SQSTM1 is overexpressed and prominently accumulated in

Acta Neuropathol (2009) 118:407–413 DOI 10.1007/s00401-009-0564-6

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 Chiara Terracciano Æ Carla D’Agostino Æ W. King Engel Æ Valerie Askanas Received: 19 May 2009 / Revised: 17 June 2009 / Accepted: 17 June 2009 / Published online: 26 June 2009 Ó Springer-Verlag 2009

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-b, 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

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: [email protected]

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. Keywords Sporadic inclusion-body myositis p62/sequestosome1 (p62/SQSTM1) Inclusions Phosphorylated tau Proteasome inhibition Lysosome inhibition Cultured human muscle fibers

Introduction There is increasing evidence that p62 protein, also known as sequestosome1 (SQSTM1), is a common component of ubiquitinated multiprotein aggregates present in proteinaggregation 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 Nterminus has an ability to directly interact with a proteasome 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].

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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 phosphorylated 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 accumulation 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 frontotemporal 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]). Pathologically, 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 containing 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 electronmicroscopic immunohistochemistry; and (b) increased both on the protein and mRNA levels. The prominent p62 immunohistochemical positivity and pattern diagnostically distinguishes s-IBM from polymyositis 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.

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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 performed 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 biopsies (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. Doubleimmunofluorescence 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 recognizes 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 developed with Streptavidin conjugated to HRP (Jackson ImmunoResearch Laboratories, West Grove, PA), diluted 1:50, followed by 50 s incubation in a DAB/H2O2 solution.


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Gold immunoelectronmicroscopy

mRNA. The results are expressed as fold-induction relative to controls. Correct PCR products were confirmed by agarose gel electrophoresis and melting curve analysis.

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% paraformaldehyde 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 agematched control patients were immunoblotted, as recently detailed [13, 27, 35]. 20 lg of protein were loaded into the gel and electrophoretically separated. After electrophoresis, 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 8 normal age-matched control muscle biopsies was isolated using an RNA isolation kit (BD Pharmingen, San Diego, CA) according to the manufacturer’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 manufacturer’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, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. Relative gene expression was calculated by using the 2-DDCT method, in which the amount of p62/SQSTM1 mRNA was normalized to an endogenous reference gene (GAPDH)

Cultured human muscle fibers Primary cultures of normal human muscle were established, 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 Meeting, 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 treatment, 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 squiggly, 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-immunoreactive cytoplasmic aggregates closely co-localized with p-tauimmunoreactive 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, and morphologically non-specific myopathies contained very tiny speckles of p62-immunoreactivity (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

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Fig. 1 Immunolocalization of p62 within s-IBM muscle fibers. a Streptavidin HRP staining shows three abnormal muscle fibers containing strongly p62immunoreactive 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

patient had slight-diffuse p62 immunoreactivity. p62 was slightly and diffusely increased within the centers of some muscle target fibers in patients with peripheral neuropathies. p62 was not immunoreactive in the mononuclear inflammatory 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 associated with paired helical filaments (Fig. 3d). Rare bundles of nuclear PHFs were also enclosed by a strong p62immunoreactivity (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).

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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


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 2.6fold (P \ 0.01) (Fig. 4c). In normal cultured human muscle fibers, a p62 immunoreactive protein band was essentially undetectable by immunoblots (Fig. 5a, b). In contrast, in both epoxomicinand 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 proposed for the neurodegenerative disorders [8, 19], that p62 might be attempting to carry ubiquitinated tau for proteasomal degradation, but has become stuck to the bundles of

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insoluble and apparently unmovable PHFs, impairing this essential task. It is unknown whether enclosure by the p62 shell might be protecting normal cytoplasmic and nucleoplasmic 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 degradation 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 subsequently influence NF-jB signaling [25]. Whether that

Fig. 3 Double-label goldimmunoelectronmicroscopy of p62 and p-tau in s-IBM muscle fibers. a–c Low-power electronmicroscopy 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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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 agematched control biopsies shows 2.6-fold (P \ 0.01) increase of p62mRNA in s-IBM muscle fibers as compared to controls ‘‘C’’. Data are presented as mean ± SEM, normalized to GAPDH expression

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.

Fig. 5 p62 protein is increased in both proteasome- and lysosomeinhibited 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

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 overexpression 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 commercial 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

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Acknowledgments Dr. Nogalska is on leave from the Department 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 electronmicroscopy. 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 Association, and The Myositis Association.

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 multifactorial muscle disease associated with aging: current concepts of pathogenesis. Curr Opin Rheumatol 19:550–559 3. Askanas V, Engel WK (2008) Inclusion-body myositis: musclefiber 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 fluorescence 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-


8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

muscle-fiber multiprotein aggregates, proteasome inhibition, endoplasmic reticulum stress, and decreased lysosomal degradation. Brain Pathol 19:493–506. doi:10.1111/j.1750-3639. 2009.00290.x Babu JR, Geetha T, Wooten MW (2005) Sequestosome 1/p62 shuttles polyubiquitinated tau for proteasomal degradation. J Neurochem 94:192–203 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 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 Bjorkoy G, Lamark T, Johansen T (2006) p62/SQSTM1: a missing link between protein aggregates and the autophagy machinery. Autophagy 2:138–139 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 Fratta P, Engel WK, McFerrin J, Davies KJ, Lin SW, Askanas V (2005) Proteasome inhibition and aggresome formation in sporadic inclusion-body myositis and in amyloid-b precursor protein-overexpressing cultured human muscle fibers. Am J Pathol 167:517–526 Goedert M, Jakes R, Vanmechelen E (1995) Monoclonal antibody AT8 recognises tau protein phosphorylated at both serine 202 and threonine 205. Neurosci Lett 189:167–170 Ishii T, Yanagawa T, Yuki K, Kawane T, Yoshida H, Bannai S (1997) Low micromolar levels of hydrogen peroxide and proteasome inhibitors induce the 60-kDa A170 stress protein in murine peritoneal macrophages. Biochem Biophys Res Comm 232:33–37 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 Neuropathol 116:159–167 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 Kuusisto E, Kauppinen T, Alafuzoff I (2008) Use of p62/ SQSTM1 antibodies for neuropathological diagnosis. Neuropathol Appl Neurobiol 34:69–80 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 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 Kuusisto E, Suuronen T, Salminen A (2001) Ubiquitin-binding protein p62 expression is induced during apoptosis and proteasomal inhibition in neuronal cells. Biochem Biophys Res Comm 280:223–228 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 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

413 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 inclusion-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 myositis 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 Alzheimer 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-Escribano 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 ubiquitinated protein aggregates by autophagy. J Biol Chem 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-associated 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 myositis. NeuroReport 8:153–158 37. Yang CC, Askanas V, Engel WK, Alvarez RB (1998) Immunolocalization 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 conformation. Eur J Biochem 252:542–552

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