EMBO/soda-nel huggingface dataset:
Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model (2018)
Figure 1

Figure 1 - Stat3 activation in an AD model is efficiently reduced using a conditional knockout strategy

(A-B) To verify the specificity of Cx43-CreERT mice for astrocytes, mice were crossed to a tdTomato reporter line to induce Cre-dependent tdTomato expression after tamoxifen administration. TdTomato fluorescence was enhanced with an antibody against red fluorescent protein, and astrocytes were stained with antibodies against GFAP or S100β (not shown). Merged images and quantification show efficient and widespread tdTomato expression specific for astrocytes (arrowheads) in 8-month and 11-month old animals. Expression was small in neurons (stained with NeuN, not shown) and negligible in NG2 cells and oligodendrocytes. Scale bars, 250 µm (upper panel) and 50 µm (lower panel).

(C) The majority of peri-plaque astrocytes in APP/PS1-Stat3WT were positive for activated/phosphorylated Stat3 (pStat3) in the cortex (Cx) and hippocampus (HC), whereas the fraction of pStat3-positive astrocytes was significantly lower in APP/PS1-Stat3KO mice in both regions (n = 8 mice (3 females and 5 males) per group; age, 8 months; Mann-Whitney test). The occurrence of pStat3 in WT-Stat3KO and WT-Stat3WT mice was negligible (n = 8 mice (4 females and 4 males) per group; age, 8 months; Mann-Whitney test).

(D-E) Examples of Stat3 activation in APP/PS1-Stat3KO and APP/PS1-Stat3WT. D, pStat3 (arrowheads) was abundantly present in reactive astrocytes (identified by GFAP) around plaques (identified by IC16). E, only few astrocytes were positive for pStat3 in APP/PS1-Stat3WT mice. Scale bars, 50 µm.

(F-G) in postmortem brain sections from AD patients, we detected nuclear pStat3 (arrows) in the majority of peri-plaque reactive astrocytes (identified by GFAP) in the cortex and hippocampus (plaques were stained with methoxy-XO4, arrowheads). Scale bar, 50 µm.
Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model (2018)
Figure 2

Figure 2 - Astrocyte-specific Stat3 deletion induces greater astrocytic and microglial complexity

(A-B) Overall astrocytic and microglial coverage, as assessed by GFAP and Iba1 stainings, remained unchanged in APP/PS1-Stat3WT vs. APP/PS1-Stat3KO mice (n = 8 mice (4 females and 4 males) per group; age, 8 months; Mann-Whitney test for each comparison). Scale bars, 500 µm.

(C-D) The volume of peri-plaque reactive astrocytes was increased by the Stat3 deletion (Mann-Whitney test), and there was a nonsignificant trend towards more astrocytes branches and junctions and longer total process length (Mann-Whitney test; APP/PS1-Stat3WT, n = 5 (2 female and 3 male) mice; APP/PS1-Stat3KO, n = 7 (3 female and 4 male) mice; age, 8-10 months). Scale bar, 20 µm.

(E-F) Microglial volume was not affected by the Stat3 deletion (Mann-Whitney test), but peri-plaque microglia had significantly more microglial branches and junctions per plaque, and the total process length of peri-plaque microglia was increased (Mann-Whitney test; same mice as in C-D). Scale bar, 20 µm.
Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model (2018)
Figure 3

Figure 3 - Astrocyte-specific Stat3 deletion reduces Aβ plaque burden and Aβ levels without altering APP metabolism

(A-B) Aβ plaque burden, as assessed by plaque load and size using an anti-Aβ antibody, was strongly reduced in APP/PS1-Stat3KO vs. APP/PS1-Stat3WT mice (Mann-Whitney test; scale bar, 300 µm).

(C) Plaque density, assessed by staining brain sections with thioflavin (yellow), remained unchanged in APP/PS1-Stat3KO vs. APP/PS1-Stat3WT mice (Mann-Whitney test; scale bar, 250 µm).

(D-E) Electrochemiluminescence ELISA after sequential extraction from whole-brain homogenates using RIPA and SDS buffer revealed that soluble Aβ1-42 and Aβ1-40 were significantly reduced (Mann-Whitney test), whereas there was a nonsignificant trend towards reduced levels of insoluble Aβ1-42 and Aβ1-40 (Mann-Whitney test).

(F-H) Western Blot quantification of full-length amyloid precursor protein (APP) and its C-terminal fragments (CTF) showed no differences between both groups (Mann-Whitney test; for all datasets, n = 9 male mice for both groups (age, 11 months)).
Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model (2018)
Figure 4

Figure 4 - Astrocyte-specific Stat3 deletion increases microglial Aβ internalization and degradation, and reduces ApoE expression, dystrophic neurites and detrimental cytokines

(A) Internalization of Aβ (stained with IC16 antibody or methoxy-XO4) was assessed using an engulfment assay, in which glial and Aβ structures were surface-rendered and Aβ volumes co-localized with glial volumes were quantified. Scale bars, 10 µm.

(B-C) Microglia (left Y axes) from APP/PS1 mice internalized significantly more Aβ positive for IC16 or methoxy-XO4 when Stat3 was deleted in astrocytes (Mann-Whitney test), whereas no changes were seen in astrocytes (right axes; APP/PS1-Stat3WT, n = 8 (4 female and 4 male) mice; APP/PS1-Stat3KO, n = 11 (5 female and 6 male) mice; age, 11 months; Mann-Whitney test).

(D-G) Western Blot quantification of protein levels of the Aβ-degrading enzymes neprilysin/CD10 and CD36, as well as the Aβ-binding apoliprotein E (apoE) revealed a significantly increased expression of neprilysin and CD36 and a decreased expression of apoE (APP/PS1-Stat3WT, n = 9 (5 female and 4 male) mice; APP/PS1-Stat3KO, n = 9 (5 female and 4 male) mice; age, 11 months; Mann-Whitney test for all comparisons). In contrast, TREM2 expression remained unchanged (APP/PS1-Stat3WT, n = 8 (4 female and 4 male) mice; APP/PS1-Stat3KO, n = 7 (4 female and 3 male) mice; age, 11 months; Mann-Whitney test).
Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model (2018)
Figure 5

Figure 5 - Stat3 deletion triggers a phenotypical switch in reactive astrocytes

(A-B) Quantitative PCR from cortex of APP/PS1-Stat3KO compared to APP/PS1-Stat3WT mice revealed lower expression of 'A1' markers Amigo2 and C3, whereas Ggta1 remained unchanged. In turn, the 'A2' marker Tm4sf1 was upregulated and there was a nonsignificant trend for a higher expression of B3gnt5 (n = 6 mice (3 females and 3 males) per group; age, 8 months; Mann-Whitney test).

(C-F) Confirming lower expression of the 'A1' marker C3d, western blot analysis indicated lower protein levels of C3d in APP/PS1-Stat3KO. Immunohistochemistry using an antibody against C3d revealed that lower expression of C3d particularly occurred in peri-plaque reactive astrocytes (arrows; plaques were visualized with methoxy-XO4, arrowheads; scale bars, 50 µm;

APP/PS1-Stat3WT, n = 6 (2 female and 4 male) mice; APP/PS1-Stat3KO, n = 6 (3 female and 3 male) mice; age, 8 months; Mann-Whitney test).

(G) Whole-brain levels of the pro-inflammatory cytokines IL-1β and TNF-α were significantly reduced in APP/PS1-Stat3KO mice (Mann-Whitney test), whereas no changes were seen for IL-10 (APP/PS1-Stat3WT, n = 13 (6 female and 7 male) mice; APP/PS1-Stat3KO, n = 13 (8 female and 5 male) mice; age, 11 months; Mann-Whitney test).

(H) These changes were paralleled by a decrease in the area covered by dystrophic neurites in APP/PS1-Stat3KO compared to APP/PS1-Stat3WT mice, as assessed by LAMP1 staining (Mann-Whitney test; n = 10 male mice for both groups; age, 8 months; scale bars, 300 µm).
Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model (2018)
Figure 6

Figure 6 - Astroglial Stat3 modulates network imbalance in APP/PS1 mice

(A) For in vivo two-photon imaging, astrocytes (arrows) and neurons (arrowheads) were labeled with the calcium indicator OGB-1, and astrocytes were co-labeled with sulforhodamine 101 (SR101; arrows). Aβ plaques were labeled with the intravital dye methoxy-XO4 (open arrowheads). Scale bar, 50 µm.

(B) Calcium imaging of anesthetized animals showed that the hyperactivity of astrocytes in APP/PS1-Stat3KO mice was reduced to levels comparable to WT-Stat3WT mice, but significantly increased in APP/PS1-Stat3WT mice (One-Way ANOVA followed by Bonferroni's multiple comparison test; n = 6 mice (3 female and 3 male) for each group; age, 8 months).

(C-D) Similarly, neuronal activity was also reduced to levels comparable to WT-Stat3WT mice in APP/PS1-Stat3KO mice, but significantly increased in APP/PS1-Stat3WT mice (One-Way ANOVA followed by Bonferroni's multiple comparison test; same mice as in B). The cumulative distributions of neuronal calcium transients in APP/PS1-Stat3KO mice were not different from those of WT-Stat3WT mice (p = 0.31, Kolmogorov-Smirnov test), but significantly different from those of APP/PS1-Stat3WT mice (p = 0.001, Kolmogorov-Smirnov test).

(E) To investigate a reciprocal scenario in which astroglial hyperactivity reduces Stat3 activation, we implanted osmotic minipumps into APP/PS1 mice and treated them with the network-normalizing P2Y1R inhibitor MRS2179 or vehicle for 6 weeks.

(F-I) Two-photon imaging of these mice confirmed that astrocytic hyperactivity and propagating calcium waves were reduced by MRS2179 (APP/PS1-Stat3WT, n = 6 (4 female and 2 male) mice; APP/PS1-Stat3KO, n = 6 (2 female and 4 male) mice; age, 8 months; Mann-Whitney test). This network normalization induced a reduction of activated Stat3 (pStat3) in astrocytes assessed in fixed brain sections of the same mice (Mann-Whitney test). Scale bars, 50 µm.
Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model (2018)
Figure 7

Figure 7 - Stat3 deletion in astrocytes protects from spatial memory and learning deterioration

(A) Spatial learning and memory were assessed in the Morris Water Maze paradigm. APP/PS1-Stat3KO mice showed faster latencies to reach the hidden platform compared with APP/PS1-Stat3WT on days 4 and 5, but were similar to WT-Stat3WT and WT-Stat3KO mice (Two-way repeated-measures ANOVA followed by Bonferroni post-hoc test; P values are for APP/PS1-Stat3KO vs. APP/PS1-Stat3WT mice).

(B) The area under the curve (AUC) for the latency to reach the hidden platform was similar in APP/PS1-Stat3WT compared to WT-Stat3WT and WT-Stat3KO mice, but significantly higher in APP/PS1-Stat3KO mice (Kruskal-Wallis test followed by Dunn's multiple comparisons test).

(C) The swimming velocity was similar in all groups (Kruskal-Wallis test followed by Dunn's multiple comparisons test).

(D-E) In the probe trial 24 h after the last training day, the time mice spent in the target quadrant (TQ) was different from chance in all groups except for APP/PS1-Stat3WT mice (p < 0.05, one-tailed one-sample t test). APP/PS1-Stat3KO, WT-Stat3WT and WT-Stat3KO mice spent significantly more time in the target quadrant compared to the mean of all other quadrants (AO), whereas APP/PS1-Stat3WT spent equal times in the target and all other quadrants (Wilcoxon matched-pairs signed rank test for each comparison;

APP/PS1-Stat3WT, n = 15 (8 female and 7 male) mice; APP/PS1-Stat3KO, n = 15 (9 female and 6 male) mice; age, 8-9 months).
Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model (2018)
Figure 8

Figure 8 - The systemic Stat3 inhibitor SH-4-54 confers protection from cognitive decline in APP/PS1 mice

(A) APP/PS1 mice were systemically treated with SH-4-54 for 6 weeks and compared to age-matched APP/PS1 mice treated with vehicle. In the Morris Water Maze test, treatment with SH-4-54 led to significantly faster latencies to reach the hidden platform on the last training day (Two-way repeated-measures ANOVA followed by Bonferroni post-hoc test).

(B) The area under the curve (AUC) for the latency to reach the hidden platform was also smaller in APP/PS1 mice treated with the Stat3 inhibitor (Mann-Whitney test).

(C) The swimming velocity was not affected by the treatment (Mann-Whitney test).

(D) In the probe trial test, APP/PS1 mice treated with the Stat3 inhibitor spent significantly more time in the target quadrant (TQ) compared to the mean of all other quadrants (AO), whereas APP/PS1 mice treated with vehicle spent equal times in the target and all other quadrants (Wilcoxon matched-pairs signed rank test for all comparisons; APP/PS1-Stat3WT, n = 12 (5 female and 7 male) mice; APP/PS1-Stat3KO, n = 12 (4 female and 8 male) mice; age, 8 months).

(E) To verify target engagement in the brain, the same mice assessed in the Morris Water Maze were anesthetized and imaged using in vivo two-photon microscopy of calcium activity. Systemic treatment with the Stat3 inhibitor reduced the hyperactive phenotype of cortical neurons (Mann-Whitney test).
Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model (2018)
Figure 9

Figure 9 - Target engagement of the systemic Stat3 inhibitor in APP/PS1 mice.

(A-D) SH-4-54 significantly decreased plaque growth, as assessed by IC16 immunohistochemistry, while plaque load remained unchanged. There was also no significant change in dystrophic neurite area during the treatment time (Mann-Whitney test for all comparisons; scale bars, 500 µm).

(E-G) The fraction of pStat3-positive astrocytes in the peri-plaque region was strongly reduced by the treatment with the Stat3 inhibitor (arrowheads indicate pStat3 signals; scale bars, 100 µm; Mann-Whitney test).

(H-K) While no changes were seen in morphological parameters of peri-plaque astrocytes, there was a significant increase in the process length of near-plaque microglia, indicating higher microglial complexity (Mann-Whitney test for all comparisons; APP/PS1-Stat3WT, n = 12 (6 female and 6 male) mice; APP/PS1-Stat3KO, n = 12 (7 female and 5 male) mice; age, 8 months).

EXPANDED VIEW FIGURE LEGENDS
NEDD4L-mediated Merlin ubiquitination facilitates Hippo pathway activation (2020)
Figure 1

Figure 1. Merlin ubiquitination commonly exists and associates with activation of the Hippo pathway.

(A) Total lysates from the indicated cell lines cultured in regular conditions at a steady state were subjected to western blotting. s.e., short exposure; l.e., long exposure. The major Merlin band is indicated by an asterisk. The ratio of mono-ubiquitinated to native Merlin in each lane of the short exposure blot was quantified by Image J and is shown under the blot. The same lysates were also used in Figure EV1A. Asterisks indicate native Merlin, arrows show ubiquitinated Merlin.

(B) Total lysates from LN229 cells in which Merlin was or was not knocked out (KO) were subjected to western blotting.

(C) and (D) LN229 (C) or Met5-A (D) cells were detached (denoted by sus., referring to suspension) by trypsinization (see Methods) and reseeded (attach) for two (LN229) or four (Met5-A) hours. Total lysates from these cells were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Ubiquitinated Merlin is marked with arrows.

(E) LN229 cells transduced with a Merlin or scrambled shRNA were treated with DMSO, ionomycin (Iono) or thapsigargin (TG). Total lysates of these cells were subjected to western blotting. The major Merlin band is indicated by an asterisk. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Ubiquitinated Merlin is marked with an arrow.

(F) LN229 cells treated with DMSO or thapsigargin (TG) were lysed and subjected to immunoprecipitation with a Merlin antibody or IgG. The immunoprecipitated products were subjected to western blotting. The number of conjugated ubiquitin (Ub) moieties was marked. Tetra-ubiquitinated Merlin is indicated by an arrow.

(G) LN229 cells stably transduced with 6x-Histidine-tagged ubiquitin (His-Ubi) were treated with DMSO or thapsigargin (TG). These cells were lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. Both arrowhead and arrow point to the mono-ubiquitinated Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.

(H) LN229 cells stably transduced with 6x-Histidine-tagged ubiquitin (His-Ubi) were detached or reseeded as in (C). These cells were lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. Arrowheads and arrows point to the mono- or di-ubiquitinated Merlin (see a longer exposed blotting results in Figure EV1D). The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.
NEDD4L-mediated Merlin ubiquitination facilitates Hippo pathway activation (2020)
Figure 2

Figure 2. Dephosphorylation of serine 518 promotes Merlin ubiquitination.

(A) LN229 cells were detached (denoted by sus., referring to suspension) by trypsinization and reseeded (denoted by attach) for two hours. Total lysates from these cells were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.

(B) LN229 cells treated with DMSO, ionomycin (Iono) or thapsigargin (TG) were lysed and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.

(C) LN229 cells treated with DMSO or thapsigargin (TG) were subjected to active RAC1 pulldown assay followed by western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.

(D) and (E) LN229 cells transduced with vector or EGFP-RAC1(Q61L) (D) or Myc-PAK1(T423E) (E) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.

(F) LN229 cells stably transduced with wild-type Merlin or the indicated mutants were treated with DMSO or thapsigargin (TG) and analyzed by western blot. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Native Merlin (asterisk); ubiquitinated Merlin (arrow).

(G) Merlin-depleted LN229 cells stably transduced with Flag-tagged wild-type Merlin or the indicated mutants were treated with DMSO or thapsigargin (TG). The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.
NEDD4L-mediated Merlin ubiquitination facilitates Hippo pathway activation (2020)
Figure 3

Figure 3. NEDD4L is the E3 ubiquitin ligase of Merlin.

(A) LN229 cells stably transduced with Flag-tagged Merlin or empty vector were treated with DMSO or thapsigargin (TG) and subjected to Flag immunoprecipitation followed by mass spectrometry. Numbers of unique peptides of each protein in the immunoprecipitated products are shown.

(B) LN229 cells transduced with a pool of four siRNAs against NEDD4L (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the short exposure blot was quantified by Image J and is shown under the blot.

(C) LN229 cells stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.

(D) LN229 cells stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were detached by trypsinization (see Methods) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.

(E) LN229 cells stably transduced with 6x-Histidine-tagged Ubiquitin (His-Ubi) were then stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA. These cells were detached as in (D), lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.

(F) HEK293T cells were transfected with indicated genes and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.

(G) HEK293T cells were transfected with indicated genes. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.

(H) LN229 cells were lysed and subjected to immunoprecipitation with a Merlin antibody, a NEDD4L antibody, or IgG. The immunoprecipitated products were subjected to western blotting.
NEDD4L-mediated Merlin ubiquitination facilitates Hippo pathway activation (2020)
Figure 4

Figure 4. AMOTL1 promotes Merlin ubiquitination by mediating the interaction between NEDD4L and Merlin.

(A) LN229 cells transduced with a pool of four siRNAs against AMOTL1 (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.

(B) LN229 cells stably transduced with 6x-Histidine-tagged Ubiquitin (His-Ubi) were then stably transduced with indicated shRNAs against AMOTL1 or a scrambled shRNA control (sh-Co.). These cells were detached, lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.

(C) A diagram illustrating that AMOTL1 mediates the NEDD4L-Merlin interaction.

(D) Merlin-depleted LN229 cells stably expressing Flag-HA-tagged Merlin were transduced with scrambled shRNA (sh-Co.) or two distinct shRNAs targeting AMOTL1, treated with DMSO or thapsigargin (TG) and subjected to Flag immunoprecipitation followed by western blotting.

(E) HEK293T cells were transfected with the indicated constructs. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. PY: PPEY motif; LY: LPTY motif. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.

(F) Merlin-depleted LN229 cells stably transduced with Flag-tagged wild-type Merlin or the indicated mutants were treated with DMSO or thapsigargin (TG). The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.

(G) HEK293T cells were transfected with the indicated constructs. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.