devoted to bone elongation. mTORC1 hyperphosphorylates the protein UV radiation resistance-associated gene (UVRAG), reducing the activity of the associated Beclin 1-Vps34 complex and thereby inhibiting phosphoinositide production. Limiting phosphoinositide production leads to a blockage of the autophagy flux in LSD chondrocytes. As a consequence, LSD chondrocytes fail to properly secrete collagens, the main components of the cartilage extracellular matrix. In mouse models of LSD, normalization of mTORC1 signaling or stimulation of the Beclin 1-Vps34-UVRAG complex rescued the autophagy flux, restored collagen levels in cartilage, and ameliorated the bone phenotype. Taken together, these data unveil a role for mTORC1 and autophagy in the pathogenesis of skeletal disorders and suggest potential therapeutic approaches for the treatment of LSDs.
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http://dx.doi.org/10.1172/JCI94130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617676PMC
October 2017

STUB1 regulates TFEB-induced autophagy-lysosome pathway.

EMBO J 2017 09 28;36(17):2544-2552. Epub 2017 Jul 28.

Department of Medicine, Baylor College of Medicine, Houston, TX, USA

TFEB is a master regulator for transcription of genes involved in autophagy and lysosome biogenesis. Activity of TFEB is inhibited upon its serine phosphorylation by mTOR The overall mechanisms by which TFEB activity in the cell is regulated are not well elucidated. Specifically, the mechanisms of TFEB turnover and how they might influence its activity remain unknown. Here, we show that STUB1, a chaperone-dependent E3 ubiquitin ligase, modulates TFEB activity by preferentially targeting inactive phosphorylated TFEB for degradation by the ubiquitin-proteasome pathway. Phosphorylated TFEB accumulated in STUB1-deficient cells and in tissues of STUB1-deficient mice resulting in reduced TFEB activity. Conversely, cellular overexpression of STUB1 resulted in reduced phosphorylated TFEB and increased TFEB activity. STUB1 preferentially interacted with and ubiqutinated phosphorylated TFEB, targeting it to proteasomal degradation. Consistent with reduced TFEB activity, accumulation of phosphorylated TFEB in STUB1-deficient cells resulted in reduced autophagy and reduced mitochondrial biogenesis. These studies reveal that the ubiquitin-proteasome pathway participates in regulating autophagy and lysosomal functions by regulating the activity of TFEB.
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http://dx.doi.org/10.15252/embj.201796699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5579343PMC
September 2017

Transcriptional activation of RagD GTPase controls mTORC1 and promotes cancer growth.

Science 2017 06;356(6343):1188-1192

Telethon Institute of Genetics and Medicine (TIGEM), Via Campi Flegrei 34, 80078 Pozzuoli, Naples, Italy.

The mechanistic target of rapamycin complex 1 (mTORC1) is recruited to the lysosome by Rag guanosine triphosphatases (GTPases) and regulates anabolic pathways in response to nutrients. We found that MiT/TFE transcription factors-master regulators of lysosomal and melanosomal biogenesis and autophagy-control mTORC1 lysosomal recruitment and activity by directly regulating the expression of RagD. In mice, this mechanism mediated adaptation to food availability after starvation and physical exercise and played an important role in cancer growth. Up-regulation of genes in cells and tissues from patients and murine models of renal cell carcinoma, pancreatic ductal adenocarcinoma, and melanoma triggered RagD-mediated mTORC1 induction, resulting in cell hyperproliferation and cancer growth. Thus, this transcriptional regulatory mechanism enables cellular adaptation to nutrient availability and supports the energy-demanding metabolism of cancer cells.
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http://dx.doi.org/10.1126/science.aag2553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730647PMC
June 2017

Low-dose Gene Therapy Reduces the Frequency of Enzyme Replacement Therapy in a Mouse Model of Lysosomal Storage Disease.

Mol Ther 2016 Dec 23;24(12):2054-2063. Epub 2016 Sep 23.

Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy.

Enzyme replacement therapy (ERT) is the standard of care for several lysosomal storage diseases (LSDs). ERT, however, requires multiple and costly administrations and has limited efficacy. We recently showed that a single high dose administration of adeno-associated viral vector serotype 8 (AAV2/8) is at least as effective as weekly ERT in a mouse model of mucopolysaccharidosis type VI (MPS VI). However, systemic administration of high doses of AAV might result in both cell-mediated immune responses and insertional mutagenesis. Here we evaluated whether the combination of low doses of AAV2/8 with a less frequent (monthly) than canonical (weekly) ERT schedule may be as effective as the single treatments at high doses or frequent regimen. A greater reduction of both urinary glycosaminoglycans, considered a sensitive biomarker of therapeutic efficacy, and storage in the myocardium and heart valves was observed in mice receiving the combined than the single therapies. Importantly, these levels of correction were similar to those we obtained in a previous study following either high doses of AAV2/8 or weekly ERT. Our data show that low-dose gene therapy can be used as a means to rarify ERT administration, thus reducing both the risks and costs associated with either therapies.
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http://dx.doi.org/10.1038/mt.2016.181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5159621PMC
December 2016

Modelling TFE renal cell carcinoma in mice reveals a critical role of WNT signaling.

Elife 2016 Sep 26;5. Epub 2016 Sep 26.

Telethon Institute of Genetics and Medicine, TIGEM, Pozzuoli, Naples, Italy.

-fusion renal cell carcinomas (-fusion ) are caused by chromosomal translocations that lead to overexpression of the and genes (Kauffman et al., 2014). The mechanisms leading to kidney tumor development remain uncharacterized and effective therapies are yet to be identified. Hence, the need to model these diseases in an experimental animal system (Kauffman et al., 2014). Here, we show that kidney-specific overexpression in transgenic mice, resulted in renal clear cells, multi-layered basement membranes, severe cystic pathology, and ultimately papillary carcinomas with hepatic metastases. These features closely recapitulate those observed in both and -mediated human kidney tumors. Analysis of kidney samples revealed transcriptional induction and enhanced signaling of the WNT β-catenin pathway. WNT signaling inhibitors normalized the proliferation rate of primary kidney cells and significantly rescued the disease phenotype in vivo. These data shed new light on the mechanisms underlying fusion and suggest a possible therapeutic strategy based on the inhibition of the WNT pathway.
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http://dx.doi.org/10.7554/eLife.17047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5036965PMC
September 2016

Brain Disorders Due to Lysosomal Dysfunction.

Annu Rev Neurosci 2016 07 18;39:277-95. Epub 2016 Apr 18.

Telethon Institute of Genetics and Medicine (TIGEM), 80078 Pozzuoli, Italy.

Recent studies of autophagic and lysosomal pathways have significantly changed our understanding of lysosomes; once thought to be simple degradative and recycling centers, lysosomes are now known to be organelles capable of influencing signal transduction, via the mammalian target of rapamycin complex 1 (mTORC1), and regulating gene expression, via transcription factor EB (TFEB) and other transcription factors. These pathways are particularly relevant to maintaining brain homeostasis, as dysfunction of the endolysosomal and autophagic pathways has been associated with common neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's, and lysosomal storage disorders, a group of inherited disorders characterized by the intralysosomal buildup of partially degraded metabolites. This review focuses on the cellular biology of lysosomes and discusses the possible mechanisms by which disruption of their function contributes to neurodegeneration. We also review and discuss how targeting TFEB and lysosomes may offer innovative therapeutic approaches for treating a wide range of neurological conditions.
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http://dx.doi.org/10.1146/annurev-neuro-070815-014031DOI Listing
July 2016

Autophagy gets to the bone.

Cell Cycle 2016 3;15(7):871-2. Epub 2016 Mar 3.

a Dulbecco Telethon Institute , Pozzuoli ( NA ), Italy.

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http://dx.doi.org/10.1080/15384101.2016.1151724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889256PMC
April 2017

FGF signalling regulates bone growth through autophagy.

Nature 2015 Dec 23;528(7581):272-5. Epub 2015 Nov 23.

Telethon Institute of Genetics and Medicine (TIGEM), Via Campi Flegrei, 34, 80078 Pozzuoli (NA), Italy.

Skeletal growth relies on both biosynthetic and catabolic processes. While the role of the former is clearly established, how the latter contributes to growth-promoting pathways is less understood. Macroautophagy, hereafter referred to as autophagy, is a catabolic process that plays a fundamental part in tissue homeostasis. We investigated the role of autophagy during bone growth, which is mediated by chondrocyte rate of proliferation, hypertrophic differentiation and extracellular matrix (ECM) deposition in growth plates. Here we show that autophagy is induced in growth-plate chondrocytes during post-natal development and regulates the secretion of type II collagen (Col2), the major component of cartilage ECM. Mice lacking the autophagy related gene 7 (Atg7) in chondrocytes experience endoplasmic reticulum storage of type II procollagen (PC2) and defective formation of the Col2 fibrillary network in the ECM. Surprisingly, post-natal induction of chondrocyte autophagy is mediated by the growth factor FGF18 through FGFR4 and JNK-dependent activation of the autophagy initiation complex VPS34-beclin-1. Autophagy is completely suppressed in growth plates from Fgf18(-/-) embryos, while Fgf18(+/-) heterozygous and Fgfr4(-/-) mice fail to induce autophagy during post-natal development and show decreased Col2 levels in the growth plate. Strikingly, the Fgf18(+/-) and Fgfr4(-/-) phenotypes can be rescued in vivo by pharmacological activation of autophagy, pointing to autophagy as a novel effector of FGF signalling in bone. These data demonstrate that autophagy is a developmentally regulated process necessary for bone growth, and identify FGF signalling as a crucial regulator of autophagy in chondrocytes.
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http://dx.doi.org/10.1038/nature16063DOI Listing
December 2015

Lysosomal calcium signalling regulates autophagy through calcineurin and ​TFEB.

Nat Cell Biol 2015 Mar;17(3):288-99

The view of the lysosome as the terminal end of cellular catabolic pathways has been challenged by recent studies showing a central role of this organelle in the control of cell function. Here we show that a lysosomal Ca2+ signalling mechanism controls the activities of the phosphatase calcineurin and of its substrate ​TFEB, a master transcriptional regulator of lysosomal biogenesis and autophagy. Lysosomal Ca2+ release through ​mucolipin 1 (​MCOLN1) activates calcineurin, which binds and dephosphorylates ​TFEB, thus promoting its nuclear translocation. Genetic and pharmacological inhibition of calcineurin suppressed ​TFEB activity during starvation and physical exercise, while calcineurin overexpression and constitutive activation had the opposite effect. Induction of autophagy and lysosomal biogenesis through ​TFEB required ​MCOLN1-mediated calcineurin activation. These data link lysosomal calcium signalling to both calcineurin regulation and autophagy induction and identify the lysosome as a hub for the signalling pathways that regulate cellular homeostasis.
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http://dx.doi.org/10.1038/ncb3114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4801004PMC
March 2015

Insulin granules. Insulin secretory granules control autophagy in pancreatic β cells.

Science 2015 Feb;347(6224):878-82

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM, CNRS, Université de Strasbourg, 67404 Illkirch, France. Nouvel Hôpital Civil, Laboratoire de Biochimie et de Biologie Moléculaire, Université de Strasbourg, 67091 Strasbourg, France.

Pancreatic β cells lower insulin release in response to nutrient depletion. The question of whether starved β cells induce macroautophagy, a predominant mechanism maintaining energy homeostasis, remains poorly explored. We found that, in contrast to many mammalian cells, macroautophagy in pancreatic β cells was suppressed upon starvation. Instead, starved β cells induced lysosomal degradation of nascent secretory insulin granules, which was controlled by protein kinase D (PKD), a key player in secretory granule biogenesis. Starvation-induced nascent granule degradation triggered lysosomal recruitment and activation of mechanistic target of rapamycin that suppressed macroautophagy. Switching from macroautophagy to insulin granule degradation was important to keep insulin secretion low upon fasting. Thus, β cells use a PKD-dependent mechanism to adapt to nutrient availability and couple autophagy flux to secretory function.
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http://dx.doi.org/10.1126/science.aaa2628DOI Listing
February 2015

TFEB and the CLEAR network.

Methods Cell Biol 2015 19;126:45-62. Epub 2015 Jan 19.

Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy.

The general view of the lysosome as the terminal end of cellular catabolic pathways, has started to change due to the recent discoveries of a lysosomal nutrient sensing machinery and of a lysosome-to-nucleus signaling mechanism that modulate lysosomal function by way of the master transcriptional regulator TFEB. Lysosomal biogenesis and autophagy are coordinated by TFEB, whose function is regulated by phosphorylation. TFEB interacts with and is phosphorylated by mTORC1 at the lysosomal surface. Thus, conditions resulting in inhibition of mTOR, such as starvation and lysosomal stress, promote TFEB nuclear translocation. Preliminary evidences showing that the TFEB activation are able to ameliorate the phenotype of lysosomal storage disorders and more common neurodegenerative diseases have opened an extraordinary possibility for the development of innovative therapies. Research in TFEB and lysosomal function has continued to advance and attract interest due to increased understanding of the mechanisms behind lysosomal function. In this paper, we present a set of procedures that facilitate examination of TFEB function and its related processes.
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http://dx.doi.org/10.1016/bs.mcb.2014.11.011DOI Listing
October 2015

Direct conversion of fibroblasts into functional astrocytes by defined transcription factors.

Stem Cell Reports 2015 Jan 31;4(1):25-36. Epub 2014 Dec 31.

Stem Cell and Neurogenesis Unit, Division of Neuroscience, San Raffaele Scientific Institute, Milan 20132, Italy. Electronic address:

Direct cell reprogramming enables direct conversion of fibroblasts into functional neurons and oligodendrocytes using a minimal set of cell-lineage-specific transcription factors. This approach is rapid and simple, generating the cell types of interest in one step. However, it remains unknown whether this technology can be applied to convert fibroblasts into astrocytes, the third neural lineage. Astrocytes play crucial roles in neuronal homeostasis, and their dysfunctions contribute to the origin and progression of multiple human diseases. Herein, we carried out a screening using several transcription factors involved in defining the astroglial cell fate and identified NFIA, NFIB, and SOX9 to be sufficient to convert with high efficiency embryonic and postnatal mouse fibroblasts into astrocytes (iAstrocytes). We proved both by gene-expression profiling and functional tests that iAstrocytes are comparable to native brain astrocytes. This protocol can be then employed to generate functional iAstrocytes for a wide range of experimental applications.
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http://dx.doi.org/10.1016/j.stemcr.2014.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4297873PMC
January 2015

Cell metabolism: autophagy transcribed.

Nature 2014 Dec 12;516(7529):40-1. Epub 2014 Nov 12.

Telethon Institute of Genetics and Medicine, Naples 80078, Italy; in the Department of Translational Medicine, Federico II University, Naples; and in the Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

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http://dx.doi.org/10.1038/nature13939DOI Listing
December 2014
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