EMBO/soda-nel huggingface dataset:
C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation (2021)
Figure 6

Figure 6. H89 and PKA knockdown improve motility defects in a Drosophila model of C9ALS/FTD.

A, B Representative graphs showing the climbing ability flies carrying the UAS-(G4C2)x36 construct in neurons using the Elav-gal4 promoter upon treatment with H89 10 µM diluted with 0.1 % of DMSO and 5% sucrose in PBS. The wild-type (w1118) line was used as control. (A) Female flies (♀). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 6th day of treatment ****P<0.0001 (G4C2)x36 vs (G4C2)x36 + H89. (B) Male flies (♂). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 6th day of treatment. ****P<0.0001 (G4C2)x36 vs (G4C2)x36 + H89. Experiments were performed three times, N=15 adult flies were used for each genotype, sex and treatment (N tot=45).

C, D Representative graphs showing climbing activity of flies co-expressing the UAS-Pka-RNAi construct (1, v330111) in combination with UAS-(G4C2)x36 in neurons using the Elav-gal4 promoter. The wild-type (w1118) line was used as control. (C) Female flies (♀). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 9th day ****P<0.0001 (G4C2)x36 vs Pka-RNAi (1);(G4C2)x36. (D) Male flies (♂). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 7th day ****P<0.0001 (G4C2)x36 vs Pka-RNAi (1);(G4C2)x36. Experiments were performed twice with different RNA interfering sequence, N=15 adult flies were used for each genotype and sex (N tot=30).
C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation (2021)
Figure 7

Figure 7. H89 reduces DPR levels in C9 patient-derived iPSC-MNs.

A, B Three independent C9 patient iPSC-MN lines (Becker S6, M211R2, C90202) were treated with GELD, SPL, FSK, H89 or DMSO vehicle control and levels of poly-GA (A) and poly-GP (B) measured after 7 days. Values are displayed as percentages of mean DMSO signal. No significant changes in either DPR were observed (linear mixed effects model, all not significant, P>0.05; P = 0.1 DMSO vs FSK). Treatments were performed in four technical replicates.

C, D Three independent C9 patient iPSC-MN lines were treated with 10 µM H89 and levels of poly-GA (C) and poly-GP (D) were measured after 14 days. Values are displayed as percentages of mean DMSO signal. H89 significantly reduced poly-GA *P = 0.039 and poly-GP levels *P = 0.018 (linear mixed effects model). Treatments were performed in four technical replicates.

E No increased LDH release was observed after 14-day 10 µM H89 treatment compared to vehicle treatment in two independent C9 patient iPSC-MN lines (unpaired t-tests, Becker S6: significant decrease in LDH, **P = 0.0064; M211R2: not significant, P>0.05). In Becker S6, mean LDH release was 22% lower after H89 treatment compared to vehicle. Treatments were performed in eight technical replicates.

F Relative fold changes of C9orf72 variant RNA levels normalised to RPL13A, after 14-day 10 µM H89 treatment vs vehicle treatment (N=1 C9 patient line, four technical replicates). No significant changes were observed between H89 and vehicle for any of the isoforms (2-way ANOVA with Šídák's multiple comparisons test, all not significant, P>0.05).

Data information: Error bars reported as ± SD. Not significant = P>0.05, *P<0.05, **P<0.01.

TABLE

Table 1 | List of the small molecules selected from the confirmatory screening.

AUG-RFP

intensity

Mean of compounds from 4 biological replicates. All the compounds were added after 3h of reverse co-transfection and used at the final concentration of 5 µM for approximately 30 h. Mean of DMSO from 64 biological replicates (32 replicates per each 384-well plate). Data are reported as Z-score values.

The entire list of small molecules used is reported in Dataset EV1.

EXPANDED VIEW FIGURE LEGENDS
Modeling Genetic Epileptic Encephalopathies using Brain Organoids (2021)
Figure 1

Figure 1. Generation and characterization of WWOX knock-out Cerebral Organoids.

Week 10 cerebral organoids (COs) stained for the progenitor marker SOX2, neuronal marker β3-Tubulin and WWOX (WT: n=8 from 3 batches, KO: n=8 from 3 batches, W-AAV n=4 from 1 batch). Scale = 100µm (left), 75µm (right).

Week 10 COs stained for the ventricular radial glia (vRGs) marker CRYAB, together with WWOX and the pan-radial glia marker SOX2. The images on the right are enlargements of the boxed area. The dashed line defines the ventricular space. Scale= 50µm (left), 25µm (right) (WT: n=8 from 3 batches, KO: n=8 from 3 batches).

Quantification of markers that represent the different populations that compose the ventricular-like zone (VZ), sub-ventricular zone (SVZ) and the cortical plate (CP), as depicted in Appendix Figure S2B-C. NeuN marks mature neurons, TBR2 marks intermediate progenitors (IPs) and SOX2 the ventricular radial glia (vRGs). The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=8 from 3 batches, KO: n=8 from 3 batches, W-AAV n=4 from 1 batch).

qPCR analysis for the assessment of expression levels of different neural markers in 15 weeks COs: SOX2 and PAX6 (progenitor cells), TUBB3 (pan-neuronal) SLC17A6 and SLC17A7 (VGLUT2 and VGLUT1; glutamatergic neurons) and GAD1 and GAD2 (GAD67 and GAD65; GABAergic neurons). Y-axis indicates relative expression fold change. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=4 from 1 batch, KO: n=4 from 1 batch, W-AAV: n=4 from 1 batch).

Immunofluorescent (IF) staining for the glutamatergic neurons marker VGLUT1 and GABAergic neurons marker GAD67 (GAD1) in week 10 COs (WT: n=8 from 3 batches, KO: n=8 from 3 batches, W-AAV: n=4 from 1 batch). Scale = 100µm.

Quantification of the images represented in figure 1E. VGLUT1 and GAD67 were quantified as the surface area covered by the staining and were normalized to the number of nuclei in each image. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. As the samples were not normally distributed, statistical significance was determined using Kruskal-Wallis test with Dunn's multiple comparisons test. Outliers were removed using the ROUT test (Q=1%). Data information: n.s (non-significant), *p ≤ 0.05, ****p ≤ 0.0001.
Modeling Genetic Epileptic Encephalopathies using Brain Organoids (2021)
Figure 2

Figure 2. WWOX-KO cerebral organoids demonstrated hyper-excitability and epileptiform activity.

Sample recordings from 7-week-old hESC-derived Cerebral Organoids (COs).

Sample traces show visible differences in local field potential, with WWOX-KO COs (Red) showing increased activity compared to WT (Blue) in baseline condition (Left) and in the presence of 100µM 4AP (Right).

Mean spectral power of week 7 WT and KO COs in baseline conditions. The line marks 0.25-1 Hz frequency range. Statistical significance was determined using two-tailed unpaired Welch's t-test (WT: n=14 slices, 5 organoids, 3 batches. KO: n=14 slices, 8 organoids, 3 batches).

Normalized area under the curve of the mean spectral power in (B) for the 0.25-1 Hz frequency range. Data represented by mean ± SEM. Statistical significance was determined using two-tailed unpaired Welch's t-test. The numerals in all bars indicate the number of analyzed slices and organoids (i.e. slices (organoids)).

CFC analysis shows increased coupling in the δ: HFO frequency pairs. The colour scale represents the modulation index - a measure of the coupling between the phase of low-frequency oscillations and amplitude of high-frequency oscillations in a given signal.

Sample spectrogram of WWOX KO slice shows a gradual increase in activity upon addition of 4AP (marked by red arrow) for up to 2 min, and a decrease after 25 min. All traces were filtered with a 60 Hz notch filter and 0.5 Hz high-pass filter.

(F,G) WWOX's coding sequence was re-introduced into week 6 WWOX-KO COs using lentiviral transduction (lenti-WWOX) (See methods). (F) Immunofluorescent staining showing WWOX expression in different populations in WWOX-KO organoids following infection with lentivirus. NT= non-treated. Scale = 50µm . (G) Normalized area under the curve of the mean spectral power of WT line, 2 KO lines and 2 KO lines infected with lenti-WWOX at week 7 in baseline condition, for the 0.25-1 Hz frequency range. Data represented by mean ± SEM. The numerals in all bars indicate the number of analyzed slices and organoids (i.e. slices (organoids)). Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test. Data information: ***p ≤ 0.001, ****p ≤ 0.0001.
Modeling Genetic Epileptic Encephalopathies using Brain Organoids (2021)
Figure 3

Figure 3. WWOX-KO cerebral organoids Showed Impaired Astrogenesis and DNA-damage Response.

Week 15 and week 24 COs stained for the astrocytic and radial glia marker GFAP, and the astrocyte-specific marker S100β (WT W15: n=9 from 3 batches, KO W15: n=16 from 3 batches, W-AAV W15: n=4 organoids from 1 batch. WT W24: n=10 from 4 batches, KO W24: n=9 from 3 batches, W-AAV W24: n=4 from 1 batch). Scale = 100µm

qPCR analysis of astrocytic markers in COs at week 15. Y-axis indicates relative expression fold change. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=4 from 1 batch, KO: n=4 from 1 batch, W-AAV: n=4 from 1 batch)

qPCR analysis of astrocytic markers in COs at week 24. Y-axis indicates relative expression fold change. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=4 from 2 batches, KO: n=3 from 2 batches, W-AAV n=3 from 1 batch).

IF staining of week 6 COs for astrocytic markers in the surrounding of VZs (WT: n=6 from 2 batches, KO: n=6 from 2 batches). Scale = 100µm (left) , 50µm (right).

Staining for the DNA damage markers γH2AX and 53BP1 in the nuclei of cells in the VZ of week 6 COs at physiological conditions, together with the pan-radial glia marker SOX2 (WT: n=8 from 3 individual batches, KO: n=12 from 3 individual batches, W-AAV: n=4 from 1 batch). Scale = 50µm (left), 25µm (right).

Quantification of γH2AX (top) and 53BP1 foci (bottom) in the nuclei of cells composing the innermost layer of the VZ, normalized to the total number of nuclei in this layer. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=8 organoids from 3 batches, KO: n=12 organoids from 3 batches, W-AAV: n=4 from 1 batch).

Data information: n.s (non-significant), *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.
Modeling Genetic Epileptic Encephalopathies using Brain Organoids (2021)
Figure 4

Figure 4. Cerebral organoids RNA-sequencing revealed major differentiation defects.

RNA sequencing (RNA-seq) of week 15 COs and transcriptome analysis (WT: n=2, KO: n=4).

Heatmap of 100 upregulated genes (left panel) and 100 downregulated genes (right panel) selected by highest fold change.

Gene-set enrichment analysis (GSEA) revealed enrichment of genes related to regionalization of the organoids, and decreased expression of genes related to negative regulation of the cell cycle.

Heatmap of Wnt-pathway related genes in 15-weeks COs.

qPCR analysis for selected Wnt target genes validating the results of the RNA- seq. Y-axis indicates relative expression fold change. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=4 from 1 batch, KO: n=4 from 1 batch, W-AAV: n=4 from 1 batch).

Week 16 COs were sub-fractionated into a cytoplasmic (C) and nuclear (N) fractions. The experiment was run twice with a total of 2 WT organoids and 4 KOs organoids (2 for each KO line). KAP-1 marks the nucleus and HSP90 marks the cytoplasm. The numbers at the bottom are a quantification of the nuclear fraction of β-catenin band intensities normalized to the cytosolic fraction [β-C (N/C)].

Heatmap showing the expression levels of markers of the six different layers of the human cortex in week 15 organoids from deepest to the most superficial: TBR1, BCL11B (CTIP2), SATB2, POU3F2 (BRN2), CUX1, RELN.

IF staining in week 15 COs validating the decreased levels of the deep-layer cortical markers CTIP2 (BCL11B) and TBR1, and superficial-layer marker SATB2 (WT: n=3, KO: n=4, W-AAV: n=4). Scale = 50µm.

Quantification of the cortical markers seen in G, normalized to the total number of nuclei. Y-axis indicates the fold change compared to the average of the WT COs. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=9 from 3 batches, KO: n=16 from 3 batches, W-AAV: n=4 organoids from 1 batch).

Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Modeling Genetic Epileptic Encephalopathies using Brain Organoids (2021)
Figure 5

Figure 5. WWOX-Related Epileptic Encephalopathy cerebral organoids recapitulate neuronal abnormalities.

Peripheral blood mononuclear cells (PBMCs) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs, and subsequently were differentiated into COs.

Week 10 WSM COs stained for the progenitor marker SOX2, neuronal marker β3-Tubulin and WWOX (WSM F1: n=2 from 1 batch, WSM M2: n=2 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.

Representative traces of spontaneous spikes recorded from neurons of WSM P (blue), WSM S (red) WSM S W-AAV (purple) week 7 COs. Each recording is 12 seconds long and the zoom-in is of 0.5 seconds (red box on the right).

Average firing rate over 24 neurons from WSM P COs (4 organoids), 41 neurons from WSM S COs (3 organoids) and 40 neurons from WSM S W-AAV organoids (3 organoids). Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test. Bars represents the mean ± SEM.

GAD67 and VGLUT1 immunostaining in week 10 WSM COs (WSM F1: n=2 from 1 batch, WSM M2: n=2 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.

Quantification of the data shown in fig 5D. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM F1: n=2 from 1 batch, WSM M2: n=2 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch).

Data information: ns (non-significant), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Modeling Genetic Epileptic Encephalopathies using Brain Organoids (2021)
Figure 6

Figure 6. WWOX-related Epileptic Encephalopathies depict molecular abnormalities similar to a complete WWOX-loss

Week 15 WSM COs stained for astrocytic markers GFAP and S100β. (WSM F1: n=2 from 1 batch, WSM M2: n=3 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.

Quantification of DNA damage foci, marked by γH2AX (left) and 53BP1 (right), in the nuclei of the cells in the VZs of week 6 WSM COs. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM F1: n=4 from 1 batch, WSM M2: n=4 from 1 batch, WSM S2: n=4 from 1 batch, WSM S5: n=4 from 1 batch, WSM S5 W-AAV3: n=4 from 1 batch, WSM S5 W-AAV6: n=4 from 1 batch).

Representative Immunofluorescence staining depicting the data summarized in 6B. SOX2 marks the radial glia in the VZ (WSM F1: n=4 from 1 batch, WSM M2: n=4 from 1 batch, WSM S2: n=4 from 1 batch, WSM S5: n=4 from 1 batch, WSM S5 W-AAV3: n=4 from 1 batch, WSM S5 W-AAV6: n=4 from 1 batch). Scale = 50µm (left), 25µm (right).

qPCR analysis for selected Wnt target genes in week 10 WSM COs. Y-axis indicates relative expression fold change. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM P: n=6 from 1 batch, WSM S: n=4 from 1 batch, WSM S W-AAV: n=4 from 1 batch).

Week 15 WSM COs stained for the cortical layers' markers CTIP2 and SATB2. (WSM F1: n=2 from 1 batch, WSM M2: n=3 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.

Quantification of the staining presented in E. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM F1: n=2 from 1 batch, WSM M2: n=3 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch).

Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Extended view (EV) figures:
Anoctamin 8 Tethers Endoplasmic Reticulum and Plasma Membrane for Assembly of Ca2+ Signaling Complexes at the ER/PM compartment (2019)
Figure 1

Fig 1: ANO8 increases interaction of STIM1-Orai1 and STIM1-STIM1 at the ER/PM junctions to control Ca2+ influx.

(A) HEK cells transfected with STIM1-CFP, Orai1-mCherry, and with (red) or without (black) untagged ANO8 were used to measure CRAC current with pipette solution (cytoplasmic buffer) containing 3 mM EGTA. The columns show the averaged effect of ANO8 on current density and on the slop of slow Ca2+-dependent inactivation (SCDI). The results are mean±s.e.m and difference were analyzed by unpaired t test.

(B) Fura2-loaded HEK cells transfected with YFP (control, black) or ANO8-YFP (red) were used to measure store-mediated Ca2+ influx. Stores were depleted by treatment with the SERCA inhibitor CPA and Ca2+ influx was measured by Ca2+ add-back. The results are mean±s.e.m and difference were analyzed by unpaired t test.

(C) FRET efficiency was measured with HEK cells transfected with STIM1-CFP, ANO8-YFP, and with (red) and without (black) Orai1-HA before and after store depletion. Here and all other FRET measurements, representative images of the FRET signal under each condition are shown next to the traces. The averages are given in (d).

(D) Summary of FRET efficiency measurements at the indicated conditions. The results are mean±s.e.m and difference were analyzed by unpaired t test.

(E) FRET efficiency was measured with HEK cells transfected with STIM1-CFP, STIM1-YFP (blue), and co-transfected with untagged ANO8 (green) or treated with siANO8 (red) before and after store depletion.

(F) Resting (R) or store-depleted cells (S) HEK cells transfected with STIM1-YFP and myc-STIM1 and with or without ANO8 or treated with siANO8, were used to immunoprecipitate (IP) STIM1-YFP and blot (B) for mys-STIM1. The inputs (In) are with anti-myc, anti-YFP or anti-ANO8.

(G) Confocal and bright field images of basal (upper) and store depleted cells (lower) of HEK cells transfected with ANO8-YFP alone.

(H) TIRF images (upper) and TIRF-Z scan (lower) of the same resting and store depleted cells HEK cells transfected with ANO8-YFP alone.

(I): Confocal images of resting cells transfected with ANO8-YFP, STIM1-CFP and Orai1-mCherry.

(J) Confocal images of store depleted cells transfected with ANO8-YFP, STIM1-CFP and Orai1-mCherry.

(K) TIRF-Z scan of resting (upper) and store depleted (lower) cells transfected with ANO8-YFP and STIM1-mCherry demonstrating the relationship between STIM1 and ANO8 puncta at the TIRF field. Cell surface is on top.

Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. The experiments in (G-K) represent at least 4 separate experiments with similar results.
Anoctamin 8 Tethers Endoplasmic Reticulum and Plasma Membrane for Assembly of Ca2+ Signaling Complexes at the ER/PM compartment (2019)
Figure 2

Fig 2: ANO8 increases native and expressed STIM1-Orai1 complexes and the number and size of ER/PM junctions.

(A) FRET ratio was measured with HEK cells transfected with STIM1-CFP, Orai1-YFP (black) and co-transfected with untagged ANO8 (red) before and after store depletion with 25 µM CPA in Ca2+-free solution.

(B-E) TIRF microscopy was used to quantify puncta formation at the TIRF field in cells transfected with STIM1-CFP, ANO8-YFP and Orai1-mCherry before and after store depletion. (A) Example images. (B) Time course of STIM1 puncta formation in cells transfected with STIM1 and Orai1 alone (black) or together with ANO8 (red). (C-E) Effect of ANO8 on the rate of STIM1-STIM1 clustering (C), number of STIM1 puncta (D) and number of Orai1 puncta (E). The results are mean±s.e.m and difference were analyzed by unpaired t test.

(F) HEK cells were used to immunoprecipitate (IP) the native ANO8 (A8) or Orai1 (O1) and blotted (B) for STIM1. R=resting, S=stimulated.

(G) HEK cells treated with scrambled (siScr) or siANO8 (siA8) were used to IP the native Orai1 (O1) or STIM1 (S1) and blotted (B) for the native STIM1, Orai1 or ANO8, as indicated. The columns show the mean±s.e.m of 4 independent experiments. The results are mean±s.e.m and difference were analyzed by unpaired t test.

(H) Co-IP of myc-STIM1 and Orai1-HA, myc-STIM1 and ANO8-YFP, and Orai1-HA and ANO8-YFP was measured in transfected HEK cells before (R, resting) and in response to store depletion (S, stimulated).

(I) Effect of ANO8-YFP on myc-STIM1 and Orai1-HA at the ER/PM junction.

(J-L) Junctional Orai1 (J), STIM1 (K) and ANO8 (L) in resting (R) and store-depleted cells (S). The results are mean±s.e.m of 4 experiments and difference were analyzed by unpaired t test. (M) Example EM images recorded from HEK cells transfected with empty vector (left) or ANO8 (right). Red arrows mark ER/PM junctions.

(N, O) The size (N) and density (O) of ER/PM junctions in vector-transfected (control) and ANO8-transfected cells.

Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.