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.
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®.
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50
100
150
200
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300
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.
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BY250 UA44 N2
Velocity(μm/S)
Strain