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

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, p21Waf1/Cip1 (p21), β-galactosidase (β-gal), Rb phosphorylation, senescence-associated (SA) β-gal activity, and lysosomal activity, were examined. The dSH 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 α-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.


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][3][4][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 alphasynuclein (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][13][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.

Isolation and Culture of Primary Cortical
Neurons from E16 Rat Embryos. Pregnant rats were euthanized with CO 2 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 20 min in a water bath. Next, the cortices were incubated with 40 μg/mL DNase I (DN25, Sigma-Aldrich), vortexed gently, and incubated for 5 min in a 37°C 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, 2.5 mg/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 5 /well) were seeded in a 12-well plate (SPL30012, SPL Life Sciences, Pochoen, South Korea) and cultured in the growth medium at 37°C in a 5% CO 2 incubator for 24 h. The culture medium was replaced with growth medium supplemented with 0.2 mg/mL of 5-fluoro-2 ′ -deoxyuridine (F0503, Sigma-Aldrich) and 96 μg/mL of uridine (U3750, Sigma-Aldrich) to inhibit mitosis. On day 6, the cells were treated with 1 μM rotenone, 1 μM GSK-KI, and 70 nM α-syn fibrils for 48 h and harvested with 1x sample buffer.

Proximity
Ligation Assay (PLA) and Immunofluorescence (IF). PLA was performed using Duolink® In Situ Detection Reagents Green (DUO92014-100RXN, Sigma-Aldrich), Duolink® In Situ PLA® 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, Kremsmünster, 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 37°C for 1 h. The blocking solution was removed, and the cells were 2 Oxidative Medicine and Cellular Longevity incubated with antibodies (50 μL) at 37°C for 3 h. The antibody mixture was comprised of an antibody diluent from the kit (antibodies used for PLA: mouse anti-LRRK2 monoclonal (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. 2.5. Immunoprecipitation. The whole-cell lysates of dSH cells treated with rotenone (1 μM) and GSK-KI (1 μM) were prepared using lysis solution (PBS, 1% Triton X-100, 1x protease inhibitor cocktail, and 1x phosphatase inhibitor cocktail). The lysates were centrifuged at 4,000g and 4°C for 5 min, and the supernatant was incubated with the anti-p53 antibody (554293, BD Biosciences) and Pierce™ Protein G Agarose (20398, Thermo Fisher Scientific) resuspended in the lysis solution for 12 h. The beads were washed twice with the lysis solution, and the bead-protein complex was denatured with 1x sample buffer.
2.6. Nuclear Fractionation. The nuclear fraction was isolated using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (78833, Thermo Fisher Scientific), following the manufacturer's instructions. μM Hoechst 33342 for examining SA β-gal activity, ROS, active lysosomes, and nucleus, respectively. The stained cells were mounted with ProLong™ Diamond Antifade Mountant (P36965, Invitrogen) and imaged using a confocal laser scanning microscope. Differential interference contrast images were obtained using a confocal laser scanning microscope.

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/6J male mice aged 16 weeks were intraperitoneally injected with rotenone (0.75 mg/kg bodyweight) and MLi-2 (an LRRK2 kinase inhibitor; 1 mg/kg bodyweight) 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 Ca 2+ and Mg 2+ . The midbrain was dissected with microscissors and tweezers. The tissues were lysed in PBS containing 1% Triton X-100, 1x Xpert Protease Inhibitor Cocktail Solution (P3100, GenDEPOT, Katy, TX, USA), and 1x 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.  Oxidative Medicine and Cellular Longevity Multi Gauge (Fujifilm, 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 staining, and PLA in the dSH cells were analyzed using twoway 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.   [16,19]. However, the rates of apoptosis were higher than those of other cellular senescence-associated phenotypes in these studies due to the use of high rotenone concentration. Hence, the cells were treated with a low rotenone concentration (1 μM) for a , 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 nucleus were examined using western blotting. LaminB was used for the normalization of nuclear p53 levels. n = 3. (f) The mRNA levels of p21 and p16 in the treatment group were normalized to those in the vehicle-(dimethyl sulfoxide-) treated group. n = 3.   Oxidative Medicine and Cellular Longevity prolonged duration (48 h) as reported previously [15]. Rotenone upregulated the LRRK2 levels. However, cotreatment with rotenone and GSK-KI did not result in the upregulation of LRRK2 levels (Figures 1(a) and 1(b)). Treatment with GSK-KI mitigated the rotenone-induced S1292 phosphorylation of LRRK2 (Figures 1(a)-1(c)). Similarly, treatment with GSK-KI nonsignificantly mitigated the rotenone-induced upregulation of p53 levels (Figures 1(a) and 1(d)). LRRK2      Oxidative Medicine and Cellular Longevity has been reported to phosphorylate p53 at the TXR site [20]. Treatment with rotenone enhanced the nuclear localization of p53, which was mitigated upon cotreatment with GSK-KI (Figures 2(a)-2(e)). Additionally, GSK-KI mitigated the rotenone-induced upregulation of p21 (Figures 1(a) and 1(e)). Rotenone downregulated the phosphorylation of Rb, which is involved in the last stage of the senescent p53-p21 pathway. Treatment with GSK-KI mitigated the rotenoneinduced phosphorylation of Rb (Figures 1(a) and 1(f)). As the p53-p21 pathway is not the only signaling pathway mediating senescence, the mRNA levels of p21 and p16 were examined. Treatment with GSK-KI mitigated the rotenone-induced upregulation of p21. The p16 mRNA levels were not significantly different between rotenonetreated and rotenone/GSK-KI-treated groups (Figure 2(f )). Rotenone upregulated the activity of β-gal, a senescence marker, which was mitigated upon cotreatment with GSK-KI (Figures 1(a) and 1(g)). Moreover, the results of PLA of pS1292 LRRK2 and total LRRK2 in the dSH cells revealed that rotenone increased the β-gal levels and the LRRK2 kinase activity (Figures 1(h)-1(j)). These results indicate that rotenone promotes cellular senescence through the p53, p21, Rb, and β-gal pathways by upregulating the LRRK2 levels and its kinase activity and that LRRK2 inhibition mitigated rotenone-induced cellular senescence in the dSH cells.

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Oxidative Medicine and Cellular Longevity β-gal activity ( Figure 5(j)) in the rat primary cortical neurons as those observed in the dSH cells. Thus, rotenone-induced cellular senescence promotes the upregulation of LRRK2 kinase and impairs autophagy-lysosomal activities, which leads to enhanced α-syn aggregation.

LRRK2 Kinase Inhibition Alleviates Rotenone-Induced
Cellular Senescence in the Mouse Midbrain. To verify the in vitro findings, an in vivo assay was performed. Mice were intraperitoneally injected with rotenone (0.75 mg/kg bodyweight) and MLi-2 (1 mg/kg bodyweight), a blood-brain barrier-permeable LRRK2 kinase inhibitor, once every two days for two weeks (Figure 6(a)). In experiments performed using high rotenone concentrations, apoptosis was the predominant cellular senescence-associated phenotype. Hence, the dSH cells and rat primary neurons were treated with low concentrations of rotenone for a prolonged duration. Rotenone nonsignificantly decreased the locomotor activity of mice as evidenced by the decreased time of falling from the rotarod (Figure 6(b)). However, rotenone upregulated the SA β-gal activity in the midbrain, which was significantly mitigated upon cotreatment with MLi-2 ( Figure 6(c)). Additionally, treatment with MLi-2 nonsignificantly mitigated the rotenone-induced activation of the LRRK2 kinase and p53-p21 pathway in the midbrain (Figures 6(d) and 6(e)). Furthermore, rotenone significantly upregulated the levels of βgal, which was mitigated upon cotreatment with MLi-2. ELISA was performed to measure the levels of fibrillar αsyn oligomers in the midbrain lysate [17]. Treatment with MLi-2 mitigated the rotenone-induced upregulation of fibrillar α-syn oligomers ( Figure 5(f)). Thus, the intraperitoneal administration of low-dose rotenone promoted cellular senescence in the mouse midbrain, which resulted in α-syn aggregation in the midbrain. Treatment with LRRK2 kinase inhibitor mitigated rotenone-induced cellular senescence in the midbrain.

Discussion
The mechanism underlying cellular senescence in the dopaminergic neurons has not been elucidated. Aging is a risk factor for PD. Hence, the correlation between aging and cellular senescence must be considered. Recent studies have suggested that PD-associated cellular senescence is related to the loss of cellular functions [14,22,23], such as the loss of LRRK2 function in the neurons resulting from deregulation of the autophagy-lysosomal pathway [24,25]. Previously, we had demonstrated that the G2019S LRRK2 mutant impaired autophagy in a mouse dopaminergic cell line by phosphorylating leucine-tRNA synthetase [26]. An impaired autophagy-lysosomal pathway promotes α-syn aggregation [11,27]. Hence, LRRK2 activity may mediate α-syn aggregation. The inhibition or prevention of α-syn aggregation using an anti-α-syn antibody is a potential therapeutic strategy for PD [28][29][30]. However, the anti-α-syn antibody did not exert a marked therapeutic effect on PD in humans. This may be due to the incomplete clearance of α-syn aggregates, including fibrils, protofibrils, and toxic oligomers. Conformationspecific antibodies for α-syn oligomers may not clear all toxic or LB-prone oligomers in the brain of patients with PD. LRRK2 kinase inhibition can activate the autophagylysosomal pathway and promote α-syn clearance. Hence, the coadministration of an LRRK2 kinase inhibitor and an anti-α-syn antibody exerts a synergistic therapeutic effect on PD.

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Oxidative Medicine and Cellular Longevity The p53-p21 pathway is involved in several cellular processes, including apoptosis and senescence [31]. The phosphorylation of Rb is the gating step of the cellular senescence signaling pathway [32]. Additionally, the activation of p16 expression mediates cellular senescence [33]. The expression levels of p16 and p21 are upregulated in the dorsal root ganglion neurons of cisplatin-injected mice [34]. Additionally, postmortem analysis of patients with amyotrophic lateral sclerosis/motor neuron disease revealed that the expression of p16 and p21 was upregulated in the astrocytes of the frontal associated cortex. However, the neurons exhibited upregulation of p21 with undetectable levels of p16 [35]. The findings of this study indicated that cellular senescence was mainly mediated by the p53-p21 pathway rather than the upregulated p16 (Figures 1, 2, and 4). Inflammatory responses are also associated with senescence. Astrocytes are involved in neuroinflammation in the brain [36]. Astrocyte senescence contributes to the progression of cognitive abnormalities [37]. Thus, neuroinflammation may be related to senescence in astrocytes. Future studies must investigate the correlation between neuroinflammation and cellular senescence and its distinct signaling pathways.
In this study, rotenone upregulated LRRK2 kinase activity. However, this study did not demonstrate the effect of the LRRK2 kinase inhibitor on the G2019S mutant. Cellular senescence was examined after the transfection of the G2019S LRRK2 mutant in the dSH cells and rat primary neurons. The prolonged expression of G2019S LRRK2 in the dSH cells and rat primary neurons significantly increased β-gal levels and SA β-gal activity. Treatment of G2019S LRRK2-transfected cells with the LRRK2 kinase inhibitor or the expression of D1994A LRRK2 did not upregulate the βgal levels and SA β-gal activity (Supplementary Figure 2). Previously, we had reported that the levels of β-gal and αsyn aggregates in the brain lysate of 24-week-old G2019Sexpressing mice were higher than those in the age-matched littermates [10]. Treatment with an LRRK2 inhibitor did not markedly mitigate low-dose rotenone-induced cellular senescence in mice. However, pharmacological advances may enhance the therapeutic effect of LRRK2 kinase inhibitors in mice ( Figure 6). Future studies must focus on improving the pharmacological effects and safety profile of LRRK2 kinase inhibitors. The administration of an LRRK2 kinase inhibitor can serve as a feasible therapeutic strategy for PD with high LRRK2 kinase activity as it can mitigate oxidative stress or the toxic effects of LRRK2 mutants (Figure 7). LRRK2 kinase activity in the human biofluids, such as the serum, cerebrospinal fluid, and urine can serve as a prognostic biomarker to determine the optimal treatment for patients with PD exhibiting enhanced LRRK2 kinase activity.

Conclusions
The findings of this study suggest that treatment with LRRK2 kinase inhibitors mitigates cellular senescence induced by mild and long-term dosing of rotenone. This study demonstrated the critical role of LRRK2 in the p53-p21 pathway, which mediates cellular senescence, and provided novel insights for developing PD therapy using LRRK2 kinase inhibitors.

Data Availability
The datasets generated and/or analyzed during the present study are available from the corresponding authors upon reasonable request.

Ethical Approval
All animal experiments were performed according to the guidelines of the Dankook Animal Ethics Committee (Dankook IACUC, 18-026).

Conflicts of Interest
The authors declare no competing interests.