Presentation_1_Calcitonin gene-related peptide is a potential autoantigen for CD4 T cells in type 1 diabetes.pdf

The calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide with critical roles in the development of peripheral sensitization and pain. One of the CGRP family peptides, islet amyloid polypeptide (IAPP), is an important autoantigen in type 1 diabetes. Due to the high structural and chemical similarity between CGRP and IAPP, we expected that the CGRP peptide could be recognized by IAPP-specific CD4 T cells. However, there was no cross-reactivity between the CGRP peptide and the diabetogenic IAPP-reactive T cells. A set of CGRP-specific CD4 T cells was isolated from non-obese diabetic (NOD) mice. The T-cell receptor (TCR) variable regions of both α and β chains were highly skewed towards TRAV13 and TRBV13, respectively. The clonal expansion of T cells suggested that the presence of activated T cells responded to CGRP stimulation. None of the CGRP-specific CD4 T cells were able to be activated by the IAPP peptide. This established that CGRP-reactive CD4 T cells are a unique type of autoantigen-specific T cells in NOD mice. Using IAg7-CGRP tetramers, we found that CGRP-specific T cells were present in the pancreas of both prediabetic and diabetic NOD mice. The percentages of CGRP-reactive T cells in the pancreas of NOD mice were correlated to the diabetic progression. We showed that the human CGRP peptide presented by IAg7 elicited strong CGRP-specific T-cell responses. These findings suggested that CGRP is a potential autoantigen for CD4 T cells in NOD mice and probably in humans. The CGRP-specific CD4 T cells could be a unique marker for type 1 diabetes. Given the ubiquity of CGRP in nervous systems, it could potentially play an important role in diabetic neuropathy.</p

The application of BiLC strategy to image the three main members of Rho GTPases.

<p>(A) The results of optical imaging of three kinds of BiLC RhoGTPase biosensors. The relative luminescence was calculated by the ratio of luminescent intensity of firefly luciferase (FL) at 600 nm to that of renilla luciferase (RL) at 500 nm (n = 4, representative of 4 independent experiments). Error bars denote standard deviations. Asterisks (*) denotes samples that show a difference from the nonspecific complementation (the non-interactive GTPase-effector pairs or the effector loop mutants) with statistical significance by analysis of variance (ANOVA) (<i>p</i>≤0.01). This result indicates that the nonspecific complementation does not impede the correct interpretation of effective interactions induced by GTPase activation. WYJH (#) denotes samples that show a difference from the wild-type biosensor with statistical significance by ANOVA (<i>p</i>≤0.01). This result indicates that the BiLC sensors possess the discriminatory power among different GTPase activity states. (B) The results of coimmunoprecipitation. The results show that the expressions of the BiLC biosensors had no significant discrimination among different alleles of Rho biosensor, but the intensities of the PPIs displayed obvious diversities, which were in accordance with the results obtained by our optical imaging. (C) In vivo optical CCD imaging of BiLC Rho GTPase biosensors. The pseudotumors in living mice were generated by engrafting with transiently transfected 293 cells. The pRL-tk plasmid was cotransfected and RL activity was detected to normalize the planted cell number. 24 h after implantation, the mice were imaged using IVIS spectrum. A significant discrimination of luciferase activity was detected among different alleles of Rho GTPase. (I: the dominant active mutants; II: the wild-type Rho GTPases; III: the effector-loop mutants; IV: the dominant negative mutants).</p

The sensitivity analysis of BiLC Rho GTPase biosensors to GEFs and GAPs.

<p>(A) The results of optical imaging among different upstream regulatory proteins. The luminescent signals were normalized using cotransfection of renilla luciferase plasmid (pRL-tk) and represented by the ratio of luminescent intensity of firefly luciferase (FL) at 600 nm to that of renilla luciferase (RL) at 500 nm. The final results were normalized by the luminescence ratio of the control vehicles, which were designated with “1”. Data is reported as the fold increase in luminescence ratio (FL/RL) relative to control. Error bars denote standard deviations. Asterisks (*) denotes samples that show a difference from the control vector with statistical significance by analysis of variance (ANONA) (<i>p</i>≤0.01). The data shown was obtained by three separate experiments performed with quadruplicate culture wells. The results highly accord with the well-known experimental data, indicating that the BiLC biosensors can response to the upstream regulatory molecules. And this ability of BiLC GTPase sensors can be used to examine the substrate selectivity of GEFs and GAPs and quantify their catalytic activities in intact living cells. (B and C) The western blots carried out in parallel to demonstrate protein expression among different GEFs and GAPs. The figure only shows the results of the CDC42 biosensors as a representative.</p

Optimizing the appropriate configuration (or domain arrangement) for BiLC Rho GTPase biosensor.

<p>(A) The optical results of eight different configurations. Relative luciferase activities were detected in living 293 cells cotransfected with the different configurations of N- and C-terminal FL fragments with interacting proteins CDC42 and WASP constructed with different orientations. Correct configuration is very critical to achieving efficient reconstruction of luciferase activity with PCA strategy. In each configuration, luciferase activity was compared among CDC42 WT, G12V, T17N and F37A mutants, which represent different levels of CDC42 activity and different degrees of the interaction. WT, G12V, T17N and F37A indicate wild type, constitutively active mutant, dominant negative mutant and effector mutant, respectively. The results were normalized using cotransfection of RL and represented by the ratio of luminescent intensity of FL at 600 nm to that of RL at 500 nm. The data shown are representative of three separate experiments performed with quadruplicate culture wells. The results show that the combinations containing Nfluc416-WASP/CDC42-Cfluc398 and Nfluc416-WASP/Cfluc398-CDC42 produced a greater level of luminescent signal and wider dynamic rang for different levels of CDC42 activity. (B) The western blots carried out in parallel to demonstrate the protein expression among CDC42 WT, G12V, T17N and F37A biosensors. This figure only shows the results of Nfluc416-WASP/Cfluc398-CDC42 as representative.</p

Optimizing appropriate dissection site of firefly luciferase for BiLC Rho GTPase biosensor.

<p>(A) The optical imaging results among different split-sites of firefly luciferase. Relative luciferase activities were detected in living 293 cells cotransfected with the five different combinations of split firefly luciferase fragments (Nfluc416-WASP/Cfluc398-CDC42, Nfluc416-WASP/Cfluc417-CDC42, Nfluc437-WASP/Cfluc438-CDC42, Nfluc398-WASP/Cfluc384-CDC42, Nfluc445-WASP/Cfluc446-CDC42), respectively. In each luciferase fragments combination, luciferase activity was compared among CDC42 WT, G12V, and F37A mutants. WT, G12V, and F37A indicate wild type, the constitutively active mutant, and the effector-loop mutant, respectively. The results were normalized using cotransfection of renilla luciferase plasmid (pRL-tk) and represented by the ratio of luminescent intensity of firefly luciferase (FL) at 600 nm to that of renilla luciferase (RL) at 500 nm (FL/RL). The data shown are representative of four separate experiments performed with quadruplicate culture wells. The result shows that the combination (Nfluc416/Cfluc398) had the widest dynamic range and the highest luciferase activity restoration. (B) The western blots carried out in parallel to demonstrate the protein expression among CDC42 WT, G12V, and F37A biosensors. This figure only shows the results of Nfluc416-WASP/CDC42-Cfluc398 as representative.</p

The sensitivity analysis of BiLC Rho GTPase sensors to extracellular ligands.

<p>(A) The temporal response of BiLC Rho GTPase sensors stimulated by extracellular ligands. After being serum-starved in serum-free DMEM medium for 6 h, mouse fibroblast NIH3T3 cells were detected the luminescent signals by adding D-luciferin until the intensities became steady, then stimulated with insulin (2 mg/mL), lysophosphatidic acid (40 ng/mL) and bradykinin (100 ng/mL), which are the known activator of Rac1, RhoA and Cdc42 respectively, and then immediately acquired the sequence image (1-min exposure; emission filter, open; f-stop, 1; binning, 8; field of view, 15 cm) for 30 min using IVIS spectrum. The data shown were obtained by three separate experiments performed with quadruplicate culture wells. The result shows that not only the activation signals of Rho GTPases from upstream pathways but also the subsequent decrease following the hydrolysis of GTP can be displayed and quantified by the BiLC-based biosensors. And the optical results (left) accord with that of ‘pull-down’ in our previous work (right). (B) The responses of BiLC Rho GTPase sensors to different concentration of extracellular ligands. The cells were stimulated with different concentrations of the stimulators and the luciferase activity was acquired after 3 min by IVIS spectrum (1-min exposure; emission filter, open; f-stop, 1; binning, 8; field of view, 15 cm). The data shown was obtained by three separate experiments performed with quadruplicate culture wells. The optical results (upper) were in accordance with that of our previous ‘pull-down’ (under).</p

The schematic diagrams of the constructs and the mechanism for BiLC-based Rho GTPase biosensors.

<p>(A) The graphic schemes of the constructs used in process of optimizing the appropriate dissection sites of firefly luciferase. For the construct of CDC42-Cfluc, CDC42 (AA 1–176) was used and the carboxy-terminal region of CDC42 (AA171–191) was added to the downstream of the fusion protein, making sure the correct localization to the plasma membrane and the regulation of GDIs. (B) The graphic schemes of the constructs used in process of optimizing the appropriate orientation of the reporter fragments (FN and FC) and the interacting proteins (WASP and CDC42). If CDC42 was fused to the N-terminal of the reporter fragment, CDC42 (AA 1–176) was used and the carboxy-terminal region of CDC42 (AA 171–191) was added downstream of the reporter fragment. (C) The schematic diagram of the optimal configuration and the mechanism for BiLC-based Rho GTPase biosensors. We use the optimal configuration as representative to describe the mechanism of the biosensors. In this strategy, two non-functional luciferase fragments are respectively fused, in fame and with a short flexible linker (G₂S)_2∼4 or (G₄S)_1∼2, to Rho GTPase and the GBD of the specific effector. Once Rho GTPase is activated by upstream stimulating factors, the two luciferase fragments (luc1 and luc2) are brought into close proximity by the active Rho GTPase binding to the GBD of the effector, leading to the restoration of luciferase activity and photon production in presence of the substrate.</p

Global splicing changes in <i>Mecp2</i> KO mouse cortex and enrichment of MeCP2 around the exon/intron boundary.

<p>(a) Number of misregulated alternative splicing (AS) events by category in <i>Mecp2</i> KO cortex. (b) Relative fraction of each AS events type positively or negatively affected by loss of MeCP2. (c) Histogram of total gene expression changes in <i>Mecp2</i> KO cortex for those 232 genes associated with splicing changes in <i>Mecp2</i> KO cortex. FC: fold change. (d) Functional enrichment analysis of genes with MeCP2 peak(s) identified in ChIP-Seq data from WT mouse cortex. Top six terms are shown and –log₁₀ Benjamini <i>P</i>-value is plotted for each GO term. (e) Read counts per millions of mapped reads (ChIP minus input) across regions spanning from 500bp upstream and 500bp downstream of all exons. Coverage for ChIP and input data are calculated for each biological replicate and normalized to be equal length across all the mm9 exons by sampling at equal intervals. Displayed is the average ChIP minus input profile of two biological replicates. The graph is generated using ngs.plot[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006129#pgen.1006129.ref041" target="_blank">41</a>].</p

Altered AMPA receptor decay kinetics and synaptic transmission in <i>Mecp2</i> KO cortical neurons can be reversed by ESF.

<p>(a) Representative sample trace of glutamate-evoked current in WT and <i>Mecp2</i> KO cortical neurons. Scale bar = 15ms. (b) Quantification of decay time constant τ of glutamate-evoked current in WT and <i>Mecp2</i> KO cortical neurons. Mean ± S.E.M is plotted. n = 37 for WT, n = 33 for KO. ** <i>P</i> < 0.01; two tailed <i>t</i>-test. (c) Representative mEPSC sample trace in WT and <i>Mecp2</i> KO cortical neurons with or without CTZ treatment. Scale bar = 10ms. (d) Quantification of decay time constant τ of mEPSC. Mean ± S.E.M is plotted. n = 42 for WT, n = 46 for KO, n = 38 for KO + CTZ. ** <i>P</i> < 0.01; one-way ANOVA with Tukey’s multiple comparisons test. (e-f) qRT-PCR analysis the flip/flop ratio (e) and total <i>Gria2</i> minigene (f) expression in HEK 293 cells co-transfected with indicated ESF and the <i>Gria2</i> minigene. Mean ± S.E.M is plotted. n = 4 for each group. * <i>P</i> < 0.05, *** <i>P</i> < 0.001; two tailed <i>t</i>-test. (g) Quantification of decay time constant τ of glutamate-evoked current in WT, <i>Mecp2</i> KO (KO+Ctrl) and <i>Mecp2</i> KO cortical neurons infected with ESF-flop-Gly lentivirus (KO+ESF). Recording was done on acute slice from mouse infected with lentivirus two weeks after stereotaxic injection. Mean ± S.E.M is plotted. n = 50 for WT, n = 26 for KO+Ctrl, n = 27 for KO+ESF. ** <i>P</i> < 0.01, *** <i>P</i> < 0.001; one-way ANOVA with Tukey’s multiple comparisons test. (h) Representative sample trace of AMPAR-mediated current in response to repetitive stimulations in WT and <i>Mecp2</i> KO neurons infected with control or ESF-flop-Gly lentivirus (KO+Ctrl or KO+ESF). Scale bar, 100ms. (i) Relative current amplitude of the xth stimulation to first stimulation in WT and <i>Mecp2</i> KO neurons infected with either control or ESF-flop-Gly lentivirus (KO+Ctrl or KO+ESF). Mean ± S.E.M is plotted. n = 36 for WT, n = 18 for KO+Ctrl, n = 18 for KO+ESF. Asterisk denotes <i>P</i>-value for comparison between WT and KO+Ctrl, *** <i>P</i> < 0.001. Pound sign denotes <i>P</i>-value for comparison between KO+ESF and KO+Ctrl, # <i>P</i> < 0.05, ## <i>P</i> < 0.01; repeated measures two-way ANOVA with Tukey’s multiple comparisons test. (j) Quantification of decay time constant τ of glutamate-evoked current in neurons infected with shCtrl or shLedgf lentivirus. Mean ± S.E.M is plotted. n = 13 for shCtrl, n = 12 for shLedgf, * <i>P</i> < 0.05; two-tailed <i>t</i>-test. (k) Representative sample trace of AMPAR-mediated current in response to repetitive stimulations in neurons infected with shCtrl or shLedgf lentivirus. Scale bar, 100ms. (l) Relative current amplitude of xth stimulation to first stimulation in neurons infected with shCtrl or shLedgf. Mean ± S.E.M is plotted. n = 12 for each group, *** <i>P</i> < 0.001; repeated measures two-way ANOVA with Tukey's multiple comparisons test.</p

RTT mutations disrupt MeCP2 interaction with LEDGF and DHX9.

<p>(a) Schematic diagram of HA-MeCP2 constructs used in this study. The methyl-CpG-binding domain (MBD) domain is shown in yellow and the transcription repression domain (TRD) in blue. (b) Western blot analysis of Myc-LEDGF/p52 in anti-HA immunoprecipitate (top panel) from cells transfected with different combinations of plasmids as labeled on top. HA-MeCP2 (middle panel) and Myc-LEDGF/p52 (bottom panel) in input were also analyzed by western blot. (c) Western blot analysis of Myc-DHX9 in anti-HA immunoprecipitate (top panel) from cells transfected with different combinations of plasmids as labeled on top. HA-MeCP2 (middle panel) and Myc-DHX9 (bottom panel) in input were also analyzed by Western blot. (d) Representative western blot analysis of Myc-LEDGF/p52 in HA immunoprecipitate (top panel) from cells transfected with different combinations of plasmids as labeled on top. HA-MeCP2 (middle panel) and Myc-LEDGF/p52 (bottom panel) in input were also analyzed by Western blot. (e) Quantification of Myc-LEDGF/p52 IP over input signal intensity of three independent experiments. Error bar represents S.E.M, **<i>P</i> < 0.01, *<i>P</i> < 0.05; two-tailed <i>t</i>-test. (f) Representative Western blot analysis of Myc-DHX9 in HA immunoprecipitate (top panel) from cells transfected with different combinations of plasmids as labeled on top. HA-MeCP2 (middle panel) and Myc-DHX9 (bottom panel) in input were also analyzed by Western blot. (g) Quantification of Myc-DHX9 IP over input signal intensity of four independent experiments. Error bar represents S.E.M, **<i>P</i> < 0.01, *<i>P</i> < 0.05; two-tailed <i>t</i>-test.</p