Pivotal roles of mitochondria in linking dopamine catabolism to axonal myelination: Implication for the pathogenesis and treatment of schizophrenia

Pivotal roles of mitochondria in linking dopamine catabolism to axonal myelination: Implication for the pathogenesis and treatment of schizophrenia | 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 Article Pivotal roles of mitochondria in linking dopamine catabolism to axonal myelination: Implication for the pathogenesis and treatment of schizophrenia Haiyun Xu, Fan Yang, Yi Zhang, Cuiting Jiang, Na Ouyang, Qianqian Wang, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3875841/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Schizophrenia is one of the most complex and debilitating brain diseases. Patients with schizophrenia may present various clinical manifestations that have been categorized into positive symptoms , negative symptoms, and cognitive deficits. In relation to these complex clinical manifestations, multiple hypotheses have been proposed to understand the pathogenesis of schizophrenia, such as the so-called dopamine (DA) hypothesis, mitochondrion hypothesis, oligodendrocyte (OL) hypothesis, etc. The concurrent existence of multiple hypotheses about one brain disease suggests a possible common neurobiological mechanism linking some of these hypotheses. This possible neurobiological mechanism has been demonstrated in this study with animal models of schizophrenia, cultured OLs, and neuron-OL co-cultures. Adolescent C57BL/6 mice given tolcapone (TOL) for two weeks showed DA elevation in prefrontal cortex (PFC), functional impairment of mitochondria in brain cells, and hypomyelination in PFC, hippocampus, and caudate putamen (CPu) in a dose-dependent manner, in addition to schizophrenia-related behaviors. The catechol-O-methyltransferase (COMT) gene knock-out (COMT-ko) mice presented dopaminergic dysfunctions in PFC and CPu, functional deficit of mitochondria, mature OL decrease, and hypomyelination in the same brain regions as those in TOL-treated mice. In cultured OLs, DA inhibited the cell development in a concentration-dependent manner while impairing mitochondrial functions. These effects of DA on cultured cells were ameliorated by the antioxidant N-acetyl-L-cysteine (NAC) and trans-2-phenylcyclopropy (TCP), an inhibitor of mitochondrial monoamine oxidases (MAOs). Moreover, DA inhibited axonal myelination in neuron-OL co-cultures while impairing mitochondrial functions. These data demonstrate the pivotal roles of mitochondria in linking DA catabolism to axonal myelination in the brain and provide a novel insight into the pathogenesis and therapeutic strategy for schizophrenia. Health sciences/Diseases/Psychiatric disorders/Schizophrenia Biological sciences/Neuroscience dopamine mitochondria oligodendrocyte hypomyelination schizophrenia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Schizophrenia is one of the most complex and debilitating brain diseases. Patients with schizophrenia may present various clinical manifestations that have been categorized into positive symptoms including hallucinations, delusions, disorganized thinking, and grossly disorganized or abnormal motor behavior, negative symptoms of apathy, anhedonia, and social withdrawal, as well as cognitive deficits encompassing deficits in attention, working memory, and executive function. While positive symptoms are often the reason for schizophrenia patients to see a psychiatrist, negative and cognitive symptoms are largely responsible for the long-term burden associated with the disease [ 1 ]. Indeed, the existing antipsychotics do not markedly improve negative symptoms and cognitive impairment although they show therapeutic efficacy on positive symptoms in most of patients with schizophrenia [ 2 , 3 ]. About 70% of patients with schizophrenia require long-term, even lifetime, medication to control their symptoms but do not achieve complete recovery [ 4 ]. These unmet clinical challenges are attributed to an incomplete understanding of the pathogenesis behind the complex clinical manifestations of schizophrenia. In the effort to understand the pathogenesis of schizophrenia, researchers have proposed several hypotheses over the past decades based on findings from preclinical and clinical studies. Of these hypotheses, the first one is the so-called dopamine (DA) hypothesis, the most enduring theory in schizophrenia research. It initially emphasized a role of hyperdopaminergia in the etiology of schizophrenia [ 5 ], followed by a modified version which specifies a subcortical hyperdopaminergia along with the prefrontal hypodopaminergia [ 6 , 7 ]. The others include the mitochondrion hypothesis [ 8 , 9 ], neurodevelopment hypothesis [ 10 ], and oligodendrocyte (OL) hypothesis [ 11 ], to name a few. The coexistence of multiple hypotheses about schizophrenia not only reflects the extreme complexity of this brain disease, but also indicates the existence of a biological mechanism being able to link these hypotheses and form an inclusive theory about the pathogenesis of schizophrenia. In line with this suggestion, previous studies have shown that, on the one hand, DA oxidation products disrupt mitochondrial function [ 12 , 13 ], and on the other hand, DA catabolism may be inhibited due to mitochondrial dysfunction as monoamine oxidases (MAO-A and MAO-B) locate in mitochondria and are responsible for the oxidative inactivation of neurotransmitter amines [ 12 , 14 , 15 ]. As such, the interplay between DA metabolism and mitochondrial defects has been considered an important neurobiological mechanism involved in the pathogenesis of schizophrenia [ 16 ]. Moreover, recent studies have linked the co-working of neuronal and oligodendroglial mitochondria to axonal myelination in the brain of mouse subjected to social defeat stress [ 17 ] or maternal separation plus early weaning [ 18 ]. The experimental paradigms employed in these recent studies simulate environmental risk factors involved in the etiology of psychotic disorders [ 19 ]. Here we demonstrate the pivotal roles of mitochondria in linking DA catabolism to axonal myelination using animal models of schizophrenia, cultured OLs, and the neuron-OL co-culture. We asked whether dopaminergic changes (in mouse brain) could lead to hypomyelination and mitochondrial dysfunction while leading to behavioral abnormalities related to some of clinical manifestations in patients with schizophrenia, whether DA addition into cultured OLs and neuron-OL co-cultures could impair mitochondrial function and impact OL development/myelination, and whether an anti-oxidative treatment or manipulation of DA catabolism could protect against OL development retardation while attenuating mitochondrial dysfunction induced by DA elevation. Materials & methods Animals All experimental procedures applied to the animals in this study were reviewed and approved by the Institutional Animal Care and Use Committees at the Wenzhou Medical University and Shantou University Medical College (SUMC2019-53). The C57BL/6 mice used in the first animal experiment and pregnant Sprague–Dawley rats used for primary OLs culture and neuron-OL co-culture experiments were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd, China. The COMT -ko mice and wild type littermates in the second animal experiment were purchased from Shanghai Southern Model Biotechnology Co., Ltd. (Shanghai, China). The gene knockout mice were produced by means of CRISPR/Cas9-based homology-directed repair and confirmed by Western blot analysis showing undetectable COMT protein in brain tissue of them. The animals were group-housed under controlled temperature (22° to 25°C) and relative humidity (50–60%) with a 12-hour light-dark cycle (lights on at 08:00) and free accesses to chow food and water. Drug administration In the first animal experiment, C57BL/6 mice were intraperitoneally given the vehicle consisting of 1% DMSO and 0.1% Tween-80 in sterilized saline, or TOL (Med Chem Express, HY-17406) in the vehicle at 15, 30, or 60 mg/kg for consecutive 14 days starting on PD 22. The intraperitoneal injection was done once a day at a same volume of 2 mL/100 g. The four groups of mice were referred to as VEH, TOL15, TOL30, and TOL60, respectively. Animal behavioral tests The open-field test (OFT) was done with the mice in the both animal experiments to measure locomotor activity and anxiety level of them. Briefly, a mouse was placed in the center of an open-field box (50 cm × 50 cm × 35 cm) and tested for 10 min during which period the mouse was allowed to move freely. The moving trajectory of the mouse on the floor of the box was recorded by a video-camera placed above the arena. A video tracking software (Noldus Information Technology, Wageningen, Netherlands) was used to record the total distance (TD) moved on the whole arena and that on the central (CD) and peripheral zones. The same software was also used for the other behavioral tests in this study. The Y-maze test was performed with the mice in the first animal experiment to assess the exploring behavior and spatial working memory of them. In brief, a mouse was placed at the converging area of a symmetrical Y-maze (30 cm × 8 cm × 5 cm) and allowed to move freely through the maze for 8 min. The total number and series of arm entry were recorded. Alternation is defined as successive entries into the three arms. Spontaneous alternation is calculated as the percentage of actual to possible alternations (defined as the total number of arm entries minus 2). Novel object recognition (NOR) test consists of three phases. During the adaptation phase, a mouse was allowed to move freely in the open field box for 5 min. Twenty-four hours later, the same mouse was put back to the open field box to explore the arena with two identical wood blocks for 5 min (training phase). Three hours later, the mouse was allowed to explore two different wood blocks for additional 5 min (discrimination phase). One block was the same one used in the training phase (familiar object), another one was new with a different color and shape (novel object). The recognition index (RI) is calculated according to the formula RI = 100 × Tn/(Tn + Tf), where Tn represents the exploration time (S) for a novel object and Tf represents the exploration time (S) for a familiar object. The puzzle box test (POT) was conducted with the mice in a Plexiglass white box which is divided by a removable barrier into two compartments: a brightly lit start zone (58 cm × 28 cm) and a smaller covered goal zone (15 cm × 28 cm). A mouse was introduced into the start zone and trained to move into the goal zone through a narrow underpass (~ 4 cm wide) located under the barrier. Then the mouse underwent a total of nine trials (T1–T9) over 3 consecutive days, with three trials per day. This sequence allows assessment of problem-solving ability (T5 and T8), and learning/short-term memory of instrumental responses (T3, T6 and T9), while the next day repetition provides a measure of long-term memory (T4 and T7). Performance of a mouse in the POT is assessed by measuring the time in seconds for the subject to arrive at the goal zone from the start zone. The POT has been used successfully to assess the problem solving ability of mice, as well as cognitive deficits exhibited in murine models of schizophrenia [ 20 ]. The elevated-plus maze (EPM) test with the mice in the second animal experiment. The EPM consists of four radial arms (two closed, 50 × 10 × 40 cm; two open, 50 × 10 × 2 cm) elevated 60 cm above the floor. Under the same lighting condition as that in the open-field test, a mouse was placed at the central zone, facing a closed arm, and the activity of the mouse on the EPM was recorded during the subsequent 10 min. The first 2 min were defined as the adaptation period and the performance of the mouse in the remaining 8 min was analyzed. The time spent by a tested mouse on the central zone, open and closed arms, and the number of entries to these locations were recorded. The social interaction test (SIT) was carried out with the mice in the second animal experiment. It consists of two sessions of 150 second (S) and a one minute interval between the two sessions as described previously [ 21 ]. During the first session, an empty (E session) wire mesh cage (12 × 12 × 18 cm) was placed at one end of an open-field arena (100 × 100 cm) where a tested mouse was allowed to move freely. During the second session, the conditions were identical except that an unfamiliar conspecific partner (C session) had been introduced into the cage before a tested mouse was placed in the open-field box. Between the two test sessions, the tested mouse was removed from the box and placed back into his/her home cage for 60 S. The time spent by the tested mouse at the interaction zone (a 16-cm-wide corridor around a cage) was recorded. High-pressure liquid chromatograph Under a deep anesthesia with 1.25% avertin (Sigma-Aldrich, T48402; i.p.), the mouse brain was removed out of the skull. Following the protocol described in a previous study [ 22 ], concentrations of the neurotransmitters of DA, norepinephrine (NE), and serotonin (5-HT) in the brain regions of PFC, hippocampus, and CPu (n = 6/group) were measured by means of high-pressure liquid chromatograph (HPLC) and quantified using known standard concentrations of chemically pure DA (Sigma-Aldrich, H8502), NE (Sigma-Aldrich, A7257), and 5-HT (Solarbio, SS9080). Histological and immunohistochemical staining Under a deep anesthesia with 1.25% avertin (i.p.), the mouse was transcardially perfused with the phosphate buffer (PB, pH = 7.4) followed by 4% paraformaldehyde in the PB. Then the whole mouse brain was removed out of the skull and immersed in the fixative for additional 2 days. After dehydration in graded concentrations of alcohol and xylene, tissue blocks were embedded in paraffin and sectioned at 5 µm thickness. For Nissl staining, sections were stained in warmed cresyl violet acetate solution (Solarbio, G1430). Immunohistochemical staining was done with brain sections of mice in the two animal experiments separately. In the first animal experiment, the paraffin sections (5 µm thickness) were incubated with the primary antibody to APC (Abcam, ab16794, 1:500) to label mature OLs. In the second experiment, frozen sections (20 µm) were incubated with the primary antibody to GST-π (1:100; Boster Biological Technology Co. Ltd; Wuhan, China). The primary antibody to MBP (1:200; Boster Biological Technology Co. Ltd; Wuhan, China) was used to label myelin sheath. Images of fixed specific areas in PFC, CPu and hippocampus were digitally recorded using a Nikon ECLIPSE Ni light microscope (Nikon, Japan) equipped with a digital capture system. The mature OLs and MBP-immunoreactivity intensity were measured using the Image-J software (version win64, National Institutes of Health, US). Transmission electron microscopy Three mice from each of VEH and TOL60 groups in the first animal experiment were used for transmission electron microscopy (TEM) analysis. The mice were anesthetized with 1.25% avertin and subjected to transcardial perfusion of 0.1 M PB and the fixative of 2.5% glutaraldehyde and 4% paraformaldehyde in PB. The cerebral cortex was dissected and immersed in 2.5% glutaraldehyde. Then the cerebral cortex was cut in 500 µm thick coronary sections using two thin blades. PFC was identified as the brain region of interest and was washed in 0.1 M PB followed by postfixed in 1% osmium tetroxide for 1 h. After contrasting in uranyl acetate for 1 h, the tissue was dehydrated in acetone and embedded in Epoxy Resin (SPI Supplies, 90529-77-4). One µm semithin sections were cut using a ultramicrotome (PowerTome-XL, RMC, US) and examined by light microscopy. Then, the selected and comparable regions of the semithin sections were trimmed and 70 nm ultrathin sections were made, contrasted with lead citrate and uranyl acetate and examined under a transmission electron microscope Hitachi H-7500. To quantify the myelinated axons, a total of 27 microphotographs were recorded for each group (3 mice × 3 sections × 3 photographs) and analyzed using the Image-J software. Primary OLs culture Primary culture of oligodendroglial lineage cells was done by referring to a previous study [ 23 ]. The cerebral hemispheres of S-D rat embryos at E16-18 were dissociated and transferred to the Accutase detachment solution (Sigma-Aldrich, A6964) at 37°C for 10 min. Then the tissue solution was moved into a centrifuge tube containing 5 mL of DMEM/F12 (Gbico,11330032), gently blowed, and stood for a few min followed by filtration through a 70 µm Nitex mesh to form a suspension of isolated cells in the medium. After cell counting, cells were seeded (at a density of 1 × 10 5 /cm 2 ) into poly-D-Lysine (Sigma-Aldrich, P6407) pre-coated T75 culture flasks containing the medium. The medium was changed every 3 days until DIV9. The cultured cells in the T75 flask were shaken at 150 rpm for 60 min to isolate microglia, and subsequently shaken at 250 rpm for 15–20 h to isolate the oligodendrocyte precursor cells (OPC) from astrocytes. After centrifugation at 1,800 rpm for 6 min, the precipitate was resuspended and seeded onto chamber slides or well plates at appropriate densities within the OPC cultivation medium [DMEM/F12 supplemented with 2% B27 (Gbico, A35828-01), 10 ng/mL fibroblast growth factor-2 (FGF-2; Peprotech, 100-18B), 1% Pen/Strep (Gbico,15140122), 10 ng/mL PDGF-AA (Peprotech, 100-13A), and 0.5% FBS (Gbico,16000-044)] for 2 days. Then the OPC cultivation medium was replaced with fresh OPC differentiation medium [DMEM/F12 supplemented with 1% N2 (Gbico,17502-048), 0.5% FBS, 1% Pen/Strep, 10 nM corticosterone (Amresco, IC0550), 10 nM D-biotin (Thermo Fisher Scientific, B20656), and 30 nM triiodothyronine (Solarbio, IT1110)] for further culture for additional 2 days. Various concentrations of DA (vehicle, 50, 100, 200 µM) were added into the medium in three different schedules of the first two days, the last two days, and the first two days followed by culture with replaced medium having no DA, to examine effects of DA on the differentiation and maturation of oligodendroglial lineage cells. In the NAC (N-acetyl-L-cysteine) experiment, cultured OLs were pretreated with DA at 50 µM for 4 h followed by subsequent incubation for 48 h in the presence of NAC (Beyotime Biotechnology, ST1546) at concentrations of 250 µM (NAC-l) or 500 µM (NAC-h). Controls include those treated with DA (50 µM) alone, NAC-h alone, and negative control lacking both DA and NAC. In the TCP (trans-2-phenylcyclopropy) experiment, primary OLs were cultured at various concentrations of DA (0, 100 µM, 200 µM) in the absence or presence of TCP (100 µM) during DIV 15–17. At the end, the mature OLs were analyzed for cell viability, ΔψM, and the production of mitochondrial ROS and ATP, in addition to Western blot analysis measuring CNP and MBP levels. Neuron-OL co-culture For neuron-OL co-culture, the neocortex rotation-mediated aggregate cell culture protocol was followed as described previously with modifications [ 24 ]. The cerebral hemispheres of E16–18 SD rat fetuses were dissociated as described previously. The culture medium for the co-culture was DMEM/F12 supplemented with 2% B27 Plus,1% FBS,1% penicillin/streptomycin, 50 ng/mL β-NGF, and 50 µg/mL vitamin C. The medium was replaced every three days until DIV 42. During DIV14-42, various concentrations of DA (vehicle, 50, 100, 200 µM) were added into the medium to examine effect of DA on axonal myelination in the neuron-OL co-cultures. Cell culture immunofluorescence Cultured cells were fixed with 4% paraformaldehyde for 15 min and then washed three times in PBS. Then, the fixed cells were permeabilized with 0.1% Triton-X in PBS for 10 min and later washed with PBS three times, and the cells were then blocked with 10% goat serum (Gibco, USA) in PBS for 60 min at room temperature. After removal of the blocking reagent, the cells were incubated with one or two of the following primary antibodies including rabbit anti-Oligo-2 (1:500, Ab9610, Sigma-Aldrich), mouse anti-Oligo-4 (1:100, O7139, Sigma), mouse anti-CNP (1: 200, ab6319, Abcam), and rabbit anti-MBP (1:200, ab40390, Abcam), mouse anti-MBP (1:200, MA5-35001, Thermo Fisher Scientific) and rabbit anti-NF-H (1:500, 18934-1-AP, Proteintech) at 4°C overnight. After rinsing, the cells were incubated with goat anti-rabbit IgG H&L (Alexa Fluor 594, 1:2000, A-11012, Thermo Fisher Scientific) or goat anti-mouse IgG H&L (Alexa Fluor 488, 1:2000, A-11001, Thermo Fisher Scientific) for 1 h at room temperature. Then, the cells were washed with PBS, and the nucleus was stained with the fluorescent dye DAPI for 5 min. In addition, the primary antibody mouse anti-cleaved Caspase-3 (1:200, 66470-2-Ig, Proteintech) was used to label apoptotic OLs due to the cytotoxicity of DA. All the cultured cells were visualized by indirect fluorescence under the fluorescent microscope (Nikon ECLIPSE Ni, Nikon, Japan). Western blot analysis Proteins were extracted from brain tissue or cultured cells using a Tris-ethylenediaminetetraacetic acid (EDTA) lysis buffer (1% Triton X-100, 10% glycerol, 20 mM Tris, pH 7.5, and 1 mM EDTA) with freshly added Protease Inhibitor Cocktail (Sigma-Aldrich). After measurement of protein concentrations using a BCA kit (Sangon Biotech, C503021), sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting were carried out. The antibodies used for all the western blot analyses in this study are shown in Supplemental Table 1. The antigen-antibody complexes were visualized by using an ECL detection kit (P10100, NCM Biotech). Quantification of the immunoblots was carried out by densitometric analysis of chemiluminescence exposed films, using Image-Lab software (version 4.0) and the results recorded as arbitrary densitometric units. Functional assessments of mitochondria Mitochondrial functions of brain cells and cultured OLs and neurons were assessed by various techniques. First, Western blot analysis was done with PFC tissue samples and neuron-OLs co-cultures to measure expression levels of mitochondrial complexes (CI, CII, CIII, CIV, and CV) as described before. Second, the cell viability of cultured OLs was assessed using the Cell Counting Kit-8 (CCK-8, HY-K0301, MedChem Express) following the protocol provided by the manufacturer. This sensitive colorimetric assay allows accurate live cell counting in a cell proliferation or cytotoxicity assays. Third, mitochondrial membrane potential (ΔψM) was measured using the JC-1staining assay kit (M8650, Solarbio). In healthy cells, JC-1 selectively enters into mitochondria and forms J-aggregates with intense red fluorescence. In apoptotic or unhealthy cells, JC-1 remains in the monomeric form showing only green fluorescence. Fourth, ROS in cultured cells were assessed using the DCFDA cellular ROS assay kit (S0033S, Beyotime Biotechnology) and following the instructions provided by the manufacturer. Fifth, ATP concentration in brain cells of mice was assessed using a commercial ATP assay kit (Beyotime Biotechnology, Nanjing, China). In the presence of magnesium, oxygen and ATP, the protein luciferase catalyzes oxidation of the substrate luciferin, which is associated with light emission. RT-qPCR Total RNA was extracted from the caudate putamen and PFC of mouse brain using TriZol (Invitrogen, Shanghai, China). cDNA was generated from total RNA samples using the ExScript RT-PCR Kit (cat.# RR047A, TaKaRa, Japan) according to manufacturer’s protocol. qPCR was performed using SYBR® Premix Ex Taq™ (Tli RNaseH Plus; cat.#RR820A, TaKaRa, Japan) following the instruction recommended by the manufacturer. β-actin mRNA was served as the internal control. Template RNA was replaced with PCR-grade water as a negative control. After amplification, melting curve analysis and length verification by gel electrophoresis were carried out to confirm the specificity of PCR products. Each sample was analyzed in triplicate. The relative levels of tested mRNA were calculated by normalization to the endogenous β-actin mRNA expression prior to comparative analysis using the comparative threshold cycle (2 –∆∆Ct ) method. The primers employed for aforementioned PCRs are shown in Supplemental Table 2. Statistical analysis Graphpad prism (GraphPad Software, version 8.0) was used for statistical analysis. All data were expressed as mean ± standard deviation (Mean ± SD) and analyzed by independent sample Student’s t -test or one-way ANOVA followed by Tukey's multiple comparisons. When a p-value was less than 0.05, the difference was considered significant. Results Tolcapone-treated mice exhibit DA elevation, hypomyelination, mitochondrial dysfunction in brain cells, and behavioral abnormalities To determine if DA elevation influences white matter development and impairs mitochondrial functions of brain cells while inducing behavioral anomalies, we administered TOL to C57BL/6 mice intraperitoneally at the dose of 0, 15, 30, or 60 mg/kg, for consecutive 14 days starting on postnatal day (PD) 22. TOL is a brain penetrant selective inhibitor of COMT devoid of psychostimulant properties [ 25 ]. The COMT enzyme plays a pivotal role in DA metabolism, specifically in PFC [ 26 ]. The four groups of mice were referred to as VEH, TOL15, TOL30, and TOL60 (n = 17/group), respectively. One day after the last TOL administration, the animals were subjected to OFT measuring their locomotor activity and anxiety-like behavior, the Y-maze test assessing spatial working memory, NOR test evaluating recognition memory, and POT examining general cognition and executive function, in the order. Compared to VEH group, the groups TOL30 and TOL60 showed higher anxiety levels indicated by significantly lower CD/TD (p < 0.01, p < 0.05, respectively, Fig. 1A). The TOL60 group also presented spatial working memory impairment indicated by significantly lower spontaneous alternation in the Y-maze test (p < 0.05, Fig. 1B). In addition, the TOL60 group showed a significantly lower RI as compared to the VEH group (p < 0.05, Fig. 1C), suggesting a recognition memory impairment. Moreover, TOL30 and TOL60 groups showed impairment in problem solving ability and executive function revealed by POT in which mice in TOL30 and TOL60 groups took longer duration to complete the task in T5 and T8 (p < 0.001, p < 0.05, respectively, Fig. 1D). One day after the last behavioral test, five mice in each group were euthanized and the brain regions of PFC, CPu, and hippocampus were isolated and used for HPLC analysis to measure levels of DA, norepinephrine (NE), and 5-hydroxytryptamine (5-HT). As for DA levels, one-way ANOVA revealed significant effects of TOL on DA level in PFC (p < 0.01). Post-hoc comparisons showed significantly higher DA level in PFC (p < 0.05) of the mice in TOL60 group, compared to the VEH group (Fig. 1E). Regarding NE level, one-way ANOVA revealed a significant effect of TOL on this index in the hippocampus (p < 0.01). Post-hoc comparisons showed significantly higher NE levels in hippocampus of the mice in TOL30 and TOL60 groups (p < 0.05 in both comparisons, Fig. 1F). Also, TOL30 group had a higher level of 5-HT in hippocampus compared to the VEH group (p < 0.05, Fig. 1G). Immunohistochemical staining with the primary antibody to APC (adenomatous polyposis coli gene clone CC1 used as the biomarker of mature OL cell body) showed dose-dependent decreases in the number of APC + cells in PFC of TOL-treated mice (Fig. 2A & B). Relevantly, Western blot analysis revealed dose-dependent decreases in protein levels of CNP, MAG, MBP, and MOG in PFC of TOL groups compared to VEH group (Fig. 2C & D). To provide further evidence for the hypomyelination in TOL-treated mice, PFC samples of mice in VEH and TOL60 groups were prepared for TEM analysis. The two groups look seemingly different in numeral density of myelinated axon and myelin sheath thickness as shown in Fig. 2E. Indeed, quantitative data revealed a lower numeral density of myelinated axon in the TOL60 group (p < 0.001, Fig. 2F), a higher G-ratio (the ratio of the inner to the outer diameter of the myelin sheath of a myelinated axon) in the TOL60 group (p < 0.001, Fig. 2G), indicating thinner myelin sheath of the myelinated axons in this group. But, the two groups were comparable in axon diameter (p = ns, Fig. 2H). Moreover, the TOL60 group showed a smaller slope of the correlation between axonal diameter and G-ratio (p < 0.001, Fig. 2I) compared to the VEH group, suggesting that the smaller axons were more susceptible to DA elevation in PFC of the TOL-treated mice. Similar to the findings in PFC, TOL administration decreased the number of APC + cells (Supplemental Fig. 1A & B) and expression levels of CNP, MBP, and MOG in the mouse hippocampus (Supplemental Fig. 1C & D). As for CPu, TOL30 and TOL60 groups showed fewer APC + cells relative to VEH group (Supplemental Fig. 1E & F), but the four groups were comparable in protein levels of CNP and MBP in this brain region (Supplemental Fig. 1G & H). The coexistence of DA elevation, APC + cell decrease, and hypomyelination in the TOL-treated mice raised a question, namely, how did DA elevation lead to mature OL decrease and hypomyelination in the mouse brain? We hypothesized that DA elevation impaired mitochondrial function thus inhibited OL maturation and axonal myelination as intact mitochondria in both neurons and OLs are essential for axonal myelination [ 27 , 28 ]. To test this hypothesis, we assessed mitochondrial functions of brain cells of the mice in this experiment. Western blot analysis showed significantly lower levels of mitochondrial complexes I, II and IV in neural cells of mouse PFC in TOL30 and TOL60 groups relative to the VEH group, while the other two complexes (III & V) were comparable across the groups (Fig. 3A & B). In addition, TOL groups show lower levels of NAT8L (N-acetyltransferase 8-like) in mouse PFC (Fig. 3C & D). This enzyme catalyzes the synthesis of NAA from aspartate and acetyl-CoA in neuronal mitochondria. The neuronal NAA is then transported to the cytoplasm of OLs, where aspartoacylase (ASPA) cleaves the acetate moiety of NAA for use in the synthesis of fatty acid and steroid, the building blocks for myelin lipid synthesis [ 29 , 30 ]. Furthermore, the TOL30 and TOL60 groups had significantly lower levels of ATP compared to VEH group (Fig. 3E). However, no difference was found between VEH and TOL groups in neuron number in the PFC (Supplemental Fig. 2), suggesting that neurons are more tolerable to mitochondrial dysfunction than OLs, the number of which decreased in TOL-treated mice as described above. This statement is consistent with a recent study reporting that neurons among the neural cells in mouse brain are the most tolerable to mitochondrial damage by cuprizone [ 31 ], which is a copper chelator and toxic to mitochondria [ 31 , 32 ]. COMT -ko mice exhibit dopaminergic changes, hypomyelination, mitochondrial dysfunction in brain cells, and behavioral abnormalities To substantiate the aforementioned changes in TOL-treated mice, we assessed and compared dopaminergic measurements of wild type (wt) and COMT -ko mice by means of various techniques. The COMT gene is located in a fragment of chromosome 22q11 which when deleted results in a complex syndrome including the psychiatric manifestations such as schizophrenia. As such, the COMT gene has been placed near the top of a list of plausible candidate genes for schizophrenia [ 33 ] and the gene variants may be involved in the pathogenesis of psychotic symptoms, and associated especially with negative symptom in schizophrenia [ 34 , 35 ]. We assessed protein levels of the enzymes relevant to DA metabolism including monoamine oxidase-A (MAO-A), MAO-B, and Dopa decarboxylase (DDC), in addition to COMT in wild type and COMT- ko mice (n = 7/group). Western blot analysis detected the presence of COMT protein in both PFC and CPu of the wt mice, but the absence in COMT -ko mice (Fig. 4A). Compared to the wt mice, COMT -ko mice showed lower MAO-A level in CPu (p < 0.05, Fig. 4B & C). Interestingly, COMT -ko mice showed MAO-B level changes in opposite directions in PFC and CPu, i.e. higher level in PFC (p < 0.05) but lower level in CPu (p < 0.01) as compared to wt mice (Fig. 4D & E). The two groups were comparable in DDC levels in both PFC and CPu (Fig. 4F & G). HPLC results showed that COMT -ko mice had a significantly lower DA level in CPu compared to the wt mice (p < 0.05), whereas the two groups were comparable in DA level in PFC (Fig. 4H). The RT-qPCR analysis showed a higher level of DR1 mRNA in PFC (p < 0.01), but lower levels of DR2 (p < 0.05) and DAT (p < 0.01) mRNAs in CPu of COMT -ko mice compared to the wt mice (Fig. 4I). Immunohistochemical staining showed decreased number of mature OLs in PFC of COMT -ko mice compared to wt mice (p < 0.05, Fig. 5A & B), but comparable MBP-immunostaining intensity between the two groups (p = ns, Fig. 5C & D). Western blot analysis revealed lower level of MBP protein in the same brain region relative to that of wt mice (p < 0.05, Fig. 5E & F). Biochemical analysis with the PFC tissue revealed a marginal significant difference (p = 0.07) in ATP level between the wt and COMT -ko mice (Fig. 5G), but no difference in ROS levels (p = ns, Fig. 5H). The changes in CPu of COMT -ko mice are similar to those in PFC, except that both ATP and ROS levels were significantly lower in COMT -ko mice compared to the wt mice (Supplemental Fig. 3). We also assessed behavioral performances of the mice in the second animal experiment. Compared to wt mice, the COMT -ko mice showed behavioral anomalies indicated by a higher level of locomotor activity and a higher CD/TD in OFT (Supplemental Fig. 4A & B), longer duration on open arms and the central zone, but shorter duration in the closed arms of the EPM (Supplemental Fig. 4C) while visiting both the closed arms and central zone more frequently (Supplemental Fig. 4D). In the SIT, COMT -ko mice were unable to tell an empty cage from an identical cage with a novel conspecifics (Supplemental Fig. 4E & F). DA inhibits the development of cultured OLs and induces OLs apoptosis via inhibiting mitochondrial functions of the cells All data from the above animal experiments strongly suggest a neurobiological mechanism in which DA elevation impairs mitochondrial function in brain cells thus inhibiting OL development/myelination process. To substantiate this neurobiological mechanism, we did in vitro experiments in which purified oligodendrocyte precursor cells (OPCs) were cultured in the absence or presence of DA at the concentrations of 50, 100, or 200 µM starting on DIV (day in vitro ) 12 and continuing for 48 hrs. Dual immunofluorescent staining with the primary antibodies to Olig 2 (O2) and O4, O2 and CNP (2',3'-cyclic nucleotide phosphodiesterase), or CNP and MBP, was done to label immature and mature OLs while nuclei of cells were stained with DAPI dye. As shown in Fig. 6A, all differentiated OLs at specific developmental stages appear in distinctive morphology (size and shape) and labeling (color). Cell counting revealed: 1) no difference between VEH and DA groups in numbers of DAPI + cell nuclei and of cells labeled by the antibody to O2 (Fig. 6B), which is a sustained marker of OLs and expressed in all stages of OL development, from OPC to mature OL; 2) the ratio O4 + /O2 + cells (expressed as percentage, the same below) decreased in DA groups in a concentration-dependent manner (p < 0.001; Fig. 6C), indicating that DA inhibited the differentiation of OPC into immature OLs; 3) the ratios CNP + /O2 + (p < 0.001) cells and MBP + cells/DAPI + nuclei (p < 0.001) decreased in DA groups, but the ratio MBP + /CNP + cells did not change across VEH and all DA groups (p = ns, Fig. 6C). These data demonstrate that DA elevation retards the maturation of O4 + cells into CNP + and MBP + cells but has no impact on the further maturation from CNP + cells to MBP + cells. Furthermore, we analyzed effects of DA on mitochondrial functions of the cultured OLs. The mitochondrial membrane potential (ΔψM) of cultured OLs from the VEH and DA groups was assessed using an assay kit with JC-1. Compared to VEH group, JC-1 aggregates decreased in DA groups in a concentration-dependent manner indicated by gradual decreases in red signal which almost completely disappeared in the cells treated with the highest concentration of DA at 200 µM. In contrast, JC-1 monomers increased in DA-treated cells relative to VEH group indicated by green signal increase which was strongest in cells treated with DA at 100 µM (Fig. 6D). Therefore, ΔψM (FL590/FL530) values decreased in a DA concentration-dependent manner (p < 0.001, Fig. 6E). These data demonstrate the disruption of mitochondrial membrane potential in OLs of DA groups. In contrast, DA increased intracellular level of reactive oxygen species (ROS) in cultured OLs in a concentration-dependent manner (p < 0.001, Fig. 6F & G). The foregoing ROS and ΔψM data strongly suggest a cytotoxic effect (lethal effect at the highest concentration) of DA on cultured OLs. To verify this suggestion, the immunofluorescent staining with the antibody to cleaved Caspase-3 was done to label apoptotic OLs while the antibody to O2 was used to label nuclei of all OLs in the cultures without or with DA at the indicated concentrations. As shown in Fig. 6H & I, Caspase-3 + cells increased in DA-treated cultures in a concentration-dependent manner (p < 0.001) and the O2 + nuclei in cells treated with DA at 100 and 200 µM look much smaller than those in the VEH group, indicating the nuclear pyknosis of these damaged OLs and confirming the apoptotic OLs induced by the higher concentrations of DA. DA inhibits axonal myelination in neuron-OL co-cultures while decreasing NAT8L level in neurons To simulate the hypomyelination seen in the in vivo experiments, we did neuron-OL co-culture experiments by referring to the protocol in a previous study [ 24 ], without or with DA at the indicated concentrations. Neuronal axons were labeled by the antibody to neurofilament (NF-H) while myelin sheath was labeled by the antibody to MBP. Immunofluorescent staining of the co-cultures showed myelin sheath wrapping around neuronal axons in VEH group and the group of 20 µM DA, but rare or no myelin sheath in cultures exposed to 100 µM or 500 µM DA (Fig. 7A), indicating that DA did inhibit axonal myelination in the neuron-OL co-cultures. In line with this indication, Western blot analysis showed decreased levels of CNP and MBP in the co-cultures treated with DA in a concentration-dependent manner (p < 0.001, Fig. 7B & C). Moreover, DA decreased NAT8L level in the co-cultures at a concentration-dependent manner (p < 0.001, Fig. 7D & E). NAC and TCP ameliorate the adverse effects of DA on cultured OLs The above in vitro data strongly indicate that the inhibitory effects of DA on cultured OLs development and axonal myelination in neuron-OL co-cultures are achieved via impairing mitochondrial function. If so, mitochondrial protection approaches should be able to ameliorate the inhibitory effects of DA. To substantiate this possibility, we did another two in vitro experiments in which NAC (N-acetyl-L-cysteine, a well-established antioxidant) or TCP (trans-2-phenylcyclopropy, an inhibitor of mitochondrial MAOs) was used, respectively. In the TCP experiment, primary OLs were cultured at the indicated concentrations of DA (0, 100 µM, 200 µM) in the absence or presence of TCP (100 µM) during DIV 15–17. At the end, the mature OLs were analyzed for cell viability, ΔψM, and the production of mitochondrial ROS and ATP, in addition to Western blot analysis measuring CNP and MBP levels. As shown in Fig. 8A & B, both 100 µM and 200 µM DA significantly decreased CNP and MBP levels in cultured OLs as compared to CNT group, but these effects were prevented or ameliorated in the presence of 100 µM TCP. 200 µM DA significantly decreased cell viability of cultured OLs as compared to CNT group (p < 0.01), but this effect was effectively ameliorated in the presence of 100 µM TCP (p < 0.05, Fig. 8C). Moreover, 200 µM DA significantly decreased mitochondrial ΔψM compared to CNT group (p < 0.001) and this damaging effect was effectively ameliorated by TCP (p < 0.01, Fig. 8D). Relevantly, DA significantly decreased the production of ATP at both 100 µM (p < 0.01) and 200 µM (p < 0.001) as compared to CNT group, but these decreases were effectively ameliorated by TCP (p < 0.05 and p < 0.01 in the two cases, respectively; Fig. 8E). In contrast, addition of 200 µM DA significantly increased ROS production in cultured OLs relative to CNT group (p < 0.01), and this increase was not seen in the presence of TCP (p < 0.05, Fig. 8F). TCP alone did not impact any of the above measurements. These data indicate that the protection of TCP against the toxic effects of DA on OLs is achieved by inhibiting the catabolism of DA and consequently decreasing ROS production. In the NAC experiment, 250 or 500 µM NAC (the two concentrations were referred to as NAC-l and NAC-h, respectively) was provided to the primary OLs 4 h before addition of 50 µM DA. Forty-eight hours later, the cell viability of cultured OLs was assessed using the CCK-8 assay kit, in addition to dual immunofluorescent staining with the primary antibodies to CNP and MBP. The other OLs were treated with 50 µM DA or 500 µM NAC alone, and the cells in Control group were treated with the vehicle in the absence of DA and NAC. As shown in Supplemental Fig. 5A, CNP + and MBP + cells appear in all groups at various proportions. CCK-8 assay showed that NAC alone had no effect on cell viability of cultured OLs (p = ns), but 50 µM DA significantly decreased cell viability, compared to the Control group (p < 0.001). Importantly, NAC pre-treatment at both 250 and 500 µM concentrations completely prevented the DA-induced cell viability decrease (Supplemental Fig. 5B). Furthermore, DA alone (50 µM) significantly decreased numbers of CNP + and MBP + cells compared to VEH group (p < 0.01, p < 0.001, respectively), but these inhibiting effects were significantly ameliorated by NAC-l (p < 0.01) and NAC-h (p < 0.001) (Supplemental Fig. 5C). These data add further evidence for the antioxidative action of NAC which is effective in protecting OLs against the toxicity of DA. Discussion For the past several decades, dopaminergic dysfunction has been considered a chief culprit for clinical manifestations of patients with schizophrenia. White matter abnormality in patients with schizophrenia, however, was not reported in living patients until the development and application of magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI) techniques in schizophrenia research and clinical practice [ 36 ]. The widely replicated DTI findings in schizophrenia patients suggest that white matter abnormalities may be at least partially associated with dopaminergic dysfunction as exemplified by a neuroimaging study reporting that the COMT genotype was associated with altered diffusion parameters in subcortical white matter in a sample of children and adolescents [ 37 ]. Moreover, a recent study reported the absence of predominantly inverse associations between D2/D3 receptor availability in the cortical and subcortical gray matter and axonal integrity in brain white matter of unmedicated patients with schizophrenia [ 38 ]. In the present study, TOL-induced DA elevation in mouse PFC was accompanied with mature OL decrease and hypomyelination indicated by lower levels of myelin related proteins including CNP, MAG, MBP, and MOG, as well as decreased myelinated axons but increased G-ratio of myelinated axons, as compared to healthy controls in the VEH group. Moreover, mitochondrial functions of neural cells in this brain region of TOL-treated mice were impaired as indicated by decreased protein levels of the complex I, II, and IV, as well as of NAT8L, in addition to ATP decrease. Of note is NAT8L, a neuron-specific protein in the brain and responsible for NAA synthesis from aspartate and acetyl-CoA in neurons [ 39 ]. In such a way, NAT8L involves in myelination in the juvenile mice via supplementation of acetate derived from NAA [ 40 ]. Taken together, all these data for the first time demonstrate a pathway from DA elevation through mitochondrial dysfunction to hypomyelination in PFC of TOL-treated mice. In COMT -ko mice, dopaminergic homeostatic responses happened in PFC and CPu. Interestingly, the responses in these two brain regions are different. In PFC, levels of MAO-B protein and DR1 mRNA increased while DA level was comparable to that in wt mice, suggesting the existence of compensatory changes in this brain region in response to COMT knockout. In CPu, levels of DA, MAO-A and MAO-B proteins, as well as mRNA levels of DR2 and DAT decreased significantly as compared to wt mice, indicating the changes in this brain region are decompensated. In line with this suggestion, the CPu of COMT -ko mice showed mature OL decrease, lower MBP immunostaining intensity and MBP protein level, as well as lower levels of ATP and ROS compared to wt mice. From all these data, an inference can be drawn that neural cells (including dopaminergic axons and OL processes wrapping around the axons) in CPu are more vulnerable to DA elevation than those in PFC. This inference is in line with the findings from previous studies that systemically administration of 10 mg/kg TOL induced an increase in hydroxyl radical in the striatum of anaesthetized rats following treatment with L-DOPA/carbidopa [ 41 ] and that methamphetamine treatment depleted striatal DA, generated ROS, and decreased activity of complex I of the mitochondria [ 42 ]. The demonstration of this inference is of great significance for the pathogenesis of extrapyramidal syndromes (EPS), which is one of the most challenging adverse effects in patients with schizophrenia when using antipsychotic drugs. Previous studies suggest that a high degree of D2R occupancy is necessary for the occurrence of EPS and schizophrenia patients with EPS have higher D2 R occupancy (above 80%) than patients free of EPS (65–80%) [ 43 , 44 ]. However, the underpinning molecular mechanisms for EPS remain to be elucidated. According to the DA elevation - mitochondrial dysfunction - hypomyelination pathway demonstrated in this study, chronic administration of antipsychotics leads to DA elevation in extracellular space and synaptic cleft due to the higher D2 R occupancy of these drugs. Consequently, increased DA may enter the cytoplasm of neural cells, where it adversely impacts mitochondrial function (as demonstrated in the present study), and/or leads to DA auto-oxidation in the extracellular space [ 45 ]. The both intracellular and extracellular events lead to the same ultimate results of ROS increase which consequently damages neural cells (as demonstrated in the present study). Not surprisingly, the TOL-treated mice and COMT -ko mice did not present same behavioral anomalies. Specifically, the mice in TOL30 and TOL60 groups showed higher anxiety level in OFT, spatial working memory impairment detected by Y-maze test, deficit in recognition memory and less interest in exploring objects throughout the NOR test, as well as impaired executive function measured in the POT. As for COMT -ko mice, they had difficulty deciding whether to enter an open or a closed arm of the EPM, suggesting a somewhat cognitive impairment. In this context, that the COMT -ko mice presented a higher CD/TD in OFT should be interpreted as a behavioral disorganization rather than a lower level of anxiety. In addition, the COMT -ko mice showed social deficits as indicated by less interest in socializing with a novel conspecifics in SIT, which is considered a reliable method to test the negative symptoms-related behaviors in animal models of schizophrenia [ 46 ]. Notably, the behavioral abnormalities co-exist with decreased levels of MAO-B in CPu of the COMT -ko mice. This phenomenon is in line with previous studies in which MAO-B KO mice exhibited behavioral dis-inhibition such as novelty seeking behavior and reduced anxiety-like behaviors but had comparatively less aggressive behavior compared with MAO-A KO mice in several behavioral paradigms targeting emotional reactivity (47–49). Taken together, all these behavioral anomalies are reminiscent of the negative symptoms and cognitive impairment seen in patients with schizophrenia. Importantly, these behavioral anomalies coexist with mature OL decrease and hypomyelination in TOL-treated mice and COMT -ko mice, suggesting a causal relationship between these two types of phenotypes. In line with this suggestion, myelin defect was shown to cause cognitive dysfunction and increase vulnerability to social withdrawal in adult mice of a recent study [ 50 ]. In another animal study, myelin degeneration and diminished myelin renewal contributed to age-related deficits in memory [ 51 ]. Moreover, a recent clinical study demonstrated that chronic schizophrenia is characterized by global microscopic brain hypomyelination in both white matter and gray matter associated with the disease duration and negative symptoms [ 52 ]. The pathway from DA elevation through mitochondrial dysfunction to hypomyelination demonstrated in animal models of schizophrenia was further substantiated by the following findings from the in vitro experiments: 1) DA inhibited the differentiation of OPC to O4 + cells and delayed the maturation of OLs in a concentration-dependent manner; 2) DA disrupted mitochondrial functions of OLs as evidenced by decreased ΔψM and increased ROS in DA-treated cells, which consequently led to OL apoptosis. These data strongly suggest that extracellular DA can enter the cytoplasm of OLs where it impacts mitochondrial functions, thereby demonstrating the molecular mechanism for the inhibiting effects of DA on OLs. In support of this inference, 3) the MAOs inhibitor TCP effectively attenuated DA-induced CNP and MBP decrease in cultured OLs in the presence of DA, and protected the OLs against the toxic effects of DA on mitochondrial functions. Moreover, 4) the antioxidant NAC effectively improved the developmental delay of cultured OLs in the presence of DA with a concentration dependent manner, providing further evidence that ROS is the culprit of the adverse effects of DA on OLs. Last but not least, DA inhibited axonal myelination in the neuron-OL co-cultures at a concentration-dependent manner, elegantly simulated the hypomyelination seen in TOL-treated mice and COMT -ko mice. As mentioned earlier, the existing antipsychotics have little or no therapeutic effect on negative symptoms and cognitive impairment in patients with schizophrenia. This predicament is due to an incomplete or even incorrect understanding of the pathogenesis of schizophrenia. According to the pathway from DA elevation through mitochondrial dysfunction to hypomyelination demonstrated in this study, antipsychotic drugs block DA receptors and increase DA levels in synaptic cleft and other parts of the extracellular space, thereby increasing ROS levels and damaging neural cells, leading to cognitive deficits and negative symptoms in patients with schizophrenia. Therefore, the correct strategy for antipsychotic treatment should deal with DA elevation and its downstream events rather than blocking DA receptors. If DA level in the brain of a schizophrenia patient can be returned to normal at time, the psychotic behaviors, i.e. the positive symptoms, of the patient would be normalized as the immediate therapeutic effect without the side effect of EPS. Moreover, the cognitive function and negative symptoms of the patient would be improved following the recovery of OLs damage resulted from high level of DA. In line with this inference, SNPs in COMT and MAO A/B genes, with reduced activity in the corresponding enzymes, are associated with a decrease in DA degradation and hence dopaminergic hyperactivity occurred via D2 receptors [ 53 ]. And there are increasing clinical studies applying antioxidant addition to antipsychotic treatment for patients with schizophrenia although results are inconsistent [ 54 , 55 ]. Moreover, targeting myelin and OL dysfunction in schizophrenia has been viewed as a novel treatment strategy [ 56 ]. In conclusion, data from in vivo experiments demonstrate a pathway from DA elevation through mitochondrial dysfunction to hypomyelination in the mouse brain. The in vitro experiments with primary OL culture and neuron-OL co-culture system elucidate the mechanism of the aforementioned pathway, wherein mitochondria play pivotal roles in DA catabolism and axonal myelination. The co-existence of behavioral anomalies and hypomyelination in mice subsequent to the disruption of this pathway provides a novel insight into the negative symptoms and cognitive impairment seen in patients with schizophrenia. More importantly, amelioration of DA-induced mitochondrial dysfunction and hypomyelination by NAC and TCP in cultured OLs offers a theoretical basis for targeting mitochondria and OLs in treating patients with schizophrenia. Declarations COMPETING INTERESTS The authors declare no competing interests. Author Contributions FY, YZ, CJ, and NO designed and carried out experiments, recorded and analyzed experimental data, and participated manuscript preparation. QW, PW, and PZ provided assistants for cell culture experiments. WW, JH, YL, HZ, and LL instructed experiments. HX conceptualized the study, wrote the draft, and edited the manuscript. HX and XY supervised the research project. All authors read and approved the submitted version of the manuscript. Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (# 81971256), Natural Science Foundation of Guangdong Province (# 2016A030313067), and Li Ka-Shing Foundation (# 43209502). Data Availability The data that support the findings of this study are available from the cor-responding author upon a reasonable request. References McCutcheon RA, Reis Marques T, Howes OD. Schizophrenia-An overview. JAMA Psychiatry. 2020; 77: 201–10. Fusar-Poli P, Papanastasiou E, Stahl D, Rocchetti M, Carpenter W, Shergill S, et al. Treatments of negative symptoms in schizophrenia: Meta-analysis of 168 randomized placebo-controlled trials. Schizophr Bull. 2015; 41: 892–9. Harvey RC, James AC, Shields GE. A Systematic review and network meta-analysis to assess the relative efficacy of antipsychotics for the treatment of positive and negative symptoms in early-onset schizophrenia. CNS Drugs. 2016; 30: 27–39. Lähteenvuo M, Tiihonen J. 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Additional Declarations The authors have declared there is NO conflict of interest to disclose Supplementary Files SupplementalTable1.docx SupplementalTable2.docx SFig1.tif SFig2.tif SFig3.tif SFig4.tif SFig5.tif Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3875841","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":317159013,"identity":"6660b9ca-bea7-4468-925c-f22969596313","order_by":0,"name":"Haiyun Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqklEQVRIiWNgGAWjYBACAxDxAYlNnBbGGSRrYeYhSYu5RI6ZtG3btsQG9uZtEgw1dwhrsZyRliad23Y7sYHnWJkEw7FnRDjsRvIx6dxtQC1A6yQYGw4ToyWxTdoSpEX+DdFagLYwgm3hIVbLmWfJlr3/bhu38aQVWyQcI0bL8RzDGz/O3JbtZz+88caHGiK0MAgkQGg2EJFAhAYGBv4DRCkbBaNgFIyCkQwAaNk5yzveVNcAAAAASUVORK5CYII=","orcid":"","institution":"Zhejiang Provincial Clinical Research Center for Mental Disorders, the Affiliated Wenzhou Kangning Hospital, School of Mental Health, Wenzhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Haiyun","middleName":"","lastName":"Xu","suffix":""},{"id":317159014,"identity":"814a9e87-3a98-44e3-976d-764aeed35fc5","order_by":1,"name":"Fan Yang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Fan","middleName":"","lastName":"Yang","suffix":""},{"id":317159015,"identity":"56000c41-cb87-4eae-84c5-92ccd4b40d4f","order_by":2,"name":"Yi Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Zhang","suffix":""},{"id":317159016,"identity":"1bcea27e-c8b3-41c0-8e8b-a708f030e415","order_by":3,"name":"Cuiting Jiang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Cuiting","middleName":"","lastName":"Jiang","suffix":""},{"id":317159017,"identity":"d8274474-7a7b-42ce-9828-62cd5cdb91d0","order_by":4,"name":"Na Ouyang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Na","middleName":"","lastName":"Ouyang","suffix":""},{"id":317159018,"identity":"2cc250dc-d881-4625-809e-637fd4aa267c","order_by":5,"name":"Qianqian Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Qianqian","middleName":"","lastName":"Wang","suffix":""},{"id":317159019,"identity":"53cf6959-c8bd-4382-9d9e-39d9a0c5534b","order_by":6,"name":"Ping Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Wang","suffix":""},{"id":317159020,"identity":"2c948d7c-48f0-4c16-b5dc-c83468e61b3b","order_by":7,"name":"Peiwen Zheng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Peiwen","middleName":"","lastName":"Zheng","suffix":""},{"id":317159021,"identity":"e4cdb9ee-743b-4917-ad3a-ce3257e4f77c","order_by":8,"name":"Wei Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Wang","suffix":""},{"id":317159022,"identity":"7f8d9fa4-0196-4a8e-a24b-8d72e040c7e8","order_by":9,"name":"Handi Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Handi","middleName":"","lastName":"Zhang","suffix":""},{"id":317159023,"identity":"5e20c6f6-2442-44e1-9159-837d43118438","order_by":10,"name":"Jue He","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jue","middleName":"","lastName":"He","suffix":""},{"id":317159024,"identity":"cb37235a-995e-4ae6-afe4-2ad689137b8f","order_by":11,"name":"Yanlong Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yanlong","middleName":"","lastName":"Liu","suffix":""},{"id":317159025,"identity":"92519fd1-d7da-4b73-9318-347e79f6bebb","order_by":12,"name":"Lingyun Lin","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lingyun","middleName":"","lastName":"Lin","suffix":""},{"id":317159026,"identity":"7c445dc0-f539-498f-bb9b-406e457c422a","order_by":13,"name":"Zhiqian Tong","email":"","orcid":"https://orcid.org/0000-0002-0511-0386","institution":"Beijing Institute of Brain Disorders, Laboratory of Brain Disorders, Ministry of Science and Technology, Collaborative Innovation Center for Brain Disorders, Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhiqian","middleName":"","lastName":"Tong","suffix":""},{"id":317159027,"identity":"7ded306c-a3f4-4d35-9351-5b39e6f760b1","order_by":14,"name":"Xin Yu","email":"","orcid":"https://orcid.org/0000-0003-3983-4937","institution":"Peking University Institute of Mental Health (Sixth Hospital)","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2024-01-18 13:05:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3875841/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3875841/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60600774,"identity":"0a2c0a04-9a85-476f-81b6-3fbcd6d4c4d5","added_by":"auto","created_at":"2024-07-18 16:02:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":109467,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAbnormalities in behaviors and brain monoamines in C57BL/6 mice given TOL\u003c/strong\u003e.\u003cstrong\u003e A\u003c/strong\u003e Abnormal performance in the open field test ( n =17/group). One-way ANOVA indicates a significant effect of TOL on CD/TD value (F\u003csub\u003e(3,64)\u003c/sub\u003e = 3.418, p = 0.022). Compared to the VEH group, the CD/TD values in TOL30 (p \u0026lt;0.01) and TOL60 (p\u0026lt;0.05) groups were significantly lower. \u003cstrong\u003eB \u003c/strong\u003eAbnormal performance in the Y-maze test (n =17/group). One-way ANOVA indicates a significant effect of TOL on spontaneous alternation value (F\u003csub\u003e(3,64)\u003c/sub\u003e = 5.361, \u003cem\u003ep \u003c/em\u003e= 0.002). Compared to the VEH group, the spontaneous alternation values in TOL60 (p\u0026lt;0.05) was significantly lower. \u003cstrong\u003eC\u003c/strong\u003e Abnormal performance in the NOR test (n = 13/group). One-way ANOVA indicates a significant effect of TOL on recognition index Tn/(T\u003csub\u003en\u003c/sub\u003e + T\u003csub\u003ef\u003c/sub\u003e) [F\u003csub\u003e(3,48)\u003c/sub\u003e = 3.284, p = 0.014]. Compared to the VEH group, the Tn/(T\u003csub\u003en\u003c/sub\u003e + T\u003csub\u003ef\u003c/sub\u003e) value in TOL60 was significantly lower (p\u0026lt;0.05). \u003cstrong\u003eD\u003c/strong\u003e Abnormal performance in the puzzle box test (n = 12/group). The Kruskal-Wallis H test (The data from the tests showed uneven variance) indicates significant effects of TOL on the performance of mice in T5 (\u003cem\u003eX\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e(3,44)\u003c/sub\u003e = 20.17, p \u0026lt;0.0001) and T8 (\u003cem\u003eX\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e(3,44)\u003c/sub\u003e = 11.50, p \u0026lt;0.01) trials. \u003cstrong\u003eE\u003c/strong\u003e DA level changes in PFC and CPu (n = 7 or 8/group). One-way ANOVA indicates significant effects of TOL on DA levels in PFC (F\u003csub\u003e3,25\u003c/sub\u003e = 5.492, p = 0.006). Post-hoc comparisons show significantly higher DA level in PFC of the mice in TOL60 group (p\u0026lt;0.05) relative to the VEH group. \u003cstrong\u003eF\u003c/strong\u003e NE increases in PFC and the hippocampus (HP) (n = 7 - 8/group). One-way ANOVA indicates a significant effect of TOL on NE level in HP (F\u003csub\u003e(3,25)\u003c/sub\u003e = 5.361, p \u0026lt; 0.01). Post-hoc comparisons show significantly higher NE level in PFC (p\u0026lt;0.05) of TOL30 group (p\u0026lt;0.05) and in HP of the mice in TOL30 (p\u0026lt;0.05) and TOL60 (p\u0026lt;0.05) groups relative to VEH group. \u003cstrong\u003eG\u003c/strong\u003e TOL increased 5-HT level in HP of TOL30 group (p\u0026lt;0.05) relative to VEH group. Data are expressed as mean ± SD. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, compared to VEH group.\u003c/p\u003e","description":"","filename":"OnlineFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3875841/v1/f5700e10771bde242fbb29b4.png"},{"id":60600777,"identity":"31410e3b-1429-4ac7-8eba-6a78b41b6d49","added_by":"auto","created_at":"2024-07-18 16:02:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":382422,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMature OL decrease and hypomyelination in the PFC of mice given TOL\u003c/strong\u003e.\u003cstrong\u003e A\u003c/strong\u003e Representative immunohistochemical staining images showing mature OLs in PFC labeled by the primary antibody to APC. The images in the top row were taken under a lower magnification while those in the bottom row were taken under a higher magnification.\u003cstrong\u003e B\u003c/strong\u003e Quantitative data of APC\u003csup\u003e+\u003c/sup\u003e cell counting (n/mm\u003csup\u003e2\u003c/sup\u003e). One-way ANOVA reveals a significant effect of TOL (F\u003csub\u003e(3,28) \u003c/sub\u003e= 13.20, \u0026nbsp;p\u0026lt;0.001). Post-hoc comparisons show significantly decreased APC\u003csup\u003e+\u003c/sup\u003e cells\u0026nbsp; in TOL30 (p = 0.03) and TOL60 (p = 0.002) groups as compared to the VEH group.\u003cstrong\u003e C\u003c/strong\u003e Representative Western blot images showing expression of CNP, MBP, MAG, and MOG in PFC. GADPH served as the internal control.\u003cstrong\u003e D\u003c/strong\u003e Quantitative data of levels of the myelin-associated proteins. One-way ANOVA reveals significant effects of TOL on the levels of the myelin-associated proteins (F\u003csub\u003e(3,16) \u003c/sub\u003e= 13.66, p\u0026lt;0.001; F\u003csub\u003e(3,16)\u003c/sub\u003e=15.93, p\u0026lt;0.001; F\u003csub\u003e(3,16)\u003c/sub\u003e=15.03, p\u0026lt;0.001; F\u003csub\u003e(3,16)\u003c/sub\u003e = 19.63, p\u0026lt;0.001, respectively). Post-hoc comparisons show significantly lower levels of the myelin-associated proteins in PFC of all TOL-treated mice compared to the VEH group.\u003cstrong\u003e E\u003c/strong\u003e Representative TEM images showing profiles of myelinated and unmyelinated axons in PFC of VEH and TOL60 groups.\u003cstrong\u003e F\u003c/strong\u003e The TOL60 group has significantly fewer myelinated axon (n/100 µm\u003csup\u003e2\u003c/sup\u003e) than the VEH group (t = 4.486, p \u0026lt;0.001). \u003cstrong\u003eG\u003c/strong\u003e The TOL60 group\u0026nbsp; has a significantly higher G-ratio than the VEH group (t = 9.126, p \u0026lt;0.001).\u003cstrong\u003e H \u003c/strong\u003eThere is no difference between the VEH and TOL60 groups in axon diameter (t = 1.026, p = 0.289).\u003cstrong\u003e I\u003c/strong\u003e The slope of correlation line between axonal diameter and G-ratio in TOL60 group is significantly smaller than that in the VEH group (p\u0026lt;0.001). Data are expressed as mean ± SD. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, compared to VEH group.\u003c/p\u003e","description":"","filename":"OnlineFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3875841/v1/8fd2d3d235ec482ba9121134.png"},{"id":60600780,"identity":"1742c38f-9f8c-4280-b73a-0ccbc0fd662b","added_by":"auto","created_at":"2024-07-18 16:02:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90839,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial impairment of brain cells in PFC of mice given TOL\u003c/strong\u003e.\u003cstrong\u003e A \u003c/strong\u003eRepresentative western blot images showing expression of mitochondrial complexes (I-V). ß-actin served as the internal control. \u003cstrong\u003eB\u003c/strong\u003e Quantitative data comparing expression levels of the mitochondrial complexes. One-way ANOVA reveals significant effects of TOL on levels of the complexes I (F\u003csub\u003e(3,26) \u003c/sub\u003e= 12.30, p\u0026lt;0.001), II (F\u003csub\u003e(3,19) \u003c/sub\u003e= 5.29, p = 0.008), and IV (F\u003csub\u003e(3,27) \u003c/sub\u003e= 7.41, p\u0026lt;0.001). Post-hoc comparisons show significantly lower levels of the complexes I, II, and IV in TOL30 and TOL60 groups compared to the VEH group. \u003cstrong\u003eC\u003c/strong\u003e A representative western blot image showing the expression of NAT8L in PFC tissue. GAPDH served as the internal control. \u003cstrong\u003eD\u003c/strong\u003e Quantitative data comparing NAT8L levels across the groups. One-way ANOVA reveals a significant effect of TOL on this measure (F\u003csub\u003e(3,18) \u003c/sub\u003e= 12.85, p = 0.002). Post-hoc comparisons show significantly lower levels of NAT8L levels in all TOL groups compared to the VEH group. \u003cstrong\u003eE\u003c/strong\u003e Quantitative data comparing ATP levels across the groups. One-way ANOVA reveals a significant effect of TOL (F\u003csub\u003e(3,18) \u003c/sub\u003e= 3.43, p = 0.04) on this index. Post-hoc comparisons show significantly lower levels of ATP in TOL30 and TOL60 groups compared to the VEH group. Data are expressed as mean ± SD. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, compared to VEH group.\u003c/p\u003e","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3875841/v1/06d4bb9a5cae2af18e3c126c.png"},{"id":60602835,"identity":"8a376929-6fc1-4645-b125-f190386dde5a","added_by":"auto","created_at":"2024-07-18 16:18:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":134973,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDopaminergic changes in PFC and CPu of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecomt\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-ko mice.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Western blot images showing the presence and absence of COMT protein in PFC and CPu of \u003cem\u003ecomt\u003c/em\u003e-ko and wt mice, respectively. α-tubulin served as the internal control. \u003cstrong\u003eB\u003c/strong\u003e Western blot images showing the expression of MAO-A. ß-actin served as the internal control. \u003cstrong\u003eC\u003c/strong\u003e Quantitative data comparing MAO-A levels between the \u003cem\u003ecomt\u003c/em\u003e-ko and wt mice. The \u003cem\u003ecomt\u003c/em\u003e-ko mice show a significantly lower MAO-A in CPu compared to the wt mice (t = 2.435, p \u0026lt; 0.05). \u003cstrong\u003eD\u003c/strong\u003eWestern blot images showing the expression of MAO-B. ß-actin served as the internal control\u003cstrong\u003e. E\u003c/strong\u003e Quantitative data comparing MAO-B levels between the \u003cem\u003ecomt\u003c/em\u003e-ko and wt mice. The \u003cem\u003ecomt\u003c/em\u003e-ko mice show a significantly higher level of MAO-B in PFC (t = 2.465, p \u0026lt;0.05) but a lower level in CPu (t = 3.641, p\u0026lt;0.01) compared to the wt mice. \u003cstrong\u003eF\u003c/strong\u003e Western blot images showing the expression of DDC. ß-actin served as the internal control. \u003cstrong\u003eG\u003c/strong\u003eQuantitative data comparing DDC levels between the \u003cem\u003ecomt\u003c/em\u003e-ko and wt mice. The two groups are comparable in this measurement (p = NS). \u003cstrong\u003eH\u003c/strong\u003eQuantitative data comparing DA levels between the \u003cem\u003ecomt\u003c/em\u003e-ko and wt mice. The \u0026nbsp;\u003cem\u003ecomt\u003c/em\u003e-ko mice show a significantly lower DA level in their CPu compared to wt mice (t = 2.495, p\u0026lt;0.05). \u003cstrong\u003eI\u003c/strong\u003e Quantitative data comparing mRNA levels of DR1, DR2, and DAT. \u0026nbsp;The \u0026nbsp;\u003cem\u003ecomt\u003c/em\u003e-ko mice show a significantly higher DR1 mRNA level in PFC (t = 3.424, p\u0026lt;0.05) but lower levels of DR2 mRNA (t = 2.616, p\u0026lt;0.05) and DAT mRNA (t = 4.895, p\u0026lt;0.01) in CPu compared to wt mice. n = 7/group. Data are expressed as mean ± SD. *p\u0026lt;0.05, **p\u0026lt;0.01, compared to wt mice.\u003c/p\u003e","description":"","filename":"OnlineFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3875841/v1/e70c03771e51a84a247763b6.png"},{"id":60600781,"identity":"7e30d6b9-1260-454e-8fc6-662f4ec5f392","added_by":"auto","created_at":"2024-07-18 16:02:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":289974,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDecreased mature OLs and hypomyelination in PFC of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecomt\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-ko mouse\u003c/strong\u003e. \u003cstrong\u003eA \u003c/strong\u003eRepresentative\u003cstrong\u003e \u003c/strong\u003eimmunohistochemical staining images showing mature OLs labeled by the antibody to GST-π. \u003cstrong\u003eB\u003c/strong\u003eQuantitative data showing a significantly decrease in GST-π positive cells in c\u003cem\u003eomt-\u003c/em\u003eko mice compared to the wt mice (t = 2.526, p \u0026lt;0.05). \u003cstrong\u003eC \u003c/strong\u003eRepresentative\u003cstrong\u003e \u003c/strong\u003eimmunohistochemical staining images showing MBP positive immunostaining. \u003cstrong\u003eD\u003c/strong\u003eQuantitative data showing comparable intensities of MBP immunostaining in PFC of mice in the two groups (p =NS). \u003cstrong\u003eE\u003c/strong\u003e A representative Western blot image showing MBP in PFC of the mice. a-tubulin served as the internal control. \u003cstrong\u003eF\u003c/strong\u003eQuantitative data showing a significantly lower MBP level in PFC of c\u003cem\u003eomt-\u003c/em\u003eko mice compared to the wt mice (t = 2.980, t\u0026lt;0.05). \u003cstrong\u003eG\u003c/strong\u003e Quantitative data showing a marginal decrease in ATP level in the \u003cem\u003ecomt\u003c/em\u003e-ko mice compared to the wt mice (t = 1.921, p = 0.07). \u003cstrong\u003eH\u003c/strong\u003e Quantitative data showing comparable ROS levels in PFC of mice in the two groups (p =NS). n = 7/group. Data are expressed as mean ± SD. *p\u0026lt;0.05, compared to wt mice. Scale bars in image groups a \u0026amp; c are equal to 100 µm.\u003c/p\u003e","description":"","filename":"OnlineFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3875841/v1/9b977136a72247b078f4d2ba.png"},{"id":60600776,"identity":"a612477d-d8cf-454c-8dbc-1dff4605e772","added_by":"auto","created_at":"2024-07-18 16:02:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":889922,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDA addition inhibits development of oligodendroglial lineage cells via impairing mitochondrial functions in cultured OLs\u003c/strong\u003e.\u003cstrong\u003e A \u003c/strong\u003eRepresentative\u003cstrong\u003e \u003c/strong\u003eimmunofluorescence staining images of oligodendroglial lineage cells\u003cstrong\u003e \u003c/strong\u003elabeled with the antibodies to O2, O4, CNP, and MBP, respectively. The nuclei of the cells were labeled with the fluorescent dye DAPI. The cells were cultured in the absence or presence of DA at the indicated concentrations. \u003cstrong\u003eB\u003c/strong\u003e Quantitative data showing numbers of O2\u003csup\u003e+\u003c/sup\u003e cells and DAPI labeled nuclei of cultured cells in the absence or presence of DA at the indicated concentrations. One-way ANOVA shows no effect of DA on the measures (p = NS). \u003cstrong\u003eC \u003c/strong\u003eQuantitative data showing proportions of O4/O2, CNP/O2, MBP/DAPI, and MBP/CNP in the cultured cells in the absence or presence of DA at the indicated concentrations. One-way ANOVA shows a significant effect of DA on the proportion of O4\u003csup\u003e+\u003c/sup\u003e/O2\u003csup\u003e+\u003c/sup\u003e cells (F\u003csub\u003e(3,42) \u003c/sub\u003e= 82.29, p\u0026lt;0.001). Post hoc comparisons indicate significantly decreased proportions of O4\u003csup\u003e+\u003c/sup\u003e/O2\u003csup\u003e+\u003c/sup\u003e cells in all the DA-treated cultures compared to Control group. Likewise, DA at all the indicated concentrations decreased the proportions of CNP\u003csup\u003e+\u003c/sup\u003e/O2\u003csup\u003e+\u003c/sup\u003e cells (F\u003csub\u003e(3,44) \u003c/sub\u003e= 528.9, p\u0026lt;0.001) and MBP\u003csup\u003e+\u003c/sup\u003e/DAPI\u003csup\u003e+\u003c/sup\u003e cells (F\u003csub\u003e(3,34) \u003c/sub\u003e= 24.78, p\u0026lt;0.001) in cell cultures. However, the DA treatments had no effect on the proportion of MBP/CNP (p = NS). \u003cstrong\u003eD\u003c/strong\u003e Representative images of OLs stained with the JC-1detection kit. The cells were cultured in the absence or presence of DA at the indicated concentrations. The mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used in the positive control group. \u003cstrong\u003eE\u003c/strong\u003e Quantitative data showing mitochondrial membrane potential (∆ᴪM FL590/FL530) of cultured OLs from all five groups. One-way ANOVA shows a significant effect of the treatment on ∆ᴪM of the cells (F\u003csub\u003e(3,60)\u003c/sub\u003e = 8.009, p\u0026lt;0.001). Post-hoc comparisons indicate significant differences between the Control group and either one of DA-treated groups or CCCP group. Of note is the 200 µM group showing a ∆ᴪM value of nearly zero. \u003cstrong\u003eF\u003c/strong\u003e Representative images of OLs labeled by means of the oxidized DCFDA and flow-cytometry technique evaluating ROS levels in the cells cultured in the absence or presence of DA at the indicated concentrations. \u003cstrong\u003eG\u003c/strong\u003e Quantitative data showing ROS levels. One-way ANOVA shows a significant effect of DA (F\u003csub\u003e(3,60) \u003c/sub\u003e= 17.83, p\u0026lt;0.001) on intracellular ROS level. Post-hoc comparisons indicate significant differences between the Control group and either one of DA-treated groups. \u003cstrong\u003eH\u003c/strong\u003e Representative immunofluorescence images of OLs labeled by the antibody to caspase-3 detecting apoptotic cells in cultures treated without or with DA at the indicated concentrations. \u003cstrong\u003eI \u003c/strong\u003eQuantitative data showing caspase-3 positive cells. One-way ANOVA shows a significant effect of DA (F\u003csub\u003e(3,60) \u003c/sub\u003e= 24.05, p\u0026lt;0.001) on caspase-3 positive cells. Post-hoc comparisons indicate significant differences between the Control group and either one of DA-treated groups. Error bars are mean ± SD. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 in all post-hoc comparisons using Control group as the comparator. Each in vitro experiment was repeated at least three times.\u003c/p\u003e","description":"","filename":"OnlineFig6XXXacXXX.png","url":"https://assets-eu.researchsquare.com/files/rs-3875841/v1/a1af5aca82b6398421500b22.png"},{"id":60602169,"identity":"f451fdf8-78b5-412b-8533-606308d8538b","added_by":"auto","created_at":"2024-07-18 16:10:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":190619,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDA addition inhibits axonal myelination and decreases NAT8L level in neurons of the neuron-OL co-cultures\u003c/strong\u003e. \u003cstrong\u003eA\u003c/strong\u003e Representative immunofluorescence images of the neuron-OL co-cultures showing rare myelin sheaths in DA-treated cultures relative to the Control. The antibodies to NF-H and MBP were used to label axons and mature OLs, respectively, while the nuclei of cells were labeled by DAPI. The white arrows point to myelin sheaths.\u0026nbsp; \u003cstrong\u003eB\u003c/strong\u003e Western blot images showing CNP and MBP in the neuron-OLs co-cultures. GAPDH was used as the internal control. \u003cstrong\u003eC\u003c/strong\u003e Quantitative data showing the relative levels of CNP and MBP in cultured OLs. One-way ANOVA shows significant effects of DA on CNP levels (F\u003csub\u003e(3,16) \u003c/sub\u003e= 12.06, p \u0026lt; 0.01) and MBP (F\u003csub\u003e(3,16) \u003c/sub\u003e= 30.46, p\u0026lt;0.001). Post-hoc comparisons indicate significant differences between the Control group and either one of DA-treated groups in CNP and MBP expression level. \u003cstrong\u003eD\u003c/strong\u003e A representative Western blot image showing NAT8L expression in the neuron-OLs co-cultures. GAPDH was used as the internal control. \u003cstrong\u003eE\u003c/strong\u003e Quantitative data showing the relative levels of NAT8L in neurons of the co-cultures in the absence or presence of DA at the indicated concentrations. One-way ANOVA shows a significant effect of DA (F\u003csub\u003e(3,16) \u003c/sub\u003e= 16.04, p\u0026lt;0.001) on NAT8L level. Post-hoc comparisons indicate significant differences between the Control group and either one of DA-treated groups.\u003cstrong\u003e \u003c/strong\u003eError bars are mean ± SD. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 in all post-hoc comparisons using Control group as the comparator. Each in vitro experiment was repeated at least three times.\u003c/p\u003e","description":"","filename":"OnlineFig7.png","url":"https://assets-eu.researchsquare.com/files/rs-3875841/v1/3e53221931c700337cd16977.png"},{"id":60602171,"identity":"76d20359-fe4f-471b-a4d7-37818b6336a4","added_by":"auto","created_at":"2024-07-18 16:10:50","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":117775,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTCP protects cultured OLs against DA effects on mitochondrial function and expression of CNP and MBP\u003c/strong\u003e.\u003cstrong\u003e A\u003c/strong\u003e Representative western blot images showing CNP and MBP expression in cultured OLs under various conditions as indicated. GAPDH served as the internal control. \u003cstrong\u003eB\u003c/strong\u003e Quantitative data of relative levels of CNP and MBP shown on western blot images. Regarding the CNP expression level, DA alone (0.1 mM, or 0.2 mM) decreased CNP expression as compared to CNT group (P\u0026lt;0.05 in both comparisons), but this inhibiting effect was blocked in the presence of TCP (0.1 mM). As for MBP, DA alone (0.1 mM, or 0.2 mM) decreased MBP expression in a concentration dependent manner, as compared to CNT group (p\u0026lt;0.05 in both comparisons). The inhibiting effect in the case of 0.1 mM DA was not blocked by TCP (0.1 mM), although the inhibiting effect in the case of 0.2 mM DA was effectively ameliorated by TCP (p\u0026lt;0.05). \u003cstrong\u003eC\u003c/strong\u003e Cell viability of cultured OLs under various conditions as indicated. Compared to Control group (dotted line), 0.2 mM DA significantly decreased cell viability of OLs (p\u0026lt;0.01), but this decrease was effectively ameliorated in the presence of TCP (p\u0026lt;0.05). \u003cstrong\u003eD\u003c/strong\u003e Mitochondrial membrane potential (∆ᴪM) of cultured OLs under various conditions as indicated. Compared to Control group (dotted line), 0.2 mM DA significantly decreased ∆ᴪM of OLs (P\u0026lt;0.001), but this decrease was effectively ameliorated in the presence of TCP (P\u0026lt;0.01). \u003cstrong\u003eE\u003c/strong\u003e ATP levels in cultured OLs under various conditions as indicated. Compared to Control group (dotted line), both 0.1 mM and 0.2 mM DA significantly decreased ATP level in OLs (P\u0026lt;0.01), but these decreases were effectively ameliorated in the presence of TCP (P\u0026lt;0.05). \u003cstrong\u003eF\u003c/strong\u003e ROS levels in cultured OLs under various conditions as indicated. Compared to Control group (dotted line), 0.2 mM DA significantly increased ROS level in OLs (P\u0026lt;0.01), but this effect was effectively ameliorated in the presence of TCP (P\u0026lt;0.05). Error bars are mean ± SD. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 compared to CNT. #p\u0026lt;0.05, ##p\u0026lt;0.01 compared to 0.1 mM DA or 0.2 mM DA group. Each of the above evaluations were repeated at least five times.\u003c/p\u003e","description":"","filename":"OnlineFig8.png","url":"https://assets-eu.researchsquare.com/files/rs-3875841/v1/b35c5028fc0c781f6339d2d9.png"},{"id":62879011,"identity":"ecf50372-fe38-45bf-b66c-0d2713bf0b02","added_by":"auto","created_at":"2024-08-20 14:27:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3426546,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3875841/v1/49a6300f-c9a2-4a40-bab2-274ae2f574f1.pdf"},{"id":60602168,"identity":"897d1924-d9bb-4cc4-80ab-15458e009b8c","added_by":"auto","created_at":"2024-07-18 16:10:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12717,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3875841/v1/5048a8217dc200f922411c56.docx"},{"id":60600773,"identity":"03ab187e-0014-4099-bc45-1271693e1d94","added_by":"auto","created_at":"2024-07-18 16:02:50","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12170,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-3875841/v1/f7f65a5c9d92c307571f2826.docx"},{"id":60602172,"identity":"fdf49e54-ba23-4710-8eb9-7c0f34ebcafd","added_by":"auto","created_at":"2024-07-18 16:10:50","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":23294940,"visible":true,"origin":"","legend":"","description":"","filename":"SFig1.tif","url":"https://assets-eu.researchsquare.com/files/rs-3875841/v1/8beecc98bf86bc8212305ffa.tif"},{"id":60602833,"identity":"8ac16c17-2811-486b-be81-df50a7bbdfee","added_by":"auto","created_at":"2024-07-18 16:18:50","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":13680244,"visible":true,"origin":"","legend":"","description":"","filename":"SFig2.tif","url":"https://assets-eu.researchsquare.com/files/rs-3875841/v1/6eb02823ffed89f2ed987aff.tif"},{"id":60602834,"identity":"036fd5e5-3da2-4978-8344-66a7c017f8bc","added_by":"auto","created_at":"2024-07-18 16:18:50","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":15476356,"visible":true,"origin":"","legend":"","description":"","filename":"SFig3.tif","url":"https://assets-eu.researchsquare.com/files/rs-3875841/v1/78a6864c5f02fe1b93b807f8.tif"},{"id":60600784,"identity":"5d4fb6cb-e5e9-4c43-b938-e441e9f81b01","added_by":"auto","created_at":"2024-07-18 16:02:50","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":18336576,"visible":true,"origin":"","legend":"","description":"","filename":"SFig4.tif","url":"https://assets-eu.researchsquare.com/files/rs-3875841/v1/8896a09e6b5bc243d06235b1.tif"},{"id":60600787,"identity":"9d9f6f76-e4e9-49b0-a612-6b6f095aedb7","added_by":"auto","created_at":"2024-07-18 16:02:51","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":10962164,"visible":true,"origin":"","legend":"","description":"","filename":"SFig5.tif","url":"https://assets-eu.researchsquare.com/files/rs-3875841/v1/a857655ed582b473ed66c5ef.tif"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Pivotal roles of mitochondria in linking dopamine catabolism to axonal myelination: Implication for the pathogenesis and treatment of schizophrenia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSchizophrenia is one of the most complex and debilitating brain diseases. Patients with schizophrenia may present various clinical manifestations that have been categorized into positive symptoms including hallucinations, delusions, disorganized thinking, and grossly disorganized or abnormal motor behavior, negative symptoms of apathy, anhedonia, and social withdrawal, as well as cognitive deficits encompassing deficits in attention, working memory, and executive function. While positive symptoms are often the reason for schizophrenia patients to see a psychiatrist, negative and cognitive symptoms are largely responsible for the long-term burden associated with the disease [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Indeed, the existing antipsychotics do not markedly improve negative symptoms and cognitive impairment although they show therapeutic efficacy on positive symptoms in most of patients with schizophrenia [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. About 70% of patients with schizophrenia require long-term, even lifetime, medication to control their symptoms but do not achieve complete recovery [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These unmet clinical challenges are attributed to an incomplete understanding of the pathogenesis behind the complex clinical manifestations of schizophrenia.\u003c/p\u003e \u003cp\u003eIn the effort to understand the pathogenesis of schizophrenia, researchers have proposed several hypotheses over the past decades based on findings from preclinical and clinical studies. Of these hypotheses, the first one is the so-called dopamine (DA) hypothesis, the most enduring theory in schizophrenia research. It initially emphasized a role of hyperdopaminergia in the etiology of schizophrenia [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], followed by a modified version which specifies a subcortical hyperdopaminergia along with the prefrontal hypodopaminergia [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The others include the mitochondrion hypothesis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], neurodevelopment hypothesis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and oligodendrocyte (OL) hypothesis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], to name a few. The coexistence of multiple hypotheses about schizophrenia not only reflects the extreme complexity of this brain disease, but also indicates the existence of a biological mechanism being able to link these hypotheses and form an inclusive theory about the pathogenesis of schizophrenia. In line with this suggestion, previous studies have shown that, on the one hand, DA oxidation products disrupt mitochondrial function [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and on the other hand, DA catabolism may be inhibited due to mitochondrial dysfunction as monoamine oxidases (MAO-A and MAO-B) locate in mitochondria and are responsible for the oxidative inactivation of neurotransmitter amines [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. As such, the interplay between DA metabolism and mitochondrial defects has been considered an important neurobiological mechanism involved in the pathogenesis of schizophrenia [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Moreover, recent studies have linked the co-working of neuronal and oligodendroglial mitochondria to axonal myelination in the brain of mouse subjected to social defeat stress [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] or maternal separation plus early weaning [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The experimental paradigms employed in these recent studies simulate environmental risk factors involved in the etiology of psychotic disorders [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere we demonstrate the pivotal roles of mitochondria in linking DA catabolism to axonal myelination using animal models of schizophrenia, cultured OLs, and the neuron-OL co-culture. We asked whether dopaminergic changes (in mouse brain) could lead to hypomyelination and mitochondrial dysfunction while leading to behavioral abnormalities related to some of clinical manifestations in patients with schizophrenia, whether DA addition into cultured OLs and neuron-OL co-cultures could impair mitochondrial function and impact OL development/myelination, and whether an anti-oxidative treatment or manipulation of DA catabolism could protect against OL development retardation while attenuating mitochondrial dysfunction induced by DA elevation.\u003c/p\u003e"},{"header":"Materials \u0026 methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e All experimental procedures applied to the animals in this study were reviewed and approved by the Institutional Animal Care and Use Committees at the Wenzhou Medical University and Shantou University Medical College (SUMC2019-53).\u003c/p\u003e \u003cp\u003eThe C57BL/6 mice used in the first animal experiment and pregnant Sprague\u0026ndash;Dawley rats used for primary OLs culture and neuron-OL co-culture experiments were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd, China. The \u003cem\u003eCOMT\u003c/em\u003e-ko mice and wild type littermates in the second animal experiment were purchased from Shanghai Southern Model Biotechnology Co., Ltd. (Shanghai, China). The gene knockout mice were produced by means of CRISPR/Cas9-based homology-directed repair and confirmed by Western blot analysis showing undetectable COMT protein in brain tissue of them. The animals were group-housed under controlled temperature (22\u0026deg; to 25\u0026deg;C) and relative humidity (50\u0026ndash;60%) with a 12-hour light-dark cycle (lights on at 08:00) and free accesses to chow food and water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDrug administration\u003c/h2\u003e \u003cp\u003eIn the first animal experiment, C57BL/6 mice were intraperitoneally given the vehicle consisting of 1% DMSO and 0.1% Tween-80 in sterilized saline, or TOL (Med Chem Express, HY-17406) in the vehicle at 15, 30, or 60 mg/kg for consecutive 14 days starting on PD 22. The intraperitoneal injection was done once a day at a same volume of 2 mL/100 g. The four groups of mice were referred to as VEH, TOL15, TOL30, and TOL60, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAnimal behavioral tests\u003c/h2\u003e \u003cp\u003eThe open-field test (OFT) was done with the mice in the both animal experiments to measure locomotor activity and anxiety level of them. Briefly, a mouse was placed in the center of an open-field box (50 cm \u0026times; 50 cm \u0026times; 35 cm) and tested for 10 min during which period the mouse was allowed to move freely. The moving trajectory of the mouse on the floor of the box was recorded by a video-camera placed above the arena. A video tracking software (Noldus Information Technology, Wageningen, Netherlands) was used to record the total distance (TD) moved on the whole arena and that on the central (CD) and peripheral zones. The same software was also used for the other behavioral tests in this study.\u003c/p\u003e \u003cp\u003eThe Y-maze test was performed with the mice in the first animal experiment to assess the exploring behavior and spatial working memory of them. In brief, a mouse was placed at the converging area of a symmetrical Y-maze (30 cm \u0026times; 8 cm \u0026times; 5 cm) and allowed to move freely through the maze for 8 min. The total number and series of arm entry were recorded. Alternation is defined as successive entries into the three arms. Spontaneous alternation is calculated as the percentage of actual to possible alternations (defined as the total number of arm entries minus 2).\u003c/p\u003e \u003cp\u003eNovel object recognition (NOR) test consists of three phases. During the adaptation phase, a mouse was allowed to move freely in the open field box for 5 min. Twenty-four hours later, the same mouse was put back to the open field box to explore the arena with two identical wood blocks for 5 min (training phase). Three hours later, the mouse was allowed to explore two different wood blocks for additional 5 min (discrimination phase). One block was the same one used in the training phase (familiar object), another one was new with a different color and shape (novel object). The recognition index (RI) is calculated according to the formula RI\u0026thinsp;=\u0026thinsp;100 \u0026times; Tn/(Tn\u0026thinsp;+\u0026thinsp;Tf), where Tn represents the exploration time (S) for a novel object and Tf represents the exploration time (S) for a familiar object.\u003c/p\u003e \u003cp\u003eThe puzzle box test (POT) was conducted with the mice in a Plexiglass white box which is divided by a removable barrier into two compartments: a brightly lit start zone (58 cm \u0026times; 28 cm) and a smaller covered goal zone (15 cm \u0026times; 28 cm). A mouse was introduced into the start zone and trained to move into the goal zone through a narrow underpass (~\u0026thinsp;4 cm wide) located under the barrier. Then the mouse underwent a total of nine trials (T1\u0026ndash;T9) over 3 consecutive days, with three trials per day. This sequence allows assessment of problem-solving ability (T5 and T8), and learning/short-term memory of instrumental responses (T3, T6 and T9), while the next day repetition provides a measure of long-term memory (T4 and T7). Performance of a mouse in the POT is assessed by measuring the time in seconds for the subject to arrive at the goal zone from the start zone. The POT has been used successfully to assess the problem solving ability of mice, as well as cognitive deficits exhibited in murine models of schizophrenia [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe elevated-plus maze (EPM) test with the mice in the second animal experiment. The EPM consists of four radial arms (two closed, 50 \u0026times; 10 \u0026times; 40 cm; two open, 50 \u0026times; 10 \u0026times; 2 cm) elevated 60 cm above the floor. Under the same lighting condition as that in the open-field test, a mouse was placed at the central zone, facing a closed arm, and the activity of the mouse on the EPM was recorded during the subsequent 10 min. The first 2 min were defined as the adaptation period and the performance of the mouse in the remaining 8 min was analyzed. The time spent by a tested mouse on the central zone, open and closed arms, and the number of entries to these locations were recorded.\u003c/p\u003e \u003cp\u003eThe social interaction test (SIT) was carried out with the mice in the second animal experiment. It consists of two sessions of 150 second (S) and a one minute interval between the two sessions as described previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. During the first session, an empty (E session) wire mesh cage (12 \u0026times; 12 \u0026times; 18 cm) was placed at one end of an open-field arena (100 \u0026times; 100 cm) where a tested mouse was allowed to move freely. During the second session, the conditions were identical except that an unfamiliar conspecific partner (C session) had been introduced into the cage before a tested mouse was placed in the open-field box. Between the two test sessions, the tested mouse was removed from the box and placed back into his/her home cage for 60 S. The time spent by the tested mouse at the interaction zone (a 16-cm-wide corridor around a cage) was recorded.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eHigh-pressure liquid chromatograph\u003c/h2\u003e \u003cp\u003eUnder a deep anesthesia with 1.25% avertin (Sigma-Aldrich, T48402; i.p.), the mouse brain was removed out of the skull. Following the protocol described in a previous study [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], concentrations of the neurotransmitters of DA, norepinephrine (NE), and serotonin (5-HT) in the brain regions of PFC, hippocampus, and CPu (n\u0026thinsp;=\u0026thinsp;6/group) were measured by means of high-pressure liquid chromatograph (HPLC) and quantified using known standard concentrations of chemically pure DA (Sigma-Aldrich, H8502), NE (Sigma-Aldrich, A7257), and 5-HT (Solarbio, SS9080).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHistological and immunohistochemical staining\u003c/h2\u003e \u003cp\u003eUnder a deep anesthesia with 1.25% avertin (i.p.), the mouse was transcardially perfused with the phosphate buffer (PB, pH\u0026thinsp;=\u0026thinsp;7.4) followed by 4% paraformaldehyde in the PB. Then the whole mouse brain was removed out of the skull and immersed in the fixative for additional 2 days. After dehydration in graded concentrations of alcohol and xylene, tissue blocks were embedded in paraffin and sectioned at 5 \u0026micro;m thickness. For Nissl staining, sections were stained in warmed cresyl violet acetate solution (Solarbio, G1430).\u003c/p\u003e \u003cp\u003eImmunohistochemical staining was done with brain sections of mice in the two animal experiments separately. In the first animal experiment, the paraffin sections (5 \u0026micro;m thickness) were incubated with the primary antibody to APC (Abcam, ab16794, 1:500) to label mature OLs. In the second experiment, frozen sections (20 \u0026micro;m) were incubated with the primary antibody to GST-π (1:100; Boster Biological Technology Co. Ltd; Wuhan, China). The primary antibody to MBP (1:200; Boster Biological Technology Co. Ltd; Wuhan, China) was used to label myelin sheath. Images of fixed specific areas in PFC, CPu and hippocampus were digitally recorded using a Nikon ECLIPSE Ni light microscope (Nikon, Japan) equipped with a digital capture system. The mature OLs and MBP-immunoreactivity intensity were measured using the Image-J software (version win64, National Institutes of Health, US).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy\u003c/h2\u003e \u003cp\u003eThree mice from each of VEH and TOL60 groups in the first animal experiment were used for transmission electron microscopy (TEM) analysis. The mice were anesthetized with 1.25% avertin and subjected to transcardial perfusion of 0.1 M PB and the fixative of 2.5% glutaraldehyde and 4% paraformaldehyde in PB. The cerebral cortex was dissected and immersed in 2.5% glutaraldehyde. Then the cerebral cortex was cut in 500 \u0026micro;m thick coronary sections using two thin blades. PFC was identified as the brain region of interest and was washed in 0.1 M PB followed by postfixed in 1% osmium tetroxide for 1 h. After contrasting in uranyl acetate for 1 h, the tissue was dehydrated in acetone and embedded in Epoxy Resin (SPI Supplies, 90529-77-4). One \u0026micro;m semithin sections were cut using a ultramicrotome (PowerTome-XL, RMC, US) and examined by light microscopy. Then, the selected and comparable regions of the semithin sections were trimmed and 70 nm ultrathin sections were made, contrasted with lead citrate and uranyl acetate and examined under a transmission electron microscope Hitachi H-7500. To quantify the myelinated axons, a total of 27 microphotographs were recorded for each group (3 mice \u0026times; 3 sections \u0026times; 3 photographs) and analyzed using the Image-J software.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003ePrimary OLs culture\u003c/h2\u003e \u003cp\u003ePrimary culture of oligodendroglial lineage cells was done by referring to a previous study [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The cerebral hemispheres of S-D rat embryos at E16-18 were dissociated and transferred to the Accutase detachment solution (Sigma-Aldrich, A6964) at 37\u0026deg;C for 10 min. Then the tissue solution was moved into a centrifuge tube containing 5 mL of DMEM/F12 (Gbico,11330032), gently blowed, and stood for a few min followed by filtration through a 70 \u0026micro;m Nitex mesh to form a suspension of isolated cells in the medium. After cell counting, cells were seeded (at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e/cm\u003csup\u003e2\u003c/sup\u003e) into poly-D-Lysine (Sigma-Aldrich, P6407) pre-coated T75 culture flasks containing the medium. The medium was changed every 3 days until DIV9. The cultured cells in the T75 flask were shaken at 150 rpm for 60 min to isolate microglia, and subsequently shaken at 250 rpm for 15\u0026ndash;20 h to isolate the oligodendrocyte precursor cells (OPC) from astrocytes. After centrifugation at 1,800 rpm for 6 min, the precipitate was resuspended and seeded onto chamber slides or well plates at appropriate densities within the OPC cultivation medium [DMEM/F12 supplemented with 2% B27 (Gbico, A35828-01), 10 ng/mL fibroblast growth factor-2 (FGF-2; Peprotech, 100-18B), 1% Pen/Strep (Gbico,15140122), 10 ng/mL PDGF-AA (Peprotech, 100-13A), and 0.5% FBS (Gbico,16000-044)] for 2 days. Then the OPC cultivation medium was replaced with fresh OPC differentiation medium [DMEM/F12 supplemented with 1% N2 (Gbico,17502-048), 0.5% FBS, 1% Pen/Strep, 10 nM corticosterone (Amresco, IC0550), 10 nM D-biotin (Thermo Fisher Scientific, B20656), and 30 nM triiodothyronine (Solarbio, IT1110)] for further culture for additional 2 days.\u003c/p\u003e \u003cp\u003eVarious concentrations of DA (vehicle, 50, 100, 200 \u0026micro;M) were added into the medium in three different schedules of the first two days, the last two days, and the first two days followed by culture with replaced medium having no DA, to examine effects of DA on the differentiation and maturation of oligodendroglial lineage cells. In the NAC (N-acetyl-L-cysteine) experiment, cultured OLs were pretreated with DA at 50 \u0026micro;M for 4 h followed by subsequent incubation for 48 h in the presence of NAC (Beyotime Biotechnology, ST1546) at concentrations of 250 \u0026micro;M (NAC-l) or 500 \u0026micro;M (NAC-h). Controls include those treated with DA (50 \u0026micro;M) alone, NAC-h alone, and negative control lacking both DA and NAC. In the TCP (trans-2-phenylcyclopropy) experiment, primary OLs were cultured at various concentrations of DA (0, 100 \u0026micro;M, 200 \u0026micro;M) in the absence or presence of TCP (100 \u0026micro;M) during DIV 15\u0026ndash;17. At the end, the mature OLs were analyzed for cell viability, ΔψM, and the production of mitochondrial ROS and ATP, in addition to Western blot analysis measuring CNP and MBP levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eNeuron-OL co-culture\u003c/h2\u003e \u003cp\u003eFor neuron-OL co-culture, the neocortex rotation-mediated aggregate cell culture protocol was followed as described previously with modifications [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The cerebral hemispheres of E16\u0026ndash;18 SD rat fetuses were dissociated as described previously. The culture medium for the co-culture was DMEM/F12 supplemented with 2% B27 Plus,1% FBS,1% penicillin/streptomycin, 50 ng/mL β-NGF, and 50 \u0026micro;g/mL vitamin C. The medium was replaced every three days until DIV 42. During DIV14-42, various concentrations of DA (vehicle, 50, 100, 200 \u0026micro;M) were added into the medium to examine effect of DA on axonal myelination in the neuron-OL co-cultures.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell culture immunofluorescence\u003c/h2\u003e \u003cp\u003eCultured cells were fixed with 4% paraformaldehyde for 15 min and then washed three times in PBS. Then, the fixed cells were permeabilized with 0.1% Triton-X in PBS for 10 min and later washed with PBS three times, and the cells were then blocked with 10% goat serum (Gibco, USA) in PBS for 60 min at room temperature. After removal of the blocking reagent, the cells were incubated with one or two of the following primary antibodies including rabbit anti-Oligo-2 (1:500, Ab9610, Sigma-Aldrich), mouse anti-Oligo-4 (1:100, O7139, Sigma), mouse anti-CNP (1: 200, ab6319, Abcam), and rabbit anti-MBP (1:200, ab40390, Abcam), mouse anti-MBP (1:200, MA5-35001, Thermo Fisher Scientific) and rabbit anti-NF-H (1:500, 18934-1-AP, Proteintech) at 4\u0026deg;C overnight. After rinsing, the cells were incubated with goat anti-rabbit IgG H\u0026amp;L (Alexa Fluor 594, 1:2000, A-11012, Thermo Fisher Scientific) or goat anti-mouse IgG H\u0026amp;L (Alexa Fluor 488, 1:2000, A-11001, Thermo Fisher Scientific) for 1 h at room temperature. Then, the cells were washed with PBS, and the nucleus was stained with the fluorescent dye DAPI for 5 min. In addition, the primary antibody mouse anti-cleaved Caspase-3 (1:200, 66470-2-Ig, Proteintech) was used to label apoptotic OLs due to the cytotoxicity of DA. All the cultured cells were visualized by indirect fluorescence under the fluorescent microscope (Nikon ECLIPSE Ni, Nikon, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eProteins were extracted from brain tissue or cultured cells using a Tris-ethylenediaminetetraacetic acid (EDTA) lysis buffer (1% Triton X-100, 10% glycerol, 20 mM Tris, pH 7.5, and 1 mM EDTA) with freshly added Protease Inhibitor Cocktail (Sigma-Aldrich). After measurement of protein concentrations using a BCA kit (Sangon Biotech, C503021), sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting were carried out. The antibodies used for all the western blot analyses in this study are shown in Supplemental Table\u0026nbsp;1. The antigen-antibody complexes were visualized by using an ECL detection kit (P10100, NCM Biotech). Quantification of the immunoblots was carried out by densitometric analysis of chemiluminescence exposed films, using Image-Lab software (version 4.0) and the results recorded as arbitrary densitometric units.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFunctional assessments of mitochondria\u003c/h2\u003e \u003cp\u003eMitochondrial functions of brain cells and cultured OLs and neurons were assessed by various techniques. First, Western blot analysis was done with PFC tissue samples and neuron-OLs co-cultures to measure expression levels of mitochondrial complexes (CI, CII, CIII, CIV, and CV) as described before. Second, the cell viability of cultured OLs was assessed using the Cell Counting Kit-8 (CCK-8, HY-K0301, MedChem Express) following the protocol provided by the manufacturer. This sensitive colorimetric assay allows accurate live cell counting in a cell proliferation or cytotoxicity assays. Third, mitochondrial membrane potential (ΔψM) was measured using the JC-1staining assay kit (M8650, Solarbio). In healthy cells, JC-1 selectively enters into mitochondria and forms J-aggregates with intense red fluorescence. In apoptotic or unhealthy cells, JC-1 remains in the monomeric form showing only green fluorescence. Fourth, ROS in cultured cells were assessed using the DCFDA cellular ROS assay kit (S0033S, Beyotime Biotechnology) and following the instructions provided by the manufacturer. Fifth, ATP concentration in brain cells of mice was assessed using a commercial ATP assay kit (Beyotime Biotechnology, Nanjing, China). In the presence of magnesium, oxygen and ATP, the protein luciferase catalyzes oxidation of the substrate luciferin, which is associated with light emission.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRT-qPCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from the caudate putamen and PFC of mouse brain using TriZol (Invitrogen, Shanghai, China). cDNA was generated from total RNA samples using the ExScript RT-PCR Kit (cat.# RR047A, TaKaRa, Japan) according to manufacturer\u0026rsquo;s protocol. qPCR was performed using SYBR\u0026reg; Premix Ex Taq\u0026trade; (Tli RNaseH Plus; cat.#RR820A, TaKaRa, Japan) following the instruction recommended by the manufacturer. \u003cem\u003eβ-actin\u003c/em\u003e mRNA was served as the internal control. Template RNA was replaced with PCR-grade water as a negative control. After amplification, melting curve analysis and length verification by gel electrophoresis were carried out to confirm the specificity of PCR products. Each sample was analyzed in triplicate. The relative levels of tested mRNA were calculated by normalization to the endogenous \u003cem\u003eβ-actin\u003c/em\u003e mRNA expression prior to comparative analysis using the comparative threshold cycle (2\u003csup\u003e\u0026ndash;∆∆Ct\u003c/sup\u003e) method. The primers employed for aforementioned PCRs are shown in Supplemental Table\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphpad prism (GraphPad Software, version 8.0) was used for statistical analysis. All data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) and analyzed by independent sample \u003cem\u003eStudent\u0026rsquo;s t\u003c/em\u003e-test or one-way ANOVA followed by Tukey's multiple comparisons. When a p-value was less than 0.05, the difference was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eTolcapone-treated mice exhibit DA elevation, hypomyelination, mitochondrial dysfunction in brain cells, and behavioral abnormalities\u003c/h2\u003e \u003cp\u003eTo determine if DA elevation influences white matter development and impairs mitochondrial functions of brain cells while inducing behavioral anomalies, we administered TOL to C57BL/6 mice intraperitoneally at the dose of 0, 15, 30, or 60 mg/kg, for consecutive 14 days starting on postnatal day (PD) 22. TOL is a brain penetrant selective inhibitor of COMT devoid of psychostimulant properties [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The COMT enzyme plays a pivotal role in DA metabolism, specifically in PFC [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The four groups of mice were referred to as VEH, TOL15, TOL30, and TOL60 (n\u0026thinsp;=\u0026thinsp;17/group), respectively. One day after the last TOL administration, the animals were subjected to OFT measuring their locomotor activity and anxiety-like behavior, the Y-maze test assessing spatial working memory, NOR test evaluating recognition memory, and POT examining general cognition and executive function, in the order. Compared to VEH group, the groups TOL30 and TOL60 showed higher anxiety levels indicated by significantly lower CD/TD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, respectively, Fig.\u0026nbsp;1A). The TOL60 group also presented spatial working memory impairment indicated by significantly lower spontaneous alternation in the Y-maze test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;1B). In addition, the TOL60 group showed a significantly lower RI as compared to the VEH group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;1C), suggesting a recognition memory impairment. Moreover, TOL30 and TOL60 groups showed impairment in problem solving ability and executive function revealed by POT in which mice in TOL30 and TOL60 groups took longer duration to complete the task in T5 and T8 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, respectively, Fig.\u0026nbsp;1D).\u003c/p\u003e \u003cp\u003eOne day after the last behavioral test, five mice in each group were euthanized and the brain regions of PFC, CPu, and hippocampus were isolated and used for HPLC analysis to measure levels of DA, norepinephrine (NE), and 5-hydroxytryptamine (5-HT). As for DA levels, one-way ANOVA revealed significant effects of TOL on DA level in PFC (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Post-hoc comparisons showed significantly higher DA level in PFC (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) of the mice in TOL60 group, compared to the VEH group (Fig.\u0026nbsp;1E). Regarding NE level, one-way ANOVA revealed a significant effect of TOL on this index in the hippocampus (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Post-hoc comparisons showed significantly higher NE levels in hippocampus of the mice in TOL30 and TOL60 groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 in both comparisons, Fig.\u0026nbsp;1F). Also, TOL30 group had a higher level of 5-HT in hippocampus compared to the VEH group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;1G).\u003c/p\u003e \u003cp\u003eImmunohistochemical staining with the primary antibody to APC (adenomatous polyposis coli gene clone CC1 used as the biomarker of mature OL cell body) showed dose-dependent decreases in the number of APC\u003csup\u003e+\u003c/sup\u003e cells in PFC of TOL-treated mice (Fig.\u0026nbsp;2A \u0026amp; B). Relevantly, Western blot analysis revealed dose-dependent decreases in protein levels of CNP, MAG, MBP, and MOG in PFC of TOL groups compared to VEH group (Fig.\u0026nbsp;2C \u0026amp; D). To provide further evidence for the hypomyelination in TOL-treated mice, PFC samples of mice in VEH and TOL60 groups were prepared for TEM analysis. The two groups look seemingly different in numeral density of myelinated axon and myelin sheath thickness as shown in Fig.\u0026nbsp;2E. Indeed, quantitative data revealed a lower numeral density of myelinated axon in the TOL60 group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;2F), a higher G-ratio (the ratio of the inner to the outer diameter of the myelin sheath of a myelinated axon) in the TOL60 group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;2G), indicating thinner myelin sheath of the myelinated axons in this group. But, the two groups were comparable in axon diameter (p\u0026thinsp;=\u0026thinsp;ns, Fig.\u0026nbsp;2H). Moreover, the TOL60 group showed a smaller slope of the correlation between axonal diameter and G-ratio (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;2I) compared to the VEH group, suggesting that the smaller axons were more susceptible to DA elevation in PFC of the TOL-treated mice.\u003c/p\u003e \u003cp\u003eSimilar to the findings in PFC, TOL administration decreased the number of APC\u003csup\u003e+\u003c/sup\u003e cells (Supplemental Fig.\u0026nbsp;1A \u0026amp; B) and expression levels of CNP, MBP, and MOG in the mouse hippocampus (Supplemental Fig.\u0026nbsp;1C \u0026amp; D). As for CPu, TOL30 and TOL60 groups showed fewer APC\u003csup\u003e+\u003c/sup\u003e cells relative to VEH group (Supplemental Fig.\u0026nbsp;1E \u0026amp; F), but the four groups were comparable in protein levels of CNP and MBP in this brain region (Supplemental Fig.\u0026nbsp;1G \u0026amp; H).\u003c/p\u003e \u003cp\u003eThe coexistence of DA elevation, APC\u003csup\u003e+\u003c/sup\u003e cell decrease, and hypomyelination in the TOL-treated mice raised a question, namely, how did DA elevation lead to mature OL decrease and hypomyelination in the mouse brain? We hypothesized that DA elevation impaired mitochondrial function thus inhibited OL maturation and axonal myelination as intact mitochondria in both neurons and OLs are essential for axonal myelination [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. To test this hypothesis, we assessed mitochondrial functions of brain cells of the mice in this experiment. Western blot analysis showed significantly lower levels of mitochondrial complexes I, II and IV in neural cells of mouse PFC in TOL30 and TOL60 groups relative to the VEH group, while the other two complexes (III \u0026amp; V) were comparable across the groups (Fig.\u0026nbsp;3A \u0026amp; B). In addition, TOL groups show lower levels of NAT8L (N-acetyltransferase 8-like) in mouse PFC (Fig.\u0026nbsp;3C \u0026amp; D). This enzyme catalyzes the synthesis of NAA from aspartate and acetyl-CoA in neuronal mitochondria. The neuronal NAA is then transported to the cytoplasm of OLs, where aspartoacylase (ASPA) cleaves the acetate moiety of NAA for use in the synthesis of fatty acid and steroid, the building blocks for myelin lipid synthesis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Furthermore, the TOL30 and TOL60 groups had significantly lower levels of ATP compared to VEH group (Fig.\u0026nbsp;3E). However, no difference was found between VEH and TOL groups in neuron number in the PFC (Supplemental Fig.\u0026nbsp;2), suggesting that neurons are more tolerable to mitochondrial dysfunction than OLs, the number of which decreased in TOL-treated mice as described above. This statement is consistent with a recent study reporting that neurons among the neural cells in mouse brain are the most tolerable to mitochondrial damage by cuprizone [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], which is a copper chelator and toxic to mitochondria [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eCOMT\u003c/b\u003e \u003cb\u003e-ko mice exhibit dopaminergic changes, hypomyelination, mitochondrial dysfunction in brain cells, and behavioral abnormalities\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo substantiate the aforementioned changes in TOL-treated mice, we assessed and compared dopaminergic measurements of wild type (wt) and \u003cem\u003eCOMT\u003c/em\u003e-ko mice by means of various techniques. The \u003cem\u003eCOMT\u003c/em\u003e gene is located in a fragment of chromosome 22q11 which when deleted results in a complex syndrome including the psychiatric manifestations such as schizophrenia. As such, the \u003cem\u003eCOMT\u003c/em\u003e gene has been placed near the top of a list of plausible candidate genes for schizophrenia [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and the gene variants may be involved in the pathogenesis of psychotic symptoms, and associated especially with negative symptom in schizophrenia [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe assessed protein levels of the enzymes relevant to DA metabolism including monoamine oxidase-A (MAO-A), MAO-B, and Dopa decarboxylase (DDC), in addition to COMT in wild type and \u003cem\u003eCOMT-\u003c/em\u003eko mice (n\u0026thinsp;=\u0026thinsp;7/group). Western blot analysis detected the presence of COMT protein in both PFC and CPu of the wt mice, but the absence in \u003cem\u003eCOMT\u003c/em\u003e-ko mice (Fig.\u0026nbsp;4A). Compared to the wt mice, \u003cem\u003eCOMT\u003c/em\u003e-ko mice showed lower MAO-A level in CPu (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;4B \u0026amp; C). Interestingly, \u003cem\u003eCOMT\u003c/em\u003e-ko mice showed MAO-B level changes in opposite directions in PFC and CPu, i.e. higher level in PFC (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but lower level in CPu (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) as compared to wt mice (Fig.\u0026nbsp;4D \u0026amp; E). The two groups were comparable in DDC levels in both PFC and CPu (Fig.\u0026nbsp;4F \u0026amp; G). HPLC results showed that \u003cem\u003eCOMT\u003c/em\u003e-ko mice had a significantly lower DA level in CPu compared to the wt mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas the two groups were comparable in DA level in PFC (Fig.\u0026nbsp;4H). The RT-qPCR analysis showed a higher level of DR1 mRNA in PFC (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), but lower levels of DR2 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and DAT (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) mRNAs in CPu of \u003cem\u003eCOMT\u003c/em\u003e-ko mice compared to the wt mice (Fig.\u0026nbsp;4I).\u003c/p\u003e \u003cp\u003eImmunohistochemical staining showed decreased number of mature OLs in PFC of \u003cem\u003eCOMT\u003c/em\u003e-ko mice compared to wt mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;5A \u0026amp; B), but comparable MBP-immunostaining intensity between the two groups (p\u0026thinsp;=\u0026thinsp;ns, Fig.\u0026nbsp;5C \u0026amp; D). Western blot analysis revealed lower level of MBP protein in the same brain region relative to that of wt mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;5E \u0026amp; F). Biochemical analysis with the PFC tissue revealed a marginal significant difference (p\u0026thinsp;=\u0026thinsp;0.07) in ATP level between the wt and \u003cem\u003eCOMT\u003c/em\u003e-ko mice (Fig.\u0026nbsp;5G), but no difference in ROS levels (p\u0026thinsp;=\u0026thinsp;ns, Fig.\u0026nbsp;5H). The changes in CPu of \u003cem\u003eCOMT\u003c/em\u003e-ko mice are similar to those in PFC, except that both ATP and ROS levels were significantly lower in \u003cem\u003eCOMT\u003c/em\u003e-ko mice compared to the wt mice (Supplemental Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eWe also assessed behavioral performances of the mice in the second animal experiment. Compared to wt mice, the \u003cem\u003eCOMT\u003c/em\u003e-ko mice showed behavioral anomalies indicated by a higher level of locomotor activity and a higher CD/TD in OFT (Supplemental Fig.\u0026nbsp;4A \u0026amp; B), longer duration on open arms and the central zone, but shorter duration in the closed arms of the EPM (Supplemental Fig.\u0026nbsp;4C) while visiting both the closed arms and central zone more frequently (Supplemental Fig.\u0026nbsp;4D). In the SIT, \u003cem\u003eCOMT\u003c/em\u003e-ko mice were unable to tell an empty cage from an identical cage with a novel conspecifics (Supplemental Fig.\u0026nbsp;4E \u0026amp; F).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDA inhibits the development of cultured OLs and induces OLs apoptosis via inhibiting mitochondrial functions of the cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll data from the above animal experiments strongly suggest a neurobiological mechanism in which DA elevation impairs mitochondrial function in brain cells thus inhibiting OL development/myelination process. To substantiate this neurobiological mechanism, we did \u003cem\u003ein vitro\u003c/em\u003e experiments in which purified oligodendrocyte precursor cells (OPCs) were cultured in the absence or presence of DA at the concentrations of 50, 100, or 200 \u0026micro;M starting on DIV (day \u003cem\u003ein vitro\u003c/em\u003e) 12 and continuing for 48 hrs. Dual immunofluorescent staining with the primary antibodies to Olig 2 (O2) and O4, O2 and CNP (2',3'-cyclic nucleotide phosphodiesterase), or CNP and MBP, was done to label immature and mature OLs while nuclei of cells were stained with DAPI dye. As shown in Fig.\u0026nbsp;6A, all differentiated OLs at specific developmental stages appear in distinctive morphology (size and shape) and labeling (color). Cell counting revealed: 1) no difference between VEH and DA groups in numbers of DAPI\u003csup\u003e+\u003c/sup\u003e cell nuclei and of cells labeled by the antibody to O2 (Fig.\u0026nbsp;6B), which is a sustained marker of OLs and expressed in all stages of OL development, from OPC to mature OL; 2) the ratio O4\u003csup\u003e+\u003c/sup\u003e/O2\u003csup\u003e+\u003c/sup\u003e cells (expressed as percentage, the same below) decreased in DA groups in a concentration-dependent manner (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;6C), indicating that DA inhibited the differentiation of OPC into immature OLs; 3) the ratios CNP\u003csup\u003e+\u003c/sup\u003e/O2\u003csup\u003e+\u003c/sup\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) cells and MBP\u003csup\u003e+\u003c/sup\u003e cells/DAPI\u003csup\u003e+\u003c/sup\u003e nuclei (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decreased in DA groups, but the ratio MBP\u003csup\u003e+\u003c/sup\u003e/CNP\u003csup\u003e+\u003c/sup\u003e cells did not change across VEH and all DA groups (p\u0026thinsp;=\u0026thinsp;ns, Fig.\u0026nbsp;6C). These data demonstrate that DA elevation retards the maturation of O4\u003csup\u003e+\u003c/sup\u003e cells into CNP\u003csup\u003e+\u003c/sup\u003e and MBP\u003csup\u003e+\u003c/sup\u003e cells but has no impact on the further maturation from CNP\u003csup\u003e+\u003c/sup\u003e cells to MBP\u003csup\u003e+\u003c/sup\u003e cells.\u003c/p\u003e \u003cp\u003eFurthermore, we analyzed effects of DA on mitochondrial functions of the cultured OLs. The mitochondrial membrane potential (ΔψM) of cultured OLs from the VEH and DA groups was assessed using an assay kit with JC-1. Compared to VEH group, JC-1 aggregates decreased in DA groups in a concentration-dependent manner indicated by gradual decreases in red signal which almost completely disappeared in the cells treated with the highest concentration of DA at 200 \u0026micro;M. In contrast, JC-1 monomers increased in DA-treated cells relative to VEH group indicated by green signal increase which was strongest in cells treated with DA at 100 \u0026micro;M (Fig.\u0026nbsp;6D). Therefore, ΔψM (FL590/FL530) values decreased in a DA concentration-dependent manner (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;6E). These data demonstrate the disruption of mitochondrial membrane potential in OLs of DA groups. In contrast, DA increased intracellular level of reactive oxygen species (ROS) in cultured OLs in a concentration-dependent manner (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;6F \u0026amp; G).\u003c/p\u003e \u003cp\u003eThe foregoing ROS and ΔψM data strongly suggest a cytotoxic effect (lethal effect at the highest concentration) of DA on cultured OLs. To verify this suggestion, the immunofluorescent staining with the antibody to cleaved Caspase-3 was done to label apoptotic OLs while the antibody to O2 was used to label nuclei of all OLs in the cultures without or with DA at the indicated concentrations. As shown in Fig.\u0026nbsp;6H \u0026amp; I, Caspase-3\u003csup\u003e+\u003c/sup\u003e cells increased in DA-treated cultures in a concentration-dependent manner (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and the O2\u003csup\u003e+\u003c/sup\u003e nuclei in cells treated with DA at 100 and 200 \u0026micro;M look much smaller than those in the VEH group, indicating the nuclear pyknosis of these damaged OLs and confirming the apoptotic OLs induced by the higher concentrations of DA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eDA inhibits axonal myelination in neuron-OL co-cultures while decreasing NAT8L level in neurons\u003c/h2\u003e \u003cp\u003eTo simulate the hypomyelination seen in the \u003cem\u003ein vivo\u003c/em\u003e experiments, we did neuron-OL co-culture experiments by referring to the protocol in a previous study [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], without or with DA at the indicated concentrations. Neuronal axons were labeled by the antibody to neurofilament (NF-H) while myelin sheath was labeled by the antibody to MBP. Immunofluorescent staining of the co-cultures showed myelin sheath wrapping around neuronal axons in VEH group and the group of 20 \u0026micro;M DA, but rare or no myelin sheath in cultures exposed to 100 \u0026micro;M or 500 \u0026micro;M DA (Fig.\u0026nbsp;7A), indicating that DA did inhibit axonal myelination in the neuron-OL co-cultures. In line with this indication, Western blot analysis showed decreased levels of CNP and MBP in the co-cultures treated with DA in a concentration-dependent manner (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;7B \u0026amp; C). Moreover, DA decreased NAT8L level in the co-cultures at a concentration-dependent manner (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;7D \u0026amp; E).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eNAC and TCP ameliorate the adverse effects of DA on cultured OLs\u003c/h2\u003e \u003cp\u003eThe above \u003cem\u003ein vitro\u003c/em\u003e data strongly indicate that the inhibitory effects of DA on cultured OLs development and axonal myelination in neuron-OL co-cultures are achieved via impairing mitochondrial function. If so, mitochondrial protection approaches should be able to ameliorate the inhibitory effects of DA. To substantiate this possibility, we did another two \u003cem\u003ein vitro\u003c/em\u003e experiments in which NAC (N-acetyl-L-cysteine, a well-established antioxidant) or TCP (trans-2-phenylcyclopropy, an inhibitor of mitochondrial MAOs) was used, respectively. In the TCP experiment, primary OLs were cultured at the indicated concentrations of DA (0, 100 \u0026micro;M, 200 \u0026micro;M) in the absence or presence of TCP (100 \u0026micro;M) during DIV 15\u0026ndash;17. At the end, the mature OLs were analyzed for cell viability, ΔψM, and the production of mitochondrial ROS and ATP, in addition to Western blot analysis measuring CNP and MBP levels. As shown in Fig.\u0026nbsp;8A \u0026amp; B, both 100 \u0026micro;M and 200 \u0026micro;M DA significantly decreased CNP and MBP levels in cultured OLs as compared to CNT group, but these effects were prevented or ameliorated in the presence of 100 \u0026micro;M TCP. 200 \u0026micro;M DA significantly decreased cell viability of cultured OLs as compared to CNT group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), but this effect was effectively ameliorated in the presence of 100 \u0026micro;M TCP (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;8C). Moreover, 200 \u0026micro;M DA significantly decreased mitochondrial ΔψM compared to CNT group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and this damaging effect was effectively ameliorated by TCP (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;8D). Relevantly, DA significantly decreased the production of ATP at both 100 \u0026micro;M (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and 200 \u0026micro;M (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) as compared to CNT group, but these decreases were effectively ameliorated by TCP (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 in the two cases, respectively; Fig.\u0026nbsp;8E). In contrast, addition of 200 \u0026micro;M DA significantly increased ROS production in cultured OLs relative to CNT group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and this increase was not seen in the presence of TCP (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;8F). TCP alone did not impact any of the above measurements. These data indicate that the protection of TCP against the toxic effects of DA on OLs is achieved by inhibiting the catabolism of DA and consequently decreasing ROS production.\u003c/p\u003e \u003cp\u003eIn the NAC experiment, 250 or 500 \u0026micro;M NAC (the two concentrations were referred to as NAC-l and NAC-h, respectively) was provided to the primary OLs 4 h before addition of 50 \u0026micro;M DA. Forty-eight hours later, the cell viability of cultured OLs was assessed using the CCK-8 assay kit, in addition to dual immunofluorescent staining with the primary antibodies to CNP and MBP. The other OLs were treated with 50 \u0026micro;M DA or 500 \u0026micro;M NAC alone, and the cells in Control group were treated with the vehicle in the absence of DA and NAC. As shown in Supplemental Fig.\u0026nbsp;5A, CNP\u003csup\u003e+\u003c/sup\u003e and MBP\u003csup\u003e+\u003c/sup\u003e cells appear in all groups at various proportions. CCK-8 assay showed that NAC alone had no effect on cell viability of cultured OLs (p\u0026thinsp;=\u0026thinsp;ns), but 50 \u0026micro;M DA significantly decreased cell viability, compared to the Control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Importantly, NAC pre-treatment at both 250 and 500 \u0026micro;M concentrations completely prevented the DA-induced cell viability decrease (Supplemental Fig.\u0026nbsp;5B). Furthermore, DA alone (50 \u0026micro;M) significantly decreased numbers of CNP\u003csup\u003e+\u003c/sup\u003e and MBP\u003csup\u003e+\u003c/sup\u003e cells compared to VEH group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively), but these inhibiting effects were significantly ameliorated by NAC-l (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and NAC-h (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Supplemental Fig.\u0026nbsp;5C). These data add further evidence for the antioxidative action of NAC which is effective in protecting OLs against the toxicity of DA.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eFor the past several decades, dopaminergic dysfunction has been considered a chief culprit for clinical manifestations of patients with schizophrenia. White matter abnormality in patients with schizophrenia, however, was not reported in living patients until the development and application of magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI) techniques in schizophrenia research and clinical practice [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The widely replicated DTI findings in schizophrenia patients suggest that white matter abnormalities may be at least partially associated with dopaminergic dysfunction as exemplified by a neuroimaging study reporting that the \u003cem\u003eCOMT\u003c/em\u003e genotype was associated with altered diffusion parameters in subcortical white matter in a sample of children and adolescents [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Moreover, a recent study reported the absence of predominantly inverse associations between D2/D3 receptor availability in the cortical and subcortical gray matter and axonal integrity in brain white matter of unmedicated patients with schizophrenia [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In the present study, TOL-induced DA elevation in mouse PFC was accompanied with mature OL decrease and hypomyelination indicated by lower levels of myelin related proteins including CNP, MAG, MBP, and MOG, as well as decreased myelinated axons but increased G-ratio of myelinated axons, as compared to healthy controls in the VEH group. Moreover, mitochondrial functions of neural cells in this brain region of TOL-treated mice were impaired as indicated by decreased protein levels of the complex I, II, and IV, as well as of NAT8L, in addition to ATP decrease. Of note is NAT8L, a neuron-specific protein in the brain and responsible for NAA synthesis from aspartate and acetyl-CoA in neurons [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In such a way, NAT8L involves in myelination in the juvenile mice via supplementation of acetate derived from NAA [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Taken together, all these data for the first time demonstrate a pathway from DA elevation through mitochondrial dysfunction to hypomyelination in PFC of TOL-treated mice.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eCOMT\u003c/em\u003e-ko mice, dopaminergic homeostatic responses happened in PFC and CPu. Interestingly, the responses in these two brain regions are different. In PFC, levels of MAO-B protein and DR1 mRNA increased while DA level was comparable to that in wt mice, suggesting the existence of compensatory changes in this brain region in response to \u003cem\u003eCOMT\u003c/em\u003e knockout. In CPu, levels of DA, MAO-A and MAO-B proteins, as well as mRNA levels of DR2 and DAT decreased significantly as compared to wt mice, indicating the changes in this brain region are decompensated. In line with this suggestion, the CPu of \u003cem\u003eCOMT\u003c/em\u003e-ko mice showed mature OL decrease, lower MBP immunostaining intensity and MBP protein level, as well as lower levels of ATP and ROS compared to wt mice. From all these data, an inference can be drawn that neural cells (including dopaminergic axons and OL processes wrapping around the axons) in CPu are more vulnerable to DA elevation than those in PFC. This inference is in line with the findings from previous studies that systemically administration of 10 mg/kg TOL induced an increase in hydroxyl radical in the striatum of anaesthetized rats following treatment with L-DOPA/carbidopa [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and that methamphetamine treatment depleted striatal DA, generated ROS, and decreased activity of complex I of the mitochondria [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The demonstration of this inference is of great significance for the pathogenesis of extrapyramidal syndromes (EPS), which is one of the most challenging adverse effects in patients with schizophrenia when using antipsychotic drugs. Previous studies suggest that a high degree of D2R occupancy is necessary for the occurrence of EPS and schizophrenia patients with EPS have higher D2 R occupancy (above 80%) than patients free of EPS (65\u0026ndash;80%) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, the underpinning molecular mechanisms for EPS remain to be elucidated. According to the DA elevation - mitochondrial dysfunction - hypomyelination pathway demonstrated in this study, chronic administration of antipsychotics leads to DA elevation in extracellular space and synaptic cleft due to the higher D2 R occupancy of these drugs. Consequently, increased DA may enter the cytoplasm of neural cells, where it adversely impacts mitochondrial function (as demonstrated in the present study), and/or leads to DA auto-oxidation in the extracellular space [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The both intracellular and extracellular events lead to the same ultimate results of ROS increase which consequently damages neural cells (as demonstrated in the present study).\u003c/p\u003e \u003cp\u003eNot surprisingly, the TOL-treated mice and \u003cem\u003eCOMT\u003c/em\u003e-ko mice did not present same behavioral anomalies. Specifically, the mice in TOL30 and TOL60 groups showed higher anxiety level in OFT, spatial working memory impairment detected by Y-maze test, deficit in recognition memory and less interest in exploring objects throughout the NOR test, as well as impaired executive function measured in the POT. As for \u003cem\u003eCOMT\u003c/em\u003e-ko mice, they had difficulty deciding whether to enter an open or a closed arm of the EPM, suggesting a somewhat cognitive impairment. In this context, that the \u003cem\u003eCOMT\u003c/em\u003e-ko mice presented a higher CD/TD in OFT should be interpreted as a behavioral disorganization rather than a lower level of anxiety. In addition, the \u003cem\u003eCOMT\u003c/em\u003e-ko mice showed social deficits as indicated by less interest in socializing with a novel conspecifics in SIT, which is considered a reliable method to test the negative symptoms-related behaviors in animal models of schizophrenia [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Notably, the behavioral abnormalities co-exist with decreased levels of MAO-B in CPu of the \u003cem\u003eCOMT\u003c/em\u003e-ko mice. This phenomenon is in line with previous studies in which \u003cem\u003eMAO-B\u003c/em\u003e KO mice exhibited behavioral dis-inhibition such as novelty seeking behavior and reduced anxiety-like behaviors but had comparatively less aggressive behavior compared with \u003cem\u003eMAO-A\u003c/em\u003e KO mice in several behavioral paradigms targeting emotional reactivity (47\u0026ndash;49). Taken together, all these behavioral anomalies are reminiscent of the negative symptoms and cognitive impairment seen in patients with schizophrenia. Importantly, these behavioral anomalies coexist with mature OL decrease and hypomyelination in TOL-treated mice and \u003cem\u003eCOMT\u003c/em\u003e-ko mice, suggesting a causal relationship between these two types of phenotypes. In line with this suggestion, myelin defect was shown to cause cognitive dysfunction and increase vulnerability to social withdrawal in adult mice of a recent study [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In another animal study, myelin degeneration and diminished myelin renewal contributed to age-related deficits in memory [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Moreover, a recent clinical study demonstrated that chronic schizophrenia is characterized by global microscopic brain hypomyelination in both white matter and gray matter associated with the disease duration and negative symptoms [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe pathway from DA elevation through mitochondrial dysfunction to hypomyelination demonstrated in animal models of schizophrenia was further substantiated by the following findings from the \u003cem\u003ein vitro\u003c/em\u003e experiments: 1) DA inhibited the differentiation of OPC to O4\u003csup\u003e+\u003c/sup\u003e cells and delayed the maturation of OLs in a concentration-dependent manner; 2) DA disrupted mitochondrial functions of OLs as evidenced by decreased ΔψM and increased ROS in DA-treated cells, which consequently led to OL apoptosis. These data strongly suggest that extracellular DA can enter the cytoplasm of OLs where it impacts mitochondrial functions, thereby demonstrating the molecular mechanism for the inhibiting effects of DA on OLs. In support of this inference, 3) the MAOs inhibitor TCP effectively attenuated DA-induced CNP and MBP decrease in cultured OLs in the presence of DA, and protected the OLs against the toxic effects of DA on mitochondrial functions. Moreover, 4) the antioxidant NAC effectively improved the developmental delay of cultured OLs in the presence of DA with a concentration dependent manner, providing further evidence that ROS is the culprit of the adverse effects of DA on OLs. Last but not least, DA inhibited axonal myelination in the neuron-OL co-cultures at a concentration-dependent manner, elegantly simulated the hypomyelination seen in TOL-treated mice and \u003cem\u003eCOMT\u003c/em\u003e-ko mice.\u003c/p\u003e \u003cp\u003eAs mentioned earlier, the existing antipsychotics have little or no therapeutic effect on negative symptoms and cognitive impairment in patients with schizophrenia. This predicament is due to an incomplete or even incorrect understanding of the pathogenesis of schizophrenia. According to the pathway from DA elevation through mitochondrial dysfunction to hypomyelination demonstrated in this study, antipsychotic drugs block DA receptors and increase DA levels in synaptic cleft and other parts of the extracellular space, thereby increasing ROS levels and damaging neural cells, leading to cognitive deficits and negative symptoms in patients with schizophrenia. Therefore, the correct strategy for antipsychotic treatment should deal with DA elevation and its downstream events rather than blocking DA receptors. If DA level in the brain of a schizophrenia patient can be returned to normal at time, the psychotic behaviors, i.e. the positive symptoms, of the patient would be normalized as the immediate therapeutic effect without the side effect of EPS. Moreover, the cognitive function and negative symptoms of the patient would be improved following the recovery of OLs damage resulted from high level of DA. In line with this inference, SNPs in \u003cem\u003eCOMT\u003c/em\u003e and \u003cem\u003eMAO A/B\u003c/em\u003e genes, with reduced activity in the corresponding enzymes, are associated with a decrease in DA degradation and hence dopaminergic hyperactivity occurred via D2 receptors [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. And there are increasing clinical studies applying antioxidant addition to antipsychotic treatment for patients with schizophrenia although results are inconsistent [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Moreover, targeting myelin and OL dysfunction in schizophrenia has been viewed as a novel treatment strategy [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, data from \u003cem\u003ein vivo\u003c/em\u003e experiments demonstrate a pathway from DA elevation through mitochondrial dysfunction to hypomyelination in the mouse brain. The \u003cem\u003ein vitro\u003c/em\u003e experiments with primary OL culture and neuron-OL co-culture system elucidate the mechanism of the aforementioned pathway, wherein mitochondria play pivotal roles in DA catabolism and axonal myelination. The co-existence of behavioral anomalies and hypomyelination in mice subsequent to the disruption of this pathway provides a novel insight into the negative symptoms and cognitive impairment seen in patients with schizophrenia. More importantly, amelioration of DA-induced mitochondrial dysfunction and hypomyelination by NAC and TCP in cultured OLs offers a theoretical basis for targeting mitochondria and OLs in treating patients with schizophrenia.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCOMPETING INTERESTS\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eFY, YZ, CJ, and NO designed and carried out experiments, recorded and analyzed experimental data, and participated manuscript preparation. QW, PW, and PZ provided assistants for cell culture experiments. WW, JH, YL, HZ, and LL instructed experiments. HX conceptualized the study, wrote the draft, and edited the manuscript. HX and XY supervised the research project. All authors read and approved the submitted version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis study was supported by grants from the National Natural Science Foundation of China (# 81971256), Natural Science Foundation of Guangdong Province (# 2016A030313067), and Li Ka-Shing Foundation (# 43209502).\u003c/p\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe data that support the findings of this study are available from the cor-responding author upon a reasonable request.\u003c/p\u003e \u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMcCutcheon RA, Reis Marques T, Howes OD. Schizophrenia-An overview. JAMA Psychiatry. 2020; 77: 201\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFusar-Poli P, Papanastasiou E, Stahl D, Rocchetti M, Carpenter W, Shergill S, et al. 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Antioxidant treatments for schizophrenia. Cochrane Database Syst Rev. 2016; 2:CD008919.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoares-Weiser K, Maayan N, Bergman H. Vitamin E for antipsychotic-induced tardive dyskinesia. Cochrane Database Syst Rev. 2018; 1: CD000209.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGouv\u0026ecirc;a-Junqueira D, Falvella ACB, Antunes ASLM, Seabra G, Brand\u0026atilde;o-Teles C, Martins-de-Souza D, et al. Novel treatment strategies targeting myelin and oligodendrocyte dysfunction in schizophrenia. Front Psychiatry. 2020; 11:379.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"dopamine, mitochondria, oligodendrocyte, hypomyelination, schizophrenia","lastPublishedDoi":"10.21203/rs.3.rs-3875841/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3875841/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Schizophrenia is one of the most complex and debilitating brain diseases. Patients with schizophrenia may present various clinical manifestations that have been categorized into positive symptoms , negative symptoms, and cognitive deficits. In relation to these complex clinical manifestations, multiple hypotheses have been proposed to understand the pathogenesis of schizophrenia, such as the so-called dopamine (DA) hypothesis, mitochondrion hypothesis, oligodendrocyte (OL) hypothesis, etc. The concurrent existence of multiple hypotheses about one brain disease suggests a possible common neurobiological mechanism linking some of these hypotheses. This possible neurobiological mechanism has been demonstrated in this study with animal models of schizophrenia, cultured OLs, and neuron-OL co-cultures. Adolescent C57BL/6 mice given tolcapone (TOL) for two weeks showed DA elevation in prefrontal cortex (PFC), functional impairment of mitochondria in brain cells, and hypomyelination in PFC, hippocampus, and caudate putamen (CPu) in a dose-dependent manner, in addition to schizophrenia-related behaviors. The catechol-O-methyltransferase (COMT) gene knock-out (COMT-ko) mice presented dopaminergic dysfunctions in PFC and CPu, functional deficit of mitochondria, mature OL decrease, and hypomyelination in the same brain regions as those in TOL-treated mice. In cultured OLs, DA inhibited the cell development in a concentration-dependent manner while impairing mitochondrial functions. These effects of DA on cultured cells were ameliorated by the antioxidant N-acetyl-L-cysteine (NAC) and trans-2-phenylcyclopropy (TCP), an inhibitor of mitochondrial monoamine oxidases (MAOs). Moreover, DA inhibited axonal myelination in neuron-OL co-cultures while impairing mitochondrial functions. These data demonstrate the pivotal roles of mitochondria in linking DA catabolism to axonal myelination in the brain and provide a novel insight into the pathogenesis and therapeutic strategy for schizophrenia.","manuscriptTitle":"Pivotal roles of mitochondria in linking dopamine catabolism to axonal myelination: Implication for the pathogenesis and treatment of schizophrenia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-18 16:02:45","doi":"10.21203/rs.3.rs-3875841/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"70d79969-8254-4c32-bf48-a5945d909292","owner":[],"postedDate":"July 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33537400,"name":"Health sciences/Diseases/Psychiatric disorders/Schizophrenia"},{"id":33537401,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2024-08-20T14:19:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-18 16:02:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3875841","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3875841","identity":"rs-3875841","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}