Lysosomal dysfunction in Schwann cells is involved in bortezomib-induced peripheral neuropathy

doi:10.21203/rs.3.rs-2241499/v1

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Lysosomal dysfunction in Schwann cells is involved in bortezomib-induced peripheral neuropathy

https://doi.org/10.21203/rs.3.rs-2241499/v1

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Bortezomib (BTZ) is a proteasome inhibitor serves as a first-line drug for multiple myeloma treatment. BTZ-induced peripheral neuropathy (BIPN) is the most common adverse effect of BTZ with an incidence as high as 40%-60%. However, the pathological mechanisms underlying BIPN remain largely unclear. BTZ leads to dramatic Schwann cell demyelination in sciatic nerves. Previous studies implied that myelin debris was predominantly degraded via autophagy-lysosome pathway in Schwann cells. However, the association of autophagy with BIPN has not been made. Mice were treated with BTZ (2 mg/kg, i.v.) on Day1 and Day4 each week for continuous 4 weeks. BTZ-treated mice showed enhanced mechanical hyperalgesia, decreased tail nerve conduction and sciatic nerve demyelination. Unexpectedly, BTZ led to the accumulation of autophagic vesicles, LC3-II and p62 in the sciatic nerve. Moreover, BTZ blocked autophagic flux in RSC96 Schwann cells as determined by mcherry-GFP-LC3 assay, suggesting BTZ may impair lysosomal function rather than inducing autophagy in Schwann cells. BTZ significantly reduced the lysosomal activity in Schwann cells as determined by reduced LysoTracker Red and DQ-Red-BSA staining and increased the level of immature Cathepsin B (CTSB). Remarkably, lysosomal activators PP242 and Torin1, significantly reversed the blockage of autophagic flux by BTZ. We further verified that Torin1 rescued the demyelination, nerve conduction and reduced the mechanical hyperalgesia in BIPN mice. Additionally, Torin1 did not compromise the efficacy of BTZ in suppressing multiple myeloma RPMI8226 cell. Taken together, we identified that lysosomal dysfunction in Schwann cells caused by BTZ is involved in the BIPN pathology. Improved lysosomal function in Schwann cells can be a promising strategy for BIPN treatment.

Bortezomib

Bortezomib-induced peripheral neuropathy

Lysosome

Autophagy

Torin1

The proteasome inhibitor bortezomib (BTZ) is the first-line treatment for primary, refractory/relapsed multiple myeloma (Raab et al. 2009). BTZ inhibits the 26S proteasome activity (Aguilar et al. 2020), thereby hindering the degradation of cyclin kinases and eventually leading to the demise of myeloma cells (Baiz et al. 2009; Hideshima and Anderson 2002). BTZ-induced peripheral neuropathy (BIPN) is a common adverse effect of BTZ with an incidence of 40%-60% (Velasco et al. 2010), remaining the main cause for BTZ discontinuation (Richardson et al. 2006). BIPN is characterized with neuropathic pain, limb numbness, paresthesia, sensory loss, burning sensation, etc. However, the toxicological mechanisms underlying BTZ have not been fully elucidated and there is a paucity of effective therapy for BIPN treatment.

Hyperalgesia remains the most prominent feature of BIPN (Bloomingdale et al. 2021), which was attributable to demyelination of sensory nerves (Cavaletti et al. 2007). Schwann cells, the myelinating cell, whose damage leads to neuropathological pain (Bruna et al. 2010), and is closely associated with the toxicity of BTZ. However, it remains elusive how BTZ injures Schwann cells in a cellular level. In BIPN mice, a large number of vacuoles were observed in the cytoplasm of Schwann cells from the sciatic nerve (Cavaletti et al. 2007). Moreover, the autophagosome marker LC3-II was significantly up-regulated in BTZ-treated Schwann cells (Nakano et al. 2011). These observations suggested BTZ activated autophagy in Schwann cells. Autophagy is a process in which intracellular contents are engulfed by autophagosome and fused with lysosomes for degradation (Yu, Chen, and Tooze 2018). As a compensation for protein degradation, autophagy can be activated upon proteasomal inhibition (Pohl and Dikic 2019). Although basal autophagy was required for Schwann cells plasticity by removal of myelin debris during nerve injury, some studies indicated excessive autophagy caused nerve injury by inducing myelin loss (Belgrad, De Pace, and Fields 2020).

Lysosomes are digestive organelles for autophagic degradation. Lysosome cleared myelin debris and thus guaranteed remyelinating function of Schwann cells in the context of inflammatory segmental demyelination (Su et al. 2019; Jang et al. 2017). Lysosome exocytosis in Schwann cells promoted remyelination in regenerated sciatic nerve (Chen et al. 2012). Moreover, decreasing lysosome function was accompanied with autophagy defects and delay of myelin clearance in Schwann cells in a nerve crush model (Reed et al. 2020). These studies indicated the essential role of lysosomes in myelin homeostasis under various pathological conditions. However, it has not been identified whether lysosome function was altered in Schwann cells exposed to BTZ and how it may relate with the pathology of BIPN.

Animals

Male C57BL/6 mice weighing 23–25 g (10 weeks old) were used. Mice were raised on a 12 h light/dark cycle with ad libitum access to water and food. All experiments were approved and conducted in accordance with the ethical guidelines of the Zhejiang University Animal Experimentation Committee and were in complete compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Efforts were made to reduce any pain and the number of animals required.

Drug administration

Male C57BL/6 mice were randomly divided into a control group and administration groups (n = 8). BTZ (obtained from Xian Janssen Pharmaceutical Ltd.) was dissolved in sterile saline solution and intravenously (i.v.) injected at doses of 1 mg/kg twice per week (at 1st and 4th day of each week) for four weeks (Carozzi et al. 2010). The mice in the control group were intravenously injected with an equal volume of saline. Corresponding behavioral tests were performed on the mice on the 7th day of each week. For Torin1 treatment, another group of mice were co-administrated with Torin1 (20 mg/kg/d, i.p. daily) during the 3rd and 4th week of BTZ administration.

Rotarod test

Mice were trained to continuously run on the spinning rod for 5 min. The spinning rod was gradually accelerated to 40 rpm in the first 2 minutes and maintained at 40 rpm in the next 3 minutes. Mice should run on the rotating rod for 5 min, the mice failed to complete the training trail were excluded from further experiments. The rotarod test was performed on Day0, Day7, Day14, Day21 and Day28. Each mouse underwent 3 parallel tests with an interval of 10 min between each experiment.

Gait Test

The gait test system (BIO-KWB-M, Bioseb) collected multiple gait behavior indicators, including stride length, step duration, and maximum footprint pressure. The mice were trained to move from the entrance to the end of the runway without stopping, exploring or moving backwards within 5 min. Each mouse was trained at least 3 to 5 times daily for a week before the drug injection. The gait test was performed on Day0, Day14, and Day28. The gait parameters were analyzed and calculated by the KWB gait test analysis system.

Hargreaves Test

The mice were placed to acclimate in a transparent Plexiglas chamber with a glass bottom for 30–60 min for two days before testing. After acclimation, thermal hyperalgesia tests were performed on Day0 and Day28. The radiant heat source (Ugo Basile) with an intensity of 20 W was positioned to the center of the bottom of the hind paws and activated with a timer. When the mouse withdraws, flicks, licks or escapes, the timer coupled with the infrared detector stopped timing, and the paw withdrawal latency (PWL) was recorded.

Von-frey Test

The mice were placed to acclimate in an elevated cage with a wire mesh floor for 30 min before testing. On Day0, Day7, Day14, Day21 and Day28, a series of von Frey filaments numbered 2 through 9 with bending force from 0.02 to 1.4 g were applied to the plantar surface of the hind paw for 3 s. Withdrawal threshold was determined by sequentially increasing and decreasing the stimulus strength (“up and down” method). The hind paw of each mouse was tested 5 times with 5 min intervals between each test. In the fifth test, if the mouse has a foot withdrawal response, the number of Von-Frey filaments is subtracted by 0.5, and if there is no response, 0.5 is added. Use the formula

\({\text{PWTforce=1}}{{\text{0}}^{\left( {{\text{x}} \times {\text{F+B}}} \right)}}\) to calculate the mechanical pain threshold of mice (F is the final number of Von-Frey fibers, x = 0.240, B=-2.00). We took the average of 10 tests for each mouse as the withdrawal threshold.

Tail Nerve Conduction Assessment

The tail nerve conduction velocity and action potential amplitude were tested on Day0, Day7, Day14, Day21 and Day28. After the mice were anesthetized by isoflurane inhalation, the mice tails were wiped and two platinum electrodes, namely stimulating electrode and recording electrode, were punctured in the root of tail and 3 cm caudally, respectively. The signal is received through the multi-channel physiological signal acquisition and processing system (RM-6240BD, Chengdu Instrument Factory), the specific parameters are (0.20 ms/div, 1 mv, 0.02 s, 3 kHz). Measure the duration of the action potential latency and the amplitude of the action potential.

Immunofluorescence Staining Of Sciatic Nerve Fibers

Mice were euthanatized by anesthesia followed by perfusion with ice-cold saline and 4% paraformaldehyde. The sciatic nerve was isolated and cut into approximately 5 mm length under a stereo microscope. After peeling off the sheath, the nerve segment was gently divided into nerve fibers and subsequently placed on adhesive-coated slides. The slides were blocked with blocking solution for 1 h, following by incubation with the primary antibodies MPZ (1:200; Abcam, Ab134439) or LC3-II (1:200; Abclonal, A 19665) overnight at 4 ºC. Secondary antibody labeled with Alex Fluor 488 or 594 (Invitrogen, A32731 and A32733) for 1 h. which were further incubated with DAPI (Abcam, ab285390), and the slides were observed with confocal microscope (Leica TCS SP8).

Transmission Electron Microscopy Analysis

The sciatic nerve was fixed in 2% glutaraldehyde at 4 ºC overnight. The sample was then fixed with 1% osmium acid for 1 h, following by staining with 2% uranyl acetate aqueous solution for 30 min. After dehydration in ethanol and clearing in 100% acetone, the samples were embedded and placed in a 37ºC oven. Semi-thin sections (1 µm) were prepared and stored at room temperature for toluidine blue staining. Ultrathin sections (500 nm) were observed under transmission electron microscope and images were collected. The G-ratio and the circularity of sciatic nerve fibers were calculated as follow: G-ratio=[(d1 + d2)/2] /[(D1 + D2)/2], Circularity=[4⋅π⋅(axonal area)]/(axonal perimeter)², where d1 and d2 refer to the diameter of axons, D1 and D2 refer to the outer diameter of myelinated fibers, both d1 & d2 and D1 & D2 are perpendicular to each other.

Rsc96 Schwann Cells Culture And Drug Administration

RSC96 Schwann cells were purchase from ATCC (CRL-2765). The cells were cultured in a humidified atmosphere with 5% CO₂ at 37°C. RSC96 cells were seeded in a 60 mm cell culture dish at a density of 5×10³ cells/mm² overnight, following by treatment with a final concentration of 25 nM, 50 nM, or 100 nM BTZ alone for 20 h. For autophagic flux analysis, a 50 µM of chloroquine diphosphate (CQ) was co-treated with BTZ for another 4 h. The control group was given an equal volume of DMSO.

Mcherry-egfp-lc3b Virus Transfection

RSC96 Schwann cells were cultured in 35 mm glass-bottom dishes. For virus transfection, the culture medium was replaced with FBS-free DMEM and 1 µl of virus solution (10⁸ TU/ml) expression mCherry-EGFP-LC3B (HANBIO, HB-LP210 0001) was added to the dish. After 4–6 h of incubation, culture medium was replaced with 10% FBS-containing DMEM. The cells were treated with indicated concentrations of BTZ for 24 h. The control group was given an equal volume of DMSO. A 63× inverted confocal microscope (Leica TCS SP8) was used for living cell observation and image acquisition at the atmosphere of 37 ºC, 5% CO₂. Image J software was applied to analyze the autophagy flux in RSC96 Schwann cells. In living cells, LC3 accumulated in autophagosomes and showed both red (mCherry⁺) and green (EGFP⁺) fluorescence. The yellow puncta (mCherry⁺ and EGFP⁺) in the merged image were characterized as autophagosomes. After fusing with lysosome, GFP was quenched in the acidic microenvironment and thus the red puncta (mCherry⁺) were characterized as autolysosomes (Klionsky et al. 2021). The autophagic flux was characterized by the change in the ratio of red puncta to yellow puncta.

Lysosome Staining

LysoTracker Red (Thermofish, L7528) are widely used to visualize lysosomes and DQ-Red-BSA (Thermofish, D12051) is employed to determine lysosomal activity (Park, Lee, and Park 2019). Schwann cells were cultured in 35 mm glass-bottom dishes. After BTZ treatment for 24 h, LysoTracker Red (75 nM) or DQ-Red-BSA (10 µg/ml) was incubated for 30 min or 2 h, respectively. Living cells were observed under the 63× inverted confocal microscope (Leica TCS SP8) at 37°C, 5% CO₂. ImageJ software was used to analyze and count the fluorescence intensity.

Western blotting

The sciatic nerves or RSC96 cells were homogenized in RIPA buffer with protease inhibitor cocktail (Roche, 04693132001). A 40 µg aliquot of protein from each sample was separated using SDS-PAGE and transferred to PVDF membrane. The following primary antibodies were used: LC3B (1:1000; Sigma-Aldrich, L7543), p62 (1:1000; MBL, PM045), Cathepsin B (1:1000; Abclonal, A0967), TFEB (1:1000; Cell Signaling Technology, 83010S), LAMP1 (1:1000; Abclonal, A16894), GAPDH (1:1000; Abclonal, AC001), β-actin (1:1000; Abclonal, AC004). Secondary antibodies conjugated with HRP against either rabbit or mouse IgG (1:3000; Cell Signaling Technology, 7071 and 7072) were applied. Digital images were quantified by densitometric measurement with the Image-Pro Plus software.

Statistical Methods

All experimental data are expressed by mean ± SD, and the software GraphpadPrism8.0 was used for statistical analysis. The significance analysis between two groups was determined by t-test. Comparations between more than two groups was determined by one-way or two-way ANOVA with Turkey's multiple-comparisons test. P < 0.05 was considered statistically significant.

Bortezomib-induced peripheral neuropathy in mice

Bortezomib (BTZ, 1 mg/kg, i.v.) was administrated every week on Day1 and Day4 for four consecutive weeks (Fig. S1A). As showed by the Von-Frey test, BTZ-treated mice showed a decreased withdrawal threshold to mechanical stimuli along with time (Fig. S1B), but the heat pain threshold by Hargreaves test was not significant changed (Fig. S1C). Peripheral nerve injury was manifested as a slowdown in conduction velocity and a decrease in the amplitude of action potentials (Bruna et al. 2020), both of which were significantly decreased along with the 4 weeks treatment of BTZ in mice (Fig. S1D-F). BTZ did not caused significant body weight loss during BTZ treatment (Fig. S1G), nor motor dysfunction as reflected by rotarod test (Fig. S1H). The gait analysis revealed that BTZ did not induce alternations in stance/step duration, swing/step duration, and stride length comparisons (Fig. S1I-K). The above results indicated that BTZ predominantly induced mechanical hyperalgesia, which mimicked the phenotype observed in BTZ-induced peripheral neuropathy (BIPN).

Bortezomib Led To Demyelination And Autophagy Flux Blockage In Schwann Cells

To further identify the BTZ-induced demyelination in mice, we observed the transverse and longitudinal sections of the sciatic nerve. As reflected by the immunofluorescent staining assay, BTZ-exposed sciatic nerve showed an irregular oval structure, which was manifested as a decrease in the fluorescence intensity of myelin protein (MPZ) (Fig. 1A). The outer layer of the dense myelin sheath showed a loose onion-like structure with BTZ treatment. Accordingly, the G-ratio increased whilst the circularity of sciatic nerve fibers decreased significantly in BTZ-treated mice (Fig. 1B), indicating BTZ induced demyelination in peripheral nerves.

We next aimed to identify whether autophagy was involved in the demyelination caused by BTZ in sciatic nerve. As reflected by immunostaining results, BTZ increased the number of LC3-positive puncta in the sciatic nerve (Fig. 1C). Increased autophagosomes may either caused by autophagy activation or lysosome dysfunction. Further immunoblot experiments showed that BTZ significantly upregulated both autophagosomal marker LC3-II and autophagy substrate p62 levels (Fig. 1D and E). These results suggested that BTZ blocked rather than activated the autophagic flux of the sciatic nerve.

To identify autophagy flux blockage by BTZ, chloroquine (CQ) was administrated to RSC96 Schwann cells. CQ is a lysosomal inhibitor widely used to neutralize lysosomal pH and block lysosomal degradation (Mauthe et al. 2018). We found BTZ accumulated LC3-II and p62 protein in a dose-dependent manner, which can be further enhanced with CQ co-treatment. However, the reinforced LC3-II and p62 accumulation by CQ decreased along with higher BTZ concentration (Fig. 1F and G). These results indicated that BTZ blocked the autophagic flux in Schwann cells in a concentration-dependent manner. To further clarify this, we transfected RSC96 cells with mCherry-EGFP-LC3B viruses. The fluorescence ratio of mCherry-EGFP-LC3B was employed to determine autophagy flux (Castillo et al. 2013). As a positive control, starvation by HBSS treatment induced prompt autolysosomes (only red puncta) formation in RSC96 cells. However, BTZ treatment led to significant accumulation of autophagosomes (merged as yellow puncta) in RSC96 Schwann cells, indicating blockage of autophagic flux (Fig. 1H and I). Overall, these results indicated that BTZ led to demyelination accompanied with blockage, rather than activation, of autophagic flux in Schwann cells, possibly by lysosomal dysfunction.

Btz Induced Lysosomal Dysfunction In Rsc96 Schwann Cells

To determine lysosomal quantity with BTZ treatment in Schwann cells, RSC96 cells were stained with LysoTracker Red (Price, Lau, and Zucker 2003). BTZ significantly reduced the fluorescence intensity of the LysoTracker Red in a concentration-dependent manner, which indicated lysosomal suppression after BTZ treatment (Fig. 2A). DQ-Red-BSA was employed to further examined whether BTZ affects lysosomal protease activity as previously described (Lao and Xu 2016). We observed a significant reduction of DQ-Red-BSA fluorescence intensity after BTZ treatment, indicating impaired protease activity (Fig. 2B). Lysosomes contain more than 50 acid hydrolases, among which Cathepsins serve crucial roles in lysosomal proteolysis (Laussmann et al. 2011). BTZ treatment upregulated the expression of immature Cathepsin B (CTSB), suggesting decreased lysosomal hydrolase activity. The decreased expression of TFEB was observed following BTZ treatment (Fig. 2C), suggesting impaired lysosome biogenesis. Furthermore, TFEB but not LAMP1 of the mouse sciatic nerve decrease significantly along with BTZ administration (Fig. 2D). These data implied that BTZ may impair lysosome biogenesis in addition to cause damaged lysosomes accumulation. These results suggested that BTZ induced lysosomal dysfunction in Schwann cell.

Torin1 Reversed Bortezomib-induced Lysosomal Dysfunction In Rsc96 Schwann Cells

Torin1 and PP242 have been reported to activate lysosomal function by inhibiting mTOR (Zhou et al. 2013). We found both Torin1 and PP242 decreased the expression of p62 and Torin1 increased the LC3-II. In addition, either co-treatment of Torin1 or PP242 significantly downregulated the elevated immature CTSB by BTZ, suggesting rescued lysosomal proteolytic enzyme activity of Schwann cells (Fig. 3A and B). These results suggested that enhanced lysosomes reversed the autophagic flux blockage by BTZ in Schwann cells. To confirm this, we found Torin1 reversed the decrease of DQ-Red-BSA fluorescence in BTZ-treated RSC96 cells, suggesting rescued lysosomal activity (Fig. 3C and D). Overall, these results indicated that Torin1 was sufficient to reverse the lysosomal dysfunction induced by BTZ.

Torin1 Ameliorated Bortezomib-induced Nerve Fiber Demyelination And Bipn

We next asked whether Torin1 was sufficient to ameliorate BIPN in mice. To this end, Torin1 (10mg/kg) was administrated daily during the last two weeks of BTZ exposure (Fig. 4A). As reflected by the TEM observation, the nerve fibers of the BTZ-treated mice were irregular, and the myelin sheath appeared loose onion-like structure. Notably, nerve fibers became regular and myelin sheath were tight by Torin1 administration (Fig. 4B). Additionally, we found Torin1 significantly reversed the increase in the G-ratio of the sciatic nerve and the decrease in the circularity ratio in BTZ-treated group (Fig. 4C). It indicated that Torin1 reversed the nerve fiber demyelination and axon degeneration caused by BTZ. We further explored whether Torin1 ameliorate BIPN. The results showed that Torin1 significantly rescued BTZ-induced mechanical allodynia, accompanied by slight amelioration of velocity and amplitude. (Fig. 4D-G) The above results indicated that Torin1, an activator of lysosomal function, alleviated BTZ-induced nerve fiber demyelination and neuropathic pain.

Torin1 Did Not Compromise The Efficacy Of Bortezomib On Multiple Myeloma

To investigate whether combining Torin1 may compromise the anti- multiple myeloma (MM) activity of BTZ, MM cells (RPMI8226) were exposed to indicated concentrations of Torin1, alone or in combination with BTZ. Cell viability was assessed and IC50 of BTZ and Torin1 were measured as 4.575 nM and 19.96 nM (Fig. 5A and B). The CompuSyn software was used to calculate the combination index (CI) of BTZ and Torin1. The results showed the CI was 0.892, 0.856, and 0.982 under the effects of IC50, IC75, and IC95 of this combination (Fig. 5C-E). These results suggested that Torin1 did not affect the anti-MM efficacy of BTZ.

The proteasome inhibitor BTZ serves as the first-line treatment for primary, refractory/relapsed multiple myeloma. BIPN remains the leading cause for BTZ dosage reduction or even discontinuation, with its toxicological mechanisms not fully understood. Biopsy analysis from BIPN patients observed that BTZ treatment induces demyelinating of sciatic nerve fibers (Stubgen 2011). BTZ induces demyelinating lesions of myelinated nerve fibers (Thawani et al. 2015; Stubgen 2011), which was a typical pathological alternation underlying mechanical hyperalgesia. In Schwann cells, lysosome function was crucial for myelin clearance after peripheral nerve injury (Reed et al. 2020). Lysosomal exocytosis in Schwann cells contributes to axon remyelination (Chen et al. 2012). As the final step of autophagy-lysosome degradation pathway, lysosomes were responsible for digesting cargo (Ballabio and Bonifacino 2020). Here we observed accumulation of both p62 and LC3-II (Fig. 1C-E) and significant increase of LC3 puncta (Fig. 1I), suggesting BTZ did not or only slightly affected the autophagosome biogenesis. This notion was unexpected because proteasome inhibition usually induced autophagic degradation as a compensation in various cells (Pan et al. 2020; Laussmann et al. 2011). Furthermore, mcherry-GFP-LC3 assay revealed enlarged and yellow puncta of LC3 in RSC96 Schwann cells (Fig. 1H-I), suggesting blockage rather than activation of autophagic flux by BTZ treatment.

Lysosomal dysfunction blocks autophagic flux (Kao et al. 2014). For this reason, we postulated lysosomal dysfunction underlying BTZ-induced autophagic blockage. Indeed, the lysosomal quantity was significantly reduced in RSC96 cells as revealed by LysoTracker Red staining. TFEB, a master transcriptional factor promoting lysosomal generation (Settembre et al. 2011), decreased in mice sciatic nerves with 4-weeks BTZ exposure (Fig. 2D). Moreover, our evidence indicating dramatic decrease of lysosomal activity by BTZ in Schwann cells as revealed by DQ-Red-BSA assay (Fig. 2B). Lysosomal hydrolysis eliminated myelin debris after peripheral nerve injury (Jang et al. 2016; Holtzman and Novikoff 1965), and the clearance of myelin debris facilitates the differentiation of myelinated Schwann cells into a repairing type, which favors myelin and axon regeneration (Jessen and Mirsky 2016). Consistently, the increased immature CTSB was observed in BIPN mice model (Fig. 2C-D). Overall, here we provide evidence both in vitro and in vivo indicating that BTZ caused lysosomal dysfunction in Schwann cells, which may underlie the toxicity of BTZ to peripheral nerves.

Bortezomib affects lysosome membrane permeability and induces lysosomal Cathepsin B release in cancer cells (Yeung, Huang, and Sinicrope 2006). In ovarian cancer and other solid tumors bortezomib also suppresses Cathepsin B and inhibits the catalytic process of autophagy (Kao et al. 2014). Additionally, BTZ inhibited the serine proteases in lysosome of neuronal cell (Arastu-Kapur et al. 2011). As a comparison, Carfilzomib, a proteasome inhibitor, failed to inhibit Cathepsin protease and showed a much lower incidence peripheral neuropathy (McCormack 2012; Arastu-Kapur et al. 2011). Thus, it seems that BIPN might be attributable to BTZ-caused lysosomal dysfunction rather than proteasomal inhibition.

Torin1 and PP242 were reported to inhibit mTOR and thus improve lysosome function (Zhou et al. 2013). We found that Torin1 reversed the up-regulation of p62 and decrease of DQ-Red-BSA fluorescence intensity caused by BTZ in Schwann cell (Fig. 3A-D), further support the autophagic flux blockage by lysosomal dysfunction in Schwann cells under BIPN conditions. Furthermore, Torin1 significantly improved BTZ-induced demyelination and mechanical pain threshold, which indicated Torin1 can alleviate BIPN (Fig. 4A-G). These results provide a causal link between lysosomal dysfunction and BIPN. Torin1 rescued lysosomal dysfunction in corneal fibroblasts (Wang et al. 2022). Although Torin1 showed disadvantages in stability and bioavailability (Chen and Zhou 2020), the present study supported the concept to rescue BIPN by improving lysosomal activity in Schwann cells. Intriguingly, Itaconate and Tubeimoside-1, were reported to enhance lysosome biogenesis by modulating mTOR signaling (Liu et al. 2021; Zhang et al. 2022). It may worth to identify their potential in rescuing BIPN. We postulated that enhancing lysosomal activity by mTOR inhibitor may have the advantage in treating BIPN. Because mTOR inhibiting prevented the proliferation of multiple myeloma cells (Frost et al. 2007) and mTOR inhibitors are often used in combination with other drugs to synergistically enhance the anti-multiple myeloma effects (Xiang et al. 2017). We found Torin1 showed a slight synergistic effect of BTZ on multiple myeloma (Fig. 5A-D). Further studies are needed to identify the efficacy of clinical available mTOR inhibitors on both multiple myeloma treatment and BIPN ameliorating in combination with BTZ.

In summary, here we found lysosomal dysfunction of Schwann cells is involved in the toxicity of BTZ to peripheral nerves. Enhanced lysosomal activity holds a promising strategy for BIPN therapy. mTOR inhibitors could be used to treat multiple myeloma in combination with BTZ.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Code availability

Not applicable.

Funding

This work was funded by the National Natural Science Foundation of China (81973402 and 82173792), Zhejiang Provincial Natural Science Foundation (LZ21H310001) and the Starry Night Science Fund of Zhejiang University Shanghai Institute for Advanced Study (SN-ZJU-SIAS-0011).

Author Contributions

Correspondence to Xiangnan Zhang and Jiaying Wu. Zhanxun Wu and Wenping Yan contributed equally to this work. All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Zhanxun Wu, Wenping Yan, Xiangnan Zhang, Jiaying Wu, Ke Wang, Genghua Xu, Danyan Zhu, Xuyun Li, Huafeng Wang and Min Yang. The first draft of the manuscript was written by Zhanxun Wu, Wenping Yan, Xiangnan Zhang and Jiaying Wu. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Ethics approval

All procedures were performed in accordance with the guidelines of the Zhejiang University Animal Experimentation Committee and were in complete compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Conflict of interest

The authors declare no competing interests

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No competing interests reported.

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Lysosomal dysfunction in Schwann cells is involved in bortezomib-induced peripheral neuropathy

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Abstract

Figures

Introduction

Materials And Methods

Animals

Drug administration

Gait Test

Hargreaves Test

Von-frey Test

Tail Nerve Conduction Assessment

Immunofluorescence Staining Of Sciatic Nerve Fibers

Transmission Electron Microscopy Analysis

Rsc96 Schwann Cells Culture And Drug Administration

Mcherry-egfp-lc3b Virus Transfection

Lysosome Staining

Western blotting

Statistical Methods

Results

Bortezomib-induced peripheral neuropathy in mice

Bortezomib Led To Demyelination And Autophagy Flux Blockage In Schwann Cells

Btz Induced Lysosomal Dysfunction In Rsc96 Schwann Cells

Torin1 Reversed Bortezomib-induced Lysosomal Dysfunction In Rsc96 Schwann Cells

Torin1 Ameliorated Bortezomib-induced Nerve Fiber Demyelination And Bipn

Torin1 Did Not Compromise The Efficacy Of Bortezomib On Multiple Myeloma

Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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