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“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
(2003), PNAS 100 (7), 4078-4083.

Paper Summary:
   Parkinson’s disease is a neurodegenerative disorder characterized by a loss of dopaminergic neurons in the substantia nigra and accumulation of Lewy bodies in humans. Although no known molecular mechanisms are known, several experiments have indicated mitochondrial dysfunction as a major causative factor. An important gene in the identification of factors causing Parkinson’s is the parkin gene, where loss of function mutations cause an early onset form of Parkinson’s known as autosomal recessive juvenile parkinsonism. The researchers in this paper decided to investigate Parkinson’s disease through Drosophila, by creating a parkin- mutant and comparing symptoms and mechanisms to those thought to occur in humans. Parkin in humans has recently been demonstrated to be an E3 ubiquitin protein ligase, interacting with ubiquitin-activating (E1) and ubiquitin-conjugating (E2) enzymes.
   Greene et al. began by finding the gene in the Drosophila genome that matched the human Parkin sequence. They searched the Berkeley Drosophila Genome Project Database with the Parkin protein sequence to query a six-way translation. Only one gene was identified, and the cDNA was then sequenced and found to have 42% amino acid identity and 59% similarity overall with human Parkin. The cDNA sequence was also compared to the genomic DNA sequence to identify splice junctions for introns and exons. The structure of the Drosophila gene was also very similar to that of the human Parkin, bearing a ubiquitin-like domain, ring-finger domains, and an IBR domain (Figure 1A).
   The parkin mutants were generated using P element insertions: genomic DNA was analyzed from approximately 5,500 flies with multiplex PCR using a primer specific to the P element terminal sequence, and other primers corresponding to sequences in the parkin gene. One line was recovered, parkEP(3)LA1, which had an insertion 71 bp upstream of the parkin start codon (Figure 1C). Imprecise excision alleles were then created by mobilizing the transposon and favoring coincident deletions, which were then determined through sequencing. Several alleles were identified as having the entire parkin sequence deleted, thus becoming null alleles, parkin13 and parkin45.   Another mutation, parkin25 had most of hte parkin gene deleted but left the latter portion intact.  A chromosome was also revealed to have precisely deleted parkEP(3)LA1, which was then used as a control chromosome (designated parkrvA). The sequence of the gene was then altered to include a polymorphism at codon 240 to match the parkin amino acid sequence predicted from the Berkeley Drosophila Genome Project. PCR was conducted to introduce sequence changes designed to improve translation, and to introduce restriction sites. Then this new construct was sequenced to ensure the integrity of the parkin coding sequence. A Northern Blot analysis was then performed with a parkin probe to ensure the construct was being made and was still identifiable with a parkin sequence probe (Figure 1B).
   The flies produced with the parkin mutations were then analyzed for the behavioral differences compared to control, wild-type flies. Flight tests were done by dispensing 20 flies through a funnel into a graduated cylinder that had a sheet covered in vacuum grease on the sides. The flies stuck to the sheet where they landed, the sheet was then removed, and the flies were counted in each region of the five regions on the sheet. Climbing assays were done using a countercurrent apparatus, and 20-30 flies were placed into a chamber then given 30 seconds to climb 10 cm. If flies completed this task successfully, they were moved to another chamber and this process was repeated 5 times. After five trials, the number of flies in each container was counted and the climbing index was calculated the same way as the flight index: the weighted average of the region or chamber where the flies had ended up, divided by four times the number of flies in the assay (Figure 3).
   The results of these experiments showed that the parkin transcripts are being made at all developmental stages, from embryos to adults, with the authors noting a particularly high abundance in adults (Figure 1B). parkin null flies showed developmental delay, significantly reduced longevity, male sterility, and severe defects in both flight and climbing ability. Each of these symptoms was analyzed for a cause, hoping to be linked to the human form of AR-JP (autosomal recessive juvenile parkinsonism). The male testes were analyzed and it was discovered that the sterility was a result of a late defect in spermatogenesis, which proceeded normally until the 64-cell germ-line cyst that normally separates into mature sperm cells failed to divide. Structural analysis of developing spermatids showed structural irregularities in the sperm tails, as well. The Nebenkern integrity, a specialized mitochondrial derivative, was severely disrupted with varying degrees: some showed reduced Nebenkern, others showed multiples (Figure 2).
   The flight problems were a result of a partially penetrant downturned wing phenotype, with the penetrance increasing with age. Climbing ability decreased at the same rate as control flies, however the parkin mutants performed consistently lower, suggesting they being adult life with a reduced ability. To address these issues, the researchers used a UAS/GAL4 system to express parkin in defined tissues. It was discovered that parkin expression in the mesoderm rescued wing posture, flight, and climbing phenotypes, demonstrating that parkin function is required in the musculature.
   Histological analysis revealed severe disruption of the muscle integrity, and another rescue was performed by expressing parkin ectopically in muscles. However, the muscles involved in the jump response of the fly and the larval body-wall muscles were morphologically and functionally normal in parkin mutants. Thus, only a subset of muscles are affected by the loss of parkin. The disruption of muscular integrity was shown to be a result of a decrease in density of myofibrils, a broadening of the myofibril Z-line, and a shortening of the sarcomere length, but the most noticeable difference from the control flies were the swollen mitochondria with severe disruption and disintegration of the cristae. Transgenic expression of parkin in the musculature restored the myofibril integrity and mitochondrial morphology (Figure 4).
   To determine if the muscle degeneration was being caused by apoptotic cell death, the indirect flight muscles were subjected to TUNEL staining. After 96 and 120 h of puparium formation, no TUNEL staining was detected in parkin mutants or control flies but a dramatic increase was observed in 1 day old adult parkin mutants compared to the control adults of the same age (Figure 5). The researchers concluded that this showed the mitochondrial defects resulted in cell death through an apoptotic mechanism.
   Experiments were also performed to check the neuropil integrity, which revealed appropriate development of the major brain centers and no neuronal loss was observed (Figure 6). The only difference between control and parkin- flies was some shrinkage of the dorsomedial dopaminergic cell cluster cell body, as well as decreased staining of the proximal dendrites.

   The first problem I see comes in the introduction, where the authors state “The finding that Parkin functions as a ubiquitin protein ligase indicates that failure to label specific cellular targets with ubiquitin is responsible for dopaminergic neuron loss in AR-JP”. While this statement may be true, there is no data supporting data, nor any other papers quoted or referenced showing that research has been conducted to lead to this statement, and this seems an unlikely conclusion with no experiments behind it. While they then go on to say that experiments premised on this hypothesis have led to several potential cellular targets of Parkin, there is no evidence supporting the view that it must be lack of specific ubiquitin labeling that causes the neuron loss.
   The second critique is that the cDNA used apparently already had a mutation when comparing the cDNA sequence to the genomic sequence, because the researchers had to alter the polymorphism (a mutation that does not change the biological function significantly) at codon 240. Also, through PCR, many different things were tagged on to the parkin coding sequence, and this may have changed the structure of the protein enough to cause a slight alteration in the function. While they did a Northern Blot test to show that the mRNA was being produced from this gene in the transgenic flies, this does not show that the parkin protein was acting in accordance with its normal function.
   With the assays testing climbing abilities, the test they used was an experiment originally designed for phototaxis experiments, not climbing experiments for flies, and they also modified the experiments used for testing flight ability. While they did explain how they performed the tests, there is no way for us to know that these tests are accurate measurements of ability.
   In the results section, parkrvA was described as the control chromosome because the mobilized transposon had deleted itself, however I wonder why they did not simply use a chromosome that had not undergone the transposon deletion at all to ensure a completely normal wild-type control.
   In Figure 1B, the Northern Blot Analysis, the lanes appear smeary or out of focus, and while the authors state that the adult fly has a significantly higher amount of parkin being produced, there is no control showing how much RNA was loaded into each lane, thus we do not know if this is an accurate representation of the concentration in various developmental stages. Also in the figure legend the researchers mention a band in adults that is larger than the 1.7 kb parkin transcript, yet this extra band is not clarified or even mentioned elsewhere in the text.
   In Figure 1C, it would have been helpful to give more accurate measurements of the deletions in the Parkin gene. While there is a schematic drawing, they do not specify which amino acids have been deleted and it is difficult to understand the parkZ472 and parkZ4437 constructs. The constructs are only explained in the diagram and nowhere in the text, thus when they are used again in later images, it is hard to tell what the experiment was testing when using the constructs.
   Figure 2 is a well-done experiment showing both control parkin+ as well as parkin-, and using arrows and arrowheads to point out the important parts of the diagram so that the reader may understand even if they are not familiar with the structures being shown, and there is quite obviously a difference between the controls and mutants, supporting their hypothesis.
   In Figure 3 E and F, it is interesting to note that Rescue 1 had a result much closer to that of the wild-type than Rescue 2, and in the case of the climbing index, Rescue 1 restores climbing to a higher ability than the control itself shows. This difference may have affected their later experiment showing the rescue of the parkin- phenotype by transgenic expression of parkin in Figure 4, because the rescue the experimenters used was Rescue 2. In Figure 4C, the authors note that some vacuoles are still present in the muscle that has been rescued, however perhaps if they had used Rescue 1, this would not have been the case.
   A critical problem in the argument of the researchers comes at Figure 6, when their image shows that loss of parkin does not cause general neuronal degeneration of dopaminergic neuron loss, such as that in AR-JP. This is only a problem for later, when they try to show that the parkin- pathway mechanisms in Drosophila are applicable to human AR-JP, because if the flies are not exhibiting a key part of the human disease, how likely are the other connections that have been made with the mitochondrial degeneration.
   For the most part, this paper displays their data well, showing the important images with clarity, yet there are a few sections that say data not shown where it might have been better to show at least a few images supporting their statements in the text.

Future Experiments:
   Since mitochondrial degeneration and cell death appear to be the main focus of this paper, perhaps a future experiment could leave parkin intact and induce mutations in other genes that affect mitochondrial formation, to see if their loss-of-function also cause similar problems to parkin null alleles. It would also be an interesting experiment to see if the human parkin protein could rescue the Drosophila parkin- phenotype. Perhaps in the hope of examining human AR-JP more closely they could attempt to find the Parkin gene ortholog in mice, using the same method to find the gene: screening the genome for sequence and structural similarities.
   Experiments should also be designed to see which proteins interact with parkin. This can be done by immunoprecipitation, then any proteins found should also be tested for their effects on the parkin- phenotype. It could be that the parkin loss causes another protein to lose its function, and this is the protein that is key to mitochondrial function. It is also important to discover the main function of the parkin protein so that we can understand the mechanism by which the mitochondrial pathology and cell death are triggered and carried through.  The Parkin gene could also be cut at varying intervals to determine which parts of the sequence are necessary for proper functioning of the protein and normal phenotypes: perhaps it is only the amino-terminus, or the ring-finer domain that interact with the necessary proteins or targets.
   Another experiment could be to over express the parkin protein and examine the phenotype that results from this, perhaps over expression of the protein could cause other dysfunctions that might be helpful in determining the role of parkin during normal functioning.

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