LRRK2 regulates ferroptosis through the system Xc‐GSH–GPX4 pathway in the neuroinflammatory mechanism of Parkinson's disease

Parkinson's disease (PD) is the most prevalent neurodegenerative disorder. Neuroinflammation mediated by activated microglia and apoptosis of dopaminergic (DA) neurons in the midbrain are its primary pathological manifestations. Leucine‐rich repeat protein kinase 2 (LRRK2) kinase has been observed to increase expression during neuroinflammation, however, the effect of LRRK2 on microglia activation remains poorly understood. In this study, we have established lipopolysaccharide (LPS) treated BV2 cells and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) models for both in vivo and in vitro investigation. Our data in vivo reveal that LRRK2 can promote microglia activation by regulating ferroptosis and activating nuclear factor‐κB. Inhibition of LRRK2 expression effectively suppressed the LPS‐induced pro‐inflammatory cytokines and facilitated the secretion of neuroprotective factors. Importantly, by co‐overexpressing LRRK2 and glutathione peroxidase 4 (GPX4), we identified the system Xc‐GSH–GPX4 pathway as a crucial component in LRRK2‐mediated microglial ferroptosis and inflammatory responses. Using a microglial culture supernatant (MCS) transfer model, we found that inhibiting LRRK2 or downregulating ferroptosis in BV2 cells prevented SH‐SY5Y cell apoptosis. Additionally, we observed abundant expression of LRRK2 and P‐P65 in the midbrain, which was elevated in the MPTP‐induced PD model, along with microglia activation. LRRK2 and P‐P65 expression inhibition with PF‐06447475 attenuated microglia activation in the nigrostriatal dense part of MPTP‐treated mice. Based on our findings, it is evident that LRRK2 plays a critical role in promoting the neuroinflammatory response during the pathogenesis of PD by regulating the system Xc‐GSH–GPX4 pathway. Taken together, our data highlights the potential research and therapeutic value of targeting LRRK2 to regulate neuroinflammatory response in PD through ferroptosis.

Parkinson's disease (PD) is an epidemic and rapidly expanding neurodegenerative disease with an estimated number of more than 10 million cases worldwide, according to data from the Global Burden of Disease Study (GDP 2016Neurology Collaborator, 2019).The hallmark of PD is the progressive loss of dopaminergic (DA) neurons in the nigrostriatal, accompanied by the formation of intracellular inclusions called Lewy bodies (Belarbi et al., 2020).Several factors, including alpha-synuclein (α-syn) protein misfolding, mitochondrial impairment, autophagy-lysosome dysfunction, and endoplasmic reticulum stress, have been associated in the apoptosis of DA neurons in PD (Bossy-Wetzel et al., 2004;Michel et al., 2016).
Despite extensive research, the pathogenesis of PD remains incompletely understood.Neuroinflammation has emerged as a crucial factor in the progression of PD (McGeer et al., 1988).
Microglia, the resident macrophage population in the brain, play a key role in sensing, monitoring, and modulating central neuroinflammation (De Virgilio et al., 2016;Prinz et al., 2011), thereby influencing the course of the disease.Activation of microglia due to various pathological stimuli is a crucial factor in the development of PD.
Studies have shown that microbial components such as lipopolysaccharide (LPS) and dsRNA can trigger microglial activation and downstream signaling pathways, resulting in a surge of proinflammatory cytokines, increased oxidative stress, and, ultimately, apoptosis of DA neurons (Kawai & Akira, 2010).In Hexb -/-mice with Sandhoff disease, microglial activation and increased proinflammatory factors were observed in the nigrostriatal region, preceding the onset of neurodegeneration and neuronal loss (Wada et al., 2000).Accordingly, targeting microglial activation represents a promising approach for elucidating the pathogenic mechanisms of PD-associated neuroinflammation and alleviating disease progression.For example, in animal models of PD, vitamin D has been shown to reduce microglial activation and enhance the production of antiinflammatory factors such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) (Kim et al., 2006).
Leucine-rich repeat protein kinase 2 (LRRK2) is the most commonly mutated gene in idiopathic and familial PD.LRRK2 protein is a 280 KD large molecular weight protein with multiple enzymebinding and protein-binding regions, suggesting that it plays a complex and critical role in regulating cellular function (Schapansky et al., 2015).
High expression of LRRK2 kinase is present in human B cells, T cells, macrophages, and other immune cells in the peripheral immune system.Its levels are strictly influenced by pathogenic microorganisms in the surrounding environment (Cook et al., 2017;Kuss et al., 2014).Furthermore, LRRK2 knockout mice show reduced tolerance to Mycobacterium tuberculosis and Salmonella typhimurium infections, indicating a critical role of LRRK2 in controlling the immune response (Liu et al., 2017;Zhang et al., 2015).Studies targeting the central nervous system have demonstrated that the LRRK2 G2385R mutation increases LRRK2 kinase activity in the brain, leading to inflammation and oxidative stress (Lee et al., 2010).Therefore, LRRK2 is likely involved in the mechanism of microglia neuroinflammation in PD.
LRRK2 mutation is the most common etiology of familial PD.
Notably, the G2385R variant is a common PD risk allele in Asian populations, and individuals, harboring this mutation exhibit elevated levels of LRRK2 kinase and pro-inflammatory factors in the brain, in addition to displaying levodopa-responsive Parkinson's syndrome at an early stage, Lewy body formation, neurofibrillary tangles, and mild neuroinflammation on histopathological examination (Tezuka et al., 2022).Similarly, It is reported that the G2019S mutation can increase LRRK2 kinase activity and aggravate neuronal death, while carriers of the G2019S mutation show elevated levels of peripheral pro-inflammatory cytokines (Gardet et al., 2010).Thus, LRRK2 is hypothesized to participate in the intrinsic regulation of microglia activation and neuroinflammation, as research has demonstrated that LPS treatment elicits robust LRRK2 expression in the mouse substantia nigra (Brockmann et al., 2016), LRRK2 kinase levels and pro-inflammatory cytokine expression are amplified in microglia of LRRK2-R1441G knock-in mice (Gillardon et al., 2012;Higashi et al., 2009;Lee et al., 2008).In a specific mechanism, LRRK2 is thought to regulate microglia activation through CX3CR1-mediated signaling pathways and phosphorylation of P53 (Muda et al., 2014).
Toll-like receptor-4 (TLR4) and TLR2 appear to be equally contributing factors to LRRK2-mediated activation of primary microglia (Moehle et al., 2012;Schapansky et al., 2014).In contrast, treatment with PF-06447475, the specificity of LRRK2 kinase inhibitors, is predicted to attenuate neuroinflammation and neurodegeneration in animals by promoting autophagy (Daher et al., 2015;Russo et al., 2019).Nonetheless, the precise mechanisms by which augmented levels of LRRK2 kinase lead to microglia activation and neuroinflammation remain elusive.
Ferroptosis is a form of iron-dependent cell death characterized by dysregulation of the intracellular oxidative stress response.The accumulation of iron and reactive oxygen species (ROS) in microglia releases inflammatory mediators.This process is primarily driven by impaired cysteine synthesis, which system Xc transports, and reduced glutathione (GSH), leading to the glutathione peroxidase 4 (GPX4) inactivation.In general, system Xc consists of SLC3A2 and SLC7A11 dimers embedded in the membrane surface.It can transport cystine into the cell while transporting glutamate to the outside, and synthesize GSH with the participation of glycine.Finally, GPX4 can convert toxic PL-PUPU-OOH into nontoxic PL-PUPU-OH with the help of GSH.Therefore, inactivation of system Xc or GPX4 causes oxidative stress and ferroptosis, On the other hand, excessive Fe 2+ , which is mediated by transferrin receptor and reduced by STEAP3, can also lead to ferroptosis (Jiang et al., 2021).Therefore, targeting ferroptosis in microglia regulation may represent a novel therapeutic strategy for PD (Subhramanyam et al., 2019).Although the investigation of ferroptosis in inflammation, particularly in microglia-mediated neuroinflammation, is still in its early stages of development (Proneth & Conrad, 2019), prior research has demonstrated that GPX4 deficiency in the brain or kidney leads to activation of astrocytes, microglia, or macrophages (Chen et al., 2015;Hambright et al., 2017).Erastin and its analogs are inhibitors of system Xc, which can lead to glutathione (GSH) depletion by blocking cystine transport across the membrane and ultimately triggering ferroptosis (Xie et al., 2016;Yang & Stockwell, 2008).In contrast, ferrostatin-1 and deferoxamine can inhibit ferroptosis by reducing intracellular iron levels and thus decreasing the release of inflammatory factors (such as IL-33) into the circulation (Martin-Sanchez et al., 2017).Another ferroptosis resistance factor is ferroptosis suppressor protein 1 (FSP1), which can be recruited to the cell membrane and acts as a reductor of coenzyme Q10 (CoQ10), enhance the lipophilic radical trapping function of CoQ10, act as an antioxidant, and prevent the diffusion of ROS (Bersuker et al., 2019).
However, the precise role of ferroptosis in pathogenesis of PD neuroinflammation and whether ferroptosis is involved in microglia activation remains unclear.Therefore, our study establishes a direct association between LRRK2 and the system Xc-GSH-GPX4 signaling pathways in the neuroinflammation pathogenesis of PD in vivo and in vitro.

| Cell culture and treatment
Mammalian microglia BV2 and DA cell line SH-SY5Y were utilized to establish a neuroinflammatory cell model and investigate DA neurons' apoptosis, respectively.These cells were obtained from the Central Laboratory of the First Affiliated Hospital of Nanchang University (Nanchang, China).BV2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.Similarly, SH-SY5Y cells were cultured in DMEM/DF12 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.Both cell types were maintained at 37°C in a humidified incubator with 5% CO 2 .To induce neuroinflammation in BV2 cells, 1 µg/mL LPS was used.Moreover, 2 µg/mL PF-06447475 was utilized to inhibit BV2 cell LRRK2 kinase, while 10 µM erastin and 0.5 µM ferrostatin-1 were employed to exacerbate and inhibit ferroptosis in BV2 cells, respectively.The agents mentioned above were obtained from Sigma-Aldrich.

| Transfection
To overexpress LRRK2 and GPX4 in BV2 cells, LRRK2 (NM_025730) Mouse Tagged ORF Clone and GPX4 (BC106147) Mouse Tagged ORF Clone were procured from OriGene Technologies (Rockville).BV2 cells were seeded at a density of 1 × 10 5 cells per well in six-well plates 1 day before transfection.After overnight adherence, cells were subjected to transfection.The old medium was replaced with a medium containing overexpression plasmids, followed by the addition of transfection reagents as per the protocol provided with the Lipofectamine 2000 kit (Invitrogen).After ensuring thorough mixing, the medium was incubated with the cells.Subsequently, cell observation and medium reFer-1shment were conducted.The efficiency of transfection was assessed via Western blot analysis.Concurrently, the pCMV6-Entry Mammalian Expression Vector (Rockville) was acquired from OriGene Technologies (Rockville) as a control plasmid.

| Experimental animals and treatments
Seventy-nine 8-week-old male c57bl/6 mice were purchased from the Laboratory Animal Center of Nanchang University and housed in a controlled environment with standard food and water.The Nanchang University Ethics Committee approved animal care and procedures.After acclimation to the environment, the mice were subjected to molding and intraperitoneally injected with 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP)-HCl (30 mg/kg; Fer-1e base; Sigma) once daily for 5 days to induce Parkinson's-like symptoms.Control mice received equivalent saline injections.At specific times, mouse midbrain samples, including the substantia nigra densa (SNpc), were collected and stored at −80°C.PF-06447475 (20 mg/kg, twice daily) was intragastric administrated.Dosing commenced 2 days before intraperitoneal injection of MPTP-HCl and continued for 14 days to achieve the best inhibitory effect.In the final analysis, we employed a total of 63 mice for our study.Out of these, 21 mice were dedicated to behavior tests and immunohistochemistry, another 21 were designated for Western blot analysis, and the remaining 21 were utilized for RNA extraction and subsequent reverse-transcription quantitative real-time PCR (RT-qPCR) analysis.To ensure the reliability of the in vitro study, we employed a rigorous approach, conducting experiments with three replicates of three mice per experimental group (Supporting Information S1: Table S1).

| Mouse behavioral scoring
-1 point: reduced escape behavior, piloerection, yellowing and soiling of fur, hunched back, decreased voluntary activity; -2 points: symptoms as described for 1 point, with a noticeable reduction in voluntary activity, lethargy, and possible tremors or unstable gait; -4 points: symptoms as described for 2 points, with unstable gait, inability to walk straight, or rotational walking; -6 points: lateral recumbency, paralysis of one side's forelimb and/ or hindlimb, difficulty walking and feeding; -8 points: complete paralysis of one side's forelimb and/or hindlimb, spasticity of limbs, significant weight loss, inability to feed; -10 points: near death or death.Scores from 2 to 6 indicate successful modeling.

| Mouse behavioral experiments
In the rotarod test, mice were initially trained on the apparatus at a constant speed of 20 rotations per minute (rpm) for 5 min, then placed on an accelerating rod (4-40 rpm) for 300 s, and the time the mice remained on the accelerating rod (latency to fall) was recorded.
In the pole test, a thick wooden pole with a diameter of 1 cm and a length of 50 cm, topped with a 2 cm diameter wooden ball, was vertically fixed on a base.Mice were placed on the ball and allowed to climb down.The time from the release of the ball until the front paws of the mouse reached the bottom of the pole was recorded.Each mouse underwent three consecutive trials, with a 30-min interval between trials, and the average time during the data processing was recorded.

| RNA extraction and RT-qPCR analysis
The total RNA of samples was extracted using EZ-press RNA Purification Kit (EZBioscience).The RNA concentration and purity were subsequently determined by NanoDrop ND-2000 spectrophotometer (Thermo Fisher Science, Inc.).A PrimeScript™ RT reagent kit with gDNA Eraser (Takara) was employed to synthesize complementary DNA (cDNA) from 1 µg of total RNA.The obtained cDNA products were then diluted to 1:10 in ddH2O.The SYBR ® Premix Ex Taq™ II and a CFX96 real-time PCR system (Bio-Rad) were utilized for RT-qPCR.The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was employed as a control for normalization.The ΔΔCt method was utilized to compute and normalize the relative expression of each gene.

| Cell viability assay
Cell viability was analyzed by cell counting kit-8 (CCK-8) assay (C0038; Beyotime).Well-grown cells were seeded in 96-well plates at a density of 5 × 10 3 per well overnight.Subsequently, a culture medium containing CCK-8 solution was added to each well.After 2 h of incubation, the optical density (OD) values of each well were measured at a wavelength of 450 nm using a microplate reader.

| Cell surface CD40 detection
Cell surface CD40 expression levels under various treatment conditions were assessed using fluorescein isothiocyanate (FITC)conjugated anti-mouse CD40 Antibody (BioLegend; 124608).BV2 cells were seeded at a density of 2 × 10 5 cells per well in 12-well plates overnight, followed by treatment with LPS, PF-06447475, erastin, and ferrostatin-1.As per the manufacturer's instructions, 1 µL of FITC-conjugated anti-mouse CD40 antibody was added to each well and incubated with BV2 cells for 15 min.Cells were then washed twice with fetal bovine serum-phosphate-buffered saline (PBS) and analyzed for cell fluorescence using an LSRII flow cytometer (BD Biosciences).Data were processed using the FlowJo software (TreeStar).

| Iron assay
BV2 cells were seeded into 12-well plates at a density of 2 × 10 5 cells per well and incubated overnight.The cells were treated with LPS, PF-06447475, erastin, and ferrostatin-1, then lysis on a shaker for 2 h.The supernatant was collected for Fe 2+ concentration analysis using an iron assay kit (Sigma-Aldrich; MAK025) following the manufacturer's instructions.The absorbance was measured at 595 nm using a microplate reader.

| Assessment of ROS production
Intracellular ROS levels in BV2 cells under different treatment conditions were assessed using DCFH-DA (Sigma-Aldrich).BV2 cells were seeded in 12-well plates at a density of 2 × 10 5 cells per well overnight and treated with LPS, PF-06447475, erastin, and ferrostatin-1.DCFH-DA was diluted to a final concentration of 10 µM according to the manufacturer's instructions and incubated with BV2 cells for 30 min.The cells were harvested, washed twice with PBS, and analyzed for cell fluorescence using an LSRII cytometer (BD Biosciences).Data were analyzed using the FlowJo software program (TreeStar).

| GSH determination
BV2 cells were seeded at a density of 2 × 10 5 cells per well in 12-well plates and incubated overnight before treatment with LPS, PF-06447475, erastin, and ferrostatin-1.To detect intracellular GSH levels, the GSH and GSSG Assay Kit (S0053; Beyotime) was used according to the manufacturer's instructions.Briefly, after treatment, the cells were lysed, and the supernatant was used for GSH detection at a wavelength of 405 nm using a microplate reader.Through the standard curve to calculate samples glutathione, specifically, use the formula GSH = total glutathione − GSSG × 2 of GSH content in the samples.
Confocal microscopy (Leica) was used to capture immunostaining images.

| Immunohistochemistry
At 7 days after MPTP administration, mouse brain tissues were removed.Then, frozen sections were made and mounted on slides.DA neurons were identified using anti-TH primary antibody (25859-1-AP; Proteintech), biotinylated secondary antibody, and streptavidin ABC solution.The resulting images were taken with a confocal microscope (Leica).

| Microglial culture supernatant transfer model
To investigate the effect of microglial inflammatory response on DA neuron apoptosis under different treatment conditions, SH-SY5Y cells were fused and cultured in complete medium, while BV2 cells were exposed to LPS, PF-06447475, erastin, and ferrostatin-1.The culture supernatant of BV2 cells was collected, centrifuged to remove cell debris, and transferred to SH-SY5Y cells for 12 h to stimulate apoptosis and death.Subsequently, cells were harvested and washed three times using PBS buffer.

| Apoptosis analysis
In the microglial culture supernatant (MCS) transfer model, apoptosis of the SH-SY5Y cells was measured using an annexin V-FITC (V-FITC)/propidium iodide (PI) apoptosis detection kit (LiankeBio).The harvested cells were pelleted and resuspended in 1 µL V-FITC.Then, 1 µL PI staining solution was added to the cells, and they were incubated at room temperature in the dark for 10 min.Flow cytometry (BD Biosciences) determined the number of apoptotic cells.

| Statistical analysis
Statistical analysis was carried out with GraphPad Prism software.
The data are presented as mean ± SD.Student's t test was employed to compare the statistical significance of two independent groups.
One-way analysis of variance was used to determine differences between groups.A p < 0.05 was considered statistically significant.
For instance, the differences between the negative control group and the positive control group are denoted by an asterisk (*), while the distinctions between the positive control group and the experimental groups are indicated by a hash sign (#).

| LRRK2 is upregulated in LPS-stimulated BV2 cells
Microglia are known to express higher levels of LRRK2 than neuronal cells, and their kinase activity is significantly increased upon stimulation by pathological factors such as TLR2 and TLR4.In this study, we studied the expression of LRRK2 in BV2 cells using RT-qPCR and to compare LRRK2 levels between resting and activated microglia.To this end, we added LPS to the microglia culture medium at increasing concentrations (0, 0.1, 0.2, 0.5, and 1 µg/mL) and stimulated for 24 h, and found that the expression of LRRK2 was significantly induced in a dose-dependent manner (Figure 1a).We also stimulated BV2 cells with LPS (1 µg/mL) for various times (0, 1, 6, 12, and 24 h), and observed a time-dependent upregulation of LRRK2 expression levels, which became more prominent at 24 h (Figure 1b).
These results demonstrate that LPS-stimulated BV2 cells have significantly higher LRRK2 expression levels compared to controls.

| Inhibition of LRRK2 can effectively reduce LPS-induced activation of BV2 microglia
We have demonstrated an increased LRRK2 expression in LPSactivated BV2 cells, which implies that LRRK2 may play a crucial role in regulating microglial neuroinflammation.To investigate the potential of downregulating LRRK2 kinase activity in attenuating microglial activation and inflammatory factor release, we employed PF-06447475 to inhibit LRRK2 kinase activity.PF-06447475 is a potent and selective inhibitor of LRRK2 kinase, with excellent brain permeability and demonstrated efficacy in blocking rotenonemediated apoptosis and oxidative stress (Henderson et al., 2015;Kim et al., 2019;Magni et al., 2012).RT-qPCR was employed to assess the inhibition of LRRK2 transcription by PF-06447475 (Figure 2a), and the inhibitory effect of PF-06447475 on LRRK2 kinase protein was further confirmed by Western blot analysis (Figure 2b) and immunofluorescence staining (Figure 2c).These results demonstrate the potent inhibitory effect of PF-06447475 on LRRK2 kinase activity.The present study has confirmed the efficacy of PF-06447475 as an LRRK2 kinase inhibitor in BV2 cells.To further investigate the impact of LRRK2 inhibition on microglial cells, morphological alterations were examined.Upon LPS stimulation, an increase in cellular activation was observed, characterized by an enlargement of cell bodies, shortening of processes, and a transition to rounded or rod-shaped activated morphologies.This effect was notably attenuated following treatment with PF-06447475 (Figure 2d).Concurrently, changes in the expression of the microglial activation marker CD40 were observed, with an upregulation induced by LPS stimulation and a subsequent downregulation post PF-06447475 treatment (Figure 2e).Microglial over-activation can lead to neurotoxicity and inflammation by producing cytotoxic factors, including IL-6, TNF-α, and IL-1β (Block et al., 2007;Tan et al., 1999).Conversely, anti-inflammatory cytokines such as TGF-β has proven mediated inhibition of microglia activation (Kim et al., 2019).The balance between cytotoxic factors and antiinflammatory cytokines indicates the degree of microglial activation.
We subsequently assessed the impact of modulating LRRK2

| Ferroptosis occurs in LPS-stimulated BV2 cells
Ferroptosis has been implicated in the pathogenesis of PD, but its mechanisms in microglia activation and neuroinflammation remain unclear.The system Xc-GSH-GPX4 pathway has been shown to play a crucial role in regulating the process of cellular ferroptosis (Lee et al., 2010).Erastin, a system Xc inhibitor that restricts cysteine input and leads to GSH depletion, can trigger ROS accumulation and GPX4 inactivation.Ferrostatin-1 is a potent non-apoptotic cellular ROS scavenger and antioxidant that effectively counteracts erastin-induced cellular ferroptosis (Zilka et al., 2017).In the present study, we found that treatment with erastin resulted in increased Fe 2+ levels (Figure 3a Further investigation revealed that the subunits of the system Xc transporter, SLC7A11 (Figure 3f,g), and the GPX4 protein (Figure 3f,h,j), as well as GSH levels (Figure 3d), were reduced in activated microglia.
To elucidate the role of the system Xc-GSH-GPX4 as a crucial mechanism regulating ferroptosis in BV2 cells, we overexpressed GPX4 and observed a decrease in cellular iron levels (Figure 3k), a reduction in oxidative stress markers ROS (Figure 3l,o) and 4-HNE (Figure 3m), accompanied by an enhancement in cell viability (Figure 3n).These findings underscore the significant pathway of system Xc-GSH-GPX4 in modulating ferroptosis.These findings suggest that the system Xc-GSH-GPX4 pathway mediates ferroptosis in BV2 microglia and that ferrostatin-1 mitigates the induced ferroptosis.

| Downregulation of ferroptosis could effectively attenuate P-P65 expression and BV2 activation
This study also demonstrated that nuclear factor-κB (NF-κB) is a key transcription factor that regulates the expression of inflammatory  (Cai et al., 2022).
While the involvement of ferroptosis in microglia activation and inflammatory factor release has been reported, the intermediate processes remain unclear.In this study, we observed a significant increase in the LRRK2 (Figure 4a-c) and expression of NF-κB p65 phosphorylation pattern (Figure 4d) in erastin-treated BV2 microglia.
Immunofluorescence staining further demonstrated an elevation in the expression level of P-P65 and its nuclear translocation (Figure 4h).Notably, ferrostatin-1 intervention effectively reversed the increase in P-P65 expression and the ensuing inflammatory response in BV2 cells.Similarly, an examination of microglial morphology and surface markers under conditions of ferroptosis activation and inhibition revealed an increase in activated phenotype cells following treatment with erastin (Figure 4i), accompanied by an elevation in CD40 expression (Figure 4j).Inhibition of ferroptosis was found to mitigate these changes.Additionally, RT-qPCR and ELISA analysis revealed that LPS-or erastin-treated BV2 microglia displayed upregulation of inflammatory cytokines, including IL-6 (Figure 4k,o), TNF-α (Figure 4l,p), and IL-1β (Figure 4m,q), and a significant downregulation of the anti-inflammatory cytokine TGF-β (Figure 4n,r).Collectively, our findings suggest that ferroptosis triggers activation and neuroinflammation in BV2 microglia by upregulating NF-κB P-P65 expression.

| Inhibition of LRRK2 downregulates ferroptosis and P-P65 expression
While previous research has shown that mutations in LRRK2 and increased levels of its protein kinase can impact microglia function and the release of inflammatory cytokines, with evidence suggesting that LRRK2 plays a crucial role in regulating microglia activation via vesicular transport, autophagy/lysosome, and other pathways, it remains unclear whether LRRK2 is involved in the process of ferroptosis (Chen et al., 2018;Cookson, 2016).Notably, our study revealed that treatment with PF-06447475 resulted in decreased Fe 2+ levels (Figure 5a), ROS formation (Figure 5b,c), 4-HNE (Figure 5e,g) and GSH (Figure 5d).The expression levels of SLC7A11 (Figure 5e,f) and GPX4 (Figure 5h-j Taken together, these results highlight the regulatory role of LRRK2 in ferroptosis and inflammation in BV2 microglia.

| Overexpression of GPX4 mitigates the ferroptosis and inflammatory response induced by the upregulation of LRRK2 protein
Previous findings have delineated that the elevated expression of LRRK2 significantly augments the ferroptotic response and neuroinflammatory levels in BV2 cells, impacting the activation of the system Xc-GSH-GPX4 pathway.This led us to hypothesize that LRRK2's neuroinflammatory alterations could be mediated through the system Xc-GSH-GPX4 pathway within the ferroptosis context.To validate this hypothesis, we initially constructed overexpression plasmids for LRRK2 and GPX4 to precisely regulate the intracellular expression levels of LRRK2 and GPX4.Following the transfection of these plasmids into BV2 cells, an upregulation of LRRK2 protein (Figure 6a) and GPX4 protein (Figure 6b) was observed.After successful transfection and precise modulation of LRRK2 and GPX4, we further F I G U R E 2 Inhibition of leucine-rich repeat protein kinase 2 (LRRK2) could effectively attenuate lipopolysaccharide (LPS)-induced BV2 microglial activation.To achieve this, 2 μg/mL of PF-06447475, a specific LRRK2 inhibitor, was cocultured with LPS-activated microglia.After 24 h, cells were harvested, and total RNA was extracted for subsequent reverse-transcription quantitative real-time PCR (RT-qPCR) analysis to quantify LRRK2 (a) expression.Furthermore, cellular proteins were collected following PF-06447475 treatment, and the levels of LRRK2 (b) protein were examined using Western blot analysis.Additionally, immunofluorescence staining was performed on PF-06447475-treated BV2 cells to visualize the distribution and expression of LRRK2 (c) following fixation.To further validate BV2 activation, morphological changes (d) and the surface marker expression of the CD40 (e) were assessed under conditions of LPS and LRRK2 inhibition.To assess the impact of LRRK2 inhibition on pro-inflammatory cytokine production, BV2 cells were treated with LPS alone or in combination with PF-06447475.After 24 h, the expression levels of pro-inflammatory cytokines interleukin-6 (IL-6) (f, j), tumor necrosis factor-⍺ (TNF-⍺) (g, k), IL-1β (h, l), and the anti-inflammatory inhibitory factor transforming growth factor-β (TGF-β) (i, m) were evaluated by RT-qPCR.The data are presented as the mean ± SD.Statistical significance was determined, with * /# p < 0.05, ** /## p < 0.01, and *** /### p < 0.001 denoting significance levels.PBS, phosphate-buffered saline; mRNA, messenger RNA.

| Downregulation of LRRK2 or ferroptosis prevented apoptosis following microglial activation in the MCS transfer model
In the present investigation, we aimed to explore the potential antiinflammatory and neuroprotective effects of modulating LRRK2 or inhibiting ferroptosis in activated microglia using BV2 cells.To this end, BV2 microglia were pretreated with PF-06447475, erastin, and ferrostatin-1 before exposure to LPS.The cells were then incubated with LPS (1 µg/mL) for 12 h.The medium was subsequently collected from the BV2 cells and mixed with Fer-1sh medium at a 1:1 (v/v) ratio.Our findings provide new insights into the potential therapeutic applications of targeting LRRK2 and ferroptosis in neuroinflammatory diseases.To assess the effects of LRRK2 inhibition and ferroptosis inhibition on neuronal apoptosis, SH-SY5Y cells were incubated with this conditioned medium for 12 h.Inhibition of LRRK2 activity in BV2 cells resulted in a significantly lower apoptotic rate in SH-SY5Y cells compared to the control group (Figure 7a,b).Similarly, inhibition of ferroptosis also led to significant differences in the apoptotic rate (Figure 7c,d).Therefore, our results suggest that elevated LRRK2 kinase can activate the ferroptosis process via the system Xc-GSH-GPX4 pathway, which increases NF-κB P-P65 to initiate an inflammatory response and ultimately regulate midbrain DA cell apoptosis.
3.8 | The levels of LRRK2, SLC7A11, GPX4, and P-P65 are increased in the SNpc of MPTP-treated mice in vivo In this study, we investigated the expression levels of LRRK2, SLC7A11, GPX4, and P-P65 in the midbrain of MPTP-treated mice, a model for PD.Firstly, we assessed the expression of LRRK2 and found that intraperitoneal injection of MPTP increased LRRK2 levels (Figure 8a,b).Next, we evaluated the expression of SLC7A11 and GPX4 at both messenger RNA (mRNA) and protein levels and observed a significant reduction in the MPTP-treated PD model (Figure 8c-f).Additionally, the protein level of P-P65 was found to be significantly elevated in the MPTP-treated PD model (Figure 8g).
Collectively, these findings suggest that LRRK2, SLC7A11, GPX4, and P-P65 may serve as neuroinflammation markers in the pathogenesis of PD.

| MPTP injection can induce mice microglia activation and apoptosis of DA
Specifically, we investigated microglial activation in a PD model using RT-qPCR analysis.Our results demonstrated that the expression of Iba1 was increased in the SNpc of MPTP-treated mice (Figure 9a).Moreover, we observed a significant upregulation in the mRNA levels of pro-inflammatory cytokines such as IL-6 (Figure 9b), TNF-α (Figure 9c), and IL-1β (Figure 9d).In contrast, the expression of the neuroprotective factor TGF-β was decreased (Figure 9e).Furthermore, we observed increased apoptosis of midbrain DA neurons in the MPTP-treated model (Figure 9f,g).
Finally, we assessed the impact of MPTP on motor ability in PD mice through behavioral experiments.We found that the motor ability of MPTP-treated mice was significantly impaired compared to the NC F I G U R E 4 Downregulation of ferroptosis could effectively attenuate P-P65 expression and BV2 activation.BV2 microglial cells were cocultured with lipopolysaccharide (LPS) and treated with either 10 μM erastin (Era) or 0.5 μM ferrostatin-1 (Fer-1).After 24 h, the cells were collected for various analyses.Western blot analysis was performed to assess the expression of leucine-rich repeat protein kinase 2 (LRRK2) (a), while reverse-transcription quantitative real-time PCR (RT-qPCR) was conducted to measure the transcript levels of LRRK2 (b).In addition, fixed BV2 cells treated with Era or Fer-1 were subjected to immunofluorescence staining to examine the distribution and expression of LRRK2 (c).Further analysis involved immunofluorescence staining of fixed BV2 cells to investigate the distribution and expression of LRRK2.Subsequently, the protein expression of P65 and PP65 (d-g) and the cellular localization and nucleation of PP65 (h) were assessed using the same methodology.To further confirm cellular activation, morphological changes (i) and CD40 surface marker expression (j) were evaluated under treatment with 10 μM Era or 0.5 μM Fer-1.Finally, the expression levels of pro-inflammatory cytokines interleukin-6 (IL-6) (k, o), tumor necrosis factor-⍺ (TNF-⍺) (l, p), IL-1β (m, q), and the inflammation suppressor transforming growth factor-β (TGF-β) (n, r) were determined by RT-qPCR.The data were presented as the mean ± SD.Statistical significance of the fold change was determined with # p < 0.05, ** /## p < 0.01, and *** /### p < 0.001.mRNA, messenger RNA; PBS, phosphate-buffered saline.
group (Figure 9h-j).Overall, these findings provide valuable insights into the pathogenesis of PD and suggest that the dysregulation of microglial activation and neuroinflammatory factors may contribute to the neurodegenerative process in PD.

| Inhibition of LRRK2 downregulates ferroptosis in the SNpc of MPTP-treated mice
Inspired by the observation that LRRK2 mitigates inflammatory responses induced by LPS through the system Xc-GSH-GPX4 signaling pathways in vitro, we explored whether LRRK2 can regulate the same pathways in MPTP-treated mice in vivo.Specifically, we orally administered PF-06447475 to MPTP-treated mice and observed a decrease in LRRK2 expression compared to the negative control (Figure 10a,b).We then noted a subsequent recovery of SLC7A11 and GPX4 expression at both the mRNA and protein levels in the MPTP-treated PD model (Figure 10c-f).Finally, we observed a significant increase in the protein level of P-P65 in the PD model (Figure 10g).Therefore, our results suggest that downregulating LRRK2 in the PD model reduces the activation of the system Xc-GSH-GPX4 pathway, reducing the ferroptosis and NF-κB P-P65 pathways.

| Inhibition of LRRK2 reduces microglial activation and midbrain DA cell apoptosis in MPTP-treated mice
Upon discovering the significant regulatory value of LRRK2 inhibition in PD models, we swiftly investigated the effects of LRRK2 inhibition on microglia activation and DA neuron apoptosis.Our results demonstrate that treatment with PF-06447475 led to a downregulation of Iba1 expression in the SNpc of mice compared to the negative control (Figure 10a).Moreover, there was a decrease in the expression of pro-inflammatory factors, including IL-6 (Figure 11b), TNF-⍺ (Figure 11c), and IL-1β (Figure 11d), while the anti-inflammatory cytokine TGF-β (Figure 11e) was significantly increased.An increased apoptosis of midbrain DA neurons in the MPTP-treated model was found (Figure 11f,g).Finally, we validated the impact of MPTP on the motor ability of PD mice via behavioral experiments.We found that the motor ability in the PF-06447475 treatment group was restored compared to the NC group (Figure 11h-j).Our findings demonstrate that PF-06447475 offers robust neuroprotection in the PD pathogenic process.

| DISCUSSION
LRRK2 is a significant gene in developing neurodegenerative diseases and plays a critical role in PD.However, the precise impact of its mutation on PD progression is somewhat controversial.Lin et al.
reported a strong protective effect of LRRK2 gene deletion in an A53Tα-syn CaMKII promoter-driven transgenic mouse (Lin et al., 2009).In contrast, Daher et al. observed that modulation of LRRK2 in A53Tα-syn transgenic mice had little effect on α-syninduced central pathology in mice (Daher et al., 2012).Consistent with this, Herzig et al. found that neither WT nor G2019S-LRRK2 mutations altered endogenous α-syn levels nor exacerbated α-syn induced disease progression [84], indicating that these events are primarily independent of LRRK2 expression (Russo et al., 2014).
However, the role of LRRK2 mutations in PD pathogenesis remains controversial, despite their association with disease progression in many PD patients (Gelders et al., 2018).To fully understand the effects of LRRK2, it is crucial to uncover the underlying mechanisms.
Previous studies have mainly focused on the role of LRRK2 in neurons, which is involved in abnormal protein processing, oxidative stress, mitochondrial dysfunction, and apoptosis (Simon et al., 2020).
However, LRRK2 is highly expressed in microglia present in the CNS (Moehle et al., 2012), and neuroinflammation is a critical process in the role of microglia in disease progression.Therefore, investigating the link between LRRK2 and microglia-mediated neuroinflammation may be essential in understanding the role of LRRK2 in PD.Our study found that LRRK2 expression was upregulated in a dose-and timedependent manner in LPS-stimulated BV2 cells, which is consistent with reports of increased LRRK2 kinase activity in response to inflammatory factors (Gloeckner et al., 2006).The elevation of LRRK2 kinase often indicates the onset of several adverse events, leading us to hypothesize that inhibiting LRRK2 kinase may alleviate neuroinflammation and PD progression.In support of our hypothesis, we found that PF-06447475 treatment downregulated the inflammatory factors IL-6, TNF-⍺, and IL-1β, while upregulating the inflammatory suppressor TGF-β in BV2 cells.This suggests that LRRK2 kinase inhibition can potentially regulate the inflammatory response in BV2 cells and can confer an anti-inflammatory protective effect.Furthermore, we observed that PF-06447475 treatment led to decreased expression of IL-6, TNF-⍺, and IL-1β, along with increased expression of TGF-β, a neuroprotective factor, in vitro.These findings indicate that LRRK2 kinase inhibition effectively attenuates LPS-or MPTPinduced microglial activation both in vitro and in vivo.
Ferroptosis is a process that typically impairs cellular functions through alterations in iron metabolism (Brown & Mercurio, 2020;Brown et al., 2019), lipid metabolism (Brown et al., 2017;Doll et al., 2017), and cellular oxidative stress (Dodson et al., 2019;Sun et al., 2016).However, despite the growing body of literature on ferroptosis, the mechanisms underlying its action in different natural immune cells, particularly microglia, remain inadequately characterized and require further investigation (Cui et al., 2021).Recent studies have demonstrated a substantial increase in iron accumulation in microglia following stimulation by inflammatory factors, which correlates with ferritin accumulation in the striatum (Mamais et al., 2021).RSL3 has been identified as a crucial regulator of ferroptosis that may influence the enhanced anti-inflammatory capacity of microglia (Yang et al., 2014).Notably, LRRK2 has been shown to drive the association of transferrin with Rab8a-positive   Immunohistochemical methods were used to assess the survival of DA neurons (f, g).Furthermore, the behavioral scores (h) of the mice were recorded, including the rod climbing test (i) and rotating rod test (j).The data are presented as the mean ± SD.The fold change was determined to be statistically significant using * /# p < 0.05, ## p < 0.01, and *** /### p < 0.001.mRNA, messenger RNA.
lysosomes and to redirect transferrin to lysosomes near the nucleus during pro-inflammatory conditions, leading to abnormal cellular iron metabolism and subsequent cell damage (Mamais et al., 2021).These findings illuminate the potential association between ferroptosis and LRRK2 in microglia.Our results demonstrated that markers of ferroptosis, including Fe 2+ and ROS, were elevated in BV2 cells, and treatment with ferrostatin-1 reduced their levels.We also observed changes in the system Xc-GSH-GPX4 pathway, which plays a critical role in ferroptosis.Specifically, we found that both SLC7A11 and GPX4 were decreased in BV2 cells in response to LPS and the ferroptosis inducer erastin, accompanied by a decrease in GSH.Treatment with ferrostatin-1 restored cell viability and reversed these changes.
Furthermore, we investigated whether LRRK2 affects the ferroptosis response induced by the system Xc-GSH-GPX4 pathway.
Our results showed that treatment with the LRRK2 kinase inhibitor NF-κB is a widely distributed family of transcription factors that govern immune cell survival signaling pathways and is crucial for the pathogenesis of neuroinflammation (Li et al., 2017).For instance, βhydroxybutyric acid has been shown to engage the NF-κB signaling pathway in immune cells through G protein-coupled receptors, leading to inhibition of pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6 (Fu et al., 2015).Studies have further indicated that the basal expression of NF-κB, which is abundant in brain tissue, surpasses that of peripheral tissues.This elevated expression plays a critical role in the development of PD (Shih et al., 2015).Phytate has been demonstrated to exhibit a long-lasting neuroprotective effect in MPTP-induced models of PD, which is closely linked to the inhibition of the NF-κB pathway.Furthermore, there is evidence of multipathway regulation of the NF-κB pathway by LRRK2, such as LRRK2's ability to modulate microglial inflammation by influencing PKA-mediated phosphorylation of NF-κB p50 in microglia (Russo et al., 2015).Notably, LRRK2 mutations have been shown to enhance the transcriptional activity of NF-κB in microglia significantly, and knockdown of LRRK2 in primary microglia was found to increase the level of phosphorylated NF-κB p50 (Yao et al., 2021) Subsequently, the mice received daily intraperitoneal injections of MPTP-HCl for a total of 5 consecutive days, while the control mice were subjected to saline injections.The treatment with PF-06447475 was initiated 2 days before the MPTP injection, and the midbrain samples were collected 7 days after the final MPTP injection.Reverse-transcription quantitative real-time PCR (RT-qPCR) and Western blot analyses were conducted to assess the levels of LRRK2 (a, b), SLC7A11 (c, d), glutathione peroxidase 4 (GPX4) (e, f), and PP65 (g), with messenger RNA (mRNA) levels normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).The obtained data were normalized to the saline control group, and the mean ± SD was calculated from three independent experiments.The statistical significance of fold changes was determined using * /# p < 0.05, ** /## p < 0.01, and *** /### p < 0.001.
neurodegenerative process.Despite this, no drugs have yet been introduced to achieve this goal (Connolly & Lang, 2014;Fox et al., 2018).Past clinical trials aimed at developing drugs to protect DA neurons have failed.Therefore, it is necessary to revisit the mechanistic studies (Hirsch & Standaert, 2021).Given the crucial role of microglial neuroinflammation in regulating PD progression, relevant clinical studies are underway.A prospective cohort study has indicated that nonsteroidal anti-inflammatory drugs may delay or prevent the onset of PD (Chen et al., 2003).Moreover, ibuprofen use is associated with a reduced risk of developing PD, suggesting that it may delay or prevent the onset of the disease (Poly et al., 2019).
Furthermore, targeted drugs directed at specific PD genes, such as GBA or LRRK2, provide a promising and hopeful perspective for the precise treatment of PD.The study of LRRK2 is expected to yield novel and effective results in therapeutic approaches for PD treatment (Jankovic & Tan, 2020).The aim and significance of our F I G U R E 11 Inhibition of leucine-rich repeat protein kinase 2 (LRRK2) reduces microglial activation and midbrain dopamine (DA) cell apoptosis in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice.Mice were subjected to gastric feeding of PF-06447475 for a period of 14 days.Subsequently, the mice received daily intraperitoneal injections of MPTP-HCl for 5 consecutive days, whereas the control mice were administered saline injections.The administration of PF-06447475 was initiated 2 days before the MPTP injection, and the midbrain samples were collected 7 days after the final MPTP injection.Following the MPTP injection, reverse-transcription quantitative real-time PCR (RT-qPCR) was conducted to assess the expression of Iba1+ (a) and the levels of interleukin-6 (IL-6) (b), tumor necrosis factor-⍺ (TNF-⍺) (c), , and the inflammation suppressor transforming growth factor-β (TGF-β) (e).Immunohistochemical methods were employed to evaluate the survival of DA neurons post-MPTP injection (f, g).Finally, the behavioral scores (h) of the mice were recorded, including the rod climbing test (i) and the rotating rod test (j).The obtained data were normalized to the saline control group, and the mean ± SD was calculated from three independent experiments.Statistical significance of the fold change was determined using * /# p < 0.05, ** /## p < 0.01, and *** /### p < 0.001.mRNA, messenger RNA.

| CONCLUSION
In summary, we observed that LPS stimulation of BV2 cells resulted in elevated expression of LRRK2, which in turn caused ferroptosis, microglia activation, and the release of inflammatory factors through modulation of the system Xc-GSH-GPX4 signaling pathway.Additionally, we found that LRRK2 and P-P65 were highly expressed in the SNpc of MPTP-induced PD model rats, with concomitant microglia activation.Treatment with the LRRK2 kinase inhibitor PF-06447475 via gastric feeding attenuated the expression of LRRK2 and P-P65 and microglia activation in MPTPinduced PD mice.Taken together, our findings suggest that inhibition of LRRK2 kinase activity can reduce the incidence of ferroptosis and attenuate neuroinflammation during PD development.Therefore, the LRRK2 kinase and system Xc-GSH-GPX4 signaling pathway-related ferroptosis process may be a promising therapeutic target for regulating the inflammatory response in PD (Figure 12).
), ROS, GSH formation and cell viability (Figure 3b-e) in BV2 microglia compared to LPS activation alone.Similarly, the level of 4-HNE, a marker of cellular lipid peroxidation, was also increased upon erastin treatment (Figure 3f,i).The findings indicate that microglial activation increases Fe 2+ levels and cellular oxidative stress damage characteristics of ferroptosis.Moreover, erastin exacerbates this damage by inducing the ferroptosis pathway.However, treatment with ferrostatin-1, a potent inhibitor of ferroptosis, rescues the aforementioned changes.

F
I G U R E 1 Leucine-rich repeat protein kinase 2 (LRRK2) is upregulated in lipopolysaccharide (LPS)-stimulated BV2 cells.LRRK2 expression was quantified using reverse-transcription quantitative real-time PCR (RT-qPCR) and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).The expression levels of LRRK2 (a) were assessed in BV2 cells treated with various concentrations (0, 0. 1, 0.2, 0.5, and 1 μg/mL) of LPS for a duration of 24 h.Additionally, the expression levels of LRRK2 (b) were analyzed in BV2 cells exposed to 1 μg/mL LPS for different time intervals (0, 1, 6, 12, and 24 h).The presented data represent the mean ± standard deviation (SD) derived from three independent experiments.Statistical significance of the fold change was determined, with **p < 0.01 and ***p < 0.001 denoting the significance levels.mRNA, messenger RNA.F I G U R E 2 (See caption on next page).ZHENG ET AL. | 7 of 24 genes, and its activation plays a crucial role in cellular immune responses.The NF-κB family consists of p105/p50 (nuclear factor-κB1), p100/52 (nuclear factor-κB2), p65 (RelA), RelB, and c-Rel.In LPS-treated PBECs, an upregulated phosphorylation pattern of NF-κB p65 and increased inflammatory factors such as IL-1β and ROS production were observed.Notably, the increased expression levels of NF-κB P-P65 phosphorylation pattern and nucleation are the primary triggering factors for inflammatory activation ) were increased, providing evidence that LRRK2 may affect ferroptosis in microglia via the system Xc-GSH-GPX4 pathway.Through the present study, we have observed that the inhibition of LRRK2 kinase activity can mitigate oxidative stress damage resulting from ferroptosis.Specifically, the expression levels of NF-κB (Figure5k) and P-P65 (Figure5l-n) were decreased, and the nucleation of P-P65 was reduced, as demonstrated by RT-qPCR analysis in BV2 cells treated with PF-06447475.These findings indicate that the abnormal elevation of LRRK2 kinase activity may activate the ferroptosis process in LPS-stimulated activated BV2 cells through the system Xc-GSH-GPX4 pathway which in turn initiates inflammation by increasing the NF-κB P-P65 response.It is noteworthy that previous studies have shown that the inhibition of LRRK2 kinase activity can lead to microglial activation and an increase in the expression of inflammatory factors.
investigated whether the effects of increased LRRK2 protein levels on ferroptosis and neuroinflammation remained as pronounced as previously described, post-inhibition of the ferroptotic pathway by overexpressing GPX4.The study revealed that co-overexpression of LRRK2 with GPX4 attenuated the oxidative stress and inflammation associated with LRRK2-induced ferroptosis, characterized by a reduction in Fe 2+ accumulation (Figure6c), a decrease in ROS (Figure6d,f), and a concomitant downregulation of the lipid peroxidation marker 4-HNE (Figure6e).Subsequently, RT-qPCR and ELISA analyses demonstrated a downregulation of proinflammatory cytokines, including IL-6 (Figure6g,k), TNF-α (Figure6h,l), and IL-1β (Figure6i,m), along with a significant upregulation of the anti-inflammatory cytokine TGF-β (Figure6j,n) following the co-overexpression of LRRK2 with GPX4.These findings underscore that the downregulation of GPX4 is a pivotal mechanism through which LRRK2 exerts its effects on ferroptosis and neuroinflammation, indicating LRRK2's regulatory role through the system Xc-

F
I G U R E 8 The levels of leucine-rich repeat protein kinase 2 (LRRK2), SLC7A11, glutathione peroxidase 4 (GPX4), and p-p65 are increased in the substantia nigra pars compacta (SNpc) of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice in vivo.Increased expression levels of LRRK2, p-p65, and activated microglia were observed in the SNpc of MPTP-treated mice in vivo.The mice received a single intraperitoneal injection of MPTP-HCl, followed by decapitation and harvesting of midbrains at various time points after MPTP-HCl administration.The time points investigated included immediately after the last MPTP injection, as well as 0, 7, and 21 days postinjection.Reverse-transcription quantitative real-time PCR (RT-qPCR) and Western blot analysis were performed to analyze the expression of LRRK2 (a, b), SLC7A11 (c, d), GPX4 (e, f), and PP65 (g).The messenger RNA (mRNA) levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) values, and β-actin was used as a loading control for normalizing the image density.The data are presented as the mean ± SD.Statistical significance of the fold change was determined using * /# p < 0.05, ## p < 0.01, and *** /### p < 0.001.F I G U R E 9 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injection can induce mice microglia activation and apoptosis of dopamine (DA).Increased expression levels of leucine-rich repeat protein kinase 2 (LRRK2), p-p65, and activated microglia were observed in the substantia nigra pars compacta (SNpc) of MPTP-treated mice in vivo.The mice received daily intraperitoneal injections of MPTP-HCl for 5 consecutive days, while the control mice received saline injections.Subsequently, the mice were decapitated, and midbrains were harvested at different time points following MPTP intoxication, including immediately after the last MPTP injection, as well as at 0, 7, and 21 days postinjection.Reversetranscription quantitative real-time PCR (RT-qPCR) was performed to detect the expression of Iba1+ (a), interleukin-6 (IL-6) (b), tumor necrosis factor-⍺ (TNF-⍺) (c),, and the inflammation suppressor transforming growth factor-β (TGF-β) (e) after MPTP injection.
PF-06447475 significantly inhibited cellular ferroptosis and restored the normal functioning of the system Xc-GSH-GPX4 pathway, leading to fixed GSH levels and reduced cellular oxidative stress, subsequently suppressing BV2 activation and the release of inflammatory factors.Upon treatment with PF-06447475, we observed a significant inhibition of cellular ferroptosis and restoration of normal functioning in the system Xc-GSH-GPX4 pathway and GSH levels, ultimately leading to a reduction in cellular oxidative stress and consequent BV2 activation.To ascertain whether LRRK2 modulates ferroptosis and neuroinflammation via the system Xc-GSH-GPX4 pathway, our study revealed that co-overexpression of LRRK2 with GPX4 attenuates the ferroptosis-induced oxidative stress and inflammatory effects initially heightened by LRRK2 overexpression.This was evidenced by a reduction in intracellular accumulation of Fe 2+ and ROS, along with a decrease in 4-HNE protein expression.Concurrently, co-overexpression of LRRK2 with GPX4 resulted in the downregulation of pro-inflammatory cytokines, including IL-6, TNF-α, and IL-1β, and a significant upregulation of the anti-inflammatory cytokine TGF-β in microglial cells.These findings suggest that the downregulation of GPX4 is a critical mechanism through which LRRK2 induces ferroptosis and neuroinflammation, thereby implicating LRRK2's regulatory role via the system Xc-GSH-GPX4 pathway in modulating ferroptosis and neuroinflammation.Furthermore, we observed a reduction in the recovery of the system Xc-GSH-GPX4 pathway and antioxidant capacity in MPTP mice, which was rescued by LRRK2 kinase inhibition, leading to a decrease in inflammatory activation in Iba1 positive cells.These findings suggest that LRRK2 is critical in regulating the ferroptosis response and subsequent neuroinflammation.
. Our study observed that LPS treatment significantly increased NF-κB activity in microglia.In contrast, inhibition of LRRK2 activity by PF-06447475 resulted in decreased NF-κB activity and reduced expression and nuclear translocation of P-P65 phosphorylation protein.Our study observed that the system Xc-GSH-GPX4 pathway in ferroptosis could modulate NF-κB activity.Specifically, erastin treatment increased NF-κB activity, P-P65 phosphorylated protein expression, and nuclear translocation.However, ferrostatin-1 reversed these effects, reducing BV2 activation and normalization of abnormal NF-κB activity.Notably, regardless of LRRK2 inhibition or ferroptosis mitigation, the level of P-P65 phosphorylation was decreased in microglia.Our findings suggest that LRRK2 aberrantly expressed in microglia regulates the LPS-induced pro-ferroptosis and NF-κB pathways via the system Xc-GSH-GPX4-associated ferroptosis pathway and secretion of inflammatory cytokines.The principal objective of PD mechanistic research is the development of drugs for disease intervention.The expectation is that a deep understanding of the molecular mechanisms will lead to the creation of drugs that can effectively slow or halt the F I G U R E 10 Inhibition of leucine-rich repeat protein kinase 2 (LRRK2) downregulates ferroptosis in the SNpc of 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP)-treated mice.Mice were orally administered PF-06447475 via gastric feeding for a duration of 14 days.
apoptosis in an MCS transfer model.Moreover, oral administration of the LRRK2 kinase inhibitor PF-06447475 in MPTP-treated mice attenuated microglial activation and prevented MPTP-induced apoptosis of DA cells in the midbrain.Our findings highlight the potential of targeting the LRRK2 gene and its associated ferroptosis process to alleviate microglia-mediated neuroinflammatory response effectively and DA neuron apoptosis, offering new insights for disease diagnosis and drug development in PD.