Immunoproteasome in animal models of Duchenne muscular dystrophy

Abstract

Increased proteasome activity has been implicated in the atrophy and deterioration associated with dystrophic muscles of Duchenne muscular dystrophy (DMD). While proteasome inhibitors show promise in the attenuation of muscle degeneration, proteasome inhibition-induced toxicity was a major drawback of this therapeutic strategy. Inhibitors that selectively target the proteasome subtype that is responsible for the loss in muscle mass and quality would reduce side effects and be less toxic. This study examined proteasome activity and subtype populations, along with muscle function, morphology and damage in wild-type (WT) mice and two murine models of DMD, dystrophin-deficient (MDX) and dystrophin- and utrophin-double-knockout (DKO) mice. We found that immunoproteasome content was increased in dystrophic muscles while the total proteasome content was unchanged among the three genotypes of mice. Proteasome proteolytic activity was elevated in dystrophic muscles, especially in DKO mice. These mice also exhibited more severe muscle atrophy than either WT or MDX mice. Muscle damage and regeneration, characterized by the activity of muscle creatine kinase in the blood and the percentage of central nuclei were equally increased in dystrophic mice. Accordingly, the overall muscle function was similarly reduced in both dystrophic mice compared with WT. These data demonstrated that there was transformation of standard proteasomes to immunoproteasomes in dystrophic muscles. In addition, DKO that showed greatest increase in proteasome activities also demonstrated more severe atrophy compared with MDX and WT. These results suggest a putative role for the immunoproteasome in muscle deterioration associated with DMD and provide a potential target for therapeutic intervention.

The ubiquitin ligase tripartite-motif-protein 32 is induced in Duchenne muscular dystrophy

“…6,8,9 To confirm the relevance of this observation in human DMD, we determined via a standard proteasome activity assay that the chymotrypsin-like 26S proteasome activity was six times higher in the muscle biopsies of six genetically determined DMD patients than in four healthy controls ( Figure 1). We next quantitated the protein levels of the Ub ligases Atrogin1, CHiP, MuRF-1, NEDD4, Ozz, and TRIM32 in muscle lysates of six DMD patients.…”

“…4 In skeletal muscle, the ubiquitin-proteasome system has an important role in the maintenance of muscle mass and proteasome dysfunctions are now linked to genetic primary myopathies. [5][6][7] We previously showed that in mdx mice, a naturally occurring mouse model of DMD, systemic treatment with the proteasome inhibitors (PIs) MG-132 or bortezomib (Velcade) up-regulated the membrane expression of DGC members and reduced the inflammatory reaction related to myofiber damage, thus improving the dystrophic phenotype. 3,8,9 Similarly, in golden retriever muscular dystrophy, bortezomib improved a few histopathological features of dystrophindeficient skeletal muscles and blocked the activation of the pro-inflammatory pathway triggered by phospho-nuclear factor kB.…”

Denervation-Induced Activation of the Standard Proteasome and Immunoproteasome

“…An enriched proteasome preparation was modified from those previously described [21,22]. One half of the frozen GAS muscle was sealed in a pouch, dipped in liquid nitrogen and crushed using a hammer and pestle.…”

“…Proteasome activities were determined using fluorogenic peptide substrates as previously described [22]. LLE-AMC (Proteasome Substrate II, Fluorogenic, EMDmillipore, Billerica, MA), LLVY-AMC (Proteasome Substrate III, Fluorogenic, EMDmillipore, Billerica, MA) and VGR-AMC (Bz-Val-Gly-Arg-AMC, ENZO) were used for caspase-, chymotrypsin-, and trypsin- like activities, respectively.…”

“…The immunoproteasome is well-known for its role in immune function, specifically generation of antigenic peptides [16] as part of immune surveillance [17–19]. Interestingly, under catabolic conditions such as aging [20,21], muscular dystrophy [22], and denervation [11], the immunoproteasome increases, which suggests a link between atrophy and the immunoproteasome may exist in skeletal muscle. A more extensive investigation is required to establish the validity of this assumption.…”