Suppression of autophagy in skeletal muscle
uncovers the accumulation of ubiquitinated
proteins and their potential role in muscle
damage in Pompe disease
Nina Raben1,?, Victoria Hill1, Lauren Shea1, Shoichi Takikita1, Rebecca Baum1,
Noboru Mizushima3, Evelyn Ralston2and Paul Plotz1
1Arthritis and Rheumatism Branch and2Light Imaging Section, Office of Science and Technology, National Institute of
Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA
and3The Department of Physiology and Cell Biology, Tokyo Medical and Dental University, Tokyo, Japan
Received July 16, 2008; Revised and Accepted September 8, 2008
The role of autophagy, a catabolic lysosome-dependent pathway, has recently been recognized in a variety of
disorders, including Pompe disease, the genetic deficiency of the glycogen-degrading lysosomal enzyme
acid-alpha glucosidase. Accumulation of lysosomal glycogen, presumably transported from the cytoplasm
by the autophagic pathway, occurs in multiple tissues, but pathology is most severe in skeletal and cardiac
muscle. Skeletal muscle pathology also involves massive autophagic buildup in the core of myofibers. To
determine if glycogen reaches the lysosome via autophagy and to ascertain whether autophagic buildup
in Pompe disease is a consequence of induction of autophagy and/or reduced turnover due to defective
fusion with lysosomes, we generated muscle-specific autophagy-deficient Pompe mice. We have demon-
strated that autophagy is not required for glycogen transport to lysosomes in skeletal muscle. We have
also found that Pompe disease involves induction of autophagy but manifests as a functional deficiency
of autophagy because of impaired autophagosomal–lysosomal fusion. As a result, autophagic substrates,
including potentially toxic aggregate-prone ubiquitinated proteins, accumulate in Pompe myofibers and
may cause profound muscle damage.
Macroautophagy (hereafter called autophagy) is an evolu-
tionarily conserved degradative pathway by which long-lived
intracellular proteins and organelles are delivered to the
lysosome for destruction and recycling. During this process,
a double-membrane vesicle called an autophagosome develops
around a portion of the cytoplasm and organelles, such as
mitochondria. The outer autophagosomal membrane fuses
with the lysosomal membrane resulting in the delivery of the
inner vesicle into the lumen of the lysosome. Within the lyso-
some, the autophagic cargo is broken down by hydrolases, and
the resulting molecules are recycled (reviewed in 1–3). Autop-
hagy is involved in the cellular response to starvation, cellular
differentiation, cell death, aging, cancer and neurodegenerative
diseases. Since the lysosomes are the final destination of the
autophagic pathway, it is not surprising that this pathway
has also been implicated in several lysosomal storage dis-
orders (4–7), including Pompe disease (glycogen storage
disease type II, GSDII), a deficiency of the glycogen-
degrading lysosomal enzyme acid-alpha glucosidase (GAA).
The deficiency of this enzyme results in the failure to meta-
bolize lysosomal glycogen to glucose leading to progressive
accumulation of glycogen and the enlargement of lysosomes
in multiple tissues. Cardiac and skeletal muscles are the major
tissues affected by the storage. Both severe cardiomyopathy
and skeletal muscle myopathy are observed in patients with
complete or near complete enzyme deficiency. Affected
infants usually die within the first year of life. In patients with
milder late-onset forms, cardiac muscle is spared, but slowly
?To whom correspondence should be addressed at: 50 South Drive, Bld. 50/1345, NIAMS, NIH, Bethesda, MD 20892-1820, USA.
Tel: þ1 3014961474; Fax: þ1 3014358017; Email: firstname.lastname@example.org
Published by Oxford University Press 2008.
Human Molecular Genetics, 2008, Vol. 17, No. 24
Advance Access published on September 9, 2008
progressive skeletal muscle weakness eventually leads to pre-
mature death due to respiratory insufficiency (8).
Pompe disease itself and the basic biochemistry of the
genetic defect have been known for decades, but the mechan-
ism of profound muscle damage has remained unclear. The
accumulation of glycogen, presumed to be transported to the
lysosomes via the autophagic pathway, is not limited to
the lysosomes in skeletal muscle, but is also found in autophagic
vacuoles containing cytoplasmic degradation products (8,9). We
have demonstrated that the great extent of autophagy and its role
in muscle damage make the autophagic process a critical player
in the pathogenesis of Pompe disease. Our findings in both
humans and GAA knockout mice (GAA KO) revealed the pre-
sence in the core of muscle fibers of huge non-contractile
inclusions containing cellular debris, fragmented mitochondria,
remnants of lysosomal membranes and a large number of
autophagosomes (10,11). In the GAA KO these inclusions
are limited to muscles rich in glycolytic fast fibers and appear
to be responsible for the disappointing response to enzyme
replacement therapy (ERT) with recombinant human enzyme
Genzyme Corp. Framingham, MA) has recently become avail-
able for ERT in Pompe patients.
To determine if glycogen reaches the lysosome via autop-
hagy and to establish if autophagic buildup in Pompe disease
is a consequence of an induction of autophagy and/or impaired
resolution of autophagosomes due to defective fusion with lyso-
somes, we have generated muscle-specific autophagy-deficient
Pompe mice. We have found that the transport of glycogen
from the cytoplasm to lysosomes does not require autophagy.
We also demonstrated that autophagy is upregulated in both
slow (type I) and fast (type II) fibers in the GAA KO, but
only in fast fibers is there also inefficient disposal of autophagic
cargo and the accumulation of ubiquitin-positive bodies, puta-
tive mediators of muscle damage. In addition, suppression of
autophagy in the wild-type (WT) control mice allowed us to
explore the role of this pathway in normal muscle, of which
little is yet known. Here we demonstrate that the consequences
of loss of autophagy in normal muscle are dependent on fiber
type, indicating that autophagy fulfills different purposes in
the diverse fiber types.
Generation of skeletal muscle-specific autophagy-deficient
GAA KO mice
We generated muscle-specific autophagy-deficient mice on the
GAA KO background (AD-GAA KO); these mice contain a
Cre recombinase transgene under the control of the human
skeletal actin promoter (HSA-Cre) and an Atg5 gene, in
which exon 3 is flanked by loxP sites (Atg5flox/flox). The
HSA promoter drives the expression of Cre recombinase in
both fast and slow muscle fibers resulting in muscle-specific
inactivation of Atg5, a critical gene in autophagosome for-
mation (14). The need for tissue-specific suppression of auto-
phagy is justified by the fact that mice with global deletion of
autophagic genes (Atg5 or Atg7) die soon after birth (15,16).
Of note is the fact that in the GAA KO mice, the GAA gene,
which is disrupted by a neo cassette in exon 6 (6neo/ 6neo),
contains two loxP sites in introns 5 and 6 (17). The expression
of Cre recombinase would therefore remove exon 6 (D6) and
the neo from the GAA gene in skeletal muscle. We have
previouslydemonstrated that in homozygous D6/D6mice,patho-
logical and biochemical changes in skeletal muscle are indistin-
guishable from those in the 6neo/6neomice (17). Nevertheless,
to obtain the most adequate control we have generated GAA
KO mice, in which Cre recombinase, driven by the HSA promo-
ter, is expressed in the muscle, resulting in the removal of exon 6
ground (AD-WT)servedasadditional controls; thedataconcern-
ing the AD-WT mice will be discussed in a separate section.
The suppression of autophagy in the skeletal muscle was
evaluated by the level of a specific autophagosomal marker,
LC3-II. The microtubule-associated protein 1 light chain 3
(LC3), the mammalian homologue of the yeast autophagoso-
mal marker, Atg8, exists as a soluble form, LC3-I, which is
modified into the PE (phosphatidylethanolamine)-conjugated
form LC3-II (18). Conversion of the soluble LC3-I to the
membrane associated LC3-II form is an Atg5-dependent
process (19,20). In AD-GAA KO only the LC3-I form is
present, indicating complete suppression of autophagy in the
skeletal muscle in these mice (Fig. 1A).
Suppression of autophagy in the skeletal muscle does not
significantly affect the level of glycogen accumulation,
but exacerbates the phenotype of the GAA KO mice
In the WT, a negligible amount of glycogen was detected in
skeletal muscle by a biochemical assay, 0.26+0.30% wet
weight (n ¼ 17). The levels of accumulated glycogen were
comparable in fast (white gastrocnemius) muscle from 4- to
7-month-old AD-GAA KO and GAA KO; 4.0+2.6% wet
weight (n ¼ 34) and 5.0+2.8% (n ¼ 36), respectively. The
same was true of slow (soleus) muscle; AD-GAA KO 9.7+
2.9%wetweight(n ¼ 9),GAAKO7.8+2.8%(n ¼ 8).Consist-
ent with these data, histological examination of muscle samples
from AD-GAA KO mice revealed multiple PAS-positive struc-
tures similar to those observed in GAA KO mice, indicating
that inactivation of autophagy does not prevent lysosomal
accumulation of glycogen (Fig. 1 B, shown for fast muscle).
Despite the similar level of glycogen accumulation in GAA
KO and AD-GAA KO, the AD-GAA KO develop a more
severe phenotype (Fig. 1B). Like GAA KO, AD-GAA KO are
born normal, but at the age of 2–3 months they already show
By the age of 4–5 months the AD-GAA KO mice exhibit severe
muscle wasting, profound kyphosis, a waddling gait and growth
retardation. The symptoms progress rapidly, and by the age of
6–7 months these mice breathe with difficulty, drag their hind
limbs in a splayed fashion while walking, cannot lift their
bellies off the ground, lose tail strength, and develop a near
paralysis of hind limbs. At this stage the AD-GAA KO mice
require daily observation and many begin to die. By 8–10
months, the surviving mice are euthanized to prevent suffering.
In contrast, the GAA KO mice remained phenotypically normal
up to 8–9 months of age, after which point they developthe first
clinical signs of muscle wasting consistent with the results
reported previously for the GAA 6neo/6neo(17).In the wire-hang
3898Human Molecular Genetics, 2008, Vol. 17, No. 24
disease: recombinant and transgenic enzymes are equipotent, but neither
completely clears glycogen from type II muscle fibers. Mol. Ther., 11,
14. Mizushima, N., Ohsumi, Y. and Yoshimori, T. (2002) Autophagosome
formation in mammalian cells. Cell Struct. Funct., 27, 421–429.
15. Kuma, A., Hatano, M., Matsui, M., Yamamoto, A., Nakaya, H.,
Yoshimori, T., Ohsumi, Y., Tokuhisa, T. and Mizushima, N. (2004) The
role of autophagy during the early neonatal starvation period. Nature, 432,
16. Komatsu, M., Waguri, S., Ueno, T., Iwata, J., Murata, S., Tanida, I.,
Ezaki, J., Mizushima, N., Ohsumi, Y., Uchiyama, Y. et al. (2005)
Impairment of starvation-induced and constitutive autophagy in
Atg7-deficient mice. J. Cell Biol., 169, 425–434.
King, C., Ward, J., Sauer, B. et al.(1998) Targeted disruption of the acid
alpha-glucosidase gene in mice causes an illness with critical features of both
infantile and adult human glycogen storage disease type II. J. Biol. Chem.,
18. Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T.,
Kominami, E., Ohsumi, Y. and Yoshimori, T. (2000) LC3, a mammalian
homologue of yeast Apg8p, is localized in autophagosome membranes after
processing. EMBO J., 19, 5720–5728.
19. Tanida, I., Ueno, T. and Kominami, E. (2004) LC3 conjugation system in
mammalian autophagy. Int. J. Biochem. Cell Biol., 36, 2503–2518.
20. Mizushima, N., Yamamoto, A., Hatano, M., Kobayashi, Y., Kabeya, Y.,
Suzuki, K., Tokuhisa, T., Ohsumi, Y. and Yoshimori, T. (2001) Dissection
of autophagosome formation using Apg5-deficient mouse embryonic stem
cells. J. Cell Biol., 152, 657–668.
21. Gomes, M.D., Lecker, S.H., Jagoe, R.T., Navon, A. and Goldberg, A.L.
(2001) Atrogin-1, a muscle-specific F-box protein highly expressed during
muscle atrophy. Proc. Natl Acad. Sci. USA, 98, 14440–14445.
22. Lecker, S.H., Jagoe, R.T., Gilbert, A., Gomes, M., Baracos, V., Bailey, J.,
Price, S.R., Mitch, W.E. and Goldberg, A.L. (2004) Multiple types of
skeletal muscle atrophy involve a common program of changes in gene
expression. FASEB J., 18, 39–51.
23. Komatsu, M., Waguri, S., Chiba, T., Murata, S., Iwata, J., Tanida, I.,
Ueno, T., Koike, M., Uchiyama, Y., Kominami, E. et al.(2006) Loss of
autophagy in the central nervous system causes neurodegeneration in
mice. Nature, 441, 880–884.
24. Hara, T., Nakamura, K., Matsui, M., Yamamoto, A., Nakahara, Y.,
Suzuki-Migishima, R., Yokoyama, M., Mishima, K., Saito, I., Okano, H.
et al. (2006) Suppression of basal autophagy in neural cells causes
neurodegenerative disease in mice. Nature, 441, 885–889.
25. Pankiv, S., Clausen, T.H., Lamark, T., Brech, A., Bruun, J.A., Outzen, H.,
Overvatn, A., Bjorkoy, G. and Johansen, T. (2007) p62/SQSTM1 binds
directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein
aggregates by autophagy. J. Biol. Chem., 282, 24131–24145.
26. Bjorkoy, G., Lamark, T., Brech, A., Outzen, H., Perander, M., Overvatn, A.,
degraded by autophagy and has a protective effect on huntingtin-induced cell
death. J. Cell Biol., 171, 603–614.
27. Ichimura, Y., Kumanomidou, T., Sou, Y.S., Mizushima, T., Ezaki, J.,
Ueno, T., Kominami, E., Yamane, T., Tanaka, K. and Komatsu, M. (2008)
Structural basis for sorting mechanism of p62 in selective autophagy.
J. Biol. Chem., 283, 22847–22857.
28. Fass, E., Shvets, E., Degani, I., Hirschberg, K. and Elazar, Z. (2006)
Microtubules support production of starvation-induced autophagosomes
but not their targeting and fusion with lysosomes. J. Biol. Chem., 281,
29. Kochl, R., Hu, X.W., Chan, E.Y. and Tooze, S.A. (2006) Microtubules
facilitate autophagosome formation and fusion of autophagosomes with
endosomes. Traffic, 7, 129–145.
30. Jahreiss, L., Menzies, F.M. and Rubinsztein, D.C. (2008) The itinerary of
autophagosomes: from peripheral formation to kiss-and-run fusion with
lysosomes. Traffic, 9, 574–587.
31. Kotoulas, O.B., Kalamidas, S.A. and Kondomerkos, D.J. (2006) Glycogen
autophagy in glucose homeostasis. Pathol. Res. Pract., 202, 631–638.
32. Schiaffino, S., Mammucari, C. and Sandri, M. (2008) The role of
autophagy in neonatal tissues: just a response to amino acid starvation?
Autophagy, 4, 727–730.
33. Kotoulas, O.B., Kalamidas, S.A. and Kondomerkos, D.J. (2004) Glycogen
autophagy. Microsc. Res. Tech., 64, 10–20.
34. Kondomerkos, D.J., Kalamidas, S.A. and Kotoulas, O.B. (2004) An
electron microscopic and biochemical study of the effects of glucagon on
glycogen autophagy in the liver and heart of newborn rats. Microsc. Res.
Tech., 63, 87–93.
35. Kondomerkos, D.J., Kalamidas, S.A., Kotoulas, O.B. and Hann, A.C.
(2005) Glycogen autophagy in the liver and heart of newborn rats. The
effects of glucagon, adrenalin or rapamycin. Histol. Histopathol., 20,
36. Schiaffino, S. and Hanzlikova, V. (1972) Autophagic degradation of
glycogen in skeletal muscles of the newborn rat. J. Cell Biol., 52, 41–51.
37. Kishnani, P.S., Nicolino, M., Voit, T., Rogers, R.C., Tsai, A.C., Waterson,
J., Herman, G.E., Amalfitano, A., Thurberg, B.L., Richards, S. et al. (2006)
Chinese hamster ovary cell-derived recombinant human acid
alpha-glucosidase in infantile-onset Pompe disease. J. Pediatr., 149, 89–97.
38. Griffin, J.L. (1984) Infantile acid maltase deficiency. I. Muscle fiber
destruction after lysosomal rupture. Virchows Arch. Cell Pathol. Incl.
Mol. Pathol., 45, 23–36.
39. Thurberg, B.L., Lynch, M.C., Vaccaro, C., Afonso, K., Tsai, A.C.,
Bossen, E., Kishnani, P.S. and O’Callaghan, M. (2006) Characterization
of pre- and post-treatment pathology after enzyme replacement therapy
for pompe disease. Lab. Invest., 86, 1208–1220.
40. Bijvoet, A.G., Van Hirtum, H., Vermey, M., Van Leenen, D., Van der Ploeg,
A.T., Mooi, W.J. and Reuser, A.J. (1999) Pathological features of glycogen
storage disease type II highlighted in the knockout mouse model. J. Pathol.,
41. Hesselink, R.P., Wagenmakers, A.J., Drost, M.R. and van der Vusse, G.J.
(2003) Lysosomal dysfunction in muscle with special reference to
glycogen storage disease type II. Biochim. Biophys. Acta, 1637, 164–170.
42. Drost, M.R., Hesselink, R.P., Oomens, C.W. and van der Vusse, G.J.
(2005) Effects of non-contractile inclusions on mechanical performance
of skeletal muscle. J. Biomech., 38, 1035–1043.
43. Fukuda, T., Ewan, L., Bauer, M., Mattaliano, R.J., Zaal, K., Ralston, E.,
Plotz, P.H. and Raben, N. (2006) Dysfunction of endocytic and autophagic
pathways in a lysosomal storage disease. Ann. Neurol., 59, 700–708.
44. Fukuda, T., Roberts, A., Plotz, P.H. and Raben, N. (2007) Acid
alpha-glucosidase deficiency (Pompe disease). Curr. Neurol. Neurosci.
Rep., 7, 71–77.
45. Klionsky, D.J., Abeliovich, H., Agostinis, P., Agrawal, D.K., Aliev, G.,
Askew, D.S., Baba, M., Baehrecke, E.H., Bahr, B.A., Ballabio, A. et al.
(2008) Guidelines for the use and interpretation of assays for monitoring
autophagy in higher eukaryotes. Autophagy, 4, 151–175.
46. Komatsu, M., Waguri, S., Koike, M., Sou, Y.S., Ueno, T., Hara, T.,
Mizushima, N., Iwata, J., Ezaki, J., Murata, S. et al. (2007) Homeostatic
levels of p62 control cytoplasmic inclusion body formation in
autophagy-deficient mice. Cell, 131, 1149–1163.
47. Yoshimori, T. (2004) Autophagy: a regulated bulk degradation process
inside cells. Biochem. Biophys. Res. Commun., 313, 453–458.
48. Klionsky, D.J. (2005) The molecular machinery of autophagy:
unanswered questions. J. Cell Sci., 118, 7–18.
49. Marmor, M.D. and Yarden, Y. (2004) Role of protein ubiquitylation in
regulating endocytosis of receptor tyrosine kinases. Oncogene, 23,
50. Dupre, S., Urban-Grimal, D. and Haguenauer-Tsapis, R. (2004) Ubiquitin
and endocytic internalization in yeast and animal cells. Biochim. Biophys.
Acta, 1695, 89–111.
51. Barriere, H., Nemes, C., Du, K. and Lukacs, G.L. (2007) Plasticity of
polyubiquitin recognition as lysosomal targeting signals by the endosomal
sorting machinery. Mol. Biol. Cell, 18, 3952–3965.
52. Bodine, S.C., Latres, E., Baumhueter, S., Lai, V.K., Nunez, L., Clarke, B.A.,
Poueymirou, W.T., Panaro, F.J., Na, E., Dharmarajan, K. et al. (2001)
Identification of ubiquitin ligases required for skeletal muscle atrophy.
Science, 294, 1704–1708.
53. Jagoe, R.T., Lecker, S.H., Gomes, M. and Goldberg, A.L. (2002) Patterns
of gene expression in atrophying skeletal muscles: response to food
deprivation. FASEB J., 16, 1697–1712.
54. Sandri, M., Sandri, C., Gilbert, A., Skurk, C., Calabria, E., Picard, A.,
Walsh, K., Schiaffino, S., Lecker, S.H. and Goldberg, A.L. (2004) Foxo
transcription factors induce the atrophy-related ubiquitin ligase atrogin-1
and cause skeletal muscle atrophy. Cell, 117, 399–412.
55. Gomes, M.D., Lecker, S.H., Jagoe, R.T., Navon, A. and Goldberg
A.L. (2001) Atrogin-1, a muscle-specific F-box protein highly
expressed during muscle atrophy. Proc. Natl Acad. Sci. USA, 98,
Human Molecular Genetics, 2008, Vol. 17, No. 243907
56. Mammucari, C., Schiaffino, S. and Sandri, M. (2008) Downstream of Akt:
FoxO3 and mTOR in the regulation of autophagy in skeletal muscle.
Autophagy, 4, 524–526.
57. Askanas, V. and Engel, W.K. (2002) Inclusion-body myositis and
myopathies: different etiologies, possibly similar pathogenic mechanisms.
Curr. Opin. Neurol., 15, 525–531.
58. Weihl, C.C., Miller, S.E., Hanson, P.I. and Pestronk, A. (2007) Transgenic
expression of inclusion body myopathy associated mutant p97/VCP
causes weakness and ubiquitinated protein inclusions in mice. Hum. Mol.
Genet., 16, 919–928.
59. Miniou, P., Tiziano, D., Frugier, T., Roblot, N., Le Meur, M. and Melki, J.
(1999) Gene targeting restricted to mouse striated muscle lineage. Nucleic
Acids Res., 27, e27.
between muscle fiber types and sizes and muscle architectural properties in
the mouse hindlimb. J. Morphol., 221, 177–190.
61. Hawes, M.L., Kennedy, W., O’Callaghan, M.W. and Thurberg, B.L.
(2007) Differential muscular glycogen clearance after enzyme
replacement therapy in a mouse model of Pompe disease. Mol. Genet.
Metab., 91, 343–351.
3908 Human Molecular Genetics, 2008, Vol. 17, No. 24