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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
Proceedings of the National Academy of Sciences (2003) 100, 4078-4083.
This paper looks at one possible way to help combat Parkinson's disease (PD). Since, PD is a neurodegenerative disorder, associated with loss of the dopamine pathways of neurons in the substantial nigra, recent experiments have found that specific toxins of dopaminergic neurons induce cell death and thus could be the cause of PD. Along with these experiments, recently a loss of function mutation in the parkin gene has been linked to autosomal recessive juvenile parkinsonism (AR-JP). AR-JP is an early onset form of PD in which the patients display many of the same clinical features of regular PD. Since, both apoptosis and mitochondrial structural problems have been shown to underlie dopaminergic neuron loss, this led the authors to further analyze whether there was a link between AR-JP and PD.
Upon creating a Drosophila model of this disease, it was found that many of the symptoms of PD were exhibited by the Drosophila including: reduced lifespan, locomoter defects and male sterility. It was found that both the sterility and the locomotor problems associated with the null mutant Drosophila resulted from defects in mitochondrial stuctural features. It was concluded that these problems in mitochondrial dysfunction along with apooptosis were important factors in neurodegeneration in idiopathic PD.
Figure 1 shows three things, the amino acid sequence (A), expression pattern (B) and mutant alleles of parkin (C). A shows the amino acid sequence for both the Drosophila Parkin (D-Park) and human Parkin (H-Park). D-Park was found by using the H-Park to query a six-way translation of the Drosophila genome sequence. From this search the authors found this 468 aa sequence that encodes a single gene, D-Park. This gene shares a 42% amino acid identity, along with a 59% similarty overall to the H-Park. D-Park is the only gene that encodes a polypeptide bearing ubiquitin-like domain, ring-finger domains and an In-Between Ring domain, demonstrating that it is the Drosophila parkin ortholog. The ubiquitin-like domain is shown by the unshaded box, while the ring-finger domain is shown by being boxed and shaded. The In-Between Domain is shown by only being shaded. B displays Northern blot analysis of the poly(A) RNA from wild-type embryos, third-instar larvae, and adults using a parkin-specific probe. The blot shows that all three expressed the poly(A) RNA at roughly 1.6 kilobases, although the adult expressed it upwards of 1.7 kilobases. There is notable degrading of the RNA, due to the upward smear. I am assuming that this degradation on some part changed the MW of the bands, because one would expect that the same gene would stay the same size even over development. Particularly high expression seemed to occur at the adult stage, however, without a loading control it is hard to determine. C displays the mutant alleles of parkin by means of a molecular map. This map is of the parkin transcript showing the park insertion (large unshaded upside down triangle), the breakpoints of park 13, park 45 and park 25 (the three parkin deletion alleles used in this experiment) and the locations of the parkin point mutations. I, however, could not locate within this paper any mention of the park 45 and park 25 mutations and feel that they should've more clearly described the used mutants. The predicted transcription intiation site is shown by a bent arrow, pointing the direction of transcription. The black boxes shown after the transcription initation site are the actual Parkin protein-coding sequences. The arrow above the park insertion shows the orientation of the P element that was introduced near parkin to create the initial disruption.
Figure 2 begins this papers look at the specific problems that underlie the D-Park mutant. These pictures show a wt parkin testes (A), a parkin- testes (B), an ultrasonic cross section of a parkin+ and parkin- (C and D), and a higher magnification of the spermatazoa of a parkin+ and parkin- (E and F). A displays the ruptured parkin+ testis, with mature sperm and spermatazoa being shown by the arrows. B shows a ruptured parkin- testis, which shows no mature sperm. Mature spermatids are formed by the parkin-, however, they simply do not differentiate from the syncytial bundle. C and D take a closer look at a late-stage 64-cell cyst. C and D show off the regular arrangement of spermatid growth, however, when one looks at the apperance of the axonemes and Nebenkern, one begins to see the differences. The apperance of the axonemes and Nebenkern in parkin+ (E) are very organized with a clear memebrane surrounding it. Looking at the parkin- (F), one sees organized axonemes, but the Nebenkern no longer shows its original shape. Now the Nebenkern shows a squashed apperance, along with its paracrystalline structure beginning to break down. These pictures I feel clearly show the detrimental affects that the parkin- has on sperm production.
Figure 3 starts off by showing wing posture of 1-day-old age-matched flies. A and A' both are that of wt flies showing off the normal positioning of the wings, while B and B' are both mutant flies, showing off the downturned wing phenotype. A and B show side pictures, while A' and B' show dorsal pictures. I am curiuos though, if A and A' along with B and B' were the same flies, just in different positions, or if the author opted to use different flies, to better his pictures. C and D look at flight and climbing abilities respectively. I feel that both of these graphs clearly show the decreased mesoderm development associated with the parkin- genotype. E and D reproduce the same experiments as C and D, but this time they rescue the expeiment. I like the increase in flight and climbing that E and F showed respectively, but it seems that the rescue was not a total one. If the rescue exmperiment had totally restored the ability of the mesoderm then one would expect that the mutant would have restored to within a closer ability level to the wt. Also, one major problem that arises is why are the controls so much lower than al the other experiments? Even though all the flies were transheterozygous for the parkin deletion, the controls used in E and F are roughly the same height as the mutants. Why would the controls be so much lower then the wt? Lastly I can not understand why the authors chose to use two controls in E and three in F. It makes me think that they are trying to hide bad data.
Figure 4 shows varying muscle and mitochondrial views of parkin+, parkin- and a transgenic rescue. In row one (A-C), A displays a normal muscle phenotype, B shows the mutant muscle phenotype and C shows the rescued muscle phenotype from a parkin mutant. As the insets show, even in the transgenic rescue, some vacuoles still exist, which demonstrates why in the last experiment the flight and climbing abilities were not restored to 100%. Row two (D-F) and three (G-I) show longitudinal and cross-sectional cuts, respectively, of the indirect flight muscles (IFMs). D and G both show how normal parkin IFMs look, while E and H both show how parkin- look. F and I rescue the parkin- and show increased myofibril and mitochondrial integrity. J through O look at how time effects the mitochondria and leads to myofibril degeneration. J and M look at IFMs from a 96-h parkin+ pupae, while K and N look at the IFMs from a parkin- pupae also at 96-h. It is clear to see how these two pupae exhibit different mitochondria due to the cristae of the mitochondria beginning to break down. The difference between the parkin+ and parkin- is further enhanced when one looks at L and O. L and O show the continued degeneration of the parkin- mitochondria cristae at 120-h, along with the mitochondria being swollen. One thing I would like to have seen in this set of pictures, is pictures of parkin+ pupae at 120-h to use as a comparison to the parkin- pupae. I feel that since the 120-h parkin- pupae were so much larger than the 96-h parkin- pupae, it is hard to say that the increased size is only do to swelling and not to actual growth.
Figure 5 shows TUNEL staining of parkin mutants at 96-h (A), 120-h (B), a 1-day-old adult parkin+ (C) and a 1-day-old parkin- (D). The main problem I have with this data is that the authors chose not to show age matched parkin+ pupae of the 96-h and 120-h wt parkin for comparison. Also, although the 1-day-old adult parkin mutant shows apoptotic nuclei, I am not sure that these nuclei arose from where the IFMs were in A and B. I feel that these apoptotic cells may be arising not from the IFMs but from some other muscle tissue. It would also be helpful to see the 96-h and 120-h parkin+ pupae that have been TUNEL stained in order to deduce where these apoptotic cells are before cell death.
Figure 6 shows staining of frontal sections of the brain. A (parkin+) and B (parkin-) show a hematoxylin and eosin-stained frontal section. Both these stainings show little to no difference in key represented areas, including the ellipsoid body, peduncle, subesophageal ganglia, medulla and lamina. Immunostaining with tyrosine hydroxylase of both parkin+ and parkin- dorsomedial clusters (C and D) reveals that the number of neurons did not noticeably change between the two groups. There was some shrinkage of overall size of the neuron's cell body along with the proximal dendrite exhibiting decreased staining. This experiment, I felt rested almost solely on this figure, for presenting a clear association between AR-JP and idiopathic PD. By the data presented throughout this paper, Drosophila parkin most definetly induces a lot of the same phenotypes in Drosophila that are associated with Parkinson's diesease but this gene does not seem to effect the main cells that should be affected. Because the cells that are primarily affected in AR-JP are the dopaminergic neurons, the cells focused on in this figure, the ones that are not significantly effected by the parkin mutant, I can not fully conclude that this gene is responsible for anything other than Parkinson like symptoms.
Since the parkin gene clearly affects muscle development, mitochondria, and sperm development, my first thoughts are that there might be multiple genes working together to form all the symptoms present in idiopathic PD or possibly another section of this gene farther downstream from the human Parkin that was not initially screened for. Thus, for future work I would start, by looking for another gene in the human that might possibly work on the dopaminergic neurons. I would begin by screening downstream from where this gene was found in humans, for a possibility that the length was underestimated. If no such candidate gene was found I would begin screening other animals genomic libraries for orthologs that caused underdeveloped growth of the neurons in the substantia nigra, since this was the area affected. If one was found, then screening for conserved domains in both the human and the Drosophila could then begin in search of a secondary gene that may be acting in conjunction with Parkin. In one last possible hope, one could inject a transposable P element that was known to transpose farther up- or downstream from the parkin gene in hopes that if the gene in question was further away, then this transposon would knock it out.
On another tract, one could start by looking in humans that do not have Parkinson's disease, but do have neuronal development problems. By using this DNA and screening for the gene that causes this neuronal under-development, one could then use a genomic library and look for an ortholog in Drosophila that causes the neurons to be affected as they might be in Parkinson's disease.
© Copyright 2003 Department of Biology, Davidson College, Davidson, NC 28035
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