EMBO/soda-nel huggingface dataset:
NPC1 enables cholesterol mobilization during LTP that can be restored in NPC by CYP46A1 activation (2019)
Figure 1

Figure 1. NPC1 localization and expression levels in wt and NPC1nmf164 mice.

A. Single grey-scale and coloured merged images of immunocytochemical staining against PSD-95, synaptophysin-1 (SYP1) and NPC1 proteins in a cultured hippocampal neuron from a wt mouse. Graphs show mean ± SEM of percentage of colocalization of NPC1 with PSD95 or SYP1 (n=12 neurons from 3 different cultures).

B. Electron microscopy image of immunogold labelling against NPC1 in the CA1 hippocampal region of a wt mouse. Presynaptic compartments are indicated in pink while postsynaptic in blue. White arrows show the 15-nm gold particles linked to NPC1 (d-dendrite, sv-synaptic vesicles, m-mitochondria).

C, D. Western blots against NPC1 and actin-β (ACTB) in total brain (C) and synaptosomes (D) from wt and NPC1nmf164 mice. Graphs show mean ± SEM NPC1 level normalized to ACTB as a percentage of control (wt) values (n=4 mice, 3 month-old, unpaired Student t test, **psynaptosomes= 0.0037).

E. Western blots against NPC1 and actin-β (ACTB) in total brain extracts from wt and Npc1-null mice (NPC1KO) containing the same amount of protein (n=2 mice, 6 week-old).

F. Graph shows mean ± SEM mRNA levels of NPC1 in total extracts and in synaptosomes from wt and NPC1nmf164 mice (n=4 mice, 3 month-old).

G. Western blots against NPC1 and actin-β (ACTB) in synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not in the presence or absence of the protein synthesis inhibitor cycloheximide (CHX). Graphs show mean ± SEM NPC1 level normalized to ACTB as a percentage of control (wt non cLTP induced) values (n=3 mice, 3 month-old, 2-way ANOVA, ***p < 0.001).

H. To the left Western blots against NPC1 and actin-β (ACTB) in synaptosomal extracts from wt and NPC1nmf164 mice used as input for the immunoprecipitation assays shown in the right. Immunoprecipitates were pulled down with an anti-ubiquitin antibody and without it for negative control and analysed by western blot against NPC1. MG132 was used as proteasome inhibitor. Graphs show mean ± SEM levels of NPC1 normalized to ACTB in the inputs (left) (n=3, one-way ANOVA, *pWT+MG-132=0.0496, pNPC1nmf1+MG-132=0.0358) and of NPC1 associated to ubiquitin (right) (n=3 mice, 3 month-old, one-way ANOVA, *pNPC1nmf164= 0.0237, pNPC1nmf164+MG-132= 0.0386.
NPC1 enables cholesterol mobilization during LTP that can be restored in NPC by CYP46A1 activation (2019)
Figure 2

Figure 2. Morphology and cholesterol level of synapses in NPC1nmf164 mice.

A. Representative electron micrographs of synapses in the hippocampal CA1 region in wt and NPC1nmf164 mice (black arrows indicate post-synaptic densities, d-dendrite, sv-synaptic vesicles, m-mitochondria, MLB-multilamellar bodies).

B. Mean ± SEM synapse density (n=3 mice, 3 month-old, unpaired Student t test, *p = 0.0377).

C. Mean ± SEM synaptic vesicle density (3,088 vesicles from 101 wt and 121 NPC1nmf164 synapses from 3 different mice (3 month-old) per group, unpaired Student t test, ***p < 0.0001).

D. Synaptic vesicle diameter (528 wt and 507 NPC1nmf164 vesicles from 3 different mice (3 month-old) per group).

E. Mean ± SEM post-synaptic density length (101 wt and 121 NPC1nmf164 post-synaptic densities from 3 different mice (3 month-old) per group, unpaired Student t test, ***p < 0.0001).

F. Mean ± SEM post-synaptic density thickness (n=3 mice, 3 month-old, unpaired Student t test, ***p < 0.0001).

G. Mean ± SEM cholesterol levels in synaptosomes (n=4 mice, 3 month-old, unpaired Student t test, *p = 0.0476) from wt and NPC1nmf164 mice expressed as percentage of wt mice.
NPC1 enables cholesterol mobilization during LTP that can be restored in NPC by CYP46A1 activation (2019)
Figure 3

Figure 3. Synaptic function and behaviour in wt and NPC1nmf164 mice.

The following properties of Schaffer collateral-CA1 synaptic responses (A-E) and behavioural tests (F-I) were recorded from wt and NPC1nmf164 mice.

A. Basal synaptic transmission expressed as mean ± SEM EPSP slope (n= 9 slices from 5 WT mice and n=8 slices from 5 NPC1nmf164 mice, all mice 10 week-old, unpaired Student t test, * p150μA = 0.0368, p200μA = 0.0138);

B. Mean ± SEM fibre volley amplitude (n=10 slices from 5 WT mice and n= 7 slices from 5 NPC1nmf164 mice, all mice 10 week-old);

C. Mean ± SEM paired pulse facilitation (n=11 slices from 5 WT mice and n=10 slices from 5 NPC1nmf164 mice, all mice 10 week-old, unpaired Student t test, **p = 0.0011);

D. LTP expressed as mean ± SEM percentage of EPSP slope over baseline (n=7 slices form 5 WT mice for WT and n=10 slices from 5 NPC1nmf164 mice, all mice 10 week-old, unpaired Student t test, ***p < 0.0001 in the last 10 min recording)

E. LTD expressed as mean ± SEM percentage of EPSP slope over baseline (n=7 slices from 5 WT mice and n=12 slices from 5 NPC1nmf164 mice, all mice 10 week-old).

F. Mean ± SEM exploration time of objects in novel and familiar location in the Object Placement Recognition test (n=10 mice, 10 week-old, 2-way ANOVA, *p = 0.0167).

G. Mean ± SEM percentage of entry to the novel arm of the Y maze test (n=10 mice, 10 week-old; unpaired Student t test, **p = 0.0015).

H. Mean ± SEM percentage of freezing time in the Contextual Fear Conditioning test (n=10 mice, 10 week-old; unpaired Student t test, ***p = 0.0002).

I. Mean ± SEM distance covered during a 5 min period in the Open Field test (n=10 mice, 10 week-old).
NPC1 enables cholesterol mobilization during LTP that can be restored in NPC by CYP46A1 activation (2019)
Figure 4

Figure 4. Cholesterol redistribution and CYP46A1 and GluA1 receptor surface delivery during LTP induction in wt and NPC1nmf164 mice.

A. Mean ± SEM cholesterol level in plasma membrane fractions of hippocampal slice cultures from wt and NPC1nmf164 mice with or without cLTP induction (n=6, unpaired Student t test, *pwt = 0.0402).

B. Western blot of NPC1 and ACTB in extracts of cultured hippocampal neurons from wt mice transfected or not with sh-scr or sh-NPC1 RNAs. Black bars indicate that the sh-NPC1 lane was not consecutive to the others but belongs to the same Western blot. Graph shows the 2 biological replicates mean ± SEM of NPC1 level normalized to ACTB as a percentage of the wt, non-transfected controls.

C. Representative images of the cholesterol binding probe mCherry-D4 before and after cLTP in cultured hippocampal neurons from wt mice transfected with sh-scr or sh-NPC1 RNAs. Z-stack images acquired using a Nikon A1R+ confocal microscope were used to generate 3D images of the neurons.

D, E. Western blots against NPC1, CYP46A1 and actin-β (ACTB) in total extracts (T) and biotin-streptavidin immunoprecipitates (S) from synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not. Graphs show mean ± SEM of the levels of biotinylated-surface NPC1 and CYP46A1 as a percentage of the total amount of each protein (n=3 mice, 3 month-old, 2-way ANOVA, *pNPC1 = 0.0251, *pCYP46A1 = 0.0371).

F. Representative images of GluA1 immunocytochemical staining before and after cLTP in non-permeabilized (surface) and permeabilized (total) cultured hippocampal neurons expressing sh-scr or sh-NPC1 RNAs. Graph shows mean ± SEM GluA1 surface staining with respect to total in cLTP conditions, as a percentage of the sh-scr baseline (n=20 images per condition from 2 different experiments, 2-way ANOVA, ***psh-scr < 0.001).
NPC1 enables cholesterol mobilization during LTP that can be restored in NPC by CYP46A1 activation (2019)
Figure 5

Figure 5. Effects of CYP46A1 activation by EFV on synaptic cholesterol redistribution and plasticity in wt and NPC1nmf164 neurons.

A. Western blots against CYP46A1 and ACTB in extracts of total brain and synaptosomes from wt and NPC1nmf164 mice as a percentage of wt values. Graphs show 3 biological replicates mean ± SEM of CYP46A1 level normalized to ACTB in arbitrary units.

B. Quantitative PCR of Cyp46 of total brain and synaptosomes from wt and NPC1nmf164 mice. 3 biological replicates mean ± SEM is shown.

C. Mean ± SEM cholesterol levels in EFV-treated or non-treated synaptosomes from wt (n=5 mice, 14-week-old, unpaired Student t test, *p = 0.0233) and NPC1nmf164 (n=4 mice, 14 week-old, unpaired Student t test, *p = 0.0188) mice. EFV values are expressed as percentage of their corresponding non-treated controls in wt and NPC1nmf164 conditions.

D. Basal synaptic transmission in EFV-treated and non-treated hippocampal slices from wt and NPC1nmf164 mice expressed as mean ± SEM EPSP slope (n=9 slices from 5 WT mice, n=11 slices EFV-treated from 5 WT mice, n=8 slices from 5 NPC1nmf164 mice and n=13 slices EFV-treated from 5 NPC1nmf164 mice, all mice 14-week-old, 2-way ANOVA, *p = 0.0117).

E. LTP in EFV-treated and non-treated hippocampal slices from wt and NPC1nmf164 mice expressed as mean ± SEM percentage of EPSP slope over baseline (n=7 slices from 5 WT mice, n=9 slices EFV-treated from 5 WT mice, n=10 slices from 5 NPC1nmf164 mice and n=11 slices EFV-tretaed from 5 NPC1nmf164 mice, all mice 14-week-old, 2-way ANOVA, ***p < 0.0001).

F. Mean ± SEM paired pulse facilitation in EFV-treated and non-treated hippocampal slices from wt and NPC1nmf164 mice (n=11 slices from 5 WT mice, n=9 slices EFV-treated from 5 WT mice, n=10 slices from 5 NPC1nmf164 mice and n=11 slices EFV-tretaed from 5 NPC1nmf164 mice, all mice 14-week-old, 2-way ANOVA, **pNPC1nmf164=0.0027, *pNPC1nmf164+EFV=0.0229).
NPC1 enables cholesterol mobilization during LTP that can be restored in NPC by CYP46A1 activation (2019)
Figure 6

Figure 6. Effects of CYP46A1 activation by EFV on cLTP-induced GluA1 surface delivery in wt and NPC1-deficient conditions.

Representative images of GluA1 immunocytochemical staining before and after cLTP induction in non-permeabilized (surface) and permeabilized (total) cultured hippocampal neurons expressing sh-scr or sh-NPC1 RNAs and treated or not with EFV. Graph shows the mean ± SEM GluA1 surface staining with respect to total after cLTP, as a percentage of the sh-scr baseline (n=20 images per condition from 2 separate experiments, 2-way ANOVA, ***psh-scr < 0.001, **psh-scr+EFV < 0.01, ***psh-NPC1+EFV < 0.001).
NPC1 enables cholesterol mobilization during LTP that can be restored in NPC by CYP46A1 activation (2019)
Figure 7

Figure 7. Effects of EFV treatment in vivo. The following analyses and behavioural tests were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV

A. Mean ± SEM 24(S)-hydroxycholesterol plasma level after 6 and 8 weeks of oral EFV treatment in wt (n=6 mice, 14 week-old and 16 week-old, 2-way ANOVA, **p6 weeks = 0.0023) and NPC1nmf164 (n=5 mice, 14 week-old and 16 week-old, 2-way ANOVA, ***p6 weeks < 0.0001; ***p8 weeks < 0.0001) mice.

B. Mean ± SEM body weight in grams in wt and NPC1nmf164 mice treated or not with EFV (n=4 mice, unpaired Student t-test, ***pNPC1nmf164 < 0.0001, **pNPC1nmf164+EFV = 0.0048)

C. Mean ± SEM discrimination index as a normalized ratio of time spent with novel and familiar objects in the Object Placement Recognition test (n=6 mice, 10 week-old, 2-way ANOVA, **pWT/NPC1nmf164 = 0.0065, ***pNPC1nmf164/NPC1nmf164+EFV = 0.0008).

D. Mean ± SEM percentage of entries in the novel arm of the Y maze test (n=10 mice, 10 week-old, 2-way ANOVA, ***p < 0.0001).

E. Mean ± SEM percentage of freezing time in the Contextual Fear Conditioning test (n=10 mice, 10 week-old, 2-way ANOVA, **pNPC1 < 0.0025, *pNPC1+EFV < 0.0181).

F. Mean ± SEM percentage of freezing time in the Cued Fear Conditioning test (n=10 mice, 10 week-old, 2-way ANOVA, *pNPC1 < 0.0453, **pNPC1+EFV < 0.0054).

G. Mean ± SEM cholesterol level in synaptosomes of wt and NPC1nmf164 mice treated or not with EFV expressed as percentage of the wt non-treated samples (n=4 mice, 14 week-old, 2-way ANOVA, *pNPC1 < 0.0476, *pNPC1+EFV < 0.0118).

H. Representative fluorescence images of the CA1 hippocampal region from wt and NPC1nmf164 mice treated or not with EFV and stained with filipin and an antibody against LAMP1. White arrows indicate lysosome enlargement in the hippocampus of non-treated NPC1nmf164 mice. Graph to the right shows mean ± SEM fluorescence intensity associated to filipin per area in arbitrary units (n=4 mice, 14-week old, unpaired Student t-test, **pNPC1nmf164 = 0.0011, *pNPC1nmf164+EFV = 0.0273)).

I. Survival graph for wt and NPC1nmf164 mice treated or not with EFV (n=5 mice, 2-way ANOVA, ***p < 0.0001).
POGZ promotes homology-directed DNA repair in an HP1-dependent manner (2021)
Figure 4

Figure 4. POGZ mediates BRCA1/BARD1 accumulation at DSBs through its HP1-binding site.

(A) Schematic diagram outlining the different domains of human POGZ and the different deletion constructs of POGZ that we generated and analyzed.

(B) U2OS stably expressing mCherry-LacR-FokI were transduced with the indicated siRNA. 24h post-transfection, cells were transfected with an empty Flag vector (EV) or a siRNA-resistant FLAG-tagged POGZ cDNA construct corresponding to indicated rescue mutant (full-length, FL; POGZ366-848, 366-848). 24h after plasmid transfection, DNA damage was induced using Shield-1 and 4-OHT. Immunofluorescence against the indicated protein was subsequently performed to monitor its respective accumulation at sites of DNA damage by confocal microscopy. Data are represented as a box-and-whisker (10-90 percentile) where the fluorescent signal at the mCherry focus was normalized to nuclear background. At least 100 cells per condition were counted. Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.005, **P<0.0001.

(C) HeLa cells transfected with the indicated construct were monitored for their capacity to form IR-induced BRCA1 foci. HeLa cells where POGZ has been targeted by CRISPR technology (POGZ∆-1) and in control HeLa cells (sgCtrl) were transfected by an empty Flag vector (EV) or a sgRNA-resistant FLAG-tagged POGZ cDNA construct corresponding to indicated rescue mutant (full-length, FL; POGZ801-848, HPZ). Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered 1h post-exposure to IR. Cells were fixed, stained, and imaged via confocal microscopy. Data are the total number of ΒRCA1 foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05, **P<0.0001.

(D) Representative images used for quantification in (C). Scale bar = 5 μm.

(E) Similar as in (C) except that γ-H2AX foci were monitored at the indicated time points by confocal microscopy. Data are the total number of γ-H2AX foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05.

(F) Representative images used for quantification in (E). Scale bar = 5 μm.
Respiratory supercomplexes enhance electron transport by decreasing cytochrome c diffusion distance (2020)
Figure 1

Fig 1. A detailed structure of the CIII-CIV interface allows to design supercomplex-disrupting mutations

(A) Side-view of the overall structure of the S. cerevisiae CIII2/CIV supercomplex and its position in the mitochondrial inner membrane (IM). IMS: intermembrane space

(B,C) Zoom-in of the CIII2/CIV interaction sites with residues and distances annotated. The inner-membrane protein-lipid-protein interactions (Rip1-CL-Cox5a), where Rip1 is marked in gold, CL in grey, and Cox5a in red (B), and the mitochondrial matrix, protein-protein interaction of Cor1 (blue), and Cox5a (red) (C) are shown.

(D) Blue-native gel electrophoresis of S. cerevisiae strains with indicated mutations in Cor1.
Respiratory supercomplexes enhance electron transport by decreasing cytochrome c diffusion distance (2020)
Figure 2

Fig 2: Characterization of strains lacking respiratory supercomplexes

(A) Blue-native gel electrophoresis and immunoblots of cells expressing the wild type form of Cor1 (Cor1WT), as well as the mutants Cor1N63A, N187A, D192A (Cor1*) and Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**) in wild type (WT) background as well as in cells lacking CRD1 (crd1Δ). Blots were probed with antibodies against Cor1 to visualize Complex III (CIII), as well as Cox1, to monitor Complex IV (CIV). CV: Complex V; Cor1Cor2: multimer consisting of Cor1 and Cor2

(B) Reduced-minus-oxidized difference spectra of strains described in (A). Heme peaks are indicated for Cor1WT in WT background.

(C-E) Steady-state protein levels of strains as described in (A) cultivated on YP media containing glycerol (C) or CM media with galactose as carbon source (D, E).
Respiratory supercomplexes enhance electron transport by decreasing cytochrome c diffusion distance (2020)
Figure 3

Fig 3. Abrogation of supercomplexes decreases competitive fitness

(A) Growth analysis of strains expressing the wild type form of Cor1 (Cor1WT) or the mutant Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**). Growth curves and calculated doubling times from the exponential phase (insert) are presented. Cells were cultivated in media containing either glucose or glycerol as carbon source.

(B) Determination of chronological lifespan via propidium iodide (PI) staining of cells described above.

(C) Scheme of the experimental setup to evaluate competitive fitness.

(D,E) Analysis of competitive fitness as described in (C). Cor1WT strains harbored a chromosomally-integrated hygromycin selection cassette, whereas Cor1** contained a clonNAT selection cassette (D). Subsequently, selection markers were switched as a control (E).

Data information: For (A),(B),(D) and (E) line graphs indicate mean ± standard error of the mean (s.e.m.). Two-Way ANOVA mixed design followed by Bonferroni post hoc test was used to analyze data from 4 individual yeast clones (n=4 biological replicates). Significances of main effects are visualized as: n.s.: not significant (P≥0.05) and ###P<0.001. For insert in (A), data is presented as mean (square) ± s.e.m., median (center line), and single data points (n=4 biological replicates). Thereby, a two-tailed independent-sample t-test was performed and significances are given as: n.s.: not significant. A detailed description of statistical analyses performed is given in Table EV6.
Respiratory supercomplexes enhance electron transport by decreasing cytochrome c diffusion distance (2020)
Figure 4

Fig 4: Lack of supercomplexes impairs electron transport from CIII to CIV

(A,B) Spectrophotometric measurement of Cyt c oxidase (CIV; A) and NADH Cyt c reductase (CIII; B) activities in solubilized mitochondrial extracts. Mitochondria were isolated from cells expressing the wild type form of Cor1 (Cor1WT) or the mutant Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**).

(C,D) Polarographic measurement of KCN-sensitive oxygen consumption, driven by NADH (C) or succinate + glycerol-3-phosphate (G3P; D) in isolated coupled mitochondria of indicated strains. Measurement was performed in the absence (basal respiration) or presence (phosphorylating condition) of ADP.

(E) Respiratory control ratio calculated from polarographic measurements of KCN-sensitive oxygen consumption driven by NADH and succinate + glycerol-3-phosphate (G3P) in isolated coupled mitochondria of indicated strains.

(F,G) Polarographic measurement of KCN-sensitive oxygen consumption driven by NADH in mitoplasts in the absence (endog. Cyt c) or presence (+ exog. Cyt c) of exogenous oxidized Cyt c. NADH oxidation (E) and the calculated ratio +Cyt c/- Cyt c of substrate oxidation (F) are visualized.

Data information: Mean (square) ± s.e.m., median (center line) and single data points are depicted. Two-tailed independent-sample t-tests were used to analyze data from at least three individual mitochondrial isolations (n≥3 biological replicates), For (A),(B) and (F endog. Cyt c) Welch correction was performed and for (E Succinate+G3P) a two-tailed Mann-Whitney U test was applied. Significances are given as: n.s.: not significant (P≥0.05), *P<0.05, **P<0.01 and ***P<0.001. A detailed description of statistical analyses performed is given in Table EV6.
Respiratory supercomplexes enhance electron transport by decreasing cytochrome c diffusion distance (2020)
Figure 5

Fig 5. Cytochrome c overexpression restores competitive fitness of strains lacking supercomplexes.

(A) Polarographic measurement of KCN-sensitive oxygen consumption, driven by NADH in isolated coupled mitochondria. Mitochondria were isolated from cells expressing the wild type form of Cor1 (Cor1WT) or the mutant Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**). Thereby, strains either overexpressed cytochrome c (Cyt cOE) or harbored the empty plasmid as a control (Empty). Measurement was performed in the absence (basal respiration) or presence (phosphorylating condition) of ADP.

(B-D) Analysis of competitive fitness of strains described in (A). Cor1WT strains harbored a chromosomally-integrated clonNAT selection cassette, whereas Cor1** contained a hygromycin selection cassette. Strains were cultivated in CM media containing glucose (B), galactose (C) or glycerol (D) as carbon source.

Data information: For (A), mean (square) ± s.e.m., median (center line) and single data points (n=6 biological replicates) are depicted and an One-Way ANOVA followed by a Bonferroni post hoc test was used for statistical analysis. For (B-D), mean ± s.e.m. (n=4 biological replicates) are depicted and data was statistically evaluated by Two-Way ANOVA mixed design followed by a Bonferroni post hoc test. Significances are visualized as: n.s.: not significant (P≥0.05), *P<0.05, **P<0.01, ***P<0.001. A detailed description of statistical analyses performed is given in Table EV6.

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