Regulation of mitochondrial dynamics and function by MT1 melatonin receptor in Parkinson’s disease

Regulation of mitochondrial dynamics and function by MT1 melatonin receptor in Parkinson’s disease | 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 Regulation of mitochondrial dynamics and function by MT1 melatonin receptor in Parkinson’s disease Xiao-Bo Wang, Li-Li Qi, Jian-Min Wang, Yan-Rui Sun, Qian-Kun Lv, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7058166/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Nov, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 5 You are reading this latest preprint version Abstract Parkinson’s disease (PD) is a neurodegenerative disease characterized by dopaminergic neurons loss and Lewy body presence in the substantia nigra. Abnormal mitochondrial function and accumulated alpha-synuclein (α-syn) are key etiology of PD. Melatonin type receptor 1 (MT1) regulates sleep upon activation by melatonin and is suggested to decrease in PD patients. However, the role of MT1 in PD pathogenesis remains elusive. In this study, we knocked down MT1 in SH-SY5Y neuroblastoma cells and found MT1 loss caused mitochondria dysfunction. Moreover, live cell imaging of MitoTracker staining and transmission electron microscope (TEM) proved that MT1 knockdown affected mitochondria morphology. The expression of mitochondria fission protein DRP1 was increased and the fusion protein OPA1, MFN1 and MFN2 was decreased. This is probably attributed to the declined phosphorylation of DRP1 at S637 by PKA and increased phosphorylation at S616 by ERK1/2. Moreover, MT1 knockdown also impaired mitophagy, manifested by declined PINK1 and Parkin. In a MPTP induced PD mouse model, MT1 deficiency altered the mitochondria fission through the same mechanism as in vitro but did not impair mitophagy, tyrosine hydroxylase (TH) expression and mice movement. However, MPTP induced autophagy inhibition was exacerbated in MT1 KO mice. Neuronal MT1 deficiency aggravated preformed fibrils (PFFs) induced autophagy inhibition and α-syn aggregation. Overexpression of MT1 reduced mitochondria fission, as well as increased LC3-II expression and decreased P62 accumulation to promote autophagy in HEK293T cells, thus mitigating the aggregation of α-syn. Autophagy flux indicated by mCherry-LC3-II-EGFP fluorescence was also enhanced after MT1 overexpression. Together, our study demonstrates the function of MT1 in mitochondria and autophagy, which sheds further light on PD prevention targeting MT1. melatonin receptor MT1 mitochondria dynamics MPTP Parkinson’s disease α-synuclein autophagy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction Parkinson’s disease (PD) is a common neurodegeneration with motor disorder, such as bradykinesia, tremor, rigidity and postural instability [ 1 ]. The pathological characteristics of PD are the loss of dopaminergic neurons in substantial nigra, descending expression of tyrosine hydroxylase (TH) and aggregation of misfolded alpha-synuclein (α-syn) in neurons [ 2 ]. Multiple mechanisms like autophagy, apoptosis, inflammation, and mitochondria dysfunction are involved in PD pathogenesis. Mitochondria dysfunction has long been believed by researchers to be a critical player to induce neuronal death in PD development [ 3 , 4 ]. PD patients affected by environmental factors accounted for more than 90% of total patients and the exposure to pesticides like MPTP, rotenone and paraquat has been applied to establish PD-related models which can mimic key features of PD [ 5 ]. Importantly, these neurotoxins all targeted mitochondria to cause cell death in which the mitochondrial complex I activity was inhibited, or oxidative stress was elevated. Mitochondria dynamics consecutively played a role in alleviating mitochondria damage by fusion or disposing disrupted mitochondria via fission. Disequilibrium of mitochondria dynamics especially the excessive fission can cause mitochondria defects, which is implicated in PD etiology [ 6 ]. Mitophagy selectively removes the dysfunctional mitochondria through autophagy to prevent deleterious effects like oxidative stress within the cell. The mutation of two essential mitophagy regulators PTEN-induced kinase 1 (PINK1) and Parkin is attributed to familial PD. Impaired mitophagy was observed in both genetic mutational and toxic PD models [ 7 , 8 ]. Therefore, maintaining mitochondria homeostasis to restore its function and enhance mitophagy to proceed with the elimination of damaged mitochondria is continually to be an effective strategy to prevent PD. α-Syn is an cytosolic protein, which locates at the end of the axon and is involved in the transport of vesicles in neurotransmitters releasing [ 9 ]. When the α-syn encoding gene, SNCA mutated or the α-syn protein misfolded, it will lead to the phosphorylation of α-syn at serine 129 site (pS129-α-syn) and formation of protein aggregates [ 10 ]. α-syn deposition not only loses the physiological function of α-syn, but also causes deteriorated intracellular response like mitochondria damage, thus eventually leading to cell death [ 11 ]. Studies revealed one of the pathogenic mechanism of PD was via the binding of α-syn with TOM20 to inhibit mitochondrial protein import [ 12 ]. Moreover, PD induced lysosomal and mitochondrial aberration also accelerates α-synuclein aggregation [ 13 ]. These properties of α-syn make it the most promising therapeutic target aimed at preventing α-syn dysfunction and subsequent protein aggregation primarily via mitochondria or autophagy-lysosome pathways. The dopamine D2 and D3 receptors reduce α-syn accumulation through inducing a BECN1-dependent autophagy activation to protect against PD [ 14 ]. Strategies to improve mitochondria function also exhibit the effects to suppress toxic α-syn accumulations [ 15 ]. Despite motor symptoms, PD patients also exhibited non-motor symptoms like sleep disorders, anxiety, pain, and depression [ 16 ]. Sleep disorder, the main prodromal symptoms of PD, was manifested as daytime sleepiness, insomnia, and rapid eye movement (REM) sleep behavior disorder (RBD) [ 17 ]. Melatonin, a natural hormone secreted by the pineal gland, can be chemically synthesized to treat sleep disorders in PD patients [ 18 ], and protect motor and pathological deficits via ameliorating neuronal loss, inflammation, mitochondria defects, and autophagy suppression in PD models [ 19 – 22 ]. For instance, melatonin protects against defects of mitochondrial respiration and ATP levels in chronic Parkinsonian mice model [ 20 ]. Melatonin rescues rotenone-induced apoptosis or MPTP-induced neurotoxicity via mediating autophagy pathway [ 21 ]. Although a preliminary study of melatonin in mitochondrial dynamics and autophagy is also implicated in MPTP treated zebrafish [ 23 ], the underlying mechanism is still not fully understood. The action of melatonin usually works through binding with a seven transmembrane G protein-coupled receptor (GPCR) on plasma membrane to activate downstream signaling despite a receptor-independent mechanism existing [ 24 ]. Two melatonin receptors, melatonin type 1 receptor (MT1) and melatonin type 2 receptor (MT2) were identified to regulate REM and non-rapid eye movement (NREM) sleep, respectively [ 25 ]. Melatonin receptor agonists, like ramelteon and agomelatine, are applied in clinical to treat sleep disorders or sleep disturbed psychiatric diseases [ 26 ]. MT1 is also reported to located on mitochondrion both in vivo and in vitro [ 27 ]. It displays a pleiotropic effect in stabilizing circadian rhythm [ 28 ], sleep [ 29 , 30 ], neuroprotection [ 27 , 31 – 33 ], antioxidative ability [ 32 , 33 ], cell survival [ 31 , 34 ], and anti-inflammation [ 35 ]. The distribution of MT1 is abundance in central nervous system, especially retina, suprachiasmatic nucleus (SCN), striatum and substantial nigra [ 36 ], and the mRNA levels of MT1 is reduced in substantial nigra of PD patients [ 37 ], which implicated a correlation of MT1 with PD. In our group, we have demonstrated the positive effects of MT1 in microglial inflammation in MPTP-induced models [ 35 ], ferroptosis and microglial phagocytosis in PFFs-induced models [ 31 , 38 ], and sleep-wake disorders in rotenone-induced models of PD [ 30 ], but how MT1 is involved in PD pathogenesis through manipulating mitochondria property and autophagy is under determined. In the present study, we determine the role of MT1 in mitochondria dynamics and mitophagy in PD, which further links to α-syn accumulation through autophagy. We identified that knockdown of MT1 causes mitochondria dysfunction, mitochondria fission and mitophagy in vitro. Loss of MT1 exacerbated mitochondria fission without influenced mitophagy, TH expression and mice movement in MPTP induced model in vivo. The increased fission may be caused by inactivation of PKA on S637-DRP1 phosphorylation and activation of ERK1/2 on S616-DRP1. Moreover, MT1 knockout exacerbated PFF-induced autophagy and potentiated α-syn aggregation in mouse primary neurons. Overexpression of MT1 reduced mitochondria fission, promoted autophagy and mitigated α-syn aggregation in HEK293T cells. In this study, we evaluated the function of MT1 in mitochondria function and autophagy regulation, which provided a potential mechanism to understand the mitochondria fragmentation and α-syn clearance in PD through MT1. 2 Materials and Methods 2.1 Animal experiments Transgenic Mtnr1a knockout (MT1-KO) mice in a C57BL/6JGpt background were purchased from GemPharmatech (China), which has been used previously [ 31 , 38 ]. Briefly, the CRISPR/Cas9 system was employed to generate the MT1 KO mice model through the deletion of exon 2 of transcript Mtnr1a-201(ENSMUST00000067984.8). The single guide RNA was designed to direct the Cas9 endonuclease to create double strand breaks at the sites of intron 1–2 and downstream of exon 2 within the MT1 gene. The sg RNA transcripts constructed in vitro were microinjected into fertilized eggs of mice together with Cas9. The fertilized eggs were then implanted to produce F0 generation mice with the desired genetic modification, which was confirmed via PCR and sequencing. Positive F0 mice were mated with C57BL/6JGpt mice to establish a stable F1 generation mouse line. Genotyping was performed by the following primers to detect wild-type (WT) MT1 and MT1-KO alleles: WT-Forword, 5’-GCTCACTTGACTCTAGGAGGGAGAC-3’; WT-Reverse, 5’-CCTGGGATAACATACAGCCAGC-3’; KO-Forword, 5’-AACTTGAAGCTCTTCAGGGTTGC-3’; KO-Reverse, 5’-CATGGTCCCTCTTGTCTTGAGTTC-3’. Homozygotes wild type (WT) littermates after cross were used as control. Mice were maintained under specific pathogen-free conditions in a 12-h light/dark cycle with unlimited water and food available. All experiments involving mice were approved by the Institutional Animal Care and Use Committee of Soochow University. Mouse models induced by MPTP were divided into four groups randomly: (1) WT + Saline group; (2) WT + MPTP group; (3) MT1-KO + Saline group; (4) MT1-KO + MPTP group. Mice in the group (2) and (4) were intraperitoneally injected with MPTP four times (first time at Zeitgeber time(ZT)0: 14 mg/kg, second time at ZT2: 16 mg/kg, third time at ZT4: 18 mg/kg and last time at ZT6: 20 mg/kg) a day with 2 h intervals for seven consecutive days to establish an acute PD model. The 4-month-old mice from group (1) and (3) received an equal volume of saline. The sample was collected at ZT6 from mice after one day of injection. 2.2 Cell culture HEK293T cells, a human embryonic kidney cell line, were maintained in Dulbecco’s modified Eagle’s medium (DMEM, 12430112, Gibco, USA) with 10% fetal bovine serum (30067334, Gibco, USA), and 1% penicillin/streptomycin antibiotics (15140122, Gibco, USA). Neuroblastoma SH-SY5Y cells were cultured in DMEM/F-12 medium supplemented with 10% fetal bovine serum, and 100 µg/ml penicillin/streptomycin at 37°C in 5% CO 2 . WT-GFP Control (TTCTCCGAACGTGTCACGT, Genechem, China) and MT1-KD-GFP (human MTNR1A-RNAi, CAGTTACTACATGGCGTAT, Genechem, China) lentivirus were used to establish stable SH-SY5Y cell lines, respectively, and single cell clone with GFP was selected by 400 µg/ml puromycin. Mouse primary cortical neurons were prepared from P1–P3 neonatal MT1-KO pups or control littermates. Cells were seeded in Poly-D-Lysine (A3890401, Gibco, USA) coated 6-well plate or 24-well plate with coverslips and cultured in a complete neurobasal-A medium (10888022, Gibco, USA) supplemented with 2% serum free B27(17504044, Gibco, USA), 1% GlutaMAX (35050061, Gibco, USA) and 1% penicillin/streptomycin. Culture medium was changed every 3 days until experiments were performed. MPP + and PFFs were added into primary neurons in a concentration of 100 µM for 6h and 1 µg/mL for 10 days, respectively. 2.3 Mitochondria membrane potential (MMP, ΔΨ) detection MMP was measured by the red-fluorescent probe tetramethyl rhodamine ethyl ester (TMRE) (Beyotime, China) in accordance with manufacturer’s protocol. TMRE was accumulated within mitochondrion but released outside as mitochondrial membrane depolarized. SH-SY5Y cells were incubated with 1×TMRE dye at 37°C for 15 min and Hoechst 33342 (Beyotime, China) for 5 min. Cells were then washed twice with prewarmed culture medium, and images were obtained by inverted fluorescent microscope (Carl Zeiss, Germany). Identical exposure times were used to take each image. Mitochondrial fluorescence intensity (OD value) was calculated by ImageJ software. 2.4 Relative oxidative species (ROS) detection SH-SY5Y cells were treated with 5µM MT1 antagonist S26131 (MedChemExpress, USA) for 24 h and subjected to ROS evaluation according to manufacturer’s protocol (Beyotime, China). DCFH-DA is a probe without fluorescence and can be loaded in cells when hydrolyzed to DCFH. Oxidized DCFH by cytosolic ROS is then converted into DCF with green fluorescence. SH-SY5Y cells were incubated with 10 nm/mL DCFH-DA at 37°C for 30 min and washed three times with culture medium without FBS to remove spare dye. The ROS fluorescent intensity was quantitated by FACScan flow cytometer (BD Biosciences, USA). 2.5 ATP determination Cellular ATP levels were measured by a commercial ATP detection kit (Beyotime, China) according to the manufacturer’s protocol. Briefly, ATP was extracted by ATP lysis buffer and centrifuged to obtain supernatant. 20 µl of supernatant or gradient ATP standard solution was mixed with 100µl of ATP working solution in 96-well plate. ATP contents were determined by a luminometer (Infinite M200, Tecan, Switzerland) and normalized with protein concentration in the supernatant. 2.6 MitoTracker Staining and Transmission electron mitochondria (TEM) WT and MT1-KD SH-SY5Y cells were cultured on 35 mm confocal dishes with glass on the bottom (NEST, China) and incubated with 100 nM Mito-Tracker Red CMXRos for 15 min at 37°C according to instructions (Beyotime, China). Images were obtained from LSM 700 confocal microscope (Carl Zeiss, Germany). The mitochondria morphology parameters were analyzed to calculate the value of form factor (perimeter 2 /4π·area) and aspect ratio (the ratio between the major and minor axis of the ellipse equivalent to the mitochondrion) as previous protocol [ 39 ]. Cells and mouse substantial nigra were obtained and preserved by using electron microscope fixative solution (G1102, Servicebio, China) in foil at 4°C. The samples were then sent to company (Servicebio, China) on ice for detection. The mitochondria morphology parameters from TEM were analyzed as indicated methods [ 40 ]. 2.7 Quantification real-time PCR TRIzol reagent (Invitrogen, USA) was used to isolate total RNA from WT-GFP, or MT1-KO-GFP stable SH-SY5Y cell lines and the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, USA) was applied to synthesized cDNA from RNA. Quantitative PCR was performed with SYBR-Green Master Mix (Vazyme, China) and measured by 7500 Real-Time PCR System (Applied Biosystems, USA). qPCR primers were obtained from GENEWIZ (China). Human MT1 Forward primer, 5’-AGGGACTCTCCAGTACGACC-3’ and Reverse primer 5’-GTTTGCGGTCAGGTTTCACC-3’; 18S Forword primer 5’-TCAACACGGGAAACCTCAC-3’ and reverse primer 5’-CGCTCCACCAACTAAGAAC-3’. mRNA levels were normalized to 18S RNA and calculated by 2ΔΔCt method. 2.8 Immunostaining Cells cultured on coverslips were washed in PBS and fixed with 4% paraformaldehyde for 30 min. After permeabilizing with 0.25% Triton X-100 for 30 min and blocking with 5% BSA for 1 h, cells were incubated with anti-Tom20 (1:250, Beyotime, China), anti-Flag (1:1000, ab1257, Abcam, USA), anti-pS129-α-syn (1:500, ab51253, Abcam, USA), anti-Tuj1 (1:1000, ab78078, Abcam) and anti-LC3 (1:200, NB100-2220, Nouvs, USA) antibodies overnight at 4°C. The secondary antibody Alexa Fluor 637 (A-21447, Thermo Fisher, USA), Alexa Fluor 488 (A28175, Thermo Fisher, USA), and Alexa Fluor 555 (A27039, Thermo Fisher, USA) was added at room temperature for 1 h. Cells were sealed with mounting medium containing DAPI (H-1200, Vector Laboratories, USA) and then observed under confocal microscope (LSM700, Carl Zeiss, Germany). 2.9 Western blot Protein from cells and tissues was extracted by strong RIPA buffer (Beyotime, China) with protease/phosphatase inhibitor (NCM, China) and sonicated in an amplitude of 20% and 30%, respectively by ultrasonic processor (SONICS, USA). The supernatants were harvested after centrifuge and the equal protein loading was determined by BCA assay kit (23227, Thermo Scientific, USA). Equal amounts of proteins in 5×Loading buffer were separated by 10% SDS-PAGE gels (NCM, China) and transferred onto a polyvinylidene fluoride membrane (Millipore, USA). Membranes were incubated with the following primary antibodies overnight at 4°C after blocking with 5% BSA: anti-β-tubulin (T0198, Sigma, USA), anti-MT1 (MTNR1A, A13030, Abclonal, USA), anti-OPA1 (ab42364, Abcam, USA), anti-Mfn1 (ab104274, Abcam, USA), anti-mfn2 (ab56889, Abcam, USA), anti-Drp1 (ab56788, Abcam, USA), anti-S637-Drp1 (AF5791, Beyotime, China), anti-S616-Drp1 (3455S, CST, USA), anti-ERK1/2 (4695S, CST, USA), anti-pERK1/2 (4370S, CST, USA), anti-PINK1 (AF7755, Beyotime, China), anti-Parkin (ab15494, Abcam, USA), anti-phosphorylated-PKA (AF1942, Beyotime, China) and anti-TH (ab193083, Abcam, USA), anti-Flag-HRP (A8592, Sigma, USA), anti-α-syn (ab1903, Abcam, USA), anti-Actin-HRP (A3854, Sigma, USA), anti-LC3 (NB100-2220, Novus, USA), anti-P62 (P0068, Sigma, USA), anti-GFP (ab290, Abcam, USA), anti-MT2 (MTNR1B, NLS932SS, Novus, USA). After incubation with HRP-conjugated anti-rabbit/mouse IgG at room temperature for 1 hour, the blots were visualized (Clinx ChemiCapture, China) by ECL detection kits (P2300, NCM, China). To quantify the content of insoluble α-syn, the lysate was centrifuged at 25,000 rpm for 30 min. Insoluble pellets were dissolved in 1 × TBS with 2% SDS and subjected to sonication. The gray value of WB bands was measured via ImageJ software (National Institutes of Health, USA) after normalization with a corresponding control. 2.10 Plasmid transfection and virus infection Human MT1(hMT1)-Flag plasmids and α-syn GFP plasmids were constructed on PCDNA3.1 from Genewiz. JetPRIME (Polyplus, France) was used to transfected plasmids into HEK293T cells according to the manufacturer’s protocol. HEK293T cells were infected with mCherry-LC3-EGFP lentivirus (HANBIO, China) in a 2.5 MOI (multiplicity of infection) for 36 h. Lysosome inhibitor, 200nm bafilomycin A1 (BafA1, A417398, Sangon Biotech, China) was added 16 h before detection. 2.11 Performed Fibrils (PFFs) preparation α-syn-PFFs was prepared according to a general protocol for primary neuronal cultures[ 31 ]. Briefly, aliquots of 5 mg/mL PFFs were stored at − 80°C and thawed at 37°C water bath before use. The PFFs were diluted into 0.5 mg/mL working solution by sterilized PBS and then sonicated for 1min with 1s intervals (total 30 times) in a 20% amplitude (SONICS, USA) to create fragmented PFFs. Neuron culture medium was added with PFFs and subjected to filtration with 0.22 µm mesh. 2.12 Rotarod test The motor function of the mice was assessed through applying the Rotarod system (ZH-300, Zhenghua Co. Ltd., China). Prior to the formal testing, the mice were trained to ensure familiarity with the task. The training was conducted on three consecutive days with three sessions per day at a fixed time from ZT6. During the test, the rotation speed was gradually raised to 15 rpm in a session. Each mouse was subjected to three trials at 30-minute intervals from ZT6, and the mean latency to fall was calculated as the final result. 2.13 Statistical analysis All the data from at least three independent experiments are presented as mean ± SEM. Statistics were performed using GraphPad Prism 9.5 software. The significance of difference was determined by unpaired t-test between two groups or One-way analysis of variance (ANOVA) for multiple-groups, while nonparametric tests were used for data with n = 3. P value < 0.05 was considered statistically significant. 3 Results 3.1 MT1-KD caused mitochondrial dysfunction in SH-SY5Y cells To observe MT1 function in vitro, we primarily established stable SH-SY5Y cell lines using WT-GFP and MT1-KD-GFP lentivirus. MT1 knockdown efficiency was validated by protein and mRNA protein levels (Fig. 1 A-C), without impacting MT2 expression (Figure S1A, B). Previous study has documented the increased ROS and declined MMP in MT1-KD retinal pigmental epithelium cells [ 41 ]. These results were also verified in our SH-SY5Y stable cell lines. We performed similar experiments and found that MT1-KD decreased MMP and cellular ATP levels (Fig. 1 D-F). To detect ROS with green fluorescent DCFH-DA probe, efficient MT1 antagonist S26131 was used to avoid GFP disturbance [ 42 ]. SH-SY5Y cells treated with S26131 intensified ROS production (Fig. 1 G). Therefore, MT1 deficiency resulted in mitochondria damage in SH-SY5Y cells. 3.2 MT1-KD impaired mitochondrial morphology in SH-SY5Y cells Alteration in mitochondrial morphology is the main aspect of its malfunction. By taking high resolution images of stained mitochondria under 100 × lens, we found that compared with WT control, MT1-KD cells exhibited more fragmented mitochondria networks and less branches (Fig. 2 A). The degree of mitochondrial branching indicated by form factor and length indicated by aspect ratio were all decreased as MT1 knocked down (Fig. 2 C, D). TEM results also corroborated the findings that MT1 knockdown decreased mitochondrial length (Fig. 2 B, E). The mitochondrion shape observed in MT1-KD SHSY-5Y cells was shorter and rounder. Interestingly, we also noted that the contacts between mitochondria and endoplasmic reticulum (ER) were decreased, implying a deterioration of the organelle function (Fig. 2 B, F). 3.3 MT1-KD increased mitochondria fission via DRP1 phosphorylation and hampered mitophagy in SH-SY5Y cells To investigate the underlying mechanisms of impaired mitochondria dysfunction and morphology, we detected the alteration of mitochondrial dynamic and mitophagy proteins. As expected, the expression of mitochondrial fusion proteins, OPA1, MFN1, and MFN2 were downregulated, and the expression of mitochondrial fission proteins DRP1 was upregulated in MT1-KD cells relative to WT control (Fig. 3 A-E). Generally, potentiated mitochondria fission may be caused by decreased DRP1 phosphorylation at S637 or increased DRP1 phosphorylation at S616. We found that MT1-KD reduced DRP1-S637 phosphorylation and increased DRP1-S616 phosphorylation (Fig. 3 F, G, I). Moreover, MT1-KD also dephosphorylated PKA, the regulator of DRP1-S637 but elevated ERK1/2 phosphorylation, which served as an upstream effector of DRP1-S616 (Fig. 3 F, H, J). The recovery of S616-DRP1 level treated with ERK1/2 inhibitor SCH772984 in MT1-KD group was identified (Figure S2A, B). The key proteins of mitophagy, PINK1 and Parkin were also impaired by MT1 knockdown (Fig. 3 K, L, M). The ascending ratio of LC3 II/I and accumulated P62 further proved the mitophagy inhibition (Fig. 3 K, N, O). Together, these results suggested MT1 deficiency caused DRP1-S637 and DRP1-S616 induced mitochondria fragmentation, and mitophagy in vitro. 3.4 MT1-KO aggravated mitochondria fission in mouse primary neurons treated with MPP+ To study in vivo, we assessed the mitochondria morphology in mice substantial nigra by TEM. The WT and MT1-KO mice were bred in a stable population from our group, and the genotypes of which has been substantiated before use [ 31 , 38 ]. In SCN where MT1 is most abundantly expressed, MT1-KO mice displayed a critical reduction of MT1 mRNA level (Figure S1C). Here, we found that both aging and MT1-KO decreased mitochondria length compared with WT mice at 4 months (Fig. 4 A, B). MT1-KO exacerbated the mitochondria length reduction induced by aging. Next, we employed MT1 expressed mouse primary cortical neurons to observe morphology of mitochondrion with Tom20 staining, in which MT2 expression was negligible (Figure S1D). Mitochondria in MT1-KO mouse primary neuronal axons also showed an apparent fragmentation relative to WT control (Fig. 4 A, B). The 1-methyl-4-phenyl pyridinium (MPP+) is the neurotoxic metabolite of MPTP. MT1-KO neurons treatment with MPP + decreased mitochondrial length compared to MT1-KO or MPP + alone group (Fig. 4 C, D). This result implied that MT1 and MPP + may work reciprocally on mitochondria morphology. 3.5 MT1-KO aggravated mitochondria fission through DRP1 phosphorylation and mitophagy in MPTP induced mouse model To further evaluate the MT1 effects on mitochondrial function in PD in vivo, we treated MT1-KO transgenic mice with or without neurotoxin MPTP. Protein from mice striatum was collected in ZT6 for subsequent experiments. Western blot results showed that not only MT1-KO but also MPTP treatment upregulated the expression of mitochondria fusion proteins (OPA1, MFN1, and MFN2) while downregulated mitochondria fission protein DRP1 (Fig. 5 A-E). Additionally, MT1-KO mice with MPTP exhibited a more profound impact on mitochondria fusion and fission protein levels than MT1-KO or MPTP groups individually (Fig. 5 A-E). Reduced DRP1 phosphorylation at S637 but increased at S616 was induced by MT1 deficiency or MPTP, which was further exacerbated in MT1-KO mice in combination with MPTP (Fig. 5 F, G, I). Consistent with the findings above, PKA was inactivated, and ERK1/2 was activated in accordance with the S637-DRP1 dephosphorylation and S616-DRP1 phosphorylation, respectively (Fig. 5 F, H, J). However, the PD marker TH expression (Fig. 5 K, L) and mouse motor ability (Fig. 5 M) were attenuated in MPTP mice but not in MT1-KO mice. PINK1 and Parkin alteration was also not observed in MT1-KO mice which may be due to the complicated in vivo environment (data not shown). Interestingly, we observed compromised autophagy after MT1 deletion, which was aggravated in MPTP mouse (Fig. 5 N-P). These results suggest that the loss of MT1 induced mitochondria fission may increase the susceptibility of PD occurrence but is not the key etiological factor. 3.6 MT1 deficiency aggravated autophagy inhibition induced by α-syn PFFs In PD, autophagy is the canonic degradation pathway of pathological α-syn clearance. To better assess MT1 functions on autophagy in α-synucleinopathy PD models, we used α-syn PFFs induced neuronal model to detect autophagy alteration, soluble and insoluble α-syn accumulation using WB. α-syn PFFs are purified misfolded α-syn aggregates, which are prion-like, self-templating and transmittable. PFFs can efficiently induce the production of α-syn aggregates, which occur within 6 days and last until 12 days [ 43 ]. After induction by PFFs for 10 days in vitro, it increased autophagy-related indicators, P62 accumulation and the ratio of LC3-II/I, indicating the autophagy inhibition by PFFs in neurons. In addition, MT1-KO exacerbated this suppression of autophagy induced by PFFs. However, the impaired autophagy in MT1-KO group was milder than PFF-induced group (Fig. 6 A, D, E). In addition, the formation α-syn significantly accelerated after PFFs induction, and MT1-KO augment PFFs induced α-syn accumulation in both forms of monomers and oligomers (Fig. 6 B, C, F, G). Moreover, we validated the aggravated pS129-α-syn expression induced by α-syn PFFs in MT1-KO mice (data not shown), which has been conducted in our group [ 31 ]. 3.7 MT1 overexpression increased mitochondria fusion and facilitated autophagy flux to promote α-syn degradation in HEK293T cells To observe the protective effects of MT1 on mitochondria and autophagy, we overexpressed hMT1-Flag in HEK293T cells for 48 h and detected the levels of related proteins. HEK293T cells exhibited a much higher transfect efficiency than SH-SY5Y cells. Western blot results showed that MT1 reduced mitochondria fission as indicated by DRP1 decline, as well as MFN1 and MFN2 increase (Fig. 7 A-D). In addition, MT1 significantly augmented the LC3-II level but reduced the level of P62 compared with PCDNA3.1 controls (Fig. 7 E-G). MT1 induced LC3-II level was further elevated in the presence of lysosome inhibitor BafA1, implying the enhancement of autophagic flux by MT1 overexpression (Fig. 7 H, I). The autophagic flux was then visually monitored by the expression of mCherry-LC3B-EGFP under confocal microscope. EGFP fluorescence quenches at the acidic condition when fused with the lysosome lumen. However, mCherry is relatively stable and keeps on fluorescing red without pH perturbation. Therefore, the yellow dots represented by colocalization of EGFP and mCherry signals implies the autophagosomes that are not fused with lysosome, whereas red dots represented by mCherry-only signals implies the autolysosomes that interprets autophagic flux results [ 14 ]. In our study, MT1 overexpression considerably boosted the number of autolysosomes per cell compared with control (Fig. 7 J, L), and slightly increased the number of autophagosomes per cell relative to control (Fig. 7 J, K). In addition, the number of autolysosomes were reduced and the autophagosomes failed to fuse with lysosomes were increased after BafA1 treatment in Flag-hMT1 overexpressed HEK293T cells (Fig. 7 J-L), which further validates the conclusion that MT1 positively regulates autophagy flux. To determine whether MT1 induced autophagy is helpful for α-syn degradation, we overexpressed hMT1-Flag for 48h and α-syn-GFP for 24h in HEK293T cells. The results showed that the expression of α-syn was decreased along with P62 reduction and LC3 II/LC3 I ratio ascendance (Fig. 7 M-P). These results suggest that MT1 can proceed α-syn degradation through autophagy. 4 Discussion Current evidence in understanding the role of MT1 associated with mitochondria and autophagy in PD is limited. In this study, we worked on MT1-KD SH-SY5Y cells and MT1-KO transgenic mice to explore its function and relationship with PD. Inhibition of MT1 blunted mitochondria function, manifested as decreased MMP and ATP, as well as ROS generation. Impaired mitochondrial morphology was observed due to the upregulation of mitochondria fission proteins and downregulation of mitochondria fusion proteins. Mechanistically, PKA signaling was inactivated to arrest DRP1 phosphorylation at S637 and ERK signaling was activated to promote DRP1 phosphorylation at S616. In vivo, MT1-KO aggravated the mitochondria fragmentation in MPTP induced PD mouse model under the same mechanism. However, PD related symptoms like TH expression and mice motor ability were not influenced by MT1 knockout. Moreover, loss of MT1 exacerbated α-syn accumulation induced by PFF due to the compromised autophagy. By overexpressing MT1, we found autophagy flux was elevated and α-syn was degraded. The study declared the impairment of mitochondria quality control and autophagy without MT1 and its relevance with PD. Mitochondria homeostasis in keeping its proper function is vital in cell survival and PD. Multifaceted mitochondria activities, such as biogenesis, dynamics, trafficking or mitophagy have impact on mitochondria function [ 44 ]. In line with previous results reported that MT1 knockdown increased ROS and decreased MMP, we confirmed the mitochondria dysfunction in our MT1 inhibition SH-SY5Y cell models [ 27 , 41 ]. Additionally, we found altered mitochondria morphology in which the mitochondria undergo excessive fission and mitophagy process. Mitochondrial morphology reflects the dynamic equilibrium of mitochondria fusion and fission, which was modulated by mitochondrial fission proteins (Drp1) and mitochondrial fusion proteins (Opa1, Mfn1, and Mfn2) [ 45 ]. Drp1 is recruited from cytosol to mitochondrion and undergoes phosphorylation on S616 and S637 during mitochondria fission. The S616-DRP1 is mostly modulated by MAPK family while S637-DRP1 is directly phosphorylated by PKA [ 46 , 47 ]. Activated S616-DRP1 or inactivated S637-DRP1 modification promotes mitochondria fission and vice versa. MT1 is a seven-fold transmembrane G protein-coupled receptor GPCR through regulating G-protein coupled signaling in diverse circumstance, like Gαs, Gαi, Gαq, and Gβ/γ. Gαs (stimulation of adenylyl cyclase) and Gαi (inhibition of adenylyl cyclase) manipulate cAMP/PKA pathway [ 48 ]. The enhancement of ERK1/2 phosphorylated S616-DRP1 and decline of PKA phosphorylated S637-DRP1 were detected in a hypoxia model of rat hippocampal neurons [ 49 ]. A study also found the phosphorylation of PKA was reduced by MT1 siRNA knock down in N2a-sw cells [ 50 ]. Here, we identified that MT1 poverty induced PKA/S637-DRP1 pathway inactivation contributed to mitochondria fission, but lack of direct demonstration in MT1 activating PKA. Moreover, the ERK/S616-DRP1 signaling pathway was activated after MT1 depletion in our experiments, and the phosphorylation of DRP1 at S616 can be rescued by ERK1/2 inhibitor. This may be due to the indirect activation of ERK1/2 by elevated ROS with mitochondria damage instead of direct GPCR signaling in MT1-KD SH-SY5Y. MPP + or MPTP induced PD models were widely used to mimic certain key features of PD like mitochondria dysfunction. Mitochondria fission was also observed in MPTP models, and drugs aimed at ameliorating mitochondria fragmentation represents a potential therapeutic for PD treatment [ 51 ]. Mitochondria fission was also observed in our MPP + or MPTP models and MT1 deficiency aggravated this phenomenon as evidenced by fragmented mitochondria morphology, and fusion and fission related proteins alteration, suggesting a converge signaling of MT1 and MPTP in regulating mitochondria dynamics. The decrease of MT1 expression was also detected with MPTP treatment, which is agreement with previous findings and clinical report, indicating a role of MT1 in PD [ 35 , 37 ]. Although the residual MT1 band is possibly due to the MT1-KO strategy in establishing transgenic mice, genetically successful knockout of MT1 has been corroborated in our group [ 31 , 38 ]. Moreover, we found the dephosphorylation of PKA and phosphorylation of ERK1/2 in MPTP mice striatum. The PKA on S637-DRP1 inactivation was not reported in MPTP mice before, and the activation of ERK1/2 was controversial in MPTP induced PD models [ 52 , 53 ]. In our acute MPTP mice, we supported the fact that the ERK1/2 was phosphorylated, thus activating S616-DRP1. Therefore, our study also provides a new insight into comprehending the PD pathogenesis via mitochondria dynamics. Mitochondrial fission could facilitate the appropriate elimination of damaged mitochondria through mitophagy. PINK1 maintains low basal levels in normal condition by translocating from the outer mitochondrial membrane (OMM) to the inner membrane for proteolytically cleavage. Upon mitochondrion damage, PINK1 is stabilized on the OMM. It recruits and phosphorylates E3 ubiquitin ligase Parkin. Phosphorylated Parkin binds with ubiquitin which is also phosphorylated by PINK1 at Ser65 residue to generate a ubiquitin chain on OMM proteins. The ubiquitin-tagged mitochondrion is recognized by autophagy adapters such as P62 and then subject to LC3 for autophagy [ 54 ]. Compromised mitophagy was confirmed in PD and the rescue of mitophagy defects has been a potential therapy of PD [ 55 ]. Here, we found the expressions of PINK1, and Parkin were decreased with MT1 deficits in vitro but not in vitro. The discrepancies between in vitro and in vivo models are probably because of the complex physiological conditions in the central nervous system, such as the interactions and signaling among neurons and glial cells. This effect is not similar to melatonin, which reported to be neuroprotective in PD by mitophagy augment [ 56 ]. Mitophagy is a selective autophagic removal process of damaged mitochondria. Since exacerbated LC3II/LC3I and P62 accumulation were observed in MT1 depleted PD mice, it is reasonable to speculate the autophagy of aggregates was also inhibited. Although we didn’t observe the TH loss in striatum and motor abnormality by MT1 knockout, the alteration of LC3II/LC3I ratio and P62 accumulation were found. This indicates the absence of MT1 may increase the susceptibility of PD occurrence through autophagy inhibition but is not the key etiological factor. Aggregation of misfolded α-syn in dopaminergic neurons in substantia nigra is called Lewy bodies, which further cause the neuronal degeneration [ 2 ]. The mechanism underlying this phenomenon is attributed to lysosome-autophagy or ubiquitin-proteasome pathway. PFFs treatment impaired autophagic flux and lysosome induction, which in turn also resulted in α-syn aggregates expansion seeding by PFFs [ 43 , 57 ]. Here, we employed α-syn modified PFFs models and identified autophagy as the potential pathway of MT1 to alleviate α-syn aggregation. LC3B-II is a marker of autophagosome formation and accumulation by adding phosphatidylethanolamine (PE) on LC3-I. As an autophagic adaptor, P62 transported ubiquitinated cargo to autophagosomes through binding with LC3-II. Failure of autophagosomes turnover or autolysosome fusion will lead to accumulation of P62. LC3-II and P62 were increased after PFFs treatment, indicating an interruption of autophagy [ 57 ]. In the present study, MT1-KO exacerbated the autophagy inhibition induced by PFFs, and overexpression of MT1 promoted autophagy by reducing P62 levels, which demonstrated the pivotal role of MT1 in autophagy induction. BafA1 was an autophagy inhibitor, used to assess the autophagy flux when LC3-II was increased [ 58 ]. Lysosome inhibition treated with BafA1 impaired fusion of autophagosomes with lysosome, causing LC3-II to elevate significantly. We not only found that MT1 overexpression induced LC3-II expression after BafA1 treatment but also corroborated MT1 function in improving autophagic flux via mCherry-LC3-EGFP lentivirus infection. Increased red dots after MT1 overexpression directly manifested the successful fusion of autophagosomes with lysosomes. Moreover, the autolysosomes were reduced in MT1 group when treated with BafA1, further confirming the regulation of MT1 in autophagic flux. The autophagy process acceleration induced by MT1 further improved α-syn degradation in lysosome. Therefore, alleviating α-syn aggregation through MT1 induced autophagy could be considered an effective way to slow PD progression instead of healing. MT1 is also present in mitochondrion but more abundant in plasma membrane [ 59 , 60 ]. It is presumably that mitochondrial MT1 works on mitochondria dynamics, but less possible to drive autophagosome formation. However, there are also some limitations within our work. First, the MT1-KO mice used here are not conditional knock out mice, it’s better to establish the DAT-cre; MT1 flox/flox mice to study the MT1 function in PD more precisely. Second, the MT1-KO mice presented the relationship between MT1 and PD, only the protective effects of MT1 in reversing mitochondria and autophagy defects induced by MPTP or PFF can corroborate the indispensable role of MT1 in PD in vivo. Third, the acute MPTP models weaken the possibility of the relevance with chronic and aging associated PD. Collectively, our findings in illustrating the MT1 function on mitochondria dynamics and autophagy related to PD, provide a favorable therapeutic target of mitigating mitochondria dysfunction and α-syn aggregation through MT1 to prevent the occurrence of PD. Declarations Conflict of Interest The authors declare no conflict of interest. Funding Our study was supported by the Science and Technology Innovation Project of Xiongan New Area (2023XAGG0073), the National Natural Science Foundation of China (82471269), Jiangsu Provincial Medical Key Discipline (ZDXK202217), Suzhou Key Laboratory (SZS2023015), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Author Contributions JY Liu and F Wang conceived and designed the study, XB Wang wrote the manuscript, XB Wang, LL Qi, JM Wang, YR Sun, QK Lv, BE Cao and SM Jiang performed the experiments and analyzed the data, QH Ma and CF Liu reviewed and edited the manuscript. All authors approved the final version of the paper. Acknowledgments The authors thank Doctor Guanghui Wang of the Soochow University for providing α-syn-GFP HEK293T stable cell lines. Data availability The datasets used in the present study are available from the corresponding author upon reasonable request. 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WANG","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYLCCCiBmY28++OCDgY0ccVrOADEfz7FkwxkFacbEa5GTyDGT5vlwOJGgaoPjZw+/OFBxh4FNIi1B2saAOYGB/fDRDXi1nMlLszhw5hkDG8/jA8Y5Bmx5DDxpaTfwaTE7kGNm/LHtMND7aQnJOQY8xQwSPGb4tZx/Y2Zw8B9QC0OOwWELA4nEBoJabuQYPzjYANTCkWPYzGBgQFiL/Y03ZgwHjgG1AAOZsccgwZiNkF8k+3OMPxyoOcwg3958/MePP//l+NkPH8OrBQjYJIBEfQOcS0A5CDB/IELRKBgFo2AUjGQAABzvTqA4v4sNAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-1348-7455","institution":"Soochow University","correspondingAuthor":true,"prefix":"","firstName":"Fen","middleName":"","lastName":"WANG","suffix":""}],"badges":[],"createdAt":"2025-07-06 13:48:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7058166/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7058166/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00018-025-05995-0","type":"published","date":"2025-11-25T15:58:47+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87811826,"identity":"bbd20583-940d-4590-b678-550a409d0870","added_by":"auto","created_at":"2025-07-29 09:29:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":242336,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMT1-KD caused mitochondrial dysfunction in SH-SY5Y cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative blots of MT1 from Control and MT1-KD SH-SY5Y cells. (B) Quantification of MT1 protein levels from western blots (n=5). (C) Statistical analysis of MT1 mRNA levels from Control or MT1-KD SH-SY5Y cells (n=5). (D) Representative images of living cell TMRE staining by 10× inverted microscope after MT1 knockdown (n=6). Scale bar, 20μm. (E) Statistical analysis of TMRE fluorescent intensity. (F) Statistical analysis of ATP levels after MT1 knockdown (n=6). (G) SH-SY5Y cells were treated with 5μM MT1 inhibitor S26131 for 24h and subjected to flow cytometry. Statistical analysis of ROS levels after S26131 treatment (n=6). Unpaired t-test analysis, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7058166/v1/a0196a965dfc070d525aad29.png"},{"id":87811831,"identity":"8c723f48-0276-4a35-b926-182f6eecda02","added_by":"auto","created_at":"2025-07-29 09:29:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":474241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMT1-KD impaired mitochondrial morphology in SH-SY5Y cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative confocal image of MitoTracker labeled mitochondria in Control and MT1-KD SH-SY5Y living cells by 100× confocal fluorescence microscopy. Scale bar, 10μm. (B) Statistical analysis of mitochondrial form factor (n=6). (C) Statistical analysis of mitochondrial aspect ratio (n=6). Scale bar, 500nm. (D) Representative TEM micrographs of mitochondria from Control and MT1-KD SH-SY5Y cells. (E) Statistical analysis of mitochondrial length (n=6). (F) Statistical analysis of mitochondria-ER contact sites (n=6). Four pictures of each independent experiment were analyzed. Unpaired t-test analysis, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7058166/v1/4774ffe477553a2b848d53d6.png"},{"id":87812544,"identity":"1a72bb95-a81b-4ef4-b8a6-3d63abf857e1","added_by":"auto","created_at":"2025-07-29 09:37:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":345892,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMT1-KD increased mitochondria fission through DRP1 phosphorylation and mitophagy in SH-SY5Y cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative blots of OPA1, MFN1, MFN2 and DRP1 protein expression from Control or MT1-KD SH-SY5Y cells. (B-E) Quantification of OPA1, MFN1, MFN2 and DRP1 protein levels, respectively (n=5). (F) Representative blots of S637-DRP1, pPKA, S616-DRP1, pERK1/2 and ERK1/2 protein expression Control or MT1-KD SH-SY5Y cells. (G-J) Quantification of S637-DRP1, pPKA, S616-DRP1, pERK1/2 and ERK1/2 phosphorylation, respectively (n=5). (K) Representative blots of PINK1, Parkin, P62 and LC3 protein expression from Control or MT1-KD SH-SY5Y cells. (L-O) Quantification of PINK1, Parkin, P62 and LC3, respectively (n=5). Two identical lanes from representative blots in each group represented two repeated experiments. Unpaired t-test analysis, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7058166/v1/e9f4c66f4bbcd6517fc5956d.png"},{"id":87814004,"identity":"8e8ebef9-ba1b-4022-abc3-01c87251a104","added_by":"auto","created_at":"2025-07-29 09:45:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":647633,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMT1-KO aggravated mitochondria fission in mouse primary neurons treated with MPP+\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative TEM micrograph of mitochondria from WT or MT1-KO transgenic mice substantial nigra with 4- or 12-month age. Scale bar, 500nm. (B) Statistical analysis of mitochondrial length by TEM. Five pictures of each independent experiment and three mitochondria of each picture were analyzed (n=5). (C) Representative immunostaining images of mitochondria with Tom20 and axons with Tuj1 from WT or MT1-KO mice primary neurons with or without MPP+. Scale bar, 1μm. (D) Statistical analysis of mitochondrial length by immunostaining (n=5). Two-way ANOVA analysis, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7058166/v1/88c3eda9cc43d1594ce6b5a0.png"},{"id":87812546,"identity":"62162231-9565-4867-81bc-c13b0f80a16c","added_by":"auto","created_at":"2025-07-29 09:37:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":862525,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMT1-KO aggravated mitochondria fission through DRP1 dephosphorylation and mitophagy in MPTP induced mouse model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative blots of OPA1, MFN1, MFN2 and DRP1 protein expression from WT or MT1-KO mice with or without MPTP. (B-E) Quantification of OPA1, MFN1, MFN2 and DRP1 protein levels, respectively (n=5). (F) Representative blots of S637-DRP1, pPKA, S616-DRP1, pERK1/2 and ERK1/2 protein expression from WT or MT1-KO mice with or without MPTP. (G-J) Quantification of S637-DRP1, pPKA, S616-DRP1, pERK1/2 and ERK1/2, respectively (n=5). (K) Representative blots of TH protein expression from WT or MT1-KO mice with or without MPTP. (L) Quantification of TH (n=5). \u0026nbsp;(M) Histogram showing the results of mouse latency to fall during the rotarod test (n=6). (N) Representative blots of P62 and LC3 protein expression from WT or MT1-KO mice with or without MPTP. (O-P) Quantification of P62 and LC3, respectively (n=5). Two-way ANOVA analysis, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01. ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7058166/v1/0ac00eddc8a0827c71170b38.png"},{"id":87812548,"identity":"670629d5-1d18-4776-b979-70140edb2cb9","added_by":"auto","created_at":"2025-07-29 09:37:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":282180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMT1 deficiency enhanced PFFs induced autophagy inhibition and α-syn aggregation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNeonatal mouse primary cortical neurons were treated with PFF (1 μg/mL) for 10 days and the protein extracted and subjected to western blot. (A) Representative blots of P62 and LC3 from three repeated experiments. (B) Representative blots of α-syn in soluble fraction. (C) Representative blots of α-syn in pellet fraction. (D-G) Quantification of protein levels of P62, LC3, α-syn monomers and high molecular weight (HMW) α-syn, respectively (n=5). Two-way ANOVA analysis, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001. ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7058166/v1/87d293be6a43e6ce89b5e008.png"},{"id":87811835,"identity":"b921ef8a-9b5c-4527-aeb0-45957b7ad911","added_by":"auto","created_at":"2025-07-29 09:29:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2303574,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMT1 overexpression increased mitochondria fusion and facilitated autophagy flux to promote α-syn degradation in HEK293T cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) HEK293T was transfected with PCDNA3.1 or hMT1-Flag and cell lysate was subjected to western blot. Representative blots of MFN1, MFN2, and DRP1 from three repeated experiments. (B-D) Statistical analysis of MFN1, MFN2, and DRP1 protein levels, respectively (n=6). Unpaired t-test. (E) Representative blots of P62 and LC3 from three repeated experiments. (F-G) Statistical analysis of P62 and LC3II protein levels, respectively (n=6). One-way ANOVA analysis. (H) HEK293T was transfected with PCDNA3.1 or hMT1-Flag for 48 h, 200nm BafA1 was added 16 h before protein detection and cell lysate was subjected to western blot. Representative blots of LC3 from three repeated experiments. (I) Statistical analysis of LC3II protein levels (n=3). Kruskal-Wallis test. (J) HEK293T cells were transfected with or without hMT1-Flag for 48 h and infected with mCherry-LC3-EGFP lentivirus for 36 h, 200nm BafA1 was added 16 h before confocal analysis. Quantification of autophagosomes (K) and autolysosomes (L) per cell using Image J (n=5). Scale bar, 5μm. Two-way ANOVA analysis. (M) α-syn-GFP HEK293T stable cell line was transfected with PCDNA3.1 or hMT1-Flag plasmids. Representative blots of P62, LC3 and α-syn-GFP. (N-P) Statistical analysis of P62, LC3II and α-syn-GFP protein levels, respectively (n=5). One-way ANOVA analysis. Three identical lanes from representative blots in each group represented three repeated experiments. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7058166/v1/0a963f1fa20b17db141952ca.png"},{"id":97179627,"identity":"e0b02d9e-2d81-4ebf-875b-e77c397fe701","added_by":"auto","created_at":"2025-12-01 16:16:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6658430,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7058166/v1/abff1ed9-cf1a-47e6-aefb-b530908716b3.pdf"},{"id":87811825,"identity":"3513d6ae-02bf-42d5-a023-85246a5177a6","added_by":"auto","created_at":"2025-07-29 09:29:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":190734,"visible":true,"origin":"","legend":"","description":"","filename":"FIGURES1andS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7058166/v1/9334e2ff45e610726a110aa5.docx"}],"financialInterests":"","formattedTitle":"Regulation of mitochondrial dynamics and function by MT1 melatonin receptor in Parkinson’s disease","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is a common neurodegeneration with motor disorder, such as bradykinesia, tremor, rigidity and postural instability [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The pathological characteristics of PD are the loss of dopaminergic neurons in substantial nigra, descending expression of tyrosine hydroxylase (TH) and aggregation of misfolded alpha-synuclein (α-syn) in neurons [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Multiple mechanisms like autophagy, apoptosis, inflammation, and mitochondria dysfunction are involved in PD pathogenesis. Mitochondria dysfunction has long been believed by researchers to be a critical player to induce neuronal death in PD development [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. PD patients affected by environmental factors accounted for more than 90% of total patients and the exposure to pesticides like MPTP, rotenone and paraquat has been applied to establish PD-related models which can mimic key features of PD [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Importantly, these neurotoxins all targeted mitochondria to cause cell death in which the mitochondrial complex I activity was inhibited, or oxidative stress was elevated. Mitochondria dynamics consecutively played a role in alleviating mitochondria damage by fusion or disposing disrupted mitochondria via fission. Disequilibrium of mitochondria dynamics especially the excessive fission can cause mitochondria defects, which is implicated in PD etiology [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Mitophagy selectively removes the dysfunctional mitochondria through autophagy to prevent deleterious effects like oxidative stress within the cell. The mutation of two essential mitophagy regulators PTEN-induced kinase 1 (PINK1) and Parkin is attributed to familial PD. Impaired mitophagy was observed in both genetic mutational and toxic PD models [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Therefore, maintaining mitochondria homeostasis to restore its function and enhance mitophagy to proceed with the elimination of damaged mitochondria is continually to be an effective strategy to prevent PD.\u003c/p\u003e\u003cp\u003eα-Syn is an cytosolic protein, which locates at the end of the axon and is involved in the transport of vesicles in neurotransmitters releasing [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. When the α-syn encoding gene, SNCA mutated or the α-syn protein misfolded, it will lead to the phosphorylation of α-syn at serine 129 site (pS129-α-syn) and formation of protein aggregates [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. α-syn deposition not only loses the physiological function of α-syn, but also causes deteriorated intracellular response like mitochondria damage, thus eventually leading to cell death [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Studies revealed one of the pathogenic mechanism of PD was via the binding of α-syn with TOM20 to inhibit mitochondrial protein import [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Moreover, PD induced lysosomal and mitochondrial aberration also accelerates α-synuclein aggregation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These properties of α-syn make it the most promising therapeutic target aimed at preventing α-syn dysfunction and subsequent protein aggregation primarily via mitochondria or autophagy-lysosome pathways. The dopamine D2 and D3 receptors reduce α-syn accumulation through inducing a BECN1-dependent autophagy activation to protect against PD [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Strategies to improve mitochondria function also exhibit the effects to suppress toxic α-syn accumulations [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite motor symptoms, PD patients also exhibited non-motor symptoms like sleep disorders, anxiety, pain, and depression [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Sleep disorder, the main prodromal symptoms of PD, was manifested as daytime sleepiness, insomnia, and rapid eye movement (REM) sleep behavior disorder (RBD) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Melatonin, a natural hormone secreted by the pineal gland, can be chemically synthesized to treat sleep disorders in PD patients [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and protect motor and pathological deficits via ameliorating neuronal loss, inflammation, mitochondria defects, and autophagy suppression in PD models [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. For instance, melatonin protects against defects of mitochondrial respiration and ATP levels in chronic Parkinsonian mice model [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Melatonin rescues rotenone-induced apoptosis or MPTP-induced neurotoxicity via mediating autophagy pathway [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Although a preliminary study of melatonin in mitochondrial dynamics and autophagy is also implicated in MPTP treated zebrafish [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], the underlying mechanism is still not fully understood.\u003c/p\u003e\u003cp\u003eThe action of melatonin usually works through binding with a seven transmembrane G protein-coupled receptor (GPCR) on plasma membrane to activate downstream signaling despite a receptor-independent mechanism existing [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Two melatonin receptors, melatonin type 1 receptor (MT1) and melatonin type 2 receptor (MT2) were identified to regulate REM and non-rapid eye movement (NREM) sleep, respectively [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Melatonin receptor agonists, like ramelteon and agomelatine, are applied in clinical to treat sleep disorders or sleep disturbed psychiatric diseases [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. MT1 is also reported to located on mitochondrion both in vivo and in vitro [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. It displays a pleiotropic effect in stabilizing circadian rhythm [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], sleep [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], neuroprotection [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], antioxidative ability [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], cell survival [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], and anti-inflammation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The distribution of MT1 is abundance in central nervous system, especially retina, suprachiasmatic nucleus (SCN), striatum and substantial nigra [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], and the mRNA levels of MT1 is reduced in substantial nigra of PD patients [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], which implicated a correlation of MT1 with PD. In our group, we have demonstrated the positive effects of MT1 in microglial inflammation in MPTP-induced models [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], ferroptosis and microglial phagocytosis in PFFs-induced models [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and sleep-wake disorders in rotenone-induced models of PD [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], but how MT1 is involved in PD pathogenesis through manipulating mitochondria property and autophagy is under determined.\u003c/p\u003e\u003cp\u003eIn the present study, we determine the role of MT1 in mitochondria dynamics and mitophagy in PD, which further links to α-syn accumulation through autophagy. We identified that knockdown of MT1 causes mitochondria dysfunction, mitochondria fission and mitophagy in vitro. Loss of MT1 exacerbated mitochondria fission without influenced mitophagy, TH expression and mice movement in MPTP induced model in vivo. The increased fission may be caused by inactivation of PKA on S637-DRP1 phosphorylation and activation of ERK1/2 on S616-DRP1. Moreover, MT1 knockout exacerbated PFF-induced autophagy and potentiated α-syn aggregation in mouse primary neurons. Overexpression of MT1 reduced mitochondria fission, promoted autophagy and mitigated α-syn aggregation in HEK293T cells. In this study, we evaluated the function of MT1 in mitochondria function and autophagy regulation, which provided a potential mechanism to understand the mitochondria fragmentation and α-syn clearance in PD through MT1.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Animal experiments\u003c/h2\u003e\u003cp\u003eTransgenic \u003cem\u003eMtnr1a\u003c/em\u003e knockout (MT1-KO) mice in a C57BL/6JGpt background were purchased from GemPharmatech (China), which has been used previously [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Briefly, the CRISPR/Cas9 system was employed to generate the MT1 KO mice model through the deletion of exon 2 of transcript Mtnr1a-201(ENSMUST00000067984.8). The single guide RNA was designed to direct the Cas9 endonuclease to create double strand breaks at the sites of intron 1\u0026ndash;2 and downstream of exon 2 within the MT1 gene. The sg RNA transcripts constructed in vitro were microinjected into fertilized eggs of mice together with Cas9. The fertilized eggs were then implanted to produce F0 generation mice with the desired genetic modification, which was confirmed via PCR and sequencing. Positive F0 mice were mated with C57BL/6JGpt mice to establish a stable F1 generation mouse line. Genotyping was performed by the following primers to detect wild-type (WT) MT1 and MT1-KO alleles: WT-Forword, 5\u0026rsquo;-GCTCACTTGACTCTAGGAGGGAGAC-3\u0026rsquo;; WT-Reverse, 5\u0026rsquo;-CCTGGGATAACATACAGCCAGC-3\u0026rsquo;; KO-Forword, 5\u0026rsquo;-AACTTGAAGCTCTTCAGGGTTGC-3\u0026rsquo;; KO-Reverse, 5\u0026rsquo;-CATGGTCCCTCTTGTCTTGAGTTC-3\u0026rsquo;. Homozygotes wild type (WT) littermates after cross were used as control. Mice were maintained under specific pathogen-free conditions in a 12-h light/dark cycle with unlimited water and food available. All experiments involving mice were approved by the Institutional Animal Care and Use Committee of Soochow University.\u003c/p\u003e\u003cp\u003eMouse models induced by MPTP were divided into four groups randomly: (1) WT\u0026thinsp;+\u0026thinsp;Saline group; (2) WT\u0026thinsp;+\u0026thinsp;MPTP group; (3) MT1-KO\u0026thinsp;+\u0026thinsp;Saline group; (4) MT1-KO\u0026thinsp;+\u0026thinsp;MPTP group. Mice in the group (2) and (4) were intraperitoneally injected with MPTP four times (first time at Zeitgeber time(ZT)0: 14 mg/kg, second time at ZT2: 16 mg/kg, third time at ZT4: 18 mg/kg and last time at ZT6: 20 mg/kg) a day with 2 h intervals for seven consecutive days to establish an acute PD model. The 4-month-old mice from group (1) and (3) received an equal volume of saline. The sample was collected at ZT6 from mice after one day of injection.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Cell culture\u003c/h2\u003e\u003cp\u003eHEK293T cells, a human embryonic kidney cell line, were maintained in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, 12430112, Gibco, USA) with 10% fetal bovine serum (30067334, Gibco, USA), and 1% penicillin/streptomycin antibiotics (15140122, Gibco, USA). Neuroblastoma SH-SY5Y cells were cultured in DMEM/F-12 medium supplemented with 10% fetal bovine serum, and 100 \u0026micro;g/ml penicillin/streptomycin at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. WT-GFP Control (TTCTCCGAACGTGTCACGT, Genechem, China) and MT1-KD-GFP (human MTNR1A-RNAi, CAGTTACTACATGGCGTAT, Genechem, China) lentivirus were used to establish stable SH-SY5Y cell lines, respectively, and single cell clone with GFP was selected by 400 \u0026micro;g/ml puromycin.\u003c/p\u003e\u003cp\u003eMouse primary cortical neurons were prepared from P1\u0026ndash;P3 neonatal MT1-KO pups or control littermates. Cells were seeded in Poly-D-Lysine (A3890401, Gibco, USA) coated 6-well plate or 24-well plate with coverslips and cultured in a complete neurobasal-A medium (10888022, Gibco, USA) supplemented with 2% serum free B27(17504044, Gibco, USA), 1% GlutaMAX (35050061, Gibco, USA) and 1% penicillin/streptomycin. Culture medium was changed every 3 days until experiments were performed. MPP\u0026thinsp;+\u0026thinsp;and PFFs were added into primary neurons in a concentration of 100 \u0026micro;M for 6h and 1 \u0026micro;g/mL for 10 days, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Mitochondria membrane potential (MMP, ΔΨ) detection\u003c/h2\u003e\u003cp\u003eMMP was measured by the red-fluorescent probe tetramethyl rhodamine ethyl ester (TMRE) (Beyotime, China) in accordance with manufacturer\u0026rsquo;s protocol. TMRE was accumulated within mitochondrion but released outside as mitochondrial membrane depolarized. SH-SY5Y cells were incubated with 1\u0026times;TMRE dye at 37\u0026deg;C for 15 min and Hoechst 33342 (Beyotime, China) for 5 min. Cells were then washed twice with prewarmed culture medium, and images were obtained by inverted fluorescent microscope (Carl Zeiss, Germany). Identical exposure times were used to take each image. Mitochondrial fluorescence intensity (OD value) was calculated by ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Relative oxidative species (ROS) detection\u003c/h2\u003e\u003cp\u003eSH-SY5Y cells were treated with 5\u0026micro;M MT1 antagonist S26131 (MedChemExpress, USA) for 24 h and subjected to ROS evaluation according to manufacturer\u0026rsquo;s protocol (Beyotime, China). DCFH-DA is a probe without fluorescence and can be loaded in cells when hydrolyzed to DCFH. Oxidized DCFH by cytosolic ROS is then converted into DCF with green fluorescence. SH-SY5Y cells were incubated with 10 nm/mL DCFH-DA at 37\u0026deg;C for 30 min and washed three times with culture medium without FBS to remove spare dye. The ROS fluorescent intensity was quantitated by FACScan flow cytometer (BD Biosciences, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 ATP determination\u003c/h2\u003e\u003cp\u003eCellular ATP levels were measured by a commercial ATP detection kit (Beyotime, China) according to the manufacturer\u0026rsquo;s protocol. Briefly, ATP was extracted by ATP lysis buffer and centrifuged to obtain supernatant. 20 \u0026micro;l of supernatant or gradient ATP standard solution was mixed with 100\u0026micro;l of ATP working solution in 96-well plate. ATP contents were determined by a luminometer (Infinite M200, Tecan, Switzerland) and normalized with protein concentration in the supernatant.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 MitoTracker Staining and Transmission electron mitochondria (TEM)\u003c/h2\u003e\u003cp\u003eWT and MT1-KD SH-SY5Y cells were cultured on 35 mm confocal dishes with glass on the bottom (NEST, China) and incubated with 100 nM Mito-Tracker Red CMXRos for 15 min at 37\u0026deg;C according to instructions (Beyotime, China). Images were obtained from LSM 700 confocal microscope (Carl Zeiss, Germany). The mitochondria morphology parameters were analyzed to calculate the value of form factor (perimeter\u003csup\u003e2\u003c/sup\u003e/4π\u0026middot;area) and aspect ratio (the ratio between the major and minor axis of the ellipse equivalent to the mitochondrion) as previous protocol [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Cells and mouse substantial nigra were obtained and preserved by using electron microscope fixative solution (G1102, Servicebio, China) in foil at 4\u0026deg;C. The samples were then sent to company (Servicebio, China) on ice for detection. The mitochondria morphology parameters from TEM were analyzed as indicated methods [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Quantification real-time PCR\u003c/h2\u003e\u003cp\u003eTRIzol reagent (Invitrogen, USA) was used to isolate total RNA from WT-GFP, or MT1-KO-GFP stable SH-SY5Y cell lines and the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, USA) was applied to synthesized cDNA from RNA. Quantitative PCR was performed with SYBR-Green Master Mix (Vazyme, China) and measured by 7500 Real-Time PCR System (Applied Biosystems, USA). qPCR primers were obtained from GENEWIZ (China). Human MT1 Forward primer, 5\u0026rsquo;-AGGGACTCTCCAGTACGACC-3\u0026rsquo; and Reverse primer 5\u0026rsquo;-GTTTGCGGTCAGGTTTCACC-3\u0026rsquo;; 18S Forword primer 5\u0026rsquo;-TCAACACGGGAAACCTCAC-3\u0026rsquo; and reverse primer 5\u0026rsquo;-CGCTCCACCAACTAAGAAC-3\u0026rsquo;. mRNA levels were normalized to 18S RNA and calculated by 2ΔΔCt method.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Immunostaining\u003c/h2\u003e\u003cp\u003eCells cultured on coverslips were washed in PBS and fixed with 4% paraformaldehyde for 30 min. After permeabilizing with 0.25% Triton X-100 for 30 min and blocking with 5% BSA for 1 h, cells were incubated with anti-Tom20 (1:250, Beyotime, China), anti-Flag (1:1000, ab1257, Abcam, USA), anti-pS129-α-syn (1:500, ab51253, Abcam, USA), anti-Tuj1 (1:1000, ab78078, Abcam) and anti-LC3 (1:200, NB100-2220, Nouvs, USA) antibodies overnight at 4\u0026deg;C. The secondary antibody Alexa Fluor 637 (A-21447, Thermo Fisher, USA), Alexa Fluor 488 (A28175, Thermo Fisher, USA), and Alexa Fluor 555 (A27039, Thermo Fisher, USA) was added at room temperature for 1 h. Cells were sealed with mounting medium containing DAPI (H-1200, Vector Laboratories, USA) and then observed under confocal microscope (LSM700, Carl Zeiss, Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Western blot\u003c/h2\u003e\u003cp\u003eProtein from cells and tissues was extracted by strong RIPA buffer (Beyotime, China) with protease/phosphatase inhibitor (NCM, China) and sonicated in an amplitude of 20% and 30%, respectively by ultrasonic processor (SONICS, USA). The supernatants were harvested after centrifuge and the equal protein loading was determined by BCA assay kit (23227, Thermo Scientific, USA). Equal amounts of proteins in 5\u0026times;Loading buffer were separated by 10% SDS-PAGE gels (NCM, China) and transferred onto a polyvinylidene fluoride membrane (Millipore, USA). Membranes were incubated with the following primary antibodies overnight at 4\u0026deg;C after blocking with 5% BSA: anti-β-tubulin (T0198, Sigma, USA), anti-MT1 (MTNR1A, A13030, Abclonal, USA), anti-OPA1 (ab42364, Abcam, USA), anti-Mfn1 (ab104274, Abcam, USA), anti-mfn2 (ab56889, Abcam, USA), anti-Drp1 (ab56788, Abcam, USA), anti-S637-Drp1 (AF5791, Beyotime, China), anti-S616-Drp1 (3455S, CST, USA), anti-ERK1/2 (4695S, CST, USA), anti-pERK1/2 (4370S, CST, USA), anti-PINK1 (AF7755, Beyotime, China), anti-Parkin (ab15494, Abcam, USA), anti-phosphorylated-PKA (AF1942, Beyotime, China) and anti-TH (ab193083, Abcam, USA), anti-Flag-HRP (A8592, Sigma, USA), anti-α-syn (ab1903, Abcam, USA), anti-Actin-HRP (A3854, Sigma, USA), anti-LC3 (NB100-2220, Novus, USA), anti-P62 (P0068, Sigma, USA), anti-GFP (ab290, Abcam, USA), anti-MT2 (MTNR1B, NLS932SS, Novus, USA). After incubation with HRP-conjugated anti-rabbit/mouse IgG at room temperature for 1 hour, the blots were visualized (Clinx ChemiCapture, China) by ECL detection kits (P2300, NCM, China). To quantify the content of insoluble α-syn, the lysate was centrifuged at 25,000 rpm for 30 min. Insoluble pellets were dissolved in 1 \u0026times; TBS with 2% SDS and subjected to sonication. The gray value of WB bands was measured via ImageJ software (National Institutes of Health, USA) after normalization with a corresponding control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Plasmid transfection and virus infection\u003c/h2\u003e\u003cp\u003eHuman MT1(hMT1)-Flag plasmids and α-syn GFP plasmids were constructed on PCDNA3.1 from Genewiz. JetPRIME (Polyplus, France) was used to transfected plasmids into HEK293T cells according to the manufacturer\u0026rsquo;s protocol. HEK293T cells were infected with mCherry-LC3-EGFP lentivirus (HANBIO, China) in a 2.5 MOI (multiplicity of infection) for 36 h. Lysosome inhibitor, 200nm bafilomycin A1 (BafA1, A417398, Sangon Biotech, China) was added 16 h before detection.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Performed Fibrils (PFFs) preparation\u003c/h2\u003e\u003cp\u003eα-syn-PFFs was prepared according to a general protocol for primary neuronal cultures[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Briefly, aliquots of 5 mg/mL PFFs were stored at \u0026minus;\u0026thinsp;80\u0026deg;C and thawed at 37\u0026deg;C water bath before use. The PFFs were diluted into 0.5 mg/mL working solution by sterilized PBS and then sonicated for 1min with 1s intervals (total 30 times) in a 20% amplitude (SONICS, USA) to create fragmented PFFs. Neuron culture medium was added with PFFs and subjected to filtration with 0.22 \u0026micro;m mesh.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12 Rotarod test\u003c/h2\u003e\u003cp\u003eThe motor function of the mice was assessed through applying the Rotarod system (ZH-300, Zhenghua Co. Ltd., China). Prior to the formal testing, the mice were trained to ensure familiarity with the task. The training was conducted on three consecutive days with three sessions per day at a fixed time from ZT6. During the test, the rotation speed was gradually raised to 15 rpm in a session. Each mouse was subjected to three trials at 30-minute intervals from ZT6, and the mean latency to fall was calculated as the final result.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13 Statistical analysis\u003c/h2\u003e\u003cp\u003eAll the data from at least three independent experiments are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistics were performed using GraphPad Prism 9.5 software. The significance of difference was determined by unpaired t-test between two groups or One-way analysis of variance (ANOVA) for multiple-groups, while nonparametric tests were used for data with n\u0026thinsp;=\u0026thinsp;3. \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.1 MT1-KD caused mitochondrial dysfunction in SH-SY5Y cells\u003c/h2\u003e\u003cp\u003eTo observe MT1 function in vitro, we primarily established stable SH-SY5Y cell lines using WT-GFP and MT1-KD-GFP lentivirus. MT1 knockdown efficiency was validated by protein and mRNA protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C), without impacting MT2 expression (Figure S1A, B). Previous study has documented the increased ROS and declined MMP in MT1-KD retinal pigmental epithelium cells [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. These results were also verified in our SH-SY5Y stable cell lines. We performed similar experiments and found that MT1-KD decreased MMP and cellular ATP levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F). To detect ROS with green fluorescent DCFH-DA probe, efficient MT1 antagonist S26131 was used to avoid GFP disturbance [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. SH-SY5Y cells treated with S26131 intensified ROS production (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Therefore, MT1 deficiency resulted in mitochondria damage in SH-SY5Y cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.2 MT1-KD impaired mitochondrial morphology in SH-SY5Y cells\u003c/h2\u003e\u003cp\u003eAlteration in mitochondrial morphology is the main aspect of its malfunction. By taking high resolution images of stained mitochondria under 100 \u0026times; lens, we found that compared with WT control, MT1-KD cells exhibited more fragmented mitochondria networks and less branches (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The degree of mitochondrial branching indicated by form factor and length indicated by aspect ratio were all decreased as MT1 knocked down (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). TEM results also corroborated the findings that MT1 knockdown decreased mitochondrial length (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, E). The mitochondrion shape observed in MT1-KD SHSY-5Y cells was shorter and rounder. Interestingly, we also noted that the contacts between mitochondria and endoplasmic reticulum (ER) were decreased, implying a deterioration of the organelle function (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.3 MT1-KD increased mitochondria fission via DRP1 phosphorylation and hampered mitophagy in SH-SY5Y cells\u003c/h2\u003e\u003cp\u003eTo investigate the underlying mechanisms of impaired mitochondria dysfunction and morphology, we detected the alteration of mitochondrial dynamic and mitophagy proteins. As expected, the expression of mitochondrial fusion proteins, OPA1, MFN1, and MFN2 were downregulated, and the expression of mitochondrial fission proteins DRP1 was upregulated in MT1-KD cells relative to WT control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-E). Generally, potentiated mitochondria fission may be caused by decreased DRP1 phosphorylation at S637 or increased DRP1 phosphorylation at S616. We found that MT1-KD reduced DRP1-S637 phosphorylation and increased DRP1-S616 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G, I). Moreover, MT1-KD also dephosphorylated PKA, the regulator of DRP1-S637 but elevated ERK1/2 phosphorylation, which served as an upstream effector of DRP1-S616 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, H, J). The recovery of S616-DRP1 level treated with ERK1/2 inhibitor SCH772984 in MT1-KD group was identified (Figure S2A, B). The key proteins of mitophagy, PINK1 and Parkin were also impaired by MT1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eK, L, M). The ascending ratio of LC3 II/I and accumulated P62 further proved the mitophagy inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eK, N, O). Together, these results suggested MT1 deficiency caused DRP1-S637 and DRP1-S616 induced mitochondria fragmentation, and mitophagy in vitro.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.4 MT1-KO aggravated mitochondria fission in mouse primary neurons treated with MPP+\u003c/h2\u003e\u003cp\u003eTo study in vivo, we assessed the mitochondria morphology in mice substantial nigra by TEM. The WT and MT1-KO mice were bred in a stable population from our group, and the genotypes of which has been substantiated before use [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In SCN where MT1 is most abundantly expressed, MT1-KO mice displayed a critical reduction of MT1 mRNA level (Figure S1C). Here, we found that both aging and MT1-KO decreased mitochondria length compared with WT mice at 4 months (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). MT1-KO exacerbated the mitochondria length reduction induced by aging. Next, we employed MT1 expressed mouse primary cortical neurons to observe morphology of mitochondrion with Tom20 staining, in which MT2 expression was negligible (Figure S1D). Mitochondria in MT1-KO mouse primary neuronal axons also showed an apparent fragmentation relative to WT control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). The 1-methyl-4-phenyl pyridinium (MPP+) is the neurotoxic metabolite of MPTP. MT1-KO neurons treatment with MPP\u0026thinsp;+\u0026thinsp;decreased mitochondrial length compared to MT1-KO or MPP\u0026thinsp;+\u0026thinsp;alone group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). This result implied that MT1 and MPP\u0026thinsp;+\u0026thinsp;may work reciprocally on mitochondria morphology.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.5 MT1-KO aggravated mitochondria fission through DRP1 phosphorylation and mitophagy in MPTP induced mouse model\u003c/h2\u003e\u003cp\u003eTo further evaluate the MT1 effects on mitochondrial function in PD in vivo, we treated MT1-KO transgenic mice with or without neurotoxin MPTP. Protein from mice striatum was collected in ZT6 for subsequent experiments. Western blot results showed that not only MT1-KO but also MPTP treatment upregulated the expression of mitochondria fusion proteins (OPA1, MFN1, and MFN2) while downregulated mitochondria fission protein DRP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-E). Additionally, MT1-KO mice with MPTP exhibited a more profound impact on mitochondria fusion and fission protein levels than MT1-KO or MPTP groups individually (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-E). Reduced DRP1 phosphorylation at S637 but increased at S616 was induced by MT1 deficiency or MPTP, which was further exacerbated in MT1-KO mice in combination with MPTP (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, G, I). Consistent with the findings above, PKA was inactivated, and ERK1/2 was activated in accordance with the S637-DRP1 dephosphorylation and S616-DRP1 phosphorylation, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, H, J). However, the PD marker TH expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eK, L) and mouse motor ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eM) were attenuated in MPTP mice but not in MT1-KO mice. PINK1 and Parkin alteration was also not observed in MT1-KO mice which may be due to the complicated in vivo environment (data not shown). Interestingly, we observed compromised autophagy after MT1 deletion, which was aggravated in MPTP mouse (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eN-P). These results suggest that the loss of MT1 induced mitochondria fission may increase the susceptibility of PD occurrence but is not the key etiological factor.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.6 MT1 deficiency aggravated autophagy inhibition induced by α-syn PFFs\u003c/h2\u003e\u003cp\u003eIn PD, autophagy is the canonic degradation pathway of pathological α-syn clearance. To better assess MT1 functions on autophagy in α-synucleinopathy PD models, we used α-syn PFFs induced neuronal model to detect autophagy alteration, soluble and insoluble α-syn accumulation using WB. α-syn PFFs are purified misfolded α-syn aggregates, which are prion-like, self-templating and transmittable. PFFs can efficiently induce the production of α-syn aggregates, which occur within 6 days and last until 12 days [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. After induction by PFFs for 10 days in vitro, it increased autophagy-related indicators, P62 accumulation and the ratio of LC3-II/I, indicating the autophagy inhibition by PFFs in neurons. In addition, MT1-KO exacerbated this suppression of autophagy induced by PFFs. However, the impaired autophagy in MT1-KO group was milder than PFF-induced group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, D, E). In addition, the formation α-syn significantly accelerated after PFFs induction, and MT1-KO augment PFFs induced α-syn accumulation in both forms of monomers and oligomers (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C, F, G). Moreover, we validated the aggravated pS129-α-syn expression induced by α-syn PFFs in MT1-KO mice (data not shown), which has been conducted in our group [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.7 MT1 overexpression increased mitochondria fusion and facilitated autophagy flux to promote α-syn degradation in HEK293T cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo observe the protective effects of MT1 on mitochondria and autophagy, we overexpressed hMT1-Flag in HEK293T cells for 48 h and detected the levels of related proteins. HEK293T cells exhibited a much higher transfect efficiency than SH-SY5Y cells. Western blot results showed that MT1 reduced mitochondria fission as indicated by DRP1 decline, as well as MFN1 and MFN2 increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D). In addition, MT1 significantly augmented the LC3-II level but reduced the level of P62 compared with PCDNA3.1 controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-G). MT1 induced LC3-II level was further elevated in the presence of lysosome inhibitor BafA1, implying the enhancement of autophagic flux by MT1 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eH, I).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe autophagic flux was then visually monitored by the expression of mCherry-LC3B-EGFP under confocal microscope. EGFP fluorescence quenches at the acidic condition when fused with the lysosome lumen. However, mCherry is relatively stable and keeps on fluorescing red without pH perturbation. Therefore, the yellow dots represented by colocalization of EGFP and mCherry signals implies the autophagosomes that are not fused with lysosome, whereas red dots represented by mCherry-only signals implies the autolysosomes that interprets autophagic flux results [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In our study, MT1 overexpression considerably boosted the number of autolysosomes per cell compared with control (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ, L), and slightly increased the number of autophagosomes per cell relative to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ, K). In addition, the number of autolysosomes were reduced and the autophagosomes failed to fuse with lysosomes were increased after BafA1 treatment in Flag-hMT1 overexpressed HEK293T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ-L), which further validates the conclusion that MT1 positively regulates autophagy flux.\u003c/p\u003e\u003cp\u003eTo determine whether MT1 induced autophagy is helpful for α-syn degradation, we overexpressed hMT1-Flag for 48h and α-syn-GFP for 24h in HEK293T cells. The results showed that the expression of α-syn was decreased along with P62 reduction and LC3 II/LC3 I ratio ascendance (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eM-P). These results suggest that MT1 can proceed α-syn degradation through autophagy.\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eCurrent evidence in understanding the role of MT1 associated with mitochondria and autophagy in PD is limited. In this study, we worked on MT1-KD SH-SY5Y cells and MT1-KO transgenic mice to explore its function and relationship with PD. Inhibition of MT1 blunted mitochondria function, manifested as decreased MMP and ATP, as well as ROS generation. Impaired mitochondrial morphology was observed due to the upregulation of mitochondria fission proteins and downregulation of mitochondria fusion proteins. Mechanistically, PKA signaling was inactivated to arrest DRP1 phosphorylation at S637 and ERK signaling was activated to promote DRP1 phosphorylation at S616. In vivo, MT1-KO aggravated the mitochondria fragmentation in MPTP induced PD mouse model under the same mechanism. However, PD related symptoms like TH expression and mice motor ability were not influenced by MT1 knockout. Moreover, loss of MT1 exacerbated α-syn accumulation induced by PFF due to the compromised autophagy. By overexpressing MT1, we found autophagy flux was elevated and α-syn was degraded. The study declared the impairment of mitochondria quality control and autophagy without MT1 and its relevance with PD.\u003c/p\u003e\u003cp\u003eMitochondria homeostasis in keeping its proper function is vital in cell survival and PD. Multifaceted mitochondria activities, such as biogenesis, dynamics, trafficking or mitophagy have impact on mitochondria function [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In line with previous results reported that MT1 knockdown increased ROS and decreased MMP, we confirmed the mitochondria dysfunction in our MT1 inhibition SH-SY5Y cell models [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Additionally, we found altered mitochondria morphology in which the mitochondria undergo excessive fission and mitophagy process. Mitochondrial morphology reflects the dynamic equilibrium of mitochondria fusion and fission, which was modulated by mitochondrial fission proteins (Drp1) and mitochondrial fusion proteins (Opa1, Mfn1, and Mfn2) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Drp1 is recruited from cytosol to mitochondrion and undergoes phosphorylation on S616 and S637 during mitochondria fission. The S616-DRP1 is mostly modulated by MAPK family while S637-DRP1 is directly phosphorylated by PKA [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Activated S616-DRP1 or inactivated S637-DRP1 modification promotes mitochondria fission and vice versa. MT1 is a seven-fold transmembrane G protein-coupled receptor GPCR through regulating G-protein coupled signaling in diverse circumstance, like Gαs, Gαi, Gαq, and Gβ/γ. Gαs (stimulation of adenylyl cyclase) and Gαi (inhibition of adenylyl cyclase) manipulate cAMP/PKA pathway [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The enhancement of ERK1/2 phosphorylated S616-DRP1 and decline of PKA phosphorylated S637-DRP1 were detected in a hypoxia model of rat hippocampal neurons [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. A study also found the phosphorylation of PKA was reduced by MT1 siRNA knock down in N2a-sw cells [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Here, we identified that MT1 poverty induced PKA/S637-DRP1 pathway inactivation contributed to mitochondria fission, but lack of direct demonstration in MT1 activating PKA. Moreover, the ERK/S616-DRP1 signaling pathway was activated after MT1 depletion in our experiments, and the phosphorylation of DRP1 at S616 can be rescued by ERK1/2 inhibitor. This may be due to the indirect activation of ERK1/2 by elevated ROS with mitochondria damage instead of direct GPCR signaling in MT1-KD SH-SY5Y.\u003c/p\u003e\u003cp\u003eMPP\u0026thinsp;+\u0026thinsp;or MPTP induced PD models were widely used to mimic certain key features of PD like mitochondria dysfunction. Mitochondria fission was also observed in MPTP models, and drugs aimed at ameliorating mitochondria fragmentation represents a potential therapeutic for PD treatment [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Mitochondria fission was also observed in our MPP\u0026thinsp;+\u0026thinsp;or MPTP models and MT1 deficiency aggravated this phenomenon as evidenced by fragmented mitochondria morphology, and fusion and fission related proteins alteration, suggesting a converge signaling of MT1 and MPTP in regulating mitochondria dynamics. The decrease of MT1 expression was also detected with MPTP treatment, which is agreement with previous findings and clinical report, indicating a role of MT1 in PD [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Although the residual MT1 band is possibly due to the MT1-KO strategy in establishing transgenic mice, genetically successful knockout of MT1 has been corroborated in our group [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Moreover, we found the dephosphorylation of PKA and phosphorylation of ERK1/2 in MPTP mice striatum. The PKA on S637-DRP1 inactivation was not reported in MPTP mice before, and the activation of ERK1/2 was controversial in MPTP induced PD models [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In our acute MPTP mice, we supported the fact that the ERK1/2 was phosphorylated, thus activating S616-DRP1. Therefore, our study also provides a new insight into comprehending the PD pathogenesis via mitochondria dynamics.\u003c/p\u003e\u003cp\u003eMitochondrial fission could facilitate the appropriate elimination of damaged mitochondria through mitophagy. PINK1 maintains low basal levels in normal condition by translocating from the outer mitochondrial membrane (OMM) to the inner membrane for proteolytically cleavage. Upon mitochondrion damage, PINK1 is stabilized on the OMM. It recruits and phosphorylates E3 ubiquitin ligase Parkin. Phosphorylated Parkin binds with ubiquitin which is also phosphorylated by PINK1 at Ser65 residue to generate a ubiquitin chain on OMM proteins. The ubiquitin-tagged mitochondrion is recognized by autophagy adapters such as P62 and then subject to LC3 for autophagy [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Compromised mitophagy was confirmed in PD and the rescue of mitophagy defects has been a potential therapy of PD [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Here, we found the expressions of PINK1, and Parkin were decreased with MT1 deficits in vitro but not in vitro. The discrepancies between in vitro and in vivo models are probably because of the complex physiological conditions in the central nervous system, such as the interactions and signaling among neurons and glial cells. This effect is not similar to melatonin, which reported to be neuroprotective in PD by mitophagy augment [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Mitophagy is a selective autophagic removal process of damaged mitochondria. Since exacerbated LC3II/LC3I and P62 accumulation were observed in MT1 depleted PD mice, it is reasonable to speculate the autophagy of aggregates was also inhibited. Although we didn\u0026rsquo;t observe the TH loss in striatum and motor abnormality by MT1 knockout, the alteration of LC3II/LC3I ratio and P62 accumulation were found. This indicates the absence of MT1 may increase the susceptibility of PD occurrence through autophagy inhibition but is not the key etiological factor.\u003c/p\u003e\u003cp\u003eAggregation of misfolded α-syn in dopaminergic neurons in substantia nigra is called Lewy bodies, which further cause the neuronal degeneration [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The mechanism underlying this phenomenon is attributed to lysosome-autophagy or ubiquitin-proteasome pathway. PFFs treatment impaired autophagic flux and lysosome induction, which in turn also resulted in α-syn aggregates expansion seeding by PFFs [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Here, we employed α-syn modified PFFs models and identified autophagy as the potential pathway of MT1 to alleviate α-syn aggregation. LC3B-II is a marker of autophagosome formation and accumulation by adding phosphatidylethanolamine (PE) on LC3-I. As an autophagic adaptor, P62 transported ubiquitinated cargo to autophagosomes through binding with LC3-II. Failure of autophagosomes turnover or autolysosome fusion will lead to accumulation of P62. LC3-II and P62 were increased after PFFs treatment, indicating an interruption of autophagy [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In the present study, MT1-KO exacerbated the autophagy inhibition induced by PFFs, and overexpression of MT1 promoted autophagy by reducing P62 levels, which demonstrated the pivotal role of MT1 in autophagy induction. BafA1 was an autophagy inhibitor, used to assess the autophagy flux when LC3-II was increased [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Lysosome inhibition treated with BafA1 impaired fusion of autophagosomes with lysosome, causing LC3-II to elevate significantly. We not only found that MT1 overexpression induced LC3-II expression after BafA1 treatment but also corroborated MT1 function in improving autophagic flux via mCherry-LC3-EGFP lentivirus infection. Increased red dots after MT1 overexpression directly manifested the successful fusion of autophagosomes with lysosomes. Moreover, the autolysosomes were reduced in MT1 group when treated with BafA1, further confirming the regulation of MT1 in autophagic flux. The autophagy process acceleration induced by MT1 further improved α-syn degradation in lysosome. Therefore, alleviating α-syn aggregation through MT1 induced autophagy could be considered an effective way to slow PD progression instead of healing. MT1 is also present in mitochondrion but more abundant in plasma membrane [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. It is presumably that mitochondrial MT1 works on mitochondria dynamics, but less possible to drive autophagosome formation.\u003c/p\u003e\u003cp\u003eHowever, there are also some limitations within our work. First, the MT1-KO mice used here are not conditional knock out mice, it\u0026rsquo;s better to establish the DAT-cre; MT1\u003csup\u003eflox/flox\u003c/sup\u003e mice to study the MT1 function in PD more precisely. Second, the MT1-KO mice presented the relationship between MT1 and PD, only the protective effects of MT1 in reversing mitochondria and autophagy defects induced by MPTP or PFF can corroborate the indispensable role of MT1 in PD in vivo. Third, the acute MPTP models weaken the possibility of the relevance with chronic and aging associated PD. Collectively, our findings in illustrating the MT1 function on mitochondria dynamics and autophagy related to PD, provide a favorable therapeutic target of mitigating mitochondria dysfunction and α-syn aggregation through MT1 to prevent the occurrence of PD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of Interest\u003c/h2\u003e\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eOur study was supported by the Science and Technology Innovation Project of Xiongan New Area (2023XAGG0073), the National Natural Science Foundation of China (82471269), Jiangsu Provincial Medical Key Discipline (ZDXK202217), Suzhou Key Laboratory (SZS2023015), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\u003cp\u003eJY Liu and F Wang conceived and designed the study, XB Wang wrote the manuscript, XB Wang, LL Qi, JM Wang, YR Sun, QK Lv, BE Cao and SM Jiang performed the experiments and analyzed the data, QH Ma and CF Liu reviewed and edited the manuscript. All authors approved the final version of the paper.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eThe authors thank Doctor Guanghui Wang of the Soochow University for providing α-syn-GFP HEK293T stable cell lines.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThe datasets used in the present study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJankovic J (2008) Parkinson\u0026rsquo;s disease and movement disorders: moving forward. Lancet Neurol 7:9\u0026ndash;11. https://doi.org/10.1016/S1474-4422(07)70302-2\u003c/li\u003e\n\u003cli\u003eAtik A, Stewart T, Zhang J (2016) Alpha-Synuclein as a Biomarker for Parkinson\u0026rsquo;s Disease. 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Proc Natl Acad Sci U S A 114:E7997\u0026ndash;E8006. https://doi.org/10.1073/pnas.1705768114\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"melatonin receptor MT1, mitochondria dynamics, MPTP, Parkinson’s disease, α-synuclein, autophagy","lastPublishedDoi":"10.21203/rs.3.rs-7058166/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7058166/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is a neurodegenerative disease characterized by dopaminergic neurons loss and Lewy body presence in the substantia nigra. Abnormal mitochondrial function and accumulated alpha-synuclein (α-syn) are key etiology of PD. Melatonin type receptor 1 (MT1) regulates sleep upon activation by melatonin and is suggested to decrease in PD patients. However, the role of MT1 in PD pathogenesis remains elusive. In this study, we knocked down MT1 in SH-SY5Y neuroblastoma cells and found MT1 loss caused mitochondria dysfunction. Moreover, live cell imaging of MitoTracker staining and transmission electron microscope (TEM) proved that MT1 knockdown affected mitochondria morphology. The expression of mitochondria fission protein DRP1 was increased and the fusion protein OPA1, MFN1 and MFN2 was decreased. This is probably attributed to the declined phosphorylation of DRP1 at S637 by PKA and increased phosphorylation at S616 by ERK1/2. Moreover, MT1 knockdown also impaired mitophagy, manifested by declined PINK1 and Parkin. In a MPTP induced PD mouse model, MT1 deficiency altered the mitochondria fission through the same mechanism as in vitro but did not impair mitophagy, tyrosine hydroxylase (TH) expression and mice movement. However, MPTP induced autophagy inhibition was exacerbated in MT1 KO mice. Neuronal MT1 deficiency aggravated preformed fibrils (PFFs) induced autophagy inhibition and α-syn aggregation. Overexpression of MT1 reduced mitochondria fission, as well as increased LC3-II expression and decreased P62 accumulation to promote autophagy in HEK293T cells, thus mitigating the aggregation of α-syn. Autophagy flux indicated by mCherry-LC3-II-EGFP fluorescence was also enhanced after MT1 overexpression. Together, our study demonstrates the function of MT1 in mitochondria and autophagy, which sheds further light on PD prevention targeting MT1.\u003c/p\u003e","manuscriptTitle":"Regulation of mitochondrial dynamics and function by MT1 melatonin receptor in Parkinson’s disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-29 09:21:42","doi":"10.21203/rs.3.rs-7058166/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2025-08-25T23:17:11+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-07-26T03:39:42+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-25T02:37:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-07T15:27:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2025-07-06T09:47:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9ff7a0f2-ae7c-4fcc-a48e-c7a4814fa24f","owner":[],"postedDate":"July 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:12:52+00:00","versionOfRecord":{"articleIdentity":"rs-7058166","link":"https://doi.org/10.1007/s00018-025-05995-0","journal":{"identity":"cellular-and-molecular-life-sciences","isVorOnly":false,"title":"Cellular and Molecular Life Sciences"},"publishedOn":"2025-11-25 15:58:47","publishedOnDateReadable":"November 25th, 2025"},"versionCreatedAt":"2025-07-29 09:21:42","video":"","vorDoi":"10.1007/s00018-025-05995-0","vorDoiUrl":"https://doi.org/10.1007/s00018-025-05995-0","workflowStages":[]},"version":"v1","identity":"rs-7058166","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7058166","identity":"rs-7058166","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}