EMBO/soda-nel huggingface dataset:
Niacin-mediated Tace activation ameliorates CMT neuropathies with focal hypermyelination (2016)
Figure 2

Figure 2. Nrg1 type III shRNA lentiviral vector transduction reduces myelin outfoldings in Mtmr2-/- cultures.

A. ShRNA targeting Nrg1 type III were validated in rat purified neurons. Western blot analysis for Nrg1 shows reduction of its expression (pro-protein at 150 KDa) using two different harpins, representative of two independent experiments. Clx is calnexin.

B. ShRNA#1 and 2 downregulate Nrg1 type III expression also in myelin-forming Schwann cell/DRG neuron co-cultures analyzed after 7 days of ascorbic acid treatment, as shown by western blot analysis, representative of two independent experiments. Clx is calnexin.

C, D. Titration of shRNA #1 (C) and of shRNA #2 (D) LVs (lentiviral vectors) on WT Schwann cell/DRG neuron co-cultures analyzed after 7 days of ascorbic acid treatment, with quantification of the mean number of Mbp positive segments and Schwann cell number. In (C) n= 20 (SCR, Scramble), n= 11 (LV #sh1, 12.5%), n= 16 (LV #sh1, 25%), n= 9 (LV #sh1, 50%) DRG/coverslips from two independent experiments. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test. In (D) n= 14 (SCR), n= 10 (LV #sh2, 12.5%), n= 12 (LV #sh2, 25%), n= 79 (LV #sh2, 50%) DRG/ coverslips, representative of two independent experiments. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test.

E. Representative confocal images of Mtmr2-/- co-cultures transduced using LV expressing Nrg1 type III shRNA #1, with quantification of the percentage of myelin outfoldings, n= number of DRG/coverslips, n= 9, 382 fibers (SCR), n=9, 368 fibers (LV #sh1, 12.5%), n= 8, 327 fibers (LV #sh1, 25%). Asterisks indicate fibers with myelin outfoldings. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test, representative of two independent experiments. Western blot analysis of Nrg1 and Akt phosphorylation (S473) shows that activation of Akt decreases following Nrg1 type III downregulation (p-Akt/Akt/Actin, ratio between Mtmr2-/- Scramble-treated and Mtmr2-/- sh#1-treated using 12.5% LV is 1 : 0.85). Each lane is a pool of at least 10 different DRG/coverslips, representative of two independent experiments. Bar in (C, D) is 100 μm; in (E) is 20 μm.
Niacin-mediated Tace activation ameliorates CMT neuropathies with focal hypermyelination (2016)
Figure 3

Figure 3. Treatment of Mtmr2-/- Schwann cell/DRG neuron co-cultures using either rhTACE or Niacin rescues myelin outfoldings.

A. Titration of rhTACE on wild-type (WT) co-cultures stained for Mbp with quantification of Mbp positive fibers and Schwann cell nuclei: n=18 (NT, not treated), n=6 (5ng/ml), n=20 (10ng/ml), n=7 (50ng/ml) DRGs/covers from two independent experiments, p=0.0053, nonparametric one-way ANOVA, followed by Dunn's post hoc test.

B. Immunohistochemistry and confocal microscopy of Mtmr2-/- cultures treated with 5ng/ml and 10ng/ml rhTACE, with quantification of the percentage of myelin outfoldings; n=10 (NT), n=10 (5ng/ml), n=11 (10ng/ml), DRGs/coverslips. A total of 547, 557, and 571 Mbp fibers were quantified, respectively, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test. Western blot analysis of lysates from Mtmr2-/- cultures treated using 10ng/ml rhTACE showing Nrg1 expression and Akt activation (S473 phosphorylation). Each lane is a lysate of at least n=10 DRGs/coverslips. In Mtmr2-/- (10ng/ml rhTACE) as compared to Mtmr2-/- (NT), Nrg1(150 KDa)/actin ratio is 0.8:1; Nrg1(65 KDa)/actin ratio is 0.48:1; and p-Akt/Akt/actin ratio is 0.6:1.

C. Titration of WT co-cultures using different amounts of Niacin, nicotinic acid, with quantification of Mbp positive fibers and Schwann cell nuclei, n=9 (NT), n=8 (1mM), n=9 (5mM), n=8 (10mM), DRGs/ coverslips. Representative of three independent experiments, p=0.0055, nonparametric one-way ANOVA, followed by Dunn's post hoc test.

D. Immunohistochemistry and confocal microscopy of Mtmr2-/- cultures treated with 1 mM and 5mM Niacin, with quantification of the percentage of myelin outfoldings; n=31 (NT), n=14 (1mM), and n=29 (5mM) DRGs/coverslips from two independent experiments. A total of 1256, 489, and 1305 fibers, respectively, were quantified, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test. Tace activity was measured from lysates of Niacin-treated cultures, n=7 (KO NT) and n=8 (KO 5mM Niacin) number of independent plates, each containing from 10-15 DRGs plated. Note that Tace activity was similar between WT and Mtmr2-/- cultures, one-tailed Mann Whitney U test, p=0.037.

E. Niacin treatment of Tace-/- explants does not rescue hypermyelination, with quantification, n=8 DRG/coverslips per condition, p=0.0056, nonparametric one-way ANOVA, followed by Dunn's post hoc test. Representative of three independent experiments.

Results are expressed as mean ± SEM. Mbp=Myelin Basic Protein; Nf=Neurofilament.

Bar in (A, C, E) is 100 μm, in (B, D) is 20 μm.
Niacin-mediated Tace activation ameliorates CMT neuropathies with focal hypermyelination (2016)
Figure 4

Figure 4. Genetic ablation of Nrg1 type III in Mtmr2-/- mice rescues myelin outfoldings.

A. Representative images of semithin section analysis of sciatic nerves from Mtmr2-/- and Mtmr2-/-;Nrg1 (III)+/- mice at 6 months. Myelin outfoldings (arrowheads) are reduced in Mtmr2-/-;Nrg1 (III)+/- nerves (n=9) as compared to Mtmr2-/- (n=8), results are mean ± SEM, p=0.0037, two-tailed Mann Whitney U test.

B. G-ratio analysis on sciatic nerves from Mtmr2-/-, Mtmr2-/-;Nrg1 (III)+/-, and Nrg1 (III)+/- mice at 6 months indicates that myelin thickness of Mtmr2-/-;Nrg1 (III)+/- is similar to Nrg1 (III)+/- nerves and different from Mtmr2-/-, which is in the normal range as already reported (Bonneick et al, 2005), n=5 animals per genotype.

Mean g-ratios: Mtmr2-/- 0.67 ± 0.03, 2129 fibers, n=5 animals; Mtmr2-/-;Nrg1 (III)+/- 0.71 ± 0.03, 2139 fibers, n=5 animals; Nrg1 (III)+/- 0.72 ± 0.03, 2152 fibers, n=4 animals. Mtmr2-/- as compared to Mtmr2-/-;Nrg1 (III)+/-, p=0.0145; Mtmr2-/- as compared to Nrg1 (III)+/-, p=0.0030, and Mtmr2-/-;Nrg1 (III)+/- as compared to Nrg1 (III)+/- p=0.3193, repeated measures ANOVA.

C. Western blot analysis on lysates from Mtmr2-/-, Mtmr2-/-;Nrg1 (III)+/-, and Nrg1 (III)+/- sciatic nerves at P10 indicates that Akt activation (S473 phosphorylation) is similar between Mtmr2-/-;Nrg1 (III)+/- and Nrg1 (III)+/- consistent with g-ratio findings, representative of three independent experiments. Phosphorylation of Akt is relative to total Akt normalized on calnexin, Clx.

Bar is 10 μm.
Niacin-mediated Tace activation ameliorates CMT neuropathies with focal hypermyelination (2016)
Figure 5

Figure 5. Niacin/Niaspan ameliorates hypermyelination in Vim-/- mouse nerves.

A. Niacin treatment of Vim-/- explants rescues hypermyelination, n=10 DRGs/ coverslips per condition, with quantification, representative of three independent experiments, p=0.0048, nonparametric one-way ANOVA, followed by Dunn's post hoc test.

Western blot analysis on lysates from treated and not treated explants (at least 10 DRGs/ coverslips per lane) shows that increased Akt activation (S473 phosphorylation) in Vim-/- explants is restored to normal levels following 5 mM Niacin treatment.

B. Niaspan administration (daily i.p. injection of 160mg/Kg Niaspan starting at P15 for 15 days) enhances Tace activity in Vim-/- nerves at P30, n=6 animals per genotype, p=0.0325, one-tailed Mann Whitney U test. Niaspan treatment of Vim-/- mice does not affect mouse growth, as the growth rate in WT (n=8, saline; n=6 Niaspan) and Vim-/- (n=10 saline and n=10 Niaspan) mice either saline or Niaspan-treated is not significantly different, Linear Mixed Effect (LME) models.

C. Semithin section and g-ratio analyses of sciatic nerves at P30 shows that Niaspan does not alter myelin thickness in WT nerves, whereas restores myelin thickness to normal values in Vim-/- nerves. G-ratio values: WT saline, 0.71 ± 0.003, 1872 fibers, n= 5 animals; WT Niaspan, 0.71 ± 0.004, 1882 fibers, n= 5 animals; Vim-/- saline 0.69 ± 0.004, 2313 fibers, n= 6 animals; Vim-/- Niaspan 0.72 ± 0.01, 1616 fibers, n=5 animals. WT saline as compared to Vim-/- saline, p=0.0588; Vim-/- saline as compared to Vim-/- Niaspan, p=0.0431, repeated measures ANOVA, representative of two independent experiments. The number of myelinated fibers is similar between the 4 groups as shown, p=0.3042, nonparametric one-way ANOVA, followed by Dunn's post hoc test.

Results are expressed as mean ± SEM.

Bar in (A) is 50 μm; in (B) is 10 μm for semithin sections and 1 μm for ultrastructural analysis images.
Niacin-mediated Tace activation ameliorates CMT neuropathies with focal hypermyelination (2016)
Figure 6

Figure 6. Niaspan reduces myelin outfoldings in the Mtmr2-/- mouse nerves.

A. Niaspan administration (daily i.p. injection of 160mg/Kg starting at P15 for 60 days) does not affect the growth of Mtmr2-/- mice. The growth rates of Mtmr2-/- and WT both saline-treated are significantly different, p<0.0001, Linear Mixed Effect (LME) models, as already reported (Bolino et al, 2004), n=8 animals per condition.

B. Niaspan administration reduces the percentage of myelin outfoldings (red asterisks) in Mtmr2-/- sciatic nerves without affecting the number of myelinated fibers as assessed by semithin section analysis at P75, n= 8 mice per genotype, results are mean ± SEM, p=0.0093, two-tailed Mann Whitney U test, representative of three independent experiments. Niaspan treatment does not alter myelin thickness in WT or Mtmr2-/- mice as assessed by g-ratio analysis (WT saline-treated, 0.67 ± 0.003, 2014 fibers; Mtmr2-/- Niaspan-treated, 0.67 ± 0.007, 1924 fibers; Mtmr2-/- saline-treated, 0.68 ± 0.003, 2244 fibers; n=4 animals per condition).

Bar is 10 μm.
Niacin-mediated Tace activation ameliorates CMT neuropathies with focal hypermyelination (2016)
Figure 7

Figure 7. Niaspan treatment ameliorates hypermyelination in Pmp22+/- mouse nerves.

A. Western blot analysis of lysates from Pmp22+/- and WT sciatic nerves at P10 shows that Akt phosphorylation (S473) is increased in Pmp22+/- nerves, results are mean ± SEM, p=0.0317, two-tailed Mann Whitney U test, representative of two independent experiments.

B. Western blot analysis of Tace using lysates from Pmp22+/- and WT sciatic nerves at different stages of development and below, Tace activity as measured in Pmp22+/- and WT sciatic nerves at P30 (n=6 mice per genotype, results are mean ± SEM, p=0.0070, two-tailed Mann Whitney U test). The arrowhead indicates the pro-protein and the arrow shows the cleaved active form of Tace. Representative of two independent experiments.

C. Representative images of semithin section analysis of sciatic nerves at P45 from Pmp22+/- saline and Pmp22+/- Niaspan-treated mice. Niaspan was administered daily by i.p. injection at 160mg/Kg starting at P15 for 30 days.

C'. The percentage of tomacula in sciatic nerves was assessed by ultrastructural analysis, results are mean ± SEM, n= 6 animals per condition, p=0.0022, two-tailed Mann Whitney U test, representative of two independent experiments.

D. G-ratio analysis on semithin sections of sciatic nerves shows that increased myelin thickness in Pmp22+/- sciatic nerves at P45 is restored to normal values following Niaspan treatment. Mean g-ratio values, WT saline-treated: 0.69 ± 0.002, 2052 fibers, n=5 animals; Pmp22+/- saline-treated: 0.66 ± 0.003, 2643 fibers, n=6 animals; Pmp22+/- Niaspan-treated, 0.68 ± 0.002, 2311 fibers, n=6 animals. WT saline as compared to Pmp22+/- saline-treated, p<0.0001; Pmp22+/- saline-treated as compared to Pmp22+/- Niaspan-treated, p=0.0002; Pmp22+/- Niaspan-treated as compared to WT saline, p=0.0899; repeated measures ANOVA.

E. The number of myelinated fibers is similar between the 3 groups analyzed, p=0.4362, nonparametric one-way ANOVA, followed by Dunn's post hoc test.

Bar is 10 μm.
Niacin-mediated Tace activation ameliorates CMT neuropathies with focal hypermyelination (2016)
Figure 8

Figure 8. Niaspan treatment does not ameliorate hypermyelination in Pmp22-/- mouse nerves.

A. Western blot analysis to detect phosphorylation levels of Akt (S473) on lysates from Pmp22-/- and WT mice at P20, with quantification. Results are mean ± SEM, p=0.6857, two-tailed Mann Whitney U test, representative of three independent experiments.

B. Western blot analysis for Mbp and P0 on lysates from Pmp22-/- and WT mice P20 shows decreased myelin protein expression levels at both time points in Pmp22-/- nerves, results are mean ± SEM, p=0.0500 with the exception of the P0 protein level at P20, for which the difference between Pmp22-/- and WT nerves is not statistically significant, p=0.1000, one-tailed Mann Whitney U test, representative of two independent experiments.

C. Representative images of semithin section analysis of WT and Pmp22-/- sciatic nerves at P30. Tomacula are abundant in Pmp22-/- nerves at this age, where both fibers with thicker myelin (in the range of <3-4 μm in diameter) and thinner myelin (with diameters greater than 4 μm, particularly in motor fasicles) can be observed.

C'. Niaspan does not ameliorate the phenotype in Pmp22-/- mice, as indicated by the number of tomacula (quantification of the percentage of tomacula in the entire sciatic nerve section, results are mean ± SEM, p=0.7400, Mann Whitney U test, n= 15 animals per condition).

D. Growth curve of Pmp22-/- mice treated with Niaspan administered by i.p. injection daily at 160mg/Kg and starting at P15 for 15 days. A significant time by group effect is noted between WT saline and Pmp22-/- saline (group effect p=0.9907 and time effect p=0.0057), indicating that the two groups start with similar weight values but then the WT saline group (n=6 animals) grows more in time. When Pmp22-/- saline (n=5) and Pmp22-/- Niaspan-treated (n=6) are compared, the group effect is statistically significant (p=0.0076) while the time effect is not (p=0.6677), suggesting that these two groups have a similar growth trend but Pmp22-/- Niaspan-treated group starts with a higher baseline weight.

E. Niaspan does not ameliorate myelin thickness as indicated by g-ratio analysis, with axonal diameter distribution: WT 0.68 ± 0.003, 2945 fibers, n=5 animals; Pmp22-/- 0.64 ± 0.003, 1557 fibers, n= 6 animals; Pmp22-/- Niaspan 0.63 ± 0.003, 1445 fibers, n=6 animals, results are mean ± SEM. WT saline as compared to Pmp22-/- saline-treated p<0.0001; WT saline as compared to Pmp22-/- Niaspan-treated p<0.0001, repeated measures ANOVA. Note the loss of myelinated fibers in Pmp22-/- nerves for all caliber axons.

Bar is 10 μm.
APOPT1/COA8 ASSISTS COX ASSEMBLY AND IS OPPOSITELY REGULATED BY UPS AND ROS (2018)
Figure 1

Figure 1 - Generation of the Apopt1 knockout mouse model

A Strategy employed for the targeted disruption of mouse chromosome 12 Apopt1 coding exon 2 by CRISPR/Cas9. The mutation selected for the characterization of the mouse model produced an indel (c.188delAinsTG) resulting in a frameshift and truncated protein (p.Asp55Valfs*20).

B Chromatograms generated by Sanger sequencing of Apopt1-/- (KO) and Apopt1+/+ (WT) genomic DNA highlighting the mutated position in comparison with the wild-type sequence.

C Relative mRNA expression of Apopt1 normalized to the expression of GAPDH in skeletal muscle and liver of wild-type, heterozygous and knock-out mice of three months of age. Data are presented as mean ± SEM (n = 6 mice per genotype). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: Muscle - ***P = 0.0001 (WT vs KO), *P = 0.0310 (WT vs het), *P = 0.0395 (het vs KO), Liver - **P = 0.0022 (WT vs KO), *P = 0.0101 (WT vs het).
APOPT1/COA8 ASSISTS COX ASSEMBLY AND IS OPPOSITELY REGULATED BY UPS AND ROS (2018)
Figure 3

Figure 3 - Morphological, biochemical and structural analysis in mouse tissues

A Representative Hematoxylin & Eosin staining of skeletal muscle in three-month old individuals.

B Histochemical reaction specific to COX and SDH in skeletal muscle, cerebellar cortex and kidney of the same individuals.

C COX (CIV) enzymatic activity normalized to the activity of citrate synthase (CS) in five animals per genotype as indicated at three and twelve months of age. Data are presented as mean ± SEM (n = 6 per group). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: Muscle - ****P < 0.0001 (het vs KO), ***P = 0.0001 (WT vs KO), *P = 0.0185 (WT vs het), Kidney - ****P < 0.0001 (het vs KO), ***P = 0.0002 (WT vs KO), Heart - ***P = 0.0003 (het vs KO), ***P = 0.0001 (WT vs KO), Brain and Liver - ****P < 0.0001.

D Western blot analysis of SDS-PAGE of total lysates from liver, skeletal muscle and brain from the indicated genotypes, each lane showing the results for one animal. The graph shows the densitometric quantification of the signals obtained in the liver. Data are presented as mean ± SEM (n=2 WT; n=2 het; n=3 KO). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons: ***P = 0.0002 and ****P < 0.0001 (WT vs KO).

E Western blot analysis of 1D-BNGE of mitochondria from skeletal muscle from the indicated genotypes, each lane showing the results from one animal.

F Western blot analysis of 2D-BNGE of mitochondria from skeletal muscle from one individual of the indicated genotype.
APOPT1/COA8 ASSISTS COX ASSEMBLY AND IS OPPOSITELY REGULATED BY UPS AND ROS (2018)
Figure 4

Figure 4 - Mitochondrial sub-localization of APOPT1

A Western blot of SDS-PAGE of different fractions from 143B cells expressing APOPT1HA. Tot: total lysate. Cyto: post-mitochondrial fraction (cytoplasm). Mt: isolated mitochondria. Mt sol: Soluble mitochondrial fraction. Mt memb: mitochondrial membranes. CO32- pellet: Pellet after carbonate extraction with 0.1 M Na2CO3, pH 10.5 for 30 minutes. CO32- sol: soluble fraction after the carbonate extraction.

B Western blot of SDS-PAGE of mitochondria used for protease protection assay after digitonin treatment. The experiment was carried out in isolated mitochondria from 143B cells expressing APOPT1HA exposed to increasing amounts of digitonin (expressed in µg) and 50 µg/ml trypsin. Complete solubilization with 1% Triton X-100 was used as a control of protease sensitivity. TOM20: Translocase of the outer membrane (OM) 20 kDa. ACO2: Aconitase 2 (mitochondrial isoform). AIF: Apoptosis inducing factor. AK2: Adenylate kinase 2. OM: outer mitochondrial membrane. IM: inner mitochondrial membrane. IMS: intermembrane space.

C Western blot of SDS-PAGE of mitochondria used for protease protection assay after hypotonic shock. The experiment was carried out in isolated mitochondria from 143B cells expressing APOPT1HA incubated either in isotonic (Iso) or hypotonic buffers for 5 minutes (Hypo 5') or 15 minutes (Hypo 15') and 50 µg/ml trypsin. Complete solubilization with 1% Triton X-100 was used as a control of protease sensitivity.

D N-SIM super-resolution micrographs showing 0.8 μm Maximum Intensity Projection (0.15 μm for each Z-stack) of 143B cells expressing APOPT1GFP shown in green, specific markers for each mitochondrial compartment: TOMM20 (OM), COX8A (IM), MitoTracker (IM+M) and matrix-target (mScarlet) shown in red. Scale bar: 5 μm. The graph shows Pearson's Coefficient in colocalized volume of different sub-compartment combinations with APOPT1GFP. Data show mean ± SEM (n = 5). One-way ANOVA (*P = 0.0298 and **P = 0.0076) was employed for the statistical analysis.
APOPT1/COA8 ASSISTS COX ASSEMBLY AND IS OPPOSITELY REGULATED BY UPS AND ROS (2018)
Figure 6

Figure 6 - Effects of proteasome inhibition and ROS overproduction on APOPT1 stability

A Western blot analysis of SDS-PAGE of MGM132-treated 143B APOPT1HA cells. The graph represents the densitometric quantification of the signals for the precursor and mature protein.

B The upper part of the panel (Input) shows Western blot analyses of APOPT1HA in the MGM132-treated cells. Note the appearance of higher molecular weight bands upon longer exposure in the samples treated with the proteasome inhibitor. The bottom part of the panel (Purified fractions) shows the analysis of fractions from the same cells immunoprecipitated with anti-HA. Note that the higher molecular weight species are cross-reacting with both anti-HA and anti-ubiquitin.

C Western blot analysis of SDS-PAGE of total lysates from 143B cells overexpressing tagged APOPT1 (as indicated) and exposed to 100 µM H2O2, as illustrated by the scheme (H2O2 treatment), for the indicated times. The graphs represent the densitometric quantification of the tagged APOPT1 signal at each time point. The graph inset shows that the increase of APOPT1 occurs in the first minutes after the exposure to H2O2.

D Western blot analysis of SDS-PAGE of total lysates from 143B cells overexpressing tagged APOPT1 (as indicated) and exposed to 5 µM MitoParaquat (MitoPQ), as illustrated by the scheme (MitoPQ treatment), for the indicated times. The graphs represent the densitometric quantification of the tagged APOPT1 signal at each time point. The graph inset shows that the increase of APOPT1 occurs in the first minutes after the exposure to MitoPQ.
APOPT1/COA8 ASSISTS COX ASSEMBLY AND IS OPPOSITELY REGULATED BY UPS AND ROS (2018)
Figure 7

Figure 7 - Effects of MitoPQ treatment in the absence or presence of APOPT1

A Western blot analysis of SDS-PAGE of different mitochondrial proteins in total lysates from the indicated cell lines treated with 5 µM MitoPQ at the indicated times. UT: untreated cells.

B Densitometric quantification of APOPT1GFP signal during the treatment in two biological replicas.

C Densitometric quantification of MT-CO1 signal in the non-complemented APOPT1-less cells (S6 GFP) vs. the complemented cells (S6 APOPT1GFP). Three biological replicas (n=3) were carried out for each cell line. The signals in UT S6 APOPT1 were considered 100%. The levels of MT-CO1 were significantly lower in the S6 GFP patient cells after 20 hours of MitoPQ treatment compared to the untreated cells (*P = 0.0202, two-tailed unpaired Student's t-test).

D Densitometric quantification of MT-CO2 signal in the non-complemented APOPT1-less cells (S6 GFP) vs. the complemented cells (S6 APOPT1GFP). Three biological replicas were carried out for each cell line. The signals in UT S6 APOPT1 were considered 100%.