Defective autophagy is a key feature of cerebral cavernous malformations
2015; Springer Nature; Volume: 7; Issue: 11 Linguagem: Inglês
10.15252/emmm.201505316
ISSN1757-4684
AutoresSaverio Marchi, Mariangela Corricelli, Eliana Trapani, Luca Bravi, Alessandra Pittaro, Simona Delle Monache, Letizia Ferroni, Simone Patergnani, Sonia Missiroli, Luca Goitre, Lorenza Trabalzini, Alessandro Rimessi, Carlotta Giorgi, Barbara Zavan, Paola Cassoni, Elisabetta Dejana, Saverio Francesco Retta, Paolo Pinton,
Tópico(s)Autophagy in Disease and Therapy
ResumoReport28 September 2015Open Access Source Data Defective autophagy is a key feature of cerebral cavernous malformations Saverio Marchi Saverio Marchi Department of Morphology, Surgery and Experimental Medicine, Section of Pathology Oncology and Experimental Biology, University of Ferrara, Ferrara, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Mariangela Corricelli Mariangela Corricelli Department of Morphology, Surgery and Experimental Medicine, Section of Pathology Oncology and Experimental Biology, University of Ferrara, Ferrara, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Eliana Trapani Eliana Trapani Department of Clinical and Biological Sciences, University of Torino, Torino, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Luca Bravi Luca Bravi IFOM, FIRC Institute of Molecular Oncology, Milano, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Alessandra Pittaro Alessandra Pittaro Department of Medical Sciences, University of Torino, Torino, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Simona Delle Monache Simona Delle Monache Department of Biotechnological and Applied Clinical Science, University of L'Aquila, L'Aquila, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Letizia Ferroni Letizia Ferroni Department of Biomedical Sciences, University of Padova, Padova, Italy Search for more papers by this author Simone Patergnani Simone Patergnani Department of Morphology, Surgery and Experimental Medicine, Section of Pathology Oncology and Experimental Biology, University of Ferrara, Ferrara, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Sonia Missiroli Sonia Missiroli Department of Morphology, Surgery and Experimental Medicine, Section of Pathology Oncology and Experimental Biology, University of Ferrara, Ferrara, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Luca Goitre Luca Goitre Department of Clinical and Biological Sciences, University of Torino, Torino, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Lorenza Trabalzini Lorenza Trabalzini Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Alessandro Rimessi Alessandro Rimessi Department of Morphology, Surgery and Experimental Medicine, Section of Pathology Oncology and Experimental Biology, University of Ferrara, Ferrara, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Carlotta Giorgi Carlotta Giorgi Department of Morphology, Surgery and Experimental Medicine, Section of Pathology Oncology and Experimental Biology, University of Ferrara, Ferrara, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Barbara Zavan Barbara Zavan Department of Biomedical Sciences, University of Padova, Padova, Italy Search for more papers by this author Paola Cassoni Paola Cassoni Department of Medical Sciences, University of Torino, Torino, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Elisabetta Dejana Elisabetta Dejana IFOM, FIRC Institute of Molecular Oncology, Milano, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Saverio Francesco Retta Corresponding Author Saverio Francesco Retta Department of Clinical and Biological Sciences, University of Torino, Torino, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Paolo Pinton Corresponding Author Paolo Pinton Department of Morphology, Surgery and Experimental Medicine, Section of Pathology Oncology and Experimental Biology, University of Ferrara, Ferrara, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Saverio Marchi Saverio Marchi Department of Morphology, Surgery and Experimental Medicine, Section of Pathology Oncology and Experimental Biology, University of Ferrara, Ferrara, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Mariangela Corricelli Mariangela Corricelli Department of Morphology, Surgery and Experimental Medicine, Section of Pathology Oncology and Experimental Biology, University of Ferrara, Ferrara, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Eliana Trapani Eliana Trapani Department of Clinical and Biological Sciences, University of Torino, Torino, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Luca Bravi Luca Bravi IFOM, FIRC Institute of Molecular Oncology, Milano, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Alessandra Pittaro Alessandra Pittaro Department of Medical Sciences, University of Torino, Torino, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Simona Delle Monache Simona Delle Monache Department of Biotechnological and Applied Clinical Science, University of L'Aquila, L'Aquila, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Letizia Ferroni Letizia Ferroni Department of Biomedical Sciences, University of Padova, Padova, Italy Search for more papers by this author Simone Patergnani Simone Patergnani Department of Morphology, Surgery and Experimental Medicine, Section of Pathology Oncology and Experimental Biology, University of Ferrara, Ferrara, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Sonia Missiroli Sonia Missiroli Department of Morphology, Surgery and Experimental Medicine, Section of Pathology Oncology and Experimental Biology, University of Ferrara, Ferrara, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Luca Goitre Luca Goitre Department of Clinical and Biological Sciences, University of Torino, Torino, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Lorenza Trabalzini Lorenza Trabalzini Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Alessandro Rimessi Alessandro Rimessi Department of Morphology, Surgery and Experimental Medicine, Section of Pathology Oncology and Experimental Biology, University of Ferrara, Ferrara, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Carlotta Giorgi Carlotta Giorgi Department of Morphology, Surgery and Experimental Medicine, Section of Pathology Oncology and Experimental Biology, University of Ferrara, Ferrara, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Barbara Zavan Barbara Zavan Department of Biomedical Sciences, University of Padova, Padova, Italy Search for more papers by this author Paola Cassoni Paola Cassoni Department of Medical Sciences, University of Torino, Torino, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Elisabetta Dejana Elisabetta Dejana IFOM, FIRC Institute of Molecular Oncology, Milano, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Saverio Francesco Retta Corresponding Author Saverio Francesco Retta Department of Clinical and Biological Sciences, University of Torino, Torino, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Paolo Pinton Corresponding Author Paolo Pinton Department of Morphology, Surgery and Experimental Medicine, Section of Pathology Oncology and Experimental Biology, University of Ferrara, Ferrara, ItalyCCM Italia Research Network (www.ccmitalia.unito.it) Search for more papers by this author Author Information Saverio Marchi1, Mariangela Corricelli1,‡, Eliana Trapani2,‡, Luca Bravi3,‡, Alessandra Pittaro4, Simona Delle Monache5, Letizia Ferroni6, Simone Patergnani1, Sonia Missiroli1, Luca Goitre2, Lorenza Trabalzini7, Alessandro Rimessi1, Carlotta Giorgi1, Barbara Zavan6, Paola Cassoni4, Elisabetta Dejana3, Saverio Francesco Retta 2 and Paolo Pinton 1 1Department of Morphology, Surgery and Experimental Medicine, Section of Pathology Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy 2Department of Clinical and Biological Sciences, University of Torino, Torino, Italy 3IFOM, FIRC Institute of Molecular Oncology, Milano, Italy 4Department of Medical Sciences, University of Torino, Torino, Italy 5Department of Biotechnological and Applied Clinical Science, University of L'Aquila, L'Aquila, Italy 6Department of Biomedical Sciences, University of Padova, Padova, Italy 7Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italy ‡These authors contributed equally to this work *Corresponding author. Tel: +39 011 6706426; E-mail: [email protected] *Corresponding author. Tel: +39 0532 455802; E-mail: [email protected] EMBO Mol Med (2015)7:1403-1417https://doi.org/10.15252/emmm.201505316 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cerebral cavernous malformation (CCM) is a major cerebrovascular disease affecting approximately 0.3–0.5% of the population and is characterized by enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhages. Cerebral cavernous malformation is a genetic disease that may arise sporadically or be inherited as an autosomal dominant condition with incomplete penetrance and variable expressivity. Causative loss-of-function mutations have been identified in three genes, KRIT1 (CCM1), CCM2 (MGC4607), and PDCD10 (CCM3), which occur in both sporadic and familial forms. Autophagy is a bulk degradation process that maintains intracellular homeostasis and that plays essential quality control functions within the cell. Indeed, several studies have identified the association between dysregulated autophagy and different human diseases. Here, we show that the ablation of the KRIT1 gene strongly suppresses autophagy, leading to the aberrant accumulation of the autophagy adaptor p62/SQSTM1, defective quality control systems, and increased intracellular stress. KRIT1 loss-of-function activates the mTOR-ULK1 pathway, which is a master regulator of autophagy, and treatment with mTOR inhibitors rescues some of the mole-cular and cellular phenotypes associated with CCM. Insufficient autophagy is also evident in CCM2-silenced human endothelial cells and in both cells and tissues from an endothelial-specific CCM3-knockout mouse model, as well as in human CCM lesions. Furthermore, defective autophagy is highly correlated to endothelial-to-mesenchymal transition, a crucial event that contributes to CCM progression. Taken together, our data point to a key role for defective autophagy in CCM disease pathogenesis, thus providing a novel framework for the development of new pharmacological strategies to prevent or reverse adverse clinical outcomes of CCM lesions. Synopsis This study provides new evidence on the mechanisms underlying cerebral cavernous malformation (CCM) disease pathogenesis, opening the prospect and offering valuable clues for the development of novel therapeutic approaches based on the regulation of the autophagic process. KRIT1 deletion in murine and human endothelial cell lines results in autophagy defects that cause aberrant accumulation of the autophagy adaptor p62/SQSTM1 and aggresome-like structures. Impaired autophagy in KRIT1 deficient endothelial cells is accompanied by over-activation of mTOR signaling pathway, suggesting novel targets for therapeutic intervention. Defective autophagy underlies major phenotypic signatures of CCM disease, including EndMt that contributes to CCM disease progression. mTOR inhibitors restore autophagy in KRIT1-depleted cells and rescue molecular and cellular disease phenotypes, thus alleviating potential causes of CCM lesion formation and progression. Defective autophagy and consequent p62/SQSTM1 accumulation are common features of loss-of-function mutations of the three known CCM genes. Introduction Cerebral cavernous malformations (CCMs; OMIM 116860), which are also known as cavernous angiomas or cavernomas, are major vascular malformations consisting of closely clustered, abnormally dilated, and leaky capillary channels (caverns) lined by a thin endothelium and devoid of normal vessel structural components (Clatterbuck et al, 2001; Gault et al, 2004; Batra et al, 2009; Cavalcanti et al, 2012). Cerebral cavernous malformation lesions are estimated to occur in 0.3–0.5% of the general population (Cavalcanti et al, 2012) and can either remain clinically silent or cause serious clinical symptoms, such as headaches, neurological deficits, seizures, strokes, and intracerebral hemorrhages (Gault et al, 2004; Batra et al, 2009). Approximately 30% of people with CCM lesions will eventually develop clinical symptoms. Cerebral cavernous malformation has a known genetic origin and may either arise sporadically or be inherited as an autosomal dominant condition with incomplete penetrance and variable expressivity. Genetic studies have identified three genes whose loss-of-function mutations cause CCM: KRIT1 (CCM1), MGC4607 (CCM2), and PDCD10 (CCM3), which account for approximately 50, 20, and 10% of CCM cases, respectively. The remaining 20% of cases have been attributed to mutations in a fourth unidentified CCM gene (Riant et al, 2010). Notably, the hereditary form of this illness is often associated with multiple cavernous angiomas, whereas the sporadic form typically presents as a solitary lesion. At present, no direct therapeutic approaches for CCM disease exist other than the surgical removal of accessible lesions in patients with recurrent hemorrhage or intractable seizures. In particular, novel pharmacological strategies are required for preventing the de novo formation of CCM lesions in susceptible individuals and the progression of the disease. Useful insights into the definition of novel approaches for CCM disease prevention and treatment could be derived from a deep understanding of the mechanisms underlying CCM pathogenesis. Macroautophagy (termed autophagy in this manuscript) is a bulk degradation process that occurs in two primary steps: (i) the sequestration of proteins and organelles into double-membrane vesicles called autophagosomes and (ii) their subsequent degradation through the fusion of autophagosomes with lysosomes (Xie & Klionsky, 2007; Feng et al, 2014). By selectively degrading harmful protein aggregates or damaged organelles, autophagy maintains intracellular homeostasis and plays essential quality control functions within the cell (Mizushima & Komatsu, 2011). Defective autophagy occurs in several pathological conditions, including cancers, neurodegenerative and cardiovascular diseases, and metabolic disorders (Levine & Kroemer, 2008; Choi et al, 2013). The suppression of autophagy causes the accumulation of proteins and potentially hazardous intracellular structures, thereby inducing high levels of metabolic stress and limiting organelle functionality. Consequently, using a pharmacological approach to re-establish physiological levels of autophagy may be beneficial in treating certain diseases. Nevertheless, several clinical trials are currently based on the employment of agents acting on autophagy induction (Choi et al, 2013; Jiang & Mizushima, 2014). In the present study, we show that human CCM lesions display increased levels of p62/SQSTM1, an autophagic marker that accumulates when autophagy is inhibited, and demonstrate that both KRIT1 and CCM3 loss-of-function impair autophagy through the up-regulation of the mechanistic target of rapamycin (mTOR) pathway, leading to a defective quality control system and the accumulation of aberrant and aggregated proteins. Our data raise the possibility that therapeutic activation of autophagy might prevent or reverse adverse clinical outcomes, thus improving the long-term prognosis of CCM patients. Results and Discussion KRIT1 deletion suppresses autophagy To study the contribution of autophagy to CCM pathogenesis, we investigated whether KRIT1 down-regulation would lead to the impairment of autophagy in endothelial cell lines. Endothelial-specific KRIT1 knockout (KO) in mice produced lesions that were identical to the CCM malformations observed in humans (Boulday et al, 2011; Maddaluno et al, 2013). We used KRIT1-KO lung endothelial cells derived from KRIT1fl/fl mice treated with Tat-Cre recombinase (Maddaluno et al, 2013). p62/SQSTM1 acts as a receptor for ubiquitinated cargoes and delivers them to the autophagosome, and p62 itself is incorporated into the autophagosome and subsequently degraded by autophagy (Komatsu et al, 2007). The autophagy protein microtubule-associated protein 1 light chain 3 (LC3) is present in the cytosol in the LC3-I form, until it is modified to a cleaved and lipidated membrane-bound form (LC3-II), which is localized to autophagosomes. Thus, in addition to p62 accumulation, another typical trait of autophagy inhibition consists of increased amounts of the cytosolic non-lipidated form of LC3 (LC3-I) and of total LC3 (Mizushima et al, 2010; Wang et al, 2012). As shown in Fig 1A, KRIT1 deficiency was associated with defective autophagy, displaying increased levels of p62 and total LC3. Figure 1. KRIT1-ablated cells display autophagy suppression Immunoblot analysis of p62 and LC3 I/II in KRIT1 wt and KRIT1-KO endothelial cells. Actin was used as a loading control. Quantification of total LC3 on actin is reported (*P = 0.02712). The results are representative of three independent experiments. Representative images of p62 dots in KRIT1 wt and KRIT1-KO endothelial cells. Scale bar, 20 μm. Magnifications in insets. Right, quantitative analysis of p62 distribution of dots is reported (four independent experiments; n = 35 cells per group). *P = 0.00542 (dotted); *P = 0.00014 (nuclear). Immunoblot analysis of p62 and LC3 I/II in KRIT1-KO and KRIT1-KO re-expressing KRIT1 (KO+KRIT1) MEFs. Left, immunoblot showing KRIT1 levels in KRIT1-KO and KO+KRIT1 cells. Right, immunoblots for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.01248). The results are representative of three independent experiments. Representative images of p62 dots in KO+KRIT1 (top) and KRIT1-KO cells (bottom). Scale bar, 50 μm. Magnifications in insets. Right, quantitative analysis of the number of p62 dots per cell is shown (four independent experiments; n = 50 cells per group). *P = 7.18e−14. Immunoblot analysis of hBMECs transiently transfected with control siRNA or KRIT1 siRNA. Left, evaluation of siRNA efficiency with antibody directed against KRIT1. Right, immunoblots for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.03071). The results are representative of three independent experiments. Immunoblot analysis of EA.hy926 cells transiently transfected with control siRNA or KRIT1 siRNA. Left, evaluation of siRNA efficiency with antibody directed against KRIT1. Right, immunoblot for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.02527). The results are representative of three independent experiments. Immunofluorescence analysis of p62 (green) and ProteoStat Aggresome staining detection reagent (red) in KRIT1 wt and KRIT1-KO lung endothelial cells. The yellow signal in the merged images represents an overlapping spatial relationship between green and red fluorescence. Magnification in insets. Scale bar, 50 μm. The images are representative of four independent experiments. Immunofluorescence analysis of p62 (green) and ProteoStat Aggresome staining detection reagent (red) in KRIT1-KO re-expressing KRIT1 (KO+KRIT1) and KRIT1-KO MEFs. The yellow signal in the merged images represents an overlapping spatial relationship between green and red fluorescence. Magnification in insets. Scale bar, 50 μm. The images are representative of four independent experiments. Source data are available online for this figure. Source Data for Figure 1 [emmm201505316-sup-0002-SdataFig1.pdf] Download figure Download PowerPoint Upon autophagy inhibition, p62 has been reported to be present in several types of cytoplasmic inclusions and to display a typical punctate pattern (Bjorkoy et al, 2005). Importantly, analysis of p62 distribution through immunofluorescence staining revealed a nuclear-enriched pattern with rare cytoplasmic dots in ~60% of KRIT1 wild-type (wt) cells. Conversely, in KO endothelial cells, the protein is primarily cytoplasmatic, forming intense perinuclear bodies with weak staining in the nucleus (Fig 1B). To investigate whether defective autophagy in CCM is a cell-autonomous process, we took advantage of KRIT1 KO (KRIT1-KO) mouse embryonic fibroblasts (MEFs), a previously established and characterized cellular model that allowed the identification of new molecules and mechanisms involved in CCM pathogenesis (Goitre et al, 2010, 2014), providing novel therapeutic perspectives (Gibson et al, 2015; Moglia et al, 2015). Compared with KRIT1-KO MEFs re-expressing KRIT1 (Fig 1C; KO+KRIT1), KRIT1-KO MEFs (Fig 1C; KO) displayed increased levels of p62 as well as significantly increased levels of total LC3 protein (Fig 1C). Moreover, immunostaining analysis revealed that KRIT1 depletion led to increases in the number of p62-containing bodies (Fig 1D), with diameters of approximately 1.5 μm. Next, we examined whether KRIT1 ablation also inhibits autophagy in human cells. The silencing of KRIT1 suppressed autophagy in both the human cerebral microvascular endothelial cell line hBMEC (Fig 1E) and the human umbilical vein cell line EA.hy926 (Fig 1F), as evidenced by increased p62 and LC3 accumulation. p62 protein expression is highly regulated at the transcriptional level via the JNK pathway (Puissant et al, 2010) or the NRF2 transcription factor, particularly under oxidative stress (He & Klionsky, 2009; Puissant et al, 2012). Considering that KRIT1 is involved in reactive oxygen species (ROS) homeostasis (Goitre et al, 2010, 2014; Jung et al, 2014), we tested whether p62 accumulation in KRIT1-KO cells was associated with autophagy inhibition rather than with ROS-dependent transcriptional effects. As expected, treatment with the antioxidant N-acetylcysteine (NAC) decreased p62 levels, but the disruption of KRIT1 still induced p62 accumulation (Appendix Fig S1A). Moreover, similar results were obtained using the protein synthesis inhibitor cycloheximide (CHX) (Appendix Fig S1B), further supporting the notion that the inhibition of autophagy-dependent protein turnover upon KRIT1 loss contributes to p62 accumulation. Consistently, no differences in p62 mRNA levels between wt and KRIT1-KO endothelial cells have been detected (Appendix Fig S1C). Importantly, when autophagy-mediated degradation is inhibited, p62 appears to be partially detergent insoluble (Klionsky et al, 2012); therefore, the lysates were divided between Triton X-100 (TX-100)-soluble and TX-100-insoluble fractions and subsequently analyzed for their protein content. The loss of KRIT1 in both endothelial cells (Appendix Fig S1D) and MEFs (Appendix Fig S1E) promoted increased levels of p62 in both the soluble and insoluble fractions, which is consistent with previous observations made under defective autophagy and high protein aggregation conditions (Waguri & Komatsu, 2009; Fujita et al, 2011; Magnaudeix et al, 2013). Autophagy is responsible for the degradation of large structures such as organelles and protein aggregates (Rabinowitz & White, 2010; Cheng et al, 2013). Consequently, we analyzed whether the defective autophagy observed upon KRIT1 loss might induce the accumulation of aggresome-like structures. As shown in Fig 1G, we observed greater colocalization between p62 and aggresomes in endothelial KRIT1-KO cells, as well as extremely high fluorescence intensity of aggresome-like inclusion bodies. The same results have been obtained in different cellular systems, such as MEFs (Fig 1H) or KRIT1-silenced hBMECs and EA.hy926 cells (Appendix Fig S1F and G), indicating that the loss of KRIT1 promotes the accumulation of aberrant proteins that could be reasonably ascribed to defective autophagy. Virtually identical observations have been reported for other autophagy-deficient scenarios (Maejima et al, 2013; Wolf et al, 2013). These findings suggest that KRIT1 ablation is sufficient to suppress autophagy in a cell-autonomous manner. Indeed, KRIT1 silencing or disruption in four different cellular contexts has been shown to result in the expression of typical markers of defective autophagy, such as increased accumulation of p62 and increased amounts of LC3-I and of total LC3. KRIT1 deletion induces up-regulation of the mTOR-ULK1 pathway The mTOR signaling network is recognized as the most important regulator of autophagy, and its implication in a wide range of diseases has been largely documented (Laplante & Sabatini, 2012). Direct selective inhibition of mTOR, through the allosteric inhibitor rapamycin or the small molecule ATP-competitive inhibitor Torin1, induces autophagy in many cell types (Kundu, 2011). Consequently, we tested whether the defective autophagy observed upon KRIT1 deletion resulted from dysregulation of the mTOR pathway. Immunoblot analysis revealed marked up-regulation of mTOR signaling in KRIT1-KO endothelial cells, as evidenced by the increased phosphorylation of both mTOR and its downstream targets p70S6k and 4E-BP1 (Fig 2A). Importantly, treatment with Torin1 suppressed mTOR activation even in KO cells, suggesting that a pharmacological approach based on mTOR inhibition might re-activate autophagy in these cells. Figure 2. KRIT1 loss-of-function activates the mTOR-ULK1 pathway Immunoblot analysis with antibodies directed against phosphorylated mTOR (Ser 2448), total mTOR, phosphorylated p70 S6 Kinase (Ser 371), total p70 S6 Kinase, phosphorylated 4E-BP1 (Thr 37/46), and total 4E-BP1; actin was used as a loading marker. Where indicated, KRIT1 wt and KRIT1-KO endothelial cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments. Immunoblot analysis of total ULK1 and actin in KRIT1 wt and KRIT1-KO endothelial cells. Where indicated, cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments. Immunoblot analysis of p62, LC3 I/II, and actin in KRIT1 wt and KRIT1-KO endothelial cells treated with 100 nM Torin1 or 500 nM rapamycin for 4 h. The results are representative of three independent experiments. Immunoblot analysis with antibodies directed against phosphorylated mTOR (Ser 2448), total mTOR, phosphorylated p70 S6 Kinase (Ser 371), total p70 S6 Kinase, phosphorylated 4E-BP1 (Thr 37/46), and total 4E-BP1; actin was used as a loading marker. Where indicated, KRIT1-KO re-expressing KRIT1 (KO+KRIT1) and KRIT1-KO MEFs were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments. Immunoblot analysis of phosphorylated ULK1 (Ser 757), total ULK1, and actin in KRIT1 KO+KRIT1, and KRIT1 KO MEFs. Where indicated, cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments. Immunoblot analysis of p62, actin, LC3 I/II in KO+KRIT1 and KRIT1-KO cells. Where indicated, cells were treated with 100 nM Torin1 for 4 h or 500 nM rapamycin for 4 h. The results are representative of three independent experiments. KRIT1 wt and KRIT1-KO endothelial cells were transiently transfected with mRFP-GFP-LC3. Where indicated, the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h. The differences in the autophagic flux were evaluated by counting the yellow LC3 I/II dots/cell (RFP+GFP+) and red LC3 dots/cell (RFP+GFP−) for each condition. Yellow dots: autophagosomes; red dots: autophagolysosomes. *P = 5.74e−5 (red dots, WT ctrl vs. WT Tor1); *P = 9.62e−5 (red dots, WT ctrl vs. WT xesto); *P = 0.00727 (red dots, WT ctrl vs. KO ctrl); #P = 0.00046 (red dots, KO ctrl vs. KO Tor1). The data are expressed as the mean ± s.e.m. KO+KRIT1 and KRIT1-KO MEFs were transiently transfected with the mRFP-GFP-LC3 tandem construct. Where indicated, the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h. The differences in th
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