Leucettine L41 Ameliorates Tau pathology and neuroinflammation in Parkinson’s disease models

Abstract

Background

Aberrant protein phosphorylation and sustained neuroinflammatory activation are key biochemical features of experimental parkinsonism. Dysregulated tau processing and excessive microglial reactivity have been implicated in motor dysfunction associated with dopaminergic injury. In the present study, we evaluated the biochemical and functional effects of Leucettine L41 using biochemical, cellular, and in vivo systems.

Results

In an in vitro tau aggregation assay, L41 reduced the ThT fluorescence, indicating the suppression of fibril formation. In SH-SY5Y neuronal cells, L41 decreased tau phosphorylation at the Ser202/Thr205 site. In lipopolysaccharide-stimulated BV-2 microglial cells, L41 suppressed the production of nitric oxide, TNF-α, IL-1β, and IL-6, demonstrating its anti-inflammatory activity. Notably, L41 did not prevent toxin-induced neuronal cytotoxicity, suggesting that its primary action may not involve direct neuroprotection. In an MPTP-induced mouse model of Parkinson’s disease, systemic L41 administration improved motor performance, accompanied by reduced tau phosphorylation and attenuated microglial activation in brain tissue.

Conclusions

These findings indicate that L41 modulates tau aggregation, phosphorylation, and neuroinflammatory signaling in experimental parkinsonism, in association with functional motor improvement.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12868-026-01026-1.

Introduction

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the selective loss of dopaminergic neurons in the substantia nigra pars compacta and the presence of intracellular Lewy bodies, primarily composed of aggregated α-synuclein [1]. Although α-synuclein aggregation is widely recognized as a defining pathological feature, PD is increasingly understood as a multifactorial disorder involving dysregulated protein homeostasis, aberrant kinase signaling, mitochondrial dysfunction, and sustained neuroinflammatory activation [2]. Chronic neuroinflammation is a significant contributor to disease progression through these mechanisms. Activated microglia release pro-inflammatory cytokines, reactive oxygen species, and nitric oxide, thereby amplifying neuronal stress and promoting a self-perpetuating cycle of neurotoxicity [3]. Persistent inflammatory signaling not only exacerbates dopaminergic degeneration but also facilitates the propagation and stabilization of misfolded protein aggregates within vulnerable neural circuits [4]. These observations highlight the importance of therapeutic approaches capable of simultaneously modulating pathological protein processing and inflammatory cascades in experimental parkinsonism.

Although PD has historically been classified as a synucleinopathy [5], accumulating evidence indicates that tau pathology also contributes to its progression. Hyperphosphorylated tau has been detected in postmortem PD brains and in toxin-induced parkinsonian models, where it is associated with cytoskeletal destabilization, impaired axonal transport, and synaptic dysfunction [6]. Experimental studies suggest that α-synuclein and tau may interact synergistically, with aberrant phosphorylation of tau promoting aggregate formation and enhancing neuronal vulnerability. Moreover, phosphorylated tau species can influence microglial activation states, linking tau dysregulation to inflammatory amplification within affected regions. Tau phosphorylation is tightly regulated by a complex network of serine/threonine kinases and dual-specificity tyrosine-phosphorylation-regulated kinase 1 A (DYRK1A). DYRK1A has been reported to phosphorylate tau at disease-relevant residues, such as Ser202, and has been implicated in signaling pathways that influence inflammatory responses [7–10]. Thus, modulation of kinase-mediated tau phosphorylation may represent a mechanistic intersection between protein pathology and neuroinflammation.

L41 is a synthetic derivative of the marine alkaloid leucettamine B and has been characterized as an inhibitor of a subset of kinases involved in phosphorylation-dependent signaling pathways [11]. Previous investigations have demonstrated that L41 modulates tau-related abnormalities and influences inflammatory signaling in cellular and experimental neurodegenerative models [12]. However, its integrated effects on tau aggregation, tau phosphorylation, and neuroinflammatory activation within a parkinsonian context remain undefined.

In addition, although many kinase inhibitors have shown promising in vitro potency, their in vivo efficacy is often limited by inadequate brain penetration or suboptimal pharmacokinetic profiles [13]. Therefore, understanding the relationship between central exposure and downstream biochemical modulation is essential when evaluating candidate compounds that target phosphorylation-dependent mechanisms in neurodegenerative disorders.

In the present study, we investigated the neurochemical and functional effects of L41 using complementary biochemical, cellular, and in vivo approaches relevant to experimental parkinsonism. We assessed its ability to influence tau aggregation in a cell-free system and modulate tau phosphorylation in neuronal cells, along with its effects on microglial activation and the production of pro-inflammatory mediators. Furthermore, we examined motor performance, tau phosphorylation, microglial activation, and DYRK1A expression in an MPTP-induced mouse model of parkinsonism. By integrating biochemical, cellular, and behavioral analyses, this study aimed to clarify the extent to which L41 modulates phosphorylation-dependent and inflammatory processes associated with parkinsonian motor dysfunction.

Materials and methods

Chemicals and reagents

All chemicals and reagents used in this study were of analytical or cell culture grade. Leucettine L41 was purchased from Med Chem Express (MCE; Monmouth Junction, NJ, USA). Dimethyl sulfoxide (DMSO) and 1-methyl-4-phenylpyridinium (MPP⁺) iodide were obtained from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) was sourced from Biosolution (Seoul, Korea). For cell culture, Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Corning (Corning, NY, USA), whereas fetal bovine serum (FBS) and penicillin–streptomycin were obtained from Hyclone (Logan, UT, USA).

Cell viability was assessed using the Cell Titer-Glo 3D assay (Promega, Madison, WI, USA). For protein analysis, radioimmunoprecipitation assay (RIPA) buffer and protein sample buffer were purchased from Elpis Biotech (Daejeon, Korea), and bicinchoninic acid (BCA) protein assay reagents, protease inhibitors, and phosphatase inhibitors were sourced from Thermo Fisher Scientific (Waltham, MA, USA). For aggregation studies, Thioflavin T (ThT) was purchased from Abcam (Cambridge, UK), and recombinant Tau (K18) monomers and fibrils were purchased from Stress Marq Biosciences (Victoria, BC, Canada).

The primary antibodies (all diluted 1:1,000) used for western blotting and immunofluorescence included mouse monoclonal antibodies against phospho-tau (Ser202/Thr205, MN1020) and total tau (MN2000), and rabbit monoclonal antibodies against DYRK1A (8765 S), GAPDH (5174 S), phospho-tau (Ser396/Ser404, ab109390), α-synuclein (4179 S), tyrosine hydroxylase (2792 S), and Iba-1 (17198 S), all of which were sourced from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies, including HRP-conjugated goat anti-rabbit IgG (31460) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (A32731), were purchased from Invitrogen (Carlsbad, CA, USA).

Cell culture

The human neuroblastoma cell line SH-SY5Y (ATCC, CRL-2266; Manassas, VA, USA) and the murine microglial cell line BV-2 (AcceGen Biotechnology, #ABC-TC212S; Fairfield, NJ, USA), were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% penicillin–streptomycin and maintained at 37 °C in a humidified incubator with 5% CO₂.

Animals

Eight-week-old male C57BL/6J and ICR mice were purchased from Orient Bio Inc. (Seongnam, Korea). The animals were housed under standard laboratory conditions (24 ± 3 °C, humidity 50 ± 3%, 12 h day/night cycles). Prior to the experiments, the animals were allowed to acclimatize to the facility for 1 week and provided with a standard chow diet and drinking water ad libitum. All animal procedures were approved by the KRICT Animal Care and Use Committee (C57BL/6J mice: 2025–07 F-02-02, approved on February 6, 2025; ICR mice: 2025–07 F-05-02, approved on May 8, 2025). At the end of the experiment, all animals were euthanised by CO₂ asphyxiation with a gradual fill rate of 30% of the chamber volume per minute.

Thioflavin T (ThT) fluorescence assay

Tau aggregation was assessed using a ThT fluorescence assay. Recombinant tau (K18) monomer (50 µM) and preformed tau (K18) fibrils (10 µM) were mixed in equal volumes with phosphate-buffered saline. L41 was added to achieve final concentrations of 3 or 10 µM. Aliquots of each reaction mixture were dispensed into black 384-well microplates, followed by the addition of ThT (10 µM) at a 1:3 ratio. Plates were incubated at 37 °C, and fluorescence intensity (excitation/emission: 440/485 nm) was recorded every 30 min for 24 h using a spectrophotometer.

Cell viability

SH-SY5Y cells were seeded at a density of 1 × 10⁵ cells/mL in white 96-well plates. Cells were pretreated with L41 for 1 h, followed by exposure to 1-methyl-4-phenylpyridinium (MPP⁺, 1 mM). After 24 h of incubation, cell viability was assessed using the Cell Titer-Glo^® 3D assay (Promega, Madison, WI, USA). The reagent was added at a 1:9 ratio to the culture medium, and luminescence was measured after a 20-min incubation at room temperature.

Immunofluorescence staining

SH-SY5Y cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 2% bovine serum albumin (BSA). Next, cells were incubated overnight at 4 °C with an anti-phospho-tau (Ser202/Thr205) antibody (1:100), followed by Alexa Fluor 488-conjugated secondary antibody (1:1000) for 1 h at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using a fluorescence microscope (Olympus, Tokyo, Japan).

Griess assay for NO production

BV-2 cells were seeded and treated as described below. After 24 h of lipopolysaccharide (LPS) stimulation, culture supernatants were mixed with an equal volume of Griess reagent in 96-well plates. Following a 10-min incubation at room temperature, absorbance was measured at 540 nm to quantify nitrite accumulation as an indicator of nitric oxide production.

Enzyme-linked immunosorbent assay (ELISA) for pro-inflammatory cytokines

BV-2 cells were pretreated with L41 (1, 3, or 10 µM) for 1 h and subsequently stimulated with LPS (10 ng/mL). Following stimulation, cytokine levels in the culture supernatant were quantified using commercial ELISA kits.

Establishment of MPTP-induced Parkinson’s disease model and drug administration

Male C57BL/6 mice were orally administered L41 (50 mg/kg) once daily for 12 consecutive days. To induce PD, MPTP (25 mg/kg) was co-administered from days 8 to 12. The mice were randomized into three groups: vehicle-treated, MPTP-only, and MPTP + L41. This protocol allowed the evaluation of L41’s protective effects against MPTP-induced neurotoxicity during the final 5 days of treatment.

Behavioral assessments

Motor coordination and locomotor activity were evaluated using the wire hang and open field tests, respectively. For the wire hang test, the latency to fall from a horizontal wire grid was recorded with a maximum cutoff time of 120 s; tests were conducted at baseline (prior to MPTP administration) and after the completion of treatment. Locomotor activity was assessed in an open field arena over a 15-min period per mouse. All movements were recorded and analyzed using EthoVision XT 14 software (Noldus Information Tech Inc., Leesburg, VA, USA).

Western blotting

For in vitro analysis, SH-SY5Y cells (1 × 10⁵ cells/mL) were pretreated with L41 (1, 3, or 10 µM) for 1 h and subsequently exposed to MPP⁺ (1 mM). BV-2 cells (5 × 10⁵ cells/mL) were pretreated with L41 (1, 3, or 10 µM) for 1 h and subsequently exposed to LPS (10 ng/mL) for 24 h. Cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors, incubated on ice for 30 min, and centrifuged at 13,000 rpm for 10 min at 4 °C.

For in vivo analysis, mice were euthanized after completion of behavioral testing, and their brains were dissected into the substantia nigra, striatum, and cortex. Brain tissues were homogenized in RIPA buffer containing protease and phosphatase inhibitors and centrifuged to obtain the supernatant for protein analysis.

Protein concentrations were determined using a BCA assay (Pierce, Rockford, IL, USA). Equal amounts of protein were mixed with loading buffer, boiled, separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% BSA in Tris-buffered saline containing Tween-20 and incubated overnight at 4 °C with primary antibodies against phospho-tau (Ser202/Thr205), phospho-tau (Ser396/Ser404), total tau, DYRK1A, Iba-1, tyrosine hydroxylase (TH), α-synuclein, and GAPDH. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were detected using enhanced chemiluminescence and visualized using a chemiluminescent imaging system (Azure Biosystems, Dublin, CA, USA).

Brain penetration study

Blood-brain barrier (BBB) penetration was assessed in male ICR mice (8 weeks old, n = 3 per time point) following a single oral dose of L41 (50 mg/kg). Blood was collected via the retro-orbital route at 1, 4, 8, and 24 h post-dose, after which the mice were sacrificed and whole brains were rapidly harvested. Plasma and brain tissues were processed using protein precipitation with acetonitrile and analyzed using LC–MS/MS. The brain-to-plasma concentration ratio was calculated to evaluate BBB permeability.

LC-MS/MS analysis

Analyses were conducted using an LC–MS/MS system consisting of an Agilent 1200 series HPLC system (Agilent Technologies, Santa Clara, CA, USA) coupled to an API 4000 QTRAP mass spectrometer (Applied Biosystems, Foster City, CA, USA) equipped with an electrospray ionization (ESI) source operated in negative ion mode. For sample preparation, 3 µL of the L41 sample solution was added to 27 µL of mouse plasma or brain homogenate, followed by the addition of 120 µL of a chlorpropamide (CPM) solution (50 ng/mL; internal standard) in acetonitrile containing 0.1% formic acid. The mixture was vortexed thoroughly and centrifuged at 15,000 rpm for 10 min at 4 °C. The resulting supernatant was collected and used for LC–MS/MS analysis. Chromatographic separation was carried out on a Unison UK-C18 column (100 × 2.0 mm, 3 μm) maintained at 40 °C, with a Security Guard C18 guard column (4 × 20 mm i.d.; Phenomenex, Torrance, CA, USA). The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), delivered at a ratio of 95:5 (v/v) under isocratic conditions. The flow rate was 0.3 mL/min, and the injection volume was 5 µL. Detection was performed in multiple reaction monitoring (MRM) mode, with a mass transition of m/z 306.045 → 277.000 for the analyte and m/z 274.816 → 189.800 for CPM (IS). Optimized values for declustering potential, collision energy, entrance potential, and collision-exit potential were 91 V, 41 eV, 10 V, and 18 V for the analyte, and 106 V, 39 eV, 10 V, and 18 V for the IS, respectively. The optimized instrument conditions were as follows: ion spray voltage, 5500 V; source temperature, 550 °C; curtain gas, 10 psi; nebulizing gas (GS1), 50 psi; and heating gas (GS2), 50 psi. Samples of mouse plasma and brain homogenate were analyzed under these conditions. Data acquisition and quantification were performed using Analyst software version 1.4.2 (Applied Biosystems, Foster City, CA, USA).

Pharmacokinetic analysis

Pharmacokinetic parameters were calculated via non-compartmental analysis using Phoenix WinNonlin, version 8.3 (Pharsight Corp., Mountain View, CA, USA). Based on the highest concentration attained, the maximum plasma concentration (C_max) and time to reach the maximum plasma concentration (T_max) were calculated directly. The linear trapezoidal approach was used to determine the area under the plasma concentration-time curve (AUC), extrapolated to an infinite time. With λ being the slope of the log-linear part of the concentration-time curve, the terminal half-life (t_1/2) was determined to be 0.693/λ.

Statistical analysis

Statistical comparisons among groups were performed using one-way analysis of variance, followed by Tukey’s post hoc test. p-values < 0.05 were considered statistically significant.

Results

L41 inhibits tau aggregation and phosphorylation

To determine whether L41 reduces tau aggregation, a ThT fluorescence assay was performed. L41 significantly inhibited tau aggregation at 3 and 10 µM, as indicated by the reduced relative fluorescence intensity compared to that of the control (Fig. 1A). AUC analysis further confirmed a dose-dependent suppression of the ThT signal (F (2, 6) = 21.78, p = 0.002) with significant inhibitory effects observed at both 3 µM (p = 0.020) and 10 µM (p = 0.001) (Fig. 1B). To assess the protective effect of L41 against MPP+-induced tau pathology, SH-SY5Y cells were pretreated with L41 (1, 3, and 10 µM) followed by MPP⁺ exposure, and phosphorylated tau (p-tau; Ser202/Thr205) and total tau levels were analyzed using western blotting. Representative western blot images are shown in Fig. 1C. Densitometric analysis revealed a significant difference in p-tau levels among groups (F (4, 10) = 13.61, p = 0.005). MPP⁺ treatment markedly increased p-tau levels compared with the control (p = 0.005), which was dose-dependently attenuated by L41, with significant reductions observed at 3 µM (p = 0.006) and 10 µM (p < 0.001) (Fig. 1D). In contrast, total tau levels were not significantly altered across experimental groups (Fig. 1E). Immunofluorescence analysis confirmed a dose-dependent reduction in p-tau levels in SH-SY5Y cells following L41 treatment, with a significant decrease observed at 10 µM, consistent with the western blot results (Fig. 1F). Collectively, these findings demonstrate that L41 effectively inhibits tau aggregation and phosphorylation at the Ser202/Thr205 epitope in vitro, supporting its potential to mitigate pathological tau accumulation in vivo.

Fig. 1.

L41 inhibits tau aggregation and reduces tau phosphorylation in neuronal cells in vitro. (A) ThT fluorescence (% control) at 3 and 10 µM. (B) AUC analysis of ThT fluorescence. (C) Representative western blots of p-tau (Ser202/Thr205) and total tau in SH-SY5Y cells treated with L41. (D, E) Densitometric quantification of p-tau and total tau, respectively, normalized to GAPDH. (F) Immunofluorescence staining of p-tau (Ser202/Thr205) in SH-SY5Y cells. Scale bar = 100 μm. Data are expressed as the mean ± SD (n = 3/group). ^##p < 0.01 vs. the control group; *p < 0.05, **p < 0.01 vs. the MPP⁺ group. Uncropped western blot images corresponding to panel (C) are provided in Supplementary Figure S1

L41 does not attenuate MPP⁺-induced cytotoxicity in SH-SY5Y cells

To evaluate whether L41 protects against MPP⁺-induced neuronal cell death, SH-SY5Y cells were treated with MPP⁺ in the presence of L41 (1, 3, and 10 µM). Cell viability differed significantly among the groups (F (4, 10) = 16.93, p < 0.001). MPP⁺ treatment significantly reduced cell viability compared to the control (p = 0.0026). However, L41 did not improve viability compared to the MPP⁺ group at any of the tested concentrations.

These results indicate that under the present experimental conditions, L41 does not exert cytoprotective effects against MPP⁺-induced neuronal death in SH-SY5Y cells (Fig. 2).

Fig. 2.

Effects of L41 on cell viability under neurotoxic conditions in SH-SY5Y cells. Cell viability following MPP⁺ exposure in the presence or absence of L41 (1, 3, and 10 µM) in SH-SY5Y cells. Data are expressed as the mean ± SD (n = 3/group). ^#p < 0.05 vs. control group

L41 attenuates LPS-induced cytokine production in BV-2 cells

To assess the anti-inflammatory effects of L41, BV-2 microglial cells were stimulated with LPS (10 ng/mL) and treated with L41 (1, 3, and 10 µM). NO production differed significantly among the groups (F (4, 10) = 71.96, p < 0.001). LPS markedly increased NO levels compared with the control (p < 0.001), which was significantly attenuated by L41 in a concentration-dependent manner (1 µM, p = 0.044; 3 µM, p < 0.001; 10 µM, p < 0.001). Similarly, TNF-α, IL-1β, and IL-6 levels differed significantly among groups (TNF-α: F (4, 10) = 54.88, p < 0.001; IL-1β: F (4, 10) = 13.77, p < 0.001; IL-6: F (4, 10) = 343.0, p < 0.001). LPS stimulation markedly increased TNF-α and IL-6 levels compared with the control (p < 0.001). L41 treatment significantly reduced TNF-α and IL-6 levels in a concentration-dependent manner (TNF-α: 1 µM, p = 0.023; 3 µM, p = 0.002; 10 µM, p = 0.001; IL-6:1 µM, p = 0.009; 3 µM, p < 0.001; 10 µM, p < 0.001; Fig. 3B, D). In contrast, although LPS markedly increased IL-1β production (p = 0.04), L41 did not significantly reduce these levels at 1 or 3 µM, whereas a significant reduction was observed only at 10 µM (p < 0.001; Fig. 3C). These findings indicate that L41 effectively suppresses key pro-inflammatory mediators, including NO, TNF-α, IL-1β, and IL-6, in LPS-stimulated BV-2 microglial cells.

Fig. 3.

L41 suppresses LPS-induced production of NO and pro-inflammatory cytokines in BV-2 cells. (A) NO, (B) TNF-α, (C) IL-1β, and (D) IL-6 levels following LPS stimulation in the presence of L41 (1, 3 and 10 µM). Data are expressed as the mean ± SD (n = 3/group). ^#p < 0.05, ^###p < 0.001 vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS group

L41 modulates LPS-induced inflammatory signaling in BV-2 cells

To further investigate the molecular mechanisms underlying the effects of L41 on LPS-induced inflammatory signaling, BV-2 cells were treated under the conditions described above. Western blot analysis was performed to assess the expression levels of NLRP3, p-IκB, and IκB (Fig. 4A). LPS treatment markedly induced NLRP3 expression. NLRP3 levels differed significantly among the groups (F (4, 10) = 67.84, p < 0.001). LPS significantly increased NLRP3 expression compared to the control (P < 0.001), which was reduced by L41 at 3 µM (p = 0.002) and 10 µM (p < 0.001), but not at 1 µM (Fig. 4B).

Fig. 4.

L41 modulates LPS-induced inflammatory signaling pathways in BV-2 microglial cells. (A) Representative western blotting of NLRP3, p-IκB, and IκB in BV-2 cells treated with LPS (10 ng/mL) in the presence of L41 (1, 3, and 10 µM). (B–D) Densitometric quantification of NLRP3, p-IκB, and IκB, respectively, normalized to GAPDH. Data are expressed as the mean ± SD (n = 3/group). ^#p < 0.05, vs. control group; *p < 0.05, vs. LPS group. Uncropped western blot images corresponding to panel (A) are provided in Supplementary Figure S2

To evaluate NF-κB pathway activation, p-IκB levels were measured. p-IκB levels differed significantly among the groups (F(4, 10) = 11.59, p = 0.028). LPS significantly increased p-IκB levels compared with the control (p = 0.028), whereas L41 treatment showed a concentration-dependent decreasing trend compared with the LPS group, although these reductions did not reach statistical significance (Fig. 4C). Total IκB levels also differed significantly among the groups (F(4, 10) = 4.54, p = 0.047). While LPS alone did not alter IκB expression compared with the control, L41 significantly reduced IκB levels at 10 µM (p = 0.047) compared with the LPS group, whereas lower concentrations (1 and 3 µM) showed no significant effects (Fig. 4D). These findings suggest that L41 modulates inflammatory signaling in BV-2 cells by inhibiting NLRP3 inflammasome activation and exerting a potent inhibitory effect on the IκB pathway at higher concentrations, thereby attenuating LPS-induced neuroinflammation.

L41 attenuates tau phosphorylation, Iba-1, α-syn levels in MPTP-induced PD mice

The experimental schedule applied to 8-week-old C57BL/6 mice is shown in Fig. 5A. L41 (50 mg/kg) was orally administered once daily for 12 days (days 1–12). To induce Parkinson’s disease-like pathology, MPTP (25 mg/kg) was administered intraperitoneally once daily from days 8 to 12. Following the treatment paradigm, neuropathological markers were analyzed by western blotting across distinct brain regions (Fig. 5B). In the striatum, DYRK1A, α-synuclein, and Iba-1 levels differed significantly among groups (DYRK1A: F (2, 6) = 53.73, p < 0.001; α-synuclein: F (2, 6) = 17.48, p = 0.003; Iba-1: F (2, 6) = 16.10, p = 0.004). MPTP administration significantly increased these protein levels compared with the vehicle-treated wild-type (WT) group (DYRK1A: p < 0.001; α-synuclein: p = 0.003; Iba-1: p = 0.01), which were attenuated by L41 treatment (DYRK1A: p < 0.001; α-synuclein: p = 0.012; Iba-1: p = 0.005) (Fig. 5C–E). In the cortex, p-tau (Ser202/Thr205) and total tau levels also differed significantly among groups (p-tau (Ser202/Thr205): F (2, 6) = 11.94, p = 0.008; total tau: F (2, 6) = 8.74, p = 0.017). MPTP treatment significantly increased total tau levels compared with the control (p = 0.014), whereas p-tau (Ser202/Thr205) levels showed a non-significant increasing trend. L41 treatment significantly reduced p-tau (Ser202/Thr205) levels (p = 0.007), while the decrease in total tau did not reach statistical significance (Fig. 5F, G). To further clarify the protective effect of L41 against progressive tau pathology, phosphorylation at the Ser396 site was additionally assessed. The levels of p-tau (Ser396/Ser404) differed significantly among groups (F (2, 6) = 15.89, p = 0.004), with MPTP administration significantly increasing p-tau (Ser396/Ser404) levels compared with the control (p = 0.028). Notably, L41 treatment significantly attenuated this MPTP-induced elevation (p = 0.003; Supplementary Fig. S3). Furthermore, TH levels in the substantia nigra differed significantly among groups (F (2, 6) = 31.94, p < 0.001). MPTP markedly reduced TH expression compared with the WT (p < 0.001), whereas L41 did not restore TH levels compared with the MPTP group (Fig. 5H). Collectively, these findings indicate that L41 effectively suppresses tau phosphorylation, α-synuclein accumulation, and neuroinflammatory markers in PD-related brain regions, although it does not rescue nigral dopaminergic neuronal loss under the present conditions.

Fig. 5.

L41 alleviates MPTP-induced pathological changes in distinct brain regions. (A) Experimental schedule of L41 treatment in the MPTP-induced PD mouse model. (B) Representative western blots of target proteins in the striatum, cortex, and substantia nigra. (C–H) Densitometric quantification of DYRK1A, α-synuclein, and Iba-1 in the striatum; p-tau (Ser202/Thr205) and total tau in the cortex; and TH in the substantia nigra, normalized to GAPDH. Data are expressed as the mean ± SD (n = 3 pooled samples/group; each sample represents tissue from two mice). ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. WT group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. MPTP group. Full-length images of the uncropped western blots (B), including the GAPDH loading WT for each brain region, are available in Supplementary Figure S4

L41 attenuates brain IL-6 levels in MPTP-induced PD mice

To evaluate the central anti-inflammatory effects of L41, cytokine concentrations were measured in the whole brains of MPTP-treated mice. TNF-α levels differed significantly among the groups (F (2, 6) = 7.16, p = 0.025). MPTP significantly increased TNF-α levels compared with those in the WT (p = 0.02), whereas L41 did not reduce TNF-α levels compared with the MPTP group (Fig. 6A). IL-1β levels did not differ significantly among the groups, and no significant changes were observed following either MPTP or L41 treatment (Fig. 6B). In contrast, IL-6 levels differed significantly among the groups (F (2, 6) = 13.02, p = 0.006). MPTP significantly increased IL-6 levels compared with those in the WT (p = 0.009), which were significantly reduced by L41 treatment (p = 0.012) (Fig. 6C). These findings indicate that L41 selectively attenuates MPTP-induced IL-6 elevation in the brain, suggesting a specific modulatory role in neuroinflammatory signaling.

Fig. 6.

L41 selectively reduces IL-6 levels in the brain of MPTP-treated mice. (A–C) Brain levels of TNF-α, IL-1β, and IL-6 following MPTP administration in the presence or absence of L41. Data are expressed as the mean ± SD (n = 3/group). #p < 0.05, ##p < 0.01 vs. WT group; *p < 0.05 vs. MPTP group

L41 improves motor impairments in MPTP-induced PD mice

To determine whether L41 alleviates MPTP-induced motor deficits, behavioral assessments were performed. In the wire hang test, hanging time differed significantly among groups (F (2, 15) = 11.96, p < 0.001). MPTP-treated mice exhibited a significant reduction in hanging time compared with the WT group (p < 0.001), whereas L41 treatment significantly improved this deficit (p = 0.029; Fig. 7A). In the open field test, total distance moved, velocity, and mobile cumulative duration differed significantly among groups (distance: F (2, 15) = 9.52, p = 0.002; velocity: F (2, 15) = 5.03, p = 0.021; duration: F (2, 15) = 5.16, p = 0.019). MPTP administration significantly reduced these locomotor parameters compared with the WT group (total distance moved: p = 0.0015; velocity: p = 0.044; mobile cumulative duration: p = 0.015). L41 treatment significantly restored movement velocity (p = 0.032), whereas changes in total distance moved and mobile duration did not reach statistical significance (Fig. 7B–D). Representative locomotion tracks illustrate the movement deficits induced by MPTP and the partial recovery following L41 administration (Fig. 7E). Collectively, these findings indicate that L41 partially but significantly improves MPTP-induced motor impairments.

Fig. 7.

L41 improves motor impairments in MPTP-induced PD mice. (A) Hanging time in the wire hang test. (B–D) Open field test parameters: (B) total distance moved, (C) velocity, and (D) mobile cumulative duration. (E) Representative locomotor trajectories from each group (WT, MPTP, and L41-treated). Data are expressed as the mean ± SD (n = 6/group). ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. WT group; *p < 0.05 vs. MPTP group

BBB penetration and pharmacokinetics of L41

To determine whether L41 crosses the BBB, its concentrations were measured in plasma and brain tissue at multiple time points following oral administration (50 mg/kg). The concentration–time profiles are presented in Fig. 8, and the corresponding PK parameters are summarized in Table 1. L41 was absorbed, reaching a plasma T_max of 2 h, and concurrently penetrated into the brain, indicating rapid BBB permeability. L41 in both plasma and brain gradually declined thereafter, exhibiting a long elimination half-life of over 7 h, indicative of sustained systemic and central exposure. The infinitive AUC of L41 was 0.09 µg·h/mL in plasma and 0.25 µg·h/g in brain tissue, yielding a calculated Kp of 2.77, which indicates preferential distribution into the brain.

Fig. 8.

Plasma and brain concentration–time profiles of L41 following oral administration in mice. Plasma and brain concentrations of L41 were measured at 1, 4, 8, and 24 h after a single oral dose (50 mg/kg). Data are expressed as the mean ± standard deviation (n = 3)

Table 1.

Plasma and brain pharmacokinetic parameters of L41 following oral administration in mice

Parameters	Plasma	Brain
Cmax (µg/mL or µg/g brain)	0.01 ± 0	0.02 ± 0.02
Tmax (h)	2 ± 1.73	2 ± 1.73
T1/2 (h)	7.9 ± 3.43	7.17 ± 4.14
AUClast (µg·h/mL or µg·h/g brain)	0.08 ± 0.03	0.16 ± 0.13
AUCinf (µg·h/mL or µg·h/g brain)	0.09 ± 0.05	0.25 ± 0.16
Kp		2.77

Discussion

We investigated the neurochemical and functional effects of L41 in experimental parkinsonism, focusing on its integrated modulation of tau pathology and neuroinflammation. While previous studies have reported the individual effects of L41 on tau and inflammatory pathways, its combined impact on these interconnected processes in the context of parkinsonism has not been fully characterized. By integrating biochemical, cellular, and in vivo analyses, the present study provides insights into how L41 simultaneously modulates multiple pathological features relevant to PD. Our findings demonstrate that L41 reduces tau phosphorylation at Ser202/Thr205 and Ser396/Ser404, suppresses tau aggregation in a cell-free system, and attenuates microglial activation, accompanied by improved motor performance in MPTP-treated mice. These results suggest that L41 influences both the proteopathic and inflammatory components of PD-related pathology.

The reduction of tau phosphorylation observed both in vitro and in vivo is consistent with the modulation of kinase-dependent signaling pathways. DYRK1A has been reported to phosphorylate tau at disease-associated residues and participate in inflammatory signaling cascades [14]. In the present study, L41 treatment was associated with decreased DYRK1A expression in the MPTP model, suggesting that modulation of DYRK1A-related pathways may contribute to the observed changes in tau phosphorylation [15]. However, given the complexity of kinase networks regulating tau, the involvement of additional kinases cannot be excluded [16].

Although L41 did not protect SH-SY5Y cells from acute MPP⁺-induced cytotoxicity, this finding indicates that its effects may be more closely related to the modulation of phosphorylation-dependent proteopathy and inflammatory signaling rather than direct mitigation of mitochondrial toxicity. Such pathway-selective modulation distinguishes L41 from nonspecific antioxidants and cytoprotective agents.

L41 exhibited anti-inflammatory effects in BV-2 microglia and reduced markers of microglial activation in vivo. Chronic neuroinflammation is known to exacerbate dopaminergic dysfunction and facilitate pathological protein accumulation; therefore, concurrent attenuation of inflammatory signaling and tau phosphorylation may underlie the functional improvements observed in the MPTP model [17]. Although specific downstream signaling pathways were not directly assessed, the observed reductions in pro-inflammatory mediators suggest the potential involvement of transcriptional programs associated with inflammatory signaling, including pathways related to NLRP3 or related cascades [18].

An important aspect of the present study is the integration of multiple pathological axes associated with PD, including tau dysregulation, neuroinflammatory activation, and kinase-dependent signaling. Rather than targeting a single pathway, L41 demonstrated coordinated modulation of these interconnected processes, including DYRK1A-mediated tau dysregulation and NLRP3-related neuroinflammation. The dissociation between the significant molecular changes and the unchanged dopaminergic markers in the substantia nigra indicates that L41 attenuated specific tau-related and neuroinflammatory pathologies without rescuing established neuronal loss. These findings suggest that L41 may modulate the acute pathological cascade in this model; however, whether it can slow disease progression or reverse established damage remains to be demonstrated in future studies.

Several limitations should be considered when interpreting these findings. The anti-aggregation activity of L41 was assessed using the K18 fragment; its direct interaction with full-length tau isoforms warrants further structural investigation. Biological differences between human and murine tau should also be considered: adult mice predominantly express 4-repeat tau and rarely form mature neurofibrillary tangles, whereas the human brain expresses comparable levels of 3R and 4R tau, which constrains the direct translatability of our findings. Nevertheless, the consistent efficacy of L41 across the human tau-derived K18 fragment, human SH-SY5Y cells, and the in vivo MPTP model supports the relevance of these findings beyond murine-specific tau properties.

From a pharmacokinetic perspective, these effects should be interpreted in the context of L41 systemic exposure and brain distribution. L41 displayed modest systemic exposure with a favorable brain-to-blood distribution profile. Given its reported nanomolar inhibitory potency against DYRK1A, moderate brain exposure may be sufficient to influence kinase-regulated signaling cascades. This suggests that L41 may retain biological activity despite limited absolute brain concentrations. Nevertheless, further studies examining free brain concentrations and direct in vivo target engagement are required to clarify this relationship.

Collectively, these findings indicate that L41 modulates tau aggregation, phosphorylation, and neuroinflammatory responses in experimental parkinsonism in association with improved motor function. Rather than acting through broad cytoprotective mechanisms, L41 appears to influence phosphorylation-dependent and inflammatory pathways that contribute to parkinsonian pathology. Our in vitro findings indicate that L41 does not directly protect MPP⁺-exposed SH-SY5Y neurons from cytotoxicity, yet robustly attenuates microglial activation and the production of pro-inflammatory mediators. This dichotomy is consistent with the pathway-selective action of L41, which targets DYRK1A-mediated tau hyperphosphorylation and neuroinflammatory cascades rather than acting as a general cytoprotective agent. In the integrated in vivo environment of the MPTP model, where microglia, astrocytes, and neurons engage in continuous multi-directional crosstalk, this selective dual action — suppression of tau pathology together with attenuation of neuroinflammation — appears to mitigate the inflammatory microenvironment surrounding dopaminergic neurons and preserve the functional output of the nigrostriatal circuit, as reflected in the significant improvement in motor performance. Notably, the dissociation between unchanged TH cell counts and preserved motor function is consistent with a therapeutic benefit of L41 mediated through modulation of the inflammatory milieu rather than direct neuronal rescue.

A direct in vitro demonstration of microglia-to-neuron protective signaling — for instance, through dual-stimulus co-culture or conditioned-media transfer paradigms — was beyond the scope of the present study. Such reductionist systems will be valuable for further delineating the mechanistic link between L41-mediated microglial modulation and neuronal outcomes, and represent an important direction for future investigation. Furthermore, while the present study primarily evaluated total α-synuclein levels, exploring the potential regulatory effects of L41 on specific α-synuclein phosphorylation sites, such as Ser129, remains a compelling direction for future studies to fully elucidate its multi-target efficacy. Future studies optimizing pharmacokinetic properties and dissecting pathway-specific mechanisms will further clarify its therapeutic potential.

Conclusion

L41 reduced tau phosphorylation, attenuated tau aggregation, and modulated central neuroinflammatory responses in a mouse model of Parkinson’s disease. In vivo, L41 treatment was associated with decreased DYRK1A expression, and these molecular changes were accompanied by improved motor performance. Although it did not protect against MPP⁺-induced cytotoxicity, L41 demonstrated consistent effects on tau-related and inflammatory pathways, supporting its potential as a therapeutic candidate for targeting key pathological processes in PD. Future studies should evaluate its long-term efficacy, optimize its pharmacokinetic profile to enhance brain exposure, and further assess its translational potential for Parkinson’s disease and related tauopathies.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

SBH: investigation, methodology, writing-original draft, HHC, HYC: investigation, methodology, SA, HY: formal analysis, resources, writing - review, JSS: conceptualization, writing - review, funding acquisition, supervision.

Funding

This research was supported by the Korea Drug Development Fund, funded by the Ministry of Science and ICT, the Ministry of Trade, Industry, and Energy, and the Ministry of Health and Welfare (RS-2025-02223246, Republic of Korea).

Data availability

The data presented in this study are available on request from the corresponding author.

Declarations

Ethics approval and consent to participate

All applicable national and institutional guidelines for the care and use of animals were followed. The protocols for animal experiments were approved by the Animal Ethics Committee of Naason Science, Inc with the code number of KBIO-IACUC-2023-053.

Consent for publication

Not applicable.

Conflict of interest

All authors declare that they have no conflicts of interest.