Darley Source Poster final
1
Development of an Assay to Detect Degenerative Dopaminergic Neurons in Caenorhabditis elegans Jacob Darley, Maria Niemuth, Eric Foss & Lucinda Carnell Biology Department, Central Washington University, Ellensburg, WA 98926, USA Results Parkinson’s disease (PK) is a disruption of motor function caused by loss of dopamine neurons. PK can be caused by environmental and genetic factors. One protein known to contribute to PK is the protein α-synuclein found in dopamine neurons. Over expression or mutations in α-synuclein can lead to PK. The soil nematode, Caenorhabditis elegans (C. elegans), has been developed as a model for PK by using a transgenically modified strain, which overexpresses the human α-synuclein protein. In this strain, the dopamine neurons, which have been labeled with a green fluorescent protein, were observed by fluorescent microscopy to degenerate after nine days of development. We have discovered that the transgenic strain expresses a locomotory behavioral defect that is indicative of deficient dopamine signaling at day three of development. When wild-type (normal) nematodes encounter their food, which is a bacterial lawn, they slow their locomotory speed. However, the transgenic strain does not exhibit the prototypical slowing behavior that stems from the excitation of the dopamine neurons. This defect was determined by utilizing an automated tracking system to quantify speed of locomotion on and off food. We can extrapolate that this behavior is correlated to nematodes that exhibit degenerative dopamine neurons, as this same behavioral defect is observed in cat-2 mutants that do not generate dopamine. In future studies, we will utilize this assay to examine the effects of environmental stressors on these neurons and their relation to PK. Abstract Model Organism: C. elegans is a free-living soil nematode whose three day reproductive cycle, relative simplicity and invariable body plan make it a prime candidate for neurobiology research. In addition to the inherent morphological and physiological traits of the organism, C. elegans is a widely studied organism, whose entire genome has been mapped and for which many mutant and transgenic strains exist. Several of these transgenic strains were used in our research. Experimental Design We have developed an model of Parkinson’s disease using C. elegans to examine loss of function of the neurons before they degenerate. This assay will decrease the amount of time required to detect genes or environmental factors that may contribute to the onset of Parkinson’s disease. Conclusion To Dr. Lucinda Carnell and Eric Foss of the Biology Department for their excellent guidance and mentorship. To the STEP program (NSF Grant DUE-0653094) for financial support. Acknowledgments Figure 2 depicts the anatomy of the nematode C. elegans. Introduction The UA44 α-synuclein transgenic strain exhibited the expected lack of slowing response when exposed to food. This behavior demonstrates deficiency in the dopaminergic mechanosensory neurons that detect the presence of the bacterial lawn. The UA44 did not however show an absolute lack of response to the presence of a bacterial lawn. This indicates that the dopaminergic neurons in question are not the only neurons which mediate locomotory response to food in C. elegans. Procedure: Strains : Three worm strains were used in this research project. N2 (wild type) strain which served as an assay control. BY250 which expresses a green fluorescent protein (GFP) to serve as an experimental control. UA44 which is essentially the transgenic strain BY250 which also contains the human α- synuclein protein in the dopamine neurons. Behavioral Assay : Eggs were collected at hour zero and transferred to maintenance plates and incubated for 72 hours. Plates were poured at hour 24 with nematode growth media (NGM). At hour 48 three of the assay plates were seeded with OP-50 bacterial broth and the bacteria was spread into an even lawn. At hour 72 the experiment was performed. Each plate had three paper corrals placed on the surface of the agar. CuCl 2 was added to the borders of the corrals. The CuCl 2 acts as a chemical repellant ensuring that the organisms do not depart from the corals. Tracking and Analysis: Animal were video taped for 15 seconds at 10 minutes and one hour. The camera used to capture the videos was mounted to a zoom system with 6x magnification. The speeds were determined using an automated tracking system running on a program “Wormtracker” written using Matlab ® . 0 50 100 150 200 250 10 Min Food 10 Min No Food 60 Min Food 60 Min No Food • Dopamine plays various roles in the human brain including motivation, motor control and reward. Of particular concern to this research is dopamine’s role in motor control. • Parkinson’s disease is characterized by the aggregation of the protein α-synuclein in dopaminergic motor neurons. This aggregation leads to loss of neural function and eventually neural degeneration • Previously the deeneration of neurons in the model organism Caenorhabditis elegans was determined via observing a fluorescent tagged protein (GFP) under fluorescent microscopy. This process was time demanding, given that the degeneration was not visible until day 9 of development. • A new assay was needed to accelerate PK research and our research sought to fill that need. Figure 4 demonstrates the results of the experiment. The UA44 (α-syn) strain demonstrates significantly faster velocity when exposed to food than both the BY250 and N2 strains. The t-value for 10 Min BY250 vs. 10 Min UA44 is 0.447. The t-value for 60 Min BY250 vs. 60 Min UA44 is 0.594. Both P-values far lower than the critical value of 2.57 and as such the data is statistically significant. Figure 3 Visualization of dopamine neurons in C elegans. There are eight dopamine neurons in C. elegans. The neurons are visualized using a transgenic strain, BY250, which expresses the green fluorescent protein in these neurons. The cell bodies and axons of the four CEP neurons located in the anterior (head) of the animal are shown. Figure 1 depicts the dopamine signaling pathways in the human brain. * * * * * * BY250 UA44 N2 Velocity (μm/S) Strain
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Transcript of Darley Source Poster final
Development of an Assay to Detect Degenerative Dopaminergic Neurons in Caenorhabditis elegans
Jacob Darley, Maria Niemuth, Eric Foss & Lucinda Carnell Biology Department, Central Washington University, Ellensburg, WA 98926, USA
ResultsParkinson’s disease (PK) is a disruption of motor function caused by loss of dopamine neurons. PK can be caused by environmental and genetic factors. One protein known to contribute to PK is the protein α-synuclein found in dopamine neurons. Over expression or mutations in α-synuclein can lead to PK. The soil nematode, Caenorhabditis elegans (C. elegans), has been developed as a model for PK by using a transgenically modified strain, which overexpresses the human α-synuclein protein. In this strain, the dopamine neurons, which have been labeled with a green fluorescent protein, were observed by fluorescent microscopy to degenerate after nine days of development. We have discovered that the transgenic strain expresses a locomotory behavioral defect that is indicative of deficient dopamine signaling at day three of development. When wild-type (normal) nematodes encounter their food, which is a bacterial lawn, they slow their locomotory speed. However, the transgenic strain does not exhibit the prototypical slowing behavior that stems from the excitation of the dopamine neurons. This defect was determined by utilizing an automated tracking system to quantify speed of locomotion on and off food. We can extrapolate that this behavior is correlated to nematodes that exhibit degenerative dopamine neurons, as this same behavioral defect is observed in cat-2 mutants that do not generate dopamine. In future studies, we will utilize this assay to examine the effects of environmental stressors on these neurons and their relation to PK.
AbstractModel Organism:
C. elegans is a free-living soil nematode whose three day reproductive cycle, relative simplicity and invariable body plan make it a prime candidate for neurobiology research. In addition to the inherent morphological and physiological traits of the organism, C. elegans is a widely studied organism, whose entire genome has been mapped and for which many mutant and transgenic strains exist. Several of these transgenic strains were used in our research.
Experimental Design
We have developed an model of Parkinson’s disease using C. elegans to examine loss of function of the neurons before they degenerate. This assay will decrease the amount of time required to detect genes or environmental factors that may contribute to the onset of Parkinson’s disease.
Conclusion
To Dr. Lucinda Carnell and Eric Foss of the Biology Department for their excellent guidance and mentorship. To the STEP program (NSF Grant DUE-0653094) for financial support.
Acknowledgments
Figure 2 depicts the anatomy of the nematode C. elegans.
Introduction The UA44 α-synuclein transgenic strain exhibited the expected lack of slowing response when exposed to food. This behavior demonstrates deficiency in the dopaminergic mechanosensory neurons that detect the presence of the bacterial lawn. The UA44 did not however show an absolute lack of response to the presence of a bacterial lawn. This indicates that the dopaminergic neurons in question are not the only neurons which mediate locomotory response to food in C. elegans.
Procedure:Strains:
Three worm strains were used in this research project. N2 (wild type) strain which served as an assay control. BY250 which expresses a green fluorescent protein (GFP) to serve as an experimental control. UA44 which is essentially the transgenic strain BY250 which also contains the human α-synuclein protein in the dopamine neurons.
Behavioral Assay:Eggs were collected at hour zero and transferred to maintenance plates
and incubated for 72 hours. Plates were poured at hour 24 with nematode growth media (NGM). At hour 48 three of the assay plates were seeded with OP-50 bacterial broth and the bacteria was spread into an even lawn. At hour 72 the experiment was performed.
Each plate had three paper corrals placed on the surface of the agar. CuCl2 was added to the borders of the corrals. The CuCl2 acts as a chemical repellant ensuring that the organisms do not depart from the corals.
Tracking and Analysis:Animal were video taped for 15 seconds at 10 minutes and one hour. The
camera used to capture the videos was mounted to a zoom system with 6x magnification. The speeds were determined using an automated tracking system running on a program “Wormtracker” written using Matlab®.
0
50
100
150
200
250
10 Min Food10 Min No Food60 Min Food60 Min No Food
• Dopamine plays various roles in the human brain including motivation, motor control and reward. Of particular concern to this research is dopamine’s role in motor control.
• Parkinson’s disease is characterized by the aggregation of the protein α-synuclein in dopaminergic motor neurons. This aggregation leads to loss of neural function and eventually neural degeneration
• Previously the deeneration of neurons in the model organism Caenorhabditis elegans was determined via observing a fluorescent tagged protein (GFP) under fluorescent microscopy. This process was time demanding, given that the degeneration was not visible until day 9 of development.
• A new assay was needed to accelerate PK research and our research sought to fill that need.
Figure 4 demonstrates the results of the experiment. The UA44 (α-syn) strain demonstrates significantly faster velocity when exposed to food than both the BY250 and N2 strains. The t-value for 10 Min BY250 vs. 10 Min UA44 is 0.447. The t-value for 60 Min BY250 vs. 60 Min UA44 is 0.594. Both P-values far lower than the critical value of 2.57 and as such the data is statistically significant.
Figure 3 Visualization of dopamine neurons in C elegans. There are eight dopamine neurons in C. elegans. The neurons are visualized using a transgenic strain, BY250, which expresses the green fluorescent protein in these neurons. The cell bodies and axons of the four CEP neurons located in the anterior (head) of the animal are shown.
Figure 1 depicts the dopamine signaling pathways in the human brain.
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BY250 UA44 N2
Velo
city
(μm
/S)
Strain
