EMBO/soda-nel huggingface dataset:
MacroH2A1.2 deficiency leads to neural stem cell differentiation defects and autism-like behaviors (2021)
Figure 3

Figure 3 Depletion of mH2A1.2 reduces neural progenitor cell differentiation.

(A) Representative images of E16.5 coronal brain sections immune stained for Tbr2 and BrdU. BrdU was injected intraperitoneally into pregnant mice at E16.5 for 2h of pulse labeling. Scale bar represents 20 μm.

(B) Statistics of TBR2+ cells per 100μm2 surface of VZ/SVZ. The number of TBR2+ cells increased significantly in KO mice compared to WT mice. n=6 mice, independent replicates.

(C) Statistics of BrdU+ cells per 100μm2 surface of VZ/SVZ. The number of BrdU+ cells increased significantly in KO mice compared to WT mice. n=6 mice, independent replicates.

(D) Percentage of TBR2+BrdU+ cells among all TBR2+ cells. TBR2+BrdU+ cells were increased in KO mice compared to WT mice. Two-tailed unpaired t-test. n=6 mice, independent replicates.

(E) Cell cycle exit was decreased in KO mice. E16.5 brain sections were stained with anti-BrdU and anti-Ki67 in WT mice and KO mice that were administered BrdU (100 mg/kg) for 24 h and harvested at E16.5. Scale bar represents 20 μm.

(F) Percentage of cell cycle exit (BrdU+Ki67−/ BrdU+) in WT mice and KO mice.

n=7 mice, independent replicates.

(G)Western blot analysis showed that the protein expression levels of neural stem cell marker, including TBR2, PCNA, and PAX6 are increased in mH2A1.2 KO mice compared with WT.

(H) Statistics of the normalized density of TBR2, PCNA, and PAX6. n=5 mice, independent replicates.

(I) Western blot analysis showed that the protein expression levels of the neuron marker TUJ1 are downregulated in mH2A1.2 KO versus WT mice. n=5 mice, independent replicates.

(J) Statistics of the normalized density of TUJ1. TUJ1 was also reduced in protein level. n=5 mice, independent replicates.

(K) Coronal brain slices of E16.5 WT mice and KO mice were immunostained using anti-TUJ1. Scale bar represents 20 μm.

(L) Relative TUJ1+ cells in the VZ of WT mice and KO mice. TUJ1 was reduced in cortical. n=6 mice, independent replicates.

(M) The apoptosis levels between WT and KO brains showed no significant difference. E16.5 brain sections of WT and KO mice were subjected to TUNEL assay. Scale bar represents 50 μm.

(N) Graph shows the numbers of apoptotic cells per field. n=4mice, independent replicates.

Date information: Representative images from at least three independent experiments.

Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.
MacroH2A1.2 deficiency leads to neural stem cell differentiation defects and autism-like behaviors (2021)
Figure 4

Figure 4 The loss of mH2A1.2 results in abnormal neuronal morphology and impaired neurogenesis.

(A) Representative images of neurons after 4 days of culture in vitro. GFP expressing plasmid was electroporated into the E13.5 cerebral cortices of WT and KO mice to label neural progenitor cells. After 24 h, the GFP+ cells were isolated and cultured in differentiation medium for 4 days. Scale bar represents 15 μm.

(B) Graph shows that the total dendritic length of neurons is decreased when

mH2A1.2 is deleted. n = 25 cells from four samples.

(C) Statistics show that the number of branch points is reduced upon mH2A1.2 deletion.

n = 25 cells from four samples.

(D) High-magnification confocal images of the upper layer show that mH2A1.2 ko results in abnormally branched processes compared to WT. The GFP plasmid was electroporated into the WT and KO embryonic brains at E13.5 and harvested at P0. Scale bar represents 10 μm.

(E) Quantification of dendritic numbers in WT and KO mouse cortices. n = 30 for all samples.

(F) The brain sections were immunostained with CUX1 and CTIP2 (marker of layer I; marker of layer V) at P0. Scale bar represents 20 μm. n=4 mice, independent replicates.

(G) Statistics of CUX1+cells in the upper layer. CUX1+cells were increased in KO mice n=4 mice, independent replicates.

(H)Statistics of CTIP2+ cells in the deep layer. CTIP2+ cells were reduced in KO mice. n=4 mice, independent replicates.

Date information: Representative images from at least three independent experiments.

Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**).
MacroH2A1.2 deficiency leads to neural stem cell differentiation defects and autism-like behaviors (2021)
Figure 6

Figure 6 mH2A1.2 inhibits neural progenitor cell proliferation by targeting the downstream gene NKX2.2.

(A) The heat map shows the gene profiling expression in E13 forebrain. Gene analysis revealed that the transcript levels of genes were upregulated or downregulated by more than two-fold between the KO and the WT cells. n= 2 biological replicates.

(B)RNA-seq analysis showing that the NKX2.2 mRNA expression level is dramatically reduced in the KO cells.

(C) Western blot analyses of the NKX2.2 expression levels in the WT and KO cortices at E13.5.

(D) NKX2.2-shRNA or control plasmid was electroporated into the brains of E13.5

embryos. At E16.5, the brains were harvested to investigate the cell distribution.

(E) Graph shows the percentage of GFP+ cells in the VZ/SVZ, IZ and CP. The number of GFP+ cells in the VZ/SVZ region increased significantly, and the number of GFP+ in the CP layer decreased significantly. n =5 mice, independent replicates.

(F) ChIP analysis revealed that mH2A binds to the NKX2.2 promoter region in NPCs. Primary NPCs were infected with control, or Flag-mH2A overexpression lentivirus. Six different regions (from −5k to CDS) of the NKX2-2 sequence were used for the analysis. n =4 independent replicates.

(G) Western blot analyses of H3K27ac, H3K56ac, H3K36me3, H3K4me3 and H3K27me3 expression protein levels in WT and KO cortices at E13.5.

(H) Quantification of H3K27ac expression in WT and KO cortices. The levels of H3K27ac decreased. n=5 mice, independent replicates.

(I) Representative images of E16.5 cortices that had been electroporated with GFP or GFP+NKX2.2 into WT and KO brains at E13.5. Scale bar represents 50 μm.

(J) Graphs of the percentage of GFP+ cells in the VZ/SVZ, IZ and CP. Overexpression of NKX2-2 restored the distribution of GFP+ cells to the normal state.

n=5 mice, independent replicates.

Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**). n.s., not significant.

EXPAND VIEW FIGURE LEGEND
Mitochondria-associated membrane collapse is a common pathomechanism in SIGMAR1- and SOD1-linked ALS (2016)
Figure 3

Figure 3 - Accelerated disease onset in Sig1R-deficient SOD1G85R mice

A. The levels of Sig1R in the lumbar spinal cord (LSC) or brain of Sig1R−/−, Sig1R+/−, or Sig1R+/+ mice at 5 months old. Representative immunoblots obtained from three independent experiments are shown. Asterisk denotes non-specific bands.

B and C. Sig1R-deficient SOD1G85R mice (SOD1G85R/Sig1R−/−) exhibited accelerated onset of the disease (B) and shortened survival time (C) compared to SOD1G85R mice with one or two copies of Sig1R (SOD1G85R/Sig1R+/−, SOD1G85R/Sig1R+/+). Open circles indicate Sig1R knockout mice (Sig1R−/−) that never developed motor neuron disease but that were sacrificed at 396 days of age (n = 8). Mean onset or survival times of mice with ±SD are shown on the top of Kaplan-Meyer curves. **: p < 0.01, *: p < 0.05, log-rank test. n = 14 (SOD1G85R/Sig1R+/+), 29 (SOD1G85R/Sig1R+/−), 17 (SOD1G85R/Sig1R−/−).

D and E. Body weights were lost earlier both in male (D) and female (E) of SOD1G85R/ Sig1R−/− mice. Data are shown as mean ± SD. *: p < 0.05 in SOD1G85R/Sig1R+/+ vs. SOD1G85R/Sig1R−/−, †: p < 0.05 in SOD1G85R/Sig1R+/− vs. SOD1G85R/Sig1R−/−. n = 8 (SOD1G85R/Sig1R+/+), 13 (SOD1G85R/Sig1R+/−), 8 (SOD1G85R/Sig1R−/−) in male (D), and n = 6 (SOD1G85R/Sig1R+/+), 16 (SOD1G85R/Sig1R+/−), 9 (SOD1G85R/Sig1R−/−) in female (E). The used animals were the same in the panels B and C. The animals used in the panels D and E were the same in the panels B and C. *: p < 0.05 in SOD1G85R/Sig1R+/+ vs. SOD1G85R/Sig1R−/−, †: p < 0.05 in SOD1G85R/Sig1R+/− vs. SOD1G85R/Sig1R−/−. Two-way ANOVA with subsequent post hoc Tukey’s test.

F. Decreased performance on the rotarod test was observed in SOD1G85R/ Sig1R−/− mice. The test was performed at 0-30 rpm with 0.1 rpm/s acceleration every week. Data are shown as mean ± SD. **: p < 0.0001 in SOD1G85R/Sig1R+/+ vs. SOD1G85R/Sig1R−/−, † †: p < 0.0001 in SOD1G85R/Sig1R+/− vs. SOD1G85R/Sig1R−/−. The used animals were the same in the panels B-E. Two-way ANOVA with subsequent post hoc Tukey’s test.

G and H. Motor function of WT (Sig1R+/+) and Sig1R knockout (Sig1R−/−) mice at 5 or 12 months old was evaluated by the rotarod test (0-30 rpm, accelerated at 0.1 rpm/s). Average time on the rotating rod was plotted. Error bars denote SD. Unpaired t-tests.
Mitochondria-associated membrane collapse is a common pathomechanism in SIGMAR1- and SOD1-linked ALS (2016)
Figure 4

Figure 4. Mutant SOD1 proteins accumulate at the MAM and mitochondria in mice and cells expressing mutant SOD1.

A and B. N2a cells expressing SOD1WT, SOD1G85R or SOD1G93A (A), or spinal cords of WT or mutant SOD1 transgenic mice at end-stage (B) were fractionated as described in Materials and methods section (Cyto: cytoplasm, P1: nuclei and debris, Mito: mitochondria, MAM: mitochondria-associate membrane, P3: microsomal fraction). Proper fractionation was confirmed by the fraction specific markers as indicated, and also shown in Fig EV1 A-E. An asterisk denotes non-specific bands.

C. Tissue and cell-type specific accumulation of mutant SOD1 at the MAM. Brain, liver, or primary astrocytes from SOD1G93A mice were fractionated and blotted as in Figures 4A and B (Upper panel). Quantification of SOD1 proteins at the MAM relative to ones in the cytoplasm was performed from immunoblotting data with an anti-human SOD1 antibody in (B) and the top panel of (C), and the data are plotted as mean ± SEM from three independent experiments (Lower panel). **: p < 0.01 vs. SOD1WT spinal cord. A one-way ANOVA with subsequent post hoc Tukey’s test.

Proper fractionation of these samples was confirmed in Fig EV 1F-H.

D. Mutant SOD1 prevented association of ER with mitochondria. Isolated ER (P3: microsomal fraction) and mitochondria of N2a cells expressing wild-type (WT) or G85R (85) SOD1 for 48 h were mixed and incubated. Mitochondrial pellets after centrifugation were analyzed by immunoblotting using anti-PDI (ER marker) and anti-VDAC (mitochondrial marker), respectively. All these results were confirmed by three independent experiments.
Mitochondria-associated membrane collapse is a common pathomechanism in SIGMAR1- and SOD1-linked ALS (2016)
Figure 5

Figure 5. Accumulation of mutant SOD1 at the MAM is most prominent around the disease onset.

A and B. Time course analyses of mutant SOD1 levels at the MAM in SOD1G93A mouse spinal cords (A) and brains (B). Immunoblots show levels of mutant SOD1 protein in the indicated fractions at various time points (left panels). Quantitative data at the right were plotted as mean ± SEM of three independent experiments. Arrows indicate a rapid reduction in SOD1 level at the MAM just following the onset of disease.

C and D. Time course analyses of mutant SOD1 accumulation at the MAM in SOD1G85R (C) and SOD1G37R (D) mouse spinal cords. Quantitative data from three independent experiments were plotted (bottom). The arrows in the bottom panel are the same as in A and B.

E. Time course analyses of MAM-specific proteins at MAM and in the whole lysates of SOD1G93A mouse spinal cords.

A-E. Fractions from a single mouse tissue at the indicated ages were loaded on each lane. Representative blots from at least three independent experiments were shown. GAPDH in the cytosolic fractions was used as a loading control.
Mitochondria-associated membrane collapse is a common pathomechanism in SIGMAR1- and SOD1-linked ALS (2016)
Figure 6

Figure 6. Loss of Sig1R or expression of mutant SOD1 compromises the MAM integrity in motor neurons.

A-I. Immunofluorescence staining of spinal cords and brains from non-transgenic (Non-Tg), SOD1 transgenic, or Sig1R−/− mice. Transverse sections of mouse spinal cords (A, D-I) or sagittal sections of mouse brains (B, C) were stained using anti-Sig1R (white), βIII-tubulin (red) and IP3R3 (green) antibodies. Note that Sig1R and IP3R3 are co-localized in the motor neurons of the anterior horn (A) and the hypoglossal nucleus (B), and IP3R3 was not expressed in hippocampal neurons (C). Mutant SOD1 induced aggregation of Sig1R and mislocalization of IP3R3 in anterior horn neurons (D-G), while their abnormalities were not observed in SOD1WT motor neurons (H). Mislocalization of IP3R3 was also observed in Sig1R−/− mouse spinal cords (I). Scale bars: 50 µm.

J and K. Representative electron micrographs of the MAM (J) (double-headed arrows) in motor neurons of 12 months-old Non-Tg, SOD1G85R or Sig1R−/− mice. Note that ER-mitochondria contacted areas were reduced in both SOD1G85R or Sig1R−/− mice. Quantification of the mitochondria surface associated to ER was calculated in K. For quantification, 13-19 motor neurons and 224-316 mitochondria with MAM in each animal (n = 2) were analyzed. Data are expressed as mean ± SEM. Double asterisk means p < 0.0001 vs Non-Tg. A one-way ANOVA with subsequent post hoc Tukey’s test. Scale bars: 300 nm.

L. The levels for IP3R3 and calreticulin were decreased in the MAM fractions of Sig1R−/− mouse brains. MAM fractions and whole tissue lysates of Sig1R+/+ or Sig1R−/− mouse brains were immunoblotted. Representative blots from three independent experiments are shown.
Mitochondria-associated membrane collapse is a common pathomechanism in SIGMAR1- and SOD1-linked ALS (2016)
Figure 7

Figure 7. Sig1R inhibits mutant SOD1-mediated, abnormal calpain activation and restores intracellular ATP levels.

A. N2a cells were transfected with WT or mutant SOD1 in the presence or absence of IP3R3. Plotted viability of the cells measured by a neurotoxicity assay revealed that IP3R3 is involved in cell vulnerability against mutant SOD1. Data are expressed as mean ± SEM from three independent experiments, triplicated in each experiment. **: p < 0.01, *: p < 0.05.

B-G. Calpain activity (B-D) and intracellular ATP levels (E-G) were measured in N2a-IP3R3 cells expressing Sig1R-FLAG (B and E), SOD1 (C and F), or both (D and G). Mean ± SEM from three independent experiments is plotted. *: p < 0.05.

H and I. Calpain activity in vivo was determined by the cleavage of spectrin α II. Calpain-cleaved 150 kDa fragment of spectrin α II in mouse lumbar spinal cord or brain (H) was normalized to β-actin (I). Quantitative data in immunoblotting analyses are plotted as mean ± SEM from three independent experiments. **: p < 0.01.

A one-way ANOVA with subsequent post hoc Tukey’s test (A-G, I).
Mitochondria-associated membrane collapse is a common pathomechanism in SIGMAR1- and SOD1-linked ALS (2016)
Figure 8

Figure 8. Pre-symptomatic administration of PRE-084, a Sig1R agonist, restores Sig1R functions at the MAM, and prevents Sig1R aggregation in vivo.

A and B. Cytoplasmic (A) or mitochondrial (B) Ca2+ flux was measured in N2a-IP3R3 cells treated with PRE-084 (5 µM) or NE-100 (5 µM) for 1 h prior to fluorescent imaging. Cytoplasmic and mitochondrial Ca2+ flux were detected by fluo-4 and Case12-mito, respectively. The fluorescent intensity was normalized by the resting state at 0 s, and plotted as mean ± SD. n = 10 each. **: p < 0.01, *: p < 0.05. Two-way ANOVA with subsequent post hoc Tukey’s test.

C and D. Calpain activity and intracellular ATP levels were measured in N2a-IP3R3 cells treated with PRE-084 (5 µM) or NE-100 (5 µM) for 24 h. Data are plotted as mean ± SEM from three independent experiments. *: p < 0.05 vs. mock control. A one-way ANOVA with subsequent post hoc Tukey’s test.

E and F. SOD1G93A mice were intraperitoneally administered with saline or PRE-084 (0.25 mg/kg, 3 times per week) from postnatal day 35 to 95. Lumbar spinal cord sections were stained by using anti-Sig1R (white), IP3R3 (green), and βIII-tubulin (red) at 95 days old. Arrowheads indicate the neurons with normal distribution of Sig1R and IP3R3. Scale bars: 50 µm.
Characterisation of the Cullin-3 mutation that causes a severe form of familial hypertension and hyperkalaemia. (2015)
Figure 2

CUL3Δ403–459 displays increased auto-ubiquitylation and ubiquitylation of KLHL3

A–D In vitro ubiquitylation assays with purified proteins. As ubiquitin is covalently attached to the substrate lysine residue, the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin. All assays contain purified UBE1, UBE2D3, ubiquitin, 0.1 mM ATP, 1 mM MgCl2 and were buffered in 50 mM HEPES, 150 mM NaCl and incubated at 30°C for the time indicated. Reactions were stopped by the addition of SDS–Laemmli buffer to a concentration of 1×. SDS–PAGE, staining with Coomassie blue, or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation. (A) to determine the relative modification of KLHL3 by CUL3WT and CUL3Δ403–459, KLHL3 was included into the reaction at 2× molar concentration over CUL3WT and CUL3Δ403–459. (B, C) Reactions serve to determine basal auto-ubiquitylation of CUL3WT or CUL3Δ403–459 and do not contain KLHL3 or other potential substrate proteins. Lysine residues on CUL3WT or CUL3Δ403–459 act as the substrate. (B) High molecular weight bands reflect ubiquitin chain linkages on CUL3 or the multiple mono-ubiquitylation of a number of CUL3 lysine residues. (C) Activity assays contain methylated ubiquitin, a form of ubiquitin incapable of forming ubiquitin chains; as such, higher band shifts reflect the attachment of mono-ubiquitin to one more lysine residue on CUL3WT or CUL3Δ403–459, respectively. (D) Activity assay performed as in (C) with methyl-ubiquitin, the boxes shown on the gel are indicative of the gel pieces excised for mass spectrometry analysis in (F).

E Schematic representation (to linear residue scale) highlighting the domains of CUL3 and schematic representation of the lysine residues of CUL3WT or CUL3Δ403–459, modified at 5 and 45 min, respectively, in the in vitro ubiquitylation assay shown in (D) and as determined by mass spectrometry.

F Structural docking model of CUL3Δ403–459 based on the structure of full-length CUL1 (1LDK) using Chimera (see Materials and Methods). The NTD is coloured green, while the CTD is red, and the four panels sample possible positioning of the CTD relative to the NTD in CUL3Δ403–459, assuming full flexibility of the linker after deletion of three helices encoded by exon 9.

Source data are available online for this figure.
Characterisation of the Cullin-3 mutation that causes a severe form of familial hypertension and hyperkalaemia. (2015)
Figure 3

CUL3Δ403–459 is unable to ubiquitylate WNK1 or WNK4 kinases in an in vitro system, and this deficiency cannot be rescued by the presence of CUL3WT

A–C In vitro ubiquitylation assays were performed as described in Figure 2, but with the addition of immunoprecipitated WNK1 in (A), or immunoprecipitated over-expressed FLAG-WNK4 in (B, C) (see Materials and Methods). The WNK kinases are modified by CUL3WT-KLHL3, with the higher molecular weight smear observed in anti-WNK1 and anti-FLAG panels representative of multiple ubiquitin molecules being covalently attached to the WNK protein. CUL3Δ403–459 is unable to modify WNKs. Samples from the same assay reactions were divided to allow immunodetection of the different protein components modified within the same assay reaction. (C) CUL3WT, CUL3Δ403–459 and an equimolar solution CUL3WT:CUL3Δ403–459 (1:1 Mix) were incubated with KLHL3 and immunoprecipitated FLAG-WNK4 in ubiquitylation reactions to determine the influence of CUL3Δ403–459 on the ubiquitylation activity of CUL3WT. Notably, the presence of CUL3Δ403–459 does not inhibit WNK ubiquitylation by CUL3WT.
Characterisation of the Cullin-3 mutation that causes a severe form of familial hypertension and hyperkalaemia. (2015)
Figure 4

CUL3WT/Δ403–459 mice recapitulate PHAII electrolyte imbalances due to over-activation of the renal WNK4-SPAK pathway

Western blot of whole kidney lysates from mice culled after a minimum 4-h fast. Following exsanguination after surgery, mouse tissues were rapidly harvested and stored, samples were then homogenised, clarified and quantified prior to SDS–PAGE. Immunodetection with the antibodies shown highlights elevated signalling through the WNK kinase pathway in CUL3WT/Δ403–459 versus CUL3WT mice. The lowest panel is an anti-CUL3 immunoprecipitation of the kidney lysate samples, whereby the CUL3 antibody was cross-linked to Protein G sepharose and used to affinity purify CUL3WT and CUL3Δ403–459 from kidney lysates. The samples were incubated together overnight to allow deneddylation of CUL3 proteins to occur and maximise CUL3 binding to the anti-CUL3 resin. Samples were thoroughly washed and eluted from the resin prior to SDS–PAGE and immunodetection for CUL3. The IP highlights that CUL3Δ403–459 is indeed present within the kidney lysate.

Fresh frozen kidney tissues from transplant patient and donor cadavers were homogenised, clarified and quantified prior to SDS–PAGE. Western blot analysis confirmed the expression of KLHL3 and CUL3 in normal healthy human kidneys.

Plasma aldosterone after a minimum 4-h fast was calculated by HTRF (homogeneous time-resolved fluorescence) aldosterone assay. The average aldosterone level per mouse was calculated from duplicate samples run in parallel on the assay. Blood was rapidly harvested in heparin-coated plasma extraction tubes following exsanguination after surgery, and samples were snap-frozen for storage. A 58% increase in aldosterone was detected in CUL3WT/Δ403–459 versus CUL3WT mice (*P = 0.0245). Two-tailed unpaired Student’s t-test; data are mean ± SEM.

Arterial blood biochemistries after a minimum 4-h fast. Under anaesthesia, the right carotid artery was cannulated to minimise atmospheric exposure of samples collected for iSTAT blood gas and electrolyte measurements. CUL3WT/Δ403–459 mice present with abnormal electrolyte homoeostasis compared to CUL3WT mice, exhibiting hyperkalaemia (***P = 0.0004) and hyperchloraemia (***P = 9.5 × 10−5) with a compensated metabolic acidosis (P = 0.7766), marked by a decrease in bicarbonate () (***P = 3.4 × 10−5), base excess (BE) (***P = 9.1 × 10−5) and partial pressure of carbon dioxide (pCO2) (***P = 0.0038). Two-tailed unpaired Student’s t-test; data are mean ± SEM.

Source data are available online for this figure.