This page has been created as part of an undergraduate course at Davidson College.
Journal article review:
Greene, JC, Whitworth, AJ, Kuo, Isabella, Andrews, LA, Feany, MB, Pallanack, LJ. (2003). Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants. PNAS 100 (7), 4078-4083.
This paper examines the biological role of Parkin by observing parkin-null Drosophila. The parkin-null Drosophila, a model for autosomal recessive juvenile parkinsonism (AR-JP), were created via targeted disruption of the highly conserved Drosophila parkin ortholog. Deletion of parkin is not lethal, but does, however, decrease longevity. Drosophila parkin null mutants also result in male sterility, locomotor defects, and structural changes in dorsomedial dopaminergic neurons.
The human Parkin sequence was used to find the Drosophila parkin ortholog. The Berkeley Drosophila Genome Project Database search yielded one genomic sequence of 468 amino acids, with 42% amino acid identity and 59% similarity overall with human Parkin. Figure 1A shows the sequence similarity and conserved domains. A parkin-specific probe was able to detect the ortholog poly(A)+ RNA in wild-type Drosophila embryos, larvae, and adults. The RNA appears to have a molecular weight of 1.7 (figure 1B). P-elements were used to make the parkin mutants. Figure 1C shows the parkin gene with the P-element, parkEP(3)LA1, insertion site, the breakpoints of the parkin null mutants, and the parkin point mutation locations.
Decreased longevity was observed in parkin null mutants. Their average lifespan was determined by two-tailed Mann-Whitney test to be 27 days, with none of the flies exceeding 50 days. Flies bearing the parkrvA chromosome (used as control in experiments), which has a precise excision of parkEP(3)LA1, had an average lifespan of 39 days, with some surviving up to 75 days.
Female parkin mutants are fertile, but male parkin mutants are sterile. Spermatogenesis appears to proceed normally until the individualization stage. The defect in the individualization stage inhibits the separation of the 64 spermatids that are contained in the cyst. Therefore, the seminal vesicle lacks mature sperm, as depicted in figure 2B. The electron microscopy reveals that the mature spermatids remain as a syncytical bundle. Structural irregularities were found in the sperm tails of parkin mutants. The Nebenkern (see mitochondrial morphogenesis during Drosophila spermatogenesis) integrity is severely disrupted, with some spermatids displaying multiple Nebenkern, and others possess significantly diminished Nebenkern (figure 2D and F). In addition, the Nebenkern outer matrix electrondensity appears diminished in comparison to the control (parkrvA/Df(3L)Pc-MK).
In addition to reduced longevity and male sterility, parkin mutants also have down turned wings with respect to control flies. The penetrance of the phenotype also increases with age, suggesting that Parkin is required for normal wing posture. Locomotor ability was tested by observing flight and climbing capabilities. Figure 3C and D clearly show that parkin mutants are less able to obtain a high flight or climbing index. Figures 3E and F demonstrate that flies that ectopically express parkin in the mesoderm regain flight and climbing abilities to nearly the same levels as those of the control. The authors deduce that Parkin function is required for musculature.
A histological analysis of the indirect flight muscles (IFMs), which are the major flight muscles, then revealed that the muscle integrity in parkin mutants is severely disrupted. Figure 4B displays parkin- IMFs with vacuole formation and cellular debris. The transgenic expression of parkin restores muscle integrity, but is not able to completely rid the muscle of vacuole formation. Moreover, the IMFs of parkin mutants appear to have irregular and dispersed myofibrillar arrangement with dispersed Z-lines and M-bands, and shortened sarcomeres. More strikingly, however, is that the mitochondria are swollen and malformed. The mitochondrial cristae also appear to be disintegrated in the 1- to 2-day-old parkin mutants. The transgenic rescue, once again, appears to structurally restore the effects of parkin-. Panels J-O show that the structural defects in parkin mutants seems to be progressive.
Similar effects have been seen in other muscles, such as the proboscis muscle. Therefore, the locomotor effects are not limited to the flight muscles. There does, however, appear to be a subset of muscles that are affected. The tergal depressor of the trochanter muscle and the larvae body wall muscles, for example, do not appear to be affected by the absence of Parkin.
TUNEL staining was used to determine if the muscle degeneration in parkin mutants proceeds through apoptotic mechanisms. One-day-old parkin mutants had many TUNEL-positive nuclei, whereas one-day-old control flies lacked TUNEL-positive nuclei (figure 5). This indicates that the muscle mitochondrial defects may result in cell death via apoptosis.
When looking at the brain, the authors noted that there is no general neuronal degeneration or dopaminergic neuron loss. Tyrosine hydroxylase immunostaining did, however, reveal that despite normal numbers of neurons in the dorsomedial cluster, cell bodies are smaller than normal (figure 6). Moreover, there appears to be decreased staining in the proximal dendrite, suggesting decreased amounts of tyrosine hydroxylase, which converts tyrosine into DOPA (see diagram).
The authors have used many experiments to describe the role of the Parkin ortholog in Drosophila. Many of the experiments seem to have been conducted cleanly, and have produced results that support the conclusions made. There are however, some slight inconsistencies within the text in regard to the data.
First, the authors gather that parkin is found in higher abundance
in adults than in embryos and larvae (figure 1B). Without a standard, quantifications
cannot be made. It is quite apparent that the band in lane A is more intense
than those found in lanes E and L. This may be due to accidental overloading,
which would also explain the upward smearing seen in lane A. The authors explain
the weaker signal in lane A as the presence of a less abundant transcript in
addition to the 1.7 transcript that is very abundant. If this is the case, then
the 1.7 transcript must be modified to give it a larger molecular weight.
A standard would give more insight to which scenario is more probable.
The behavioral tests that revealed decreased ability of flight and climb in parkin mutants demonstrated that flies with the rescue chromosome were not significantly less able than the controls. There is, however, a large discrepancy between the two rescue chromosomes constructed. Rescue 1 (w; UAS-park/+; park13/24B-GAL4, Df) shows significantly better rescue of the mutation than Rescue 2 (w; UAS-park/+; park13/Dmef2-GAL4, Df). Although both significantly increase the ability of parkin- flies, the discrepancy may make a difference in later experiments, such as the histological examination of IMFs.
The transgenic rescue used in the histological assays of IMFs was not specified. Based on behavioral rescue, it would make sense that the Rescue 1 chromosome would rescue myofibril and mitochondrial integrity. It would however be interesting to see level of pathology in a fly containing the Rescue 2 chromosome.
This paper describes observations found due to the loss of a gene. It still does not give us enough information to deduce the function of Parkin. Therefore, future experiments must be designed in order to gain more knowledge of the protein.
One experiment of interest would be determining Parkin protein interactions. Parkin can be immunoprecipitated using a monoclonal antibody. Parkin will then be isolated with any protein to which it is bound. SDS can be used to break the molecular bonds. The proteins can then be electrophoresed and stained for proteins. With the molecular weight, assumptions can be made to guess the identity of the unknown molecule. I would suspect that Parkin might bind to mitochondria, or a motor protein that is associated with mitochondria.
Once the interacting protein is identified, finding the bingind region would be of interest. This can be done by creating constructs that contain parts of the protein with which Parkin interacts. The construct to which Parkin would bind would most likely be the epitope for Parkin.
Another experiment that would give us more information on the function of Parkin would be to over express parkin. The recombinant vaccina virus system and liposomes can be used to transfect HeLa cells. Therefore, parkin will be overexpressed, and observations can be made about the effects.
It would also be interesting to see if insertion of a rescue plasmid would recover damage of a parkin mutant. This is to say that if it does then perhaps humans that lack Parkin could be injected with a plasmid containing the parkin rescue. Experiments would have to test to which degree the rescue plasmid could recover the mutant phenotype, and how late in the degradation the plasmid will successfully rescue the phenotype.
Further study of parkin in Drosophila or mice will broaden our understanding of the Parkin protein. The new understanding can then be used for understanding AR-JP in humans. A greater understanding always leads to a greater possibility for understanding how to fix the problem at hand. This is why this paper and those that will follow are of such importance, regardless of the model being used.
Links to Parkinson's Organizations
National Parkinson's Foundation
American Parkinson's Disease Association
Michael J Fox Foundation
Parkinson's Disease Foundation
Link to a paper I wrote on Herbicides and Parkinson's Disease.
Please send any questions or comments to: Monica
Molecular Biology at Davidson College
Back to my molecular biology home page