Mitochondrial Pathology and Apoptotic Muscle Degeneration in Drosophila parkin Mutants.
Jessica C. Greene, Alexander J. Whitworth, Isabella Kuo, Laurie A. Andrews, Mel B. Feany, and Leo J. Pallanck.
Department of Genome Sciences, University of Washington and Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School.
Proceedings of the National Acedemy of Sciences of the United States of America, Volume 100, Number 7, April 1, 2003 4078-4083.
This paper begins with a brief introduction to Parkinson’s disease (PD). It is characterized by the loss of dopaminergic neurons in the substantia nigra and accumulation of Lewy bodies. An early onset form of Parkinson’s disease (AR-JP) has been associated with a loss of function mutation in the gene parkin, although in this form Lewy bodies do not accumulate. They also introduce mitochondrial dysfunction as a possible underlying cause of PD. The mechanism by which the protein Parkin function is poorly understood but it is suspected that it acts as an ubiquitin protein ligase which labels specific cellular targets with ubiquitin causing dopamineric neuron loss.
In this experiment they found a high conserved parkin ortholog in Drosophila. There are many mutant phenotypes which result from parkin- mutants including reduced longevity, male steriltiy and flight and climbing defects. They found that mitochondrial dysfunction is an early warning sign of muscle degeneration and a common feature of spermatids in the process of individualization in parkin- mutants. They also prospose that mitochondrial dysfunction causes the mutant phenotypes observed in parkin- mutants and may also play a role in AR-JP.
Figure 1. Amino acid sequence, expression patterns, and mutant alleles of parkin. Part A compares the amino acid sequence of the Human parkin gene with the amino acid sequence of the Drosophila parkin gene. The authors report a 59% similarity between the two sets of sequences. They highlight certain regions of interest such as the RING finger domains, the N-terminal ubiquitin-like domain and the In-Between Ring domain. These domains are important because it demonstrates that the Drosophila parkin gene has these critical elements and therefore is further proof that it is the ortholog to the Human parkin gene. Because the Drosophila gene is the orothlog the experimental findings will be significant. Part B is a Northern Blot that demonstrates the presence of the 1.7kb Parkin in embryos, larvae and adult Drosophila. This is important because based on what the authors are proposing as a function of the parkin gene, Parkin should be present in all three developmental stages. There is an additional band in the adult Drosophila lane, which the authors do not directly address. Part C displays the molecular map of Parkin. The authors show information such as the location of the point mutation sites, the protein coding sequence and the three parkin deletion alleles sites.
Critique. There isn’t much to critique in this figure. After reading the paper it might have been interesting to see the addition of the rescued parkin- fly’s poly(A)+ RNA in part B, but I think they just included this blot to show that the parkin gene is active in all three states of the Drosophila’s life cycle. I found part C to be a little confusing, my only suggestion is to find a way to make this figure a little easier to follow. Overall the first figure provided some good background information to start the paper with. I don’t know if any other figures would have helped them here because they seemed to have shown what they intended to in this figure.
Figure 2. parkin mutants manifest a spermatid individualization
defect associated with abnormal mitochondrial derivatives. Male
parkin – mutants were found to be completely sterile. The authors hypothesized
that male sterility was caused by the lack of the parkin gene. They sequenced
mutant parkin genes for mutations and found missense and early stop codons.
This indicates that male sterility was resulting from mutations in the parkin
gene, which leads to loss of function. For A and B the authors dissected testes
then used DAPI to stain the nuclei. A, from the parkin + line, showed sperm
being released from a ruptured testis which is normal in individualization.
But B, from the parkin- line, did not show the individualized sperm resulting
from a ruptured testis. They found the sperm are still able to mature but don’t
individualize. From this the authors hypothesized that there was some change
due to the parkin – phenotype that caused the failure of individualization.
C, D, E and F all show a magnified cross-section of the testes. The authors
point out noticable differences between the parkin + and the parkin –
lines. The difference is of the Nebenkern, a specialized mitochondrial derivative
found in the tail of sperm, from the parkin – line. Compared to the Nebenkern
of the parkin + line these cells show abnormal distribution, shrinkage, and
diffuse surrounding matrix. These results suggest that the mutation of the Nebenkern
may cause the failure of the spermatid to individualize in parkin – mutants
making the male parkin – mutants sterile.
Critique. This figure does a good job of illustrating the differences between the Nebenkern in the muscle tissue of the parkin + and parkin – mutants. The authors have shown there is clear structural and distributional differences between the two types of Nebenkern. Cells E and F are more useful than cells C and D because they show the magnified detail of the Nebenkern. To the untrained eye, cells C and D probably could have been left out of figure 2 because it is hard to differentiate between normal and mutant Nebenkern at such a low magnification. The authors also did a good job illustrating the lack of individualized sperm in the parkin – mutants compared to the normal individualized sperm of the parkin + mutants. By including cells A and B in the figure, it lets the reader see the sperm maturation and the testes in parkin + and compare it to the vastly different sperm maturation and testes in parkin – mutants. By showing the differences in the two sets of testes it justifys the authors next step of magnifying the testes in an attempt to try to discover the reason behind the differences. For the purpose of this figure I don’t believe they could have included any other pictures that would have helped their argument. This figure has potential to support the authors claims that mitochondrial dysfunction is an early sign of individualizing sperm problems in parkin – mutants. But to fully convince me of this, they would have to perform some sort of functional test to prove the sperm don’t individualize in parkin – mutants because of the dysfunctional mitochonria. They have simply shown that the Nebenkern are physically different in parkin – mutants but have not shown that they are different functionally or this change in function is causing the lack of sperm individualization.
Figure 3. Parkin function is required in mesoderm for
normal wing posture, flight, and locomotion. This figure compares
the wing posture, flight ability and climbing ability of parkin + and parkin
– adult Drosophila. Parts A and B display the differences of the wing
posture. The parkin – flys have a distinct downturn to their wings when
compared to the wing posture of the parkin + flys. Part C compares the flight
ability of parkin + flys with the flight ability of three different parkin –
flys lines. The parkin – flys show consistant impairment of their flight
ability. The authors then rescued the parkin – mutants and looked at their
flight ability. After rescue the parkin – flys had their flight ability
restored close to the levels of parkin + flys, part E. Part D compares the climbing
ability of parkin + and parkin – flys. The parkin – flys had consistently
worse climbing ability over a period of 17 days than the parkin + flys. The
authors again rescued the parkin – flys and looked at their climbing ability,
part F. The rescued parkin – flys had their climbing ability restored
close to the ability of the parkin + flys.
Critique. I thought the tests the authors performed for figure three were a great idea and were very powerful in helping their argument. I liked seeing the differences of the flight wings in the parkin + and parkin – mutants but I think they should have added a picture of the flight wings of the rescued parkin – mutants. I personally would like to see if they were rescued to physically match the wild type flys or if their wings were at some intermediate level between the wild type and the parkin – mutant flight wings. It was good that before they performed the rescue of the parkin – flys that they demonstrated the flight and climbing differences between the parkin + and parkin – flys. In cell C it was smart that they included the data of three different mutant strains of parkin – flys so we could see that they all had decreased flight ability and it wasn’t just a fluke in one mutant strain. I also found it interesting that they kept track of climbing abilty over a period of 17 days. I don’t see how this helps their argument but it was interesting information. Cells E and F were the most important. Since the parkin – mutants were rescued with the parkin gene it is very intreging that these flys were able to be almost fully rescued to the wild type fly flight and climbing abilities. This is very strong evidence that the parkin gene is at least strongly involved in the flight and climbing abilites of Drosophila. It was an extremely smart choice to try to rescue parkin – mutants but now they need to try to show that there is some physiological difference between the parkin +, the parkin – and the parkin – rescued mutants that might lead to the difference in flight and climbing abilities. If they could show this than their argument would be much stronger.
Figure 4. Parkin mutants manifest muscle degeneration and mitochondrial pathology. In this figure the authors compared muscle condition and mitochondrial pathology between parkin +, parkin – and rescued parkin – mutants. The parkin + muscle tissue, A, D and G were first compared to the parkin – muscle tissue, B, E, and H. The authors pointed out differences between the two. In parkin – muscle tissue there were signs of muscle degeneration indicated by the presence of vacuole formation and cellular debris. Upon closer magnification the authors noticed that the parkin – muscle tissue had irregular myofibril arrangement and had swollen mitochondria when compared to the parkin + muscle tissue. The authors then rescued the parkin – flys and compared their muscle tissue to the tissues of the parkin + and parkin – flys. The rescued parkin – fly tissue, C, F and I, showed fewer signs of muscle degeneration than the parkin – muscle tissue because there was less vacuole formation. Upon closer magification the authors concluded that the rescue of parkin – flys is able to restore the myofibril and mitochondria. The next thing the authors did was look at the mitochondria in 96 and 120 hour old pupae. They found that in the 96 hour old pupae muscle tissue there were fewer mitochondria and fewer cristae and in the 120 hour old pupae the mitochondria were swollen with even fewer cristae than in the 96 hour old pupae. In both the 96 and 120 hour old pupae, the myofibril was still intact which lead the authors to believe mitochondrial pathology mutates first before the myofibril start degenerating and can be an early sign of muscle degeneration.
Critique. This figure also helped to show the drastic physical difference between the parkin + and parkin- mutants. Cells A through C made it easy to distinguish the accumulation of vacuoles in the parkin- mutants and the almost full recovery of the muscle tissue in the rescued parkin- fly mutants. Cells D through F were less helpful in my point of view. It was very hard to recognize what the authors were pointing out as the differences between the parkin +, parkin- and rescued parkin – flys. I think they should have found a better muscle sample that would give a better example of the characteristics the authors wanted to highlight with this figure. Cells G through I do a very good job of displaying the differences in myofibril arrangement betweent the parkin+, parkin-, and rescued parkin- flys. It was very easy to see the differences caused by the lack of the parkin gene. In cells J through O, it was very difficult to see the progressive degeneration of the mitochondria. I think if you were trained to recognize a normal mitochondria these figures would become more clear but for the untrained eye the ambiguous differences provided little support for the author’s argument. If the mitochondrial differences had been more clear the authors should have included a picture of the of the parkin+ muscle tissue at 120 hours APF so that we could compare the parkin- at 120 hours APF to something. I think the authors made a good decision to look at the muscle but they need to find clearer pictures to get their point across. Overall I believe figure 4 helped support their argument that mitochondrial dysfunction is an early sign of muscle degeneration. Although, they only proved that mitochondrial are physically different in parkin- mutants. To be full convincing they will need to devise a test to show that the mitochondria in the parkin- mutants are functionally different than the parkin+ mitochondria.
Figure 5. parkin mutants exhibit apoptotic cell death
of flight muscle. Next the authors examined apoptotic cell death
in parkin + and parkin – flight muscles. The parkin + one day old adults
showed no signs of apoptotic nuclei. The mutant parkin– didn’t show
apoptotic nuclei in 96 and 120 hour pupae but did show apoptotic nuclei in one
day old adults. These results suggest that cell death occurs by an apoptotic
mechanism due to mitochondrial mutations.
Critique. This figure is very intreguing because the appearance of apototic nuclei is so apparent. The authors may have wanted to show the gradual progression of apototic nuclei by chosing a time point between 120 hours APF and 1 day adult but I don’t think its absolutely necessary. I realize that the parkin+ muscle probably showed no apototic nuclei at 96 hour APF and 120 hour APF but it would have been nice to show these pictures so we could compare all of the parkin- pictures with a parkin+ picture of the same time point. I also think it would be very interesting to see a picture of the rescued parkin- muscle at 96 hour APF, 120 hour APF and 1 day adult. I think if they had included this information it would have greatly helped their argument assumsing the parkin- muscle showed a decrease or total absence of apototic nuclei. This figure does seem to support the author’s hypothesis that parkin- mutants undergo apototic cell death. The rescued parkin- picture would greatly improve my opinion of this experiment. I guess its possible that something else is causing this apototic cell death in parkin- mutants but from the angle presented by the authors it does seem that the lack of the parkin gene is behind this cell death.
Figure 6. Loss of parkin function does not cause general neuronal degeneration or dopaminergic neuron loss. The last thing the authors looked at was brain tissue from parkin + and parkin – adult flys. When the tissue from both parkin + and parkin – was stained with hematoxylin and eosin, both samples showed normal arrangement of all parts in the nervous system. When the tissue from both parkin + and parkin – was stained with tyrosine hydroxylase the authors noticed a slight difference. They found that the neurons were shrunken and the proximal dendrites stained less strongly in parkin – mutant brain tissue compared with parkin + brain tissue. These results did not give the authors any deep insight into the role of parkin in the brain but provided some interesting information that will inspire future experiments.
Critique. This figure was less than convincing. I did like that the authors were very honest when comparing cells A and B. They admitted that the parkin- mutant’s brain tissue seemed to lack neuronal degeneration, which was what they were projecting. The authors did use the apparent shrinkage of the neuron cell body in the parkin- brain tissue and the smaller amount of dendrite staining as evidence that the parkin gene is responsible for degeneration within the brain tissue. I did not feel that this change was very evident in the pictures they provided. I think the addition of pictures of the rescued parkin- mutant brain tissue would have been very interesting. If they had included this, it might have been easier to compare the neuron size and dendrite staining. Overall this figure was not very supportive of the author’s hypothesis especially considering that they said the disease in humans only affects neuronal cells. So the fact that little, if any, change is observed in Drosophila is troubling.
In general, Greene et al. did a good job presenting physical evidence that supports the hypothesis that the Drosophila parkin gene is involved in sperm individualization, locomotor defects including decreased ability of flight and climbing, and physical changes in dopaminergic neurons. From the data presented I am convinced that the Drosophila parkin gene is involved all of these processes although without any functional tests I am hesitant to say I believe the Drosophila parkin gene is directly responsible for the physical and structural changes observed in the data presented. The biggest problem is the lack of functional tests to support the hypothesis.
The first thing I would suggest is to demonstrate the function of Parkin in wild type Drosophila. The authors seemed to skip over this piece of information but I believe it would be helpful to have as background information when evaluating each bit of data presented in the paper. It might also be useful to show data proving Parkin isn’t transcribed or translated in parkin- Drosophila, possibly by showing its not active in these individuals using mRNA testing. In addition, the authors should provide information describing what kind of molecule Parkin interacts with and if Parkin undergos any post-transcriptional modifications using a band shift assay.
One test that might provide data to support the author’s hypothesis is to track the areas to which Parkin localizes to in wild type Drosophila. One way they could do this is to utilize green fluorescent protein with the parkin promoter. This way the green fluorescent protein would be expressed in the locations that parkin is usually expressed. If the green fluorescent protein shows up in the tissues the author’s found were affected by the parkin gene, it would give additional support to their claim that parkin was responsible for the structural and functional changes they observed. I would expect Parkin to localize in the tissues that exhibited structural changes in parkin- mutants, the flight muscle, brain tissue, and testes.
Another test that might prove useful is to use plasmids to introduce the parkin gene into parkin- mutants. The plasmid would contain the parkin gene and they could use promoters of different strengths. When the plasmids were injected into parkin- mutants they could then test and observe the affects of the varying expression levels of Parkin. I’m not sure what I would expect from this type of test but I believe the results would prove informative about the function of Parkin.
Similar to the last experiment, they could desgin plasmids which contained varying sized pieces of the parkin gene with the parkin promoter and observe the affect each plasmid has on a parkin- mutant. This would allow them to determine which part of the gene is necessary for fully functional Parkin. I would expect a fully functional Parkin to be translated when the entire parkin gene was used in the plasmid and possibly for plamids that did not contain the entire gene but contained the vast majority of the gene. I would also expect Parkin not to be functional when the important coding sequences for the conserved domains were left out of the plasmid.
They said there is a lack of an animal model of AR-JP, but there might be a different gene that when mutated or removed completely result in very similar consequences. This gene would not be an ortholog but I think it would help experimenting on an animal closer in relation to humans than a fruit fly.
Another possible test is to look at different enzyme and protein levels in the flight muscle of Drosophila by evaluating their mRNA levels which gives you an indirect measurement of protein activity in the cell. By looking at these levels it may give clues to the cause of apoptotic cell death in flight muscle in parkin- mutants.
Lastly, the author’s definitely need to perform functional tests to supplement
all of their structural tests presented in this paper. Without these tests,
the structural tests are not as strong as they could be and leave many unanswered
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