Rifampicin attenuates rotenone-treated microglia inflammation via improving lysosomal function

Journal Pre-proof Rifampicin attenuates rotenone-treated microglia inflammation via improving lysosomal function

Yanran Liang, Dezhi Zheng, Sudan Peng, Danyu Lin, Xiuna Jing, Zhifen Zeng, Ying Chen, Kaixun Huang, Yingyu Xie, Tianen Zhou, Enxiang Tao PII:

S0887-2333(19)30455-2

DOI:

https://doi.org/10.1016/j.tiv.2019.104690

Reference:

TIV 104690

To appear in:

Toxicology in Vitro

Received date:

12 June 2019

Revised date:

13 October 2019

Accepted date:

14 October 2019

Please cite this article as: Y. Liang, D. Zheng, S. Peng, et al., Rifampicin attenuates rotenone-treated microglia inflammation via improving lysosomal function, Toxicology in Vitro(2018), https://doi.org/10.1016/j.tiv.2019.104690

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© 2018 Published by Elsevier.

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Rifampicin attenuates rotenone-treated microglia inflammation via improving lysosomal function Yanran Liang1,a, Dezhi Zheng1,d, Sudan Peng1,a, Danyu Lin1,e, Xiuna Jinga, Zhifen Zenga, Ying Chena, Kaixun Huange, Yingyu Xiea, Tianen Zhou2,b, Enxiang Tao2,a,c a

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Department of Neurology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, 107 Yanjiang West Road, Guangzhou 510080, China. b Department of Emergency, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, 107 Yanjiang West Road, Guangzhou 510080, China. c Guangdong Province Key Laboratory of Brain Function and Disease, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou 510080, China d Department of Neurology, The First People’s Hospital of Foshan, Foshan, Guangdong 528000, China e Department of Neurology, The Eighth Affiliated Hospital, Sun Yat-sen University, Shenzhen, Guangdong 518033, China 1 These authors contributed equally to this work. 2 These authors are corresponding author.

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Address for correspondence Enxiang Tao, Department of Neurology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, 107 Yanjiang West Road, Guangzhou 510080, China. Email: [email protected] Tianen Zhou, Department of Emergency, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, 107 Yanjiang West Road, Guangzhou 510080, China. Email: [email protected]

Abstract 1

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Mounting evidence suggests that lysosome dysfunction promotes the progression of several neurodegenerative diseases via hampering autophagy flux. While regulation of autophagy in microglia may affect chronic inflammation involved in Parkinson’s disease (PD). Our previous studies have reported rifampicin inhibits rotenone-induced microglia inflammation by enhancing autophagy, however the precise mechanism remains unclear. Human microglia (HM) cells were pretreated with 100 μM rifampicin for 2 h followed by exposure to 0.1 µM

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rotenone. We found that rifampicin pretreatment suppressed the gene expression of IL-1β and IL-6 via inhibiting activation of JNK after rotenone induction, but the anti-inflammatory

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effect of rifampicin was reversed by chloroquine. Moreover, rifampicin pretreatment not only

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improved the ratio of LC3-II/LC3-I in rotenone-treated cells, but also increased

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autolysosomes and decreased autophagosomes in RFP-GFP-LC3B transfected HM cells

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exposed to rotenone, thus indicating rifampicin improves autophagy flux in rotenone-treated HM cells. Finally, we verified rifampicin pretreatment enhanced ATP6V0A1 expression when

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compared to that exposed to rotenone alone. ATP6V0A1 knockdown inhibited the effect of rifampicin on maintaining lysosome acidification and autophagosome-lysosome fusion in rotenone-treated microglia. Taken together, our results indicated that rifampicin attenuates rotenone-induced microglia inflammation partially via elevating ATP6V0A1. Modulation of lysosomal function by rifampicin may be a novel therapeutic strategy for PD.

Keywords Rifampicin, Rotenone, Microglia, Inflammation, Autophagy flux, Lysosomal function Highlights 2

Journal Pre-proof  Autophagy inhibitor attenuated rifampicin’s anti-inflammatory effect.  Rifampicin improved autophagy flux in rotenone-treated microglia.  Rifampicin enhanced expression of ATP6V0A1 in rotenone-treated microglia.  Rifampicin improved lysosomal function by increasing ATP6V0A1 expression.

1. Introduction

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Parkinson’s disease (PD) is a common neurodegenerative disease. Both the presence of intracytoplasmatic inclusions, such as Lewy bodies and neuritis, and progressive loss of

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dopaminergic neurons in the substantia nigra pars compacta are considered as the pathological

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hallmarks of PD. More and more evidences have indicated interactions between genetic

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defects and exposure to environmental risk factors in the pathogenesis of PD.

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Epidemiological studies have suggested that rotenone is one of the environmental toxicants triggering PD-like progression(Pan-Montojo et al., 2012). Although the molecular

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mechanisms underlying rotenone-related neurotoxicity remains elusive, growing lines of evidence reveal that rotenone can promote oxidative stress and mitochondrial dysfunction by inhibiting the complexes I of mitochondrial electron transport chains. More recent studies suggest that rotenone not only elevates α-synuclein expression levels and accelerates the accumulation of misfolded α-synuclein in neurons, but also damages neurons indirectly by continuously triggering activation of inflammation in microglia and astrocytes(Lee et al., 2002)(Yuan et al., 2013). As autophagy is one of the crucial pathways to clean degenerative, damaged, and non-functional proteins and organelles, the question whether autophagy dysfunction is 3

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involved in rotenone-induced neurotoxicity has attracted a lot of interest in scientific community in recent years. High levels of LC3-II and autophagic vacuoles were found in PC12 cells and mouse models exposed to rotenone(Wu et al., 2015). In addition, our previous studies have observed that rotenone could triggers inflammation via causing autophagy dysfunction in microglia(Liang et al., 2017). Therefore, modulation of autophagy might be a

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promising therapeutic strategy for PD.

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Autophagy is a lysosomal degradation pathway containing multiple steps: firstly, initiation with phagophore induction; secondly, autophagosome formation; finally,

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autolysosome formation and degradation. Lysosomal degradation is the ultimate step of

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autophagy. Thus, lysosomes are the key organelles in the autophagy process. Lysosomes are

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cytoplasmic organelles with a single membrane, which contains hydrolytic enzyme that

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degrade intracellular components. The acidic environment (pH4.5-5.0) in the lumen of lysosomes is the main determinant of hydrolytic enzyme activation. This acidic environment

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is maintained by vacuolar ATPases (V-ATPase) pumping proton into the lumen(Mindell, 2012). V-ATPase is the major component of lysosomal membrane and responsible to regulates lysosomal acidic environment as well as autophagosome-lysosome fusion(Mijaljica et al., 2011). Studies revealed that ATP6V0A1, one of the subunits of V0 sector of V-ATPase, participates in proton transport and autolysosome formation(Peri and Nusslein-Volhard, 2008)(Bagh et al., 2017). Apart from impaired lysosomal acidification, lysosome membrane instability and obstacle of autophagosome-lysosome fusion also contributes to defects of autophagy flux. Accumulating research shows that autophagy flux impairment is one of the pathogenesis of 4

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PD. Miquel Vila et al. demonstrated that the abnormal lysosomal membrane permeabilization plays an instrumental role in the impairment of autophagy in dopaminergic BE-M17 neuroblastoma cells exposed to 1-methyl-4-phenylpyridinium (MPP+)(Vila et al., 2011). Marija Usenovic et al. reported that loss of ATP13A2 (PARK9) in fibroblasts and primary neurons leads to impaired lysosomal degradation capacity(Usenovic et al., 2012). Both

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environmental toxicity (such as MPP+ and rotenone) and gene mutation (such as PARK9,

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LRRK2 and PINK1) may lead to PD via impairing lysosomal function(Wu et al., 2015)(Dehay et al., 2013). Thus, searching for a safe and effective drug to regulate lysosomal

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function may be an important therapeutic strategy for PD.

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Recently, mounting studies demonstrate that rifampicin, a classic and safe

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anti-tuberculous drug, has neuro-protective effects in acute and chronic brain injury.

α-synuclein,

regulating

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Rifampicin may protect neurons via upregulating GRP78, enhancing sumoylation of PI3K/Akt/GSK-3β/CREB

signaling

pathway

or

inhibiting

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neuroinflammation through TLR-4 pathway(Lin et al., 2017)(Jing et al., 2014)(Wu et al., 2018). Furthermore, rifampicin can inhibit release of IL-1β by suppressing the activation of NLRP3 inflammasome in microglia(Liang et al., 2015). In our preliminary study, we found that rifampicin could inhibit rotenone-induced microglia inflammation via enhancing autophagy to degrade damaged mitochondria(Liang et al., 2017). However, the precise mechanism of how rifampicin improved autophagy requires further studies. Our present study aims to investigate whether rifampicin attenuates rotenone-induced microglia inflammation via improving lysosomal function. We demonstrated that rifampicin attenuated inflammation and improved lysosomal function in microglia exposed to rotenone 5

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by increasing expression of ATP60V1 2. Methods 2.1. Cell culture Immortalized human microglia cells (HM cells，Human Microglia Catalog #1900) were purchased from ScienCell (Carlsbad, CA). HM cells were cultured in DMEM medium

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supplemented with 10% FBS at 37 °C in a humid air atmosphere with 5% CO2.

2.2. Materials

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Rifampicin and rotenone were purchased from Sigma-Aldrich (MO USA). LC3B

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antibody were purchased from GeneTex (Irvine, TX, USA), LAMP1 were purchased from

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Abcam Company (Cambridge, MA, USA). LysoTrackerTM Red DND-99 and LysoSensor

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2.3. Cell viability assay

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yellow/blue DND-160 were obtained from Thermo Fisher Scientific (MA, USA).

The effects of rifampicin and rotenone on HM cell viability were measured via Cell Counting Kit-8 (CCK-8) assay (Dojindo Kumamoto, Japan). HM cells were seeded in a 96-well plate at density of 1 × 104 / well for 24 h. After treatment with different drugs, the cells were cultured in 100 μl DMEM medium with 10% CCK-8 reagent for 2 h. The absorbance of each well was measured on Spectrophotometer (SpectraMax M5, Sunnyvale, CA, USA) at 450 nm.

2.4. Western-blot analysis 6

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Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris (pH 7.4), 150 mM NaCI, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS), protease inhibitor (100 mM AEBSF, 5 mM Bestatin, 1.5 mM E64, 2 mM Pepstatin A, 0.2 mM Phosphoramidon) supplemented with phosphatase inhibitor (250 mM sodium fluoride, 50 mM sodium pyrophosphate, 50 mM β-glycerophosphate, 100 mM sodium orthovanadate) for

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30 min on ice. Protein samples (30 µg) were separated by 12% SDS-PAGE gels and

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transferred to PVDF membranes. After being blocked with 5% non-fat milk for 1 h at room temperature, membranes were incubated with primary antibodies including p-JNK 1:1000

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(CST, #4668), JNK 1:1000 (CST, #9252), p-ERK 1:1000 (CST, #4370), ERK 1:1000 (CST,

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#4696), p-P38 1:1000 (CST, #4511), P38 1:1000 (CST, #8690), LC3 1:1000 (CST, #4108)

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and ATP6V0A1 1:1000 (Abnova, H00000535). And then, membranes were incubated with

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appropriate HRP–conjugated secondary antibodies 1:5000 (CST, #7074; CST, #7076). Blots

MD, USA).

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were detected using chemiluminescence and analyzed with Image-J software (NIH, Bethesda,

2.5. Measure of lysosomal acidity

For the analysis of lysosomal acidity, 0.5 μM LysoTrackerTM Red DND-99 was added and incubated with cells for 1 h at 37 °C. Then the loading solution was replaced with fresh medium and the cells were immediately observed under fluorescent microscope (Carl Zeiss, Germany). The density of LysoTrackerTM Red staining was quantitatively analyzed by Image J to determine the lysosomal acidity (n = 20 cells per group). Lysosome pH was further performed using lysosomal pH probe LysoSensor 7

Journal Pre-proof yellow/blue DND-160. LysoSensor™ Yellow/Blue DND-160 is a ratiometric probe that commonly used for sensing pH in acidic organelles. The LysoSensor dye exhibits yellow fluorescence in acidic environments and produces blue fluorescence in neutral environments. At the end of different treatments, cells were loaded with 1 mM LysoSensor™ yellow/blue DND-160 for 1 min at 37 °C. Then the cells were resuspended in fresh prewarmed medium

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and images were captured using a laser confocal microscope (Carl Zeiss, Germany) (n = 20

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cells per group).

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2.6. Real-time reverse transcription polymerase chain reaction (RT-PCR)

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At the end of the treatments, RNA samples were extracted from HM cells by TRIzol

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reagent (Takara Bio Inc, Tokyo, Japan) according to procedure. Reverse transcription was

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performed on 1 μg of RNA using reverse transcription polymerase assay (Takara bio Inc, Tokyo, Japan). The cDNA was used for SYBR green real-time quantitative PCR assay with

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specific primers (Table. 1) to analyze the expression level of targeted genes.

2.7. Determination of autophagy flux by a tandem mRFP-GFP-LC3 reporter in transfected cells MRFP-GFP-LC3 dual fluorescent indicator is widely used for the detection of autophagy flux. As GFP is an acid-sensitive protein, the green fluorescent signal gets quenched when autophagosome fuses with lysosome forming autolysosome. While mRFP stably emits red fluorescence even under the acidic condition (pH below 5) inside the lysosome. Thus autophagosomes are observed as yellow puncta and red puncta indicate autolysosomes. 8

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Briefly, the HM cells were seeded on the culture plates and allowed to reach 50%–70% confluence at the time of transfection. According to the manufacturer’s instructions, moderate mRFP-GFP-LC3 adenoviral vectors were added into the media and incubated with the cells for 2h. Then the supernatants of cell cultures were replaced with fresh media, and the cells were allowed to express the protein for 24 hours. At the end of treatments as indicated, a laser

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confocal microscope (Leica, Heidelberg, Germany) was used to observe the LC3 puncta. The

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numbers of LC3 puncta with red and yellow fluorescent signal were quantitatively analyzed with 20 cells for triplicate samples per condition per experiment, respectively. The dots with

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size from 0.2 μm to 1.0 μm in MERGE phase were counted for autophagosomes or

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autolysosomes.

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2.8. Detection of autophagosome-lysosome fusion by immunofluorescence HM cells were transfected with short interfering RNA (siRNA) using Lipofectamine 3000

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(Thermo Fisher Scientific, MA, USA) according to the manufacturer’s protocol. The target sequence to silence the ATPV0A1 gene was 5’-GCUUCGAUUUGUUGAGAAA -dTdT-3’ and 5’-UUUCUCAAACAAAUCGAAGCdTdT-3’. Prior to harvest, cells were fixed in absolute methanol for 10 min. Then, cells were incubated with 5% albumin for 20 min to block non-specific binding of antibodies. A mixture of antibodies against LAMP1 1:200 (ab62562) and LC3B 1:200 (GT3612) was applied and further incubated overnight at 4 °C. After rinsing with PBS, the samples were then incubated with the relevant secondary antibodies for 1 h at room temperature: donkey anti-mouse 488 and donkey anti-rabbit 594 (1:100; Jackson ImmunoResearch, PA, USA). And DAPI was used for nucleus staining. 9

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Finally, immunostaining of LC3 (green puncta) and LAMP1 (red puncta) were visualized in 20 well-stained and randomly selected cells per group by confocal fluorescence microscopy. Yellow

fluorescent

puncta

indicate

autolysosomes

as

LC3

signal

representing

autophagosomes and LAMP1 signal representing autophagosomes.

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2.9. Statistical analysis

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The data were analyzed employing Student's unpaired t-test for two groups and one-way ANOVA for multiple comparisons in SPSS 20.0. Data was expressed as mean ±

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standard deviation (SD). And the difference between groups was considered significant when

3. Results 3.1. Cell viability

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P value < 0.05.

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To examine the cytotoxicity of rifampicin and rotenone in human microglia cells (HM cells), Cell Counting Kit-8 (CCK-8) assay was used to detect the viability of HM cells. As shown in Fig. 1A, the treatment with rotenone for 24 h reduced cell viability in a dose-dependent manner. Treatment with 0.1 μM rotenone (Rot) reduced cell viability to 61.9 ± 3.1% (P < 0.05). Rifampicin (Rif) did not reduce cell viability at the concentration of 25 μM, 50 μM, and 100 μM, but decreased cell viability at 200 μM (*P < 0.05: vs. Con). Thus, we used 0.1 μM rotenone and 100 μM rifampicin in subsequent experiments.

3.2. Rifampicin’s anti-inflammatory effect in HM cells treated with rotenone was 10

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compromised by lysosome acidity inhibitor Real-time PCR was used to detect mRNA level of IL-1β and IL-6 (Fig. 2A, B). HM cells were either treated or not treated with 40 μM chloroquine (CQ) for 4 h. Then HM cells were either pretreated or not pretreated with 100 μM rifampicin for 2 h, before exposure to 0.1 μM rotenone for another 24 h. Treatment with rotenone significantly enhanced the expression of IL-1β and IL-6 (*P < 0.05 vs. Con), while pretreatment with rifampicin for 2 h attenuated the

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levels of these cytokines (#P < 0.05 vs. Rot.). As shown in Fig. 2A and Fig. 2B, treatment with

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40 μM chloroquine alone, the level of IL-1β and IL-6 was not significantly different from the

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control group (P > 0.05). However, chloroquine significantly enhanced the levels of IL-1β and

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IL6 in HM cells treated with rifampicin followed by rotenone (CQ + Rif + Rot vs. Rif + Rot, ##

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P < 0.05)). These results indicated the anti-inflammatory effect of rifampicin was weakened

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by chloroquine, a lysosome inhibitor.

The MAPKS signaling pathway takes part in regulating the production of

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pro-inflammatory cytokines. Here, we explored the expression of molecular markers related to the activation of inflammatory pathways in HM cells exposed to rotenone for 6h, in the presence/absence of rifampicin or autophagy inhibitors. As shown in Fig. 2C and Fig. 2D, rotenone significantly increased the phosphorylation of JNK in microglia compared to the control, which was prevented by the pretreatment of rifampicin (Rif+Rot). However, the inhibitory effect of rifampicin on the p-JNK expression in rotenone-treated HM cells (Rif+Rot) was reversed by chloroquine (CQ+Rif+Rot). Interestingly, neither rotenone nor rifampicin influenced the phosphorylation of ERK and P38. Taken together, our results indicated that the anti-inflammatory effect of rifampicin was related to autophagy flux. *P < 11

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0.05: vs. Con. #P < 0.05: vs. Rot. ## P < 0.05: vs. Rif + Rot. NS: not significant.

3.3. Rifampicin improves autophagy flux in rotenone-treated HM cells We detected the ratio of LC3-II/LC3-I by western-blot to analyze autophagy flux. As shown in Fig. 3A, rifampicin enhanced the ratio of LC3-II/LC3-I when compared to control

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group (P < 0.05). Pretreatment with chloroquine for 4 h before treatment with rifampicin

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further increased the ratio of LC3-II/LC3-I comparing to treatment with rifampicin alone (P < 0.05), implying that rifampicin could enhance autophagy rather than block the degradation

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of autophagosomes in HM cells. Rotenone increased the ratio of LC3-II/LC3-I when

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compared to control group (P < 0.05). However, no difference was seen in the conversion

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from LC3-I to LC3-II between Rot group and CQ + Rot group (P > 0.05), suggesting that

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rotenone impaired autophagy flux in HM cells (Fig. 3B). To examine whether rifampicin improved autophagy flux in rotenone-induced HM cells, cells were either treated or not

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treated with rifampicin for 2 h followed by treatment with rotenone for 24 h. As shown in Fig. 3C, rotenone markedly increased the ratio of LC3-II/LC3-I in HM cells (P < 0.05). However, this increase could be inhibited by pretreatment with rifampicin (P < 0.05). To further confirm whether rifampicin modulates autophagic dynamics, we transfected HM cells with the tandem RFP-GFP-LC3 lentivirus. Then the transfected cells were either pre-incubated or not pre-incubated with rifampicin for 2 h, followed by exposure to rotenone for 24 h. As shown in Fig. 3D, both green GFP-LC3 and red RFP-LC3 signals were mostly diffuse in control cells. Whereas rotenone significantly increased the number of yellow (merge of GFP- and RFP-fluorescent) dots per cell with little change of the number of 12

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red-only dots, thus suggesting that rotenone significantly increased autophagosomes (P < 0.05, Rot vs. Con), but not autolysosomes, when compared to control group. However, pretreatment with rifampicin markedly reduced autophagosomes and increased autolysosomes in rotenone-induced HM cells (P < 0.05, Rif + Rot vs. Rot). Taken together, these results indicated that rifampicin improved maturation of autolysosomes in HM cells treated with

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rotenone. Scale bar: 10 μm.

3.4. Rifampicin enhances expression of ATP6V0A1 in rotenone-induced HM cells.

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In Fig. 3, we demonstrated that rotenone induced autophagosome accumulation, while

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rifampicin significantly decreased this accumulation and increased autolysosomes in

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rotenone-induced HM cells. However, the anti-inflammatory effect of rifampicin was

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weakened by chloroquine, a lysosome acidity inhibitor. Lysosomal function is a critical component in the lysosomal degradation pathway. This prompted us to examine whether

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rifampicin improved autophagy flux by improving lysosome function in rotenone-induced HM cells. V-ATPase has been shown to be involved in maintaining lysosome function, which is composed of V0 domain and V1 domain, containing at least 13 subunits. Therefore, we investigated the effects of rotenone on mRNA expression of V-ATPase by PCR. As shown in Fig. 4A, we noted that only ATP6V0A1 expression was decreased among the 23 genes of V-ATPase in rotenone-induced HM cells. To examine the effects of rifampicin on the expression of ATP6V0A1 in rotenone-induced HM cells, PCR and western-blot were used to detect the expression level of ATP6V0A1. We found the gene and protein expression levels of ATP6V0A1 significantly 13

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increased in HM cells with rifampicin pre-conditioning followed by exposure to rotenone compared to those with rotenone alone (Fig. 4B, C). These data suggested that rifampicin enhanced expression of ATP6V0A1 in rotenone-induced microglia. *P < 0.05: vs. Con. #P < 0.05: vs. Rot.

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3.5 ATP6V0A1 silencing suppresses the effect of rifampicin on preserving lysosome acidity

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in rotenone-treated HM cells.

Lysosomal function is a critical component in the lysosomal degradation pathway. Since

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LysoTrackerTM Red DND-99 can accumulate within acidic vesicles and emit red

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fluorescence, we used this dye to examine the lysosomal function of HM cells. As shown in

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Fig. 5B, the fluorescent intensity of LysoTrackerTM Red staining was significantly decreased

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in rotenone-induced HM cells (*P < 0.05, vs. Con), indicating lysosomal function was clearly damaged. However, pretreatment with rifampicin markedly enhanced the fluorescent intensity

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of LysoTrackerTM Red staining (#P < 0.05, vs. Rot). Using LysoSensor yellow/blue DND-160 (a lysosomal pH probe) to detect the lysosomal acidity, we demonstrated that rotenone significantly increased lysosomal pH, whereas rifampicin markedly suppressed the increase of lysosomal pH, suggesting that rifampicin prevented injuries to lysosomal function from rotenone (Fig. 5C). To investigate whether rifampicin maintains lysosome acidity via enhancing ATP6V0A1 expression in rotenone-treated HM cells, siRNA-mediated knockdown of ATP6V0A1 was used. As shown in Fig. 5A, the expression of ATP6V0A1 was significantly reduced in cells after transfection of siRNA-ATP6V0A1 compared to treatment with 14

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scrambled siRNA control.

Then HM cells with ATP6V0A1 knockdown were either

pretreated with or without rifampicin for 2 h, followed by exposure to rotenone for 24 h. LysoTrackerTM Red DND-99 and LysoSensor yellow/blue DND-160 was used to detect the lysosome acidity. After knockdown of ATP6V0A1, LysoTracker Red DND-99 accumulation (Fig. 5B) and

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the change of fluorescent signal from yellow to blue in LysoSensor staining (Fig. 5C) in

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rotenone-treated HM cells were indistinguishable from those pretreated with rifampicin (P>0.05, siRNA-ATP6V0A1+Rif+Rot vs. siRNA-ATP6V0A1+Rot). The results above

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indicate that rifampicin improved lysosome acidity in rotenone-induced HM cells partly via

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ATP6V0A1. Scale bar: 10 μm.

rotenone-treated HM cells.

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3.6. ATP6V0A1 silencing suppressed rifampicin-mediated autophagosome-lysosome fusion in

lysosomes.

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Recent studies showed that ATP6V0A1 mediated fusion between phagosomes and To

investigate

the

function

of

ATP6V0A1

in

rifampicin-mediated

autophagosome-lysosome fusion in HM cells exposed to rotenone, morphometric analysis was performed at the end of different treatments. The colocalization of LC3B with lysosomal maker LAMP1 indicated the fusion of autophagosomes with lysosomes. As shown in Fig. 6A, pretreatment with rifampicin increased the level of autolysosomes in rotenone- induced HM cells. However, after ATP6V0A1 silencing, pretreatment with rifampicin was unable to increase the level of autolysosomes in rotenone-induced HM cells. These results suggest that ATP6V0A1 silencing suppressed rifampicin-mediated autophagosome-lysosome fusion in 15

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rotenone-induced HM cells. Western-blot is employed to detect the level of LC3-II/LC3-I to further analyze the function of ATP6V0A1 in autophagy flux. As shown in Fig. 6B, rotenone increased the level of LC3-II/LC3-I in HM cells (P < 0.05, vs. Con group), but pretreatment with rifampicin was able to reverse this increase (#P < 0.05, Rif + Rot vs. Rot). However, after ATP6V0A1

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silencing, pretreatment with rifampicin could not suppress the increase in the ratio of

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LC3-II/LC3-I induced by rotenone (P > 0.05, siRNA + Rif + Rot vs. siRNA + Rot). These results suggest that ATP6V0A1 silencing suppressed rifampicin-mediated autophagy flux in

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rotenone-induced HM cells. siRNA: short interfering RNA

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4. Discussion

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In this study, we demonstrated that rifampicin attenuated microglia inflammation via improving autophagy flux and lysosomal function in rotenone-induced PD inflammatory

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model. First, we found that rifampicin suppressed the secretion of inflammatory factors in rotenone-treated HM cells; however, the anti-inflammation effect of rifampicin was inhibited by

autophagy

inhibitor.

Then,

we

demonstrated

that

rifampicin

increased

autophagosome-lysosome fusion and improved autophagy flux in microglia exposed to rotenone using mRFP-GFP-LC3 dual fluorescent indicator. Finally, we found that rifampicin improved lysosome acidification and autophagosome-lysosome fusion by increasing ATP6V0A1 expression in rotenone-treated microglia. Numerous evidences have indicated that microglial inflammation plays an important role in the pathogenesis of PD via promoting the degeneration of dopaminergic neurons. Clinical 16

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research discovered that activated microglia existed in substantia nigra(McGeer et al., 1988). Compared to normal people, there was high level of inflammatory molecules, such as interleukins, interferon gamma (IFN-γ) and tumor necrosis factor-α (TNF-α) in blood and cerebrospinal fluid(Bartels and Leenders, 2007). Synthesis and release of pro-inflammatory factors can be regulated by mitogen-activated protein kinases (MAPKs) signal pathways,

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which might be activated by various environmental stresses including radiation, reactive

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oxygen species (ROS) and environmental toxicity. Rotenone, a mitochondrial complex I inhibitor, is one of the environmental toxins participating in the pathogenesis of PD. Gao et al.

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demonstrated that the environmental toxin rotenone can directly activate microglia through

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the P38 pathway(Gao et al., 2013). Zhou et al. reported that rotenone can induced microglial

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inflammation via disrupting mitochondrial membrane potential and increasing P38/JNK

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activation in microglia(Zhou et al., 2008). Furthermore, accumulating studies have supported that modulation of microglia inflammation may provide a novel therapeutic target for PD. Our

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previous studies reported that rifampicin enhanced cell viability of SH-SY5Y cells co-cultured with BV2 microglial cells via suppressing the release of cytokines(Liang et al., 2017). Furthermore, accumulating studies reports support that modulation of microglia inflammation may provide a novel therapeutic target for PD. Our previous studies reported that rifampicin enhanced cell viability of SH-SY5Y cells co-cultured with BV2 microglial cells via suppressing the release of cytokines(Liang et al., 2017). Here we found that rotenone activated JNK and increased gene expression of inflammatory factors. While rifampicin inhibited the activation of JNK, thereby reducing the production of inflammatory cytokines in rotenone-treated HM cells. 17

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Based on the experimental results that autophagy inhibitor (chloroquine) blocked the anti-inflammatory effect of rifampicin, it is rational to hypothesize that rifampicin may inhibit rotenone-induced microglial inflammation by enhancing autophagy. Recently, studies revealed that autophagy takes part in regulating the activation of immune cell and release of cytokines(Su et al., 2016). It has been reported that the depletion of autophagic proteins LC3 beclin

1

enhanced

NLRP3-dependent

inflammation

in

LPS-induced

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and

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macrophages(Nakahira et al., 2011). Activation of autophagy, induced by rapamycin, suppressed the expression of iNOS, IL-6 and cell death in LPS-stimulated BV2 cells(Han et

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al., 2013). Moreover, autophagy impairment-related microglia inflammation may be

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implicated in the pathogenesis of PD(Plaza-Zabala et al., 2017). Bussi et al. found that

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autophagy downregulated pro-inflammatory mediators in both LPS and alpha-synuclein

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induced BV2 microglial cells(Bussi et al., 2017). We also previously found that rifampicin could scavenge damaged mitochondria by enhancing autophagy in microglia exposed to

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rotenone. Thus, further exploration are required to clarify the exact mechanism through which rifampicin promote autophagy in microglia. Autophagy is responsible for maintaining the quality and balance of cytoplasmic material. Isolation membranes package cellular contents to form autophagosomes. Once completed, autophagosomes fuse with the lysosomes to form autolysosomes for degradation. LC3 participates in the entire process of autophagy, which makes it an important marker of autophagy flux. LC3-I attaches to lipid phosphatidylethanolamine to form LC3-II, which is recruited to autophagosomal membrane. When the autophagosome was degraded by lysosome, the LC3-II in autophagosomal lumen is degraded simultaneously. Therefore, detecting LC3 18

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conversion (LC3-I to LC3-II) has become a reliable method for monitoring autophagy. Since both autophagy induction and autophagy flux blockage can enhance the ratio of LC3-II/ LC3-I, it is necessary to add lysosomal acidification inhibitor to distinguish these two situations. With the help of chloroquine, our results indicated that the increased ratio of LC3-II/ LC3-I in rotenone-treated HM cells resulted from the impaired degradation of

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cells treated with rotenone by promoting autophagy flux.

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autophagic vesicles, and rifampicin pretreatment reduced the ratio of LC3-II/ LC3-I in HM

Autophagosomes can fuse with lysosomes to form autolysosomes, in which lysosome's

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hydrolases degrade the autophagosome-delivered components. Studies showed that

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impairment of the procedure of autolysosome maturation, resulted by abnormal expression of

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membrane fusion proteins, such as ATP6V0A1, Qa-SNARE syntaxin 17 (STX17) and Ectopic

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P granules protein 5 (EPG5), could impair the autophagy flux(Bagh et al., 2017)(Nakamura and Yoshimori, 2017). Fluorescent-tagged LC3 (RFP-GFP-LC3B) is an efficient and

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convenient assay to detect the number of autophagosomes and monitor autophagy flux(Kimura et al., 2007). In this study, using RFP-GFP-LC3B transfected cells to observe the autophagy flux, we demonstrated that rotenone impaired the maturation of autolysosomes in RFP-GFP-LC3B transfected HM cells, while pretreatment with rifampicin increased the level of autolysosomes in HM cells exposed to rotenone. Our results indicate that rifampicin inhibited microglia inflammation via improving maturation of autolysosomes in rotenone-treated HM cells. Both the impairment of autophagosome-lysosome fusion and lysosomal acidification leads to inhibition of autophagy flux. V-ATPase composes of V0 domain (membrane 19

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associated subunits) and V1 domain (peripherally associated subunits), containing at least 13 subunits. The V0 domain takes part in translocating protons across the membrane. The V1 domain is responsible for hydrolyzing ATP to supply the energy for translocating protons. Studies demonstrated that the A1 subunit of the V0 domain (ATP6V0A1) participates in lysosomal acidification and membrane fusion of vesicles. Here we demonstrated that

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rifampicin increases the expression of ATP6V0A1 in rotenone-treated microglia. Thus, we

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further investigated whether rifampicin improved maturation of autolysosomes in rotenone-treated microglia via increasing the expression of ATP6V0A1.

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Lysosomal acidification, a critical step of the autophagic process, controls the maturation

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of lysosomal hydrolases and the modification of certain cargoes delivered to further their

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biological use(Colacurcio and Nixon, 2016). The optimal lumen’s pH (pH4.5-5.0) is

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important for the digestive function of lysosomes. Several studies have reported that lysosomal acidification dysfunction existed in PD model, such as rotenone-induced PD model

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and TNF-α treated PC12 cells(Wu et al., 2015)(Wang et al., 2015). Bourdenx et al. demonstrated that the use of poly (DL-lactide-co-glycolide) (PLGA) acidic nanoparticles (aNP) restored impaired lysosomal acidification and autophagy flux in a series of toxin and genetic cellular models of PD(Bourdenx et al., 2016). Lysosomal acidity is maintained by V-ATPase (vacuolar ATPase) transporting protons across lysosome membranes to lysosome(Mindell, 2012). Studies demonstrated that ATP6V0A1 participates in lysosomal acidification. The decreases of ATP6V0A1 in lysosomes caused lysosomal acidity dysfunction in Cln1-/- mice, a neurodegenerative lysosomal storage disease model(Bagh et al., 2017). Our results demonstrated that rifampicin improved lysosomal acidity in 20

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rotenone-treated HM cells via increasing expression of ATP6V0A1. Autophagosomes fuse with lysosomes to form the single-membraned autolysosomes, which plays a key role in autophagosomes degradation system. Boland et al. showed that inhibition of the microtubule-mediated fusion of autophagosomes and lysosomes by vinblastine is associated with Alzheimer’s disease(Boland et al., 2008). Studies demonstrated

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that ATP6V0A1 participates in membrane fusion of vesicles. Peri et al. showed that

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ATP6V0A1 mediated fusion between phagosomes and lysosomes during phagocytosis in microglia of zebrafish, while knockdown of ATP6V0A1 suppressed digestion of dying

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neurons by microglia(Peri and Nusslein-Volhard, 2008). Another research demonstrated that

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ATP6V0A1 knockdown blocked autophagosome-lysosome fusion, increasing autophagic cell

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death in GMI treatment Calu-1 cells(Hsin et al., 2012). In the current study, we found that

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knockdown of ATP6V0A1 suppressed rifampicin-mediated autophagosome-lysosome fusion, which indicated that rifampicin enhanced autophagosome-lysosome fusion via upregulating

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expression of ATP6V0A1 in rotenone-treated microglia. In conclusion, our study revealed that rifampicin attenuated rotenone-induced microglia inflammation via improving lysosomal function. Further studies are required to investigate the effects of rifampicin on autophagy flux and to determine the clinical application of rifampicin in neurodegenerative diseases. Acknowledgments This work was supported by National Natural Science Foundation of China (grant numbers 81503052, 81571244, 81771378, 81971195), Basic and Applied Basic Research Project of Guangdong Province (grant numbers 2016A030313322), Natural Science 21

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Foundation of Guangdong Province (grant numbers 2017A030313840，2017A030313459， 2018A0303130205), Fundamental Research Fund for University Youth Scholars (grant numbers 17ykpy39), Key Field Research and Development Program of Guangdong Province （grant numbers 2018B030337001, and Yat-Sen Scholarship for Young Scientist.

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Conflicts of interest

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There are no conflicts of interest.

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Figures Fig. 1 A

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B

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Fig. 1. Cytotoxicity effect of rotenone and rifampicin on HM cells. (A) Rotenone reduced cell

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viability in a dose-dependent manner. Cells were stimulated with rotenone at different

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concentrations (0 μM, 0.01 μM, 0.1 μM, 1 μM) for 24 h. (B) The effect of rifampicin on cell viability. Cells were treated with rifampicin at different concentrations, ranging from 0 μM to

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200 μM for 24 h. Cell viability was detected by CCK-8 assay. Each experiment was replicated at least 3 times. *P < 0.05: vs. Con.

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Fig. 2 B

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A

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C p-JNK

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JNK p-ERK ERK p-P38 P38 β-ACTIN

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Journal Pre-proof Fig. 2. Rifampicin’s anti-inflammatory effect in rotenone-treated HM cells was compromised by lysosome acidity inhibitor. (A, B) The anti-inflammatory effect of rifampicin was weakened by chloroquine. Cells were either treated or not treated with 40 μM chloroquine for 4 h. Then HM cells were either pretreated or not pretreated with 100 μM rifampicin for 2 h, before treatment with 0.1 μM rotenone for 24 h. IL-1β and IL-6 mRNA level in culture media were detected by PCR. (C) Rifampicin inhibited rotenone-treated inflammation via

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suppressing the activation of JNK. HM cells were either treated or not treated with 40 μM

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chloroquine for 4 h. Then HM cells were either pretreated or not pretreated with 100 μM

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rifampicin for 2 h, before treatment with 0.1 μM rotenone for 6 h. Cells were lysed, and

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p-JNK, JNK, p-ERK, ERK, p-P38, and P38 were examined by western blot. Each experiment

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NS: not significant.

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was replicated for 3 times. *P < 0.05: vs. Con. #P < 0.05: vs. Rot. ##P < 0.05: vs. Rif + Rot.

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Fig. 3

A

B

C

LC3-I LC3-II

LC3-I LC3-II

β-ACTIN

β-ACTIN

β-ACTIN

D

GFP

MERGE

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RFP

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LC3-I LC3-II

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Con

Rot

Rif

Rif+Rot

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Fig. 3. Rifampicin improved autophagy flux in rotenone-treated HM cells. (A) Rifampicin enhanced autophagy in HM cells. Cells were either treated or not treated with 40 μM chloroquine for 4 h, followed by treatment with rifampicin (100 μM) for 24 h. *P < 0.05: vs. Con. #P < 0.05: vs. Rif. (B) Rotenone impaired autophagy flux in HM cells. Cells were either treated or not treated with 40 μM chloroquine for 4 h, followed by treatment with rotenone

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(0.1 μM) for 24 h. *P < 0.05: vs. Con. NS: not significant. (C) Rifampicin improved autophagy flux in rotenone-treated HM cells. Cells were either treated or not treated with 100

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μM rifampicin for 2 h, followed by treatment with rotenone (0.1 μM) for 24 h. At the end of

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different treatments，the ratio of LC3-II/LC3-I was examined by western blot and the signals

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from the corresponding bands were quantitated using Image J software. Values are the

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means±SD of three independent experiments. *P < 0.05: vs. Con. #P < 0.05: vs. Rot. (D)

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Autophagy dynamics was analyzed in HM cells transfected with RFP-GFP-LC3 lentivirus. After transfection of RFP-GFP-LC3 lentivirus, HM cells were either pretreated or not

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pretreated with rifampicin for 2 h, followed by exposure to rotenone. Then the cells were observed under a laser scanning confocal fluorescence microscope. Representative images showing GFP-LC3 and mRFP-LC3 puncta in transfected cells. As GFP rather than mRFP is sensitive to lysosomal pH, GFP fluorescence gets quenched in the acidic autolysosomes. Yellow arrows indicate autophagosomes (GFP+/mRFP+, yellow puncta) and red arrows are indicative of autolysosomes (GFP-/mRFP+, red puncta). Each experiment was replicated for 3 times. N=20 cells / per group. Scale bar: 10 μm. *P < 0.05: vs. Con. #P < 0.05: vs. Rot. +: nonspecific staining. RFP: red fluorescent protein. GFP: green fluorescent protein.

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A

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Fig. 4

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B

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C

ATP6V0A1 β-ACTIN

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Fig. 4. Rifampicin enhances expression of ATP6V0A1 in rotenone-treated HM cells. (A) ATP6V0A1 expression was decreased among the 23 genes of V-ATPase in rotenone-induced HM cells. Cells were treated with rotenone (0.1 μM) for 24 h. RNA samples were extracted from HM cells, and the mRNA expression level of V-ATPase was detected by PCR. (B, C) Rifampicin enhanced expression of ATP6V0A1 in rotenone-treated HM cells. Rifampicin improved autophagy flux in rotenone-treated HM cells. Cells were either treated or not treated

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with 100 μM rifampicin for 2 h, followed by treatment with rotenone (0.1 μM) for 24 h. ATP6V0A1 expression level was examined by PCR and western blot. Each experiment was

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replicated for 3 times. *P < 0.05: vs. Con. #P < 0.05: vs. Rot.

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Fig. 5

A

B Con

ATP6V0A1

Rif

Rot

Rif+Rot

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β-ACTIN

siRNA

NC

Rif+Rot

Rot

Rif+Rot

C

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D

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Rot

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Con

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Rif

Rot

NC Rot

Rif+Rot

Rif+Rot

siRNA Rot

Rif+Rot

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Fig. 5. Rifampicin maintains lysosome acidification in HM cells exposed to rotenone partially via ATP6V0A1. (A) The silencing effect of siRNA against ATP6V0A1 was confirmed by western blot. HM cells with ATP6V0A1 knockdown were either pretreated or not pretreated with rifampicin (100 μM) for 2 h, followed by treatment of rotenone (0.1 μM) for 24 h. (B) LysoTrackerTM Red DND-99 and LysoSensor yellow/blue DND-160 was used to detect the

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lysosome acidification. (C) Quantitation of red fluorescent intensity after LysoTrackerTM

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Red staining was performed by Image-J. Data = mean±SD, n = 20 cells per group. *P < 0.05: vs. Con. #P < 0.05: vs. Rot. ## < 0.05: vs. NC + Rot. NS: not significant. (D) Representative

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images of lysosomes in HM cells stained with LysoSensor yellow/blue DND-160. The

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fluctuation of lysosome PH can be measured by the change of fluorescence signal, as the

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presence of blue and yellow signals indicate neutral and more acidic environment in

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lysosomes, respectively. Each experiment was replicated for 3 times.

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Fig. 6 A LC3B

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LAMP1

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MERGE

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NC

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LC3-I LC3-II

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β-ACTIN

NC

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siRNA

siRNA

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Fig. 6. ATP6V0A1 silencing suppressed rifampicin-mediated autophagosome-lysosome fusion in rotenone-treated HM cells. (A) HM cell with ATP6V0A1 knockdown were exposed to

rotenone

(0.1

μM)

with

or

without

rifampicin

(100

μM)

pretreatment.

Autophagosome-lysosome fusion was analyzed by immunofluorescence assay. Representative images showing colocalization of autophagosomal marker (LC3, green) and lysosomal

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marker (LAMP1, red) in HM cells. ATP6V0A1 silencing suppressed the increase of

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rifampicin-mediated autolysosome in rotenone-treated HM cells. (B) Autophagy flux was analyzed by detection of LC3-II conversion using western blot. Data are presented as

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mean±SD. from 3 independent experiments. One-way ANOVA was carried out for the

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analysis. #P < 0.05 vs. Rot, ##P < 0.05 vs. negative control siRNA+Rot. NS: not significant.

Gene

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Table. 1 Primer sequences

Primer sequence

5’-GGTTCTGAATCAAACGGAGGAT-3’

ATP6V0A1-R

5’-GCAAACTGGATGGAGTCAAGGT-3’

ATP6V0A2-F

5’-CTATCCAAACACAGCCGAG-3’

ATP6V0A2-R

5’-TTTGTGCAGTACAGTGTAGAG-3’

ATP6V0A3-F

5’-GAGACCTCAACGAATCCGTGA-3’

ATP6V0A3-R

5’-CGATCCGTTTCCTCCTGGA-3’

ATP6V0A4-F

5’-ATGTTTGGAGACTGTGGTC-3’

ATP6V0A4-R

5’-GTTCCAAATCTCATTGTCTGTC-3’

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ATP6V0A1-F

ATP6V0B-F 33

5’-ACTTCGCCCTTCATGTGGTC-3’

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5’-CAATGATGGAGGAGCCGGTAA-3’

ATP6V0C-F

5’-ATCATCCCAGTGGTCATGG-3’

ATP6V0C-R

5’-GGAGGAAGCTCTTGTAGAGG-3’

ATP6V0D1-F

5’-CCATCCGCTAGGCAGCTTT-3’

ATP6V0D1-R

5’-ATCAAGGTCCTGCTCTGAGAT-3’

ATP6V0D2-F

5’-TTCTTGAGTTTGAGGCCGAC-3’

ATP6V0D2-R

5’-CAGCTTGAGCTAACAACCGC-3’

ATP6V0E1-F

5’-TTGTGATGAGCGTGTTCTG-3’

ATP6V0E1-R

5’-CCAACATGGTAATGATAACTCCC-3’

ATP6V0E2-F

5’-CATTCGCCCTCCCGGTCAT-3’

ATP6V0E2-R

5’-TCACTCCGCGGTTGGGTC-3’

ATP6V1A-F

5’-GAGGAGTAAACGTGTCTGC-3’

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ATP6V0B-R

ATP6V1A-R

5’-TATGACTACCAACCCGTAGG-3’ 5’-TTGTTCAGGTGTTTGAAGGG-3’

ATP6V1B1-R

5’-GGAGTTCGTAGGATGTCCC-3’

ATP6V1B2-F

5’-TAGTTCAGGTATTTGAAGGGAC-3’

ATP6V1B2-R

5’-GGTGTTCGGAGAATATCCC-3’

ATP6V1C1-F

5’-GGCAGTTGATGACTTCAGACAC-3’

ATP6V1C1-R

5’-ACGTGAATCCATGCAATAAATG-3’

ATP6V1C2-F

5’-GAAGGCCAACCTGGAGAACT-3’

ATP6V1C2-R

5’-AAGCTTGACTTGGGGACGAT-3’

ATP6V1D-F

5’-GAGCGAAGAAAGATAATGTAGC-3’

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ATP6V1B1-F

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ATP6V1D-R

5’-TCAGTTCATAACTGTCAGTTCC-3’

ATP6V1E1-F

5’-GGCGCTCAGCGATGCAGATGT-3’

ATP6V1E1-R

5’-CAAGGCGACCTTTCTCAATG-3’

ATP6V1E2-F

5’-AGAGGCATACCTGGCTGTGA-3’

ATP6V1E2-R

5’-GAGATCCAGTCGGCTTTCCA-3’ 5’-CCTCATCAACCAGTACATCGCA-3’

ATP6V1F-R

5’-CGTCATATGGGTGCTCCTTGG-3’

ATP6V1G1-F

5’-AAAGAAAGAACCGGAGGCT-3’

ATP6V1G1-R

5’-TACTGTTCAATTTCAGCCTGAG-3’

ATP6V1G2-F

5’-AGCAGAGAAACCGAGAGCG-3’

ATP6V1G2-R

5’-GGCAGAAATCCGGTAGTTGGG-3’

ATP6V1G3-F

5’-CAATCTAAGATAATGGGCTCTCAG-3’

na

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ATP6V1F-F

ATP6V1G3-R

5’-GTCCATTAAGTTCTTGTATCTTCCC-3’ 5’-CTTTCTGCCAATGTTGAATCGC-3’

ATP6V1H-R

5’-GTCACTGCCTTCCATCAGTTC-3’

β-ACTIN-F

5’-TGTCCACCTTCCAGCAGATGT-3’

β-ACTIN-R

5’-AGCTCAGTAACAGTCCGCCTAG-3’

IL-1β-F

5’-ATGATGGCTTATTACAGTGGCAA-3’

IL-1β-R

5’-GTCGGAGATTCGTAGCTGGA-3’

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ATP6V1H-F

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IL-6-F

5’-ACTCACCTCTTCAGAACGAATTG-3’

IL-6-R

5’-CCATCTTTGGAAGGTTCAGGTTG-3’

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Journal Pre-proof Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Corresponding author Enxiang Tao

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Highlights  Autophagy inhibitor attenuated rifampicin’s anti-inflammatory effect.  Rifampicin improved autophagy flux in rotenone-treated microglia.  Rifampicin enhanced expression of ATP6V0A1 in rotenone-treated microglia.

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 Rifampicin improved lysosomal function by increasing ATP6V0A1 expression.

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