By Sarah Obenrader
May 2nd, 2003
This web page was produced as an assignment for an undergraduate course at Davidson College
Greene, J. C; et al. 2003. Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants. PNAS. 100(7): 4078-4083.
Individuals suffering from autosomal recessive juvenile parkinsonism (AR-JP), a form of Parkinson’s disease (PD), experience the loss of dopaminergic neurons in the sustantia nigra pars compacta and a disruption in locomotion of their muscles, both of which seem to be linked to mitochondrial dysfunction and apoptotic cell death. The cause of this disease is related to mutations in the parkin gene, which encodes for an ubiquitin-protein ligase. However, how mitochondrial dysfunction leads to apoptosis is unknown, and so this paper attempts to elucidate a mechanism for selective cell death in AR-JP using a Drosophila model of the disease.
In order to create a Drosophila model for AR-JP, they used the human Parkin sequence to locate the Drosophila homolog, and found only one potential gene, which is depicted in Figure 1A. This was a very good candidate for the parkin gene as it had a 59% sequence similarity to the human version. It was also the only gene in the Drosophila genome that encodes for a protein with an ubiquitin-like domain that has protein ligase activity, and these proteins are suspected to play a role in the mitochondrial function. In addition the protein also contains ring-finger domains and an In-between ring domain, all of which are found in its human counterpart. After identifying a homolog, they wanted to further insure this was the correct gene by testing its presence at different developmental stages. By doing a Northern blot with an anti-parkin probe, shown in Figure 1B, they illustrated that parkin was found in wild type flies at the embryonic, larval and adult stage. This was an important test for them to perform because in order for it to be the parkin gene it must be expressed throughout the organism life to cause AR-JP or PD. The last part of figure C depicts the parkin transcript along with the P element, the different deletions used and the sites of point mutations that occurred in their experimental model.
Another phenotypic change that arises in parkin mutants is male sterility, and so this issue was further analyzed by dissecting the testes of normal and abnormal males and studying the sperm cells. Figure 2 very clearly illustrates the difference in sperm cells between wild type and mutant flies and highlights the effect that the absence of parkin can have on mitochondria. The inset in figure 2B explains why parkin- males are sterile: their mature spermatids stay in a 64-cell cluster instead of individualizing. The malformed spermatids are obviously a consequence of the misshapen and highly irregular nebenkern (a specialized mitochondria) seen in figure 2F (as compared to the wild type in 2E). Thus it seems that male sterility is due to mitochondrial dysfunction in the case of the parkin mutants.
Besides male sterility, parkin mutants often phenotypically express a slightly upturned wing posture (Figure 3A and B), which led to an investigation to assess flight and climbing ability in these organism. Figures C-D show that the parkin- Drosophila are clearly disabled compared to wild type flies when it comes to flying and climbing, and that the mutants have these deficiencies before the onset of adulthood. However, replacement of the parkin gene back into the genome results in a rescue phenotype, which is illustrated by E and F, and proves that a functional version of parkin is necessary for normal flight and climbing ability. These figures are somewhat difficult at first glance, though, because normal flies are labeled as parkrvA and mutant flies as control and park13, which creates inconsistency and confusion.
Next they determined that the abnormal flight and climbing abilities is related to parkin’s effect on muscle cells, shown in figure 4A-I. This is an amazing figure as it emphasizes just how much influence a mutation in a gene can have over a cell or even an entire organism. This figure also demonstrates that there are definite abnormalities in the mitochondria of parkin mutants, which likely leads to the loss of muscle integrity, and that reintroduction of the gene can produce an essentially phenotypically normal tissue. The authors also demonstrated that muscle degeneration proceeds mitochondrial development, which suggests that mitochondrial dysfunction leads to muscle deterioration and becomes more severe with time.
Once they proved that mitochondrial dysfunction eventually leads to the decay of muscles cells, they investigated the mechanism of cell death and found it to be an internal one. Using TUNEL method, they found that 1-day old parkin mutants exhibited TUNEL-positive nuclei (Figure 5D), whereas wild type flies and parkin- pupae lacked the positive nuclei (Figure 5A-C). From here they made the conclusion that this is indicative of an apoptotic mechanism and that this in turn leads to problems with locomotion. However, at this point I became a little skeptical. No TUNEL-staining was seen in mutants at the pupae stage, and they did not suggest the reason or significance of these results. One possible explanation is that it may be too early in the organism’s developmental for any degenerative changes to occur.
Lastly they looked at the effect of parkin on the brain (Figure 6A-D) and found that compared to a normal brain, lack of parkin does not seem to have a major impact on the brain. They did note, however, that there was decreased staining of the proximal dendrite, which is the portion of the neuron that sends the signal back to the cell. From this evidence though, it seems difficult to make any correlations between dendrite staining and mitochondrial dysfunction. The author’s conclude with the proposal that AR-JP and PD may both be caused by mitochondrial dysfunction, but their underlying mutations are different, which leads me to believe that this is the direction they will be heading in for future research.
Overall this paper has uncovered some fascinating evidence that links mitochondrial dysfunction to muscle deterioration and AR-JP; however, more research could be done in certain areas. First, it should be determined whether it is a structural or functional problem with the ubiquitin-ligase protein. In order to assess this problem, an anti-idiotype for the ubiquitin protein could be synthesized, and then one could test how well the protein from both wild type and parkin mutants binds to the anti-idiotype. If the amount of binding is greater in wild type versus mutants, then that would suggest that there is a problem with the protein structure. Perhaps if the structure is somehow flawed, then it prevents the protein from binding appropriately to the cell, thus disturbing the mitochondrial development. Second, determine the reason for why parkin affects only certain cell types. To test this issue, one could measure the concentration of the ubiquitin protein across different cell types to see if perhaps the affected cell types have a different ubiquitin concentration than unaffected tissue because there has to be something about the muscle and sperm cells that make them different from the others. No matter what direction the research progresses in, I believe that this paper has discovered significant information on the parkin gene and could very well lead to important insights into PD and eventually a cure for this debilitating disease.
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