Cisplatin induces mitochondrial deficits in Drosophila larval segmental nerve

Abstract

Cisplatin is an effective chemotherapy drug that induces peripheral neuropathy in cancer patients. In rodent dorsal root ganglion neurons, cisplatin binds nuclear and mitochondrial DNA (mtDNA) inducing DNA damage and apoptosis. Platinum-mtDNA adducts inhibit mtDNA replication and transcription leading to mitochondrial degradation. Cisplatin also induces climbing deficiencies associated with neuronal apoptosis in adult Drosophila melanogaster. Here we used Drosophila larvae that express green fluorescent protein in the mitochondria of motor neurons to observe the effects of cisplatin on mitochondrial dynamics and function. Larvae treated with 10μg/ml cisplatin had normal survival with deficiencies in righting and heat sensing behavior. Behavior was abrogated by, the pan caspase inhibitor, p35. However, active caspase 3 was not detected by immunostaining. There was a 27% decrease in mitochondrial membrane potential and a 42% increase in reactive oxygen species (ROS) in mitochondria along the axon. Examination of mitochondrial axonal trafficking showed no changes in velocity, flux or mitochondrial length. However, cisplatin treatment resulted in a greater number of stationary organelles caused by extended pausing during axonal motility. These results demonstrate that cisplatin induces behavior deficiencies in Drosophila larvae, decreased mitochondrial activity, increased ROS production and mitochondrial pausing without killing the larvae. Thus, we identified particular aspects of mitochondrial dynamics and function that are affected in cisplatin-induced peripheral neuropathy and may represent key therapeutic targets.

Keywords: Apoptosis; Axonal trafficking; Cisplatin; Drosophila; Membrane potential; Mitochondria; Motor neuron.

Figure 1

Experimental Model. (A) Schematic diagram of synchronization of larvae developmental stages and drug treatment. (B) Image of dissected larva with exposed motor neurons and segmental nerves. Larvae image was acquired using a Zeiss Axioscope (Carl Zeiss, Thornwood, NY) with a 40X lens.

Figure 2. Cisplatin induces behavior deficiencies at 10 μg/ml without affecting larval survival and behavior deficiencies are inhibited by p35

(A) Survival of cisplatin treated Drosophila larvae to pupa and adults developmental stages was determined as a percent of the number of larvae added at the beginning of the experiments. The number of larvae that became pupa and the number of pupa that hatched into adults were calculated as percent survival (n=100-180 larvae). Larvae treated with 0, 10 and 25 μg/ml cisplatin were observed for righting, crawling and heat sensing abilities. There were significant delays in righting time (B) (p<0.0001), decreases in crawling distance (C) (p<0.01) and increases in heat sensing time (D) (p<0.01-0.0001) with 10 and 25 μg/ml cisplatin (n=14-60 for righting, 30-35 for crawling and 20-27 for heat sensing behavior). Expression of p35 in motor neurons improved righting (B), crawling (C) and heat sensing (D) behavior with no significant differences between untreated and cisplatin treated p35 larvae. However, righting behavior in 25 μg/ml cisplatin treated p35 larvae was significant from untreated p35 larvae (p<0.05). (n=14-15 larvae) (means and SEM are shown for each data point)

Figure 3. No active caspase 3 was observed in motor neurons treated with 10 μg/ml cisplatin

Mito-GFP larvae treated with 0, 10 and 25 μg/ml cisplatin were dissected and immunostained for active caspase 3. Images of ventral nerve cord showed no positive staining in Mito-GFP larvae treated with 0 (A, D, G) and 10 (B, E, H) μg/ml cisplatin. Increasing cisplatin concentration to 25 μg/ml induced active caspase 3 (C, F) in mitochondria (I). Images were acquired using a LSM 780 Axio Observer microscope with a 40X lens (C-apochromat/1.20 W). Scale bar 2.5 μm.

Figure 4. Cisplatin induces a decrease in mitochondrial TMRM uptake and an increase in ROS production

Mito-GFP Drosophila larvae treated with 0 (A) and 10 μg/ml (B) cisplatin were stained with 1 μM TMRM (C, D, red). (E, F) Co-localization of green (mitochondrial GFP) and red (TMRM uptake) fluorescence in larvae segmental nerve. (G) Percent of GFP-tagged mitochondria that uptake TMRM. Analysis was done in 34-79 nerve segments from 17-38 larvae (p<0.0001). Scale bar 30 μm. Mito-GFP Drosophila larvae treated with 0 (H) and 10 μg/ml cisplatin (I) and stained for ROS using 5 nM MitoSox (J, K, red). (L) Cisplatin significantly increased mitochondrial ROS as indicated by the oxygen radical sensor, MitoSox. Analysis was done in 26-41 nerve segments from 7-16 larvae (p<0.05). Scale bar 300 μm. Images were acquired using an Olympus Multiphoton Laser Scanning microscope with a 60X lens (UPLSAPO/1.2 W). (Means and SEM are shown for each data point)

Figure 5. Treatment with cisplatin does not affect the velocity of mitochondrial motility in axons, the number of moving organelles or their length

(A) Real time imaging of mitochondrial movement within the motor neurons of live third instar larvae recorded using confocal fluorescence microscopy. All mitochondria express GFP (top image). Central area of the nerve was photo bleached (second image from the top) to visualize mitochondrial movement. The progression of the circled organelle along the axon could be seen in the consecutive images. Scale bar, 30 μm. Images were acquired using an Olympus Multiphoton Laser Scanning microscope (FV1000MPE) with a 60X lens (UPLSAPO/1.2 W). (B) Velocity of mitochondria was determined by dividing the total distance (μm) traveled by the total time spent in motion (sec). A total of 326-360 mitochondria were measured per condition for velocity. (C) Mitochondrial flux measured the number of mitochondria moving across the axon in the anterograde and retrograde direction. Flux was measured in 21-22 nerve segments from 16-17 larvae. (D) Mitochondria length was measured in 45-50 larvae (μm). (Means and SEM are shown for each data point)

Figure 6. Cisplatin increased the number and length of mitochondrial pausing along the axon

(A) Mitochondria were quantitated for the percent of total time spend moving in the anterograde and retrograde direction or paused. Duty cycle was measured in 328-360 mitochondria (p<0.05-p<0.01). Kymographs of mitochondria in untreated (B, C) and treated (D, E) motor neurons show overall movement with time (X axis) verse distance (Y axis). White arrows in (B) and (C) represent organelles moving in the net anterograde and retrograde directions, respectively. Images were acquired using an Olympus Multiphoton Laser Scanning microscope (FV1000MPE) with a 60X lens (UPLSAPO/1.2 W). Scale bar 30 μm. Larvae treated with 10 μg/ml cisplatin showed longer pauses (white brackets) in both the (D) anterograde and (E) retrograde net direction. (F) Pauses separated into net anterograde or net retrograde moving mitochondria show increased pausing in both directions. (G) The length of pauses of mitochondria moving in the anterograde and retrograde net direction (p<0.05). Pause length was calculated from 147-157 mitochondria per condition with a total of 3225-7561 pauses taken into analysis. (Means and SEM are shown for each data point)

Figure 7

Model of cisplatin-induced neurotoxicity.