Two mTOR inhibitors, rapamycin and Torin 1, differentially regulate iron-induced generation of mitochondrial ROS

Hui Huang . Jun Chen . Huiru Lu . Mengxue Zhou . Zhifang Chai .
Yi Hu

Received: 5 October 2017 / Accepted: 13 October 2017
© Springer Science+Business Media, LLC 2017

Abstract It is generally believed that gene-environ- ment interaction may contribute to neurodegeneration. Of particular note is that iron overload may be one of the risk factors for neurodegeneration. However, the mechanisms underlying iron-associated neurotoxicity are not fully understood. Here we explored the effects of mechanistic target of rapamycin (mTOR) inhibition in iron-stressed human neuroblastoma cells. Two mTOR inhibitors, rapamycin and Torin 1, had similar effects in cells exposed to a relatively low concentra- tion of iron. At a higher concentration of iron, Torin 1, instead of rapamycin, could further aggravate iron- induced cytotoxicity, and mitochondrial ROS levels were significantly higher in Torin 1-treated cells. These results suggest that mTOR inhibition may not be able to alleviate iron-induced neurotoxicity.

Keywords mTOR · Rapamycin · Torin 1 · ROS

H. Huang J. Chen H. Lu M. Zhou
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Z. Chai Y. Hu (&)
CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Multidisciplinary Research Division, Institute of High Energy Physics, Chinese Academy of Sciences (CAS), Beijing 100049,
China
e-mail: [email protected]

· · · ·
H. Huang J. Chen M. Zhou Z. Chai Y. Hu University of Chinese Academy of Sciences, Beijing 100049, China
Introduction

Parkinson’s disease (PD) is a common neurodegener- ative disease in elderly people. To date, no effective therapy exists that can halt the progression of PD. In addition to several well-known genetic risk factors, accumulating evidence indicates that environmental factors may also be implicated in the pathogenesis of PD (Ascherio and Schwarzschild 2016). Several previous studies have suggested that early-life or occupational exposure to redox-active elements, such as iron and copper, is a putative risk factor for PD (Gorell et al. 1999; Hare et al. 2015; Lan et al. 2016a). In addition, nigral iron accumulation is often seen in the brains of PD (Ayton and Lei 2014; Pyatigorskaya et al. 2015; Wang et al. 2016; Wieler et al. 2016). Nevertheless, the association of dietary iron intake with PD remains debating (Johnson et al. 1999; Miyake et al. 2011). This apparent discrepancy may be ascribed to the compounding factors in the popu- lation studies (Johnson et al. 1999; Miyake et al. 2011), such as other nutrient intakes (Logroscino et al. 2008; Powers et al. 2003, 2009), sex (Cheng et al. 2015; Powers et al. 2009) and age (Johnson et al. 1999). Therefore, further mechanistic study is required to clarify the role of iron in PD.
Iron is an essential biometal, which interacts with DNA and proteins (Duck and Connor 2016; Lu et al. 2017, 2015). On the other hand, iron overload can be cytotoxic in that iron can generate intracellular

reactive oxygen species (ROS) via Fenton reaction (Lan et al. 2016a). We have recently reported that iron-induced generation of mitochondrial ROS could be regulated by AMP-activated protein kinase (AMPK) in human neuroblastoma cells (Huang et al. 2017). Pharmacological activation of AMPK potently suppressed the production of mitochondrial ROS induced by iron, whereas AMPK inhibition further increased mitochondrial ROS levels (Huang et al. 2017). Similarly, inhibition of AMPK aggravated the neurotoxicity of another redox-active element, i.e., copper, in SH-SY5Y cells (Lan et al. 2016b). As mechanistic target of rapamycin (mTOR) is an important downstream effector of AMPK (Lan et al. 2017), we next asked whether mTOR activity is also associated with iron-induced generation of mitochon- drial ROS.
mTOR is widely implicated in devastating human diseases including cancer and diabetes (Laplante and Sabatini 2012). Recently, multiple lines of evidence have suggested that mTOR may also be associated with the risk factors for PD (Lan et al. 2017). However, molecular mechanisms underlying mTOR-mediated neurodegeneration are still largely unknown in PD and further investigation is compulsory. Here we aimed to explore whether mTOR inhibitors, including rapamy- cin and Torin 1, could modulate the toxic effect of Fe2? in human neuroblastoma cells.

Materials and methods

Materials

Dulbecco’s modified eagle medium (DMEM), peni- cillin, streptomycin and phosphate buffer saline (PBS) were purchased from Hyclone. Fetal bovine serum (FBS) was supplied by Gibco. Ferrous chloride (FeCl2) was purchased from Aladdin. To remove impurities, FeCl2 dissolved in distilled water was filtered through a 0.22 lm filter. 3-(4,5-dimethylthia- zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were obtained from Aladdin. Torin 1, rotenone and rapamycin were purchased from Calbiochem. Mito-SOX red mito- chondrial superoxide indicator and Hoechst 33342 were purchased from Invitrogen Inc.
Cell culture

SH-SY5Y cells were maintained at 37 °C with 5% CO2 in DMEM medium which contains 100 U/mL penicillin, 100 lg/mL streptomycin and 10% FBS. Prior to the experiments of iron toxicity, 5 lM rotenone was incubated with SH-SY5Y cells for 2 h. To assess the biological effect of rapamycin, SH- SY5Y cells were pretreated with 100 ng/mL rapamy- cin for 1 h and then incubated with different concen- trations of Fe2? for another 24 h. Similarly, to investigate the inhibition effect of Torin 1, cells were pretreated with 100 ng/mL Torin 1 for 1 h and then co-cultured with Fe2? for another 24 h.

Cytotoxicity

MTT assay was used to examine cell viability. Briefly, SH-SY5Y cells were grown in a 96-well plate at the density of 8000 cells per well and cultured for 24 h. Afterward, fresh medium containing different con- centrations of Fe2? or mTOR inhibitors was added and incubated with the cells. After incubation, the medium in each well was changed to 100 lL of culture medium and 10 lL of MTT, and then cells were cultured for additional 4 h. Subsequently, 200 lL DMSO was added to each well to dissolve the formazan crystals. Finally, the absorbance of the sample in each well at 490 nm was measured by a microplate reader (Biotech, MQX 200).

Detection of mitochondrial ROS

Mitochondrial ROS was assessed by Mito-SOX red mitochondrial superoxide indicator. Prior to use, Mito- SOX was prepared in Hank’s Balanced Salt Solution (HBSS) at a final concentration of 5 lM, which was incubated with SH-SY5Y cells for 10 min at 37 °C in the dark. After washing three times with PBS, cells were co-incubated with 5 mg/L Hoechst 33342 for 10 min at 37 °C in the dark. Following three times washing with PBS, fluorescence images were recorded with a fluorescence microscope (BX 50-FLA; Olym- pus, Tokyo, Japan).

Statistical analysis

All experiments data were presented as mean ± SD. Statistical significance was analyzed by two-tailed student’s t test.

Results and discussion

Human neuroblastoma SH-SY5Y cells treated with rotenone were used as a cellular model of PD in this study (Betarbet et al. 2000). As previously reported (Huang et al. 2017), the cytotoxicity of Fe2? was concentration-dependent and here we chose 0.5 mM and 5 mM of Fe2?, which are close to the physiolog- ical concentrations of iron (Gotz et al. 2004). As shown in Fig. 1, 5 mM of Fe2? inhibited cell prolif- eration, whereas 0.5 mM of Fe2? had virtually no effect. To examine the effect of mTOR inhibition, SH- SY5Y cells were pretreated with mTOR inhibitors before being incubated with Fe2?. Rapamycin is a natural product (Liu et al. 2012), while Torin 1 is a synthetic molecule firstly reported in 2009 as an mTOR inhibitor (Thoreen et al. 2009). Figure 1 shows that both inhibitors could elicit the neurotoxicity of
0.5 mM Fe2?. At a relatively high concentration of Fe2?, rapamycin had virtually no effect (Fig. 1a). By contrast, Torin 1 further aggravated the cytotoxicity of 5 mM Fe2? in SH-SY5Y cells (Fig. 1b).
The differential effects of rapamycin and Torin 1 may be explained by the inhibitory mechanisms of
these two molecules as mTOR inhibitors. mTOR is the catalytic subunit of two distinct protein complexes, i.e., mTOR complex 1 (mTORC1) and mTOR com- plex 2 (mTORC2). Rapamycin is an inhibitor mainly for mTORC1 (Laplante and Sabatini 2012). In addi- tion, inhibition of mTORC1 by rapamycin could turn on a negative feedback loop to re-activate mTORC1 (Laplante and Sabatini 2012). Therefore, rapamycin only has limited inhibitory effect on mTOR signaling, and combination of rapamycin with other protein inhibitors may be therapeutically advantageous (Hu et al. 2014). By contrast, Torin 1 is an inhibitor for both mTORC1 and mTORC2, and it is believed to be a more potent inhibitor of mTOR than rapamycin (Thoreen et al. 2009). The fact that Torin 1, instead of rapamycin, enhances the cytotoxicity of 5 mM Fe2? might be ascribed to potent inhibition of mTOR by Torin 1.
Iron overload can induce the production of mito- chondrial ROS (Huang et al. 2017). We next asked whether mTOR inhibitors could alter mitochondrial ROS levels in SH-SY5Y cells upon iron exposure. As shown in Fig. 2, 5 mM of Fe2? significantly up- regulated mitochondrial ROS levels in SH-SY5Y cells. 0.5 mM of Fe2? had virtually no effect on the production of mitochondrial ROS. Pretreatment with rapamycin further increased mitochondrial ROS levels in SH-SY5Y cells exposed to 0.5 mM or 5 mM of Fe2? (Fig. 2a, b). While rapamycin had virtually no effect on the cytotoxicity of 5 mM Fe2? (Fig. 1a), it could enhance 5 mM Fe2?-induced generation of

Fig. 1 The cytotoxicity of Fe2? is enhanced by rapamycin or Torin 1. a SH-SY5Y cells were exposed to 0, 0.5 or 5 mM Fe2? with or without 100 ng/mL rapamycin for 24 h. b SH-SY5Y
cells were exposed to 0, 0.5 or 5 mM Fe2? with or without 100 ng/mL Torin 1 for 24 h. *P \ 0.05 and **P \ 0.01 between indicated groups, n = 6

b Fig. 2 Mitochondrial ROS levels are increased by mTOR inhibition. a Fluorescence images of Mito-SOX probe in SH- SY5Y cells exposed to 0, 0.5 or 5 mM Fe2? with or without 100 ng/mL rapamycin for 24 h. b Quantitative analysis of fluorescence intensity of (a). c Fluorescence images of Mito- SOX probe in SH-SY5Y cells exposed to 0, 0.5 or 5 mM Fe2? with or without 100 ng/mL Torin 1 for 24 h. d Quantitative analysis of fluorescence intensity of (c). e Difference in fluorescence intensities between the groups treated with rapamycin and the groups treated with Torin 1. **P \ 0.01 and ***P \ 0.001 between indicated groups, n = 4. Scale bar 11 lm

mitochondrial ROS (Fig. 2b). Similarly, pretreatment with Torin 1 also further increased mitochondrial ROS levels in iron-treated SH-SY5Y cells (Fig. 2c, d). As compared with 0.5 mM Fe2?, 5 mM Fe2? induced more mitochondrial ROS in SH-SY5Y cells pretreated with Torin 1 (Fig. 2d), which was not seen in the cells pretreated with rapamycin (Fig. 2b). Further quanti- tative analysis indicated that Torin 1 induced consid- erably higher levels of mitochondrial ROS in SH- SY5Y cells than rapamycin did (Fig. 2e). These results suggest that mTOR inhibition enhances Fe2?- induced generation of mitochondrial ROS. Compared with rapamycin, Torin 1 could be a potent inducer of mitochondrial ROS in iron-treated SH-SY5Y cells, which is consistent with the results of cytotoxicity experiments (Fig. 1).
It is worth noting that mTOR inhibition could be either neuroprotective or neurotoxic in distinct PD models (reviewed in ref. (Lan et al. 2017)). mTOR inhibition by rapamycin has been reported to reduce a- synuclein accumulation (Spencer et al. 2009), prevent L-DOPA induced dyskinesia (Decressac and Bjork- lund 2013; Santini et al. 2009), and suppress dopamin- ergic neurodegeneration (Tain et al. 2009). By contrast, rapamycin could also potentiate oxidative stress-induced loss of neuronal cells (Choi et al. 2010). Moreover, PD toxins including rotenone, 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hy- droxydopamine (6-OHDA) have been reported to be able to inhibit mTOR signaling and suppress cell viability (Lan et al. 2017). It is reasonable to speculate that either hyperactive or hypoactive mTOR may facilitate neurodegeneration, and only fine-tuned mTOR activity would be crucial for neuronal survival (Lan et al. 2017).
Recently, Farshbaf and Ghaedi have proposed that mTOR inhibitors that attenuate iron overload could be considered as potential neuroprotective agents (Farsh- baf and Ghaedi 2017). However, our and others’ results imply that it may be currently immature to conclude the effects of mTOR inhibition in PD. More validation experiments in distinct PD models are needed. As rapamycin only partially inhibits mTOR activity, it is possible that the effects of rapamycin are ascribed to the inhibition of certain but not all the functions of mTOR. We suggest replication study with Torin 1 should be conducted to exclude the effects of incompletely inhibited mTOR.
Collectively, our data indicated that inhibition of mTOR could aggravate iron-induced neurotoxicity and generation of mitochondrial ROS. Compared to rapamycin, Torin 1 is a potent effector in iron-stress neuronal cells. These results may make us reconsider mTOR inhibitors as neuroprotective agents in PD with iron overload.

Acknowledgements We acknowledge the financial support from National Natural Science Foundation of China (Grant No. 11375213, 21390411), the Hundred Talents Program of the Chinese Academy of Sciences and IHEP Innovation Program.

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