1. Mitochondrial dynamics and behavioral defects in Taxol and Cisplatin treated
Drosophila melanogaster larvae: A new model for chemotherapy induced
peripheral neuropathy
Figure 3. D. melanogaster larvae behavioral assays. A. The righting
assay measures larval motor control (adapted from Mudher, 2004). B.
The heating assay measures larval sensory reception. The protocol was
adapted from Oswald, et al., 2011.
A.Righting Assay
dorsal
ventral
B. Heating Assay
Chemotherapy induced peripheral neuropathy (CIPN) affects ~ 40
percent of patients receiving chemotherapy treatment causing its
premature termination and sometimes irreversible nerve damage 1,2.
Lack of the model organisms, preferentially with intact nervous system,
to study the CIPN mechanisms prevents from the development of
efficient therapeutic strategies. Changes in neuronal mitochondria
have been observed in many neurodegenerative disorders3. Therefore,
the overall goals of this project are (1) to develop a novel model in D.
melanogaster larvae to study CIPN, (2) to establish whether treatment
with the chemotherapy drugs Taxol and Cisplatin induces changes in
mitochondrial dynamics and function, and (3) examine whether
pretreatment with the drug, code name CP2, that enhances
mitochondrial function could alleviate the effect of chemotherapy
drugs. Since chemotherapy treatments in people are delivered on a
regular schedule, drugs that have a protective effect on peripheral
nerves could potentially eliminate CIPN.
Introduction and Objectives
ã 2012 Mayo Foundation for Medical Education and Research
Steven Forsythe*, Kiley Schmidt*, Georgiy Yudintsev*, Jewel Podratz, Anthony Windebank, Eugenia Trushina
Department of Neurology
Mayo Clinic, Rochester, MN
Figure 2. Growth, treatment, and analysis schedules. A. The
A.Development cycle development cycle of D.
melanogaster. B.
Protocol A is the initially designed
experiment schedule. C. Protocol B was
adapted to include time for full
development and the
possibility of pre-treatment with CP2.
B,C. For both protocols the
concentration curve for Cisplatin was
0uM, 1uM, 5uM, 10uM, 25uM, 50uM,
for Taxol, 0uM, 0.5uM, 0.8uM, 1uM,
10uM, 30uM.
Flagg, 1979
B. Protocol A C. Protocol B
Day 1: Add adult flies to grape Day 1: Add adult flies to grape
plate apparatus. plate apparatus
Day 2: Remove adult flies. Remove Day 2: Remove adult flies. Remove
plate containing eggs from plate containing eggs from
apparatus. apparatus.
Day 3: Transfer 1st instar larvae to Day 3: Transfer 1st instar larvae to
drug treated blue food. clean blue food or CP2
pre-treated blue food.
Day 4,5: Incubate at 25°C
Day 6: Harvest early 3rd instar larvae. Day 4: Incubate at 25°C
Perform behavioral tests and Day 5: Treat 2nd instar larvae with
confocal imaging. Cisplatin or Taxol food.
Day 7+: Measure percent survival.
Day 6,7: Incubate at 25°C
Day 8: Perform behavioral tests and
confocal imaging.
Day 9+: Measure percent survival
Protocol Overview
• Breed w; +/+; D42-Gal 4, UAS-mitoGFP/TM6 Tb adult D.
melanogaster
• Treat larvae with various concentrations of Cisplatin or Taxol
• Perform behavioral assays on 3rd instar larvae to determine motor
and sensory axon dysfunction
• Dissect 3rd instar larvae to display intact nervous system
• Image motor axons using confocal microscopy
• Analyze mitochondria dynamics
Methods
measure time
Figure 4. Survival measurement A.
A. Developed protocol
to measure lethality
for each drug treatment.
B. Data recorded for Taxol and
Cisplatin percent survival.
B.
Taxol Survival Curve Cisplatin Survival Curve
1. National Cancer Institute. 2010. Chemotherapy-induced peripheral neuropathy. NCI Cancer Bulletin 7(4).
2. Marchettini P, Lacerenza M, Mauri E, Marangoni C. 2006. Painful peripheral neuropathies. Current Neuropharmacology 4(3): 175-181.
3. Filosto M, Scarpelli M, Cotelli MS, Vielmi V, Todeschini A, Gregorelli V, Tonin P, Tomelleri G, Padovani A. 2011. The role of mitochondria in
neurodegenerative diseases. Journal of Neurology 258(10): 1763-1774.
4. Flagg RO. 1979. The Carolina Drosophila manual. Carolina Biological Supply Co.
5. Mudher A, Shepherd D, Newman TA, Mildren P, Jukes JP, Squire A, Mears A, Berg S, MacKay D, Asuni AA, Bhat R, Lovestone S. 2004. GSK-3 inhibition
reverses axonal transport defects and behavioural phenotypes in Drosophila. Molecules Psychiatry 9: 522-530
6. Oswald M, Rymarczyk B, Chatters A, Sweeney S. 2011. A novel thermosensitive escape behavior in Drosophila larvae. Fly 5(4): 304-306.
References
•A successful protocol for using D. melanogaster for the study of CIPN was
developed. This included the treatment schedule, the behavioral and survival
assay, modified larval dissection and preparation methods, and confocal
imaging and analysis techniques.
•Preliminary data has been collected for the behavioral assays and survival
tests. The results display the trend that as the concentration of drug is
increased, the motor and sensory function is decreased and lethality is
increased.
•Kymographs were prepared to observe mitochondrial dynamics.
Conclusion
Figure 1. Fluorescent image of D. melanogaster nervous system. The
ventral ganglion (VG) is labeled on the left (anterior end). Motor neuron
axons extend posteriorly from VG with GFP tagged mitochondria.
0
20
40
60
80
100
0 0.5 0.8 1 10 30
PercentSurvival
Taxol Concentration (uM)
0
20
40
60
80
100
0 0.5 0.8 1 10 30
SecondstoRight
Taxol Concentration (uM)
0
20
40
60
80
100
28 29 30 31 32
NumberofLarvae
Temperature (C)
0
2
4
6
8
10
12
0 1 5 10 25 50
NumberofLarvae
Cisplatin Concentration (ug/ml)
Larva Nonreacted
Larva Reacted
0
20
40
60
80
100
0 1 5 10 25 50
PercentSurvival
Cisplatin Concentration (ug/ml)
•Replicate experiments to increase numbers
•Refine techniques
•Perform CP2 pre-treatment experiments and analyze for recovered phenotype
•Analyze numerical data from confocal videos
•Analyze mitochondrial function, fission and fusion
•Link behavioral data to mitochondrial dynamics and function
Future Directions
Figure 5. D. melanogaster segmental nerve confocal imaging.
Dissected larval
preparations were placed
On a glass slide in
hemolymph like solution
(HL6 buffer) at room
Temperature and
coverslipped. Distal region
of the 8th segmental nerve
was bleached for 15 sec
at 100% power of 488 nm
Laser and then imaged for
600 frames with 1 sec.
interval using 60x water
objective. Anterograde
and retrograde movements
of mitochondria were
tracked from frame to frame.
Figure 6. Change in mitochondrial dynamics upon treatment with
Cisplatin and Taxol. Kymographs were generated for Cisplatin 1.0
µg/ml, 5.0 µg/ml, 10.0 µg/ml and Taxol 0.8 µM, 1.0 µM, 10 µM treated 3rd
instar larvae versus untreated 3rd instar larvae. X-axis represents the
length of the region of interest (distal part of segmental nerve 8).
Y-axis represents the time (1 sec/pixel). From these treatments we have
observed that both retrograde and anterograde fluxes decrease in
treated flies, with most prominent
changes in mitochondrial transport
seen in 5.0 µg/ml of Cisplatin where no
movement of the mitochondria is seen.
This work was supported by funding through UREP and SURF programs, as well as
the Department of Molecular and Cellular biology at the University of Illinois Urbana-
Champaign. We also would like to give special thanks to Jewel Podratz, Swathi
Devireddy and Dr. Hollenbeck, Dr. Eugenia Trushina, and Han Lee for all their help
and guidance in the project and Mayo Clinic Microscopy Core facility.
Acknowledgments