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Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants: A review

Jessica C. Greene, Alexander J. Whitworth, Isabella Kuo, Laurie A. Andrews, Mel B. Feany, and Leo J. Pallanck



This paper begins by summarizing the molecular symptoms of ParkinsonÝs Disease, which is in the future labeled as PD.  PD is most characterized by its signature loss of dopaminergic neurons in the substantia nigra and its accompaniment by the accumulation of Lewey bodies.  AR-JP, which is an earlier and more juvenile form of PD, is known to be associated with a loss of function mutation in the gene known as parkin.  In this mutant form though, the Lewey body phenomenon is not present.  It was also proposed that a dysfunction in mitochondria is the underlying cause of PD.  However, the mechanism by which parkin actually works is not clear but it is most similar to a ubiquitin protein ligase.  It is able in this regard to label specific cellular targets with ubiquitin to cause dopamineric neuron loss.  The experiment yielded the finding of a highly conserved parkin ortholog in Drosophila.  Reduced longevity, male sterility, and flight and climbing defects were phenotypical results of mutants generated with a parkin defect.  It was also found that mitochondrial defects were subsequently a sign of muscle atrophy and wasting with regard to spermatids in the process of becoming individual gametes in mutants.  Their overall proposal was that mitochondrial dysfunctions cause the mutant phenotypes that were observed in the parkin mutants and that they also play a role in AR-JP and thus possibly PD. 



Figure 1: AA sequences, expression patterns, mutant alleles (parkin)

Part A of the figure is showing comparisons between the amino acid sequences of the human and drosophila parkin genes.  A 59% similarity is cited by the authors and regions are indicated that are of extreme interest including the ring finger domain, N-terminal ubiquitin-like domain, and the In-Between Ring domain.  The indication of these domains further serves the purpose of the authors in comparing these as homologues due to overall structural similarity.  Part B is a Northern Blot that is showing that 1.7kb Parkin is present in embryos, larvae, and adult Drosophila.  According to the authors, Parkin should be present in all three stages.  Part C shows a molecular map of sorts of Parkin.  The locations of the point mutations, the protein coding sequences, and the three parkin deletion allele sites are indicated. 


My only complaint with this figure is that part C is a bit too complicated and very difficult to follow, it feels as if it is just taking up space. I feel that this figure provided a solid support for the rest of their paper and is truly some good visual background.  I donÝt believe any major additions to this figure wouldÝve done anything more than provide meaningless additional information that would clutter the paper.


Figure 2: Spermatid Individualization Defect Associated with Abnormal Mitochondrial derivatives

Male parkin mutants were found to be sterile.  The second figure indicates that males undergo a spermatid individualization defect due to the influence of abnormal mitochondrial derivatives.  The different panels were visualized by electron microscopy:  Park + panels were A, C, and E while Parkin ˝ panels were B, D, and F.  The arrow in panel A indicates a sperm released from a testis as normal in the parkin + line during individualization.  Panel B indicates the absence of mature sperm from the seminal vesicle in parkin-, the sperm has formed but has failed to individualize out of a syncytial bundle.  Panels C, D, E, and F indicate, through a magnification of a cross section of a testis, that the Parkin ˝ mutants show clearly developed axonemes in developing spermatids but the oddly shaped Nebenkerns are scattered about and not nearly as dense in the same region that is dense in electrons in parkin + flies.  This diffusion of the Nebenkerns causes the male drosohpila to be sterile.  The Nebenkerns are also significant due to their mitochondria-like nature because this indicates that a disfunction of the mitochondria, as related to a mitochondria that is specialized, could directly result in sterility of male drosophila. 


It is my opinion that this was an excellent figure that did a great job at contrasting the Nebenkern and muscle tissues of the parkin mutants and the wild type flies.  This figure indicated clear differences in structure and distribution between the Nebenkerns of the two fly strains to the reader.  I feel that Cells C and D were needless.  At such a low magnification, those who are reading the paper and who are unfamiliar with drosophila would not be able to recognize what they are trying to indicate.  Cells A and B adequately show the maturation of sperm and the testes in wild type flies as compared to the mutant variant.  The magnified view to show the Nebenkerns were extremely well done in E and F and were able to lend great support to their theory of mitochondrial dysfunction.  However, it is important to also remember that the only thing they are able to indicate here are structural differences.  Functional testing of the sperm individualization as compared between the two fly strains would be required in order to finitely prove their theory through this figure.  They need to show that a change in functioning of the sperm is causing the lack of sperm individualization as well. 


Figure 3: Parkin function is required in mesoderm

Figure 3 compares wing postures, flight ability, and climbing abilities of wild type and parkin ˝ adult flies.  Parts A and B indicated differences in posture with the parkin ˝ flies having a distinct downward curvature of the wings as compared to the wild type flies.  Part C compares the flight ability of wild type flies with the abilities of three different lines of parkin ˝ flies, the parkin ˝ flies showing significant impairment.  Rescue of the parkin ˝ flies caused restoration of their flight ability to near the level of wild type flies.  Part D shows differences in climbing ability of wild type and parkin ˝ flies with the parkin ˝ flies showing significantly worse climbing ability over a period of 17 days than their wild type counterparts.  Rescue of the parkin ˝ flies resulted in restoration of their climbing ability close to the level of their wild type counterparts.


The tests performed here were both well thought out and very supportive of their argument.  My major complaint is that they do not discuss enough about the rescued flies as far as specifying phenotype and visual appearance goes.  I believe they wouldÝve been served greatly by somehow showing photographs of the rescued flies as compared to the flies before rescue.  Additionally, some sort of magnified view of their behaviors would have helped to serve their argument.  It is the rescuing of these flies that I feel gives greatest credence to their belief that the parkin gene is responsible for regulating these behaviors in drosophila and thus a great deal of detail should be included.  They were very inclusive in what they decided to show in these images and they even thought to show all three mutant variants at one point to indicate the validity of their data.  This was a strong figure that really couldÝve only been served by a more visual look at these phenotypical manifestations of parkin deficiency.


Figure 4: Parkin mutants manifest muscle degeneration and mitochondrial pathology

Panels A, B, and C indicate longitudinal sections of indirect flight muscles from wild type flies, parkin ˝ flies, and flies that have been rescued.  Severe disruption of muscle integrity is shown in panel B, an absence of parkin has a direct effect on musculature then.  Subsequently, panel C shows that the musculature can be restored to normal after rescue.  The authors say that also not all muscles in the flies were affected by an absence of parkin but that the data was not shown.  Panels D, G, E, H, F, I compare the wild type and parkin ˝ flies and show that the parkin- flies have irregular myofibrillar arrangement and that their mitochonria are malformed.  Rescue of the flies was shown to restore the wild type phenotypes as well.  Panels J,K,L,M,N, and O show that the mitochondria of 96-h pupae of parkin ˝ flies are less electron dense mitochondria than wild type mitochondria.  120-h pupae continue to indicate swollen mitochondria whereas their myofibril structure was intact.


I feel that this figure would have first been better served had they tested all the mutant variants of parkin ˝ flies available.  They only tested one variant, which leaves the question of it being a fluke, or not up in the air.  The same problem that I mentioned before with the pictures of spermatid individualization are present here, they have managed to indicate physical differences between the wild type and parkin ˝ mutant flies but they need some sort of functional test for this to be more convincing and appropriate for their argument.  I feel that some of the figures such as cells J-O that the pictures were unclear and it was difficult to see the degeneration of mitochondria for someone who is untrained in recognizing them.  Overall this was a great figure to include and they had the right idea about discussing the actual muscle development in their believe that mitochondrial dysfunction is related to muscular atrophy.  However, I believe more care needs to be given to choose pictures that are clearer.  Perhaps they shouldÝve taken the muscle tissue from a different site?


Figure 5: Parkin mutants exhibit apoptotic Cell Death of flight muscle

This figure examines apoptotic cell death in parkin + and parkin ˝ fly flight muscles through TUNEL staining.  The wild type flies show no signs of apoptopic nuclei whereas the mutant flies did show apoptotic nuclei in one-day-old adults but not before.  This suggests that cell death occurs by some sort of apoptotic mechanism due to mitochondrial dysfunction.


As with the previous figure, this figure shouldÝve included all the mutant variants of flies for more viable and believable results.  I believe that they do a good job of indicating their point but I am still unconvinced as to if this apoptotic mechanism is due to mitochodnrial dysfunction.  I believe that the authors erred here also in not including matching parkin + pictures at each time interval that the parkin ˝ pictures were taken.  It certainly was not necessary but it would have greatly buffered their experiment as far as experimental believability goes, in my opinion.  I believe it was also a mistake not to show pictures of the rescued parkin ˝ muscle at 96 hr APF, 120 hr APF, and 1 day adult flies.  I believe this would have further verified their argument and indicated further that the changes in muscle tissue was due specifically to mitochondrial dysfunction through an apoptotic mechanism.


Figure 6: Loss of parkin function does not cause general neuronal degeneration or dopaminergic neuron loss.

The authors examined brain tissue from parkin ˝ and wild type flies.  The tissue was stained with hematoxylin and eosin to indicate that both samples had normal arrangement in the nervous system.  The tissue was also stained with tyrosine hydroxylase and there was only a slight difference indicated.  The neurons were shrunken and the proximal dendrites were less strongly indicated in mutant brain tissue compared with wild type. 


This figure was experimentally disappointing.  The authors, however, were entirely forthcoming when they were comparing A and B.  It was clear that the parkin ˝ mutants brain tissue lacked degeneration of neurons, which is not what they were projecting.  The change that they indicated in shrinkage of the dendrites in parkin ˝ mutants was not very clear at all in the figures.  Again, I feel they should have included all of the mutant variants as well as a view of rescued fly brain tissue.  This is an extremely disappointing figure as well because it affectively dashes hopes of relating this to human PD (human PD only affects neuronal cells).    


General Critique-

Overall, this paper did well at presenting their case by including evidence that the parkin gene is involved in sperm individualization, flight and climbing ability, and physical changes in neurons. Personllay, I believe that the parkin gene is involved as they say it is though I believe that there are other factors that are contributing.


Future Experiments-


I believe that the chief problem of this experiment should be the springboard for further experimentation in this area. 


The fact that no neuronal degeneration was deserved in drosophila may be an organismal specific fluke.  It is for this reason that I believe that work should be put towards determining another ortholog in a different species closely related to humans such as mice or monkeys.  Once the ortholog was discovered, one could analyze the brian tissues of these organisms much in the same way that this paper did to verify whether or not mitochondrial disfunction was present.  The presence of an obvious mitochondrial disfunction could not only further validate this paper but also cause researchers to shift their attention to why this is true.  What is different about these parkin proteins within these organisms to cause these problems to appear?  I believe that further testing on more model organisms that are genetically identical to humans would either effectively answer all questions about this protein or pose more than there were before.  Either way, that is good science. 


I also believe that a wise experiment to conduct would be to test the mRNA levels in the flight muscles of the drosophila.  Perhaps looking at the different levels of intensity of RNA and comparing the wild type to parkin deficient mutants would give more clues as to the apoptotic cell death topic.


Another major problem that I had with this paper was the fact that they did not think to include any functional testing regarding the parkin protein.  I feel that a test that would fit in this vein of testing that would serve their purposes well would be to use GFP or some other fluorescent marker in combination with the appropriate promoter for parkin.  The GFP would be observed  in different regions of the body of the fly and depending on its localization, different and more finite functional conclusions could be drawn about its function.  This would also make testing with transparent worm species intriguing in addition to the other model organisms mentioned before, they are an excellent species to use in combination with GFP.  How does parkin work in that species of animal, are its application more universal than just humans and drosophila? Also along the vein of functional testing, would it be possible to do some sort of electrolysis testing for the muscle tissues?  Data could be gathered about muscle tension along with muscle lesions upon the administration of a stimulus and plotted linearly, then used to compare wild type and parkin deficient mutants.  It is this type of result that this paper was missing as I said before many times, they proved many things about differences in structure but never really in function due to the parkin mutation.


With these research suggestions in mind and the critical nitpickings aside, this paper made a genuinely good effort at answering a difficult question.


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