p62/SQSTM1 is overexpressed and prominently accumulated in inclusions of sporadic inclusion-body myositis muscle fibers, and can help differentiating it from polymyositis and dermatomyositis.

Abstract

p62, also known as sequestosome1, is a shuttle protein transporting polyubiquitinated proteins for both the proteasomal and lysosomal degradation. p62 is an integral component of inclusions in brains of various neurodegenerative disorders, including Alzheimer disease (AD) neurofibrillary tangles (NFTs) and Lewy bodies in Parkinson disease. In AD brain, the p62 localized in NFTs is associated with phosphorylated tau (p-tau). Sporadic inclusion-body myositis (s-IBM) is the most common progressive muscle disease associated with aging, and its muscle tissue has several phenotypic similarities to AD brain. Abnormal accumulation of intracellular multiprotein inclusions, containing p-tau in the form of paired helical filaments, amyloid-beta, and several other "Alzheimer-characteristic proteins", is a characteristic feature of the s-IBM muscle fiber phenotype. Diminished proteasomal and lysosomal protein degradation appear to play an important role in the formation of intra-muscle-fiber inclusions. We now report that: (1) in s-IBM muscle fibers, p62 protein is increased on both the protein and the mRNA levels, and it is strongly accumulated within, and as a dense peripheral shell surrounding, p-tau containing inclusions, by both the light- and electron-microscopy. Accordingly, our studies provide a new, reliable, and simple molecular marker of p-tau inclusions in s-IBM muscle fibers. The prominent p62 immunohistochemical positivity and pattern diagnostically distinguish s-IBM from polymyositis and dermatomyositis. (2) In normal cultured human muscle fibers, experimental inhibition of either proteasomal or lysosomal protein degradation caused substantial increase of p62, suggesting that similar in vivo mechanisms might contribute to the p62 increase in s-IBM muscle fibers.

Full text

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ORIGINAL PAPER
p62/SQSTM1 is overexpressed and prominently accumulated
in inclusions of sporadic inclusion-body myositis muscle fibers,
and can help differentiating it from polymyositis
and dermatomyositis
Anna Nogalska Æ Æ Chiara Terracciano Æ Æ
Carla D’Agostino Æ Æ W. King Engel Æ Æ
Valerie Askanas
Received: 19 May 2009/Revised: 17 June 2009/Accepted: 17 June 2009/Published online: 26 June 2009
? Springer-Verlag 2009
Abstract
protein transporting polyubiquitinated proteins for both the
proteasomal and lysosomal degradation. p62 is an integral
component of inclusions in brains of various neurodegen-
erative disorders, including Alzheimer disease (AD)
neurofibrillary tangles (NFTs) and Lewy bodies in Par-
kinson disease. In AD brain, the p62 localized in NFTs
is associated with phosphorylated tau (p-tau). Sporadic
inclusion-body myositis (s-IBM) is the most common
progressive muscle disease associated with aging, and its
muscle tissue has several phenotypic similarities to AD
brain. Abnormal accumulation of intracellular multiprotein
inclusions, containing p-tau in the form of paired helical
filaments, amyloid-b, and several other ‘‘Alzheimer-char-
acteristic proteins’’, is a characteristic feature of the s-IBM
muscle fiber phenotype. Diminished proteasomal and
lysosomal protein degradation appear to play an important
role in the formation of intra-muscle-fiber inclusions. We
now report that: (1) in s-IBM muscle fibers, p62 protein is
increased on both the protein and the mRNA levels, and it
is strongly accumulated within, and as a dense peripheral
shell surrounding, p-tau containing inclusions, by both the
light- and electron-microscopy. Accordingly, our studies
provide a new, reliable, and simple molecular marker of
p-tau inclusions in s-IBM muscle fibers. The prominent p62
immunohistochemical positivity and pattern diagnostically
p62, also known as sequestosome1, is a shuttle
distinguish s-IBM from polymyositis and dermatomyositis.
(2) In normal cultured human muscle fibers, experimental
inhibition of either proteasomal or lysosomal protein deg-
radation caused substantial increase of p62, suggesting that
similar in vivo mechanisms might contribute to the p62
increase in s-IBM muscle fibers.
Keywords
p62/sequestosome1 (p62/SQSTM1) ? Inclusions ?
Phosphorylated tau ? Proteasome inhibition ?
Lysosome inhibition ? Cultured human muscle fibers
Sporadic inclusion-body myositis ?
Introduction
There is increasing evidence that p62 protein, also known
as sequestosome1 (SQSTM1), is a common component of
ubiquitinated multiprotein aggregates present in protein-
aggregation disorders involving the central nervous system
and liver [19, 20, 38]. p62/SQSTM1, herein referred as
‘‘p62’’, is a shuttle protein transporting polyubiquitinated
proteins for their degradation by both the proteasome and
lysosome [11, 34]. p62 protein contains at its C-terminus
an ubiquitin-associated (UBA) domain that selectively
binds lysine 63 (Lys63) of polyubiquitinated proteins [11,
25, 34], and it was reported to have a critical role in
sequestering polyubiquitinated proteins [12, 34]. Its N-
terminus has an ability to directly interact with a protea-
some subunit to allow delivery of polyubiquitinated
proteins for their degradation there [34]. p62 has also been
reported to interact with the autophagosome membrane
light-chain3 (LC3) protein, which facilitates delivery of its
polyubiquitinated cargo for lysosomal degradation [10, 32].
Several years ago, p62 was found in brain cytoplasmic
inclusions in various neurodegenerative disorders [21].
A. Nogalska ? C. Terracciano ? C. D’Agostino ?
W. King Engel ? V. Askanas (&)
Department of Neurology, USC Neuromuscular Center,
Good Samaritan Hospital, University of Southern California
Keck School of Medicine, 637 S. Lucas Ave,
Los Angeles, CA 90017-1912, USA
e-mail: askanas@usc.edu
123
Acta Neuropathol (2009) 118:407–413
DOI 10.1007/s00401-009-0564-6

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Subsequently it was demonstrated to be an integral part
of neurofibrillary tangles (NFTs) in Alzheimer disease
(AD) brain [19]. In NFTs, p62 co-localizes with phos-
phorylated tau (p-tau), and it appears there early during
neurofibrillary pathogenesis [19], suggesting that p62
might be involved in the aggregation process of p-tau
[19]. p62 is considered to shuttle ubiquitinated tau for
proteasomal degradation [8]. Genetic inactivation of p62
in an adult mouse led to age-dependent brain accumula-
tion of p-tau, NFT formation, and neurodegeneration [9].
Accumulation of p62 has also been considered a sensitive
marker of argyrophylic grain pathology and was helpful
in distinguishing cases of TDP-43-negative frontotempo-
ral lobar degeneration [16, 33]. In addition to shuttling
ubiquitinated proteins for their degradation, p62 plays a
role in signaling processes, including activation of NF-jB
[25, 34].
Sporadic inclusion-body myositis (s-IBM) is the most
common muscle disease of persons aged 50 years and
older. s-IBM muscle tissue shares several phenotypic
similarities with brain tissue of AD and Lewy bodies in
Parkinson disease (recently reviewed in [3]). Pathologi-
cally, there are two characteristics of s-IBM muscle
biopsies—vacuolar degeneration and atrophy of muscle
fibers, and mononuclear cell inflammation [3, 26]. The
vacuolar degeneration is accompanied by accumulations
within those muscle fibers, mainly in their non-vacuolated
cytoplasm, of ubiquitinated, multiprotein aggregates con-
taining p-tau in the form of paired helical filaments
(PHFs), amyloid-b (Ab), and multiple other proteins [2,
3]. At least some of the proteins in these multi-protein
aggregates are in the b-pleated sheet conformation of
amyloid [5, 22], indicating their unfolded/misfolded
status.
The exact mechanisms leading to formation of these
intra-muscle-fiber ubiquitinated multiprotein aggregates
containing misfolded proteins is not well understood,
but the co-existing inhibition of the 26S proteasome
and defective lysosomal degradation likely contribute
(reviewed in [7]).
We now report our novel findings that in s-IBM muscle
fibers, p62 is: (a) accumulated in muscle fiber inclusions,
where it co-localizes with p-tau by both light- and electron-
microscopic immunohistochemistry; and (b) increased both
on the protein and mRNA levels.
The prominent p62 immunohistochemical positivity and
pattern diagnostically distinguishes s-IBM from polymyo-
sitis and dermatomyositis.
We also demonstrate that in cultured human muscle
fibers p62 is increased by experimental inhibition of either
the 26S proteasome or the lysosomal enzyme activities,
suggesting that similar mechanisms may contribute to p62
increase in s-IBM muscle fibers.
Materials and methods
Muscle biopsies
Studies were performed on fresh-frozen diagnostic muscle
biopsies obtained, with informed consent, from 15 s-IBM,
5 polymyositis, 2 dermatomyositis, 4 morphologically
nonspecific myopathy, 3 ALS, and 5 peripheral neuropathy
patients, as well as 12 normal muscle biopsies of patients
who, after all tests performed, were considered free of
muscle disease. s-IBM patients were ages 56–79 years,
median age 62; normal control patients were ages 45–86,
median age 71. Diagnoses were based on clinical and
laboratory investigations, including our routinely per-
formed 16-reaction diagnostic histochemistry of muscle
biopsies. All s-IBM biopsies met s-IBM diagnostic criteria
[4].
Light-microscopic immunocytochemistry
Immunofluorescence was performed as described [6, 13,
27, 35] on 10-lm-thick transverse sections of 5 s-IBM,
3 age-matched normal and 19 disease-control muscle biop-
sies (specified above), using well-characterized [18] rabbit
polyclonal anti-p62/SQSTM1 (H-290), diluted 1:100, or
mouse monoclonal anti-p62/SQSTM1 (D-3), diluted 1:100,
antibodies (both from Santa Cruz Biotechnology, Santa
Cruz, CA). Incubations in each antibody were either
overnight at 4?C or for 3 h in room temperature. Double-
immunofluorescence utilized an antibody against p62
combined with one of the following: (a) mouse monoclonal
antibody AT100 (Pierce, Rockford, IL) diluted 1:1,000,
which recognizes Ser212 and Thr214 of p-tau in AD PHFs
[39]; (b) mouse monoclonal antibody AT8 (Thermo
Scientific, Rockford, IL) recognizing Ser202 and Thr205 of
p-tau [14], diluted 1:5; and (c) mouse monoclonal antibody
SMI-31 (Covance, Denver, PA) diluted 1:500, which rec-
ognizes p-tau of s-IBM [24] and AD PHFs [29] between
phosphorylated-serine 396 and phosphorylated-serine 404
[17, 29]. All the above anti-p-tau antibodies have been
previously shown to be strongly immunoreactive in s-IBM
[13, 24]. To block non-specific binding of an antibody to
Fc receptors, sections were pre-incubated with normal goat
serum diluted 1:10 [6, 13, 27, 35]. Controls for staining
specificity were (a) omission of the primary antibody, or
(b) its replacement with non-immune sera or irrelevant
antibody. Those were always negative.
For immunocytochemistry using an HRP/DAB method,
the sections were processed as above, except that after
incubation in the secondary antibody the color was devel-
oped with Streptavidin conjugated to HRP (Jackson
ImmunoResearch Laboratories, West Grove, PA), diluted
1:50, followed by 50 s incubation in a DAB/H2O2solution.
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Gold immunoelectronmicroscopy
This was performed on 10-lm fresh-frozen biopsy sections
adhered to the bottom of 35-mm Petri dishes [6, 13, 24, 27,
35]. In brief, sections were prefixed in 2% paraformalde-
hyde for 2 min, preincubated with 10% normal goat serum,
followed by incubation with rabbit polyclonal antibody
against p62 used in combination with mouse monoclonal
antibody SMI-31. After incubation with the appropriate
secondary antibodies conjugated to 6 and 12 nm gold
particles (Jackson ImmunoResearch Laboratories), sections
were processed for electronmicroscopy [6, 13, 24, 27, 35].
Immunoblots
To compare the amount of p62 protein between s-IBM and
controls, muscle biopsies of 7 s-IBM and 6 normal age-
matched control patients were immunoblotted, as recently
detailed [13, 27, 35]. 20 lg of protein were loaded into the
gel and electrophoretically separated. After electrophore-
sis, samples were transferred to a nitrocellulose membrane.
All reagents were obtained from Invitrogen (Carlsbad,
CA). To prevent non-specific binding of antibodies, the
nitrocellulose membranes were blocked in Blocking
Reagent (Invitrogen); then they were incubated overnight
at 4?C with a primary anti-p62 antibody (A-6, Santa Cruz
Biotechnology). Specificity of the p62 antibody was tested
using p62-blocking peptide (Santa Cruz Biotechnology).
Blots were developed using the anti-mouse WesternBreeze
chemiluminescent kit (Invitrogen). Protein loading was
evaluated by the actin band (Santa Cruz Biotechnology).
Quantification of the immunoreactivity was performed by
densitometric analysis using NIH Image J 1.310 software.
RNA isolation and real-time PCR
Total RNA from 8 s-IBM and 8normal age-matched control
muscle biopsies was isolated using an RNA isolation kit
(BD Pharmingen, San Diego, CA) according to the manu-
facturer’s instructions and as recently described [27, 35].
1 lg of RNA was subjected to genomic DNA removal, and
to cDNA synthesis using QuantiTect Reverse Transcription
Kit (Qiagen, Valencia, CA), according to the manufac-
turer’s instructions. Real-time PCR was performed in
duplicate at total volume of 25 ll using 1 ll of cDNA,
QuantiTect Primers (Qiagen) for p62/SQSTM1 or GAPDH,
and QuantiFastSYBR Green PCR Master mix (Qiagen).
PCR runs were performed on an Eppendorf Mastercycler
Realplex2. Cycling conditions were 95?C for 5 min, fol-
lowed by 40 cycles of 95?C for 10 s and 60?C for 30 s.
Relative gene expression was calculated by using the
2-DDCTmethod,inwhichtheamountofp62/SQSTM1mRNA
was normalized to an endogenous reference gene (GAPDH)
mRNA. The results are expressed as fold-induction relative
to controls. Correct PCR products were confirmed by aga-
rose gel electrophoresis and melting curve analysis.
Cultured human muscle fibers
Primary cultures of normal human muscle were estab-
lished, as described [1], from satellite cells of portions of
diagnostic muscle biopsies from patients who, after all tests
were performed, were considered free of muscle disease.
Experiments were performed on at least five different
culture sets, each established from satellite cells derived
from a different muscle biopsy. 12–18 days after fusion of
myoblasts, well-differentiated myotubes were treated with
(a) an irreversible proteasome inhibitor epoxomicin [23]
(1 lM) (Biomol Research Laboratories, Plymouth Meet-
ing, PA) for 24 h [13], or (b) the lysosome inhibitor
chloroquine [30] (20 lM) (Sigma Co, St. Louis, MO) for
48 h. In each experiment, treated cultures were compared
to their untreated-sister-control cultures. After the treat-
ment, the cultures were processed for immunoblots,
performed as described [13, 27, 28].
Statistical analysis
The statistical significance was determined by Student’s t
test. The level of significance was set at P\0.05. Data are
presented as mean ± SEM.
Results and discussion
With light microscopic immunocytochemistry, in each
sporadic IBM muscle biopsy p62 was localized in the form
of strongly immunoreactive, various-sized, mainly squig-
gly, linear, or small rounded aggregates (Fig. 1a). These
were in the non-vacuolated cytoplasm of approximately
80% of the vacuolated muscle fibers, and in about 20–25%
of the muscle fibers non-vacuolated in the studied 10 lm
transverse section. The identical pattern was obtained with
both monoclonal and polyclonal anti-p62 antibodies. By
double-label immunofluorescence, those p62-immunoreac-
tive cytoplasmic aggregates closely co-localized with p-tau-
immunoreactive aggregates (Fig. 1b–g). The same pattern
was obtained with all anti-p-tau antibodies used.
In none of the control normal or disease muscle biopsies
were there any p62 immunoreactive aggregates present
like those found in s-IBM. Rare fibers in polymyositis,
dermatomyositis,andmorphologically
myopathies contained very tiny speckles of p62-immuno-
reactivity (Fig. 2), which were identified with the more
sensitive fluorescence and were virtually invisible with
the HRP staining technique. Occasional small-angulated
denervated muscle fibers in a peripheral neuropathy or ALS
non-specific
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patient had slight-diffuse p62 immunoreactivity. p62 was
slightly and diffusely increased within the centers of some
muscle target fibers inpatients with peripheralneuropathies.
p62 was not immunoreactive in the mononuclear inflam-
matory cells of s-IBM, polymyositis or dermatomyositis. In
all biopsies, normal myonuclei were not immunoreactive.
By gold immunoelectronmicroscopy, p62 antibodies
disclosed a strongly immunoreactive shell at the periphery
of each bundle of cytoplasmic PHFs, thereby appearing to
sequester them from the cytoplasm (Fig. 3a–c). Higher
magnification showed that p62 and p-tau were both asso-
ciated with paired helical filaments (Fig. 3d). Rare bundles
of nuclear PHFs were also enclosed by a strong p62-
immunoreactivity (Fig. 3e). Similarly, to the cytoplasmic
inclusions, in the nuclear inclusions higher magnification
revealed that p62 and p-tau were both associated with
paired helical filaments (Fig. 3f).
Fig. 1 Immunolocalization of
p62 within s-IBM muscle fibers.
a Streptavidin HRP staining
shows three abnormal muscle
fibers containing strongly p62-
immunoreactive squiggly, linear
and small rounded inclusions.
b–g Double-label
immunofluorescence illustrating
close co-localization of p62 and
p-tau (visualized with 2
different anti-p-tau antibodies,
SMI-31 and AT100. Details in
text). All 91,200
Fig. 2 Immunolocalization of p62 within s-IBM and polymyositis
(PM) muscle fibers. In s-IBM immunofluorescence demonstrates
strongly p62-immunoreactive inclusions in three muscle fibers, while
in PM, p62 is immunolocalized in one muscle fiber in the form of tiny
speckles. Both 91,000
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Immunoblots demonstrated that total p62 protein was
increased threefold (P\0.01) in s-IBM muscle biopsies as
compared to normal, age-matched controls (Fig. 4a, b).
Real-Time PCR showed that p62 mRNA was increased 2.6-
fold (P\0.01) (Fig. 4c).
In normal cultured human muscle fibers, a p62 immu-
noreactive protein band was essentially undetectable by
immunoblots (Fig. 5a, b). In contrast, in both epoxomicin-
and chloroquine-treated cultures the p62-immunoreactive
band was very evident (Fig. 5a, b).
Together, our studies demonstrate that in s-IBM, as in
AD brain, p62 immunoreactivity is associated with bundles
of paired helical filaments, which appear to be the same as
AD neurofibrillary tangles. Close co-localization of p62
with p-tau in s-IBM suggests, similarly to what was pro-
posed for the neurodegenerative disorders [8, 19], that p62
might be attempting to carry ubiquitinated tau for prote-
asomal degradation, but has become stuck to the bundles of
insoluble and apparently unmovable PHFs, impairing this
essential task. It is unknown whether enclosure by the p62
shell might be protecting normal cytoplasmic and nucleo-
plasmic components from a possible toxicity of the
misfolded, aggregating p-tau.
Another interesting aspect of this study is an increase of
total p62 on both the protein and mRNA levels. While
increase on the protein level might reflect decreased deg-
radation of p62 as suggested in our culture models,
increased p62 mRNA suggests its increased synthesis—
both might be occurring in s-IBM muscle fibers. The latter
phenomenon might result from the muscle fiber’s attempt
to increase p62 production in order to facilitate shuttling of
proteins, particularly p-tau, for its proteasomal degradation.
In other systems, some conditions that also occur within
s-IBM muscle fibers, e.g. oxidative stress [35, 36], were
reported to upregulate p62 transcription [15], and subse-
quently influence NF-jB signaling [25]. Whether that
Fig. 3 Double-label gold-
immunoelectronmicroscopy of
p62 and p-tau in s-IBM muscle
fibers. a–c Low-power electron-
microscopy illustrating that
p62 forms a strongly
immunoreactive shell around
the individual bundles of PHFs
(10-nm gold particles). d p62
(10-nm gold particles) and p-tau
(5-nm gold particles) associate
with PHFs. e A low-power
electron-micrograph
demonstrates a nuclear
inclusion encased by a shell
composed of p62 (10-nm gold
particles). f Higher
magnification of the area
indicated by the square shown
in e demonstrates that the
nuclear inclusion is composed
of PHFs associated both with
p62 (10-nm gold particles) and
p-tau (5-nm gold particles).
a 912,000; b 921,300;
c 969,000
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mechanism contributes to the previously reported increase
of NF-jB activation in s-IBM muscle fibers [28, 37] is not
known.
Aggregated p62 has been shown within muscle fibers of
patients with myotilinopathies and desminopathies [31];
however, whether its accumulation reflects p62 overex-
pression on the protein and mRNA levels has not been
reported.
In conclusion, our studies demonstrate that p62 is an
integral part of s-IBM PHF inclusions. Because (a) the p62
immunoreactivity is very specific and strong, possibly due
to its being concentrated as a peripheral ‘‘enclosure’’ of the
PHFs bundles, and (b) its apparent specificity for s-IBM,
HRP/DAB staining of p62 using easily available commer-
cial antibodies seems to provide a new diagnostic
pathologic marker of s-IBM to enable its distinction from
polymyositis and dermatomyositis. In addition, our p62 data
from both s-IBM muscle biopsies and relevant human
muscle culture models are harmonious with the concept of
defective protein degradation in the s-IBM pathogenesis.
Acknowledgments
of Biochemistry, Medical University of Gdansk, Gdansk, Poland. Dr.
Terracciano was on leave from the Department of Neuroscience,
University of Tor Vergata and Fondazione S. Lucia, Rome, Italy.
Maggie Baburyan provided excellent technical assistance in elec-
tronmicroscopy. Margherita Simonetti participated in tissue culture
experiments. We are grateful to Dr. Michael Jakowec of the USC
Department of Neurology for allowing us to use his real-time PCR
equipment. Supported by grants (to VA) from the National Institutes
of Health (AG 16768 Merit Award), the Muscular Dystrophy Asso-
ciation, and The Myositis Association.
Dr. Nogalska is on leave from the Department
References
1. Askanas V, Engel WK (1992) Cultured normal and genetically
abnormal human muscle. In: Rowland LP, Di Mauro S (eds)
The handbook of clinical neurology, myopathies, vol 18. North
Holland, Amsterdam, pp 85–116
2. Askanas V, Engel WK (2007) Inclusion-body myositis, a multi-
factorial muscle disease associated with aging: current concepts
of pathogenesis. Curr Opin Rheumatol 19:550–559
3. Askanas V, Engel WK (2008) Inclusion-body myositis: muscle-
fiber molecular pathology and possible pathogenic significance of
its similarity to Alzheimer’s and Parkinson’s disease brains. Acta
Neuropathol 116:583–595
4. Askanas V, Engel WK (2001) Inclusion-body myositis: newest
concepts of pathogenesis and relation to aging and Alzheimer
disease. J Neuropathol Exp Neurol 60:1–14
5. Askanas V, Engel WK, Alvarez RB (1993) Enhanced detection of
congo-red-positive amyloid deposits in muscle fibers of inclusion
body myositis and brain of Alzheimer’s disease using fluores-
cence technique. Neurology 43:1265–1267
6. Askanas V, Engel WK, Alvarez RB, McFerrin J, Broccolini A
(2000) Novel immunolocalization of alpha-synuclein in human
muscle of inclusion-body myositis, regenerating and necrotic
muscle fibers, and at neuromuscular junctions. J Neuropathol Exp
Neurol 59:592–598
7. Askanas V, Engel WK, Nogalska A (2009) Inclusion-body
myositis: a degenerative muscle disease associated with intra-
Fig. 4 p62 protein and mRNA are increased in s-IBM muscle fibers.
a Representative immunoblot of normal control ‘‘C’’ and ‘‘s-IBM’’
muscle biopsies—it shows a stronger p62 band in s-IBM. The actin
bands indicate protein loading in each sample. Absorption of the
primary anti-p62 antibody with a specific p62 blocking (neutralizing)
peptide resulted in a great decrease of the p62 band. b Densitometric
analysis of immunoblot of p62 protein bands, based on 6 control and
7 s-IBM muscle biopsies, shows that in s-IBM muscle fibers p62 is
increased threefold (P\0.01) as compared to the age-matched
controls (±SEM); c Real-time PCR based on 8 s-IBM and 8 age-
matched control biopsies shows 2.6-fold (P\0.01) increase of p62-
mRNA in s-IBM muscle fibers as compared to controls ‘‘C’’. Data are
presented as mean ± SEM, normalized to GAPDH expression
Fig. 5 p62 protein is increased in both proteasome- and lysosome-
inhibited cultured human muscle fibers (CHMFs). Representative
immunoblots of homogenates of (a) control ‘‘C’’ and Epoxomicin
(‘‘Epox’’)-treated; and (b) control ‘‘C’’ and Chloroquine (‘‘Chlor’’)-
treated CHMFs, showing that the p62 protein band is virtually
invisible in control cultures, while it is clearly present after both the
proteasomal and lysosomal activities are inhibited. The actin bands
indicate protein loading in each sample
412 Acta Neuropathol (2009) 118:407–413
123

Page 7

muscle-fiber multiprotein aggregates, proteasome inhibition,
endoplasmic reticulum stress, and decreased lysosomal degrada-
tion.Brain Pathol19:493–506.
2009.00290.x
8. Babu JR, Geetha T, Wooten MW (2005) Sequestosome 1/p62
shuttles polyubiquitinated tau for proteasomal degradation.
J Neurochem 94:192–203
9. Babu R, Seibenhener LM, Peng J, Strom AL, Kemppainen R,
Cox N, Zhu H, Wooten MC, Diaz-Meco MT, Moscat J, Wooten
MW (2008) Genetic inactivation of p62 leads to accumulation of
hyperphosphorylated tau and neurodegeneration. J Neurochem
106:107–120
10. Bjorkoy G, Lamark T, Brech A, Outzen H, Perander M, Overvatn
A, Stenmark H, Johansen T (2005) p62/SQSTM1 forms protein
aggregates degraded by autophagy and has a protective effect on
huntingtin-induced cell death. J Cell Biol 171:603–614
11. Bjorkoy G, Lamark T, Johansen T (2006) p62/SQSTM1: a
missing link between protein aggregates and the autophagy
machinery. Autophagy 2:138–139
12. Donaldson KM, Li W, Ching KA, Batalov S, Tsai CC, Joazeiro
CAP (2003) Ubiquitin-mediated sequestration of normal cellular
proteins into polyglutamine aggregates. Proc Natl Acad Sci USA
100:8892–8897
13. Fratta P, Engel WK, McFerrin J, Davies KJ, Lin SW, Askanas V
(2005) Proteasome inhibition and aggresome formation in spo-
radic inclusion-body myositis and in amyloid-b precursor
protein-overexpressing cultured human muscle fibers. Am J
Pathol 167:517–526
14. Goedert M, Jakes R, Vanmechelen E (1995) Monoclonal anti-
body AT8 recognises tau protein phosphorylated at both serine
202 and threonine 205. Neurosci Lett 189:167–170
15. Ishii T, Yanagawa T, Yuki K, Kawane T, Yoshida H, Bannai S
(1997) Low micromolar levels of hydrogen peroxide and protea-
some inhibitors induce the 60-kDa A170 stress protein in murine
peritoneal macrophages. Biochem Biophys Res Comm 232:33–37
16. Josephs KA, Lin WL, Ahmed Z, Stroh DA, Graff-Radford NR,
Dickson DW (2008) Frontotemporal lobar degeneration with
ubiquitin-positive, but TDP-43-negative inclusions. Acta Neuro-
pathol 116:159–167
17. Ksiezak-Reding H, Dickson DW, Davies P, Yen SH (1987)
Recognition of tau epitopes by anti-neurofilament antibodies that
bind to Alzheimer neurofibrillary tangles. Proc Natl Acad Sci
USA 84:3410–3414
18. Kuusisto E, Kauppinen T, Alafuzoff I (2008) Use of p62/
SQSTM1 antibodies for neuropathological diagnosis. Neuropa-
thol Appl Neurobiol 34:69–80
19. Kuusisto E, Salminen A, Alafuzoff I (2002) Early accumulation of
p62 in neurofibrillary tangles in Alzheimer’s disease: possible role
in tangle formation. Neuropathol Appl Neurobiol 28:228–237
20. Kuusisto E, Salminen A, Alafuzoff I (2001) Ubiquitin-binding
protein p62 is present in neuronal and glial inclusions in human
tauopathies and synucleinopathies. NeuroReport 12:2085–2090
21. Kuusisto E, Suuronen T, Salminen A (2001) Ubiquitin-binding
protein p62 expression is induced during apoptosis and prote-
asomal inhibition in neuronal cells. Biochem Biophys Res Comm
280:223–228
22. Mendell JR, Sahenk Z, Gales T, Paul L (1991) Amyloid filaments
in inclusion body myositis. Novel findings provide insight into
nature of filaments. Arch Neurol 48:1229–1234
23. Meng L, Mohan R, Kwok BH, Elofsson M, Sin N, Crews CM
(1999) Epoxomicin, a potent and selective proteasome inhibitor,
exhibits in vivo antiinflammatory activity. Proc Natl Acad Sci
USA 96:10403–10408
doi:10.1111/j.1750-3639.
24. Mirabella M, Alvarez RB, Bilak M, Engel WK, Askanas V
(1996) Difference in expression of phosphorylated tau epitopes
between sporadic inclusion-body myositis and hereditary inclu-
sion-body myopathies. J Neuropathol Exp Neurol 55:774–786
25. Moscat J, Diaz-Meco MT, Wooten MW (2007) Signal integration
and diversification through the p62 scaffold protein. Trends
Biochem Sci 32:95–100
26. Needham M, Mastaglia FL (2007) Inclusion body myositis:
current pathogenetic concepts and diagnostic and therapeutic
approaches. Lancet Neurol 6:620–631
27. Nogalska A, Engel WK, McFerrin J, Kokame K, Komano H,
Askanas V (2006) Homocysteine-induced endoplasmic reticulum
protein (Herp) is up-regulated in sporadic inclusion-body myo-
sitis and in endoplasmic reticulum stress-induced cultured human
muscle fibers. J Neurochem 96:1491–1499
28. Nogalska A, Wojcik S, Engel WK, McFerrin J, Askanas V (2007)
Endoplasmic reticulum stress induces myostatin precursor protein
and NF-jB in cultured human muscle fibers: relevance to
inclusion body myositis. Exp Neurol 204:610–618
29. Nukina N, Kosik KS, Selkoe DJ (1987) Recognition of Alzhei-
mer paired helical filaments by monoclonal neurofilament
antibodies is due to crossreaction with tau protein. Proc Natl
Acad Sci USA 84:3415–3419
30. Ohkuma S, Poole B (1978) Fluorescence probe measurement of
the intralysosomal pH in living cells and the perturbation of pH
by various agents. Proc Natl Acad Sci USA 75:3327–3331
31. Olive ´ M, van Leeuwen FW, Janue ´ A, Moreno D, Torrejo ´n-Es-
cribano B, Ferrer I (2008) Expression of mutant ubiquitin
(UBB ? 1) and p62 in myotilinopathies and desminopathies.
Neuropathol Appl Neurobiol 34:76–87
32. Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen
H, Overvatn A, Bjorkoy G, Johansen T (2007) p62/SQSTM1
binds directly to Atg8/LC3 to facilitate degradation of ubiqui-
tinated protein aggregates
282:24131–24145
33. Scott IS, Lowe JS (2007) The ubiquitin-binding protein p62
identifies argyrophilic grain pathology with greater sensitivity
than conventional silver stains. Acta Neuropathol 113:417–420
34. Seibenhener ML, Babu JR, Geetha T, Wong HC, Krishna NR,
Wooten MW (2004) Sequestosome 1/p62 is a polyubiquitin chain
binding protein involved in ubiquitin proteasome degradation.
Mol Cell Biol 24:8055–8068
35. Terracciano C, Nogalska A, Engel WK, Wojcik S, Askanas V
(2008) In inclusion-body myositis muscle fibers Parkinson-asso-
ciated DJ-1 is increased and oxidized. Free Radic Biol Med
45:773–779
36. Yang CC, Alvarez RB, Engel WK, Askanas V (1996) Increase of
nitric oxide synthases and nitrotyrosine in inclusion-body myo-
sitis. NeuroReport 8:153–158
37. Yang CC, Askanas V, Engel WK, Alvarez RB (1998) Immuno-
localization of transcription factor NF-kappaB in inclusion-body
myositis muscle and at normal human neuromuscular junctions.
Neurosci Lett 254:77–80
38. Zatloukal K, Stumptner C, Fuchsbichler A, Heid H, Schnoelzer
M, Kenner L, Kleinert R, Prinz M, Aguzzi A, Denk H (2002) p62
is a common component of cytoplasmic inclusions in protein
aggregation diseases. Am J Pathol 160:255–263
39. Zheng-Fischhe `ofer Q, Biernat J, Mandelkow EM, Illenberger S,
Godemann R, Mandelkow E (1998) Sequential phosphorylation
of Tau by glycogen synthase kinase-3beta and protein kinase A at
Thr212 and Ser214 generates the Alzheimer-specific epitope of
antibody AT100 and requires a paired-helical-filament-like con-
formation. Eur J Biochem 252:542–552
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