Impaired autophagy in sporadic inclusion-body myositis and in endoplasmic reticulum stress-provoked cultured human muscle fibers.

Abstract

The hallmark pathologies of sporadic inclusion-body myositis (s-IBM) muscle fibers are autophagic vacuoles and accumulation of ubiquitin-positive multiprotein aggregates that contain amyloid-beta or phosphorylated tau in a beta-pleated sheet amyloid configuration. Endoplasmic reticulum stress (ERS) and 26S proteasome inhibition, also associated with s-IBM, putatively aggrandize the accumulation of misfolded proteins. However, autophagosomal-lysosomal pathway formation and function, indicated by autophagosome maturation, have not been previously analyzed in this system. Here we studied the autophagosomal-lysosomal pathway using 14 s-IBM and 30 disease control and normal control muscle biopsy samples and our cultured human muscle fibers in a microenvironment modified to resemble aspects of s-IBM pathology. We report for the first time that in s-IBM, lysosomal enzyme activities of cathepsin D and B were decreased 60% (P < 0.01) and 40% (P < 0.05), respectively. We also detected two indicators of increased autophagosome maturation, the presence of LC3-II and decreased mammalian target of rapamycin-mediated phosphorylation of p70S6 kinase. Moreover, in cultured human muscle fibers, ERS induction significantly decreased activities of cathepsins D and B, increased levels of LC3-II, decreased phosphorylation of p70S6 kinase, and decreased expression of VMA21, a chaperone for assembly of lysosomal V-ATPase. We conclude that in s-IBM muscle, decreased lysosomal proteolytic activity might enhance accumulation of misfolded proteins, despite increased maturation of autophagosomes, and that ERS is a possible cause of s-IBM-impaired lysosomal function. Thus, unblocking protein degradation in s-IBM muscle fibers may be a desirable therapeutic strategy.

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Musculoskeletal Pathology
Impaired Autophagy in Sporadic Inclusion-Body Myositis
and in Endoplasmic Reticulum Stress-Provoked Cultured
Human Muscle Fibers
Anna Nogalska, Carla D’Agostino,
Chiara Terracciano, W. King Engel,
and Valerie Askanas
From the USC Neuromuscular Center, Department of Neurology,
University of Southern California Keck School of Medicine, Good
Samaritan Hospital, Los Angeles, California
The hallmark pathologies of sporadic inclusion-body
myositis (s-IBM) muscle fibers are autophagic vacuoles
and accumulation of ubiquitin-positive multiprotein ag-
gregates that contain amyloid-? or phosphorylated tau
in a ?-pleated sheet amyloid configuration. Endoplas-
mic reticulum stress (ERS) and 26S proteasome inhibi-
tion, also associated with s-IBM, putatively aggrandize
the accumulation of misfolded proteins. However, au-
tophagosomal-lysosomal pathway formation and func-
tion, indicated by autophagosome maturation, have not
been previously analyzed in this system. Here we stud-
ied the autophagosomal-lysosomal pathway using 14
s-IBMand30diseasecontrolandnormalcontrolmuscle
biopsy samples and our cultured human muscle fibers
in a microenvironment modified to resemble aspects of
s-IBM pathology. We report for the first time that in
s-IBM, lysosomal enzyme activities of cathepsin D and B
were decreased 60% (P < 0.01) and 40% (P < 0.05),
respectively. We also detected two indicators of in-
creased autophagosome maturation, the presence of
LC3-II and decreased mammalian target of rapamycin-
mediated phosphorylation of p70S6 kinase. Moreover,
in cultured human muscle fibers, ERS induction signif-
icantly decreased activities of cathepsins D and B, in-
creased levels of LC3-II, decreased phosphorylation of
p70S6 kinase, and decreased expression of VMA21, a
chaperoneforassemblyoflysosomalV-ATPase.Wecon-
clude that in s-IBM muscle, decreased lysosomal proteo-
lytic activity might enhance accumulation of misfolded
proteins, despite increased maturation of autophago-
somes, and that ERS is a possible cause of s-IBM-im-
paired lysosomal function. Thus, unblocking protein
degradation in s-IBM muscle fibers may be a desirable
therapeutic strategy.
DOI: 10.2353/ajpath.2010.100050)
(Am J Pathol 2010, 177:1377–1387;
Sporadic inclusion-body myositis (s-IBM) is the most
common progressive muscle disease of older persons
(reviewed in 1, 2). It leads to severe disability, and there
is no enduring treatment currently available.1,2
The characteristic pathologies of s-IBM muscle fibers
are autophagic vacuoles, accumulation of ubiquitin-pos-
itive multiprotein aggregates containing misfolded pro-
teins in the ?-pleated sheet conformation of amyloid and
mononuclear cell inflammation (recently reviewed in 3, 4).
In addition to their vacuolization, s-IBM muscle fibers have
other intriguing features, such as their phenotypic similari-
tiestoAlzheimer’sdiseaseandParkinson’sdiseasebrains.3
Several proteins accumulated in Alzheimer’s and Parkin-
son’s disease brains are accumulated within s-IBM muscle
fibers, such as aggregated amyloid-? (A?), phosphorylated
tau occurring in paired helical filaments, ?-synuclein, and
parkin.3,4In addition, similarly to Alzheimer’s and Parkin-
son’s disease brains,5–7endoplasmic reticulum stress, and
oxidative stress, inhibition of the 26S proteasome and mito-
chondrial abnormalities are important aspects of the s-IBM
pathology.8–12
Vacuoles in apparently living s-IBM muscle fibers are
considered autophagic, because they often contain i)
lysosomal membranous debris by light and electron mi-
croscopy, which is considered a result of indigestibility of
normally turned-over or pathologically damaged cellular
proteins and organelles, ii) acid phosphatase positivity,
Supported by grants from the National Institutes of Health (AG 16768
Merit Award), the Muscular Dystrophy Association, and the Myositis As-
sociation and the Helen Lewis Research Fund (to V.A.).
Accepted for publication April 28, 2010.
None of the authors declare any relevant financial relationships.
A.N. is on leave from Department of Biochemistry, Medical University of
Gdansk, Gdansk, Poland. C.T. was on leave from Department of Neuro-
science, University of Tor Vergata and Fondazione S. Lucia, Rome, Italy.
Address reprint requests to Valerie Askanas, M.D., Ph.D., USC Neuro-
muscular Center, Good Samaritan Hospital, 637 S. Lucas Ave., Los
Angeles, CA 90017-1912. E-mail: askanas@usc.edu.
The American Journal of Pathology, Vol. 177, No. 3, September 2010
Copyright © American Society for Investigative Pathology
DOI: 10.2353/ajpath.2010.100050
1377

Page 2

and iii) increased immunoreactivity of some lysosomal
enzymes (13, 14 also reviewed in 1, 4). Although the
pathogenesis of s-IBM seems complex and multifactori-
al,1–4accumulation of the nondegraded, misfolded pro-
teins and putatively toxic oligomers has been proposed
to be an important aspect.4
Recently, increased synthesis and accumulation of p62/
SQSTM1, a ubiquitin-binding protein allegedly shuttling
polyubiquitinated proteins for their degradation by both pro-
teasome and lysosomes,15,16have been demonstrated in i)
s-IBM muscle fibers17and ii) cultured human muscle fibers
in which activity of either the lysosomes or proteasome was
experimentally inhibited.17Increased accumulation of p62/
SQSTM1 in s-IBM muscle fibers also indicated an inade-
quate protein disposal system.
The two major pathways of cellular protein degradation
relate to the 26S proteasome and the autophagic/lysoso-
mal systems.18The 26S proteasome, also called the
ubiquitin-proteasome system, is a major degradation
mechanism for i) normal regulatory and other short-lived
proteins and ii) misfolded proteins exported from the
endoplasmic reticulum (ER) through a ubiquitin-medi-
ated ATP-independent process.6,18,19However, long-
lived, structural proteins and/or damaged or misfolded
proteins and obsolescent cellular organelles are de-
graded through “autophagy.”20–23“Macroautophagy” re-
fers to formation and maturation of autophagosomes,
which are structures carrying proteins and organelles
destined for lysosomal degradation. After an autophago-
some fuses with the lysosomal membrane, it disposes its
cargo into the lysosome, where it is then degraded by the
lysosomal enzymes.20–23In normal cells, the correctly
functioning ubiquitin-proteasome system and the auto-
phagosomal-lysosomal pathways (ALP) assure proper
control of protein quality and quantity. Abnormal accu-
mulation within a cell of misfolded or damaged proteins,
which might occur due either to their excessive produc-
tion, proteasome inhibition, oxidative stress, and/or other
stressors, increases the need for autophagic degrada-
tion, which is accompanied by increased formation and
maturation of autophagosomes.18,21However, when ly-
sosomal degradation is impaired or the amount of mate-
rial to be degraded exceeds lysosomal capability, forma-
tion of autophagosomes dramatically increases, leading
to formation of autophagic vacuoles.23In mammalian
cells, the presence on immunoblots of LC3-II protein, a
lipidated form of the autophagosomal protein LC3 (micro-
tubule-associated protein light chain 3B) is considered
the most important indicator of increased formation and
maturation of autophagosomes.21,23,24
In contrast to several neurodegenerative diseases in
which ALP functions have been extensively studied,20–23,25
ALP function in s-IBM muscle fibers is virtually unex-
plored. In the present article, we demonstrate for the first
time that i) in s-IBM muscle, increased proliferation and
maturation of autophagosomes, as indicated by the pres-
ence of LC3-II protein and a decreased ratio of phos-
phorylated p70S6 kinase to total p70S6 kinase, is asso-
ciated with impaired activities of the two major lysosomal
enzymes, cathepsin D and B and ii) in cultured human
muscle fibers, experimentally induced ER stress leads to
impaired lysosomal enzyme activities, suggesting that ER
stress, perhaps among other factors, might contribute to
malfunction of the ALP in patients with s-IBM.
Materials and Methods
Muscle Biopsy Samples
Studies were performed on fresh-frozen diagnostic mus-
cle biopsy samples obtained, with informed consent,
from 14 patients with s-IBM, 6 with polymyositis (PM), 1
with dermatomyositis, 1 with morphologically nonspecific
myopathy, 2 with amyotrophic lateral sclerosis, and 2 with
peripheral neuropathy, and on 18 control muscle biopsy
samples from patients who, after all tests were per-
formed, were considered free of muscle disease. Patients
with s-IBM were aged 46 to 79 years (median 68 years);
control patients for s-IBM were aged 49 to 84 years
(median 63 years). (Not all studies were performed on all
biopsy samples [details below].)
Patient diagnoses were based on clinical and labora-
tory investigations, including our routinely performed 16-
reaction diagnostic histochemical analysis of the muscle
biopsy samples. All s-IBM biopsy samples met s-IBM
diagnostic criteria.1,3Clinically all of our patients with
s-IBM had very weak and atrophic quadriceps muscle
(making them unlike patients with GNE mutation hereditary
inclusion-body myopathy), and none had a positive family
history for any neuromuscular disorder. In addition, none
had any family members with Paget’s disease or frontotem-
poral dementia, which often occur in patients with valosin-
containing protein (VCP) mutations. Pathologically, all of our
patients with s-IBM had various-sized foci of mononuclear
cell inflammation in their muscle biopsy samples, and they
did not have foci of myofibrillar disorganization (as occur in
myofibrillar myopathy). Accordingly, we are confident that
none of our patients had myofibrillar myopathy or hereditary
inclusion-body myopathy.
Light Microscopic Immunocytochemistry
Immunofluorescence detection was performed, as de-
scribed previously8–10,17on 10-?m-thick transverse sec-
tions of four s-IBM, four age-matched-control, two poly-
myositis, and six other disease control muscle biopsy
samples, as specified above. The sources and dilutions
of antibodies used are listed in Table 1. Incubations in
each antibody were performed overnight at 4°C. For dou-
ble immunofluorescence an antibody against LC3 (which
recognizes both LC3-I and LC3-II) combined with an
antibody against p62 was used (Table 1), as described
previously for immuno-colocalizing other proteins.8–10,17
To block nonspecific binding of an antibody to Fc recep-
tors, sections were preincubated with normal goat serum
diluted 1:10.8–10,17Controls for staining specificity were i)
omission of the primary antibody or ii) its replacement
with nonimmune sera or irrelevant antibody. Results for
these were always negative.
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Gold Immunoelectron Microscopy
This was performed on adhered to the bottom of 35-mm Petri
dishes10?mfresh-frozenbiopsysectionsfromthreedifferent
patients with s-IBM, as described.8–11,17In brief, sections
were prefixed in 2% paraformaldehyde for 2 minutes and
preincubated with 10% normal goat serum, followed by
incubation with mouse monoclonal antibody against
LC3 used in combination with rabbit polyclonal anti-
body against p62. After incubation with appropriate sec-
ondary antibodies conjugated to 6- and 12-nm gold par-
ticles (Jackson ImmunoResearch Laboratories Inc., West
Grove, PA), sections were processed for electron
microscopy.8–11,17
Immunoblots
Immunoblots were performed on muscle biopsy samples
from 7 patients with s-IBM, 6 patients with polymyositis,
and 10 control patients as recently detailed.8–11,17
Twenty to 50 ?g of protein was loaded into the gel and
electrophoretically separated. After electrophoresis, sam-
ples were transferred to a nitrocellulose membrane. All re-
agents were obtained from Invitrogen (Carlsbad, CA). To
prevent nonspecific binding of antibodies, the nitrocellulose
membranes were blocked in blocking reagent (Invitrogen);
they were then incubated overnight at 4°C with an appro-
priate primary antibody (Table 1). Blots were developed
using Western Breeze chemiluminescent kits (Invitrogen).
Protein loading was evaluated by the actin band (Santa
Cruz Biotechnology, Santa Cruz, CA). Quantification of im-
munoreactivity was performed by densitometric analysis
using NIH ImageJ 1.310 software.
Combined Immunoprecipitation/Immunoblot
Procedure
To evaluate whether LC3-I/LC3-II and p62 physically as-
sociate in s-IBM muscle fibers, a combined immunopre-
cipitation/immunoblot technique was used, as detailed
previously.8,9,26In brief, 150 ?g of total muscle protein
were immunoprecipitated in precipitation buffer contain-
ing 10 ?g of anti-p62 antibody (Table 1). The immuno-
precipitated complexes, containing IgG antibody along
with its bound target antigen and all proteins bound to
that antigen, were pulled down using Protein G Sepha-
rose 4 Fast Flow (GE Healthcare, Piscataway, NJ) during
4 hours of incubation at 4°C. The solution was then cen-
trifuged for 5 minutes (16,000 ? g at 4°C), and the su-
pernatant was removed. The precipitated immunocom-
plexes were washed five times with the precipitation
buffer by centrifuging for 5 minutes each (16,000 ? g at
4°C). Immunoprecipitates were then electrophoresed
and immunoprobed with the anti-LC3 antibody (which
recognizes both LC3-I and LC3-II), followed by an appro-
priate secondary antibody, and developed using the
Western Breeze anti-rabbit chemiluminescence kit (In-
vitrogen). To confirm specificity of the physical associa-
tion identified by the immunoprecipitation-immunoblot re-
action, the primary antibody was omitted from the
immunoprecipitation solution. To confirm the identity of
the immunoprecipitate, the immunoprecipitate was also
probed with the anti-p62 antibody.
Measurement of Cathepsin D and B Activities
Cathepsin D (EC 3.4.23.5) activity was measured using a
Cathepsin D Assay Kit (Sigma-Aldrich, St. Louis, MO)
according to the manufacturer’s instructions. In brief, 10
10-?m-thick fresh-frozen sections of 5 s-IBM, 4 PM,
and 13 control muscle biopsy samples were individu-
ally homogenized in PBS with addition of 0.05% 3-[(3-
cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid (CHAPS) (Sigma-Aldrich). The reaction mixture con-
tained a cathepsin D assay buffer, muscle extracts (30
?g of protein from each biopsy), and an internally
quenched fluorescent substrate (included in the kit). Be-
cause other enzymatic activities might possibly cleave
the substrate, the samples were also measured in the
presence of the specific inhibitor pepstatin A to inhibit
Table 1.
Antibodies: Their Dilution and Sources
Name SpecificitySource Host ApplicationDilution
LC3N terminus of LC3-B of human origin NanoTools/Axxora, LLC
(San Diego, CA)
Novus Biologicals
(Littleton, CO)
Cell Signaling
(Danvers, MA)
Cell Signaling
Santa Cruz Biotechnology
(Santa Cruz, CA)
Santa Cruz Biotechnology
Mouse IHC, EM1:100
LC3 N-terminal portion of human LC3
protein
aa surrounding Thr389 of human
p70S6K
C terminus of p70S6K of human origin
aa 151-440 of SQSTM1 of human
origin
aa 151-440 of SQSTM1 of human
origin
C terminus of cathepsin D of human
origin
aa 1-339 representing full-length
cathepsin B of human origin
LOC203547 of human origin
C terminus of actin of human origin
RabbitIB1:500
Phosphorylated-p70S6
kinase
p70S6 kinase
p62/SQSTM1
RabbitIB1:500
Rabbit
Mouse
IB
IB, IP
1:100
1:100
p62/SQSTM1 Rabbit IHC, EM1:100
Cathepsin D (C-20)Santa Cruz BiotechnologyGoat IB1:5000
Cathepsin B (FL-339)Santa Cruz Biotechnology Rabbit IB1:100
VMA21 (M-22)
Actin (C-2)
Santa Cruz Biotechnology
Santa Cruz Biotechnology
Rabbit
Mouse
IB
IB
1:100
1:500
IHC, immunohistochemistry; EM, electron microscopy; IB, immunoblotting; aa, amino acids; IP, immunoprecipitation.
Impaired Autophagy in s-IBM
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cathepsin D activity. The activity in the presence of pep-
statin A (equivalent to the non-cathepsin D activity in the
samples) was subtracted from the total activity measured
to obtain cathepsin D activity. The positive control was
purified cathepsin D from bovine spleen (Sigma-Aldrich
kit) used instead of our muscle homogenates; and the
negative control was that reaction with pepstatin A. All
samples were measured in duplicate. They were incu-
bated at 37°C, and the fluorescence emission excited at
355 nm was recorded at 444 nm every 2 minutes. Activity
of cathepsin D measured in the linear phase of the reac-
tion was expressed as fluorescence intensity per minute
and was normalized to the amount of cathepsin D protein
in the same homogenate as measured by immunoblotting
and expressed per actin (actin was used as the protein
loading reference).
Cathepsin B (EC 3.4.22.1) activity was measured as
described.27Ten 10-?m-thick fresh-frozen sections of 3
s-IBM, 6 PM, and 11 control muscle biopsy samples were
homogenized in the PBS with addition of 0.05% CHAPS.
The reaction mixture contained cathepsin B assay buffer
(0.1 mol/L 4-morpholineethanesulfonic acid, 1 mmol/L
EDTA, and 2 mmol/L dithiothreitol, pH 6.0), muscle ex-
tracts (50 ?g of protein from each biopsy), and 1 mmol/L
cysteine. The reaction was initiated by addition of the
specific synthetic substrate Z-ARG-ARG-AMC (1 ?mol/
L). Because other enzymatic activities might cleave the
substrate, the specific inhibitor CA-074 (10 ?mol/L) (Enzo
Life Sciences, Plymouth Meeting, PA) was added to the
samples to inhibit cathepsin B activity. The activity in the
presence of CA-074 (equivalent to the non-cathepsin B
activity present in the samples) was subtracted from the
total activity measured. Samples were measured in du-
plicate. All samples were incubated for 30 minutes at
37°C, and the fluorescence emission excited at 355 nm
was recorded at 444 nm. The positive control was a
purified cathepsin B from bovine spleen used instead of
the muscle homogenates, and the negative control was
the reaction with CA-074. Activity of cathepsin B was
expressed as arbitrary fluorescence units. Because ca-
thepsin B protein was not studied in the same biopsy
homogenate as activity, we do not express cathepsin B
activity per its protein.
All reagents used for this assay were purchased from
Sigma-Aldrich, unless indicated otherwise.
Cultured Human Muscle Fibers
Primary cultures of normal human muscle were estab-
lished as routinely performed in our laboratory,28from
archived satellite cells of portions of diagnostic muscle
biopsy samples from patients who, after all tests had
been performed, were considered free of muscle dis-
ease. Each experiment was performed on at least five
different culture sets, each established from satellite cells
derived from a different muscle biopsy sample. Twelve to
18 days after fusion of myoblasts, well differentiated myo-
tubes were exposed for 24 hours to either i) an estab-
lished ER stress inducer tunicamycin, which is an N-
glycosylation inhibitor29(4 ?g/ml; Sigma-Aldrich) that in
our previous studies was shown to induce ER stress and
its consequences,10,30,31ii) an irreversible proteasome
inhibitor epoxomicin8,26,32(1 ?mol/L; BIOMOL Research
Laboratories, Plymouth Meeting, PA), iii) bafilomycin A1,
an inhibitor of lysosomal V-ATPase, causing an in-
crease of lysosomal pH33and inhibiting activities of
cathepsins (25 nmol/L; Sigma-Aldrich), or iv) chloro-
quine (20 ?mol/L; Sigma-Aldrich) for 24 and 48 hours,
a lysosomotropic agent raising lysosomal pH33and
inhibiting activities of cathepsins. After these various
treatments, cultures were processed for immunoblots as
described previously9,10,26,30or measurement of enzy-
matic activities as above for muscle biopsy samples. In
each experiment, treated cultures were compared with
their untreated sister control cultures.
Statistical Analysis
Statistical significance was determined by Student’s t-
test. The level of significance was set at P ? 0.05. Data
are presented as means ? SEM.
Results
Macroautophagy is Activated in s-IBM Muscle
Fibers
Morphology
Light microscopic immunohistochemistry was used to
evaluate the distribution and intracellular localization of
LC3-I/LC3-II protein in s-IBM and control muscle fibers. In
normal human muscle biopsy samples there was no de-
tectable LC3-I/LC3-II immunoreactivity, except for occa-
sional slight sarcolemmal staining on a rare muscle fiber
(Figure 1A). In contrast, in s-IBM muscle fibers, LC3-I/
LC3-II was accumulated in the cytoplasm in the form of
aggregates that were often very large (Figure 1, B and C)
and were commonly adjacent to or surrounding vacuoles;
these were not evident in PM. In PM there were very rare
muscle fibers that contained one or several small areas of
increased LC3-I/LC3-II immunoreactivity (Figure 1D). In
PM, dermatomyositis, and morphologically nonspecific
myopathy, there were occasional small muscle fibers that
Figure 1. LC3 in control, s-IBM and PM muscle fibers. Although a represen-
tative fiber in a control biopsy (A) does not contain any inclusions, within
s-IBM muscle fibers (B and C) there are large LC3-immunoreactive cytoplas-
mic inclusions. D: In PM there are small various-sized LC3-immunoreactive
inclusions. In A, B, and C there is some degree of muscle-fiber sarcolemmal
staining. Original magnification, ?900.
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Page 5

contained slight diffuse LC3-I/LC3-II immunoreactivity
(not shown). Endomysial and perivascular mononucle-
ated cells in s-IBM, polymyositis, and dermatomyositis
muscle biopsy samples were not immunoreactive for
LC3-I/LC3-II. None of the normal or disease control bi-
opsy samples had muscle fibers containing large LC3-I/
LC3-II immunoreactive aggregates like those present in
s-IBM.
Immunoblots
In s-IBM muscle biopsy samples, we studied by immu-
noblot two important markers of activated macroautoph-
agy, the process that involves proliferation and matura-
tion of autophagosomes. Those markers were i) LC3-II, a
lipidated form of LC324,25that is distinguished from LC3-I
by its electrophoretic mobility, and ii) the ratio of phos-
pho-p70S6 kinase (phosphorylated on threonine 389) to
total p70S6 kinase.24,25A decrease in the ratio of mam-
malian target of rapamycin-mediated phosphorylation of
p70S6 kinase to total p70S6 kinase is considered an
important marker of macroautophagy induction.24,25On
immunoblots, s-IBM muscle showed a distinct LC3-II
band, whereas normal age-matched controls did not
manifest an LC3-II band (Figure 2A). In addition, in s-IBM
the ratio of phospho-p70S6 kinase to total p70S6 kinase
was decreased by 60% (P ? 0.001) (Figure 2, B and C).
Both the presence of the LC3-II and the decreased ratio
of the phosphorylated p70S6 to total p70S6 kinase indi-
cate that macroautophagy and mechanisms leading to
autophagosome formation are activated in s-IBM.
Decreased Activity of Lysosomal Cathepsin D
and Cathepsin B in s-IBM
To evaluate whether the lysosomal enzymatic system
functions normally in s-IBM muscle, we studied two major
lysosomal proteases, cathepsin D and B. By immuno-
blots, in s-IBM muscle cathepsin D protein was increased
approximately fourfold (P ? 0.05) (Figure 2, D and E),
and cathepsin B protein was increased twofold (P ?
0.05) (Figure 2, G and H).
However, in s-IBM muscle biopsy samples, despite
increased cathepsin D and B protein, cathepsin D enzy-
matic activity, compared with age-matched controls, was
surprisingly decreased by 60% (P ? 0.01) (Figure 2F),
and cathepsin B activity was decreased by 40% (P ?
0.05) (Figure 2I). This decreased enzymatic activity could
impair lysosomal protein degradation in s-IBM.
Increased Macroautophagy but Unimpaired
Lysosomal Enzymatic Activity in Polymyositis
To assess whether the abnormalities we observed in the
ALP are specific to s-IBM, we studied LC3-II and cathep-
sin D and B activities in PM. In PM, but not in control
muscle biopsy samples, an occasional muscle fiber con-
tained very small LC3-I/LC3-II immunoreactive inclusions
(Figure 1D), and LC3-II was evident in immunoblots (Fig-
ure 3A). In PM the protein levels of cathepsin D and B
were increased compared with those in PM age-matched
controls (Figure 3, B, C, E, and F), similarly to s-IBM. But,
in contrast to s-IBM, in PM muscle biopsy samples the
enzymatic activities of cathepsin D and B were not de-
creased; in fact, the activity of cathepsin D was slightly
increased (Figure 3D) and that of cathepsin B was sig-
nificantly increased (Figure 3G). Hypothetically, the in-
creased macroautophagy in PM may result from an in-
creased demand for lysosomal degradation of damaged
or excessively produced proteins resulting from the mus-
cle fiber degeneration and regeneration characteristic of
PM muscle biopsy samples. In PM, probably because the
lysosomal degradation itself seems to be functioning
properly, autophagic vacuoles do not form.
Lack of Colocalization and Physical Association
between LC3 and p62/SQSTM1 in s-IBM
p62/SQSTM1 is a shuttle protein shown to deliver ubiqui-
tinated proteins to both the proteasomal and autophagic
degradation systems. It was shown to bind to LC3 in
Figure 2. LC3-II, p70S6 kinase, and cathepsins D and B in control and s-IBM
muscle biopsies. A: On representative immunoblots of four s-IBM and four
control muscle biopsies, there is a LC3-II band in s-IBM but not in control
samples. B: Representative immunoblot of phosphorylated p70S6 kinase
(P-p70S6K) shows a clearly decreased band of a P-p70S6K in s-IBM. C:
Densitometric analysis of P-p70S6K per total p70S6K, based on six s-IBM and
four control muscle biopsy samples, indicates a 60% decrease of P-p70S6K in
s-IBM. Representative immunoblots (D) and a densitometric analysis (E) of
cathepsin D (CatD) in five s-IBM and six control muscle biopsy samples show
a fourfold increase in cathepsin D protein in s-IBM, whereas its activity (F) is
decreased by 60% compared with that of control biopsy samples. Represen-
tative immunoblots of cathepsin B (CatB) protein (G) and densitometric
analysis (H) based on seven s-IBM and five control muscle biopsy samples
show a twofold increase in cathepsin B compared with that in control
samples, but its activity (I) is decreased by 40% compared with controls.
Actin bands in D and E are used as protein loading controls. AFU, arbitrary
fluorescence units. *P ? 0.05, **P ? 0.01, ***P ? 0.001.
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various LC3-overexpressing cultured cells13,34and mouse
liver.35In the present study, we investigated immunohis-
tochemical colocalization and physical association be-
tween LC3 and p62 in s-IBM muscle fibers. By light
microscopy, LC3 and p62 were in close proximity but
did not colocalize with each other (Figure 4, A–C). In
addition, there were fibers that contained strongly immu-
noreactive p62, but were LC3-negative (Figure 4, D–F,
arrows).
Our double immunolocalization of p62 and LC3 by
gold-immunoelectron microscopy showed that they were
in very close proximity to each other but were not immu-
nodecorating the same structures (Figure 5, A–C). LC3
seemed to immunodecorate autophagosomal fragments,
and, as we have shown previously,17p62 was immuno-
decorating paired helical filaments.
In our immunoprecipitation of s-IBM biopsy samples
using an antibody against p62 followed by immunoblot-
ting with an antibody recognizing LC3-I/LC3-II, we were
not able to detect an immunoprecipitation band in our
material (Figure 4G); this result suggests that there is no
physical association between p62 and LC3-I or LC3-II in
s-IBM muscle fibers. Immunoblotting of p62 immunopre-
cipitates using an anti-p62 antibody, performed as a
positive control of the immunoprecipitation reaction, re-
vealed a 62-kDa p62 band, confirming the validity of the
immunoprecipitation experiment (Figure 4G).
Experimentally Evoked ER Stress in Cultured
HumanMuscleFibersInducedMacroautophagybut
ImpairedCathepsinDandBActivities
To study possible molecular mechanisms involved in
the s-IBM pathogenesis, we routinely modify the cellu-
lar microenvironment of cultured human muscle fibers
(CHMFs) to mimic various aspects of the s-IBM patho-
genesis, thereby providing experimental “IBM-cultured
Figure 3. LC3-II and cathepsins D and B in PM muscle biopsy samples. A:
By immunoblots, there is a LC3-II band present in PM samples, whereas the
control samples do not manifest a LC3-II band. Representative immunoblots
(B) and (C) a densitometric analysis of cathepsin D (CatD), based on four PM
and eight control muscle biopsy samples show a threefold increase of
cathepsin D protein in PM, and its activity is slightly increased (D). In PM
muscle biopsy samples, representative immunoblots of cathepsin B (CatB)
protein (E) and their densitometric analysis (F) based on six PM and seven
control muscle biopsy samples show a 1.7-fold increase of cathepsin B
compared with control samples; its enzymatic activity is increased sixfold
(G). AFU, arbitrary fluorescence units. **P ? 0.01.
Figure 4. Light microscopic immunohistochemical colocalization and immunoprecipitation of p62/SQSTM1 and LC3/LC3-II in s-IBM muscle biopsy samples. A–F:
p62- and LC3-immunoreactive inclusions seem to be near each other, but they do not absolutely colocalize; there are a number of inclusions that are positive only
for one or the other protein (A–C). In E there is a muscle fiber that contains p62-immunoreactive inclusions but is LC3-negative (D–F, arrows). Original
magnification, ?1100 (A–F). G: Immunoprecipitation of s-IBM biopsy sample using an antibody against p62 followed by immunoblotting (IB) with an antibody
recognizing LC3 or p62. There is no immunoprecipitation band visible using an antibody against LC3, but there is a clear band present using an antibody against
p62. This results indicates a successful immunoprecipitation reaction and a lack of association between p62 and LC3. ?, omitting the primary antibody in the
immunoprecipitation reaction; #, omitting the secondary antibody in the immunoblot.
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human muscle models.” Those models have proved use-
ful in our previous studies.8–10,26,30,31,36
In the present study, to seek mechanisms that might
lead to the lysosomal impairment in s-IBM, we used our
IBM culture models with perturbed protein degradation,
achieved experimentally by i) inhibiting proteasome ac-
tivity and ii) inducing ER stress. For each study, the
proteasome-inhibited and ER stress-provoked cultures
were compared with their sister nonaltered cultures and
with sister cultures treated with either chloroquine or
bafilomycin, both being known inhibitors of lysosomal
function.
Macroautophagy
Compared with controls, all four experimental condi-
tions led to increased LC3-II protein by immunoblots; the
strongest increase was achieved after treatment with
chloroquine or bafilomycin (Figure 6A). The ratio of phos-
phorylated p70S6 kinase to total p70S6 kinase was sig-
nificantly decreased after all treatments: tunicamycin by
70% (P ? 0.001), epoxomycin by 70% (P ? 0.05), chlo-
roquine by 40% (P ? 0.05), and bafilomycin by 70% (P ?
0.01) (Figure 6B). These data provide evidence of acti-
vated macroautophagy.
Cathepsins D and B
Both chloroquine and bafilomycin significantly de-
creased cathepsin D and B enzymatic activities (Figure
6C). In tunicamycin-treated CHMFs, cathepsin D and B
activities were decreased by 17% (P ? 0.001) and 50%
(P ? 0.001), respectively (Figure 6C). Proteasome inhi-
bition did not influence cathepsin D activity (Figure 6C);
thus, we did not measure cathepsin B activity under this
condition because of the scarcity of human muscle ma-
terial for the primary cultures.
Morphological Evaluation
By phase-contrast light microscopy, tunicamycin-,
chloroquine-, and bafilomycin-treated cultures were
highly vacuolated (Figure 7, A–C). Transmission electron
microscopy revealed vacuolar structures containing par-
tially digested cytoplasmic contents, electron-dense ma-
terial, and membranous whorls (Figure 7, E–G), abnor-
malities resembling those observed in s-IBM muscle
fibers (Figure 7, D and H).38
ER Stress Decreases VMA21 Protein in CHMFs
A mutation in VMA21, a chaperone essential for assembly
of lysosomal V-ATPase, which is responsible for maintain-
ing lysosomal pH, was recently described in a human
X-linked myopathy with excessive autophagy.38,39It was
Figure 5. Double-label gold-immunoelectron microscopy of p62 and LC3 in
s-IBM muscle biopsies. p62 and LC3 were in a very close proximity to each
other but were not immunodecorating the same subcellular structures. A: p62
antibody (6-nm gold particles) is immunodecorating a bundle of paired
helical filaments (PHFs). However, LC3 antibody (12-nm gold particles)
seems to be associated with autophagosomal fragments (A–C), some of
which seem to have an autophagosome-characteristic double membrane (B
and C, arrowheads). Original magnification: ?79,000 (A); ?63,000 (B);
?95,000 (C).
Figure 6. Macroautophagy and activities of cathepsins D and B in CHMFs
studied under different conditions. A: Treatments with tunicamycin (Tm),
epoxomycin (Epox), chloroquine (Chlor), or bafilomycin (Baf) significantly
increase LC3-II compared with their untreated sister control cultures (details
in the text). Densitometric analysis based on 10 different experimental cul-
ture sets is shown above the representative blots. *P ? 0.05; **P ? 0.01;
***P ? 0.001. B: Phosphorylated p70S6K (P-p70S6K), expressed per total
p70S6K, is significantly decreased under all four experimental conditions.
*P ? 0.05; **P ? 0.01; ***P ? 0.001. A and B: Activated macroautophagy. C:
Treatments with tunicamycin, chloroquine, or bafilomycin significantly de-
crease cathepsin D and B activities. *P ? 0.05; **P ? 0.01; ***P ? 0.001
(details in the text). Because exposure to epoxomycin did not influence
cathepsin D activity, cathepsin B was not measured under this experimental
condition. AFU, arbitrary fluorescence units.
Impaired Autophagy in s-IBM
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associated with muscle fiber vacuolization, increased
macroautophagy, and impaired lysosomal degradation.
In regard to s-IBM and the possible mechanisms causing
impaired lysosomal function associated with ER stress,
we studied VMA21 protein in our IBM culture model.
Treatment with tunicamycin decreased VMA21 protein by
25% (P ? 0.01) (Figure 8), whereas this effect was not
induced by inhibition of proteasome or inhibition of lyso-
somal functions with either chloroquine or bafilomycin
(not shown).
Discussion
In addition to s-IBM, impaired lysosomal degradation has
been implicated in an X-linked myopathy with excessive
autophagy,38,39in hereditary inclusion-body myopathy
with VCP mutation,40and in other myopathies containing
autophagic vacuoles, such as Danon disease and acid
maltase deficiency (reviewed in 41). Even though the
existence of autophagic vacuoles associated with accu-
mulated lysosomal-membranous structures in s-IBM
muscle biopsy samples is well known (13, 14, 42 and
reviewed in 1, 4), the mechanism of their formation is not
well understood. We now demonstrate, for the first time,
increased formation and maturation of vacuolar auto-
phagosomes in s-IBM muscle fibers, as indicated by i)
the autophagosomal marker LC3-II24,25and ii) mamma-
lian target of rapamycin-mediated phosphorylation of
p70S6K.23–25These observations strongly suggest that
activated macroautophagy might be an important factor
leading to formation of the vacuoles.
We also demonstrate for the first time that enzymatic
activities of the two major lysosomal proteases, cathepsin
D and B, are decreased in s-IBM muscle fibers. Accord-
ingly, our studies strongly suggest that total autophagy,
composed of the autophagosomal-lysosomal pathway, is
impaired in s-IBM muscle fibers.
A?-immunoreactive inclusions were previously found
by immunohistochemistry to colocalize with LC3 in s-IBM
muscle fibers; however, LC3-II, an indicator of autopha-
gosome proliferation and maturation, was not exam-
ined.43Our present study, in addition to immunohisto-
chemically identifying large LC3-I/LC3-II-imunoreactive
inclusions, also demonstrated, by immunoblots of s-IBM
muscle, the presence of LC3-II protein, a reliable marker
of increased macroautophagy. The LC3-II increase re-
flects increased synthesis and formation of autophago-
somes, which might occur either when i) there is an
increased burden of undigested, misfolded, or damaged
proteins and organelles for the delivery to the lysosomes
or ii) when there is an impairment (block) at a later step,
for example, involving impaired lysosomal activity, a de-
layed/impaired trafficking to lysosomes, or reduced fu-
sion between autophagosomes and lysosomes.24Our
studies strongly suggest that in s-IBM increased forma-
tion and maturation of autophagosomes are, at least par-
tially, due to impaired lysosomal enzymatic activity. Thus,
in s-IBM muscle fibers, there is i) impaired autophagy
involving at least two key lysosomal enzymes, cathepsin
D and B, and ii) attempted compensation by excessive
proliferation of autophagosomes and lysosomes, as indi-
cated by the increase in the lysosomal enzymes cathep-
sin D and cathepsin B protein per immunoblots (but not of
their enzymatic activity) and increased acid phosphatase
by histochemistry. The decreased lysosomal cathepsin D
and B enzymatic activities seem to be specific to s-IBM,
because in PM muscle fibers, cathepsin D and B activi-
ties were found to be increased in this study and by
others.44,45Our demonstration of a macroautophagy in-
crease in PM suggests a need of the PM muscle for
increased protein degradation but, perhaps because the
lysosomal system may be functioning adequately, auto-
phagic vacuoles and inclusions do not form. Our results
also suggest that inflammation, which is present in both
s-IBM and PM, does not contribute to impairment of
autophagic/lysosomal degradation in s-IBM. Possible ex-
Figure 7. Morphology of CHMFs exposed to
tunicamycin, bafilomycin, or chloroquine and of
biopsied s-IBM muscle. A–C: Phase-contrast
light microscopy illustrating vacuolization of
CHMFs under all these challenges. E–G: Trans-
mission electron microscopy of CHMFs shows
that all these treatments induced pronounced
abnormalities, as evidenced by various lysoso-
mal debris, myelin-like whorls, and autophago-
some-like structures. D and H: Biopsied s-IBM
muscle. D: Bright-field light microscopy of an
abnormal muscle fiber stained with Engel-Go-
mori trichrome37illustrates various-sized vacu-
oles. H: Electron microscopy shows a vacuole
containing inclusions consisting of numerous
various-sized membranous whorls of autopha-
gosomal/lysosomal debris. Original magnifica-
tion: ?1800 (A–C); ?2100 (D); ?95,000 (E–G);
?50,000 (H).
Figure 8. VMA21 in tunicamycin-treated cultures. On the representative
immunoblots of three different culture sets, VMA21 is clearly decreased in
tunicamycin (Tm)-treated cultures compared with controls (C). Densitomet-
ric analysis based on 10 culture sets indicates a 25% decrease of VMA21 in
Tm-treated cultures. *P ? 0.01.
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planations for the decreased enzymatic activity of the
increased cathepsin D and B proteins in s-IBM might
include their inhibition due to i) changes in lysosomal pH
or ii) a detrimental influence on cathepsins by the prod-
ucts of oxidative stress,46which is known to occur in
s-IBM11,47,48or, in the case of cathepsin B, increased
cystatin C, a recognized inhibitor of serine proteases49
that is increased in s-IBM muscle fibers.50
It has been reported that when the ALP functions prop-
erly, p62, a receptor for ubiquitinated proteins,51is incor-
porated into autophagosomes and subsequently de-
graded by lysosomes.15,24,34One possible explanation
for the increased p62 is impaired autophagic degrada-
tion.15,24,34We previously demonstrated that p62 is
significantly increased and aggregated in s-IBM mus-
cle fibers, but not in PM.17In contrast to studies by
others in various LC3-overexpressing cells and in
mouse liver,15,34,35we were not able to demonstrate a
physical association between p62 and LC3 in the present
study of s-IBM muscle fibers. Whether this lack of asso-
ciation, which conceivably could contribute to accumu-
lation of ubiquitinated proteins i) reflects a characteristic
of s-IBM muscle or ii) is specific for human muscle tissue
is not known.
Another interesting and novel aspect of our study is the
identification of ER stress as a possible cause of the
impaired lysosomal degradation: ER stress is known to
be an important aspect of the s-IBM pathogenesis.9,10
Previously, in various species including mammals, ER
stress was reported to induce macroautophagy, as evi-
denced by increased LC3-II,51–54putatively to eliminate
misfolded/unfolded proteins accumulated in the ER lu-
men.54In agreement with those reports, ER stress in-
duced in our primary cultures of human muscle in-
creased macroautophagy as reflected by the LC3-II
increase and phosphorylated p70S6 kinase decrease;
however, concurrently, ER stress impaired enzymatic ac-
tivities of the lysosomal proteases cathepsin D and B.
Even though the exact mechanism involving the ER
stress reduction of cathepsin D and B enzymatic activi-
ties in CHMFs is not known, our demonstrated ER stress-
dependent decrease of VMA21 in them might raise the
lysosomal pH, thereby decreasing cathepsin D and B
activities and perhaps activities of other unexamined ly-
sosomal enzymes. Whether or not a similar mechanism
occurs in s-IBM muscle fibers is not known. (Because of
an inherently small amount of VMA21 in human muscle,
we were not able, with the antibody used, to adequately
evaluate it by immunoblots in either normal or s-IBM
biopsy samples.)
Cathepsin D and B are two major proteases participat-
ing in normal lysosomal degradation. Detrimental conse-
quences of their impaired activities might be several,
including abnormalities known to occur in s-IBM muscle
fibers. For example, autophagy-lysosomal perturbations
have been shown to enhance tau aggregation,55and
autophagosomes have been demonstrated as an impor-
tant locus for A? production.21,56,57Accordingly, the im-
paired ALP system in s-IBM muscle fibers might contribute
to the well known increase in A? and aggregation of tau
within s-IBM muscle fibers (58, 59 and reviewed in 3, 4).
Interestingly, the skeletal muscle of chloroquine-treated
rats was reported to accumulate A?60,61and to display
some aspects of ER stress.61However, our preliminary
studies of CHMFs treated with either chloroquine or
bafilomycin have not revealed evidence of ER stress.
Several additional proteins accumulated in s-IBM mus-
cle fibers; for example, BACE1, ?-synuclein and tau,
have been reported to be degraded by both lysosomes
and the 26S proteasome.19,20,55,62,63A?42, a more toxic
form of A? that is preferentially increased in s-IBM muscle
fibers,58was shown to be degraded in lysosomes by
cathepsin B.21,64
In CHMFs, A?PP and its phosphorylated form are both
degraded by proteasome.26Accordingly, inhibition of
both proteasomal and lysosomal systems probably has
very detrimental consequences. Moreover, both A?PP
and A? have been shown to inhibit proteasome.8,65,66
In summary, we propose that a double impairment of
protein disposal, caused by inhibition of both lysosomal
and proteasomal degradation, contributes importantly to
s-IBM pathogenesis. Although the igniting mechanisms
leading to the s-IBM muscle fiber degeneration are still
not known, our studies suggest that attempts to unblock
protein degradation might be a therapeutic strategy for
patients with s-IBM.
Acknowledgments
We thank Dr. Kelvin J. Davies from the USC Andrus
Gerontology Center for allowing us to use his fluorometer
to measure enzyme activities. Maggie Baburyan pro-
vided technical assistance in electron microscopy. Mar-
gherita Simonetti participated in preliminary tissue culture
experiments.
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