PINK1 alleviates thermal hypersensitivity in a paclitaxel-induced Drosophila model of peripheral neuropathy.

Paclitaxel is a representative anticancer drug that induces chemotherapy-induced peripheral neuropathy (CIPN), a common side effect that limits many anticancer chemotherapies. Although PINK1, a key mediator of mitochondrial quality control, has been shown to protect neuronal cells from various toxic treatments, the role of PINK1 in CIPN has not been investigated. Here, we examined the effect of PINK1 expression on CIPN using a recently established paclitaxel-induced peripheral neuropathy model in Drosophila larvae. We found that the class IV dendritic arborization (C4da) sensory neuron-specific expression of PINK1 significantly ameliorated the paclitaxel-induced thermal hyperalgesia phenotype. In contrast, knockdown of PINK1 resulted in an increase in thermal hypersensitivity, suggesting a critical role for PINK1 in sensory neuron-mediated thermal nociceptive sensitivity. Interestingly, analysis of the C4da neuron morphology suggests that PINK1 expression alleviates paclitaxel-induced thermal hypersensitivity by means other than preventing alterations in sensory dendrites in C4da neurons. We found that paclitaxel induces mitochondrial dysfunction in C4da neurons and that PINK1 expression suppressed the paclitaxel-induced increase in mitophagy in C4da neurons. These results suggest that PINK1 mitigates paclitaxel-induced sensory dendrite alterations and restores mitochondrial homeostasis in C4da neurons and that improvement in mitochondrial quality control could be a promising strategy for the treatment of CIPN.

Introduction

Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect that limits many effective anticancer chemotherapies [1]. CIPN is observed in over 68% of patients that receive chemotherapy [2]. Although various symptoms arise depending on the type, amount, and duration of the anticancer drug, typical clinical features of CIPN include sensory nerve abnormalities such as paresthesia, allodynia and hyperalgesia [2]. These symptoms may persist for several months to several years after the completion of chemotherapy in up to 30% of patients, and in severe cases, CIPN adversely affects the quality of life of cancer survivors for the lifetime [2]. However, although CIPN has a negative impact on the quality of life of cancer patients, there is currently no effective and established preventive treatment. Paclitaxel, a microtubule-stabilizing agent, is widely used to treat various solid tumors and is a representative anticancer drug that induces CIPN [3]. The major dose-limiting side effect of paclitaxel treatment is painful peripheral neuropathy, which is predominantly sensory [4, 5].

Drosophila models have been shown to be useful for identifying essential genes required for the thermal nociception response [6, 7]. Class IV multidendritic sensory neurons are responsible for sensory nociception and behavioral responses to various noxious stimuli, including mechanical forces and high temperatures, in Drosophila larvae [7, 8]. Upon noxious thermal challenge, third instar (L3) larvae show a characteristic corkscrew-like rolling motion that can be easily analyzed [7]. Recent studies that used the Drosophila larval CIPN model for paclitaxel-induced peripheral neuropathy have recapitulated CIPN sensory dysfunction [9, 10]. These studies established that paclitaxel treatment induces hypersensitivity to noxious thermal stimulation, that is, thermal hyperalgesia in L3 larvae. More importantly, these studies also revealed that alterations in the sensory dendrites of class IV dendritic arborization (C4da) sensory neurons are associated with paclitaxel-induced peripheral neuropathy [9, 10]. These results proved that the Drosophila larva model of paclitaxel-induced peripheral neuropathy is a robust model to investigate the molecular mechanism of CIPN.

Phosphatase and tensin homologue (PTEN)-induced putative kinase 1 (PINK1) is a mitochondrial Ser/Thr kinase and is known as a key regulator of mitochondrial quality control and mitochondria homeostasis [11-13]. PINK1 works as a molecular sensor for mitochondrial damage [11] and controls critical mechanisms for mitochondria quality control including mitochondria fission, fusion, transport, biogenesis and mitophagy [14]. Consistent with the essential role of PINK1 in mitochondrial function, the loss of PINK1 results in mitochondrial dysfunction and hypersensitivity to toxic stress [15, 16]. In contrast, PINK1 protects neuronal cells against various toxic insults including MPTP and, α-synuclein [17, 18]. However, whether PINK1 expression also has a protective effect on CIPN has not been investigated.

In this study, we examined the effect of PINK1 expression on the paclitaxel-induced CIPN model in Drosophila larvae. We found that PINK1 expression significantly ameliorated the paclitaxel-induced thermal hyperalgesia phenotype. Our analysis revealed that PINK1 expression suppressed aberrant paclitaxel-induced alterations in sensory dendrite C4da neurons and mitophagy induction.

Materials and methods

Drosophila strains

The ppk-GAL4, ppk-GAL4 and UAS-CD4-tdTomato (CD4-tdTom) lines were kindly provided by Y.N. Jan (University of California, San Francisco, CA). The UAS-mt-Keima line was generated previously [19]. The UAS-PINK1 and PINK1 RNAi lines were generated previously [16]. w and (UAS-GFP dsRNA; BL9331) lines were obtained from the Bloomington Stock Center (Indiana University, Bloomington, IN). The UAS-mito-roGFP2-Orp1 line was a gift from Dr. Tobias Dick (German Cancer Research Center, Heidelberg, Germany).

Larval thermal nociception assays

The larval thermal nociception assays were performed as described previously [6, 9, 10]. Briefly, L3 larvae (120 h after egg laying [AEL]) were rinsed with distilled water and gently placed on a petri dish. After 10 sec acclimation, the larval abdominal A4-A5 segments were touched under a microscope with a custom-built 0.6-mm-wide thermal probe whose temperature was controlled by a microprocessor. The time required to induce the aversive corkscrew-like rolling response was measured as the withdrawal latency up to the 20-sec cut off. The larvae showing no rolling response within 20 sec were considered to have no response. For each thermal nociception assay, at least 50 larvae were analyzed, and the results are presented as the mean values with standard deviation (SD).

Paclitaxel treatment

Paclitaxel was administered following the feeding regimen described previously [9]. Briefly, twenty virgin female files were mated with fifty male flies for 48–72 h, and the embryos were collected for 2–4 h on grape juice agar plates (15 ml ddH2O, 5 ml grape juice, 0.6 g agar, 1.1 g sucrose, 0.5 ml ethanol, 0.25 ml acetic acid, supplemented with yeast paste (700 mg baker’s yeast in 1 ml ddH2O)). The embryos were grown for 72 h to develop into L3 larvae. Larvae were rinsed with distilled water and transferred to freshly made grape juice agar plates containing either 20 μM paclitaxel or 0.2% DMSO. Larvae were grown for another 48 h before performing the thermal nociception assay.

Measurement of larval size

To measure larval size, images of larvae were captured using a dissection microscope (OLYMPUS MVX10, Olympus Co., Tokyo, Japan) after the thermal nociception assay. The larval area was calculated using ImageJ software (NIH, Bethesda, MD). At least 30 larvae were analyzed per genotype, and the results are presented as the mean values with SD.

Analysis of images of C4da neuron dendrites

To determine the dendritic structure of C4da neurons at abdominal segment A4 of L3 larvae, images of the fluorescent plasma membrane marker CD4-tdTomato (CD4-tdTom) [20] were obtained using a Zeiss LSM 800 confocal microscope (Carl Zeiss, Oberkochen, Germany). Confocal image stacks of C4da neuron dendrites were converted to maximum intensity projections using Zeiss Zen software. Dendrite length and the number of dendritic branches were analyzed in ImageJ software using the skeleton plugin function (NIH, Bethesda, MD). At least 5 larvae were analyzed per genotype. The dendrite image analysis was repeated three times, and similar results were observed. The results are presented as the mean values with SD.

In vivo measurement of mitochondrial ROS of C4da neurons

For in vivo ROS imaging, ppk>mito-roGFP2-Orp1 L3 larvae were examined with a Zeiss LSM 800 confocal microscope (Carl Zeiss) with a 405-nm (oxidized) or 488-nm (reduced) excitation laser using 520-nm emission as previously described [21]. At least 4–5 larvae were imaged for each group and the 405-nm/ 488-nm fluorescence intensity was obtained using Zeiss Zen software. The results are presented as the mean values with SD.

Measurement of mitophagy levels

Mitophagy levels were examined using the pH-dependent, fluorescent probe mt-Keima by confocal microscopy as previously described [19, 22]. To acquire the mt-Keima fluorescence images, ppk>mt-Keima larvae were examined with a Zeiss LSM 800 confocal microscope (Carl Zeiss) equipped with a Plan-Apochromat 10×/0.45 M27, Plan-Apochromat 20×/0.8 M27, and c-Apochromat 40×/1.20 W Korr lens. mt-Keima fluorescence was imaged with two sequential excitation lasers (488 nm and 555 nm) using a 595–700 nm emission bandwidth. Mitophagy was quantified based on the analysis of the mt-Keima confocal images using Zeiss Zen software on a pixel-by-pixel basis, as described previously [19, 22]. The mitophagy level (% of mitophagy) was defined as the number of pixels that have a high red/green ratio divided by the total number of pixels. To quantify the mitophagy level in C4da neurons, at least five larvae samples were used for quantification, and the average values were calculated. In all confocal microscopy analyses, all imaging parameters remained constant, and only the gain level was adjusted to avoid the saturation of any pixel. The results are presented as the mean values with SD.

Statistical analysis

All data are presented as the means ± SDs. Differences between two experimental groups were analyzed using Student’s t-test. To compare three or more groups, we used a one-way ANOVA with Sidák correction. A P-value of < 0.05 was considered statistically significant.

Genotypes

The following genotypes were used: ppk>w (ppk-GAL4/+); ppk > CD4-tdTom (ppk-GAL4/UAS-CD4-tdTomato); ppk>PINK1 (UAS-PINK1/+; ppk-GAL4/+); ppk >GFP RNAi (ppk-GAL4/UAS-GFP RNAi), ppk>PINK1 RNAi (ppk-GAL4/UAS-PINK1 RNAi); ppk>CD4-tdTom, PINK1 (UAS-PINK1/+; ppk-GAL4,UAS-CD4-tdTomato/+); ppk>mt-Keima (ppk-GAL4,UAS-mt-Keima/+), ppk>mt-Keima, PINK1 (UAS-PINK1/+; ppk-GAL4,UAS-mt-Keima/+). ppk>mito-roGFP2-Orp1 (ppk-GAL4/UAS-mito-roGFP2-Orp1).

Results

Paclitaxel induces a peripheral neuropathy phenotype in Drosophila larvae

To study paclitaxel-induced peripheral neuropathy, we adopted a recently established Drosophila thermal nociceptive model [6, 9, 10]. We first measured the time required to induce the corkscrew-like rolling withdrawal response after touching the A4-A5 segment region of the third instar (L3) larvae using a heat probe set to different temperatures. Larvae that did not show the rolling response after 20 sec were considered to have no response. As shown in Fig 1A, all larvae showed no response to the 36°C heat probe, whereas all larvae showed a rapid rolling response to the 46°C probe (mean withdrawal latency [MWL] = 1.9 sec). We chose 40°C for the following heat probe assay because the most dynamic thermal nociception response was observed with this heat probe temperature.

Fig 1

Paclitaxel treatment induces a heat-hyperalgesia phenotype in Drosophila larvae.

(A) Thermal nociceptive response of L3 larvae (ppk>w) to heat probes at different temperatures. Each data point represents the withdrawal latency of an individual larva. The absence of an aversive rolling response within 20 sec was considered no response. n = 50 larvae were tested at each temperature. (B) Experimental design for paclitaxel treatment and the thermal nociception assay. The early L3 larvae were transferred to medium containing either DMSO vehicle or paclitaxel (20 μM) at 72 h AEL. The larvae were treated for 48 h, and the thermal nociception response was observed at 120 AEL. (C) Thermal nociceptive withdrawal upon 40°C stimulation was assessed after either paclitaxel (20 μM) or DMSO treatment as in (B) (n = 50 per sample). (D) Representative images of C4da neurons at abdominal segment A4 in L3 larvae (ppk > CD4-tdTom) expressing the plasma membrane marker CD4-tdTom after 48 h exposure to either vehicle (DMSO) or 20 μM paclitaxel according to the paclitaxel treatment regimen in (B). The boxed regions are shown enlarged in the bottom panel. Scale bars, 50 μm. (E) Quantification of the length of dendrites and the number of dendritic branch points of C4da neurons. n = 5 per sample. The results are presented as the mean values, and the error bars represent the SD. *P <0.05; ***P <0.001 as determined by Student’s t-test.

Recently, feeding paclitaxel to L3 larvae was shown to induce hypersensitivity to thermal nociception and dendritic structure alteration of C4da sensory neurons [9, 10]. Similar to these studies, we treated L3 larvae with 20 μM paclitaxel for 48 h and performed the thermal nociception assay (Fig 1B). Consistent with previous studies [9, 10], paclitaxel (20 μM) treatment was sufficient to induce significant hypersensitivity to noxious heat (40°C) (Fig 1C). MWL was reduced to approximately 58% (from 5.88 sec to 3.41 sec) upon paclitaxel treatment. Next, we compared terminal dendrite morphology and branch points of the control C4da neurons after paclitaxel treatment by visualizing C4da neuron dendrites using the plasma membrane marker CD4-tdTomato, which efficiently labels terminal dendrite branches [20]. In addition to the increased thermal nociception in the heat probe assay, paclitaxel treatment has been shown to lead to alterations in sensory neuron structure [9, 10]. Consistent with these reports, we observed a significant increase in dendrite length and in the number of dendrite branch points (Fig 1D and 1E). These results indicate that our paclitaxel feeding regimen is feasible to develop a peripheral neuropathy phenotype in Drosophila larvae.

Ectopic expression of PINK1 rescues the thermal hypersensitivity phenotype induced by paclitaxel treatment

To examine the effect of PINK1 on paclitaxel-induced thermal sensory hypersensitivity, we expressed PINK1 specifically within C4da neurons using the ppk-GAL4 driver [20] and examined thermal nociception upon paclitaxel treatment using a ppk-GAL4 as a control. We found that PINK1 overexpression significantly reduced thermal sensitivity (Fig 2A). Larvae expressing PINK1 exhibited approximately 13% to 48% increased withdrawal latency with a different heat probe temperature, suggesting that PINK1 is implicated in thermal nociception. Interestingly, we observed that PINK1 overexpression in C4da neurons significantly suppressed the sensitivity to heat upon paclitaxel treatment. While paclitaxel reduced the MWL by 2.41 sec (from 5.88 sec to 3.47 sec) in the control larvae (ppk>w), the MWL was decreased by0.99 sec (from 8.08 sec to 7.09 sec) upon paclitaxel treatment in the larvae expressing PINK1 (ppk>PINK1) (Fig 2B). In terms of the relative changes, paclitaxel reduced the MWL by 42% in control larvae, while the MWL was decreased by only 12% in larvae expressing PINK1 upon paclitaxel treatment (Fig 2C). These results suggest that PINK1 expression significantly alleviated paclitaxel-induced thermal hypersensitivity in a thermal nociception assay.

Fig 2

PINK1 mitigates the heat-hyperalgesia phenotype induced by paclitaxel treatment.

(A) The thermal nociceptive response of ppk-GAL4 control L3 larvae (ppk>w) and larvae expressing PINK1 in C4da sensory neuron (ppk>PINK1) to heat probes at different temperatures. Each data point represents the withdrawal latency of an individual larva. The absence of an aversive rolling response within 20 sec was considered no response. n = 50 larvae were tested at each temperature. (B) Thermal nociceptive withdrawal of ppk>w and ppk>PINK1 larvae from heat probe (40°C) after 48 h of exposure to either vehicle (DMSO) or 20 μM paclitaxel (n = 50 per sample). (C) The relative withdrawal latency of ppk>w and ppk>PINK1 larvae upon paclitaxel treatment was calculated according to that of the vehicle sample of each genotype, which was considered to be 100%. (D) Representative images of C4da neurons at abdominal segment A4 of control L3 larvae (ppk > CD4-tdTom) and L3 larvae expressing PINK1 (ppk > CD4-tdTom,PINK1). Larvae were treated with paclitaxel (20 μM) for 48 h. Right images are enlargements of the boxed regions in the left images. Scale bars, 50 μm. (E) Quantification of the length of dendrites (left) and the number of dendrite branch points (right) in C4da neurons. n = 5 per sample. (F) Representative images of larvae from each genotype at 120 AEL treated with either DMSO or paclitaxel for 48 h (left). The larval areas calculated by the length multiplied by the width of each larva of each genotype after either DMSO or paclitaxel treatment were plotted (n≧50 per sample) (right). Scale bars, 1 mm. The results are presented as the mean values, and the error bars represent the SD. Significance was determined by one-way ANOVA with Sidák correction. *P <0.05; **P <0.01; ***P <0.001. NS; not significant.

We then assessed the dendritic arborization of C4da neurons, which is known to be associated with the thermal nociception [8]. We observed significant decreases in dendrite length and the number of branch points in larvae expressing PINK1 (Fig 2D and 2E). Ectopic expression of PINK1 reduced the dendrite length and the number of branch points of C4da neurons by 51% and 53%, respectively, compared to those of the control larvae, suggesting that the reduced thermal sensitivity upon PINK1 expression may be due to decreased dendritic arborization of C4da neurons. Because PINK1 expression suppressed paclitaxel-induced thermal hypersensitivity, we expected that dendritic arborization would increase less in larvae expressing PINK1 than in control larvae. However, paclitaxel treatment further increased dendritic arborization in larvae expressing PINK1 compared with control larvae (Fig 2D and 2E). Quantitative analysis of multiple C4da neurons (n≧ 5) revealed that the dendrite length was increased upon paclitaxel treatment by 32% in control larvae and by 42% in larvae expressing PINK1 (Fig 2E). The number of dendrite branch points was increased upon paclitaxel treatment by 49% and 70% in the control larvae and in the larvae expressing PINK1 respectively (Fig 2E). These results suggest that suppressing alteration of dendrite arborization may not be the only mechanism through which PINK1 alleviates the thermal hypersensitivity induced by paclitaxel treatment.

A previous study has shown that paclitaxel treatment in L3 larvae slightly inhibited larval growth [9]. The size of larvae expressing PINK1 in C4da neurons was comparable to that of the control larvae (Fig 2F), indicating that PINK1 overexpression in C4da neurons does not interfere with larval growth. In addition, paclitaxel induced a similar level of inhibition of larval growth in both control and PINK1-expressing larvae (Fig 2F). The size of the larvae was reduced upon paclitaxel treatment in control larvae (ppk>w) and in larvae expressing PINK1 (ppk>PINK1) to 58% and 67%, respectively. These results also suggest that PINK1 expression does not significantly interfere with the inhibitory effect of paclitaxel on larval growth, although the basal MWL was increased.

PINK1 knockdown induces thermal hypersensitivity

To further test the role of PINK1 in thermal nociception, we next examined the effect of specific knockdown of PINK1 expression in C4da neurons. Whereas ectopic PINK1 expression reduced thermal sensitivity in the heat probe assay, C4da neuron-specific knockdown of PINK1 using the ppk-GAL4 driver resulted in significant sensitization of L3 larvae to different heat probe temperatures in the thermal nociception assay (Fig 3A). PINK1 knockdown decreased the withdrawal latency to the 40°C heat probe to 41% of the control level (Fig 3B), confirming that PINK1 plays an important role in thermal nociception.

Fig 3

Effect of PINK1 knockdown on thermal nociception in Drosophila larvae.

(A) The thermal nociceptive profiles of L3 ppk>GFP RNAi and ppk>PINK1 RNAi larvae in response to heat probes at different temperatures. Each data point represents the withdrawal latency of an individual larva. The absence of an aversive rolling response within 20 sec was considered no response. n = 50 larvae were tested at each temperature. (B) Thermal nociceptive withdrawal of ppk>GFP RNAi and ppk>PINK1 RNAi to a heat probe (40°C) was examined in the L3 larvae stage (120 h AEL). (n = 50 per sample). (C) Representative images of C4da neurons at abdominal segment A4 of control L3 larvae (ppk > CD4-tdTom, GFP RNAi) and L3 larvae expressing PINK1 (ppk > CD4-tdTom, PINK1 RNAi). Larvae were treated with paclitaxel (20 μM) for 48 h. Right images are enlargements of the boxed regions in the left images. The boxed regions are shown enlarged in the bottom panel. Scale bars, 50 μm. (D) Quantification of the length of the dendrites (left) and the number of dendrite branch points (right) in C4da neurons. n = 5 per sample. Scale bars, 1 mm. (E) Representative images of larvae from each genotype at 120 h AEL (left). The larval areas were calculated by the length multiplied by the width of each larva of each genotype (n = 50 per sample) (right). The results are presented as the mean values, and the error bars represent the SD. ***P <0.001 as determined by Student’s t-test. NS; not significant.

To understand the role of PINK1 in the sensory dendrites of C4da neurons, we also examined the dendritic structure of C4da neurons upon C4da neuron-specific knockdown of PINK1. Interestingly, we observed no change in the dendritic structure of C4da neurons upon PINK1 knockdown (Fig 3C). The quantitative analysis of multiple C4da neurons (n≧ 5) also showed that both dendrite length and the number of dendrite branches (Fig 3D) were not significantly changed upon PINK1 knockdown. These results indicated that knockdown of PINK1 in C4da neurons induces an increase in thermal nociception without changes in the dendritic structure. We also found that the size of larvae was not significantly changed upon PINK1 knockdown (Fig 3E), confirming that PINK1 has no effect on larval growth. These results suggest that PINK1 may separately regulate thermal nociception and dendrite structure through independent mechanisms.

PINK1 reduced paclitaxel-induced increases in mitophagy levels in C4da sensory neurons

Recent studies showed that paclitaxel induces mitochondrial dysfunction and this mitochondrial dysfunction could be a possible cause of paclitaxel-induced sensory peripheral neuropathy [23, 24]. To understand how PINK1 mitigates paclitaxel-induced thermal hypersensitivity, we next examined whether paclitaxel induces mitochondrial dysfunction in our paclitaxel-induced peripheral neuropathy model. It has been shown that mitochondrial ROS levels correlate with mitochondrial dysfunction in Drosophila [25]. To examine mitochondrial dysfunction upon paclitaxel treatment, we measured the mitochondrial ROS levels of C4da neurons by expressing the in vivo mitochondrial H2O2 probe mito-roGFP2-Orp1 [21] specifically within C4da neurons. As shown in Fig 4, we observed that the mitochondrial ROS level was significantly increased upon paclitaxel treatment in C4da neurons. These results suggest that paclitaxel induces mitochondrial dysfunction in C4da neurons.

Fig 4

Increased mitochondrial ROS upon paclitaxel treatment in C4da neurons.

(A) Representative fluorescence images of C4da sensory neurons at abdominal segment A4 in L3 larvae expressing the in vivo mitochondrial H2O2 probe mito-roGFP2-Orp1 (ppk>mito-roGFP2-Orp1) with either DMSO or paclitaxel (20 μM) for 48 h. Scale bars, 10 μm. (B) Quantitative analysis of the mitochondrial ROS levels of the C4da sensory neurons in each group (n = 4 or 5 per group). The results are presented as the mean values, and the error bars represent the SD. *P <0.05 as determined by Student’s t-test.

Mitochondrial dysfunction is known to induce mitophagy, a selective process for the degradation of damaged or dysfunctional mitochondria [26, 27]. We also previously showed that various insults inducing mitochondrial dysfunction such as the loss of the mitochondrial polymerase γ (POLG) gene, hypoxia, or rotenone treatment results in increased mitophagy [19, 22]. Then, we next examined the level of mitophagy in C4da sensory neurons upon paclitaxel treatment. To measure the mitophagy level in C4da sensory neurons, we specifically expressed a pH-dependent fluorescent protein probe, mitochondria-targeted Keima (mt-Keima) [19, 22], using the ppk-GAL4 driver and quantitatively measured mitophagy activity as described recently [19]. Red puncta, an indicator of mitophagic mitochondria, were increased upon paclitaxel treatment (Fig 5A). The quantitative analysis of multiple C4da neurons revealed that the mitophagy level was increased by approximately 2.5-fold upon paclitaxel treatment (Fig 5B), suggesting paclitaxel induces mitochondrial dysfunction in C4da sensory neurons. PINK1 has been shown to reduce the aberrant increase in mitophagy in response to toxic treatment in SH-SY5Y cells [28]. Consistently, the paclitaxel-induced increase in mitophagy in C4da sensory neurons was significantly suppressed by PINK1 expression (Fig 5A and 5B). We did not observe significant differences in the levels of basal mitophagy in the C4da neurons after either overexpression or knockdown of PINK1 (Fig 5A and S1 Fig), suggesting the PINK1 function is dispensable for basal mitophagy as reported by previous studies [19, 29]. Together, these results suggest that PINK1 expression reduced mitochondrial dysfunction and restored mitochondria homeostasis in the paclitaxel-induced CIPN model in Drosophila larvae.

Fig 5

PINK1 restores mitochondrial homeostasis in paclitaxel-treated sensory neurons.

(A) Representative mt-Keima fluorescence images of C4da sensory neurons at abdominal segment A4 in control L3 larvae (ppk > mt-Keima) and L3 larvae expressing PINK1 (ppk > mt-Keima, PINK1) treated with either DMSO or paclitaxel (20 μM) for 48 h. Scale bars, 10 μm. (B) Quantitative analysis of the mitophagy of C4da sensory neurons in each group (n = 10 per group). The results are presented as the mean values, and the error bars represent the SD. Significance was determined by one-way ANOVA with Sidák correction. **P <0.01.

Discussion

In this study, we report for the first time the neuroprotective function of PINK1 in a paclitaxel-induced peripheral neuropathy model. First, we found that the overexpression of PINK1 in C4da sensory neurons significantly ameliorates paclitaxel-induced thermal hypersensitivity in Drosophila larvae. The critical role of PINK1 in sensory nociception was further confirmed by an increase in thermal hypersensitivity upon the knockdown of PINK1 in C4da neurons.

PINK1 has been shown to protect neuronal cells from various toxic reagents, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [30], staurosporin [31], propofol [32], chlorpyrifos [33], and thapsigargin [34]. PINK1 has also shown protective effects in various neuronal disease models, including an α-synuclein-induced Parkinson’s disease model [17, 18, 35, 36], a Huntington’s disease model [37], and an Alzheimer’s disease model [38]. These studies showed that PINK1 plays critical prosurvival and antiapoptotic functions in neuronal cells. Interestingly, we observed that the paclitaxel-induced larval growth inhibition was not affected by PINK1 expression, suggesting that PINK1 ameliorates the paclitaxel-induced hyperalgesia phenotype through methods other than the inhibition of cell death.

Drosophila terminal sensory dendrites have dynamic plasticity, showing both retraction and extension events [39] similar to mammalian skin sensory axons [40]. Brazill et al. recently revealed that paclitaxel treatment increases the stability of terminal dendrites and inhibits terminal branch retraction, leading to increased terminal dendrite density [9]. In the present study, we observed that PINK1 expression induces changes in the dendrite density of C4da sensory neurons, suggesting that PINK1 modulates the dynamic plasticity of sensory neurons. Recent studies have also shown that PINK1 regulates dendrite morphogenesis and neuronal plasticity [41, 42]. Thus, the results from our group and others suggest that PINK1 is an important regulator of dendrite plasticity. Interestingly, knockdown of PINK1 in C4da neurons resulted in increased thermal nociception without changes in dendrite structure. Therefore, our results from ectopic expression of PINK1 and knockdown experiments suggest that PINK1 regulates thermal nociception through a different mechanism than the controlling dendrite structure of C4da neurons.

Beyond its antimicrotubule effects, it has long been known that paclitaxel also acts on mitochondria. [43]. Previous in vivo studies also indicated that mitochondrial dysfunction is associated with paclitaxel-induced peripheral neuropathy. Xiao et al and Zheng et al revealed that paclitaxel treatment resulted in mitochondrial dysfunction in a rat model, as evidenced by reduced mitochondrial respiration and swollen and vacuolated mitochondria in sensory neurons [23, 24]. Paclitaxel caused an increase in mitochondrial ROS, increased mitochondrial volume and dysregulated intracellular Ca2+ [44, 45]. These studies suggest that the neuronal damage induced by paclitaxel treatment is closely associated with mitochondrial dysfunction. Previous studies have shown that the protective effect of PINK1 relies on its role in ameliorating mitochondrial dysfunction [37, 38]. In contrast, the loss of PINK1 resulted in severe mitochondrial dysfunction in a Drosophila model [16], a cell line [28] and a mouse model [46]. These results indicate that PINK1 plays a critical role in maintaining mitochondrial homeostasis.

PINK1 may control thermal nociception by reducing mitochondrial dysfunction in C4da neurons. Dagda et al. previously showed that the overexpression of PINK1 suppressed toxin-induced mitophagy, while the knockdown of PINK1 induced mitochondrial ROS and morphological changes [28]. Consistent with this study, we also observed that PINK1 expression suppressed the paclitaxel-induced increase of mitophagy in C4da sensory neurons. The restoration of mitophagy levels in C4da neurons to normal levels upon PINK1 expression suggests that mitochondrial homeostasis is restored by PINK1. Recent studies have shown that loss of PINK1 had no significant effect on the base mitophagy levels in different tissues such as muscle and DA neurons [19, 29, 47]. In the present study, we also observed that the mitophagy level of C4da neurons in L3 larvae was not significantly changed by either overexpression or knockdown of PINK1. Given the significant increase in thermal sensitivity upon PINK1 knockdown, the unchanged mitophagy activity suggests that PINK1 may restore mitochondrial homeostasis through mechanisms other than mitophagy. Previous studies have indicated that PINK1 controls mitochondrial quality through additional ways such as regulating complex I activity and mitochondrial transport [13, 48]. A body of studies even suggest that PINK1 suppresses the mitophagy activity in certain cellular contexts in a direct or indirect manner [48]. Therefore, the exact mechanism by which PINK1 ameliorates paclitaxel-induced mitochondrial dysfunction in C4da neurons should be determined by further studies. Further studies investigating how PINK1 restores mitochondrial function in paclitaxel-treated sensory neurons and the functional relationship between mitochondrial function and thermal nociception upon paclitaxel treatment will provide valuable information about the molecular mechanism responsible for paclitaxel-induced peripheral neuropathy. In addition, because PINK1 overexpression induces alteration of the dendrite structure of C4da neuron as well as thermal nociception, pharmacological transient activation of PINK1 may be an efficient strategy to control paclitaxel-induced thermal hypersensitivity while mitigating possible side effects from alteration of the dendrite structure of sensory neurons.

In conclusion, our study provides evidence that the ectopic expression of PINK1 ameliorates the thermal hypersensitive phenotype of paclitaxel-induced peripheral neuropathy. Although the precise mechanism by which PINK1 expression suppresses paclitaxel-induced mitochondrial dysfunction in sensory neurons remains to be explored, our results highlight PINK1 as a potential target for the treatment of paclitaxel-induced peripheral neuropathy.

Effect of PINK1 knockdown on the mitophagy level in C4da neuron of L3 larvae.

Quantitative analysis of the mitophagy of C4da sensory neurons at abdominal segment A4 in L3 ppk>GFP RNAi and ppk>PINK1 RNAi larvae (n = 5 per group). The results are presented as the mean values, and the error bars represent the SD. Significance was determined by Student’s t-test. NS; not significant.

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9 Jan 2020

PONE-D-19-31363

PINK1 alleviates thermal hypersensitivity in a paclitaxel-induced Drosophila model of peripheral neuropathy

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This study focuses on the role of PINK1 in chemotherapy induced pain. The study is clearly presented and executed reasonably. The results are interesting to both the pain field and probably also the neurodegeneration community.

I have only minor comments:

1. Please show temperature dose response profiles similar to Fig 1 a for PPK-GFP vs PPK-PINK1 overexpression or control vs RNAi so we understand the baseline difference in nociception.

2. PDF figures are of low quality please improve.

Reviewer #2: Kim et al, in this manuscript found that overexpressing pink1 rescue paclitaxel-induced Drosophila model of peripheral neuropathy. This CIPN) model was previously established by other labs. Overall, the phenotype sounds interesting, while the underlying molecular mechanism is elusive. The authors need address the following questions for further consideration.

Point 1, data showed in Figure1 is quite similar with a previous publication in DMM PMID: 6031360, where the assay was developed. They just verified this assay and no point to present as a main figure.

Point 2. Fig2 and Fig4 showed that overexpressing pink1 can rescue branch numbers, dendrite length and relative withdrawal for CIPN, since they addressed the same point, they could be merged in a single figure.

Point 3. Phenotypical analysis such as branch numbers, dendrite length should also be done for pink1 RNAi in Fig 3. Also, the phenotype when knocking down pink1 in this CIPN model need to be addressed.

Point 4. In Fig4, the authors showed that mitophagy defects in this CIPN model can also be restored by overexpressing pink1.

This correlation study failed to provide in-depth information about the role of pink1 in this context. A couple of experiments may be helpful to address this question:

For instance, is parkin or other autophagic genes involved?

Is mitochondrial function normal?

Mitochondrial membrane potential or ROS level can be easily tested.

In addition, since paclitaxel targets microtube, mitochondrial transport might also be affected. They should test it.

Point 5. Loss of Pink1/parkin cause neurodegeneration, whether pink1 cause C4da neurons death was not tested in this context.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

11 Mar 2020

March 9, 2020

Dear Editor,

Thank you very much for reviewing our manuscript entitled “PINK1 alleviates thermal hypersensitivity in a paclitaxel-induced Drosophila model of peripheral neuropathy” (PONE-D-19-31363). We greatly appreciate the reviewers’ valuable comments and suggestions. We have modified the manuscript extensively and have included additional data based on the reviewers’ suggestions, which we believe have strengthened the manuscript.

We hope that we have fully addressed the concerns of each reviewer and that the revised manuscript now meets the standards for publication in PLoS One.

Each concern raised by the reviewers is carefully addressed point-by-point below.

Reviewer #1:

This study focuses on the role of PINK1 in chemotherapy induced pain. The study is clearly presented and executed reasonably. The results are interesting to both the pain field and probably also the neurodegeneration community.

I have only minor comments:

1. Please show temperature dose response profiles similar to Fig 1 a for PPK-GFP vs PPK-PINK1 overexpression or control vs RNAi so we understand the baseline difference in nociception.

Response: We appreciate the reviewer for their positive comments. According to the reviewer’s suggestion, we measured the thermal nociceptive response of ppk>w1118 and ppk>PINK1 L3 larvae to heat probes at different temperatures. We also measured the thermal nociceptive response of ppk>GFP RNAi and ppk>PINK1 RNAi L3 larvae.

We have added these data as Supplementary Fig. S1 and Fig. S2, respectively, and have modified the Results section accordingly.

2. PDF figures are of low quality please improve.

Response: The low resolution figures in the PDF file are probably caused by the conversion of the original images to the PDF files. All of the submitted figure images were of 300 dpi resolution. In this revision, we have prepared all the original figure images carefully according to the figure preparation guidelines of PLoS One.

Reviewer #2:

Kim et al, in this manuscript found that overexpressing pink1 rescue paclitaxel-induced Drosophila model of peripheral neuropathy. This CIPN) model was previously established by other labs. Overall, the phenotype sounds interesting, while the underlying molecular mechanism is elusive. The authors need address the following questions for further consideration.

Point 1, data showed in Figure1 is quite similar with a previous publication in DMM PMID: 6031360, where the assay was developed. They just verified this assay and no point to present as a main figure.

Response: As we have described in the Results section, we established a paclitaxel-induced peripheral neuropathy model and phenotype analysis system referring to several recent studies. Although we adopted the paclitaxel treatment paradigm from Zhai’s groups study (Brazil J.M. et al, 2018, Dis Model Mech, doi: 10.1242/dmm.032938), we established the heat probe assay and dendrite structure analysis assays using different system such as different custom-built thermal probes, different fluorescent membrane markers (CD4-tdTomato), and a dendrite analysis system. We believe that verifying the paclitaxel-induced peripheral neuropathy model is an important step for our study and thus, we want keep Figure 1 as a main figure in our manuscript.

Point 2. Fig2 and Fig4 showed that overexpressing pink1 can rescue branch numbers, dendrite length and relative withdrawal for CIPN, since they addressed the same point, they could be merged in a single figure.

Response: According to the reviewer’s suggestion, we have combined previous Figure 2 and Figure 4 and corrected the manuscript accordingly.

Point 3. Phenotypical analysis such as branch numbers, dendrite length should also be done for pink1 RNAi in Fig 3. Also, the phenotype when knocking down pink1 in this CIPN model need to be addressed.

Response: According to the reviewer’s suggestion, we have examined the dendrite structure of C4da neurons upon PINK1 knockdown. We found that that both the dendrite length and the number of dendrite branches were not significantly changed upon PINK1 knockdown. These results suggest that knockdown of PINK1 has no effect on the dendrite structure of C4da neurons as well as larval growth.

We have added these data to Figure 3(C, D) and have modified the Results section.

Point 4. In Fig4, the authors showed that mitophagy defects in this CIPN model can also be restored by overexpressing pink1.

This correlation study failed to provide in-depth information about the role of pink1 in this context. A couple of experiments may be helpful to address this question:

For instance, is parkin or other autophagic genes involved? Is mitochondrial function normal?

Mitochondrial membrane potential or ROS level can be easily tested.

In addition, since paclitaxel targets microtube, mitochondrial transport might also be affected. They should test it.

Response: We agree with the reviewer that the analysis of the involvement of Parkin or other autophagy genes would provide valuable information for understanding the role of PINK1. Regrettably, we were not able to perform an additional genetic study because the generation of the Drosophila line is not possible within the limited time frame of this revision. Mitochondrial transport was also not examined during this revision due to a lack of a system for the analysis.

During this revision, we further confirmed paclitaxel-induced mitochondrial dysfunction. Because the analysis of C4da neuron-specific changes in mitochondrial membrane potential and mitochondrial ROS is not possible using conventional fluorescence dyes such as TMRM, TMRE, or mitoSOX, we adopted the mitochondrial H2O2 probe mito-roGFP2-Orp1 (Albrecht, S.C. et al, 2011, Cell Metab, doi: 10.1016/j.cmet.2011.10.010). By expressing mito-roGFP2-Orp1 using a C4da-specific ppk-GAL4 driver, we were able to analyze mitochondrial ROS changes upon paclitaxel treatment in C4da neurons. We observed that the level of mitochondrial ROS was significantly increased in C4da neurons upon paclitaxel treatment.

We have added these data as Figure 4 and have modified the Results section.

Point 5. Loss of Pink1/parkin cause neurodegeneration, whether pink1 cause C4da neurons death was not tested in this context.

Response: We understand the reviewer’s concern about the toxic effect of PINK1 in C4da neurons. However, we observed no significant change in larval growth upon either overexpression or knockdown of PINK1. In addition, the dendrite structure of C4da neurons was not significantly changed upon knockdown of PINK1. Thus, these results suggest that the growth and survival of C4da neuron was not affected by PINK1 at least in our experimental setting. We have mentioned this point in the Results section.

Submitted filename: Revision letter.docx

Click here for additional data file.

2 Apr 2020

PONE-D-19-31363R1

PINK1 alleviates thermal hypersensitivity in a paclitaxel-induced Drosophila model of peripheral neuropathy

PLOS ONE

Dear Prof Yun,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by May 17 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

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Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

David Chau

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I think this manuscript is almost finished, but a few issues need to be addressed.

1. I think it is essential to show the raw control vs pink1 (overexpression or RNAi) withdrawal responses side by side with significance assessed to highlight if there is a significant baseline difference in the main figures of the manuscript, then show the relative changes from there. The issue is the baseline looks different, and the response to paclitaxel is different, and both messages should be clearly presented in the main figures to help the reader fully understand the results.

2. Figure 2a as presented makes it look like there is no baseline change in nociceptive response to 40C, but figure S1 40C shows what looks like a strong difference at 40C, and I feel this needs to be dealt with upfront so that the data and normalized differences don’t get misinterpreted. Similar issue with Figure 2a vs S2. Then for both the authors should discuss how the baseline difference could confound interpretation of the paclitaxel data so the reader is led toward a more complete understanding of the results.

3. The figures are out of order in the PDF for some reason, also the figure quality is still poor for some reason, please make sure the final published figures are legible, I understand the PDF conversion process can cause this.

4. Figure 1C etc, “relative withdrawal (%)” is sort of confusing, as presented it at first glance seems like paclitaxel reduces sensitivity. It might make more sense to present the data as % sensitization or similar. In general I find “decreased MWL” difficult to conceptualize and I feel “sensitization” is easier for the non-pain expert to understand.

Reviewer #2: The authors have addressed all the concerns I have. It would be great if they had some mechanistic analysis rahter than the descriptive data only.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

1 Jun 2020

May 31, 2020

Dear Editor,

Thank you very much for reviewing our manuscript entitled “PINK1 alleviates thermal hypersensitivity in a paclitaxel-induced Drosophila model of peripheral neuropathy” (PONE-D-19-31363R1). We sincerely appreciate the reviewers’ valuable comments and suggestions. We have modified the manuscript extensively and have included additional data based on the reviewers’ suggestions, which we believe have strengthened the manuscript.

We hope that we have fully addressed the concerns of each reviewer, and that the revised manuscript now meets the standards for publication in PLoS One.

Each concern raised by the reviewers is carefully addressed point by point below.

Reviewer #1: I think this manuscript is almost finished, but a few issues need to be addressed.

1. I think it is essential to show the raw control vs pink1 (overexpression or RNAi) withdrawal responses side by side with significance assessed to highlight if there is a significant baseline difference in the main figures of the manuscript, then show the relative changes from there. The issue is the baseline looks different, and the response to paclitaxel is different, and both messages should be clearly presented in the main figures to help the reader fully understand the results.

Response: We agree with the reviewer that we should show the raw thermal nociceptive response in the main figure before we show the relative withdrawal response. According to the reviewer’s suggestion, we now show the thermal nociceptive responses of control (ppk>w1118) and PINK1-overexpressing (ppk>PINK1) larvae in Fig. 2A. The thermal nociceptive responses of control (ppk>GFP RNAi) and PINK1 RNAi (ppk>PINK1 RNAi) larvae are also shown in Fig. 3A.

In addition, we show the raw heat probe assay results of control (ppk>w1118) and PINK1-overexpressing (ppk>PINK1) larvae in Fig. 2B, and the relative withdrawal changes are shown in Fig. 2C.

We appreciate the reviewer for pointing out this issue. By considering the effects of PINK1 overexpression and knockdown on thermal nociception and the dendrite structure of C4da neurons, we found that PINK1 may alleviate paclitaxel-induced thermal hypersensitivity by means other than preventing alterations in the sensory dendrites of C4da neurons. Thus, we modified the manuscript as we discuss in our next response below.

2. Figure 2a as presented makes it look like there is no baseline change in nociceptive response to 40C, but figure S1 40C shows what looks like a strong difference at 40C, and I feel this needs to be dealt with upfront so that the data and normalized differences don’t get misinterpreted. Similar issue with Figure 2a vs S2. Then for both the authors should discuss how the baseline difference could confound interpretation of the paclitaxel data so the reader is led toward a more complete understanding of the results.

Response: We agree with the reviewer that the effect of PINK1 overexpression should be considered carefully. By analyzing the results upon PINK1 overexpression, we found that PINK1 expression significantly changed thermal nociception as well as the dendrite structure of C4da neurons. Nevertheless, our results suggest that PINK1 expression significantly alleviates paclitaxel-induced thermal hypersensitivity in larvae, and that PINK1 may regulate thermal nociception and dendrite structure through different mechanisms. We believe that the results obtained from PINK1-knockdown experiments further support this notion. Therefore, we have carefully revised the manuscript to clearly state our interpretation of the results. The major changes are shown below.

(Page 9 line 1- Page 10 line 3)

Ectopic expression of PINK1 rescues the thermal hypersensitivity phenotype induced by paclitaxel treatment

To examine the effect of PINK1 on paclitaxel-induced thermal sensory hypersensitivity, we expressed PINK1 specifically within C4da neurons using the ppk-GAL4 driver [20] and examined thermal nociception upon paclitaxel treatment. We found that PINK1 overexpression significantly reduced thermal sensitivity (Fig. 2A). Larvae expressing PINK1 exhibited approximately 13% to 48 % increased withdrawal latency with different heat probe temperature, suggesting that PINK1 is implicated in thermal nociception. Interestingly, we observed that PINK1 overexpression within C4da neurons significantly suppressed the sensitivity to heat upon paclitaxel treatment. While paclitaxel reduced the MWL by 2.41 sec (from 5.88 sec to 3.47 sec) in control larvae (ppk>w1118), the MWL was decreased by 0.99 sec (from 8.08 sec to 7.09 sec) upon paclitaxel treatment in larvae expressing PINK1 (ppk>PINK1) (Fig. 2B). In terms of the relative changes, paclitaxel reduced the MWL by 42% in control larvae, while the MWL was decreased by only 12% in larvae expressing PINK1 upon paclitaxel treatment (Fig. 2C). These results suggest that PINK1 expression significantly alleviated paclitaxel-induced thermal hypersensitivity in a thermal nociception assay.

We then assessed the dendritic arborization of C4da neurons, which is known to be associated with the thermal nociception [8]. We observed significant decreases in dendrite length and the number of branch points in larvae expressing PINK1 (Fig. 2D and E). Ectopic expression of PINK1 reduced the dendrite length and the number of branch points of C4da neurons by 51% and 53%, respectively, compared to control larvae, suggesting that the reduced thermal sensitivity upon PINK1 expression may be due to decreased dendritic arborization of C4da neurons. Because PINK1 expression suppressed paclitaxel-induced thermal hypersensitivity, we expected that dendritic arborization would increase less in larvae expressing PINK1 than in control larvae. However, paclitaxel treatment further increased dendritic arborization in larvae expressing PINK1 compared with control larvae (Fig. 2D and E). Quantitative analysis of multiple C4da neurons (n≧ 5) revealed that the dendrite length was increased upon paclitaxel treatment by 32% in control larvae and by 42% in larvae expressing PINK1 (Fig. 2E). The number of dendrite branch points was increased upon paclitaxel treatment by 49% and 70% in control larvae and in larvae expressing PINK1 respectively (Fig. 2E). These results suggest that suppressing alteration of dendrite arborization may not be the only mechanism through which PINK1 alleviates the thermal hypersensitivity induced by paclitaxel treatment.

(Page 10 line 13- Page 11 line 5)

PINK1 knockdown induces thermal hypersensitivity.

To further test the role of PINK1 in thermal nociception, we next examined the effect of specific knockdown of PINK1 expression in C4da neurons. Whereas ectopic PINK1 expression reduced thermal sensitivity in the heat probe assay, C4da neuron-specific knockdown of PINK1 using the ppk-GAL4 driver resulted in significant sensitization of L3 larvae to different heat probe temperatures in the thermal nociception assay (Fig. 3A). PINK1 knockdown decreased the withdrawal latency to the 40 ºC heat probe to 41% of the control level (Fig. 3B), confirming that PINK1 plays an important role in thermal nociception.

To understand the role of PINK1 in the sensory dendrites of C4da neurons, we also examined the dendritic structure of C4da neurons upon C4da neuron-specific knockdown of PINK1. Interestingly, we observed no change in the dendritic structure of C4da neurons upon PINK1 knockdown (Fig. 3C). The quantitative analysis of multiple C4da neurons (n≧ 5) also showed that both dendrite length and the number of dendrite branches (Fig. 3D) were not significantly changed upon PINK1 knockdown. These results indicate that knockdown of PINK1 in C4 da neurons induces an increase in thermal nociception without changes in the dendritic structure. We also found that the size of larvae was not significantly changed upon PINK1 knockdown (Fig. 3E), confirming that PINK1 has no effect on larval growth. These results suggest that PINK1 may separately regulate thermal nociception and dendrite structure through independent mechanisms.

3. The figures are out of order in the PDF for some reason, also the figure quality is still poor for some reason, please make sure the final published figures are legible, I understand the PDF conversion process can cause this.

Response: We apologize for this error. We carefully checked the order of the figures this revision.

Regarding the quality of the figures, we have prepared all the original images carefully according to the figure preparation guidelines of PLoS One. All of the submitted figure images are of 300 dpi resolution. Thus, we believe that the quality of original figures will meet the standard of the journal.

4. Figure 1C etc, “relative withdrawal (%)” is sort of confusing, as presented it at first glance seems like paclitaxel reduces sensitivity. It might make more sense to present the data as % sensitization or similar. In general I find “decreased MWL” difficult to conceptualize and I feel “sensitization” is easier for the non-pain expert to understand.

Response: To address the reviewer’s concern about the term “relative withdrawal (%)”, we instead use the term “withdrawal latency (% baseline)”, which is commonly used in pain experiments, in Fig. 2C. The results in Fig. 1C are now expressed as “withdrawal latency” instead of the relative values.

To facilitate readers’ understanding, we have reduced the use of the term “mean withdrawal latency (MWL)” and revised the manuscript by using the term “thermal sensitivity” or “sensitization” as shown below.

(Page 9, line 5)

We found that PINK1 overexpression significantly reduced thermal sensitivity (Fig. 2A). Larvae expressing PINK1 exhibited approximately 13% to 48% increased withdrawal latency with different heat probe temperatures,…

(Page 10, line 15)

Whereas ectopic PINK1 expression reduced thermal sensitivity in the heat probe assay, C4da neuron-specific knockdown of PINK1 using the ppk-GAL4 driver resulted in significant sensitization of L3 larvae to different heat probe temperatures in the thermal nociception assay (Fig. 3A). PINK1 knockdown decreased the withdrawal latency to the 40 ºC heat probe to 41% of the control level (Fig. 3B),…

Reviewer #2: The authors have addressed all the concerns I have. It would be great if they had some mechanistic analysis rahter than the descriptive data only.

Response: We agree with the reviewer that understanding the precise mechanism through which PINK1 expression suppresses paclitaxel-induced thermal hypersensitivity is important. In particular, how PINK1 reduces paclitaxel-induced mitochondrial dysfunction in sensory neurons needs to be explored in further studies. We are currently conducting experiments to understand the molecular mechanisms and hope that we are able to report the results soon.

Submitted filename: Revision letterR2.docx

Click here for additional data file.

24 Jun 2020

PONE-D-19-31363R2

PINK1 alleviates thermal hypersensitivity in a paclitaxel-induced Drosophila model of peripheral neuropathy

PLOS ONE

Dear Dr. Yun,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Aug 08 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

David Chau

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have addressed all of my concerns and I believe the manuscript is suitable for publicaiton.

Reviewer #2: The current manuscript is significantly improved, however, it is overall descriptive, mechanistic analysis is largely missing.

In Figure 5, the authors observed that paclitaxel induced mitophagy was suppressed by pink1 OE using mitoKeima reporter. The authors then claimed that PINK1 reduced paclitaxel-induced increases in mitophagy levels.

Whether pink1 suppress or accelerate mitophagy in this context needs to be clarified, since previously reports showed that pink1/parkin KD muscles or DA neurons have less mitophagy activity in Drosophila (eLife 2018;7:e35878 doi: 10.7554/eLife.35878).

The authors can easily address this by manipulating downstream events of mitophagy, such as lysosome and proteasome activity in pink1 LOF background.

**********

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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6 Aug 2020

August 7, 2020

Dear Editor,

Thank you very much for reviewing our manuscript entitled “PINK1 alleviates thermal hypersensitivity in a paclitaxel-induced Drosophila model of peripheral neuropathy” (PONE-D-19-31363R2). We sincerely appreciate the reviewers’ valuable comments and suggestions. We have modified the manuscript and have included additional data based on the reviewers’ suggestions, which we believe have strengthened the manuscript.

We hope that we have fully addressed the concerns of each reviewer, and that the revised manuscript now meets the standards for publication in PLoS One.

Each concern raised by the reviewers is carefully addressed point by point below.

Reviewer #1: The authors have addressed all of my concerns and I believe the manuscript is suitable for publication.

Reviewer #2: The current manuscript is significantly improved, however, it is overall descriptive, mechanistic analysis is largely missing.

In Figure 5, the authors observed that paclitaxel induced mitophagy was suppressed by pink1 OE using mitoKeima reporter. The authors then claimed that PINK1 reduced paclitaxel-induced increases in mitophagy levels.

Whether pink1 suppress or accelerate mitophagy in this context needs to be clarified, since previously reports showed that pink1/parkin KD muscles or DA neurons have less mitophagy activity in Drosophila (eLife 2018;7:e35878 doi: 10.7554/eLife.35878).

The authors can easily address this by manipulating downstream events of mitophagy, such as lysosome and proteasome activity in pink1 LOF background.

Response: We understand the reviewer’s concern about the mitophagy change upon PINK1 overexpression. We repeated the Fig. 5 experiment and mitophagy analysis with larger number of samples and found that the basal mitophagy level of the C4da neuron was not significantly changed upon PINK1 expression. As the reviewer mentioned, Wim Vandenberghe’s group has shown that loss of PINK1 and knockdown of parkin abolish the age-dependent increase of mitophagy in muscle while basal mitophagy is unchanged (Cornelissen T et al, 2018, eLife, 7:e35878). We have also previously shown that the mitophagy level of DA neurons was not changed by knockdown of PINK1, but mitophagy inductions upon hypoxia, and rotenone treatment were abolished (Kim YY et al, 2019, FASEB J, 33:9742-9751. doi: 10.1096/fj.201900073R), suggesting that PINK1 function is dispensable for basal mitophagy. We also observed that the mitophagy level of C4da neurons was not changed by knockdown of PINK1. Given the significant increase in thermal sensitivity upon PINK1 knockdown, the unchanged mitophagy activity suggested that PINK1 may restore mitochondrial homeostasis through mechanisms other than mitophagy. Previous studies have indicated that PINK1 controls mitochondrial quality through additional methods such as regulating complex I activity and mitochondrial transport (Reviewed in Voigt A et al, 2016, J Neurochem, 139:232-239, doi: 10.1111/jnc.13655; Steer EK et al, 2015, Antioxid Redox Signal, 22(12):1047-1059 doi: 10.1089/ars.2014.6206). A body of studies even suggest that PINK1 suppresses mitophagy activity in certain cellular contexts through a direct or indirect manner (Reviewed in Steer EK et al, 2015, Antioxid Redox Signal, 22(12):1047-1059 doi: 10.1089/ars.2014.6206). Therefore, the exact mechanism by which PINK1 ameliorates paclitaxel-induced mitochondrial dysfunction in C4da neuron should be carefully determined in further studies.

We have revised the discussion to clearly state these points.

We replaced Fig. 5B with a new results and added the mitophagy results upon PINK1 knockdown as Supplementary FigS1.

Submitted filename: Revision letterR3.docx

Click here for additional data file.

26 Aug 2020

PONE-D-19-31363R3

PINK1 alleviates thermal hypersensitivity in a paclitaxel-induced Drosophila model of peripheral neuropathy

PLOS ONE

Dear Dr. Yun,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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David Chau

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: No

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: No

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: In this version, the authors have addressed all my concerns, and is significantly improved. As the authors discussed, the underlying mechanism need to be adressed in the future.

Reviewer #3: In the present well written study the researchers aim to shed light on the in vivo mechanism of chemotherapy-induced peripheral neuropathy (CIPN), a common side-effect of chemotherapy treatment, and induced by the anticancer drug paclitaxel. They use a recently published drosophila model of paclitaxel-induced thermal hyperalgesia in L3 larvae. Whilst some aspects of paclitaxel mechanism of action are known, how it induces peripheral neuropathy is less well understood. Here, the authors examined the role of PINK1, a protein implicated in mitochondrial homeostasis and quality control in paclitaxel-induced thermal hyperalgesia.

The authors' conclusion that PINK1 plays a role in thermal sensitivity is supported by their data showing that ectopic expression of PINK1 in class IV dendritic aborization sensory neurons under basal conditions led to increased withdrawal latency times to the heat stimulus compared with background controls. Moreover, KD of PINK1 induced a reduced withdrawal latency compared with a control RNAi genotype.

PINK1 was further implicated in thermal hyperalgesia as ectopic expression of PINK1 (as above), dampened the ability of paclitaxel to induce increased withdrawal latency times compared to vehicle control. Thus, the authors’ conclusion that PINK1 has a neuroprotective function in a paclitaxel-induced peripheral neuropathy model is substantiated by the data.

The authors’ conclusion that the neuroprotective role of PINK in paclitaxel-induced thermal hyperalgesia is via mechanisms independent of dendritic organisation is supported by their findings that PINK1 expression reduced measures of dendritic arborization (dendritic branching and length) but did not suppress paclitaxel-induced increases, whilst PINK1 KD did not change these parameters.

The authors attempted to address the underlying mechanism of this neuroprotective effect by examining aspects of mitochondrial dysfunction. They found that paclitaxel treatment induced mitochondrial reactive oxygen species production and mitophagy, using established in vivo tools, and that PINK1 expression ameliorated the induction of mitophagy by paclitaxel. Additionally, basal levels of mitophagy were not changed by either ectopic expression or knockdown of PINK1. The authors concluded that PINK1 modulates thermal sensitivity by regulating mitochondrial homeostasis, by mechanisms independently of mitophagy. Whilst the findings can be supported by appropriate citations, they are observational in manner and further mechanistic insight was not gained. The authors acknowledge that further studies are necessary to understand the current findings within the context of the in vivo work within the field.

The work presented in this manuscript investigates an important area in the field by using established reagents and methodologies, thus generating robust results. Although the data which sought to uncover a mitochondrial homeostasis mechanism is mainly observational and somewhat inconclusive, the overall conclusion of the manuscript that PINK1 plays a role in thermal sensitivity is clear. The findings support the conclusion that PINK1 may be a bonafide therapeutic target for chemotherapy-induced peripheral neuropathy worthy of future investigation. Since further work to uncover mechanistic insights is beyond the scope of the manuscript, if the authors address the points below it would be suitable for publication in PLOS ONE.

Major point:

1. The observation that ectopic expression of PINK1 ameliorates paclitaxel-induced thermal hyperalgesia and mitophagy would be more compelling if a benign UAS control gene was used to account for the use of the GAL4-UAS system for expression of PINK. However, this is not necessarily a requirement for publication since there are conflicting conventions in the field regarding the use of such controls. Please can the authors justify the controls used in Figure 2 and Figure 5.

Minor points:

1. Consistency with GAL4 notation on page 4 Material and Methods section Drosophila strains.

2. Figure 1D, Figure 2D and Figure 3C - please add a scale bar to the representative images and detail in the legends.

3. Please add in the citation Lee et al, J Cell Biol. 2018 May 7; 217(5): 1613–1622. doi: 10.1083/jcb.201801044 to page 14 in relevance to loss of PINK1 minimally affecting basal mitophagy in Drosophila

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

28 Aug 2020

August 28, 2020

Dear Editor,

Thank you very much for reviewing our manuscript entitled “PINK1 alleviates thermal hypersensitivity in a paclitaxel-induced Drosophila model of peripheral neuropathy” (PONE-D-19-31363R3). We sincerely appreciate the reviewers’ valuable comments and suggestions. We have modified the manuscript and have included additional data based on the reviewers’ suggestions, which we believe have strengthened the manuscript.

We hope that we have fully addressed the concerns of each reviewer, and that the revised manuscript now meets the standards for publication in PLoS One.

Each concern raised by the reviewers is carefully addressed point by point below.

Reviewer #2: In this version, the authors have addressed all my concerns, and is significantly improved. As the authors discussed, the underlying mechanism need to be adressed in the future.

Reviewer #3: In the present well written study the researchers aim to shed light on the in vivo mechanism of chemotherapy-induced peripheral neuropathy (CIPN), a common side-effect of chemotherapy treatment, and induced by the anticancer drug paclitaxel. They use a recently published drosophila model of paclitaxel-induced thermal hyperalgesia in L3 larvae. Whilst some aspects of paclitaxel mechanism of action are known, how it induces peripheral neuropathy is less well understood. Here, the authors examined the role of PINK1, a protein implicated in mitochondrial homeostasis and quality control in paclitaxel-induced thermal hyperalgesia.

The authors' conclusion that PINK1 plays a role in thermal sensitivity is supported by their data showing that ectopic expression of PINK1 in class IV dendritic aborization sensory neurons under basal conditions led to increased withdrawal latency times to the heat stimulus compared with background controls. Moreover, KD of PINK1 induced a reduced withdrawal latency compared with a control RNAi genotype.

PINK1 was further implicated in thermal hyperalgesia as ectopic expression of PINK1 (as above), dampened the ability of paclitaxel to induce increased withdrawal latency times compared to vehicle control. Thus, the authors’ conclusion that PINK1 has a neuroprotective function in a paclitaxel-induced peripheral neuropathy model is substantiated by the data.

The authors’ conclusion that the neuroprotective role of PINK in paclitaxel-induced thermal hyperalgesia is via mechanisms independent of dendritic organisation is supported by their findings that PINK1 expression reduced measures of dendritic arborization (dendritic branching and length) but did not suppress paclitaxel-induced increases, whilst PINK1 KD did not change these parameters.

The authors attempted to address the underlying mechanism of this neuroprotective effect by examining aspects of mitochondrial dysfunction. They found that paclitaxel treatment induced mitochondrial reactive oxygen species production and mitophagy, using established in vivo tools, and that PINK1 expression ameliorated the induction of mitophagy by paclitaxel. Additionally, basal levels of mitophagy were not changed by either ectopic expression or knockdown of PINK1. The authors concluded that PINK1 modulates thermal sensitivity by regulating mitochondrial homeostasis, by mechanisms independently of mitophagy. Whilst the findings can be supported by appropriate citations, they are observational in manner and further mechanistic insight was not gained. The authors acknowledge that further studies are necessary to understand the current findings within the context of the in vivo work within the field.

The work presented in this manuscript investigates an important area in the field by using established reagents and methodologies, thus generating robust results. Although the data which sought to uncover a mitochondrial homeostasis mechanism is mainly observational and somewhat inconclusive, the overall conclusion of the manuscript that PINK1 plays a role in thermal sensitivity is clear. The findings support the conclusion that PINK1 may be a bonafide therapeutic target for chemotherapy-induced peripheral neuropathy worthy of future investigation. Since further work to uncover mechanistic insights is beyond the scope of the manuscript, if the authors address the points below it would be suitable for publication in PLOS ONE.

Major point:

1. The observation that ectopic expression of PINK1 ameliorates paclitaxel-induced thermal hyperalgesia and mitophagy would be more compelling if a benign UAS control gene was used to account for the use of the GAL4-UAS system for expression of PINK. However, this is not necessarily a requirement for publication since there are conflicting conventions in the field regarding the use of such controls. Please can the authors justify the controls used in Figure 2 and Figure 5.

Response: We understand the reviewer’s concern about the control for PINK1 expression. As stated in the beginning of the result section (page 8), we adopted a Drosophila thermal nociception assay from recently performed studies. In particular, experiments investigating the effect of PINK1 expression were conducted in accordance with the experimental setting of Grace Zhai group’s recent paper (Grazil J et al. 2018, Dis Model Mech, DOI: 10.1242/dmm.032938). We used a ppk-GAL4 fly as a control for the ppk-GAL4-drived expression of PINK1, because Grazil J et al also used ppk-GAL4 as a control when they examined the effect of Nmnat expression on paclitaxel-induced hyperalgesia in their study.

We have revised the result and figure legends to state more clearly the control for PINK1 expression.

(Page 9 line 2) To examine the effect of PINK1 on paclitaxel-induced thermal sensory hypersensitivity, we expressed PINK1 specifically within C4da neurons using the ppk-GAL4 driver [20] and examined thermal nociception upon paclitaxel treatment using a ppk-GAL4 as a control.

(Page 17 line 18) Figure 2. PINK1 mitigates the heat-hyperalgesia phenotype induced by paclitaxel treatment.

(A) The thermal nociceptive response of ppk-GAL4 control L3 larvae (ppk>w1118) and larvae expressing PINK1 in C4da sensory neuron (ppk>PINK1) to heat probes at different temperatures.

Minor points:

1. Consistency with GAL4 notation on page 4 Material and Methods section Drosophila strains.

Response: We thank the reviewer for correct this mistake. We changed the “ppk-Gal4” to “ppk-GAL4” for the consistent description.

2. Figure 1D, Figure 2D and Figure 3C - please add a scale bar to the representative images and detail in the legends.

Response: Thank you again for this comment. We added scale bars at the pictures in Figure 1D, Figure 2D and Figure 3C. We have revised Figure legends accordingly.

3. Please add in the citation Lee et al, J Cell Biol. 2018 May 7; 217(5): 1613–1622. doi: 10.1083/jcb.201801044 to page 14 in relevance to loss of PINK1 minimally affecting basal mitophagy in Drosophila

Response: According to the reviewer’s suggestion, we have added the recommended paper to the discussion section.

1 Sep 2020

PINK1 alleviates thermal hypersensitivity in a paclitaxel-induced Drosophila model of peripheral neuropathy

PONE-D-19-31363R4

Dear Dr. Yun,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

David Chau

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: In the revised manuscript the authors have satisfactorily address the concerns and comments raised.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No