Review Paper
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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*§
*Department of Genome Sciences, University of Washington, P.O. Box 357730, Seattle, WA 98195; and ‡Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
Edited by Kathryn V. Anderson, Sloan-Kettering Institute, New York, NY, and approved January 30, 2003 (received for review December 11, 2002)
Summary and Critique:
Parkinson’s disease is a neurological disorder leading to the breakdown of dopaminergic neurons in affected individuals. This paper describes how Drosophila flies were made lacking the parkin gene, which if missing in humans can cause autosomal recessive juvenile parkinsonism (AR-JP). The parkin protein serves as an E3 ubiquitin protein ligase, labeling targets within the cell with ubiquitin. Creating parkin- flies gave this research team a comparison of fly and human parkin- disorders, and once they found mechanistic similarities, provided them with a useful model organism to conduct future experiments.
After finding the parkin ortholog of flies in the Drosophila Genome Project Database, they used the EP(3)3515 P element to insert itself randomly within the genome of 5,500 flies (for more information on P element use click here). Once they probed the fly lines with primers for both the P element and parkin, they excised parkin to create three different parkin- flies (using different excision sites). The parkin- flies were either homozygous parkin- or transheterozygous to df(3L)Pc-MK. These experiments then included transgenic flies that expressed inserted parkin cDNA. In setting up these experiments they were able to see what happened in flies when they had parkin, deleted parkin, then put parkin back in. These experiments demonstrated that when this gene is disrupted, parkin- flies have decreased flight and climbing, shorter life spans, and male sterility. They conclude that mitochondrial deformity in parkin- flies is the cause of the observed defects, and these findings may relate to human idiopathic Parkinson’s disease.
Figure 1A compares the amino acid sequence of Drosophila parkin (D-Park) to human parkin (H-Park). This figure highlights three important structural properties of the parkin protein that both D-Park and H-Park contain: an ubiquitin-like domain, a RING finger domain, and an In-Between Ring domain. The human and Drosophila sequences had a 59 % similarity and D-Park was the only gene in the fly genome that encoded for all three of those important domains. Figure 1B is a Northern blot trying to show the increasing levels of parkin mRNA with increasing age in wild-type flies. However, this general trend would have been better demonstrated with the use of a loading standard such glycogen synthase in order to compare band intensities of different lanes. They also claim that only the adult flies express a larger parkin transcript (above 1.7 kb band), but faint bands appear for this transcript in all three lanes. It would have been helpful to include lanes probing parkin- flies for both bands to make sure they are in fact deleted. Figure 1C maps out the P element insertion site and the coding (black boxes) and non-coding sections (dashes) of the parkin gene. They include the deletion sites of all three parkin- mutants used. Their control or parkin+ flies contained a deletion of the just the P element, which provided a good control to rule out any question of whether or not their excision process influenced the data.
Figure 2 focuses on ruptured testes from control and parkin-, showing that mature sperm are formed but are unable to individualize or separate. The parkin- flies also show irregular Nebenkern (mitochondrial derivative) formation, where some have reduced Nebenkern size and others have multiple Nebenkern. The change in size is less noticeable than the more diffuse matrix surrounding the Nebenkern in parkin- flies (Nebenkern in upper left corner of control flies appears to be same size of parkin- flies). The authors claim that the transgenic parkin flies regain flight and climbing abilities along with normal life spans. However, they do not mention transgenic loss of sterility. In both figures 3 and 4, the authors compare data of parkin- flies to the transgenic rescue flies, both leave it out in figure 2. Unless the transgenic flies were unable to regain fertility, it is strange why the authors would exclude the rescue data from this figure.
Figure 3 shows the flight index and climbing index data for control, parkin-, and rescued flies. The flight data was obtained using a sticky sheet that caught the flies at different regions to evaluate the average flight capability of each group. For all three mutants there was a significant decrease in flight ability. The rescued flies showed increases in flight ability from mutants, but did not regain full flight capability compared to control flies. Rescue 2 flies were less efficient in restoring both flight and climbing ability compared to rescue 1 flies, which may demonstrate a greater efficiency of 24B-GAL4 (rescue 1) over Dmef2-GAL4(21) (rescue 2). The climbing index was determined by the ability of flies to climb to a certain height in a given amount of time after being tapped to the bottom of a chamber. Once again mutants showed a significant decrease in climbing ability compared to the controls. The rescued flies showed increases in climbing ability from mutants, where rescue 1 flies regained wild-type climbing ability, but rescue 2 flies did not. These functional experiments were valuable to the general purpose of this experiment; to investigate the functional abnormalities of parkin- flies and to compare those abnormalities with human Parkinson’s disease. The authors also included pictures comparing parkin- wing posture to control flies, where parkin- wings demonstrate downturned wing posture. However, this deformity does not necessarily account for the lowered flight index of parkin- flies, because even parkin- flies with normal wings had lowered flight ability. Because the wing deformity does not appear in all parkin- flies, it would have been appropriate for the authors to include the percentage of mutant flies with deformed wings, and if the three different mutant lines differed in wing deformity.
Figure 4 provides various stainings of sectioned indirect flight muscles (IFMs). The IFMs of parkin- flies severely degenerated compared to control flies, where the rescue flies regained wild-type muscle formation except for a few abnormal vacuoles. This figure also compares the separated myofibrillar arrangements and degenerated mitochondria of parkin- flies compared to controls, where the rescue flies are able to restore most wild-type characteristics. The mitochondria of parkin- flies are much larger and have cristae disintegration (most evident in panels J-O). The scale bars in M-O are different sizes, showing that O is zoomed out, which makes size comparison confusing for the reader. This figure would also be enhanced with additional panels showing control mitochondria at stage 120h, so that normal growth of mitochondria from 96h to 120h can be ruled out as the main cause of parkin- swollen mitochondria formation.
Figure 5 incorporates the terminal deoxynucleotidyltransferase-mediated dUTP end labeling (TUNEL) to stain any cells with apoptotic nuclei. How this method stains only for apoptotic cells is unclear from the paper, but this figure shows TUNEL markers for parkin- 1 day-old adults, where these cells are active in apoptosis. TUNEL markers are not detected for younger parkin- pupae at 96h and 120h or for parkin+ 1 day-old adults. Again, the authors would have enhanced this figure with panels showing the staining of muscle tissue from rescue flies, where TUNEL markers should be absent.
Figure 6 attempts to demonstrate that parkin- flies have normal nervous systems except for slightly decreased cell bodies and proximal dendrites in the dorsomedial cluster. The background level of staining in C is higher than in D, which causes a problem when the authors claim that the dendrite staining in D is lower than in C. There are also no scale bars for us to compare relative sizes within these panels. Although this figure lacks any blaring distinctions between parkin+ and parkin- nervous tissues, there is a central object in the parkin+ section (panel A- two circular rings in lower white area) that does not appear in the parkin- section. This difference may be an individual occurrence and not representative of other parkin- flies, but it is a clear difference between the two panels that should have been addressed.
The authors of this paper claim that there may be a similar mechanism relating dopaminergic neuron loss in AR-JP and mitochondrial abnormalilties in parkin- flies, however if this were the case we would expect to see a greater change in neuron count from control to parkin- flies in figure 6 (A-B and C-D). Because the parkin- neurons do not change considerably from control, it is unreasonable for the authors to tie the mitochondrial deformities found in parkin- flies to dopaminergic neuron loss in AR-JP based on this data alone. Futhermore, earlier in the paper the authors admit that AR-JP and idiopathic Parkinson’s disease may result from completely separate mechanisms because of the lack of Lewy bodies in parkin- AR-JP individuals. However they conclude the paper claiming their findings provide evidence for a link between the two forms of Parkinson’s disease, related in their mitochondrial deformity. Although there does appear to be a link between idiopathic Parkinson’s disease and parkin- flies through mitochondrial deformity (as stated early in the paper describing PD-inducing compounds), a clear connection between the two human diseases remains unclear because of the weak link between AR-JP and parkin- flies.
Future Experiments:
The authors hint at their future work to find a link between the spermatid individualization defect in parkin- flies and mitochondrial deformity. They also plan to investigate the mechanical process by which a loss of parkin leads to mitochondrial deformities by comparing parkin- mitochondria to those affected by various chemicals producing similar deformities. Although their future investigations may lead to a better understanding of this particular mitochondrial deformity, the relation between parkin- and AR-JP would remain unclear. An alternative future experiment could study the loss of parkin in a vertebrate model such as mice. With only 59 % of the fly and human parkin matching, the exact mechanism resulting from a loss of function could differ between these two distant species. Creating a knockout mouse for parkin would provide a more useful model to study parkin- phenotypes to correlate any similarities between parkin- mice and parkin- humans. Mouse musculature and nervous development is also much closer to human structures. Once knockout mice missing parkin have been created, we can study the nervous system in more depth, as Greene et. al only devoted one figure to this aspect of the disorder. Because dopaminergic neuron loss is the most recognizable defect in AR-JP patients, more data concerning these neurons in the knockout mice should be collected. One such test could include a staining similar to the TUNEL markers used in figure D, but specific to neuronal loss in the nervous tissue of the knockout mice. A simple glance at the tissue without any specific markers (figure 6) may not be sufficient to identify dopaminergic neuron loss. If we were to find dopaminergic neuron loss in the knockout mice (which should be expected since it occurs in humans with loss-of-function in parkin), then we could better relate AR-JP and idiopathic Parkinson’s disease.