β-amyloid in a human rhabdomyosarcoma cell line

Journal of the Neurological Sciences 325 (2013) 103–107

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TNF-α upregulates macroautophagic processing of APP/β-amyloid in a human rhabdomyosarcoma cell line Christian W. Keller a, b, Matthias Schmitz a, Christian Münz c, 1, Jan D. Lünemann b, 1, Jens Schmidt a, d,⁎, 1 a

Department of Neurology, University Medical Center Göttingen, Göttingen, Germany Institute of Experimental Immunology, Department of Neuroinflammation, University of Zürich, Zürich, Switzerland Institute of Experimental Immunology, Department of Viral Immunobiology, University of Zürich, Zürich, Switzerland d Department of Neuroimmunology, Institute for Multiple Sclerosis Research and Hertie Foundation, University Medical Center Göttingen, Göttingen, Germany b c

a r t i c l e

i n f o

Article history: Received 11 July 2012 Received in revised form 11 December 2012 Accepted 11 December 2012 Available online 5 January 2013 Keywords: Macroautophagy Skeletal muscle TNF-α Myositis β-Amyloid

a b s t r a c t Sporadic inclusion body myositis is a chronic progressive, inflammatory disorder of the skeletal muscle. No effective treatment is available for this debilitating condition and the complex disease pathology is far from being understood. The major hallmark of the pathomechanisms is the co-occurrence of inflammatory as well as degenerative cascades including aggregates consisting of β-amyloid within skeletal muscle fibers. Macroautophagy, a homeostatic process that shuttles cytoplasmic constituents into endosomal and lysosomal compartments, has recently been shown to be upregulated via the proinflammatory cytokine TNF-α in human skeletal muscle cells. In a human cell line from rhabdomyosarcoma as a model to study muscle cells, we here show that TNF-α-mediated upregulation of macroautophagy modulates APP and β-amyloid load and can be blocked by inhibition of macroautophagy. Thus, macroautophagy may be a crucial mediator between inflammation and β-amyloid-associated degeneration in skeletal muscle. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Autophagy is a set of highly conserved catabolic pathways during which cell organelles and long-lived proteins are degraded in lysosomes in order to recycle nutrients for cell survival when facing starvation [1]. During macroautophagy cytosolic material is sequestered within a double-membraned organelle called the autophagosome, which subsequently fuses with lysosomes and late endosomes to form the autolysosome in which the cargo faces degradation via acidic lysosomal hydrolases [2]. Aside from its involvement in homeostasis, cell death [3] and immunity [4], autophagy appears to play a crucial role in the elimination of abnormal intracellular protein aggregates of numerous neurodegenerative disorders [5,6]. Apart from other stimuli, macroautophagy can be regulated via cytokines [7]. Amongst them, tumor necrosis factor (TNF)-α has proven to be involved in the induction of macroautophagic activity in several cell types [8,9].

Abbreviations: sIBM, sporadic inclusion body myositis; APP, amyloid precursor protein; 3MA, 3-methyladenine; CQ, chloroquine; FKBP12, FK-binding protein 12; mTORC1, mammalian target of rapamycin complex1; BACE1, β-site APP-cleaving enzyme 1; AD, Alzheimer's dementia. ⁎ Corresponding author at: Dept. of Neurology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany. Tel.: +49 551 39 8484; fax: +49 551 39 8405. E-mail address: [email protected] (J. Schmidt). 1 These authors contributed equally to this work and should be considered senior authors. 0022-510X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jns.2012.12.011

Sporadic inclusion body myositis (sIBM) is the most common inflammatory myopathy in patients above the age of 50 and is characterized by the concurrent presence of both immune mediated as well as degeneration driven pathomechanisms [10–12]. Aside from CD8+ T cell mediated cytotoxicity and upregulation of proinflammatory cytokines like TNF-α and interleukin (IL)-1β, muscle fibers from sIBM patients display distinctive accumulations of aberrant molecules including aggregates of β-amyloid as well as overexpression of the amyloid precursor protein (APP) [13]. It has been previously reported that muscle fibers of sIBM patients feature increased frequencies of autophagosomes in comparison with nonmyopathic muscle and that intracellular APP as well as β-amyloid showed a significant level of co-occurrence with the autophagosomal marker LC3 [14]. We could recently show that the proinflammatory cytokine TNF-α induced macroautophagic activity and regulates MHC expression in human skeletal muscle cells [15]. In this study we investigated whether TNF-α mediated upregulation of macroautophagy was associated with increased APP expression and intracellular β-amyloid load in a human cell line from rhabdomyosarcoma as a model to study muscle cells. 2. Materials and methods 2.1. Cell culture Human rhabdomyosarcoma CCL136 cells (ATCC; American Type Culture Collection, Manassas, USA), a standard in vitro model to study

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muscle cells [13–15], were cultured in Dulbecco's modified eagle medium with 10% fetal calf serum, 1 mM glutamine, 110 μg/ml sodium pyruvate, and 2 μg/ml gentamycin (all cell culture reagents from Invitrogen, Carlsbad, USA). Chamber slides and culture wells in duplicates were exposed to the cytokine TNF-α (5–10 ng/ml, R&D Systems, Minneapolis, USA) and the reagents chloroquine (50 μM, Sigma, St. Louis, USA) and rapamycin (1 μg/ml, Sigma, St. Louis, USA) in serum free X-vivo medium (Cambrex Bio Science, Wiesbaden, Germany).

mouse anti-β-amyloid antibody clone 1A10 as a capture antibody and an HRP-conjugated polyclonal rabbit anti-human β-amyloid as a detection antibody. Samples of 20–40 μg total protein were sonicated and diluted in EIA buffer to a volume of 100 μl and treated according to the manufacturer's instructions. The colorimetric reaction was measured at 450 nm with a 1420 Multilabel Counter Victor 2 (PerkinElmer, Rodgau, Germany). 3. Results

2.2. Thioflavin-S-fluorescence For fluorescent thioflavin-S-staining, cultured CCL136 muscle cells were seeded in 8-chamber slides (Nunc, Rochester, USA). Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, USA) in PBS for 10 min at room temperature, followed by further fixation in methanol at −20 °C for 10 min. Staining of amyloid aggregation was achieved by thioflavin-S (Sigma, St. Louis, USA) at 1% in distilled H2O for 5 min at room temperature. Nuclei were counterstained by 4,6-diamidino-2-phenylindole (0.5 μg/ml, Invitrogen/Molecular Probes, Carlsbad, USA) for 1 min; slides were mounted in Fluoromount G (Electron Microscopy Sciences, Hatfield, USA). Digital photography was performed on an Axiophot microscope (Zeiss, Göttingen, Germany). Appropriate filters for green (488 nm) and blue (350 nm) fluorescence, a cooled charge-coupled device digital camera (Retiga 1300; Qimaging, Burnaby, Canada) and the Image-Pro software (Media Cybernetics, Inc., Bethesda, USA) were used. For quantitative assessment a grayscale analysis was performed using the Scion image software (Scioncorp., Frederick, USA) and the value was expressed as arbitrary units. 2.3. Western blot CCL136 cells were lysed in lysis buffer (20 mM Hepes, 150 mM NaCl, 2 mM EDTA, 1% NP40, pH 7.9) containing protease inhibitors (Roche, Mannheim, Germany). Proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). After blocking with 5% skimmed milk in TBS for 1 h, membranes were incubated over night at 4 °C with the primary antibodies anti-APP/ β-amyloid (mouse monoclonal 6E10, diluted 1/1000; Signet, Dedham, USA) and anti-β-actin (mouse monoclonal, diluted 1/10,000; Sigma, St. Louis, USA). Horseradish peroxidase-conjugated goat anti-mouse antibodies (Jackson Immuno Research, Suffolk, UK) were used as secondary reagents. Blots were developed with the enhanced chemiluminescence technique (ChemiGlow West; Alpha Innotech, San Leandro, USA) following the supplier's protocol. 2.4. Inhibition of macroautophagy 3-Methyladenine (3MA) (Sigma, St. Louis, USA) and Atg12 siRNA (Qiagen Inc., Valencia, USA) together with respective control siRNA (Applied Biosystems, Foster City, USA) were used to inhibit autophagic activity. For 3MA treatment CCL136 myoblasts were plated in 8-chamber slides in serum free X-vivo medium supplemented with 10 mM 3MA with or without TNF-α. All experiments were terminated after 48 h and cells were analyzed using fluorescence microscopy. For macroautophagy inhibition by RNA interference, Atg12 specific siRNA or negative siRNA controls were used. Cells were seeded in 8-chamber wells in serum free X-vivo medium and transfected with Atg12 siRNA and negative control siRNA respectively via Nanofectin (PAA, Pasching, Austria) following the manufacturer's protocol. 2.5. β-Amyloid1–40 ELISA All samples were analyzed at least in triplicates using the β-amyloid (1–40) assay (IBL, Minneapolis, USA). This kit is designed to measure full-length β-amyloid peptides with an intact N terminus. It uses the

3.1. TNF-α induced macroautophagy regulates APP- and β-amyloid load in human skeletal muscle cells To analyze if TNF-α mediated upregulation of macroautophagy leads to modulation of APP-processing in human skeletal muscle cells CCL136 from rhabdomyosarcoma were incubated with TNF-α compared to rapamycin, which induces autophagosome formation via binding the cytosolic FK-binding protein 12 (FKBP12) and subsequent inhibition and binding of the mammalian target of rapamycin complex1 (mTORC1). Cells were left untreated or treated with the lysosomal acidification inhibitor chloroquine (CQ). CQ raises the lysosomal pH, which leads to inhibition of lysosomal protein degradation; thus, the accumulation of autophagic vesicles under CQ treatment indicates autophagic flux. For these experiments concentrations of TNF-α and rapamycin were used which have previously been proven to upregulate macroautophagy in this cell line [15]. Upon 48 h incubation with TNF-α or rapamycin muscle cells showed an increase in the APP-signal compared to untreated controls, particularly with the lysosomal acidification inhibitor CQ (Fig. 1A), which reached statistical significance upon rapamycin. The observed increase in the APP-signal upon TNF-α treatment was concentration dependent (data not shown). To determine whether TNF-α-mediated stimulation of CCL136 cells would also lead to accumulation of the APP cleaving-product β-amyloid, the muscle cells were incubated for 48 h with TNF-α. Upon exposure to TNF-α or rapamycin, a significant signal increase of a size of 7 kD was detected by western blot, which likely represents dimers of β-amyloid (Fig. 1B). 3.2. Thioflavin-S-fluorescence upon inhibition of macroautophagy in human skeletal muscle cells To further identify protein aggregation of β-amyloid, a fluorescent thioflavin-S staining was carried out to visualize β-sheet structures [13]. An increase in thioflavin-S-fluorescence could be detected upon a 48 h incubation of muscle cells with TNF-α (Fig. 2). To analyze if the TNF-α-mediated increase in the thioflavin-S-signal was dependent on macroautophagy, we analyzed fluorescence upon specific blockade. Knockdown of Atg12, a gene essential for autophagosome formation, significantly decreased the thioflavine-S signal and completely abolished the TNF-α-induced upregulation of autophagic activity (Fig. 2). Comparable findings were observed with 3MA as a pharmacological inhibitor of macroautophagy (data not shown). An ELISA-assay was used to complement the analysis by immunohistochemistry: the concentration of β-amyloid1–40 in supernatants from muscle cells was elevated upon TNF-α and downmodulated after pharmacological inhibition of macroautophagy using 3MA (Fig. 3). Although no statistical significance was reached, the trend of these data was in line with the results obtained by Western blot and immunocytochemistry. 4. Discussion In this study we show that incubation of rhabdomyosarcomaderived human skeletal muscle cells with TNF-α led to an overexpression of APP and accumulation of β-amyloid. Autophagy appears to have a protective role against diverse pathologies owing to its cellular clearance function and removal of damaged or aggregate-prone proteins

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Fig. 1. A) Increased accumulation of APP after incubation of human skeletal muscle cells with TNF-α or rapamycin in immunoblot analysis. 48 h exposure of muscle cells (CCL136) to TNF-α (10 ng/ml) or macroautophagy inductor rapamycin (1 μg/ml) led to an increase in the APP-signal in western blot analysis both with and without inhibition of lysosomal acidification via chloroquine (50 μM, 10 h). Data are representative of 3 experiments with similar results (P values: w/o vs. TNF-α: P=0.0672; w/o vs. rapamycin: *P=0.0342; +CQ vs. TNF-α+CQ: P=0.1430; +CQ vs. rapamycin+CQ: *P=0.0338). B) Increase of APP cleaving products upon TNF-α exposure of CCL136 cells in immunoblot analysis. 48 h exposure of human skeletal muscle cells to TNF-α (10 ng/ml) or rapamycin (1 μg/ml) resulted in an increase of APP cleaving products compared to control lysates. β-Actin was used as a loading control. Data are representative of 3 experiments with similar results. (P values: w/o vs. TNF-α: *P=0.0223; w/o vs. rapamycin: **P=0.0022; TNF-α vs. rapamycin: *P=0.0214.).

[16,17]. Surprisingly, the increase of β-amyloid aggregate formation in muscle cells was abolished after inhibition of macroautophagy. A potential role for macroautophagy in the production of β-amyloid has been proposed and a body of recent work points towards an active role of autophagosomes in β-amyloid generation: Upregulation of autophagy via inhibition of mTOR and deprivation of amino acids led to a significant increase in the intracellular β-amyloid load as well as increased β-amyloid secretion in a fibroblast-like cell line [18]. Furthermore, inhibition of autophagic activity via 3MA resulted in fewer β-amyloid in

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these cells, indicating a direct relationship between modulation of autophagy and β-amyloid production. Another report showed that inhibition of autophagy via 3MA significantly reduced thalamic neuronal damage, β-amyloid deposits and β-site APP-cleaving enzyme 1 (BACE1) activity [19]. Also, knockdown of Beclin-1, a mammalian homolog of yeast ATG6 and critical regulator for autophagic induction as well as treatment with 3MA markedly reduced β-amyloid deposits following cerebral infarction in rats [20]. Moreover, it has been recently reported that upregulation of macroautophagy resulted in increased

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Fig. 2. Increased thioflavin-S-fluorescence in human skeletal muscle cells upon TNF-α exposure is reversed after inhibition of macroautophagy. Muscle cells that were incubated with TNF-α (10 ng/ml) for 48 h showed increased intracellular β-amyloid accumulation reflected by increased thioflavin-S-fluorescence (green). This effect was reversed upon inhibition of macroautophagy by Atg12 specific siRNA. Photos were taken by a CCD-camera using a conventional fluorescent microscope with a 20× objective. All photomicrographs in this figure have been acquired with the same settings of camera and microscope and a grayscale analysis of the thioflavin-S staining is shown on the right. Data are depicted as mean + SD from 3 experiments (**P b 0.01; ***P b 0.001).

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Fig. 3. β-Amyloid levels in supernatant are increased upon TNF-α and blocked by 3MA. The extracellular levels of β-amyloid1-40 upon exposure to TNF-α (10 ng/ml) vs. controls for 48 h were analyzed by ELISA. The upregulation of β-amyloid upon TNF-α was downmodulated by blocking macroautophagic activity with 3MA (10 mM). Data are shown as mean+SD of three wells from one representative experiment. Significant P-values were not observed.

lysosomal β-amyloid in cultured neuroblastoma cells [21]. These reports are consistent with our findings in muscle cells that show a higher intracellular load of β-amyloid as well as an increased β-amyloid secretion upon TNF-α incubation, an effect that was blocked by inhibition of macroautophagy. Aside from the inflammatory mechanisms present in sIBM muscle, degeneration-associated inclusion bodies and vacuoles are another hallmark of this myopathy. Posttranslational mechanisms are likely to be responsible for the protein deposits observed in sIBM muscle fibers rather than uncontrolled gene activation or transcriptional malregulation. In comparison with other inflammatory myopathies, only in sIBM a positive correlation between mRNA-expression of APP and the mRNAexpression of numerous cytokines has been observed [13]. We show that exposure of human muscle cells to TNF-α in concentrations, which have previously shown to successfully upregulate autophagy in these cells, led to an overexpression of APP. In addition to this, exposure of these cells to TNF-α led to a signal increase of a 7 kD band by western blot analysis, which is indicative of β-amyloid dimers/oligomers. Fluorescence microscopy analysis revealed an increase in the staining signal for thioflavin-S, which is commonly used to visualize aggregates consisting of β-sheets [13]. The increase in thioflavinS-positive aggregates in muscle cells was prevented upon inhibition of macroautophagy via Atg12 specific siRNA or the pharmacological inhibitor 3MA. Apart from the characteristic morphological hallmarks, in Alzheimer's dementia (AD), one finds upregulation of proinflammatory cytokines including TNF-α, particularly in early disease stages. It is conceivable that these molecules function as regulating factors by increasing susceptibility towards degenerative changes at a rather early stage in AD brains [22]. Given the similarities concerning the degenerative changes between sIBM and AD, it is possible that in line with the mechanism proposed for AD, in sIBM an early cytokine-mediated upregulation of macroautophagy leads to excessive production of β-amyloid. Therefore, macroautophagy could be an important mediator between inflammatory and degenerative mechanisms in human skeletal muscle, e.g. during chronic muscle inflammation. However, the potential relevance of our findings to sIBM is yet unresolved and could be addressed e.g. by studying the co-occurrence of TNF-α and LC3. The predominant source of TNF-α are macrophages and T-cells; it is possible that secreted TNF-α acts upon muscle fibers and upregulates macroautophagic activity. Taken together we showed that TNF-α-mediated upregulation of macroautophagy modulated APP and β-amyloid load in human muscle cells. Thus, TNF-α may be a relevant cytokine during chronic muscle

inflammation, potentially also in the pathogenesis of sIBM, both maintaining an immune response as well as initiating degenerative changes via induction of macroautophagy. It is rather unlikely that blockade of TNF-α would be sufficient to reduce the autophagic activity in skeletal muscle and no other agent is available to accomplish this. A drug that could potentially block autophagic activity may be able to diminish the ongoing production/processing of APP/β-amyloid in skeletal muscle, but a clearance of protein aggregations of β-amyloid is rather not expected. A pilot study on sIBM with the TNF-α fusion protein etanercept did not show a significant effect between treatment and control group after 6 months [23]. The lack of efficacy could be explained in that elevated TNF-α levels may be an early change and that, once degeneration has evolved, the disease progression cannot be halted. It is speculative if a TNF-α block during a very early phase of the disease would be more efficient. Yet, treatment during early stages of the disease is hampered by the fact that sIBM is typically diagnosed after several years of its presumed onset with inflammatory and/or degenerative changes in the skeletal muscle. On the other hand, it is possible that other cytokines such as IL-1β or downstream cascades like nitric oxide are more relevant to the disease pathology [24]. It will be of interest to further explore the complex network of pathomechanisms in sIBM. Collectively, our data suggest a functional interrelationship between TNF-α-mediated regulation of macroautophagy and accumulation of aberrant molecules in human skeletal muscle cells. Further experiments will be needed to study the relevance of these mechanisms to sIBM. Conflict of interest None. Acknowledgment We gratefully acknowledge Claudia Fokken and Konstanze Kleinschnitz for the technical advice on immunoblotting and immunofluorescent staining. The excellent technical support by Nicole Tasch and Fatma Betül Agdas is gratefully acknowledged. JS was supported by the Deutsche Forschungsgemeinschaft (DFG, SCHM 1669/2-1) and this study was funded by the Fritz Thyssen Stiftung (Az 10.08.2.168 to JS). References [1] Klionsky DJD, Emr SDS. Autophagy as a regulated pathway of cellular degradation. Science 2000;290:1717–21. [2] Mizushima N, Klionsky DJ. Protein turnover via autophagy: implications for metabolism. Annu Rev Nutr 2007;27:19–40. [3] Wang Y, Singh R, Massey AC, Kane SS, Kaushik S, Grant T, et al. Loss of macroautophagy promotes or prevents fibroblast apoptosis depending on the death stimulus. J Biol Chem 2008;283:4766–77. [4] Münz CC. Macroautophagy during innate immune activation. Front Microbiol 2011;2:72. [5] Cuervo AM, Stefanis L, Fredenburg R, Lansbury PT, Sulzer D. Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy. Science 2004;305: 1292–5. [6] Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, et al. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet 2004;36:585–95. [7] Harris J. Autophagy and cytokines. Cytokine 2011;56:140–4. [8] Andrade RMR, Wessendarp MM, Gubbels M-JM, Striepen BB, Subauste CSC. CD40 induces macrophage anti-Toxoplasma gondii activity by triggering autophagydependent fusion of pathogen-containing vacuoles and lysosomes. J Clin Invest 2006;116:2366–77. [9] Jia G, Cheng G, Gangahar DM, Agrawal DK. Insulin-like growth factor-1 and TNF-alpha regulate autophagy through c-jun N-terminal kinase and Akt pathways in human atherosclerotic vascular smooth cells. Immunol Cell Biol 2006;84: 448–54. [10] Needham M, Mastaglia FL. Sporadic inclusion body myositis: a continuing puzzle. Neuromuscul Disord 2008;18:6–16. [11] Schmidt J, Dalakas MC. Inclusion-body myositis in the elderly: an update. Aging Health 2010;6:687–94. [12] Askanas V, Engel WK, Nogalska A. Inclusion body myositis: a degenerative muscle disease associated with intra-muscle fiber multi-protein aggregates, proteasome

C.W. Keller et al. / Journal of the Neurological Sciences 325 (2013) 103–107

[13]

[14]

[15]

[16]

[17] [18]

[19]

inhibition, endoplasmic reticulum stress and decreased lysosomal degradation. Brain Pathol 2009;19:493–506. Schmidt J, Barthel K, Wrede A, Salajegheh M, Bähr M, Dalakas MC. Interrelation of inflammation and APP in sIBM: IL-1 beta induces accumulation of beta-amyloid in skeletal muscle. Brain 2008;131:1228–40. Lünemann JD, Schmidt J, Schmid D, Barthel K, Wrede A, Dalakas MC, et al. β-Amyloid is a substrate of autophagy in sporadic inclusion body myositis. Ann Neurol 2007;61: 476–83. Keller CW, Fokken C, Turville SG, Lünemann A, Schmidt J, Münz C, et al. TNF-alpha induces macroautophagy and regulates MHC class II expression in human skeletal muscle cells. J Biol Chem 2011;286:3970–80. Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y, Suzuki-Migishima R, et al. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature 2006;441:885–9. Martínez-Vicente M, Cuervo AM. Autophagy and neurodegeneration: when the cleaning crew goes on strike. Lancet Neurol 2007;6:352–61. Yu WH, Cuervo AM, Kumar A, Peterhoff CM, Schmidt SD, Lee J-H, et al. Macroautophagy—a novel beta-amyloid peptide-generating pathway activated in Alzheimer's disease. J Cell Biol 2005;171:87–98. Zhang J, Zhang Y, Li J, Xing S, Li C, Li Y, et al. Autophagosomes accumulation is associated with β-amyloid deposits and secondary damage in the thalamus after focal cortical infarction in hypertensive rats. J Neurochem 2012;120:564–73.

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[20] Xing S, Zhang Y, Li J, Zhang J, Li Y, Dang C, et al. Beclin 1 knockdown inhibits autophagic activation and prevents the secondary neurodegenerative damage in the ipsilateral thalamus following focal cerebral infarction. Autophagy 2012;8: 63–76. [21] Zheng L, Terman A, Hallbeck M, Dehvari N, Cowburn RF, Benedikz E, et al. Macroautophagy-generated increase of lysosomal amyloid β-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells. Autophagy 2011;7: 1528–45. [22] Yamamoto M, Kiyota T, Horiba M, Buescher JL, Walsh SM, Gendelman HE, et al. Interferon-gamma and tumor necrosis factor-alpha regulate amyloid-beta plaque deposition and beta-secretase expression in Swedish mutant APP transgenic mice. Am J Pathol 2007;170:680–92. [23] Barohn RJ, Herbelin L, Kissel JT, King W, McVey AL, Saperstein DS, et al. Pilot trial of etanercept in the treatment of inclusion-body myositis. Neurology 2006;66: 123–4. [24] Schmidt J, Barthel K, Zschüntzsch J, Muth IE, Swindle EJ, Hombach A, et al. Nitric oxide stress in sporadic inclusion body myositis muscle fibres: inhibition of inducible nitric oxide synthase prevents interleukin-1β-induced accumulation of β-amyloid and cell death. Brain 2012;135:1102–14.