Proteasome Inhibition and Aggresome Formation in Sporadic Inclusion-Body Myositis and in Amyloid-β Precursor Protein-Overexpressing Cultured Human Muscle Fibers

Abstract

The 26S proteasome system is involved in eliminating various proteins, including ubiquitinated misfolded/unfolded proteins, and its inhibition results in cellular accumulation of protein aggregates. Intramuscle-fiber ubiquitinated multiprotein-aggregates are characteristic of sporadic inclusion-body myositis (s-IBM) muscle fibers. Two major types of aggregates exist, containing either amyloid-beta (Abeta) or phosphorylated tau (p-tau). We have now asked whether abnormalities of the 26S proteasome contribute to s-IBM pathogenesis and whether the multiprotein aggregates have features of aggresomes. Using cultured human muscle fibers we also studied the effect of amyloid-beta precursor protein (AbetaPP) overexpression on proteasome function and the influence of proteasome inhibition on aggresome formation. We report that in s-IBM muscle biopsies 26S proteasome subunits were immunodetected in the gamma-tubulin-associated aggresomes, which also contained Abeta, p-tau, ubiquitin, and HSP70. In addition, a) expression of proteasome subunits was greatly increased, b) the 20Salpha proteasome subunit co-immunoprecipitated with AbetaPP/Abeta, and c) the three major proteasomal proteolytic activities were reduced. In cultured muscle fibers, AbetaPP-overexpressing fibers displayed diminished proteasomal proteolytic activities, and addition of proteasome inhibitor strikingly increased aggresome formation. Accordingly, proteasome dysfunction in s-IBM muscle fibers may play a role in accumulation of misfolded, potentially cytotoxic proteins and may be induced by increased intracellular AbetaPP/Abeta.

AβPP-overexpression and proteasome inhibition increase αB-crystallin in cultured human muscle: Relevance to inclusion-body myositis

“…In six culture sets, each established from a different biopsy, a 3 Kb 751 AβPP-cDNA, in either sense or anti-sense orientation, was transferred into three-week-old cultured muscle fibers, using a replication-deficient adenovirus (RDAV) vector, at 0.3×10 8 pfu/ml culture medium, as detailed [4][5][6][7]. (In this culture model, an increase of AβPP and Aβ is visible by immunoblots and Elisa within 24-48 hours after AβPP gene-transfer, and morphologic aspects of IBM appear approximately 14-21 days after the transfer.)…”

“…Three days after transfer, experimental and control (non-AβPP-overexpressing) cultures were treated with 1μM epoxomicin (Biomol Research Laboratories, Plymouth Meeting, PA), an irreversible proteasome inhibitor [22]. 24 hours thereafter, the cultures were processed for light-microscopic immunocytochemistry, immunoblotting, and combined immunoprecipitation/immunoblotting, as described [4][5][6][7]11,23,24].…”

“…Rabbit polyclonal antibody against γ-tubulin (Santa Cruz, CA), diluted 1:150, was used to identify aggresomes [4].…”

“…Two hypotheses regarding the key pathogenic mechanisms involved in s-IBM are: a) an amyloid-β-related myodegenerative process, and b) an immune dysregulation [reviewed in 2,3]. Intriguingly, the s-IBM muscle-fiber phenotype has similarity to the Alzheimer-disease brain, such as accumulations of multiproteinaggregates containing proteins in congophilic alternate conformation, including amyloid-β (Aβ) [2].Reduction of 26S proteasome activity and features of aggresomes were recently demonstrated within s-IBM muscle fibers, and these were modeled in cultured human muscle by experimental overexpression of AβPP [4]. Abnormal increase of AβPP/Aβ appears to be an early upstream step in the IBM pathogenesis because a) AβPP/Aβ accumulation appears to precede other detected abnormalities ins-IBM muscle fibers [2], and b) several aspects of the IBM phenotype were produced in cultured normal human muscle fibers (CHMFs) after experimental long-term overexpression of AβPP [5][6][7].…”

Immune and myodegenerative pathomechanisms in inclusion body myositis

“…Fratta et al have reported that the 26S proteasome co‐stains with A β , phosphorylated tau, ubiquitin, and heat shock protein 70 (Hsp70) in muscle biopsies from IBM patients 242. While protein expression of proteasomal subunits 19S, 20S α , and 20S β is greatly increased in IBM muscle compared to age‐matched controls, proteolytic activity of the proteasomal machinery is significantly impaired in IBM muscle.…”