Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants

Jessica C. Green, 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

Proceedings of the National Academy of Sciences (2003) 100, 4078-4083.

Critique and Summary:

The molecular causes of Parkinson’s disease (PD) are not fully known; however, research has shown that it is associated with dysfunctional mitochondria. The link between dysfunctional mitochondria and characteristic loss of dopaminergic neurons in PD has not been elucidated yet. Recently, the identification of rare monogenic form of PD has given insight into the molecular mechanisms of the disease. Autosomal recessive juvenile parkinsonism (AR-JP) is caused by a loss-of-function mutation in the parkin gene. This results in symptoms similar to PD.

The parkin gene product is a component in the ubiquitin protein ligase pathway, and as such loss-of-function mutations cause cellular targets not to be labeled and thus cell death. In this paper, the link between loss-of-function mutations in the parkin gene in Drosophila and PD symptoms is explored.

The Drosophila parkin ortholog was identified using the human polypeptide sequence, and then used to create several lines of transgenic flies with knock-out mutations of the parkin loci. The flies were tested for longevity, flight capability, climbing capability, muscle structure, neuronal degeneration, and apoptosis. Males were also screened for spermatogenesis dysfunction.

Fig. 1 shows the comparison of the amino acid sequence of the Drosophila Parkin and human Parkin proteins. The Drosophila homolog has 59% similarity with the human form, and exhibits similar ubiquitin-like domains. Several lines of parkin mutants were created using transposon mutagenisis. These lines include null mutations, and a control line parkEP(3)LA1. The null mutation lines are transheterozygous with the Df(3L)Pc-MK deletion chromosome, which produces viable flies through adulthood.

Fig. 2 shows the histological differences between parkin+ flies and parkin13 mutants. In parkin mutants, mature sperm do not develop. Further analysis shows that spermatogenesis arrests right before the spermatids individualize. This is due to irregular formation of the Neberken, a mitochondrial-like structure in the tail of the sperm.

Fig. 3 shows the affect the parkin mutant gene has on wing phenotype. Mutant flies do not hold their wings in the proper posture. parkin mutants also showed decreased flight and climbing ability. Various parkin mutants were tested against wild-type flies, as well as parkin-rescued flies, and all parkin mutants showed a reduced flying and climbing ability.

Fig. 4 is a histological analysis of muscle tissue. Muscle tissue from the indirect flight muscles (IFMs) of parkin+, parkin-, and parkin-rescued flies was compared and showed that the parkin- mutants had considerable muscle degeneration. The mitochondria in these mutants were swollen and the cristae were disrupted. The transgenic rescue flies had phenotypes similar to the wild-type flies. Mitochondrial degeneration was shown to be degenerative, as indicated by comparison of pupae mitochondria of wild type and mutant flies at 96 h and 120 h after pupae formation. This suggests that mitochondrial pathology is an indicator of muscle degeneration.

Fig. 5 is a TUNEL stain of the IFMs in parkin mutants and age-matched control flies. No TUNEL staining was seen in pupae of either line at 96 h APF and 120 h APF. There was TUNEL staining in 1-day-old parkin mutant, but not the control flies, showing that mitochondrial dysfunction leads to apoptosis.

Fig. 6 is a histological analysis of brain tissue from parkin+ flies and parkin- flies at 1-, 10-, and 30-days-old. There was no significant difference was found between the two lines when the frontal sections of the brain were compared. Tyrosine hydroxylase immunostaining showed that the number of dorsomedial cluster neurons was similar in the parkin mutants and control flies; however, the cell bodies in the parkin mutants were smaller.

The results of this investigation have shown that the parkin mutation causes lower longevity in flies, defective spermatogenesis, impaired fliying and climbing ability, mitochondrial dysfunction, and muscle degeneration, and ultimately apoptosis in muscle cells. However, the Drosophila parkin mutants do not show the same symptoms as humans with PD. In Drosophila the muscle tissue, and sperm of the males is affected. In individuals with PD and AR-JP, the tissue that is affected are the dopaminergic neurons in the substantia nigra. The next step in this line of research is to see if the same underlying pathways that cause muscle degeneration and defective spermatogenesis in Drosophila, i.e. mitochondrial dysfunction, also cause dopaminergic neuron loss in humans.
This could be accomplished by creating parkin mutations in a line of mice, and then looking at the histological effects in various tissues, as well as observing their behavior. If mitochondrial dysfunction is the root of PD, then abnormalities should be observed. Furthermore, the role parkin plays in mitochondrial functioning should be investigated. Where in the cell is parkin mRNA concentrated? What type of molecules does it bind with?

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