This page was produced as an assignment for an undergraduate course at Davidson College by Jessica Austin under the supervison of Dr. Campbell
Parkinson’s Disease (PD) is a neurodegenerative disorder that results in malfunction of dopaminergic neurons in the substantia nigra. Resent studies, involving the compound 1-methyl-4-phenylpyridinium (MPP+), which inhibits the mitochondria through cell death, have suggested that mutations in mitochondria that lead to loss of function are partially responsible for some of the symptoms of Parkinson’s disease. Both genetic factors and environmental conditions are thought to play a role in expression of PD phenotypes. Current studies center around identifying loci for extremely rare monogenetic forms of PD. One of these trials identified a gene called parkin thought to be important in autosomal recessive juvenile parkinsonism (AR-JP), which is a juvenile form of PD that exhibits many of the features of idiopathic PD. Parkin is believed to be responsible dopaminergic neuron loss due to its failure to properly label cellular targets with ubiquitin. This study endeavors to show the importance of parkin in motor control, neuron function, and fertility through the use of drosophila parkin- mutants. A summarization and critique of the data obtained in this study in addition to suggestions for future avenues of study are as follows.
An integral aspect of this study revolved around the identification of a human parkin gene homolog in Drosophila. This was done by comparing known human parkin protein sequences to the Berkely Drosophila Geneome Project Database. This query resulted in a gene that encoded a protein containing 488 amino acid and an overall parallel of 59% to the human parkin sequence. A comparions of the genes shows several similarities which include an ubiquitin-like domain at the N-terminal in addition to Ring finger domains and in-between ring domains. The gene found to have 50% homology with the human parkin gene was the only gene found to have an ubiquitin like domain, ring finger structures, as well as an IRB structure.
In order to establish normal expression of the parkin protein in drosophila poly(A) RNA was isolated from wildtype embryos, larvae, and adults. A northern blot utilizing a parkin-specific probe, identified parkin in all stages of development, although it was found to be expressed mostly in adults.
An integral part of this study involved parkin null mutants, which were developed using a transposon mutagenes screen that utilized P element mapping similar to parkin. This methodology resulted in the generation of an insertion near the start codon of the parkin gene, several parkin deletion alleles, as well as point mutations in the parkin gene. The molecular map in Fig 1C shows the location of all insertions, deletions, and point mutations generated in this experiment.
Results of this study show that parkin null phenotypes exhibit slightly arrested developmental stages, significantly reduced longevity in addition to complete sterility in adult males (females are fertile and produce normal offspring). Males were found to express parkin proteins that contained missense mutations and premature stop codons. Average lifespan of parkin mutants is 27 days, whereas the average lifespan of wild types 39 days.
Analysis of testes harvested from parkin mutants show that sterility is the result of a late defect in spermatogenesis. Parkin mutants were shown to have absent mature sperms. The results of the tests analysis showed that the germ line cyst that under normal circumstances develops into individual sperm fail to separate. Further analysis of structural abnormalities in parkin- sperm revealed that the Nebenkern (sperm mitochondria) were found in abnormal amounts.
As stated above loss of motor control is one of the phenotypes most commonly associated with PD. In order to establish the degree to which motor control was impaired scientists utilized climbing and flying tests in addition to recording obvious physical abnormalities in drosophila mutants. Analysis of physical abnormalities revealed deformation in the wings (wings were down-turned) of parkin mutants. Wing Abnormalities were relatively nonexistent in juvenile drosophila, but the degree of deformation increased as the flies aged. Analysis of flight and climbing ability revealed that mutants had an severely impaired ability to fly that was consistent in all mutants in addition decreased climbing abilities. A functional tests showed that by expressing the parkin protein in the mesoderm of mutant drosophila rescued mutants and restored the ability to fly and climb
Because muscle control is such a large factor in PD, researchers sought to show that the parkin protein was directly related to loss of motor control due to abnormal musculature. Analysis of indirect flight muscles showed severe abnormalities in the muscle quality of parkin mutants. In wild-type dorosophila muscles are compact and contain election dense mitochondria. In parkin mutants muscles are irregular and dispersed and mitochondria are misshapen and swollen, showing disintegration of the cristae. Mutant phenotypes can be saved by expression of the normal parkin protein through transgenic gene expression in muscles tissues. Comparison of mutants and wild-type alleles show that disintegration of mitochondria directly correlates with the age of the dorsophila mutants.
The tunnel assay was performed on the indirect flight muscle (IFM) of parkin mutants as well as wild-types in order to determine the presence or absence of apoptosis. Parkin mutants were found to exhibit a high degree of apoptosis in IFM in comparison to their wild-type counterparts. Results of the experiment suggest that apoptosis is largely responsible for mitochondrial defects in parkin mutants.
Neurological degradation of neurons is a defining characteristic PD in human phenotypes. However analysis of brain tissues in parkin mutant flies showed no abnormal development of dopaminergic neurons. For the most part, there was no difference in neuronal development of parkin mutants and wild-type flies. Only cells of the dorsaomedial dopaminergic cell cluste showed any abnormality in parkin mutants when compaired to control flies.
One of the major critiques I have of this paper deals with the longevity data.
I think than an important part of the research center around how long the parkin
mutant flies lived. I would like to see data involving the longevity of drosophila
that included what developmental stage the drosophila died at, how many of the
mutants and wild-type flies died at this stated, and a distinction between male
and female longevity. While not exactly necessary, I think it would have made
the paper better to have such an analysis. Also distinguishing between male
and female among both wild-type and mutant phenotypes would allow a comparison
on the severity of the disease.
Also I was bothered by the lack of neurological degradation exhibited by parkin mutants. Neurological impairment is a major characteristic of PD and I would like to see more experiments performed to establish why no degradation is seen in parkin mutants. I also feel that Fig. 6 would be stronger if it showed a quantification of protein taken at these stages, and the stages where abnormalities in dorsomedial dopaminergic cells began, in addition to how it progressed. The data shown presents a strong case in favor of parkin being responsible for some forms of PD, but because of the small amount neurological impairment I am not 100% convinced that it is necessary for establishement of PD.
Because I am skeptical about the amount of neurological impairment expressed by parkin mutants, I would like to see a similar series of experiments performed on mice. I question whether or not the lack of neurological impairment is a consequence of lower brain function in flies. I felt that by identifying a homologous parkin gene in mice I might determine whether or not parkin was really a protein responsible for PD. Homologous mouse parkin gene would be identified using the mouse genome project, in a similar fashion to the one described above. I would generate both knockout mice and mutated proteins to determine which phenotype is most consistent with human PD characteristics. Using northern blots I would then analyze the mouse parkin gene with a parkin specific probe. I hypothesize that the results would be similar in loss of motor control and hopefully I would be able to see some neurological degradation, which would help prove definitively that the parkin gene was directly responsible for some cases of PD. I would also preform a functional test similar to the one performed in the paper discussed above using transgenic elements to rescue both parkin knockouts and mutants. The purpose of using both knockouts and naturally occuring mutants is to assess whether or not there is a difference in expression of the disease with total deletion of the genome sequence encoding the parkin homologe.