Chronic activation of the classical NF-κB signaling pathway in DMD muscle leads to many of the pathological symptoms in DMD (6
), and it has been shown that inhibition of NF-κB activation ameliorates dystrophic muscle pathology (17
). We show, for the first time, the role of A20 overexpression as a potential therapeutic target for DMD in mdx
mice. We assessed the effect on skeletal muscle pathology in mdx
mice of muscle-specific overexpression of A20. We found that increased protein expression of A20 led to a significant decrease in NF-κB pathway activation in quadriceps of mdx
mice. We also observed an increase in protein levels of RelB, a subunit of the alternate pathway of NF-κB activation, and Myf-5, a muscle transcription factor required for differentiation. Moreover, we observed a decrease in the number of fibers with centrally placed nuclei and a decrease in markers of regeneration in the quadriceps of mdx
mice. Lastly, we detected a reduction in the number of infiltrating inflammatory CD4 T cells in the AAV8-tMCK-A20–treated quadriceps. Taken together, we can conclude that A20 overexpression can reduce the activation of NF-κB and is thus an attractive candidate to further explore as a therapeutic target for the treatment of DMD.
Expression of A20 after systemic delivery with an AAV8 vector carrying the murine A20 cDNA driven by the muscle-specific tMCK promoter was increased 1.4-fold in quadriceps. One possible reason for this modest increase in A20 protein levels may have been due to delivery of an insufficient number of viral genomes. Also, levels of A20 may be affected by its expression by a single-stranded AAV (ssAAV) and not double-stranded AAV (dsAAV) vector. Our studies confirmed prior findings that the dsAAV vector and the tMCK promoter were able to drive robust GFP expression in the heart and skeletal muscles, but not in the liver (26
). We confirmed, however, that the tMCK promoter provided transgene expression levels in skeletal muscle that was superior to the ubiquitous CMV promoter. Thus, even with the modest 1.4-fold increase of A20 expression, we provide proof-of-concept by the observation of a significant reduction in the activation of the NF-κB pathway in skeletal muscle. This result supports the hypothesis that overexpression of A20 would regulate the NF-κB pathway in muscle.
Interestingly, we also observed an increase in the expression levels of RelB, which reflects the NF-κB alternate pathway. We previously showed that RelB is overexpressed in regenerating fibers of dystrophic muscle and that A20 plays a role in muscle regeneration by inhibiting the classical but not the alternate pathway of NF-κB activation. Also, the alternate pathway of NF-κB activation was shown by others to be required for myogenesis and maintenance of mature myofibers (31
). Thus, we can speculate that overexpression of A20 inhibits the classical pathway of NF-κB activation, which leads to the upregulation of the alternate pathway, promoting muscle regeneration. We also observed an increase in Myf-5, but not MyoD, levels in quadriceps of AAV8-A20–treated mice. Myf-5 and MyoD play essential but redundant roles in muscle development (14
), which might explain our findings of increased expression of Myf-5 but not MyoD. However, the dose-dependent effect of AAV8-A20 on the levels of these transcription factors remains to be evaluated.
Absence of dystrophin in skeletal muscle leads to loss of integrity of the muscle membrane, causing muscle fibers to undergo cycles of degeneration and regeneration (32
). Because of this constant damage to muscle, myofibers become unstable, ultimately leading to protein degradation and muscle atrophy (32
). We analyzed the effect of overexpression of recombinant A20 in quadriceps of AAV8-tMCK-A20–treated mdx
mice on this cycle of damage and repair and observed a significant decrease in the number of fibers with centrally placed nuclei, a well-studied marker of continuous degeneration and regeneration. Earlier studies have also shown that reduced activation of the classical NF-κB pathway causes decreased regeneration and increased stability of muscle (17
). Consistent with these studies, we also observed a decrease in the number of regenerating fibers in quadriceps of AAV8-A20–treated mdx
mice. This result suggests a decrease in the damaging cycles of myofiber degeneration and regeneration and an overall improvement in muscle health and stability in the skeletal muscles.
The muscle pathology in DMD is thought to be caused by an imbalance between the amount of regeneration and the amount of necrosis in muscle tissue (32
). One of the reasons muscle fibers degenerate is because of NF-κB–induced activation of transcription targets such as MuRF1 and atrogin-1 that mediate up-regulation of the ubiquitin-proteasome pathway causing degradation of muscle fibers (28
). We analyzed the effect of A20 overexpression on the number of necrotic fibers in skeletal muscle of treated mdx
mice. Surprisingly, overexpression of A20 did not have any effect on necrosis in quadriceps muscle collected at 8 wks of age. We observed no change in the number of necrotic fibers in AAV8-tMCK-A20–treated compared with saline-treated mdx
mice. Thus, although we observe a reduction in the markers for regeneration in quadriceps muscles, there is no change in degeneration in the muscles of the treated mice. One explanation for this could relate to the relatively low dose of the A20 transgene delivered to each mouse. Because the A20 transgene was delivered to neonates, its early expression during development could help to maintain the health and stability of muscle fibers by inhibiting activation of the NF-κB pathway. However, the relatively low A20 transgene expression may not have been sufficient to sustain its function to completely inhibit NF-κB activation leading to the transcription of its downstream targets, thus causing necrosis of muscle fibers. Future studies to determine a dose effect of the delivered transgene and the effect of mouse age at the time of gene transfer could aid in the further understanding of the dynamic relationship between necrosis and regeneration in diseased skeletal muscle.
Chronic activation of the NF-κB pathway in skeletal muscle leads to increased concentrations of cytokines and chemokines and the infiltration of inflammatory cells such as macrophages (37
). Specifically, CD4 and CD8 T cells were shown to play a role in dystrophic pathology, and depletion of these cells led to improvement of histopathology in mdx
). Because A20 causes reduction of the classical NF-κB pathway activation, we speculated that this reduction would decrease the amount of inflammation in muscle. We observed a reduction in the number of infiltrating CD4 T cells in the quadriceps of AAV8-tMCK-A20–treated mdx
mice. We did not, however, observe any decrease in the number of CD8 T cells in these mice. Although, both CD4 and CD8 T cells are known to play a role in inflammation in mdx
mice, CD4 T cells are more prevalent in mdx
). Furthermore, muscle biopsies from DMD patients showed a predominance of CD4 T cells compared with CD8 T cells (32
). Also, CD4 T cells were found to colocalize with macrophages and degenerating muscle fibers, whereas CD8 T cells were scattered throughout muscle tissue (39
). Hence, it was speculated that CD4 T cells, and not CD8 T cells, played a major role in causing cytotoxic muscle damage (39
). A recent study showed that AAV vectors alone were capable of activating the alternate NF-κB pathway in HeLa cells (41
). This was an interesting observation from a therapeutic point of view, since AAV-mediated transfer would be able to not only inhibit the classical pathway, reducing inflammation, but also promote activation of the alternate pathway, promoting improved muscle health.