Thant_Investigating the Effects of Paraquat Induced Oxidative Stress on Axonal Transport
1. www.buffalo.edu
Abstract
High levels of oxidative stress can be detected in neurons affected by
neurodegenerative diseases such as Parkinson’s, Huntington’s, and Alzheimer’s
diseases. In addition to oxidative stress, axonal transport defects are seen in these
disease backgrounds. However, no research exists testing whether there is a direct
relationship between oxidative stress and axonal transport defects. Here, we test the
hypothesis that elevated levels of oxidative stress within neurons will cause defects in
overall axonal transport. In order to test this hypothesis, the model system Drosophila
melanogaster will be utilized. Drosophila larvae will be raised on food laced with
Paraquat, a chemical known to induce oxidative stress. These larvae will then be
dissected, and analyzed for axonal transport defects using immunofluorescence
microscopy. Interestingly we find that the number of blockages increase with increased
concentration of Paraquat suggesting that increased oxidation and the viability of adult
flies decline. Suggesting that oxidative stress can lead to axonal transport defects.
Project Aims
Investigating the Effects of Paraquat Induced Oxidative Stress on Axonal
Transport
Claire Thant, Megan Lamb, Timothy Hansen, Shermali Gunawardena
University at Buffalo Department of Biological Sciences
Aim 1: Examine the role of Paraquat induced stress in causing axonal
blockages
Aim 2: Examine the role the PI3K-Akt signaling pathway plays in suppressing
Paraquat induced axonal blockages.
Experiment Concentration Expected
Result
Observed
Result
Aim 1: Effect of
Paraquat Induced Stress
on Axonal Transport
0 mM Paraquat No Axonal
Blocks
No Axonal
Blocks
10 mM Paraquat Axonal
Blocks
Axonal Blocks
20 mM Paraquat Axonal
Blocks
Axonal Blocks
Aim 2: Effect of
PI3K.CAAX on Paraquat
Induced Axonal
Transport Defects
0 mM Paraquat,
PI3K.CAAX
No Axonal
Blocks
TBD
20 mM Paraquat,
PI3K.CAAX
No Axonal
Blocks
TBD
Paraquat causes high levels of oxidative stress in cells. Our hypothesis is that in
neurons this stress causes defects in overall axonal transport since this pathway is
needed for viability. To test this hypothesis, the model system Drosophila
melanogaster (fruit fly) is utilized. Paraquat is fed to Drosophila larvae from the time
they hatch from eggs, 0 mM, 10mM, & 20mM, and then these larvae are dissected,
fixed, and stained with antibodies for synaptic vesicles and imaged to evaluate the
presence of axonal transport defects identified as axonal blockages containing
accumulations of synaptic vesicles.
The PI3K-Akt signaling pathway has been shown to reduce oxidative stress when over
activated (Martindale et al, 2002). Our hypothesis is that overexpression of a
constitutively active form of the PI3K protein will reduce oxidative stress in neurons;
thereby suppressing Paraquat induced axonal transport defects. To test this hypothesis
larvae that express a constitutively active form of the protein PI3K will be generated.
These larvae will then be subjected to the same doses of Paraquat as the wild type
larvae, and will be analyzed for axonal blockages.
References
Acknowledgements
Special thanks to everyone at the Gunawardena Lab, as well as the UB Center for
Undergraduate Research and Creative Activities for funding this project.
Third Instar Larvae Fixed Dissection
Brain & Ventral Ganglion Nerves
Superior view
A.
Cranial Caudal
Fly Cage Apparatus:
Apple Juice Agar Plates
B.
Fly Condo #1: Wild Type: ApplGal4;+;+C.
Fly Condo # 2: Constitutively Active PI3K:
ApplGal4;+;+ x UAS-PI3K92E.CAAX;+;+☿ ♂
D.
Control 20x
Control 40x
Control 63x
10 mM Paraquat 20x
10 mM Paraquat 40x
10 mM Paraquat 63x
20 mM Paraquat 20x
20 mM Paraquat 40x
20 mM Paraquat 63x
ApplGal4;+;+
Arvind K. Shukla, Prakash Pragya, Hitesh S. Chaouhan, D.K. Patel, M.Z. Abdin,
Debapratim Kar Chowdhuri, “A mutation in Drosophila methuselah resists paraquat
induced Parkinson-like phenotypes.” Neurobiology of Aging, Volume 35, Issue 10,
October 2014, Pages 2419.e1-2419.e16
Gunawardena, S. and Goldstein, L.S.B. (2001).
"Disruption of axonal transport and neuronal viability by amyloid precursor protein mutations in
Neuron 32:389-401.
Gunawardena, S., Her, L., Laymon, R.A., Brusch, R.G., Niesman, I.R., Sintasath, L.,
Bonini, N.M., and Goldstein, L.S.B. (2003) "Disruption of axonal transport by loss of
huntingtin or expression of poly Q protein in Drosophila." Neuron 40:25-40.
Martindale, J.L., Holbrook, N.J. (2002) “Cellular response to oxidative stress: Signaling
for suicide and survival” J. Cel.. Physiol. 192: 1-15.
Methods
Figure 1. Experimental
setup for inducing oxidative
stress in Drosophila larvae
via a Paraquat feeding
assay. A. Third Instar
Drosophila larvae dissected
so the nervous system is
visible to image for axonal
blockages. B. Fly Cage
Apparatus for collection of
embryos. C. Fly Condo #1
setup for raising Wild Type
Drosophila on 0 mM,
10mM, 20 mM Paraquat
concentrations. D. Fly
Condo #2 setup for raising
Drosophila with
Constitutively Active PI3K
with 0 mM, and 20 mM
Paraquat concentrations.
Results
CSP
A B C
Conclusion
p = 0.12904
p = 0.08745
Figure 2: Paraquat induces axonal transport defects in third instar Drosophila
larvae. A. Control larvae raised on food that did not contain Paraquat show no
defects in axonal transport as analyzed by an antibody against synaptic vesicles
(CSP). B-C. Larvae raised on food containing either 10 mM, or 20 mM Paraquat
show axonal tranpsort defects (Arrows). D. Quantitative analysis reveals the extent
of blockages in 0 mM, 10 mM, and 20 mM Paraquat concentration fed larvae are not
significant when compared to the wild type control, however are trending towards
significance. N=5 larvae. E. Quantitative analysis of viable adult flies from each
concentration wells of Fly Condo #1, shows significant decrease in adults in the 20
mM Paraquat concentration when compared with the control, although the adults from
10 mM concentration are not significant when compared to the control, there appears
to be a trend in decline of viable adults with increased concentration.
D. E.
p = 0.06158
*
p = 0.02888
• Oxidative Stress induced by 10 mM and 20 mM Paraquat concentrations have an
effect on axonal transport. (Figure 2 B-C.)
• 10 mM and 20 mM concentration of Paraquat induces blockages trending towards
significance when compared to the control. (Figure 2 D.)
• Significant decrease in adult fly viability is seen in 20 mM Paraquat concentration
when compared to the control. (Figure 2 E.)
In a repeat experiment of Fly Condo #1 are Paraquat induced axonal blockages seen?
If so, what is the role of PI3k-Akt signaling pathway in suppressing Paraquat
induced axonal blockages?