LRRK2 Kinase Inhibitor Rejuvenates Oxidative Stress-Induced Cellular Senescence in Neuronal Cells Part 1

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Background. Leucine-rich repeat kinase 2(LRRK2)plays a critical role in the pathogenesis of Parkinson's disease (PD). Aging is the most critical risk factor for the progression of PD. The correlation between aging and cellular senescence has been established. Cellular senescence is correlated with the dysregulation of the proteolytic pathway and mitochondrial dysfunction, which are also associated with the aggregation of α-synuclein (α-syn).Methods. Human dopaminergic neuron-like cells(differentiated SH-SY5Y cells)were treated with rotenone in the presence or absence of the LRRK2 kinase inhibitor GSK2578215A(GSK-KI) for 48 h. The markers of cellular senescence, including p53, p21Wafi/Cip1(p21),β-galactosidase(β-gal), Rb phosphorylation, senescence-associated (SA)β-gal activity, and lysosomal activity, were examined. The dash cells and rat primary cortical neurons were treated with α-syn fibrils 30 min before treatment with rotenone in the presence or absence of GSK-KI for 48 h. Mice were intraperitoneally injected with rotenone and MLi-2 (LRRK2 kinase inhibitor)once every two days for two weeks. Results. Rotenone upregulated LRRK2 phosphorylation and β-gal levels through the activation of the p53-p21 signaling axis and downregulated Rb phosphorylation. Additionally, rotenone upregulated SA β-gal activity, reactive oxygen species levels, and LRRK2 phosphorylation and inhibited lysosome activity. Rotenone-induced LRRK2 upregulation impaired the clearance of a-syn fibrils. Treatment with LRRK2 inhibitor mitigated rotenone-induced cellular senescence and α-syn accumulation. Conclusions. Rotenone-induced upregulation of LRRK2 kinase activity promoted cellular senescence, which enhanced α-syn accumulation. However, the administration of an LRRK2 kinase inhibitor rejuvenated rotenone-induced cellular senescence.

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1. Introduction

Parkinson's disease(PD), which is the most common neurodegenerative disease, is characterized by impaired motor control[1]. Several genetic and environmental factors contribute to the pathogenesis of PD[2-5].LRRK2, a major genetic risk factor for PD, exhibits both GTPase and kinase activities [6]. The LRRK2 G2019S mutant, which exhibits enhanced kinase activity, promotes PD progression [7]. The risk of developing PD in patients harboring the G2019S mutant increases with age [8] as aging is associated with the progression of neurodegenerative diseases [9]. Previously, we had reported that LRRK2 kinase activity promoted cellular senescence and inhibited the degradation of alpha-synuclein (α-syn) aggregates [10].α-Syn is a major component of Lewy bodies (LB)or Lewy neurites, which are the postmortem markers of PD. Impaired degradation of α-syn results in its aggregation [11].

Cellular senescence impairs the protein degradation machinery[12-14]. Exposure to low-dose rotenone, which is reported to induce PD, promotes cellular senescence in the human trabecular meshwork cell line [15]. Additionally, rotenone upregulates LRRK2 kinase activity in the neurons [16]. Therefore, we hypothesized that rotenone promotes cellular senescence through the activation of LRRK2 kinase and consequently enhances α-syn aggregation.

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2. Methods and Materials

2.1. Cell Culture and Treatment.

The human neuroblastoma cell line(SH-SY5Y cells) was cultured in a growth medium (Dulbecco's modified Eagle's medium (DMEM;10-013-CV; Corning cellgro, Thermo Fisher Scientific, Waltham, MA, USA)supplemented with 10% fetal bovine serum (FBS, 35-010-CV, Corning cellgro),1% penicillin/streptomycin(P/S, 10378-016, Gibco, Thermo Fisher Scientific), and 1% mycoplasma removal reagent(KOMA)) at 37°Cin a 5%CO, incubator (Thermo Fisher Scientific). The differentiated SH-SY5Y cells(dSH cells) were obtained after treatment with 10μM all-trans retinoic acid （R2625-100MG， Sigma-Aldrich， St. Louis, MO, USA)prepared in a growth medium once every two days for seven days. On day 7 post retinoid acid treatment, the cells were cotreated with rotenone(1 uM) and GSK2572815A（GSK-KI; LRRK2 kinase inhibitor;1μM）pre-pared in a growth medium for 48 h. Dimethyl sulfoxide (DMSO) was used as a vehicle for rotenone and GSK-KI. The α-syn fibrils were purified as described previously [10]. The rotenone- and GSK-KI-treated cells were treated with a-syn fibrils (70nM). The rat primary cortical neurons were treated with rotenone（1μM）， GSK-KI（l μM）， and a-syn fibril(70nM)for 48h. The treatment conditions for the rat primary cortical neurons were identical to those employed for dSH. The cells were washed twice with ice-cold phosphate-buffered saline(PBS) and lysed in 1x sample buffer(50mM Tris-HCl(pH6.8),2% sodium dodecyl sulfate, 10% glycerol,1% β-mercaptoethanol, and 0.02% bromophenol blue).

2.2.Isolation and Culture of Primary Cortical Neurons from E16 Rat Embryos.

Pregnant rats were euthanized with CO, gas. The uterus was dissected, and the fetuses from the embryonic sacs were placed in ice-cold HBSS-/-(14175-079, Gibco, Thermo Fisher Scientific). The skull was peeled using forceps, and the cortices were dissected from the whole brain. After the removal of the meninges, the cortices were incubated with 0.25% Trypsin-EDTA(25200-056, Gibco, Thermo Fisher Scientific) at 37℃C for 20min in a water bath. Next， the cortices were incubated with 40μg/mL DNase I (DN25, Sigma-Aldrich), vortexed gently, and incubated for 5min in a 37℃ water bath. Most of the supernatant was removed by suction, and the samples were incubated with a serum inhibitor-containing MEM(11090-08,Gibco, Thermo Fisher Scientific),1.5% DMEM,5% FBS,2.5mg/mL bovine serum albumin (BSA; A7906, Sigma-Aldrich), and 2.5 mg/mL trypsin inhibitor(T9253, Sigma-Aldrich). The serum inhibitor was removed, and the cell pellet was resuspended in 10 times its volume with the growth medium. The composition of the growth medium was as follows: neurobasal medium(21103-049, Corning cellgro, Thermo Fisher Scientific),2% B-27(17504044, Corning cellgro, Thermo Fisher Scientific), and 1x GlutaMAX-1(35050-061, Corning cellgro, Thermo Fisher Scientific). The isolated rat primary cortical neuron cells(3×10°/well) were seeded in a 12-well plate(SPL30012, SPL Life Sciences, Pochoen, South Korea)and cultured in the growth medium at 37°Cin a 5% CO, incuba-tor for 24h. The culture medium was replaced with growth medium supplemented with 0.2mg/mL of 5-fluoro-2'-deoxyuridine(F0503, Sigma-Aldrich)and 96ug/mL of uridine(U3750, Sigma-Aldrich)to inhibit mitosis. On day 6, the cells were treated with 1uM rotenone,1uM GSK-KI, and 70nM α-syn fibrils for 48h and harvested with lx sample buffer.

2.3. Western Blot.

Western blotting was performed as described previously [17]. The following antibodies were used for the western blotting: rabbit anti-LRRK2 phospho-S935 monoclonal(ab133450,Abcam, Cambridge,UK),rab-bit anti-LRRK2 phospho-S1292 monoclonal (ab203181, Abcam),mouse anti-LRRK2 monoclonal (N241A/34, Neu-roMab,UC Davis, CA, USA), mouse anti-p53 monoclonal (for human p53:sc-126; for mouse p53:sc-393031;Santa Cruz Biotechnology, Dallas, TX, USA),mouse anti-p21wafi/-Ccip1(p21) monoclonal(CM5131, ECM Biosciences,Ver-sailles, KY, USA), mouse anti-Rb monoclonal(#9309S, Cell Signaling Technology(CST), Danvers,MA, USA), anti-phosphoRb(Ser807/811)(#9308S, CST),mouse anti-β-galactosidase monoclonal (sc-377257,Santa Cruz Biotech-nology),mouse anti-p62 monoclonal(ab56416,Abcam), mouse anti-β-actin monoclonal(sc-47778; Santa Cruz Bio-technology),rabbit anti-LC3B polyclonal(#2775S; CST), mouse anti-α-syn (clone 42)monoclonal(610786; BD Bio-sciences, San Jose, CA, USA), anti-phospho-threonine-argi-nine(#2351S, CST), anti-p53(SC-99, Santa Cruz Biotechnology),anti-LaminB(SC-6216,Santa Cruz Biotech-nology), goat peroxidase-conjugated AffiniPure anti-mouse IgG(H+L)(#115-035-003;Jackson Immunoresearch Labo-ratories Inc., West Grove, PA, USA), and goat peroxidase-conjugated AffiniPure anti-rabbit IgG(H+L)(#1l1-035-144; Jackson Immunoresearch Laboratories Inc.)antibodies. Immunoreactive signals in the nitrocellulose membrane were developed with Luminata Crescendo Western HRP (#WBLUR0500, Merck & Co. Inc.,Kenilworth, NJ, USA), and the images were captured with a MicroChemi4.2 camera (Shimadzu, Kyoto, Japan).

2.4.Proximity Ligation Assay (PLA)and Immunofluorescence (IF).

PLA was performed using Duolink⑨ In Situ Detection Reagents Green (DUO92014-100RXN, Sigma-Aldrich), DuolinkeIn in Situ PLA四Probe Anti-Mouse MINUS antibody (DUO92002-100RXN, Sigma-Aldrich), and Anti-Rabbit Plus antibody (DUO92004-100RXN, Sigma-Aldrich), following the manufacturer's instructions. The SH-SY5Y cells seeded in 96-well black plates(655077, Greiner Bio-One, Krems-münster, Austria)were differentiated, and the differentiated cells were treated with rotenone and GSK-KI on day 7. The cells were rinsed twice with ice-cold Dulbecco's PBS(DPBS), fixed with 4% paraformaldehyde for 30 min at room temperature(RT), and rinsed thrice with ice-cold DPBS. Next, the cells were permeabilized using 0.1% Triton X-100 prepared in DPBS. After washing thrice with cold DPBS, the cells were incubated with a drop of blocking solution at 37C for 1h. The blocking solution was removed, and the cells were incubated with antibodies（50μL） at 37℃ for 3h. The anti-body mixture was comprised of an antibody diluent from the kit (antibodies used for PLA: mouse anti-LRRK2 mono-clonal(1:20) antibody and rabbit anti-LRRK2 phospho-S1292 monoclonal(1:20) antibody; antibody used for the IF assay: chicken anti-β-gal polyclonal antibody(1:400, ab9361, Abcam)).In the IF assay, the antibody was added throughout the entire process until the PLA application step. The samples were then washed twice with 1x wash buffer A for5min.To prepare the probe solution, the Duolink⑧In Situ PLA· Probe Anti-Mouse Minus and Anti-Rabbit Plus were mixed with the antibody diluent(1:5). For the IF assay, the anti-β-gal antibody was also added(1:400) to the mixture. The cells were incubated with 50μL of the probe solution at 37C for 1h. Next, the cells were washed twice with lx wash buffer A for 5min. For the IF assay, ligase(40:1)and anti-β-gal antibody(400:1)were mixed in the ligation buffer. The cells were incubated with the ligation solution（50μL） at 37°C for 30min and washed twice with lx wash buffer A for 5min. For the IF assay, polymerase(1:40)was incubated with Goat Anti-Chicken IgY H+L(Alexa Fluor② 594; ab150172)secondary antibody(1:500)for 100min at 37C. The cells were washed twice with 1x wash buffer B for 10 min, followed by washing with 0.01x wash buffer B for 1min. To stain the nucleus, the cells were incubated with Hoechst 33342(1:1000;62249, Thermo Fisher Scientific) prepared in DPBS at RT for 10min. The images of the cells in the 96-well black plates were captured using a confocal laser scanning microscope (LSM 700, Carl Zeiss Jena, Germany).

2.5.Immunoprecipitation.

The whole-cell lysates of dSHcells treated with rotenone（1 μM） and GSK-KI （1 uM） were pre-pared using lysis solution(PBS,1% Triton X-100,1x protease inhibitor cocktail, and lx phosphatase inhibitor cocktail). The lysates were centrifuged at 4,000g and 4°℃ for 5min, and the supernatant was incubated with the anti-p53 antibody(554293, BD Biosciences) and PierceProtein G Aga-rose(20398, Thermo Fisher Scientific) resuspended in the lysis solution for 12h. The beads were washed twice with the lysis solution, and the bead-protein complex was denatured with lx sample buffer.

2.6. Nuclear Fractionation.

The nuclear fraction was isolated using NE-PERr Nuclear and Cytoplasmic Extraction Reagents(78833, Thermo Fisher Scientific), following the manufacturer's instructions.

2.7. mRNA Preparation and Quantitative Real-Time Polymerase Chain Reaction(qRT-PCR).

mRNA was isolated from rotenone/GSK-KI-treated dSH cells using the RNeasy Plus Mini kit(74134, QIAGEN, Hilden, Germany). Next, mRNA was reverse transcribed into complementary DNA (cDNA) using the TOPscript cDNA Synthesis Kit (EZ005S, Enzynomics, Daejeon, Republic of Korea).cDNA was subjected to qRT-PCR using TOPreal"qPCR 2x Pre-MIX(SYBR Green with low ROX, UDG plus)(RT500M, Enzynomics). The following primers were used for qRT-PCR: human p21,5'-ATG AAA TTC ACC CCC TTT CC-3'(forward)) and 5'-CCC TAG GCT GTG CTC ACT TC-3'(reverse); human p16INK4(p16),5'-CCC AAC GCA CCG AAT AGT TAC-3'(forward) and 5'-CAC GGG TCG GGT GAG AGT-3'(reverse).qRT-PCR was performed on the Mic qPCR cycler(MIC-4,BioMolecular Systems,Upper Coomera, Australia).

2.8. Measurement of Senescence-Associated (SA)Galactosidase (β-Gal) Activity, Reactive Oxygen Species (ROS), and Lysosome Activity.

At 30min posttreatment, live dSH cells were stained with 2μM GlycoGREEN-βGal (GC611, Goryo Chemical Inc., Hokkaido, Japan),5uM 2',7'-dichlorofluorescein diacetate(DCFDA;287810, Calbio-chem， San Diego， CA， USA），2μM LysoSensorm Blue DND-167 (L7533, Invitrogen, Carlsbad, CA, USA), and 1 μM Hoechst 33342 for examining SA β-gal activity， ROS， active lysosomes, and nucleus, respectively. The stained cells were mounted with ProLongDiamond Antifade Mountant (P36965, Invitrogen)and imaged using a confocal laser scanning microscope. Differential interference contrast images were obtained using a confocal laser scanning microscope. 2.9.SA β-Gal Activity Assay. The SAβ-gal activity was examined using a 96-well cellular senescence assay kit(CBA-213, Cell Biolabs Inc., San Diego, CA, USA), following the manufacturer's instructions.

2.10.Mouse Handling and Drug Administration.

Mice were handled as described previously [18] according to the guidelines of the Dankook Animal Ethics Committee(Dankook IACUC,18-026).C57BL/6] male mice aged 16 weeks were intraperitoneally injected with rotenone (0.75 mg/kg body-weight) and MLi-2 (an LRRK2 kinase inhibitor; 1mg/kg body weight) once every two days for two weeks.

2.11.Rotarod Test and Brain Tissue Preparation.

On day 14 posttreatment, the mice were placed on a rotatable cylinder-shaped rod. The time required to fall to the floor was recorded. The details of the rotarod test are described elsewhere [18]. Next, the mice were euthanized and transcardially perfused with ice-cold HBSS containing Ca2 and Mg. The midbrain was dissected with micro scissors and tweezers. The tissues were lysed in PBS containing 1% Triton X-100,1xXpert Protease Inhibitor Cocktail Solution(P3100, GenDEPOT, Katy, TX, USA), and lx Xpert Phosphatase Inhibitor Cocktail Solution(P3200,GenDEPOT). The samples were homogenized using a Pellet Pestle Cordless Motor (Sigma-Aldrich). The supernatant was subjected to western blotting, SA β-gal activity assay, and enzyme-linked immunosorbent assay (ELISA).

2.12.ELISA for Oligomeric α-Syn.

Previously, we had established a sandwich ELISA for the analysis of fibrillar α-syn oligomers using an antibody that recognizes the filamentous conformation of α-syn aggregates [17]. The crude midbrain lysates were subjected to ELISA, and the α-syn aggregates were quantified using α-syn fibril standards.

2.13. Image and Data Analyses.

The intensities of PLA and live-cell staining were measured using Zen 2012(Carl Zeiss). Densitometry of the target proteins was performed using

FiGURE 1:Rotenone-induced LRRK2 kinase activation promotes cellular senescence in the differentiated human neuroblastoma cel line. Western blotting analysis of dSH cells treated with 1 uM rotenone or 1 uM GSK2578215A(GSK-K), an LRRK2 kinase inhibitor, for 48 h using antibodies against the target proteins(a). The densities of the proteins were normalized to those of β-actin (b-g).n =3.(h) dSH cells treated with 1 uM rotenone or 1uM GSK-KI for 48h were subjected to proximity ligation assay (PLA) of phospho-S1292 LRRK2 and total IRRK2(PIA-pS1292)and immunofluorescence (TF) analysis of β-galactosidase, Nuclei were stained with Hoechst 33342.The controls used in PLA and IF staining revealed the validity of the results (right two panels). The intensities of PLA-pS1292(i) and β-galactosidase (j) were normalized to those of 4',6-diamidino-2-phenylindole.n= 4; number of cells= 12-17.

Multi Gauge(Fujilm, Tokyo, Japan).All datasets were analyzed and graphed using Prism 8(GraphPad Software, San Diego, CA, USA).Data from experiments performed using dSH cells and rat primary cortical neurons were analyzed using two-way analysis of variance (ANOVA), followed by Bonferroni's post hoc test(n=3). Meanwhile, the data obtained from mouse experiments, live-cell stain-ing,and PLA in the dSH cells were analyzed using two-way ANOVA, followed by Tukey's post hoc test (n>3). All data are represented as mean ± standard error of mean. *p<0.05,**p<0.01,***p<0.001,***p<0.0001,and n.s. = not significant.

FIGURE 2: LRRK kinase inhibitor inhibits the activation of p53 in the differentiated human neuroblastoma cell line. (a)Whole-cell lysates (20%), which were used as the input of immunoprecipitation(IP), and nuclear fraction were collected. The analysis revealed that 20%input exhibited results similar to those observed in Figure 1. (b, c) The IP samples were denatured and subjected to western blotting. The TXR site phosphorylation levels in p53 were normalized to total p53 levels. n=3.(d-e)Total p53 levels in the nuceus were examined using western blotting. LaminB was used for the normalization of nuclear p53 levels. n=3.(f) The mRNA levels of p21 and pl6 in the treatment group were normalized to those in the vehicle-(dimethyl sulfoxide-) treated group,n =3.

This article is extracted from Hindawi Oxidative Medicine and Cellular Longevity Volume 2021, Article ID 9969842, 16 pages https://doi.org/10.1155/2021/9969842