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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
Reviewed by Melissa Millar
Introduction and Summary
One of the remarkable features of molecular biology is sheer number of tools and methods available to aid in unraveling genetic mysteries. This is of particular interest in the biomedical field as more and more diseases are found to be genetically linked. The paper reviewed is one in which researchers used molecular methods to examine the potential genetic cause of Parkinson’s Disease (PD). More specifically, Leo J. Pallanck and his team explore the function of the parkin gene, which has been associated with a familial form of PD called autosomal recessive juvenile parkinsonism (AR-JP).
According to Greene et al., “several lines of evidence strongly implicate mitrochondrial dysfunction as a major causative factor in PD.” Furthermore, in studying Drosophila with a non-functional parkin gene, researchers observed phenotypes that are connected to mitochondrial pathology. This evidence suggests a connection between AR-JP and PD in regards to the effects of mitochondrial impairment.
In order to investigate the biological role of the parkin gene, researchers first identified a Drosophilia parkin ortholog. Then they did a Northern Blot analysis on wild-type flies to identify parkin RNA. Finally, they identified a P element within the parkin gene, which they used to yield deletion mutations of null alleles. From their sequencing, they were also able to identify two mutant alleles from a collection of homozygous viable male sterile Drosophilia. Finally, they constructed positive control Drosophilia by excising the P element from the orginal transcript. Null alleles were used in transgenic parkin- Drosophilia. See description of Figure 1.
The next step researchers took was to analyze specific tissues associated with phenotypes from parkin- Drosophilia (Figures 2 and 3). These phenotypes include male sterility, locomotor defects, and a reduced lifespan. Upon examination of testes tissue under an electron microscope, they concluded that sterility resulted from a spermatid individualization defect, which is associated with abnormal mitochondrial derivatives. The spermatid defect was determined to occur at the individulization stage, “at which point a 64-germ-line cyst that normally separates into mature sperm cells fails to do so, resulting in an absence of mature sperm cells in the seminal vesicle.” Additionally, degeneration of muscle was also observed in a histological analysis (Figure 4). To determine whether the source of muscle degeneration was apoptosis, researchers used TUNEL staining (Figure 5).
Though phenotypes seemed to be cell specific, researchers wanted to examine the role of parkin in the brain, and did histologic analysis of cross sections of the brain. They looked for neuronal degenerational or dopaminergic neuron loss. See description of Figure 6.
Description and Analysis of Figures
Primary steps are depicted in Figure 1. Figure 1 A gives the amino acid sequence of the Drosophilia parkin aligned with the human parkin sequence in order to show the degree of similarity. Figure 1 B is a Northern Blot analysis. Biologists used a poly-A RNA probe on Drosophilia of varying ages, including the embryonic stage, the larvae stage and on adults. Figure 1 C shows a molecular map of different alleles of the parkin transcript, specifying the location of the P element upstream of the start codon, the location of a missense and a premature stop codon in two mutated genes, as well as the null alleles resulting from deletions made by mobilizing the P element. There was 59% similarity between human and Drosophilia parkin genes. Researchers attempted to quantify the amount of parkin gene in the northern blot; and clearly the band is darker at the adult stage than either the embryo or larvae stages. However, there is no loading control, which is needed to provide a loading standard. The molecular map is a good visual representation of the different alleles used in the experiment.
Figure 2 is a collection of photographs of the testes, with a positive control designated parkin+ and the null allele, parkin-. The testes were stained with DAPI to designate nuclei. Panels A and B show the presence and absence of mature individual sperm in parkin+ and parkin-, respectively. Panels C and D are ultrastructural analyses of cross sections of 64-cell cysts, and E and F are higher magnification cross sections showing the axoneme (Ax) and a mitochondrial derivative, the Nebenkern (N). The parkin- mutants show an abnormal distribution of N; some spermatids have several large N, others none at all. Controls have a normal sized N. Also, the electron-dense matrix surrounding N is diffuse in the parkin-. The abnormalities of N are a strong indication of failure at some point in the mitochondrial mechanism.
Figure 3 concerns wing posture, and flight and climbing ability. Photographs of day-old control Drosophilia (panels A/A’) and age matched null alleles (panels B/B’) showed partially penetrant downturned wings in homozygotes and transheterozygotes with null parkin. Graphs C and D show the decreased flight index for various null parkin, and decreased climbing ability for null alleles in comparison to the controls.. Graph E is interesting; it is a graph of flight index of control and null alleles, as well as alleles rescued via the UAS/GAL4 system. Graph F is the climbing index for the same alleles as in graph E. These data demostrate that rescued Parkin- alleles had almost full recovery. Moreover, the logical conclusion can be drawn that “parkin function is required in the musculature”, and in light of the decreased physical ability of Drosophilia, the data support the idea of cell selective apoptosis as responsible for phenotypes observed in AR-JP.
Figure 4 is a collection of panels depicting photographs taken under electron microscopy. Researchers compare muscle degeration between control (parkin+) flies, parkin- flies, and transgenically rescued Drosophilia. In parkin-, they observe disruption of muscle integrity, a decrease in density of myofibrils, disruption and disintegration of the cristae, and disintegration of the mitochondrial matrix. However, when rescued, it was evident that muscle integrity and mitochondrial integrity was restored. These panels are compelling; the data is clear and easy to read. This data is evidence that supports the authors’ hypothesis that mitochodrial failure at some point is implicit in AR-JP.
Figure 5 is a collection of photographs taken under electron microscopy. In panels A and B, indirect flight muscles from varying aged parkin muntant pupae lack TUNEL-positive nuclei. However, TUNEL staining is not detected in day old control flies (panel C), but is clearly detected in aged matched parkin mutants (panel D). TUNEL staining exposes fragmental DNA; therefore, in day-old parkin mutants there are many apoptotic nuclei. This data is well controlled and gives convincing evidence of cell death via apoptosis, which can also be attributed to muscle mitochodrial deficiency.
Figure 6 includes photographs of histological analysis of brain sections from parkin+ and parkin- Drosophilia. Researchers used hematoxylin and eosin to stain frontal sections of flies at 30 days of age. There was no difference between the controls and null parkin when looking at the organization of the nervous system, nor was there difference in age-related neurodegeneration in the cell cortex or neuropil (panels A and B). In contrast, tyrosine hydroxlyase immunostaining exposed similar numbers of neurons in the dorsomedial cluster of control and parkin- flies, although there was shrinkage in cell body and decreased staining in the proximal dendrite in the parkin- (panels C and D). Decreased staining indicates that there are less dopaminergic neurons in this site. This was not a surprise because the authors pointed out that “dopaminergic neurons are a preferential target in AR-JP”. This figure is impressive because of the detail taken by labeling important components of the nervous system. However, panels C and D were less clear than A and B.
Greene et al. present strong evidence for the implication of mitochondrial pathology and apoptotic muscle cells in AR-JP phenotypes. They demonstrate that a faulty gene is linked to muscle degeneration, which is responsible for decreased flight and climbing ability. They show that the formation of spermatids, associated with abnormal mitochondrial derivatives, are adversely affected by the null allele. They present data that allow for apoptosis to be observed in the null allele model. Furthermore, they identify the preferentially affected tissues in Drosophilia mutants as derivatives of the muscle and germ-line pathology, as opposed to dopaminergic neurons. Finally, they point out that “dopaminergic neurons in the substantia nigra appear to be the primary tissues affected in AR-JP individuals”.
Given this last observation, one follow-up experiment could be done examining brain cells from an AR-JP patient. Using the same techniques as before, dopaminergic neuron loss could be studied. Mitochondrial pathways and molecular mechanisms between the parkin Drosophilia and the AR-JP would be the next subject of interest. Examination of the function of mitochondria found in parkin- individuals would be a good starting point. This would be accomplished by using wild type fly mitochondria as positive controls and parkin- mitochondria as subjects of study.
The authors mention that mitochondrial pathology in relation to spermatid individualization defects is the next point of experimentation. Further work is needed to determine what the connection is and how it proceeds in this pathway. By the same token, in light of the apparent connections between mitochondrial dysfunction and the observed phenotypes in mutated Drosophilia, more follow-up work should be done to determine how exactly the mitochondria is altered. The authors revealed that a variety of cellular insults exist that are able to alter the structure of mitochondria. Examining the structure of mutant and wild-type mitochondria would be ideal. One method would involve a Western Blot analysis, without denaturing the proteins. If mitochondrial proteins are structurally different, they would move with different speeds though the gel.
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