Cervical cancer (CC) is a prevalent gynecological cancer, and the patients with CC usually suffer from dismal prognosis. Long non-coding RNAs (lncRNAs) are demonstrated to serve as promising biological targets in human cancers. Gastric carcinoma proliferation enhancing transcript 1 (GHET1) has been revealed to function as an oncogene in several cancers, but it has never been investigated in CC. We proposed to examine the biological role of GHET1 in CC and the underlying mechanism and validated the up-regulated expression of GHET1 in CC cell lines. Loss-of-function assays demonstrated that down-regulation of GHET1 inhibited cell growth, migration and epithelial-to-mesenchymal transition (EMT) in CC. Furthermore, we validated that GHET1 down-regulation could inactivate AKT/mTOR and Wnt/β-catenin pathways, and that respective activation of these two pathways abrogated the inhibitive effect of GHET1 knockdown on CC cell growth, migration and EMT. Moreover, we unfolded a preliminary investigation on the modulation of GHET1 on AKT/mTOR and Wnt/β-catenin pathways. We found that GHET1 stabilized E2F6 mRNA through interacting with IGF2BP2, so as to regulate the activity of AKT/mTOR and Wnt/β-catenin pathways. Rescue assays also proved that GHET1 regulated these two pathways and CC cell growth, migration and EMT through E2F6. In conclusion, we revealed that down-regulation of GHET1 suppresses cervical cancer progression through regulating AKT/mTOR and Wnt/β-catenin signaling pathways, indicating GHET1 as a promising molecular biomarker for CC treatment improvement. As a familiar gynecological cancer, cervical cancer (CC) accounts for a large proportion of tumor-resulted deaths globally ,2. AlthoLong non-coding RNAs (lncRNAs), a set of transcribed RNAs with over 200 nucleotides in length, are incapable of translating proteins . MountinThe tumor development involves the activation and inactivation of multiple pathways. AKT/mTOR is an essential signaling pathway responsible for the modulation of cancer cell biological activities such as proliferation and migration . The actE2F transcription factor 6 (E2F6) belongs to the family of E2F transcription factors that regulate gene transcription and influence cell differentiation, proliferation, cell cycle and apoptosis . MultiplPresent study planned to investigate the function of GHET1 in cervical cancer and its mechanism in regulating AKT/mTOR and Wnt/β-catenin signaling pathways.Forty patients from Wuhan Children’s Hospital , Tongji Medical College, Huazhong University of Science and Technology were participated in the present study. None of them experienced chemo- or radiation therapy. After surgical resection, tumor tissues were kept in liquid nitrogen at once and were reserved under −80°C for following use. This research was approved by the Ethics Committee of Wuhan Children’s Hospital , Tongji Medical College, Huazhong University of Science and Technology. Informed consent was attained from all the patients.2. Cells that were exponentially growing were collected for later experiments.Four human CC cell lines  and the normal cervical cell line (Crl-2614) were provided by American Type Culture Collection . The culture medium of all cell lines was Dulbecco’s Modified Eagle’s Medium . Media were with the supplementation of 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin , and were maintained at 37°C in a humidified incubator with 5% COGHET1/IGF2BP2 was knocked down by two specific small interfering RNAs (siRNAs) targeting GHET1 (siGHET1#1/siIGF2BP2#1 and siGHET1#2/siIGF2BP2#2), with the scramble siRNA  as negative control. IGF2BP2 and E2F6 were overexpressed by inserting the sequences of IGF2BP2 or E2F6 into pcDNA3.1, with vector pcDNA3.1  acting as negative control. The transfection of these plasmids into CC cells was conducted by Lipofectamine 2000 .C−ΔΔt method. The internal control was GAPDH. The primer sequences are presented in The RNA extracts were obtained from the harvested CC cells by the use of TRIzol® reagent , and the RNase-Free DNase Set  was used to purify the RNAs. Subsequently, purified RNAs were subjected to the reverse transcription into complementary DNA (cDNA) with the use of SuperScript III Reverse Transcriptase . Then, qPCR was implemented on the ABI 7300 Real-Time PCR System  by the application of Power SYBR Green Master Mix Kit . The calculation of gene expressions was conducted on the basis of the 23/well). At 0, 24, 48, 72 and 96 h after incubation respectively, each well was added with CCK-8 solution (10 μl) followed by the incubation of CC cells at 37°C for 2 h. Measurement of the absorbance (wavelength: 450 nm) in each well was carried out applying a microplate absorbance reader .The proliferation of CC cells was examined applying the Cell Counting Kit-8  reagent. In short, CC cells were plated into the 96-well plates  for migration assay. The media with 10% FBS was added to lower chamber. After that, CC cells that had migrated through the membrane were subjected to the staining in methanol with 0.1% Crystal Violet. Then, IX71 inverted microscope  was applied to observe and count cells.The construction of β-catenin reporter plasmid (TOP-flash) as well as the mutant control (FOP-flash) was accomplished by Millipore Corporation . Then, TOP-flash or the FOP flash expression plasmids were co-transfected with pRL-TK  into CC cells that were previously serum-starved for 12 h by means of Lipofectamine 2000 (Invitrogen). Determination of firefly or Renilla luciferase activities was carried out at 48 h following transfection applying a Dual Luciferase Assay Kit . The activity of TOP-Flash reporter was presented as the relative proportion of firefly luciferase activity to the Renilla luciferase activity. TOP/FOP ratio was utilized to examine the β-catenin-driven transcription.After the 1 h incubation of CC cells with blocking buffer , the primary antibodies against β-catenin  were added and incubated with the CC cells overnight. Following rinsing for three times with PBST, cells were then incubated with secondary antibodies for 1 h under room temperature. DAPI  was then used to stain the cell nuclei. Pictures were taken applying the Nikon Ti inverted fluorescence microscope.de novo RNA. Following the harvest of total RNA at indicated time points, the expression of E2F6 mRNA was evaluated by qRT-PCR. The half-life of E2F6 mRNA was examined by comparing the mRNA expression of E2F6 to that of E2F6 before adding ActD.After the transfection for 48 h, the Actinomycin D (ActD)  was used to inhibit Magna RIP RNA-Binding Protein Immunoprecipitation Kit  was applied for RIP assay. After CC cells were lysed in the complete RIP lysis buffer, the whole cell extracts were subjected to the overnight incubation with RIP buffer magnetic beads with antibodies against IGF2BP2  at 4°C, with IgG (Abcam) as negative control. Then, the purified RNAs in the precipitates were evaluated by RT-qPCR.After being lysed with RIPA Lysis Buffer , the protein density of CC cells was examined using Bradford Protein Assay Kit (Beyotime). Subsequently, proteins were subjected to the separation by 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Then, proteins were transferred onto the polyvinylidene difluoride (PVDF) membranes , followed by the blocking in 10% non-fat milk at 37°C for 1.5 h. Thereafter, membranes were washed and detected with the primary antibodies for 12 h at 4°C and were subsequently incubated with the secondary antibodies for 2 h. Examination of protein bands was carried out utilizing the enhanced chemiluminescence with imaging system (Bio-Rad). The primary antibodies against E-cadherin, N-cadherin, p-AKT, AKT, p-mTOR, mTOR, p-GSK3β, total p-GSK3β, β-catenin, Cyclin D1, c-Myc, E2F6 and GAPDH were purchased from Abcam .t-test or one-way ANOVA. P < 0.05 suggested significance at a statistical level.All assays were conducted ≥3 times. The data presentation was carried out as mean ± standard deviation. Data analysis was carried out utilizing SPSS 16.0 software . The determination of statistical differences between two groups or among multiple groups was performed utilizing the Student’s We first investigated how GHET1 affected the biological activities of CC cells. As shown in It has been acknowledged that AKT/mTOR pathway performs essential part in regulating cancer cell biological activities such as proliferation and migration , includiThen, rescue assays were implemented to examine whether GHET1 regulated CC cell proliferation and migration through AKT/mTOR and Wnt/β-catenin patterns. We found that the treatment of IGF-1 (activator of AKT/mTOR pathway) impaired repressive effect of siGHET1#1 on levels of p-AKT and p-mTOR in SiHa cells A. Also, http://starbase.sysu.edu.cn/index.php) that IGF2BP2, another member of IGF-2 binding protein family, was a RNA-binding protein potentially interacting with both GHET1 and E2F6 (Finally, we sought to investigate how GHET1 regulated AKT/mTOR and Wnt/β-catenin pathways. E2F6 could regulate AKT/mTOR and Wnt/β-catenin pathways in Ewing’s sarcoma . Also, Eand E2F6 B. IGF2BPand E2F6 C. Subseqand E2F6 D. Besideand E2F6 E. Westerand E2F6 F. In addFinally, we probed whether GHET1 regulated CC progression through E2F6. We overexpressed E2F6 in SiHa cells G. OverexLncRNAs are increasingly illustrated by mounting studies to have significant impact on cancer regulation ,7, incluFormer studies have delineated that AKT/mTOR pathway and Wnt/β-catenin pathway are key pattern regulating cancer progression ,26, inclFurthermore, we probed the mechanism whereby GHET1 regulated AKT/mTOR pathway and Wnt/β-catenin pathway. A former study has shown that E2F6 could regulate both AKT/mTOR and Wnt/β-catenin pathways in Ewing’s sarcoma . E2F6 beTo be concluded, our findings first showed that knockdown of GHET1 inhibited proliferation, migration and EMT through AKT/mTOR and Wnt/β-catenin pathways, suggesting GHET1 as a novel biological target in CC and providing new thoughts for the improvement of treatment of CC.Click here for additional data file.


Conversely, it was reported that AhR negatively regulates the β-catenin signal via ubiquitination and subsequent degradation in a ligand dependent manner. However, there have been conflicting data among previous studies regarding the relationship between these two proteins. In this report, we conducted confirmatory studies dissecting the relationship between AhR and β-catenin. We did not observe β-catenin degradation by AhR ligands in several colon cancer cell lines. Reporter assays revealed that the AhR ligand did not alter TcF/β-catenin dependent transcription. Yeast and mammalian two-hybrid assays failed to reconstruct the interaction of β-catenin and AhR even when other factors, Arnt, CUL4B, and DDB1, were co-expressed additionally. Independently to induction of AhR expression, β-catenin enhanced AhR-dependent transcriptional activation via the xenobiotic response element (XRE). Coimmunoprecipitation detected the formation of a β-catenin and ligand-activated AhR complex, which was thought to reflect the β-catenin mediated enhancement of the AhR signaling. Overall, we could only confirm unidirectional interaction, which is positive regulation of the AhR signal by β-catenin. These results suggested that data from previous reports on the degradation of β-catenin via liganded AhR warrants further investigation to yield clarity in the field. Wnt family signals are triggered by extracellular glycolipoprotein and mediated by intracellular β-catenin. β-Catenin is a component of the cadherin protein complex that regulates cell–cell adhesion. In the nucleus, β-catenin also acts as a transcriptional coactivator of the T-cell factor and lymphoid enhancer-binding factor 1 (Tcf/Lef) family . IntraceThe aryl-hydrocarbon receptor (AhR) is associated with tumorigenesis in a distinct way from β-catenin . AhR is Interestingly, numerous studies have reported on crosstalk of β-catenin and AhR . It has This mutual regulation between AhR and β-catenin looks like a well-crafted feedback loop: β-catenin enhances the AhR signal, and AhR down-regulates β-catenin signal. However, data from other studies indicated several discrepancies regarding the interaction between β-catenin and AhR. In the hepatoma cell line, TCDD failed to alter β-catenin/TcF reporter activity as well as their target genes such as Axin2 and Gpr49 . In rat v/v) in culture media.3-Methylcholanthrene (3MC), indole-3-carbinol (I3C), indole-3-acetate (IAA), cycloheximide (CHX), and β-naphthoflavone (βNF) was purchased from FUJIFILM Wako Pure Chemical Corporation . Indirubin was kindly provided by Dr. Tomonari Matsuda . MG-132 was purchased from Abcam . All other chemicals and solvents used were purchased from Sigma-Aldrich . Ligands were dissolved in dimethyl sulfoxide (DMSO) and added to media. The final concentration of DMSO was adjusted to 0.1% . Expression plasmids for the AhR subdomain and deleted AhR were constructed using PCR. Supporting information The reporter plasmid pX4TK-Luc, including the firefly luciferase gene under the control of four copies of XRE and the thymidine kinase promoter, was a gift from K. Sogawa . Both TOPflash and FOPflash plasmids used for the TcF reporter assay were obtained from Millipore . The mammalian expression plasmid containing mouse (C57BL) and human AhR cDNAs as well as human Arnt and human SRC-1 cDNA were constructed in our previous study . Human β2HPO4, 40 mM NaH2PO4, 1 mM MgCl2, 10 mM KCl, 2 mM dithiothreitol, and 0.2% sarcosyl, adjusted to pH 7.0) containing 1 mg/ml o-nitrophenol-β-D-galactopyranoside and subsequently incubated for 60 min at 37°C. The absorbance at 405 and 595 nm was measured to indicate the amount of generated o-nitrophenol and yeast cell density, respectively.The plasmid vector pGADT7, pGBKT7, and their derivative plasmids were co-expressed in yeast strain Y187. Each yeast strain was cultured overnight at 30°C in synthetic medium lacking tryptophan, uracil, and/or leucine. Then, 5 μL of yeast culture was added to 100 μL of synthetic medium and exposed to 1 μM of 3MC, and the 96-well microtiter plates were incubated for 12 h at 30°C. After incubation, yeast cell suspension (10 μL) was added to 100 μL of Z-buffer  were combined in 200 μL Opti-MEM (Thermo Fisher Scientific). After incubation for 15 min, 2 μL Lipofectamine 2000 reagent (Thermo Fisher Scientific), diluted with 200 μL Opti-MEM, was added and incubated for an additional 15 min. The cells were plated in 6-well tissue culture plates at 30–40% confluence a day before transfection. Liposomes were added in serum-free medium for 3 h and then replaced with MEM or DMEM containing 10% charcoal-stripped FBS without 1× Antibiotic-Antimycotic. In AhR knockdown experiments, HCT116 cells were transfected with human AhR  or control (Ambion) siRNA at 10 nM in a culture medium using Lipofectamine RNA iMAX .All cell lines used in this study were obtained from the American Type Culture Collection . DLD-1 cells were also purchased from the Health Science Research Resources Bank . The human epithelial carcinoma line, HeLa, was grown in MEM containing 10% charcoal-stripped FBS and 1× Antibiotic-Antimycotic . Human colon cancer cell lines, Caco-2, DLD-1, HCT116, LS174T, SW480, and WiDr; human hepatoma cell line HepG2; and human breast adenocarcinoma cell line MCF-7 were grown in phenol red free DMEM containing 10% charcoal-stripped FBS and 1× Antibiotic-Antimycotic. All cultures were incubated at 37°C in 5% COforward primer, 5'‐CATAGACACTGATCTGGCTGCAG‐3' and reverse primer, 5'‐GGGAAGGCTCCATCAGCATC‐3'. For semi-quantification of the amplified PCR products, samples were collected every 3 cycles between 24–36 cycles and applied to electrophoresis.Total RNA was isolated using Isogen  according to the manufacturer’s instructions. An aliquot (2 µg) of total RNA was subjected to reverse transcription using SuperScriptIII reverse transcriptase (Thermo Fisher Scientific) and oligo-dT primers. cDNA was diluted and amplified by Ex Taq polymerase  with gene-specific primers. Primers for human CYP1A1 cDNA amplification were: Renilla luciferase (Rluc) expression vector pRL-CMV (Promega). The transfected cells were washed with cold PBS and lysed in 25 μL 1× passive lysis buffer (Promega). Aliquots (10 μL) of the lysates were transferred to microcentrifuge tubes, and firefly luciferase (Luc+) and Renilla luciferase (Rluc) activities were measured using the Dual-Luciferase Reporter Assay System (Promega) in the GloMax 20/20 Single Tube Luminometer (Promega). Transfection and translation efficiencies varied between independent experiments, and the results were normalized by calculating Luc+/Rluc ratios.The effects of AhR ligands on TcF-dependent transcription were elucidated by transfection of the TOPflash or FOPflash plasmid with other expression plasmid into cells. The effects of AhR ligands on XRE-dependent transcriptional activity were evaluated by the reporter plasmid pX4TK-Luc . ReporteTo prepare the sample for immunoblotting, TNE buffer  was used for cell lysis. Cells were washed with PBS and lysed by adding TNE buffer on ice. Then, cell lysates were sonicated moderately and centrifuged at 4°C for 30 min at 20,000×g. For the fractionation of cell lysates, NE-PER Nuclear and Cytoplasmic Extraction Reagents  were used. According to the manufacturer's protocol, nuclear or cytoplasmic proteins were extracted. Protein concentration in cell lysates was measured by the Bradford method using a Bio-Rad Protein Assay , and 10- or 20 μg aliquot of each sample was separated on an SDS-polyacrylamide gel. Immunoblotting was performed with an anti-human AhR antibody  or with an anti-β-catenin antibody . Anti-actin antibody , anti-MEK1/2 , and anti-Lamine B  were also used for control experiments. Signals were detected with the peroxidase-labeled secondary antibody contained in the ECL Plus detection system  or anti-goat IgG . Specific protein bands were visualized by the ECL Plus detection system and imaged using ImageQuant LAS-4000 Chemiluminescence & Fluorescence Imaging System .2, 10 mM KCl, 0.2 mM EDTA, 0.1% NP-40, and CompleteTM Protease Inhibitor Cocktail . Diluted samples were precleared by adding 25 μL (50% slurry) of protein G Sepharose CL-4B . After 1 hour incubation at 4°C, the CL-4B beads were removed by centrifugation and filtration, and samples were incubated with antibodies (2.5 μL for anti-AhR antibody and 2 μL for anti-β-catenin antibody) for 2-hours at 4°C with rotation. Then, 25 μL of protein G Sepharose CL-4B were newly added to samples and incubated for another one-hour at 4°C. The beads were washed twice with sample buffer mentioned above and finally eluted with 25 μL of sample buffer containing 100 mM Tris-HCl (pH 7.5), 4% 2-mercaptoethanol, and 2% SDS. Immunoblotting was performed with anti-β-catenin antibody, anti-CUL4B antibody  and anti-Arnt antibody .To prepare samples for coimmunoprecipitation, HCT-116 cells were induced to overexpress AhR and β-catenin via pCI-mAhR and pCI-βcat transfection. Twenty-four hours after transfection, cells were exposed to 1 μM 3MC with or without 100 μM MG-132. After 2 h of incubation, the nuclear or cytoplasmic fraction was isolated using a Dounce homogenizer. Nuclear proteins were extracted using the Nuclear Complex Co-IP Kit  according to the manufacturer’s protocol. The extracted protein was measured via Bradford method, and 100 μg aliquot of each sample was diluted with 400 μL of binding buffer containing 10 mM HEPES (pH 7.9), 1.5 mM MgClStudent’s t test was used to evaluate significant differences between two groups.de novo synthesis of β-catenin could result in the degradation of β-catenin, rendering it undetectable. The reduction of the β-catenin was not obtained in the nucleus protein as well as cytosol protein after ligand exposure  reporter vector or a β-catenin-LEF/TCF insensitive (FOPflash) reporter vector and expression vector of the hyperactive form of β-catenin (S37A mutant) were co-transfected into DLD-1 cells. As We evaluated ligand-dependent AhR activation in several colorectal cancer cell lines by CYP1A1 mRNA induction, as shown in siRNA treatment reduced AhR expression to approximately 30% of the basal levels, resulting in slight downregulation of β-catenin expression compared with the control . Both thWe investigated protein-protein interactions by using the yeast two-hybrid assay. As Next, we investigated the interaction of β-catenin and AhR or CUL4B by using the mammalian two-hybrid assay. The S37A mutant of β-catenin fused with the GAL4 DNA-binding domain (pBIND-βcatS37A) in HeLa cells induced reporter gene activity . AhR or In the experiment with exchanging prey and bait protein, full length mouse AhR fused to the Gal4 DNA-binding domain (pBIND-mAhR) showed ligand-dependent induction of the reporter gene . Co-exprSince the acidic domain fused to Gal4-DBD (pBIND-mAhR-acid) seemed to have transcriptional activation function, we constructed AhR lacking the acidic domain (pCI-mAhRΔacid) or transcriptional activation domain (pCI-mAhRΔTAD) as outlined previously . The XRAlthough the transactivation and acidic domains of AhR are responsible for the enhancement of AhR-mediated transactivation via coactivator binding, each of these domains alone is insufficient for the enhancement of transactivation by β-catenin.Several reports have investigated β-catenin and AhR interaction by co-immunoprecipitation methods, presenting conflicting data illustrating both detection and non-detection. By using specific antibodies against AhR and β-catenin, we detected a complex of these proteins from HCT116 cells after transfection of pCI-mAhR and pCI-βcat . β-CatenProtein-protein interactions are essential for regulating most cellular functions. Among an enormous number of combinations between proteins, protein interactions involving β-catenin are recognized to be particularly important for colorectal cancer. AhR was reported as an interacting protein with β-catenin, but studies dissecting their crosstalk have generated conflicting outcomes. In this study, we focused on ligand-dependent β-catenin degradation via AhR, and we attempted to verify previous data about AhR and β-catenin interactions. In conclusion, we found several inconsistencies in results reported previously, such as studies illustrating that AhR acts as an ubiquitin ligase and degrades β-catenin.de novo synthesis was inhibited , 1 or 3 μM 3-methylcholanthrene (3MC), 1 μM β-naphthoflavone (βNF), 3 μM indirubin (Ind), 100 μM indole-3-carbinol (I3C), or 100 μM or 1 mM indole-3-acetate (IAA). After 16 h of incubation, the cells were lysed, and firefly and Renilla luciferase activities were measured. Data represent the average of normalized firefly luciferase/Renilla luciferase activities from experimental duplicates.(PDF)Click here for additional data file.S2 FigUpper panel indicates scheme of each hybrid construct. Various mouse AhR cDNA fragments were fused to the Gal4 DNA-binding domain in the plasmid vector pBIND. pBIND empty, parental empty vector; pBIND-mAhR, containing full length mouse AhR; pBIND-mAhR-TAD, containing mouse AhR trans-activating domain (aa 425–805); pBIND-mAhR-acid, containing mouse AhR c-terminal acidic domain (aa 524–583). Each of these plasmids and reporter vector pG5luc were transfected into HeLa cells together with pCI-SRC1, an expression vector for human SRC-1, or pCI-empty vector. After transfection, cells were exposed to 3MC (1 µM) or DMSO  for 16 h. Data represent the average of firefly luciferase activity normalized to Renilla luciferase activity expressed from experimental duplicates.(PDF)Click here for additional data file.S3 FigAn experiment similar to (PDF)Click here for additional data file.S4 FigUpper panel indicates scheme of each hybrid construct. Plasmid vector containing mouse AhR cDNA (pCI-mRNA), lacking acidic domain (pCI-mAhRΔacid: Δ aa 524–583) or c-terminal trans-activating domain (pCI-mAhRΔTAD: Δ aa 424–805) were transfected together with Renilla luciferase expression vector pRL-CMV and reporter vector pX4TK-Luc into MCF-7 cells. Mock plasmid, pCI-empty vector, is the parental plasmid without cDNA insertion. After transfection, cells were exposed to 1 μM 3MC, and luciferase activity was measured. Data represent the average of experimental duplicates.(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


TGF-β1, and HMGB-1 expressions, and that could be attenuated by NS398 or DKK-1 addition. LPS induced the PGE2 production, and PGE2 increased the expression and nuclear translocation of β-catenin, while EP2 blocker, AH6809, alleviated those effects. TWS119 increased the luciferase activity in the COX-2 promoter. In conclusion, LPS stimulated the NIH3T3 fibroblasts migration through a positive feedback between β-catenin and COX-2, in which PGE2, EP2, TGF-β1, and HMGB-1 played as signal molecules.How β-catenin/COX-2 contribute to inflammation-induced fibroblasts migration remains poorly understood. Therefore, in this study, lipopolysaccharide (LPS) was used as a stimulus to accelerate the migration of NIH3T3 cells, which mimicked the tissue repair process. LPS treatment increased the cell migration in concentration-and time-dependent manner. And NS398, a COX-2 inhibitor, inhibited LPS-induced NIH3T3 cells migration. DKK-1, an antagonist of the Wnt/β-catenin signaling, also inhibited that migration. However, TWS119, an inducer of β-catenin via GSK-3β, increased the cell migration. LPS or TWS119 treatment increased COX-2, β-catenin,  Fibroblasts comprise the most abundant cell type in connective tissues and are central players in organ homeostasis. Their main function is to maintain the structural integrity of connective tissue by secreting collagen and fibronectin, which are principal components of the extracellular matrix. Synthesis of new tissue by fibroblasts is required for tissue rebuilding in response to injury. The recruitment of fibroblasts is essential in wound healing but are also central features in pathological mechanisms leading to tissue fibrosis and remodeling .Inflammation is part of the complex biological response of body tissues to harmful stimuli. One of the most important function of inflammation is to initiate tissue repair . Cycloox2 receptor 2 (EP2)]. The results give us a better interview of the roles of COX-2 and β-catenin in inflammation-induced fibroblasts migration, and might be useful for tissue repair research.The aforementioned observations lead us to the hypothesis that the crosstalk between COX-2 and β-catenin could be an important regulator of inflammation-induced fibroblasts migration. To clarify that point, fibroblasts migration was induced by lipopolysaccharide (LPS) in NIH3T3 cells with or without selective tools [NS-398, a COX-2 inhibitor; DKK-1, an antagonist of the β-catenin signaling; AH6908, an antagonist of PGE2. The media were supplemented with 10% heat-inactivated calf serum and 1% penicillin/streptomycin. The cells were plated on 35-mm dishes or μ-slides with eight wells. LPS from Escherichia coli O111:B4 was purchased from sigma (#L4130). All the cells were passaged 1:4 (35 mm) using 0.25% trypsin when they reached 80–90% confluence. Then, these cells were randomly divided into seven groups: the control group (n = 5); the LPS group ; the LPS plus NS398 group ; the LPS plus DKK-1 group ; the TWS119 group ; and the TWS119 plus NS398 group ; the TWS119 plus DKK-1 group . Recombinant human DKK-1  was purchased from PeproTech.NIH3T3 cells were obtained from the China Center for Type Culture Collection . The cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) at 37°C in a humidified atmosphere of 5% COThe scratch assay was used to evaluate the migration ability of NIH3T3 cells, as described previously . BrieflyFor Western blotting analysis, protein extraction from cultured NIH3T3 cells was conducted according to previously described methods . Whole c2 in cell-culture supernatants and cell were quantified by commercial ELISA kits (Bioss Antibodies) in accordance with the manufacturer’s instructions.Prostaglandin E® RT-for-PCR Kit  based on the SYBR Premix Ex Taq II  with 30–40 cycles of denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s. Furthermore, the mRNA expression levels of the target genes were normalized to that of GAPDH where the sequences of both forward and reverse primers are listed in Table Total RNA was extracted from cultured NIH3T3 cells using RNAiso Plus , followed by cDNA synthesis according to the manufacturer’s instructions with the Advantage2, LPS, PGE2 (1 μg/mL)+AH6809 (1 μg/mL), or LPS (400 ng/mL)+AH6809 (1 μg/mL) for 24 h. Immunofluorescence staining was performed according to previously described methods  without antibiotics. After 24 h, transfections were carried out using Lipofectamine 2000 reagent  according to the manufacturer’s instructions. The efficiency of transfection was evaluated by fluorescence intensity using inverted fluorescence microscopy .It has been previously reported that COX-2 promoter contained a functional TCF/LEF-response element for the enhancement of β-catenin signaling . Here, w3T cells . Dual lut-test. The analyses were performed using InStat software . P < 0.05 was considered to be statistically significant.All data were expressed as mean ± standard deviation (SD) and analyzed by one way analysis of variance (ANOVA) with Tukey test to determine group differences. Difference between two groups was compared by unpaired Student’s P < 0.05). Furthermore, to investigate the time-effect relationship, the scratch assay was conducted with 400 ng/ml LPS. Photographs of cellular migrations were taken at 4, 12, 24, and 36 h after scratching. Representative photomicrographs were shown in Figures P < 0.01; 36 h, 129 ± 1% of control, P < 0.01). Thus, 400 ng/ml LPS treatment for 24 h was used for further scratch assay.Lipopolysaccharide  did not affect the viability of NIH3T3 cells significantly. As shown in Figures P < 0.001; 153 ± 1% of the control for β-catenin, P < 0.001). These results suggested that the activation of fibroblast migration by LPS was associated with increases of COX-2 and β-catenin expressions in fibroblasts.As shown in Figure P < 0.001; 133 ± 3%, P < 0.001, respectively). LPS induced the mRNA and protein levels of COX-2 and β-catenin . Furthermore, the protein and mRNA expressions of COX-2 in the TWS119 + NS398 treated group were significantly decreased in comparison with that of the TWS119 treated group. There was a same trend in the TWS119 + DKK-1 treated group. As shown in Figures As shown in Figures 2 production in a concentration-dependent manner , alleviated the effects of PGE2, or LPS  was 1.98 ± 0.48, P < 0.05; 3.04 ± 0.23, P < 0.0001; 1.49 ± 0.25, respectively. TWS119 increased the luciferase expression of PGL3-mCOX-2-pro200 significantly, but not control, pro500, or pro200mu.An obviously increased luciferase activity was observed in the in vivo wound repair, where fibroblasts play an important role in forming the granulation tissue. In accordance with veritable scar formation, the scratch assay functions are as an appropriate model to mimic the process of fibroblast migrations under the condition of destroyed skin (Wound healing assays are often studied as a model system for yed skin . In wounin vitro scratch assay. LPS treatment increased the cell migration in concentration-and time-dependent manner. To evaluate whether the LPS-induced migration of NIH3T3 cells was associated with COX-2 and β-catenin, we used inhibitors. And NS398, a COX-2 inhibitor, inhibited LPS-induced NIH3T3 cells migration. DKK-1, an antagonist of the Wnt/β-catenin signaling, also inhibited that migration. However, TWS119, an inducer of β-catenin via GSK-3β, increased the cell migration. We further quantified the protein expressions of COX-2 and β-catenin using RT-PCR, Western blot, and immunofluorescence staining methods. LPS or TWS119 treatment increased COX-2 and β-catenin expressions, and that could be attenuated by NS398 or DKK-1 addition. There is a classic signal pathway between COX-2 and β-catenin in cancer cell migration: COX-2 increases the PGE2 production, and PGE2 binds to EP2. This leads to the inactivation of GSK-3β, thereby relieving the inhibitory phosphorylation of β-catenin and activating its signaling pathway (2 production, and PGE2 increased the expression and nuclear translocation of β-catenin, while EP2 blocker alleviated those effects.Since migration of fibroblasts was a major process of wound healing, we first checked the effects of LPS on NIH3T3 cells using the  pathway . In the TGF-β1 and HMGB-1 are important regulators of both inflammation and fibroblasts migration (TGF-β1 and HMGB-1 mRNA were increased by LPS or TWS119, and reduced by COX-2/β-catenin inhibitor. Thus, TGF-β1 and HMGB-1 might be the downstream of COX-2/β-catenin pathway and contribute to the LPS-induced migration of NIH3T3 cells. As a target of the β-catenin signaling pathway, HMGB-1 could directly bind to the promoter regions and subsequently activate the transcription of COX-2 in human umbilical vein endothelial cells (igration . Our resal cells  and pancal cells , suggestMoreover, DKK-1, an antagonist of the Wnt/β-catenin signaling, abolishes the expressions of β-catenin proteins and translocations induced by LPS or TWS119. That further revealed the possibility of a positive feedback between COX-2 and β-catenin. This crosstalk between COX-2 and β-catenin could be explained by the COX-2 promoter containing a functional TCF/LEF-response element for the enhancement of β-catenin signaling . FurtherIn order to investigate the pathway, we indeed used inhibitors. These inhibitors are carefully selected and we have checked their action points, and the inhibitor controls were set for every experiment . However2, EP2, TGF-β1, and HMGB-1 played as signal molecules (Figure Within this work, wound healing assays, performed on fibroblast monolayers, revealed an interesting crosstalk between β-catenin and COX-2. LPS stimulated the NIH3T3 fibroblasts migration through a positive feedback between β-catenin and COX-2, in which PGEs Figure .H-BT conceived and designed the experiments. X-JL, F-ZH, YW, YX, and H-BT performed the experiments. H-BT, F-ZH, Y-SL, X-JL, WZ, YX, and G-HT analyzed the data and wrote the paper. H-BT, F-ZH, and YW contributed reagents, materials, and analysis tools.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.


Here, we investigated the role of CX3CR1 in the neuroinflammation induced by infection with Japanese encephalitis virus (JEV), a neurotrophic virus. Interestingly, CX3CR1 deficiency strongly enhanced susceptibility to JEV only after peripheral inoculation via footpad. By contrast, both CX3CR1+/+ and CX3CR1−/− mice showed comparable susceptibility to JEV following inoculation via intranasal and intraperitoneal routes. CX3CR1−/− mice exhibited lethal neuroinflammation after JEV inoculation via footpad route, showing high mortality, morbidity, pro-inflammatory cytokine expression, and uncontrolled CNS-infiltration of peripheral leukocytes including Ly-6Chi monocytes and Ly-6Ghi granulocytes. Furthermore, the absence of CX3CR1+CD11c+ DCs appeared to enhance susceptibility of CX3CR1−/− mice to JE after peripheral JEV inoculation. CX3CR1 ablation impaired the migration of CX3CR1+CD11c+ DCs from JEV-inoculated sites to draining lymph nodes (dLNs), resulting in decreased NK cell activation and JEV-specific CD4+/CD8+ T-cell responses. However, CX3CR1-competent mice showed rapid temporal expression of viral Ags in dLNs. Subsequently, JEV was rapidly cleared, with concomitant generation of antiviral NK cell activation and T-cell responses mediated by rapid migration of JEV Ag+CX3CR1+CD11c+ DCs. Using biallelic functional CX3CR1 expression system, the functional expression of CX3CR1 on CD11chi DCs appeared to be essentially required for inducing rapid and effective responses of NK cell activation and Ag-specific CD4+ T cells in dLNs. Strikingly, adoptive transfer of CX3CR1+CD11c+ DCs was found to completely restore the resistance of CX3CR1−/− recipients to JEV, as corroborated by the rapid delivery of JEV Ags in dLNs and attenuation of neuroinflammation in the CNS. Collectively, these results indicate that CX3CR1+CD11c+ DCs play an important role in generating rapid and effective responses of antiviral NK cell activation and Ag-specific T cells after peripheral inoculation with the virus, thereby resulting in conferring resistance to viral infection by reducing the peripheral viral burden.A coordinated host immune response mediated via chemokine network plays a crucial role in boosting defense mechanisms against pathogenic infections. The speed of Ag presentation and delivery by CD11c Japanese encephalitis (JE) is a leading cause of viral encephalitis characterized by extensive neuroinflammation in the central nervous system (CNS) and disruption of the blood-brain barrier (BBB) following infection with JE virus (JEV). The zoonotic, mosquito-borne JEV is a single-stranded, positive-sense RNA virus and is endemic to the Asia-Pacific region, including China, India, and northern Australia . Compete+ Th1 cells has a positive effect on disease outcome after JEV infection  are professional antigen-presenting cells (APCs) and key instigators of protective immunity (+ DCs to initiate protective immunity has solidified their roles in determining the outcomes of infectious diseases (+ DCs deliver and present Ags to cognate T cells in lymph nodes (LNs) through their migration from inflammatory sites may decide the severity of infectious disease. Therefore, understanding the sensitivity of CD11c+ DCs in detecting peripheral pathogen invasion and relay of Ags to adaptive immune cells in draining LNs (dLNs) is needed to develop strategies for effective induction of protective immunity.Coordination between host's innate and adaptive immune response is crucial in regulating infectious diseases caused by various pathogens, including JEV. In particular, the speed at which the host innate and adaptive immune cells respond to infection is critical to the clinical outcome . CD11c+ immunity . Detailediseases . The spe+ DCs to peripheral pathogen infection is controlled by inflammatory mediators such as chemokines  belonging to CX3C subfamily is unique in that the first two conserved cysteine residues in the chemokine are separated by three non-conserved amino acids  of Chonbuk National University, and were pre-approved by the Ethics Committee for Animal Experiments of Chonbuk National University . The animal research protocol used in this study followed the guidelines set up by the nationally recognized Korea Association for Laboratory Animal Sciences (KALAS). All experimental protocols requiring biosafety were approved by the Institutional Biosafety Committee (IBC) of Chonbuk National University.b) control mice  were purchased from SAMTAKO , and CX3CR1-deficient  mice were obtained from Taconic Biosciences . The CX3CR1gfp/gfp (H-2b) mice originally obtained from Jackson laboratory  were generously provided by Dr. Doo Hyun Jung  and crossed with C57BL/6 mice to generate CX3CR1+/gfp heterozygous mice. The JEV Beijing-1 strain was propagated in a mosquito cell line C6/36 using DMEM supplemented with 2% FBS, penicillin (100 U/ml), and streptomycin (100 U/ml) as described previously (Wild-type C57BL/6 (H-2eviously . The receviously . Virus s3CR1+/+ and CX3CR1−/− mice were infected with JEV [5.0 × 107 plague-forming units (PFU)] via footpad , intranasal (20 μl), and intraperitoneal routes (200 μl). Infected mice were monitored daily for mortality, morbidity (weight loss), and neurological disorders . Mice were also scored daily for encephalitis signs and symptoms as described previously ; PE-labeled anti-granzyme B (16G6), CD40L (MR1), CD8 (53–6.7), F4/80 (BM8), IFN-γ (XMG1.2), and CD11c (M1/70); PerCP/Cy5.5-labeled anti-mouse Ly-6C antibody (HK1.4) and IFN-γ (XMG1.2); PE-Cy7-lableled anti-NK1.1 (PK136); APC-labeled anti-Ly6-G (1A8), TNF-α (MP6-XT22), and CD49b-integrin alpha 2 (DX5); and biotin-labeled anti-IL-6 (MP5-32C11) and TNF-α (MP6-XT22). PE-labeled anti-mouse Tmem119 (106-6) was obtained from Abcam . The mAbs against non-structural protein 1 (NS1) and envelope glycoprotein protein (E) of JEV were also obtained from Abcam. The JEV epitope peptide of CD4+ T cells [NS3563−574 (WCFDGPRTNAIL)] or CD8+ T cells [NS4B215−223 (9SAVWNSTTA)] was chemically synthesized at Peptron . Phorbol-12-Myristate-13-Acetate (PMA) and ionomycin were purchased from Sigma-Aldrich .The following mAbs were obtained from eBioscience  or BioLegend  for FACS analysis and other experiments: FITC-labeled anti-CD4 (RMA4-5), CD45 (30-F11), CD11b (M1/70), CD3 (145-2C11), and CX7 PFU) via footpad inoculation and various tissues including popliteal LNs, spleen, and brain were harvested at different time points post-infection (pi). Total RNAs were extracted from the collected tissues using easy-BLUE  and subjected to real-time qRT-PCR using a CFX96 Real-Time PCR Detection system . Following reverse transcription of total RNA with High-Capacity cDNA Reverse Transcription Kits , the reaction mixture  contained 2 μl of template cDNA, 10 μl of 2× SYBR Premix Ex Taq, and 200 nM primers . The sections were then washed with PBS for 4 min. Primary antibodies  were applied for overnight at 4°C in a humidified chamber. After rinsing the slides in PBS, they were incubated in secondary antibody  for 30 min at room temperature. After washing with PBS for 5 min, color development was achieved by applying diaminobenzidine tetrahydrochloride (DAB) solution (Vector Laboratories) for 0.5–1 min. After washing in distilled water, the sections were counterstained with VECTOR methyl Green (Vector Laboratories), and cover-slipped using a mounting medium (Fisher Scientific). Sections were analyzed using a Nikon Eclipse E600 microscope . For confocal microscopy staining, popliteal LNs and brain were collected at 2 and 5 dpi, respectively, and frozen in optimum cutting temperature (OCT) compound. Sections of 6–7 μm in thickness were cut, air-dried, and fixed with 1:1 mixture of acetone and methanol for 15 min at −20°C. After washing with PBS three times, non-specific binding was blocked with 10% normal goat serum and cells were permeabilized with 0.1% Triton X-100. Staining was performed by incubating sections overnight in moist chambers at 4°C with FITC-conjugated anti-mouse CX3CR1, APC-conjugated anti-mouse CD11c, and anti-JEV NS1 and E. Primary antibodies were detected with secondary PE-conjugated goat anti-mouse IgG . Nuclei were counterstained with DAPI . Fluorescence was observed using a confocal laser scanning microscope .Histopathological examination was performed using brains derived from CXSandwich ELISA was used to determine the levels of IL-6 and TNF-α cytokines in sera. ELISA plates were coated with IL-6 (MP5-20F3) and TNF-α (1F3F3D4) antibodies (eBioscience), and incubated at 4°C overnight. After plates were washed three times with PBS containing 0.05% Tween 20 (PBST), they were blocked with 3% non-fat-dried milk at 37°C for 2 h. Sera and standards for recombinant cytokine proteins  were added to these plates and incubated at 37°C for 2 h. Plates were washed again with PBST, and then biotinylated IL-6 (MP5-32C11) and TNF-α  antibodies were added. The mixture was incubated overnight at 4°C followed by washing with PBST and subsequent incubation with peroxidase-conjugated streptavidin (eBioscience) at 37°C for 1 h. Color was then developed by adding a substrate (ABTS) solution. Cytokine concentrations were determined using an automated ELISA reader and SoftMax Pro4.3 by comparison with two concentrations of standard cytokine proteins.g for 30 min  using Opti-prep density gradient (18/10/5%), and the cells were collected from 18 to 10% interface and washed twice with PBS. Leukocytes derived from popliteal LNs and spleen were prepared by gently pressing lymphoid tissues through 100-mesh tissue dishes. The cells were then counted and stained for CD45, CD11b, CD11c, Ly-6C, CX3CR1, and Ly-6G with directly conjugated antibodies for 30 min at 4°C. Finally, cells were fixed with 1% formaldehyde. Data collection and analysis were performed using a FACS Calibur flow cytometer  with FlowJo software .Mice infected with JEV were perfused with 30 ml of HBSS at 2, 3, and 4 dpi via cardiac puncture of the left ventricle. Brains were then harvested and homogenized by gently pressing them through a 100-mesh tissue sieve, followed by digestion with 25 mg/ml of collagenase type IV , 10 mg/ml DNase I , and incubation with RPMI medium for 1 h at 37°C with shaking. Cells were separated by centrifugation at 800×3CR1+/+ and CX3CR1−/− mice at 2 dpi and stimulated with PMA and ionomycin in the presence of monensin (2 μM) to induce the expression of IFN-γ (PMA 50 ng/ml plus ionomycin 750 ng/ml for 2 h) or granzyme B (PMA 50 ng/ml plus ionomycin 750 ng/ml for 4 h). The stimulated cells were washed twice with PBS containing monensin and surface-stained with CD3, NK1.1, and DX5 antibodies for 30 min at 4°C. After fixation, cells were washed twice with 1× Permeabilization Buffer (eBioscience) and subjected to intracellular IFN-γ and GrB staining in the buffer for 30 min at room temperature. Stained cells were washed twice with 1× Permeabilization Buffer (eBioscience) and FACS buffer. Analysis was then performed using a FACSCalibur flow cytometer  with FlowJo software (Tree Star).The activity of NK cells was assessed by their capacity to produce IFN-γ and granzyme B (GrB) following brief stimulation with PMA and ionomycin (Sigma-Aldrich). Cells were obtained from popliteal LNs of CX+ and CD8+ T-cell responses were determined by intracellular CD154 , IFN-γ, and TNF-α staining in response to stimulation with JEV epitope peptides. Surviving mice infected with 5.0× 107 PFU JEV were sacrificed on day 7 pi and leukocytes were prepared from popliteal LNs. These leukocytes were cultured in 96-well-culture plates (5 × 105 cells/well) in the presence of synthetic peptide epitopes (NS1132−145 and NS4B215−225) for 12 h and 6 h to observe CD4 + and CD8 + T cell responses, respectively. Monensin at concentration of 2 μM was added to antigen-stimulated cells 6 h before harvest. Cells were washed twice with FACS buffer containing monensin, surface-stained with FITC-anti-CD4 or CD8 antibodies for 30 min at 4°C, and then washed twice with PBS containing monensin. After fixation, cells were washed twice with 1× Permeabilization Buffer (eBioscience) and stained with PepCP-Cy5.5 anti-IFN-γ or APC-anti-TNF-α in the permeabilization buffer for 30 min at room temperature. Intracellular CD154 was detected by addition of CD154 mAb to culture media during peptide stimulation, as described previously . JEV-specific CD43CR1+CD11c+ DCs were purified from spleens of CX3CR1+/gfp or CX3CR1gfp/gfp mice  after surface-staining with PE-conjugated anti-CD11c mAb according to the manufacturer's instructions. Enriched CD11c+ cells were then applied to a FACS sorter to purify CX3CR1+CD11c+ DCs. Purified CX3CR1+CD11c+ DCs contained CD11b+ cells as well. CX3CR1+/gfp and CX3CR1gfp/gfpCD11c+ DCs  were injected into left and right footpads of CX3CR1−/− mice, respectively. CX3CR1−/− recipients were immediately infected with JEV (5.0 × 107 PFU) via footpad, and leukocytes were obtained from left and right popliteal LNs at 3 dpi. Popliteal LN cells were surface-stained with PE-conjugated anti-mouse CD11c for CX3CR1+/gfp and CX3CR1gfp/gfp CD11c+ DCs. In some challenge experiments, purified CD11c+ DCs  were injected i.v. into CX3CR1−/− mice. Flow cytometric analysis was performed using a FACS Calibur flow cytometer  with FlowJo software (Tree Star).CXgfp mice . Splenoc+ T cell responses in popliteal LNs were assessed by CFSE cell division following footpad injection of CX3CR1+/gfp or CX3CR1gfp/gfp CD11c+ DCs. Briefly, OVA323−339-specific CD4+ T cells were purified from OT-II mice using a MACS LS column (Miltenyi Biotec), according to the manufacturer's instructions. Purified OT-II CD4+ T cells were labeled with 2.5 μM CFSE and adoptively transferred into CX3CR1−/− mice (1 × 106 cells/mouse) injected with CX3CR+/gfp and CX3CR1gfp/gfp CD11c+ DCs in the left and the right footpads, respectively. Three days following infection of CX3CR1−/− recipients with recombinant vaccinia virus expressing OVA (1 × 106 PFU/mouse) via footpad route, leukocytes were obtained from left and right popliteal LNs of the recipients and subjected to surface-staining for CD4 and Vα2 using PE-conjugated anti-mouse CD4 and PerCP-conjugated anti-mouse Vα2. Flow cytometric analysis was performed on a FACS Calibur flow cytometer  with FlowJo software (Tree Star).Ag-specific CD4t-test for ex vivo experiments and immune cell analysis. For multiple comparisons, statistical significance was determined using one-way or two-way analysis of variance (ANOVA) with repeated measures followed by Bonferroni post-hoc tests. Statistical significance of viral burden and in vivo cytokine gene expression were evaluated by Mann-Whitney test or unpaired two-tailed Student's t-test. Kaplan-Meier survival curves were analyzed by log-rank test. A p ≤ 0.05 was considered significant. All data were analyzed using GraphPadPrism4 software .All data were expressed as average ± standard error of the mean (SEM). Statistically significant differences between groups were analyzed using an unpaired two-tailed Student's 3CR1 in JE progression, we infected CX3CR1-competent and -deficient mice  with JEV via footpad, intranasal, and intraperitoneal routes. We then compared the susceptibilities of both strains to JE progression. Our data revealed that the ablation of CX3CR1 resulted in markedly enhanced susceptibility to JE, with mortality of around 80% after JEV infection via footpad inoculation compared to 10% mortality in CX3CR1+/+ mice (left graph). In contrast, both CX3CR1-competent and deficient mice all succumbed to JE progression following intranasal inoculation of JEV infection, although the survival of CX3CR1+/+ mice was moderately prolonged (middle graph). A mortality of 80% was observed among CX3CR1+/+ and CX3CR1−/− mice exposed to intraperitoneal inoculation of JEV infection (right graph). These results indicate that CX3CR1-ablated mice were highly susceptible to JE progression only after JEV inoculation via footpad route. In support of this finding, the CX3CR1−/− mice showed a rapid and higher proportion of neurological disorders starting at 4–5 dpi, compared to CX3CR1+/+ mice that displayed the delayed signs of neurological disorder around 6–7 days after footpad inoculation of JEV (left graph). However, the proportions of CX3CR1−/− mice showing neurological disorder were similar to those of CX3CR1+/+ mice following intranasal and intraperitoneal inoculation with JEV (middle and right graphs). Furthermore, CX3CR1−/− mice scored higher for clinical signs of encephalitis than CX3CR1+/+ mice after peripheral JEV inoculation (left graph). In contrast, CX3CR1+/+ and CX3CR1−/− mice showed similar kinetics for encephalitis score after intranasal and intraperitoneal inoculation, although CX3CR1−/− mice showed rapid clinical signs of encephalitis compared with CX3CR1+/+ mice (middle and right graphs). CX3CR1 ablation resulted in marked changes in body weight during JE progression following footpad inoculation of JEV. However, CX3CR1−/− and CX3CR1+/+ mice showed similar reductions in body weight after intranasal and intraperitoneal JEV administration, except that CX3CR1−/− mice showed a slightly higher reduction in body weight at a later stage after intranasal exposure to JEV compared with CX3CR1+/+ mice (3CR1gfp/gfp mice with green fluorescent protein (GFP) inserted into two allele of CX3CR1 locus showed highly increased susceptibility to JE progression only after peripheral inoculation of JEV via footpad, compared to CX3CR1+/gfp mice (Data not shown). To better understand the severity of JE progression in CX3CR1−/− mice following peripheral JEV inoculation, we performed histopathological analysis of brains derived from CX3CR1+/+ and CX3CR1−/− mice after JEV inoculation via footpad, intranasal, and intraperitoneal routes. As expected, CX3CR1+/+ mice showed reduced inflammation involving blood vessels, meninges, and ventricles in the brain compared with CX3CR1−/− mice exposed to JEV inoculation via footpad, based on CNS infiltration of peripheral leukocytes  depending on the expression of Ly-6C and Ly-6G  compared with CX3CR1-competent mice  4 dpi compared with those in CX3CR1+/+ mice (0.96%). In addition, CX3CR1−/− mice contained accumulated number of CD11b+CD45hiTmem119int activated microglia in the CNS with markedly higher levels up to 4 dpi, compared to CX3CR1+/+ mice . This indicates that CX3CR1 ablation increased both activated and resting microglia during JE progression.JE is a lethal neuroinflammation characterized by extensive CNS infiltration of myeloid-derived cells including Ly-6Culocytes . Notablyicroglia , 53. Altammation . To furt+/+ mice . The CNSnd Ly-6G . To delient mice . Similarle virus  and CNS-icroglia . Ly-6Cinia cells . Therefoia cells . As show+/+ mice . CD11b+C3CR1−/− mice following peripheral JEV inoculation via footpad, the expression of cytokines and chemokines within the CNS may further explain encephalitis because neuroinflammation triggered by neurotrophic viruses is indirectly attributed to CNS degeneration by robust immunological responses such as uncontrolled secretion of cytokines and chemokines and the activation of microglia and astrocytes   compared to CX3CR1gfp/gfp mice (upper and lower pictures). Viral Ags were mostly detected within interfollicular and sinus adjacent area near germinal center (upper pictures). Notably, many JEV Ags were co-localized with CX3CR1+ DCs in interfollicular and T-cell zone in popliteal LNs of CX3CR1+/gfp mice. In contrast, JEV Ags were detected in the brains of CX3CR1gfp/gfp mice with a high frequency at the late stage (5 dpi) compared to the brains of CX3CR1+/gfp mice  at early stage following viral inoculation via footpad. It also plays an important role in viral clearance in the peripheral lymphoid tissues and the CNS at later stage.Neurotrophic viruses such as WNV and JEV are thought to replicate in keratinocytes and cutaneous DCs and Langerhans cells following inoculation at peripheral sites such as footpad –61. Infeal sites . However+/+ mice . Notablygfp mice . Collect3CR1-ablated mice showed an impaired clearance of footpad-inoculated JEV in dLNs. Therefore, we compared NK-cell and JEV-specific T-cell responses in popliteal LNs of both wild-type CX3CR1+/+ and CX3CR1−/− mice following footpad JEV inoculation. Both CX3CR1+/+ and CX3CR1−/− mice contained comparable numbers of CD3−NK1.1+DX5+ NK cells in popliteal LNs with increased levels at 24 and 48 h after JEV infection compared to mock-infected mice     . Consistted mice . CX3CR1-215−223) . In orde footpad . Similar AZD8797 , which i+/+ mice . Also, C+/+ mice . However antigen . This fi3CR1 and its ligand CX3CL1 contribute to immune cell recruitment during inflammation via either chemotaxis or adhesion because the CX3CR1-CX3CL1 axis is known to play a role in the migration of NK cells, T cells, monocytes, and mast cells   . Similar11c+ DCs . Consist11c+ DCs . Ultimat3CR1−/− recipients injected with CX3CR1gfp/gfpCD11c+ DCs, we determined whether functional deficiency of CX3CR1 expression affected their migration from injection site (footpad) to popliteal LNs following JEV infection, resulting in impaired NK cell activation and JEV-specific T-cell responses. CX3CR1+/gfp and CX3CR1gfp/gfpCD11c+ DCs were sorted from spleens of heterozygous CX3CR1+/gfp and homozygous CX3CR1gfp/gfp mice, and injected into left and right footpads of CX3CR1−/− mice, respectively. Recruitment of CX3CR1+/gfp and CX3CR1gfp/gfpCD11c+ DCs in popliteal LNs of CX3CR1−/− recipients was then observed at 3 days after footpad inoculation of JEV. Our results revealed that functional deficiency of CX3CR1 expression delayed the migration of CD11c+ DCs from the footpad to popliteal LNs, as the lower frequency of CX3CR1gfp/gfpCD11c+ DCs was observed in popliteal LNs of the right footpad injected with CX3CR1gfp/gfpCD11c+ DCs compared to popliteal LNs of the left footpad injected with CX3CR1+/gfpCD11c+ DCs . The CFSE-labeled OT-II CD4+ T cells purified from OT-II mice were adoptively transferred into CX3CR1−/− mice, followed by footpad inoculation with vaccinia virus expressing OVA after injection of CX3CR1+/gfp and CX3CR1gfp/gfpCD11c+ DCs into the left and right footpads, respectively. It was found that OT-II CD4+ T cells in popliteal LNs of the left footpad injected with CX3CR1+/gfpCD11c+ DCs proliferated rapidly compared to OT-II CD4+ T cells in popliteal LNs of the right footpad injected with CX3CR1gfp/gfpCD11c+ DCs  and chemokines  in the CNS during JE progression . Although CX3CR1+/+ mice showed higher viral burdens temporally in lymphoid tissues at the early stage (1–3 dpi), JEV was rapidly cleared in the peripheral tissues. This viral clearance in the peripheral lymphoid tissues of CX3CR1+/+ mice appeared to be mediated by rapid and effective NK cell activation and JEV-specific CD4+ and CD8+ T cell response. The rapid delivery of viral Ag to cognate T cells might induce the prompt proliferation of viral Ag-specific CD4+ and CD8+ T cells in dLNs  during JE progression.DCs are key players in the initiation and generation of Ag-specific CD4rosstalk , 17, 66. T cells –35, 68.  tissues . We anal in dLNs . IFN-γ pwith JEV . NK cellwith JEV . Also, Cammation , 73, eve3CR1-competent mice, the expression of CX3CR1 on various leukocytes in both dLNs and inoculation site was analyzed. The results showed that ~90% of CD11b+CD11c+ DC populations at the inoculation site (footpad) were CX3CR1-positive. The CX3CR1+CD11b+CD11c+ DC population was rapidly recruited to dLNs following peripheral JEV inoculation, because the proportion of CX3CR1+ cells in CD11b+CD11c+ DC population was increased from 40% to around 70–80%. CX3CR1 ablation also interfered with the migration of CD11c+ DCs and CD11b+ myeloid cells into dLNs . The impaired migration of CX3CR1+CD11c+ DCs from the inoculation site to dLNs in CX3CR1−/− mice was likely to induce delayed and diminished responses of JEV-specific CD4+ and CD8+ T cells. The CX3CR1−/− mice also showed a weak activation of NK cells without changes in the absolute number of CD3−NK1.1+DX5+ NK cells in dLNs. This finding was corroborated by IFN-γ and granzyme B-producing NK cells. It is plausible that the impaired migration of CD11c+ DCs affected the activation of NK cells in dLNs because DCs play a crucial role in activating NK cells via DC-NK crosstalk (+ DCs (cDC) originating in common DC precursors (CDPs) via cDC-restricted progenitors (pre-cDCs) have been detected in lymphoid and non-lymphoid tissues. They are strategically located in areas to actively detect signs of pathogens and damage in the cellular and physiological environment  and transgenic OT-II CD4+ T cells, we analyzed the role of CX3CR1+CD11c+ DCs in generating rapid and effective NK cell activation and Ag-specific CD4+ Th1 responses. CX3CR1-CX3CL1 axis not only mediates the migration of leukocytes to promote cell-to-cell interaction with an inflamed endothelium, but also regulates the development of monocytes and their survival  in dLNs. Also, CX3CR1+/gfp, and CX3CR1gfp/gfpCD11c+ DCs show comparable expression of phenotype markers related to Ag-presentation CD80, CD86, MHC I, and MHC II), indicating that both CX3CR1-competent and incompetent DCs have the same ability to present Ags. Therefore, CX3CR1 expression on CD11c+ DCs appeared to involve in their migration from inoculation site to dLNs, which subsequently generated effective NK cell activation and Ag-specific T-cell responses. CX3CR1+/gfpCD11c+ DCs were detected at a higher frequency in the corresponding dLNs, compared to CX3CR1gfp/gfpCD11c+ DCs. Enhanced migration of CX3CR1+CD11c+ DCs carrying JEV Ags might increase the binding frequency to cognate CD4+ and CD8+ T cells in dLNs, thereby inducing rapid and robust T-cell responses as well as NK cell activation. The striking evidence supporting the regulatory role of CX3CR1+CD11c+ DCs in JE progression was based on the adoptive transfer of purified CX3CR1+CD11c+ DCs into CX3CR1−/− recipients. CX3CR1−/− mice injected with CX3CR1+CD11c+ DCs displayed resistance to JE with a survival rate comparable to CX3CR1+/+ wild-type mice after peripheral JEV inoculation. Our data revealed that CX3CR1−/− mice injected with CX3CR1+CD11c+ DCs showed rapid expression of JEV RNA in dLNs and the spleen at the early stage after JEV inoculation. Subsequently, these viruses were rapidly cleared from the peripheral lymphoid tissues as shown in CX3CR1+/+ wild-type mice. This finding strongly suggests that CX3CR1+CD11c+ DCs provides resistance to JE via rapid and effective NK cell activation and Ag-specific CD4+/CD8+ T-cell responses with rapid delivery of viral Ag in peripheral lymphoid tissues.To assess the cell type involved in rapid NK cell activation and JEV-specific T-cell response in dLNs of CXrosstalk . DCs locironment . Until nnt  of Chonbuk National University, and were pre-approved by the Ethics Committee for Animal Experiments of Chonbuk National University . The animal research protocol used in this study followed the guidelines set up by the nationally recognized Korea Association for Laboratory Animal Sciences (KALAS). All experimental protocols requiring biosafety were approved by the Institutional Biosafety Committee (IBC) of Chonbuk National University.JC, JK, and SE conceived and designed this research. JC, JK, FH, EU, and SP performed animal study design, analysis, and interpretation. JC and EU performed Ag-specific T-cell responses. BK and KK provided critical discussion for histopathological investigations and key resources. JC, JK, and SE performed data interpretation and wrote the draft of the manuscript. All authors reviewed the manuscript.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.


Phoma species are phytopathogens that are widely distributed in the environment, most commonly found in aquatic systems and soil. Phoma spp. have the potential to be pathogenic in plants, animals and humans; the latter is a rare occurrence. However, as our immunocompromised population increases, so do the reports of these infections. Medical advances have allowed for the increase in solid organ transplantation; chemotherapies to treat malignancies; and the use of other immunosuppressive agents, which have resulted in a greater population at risk when exposed to diverse fungi including Phoma spp. These fungi have been isolated from water sources, food, and crops; thus acting as opportunistic pathogens when the right host is exposed. Phoma spp. contaminates common food sources such as potatoes and maize, a common species isolated being Phoma sorghina. Though there is potential for causing infection via consumption of contaminated foods, there is insufficient data detailing what levels of organism can lead to an infection, and a regulated process for detecting the organism. The spectrum of disease is wide, depending on the host, ranging from cutaneous infections to invasive diseases. Mortality, however, remains low. Phoma is a polyphyletic genus of fungal organisms belonging to the phylum Ascomycota, class Dothideomycetes, order Pleosporales, and family Didymellaceae, as depicted in Phoma spp. was conceptualized in the 19th century by Italian mycologist Pier Andrea Saccardo (1880), and almost 100 years later updates were made to the definition and classification of the genus by Gerhard Boerema and Gerrit Bollen (1975) .,77.34,77n MIC 32 . Everettn MIC 32 . TreatmePhoma spp. remains a taxonomically controversial genus; morphological and molecular data has been generated, but many questions remain. Molecular tools will aid in the identification and classification or re-classification of the Phoma complexities. Infections due to Phoma spp. remain low when compared to other fungi. However, the majority of these cases  were reported in the literature over the last two decades. Phoma spp. are found in water distribution systems, public bathrooms and other public areas such as swimming pools, bodies of water, plants, soil, air and food. This wide distribution allows for high exposure in patients who are both immunocompetent and immunocompromised. Inoculation through trauma is common in the immunocompetent but exposure to the immunocompromised is harder to elucidate and subsequently prevent. Perhaps, new guidelines need to be instituted for filamentous and yeast monitoring of water distribution systems, given our growing immunocompromised population.Penicillum spp. (22/42), Aspergillus spp. and Cladosporium spp. were the most frequent genera isolated [Phoma sorghina has similarly been isolated from similar types of food such as sorghum and bananas [In addition to surveillance of water distribution systems, healthcare providers and individuals who are immunosuppressed should also be aware of, and possibly monitor, specific foods for fungal contaminants. Analogous to water sources, foods and crops similarly pose a risk of developing opportunistic fungal infections in immunocompromised patients as fungal contaminants in food have the potential to be pathogenic. In a 10-year surveillance of fungal contamination of food within a hematological unit, a protocol of food management was analyzed, revealing that filamentous fungi were isolated from 37/456 (8.1%) types of food . Processisolated . Phoma s bananas . The potPhoma spp. versus non-Phoma spp. fungal organisms will lead to timelier identification and possibly improved treatment. Education about the hazards of exposure to Phoma spp. for susceptible populations as well as for medical facilities that treat such patient populations will hopefully lead to decreasing the incidence of filamentous fungal infections in the at-risk populations.As technology advances, a clearer delineation of


Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the death of both upper and lower motor neurons (MNs) in the brain, brainstem and spinal cord. The neurodegenerative mechanisms leading to MN loss in ALS are not fully understood. Importantly, the reasons why MNs are specifically targeted in this disorder are unclear, when the proteins associated genetically or pathologically with ALS are expressed ubiquitously. Furthermore, MNs themselves are not affected equally; specific MNs subpopulations are more susceptible than others in both animal models and human patients. Corticospinal MNs and lower somatic MNs, which innervate voluntary muscles, degenerate more readily than specific subgroups of lower MNs, which remain resistant to degeneration, reflecting the clinical manifestations of ALS. In this review, we discuss the possible factors intrinsic to MNs that render them uniquely susceptible to neurodegeneration in ALS. We also speculate why some MN subpopulations are more vulnerable than others, focusing on both their molecular and physiological properties. Finally, we review the anatomical network and neuronal microenvironment as determinants of MN subtype vulnerability and hence the progression of ALS. Amyotrophic lateral sclerosis (ALS) is a late-onset, progressive and fatal neurodegenerative disease which primarily affects motor neurons (MNs) of the motor cortex of the brain, brainstem motor nuclei and anterior horn of the spinal cord . ALS comNeurons have complex and extended morphologies compared to other cell types, and within the CNS, neurons can vary greatly in their properties. MNs are unique cells amongst neurons because they are large, even by neuronal standards, with very long axons, up to 1 m in length in an adult human. MNs can be distinguished into two main categories according to their location in the CNS: upper MNs (UMNs) located in the cortex, and lower MNs (LMNs) located in the brainstem and spinal cord. The spinal MNs comprise both visceral MNs of the thoracic and sacral regions, which control autonomic functions, and somatic MNs, which regulate the contraction of skeletal muscles and thus control movement. The diversity of MNs reflects the variety of targets they innervate, including a wide range of muscle fiber types. UMNs and LMNs differ in the location of their cell bodies, the neurotransmitters released, their targeting and symptoms resulting from their injury.It is unknown why MNs are specifically targeted in ALS and remarkably, MNs are not equally affected . Whilst ALS shares clinical and pathological features with frontotemporal dementia (FTD), a type of dementia that involves impaired judgment and executive skills. In FTD, the loss of cortical MNs is accompanied by loss of neurons in the frontal and temporal cortices, which correlates clinically with the symptoms of FTD . The relOne of the main pathological characteristics of ALS is the presence of insoluble protein inclusions in the soma of MNs. TAR DNA binding protein-43 (TDP-43) is the major component of these inclusions  in almosWhilst our understanding of the etiology of ALS has increased significantly in recent years, major gaps in our knowledge remain. In this review, we address several unanswered questions regarding the unique susceptibility of specific types of MNs in ALS: Why does neurodegeneration spread throughout specific neural networks? How can ubiquitously expressed genes be selectively toxic to MNs? Why are some MN subtypes more vulnerable to degeneration than others? We also discuss the role of the neuronal network and the specific cellular microenvironment in driving cell-to-cell disease progression, plus the importance of genetics in influencing susceptibility of specific neuronal subpopulations. Finally, we discuss the role of aging as a potential risk factor for the susceptibility of specific MN subtypes. A thorough comprehension of why specific cell types degenerate is imperative to our understanding of ALS because it provides important clues as to what initiates neurodegeneration, and how this knowledge may be harnessed therapeutically.In the CNS, the motor cortex, basal ganglia, cerebellum, and parts of the brainstem, are directly involved in the planning and initiation of movement. In contrast, the precise timing and pattern of movement is generated by MNs located in the spinal cord . The corGlutamate  and acetylcholine  modulate excitatory input within neurons, whereas GABA and glycine facilitate inhibitory neurotransmission . At the However, MNs are not homogeneous throughout the CNS because they exhibit distinct morphologies and patterns of connectivity, which underlie their different physiological functions. Hence, within a single region, MNs that perform closely related functions can be further subdivided, both anatomically and physiologically. The identities of specific MN subtypes and their target projections are controlled by selective cell-type expression of transcription factors, notably members of the Hox, LIM, Nkx6, and ETS families . This prA MN located in the spinal cord innervates up to several hundred fibers within one muscle, which together form the motor unit. Trains of action potentials within the axon cause the release of acetylcholine at the NMJ, which activates nicotinic receptors on the muscle fibers the MN innervates. This initiates a cascade of signaling events in the muscle fiber that leads to its contraction. A motor pool consists of all the individual MNs that innervate a single muscle. A muscle unit (one muscle and its motor pool) is composed of three different types of functional motor units consisting of alpha (α), beta (β), and gamma (γ) MNs, which are classified according to the contractile activity of the muscle fiber innervated. We will now discuss in more detail the anatomy of those structures involved in movement.In the spinal cord, MNs are organized into columns  based onA distinct group of MNs in the sacral spinal cord termed ‘Onuf’s’ neurons, innervate the striated muscles of the external urethra, external anal sphincter via the pudental nerve, and the ischiocavernosus and bulbocavernosus muscles in males . These Mα-motor units can be subdivided according to their contractile properties, into fast-twitch (F) and slow-twitch (S) fatigue-resistant types  Burke e. In addiD, V0V, V1, V2b, Ia and Renshaw cells (belonging to the V1 interneuron subclass), which display specific locations and projections within the spinal cord  innervation to the structures of the head and neck . The morThe motor cortex is the region of the cerebral cortex responsible for mediating voluntary movements. In rodents, the primary cortex (M1) is large and comprises almost all of the frontal cortex , whereasLesions to motor structures in humans and experimental animals lead to impairments in normal movement. In ALS, as MNs degenerate, the ability to control movement of the muscles is progressively lost. Specific MNs in the brain, brainstem and spinal cord are selectively targeted, and pathology appears first in these restricted MN populations. In fact, the name “Amyotrophic Lateral Sclerosis” reflects the strikingly selective degeneration of MNs in ALS. It is derived from a combination of three words; “Lateral” refers to the lateral spinal cord, given that corticospinal MNs are particularly vulnerable to degeneration; “Amyotrophic” is from the Greek “amyotrophia,” meaning lacking muscle nourishment; and “Sclerosis” (fibrosis) refers to gliosis of the crossed corticospinal tract in the dorsolateral quadrant of the spinal cord . In the MRI studies of ALS patients has revealed that very specific neuronal networks are vulnerable to degeneration in ALS . HoweverMost ALS cases occur without a clearly identified cause and are therefore referred to as sporadic ALS (SALS). In contrast, a positive family history is present in ∼10% of all patients   and thesSOD1), an enzyme that detoxifies superoxide radicals  gene are the most common cause of FALS and FTD  , TARDPB encoding TDP-43   (CCNF encoding cyclin F (0.6–3.3% of FALS-FTD) are more frequent than the remaining 20 genes mutated in the much rarer forms of FALS   (Supplemand FTD) , in bothand FTD) . Howeverand FTD) . Healthy , Fused iof FALS) , and CCN of FALS . The phy of FALS . Consist of FALS .TARDBP have now been associated with FALS, located within all but one reside of the C-terminal domain of TDP-43 [http://alsod.iop.kcl.ac.uk/]. Pathological forms of TDP-43 – phosphorylated, fragmented, aggregated, ubiquitinated TDP-43 – were identified as the major component of MN inclusions  and FUS colocalises with TDP-43 in protein aggregates in MNs of a proportion of SALS and FALS patients (TDP-43 is an ubiquitously expressed RNA-binding protein belonging to the heterogeneous nuclear ribonucleoprotein (hnRNP) family. Fifty three mutations in clusions  in almosclusions . TDP-43 midbrain , highligmidbrain . TDP-43 patients .A wide range of cellular pathways have been implicated in ALS pathogenesis, as reviewed recently . These iG93A) has been used most extensively dl1Gur, possesses fewer copies of the transgene; 8 ± 1.5 , develops a slower disease course in comparison, where paralysis begins at 6–8.5 months of age  have been produced, with variable phenotypes (The B6SJL-TgN(SOD1-G93A)1Gur mouse  carries G93A . Whilst G93A , these aG93A . These S 8 ± 1.5 2. This “s of age . In addipy lines  -α -α . SOD1G93ogliosis . This raogliosis .Together these studies suggest the involvement of astrocytes in the selective degeneration of MNs in ALS. Under normal conditions, astrocytes may be able to cope with the expression of low levels of misfolded proteins, but, during cell stress or in the context of MN degeneration, they become more vulnerable, and release factors toxic to MNs, thus producing a vicious cycle. However, the relative resistance of neuronal populations surrounded by reactive astrocytes indicates that the vulnerability of MNs is also determined by cell-autonomous components, such as their genetic background and transcriptional/translational profiles .The two glial cell types responsible for myelination of axons have also been investigated in the context of ALS. Oligodendrocytes myelinate axons in the CNS whereas Schwann cells are responsible for myelination in the peripheral nervous system (PNS). Whilst they perform similar functions, there are also important differences between these two cell types. Schwann cells form a single myelin sheath around one single axon, whereas oligodendrocytes myelinate many different axons. Furthermore, there are differences in the protein composition of CNS and PNS myelin.R521C or FUSP525L mutations , which is protective to MNs (see section “Neuroprotective and Neurotoxic Factor Expression in MN Subpopulations” below)  . This susurvival . Furthersurvival , in contsurvival . Hence, in vivo and in vitro models of FALS suggest that the intrinsic properties of MNs are crucial for degeneration and/or protection  are involved in axon guidance during neural development . Sema3A 93A mice . Nrp1 is93A mice . Further93A mice , but not93A mice , but not93A mice . Similar93A mice . Further93A mice . These d93A mice . EPHA4, n adults . The morn adults .G93A mice  has been recently identified as another determinant of selective neuronal vulnerability in SOD193A mice . MMP-9 w93A mice . Deliver93A mice . Similar neurons . Edaravo neurons . FurtherUNC13A are associated with greater susceptibility to disease and shorter survival , which cause spinocerebellar ataxia type 2 (SCA2), are also increased in ALS patients compared to healthy controls , which generates the Na+/K+ gradients that drive the action potential, are associated with the specific vulnerability of MN subtypes. Misfolded mutant SOD1 forms a complex with the α3 isoform of Na+/K+ATPase, and this leads to impairment in its ATPase activity. Altered levels of this isoform were also observed in spinal cords of SALS and non-SOD1 FALS patients   . This inG93A mice reversed misfolded mutant protein accumulation, delayed pathology and extended survival, whereas reducing MN excitability by antagonist CNQX accelerated disease and induced early denervation, even in the more resistant S-MNs , which mediates the generation of persistent inward currents  in the GluA2 mRNA, and the AMPA receptor is Ca2+-impermeable if it contains the edited GluA2(R) subunit. Conversely, the receptor is Ca2+-permeable if it lacks GluA2 or if it contains the unedited GluA2(Q) subunit. Interestingly, spinal MNs in human ALS patients display less GluR2 Q/R site editing  were detected in vulnerable FF-MNs of the tibialis anterior muscle, compared to resistant MNs of the soleus . This aims to improve the cellular protein folding capacity by inhibiting translation, upregulating ER chaperones – such as immunoglobulin binding protein (BiP) and protein disulfide isomerase (PDI) – and stimulating protein degradation . NumerouLS α-MNs . Hence, in vivo longitudinal analysis of MNs revealed that ER stress influences disease manifestations in SOD1G93A and SOD1G85R mouse models of FALS  is not as well characterized as other cell types. In fact, most studies examining UPR mechanisms have involved non-neuronal cells. Neurons possess extensive ER which is distributed continuously throughout the axonal, dendritic and somatic compartments, implying that neurons make unique demands on the ER compared to other cell types . Hence, +/K+ pump is estimated to account for 20–40% of the brain’s energy consumption  or nitrogen species (RNS) accumulate within cells. This can lead to oxidative modifications and altered functional states of proteins, nucleic acids and lipids. Oxidative stress is linked to neurodegeneration in ALS  and oxidSome neurons exhibit differential vulnerability to oxidative damage. Cerebellar granule and hippocampal CA1 neurons are more sensitive to oxidative stress than cerebral cortical and hippocampal CA3 neurons . Hence, Efficient intracellular trafficking is required to maintain the structure and function of MNs, particularly because MNs have very long axons that connect the soma with distant synaptic sites [reviewed in The existence of mutations in genes encoding cytoskeletal proteins or the cellular transport machinery highlights the involvement of these processes in ALS/FTD. These include tubulin α4A , a majorAxonal transport defects may be an important factor underlying the selective vulnerability of MNs or MN subtypes in ALS/FTD. Abnormal accumulation of phosphorylated neurofilaments, mitochondria and lysosomes in the proximal axon of large MNs and axonal spheroids, are present in SALS and FALS patients . Mutant G93A mice compared to wild type mice although its expression in MNs was equivalent , implying that the normal aging process renders MNs vulnerable to degeneration. However, there is considerable variability in disease progression amongst mutation carriers, even within the same families. Hence, this implies that there is no simple correlation between genetics and disease phenotypes, suggesting that environmental factors and the normal aging process are relevant to understand neuronal vulnerability in ALS/FTD.Aging results in the accumulation of detrimental biological changes over time. The reduction of muscle mass and strength (sarcopenia) is one of the major causes of disability in older persons , which aAging-associated muscle weakness also results from impairment of the activity of MNs contacting skeletal muscles . High reA major component of aging-related muscle weakness is breakdown in communication between the brain and NMJ. This is related to increased neural noise which reduces the accuracy of neural transmission . This caDuring the aging process, a decline in the normal cellular ability to maintain proteostasis is observed and, as a result, damaged proteins accumulate . Thus th2+ storage (2+ in the cortex and hippocampus is greater than that of the striatum and the cerebellum in aged rats (2+, unlike those located in the soma (Mitochondria play a crucial role in neuronal aging. Normal features observed in the aging brain include the accumulation of mutations in mitochondrial DNA, the production of ROS, mitochondrial metabolic abnormalities and altered Ca storage . Remarkaged rats . The celged rats . In addithe soma . Furtherthe soma . These sG93A does not impact on DNA strand integrity, implying that DNA damage is not present in all forms of ALS (The mammalian genome is under constant attack from both endogenous and exogenous sources. This can result in DNA damage, mutations and impaired cellular viability if not repaired correctly . There is of ALS .2+ as well as the more resistant MN types. These properties may confer unique sensitivity to neurodegeneration in ALS. Interestingly, even within spinal MNs, there are distinct differences in vulnerability, because FF-MNs degenerate first, followed by FR-MNs, and the more resistant S-MNs degenerate later. Similarly, these cells also display differences in excitability and ER stress.Motor neurons are unique cells compared to other neurons. They are large cells, with extraordinarily long axons, and very high energetic requirements, which may render them uniquely susceptible to degeneration in ALS. Remarkably, however, not all MNs are equally affected, and there are marked differences in vulnerabilities between MN subtypes, even within the same motor unit. The resistant MNs possess distinct morphological and functional characteristics, as well as different gene expression profiles, compared to the more vulnerable groups . ImportaA hypothetical model is presented in The susceptibility of specific MN groups, however, is further complicated by the heterogeneous nature of ALS, even within the same families, and the different patterns of motor involvement. Stratification of ALS patients into distinct subtypes and investigations into MNs susceptibilities may reveal more insights why specific groups of MNs degenerate first in ALS in the future. However, the blurring of some neurodegenerative disorders, including ALS and FTD, and the presence of C9orf72 mutations in several other neurodegenerative conditions as well as ALS, is another confounding factor. Understanding the fundamental mechanisms dictating MN vulnerability in ALS is central to our understanding of this devastating disorder. Hence, studies in this area may lead to novel therapeutic insights in the future.MV wrote the “Site-Specific Onset and Spread of Neurodegeneration in ALS” section. MJ wrote the “Role of Glial Cells in Driving Disease Progression” section. SS wrote the “Aging” section. AR conceived and prepared the figures, and wrote the “Introduction,” and “Anatomy of the Motor System,” “Genetic Mutations and Risk Factors in ALS,” and “Intrinsic Factors Specific to MN Subpopulations” sections. JA conceived the article, wrote the “Conclusion” section, contributed text in numerous sections, and edited the manuscript throughout for content and style consistency.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.