Abstract
X-linked Myopathy with Excessive Autophagy (XMEA) is a childhood onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p, VMA21 is an essential assembly chaperone of the vacuolar ATPase (V-ATPase), the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids which leads to downregulation of the mTORC1 pathway, and consequent increased macroautophagy resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge, and vacuolate the cell. Our results uncover a novel mechanism of disease, namely macroautophagic overcompensation leading to cell vacuolation and tissue atrophy.
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References
Beugnet A, Tee AR, Taylor PM, Proud CG (2003) Regulation of targets of mTOR (mammalian target of rapamycin) signalling by intracellular amino acid availability. Biochem J 372:555–566
Blommaart EF, Luiken JJ, Blommaart PJ, van Woerkom GM, Meijer AJ (1995) Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes. J Biol Chem 270:2320–2326
Brummelkamp TR, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550–553
Cao Y, Klionsky DJ (2007) Physiological functions of Atg6/Beclin 1: a unique autophagy-related protein. Cell Res 17:839–849
Cuervo AM, Stefanis L, Fredenburg R, Lansbury PT, Sulzer D (2004) Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy. Science 305:1292–1295
Demaurex N, Furuya W, D’Souza S, Bonifacino JS, Grinstein S (1998) Mechanism of acidification of the trans-Golgi network (TGN). In situ measurements of pH using retrieval of TGN38 and furin from the cell surface. J Biol Chem 273:2044–2051
Drose S, Altendorf K (1997) Bafilomycins and concanamycins as inhibitors of V-ATPases and P-ATPases. J Exp Biol 200:1–8
Forgac M (2007) Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology. Nat Rev Mol Cell Biol 8:917–929
Frattini A, Orchard PJ, Sobacchi C et al (2000) Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis. Nat Genet 25:343–346
Fukuda K, Hirai Y, Yoshida H, Nakajima T, Usui T (1982) Free amino acid content of lymphocytes and granulocytes compared. Clin Chem 28:1758–1761
Huss M, Sasse F, Kunze B (2005) Archazolid and apicularen: novel specific V-ATPase inhibitors. BMC Biochem 6:13
Kalimo H, Savontaus ML, Lang H et al (1988) X-linked myopathy with excessive autophagy: a new hereditary muscle disease. Ann Neurol 23:258–265
Karet FE, Finberg KE, Nelson RD et al (1999) Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness. Nat Genet 21:84–90
Kornak U, Reynders E, Dimopoulou A et al (2008) Impaired glycosylation and cutis laxa caused by mutations in the vesicular H+-ATPase subunit ATP6V0A2. Nat Genet 40:32–34
Kovacs AL, Reith A, Seglen PO (1982) Accumulation of autophagosomes after inhibition of hepatocytic protein degradation by vinblastine, leupeptin or a lysosomotropic amine. Exp Cell Res 137:191–201
Li JB, Goldberg AL (1976) Effects of food deprivation on protein synthesis and degradation in rat skeletal muscles. Am J Physiol 231:441–448
Malicdan MC, Noguchi S, Nonaka I, Saftig P, Nishino I (2008) Lysosomal myopathies: an excessive build-up in autophagosomes is too much to handle. Neuromuscul Disord 18:521–529
Malkus P, Graham LA, Stevens TH, Schekman R (2004) Role of Vma21p in assembly and transport of the yeast vacuolar ATPase. Mol Biol Cell 15:5075–5091
Mammucari C, Schiaffino S, Sandri M (2008) Downstream of Akt: foxO3 and mTOR in the regulation of autophagy in skeletal muscle. Autophagy 4:524–526
Massey AC, Kaushik S, Sovak G, Kiffin R, Cuervo AM (2006) Consequences of the selective blockage of chaperone-mediated autophagy. Proc Natl Acad Sci USA 103:5805–5810
Mizushima N (2007) Autophagy: process and function. Genes Dev 21:2861–2873
Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y (2004) In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell 15:1101–1111
Munteanu I, Ramachandran N, Mnatzakanian GN et al (2008) Fine-mapping the gene for X-linked myopathy with excessive autophagy. Neurology 71:951–953
Nishino I, Fu J, Tanji K et al (2000) Primary LAMP-2 deficiency causes X-linked vacuolar cardiomyopathy and myopathy (Danon disease). Nature 406:906–910
Ostenfeld MS, Hoyer-Hansen M, Bastholm L et al (2008) Anti-cancer agent siramesine is a lysosomotropic detergent that induces cytoprotective autophagosome accumulation. Autophagy 4:487–499
Paroutis P, Touret N, Grinstein S (2004) The pH of the secretory pathway: measurement, determinants, and regulation. Physiology (Bethesda) 19:207–215
Salvador N, Aguado C, Horst M, Knecht E (2000) Import of a cytosolic protein into lysosomes by chaperone-mediated autophagy depends on its folding state. J Biol Chem 275:27447–27456
Sandri M (2010) Autophagy in skeletal muscle. FEBS Lett 584:1411–1416
Scornik OA, Howell SK, Botbol V (1997) Protein depletion and replenishment in mice: different roles of muscle and liver. Am J Physiol 273:E1158–E1167
Scott JA, North ML, Rafii M et al (2011) Asymmetric dimethylarginine is increased in asthma. Am J Respir Crit Care Med 184:779–785
Shacka JJ, Klocke BJ, Shibata M et al (2006) Bafilomycin A1 inhibits chloroquine-induced death of cerebellar granule neurons. Mol Pharmacol 69:1125–1136
Sharma R, Deng H, Leung A, Mahuran D (2001) Identification of the 6-sulfate binding site unique to alpha-subunit-containing isozymes of human beta-hexosaminidase. Biochemistry 40:5440–5446
Stauber WT, Hedge AM, Trout JJ, Schottelius BA (1981) Inhibition of lysosomal function in red and white skeletal muscles by chloroquine. Exp Neurol 71:295–306
Tanaka Y, Guhde G, Suter A et al (2000) Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice. Nature 406:902–906
Taussky HH, Shorr E (1953) A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem 202:675–685
Villanova M, Louboutin JP, Chateau D et al (1995) X-linked vacuolated myopathy: complement membrane attack complex on surface membrane of injured muscle fibers. Ann Neurol 37:637–645
Webber JL, Tooze SA (2010) Coordinated regulation of autophagy by p38alpha MAPK through mAtg9 and p38IP. EMBO J 29:27–40
Zoncu R, Bar-Peled L, Efeyan A, Wang S, Sancak Y, Sabatini DM (2011) mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(+)-ATPase. Science 334:678–683
Acknowledgments
We wish to thank all the XMEA families. We are grateful to Drs. G. Israelian, V.C. Juel, M. Villanova, the late G. Karpati, S. Carpenter, and D. Figarella-Branger for the clinicopathologic diagnosis of some of the patients included in this study, previously published in clinical journals. We thank Drs. S. Grinstein for helpful discussions and Drs. J Rommens and S Meyn for their review of our manuscript, T. Sarkisyan, J. Kere, E. Heon, F. Zara and H. Lohi for ethnic control DNA samples, A. Leung for assistance with experiments in Supplemental Fig. 12, N. Ochtony for Supplemental Fig. 1A, P. Bradhsaw for assistance in deconvolution microscopy. Principal funding was from the Canadian Institutes of Health Research. The Association Française contre les Myopathies provided support to N. Levy, EVO Funds of Helsinki and Turku University Hospitals (Finland) and sports research grant from Ministry of Education of Finland to H. Kalimo, and the Natural Sciences and Engineering Research Council (Canada) to I. Munteanu.
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The authors declare that they have no conflicts of interests.
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This study was approved by the Research Ethics Board of the Hospital for Sick Children Toronto and informed consent was obtained from all subjects.
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N. Ramachandran and I. Munteanu are both first-authors of this paper.
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Ramachandran, N., Munteanu, I., Wang, P. et al. VMA21 deficiency prevents vacuolar ATPase assembly and causes autophagic vacuolar myopathy. Acta Neuropathol 125, 439–457 (2013). https://doi.org/10.1007/s00401-012-1073-6
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DOI: https://doi.org/10.1007/s00401-012-1073-6