Download Patti Weed – DMD

Patti Weed
Duchenne muscular dystrophy and the attempts at finding a cure
Duchenne muscular dystrophy (DMD) is a sex-linked disease on the X-chromosome, which
causes it to be more common in males. DMD is found in one out of every 3,500 boys (Campbell & Reece,
2005). Boys with DMD are dependent on a wheelchair between the ages of 6-12 and die in their 20s
(Acharyya et al., 2007). DMD is a genetic disease coupled with progressive muscle degeneration and
premature death (Acharyya et al., 2007). The muscle degeneration is caused by the lack of a vital
muscular protein called dystrophin (Campbell & Reece, 2005). Without this protein, muscle cells pull
away from the membrane during simple movements causing the muscle cells to become severely
damaged, such that they cannot repair themselves properly (Grounds, 2008). The process of muscle
damage and repair normally occurs in response to exercise induced damage; however, due to a gene
mutation and the chronic inflammation that coincides with the disease, muscle repair is inhibited.
One solution to DMD is to fix the mutation in the dystrophin gene that causes DMD through gene
therapy. As one of the following papers will show, this approach has worked in the mdx mouse model, but
gene therapy in humans has been attempted in numerous studies and has so far been unsuccessful. As a
side note, mdx mice are good models to use because mdx mice have the same dystrophin lacking mutation
as humans causing DMD to be very similar between mdx mice and humans (Bulfield et al., 1984).
Because human gene therapies are unavailable and inflammation seems to inhibit muscle growth, the
inflammatory pathway is currently being targeted by therapeutic interventions.
Using pharmaceutical drugs to target certain pathways can lead to an amelioration of the severity
of the disease. Specifically by using therapeutic drugs to inhibit the IKK/NF-ƘB signaling pathway,
DMD muscle pathology improved. Deleting IKKβ altogether reduces inflammation in DMD muscle
(Acharyya et al., 2007). The problem associated with the therapeutic drugs used is the severe adverse side
effects, such as weight gain, behavioral changes, facial puffiness, bruising, and osteoporosis (Manzur et
al., 2004). Learning more about DMD can help scientists and doctors control and stabilize the
deterioration of muscles to prolong the life of boys with DMD.
The primary cause of DMD is the lack of dystrophin production due to a mutation or deletion in
the dystrophin gene. Haslett et al. (2002) compared gene expression in DMD muscle and normal skeletal
muscle to understand better the difference of protein composition in DMD muscle. The results also will
assist in comparing the pathophysiological pathways in DMD and normal muscle. In this study, twelve
quadriceps biopsies were taken from DMD patients and another twelve were taken from unaffected
patients. Each biopsy was analyzed individually by immunofluorescence and then two statistical tests
were done to compile an acceptable list of differentially expressed genes between the DMD and normal
skeletal muscle (Haslett et al., 2002).
The first statistical test was a t-test. Two hundred thirty-five genes were recognized with 173
overexpressed and 62 underexpressed genes in DMD muscle. The next test was a geometric fold change
analysis in which 112 genes were identified as overexpressed and 21 genes were underexpressed in DMD
muscle. After comparing the two statistical tests and eliminating the false positives, a final list of 105
genes was considered differentially expressed (Haslett et al, 2002).
Dystrophin was significantly underexpressed in the DMD muscle and normally expressed in the
unaffected muscle. Genes that were overexpressed in DMD muscle included immune response signals
and extracellular matrix genes, which relates to the abundance of inflammatory cells in DMD muscle.
Also overexpressed were muscle structure genes because of the regenerating muscle characteristic of
DMD. Putting these genes in pathophysiological pathways will help us to understand the molecular
pathology better (Haslett et al., 2002).
The molecular pathology of DMD is not well understood, but in an experiment by Acharyya et al.
(2007) the IKK/NF-ƘB signaling pathway was examined and determined to be a good therapeutic target
for DMD. Again in this experiment, the mdx mouse model was used and biopsies of DMD muscle were
extracted. Multiple tests were done in order to analyze the IKK/NF-ƘB signaling pathway. First, an
EMSA (electrophoretic mobility shift assay) analysis of DMD muscle was done, which determined that
NF-ƘB binding is activated in immune cells. Therefore, NF-ƘB activity is related to the degeneration of
DMD muscle. NF-ƘB is a protein complex that regulates the immune response. In addition, it was found
that if there is more NF-ƘB activity, then the severity of DMD increases in the patient. Next, in a Western
blot analysis, it was determined that IKK activity increased in DMD muscle. IKK is IκB kinase, which
is an enzyme complex related to NF-ƘB (Acharyya et al., 2007).
After determining the importance of NF-ƘB and IKK in DMD, more tests were completed to find
the consequences of eliminating them. It was discovered that deleting IKKβ reduced inflammation.
Reducing inflammation is vital for DMD muscle because it allows the muscles to regenerate like normal
skeletal muscle after injury. Finally, an IKK inhibiting peptide and a NF-ƘB inhibiting peptide were
injected into the mice and showed that pharmacological inhibition could create the same results as
eliminating the IKK/NF-ƘB signaling pathway. Overall, Acharyya et al. (2007) discovered that the
IKK/NF-ƘB signaling pathway is a good therapeutic aim for the mitigation of DMD. Inhibiting this
pathway will allow for the inflammation to decrease and for the DMD muscle to regenerate healthily
(Acharyya et al., 2007).
A common drug used to hinder the IKK/NF-ƘB signaling pathway is prednisone, a type of
glucocorticoid or steroid hormone. This drug slows the progression of muscle wasting, which over time
increases the mass and strength of the muscle (Moxley III et al., 2005). The main problem with
prednisone is the side effects associated with the drug, which include weight gain, behavioral changes,
facial puffiness, bruising, and osteoporosis (Manzur et al., 2004). Deflazacort, a prednisone-derivative,
yields similar results as prednisone, but with fewer side effects. Deflazacort has its main effects on
improving the diaphragm muscle (Moxley III et al., 2005).
St-Pierre et al. (2004) showed how deflazacort works and what pathways it affects were
specifically studied. Once again, the mdx mouse model and DMD muscle biopsies were examined.
Various doses of deflazacort were injected into the mice, and after their death, muscle biopsies were
removed. After analyzing the biopsies, it was found that deflazacort activates calcineurin, which then
activates NF-ATc1 dephosphorylation and creates the correct gene expression in the muscle. Calcineurin
is a vital protein phosphatase (St-Pierre et al., 2004).
It is known that the JNK1 signaling pathway is partially accountable for DMD, and it has been
debated whether deflazacort affects the JNK1 pathway. St-Pierre et al. (2004) discovered that deflazacort
affects the calcineurin/NF-AT pathway, which counteracts the harmful JNK1 pathway allowing the drug
to alleviate DMD. This experiment suggests that perhaps a better drug could be found that would
specifically increase calcineurin, and at the same time inhibit the JKN1 pathway. Alternatively, gene
therapy could be used to activate calcineurin (St-Pierre et al., 2004).
Gene therapy to replace a defective dystrophin gene has been successful in the mdx mouse model
Denti et al. (2006), but not yet in human patients. The mutated dystrophin lacking gene can be fixed by
skipping over the mutated exon 23 at the posttranscriptional level. In this study, exon 23 was skipped by
creating an antisense sequence in RNA into an AVV vector injection and injecting it into the mdx mice.
Six weeks after injection, biopsies from the DMD muscle were examined by immunofluorescence and
showed that dystrophin was expressed (Denti et al., 2006). Two exercise tests were demonstrated that
DMD muscle could regenerate after exercise. In fact, the treated muscle fibers were stronger after
exercise indicating that the treatment caused the cells to produce functional dystrophin (Denti et al.,
2006).
Duchenne muscular dystrophy is a fatal disease, which takes the lives of young boys early in their
lives. It is caused by the lack of the protein dystrophin and chronic inflammation that does not allow the
muscle to regenerate even after simple movements (Acharyya et al., 2007). The papers reviewed here
suggest that effective treatments may soon be available.
References
Acharyya, S., Villalta, S.A., Bakkar, N., Bupha-Intr, T., Janssen, P.M.L., Carathers, M., et al. (2007).
Interplay of IKK/NF-κB signaling in macrophages and myofibers promotes muscle degeneration
in Duchenne muscular dystrophy. The Journal of Clinical Investigations, 117(4): 889-901.
Bulfield, G., Siller, W.G., Wight, P.A., & Moore, K.J. (1984). X chromosome-linked muscular dystrophy
(mdx) in the mouse. Proc. Natl. Acad. Sci. U. S. A., 81(4): 1189–92.
Campbell, N.A., & Reece, J.B. (2005). Biology (7th ed.). San Francisco: Pearson Education, Inc.
Denti, M.A., Rosa, A., D’Antona, G., Sthandier, O., De Angelis, F.G., Nicoletti, C., et al. (2006). Bodywide gene therapy of Duchenne muscular dystrophy in the mdx mouse model. The National
Academy of Sciences of the USA, 103(10): 3758-63.
Grounds, M.D. (2008). Two-tiered hypotheses for Duchenne muscular dystrophy. Cellular and Molecular
Life Sciences, 65(11): 1621-5.
Haslett, J.N., Sanoudou, D., Kho, A.T., Bennett, R.R., Greenberg, S.A., Kohane, I.S., et al. (2002). Gene
Expression comparison of biopsies from Duchenne muscular dystrophy (DMD) and normal
skeletal muscle. Proceedings of the National Academy of Sciences, 99(23): 15000-15005.
Manzur, A.Y., Kuntzler, T., Pike, M., & Swan, A. (2004). Glucocorticoid corticosteroids for Duchenne
muscular dystrophy. Cochrane Database Syst Rev, 2: CD003725.
Moxley III, R.T., Ashwal, S., Pandya, S., Connolly, A., Florence, J., Mathews, K., et al. (2005). Practice
Parameter: Corticosteroid Treatment of Duchenne Dystrophy. American Academy of Neurology,
64: 13-20.
St-Pierre, S.J.G., Chakkalakal, J.V., Kolodziejczyk, S.M., Knudson, J.C., Jasmin, B.J., Megeney, L.A.
(2004). Glucocorticoid treatment alleviates dystrophic myofiber pathology by activation of the
calcineurin/NF-AT pathway. The FASEB Journal, 18:1937-39.