Repetitive transcranial magnetic stimulatation alleviates motor impairment in Parkinson's disease: association with peripheral inflammatory regulatory T-cells and SYT6

Repetitive transcranial magnetic stimulatation alleviates motor impairment in Parkinson's disease: association with peripheral inflammatory regulatory T-cells and SYT6 | 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 Repetitive transcranial magnetic stimulatation alleviates motor impairment in Parkinson's disease: association with peripheral inflammatory regulatory T-cells and SYT6 Fen Xie, BIbiao Shen, Yuqi Luo, Hang Zhou, Zhenchao Xie, Shuzhen Zhu, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4959031/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Oct, 2024 Read the published version in Molecular Neurodegeneration → Version 1 posted 5 You are reading this latest preprint version Abstract Background Repetitive transcranial magnetic stimulation (rTMS) has been used to treat various neurological disorders. However, the molecular mechanism underlying the therapeutic effect of rTMS on Parkinson’s disease (PD) has not been fully elucidated. Neuroinflammation like regulatory T-cells (Tregs) appears to be a key modulator of disease progression in PD. If rTMS affects the peripheral Tregs in PD remains unknown. Methods Here, we conducted a prospective clinical study (Chinese ClinicalTrials. gov: ChiCTR 2100051140) involving 54 PD patients who received 10-day rTMS (10 Hz) stimulation on the primary motor cortex (M1) region or sham treatment. Clinical and function assessment as well as flow cytology study were undertaken in 54 PD patients who were consecutively recruited from the department of neurology at Zhujiang hospital between September 2021 and January 2022. Subsequently, we implemented flow cytometry analysis to examine the Tregs population in spleen of MPTP-induced PD mice that received rTMS or sham treatment, along with quantitative proteomic approach reveal novel molecular targets for Parkinson's disease, and finally, the RNA interference method verifies the role of these new molecular targets in the treatment of PD. Results We demonstrated that a 10-day rTMS treatment on the M1 motor cortex significantly improved motor dysfunction in PD patients. The beneficial effects persisted for up to 40 days, and were associated with an increase in peripheral Tregs. There was a positive correlation between Tregs and motor improvements in PD cases. Similarly, a 10-day rTMS treatment on the brains of MPTP-induced PD mice significantly ameliorated motor symptoms. rTMS reversed the downregulation of circulating Tregs and tyrosine hydroxylase neurons in these mice. It also increased anti-inflammatory mediators, deactivated microglia, and decreased inflammatory cytokines. These effects were blocked by administration of a Treg inhibitor anti-CD25 antibody in MPTP-induced PD mice. Quantitative proteomic analysis identified TLR4, TH, Slc6a3 and especially Syt6 as the hub node proteins related to Tregs and rTMS therapy. Lastly, we validated the role of Treg and rTMS-related protein syt6 in MPTP mice using the virus interference method. Conclusions Our clinical and experimental studies suggest that rTMS improves motor function by modulating the function of Tregs and suppressing toxic neuroinflammation. Hub node proteins (especially Syt6) may be potential therapeutic targets. Trial registration: Chinese ClinicalTrials, ChiCTR2100051140. Registered 15 December 2021, https://www.chictr.org.cn/bin/project/edit?pid=133691 Parkinson's disease repetitive transcranial magnetic stimulation regulatory T cells neuro-inflammation motor dysfunction syt6 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Parkinson’s disease (PD) is a progressive neurodegenerative disorder in which Dopamine (DA) neurons degenerate and neuroinflammatory responses play crucial roles in its pathogenesis [ 1 – 4 ]. There is increasing evidence that the abnormal activation of peripheral immune cells and circulating mediators, including microglia and T cells, actively contribute to neurodegeneration and disease progression in PD[ 5 – 9 ]. Microglia are the most abundant resident immune cells in the brain, and these cells are activated in response to chronic inflammation and produce proinflammatory cytokines in PD[ 3 , 10 ]. In the immune system, regulatory T cells (Tregs) are an immunosuppressive T cell subset that are important regulators of neuroinflammatory tolerance and preserve immune homeostasis in the central nervous system (CNS) and peripheral circulation[ 11 – 14 ]. Recent studies have shown that Tregs exert neuroprotective effects on some neurological diseases, such as amyotrophic lateral sclerosis (ALS) and ischemic stroke, possibly by mediating the suppression of toxic neuroinflammation and regulating neurotrophic factors in the CNS and peripheral circulation[ 15 – 18 ]. It has been shown that an increase in Tregs in PD models significantly prevents dopaminergic neuronal loss and behavioral changes, and attenuates inflammatory reactions in the CNS[ 11 , 13 , 19 ]. One recent study by Park et al., also suggest that co-transplanting autologous Treg cells could effectively provide a potential strategy to achieve better clinical outcomes for cell therapy in Parkinson's disease[ 20 ]. Hence, the role of Tregs in PD needs to be further investigated. Repetitive transcranial magnetic stimulation (rTMS) is a safe and noninvasive neuromodulation technique that has been used to improve motor symptoms in PD[ 21 – 24 ]. However, the reported outcomes of rTMS in PD have been inconsistent[ 22 ]. A recent study has suggested that multi-session of high-frequency rTMS over the primary motor cortex (M1, especially bilateral M1) with a total of 18, 000–20, 000 pulses may be optimal for motor improvement in PD[ 25 ]. However, the molecular mechanisms underpinning the therapeutic effects of rTMS on PD have not been elucidated. Animal studies have shown that rTMS can play a neuroprotective role in PD animal models through anti-apoptosis and anti-inflammation[ 26 , 27 ]. There is a suggestion that proinflammatory cytokines (IFNγ and IL-17A) in the peripheral blood of PD patients may change after TMS treatment[ 28 ]. A decrease in the proportion of Tregs in the peripheral blood of PD may be an important feature of the peripheral immune response in PD[ 13 , 29 , 30 ]. Therefore, we hypothesize that rTMS may exert its modulatory effects on Tregs in PD. To test this hypothesis and address current gaps in knowledge, we first designed a prospective clinical trial in PD patients to evaluate the effect of rTMS on motor dysfunction and investigated the relationship between rTMS and changes in circulating Tregs. We next validated the clinical findings using an experimental MPTP mouse model. In addition, we also investigated the mechanisms of Tregs in regulating the therapeutic effects of rTMS. Last, we screened and identified Treg-related hub node proteins in the MPTP model. Materials and methods Ethics Statement The human studies were approved by the Ethics Committee of Zhujiang Hospital of Southern Medical University, and all patients provided written informed consent prior to recruitment into the study and was conducted in accordance with the principles of the Helsinki Declaration revised in 1975 and the National Institutes of Health Policy and Guidelines for Human Subjects issued in 1999. All mouse experiments were approved by the Experimental Animal Ethics Committee of Zhujiang Hospital of Southern Medical University. Clinical Study From Sep 2021 to Jan 2022, 60 PD patients were prospectively recruited in a randomized, double-blinded study (the main researchers, raters, experienced neurologists and patients involved in the study were not aware of the rTMS treatment protocol, except the therapists who did rTMS treatment for PD) from Zhujiang Hospital (Trial Registration Chinese ClinicalTrials. gov Identifier: ChiCTR2100051140). All participants provided written consent to participate in the investigation and allowed investigators to examine their blood samples. Informed written consent was obtained from patients and family members. The enrolled PD patients were interviewed by two experienced neurologists. Characteristics of PD patients, clinical ratings and neuropsychological tests were performed in Table 1 . The exclusion Criteria include: 1.Patients with secondary Parkinson's syndrome or Parkinson's overlap syndrome caused by vascular factors, toxins, medications, etc.; 2. PD patients with persistent head tremors; 3. History of Deep Brain Stimulation (DBS) or ablative surgery in the past;4. Other contraindications for rTMS, such as history of epileptic seizures, pregnant women, individuals with cardiac pacemakers, those with metal implants in the body, intracranial hypertension, or severe bleeding tendencies;5. Individuals who have received rTMS treatment within the last 6 months;6. As assessed by the researchers, other factors that make the patient unsuitable for participation in the study or those who are unable to commit to completing follow-up visits. All participants with PD received an EEG before inclusion and fulfilled the Movement Disorder Society Clinical Diagnostic Criteria for Idiopathic PD[ 31 ] and underwent extensive clinical examinations. 54 PD patients were ultimately recruited (6 PD patients were ultimately excluded due to four individuals elected not to partake in the study and two subjects were identified as having secondary parkinsonism) and received rTMS treatment or sham rTMS treatment according to random numbers. Random numbers were performed as follows. Computer SAS software was used to randomly group according to a single center, generate random numbers, and the treatment scheme is hidden using opaque envelopes. The envelopes are kept by the treatment provider and opened and sealed by the treatment provider in the order of the patient's visit. Unless the conditions for unblinding are met, the treatment plan cannot be disclosed under any circumstances. The follow-up examinations were done by the same rater and under the same time and all the patients were ON-condition. Sample size was based on a calculation of the results of pre-experiments. The rTMS were delivered by MagPro R30 (Tonica Elektronik A/S, Denmark). The specific rTMS treatment parameters are as follows: initially, high-frequency rTMS (100% resting motor threshold, 10 Hz, 1000 pulses) is administered to the primary motor cortex of the left hemisphere. Subsequently, after a 5-minute treatment interval, high-frequency rTMS (100% resting motor threshold, 10 Hz, 1000 pulses) is applied to the primary motor cortex of the right hemisphere. This bilateral hemispheric treatment is conducted daily. The treatment protocol spans over two weeks, with a consecutive 10 working days of treatment for each hemisphere. During the 2-week rTMS treatment period, the dosage of the original treatment drug remained unchanged. The sham rTMS treatment protocol was the same as the rTMS treatment protocol, except that a Cool-B65 P CO coil was used for the sham treatment, while a Cool-B65 A CO coil was used for the rTMS treatment. The alternative sham stimulation was also proceeded for two-week. At the start of each treatment, the resting motor threshold (RMT) of the patient's cortex was measured according to relative frequency method[ 32 ]. Specifically, the patients were seated in an armchair with a silversilver chloride surface electrode placed over the abductor pollicis brevis muscle contralateral to each hemisphere. The hot spot was determined using the MagPro R30 Stimulator TMS System (Tonica Elektronik A/S, Denmark) and octagonal coil. The octagonal coil was placed over the scalp and repositioned until the maximal motor evoked potential (MEP) was elicited. After determining the hot spot, the RMT was obtained by delivering single pulse transcranial magnetic stimulation to the hot spot. The RMT was defined as the lowest TMS intensity capable of eliciting a MEP greater than a 50 µV peak-to-peak amplitude in five of the ten subsequent trials. CD4 + CD25 + CD127- cells, CD4 + CD25(low)CD45RA+ [naive Treg (nTreg) cells] and CD4 + CD25(high)CD45RA- [activated Treg (aTreg) cells] subsets in the peripheral blood were assessed by flow cytometry and IFN-γ, TNF-α, IL-17α, IL4, IL10, TGF-β1 in the peripheral blood were assessed by Enzyme-linked immunosorbent assay (ELISA) prior to treatment and day 13. Hoehn and Yahr and MDS-UPDRS III were performed prior to treatment and on Days 13, 19, and 40 following the culmination of the two-week rTMS therapy. Table 1 Baseline characteristics of PD patients in sham and rTMS group Characteristics Sham rTMS P value Sham vs rTMS n 27 27 Gender (M/F) 14/13 14/13 > 0.9999 Age (years) 63.48 ± 9.57 63.26 ± 8.17 0.9273 MDS-UPDRS Ⅲ (score) 51.33 ± 11.68 50.67 ± 10.47 0.8260 20–30 (n) 1 1 30–50 (n) 10 9 > 50 (n) 16 17 H & Y scale 2.61 ± 0.68 2.67 ± 0.65 0.7610 1(n) 0 1 1.5(n) 6 2 2(n) 1 4 2.5(n) 3 2 3(n) 16 17 4(n) 1 1 LED (mg/day) 838.0 ± 484.0 830.8 ± 412.5 0.9534 A(n) 2 2 F(n) 1 2 A + C(n) 0 1 A + D(n) 2 2 A + E(n) 1 1 A + B + C(n) 1 1 A + B + D(n) 1 0 A + C + D(n) 4 4 A + C + E(n) 2 3 A + C + G(n) 1 2 A + D + E(n) 2 1 A + D + F(n) 1 0 A + B + C + D(n) 3 2 A + B + C + E(n) 1 0 A + C + D + E(n) 1 1 A + C + D + F(n) 1 1 A + C + E + G(n) 1 1 A + D + E + G(n) 0 1 A + B + C + D + E(n) 1 1 A + C + D + E + G(n) 1 1 Duration since symptom onset(month) 71.26 ± 46.54 86.81 ± 69.53 0.3385 Motor subtype > 0.9999 Mixed type 10 11 Tremor type 12 10 akinetic-rigid type 5 6 The presence of motor fluctuations(Yes/No) 10/17 8/19 0.5723 LED refers to the equivalent dose of levodopa; A = Dobassrazide tablets; B = Carlevodopa controlled-release tablets; C = Entacapone; D = Pramipexole hydrochloride; E = Amantadine hydrochloride; F = Rasagiline; G = Ropinirole hydrochloride sustained-release tablets. A indicates the use of dobasilazine tablets as monotherapy; F indicates the use of rasagiline monotherapy; A + D indicates the combined use of dobasilazine tablets and pramipexole hydrochloride; the other combination drugs can be deduced by analogy. Data are means ± SD unless otherwise indicated. The peripheral blood was obtained from PD patients[ 33 ] and lysed in ACK lysis buffer (Biosharp, China).CD4 T cells were then purified using magnetic bead separation (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The cells were stained with anti-CD25 and anti-CD127 (all from BD Biosciences) for analysis by flow cytometry. Following the acquisition of sample data on a FACSCalibur flow cytometer (BD Biosciences), the results were generated in graphical and tabular formats using FlowJo V10 software (TreeStar Inc., Ashland, USA). Animal Study Based on our clinical findings of an association between rTMS and changes in circulating Tregs of PD patients, we next conducted studies in a PD mouse model to validate the results. We determined whether the Tregs in the spleen of the PD mouse model changed after rTMS treatment and investigated the potential mechanisms that led to these changes. We applied a similar rTMS treatment protocol in MPTP mice. Male C57BL/6 mice (8 weeks old, 22–24 g) received intraperitoneal (i.p.) injection of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) (Sigma–Aldrich, USA) (30 mg/kg) for 5 days as PD animal model, while control mice received the same dose of normal saline. Another group of MPTP mice received i. p. injection of anti-CD25 monoclonal antibody (1 mg/kg; Thermo, USA) for three consecutive days to block Tregs in the spleen, to investigate whether the therapeutic effect of rTMS on MPTP mice is related to changes in Treg level in the spleen. In addition, we also evaluated the behavior of mice by pole climbing test and detected the expression of dopamine cells, neurotrophic factors BDNF, GDNF, microglia and inflammatory factors in the SN of mice. We next screened the hub node proteins related to rTMS on MPTP mice by quantitative proteomics analysis with TMT labeled of whole proteins. Finally, to better understand the role of hub proteins in MPTP mice, RNA interference (RNAi) mediated Adeno-associated virus (AAV) vector was injected into midbrain via a stereotactic midbrain approach to block hub proteins level in SN. Immunofluorescent staining and western blots (WB) were used to detect the transfection efficiency. The changes of hub protein levels in SN were detected by immunofluorescence staining and the inflammatory cytokines in the ventral midbrain were detected by ELISA after the hub proteins were interfered by the virus. For the sample size in animal experiments, a power analysis was performed. A sample size of at least n = 8 per group was determined to reach a statistical significance of 0.05, in detecting an effect size of at least 1.06 with 80% power. Mice were assigned randomly to the experimental groups. The investigators were blinded to the identities of treatment groups. Animals and MPTP-PD Model Male C57BL/6 mice (8 weeks old, 22–24 g) were obtained from Liaoning Changsheng Biotechnology Co., Ltd. (Liaoning, China). Before the experiment, all mice were housed at 20–22°C with a 12 h: 12 h light/dark cycle and food and water provided ad libitum for 7 days. The CD25 receptor is a key site for activating Treg, and as the hypothesis is that rTMS may exert its modulatory effects on Treg in PD, we used anti CD25 injections to specifically block Treg to determine if rTMS still has an impact on PD. A total of 189 male mice were randomly divided into 9 groups as follows: (1) the saline normal control group (NC group, n = 21 mice) was only administered saline by intraperitoneal (i. p. ) injection for 5 days; (2) the NC + sham rTMS group (NC + sham group, n = 21 mice), which were treated with i. p. injections of saline (30 mg/kg) for 5 days followed by sham rTMS treatment; (3) the NC + rTMS (10 Hz) group (n = 21 mice), which were administered i. p. doses of saline (30 mg/kg) for 5 days before receiving rTMS (10 Hz) therapy; (4) the MPTP group (n = 21 mice), which received i. p. injections of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) (Sigma–Aldrich, USA) (30 mg/kg) for 5 days; (5) the MPTP + sham rTMS group (n = 21 mice), which were treated with i. p. injections of MPTP (30 mg/kg) for 5 days followed by sham rTMS treatment; (6) MPTP + rTMS (10 Hz) group (n = 21 mice), which were administered i. p. doses of MPTP (30 mg/kg) for 5 days before receiving rTMS (10 Hz) therapy; (7) the MPTP + block group (n = 21 mice), which received 3 days of continuous i. p. injections of anti-CD25 monoclonal antibodies (1 mg/kg) followed by 5 days of i. p. injections of MPTP (30 mg/kg); (8) the MPTP + block + sham (10 Hz) group (n = 21 mice), which was administered i. p. injections of anti-CD25 monoclonal antibodies (1 mg/kg, Thermo, USA) for 3 days before receiving i. p. injections of MPTP (30 mg/kg) for 5 days before receiving shamtherapy; and (9) the MPTP + block + rTMS (10 Hz) group (n = 21 mice), which was administered i. p. injections of anti-CD25 monoclonal antibodies (1 mg/kg, Thermo, USA) for 3 days before receiving i. p. injections of MPTP (30 mg/kg) for 5 days before receiving sham therapy. rTMS treatment A transcranial magnetic stimulator (MagVenture A/S, Denmark) connected to a circular coil (Cool-40 Rat Coil) with a diameter of 40 mm was used to deliver rTMS. the rTMS treatment regimen for mice is consistent with the treatment regimen for PD patients. In the sham rTMS group, the stimulus was delivered by fixing the coil 10 cm above the heads of the mice to ensure that the mice felt the clicking sound or vibrations generated by the rTMS coil but did not receive brain stimulation. To minimize the discomfort of the mice, their movements were carried out in a soft plastic funnel, therefore, on the first day, they were restrained without stimulation for 10s to familiarize them with the experimental procedure. At the start of each treatment, the resting motor threshold was measured as previously described[ 34 ]. Briefly, a standard electromyographic (EMG) machine (Counterpoint, Dantec Medical Inc., Denmark) was used for recording of the motor evoked potentials (MEPs). Sampling frequency was 51.4kHz, high and low pass filter settings were 10 and 3000Hz. A 0.5mm bipolar EMG needle (DANTEC) placed in the right hindlimb biceps femoris muscle with a ground electrode 10 mm proximal to the recording electrode was used for recording the muscle activity. The motor threshold was defined as a reproducible motor evoked potential in five consecutive stimuli with an interstimulus interval > 3s and an amplitude > 50mV. The real and sham rTMS treatments did not induce seizures or other behavioral changes during the treatment period. Pole climbing test We assessed motor dysfunction by performing pole tests[ 35 ] Briefly, the mice were placed head-up on the top of the pole (height 50 cm, diameter 1 cm), and their return to the bottom was timed. Timing began when the mouse was released and stopped when one hindlimb reached the bottom. The test was repeated 3 times for each mouse after they were trained once at 30 min intervals, and the average time was used for data analysis. The raters were blinded for the treatment group. Flow cytometric analysis of CD4 + CD25 + Foxp3 + Tregs in the spleen The spleens of male C57BL/6J mice[ 19 ] were removed and homogenized by a tissue grinder and a wire mesh screen. Red blood cells were then lysed in ACK lysis buffer. Then CD4 + T cells were then purified using magnetic bead separation (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The cells were stained with anti-CD25 and anti-Foxp3 (BD Biosciences, USA, 567456) for flow cytometric analysis. Following the acquisition of sample data on a FACSCalibur flow cytometer (BD Biosciences), the results were generated in graphical and tabular formats using FlowJo V10 software (TreeStar Inc., Ashland, USA). Immunohistochemical staining The mice were anesthetized with sodium pentobarbital, sacrificed, and transcardially perfused with normal saline containing 0.5% sodium nitrate and heparin (10 U/ml) followed by 4% paraformaldehyde (PFA) dissolved in 0.1 M phosphate buffer. The brain was dissected and postfixed in 4% PFA at 4°C for 18–24 h and then immersed in 20% sucrose at 4°C until the brain dropped to the bottom, followed by immersion in 30% sucrose at 4°C for 24 h. The brains were embedded in optimal cutting temperature compound (O. C. T. Compound, Tissue-Tek, USA.) We prepared coronal sections of the frozen brains with a cryostat microtome (CM1950, Leica, Germany). Frozen brains were sectioned into 40-µm-thick coronal sections. All sections were collected and processed for immunohistochemical staining. For antigen retrieval, the tissue slices were submerged in 0.01 M sodium citrate buffer (pH 6.0) and rinsed twice in PBS. The tissue slices were then treated for 1 hour at 37°C in PBS containing 0.2% v/v Triton X-100, 0.02% w/v sodium azide, and 5% v/v goat serum. Primary antibodies against tyrosine hydroxylase TH (1: 800, Proteintech, USA, 25859-1-AP), BDNF (1: 500, Abcam, England, ab108319), GDNF (1: 500, Abcam, England, ab18956) and Iba-1 (1: 500, Abcam, England, ab178846) SYT6 (1: 250, SANTA CRUZ, sc390321), TLR4 (1: 250, Invitrogen, 710185) were added to the slices and incubated overnight at 4°C. Then, the sections were treated with the appropriate biotinylated secondary antibody, followed by Vectastain ABC reagent (Vector Laboratories, Burlingame, CA, USA) and 3, 3’-diaminobenzidine (Sigma–Aldrich, Vienna, Austria). The stained slices were dehydrated and coverslipped with Entellan before being mounted on slides (Merck, Darmstadt, Germany). IgG conjugated with Alexa 594 (1: 500, Abcam, England, ab150080) was used for immunofluorescent staining. Mounting medium was used to cover the sections (Dianova, Hamburg Germany). The numbers of TH+, SYT6, TLR4 in the substantia nigra pars compacta (SNc) were estimated using unbiased stereological methods (Stereo Investigator, MicroBrightField, VT). The numbers of activated Iba-1-positivemicroglia in the SNc were estimated using the optical fractionator probe with the stereologist. Microglia were classified as activated if the cell body was visibly increased in diameter and the cell had shortened and thickened processes. The three stereologists were blinded to the treatment received. Additionally, we determined the average optical density value of BDNF and GDNF immunoreactivity through densitometry, employing the freely available ImageJ software (National Institutes of Health, Bethesda, MD, USA). All histochemical data statistics was conducted in a single-blind manner by four experimenters. FX renumbers each group of fluorescent films in an experiment and randomly divides them into three groups (random number method) and assigns them to three investigators (YQL, ZCX and Q.W). Each film takes 5 fields of view for statistical averaging, and finally the data is summarized with FX. Quantitative proteomics technology with TMT labeling of whole protein Twelve brain tissue from 4 groups (saline normal control, MPTP, MPTP + rTMS, MPTP + block + rTMS) were collected for proteome Tandem Mass Tag (TMT) analysis. The collected samples were processed for TMT analysis in JingJie Bio-tech. Output was visualized using R and CummeRbund. Heatmaps were generated in R using pHeatmap and R ColorBrewer. Result was analyzed according to Maxquant (v1.6.15.0) database. Molecular docking method The protein data bank (PDB) database ( https://www.rcsb.org/ ) was used to obtain the PDB file which has the active structure of the target protein, The smaller Resolution value and complete protein binding pockets were used as the screening conditions to select the crystal structure for subsequent analysis. Refer to the previous literature[ 36 – 40 ], according to the amino acid sequence provided by Unirpot, SWISS-MODEL was used ( https://swissmodel.expasy.org/interactive ) to perform online homology modeling of proteins without ready-made protein crystal structures in the PDB database. Select the result with homology ≥ 30%, and then according to the scoring screen to evaluate the model quality with GMQE and QMEAN values when the homology reaches the standard, the template with larger GMQE and QMEAN close to 0 was chose. Then, online protein-protein docking is performed through Z-dock ( http://zdock.umassmed.edu/ ), the version is selected as V3.0.2[ 41 , 42 ], and the docking result image uses PyMol 2. 3.5 for image rendering. Path enrichment analysis and PPI network construction GO database ( http://geneontology.org/ ) was used for pathway enrichment analysis[ 43 ], obtain immune-related pathway genes, and String database ( https://www . string-db. org/) was used to construct protein online Protein interaction network (PPI network)[ 44 ], the species is selected as Mus musculus, the minimum required interaction score is set to 0.4, and the other conditions are the default settings. Western blotting Tissues were lysed in a detergent-based lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and 1 mM PMSF). Samples were then centrifuged for 10 min at 13, 000 g at 4°C, and the supernatant was collected and quantified with the BCA Protein Assay Kit (Thermo Scientific, USA). Equal amounts of protein sample were separated by 10% SDS-PAGE. Antibodies used for western blotting were: Syt6 (1: 1000, abcam, USA), TLR4 (1: 1000, abcam, USA), TH (1: 1000, proteintech, USA), Slc6a3 (1: 1000, abcam, USA) and β-actin (1: 10000, Thermo, USA). Antibody binding was detected using the secondary antibodies (Peroxidase-Conjugated AffiniPure Goat Anti-Rabbit/Mouse IgG (H + L), 1: 5000, thermofisher, USA). All samples were normalized to β-actin. All quantification was calculated with Alpha Ease FC software (Alpha Innotech Corporation, USA). ELISA assay Cytokine levels of IFN-γ, TNF-α, IL-17α, IL4, IL10 and TGF-β1 in the serum of PD patients were measured using commercial ELISA kits (IFN-γ ELISA kit (Abcam, England, ab46025), TNF-α ELISA kit (Abcam, England, ab181421), IL-17α ELISA kit (Abcam, England, ab216167), IL4 ELISA Kit (Abcam, England, ab215089), IL-10 ELISA kit (Abcam, England, ab100549), TGF-β1 ELISA kit (Sigma–Aldrich, USA,RAB0460) in accordance with the manufacturer’s protocols. Brain tissue was collected from the different groups of mice, and then the tissue from the ventral midbrain was separated and homogenized with lysis buffer on ice. The supernatant was collected at 12, 000 rpm at 4°C and centrifuged for 10 min for subsequent experiments. The proinflammatory cytokines IL-6, IFN-γ, TNF-α, IL-1β, IL-10 and TGF-β1 were measured using commercial ELISA kits (IL-6 ELISA kit (Abcam, England, ab100713), IFN-γ ELISA kit (Abcam, England, ab100690), TNF-α ELISA kit (Abcam, England, ab229393), IL-1β ELISA Kit (Sigma–Aldrich, USA, RAB0275), IL-10 ELISA kit (Abcam, England, ab100697), TGF-β1 ELISA kit (Abcam, England, ab119557) in accordance with the manufacturer’s protocols. Virus Constructs and stereotaxic surgery The scramble short hairpin RNA (shRNA) (5’-CCTAAGGTTAAGTCGCCCTCG-3’) and shRNA coding sequences targeting mous syt6 (5’-ATGAAAGCGAGACGCTGATTG-3’), (5’- ATGTCTCCAGCGTGGACTATG-3’) and (5’- GCGGAAGTTCTGACCCTTA-3’) were cloned into the PAAV-U6-shRNA(Syt6)-CMV-EGFP-WPRE vector (Obio Technology, Shanghai, China). Adeno-associated viruses (AAVs) described above were packaged by Obio Technology into serotype 2/9.After mice were anesthetized and placed in the stereotaxic frame, 2 µl of purified and concentrated AAV(10 12 IU/mL) was unilateral injected into the right SNc (AP = − 3.2 mm; ML = ± 1.2 mm, DV = − 4.6 mm) at a rate of 0.2 µl/min according to previously described protocols[ 45 ]. The 5-µl Hamilton syringe was kept in place for 10 min before being slowly retracted within 5 min. Statistical Analysis Statistical analyses were performed using GraphPad Prism 8.0 software. Data with a normal distribution are presented as the mean ± SD. Intra-group comparisons of squared differences were conducted using paired sample t-tests, while inter-group comparisons were made using two independent samples t-tests. Data that did not conform to a normal distribution are presented with comparisons made using non-parametric tests. Counting data are presented as frequencies, with comparisons made using the χ2 test. A two-sided test was chosen, and a P-value less than 0.05 was considered statistically significant. Correlation between the change of Treg ratio in peripheral blood and the change of MDS-UPDRSIII before and after treatment with rTMS were calculated by the Pearson correlation. After flowcytometry, CD4 subsets were Determined as a percentage of all lymphocytes gated using forward and side scatter, and as a percentage of all leukocytes bydual-platform analysis using full blood cell counts from the same blood collection. Mouse flow cytometry data, total times of pole climbing test, TH, BDNF, GDNF, Syt6, TLR4 immunohistochemistry, IL-6, IFN-γ, TNF-α, IL-1β, IL-10 and TGF-β1 enzyme-linked immunosorbent assay and Syt6, TLR4, Slc6a3 and TH western blotting were analyzed using One-way ANOVA followed by the least significant difference (LSD) for post hoc comparisons. The data are representative of at least three independent experiments. Results rTMS increased the proportion of Tregs, attenuated inflammation and improved motor impairment in PD patients We conducted a clinical trial that included a total of 60 PD patients (6 PD patients were ultimately excluded). Fifty-four PD patients were divided into two groups; one group was treated with 10-day rTMS (10 Hz) in the M1 area, and the other group received sham rTMS treatment (Fig. 1 A, B), followed by Hoehn and Yahr and MDS-UPDRS III assessments at Days 1, 13, 19, and 40 ( Fig. 1 A, B; Table 1 ) . Our clinical trial showed that the MDS-UPDRS Part III score was significantly reduced in PD patients receiving rTMS as compared to the sham control group on the day following the completion of a two-week rTMS treatment protocol (-4.15 ± 1.689, P < 0.0001). Furthermore, the MDS-UPDRS Part III score sustained a lower score for up to 40 days post-treatment with rTMS (-4.25 ± 1.716, P < 0.0001) (Fig. 1 C, D). The baseline clinical and demographic characteristics were not different between the rTMS and sham groups (Table 1 ). We performed flow cytometry with anti-CD4, CD25 and CD127 antibodies to examine the proportions of CD4 + CD25 + CD127- Treg cells and CD4 + T cells in sham and rTMS groups. The results showed no difference in the proportion of Tregs (% CD4 + T cell population) between the sham and rTMS groups at baseline ( Fig. 1 E ) . However, 10-day rTMS treatment (9.0 ± 0.64%) elevated peripheral Treg levels in PD patients (6.87 ± 0.69%) (p < 0.0001; Fig. 1 G, H), but there was no difference in the peripheral Treg levels in sham group (Fig. 1 F). Pearson’s correlation was used to analyze the association between MDS-UPDRS III scores and the proportion of Tregs. We found that the increased proportion of Tregs was negatively correlated with the change in the MDS-UPDRS III scores in the rTMS PD group (P < 0.0001; Fig. 1 J) but not in the sham treatment group ( Fig. 1 I ) . Furthermore, there was an observed increase in the proportion of aTreg/nTreg cells within the peripheral blood (Supplementary Fig. 1A, B, C). Concurrently, levels of IL4, IL10, and TGF-β1 were elevated, while pro-inflammatory markers such as IFN-γ, TNF-α, and IL-17α demonstrated a decrease (Supplementary Fig. 1D, E, F). The MFI of CD25 also shown no difference in the proportion of CD25 between the sham and rTMS groups at baseline (Supplementary Fig. 2A). In addition, the median fluorescence intensity (MFI) of CD25 was increased after the treatment of rTMS compared with sham treatment (Supplementary Fig. 2C). rTMS ameliorated behavioral impairment in MPTP-induced mice We induced PD-like symptoms and analyzed the impact of rTMS on motor function in MPTP mice. Based on the results of the pole climbing test, MPTP significantly impaired motor function, and rTMS ameliorated this impairment ( Fig. 2 A, B ) . Compared to saline normal control mice, MPTP-induced mice took 121.14% more time to turn around and climb down in the pole climbing test (p < 0.0001, Fig. 2 A, B ) . However, compared with MPTP and sham rTMS mice, MPTP + rTMS (10 Hz)-treated mice spent less time and displayed considerably improved performance on the pole test (34.97% decrease in rTMS-treated PD mice vs. sham-treated PD mice, p < 0.0001, Fig. 2 A, B). However, the Treg inhibitor anti-CD25 significantly abolished this improvement (Fig. 2 A, B). rTMS rescued dopaminergic neurons, reversed the decrease in the circulating proportion of Tregs and attenuated inflammatory cytokines in MPTP-induced mice We performed immunohistochemical staining to examine the loss of dopaminergic neurons, BDNF and GDNF in the SN after MPTP administration. The immunohistochemical staining results showed that MPTP-induced mice showed 50.15%, 38.38% and 21.82% reductions in TH-positive neurons and fibers, BDNF and GDNF in the SN compared with saline normal control (NC) mice, respectively (p < 0.001 for TH, p < 0.001 for BDNF and GDNF; Supplementary Fig. 3 ). Compared with those in the MPTP and sham rTMS groups, TH, BDNF and GDNF were considerably increased in the MPTP + rTMS (10 Hz) group ( Supplementary Fig. 3 ). Compared with that in saline NC mice, numerous microglia were activated in the SN of MPTP mice, and the proinflammatory cytokines IL-6, IFN-γ, TNF-α and IL-1β in the ventral midbrain of MPTP mice were significantly increased by 75.37%, 185.87%, 91.66%, 140.36%, respectively, while the anti-inflammatory cytokines IL-10 and TGF-β1 were significantly reduced by 59.90% and 17.02%, respectively (Fig. 2 E-G). The results showed a significant decline in the circulating proportion of Treg cells (% CD4 + T cell population) in PD mice (1.98 ± 0.33%) compared to NC mice (4.54 ± 0.37%) (Fig. 2 C, D). Compared with that in sham mice, rTMS (10 Hz) therapy significantly reversed the decrease in the circulating proportion of Tregs in PD mice (302.44% increase in rTMS-treated PD mice vs. sham-treated PD mice, p < 0.0001 for Tregs, Fig. 2 C, D), increased the anti-inflammatory mediators IL-10 and TGF-β1 by 69.44% and 12.34%, respectively, deactivated microglia and decreased the inflammatory cytokines IL-6, IFN-γ, TNF-α and IL-1β by 28.41%, 24.40%, 30.48% and 22.78% in the ventral midbrain of PD mice, respectively (Fig. 2 E-G). This finding indicated that rTMS treatment of PD mice could attenuate inflammation in the brain. Injection of the Treg inhibitor anti-CD25 (1 mg/kg/d, for 3 days) into PD mice significantly abolished the rTMS-mediated increase in circulating Tregs and anti-inflammatory cytokines and reversed the downregulation of these inflammatory cytokines, as well as microglial activation in the SN (p < 0.0001 for Tregs, p < 0.0001 for microglia, p < 0.0001 for inflammatory and anti-inflammatory cytokines; Fig. 2 C-G). Quantitative proteomics analysis of Treg- and rTMS-related proteins To further identify the molecular targets and hub proteins related to Tregs and rTMS therapy in PD, we used tandem mass tag (TMT) technology to label 12 samples (4 groups: saline normal control, MPTP, MPTP + rTMS, and MPTP + block + rTMS, 3 samples per group) for quantitative proteomics analysis. We first identified 7, 817 proteins ( Supplementary Table 1 ), and proteins that were significantly upregulated and downregulated in all four clustered groups are shown in Supplementary Table 2A and Fig. 3 A-B. We next identified 2 proteins (Syt6 and Slc6a3) through overlapping pairwise comparisons (Fig. 3 C). Analysis of the protein–protein interaction (PPI) network also confirmed that Syt6 and Slc6a3 were involved in multiple pathways and associated with the activation of T cells (Fig. 3 D). We performed full rigid-body docking orientation analysis of the two proteins with the ZDOCK algorithm and found that Syt6 docked with TLR4. This proteomics analysis suggests that there may be protein interactions between TLR4 and SYT6, and the two protein interactions may exert biological effects through multiple binding sites. The main sites of TLR4 are LYS-47, SEP-71 and ASP-95, and the main sites of SYT6 are ARG − 321 and ARG-386 ( Supplementary Table 2B, C ). These proteins are considered hub proteins and may serve as novel targets of PD. To further characterize the role of rTMS and identify its association with Tregs in PD, we investigated these screened hub proteins by Western blotting (Fig. 3 F-G). Compared with those in saline normal controls, the levels of Syt6 and TLR4 were significantly increased by 164.19% and 85.55%, respectively, and TH and Slc6a3 were significantly decreased by 41.08% and 45.97% in the PD mouse brain, respectively, while rTMS therapy reversed these changes (P < 0.0001 for Syt6, TLR4, TH and Slc6a3; Fig. 3 A-E). More interestingly, after inhibiting Tregs with anti-CD25 and stimulating PD mice with rTMS, the levels of Syt6, TLR4, TH and Slc6a3 in PD mouse brains were significantly reversed compared with those in the PD + rTMS group (p < 0.0001; Fig. 3 F-G ) . Verification of the role of Treg and rTMS-related protein syt6 in MPTP mice by virus interference method Adeno-associated virus (AAV) vector was injected into midbrain via a stereotactic midbrain approach to block syt6 level. Immunofluorescent staining and WB were used to detect the efficiency of transfection (Fig. 4 A, C, E). Compared with those in MPTP + scramble control group, the SYT6/DAPI expression was significantly decreased by 82.2% in the SN of MPTP + syt6-shRNA3 group, and the syt6 relative expression level was similar to that in WB. Similarly, mice in the MPTP + Syt6-shRNA3 group showed significantly improved performance in pole test after Syt6 knockdown in SN (Fig. 4 B, D, F). Therefore, we then used syt6-shRNA3 as an AAV to block the expression of syt6 protein in SN. Interestingly, rTMS did not change the total climbing time of MPTP mice with syt6 AAV. On the contrary, rTMS treatment significantly reduced the total climbing time of MPTP mice with AAV empty vector (Fig. 5 A, B). We next examined the expression of syt6, TLR4, TH and inflammatory cytokines with syt6 AAV intervention in MPTP-induced mice with or without rTMS treatment ( Fig. 5 C, E, G, I ) . There was a significant increase by 47.72% in TH expression in MPTP mice with syt6 AAV intervention but rTMS treatment did not change the TH level in MPTP mice with syt6 AVV intervention. On the contrary, rTMS treatment significantly increased TH level in MPTP mice with AAV empty vector by 39.51% ( Fig. 5 E, F ) . There was a significant decrease by 80.47% of syt6 expression in MPTP mice with syt6 AAV intervention ( Fig. 5 C, D ) ; however, rTMS did not change the syt6, TLR4, IL-β1, TNF-α and IL-6 levels in MPTP mice with syt6 AAV. On the contrary, rTMS significantly decreased TLR4, IL-β1, TNF-α and IL-6 level by 41.80%, 25.52%, 32.12% and 10.79% respectively in MPTP mice with AAV empty vector ( Fig. 5 G, H, I ) . In addition, the levels of anti-inflammatory cytokines, such as IL-10 and TGF-β, were significantly elevated in MPTP-induced mice following rTMS treatment, which was not altered by Syt6 knockdown (Fig. 5 I), Furthermore, Syt6 expression was detected in Iba1-positive cells ( Supplementary Fig. 4 ). These data suggest that rTMS treatment affects syt6, a hub protein, to increase the proportion of Tregs in MPTP mice. Discussion Our randomized sham controlled clinical trial demonstrated that 10 days of rTMS stimulation improved motor dysfunction in PD subjects after 13 days, and the effect lasted for 40 days. Importantly, rTMS treatment elevated peripheral Treg levels in PD patients (p < 0.0001), but not in sham group (Fig. 1 G). In addition, we found a significant negative correlation between the increased proportion of peripheral blood Tregs and MDS-UPDRS III scores (Fig. 1 J), suggesting that motor improvement induced by rTMS might be due to an increase in circulating Tregs. rTMS is a non-invasive neuromodulation technique that utilizes magnetic fields to stimulate nerve cells in the brain. Extensive research has been conducted on its potential therapeutic effects in various neurological and psychiatric disorders, including Alzheimer's disease, PD, stroke, and multiple sclerosis. Preclinical studies have suggested that rTMS may improve cognitive function in Alzheimer's disease by enhancing synaptic plasticity and reducing amyloid-beta plaques. rTMS has also been studied for its potential to improve motor function and reduce fatigue in multiple sclerosis patients by modulating the excitability of the motor cortex [ 46 ]. A systematic review has shown that rTMS intervention may have the potential to modify apathy among patients with chronic stroke[ 46 ]. The exact mechanisms by which rTMS exerts its effects are not fully understood but are normally thought to involve changes in neuronal activity, synaptic plasticity, and neurotransmitter release. In our study, we found that rTMS improves motor function by modulating the function of Tregs and suppressing toxic neuroinflammation in PD, which is consistent with previous studies[ 47 – 49 ]. Ongoing research is exploring the use of rTMS in combination with other neuromodulation techniques to enhance therapeutic effects, indicating the potential therapeutic effects of rTMS in the treatment of neurodegenerative diseases. Evidence-based guidelines for the therapeutic use of rTMS have demonstrated that high-frequency rTMS (HF-rTMS) applied to bilateral M1 regions or the left dorsolateral prefrontal cortex (DLPFC) can ameliorate motor impairment and depression, respectively, in Parkinson's disease, with Level B evidence indicating probable efficacy[ 24 ]. Furthermore, a randomized controlled trial published in a neurology journal provides Class I evidence that, in patients with Parkinson's disease experiencing depression, bilateral M1 rTMS (comprising 1,000 stimuli for each M1; 50 trains of 4 seconds at 10 Hz with 40 stimuli per train) results in enhanced motor function compared to sham rTMS[ 50 ]. Additionally, a meta-analysis has shown that multiple sessions of HF-rTMS over the M1, particularly when administered bilaterally, with a cumulative total of 18,000–20,000 pulses, appears to be the optimal parameters for improving motor symptoms in Parkinson's disease[ 25 ]. Informed by these references, our clinical trial was designed to ameliorate the motor symptoms of Parkinson's disease using HF-rTMS applied to both M1 areas, delivering 1,000 stimuli to each side. The treatment spanned 10 days, accumulating a total of 20,000 stimuli. To take our findings further, we next attempted to validate our clinical findings in a PD animal model. Previous studies have indicated that induction of Tregs promoted neuronal survival through suppression of microglial activation in MPTP-induced PD mouse model[ 19 , 51 ]. Here, we utilized MPTP PD mice (Fig. 2 A, B; Supplementary Fig. 1A, B) and found that Tregs in the spleen were dysregulated in these mice compared with NC mice (Fig. 2 C, D), which is consistent with previous reports[ 19 , 52 ]. Compared with that in NC mice, numerous microglia in the SN of PD mice were activated, and the proinflammatory cytokines IL-6, IFN-γ, TNF-α and IL-1β were significantly increased, while the anti-inflammatory cytokines IL-10 and TGF-β1 were significantly reduced ( Fig. 2 E-G ) . rTMS has been shown to ameliorate inflammation and regulate neurotrophic factors in depression, anxiety, cerebral ischemia and spinal cord lesions[ 53 – 56 ]. To evaluate whether rTMS could influence PD by impacting circulating Tregs and regulating inflammation in the brain, we applied rTMS to the brains of MPTP mice for 10 days and identified that a 10 Hz stimulation led to consistent and immediate improvements in motor symptoms at 1 day after rTMS therapy ( Fig. 2 A, B ) . In comparison to the sham-treated mice, our findings revealed that rTMS at 10 Hz significantly enhanced the circulating proportion of Tregs and TH + neurons within the SN (Fig. 2 C, D, Supplementary Fig. 1A, B) , increased the anti-inflammatory mediators IL-10 and TGF-β1, deactivated microglia and decreased the inflammatory cytokines IL-6, IFN-γ, TNF-α and IL-1β1 in the ventral midbrain of PD mice (p < 0.0001, Fig. 2 E-G ). These results suggest that rTMS therapy could attenuate inflammation in the brains in PD mice. One previous study showed that the transfer of Tregs to MPTP mice prevented DA neuronal degeneration by suppressing microglial activation and attenuating neuroinflammation, suggesting that the increased Tregs exerted neuroprotective effects on PD[ 57 ]. We next injected the Treg inhibitor anti-CD25 into MPTP mice and observed that this treatment significantly attenuated the rTMS-mediated improvements in motor symptoms, abolished the rTMS-mediated upregulation of circulating Tregs, and reversed the downregulation of inflammatory cytokines in the ventral midbrain and microglial activation in the SN (Fig. 2 C-G ) . These results indicate that the restoration of peripheral Tregs may be pivotal following rTMS stimulation in the brain, and rTMS may contribute to neuroprotection in PD at least partially by upregulating peripheral Tregs, subsequently reducing neuroinflammation and deactivating microglia in the SN. rTMS also profoundly upregulated the levels of TH, BDNF and GDNF in the SN ( Supplementary Fig. 3) , and these effects were partially abolished by anti-CD25 ( Supplementary Fig. 3 ). To further identify the hub proteins related to Tregs and to screen biological indicators, tandem mass tags (TMT) technology was used to label 12 samples that were used for quantitative proteomic analysis. After intersection analysis of differentially expressed genes in the MPTP + block + rTMS treatment group and MPTP + rTMS group was performed, 30 differentially expressed genes were selected for pathway enrichment analysis, and a protein–protein interaction (PPI) network was constructed and identified the Treg-related downstream targets Syt6, TLR4, TH and Slc6a3 ( Fig. 3 A-E ) , which were closely associated with the activation of T cells after rTMS stimulation in MPTP mouse brain, Compared with those in the saline normal controls, the levels of Syt6 and TLR4 were significantly increased, and TH and Slc6a3 were significantly decreased in the MPTP mouse brain, while rTMS therapy reversed these effects (Fig. 3 A-E ) . Interestingly, after inhibiting Tregs with anti-CD25 and administering rTMS stimulation, the changes in the levels of Syt6, TLR4, TH and Slc6a3 in MPTP mouse brains were significantly reversed compared with those in the PD + rTMS group (Fig. 3 F-G). These results suggest that Syt6, TLR4, and Slc6a3 are key targets of Tregs in regulating MPTP following rTMS stimulation, further indicating an important pathophysiologic role of Tregs in rTMS treatment in PD. We also performed a full rigid-body docking orientation between two proteins by ZDOCK algorithm, and found that Syt6 was docking with TLR4 (Fig. 3 E ) . These four proteins were recognized as the hub node proteins and may serve as novel targets in regulating PD. Four identified proteins in our study exhibit close intersection with neuroinflammation, microglia and Tregs in neurodegeneration. Microglia and Tregs are both key players in the immune response and homeostasis maintain within the CNS. Recent studies have highlighted that infiltrating Treg cells is essential for behavioral recovery and brain repair with their immunomodulatory effects on microglia after stoke[ 58 ]. A clinical study revealed a diminished proportion of Th2, Th17, and Treg cells in the peripheral blood of PD patients, along with a reduction in circulating CD4 + T lymphocytes[ 29 ]. Collectively, Treg cells can modulate microglial responses, potentially leading to improved outcomes in neurodegenerative diseases. Here in our study, following rTMS treatment, we observed an increased proportion of aTreg/nTreg cells in the peripheral blood, accompanied by a decrease in pro-inflammatory factors such as IFN-γ, TNF-α, and IL-17α. Tyrosine hydroxylase (TH), a tetrahydrobiopterin (BH4)-requiring monooxygenase that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines (CAs), such as dopamine, has been suggested as the enzyme being a source of reactive oxygen species (ROS) in vitro and a target for radical-mediated oxidative injury[ 59 ]. A translational study highlighted that TLR4-mediated neuroinflammation plays an important role in intestinal and/or brain inflammation, which may be one of the key factors leading to neurodegeneration[ 60 ]. An animal study also revealed that TLR4 stimulates release of cytokine through NF-kB by activating glial cells, thus resulting in the death of dopaminergic neurons in the MTPT-induced mice[ 61 ]. One recent study showed that ex vivo expanded Tregs suppressed neuroinflammation, down-regulated the expression of TLR4 and alleviated AD pathology in vivo[ 62 ]. Syt6 (Synaptotagmin 6), one isoform of Synaptotagmins and a brain-specific Ca2+/phospholipid-binding protein, has been shown to be a key component of the secretory machinery involved in acrosomal exocytosis and regulation of membrane trafficking in neurodegeneration[ 63 , 64 ]. Its function is seldom investigated in neurological diseases; one recent report suggests that it is a key mediator of endocytic pathways in BDNF release[ 65 ]. SLC6A3, the gene encoding the dopamine transporter (DAT), is deeply influenced by neuroinflammation in Parkinson’s disease[ 30 , 66 ]. Our results strongly suggest that rTMS exerts a beneficial effect on the peripheral immune system in PD via Tregs. Next, to better understand the role of hub node proteins related to the therapeutic effect of rTMS in MPTP mice, we injected RNAi mediated AAV vector into the midbrain to block the hub protein in SN. Among Treg-related downstream targets Syt6, TLR4, TH and Slc6a3, previous studies have shown TLR4, TH and Slc6a3 have been associated with the pathogenesis of PD[ 6 , 66 – 68 ]. Using the ZDOCK algorithm method, we found that Syt6 docked with TLR4, which has been reported to be associated with inflammation in SN of MPTP mice in previous studies[ 67 – 69 ]. We next selected virus interference with syt6 to verify if syt6 is the hub node protein related to the role of Treg in MPTP mice. We found that rTMS treatment did not change the total climbing time of MPTP mice, neither nigral IL-β1, TNF-α and IL-6 levels following syt6 virus interference, further verifying our hypothesis that syt6 participates in the therapeutic effect of rTMS in MPTP mice. To our knowledge, this is the first study directly showing SYT6 is involved in the neuro-pathogenesis of PD, especially modulating Treg-related neuro-inflammation. Proinflammatory mediators and reactive oxygen species (ROS) in the circulation are closely associated with PD severity[ 5 , 6 , 70 – 72 ]. It is possible that the infiltration of circulating immune cells into the brain leads to extensive BBB breakdown, which in return further exacerbates peripheral inflammatory cytokines crossing the BBB and damaging neurons[ 13 ]. Among immune cells, Tregs play a vital role in regulating inflammatory responses and maintaining homeostasis in the CNS microenvironment[ 13 ]. One recent clinical trial with long-term sargramostim treatment for PD showed that the drug (a recombinant granulocyte macrophage colony-stimulatory factor) was able to stabilize immune homeostasis, restore peripheral immune function and increase the numbers of Tregs in the circulation[ 73 ]. Taken together, our findings ( Fig. 1 J ) suggest that this restoration of peripheral blood Tregs in PD patients by rTMS stimulation may partially contribute to the improvement in motor dysfunction in PD patients. Limitations Our data did not examine a prospective temporal relationship between motor improvement and the proportion of Tregs and hence longitudinal studies are needed to determine if the circulating Treg level following rTMS stimulation can be used to monitor the clinical progression in PD. Randomized trials of potent reversible pharmacological inhibitors or agonists of Tregs may help to clarify whether modifying Tregs can delay the progression of sporadic PD in patients. Conclusion Our clinical and laboratory studies in PD patients and PD animal model suggest that rTMS improved motor function through modulating the function of Tregs and suppressing toxic neuroinflammation. We also identified several hub node proteins (especially Syt6) that may be potential therapeutic targets. Our findings provide pathophysiologic insights into the neuromodulatory effect of rTMS therapy in PD. Strategies to enhance peripheral Tregs and candidate molecular targets should be further explored. Abbreviations AAV = Adeno-associated virus; AD = Alzheimer’s disease; ALS = amyotrophic lateral sclerosis; aTreg = activated Treg; BBB = blood-brain barrier; BDNF = brain derived neurotrophic factor; CNS = Central Nervous System; DA = dopaminergic; DAT = dopamine transporter ; ELISA = Enzyme-linked immunosorbent assay ; PFA = 4% paraformaldehyde; GDNF = glial cell line derived neurotrophic factor; H & Y = Hoehn-Yahr ; IFN-γ = Interferon-γ; IL-1β = Interleukin-1β; IL-10 = Interleukin-10; IL-6 = Interleukin-6; LSD = least significant difference; M1 =primary motor cortex; MDS - UPDRS III = Movement Disorder Society Unified Parkinson's Disease Rating Scale part III; MFI = Median Fluorescence Intensity; MPTP = 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine; NC = normal control ; nTreg = naive Treg ; PD = Parkinson disease ; PDS = Parkinson’s Disease Syndrome ; PDB = protein data bank ; PPI = protein-protein interaction ; rTMS = repetitive transcranial magnetic stimulation; ROS = reactive oxygen species; RMT = resting motor threshold ; RNAi = RNA interference SNc = substantia nigra pars compacta; Syt6 = Synaptotagmin IV; SLC6A3 = Solute carrier family 6 member 3; SD = Standard Deviation; Tregs = Regulatory T-cells; TNF-α = tumor necrosis factor-alpha; TGF-β1 = Transforming growth factor-β1; TH = tyrosine hydroxylase; TMT = Tandem Mass Tags; TLR4 = Toll-like Receptor 4. Declarations Data and materials availability The data that support the findings of this study are available from the corresponding author, upon reasonable request. Conflict of Interests The authors declare that they have no conflict of interest. Acknowledgement We would like to thank Dr. Shengmin Lai, Guangdong Pharmaceutical University to provide valuable comments in the quantitative proteomics analysis. We also would like to thank Drs. Bin Deng, Zifeng Huang and Zhe Zheng from Zhujiang Hospital to assist the immunohistochemistry staining. Funding National Natural Science Foundation of China 81873777, 82071414, 82471433(QW) Initiated Foundation of Zhujiang Hospital 02020318005 (QW) Scientific Research Foundation of Guangzhou 202206010005 (QW) Science and Technology Program of Guangdong of China 2020A0505100037 (QW) Guangdong Medical Science and Technology Research Foundation Project A2020305(FX) National Medical Research Council Singapore (EKT) Author Contributions Conceived and designed the study: FX, EKT and Q. W. Performed the study: FX, BBS, HZ and Q. W. Revised the paper for intellectual content: YQL, HZ, SZZ, XBW, ZHC, ZHZ, CHD, CWY, KRC, BL, KLJ, LLC and EKT. Data statistics and analysis: FX, YQL, ZCX and Q. W. Wrote the paper: FX, EKT and Q. W. References Tan EK, Chao YX, West A, Chan LL, Poewe W, Jankovic J: Parkinson disease and the immune system - associations, mechanisms and therapeutics . Nat Rev Neurol 2020, 16 (6):303-318. Hirsch EC, Hunot S: Neuroinflammation in Parkinson's disease: a target for neuroprotection? Lancet Neurol 2009, 8 (4):382-397. Geng L, Gao W, Saiyin H, Li Y, Zeng Y, Zhang Z, Li X, Liu Z, Gao Q, An P et al : MLKL deficiency alleviates neuroinflammation and motor deficits in the α-synuclein transgenic mouse model of Parkinson's disease . Mol Neurodegener 2023, 18 (1):94. Msackyi M, Chen Y, Tsering W, Wang N, Zhang H: Dopamine Release Neuroenergetics in Mouse Striatal Slices . Int J Mol Sci 2024, 25 (9). Yang W, Chang Z, Que R, Weng G, Deng B, Wang T, Huang Z, Xie F, Wei X, Yang Q et al : Contra-Directional Expression of Plasma Superoxide Dismutase with Lipoprotein Cholesterol and High-Sensitivity C-reactive Protein as Important Markers of Parkinson's Disease Severity . Front Aging Neurosci 2020, 12 :53. Wang Q, Luo Y, Ray Chaudhuri K, Reynolds R, Tan EK, Pettersson S: The role of gut dysbiosis in Parkinson's disease: mechanistic insights and therapeutic options . Brain 2021, 144 (9):2571-2593. Que R, Zheng J, Chang Z, Zhang W, Li H, Xie Z, Huang Z, Wang HT, Xu J, Jin D et al : Dl-3-n-Butylphthalide Rescues Dopaminergic Neurons in Parkinson's Disease Models by Inhibiting the NLRP3 Inflammasome and Ameliorating Mitochondrial Impairment . Front Immunol 2021, 12 :794770. Marshall LL, Killinger BA, Ensink E, Li P, Li KX, Cui W, Lubben N, Weiland M, Wang X, Gordevicius J et al : Epigenomic analysis of Parkinson's disease neurons identifies Tet2 loss as neuroprotective . Nat Neurosci 2020, 23 (10):1203-1214. Huang Y, Liu B, Sinha SC, Amin S, Gan L: Mechanism and therapeutic potential of targeting cGAS-STING signaling in neurological disorders . Mol Neurodegener 2023, 18 (1):79. Alam Q, Alam MZ, Mushtaq G, Damanhouri GA, Rasool M, Kamal MA, Haque A: Inflammatory Process in Alzheimer's and Parkinson's Diseases: Central Role of Cytokines . Curr Pharm Des 2016, 22 (5):541-548. Schutt CR, Gendelman HE, Mosley RL: Tolerogenic bone marrow-derived dendritic cells induce neuroprotective regulatory T cells in a model of Parkinson's disease . Mol Neurodegener 2018, 13 (1):26. Beers DR, Zhao W, Appel SH: The Role of Regulatory T Lymphocytes in Amyotrophic Lateral Sclerosis . JAMA Neurol 2018, 75 (6):656-658. Thome AD, Atassi F, Wang J, Faridar A, Zhao W, Thonhoff JR, Beers DR, Lai EC, Appel SH: Ex vivo expansion of dysfunctional regulatory T lymphocytes restores suppressive function in Parkinson's disease . NPJ Parkinsons Dis 2021, 7 (1):41. Haider A, Elghazawy NH, Dawoud A, Gebhard C, Wichmann T, Sippl W, Hoener M, Arenas E, Liang SH: Translational molecular imaging and drug development in Parkinson's disease . Mol Neurodegener 2023, 18 (1):11. Sheean RK, McKay FC, Cretney E, Bye CR, Perera ND, Tomas D, Weston RA, Scheller KJ, Djouma E, Menon P et al : Association of Regulatory T-Cell Expansion With Progression of Amyotrophic Lateral Sclerosis: A Study of Humans and a Transgenic Mouse Model . JAMA Neurol 2018, 75 (6):681-689. Gonzц║lez H, Pacheco R: T-cell-mediated regulation of neuroinflammation involved in neurodegenerative diseases . J Neuroinflammation 2014, 11 :201. Santamarц╜a-Cadavid M, Rodrц╜guez-Castro E, Rodrц╜guez-Yц║ц╠ez M, Arias-Rivas S, LцЁpez-Dequidt I, Pц╘rez-Mato M, Rodrц╜guez-Pц╘rez M, LцЁpez-Loureiro I, Hervella P, Campos F et al : Regulatory T cells participate in the recovery of ischemic stroke patients . BMC Neurol 2020, 20 (1):68. Wang M, Thomson AW, Yu F, Hazra R, Junagade A, Hu X: Regulatory T lymphocytes as a therapy for ischemic stroke . Semin Immunopathol 2023, 45 (3):329-346. Chung ES, Kim H, Lee G, Park S, Kim H, Bae H: Neuro-protective effects of bee venom by suppression of neuroinflammatory responses in a mouse model of Parkinson's disease: role of regulatory T cells . Brain Behav Immun 2012, 26 (8):1322-1330. Park TY, Jeon J, Lee N, Kim J, Song B, Kim JH, Lee SK, Liu D, Cha Y, Kim M et al : Co-transplantation of autologous T(reg) cells in a cell therapy for Parkinson's disease . Nature 2023, 619 (7970):606-615. Priori A, Lefaucheur JP: Chronic epidural motor cortical stimulation for movement disorders . Lancet Neurol 2007, 6 (3):279-286. Chou YH, Hickey PT, Sundman M, Song AW, Chen NK: Effects of repetitive transcranial magnetic stimulation on motor symptoms in Parkinson disease: a systematic review and meta-analysis . JAMA Neurol 2015, 72 (4):432-440. Chung CL, Mak MK, Hallett M: Transcranial Magnetic Stimulation Promotes Gait Training in Parkinson Disease . Ann Neurol 2020, 88 (5):933-945. Lefaucheur JP, Aleman A, Baeken C, Benninger DH, Brunelin J, Di Lazzaro V, Filipović SR, Grefkes C, Hasan A, Hummel FC et al : Evidence-based guidelines on the therapeutic use of repetitive transcranial magnetic stimulation (rTMS): An update (2014-2018) . Clin Neurophysiol 2020, 131 (2):474-528. Yang C, Guo Z, Peng H, Xing G, Chen H, McClure MA, He B, He L, Du F, Xiong L et al : Repetitive transcranial magnetic stimulation therapy for motor recovery in Parkinson's disease: A Meta-analysis . Brain Behav 2018, 8 (11):e01132. Ba M, Ma G, Ren C, Sun X, Kong M: Repetitive transcranial magnetic stimulation for treatment of lactacystin-induced Parkinsonian rat model . Oncotarget 2017, 8 (31):50921-50929. Yang X, Song L, Liu Z: The effect of repetitive transcranial magnetic stimulation on a model rat of Parkinson's disease . Neuroreport 2010, 21 (4):268-272. Aftanas LI, Gevorgyan MM, Zhanaeva SY, Dzemidovich SS, Kulikova KI, Al'perina EL, Danilenko KV, Idova GV: Therapeutic Effects of Repetitive Transcranial Magnetic Stimulation (rTMS) on Neuroinflammation and Neuroplasticity in Patients with Parkinson's Disease: a Placebo-Controlled Study . Bull Exp Biol Med 2018, 165 (2):195-199. Kustrimovic N, Comi C, Magistrelli L, Rasini E, Legnaro M, Bombelli R, Aleksic I, Blandini F, Minafra B, Riboldazzi G et al : Parkinson's disease patients have a complex phenotypic and functional Th1 bias: cross-sectional studies of CD4+ Th1/Th2/T17 and Treg in drug-naïve and drug-treated patients . J Neuroinflammation 2018, 15 (1):205. Ng J, Barral S, De La Fuente Barrigon C, Lignani G, Erdem FA, Wallings R, Privolizzi R, Rossignoli G, Alrashidi H, Heasman S et al : Gene therapy restores dopamine transporter expression and ameliorates pathology in iPSC and mouse models of infantile parkinsonism . Sci Transl Med 2021, 13 (594). Postuma RB, Berg D, Stern M, Poewe W, Olanow CW, Oertel W, Obeso J, Marek K, Litvan I, Lang AE et al : MDS clinical diagnostic criteria for Parkinson's disease . Mov Disord 2015, 30 (12):1591-1601. Groppa S, Oliviero A, Eisen A, Quartarone A, Cohen LG, Mall V, Kaelin-Lang A, Mima T, Rossi S, Thickbroom GW et al : A practical guide to diagnostic transcranial magnetic stimulation: report of an IFCN committee . Clin Neurophysiol 2012, 123 (5):858-882. Yu N, Li X, Song W, Li D, Yu D, Zeng X, Li M, Leng X, Li X: CD4(+)CD25 (+)CD127 (low/-) T cells: a more specific Treg population in human peripheral blood . Inflammation 2012, 35 (6):1773-1780. Jennum P, Klitgaard H: Repetitive transcranial magnetic stimulations of the rat. Effect of acute and chronic stimulations on pentylenetetrazole-induced clonic seizures . Epilepsy Res 1996, 23 (2):115-122. Sun MF, Zhu YL, Zhou ZL, Jia XB, Xu YD, Yang Q, Cui C, Shen YQ: Neuroprotective effects of fecal microbiota transplantation on MPTP-induced Parkinson's disease mice: Gut microbiota, glial reaction and TLR4/TNF-α signaling pathway . Brain Behav Immun 2018, 70 :48-60. Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L et al : SWISS-MODEL: homology modelling of protein structures and complexes . Nucleic Acids Res 2018, 46 (W1):W296-W303. Guex N, Peitsch MC, Schwede T: Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: a historical perspective . Electrophoresis 2009, 30 Suppl 1 :S162-173. Bertoni M, Kiefer F, Biasini M, Bordoli L, Schwede T: Modeling protein quaternary structure of homo- and hetero-oligomers beyond binary interactions by homology . Sci Rep 2017, 7 (1):10480. Bienert S, Waterhouse A, de Beer TA, Tauriello G, Studer G, Bordoli L, Schwede T: The SWISS-MODEL Repository-new features and functionality . Nucleic Acids Res 2017, 45 (D1):D313-D319. Studer G, Rempfer C, Waterhouse AM, Gumienny R, Haas J, Schwede T: QMEANDisCo-distance constraints applied on model quality estimation . Bioinformatics 2020, 36 (6):1765-1771. Pierce BG, Wiehe K, Hwang H, Kim BH, Vreven T, Weng Z: ZDOCK server: interactive docking prediction of protein-protein complexes and symmetric multimers . Bioinformatics 2014, 30 (12):1771-1773. Pierce BG, Hourai Y, Weng Z: Accelerating protein docking in ZDOCK using an advanced 3D convolution library . PLoS One 2011, 6 (9):e24657. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT et al : Gene ontology: tool for the unification of biology. The Gene Ontology Consortium . Nat Genet 2000, 25 (1):25-29. Szklarczyk D, Gable AL, Lyon D, Junge A, Wyder S, Huerta-Cepas J, Simonovic M, Doncheva NT, Morris JH, Bork P et al : STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets . Nucleic Acids Res 2019, 47 (D1):D607-D613. Williams GP, Schonhoff AM, Jurkuvenaite A, Gallups NJ, Standaert DG, Harms AS: CD4 T cells mediate brain inflammation and neurodegeneration in a mouse model of Parkinson's disease . Brain 2021, 144 (7):2047-2059. Ji Y, Yang C, Pang X, Yan Y, Wu Y, Geng Z, Hu W, Hu P, Wu X, Wang K: Repetitive transcranial magnetic stimulation in Alzheimer's disease: effects on neural and synaptic rehabilitation . Neural Regen Res 2025, 20 (2):326-342. Huang P, Zhu Z, Li W, Zhang R, Chi Y, Gong W: rTMS improves dysphagia by inhibiting NLRP3 inflammasome activation and caspase-1 dependent pyroptosis in PD mice . NPJ Parkinsons Dis 2024, 10 (1):156. Chen XY, Feng SN, Bao Y, Zhou YX, Ba F: Identification of Clec7a as the therapeutic target of rTMS in alleviating Parkinson's disease: targeting neuroinflammation . Biochim Biophys Acta Mol Basis Dis 2023, 1869 (8):166814. Sun L, Wang F, Han J, Bai L, Du J: Repetitive Transcranial Magnetic Stimulation Reduces Neuronal Loss and Neuroinflammation in Parkinson?s Disease Through the miR-195a-5p/CREB Axis . Turk Neurosurg 2023, 33 (2):229-237. Brys M, Fox MD, Agarwal S, Biagioni M, Dacpano G, Kumar P, Pirraglia E, Chen R, Wu A, Fernandez H et al : Multifocal repetitive TMS for motor and mood symptoms of Parkinson disease: A randomized trial . Neurology 2016, 87 (18):1907-1915. Reynolds AD, Banerjee R, Liu J, Gendelman HE, Mosley RL: Neuroprotective activities of CD4+CD25+ regulatory T cells in an animal model of Parkinson's disease . J Leukoc Biol 2007, 82 (5):1083-1094. Theodore S, Maragos W: 6-Hydroxydopamine as a tool to understand adaptive immune system-induced dopamine neurodegeneration in Parkinson's disease . Immunopharmacol Immunotoxicol 2015, 37 (4):393-399. Tian L, Sun SS, Cui LB, Wang SQ, Peng ZW, Tan QR, Hou WG, Cai M: Repetitive Transcranial Magnetic Stimulation Elicits Antidepressant- and Anxiolytic-like Effect via Nuclear Factor-E2-related Factor 2-mediated Anti-inflammation Mechanism in Rats . Neuroscience 2020, 429 :119-133. Lenz M, Eichler A, Kruse P, Strehl A, Rodriguez-Rozada S, Goren I, Yogev N, Frank S, Waisman A, Deller T et al : Interleukin 10 Restores Lipopolysaccharide-Induced Alterations in Synaptic Plasticity Probed by Repetitive Magnetic Stimulation . Front Immunol 2020, 11 :614509. Zhao CG, Qin J, Sun W, Ju F, Zhao YL, Wang R, Sun XL, Mou X, Yuan H: rTMS Regulates the Balance Between Proliferation and Apoptosis of Spinal Cord Derived Neural Stem/Progenitor Cells . Front Cell Neurosci 2019, 13 :584. Li H, Shang J, Zhang C, Lu R, Chen J, Zhou X: Repetitive Transcranial Magnetic Stimulation Alleviates Neurological Deficits After Cerebral Ischemia Through Interaction Between RACK1 and BDNF exon IV by the Phosphorylation-Dependent Factor MeCP2 . Neurotherapeutics 2020, 17 (2):651-663. Reynolds AD, Stone DK, Hutter JA, Benner EJ, Mosley RL, Gendelman HE: Regulatory T cells attenuate Th17 cell-mediated nigrostriatal dopaminergic neurodegeneration in a model of Parkinson's disease . J Immunol 2010, 184 (5):2261-2271. Shi L, Sun Z, Su W, Xu F, Xie D, Zhang Q, Dai X, Iyer K, Hitchens TK, Foley LM et al : Treg cell-derived osteopontin promotes microglia-mediated white matter repair after ischemic stroke . Immunity 2021, 54 (7):1527-1542 e1528. Haavik J, Toska K: Tyrosine hydroxylase and Parkinson's disease . Mol Neurobiol 1998, 16 (3):285-309. Zhu M, Huang Y, Wang Z, Jin Z, Cao J, Zhong Q, Xiong Z: Fecal Microbiota Transplantation Attenuates Frailty via Gut-Muscle Axis in Old Mice . Aging Dis 2024. Yildirim S, Ozkan A, Aytac G, Agar A, Tanriover G: Role of melatonin in TLR4-mediated inflammatory pathway in the MTPT-induced mouse model . Neurotoxicology 2022, 88 :168-177. Faridar A, Vasquez M, Thome AD, Yin Z, Xuan H, Wang JH, Wen S, Li X, Thonhoff JR, Zhao W et al : Ex vivo expanded human regulatory T cells modify neuroinflammation in a preclinical model of Alzheimer's disease . Acta Neuropathol Commun 2022, 10 (1):144. Glavan G, Schliebs R, Zivin M: Synaptotagmins in neurodegeneration . Anat Rec (Hoboken) 2009, 292 (12):1849-1862. Xie Y, Zhi K, Meng X: Effects and Mechanisms of Synaptotagmin-7 in the Hippocampus on Cognitive Impairment in Aging Mice . Mol Neurobiol 2021, 58 (11):5756-5771. Wong YH, Lee CM, Xie W, Cui B, Poo MM: Activity-dependent BDNF release via endocytic pathways is regulated by synaptotagmin-6 and complexin . Proc Natl Acad Sci U S A 2015, 112 (32):E4475-4484. Mackie PM, Gopinath A, Montas DM, Nielsen A, Smith A, Nolan RA, Runner K, Matt SM, McNamee J, Riklan JE et al : Functional characterization of the biogenic amine transporters on human macrophages . JCI Insight 2022, 7 (4). Campolo M, Paterniti I, Siracusa R, Filippone A, Esposito E, Cuzzocrea S: TLR4 absence reduces neuroinflammation and inflammasome activation in Parkinson's diseases in vivo model . Brain Behav Immun 2019, 76 :236-247. Hughes CD, Choi ML, Ryten M, Hopkins L, Drews A, Botía JA, Iljina M, Rodrigues M, Gagliano SA, Gandhi S et al : Picomolar concentrations of oligomeric alpha-synuclein sensitizes TLR4 to play an initiating role in Parkinson's disease pathogenesis . Acta Neuropathol 2019, 137 (1):103-120. Conte C, Roscini L, Sardella R, Mariucci G, Scorzoni S, Beccari T, Corte L: Toll Like Receptor 4 Affects the Cerebral Biochemical Changes Induced by MPTP Treatment . Neurochem Res 2017, 42 (2):493-500. Wang T, Yuan F, Chen Z, Zhu S, Chang Z, Yang W, Deng B, Que R, Cao P, Chao Y et al : Vascular, inflammatory and metabolic risk factors in relation to dementia in Parkinson's disease patients with type 2 diabetes mellitus . Aging (Albany NY) 2020, 12 (15):15682-15704. Zhu S, Li H, Xu X, Luo Y, Deng B, Guo X, Guo Y, Yang W, Wei X, Wang Q: The Pathogenesis and Treatment of Cardiovascular Autonomic Dysfunction in Parkinson's Disease: What We Know and Where to Go . Aging Dis 2021, 12 (7):1675-1692. Xie Z, Zhang M, Luo Y, Jin D, Guo X, Yang W, Zheng J, Zhang H, Zhang L, Deng C et al : Healthy Human Fecal Microbiota Transplantation into Mice Attenuates MPTP-Induced Neurotoxicity via AMPK/SOD2 Pathway . Aging Dis 2023, 14 (6):2193-2214. Olson KE, Namminga KL, Lu Y, Schwab AD, Thurston MJ, Abdelmoaty MM, Kumar V, Wojtkiewicz M, Obaro H, Santamaria P et al : Safety, tolerability, and immune-biomarker profiling for year-long sargramostim treatment of Parkinson's disease . EBioMedicine 2021, 67 :103380. Supplementary Files SupplementaryTable1.doc SupplementaryTable2.doc Supplementaryfigurelegendsfinal.docx Supplementaryfigure1.pdf Supplementaryfigure2.pdf Supplementaryfigure3.pdf Supplementaryfigure4.pdf GraphicalAbstract.pdf Graphical abstract. rTMS is a safe and non-invasive method for Parkinson's disease. In this study, we showed the proportion of CD4+CD25+CD127- regulatory T-cells (Tregs) in the peripheral blood was significantly increased after rTMS treatment. Similar effects of rTMS treatment were verified in MPTP-induced PD mice. Proteomic analysis and RNA interference analyses identified TLR4, TH, Slc6a3 and especially Syt6 as hub node proteins that can be modulated by rTMS therapy in PD. Cite Share Download PDF Status: Published Journal Publication published 25 Oct, 2024 Read the published version in Molecular Neurodegeneration → Version 1 posted Editorial decision: Accept 12 Oct, 2024 Reviewers agreed at journal 11 Oct, 2024 Reviewers invited by journal 27 Sep, 2024 Editor assigned by journal 26 Sep, 2024 First submitted to journal 21 Sep, 2024 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4959031","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":359940184,"identity":"148309b7-87ac-4d87-a0b1-764b8b4afebd","order_by":0,"name":"Fen Xie","email":"","orcid":"","institution":"Zhujiang Hospital of Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fen","middleName":"","lastName":"Xie","suffix":""},{"id":359940185,"identity":"f6cbd584-20a2-4ff8-b1af-20457b261feb","order_by":1,"name":"BIbiao Shen","email":"","orcid":"","institution":"Zhujiang Hospital of Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"BIbiao","middleName":"","lastName":"Shen","suffix":""},{"id":359940186,"identity":"b6658ed4-e7b6-4888-ba22-d33cfad83d7e","order_by":2,"name":"Yuqi Luo","email":"","orcid":"","institution":"Zhujiang Hospital of Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuqi","middleName":"","lastName":"Luo","suffix":""},{"id":359940187,"identity":"606e9471-165e-49b7-ace3-59b9c551f722","order_by":3,"name":"Hang Zhou","email":"","orcid":"","institution":"Zhujiang Hospital of Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hang","middleName":"","lastName":"Zhou","suffix":""},{"id":359940188,"identity":"47e5bf36-de5f-4d25-afa4-c9e165a3e23c","order_by":4,"name":"Zhenchao Xie","email":"","orcid":"","institution":"Zhujiang Hospital of Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhenchao","middleName":"","lastName":"Xie","suffix":""},{"id":359940189,"identity":"be6fd1cd-8fa5-4645-8b53-acee893c1022","order_by":5,"name":"Shuzhen Zhu","email":"","orcid":"","institution":"Zhujiang Hospital of Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuzhen","middleName":"","lastName":"Zhu","suffix":""},{"id":359940190,"identity":"faad4670-6438-448f-8044-c6a7639ffb24","order_by":6,"name":"Xiaobo Wei","email":"","orcid":"","institution":"Zhujiang Hospital of Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaobo","middleName":"","lastName":"Wei","suffix":""},{"id":359940191,"identity":"0eb86b0b-a385-49e4-8e19-e797493cf906","order_by":7,"name":"Zihan Chang","email":"","orcid":"","institution":"Zhujiang Hospital of Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zihan","middleName":"","lastName":"Chang","suffix":""},{"id":359940192,"identity":"bc140d83-f9c6-4650-a016-99d347e1352d","order_by":8,"name":"Zhaohua Zhu","email":"","orcid":"","institution":"Zhujiang Hospital of Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhaohua","middleName":"","lastName":"Zhu","suffix":""},{"id":359940193,"identity":"9827346d-6d86-4e7b-a116-9ffaa76b302c","order_by":9,"name":"Changhai Ding","email":"","orcid":"","institution":"Zhujiang Hospital of Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Changhai","middleName":"","lastName":"Ding","suffix":""},{"id":359940194,"identity":"edf885d9-ab6f-4715-ad29-5ab4fd52cefc","order_by":10,"name":"Kunlin Jin","email":"","orcid":"","institution":"University of North Texas Health Science Center","correspondingAuthor":false,"prefix":"","firstName":"Kunlin","middleName":"","lastName":"Jin","suffix":""},{"id":359940195,"identity":"88684a44-bc5f-4b6d-bbe7-f10edaa9d083","order_by":11,"name":"Chengwu Yang","email":"","orcid":"","institution":"UMass Chan Medical School TH Chan School of Medicine: University of Massachusetts Chan Medical School TH Chan School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chengwu","middleName":"","lastName":"Yang","suffix":""},{"id":359940196,"identity":"0e73485f-09aa-4027-85cf-f88e8ca36535","order_by":12,"name":"Lucia Batzu","email":"","orcid":"","institution":"King’s Foundations: King's College London International Foundation","correspondingAuthor":false,"prefix":"","firstName":"Lucia","middleName":"","lastName":"Batzu","suffix":""},{"id":359940197,"identity":"ada8913b-b4f2-41c6-82bb-598cce2663d7","order_by":13,"name":"K Ray Chaudhuri","email":"","orcid":"","institution":"Parkinson's UK","correspondingAuthor":false,"prefix":"","firstName":"K","middleName":"Ray","lastName":"Chaudhuri","suffix":""},{"id":359940198,"identity":"b4a17d5e-cf12-436f-b5f7-b173f5115a56","order_by":14,"name":"Ling-Ling Chan","email":"","orcid":"","institution":"Singapore General Hospital Department of Neurology","correspondingAuthor":false,"prefix":"","firstName":"Ling-Ling","middleName":"","lastName":"Chan","suffix":""},{"id":359940199,"identity":"c67b0d72-684d-4e12-97ea-1002c5d86557","order_by":15,"name":"Eng-King Tan","email":"","orcid":"","institution":"Singapore General Hospital Department of Neurology","correspondingAuthor":false,"prefix":"","firstName":"Eng-King","middleName":"","lastName":"Tan","suffix":""},{"id":359940200,"identity":"8d25ed9b-36ae-454a-aaea-4acf90d458bd","order_by":16,"name":"Qing Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYHACAyCWYOCXAHMkZIjXIjmDgbEByOAhVguQvAHWwkBYi8Hx5I2PC3dY2Bvfbj7+6EaNBQ8D++GjG/BqOfOs2HjmGYnEbXeOJTbnHAM6jCct7QY+LWY3csykedskEoAMw+YcNqAWCR4zQlrMfwO12BvPAGn5R5wWM2agFsYNEkAtuW1EaLEH+gXksMQZN9ISZ+f2SfCwEfKLZHvyxs+8bXX2/DOSD3zO+VYnx89++BheLQwMCWh8NvzKsWkZBaNgFIyCUYAOAC7aRQy2WR3AAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-3882-6540","institution":"Zhujiang Hospital of Southern Medical University","correspondingAuthor":true,"prefix":"","firstName":"Qing","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-08-22 15:08:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4959031/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4959031/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13024-024-00770-4","type":"published","date":"2024-10-25T15:58:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66941428,"identity":"3df94916-d998-4115-8aa6-0a8d1c26cac9","added_by":"auto","created_at":"2024-10-18 08:54:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":749582,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003erTMS treatment improved motor functions and increased the proportion of peripheral Tregs in PD patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Study flow chart. Sixty patients with PD were screened for eligibility in this clinical study between Sep 2021 and Jan 2022. A total of 6 PD patients were ultimately excluded. Finally, the 54 PD patients were randomized (1: 1) to divided into sham and rTMS group. Demographic data, basic examinations including electroencephalography, medication dosage before and after rTMS or sham treatment were carried out in both groups of patients. Tregs, aTreg, nTreg, proportion and the levels of IFN-γ, TNF-α, IL-17α, IL4, IL10 and TGF-β1 in the peripheral blood were measured, H\u0026amp;Y and MDS-UPDRS III scores were performed at the same time point before and after rTMS or sham treatment.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Tregs, and aTreg/nTreg proportion in the peripheral blood were assessed by flow cytometry prior to treatment and 1 day after the treatment. Hoehn and Yahr and MDS-UPDRS III were performed prior to treatment and on Days 13, 19, and 40 after the treatment.\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; No significant difference between the Mean H \u0026amp; Y ± SD on Days -1, 13, 19 and 40 in the sham and rTMS groups. (sham vs. rTMS groups on days -1: n = 27, P = 0.761, t = -0.306, d. f. = 52; sham vs. rTMS groups on days 13: n = 27, P = 0.761, t = -0.306, d. f. = 52; sham vs. rTMS groups on days 19: n = 27, P = 0.761, t = -0.306, d. f. = 52; sham vs. rTMS groups on days 40: n = 27, P = 0.761, t = -0.306, d. f. = 52).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; There was a significant difference in the difference between the MDS-UPDRS III scores of the rTMS group and the sham group on day 13, 19, and 40 and their respective MDS-UPDRSIII scores on day -1.(rTMS vs. sham groups between days 13 and -1: n = 27, P \u0026lt; 0.0001, t = 15.64, d. f. = 208; rTMS vs. sham groups on 19 and -1: n = 27, P \u0026lt; 0.0001, t = 15.38, d. f. = 208; rTMS vs. sham groups on on 40 and -1: n = 27, P \u0026lt; 0.0001, t = 15.77, d. f. = 208).\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; The baseline of the proportion of CD4+CD25+CD127- Treg cells in the peripheral blood displays no significant difference between sham and rTMS group. (sham vs. rTMS groups: n = 27, P = 0.796, t = 0.26, d. f. = 52).\u003c/p\u003e\n\u003cp\u003eF.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; The peripheral blood CD4+CD25+CD127- Treg cells before and after sham rTMS treatment in the sham treatment group shows no significant difference. (before vs. after: n = 27, P = 0.185, t = 1.363, d. f. = 26).\u003c/p\u003e\n\u003cp\u003eG.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; The peripheral blood CD4+CD25+CD127-Treg cells before and after rTMS treatment in the rTMS treatment group shows significant difference. (before vs. after: n = 27, P \u0026lt; 0.000, t = -14.88, d. f. = 26).\u003c/p\u003e\n\u003cp\u003eH.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Mean change in the proportion of Tregs/CD4+T cells in the peripheral blood± SD between days -1 and 13 in the sham and rTMS groups. (n = 27, P \u0026lt; 0.0001, t = 14.98, d. f. = 52).\u003c/p\u003e\n\u003cp\u003eI.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; No correlation\u0026nbsp;between the change in the proportion of Tregs/CD4+ T cells and the change in MDS-UPDRSIII scores (between days -1 and 13) in sham group (n = 27, r\u003csup\u003e2 \u003c/sup\u003e= 0. 0007, p = 0.895).\u003c/p\u003e\n\u003cp\u003eJ.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Negative\u0026nbsp;correlation\u0026nbsp;between the change in the proportion of Tregs/CD4+ T cells. and the change in MDS-UPDRSIII scores (between days -1 and 13) in rTMS group. (n = 27, r\u003csup\u003e2 \u003c/sup\u003e= 0.8261, p\u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003erTMS = repetitive transcranial magnetic stimulation; H \u0026amp; Y = Hoehn-Yahr; MDS UPDRS III = Movement Disorder Society Unified Parkinson's Disease Rating Scale part III; SD = Standard Deviation; Data are presented as mean ± SD; two-tailed unpaired t test (C, D, E, H); paired sample t test (F, G); linear regression analysis (I, J). ****P \u0026lt; 0.0001. Each data point represents an individual subject. Comparisons with no asterisk had a P \u0026gt; 0.05 and were considered not significant.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4959031/v1/39dde2865b91e9fd302fd6f0.png"},{"id":66941384,"identity":"004afaea-c02e-446d-8b05-5ea5aae3390d","added_by":"auto","created_at":"2024-10-18 08:53:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1930900,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003erTMS treatment ameliorated behavioral impairments, increased the proportion of Tregs, suppressed the neuroinflammatory responses in MPTP-induced PD mice, and regulated the potential targets of Tregs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Animal experimental design and rTMS treatment scheme to explore the role and mechanism of rTMS in the MPTP-induced PD mouse model.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Quantification of total the times for each group mice that spent in pole climbing test. (F\u003csub\u003e8,99 \u003c/sub\u003e= 8257, NC vs. NC+sham: P \u0026gt; 0.9999; NC vs. MPTP: P \u0026lt; 0.0001; NC+sham vs. NC+rTMS: P \u0026gt; 0.9999; MPTP vs. MPTP+sham: P \u0026gt; 0.9999; MPTP vs. MPTP+rTMS: P\u0026lt; 0.0001; MPTP+sham vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP vs. MPTP+block: P \u0026lt; 0.0001; MPTP+block+sham vs. MPTP+block+rTMS: P = 0.9494, n = 12 mice per group).\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Flow cytometry–based analysis of CD3+CD4+CD25+Foxp3+Treg cells represen-\u003c/p\u003e\n\u003cp\u003etative fluorescence-activated cell sorting (FACS) plots of each group of mice.\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; The proportions of CD3+CD4+CD25+Foxp3+ Treg cells/CD4+ T cells in the spleens of mice in each group. (F\u003csub\u003e8, 45 \u003c/sub\u003e= 608.8, NC vs. NC+sham: P \u0026gt; 0.9999; NC vs. MPTP: P \u0026lt; 0.0001; NC+sham vs. NC+rTMS: P = 0.9998; MPTP vs. MPTP+sham: P \u0026gt; 0.9999; MPTP vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP+sham vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP vs. MPTP+block: P \u0026lt; 0.0001; MPTP+block+sham vs. MPTP+block+rTMS: P \u0026gt; 0.9999, n = 6 mice per group).\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Representative images of microglia in the SN. microglia were visualized by iba-1 staining. Scale bar, 50 μm.\u003c/p\u003e\n\u003cp\u003eF.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Mean microglial immunostaining of Iba-1-positive cells in the SN in each group of mice ± SD. (F\u003csub\u003e8, 81\u003c/sub\u003e = 1803, NC vs. NC+sham: P = 0.9985; NC vs. MPTP: P \u0026lt; 0.0001; NC+sham vs. NC+rTMS: P \u0026gt; 0.9999; MPTP vs. MPTP+sham: P = 0.9985; MPTP vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP+sham vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP vs. MPTP+block: P \u0026lt; 0.0001; MPTP+block+sham vs. MPTP+block+rTMS: P = 0.9985, n = 10 mice per group).\u003c/p\u003e\n\u003cp\u003eG.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; ELISA was used to analyze the protein expression of IL-10, TGF-β1, IL-6, IFN-γ, TNF-α and IL-1β in the ventral midbrain in each group of mice. (\u003cstrong\u003eIL-1β:\u003c/strong\u003e F\u003csub\u003e8, 45\u003c/sub\u003e = 30632, NC \u0026nbsp;vs. NC+sham: P \u0026gt; 0.9999; NC vs. MPTP: P \u0026lt; 0.0001; NC+sham vs. NC+rTMS: P \u0026gt; 0.9999; MPTP vs. MPTP+sham: P \u0026gt; 0.9999; MPTP vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP+sham vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP vs. MPTP+block: P \u0026lt; 0.0001; MPTP+block+sham vs. MPTP+block+rTMS: P \u0026gt; 0.9999;\u003cstrong\u003e IL-10:\u003c/strong\u003e F\u003csub\u003e8, 45\u003c/sub\u003e = 36052, NC vs. NC+sham: P \u0026gt; 0.9999; NC vs. MPTP: P \u0026lt; 0.0001; NC+sham vs. NC+rTMS: P = 0.9894; MPTP vs. MPTP+sham: P = 0.9493; MPTP vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP+sham vs. MPTP+rTMS: P \u0026lt;= 0.0001; MPTP vs. MPTP+block P \u0026lt; 0.0001; MPTP+block+sham vs. MPTP+block+rTMS: P = 0.9999; \u003cstrong\u003eTNF-α: \u003c/strong\u003eF\u003csub\u003e8, 45\u003c/sub\u003e = 1767, NC vs. NC+sham: P \u0026gt; 0.9999; NC vs. MPTP: P \u0026lt; 0.0001; NC+sham vs. NC+rTMS: P \u0026gt; 0.9999; MPTP vs. MPTP+sham: P = 0.5829; MPTP vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP+sham vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP vs. MPTP+block: P \u0026lt; 0.0001; MPTP+block+sham vs. MPTP+ block +rTMS: P \u0026gt; 0.9999;\u003cstrong\u003e IL-6:\u003c/strong\u003e F\u003csub\u003e8, 45\u003c/sub\u003e = 7443, NC vs. NC+sham: P = 0.9987; NC vs. MPTP: P \u0026lt; 0.0001; NC+sham vs. NC+rTMS: P \u0026gt; 0.9999; MPTP vs. MPTP+sham: P = 0.7244; MPTP vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP+sham vs. MPTP+rTMS: P\u0026lt; 0.0001; MPTP vs. MPTP+block: P \u0026lt; 0.0001; MPTP+block+sham vs. MPTP+block+rTMS: P = 0.6587; \u003cstrong\u003eIFN-γ:\u003c/strong\u003e F\u003csub\u003e8, 45\u003c/sub\u003e = 25766, NC vs. NC+sham: P \u0026gt; 0.9999; NC vs. MPTP: P \u0026lt; 0.0001; NC+sham vs. NC+rTMS: P \u0026gt; 0.9999; MPTP vs. MPTP+sham: P = 0.9798; MPTP vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP+sham vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP vs. MPTP+block: P \u0026lt; 0.0001; MPTP+block+sham vs. MPTP+block+rTMS: P \u0026gt; 0.9999; \u003cstrong\u003eTGF-β1:\u003c/strong\u003e F\u003csub\u003e8, 45\u003c/sub\u003e = 22257, NC vs. NC+sham: P \u0026gt; 0.9999; NC vs. MPTP: P \u0026lt; 0.0001; NC+sham vs. NC+rTMS: P \u0026gt; 0.9999; MPTP vs. MPTP+sham: P \u0026gt; 0.9999; MPTP vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP+sham vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP vs. MPTP+block: P \u0026lt; 0.0001; MPTP+block+sham vs. MPTP+block+rTMS: P = 0.9998; n = 6 mice per group).\u003c/p\u003e\n\u003cp\u003eSN = substantia nigra; LSD = least significant difference; MPTP = 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine; NC = normal control; Data are presented as mean ± SD; One-way ANOVA with Fisher’s LSD multiple comparison post hoc test (B, D, E, G). ****P \u0026lt; 0.0001. Each data point represents an individual mouse. Comparisons with no asterisk had a P \u0026gt; 0.05 and were considered not significant.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4959031/v1/3160bbeb5cf7cb576fc9e7b8.png"},{"id":66941461,"identity":"4950ab6c-7eaa-4200-9e89-1d4c7bbc114a","added_by":"auto","created_at":"2024-10-18 08:54:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1828888,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantitative proteomic analysis identified the Treg-related downstream targets Syt6, TLR4, TH and Slc6a3 that were associated with rTMS stimulation in PD.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Principal component analysis (PCA). The results showed an increased degree of aggregation among replicate samples and improved quantitative repeatability, with a large significant difference between the groups (n=3, Con=saline normal control group, MPTP=MPTP-induced PD group, MPTP + rTMS=MPTP + rTMS treatment group, MPTP + block + rTMS = MPTP+ Treg block + rTMS treatment group).\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Heatmap showing the gene expression level per sample relative to the average expression of all samples. Red represents higher expression, and green represents lower expression (n=3, Con=saline normal control group, MPTP=MPTP-induced PD group, MPTP + rTMS=MPTP + rTMS treatment group, MPTP + block + rTMS = MPTP+ Treg block + rTMS treatment group).\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; All differentially expressed genes in each group were analyzed by intersection analysis, and two common differentially expressed genes, Slc6a3 and Syt6, were screened out (n=3, Con=saline normal control group, MPTP=MPTP-induced PD group, MPTP + rTMS = MPTP + rTMS treatment group, MPTP + block + rTMS = MPTP+ Treg block + rTMS treatment group).\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Protein–protein interaction (PPI) network of differentially expressed proteins in the MPTP + rTMS and MPTP + block + rTMS groups (PPI enrichment p value\u0026lt; 1.0e-16); (n=3, MPTP + rTMS = MPTP + rTMS treatment group, MPTP + block + rTMS = MPTP+ Treg block + rTMS treatment group).\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; The SYT6 protein has a binding and interaction relationship with TLR4.\u003c/p\u003e\n\u003cp\u003eF.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Representative western blot bands of the protein expression of Syt6, TLR4, TH, and Slc6a3 in the ventral midbrain of each group of mice.\u003c/p\u003e\n\u003cp\u003eG.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Mean protein expression of Syt6, TLR4, TH, and Slc6a3 in the ventral midbrain in each group of mice ± SD. (\u003cstrong\u003eSyt6: \u003c/strong\u003eF\u003csub\u003e8, 18 \u003c/sub\u003e= 7696, NC vs. NC+sham: P \u0026gt; 0.9999; NC vs. MPTP: P \u0026lt; 0.0001; NC+sham vs. NC+rTMS: P \u0026gt; 0.9999; MPTP vs. MPTP+sham: P \u0026gt; 0.9999; MPTP vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP+sham vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP vs. MPTP+bock: P \u0026lt; 0.0001; MPTP+block+sham vs. MPTP+block+rTMS: P \u0026gt; 0.9999; \u003cstrong\u003eTLR4: \u003c/strong\u003eF\u003csub\u003e8, 18 \u003c/sub\u003e= 3511, NC vs. NC+sham: P \u0026gt; 0.9999; NC vs. MPTP: P \u0026lt; 0.0001; NC+sham vs. NC+rTMS: P \u0026gt; 0.9999; MPTP vs. MPTP+sham: P \u0026gt; 0.9999; MPTP vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP+sham vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP vs. MPTP+block: P \u0026lt; 0.0001; MPTP+block+sham vs. MPTP+block+rTMS: P = 0.9628; \u003cstrong\u003eSlc6a3:\u003c/strong\u003e F\u003csub\u003e8, 18 \u003c/sub\u003e= 626.5, NC vs. NC+sham: P \u0026gt; 0.9999; NC vs. MPTP: P \u0026lt; 0.0001; NC+sham vs. NC+rTMS: P \u0026gt; 0.9999; MPTP vs. MPTP+sham: P \u0026gt; 0.9999; MPTP vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP+sham vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP vs. MPTP+block: P \u0026lt; 0.0001; MPTP+block+sham vs. MPTP+block+rTMS: P \u0026gt; 0.9999; \u003cstrong\u003eTH: \u003c/strong\u003eF\u003csub\u003e8, 18\u003c/sub\u003e = 334.50, NC vs. NC+sham: P = 0.9999; NC vs. MPTP: P \u0026lt; 0.0001; NC+sham vs. NC+rTMS: P = 0.9998; MPTP vs. MPTP+sham: P \u0026gt; 0.9999; MPTP vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP+sham vs. MPTP+rTMS: P \u0026lt; 0.0001; MPTP vs. MPTP+block: P \u0026lt; 0.0001; MPTP+block+sham vs. MPTP+block+rTMS: P \u0026gt; 0.9999, n = 3 mice per group).\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SD; One-way ANOVA with Fisher’s LSD multiple comparison post hoc test (G). ****P \u0026lt; 0.0001. Each data point represents an individual mouse. Comparisons with no asterisk had a P \u0026gt; 0.05 and were considered not significant.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4959031/v1/02b7349b7883156830bf704c.png"},{"id":66941406,"identity":"c6c2ff90-4de9-475b-a4b8-b9ecee8af004","added_by":"auto","created_at":"2024-10-18 08:54:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":438588,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening out effective virus interference bands in syt6 to ensure interference efficiency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Adeno-associated virus (AAV) vector was injected into midbrain via a stereotactic midbrain approach to block syt6 level. Pole climbing pole test, immunofluorescent staining and WB were used to detect the efficiency of transfection.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Quantification of total the times for each group mice that spent in pole climbing test. (F\u003csub\u003e3,8 \u003c/sub\u003e= 3691, MPTP+scramble vs. MPTP+syt6-shRNA1: P = 0.8070; MPTP+ scramble vs. MPTP+syt6-shRNA2:P = 0.0022; MPTP+scramble vs. MPTP+syt6- shRNA3: P\u0026lt; 0.0001; n = 3 mice per group).\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Immunofluorescent staining images of Syt6 expression in the SN of each group mice. Scale bar, 10μm.\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Quantitative analyses of Syt6 immunostaining in the SN. (F\u003csub\u003e3,8 \u003c/sub\u003e= 4701, MPTP+scramble vs. MPTP+syt6-shRNA1: P = 0.3061; MPTP+scramble vs. MPTP+ syt6-shRNA2: P = 0.0040; MPTP+scramble vs. MPTP+syt6-shRNA3: P \u0026lt; 0.0001; n = 3 mice per group).\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Representative western blot bands of the protein expression of Syt6 in the ventral midbrain of each group mice.\u003c/p\u003e\n\u003cp\u003eF.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Quantitative analysis of the protein expression of Syt6 in the ventral midbrain of each group mice. (F\u003csub\u003e3,8 \u003c/sub\u003e= 275.9, MPTP+scramble vs. MPTP+syt6-shRNA1: P = 0.8026; MPTP+ scramble vs. MPTP+syt6-shRNA2: P = 0.0018; MPTP+scramble vs. MPTP+syt6- shRNA3: P \u0026lt; 0.0001; n = 3 mice per group).\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SD; One-way ANOVA with Fisher’s LSD multiple comparison post hoc test (G). ****P \u0026lt; 0.0001, *P \u0026lt; 0.05. Each data point represents an individual mouse. Comparisons with no asterisk had a P \u0026gt; 0.05 and were considered not significant.\u0026nbsp;\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4959031/v1/67bdf779ffd495f056860a25.png"},{"id":66941456,"identity":"4e5d34f8-b50b-4789-b7ce-c2a992e247ec","added_by":"auto","created_at":"2024-10-18 08:54:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3128675,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVerification of the role of Treg and rTMS-related protein syt6 in MPTP mice by virus interference syt6 method\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Animal experimental design and virus interference syt6 scheme to explore the role of Treg and rTMS-related protein syt6 in the MPTP-induced PD mouse model.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Quantification of total the times for each group mice that spent in pole climbing test. (F\u003csub\u003e7,112\u003c/sub\u003e = 18690, NC+scramble+sham vs. NC+scramble+rTMS: P \u0026gt; 0.9999; NC+sramble+sham vs. MPTP+scramble+sham: P \u0026lt; 0.0001; NC+syt6-shRNA+sham vs. NC+syt6-shRNA+rTMS: P = 0.9999; MPTP+scramble+sham vs. MPTP+ scramble+rTMS: P\u0026lt; 0.0001; MPTP+scramble+sham vs. MPTP+ syt6-shRNA+sham: P \u0026lt; 0.0001; MPTP+ syt6-shRNA+sham vs. MPTP+syt6-shRNA+rTMS: P = 0.9999, n = 15 mice per group)\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Representative images of SYT6 expression in the SN. Scale bar, 10 μm.\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Quantitative analyses of SYT6 immunostaining in the SN. (F\u003csub\u003e7,72 \u003c/sub\u003e= 2866, NC+scramble+sham vs. NC+scramble+rTMS: P \u0026gt; 0.9999; NC+scramble+sham vs. NC+syt6-shRNA+sham: P\u0026lt;0.0001; NC+scramble+sham vs. MPTP+scramble+sham\u003c/p\u003e\n\u003cp\u003e: P \u0026lt; 0.0001; NC+syt6-shRNA+sham vs. NC+syt6-shRNA+rTMS: P \u0026gt; 0.9999; MPTP+scramble+sham vs. MPTP+ scramble+rTMS: P \u0026lt; 0.0001; MPTP+scramble+\u003c/p\u003e\n\u003cp\u003esham vs. MPTP+syt6-shRNA+sham: P \u0026lt; 0.0001; MPTP+syt6-shRNA+sham vs. MPTP+syt6- shRNA+rTMS: P \u0026gt; 0.9999, n = 10 mice per group).\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Representative images of TH expression in the SN. Scale bar, 50 μm.\u003c/p\u003e\n\u003cp\u003eF.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Quantitative analyses of TH immunostaining in the SN. (F\u003csub\u003e7,72 \u003c/sub\u003e= 649.7, NC+sramble+sham vs. NC+scramble+rTMS: P = 0.9426; NC+sramble+sham vs. NC+syt6-shRNA+sham: P\u0026lt;0.0001; NC+scramble+sham vs. MPTP+scramble+sham\u003c/p\u003e\n\u003cp\u003em: P \u0026lt; 0.0001; NC+syt6-shRNA+sham vs. NC+syt6-shRNA+rTMS: P = 0.9993; MPTP+ scramble+sham vs. MPTP+ scramble+rTMS: P \u0026lt; 0.0001; MPTP+scramble+\u003c/p\u003e\n\u003cp\u003esham vs. MPTP+syt6-shRNA+sham: P\u0026lt;0.0001; MPTP+syt6-shRNA+sham vs. MPTP+syt6- shRNA+rTMS: P \u0026gt; 0.9999, n = 10 mice per group).\u003c/p\u003e\n\u003cp\u003eG.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Representative images of TLR4 expression in the SN. Scale bar, 10 μm.\u003c/p\u003e\n\u003cp\u003eH.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Quantitative analyses of TLR4 immunostaining in the SN. (F\u003csub\u003e7,72\u003c/sub\u003e = 1923, NC+sramble+sham vs. NC+scramble+rTMS: P \u0026gt; 0.9999; NC+scramble+sham vs. NC+syt6-shRNA+sham: P \u0026lt; 0.0001; NC+scramble+sham vs. MPTP+scramble+sha-\u003c/p\u003e\n\u003cp\u003em: P \u0026lt; 0.0001; NC+syt6-shRNA+sham vs. NC+syt6-shRNA+rTMS: P \u0026gt; 0.9999; MPTP+scramble+sham vs. MPTP+ sramble+rTMS: P \u0026lt; 0.0001; MPTP+scramble+\u003c/p\u003e\n\u003cp\u003esham vs. MPTP+syt6-shRNA+sham: P \u0026lt; 0.0001; MPTP+syt6-shRNA+sham vs. MPTP+syt6- shRNA +rTMS: P\u0026gt;0.9999, n = 10 mice per group).\u003c/p\u003e\n\u003cp\u003eI.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; ELISA was used to analyze the protein expression of IL-1β, TNF-α, IL-6, IL-10, TGF-β1 and IFN-γ in the ventral midbrain in each group of mice. \u003cstrong\u003e(IL-1β: \u003c/strong\u003eF\u003csub\u003e7,40 \u003c/sub\u003e= 6230, NC+scramble+sham vs. NC+scramble+rTMS: P = 0.9958; NC+scramble+sham vs. NC+syt6-shRNA+sham: P\u0026lt;0.0001; NC+scramble+sham vs. MPTP+scramble+sham: P \u0026lt; 0.0001; NC+syt6-shRNA+sham vs. NC+syt6-shRNA+rTMS: P= 0.9279; MPTP+scramble+sham vs. MPTP+ scramble+rTMS: P \u0026lt; 0.0001; MPTP+scramble+sham vs. MPTP+syt6-shRNA+sham: P \u0026lt; 0.0001; MPTP+syt6-shRNA+sham vs. MPTP+syt6-shRNA+rTMS: P = 0.9998; \u003cstrong\u003eTNF-α: \u003c/strong\u003eF\u003csub\u003e7,40 \u003c/sub\u003e=347.7, NC+scramble+sham vs. NC+scramble+rTMS: P = 0.9651; NC+scramble+sham vs. NC+syt6-shRNA+sham: P \u0026lt; 0.0001; NC+scramble+sham vs. MPTP+scramble+sham: P \u0026lt; 0.0001; NC+syt6-shRNA+sham vs. NC+syt6-shRNA+rTMS: P\u0026gt; 0.9999; MPTP+scramble+sham vs. MPTP+scramble+rTMS: P \u0026lt; 0.0001; MPTP+scramble+sham vs. MPTP+syt6-shRNA\u003c/p\u003e\n\u003cp\u003e+sham: P \u0026lt; 0.0001; MPTP+syt6-shRNA+sham vs. MPTP+syt6-shRNA+rTMS: P = 0.8441; \u003cstrong\u003eIL-6: \u003c/strong\u003eF\u003csub\u003e7,40 \u003c/sub\u003e= 1523, NC+scramble+sham vs. NC+scramble+rTMS: P = 0.8101; NC+scramble+sham vs. NC+syt6-shRNA+sham: P \u0026lt; 0.0001; NC+scramble+sham vs. MPTP+scramble+sham: P \u0026lt; 0.0001; NC+syt6-shRNA+sham vs. NC+syt6-shRNA\u003c/p\u003e\n\u003cp\u003e+rTMS: P = 0.9999; MPTP+scramble+sham vs. MPTP+scramble+rTMS: P \u0026lt; 0.0001; MPTP+scramble+sham vs. MPTP+syt6-shRNA+sham: P\u0026lt; 0.0001; MPTP+syt6-RNA\u003c/p\u003e\n\u003cp\u003e+sham vs. MPTP+syt6-shRNA+rTMS: P = 0.4726; \u003cstrong\u003eIL-10:\u003c/strong\u003e F\u003csub\u003e7,40\u003c/sub\u003e =4702, NC+scramble\u003c/p\u003e\n\u003cp\u003e+sham vs. NC+scramble+rTMS:P\u0026gt;0.9999; NC+scramble+sham vs. NC+syt6-shRNA\u003c/p\u003e\n\u003cp\u003e+sham: P \u0026lt; 0.0001; NC+scramble+sham vs. MPTP+ scramble+sham: P \u0026lt; 0.0001; NC+syt6-shRNA+sham vs. NC+syt6-shRNA+rTMS: P = 0.9894; MPTP+scramble+\u003c/p\u003e\n\u003cp\u003esham vs. MPTP+scramble+rTMS: P \u0026lt; 0.0001; MPTP+scramble+sham vs. MPTP+\u003c/p\u003e\n\u003cp\u003esyt6-shRNA+sham: P \u0026lt; 0.0001; MPTP+syt6-shRNA+sham vs. MPTP+syt6-shRNA+\u003c/p\u003e\n\u003cp\u003erTMS: P = 0.9997;\u003cstrong\u003e TGF-β1:\u003c/strong\u003e F\u003csub\u003e7,40\u003c/sub\u003e = 482.6, NC+scramble+sham vs. NC+scramble+\u003c/p\u003e\n\u003cp\u003erTMS: P \u0026gt; 0.9999; NC+scramble+sham vs. NC+syt6-shRNA+sham: P = 0.0004; NC\u003c/p\u003e\n\u003cp\u003e+scramble+sham vs. MPTP+scramble+sham: P \u0026lt; 0.0001; NC+syt6-shRNA+sham vs. NC+syt6-shRNA+rTMS:P\u0026gt;0.9999; MPTP+scramble+sham vs. MPTP+scramble+\u003c/p\u003e\n\u003cp\u003erTMS: P \u0026lt; 0.0001; MPTP+scramble+sham vs. MPTP+syt6-shRNA+sham: P \u0026lt; 0.0001; MPTP+syt6-shRNA+sham vs. MPTP+syt6-shRNA+rTMS: P \u0026gt; 0.9999;\u003cstrong\u003e IFN-γ:\u003c/strong\u003e F\u003csub\u003e7,40\u003c/sub\u003e = 6016, NC+scramble+sham vs. NC+scramble+rTMS: P = 0.9784; NC+scramble+sham vs. NC+syt6-shRNA+sham: P \u0026lt; 0.0001; NC+scramble+sham vs. MPTP+scramble+sham: P \u0026lt; 0.0001; NC+syt6-shRNA+sham vs. NC+syt6-shRNA\u003c/p\u003e\n\u003cp\u003e+rTMS: P \u0026gt; 0.9999; MPTP+scramble+sham vs. MPTP+scramble+rTMS: P \u0026lt; 0.0001; MPTP+scramble+sham vs. MPTP+syt6-shRNA+sham: P\u0026lt; 0.0001; MPTP+syt6-shR-\u003c/p\u003e\n\u003cp\u003eNA+sham vs. MPTP+syt6-shRNA+rTMS: P = 0.9890; n = 6 mice per group).\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SD; One-way ANOVA with Fisher’s LSD multiple comparison post hoc test (B, D, F, H, I). ****P \u0026lt; 0.0001. Each data point represents an individual mouse. Comparisons with no asterisk had a P \u0026gt; 0.05 and were considered not significant.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4959031/v1/521ac815e36a348dae0dc812.png"},{"id":67682045,"identity":"51a9cf60-5cb9-4aa4-91e1-363b69ef914e","added_by":"auto","created_at":"2024-10-28 16:12:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10444267,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4959031/v1/db2b2066-fa89-4cca-981c-628dfa271160.pdf"},{"id":66941386,"identity":"ec4f6ae3-d499-4165-a10b-523a9c6a2de5","added_by":"auto","created_at":"2024-10-18 08:53:58","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":30208,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.doc","url":"https://assets-eu.researchsquare.com/files/rs-4959031/v1/3fc36abf642f0e06db8a700b.doc"},{"id":66941455,"identity":"137e8868-baf7-41ef-a3ad-8bfe8802bb18","added_by":"auto","created_at":"2024-10-18 08:54:02","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":57856,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.doc","url":"https://assets-eu.researchsquare.com/files/rs-4959031/v1/754df0f2f43d4eec50b8295f.doc"},{"id":66941454,"identity":"454ae8a0-c618-45df-9fdb-68c5bf6fee2b","added_by":"auto","created_at":"2024-10-18 08:54:02","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":22021,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigurelegendsfinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-4959031/v1/a416f12a50a5ba317d0dc95d.docx"},{"id":66941457,"identity":"21446058-92a4-429e-8e40-72364cce7429","added_by":"auto","created_at":"2024-10-18 08:54:02","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":675790,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4959031/v1/7e5eb9c9e6c6452d86dde52f.pdf"},{"id":66941387,"identity":"b21db732-b9b6-4688-ad4c-34f2894b9e33","added_by":"auto","created_at":"2024-10-18 08:53:59","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":573844,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4959031/v1/8b3de89e8dbb9ce1048f4c4a.pdf"},{"id":66941452,"identity":"c540b1a3-ebb4-42ec-912d-17bf0a7691e3","added_by":"auto","created_at":"2024-10-18 08:54:01","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1804522,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4959031/v1/e34bbb749b110b1d2e51a98d.pdf"},{"id":66941453,"identity":"32a29143-d350-4c8e-ba59-11111b400ef0","added_by":"auto","created_at":"2024-10-18 08:54:01","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":5470162,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4959031/v1/715d3387b952e6743102a585.pdf"},{"id":66941437,"identity":"0d77100c-5353-49ed-b9ef-6aec34d3a9e3","added_by":"auto","created_at":"2024-10-18 08:54:01","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":2109557,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003erTMS is a safe and non-invasive method for Parkinson's disease. In this study, we showed the proportion of CD4+CD25+CD127- regulatory T-cells (Tregs) in the peripheral blood was significantly increased after rTMS treatment. Similar effects of rTMS treatment were verified in MPTP-induced PD mice. Proteomic analysis and RNA interference analyses identified TLR4, TH, Slc6a3 and especially Syt6 as hub node proteins that can be modulated by rTMS therapy in PD.\u003c/p\u003e","description":"","filename":"GraphicalAbstract.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4959031/v1/f52651f96b5a54ad9a29c121.pdf"}],"financialInterests":"","formattedTitle":"Repetitive transcranial magnetic stimulatation alleviates motor impairment in Parkinson's disease: association with peripheral inflammatory regulatory T-cells and SYT6","fulltext":[{"header":"Background","content":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is a progressive neurodegenerative disorder in which Dopamine (DA) neurons degenerate and neuroinflammatory responses play crucial roles in its pathogenesis [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. There is increasing evidence that the abnormal activation of peripheral immune cells and circulating mediators, including microglia and T cells, actively contribute to neurodegeneration and disease progression in PD[\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Microglia are the most abundant resident immune cells in the brain, and these cells are activated in response to chronic inflammation and produce proinflammatory cytokines in PD[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the immune system, regulatory T cells (Tregs) are an immunosuppressive T cell subset that are important regulators of neuroinflammatory tolerance and preserve immune homeostasis in the central nervous system (CNS) and peripheral circulation[\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Recent studies have shown that Tregs exert neuroprotective effects on some neurological diseases, such as amyotrophic lateral sclerosis (ALS) and ischemic stroke, possibly by mediating the suppression of toxic neuroinflammation and regulating neurotrophic factors in the CNS and peripheral circulation[\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It has been shown that an increase in Tregs in PD models significantly prevents dopaminergic neuronal loss and behavioral changes, and attenuates inflammatory reactions in the CNS[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. One recent study by Park et al., also suggest that co-transplanting autologous Treg cells could effectively provide a potential strategy to achieve better clinical outcomes for cell therapy in Parkinson's disease[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Hence, the role of Tregs in PD needs to be further investigated.\u003c/p\u003e \u003cp\u003eRepetitive transcranial magnetic stimulation (rTMS) is a safe and noninvasive neuromodulation technique that has been used to improve motor symptoms in PD[\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, the reported outcomes of rTMS in PD have been inconsistent[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A recent study has suggested that multi-session of high-frequency rTMS over the primary motor cortex (M1, especially bilateral M1) with a total of 18, 000\u0026ndash;20, 000 pulses may be optimal for motor improvement in PD[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, the molecular mechanisms underpinning the therapeutic effects of rTMS on PD have not been elucidated. Animal studies have shown that rTMS can play a neuroprotective role in PD animal models through anti-apoptosis and anti-inflammation[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. There is a suggestion that proinflammatory cytokines (IFNγ and IL-17A) in the peripheral blood of PD patients may change after TMS treatment[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A decrease in the proportion of Tregs in the peripheral blood of PD may be an important feature of the peripheral immune response in PD[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, we hypothesize that rTMS may exert its modulatory effects on Tregs in PD.\u003c/p\u003e \u003cp\u003eTo test this hypothesis and address current gaps in knowledge, we first designed a prospective clinical trial in PD patients to evaluate the effect of rTMS on motor dysfunction and investigated the relationship between rTMS and changes in circulating Tregs. We next validated the clinical findings using an experimental MPTP mouse model. In addition, we also investigated the mechanisms of Tregs in regulating the therapeutic effects of rTMS. Last, we screened and identified Treg-related hub node proteins in the MPTP model.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics Statement\u003c/h2\u003e \u003cp\u003e The human studies were approved by the Ethics Committee of Zhujiang Hospital of Southern Medical University, and all patients provided written informed consent prior to recruitment into the study and was conducted in accordance with the principles of the Helsinki Declaration revised in 1975 and the National Institutes of Health Policy and Guidelines for Human Subjects issued in 1999. All mouse experiments were approved by the Experimental Animal Ethics Committee of Zhujiang Hospital of Southern Medical University.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eClinical Study\u003c/h3\u003e\n\u003cp\u003eFrom Sep 2021 to Jan 2022, 60 PD patients were prospectively recruited in a randomized, double-blinded study (the main researchers, raters, experienced neurologists and patients involved in the study were not aware of the rTMS treatment protocol, except the therapists who did rTMS treatment for PD) from Zhujiang Hospital (Trial Registration Chinese ClinicalTrials. gov Identifier: ChiCTR2100051140). All participants provided written consent to participate in the investigation and allowed investigators to examine their blood samples. Informed written consent was obtained from patients and family members. The enrolled PD patients were interviewed by two experienced neurologists. Characteristics of PD patients, clinical ratings and neuropsychological tests were performed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The exclusion Criteria include: 1.Patients with secondary Parkinson's syndrome or Parkinson's overlap syndrome caused by vascular factors, toxins, medications, etc.; 2. PD patients with persistent head tremors; 3. History of Deep Brain Stimulation (DBS) or ablative surgery in the past;4. Other contraindications for rTMS, such as history of epileptic seizures, pregnant women, individuals with cardiac pacemakers, those with metal implants in the body, intracranial hypertension, or severe bleeding tendencies;5. Individuals who have received rTMS treatment within the last 6 months;6. As assessed by the researchers, other factors that make the patient unsuitable for participation in the study or those who are unable to commit to completing follow-up visits. All participants with PD received an EEG before inclusion and fulfilled the Movement Disorder Society Clinical Diagnostic Criteria for Idiopathic PD[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and underwent extensive clinical examinations. 54 PD patients were ultimately recruited (6 PD patients were ultimately excluded due to four individuals elected not to partake in the study and two subjects were identified as having secondary parkinsonism) and received rTMS treatment or sham rTMS treatment according to random numbers. Random numbers were performed as follows. Computer SAS software was used to randomly group according to a single center, generate random numbers, and the treatment scheme is hidden using opaque envelopes. The envelopes are kept by the treatment provider and opened and sealed by the treatment provider in the order of the patient's visit. Unless the conditions for unblinding are met, the treatment plan cannot be disclosed under any circumstances. The follow-up examinations were done by the same rater and under the same time and all the patients were ON-condition. Sample size was based on a calculation of the results of pre-experiments. The rTMS were delivered by MagPro R30 (Tonica Elektronik A/S, Denmark). The specific rTMS treatment parameters are as follows: initially, high-frequency rTMS (100% resting motor threshold, 10 Hz, 1000 pulses) is administered to the primary motor cortex of the left hemisphere. Subsequently, after a 5-minute treatment interval, high-frequency rTMS (100% resting motor threshold, 10 Hz, 1000 pulses) is applied to the primary motor cortex of the right hemisphere. This bilateral hemispheric treatment is conducted daily. The treatment protocol spans over two weeks, with a consecutive 10 working days of treatment for each hemisphere. During the 2-week rTMS treatment period, the dosage of the original treatment drug remained unchanged. The sham rTMS treatment protocol was the same as the rTMS treatment protocol, except that a Cool-B65 P CO coil was used for the sham treatment, while a Cool-B65 A CO coil was used for the rTMS treatment. The alternative sham stimulation was also proceeded for two-week. At the start of each treatment, the resting motor threshold (RMT) of the patient's cortex was measured according to relative frequency method[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Specifically, the patients were seated in an armchair with a silversilver chloride surface electrode placed over the abductor pollicis brevis muscle contralateral to each hemisphere. The hot spot was determined using the MagPro R30 Stimulator TMS System (Tonica Elektronik A/S, Denmark) and octagonal coil. The octagonal coil was placed over the scalp and repositioned until the maximal motor evoked potential (MEP) was elicited. After determining the hot spot, the RMT was obtained by delivering single pulse transcranial magnetic stimulation to the hot spot. The RMT was defined as the lowest TMS intensity capable of eliciting a MEP greater than a 50 \u0026micro;V peak-to-peak amplitude in five of the ten subsequent trials. CD4\u0026thinsp;+\u0026thinsp;CD25\u0026thinsp;+\u0026thinsp;CD127- cells, CD4\u0026thinsp;+\u0026thinsp;CD25(low)CD45RA+ [naive Treg (nTreg) cells] and CD4\u0026thinsp;+\u0026thinsp;CD25(high)CD45RA- [activated Treg (aTreg) cells] subsets in the peripheral blood were assessed by flow cytometry and IFN-γ, TNF-α, IL-17α, IL4, IL10, TGF-β1 in the peripheral blood were assessed by Enzyme-linked immunosorbent assay (ELISA) prior to treatment and day 13. Hoehn and Yahr and MDS-UPDRS III were performed prior to treatment and on Days 13, 19, and 40 following the culmination of the two-week rTMS therapy.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBaseline characteristics of PD patients in sham and rTMS group\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSham\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003erTMS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSham vs rTMS\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender (M/F)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14/13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14/13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63.48\u0026thinsp;\u0026plusmn;\u0026thinsp;9.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e63.26\u0026thinsp;\u0026plusmn;\u0026thinsp;8.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9273\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMDS-UPDRS Ⅲ (score)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51.33\u0026thinsp;\u0026plusmn;\u0026thinsp;11.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50.67\u0026thinsp;\u0026plusmn;\u0026thinsp;10.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.8260\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u0026ndash;30 (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u0026ndash;50 (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50 (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH \u0026amp; Y scale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.7610\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.5(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.5(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLED (mg/day)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e838.0\u0026thinsp;\u0026plusmn;\u0026thinsp;484.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e830.8\u0026thinsp;\u0026plusmn;\u0026thinsp;412.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9534\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;C(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;D(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;E(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;D(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;C\u0026thinsp;+\u0026thinsp;D(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;C\u0026thinsp;+\u0026thinsp;E(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;C\u0026thinsp;+\u0026thinsp;G(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;D\u0026thinsp;+\u0026thinsp;E(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;D\u0026thinsp;+\u0026thinsp;F(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C\u0026thinsp;+\u0026thinsp;D(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C\u0026thinsp;+\u0026thinsp;E(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;C\u0026thinsp;+\u0026thinsp;D\u0026thinsp;+\u0026thinsp;E(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;C\u0026thinsp;+\u0026thinsp;D\u0026thinsp;+\u0026thinsp;F(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;C\u0026thinsp;+\u0026thinsp;E\u0026thinsp;+\u0026thinsp;G(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;D\u0026thinsp;+\u0026thinsp;E\u0026thinsp;+\u0026thinsp;G(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C\u0026thinsp;+\u0026thinsp;D\u0026thinsp;+\u0026thinsp;E(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;C\u0026thinsp;+\u0026thinsp;D\u0026thinsp;+\u0026thinsp;E\u0026thinsp;+\u0026thinsp;G(n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuration since symptom onset(month)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e71.26\u0026thinsp;\u0026plusmn;\u0026thinsp;46.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e86.81\u0026thinsp;\u0026plusmn;\u0026thinsp;69.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.3385\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMotor subtype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMixed type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTremor type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eakinetic-rigid type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThe presence of motor fluctuations(Yes/No)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10/17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8/19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.5723\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eLED refers to the equivalent dose of levodopa; A\u0026thinsp;=\u0026thinsp;Dobassrazide tablets; B\u0026thinsp;=\u0026thinsp;Carlevodopa controlled-release tablets; C\u0026thinsp;=\u0026thinsp;Entacapone; D\u0026thinsp;=\u0026thinsp;Pramipexole hydrochloride; E\u0026thinsp;=\u0026thinsp;Amantadine hydrochloride; F\u0026thinsp;=\u0026thinsp;Rasagiline; G\u0026thinsp;=\u0026thinsp;Ropinirole hydrochloride sustained-release tablets. A indicates the use of dobasilazine tablets as monotherapy; F indicates the use of rasagiline monotherapy; A\u0026thinsp;+\u0026thinsp;D indicates the combined use of dobasilazine tablets and pramipexole hydrochloride; the other combination drugs can be deduced by analogy. Data are means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD unless otherwise indicated.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe peripheral blood was obtained from PD patients[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and lysed in ACK lysis buffer (Biosharp, China).CD4 T cells were then purified using magnetic bead separation (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The cells were stained with anti-CD25 and anti-CD127 (all from BD Biosciences) for analysis by flow cytometry. Following the acquisition of sample data on a FACSCalibur flow cytometer (BD Biosciences), the results were generated in graphical and tabular formats using FlowJo V10 software (TreeStar Inc., Ashland, USA).\u003c/p\u003e\n\u003ch3\u003eAnimal Study\u003c/h3\u003e\n\u003cp\u003eBased on our clinical findings of an association between rTMS and changes in circulating Tregs of PD patients, we next conducted studies in a PD mouse model to validate the results. We determined whether the Tregs in the spleen of the PD mouse model changed after rTMS treatment and investigated the potential mechanisms that led to these changes. We applied a similar rTMS treatment protocol in MPTP mice. Male C57BL/6 mice (8 weeks old, 22\u0026ndash;24 g) received intraperitoneal (i.p.) injection of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) (Sigma\u0026ndash;Aldrich, USA) (30 mg/kg) for 5 days as PD animal model, while control mice received the same dose of normal saline. Another group of MPTP mice received i. p. injection of anti-CD25 monoclonal antibody (1 mg/kg; Thermo, USA) for three consecutive days to block Tregs in the spleen, to investigate whether the therapeutic effect of rTMS on MPTP mice is related to changes in Treg level in the spleen. In addition, we also evaluated the behavior of mice by pole climbing test and detected the expression of dopamine cells, neurotrophic factors BDNF, GDNF, microglia and inflammatory factors in the SN of mice. We next screened the hub node proteins related to rTMS on MPTP mice by quantitative proteomics analysis with TMT labeled of whole proteins. Finally, to better understand the role of hub proteins in MPTP mice, RNA interference (RNAi) mediated Adeno-associated virus (AAV) vector was injected into midbrain via a stereotactic midbrain approach to block hub proteins level in SN. Immunofluorescent staining and western blots (WB) were used to detect the transfection efficiency. The changes of hub protein levels in SN were detected by immunofluorescence staining and the inflammatory cytokines in the ventral midbrain were detected by ELISA after the hub proteins were interfered by the virus.\u003c/p\u003e \u003cp\u003eFor the sample size in animal experiments, a power analysis was performed. A sample size of at least n\u0026thinsp;=\u0026thinsp;8 per group was determined to reach a statistical significance of 0.05, in detecting an effect size of at least 1.06 with 80% power. Mice were assigned randomly to the experimental groups. The investigators were blinded to the identities of treatment groups.\u003c/p\u003e\n\u003ch3\u003eAnimals and MPTP-PD Model\u003c/h3\u003e\n\u003cp\u003eMale C57BL/6 mice (8 weeks old, 22\u0026ndash;24 g) were obtained from Liaoning Changsheng Biotechnology Co., Ltd. (Liaoning, China). Before the experiment, all mice were housed at 20\u0026ndash;22\u0026deg;C with a 12 h: 12 h light/dark cycle and food and water provided ad libitum for 7 days. The CD25 receptor is a key site for activating Treg, and as the hypothesis is that rTMS may exert its modulatory effects on Treg in PD, we used anti CD25 injections to specifically block Treg to determine if rTMS still has an impact on PD. A total of 189 male mice were randomly divided into 9 groups as follows: (1) the saline normal control group (NC group, n\u0026thinsp;=\u0026thinsp;21 mice) was only administered saline by intraperitoneal (i. p. ) injection for 5 days; (2) the NC\u0026thinsp;+\u0026thinsp;sham rTMS group (NC\u0026thinsp;+\u0026thinsp;sham group, n\u0026thinsp;=\u0026thinsp;21 mice), which were treated with i. p. injections of saline (30 mg/kg) for 5 days followed by sham rTMS treatment; (3) the NC\u0026thinsp;+\u0026thinsp;rTMS (10 Hz) group (n\u0026thinsp;=\u0026thinsp;21 mice), which were administered i. p. doses of saline (30 mg/kg) for 5 days before receiving rTMS (10 Hz) therapy; (4) the MPTP group (n\u0026thinsp;=\u0026thinsp;21 mice), which received i. p. injections of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) (Sigma\u0026ndash;Aldrich, USA) (30 mg/kg) for 5 days; (5) the MPTP\u0026thinsp;+\u0026thinsp;sham rTMS group (n\u0026thinsp;=\u0026thinsp;21 mice), which were treated with i. p. injections of MPTP (30 mg/kg) for 5 days followed by sham rTMS treatment; (6) MPTP\u0026thinsp;+\u0026thinsp;rTMS (10 Hz) group (n\u0026thinsp;=\u0026thinsp;21 mice), which were administered i. p. doses of MPTP (30 mg/kg) for 5 days before receiving rTMS (10 Hz) therapy; (7) the MPTP\u0026thinsp;+\u0026thinsp;block group (n\u0026thinsp;=\u0026thinsp;21 mice), which received 3 days of continuous i. p. injections of anti-CD25 monoclonal antibodies (1 mg/kg) followed by 5 days of i. p. injections of MPTP (30 mg/kg); (8) the MPTP\u0026thinsp;+\u0026thinsp;block\u0026thinsp;+\u0026thinsp;sham (10 Hz) group (n\u0026thinsp;=\u0026thinsp;21 mice), which was administered i. p. injections of anti-CD25 monoclonal antibodies (1 mg/kg, Thermo, USA) for 3 days before receiving i. p. injections of MPTP (30 mg/kg) for 5 days before receiving shamtherapy; and (9) the MPTP\u0026thinsp;+\u0026thinsp;block\u0026thinsp;+\u0026thinsp;rTMS (10 Hz) group (n\u0026thinsp;=\u0026thinsp;21 mice), which was administered i. p. injections of anti-CD25 monoclonal antibodies (1 mg/kg, Thermo, USA) for 3 days before receiving i. p. injections of MPTP (30 mg/kg) for 5 days before receiving sham therapy.\u003c/p\u003e\n\u003ch3\u003erTMS treatment\u003c/h3\u003e\n\u003cp\u003eA transcranial magnetic stimulator (MagVenture A/S, Denmark) connected to a circular coil (Cool-40 Rat Coil) with a diameter of 40 mm was used to deliver rTMS. the rTMS treatment regimen for mice is consistent with the treatment regimen for PD patients. In the sham rTMS group, the stimulus was delivered by fixing the coil 10 cm above the heads of the mice to ensure that the mice felt the clicking sound or vibrations generated by the rTMS coil but did not receive brain stimulation. To minimize the discomfort of the mice, their movements were carried out in a soft plastic funnel, therefore, on the first day, they were restrained without stimulation for 10s to familiarize them with the experimental procedure. At the start of each treatment, the resting motor threshold was measured as previously described[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Briefly, a standard electromyographic (EMG) machine (Counterpoint, Dantec Medical Inc., Denmark) was used for recording of the motor evoked potentials (MEPs). Sampling frequency was 51.4kHz, high and low pass filter settings were 10 and 3000Hz. A 0.5mm bipolar EMG needle (DANTEC) placed in the right hindlimb biceps femoris muscle with a ground electrode 10 mm proximal to the recording electrode was used for recording the muscle activity. The motor threshold was defined as a reproducible motor evoked potential in five consecutive stimuli with an interstimulus interval\u0026thinsp;\u0026gt;\u0026thinsp;3s and an amplitude\u0026thinsp;\u0026gt;\u0026thinsp;50mV. The real and sham rTMS treatments did not induce seizures or other behavioral changes during the treatment period.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePole climbing test\u003c/h2\u003e \u003cp\u003eWe assessed motor dysfunction by performing pole tests[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] Briefly, the mice were placed head-up on the top of the pole (height 50 cm, diameter 1 cm), and their return to the bottom was timed. Timing began when the mouse was released and stopped when one hindlimb reached the bottom. The test was repeated 3 times for each mouse after they were trained once at 30 min intervals, and the average time was used for data analysis. The raters were blinded for the treatment group.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFlow cytometric analysis of CD4 + CD25 + Foxp3 + Tregs in the spleen\u003c/h3\u003e\n\u003cp\u003eThe spleens of male C57BL/6J mice[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] were removed and homogenized by a tissue grinder and a wire mesh screen. Red blood cells were then lysed in ACK lysis buffer. Then CD4\u0026thinsp;+\u0026thinsp;T cells were then purified using magnetic bead separation (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The cells were stained with anti-CD25 and anti-Foxp3 (BD Biosciences, USA, 567456) for flow cytometric analysis. Following the acquisition of sample data on a FACSCalibur flow cytometer (BD Biosciences), the results were generated in graphical and tabular formats using FlowJo V10 software (TreeStar Inc., Ashland, USA).\u003c/p\u003e\n\u003ch3\u003eImmunohistochemical staining\u003c/h3\u003e\n\u003cp\u003eThe mice were anesthetized with sodium pentobarbital, sacrificed, and transcardially perfused with normal saline containing 0.5% sodium nitrate and heparin (10 U/ml) followed by 4% paraformaldehyde (PFA) dissolved in 0.1 M phosphate buffer. The brain was dissected and postfixed in 4% PFA at 4\u0026deg;C for 18\u0026ndash;24 h and then immersed in 20% sucrose at 4\u0026deg;C until the brain dropped to the bottom, followed by immersion in 30% sucrose at 4\u0026deg;C for 24 h. The brains were embedded in optimal cutting temperature compound (O. C. T. Compound, Tissue-Tek, USA.) We prepared coronal sections of the frozen brains with a cryostat microtome (CM1950, Leica, Germany). Frozen brains were sectioned into 40-\u0026micro;m-thick coronal sections. All sections were collected and processed for immunohistochemical staining. For antigen retrieval, the tissue slices were submerged in 0.01 M sodium citrate buffer (pH 6.0) and rinsed twice in PBS. The tissue slices were then treated for 1 hour at 37\u0026deg;C in PBS containing 0.2% v/v Triton X-100, 0.02% w/v sodium azide, and 5% v/v goat serum. Primary antibodies against tyrosine hydroxylase TH (1: 800, Proteintech, USA, 25859-1-AP), BDNF (1: 500, Abcam, England, ab108319), GDNF (1: 500, Abcam, England, ab18956) and Iba-1 (1: 500, Abcam, England, ab178846) SYT6 (1: 250, SANTA CRUZ, sc390321), TLR4 (1: 250, Invitrogen, 710185) were added to the slices and incubated overnight at 4\u0026deg;C. Then, the sections were treated with the appropriate biotinylated secondary antibody, followed by Vectastain ABC reagent (Vector Laboratories, Burlingame, CA, USA) and 3, 3\u0026rsquo;-diaminobenzidine (Sigma\u0026ndash;Aldrich, Vienna, Austria). The stained slices were dehydrated and coverslipped with Entellan before being mounted on slides (Merck, Darmstadt, Germany). IgG conjugated with Alexa 594 (1: 500, Abcam, England, ab150080) was used for immunofluorescent staining. Mounting medium was used to cover the sections (Dianova, Hamburg Germany). The numbers of TH+, SYT6, TLR4 in the substantia nigra pars compacta (SNc) were estimated using unbiased stereological methods (Stereo Investigator, MicroBrightField, VT). The numbers of activated Iba-1-positivemicroglia in the SNc were estimated using the optical fractionator probe with the stereologist. Microglia were classified as activated if the cell body was visibly increased in diameter and the cell had shortened and thickened processes. The three stereologists were blinded to the treatment received. Additionally, we determined the average optical density value of BDNF and GDNF immunoreactivity through densitometry, employing the freely available ImageJ software (National Institutes of Health, Bethesda, MD, USA). All histochemical data statistics was conducted in a single-blind manner by four experimenters. FX renumbers each group of fluorescent films in an experiment and randomly divides them into three groups (random number method) and assigns them to three investigators (YQL, ZCX and Q.W). Each film takes 5 fields of view for statistical averaging, and finally the data is summarized with FX.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative proteomics technology with TMT labeling of whole protein\u003c/h2\u003e \u003cp\u003eTwelve brain tissue from 4 groups (saline normal control, MPTP, MPTP\u0026thinsp;+\u0026thinsp;rTMS, MPTP\u0026thinsp;+\u0026thinsp;block\u0026thinsp;+\u0026thinsp;rTMS) were collected for proteome Tandem Mass Tag (TMT) analysis. The collected samples were processed for TMT analysis in JingJie Bio-tech. Output was visualized using R and CummeRbund. Heatmaps were generated in R using pHeatmap and R ColorBrewer. Result was analyzed according to Maxquant (v1.6.15.0) database.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMolecular docking method\u003c/h2\u003e \u003cp\u003eThe protein data bank (PDB) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to obtain the PDB file which has the active structure of the target protein, The smaller Resolution value and complete protein binding pockets were used as the screening conditions to select the crystal structure for subsequent analysis. Refer to the previous literature[\u003cspan additionalcitationids=\"CR37 CR38 CR39\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], according to the amino acid sequence provided by Unirpot, SWISS-MODEL was used (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/interactive\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org/interactive\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to perform online homology modeling of proteins without ready-made protein crystal structures in the PDB database. Select the result with homology\u0026thinsp;\u0026ge;\u0026thinsp;30%, and then according to the scoring screen to evaluate the model quality with GMQE and QMEAN values when the homology reaches the standard, the template with larger GMQE and QMEAN close to 0 was chose. Then, online protein-protein docking is performed through Z-dock (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://zdock.umassmed.edu/\u003c/span\u003e\u003cspan address=\"http://zdock.umassmed.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the version is selected as V3.0.2[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and the docking result image uses PyMol 2. 3.5 for image rendering.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePath enrichment analysis and PPI network construction\u003c/h2\u003e \u003cp\u003eGO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://geneontology.org/\u003c/span\u003e\u003cspan address=\"http://geneontology.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used for pathway enrichment analysis[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], obtain immune-related pathway genes, and String database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www\u003c/span\u003e\u003cspan address=\"https://www\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. string-db. org/) was used to construct protein online Protein interaction network (PPI network)[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], the species is selected as Mus musculus, the minimum required interaction score is set to 0.4, and the other conditions are the default settings.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eTissues were lysed in a detergent-based lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and 1 mM PMSF). Samples were then centrifuged for 10 min at 13, 000 g at 4\u0026deg;C, and the supernatant was collected and quantified with the BCA Protein Assay Kit (Thermo Scientific, USA). Equal amounts of protein sample were separated by 10% SDS-PAGE. Antibodies used for western blotting were: Syt6 (1: 1000, abcam, USA), TLR4 (1: 1000, abcam, USA), TH (1: 1000, proteintech, USA), Slc6a3 (1: 1000, abcam, USA) and β-actin (1: 10000, Thermo, USA). Antibody binding was detected using the secondary antibodies (Peroxidase-Conjugated AffiniPure Goat Anti-Rabbit/Mouse IgG (H\u0026thinsp;+\u0026thinsp;L), 1: 5000, thermofisher, USA). All samples were normalized to β-actin. All quantification was calculated with Alpha Ease FC software (Alpha Innotech Corporation, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eELISA assay\u003c/h2\u003e \u003cp\u003eCytokine levels of IFN-γ, TNF-α, IL-17α, IL4, IL10 and TGF-β1 in the serum of PD patients were measured using commercial ELISA kits (IFN-γ ELISA kit (Abcam, England, ab46025), TNF-α ELISA kit (Abcam, England, ab181421), IL-17α ELISA kit (Abcam, England, ab216167), IL4 ELISA Kit (Abcam, England, ab215089), IL-10 ELISA kit (Abcam, England, ab100549), TGF-β1 ELISA kit (Sigma\u0026ndash;Aldrich, USA,RAB0460) in accordance with the manufacturer\u0026rsquo;s protocols. Brain tissue was collected from the different groups of mice, and then the tissue from the ventral midbrain was separated and homogenized with lysis buffer on ice. The supernatant was collected at 12, 000 rpm at 4\u0026deg;C and centrifuged for 10 min for subsequent experiments. The proinflammatory cytokines IL-6, IFN-γ, TNF-α, IL-1β, IL-10 and TGF-β1 were measured using commercial ELISA kits (IL-6 ELISA kit (Abcam, England, ab100713), IFN-γ ELISA kit (Abcam, England, ab100690), TNF-α ELISA kit (Abcam, England, ab229393), IL-1β ELISA Kit (Sigma\u0026ndash;Aldrich, USA, RAB0275), IL-10 ELISA kit (Abcam, England, ab100697), TGF-β1 ELISA kit (Abcam, England, ab119557) in accordance with the manufacturer\u0026rsquo;s protocols.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eVirus Constructs and stereotaxic surgery\u003c/h2\u003e \u003cp\u003eThe scramble short hairpin RNA (shRNA) (5\u0026rsquo;-CCTAAGGTTAAGTCGCCCTCG-3\u0026rsquo;) and shRNA coding sequences targeting mous syt6 (5\u0026rsquo;-ATGAAAGCGAGACGCTGATTG-3\u0026rsquo;), (5\u0026rsquo;- ATGTCTCCAGCGTGGACTATG-3\u0026rsquo;) and (5\u0026rsquo;- GCGGAAGTTCTGACCCTTA-3\u0026rsquo;) were cloned into the PAAV-U6-shRNA(Syt6)-CMV-EGFP-WPRE vector (Obio Technology, Shanghai, China). Adeno-associated viruses (AAVs) described above were packaged by Obio Technology into serotype 2/9.After mice were anesthetized and placed in the stereotaxic frame, 2 \u0026micro;l of purified and concentrated AAV(10\u003csup\u003e12\u003c/sup\u003e IU/mL) was unilateral injected into the right SNc (AP\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;3.2 mm; ML\u0026thinsp;=\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 mm, DV\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;4.6 mm) at a rate of 0.2 \u0026micro;l/min according to previously described protocols[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The 5-\u0026micro;l Hamilton syringe was kept in place for 10 min before being slowly retracted within 5 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism 8.0 software. Data with a normal distribution are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Intra-group comparisons of squared differences were conducted using paired sample t-tests, while inter-group comparisons were made using two independent samples t-tests. Data that did not conform to a normal distribution are presented with comparisons made using non-parametric tests. Counting data are presented as frequencies, with comparisons made using the χ2 test. A two-sided test was chosen, and a P-value less than 0.05 was considered statistically significant. Correlation between the change of Treg ratio in peripheral blood and the change of MDS-UPDRSIII before and after treatment with rTMS were calculated by the Pearson correlation. After flowcytometry, CD4 subsets were Determined as a percentage of all lymphocytes gated using forward and side scatter, and as a percentage of all leukocytes bydual-platform analysis using full blood cell counts from the same blood collection. Mouse flow cytometry data, total times of pole climbing test, TH, BDNF, GDNF, Syt6, TLR4 immunohistochemistry, IL-6, IFN-γ, TNF-α, IL-1β, IL-10 and TGF-β1 enzyme-linked immunosorbent assay and Syt6, TLR4, Slc6a3 and TH western blotting were analyzed using One-way ANOVA followed by the least significant difference (LSD) for post hoc comparisons. The data are representative of at least three independent experiments.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003erTMS increased the proportion of Tregs, attenuated inflammation and improved motor impairment in PD patients\u003c/h2\u003e \u003cp\u003eWe conducted a clinical trial that included a total of 60 PD patients (6 PD patients were ultimately excluded). Fifty-four PD patients were divided into two groups; one group was treated with 10-day rTMS (10 Hz) in the M1 area, and the other group received sham rTMS treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B), followed by Hoehn and Yahr and MDS-UPDRS III assessments at Days 1, 13, 19, and 40 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Our clinical trial showed that the MDS-UPDRS Part III score was significantly reduced in PD patients receiving rTMS as compared to the sham control group on the day following the completion of a two-week rTMS treatment protocol (-4.15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.689, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Furthermore, the MDS-UPDRS Part III score sustained a lower score for up to 40 days post-treatment with rTMS (-4.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.716, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). The baseline clinical and demographic characteristics were not different between the rTMS and sham groups (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We performed flow cytometry with anti-CD4, CD25 and CD127 antibodies to examine the proportions of CD4\u0026thinsp;+\u0026thinsp;CD25\u0026thinsp;+\u0026thinsp;CD127- Treg cells and CD4\u0026thinsp;+\u0026thinsp;T cells in sham and rTMS groups. The results showed no difference in the proportion of Tregs (% CD4\u0026thinsp;+\u0026thinsp;T cell population) between the sham and rTMS groups at baseline \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. However, 10-day rTMS treatment (9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64%) elevated peripheral Treg levels in PD patients (6.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69%) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, H), but there was no difference in the peripheral Treg levels in sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Pearson\u0026rsquo;s correlation was used to analyze the association between MDS-UPDRS III scores and the proportion of Tregs. We found that the increased proportion of Tregs was negatively correlated with the change in the MDS-UPDRS III scores in the rTMS PD group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ) but not in the sham treatment group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI\u003cb\u003e)\u003c/b\u003e. Furthermore, there was an observed increase in the proportion of aTreg/nTreg cells within the peripheral blood (Supplementary Fig.\u0026nbsp;1A, B, C). Concurrently, levels of IL4, IL10, and TGF-β1 were elevated, while pro-inflammatory markers such as IFN-γ, TNF-α, and IL-17α demonstrated a decrease (Supplementary Fig.\u0026nbsp;1D, E, F). The MFI of CD25 also shown no difference in the proportion of CD25 between the sham and rTMS groups at baseline (Supplementary Fig.\u0026nbsp;2A). In addition, the median fluorescence intensity (MFI) of CD25 was increased after the treatment of rTMS compared with sham treatment (Supplementary Fig.\u0026nbsp;2C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003erTMS ameliorated behavioral impairment in MPTP-induced mice\u003c/h2\u003e \u003cp\u003eWe induced PD-like symptoms and analyzed the impact of rTMS on motor function in MPTP mice. Based on the results of the pole climbing test, MPTP significantly impaired motor function, and rTMS ameliorated this impairment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B\u003cb\u003e)\u003c/b\u003e. Compared to saline normal control mice, MPTP-induced mice took 121.14% more time to turn around and climb down in the pole climbing test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B\u003cb\u003e)\u003c/b\u003e. However, compared with MPTP and sham rTMS mice, MPTP\u0026thinsp;+\u0026thinsp;rTMS (10 Hz)-treated mice spent less time and displayed considerably improved performance on the pole test (34.97% decrease in rTMS-treated PD mice vs. sham-treated PD mice, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). However, the Treg inhibitor anti-CD25 significantly abolished this improvement (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003erTMS rescued dopaminergic neurons, reversed the decrease in the circulating proportion of Tregs and attenuated inflammatory cytokines in MPTP-induced mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe performed immunohistochemical staining to examine the loss of dopaminergic neurons, BDNF and GDNF in the SN after MPTP administration. The immunohistochemical staining results showed that MPTP-induced mice showed 50.15%, 38.38% and 21.82% reductions in TH-positive neurons and fibers, BDNF and GDNF in the SN compared with saline normal control (NC) mice, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for TH, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for BDNF and GDNF; \u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e). Compared with those in the MPTP and sham rTMS groups, TH, BDNF and GDNF were considerably increased in the MPTP\u0026thinsp;+\u0026thinsp;rTMS (10 Hz) group (\u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e). Compared with that in saline NC mice, numerous microglia were activated in the SN of MPTP mice, and the proinflammatory cytokines IL-6, IFN-γ, TNF-α and IL-1β in the ventral midbrain of MPTP mice were significantly increased by 75.37%, 185.87%, 91.66%, 140.36%, respectively, while the anti-inflammatory cytokines IL-10 and TGF-β1 were significantly reduced by 59.90% and 17.02%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-G). The results showed a significant decline in the circulating proportion of Treg cells (% CD4\u0026thinsp;+\u0026thinsp;T cell population) in PD mice (1.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33%) compared to NC mice (4.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). Compared with that in sham mice, rTMS (10 Hz) therapy significantly reversed the decrease in the circulating proportion of Tregs in PD mice (302.44% increase in rTMS-treated PD mice vs. sham-treated PD mice, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for Tregs, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D), increased the anti-inflammatory mediators IL-10 and TGF-β1 by 69.44% and 12.34%, respectively, deactivated microglia and decreased the inflammatory cytokines IL-6, IFN-γ, TNF-α and IL-1β by 28.41%, 24.40%, 30.48% and 22.78% in the ventral midbrain of PD mice, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-G). This finding indicated that rTMS treatment of PD mice could attenuate inflammation in the brain. Injection of the Treg inhibitor anti-CD25 (1 mg/kg/d, for 3 days) into PD mice significantly abolished the rTMS-mediated increase in circulating Tregs and anti-inflammatory cytokines and reversed the downregulation of these inflammatory cytokines, as well as microglial activation in the SN (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for Tregs, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for microglia, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for inflammatory and anti-inflammatory cytokines; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-G).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative proteomics analysis of Treg- and rTMS-related proteins\u003c/h2\u003e \u003cp\u003eTo further identify the molecular targets and hub proteins related to Tregs and rTMS therapy in PD, we used tandem mass tag (TMT) technology to label 12 samples (4 groups: saline normal control, MPTP, MPTP\u0026thinsp;+\u0026thinsp;rTMS, and MPTP\u0026thinsp;+\u0026thinsp;block\u0026thinsp;+\u0026thinsp;rTMS, 3 samples per group) for quantitative proteomics analysis. We first identified 7, 817 proteins (\u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e), and proteins that were significantly upregulated and downregulated in all four clustered groups are shown in \u003cb\u003eSupplementary Table\u0026nbsp;2A\u003c/b\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B. We next identified 2 proteins (Syt6 and Slc6a3) through overlapping pairwise comparisons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Analysis of the protein\u0026ndash;protein interaction (PPI) network also confirmed that Syt6 and Slc6a3 were involved in multiple pathways and associated with the activation of T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). We performed full rigid-body docking orientation analysis of the two proteins with the ZDOCK algorithm and found that Syt6 docked with TLR4. This proteomics analysis suggests that there may be protein interactions between TLR4 and SYT6, and the two protein interactions may exert biological effects through multiple binding sites. The main sites of TLR4 are LYS-47, SEP-71 and ASP-95, and the main sites of SYT6 are ARG \u0026minus;\u0026thinsp;321 and ARG-386 (\u003cb\u003eSupplementary Table\u0026nbsp;2B, C\u003c/b\u003e). These proteins are considered hub proteins and may serve as novel targets of PD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further characterize the role of rTMS and identify its association with Tregs in PD, we investigated these screened hub proteins by Western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-G). Compared with those in saline normal controls, the levels of Syt6 and TLR4 were significantly increased by 164.19% and 85.55%, respectively, and TH and Slc6a3 were significantly decreased by 41.08% and 45.97% in the PD mouse brain, respectively, while rTMS therapy reversed these changes (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for Syt6, TLR4, TH and Slc6a3; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-E). More interestingly, after inhibiting Tregs with anti-CD25 and stimulating PD mice with rTMS, the levels of Syt6, TLR4, TH and Slc6a3 in PD mouse brains were significantly reversed compared with those in the PD\u0026thinsp;+\u0026thinsp;rTMS group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-G\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eVerification of the role of Treg and rTMS-related protein syt6 in MPTP mice by virus interference method\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAdeno-associated virus (AAV) vector was injected into midbrain via a stereotactic midbrain approach to block syt6 level. Immunofluorescent staining and WB were used to detect the efficiency of transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, C, E). Compared with those in MPTP\u0026thinsp;+\u0026thinsp;scramble control group, the SYT6/DAPI expression was significantly decreased by 82.2% in the SN of MPTP\u0026thinsp;+\u0026thinsp;syt6-shRNA3 group, and the syt6 relative expression level was similar to that in WB. Similarly, mice in the MPTP\u0026thinsp;+\u0026thinsp;Syt6-shRNA3 group showed significantly improved performance in pole test after Syt6 knockdown in SN (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, D, F). Therefore, we then used syt6-shRNA3 as an AAV to block the expression of syt6 protein in SN. Interestingly, rTMS did not change the total climbing time of MPTP mice with syt6 AAV. On the contrary, rTMS treatment significantly reduced the total climbing time of MPTP mice with AAV empty vector (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). We next examined the expression of syt6, TLR4, TH and inflammatory cytokines with syt6 AAV intervention in MPTP-induced mice with or without rTMS treatment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, E, G, I\u003cb\u003e)\u003c/b\u003e. There was a significant increase by 47.72% in TH expression in MPTP mice with syt6 AAV intervention but rTMS treatment did not change the TH level in MPTP mice with syt6 AVV intervention. On the contrary, rTMS treatment significantly increased TH level in MPTP mice with AAV empty vector by 39.51% \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F\u003cb\u003e)\u003c/b\u003e. There was a significant decrease by 80.47% of syt6 expression in MPTP mice with syt6 AAV intervention \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D\u003cb\u003e)\u003c/b\u003e; however, rTMS did not change the syt6, TLR4, IL-β1, TNF-α and IL-6 levels in MPTP mice with syt6 AAV. On the contrary, rTMS significantly decreased TLR4, IL-β1, TNF-α and IL-6 level by 41.80%, 25.52%, 32.12% and 10.79% respectively in MPTP mice with AAV empty vector \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, H, I\u003cb\u003e)\u003c/b\u003e. In addition, the levels of anti-inflammatory cytokines, such as IL-10 and TGF-β, were significantly elevated in MPTP-induced mice following rTMS treatment, which was not altered by Syt6 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI), Furthermore, Syt6 expression was detected in Iba1-positive cells (\u003cb\u003eSupplementary Fig.\u0026nbsp;4\u003c/b\u003e). These data suggest that rTMS treatment affects syt6, a hub protein, to increase the proportion of Tregs in MPTP mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur randomized sham controlled clinical trial demonstrated that 10 days of rTMS stimulation improved motor dysfunction in PD subjects after 13 days, and the effect lasted for 40 days. Importantly, rTMS treatment elevated peripheral Treg levels in PD patients (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), but not in sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). In addition, we found a significant negative correlation between the increased proportion of peripheral blood Tregs and MDS-UPDRS III scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ), suggesting that motor improvement induced by rTMS might be due to an increase in circulating Tregs.\u003c/p\u003e \u003cp\u003erTMS is a non-invasive neuromodulation technique that utilizes magnetic fields to stimulate nerve cells in the brain. Extensive research has been conducted on its potential therapeutic effects in various neurological and psychiatric disorders, including Alzheimer's disease, PD, stroke, and multiple sclerosis. Preclinical studies have suggested that rTMS may improve cognitive function in Alzheimer's disease by enhancing synaptic plasticity and reducing amyloid-beta plaques. rTMS has also been studied for its potential to improve motor function and reduce fatigue in multiple sclerosis patients by modulating the excitability of the motor cortex [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. A systematic review has shown that rTMS intervention may have the potential to modify apathy among patients with chronic stroke[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The exact mechanisms by which rTMS exerts its effects are not fully understood but are normally thought to involve changes in neuronal activity, synaptic plasticity, and neurotransmitter release. In our study, we found that rTMS improves motor function by modulating the function of Tregs and suppressing toxic neuroinflammation in PD, which is consistent with previous studies[\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Ongoing research is exploring the use of rTMS in combination with other neuromodulation techniques to enhance therapeutic effects, indicating the potential therapeutic effects of rTMS in the treatment of neurodegenerative diseases.\u003c/p\u003e \u003cp\u003eEvidence-based guidelines for the therapeutic use of rTMS have demonstrated that high-frequency rTMS (HF-rTMS) applied to bilateral M1 regions or the left dorsolateral prefrontal cortex (DLPFC) can ameliorate motor impairment and depression, respectively, in Parkinson's disease, with Level B evidence indicating probable efficacy[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Furthermore, a randomized controlled trial published in a neurology journal provides Class I evidence that, in patients with Parkinson's disease experiencing depression, bilateral M1 rTMS (comprising 1,000 stimuli for each M1; 50 trains of 4 seconds at 10 Hz with 40 stimuli per train) results in enhanced motor function compared to sham rTMS[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Additionally, a meta-analysis has shown that multiple sessions of HF-rTMS over the M1, particularly when administered bilaterally, with a cumulative total of 18,000\u0026ndash;20,000 pulses, appears to be the optimal parameters for improving motor symptoms in Parkinson's disease[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Informed by these references, our clinical trial was designed to ameliorate the motor symptoms of Parkinson's disease using HF-rTMS applied to both M1 areas, delivering 1,000 stimuli to each side. The treatment spanned 10 days, accumulating a total of 20,000 stimuli.\u003c/p\u003e \u003cp\u003eTo take our findings further, we next attempted to validate our clinical findings in a PD animal model. Previous studies have indicated that induction of Tregs promoted neuronal survival through suppression of microglial activation in MPTP-induced PD mouse model[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Here, we utilized MPTP PD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B; Supplementary Fig.\u0026nbsp;1A, B) and found that Tregs in the spleen were dysregulated in these mice compared with NC mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D), which is consistent with previous reports[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Compared with that in NC mice, numerous microglia in the SN of PD mice were activated, and the proinflammatory cytokines IL-6, IFN-γ, TNF-α and IL-1β were significantly increased, while the anti-inflammatory cytokines IL-10 and TGF-β1 were significantly reduced \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-G\u003cb\u003e)\u003c/b\u003e. rTMS has been shown to ameliorate inflammation and regulate neurotrophic factors in depression, anxiety, cerebral ischemia and spinal cord lesions[\u003cspan additionalcitationids=\"CR54 CR55\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. To evaluate whether rTMS could influence PD by impacting circulating Tregs and regulating inflammation in the brain, we applied rTMS to the brains of MPTP mice for 10 days and identified that a 10 Hz stimulation led to consistent and immediate improvements in motor symptoms at 1 day after rTMS therapy \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B\u003cb\u003e)\u003c/b\u003e. In comparison to the sham-treated mice, our findings revealed that rTMS at 10 Hz significantly enhanced the circulating proportion of Tregs and TH\u0026thinsp;+\u0026thinsp;neurons within the SN (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D, \u003cb\u003eSupplementary Fig.\u0026nbsp;1A, B)\u003c/b\u003e, increased the anti-inflammatory mediators IL-10 and TGF-β1, deactivated microglia and decreased the inflammatory cytokines IL-6, IFN-γ, TNF-α and IL-1β1 in the ventral midbrain of PD mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-G\u003cb\u003e).\u003c/b\u003e These results suggest that rTMS therapy could attenuate inflammation in the brains in PD mice. One previous study showed that the transfer of Tregs to MPTP mice prevented DA neuronal degeneration by suppressing microglial activation and attenuating neuroinflammation, suggesting that the increased Tregs exerted neuroprotective effects on PD[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. We next injected the Treg inhibitor anti-CD25 into MPTP mice and observed that this treatment significantly attenuated the rTMS-mediated improvements in motor symptoms, abolished the rTMS-mediated upregulation of circulating Tregs, and reversed the downregulation of inflammatory cytokines in the ventral midbrain and microglial activation in the SN (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-G\u003cb\u003e)\u003c/b\u003e. These results indicate that the restoration of peripheral Tregs may be pivotal following rTMS stimulation in the brain, and rTMS may contribute to neuroprotection in PD at least partially by upregulating peripheral Tregs, subsequently reducing neuroinflammation and deactivating microglia in the SN. rTMS also profoundly upregulated the levels of TH, BDNF and GDNF in the SN (\u003cb\u003eSupplementary Fig.\u0026nbsp;3)\u003c/b\u003e, and these effects were partially abolished by anti-CD25 (\u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTo further identify the hub proteins related to Tregs and to screen biological indicators, tandem mass tags (TMT) technology was used to label 12 samples that were used for quantitative proteomic analysis. After intersection analysis of differentially expressed genes in the MPTP\u0026thinsp;+\u0026thinsp;block\u0026thinsp;+\u0026thinsp;rTMS treatment group and MPTP\u0026thinsp;+\u0026thinsp;rTMS group was performed, 30 differentially expressed genes were selected for pathway enrichment analysis, and a protein\u0026ndash;protein interaction (PPI) network was constructed and identified the Treg-related downstream targets Syt6, TLR4, TH and Slc6a3 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-E\u003cb\u003e)\u003c/b\u003e, which were closely associated with the activation of T cells after rTMS stimulation in MPTP mouse brain, Compared with those in the saline normal controls, the levels of Syt6 and TLR4 were significantly increased, and TH and Slc6a3 were significantly decreased in the MPTP mouse brain, while rTMS therapy reversed these effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-E\u003cb\u003e)\u003c/b\u003e. Interestingly, after inhibiting Tregs with anti-CD25 and administering rTMS stimulation, the changes in the levels of Syt6, TLR4, TH and Slc6a3 in MPTP mouse brains were significantly reversed compared with those in the PD\u0026thinsp;+\u0026thinsp;rTMS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-G). These results suggest that Syt6, TLR4, and Slc6a3 are key targets of Tregs in regulating MPTP following rTMS stimulation, further indicating an important pathophysiologic role of Tregs in rTMS treatment in PD. We also performed a full rigid-body docking orientation between two proteins by ZDOCK algorithm, and found that Syt6 was docking with TLR4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. These four proteins were recognized as the hub node proteins and may serve as novel targets in regulating PD.\u003c/p\u003e \u003cp\u003eFour identified proteins in our study exhibit close intersection with neuroinflammation, microglia and Tregs in neurodegeneration. Microglia and Tregs are both key players in the immune response and homeostasis maintain within the CNS. Recent studies have highlighted that infiltrating Treg cells is essential for behavioral recovery and brain repair with their immunomodulatory effects on microglia after stoke[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. A clinical study revealed a diminished proportion of Th2, Th17, and Treg cells in the peripheral blood of PD patients, along with a reduction in circulating CD4\u003csup\u003e+\u003c/sup\u003eT lymphocytes[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Collectively, Treg cells can modulate microglial responses, potentially leading to improved outcomes in neurodegenerative diseases. Here in our study, following rTMS treatment, we observed an increased proportion of aTreg/nTreg cells in the peripheral blood, accompanied by a decrease in pro-inflammatory factors such as IFN-γ, TNF-α, and IL-17α.\u003c/p\u003e \u003cp\u003eTyrosine hydroxylase (TH), a tetrahydrobiopterin (BH4)-requiring monooxygenase that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines (CAs), such as dopamine, has been suggested as the enzyme being a source of reactive oxygen species (ROS) in vitro and a target for radical-mediated oxidative injury[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. A translational study highlighted that TLR4-mediated neuroinflammation plays an important role in intestinal and/or brain inflammation, which may be one of the key factors leading to neurodegeneration[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. An animal study also revealed that TLR4 stimulates release of cytokine through NF-kB by activating glial cells, thus resulting in the death of dopaminergic neurons in the MTPT-induced mice[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. One recent study showed that ex vivo expanded Tregs suppressed neuroinflammation, down-regulated the expression of TLR4 and alleviated AD pathology in vivo[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSyt6 (Synaptotagmin 6), one isoform of Synaptotagmins and a brain-specific Ca2+/phospholipid-binding protein, has been shown to be a key component of the secretory machinery involved in acrosomal exocytosis and regulation of membrane trafficking in neurodegeneration[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Its function is seldom investigated in neurological diseases; one recent report suggests that it is a key mediator of endocytic pathways in BDNF release[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. SLC6A3, the gene encoding the dopamine transporter (DAT), is deeply influenced by neuroinflammation in Parkinson\u0026rsquo;s disease[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Our results strongly suggest that rTMS exerts a beneficial effect on the peripheral immune system in PD via Tregs.\u003c/p\u003e \u003cp\u003eNext, to better understand the role of hub node proteins related to the therapeutic effect of rTMS in MPTP mice, we injected RNAi mediated AAV vector into the midbrain to block the hub protein in SN. Among Treg-related downstream targets Syt6, TLR4, TH and Slc6a3, previous studies have shown TLR4, TH and Slc6a3 have been associated with the pathogenesis of PD[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR67\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Using the ZDOCK algorithm method, we found that Syt6 docked with TLR4, which has been reported to be associated with inflammation in SN of MPTP mice in previous studies[\u003cspan additionalcitationids=\"CR68\" citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. We next selected virus interference with syt6 to verify if syt6 is the hub node protein related to the role of Treg in MPTP mice. We found that rTMS treatment did not change the total climbing time of MPTP mice, neither nigral IL-β1, TNF-α and IL-6 levels following syt6 virus interference, further verifying our hypothesis that syt6 participates in the therapeutic effect of rTMS in MPTP mice. To our knowledge, this is the first study directly showing SYT6 is involved in the neuro-pathogenesis of PD, especially modulating Treg-related neuro-inflammation.\u003c/p\u003e \u003cp\u003eProinflammatory mediators and reactive oxygen species (ROS) in the circulation are closely associated with PD severity[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR71\" citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. It is possible that the infiltration of circulating immune cells into the brain leads to extensive BBB breakdown, which in return further exacerbates peripheral inflammatory cytokines crossing the BBB and damaging neurons[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Among immune cells, Tregs play a vital role in regulating inflammatory responses and maintaining homeostasis in the CNS microenvironment[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. One recent clinical trial with long-term sargramostim treatment for PD showed that the drug (a recombinant granulocyte macrophage colony-stimulatory factor) was able to stabilize immune homeostasis, restore peripheral immune function and increase the numbers of Tregs in the circulation[\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Taken together, our findings \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ\u003cb\u003e)\u003c/b\u003e suggest that this restoration of peripheral blood Tregs in PD patients by rTMS stimulation may partially contribute to the improvement in motor dysfunction in PD patients.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eLimitations\u003c/h2\u003e \u003cp\u003eOur data did not examine a prospective temporal relationship between motor improvement and the proportion of Tregs and hence longitudinal studies are needed to determine if the circulating Treg level following rTMS stimulation can be used to monitor the clinical progression in PD. Randomized trials of potent reversible pharmacological inhibitors or agonists of Tregs may help to clarify whether modifying Tregs can delay the progression of sporadic PD in patients.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur clinical and laboratory studies in PD patients and PD animal model suggest that rTMS improved motor function through modulating the function of Tregs and suppressing toxic neuroinflammation. We also identified several hub node proteins (especially Syt6) that may be potential therapeutic targets. Our findings provide pathophysiologic insights into the neuromodulatory effect of rTMS therapy in PD. Strategies to enhance peripheral Tregs and candidate molecular targets should be further explored.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eAAV\u0026nbsp;\u003c/strong\u003e= Adeno-associated virus;\u0026nbsp;\u003cstrong\u003eAD\u0026nbsp;\u003c/strong\u003e=\u0026nbsp;Alzheimer\u0026rsquo;s disease;\u0026nbsp;\u003cstrong\u003eALS\u0026nbsp;\u003c/strong\u003e=\u0026nbsp;amyotrophic lateral sclerosis; \u003cstrong\u003eaTreg\u003c/strong\u003e = activated Treg;\u0026nbsp;\u003cstrong\u003eBBB\u0026nbsp;\u003c/strong\u003e=\u0026nbsp;blood-brain barrier;\u0026nbsp;\u003cstrong\u003eBDNF\u003c/strong\u003e =\u0026nbsp;brain derived neurotrophic factor; \u003cstrong\u003eCNS\u0026nbsp;\u003c/strong\u003e=\u0026nbsp;Central Nervous System; \u003cstrong\u003eDA\u0026nbsp;\u003c/strong\u003e= dopaminergic; \u003cstrong\u003eDAT\u003c/strong\u003e = dopamine transporter ;\u003cstrong\u003eELISA\u003c/strong\u003e=\u0026nbsp;Enzyme-linked immunosorbent assay ; \u003cstrong\u003ePFA\u0026nbsp;\u003c/strong\u003e= 4% paraformaldehyde;\u0026nbsp;\u003cstrong\u003eGDNF\u003c/strong\u003e= glial cell line derived neurotrophic factor; \u003cstrong\u003eH \u0026amp; Y\u0026nbsp;\u003c/strong\u003e=\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eHoehn-Yahr\u003cstrong\u003e; IFN-\u0026gamma;\u0026nbsp;\u003c/strong\u003e= Interferon-\u0026gamma;;\u0026nbsp;\u003cstrong\u003eIL-1\u0026beta;\u0026nbsp;\u003c/strong\u003e=\u0026nbsp;Interleukin-1\u0026beta;;\u0026nbsp;\u003cstrong\u003eIL-10\u0026nbsp;\u003c/strong\u003e=\u0026nbsp;Interleukin-10;\u0026nbsp;\u003cstrong\u003eIL-6\u0026nbsp;\u003c/strong\u003e=\u0026nbsp;Interleukin-6;\u0026nbsp;\u003cstrong\u003eLSD\u0026nbsp;\u003c/strong\u003e=\u0026nbsp;least significant difference;\u0026nbsp;\u003cstrong\u003eM1\u003c/strong\u003e=primary motor cortex;\u0026nbsp;\u003cstrong\u003eMDS\u003c/strong\u003e-\u003cstrong\u003eUPDRS III\u0026nbsp;\u003c/strong\u003e=\u0026nbsp;Movement Disorder Society Unified Parkinson\u0026apos;s Disease Rating Scale part III; \u003cstrong\u003eMFI\u003c/strong\u003e= Median Fluorescence Intensity; \u003cstrong\u003eMPTP\u0026nbsp;\u003c/strong\u003e= 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine; \u003cstrong\u003eNC\u003c/strong\u003e= normal control\u003cstrong\u003e; nTreg\u0026nbsp;\u003c/strong\u003e= naive Treg\u003cstrong\u003e;\u0026nbsp;\u003c/strong\u003e \u003cstrong\u003ePD\u0026nbsp;\u003c/strong\u003e= Parkinson disease\u003cstrong\u003e; PDS\u0026nbsp;\u003c/strong\u003e= Parkinson\u0026rsquo;s Disease Syndrome\u003cstrong\u003e; PDB\u0026nbsp;\u003c/strong\u003e= protein data bank\u003cstrong\u003e;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePPI\u0026nbsp;\u003c/strong\u003e=\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eprotein-protein interaction\u003cstrong\u003e; rTMS\u0026nbsp;\u003c/strong\u003e=\u0026nbsp;repetitive transcranial magnetic stimulation;\u0026nbsp;\u003cstrong\u003eROS\u0026nbsp;\u003c/strong\u003e=\u0026nbsp;reactive oxygen species;\u0026nbsp;\u003cstrong\u003eRMT\u0026nbsp;\u003c/strong\u003e=\u0026nbsp;resting motor threshold\u0026nbsp;; \u003cstrong\u003eRNAi\u003c/strong\u003e= RNA interference \u003cstrong\u003eSNc\u0026nbsp;\u003c/strong\u003e= substantia nigra pars compacta; \u003cstrong\u003eSyt6\u0026nbsp;\u003c/strong\u003e= Synaptotagmin IV; \u003cstrong\u003eSLC6A3\u0026nbsp;\u003c/strong\u003e=\u0026nbsp;Solute carrier family 6 member 3;\u0026nbsp;\u003cstrong\u003eSD\u003c/strong\u003e = Standard Deviation;\u0026nbsp;\u003cstrong\u003eTregs\u0026nbsp;\u003c/strong\u003e= Regulatory T-cells; \u003cstrong\u003eTNF-\u0026alpha;\u0026nbsp;\u003c/strong\u003e=\u0026nbsp;tumor necrosis factor-alpha; \u003cstrong\u003eTGF-\u0026beta;1\u0026nbsp;\u003c/strong\u003e= Transforming growth factor-\u0026beta;1; \u003cstrong\u003eTH\u003c/strong\u003e = tyrosine hydroxylase; \u003cstrong\u003eTMT\u0026nbsp;\u003c/strong\u003e=\u0026nbsp;Tandem Mass Tags; \u003cstrong\u003eTLR4\u003c/strong\u003e = Toll-like Receptor 4.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData and materials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author, upon reasonable request. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConflict of Interests \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe would like to thank Dr. Shengmin Lai, Guangdong Pharmaceutical University to provide valuable comments in the quantitative proteomics analysis. We also would like to thank Drs. Bin Deng, Zifeng Huang and Zhe Zheng from Zhujiang Hospital to assist the immunohistochemistry staining. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNational Natural Science Foundation of China 81873777, 82071414, 82471433(QW)\u003c/p\u003e\n\u003cp\u003eInitiated Foundation of Zhujiang Hospital 02020318005 (QW)\u003c/p\u003e\n\u003cp\u003eScientific Research Foundation of Guangzhou 202206010005 (QW)\u003c/p\u003e\n\u003cp\u003eScience and Technology Program of Guangdong of China 2020A0505100037 (QW)\u003c/p\u003e\n\u003cp\u003eGuangdong Medical Science and Technology Research Foundation Project A2020305(FX)\u003c/p\u003e\n\u003cp\u003eNational Medical Research Council Singapore (EKT)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConceived and designed the study: FX, EKT and Q. W.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePerformed the study: FX, BBS, HZ and Q. W.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRevised the paper for intellectual content: YQL, HZ, SZZ, XBW, ZHC, ZHZ, CHD, CWY, KRC, BL, KLJ, LLC and EKT.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData statistics and analysis: FX, YQL, ZCX and Q. W.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWrote the paper: FX, EKT and Q. W. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTan EK, Chao YX, West A, Chan LL, Poewe W, Jankovic J: \u003cstrong\u003eParkinson disease and the immune system - associations, mechanisms and therapeutics\u003c/strong\u003e. \u003cem\u003eNat Rev Neurol \u003c/em\u003e2020, \u003cstrong\u003e16\u003c/strong\u003e(6):303-318.\u003c/li\u003e\n\u003cli\u003eHirsch EC, Hunot S: \u003cstrong\u003eNeuroinflammation in Parkinson\u0026apos;s disease: a target for neuroprotection?\u003c/strong\u003e \u003cem\u003eLancet Neurol \u003c/em\u003e2009, \u003cstrong\u003e8\u003c/strong\u003e(4):382-397.\u003c/li\u003e\n\u003cli\u003eGeng L, Gao W, Saiyin H, Li Y, Zeng Y, Zhang Z, Li X, Liu Z, Gao Q, An P\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eMLKL deficiency alleviates neuroinflammation and motor deficits in the \u0026alpha;-synuclein transgenic mouse model of Parkinson\u0026apos;s disease\u003c/strong\u003e. \u003cem\u003eMol Neurodegener \u003c/em\u003e2023, \u003cstrong\u003e18\u003c/strong\u003e(1):94.\u003c/li\u003e\n\u003cli\u003eMsackyi M, Chen Y, Tsering W, Wang N, Zhang H: \u003cstrong\u003eDopamine Release Neuroenergetics in Mouse Striatal Slices\u003c/strong\u003e. \u003cem\u003eInt J Mol Sci \u003c/em\u003e2024, \u003cstrong\u003e25\u003c/strong\u003e(9).\u003c/li\u003e\n\u003cli\u003eYang W, Chang Z, Que R, Weng G, Deng B, Wang T, Huang Z, Xie F, Wei X, Yang Q\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eContra-Directional Expression of Plasma Superoxide Dismutase with Lipoprotein Cholesterol and High-Sensitivity C-reactive Protein as Important Markers of Parkinson\u0026apos;s Disease Severity\u003c/strong\u003e. \u003cem\u003eFront Aging Neurosci \u003c/em\u003e2020, \u003cstrong\u003e12\u003c/strong\u003e:53.\u003c/li\u003e\n\u003cli\u003eWang Q, Luo Y, Ray Chaudhuri K, Reynolds R, Tan EK, Pettersson S: \u003cstrong\u003eThe role of gut dysbiosis in Parkinson\u0026apos;s disease: mechanistic insights and therapeutic options\u003c/strong\u003e. \u003cem\u003eBrain \u003c/em\u003e2021, \u003cstrong\u003e144\u003c/strong\u003e(9):2571-2593.\u003c/li\u003e\n\u003cli\u003eQue R, Zheng J, Chang Z, Zhang W, Li H, Xie Z, Huang Z, Wang HT, Xu J, Jin D\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eDl-3-n-Butylphthalide Rescues Dopaminergic Neurons in Parkinson\u0026apos;s Disease Models by Inhibiting the NLRP3 Inflammasome and Ameliorating Mitochondrial Impairment\u003c/strong\u003e. \u003cem\u003eFront Immunol \u003c/em\u003e2021, \u003cstrong\u003e12\u003c/strong\u003e:794770.\u003c/li\u003e\n\u003cli\u003eMarshall LL, Killinger BA, Ensink E, Li P, Li KX, Cui W, Lubben N, Weiland M, Wang X, Gordevicius J\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eEpigenomic analysis of Parkinson\u0026apos;s disease neurons identifies Tet2 loss as neuroprotective\u003c/strong\u003e. \u003cem\u003eNat Neurosci \u003c/em\u003e2020, \u003cstrong\u003e23\u003c/strong\u003e(10):1203-1214.\u003c/li\u003e\n\u003cli\u003eHuang Y, Liu B, Sinha SC, Amin S, Gan L: \u003cstrong\u003eMechanism and therapeutic potential of targeting cGAS-STING signaling in neurological disorders\u003c/strong\u003e. \u003cem\u003eMol Neurodegener \u003c/em\u003e2023, \u003cstrong\u003e18\u003c/strong\u003e(1):79.\u003c/li\u003e\n\u003cli\u003eAlam Q, Alam MZ, Mushtaq G, Damanhouri GA, Rasool M, Kamal MA, Haque A: \u003cstrong\u003eInflammatory Process in Alzheimer\u0026apos;s and Parkinson\u0026apos;s Diseases: Central Role of Cytokines\u003c/strong\u003e. \u003cem\u003eCurr Pharm Des \u003c/em\u003e2016, \u003cstrong\u003e22\u003c/strong\u003e(5):541-548.\u003c/li\u003e\n\u003cli\u003eSchutt CR, Gendelman HE, Mosley RL: \u003cstrong\u003eTolerogenic bone marrow-derived dendritic cells induce neuroprotective regulatory T cells in a model of Parkinson\u0026apos;s disease\u003c/strong\u003e. \u003cem\u003eMol Neurodegener \u003c/em\u003e2018, \u003cstrong\u003e13\u003c/strong\u003e(1):26.\u003c/li\u003e\n\u003cli\u003eBeers DR, Zhao W, Appel SH: \u003cstrong\u003eThe Role of Regulatory T Lymphocytes in Amyotrophic Lateral Sclerosis\u003c/strong\u003e. \u003cem\u003eJAMA Neurol \u003c/em\u003e2018, \u003cstrong\u003e75\u003c/strong\u003e(6):656-658.\u003c/li\u003e\n\u003cli\u003eThome AD, Atassi F, Wang J, Faridar A, Zhao W, Thonhoff JR, Beers DR, Lai EC, Appel SH: \u003cstrong\u003eEx vivo expansion of dysfunctional regulatory T lymphocytes restores suppressive function in Parkinson\u0026apos;s disease\u003c/strong\u003e. \u003cem\u003eNPJ Parkinsons Dis \u003c/em\u003e2021, \u003cstrong\u003e7\u003c/strong\u003e(1):41.\u003c/li\u003e\n\u003cli\u003eHaider A, Elghazawy NH, Dawoud A, Gebhard C, Wichmann T, Sippl W, Hoener M, Arenas E, Liang SH: \u003cstrong\u003eTranslational molecular imaging and drug development in Parkinson\u0026apos;s disease\u003c/strong\u003e. \u003cem\u003eMol Neurodegener \u003c/em\u003e2023, \u003cstrong\u003e18\u003c/strong\u003e(1):11.\u003c/li\u003e\n\u003cli\u003eSheean RK, McKay FC, Cretney E, Bye CR, Perera ND, Tomas D, Weston RA, Scheller KJ, Djouma E, Menon P\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eAssociation of Regulatory T-Cell Expansion With Progression of Amyotrophic Lateral Sclerosis: A Study of Humans and a Transgenic Mouse Model\u003c/strong\u003e. \u003cem\u003eJAMA Neurol \u003c/em\u003e2018, \u003cstrong\u003e75\u003c/strong\u003e(6):681-689.\u003c/li\u003e\n\u003cli\u003eGonzц║lez H, Pacheco R: \u003cstrong\u003eT-cell-mediated regulation of neuroinflammation involved in neurodegenerative diseases\u003c/strong\u003e. \u003cem\u003eJ Neuroinflammation \u003c/em\u003e2014, \u003cstrong\u003e11\u003c/strong\u003e:201.\u003c/li\u003e\n\u003cli\u003eSantamarц╜a-Cadavid M, Rodrц╜guez-Castro E, Rodrц╜guez-Yц║ц╠ez M, Arias-Rivas S, LцЁpez-Dequidt I, Pц╘rez-Mato M, Rodrц╜guez-Pц╘rez M, LцЁpez-Loureiro I, Hervella P, Campos F\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eRegulatory T cells participate in the recovery of ischemic stroke patients\u003c/strong\u003e. \u003cem\u003eBMC Neurol \u003c/em\u003e2020, \u003cstrong\u003e20\u003c/strong\u003e(1):68.\u003c/li\u003e\n\u003cli\u003eWang M, Thomson AW, Yu F, Hazra R, Junagade A, Hu X: \u003cstrong\u003eRegulatory T lymphocytes as a therapy for ischemic stroke\u003c/strong\u003e. \u003cem\u003eSemin Immunopathol \u003c/em\u003e2023, \u003cstrong\u003e45\u003c/strong\u003e(3):329-346.\u003c/li\u003e\n\u003cli\u003eChung ES, Kim H, Lee G, Park S, Kim H, Bae H: \u003cstrong\u003eNeuro-protective effects of bee venom by suppression of neuroinflammatory responses in a mouse model of Parkinson\u0026apos;s disease: role of regulatory T cells\u003c/strong\u003e. \u003cem\u003eBrain Behav Immun \u003c/em\u003e2012, \u003cstrong\u003e26\u003c/strong\u003e(8):1322-1330.\u003c/li\u003e\n\u003cli\u003ePark TY, Jeon J, Lee N, Kim J, Song B, Kim JH, Lee SK, Liu D, Cha Y, Kim M\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eCo-transplantation of autologous T(reg) cells in a cell therapy for Parkinson\u0026apos;s disease\u003c/strong\u003e. \u003cem\u003eNature \u003c/em\u003e2023, \u003cstrong\u003e619\u003c/strong\u003e(7970):606-615.\u003c/li\u003e\n\u003cli\u003ePriori A, Lefaucheur JP: \u003cstrong\u003eChronic epidural motor cortical stimulation for movement disorders\u003c/strong\u003e. \u003cem\u003eLancet Neurol \u003c/em\u003e2007, \u003cstrong\u003e6\u003c/strong\u003e(3):279-286.\u003c/li\u003e\n\u003cli\u003eChou YH, Hickey PT, Sundman M, Song AW, Chen NK: \u003cstrong\u003eEffects of repetitive transcranial magnetic stimulation on motor symptoms in Parkinson disease: a systematic review and meta-analysis\u003c/strong\u003e. \u003cem\u003eJAMA Neurol \u003c/em\u003e2015, \u003cstrong\u003e72\u003c/strong\u003e(4):432-440.\u003c/li\u003e\n\u003cli\u003eChung CL, Mak MK, Hallett M: \u003cstrong\u003eTranscranial Magnetic Stimulation Promotes Gait Training in Parkinson Disease\u003c/strong\u003e. \u003cem\u003eAnn Neurol \u003c/em\u003e2020, \u003cstrong\u003e88\u003c/strong\u003e(5):933-945.\u003c/li\u003e\n\u003cli\u003eLefaucheur JP, Aleman A, Baeken C, Benninger DH, Brunelin J, Di Lazzaro V, Filipović SR, Grefkes C, Hasan A, Hummel FC\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eEvidence-based guidelines on the therapeutic use of repetitive transcranial magnetic stimulation (rTMS): An update (2014-2018)\u003c/strong\u003e. \u003cem\u003eClin Neurophysiol \u003c/em\u003e2020, \u003cstrong\u003e131\u003c/strong\u003e(2):474-528.\u003c/li\u003e\n\u003cli\u003eYang C, Guo Z, Peng H, Xing G, Chen H, McClure MA, He B, He L, Du F, Xiong L\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eRepetitive transcranial magnetic stimulation therapy for motor recovery in Parkinson\u0026apos;s disease: A Meta-analysis\u003c/strong\u003e. \u003cem\u003eBrain Behav \u003c/em\u003e2018, \u003cstrong\u003e8\u003c/strong\u003e(11):e01132.\u003c/li\u003e\n\u003cli\u003eBa M, Ma G, Ren C, Sun X, Kong M: \u003cstrong\u003eRepetitive transcranial magnetic stimulation for treatment of lactacystin-induced Parkinsonian rat model\u003c/strong\u003e. \u003cem\u003eOncotarget \u003c/em\u003e2017, \u003cstrong\u003e8\u003c/strong\u003e(31):50921-50929.\u003c/li\u003e\n\u003cli\u003eYang X, Song L, Liu Z: \u003cstrong\u003eThe effect of repetitive transcranial magnetic stimulation on a model rat of Parkinson\u0026apos;s disease\u003c/strong\u003e. \u003cem\u003eNeuroreport \u003c/em\u003e2010, \u003cstrong\u003e21\u003c/strong\u003e(4):268-272.\u003c/li\u003e\n\u003cli\u003eAftanas LI, Gevorgyan MM, Zhanaeva SY, Dzemidovich SS, Kulikova KI, Al\u0026apos;perina EL, Danilenko KV, Idova GV: \u003cstrong\u003eTherapeutic Effects of Repetitive Transcranial Magnetic Stimulation (rTMS) on Neuroinflammation and Neuroplasticity in Patients with Parkinson\u0026apos;s Disease: a Placebo-Controlled Study\u003c/strong\u003e. \u003cem\u003eBull Exp Biol Med \u003c/em\u003e2018, \u003cstrong\u003e165\u003c/strong\u003e(2):195-199.\u003c/li\u003e\n\u003cli\u003eKustrimovic N, Comi C, Magistrelli L, Rasini E, Legnaro M, Bombelli R, Aleksic I, Blandini F, Minafra B, Riboldazzi G\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eParkinson\u0026apos;s disease patients have a complex phenotypic and functional Th1 bias: cross-sectional studies of CD4+ Th1/Th2/T17 and Treg in drug-na\u0026iuml;ve and drug-treated patients\u003c/strong\u003e. \u003cem\u003eJ Neuroinflammation \u003c/em\u003e2018, \u003cstrong\u003e15\u003c/strong\u003e(1):205.\u003c/li\u003e\n\u003cli\u003eNg J, Barral S, De La Fuente Barrigon C, Lignani G, Erdem FA, Wallings R, Privolizzi R, Rossignoli G, Alrashidi H, Heasman S\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eGene therapy restores dopamine transporter expression and ameliorates pathology in iPSC and mouse models of infantile parkinsonism\u003c/strong\u003e. \u003cem\u003eSci Transl Med \u003c/em\u003e2021, \u003cstrong\u003e13\u003c/strong\u003e(594).\u003c/li\u003e\n\u003cli\u003ePostuma RB, Berg D, Stern M, Poewe W, Olanow CW, Oertel W, Obeso J, Marek K, Litvan I, Lang AE\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eMDS clinical diagnostic criteria for Parkinson\u0026apos;s disease\u003c/strong\u003e. \u003cem\u003eMov Disord \u003c/em\u003e2015, \u003cstrong\u003e30\u003c/strong\u003e(12):1591-1601.\u003c/li\u003e\n\u003cli\u003eGroppa S, Oliviero A, Eisen A, Quartarone A, Cohen LG, Mall V, Kaelin-Lang A, Mima T, Rossi S, Thickbroom GW\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eA practical guide to diagnostic transcranial magnetic stimulation: report of an IFCN committee\u003c/strong\u003e. \u003cem\u003eClin Neurophysiol \u003c/em\u003e2012, \u003cstrong\u003e123\u003c/strong\u003e(5):858-882.\u003c/li\u003e\n\u003cli\u003eYu N, Li X, Song W, Li D, Yu D, Zeng X, Li M, Leng X, Li X: \u003cstrong\u003eCD4(+)CD25 (+)CD127 (low/-) T cells: a more specific Treg population in human peripheral blood\u003c/strong\u003e. \u003cem\u003eInflammation \u003c/em\u003e2012, \u003cstrong\u003e35\u003c/strong\u003e(6):1773-1780.\u003c/li\u003e\n\u003cli\u003eJennum P, Klitgaard H: \u003cstrong\u003eRepetitive transcranial magnetic stimulations of the rat. Effect of acute and chronic stimulations on pentylenetetrazole-induced clonic seizures\u003c/strong\u003e. \u003cem\u003eEpilepsy Res \u003c/em\u003e1996, \u003cstrong\u003e23\u003c/strong\u003e(2):115-122.\u003c/li\u003e\n\u003cli\u003eSun MF, Zhu YL, Zhou ZL, Jia XB, Xu YD, Yang Q, Cui C, Shen YQ: \u003cstrong\u003eNeuroprotective effects of fecal microbiota transplantation on MPTP-induced Parkinson\u0026apos;s disease mice: Gut microbiota, glial reaction and TLR4/TNF-\u0026alpha; signaling pathway\u003c/strong\u003e. \u003cem\u003eBrain Behav Immun \u003c/em\u003e2018, \u003cstrong\u003e70\u003c/strong\u003e:48-60.\u003c/li\u003e\n\u003cli\u003eWaterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eSWISS-MODEL: homology modelling of protein structures and complexes\u003c/strong\u003e. \u003cem\u003eNucleic Acids Res \u003c/em\u003e2018, \u003cstrong\u003e46\u003c/strong\u003e(W1):W296-W303.\u003c/li\u003e\n\u003cli\u003eGuex N, Peitsch MC, Schwede T: \u003cstrong\u003eAutomated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: a historical perspective\u003c/strong\u003e. \u003cem\u003eElectrophoresis \u003c/em\u003e2009, \u003cstrong\u003e30 Suppl 1\u003c/strong\u003e:S162-173.\u003c/li\u003e\n\u003cli\u003eBertoni M, Kiefer F, Biasini M, Bordoli L, Schwede T: \u003cstrong\u003eModeling protein quaternary structure of homo- and hetero-oligomers beyond binary interactions by homology\u003c/strong\u003e. \u003cem\u003eSci Rep \u003c/em\u003e2017, \u003cstrong\u003e7\u003c/strong\u003e(1):10480.\u003c/li\u003e\n\u003cli\u003eBienert S, Waterhouse A, de Beer TA, Tauriello G, Studer G, Bordoli L, Schwede T: \u003cstrong\u003eThe SWISS-MODEL Repository-new features and functionality\u003c/strong\u003e. \u003cem\u003eNucleic Acids Res \u003c/em\u003e2017, \u003cstrong\u003e45\u003c/strong\u003e(D1):D313-D319.\u003c/li\u003e\n\u003cli\u003eStuder G, Rempfer C, Waterhouse AM, Gumienny R, Haas J, Schwede T: \u003cstrong\u003eQMEANDisCo-distance constraints applied on model quality estimation\u003c/strong\u003e. \u003cem\u003eBioinformatics \u003c/em\u003e2020, \u003cstrong\u003e36\u003c/strong\u003e(6):1765-1771.\u003c/li\u003e\n\u003cli\u003ePierce BG, Wiehe K, Hwang H, Kim BH, Vreven T, Weng Z: \u003cstrong\u003eZDOCK server: interactive docking prediction of protein-protein complexes and symmetric multimers\u003c/strong\u003e. \u003cem\u003eBioinformatics \u003c/em\u003e2014, \u003cstrong\u003e30\u003c/strong\u003e(12):1771-1773.\u003c/li\u003e\n\u003cli\u003ePierce BG, Hourai Y, Weng Z: \u003cstrong\u003eAccelerating protein docking in ZDOCK using an advanced 3D convolution library\u003c/strong\u003e. \u003cem\u003ePLoS One \u003c/em\u003e2011, \u003cstrong\u003e6\u003c/strong\u003e(9):e24657.\u003c/li\u003e\n\u003cli\u003eAshburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eGene ontology: tool for the unification of biology. The Gene Ontology Consortium\u003c/strong\u003e. \u003cem\u003eNat Genet \u003c/em\u003e2000, \u003cstrong\u003e25\u003c/strong\u003e(1):25-29.\u003c/li\u003e\n\u003cli\u003eSzklarczyk D, Gable AL, Lyon D, Junge A, Wyder S, Huerta-Cepas J, Simonovic M, Doncheva NT, Morris JH, Bork P\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eSTRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets\u003c/strong\u003e. \u003cem\u003eNucleic Acids Res \u003c/em\u003e2019, \u003cstrong\u003e47\u003c/strong\u003e(D1):D607-D613.\u003c/li\u003e\n\u003cli\u003eWilliams GP, Schonhoff AM, Jurkuvenaite A, Gallups NJ, Standaert DG, Harms AS: \u003cstrong\u003eCD4 T cells mediate brain inflammation and neurodegeneration in a mouse model of Parkinson\u0026apos;s disease\u003c/strong\u003e. \u003cem\u003eBrain \u003c/em\u003e2021, \u003cstrong\u003e144\u003c/strong\u003e(7):2047-2059.\u003c/li\u003e\n\u003cli\u003eJi Y, Yang C, Pang X, Yan Y, Wu Y, Geng Z, Hu W, Hu P, Wu X, Wang K: \u003cstrong\u003eRepetitive transcranial magnetic stimulation in Alzheimer\u0026apos;s disease: effects on neural and synaptic rehabilitation\u003c/strong\u003e. \u003cem\u003eNeural Regen Res \u003c/em\u003e2025, \u003cstrong\u003e20\u003c/strong\u003e(2):326-342.\u003c/li\u003e\n\u003cli\u003eHuang P, Zhu Z, Li W, Zhang R, Chi Y, Gong W: \u003cstrong\u003erTMS improves dysphagia by inhibiting NLRP3 inflammasome activation and caspase-1 dependent pyroptosis in PD mice\u003c/strong\u003e. \u003cem\u003eNPJ Parkinsons Dis \u003c/em\u003e2024, \u003cstrong\u003e10\u003c/strong\u003e(1):156.\u003c/li\u003e\n\u003cli\u003eChen XY, Feng SN, Bao Y, Zhou YX, Ba F: \u003cstrong\u003eIdentification of Clec7a as the therapeutic target of rTMS in alleviating Parkinson\u0026apos;s disease: targeting neuroinflammation\u003c/strong\u003e. \u003cem\u003eBiochim Biophys Acta Mol Basis Dis \u003c/em\u003e2023, \u003cstrong\u003e1869\u003c/strong\u003e(8):166814.\u003c/li\u003e\n\u003cli\u003eSun L, Wang F, Han J, Bai L, Du J: \u003cstrong\u003eRepetitive Transcranial Magnetic Stimulation Reduces Neuronal Loss and Neuroinflammation in Parkinson?s Disease Through the miR-195a-5p/CREB Axis\u003c/strong\u003e. \u003cem\u003eTurk Neurosurg \u003c/em\u003e2023, \u003cstrong\u003e33\u003c/strong\u003e(2):229-237.\u003c/li\u003e\n\u003cli\u003eBrys M, Fox MD, Agarwal S, Biagioni M, Dacpano G, Kumar P, Pirraglia E, Chen R, Wu A, Fernandez H\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eMultifocal repetitive TMS for motor and mood symptoms of Parkinson disease: A randomized trial\u003c/strong\u003e. \u003cem\u003eNeurology \u003c/em\u003e2016, \u003cstrong\u003e87\u003c/strong\u003e(18):1907-1915.\u003c/li\u003e\n\u003cli\u003eReynolds AD, Banerjee R, Liu J, Gendelman HE, Mosley RL: \u003cstrong\u003eNeuroprotective activities of CD4+CD25+ regulatory T cells in an animal model of Parkinson\u0026apos;s disease\u003c/strong\u003e. \u003cem\u003eJ Leukoc Biol \u003c/em\u003e2007, \u003cstrong\u003e82\u003c/strong\u003e(5):1083-1094.\u003c/li\u003e\n\u003cli\u003eTheodore S, Maragos W: \u003cstrong\u003e6-Hydroxydopamine as a tool to understand adaptive immune system-induced dopamine neurodegeneration in Parkinson\u0026apos;s disease\u003c/strong\u003e. \u003cem\u003eImmunopharmacol Immunotoxicol \u003c/em\u003e2015, \u003cstrong\u003e37\u003c/strong\u003e(4):393-399.\u003c/li\u003e\n\u003cli\u003eTian L, Sun SS, Cui LB, Wang SQ, Peng ZW, Tan QR, Hou WG, Cai M: \u003cstrong\u003eRepetitive Transcranial Magnetic Stimulation Elicits Antidepressant- and Anxiolytic-like Effect via Nuclear Factor-E2-related Factor 2-mediated Anti-inflammation Mechanism in Rats\u003c/strong\u003e. \u003cem\u003eNeuroscience \u003c/em\u003e2020, \u003cstrong\u003e429\u003c/strong\u003e:119-133.\u003c/li\u003e\n\u003cli\u003eLenz M, Eichler A, Kruse P, Strehl A, Rodriguez-Rozada S, Goren I, Yogev N, Frank S, Waisman A, Deller T\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eInterleukin 10 Restores Lipopolysaccharide-Induced Alterations in Synaptic Plasticity Probed by Repetitive Magnetic Stimulation\u003c/strong\u003e. \u003cem\u003eFront Immunol \u003c/em\u003e2020, \u003cstrong\u003e11\u003c/strong\u003e:614509.\u003c/li\u003e\n\u003cli\u003eZhao CG, Qin J, Sun W, Ju F, Zhao YL, Wang R, Sun XL, Mou X, Yuan H: \u003cstrong\u003erTMS Regulates the Balance Between Proliferation and Apoptosis of Spinal Cord Derived Neural Stem/Progenitor Cells\u003c/strong\u003e. \u003cem\u003eFront Cell Neurosci \u003c/em\u003e2019, \u003cstrong\u003e13\u003c/strong\u003e:584.\u003c/li\u003e\n\u003cli\u003eLi H, Shang J, Zhang C, Lu R, Chen J, Zhou X: \u003cstrong\u003eRepetitive Transcranial Magnetic Stimulation Alleviates Neurological Deficits After Cerebral Ischemia Through Interaction Between RACK1 and BDNF exon IV by the Phosphorylation-Dependent Factor MeCP2\u003c/strong\u003e. \u003cem\u003eNeurotherapeutics \u003c/em\u003e2020, \u003cstrong\u003e17\u003c/strong\u003e(2):651-663.\u003c/li\u003e\n\u003cli\u003eReynolds AD, Stone DK, Hutter JA, Benner EJ, Mosley RL, Gendelman HE: \u003cstrong\u003eRegulatory T cells attenuate Th17 cell-mediated nigrostriatal dopaminergic neurodegeneration in a model of Parkinson\u0026apos;s disease\u003c/strong\u003e. \u003cem\u003eJ Immunol \u003c/em\u003e2010, \u003cstrong\u003e184\u003c/strong\u003e(5):2261-2271.\u003c/li\u003e\n\u003cli\u003eShi L, Sun Z, Su W, Xu F, Xie D, Zhang Q, Dai X, Iyer K, Hitchens TK, Foley LM\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eTreg cell-derived osteopontin promotes microglia-mediated white matter repair after ischemic stroke\u003c/strong\u003e. \u003cem\u003eImmunity \u003c/em\u003e2021, \u003cstrong\u003e54\u003c/strong\u003e(7):1527-1542 e1528.\u003c/li\u003e\n\u003cli\u003eHaavik J, Toska K: \u003cstrong\u003eTyrosine hydroxylase and Parkinson\u0026apos;s disease\u003c/strong\u003e. \u003cem\u003eMol Neurobiol \u003c/em\u003e1998, \u003cstrong\u003e16\u003c/strong\u003e(3):285-309.\u003c/li\u003e\n\u003cli\u003eZhu M, Huang Y, Wang Z, Jin Z, Cao J, Zhong Q, Xiong Z: \u003cstrong\u003eFecal Microbiota Transplantation Attenuates Frailty via Gut-Muscle Axis in Old Mice\u003c/strong\u003e. \u003cem\u003eAging Dis \u003c/em\u003e2024.\u003c/li\u003e\n\u003cli\u003eYildirim S, Ozkan A, Aytac G, Agar A, Tanriover G: \u003cstrong\u003eRole of melatonin in TLR4-mediated inflammatory pathway in the MTPT-induced mouse model\u003c/strong\u003e. \u003cem\u003eNeurotoxicology \u003c/em\u003e2022, \u003cstrong\u003e88\u003c/strong\u003e:168-177.\u003c/li\u003e\n\u003cli\u003eFaridar A, Vasquez M, Thome AD, Yin Z, Xuan H, Wang JH, Wen S, Li X, Thonhoff JR, Zhao W\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eEx vivo expanded human regulatory T cells modify neuroinflammation in a preclinical model of Alzheimer\u0026apos;s disease\u003c/strong\u003e. \u003cem\u003eActa Neuropathol Commun \u003c/em\u003e2022, \u003cstrong\u003e10\u003c/strong\u003e(1):144.\u003c/li\u003e\n\u003cli\u003eGlavan G, Schliebs R, Zivin M: \u003cstrong\u003eSynaptotagmins in neurodegeneration\u003c/strong\u003e. \u003cem\u003eAnat Rec (Hoboken) \u003c/em\u003e2009, \u003cstrong\u003e292\u003c/strong\u003e(12):1849-1862.\u003c/li\u003e\n\u003cli\u003eXie Y, Zhi K, Meng X: \u003cstrong\u003eEffects and Mechanisms of Synaptotagmin-7 in the Hippocampus on Cognitive Impairment in Aging Mice\u003c/strong\u003e. \u003cem\u003eMol Neurobiol \u003c/em\u003e2021, \u003cstrong\u003e58\u003c/strong\u003e(11):5756-5771.\u003c/li\u003e\n\u003cli\u003eWong YH, Lee CM, Xie W, Cui B, Poo MM: \u003cstrong\u003eActivity-dependent BDNF release via endocytic pathways is regulated by synaptotagmin-6 and complexin\u003c/strong\u003e. \u003cem\u003eProc Natl Acad Sci U S A \u003c/em\u003e2015, \u003cstrong\u003e112\u003c/strong\u003e(32):E4475-4484.\u003c/li\u003e\n\u003cli\u003eMackie PM, Gopinath A, Montas DM, Nielsen A, Smith A, Nolan RA, Runner K, Matt SM, McNamee J, Riklan JE\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eFunctional characterization of the biogenic amine transporters on human macrophages\u003c/strong\u003e. \u003cem\u003eJCI Insight \u003c/em\u003e2022, \u003cstrong\u003e7\u003c/strong\u003e(4).\u003c/li\u003e\n\u003cli\u003eCampolo M, Paterniti I, Siracusa R, Filippone A, Esposito E, Cuzzocrea S: \u003cstrong\u003eTLR4 absence reduces neuroinflammation and inflammasome activation in Parkinson\u0026apos;s diseases in vivo model\u003c/strong\u003e. \u003cem\u003eBrain Behav Immun \u003c/em\u003e2019, \u003cstrong\u003e76\u003c/strong\u003e:236-247.\u003c/li\u003e\n\u003cli\u003eHughes CD, Choi ML, Ryten M, Hopkins L, Drews A, Bot\u0026iacute;a JA, Iljina M, Rodrigues M, Gagliano SA, Gandhi S\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003ePicomolar concentrations of oligomeric alpha-synuclein sensitizes TLR4 to play an initiating role in Parkinson\u0026apos;s disease pathogenesis\u003c/strong\u003e. \u003cem\u003eActa Neuropathol \u003c/em\u003e2019, \u003cstrong\u003e137\u003c/strong\u003e(1):103-120.\u003c/li\u003e\n\u003cli\u003eConte C, Roscini L, Sardella R, Mariucci G, Scorzoni S, Beccari T, Corte L: \u003cstrong\u003eToll Like Receptor 4 Affects the Cerebral Biochemical Changes Induced by MPTP Treatment\u003c/strong\u003e. \u003cem\u003eNeurochem Res \u003c/em\u003e2017, \u003cstrong\u003e42\u003c/strong\u003e(2):493-500.\u003c/li\u003e\n\u003cli\u003eWang T, Yuan F, Chen Z, Zhu S, Chang Z, Yang W, Deng B, Que R, Cao P, Chao Y\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eVascular, inflammatory and metabolic risk factors in relation to dementia in Parkinson\u0026apos;s disease patients with type 2 diabetes mellitus\u003c/strong\u003e. \u003cem\u003eAging (Albany NY) \u003c/em\u003e2020, \u003cstrong\u003e12\u003c/strong\u003e(15):15682-15704.\u003c/li\u003e\n\u003cli\u003eZhu S, Li H, Xu X, Luo Y, Deng B, Guo X, Guo Y, Yang W, Wei X, Wang Q: \u003cstrong\u003eThe Pathogenesis and Treatment of Cardiovascular Autonomic Dysfunction in Parkinson\u0026apos;s Disease: What We Know and Where to Go\u003c/strong\u003e. \u003cem\u003eAging Dis \u003c/em\u003e2021, \u003cstrong\u003e12\u003c/strong\u003e(7):1675-1692.\u003c/li\u003e\n\u003cli\u003eXie Z, Zhang M, Luo Y, Jin D, Guo X, Yang W, Zheng J, Zhang H, Zhang L, Deng C\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eHealthy Human Fecal Microbiota Transplantation into Mice Attenuates MPTP-Induced Neurotoxicity via AMPK/SOD2 Pathway\u003c/strong\u003e. \u003cem\u003eAging Dis \u003c/em\u003e2023, \u003cstrong\u003e14\u003c/strong\u003e(6):2193-2214.\u003c/li\u003e\n\u003cli\u003eOlson KE, Namminga KL, Lu Y, Schwab AD, Thurston MJ, Abdelmoaty MM, Kumar V, Wojtkiewicz M, Obaro H, Santamaria P\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eSafety, tolerability, and immune-biomarker profiling for year-long sargramostim treatment of Parkinson\u0026apos;s disease\u003c/strong\u003e. \u003cem\u003eEBioMedicine \u003c/em\u003e2021, \u003cstrong\u003e67\u003c/strong\u003e:103380.\u003c/li\u003e\n\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":"molecular-neurodegeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mond","sideBox":"Learn more about [Molecular Neurodegeneration](http://molecularneurodegeneration.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mond/default.aspx","title":"Molecular Neurodegeneration","twitterHandle":"@MolNeuro","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Parkinson's disease, repetitive transcranial magnetic stimulation, regulatory T cells, neuro-inflammation, motor dysfunction, syt6","lastPublishedDoi":"10.21203/rs.3.rs-4959031/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4959031/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepetitive transcranial magnetic stimulation (rTMS) has been used to treat various neurological disorders. However, the molecular mechanism underlying the therapeutic effect of rTMS on Parkinson’s disease (PD) has not been fully elucidated. Neuroinflammation like regulatory T-cells (Tregs) appears to be a key modulator of disease progression in PD. If rTMS affects the peripheral Tregs in PD remains unknown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHere, we conducted a prospective clinical study (Chinese ClinicalTrials. gov: ChiCTR 2100051140) involving 54 PD patients who received 10-day rTMS (10 Hz) stimulation on the primary motor cortex (M1) region or sham treatment. Clinical and function assessment as well as flow cytology study were undertaken in 54 PD patients who were consecutively recruited from the department of neurology at Zhujiang hospital between September 2021 and January 2022. Subsequently, we implemented flow cytometry analysis to examine the Tregs population in spleen of MPTP-induced PD mice that received rTMS or sham treatment, along with quantitative proteomic approach reveal novel molecular targets for Parkinson's disease, and finally, the RNA interference method verifies the role of these new molecular targets in the treatment of PD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe demonstrated that a 10-day rTMS treatment on the M1 motor cortex significantly improved motor dysfunction in PD patients. The beneficial effects persisted for up to 40 days, and were associated with an increase in peripheral Tregs. There was a positive correlation between Tregs and motor improvements in PD cases. Similarly, a 10-day rTMS treatment on the brains of MPTP-induced PD mice significantly ameliorated motor symptoms. rTMS reversed the downregulation of circulating Tregs and tyrosine hydroxylase neurons in these mice. It also increased anti-inflammatory mediators, deactivated microglia, and decreased inflammatory cytokines. These effects were blocked by administration of a Treg inhibitor anti-CD25 antibody in MPTP-induced PD mice. Quantitative proteomic analysis identified TLR4, TH, Slc6a3 and especially Syt6 as the hub node proteins related to Tregs and rTMS therapy. Lastly, we validated the role of Treg and rTMS-related protein syt6 in MPTP mice using the virus interference method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur clinical and experimental studies suggest that rTMS improves motor function by modulating the function of Tregs and suppressing toxic neuroinflammation. Hub node proteins (especially Syt6) may be potential therapeutic targets.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial registration: Chinese ClinicalTrials,\u003c/strong\u003e \u003cstrong\u003eChiCTR2100051140. Registered 15 December 2021, https://www.chictr.org.cn/bin/project/edit?pid=133691\u003c/strong\u003e\u003c/p\u003e","manuscriptTitle":"Repetitive transcranial magnetic stimulatation alleviates motor impairment in Parkinson's disease: association with peripheral inflammatory regulatory T-cells and SYT6","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-18 08:53:16","doi":"10.21203/rs.3.rs-4959031/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2024-10-12T20:44:01+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-10-11T12:52:56+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-27T23:00:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-26T09:53:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurodegeneration","date":"2024-09-22T00:46:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurodegeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mond","sideBox":"Learn more about [Molecular Neurodegeneration](http://molecularneurodegeneration.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mond/default.aspx","title":"Molecular Neurodegeneration","twitterHandle":"@MolNeuro","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bb76e943-326f-42bc-b150-5bcfd6b3d72e","owner":[],"postedDate":"October 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-28T16:05:49+00:00","versionOfRecord":{"articleIdentity":"rs-4959031","link":"https://doi.org/10.1186/s13024-024-00770-4","journal":{"identity":"molecular-neurodegeneration","isVorOnly":false,"title":"Molecular Neurodegeneration"},"publishedOn":"2024-10-25 15:58:17","publishedOnDateReadable":"October 25th, 2024"},"versionCreatedAt":"2024-10-18 08:53:16","video":"","vorDoi":"10.1186/s13024-024-00770-4","vorDoiUrl":"https://doi.org/10.1186/s13024-024-00770-4","workflowStages":[]},"version":"v1","identity":"rs-4959031","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4959031","identity":"rs-4959031","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}