Dual-specificity phosphatase (DUSP)14  inhibits T-cell receptor (TCR) signaling and T-cell–mediated immune responses by inactivation of the TGF-β activated kinase 1 binding protein (TAB1)–TGF-β activated kinase 1 (TAK1) complex and ERK. DUSP14 phosphatase activity is induced by the E3 ligase TNF receptor associated factor (TRAF)2-mediated Lys63-linked ubiquitination. Here we report an interaction between DUSP14 and protein arginine methyltransferase (PRMT)5 by proximity ligation assay; similarly, DUSP14 directly interacted with TAB1 but not TAK1. DUSP14 is methylated by PRMT5 at arginine 17, 38, and 45 residues. The DUSP14 triple-methylation mutant was impaired in PRMT5-mediated arginine methylation, TRAF2-mediated lysine ubiquitination, and DUSP14 phosphatase activity. Consistently, DUSP14 methylation, TRAF2 binding, and DUSP14 ubiquitination were attenuated by PRMT5 short hairpin RNA knockdown. Furthermore, DUSP14 was inducibly interacted with PRMT5 and was methylated during TCR signaling in T cells. Together, these findings reveal a novel regulatory mechanism of DUSP14 by which PRMT5-mediated arginine methylation may sequentially stimulate TRAF2-mediated DUSP14 ubiquitination and phosphatase activity, leading to inhibition of TCR signaling.—Yang, C.-Y., Chiu, L.-L., Chang, C.-C., Chuang, H.-C., Tan, T.-H. Induction of DUSP14 ubiquitination by PRMT5-mediated arginine methylation. Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the transfer of methyl groups from S-adenosyl methionine to the guanidine nitrogen of arginine residues . Proteinin vitro (in situ proximity ligation assay (PLA) (in this report). DUSP14 is also reported to directly interact with TAK1 in the pull-down assay using immunopurified proteins  remove phosphate groups from phosphoserine/phosphothreonine and/or phosphotyrosine residues from their substrates . The DUS12in vitro . DUSP14 in vitro , 17 and in vitro . If TAB1in vitro  as well proteins ; howeverproteins . Thus, Dproteins . We furtproteins . Here weMyc-DUSP14, Flag-DUSP14, Flag-DUSP14 (C111S) mutant, Flag-TAB1, Flag-TAK1 , Flag-DU181IPDVYEKESRHLMPYWGI198 (corresponding to human DUSP14 protein sequence 181VPDVYEKESRHLMPYWGI198) (Anti-DUSP14 antibody was generated by immunization of rabbits with the murine DUSP14 peptide YWGI198) . Anti–phYWGI198) . Anti–ph2, 25 mM NaF, 1 mM Na3VO4). The cell lysates were incubated with anti-Myc beads for 60 min and then incubated with anti-Flag beads for another 60 min. The Alpha signals from each pair of anti-Flag and anti-Myc beads in proximity (<200 nm)   were detIn Situ Red Starter Kit (MilliporeSigma) according to the manufacturer’s instructions. Briefly, Flag-DUSP14 plus Myc-PRMT5, Myc-DUSP14 plus Flag-TAK1, Myc-DUSP14 plus Flag-TAB1, or Flag-TAK1 plus Myc-TAB1 coexpressing HEK293T cells were incubated with anti-Flag  and anti-Myc (MilliporeSigma) antibodies, followed by rabbit- and mouse-specific secondary antibodies conjugated with oligonucleotides (PLA probes). After ligation and amplification reactions, the PLA signals from each pair of PLA probes in proximity (<40 nm) were visualized as individual red dots by a fluorescence microscope .PLA assays  were perMethylation assays were carried out at 37°C for 1 h in 30 µl of buffer containing 50 mM Tris (pH 7.5), 0.1 mM EDTA, 50 mM NaC1, 0.8 mM S-adenosyl methionine, purified Flag-PRMT5 proteins, and purified Flag-DUSP14 wild-type or mutant proteins. Reactions were stopped by boiling in SDS sample buffer; proteins were resolved by SDS-PAGE and then immunoblotted with specific antibodies.Sample preparation and liquid chromatography–tandem mass spectrometry (MS) analysis were performed as previously described , 23.These DUSP14 experiments were used or performed as previously described . Cell lisem; the mean ± sem values were calculated using SPSS v.19 software .All experiments were repeated at least 3 times. Bar graphs are presented as mean ± Mr of about 70 kDa coimmunoprecipitated with DUSP14; this protein was identified as the protein arginine methyltransferase PRMT5 by MS  in cells with paired antibody-conjugated probes  of DUSP14 were identified to be methylated A and dimin vitro by methylation assays using purified PRMT5 and DUSP14 proteins  was generated in which Arg17, Arg38, and Arg45 residues were altered to lysine residues. Flag-tagged DUSP14 wild-type or mutant (3R→K) was transfected into HEK293T cells. The methylation of immunoprecipitated DUSP14 was detected by immunoblotting using an anti–methyl-arginine antibody, which has been successfully used for detecting the arginine methylation of proteins . The metproteins E. When wproteins E, lane 2in vitro phosphatase assays. HEK293T cells were transfected with Flag-DUSP14 wild-type, C111S mutant, or 3R→K mutant. The cell lysates were immunoprecipitated with anti-Flag antibody. The anti–Flag-DUSP14 immunoprecipitates were subjected to in vitro phosphatase assays using p-nitrophenyl phosphate as the substrate. For DUSP14 mutant (C111S), the Cys-to-Ser substitution resulted in a reduction of phosphatase activity  was immunoprecipitated with anti-Flag antibody from transfected HEK293T cells, followed by immunoblotting with anti–methyl-arginine antibody. We found that DUSP14 wild-type and ubiquitination-defective mutant (K103R) were methylated equally well in cells , we cannWe next studied whether DUSP14 methylation regulates its ubiquitination. HEK293T cells were transfected with HA-ubiquitin plus either Flag-DUSP14 wild-type or methylation-defective mutant (3R→K). Flag-tagged DUSP14 proteins were immunoprecipitated with anti-Flag antibody from the transfected cells and then immunoblotted with anti-HA antibody for detecting ubiquitinated proteins. Wild-type DUSP14 was ubiquitinated, whereas methylation-defective mutant (3R→K) was only slightly ubiquitinated B, lane 527IAQIT31, near the putative DUSP14 methylation sites. Thus, we studied whether PRMT5-mediated DUSP14 methylation regulates TRAF2 binding to DUSP14 using PRMT5 shRNA and coimmunoprecipitation assays. HEK293T cells were cotransfected with Myc-TRAF2, Flag-DUSP14, and either PRMT5 shRNA #2 or empty vector. The data showed that the interaction between DUSP14 and TRAF2 was reduced by PRMT5 shRNA knockdown  did not interact with TRAF2  T cells were transfected with either Flag-DUSP14 wild-type or mutant (3R→K). Stimulation with anti-CD3 antibody induced the interaction between DUSP14 and PRMT5 in transfected Jurkat T cells B, lane 327I-A-Q-I-T31, which is close to 3 putative methylation sites . Our data also showed that double mutations  on the putative TRAF2-binding motif of DUSP14 abolished its TRAF2 binding. Given the above observations, we propose a model (Here we report that DUSP14 was methylated at arginine 17, 38, and 45 residues by the arginine methyltransferase PRMT5. Furthermore, PRMT5-mediated arginine methylation of DUSP14 promotes its ubiquitination, which may lead to DUSP14 activation during TCR signaling. The regulation of DUSP14 ubiquitination by its methylation may be due to enhancement of the interaction between the methylated DUSP14 and the E3 ligase TRAF2. Notably, DUSP14 contains the putative TRAF2-binding motif  a model  that PRM a model , 16. ColArginine methylation can modulate protein-protein interactions by disrupting hydrogen bonding or by increasing hydrophobicity . PRMT1-mProtein methylation also regulates protein stability through inducing Lys48-linked ubiquitination –29. For The activity of PRMT5 is regulated by phosphorylation . The ser


Prediction of antigen-specific HLA-binding peptides generates testable hypotheses for T cell identification.The mechanisms responsible for the generation of LGI1- or CASPR2-antibodies are unknown. Binks  n = 5553) and 111 Caucasian patients with VGKC-complex autoantibodies. In patients with LGI1 antibodies (n = 68), HLA-DRB1*07:01 was strongly represented . In contrast, patients with CASPR2 antibodies (n = 31) showed over-representation of HLA-DRB1*11:01 . Other allelic associations for patients with LGI1 antibodies reflected linkage, and significant haplotypic associations included HLA-DRB1*07:01-DQA1*02:01-DQB1*02:02, by comparison to DRB1*11:01-DQA1*05:01-DQB1*03:01 in CASPR2-antibody patients. Conditional analysis in LGI1-antibody patients resolved further independent class I and II associations. By comparison, patients with both LGI1 and CASPR2 antibodies (n = 3) carried yet another complement of HLA variants, and patients with intracellular VGKC antibodies (n = 9) lacked significant HLA associations. Within LGI1- or CASPR2-antibody patients, HLA associations did not correlate with clinical features. In silico predictions identified unique CASPR2- and LGI1-derived peptides potentially presented by the respective over-represented HLA molecules. These highly significant HLA associations dichotomize the underlying immunology in patients with LGI1 or CASPR2 antibodies, and inform T cell specificities and cellular interactions at disease initiation.The recent biochemical distinction between antibodies against leucine-rich, glioma-inactivated-1 (LGI1), contactin-associated protein-2 (CASPR2) and intracellular epitopes of voltage-gated potassium-channels (VGKCs) demands aetiological explanations. Given established associations between human leucocyte antigen (HLA) alleles and adverse drug reactions, and our clinical observation of frequent adverse drugs reactions in patients with LGI1 antibodies, we compared HLA alleles between healthy controls ( The discovery of autoantibodies against leucine-rich, glioma-inactivated 1 (LGI1), contactin-associated protein 2 (CASPR2)  and, less so, corticosteroids , we hypoin silico to identify peptides that may be presented by these HLA molecules.To extend these early observations, and given the hypothesis that the VGKC complex may be the initiating immunizing agent, we sought to compare and contrast HLA-associations in a sizeable cohort of clinically well-characterized patients with antibodies against LGI1, CASPR2, both LGI1 and CASPR2, and VGKCs, and n = 51) (n = 46) or from the Autoimmune Encephalopathy Clinic, University of California San Francisco (n = 14). These patients had serum antibodies against LGI1 only (n = 68), CASPR2 only (n = 31), both LGI1 and CASPR2 (n = 3) or intracellular aspects of VGKCs (n = 9), as determined by previously described antigen-specific cell-based assays  at two-field resolution . FurtherP-values < 0.05 were considered significant, and are presented. Odds ratios (ORs) were calculated using the median-unbiased estimation method.For each antibody group, Fisher’s exact test (two-tailed) was used to compare the HLA allele and haplotype carrier frequencies between patients and the healthy control dataset. Hochberg’s method was used to correct for multiple comparisons. Corrected The NetMHCIIpan 3.1 server model based on artificial neural networks (P < 0.0001) who had more seizures (P = 0.01) than patients with CASPR2 antibodies, where peripheral nerve features of neuromyotonia (P = 0.0003) and neuropathic pain (P < 0.0001) were preferentially associated. As expected, the nine patients with antibodies to intracellular VGKC epitopes had heterogeneous, often non-immune, clinical syndromes. By contrast, likely non-immune syndromes were noted in only one patient with LGI1 antibodies (stroke) and in four with CASPR2 antibodies .n = 8), psoriasis (n = 7) and pernicious anaemia (n = 2). Moreover, the LGI1-antibody cohort was distinctive for a 47% rate of adverse drug reactions from corticosteroids  and a significantly higher rate of drug-induced rashes . The reported rashes were secondary to AEDs , antibiotics [n = 6: penicillins (n = 5) and metronidazole (n = 1)] and immunosuppressants . Thus, the LGI1- and CASPR2-antibody groups displayed differing clinical autoimmune features suggesting divergent immunogenetic pathways.Of greater relevance to a HLA study, patients with LGI1 or CASPR2 antibodies often had co-existent autoimmune conditions , including Hashimoto’s thyroiditis  were homozygous for DRB1*07:01, compared to 2% (115/5553) healthy controls . Alleles recognized to be part of haplotypes involving HLA-DRB1*07:01 (P = 0.014] and HLA-C*06:02 . After conditioning on the commonest allele, HLA-DRB1*07:01, two other DQ alleles reached statistical significance consistent with evidence of an independent association, HLA-DQA1*01:03  and HLA-DRB1*01:03 .We proceeded to analyse HLA associations, as summarized in P = 5.7 × 10−6]. One CASPR2-antibody patient was homozygous for HLA-DRB1*11:01. Interestingly, the four patients with non-immune conditions and CASPR2 antibodies ; tibodies  did not Intriguingly, of the three patients with co-existent CASPR2 and LGI1 antibodies, only one carried HLA-DRB1*07:01 and none carried HLA-DRB1*11:01. However, all three carried HLA-B*44:02, HLA-C*05:01, HLA-DQA1*03:01 and HLA-DQB1*03:01, a different complement of alleles to the patients with antibodies to either LGI1 or CASPR2 . There wen bloc allelic inheritance and in vivo relevance of HLA combinations that may present LGI1 and CASPR2 antigens, we explored associations involving HLA haplotypes  ; P = 0.02] and DPA1*02:01-DPB1*11:01 . In addition, LGI1-antibody status was associated with a HLA class I haplotype, HLA-C*06:02-B*57:01 . By contrast, only one HLA class II haplotype was associated with CASPR2 antibodies: DRB1*11:01-DQA1*05:01-DQB1*03:01 .Next, to understand the plotypes . We notectively) . This waGiven these significant and distinct allelic and haplotypic HLA associations, for each serologically-defined group, we evaluated their value in explaining sub-phenotypes , long-term outcomes or adverse drug reactions , and fouHLA-DRB1 paralogue HLA-DRB4 was sequenced and observed to be absent or a null allele in 61% (19/31) of the CASPR2-antibody cohort and 44% (4/9) of the intracellular VGKC-antibody cohort, consistent with the healthy control frequencies. However, only 16% (11/68) of the LGI1-antibody cohort showed an absent or null HLA-DRB4 allele, and these 11 patients all carried HLA-DRB1*07:01 (To extend a previous report (B1*07:01 .+ T cells in the pathogenesis of both LGI1- and CASPR2-antibody-associated diseases. To locate potentially high-affinity peptides that complex with HLA class II heterodimers, and may interact with patient T cells, in silico modelling was used and focused on all the class II haplotypes identified above (These robust HLA class II associations strongly implicate CD4ed above .Overall, many peptides from both LGI1 and CASPR2 ranked highly for potential binding to several HLA-DR, HLA-DP and HLA-DQ variants A and C, As expected for the shared core sequences between consecutive 15-mers, many highly ranked peptides were from tightly clustered locations within the full-length protein . Most pein silico data may help identify these interacting T cells.This study is the first comparative HLA analysis of LGI1- and CASPR2-autoantibody mediated diseases, and shows marked and strikingly different HLA associations for these patients, at both allelic and haplotypic levels. Given the frequently overlapping clinical features in patients with LGI1 and CASPR2 antibodies, and their co-expression in VGKC complexes, these findings indicate that dichotomous predisposing HLA variants govern the generation of LGI1 versus CASPR2 antibodies. Furthermore, they strongly implicate T cells in disease initiation and the candidate HLA-binding peptide partners generated by our While HLA-DRB1*07:01 and linked class II alleles, including the haplotype HLA-DRB1*07:01-DQA1*02:01-DQB1*02:02, showed very strong associations with LGI1-antibody patients, this was not observed among CASPR2-antibody patients in whom we found clear associations with HLA-DRB1*11:01 only. Among LGI1-antibody patients, DRB1*11:01 was observed at around healthy control rates, DRB4 was less frequently detected than DRB1*07:01, homozygosity for HLA-DRB1*07:01 was recognized, and other independent associations involved HLA class I alleles HLA-B*57:01 and HLA-C*06:02. Albeit limited by their intrinsic rarity, intriguingly, the three patients with both LGI1 and CASPR2 antibodies had yet another complement of HLA alleles. Perhaps this implicates further divergence in molecular mechanisms responsible for the generation of both autoantibody specificities within an individual. However, the HLA associations do not appear to distinguish between sub-phenotypes, outcomes or, in contrast to a previous observation, the presence of associated tumours (in silico predictions suggest that HLA-DQA1*02:01-DQB1*03:03 is unlikely to mediate presentation of LGI1-derived peptides, whereas the HLA-DQA1*05:01-DQB1*03:01 heterodimer may be implicated in the CASPR2-antibody phenotype. Also, the promiscuity of both CASPR2 and LGI1 peptides for some HLA variants, including HLA-DRB1*07:01 (in silico approach is inherently limited by the possibility that high affinity peptides are more effectively deleted through central tolerance. Nevertheless, taken together, the range of antigen-restricted peptides derived herein, and the relative HLA variant frequencies in disease versus control populations, generate hypothesis-driven approaches to expand disease-specific T cells in vitro and complement recent clinical and laboratory observations which strongly implicate T cell dependence of antibody-mediated diseases (Furthermore, our data also provide several intriguing insights into the immunopathogenesis of these diseases. First, they extend the frequent HLA associations in IgG4-related diseases , but thein silico predictions, offers potential to better understand the likely initiating T–B cell interactions. Further work should focus on the environmental factors that influence the presentation of peptides in genetically predisposed individuals.In summary, the distinct HLA associations in patients with LGI1 and CASPR2 autoantibodies, together with differing clinical features relating to autoimmunity, support an immunological dissociation in generation of these clinically-overlapping autoantibody-mediated syndromes. The dominant class II HLA involvement combined with Supplementary DataClick here for additional data file.


Chimeric antigen receptor (CAR)-based T cell therapy is in early clinical trials to target the neuroectodermal tumor, neuroblastoma. No preclinical or clinical efficacy data are available for retinoblastoma to date. Whereas unilateral intraocular retinoblastoma is cured by enucleation of the eye, infiltration of the optic nerve indicates potential diffuse scattering and tumor spread leading to a major therapeutic challenge. CAR-T cell therapy could improve the currently limited therapeutic strategies for metastasized retinoblastoma by simultaneously killing both primary tumor and metastasizing malignant cells and by reducing chemotherapy-related late effects.+) was immunohistochemically assessed in retrospectively collected primary retinoblastomas. The efficacy of CAR-T cells targeting the CD171 and GD2 tumor-associated antigens was preclinically tested against three antigen-expressing retinoblastoma cell lines. CAR-T cell activation and exhaustion were assessed by cytokine release assays and flow cytometric detection of cell surface markers, and killing ability was assessed in cytotoxic assays. CAR constructs harboring different extracellular spacer lengths (short/long) and intracellular co-stimulatory domains (CD28/4-1BB) were compared to select the most potent constructs.CD171 and GD2 expression was flow cytometrically analyzed in 11 retinoblastoma cell lines. CD171 expression and T cell infiltration (CD3All retinoblastoma cell lines investigated expressed CD171 and GD2. CD171 was expressed in 15/30 primary retinoblastomas. Retinoblastoma cell encounter strongly activated both CD171-specific and GD2-specific CAR-T cells. Targeting either CD171 or GD2 effectively killed all retinoblastoma cell lines examined. Similar activation and killing ability for either target was achieved by all CAR constructs irrespective of the length of the extracellular spacers and the co-stimulatory domain. Cell lines differentially lost tumor antigen expression upon CAR-T cell encounter, with CD171 being completely lost by all tested cell lines and GD2 further down-regulated in cell lines expressing low GD2 levels before CAR-T cell challenge. Alternating the CAR-T cell target in sequential challenges enhanced retinoblastoma cell killing.Both CD171 and GD2 are effective targets on human retinoblastoma cell lines, and CAR-T cell therapy is highly effective against retinoblastoma in vitro. Targeting of two different antigens by sequential CAR-T cell applications enhanced tumor cell killing and preempted tumor antigen loss in preclinical testing.Supplementary information accompanies this paper at 10.1186/s12885-019-6131-1. RB1. × 100 in relation to untreated cells. For sequential treatment, the additive amount of tumor lysis was calculated related to viable tumor cells at day 3.For cytokine release assays, 2 × 10Cell-surface expression of GD2 , CD8  and CD171  was detected by fluorophore-conjugated monoclonal antibodies. EGFRt expression was detected using biotinylated cetuximab (Bristol-Myers Squibb) and a phycoerythrin (PE)-conjugated streptavidin antibody . Activation and exhaustion were assessed by fluorophore-conjugated monoclonal antibodies detecting CD137 , CD25 , PD1 , TIM3  and LAG3 . Flow cytometry was performed on a Fortessa X-20 (BD Biosciences) and data processed using FlowJo software (Tree Star Inc.). Dead cells were excluded from analyses using LIVE/DEAD™ Fixable Green Dead Cell Stain Kit . QuantiBRITE PE calibration beads (BD Biosciences) were used to determine GD2 and CD171 antigen density on retinoblastoma cells according to the manufacturer’s instructions.P values < 0.05 were considered statistically significant.To determine significance of differences in cytotoxic activity and cytokine release of GD2-and CD171-specific CAR-T cells compared with negative control mock T-cells, unpaired Student’s t-test was performed using GraphPad prism (GraphPad Software). + cells) ranged from 16.3 CD3+ cells/cm2 to 160.1 CD3+ cells/cm2. We next assessed CD171 expression in 11 retinoblastoma cell lines by flow cytometry. CD171 was expressed on almost all cells (80–90%) in 6/11 of the retinoblastoma cell lines analyzed, with 3 cell lines moderately expressing CD171 (39–58%) and 2 cell lines exhibiting low-level (< 20%) expression  released the most IFNG and IL2 after encounters with any tested cell line Additional file 2: Supplementary Method Description. Detailed description of immunohistochemically methods including detection of CD171- and CD3-positive cells and quantification of CD3+ cells in primary retinoblastoma tissue sections. (DOCX 20 kb)Additional file 3: Figure S2. Classification of CD171-positive primary retinoblastoma tissue sections in various frequencies. Samples were categorized in < 5%, 5–50%, 51–90 and > 90% CD171-positive samples. (JPG 100 kb)Additional file 4: Figure S3. CAR-transduction efficacy after enrichment. CD8+ bulk cells were lentivirally transduced with CD171- (A) and GD2-specific (B) CAR-constructs, respectively. After enrichment, detection of CAR positive CD8+ cells was performed with fluorochrome-conjugated cetuximab antibody. Untransduced T cells serve as negative control (labeled as mock). (JPG 261 kb)Additional file 5: Figure S4. B-cell lymphoma cell line NALM-6 serves as negative control. A. NALM-6 cells do not express antigens CD171 and GD2 as analyzed by flow cytometry. IFNG and IL2 release of CD171- (B) and GD2-specific CAR-T cells (C) following a 24-h co-culture at a 2:1 E:T ratio with NALM-6 cells compared to RBL15 retinoblastoma cells . D. Cytotoxicity of CD171-and GD2-specific CAR-T cells is displayed in comparison to mock-transduced T cells. Cytotoxicity was measured by a luciferase-based killing assay following a 24 h co-culture at a 2:1 E:T ratio compared to RBL15 cells . Color-coding is the same as used in B. (JPG 128 kb)Additional file 6: Figure S5. CD171- and GD2-specific CAR-T cells show dose-dependent cytotoxicity. Tumor lysis of CD171- and GD2-specific CAR-T cells (CD28 long-spacer) as well as simultaneous treatment with both constructs in comparison to mock-transduced CAR-T cells after co-culture with RBL15. Tumor lysis was measured by a luciferase-based killing assay following a 24 h co-culture at different effector:target ratios . (JPG 44 kb)Additional file 7: Figure S6. CD171-specific CAR-T cell treatment leads to reduction of CD171-positive cells. CD171 expression on WERI-Rb1 and RB355 cells on day 3 of co-culture with CD171- and GD2-specific CAR-T cells compared to untreated cells and treatment with untransduced T cells (mock) as analyzed by flow-cytometry. (JPG 88 kb)Additional file 8: Figure S7. GD2-specific CAR-T cell treatment effects CD171 and GD2 expression on RB355 cells while antigen expression on WERI-Rb1 cells remains unchanged. GD2 expression in WERI-Rb1 and RB355 cells on day 3 of co-culture with CD171- and GD2-specific CAR-T cells compared to untreated cells and treatment with untransduced T cells (mock) as analyzed by flow-cytometry. (JPG 96 kb)Additional file 9: Figure S8. CD171 and GD2 expression profiles on retinoblastoma cell lines. FACS-based data of CD171 and GD2 expression for all analyzed retinoblastoma cell lines. (JPG 79 kb)


Scientific Reports 10.1038/s41598-019-47982-z, published online 08 August 2019Correction to: This Article contains an error in Table 2 where the frequency of hybrid (H1) haplotype detection before FDNPP accident and after (2014–2018) partitioned by Difficult-to-Return zones (20–40 km) is incorrect.The correct value should read 4(14%).


Upon EV71 infection, wild-type (WT) mice displayed more significant body weight loss, higher clinical scores, and lower survival rates as compared with TLR7-/- mice. In the cerebral cortex of EV71-infected mice, neurofilament integrity was disrupted, and inflammatory cell infiltration and neurodegeneration were induced in WT mice, whereas these were largely absent in TLR7-/- mice. Similarly, IL-6 production, Caspase-3 cleavage, and cell apoptosis were significantly higher in EV71-infected WT mice as compared with TLR7-/- mice. Moreover, EV71 preferentially infected and induced IL-6 in astrocytes of mice brain. In U251 cells, EV71-induced IL-6 production and cell apoptosis were suppressed by shRNA-mediated knockdown of TLR7 (shTLR7). Moreover, in the cerebral cortex of EV71-infected mice, the blockade of IL-6 with anti-IL-6 antibody (IL-6-Ab) restored the body weight loss, attenuated clinical scores, improved survival rates, reduced the disruption of neurofilament integrity, decreased cell apoptotic induction, and lowered levels of Caspase-3 cleavage. Similarly, in EV71-infected U251 cells, IL-6-Ab blocked EV71-induced IL-6 production and cell apoptosis in response to viral infection. Collectively, it’s exhibited TLR7 upregulation, IL-6 induction and astrocytic cell apoptosis in EV71-infected human brain. Taken together, we propose that EV71 infects astrocytes of the cerebral cortex in mice and human and triggers TLR7 signaling and IL-6 release, subsequently inducing neural pathogenesis in the brain.As a neurotropic virus, human Enterovirus 71 (EV71) infection causes hand-foot-and-mouth disease (HFMD) and may develop severe neurological disorders in infants. Toll-like receptor 7 (TLR7) acts as an innate immune receptor and is also a death receptor in the central nervous system (CNS). However, the mechanisms underlying the regulation of TLR7-mediated brain pathogenesis upon EV71 infection remain largely elusive. Here we reveal a novel mechanism by which EV71 infects astrocytes in the brain and induces neural pathogenesis  via TLR7 and interleukin-6 (IL-6). Upon EV71 infection, TLR7-/- mice displayed less body weight loss, lower clinical score, and higher survival rate as compared with wild-type (WT) mice. Meanwhile, a severer histopathologic neurofilaments disruption, neurodegeneration and cell apoptosis were observed in brain of EV71-infected WT mice. IL-6 release, cell apoptosis, and Caspase-3 cleavage were attenuated by shRNA targeting TLR7 (shTLR7) in EV71-infected U251 cells. Moreover, anti-IL-6 antibody (IL-6-Ab) suppressed EV71-induced body weight loss, clinical score increase, and survival rate decrease as well as neurofilaments disruption and neurodegeneration in mice, and it also attenuated EV71-induced cell apoptosis and Caspase-3 cleavage in U251 cells. It’s retrospectively observed that TLR7 upregulation, IL-6 induction and astrocytic cell apoptosis in EV71-infected human brain. Therefore, TLR7 is required for neural pathogenesis by IL-6 induction upon EV71 infection.Enterovirus 71 (EV71) infection causes aseptic meningitis, poliomyelitis-like paralysis and fatal encephalitis in infants. Besides an immune receptor, toll-like receptor 7 (TLR7) serves as a death receptor in central nervous system (CNS). However, the role of TLR7 in EV71-induced neural pathogenesis remains ambiguous. This study reveals a distinct mechanism by which EV71 induces neurodegeneration  Enterovirus 71 (EV71) is an RNA virus that causes hand-foot-mouth disease (HFMD), commonly causing mild symptom in infants, but in some cases also leading to severe diseases such as aseptic meningitis (AM), brain stem encephalitis (BSE), acute flaccid paralysis (AFP), and even fatal encephalitis in neonates . As a neToll-like receptor 7 (TLR7) is widely expressed in immune cells, intestinal cells, lung cells, and neural cells. As a pattern-recognition receptor (PRR), TLR7 can recognize viral RNA, oligomeric RNA, short interfering RNA (siRNA), and microRNA (miRNA) from pathogenic sources to activate intracellular signaling pathways and release appropriate cytokines –12. EmerIn infectious contexts, TLR7 plays multiple roles relevant to neurological disorders and inflammatory responses in the CNS. When simian immunodeficiency virus (SIV) infects the brains of rhesus macaques, the secretion of microRNA21 activates the TLR7 pathway, leading to neurological disease . Japanes-/-) mice. Compared with EV71-infected WT mice, there was an obvious alleviation of neurological injury-related symptoms, histopathologic damage, and cell apoptosis, as well as lower brain IL-6 levels in EV71-infected TLR7-/- mice. Further investigations revealed that intracranial IL-6 neutralization significantly relieved neurological injury-related symptoms and neural cell apoptosis in the brains of EV71-infected WT mice. Moreover, we demonstrated that EV71 preferentially infected and induced IL-6 production in murine brain astrocytes, and we further verified that TLR7 silencing and IL-6 blockade attenuated EV71-infected cell apoptosis in vitro. Finally, we retrospectively found TLR7 upregulation, IL-6 induction and astrocytic cell apoptosis in EV71-infected human brain. Thus, we identified a novel mechanism underlying TLR7-mediated neural pathogenesis involved in IL-6 induction in astrocytes of the brain upon EV71 infection.In this study, we conducted experimental intracranial infections with EV71 in neonatal mice, and found a non-significant viral load in the cerebral cortex of both wild-type (WT) and TLR7 knockout  and Japanese encephalitis virus (JEV) is generally associated with neural cell apoptosis ,22. We fin vivo utilizing a TLR7 knock-out (TLR7-/-) mouse model. Initially, the genetic characteristics of the TLR7 gene in brain tissues were determined. By assessing the TLR7 gene  staining indicated that TLR7 protein was present in the cerebral cortex of WT mice, whereas it was not produced in the cerebral cortex of TLR7-/- mice  was barely detected in the cerebral cortex of both mock-infected WT and TLR7-/- mice , left; w-/- mice , right. -/- mice . These d-/- mice , left; hsections , right, -/- mice with or without EV71 infection  and blood circulation , the IL-Moreover, immunohistochemical staining for neurofilaments revealed that in mock-infected mice, the integrity of neurofilaments was intact in the cerebral cortex of both IgG-treated and IL-6-Ab-treated mice, whereas upon EV71 infection, the integrity of neurofilaments was disrupted and neurofilament levels were reduced with the progressive viral replication in the cerebral cortex of IgG-treated mice relative to those treated with IL-6-Ab . These rin vivo.Next, the effects of IL-6 on EV71-induced cell apoptosis were assessed. TUNNEL staining revealed that in mock-infected mice, cell apoptosis was barely detectable in the cerebral cortex of IgG-treated and IL-6-Ab treated mice , left. H-/- mice based on neural markers. Immunohistochemical analysis revealed that there were no significant differences in numbers and distributions of astrocytes (GFAP-positive), neurons (NeuN-positive), or microglia (IBA-1 positive) between cerebral cortexes or hippocampal areas of WT and TLR7-/- mice  was highly expressed in the cytoplasm of U251 cells . In addition, we found that an appropriate concentration of IL-6 (ranged from 20 to 500 pg/ml) activated Caspase-3 cleavage in U251 cells  or anti-IL-6 antibody (IL-6-Ab) . ELISA red cells . Notablyed cells . CollectEV71 is neurotropic that has been demonstrated in human and primate pathological specimens, and the viral distribution is distinct and stereotyped . To extrTaken together, we propose that the neurotropic EV71 preferentially infects and replicates in astrocytes of the murine and human cerebral cortex. EV71 RNA is recognized by TLR7 to trigger the signaling events and to promote IL-6 production and release, which results in the induction of cell apoptosis and in the development of neuroinflammation and brain injury .Toll-like receptors (TLRs) act as innate immune receptors to recognize conserved pathogen-associated molecules in certain immune cell types. Various TLRs are also expressed in neurons, astrocytes, and in microglial cells, and they are conducive to the immunological responses of the CNS –34. Emer-/- mice. Compared with WT mice, TLR7-/- mice were protected from EV71-induced weight loss, hind-limb paralysis, and lethality. Further histological analyses revealed a reduction in EV71-induced neurodegeneration in TLR7-/- mice, suggesting TLR7 was essential for EV71 neurovirulence in mice. Unexpectedly, further findings revealed EV71 replication and tissue distribution were unaffected in the cerebral cortex of WT and TLR7-/- mice. This suggested that TLR7 failed to act as an antiviral immune receptor in these tissues, instead of serving as a death receptor in the context of murine brain neuropathogenesis upon EV71 infection. Thus, we uncovered an additional biological function of TLR7 in the context of EV71-induced neurological lesion-related symptoms in the brains of mice. TLR3, TLR7, and TLR9 are known to be important host factors involved in restricting EV71 infection in diverse infection models in vivo and in vitro [We have previously shown that TLR7 orchestrates immune and inflammatory responses in macrophages during EV71 infection . Given tin vitro , 39, 40.in vitro . In this-/- mice. Our observations revealed that the induction of apoptosis upon EV71-infection was significantly reduced in TLR7-/- mice. The coordinated neuroinflammatory response may be an important factor in neural injury [in vivo.In the CNS, the TLR7-mediated apoptosis pathway leads to cell death under either physiological (eg. neurodegeneration) or pathological (eg. neurotropic virus infection) conditions ,12,42. Tl injury . In TLR7l injury . Besidesl injury , 44, we Neuroinflammation has been described in various neural cells of the brain, including astrocytes, neurons, and microglia, which contribute to disease development and progression . As TLR7in vivo, and therefore the neurotoxic reactive astrocytes are also induced by activated microglia or damaged neurons in neurodegenerative diseases [in vivo brain environment [Astrocytes  are a class of neural cells of ectodermal and neuroepithelial origin that maintain homeostasis and provide for the defense of the CNS . They ardiseases , 50. In ironment , which aViral infections of the CNS can manifest in various forms of inflammation to influence the CNS homeostasis, including tick-borne encephalitis virus (TBEV), Zika virus (ZIKV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) , 52, 53,-/- mice were originally obtained from The Jackson Laboratory . The mice were housed under specific pathogen-free (SPF) conditions in individually ventilated cages. Three-day-old suckling mice were intracranially injected with 10 μl PBS containing 1×109 plaque-forming units (PFU)/ml of EV71 with a 50-μl gastight microsyringe . Following EV71 infection, mouse weights and clinical scores were recorded every day until one-week post-treatment. The clinical scores were defined as follows: 0, healthy; 1, ruffled hair and hunched back; 2, limb weakness; 3, paralysis in one limb; 4, paralysis in both limbs; and 5, death.C57BL/6 WT mice were purchased from Shanghai Laboratory Animal Center. The TLR7in vivo, the suckling mice were intracranially injected with 10 μl PBS containing R837  at a dose of 50 μg/per mouse.For intracranial IL-6 neutralization, neonatal WT mice were intracranially injected with 10 μl PBS or EV71 per mouse. Eight hours later, the mice were divided into groups and separately intracranially injected with IgG isotype or anti-IL-6 at a dose of 8 μg/per mouse, and this injection was repeated the following day. For R837 administration fluid (CSF), there-day-old WT mice were sacrificed and removed the entire spine of the mouse, 10 μl of CSF was aspirated with microsyringe, and then diluted in 1ml PBS. Finally, 200 μl diluted CSF was subjected to ELISA assay to detect IL-6 protein expression using mouse IL-6 ELISA kit , or RNA extraction to measure EV71 RNA copies.To collect cerebrospinal All animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . All procedures involving mice and experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the College of Life Sciences, Wuhan University (Permit numbers: 2017–016).Brain tissue samples from six deceased patients (three were confirmed to be EV71-infected and the other three were uninfected as the negative controls) were obtained from the School of Forensic Medicine, Kunming Medical University. The tissues were fixed in formalin, and embedded in paraffin. The infected group included two males and one female, and the average age was 27.3 months. The average age of control group was 16 months .2 incubator.Human astroglioma U251 cells were purchased from American Type Culture Collection (ATCC) . Human embryonal rhabdomyosarcoma (RD) cells were obtained from the China Center for Type Culture Collection (CCTCC) . All cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) , supplemented with 10% fetal bovine serum , 100 U/ml penicillin, and 100 mg/ml streptomycin sulfate at 37°C in a 5% CO6) were cultured in 6-plate well in 2 ml medium, and then 2 μg plasmids transcribing shRNA were transiently transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.For cell transfection, U251 cells (2×10The neurotropic Enterovirus 71 (EV71) strain (Xiangyang-Hubei-09) was originated from the brain tissue of a one-year-old dead EV71-infected infant in Xiangyang city, Hubei province in China, and isolated previously in our laboratory (GenBank accession no. JN230523.1). The virus stock was propagated in RD cells, and virus titration and inoculation with EV71 were performed as described previously ,55. The In vitro, U251 cells were infected with the virus at the indicated multiplicities of infection (MOIs) and unbound virus was washed away 2 h later, after which samples were incubated at 37°C for an additional 24 or 48 h.Aliquots were stored at -80°C prior to usage. Mouse anti-TLR7  was purchased from Novus Biologicals . Mouse anti-Neurofilament-L (DA2), as well as rabbit antibodies against Hexaribonucleotide binding protein-3 (NeuN) , Glial fibrillary acidic protein (GFAP) , Caspase-3 , Cleaved Caspase-3 (Asp175) , and Poly(ADP-ribose) polymerase (PARP)  were purchased from Cell Signaling Technology . Rabbit anti-Ionized calcium binding adaptor molecule 1 (IBA-1) was purchased from Abcam . Mouse anti-EV71 VP1 was purchased from Abnova Company . Mouse anti-dsRNA monoclonal antibody J2 was obtained from Scicons (Hungary). Rabbit anti-TLR8 (abs102571) was obtained from Absin Bioscience Inc. . Rabbit anti-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from ProteinTech Group . Mouse anti-β-actin was purchased from Santa Cruz Biotechnology . Rat antibodies against mouse IL-6  or isotype IgG1  were purchased from BioXcell . A mouse monoclonal antibody against human/primate IL-6  and isotype IgG1  was purchased from R&D systems . Recombinant human IL-6 protein  was purchased from Pepro Tech Inc. . Cell apoptosis inducer, etoposide , and TLR8 agonist, VTX-2337  were purchased from MedchemExpress .The shTLR7 and shGFP plasmids expressing a short-hairpin RNA (shRNA) targeting TLR7 or Green fluorescent protein (GFP) as a negative control were generated by inserting the appropriate DNA fragments into the pSilencer 2.1-U6 neo vector , respectively, as described previously . The spe2O2 and 10 mM citrate buffer (pH6) was used for antigen retrieval. Nonspecific peroxidase activity was blocked for 30 min with 5% bovine serum albumin (BSA). The slides were then incubated with the primary antibody overnight at 4°C and then washed with PBS for 10 min. The biotinylated secondary antibody was initially applied for 30 min, after which an avidin biotin complex kit  was used for an additional 30 min. 3,3’-diaminobenzidine tetrahydrochloride hydrate (DAB) with 5% H2O2 was used for detection. Slides were then counterstained with hematoxylin and eosin (H&E) to stain nuclei. Immunohistochemistry was conducted at each site using specific antibodies, and samples were visualized by microscopy .Tissue samples were mounted on slides from paraffin blocks (5-μm sections), deparaffinized three times in xylene for 5 min, and hydrated in a methanol gradient . 3% HFor immunohistochemistry analysis, the IHC reaction was assessed using light microscopy following scoring criteria: 0, no or faint staining intensity; 1+, faint cytoplasmic staining; 2+, moderate and incomplete membranous staining; and 3+, strong membranous staining. The intensity of immunohistochemistry or immunofluorescence staining was generated using Image J software from three representative images.ΔΔCt method. Primer sequences are listed in RNA was extracted from homogenized mouse brain tissues or cultured cells using the TRIzol reagent . Quantitative real-time PCR (qPCR) analyses were performed using the Roche LightCycler 480  and SYBR RT-PCR kits (Roche) according to the manufacturer's instructions. The data represent absolute mRNA copy numbers normalized to GAPDH used as a reference gene. Relative fold expression values were determined by using the Radio-immunoprecipitation assay (RIPA) buffer containing protease inhibitors was used in order to lyse and extract protein from murine brain homogenates and cultured cells. Supernatant protein concentrations were then determined using a BCA assay kit, and protein lysates (100 μg) were resolved by SDS-PAGE and transferred onto nitrocellulose (NC) membranes . Nonspecific binding was blocked with 5% nonfat dried milk  for 1 h at room temperature. After three washes in PBS, the NC membranes were incubated with appropriate primary and secondary antibodies. Blots were then analyzed using a Luminescent Image Analyzer . The relative target protein expression to internal control is quantified using Image J software.6 per well) were seeded in 6-well culture plates with 10% FBS, and were transfected with shGFP or shTLR7 plasmids. At 24 h post-transfection, cells were infected with EV71 at a MOI = 0.5 for 2 h, and were then washed with PBS to remove the unbound virus. Then, cells were cultured in 2 ml of FBS-free DMEM for an additional 12 h. Finally, the cell supernatants were harvested after centrifugation at 10,000 g for 5 min for ELISAs. Supernatant cytokine levels were assessed using human IL-1β immunoassay , human IL-6 immunoassay kits , and human IL-8 ELISA kit  following the manufacturer’s provided instructions.U251 cells (1×105 per well) were seeded in 24-well plates after treatment. A cell viability assay was then performed using the Cell Counting Kit 8  according to the manufacturer's instructions. Cell injury was determined by measuring the lactate dehydrogenase (LDH) activity in FBS-free culture medium at 30 min after restoration using an LDH detection kit  according to the manufacturer’s instructions. Briefly, medium was harvested from a 24-well plate and centrifuged at 5000 rpm for 10 min. This cell-free supernatant was then incubated with the provided reaction mixture, and LDH activity was determined based on absorbance at 450 nm.Cells . Survival curves were plotted using the Kaplan-Meier method, and significant differences in survival were calculated via the log-rank test. Data are means ± the standard deviation (SD), and statistical significance was evaluated using the following P values: P <0.05 (*), P <0.01 (**) or P <0.001 (***).All experiments were reproducible, and each set of experiments was repeated at least three times with similar results. Statistical significance for comparison of two means was assessed via unpaired Student’s S1 FigA and B) The EV71 virus RNA copies in cerebral cortex (A) or cerebellum (B) were determined by absolute quantitative PCR. Data are shown as mean ± SD. ***, P < 0.001. (C and D) The cerebral cortex sections of mice on day 3 post-infection from different groups were fixed and subjected to immunostaining with cl-Caspase-3 (Red), dsRNA (Green), and DAPI (Blue) (C). The presentative images were acquired using fluorescence microscopy. Bar = 20 μm. The relative expression of cl-Caspase-3 and dsRNA was quantified using Image J software (D). Data are shown as mean ± SD. (E and F) The cerebral cortex sections of mice on day 5 post-infection from different groups were immunostained with cl-Caspase-3 (Red), dsRNA (Green), and DAPI (Blue) (E). The presentative images were acquired using fluorescence microscopy. Bar = 20 μm. The relative expression of cl-Caspase-3 and dsRNA was quantified using Image J software (F). Data are shown as mean ± SD.There-day-old WT mice were intracranially injected with 10 μl PBS, EV71-UV, EV71- Heated or EV71 per mouse  and sacrificed on day 1, 3 or 5 post-infection, respectively. ((TIF)Click here for additional data file.S2 FigA and B) The cerebellum sections of mice on day 3 post-infection from different groups were fixed and subjected to immunostaining with cl-Caspase-3 (Red), dsRNA (Green), and DAPI (Blue) (A). The presentative images were acquired using fluorescence microscopy. Bar = 20 μm. The relative expression of cl-Caspase-3 and dsRNA was quantified using Image J software (B). Data are shown as mean ± SD. (C and D) The cerebellum sections of mice on day 5 post-infection from different groups were immunostained with cl-Caspase-3 (Red), dsRNA (Green), and DAPI (Blue) (C). The presentative images were acquired using fluorescence microscopy. Bar = 20 μm. The relative expression of cl-Caspase-3 and dsRNA was quantified using Image J software (D). Data are shown as mean ± SD.There-day-old WT mice were intracranially injected with 10 μl PBS, EV71-UV, EV71- Heated or EV71 per mouse  and sacrificed on day 1, 3 or 5 post-infection, respectively. ((TIF)Click here for additional data file.S3 FigA and B) WT mice and TLR7-/- mice mock-infected or EV71-infected were sacrificed on 2, 3, 5, and 7 days post-infection . The mice cerebral cortex sections (A) and cerebellum sections (B) were fixed and subjected to IHC staining with EV71 VP1 antibody (Brown), respectively. The presentative images were acquired using light microscopy. Bar = 100 μm. EV71 VP1 relative expression was shown as VP1 positive index and quantified with Image J software. Data are shown as mean ± SD. ns, non-significant.((TIF)Click here for additional data file.S4 FigA and B) The proteins were extracted from individual mice cerebral cortex (A) or cerebellum (B) tissues and then the IL-6 protein level in tissues (per gram) was determined by ELISA assay. (C and D) IL-6 secretion in cerebrospinal fluid (CSF) (C) and peripheral blood (D) were determined by ELISA assay. (E-H) EV71 RNA was extracted from mice cerebral cortex (E), cerebellum (F), CSF (G) and peripheral blood (H). EV71 viral RNA copies were determined by absolute quantitative PCR. Data are shown as mean ± SD. ns, non-significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.Neonatal WT mice were intracranially injected with 10 μl PBS or EV71 per mouse, and separately intracranially treated with IgG isotype or anti-IL-6 antibody. The different sections of mice on day 1 in different groups were subjected to IL-6 protein and EV71 load detection. ((TIF)Click here for additional data file.S5 FigA) The presentative images of cerebral cortex sections were acquired using fluorescence microscopy. Bar = 20 μm. (B) The relative expression of IL-6 and EV71 VP1 in cerebral cortex was quantified using Image J software. (C) The presentative images of cerebellum sections were acquired using fluorescence microscopy. Bar = 20 μm. (D) The relative expression of IL-6 and EV71 VP1 in cerebellum was quantified using Image J software. Data are shown as mean ± SD. ns, non-significant; *, P < 0.05.Neonatal WT mice were intracranially injected with PBS or EV71 per mouse, and separately intracranially treated with IgG isotype or anti-IL-6 antibody. The cerebral cortex and cerebellum sections of mice on day 1 in different groups were immunostained with IL-6 (Red), EV71 VP1 (Green), and DAPI (Blue). ((TIF)Click here for additional data file.S6 FigA) The presentative images of spinal cord sections were acquired using fluorescence microscopy. Bar = 20 μm. (B) The relative expression of IL-6 and EV71 VP1 in spinal cord was quantified using Image J software. (C) The presentative images of skeletal muscle sections were acquired using fluorescence microscopy. Bar = 20 μm. (D) The relative expression of IL-6 and EV71 VP1 in skeletal muscle was quantified using Image J software. Data are shown as mean ± SD. ns, non-significant; *, P < 0.05.Neonatal WT mice were intracranially injected with PBS or EV71 per mouse, and separately intracranially treated with IgG isotype or anti-IL-6 antibody. The spinal cord and skeletal muscle sections of mice on day 1 in different groups were immunostained with IL-6 (Red), EV71 VP1 (Green), and DAPI (Blue). ((TIF)Click here for additional data file.S7 Fig-/- mice were intracranially injected with PBS or EV71. After 3 days EV71 incubation, brain sections from mice were subjected to immunostaining. (A and B) The cerebral cortex sections were immunostained with DAPI (Blue), IBA-1 (Green) and IL-6 (Red). Bar = 20 μm. Cells were observed using fluorescence confocal microscopy (A) and the IL-6 expression in IBA-1-positive (IBA-1+) cells was calculated (B). (C and D) The cerebral cortex sections were immunostained with DAPI (Blue), NeuN (Green) and IL-6 (Red). Bar = 20 μm. Cells were observed using fluorescence confocal microscopy (C) and the IL-6 expression in NeuN-positive (NeuN+) cells was calculated (D). Graphs show mean ± SD. ns, non-significant; *, P < 0.05.Neonatal WT and TLR7(TIF)Click here for additional data file.S8 FigA) Immunostaining of the brain’s cortex from day 3 or 5 post-R837 administration was probed with IL-6 (Red), GFAP (Green) and stained with DAPI (Blue). The presentative images were acquired using fluorescence microscopy. Bar = 20 μm. (B) The mice cerebral cortex sections were subjected to cl-Caspase-3 (Red), GFAP (Green) and DAPI (Blue) staining. The presentative images were captured using fluorescence microscopy. Bar = 20 μm. (C) The IL-6 protein level in CSF from mice was detected by ELISA. Graphs show mean ± SD. *, P < 0.05; **, P < 0.01.There-day-old WT mice were intracranially injected with 10 μl PBS or 10 μl PBS containing 50 μg R837 per mouse, and then sacrificed on Day 1, 3 or 5 post-R837 administration. ((TIF)Click here for additional data file.S9 FigA and B) U251 cells (2×106) were seeded on a 6-well plate, then mock-infected or infected with EV71 (MOI = 0.5) for different periods. The total RNA was extracted from cells and EV71 RNA level was determined by qPCR. The GAPDH mRNA is used as an internal control (A). The total protein was extracted from cells. EV71 VP1 and 3C expression were detected by Western blotting analysis (B). (C) U251 cells (2×106) were transfected with 2 μg plasmid transcribing siRNA specific to TLR7 (shTLR7) or its control (shGFP) and then mock-infected or infected with EV71 (MOI = 0.5) for 12 or 24 h. IL-8 secretion in supernatants of the cell cultures was analyzed by ELISA. Graphs show mean ± SD. ns, non-significant.((TIF)Click here for additional data file.S10 FigA) WT or TLR7-/- mice cerebral cortex sections were stained with TLR8 (Red), GFAP (Green), and DAPI (Blue). The presentative images were captured using fluorescence microscopy. Bar = 20 μm. (B and C) U251 cells (2×106) were transfected with siRNA (50 nM) target to TLR8 (siRNA-TLR8) or its control (siRNA-NC) for 24 h, and then treated with EV71 (MOI = 0.5) or R837 (10 μM) for another 24 h, respectively. The cell lysates were harvested for Western blotting to examine the expression level of TLR8, TLR7, and GAPDH (B). Cell viability and LDH release of treated cells were examined using CCK8 and LDH assay, respectively (C). (D and E) U251 cells were treated with different concentrations of VXT-2337 (TLR8 agonist)  for 24 h. The protein was extracted and then detected by Western blotting with targeted antibodies (D). Cell viability and LDH release of treated cells were examined using CCK8 and LDH assay (E).((TIF)Click here for additional data file.S11 FigA and B) U251 cells were treated with different concentrations of R837  for 24 h. Cell viability (A) and LDH release (B) of treated cells were examined using CCK8 and LDH assay. (C and D) U251 cells were treated with R837 (10 μM) or EV71 (MOI = 0.5) for 24 h. The supernatants of treated cells were collected and IL-6 protein level was measured by ELISA (C). The protein was extracted and then detected by Western blotting with targeted antibodies (D). (E and F) U251 cells were seeded on 20-mm cover slips and treated with different concentrations of human IL-6 protein  or Etoposide (150 μM) for 24 h, and then probed with cl-Caspase-3 (Green) and DAPI (Blue) (E). Cells were observed using fluorescence confocal microscopy. Bar = 20 μm. The relative cl-Caspase-3 expression was calculated (F). Graphs show mean ± SD. *, P < 0.05; **, P < 0.01.((TIF)Click here for additional data file.S12 FigA) The hematoxylin-eosin (H&E) staining of cerebellum sections from human brain tissues . The presentative images were acquired using light microscopy. Bar = 100 μm. (B–D) Cerebellum sections from EV71-infected or uninfected humans were subjected to IHC staining with anti-EV71 VP1 antibody (B), anti-dsRNA antibody (C) or anti-TLR7 antibody (D). The presentative images were acquired using light microscopy. Bar = 100 μm. The relative expression of indicated was shown as a positive index and quantified with Image J software. (E and F) Human cerebellum tissue sections were fixed and stained with GFAP (Green) and IL-6 (Red) (E) or cl-Caspase-3 (Red) (F). The presentative images were acquired using fluorescence microscopy. Bar = 100 μm. The relative expression of IL-6 or cl-Caspase-3 in GFAP positive (GFAP+) cells was calculated with Image J software. Data are shown as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.((TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


It remains largely unknown whether gene-editing of endogenous genes can genetically empower HSC to endure the culture stress and preserve stemness. In this study, we revealed that both NUP98-HOXA10HD fusion and endogenous Nras mutation modifications (NrasG12D) promoted the engraftment competitiveness of HSC. Furthermore, the synergy of these two genetic modifications endowed HSC with super competitiveness in vivo. Strikingly, single NAV-HSC successfully maintained its stemness and showed robust multi-lineage engraftments after undergoing the in vitro culture. Mechanistically, NUP98-HOXA10HD fusion and NrasG12D mutation distinctly altered multiple pathways involving the cell cycle, cell division, and DNA replication, and distinctly regulated stemness-related genes including Hoxa9, Prdm16, Hoxb4, Trim27, and Smarcc1 in the context of HSC. Thus, we develop a super-sensitive transgenic model reporting the existence of HSC at the single cell level on culture condition, which could be beneficial for protocol screening of bona fide HSC regeneration from pluripotent stem cells in vitro.Natural hematopoietic stem cells (HSC) are susceptible and tend to lose stemness, differentiate, or die on culture condition in vitro, which adds technical challenge for maintaining  Single cell transplantation of fresh wild type hematopoietic stem cells (HSC) can successfully repopulate multi-lineage hematopoiesis in myeloid-ablated mice ,2,3. HowNatural HSC tend to lose their stemness, differentiate to lineage offspring, or die on the condition of in vitro culture stress. Although there have been many reports that HSC proliferated in vitro in the presence of certain cytokines, chemical compounds, and feeder cells ,5,6,7,8,Hoxb4 has been reported to be the key element in HSC stemness and was used to mediate pluripotent stem cell differentiation toward HSC [ward HSC ,10. Sincward HSC . Moreoveward HSC . Howeverbona fide HSC regeneration from pluripotent stem cells in vitro.In this study, we compared the effects of the NrasG12D mutation and the NUP98-HOXA10HD fusion gene on the engraftment competitiveness of HSC and their combinative role in preserving the stemness of HSC after in vitro culture stress. Despite that both the NUP98-HOX10HD fusion gene and the NrasG12D mutation enhance the competitiveness of HSC engraftment, they employ distinct signaling mechanisms and the synergy of these two factors result in super competitiveness in vivo in modified HSC (NAV-HSC). The single NAV-HSC preserved their stemness after a 10-day feeder-free culture in vitro and showed robust multi-lineage engraftments in vivo upon transplantation. Thus, we developed a super-sensitive model reporting the existence of HSC at the single cell resolution, which is beneficial for protocol screening of +). A Western blot using antibodies and recognizing the 3xFlag confirmed the expression of NA10hd-3xFlag protein in the bone marrow nucleated cells of NA10hd mice  treated mice (CD45.1) together with an equal number of bone marrow competitor cells from littermate control (NA10hdLSL/+) mice (CD45.2) (p < 0.001)  A. These (CD45.2) B startin< 0.001) C,D. Thes5 NrasG12D-expressing total bone marrow cells (NV) together with an equal number of NA10hd cell counterparts into lethal irratidation (2 × 5.0 Gy) treated mice (CD45.1). Donor-cell chimerism was analyzed every four weeks post-transplantation. NV cells dominated the peripheral blood from week 4 and contributed significantly higher (>95%) than NA10hd cells (<5%) in the peripheral blood of recipients 20 weeks post-transplantation (NA10hd-HSC (<10%) dominantly occupied the HSC compartment in bone marrow of recipients 20 weeks post-transplantation (p < 0.001)  B. Thus, 5 bone marrow nuleated cells from NAV mice, together with an equal number of bone marrow competiter cells from NV mice, into lethally irratidated (2 × 5.0 Gy) recipients (CD45.1). The conribution of NAV-derived cells in peripheral blood was stable and higher than NV and exceeded 60% post transplantation (p < 0.001)  mice, which simultaneously express NUP98-HOXA10HD and NrasG12D proteins in the circulatory system. We subsequently transplanted 2.5 × 10antation C. In bon< 0.001) D. NV-der−CD48−c-Kit+Sca1+CD135−CD150+ ). There were only 59 overlapping DEGs in NA10hd and NV 5−CD150+  HSC from5−CD150+  and 998 5−CD150+  when comd and NV A. GO enrd and NV B,C. Onlyd and NV D. Howeveiescence   and cell division cycle (Cdc) family genes  were upregulated in NV HSC but down-regulated in NA10hd HSC  and Origin Recognition Complex (ORC) family genes   analysis showed that NA10hd and NV regulated the cell cycle, MCM_Pathway, and Apoptosis_by_CDKN1A_Via_TP53 signaling pathways in an opposite manner A. Furthe10hd HSC B. At the4, Orc6) C had the) ). To evaluate the stemness preservation, colony-forming unit (CFU) and transplantation assay were performed using the single cell-derivatives after a 10-day culture . Of the transplanted nine colonies, five were homogenous and four were heterogeneous. Cells from six individual wells, including five homogenous colonies and one heterogeneous colony, successfully reconstituted multi-lineage hematopoiesis over 16 weeks after transplantation A,B. To fLSL/+ mice were generated by targeting a mouse ES line  through homologous recombination at the ROSA26 locus. NALSL/+ mice were subsequently bred with Vav-Cre mice to generate NA10hdLSL/+ and Vav-Cre (NA10hd) compound mice. Mice expressing the conditional oncogenic NrasG12D mutation  were crossed with Vav-Cre mice to generate LSL NrasG12D/+ and the Vav-Cre compound (NV) mice. Mice were housed in the SPF grade animal facility of the Guangzhou Institution of Biomedicine and Health, Chinese Academy of Science . All animal experiments were approved by the Institutional Animal Care and Use Committee of Guangzhou Institutes of Biomedicine and Health (IACUC-GIBH).C57BL/6 (CD45.1) and Vav-Cre strain (CD45.2) mice were purchased from the Jackson Laboratory. C57BL/6 (CD45.2) were from the Beijing Vital River Laboratory Animal Technology Ltd. NA10hdAll the competitive transplantation of total bone marrow were performed at the ratio of 1:1 (0.25 million to 0.25 million). The recipients (CD45.1) of all competitive transplantation were lethal irradiated (2 × 5.0 Gy). The donor-derived chimeric ratios of peripheral blood cells were analyzed regularly and the HSC compartment was analyzed 20 weeks post-transplantation. Donor-derived cells from NA10hd and NAV were identified with Tdtomato.Antibodies for the hematopoietic lineage and hematopoietic progenitors or stem cells analysis: CD45.2 (104), CD2 (RM2-5), CD3e (145-2C11), CD4 (RM4-5), CD8a (53-6.7), Ter119 (TER-119), Mac1 (M1/70), B220 (6B2), Gr1 (RB6-8C5), CD19 (1D3/CD19), CD90.2 (53-2.1), CD48 (HM48-1), IL-7R (A7R34), Sca1 (E13-161.7), c-Kit (2B8), CD34 (RAM34), and CD16/32 (93) antibodies were purchased from eBiosciences, and CD150 (TC15-12F12.2) was purchased from Biolegend. DAPI (#D9542-50MG) was purchased from Sigma.® 780, CD34-Alex700, and CD16/32-PE-Cy7. For lymphocyte progenitors staining, BM cells were stained with the antibodies mixture of Lin -FITC, IL-7R-APC, Sca1-PerCP-Cy5.5, c-Kit-APC-eFluor® 780. For hematopoietic stem cell (HSC) and multipotent progenitor (MPP) staining, BM cells were stained with the antibodies mixture of Lin -FITC, CD48-FITC, Sca1-PerCP-Cyanine5.5, c-Kit-APC-eFluor® 780, CD45.2-PE (only in NV and CD45.1 competitive transplantation recipients), and CD150-PE-Cy7. Lastly, all the cells were resuspended in DAPI solution.All the nucleated cells harvested from moue tissues were first treated with ACK solution, and then blocked with the CD16/32 antibody. For lineages staining, blocked nucleated cells were incubated with antibodies mixture of CD19-FITC, Mac1-PE-Cy7, and Thy1.2-APC. For myeloid progenitors staining, bone marrow cells were stained with antibodies mixtures of Lin -FITC, Sca1-PerCP-Cy5.5, c-Kit-APC-eFluorThe stained cells were analyzed on LSR Fortessa (BD Bioscience), and the data were analyzed using Flowjo software (FlowJo).Individual NAV or wild-type (WT) HSC was sorted into a 96-well low adhesion plate containing 150 µL culture medium  and 100 ng/mL mSCF + 20 ng/mL human IL11 + β-mercaptoethanol (2000 fold diluted)). A half-medium change was performed every other day. Colonies with more than 100 cells in each well were divided into equal aliquots. One aliquot of around 50 cells were plated for CFU-C colony-forming assay following the manufacturer’s instructions (STEMCELL Technologies) and the other aliquot of 50 cells were transplanted into sub-lethally irradiated (6.5 Gy) recipients (CD45.1) after a 10-day in-vitro culture.NA10hd, NV, and littermate control (Ctrl) HSC were sorted separately into 200 µL DPBS-BSA buffer (0.5% BSA) using a 1.5 mL Eppendorf tube. The cDNA of sorted 1000-cell aliquots were generated and amplified, as described previously . Then qup < 0.05, ** p < 0.01, *** p < 0.001).The data were represented as mean ± SD. Two-tailed independent Student’s t-tests were performed for a comparison of two groups of data . P values of less than 0.05 were considered statistically significant (* The data that support the findings of this study are available from the corresponding author upon request. All RNA-Seq data are in the GEO database with accession code GSE118809.Pbx1 improves the competitiveness of HoxB4-overexpressing HSC and that the capacity of NUP98-HoxA10HD to induce HSC expansion does not require a PBX-interacting motif [Hoxa10 in the fusion improved the competitiveness of hematopoietic stem cells. This competitive advantage may be due to dominance of NA10hd HSC in the HSC compartment. Moreover, recipient mice with NA10hd HSC transplantation did not develop myeloproliferative disease until the end of the experiment. This is consistent with a previous study, which shows restriction of NUP98-fusion to the homeodomain of Hoxa10 blunts lineage bias and leukemogenic potential of Nup98-Hoxa10 [NA10hd transgenic mice had normal multi-lineage hematopoiesis, demonstrated by normal lineage and progenitor contribution indistinguishable from control mice. A previous study reported that knockdown of ng motif . ConsistFlt3l, which is one of these signature genes, has been reported to regulate multipotent stem and progenitor cells and enhance their proliferation [It was previously reported that NrasG12D could sustainably increase the competitiveness of HSC ,15. It iferation .The co-expression of NA10hd and Nras in hematopoietic stem cells conferred the strongest engraftment advantage in vivo than NV or NA10hd alone. In addition, a transplantation assay of single NAV HSC-derived cells after 10-day feeder-free in vitro culture showed that the NAV HSC derivatives could efficiently reconstitute the whole blood lineages in all the recipients. These results suggested that NA10hd and NrasG12D gene could synergistically enhance the competitiveness and preserve stemness of hematopoietic stem cells. Since NA10hd and NV HSC employs mostly distinct signaling pathways yet share a similar trend for a group of genes to confer competitiveness, how NA10hd and NV collaborate to eventually confer a competitive advantage in HSC deserves further investigation. In addition, it would be of great interest to investigate whether the NAV modification could be introduced into pluripotent stem cells to capture bona fide HSC regenerated from pluripotent stem cells.In conclusion, HSC with NrasG12D and NUP98-HOXA10HD gene-modifications possess a super engraftment advantage and preserved their multi-lineage potential after in vitro culture. Thus, we established a super-sensitive readout system reporting the existence of HSC on the culture condition at single cell resolution, which could be beneficial for screening induced HSC from pluripotent stem cells.


Therefore, we hypothesized that inhibition of Notch signaling would promote LPC-to-hepatocyte differentiation by repressing sox9b expression in zebrafish. We ablated the hepatocytes of Tg(fabp10a:CFP-NTR) larvae and blocked Notch activation during liver regeneration through treatment with γ-secretase inhibitor LY411575 and demonstrated enhanced induction of Hnf4a in LPCs. Alternatively, enhancing Notch signaling via Notch3 intracellular domain (N3ICD) overexpression impaired Hnf4a induction. Hepatocyte ablation in sox9b heterozygous mutant embryos enhanced Hnf4a induction, while BEC-specific Sox9b overexpression impaired LPC-to-hepatocyte differentiation. Our results establish the Notch-Sox9b signaling axis as inhibitory to LPC-to-hepatocyte differentiation in a well-established in vivo LPC-driven liver regeneration model.Liver regeneration after most forms of injury is mediated through the proliferation of hepatocytes. However, when hepatocyte proliferation is impaired, such as during chronic liver disease, liver progenitor cells (LPCs) arising from the biliary epithelial cell (BEC) compartment can give rise to hepatocytes to mediate hepatic repair. Promotion of LPC-to-hepatocyte differentiation in patients with chronic liver disease could serve as a potentially new therapeutic option, but first requires the identification of the molecular mechanisms driving this process. Notch signaling has been identified as an important signaling pathway promoting the BEC fate during development and has also been implicated in regulating LPC differentiation during regeneration. SRY-related HMG box transcription factor 9 (Sox9) is a direct target of Notch signaling in the liver, and Sox9 has also been shown to promote the BEC fate during development. We have recently shown in a zebrafish model of LPC-driven liver regeneration that inhibition of Hdac1 activity through MS-275 treatment enhances  The liver is the only human internal organ capable of regeneration, and after most forms of acute injury, this regeneration is mediated by the proliferation of the differentiated epithelial cells of the liver, namely, hepatocytes or biliary epithelial cells (BECs). However, during prolonged chronic liver injury, this innate regenerative capacity may be exhausted, leading to progression to end-stage liver disease, cirrhosis, and liver failure . A commoγ-secretase. The NICD translocates to the nucleus, where it interacts with the DNA-binding protein recombination signal-binding protein immunoglobulin kappa J (Rbpj) to mediate target gene transcription [in vitro [in vitro [in vivo inhibition of Notch signaling impaired BEC proliferation after bile duct ligation [The Notch signaling pathway is an important pathway during liver development, where its activation in fetal hepatoblasts promotes biliary differentiation and bile duct morphogenesis –16. Thercription . In addiin vitro , 19. Simin vitro –20, whilligation  and inhiligation . Work inligation , while hligation . TogetheSox9 in mice results in delayed biliary development [sox9b mutants exhibit improper differentiation of progenitors into the hepatic or pancreatic fates and also display cholestasis from impaired intrahepatic duct formation during early liver development [in vivo [During liver development, SRY-related HMG box transcription factor 9 (Sox9) is considered to be the earliest marker of commitment to the biliary fate. Liver-specific deletion of elopment , while zelopment . Sox9 iselopment  and ornielopment  in humanelopment , 29 and elopment  in rodenelopment , 30, alt[in vivo , and CD2[in vivo . These dSox9 expression in the livers of newborn pups [Sox9 promoter [sox9b mutant zebrafish embryos, it was shown that Sox9b was required for the maintenance, but not the initiation, of Notch signaling in intrahepatic biliary cells during liver development [in vivo zebrafish model of extreme hepatocyte ablation such that liver regeneration is mediated by LPC-to-hepatocyte differentiation [sox9b expression [in vitro data suggest a role for Notch signaling in LPC differentiation, we hypothesized that inhibition of Notch signaling would promote LPC-to-hepatocyte differentiation through the repression of Sox9 expression. Here, we utilized LY411575, a small molecule inhibitor of γ-secretase which blocks Notch activation, in our zebrafish hepatocyte ablation model to show that inhibition of Notch signaling promotes Hnf4a induction in LPCs, while overexpression of NICD impairs Hnf4a induction. We further show that deletion of one allele of sox9b in zebrafish augments Hnf4a induction in LPCs, while BEC-specific overexpression of Sox9b impairs LPC-to-hepatocyte differentiation. Finally, we show that inhibition of Notch signaling is sufficient to rescue the impaired LPC-to-hepatocyte differentiation in larvae with inhibition of Hdac1 activity, but does not rescue the defects in BET inhibitor or DMH1-treated larvae. Our work clearly establishes the importance of the Notch-Sox9 signaling axis during in vivo LPC differentiation.Sox9 has also been shown to be a direct target of Notch signaling in the liver. When NICD was overexpressed during mouse liver development, there was dramatic upregulation in orn pups . Additiopromoter . In sox9elopment . Despitentiation . This montiation  and selentiation , and we pression . Given tTg(fabp10a:CFP-NTR) line, which expresses nitroreductase (NTR) under the hepatocyte-specific fabp10a promoter (ref). When these larvae are exposed to nontoxic metronidazole (MTZ) for 36 hours (A36h) from 3.5 to 5 days postfertilization (dpf), NTR expression in hepatocytes converts MTZ into a cytotoxic compound, leading to near-total hepatocyte ablation. We also used the Tg(Tp1:H2B-mCherry) line to mark BECs, as the mCherry expression is driven by the Tp1 promoter containing Notch-responsive elements [γ-secretase inhibitor LY411575 for up to 22 hours, starting from 20 hours into MTZ ablation (A20h) until 6 hours after MTZ washout (R6h) (Tp1:H2B-mCherry+/Hnf4a+ cells in LY411575-treated livers compared to dimethyl sulfoxide- (DMSO-) treated controls . The total number of Tp1:H2B-mCherry+ cells were comparable between DMSO- and LY411575-treated regenerating livers at A33h  . After 1 Figures . Hnf4a i at A33h , suggestcontrols . TogetheTg(hs:N3ICD) line, where the inducible expression of N3ICD is under the control of the heat shock protein 70l promoter [Tp1:H2B-mCherry+ cells, but the number of Tp1:H2B-mCherry+/Hnf4a+ cells was dramatically reduced in N3ICD-expressing larvae ; Tg(Tp1:CreERT2); Tg(ubb:loxP-CFP-loxP-sox9b-2A-mCherry) larvae, which upon administration of 4-hydroxytamoxifen (4-OHT) overexpresses Sox9b specifically in BECs [fabp10a:CFP-NTR transgene) in livers at the R24h time point, at which LPC-derived hepatocytes are evident [+ cells between CFP+/mCherry− and CFP+/mCherry+ populations revealed significantly fewer Bhmt+ cells in the CFP+/mCherry+ population ;Tg(Tp1:H2B-mCherry) larvae with a various combination of compounds and assessed the extent of LPC-to-hepatocyte differentiation at the R24h time point  [s939Tg(EPV.TP1-Mmu.Hbb:hist2h2l-mCherry) [co17Tg(hsp70l:canotch3-EGFP) [s959Tg [jh4 37and Tg(ubb:loxP-eCFP-loxP-sox9b-2A-mCherry) (referred to here as Tg(fabp10a:CFP-NTR), Tg(Tp1:H2B-mCherry), Tg(hs:N3ICD), Tg(Tp1:CreERT2), and Tg(ubb:loxP-CFP-loxP-sox9b-2A-mCherry), respectively).Experiments were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) at the University of Pittsburgh. Embryos and adult fish were raised and maintained under standard laboratory conditions. We used the fsB)s931 , Tg(EPV.rry)s939 , Tg(hsp7GFP)co17 , Tg(EPV.rry)s959 , and Tg(Tg(fabp10a:CFP-NTR) larvae with 10 mM MTZ in egg water supplemented with 0.2% DMSO and 0.2 mM 1-phenyl-2-thiourea (PTU).Hepatocyte ablation was performed by treating sox9b genotyping, genomic DNA was amplified with either wild-type allele- (5′-AGACCAGTCGTAGCCCTT-3′) or mutant allele-specific (5′-AGACCAGTCGTAGCCCTA-3′) reverse primer and a common forward primer (5′-TGAGTGTGTCCGGAGCTCCGA-3′).For μM was used for its final concentration. For JQ1 , 3 μM was used for final concentration as previously described [μM was used as previously reported [For MS-275  treatment, 25 escribed . For LY4reported .Whole-mount immunostaining was performed as previously described , using tt-test was performed, with P < 0.05 considered significant. For analyses concerning more than two groups, a one-way ANOVA test was performed, with P < 0.05 considered significant. Quantitative data were shown as means ± SEM.A Zeiss LSM700 confocal microscope was used to obtain image data. Confocal stacks were analyzed using the Zen 2009 software. All figures, labels, arrows, scale bars, and outlines were assembled or drawn using the Adobe Illustrator software. For analyses concerning only two groups, a two-tailed Student's Tg(hs:N3ICD) larvae were heat-shocked by transferring them into egg water prewarmed to 37°C and kept at this temperature for 20 minutes as previously described [escribed .Tp1:CreERT2 transgene were crossed to Tg(ubb:loxP-CFP-loxP-sox9b-2A-mCherry) fish. Larvae from the crosses were treated with MTZ from 3.5 to 5 dpf and additionally treated with both 5 μM 4-OHT from 2 to 5 dpf for 3 days. At 6 dpf, 24 hours after 4-OHT and MTZ washout, the larvae were harvested and processed for immunostaining to reveal mCherry+ cells that express Sox9b, as previously described [Fish carrying the escribed .eef1a1l1 was used for normalization as previously described [Total RNA was extracted from 50 dissected livers (pooled) using the RNeasy Mini Kit ; cDNA was synthesized from the RNA using the SuperScript® III First-Strand Synthesis SuperMix  according to the kit protocols. qPCR was performed as previously described , using tescribed . Techniceef1a1l1 forward (5′-CTGGAGGCCAGCTCAAACAT-3′), eef1a1l1 reverse (5′-ATCAAGAAGAGTAGTACCGCTAGCATTAC-3′), fabp10a forward (5′-GCAGGTTTACGCTCAGGAGA-3′), fabp10a reverse (5′-TCCTGATCATGGTGGTTCCT-3′); bhmt forward (5′-CTGATCGCTGAGTACTTTG-3′), bhmt reverse (5′-CAATGAAGCCCTGGCAGC-3′), sox9b forward (5′-CTGATCGCTGAGTACTTTG-3′); sox9b reverse (5′-CACACCGGCAGATCTGTTT-3′), tfa forward (5′-ACTACGCTGTGGCTGTTGTG-3′); tfa reverse (5′-AATCCTTTGCCCAGTCCTTT-3′), her9 forward (5′-AATGCCAGCGAGCATAGAAAGTC-3′), and her9 reverse (5′-TGCCCAAGGCTCTCGTTGATTC-3′).


Caenorhabditis elegans by asking how neuron morphological aging is arrested in the long-lived, alternate organismal state, the dauer diapause. We find that a hormone signaling pathway, the abnormal DAuer Formation (DAF) 12 nuclear hormone receptor (NHR) pathway, functions cell-intrinsically in the dauer diapause to arrest neuron morphological aging, and that same pathway can be cell-autonomously manipulated during normal organismal aging to delay neuron morphological aging. This delayed aging is mediated by suppressing constitutive endocytosis, which alters the subcellular localization of the actin regulator T cell lymphoma Invasion And Metastasis 1 (TIAM-1), thereby decreasing age-dependent neurite growth. Intriguingly, we show that suppressed endocytosis appears to be a general feature of cells in diapause, suggestive that this may be a mechanism to halt the growth and other age-related programs supported by most endosome recycling.Neurons have a lifespan that parallels that of the organism and are largely irreplaceable. Their unusually long lifespan predisposes neurons to neurodegenerative disease. We sought to identify physiological mechanisms that delay neuron aging in  Neurons have a lifespan that parallels that of the organism and are largely irreplaceable. Analysis of the nematode “dauer” diapause reveals that state-dependent hormone signaling halts constitutive endocytosis, altering the localization of an actin regulator, thereby suspending dendrite morphogenesis and aging. Neurons are largely irreplaceable and must therefore survive and function for the lifespan of the organism. In mammals, aging is associated with cognitive decline, structural alterations in neurons such as sprouting, and synaptic deterioration . FurtherThe endomembrane system is the central mechanism by which metazoan cells interact with their organismal environment. The endosomal pathways of this system promote constant sampling of the environment—evocatively, mouse embryonic fibroblasts recycle their entire cell surface in under 15 minutes . EndosomIn neurons, endosomes likewise regulate plasma membrane proteins to modulate cellular responses to physiological changes , and theOn top of these cell-intrinsic and local regulatory mechanisms, the morphology and function of the nervous system is dramatically impacted by endocrine signaling. Sex-specific hormones organize distinctive male or female connectivity during development, govern animal behavior, and alter long-term and acute neuronal activity at the cellular level . EndocriC. elegans uses endocrine signaling to choose between 2 states: in favorable environmental conditions, animals undergo “reproductive development” (RD), in which they grow continuously to adulthood through 4 larval stages (L1–L4) and have a lifespan of about 2 weeks; in response to adverse environmental conditions, animals instead enter the dauer diapause—an ageless, stress-resistant alternative to the L3 larval stage—in which they can survive for months . We. We29]. C. elegans [din-1 functions to instruct PVD to adopt the dauer morphology rather than to limit dendrite growth per se, the din-1(o) mutant exhibits a wild-type PVD dendrite morphology in RD  versus dauer-like (without 4°s) morphology. The transcriptional corepressor daf-12 interacting protein (DIN-1)/split ends (SPEN) regulates organismal dauer entry but has not previously linked to neurodevelopment in  elegans . We foungy in RD . Genes idin-1 specifically in the PVD neuron rescues the din-1 mutant dauer dendrite growth arrest  null mutants cannot enter dauer and exhibit a shortened adult lifespan in RD, the previously identified gain-of-function allele daf-12(r273) promotes constitutive dauer entry and extends adult lifespan in RD [To determine whether a cell-autonomous dauer decision could also be sufficient to induce dendrite growth arrest, we turned to the DAF-12 NHR, which binds DIN-1 to promote dauer entry ,33,34. Aan in RD –38 daf-12(o)an in RD . Interesan in RD .daf-12(o) mutants exhibit a PVD dendrite morphology in RD indistinguishable from that of wild type, the daf-12(−ligand) mutant grown in RD shows a reduction in 4° branches reminiscent of the dauer dendrite morphology (daf-12(−ligand) cDNA in a wild-type genetic background is sufficient to induce a dauer-like dendrite morphology (daf-12(−ligand) in muscles does not alter PVD dendrites, nor does overexpression of wild-type daf-12 in PVD  dendrite morphogenesis defect in RD is completely suppressed in dauer  mutants have a severely stunted PVD dendritic arbor with almost no 2° or 4° branches, and TIAM-1::GFP exhibits a vesicular localization in the PVD dendrite [How does suppressing general endosome production lead to arrested dendrite development and aging? Our previous work showed that PVD dendrite morphogenesis is mainly driven by the actin cytoskeleton through the activation of regulators such as WASP-family verprolin homologous protein 1 (WAVE) Regulatory Complex and TIAM-1 . TIAM-1 dendrite ,52  versus wild-type worms at the RD developmental stage in which 2° and 3° PVD branches are forming. Both neurite outgrowth frequency and growth speed in the tiam-1(o) mutant are indistinguishable from that of wild type, whereas neurite retraction is increased in the tiam-1(o) mutant, resulting in fewer 3° dendrite growths  mutant, again to a dauer-like morphology with wild-type 2°s but few 4°s (tiam-1(o) mutant, these data indicate that diffusely localized TIAM-1 promotes dendrite stabilization but inhibits 4° branch growth via the TIAM-1(dhph) domain.Next, to understand the function of vesicular versus diffusely localized TIAM-1, we performed structure-function analyses. We divided TIAM-1 into the C-terminal portion, “TIAM-1(dhph),” which contains Rac GEF Dbl homology (DH) and pleckstrin homology (PH) domains, and the N-terminal portion, “TIAM-1(N-term),” which is required for membrane localization and numerous protein interactions and is auto-inhibitory for the GEF activity in vitro  Fig 4H)Fig 4H).  few 4°s . Further few 4°s . It is t few 4°s  (S3 Movi few 4°s . Togethetiam-1(o) mutant (In contrast to TIAM-1(dhph), the endosome-localized TIAM-1(N-term) has no effect on dendrite morphology in the wild-type background . Interes) mutant , indicat) mutant . In daueHere, we have elucidated a hormone-regulated mechanism that can cell-autonomously delay neuronal morphological development and aging. In addition, we propose a surprising modification to the current model for how neurite outgrowth is effected . In summary of the first point, cells in the slowed-aging state broadly suppress endosome production, though SV cycling is uniquely preserved. In the PVD dendrite, endosomes localize the actin regulator TIAM-1 to permit dendrite growth in RD or stall it in dauer . Future daf-12(−ligand) would lead to suppressed endosome generation, and we were unable to inhibit PVD dendritic endosomes through manipulation of known endocytosis effectors  mutant [C. elegans to not only halt the growth and other age-related programs supported by most endosome recycling but also conserve energy while maintaining neurotransmission, and thus behaviors, during dauer diapause.Animals in dauer diapause do not eat, so many of the cellular changes associated with dauer diapause, such as decreased transcription, altered metabolism, and increased autophagy, are thought to be energy-conservation mechanisms . Broadly) mutant . Still, ) mutant , 55. We tiam-1 in vivo has no effect on PVD morphology [In neuron morphogenesis and remodeling, it is generally thought that the mechanisms producing the actin-mediated filopodial and lamellipodial protrusions as well as those stabilizing the growth cone are both forces that promote growth cone extension . By examrphology ,52. How C. elegans strains were cultured on Escherichia coli OP50 as described by Brenner [daf-2(e1370) genetic background, which grows though RD at 16°C and into dauer at 25°C. Dauers were grown from eggs at 25°C for 5 days, and they were compared against RD worms of the same strain grown at 16°C to the L4 stage. In all transgenes expressing daf-12 variants, daf-12 was translationally tagged with gfpnovo2, allowing verification of expression and nuclear localization of the construct. Several strains were provided by the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The following strains were used in this study: TV15921 wyIs594, TV20384 daf-2(e1370); wyIs594, TV22406 din-1(dh149); daf-2(e1370); wyIs594, TV23318 din-1(dh149); daf-2(e1370); wyIs594; wyEx9453, TV22125 wyIs594; daf-12(rh61rh411), TV23204 wyIs594; daf-12(rh273), TV22536 wyIs594; wyEx9238, TV22537 wyIs594; wyEx9239, TV22833 wyIs840(ser2prom3::daf-12a(rh273)::gfpnovo2, Pmyo-2::gfp); wyIs592(ser2prom3::myr-gfp), TV22534 wyIs594; wyEx9236(Phlh-1::daf-12a(rh273)::gfpnovo2, Pmyo-2::gfp, TV11461 wyEx4684, TV24585 wyIs1178(Prgef-1::daf-12a(rh273)::gfpnovo2, ser2prom3::myr-gfp); wyEx4684, TV23226 wyIs594; daf-12(rh61rh411); wyEx9413(ser2prom3::EnR::daf-12(DNA-binding-domain)::gfpnovo2, Punc-122::rfp), TV23225 wyIs594; daf-12(rh61rh411); wyEx9412(ser2prom3::VP64::daf-12(DNA-binding-domain)::gfpnovo2, Punc-122::rfp), TV21537 wyIs50025, TV23485 wyIs840; wyIs50025, NK1351 qyIs296), TV23069 wyIs840; qyIs296, TV24940 wyIs235006(ser2prom3::gfp::tomm-20)), TV23354 wyIs840; wyIs235006, MAH242 sqIs24, TV23205 wyIs840; sqIs24, TV23488 daf-2(e1370); wyEx7701, TV23337 wyIs840; wyEx7701, TV17379 qyIs369; wyEx7137, TV24096 wyIs840; wyEx9514, TV12922 wyEx5216, TV23481 wyIs840; wyEx5216, TV17207 wyIs581, wyEx7069, TV23567 wyIs856, TV23258 wyIs840; wyIs856, TV23698 smg-1(r861); wyEx9576::mcd8::tagrfp, Pmyo-2::rfp), TV24551 rab-10(ok1494); daf-2(e1370); wyIs594, TV24540 daf-2(e1370); wyEx9853, TV24432 daf-2(e1370), pwIs206, daf-2(e1370); arIs37(Pmyo-3::sp-gfp); wyIs93, TV24912 daf-2(31370); wyEx9934, TV24514 dma-1(tm5159); wyEx1240::superfoldGFP, Podr-1::rfp), TV21000 daf-2(e1370); wyIs740, TV22165 wyIs1139, TV17428 tiam-1(tm1556); wyIs594, TV24791 wyIs592; wyEx9908(ser2prom3::tiam-1(dhph), Podr-1::gfp, TV24877 wyIs592; wyEx9929(ser2prom3::myr-tiam-1(dhph)::tagrfp, Podr-1::gfp), TV24979 tiam-1(tm1556); wyIs592; wyEx9929, TV24880 wyIs592; wyEx9930(ser2prom3::myr-tiam-1::dhph)::tagrfp::sp12, Podr-1::gfp), TV20605 dyn-1(wy1150); wyIs740, EG9408 sng-1(ox706), TV24431 daf-2(e1370); pwIs72(Pvha-6::gfp::rab-5). Brenner . Data fotiam-1 truncation constructs were as follows: pCER255(ser2prom3::myr-tiam-1(dhph)(aa519-889)::tagrfp); pCER258(ser2prom3::tiam-1N-term(aa1-540)::tagrfp); pCER261(ser2prom3::tiam-1(dhph)(aa519-889); and pCER269(ser2prom3::myr-tiam-1(dhph)(aa519-889)::tagrfp::sp12).Expression vectors were made in the pSM vector, a derivative of pPD49.26, or pPD117.01  using standard techniques. Transgenes expressed from extrachromosomal or integrated arrays were generated using standard gonad transformation by injection . tiam-1 Visual inspections of fluorescence were performed using a Zeiss Axioplan 2 microscope with a 63×/1.4NA objective and Chroma HQ filter sets for GFP, YFP, and RFP. Animals were immobilized in 10 mM levamisole  in M9 buffer. Images were acquired with either a Zeiss LSM710 confocal microscope using a Plan Apochromat 63×/1.4 objective or an inverted Zeiss Azio Observer Z1 spinning disk confocal microscope with a 63×/1.4NA or 40× objective attached to a QuantEM:512SC camera. Images were analyzed in ImageJ. Light artifacts due to scatter from the spinning disk were removed from images of PVD morphology with ImageJ. Puncta were quantified from images using either “find maxima” Figs  and 4B oSynchronized L4 worms with GFP-labeled neurons (wyIs592) were prepared for cell isolation as previously described . Cells wSequencing samples were initially analyzed by FastQC to assess sequencing quality and sequencing bias. TrimGalore (0.5.0), a tool that combines Cutadapt and FastQC, was used to remove low-quality base calls and the Illumina adapter from the 3′ end of reads. The reads for each sample were mapped to the reference genome (ce10) using TopHat (version 2.1.1), and expression levels and statistical significance of observed changes were analyzed using the complete Cufflinks pipeline (version 2.2.1). Lists of differentially expressed genes were analyzed for GO term enrichment using DAVID (version 6.8).S1 Data(XLSX)Click here for additional data file.S1 Figdaf-2/insulin like-receptor, daf-3/SMAD, daf-7/TGFβ, and daf-16/FOXO. The daf-3 and daf-16 mutants are resistant to entering dauer, like the din-1 mutant, so either the daf-2 or the daf-7 mutation was added to their genetic background to enable dauer entry. Data for wild type are the same as that shown in Shown are loss-of-function mutants in (TIF)Click here for additional data file.S2 Figdaf-12(o) animals expressing the DAF-12 DBD attached to a trans-repressive (EnR) (panel A) or trans-activating (VP64) (panel B) domain driven from a PVD-specific promoter. (C) The transcriptionally repressive DAF-12 chimera cell-autonomously reduces dendrite growth in RD. ***P < 0.001, one-way ANOVA with Tukey post test. DBD, DNA-binding domain.(A–B) (TIF)Click here for additional data file.S3 FigPVD>daf-12(−ligand)) PVD. (A) Representative image of endoplasmic reticulum morphology in a wild-type day 1 adult: it fills the primary dendrite and a (seemingly arbitrary) subset of secondary and tertiary branches [P < 0.001, two-tailed t test. 1. Liu X, Guo X, Niu L, Li X, Sun F, Hu J et al. Atlastin-1 regulates morphology and function of endoplasmic reticulum in dendrites. Nat Commun. 2019;10: 568.Endoplasmic reticulum morphology (A–C), Golgi morphology (D), mitochondria (E–G), and autophagy (H) all appear similar in wild type versus dauerized ((TIF)Click here for additional data file.S4 FigP < 0.01, two-tailed t test. (F) Strategy for constructing the DMA-1 guidance receptor endosome reporter. DMA-1 is fused to RFP on the cytosolic side and the GCN4 peptide epitope on the extracellular side. GFP fused to anti-GCN4 nanobody is secreted from the muscle. (G) Most DMA-1-containing vesicles in the PVD dendrite have been recycled off the plasma membrane. (H–I) DMA-1 exhibits both diffuse and endosomal localization in RD but is predominantly diffuse in dauer. In the rab-10 mutant, there is little endosomal DMA-1 in RD but much more diffuse DMA-1 in dauer, consistent with the model that RAB-10 is required to recycle endosomes to the plasma membrane and that endosome production is shut down in dauer. ***P < 0.001, one-way ANOVA with Tukey post test. (J–L) Example images of the intestinal and coelomocyte endocytosis reporters quantified in (A–C) Localization of RAB-10, RAB-7, and RAB-6.2 is less vesicular and more diffuse in dauerized dendrite compared to wild type. (D–E) Localization of RAB-3 is similar, but with increased accumulation, in dauerized versus wild-type PVD. **(TIF)Click here for additional data file.S5 Figdyn-1(wy1150) mutant. This CRISPR-generated allele produces the same temperature-sensitive lesion as Drosophila shibire(ts1), at conserved reside G273D [arf-6(tm1447) (a deletion causing a putative null), clathrin adaptor AP-1μ—unc-101(wy50042) , and EpsI5-homology domain (EHD) protein—rme-1(b1045) (a deletion causing a putative null) [drosophila shibire gene associated with vesicular traffic. Nature. 1991;351:411–414. 2. Grant B, Zhang Y, Paupard MC, Hall DH, Hirsh D, Lin SX. Evidence that RME-1, a conserved C. elegans EH-domain protein, functions in endocytic recycling. Nat Cell Biol. 2001;3: 573–579.(A) Examples of DMA-1::GFP in the PVD anterior primary dendrite and cell body in the dynamin (TIF)Click here for additional data file.S6 FigPVD>daf-12(−ligand)) PVD . Scale = 10 um. ***P < 0.001, two-tailed t test.In wild-type RD, GFP::TIAM-1 localizes to vesicles throughout the dendrite , and it is less vesicular and more diffuse in dauerized ((TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S1 MovieMovie shows maximum projection from image stacks acquired every 3 minutes for 3 hours and are played at 2 frames per second. Ventral is down; anterior is left.(AVI)Click here for additional data file.S2 MoviePVD development is shown from 2 animals laying side by side. Movie acquired as in (AVI)Click here for additional data file.S3 MovieTwo animals are shown, but PVD is visible only in the lower worm. Movie acquired as in (AVI)Click here for additional data file.