EMBO/soda-nel huggingface dataset:
Aut2p and Aut7p, two novel microtubule‐associated proteins are essential for delivery of autophagic vesicles to the vacuole (1998)
Figure 7

(A) Autophagosome‐like vesicles (arrowhead) are accumulating in the cytoplasm of aut2Δ cells starved for 4 h on 1% K‐acetate in the presence of PMSF. Bar represents 100 nm. (B) Electron micrographs unravels the accumulation of double membrane‐layered autophagosome‐like vesicles in the cytoplasm of aut7Δ cells starved for 3 h in nitrogen‐free medium in the presence of PMSF. Arrowheads indicate the clearly distinguishable two membrane layers. Bar represents 100 nm. (C and D) Cytosol containing autophagosome‐like vesicles accumulating in the cytosol of aut2Δ cells (D) can be isolated with an additional 100 000 g centrifugation step yielding a P100 pellet fraction. In wild‐type cells (C), no P100 is detectable. Fatty acid synthase (Fas) was used as a cytosolic protein. Further details are given in the text.
Nec-1 alleviates cognitive impairment with reduction of Aβ and tau abnormalities in APP/PS1 mice (2016)
Figure 1

Figure 1. Nec-1 inhibits neural cell death in response to Aβ aggregates.

A. Chemical structure of Nec-1. The schematic diagram was drawn by S. Lee using ChemDraw Professional 15.0. software.

B, C. MTT cytotoxicity assay. (B) HT22 cell line was treated with various concentrations of Nec-1 (0 - 200 μM). Aggregated Aβ(1-42) was used as the positive control for the induction of neural cell death. (C) HT22, BV2 cell lines and primary astrocytes were pre-treated with Nec-1 (50 μM) 15 minutes prior to application of 10 μM of pre-aggregated Aβ(1-42) for indicated times. Cell proliferation was then measured by MTT assay. All data represented the mean ± standard deviation of three independent experiments performed in triplicate.

D, E. Neural cell death levels measured by staining with the Live/Dead Viability/Cytotoxicity Assay Kit (Molecular Probes). Pre-treatment of cells with 50 μM Nec-1 preceded the application of 10 μM pre-aggregated Aβ(1-42) by 15 minutes. The assay was performed 24 hours after Aβ(1-42) application. (D) Representation of the stained cells. Scale bar = 500 μm (E) Quantification of dead cells stained by EthD-1.

F. Internalization of Aβ(1-42) into HT22 and BV2 cell lines in the presence or the absence of Nec-1 pre-treatment for indicated periods.

G. Cellular expression levels of indicated proteins in HT22, BV2 cell lines, and primary astrocyte stimulated with Aβ(1-42) aggregates in the presence or the absence of Nec-1 pre-treatment for 24 hours.

Data information: In (B, C, and E), data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (One-way ANOVA followed by Bonferroni’s post-hoc comparisons tests). Exact p-values are shown in Table EV1. NT, non-treated cells.
Nec-1 alleviates cognitive impairment with reduction of Aβ and tau abnormalities in APP/PS1 mice (2016)
Figure 2

Figure 2. Nec-1 inhibits spatial working memory and hippocampal memory declines in AD-like mouse models.

A-E. Behavioural tests of APP/PS1 mice. (A) Schedule of behavioural tests. Four-month-old male APP/PS1 mice were intravenously injected with Nec-1 (6.25 mg/kg, n = 7) or vehicle (2.5% DMSO in PBS, n = 6) for 12 weeks (2 times per week). As controls, age-matched male wild-type (Wt) mice were injected with Nec-1 (6.25 mg/kg, n = 7) or vehicle (2.5% DMSO in PBS, n = 7) for 12 weeks. (B-D) Y-maze and passive avoidance tests on 7-month-old APP/PS1 mice after Nec-1 administration for 12 weeks. Average alternation (%) for each test group (B) and total entry number into each arm (C) on Y-maze. (D) Average latency time in seconds for each test group on passive avoidance test. (E) Survival of Wt and APP/PS1 mice after injection of Nec-1 (6.25 mg/kg).

F-I. Behavrioural tests of Aβ(1-42) infusion mice. (F) Schedule of behavioural tests. In first week, 8-week-old male ICR mice (n = 8 per each group) were intravenously injected with either Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS) twice. Then 5 μl of Aβ(1-42) (100 μM) aggregates or vehicle (10% DMSO in PBS) were injected in intracerebroventricular region to generate Aβ(1-42) infusion mice model. In second week, same concentrations of Nec-1 or vehicle were intravenously injected twice again. (G) Intracerebroventricular (i.c.v.) injection site brain schematic diagram. (H, I) Y-maze tests on Aβ(1-42) infusion mice model injected with Nec-1. Average alternation (%) for each test group (H) and total entry number into each arm (I) on Y-maze.

Data information: In (B-D, H, and I), data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (One-way ANOVA followed by Bonferroni’s post-hoc comparisons tests). Exact p-values are shown in Table EV2.
Nec-1 alleviates cognitive impairment with reduction of Aβ and tau abnormalities in APP/PS1 mice (2016)
Figure 3

Figure 3. Nec-1 binds to Aβ aggregates.

A. Co-immunoprecipitation of RIPK3 with Aβ(1-42) in HT22, BV2 cell lines, and primary astrocytes in response to Aβ(1-42) aggregates. IP, immunoprecipitation; IB, immunoblot.

B. A close-up view of the interactions between Nec-1 and surrounding residues in RIPK1. Nec-1 is shown in green. The hydrophobic residues around Nec-1 in RIPK1 kinase domain are shown in sticks. Hydrogen bonds in this and all other figures are represented by yellow dashed lines. Docking score of Nec-1 in RIPK1 was -9.550.

C. Surface treated view of 12-mer Aβ fibrils (2LMO, VDW radius = 0.55 Å) having hydrophobic pockets plausible for Nec-1 binding.

D. Docked structure of 12-mer Aβ fibrils (2LMO) with 4 molecules of Nec-1. Docking scores for four top-ranked Glide docking poses of Nec-1 in Aβ plaques; -8.338, -4.755, -8.493, and -6.754 in clockwise order starting from the top-left corner.

E. Surface plasmon resonance sensorgrams of Nec-1 targeting Aβ(1-42) aggregates (left), and the corresponding dissociation fitting curve from the saturated region (right) under various concentrations of Nec-1.
Nec-1 alleviates cognitive impairment with reduction of Aβ and tau abnormalities in APP/PS1 mice (2016)
Figure 4

Figure 4. Nec-1 reduces insoluble Aβ plaques in the brains of APP/PS1 mice.

4-month-old APP/PS1 (n = 13, male) and age-matched wild-type (Wt, n = 14, male) mice were intravenously injected with Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS) for 12 weeks. After the completion of behavioural tests, the brains of each group of mice were subjected to analysis.

A. ThS-stained insoluble Aβ plaques in whole brain and the hippocampal region of each group. Scale bars = 1 mM (upper), 200 μM (lower).

B-G. Statistics of ThS-positive Aβ plaques in the brains of APP/PS1 mice. Total numbers and areas of ThS-positive Aβ plaques in the whole brains (B and C), cortex (D and E) and hippocampus (F and G).

Data information: In (B-G), data are presented as mean ± SEM. **P ≤ 0.01 and ***P ≤ 0.001 (One-way ANOVA followed by Bonferroni’s post-hoc comparisons tests). Exact p-values are shown in Table EV3.
Nec-1 alleviates cognitive impairment with reduction of Aβ and tau abnormalities in APP/PS1 mice (2016)
Figure 5

Figure 5. Nec-1 decreases diffuse plaques and Aβ oligomers in the brains of APP/PS1 mice.

A. Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild-type (Wt) and APP/PS1 mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS). Diffuse plaques in the brain sections were stained by anti-Aβ antibody (6E10 clone, green colour) and anti-GFAP antibody (red colour). Hoechst 33342 (blue colour) was applied for nuclear counterstaining. Scale bars = 200 μM.

B-D. Dot blot analysis of total Aβ (anti-Aβ: 6E10 clone, also recognizes APP) and protein oligomer (anti-amyloidogenic protein oligomer A11) in the cortical (B) and hippocampal (C) region of indicated groups. (D) Quantification analysis of total Aβ and protein oligomer in the cortical and hippocampal region of indicated groups. Relative intensity of each band was determined by densitometry of dot blots (B and C) using ImageJ software and normalized to vehicle-administered APP/PS1 mouse group.

E. Sandwich ELISA of Aβ-soluble and –insoluble fractions from cortical and hippocampal lysates of APP/PS1 mice.

F. Western blot analyses of cortical and hippocampal lysates from APP/PS1 mice for APP and sAPP expressions.

G. ThT assays for the inhibition of Aβ aggregation. Fluorescence intensity was normalized to Aβ aggregates (100%, day 5).

H. SDS-PAGE analysis of PICUP cross-linked Aβ aggregates visualized by silver staining.

Data information: In (D, E, and G), data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (One-way ANOVA followed by Bonferroni’s post-hoc comparisons tests). Exact p-values are shown in Table EV4.
Nec-1 alleviates cognitive impairment with reduction of Aβ and tau abnormalities in APP/PS1 mice (2016)
Figure 6

Figure 6. Nec-1 inhibits the phosphorylation and aggregation of tau.

A. Western blot analysis of phosphorylation levels of tau and expression levels of indicated proteins in the cortical and hippocampal regions of the brain in APP/PS1 mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS). The membranes analysed in (A) are identical to those in Fig 5F.

B. Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild-type (Wt) and APP/PS1 mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS) for phosphorylated tau expression levels. Anti-tau (phospho Ser199) antibody (red colour) was applied for staining tau phosphorylation on Serine-199 and Hoechst 33342 (blue colour) for nuclear counterstaining in the brain sections. Scale bars = 200 μM.

C. Western blot analyses of phosphorylated RIPK3 expressions in cortex and hippocampus of APP/PS1 mice injected with Nec-1 (upper), and primary cultured astrocytes treated with Nec-1 (lower). The membranes analysed in (C) are identical to those in Fig 1G and 5F.

D, E. ThT assays for the inhibition of tau aggregation (D) and for disaggregation of tau (E). Fluorescence intensity was normalized to tau aggregates (100%, day 5).

F. Surface plasmon resonance sensorgrams of Nec-1 targeting tau (left), and the corresponding dissociation fitting curve from the saturated region (right) under various concentrations of Nec-1.

Data information: In (D and E), data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (One-way ANOVA followed by Bonferroni’s post-hoc comparisons tests). Exact p-values are shown in Table EV5.
Steric Hindrance of SNARE Transmembrane Domain Organisation Impairs the Hemifusion-to-Fusion Transition (2016)
Figure 2

Figure 2. SNARE forces

(A) Estimation of the intrinsic force that the vacuolar SNARE complex can exert on a stalk-like fusion intermediate via its TMD C-termini (Nyv1 and Vam3). At this stage, the distance between the C-termini is about 8 nm. The force that the C-termini exert on the membrane, about 9 pN, is estimated from the difference in work required to induce a small indentation in, respectively, the presence of structured or unstructured Nyv1 and Vam3 linker regions (the slope of this profile defines the force). The additional presence of a structured Vti1 linker doubles the exerted force from 9 pN to 18 pN. (B) The inherent elastic response of a lipid membrane (POPC) was studied by enforcing indentations via the isolated SNARE C-termini in the presence or absence of a short linker peptide, or the linker plus a fluorescent protein tag (FP). We define "relative indentation" as the change in C-terminus (Nyv1) - C-terminus (Vam3) distance that occurs when the SNARE complex performs mechanical work. It reflects the depth of the formed well upon squeezing. The slope of the obtained work profile estimates the required force (thin black line). Perforation (red arrow) requires a force of 80 pN for the wild-type C-termini. The plateau within the work profile indicates that the membrane gives up its elastic response (perforation transition). C-terminal tags oppose such a perforation transition but do not significantly affect the forces observed within the indentation regime. Furthermore, if the unstructured peptide-spacer would be alpha-helical – and thus much shorter – indentation forces would still be only slightly affected.
Steric Hindrance of SNARE Transmembrane Domain Organisation Impairs the Hemifusion-to-Fusion Transition (2016)
Figure 3

Figure 3. Effect of small peptide tags on Vam3 and Nyv1 on vacuole morphology and fusion activity.

(A) Schematic view of the constructs for genomically tagging Vam3 and Nyv1 at their C-termini. (B) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS-PAGE and Western blotting. (C) In vivo vacuole morphology was assayed by FM4-64 staining. Scale bar: 5 µm. (D) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments. (E) Vacuoles were isolated from BJ3505 strains expressing the indicated tagged versions of Vam3 and Nyv1 and Pho8-EGFP or Pho8-mCherry. 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy. Arrows indicate examples of fusion products. Scale bar: 5 µm. (F) Fusion activity: vacuole fusion was assayed by measuring the percentage of co-localization of the two Pho8-EGFP and Pho8-mCherry signals. Means s.d. are shown for at least 100 stained vacuoles from 3 experiments.
Steric Hindrance of SNARE Transmembrane Domain Organisation Impairs the Hemifusion-to-Fusion Transition (2016)
Figure 4

Figure 4. C-terminal tagging of the SNAREs Vam3 and Nyv1 with large tags.

(A) Vacuoles were isolated from BJ3505 cells expressing the indicated versions of Vam3 and/or Nyv1. 10 µg of the organelles were analyzed by SDS-PAGE and Western blotting using antibodies to Vti1, Vam3 and Nyv1. (B) Localization in vivo. The indicated strains were grown logarithmically in synthetic complete (SC) medium. After 16 h at 30°C, when the final OD of each culture was about 1, the cells were observed by confocal fluorescence and differential interference (DIC) microscopy. Scale bar: 5 µm.
Steric Hindrance of SNARE Transmembrane Domain Organisation Impairs the Hemifusion-to-Fusion Transition (2016)
Figure 5

Figure 5. Effect of tags on vacuole morphology and cis-SNARE activation.

(A) In vivo morphology. Cells expressing the indicated SNARE variants were grown in SC medium (16 h, 30°C), stained with FM4-64 and their relative morphology was assessed by fluorescence and DIC microscopy. Scale bar: 5 µm. The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments. (B) and (C) SNARE activation on isolated vacuoles. Vacuoles were isolated from a strain co-expressing NYV1-S9-EGFP and VAM3-S9-mCitrine and from an isogenic wild-type. 150 µg of the organelles were incubated in fusion reactions in the presence or absence of an ATP-regenerating system and recombinant, purified Sec18/NSF (rSec18, 50 µg/ml). After 10 min of incubation at 27°C, the vacuoles were solubilized and immunoprecipitated with antibodies to Nyv1. Co-immunoprecipitated proteins analyzed by SDS-PAGE and Western blotting. The histograms provide quantifications of the band intensities as the means and s.d. from three independent experiments.
Steric Hindrance of SNARE Transmembrane Domain Organisation Impairs the Hemifusion-to-Fusion Transition (2016)
Figure 6

Figure 6. Effect of large protein tag on lipid and content mixing.

(A) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS-PAGE and Western blotting. (B) Vacuole morphology was assessed for the indicated cells as in Fig. 3C. Scale bar: 5 µm. (C) Vacuoles were isolated from BJ3505 and DKY6281 cells with SNAREs tagged or deleted as indicated. Note that presence of Nyv1 on only one fusion partner still permits efficient fusion [65] but ensures that trans-SNARE complexes can only form in one orientation, between the Q-SNARE in DKY6281 and the R-SNARE in BJ3505. The vacuoles were used in standard fusion reactions and content mixing was measured via the alkaline phosphatase assay. In parallel, identical samples were incubated either incubated on ice, which prevents fusion, or in the presence of 0.5% Triton X-100, which allows fusion-independent access of the maturase Pep4 to pro-ALP and controls for the levels of these two reporter enzymes in the samples. (D) Using strains from C, lipid and content mixing (left and right panel, respectively) were performed in parallel in the presence or absence of ATP. Anti-Vam3 antibody (3 µg) was added to some reactions in order to inhibit trans-SNARE pairing and fusion. Means and the s.d. are shown for three independent experiments.
Steric Hindrance of SNARE Transmembrane Domain Organisation Impairs the Hemifusion-to-Fusion Transition (2016)
Figure 7

Figure 7. Effect of extending the spacers between the SNARE C-termini and the fluorescent protein tags.

Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig. 3A but the spacer was extended (S34) by an additional 25-amino acid sequence (SGGGGSGGGGSGGGGSGGGGAAAGG) to the previous short spacer.

(A) Protein levels on isolated vacuoles from these strains were compared as in Fig. 3B. (B) Vacuole morphology was assessed as in Fig. 3C. Scale bar: 5 µm. (C) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments. (D) Fusion activity: vacuoles were isolated from BJ3505 strains expressing the indicated versions of Vam3 and Nyv1 and Pho8-mCFP or Pho8-mCherry. 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy. Arrows indicate examples of fusion products. Means s.d. are shown for at least 100 stained vacuoles from 3 experiments. Scale bar: 5 µm. (E) Vacuole fusion was assayed by measuring the percentage of co-localisation of the two Pho8-mCFP and Pho8-mCherry signals.
Steric Hindrance of SNARE Transmembrane Domain Organisation Impairs the Hemifusion-to-Fusion Transition (2016)
Figure 8

Figure 8. Effect of chlorpromazine on fusion activity and morphology of double-tagged vacuoles.

(A) Yeast strain expressing a plasmid with Vph1-GFP was grown in SC-URA medium for 16 h at 30°C. The mobility of Vph1-GFP in the absence (upper panels) or presence (lower panel) of CPZ (150 µM) was assessed by FRAP. (B) The histogram shows the GFP signals in the bleached area during the FRAP procedure. (C) The half-time is shown. Means and s.d. are shown for 20 cells of 3 independent experiments. (D) Yeast cells were grown in SC medium for 16 h at 30°C. After staining with FM4-64, cells were treated with CPZ (150 µM). Vacuole morphology was assessed by confocal microscopy before and after 5 and 20 min of CPZ addition. Scale bar: 5 µm. (E) Fusion activity. Vacuoles were isolated from BJ cells carrying Nyv1-S9-EGFP and DKY cells deleted for Nyv1 and carrying Vam3-S9-mCitrine or from wild-type cells and incubated in standard fusion reactions in the presence or absence of chlorpromazine (CPZ, 150 µM) and anti-Vam3 (3 µg). Content mixing was assayed via alkaline phosphatase activity. Means and s.d. are shown for three independent experiments.
Impaired GAPDH-induced mitophagy contributes to the pathology of Huntington's disease. (2015)
Figure 1

GAPDH selectively associates with mitochondria in cells expressing expanded polyglutamine repeats

Representative Western blots showing GAPDH association with mitochondria-enriched fractions in different HD models. The levels of GAPDH were normalized to mitochondrial matrix protein, aconitase. n = 3. *P = 0.0005; **P = 0.019; #P = 0.042.

Measurement of GAPDH activity in the mitochondria-enriched fraction isolated from PC12 cells. n = 3. *P = 0.0005.

Western blots showing oxidized GAPDH in the total lysates of PC12 cells and isolated mitochondria. The levels of oxidized GAPDH were normalized to beta-actin (total lysate) and aconitase (mitochondrial fraction), respectively. n = 2. *P = 0.023; **P = 0.039.

Western blot showing co-immunoprecipitation of GAPDH from the total lysates of PC12 cells in RIPA buffer without detergent (Triton X-100). Immunoprecipitated samples were immunoblotted with antibodies against mitochondrial (VDAC and Tom20), endoplasmic reticulum (calnexin), and lysosomal markers (LAMP1). ctr: control. n = 2.

Data information: The data are presented as mean ± SEM. Statistical significance was assessed by Student’s t-test.

Source data are available online for this figure.
Impaired GAPDH-induced mitophagy contributes to the pathology of Huntington's disease. (2015)
Figure 2

Expanded polyglutamine repeats cause mitochondrial dysfunction

Representative electron micrographs showing mitochondrial morphology in PC12 cells expressing Q23 (top panel, left) and Q74 (bottom panel, left) and in brain tissues from wild-type (WT) (top panel, right) and HD transgenic mice (bottom panel, right). An indicated portion of each image (in yellow) is expanded to illustrate the mitochondrial morphology. Scale bars: 1 μm.

Measurement of mitochondrial ROS production (using Mitosox) in different HD models. Results are presented as percent of control [Q23, normal fibroblast (Nor), Q7]. n = 3. *P = 0.001; **P = 0.0001; ***P = 0.0001; #P = 0.0045.

Mitochondrial membrane potential measured by using JC-1 dye in models of HD and control cells (MMP, ΔΨm). n = 3. *P = 0.008; **P = 0.044; ***P = 0.025; #P = 0.005.

Mitochondrial respiratory function in PC12 cells, represented by respiratory control ratio (RCR; [state3]/[state4]). n = 2. *P = 0.028.

ATP levels in HD patient-derived fibroblast cells. n = 2. *P = 0.003; **P = 0.004.

Western blots showing cytochrome c release from mitochondria. n = 2. *P = 0.003; **P = 0.003; ***P = 0.01; #P = 0.008.

Cell viability determined by colorimetric assays utilizing WST8 conversion to WST8 formazan in different HD models. n = 3. *P, **P, ***P, #P < 0.005.

Representative immunofluorescence of normal and HD patient-derived fibroblasts stained for GAPDH and mitochondrial ROS with Mitosox. Three independent images per condition were analyzed to calculate Mitosox intensity and the Pearson’s correlation coefficient for quantification of colocalization between GAPDH and mitochondrial ROS. #P = 0.03; ##P = 0.02; *P = 0.006; **P = 0.005. Images were acquired at 63× magnification, and brightness and contrast of images were adjusted by 50%.

Data information: The data are presented as mean ± SEM. Statistical significance was assessed by Student’s t-test.

Source data are available online for this figure.