Anna Chiara Nascimbeni

Phosphatidylinositol‐3‐phosphate ( PI 3P) is a key player in membrane dynamics and trafficking regulation. Most PI 3P is associated with endosomal membranes and with the autophagosome preassembly machinery, presumably at the endoplasmic reticulum. The enzyme responsible for most PI 3P synthesis, VPS 34 and proteins such as Beclin1 and ATG 14L that regulate PI 3P levels are positive modulators of autophagy initiation. It had been assumed that a local PI 3P pool was present at autophagosomes and preautophagosomal structures, such as the omegasome and the phagophore. This was recently confirmed by the demonstration that PI 3P‐binding proteins participate in the complex sequence of signalling that results in autophagosome assembly and activity. Here we summarize the historical discoveries of PI 3P lipid kinase involvement in autophagy, and we discuss the proposed role of PI 3P during autophagy, notably during the autophagosome biogenesis sequence.

Article26 May 2017free access Transparent process ER–plasma membrane contact sites contribute to autophagosome biogenesis by regulation of local PI3P synthesis Anna Chiara Nascimbeni Anna Chiara Nascimbeni Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Francesca Giordano Francesca Giordano orcid.org/0000-0002-5942-1753 Institut Jacques Monod, CNRS UMR 7592, Paris, France Université Paris Diderot-Sorbonne Paris Cité, Paris, France Search for more papers by this author Nicolas Dupont Nicolas Dupont Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Daniel Grasso Daniel Grasso Department of Pathophysiology, Institute of Biochemistry and Molecular Medicine, National Council for Scientific and Technological Research, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina Search for more papers by this author Maria I Vaccaro Maria I Vaccaro Department of Pathophysiology, Institute of Biochemistry and Molecular Medicine, National Council for Scientific and Technological Research, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina Search for more papers by this author Patrice Codogno Patrice Codogno Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Etienne Morel Corresponding Author Etienne Morel [email protected] orcid.org/0000-0002-4763-4954 Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Anna Chiara Nascimbeni Anna Chiara Nascimbeni Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Francesca Giordano Francesca Giordano orcid.org/0000-0002-5942-1753 Institut Jacques Monod, CNRS UMR 7592, Paris, France Université Paris Diderot-Sorbonne Paris Cité, Paris, France Search for more papers by this author Nicolas Dupont Nicolas Dupont Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Daniel Grasso Daniel Grasso Department of Pathophysiology, Institute of Biochemistry and Molecular Medicine, National Council for Scientific and Technological Research, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina Search for more papers by this author Maria I Vaccaro Maria I Vaccaro Department of Pathophysiology, Institute of Biochemistry and Molecular Medicine, National Council for Scientific and Technological Research, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina Search for more papers by this author Patrice Codogno Patrice Codogno Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Etienne Morel Corresponding Author Etienne Morel [email protected] orcid.org/0000-0002-4763-4954 Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Author Information Anna Chiara Nascimbeni1,2, Francesca Giordano3,4, Nicolas Dupont1,2, Daniel Grasso5, Maria I Vaccaro5, Patrice Codogno1,2 and Etienne Morel *,1,2 1Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France 2Université Paris Descartes-Sorbonne Paris Cité, Paris, France 3Institut Jacques Monod, CNRS UMR 7592, Paris, France 4Université Paris Diderot-Sorbonne Paris Cité, Paris, France 5Department of Pathophysiology, Institute of Biochemistry and Molecular Medicine, National Council for Scientific and Technological Research, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina *Corresponding author. Tel: +33 172606474; Fax: +33 172606399; E-mail: [email protected] The EMBO Journal (2017)36:2018-2033https://doi.org/10.15252/embj.201797006 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 The double-membrane-bound autophagosome is formed by the closure of a structure called the phagophore, origin of which is still unclear. The endoplasmic reticulum (ER) is clearly implicated in autophagosome biogenesis due to the presence of the omegasome subdomain positive for DFCP1, a phosphatidyl-inositol-3-phosphate (PI3P) binding protein. Contribution of other membrane sources, like the plasma membrane (PM), is still difficult to integrate in a global picture. Here we show that ER–plasma membrane contact sites are mobilized for autophagosome biogenesis, by direct implication of the tethering extended synaptotagmins (E-Syts) proteins. Imaging data revealed that early autophagic markers are recruited to E-Syt-containing domains during autophagy and that inhibition of E-Syts expression leads to a reduction in autophagosome biogenesis. Furthermore, we demonstrate that E-Syts are essential for autophagy-associated PI3P synthesis at the cortical ER membrane via the recruitment of VMP1, the stabilizing ER partner of the PI3KC3 complex. These results highlight the contribution of ER–plasma membrane tethers to autophagosome biogenesis regulation and support the importance of membrane contact sites in autophagy. Synopsis Early autophagic markers are recruited to endoplasmic reticulum-plasma membrane (ER-PM) contact sites established by tethering factors extended synaptotagmins, allowing for local phosphatidylinositol-3-phosphate synthesis and autophagosome biogenesis. Autophagy induction is accompanied by ER-PM contact site mobilization. E-Syt2, a major tethering protein of ER-PM contact sites, forms a complex with VMP1 and Beclin1, two regulators of PI3KC3 complex activity. Local autophagosome biogenesis is initiated by local PI3P synthesis via the targeting of PI3KC3 complex at ER-PM contact sites. Introduction Macro-autophagy (hereafter referred to as autophagy) is a highly regulated intracellular degradation pathway necessary for cellular homeostasis (Boya et al, 2013). Autophagy is initiated by the formation of a specific double-membrane organelle called the autophagosome. The biogenesis of autophagosome is orchestrated by multiple signalling pathways and complexes that regulate membrane dynamics that contain autophagy-related (ATG) proteins. Autophagy initiates with biogenesis of a pre-autophagosomal double-membrane structure, termed the phagophore, which emanates from the omegasome, a subdomain of the endoplasmic reticulum (ER) membrane positive for PI3P (phosphatidyl-inositol-3-phosphate) and PI3P-binding proteins (Axe et al, 2008). The PI3P pool engaged in autophagosome biogenesis is synthesized by the class 3 PI3kinase complex (PI3KC3), comprised of VPS34, VPS15, ATG14L, Beclin1, and regulating adaptors, such as VMP1, NRBF2 and Ambra1, and is dependent on ULK1 complex signalling (Nascimbeni et al, 2017). The phagophore then elongates and is close to form a mature autophagosome that will latter fuse with the lysosome. Although the ER membrane requirement is well established, other membrane sources, like the Golgi apparatus, endosomes, the mitochondria and the plasma membrane (PM), have been proposed to participate, directly, indirectly or partially, in autophagosome biogenesis (Molino et al, 2017), from phagophore generation to growth of the organelle (Ravikumar et al, 2010a,b; Rubinsztein et al, 2012). The ER is a dynamic and complex membranous network that extends throughout the cell impacting a multiplicity of cellular functions (Friedman & Voeltz, 2011). There is growing evidence that close appositions between the ER and the membranes of virtually all other organelles play major roles in cell physiology (Helle et al, 2013). Notably, ER–mitochondria contact sites actively participate in autophagosome biogenesis via the regulation of PI3KC3 complex (Hamasaki et al, 2013). ER-PM contact sites are important for lipid metabolism and transport, notably of phosphoinositides, and these domains have the potential to affect membrane trafficking and signalling events that occur at the PM (Stefan et al, 2013). In higher eukaryotes, three ER-localized proteins, the extended synaptotagmins (E-Syts 1, 2 and 3), play crucial roles in tethering the ER to the PM and are thus considered as key regulators, as well as precise markers, of ER-PM tethering zones (Giordano et al, 2013). Because both ER and PM have been directly associated with autophagy regulation and because ER tethering could be important for membrane remodelling, we hypothesized that the ER cooperates with plasma membrane during the very first steps of the autophagosome biogenesis via the establishment of ER-PM specialized contact sites. Indeed, we show here that stress situations that induce autophagy lead as well to ER-PM contact site mobilization, highlighting a connection between ER-PM tethering and the autophagy machinery. We observed local recruitment of autophagic and pre-autophagic markers at E-Syts domains of the cortical ER during autophagy initiation. Further, autophagy was enhanced in E-Syt-overexpressing cells, whereas inhibition of E-Syts expression reduced autophagosome biogenesis. Finally, we demonstrated that ER-PM contact sites are required for local PI3P synthesis by the PIK3C3 complex. We found that the PIK3C3 complex at ER-PM contact sites is mobilized at the ER membrane via the binding of VMP1 (Molejon et al, 2013a), the ER partner of Beclin1, the major regulator of autophagy-associated PI3P synthesis. Results We first studied the behaviour of ER-PM contact sites in conditions that promote autophagy using a HRP-myc-KDEL reporter that allows indirect visualization of ER lumen by electron microscopy (EM; Giordano et al, 2013). We analysed and quantified ER-PM contact zones in control and in HeLa cells starved to induce autophagy. We observed a massive increase in the number of ER-PM contact sites compared to control situation (Fig 1A and B), and this increase correlated with autophagy induction (Fig 1C). Levels of E-Syt2 and 3 proteins increased after 1 and 4 h of starvation, whereas levels of calnexin (an ER marker), syntaxin 17 [STX17, previously identified as a marker of an autophagy-related ER–mitochondria contact sites (Hamasaki et al, 2013)] or PTPIP51 [an ER–mitochondria tethering protein recently shown to participate in autophagy regulation (Gomez-Suaga et al, 2017)] were not affected (Fig 1D and E). The increase in E-Syt2 was also induced by mechanical stress in KEC cells (Fig EV1A), a condition that promotes autophagy (Orhon et al, 2016), and by serum starvation or mTOR chemical inhibition (Fig EV1B). Figure 1. Starvation increases ER-PM contact sites density A. Electron micrographs of HeLa cells grown under complete medium conditions (control, ctrl) or starved for 1 h (1 h STV). HeLa cells were transfected with the ER luminal marker ssHRP-myc-KDEL (which enables ER identification via an electron-dense (dark) HRP reaction) to allow detection of ER-PM contact sites (black arrows). Scale bar, 2 μm. Representative images from one of three independent experiments are shown. B. Quantification of ER-PM contact sites visualized in electron micrographs with 20 cells analysed per condition in each of three experiments. Means ± s.e.m. are plotted. ***P < 0.001, unpaired two-tailed t-test. C–E. HeLa cells were grown under control and starvation conditions for 1 and 4 h. Representative Western blots of lysates for (C) p62 and lipidated LC3 and (D) E-Syts, calnexin (CLNX), STX17, and PTPIP51 are shown. Actin was used as a loading control. (E) Quantification of Western blots from three independent experiments. Means ± s.e.m. are plotted. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Multiple autophagy stimuli induce E-Syt2 expression Mechanical stress: Western blots and protein quantifications of protein lysates from KEC cells under basal (ctrl) and mechanical stress (flow) conditions for 4–72 h (n = 3). Plotted are mean ± s.d. mTOR inhibition and serum starvation: Western blots and protein quantifications (compared to ctrl) of protein lysates from HeLa cells grown under basal (ctrl) and autophagy-inducing conditions. Cells were cultured with 1.5 μM Torin1, an mTOR inhibitor, for 2 h or in a medium without serum for 1 h, respectively (n = 3). Plotted are mean ± s.d. Download figure Download PowerPoint Since our data suggested that autophagy and formation of ER-PM contact sites are stimulated in the same time frame, we next investigated whether autophagosomal markers could be detected at the cortical ER, near the PM. Indeed, in HeLa cells, we detected the phagophore, autophagosome and autolysosome marker LC3 near the cell boundary as early as 15 min post-starvation, a time at which most of LC3 was associated with the ER marker Sec61β (Fig 2A). The presence of LC3 in the vicinity of the plasma membrane was further confirmed by total internal reflection fluorescence (TIRF) microscopy (Fig 2B). The number of LC3 puncta in the TIRF zone increased with time under starvation conditions (Fig 2B and C), showing that LC3 autophagic structures appear at the immediate vicinity of the PM during starvation-induced autophagy. A similar LC3 pattern was observed in MDCK cells under mechanical stress (Orhon et al, 2016; Fig 2D). These results indicated that in different cell types treated with different autophagy inducers, LC3 staining was detected very close to the PM, suggesting that these autophagic structures might be associated with ER-PM contact sites. Figure 2. Early autophagic structures are detected in the vicinity of the plasma membrane HeLa cells were transfected with RFP-Sec61β (an ER marker) and immunostained for the autophagosome marker LC3. The two markers co-distribute in the vicinity of the plasma membrane (empty arrowheads) after 15 min of starvation (STV 15 min) as shown by confocal microscopy. TIRF analysis after 0 (control), 15 (STV 15 min) and 60 (STV 60 min) min of starvation. Quantification of LC3 puncta per TIRF section (n = 3; 20 cells analysed per condition). **P < 0.01, ***P < 0.001, unpaired two-tailed t-test. Means ± s.e.m. are plotted. Representative confocal microscopy images and 3D reconstructions of MDCK cells immunostained for LC3, Na/K-ATPase and DAPI under mechanical stress conditions. Arrowheads indicate LC3 puncta at vicinity of plasma membrane. Data information: Scale bars, 10 and 4 μm (magnified area in A). Download figure Download PowerPoint To analyse further whether ER-PM contact zones are sites of autophagosome formation, we used tagged E-Syt2 and E-Syt3 proteins as markers of ER-PM contact sites: while E-Syt1 can be detected on perinuclear as well as cortical ER structures, E-Syt2 and E-Syt3 localizations are restricted only to cortical ER engaged in ER-PM contact sites (Appendix Fig S1A and Fernández-Busnadiego et al (2015); Giordano et al (2013)). In HeLa cells starved for 15 min, we observed co-distribution of LC3 with the ER markers Sec61βRFP and E-Syt2GFP by confocal microscopy (Fig EV2A and C) at the basal level of the cells and by super-resolution two-colour stimulated emission depletion (STED) microscopy (Fig 3A). 3D reconstructions showed that LC3 was often directly connected to the ER membrane via E-Syt2-positive ER domains (Fig 3B) and sometimes appeared within a membranous niche positive for Sec61βRFP and E-Syt2GFP (Fig EV2A). Similar results were obtained when we used an antibody to ATG16L1 (Fig EV2B and C), a regulator of autophagosome biogenesis known to participate in the early events of LC3 recruitment to omegasome/phagophore structures (Wilson et al, 2014). The LC3-positive structures that were in the vicinity of the PM were negative for Rubicon (Appendix Fig S2), excluding the possibility of a non-autophagy-related LC3-associated phagocytosis (Levine et al, 2015). Using immunogold EM, we clearly observed E-Syt2myc and LC3GFP co-distribution on ER-PM contact sites in HeLa cells starved for 60 min (Fig EV2D); these are likely the same autophagic structures that we observed directly by electron microscopy in the immediate vicinity of cortical ER and PM in the same conditions (Fig 3C). Click here to expand this figure. Figure EV2. LC3 and ATG16L1 reside at ER-PM contact sites under starvation conditions A, B. Confocal microscope images and 3D reconstructions of HeLa cells co-transfected with GFP-E-Syt2 and RFP-Sec61β and immunostained for LC3 or ATG16L1 and DAPI. Arrowheads denote LC3 or ATG16L1 puncta near the E-Syt2-positive niche of the ER. Scale bars, 5 and 2.5 μm (magnified areas). C. Quantification of LC3 and ATG16L1 co-distribution with E-Syt2 at basal plan of the cell n = 80 cells. Mean ± s.e.m. shown. D. Immunogold electron micrographs of starved HeLa cells, showing co-distribution of the myc-E-Syt2 and anti-LC3 antibody at ER (empty arrowheads) and PM (black arrowheads) juxtaposition sites. Scale bar, 250 nm. Download figure Download PowerPoint Figure 3. Autophagosomes can form at ER-PM contact sites STED images of the basal plane of a HeLa cell co-transfected with vectors for expression of the ER marker Sec61βGFP and the ER-PM contact marker mCherry-E-Syt3 and immunostained for the autophagosome marker LC3. Scale bars, 5 μm. Arrowheads indicate autophagic structures (LC3) arising from ER niches (Sec61β-positive) at ER-PM contact sites (E-Syt3-positive). Three-dimensional reconstruction from confocal microscope images of HeLa cells co-transfected with vectors for expression of Sec61βRFP and E-Syt2GFP and immunostained for LC3. Scale bar, 2.5 μm. The arrowheads indicate the LC3-positive structures. Electron micrographs of HeLa cells starved 1 h and transfected with a vector for expression of the ER luminal marker ssHRP-myc-KDEL, showing early autophagic structures in the proximity of the PM. Scale bar, 400 nm. Download figure Download PowerPoint We next analysed distribution of the omegasome-marker (Axe et al, 2008) DCFP1GFP in HeLa cells after a short time of starvation, to maximize detection of autophagosome biogenesis-related events. We clearly observed co-distribution of DFCP1 with membranes positive for E-Syts, LC3 and Sec61βRFP by confocal microscopy (Fig 4A and B) and by time-lapse microscopy (Fig 4C). These results strongly suggest that at least some autophagosome biogenesis occurs at ER-PM contact sites. We then quantified the E-Syt2-positive omegasome structures and the omegasome structures at ER-mitochondria contact sites identified by mitochondrial protein TOM20 (Hamasaki et al, 2013). Our results indicate that, within 15 min of autophagy induction, approximately 30% of DFCP1-positive structures were associated with E-Syt2-positive domains (Fig EV3A and C), a ratio very close to the one we observed for DFCP1-positive ER–mitochondria contact sites (Fig EV3B and C). These results were further confirmed by electron microscopy analyses (Fig EV3D). Together, these data demonstrate that autophagosome assembly at ER-PM contact sites domains accounts for approximately 30% of total autophagosomes observed after a 15-min starvation of HeLa cells. Figure 4. LC3 and DFCP1 are present at ER-PM contact sites under starvation conditions Representative confocal microscopy images taken in the basal plane of a HeLa cell expressing myc-E-Syt2, RFP-Sec61β and GFP-DFCP1 and immunostained for LC3. Scale bars, 10 and 3 μm (magnified areas). 3D reconstructions of representative HeLa cell expressing myc-E-Syt2, RFP-Sec61β and GFP-DFCP1 and immunostained for LC3. Arrowheads denote DFCP1 and LC3 puncta connected with E-Syt2-positive niches of the ER. Scale bar, 5 μm. Time-lapse confocal images of HeLa cells expressing mCherry-E-Syt3 and GFP-DFCP1 after cells were starved for 15 min. Two channels were observed simultaneously using two cameras. Arrowheads denote DFCP1 puncta in E-Syt2-positive niches of the ER. Scale bar, 5 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Early autophagic structures form at ER-PM and ER–mitochondria contact sites A, B. Confocal microscope images of HeLa cells co-transfected with GFP-DFCP1 (an omegasome marker), RFP-Sec61β (an ER marker) and myc-E-Syt2 (an ER-PM contact sites marker) or immunostained for TOM20 (a mitochondria marker) under starved conditions (15 min). Arrowheads denote omegasome formation at (A) ER-PM or (B) ER–mitochondria contact sites. Scale bars, 10 μm. C. Quantification of percent DFCP1-positive structures at ER-PM (Sec61β/E-Syt2 interface) and ER–mitochondria (Sec61β/TOM20 interface) contact sites. Plotted are mean ± s.e.m., n = 80. D. Electron microscopy images of HeLa cells transfected with the ER luminal marker ssHRP-myc-KDEL and starved for 1 h showing autophagic structures adjacent (within 1 μm) to ER-PM contact sites (ER-PM), ER-mitochondria contact sites (ER-mito) and to neither organelle (cytoplasm). The quantification of these autophagic structures is shown as well. Plotted are means ± s.e.m., n = 80 cells. Download figure Download PowerPoint As previously reported (Giordano et al, 2013), overexpression of E-Syt2 or E-Syt3 stabilized and increased the density of ER-PM contact sites (Appendix Fig S2). In cells overexpressing E-Syts, the lipidation of LC3 was increased and more LC3-positive structures were observed both in fed and starved conditions compared to mock-transfected cells (Fig 5A and B). Interestingly, LC3 puncta were significantly increased in the vicinity of the PM in E-Syt3-overexpressing cells (Fig 5C). Electron microscopy analyses showed twice as many autophagic structures in HeLa cells overexpressing E-Syt2 as in control cells (Fig EV4A). In functional tests monitoring long-lived protein degradation, which depends on autophagy, we observed a significant increase of protein degradation in E-Syt2- and E-Syt3-overexpressing cells, as compared to control cells (Fig EV4B). Thus, the observed autophagic structures originating from the ER-PM contact zones appear to be functional. Figure 5. Overexpression of E-Syt2 and E-Syt3 induces autophagosome formation Western blot analysis of the autophagic flux in cell lysates from control (mock) and GFP-E-Syt2 or GFP-E-Syt3-expressing HeLa cells, under complete medium and starvation (1 h EBSS) conditions, without or with Bafilomycin A1 (+BAF. A1). HeLa cells expressing GFP-E-Syt2 or GFP-E-Syt3 were immunostained for LC3. Compared to control (mock), transfected cells showed a dramatic increase in LC3 puncta, in both basal (complete medium) and starved (1 h) conditions, as evidenced by counting of LC3 puncta (n = 3; 20 cells per condition). The increase in LC3 puncta observed in cells overexpressing E-Syt3GFP (similar results were obtained with E-Syt2GFP, data not shown) involves mainly peripheral rather than perinuclear cellular regions (n = 3). Arrowheads indicate peripheral LC3 puncta (n = 3; 20–70 cells per condition). Data information: Means ± s.e.m. are plotted. NS, non-significant, ***P < 0.001, unpaired two-tailed t-test. Scale bars, 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Overexpression of E-Syt2 and E-Syt3 enhances autophagy An electron micrograph of GFP-E-Syt2-expressing HeLa cells starved for 1 h, showing an increased number of autophagic structures (arrowheads) compared to starved control cells. Scale bar, 1 μm. Wortmannin (wort, 100 nM) was used as a negative control, and autophagic structures were counted in 15 μm2 areas (n = 10 cells). Proteolysis analysis showing an increased protein degradation rate in GFP-E-Syt2- and GFP-E-Syt3-expressing cells (n = 3). 3-methyladenine (3-MA) was used at 10 mM. Data information: Means ± s.e.m. are plotted. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed t-test. Download figure Download PowerPoint We then sought to test how impairing ER-PM contact sites formation would influence autophagy. To do this, we inhibited expression of the three E-Syt proteins (E-Syt1, E-Syt2 and E-Syt3) simultaneously using siRNAs targeting the mRNAs encoding each of these proteins (Fig 6A and B). Interestingly, in the E-Syt-deficient cells the total number of LC3GFP structures was decreased compared to control cells (Fig 6C and D). The difference was even more striking when we quantified the peripheral to perinuclear ratio of LC3 puncta in cells treated with Bafilomycin A1 (a V-ATPase inhibitor preventing fusion between autophagosome and lysosome) to maximize the number of autophagic structures (Fig 6C and D). The decrease observed in siE-Syt-treated cells was primarily due to decreases in numbers of peripheral puncta rather than to decreases in perinuclear LC3. Figure 6. Autophagosome biogenesis is reduced in E-Syt-deficient cells A, B. HeLa cells were treated with control siRNA (siCTRL) or with siRNAs targeting mRNAs encoding each of the E-Syts (siE-Syts). (A) Western blots of cell lysates. Actin is used as a loading control. (B) E-Syts mRNAs were quantified. Means ± s.d. are plotted (n = 3). C. HeLa cells stably transfected with GFP-LC3 and treated with siE-Syts or siCTRL were not treated (−BAF. A1) or were treated with Bafilomycin A1 (+BAF. A1). Representative images are shown. Empty arrowheads indicate peripheral LC3 puncta. Scale bars, 10 μm. D. Quantification of experiments shown in panel (C) (n = 3; 20 cells per condition). Means ± s.e.m. are plotted. E. HeLa cells treated with control siCTRL or with siE-Syts were grown in complete medium or were starved for 1 or 4 h, and cells lysates were subjected to Western blot for indicated proteins. F. Quantification of Western blot shown in panel (E), with 20 cells analysed per condition (n = 5). Data information: NS, non-significant, *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed t-test. Download figure Download PowerPoint Western blot analyses performed following a time course of starvation-induced autophagy revealed a decrease in LC3 lipidation as well as decreases of the amounts of the autophagosome biogenesis regulators ATG16L1 and ATG5—ATG12 in E-Syt-deficient cells (Fig 6E and F). Moreover, using the LC3GFP-RFP tandem dye, which is widely used to measure autophagic flux (Klionsky et al, 2016), we observed that the GFP/RFP ratio was not modified in the E-Syt-deficient cells compared to control cells (Fig EV5). Together, these data suggest that, although the number of autophagic structures was diminished when ER-PM contact sites were reduced, the maturation and transport to lysosomes of the remaining autophagosomes were not altered. Click here to expand this figure. Figure EV5. Autophagosome maturation is not affected in E-Syt-deficient cells A, B. HeLa cells stably transfected with mRFP-GFP-LC3 and treated with siE-Syts have fewer autophagosomes, but autophagosomes have normal functionality, as evidenced by (A) microscopy and (B) by RFP+/GFP+ and RFP+/GFP− LC3-puncta counting. Cells treated with Bafilomycin A1 (+BAF. A1) and siCTRL were used as a positive functionality control and showed decreased autophagosome maturation (i.e. reduced RFP+/GFP− LC3-puncta compared to siCTRL-treated cells) as expected (n = 3). ***P < 0.001, unpaired two-tailed t-test. Scale bar, 10 μm. Plotted are mean ± s.e.m. Download figure Download PowerPoint One of the major molecular events responsible for autophagosome biogenesis is the synthesis of PI3P at the omegasome on the ER membrane (Axe et al, 2008; Lamb et al, 2013; Roberts & Ktistakis, 2013). PI3P is synthesized not only at the omegasome membrane but also on early endosomes as well (Di Paolo & De Camilli, 2006; Marat & Haucke, 2016). We observed that the omegasome-marker and PI3P binding protein DFCP1 co-distributed with E-Syt2 domains on the ER (Fig 4). Therefore, we looked directly for PI3P lipid in proximity to these ER-PM contact sites during short-term starvation. Interestingly, we observed PI3P-positive structures [detected by FYVEGST/fluorescent anti-GST antibody indirect staining (Khaldoun et al, 2014)] in the immediate vicinity of ER membrane regions positive for E-Syt3 and LC3 but not in regions stained by endosomal marker EEA1 after 15 min of starvation (Appendix Fig S3A and B). We obtained similar results using 2x-FYVEGFP dye to stain for PI3P (Appendix Fig S3C) and when cells were stained using ATG16L1 and VPS35 (Seaman et al, 1998) to mark early autophagic structures and early endosomes, respectively (Appendix Fig S3D). Our results suggest that E-Syts directly or indirectly participate in autophagosome biogenesis. Because we observed PI3P at E-Syts domains after autophagy induction, we speculated that these proteins are involved in regulation of PI3P synthesis at ER-PM contact site-associated autophagosome biogenesis. To assess this hypothesis, we quantified PI3P puncta in control cells and cells deficient in all three E-Syt proteins under both fed and starved conditions. We used wort

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.

Macroautophagy (hereafter called autophagy) is a vacuolar, lysosomal pathway for catabolism of intracellular material that is conserved among eukaryotic cells. Autophagy plays a crucial role in tissue homeostasis, adaptation to stress situations, immune responses, and the regulation of the inflammatory response. Blockade or uncontrolled activation of autophagy is associated with cancer, diabetes, obesity, cardiovascular disease, neurodegenerative disease, autoimmune disease, infection, and chronic inflammatory disease. During the past decade, researchers have made major progress in understanding the three levels of regulation of autophagy in mammalian cells: signaling, autophagosome formation, and autophagosome maturation and lysosomal degradation. As we discuss in this review, each of these levels is potentially druggable, and, depending on the indication, may be able to stimulate or inhibit autophagy. We also summarize the different modulators of autophagy and their potential and limitations in the treatment of life-threatening diseases.

Abstract Cells subjected to stress situations mobilize specific membranes and proteins to initiate autophagy. Phosphatidylinositol-3-phosphate (PI3P), a crucial lipid in membrane dynamics, is known to be essential in this context. In addition to nutriments deprivation, autophagy is also triggered by fluid-flow induced shear stress in epithelial cells, and this specific autophagic response depends on primary cilium (PC) signaling and leads to cell size regulation. Here we report that PI3KC2α, required for ciliogenesis and PC functions, promotes the synthesis of a local pool of PI3P upon shear stress. We show that PI3KC2α depletion in cells subjected to shear stress abolishes ciliogenesis as well as the autophagy and related cell size regulation. We finally show that PI3KC2α and VPS34, the two main enzymes responsible for PI3P synthesis, have different roles during autophagy, depending on the type of cellular stress: while VPS34 is clearly required for starvation-induced autophagy, PI3KC2α participates only in shear stress-dependent autophagy.

The purpose of this study is to identify novel proteins released by cancer cells that are involved in extracellular matrix (ECM) remodeling using small-volume samples and automated technology. We applied multidimensional protein identification technology (MudPIT), which incorporates two-dimensional capillary chromatography coupled to tandem mass spectrometry to small quantities of serum-free supernatants of resting or phorbol ester-activated Suit-2 pancreatic cancer cells. Selected markers were validated in additional pancreatic cancer cell lines, primary cancers, and xenografted cancer cells. MudPIT analysis of 10 microl of supernatants identified 46 proteins, 21 of which are classified as secreted, and 10 have never been associated with pancreatic cancer. These include CSPG2/versican, Mac25/angiomodulin, IGFBP-1, HSPG2/perlecan, syndecan 4, FAM3C, APLP2, cyclophilin B, beta2 microglobulin, and ICA69. Evidence that cancer cells release these proteins in vivo was obtained for CSPG2/versican and Mac25/angiomodulin by immunohistochemistry on both primary pancreatic cancers and in a model consisting of Suit-2 cells embedded in an amorphous matrix and implanted in athymic mice. MudPIT allowed efficient and rapid identification of proteins released by cancer cells, including molecules previously undescribed in the type of cancer analyzed. Our finding that pancreatic cancer cells secrete a series of proteoglycans, including versican, perlecan, syndecan 1 and 4, challenges the common view that fibroblasts of tumor stroma are the sole source of these molecules.

Limb girdle muscular dystrophy (LGMD) type 2A (LGMD2A) is caused by mutations in the CAPN3 gene encoding for calpain-3, a muscle specific protease. While a large number of CAPN3 gene mutations have already been described in calpainopathy patients, the diagnosis has recently shifted from molecular genetics towards biochemical assay of defective protein. However, an estimate of sensitivity and specificity of protein analysis remains to be established. Thus, we first correlated protein and molecular data in our large LGMD2A patient population. By a preliminary immunoblot screening for calpain-3 protein of 548 unclassified patients with various phenotypes (LGMD, myopathy, or elevated levels of serum creatine kinase [hyperCKemia]), we selected 208 cases for CAPN3 gene mutation analysis: 69 had protein deficiency and 139 had normal expression. Mutation search was conducted using SSCP, denaturing high performance liquid chromatography (DHPLC), amplification refractory mutation system (ARMS-PCR), and direct sequencing methods. We identified 58 LGMD2A mutant patients: 46 (80%) had a variable degree of protein deficiency and 12 (20%) had normal amount of calpain-3. We calculated that the probability of having LGMD2A is very high (84%) when patients show a complete calpain-3 deficiency and progressively decreases with the amount of protein; this new data offers an important tool for genetic counseling when only protein data are available. A total of 37 different CAPN3 gene mutations were detected, 10 of which are novel. In our population, 87% of mutant alleles were concentrated in seven exons (exons 1, 4, 5, 8, 10, 11, and 21) and 61% correspond to only eight mutations, indicating the regions where future molecular analysis could be restricted. This study reports the largest collection of LGMD2A patients so far in which both protein and gene mutations were obtained to draw genotype–protein–phenotype correlations and provide insights into a critical protein domain. Hum Mutat 24:52–62, 2004. © 2004 Wiley-Liss, Inc.

The frequency of various limb-girdle muscular dystrophy (LGMD) molecular diagnoses has previously been investigated only in cohorts of patients presenting LGMD phenotype.A total of 550 muscle biopsies underwent multiple protein screening (including calpain-3 functional assay) and extensive gene mutation analysis to examine the frequency of LGMD subtypes in patients with distinct clinical phenotypes (severe childhood-onset LGMD, adult-onset LGMD, distoproximal myopathy, and asymptomatic hyperCKemia).The percentage of molecularly ascertained cases directly relates with the degree of clinical involvement: 60% of total LGMD (77% of childhood-onset, 46% of adult-onset, 66% of distoproximal myopathy) and 14% of hyperCKemia. The higher number of molecular diagnoses in severe phenotypes might suggest that genes selected for our screening are those more frequently associated with severe LGMD, and that the hyperCKemia group includes heterogeneous diagnoses. The probability of obtaining a molecular diagnosis increases when a protein defect is found in a muscle biopsy: in such cases, we diagnosed 87% of LGMD and 76% of hyperCKemia.Diagnosing 77% of childhood-onset limb-girdle muscular dystrophy (LGMD) and 60% of total LGMD is an important result. The missing identification of gene mutations in about 40% of patients with typical LGMD phenotype suggests that unknown genetic or nongenetic etiologies are still to be recognized. Dysferlin, caveolin-3, and emerin protein defects invariably corresponded to primary disorders (100%), whereas a lower correlation was found for sarcoglycans (77%) and calpain-3 (84%). The different efficiency of genetic diagnosis after the identification of a protein defect in the various disorders is possibly due to different pathogenetic effects of mutations.

Abstract The autophagy–lysosome system is critical for muscle homeostasis and defects in lysosomal function result in a number of inherited muscle diseases, generally referred to as autophagic vacuolar myopathies (AVMs). Among them, Danon Disease (DD) and glycogen storage disease type II (GSDII) are due to primary lysosomal protein defects. DD is characterized by mutations in the lysosome-associated membrane protein 2 ( LAMP2 ) gene. The DD mouse model suggests that inefficient lysosome biogenesis/maturation and impairment of autophagosome-lysosome fusion contribute to the pathogenesis of muscle wasting. To define the role of autophagy in human disease, we analyzed the muscle biopsies of DD patients and monitored autophagy and several autophagy regulators like transcription factor EB (TFEB), a master player in lysosomal biogenesis, and vacuolar protein sorting 15 (VPS15), a critical factor for autophagosome and endosome biogenesis and trafficking. Furthermore, to clarify whether the mechanisms involved are shared by other AVMs, we extended our mechanistic study to a group of adult GSDII patients. Our data show that, similar to GSDII, DD patients display an autophagy block that correlates with the severity of the disease. Both DD and GSDII show accumulation and altered localization of VPS15 in autophagy-incompetent fibers. However, TFEB displays a different pattern between these two lysosomal storage diseases. Although in DD TFEB and downstream targets are activated, in GSDII patients TFEB is inhibited. These findings suggest that these regulatory factors may have an active role in the pathogenesis of these diseases. Therapeutic approaches targeted to normalize these factors and restore the autophagic flux in these patients should therefore be considered.

The diagnosis of limb girdle muscular dystrophy (LGMD) type 2A (due to mutations in the gene encoding for calpain-3) is currently based on protein analysis, but mutant patients with normal protein expression have also been identified. In this study we investigated 150 LGMD patients with normal calpain-3 protein expression, identified gene mutations by an allele-specific polymerase chain reaction test, and analyzed the mutant calpain-3 catalytic activity. Four different mutations were found in eight patients (5.5%): a frame-shifting deletion (550 A del) and three missense (R490Q, R489Q, R490W). Patients with normal calpain-3 protein expression on Western blot are a considerable proportion (20%) of our total LGMD2A population. While in control muscle the calpain-3 Ca(++)-dependent autocatalytic activity was evident within 5 minutes and was prevented by ethylene diaminetetraacetic acid, in all mutant patient samples the protein was not degraded, indicating that the normal autocatalytic function had been lost. By this new functional test, we show that conventional protein diagnosis fails to detect some mutant proteins, and prove the pathogenetic role of R490Q, R489Q, R490W missense mutations. We suggest that these mutations impair protein activity by affecting interdomain protein interaction, or reduce autocatalytic activity by lowering the Ca(++) sensitivity.

Limb-girdle muscular dystrophy 2A (LGMD2A) is considered to be the most frequent LGMD. Our study surveyed an area in northeastern Italy where an almost complete ascertainment was possible. To identify LGMD2A patients we used a new diagnostic approach, including several molecular and biochemical methods. In 84 screened patients from northeastern Italy, we identified 39 LGMD2A patients, the prevalence of LGMD2A being 9.47 per million. In the Venezia district it appears higher than in other districts of the Veneto region, and in the Friuli region it is three times higher than in Veneto, due to the recurrence of single mutation. Haplotype analysis suggested a founder effect. The population from Venezia and Friuli has a higher risk of being heterozygote for these two mutant alleles than people from the rest of northeastern Italy. Our results indicate that LGMD2A is one of the most frequent autosomal recessive disorders, thus finding its molecular characterization becoming increasingly important.

The autophagy-lysosome system is essential for muscle cell homeostasis and its dysfunction has been linked to muscle disorders that are typically distinguished by massive autophagic buildup. Among them, glycogen storage disease type II (GSDII) is characterized by the presence of large glycogen-filled lysosomes in the skeletal muscle, due to a defect in the lysosomal enzyme acid α-glucosidase (GAA). The accumulation of autophagosomes is believed to be detrimental for myofiber function. However, the role of autophagy in the pathogenesis of GSDII is still unclear. To address this issue we monitored autophagy in muscle biopsies and myotubes of early and late-onset GSDII patients at different time points of disease progression. Moreover we also analyzed muscles from patients treated with enzyme replacement therapy (ERT). Our data suggest that autophagy is a protective mechanism that is required for myofiber survival in late-onset forms of GSDII. Importantly, our findings suggest that a normal autophagy flux is important for a correct maturation of GAA and for the uptake of recombinant human GAA. In conclusion, autophagy failure plays an important role in GSDII disease progression, and the development of new drugs to restore the autophagic flux should be considered to improve ERT efficacy.

Danon disease, an X-linked dominant disorder, results from mutations in the lysosome-associated membrane protein-2 (LAMP2) gene and presents with hypertrophic cardiomyopathy, skeletal myopathy, and mental retardation. To investigate the effects of LAMP2 gene mutations on protein expression in different tissues, we screened LAMP2 gene mutations and LAMP-2 protein deficiency in the skeletal muscle of nine unrelated patients with hypertrophic cardiomyopathy and vacuolar myopathy. We identified three novel families (including one affected mother) with unreported LAMP2 gene null mutations and LAMP-2 protein deficiency in skeletal and myocardial muscle, leukocytes, and fibroblasts. LAMP-2 protein deficiency was detectable in various tissues, including leukocytes, explaining the multisystem clinical involvement. Skeletal muscle immunopathology showed that mutant protein was not localized in the Golgi complex, vacuolar membranes expressed sarcolemmal-specific proteins, and the degree of muscle fiber vacuolization correlated with clinical muscle involvement. In our female patient, muscle histopathology and LAMP-2 protein analysis was inconclusive, indicating that diagnosis in females requires mutation identification. The random X-chromosome inactivation found in muscle and leukocytes excluded the possibility that selective involvement of some tissues in females is due to skewed X-chromosome inactivation. Therefore, biochemical analysis of leukocytes might be used for screening in male patients, but genetic screening is required in females.

AbstractObjective: Autosomal recessive limb girdle muscular dystrophies (LGMD type 2) are a clinically and genetically heterogeneous group of disorders, characterized by progressive involvement and wasting of limb girdle muscles. In order to describe the peculiar clinical features of LGMD2A (calpainopathy) and LGMD2B (dysferlinopathy), the most frequent forms of LGMD in European countries, we analysed and compared the phenotype and the clinical course in two relatively large groups of these patients.Methods: We selected 22 patients with a molecular diagnosis of LGMD2A and 21 patients with LGMD2B and reported their clinical data collected from both clinical history and during periodical neuromuscular examinations: age and distribution of muscle involvement at onset, clinical functional score by the use of ten-point modified scale of Gardner–Medwin and Walton at onset and at last clinical examination, and the rate of disease progression.Results: LGMD2A group included patients with different ages at onset (early-onset or late-onset), different phenotypes (upper girdle in Erb LGMD or lower girdle in Leyden–Moebius LGMD) and different disease progressions (rapid or slow course). LGMD2B patients differed for pattern of muscle involvement at onset (distal in Miyoshi dystrophy or proximal in Leyden–Moebius LGMD) but they had a rather homogeneous age at onset (in the second/third decade) and rate of disease progression.Discussion: Our data show that besides the clinical differences within each group of patients, the two forms of LGMD present distinctive clinical features. The various phenotypes and courses can be attributed to specific pathogenetic mechanisms and might suggest differential therapeutic strategies.Keywords: CALPAIN-3CLINICAL COURSEDYSFERLINLGMD

<b>Objectives: </b> To examine at molecular, biochemical, and muscle pathology level the striking clinical heterogeneity resulting from acid α-glucosidase deficiency. <b>Methods: </b> We investigated 23 patients with infantile-onset or late-onset glycogen storage disease type II by enzyme activity, protein expression by immunoblotting, <i>GAA</i> gene mutations, and muscle pathology including immunolabeling for Golgi and sarcolemmal proteins. <b>Results: </b> The enzyme activity was absent or minimal in infantile-onset cases and variably reduced in late-onset patients. Genotype-phenotype correlation (seven novel mutations were found) showed that most late-onset patients had the heterozygous IVS1 leaky splicing mutation (one patient was homozygous), but the course of the disease was often difficult to predict on the basis of the mutations alone. All patients showed an abnormal pattern of enzyme protein processing, with increased amounts of the inactive forms and very low or absent amounts of the mature forms. The molecular weight of the mature and the intermediate forms appeared higher in patients’ samples than in the control muscle. We observed a Golgi proliferation in muscle fibers possibly caused by the retention of inactive forms of enzyme protein that cannot be correctly targeted from Golgi to lysosomes. The vacuolar membranes expressed sarcolemmal proteins in late-onset but not in infantile-onset patients, suggesting an extensive autophagy and vacuolar membrane remodeling in late-onset patients. <b>Conclusions: </b> The different protein molecular weight between patients and controls could be due to an excessive sialylation of mutant enzyme: this might be possibly caused by a delayed transport and longer transit of the inactive proteins in the Golgi apparatus.

ABSTRACT Introduction : Muscle fiber atrophy and the molecular pathways underlying this process have not been investigated in dysferlinopathy patients. Methods : In 22 muscles from dysferlinopathy patients we investigated fiber atrophy by morphometry and ubiquitin–proteasome and autophagic pathways using protein and/or transcriptional analysis of atrophy‐ and autophagy‐related genes ( MuRF1 , atrogin1 , LC3 , p62 , Bnip3 ). Results : Dysferlinopathy showed significant fiber atrophy and higher MuRF‐1 protein and mRNA levels, which correlated with fiber size, suggesting activation of the atrophy program by proteasome induction. Conclusions : Some of the MuRF‐1 upregulation and proteasome induction may be attributed to the prominent regeneration found. A potential role of impaired autophagy was suggested by p62‐positive protein aggregates in atrophic fibers and significantly higher levels of LC3‐II and p62 proteins and overexpression of p62 and Bnip3 mRNA. Damaged muscle fibers and prominent inflammatory changes may also enhance autophagy due to the insufficient level of proteasomal degradation of mutant dysferlin. Muscle Nerve 50 : 340–347, 2014

Figure S1. Acid phosphatase histoenzymatic staining in the two muscle biopsies obtained from the treated case before (A) and after (B) ERT treatment. After ERT, muscle shows improved muscle pathology, consisting in a lower proportion of fibers with increased lysosomal activity and a generalized increase in fibers' size. Microscope magnification × 200. Table S1. Patients' clinical and molecular data. Appendix S1. Supplementary material. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Macroautophagy (hereafter called autophagy) is a vacuolar lysosomal pathway for degradation of intracellular material in eukaryotic cells. Autophagy plays crucial roles in tissue homeostasis, in adaptation to stress situations, and in immune and inflammatory responses. Alteration of autophagy is associated with cancer, diabetes and obesity, cardiovascular disease, neurodegenerative disease, autoimmune disease, infection, and chronic inflammatory disease. Autophagy is controlled by autophagy-related (ATG) proteins that act in a coordinated manner to build up the initial autophagic vacuole named the autophagosome. It is now known that the activities of ATG proteins are modulated by posttranslational modifications such as phosphorylation, ubiquitination, and acetylation. Moreover, transcriptional and epigenetic controls are involved in the regulation of autophagy in stress situations. Here we summarize and discuss how posttranslational modifications and transcriptional and epigenetic controls regulate the involvement of autophagy in the proteostasis network.

Macroautophagy is a lysosomal catabolic process that maintains the homeostasis of eukaryotic cells, tissues, and organisms. Macroautophagy plays important physiological roles during development and aging processes and also contributes to immune responses. The process of macroautophagy is compromised in diseases, such as cancer, neurodegenerative disorders, and diabetes. The autophagosome, the double-membrane-bound organelle that sequesters cytoplasmic material to initiate macroautophagy, is formed by the hierarchical recruitment of about 15 autophagy-related (ATG) proteins and associated proteins, such as DFCP1, AMBRA1, the class III phosphatidyl-inositol 3-kinase VPS34, and p150/VPS15. Evidence suggests that in addition to the canonical pathway, noncanonical pathways that do not require the entire repertoire of ATGs can also result in formation of autophagosomes. Here we will discuss recent discoveries concerning the molecular regulation of these noncanonical forms of macroautophagy and their potential roles in cellular responses to stressful situations.

Mutations in the caveolin-3 gene (CAV3) cause limb girdle muscular dystrophy (LGMD) type 1C (LGMD1C) and other muscle phenotypes. We screened 663 patients with various phenotypes of unknown etiology, for caveolin-3 protein deficiency, and we identified eight unreported caveolin-deficient patients (from seven families) in whom four CAV3 mutations had been detected (two are unreported). Following our wide screening, we estimated that caveolinopathies are 1% of both unclassified LGMD and other phenotypes, and demonstrated that caveolin-3 protein deficiency is a highly sensitive and specific marker of primary caveolinopathy. This is the largest series of caveolinopathy families in whom the effect of gene mutations has been analyzed for protein level and phenotype. We showed that the same mutation could lead to heterogeneous clinical phenotypes and muscle histopathological changes. To study the role of the Golgi complex in the pathological pathway of misfolded caveolin-3 oligomers, we performed a histopathological study on muscle biopsies from caveolinopathy patients. We documented normal caveolin-3 immunolabeling at the plasmalemma in some regenerating fibers showing a proliferation of the Golgi complex. It is likely that caveolin-3 overexpression occurring in regenerating fibers (compared with caveolin-deficient adult fibers) may lead to an accumulation of misfolded oligomers in the Golgi and to its consequent proliferation. Hum Mutat 25:82–89, 2005. © 2004 Wiley-Liss, Inc.

Phosphatidylinositol 3-phosphate (PtdIns3P) is a key player of membrane trafficking regulation, mostly synthesized by the PIK3C3 lipid kinase. The presence of PtdIns3P on endosomes has been demonstrated; however, the role and dynamics of the pool of PtdIns3P dedicated to macroautophagy/autophagy remains elusive. Here we addressed this question by studying the mobilization of PtdIns3P in time and space during autophagosome biogenesis. We compared different dyes known to specifically detect PtdIns3P by fluorescence microscopy analysis, based on PtdIns3P-binding FYVE and PX domains, and show that these transfected dyes induce defects in endosomal dynamics as well as artificial and sustained autophagosome formation. In contrast, indirect use of recombinant FYVE enabled us to track and discriminate endosomal and autophagosomal pools of PtdIns3P. We used this method to analyze localization and dynamics of PtdIns3P subdomains on the endoplasmic reticulum, at sites of pre-autophagosome associated protein recruitment such as the PtdIns3P-binding ZFYVE1/DFCP1 and WIPI2 autophagy regulators. This approach thus revealed the presence of a specific pool of PtdIns3P at the site where autophagosome assembly is initiated.

Endoplasmic Reticulum (ER), spreading in the whole cell cytoplasm, is a central player in eukaryotic cell homeostasis, from plants to mammals. Beside crucial functions, such as membrane lipids and proteins synthesis and outward transport, the ER is able to connect to virtually every endomembrane compartment by specific tethering molecular machineries, which enables the establishment of membrane-membrane contact sites. ER-mitochondria contact sites have been shown to be involved in autophagosome biogenesis, the main organelle of the autophagy degradation pathway. More recently we demonstrated that also ER-plasma membrane contact sites are sites for autophagosomes assembly, suggesting that more generally ER-organelles contacts are involved in autophagy and organelle biogenesis. Here we aim to discuss the functioning of ER-driven contact sites in mammals and plants and more in particular emphasize on their recently highlighted function in autophagy to finally conclude on some key questions that may be useful for further research in the field.

Triglycerides droplets are massively stored in muscle in Lipid Storage Myopathies (LSM). We studied in muscle regulators of lipophagy, the expression of the transcription factor-EB (TFEB) (a master regulator of lysosomal biogenesis), and markers of autophagy which are induced by starvation and exert a transcriptional control on lipid catabolism.We investigated the factors that regulate lipophagy in muscle biopsies from 6 patients with different types of LSM: 2 cases of riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (MADD), 1 case of primary carnitine deficiency (CD), 2 cases of neutral lipid storage myopathy (NLSD-M), 1 case of carnitine-palmitoyl-transferase-II (CPT) deficiency.Conventional morphology and electron microscopy documented the lipid accumulation and its dramatic resolution after treatment. Muscle immunofluorescence showed that while in MADD and NLSD-M there was a co-localized expression of TFEB and p62-SQSTM1 (marker of protein aggregates) in some atrophic fibers, in CD and CPT-II deficiency the reaction was almost normal. In regenerating fibers, TFEB localized in the cytoplasm (inactive form), whereas in atrophic fibers it localized in the nuclei (active form). Lipid-accumulated/atrophic fibers did not display p62-positive protein aggregates, indicating, together with the LC3-II (marker of autophagosomes) and p62-SQSTM1 analysis, that the autophagic flux is often preserved and lipophagy occurs.In atrophic and regenerating fibers of patients with NLSD-M we observed TFEB over-expression; in other conditions autophagy markers are increased, suggesting lipophagy active role on human lipid metabolism.

Limb-girdle muscular dystrophy (LGMD) 2A (calpainopathy) is the most frequent form of LGMD in many European countries. The increasing demand for a molecular diagnosis makes the identification of strategies to improve gene mutation detection crucial. We conducted both a quantitative analysis of calpain-3 protein in 519 muscles from patients with unclassified LGMD, unclassified myopathy and hyperCKemia, and a functional assay of calpain-3 autolytic activity in 108 cases with LGMD and normal protein quantity. Subsequently, screening of CAPN3 gene mutations was performed using allele-specific tests and simplified SSCP analysis. We diagnosed a total of 94 LGMD2A patients, carrying 66 different mutations (six are newly identified). The probability of diagnosing calpainopathy was very high in patients showing either a quantitative (80%) or a functional calpain-3 protein defect (88%). Our data show a high predictive value for reduced-absent calpain-3 or lost autolytic activity. These biochemical assays are powerful tools for otherwise laborious genetic screening of cases with a high probability of being primary calpainopathy. Our multistep diagnostic approach is rational and highly effective. This strategy has improved the detection rate of the disease and our extension of screening to presymptomatic phenotypes (hyperCKemia) has allowed us to obtain early diagnoses, which has important consequences for patient care and genetic counseling.

Aims Pompe disease is an autosomal recessive lysosomal storage disorder resulting from deficiency of acid α‐glucosidase (GAA) enzyme. Histopathological hallmarks in skeletal muscle tissue are fibre vacuolization and autophagy. Since 2006, enzyme replacement therapy (ERT) is the only approved treatment with human recombinant GAA alglucosidase alfa. We designed a study to examine ERT‐related skeletal muscle changes in 18 modestly to moderately affected late onset Pompe disease (LOPD) patients along with the relationship between morphological/biochemical changes and clinical outcomes. Treatment duration was short‐to‐long term. Methods We examined muscle biopsies from 18 LOPD patients at both histopathological and biochemical level. All patients underwent two muscle biopsies, before and after ERT administration respectively. The study is partially retrospective because the first biopsies were taken before the study was designed, whereas the second biopsy was always performed after at least 6 months of ERT administration. Results After ERT, 15 out of 18 patients showed improved 6‐min walking test (6MWT; P = 0.0007) and most of them achieved respiratory stabilization. Pretreatment muscle biopsies disclosed marked histopathological variability, ranging from an almost normal pattern to a severe vacuolar myopathy. After treatment, we detected morphological improvement in 15 patients and worsening in three patients. Post‐ERT GAA enzymatic activity was mildly increased compared with pretreatment levels in all patients. Protein levels of the mature enzyme increased in 14 of the 18 patients (mean increase = +35%; P &lt; 0.05). Additional studies demonstrated an improved autophagic flux after ERT in some patients. Conclusions ERT positively modified skeletal muscle pathology as well as motor and respiratory outcomes in the majority of LOPD patients.

Limb girdle muscular dystrophies (LGMD), a genetically and clinically heterogeneous group of neuromuscular disorders, may show gender differences in the disease severity. We aimed to measure the extent of muscle fiber atrophy and evaluate possible gender differences at fiber level.We conducted a thorough morphometric analysis of muscle fiber size and fiber area in 101 muscles from patients with various forms of LGMD (43 LGMD2A, 30 LGMD2B, 21 LGMD2C-2D-2E, 7 LGMD1C) and 12 normal controls.Reduced fiber size (atrophy) was pronounced in LGMD2A and LGMD2B, while LGMD1C showed a significant fiber hypertrophy. When we compared LGMD patients and controls of the same gender, males with LGMD2A and LGMD2B showed significantly higher fiber atrophy than control males, whereas female LGMD patients had similar values to female controls, suggesting a gender difference in muscle fiber atrophy.Less recovery to disuse atrophy in men than in women has been attributed to the possibility that in women a smaller initial muscle size associated to endocrine factors could attenuate gender-specific muscle loss. The possibility that males with LGMD may be clinically more severely affected than females has been explored, but the mechanism remains elusive.

Variants of unknown significance in the CAPN3 gene constitute a significant challenge for genetic counselling. Despite the frequency of intronic nucleotide changes in this gene (15-25% of all mutations), so far their pathogenicity has only been inferred by in-silico analysis, and occasionally, proven by RNA analysis. In this study, 5 different intronic variants (one novel) that bioinformatic tools predicted would affect RNA splicing, underwent comprehensive studies which were designed to prove they are disease-causing. Muscle mRNA from 15 calpainopathy patients was analyzed by RT-PCR and splicing-specific-PCR tests. We established the previously unrecognized pathogenicity of these mutations, which caused aberrant splicing, most frequently by the activation of cryptic splicing sites or, occasionally, by exon skipping. The absence or severe reduction of protein demonstrated their deleterious effect at translational level. We concluded that bioinformatic tools are valuable to suggest the potential effects of intronic variants; however, the experimental demonstration of the pathogenicity is not always easy to do even when using RNA analysis (low abundance, degradation mechanisms), and it might not be successful unless splicing-specific-PCR tests are used. A comprehensive approach is therefore recommended to identify and describe unclassified variants in order to offer essential data for basic and clinical geneticists.

Muscle & NerveVolume 51, Issue 1 p. 145-147 Letter to the Editor Dominant muscular dystrophy with a novel SYNE1 gene mutation Marina Fanin PhD, Marina Fanin PhD Department of Neurosciences, University of Padova, Padova, ItalySearch for more papers by this authorMarco Savarese PhD, Marco Savarese PhD Department of Biochemistry, Biophysics and General Pathology, II University of Naples, Naples, Italy Telethon Institute of Genetics and Medicine, Naples, ItalySearch for more papers by this authorAnna C. Nascimbeni PhD, Anna C. Nascimbeni PhD Department of Neurosciences, University of Padova, Padova, ItalySearch for more papers by this authorGiuseppina Di Fruscio PhD, Giuseppina Di Fruscio PhD Department of Biochemistry, Biophysics and General Pathology, II University of Naples, Naples, Italy Telethon Institute of Genetics and Medicine, Naples, ItalySearch for more papers by this authorEbe Pastorello MD, Ebe Pastorello MD ULSS 16 Padova, ItalySearch for more papers by this authorElisabetta Tasca PhD, Elisabetta Tasca PhD IRCCS Fondazione San Camillo Hospital, Venice, ItalySearch for more papers by this authorCarlo P. Trevisan MD, Carlo P. Trevisan MD Department of Neurosciences, University of Padova, Padova, ItalySearch for more papers by this authorVincenzo Nigro MD, PhD, Vincenzo Nigro MD, PhD Department of Biochemistry, Biophysics and General Pathology, II University of Naples, Naples, Italy Telethon Institute of Genetics and Medicine, Naples, ItalySearch for more papers by this authorCorrado Angelini MD, Corrado Angelini MD Department of Neurosciences, University of Padova, Padova, Italy IRCCS Fondazione San Camillo Hospital, Venice, ItalySearch for more papers by this author Marina Fanin PhD, Marina Fanin PhD Department of Neurosciences, University of Padova, Padova, ItalySearch for more papers by this authorMarco Savarese PhD, Marco Savarese PhD Department of Biochemistry, Biophysics and General Pathology, II University of Naples, Naples, Italy Telethon Institute of Genetics and Medicine, Naples, ItalySearch for more papers by this authorAnna C. Nascimbeni PhD, Anna C. Nascimbeni PhD Department of Neurosciences, University of Padova, Padova, ItalySearch for more papers by this authorGiuseppina Di Fruscio PhD, Giuseppina Di Fruscio PhD Department of Biochemistry, Biophysics and General Pathology, II University of Naples, Naples, Italy Telethon Institute of Genetics and Medicine, Naples, ItalySearch for more papers by this authorEbe Pastorello MD, Ebe Pastorello MD ULSS 16 Padova, ItalySearch for more papers by this authorElisabetta Tasca PhD, Elisabetta Tasca PhD IRCCS Fondazione San Camillo Hospital, Venice, ItalySearch for more papers by this authorCarlo P. Trevisan MD, Carlo P. Trevisan MD Department of Neurosciences, University of Padova, Padova, ItalySearch for more papers by this authorVincenzo Nigro MD, PhD, Vincenzo Nigro MD, PhD Department of Biochemistry, Biophysics and General Pathology, II University of Naples, Naples, Italy Telethon Institute of Genetics and Medicine, Naples, ItalySearch for more papers by this authorCorrado Angelini MD, Corrado Angelini MD Department of Neurosciences, University of Padova, Padova, Italy IRCCS Fondazione San Camillo Hospital, Venice, ItalySearch for more papers by this author First published: 05 August 2014 https://doi.org/10.1002/mus.24357Citations: 20Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume51, Issue1January 2015Pages 145-147 RelatedInformation

The diagnosis of calpainopathy is obtained by identifying calpain-3 protein deficiency or CAPN3 gene mutations. However, in many patients with limb girdle muscular dystrophy type 2A (LGMD2A), the calpain-3 protein quantity is normal because loss-of-function mutations cause its enzymatic inactivation. The identification of such patients is difficult unless a functional test suggests pursuing a search for mutations.A functional in vitro assay, which was able to test calpain-3 autolytic function, was used to screen a large series of muscle biopsy specimens from patients with unclassified LGMD/hyperCKaemia who have previously shown normal calpain-3 protein quantity.Of 148 muscle biopsy specimens tested,17 samples (11%) had lost normal autolytic function. CAPN3 gene mutations were identified in 15 of 17 patients (88%), who account for about 20% of the total patients with LGMD2A diagnosed in our series.The loss of calpain-3 autolytic activity is highly predictive of primary calpainopathy, and the use of this test as part of calpainopathy diagnosis would improve the rate of disease detection markedly. This study provides the first evidence of the pathogenetic effect of specific CAPN3 gene mutations on the corresponding protein function in LGMD2A muscle and offers new insights into the structural-functional relationship of the gene and protein regions that are crucial for the autolytic activity of calpain-3.

Torpedo maculopathy (TM) is a rare congenital anomaly firstly reported by Roseman and Gass in 1992 as a solitary hypopigmented nevus of the retinal pigment epithelium (RPE) [1]. It typically presents as a well-defined, flat, bullet-shaped lesion, located temporal to the macula and pointing towards it [2]. In the vast majority of cases, this anomaly is unilateral, asymptomatic, and nonprogressive and most of the time, the lesion is discovered incidentally and remains benign over time, although rare cases of neovascularization have been described [3].

Several endocrine pathologies are commonly associated with ophthalmic involvement, such as diabetes and Graves' disease [1]. The parathyroid glands produce parathyroid hormone (PTH), which plays a key role in the regulation of calcium and phosphate levels in the blood. Dysfunction of these small endocrine glands can lead to various systemic manifestations [2]. However, there is limited clinical information regarding a possible association between ocular manifestations and parathyroid gland disorders [3], [4].

The diagnosis of isolated heterozygotes for recessive LGMD is quite difficult because no specific biochemical or protein assays are available, and the molecular analysis is not feasible due to the wide genetic heterogeneity. We investigated a series of definite heterozygotes for different forms of LGMD to determine whether the carrier status will result in a detectable protein defect in muscle biopsy. Definite heterozygotes from 4 families (3 LGMD2B and 1 LGMD2E) underwent quantitative immunoblot analysis of mutant protein in their muscle. While the quantity of beta-sarcoglycan was nearly normal in the LGMD2E carrier, the levels of dysferlin protein were reduced to 50% of controls in the carriers of LGMD2B. We have demonstrated the value of protein analysis in the identification of both familial and isolated LGMD2B heterozygotes, and suggested the use of dysferlin protein testing to select muscle biopsies from suspected carriers for a subsequent mutation analysis. Muscle protein analysis would be used to screen asymptomatic patients who underwent muscle biopsy because of unexplained hyperCKemia.

Reduction of neuronal nitric oxide synthase (nNOS) has been associated with the pathogenesis and clinical expression of inherited myopathies. To determine whether a defect in nNOS might be an adverse modulating factor in the course of limb-girdle muscular dystrophy, we investigated cytosolic and sarcolemmal nNOS expression in muscle biopsies from 32 patients with 7 forms of limb-girdle muscular dystrophy. Primary calpainopathy, dysferlinopathy, and caveolinopathy biopsies showed normal levels of cytosolic nNOS and preserved sarcolemmal nNOS immunoreactivity. By contrast, the cytosolic nNOS levels in sarcoglycanopathy muscles were variably reduced. Sarcolemmal nNOS immunoreactivity varied from absent to reduced, depending on the integrity of the sarcoglycan complex. In muscles with loss of the entire sarcoglycan complex, sarcolemmal nNOS was absent; it otherwise depended on the specific sarcoglycan gene and type of mutation. The integrity of the entire sarcoglycan complex is, therefore, essential for the stabilization of nNOS to the sarcolemma. Absence of sarcolemmal nNOS in sarcoglycanopathy muscle was always associated with severe muscular dystrophy and sometimes with dilated cardiomyopathy, supporting the hypothesis that nNOS defect might contribute to skeletal and cardiac muscle disease progression. These results emphasize the value of nNOS immunohistochemical analysis in limb-girdle muscular dystrophy and provide additional insights for future therapeutic interventions in these disorders.

Muscle fatigability and atrophy are frequent clinical signs in limb girdle muscular dystrophy (LGMD), but their pathogenetic mechanisms are still poorly understood. We review a series of different factors that may be connected in causing fatigue and atrophy, particularly considering the role of neuronal nitric oxide synthase (nNOS) and additional factors such as gender in different forms of LGMD (both recessive and dominant) underlying different pathogenetic mechanisms. In sarcoglycanopathies, the sarcolemmal nNOS reactivity varied from absent to reduced, depending on the residual level of sarcoglycan complex: in cases with complete sarcoglycan complex deficiency (mostly in beta-sarcoglycanopathy), the sarcolemmal nNOS reaction was absent and it was always associated with early severe clinical phenotype and cardiomyopathy. Calpainopathy, dysferlinopathy, and caveolinopathy present gradual onset of fatigability and had normal sarcolemmal nNOS reactivity. Notably, as compared with caveolinopathy and sarcoglycanopathies, calpainopathy and dysferlinopathy showed a higher degree of muscle fiber atrophy. Males with calpainopathy and dysferlinopathy showed significantly higher fiber atrophy than control males, whereas female patients have similar values than female controls, suggesting a gender difference in muscle fiber atrophy with a relative protection in females. In female patients, the smaller initial muscle fiber size associated to endocrine factors and less physical effort might attenuate gender-specific muscle loss and atrophy.

Clinical GeneticsVolume 73, Issue 4 p. 388-390 Cardioembolic stroke in Danon disease M Spinazzi, Corresponding Author M Spinazzi Department of NeurosciencesDr Marco SpinazziDepartment of NeurosciencesUniversity of PadovaClinica Neurologica IIVia Facciolati 7135100 PadovaItalyTel.: 0039 049 8215315Fax: 0039 049 8215310e-mail: [email protected]Search for more papers by this authorM Fanin, M Fanin Department of NeurosciencesSearch for more papers by this authorP Melacini, P Melacini Department of Cardiac, Thoracic and Vascular Sciences, University of Padova, Padova, ItalySearch for more papers by this authorAC Nascimbeni, AC Nascimbeni Department of NeurosciencesSearch for more papers by this authorC Angelini, C Angelini Department of NeurosciencesSearch for more papers by this author M Spinazzi, Corresponding Author M Spinazzi Department of NeurosciencesDr Marco SpinazziDepartment of NeurosciencesUniversity of PadovaClinica Neurologica IIVia Facciolati 7135100 PadovaItalyTel.: 0039 049 8215315Fax: 0039 049 8215310e-mail: [email protected]Search for more papers by this authorM Fanin, M Fanin Department of NeurosciencesSearch for more papers by this authorP Melacini, P Melacini Department of Cardiac, Thoracic and Vascular Sciences, University of Padova, Padova, ItalySearch for more papers by this authorAC Nascimbeni, AC Nascimbeni Department of NeurosciencesSearch for more papers by this authorC Angelini, C Angelini Department of NeurosciencesSearch for more papers by this author First published: 26 February 2008 https://doi.org/10.1111/j.1399-0004.2008.00971.xCitations: 12Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume73, Issue4April 2008Pages 388-390 RelatedInformation

Muscle & NerveVolume 52, Issue 2 p. 305-306 Letter to the Editor Incomplete penetrance in limb-girdle muscular dystrophy type 1F Marina Fanin PhD, Marina Fanin PhD Department of Neurosciences, University of Padova, Padova, ItalySearch for more papers by this authorEnrico Peterle MD, Enrico Peterle MD Department of Neurosciences, University of Padova, Padova, ItalySearch for more papers by this authorChiara Fritegotto PhD, Chiara Fritegotto PhD Department of Neurosciences, University of Padova, Padova, ItalySearch for more papers by this authorAnna C. Nascimbeni PhD, Anna C. Nascimbeni PhD Department of Neurosciences, University of Padova, Padova, ItalySearch for more papers by this authorElisabetta Tasca PhD, Elisabetta Tasca PhD IRCCS Fondazione San Camillo Hospital, Venice, ItalySearch for more papers by this authorAnnalaura Torella PhD, Annalaura Torella PhD Department of Biochemistry, Biophysics and General Pathology, II University of Naples, Naples, Italy Telethon Institute of Genetics and Medicine, Naples, ItalySearch for more papers by this authorVincenzo Nigro MD, PhD, Vincenzo Nigro MD, PhD Department of Biochemistry, Biophysics and General Pathology, II University of Naples, Naples, Italy Telethon Institute of Genetics and Medicine, Naples, ItalySearch for more papers by this authorCorrado Angelini MD, Corrado Angelini MD Department of Neurosciences, University of Padova, Padova, Italy IRCCS Fondazione San Camillo Hospital, Venice, ItalySearch for more papers by this author Marina Fanin PhD, Marina Fanin PhD Department of Neurosciences, University of Padova, Padova, ItalySearch for more papers by this authorEnrico Peterle MD, Enrico Peterle MD Department of Neurosciences, University of Padova, Padova, ItalySearch for more papers by this authorChiara Fritegotto PhD, Chiara Fritegotto PhD Department of Neurosciences, University of Padova, Padova, ItalySearch for more papers by this authorAnna C. Nascimbeni PhD, Anna C. Nascimbeni PhD Department of Neurosciences, University of Padova, Padova, ItalySearch for more papers by this authorElisabetta Tasca PhD, Elisabetta Tasca PhD IRCCS Fondazione San Camillo Hospital, Venice, ItalySearch for more papers by this authorAnnalaura Torella PhD, Annalaura Torella PhD Department of Biochemistry, Biophysics and General Pathology, II University of Naples, Naples, Italy Telethon Institute of Genetics and Medicine, Naples, ItalySearch for more papers by this authorVincenzo Nigro MD, PhD, Vincenzo Nigro MD, PhD Department of Biochemistry, Biophysics and General Pathology, II University of Naples, Naples, Italy Telethon Institute of Genetics and Medicine, Naples, ItalySearch for more papers by this authorCorrado Angelini MD, Corrado Angelini MD Department of Neurosciences, University of Padova, Padova, Italy IRCCS Fondazione San Camillo Hospital, Venice, ItalySearch for more papers by this author First published: 09 December 2014 https://doi.org/10.1002/mus.24539Citations: 11Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume52, Issue2August 2015Pages 305-306 RelatedInformation

Table S1. Targeted next-generation sequencing. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Clinical GeneticsVolume 71, Issue 4 p. 374-375 Late-onset GSDII with novel GAA gene mutation C Angelini, Corresponding Author C Angelini Department of Neurosciences University of Padova Research Foundation, Venetian Institute of Molecular Medicine, Padova, ItalyCorrado Angelini, MDDepartment of NeurosciencesUniversity of PadovaVia Giustiniani 535128 PadovaItalyTel.: +39 49 8213625Fax: +39 49 8751770e-mail: corrado.angelini@unipd.itSearch for more papers by this authorAC Nascimbeni, AC Nascimbeni Department of Neurosciences University of Padova Research Foundation, Venetian Institute of Molecular Medicine, Padova, ItalySearch for more papers by this author C Angelini, Corresponding Author C Angelini Department of Neurosciences University of Padova Research Foundation, Venetian Institute of Molecular Medicine, Padova, ItalyCorrado Angelini, MDDepartment of NeurosciencesUniversity of PadovaVia Giustiniani 535128 PadovaItalyTel.: +39 49 8213625Fax: +39 49 8751770e-mail: corrado.angelini@unipd.itSearch for more papers by this authorAC Nascimbeni, AC Nascimbeni Department of Neurosciences University of Padova Research Foundation, Venetian Institute of Molecular Medicine, Padova, ItalySearch for more papers by this author First published: 25 April 2007 https://doi.org/10.1111/j.1399-0004.2007.00785.xCitations: 8Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume71, Issue4April 2007Pages 374-375 RelatedInformation

Pompe disease (PD) is a metabolic myopathy caused by a deficiency of acid-alpha glucosidase (GAA), a lysosomal enzyme that cleaves glycogen. The classic infantile-onset form is characterised by severe hypotonia and cardiomyopathy. Untreated patients usually die within the first year of life due to cardiorespiratory failure. Several studies involving patients with infantile-onset PD have shown that enzyme replacement therapy (ERT) with alglucosidase alfa, recombinant human GAA (rhGAA), significantly prolongs survival, decreases cardiomegaly, and improves cardiac function and conduction abnormalities. However, the efficacy on motor, cognitive and social milestones appears to be more related to the condition of the patient before the start of treatment. To date, the sample of early diagnosed and treated patients is small and the length of follow-up is still limited. We report the results of a long-term follow-up of one patient presenting severe bradycardia and cardiomyopathy at birth, diagnosed in the third day of life and successfully treated by ERT. Serum muscle enzymes at diagnosis were AST 200 U/L, ALT 99 U/L and CPK 731 U/L (n.v. 0-295); the molecular study identified the homozygous missense mutation c.1933 G> A p.Asp645Asn (GAA exon 14). Left Ventricular Mass Index (LVMI) at baseline was 171 g/m(2) (Z-score = 4.3) and decreased to normal values since the 3-month follow-up. A muscle biopsy performed at 18 months after the start of therapy, showed only a low degree of muscle involvement. To our knowledge, this is the longest ERT treatment follow-up in a symptomatic neonatal patient with Pompe disease.

The world of metabolic myopathies has been dramatically modified by the advent of enzyme replacement therapy (ERT), the first causative treatment for glycogenosis type II (GSDII) or Pompe disease, which has given new impetus to research into that disease and also other pathologies. This article reviews new advances in the treatment of GSDII, the consensus about ERT, and its limitations. In addition, the most recent knowledge regarding the pathophysiology, phenotype, and genotype of the disease is discussed. Pharmacological, immunotherapy, nutritional, and physical/rehabilitative treatments for late-onset Pompe disease and other metabolic myopathies are covered, including treatments for defects in glycogen metabolism, such as glycogenosis type V (McArdle disease), and glycogenosis type III (debrancher enzyme deficiency), and defects in lipid metabolism, such as carnitine palmitoyltransferase II deficiency and electron transferring flavoprotein dehydrogenase deficiency, or riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency.

Although cells are a part of the whole organism, classical dogma emphasizes that individual cells function autonomously. Many physiological and pathological conditions, including cancer, and metabolic and neurodegenerative diseases, have been considered mechanistically as cell-autonomous pathologies, meaning those that damage or defect within a selective population of affected cells suffice to produce disease. It is becoming clear, however, that cells and cellular processes cannot be considered in isolation. Best known for shuttling cytoplasmic content to the lysosome for degradation and repurposing of recycled building blocks such as amino acids, nucleotides, and fatty acids, autophagy serves a housekeeping function in every cell and plays key roles in cell development, immunity, tissue remodeling, and homeostasis with the surrounding environment and the distant organs. In this review, we underscore the importance of taking interactions with the microenvironment into consideration while addressing the cell autonomous and non-autonomous functions of autophagy between cells of the same and different types and in physiological and pathophysiological situations.

Primary cilium-dependent macroautophagy/autophagy is induced by the urinary flow in epithelial cells of the kidney proximal tubule. A major physiological outcome of this cascade is the control of cell size. Some components of the ATG machinery are recruited at the primary cilium to generate autophagic structures. Shear stress induced by the liquid flow promotes PtdIns3P synthesis at the primary cilium, and this lipid is required both for ciliogenesis and initiation of autophagy. We showed that PtdIns3P is generated by PIK3C2A, but not by PIK3C3/VPS34, during flow-associated primary cilium-dependent autophagy, in a ULK1-independent manner. Along the same line BECN1 (beclin 1), a partner of PIK3C3 in starvation-induced autophagy, is not recruited at the primary cilium under shear stress. Thus, kidney epithelial cells mobilize different PtdIns 3-kinases, i.e., PIK3C2A or PIK3C3, to produce PtdIns3P in order to initiate autophagy depending on the stimuli (shear stress or starvation).Abbreviations PtdIns3P: phosphatidylinositol-3-phosphate; PIK3C2A: class two alpha phosphatidylinositol 3-kinase; PIK3C3/VPS34: class three phosphatidylinositol 3-kinase; ATG: autophagy associated genes.

We studied the role of autophagy in a series of 10 infantile-, juvenile-, and adult-onset GSDII patients and investigated autophagy blockade in successive biopsies of adult cases during disease natural history. We also correlated the autophagosome accumulation and efficiency of enzyme replacement therapy (ERT) in four treated cases (two infantile and two juvenile-adult onsets). The autophagic flux was monitored by measuring the amount of p62-positive protein aggregates and compared, together with fibre vacuolisation, to fibre atrophy. A blocked autophagic flux resulted in p62 accumulation, increased vacuolisation, and progressive atrophy of muscle fibres in biopsies collected from patients during natural history. On the contrary, in the GSDII cases early treated with ERT, the autophagic flux improved and muscle fibre atrophy, fibre vacuolisation, and acid phosphatase activity decreased. The functionality of the autophagy-lysosome system is essential in GSDII muscle, which is characterised by the presence of swollen glycogen-filled lysosomes and autophagic build-up. Defining the role of autophagy and its relationship with muscle loss is critical for understanding the disease pathogenesis, for developing new therapies, and for improving ERT efficacy in GSDII.

The biogenesis of autophagosome, the double membrane bound organelle related to macro-autophagy, is a complex event requiring numerous key-proteins and membrane remodeling events. Our recent findings identify the extended synaptotagmins, crucial tethers of Endoplasmic Reticulum-plasma membrane contact sites, as key-regulators of this molecular sequence.

Skeletal muscle is one of the tissues with the highest rate of autophagosome formation and degradation.A group of congenital myopathies, characterised by lysosomal alteration, are described by massive autophagic build-up and are named Autophagic Vacuolar Myopathies (AVM).Among these disorders, Danon disease (DD) and Pompe (GSD2) are characterised by the presence of large glycogen-fi lled lysosomes in the skeletal muscle.We have recently shown that autophagy impairment contributes to disease progression in GSD2 patients.However, the mechanism that leads to autophagy inhibition, and whether this impairment is shared in other AVM in humans is an open issue. MATERIAL AND METHODSWe have analysed muscle biopsies of GSD2 and DD patients for markers of autophagy and signalling pathways related to lysosomal biogenesis and endosome traffi cking by immunofl uorescence, Western blotting and quantitative RT-PCR techniques.

The highly complex proprioceptive system provides neuromuscular control of the mobile cervical spine. Static neck flexion can induce the elongation of posterior tissues and altered afferent input from the mechanoreceptors. The purpose of this study was to examine the effect of prolonged static neck flexion on neck proprioception and anticipatory postural adjustments. Thirty-eight healthy participants (20 females and 18 males) between the ages of 20–35 years with no history of neck, low back, and shoulder pain enrolled in this study. Neck proprioception and anticipatory muscle activity were tested before and after 10-min static neck flexion. For assessment of neck proprioception, each participant was asked to perform 10 trials of the cervicocephalic relocation test to the neutral head position after active neck rotation to the left and right sides. Anticipatory postural adjustments were evaluated during a rapid arm flexion test. Following the flexion, the absolute and variable errors in head repositioning significantly increased (p < 0.05). The results also showed that there was a significant delay in the onset of myoelectric activity of the cervical erector spinae muscles after flexion (p = 0.001). The results of this study suggested that a 10-min static flexion can lead to changes in the neck proprioception and feed-forward control due to mechanical and neuromuscular changes in the viscoelastic cervical spine structures. These changes in sensory-motor control may be a risk factor for neck pain and injury.

The aim of this study is to evaluate the foot-contact gait patterns in spastic hemiplegic children of Winters type I and II vs. healthy controls. By means of ‘statistical gait analysis' we analysed 100-200 gait cycles for each child, obtaining the relative frequency of the main foot-floor gait sequences observed during a walk at self-selected speed. Type I hemiplegic children show 27 % of strides with a ‘normal' foot-floor contact pattern, while for type II this is observed only in 5% of strides. The more frequently observed abnormal gait cycles are described

Danon disease is an X-linked dominant disorder due to mutations in the LAMP2 gene, presenting with hypertrophic cardiomyopathy, skeletal myopathy and mental retardation. To investigate the effects of LAMP2 gene mutations on protein expression in different tissues, we screened LAMP2 gene mutations and LAMP-2 protein deficiency in the skeletal muscle of 9 unrelated patients with hypertrophic cardiomyopathy and vacuolar myopathy. We identified 3 novel families with unreported LAMP2 gene null mutations and LAMP-2 protein deficiency in skeletal and myocardial muscle, leukocytes and fibroblasts. LAMP-2 protein deficiency was detectable in various tissues indicating that the biochemical diagnosis can be obtained on leukocytes and might be used for screening in male patients, and that the multiorgan protein deficiency would explain the multisystem clinical involvement. In our female patient, muscle histopathology and LAMP-2 protein analysis was inconclusive, indicating that the diagnosis in females can be obtained only by mutation identification.

Background: Polyglucosan body myopathies are due to heterogeneous causes. Objectives. Mutations in the newly identified RBCK1 gene have been recognized to be a rare cause of polyglucosan body myopathy. Here we report a novel family in which a form of adult-onset limb-girdle myopathy was associated with polyglucosan storage in muscle. Methods and patients: In 3 affected relatives we investigated the clinical phenotype, the muscle morphological and ultrastructural features, the RBCK1 genotype and the role of ubiquitin-proteasome and lysosomal-autophagic degradation pathways by analysing ubiquitin, p62/SQSTM1, LC3 and MuRF-1 proteins. Results: The phenotype in all 3 patients was characterized by limb-girdle muscular dystrophy with onset in the fifth decade, associated with autoimmune disorders in 2 cases (generalized vitiligo, chronic inflammatory demyelinating polyneuropathy), without overt cardiac impairment. Accumulation of PAS-positive, amylase-resistant polyglucosan bodies was detectable in 2 of the 4 muscle biopsies studied. Immunofluorescence analysis showed that the inclusions were strongly labelled for ubiquitin and p62/SQSTM1, and immunoblotting revealed increased levels of MuRF-1 and LC3-II proteins in muscles from patients as compared to controls. Conclusions: As compared with cases with RBCK1-related myopathy, this family shows late-onset of limb girdle muscular dystrophy and lack of cardiac involvement, whereas muscle histopathological features are completely matching. We offered evidence supporting the role of both the ubiquitin–proteasome and the lysosomal–autophagic degradation pathways in this family, although the mechanism generating polyglucosan bodies in this family is most likely in glycogenin-1 (under investigation).

April 21, 2015April 6, 2015Free AccessRegulatory Factors Of Autophagy And Triglyceride Accumulation In Lipid Storage Myopathies (S17.004)Corrado Angelini, Anna Nascimbeni, and Elisabetta TascaAuthors Info & AffiliationsApril 6, 2015 issue84 (14_supplement)https://doi.org/10.1212/WNL.84.14_supplement.S17.004 Letters to the Editor

INTRODUCTION and AIM In children, hemiplegia is a common consequence of cerebral palsy (CP) and causes altered selective motor control, weakness and spasticity. A correct classification of children with CP is important to assist diagnosis and clinical decision-making. The classification of spastic hemiplegia proposed by Winters et al. is widely accepted in literature. Type I is defined by the presence of drop foot in swing, type II by the persistence of equinism throughout the gait cycle, with a possible knee hyperextension in stance. Foot-contact event detection is fundamental in clinical gait analysis, but it is particularly challenging in children with CP due to initial toecontact. In a recent work, we described an algorithm for the automatic segmentation of gait cycles from the foot-switch signal that it is applicable also to pathological gait. The aim of this contribution is to apply this method to a population of CP children to study their foot-floor contact sequences, considering also the sub-phases of stance. The activation patterns of tibialis anterior (TA) and gastrocnemius lateralis (GL) helped us in the interpretation of the results

As reported recently, massive accumulation of autophagic debris appears to contribute greatly to skeletal muscle weakness in Pompe disease.

The objective of this study was to examine at molecular, biochemical and muscle pathology level two groups of patients affected with Danon disease and GSDII, in order to get new insights that might help in tracing genotype-phenotype correlations and to delineate their pathological mechanisms. Glycogen storage disease type II (GSDII) is an autosomal recessive disorder (OMIM # 232300) caused by the deficiency of the lysosomal enzyme acid ?-glucosidase or acid maltase (EC 3.2.1.20/3), which catalyses the hydrolysis of ?-1,4 and ?-1,6 links of glycogen. The enzyme deficiency leads to lysosomal accumulation of glycogen that results in different clinical phenotypes, ranging from the : the severe infantile-onset form to the childhood, juvenile or adult-onset forms (late-onset forms). We investigated 23 patients with infantile-onset or late-onset glycogen storage disease type II by enzyme activity, protein expression by immunoblotting, GAA gene mutations, and muscle pathology including immunolabeling for Golgi and sarcolemmal proteins. The enzyme activity resulted absent or minimal in infantile-onset cases and variably reduced in late-onset patients. Genotype-phenotype correlation (seven novel mutations were found) showed that most late-onset patients had the heterozygous c.–32-13T>G leaky splicing mutation (one patient was homozygous), but the course of the disease was often difficult to predict on the basis of the mutations alone. One important and novel result from our study came from the Western blot analysis of the different maturative forms of acid ? –glucosidase protein in the muscle from patients with GSDII. We have demonstrated that the muscle from patients with GSDII has a predominant expression of inactive forms of acid ?-glucosidase protein and severely reduced or absent levels of the mature forms. Furthermore, the residual amount of the mature forms of the protein on blotting correlated with the level of enzyme activity in muscle. We first report a different molecular weight of the mature and the intermediate forms of the protein between patients and controls that we attribute to an excessive sialylation of mutant proteins. This is likely caused by a delayed transport and longer transit of the inactive proteins in the Golgi where the sialyltransferases are localized. Supporting this hypothesis, we observed that, in both infantile and late-onset patients, there is an enhanced proliferation of the Golgi apparatus. On the other hand, we did not find any increased expression of LAMP-1 in patients with GSDII, possibly due to the fact that only a minor proportion of mutant enzyme protein is able to reach the lysosomes. Another interesting data rises from the morphologic analysis of the different cellular organelles. Interestingly, we observed a differential degree of dysfunction of endocytic and autophagic pathways in patients with infantile and late-onset GSDII. In late-onset acid maltase deficient muscle, vacuolar membranes expressed sarcolemmal proteins, such as caveolin-3 and dystrophin (previously classified as type 2 vacuoles) and not in the infantile form of the disease (type 4 vacuoles, lakes of glycogen). These features are possibly due to reduced membrane proliferation and vesicular movement in the overcrowded muscle fibers of Pompe disease, and to the membrane remodelling occurring only in patients with late-onset GSDII, which would be a protective mechanism to prevent membrane rupture during fiber contraction. This observation is important because the pathogenesis of the autophagosomes has not yet been fully investigated. Autophagy and membrane remodelling, which is peculiar to late onset disease, might modify a clear response to enzyme replacement therapy and, also, compartmentalize the delivery of the recombinant enzyme. Danon disease, an X-linked dominant disorder, results from mutations in the lysosome-associated membrane protein-2 (LAMP2) gene and presents with hypertrophic cardiomyopathy, skeletal myopathy, and mental retardation. To investigate the effects of LAMP2 gene mutations on protein expression in different tissues, we screened LAMP2 gene mutations and LAMP-2 protein deficiency in the skeletal muscle of nine unrelated patients with hypertrophic cardiomyopathy and vacuolar myopathy. We identified three novel families (including one affected mother) with unreported LAMP2 gene null mutations and LAMP-2 protein deficiency in skeletal and myocardial muscle, leukocytes, and fibroblasts. LAMP-2 protein deficiency was detectable in various tissues, including leukocytes, explaining the multisystem clinical involvement. Skeletal muscle immunopathology showed that mutant protein was not localized in the Golgi complex, vacuolar membranes expressed sarcolemmal- specific proteins, and the degree of muscle fiber vacuolization correlated with clinical muscle involvement. In our female patient, muscle histopathology and LAMP-2 protein analysis was inconclusive, indicating that diagnosis in females requires mutation identification. The random X-chromosome inactivation found in muscle and leukocytes excluded the possibility that selective involvement of some tissues in females is due to skewed X-chromosome inactivation. Therefore, biochemical analysis of leukocytes might be used for screening in male patients, but genetic screening is required in females.

The use of surface electromyography (sEMG) recorded during ambulation has provided valuable insight into motor development and changes with age in the pediatric population. However, no studies have reported sEMG differences with age in the children with cerebral palsy (CP). In this study, data from 50 children were divided retrospectively into four groups, representing either an older (above the age of seven years) or younger (below the age of seven years) age group with either typical development (TD) or CP. Data were analyzed from 16 children in the younger age group with TD, and eight in the older age group with TD. Data were also available from 14 in the younger age group with CP, and 12 in the older age group with CP. SEMG signals from the rectus femoris (RF) and medial hamstring (MH) were analyzed using wavelet techniques to examine time–frequency content. RF muscle activity was statistically different between all groups (p < 0.001), with an elevated instantaneous mean frequency (IMNF) in the older TD group than the younger TD group, an elevated IMNF in the younger CP group than the older CP group, and elevated IMNF in both CP groups compared to both TD groups. Activity for the MH muscle followed the same pattern except for the CP young and old group comparison, which indicated no difference. The results indicate that differences in neuromuscular activation exist between younger and older groups of children with both TD and CP, and may provide new insight into muscle activity pattern changes during the development of walking.

To evaluate local joint variables after intra-articular injection with triamcinolone hexacetonide in rheumatoid arthritis patients.We blindly and prospectively (baseline, 1, 4, 12 and 24 weeks) evaluated metacarpophalangeal, wrist, elbow, shoulder, knee and ankle joints after triamcinolone hexacetonide intra-articular injection by the following outcome measures: visual analogue scale 0–10 cm (VAS) for rest pain (VASR); VAS for movement pain (VASM); VAS for joint swelling (VASSw); flexion (FlexG) and extension (ExtG).289 patients (635 joints) were studied. VASSw (p < 0.001) and VASR (0.001 < p < 0.016) improved from T0 to T4, T12 and T24 for all joints. VASM improved from T0 to T4 (p < 0.021) for all joints; T0 to T12 (p < 0.023) for MCF and knee; T0 to T24 (p < 0.019) only for MCF and knee. FlexG improved from T0 to T4 (p < 0.001) for all joints; T0 to T12 (p < 0.001) and T0 to T24 (p < 0.02) only for MCF and knee. ExtG improved from T0 to T4 (p < 0.001) for all joints except for elbow; T0 to T12 (p = 0.003) for wrist, metacarpophalangeal and knee; and T0 to T24 (p = 0.014) for MCF and knee.VASSw responded better at short and medium term after IAI with triamcinolone hexacetonide in our sample of RA patients.Avaliar variáveis articulares locais após a infiltração intra-articular (IIA) de hexacetonido de triancinolona (HT) em pacientes com artrite reumatoide (AR).Foram avaliadas, de modo cego e prospectivo (nos tempos inicial, 1, 4, 12 e 24 semanas), as articulações metacarpofalângica (MCF), punho, cotovelo, ombro, joelho e tornozelo após a IIA de HT utilizando-se das seguintes medidas de desfecho: escala visual analógica (EVA) de 0 a 10 cm para dor em repouso (EVAr); EVA para dor ao movimento (EVAm); EVA para edema articular (EVAe); flexão (FlexG) e extensão (ExtG).Estudaram-se 289 pacientes (635 articulações). A EVAe (p < 0,001) e a EVAr (0,001 < p < 0,016) melhoraram de T0 a T4, T12 e T24 em todas as articulações. A EVAm melhorou de T0-T4 (p < 0,021) em todas as articulações; T0-T12 (p < 0,023) na MCF e no joelho; T0-T24 (p < 0,019) apenas na MCF e no joelho. A FlexG melhorou de T0-T4 (p < 0,001) em todas as articulações; T0-T12 (p < 0,001) e T0-T24 (p < 0,02) apenas na MCF e no joelho. A ExtG melhorou de T0-T4 (p < 0,001) em todas as articulações, exceto no cotovelo; T0-T12 (p = 0,003) no punho, na MCF e no joelho; e T0-T24 (p = 0,014) na MCF e no joelho.A EVAe respondeu melhor em curto e médio prazos após a IIA de HT nessa amostra de pacientes com AR.