Why Drosophila as a model? The Drosophila melanogaster is a powerful model system. Drosophila is one of the most widely studied organisms in biology. Many genes and signaling pathways are conserved between human and flies. Flies have simpler genetics, only 4 pairs of homologous chromosomes to manipulate. They have 200,000 neurons compared to 100 billion neurons in humans. They can perform motor behaviors, such as walking, climbing, and flying, and they can be trained using learning and memory. Many pathways and behaviors are conserved between humans and drosophila.
Another advantage is their short reproductive and developmental cycles. It takes 10 days to produce a mature adult from embryo. This makes observing genetic manipulations faster when compared to using mammals. Manipulation like deletion, insertion, knockdown or transgenics in their genes are easily perform in drosophila compare to mammalian models. Maintaining its stock is simple and inexpensive. Many different mutants can be store in a small area. This makes Drosophila an ideal laboratory animal.
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The simplicity of Drosophila systems can be use to research neurodegenerative diseases like Parkinson. Many researchers use the drosophila model system to understand neurodegenerative disease.
Parkinson disease is a common neurodegenerative disorder. It is estimated to affect 1 in 50 people over the ages of 60 years. It is also a hereditary disease, which observation through different generation should be made with this disease. One observation made on people with Parkinson is the result of a major loss of dopaminergic neurons in the substantia nigra. This led to formation of Lewy bodies in neurons. Lewy bodies have abnormal a-synuclein protein in nerve cells. Other protein such as ubiquitin, tau protein, pink, and park maybe presents in the lewy bodies causing Parkinson, but a-synuclein is the most studied in drosophila. They appear as spherical masses cluster in areas that are affected. Lewy bodie are also seen in people with alzheimer's. Parkinson causes impaired locomotor function. Mutations in a-synuclein gene are linked to Parkinson's disease. It causes abnormal amounts of a-synuclein accumulate in Lewy bodies. There are other mutated genes that are linked to Parkinson but α-synuclein and have been most studied. The exact function of alpha-synuclein remains a mystery, but it has been linked to increase in oxidative stress in cells. When cells undergo oxidative stress, free radical can damage the cells.
figure 1: (Feany, 2000)
The figure 1 show nerve cells with parkinson. Here on the left of figure 1 shows a-synuclein in Drosophila. On the right of figure 1 shows the lewy body containing a-synuclein in human. The lewy body and a mutated a-synuclein gene will cause damage to this of dopaminergic neurons in the future.
One of the causes of Parkinson disease is a mutated a-synuclein gene. If we place this mutated human a-synuclein gene in the drosophila model system similar of human Parkinson disease affects will be observe in drosophila.
Many experiments were done on drosophila. The sectioning and immunostaining experiment involves using a standard electron microscope. Adult flies are fixed in formalin and embedded in paraffin. Antiboies was used in the drosophila call avidin-biotin peroxidase which labels the neurons. The stain also includes anti tyrosine hydroxylase. Observations were made in days 1, 30 and 60 intervals for activity of the region of the dopaminergic cells.
A confocal microscope can also be used to view the bigger picture of the cross section of the drosophila's brain. This method uses a anti-TH antiserum co-staining. It will review most of the dopaminergic neuron.
The methodology of the climbing assay involves using the fruit fly tap in a vial. The flies were observed traveling from the start line to the finish line in the tube.
Figure 2: Withwortth, A.J, 2006. This represents the dopamingergic systemin the nervous system of a adult drosophila affected for Parkinson.
This picture on figure 2 represents distribution of dopaminergic neurons in the brain of adult Drosophila. They are revealed by confocal microscopy. DA neurons are grouped in small clustersin different spots of the brain.The PPM1/2 is protocerebral posterior medial show cluster of a-synuclein is reported to be affected in the α-synuclein in Parkinson.(Whitworth1, 2006) They are lewy bodies like structures. Loss of function due to Parkinson is shown here.
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Figure 3: (Feany, 2000) Tyrosine hydroxylase immunostaining of a brain tissue cross section. C shows control and d shows experimental
To observe Parkinson in a fly, we have to transfer the Expression of human a-synuclein to the Drosophila model. Figure 3 shows a part of a brain of adult transgenic fly which is similar to Parkinson's disease. To create this fly the mutated a-synuclein gene was inserted in the fly during the embryonic stage.
These are stained tissue sections through the entire brain of the adult fly. They are stained with an antibody against tyrosine hydroxylase. It reveals the dopaminergic neurons. In this cross section on the left, is a one-day-old control fly and dopaminergic neurons are still present. Figure 3 on the right express mutated a-synuclein. It is 30-day-old shows no dopaminergic neuron immunostaining in the same area. This is similar to the results in human parkinson disease. This confirms that Parkinson disease does in fact destroy dopaminergic neurons. The lifespan of the flies are about 60 days.
Figure 4: Macmillian Magazine Ltd. www.nature.com 2000. Climbing assay
Another research experiment call the climbing assay was performed to test their locomotor ability in the presents of Parkinson. Forty flies were placed in a plastic vial, and gently tapped to the bottom of the vial. The files are then travel to the top. The number of flies at the top of the vial was counted after 18 s of climbing. The data shown represent results of 55 days. The experiment was carried out under red light so the flies can travel to the light.
Control flies and Experimental mutant were used. The experimental groups are the transgenic flies that produce inserting normal human a-synuclein and two other is a mutant a-synuclein A30P and A53T a-synuclein. They were produce using missense mutation.
In figure 4 the A30P drosophilas lose their climbing ability earlier than flies expressing wild-type or A53T. The climbing ability of the experimental group decline rapidly compared to the control group from days 23 to 45. Climbing scores for A30P shows more biological toxicity in this mutant protein and a greater damage done in this gene from mutation.
Drosophila as a Model System for Therapeutic Discovery
Drosophila can be use as a rapid model system to test the efficacy of neuroprotective compounds. For example; A discovery was found that overexpression of Hsp70 a 70 kilodalton heat shock proteins can prevent the loss of dopaminergic neurons in this Drosophila Parkinson model. (Celotto, 2005) Geldanamycin is a drug increase Hsp70 protein expression in vivo during cellular stress, which suggests that this compound may prove a viable therapy in humans.
Limitation of drosophila model system
Although Drosophila may provide many key features of human neurological diseases, there are limitations. Our understanding of the fruit fly's neuroanatomy is still developing. The fly has a simpler blood brain barrierand immune system compared to mammalian.(Celotto, 2005) So neuroprotective compounds can easily pass through blood-brain barrier and immune system of the Drosophila. Although we can use Drosophila to make new discoveries in the field neurodegeneration, the discorvery would still need to be validated in a mammalian model system.
Using the drosophila model system is beneficial in experiment. This study is important to help us understand parkinson disease which will lead to more studies of finding a cure for Parkinson disease. We can study animal behaviors and activity with the effects of Parkinson. By putting Parkinson gene in drosophila, we can utilize different neuroprotective compounds that can alleviate the affects of Parkinson.