In this study, we investigate the protective effect of hyperoside on neuroinflammation and its possible molecular mechanism. Lipopolysaccharide (LPS) and hyperoside were used to treat HT22 cells. The cell viability was measured by MTT assay. The cell apoptosis rate was measured by flow cytometry assay. The mRNA expression levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) were determined by quantitative reverse transcription polymerase chain reaction. The levels of oxidative stress indices superoxide dismutase (SOD), reactive oxygen species (ROS), catalase (CAT), glutathione (GSH), and malondialdehyde (MDA) were measured by the kits. The expression of neurotrophic factor and the relationship among hyperoside, silent mating type information regulation 2 homolog-1 (SIRT1) and Wnt/β-catenin, and sonic hedgehog was examined by western blotting. In the LPS-induced HT22 cells, hyperoside promotes cell survival; alleviates the level of IL-1β, IL-6, IL-8, TNF-α, ROS, MDA, Bax, and caspase-3; and increases the expression of CAT, SOD, GSH, Bcl-2, BDNF, TrkB, and NGF. In addition, hyperoside upregulated the expression of SIRT1. Further mechanistic investigation showed that hyperoside alleviated LPS-induced inflammation, oxidative stress, and apoptosis by upregulating SIRT1 to activate Wnt/β-catenin and sonic hedgehog pathways. Taken together, our data suggested that hyperoside acts as a protector in neuroinflammation.Neuroinflammation plays important roles in the pathogenesis and progression of altered neurodevelopment, sensorineural hearing loss, and certain neurodegenerative diseases. Hyperoside (quercetin-3-O- Neuroinflammation is a chronic inflammation of brain tissue, which plays an important role in the pathogenesis and progression of altered neurodevelopment, sensorineural hearing loss, and certain neurodegenerative diseases –5. In thβ-D-galactoside) is an active compound isolated from Hypericum plants. It has antioxidant and anti-inflammatory activities, decreasing calcium overload and inhibiting apoptosis [α/NF-κB/caspase-3 signaling pathway [Hyperoside  is widely used to activate the innate immune system. Previous studies have shown that LPS is usually used to prepare neuroinflammation models induced by inflammatory response , 14. In β-catenin, Shh, Patch, and GAPDH and secondary antibodies were all purchased from Cell Signaling .LPS, hyperoside, and 3--2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich . Dulbecco's modified Eagle medium containing 10% fetal bovine serum (FBS) was purchased from Gibco . 100 U/ml penicillin and 100 mg/ml streptomycin were purchased from Sigma-Aldrich . LiCl and sonic hedgehog agonist SAg were purchased from Merck . Primary antibodies against Bcl-2, Bax, caspase-3, BDNF, NGF, SIRT1, Wnt1, 4 cells/well in a 6-well plate and cultured in the DMEM with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a 5% CO2 humidified incubator. When HT22 reached 70% confluency after 24 h, cell transfection pretreated with different concentrations of hyperoside and LPS (1 μg/ml) was performed.The HT22 murine neuronal cell line was purchased from Kunming Cell Bank of the Chinese Academy of Sciences . HT22 cells were seeded at 2 × 104 cells/ml for 24 h. Cells were starved overnight and then pretreated with different concentrations of hyperoside for 24 h followed by incubation with LPS (1 μg/ml) for another 24 h. The medium was refreshed and incubated with 50 μl of MTT  for 4 h at 37°C. Next, the solution was removed and added DMSO to plates. The absorbance at 570 nm was measured using a plate reader.Cell viability was determined by MTT assay. Briefly, the HT22 cells seeded into 24-well plates at a density of 2 × 10β-catenin, Shh, Patch, and GAPDH) at 4°C overnight. Then, the next day, the membranes were washed with PBS and incubated with secondary antibodies for 1 h. Finally, the protein bands were measured using the ImageJ software. The data were collected from at least three independent experiments.RIPA buffer added to HT22 cells  to collect the total protein; then the protein concentrations were determined assessed using the BCA method . The protein samples were mixed with a 5x loading buffer and denatured at the boil. The proteins were separated by 15% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Next, the membranes were incubated with primary antibodies , and the isolated total RNA was reverse-transcribed to cDNA using a reverse transcription kit . Next, SYBR Premix Ex Taq II  was used to perform qRT-PCR amplification. The IL-1We measured oxygen species (ROS), catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) levels activity using the corresponding assay kits .Flow cytometry assay was used to measure the cell apoptosis rate according to a previous report . HT22 cet-test, and that among groups was analyzed by one-way analysis of variance (ANOVA). All data were presented as the mean values ± standard deviation (SD). Statistical analyses were performed by GraphPad Prism 7.0 software. P < 0.05 was considered to indicate a statistically significant difference.In this study, the difference between two groups was compared by using a μg/ml) significantly reduced HT22 cell viability ) were used to treat cells. Western blotting showed that SITR1 expression decreased in LPS-induced cells; simultaneously, compared with LPS induction, hyperoside treatment upregulated the expression level of SITR1. In addition, NAM treatment decreased the expression of SIRT1 in HT22 cells compared with the treatment of cells with LPS+hyperoside treatment . Likewisreatment . These rWe studied the effect of SIRT1 on oxidative stress and neurotrophic factors in LPS-induced HT22 cells. The results showed that NAM treatment decreased the levels of SOD, GSH, CAT, and neurotrophic factors BDNF and NGF and increased the levels of ROS and MDA in HT22 cells, compared with the treatment of cells with LPS+hyperoside treatment Figures . These rβ-catenin and sonic hedgehog pathways, we measured the expression levels of signaling molecules in the Wnt/β-catenin and sonic hedgehog pathways. The results showed that LPS significantly decreased the expression levels of Wnt1, β-catenin, Shh, and Patch; hyperoside treatment significantly increased the expression levels of Wnt1, β-catenin, Shh, and patch; in addition, NAM treatment decreased the expression levels of Wnt1, β-catenin, Shh, and patch. In addition, in HT22 cells, compared with NAM-treated cells, Wnt/β-catenin agonist (LiCl) and sonic hedgehog agonist (SAg) inhibited the inhibitory effect of NAM  and apoptosis proteins Bax and xaspase-3, and activated oxidative stress. However, pretreatment with hyperoside significantly protected HT22 cells from LPS-induced cell growth inhibition by inhibiting apoptosis; downregulating TNF-α, IL-1β, IL-6, IL-8, Bax, and caspase-3 levels; and inhibiting oxidative stress. These results are consistent with previous studies on hyperoside as antioxidants, anti-inflammatory agents, and antiapoptotic agents.Neurons are the basic structural and functional units of the nervous system . ChangesRegulation that protects neuronal survival is essential in the pathological process of alleviating neurological complications caused by neuroinflammation , 31. In β-catenin and sonic hedgehog pathways are confirmed to be regulated by SIRT1; SIRT1-mediated deacetylation in the process of c-myc degradation, which affected the stability of c-myc and increased the transcriptional activity of β-catenin, activates Wnt signaling through β-catenin [β-catenin and sonic hedgehog signaling pathways are inhibited in neuroinflammation [β-catenin and sonic hedgehog signaling pathways are involved in the development of neuroinflammation-mediated hearing loss and other neurological diseases [β-catenin, Shh, and patch by upregulating SIRT1.SIRT1 is the main regulator of neurogenesis and plays a neuroprotective role in neurological diseases . Previou-catenin ; simulta-catenin . In addiammation , 45. Simdiseases –55. In tβ-catenin and sonic hedgehog signaling pathways. In conclusion, based on our findings, the therapeutic effect of hyperoside on neuroinflammation is further clarified, providing possible treatment basis for its clinical application for neuroinflammation.In summary, the present study showed that hyperoside alleviated apoptosis, inflammation, oxidative stress, and reduction of neurotrophic factor in the LPS-induced HT22 cells. We further found that hyperoside alleviated nerve damage by upregulating SIRT1 to activate Wnt/


Glioblastoma multiforme (GBM) is one of the most lethal forms of primary brain tumors. Glioblastoma stem cells (GSCs) play an undeniable role in tumor development by activating multiple signaling pathways such as Wnt/β-catenin and PI3K/AKT/mTOR that facilitate brain tumor formation. CD133, a transmembrane glycoprotein, has been used to classify cancer stem cells (CSCs) in GBM. The therapeutic value of CD133 is a biomarker of the CSC in multiple cancers. It also leads to growth and recurrence of the tumor. More recent findings have confirmed the association of telomerase/TERT with Wnt/β-catenin and the PI3K/AKT/mTOR signaling pathways. Advance studies have shown that crosstalk between CD133, Wnt/β-catenin, and telomerase/TERT can facilitate GBM stemness and lead to therapeutic resistance. Mechanistic insight into signaling mechanisms downstream of surface biomarkers has been revolutionized by facilitating targeting of tumor-specific molecular deregulation. This review also addresses the importance of interplay between CD133, Wnt/β-catenin and TERT signaling pathways in GSCs and outlines the future therapeutic goals for glioblastoma treatment. Glioblastoma multiform (GBM) is one of the calamitous kinds of aggressive primary glial brain tumor in adults, with a median overall survival between 10 to 20 months. Intriguingly, the recent study has demonstrated that outer radial glia-like cancer stem cells could impart to heterogenicity of GBM .GSCs have been identified by many biomarkers of CSCs. CD133, also known as prominin-1, has been used as an essential marker for the detection of GSCs. Additionally, CD133 has been shown to be associated with GBM development, recurrence, and poor overall survival . EmerginEach component of the CD133, Wnt/β-catenin, and TERT signaling pathways play a crucial role in the normal brain. CD133 is present in epithelial cells throughout the body, including the mammary gland, testis, digestive tract, trachea, and placenta, and is expressed in stem cells. CD133 is also present in non-epithelial cells, such as rod photoreceptor cells, and in many cancers . The WntThe current review focuses on the significance of interplaying between CD133, Wnt/β-catenin and TERT signaling pathways in normal brain and GSCs. From a therapeutic perspective, targeting the interaction between CD133, Wnt/β-catenin and TERT will lead to the development of novel anti-cancer strategies and it would be important to examine if all these three signaling cascades can be inhibited concurrently by targeting CD133, thus killing many birds with one stone Figure 1in vitro and in vivo CD133POS-marker cells. Furthermore, a study showed that CD133 knockdown could inhibit cell proliferation of glioma , a 5-transmembrance glycoprotein, is one of these biomarkers and also commonly used in identification and isolation of GSCs . Highly f glioma . In lighgnancies . It was gression . In suppr masses .Pos ependymal stem cells in the adult SVZ/RMS in vivo and it was reported that CD133 is specifically localized for ependyma, albeit not all ependymal cells are CD133Pos  of mammals, neurons, astrocytes and oligodendrocytes occurs from a reservoir of murine neuroepithelial (NE) cells, which are neural progenitor cells. Proliferative cell division changes to differentiating cell division as neurogenesis starts and it was shown that membrane particles containing CD133 have been shown to help cell differentiation. In addition to being restricted to NE progenitors, the presence of CD133 is also present in both epithelial and non-epithelial cell forms . Given tCD133Pos . In suppts brain .Wnt signaling pathway is mediated by Wnts proteins that are secreted by glycoproteins. This Wnts proteins are critical for cell proliferation, and differentiation of both normal and cancer stem cells , 29. TwoIt has been accepted that Wnt/β-catenin plays an inevitable role in neurogenesis and gliogenesis in the neocortex. Neurogenesis in neocortex occurs in the ventricular zone (VZ) of the dorsal telencephalon from radial glia (RG) and in the subventricular zone (SVZ) from neural intermediate progenitors (Ips) . During It subsists ambiguous what self-defense mechanism are applied by GSCs against chemotherapeutic drugs. As of lately, it is believed that the DNA damage response (DDR) and DNA repair pathway have undeniable role in self-defense mechanism of cancer stem cells (CSCs) . GrowingIn the majority of human primary cancers (~90%), TERT expression/telomerase activity is perceptible. Additionally, induced-TERT or activated-telomerase bestows immortality properties to cancer cells by stabilizing their telomere length (TL). However, current surveys have indicated that its oncogenic features independently of TL, which include DNA damage repair, gene transcription and microRNA expression . MoreoveDoes activated-telomerase and expressed-TERT guard the neurons in our brain? Activated-telomerase is conjoined with cell proliferation during mammalian embryonic development, cell transformation, and in cancers. In adult animals the expression of telomerase is limited to the subventricular zone (SVZ) and olfactory bulb . It was Meanwhile, CD133, also known as prominin-1, is one of the most used biomarkers for the isolation of cancer stem cells (CSCs). The rigorous function of CD133 maintains unidentified, but it has been proposed that it would perform as a cell membrane topology organizer. Furthermore, by activating the Wnt/β-catenin pathway, CD133 could enhance growth, differentiation of nerve cells, and neurogenesis in the brain . The actPosCSC and CD133Neg differentiated glioblastoma cells (DGCs) were compared (P ≤ 0.05). The expression of eight Wnt proteins  was substantially increased. Interestingly, the expression of CTNNB1 (β-catenin) was enhanced 13.98-fold in CD133PosCSC relative to CD133Neg DGCs , Nanog, Octamer-binding transcription factor-4 (Oct-4), and Nestin, which proposes the role of biomarkers-Wnt/β-catenin axis in remaining stemness traits of GBM . Additio in GSCs . Consequ in GSCs –77. The  in GSCs . Activat in GSCs . Some in in GSCs , 81.in vitro research findings showed that TERT expression levels were associated with Snail1 and vimentin expression levels  may be regulated by Wnt/β-catenin. On one hand, TERT may be regarded as one of the key targets of the Wnt/β-catenin signaling pathway. It has been shown that β-catenin can interact directly with the mouse and human cancer cells or cancer stem cells TERT promoter. In plain terms, these findings indicate that β-catenin controls the length of telomere by activating the transcription of TERT. Another research stated that TERT could stimulate EMT using Wnt/β-catenin. In addition, n levels –86. TERTn levels , 87. Of n levels , 86, 88.n levels , 89. In pos/high sub-population of GSCs correlates to invasiveness and progression of tumor and besides, CD133-Wnt/β-catenin axis has an inevitable role in stemness of GBM. Recent evidence also demonstrated that interplay between Wnt/β-catenin pathway and TERT can be modulating the stemness of CSCs. Hence, activation of the above axis in the brain might be considered as a gate between “tranquility and turmoil” and targeting this pathway might shed a light on GBM therapy and prompt further investigations in this area. However, the bulk of these targeting techniques are of an experimental nature and their medical use is minimal. The biggest challenge is the heterogeneity of GSC’s surface biomarkers, which render it challenging to detect and therefore aim therapy. In addition, answering the challenge of similarities between normal neural stem cells and GSCs makes treatment-associated toxicity possible.In a nutshell, in the normal brain, each element of signaling pathways of CD133, Wnt/β-catenin, and TERT has a critical role in the integrity of the brain It is generally accepted that the expression of CD133 in normal cell is significantly lower compared to cancerous cells. This is also true and when comparing normal cancer cells to GSCs , especially in GSCs which has the highest expression. Upon minimizing toxicity effect towards normal cells targeting therapy is important. One can target CD133 surface receptor to specifically bring drugs to CSCs leveraging on its significantly high expression in CSCs. The overexpressed-TERT would increase cell proliferation during embryonic development, whilst CD133 biomarker by activating Wnt/β-catenin pathway leads to neurogenesis. Thus, might be the cross-talk between each component of the mentioned axis has a neuroprotective effect on the normal brain. On the one hand, CD133AB: Conceptualization, Writing – Original draft. AS: Supervision, Writing – Reviewing, and Editing, Funding Acquisition. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The incidence of migraines is higher among individuals with epilepsy than in healthy individuals, and these two diseases are thought to shared pathophysiological mechanisms. Excitation/inhibition imbalance plays an essential role in the comorbidity of epilepsy and migraine. Microglial activation is crucial for abnormal neuronal signal transmission. However, it remains unclear whether and how microglia are activated and their role in comorbidities after being activated. This study aimed to explore the characteristics and mechanism of microglial activation after seizures and their effect on migraine.Model rats of status epilepticus (SE) induced by intraperitoneal injection of lithium chloride (LiCl)-pilocarpine and migraine induced by repeated dural injections of inflammatory soup (IS) were generated, and molecular and histopathologic evidence of the microglial activation targets of fractalkine (FKN) signalling were examined. HT22-BV2 transwell coculture assays were used to explore the interaction between neurons and microglia. LPS  and FKN stimulation of BV2 microglial cells were used to evaluate changes in BDNF levels after microglial activation.Microglia were specifically hyperplastic and activated in the temporal lobe cortex, thalamus, and spinal trigeminal nucleus caudalis (sp5c), accompanied by the upregulation of FKN and CX3CR1 four days after seizures. Moreover, SE-induced increases in nociceptive behaviour and FKN/CX3CR1 axis expression in migraine model rats. AZD8797 (a CX3CR1 inhibitor) prevented the worsening of hyperalgesia and microglial activation in migraine model rats after seizures, while FKN infusion in migraine model rats exacerbated hyperalgesia and microglial activation associated with BDNF-Trkb signalling. Furthermore, in neuron-microglia cocultures, microglial activation and FKN/CX3CR1/BDNF/iba1 expression were increased compared with those in microglial cultures alone. Activating microglia with LPS and FKN increased BDNF synthesis in BV2 microglia.Our results indicated that epilepsy facilitated migraine through FKN/CX3CR1 axis-mediated microglial activation in the cortex/thalamus/sp5c, which was accompanied by BDNF release. Blocking the FKN/CX3CR1 axis and microglial activation are potential therapeutic strategies for preventing and treating migraine in patients with epilepsy.The online version contains supplementary material available at 10.1186/s10194-022-01416-w. Migraine and epilepsy are among the most frequently encountered nervous system diseases. A large number of studies have highlighted the complex relationship between migraine and epilepsy. Patients with epilepsy have a 52% greater lifetime prevalence of migraines than those without epilepsy . The undResting-state functional magnetic resonance imaging (fMRI) has revealed diverse connections between the thalamus and cortex in humans . EpilepsMicroglia and neuron-microglia interactions are crucial in the central nervous system (CNS) and are characterized by altered neural network excitability . Many stFKN is a chemokine expressed by both neurons and glia, and its receptor, CX3CR1, is primarily expressed on microglia . Given tBrain-derived neurotrophic factor (BDNF), including the precursor molecule (proBDNF) and a mature form, is mainly expressed in the CNS. BDNF is released by neurons and microglia and plays a crucial role in neuroinflammation . Many stOur research aimed to investigate whether microglia and their interactions with neurons via the FKN/CX3CR1 axis in the thalamic cortical network are involved in the comorbidities of epilepsy and migraine by regulating the expression of BDNF. Here, we used a comorbidity model of epilepsy and migraine established in our previous research . MigrainAdult male Sprague–Dawley (SD) rats weighing 200–250 g were procured from the Laboratory Animal Centre of Renmin Hospital, Wuhan University. All rats were housed under specific pathogen-free (SPF) laboratory conditions with a 12-h light/dark cycle  and ad libitum access to food and water. The protocol was reviewed and approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University. Animal studies were performed in compliance with the ARRIVE guidelines and the Basel Declaration, including the “3R” concept .The induction of status epilepticus using pilocarpine was performed as previously described . ExperimThe experimental procedures were carried out as previously reported . After tNociceptive behaviour was assessed by counting the number of head-scratching actions and measuring the periorbital region mechanical sensitivity threshold. The head-scratching counts were recorded for one hour immediately after the last IS administration to assess nociceptive behaviours. Only rats scratching the face above the eye, which is innervated by the first division of the trigeminal nerve, were counted . To asseAZD8797 (an antagonist of CX3CR1) administration was carried out as described previously . AZD8797After one week of acclimatization, a total of 80 rats were randomly divided into ten groups of eight rats each according to experimental needs.Experiment 1 was designed to assess the effect of seizures on migraine and the expression of the FKN/CX3CR1 axis in SE and migraine rats. The rats were randomly divided into four groups: sham group, migraine group, SE group, and comorbidity group. The sham group received PBS. Rats in the migraine group underwent IS infusion. LiCl-Pilo was used in the SE group. Repeated IS injection into the dura mater of SE model rats was performed once per day for four consecutive days to establish the comorbidity group . The protein levels of proBDNF/BDNF were also measured to examine the potential molecular pathway associated with the FKN/CX3CR1 axis. Moreover, IF staining was performed to examine changes in microglia (n = 3 ~ 4/group).Experiment 2 was performed to investigate whether the effect of seizures on migraine was mediated by FKN/CX3CR1. AZD8797 was administered to rats in the comorbidity group. The groups included the sham-PBS group, the comorbidity-PBS group, and the comorbidity-AZD group Fig. A. After n = 3 ~ 4/group), and IF staining (n = 3 ~ 4/group) were conducted in this experiment.To further validate the effect of FKN/CX3CR1 on facilitating migraine-related behaviours and the specific mechanisms, FKN was infused into rats with migraine. There were three groups: the PBS-sham group, the PBS-migraine group, and the FKN-migraine group Fig. A. BehaviThe observers were blinded to group allocations during the animal experiments.2 incubator at 37 °C. BV2 cells were activated with lipopolysaccharide  for 90 min . C. C25]. CThe number and activation state of microglia was increased in the cortex/thalamus/sp5c after seizuresP < 0.01), more synapses , and shorter branches  than the sham group . These results suggest that the FKN/CX3CR1 axis may be linked to the pathogenesis of migraine and that epileptic seizures may exacerbate migraine by promoting the expression of FKN/CX3CR1 axis. Furthermore, immunofluorescence staining was conducted to examine the FKN/CX3CR1 expression in the sp5c. With iba1 immunoreactivity serving as a marker for microglia, a marked visual overlap between iba1 and CX3CR1 immunoreactivities was observed in sp5c sections . Correspondingly, FKN was mainly expressed in the cytoplasm of neurons and largely colocalized with Neun, a neuronal marker . We next explored the protein expression of CX3CR1/FKN/proBDNF/BDNF to examine the effects of AZD8797 on migraine model rats after seizures at the molecular level. The expression of FKN/CX3CR1 axis factors decreased by 1/3 in the comorbidity-AZD group compared with the comorbidity-PBS group , indicating that AZD8797 could block the FKN/CX3CR1 axis. Additionally, the expression of proBDNF and BDNF decreased by 45% and 41%, respectively, after AZD8797 treatment (Fig. AZD8797 is one of the most commonly used antagonists of CX3CR1. To furtFKN infusion increased nociceptive behaviour and proBDNF/BDNF/iba1 expression in migraine model ratsP < 0.001; Fig. P < 0.01; Fig. To further investigate whether seizures facilitate migraines through the FKN/CX3CR1 axis, we injected FKN into the sp5c of migraine model rats using stereotactic brain injection, and the sham groups received the same amount of PBS. The experimental procedure is shown in Fig. Migraine comorbidity with epilepsy significantly increases disease burden. Although numerous studies have been published on the association of microglia with migraine and epilepsy, few studies have investigated whether microglial activation is one of the causes of migraine susceptibility after epileptic seizures. In line with this hypothesis, our study supports this idea by showing that neurons activate microglia in the cortex/thalamic/sp5c via the FKN/CX3CR1 axis, which leads to enhanced BDNF production in migraine model rats following an epileptic episode. Furthermore, we explored whether the FKN/CX3CR1 axis intervention regulated nociceptive behaviour in migraine model rats, further revealing its role in migraine. These findings contribute to advancing our understanding of the pathophysiological process and provide a potential preventative target for migraine after seizures.In accordance with previous reports , we confEven more unexpected was that the activation and proliferation of microglia after epileptic seizures are region-specific, and these cells were mainly concentrated in the cortex, and the thalamic nucleus Fig. A, which Our previous studies have shown that BDNF is involved in comorbidities of epilepsy and migraine . This stThe present study provides evidence that modulating microglial activation can affect epileptic seizure-induced migraine-relevant phenotypes. These results deepen the understanding of the role of microglia in epilepsy, fill the gap in the research of microglia in the comorbidity of epilepsy and migraine, and provide a new approach for managing migraines after epilepsy. However, this study has limitations that must be acknowledged. First, microglia may play dual roles role in the pathogenesis of epilepsy. Wu et al. discovered that microglial depletion exacerbates the severity of acute and chronic seizures in Cx3cr1-KO mice . This fiIn summary, through a rat model of comorbid migraine and epilepsy, we explored the characteristics and mechanism of microglial activation after seizures and their effects on migraine. These results indicate that SE may facilitate migraine through BDNF release induced by microglial activation in the cortex/thalamus/sp5c mediated by the FKN/CX3CR1 axis. Future studies are needed to illustrate the feasibility of targeting the FKN/CX3CR1 axis in migraine patients, especially in female patients.Additional file1: Fig. S1. Schematic representation of the FKN injection site.


In the present study, we investigated the difference between monocular augmented reality (AR) and binocular AR in terms of perception and cognition by using a task that combines the flanker task with the oddball task. A right- or left-facing arrowhead was presented as a central stimulus at the central vision, and participants were instructed to press a key only when the direction in which the arrowhead faced was a target. In a small number of trials, arrowheads that were facing in the same or opposite direction (flanker stimuli) were presented beside the central stimulus binocularly or monocularly as an AR image. In the binocular condition, the flanker stimuli were presented to both eyes, and, in the monocular condition, only to the dominant eye. The results revealed that participants could respond faster in the binocular condition than in the monocular one; however, only when the flanker stimuli were in the opposite direction was the response faster in the monocular condition. Moreover, the results of event-related brain potentials (ERPs) showed that all stimuli were processed in both the monocular and the binocular conditions in the perceptual stage; however, the influence of the flanker stimuli was attenuated in the monocular condition in the cognitive stage. The influence of flanker stimuli might be more unstable in the monocular condition than in the binocular condition, but more precise examination should be conducted in a future study. In the field of cognitive psychology, there are a great number of studies on the perceptual and cognitive differences between the binocular and monocular presentation of stimuli. For binocular presentation, better task performances are reported in some tasks, such as shorter reaction time, higher hit rate, and lower threshold, and this phenomenon is known as “binocular summation” . AR is an emerging technology in which information is superimposed onto the real world directly Azuma, . AR is eTwo types of methods are used in AR systems: binocular presentation and monocular presentation  are presented in the central vision as central stimuli, and participants are asked to respond depending on the stimulus  and analyzed event-related brain potentials (ERPs) in a Go/No-go task. In this case, as a physiological indicator, Go P3 at the central to parietal regions  is evoked for targets, and No-go P3 at the frontal to central regions  is evoked for non-targets  elicit visual mismatch negativity . We hypothesized that participants could ignore information from the eye to which AR flanker stimuli were presented in the perceptual stage. If they could focus on only the required information in the monocular condition, it would mean that the flanker stimuli might not prevent the central stimulus from being seen; therefore, there might be no difference in reaction time between the flanker condition and the void condition, in which no flanker is presented.In addition, if the hypothesis is true, the flanker stimuli would not be processed in the monocular condition; therefore, vMMN would not be evoked in the flanker as well as the void condition. vMMN would be evoked in the flanker condition only in the binocular condition, in which the flanker stimuli would inevitably be processed. Furthermore, in the monocular condition, N2 would not be evoked in the incongruent flanker condition as well as the void condition because the flanker itself would not be processed, resulting in reaction competition not occurring. In comparison, in the binocular condition, N2 would be evoked in the incongruent condition. Last, in the monocular condition, No-go P3 and Go P3 would be similar in the flanker condition to the void condition, and, in the binocular condition, the flanker stimuli would influence P3.However, contrary to the assumption that the flanker stimuli would not be processed in the monocular condition, there is enough room to assume that the flanker stimuli would be processed not only in the binocular condition but also in the monocular condition because once the light of stimuli hits the retina, the stimuli are processed to some degree, though a specific percept might not be produced. In this case, it might be possible for ERPs to also be evoked in the monocular condition and that each ERP could be comprehended as below. If vMMN were evoked in the monocular condition as well as in the binocular condition, it would reveal that at least the flanker stimuli would be processed at the perceptual stage in both conditions. Moreover, if there were a difference between the monocular and binocular conditions for N2, it would reveal that the cognitive process is different between the two conditions at the stimuli evaluation stage. Last, if there were a difference between the monocular and binocular conditions for P3, it would reveal that the attentional cognitive process for selecting a reaction would be different between the two conditions.This experiment was approved by the Behavioral Research Ethics Committee of the Osaka University School of Human Sciences. We acquired written informed consent from all of participants before the experiment started.SD = 1.79). All participants had normal or corrected-to-normal vision . Nine participants had a right dominant eye, and nine had a left dominant eye. By using Ishihara color test II (24 plates), all participants were confirmed to have normal color vision.Six students at Osaka University and 16 people arranged by a worker dispatch company participated in the experiment. Four of the participants were excluded from the analysis because of mechanical malfunction, misunderstanding the instructions, and low quality of data for ERPs, that is, the number of trials available for analyzing of P300 amplitude was less than 20 , a semi-transparent mirror, a pen-tablet monitor , an LCD , AX220 model, resolution: 1,920 × 1,080), a computer , and a numeric keypad . Figure EEG data were recorded by using Polymate AP1132  and an electrode cap  using Ag/AgCl electrodes at 26 sites  according to the modified 10–20 System. In addition, electrodes were also placed on both earlobes (A1 and A2). The reference electrode was on the tip of the nose, and the ground electrode site was AFz. The data from all channels were recorded using the Mobile Acquisition Monitor Program . The electrode impedances were kept below 10 kΩ. A DC filter was used during recording. The sampling rate was 1,000 Hz.Right- and left-facing arrowheads (< >) were used as target, non-target, and flanker stimuli see Fig. . The sizTo emphasize the difference between the binocular and monocular presentation of AR, an AR Mondrian stimulus (an image that has various colors and numerous edges) covered all stimuli. The size of the AR Mondrian stimulus was 8.8° high × 13.2° wide.Figure After 120 trials were finished, “Ready?” was presented again. During this section, participants were free to take a break and press the 5 key to start the next trial. These 120 trials were treated as a block, and five blocks were conducted sequentially for one observation condition. After five blocks were finished, an experimenter changed the observation condition, and the next five blocks then started. The order of observation conditions was randomized between participants. Before the monocular condition started, participants were instructed to direct their attention to the eye to which the AR image was not presented.Between the fifth and sixth blocks , the participants answered a questionnaire orally  × flanker condition  × target condition  × repeat 5 blocks, equaling 1,200 trials. All factors were within-participant.As a subjective measure, the answers to the questionnaire were recorded Table .As a behavioral measure, the reaction time to the Go-target was recorded. In addition, the response to each target (whether the 5 key was pressed or not) was recorded. In a Go trial, pressing the button was the correct response, and in the No-go trial, the opposite was correct.As a physiological measure, the mean amplitudes of ERPs were analyzed from EEGs. To analyze the data, the EEGLAB toolbox . In general, d’ and the criterion based on the signal detection theory . Post hoc comparisons were made using Shaffer’s modified sequentially rejective multiple test procedure, which extends Bonferroni t tests in a stepwise fashion . The ANOVAs for the analysis of all ERPs were conducted by applying Greenhouse-Geisser corrections to the degrees of freedom . Moreover, the interaction of the observation and target conditions was significant. Post hoc comparisons indicated that the mean amplitude of the Go condition was larger in the negative direction than the No-go condition in the monocular condition (p < .05). The other main effects and interactions were not significant. In summary, the flanker stimuli elicited vMMN in both conditions, and the amplitude of vMMN was different between the Go and No-go conditions in the monocular condition.Figure The mean amplitudes of N2 for the Go condition at Fz, Cz, and Pz were assessed with a three-way repeated-measures ANOVA ) in each target condition to eliminate the influence of Go P3 and No-go P3.ps < .05). Moreover, the main effect of the electrodes was significant. Post hoc comparisons showed that Fz had the largest mean amplitude in the negative direction, followed in order by Cz and Pz (ps < .05). Furthermore, the main effect of the observation condition was significant, and the mean amplitude of N2 in the monocular condition was larger in the negative direction than the binocular condition (p < .05). In addition, the interaction of the flanker condition and electrodes was significant. Post hoc comparisons showed the same result for the main effect of the flanker condition and electrodes in the all electrode and flanker conditions (ps < .05). Finally, the interaction of the observation condition and electrodes was significant. Post hoc comparisons showed the same result for the main effect of the observation condition and electrodes in the all the electrode and observation conditions (ps < .05). In summary, N2 in the incongruent condition was elicited in both the binocular and the monocular conditions, and the amplitude of N2 at the frontal electrode was larger in the negative direction than at the central and parietal electrodes. In addition, the amplitude of N2 was different between the binocular and monocular conditions.Figure p < .05). Moreover, the main effect of the electrodes was significant. Post hoc comparisons showed that Pz had the largest mean amplitude, followed in order by Cz and Fz (ps < .05). Furthermore, the interaction of the observation condition and electrodes was significant. Post hoc comparisons showed the same result for the main effect of the observation condition and electrodes in all the electrode and observation conditions (ps < .05). In summary, the P3 amplitude elicited by the binocular condition was larger than the monocular condition, and the amplitude of P3 at the parietal electrode was larger than at the frontal and central electrodes.Figure ps < .05). Moreover, the main effect of the electrodes was significant. Post hoc comparisons showed that Fz had the largest mean amplitude, followed in order by Cz and Pz (ps < .05). Furthermore, the interaction of the flanker condition and electrodes was significant. Post hoc comparisons showed the same result for the main effect of the flanker condition at Fz and Cz and the electrodes in the congruent and incongruent conditions (ps < .05). In the void condition, the mean amplitude of N2 at Fz was larger than at Cz and Pz (ps < .05). Finally, the interaction of the observation condition and electrodes was significant. Post hoc comparisons showed the same result for the main effect of the electrodes in all observation conditions (ps < .05). For Pz, the mean amplitude of the monocular condition was larger than the binocular condition (ps < .05). In summary, the incongruent condition elicited N2 in both conditions, and the amplitude of N2 at the frontal electrode was larger than the central and parietal electrodes.Figure p < .05). Moreover, the main effect of the electrodes was significant. Post hoc comparisons showed that the mean amplitude of Pz and Cz were larger than Fz (ps < .05). Furthermore, the interaction of the flanker condition and electrodes was significant. Post hoc comparisons showed the same result for the main effect of the electrodes in the void and incongruent conditions (ps < .05). In the congruent condition, Pz had the largest mean amplitude, followed in order by Cz and Fz (ps < .05).Figure In addition, the interaction of the observation condition, flanker condition, and electrodes was significant. Table ps < .05). In the congruent condition, Pz had the largest mean amplitude, followed in order by Cz and Fz (ps < .05). In addition, the simple-simple main effect of the electrodes was significant, and the mean amplitudes of Pz and Cz were larger than Fz (ps < .05). For the factor of the monocular condition, the simple-simple main effect of the electrodes was significant. Post hoc comparisons showed that Pz had the largest mean amplitude, followed in order by Cz and Fz (ps < .05).For the factor of binocular condition, the simple interaction of the flanker condition and electrodes was significant. Post hoc comparisons showed that the mean amplitudes of Pz and Cz were larger than Fz in the void and incongruent conditions (ps < .05). Pz had the largest mean amplitude, followed in order by Cz and Fz in the binocular and monocular conditions (ps < .05). In addition, the simple-simple main effect of the observation condition was significant, and the mean amplitude of the binocular condition was larger than the monocular condition (p < .05). Additionally, the simple-simple main effect of the electrodes was significant. Post hoc comparisons showed that the mean amplitudes of Pz and Cz were larger than Fz (ps < .05). For the factor of congruent condition, the simple-simple main effect of the electrodes was significant. Post hoc comparisons showed that Pz had the largest mean amplitude, followed in order by Cz and Fz (ps < .05). For the factor of incongruent condition, the simple-simple main effect of the electrodes was significant. Post hoc comparisons showed that the mean amplitudes of Pz and Cz were larger than Fz (ps < .05).For the factor of the void condition, the simple interaction of the observation condition and electrodes was significant. Post hoc comparisons showed that the mean amplitude of the binocular condition was larger than the monocular condition for all electrodes (ps < .05).For the electrodes of Fz, Cz, and Pz, the simple-simple main effect of the observation condition was significant, and the mean amplitude of the binocular condition was larger than the monocular condition .For Q1, the subjective attentional control was measured, and the mean was 5.70  than in the monocular condition  (t (17) = 3.00, p = .008**).Q2 and Q4 were questions for measuring the subjective perception of the flanker stimuli in both the monocular and binocular conditions. Hence, a paired t test was conducted. The result revealed that the subjective annoyance of the flanker stimuli was not significantly different between the binocular  and monocular conditions  (t (17) = 0.15, p = .882).Q3 and Q5 were questions for measuring the subjective annoyance of the flanker stimuli in both the monocular and binocular conditions. Hence, a paired In the present study, the difference between monocular and binocular AR presentation in information processing was investigated by using a task that combined the flanker task with the oddball task.The results for the reaction times Figs.  revealedHowever, in the incongruent condition, the influence of the flanker stimuli was smaller in the monocular condition than in the binocular condition; at the least, for this point, the result partially supported the hypothesis. A histogram of the reaction times Fig.  revealedThis result could suggest that the flanker stimuli are processed less in the monocular condition, though not perfectly ignored. For example, in one trial the effect of the incongruent flanker stimuli was very strong, and in another trial, the effect becomes weak; in this way, the effect of flankers might be different among each trial. The subjective data also revealed that the perception of the flanker stimuli was weaker in the monocular condition than in the binocular condition, but the stimuli were not perfectly ignored. In addition, previous studies revealed that binocular rivalry is influenced by endogenous attention; however, observers cannot perfectly control their perception in binocular rivalry by their own will , were evoked for the target, and the No-go P3s caused by No-go stimuli in the central to parietal regions  were evoked for non-targets  and stimulus conflict stage , the physiological measure revealed that the flanker stimuli were processed both in the monocular and binocular conditions. However, the measures reflecting the reaction process, such as the P3s, reaction times, and hit rates, were different between the two conditions, and this implies that participants might have been able to distribute more attention to the task in the binocular condition than in the monocular condition. The mechanism that influences these reactions was not clarified in the present study, so more precise scrutiny is needed.


The objective of this study is to investigate the effects of different nanoparticles as reinforcement in a polymeric matrix on the mechanical and tribological properties of the composite. The different particles including nanoclay (NC), nano-graphene oxide (NGO), and carbon nanotubes (CNT) with various weight percentages were incorporated into the epoxy matrix. Young’s modulus, ultimate tensile strength, strain at the fracture point, and the fracture toughness of nanocomposite samples were investigated. Besides, the tribological performance of these fabricated nanocomposites was evaluated and discussed. The results show that a significant change in the mechanical properties of the nanocomposites compared to the epoxy matrix. Also, the results reveal that the combination of NC with NGO improves the mechanical properties of graphene nanocomposites. It is found that adding NC to the NG/epoxy composite, may increase the fracture toughness up to 2 times as well as improve the ultimate tensile strength and strain at the fracture point. However, there was no significant change in Young’s modulus. Due to the prominent combination of low shrinkage, dimensional stability, adhesion, dielectric properties, and processing versatility, epoxies are the most common thermosetting polymer for high-performance composites and adhesives –3. FurthResearchers have considered the effects of NGO on epoxy. Zhao et al.  developeTo reinforce epoxy some studies investigate the role of NC on epoxy. Chan et al.  preparedSeveral experimental researches are also directed to find the better compatibility between CNTs and polymer matrices to make a better composite. Jagtap and Ratna  FabricatOn the other hand, many researchers used different approaches of assembling two or three different nano fillers to improve some of the poor properties presented by single filler/epoxy. Ruiz-Hitzky et al.  develope2 through the hydrothermal method, and its microstructure, phase composition. Results revealed EP-CNTs/GO/MoS2 possessed the lowest friction coefficient and wear rate than EP and its other composite coatings reinforced by single filler or binary hybrids.Jen et al.  preparedShubham et al.  concludeAs mentioned above many studies provide samples in which the mechanical properties of materials are significantly improved by adding small amounts of nano-particles. In many cases, the addition of such nano-particles is performed on polymer-matrix composites, with reported improvements in mechanical, optical, thermal or electrical properties. However, some research has criticized these results. Cicero and his colleagues  introducIn the present study, the effects of the addition of different contents of NC, NGO, and CNT on the mechanical and tribological properties of the polymer matrix nanocomposites were investigated. Mechanical stirring with ultrasonication was utilized to achieve uniform dispersion of nanoparticles into the epoxy matrix. Young’s modulus, ultimate tensile strength, strain at the breakpoint, and fracture toughness of the nanocomposite specimens were extracted from stress-strain graphs. Furthermore, the fracture and wear mechanisms were determined by studying the surface morphology.3. Research grade NGO nanoplatelets powder  was provided from US Research Nanomaterials. NC montmorillonite  was purchased from Sigma–Aldrich Chemical Company and MWCNT-COOH functionalized  was used.The resin used in this study was KER 828 composed of bisphenol A and epichlorohydrin supplied by Kumho P&B Chemicals, Inc. A 10% wt triethylenetetramine hardener was used as a curing agent. The dynamic viscosity of KER 828 was ranged from 12 to 14 Pa.s at 25°C with density of 1160 kg/mThe weight percentages of epoxy resin, hardener, and nanostructured fillers in the fabricated nanocomposite samples were 89.50, 8.95, and 1.50% respectively. The combinations of nanostructured fillers applied in the fabrication of the samples are listed in The process of preparing epoxy composite and nanocomposite samples was performed in two steps: first, the dispersion of nanostructured fillers into epoxy resin, and second curing of the mixtures. To execute the first step, the intended amount of each type of nanostructured filler for fabricating the composite samples  with a crosshead speed of 2 mm/min. The tensile tests were performed for at least 3 samples of each material at room temperature.The friction and wear experiments were performed by using a pin-on-disc tribometer and conducted under dry contact conditions at room temperature according to ASTM G99. The tribometer was accurately calibrated for wear measurements. The 100 cr6 steel pin with a hardness of 58 HRC and spherical head with a diameter of 10 mm and length of 55 mm was employed as counterface. Prior to testing and measuring the weights of the samples, they were first cleaned and dried. For this purpose, acetone was used as a non-chlorinated, non-film-forming cleaning agent and solvent. Then, the pin was held by the pin holder and composite disc samples were screwed to the machine. Vertical loads with different values  were applied to the specimens at each stage. The tests were carried out at a constant sliding velocity of 0.1 m/s and sliding distance of 1000 m and for wear track radius of 15, 20, and 25 mm in dry condition and room temperature. Each experiment was repeated 3 times to ensure the repeatability of the results and obtain the average values. The initial and final weights of the samples were measured using a balance with a precision of 0.0001 g (0.1 mg) to determine the weight loss. The friction force was measured using the wear test machine and then, the friction coefficient was obtained by dividing the friction force by the applied normal load.One of the main goals of the present study was to investigate the changes in mechanical properties due to the addition of NC, NGO and CNT with different weight percentages  comparedGenerally, it can be said that NC/epoxy and CNT/epoxy samples present better results compared to other composites while NGO/epoxy and NGO/CNT/Epoxy ones show a remarkable reduction in their most mechanical properties.As shown in Thus, only adding NGO) to epoxy exhibits more brittle behavior and consequently decreases all mechanical properties compared to pure epoxy, while adding NGO along with NC to epoxy has a profound impact on improving composite mechanical properties. This can be due to a higher degree of intercalation/exfoliation and better dispersion of NC particles that can modify the undesirable effects of NGO aggregation. Additionally, adhesively bonded joint of NC prevented interfacial failure and effectively increased its tensile strength. Moreover, some studies indicated that the addition of NC to composite can retard crack propagation due to bifurcation around the NCs and consequently enhance its fracture toughness and strength .In return, it seems that the presence of CNT in epoxy structure enhances output results and there is no need to add additional fillers.The weight loss of the various nanocomposites was studied in this research. The results showed that at three different loads of 20, 60, and 100 N, most nanocomposites had significantly lower weight loss than that of pure epoxy . The nanThe weight loss of NGO/epoxy compared to pure epoxy is about 12.5, 35, and 46% for loads of 20, 60, and 100N, respectively. The wear rate of NC/epoxy for the aforementioned loads compared to pure epoxy is 12.5, 30, and ~40%, and these values for NC/NGO/epoxy are 25, 45, and 49%, compared to pure epoxy for the same conditions, respectively. This phenomenon might be due to the effect of the formation of the transfer film on the counterface during the wear process of the nanocomposites, which protects the soft polymeric material against the hard metallic material. Generally, the NC/epoxy nanocomposite samples show minimum weight loss, compared to other nanocomposites. The mechanical properties of the nanocomposites might be increased by the homogeneous distribution of the studied nanoparticles in the epoxy matrix. The increase in the interface local temperature will result in the adhesion of the materials to the steel pin and increasing the wear rate. The increase in weight loss by increasing load may also be attributed to the increase in the interface local temperature.rW) of pure epoxy and nanocomposite samples are calculated with different applied normal loads under dry conditions. It is observed that weight loss increases as applied normal loads increase. The specific wear rate of samples is calculated as [The weight loss and specific wear rate  with a constant velocity of 0.1m/s and a sliding distance of 1000m are shown in At the load of 20N, CNT/epoxy, NC/epoxy, NGO/epoxy, and NC/CNT/epoxy samples had the same wear rate. Among nanocomposite samples, the nanocomposite containing 1.5 wt% CNT at the load of 60N, and the sample with 1.5 wt% NC at the load of 100N had the lowest wear rate, compared to other samples. Among all nanocomposites, the highest wear rates at the studied loads were recorded by NGO/CNT/epoxy sample.μ is the friction coefficient, Fp and FN are the rate of angular friction force and applied normal load, respectively. dp and dN are also the distance between the center to the pin and distance the center to the normal force, respectively. As shown, the samples consisting of a combination of two or three nanostructured fillers have a higher coefficient of friction while epoxy with just single-type filler presents less coefficient of friction value as sliding time exceeded 30 min compared to pure epoxy. As the experiment time increases, the coefficient of friction may increase due to the agglomeration of the nanostructured fillers and their accumulation in the epoxy matrix.Fracture and wear surfaces of the samples are examined to determine the failure and wear mechanisms using images obtained from FESEM using sputter-coating of the samples with a thin layer of gold.FESEM images of the fractured surfaces of pure epoxy and other nanocomposites at three different magnifications for each sample are shown in For NGO/epoxy, the fracture surface is rough . The imaThe mechanism of crack formation in a sample containing NC is completely different from that of pure epoxy due to the cohesion of the particles , TherefoFor CNT/epoxy sample, the mode of the CNTs protrusion after loading indicates a strong bond between the epoxy and the CNTs. As shown in Regarding the NGO/CNT/epoxy sample, unlike CNT/epoxy sample, no uniform distribution of CNTs can be observed . It seemIn the NC/CNT/epoxy sample, although CNTs are much more uniformly distributed in the epoxy sample than the NGO/CNT/epoxy without agglomeration around the NC , the exiOn the other hand, the addition of NC to NGO increases the strength of the NGO/NC/epoxy sample and also may reduce the slip of graphene layers in the epoxy matrix . NC incrThe proper dispersion of the nanostructured fillers is seen in the NGO/NC/CNT/epoxy sample images in Debris particles produced during the abrasion process for the NGO/epoxy sample are smaller than other samples . The maiThe abrasion groove in the NC/epoxy sample is difficult to be detected because the wear depth in this sample is very small .By adding CNTs to the resin, wear on the abrasive surfaces is significantly reduced and the abrasion resistance of pure epoxy is significantly increased . This caThe composition of the NGO and CNTs increase the coefficient of friction so that the coefficient of friction of NGO/CNT/epoxy is even higher than that of pure epoxy. Increasing the coefficient of friction causes more weight loss of the sample due to wear peeling. Particles separated by wear are a factor in increasing the wear rate ,45. Due The NC/CNT/epoxy sample has a higher coefficient of friction than the NC/epoxy and CNT/epoxy samples . The bouHoles and micro-cracks are more visible in the NGO/NC/epoxy sample . ParalleThe worn surface of the NGO/NC/CNT/epoxy sample has signs of adhesion and abrasion . The morDue to the importance of studying the abrasion damages on various engineering structures, in this research, the effect of nanostructured fillers with different weight percents on the mechanical and wear resistance properties of polymer matrix nanocomposites was investigated. For this purpose, NGO, CNTs, and NC with different weight percentages were used. Young’s modulus, ultimate tensile strength, strain at the breakpoint, and fracture toughness, coefficient of friction, weight loss, and specific wear rate were studied. It was found that NGO/epoxy had the lowest ultimate tensile strength, strain at the breakpoint, and toughness while NGO/epoxy nanocomposite showed more brittle fracture behavior than even the pure epoxy. The addition of CNTs to the epoxy resulted in the highest ultimate strength and the lowest tensile strength. It was observed that the highest amount of toughness was related to the NGO/NC/epoxy sample and the lowest amount of toughness was recorded for NGO/epoxy. This means that the synergy of NC and NGO had a significant effect on increasing toughness.NC/epoxy nanocomposite demonstrated minimal weight loss compared to other nanocomposite samples. Among all nanocomposites, the highest wear rate was recorded for the NGO/CNT/epoxy sample. The NGO/CNT/epoxy sample showed the worst performance in terms of abrasion due to the agglomeration of the nanostructured fillers.


P38α, JNK1, ERK1, ERK2, and AdipoRs as well as pharmacological MAPK inhibition were performed. We found that compressive forces increase the expression of AdipoRs. Adiponectin and compression up-regulate P38α,JNK1, ERK1, and ERK2 as well as β-catenin gene expression. Western blots showed that co-stimuli activate the MAPK and β-catenin signaling pathways. MAPK inhibition alters the compression-induced β-catenin activation and the siRNAs targeting AdipoRs, P38α, and JNK1, showing the interaction of single MAPK molecules and β-catenin signaling in response to compression or adiponectin. Silencing by a dominantly negative version of P38α and JNK1 attenuates adiponectin-induced TCF/LEF reporter activation. Together, we found that light compressive forces activate β-catenin and MAPK signaling pathways. Adiponectin regulates β-catenin signaling principally by inactivating the GSK-3β kinase activity. β-Catenin expression was partially inhibited by MAPK blockade, indicating that MAPK plays a crucial role regulating β-catenin during cementogenesis. Moreover, adiponectin modulates GSK-3β and β-catenin mostly through AdipoR1. P38α is a key connector between β-catenin, TCF/LEF transcription, and MAPK signaling pathway.We aimed to investigate the molecular effect that adiponectin exerts on cementoblasts especially in the presence of compressive forces. OCCM-30 cells  were used. Real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and western blots were employed to verify if the mRNA and protein levels of adiponectin receptors (AdipoRs), mitogen-activated protein kinase (MAPK), and β-catenin signaling were influenced by compressive forces or adiponectin. Moreover, siRNAs targeting  OIIRR iin (OPG) .It has been highlighted that β-catenin signaling regulates cementum homeostasis, and a down-regulation of WNT causes root resorptions . MechaniDuring orthodontic force application, compressive forces also induce human mandibular-derived osteoblast differentiation via WNT/β-catenin signaling . WNT sigAdiponectin, a secreted protein produced mainly by adipocytes , binds tIt is now of interest how the interaction between the MAPK pathway and β-catenin signaling occurs. Additionally, the mechanism of adiponectin regulation in cementoblasts remains unclear. The aim of the current study is to assess whether β-catenin signaling modulation is regulated via the activation of MAPK signaling. Furthermore, it is aimed to identify the molecular mechanism by which adiponectin-induced MAPK interacts with compression-induced β-catenin.2 at 37°C. Cells were stimulated using different concentrations of mouse adiponectin/Acrp30/ADIPOQ protein (His Tag) from Sino Biological Inc. (Cat. No.: 113 50636-M08H) with or without compressive force application as described by 2, respectively. The cells were seeded into six-well plates at a density of 3 × 104 cells/well until confluence and covered with the glass cylinders afterward.The OCCM-30 cementoblast cell line was kindly provided by Prof. M. Somerman  and maintained in α-MEM  containing 10% fetal bovine serum (FBS)  and 1% penicillin/streptomycin  and incubated in a humidified atmosphere of 5% COTo induce cementogenesis, the cell culture medium was supplemented with 10 mM β-glycerophosphate  and 50 μg/ml ascorbic acid .Inhibitors for P38 (SB203580) , ERK1/2 (FR180204) , and JNK (SP600125)  were used.@ Transfection Reagent  in 100 μl Opti-MEM medium  at room temperature for 10 min, and then, each transfection mixture was added into 2.3 ml growth medium in the six-well plate in which OCCM-30 cells were cultured at 60–70% confluence. After siRNA transfection for 24 h, the cells were kept in starvation medium [ containing 0.5% FBS  and 1% penicillin/streptomycin ] for 2 h, and afterward, 100 ng/ml adiponectin  was added.The siRNAs targeting mouse AdipoR1 (SI00890295), AdipoR2 (SI00890323), MAPK1 (SI02672117), MAPK3 (SI01300579), MAPK8 (SI1300691), MAPK14 (SI01300523), negative control (1027280), and cell death control (SI04939025) were purchased from QIAGEN Inc. siRNAs were incubated with 12 μl HiPerFect2). Total RNA was extracted using NucleoSpin® RNA Kit . RNA concentrations were measured using a spectrophotometer . Using commercial innuSCRIPT Reverse Transcriptase kit , 1.0 μg RNA was transcribed, and 1.0 μl of the resulting cDNA was used at the final reaction volume of 20 μl/well in a CFX96TM Real-Time System Cycler (Bio-Rad). Real-time reverse transcriptase–polymerase chain reaction (RT-PCR) amplification was carried out using the SsoAdvancedTM Universal SYBR@ Green Supermix . The primers for mouse AdipoR1&2 (qMmuCID0023619 and qMmuCID0010157) and MAPK3 (qMmuCED0025043) were purchased from Bio-Rad. The primers for MAPK1, MAPK8, MAPK14, GSK-3β, and β-catenin were purchased from Eurofins Genomics Inc. Primer sets used in the analysis are listed in Cells were grown to confluency and kept overnight in starvation medium [α-MEM  containing 0.5% FBS  and 1% penicillin/streptomycin ]. Afterward, cells were either stimulated with adiponectin (100 ng/ml)  or cultivated under compression  on a DR/2000 Spectrophotometer . Then, 20 μg lysate/lane was diluted in a sample loading buffer  and separated by 10–12% SDS-PAGE gels and then the resolved proteins were transferred electrophoretically to nitrocellulose membranes . Protein loading was verified by Ponceau S staining . Membranes were blocked with 5% non-fat milk  for 1 h at room temperature and further incubated with the primary antibodies for ERK1/2 ; phospho-ERK1/2  ; p54/p56 JNK ; phosphor-SAPK/JNK (Thr183/Tyr185) ; P38 MAPK ; phospho-P38 MAPK alpha ; GSK-3β ; phospho-GSK-3β (Ser9) ; β-catenin ; phospho-β-catenin (Ser33/37/Thr41) ; and β-actin  followed by peroxidase-conjugated secondary antibodies including polyclonal goat anti-rabbit ; rabbit anti-goat , and polyclonal goat anti-mouse  in 2.5% non-fat milk  for 1 h at room temperature. The band signal detection was then performed with X-ray Amersham Hyperfilm  utilizing Amersham ECL Western Blotting Detection Reagents  and detected with an OPTIMAX X-Ray Film Processor  in a dark room.The OCCM-30 cells were lysed in RIPA buffer  supplied with 3% protease inhibitor . The insoluble material was removed by centrifugation at 14,000 rpm/min for 15 min. Protein concentrations were measured using PierceTM Chambered Cell Culture Slides  until 50% confluence and afterward were fixed with 4% paraformaldehyde  dissolved in 1X phosphate-buffered saline (PBS) , adjusted to pH 7.4, for 10 min at room temperature, and permeabilized with 0.5% TritonTM X-100 Surfact-AmpsTM Detergent Solution  for 20 min. Then, cells were kept in a blocking buffer containing 10% goat serum, 0.3 M glycine, 1% BSA , and 0.1% Tween-20  for 30 min at room temperature and incubated with primary antibodies for GSK-3β  and β-catenin  at 4°C overnight. After washing three times with 1X PBS –0.1% TRITON X-100  for 5 min, the cells were incubated with DyLight 488 goat anti-rabbit polyclonal secondary antibody , which conjugated to fluorescein isothiocyanate for 1 h. After washing with 1X PBS-Tween-20, DNA was stained using a fluorescent mounting medium with 4′,6-diamidino-2-phenylindole (DAPI)  for 15 min. Staining was analyzed using a high-resolution fluorescence microscope  and photographed.Cementoblasts were cultured on sterile Falcon4 cells per well in 100 μl growth medium [α-MEM  containing 10% FBS  and 1% penicillin/streptomycin ] overnight. The reverse transfection of OCCM-30 cells with silencing RNA was performed with specific siRNAs to knock-down AdipoR1, AdipoR2, MAPK1, MAPK3, MAPK8, and MAPK14. Briefly, cells were incubated with 24.25 μl Opti-MEM antibiotic-free medium  and 0.75 μl HiPerFect@ Transfection Reagent  supplemented with 12.5 ng specific siRNA for incubation for 24 h. The cells were simultaneously transfected with the TCF/LEF Reporter Kit . For control transfection, cells were transfected with DNA mixture by 1 μl of TCF/LEF luciferase reporter vector  plus negative control siRNA ; 1 μl of non-inducible luciferase vector  plus negative control siRNA ; and 1 μl of non-inducible luciferase vector  plus specific siRNA in 15 μl Opti-MEM antibiotic-free medium . For experimental transfection, the DNA mixture of 1 μl TCF/LEF luciferase reporter vector  plus specific siRNA was incubated in 15 μl Opti-MEM antibiotic-free medium . All the DNA mixtures were then mixed with 0.35 μl LipofectamineTM 2000 Transfection Reagent  in 15 μl Opti-MEM antibiotic-free medium  at room temperature for 25 min. After 24 h of transfection, the medium was changed to fresh growth medium. Following incubation for another 23 h, adiponectin (100 ng/ml) was added to stimulate cells for 1 h. We set up each treatment in triplicate.OCCM-30 cells were seed into a 96-well plate at a density of 3 × 10After 48 h of transfection, the firefly luciferase activities were performed using the BPS Dual Luciferase (Firefly/Renilla) Assay system  using a Mithras LB 940 Luminometer  and analyzed by MikroWin 2000 (Mikrotek Laborsysteme GmbH), which normalized to the Renilla luciferase activities.t-test followed by Tukey’s post-hoc test for unpaired samples to determine the statistically significant differences for multiple comparisons. Differences were considered statistically significant at a p-value of < 0.05 and p-values are shown with respect to controls, unless otherwise indicated. Data distribution was analyzed using the Kolmogorov–Smirnov and the Shapiro–Wilk test and visually using QQ plots. All experiments were successfully performed in triplicate.Statistical analyses were plotted using the GraphPad Prism 6.0 software . Quantitative values are expressed as means ± standard deviation (SD) and analyzed using independent one-way in vitro on cementoblasts, cells were additionally stimulated with 100 ng/ml adiponectin. Next, we performed a dynamic analysis of receptor expression, cultivating the cells in the presence of different compressive forces: OCCM-30 cells underwent compressive forces of 1.2, 2.4, and 3.6 gf/cm2. The RT-PCR analysis revealed that the addition of adiponectin did not significantly influence AdipoRs mRNA expression (p > 0.05), while the application of compressive forces of 2.4 or 3.6 gf/cm2 significantly increased its expression  at mRNA and protein levels on OCCM-30 cells. To evaluate the effect that adiponectin as well as compressive forces exert P38, JNK, and ERK. RT-PCR results show that the expression of P38α and JNK1 was strongly up-regulated by adiponectin (∗p < 0.05) or compression (∗p < 0.05), whereas cells showed a slight up-regulation (∗p < 0.05) of ERK1 and ERK2 in the presence of adiponectin or compressive forces (GSK-3β mRNA expression (∗p < 0.05), while it positively up-regulates β-catenin expression (∗∗p < 0.01) after 1 h of stimulation  and increase β-catenin expression  resulted in an increased and sustained phosphorylation stage of P38, ERK1/2, and JNK, at different degrees. Western blots (WBs) revealed that the phosphorylation of P38 and ERK1/2 was maintained from 0.5 to 24 h, reaching a peaking during 2–6 h of stimulation. The phosphorylation of JNK occurred after 4 h, reaching a peak at time point 6 h (The co-cultivation of cementoblasts with adiponectin (100 ng/ml) under compressive forces  over a time period from 30 min to 3 h . Adipone2) alone can have on the cellular accumulation of β-catenin. The time course experiments showed that the activation of β-catenin protein was transiently increased during 10 min to 2 h during compression exposure  upregulates the expression of dephosphorylated β-catenin 10 min after exposure. This effect was sustained over a period of 3 h  in combination with compressive forces , such effect was reversed . In the The expression of total β-catenin was down-regulated in the presence of P38 or ERK1/2 inhibitors, but rescued by adiponectin (100 ng/ml) addition, a fact that suggests that P38, ERK1/2, and JNK modulate GSK-3β and subsequent cellular events such as the cytoplasmic accumulation of β-catenin .2 for 60 min. Afterward, we examined by WB the expression of GSK-3β and β-catenin. The results show that the suppression of the MAPK pathway has a significant blockade effect on β-catenin expression. This effect was restored when cells were exposed to compression for 1-h stimulation  for another 1 h.Furthermore, to determine whether the activation of P38, ERK1/2, and JNK is involved in the stimulation of β-catenin by adiponectin or compression, the cells were preincubated with the pharmacological MAPK inhibitors SB203580 (P38), FR180204 (ERK1/2), and SP600125 (JNK) for 1 h and then cultivated under compressive forces of 2.4 gf/cmAs a result, we observed that ERK1/2 inhibition as well as JNK inhibition in combination with compression inhibit GSK-3β expression, whereas the total GSK-3β expression was promoted in the group treated with P38 inhibitor in the presence of compression and adiponectin . The totAdipoR1, AdipoR2, P38α, ERK1, ERK2, and JNK1 on adiponectin-stimulated TCF/LEF-sensitive transcription in cementoblasts , ERK2 (MAPK1), JNK1 (MAPK8), and P38α (MAPK14) using siRNA transfection. The efficacy of the gene knock-down by siRNA transfections was analyzed by RT-PCR. After a transfection period of 48 h, we could observe effective down-regulation of the target genes  and β-catenin mRNA levels (∗p < 0.05), whereas mock treatment did not alter either GSK-3β or β-catenin mRNA levels significantly. Conversely, the group treated with siRNA against AdipoR1 did not show significant differences in the expression of GSK-3β or β-catenin , and we observed that the group treated with siRNA against -catenin .ERK1, ERK2, JNK1, and P38α mRNA or mock treatment in the presence or absence of adiponectin, RT-PCR was performed to analyze GSK-3β or β-Catenin mRNA expression. Adiponectin treatment in JNK1 and P38α siRNA groups has a down-regulation effect on the GSK-3β mRNA expression. The silencing of ERK2 but not that of ERK1 causes the GSK-3β mRNA up-regulation after adiponectin addition (∗∗p < 0.01). The single knock-down of JNK1 or P38α results in the reduction of β-catenin mRNA expression. This downregulation was rescued after adiponectin addition  and osteoprotegerin (OPG) mRNA expression at varying degrees .After cementogenesis induction, knock-down of In the present study, we demonstrated that adiponectin as well as compressive forces activate the MAPK and β-catenin pathways on cementoblasts. Through a cross-link mechanism, compression as well as adiponectin induce β-catenin accumulation on OCCM-30 cells by MAPK pathway commitment principally via AdipoR1. This effect involves multiple mediators including JNK1 and P38α that are key players in this process.We observed that adiponectin can interact with cementoblasts and influence their biological response to compressive forces promoting the expression of p-P38, p-ERK1/2, and p-JNK. Interestingly, we found a longer up-regulation period of p-P38 and p-ERK1/2 by co-stimulation of adiponectin combined with compression, while the expression levels of p-JNK were altered after co-stimulation and reached an expression peak later. This indicates that adiponectin additionally triggers the MAP kinase pathway. In accordance with our previous data (2020), adiponectin strongly induced the expression of phosphorylated P38, ERK1/2, and JNK on cementoblasts .The cooperative interaction between the β-catenin and MAPK signaling pathways has been reported in several studies . On MC3TERK1 and ERK2 as well as P38α and JNK1 showed that adiponectin-induced β-catenin activation on OCCM-30 cementoblasts was especially sensitive to P38α and JNK1 knock-down. JNK1 and P38α silencing negatively regulates β-catenin, whereas ERK1 has a significant positive effect on GSK-3β expression. Furthermore, β-catenin expression was up-regulated after adiponectin addition. In these contexts, our results reveal the role of both molecules triggering adiponectin–MAPK and β-catenin signaling interactions. Our data is consistent with previous studies  and FR180204 (ERK1/2) antagonists. Furthermore, the single gene silencing of  studies . ThorntoIn the current study, we verified that compression forces activate β-catenin signaling in OCCM-30 cells. This finding is in accordance with the results of An important observation that warrants discussion is that adiponectin addition combined with compression to OCCM-30 cells did not significantly affect β-catenin expression, even under the inhibition of MAPKs. This fact suggests that adiponectin addition exerts a multistep process in β-catenin modulation that needs to be further investigated. However, all present results imply that MAPK signaling participates in the process of the β-catenin signaling on cementoblasts.in vivo and in vitro that adiponectin could facilitate bone mesenchymal stem cell (BMSC) osteogenic differentiation and osteogenesis by the WNT/β-catenin pathway. ALP, bone sialoprotein (BSP), and osteocalcin (OCN) but promotes cell proliferation.AdipoR2 in OCCM-30 cells induces β-catenin up-regulation after adiponectin addition. Furthermore, our findings showing that the up-regulation of GSK-3β mRNA expression occurred after adiponectin addition in AdipoR2-silenced group strongly suggest that the modulation effect that adiponectin exerts on β-catenin expression is mostly mediated by AdipoR1. These data raise the possibility that adiponectin indirectly up-regulated β-catenin through the adiponectin/adipoR1 pathway.In our experiment, knock-down of OCN and OPG mRNA levels, which were identified as cementogenesis biomarkers (P38α knock-down in the presence of adiponectin. Hence, there is a possibility that OCN and OPG can be altered by the interplays between MAPK and β-catenin signaling in the cellular responses of cementoblasts under adiponectin and compression forces.omarkers , were deOPG and OCN that form extracellular matrices in OCCM-30 cells (Clinical studies have described that the level of adiponectin is reduced in the serum of obese individuals . In this30 cells . HoweverIn conclusion, we demonstrated that adiponectin as well as compressive forces stimulate both the MAPK and β-catenin signaling pathways on cementoblasts. The inhibition of P38, JNK, and ERK1/2 differently regulates β-catenin expression as well as TCF/LEF transcription. Furthermore, adiponectin regulates the compression-induced β-catenin signaling via cross-interacting with the MAPK signaling pathway.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.JY acquired and analyzed the data. JY, GR-H, and JB interpreted the data and wrote the manuscript. JY, GR-H, and SR conceived, designed, and supervised the study. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The protein arginine methyl transferase PRMT5 is an enzyme expressed in oligodendrocyte lineage cells and responsible for the symmetric methylation of arginine residues on histone tails. Previous work from our laboratory identified PRMT5 as critical for myelination, due to its transcriptional regulation of genes involved in survival and early stages of differentiation. However, besides its nuclear localization, PRMT5 is found at high levels in the cytoplasm of several cell types, including oligodendrocyte progenitor cells (OPCs) and yet, its interacting partners in this lineage, remain elusive. By using mass spectrometry on protein eluates from extracts generated from primary oligodendrocyte lineage cells and immunoprecipitated with PRMT5 antibodies, we identified 1196 proteins as PRMT5 interacting partners. These proteins were related to molecular functions such as RNA binding, ribosomal structure, cadherin and actin binding, nucleotide and protein binding, and GTP and GTPase activity. We then investigated PRMT5 substrates using iTRAQ-based proteomics on cytosolic and nuclear protein extracts from CRISPR-PRMT5 knockdown immortalized oligodendrocyte progenitors compared to CRISPR-EGFP controls. This analysis identified a similar number of peptides in the two subcellular fractions and a total number of 57 proteins with statistically decreased symmetric methylation of arginine residues in the CRISPR-PRMT5 knockdown compared to control. Several PRMT5 substrates were in common with cancer cell lines and related to RNA processing, splicing and transcription. In addition, we detected ten oligodendrocyte lineage specific substrates, corresponding to proteins with high expression levels in neural tissue. They included: PRC2C, a proline-rich protein involved in methyl-RNA binding, HNRPD an RNA binding protein involved in regulation of RNA stability, nuclear proteins involved in transcription and other proteins related to migration and actin cytoskeleton. Together, these results highlight a cell-specific role of PRMT5 in OPC in regulating several other cellular processes, besides RNA splicing and metabolism. The protein arginine methyltransferases (PRMTs) family of enzymes catalyzes the transfer of methyl groups from S-adenosyl methionine (SAM) to guanidine nitrogen of peptidyl arginine residues in mammals  and theiPrmt5 failed to differentiate in vitro (Id2 and Id4)   and moreand Id4) , a findiand Id4) . Howeverand Id4)  and stemand Id4) . In thesand Id4) , RNA spland Id4) , ribosomand Id4)  and RNA  hnRNPA1 . Cytopla hnRNPA1 .Therefore, many of the previous studies focused on PRMT5 as critical regulator of RNA biology, while our previous study addressed the nuclear function of PRMT5, despite its predominant cytosolic localization in OPCs. In this study we use mass spectrometry-based proteomic approaches and iTRAQ labeling of nuclear and cytosolic protein extracts, to broaden our understanding of the role of PRMT5 in OPC by identifying its protein interactors and substrates in oligodendrocyte lineage cells.Primary mouse OPCs were generated from the cortices of C57BL/6 mice, purchased from Jackson Laboratories and euthanized at postnatal day 7 according to IACUC approved protocols at Icahn School of Medicine. Progenitors were immunopanned with a rat anti-mouse CD140a antibody , as previously described J. . The isoImmortalized oligodendrocyte progenitors were received as a gift from Jackie Trotter  and were1 was used in designing the sgRNA targets. The two sgRNA target sequences of PRMT5-CRISPR (GAATTGC GTCCCCGAAATAG & CCCGCGTTTCAAGAGGGAGT) used in the study were directed to exon 1 and 2, respectively, of the Prmt5 gene. The other two sgRNAs targeted EGFP gene (GGG CGAGGAGCTGTTCACCG & GAGCTGGACGGCGACGTA AA) and these were used as controls. Both the Prmt5 and EGFP sgRNAs were cloned into the same vector backbone. Cloning was performed according to the Addgene guidelines and as previously described (TM concentrator kit (Clontech) was used in concentrating viruses.The lentiCRISPR-v2 vector was received as a gift from Feng Zhang (Addgene plasmid #52961). Feng Zhang’s laboratory online programescribed . The lenImmortalized oligodendrocyte progenitors (0.5 million cells) were split into each 10-cm dish 24 h prior to viral infection. Concentrated virus was added into the tissue culture medium of the cells to be infected. The culture medium was supplemented with 4 μg/ml of polybrene. Virus-containing media were replaced by fresh media 8 h post infection. To select for transfected cells, 1 μg/ml of puromycin was added to the medium 2 days post transfection. For experimental analysis, the infected cells were harvested 6 days after infection.Western blotting was done by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) followed by a wet transfer of the proteins onto a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked for 1 h in 10% milk/0.1% Tween/TBS. Primary antibodies were incubated overnight at 4°C in 5% milk/0.1% Tween/TBS. Primary antibodies used include: rabbit-PRMT5 , mouse-GAPDH , total H3 rabbit-, rabbit anti-symmetric Di-Methyl Arginine Motif  and rabbit anti-asymmetric Di-Methyl Arginine Motif . Membranes were washed with 0.1% Tween/TBS and incubated at room temperature for 1 h with horseradish peroxidase conjugated secondary antibodies  5% milk/0.1% Tween/TBS. ECL Prime Wester Blotting Detection Reagent kit  were then used to develop the membrane. Ponceau staining was performed with Ponceau S solution (Sigma) according to the manufacturer’s instruction. Image J was used to quantify the protein bands.2, 10 mM KCl) supplemented with 0.5 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitor cocktail. After incubation of the re-suspended cells (by rotation at 4°C for 15 min), the membranes of the cells were disrupted by the addition of 0.5% NP40 and vortexed for 10 sec. Cell lysates were centrifuged at 1,500 × g for 10 min at 4°C to separate cytoplasmic components (supernatant) from nuclei-enriched fractions (pellets). Cytoplasmic extraction buffer (10×)  was added to the supernatant and sonicated for 5 min (30 s ON/OFF) at high power in the Bioruptor (Diagenode). The soluble fraction was collected as cytoplasmic extract after centrifugation at 16,000 × g for 10 min at 4°C. The nuclei-enriched pellet initially obtained was then washed twice with hypotonic buffer + 0.5% NP40. The washed nuclei pellets were re-suspended in mild salt buffer  supplemented with 0.5 mM PMSF and protease inhibitor cocktail. The re-suspended pellets were rotated in 4°C for 20 min. The solution was centrifuged at 10,000 × g for 10 min at 4°C. The soluble fraction was collected as soluble nuclear protein extract.To sperate cellular extracts into cytosolic and nuclear fractions, cell pellets were treated with hypotonic buffer  into 1–2 mg/ml with lysis buffer. For immunoprecipitation, 5 mg of total proteins for each IP reaction were used. The lysates were pre-cleared with 10 μg of normal IgG for 2 h at 4°C, followed by addition of 50 μL of protein-A. Each sample was divided into two aliquots. One aliquot was immunoprecipitated with 10 μg of PRMT5 antibody  while 10 μg of normal IgG were added to the other aliquot and served as control. The samples were left in rotation at 4°C overnight. Next, 50 μl of protein-A beads were added into each sample and left in rotation for 2 h at 4°C. Following centrifugation at 3,000 × g for 30 s at 4°C, the immunoprecipitated complexes were washed with 500 μl of lysis buffer supplemented with the inhibitors for a total of five times. Elution was achieved by boiling the samples at 95°C for 10 min in 50 μl of 5× WB loading buffer supplemented with beta-mercaptoethanol. Upon rapid cooling of the samples on ice followed by centrifugation at 15,000 × g for 2 min, the supernatant was used for in gel tryptic digestion and LC-MS/MS.Primary oligodendrocyte progenitors kept either in the presence or absence of mitogens supplemented with T3 for 48 h (four preparations) were lysed in NP-40 lysis buffer  supplemented with protease inhibitor cocktail, PMSF, phosphatase inhibitor and TSA. The re-suspended cells were then incubated and rotated at 4°C for 2 h followed by light sonication at low power for 5 min (30 s ON/OFF). After sonication, the samples were centrifuged at 15,000 × The immunoprecipitated samples were run ∼1 cm into SDS-PAGE (Invitrogen NuPAGE Bis-Tris 1.5 mm 10% gel) and stained with Coomassie R250. Gel plugs were subjected to in-gel reduction, alkylation, tryptic digestion, and peptide extraction as described in Proteome Discoverer was used to generate mgf files and data were searched against the Ensembl mouse database  using an in house version of X!Tandem  . PrecursThe protein samples included cytosolic and nuclear fractions, which were independently processed and 0.7 mg/sample for cytosolic fractions and 0.9 mg/sample for nuclear fractions were digested with trypsin, labeled with the iTRAQ reagents following manufacturer’s instruction (ABSciex). iTRAQ samples from each genotype were pooled together and a small fraction (5%) was initially analyzed by quantitative nanoLC-MS/MS, thereby verifying that similar protein amounts were present in both samples. Total reporter ion intensities for each iTRAQ label were determined to be in a similar range in both samples, further validating the similar protein amounts and used as normalization factors in analysis of enriched methylated peptides. This served as control for minor variations in amounts of protein analyzed along with digestion and labeling efficiency. The rest of the samples from each fraction from PRMT5 silenced cells and controls, were subjected to an affinity enrichment using an immobilized antibody recognizing symmetric dimethyl arginine residues  following manufacturer’s standard protocol. At the elution stage, the beads were eluted with 0.15% TFA (trifluroracetic acid) and desalted/concentrated using STAGE trips . Each ofMSV000088396. They can be accessed with the following URL: https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp.Proteomics MS data (interactome and iTRAQ) have been deposited at Mass Spectrometry Interactive Virtual Environment (MassIVE) with accession number Mep50/Wdr77), a molecule that forms a complex with PRMT5 and correctly positions substrates for increased binding affinity (Epb41l3) (Snrpd1) (Traf4) (Znf326)  , the sma(Snrpd1) , the TNF (Traf4) , and the(Znf326) . In addi(Znf326) . Gene on(Znf326) . Consist(Znf326) , the lar(Znf326) . Togethep-value < 0.05) (n = 216) or nuclear fraction (n = 230) of the PRMT5 knockdown cells compared to the EGFP controls. These peptides corresponded to 60 putative protein substrates with significantly different arginine methylation in knockdown cells compared to controls. We then subtracted the proteins whose transcript levels were downregulated by PRMT5 knockdown [as we published in p-values are shown in the volcano plot (Cnbp), the temperature responsive mRNA binding protein RBM3 (encoded by Rbm3), the mRNA trafficking protein FMR1 (encoded by Fmr1), the chromatin remodeling complex BAZ1A (Encoded by Baz1a) and the repressor of translation initiation GGYF2 (encoded by Gigyf2). Substrates identified in the nuclear fraction included: the multi-functional ATP binding helicase DHX9 (encoded by Dhx9) expressed at high levels in the oligodendrocyte lineage and involved in the positive regulation of nuclear export of constitutive transport element (CTE)-containing unspliced mRNA (H2az1), and the transcription factor ZN658 (encoded by Zfp658). Substrates identified in both cytosolic and nuclear fractions included: proteins regulating pre-mRNA splicing , brain-specific RNA splicing , regulation or packaging of pre-mRNA into hnRNP particles  and transport from the nucleus to the cytoplasm , RNA decay and mRNA turnover  as well as the methylated RNA binding proline-rich protein PRC2C (encoded by Prrc2c)  of cytosolic or nuclear extracts from immortalized oligodendrocyte progenitors either silenced with lentiviral-mediated PRMT5 CRISPR-Cas9 (PRMT5-CRISPR) or transduced with EGFP-CRISPR, as control. The efficiency of the knockdown was validated using western blot analysis . Decreas < 0.05) . Of thos < 0.05) . Therefoano plot . The ideano plot . These dano plot , with veced mRNA , the his Prrc2c) . Next, w Prrc2c) . RegardlFunctional interactions among the 57 PRMT5 substrates were analyzed using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and validated the importance of PRMT5 substrates in regulating three main processes: overwhelmingly mRNA splicing and related pathways, but also DNA binding and other cellular processes involved in migration and other functions . We thenPrmt5 impacted survival of newly formed oligodendrocytes and impaired differentiation (Znf326)  . At odds(Znf326)  or by di(Znf326) , we did knockout  and furtWe then searched for PRMT5 substrates using iTRAQ analysis of cytosolic and nuclear protein extracts from oligodendrocyte lineage cells with PRMT5 knocked down compared to control cells, immunoprecipitated with antibodies recognizing symmetrically methylated arginine residues. We only analyzed peptides with symmetrically methylated arginine residues in control, but not in PRMT5 knockdown cells. We then obtained the proteins corresponding to those peptides and eliminated from the results any molecule whose transcripts we had previously reported to be downregulated in Prmt5 knockdown cells . This leCry and Per  into messenger RNAs (mRNA) and their subsequent translation . Due to  and Per .6-methyladenosine (m6A), a methylated adenosine modification in mRNA . A notable example is the membrane associated PDGFRα, previously identified as PRMT5 target in OPCs . OverallMSV000088396.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: CCMS MassIVE, accession no: This animal study was reviewed and approved by IACUC at Icahn School of Medicine.DD analyzed the data relative to interacting partners and substrates, prepared the figures, wrote the first draft of the text, and edited the manuscript with PC. JL prepared the cell extracts and worked with PC and HZ in experimental design. IS performed the substrate data analysis and comparative analysis with other studies and helped with figure preparation and text editing. HZ and DM helped with the design and performance of the iTRAQ experiment and mass spectrometry, analyzed the raw data, and contributed to manuscript editing. PC worked with JL on the initial experimental design and conceptualization, wrote the text with DD, and supervised the overall workflow. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.