Necrosulfonamide exerts neuroprotective effect by inhibiting necroptosis, neuroinflammation, and α-synuclein oligomerization in a subacute MPTP mouse model of Parkinson’s disease

Parkinson’s disease (PD) is an incurable movement disorder characterized by dopaminergic cell loss, neuroinflammation, and α-synuclein pathology. Herein, we investigated the therapeutic effects of necrosulfonamide (NSA), a specific inhibitor of mixed lineage kinase domain-like protein (MLKL), in a subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. MLKL is an executor of necroptosis, a programmed cell death pathway that causes inflammation. Repeated administration of NSA resulted in the recovery of impaired motor performance and dopaminergic degeneration. Furthermore, NSA inhibited the phosphorylation, ubiquitylation, and oligomerization of MLKL, all of which are associated with MLKL cell death-inducing activity in dopaminergic cells in the substantia nigra (SN). NSA also inhibited microglial activation and reactive astrogliosis as well as the MPTP-induced expression of proinflammatory molecules such as tumor necrosis factor-α, interleukin-1β, inducible nitric oxide synthase, and cystatin F. Furthermore, NSA inhibited α-synuclein oligomerization and phosphorylation in the SN of MPTP-treated mice by inhibiting the activity of glycogen synthase kinase 3β and matrix metalloproteinase-3. In conclusion, NSA has anti-necroptotic, anti-inflammatory, and anti-synucleinopathic effects on PD pathology. Therefore, NSA is a potential therapeutic candidate for PD.


PTM
Post-translational modification SN Substantia nigra Thio-S Thioflavin-S TNF-α Tumor necrosis factor-alpha Ub Ubiquitin Necroptosis is a type of programmed cell death implicated in various pathological conditions, including infection, inflammation, ischemic injury, spinal cord injury, and neurodegeneration [1][2][3][4] . Necroptosis is indicated by plasma membrane disruption and leakage of damage-associated molecular patterns (DAMPs), such as the high mobility group box 1 protein and mitochondrial DNA, which can lead to a robust immune response and inflammation [5][6][7] .
Necrosulfonamide (NSA) is a specific MLKL inhibitor that suppresses necroptosis 9 . Recent studies have shown that NSA has a therapeutic window in rodent models of neurological disorders. Besides suppressing MLKL activity, NSA ameliorated neurological impairment by improving antioxidant capacity in a mouse model of spinal cord injury 30 . NSA also alleviated amyloidopathy and tauopathy in a rat model of Alzheimer's disease 31 . Furthermore, NSA exhibited neuroprotective effects after ischemic brain injury in mice by inducing MLKL ubiquitination and degradation 32 . Moreover, NSA alleviated acute brain injury in a mouse intracerebral hemorrhage model by inhibiting inflammation and necroptosis 4 . However, the potential effect of NSA on the pathophysiology of PD has not yet been reported.
To test an available option for the amending role of NSA in PD pathology, we empirically investigated the effects of NSA on neuroinflammation, α-synuclein pathology, dopaminergic degeneration, and neurobehavioral outcomes in a PD mouse model.

Materials and methods
Reagents and antibodies. Necrosulfonamide (NSA) was obtained from Merck Millipore (Billerica, MA).
MPTP was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). Avidin-biotin horseradish peroxidase (HRP) complex reagent, biotinylated secondary antibodies, diaminobenzidine tetrahydrochloride, and antifade reagent were obtained from Vector Laboratories (Burlingame, CA, USA). Thioflavin S was obtained from Sigma-Aldrich (St. Louis, MO, USA). The primary antibodies used were as follows: anti-TH (Cat# 58844), anti-TNF-α (Cat# 11948), anti-p-α-synuclein (Ser129, Cat# 23706), anti-α-synuclein (Cat# 2642) Animals. Adult male C57BL/6 mice (7 weeks old) were purchased from Orient Bio, Inc. (Seongnam, South Korea), a branch of the Charles River Laboratories. The mice were maintained at 21 °C under a 12-h light: dark cycle and had ad libitum access to water and rodent chow. Every effort was made to minimize animal suffering. All experiments were performed in accordance with the National Institutes of Health and Ewha Womans University guidelines for laboratory animal care and use, and the study was approved by the Institutional Animal Care and Use Committee of the Medical School of Ewha Womans University (#EUM 20-022). The study was carried out in compliance with the ARRIVE guidelines.
MPTP administration and experimental procedure. The mice were randomly divided into four groups: CON, control; M/P, MPTP + probenecid; M/P + NSA; and NSA (CON, N = 49; M/P, N = 50; M/P + NSA, N = 50; NSA, N = 49). MPTP and probenecid were administered intraperitoneally for five consecutive days. In brief, mice were administered MPTP (25 mg/kg i.p.) twice a day with a 6-h interval on day 1, and once a day on days 2-5. The mice were treated with probenecid (250 mg/kg/day, s.c.) 30 min before MPTP injection to promote the prolonged neuronal retention of MPTP. Thereafter, NSA was intraperitoneally injected for three weeks (0.5 mg/kg/day for three days and 1 mg/kg/day for seven days every two days), and the animals were subjected to behavioral tests two and three days after the last NSA treatment. The mice were decapitated two days after the completion of the behavioral tests (Fig. 1a).
Behavioral tests. An accelerated rotarod test was performed to assess motor coordination in the mice.
Five days before drug treatment, all mice were trained on the rotarod (4-13 rpm) until they remained on the apparatus for 300 s without falling. The rotarod was accelerated from 4 to 40 rpm over a 300-s period. The mice were subjected to three trials at 15-min intervals. The retention times on the rods in each trial were recorded. To evaluate akinesia, a pole test was implemented 33 . The time taken for each mouse to descend the pole was recorded. Each mouse was subjected to three trials, and the average was recorded. For the open field test (OFT), mice were placed in the center of a clear Plexiglas box made of white nonporous plastic (50 cm × 50 cm × 38 cm). The total distance traveled and velocity within 5 min were measured using the EthoVision 17 program (Noldus, Wageningen, Netherlands).
Histological analysis. Brain sections were obtained according to a previously described method (striatum: every six sections/brain; substantia nigra [SN]: every three sections/brain) 33 . The sections were subjected to endogenous peroxidation inactivation with 3% hydrogen peroxide, and non-specific binding was blocked with 4% bovine serum albumin. The sections were incubated overnight with primary antibodies and then with biotinylated secondary antibodies for 1 h at 25 °C on the following day. The sections were subsequently incubated with an avidin-biotin-HRP complex reagent solution for 1.5 h, and a peroxidase reaction was performed using diaminobenzidine tetrahydrochloride. For double immunofluorescence, non-specific binding was blocked, and the sections were incubated with primary antibodies, followed by fluorochrome-conjugated secondary antibodies. For thioflavin-S (Thio-S) staining, sections were incubated with 0.005% Thio-S for 8 min, washed twice with 50% ethanol for 5 min, and washed with phosphate-buffered saline. The tissue was then mounted using an antifade reagent (Vector Laboratories). Digital images of immunohistochemical and immunofluorescence staining were captured using a Leica DM750 microscope (Leica Microsystems). Quantification was performed using the ImageJ software (NIH, Bethesda, MD, USA).

Western blot analysis.
Tissues collected from the striatum and SN were homogenized in an ice-cold lysis buffer 34 . Subsequently, the samples were vortexed vigorously at 10-min intervals and then incubated for 30 min at 4 °C. The samples were then centrifuged at 20,000×g for 30 min, and the supernatant was collected (S1). The pellet was resuspended in lysis buffer, and the supernatant (S2) was obtained by repeated centrifugation. These protein extracts (S1 and S2) represented the Triton X-100-soluble fraction. Protein samples (50-100 μg) were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with primary antibodies according to the manufacturer's instructions. To detect MLKL tetramer, the final pellet (insoluble fraction) obtained after S1 and S2 fractionation was resuspended through sonication in 2% SDS lysis buffer, and the lysate was loaded with non-reducing sample buffer. The membranes were thoroughly washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies. Subsequently, the blots were developed using an enhanced chemiluminescence detection kit (Thermo Fisher Scientific, Waltham, MA, USA). Using ImageJ software, the density of specific target bands was normalized against that of β-actin for quantification.
Co-immunoprecipitation assay. Immunoprecipitation of pMLKL was performed according to the manufacturer's instructions (Dynabeads Protein G Immunoprecipitation Kit; Cat# 10007D; Thermo Fisher Scientific, Waltham, MA, USA). Four samples were pooled per group (N = 4). The tissue samples were homogenized in RIPA lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, and a proteinase inhibitor cocktail). Anti-pMLKL (3 μg) antibodies were captured through incubation with binding buffer containing the magnetic bead for 6 h at 4 °C. The immune complexes were incubated with cell lysates (1 mg) from the SN overnight at 4 °C. Protein G complexes were precipitated, washed three times in washing buffer, and eluted through boiling in the elution buffer and 5 × reducing SDS sample buffer for 10 min at 95 °C. Finally, the immunoprecipitated samples were immunoblotted using an anti-ubiquitin antibody (1:500) with 10% SDS-PAGE. Statistical analysis. Statistical analyses were performed using SPSS for Windows (version 18.0; SPSS Inc., Chicago, IL, USA). Differences among groups were analyzed using one-way analysis of variance. Post hoc comparisons were conducted using Tukey's test. Pearson's correlation coefficient was used to analyze the correlations between variables. All values were presented as the means ± standard errors of the mean (SEMs). At p < 0.05, differences were considered statistically significant.

NSA restored dopaminergic degeneration in nigrostriatal pathway and improved behavioral outcomes.
To examine the effects of NSA on dopaminergic degeneration in the striatum and SN and motor performance in a PD mouse model, mice were injected with MPTP for 5 days and then treated with NSA (0.5 mg/kg/day for 3 days and 1 mg/kg/day for 7 days, every second day of i.p. injection), followed by behavioral tests and sacrifice, as shown in Fig. 1a. Motor coordination and akinesia were assessed using accelerating-speed rotarod and pole tests to investigate the motor function-restoring role of NSA in the MPTP-treated mice. In the  NSA inhibited MLKL expression, oligomerization, and phosphorylation in the SN region of MPTP-treated mice. Next, we verified the involvement of necroptotic signaling in dopaminergic neuronal cell death, and the potential role of NSA in the SN of a subacute MPTP mouse model of PD. Since MLKL undergoes oligomerization and phosphorylation during necroptotic signaling 9,12 , we examined the effect of NSA on the PTMs of MLKL. MLKL protein expression significantly increased in response to MPTP toxicity, which was mitigated by NSA treatment (Fig. 2a,b; F 3, 20 = 52.80, p < 0.01). The smeared bands appear to be the ubiquitylated form of MLKL. In the insoluble fraction, MLKL tetramer expression was increased by MPTP and inhibited by NSA (Fig. 2c,d; F 3, 20 = 15.36, p < 0.01). Accordingly, western blot analysis using a conformation-sensitive A11 antibody (specifically recognizing oligomeric proteins) revealed that MPTP treatment increased MLKL oligomer formation, which was reduced by NSA (Fig. 2e,f; F 3, 20 = 16.35, p < 0.01). The expression of RIPK1 and RIPK3 proteins was significantly enhanced in the SN region of MPTP mice, but NSA did not reduce these levels (Fig. S1a,b). Moreover, pMLKL + (Ser345-p) intensities and the number of pMLKL + /TH + cells in the MPTP group were higher than those in the control group, and NSA treatment significantly reduced the MPTP-induced increase in the pMLKL population ( Fig. 2g-

NSA interrupted the ubiquitylation of MLKL in the SN region of MPTP-treated mice.
Endogenous MLKL is ubiquitylated at some lysine residues, which is required for retaining the cell death-inducing activity of MLKL 13 . To examine the ubiquitylation state of phosphorylated MLKL protein and the impact of NSA in the MPTP-intoxicated SN area, double immunofluorescence staining with anti-pMLKL and anti-Ub antibodies was performed. Ub + punctae intensity and Ub + /pMLKL + area were significantly enhanced in the SN region of MPTP mice, and NSA significantly diminished both Ub + intensity and Ub + /pMLKL + area (Fig. 3a-d; for Ub + , F 3, 26 = 8.67, p < 0.01; for Ub + /pMLKL + , F 3, 26 = 13.86, p < 0.01). Furthermore, we verified that NSA suppressed ubiquitylated pMLKL levels by performing a co-immunoprecipitation assay (four samples per group were pooled; Fig. 3e). These results indicate that NSA reduces the ubiquitylation of p-MLKL, which is necessary for necroptotic execution in PD pathology.

Discussion
PD causes motor performance defects due to basal ganglion dysfunction, which is characterized by dopaminergic cell death, neuroinflammation, and α-synucleinopathy. Heterogeneous cell death, such as apoptosis and necroptosis, occurs during PD progression 22,23,43,44 . Necroptosis is an inflammatory form of programmed cell death tightly regulated by the RIPK1/RIPK3/MLKL axis. As MLKL is an executor in necroptotic signaling, targeting MLKL has been suggested as an attractive strategy for restraining the pathological progression of PD. In this study, we tested the therapeutic efficacy of NSA, a pharmacological inhibitor of MLKL, in a mouse model of PD. NSA restored motor coordination and akinesia, and reversed dopaminergic cell loss in the SN area and the blunted neurotransmission toward the striatum. These results suggest the involvement of necroptosis in dopaminergic cell loss and the protective role of NSA in mice with PD. PTMs of MLKL, including phosphorylation and ubiquitylation, are required for its cell lysis-inducing activity [11][12][13] . RIPK3-dependent phosphorylation at Ser345, Ser347, and Thr349 in the pseudokinase domain within the C-terminal region of murine MLKL occurs upon necroptotic stimulation, and MLKL phosphorylation at Ser345 promotes oligomerization, facilitating membrane targeting [45][46][47] . MLKL is conjugated to the K63-linked ubiquitin chain under necroptotic signaling, and ubiquitylation of MLKL at Lys219 induces optimal oligomerization at the plasma membrane and augments MLKL activity to potentiate cytotoxic effects 11,13 . This study revealed more pMLKL + /TH + and pMLKL + /Ub + cells measured through histological data, and higher ubiquitylated pMLKL levels measured using co-immunoprecipitation assay in MPTP-intoxicated SN than in the control group, and the reversal effects of NSA exposure implied that repeated treatment with NSA hinders the cell death-inducing activity of MLKL in PD mice. Furthermore, our finding that NSA had no effect on the MPTP-induced increase in RIPK1 and RIPK3 protein levels corresponded to previous findings in rodent models 4,31 , indicating that NSA acts specifically on MLKL in vivo. NSA treatment reduced the MPTP-induced increase in MLKL protein levels in the MPTP mice. In protein homeostasis, functional proteins are retained in their proper contents and cellular compartments, where misfolded, damaged, or redundant proteins undergo protein degradation via proteolytic machinery 48 . Moreover, active MLKL reportedly forms amyloid-like polymers and attenuates autophagic flux, rendering MLKL resistant to protease digestion 49,50 . In this study, we demonstrated that NSA reduced MLKL tetramer levels in the insoluble fraction and MLKL oligomer formation in the SN of MPTP-treated mice. Based on previous findings, the inhibition of active MLKL by NSA may promote protein turnover and block the further expansion of necroptotic signaling.
The study's main finding was that NSA inhibits α-synuclein oligomerization and neuroinflammation in a mouse model of PD. Some research has suggested a link between α-synuclein aggregation and neuroinflammation in PD pathology, in which α-synuclein oligomers activate microglia via toll-like receptor 2, and reactive oxygen species released by activated microglia facilitate α-synuclein aggregation [51][52][53] . On the other hand, necroptosis can trigger inflammation via the release of proinflammatory factors such as DAMPs [5][6][7] . Moreover, active MLKL induces NLRP3 inflammasome activation, leading to secretion of IL-1β 1 . In this study, NSA-mediated inhibition of active MLKL alleviated neuroinflammation in the MPTP-intoxicated SN area, as indicated by decreased microglial reactivity and proinflammatory mediators such as iNOS, IL-1β, TNF-α, and CST7. Moreover, the detrimental role of reactive astrocytes in PD pathogenesis is reportedly associated with overexpression and release of S100β in PD animal models and patients with PD [54][55][56] . In this study, NSA suppressed reactive astrogliosis in MPTP-intoxicated SN and striatum, as indicated through reduced S100β + intensity and S100β + /GFAP + area. These results imply that NSA ameliorates microglial activation-and reactive astrogliosis-mediated neuroinflammation, thereby restoring neurodegenerative phenotypes. Furthermore, NSA inhibited the MPTP-induced expression of active GSK3β and MMP3, which are increased under neuroinflammatory conditions 33,57,58 . Thus, by inhibiting GSK3β and MMP3 activity, NSA's anti-inflammatory effects may contribute to the relief of α-synuclein pathology. Previous research and our findings suggest that repeated NSA administration may disrupt the forward The proposed mechanism of the NSA is shown in Fig. 8b.
In conclusion, this is the first study to show that NSA has a therapeutic effect on PD-like behavioral defects and pathophysiology, such as necroptosis, neuroinflammation, and α-synuclein oligomerization, in a subacute MPTP mouse model of PD. Consequently, our findings add to the evidence that necroptosis plays a role in PD pathogenesis and support the anti-parkinsonian effect of NSA.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.