EMBO/soda-nel huggingface dataset:
Disease-specific phenotypes in iPSC-derived neural stem cells with POLG mutations (2020)
Figure 4

Figure 4: NSCs retained the original genotype and sequence.

A: Quantification of gene expression for NSC markers PAX6, NESTIN and SOX2 for all NSCs from RT-qPCR analysis. The expression of the neural stem cell markers is assessed with fold change using the comparative ΔΔCt method by normalizing NSCs to iPSCs (n=3, technical replicates per ESC line or iPSC clone).

B, C: Quantification of protein expression level for PAX6 (B, n=6, technical replicates per line for ESCs; n=7, technical replicates per clone for control; n=8, technical replicates per clone for WS5A; n=9, technical replicates per clone for CP2A) and NESTIN (C, n=6, technical replicates per line for ESCs; n=7, technical replicates per clone for control; n=8, technical replicates per clone for WS5A; n=9, technical replicates per clone for CP2A) for iPSC-derived NSCs using flow cytometry.

D: Sequencing chromatogram showing the homozygous c.2243G>C variation in POLG in WS5A iPSC-derived NSCs and the heterozygous c.1399G>A and c.2243G>C variation in POLG in CP2A iPSC-derived NSCs.

E: Representative confocal images showing glial and neuronal lineages derived from NSCs. a, Immunostaining of NSC-derived astrocytes with GFAP (red) staining (scale bar, 50 µm). b, Immunostaining of oligodendrocytes showing GALC (red) positive labeling (scale bar, 50 µm). c, Dopaminergic neurons showing TH (green) and TUJ1 (red) positive staining (scale bar, 25 µm). Nuclei are stained with DAPI (blue).

Data information: The data points in A-C represent NSCs generated from 2 ESC lines, 3 different control iPSCs including 2 different clones from Detroit 551 control, one clone from control AG05836, 3 different clones from WS5A patient and 2 different clones from CP2A patient iPSCs. Data are presented as mean ± SEM for the number of samples.
Disease-specific phenotypes in iPSC-derived neural stem cells with POLG mutations (2020)
Figure 7

Figure 7: POLG mutations induced defects in respiratory chain complex I.

A: Complex I immunohistochemistry in the occipital cortex of a neurologically healthy control (a) and two patients with POLG-disease (b and c) (Scale bar, 20 µm). Patients have numerous complex-I negative neurons (examples marked by arrows). (d) mtDNA relative quantification in microdissected neurons from the occipital cortex of patients with POLG diseases (n=5) and neurologically healthy controls (n=5). Each point represents the mean value of three technical replicates from a pooled sample of 10 neurons. For the purposes of comparison, a control sample has been arbitrarily set to one. The medians of the two groups are compared by Mann-Whitney U test. Data are presented as mean (horizontal bars) ± SEM (vertical bars).

B: Representative confocal images of immunostaining for mitocondrial complex I subunit NDUFB10 (green) and TOMM20 (red) in control, WS5A and CP2A NSCs (scale bars, 50 µm). Nuclei are stained with DAPI (blue).

C-E: Flow cytometric measurements of mitochondrial complex I (C, n=4, technical replicates per clone), II (D, n=3, technical replicates per clone for control; n=4, technical replicates per clone for WS5A and CP2A) and IV (E, n=3, technical replicates per clone for control and CP2A; n=4, technical replicates per clone for WS5A) protein level in iPSC-derived NSCs. Expressed as specific complex I, II and IV level (total complex I, II, IV level/TOMM20).

F-H: Flow cytometric measurements of mitochondrial complex I (F, n=4, technical replicates per clone), II (G, n=4, technical replicates per clone) and IV (H, n=4, technical replicates per clone) protein level in iPSCs. Expressed as specific complex I, II and IV level.

I-K: Flow cytometric measurements of mitochondrial complex I (I, n=4, technical replicates), II (J, n=4, technical replicates) and IV (K, n=4, technical replicates) protein level in parental fibroblasts expressed as specific complex I, II and IV level.

Data information: The data points in C represent NSCs generated from 5 different controls, including 3 clones from Detroit 551 control, 1 clone from control AG05836, 1 clone from control CCD-1079Sk, 3 clones from WS5A patient iPSCs and 2 clones from CP2A patient iPSCs. The data points in C represent NSCs generated from 3 clones from Detroit 551 control, 3 clones from WS5A patient iPSCs and 2 clones from CP2A patient iPSCs. The data points in F-H represent 2 clones from Detroit 551 iPSCs, 3 clones from WS5A patient iPSCs and 2 clones for CP2A iPSCs. Data are presented as mean ± SEM for the number of samples. Mann-Whitney U test was used for the data presented in F and J. Two-sided Student's t-test was used for the data presented in C-E, G-I and K. Significance is denoted for P values of less than 0.05. * P<0.05; *** P<0.001; **** P<0.0001.
Disease-specific phenotypes in iPSC-derived neural stem cells with POLG mutations (2020)
Figure 8

Figure 8: NAD+ metabolism, ROS overproduction, exhibition of a senescence phenotype through UCP2/SirT1 and mitophagy activation via BNIP3.

A-C: LCMS-based metabolomics for quantitative measurements of NAD+/NADH ratio (A, n=3, technical replicates per clone), NAD+ (B, n=3, technical replicates per clone) and NADH (C, n=3, technical replicates per clone) level in NSCs.

D-F: LCMS-based metabolomics for quantitative measurement of NAD+/NADH ratio (D, n=3, technical replicates per clone), NAD+ (E, n=3, technical replicates per clone) and NADH (F, n=3, technical replicates per clone) level in iPSCs.

G: Intracellular ROS production measurements of the specific ROS level calculated by total ROS/MTDR in control, WS5A and CP2A iPSC-derived NSCs using DCFDA and MTDR (n=6, technical replicates per clone for control; n=5, technical replicates per clone for WS5A; n=3, technical replicates per clone for CP2A).

H: Flow cytometric measurements of mitochondrial ROS production at the specific ROS level in Detroit 551, WS5A and CP2A iPSC-derived NSCs calculated by total ROS (MitoSox Red)/MTG (n=4, technical replicates per clone for control and CP2A; n=3, technical replicates per clone for WS5A).

I-J: Representative images of senescence β-galactosidase staining (scale bars, 20 µm) (I) and quantification by calculating the percentage of positively stained cells by division of the negative cells from I (J, n=3, technical replicates per clone).

K-L: Representative images (K) and quantification (L) for western blotting with Phospho-SirT1 (Ser47), LC3B, UCP2, PINK1, Parkin, BNIP3 and β-ACTIN. Three independent experiments are included.

Data information: The data points in A-F and H represent iPSCs or iPSC-derived NSCs from 2 different clones from Detroit 551 control, 3 different clones from WS5A patient iPSCs and 2 different clones from CP2A patient iPSCs. The data points in G represent NSCs generated from 5 different control iPSCs including 4 clones from Detroit 551 control and one clone from control AG05836, 3 clones from WS5A patient iPSCs and 2 clones from CP2A patient iPSCs. The data points in J represent NSCs generated from 2 different clones from Detroit 551 control, 2 different clones from WS5A patient iPSCs and 2 different clones from CP2A patient iPSCs. The data points in L represent NSCs generated from 2 to 3 different clones from Detroit 551 control and 3 different clones from WS5A patient iPSCs and 2 to 3 different clones from CP2A patient iPSCs. Data are presented as mean ± SEM for the number of samples. Mann-Whitney U test was used for the data presented in G, L, e and L, f. Two-sided Student's t-test was used for the data presented in A-F, J, H and L, a-d. Significance is denoted for P values of less than 0.05. *P<0.05; ** P<0.01; ***P<0.001; **** P<0.0001.
Rapamycin activates autophagy and lysosomal biogenesis in a mitochondrial myopathy model (2018)
Figure 1

Figure 1. Rapamycin treatment improves the myopathic phenotype of Cox15sm/sm mice

A) Treadmill analysis (n=8/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by unpaired, two-tailed Student's t test ***p=0.005; ***p=0.0007 (T3) and ****p<0.0001 (T4). Only p values for rapamycin-treated vs. untreated Cox15sm/sm mice are shown.

B) Histological and histochemical characterization of skeletal muscle in rapamycin-treated and untreated Cox15sm/sm and WT mice. H&E: hematoxylin and eosin; PAS: periodic acid Schiff reaction; COX: cytochrome c oxidase; SDH: succinate dehydrogenase. White bars correspond to 50µm.

C) Analysis of the cross-sectional area of muscle fibers in the different genotypes (n=3/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test: ***p=0.001; ***p=0.006, ****p<0.0001.

D) Analysis of the number of centralized nuclei in muscle fibers (n=3/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0018, **p=0.0052.

E) Analysis of PAS-reaction intensity in muscle fibers (n=4/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test *p=0.0293 (WT vs. Cox15sm/sm) and *p=0.0150 (Cox15sm/sm vs. Cox15sm/sm Rapa).

F) Analysis of COX-reaction intensity in muscle fibers (n=3). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test ****p<0.0001; **p=0.0031.

G) Analysis of SDH-reaction intensity in muscle fibers (n=3). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test *p=0.0199, **p=0.0091.
Rapamycin activates autophagy and lysosomal biogenesis in a mitochondrial myopathy model (2018)
Figure 2

Figure 2. Rapamycin improves the biochemical phenotype of Cox15sm/sm skeletal muscles.

A) Spectrophotometric activities of the respiratory chain (n=4-5/group). CS: citrate synthase; CIV: complex IV. Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by two way ANOVA with Tukey's correction ****p<0.0001 (CS: WT vs. Cox15sm/sm), ***p=0.0010 (CS: Cox15sm/sm vs. Cox15sm/sm rapamycin), ****p<0.0001 (CIV: WT vs. Cox15sm/sm), **p=0.0060 (CIV/CS: WT vs. Cox15sm/sm).

B) Western blot immunovisualization of subunits of the respiratory complexes. Note the increased protein levels in Cox15sm/sm vs. WT samples, which are reduced to normal levels upon rapamycin treatment. Additional samples were run on a separate gel (not shown).

C) BNGE in gel activity for cIV. Sc: supercomplexes. Note that the COX reaction is slightly increased in rapamycin-treated vs. untreated Cox15sm/sm samples.

D) Immunoblot of 1D-BNGE using an anti-cIV antibody (COX1). Note that COX amount is slightly increased in rapamycin treated vs. untreated Cox15sm/sm muscles. SDHB was used as a loading control.

E) State III (succinate-driven) oxygen consumption (n=3/group). Error bars represent SEM. The asterisks represent the significance levels calculated by unpaired, two tail Student's t test **p=0.0016 (WT vs. Cox15sm/sm); *p=0.047 (Cox15sm/sm vs. Cox15sm/sm rapamycin).

F) Analysis of mtDNA amount (n=9/group). Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey's correction: **p=0.0020; *p=0.0296.
Rapamycin activates autophagy and lysosomal biogenesis in a mitochondrial myopathy model (2018)
Figure 3

Figure 3. Rapamycin improves mitochondrial ultrastructure in Cox15sm/sm mice.

Note the accumulation of profoundly altered mitochondria in Cox15sm/sm muscles (white arrows), possibly as a result of partially digested organelles within endolysosomes. Black arrows indicate examples of normal mitochondria. In rapamycin-treated Cox15sm/sm animals only a few of these structures are present, which can be detected also in rapamycin-treated WT samples. The bar corresponds to 1136 nm (4400X).
Rapamycin activates autophagy and lysosomal biogenesis in a mitochondrial myopathy model (2018)
Figure 4

Figure 4. Rapamycin induces autophagy in Cox15sm/sm muscles

A) Representative western blot of autophagy markers at steady state in skeletal muscle. GAPDH was used as a loading control.

B) Densitometric analysis of immunoblots similar to that shown in A (n=4/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0068 (LC3-II WT vs. Cox15sm/sm), **p=0.0063 (P62 WT vs. Cox15sm/sm) *p=0.025 (P62 Cox15sm/sm vs. Cox15sm/sm+rapamycin), **p=0.0098 (S6 WT vs. Cox15sm/sm), *p=0.034 (P-S6 WT vs WT+rapamycin), ***p=0.0002 (Cox15sm/sm vs. Cox15sm/sm+rapamycin), *p=0.014 (P-S6 WT vs. WT+rapamycin) ***p=0.0007 (Cox15sm/sm vs. Cox15sm/sm+rapamycin).

C) Analysis of the autophagic flux (n=3/group). Upper panel: representative western blot of Cox15sm/sm and WT muscles treated with colchicine, rapamycin, or rapamycin plus colchicine. Note that LC3-II was increased in Cox15sm/sm vs. WT samples. Colchicine did not increase LC3-II levels suggesting a block in the autophagic flux; rapamycin plus colchicine treated Cox15sm/sm mice showed higher levels of LC3-II, suggesting that rapamycin increased the autophagic flux in Cox15sm/sm muscles.

Black: WT; dark grey: colchicine-treated WT; middle grey: rapamycin-treated WT; light grey: colchicine plus rapamycin treated mice; red: untreated Cox15sm/sm; orange: colchicine-treated; blue: rapamycin-treated Cox15sm/sm; purple: colchicine plus rapamycin treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0049 (WT vs. WT+colchicine), **p=0.0046 (WT+rapamycin vs. WT+rapamycin+colchicine), *p=0.0225 (WT vs. Cox15sm/sm), **p=0.0057 (Cox15sm/sm colchicine vs. Cox15sm/sm+rapamycin+colchicine), ***p=0.0003 (Cox15sm/sm rapamycin vs. Cox15sm/sm+rapamycin+colchicine).
Rapamycin activates autophagy and lysosomal biogenesis in a mitochondrial myopathy model (2018)
Figure 5

Figure 5. Rilmenidine induces autophagic flux, but does not improve the phenotype of Cox15sm/sm mice

A) Analysis of the autophagic flux (n=4/group). Upper panel: representative western blot of Cox15sm/sm and WT muscles treated with colchicine, rilmenidine, or rilmenidine plus colchicine. Colchicine alone increased LC3-II in WT but not Cox15sm/sm mice; rilmenidine had no effect on WT LC3-II levels, but reduced them in Cox15sm/sm mice. Rilmenidine plus colchicine increased LC3-II levels in both WT and Cox15sm/sm mice.

Black: WT; dark grey: colchicine-treated WT; middle grey: rapamycin-treated WT; light grey: colchicine plus rapamycin treated mice; red: untreated Cox15sm/sm; orange: colchicine-treated; blue: rapamycin-treated Cox15sm/sm; purple: colchicine plus rapamycin treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test: **p=0.0093 (WT vs. WT+colchicine), ****p<0.0001 (WT+rilmenidine vs. WT+rilmenidine+colchicine), **p=0.0001 (WT vs. Cox15sm/sm), **p=0.0083 (Cox15sm/sm vs. Cox15sm/sm+rilmenidine), *p=0.0112 (Cox15sm/sm vs. Cox15sm/sm+rilmenidine+colchicine), ****p<0.0001

B) Treadmill analysis (n=4/group). Black: untreated WT; grey: rilmenidine-treated WT; red: untreated Cox15sm/sm; blue: rilmenidine-treated Cox15sm/sm. Error bars represent SEM.

C) Histological and histochemical characterization of skeletal muscle in rapamycin-treated and untreated Cox15sm/sm and WT mice. H&E: hematoxylin and eosin; GT: Gomori trichrome; PAS: periodic acid Schiff reaction.

D) Analysis of the cross-sectional area of muscle fibers in the different genotypes (n=3/group). Black: untreated WT; grey: rilmenidine-treated WT; red: untreated Cox15sm/sm; blue: rilmenidine-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test ***p=0.0002, ****p<0.0001.

E) Analysis of the number of centralized nuclei in muscle fibres (n=3/group). Black: untreated WT; grey: rilmenidine-treated WT; red: untreated Cox15sm/sm; blue: rilmenidine-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0091.
Rapamycin activates autophagy and lysosomal biogenesis in a mitochondrial myopathy model (2018)
Figure 6

Figure 6. Rapamycin, but not rilmendine, induces TFEB translocation to the nucleus.

A) anti-TFEB immunofluorescence on rapamycin- and rilmenidine-treated WT and Cox15sm/sm muscles vs. untreated (UT) samples. CD11C (blue signal) indicates inflammatory cells. Right panel: quantification of n=3 animals/group. The scale bars correspond to 30µm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test

B) Quantification of the TFEB-positive nuclei (n=3/group). Quantification was performed using Imaris spots surface excluding the CD11C positive nuclei. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test *p=0.0199 (WT vs. WT+rapamycin), *p=0.0150 (Cox15sm/sm vs. Cox15sm/sm+rapamycin), ***p=0.0001.
Rapamycin activates autophagy and lysosomal biogenesis in a mitochondrial myopathy model (2018)
Figure 7

Figure 7. Rapamycin, but not rilmendine, increases lysosomal biogenesis

A) Real-time PCR analysis of Lamp1 transcript (n=3/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by Student's t test **p=0.008, *p=0.038.

B) Anti-LAMP1 staining in rapamycin- and rilmenidine-treated WT and Cox15sm/sm muscles vs. untreated (UT) samples. Rapamycin, but not rilmendine, increases the number of LAMP1-positive vesicles in Cox15sm/sm samples (inset). The scale bars correspond to 30µm.

C) Quantification of LAMP1-positive vesicles (n=3/group). Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test ****p<0.0001.

D) Cathepsin L specific activity (n=3/group). Error bars represent SEM. The asterisks represent the significance levels calculated by Student's t test *p=0.0133.
Yeast and mammalian autophagosomes exhibit distinct phosphatidylinositol 3-phosphate asymmetries (2014)
f2

(a) Outline of freeze-fracture EM. Replicas representing the outer (luminal) and inner (cytoplasmic) membrane leaflets are called the EF and the PF, respectively. Fracture planes passing through the cytoplasm and the extracellular space are shown by a dotted line. (b) QF-FRL in wild-type yeast in log phase. PtdIns(3)P was labelled with GFP-PX and marked with colloidal gold (10 nm). The vacuolar membrane PF was labelled intensely, whereas other organellar membranes did not exhibit significant labelling. Scale bar, 0.5 μm. (c) Quantification of the labelling intensity of PtdIns(3)P (mean±s.e.m.). The experiment was repeated three times and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images. (d,e) Negative controls. Both wild-type yeast incubated with GST-PXR58A (d) and vps34Δ yeast incubated with GST-PX (e) were virtually unlabelled. Scale bar, 0.5 μm.
Yeast and mammalian autophagosomes exhibit distinct phosphatidylinositol 3-phosphate asymmetries (2014)
f5

(a) Huh7 cells expressing GFP-LC3 treated with 0.25 μM Torin1 and 0.4 μM bafilomycin A1 for 1 h. The autophagosome was identified as the IMP-poor double-membrane structure labelled with anti-GFP antibody (large gold, 10 nm). Both PtdIns(3)P (small gold, 6 nm) and GFP-LC3 were seen exclusively in the PF in both convex and concave structures. The small and large gold particles are shown as green and red dots, respectively, in the figures at right. Scale bars, 0.1 μm. Figures displaying wider areas, as well as additional examples, are presented in Supplementary Fig. 4c. (b) NIH3T3 cells expressing Atg4C78A were incubated in Earle’s balanced salt solution for 1 h. The isolation membrane was observed as parallel membranes with few IMPs. PtdIns(3)P was labelled only in the PF. Scale bar, 0.1 μm. Other examples are presented in Supplementary Fig. 5. (c) Labelling density of PtdIns(3)P in mammalian autophagosomal membrane (mean±s.e.m.). The experiment was repeated three times and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images. Some labelling was observed in the EF but the majority of gold particles in the EF occurred near the EF-PF boundary (white circles; see Supplementary Fig. 6c for more examples), suggesting that many of them actually represent PtdIns(3)P in the PF.
Yeast and mammalian autophagosomes exhibit distinct phosphatidylinositol 3-phosphate asymmetries (2014)
f6

(a) ymr1Δsjl3Δ cells cultured in S(-NC) for 4 h. PtdIns(3)P was labelled in comparable densities in PF and EF of autophagosomal membranes. Scale bar, 0.2 μm. (b) The PF/EF ratio of the PtdIns(3)P labelling density in autophagosomal membrane (mean±s.e.m.). The ratio was small (that is, PF) in WT, but close to 1 (that is, PF≅EF) in ymr1Δsjl3Δ. The experiment was repeated three times and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images. (c) A hypothetical mechanism to generate the PtdIns(3)P asymmetry in yeast autophagic membranes. PtdIns(3)P is formed not only in the cytoplasmic leaflet but also in the luminal leaflet of the isolation membrane and/or the autophagosome by an unknown mechanism(s). PtdIns(3)P in the cytoplasmic leaflet, but not that in the luminal leaflet, is hydrolysed by phosphatases, resulting in the observed PtdIns(3)P asymmetry.