Cacciottolo M, Nogalska A, D'Agostino C, Engel W K, Askanas V
USC Neuromuscular Center, Department of Neurology, University of Southern California Keck School of Medicine, Good Samaritan Hospital, Los Angeles, CA, USA.
Neuropathol Appl Neurobiol. 2013 Dec;39(7):750-61. doi: 10.1111/nan.12038.
Sporadic inclusion-body myositis (s-IBM) is an age-associated degenerative muscle disease. Characteristic features are muscle-fibre vacuolization and intramuscle-fibre accumulations of multiprotein aggregates, which may result from the demonstrated impairments of the 26S proteasome and autophagy. Chaperone-mediated autophagy (CMA) is a selective form of lysosomal degradation targeting proteins carrying the KFERQ motif. Lysosome-associated membrane protein type 2A (LAMP2A) and the heat-shock cognate protein 70 (Hsc70) constitute specific CMA components. Neither CMA components nor CMA activity has been studied in normal or disease human muscle, to our knowledge.
We studied CMA components by immunocytochemistry, immunoblots, real-time PCR and immunoprecipitation in: (a) 16 s-IBM, nine aged-matched normal and nine disease control muscle biopsies; and (b) cultured human muscle fibres (CHMFs) with experimentally inhibited activities of either the 26S proteasome or autophagy.
Compared with age-matched controls, in s-IBM muscle, LAMP2A and Hsc70 were on a given transverse section accumulated as aggregates in approximately 5% of muscle fibres, where they (a) colocalized with each other and α-synuclein (α-syn), a CMA-targeted protein; and (b) were bound to each other and to α-syn by immunoprecipitation. By immunoblots, LAMP2A was increased sevenfold P < 0.001 and Hsc70 2.6-fold P < 0.05. LAMP2A mRNA was increased 4.4-fold P < 0.001 and Hsc70 mRNA 1.9-fold P < 0.05. In CHMFs inhibition of either the 26S proteasome or autophagy induced CMA, evidenced by a significant increase of both LAMP2A and Hsc70.
Our study demonstrates, for the first time, up-regulation of CMA components in s-IBM muscle, and it provides further evidence that altered protein degradation is likely an important pathogenic aspect in s-IBM.
散发性包涵体肌炎（s-IBM）是一种与年龄相关的退行性肌肉疾病。其特征性表现为肌纤维空泡化以及肌纤维内多蛋白聚集体的积累，这可能是由已证实的26S蛋白酶体和自噬功能受损所致。伴侣介导的自噬（CMA）是一种溶酶体降解的选择性形式，靶向携带KFERQ基序的蛋白质。2型溶酶体相关膜蛋白（LAMP2A）和热休克同源蛋白70（Hsc70）构成特定的CMA组分。据我们所知，在正常或患病的人类肌肉中，尚未对CMA组分和CMA活性进行研究。
我们通过免疫细胞化学、免疫印迹、实时PCR和免疫沉淀法研究了CMA组分，研究对象包括：（a）16例s-IBM患者、9例年龄匹配的正常人和9例疾病对照的肌肉活检样本；（b）26S蛋白酶体或自噬活性受到实验性抑制的培养人肌纤维（CHMFs）。
与年龄匹配的对照组相比，在s-IBM肌肉中，LAMP2A和Hsc70在给定的横切面上以聚集体形式在约5%的肌纤维中积累，在这些肌纤维中它们：（a）彼此共定位，且与α-突触核蛋白（α-syn）共定位，α-syn是一种CMA靶向蛋白；（b）通过免疫沉淀法彼此结合并与α-syn结合。通过免疫印迹法，LAMP2A增加了7倍（P<0.001），Hsc70增加了2.6倍（P<0.05）。LAMP2A mRNA增加了4.4倍（P<0.001），Hsc70 mRNA增加了1.9倍（P<0.05）。在CHMFs中，26S蛋白酶体或自噬的抑制诱导了CMA，表现为LAMP2A和Hsc70均显著增加。
我们的研究首次证明了s-IBM肌肉中CMA组分的上调，并进一步证明蛋白质降解改变可能是s-IBM的一个重要致病因素。
