EMBO/soda-nel huggingface dataset:
Deletions in CWH43 Cause Idiopathic Normal Pressure Hydrocephalus (2020)
Figure 3

Figure 3: CWH43 mutation increases ventricular volume and causes gait dysfunction in mice.

A. Scatter plot comparing ventricular volume from CWH43WT/WT (wild type, WT), heterozygous CWH43WT/M533, homozygous CWH43M533/M533 and CWH43M533/A530 mice at 6 months. Ventricular volume was calculated from T2-weighted MR images of the mouse brain using a custom automated computer algorithm. Horizontal bars indicate the mean of the measurements in each column. Statistical significance for each mutant mouse line compared to WT was determined using the unpaired t-test (P=0.0015 for CWH43WT/M533; P=0.0014 for CWH43M533/M533; P=0.006 for CWH43M533/A530).

B. Representative 3D volumetric MR images of mouse brains from 6 month old CWH43WT/WT, CWH43M533/M533 and CWH43M533/A530 mice. LV = lateral ventricle, 3rd V = third ventricle, 4th V = fourth ventricle. Scale bar is approximately 1 mm.

C. Quantitative gait analysis at 7 months of age revealed increased sway among homozygous CWH43M533/M533 mice, (*P=0.03, n=5) and compound heterozygous CWH43M533/A530 (*P=0.034, n=4) mice when compared to wild type CWH43WT/WT mice (n=5). Sway (the distance between the hind paws during walking) was measured repeatedly for individual mice in each group during a constrained unidirectional walk. Data shown are the mean ± SD for each group. Statistical significance was determined using the unpaired t-test.

D. Box plot showing rotarod performance data for CWH43WT/WT, CWH43WT/M533, CWH43M533/M533 and CWH43M533/A530 mice at 7 months of age. Data shown are the mean, 1st quartile, 3rd quartile, minimum and maximum for each group of mice. Statistical significance was determined using the unpaired t-test. When compared to wild type CWH43WT/WT mice (n=10), balance time on the rotarod was decreased significantly among heterozygous CWH43WT/M533 mice (*P=0.04, n=8), homozygous CWH43M533/M533 mice (*P=0.03, n=7), and compound heterozygous CWH43M533/A530 mice (*P=0.03, n=4).
Deletions in CWH43 Cause Idiopathic Normal Pressure Hydrocephalus (2020)
Figure 4

Figure 4: CWH43 mutation decreases cilia number and alters the distribution of GPI-anchored proteins in mouse ventricular ciliated epithelia.

Scanning electron micrographs of the ependymal surface of the lateral ventricle of CWH43 WT and CWH43M533/M533 mice. Scale bar is approximately 40 µm. The graph on the right quantifies the data from the electron micrographs on the left. Data shown are the mean ± SEM. * = P=0.004, n = 4, unpaired t-test.

Western blot analysis of total membrane, aqueous and lipid (GPI-anchor-containing) Triton X-114 extracts derived from wild type, CWH43M533/M533 and CWH43WT/M533mouse brain or kidney. The Western blot was stained using an antibody directed against CD59, a GPI-anchored protein.

Fluorescence immunohistochemistry for CD59 in the ependymal layer and choroid plexus of the lateral ventricle from CWH43WT/WT, CWH43M533/M533 and CWH43M533/A530 mice. Arrowheads point to apical surfaces of ependymal and choroid plexus cells. Nuclei are counterstained using DAPI (blue). Scale bar is approximately 5 µm.
Prevention of excitotoxicity-induced processing of BDNF receptor TrkB-FL leads to stroke neuroprotection (2019)
Figure 1

Figure 1. TrkB-FL juxtamembrane region is a rational target for design of CPPs interfering calpain-processing

(A) Region details and sequence selection. Common residues to TrkB-FL and TrkB-T1 are in light blue and include a KFG sequence completely conserved in Trk proteins (filled black box). In the heatmap representation, dark red, white and green represent respectively strong, weak or no effect of point mutations while black corresponds to wild type residues. Black arrows denote theoretical calpain cleavage sites while the red arrow points an experimentally established site. Sequences included in CPPs are emphasized by black rectangles. TM, transmembrane; TK, tyrosine kinase.

(B) CPP design. Peptides contain Tat aa 47-57 (italic) followed by the indicated rat TrkB-FL (green) or c-Myc (black) sequences.

(C) Confirmation of peptide entry into neurons. Cultures were incubated with Bio-TMyc (25 µM, 1 h) (panel b) or left untreated (panel a). Arrowheads highlight peptide permeability, detected by fluorescein Avidin D (green), into neurons labeled with neuronal-specific antibody NeuN (red). Peptide is not detected in some neurons (arrow) and non-neuronal cells (asterisk). Confocal microscopy images correspond to single sections and are representative of five independent experiments. Scale bar, 10 μm.

(D) Effect of TMyc on neuronal survival. Primary cultures were incubated with TMyc (5, 15 or 25 μM, 30 min) and subjected to treatment with NMDA (100 µM) and glycine (10 µM) for 4 h. Specific neuronal viability was established and expressed relative to values in cultures with no treatment. Means ± SEM are represented (n = 8) and statistical analysis was performed by ANOVA test followed by post-hoc Tukey HSD test.
Prevention of excitotoxicity-induced processing of BDNF receptor TrkB-FL leads to stroke neuroprotection (2019)
Figure 2

Figure 2. TFL457 interferes TrkB-FL downregulation and prevents acute and chronic excitotoxicity

(A) Effect of TFL457, TFL482 and TFL541 on TrkB-FL levels. Cultures preincubated with TrkB-FL peptides or TMyc (25 µM, 30 min), followed by chronic NMDA treatment (2 h), were compared to cells without peptide. Levels of full-length (FL) TrkB were established with panTrkB, an antibody for the extracellular domain that also detects the truncated forms (tTrkB).

(B) Quantitation of peptide interference of TrkB-FL downregulation. Receptor levels were normalized to NSE and expressed relative to values obtained in cultures without peptide or NMDA. Means ± SEM are represented and analysis was performed by ANOVA test followed by post-hoc Tukey HSD test (*p = 0.02; n = 4).

(C) Effect of TFL457, TFL482 and TFL541 on neuronal viability after chronic excitotoxicity. Cultures were preincubated with peptides and treated with NMDA for 0-6 h as before. Values for each time point were represented relative to those of neurons incubated with the same peptide but no NMDA. Mean ± SEM of eleven (TFL457), three (TFL482) and seven (TFL541) independent experiments is given. Data were analyzed by a two-way ANOVA test followed by post-hoc Bonferroni test comparing values for each time point (***p = 0.0002, ***p = 0.00009 and *p = 0.0357, respectively for 2, 4 or 6 h).

(D) Effect of TFL518 on TrkB-FL levels. Cultures incubated with TFL518 as before were compared to those treated with TMyc or TFL457.

(E) TFL518 effect on neuronal viability. Cultures were treated and analyzed as above. No significant differences were found for TFL518 while neuroprotection by TFL457 was confirmed (***p = 0.0002 and, **p = 0.002, respectively for 2 or 4 h compared to TMyc; n = 3).

(F) Effect of TFL457 on neuronal viability after acute excitotoxicity. Cells were induced with NMDA (50 μM) and glycine (10 μM) for 1 h and, after agonists removal, culture proceeded for 20 h with DL-AP5 (200 μM) and TMyc or TFL457 (15 μM). Mean ± SEM relative to cultures incubated with TMyc but no NMDA is represented. Analysis was performed by unpaired Student t test (*p = 0.04; n = 5).
Prevention of excitotoxicity-induced processing of BDNF receptor TrkB-FL leads to stroke neuroprotection (2019)
Figure 3

Figure 3. TrkB-FL preserved by TFL457 action maintains Y816 phosphorylation and PLCγ-dependent pathways required for neuroprotection

(A) TrkB-FL main signaling cascades with indication of anchoring residues, tested drugs and their targets.

(B) Effect of TFL457 on Y515 and Y816 phosphorylation. Cultures preincubated with TMyc or TFL457 (25 μM, 30 min) were treated with NMDA (0-6 h) and analyzed with panTrkB or phosphospecific antibodies.

(C) Quantitation of pY515 and pY816. Mean ± SEM of normalized pY515 (n = 7) or pY816 (n = 4) levels obtained after NMDA-treatment (2 h) are represented relative to those found in cells with the same peptide but no NMDA. Statistical analysis was performed by unpaired Student t test (*p = 0.046; n.s. = non-significant).

(D) Analysis by immunoprecipitation of TFL457 effect on pY816 levels. Cultures preincubated and treated as before with NMDA (2 h), were immunoprecipitated with antibodies specific for phosphorylated tyrosine (pY). pY816 was analyzed by WB in immunoprecipates.

(E) TFL457 effects on neuronal viability after inhibition of TrkB-FL signaling. Cultures were preincubated (30 min) with inhibitors specific for PI3K (Wortmannin, 100 nM), MAPK/ERK (UO126, 300 nM) or PLCγ (U-73122, 5 μM) before incubation with TMyc or TFL457 (25 μM, 30 min). Viability was established 4 h after NMDA treatment. Means ± SEM relative to untreated cultures are represented and data were analyzed by ANOVA test followed by post-hoc Tukey HSD test (***p = 0.0001, **p = 0.009, *p = 0.02, n.s. = non-significant, respectively for TMyc vs TFL457 in untreated or Wortmannin, UO126 or U-73122-treated cells; **p = 0.008 for untreated vs U-73122-treated cells preincubated with TFL457; n = 5).
Prevention of excitotoxicity-induced processing of BDNF receptor TrkB-FL leads to stroke neuroprotection (2019)
Figure 4

Figure 4. TFL457 preserves additional survival proteins downstream TrkB signaling in excitotoxicity

(A) Dose-dependent TFL457 effects. Cultures incubated with TMyc o TFL457 (5, 15 o 25 μM, 30 min) were treated with NMDA (2 h) and compared to cells without peptide or NMDA.

(B) Time-course TFL457 effects. Cells preincubated with peptides (25 μM, 30 min) were treated with NMDA (0-6 h).

(C) Quantitation of peptide effects. Normalized protein levels are presented relative to values obtained in cells incubated with the same peptide but no NMDA. Means ± SEM are given. Results were analyzed by a two-way ANOVA test followed by post-hoc Bonferroni test, comparing TMyc or TFL457-treated cells for each time-point (TrkB-FL, ***p = 0.0009 and *p = 0.02; CREB, *p = 0.0149 and *p = 0.0148; pCREB, ***p = 0.0007 and **p = 0.0012; MEF2D, **p = 0.0023 and **p = 0.0047. All values are respectively for 2 or 4 h; n = 6).
Prevention of excitotoxicity-induced processing of BDNF receptor TrkB-FL leads to stroke neuroprotection (2019)
Figure 5

Figure 5. Promoter activity and mRNA levels of CREB and MEF2-regulated genes are preserved by TFL457 in excitotoxicity

(A) Effect of TFL457 on MEF2-promoter activity. Cultures transfected with pMEF2 (two minimal wild type MEF2 elements) or pMEF2mut (mutant) were preincubated with CPPs (25 μM, 30 min) and treated with NMDA (100 µM, 2 h). Means ± SEM of luciferase activities obtained in excitotoxicity, relative to values found in cells treated with same peptide and no NMDA, are presented. Significance was analyzed by Student's t-test (*p = 0.039; n.s. = non-significant, p = 0.79; n = 8).

(B) Effect on CRE-promoter activity. pCRE contains two minimal CREs. Peptide preincubation was as above with or without KG-501 (10 µM). Mean ± SEM luciferase activities are given relative to values in cells treated with the same peptide but no NMDA or KG-501. Differences found in excitotoxicity were analyzed by ANOVA test followed by post-hoc Tukey HSD test (*p = 0.023; **p = 0.003; n.s. = non-significant, p = 0.20; n = 6).

(C) Effect on NMDAR-subunit promoters. Cells transfected with pGluN1 or pGluN2A were treated and analyzed as in panel A (pGluN1, *p = 0.016; pGluN2A, *p = 0.029; n = 14).

(D) Effects on BDNF promoter III or TrkB promoter. Cells transfected with pIIIBDNF (n = 9), pTrkB (n = 7) or pCRE (n = 5), as a control, were processed as above but using 20 µM NMDA for 4 h. Data are presented and analyzed as in panel A (pCRE, **p = 0.0098; pIIIBDNF, *p = 0.029; pTrkB, *p = 0.033).

(E) Effects of TFL457 on mRNA levels of CREB/MEF2-regulated genes. Total RNA was extracted from cultures preincubated with CPPs (25 μM, 30 min) and treated or not with NMDA (100 µM, 4 h). Levels of mRNA were normalized to NSE (genes expressed in neurons, left panel) or GAPDH (neuronal and glial expression, right panel). Means ± SEM of levels obtained in excitotoxicity relative to values found in cells treated with same peptide and no NMDA are presented. Differences found in excitotoxicity were analyzed by ANOVA test followed by post-hoc Tukey HSD test (GluN1, ***p = 0.00096; GluN2A, p = 0.98; TrkB-FL, ***p = 0.00037; BDNF, *p = 0.045; n = 8).
Prevention of excitotoxicity-induced processing of BDNF receptor TrkB-FL leads to stroke neuroprotection (2019)
Figure 6

Figure 6. Preservation of TrkB-FL in the cell-surface by TFL457 precedes interference of RIP and calpain-processing in excitotoxicity

(A) TFL457 interference of RIP and calpain-processing in excitotoxicity. Cultures were preincubated with peptides (25 µM, 30 min) or left untreated before NMDA addition (2 h). Different exposures are shown to facilitate visualization of FL, f42/39 or f32.

(B) Quantitation of normalized f32 and f42/39 levels. Means ± SEM are shown relative to cultures treated with NMDA and no peptide. Analysis was performed by unpaired Student t test (*p = 0.014, **p = 0.00198; n = 6).

(C) Effect of TFL457 on neuronal viability after calpain inhibition. After 30 min preincubation with calpeptin (Calp, 10 µM) and calpain inhibitor III (CiIII, 10 µM), cultures were treated with CPPs and NMDA (4 h) as before. Means ± SEM relative to those obtained in untreated cultures are given. We performed ANOVA test followed by post-hoc Tukey HSD test (*p = 0.035, **p = 0.0058, ***p = 0.00009; n=12).

(D) Effect of TFL457 on neuronal viability after metalloproteinase inhibition. Cultures preincubated with GM6001 (10 µM, 30 min) were treated with CPPs and NMDA as before. Analysis was performed as above (without GM6001, ***p = 0.0007; with GM6001, ***p = 0.0006; n = 4).

(E) Effect of NMDA on total and cell-surface TrkB-FL levels. Cultures were incubated with NMDA (100 µM) or BDNF (100 ng/ml) for 1 h. Membrane proteins were labeled and purified and compared to corresponding total lysates. TrkB-FL levels are expressed relative to untreated cells. Mean ± SEM are presented, analyzed by Student's t-test (*p = 0.027; n.s. = non-significant, p = 0.1; n = 7).

(F) Effect of TFL457 on the decrease of TrkB-FL surface levels induced by NMDA. Analysis was performed as before in cells incubated with CPPs (25 μM, 30 min) and NMDA-treated (1 h). Results obtained in excitotoxic cultures are expressed relative to cells treated with the same peptide but no NMDA. Data are analyzed as in panel E (*p = 0.019; n.s. = non-significant, p = 0.44; n = 4).

(G) Experimental design to study the effect of endocytosis inhibition on TrkB-FL downregulation in ischemia. Mice were i.p. injected with vehicle or dynasore followed by Rose Bengal. Vessel occlusion and brain damage were induced by cold-light irradiation. Animals were sacrificed early after damage initiation.

(H) Effect of endocytosis inhibition on TrkB-FL downregulation. The infarcted area of the ipsilateral hemisphere (I) was compared to the corresponding region of the contralateral area (C). Results from representative mice injected with dynasore or vehicle are shown. Different exposures are presented to facilitate visualization of dynasore effects on TrkB-FL and f32.

(I) Quantitation of TrkB-FL and TrkB-T1 in the infarcted area. Normalized protein levels are expressed relative to those of the corresponding contralateral region. Mean ± SEM are presented and analyzed by Student's t-test (*p = 0.04; n.s. = non-significant, p = 0.14; n = 9).
Prevention of excitotoxicity-induced processing of BDNF receptor TrkB-FL leads to stroke neuroprotection (2019)
Figure 7

Figure 7. TFL457 counteracts TrkB-FL downregulation in ischemia and reduces infarct volume and neurological damage

(A) Confirmation of Bio-TFL457 delivery to mice cortex. Biotinylated TFL457 (Bio- TFL457, 4 nMole/g, retro-orbitally injected) was detected in coronal sections stained with NeuN and DAPI. Representative confocal microscopy images of cortical areas correspond to single sections. Scale bar, 10 µm.

(B) Timeline to analyze in vivo effects of MTMyc and MTFL457. Mice subjected to ischemia as before were retro-orbitally injected with CPPs (10 nMole/g) 10 min after damage initiation.

(C) Representative analysis of TrkB-FL at 3 h of damage. Comparison of the infarcted area (I) and the corresponding contralateral region (C) is shown.

(D) Quantitation of TrkB-FL levels. Means ± SEM of normalized TrkB-FL levels in the infarcted area relative to the corresponding contralateral region are presented and results analyzed by Student's t-test (*p = 0.021; n = 3).

(E) Representative 1 mm brain coronal slices stained with TTC after 24 h of insult corresponding to animals injected with MTMyc or MTFL457.

(F) Infarct volume of animals injected with MTMyc or MTFL457 expressed as a percentage of the hemisphere volume. Means ± SEM are given. Differences were analyzed by Student's t-test (***p = 0.00008; n = 14). Results for MTFL457 are also expressed as percentage of the infarct volume in animals injected with MTMyc.

(G) Evaluation of balance and motor coordination. Number of contralateral hind paw slips were measured after 24 h of damage. Data spread is presented by box and whisker plots showing interquartile range, median, minimum and maximum values. Means (dots) ± SEM were also calculated and analyzed by Student's t-test (*p = 0.04; n = 11).

Expanded View Figure legends
Nlrp3 inflammasome activation in macrophages suffices for inducing auto-inflammation in mice (2022)
Figure 3

Figure 3. CAPS in Nlrp3Mac-A350V/WT mice is associated with inflammasome activity in spleen and liver and is mediated by both IL-1β and IL-18 cytokines.

(A-C) Tissue lysates from (A) spleen, (B) liver and (C) kidney from 6- to 9-day old indicated mice were immunoblotted to determine cleavage of caspase-1. Each lane represents a lysate from a different mouse. Positive control lanes (+) are BMDMs stimulated with LPS/ATP; negative control lanes (-) are respective organ homogenates from caspase-1-/- mice.

(D-G) Tissue lysates from (D, F) spleen and (E, G) liver from 6- to 9-day old indicated mice were immunoblotted to determine pro-IL-1β cleavage to IL-1β (D-E) or GSDMD cleavage to its p30 and p20 fragments (F-G). Each lane represents a lysate from a different mouse. Positive control lanes (+) are BMDMs stimulated with LPS/ATP; negative control lanes (-) are respective organ homogenates from (D-E) caspase-1-/- or (F-G) Gsdmd-/- mice.

(H-K) Growth (H, J) and survival (I, K) curves of Nlrp3NeoR-A350V/WT and Nlrp3Mac-A350V/WT littermates injected subcutaneously with PBS or Anakinra (H-I) or with anti-IL-18 or isotype antibodies (J-K). Injection days are indicated with arrows.

Data information: Statistics in (H, J) were analyzed by student t-test, error bars represent means ± SD of indicated numbers of biological replicates. Statistics in (I, K) were analyzed by log-rank Mantel-Cox test. ns not significant; * p<0.05; ** p<0.01.
Multiple cancer pathways regulate telomere protection (2019)
Figure 1

Figure 1. Identification of novel compounds with the ability to downregulate TRF1 protein levels. (A) Experimental procedure: 114 compounds approved by the FDA or in clinical trials are assessed by the Opera High Screening system for their ability to reduce TRF1 protein levels in CHA9.3 lung cancer mouse cells. (B) Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence of H676 cells treated with the indicated compounds for 24h at 1 μM. Scale bars, 5μm. Data are representative of n=3 biological replicates. (C) Western blot images (top) and TRF1 protein levels (bottom) of h676 cells treated with the indicated compounds for 24h at 1 μM. Data are representative of n=3 (PLKi, HSP90i and RTKi) and n=4 (AuroraI, mTOR, CDKi, Docetaxel, Gemcitabine, ERKi, MEKi) biological replicates. (D) Schematic representation of the novel TRF1 regulatory pathways. Data are represented as mean ± SEM. n represents biological replicates. Significant differences using unpaired t‐test are indicated by *P < 0.05, **P < 0.01, ***P < 0.001.
Multiple cancer pathways regulate telomere protection (2019)
Figure 2

Figure 2. New TRF1 chemical inhibitors induce DNA damage in lung cancer and glioblastoma cells. (A) Representative images (left) and percentage (right) of cells presenting 1 or more γH2AX and RAP1 colocalizing foci (TIFs) upon treatment of CHA9-3 lung cancer cells with the indicated compounds. White arrowheads point to colocalization of γH2AX and RAP1. Scale bars, 5μm. Data are representative of n=6 (DMSO) and n=3 (mTORi, PI3Ki, RTKi, MEKi, ERKi, HSPO90i, CDKi, Docetaxel) biological replicates (B) Representative images (left) and percentage (right) of γH2AX-positive cells per field in DMSO or compound treated patient-derived h676 GSCs. Scale bars, 50μm. Data are representative of 6 (DMSO) and 3 (mTORi, Docetaxel, ERKi, MEKi, RTKi, HSP90i, Gemcitabine, CDKi) biological replicates (C) Quantification of multitelomeric signals (MTS) in h676 GSC metaphases upon treatment with the indicated compounds. Representative images of the qFISH in the metaphases (left). Multitelomeric signals are indicated by arrowheads. Scale bars, 1μm. Data are representative of n=31 (DMSO), n=18 (mTORi), n=11 (MEKi) and n=24 (RTKi) biological replicates. Data are represented as mean ± SEM. Significant differences using unpaired t‐test are indicated by *P < 0.05, **P < 0.01, ***P < 0.001.
Multiple cancer pathways regulate telomere protection (2019)
Figure 4

Figure 4. ERK2, bRaf and mTOR kinases phosphorylate TRF1 in vitro. (A-D) 1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of mouse ERK2 kinase (A), human b-Raf kinase (WT or V600E) (B,C), or mouse MEK1 kinase (D) in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography. (E) 1 μM of GST-TRF1 and 0.2 μM of mouse ERK2 kinase were incubated in the presence of ERK and MEK inhibitors. (F) 2 μM of GST-TRF1 and 0.1 μM of human b-Raf kinase were incubated in the presence of the bRaf inhibitors dabrafenib and vemurafenib. (G-H) 1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human mTOR kinase (G) in the presence of the mTOR inhibitors rapamycin and Ku0063794 (I-K) Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 or TRF1 plus ERK2 (I), TRF1 plus bRAFWT or bRAFV600E (J) and TRF1 plus mTOR (K); Data are representative of n=2 independent experiments. (L) Schematic representation of TRF1 protein with the phosphorylation sites by ERK2, bRAF, mTOR and AKT. (M-O) Representative image (down) and quantification (up) of in vitro phosphorylation assays with the indicated GST-TRF1 wild-type or mutated forms in the presence of mouse ERK2 kinase. Data are representative of n=4 independent experiments. Data are represented as mean ± SEM. Significant differences using unpaired t‐test are indicated by *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001