Gliomas are the most common primary brain malignancies and are associated with a poor prognosis. Here, we showed that the PDZ domain-containing protein membrane-associated guanylate kinase inverted 3 (MAGI3) was downregulated at the both mRNA and protein levels in human glioma samples. MAGI3 inhibited proliferation, migration, and cell cycle progression of glioma cells in its overexpression and knockdown studies. By using GST pull-down and co-immunoprecipitation assays, we found that MAGI3 bound to β-catenin through its PDZ domains and the PDZ-binding motif of β-catenin. MAGI3 overexpression inhibited β-catenin transcriptional activity via its interaction with β-catenin. Consistently, MAGI3 overexpression in glioma cells C6 suppressed expression of β-catenin target genes including Cyclin D1 and Axin2, whereas MAGI3 knockdown in glioma cells U373 and LN229 enhanced their expression. MAGI3 overexpression decreased growth of C6 subcutaneous tumors in mice, and inhibited expression of β-catenin target genes in xenograft tumors. Furthermore, analysis based on the Gene Expression Omnibus (GEO) glioma dataset showed association of MAGI3 expression with overall survival and tumor grade. Finally, we demonstrated negative correlation between MAGI3 expression and activity of Wnt/β-catenin signaling through GSEA of three public glioma datasets and immunohistochemical staining of clinical glioma samples. Taken together, these results identify MAGI3 as a novel tumor suppressor and provide insight into the pathogenesis of glioma. Gliomas are the most common primary brain tumors, comprising heterogeneous entities from low-grade to high-grade malignancies. However, nearly all low-grade tumors eventually progress to high-grade malignancies with highly proliferative and invasive phenotypes . DespiteGliomas exhibit a vast array of genetic changes including loss-of-function mutations of the p53 and PTEN tumor suppressors, and hyperactivation of receptor tyrosine kinase signaling . In the Many studies contributed to the elucidation of the molecular mechanisms underlying the regulation of β-catenin signaling, such as destabilization of β-catenin or blockage of its nuclear translocation . An incrIn the present study, we identified a PDZ protein, membrane-associated guanylate kinase inverted 3 (MAGI3) as a novel inhibitor of Wnt/β-catenin signaling and showed that it suppressed the malignant phenotypes of glioma cells. MAGI3 expression was negatively correlated with tumor grade and poor prognosis.http://www.ncbi.nlm.nih.gov/geo/). The gene expression profile of various PDZ proteins in the top 1 largest database GSE4290 was analyzed to identify differentially expressed PDZ genes in human glioma. GSE4290 is a publicly available microarray gene expression dataset comprising 81 glioblastomas and 23 non-tumor brain samples [P < 0.01; Several lines of evidence suggest that aberrant expression of PDZ proteins is associated with tumorigenesis and/or metastasis . Dysregu samples . Among gP < 0.01; Figure To verify these findings, MAGI3 expression was examined by immunohistochemistry in a tissue microarray containing 35 human glioma specimens and 5 normal brain tissue samples. MAGI3 protein expression was significantly downregulated in glioma tissues compared with the normal controls (P < 0.01), whereas MAGI3 knockdown in U373 or LN229 cells dramatically promoted cell proliferation  than in grade IV gliomas (n = 59)  containing 85 high-grade gliomas were examined. The results showed that the expression level of MAGI3 was higher in grade III  Figure . The dathttp://www.stanford.edu/group/nusselab/cgi-bin/wnt/target_genes), was highly enriched in the low MAGI3 expression group (FDR < 0.05). These data suggest a negative correlation between MAGI3 expression and activity of β-catenin signaling in clinical glioma sample. To confirm the relationship of MAGI3 expression and activity of β-catenin signaling in glioma and glial cells, serial sections of glioma and adjacent normal tissue were respectively stained for MAGI3, Cyclin D1 and GFAP by IHC. Similar histological feature was observed in GFAP-, MAGI3- and Cyclin D1-staining sections. GFAP-positive cells were widely distributed in either glioma or adjacent tissues. MAGI3 immunoreactivity was downregulated concomitant with upregulation of Cyclin D1 in glioma cells compared with adjacent normal cells  were divided into high MAGI3 expression and low MAGI3 expression groups, and further analyzed using GSEA method. As shown in Figure s Figure .The Wnt/β-catenin signaling pathway plays an essential role in regulating the self-renewal, proliferation and differentiation of neural stem cells and progenitor cells in the brain. However, overactivation of β-catenin signaling has been associated with the development of brain tumors including gliomas . Here, wMAGI3 belongs to the MAGUK (membrane-associated guanylate kinase) family, which includes two closely related members, MAGI1 and MAGI2. Similar to the other two family members, MAGI3 contains five PDZ domains (PDZ1–5), one guanylate kinase (GK) domain, and two WW domains . We and Our study demonstrated that MAGI3 inhibited the transcriptional activity of β-catenin through a direct interaction between the PDZ domains of MAGI3 and the C-terminus of β-catenin. This conclusion is supported by the finding that MAGI3 exerted little effect on the activation of β-catenin mutants, in which MAGI3 binding was impaired Figures  and 4F. MAGI3 is localized to different subcellular compartments including the cell surface and cytoplasm . Previouin vivo. The inhibitory effect of MAGI3 on the malignant behaviors of glioma cells could be attributed to its capacity for suppressing β-catenin signaling in glioma. This hypothesis is supported by the observations: MAGI3 overexpression downregulated the expression of c-Myc and Cyclin D1 , a main product of cyclooxgenase-2 (COX-2) . COX-2 iIn conclusion, the present study identified MAGI3 as a novel tumor suppressor in glioma and showed its inhibitory effect on Wnt/β-catenin signaling. Our findings provide novel clues to improve our understanding of the pathogenesis of glioma.The anti-His antibody was purchased from MBL . Anti-MAGI3, anti-Cyclin D1 and anti-GFAP antibodies from Abcam ; Anti-β-catenin and anti-GFP antibodies from Cell Signaling Technology ; Anti-Flag M2 antibody and anti-Flag M2 affinity gels from Sigma ; Anti-β-actin, anti-GAPDH and HRP-conjugated secondary antibodies from ZSGB-BIO .Glioma samples were collected from the First Hospital of Shanxi Medical University of Taiyuan during 2010. Patients were included after signing written informed consents. The study was approved by the Ethics Committee of First Hospital of Shanxi Medical University. Diagnosis and classification of glioma were based on the World Health Organization classification. None of the patients included in the study had a family history of glioma or secondary malignancies, and none had received radiotherapy or chemotherapy before surgery. A glioma tissue microarray, containing biopsies from 35 different gliomas and 5 normal human brain specimens was obtained from US Biomax .Glioma and adjacent normal tissues were fixed with 4% formalin, prepared as paraffin-embedded section. Immunohistochemistry was performed as described previously . For tisTotal RNA was isolated using the Trizol reagent  and reverse transcription was performed with a Protoscript M-Mulv First strand cDNA Synthesis Kit . The cDNAs obtained were mixed with polymerase chain reaction (PCR) solution containing the primers indicated in 2 at constant humidity. The Wnt3a-conditioned medium (CM) and L cells control CM were prepared as described previously [Human or rat glioblastoma cell lines (U373 and C6), the HEK293 and COS-7 cell lines were from the European Collection for Animal Cell Culture . The human glioblastoma cell line LN229 was purchased from American Type Culture Collection . Parental and Wnt3a-expressing L cells were kindly provided by Dr. Clever Hans . All cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and grown at 37°C in 5% COeviously .The constructs encoding Flag-tagged β-catenin, Renilla luciferase, TOP-Flash or FOP-Flash luciferase reporters and the pGEX-4T-1-β-catenin vector were kindly provided by Dr. Wei Wu . Point mutations of β-catenin were introduced into the Flag-β-catenin construct by PCR and verified by bidirectional sequencing. The plasmid for Myc-tagged Dvl-2 was kind gift from Dr. Yeguang Chen , and pEGFP-N4-MAGI3 and pcDNA3-V5/His-MAGI3 were kindly provided by Dr. Randy Hall . pET30A plasmids containing the PDZ domains of MAGI3 were generated as described previously .SiRNAs and their sources were as follows: MAGI3 siRNA-1 targeted the sequence 5′-GGUCCACCAUCAGGAACAAACUCAG-3′; MAGI3 siRNA-2 targeted the sequence 5′-GAGAUGGACC UGACCAGUCUAUAUA-3′ . β-catenin siRNA (sc-29209) was obtained from Santa Cruz Biotechnology. The control siRNA (si-control) was 5′-UUCUCCGAACGUGUCACGUTT-3′ (Invitrogen).Cell transfection was performed using Lipofectamine 2000 (Invitrogen). For siRNA knockdown and transient transfection experiments, RNA or protein extraction was performed 48 h post-transfection. For establishment of stable GFP-MAGI3-expressing cells, C6 cells were transfected with constructs of pEGFP-N4-MAGI3. At 24 h after transfection, the cells were selected with 1 mg/mL G418  in culture medium for an additional 14 days. Expression of the GFP-MAGI3 protein was verified by western blotting.The assay was performed as described previously . LuminesCells were cultured in 96-well microplates at a density of 3000 per well. MAGI3-overexpressing or siRNA-transfected cells were seeded in DMEM medium containing 5% FBS 24 h post-transfection. At 48 h (C6 cells) or 96 h (U373 and LN229 cells) after plating, CCK-8  was added. Viable cells were quantified by measuring absorbance at 450 nm with an EnVision 2104 multilabel reader .5 cells/well in a 6-well plate and allowed to reach confluence. The cell monolayer was carefully scratched using a sterile 200 μL pipette tip across the center of the well. Images of the wound were recorded under a phase contrast microscope at 0 and 16 h after culture. The widths of three different wound surfaces in each group were noted and measured using Image J analysis software.Control and stable GFP-MAGI3-transfected C6 cells were cultured at a density of 5 × 10Control and stable GFP-MAGI3-expressing C6 cells were seeded into an eight-chambered cover glass  and cultured in DMEM. The random migration of the cells was recorded every 5 min for 8 h under an inverted fluorescence microscope  with the confocal imaging system Ultra VIEW Vox . The traces and migration speed of cells were analyzed with the velocity as described previously .Purification of GST- or His-tagged proteins and GST pull-down assay, lysis of cells and tumor samples, co-immunoprecipitation and western blotting were performed as described previously .5 cells were analyzed for their DNA content using an EPICS@XL flow cytometer . Data acquisition was performed with EXPO32 ADCv1.1C MultiCycle software .Cells in the log phase of growth were harvested and fixed with 70% ethanol overnight at 4°C. The cells were then incubated with 0.1% Triton X-100 solution containing 50 μg/mL propidium iodide  and RNase in the dark for 1 h at room temperature. A total of 1 × 105) in 0.1 mL PBS were subcutaneously injected into the right flank of male Balb/c nude mice (4–5 weeks). Each group included 7 mice. Tumor size was measured daily with calipers and tumor volume was estimated using the following formula: volume = (length × width2)/2. The mice were sacrificed on day 17 post-injection, and tumors were harvested and individually weighed. Following resection, a portion of each tumor sample was prepared for isolation of RNA.All animal experiments were performed following the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Use and Care Committee of Capital Medical University. Control or stable GFP-MAGI3-expressing C6 cells (1 × 10http://www.broad.mit.edu/gsea/). GSEA calculates a pathway Enrichment Score (ES) that estimates whether genes from pre-defined gene set of Wnt/β-catenin target genes (http://www.stanford.edu/group/nusselab/cgi-bin/wnt/target_genes) are enriched among the highest- (or lowest-) ranked genes or distributed randomly. Default settings were used. Thresholds for significance were determined by permutation analysis (1000 permutations). False Discovery Rate (FDR) was calculated. A gene set is considered significantly enriched when the FDR score is <0.05.The association between expression of MAGI3 and biological processes was analyzed using Gene Set Enrichment Analysis  and Graphpad Prism 5 . Group distributions were compared using the Student's


TARDBP or C9ORF72 ALS-causing mutations are able to develop appropriate physiological properties. However, patient iPSC-derived MNs, independent of genotype, display an initial hyperexcitability followed by progressive loss of action potential output and synaptic activity. This loss of functional output reflects a progressive decrease in voltage-activated Na+ and K+ currents, which occurs in the absence of overt changes in cell viability. These data implicate early dysfunction or loss of ion channels as a convergent point that may contribute to the initiation of downstream degenerative pathways that ultimately lead to MN loss in ALS.Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease for which a greater understanding of early disease mechanisms is needed to reveal novel therapeutic targets. We report the use of human induced pluripotent stem cell (iPSC)-derived motoneurons (MNs) to study the pathophysiology of ALS. We demonstrate that MNs derived from iPSCs obtained from healthy individuals or patients harbouring  Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects spinal cord motor neurons. Here the authors use induced pluripotent stem cell-derived motor neurons obtained from patients with ALS-linked mutations, and find functional deficits resulting from a progressive decrease in voltage-activated Na+ and K+ currents that occur in the absence of cell death. TARDBP and C9ORF72 in familial and sporadic forms of these diseases, has become increasingly influential in shaping our understanding of ALS and related disorders345891011121314Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, fatal neurodegenerative disease for which no effective treatment exists. Although the mechanisms underlying motoneuron (MN) loss in ALS remain unclear, recent pathological and genetic discoveries have provided important insights that implicate common mechanisms across both ALS and fronto-temporal dementia, as well as a link between familial and sporadic ALS1+ currents161718192021+ currents, has also been shown in studies of ALS patients23Interestingly, physiological analyses have already revealed changes in the functional properties of MNs at very early stages  in transgenic rodent models of ALS. Several studies have demonstrated pre-symptomatic hyperexcitability of MNs due to perturbations in their intrinsic properties, particularly NaTARDBP and C9ORF72. We demonstrate the development of appropriate functional properties in both control and patient iPSC-derived MNs but reveal a progressive loss of action potential output, spontaneous synaptic activity and ionic conductances in patient-derived MNs, regardless of their genotype, which occurs before any overt changes in cell viability.In the present study, we have undertaken detailed, temporal electrophysiological analyses of human iPSC-derived MNs, to investigate whether MN dysfunction represents an early feature of ALS pathogenesis common to neurons carrying mutations in TARDBP patient (lines D1 and D3); single clones from two different C9ORF72 patients (lines S6 and R2); two clones from one healthy control (lines R6 and M2); and single clones from two additional healthy controls , with each line differentiated a minimum of four times.iPSCs were generated from fibroblasts of ALS patients and healthy individuals as previously described9TARDBP D1 and C9ORF72 S6) iPSC lines , two C9ORF72 lines (S6 and R2) and three control lines  revealed no greater LDH activity in patient compared with control lines at any time point during the 10 weeks in culture (TARDBP lines at 9–10 weeks post plating (P<0.05). Finally, we also assessed nuclear morphology at 9–10 weeks post plating in one control (D6), one TARDBP (D1) and one C9ORF72 (S6) line. We found no difference in the percentage of cells with pyknotic nuclei in patient lines compared with controls . In summary, cell counts, LDH assays and counts of pyknotic nuclei failed to reveal any differences in viability between neurons derived from iPSC lines harbouring TARDBP or C9ORF72 mutations compared with controls.Having established equivalent MN-enriched cultures from both control and patient iPSC lines, we first investigated whether there were any differences in cell viability between neurons derived from control and ALS patient iPSCs. Our initial observations suggested that control and patient iPSC-derived neurons with either rphology , up to trphology . Analysi plating , P<0.05.TARDBP or C9ORF72 mutations on iPSC-derived cell viability, we next turned to more sensitive electrophysiological analyses to uncover any signs of neuronal dysfunction.Given that standard cell viability assays failed to reveal overt effects of post-hoc SMI-32 labelling of neurons filled with an Alexa Fluor dye during recordings  and C9ORF72 (R2 and S6) iPSC lines (see Cm) values were similar across control and patient iPSC-derived MNs , and control MNs had lower Cm compared with MNs harbouring TARDBP mutations at weeks 7–8 and weeks 9–10 (P<0.0001). We found no significant differences in the input resistance (RN) of MNs derived from control, TARDBP or C9ORF72 iPSCs from weeks 3 to 10 post plating  compared with controls at weeks 3–4 (P<0.001) and weeks 9–10 (P<0.0001), while the RMPs of MNs with a C9ORF72 mutation were more depolarized than controls at weeks 7–8 (P<0.0001). The largest differences in RMP between control and patient iPSC-derived MNs were apparent towards the end of the time course studied. It should be noted that these numbers may represent an underestimation of the extent of membrane potential depolarization in patient iPSC-derived MNs, because cells that were more depolarized than −20 mV were excluded from all analyses, to avoid the inclusion of cells that were depolarized due to damage associated with the establishment of recordings.We began by comparing the passive membrane properties of MNs derived from control and patient iPSCs. For these and all other electrophysiological analyses, data were pooled for control ,  plating . MNs harf–I) relationships were generated from responses to a series of injected current steps . Comparisons were performed on data pooled from recordings of repetitively firing cells at weeks 2–6 post plating . However, data demonstrated hyperexcitability in both the TARDBP and C9ORF72 lines compared with controls, as indicated by the greater slopes of their f–I relationships . No significant difference was detected between the excitability of MNs derived from the TARDBP and C9ORF72 lines. Thus, in parallel with findings in animal models of ALS, human MNs harbouring two different ALS-related mutations are initially more excitable than controls.Current-clamp mode recordings of iPSC-derived MNs demonstrated that by 2 weeks post plating, all lines developed the ability to repetitively fire trains of action potentials in response to current injections. To compare the excitability of repetitively firing patient and control iPSC-derived MNs, frequency–current , while the ratio of firing versus non-firing cells remaining unchanged throughout the 10 weeks in control lines  relationships or peak Na+ currents between MNs derived from patient and control iPSCs at weeks 3–4 post plating  and from weeks 7–10 post plating in TARDBP iPSC-derived MNs (P<0.01), while peak currents were also smaller in neurons derived from C9ORF72 iPSCs compared with those derived from TARDBP iPSCs (P<0.05) from week 7 onwards  and MNs derived from TARDBP iPSCs having smaller K+ currents compared with controls from weeks 7–8 post plating onwards .Having revealed a progressive loss of Nacurrents  elicitedcurrents . At week+ currents and persistent, voltage-activated K+ currents in patient iPSC-derived MNs. Given the similar time courses of current loss and action potential loss, and the observation that current loss precedes changes in the probability of firing, it is likely to be that reductions in voltage-activated currents underlie the progressive loss of functional output in MNs harbouring ALS-related mutations.Taken together, these data demonstrate a progressive reduction in both fast, inactivating Na+ and K+ currents, and the type of output produced by iPSC-derived MNs for which both voltage- and current-clamp data were available . Plots of raw data indicated similar relationships between peak currents and firing categories in all iPSC lines 3–6 weeks post plating; larger currents were associated with greater output (TARDBP or C9ORF72) and peak current (Na+ or K+) as the predictor variables. Using these models, we assessed the predicted probabilities for each firing category based on the measured currents. Models showed that both Na+ and K+ currents were strong predictors of firing category . In addition, the type of iPSC line contributed to the predicted firing outcome . As expected, we found that the probability of a cell failing to spike in response to current injection decreases with increasing peak Na+ or K+ currents  is governed by the size of Naived MNs  is likelTARDBP or C9ORF72 ALS mutations develop appropriate physiological properties. However, temporal analysis revealed that patient iPSC-derived MNs display an initial hyperexcitability, followed by a progressive loss in action potential output and synaptic activity. This loss of functional output appears to result from a progressive decrease in voltage-activated Na+ and K+ currents that occurs in the absence of overt changes in cell viability. These novel data from ALS-affected human MNs indicate that early dysfunction or loss of ion channels may contribute to the initiation of downstream degenerative pathways that ultimately lead to MN loss in ALS.We have demonstrated that MNs derived from human iPSCs obtained from healthy individuals or patients harbouring Given that all neurons must be by definition excitable, it is critical that protocols established for the derivation of MNs from iPSCs are validated via the electrophysiological demonstration of appropriate functional properties. Previous studies have demonstrated the ability of human iPSC-derived MNs to fire action potentials in response to current injection and to receive spontaneous synaptic inputs9121314269111213141534TARDBP or C9ORF72 mutations also exhibit hyperexcitability at early stages in culture. Although hyperexcitability was transient in our study and does not persist into symptomatic stages in animal models of ALS222+ handling382+ and the eventual activation of cell death pathways. In opposition to this, however, recent work supports a link between hyperexcitability and neuroprotection4143Previous studies of ALS patients2324161718192021C9ORF72 mutationin vivo recordings from presymptomatic mSOD1 mice, showed that a significant proportion of MNs could not discharge repetitively in response to current ramps, although their neuromuscular junctions remained functional. Thus, dysfunction at the MN cell body appears to precede that at the neuromuscular junction. Taken together, these findings highlight the importance of addressing early perturbations in mechanisms underlying spike generation at the MN soma when considering disease pathogenesis and potential treatment strategies for ALS.Following an early stage of hyperexcitability, we found that patient iPSC-derived MNs progressively lost their ability to generate action potentials. This was evidenced by a reduction in the proportion of patient iPSC-derived MNs, which were able to fire repetitive or even single spikes. In comparison, the proportion of control iPSC-derived MNs in each of the firing categories remained unchanged throughout the 10 weeks studied. Thus, despite remaining viable in culture, ALS-affected MNs were gradually rendered non-functional. Our findings of a progressive loss of MN output are consistent with recent reports of hypoexcitability of iPSC-derived MNs harbouring a et al.SOD-1 mutation at ~4 weeks post plating, a time point equivalent to when we also observed hyperexcitability in iPSC-derived MNs harbouring TARDBP or C9ORF72 mutations. In contrast, Sareen et al.C9ORF72 mutations at a time point comparable to our week 7–8 post plating when we first observed loss of MN output. Thus, these recent studies support our observation of a progression from hyperexcitability to hypoexcitability. Although this progression is consistent with work in animal modelsin vivo. It also remains unclear whether mechanistic links exist between hyperexcitability and hypoexcitability, which will be important to address in future studies.Importantly, we believe our work has addressed an apparent contradiction arising from recent studies performing similar electrophysiological analysis of ALS patient iPSC-derived MNs. Wainger Although our focus was on the intrinsic properties of MNs, we also observed a concomitant reduction in the proportion of patient iPSC-derived MNs that received synaptic inputs. Loss of synaptic activity may simply reflect a general loss of action potential generation in culture. However, given evidence of loss and dysfunction of synapses in ALS2829303132+ and K+ channels in animal models of ALS161721364612+  and K+ (persistent) currents in patient iPSC-derived MNs. Given that current loss began before and continued in parallel with changes in firing probability, and that Na+ and K+ current magnitudes are excellent predictors of firing patterns, reductions in voltage-activated currents most likely underlie the loss of functional output observed in patient iPSC-derived MNs. Mechanisms underlying the initial hyperexcitability of MNs are less clear. Wainger et al.+ currents underlie hyperexcitability in iPSC-derived MNs harbouring SOD-1 mutations. Although we also observed lower K+ current magnitude in MNs harbouring C9ORF72 mutations at hyperexcitable stages, the same was not true for MNs harbouring TARDBP mutations. Thus, reductions in K+ currents are unlikely to be the sole mechanism underlying hyperexcitability in patient iPSC-derived MNs. Either loss or dysfunction of channels may underlie the loss of current observed in patient iPSC-derived MNs. Regardless of the exact effect on ion channels, our data are consistent with the recent proposal that ALS involves channelopathy-like mechanisms, a view which stemmed from the demonstration that mutant SOD1 inhibits the conductance of a mitochondrial ion channel+/K+ pump for which reduced expression has been shown in mSOD1 miceFollowing the demonstration of reduced output from patient iPSC-derived MNs, we used voltage-clamp recordings to investigate the mechanisms likely to underlie this loss of function. Previous studies have demonstrated changes in both the density and function of NaTARDBP and C9ORF72. TARDBP encodes the RNA-binding protein TDP-43, which can act as a transcription repressor and a splicing regulator, and can also contribute to RNA stability and transport5051TARDBP are associated with deficits in RNA processing53C9ORF72 remains unknown, its mutant form also appears to cause aberrant RNA processing56TARDBP and C9ORF72-mediated ALS and may contribute to loss of voltage-activated currents in patient iPSC-derived MNs. In support of this, recent work has shown that the expression of genes involved in neuronal excitability, including those encoding ion channels, is altered in iPSC-derived MNs with C9ORF72 mutationsTARDBP and C9ORF72 iPSC lines exhibited similar pathophysiology; hyperexcitability, followed by a loss of functional output due to a reduction in the magnitude of voltage-activated currents. Alternative interpretations include the possibility that perturbations in ion channels and MN output represent core disease mechanisms common to many forms of ALS, including both familial and sporadic ALS given the demonstration of TARDBP and C9ORF72 mutations in both of these forms of the disease. Further experiments using a variety of patient iPSC lines will be needed to confirm the relevance of our findings to a wide spectrum of ALS cases.In this study, we used iPSC-derived MNs harbouring mutations in two different genes associated with ALS, In summary, our study has provided important new insight into early ALS disease mechanisms by investigating pathophysiological changes in iPSC-derived MNs from ALS patients with two different genetic mutations. We have shown that MN output is severely compromised, due to the loss of voltage-activated currents, before any other overt signs of neurodegeneration. These data suggest that ‘functional loss’ of MNs may render the motor system inactive before neurodegeneration of MNs, highlighting the importance of addressing MN function, perhaps by targeting ion channels, when designing new treatment strategies for ALS. Furthermore, our findings demonstrate the usefulness of sensitive physiological studies of human iPSC-derived MNs for future work aiming to develop much needed therapeutics for this devastating disease.C9ORF72 ALS patient ; one male C9ORF72 ALS patient ; two female healthy controls  and one male healthy control . All participants provided written signed consent to donate their skin sample to derive iPSCs and their use was approved by the Ethics Committee from the King’s College Hospital, a national Medical Research Ethics Committee (MREC). iPSCs were generated from fibroblasts as previously described9TARDBP: D1=6, D3=8; C9ORF72: S6=4, R2=4), with control and patient iPSC differentiations always running in parallel. iPSCs were established by virally transducing 105 fibroblasts with the Yamanaka reprogramming factors OCT4, SOX2, KLF4 and c-MYC using either lentiviral  or Sendai (Life technologies) reprogramming kits. Cells were maintained in murine embryonic fibroblast (MEF) media at 37 °C and 5% CO2. MEF media consisted of KO-DMEM (Invitrogen), 2 mM L-glutamine (Invitrogen), 10% FBS (Invitrogen) and 1% penicillin/streptomycin (Invitrogen). After 5 days, the cells were split and re-plated as single cells on to an MEF feeder plate. On day 7, media was changed to Knockout serum replacement (KSR) and fibroblast growth factor 2 (FGF2) consisting of advanced DMEM (Invitrogen), 20% serum replacer (Invitrogen), 1 μl ml−1 FGF2 (PeproTech), 1% penicillin/streptomycin (Invitrogen), 1% L-glutamine (Invitrogen) and 0.007 μl ml−1 2-mercaptoethanol (Invitrogen). After ~21 days, colonies with compact human embryonic cell-like morphology were expanded and clonal lines were established. Human iPSC lines were maintained long term on CF-1-irradiated mouse embryonic fibroblasts, with KO-DMEM (Invitrogen) supplemented with 20% knockout serum replacement (Invitrogen), 10 ng ml−1 basic FGF2 (PeproTech), 1% L-glutamine (Invitrogen), 0.007 μl ml−1 2-mercaptoethanol (Invitrogen) and 1% penicillin/streptomycin (Invitrogen) at 37 °C and 5% CO2. For the neural conversion, the cells were transitioned to feeder-free culture conditions in MTESR1 media (Stem Cell Technologies) and passaged three times before use.For this study, we used eight iPSC lines from six individuals: one male M337V TDP-43 ALS patient ; one female TARDBP lines and hexanucleotide 5′- GGGGCC -3′ repeat expansions in C9ORF72 lines shown by repeat prime PCR , 1% chemically defined Lipid 100 × (Invitrogen), 450 mM monothioglycerol (Sigma), 7 mg ml−1 insulin (Roche), 15 mg ml−1 transferrin (Roche), 1% penicillin/streptomycin), supplemented with 1 mM N-acetyl cysteine (Sigma), 10 μM Activin Inhibitor (R&D Systems) and 2 μM Dorsomorphin (Merck Millipore)). This medium was changed every 2–3 days for 4–10 days. Neurospheres were patterned to a caudal, spinal cord identity in CDM, 1 mM N-acetyl cysteine, 5 ng ml−1 FGF (PeproTech)/heparin  and 0.1 μM retinoic acid (Sigma) for 4–10 days, changing medium every 2–3 days. Caudalized neural stem cells were ventralized in the presence of Advanced DMEM Nutrient Mixture F12 (Invitrogen), 1% penicillin/streptomycin, 0.5% GlutaMAX, 1% B-27, 0.5% N-2 supplement, 5 ng ml−1 FGF+Heparin, 1 μM retinoic acid and 1 μM purmorphamine (Merck Millipore) for 4–10 days, changing every 2–3 days. The FGF/Heparin was removed from the medium and replaced with 0.5 μM purmorphamine, with the cells cultured for another 4–14 days, changing the medium every 2–3 days. Caudalized and ventralized neural stem cells were transitioned to MN maturation medium containing advanced DMEM/F12, 1% penicillin/streptomycin, 0.5% B-27, 0.5% N-2 supplement, 2 ng ml−1 Heparin, 10 ng ml−1 brain- derived neurotrophic factor (R&D systems), 10 ng ml−1 glial cell line-derived neurotrophic factor (R&D systems), 10 μM forskolin (R&D systems), 0.1 μM retinoic acid and 0.1 μM purmorphamine for 2–6 weeks, changing the medium every 2–3 days. MN progenitors were next dissociated with the Papain Dissociation System , plated at 50 × 103 cells per well in 24-well plates, with 13 mm glass coverslips coated with poly-ornithine (Sigma), laminin (Sigma), fibronectin (Sigma) and Matrigel (BD Biosciences). Plate down medium consisted of Neurobasal medium (Invitrogen), 1% penicillin/streptomycin, 0.5% GlutaMAX, 0.5% B-27, 0.5% N-2 Supplement, 20 ng ml−1 basic FGF, 1 μM retinoic acid, 1 μM purmorphamine, 1 μM mouse Smo agonist SAG (Merck Millipore)−1 ciliary neurotrophic factor (R&D systems), 10 ng ml−1 glial cell line-derived neurotrophic factor and 10 μM forskolin, which promotes cell cycle exit, were added, and this medium was used until day 14, feeding every 3 days. From day 14, RA, SAG, purmorphamine and forskolin was removed from the medium, with cells then maintained for up to 10 weeks. As an indication that neuronal differentiation was successful, with few mitotic cells present, we performed immunostaining for the mitotic marker Ki67 (3–4 weeks post plating). Less than 7% of cells expressed Ki67 in control and patient iPSC-derived cultures.Differentiation of iPSCs into a neuronal and MN lineage was performed using modifications of previously established and validated protocols9Cells were fixed in 4% (wt/vol) paraformaldehyde for 10 min, permeabilized in 0.1% Triton X-100 at room temperature for 10 min and blocked in 3% (vol/vol) goat or donkey serum for 45 min. They were then incubated in primary antibodies for 45 min  followedC9ORF72 hexanucleotide repeat expansion in control or carrier neurons. PCR amplification was carried out using Multiplex Mastermix (Qiagen); 7% dimethylsulfoxide, 0.6 M Betaine, 7.6 μM Primer F (5′- CTGTAGCAAGCTCTGGAACTCAGGAGTCG -3′), 3.6 μm Primer Repeat R (5′- TACGCATCCCAGTTTGAGACGCCCCGGCCCCGGCCCCGGCCCC -3′), 11.6 μM Tail R (5′- TACGCATCCCAGTTTGAGACG -3′), 8 μM 7-deaza-2′-dGTP and 200 ng DNA. Cycling conditions were performed as per the manufacturer’s recommendations, except for the annealing temperature, which was 68 °C for 15 cycles, then 60 °C for a further 20 cycles. PCR products were separated on an ABI 3130 × l analyser (Life Technologies) and data were analysed using GeneMarker software (Soft Genetics).Repeat primed–PCR was used to confirm the presence or absence of the 4 oligoneucleotide probe (IDT). Briefly, cells on glass coverslips were fixed in 4% paraformaldehyde for 30 min, permeabilized in 70% ethanol at 4 °C, incubated with 50% formamide/2 × SSC for 10 min at room temperature and hybridized for 2 h at 37 °C with the oligoneucleotide probe (0.16 ng μl−1) in hybridization buffer consisting of 50% formamide, 2 × SSC, 10% dextran sulphate, yeast transfer RNA (1 mg ml−1), salmon sperm DNA (1 mg ml−1) and 0.2% Tween-20. The cells were washed twice with 50% formamide/1 × SSC for 30 min at 37 °C and once with 2 × SSC at room temperature for 30 min. Immunostaining was performed as described above.FISH analysis was performed using an Alexa 546-conjugated (GGCCCC)TARDBP and C9ORF72) at each 2 week time point throughout the 10 weeks cells were maintained. Infrared-DIC images were chosen at random from a database of images (× 40 magnification) that were obtained whenever whole-cell patch-clamp recordings were attempted. Cell counts were fitted with a negative binomial generalized linear model and the effect of time assessed with a likelihood ratio test.Cell counts were performed, with the observer blinded to cell type, on 20 IR-DIC images for each cell type , C9ORF72 (S6: 2 experiments and R2) and control lines . LDH activity (mU ml−1) was calculated for each cell type using LDH assay kits (Abcam). LDH activity was plotted versus postplating date and compared between cell types using linear models.For LDH assays, cell culture medium was collected from control and patient-derived lines, which were differentiated in parallel to enable direct comparisons. Medium was collected twice per week across weeks 3–10 post plating from post-hoc test.Nuclear morphology was assessed, with the observer blinded to cell type, by counting the number of pyknotic nuclei in 40 images taken from control and patient-derived lines at 9–10 weeks post plating. Statistical analysis was performed using a single factor ANOVA followed by Tukey’s Cm), input resistance (RN), series resistance (RS) and RMP values were calculated using pClamp10 software. Only cells with an Rs<20 MΩ, a RMP more hyperpolarized than −20 mV and RN>100 MΩ were included in data analysis. Rs values were not significantly different between control, C9ORF72 and TARDBP lines. Cells were defined as neurons if they had clear fast-inactivating inward currents (≥50 pA). In a subset of experiments, such currents were found to be tetrodotoxin sensitive (data not shown). Recordings from glial cells, which were clearly distinguishable based on their hyperpolarized RMP and absence of inward currents, were excluded from all analyses. An on-line P4 leak subtraction protocol was used for all recordings of voltage-activated currents. Descriptions of voltage- and current-clamp protocols are provided in the Results section.Whole-cell patch-clamp recordings were used to assess the functionality of iPSC-derived MNs. Voltage-clamp mode was used to investigate intrinsic membrane properties. Current-clamp mode was used to investigate the firing properties of MNs. Experiments were carried out in a recording chamber, which was perfused continuously with oxygenated artificial cerebral spinal fluid (aCSF) at room temperature (22–24 °C). Whole-cell patch-clamp recordings were made from cells visualized by IR-DIC microscopy using an Olympus upright BX51WI microscope with a × 40 submersion lens. Patch electrodes (4.0–5.0 MΩ resistance) were pulled on a Sutter P-97 horizontal puller  from borosilicate glass capillaries . Recorded signals were amplified and filtered (4 kHz low-pass Bessel filter) using a MultiClamp 700B amplifier  and acquired at ≥10 kHz using a Digidata 1440A analogue-to-digital board and pClamp10 software (Axon Instruments). Whole-cell capacitance . The pipette solution contained (in mM): 140 potassium methane-sulfonate, 10 NaCl, 1 CaCl2, 10 HEPES, 0.2 EGTA, 3 ATP-Mg, 0.4 GTP, . All drugs were made up as concentrated stock solutions in single-use vials and stored at −20 °C. The final concentrations were achieved by diluting stock solutions in aCSF. Stock solutions of GABA and glycine were made up in distilled water. Stock solutions of glutamate were made up in 0.1 M NaOH. Drug application was via addition to the perfusate.The aCSF used for all electrophysiological recordings contained the following in mM; 127 NaCl, 3 KCl, 2 CaClTARDBP and C9ORF72) were pooled for all analyses. Peak Na+ currents and peak K+ currents (log10 transformed), Cm, RN and RMP were compared across the three different types of cell lines using factorial ANOVAs with postplating date  and cell line type  as factors. f–I relationships were compared across cell lines using linear models fitted to the initial, most linear portion of the f–I relationship (≤50 pA above rheobase). Pair-wise comparisons were made using Tukey’s honest significant difference test, where necessary.Electrophysiological data were analysed using Clampfit10 software (Axon Instruments) or Dataview . Data from each type of iPSC line  as factors. Using these models, predicted probabilities for each firing mode were calculated over a range of K+ and Na+ current magnitudes for all types of iPSC lines. Factors within the multinomial logistic regression were assessed with likelihood ratio tests.Firing properties were further analysed by assigning cells into four categories (No Spike/Single/Adaptive/Repetitive). These data were then fitted with a multinomial logistic regression using type of iPSC line and either peak KStatistical analyses were performed using SPSS, R and the software packages MASS, multcomp and nnet. All data are presented as mean±s.e.m. Sample sizes were similar to those used in previous studies.How to cite this article: Devlin, A.-C. et al. Human iPSC-derived motoneurons harbouring TARDBP or C9ORF72 ALS mutations are dysfunctional despite maintaining viability. Nat. Commun. 6:5999 doi: 10.1038/ncomms6999 (2015).A.-C.D., S.C. and G.B.M. designed the study. A.C.D. performed experiments and analysed the data. G.B.M. supervised the project. K.B. maintained iPSC lines. K.B. and E.M.C. validated iPSC lines. S.B., I.G., L.V. and C.E.S. generated and provided iPSC lines. A.-C.D. and G.B.M. interpreted the data and wrote the manuscript. J.D.F. performed statistical analyses and modelling, and drafted text for these parts of the manuscript. A.C.D., C.E.S., S.C. and G.B.M. edited and revised the manuscript.Supplementary Figures 1-3, Supplementary Tables 1-2, and Supplementary References


To our surprise, melanoma tumors formed from β‐catenin‐deficient group (B16F10 mixed with β‐catenin‐deficient fibroblasts) appeared earlier than tumors formed from control group . At the end point when tumors were collected, mutant tumors were bigger and heavier than control tumors. Further analysis showed that there were fewer amounts of stromal fibroblasts and myofibroblasts inside mutant tumor stroma. Melanoma tumors from control group showed reduced proliferation, down‐regulated expression of cyclin D1 and increased expression of cyclin‐dependent kinase inhibitor p16, suggesting dermal fibroblasts blocked the onset of melanoma tumor formation by inducing a cell cycle arrest in B16F10 melanoma cells. Furthermore, we discovered that dermal fibroblasts prevented epithelial‐mesenchymal transition in melanoma cells. Overall, our findings demonstrated that dermal fibroblasts crosstalk with melanoma cells to regulate in vivo tumor development via multiple mechanisms, and the outcomes of their reciprocal interactions depend on activation states of stromal fibroblasts and stages of melanoma development.Stromal fibroblasts are an integral part of the tumor stroma and constantly interact with cancer cells to promote their initiation and progression. However, the role and function of dermal fibroblasts during the early stage of melanoma development remain poorly understood. We, therefore, designed a novel genetic approach to deactivate stromal fibroblasts at the onset of melanoma formation by targeted ablation of  Cutaneous melanoma consists of not only melanoma cells but also a heterogeneous mix of genetically stable non‐cancer cells, including endothelial cells, immune cells, and fibroblasts, embedded in a tumor‐specific extracellular matrix (ECM) One major source of CAFs in melanoma is resident normal skin fibroblasts (NFs), which are recruited and activated by chemical factors released from melanoma cells β‐catenin signaling is one of the key signaling pathways that are deregulated in a variety of human cancer cells, including stromal fibroblasts β‐catenin signaling in fibroblasts leads to diseases such as skin fibrosis β‐catenin not only functions as a transcriptional coactivator to regulate the proliferation and differentiation of fibroblasts, and also constitutes as a critical component of β‐catenin/E‐cadherin complex to control fibroblast motility and migration, which greatly influence the wound healing speed and wound size Wnt/in vivo by targeted ablation of β‐catenin. Our results demonstrated that dermal fibroblasts function as physical and signaling barriers to block melanoma formation at the initiation stage before melanoma cells stimulate them to transdifferentiate into CAFs.In this study, we developed a novel genetic approach to deactivate stromal fibroblasts Col1α2‐CreER mice were generated as described previously Ctnnb1fl/fl mice were obtained from the Jackson laboratory . Ctnnb1fl/fl mice were crossed with Col1α2‐CreER mice for several generations to generate Ctnnb1fl/fl; Col1α2‐CreER mice. Ctnnb1fl/fl; Col1α2‐CreER mice were further back crossed with B6 mice for ten generations. Mice were genotyped by polymerase chain reaction (PCR) analysis of genomic DNA extracted from tail biopsies. The β‐catenin alleles were genotyped with the following primer pair: Forward: 5′‐AAGGTAGAGTGATGAAGGTTGTT‐3′ and Reverse: 5′‐CACCATGTCCTCTGTCTA‐3′. The presence of Cre transgene was identified by PCR using the following primer pair: Forward: 5′‐CGGTCTGGCAGTAAAAACTAT‐3′ and Reverse: 5′‐CAGGGTGTTATAAGCAATCCC‐3′. All mice were housed in the Laboratory Animal Services Facility of the University of Cincinnati under an artificial 12/12 light–dark cycle and were allowed free access to normal mouse feedings and water. The Institutional Animal Care and Use Committee of the University of Cincinnati approved all experimental procedures involving mice.μL bleomycin sulfate (Sigma‐Aldrich) was injected subcutaneously into each mouse every day for 30 days to induce the activation of fibroblasts as described in the text. Back skin of induced mice was harvested at postnatal day 60 (P60) for histological analysis.To induce Cre activity, tamoxifen  in corn oil (10 mg/ml) was administered to adult mice by intraperitoneal (i.p.) injection at 1 mg/g body weight for 7 consecutive days. 100 Ctnnb1fl/fl; Col1a2‐CreER mice and control littermates of genotype Ctnnb1+/fl; Col1a2‐CreER. They were then washed in phosphate buffered saline (PBS) twice, minced with curved scissors into small pieces, and incubated with 5 mg/ml collagenase I for 30 minutes under stirring. Digested skin tissue was further washed twice with calcium‐free Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). After washing, skin pieces were cultured in DMEM supplemented with 10% FBS, 1% MEM nonessential amino acid, 1% Hank's balanced salt solution (HBSS), and 1% Penicillin/Streptomycin (PS) at 37°C with 5% CO2 to let fibroblasts crawl out of the tissue fragments and attach to the plate. After 2 passages, the purity of dermal fibroblasts was examined by flow cytometry analysis. Fibroblasts were then seeded at a concentration of 3x104 cells/m for further analysis. β‐catenin null mutation in fibroblasts were induced by adding 100 ng/mL 4‐hydroxytamoxifen (4‐OHT) into the culture medium and incubated for 48 h before each experiment. Murine B16F10 melanoma cell line was obtained from American Type Culture Collection . Cells were maintained in DMEM supplemented with 10% FBS and 1% PS. All cells were incubated at 37°C in a 5% CO2, 95% air‐humidified incubator. All cell culture media were purchased from Invitrogen  unless otherwise indicated.Fibroblast cultures were established according to published protocol Ctnnb1fl/fl, Col1a2‐CreER fibroblasts and control Ctnnb1+/fl, Col1a2‐CreER fibroblasts were seeded at 300,000 cells/dish in a 10 cm tissue culture dish. 4‐OHT was then added to culture medium to induce Cre recombinase expression and the ablation of β‐catenin. After 48‐h incubation, fibroblasts were disassociated with 0.5% trypsin‐EDTA (Invitrogen) and collected by centrifuge. Cell numbers were counted manually under light‐phase microscope and reseeded for further experiments. To perform cell cycle analysis, cells were washed with ice‐cold PBS and fixed with ice‐cold 70% ethanol for 24 h. After fixation, ethanol was removed by centrifugation, and cells were suspended in 0.5 mL propidium iodide (PI)/RNase solution and incubated at 37°C for 30 min in the dark. Stained cells were analyzed using a BD FACSCanto flow cytometer  to quantify DNA content in cells. Cell cycle histograms were generated from flow cytometry data, and the percentage of fibroblasts in each cell cycle phase was determined. Data analysis was performed using the Modfit LT software .™ Real‐Time PCR system  as previously published. qPCR primers for Egf, Fn1, Hgf, MMP2, Tgfα, Tgfβ1, TNC, were purchased from Qiagen. All qPCR data were normalized to GAPDH expression.Total RNA was isolated from mouse dermis using RNeasy Fibrous Tissue mini kit or from cultured fibroblasts using RNeasy Mini Kit from Qiagen . Subsequently total RNA was reverse transcribed to complementary DNA (cDNA) using Superscript III kit (Invitrogen). Quantitative PCR (qPCR) reactions were performed using the Power SYBR green dye in the StepOnePlusCtnnb1fl/fl; Col1α2‐CreER fibroblasts or control Ctnnb1+/fl; Col1α2‐CreER fibroblasts at a ratio of 1:2 were injected intradermally into the flanks of B6 wild‐type mice with a total cell number of 6 × 104 for each tumor. Size of tumors was measured and recorded every other day for a maximum of 7 days after tumor appeared. All mice were then sacrificed, and the tumors were harvested and fixed with 4% (w/w) formaldehyde in PBS for paraffin sections or prepared for western blot analysis.All animal experiments were conducted with approval from the University of Cincinnati Institutional Animal Care and Use Committee. To induce melanoma formation, B16F10 murine melanoma cells mixed with either mutant β‐gal) staining and immunostaining according to published protocols β‐catenin , anti‐PDGFR‐α , anti‐prolyl‐4‐hydroxylase‐ β(P4HB) , anti‐BrdU , anti‐Ki67 , anti‐Cyclin D1 , anti‐E‐cadherin , anti‐Vimentin , anti‐N‐cadherin , anti‐phospho‐pRb . The images were taken under a Nikon Eclipse 80i fluorescence microscope.Paraffin or frozen tumor tissue sections were prepared for collagen sirius red staining, histological analysis, BrdU incorporation assays, TUNEL assays, senescence‐associated‐beta‐galactosidease , anti‐pERK1/2 MAPK , anti‐pospho‐pERK1/2MAPK , anti‐pRb , and anti‐p16 . To verify equal loading of samples, the membranes were incubated with monoclonal β‐actin antibody, followed by an HRP‐conjugated goat anti‐mouse IgG.Immunoblotting was performed as described elsewhere. The following primary antibodies were used: anti‐t‐test. Data were analyzed using a GraphPad Prism 5.01 software package  and expressed as the mean ± SEM. Differences are considered significant at P < 0.05.Statistical analysis of difference was carried out by student's β‐catenin is involved in many different cellular processes, such as cell growth and differentiation. It has been reported that stromal fibroblasts located in and around breast carcinomas frequently expressed nuclear β–catenin β‐catenin activity in melanoma‐associated fibroblasts, we co‐stained human melanoma tissue paraffin sections with an anti‐β‐catenin antibody and an anti‐fibroblast antibody TE7 β‐catenin. As shown in Figure β‐catenin while 32.47 ± 3.18% of them were nuclear localized, suggesting stromal β‐catenin activity is involved during melanoma formation and development.β‐catenin loss could potentially deactivate stromal fibroblasts and ablate their biological activities, we have introduced a conditional gene knockout mouse model, Col1α2‐CreER mouse , compared with 0.35 mm ± 0.05 mm in Ctnnb1fl/fl; Col1α2‐CreER mutant mice (n ≥ 3) . Collagen protein is mainly produced by skin fibroblasts. qPCR showed a significant decrease in production of representative cytokines, growth factors, and ECM proteins in dermis of mutant mice when compared with control mice , transforming growth factor beta1 (TGF‐β1), matrix metalloproteinase 2 (MMP2), tenascin C (TN‐C), and fibronectin 1 (FN1). TN‐C and FN1 are pro‐survival ECM proteins normally secreted by stromal fibroblasts β‐catenin in fibroblasts in vivo indeed prevented dermal fibroblasts from being activated by bleomycin stimulation and abrogated their biological functions.We generated a double transgenic mouse model, in vitro and in vivo tumor microenvironment investigation, dermal fibroblasts were isolated from neonatal mice carrying either Ctnnb1fl/fl; Col1α2‐CreER (bcat/Fb) or Ctnnb1+/fl; Col1α2‐CreER (Fb) alleles for culture as described α), which is a surface marker for dermal fibroblasts α‐positive fibroblasts. Fibroblasts were then seeded at a density of 3 × 105 cells/dish in 10 cm tissue culture dish. Ablation of β‐catenin in Ctnnb1fl/fl; Col1α2‐CreER fibroblasts was induced by adding 4‐OHT to the culture medium at a concentration of 100 ng/mL. Control Ctnnb1fl/+; Col1α2‐CreER fibroblasts underwent the same treatment. After 48‐h incubation, 4‐OHT was removed and replaced with fresh medium with 10% FBS. Ablation of β‐catenin expression in mutant fibroblasts was confirmed by western blotting  phase to G2 (second gap phase) of the cell division cycle  or Fb fibroblasts  can be injected intradermally into the flanks of B6 mice to induce melanoma tumor formation without immune rejection. The B16F10 mouse melanoma line is widely used as a model system for cancer biology research because of its highly invasive and metastatic potential Based on the above data, we decided to use targeted ablation of β‐catenin‐deficient group and the control group was compared using student's t‐test. The mice were sacrificed 2 weeks after tumor grafting, and tumors were collected for analysis. All tumors were encapsulated. We did not detect any local invasion or remote metastasis because the period of tumor development was limited – within a 2‐week time frame.To determine the potential effects of deactivated stromal fibroblasts on melanoma onset, the B16F10 mice melanoma cells were mixed with 4‐OHT pretreated Fb  or bcat/Fb fibroblasts  at the ratio of 1:2 and injected intradermally into left or right flank of wild‐type B6 mice to induce melanoma formation as illustrated in Figure αIn general, it took 5 days for B16F10; bcat/Fb mixture to form a melanoma tumor while mixture of B16F10; Fb needed an average of 7–8 days. For the convenience of tumor size comparison and calculation, we consider the day when the B16F10; Fb tumor appeared as day 1. As shown in Figure β‐Gal staining. In line with the slow melanoma formation from B16F10; Fb cell mixture, B16F10; Fb tumors had more senescent cells than B16F10; bcat/Fb tumors (data now shown).To determine the underlying cause of the suppressed melanoma formation by normal dermal fibroblasts, we assayed for changes in cell proliferation and apoptosis. As shown in Figure β‐catenin ablation. Cyclin D1 forms a complex with cyclin‐dependent kinase CDK4/6 resulting in the sequestration of the CDK inhibitors, p16(INK4a), p21(cip1), and p27(kip1) β‐catenin‐deficient fibroblasts was confirmed by western blots  occurs in most tumorigenic cells including melanoma cells, resulting in increased tumor invasion and metastasis Melanomas are well known for their aggressive clinical behavior, high propensity to metastasize, and resistance to therapeutic treatments. Clinical studies have demonstrated that melanomas arise from pigment‐producing melanocytes due to genetic mutations. Normal melanocytes reside predominantly at the junction of the epidermis and the dermis forming an “Epidermal Melanin Unit” with up to thirty‐six keratinocytes. It has been reported that melanocytes are restricted in the epidermis due to the E‐cadherin‐mediated interactions with keratinocytes Dermal fibroblasts generally appear to be quiescent under normal healthy conditions and function as required to maintain tissue homeostasis β‐catenin signaling, TGF signaling, Notch signaling, etc. Wnt/β‐catenin signaling is one of the key signaling pathways that are involved in a variety of cellular processes such as proliferation, apoptosis, migration, and inflammation. It has been shown that Wnt/β‐catenin signaling plays an important role in fibroblast growth β‐catenin activity in mouse dermal fibroblasts resulted in abnormally activated phenotype that is a resemblance to human fibrosis β‐catenin expression was detected in stromal fibroblasts surrounding and infiltrating human melanoma stroma, suggesting β‐catenin signaling possibly plays an important role in stromal fibroblasts in the melanoma tumor microenvironment.In order to determine the role and function of normal dermal fibroblasts at the initiation stage of melanomagenesis, we have designed a novel genetic approach to deactivate dermal fibroblasts that could be manipulated at different stages of melanoma formation. The biological activities of dermal fibroblasts can be regulated by an array of cellular signaling pathways, including Wnt/Col1α2‐CreER mouse model drives the unique expression of Cre recombinase in dermal fibroblasts under the control of the fibroblast‐specific Col1α2 promoter, which is most active in dividing fibroblasts such as during wound healing, fibrosis, and tumor formation in vivo to remove a specific DNA fragment that is flanked by loxP sites but is not active unless mouse is treated with tamoxifen or 4‐OHT. The combination of this Col1α2‐CreER mouse with β‐catenin loss‐of‐function Ctnnb1fl/fl mouse enables the inducible ablation of β‐catenin activity in dermal fibroblasts in vitro or in vivo.Transgenic β‐catenin‐deficient mice appeared much thinner than that of control littermates after bleomycin stimulation, suggesting proliferation of dermal fibroblasts was inhibited. Interestingly, the production of collagen fiber, chemical factors, and ECM proteins that are normally generated by dermal fibroblasts were also greatly affected. In vitro culture of β‐catenin‐deficient fibroblasts and normal fibroblasts showed same phenotypes as in vivo observation. β‐catenin‐deficient fibroblasts grew much slower than normal fibroblasts. Further cell cycle analysis indicates that there was a cell cycle arrest in dermal fibroblasts upon β ‐catenin ablation. Our results suggest that β‐catenin depletion is a useful tool to deactivate dermal fibroblasts in vivo. Targeted ablation of stromal β‐catenin could be applied to study the cellular role and function of stromal fibroblasts at different stages of melanoma formation.By studying this mouse model in bleomycin‐induced skin fibrosis, we discovered that the dermis of β‐catenin in dermal fibroblasts associated with melanoma cells indeed deactivated them, and this deactivation actually allowed B16F10 melanoma tumor to grow faster as compared with melanoma tumor formed with normal fibroblasts. This result is possibly contrary to existing concepts but not completely unexpected. Although it is widely accepted that stromal fibroblasts are involved in promoting tumor initiation, growth and metastasis in many different types of solid tumors, including melanoma, some papers suggested that stromal fibroblasts initially were not cancer‐supportive and exhibited suppressive effects, and only promoted tumor progression when abnormally activated to become CAFs in vivo and in vitro β‐catenin ablation in fibroblasts experiments, few fibroblasts could be detected in B16F10; bcat/Fb tumor samples. This reduction consequently diminished the inhibitory effects of dermal fibroblasts on melanoma tumor formation. Our findings provide clear evidences that dermal fibroblasts fight against melanoma tumor growth at the onset stage.By grafting mixture of B16F10 melanoma cells and dermal fibroblasts, we have a valid approach to determine how resident fibroblasts function at the initial stage of melanoma formation. Our work showed that disruption of β‐catenin ablation and no longer able to release enough inhibitory signals, melanoma showed increased proliferation, decreased cell senescence, and apoptosis. Uncontrolled cell cycle is a major driver of unrestricted tumor cell growth. In our tumor grafting experiment, B16F10; bcat/Fb melanoma tumors showed upregulated expression of cyclin D1 and decreased expression of CDK inhibitor p16 as compared with B16F10; Fb melanoma. Cyclin D1 is a direct target of the MAPK/ERK signaling pathways, which controls cellular processes such as cell proliferation and migration in cancer development Melanoma cells and dermal fibroblasts communicate throughout the tumorigenic process. Although genetic mutations in melanoma cells cause regulatory mechanisms that monitor DNA damage, repair and cell cycle progression to malfunction, our experimental results also revealed that the tumor microenvironment including CAFs could potentially send positive or negative signals to tumor cells to influence their biological behaviors. When dermal fibroblasts were deactivated by One hallmark of cancer is that tumor cells progressively acquire the abilities to escape primary tumor sites, invade adjacent tissues and colonize at distant sites β‐catenin signaling may also exert different roles in the biological activities of normal fibroblasts and CAFs. It would be very interesting to understand in future whether deactivation of CAFs during later stage of melanoma development could effectively inhibit malignant expansion of tumor cells and bypass genomic instability considering β‐catenin ablation deprives stromal fibroblasts of their biological functions. β‐catenin may serve as a potential target for a combination treatment targeting both the host and melanoma tumor cells.Stromal fibroblasts may play distinguished roles before and after interaction with melanoma cells. Our findings provided clear evidence that normal dermal fibroblasts interact with melanoma cells and play a defensive role at the onset of melanoma formation, which happens before they are abnormally activated by stimulatory signals from melanoma cells and acquire tumor‐promoting properties. Upon reciprocal crosstalk with tumor cells, their role and functions may change and become supportive of tumor progression and invasion. On the other hand, The authors declare no conflict of interest.


The technique of bedside percutaneous tracheostomy (BPT) has become a routine procedure in the ICU. As a common standard, a bronchoscope is used during the BPT in order to assess the upper airway anatomy and to assist guiding the needle while penetrating the tracheal inter-ring space into the correct central positioning. However, using the bronchoscope during the puncture of the trachea holds a potential patient safety risk due to the possibility of inaccurate device placement and associated temporary hypoventilation. Moreover, an accidental needle puncture of the bronchoscope is a frequent problem and leads to high repair costs as well as to prolonged downtime periods of the damaged device. It is therefore important to further improve the BPT technique aiming towards minimizing patient safety risks as well as eliminating avoidable technical and financial burdens for healthcare institutions.We have recently reported a series of 25 patients subjected to BPT combining realtime ultrasound guidance with a balloon dilatational device . The tecIn order to compare our own previously reported group  of realtime ultrasound guided BPT procedures with a conventional ETT in place, we changed the protocol to preprocedural LMA ventilation in another group of critically ill patients  scheduled for tracheostomy.As control data, we used our own ETT group , who underwent BPT with ultrasound guidance and applying a balloon dilatational technique. The average time of the procedure in the ETT group was 15.9 minutes and no complications have occured. In the LMA group , the same procedural approach was used with the exception of inserting a LMA before the procedure. In the LMA group, the average time of the procedure was 12.4 minutes (p=0.004) and likewise, no complications have occurred.We have demonstrated in a previous serious of patients, that combining realtime ultrasound guidance and balloon dilatational technique in performing BPT is easily feasible and safe. By inserting a LMA before the BPT – instead of keeping the ETT in place - we successfully further improved this concept by adding additional beneficial features such as procedural time reduction and improved ultrasound imaging of sublaryngeal anatomical structures. These additional advantages were achieved without compromising the already established patient safety features of the previous technique. In our practise, this technique has become the standard BPT strategy due to its combination of excellent features, e.g. avoidance of intraprocedural tube misplacement and hypoventilation, improved assessment of sublaryngeal anatomical structures, elimination of accidental bronchoscope damage and minimization of procedural time even in technically challenging patients with obesity and difficult neck anatomy.In our opinion, the strategy of combining realtime ultrasound guided identification of correct tracheal structures, preprocedural insertion of a LMA and the balloon dilatational approach represents the least invasive technique of performing BPT. It can be done easily, safely and significantly further shortened the procedural time.


Canonical Wnt signaling pathway, also known as Wnt/β-catenin signaling pathway, is a crucial mechanism for cellular maintenance and development. It regulates cell cycle progression, apoptosis, proliferation, migration, and differentiation. Dysregulation of this pathway correlates with oncogenesis in various tissues including breast, colon, pancreatic as well as head and neck cancers. Furthermore, the canonical Wnt signaling pathway has also been described as one of the critical signaling pathways for regulation of normal stem cells as well as cancer cells with stem cell-like features, termed cancer stem cells (CSC). In this review, we will briefly describe the basic mechanisms of Wnt signaling pathway and its crucial roles in the normal regulation of cellular processes as well as in the development of cancer. Next, we will highlight the roles of canonical Wnt signaling pathway in the regulation of CSC properties namely self-renewal, differentiation, metastasis, and drug resistance abilities, particularly in head and neck squamous cell carcinoma. Finally, we will examine the findings of several recent studies which explore druggable targets in the canonical Wnt signaling pathway which could be valuable to improve the treatment outcome for head and neck cancer. Head and neck squamous cell carcinoma (HNSCC) is the most frequent type of head and neck cancer that usually arises from the mucosal surfaces of several organs including nasal cavity, paranasal sinuses, oral cavity, tongue, pharynx, and larynx . GloballRecently, it has been proposed that the aberrant activation of canonical Wnt signaling pathway in CSC is one of the underlying mechanisms of the progression of cancers as demonstrated in several cancer studies such as colon , lung Z, breast Stem cells can be classified as a group of cells which are able to self-renew through indefinite cell division over a long period of time and have the potential to differentiate into several specific cell types. They do not perform any specialized function in the body as they do not have any tissue-specific structures. When triggered by either intrinsic or extrinsic signals, they undergo cell division to produce daughter cells where each daughter cell could remain as a stem cell or become a progenitor cell that further differentiates into a mature cell with more specialized functions . In geneRecently, the theory of CSC has rapidly evolved owing to the first discovery of similarities in the stemness properties between normal hematopoietic stem cells (HSC) and a rare population of cells isolated from acute myeloid leukemic (AML) cells. In xenotransplantation of immunodeficient mice, these stem-like cells, termed severe combined immunodeficiency disease leukemia-initiating cells (SL-IC), proliferate, differentiate, and eventually initiate AML, a disease that was identical to the original human donor. These SL-IC also exhibit a remarkable self-renewing capacity whereby they promote reestablishment of AML in a secondary recipient mouse injected with SL-IC derived from the primary immunodeficient mice . FollowiGenerally, the regulatory mechanisms of stem cell features for both normal and CSCs are mainly associated with the homeostatic regulation by neighboring cells named niche, which provides a specialized microenvironment by generating extrinsic signals to regulate stem cell proliferation and cell fate determination . SeveralWnt signaling pathway is an important mechanism for embryonic development and homeostasis of mature tissues . It playNon-canonical Wnt signaling pathways, which are independent of β-catenin, rely on the signal transduction of Wnt through Fzd as well as its coreceptors such as receptor tyrosine kinase-like orphan receptor 2 (ROR2) or receptor-like tyrosine kinase (RYK) . The sigFigure 1), Axin-GSK3-CK1 complex is formed when GSK3 and CK1 bind to the separate domains of the Axin. Meanwhile, cytoplasmic β-catenin directly binds to adenomatous polyposis coli , the binding of Wnt to Fzd and its co-receptor, lipoprotein receptor-related protein 5 or 6 (LRP 5/6), leads to the formation of Wnt-Fzd-LRP 5/6 complex. This is followed by the recruitment of Dvl to Fzd , bone morphogenetic protein 4 (BMP4), matrix metalloproteinases 7 (MMP7), AXIN2, multi-drug resistance 1 (MDR1), β-TRCP, and endothelial growth factor . For instance, alteration in CTNNB1, a gene encoding β-catenin, causes prolonged half-life of β-catenin due to changes either at the APC recognition site or GSK3 phosphorylation site of β-catenin, by which both sites are important for degradation of β-catenin , Fzd homolog 3 , and Dvl homolog (GenBank Accession No. U46461) genes, which are functionally important in the canonical Wnt signaling pathway, were highly expressed in HNSCC by two- to fivefold compared to normal tissues . Furtherin vitro studies of breast, colon and head and neck cancers demonstrated that the pathway is highly activated in CSC compared to normal stem cells. For instance, overactivation of this pathway was demonstrated in breast tumor sphere growth, a method that enriches CSCs, compared to enriched normal breast stem cells (AXIN2 and LEF1 as well as a reduction in Dickkopf 1 (DKK1), which is the Wnt-induced Fzd-LRP 5/6 complex inhibitor . This pathway has been demonstrated in many studies as a regulator of CSC traits in several cancers such as breast, colon, lung, pancreatic, and head and neck cancers, thus suggesting its critical roles in tumor recurrence. In this review, the roles of the pathway in regulating the stemness properties of CSC in a common type of head and neck cancer, which is HNSCC, will be further discussed in details.All these recent findings implied the critical roles of canonical Wnt signaling pathway in cell regulation in normal condition of the human body as well as in cancer development through regulation of cell expansion and survival of both mature and stem cells by their direct and/or indirect target genes , CSC proliferation rate has shown to be influenced by the modulation of canonical Wnt signaling pathway through Wnt activator, 6-bromoin-dirubin-3′-oxime (BIO) and Wnt inhibitor, DKK1. Neither BIO nor DKK1 showed significant differences in its proliferative effects in parental OSCC cells, suggesting that the influence of the pathway on cell proliferation is limited to CSC only . Increasin vivo when induced with HNE1 cells which presumably contains CSC. In contrast, the control group without the inhibitor formed progressive growing tumor rapidly after a long latency period  process. In cancer, EMT process involves the disruption of cell adhesion and loss of cell polarity which eventually allows cells to migrate from the primary tumor, metastasize to distant sites, and undergo mesenchymal-epithelial transition (MET) at the metastatic sites in the body . SeveralMMP7 gene expression in OSCC cells with highly activated canonical Wnt signaling pathway. The increase in MMP7 expression resulted in redistribution of E-cadherin and cytoskeleton changes, which are often observed in early EMT , a family of enzymes that acts as proteolytic destructor of basement membrane and extracellular matrix (ECM) components including E-cadherin, is often correlated with the invasiveness and metastasis of cancer . The levarly EMT .CDH1, which is the gene that encodes for E-cadherin  cells whereby the activation of canonical Wnt signaling pathway promotes the increase in EMT-related protein expression, particularly Twist, and eventually contributes to cell metastasis through the loss of E-cadherin , which is an enzyme necessary for Wnt palmitoylation, secretion and biologic activity. For instance, inhibition of PORCN leads to reduction in palmitoylation of serine residue of Wnt to suppress Wnt secretion and its binding interaction to Fzd . In receIn vivo studies of those PORCN inhibitors have shown significant tumor growth regression with evident downregulation of Wnt target genes. Furthermore, the toxicity study also displayed no pathological changes in other normal Wnt-dependent tissues, including the intestine, stomach, brain, and skin, with no significant body weight loss at effective and well-tolerated dose of those inhibitors. In contrast, a very high dose promotes excessive inhibition of Wnt signaling pathway and causes significant effects to those Wnt-dependent tissues, including alopecia, intestinal damage, body weight loss, neurologic impairment, and increased neutrophils secondary to inflammation , a regulator enzyme for Axin homeostasis, had been studied in colorectal cancer. XAV939 causes upregulation of Axin level and prolongs the half-life of Axin. An increase in Axin, which is the concentration-limiting component of the destruction complex in canonical Wnt signaling pathway, stimulates β-catenin degradation and eventually diminishes the pathway activation . BesidesOther than that, several non-steroidal anti-inflammatory drugs, including aspirin and indomethacin, commonly used for treatment of pain, inflammation and fever, have been reported to inhibit the oncogenic canonical Wnt signaling pathway activity. It has been reported that indomethacin reduces canonical Wnt signaling pathway activity in a time- and dose-dependent manner through phosphorylation of NH2-terminal serine/threonine residues of β-catenin in colorectal cancer cells . AspirinWIF1). Subsequently, this will restore the expression of WIF1 protein which acts as a Wnt antagonist by direct binding to Wnt molecules for downregulation of canonical Wnt signaling pathway , wild type β-catenin plasmid or mutant S33Y β-catenin plasmid (containing gene of mutant β-catenin which is resistant to Axin-APC-GSK3β destructive complex) in a dose dependent manner. Furthermore, they also found that the amount of nuclear β-catenin was reduced while no changes in the level of cytoplasmic β-catenin were seen after curcumin treatment. These findings suggested that the action of curcumin is independent of Axin-APC-GSK3β multiprotein destructive complex machinery. Rather, it involves the inhibition of nuclear translocation of β-catenin for further β-catenin/TCF transcriptional activities. However, the interaction of curcumin and β-catenin was not further investigated in the study , 2014. O pathway .in vitro inhibition of canonical Wnt signaling pathway and eventually in vivo suppression of tumor growth in pancreatic cancer. OMP54F28 in combination with gemcitabine also showed synergistic reduction of CSC frequency in pancreatic cancer  receptor and human IgG1 Fc fragment, is advancing into the first phase of clinical trial. The binding of OMP54F28 to Fzd8 prevents binding of Wnt to its receptor for downstream Wnt signaling activation. Promising preclinical findings of OMP54F28 showed significant c cancer . Ongoingc cancer .CYCLIN D1 and AXIN2 as compared to a low dose of resveratrol alone. Hence, this finding suggested that resveratrol or resveratrol-containing GP could act as potential colon cancer preventive agents rather than treatment of colon cancer via targeting Wnt signaling pathway , have been examined for their effects on Wnt signaling pathway in colon cancer and normal colonic mucosa of eight volunteer colon cancer patients in a phase 1 pilot clinical trial. No significant inhibitory effect on Wnt signaling pathway was seen in colon cancer tissue for both treatments. Nevertheless, GP with a low dose of resveratrol in combination with other bioactive compounds demonstrated inhibition of Wnt signaling in normal colonic mucosa as indicated by depletion in the expression of Wnt target genes such as  pathway .Promising findings of preclinical studies and early phase of clinical trial of targeted therapy against canonical Wnt signaling-driven tumor have led to new insight into attractive therapeutic interventions for targeting CSC which are also principally regulated by canonical Wnt signaling pathway. As discussed previously, the properties of CSC of HNSCC, such as cell self-renewal, differentiation, metastasis, and chemotherapy resistance, are mainly regulated by the abnormal canonical Wnt signaling pathway activity and hence implicated in the development and progression of HNSCC. Although the crosstalk between multiple signaling pathways plays the vital role in the development of cancer, further understanding of one important molecular mechanism, which is canonical Wnt signaling pathway, is critical as targeting the pathway could be a promising approach in eradicating the treatment failure and relapse in HNSCC.As complex as cancer disease, it is important to note that targeting aberrant canonical Wnt signaling pathway is challenging. Several major obstacles of clinical drug development of Wnt inhibitors are due to either insufficient efficacy, unspecific binding target, or the concern of the therapeutic window of the inhibitors. For instance, the efficacy of inhibitors may be insufficient as a single-agent treatment or be limited by the low bioavailability. Thus, combination treatment with other drugs and development of a good drug delivery system can be a good approach to be applied. Furthermore, since Wnt signaling pathway is also essential for normal cell homeostasis, selective binding of inhibitors and eventually inhibition of the pathway limited to cancer cells is crucial in order to circumvent any unfavorable effects in human body. The other major concern includes the challenge in obtaining the range of doses that produces therapeutic response without causing significant toxicity in patients. Complex and profound study on the efficacy, safety, and toxicity of the candidate drugs is fundamental in pursuing a novel agent that potentially having low side effects and exhibits greater medical benefits outweigh the risks in the patient. Recently, drug repurposing efforts have becoming a prominent alternative to traditional drug discovery. Exploring other uses of the existing drugs is promising as tolerability and safety profiles of these drugs are already well established.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Saccharomyces cerevisiae. Failure to recruit condensin results in an abnormal conformation of the pericentric region and impairs the correction of tensionless chromosome attachments. Moreover, we found that shugoshin is required for maintaining Aurora B/Ipl1 localization on kinetochores during metaphase. Thus, shugoshin has a dual function in promoting biorientation in budding yeast: first, by its ability to facilitate condensin recruitment it modulates the conformation of the pericentric chromatin. Second, shugoshin contributes to the maintenance of Aurora B/Ipl1 at the kinetochore during gradual establishment of bipolarity in budding yeast mitosis. Our findings identify shugoshin as a versatile molecular adaptor that governs chromosome biorientation.Correct chromosome segregation is essential in order to prevent aneuploidy. To segregate sister chromatids equally to daughter cells, the sisters must attach to microtubules emanating from opposite spindle poles. This so-called biorientation manifests itself by increased tension and conformational changes across kinetochores and pericentric chromatin. Tensionless attachments are dissolved by the activity of the conserved mitotic kinase Aurora B/Ipl1, thereby promoting the formation of correctly attached chromosomes. Recruitment of the conserved centromeric protein shugoshin is essential for biorientation, but its exact role has been enigmatic. Here, we identify a novel function of shugoshin (Sgo1 in budding yeast) that together with the protein phosphatase PP2A-Rts1 ensures localization of condensin to the centromeric chromatin in yeast  Accurate chromosome segregation is required for the equal distribution of genetic information to progeny. Failure to equally segregate chromosomes leads to aneuploidy, cell death or cancer. Proteins of the conserved shugoshin family contribute to accurate chromosome segregation in both meiosis and mitosis. The role of shugoshin in protection of centromeric cohesion during meiosis is well understood, but only little is known about shugoshin's function during mitosis. We show that Sgo1 mediates localization of the heterotrimeric phosphatase PP2A-Rts1 to the centromere and that this is in turn important for the efficient recruitment of condensin to the centromere. The failure to load centromeric condensin results in a defect during correction of improper microtubule-kinetochore attachments. Moreover, Sgo1 facilitates the maintenance of a centromeric pool of Aurora B/Ipl1, a conserved mitotic kinase essential for the correction of faulty microtubule-kinetochore attachments. Our results show that Sgo1 operates as a multifunctional hub that coordinates two centromeric functions essential for correct chromosome segregation. Xenopus egg extracts is impaired upon depletion of Sgo2 Accurate chromosome segregation into daughter cells requires the formation of correct chromosome attachments to the mitotic spindle. Each of the sister kinetochores has to attach to microtubules (MTs) emanating from opposite spindle poles in order to achieve biorientation of sister chromatids. Correctly attached and bioriented kinetochores (KTs) generate tension caused by attached MTs, which is, in turn, opposed by sister chromatid cohesion C. elegans is required for centromere resolution Kinetochores, large proteinaceous complexes assembled on centromeric DNA, are crucial interaction hubs for events necessary for accurate chromosome segregation. KTs are flexible and dynamic structures that become stretched when MTs attach and bipolarity is achieved Here, we show that Sgo1 function is essential for the recruitment of condensin to the pericentric region. Moreover, Sgo1 localizes the PP2A subunit Rts1 to the same region, which facilitates the binding of condensin to centromeric chromatin. The failure to load functional condensin onto pericentric chromatin impairs the cellular response to tensionless attachments. Additionally, Sgo1 is required for the maintenance of the Ipl1 kinase on centromeres, which, in turn, allows for correction of erroneous MT-KT attachments. We propose that Sgo1 is a scaffold protein that via spatio-temporal modulation of kinase and phosphatase activity on KTs ensures a dual function - organization of the pericentric chromatin that mediates tension sensing and the maintenance of centromeric Ipl1 that facilitates correction of tensionless attachments.SGO1 with the sgo1-N51I allele resulted in the reduction of the Rts1-GFP signal intensity between the spindle pole bodies (SPBs)  . Chromats (SPBs) , similarI allele . Thus, psgo1-N51I mutation causes sensitivity to the microtubule-destabilizing drug benomyl, indicating that the recruitment of PP2A-Rts1 is crucial for Sgo1's functions in mitosis  and only 10% of cells contained stretched centromeres  was comparable in both wild type and sgo1Δ mutant cells  via its N-terminal coiled-coil domain ng drugs . We alsong drugs , B. VeryThe correct conformation of pericentric chromatin is essential for bioriented MT-KT attachments because it provides the rigidity and at the same time elasticity necessary for chromatin to withstand the opposing forces exerted by spindle MTs What is the function of condensin on centromeres? One possibility is that condensin maintains the conformation of centromeric regions, thereby facilitating the intrinsic bias of budding yeast KTs to biorient on the mitotic spindle sgo1Δ cells and centromeric localization of Ipl1 in rts1Δ cells  or BY4741 (his3Δ1 leu2Δ0 met15Δ0 ura3Δ0). Gene deletions and epitope-tagging were performed using standard PCR-based methods. Cells were grown in either full  or synthetic complete  medium supplemented with 2% glucose (YPD), 2% raffinose (YPR) or 2% galactose (YPG). Cells were arrested in G1 using 10 µM α-factor  or in mitosis using 30 µg/ml nocodazole (Santa Cruz Biotechnology). Mutants were grown at 25 °C to reduce chromosome missegregation. For viability assays cells were grown overnight in full medium, diluted to an OD600 of 0.3 and tenfold serial dilutions were spotted on YPD/YPG plates or on SC plates containing indicated concentrations of benomyl and nocodazole, respectively. The phosphatase inhibitor okadaic acid was used at concentrations of 10 or 20 µM as stated for the individual experiments.All yeast strains used in this study are listed in Supporting GAL1 promoter with a C-terminal TAP- or GFP-tag. SGO1 was cloned with its endogenous promoter into pRS405 with a C-terminal TAP-tag. Plasmids encoding the Mtw1-fusion proteins were constructed by cloning MTW1 with its endogenous promoter adjacent to the corresponding gene into the MCS of pRS405. Chromosomal integration of plasmids encoding Sgo1- or Mtw1-fusion proteins was targeted into the endogenous locus using restriction enzymes cutting in the respective promoter region. Point mutations were introduced using the PCR-based Quick Change (Stratagene) site-directed mutagenesis approach.All plasmids used in this study are listed in Supporting S. cerevisiae were prepared either by glass bead lysis or alkaline lysis followed by TCA precipitation. Proteins were separated by SDS-PAGE, transferred to PVDF or nitrocellulose membranes and detected using antibodies according to standard protocols. Commercially available antibodies were used to detect individual proteins: myc , HA , PAP (Sigma-Aldrich), Clb2 , Pgk1 (Invitrogen).Protein extracts of S. cerevisiae were prepared by glass bead lysis in a lysis buffer  followed by centrifugation . Sgo1-TAP was purified by incubation of approx. 3.5 mg protein extract with pre-washed calmodulin-coated beads . Beads were washed using lysis buffer containing increasing salt concentrations (up to 600 mM KCl) and finally eluted in the presence of 5 mM EGTA. Proteins were precipitated with TCA and co-purified proteins were analyzed by SDS-PAGE followed by immunoblotting.Protein extracts from S. cerevisiae via Rts1- or Cdc55-TAP fusion proteins using calmodulin-coupled agarose as described above. Equal amounts of PP2A complexes were incubated with recombinant Ni-NTA agarose-bound His-Sgo1 fragments containing the conserved coiled-coil domain (amino acids 1–340) in a pulldown buffer  for 2 hours. Proteins were eluted by boiling in SDS sample buffer and subjected to SDS-PAGE and immunoblotting to detect the bound PP2A subunits.Heterotrimeric PP2A complexes were purified from mitotic lysates of Images of cells were obtained using a fully automated Zeiss inverted microscope (AxioObserver Z1) equipped with a MS-2000 stage , a CSU-X1 spinning disk confocal head , LaserStack Launch with selectable laser lines  and an X-CITE Fluorescent Illumination System. Images were captured using a CoolSnap HQ camera  under the control of the Slidebook software . All fluorescence signals were imaged with a 63x oil objective. A total of 10 z-stacks were collected and each optical section was 0.4 µm thick. Projected images were used for display. Only cells with spindles shorter than 2 µm and fully localized in the mother cell were scored to evaluate the localization in pre-anaphase. For Ipl1-GFP microscopy we defined three types of Ipl1-GFP localization: “centromeric” – fluorescence signal only in the area between the SPBs, “partially diffuse” – diffuse nuclear fluorescence signal and increased fluorescence intensity between the SPBs and “diffuse” – only diffuse nuclear fluorescence signal.Chromatin immunoprecipitation (ChIP) was performed as previously described Figure S1Functional interactions between Sgo1 and PP2A. (A) The heterotrimeric PP2A-Rts1 complex was purified from mitotic protein lysates via Rts1-TAP. To analyze the interaction between Rts1 and Sgo1, the purified PP2A-Rts1 complex was loaded either on the Ni-NTA-bound wild type or mutant His-N-terminal fragment of Sgo1 (+) or on empty Ni-NTA beads (-) as a negative control. (B) The heterotrimeric PP2A-Cdc55 complex was purified from mitotic protein lysates via Cdc55-TAP . To analyze the interaction between Cdc55 and Sgo1, the purified PP2A-Cdc55 complex was loaded either on the Ni-NTA-bound wild type or mutant His-N-terminal fragment of Sgo1 (+) or on empty Ni-NTA beads (-) as a negative control. (C) Enrichment of Rts1-FLAG on centromeric DNA  and on rDNA (NTS1-2) normalized to the levels of Rts1-FLAG bound to the arm of chromosome 10 in mitotic cells. ChIP-qPCR experiments of Rts1-FLAG were performed using wild type and sgo1Δ cells arrested with nocodazole. Error bars represent the standard error of the mean. (D) Genetic interactions between SGO1, CDC55, RTS1 and sgo1-N51I mutants.(EPS)Click here for additional data file.Figure S2Genetic interactions between Sgo1 and PP2A. (A) Sensitivity of SGO1-deficient cells and mutants to the microtubule depolymerizing drug benomyl. (B) Schematic depiction of Sgo1 and its domains. sgo1-N51I impairs interaction with PP2A-Rts1, sgo1-T379D mutation impairs localization to the centromere. (C) Sensitivity of strains with Mtw1-fusion proteins to the microtubule depolymerizing drug benomyl. (D) Schematic representation of the fusion proteins used in GAL1 promoter to the microtubule depolymerizing drug benomyl. (G) Expression levels of the overexpressed pGAL1-Rts1-TAP.(EPS)Click here for additional data file.Figure S3Recruitment of Rts1 to the kinetochores via Sgo1 is required for correction of erroneous (tensionless) attachments. (A) Chromosome segregation (centromeres of chromosome 4 labeled by tetO/TetR-GFP) in cells released from nocodazole-induced arrest. Cells were collected 75 min after the release from nocodazole arrest into fresh medium containing α-factor (to prevent entry into another cell cycle), fixed with 3.7% formaldehyde and imaged. A mean of three experiments with standard deviation is shown. Top: Schematic depiction of the experiment. (B) Time course of the establishment of biorientation after the release from nocodazole-induced arrest. Cells were released from cell cycle arrest into fresh medium containing α-factor (to prevent entry into another cell cycle) and samples were collected at indicated time points, fixed with 3.7% formaldehyde and imaged. A mean of four experiments with standard deviation is shown. Red line – rate of SPB separation; green line – rate of chromosome biorientation. Micrographs: left: examples of cells with and without bioriented chromosomes; right: examples of cells correctly segregating and missegregating chromosome 4. Bar – 1 µm. (C) Progression through the cell cycle upon induction of the spindle assembly checkpoint by nocodazole. Top – Schematic depiction of the experimental setup. Cells were synchronized in G1 by α-factor and released into medium containing nocodazole. Samples were collected at indicated time points.(EPS)Click here for additional data file.Figure S4sgo1Δ and rts1Δ cellsLocalization of condensin and cohesin subunits in . (A) Localization of the cohesin subunit Smc3-GFP in pre-anaphase wild type, sgo1Δ and rts1Δ cells. SPBs are marked with Spc29-RFP. Bar – 5 µm. (B) Quantification of the localization of the cohesin subunit Smc3-GFP in wild type, sgo1Δ and rts1Δ cells. Means with SD of three independent experiments are shown. At least 150 cells were scored in each experiment. (C) Enrichment of Mcd1-FLAG on centromeric DNA  and on rDNA (NTS1-2) normalized to the levels of Mcd1-FLAG bound to the arm of chromosome 10 in mitotic cells. ChIP-qPCR experiments of Mcd1-FLAG were performed using wild type and sgo1Δ cells arrested with nocodazole. Error bars represent the standard error of the mean. (D) Localization of the condensin subunit Smc2-GFP in wild type, sgo1Δ and rts1Δ cells. SPBs are marked with Spc29-RFP. Yellow arrowhead - centromeric Smc2-GFP. Bar – 5 µm. (E) Quantification of the localization of the condensin subunit Smc2-GFP in wild type and sgo1Δ cells. Means with SD of three independent experiments are shown. At least 150 cells were scored in each experiment.(EPS)Click here for additional data file.Figure S5The Lrs4-Csm1 complex is not involved in the establishment of biorientation. (A) Localization of Csm1-GFP. Yellow arrowheads indicate the absence of Csm1-GFP from the centromere/kinetochore. White arrowheads indicate rDNA localization of Csm1-GFP. Bar – 5 µm. (B) Deletion of the CSM1 and LRS4 genes does not lead to a growth defect in the presence of high levels of syntelic attachments.(EPS)Click here for additional data file.Figure S6Localization of Kin4-GFP upon treatment with a PP2A inhibitor. (A) Localization of Kin4-GFP in cells with and without treatment with okadaic acid. White arrow indicates the correct localization of Kin4-GFP to the mother spindle pole during anaphase. Bar – 5 µm. (B) Quantification of the changes in the Kin4 localization upon treatment with okadaic acid (20 µM). Mean of three independent experiments with standard deviation is shown.(EPS)Click here for additional data file.Figure S7SGO1.Analysis of Ipl1 localization in cells lacking (A) Localization of Ipl1-GFP in small budded cells in wild type and in cells lacking Sgo1. Bar – 5 µm. (B) Dependency of Ipl1-GFP localization on the length of the mitotic spindle (measured as distance between SPBs marked with Spc29-RFP). Only mitotic spindles shorter than 2 µm were counted. Means of two independent experiments are shown. At least 100 cells were counted in each experiment.(EPS)Click here for additional data file.Figure S8smc2-8 cellsLocalization of Sgo1-GFP, Rts1-GFP, Ipl1-GFP and Ycg1-GFP in . (A) Control analysis of Ycg1-GFP at the pre-anaphase spindles confirms the condensin deficiency in smc2-8 mutants at high temperature. (B) Localization of Sgo1-GFP at the pre-anaphase spindles is not markedly altered in cells with defective condensin. (C) Localization of Rts1-GFP at the pre-anaphase spindles is only slightly altered in cells with defective condensin, but the effect is not temperature-dependent. (D) Localization of Ipl1-GFP at the pre-anaphase spindles is impaired in cells with defective condensin. Means of three independent experiments (two in Ipl1-GFP) and standard deviation (not in Ipl1-GFP) are shown.(EPS)Click here for additional data file.Table S1Yeast strains used in this study.(DOC)Click here for additional data file.Table S2Plasmids used in this study.(DOC)Click here for additional data file.


These results suggest that GSK-3β-mediated β-catenin degradation in adherens junctions is one of the key mechanisms of vascular hyperpermeability in severe influenza.Multiorgan failure with vascular hyperpermeability is the final outcome in the progression of seasonal influenza virus pneumonia and influenza-associated encephalopathy, and it is also common in infection with highly pathogenic avian influenza virus. However, the precise molecular mechanism by which influenza virus infection causes vascular endothelial cell hyperpermeability remains poorly defined. We investigated the mechanisms of hyperpermeability of human umbilical vein endothelial cells infected with influenza A virus (IAV)/Puerto Rico/8/34 (PR8) (H1N1). The levels of β-catenin, a key regulatory component of the vascular endothelial-cadherin cell adhesion complex, were markedly decreased during infection for 28 h, with increments of vascular hyperpermeability measured by transendothelial electrical resistance. Lactacystin (at 2 μM), a proteasome inhibitor, inhibited the decrease in β-catenin levels. Since the N-terminal phosphorylation of β-catenin by glycogen synthase kinase (GSK)-3β is the initiation step of proteasome-dependent degradation, we examined the effects of GSK-3β suppression by RNA interference in endothelial cells. IAV-infection-induced β-catenin degradation was significantly inhibited in GSK-3β-knockdown cells, and transfection of cells with recombinant β-catenin significantly suppressed IAV-induced hyperpermeability. These findings suggest that IAV infection induces GSK-3β-mediated β-catenin degradation in the adherens junctional complexes and induces vascular hyperpermeability. The  Influenza A virus (IAV) is the most common infectious pathogen in humans and causes significant morbidity and mortality, particularly in infants and the elderly population , 2. Mult2+]i) and loss of tight-junction protein ZO-1 via PAR-2 in the BBB i and loss of ZO-1 via proteinase-activated receptor-2 signaling i i .Francisella tularensis, the causative agent of highly virulent tularemia [Burkholderia pseudomallei [GSK-3β is a serine/threonine protein kinase that has been shown recently to play a key role during the inflammatory response induced by various pathogenic bacteria, such as ularemia , Burkholdomallei  and groudomallei . Bacteridomallei . In the domallei , TLR7/8 domallei , furtherIn summary, our results show that infection of HUVECs with IAV PR8 markedly decreases β-catenin levels in the VE-cadherin-β-catenin adhesive complex of HUVECs, together with hyperpermeability of HUVEC monolayers. The level of β-catenin in the adhesive complex is tightly regulated by activation of GSK-3β and the phosphorylation-dependent ubiquitin-proteasome degradation pathway. Destruction of the VE-cadherin-β-catenin adhesive complex could be one of the main pathomechanisms of MOF after severe IAV infection.