The Myelin-Derived Peptide NSDP1 Promotes Remyelination and Attenuates Neuroinflammation in Cuprizone-Induced Demyelination via Suppression of the cGAS- STING Pathway

The Myelin-Derived Peptide NSDP1 Promotes Remyelination and Attenuates Neuroinflammation in Cuprizone-Induced Demyelination via Suppression of the cGAS- STING Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Myelin-Derived Peptide NSDP1 Promotes Remyelination and Attenuates Neuroinflammation in Cuprizone-Induced Demyelination via Suppression of the cGAS- STING Pathway Junjie Yang, Qinze Chi, Minhao Huang, Jingcong Lu, Xiaohua Dong, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8298082/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Multiple sclerosis (MS) involves demyelination and neuroinflammation. Proteomic analysis identified significant downregulation of the myelin basic protein-derived peptide NSDP1 in the cuprizone (CPZ)-induced demyelination mouse model. In vitro , NSDP1 suppressed LPS-induced microglial activation in BV2 cells, reducing reactive oxygen species (ROS) production, downregulating pro-inflammatory markers (iNOS, TNF-α, IL-1β), and upregulating the expression of anti-inflammatory marker Arg-1. In vivo , NSDP1 administration via intracerebroventricular injection significantly mitigated CPZ-induced weight loss and demyelination in the corpus callosum. NSDP1 promoted functional remyelination, restoring expression of myelin proteins (MAG, MOG), increasing oligodendrocyte precursor cell (OPC) density, improving myelin sheath ultrastructure, and enhancing axonal myelination efficiency. Furthermore, NSDP1 attenuated CPZ-induced reactive gliosis, reducing both microglial activation and astrocytic reactivity in the corpus callosum. RNA sequencing revealed that NSDP1 modulated myelination-related pathways and correlated with improved locomotor recovery. Mechanistically, NSDP1 exerted its anti-inflammatory effects by inhibiting the cGAS-STING signaling pathway, as shown by reduced cGAS and STING expression in LPS-stimulated BV2 cells. The effects of NSDP1 on ROS and pro-inflammatory cytokine release were reversed by the STING activator DMX and mimicked by the STING inhibitor SN-011. Collectively, these findings identify NSDP1 as a downregulated myelin-derived peptide with potent therapeutic potential, which promotes remyelination and suppresses neuroinflammation in demyelinating diseases by inhibiting the cGAS-STING pathway. NSDP1 Peptide neuroinflammation Multiple Sclerosis cGAS-STING Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Multiple Sclerosis (MS) is an autoimmune disease affecting the central nervous system (CNS), specifically targeting the myelin sheath-the protective covering of nerve fibers ( 1 ). The global incidence of MS exhibits significant geographical variation, with reported rates approximating 5 to 300 per 100,000 individuals, and a female predominance with an incidence over two-fold higher than that in males( 2 ). Inflammatory demyelinating lesions disrupt the periventricular region, proximal cortex, spinal cord, optic nerve, and cerebellum, leading to a wide range of symptoms( 3 ). These symptoms include eye pain, vision loss, weakness or sensory changes in the body, dizziness, balance difficulties, and memory impairment ( 4 ). Newer disease-modifying therapies (DMTs) are available for treating different types of MS, including relapsing–remitting MS, active secondary progressive MS, and primary progressive MS. However, the efficacy of current DMTs ranges only from 29% to 68%, and they are associated with various adverse effects such as bradycardia, infections, heart blocks, and infusion reactions( 5 ). Therefore, there is an urgent need to identify more effective therapeutic targets and treatments for MS. Neuroinflammation plays a vital role in the pathogenesis of MS. The infiltration of immune cells into the CNS and activation of CNS-intrinsic microglia and astrocytes is the dominant feature of MS( 6 , 7 ). Although early intervention with peripherally acting therapeutics that deplete immune cells or impede their trafficking into the CNS can effectively mitigate lesion formation and disease relapses ( 8 ), the persistent activation of microglia emerges as a pivotal driver of pathogenic processes( 9 ). It was reported that BTK (Bruton's tyrosine kinase) inhibitors targeting microglia can effectively suppress neuroinflammation and slow the progression of progressive MS( 10 ). Therefore, modulating the overactivation of immune and inflammatory responses represents a promising therapeutic strategy against MS ( 11 ). Peptides are a class of short chains of amino acids which play an important role in a multitude of processes in human development, tissue homeostasis, and cell metabolism. The peptide therapeutics started in 1922 with the use of insulin for treatment of type 1 diabetes( 12 ). Until now, over 80 peptide drugs have been available on the global market, and more than 150 peptide drugs are in clinical trials ( 13 , 14 ). It was reported that MBP fragments suppressed the disease significantly ( 15 , 16 ). However, whether other MBP-derived peptides participate in the pathogenesis of MS remains largely unknown. In our previous study, we identified dysregulated peptides in the corpus callosum of mouse models of MS subjected to a cuprizone (CPZ) diet, which including the MBP. However, the specific role of these MBP derived dysregulated peptides remains need for further investigation. In this study, we found that MBP-derived peptide NSDP1, which is significantly downregulated in the corpus callosum of CPZ diet MS mice, plays a critical role in inhibiting neuroinflammation and promoting myelin remyelination. Administration of NSDP1 exerts potent anti-inflammatory effects in vitro by suppressing LPS-induced microglial activation. Moreover, in vivo administration of NSDP1 effectively mitigated CPZ-induced demyelination and promoted functional remyelination in the corpus callosum of MS mice model. Furthermore, NSDP1 treatment enhanced locomotor recovery and modulated myelination-related gene pathways. Mechanistically, NSDP1 exerts its remyelination effects by regulating cGAS-STING signaling, thereby reducing microglia activation and inhibiting neuroinflammation. These findings suggest NSDP1 as a novel and promising therapeutic candidate for MS treatment. Materials and methods Cell culture and treatment The mouse microglia cell line BV2 cells were obtained from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd (ZQ0397, Shanghai, China). The cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM, Gibco,11965-092, USA) supplemented with 1% penicillin-streptomycin (PS) and 10% fetal bovine serum (FBS, Gibco, 10099141C, USA). BV2 cells were cultured in the incubator with 5% CO₂ at 37°C. The BV2 cells were treated with 100 ng/mL LPS (Sigma, L2880, USA) for 24 hours. 100 nM, 200 nM, or 400 nM NSDP1 peptides were combined with 100 ng/mL LPS-treated BV2 cells for 24 hours. 400 nM Scr peptides were combined with 100 ng/mL LPS-treated BV2 cells for 24 hours. 1 µM SN-011 was exposed to LPS-treated BV2 cells for 6 hours. 100 µg/mL Vadimezan (DMX, MedChemExpress, China) was exposed to NSDP1 peptides treated BV2 cells for 24 hours. NSDP1 Peptide Synthesis The NSDP1 peptide, which derived from mouse Myelin basic protein isoform 1(MBP, NCBI Reference Sequence: Np_001020422.1) 33–45 (Sequence: DTGILDSIGRFFS), Scrambled peptides (Scr, Sequence: ARFGGDLGEGE) were used in this study. NSDP1 peptide and the Scr peptides were obtained from Shanghai Kaitaibio Co., Ltd. Briefly, rink amide resin (3 g) was first equilibrated in dichloromethane for two hours. After draining, the support was washed repeatedly with N, N-dimethylformamide and dried. For the initial attachment, Glycine and HOBt were dissolved in DMF. DIC was introduced, and the activated mixture was combined with the solid support. Three volumes of DMAP were added, and the conjugation proceeded at 30°C for 4 hours. Remaining reactive sites were subsequently capped using a solution of acetic anhydride and DIEA in DCM. The Fmoc protecting group was removed with 20% piperidine in DMF. Following this deprotection step, the resin was thoroughly washed. Subsequent amino acid residues were connected analogously. HOBt, DIC, and DMAP were employed for activation. Completion of each coupling was verified via the ninhydrin assay. After every successive amino acid incorporation, the Fmoc group was cleaved off. The final product was liberated from the solid support using a TFA-based cleavage cocktail containing TIS, EDT, and water. The crude material was precipitated in cold diethyl ether. Purification was performed using preparative HPLC, and the pure product was obtained as a lyophilized powder. A cell-penetrating sequence (9x Arg) was incorporated at the C-terminus of all analogues. CCK-8 assay Cell viability was assessed using a CCK-8 assay kit (Beyotime, China). In brief, BV2 cells were plated in 96-well plates at 5×10⁴ cells per well and cultured under standard conditions (37°C, 5% CO₂). Following experimental treatments, the cells were supplemented with 10 µL of CCK-8 solution per well and incubated for 1 hours. Absorbance at 450 nm was then recorded using the microplate reader. LC-MS/MS Analysis Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on a TripleTOF 5600 system (SCIEX, USA). Peptide extracts from the mouse corpus callosum, obtained from both CPZ-induced and control diet-fed groups, were dissolved in 2% acetonitrile containing 0.1% formic acid, loaded onto a C18 trap column (5 µm, 10 µm × 20 mm), and separated on a C18 analytical column (3 µm, 75 µm × 150 mm) at a flow rate of 30 nL/min over a 90-minute gradient. Mass spectrometry analysis employed an information-dependent acquisition (IDA) mode. Full-scan MS1 spectra (350–1500 m/z) were acquired with 250 ms accumulation time, followed by the collection of MS2 spectra for up to 30 precursor ions (100–1500 m/z) with 50 ms accumulation time. A dynamic exclusion window of 15 seconds was applied to prevent repeated sequencing. Animals Six-week-old male C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed for two weeks under controlled conditions (22°C, 40–60% humidity, 12 h light/dark cycle). At eight weeks of age, mice were randomly assigned to three experimental groups (n = 15/group): the control group received standard rodent chow, while the cuprizone (CPZ) model group was fed a 0.2% CPZ-containing diet (Sigma, St. Louis, MO, USA) for eight weeks to induce demyelination. For the NSDP1 and relative control Scr peptide-treated group, NSDP1 and Scr peptide were administered via weekly intracerebroventricular injection at a concentration of 0.2 µmol in a 5 µL volume from the sixth to eighth week. All mice were weighed weekly throughout the study period. The experimental protocol received approval from the Animal Ethics Committee of Tongren Hospital, China (Approval No. 2022-045-01). Luxol Fast Blue (LFB) staining Paraffin-embedded sections were dewaxed and rehydrated through sequential immersion in Xylene I (20 min), Xylene II (20 min), absolute ethanol I (5 min), absolute ethanol II (5 min), and 75% ethanol (5 min), followed by rinsing in water. For myelin staining, sections were immersed in pre-warmed (65°C, 30 min) Myelin Stain Solution A (G1030, Servicebio, China) within a covered staining chamber for 60 min at 65°C, then rapidly rinsed in tap water. Differentiation was performed by briefly immersing sections (< 2 s) in pre-heated Myelin Stain Solution B (G1030, Servicebio, China), immediately transferring to Myelin Stain Solution C (15 s) (G1030, Servicebio, China), and water-rinsing to terminate differentiation. This differentiation cycle was repeated under microscopic monitoring until myelin sheaths appeared blue against a near-colorless background. Sections were dehydrated through absolute ethanol I (5 min), absolute ethanol II (5 min), absolute ethanol III (5 min), cleared in Xylene I (5 min) and Xylene II (5 min), and mounted with neutral balsam. Stained sections were microscopically examined, and images were acquired for analysis. Measurement of Intracellular Reactive Oxygen Species (ROS) Levels ROS generation was quantified using the DHE-based ROS Assay Kit (Applygene Technologies Inc., C1300-2, China) following the manufacturer's protocol. Briefly, cells were loaded with 10 µM dihydroethidium (DHE) probe and incubated for 30 minutes at 37°C under dark, humidified conditions. Fluorescence images were captured using a Leica SP8 confocal laser scanning microscope, with subsequent quantitative analysis performed in ImageJ. Immunofluorescence Staining Cultured BV2 microglia and brain tissue sections underwent immunofluorescence staining using standardized protocols. BV2 cells were fixed with 4% paraformaldehyde (PFA), blocked in 5% bovine serum albumin (BSA), and incubated overnight at 4°C with primary antibodies against iNOS (Proteintech, 22226-1-AP, China) and Arg1 (Novus Biologicals, NB100-59740, USA), cGAS (Proteintech, 26416-1-AP, China), and STING (Abcam, AB288157, USA). Following PBS washes, species-matched Alexa Fluor-conjugated secondary antibodies (Proteintech, SA00013 series, China) were applied for 2 hr at room temperature. Nuclei were counterstained with DAPI (Beyotime, P0131, China). For brain tissue analysis, 30-µm cryosections were prepared from tissues post-fixed in 4% PFA for 24 hr and cryoprotected in 30% sucrose. Sections were immunolabeled with GFAP (Cell Signaling Technology, 3670), Iba1 (Cell Signaling Technology, 17198), MAG (Cell Signaling Technology, 9043S), MOG (Cell Signaling Technology, 45268S), and Pdgfrα (Abcam, AB203491) antibodies using identical secondary antibody and DAPI protocols. All samples were imaged using the Leica SP8 confocal microscope. Transmission Electron Microscopy (TEM) of Corpus Callosum Myelin Corpus callosum tissues were processed for ultrastructural analysis using TEM. Mice underwent transcardial perfusion with heparinized saline followed by primary fixation in 3% glutaraldehyde/0.1 M phosphate buffer (pH 7.4) for 24 hr at 4°C. Tissues were post-fixed in 1% osmium tetroxide for 2 hr, dehydrated through graded ethanol series and embedded. Myelinated axons in the callosal midline were examined using the Tecnai G2 20 Twin TEM (FEI). Myelin integrity was quantified by measuring G-ratios (inner axon diameter/total myelinated fiber diameter). RNA-sequencing (RNA-seq) analysis RNA-seq was performed as we previously reported ( 17 ). Corpus callosum tissues from NSDP1-treated mice and CPZ model mice were harvested for RNA-seq analysis. RNA-seq analysis was conducted by OE Biotech Co., Ltd. (Shanghai, China) according to their stranded protocol. Differential gene expression analysis used the DESeq2 algorithm, with significant differentially expressed genes (DEGs) identified under thresholds of |log₂(fold change) | > 1 and adjusted p-value < 0.05. Functional enrichment analysis of DEGs interrogated KEGG, Reactome, and WikiPathways databases using clusterProfiler in R (version 3.2.0). ELISA Concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in BV2 lysates were quantified using mouse-specific ELISA kits (IL-1β: Mlbio ml098416; TNF-α: Mlbio ml002095) per manufacturer's protocol. Following centrifugation of cell lysates (12,000 × g, 20 min, 4°C), supernatants were analyzed in technical duplicates. Absorbance at 450 nm (reference 570 nm) was measured using the Multiskan FC microplate reader (Thermo Fisher), with analyte concentrations calculated against standardized curves. Quantification and statistical analysis All datasets were analyzed in GraphPad Prism 9.0 using unpaired Student's t-tests for dual-group comparisons and one-way ANOVA with Tukey's post-hoc testing for multi-group analyses, with statistical significance defined as p < 0.05, data are presented as mean ± standard deviation (SD) with error bars. Results NSDP1 is Downregulated in CPZ- induced MS mice model and Suppresses LPS-Induced Microglial Activation In Vitro Proteomic analysis of the corpus callosum in the cuprizone-induced demyelination mouse model identified that the peptide DTGILDSIGRFFS, derived from residues 33–45 of myelin basic protein (MBP), was significantly downregulated. We designated this nervous system-derived peptide as NSDP1 (nervous system derived peptide 1) hereafter (Fig. 1 A-B). To determine whether NSDP1 involved in the pathogenesis of MS, NSDP1 was treated to BV2 cells with the concentrations 100 nM, 200 nM, and 400 nM. CCK-8 assay showed that NSDP1 exerted no cytotoxicity on BV2 cells (Fig. 1 C). In addition, administration of the NSDP1 peptide (400 nM) significantly suppressed LPS-induced ROS production compared with the Scr peptide control (Fig. 1 D-E). Moreover, NSDP1 significantly decreased the expression of the pro-inflammatory marker iNOS and increased the expression of the anti-inflammatory marker Arg-1 (Fig. 1 F-H). Furthermore, NSDP1 treatment also markedly inhibited the LPS-induced secretion of pro-inflammatory cytokines TNF-α and IL-1β (Fig. 1 I). Taken together, these findings suggest that identify NSDP1 as a myelin-derived peptide deficient in demyelination, which exhibits potent anti-inflammatory effects by modulating microglial activation and polarization in vitro . NSDP1 Treatment mitigate Cuprizone-Induced Demyelination To evaluate the therapeutic potential of NSDP1 in vivo , we utilized the CPZ-induced demyelination model. Mice received a 0.2% CPZ diet for 8 weeks and were administered NSDP1 or Scr peptides via intracerebroventricular (i.c.v.) injection at 6 and 7 weeks (twice total; Fig. 2 A). The results shown that CPZ treatment induced the significant decrease in body weight starting at week 5. However, NSDP1 administration significantly reversed this weight loss by week 8 compared with the Scr-treated CPZ group (Fig. 2 B). Furthermore, Luxol Fast Blue (LFB) staining of the corpus callosum revealed severe demyelination in CPZ-treated mice after 8 weeks of CPZ exposure. This demyelination was significantly attenuated by NSDP1 treatment (Fig. 2 C). Quantitative analysis confirmed that NSDP1 significantly restored myelination ( p < 0.01) compared to CPZ controls (Fig. 2 D). To further determine whether NSDP1 promotes remyelination in the cuprizone-induced demyelination model, we quantitatively assessed myelin-associated proteins and myelin sheath ultrastructure. Immunofluorescence analysis revealed significant recovery of myelin-associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG) expression in the corpus callosum of NSDP1-treated CPZ mice versus CPZ + Scr groups (Fig. 3 A-C). Notably, NSDP1 administration significantly increased Pdgfrα⁺ oligodendrocyte precursor cell (OPC) density compared to CPZ + Scr mice (Fig. 3 D-E). In addition, Ultrastructural analysis by TEM demonstrated that cuprizone feeding induced characteristic pathologies including demyelination, myelin decompaction, axonal swelling, and distorted myelin profiles (Fig. 3 F). In contrast, NSDP1 treatment restored compact myelin lamellae and normalized myelinated axon morphology in the corpus callosum. Consistent with this finding, g-ratio quantification confirmed significantly improved axonal myelination efficiency in NSDP1-treated mice (Fig. 3 G). Collectively, these results indicate that NSDP1 administration promotes functional remyelination through increased OPC number and myelin restoration. NSDP1 Attenuated Glial overactivity in the CPZ Mice Accumulating evidence suggests that microglial and astrocytic activation within the corpus callosum contributes to demyelination in the CPZ mouse model. To assess the effects of NSDP1 administration on reactive gliosis in MS mice, we performed IF staining for Iba1 (microglia) and GFAP (astrocytes) in control, CPZ-fed, and CPZ-fed + NSDP1-treated mice. CPZ exposure significantly increased the density of Iba1 + microglia within the corpus callosum, which exhibited characteristic morphological activation features, including increased branch endpoints and enlarged somata. NSDP1 treatment significantly attenuated these morphological changes, reducing both branch complexity and somatic hypertrophy, indicative of suppressed microglial activation (Fig. 4 A-B). Furthermore, CPZ feeding dramatically upregulated GFAP expression, reflecting astrocyte reactivity, whereas NSDP1 administration significantly reduced GFAP expression in the corpus callosum (Fig. 4 A, C). These findings demonstrate that NSDP1 effectively mitigates CPZ-induced neuroinflammation by attenuating both microglial and astrocytic activation. NSDP1 involved in the functional recovery by RNA-seq detection To further elucidate the functional role of NSDP1 in cuprizone-induced demyelination, RNA sequencing was performed on corpus callosum tissue from NSDP1-treated mice compared to CPZ-exposed controls. Analysis revealed 61 significantly dysregulated genes ( p 1), comprising 31 downregulated and 30 upregulated transcripts (Fig. 5 A-B). KEGG pathway enrichment demonstrated that NSDP1 treatment potentiated myelination-related pathways (Fig. 5 C) and enhanced locomotor functional recovery, as evidenced by improved adult walking behavior (Fig. 5 D). These findings indicate that NSDP1 promotes functional restoration in CPZ-demyelinated mice and critical for remyelination and neural repair. NSDP1 suppresses neuroinflammation via modulating the cGAS-STING signaling pathway It has been reported that the cGAS-STING signaling pathway plays a crucial role in neuroinflammation. To determine whether NSDP1 reduces microglial activation by modulating the cGAS-STING signaling pathway, the expression levels of cGAS and STING were detected in LPS-induced BV2 cells incubated with NSDP1. The results demonstrated that the expression of cGAS and STING was significantly downregulated in NSDP1-treated LPS-induced BV2 cells (Fig. 6 A-D).​ To further verify whether NSDP1 regulates inflammatory responses through the cGAS-STING signaling pathway, the STING inhibitor SN-011 and STING activator Vadimezan (DMX) were employed in LPS-induced BV2 cells. ROS staining revealed a marked reduction in ROS levels in both NSDP1-treated and SN-011-treated BV2 cells. In contrast, ROS staining was significantly enhanced in BV2 cells cotreated with NSDP1 and DMX (Fig. 6 E-F). Moreover, ELISA results showed that the release of proinflammatory factors TNF-α and IL-1β was significantly downregulated in SN-011-treated LPS-induced BV2 cells, while their release was upregulated in LPS-induced BV2 cells cotreated with NSDP1 and DMX (Fig. 6 G-H).​ Collectively, these results indicate that cGAS-STING signaling pathway serves as a critical downstream mediator through which NSDP1 exerts its anti-inflammatory effects in microglia. Discussion Multiple sclerosis (MS), a chronic immune-mediated disorder of the central nervous system and a leading global cause of neurological disability in young adults, remains a major clinical challenge. In this study, we identified an MBP-derived peptide—designated NSDP1-that demonstrates significant anti-neuroinflammatory activity in MS. NSDP1 treatment markedly attenuated LPS-induced ROS generation, downregulated pro-inflammatory factor expression, and promoted M2 microglial polarization. Furthermore, NSDP1 effectively alleviated corpus callosum demyelination by suppressing CPZ-activated microglial and astrocytic responses, concomitant with upregulated Pdgfrα, MAG, and MOG expression. Mechanistic investigations revealed that NSDP1-mediated remyelination critically depends on modulation of the cGAS-STING signaling axis. These findings collectively establish NSDP1 as a promising therapeutic candidate for MS, whose modulation of the cGAS-STING pathway may address the pressing need for targeted remyelination strategies. Peptides typically consist of fewer than 50 amino acids, with a molecular weight ranging from 500 to 5000 Da ( 18 , 19 ). Endogenous peptides originate either from proteolytic degradation of intracellular proteins or via direct translation of RNA-encoded sequences( 20 ). Peptides possess intrinsic advantages, such as high specificity, good efficacy, low immunogenicity, and membrane permeability. Over 80 therapeutic peptides act as hormones, neurotransmitters, growth factors, ion channel ligands, or anti-infective agents, targeting conditions including cancer, metabolic diseases, and neurodegenerative disorders ( 21 ). In our previous study, label-free liquid chromatography-mass spectrometry was employed to detect endogenous peptides that might be involved in the progression of MS. A total of 217 peptides with a fold change ≥ 2 and p ≤ 0.05 were identified in CPZ-induced mice, including 36 down-regulated and 181 up-regulated peptides( 22 ). We identified 11 differentially expressed peptides derived from MBP. However, whether these differentially expressed peptides play a role in the pathophysiology of MS remains unclear. In this study, we identified an MBP-derived peptide (33–45) (sequence: DTGILDSIGRFFS), designated as NSDP1, which plays an important role in MS-induced neuroinflammation. In MS, the immune system erroneously attacks the myelin sheath, leading to demyelination, inflammation, and nerve damage( 23 , 24 ). MBP is one of the major components of myelin and is thus a key target of this autoimmune response( 25 , 26 ). However, peptides derived from MBP have been developed as potential immunomodulators for the treatment of MS( 27 – 29 ). These peptides aim to regulate immune responses in MS by inducing immune tolerance, altering T cell responses, or blocking autoimmune responses ( 30 , 31 ). Administration of MBP peptides can induce antigen-specific immune tolerance, thereby reducing autoimmune attacks on the myelin sheath( 27 – 29 ). For example, studies have shown that administration of MBP peptides to MS patients can neutralize free anti-MBP antibodies in cerebrospinal fluid, thereby alleviating inflammatory responses( 27 ). Moreover, MBP peptides can alter the responses of MBP-specific T cells, thereby alleviating MS symptoms( 30 , 32 ). Some studies have indicated that MBP peptides can increase the number of regulatory T cells (Treg), which are capable of suppressing autoimmune responses and promoting immune tolerance( 30 ). However, whether other MBP-derived peptide participated in the pathogenesis of MS remain unknown. In our study, we identified a novel MBP-derived peptide (166–177), NSDP1, which is significantly downregulated in the corpus callosum of cuprizone-diet-fed mice. Treatment with NSDP1 reduced LPS-induced microglial ROS generation and pro-inflammatory cytokine secretion, including IL-1β and TNF-α. Moreover, NSDP1 administration effectively increased body weight in CPZ-diet mice, mitigated CPZ-induced demyelination, promoted axonal remyelination, and facilitated motor function recovery, which indicated the potential therapeutic role of NSDP1 for demyelinating disorders. Microglia mediated neuroinflammation play a vital role in MS. In our study, we found that NSDP1 effectively inhibited microglia pro-inflammation and promote neuroprotective M2 phenotype. Mechanistic studies have shown that NSDP1-mediated anti-inflammation critically depends on the regulation of the cGAS-STING signaling pathway. The cGAS-STING pathway is an intracellular DNA sensor that can recognize cytoplasmic DNA and activate downstream inflammatory responses( 33 – 35 ). In neurodegenerative diseases, aberrant activation of the cGAS-STING pathway leads to neuroinflammation and neuronal damage( 36 ). For example, in Parkinson's disease models, activation of cGAS in microglia exacerbates neuroinflammation and neurodegeneration ( 37 ). In ischemic stroke models, knockdown of cGAS can promote M2 polarization of microglia and alleviate neuroinflammation ( 38 ). Our study suggests that NSDP1 may promote myelination by inhibiting the cGAS-STING pathway and alleviating neuroinflammation. Inhibition of STING can reduce the release of pro-inflammatory cytokines, thereby alleviating neuroinflammation and neuronal degeneration. In summary, this study identifies the MBP-derived peptide NSDP1 (166–177) as a potent modulator of neuroinflammation and a significant promoter of remyelination in models relevant to MS. NSDP1 treatment effectively suppressed detrimental microglial activation, as evidenced by reduced LPS-induced ROS generation and pro-inflammatory cytokine (IL-1β, TNF-α) secretion, while promoting a neuroprotective M2 phenotype. Crucially, NSDP1 administration demonstrated substantial therapeutic efficacy in vivo , mitigating cuprizone-induced demyelination in the corpus callosum, promoting axonal remyelination, facilitating motor function recovery, and ameliorating weight loss. Mechanistically, we reveal that the anti-inflammatory and pro-remyelination effects of NSDP1 critically depend on its modulation of the cGAS-STING signaling pathway, a key driver of neuroinflammation in demyelinating disorders. Collectively, these findings establish NSDP1 as a highly promising therapeutic candidate for MS, offering a novel, peptide-based strategy to combat neuroinflammation and critically effective for remyelination therapies. Declarations Author contributions JY wrote the manuscript, performed the experiments, and analyzed the data; JC and MH performed bioinformatics and statistical analyses; YL and XD designed the project, and reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript. Data availability The data supporting the findings of this study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate The experimental protocols received ethical approval from the Institutional Animal Care Committee at Tongren Hospital of Chinese Medicine (Approval ID: 2022-045-01) Disclosure statement The authors report no conflict of interest. Finding This work was supported by the Natural Science Foundation of Shanghai (22ZR1457100 and 24ZR1463600). References Wang Y, Wang J, Feng J (2023) Multiple sclerosis and pregnancy: Pathogenesis, influencing factors, and treatment options. Autoimmun Rev 22:103449 Graf J, Akmatov MK, Meuth SG, Tremlett H, Holstiege J (2024) Updated Multiple Sclerosis Incidence, 2015–2022. JAMA Neurol 81:1100–1102 Koch-Henriksen N, Magyari M (2021) Apparent changes in the epidemiology and severity of multiple sclerosis. 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Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 25 Jan, 2026 Reviews received at journal 19 Jan, 2026 Reviewers agreed at journal 22 Dec, 2025 Reviews received at journal 19 Dec, 2025 Reviewers agreed at journal 19 Dec, 2025 Reviewers agreed at journal 19 Dec, 2025 Reviews received at journal 14 Dec, 2025 Reviewers agreed at journal 13 Dec, 2025 Reviewers invited by journal 12 Dec, 2025 Editor assigned by journal 10 Dec, 2025 Submission checks completed at journal 10 Dec, 2025 First submitted to journal 07 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8298082","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":560688816,"identity":"19fb7392-23c3-45e1-bb1c-c7f14485a033","order_by":0,"name":"Junjie Yang","email":"","orcid":"","institution":"Tongren Hospital","correspondingAuthor":false,"prefix":"","firstName":"Junjie","middleName":"","lastName":"Yang","suffix":""},{"id":560688818,"identity":"d2c2a33d-3aaa-48c7-9951-fb80cd33e50b","order_by":1,"name":"Qinze Chi","email":"","orcid":"","institution":"Tongren 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17:34:16","extension":"xml","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":100522,"visible":true,"origin":"","legend":"","description":"","filename":"20d6bd330a3a44c39f2ba0d43fb8a6d91structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8298082/v1/1a71cc1cebe71c620b369064.xml"},{"id":98451920,"identity":"172ccc83-0404-46d8-941e-6b0f2396d0cc","added_by":"auto","created_at":"2025-12-17 17:34:11","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":109299,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8298082/v1/43ff00865db655240e1724e1.html"},{"id":98451973,"identity":"8500a19a-99d5-4fdb-b348-f43ed7039279","added_by":"auto","created_at":"2025-12-17 17:34:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4474144,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeuroprotective peptide NSDP1 suppresses LPS-induced microglial activation. \u003c/strong\u003e(A) Schematic illustration of identifying differential peptides in the CPZ-induced demyelination mouse model. (B) Reduced levels of NSDP1 in CPZ-treated mouse brains compared to controls (n=3/group). (C) CCK-8 assay of NSDP1 in BV2 microglial cells at 100 nM, 200 nM, and 400 nM concentrations (n=6/group). (D) Representative images of ROS staining (red) and (E) quantitative analysis of ROS fluorescence intensity in control, Scr and NSDP1-treated LPS-induced BV2 cells (n=6/group). (F) Immunofluorescence staining of pro-inflammatory marker iNOS (green) and anti-inflammatory marker Arg-1 (red) in control, Scr and NSDP1-treated LPS-induced BV2 cells. (G-H) Quantification of iNOS and Arg-1 fluorescence intensity of F (n=6/group). (I) ELISA analysis of pro-inflammatory cytokines (TNF-α and IL-1β) in BV2 cell supernatants (n=3). Data presented as relative intensity (mean ± SD), * p\u0026lt;0.05, ** p\u0026lt;0.01, and *** p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8298082/v1/57af409d0ee60666d01f0803.jpg"},{"id":98451832,"identity":"8e5c3b55-32e3-4e2e-8b74-cac5df81b73e","added_by":"auto","created_at":"2025-12-17 17:33:39","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1114546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNSDP1 administration ameliorates cuprizone-induced demyelination. (A) \u003c/strong\u003eExperimental design showing the control group (normal diet), CPZ+Scr group (0.2% cuprizone diet for 8 weeks), and CPZ+NSDP1 group (cuprizone diet with NSDP1 treatment). (B) Body weight changes in control, CPZ+Scr, and CPZ+NSDP1 groups during the 8-week experimental period (n=6/group). (C) Representative LFB staining images of the corpus callosum in control, CPZ+Scr, and CPZ+NSDP1 groups. (D) Quantification of myelinated area percentage (n=6/group). Data are presented as mean ± SD; *p\u0026lt;0.05, **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8298082/v1/4a45082a65859437fc8d596a.jpg"},{"id":98451833,"identity":"88750b5d-2027-4ed6-b1f7-b3a9dc906ab2","added_by":"auto","created_at":"2025-12-17 17:33:39","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1249310,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNSDP1 administration ameliorates cuprizone-induced demyelination. (A) \u003c/strong\u003eRepresentative images of MAG (green) and MOG (red) in the corpus callosum of control, CPZ+Scr, and CPZ+NSDP1 groups. (B-C) Quantification analysis of MAG and MOG fluorescence intensity in A (n=6/group). (D) Representative images of Pdgfrα staining in the corpus callosum of control, CPZ+Scr, and CPZ+NSDP1 groups. (E) Quantification of Pdgfrα fluorescence intensity in D (n=6/group). (F) Representative TEM images of myelin sheath ultrastructure in control, CPZ+Scr, and CPZ+NSDP1 groups. (G) g-ratio analysis of F (n=6/group). Data are presented as mean ± SD; *p\u0026lt;0.05, **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8298082/v1/d1fe38f7b96ad6da3431fdcc.jpg"},{"id":98451925,"identity":"7a2a723f-7210-443c-ac05-773522296c5e","added_by":"auto","created_at":"2025-12-17 17:34:14","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1087827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNSDP1 administration ameliorates cuprizone-induced neuroinflammation. \u003c/strong\u003e(A) Representative images of IBA1 (green) and GFAP (red) in the corpus callosum of control, CPZ+Scr, and CPZ+NSDP1 groups. (B-C) Quantification of IBA1 and GFAP fluorescence intensity in A (n=6/group). Data are presented as mean ± SD; **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8298082/v1/7f71b98194e202f7bc375076.jpg"},{"id":98451896,"identity":"5bb14026-cf29-41b0-889f-7c8dd4525904","added_by":"auto","created_at":"2025-12-17 17:34:03","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":859549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNSDP1 promotes myelination and improves behavioral deficits in CPZ-induced demyelination mice.\u003c/strong\u003e (A) Heatmap clustering of differentially expressed genes in the corpus callosum of NSDP1-treated versus untreated CPZ mice (n=3/group).(B) Volcano plot analysis of differentially expressed genes in the corpus callosum of NSDP1-treated versus untreated CPZ mice. (C) GSEA shows significant enrichment of myelination-related pathways in NSDP1-treated CPZ mice. (D) GSEA reveals enrichment of locomotor behavior-related genes in NSDP1-treated CPZ mice.\u003c/p\u003e","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8298082/v1/ccfc9d447f9c13dbbc553f11.jpg"},{"id":98451975,"identity":"998d99ae-e65a-4267-8c1a-743bf21bc6ae","added_by":"auto","created_at":"2025-12-17 17:34:33","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2147790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNSDP1 attenuates neuroinflammation by suppressing cGAS-STING signaling pathway. \u003c/strong\u003e(A-B) Representative images of cGAS and STING in control, LPS+Scr, and LPS+NSDP1 BV2 cells. (C-D) Quantification analysis of cGAS and STING fluorescence intensity in A and B (n=6/group). (E) Representative images of ROS staining in control, LPS+Scr, LPS+NSDP1, LPS+SN, LPS+NSDP1+DMX groups. (F) Quantification analysis of fluorescence intensity of E (n=6/group). (G-H) ELISA analysis of pro-inflammatory cytokines (TNF-α and IL-1β) in BV2 cell supernatants (n=3/group). Data are presented as mean ± SD; **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8298082/v1/09873ec35c3de7c04ec356a9.jpg"},{"id":98775805,"identity":"b70f354c-2285-4155-907c-d73677f5d720","added_by":"auto","created_at":"2025-12-22 12:21:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11861311,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8298082/v1/2bf94ac2-8482-4c72-9011-fd1ee7261bcd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Myelin-Derived Peptide NSDP1 Promotes Remyelination and Attenuates Neuroinflammation in Cuprizone-Induced Demyelination via Suppression of the cGAS- STING Pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMultiple Sclerosis (MS) is an autoimmune disease affecting the central nervous system (CNS), specifically targeting the myelin sheath-the protective covering of nerve fibers (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The global incidence of MS exhibits significant geographical variation, with reported rates approximating 5 to 300 per 100,000 individuals, and a female predominance with an incidence over two-fold higher than that in males(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Inflammatory demyelinating lesions disrupt the periventricular region, proximal cortex, spinal cord, optic nerve, and cerebellum, leading to a wide range of symptoms(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). These symptoms include eye pain, vision loss, weakness or sensory changes in the body, dizziness, balance difficulties, and memory impairment (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Newer disease-modifying therapies (DMTs) are available for treating different types of MS, including relapsing\u0026ndash;remitting MS, active secondary progressive MS, and primary progressive MS. However, the efficacy of current DMTs ranges only from 29% to 68%, and they are associated with various adverse effects such as bradycardia, infections, heart blocks, and infusion reactions(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Therefore, there is an urgent need to identify more effective therapeutic targets and treatments for MS. Neuroinflammation plays a vital role in the pathogenesis of MS. The infiltration of immune cells into the CNS and activation of CNS-intrinsic microglia and astrocytes is the dominant feature of MS(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Although early intervention with peripherally acting therapeutics that deplete immune cells or impede their trafficking into the CNS can effectively mitigate lesion formation and disease relapses (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), the persistent activation of microglia emerges as a pivotal driver of pathogenic processes(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). It was reported that BTK (Bruton's tyrosine kinase) inhibitors targeting microglia can effectively suppress neuroinflammation and slow the progression of progressive MS(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Therefore, modulating the overactivation of immune and inflammatory responses represents a promising therapeutic strategy against MS (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePeptides are a class of short chains of amino acids which play an important role in a multitude of processes in human development, tissue homeostasis, and cell metabolism. The peptide therapeutics started in 1922 with the use of insulin for treatment of type 1 diabetes(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Until now, over 80 peptide drugs have been available on the global market, and more than 150 peptide drugs are in clinical trials (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). It was reported that MBP fragments suppressed the disease significantly (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). However, whether other MBP-derived peptides participate in the pathogenesis of MS remains largely unknown. In our previous study, we identified dysregulated peptides in the corpus callosum of mouse models of MS subjected to a cuprizone (CPZ) diet, which including the MBP. However, the specific role of these MBP derived dysregulated peptides remains need for further investigation.\u003c/p\u003e \u003cp\u003eIn this study, we found that MBP-derived peptide NSDP1, which is significantly downregulated in the corpus callosum of CPZ diet MS mice, plays a critical role in inhibiting neuroinflammation and promoting myelin remyelination. Administration of NSDP1 exerts potent anti-inflammatory effects \u003cem\u003ein vitro\u003c/em\u003e by suppressing LPS-induced microglial activation. Moreover, \u003cem\u003ein vivo\u003c/em\u003e administration of NSDP1 effectively mitigated CPZ-induced demyelination and promoted functional remyelination in the corpus callosum of MS mice model. Furthermore, NSDP1 treatment enhanced locomotor recovery and modulated myelination-related gene pathways. Mechanistically, NSDP1 exerts its remyelination effects by regulating cGAS-STING signaling, thereby reducing microglia activation and inhibiting neuroinflammation. These findings suggest NSDP1 as a novel and promising therapeutic candidate for MS treatment.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and treatment\u003c/h2\u003e \u003cp\u003eThe mouse microglia cell line BV2 cells were obtained from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd (ZQ0397, Shanghai, China). The cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM, Gibco,11965-092, USA) supplemented with 1% penicillin-streptomycin (PS) and 10% fetal bovine serum (FBS, Gibco, 10099141C, USA). BV2 cells were cultured in the incubator with 5% CO₂ at 37\u0026deg;C. The BV2 cells were treated with 100 ng/mL LPS (Sigma, L2880, USA) for 24 hours. 100 nM, 200 nM, or 400 nM NSDP1 peptides were combined with 100 ng/mL LPS-treated BV2 cells for 24 hours. 400 nM Scr peptides were combined with 100 ng/mL LPS-treated BV2 cells for 24 hours. 1 \u0026micro;M SN-011 was exposed to LPS-treated BV2 cells for 6 hours. 100 \u0026micro;g/mL Vadimezan (DMX, MedChemExpress, China) was exposed to NSDP1 peptides treated BV2 cells for 24 hours.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNSDP1 Peptide Synthesis\u003c/h3\u003e\n\u003cp\u003eThe NSDP1 peptide, which derived from mouse Myelin basic protein isoform 1(MBP, NCBI Reference Sequence: Np_001020422.1) 33\u0026ndash;45 (Sequence: DTGILDSIGRFFS), Scrambled peptides (Scr, Sequence: ARFGGDLGEGE) were used in this study. NSDP1 peptide and the Scr peptides were obtained from Shanghai Kaitaibio Co., Ltd. Briefly, rink amide resin (3 g) was first equilibrated in dichloromethane for two hours. After draining, the support was washed repeatedly with N, N-dimethylformamide and dried. For the initial attachment, Glycine and HOBt were dissolved in DMF. DIC was introduced, and the activated mixture was combined with the solid support. Three volumes of DMAP were added, and the conjugation proceeded at 30\u0026deg;C for 4 hours. Remaining reactive sites were subsequently capped using a solution of acetic anhydride and DIEA in DCM. The Fmoc protecting group was removed with 20% piperidine in DMF. Following this deprotection step, the resin was thoroughly washed. Subsequent amino acid residues were connected analogously. HOBt, DIC, and DMAP were employed for activation. Completion of each coupling was verified via the ninhydrin assay. After every successive amino acid incorporation, the Fmoc group was cleaved off. The final product was liberated from the solid support using a TFA-based cleavage cocktail containing TIS, EDT, and water. The crude material was precipitated in cold diethyl ether. Purification was performed using preparative HPLC, and the pure product was obtained as a lyophilized powder. A cell-penetrating sequence (9x Arg) was incorporated at the C-terminus of all analogues.\u003c/p\u003e\n\u003ch3\u003eCCK-8 assay\u003c/h3\u003e\n\u003cp\u003eCell viability was assessed using a CCK-8 assay kit (Beyotime, China). In brief, BV2 cells were plated in 96-well plates at 5\u0026times;10⁴ cells per well and cultured under standard conditions (37\u0026deg;C, 5% CO₂). Following experimental treatments, the cells were supplemented with 10 \u0026micro;L of CCK-8 solution per well and incubated for 1 hours. Absorbance at 450 nm was then recorded using the microplate reader.\u003c/p\u003e\n\u003ch3\u003eLC-MS/MS Analysis\u003c/h3\u003e\n\u003cp\u003eLiquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on a TripleTOF 5600 system (SCIEX, USA). Peptide extracts from the mouse corpus callosum, obtained from both CPZ-induced and control diet-fed groups, were dissolved in 2% acetonitrile containing 0.1% formic acid, loaded onto a C18 trap column (5 \u0026micro;m, 10 \u0026micro;m \u0026times; 20 mm), and separated on a C18 analytical column (3 \u0026micro;m, 75 \u0026micro;m \u0026times; 150 mm) at a flow rate of 30 nL/min over a 90-minute gradient. Mass spectrometry analysis employed an information-dependent acquisition (IDA) mode. Full-scan MS1 spectra (350\u0026ndash;1500 m/z) were acquired with 250 ms accumulation time, followed by the collection of MS2 spectra for up to 30 precursor ions (100\u0026ndash;1500 m/z) with 50 ms accumulation time. A dynamic exclusion window of 15 seconds was applied to prevent repeated sequencing.\u003c/p\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eSix-week-old male C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed for two weeks under controlled conditions (22\u0026deg;C, 40\u0026ndash;60% humidity, 12 h light/dark cycle). At eight weeks of age, mice were randomly assigned to three experimental groups (n\u0026thinsp;=\u0026thinsp;15/group): the control group received standard rodent chow, while the cuprizone (CPZ) model group was fed a 0.2% CPZ-containing diet (Sigma, St. Louis, MO, USA) for eight weeks to induce demyelination. For the NSDP1 and relative control Scr peptide-treated group, NSDP1 and Scr peptide were administered via weekly intracerebroventricular injection at a concentration of 0.2 \u0026micro;mol in a 5 \u0026micro;L volume from the sixth to eighth week. All mice were weighed weekly throughout the study period. The experimental protocol received approval from the Animal Ethics Committee of Tongren Hospital, China (Approval No. 2022-045-01).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLuxol Fast Blue (LFB) staining\u003c/h2\u003e \u003cp\u003eParaffin-embedded sections were dewaxed and rehydrated through sequential immersion in Xylene I (20 min), Xylene II (20 min), absolute ethanol I (5 min), absolute ethanol II (5 min), and 75% ethanol (5 min), followed by rinsing in water. For myelin staining, sections were immersed in pre-warmed (65\u0026deg;C, 30 min) Myelin Stain Solution A (G1030, Servicebio, China) within a covered staining chamber for 60 min at 65\u0026deg;C, then rapidly rinsed in tap water. Differentiation was performed by briefly immersing sections (\u0026lt;\u0026thinsp;2 s) in pre-heated Myelin Stain Solution B (G1030, Servicebio, China), immediately transferring to Myelin Stain Solution C (15 s) (G1030, Servicebio, China), and water-rinsing to terminate differentiation. This differentiation cycle was repeated under microscopic monitoring until myelin sheaths appeared blue against a near-colorless background. Sections were dehydrated through absolute ethanol I (5 min), absolute ethanol II (5 min), absolute ethanol III (5 min), cleared in Xylene I (5 min) and Xylene II (5 min), and mounted with neutral balsam. Stained sections were microscopically examined, and images were acquired for analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeasurement of Intracellular Reactive Oxygen Species (ROS) Levels\u003c/h3\u003e\n\u003cp\u003eROS generation was quantified using the DHE-based ROS Assay Kit (Applygene Technologies Inc., C1300-2, China) following the manufacturer's protocol. Briefly, cells were loaded with 10 \u0026micro;M dihydroethidium (DHE) probe and incubated for 30 minutes at 37\u0026deg;C under dark, humidified conditions. Fluorescence images were captured using a Leica SP8 confocal laser scanning microscope, with subsequent quantitative analysis performed in ImageJ.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence Staining\u003c/h3\u003e\n\u003cp\u003eCultured BV2 microglia and brain tissue sections underwent immunofluorescence staining using standardized protocols. BV2 cells were fixed with 4% paraformaldehyde (PFA), blocked in 5% bovine serum albumin (BSA), and incubated overnight at 4\u0026deg;C with primary antibodies against iNOS (Proteintech, 22226-1-AP, China) and Arg1 (Novus Biologicals, NB100-59740, USA), cGAS (Proteintech, 26416-1-AP, China), and STING (Abcam, AB288157, USA). Following PBS washes, species-matched Alexa Fluor-conjugated secondary antibodies (Proteintech, SA00013 series, China) were applied for 2 hr at room temperature. Nuclei were counterstained with DAPI (Beyotime, P0131, China).\u003c/p\u003e \u003cp\u003eFor brain tissue analysis, 30-\u0026micro;m cryosections were prepared from tissues post-fixed in 4% PFA for 24 hr and cryoprotected in 30% sucrose. Sections were immunolabeled with GFAP (Cell Signaling Technology, 3670), Iba1 (Cell Signaling Technology, 17198), MAG (Cell Signaling Technology, 9043S), MOG (Cell Signaling Technology, 45268S), and Pdgfrα (Abcam, AB203491) antibodies using identical secondary antibody and DAPI protocols. All samples were imaged using the Leica SP8 confocal microscope.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTransmission Electron Microscopy (TEM) of Corpus Callosum Myelin\u003c/h2\u003e \u003cp\u003eCorpus callosum tissues were processed for ultrastructural analysis using TEM. Mice underwent transcardial perfusion with heparinized saline followed by primary fixation in 3% glutaraldehyde/0.1 M phosphate buffer (pH 7.4) for 24 hr at 4\u0026deg;C. Tissues were post-fixed in 1% osmium tetroxide for 2 hr, dehydrated through graded ethanol series and embedded. Myelinated axons in the callosal midline were examined using the Tecnai G2 20 Twin TEM (FEI). Myelin integrity was quantified by measuring G-ratios (inner axon diameter/total myelinated fiber diameter).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRNA-sequencing (RNA-seq) analysis\u003c/h2\u003e \u003cp\u003eRNA-seq was performed as we previously reported (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Corpus callosum tissues from NSDP1-treated mice and CPZ model mice were harvested for RNA-seq analysis. RNA-seq analysis was conducted by OE Biotech Co., Ltd. (Shanghai, China) according to their stranded protocol. Differential gene expression analysis used the DESeq2 algorithm, with significant differentially expressed genes (DEGs) identified under thresholds of |log₂(fold change) | \u0026gt; 1 and adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Functional enrichment analysis of DEGs interrogated KEGG, Reactome, and WikiPathways databases using clusterProfiler in R (version 3.2.0).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eConcentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in BV2 lysates were quantified using mouse-specific ELISA kits (IL-1β: Mlbio ml098416; TNF-α: Mlbio ml002095) per manufacturer's protocol. Following centrifugation of cell lysates (12,000 \u0026times; g, 20 min, 4\u0026deg;C), supernatants were analyzed in technical duplicates. Absorbance at 450 nm (reference 570 nm) was measured using the Multiskan FC microplate reader (Thermo Fisher), with analyte concentrations calculated against standardized curves.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eQuantification and statistical analysis\u003c/h2\u003e \u003cp\u003eAll datasets were analyzed in GraphPad Prism 9.0 using unpaired Student's t-tests for dual-group comparisons and one-way ANOVA with Tukey's post-hoc testing for multi-group analyses, with statistical significance defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) with error bars.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eNSDP1 is Downregulated in CPZ- induced MS mice model and Suppresses LPS-Induced Microglial Activation In Vitro\u003c/b\u003e \u003c/p\u003e \u003cp\u003eProteomic analysis of the corpus callosum in the cuprizone-induced demyelination mouse model identified that the peptide DTGILDSIGRFFS, derived from residues 33\u0026ndash;45 of myelin basic protein (MBP), was significantly downregulated. We designated this nervous system-derived peptide as NSDP1 (nervous system derived peptide 1) hereafter (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). To determine whether NSDP1 involved in the pathogenesis of MS, NSDP1 was treated to BV2 cells with the concentrations 100 nM, 200 nM, and 400 nM. CCK-8 assay showed that NSDP1 exerted no cytotoxicity on BV2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In addition, administration of the NSDP1 peptide (400 nM) significantly suppressed LPS-induced ROS production compared with the Scr peptide control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E). Moreover, NSDP1 significantly decreased the expression of the pro-inflammatory marker iNOS and increased the expression of the anti-inflammatory marker Arg-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-H). Furthermore, NSDP1 treatment also markedly inhibited the LPS-induced secretion of pro-inflammatory cytokines TNF-α and IL-1β (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Taken together, these findings suggest that identify NSDP1 as a myelin-derived peptide deficient in demyelination, which exhibits potent anti-inflammatory effects by modulating microglial activation and polarization \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eNSDP1 Treatment mitigate Cuprizone-Induced Demyelination\u003c/h2\u003e \u003cp\u003eTo evaluate the therapeutic potential of NSDP1 \u003cem\u003ein vivo\u003c/em\u003e, we utilized the CPZ-induced demyelination model. Mice received a 0.2% CPZ diet for 8 weeks and were administered NSDP1 or Scr peptides via intracerebroventricular (i.c.v.) injection at 6 and 7 weeks (twice total; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The results shown that CPZ treatment induced the significant decrease in body weight starting at week 5. However, NSDP1 administration significantly reversed this weight loss by week 8 compared with the Scr-treated CPZ group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Furthermore, Luxol Fast Blue (LFB) staining of the corpus callosum revealed severe demyelination in CPZ-treated mice after 8 weeks of CPZ exposure. This demyelination was significantly attenuated by NSDP1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Quantitative analysis confirmed that NSDP1 significantly restored myelination (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to CPZ controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further determine whether NSDP1 promotes remyelination in the cuprizone-induced demyelination model, we quantitatively assessed myelin-associated proteins and myelin sheath ultrastructure. Immunofluorescence analysis revealed significant recovery of myelin-associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG) expression in the corpus callosum of NSDP1-treated CPZ mice versus CPZ\u0026thinsp;+\u0026thinsp;Scr groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). Notably, NSDP1 administration significantly increased Pdgfrα⁺ oligodendrocyte precursor cell (OPC) density compared to CPZ\u0026thinsp;+\u0026thinsp;Scr mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E). In addition, Ultrastructural analysis by TEM demonstrated that cuprizone feeding induced characteristic pathologies including demyelination, myelin decompaction, axonal swelling, and distorted myelin profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). In contrast, NSDP1 treatment restored compact myelin lamellae and normalized myelinated axon morphology in the corpus callosum. Consistent with this finding, g-ratio quantification confirmed significantly improved axonal myelination efficiency in NSDP1-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Collectively, these results indicate that NSDP1 administration promotes functional remyelination through increased OPC number and myelin restoration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eNSDP1 Attenuated Glial overactivity in the CPZ Mice\u003c/h2\u003e \u003cp\u003eAccumulating evidence suggests that microglial and astrocytic activation within the corpus callosum contributes to demyelination in the CPZ mouse model. To assess the effects of NSDP1 administration on reactive gliosis in MS mice, we performed IF staining for Iba1 (microglia) and GFAP (astrocytes) in control, CPZ-fed, and CPZ-fed\u0026thinsp;+\u0026thinsp;NSDP1-treated mice. CPZ exposure significantly increased the density of Iba1\u003csup\u003e+\u003c/sup\u003e microglia within the corpus callosum, which exhibited characteristic morphological activation features, including increased branch endpoints and enlarged somata. NSDP1 treatment significantly attenuated these morphological changes, reducing both branch complexity and somatic hypertrophy, indicative of suppressed microglial activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). Furthermore, CPZ feeding dramatically upregulated GFAP expression, reflecting astrocyte reactivity, whereas NSDP1 administration significantly reduced GFAP expression in the corpus callosum (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, C). These findings demonstrate that NSDP1 effectively mitigates CPZ-induced neuroinflammation by attenuating both microglial and astrocytic activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eNSDP1 involved in the functional recovery by RNA-seq detection\u003c/h2\u003e \u003cp\u003eTo further elucidate the functional role of NSDP1 in cuprizone-induced demyelination, RNA sequencing was performed on corpus callosum tissue from NSDP1-treated mice compared to CPZ-exposed controls. Analysis revealed 61 significantly dysregulated genes (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, |log₂FC| \u0026gt; 1), comprising 31 downregulated and 30 upregulated transcripts (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). KEGG pathway enrichment demonstrated that NSDP1 treatment potentiated myelination-related pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and enhanced locomotor functional recovery, as evidenced by improved adult walking behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These findings indicate that NSDP1 promotes functional restoration in CPZ-demyelinated mice and critical for remyelination and neural repair.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eNSDP1 suppresses neuroinflammation via modulating the cGAS-STING signaling pathway\u003c/h2\u003e \u003cp\u003eIt has been reported that the cGAS-STING signaling pathway plays a crucial role in neuroinflammation. To determine whether NSDP1 reduces microglial activation by modulating the cGAS-STING signaling pathway, the expression levels of cGAS and STING were detected in LPS-induced BV2 cells incubated with NSDP1. The results demonstrated that the expression of cGAS and STING was significantly downregulated in NSDP1-treated LPS-induced BV2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D).​ To further verify whether NSDP1 regulates inflammatory responses through the cGAS-STING signaling pathway, the STING inhibitor SN-011 and STING activator Vadimezan (DMX) were employed in LPS-induced BV2 cells. ROS staining revealed a marked reduction in ROS levels in both NSDP1-treated and SN-011-treated BV2 cells. In contrast, ROS staining was significantly enhanced in BV2 cells cotreated with NSDP1 and DMX (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-F). Moreover, ELISA results showed that the release of proinflammatory factors TNF-α and IL-1β was significantly downregulated in SN-011-treated LPS-induced BV2 cells, while their release was upregulated in LPS-induced BV2 cells cotreated with NSDP1 and DMX (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-H).​ Collectively, these results indicate that cGAS-STING signaling pathway serves as a critical downstream mediator through which NSDP1 exerts its anti-inflammatory effects in microglia.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMultiple sclerosis (MS), a chronic immune-mediated disorder of the central nervous system and a leading global cause of neurological disability in young adults, remains a major clinical challenge. In this study, we identified an MBP-derived peptide\u0026mdash;designated NSDP1-that demonstrates significant anti-neuroinflammatory activity in MS. NSDP1 treatment markedly attenuated LPS-induced ROS generation, downregulated pro-inflammatory factor expression, and promoted M2 microglial polarization. Furthermore, NSDP1 effectively alleviated corpus callosum demyelination by suppressing CPZ-activated microglial and astrocytic responses, concomitant with upregulated Pdgfrα, MAG, and MOG expression. Mechanistic investigations revealed that NSDP1-mediated remyelination critically depends on modulation of the cGAS-STING signaling axis. These findings collectively establish NSDP1 as a promising therapeutic candidate for MS, whose modulation of the cGAS-STING pathway may address the pressing need for targeted remyelination strategies.\u003c/p\u003e \u003cp\u003ePeptides typically consist of fewer than 50 amino acids, with a molecular weight ranging from 500 to 5000 Da (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Endogenous peptides originate either from proteolytic degradation of intracellular proteins or via direct translation of RNA-encoded sequences(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Peptides possess intrinsic advantages, such as high specificity, good efficacy, low immunogenicity, and membrane permeability. Over 80 therapeutic peptides act as hormones, neurotransmitters, growth factors, ion channel ligands, or anti-infective agents, targeting conditions including cancer, metabolic diseases, and neurodegenerative disorders (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). In our previous study, label-free liquid chromatography-mass spectrometry was employed to detect endogenous peptides that might be involved in the progression of MS. A total of 217 peptides with a fold change\u0026thinsp;\u0026ge;\u0026thinsp;2 and p\u0026thinsp;\u0026le;\u0026thinsp;0.05 were identified in CPZ-induced mice, including 36 down-regulated and 181 up-regulated peptides(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). We identified 11 differentially expressed peptides derived from MBP. However, whether these differentially expressed peptides play a role in the pathophysiology of MS remains unclear. In this study, we identified an MBP-derived peptide (33\u0026ndash;45) (sequence: DTGILDSIGRFFS), designated as NSDP1, which plays an important role in MS-induced neuroinflammation.\u003c/p\u003e \u003cp\u003eIn MS, the immune system erroneously attacks the myelin sheath, leading to demyelination, inflammation, and nerve damage(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). MBP is one of the major components of myelin and is thus a key target of this autoimmune response(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). However, peptides derived from MBP have been developed as potential immunomodulators for the treatment of MS(\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). These peptides aim to regulate immune responses in MS by inducing immune tolerance, altering T cell responses, or blocking autoimmune responses (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Administration of MBP peptides can induce antigen-specific immune tolerance, thereby reducing autoimmune attacks on the myelin sheath(\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). For example, studies have shown that administration of MBP peptides to MS patients can neutralize free anti-MBP antibodies in cerebrospinal fluid, thereby alleviating inflammatory responses(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Moreover, MBP peptides can alter the responses of MBP-specific T cells, thereby alleviating MS symptoms(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Some studies have indicated that MBP peptides can increase the number of regulatory T cells (Treg), which are capable of suppressing autoimmune responses and promoting immune tolerance(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). However, whether other MBP-derived peptide participated in the pathogenesis of MS remain unknown. In our study, we identified a novel MBP-derived peptide (166\u0026ndash;177), NSDP1, which is significantly downregulated in the corpus callosum of cuprizone-diet-fed mice. Treatment with NSDP1 reduced LPS-induced microglial ROS generation and pro-inflammatory cytokine secretion, including IL-1β and TNF-α. Moreover, NSDP1 administration effectively increased body weight in CPZ-diet mice, mitigated CPZ-induced demyelination, promoted axonal remyelination, and facilitated motor function recovery, which indicated the potential therapeutic role of NSDP1 for demyelinating disorders.\u003c/p\u003e \u003cp\u003eMicroglia mediated neuroinflammation play a vital role in MS. In our study, we found that NSDP1 effectively inhibited microglia pro-inflammation and promote neuroprotective M2 phenotype. Mechanistic studies have shown that NSDP1-mediated anti-inflammation critically depends on the regulation of the cGAS-STING signaling pathway. The cGAS-STING pathway is an intracellular DNA sensor that can recognize cytoplasmic DNA and activate downstream inflammatory responses(\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). In neurodegenerative diseases, aberrant activation of the cGAS-STING pathway leads to neuroinflammation and neuronal damage(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). For example, in Parkinson's disease models, activation of cGAS in microglia exacerbates neuroinflammation and neurodegeneration (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In ischemic stroke models, knockdown of cGAS can promote M2 polarization of microglia and alleviate neuroinflammation (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Our study suggests that NSDP1 may promote myelination by inhibiting the cGAS-STING pathway and alleviating neuroinflammation. Inhibition of STING can reduce the release of pro-inflammatory cytokines, thereby alleviating neuroinflammation and neuronal degeneration.\u003c/p\u003e \u003cp\u003eIn summary, this study identifies the MBP-derived peptide NSDP1 (166\u0026ndash;177) as a potent modulator of neuroinflammation and a significant promoter of remyelination in models relevant to MS. NSDP1 treatment effectively suppressed detrimental microglial activation, as evidenced by reduced LPS-induced ROS generation and pro-inflammatory cytokine (IL-1β, TNF-α) secretion, while promoting a neuroprotective M2 phenotype. Crucially, NSDP1 administration demonstrated substantial therapeutic efficacy \u003cem\u003ein vivo\u003c/em\u003e, mitigating cuprizone-induced demyelination in the corpus callosum, promoting axonal remyelination, facilitating motor function recovery, and ameliorating weight loss. Mechanistically, we reveal that the anti-inflammatory and pro-remyelination effects of NSDP1 critically depend on its modulation of the cGAS-STING signaling pathway, a key driver of neuroinflammation in demyelinating disorders. Collectively, these findings establish NSDP1 as a highly promising therapeutic candidate for MS, offering a novel, peptide-based strategy to combat neuroinflammation and critically effective for remyelination therapies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJY wrote the manuscript, performed the experiments, and analyzed the data; JC and MH performed bioinformatics and statistical analyses; YL and XD designed the project, and reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental protocols received ethical approval from the Institutional Animal Care Committee at Tongren Hospital of Chinese Medicine (Approval ID: 2022-045-01)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of Shanghai (22ZR1457100 and 24ZR1463600).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang Y, Wang J, Feng J (2023) Multiple sclerosis and pregnancy: Pathogenesis, influencing factors, and treatment options. 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Immunity 57:790\u0026ndash;814\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa C, Liu Y, Li S, Ma C, Huang J, Wen S, Yang S, Wang B (2023) Microglial cGAS drives neuroinflammation in the MPTP mouse models of Parkinson's disease. CNS Neurosci Ther 29:2018\u0026ndash;2035\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang GL, Yang XL, Zhou HJ, Long J, Liu B, Zhang LM, Lu D (2021) cGAS knockdown promotes microglial M2 polarization to alleviate neuroinflammation by inhibiting cGAS-STING signaling pathway in cerebral ischemic stroke. Brain Res Bull 171:183\u0026ndash;195\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"NSDP1, Peptide, neuroinflammation, Multiple Sclerosis, cGAS-STING","lastPublishedDoi":"10.21203/rs.3.rs-8298082/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8298082/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMultiple sclerosis (MS) involves demyelination and neuroinflammation. Proteomic analysis identified significant downregulation of the myelin basic protein-derived peptide NSDP1 in the cuprizone (CPZ)-induced demyelination mouse model. \u003cem\u003eIn vitro\u003c/em\u003e, NSDP1 suppressed LPS-induced microglial activation in BV2 cells, reducing reactive oxygen species (ROS) production, downregulating pro-inflammatory markers (iNOS, TNF-α, IL-1β), and upregulating the expression of anti-inflammatory marker Arg-1. \u003cem\u003eIn vivo\u003c/em\u003e, NSDP1 administration via intracerebroventricular injection significantly mitigated CPZ-induced weight loss and demyelination in the corpus callosum. NSDP1 promoted functional remyelination, restoring expression of myelin proteins (MAG, MOG), increasing oligodendrocyte precursor cell (OPC) density, improving myelin sheath ultrastructure, and enhancing axonal myelination efficiency. Furthermore, NSDP1 attenuated CPZ-induced reactive gliosis, reducing both microglial activation and astrocytic reactivity in the corpus callosum. RNA sequencing revealed that NSDP1 modulated myelination-related pathways and correlated with improved locomotor recovery. Mechanistically, NSDP1 exerted its anti-inflammatory effects by inhibiting the cGAS-STING signaling pathway, as shown by reduced cGAS and STING expression in LPS-stimulated BV2 cells. The effects of NSDP1 on ROS and pro-inflammatory cytokine release were reversed by the STING activator DMX and mimicked by the STING inhibitor SN-011. Collectively, these findings identify NSDP1 as a downregulated myelin-derived peptide with potent therapeutic potential, which promotes remyelination and suppresses neuroinflammation in demyelinating diseases by inhibiting the cGAS-STING pathway.\u003c/p\u003e","manuscriptTitle":"The Myelin-Derived Peptide NSDP1 Promotes Remyelination and Attenuates Neuroinflammation in Cuprizone-Induced Demyelination via Suppression of the cGAS- STING Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-17 17:30:09","doi":"10.21203/rs.3.rs-8298082/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-26T04:05:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-19T08:50:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"144227695803283461321074429823577517960","date":"2025-12-22T18:11:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-19T19:33:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"320526525719883663744516438244130663896","date":"2025-12-19T12:44:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"262249494923496915345030270092345474274","date":"2025-12-19T07:31:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-15T03:06:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"274309518263952822771337724749249262269","date":"2025-12-13T12:23:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-12T07:14:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-10T18:35:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-10T06:57:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Neurochemical Research","date":"2025-12-07T06:28:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a7103176-b170-4fae-b07d-7a0c7d5f4bf2","owner":[],"postedDate":"December 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-14T20:08:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-17 17:30:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8298082","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8298082","identity":"rs-8298082","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}