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Assignment #4: Review Paper

Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants

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

PNAS (2003) 100 (7); 4078-4083


Parkinson’s Disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons and the accumulation of intraneural inclusions, called Lewy bodies. There is no cure for this disease and there is only a small amount of information known about the disease’s mechanism of action although it is believed that environmental and genetic factors contribute to the disease. It has been shown that 1-methyl-4-phenylpyridinium (MPP+) is a toxin of dopaminergic neurons and that MPP+ induces these cells to die by inhibiting mitochondrial complex I. This observation led to further speculation about the correlation between mitochondrial dysfunction and PD and is now a prominent area of PD research. Genes have been discovered which are known to cause PD, such as the parkin gene. Mutations of the parkin gene resulting in a loss of function of the gene results in an early onset form of PD known as autosomal recessive juvenile parkinsonism (AR-JP). Patients with AR-JP display most of the same symptoms as patients with idiopathic PD, but these cases lack Lewy body formation. The parkin gene encodes a protein with a ubiquitin-like domain, and it has been shown that Parkin functions as a ubiquitin protein ligase and that when the parkin gene is mutated, the ubiquitin protein ligase cannot attach to its cellular targets and this is what causes dopaminergic neuron loss in AR-JP. One potential target of this ligase is components of Lewy body formation. This model would explain why patients with AR-JP have no Lewy body formation; if the parkin gene is mutated, then Parkin cannot attach to Lewy body components and Lewy body formation does not occur. In this paper, The biological function of parkin was analyzed by mutating a Drosophila parkin analog and looking for the subsequent phenotypes in flies and then comparing this phenotype to that of wt parkin+ flies.


Figure 1A shows the similarities between the human (H-Park 1) and Drosophila (D-Park 1) Parkin sequence. The human Parkin sequence was derived from its cDNA sequence and the corresponding Drosophila analog was searched for using the Berkeley Drosophila Genome Project Database. Both sequences show an ubiquitin-like domain (boxed), RING finger domains (boxed and shaded), and an In-Between Ring domain (shaded). The Drosophila sequence depicted in this figure was the only sequence detected in the fly with these PD domains, and this fly sequences also exhibits a 59% similarity overall with the human Parkin sequence. Because of these similarities, it is assumed that the human Parkin analog in Drosophila has been established and is as shown in this figure. Figure 1B shows a Northern Blot using poly(A)+ RNA from Drosophila embryos (E), third-instar larvae (L), and adults (A) using a probe specific to parkin. As is shown by this figure, the parkin transcript is expressed at each stage of development but is very abundant in adults. Figure 1C shows the Drosophila parkin gene sequence with a 71 bp insertion upstream from the parkin start codon (line is called parkEP(3)LA1, designated parkrvA later in paper) and also shows three deletion constructs, park13, park45, and park25 that are made from varying pieces of the parkin gene as is shown by the black bars. The bent arrow shows where the predicted transcription initiation site of parkin is located. Some deletion constructs contained no sequence from the parkin coding sequence and represent null alleles of parkin (park13 and park45). All constructs containing the parkin null allele are viable through the adult stage, but have a significantly decreased average lifespan and develop at a slower rate than their wt countertypes. After observing that all female parkin mutants were fertile but that all male parkin mutants were sterile, it was discovered that the Parkin sequence of male parkin mutants contained two missense mutations, one at amino acid 46 that changes A to T, and another at amino acid 431 that changes R to a stop codon (Figure 1C).

Figure 2 shows that spermatogenesis is also affected by parkin mutations. At the individualization stage, a germ-line cyst that normally separates into mature sperm cells fails to do so in parkin mutants, as is shown by the arrow in figure 2B. Figure 2A shows wt parkin flies that show mature sperm cells, as is depicted by the arrowhead, and these cells are smaller and less clumped together than those cells in figure 2B. Figures 2E and 2F are higher magnification pictures of figures 2C and 2D respectively and show other discrepancies between parkin mutants and wt flies. As is shown in figure 2E, normal mature spermatozoa show the axoneme (A) and the Nebenkern (N), a mitochondrial derivative. The Nebenkern is surrounded by a uniform and dense matrix, as is depicted by the black arrow. In figure 2F, the spermatozoa of parkin mutants show a normal axoneme, but the Nebenkern is practically absent in some spermatids (arrowhead) and is too abundant in other spermatids (white arrows). In addition, the matrix surrounding the Nebenkern in parkin mutants is not nearly as uniform or as dense as that shown in 2E. These results are consistent with the hypothesis that parkin mutations are due to defective Nebenkern formation.

Figure 3 shows that warped wing posture is also a characteristic of parkin mutants. Figures 3A and 3A’ both show 1-day old wt flies with normal wing posture. Figures 3B and 3B’ show age-matched parkin mutant flies (parkin25) and from these figures it is easily seen that the mutants have a warped wing posture. Because of this abnormality, the flight and climbing ability of parkin mutants is compromised as is shown by Figures 3C-3F. Figure 3C shows that for the control parkin allele, parkrvA, the flight index is much higher than for the parkin mutants. Figure 3D shows that as the age of the fly increases, the climbing index decreases. This is true for the control allele (parkrvA) and for the mutated allele (park13), although the climbing index is larger at each point for the control allele than for the mutated allele. Figures 3E and 3F show that when the UAS/GAL4 system is used to express parkin in defined tissues, the mutated alleles show a rescue effect in that the flight ability and climbing index of the mutants increases. Rescue 1 and 2 are different in that each rescue uses a different enhancer sequence upstream of the GAL4 sequence. Rescue 1 uses a 24B-GAL4 sequence and rescue 2 uses a Dmef2-GAL4 sequence. The controls depict parkin mutant flies in which the rescue effect is not allowed to take place because of the lack of either the UAS or the GAL4 sequence (control 1- no UAS, control 2 and 3- no GAL4). This clarifies that Parkin is indeed necessary for wing posture and flight and climbing capabilities and for the musculature in general.

Figure 4 looks at the musculature defects in parkin mutants in more detail. Figures 4A-4C show longitudal sections of the indirect flight muscles (IFMs) from wt parkin flies, parkin mutants, and mutants that have undergone transgenic rescue. As is shown, parkin mutants show decreased muscle integrity as is depicted by the vacuole formation (arrow) and the accumulation of cellular debris (arrowhead). Rescued alleles show muscle integrity between that of the wt flies in 4A and the mutant flies in 4B. Figures 4D-4L show that parkin mutants show decreased myofibril density, decreased sarcomere length, and swollen and degenerating mitochondria as compared to the wt parkin flies and the parkin-rescued flies. These figures basically show that parkin mutants generally show mitochondrial pathology that most likely leads to muscle dysfunction and degeneration and that differs from normal myofibril and mitochondrial integrity.

Figure 5 shows that muscle dysfunction and degeneration proceeds through an apoptotic mechanism. The IFM in age-matched wt parkin flies and mutant flies underwent terminal deoxynucleotidyltransferase-mediated dUTP end labeling (TUNEL) staining, and it was found at 96 and 120 hours after puparium formation that no TUNEL staining was observed in parkin wt and mutant flies (Figures 5A and 5B) but that there was a significant amount of staining in 1-day old mutants (5D) as compared to 1-day old control flies (5C). The increased staining in parkin mutants suggests that the IFM is indeed formed but that it disintegrates later on in development. This ultimately means that muscle mitochondrial defects result in cell death by an apoptotic mechanism.

The goal of figure 6 is to determine the role of Parkin in the brain. Histologic analysis of brain sections from 30-day old parkin wt and mutant flies is shown in figures 6A and 6B respectively. As is shown in these figures, there are no structural abnormalities in the parkin mutant, and also that no increase in neurodegeneration is evident in the mutants as compared to the wt flies. Figures 6A and 6B look practically identical. Figures 6C and 6D show that there is no difference in the number of neurons in the dorsomedial cluster between parkin wt (6C) and parkin mutant (6D) flies. The neurons were stained with tyrosine hydroxylase. In figure 6D the cell body is smaller for the parkin mutant than for the wt fly (shown by arrow), and that the proximal dendrite shows less staining for the mutant than the wt fly (shown by arrowhead), but these defects do not effect on the number of neurons present in the parkin mutant fly.


The authors restate the findings that loss of parkin function in flies results in male sterility, deformed wing posture, impaired flight and climbing abilities, muscle degeneration, mitochondrial pathology, and structural changes in dopaminergic neurons. One difference between the Drosophila parkin mutants and AR-JP individuals is that in the fly mutants, the most striking phenotype is the muscle degeneration and germ-line pathology, but in humans, the most striking phenotype is the loss of dopaminergic neurons. They proceed to say that despite these phenotypic differences, the affected tissues undergo alterations and deformations by the same mechanism. The differences that are observed in parkin mutant flies and humans as compared to their wt counterparts arises through mitochondrial dysfunction. They conclude that further work will need to be done to elucidate the mechanism by which mitochondrial dysfunction affects cell death and the mechanism by which loss of parkin affects the mitochondrial system.


I believe that all of the experiments comparing the phenotypes of parkin mutant flies to their counterpart wt flies are very well done and well controlled. For every figure of a parkin- fly, there is a corresponding figure of a parkin+ fly. I was forced sometimes to trust the author’s conclusions about figures and trust that the differences between the wt and mutant flies corresponds to what they say it does, which is frustrating, but is necessary since I am not an expert in Drosophila eye, muscle, and brain structure and pathology. For instance, in Figures 2A and 2B, I see a difference between these figures, but I must trust the authors’ claim that this difference is attributed to the presence or lack of mature sperm cells. Despite having to blindly believe that their interpretation of the data is accurate, I was satisfied with the results and presentation of the results and thought that the data were well-controlled and explained well.

My only actual criticism of this paper is that they fail to go into detail about something seen in figure 6 that is particularly striking to me. The authors state in the introduction that AR-JP is characterized primarily by a loss of dopaminergic neurons, but in figure 6, the parkin- flies show absolutely no dopaminergic neuron loss. The authors briefly mention this observation as is seen in figure 6, but do not focus much on it. I just have a hard time believing that the mechanisms by which the parkin mutation acts on the fly and human system are that similar when the outcomes seem drastically different. Yes, the parkin mutant fly exhibits traits that derive from mitochondrial defects as is shown in this paper (figure 4), and yes, the parkin mutant humans exhibit traits that derive from the inhibition of mitochondrial complex I, but since the phenotypic expression of the loss of parkin seems so different between the two systems, I am finding it difficult to assume so many similarities between the mechanisms underlying these very different phenotypic outcomes. If it had been found that at least some dopaminergic neuron loss had been found in parkin mutant flies but that the muscle degeneration and mitochondrial pathology was just more prominent in mutant flies, I would be more satisfied with the conclusion that the phenotypic expression of parkin mutations in these systems result from similar mechanisms because then there would be dopaminergic neuron loss in both systems. After all, PD is primarily characterized by neuron loss.

I would also have liked to see some other data from AR-JP individuals, such as spermatogenesis development and muscle and mitochondrial pathology to determine if these characteristics are common to both flies and humans. I do not know if any of the phenotypic expression that results from this mutation is common to both species, but it would have been nice to see if there was any similarities so that the argument that the phenotypic mutations result from the same mechanism would have been stronger.

Future Work:

As I just mentioned, I would like to see some data comparing the phenotypic expressions of parkin mutants in humans and flies to see if there are any similarities that exist between these species. I would like to see a figure such as figure 1, but using AR-JP individuals as well, and I also think it would be nice to see a figure like figure 6 using AR-JP individuals (along with the appropriate controls like in this paper). I do not know if human biological structure and function is similar to those in flies, but I think it would be interesting to see if the extent of sperm maturation, muscle degeneration, mitochondrial pathology, and brain development in AR-JP individuals. Then, with this data from AR-JP individuals, it would be possible to compare the phenotypic expression of the parkin mutant flies to that of AR-JP humans to see if any other phenotypic similarities exist besides the proposed mitochondrial mechanism.

As the authors suggest, I think the next big step in this research is to determine the specific mechanism by which the loss of the parkin gene affects mitochondrial structure and function. I am having a hard time with the authors assumption that the loss of parkin function in the fly and human is extremely similar, and so I would feel more confident about this conclusion if data were presented that elucidated the mechanism by which loss of parkin affects sperm maturation, muscle degeneration, mitochondrial pathology, and brain structure data for both flies and humans. In order to accomplish this goal, it seems necessary to look for transcription factors or other proteins that are affected by the parkin mutation in humans and see if these equivalent proteins are affected in flies. This isolation of transcription factors or other proteins involved in this mechanism might be accomplished by producing a radioactive or labeled Parkin antibody and inserting it into various tissues (muscle, mitochondria, brain) and seeing where it stuck. Once the Parkin receptor was identified and sequenced for both humans and flies, it could be determined if the receptor protein is similar for both species. Then the same process can be repeated by making a labeled antibody for the receptor protein and see what protein the first protein stuck to. In this way, the mechanism for how Parkin affects proteins in the body can be elucidated. In addition, at the end of every step and once the affected protein is sequenced, the amino acid sequence can be compared between flies and humans to see if the Parkin protein affects the same sorts of proteins and if the process is similar. In this way, the mechanism for how the expression or nonexpression of the Parkin protein affects bodily processes can be elucidated for humans and for flies and the mechanisms can be compared to each other to determine if, in fact, they both involve a mitochondrial pathway.


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