EMBO/SourceData huggingface dataset:
Figure 1Relative quantification of mRNA for Ppcs, Ppcdc, and Coasy by genotype from each of the three study regions. n=11,4,4 for WT and n=8,4,4 for KO (GP, SN, Cerebellum, respectively)Western blot and quantification for Coasy from GP by genotype and treatment status (pPanSH = 4'-phosphopantetheine). Ponceau S was used as a loading control. n=8, 4 for WT and n=8, 6 for KO (vehicle and pPanSH groups, respectively).
Figure 2Relative mRNA expression of Tfrc, Ireb2, and Hamp by genotype and brain region. n=11,4,4 for WT and n=8,4,4 for KO (GP, SN, Cerebellum, respectively)Total iron quantity in cytosol and mitochondria from GP using ICP-MS. The data distribution is presented using a box plot, including minimum, first quartile, median, third quartile, and maximum. The magenta-colored line represent mean. n=6 for both genotypesAssay of mitochondrial versus cytosolic aconitase activity in brain compared by genotype. Quantification of aconitase activity in two tissues (brain and liver) from animals of each genotype by band density. n=4 for WT, n=5 for KO (D-E) Activity of pyruvate dehydrogenaseAssay of mitochondrial versus cytosolic aconitase activity in brain compared by genotype. Quantification of aconitase activity in two tissues (brain and liver) from animals of each genotype by band density. n=4 for WT, n=5 for KO (D-E) Activity of pyruvate dehydrogenase (D) and complex I (E) in GP by genotype and treatment status (veh = vehicle; pPanSH = 4'-phosphopantetheine). n=5 for both genotypes and treatment groups.Western blot and quantification of TfR1 and PDHE1α1 from GP by genotype and treatment status (pPanSH = 4'-phosphopantetheine). Ponceau S was used as total protein loading control and VDAC for the mitochondrial protein loading control.
Figure 3Relative mRNA and protein expression of the dopamine receptor Drd1 compared by genotype, brain region and treatment status (pPanSH = 4'-phosphopantetheine). n=11,4,4 for WT and n=8,4,4 for KO (GP, SN, Cerebellum, respectively) QRT-PCR. n=5 for Western blot for all groupsRelative mRNA expression of Drd2 and GABA receptor Gabra3 expression by genotype and brain region. n= same as 3A.Western blot quantification from whole brain of tyrosine hydroxylase compared by genotype. n=5 for WT and n=3 for KO.
Figure 4Relative quantification of COASY mRNA in human PKAN primary cell lines. n= 5 for both genotypes and cell typesRelative quantification of COASY/Coasy in fresh, blood-derived lymphocytes from human and mouse compared by genotype. n=51,35 for human (control, PKAN, respectively) and n=5 for both mouse genotypes.Extracellular flux analysis in human fibroblasts showing differences in OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) by genotype. Oligom; Oligomycin, FCCP; Carbonyl cyanide-ptrifluoromethoxyphenylhydrazone, 2-DG; 2-deoxy-glucose. Quantification of baseline OCR and ECAR is shown, as well. n=3 per genotype with four technical replicates.Complex I activity assay using fibroblast lysates. Cells were either treated with vehicle or 50μM pPanSH for three days and total protein extract was used for the Complex I Dipstick assay n=4 for both genotypes and treatment groups.Relative mRNA expression of mitochondrially-encoded genes important for oxidative phosphorylation from complex I (MT-ND1), complex III (MT-CYB), cytochrome C (MT-CO1), and ATP synthase (MT-ATP6) in human mutant fibroblasts versus control cells. n=3 for both genotypes.Calculation of active mitochondria using the JC-1 assay by genotype in control and PKAN lymphoblasts.
Figure 5Relative expression of Pank1α, Pank1β, total Pank1 and Pank3 in mouse brain regions. n=8,8,4 for both genotypes (GP, SN, Cerebellum, respectively).Relative expression of Pank1α and Coasy in GP from WT and KO mice treated for 14 days with bezafibrate (0.8 mg/g body weight) or gemfibrozil (1.2 mg/g body weight). n=8 for both genotypes treated with vehicle, n=5 for both KO groups treated with either bezafibrate or gemfibrozil.
Figure 6Relative quantification of Coasy mRNA from GP compared by treatment status using four compounds: 4'-phosphopantetheine (pPanSH), pantetheine, vitamin B5, and CoA. n=8 (WT-veh), 5 (KO-veh), 5 (KO-pPanSH), 4 (KO-all other intermediates). Correction of Coasy, Tfrc, Ireb2, and Drd1 expression in GP by 4'-phosphopantetheine over a range of doses. n=8 (WT-veh), 5 (KO-veh), 5 (KO-0.82), 4 (WT-5), 5 (KO-5), 5 (KO-8.2), 5 (KO-10). 5 (WT-20), 5 (KO-20)Relative quantification of COASY mRNA from human fibroblasts treated with pPanSH. n=3 for both genotypes.ECAR measured in cultured human fibroblasts by genotype and treatment status. Cells were treated for three days with 4'-phosphopantetheine (pPanSH) or vehicle (veh). n=3 for both genotypes.
Figure 7Relative quantification of Coasy, Tfrc and Drd1 mRNA from GP by number of days post-cessation of treatment with 5μg of 4'-phosphopantetheine per g of body weight. n= 11 (WT-veh-D0), 5 (KO-veh-D0), 4 (WT-pPanSH-D0), 5 (KO-pPanSH-D0), 5 (KO-pPanSH-D1), 5 (KO-pPanSH-D2), 5 (KO-pPanSH-D3), 5 (WT-pPanSH-D7), 4 (KO-pPanSH-D7),
Figure 1A OT-I CD8+ CTLs were conjugated with 10 nM OVA257-264-pulsed EL-4 cells for 30 min, fixed, and stained with anti-SKAP55 (red), anti-LFA-1 (green) and Hoechst (blue).B, C WT and SKAP55−/−OT-ICD8+CTLs were generated from WT or SKAP55−/−OT-I Tg mice, then incubated with 10 nM OVA257-264-pulsed EL4 targets for 4 h to assess the in vitro cytotoxicity at different Effector:Target ratios (B; mean of triplicates ± SD); surface expression and the mRNA levels of PD-1 (C). Graphs are representative of at least three independent experiments.D WT and SKAP55−/− OT-I CD8+ CTLs (3 × 106) were injected into C57BL/6 mice, followed by injection of non-pulsed (CFSEhi) or 10 nM OVA257-264-pulsed (CFSElo) splenocytes (5 × 106) to measure in vivo cytotoxicity (mean ± SD, n = 3 mice per group). Representative data of three independent experiments.E WT and SKAP55−/− OT-I CD8+ CTLs were pretreated with anti-PD-1 antibody or IgG control, followed by incubation with OVA257-264-pulsed EL4 cells to examine in vitro killing ability (mean of triplicates ± SD). Graphs are representative of three independent experiments.F CD8+ CTLs were transfected with plasmids expressing SKAP55-GFP or EGFP, then treated with non-pulsed or 10 nM OVA257-264-pulsed EL4 cells to examine surface PD-1 expression (left panel) or the in vitro killing assay (mean of triplicates ± SD) (right panel).
Figure 2A OT-I CD8+ CTLs were conjugated with CFSE-labeled OVA257-264-pulsed or non-pulsed EL-4 cells for 30 min, fixed, and stained with anti-ADAP (red).B OT-I CD8+ CTLs were transfected with plasmids expressing ADAP or GFP, then stimulated with 10 nM OVA257-264-pulsed EL-4 cells to detect surface PD-1 expression. Representative of three independent experiments.C, D WT and ADAP−/− OT-I CD8+ CTLs were stimulated with 10 nM OVA257-264-pulsed or unpulsed EL-4 cells for 4 h to examine surface expression and the mRNA levels of PD-1 (C), in vitro cytotoxicity at different Effector:Target ratios (D; mean of triplicates ± SD). Graphs are representative of at least three independent experiments.E WT and ADAP−/− OT-I CD8+ CTLs were pretreated with 10 μg/ml anti-PD-1 antibody or IgG control, followed by incubation with 10 nM OVA257-264-pulsed EL4 cells to examine in vitro killing ability (mean of triplicates ± SD).F Naïve CD8+ T cells were isolated from WT, SKAP55−/−, ADAP−/−, and SKAP55−/−ADAP−/− splenocytes, then stimulated with plate-bound anti-CD3/CD28 for 12 h to assess PD-1 mRNA levels, and for 48 h to check surface PD-1 expression (mean of triplicates ± SD).
Figure 3A-C WT, ADAP−/−, or SKAP55−/− OT-I CD8+ CTLs were stimulated with OVA257-264-pulsed or unpulsed EL4 cells to detect the levels of NFATc1 expression, activation and nuclear entry by immunoblotting (A) or immunostaining (B). Alternatively, nuclear extracts from these CTLs were incubated with the N1 DNA probes containing NFAT-binding sites for an EMSA. The unlabeled competitor probe (Comp. N1) and the mutant probe that contains the same sequence except for carrying a mutated NFAT-binding site (Comp. mutN1) were included as controls (C).D WT, ADAP−/−, or SKAP55−/− OT-I CD8+ CTLs were pretreated with DMSO or 5 μM CsA, then stimulated with OVA257-264-pulsed EL-4 cells for 4 h to examine surface PD-1 expression (mean of triplicates ± SD).E WT, ADAP−/−, or SKAP55−/− OT-I CTLs were pretreated with the DMSO control, 10 μM PP2 or 75 μM 2APB, then stimulated with OVA257-264-pulsed EL-4 cells for 4 h to examine surface PD-1 expression (mean of triplicates ± SD).F GFP, ADAP, or SKAP55 was overexpressed with pGL3-NFAT Luciferase reporter plasmid into Jurkat cells. Cells were stimulated with anti-CD3 and anti-CD28 in the presence or absence of PP2 or 2APB for 6 h, followed by measuring luciferase readings (mean of triplicates ± SD).
Figure 4A WT and SKAP55 KO C57BL/6 mice were s.c. immunized on day −14 and day −7 by DCs that were prepulsed with B16F10 tumor lysates. On day 0, the immunized mice were i.v. inoculated with B16F10 cells. Number of lung tumors were counted 26 days after B16F10 challenge (n ≥ 5 mice).B, C At day 0 (B, before tumor injection) or at day 26 (C, after tumor injection), surface PD-1 expression in CD44hi CD8+ or CD44hi CD4+T cells, and the percentages of CD44hi CD8+T cells or Foxp3+ CD4+ cells were analyzed (mean ± SD, n ≥ 5 mice).D, E The mRNA levels of PD-1, NFATc1, or the mRNA and protein levels of granzyme B in lung infiltrating CD8+ T cells were examined at day 26 (mean of triplicates ± SD, and the mRNA samples were prepared from at least 3 mice per group).F CTLA-4 levels in the lung-infiltrated CD44hiCD8+ T cells or the percentages of CD4+Foxp3+ T cells in lungs, MLN, and spleen were measured at day 26 (mean ± SD, n ≥ 5 mice).
Figure 5A The same procedures as in Fig4A were performed with WT and ADAP−/− C57BL/6 mice. HE staining of lung tissue was examined on day 26 (representative graph of 5 mice).B, C PD-1 expression in CD44hiCD8+ or CD44hiCD4+ T cells (mean ± SD, n ≥ 5 mice per group) or granzyme B expression in CD44hiCD8+ T cells were examined from the lungs at day 26 (representative data of 3 mice per group).D, E CTLA-4 levels in lung infiltrating CD44hiCD8+ T cells and the percentages of CD4+Foxp3+ T cells in the lungs or MLN were measured at day 26 (mean ± SD, n ≥ 5 mice per group).
Figure 6A, B WT and SKAP55−/− mice were s.c. injected with MO5 melanoma cells followed by two injection at day 7 and day 14 of OVA257-264-pulsed DCs or the PBS control respectively (n ≥ 5). The growth of tumors under skin was measured every three days according to tumor diameter (mean ± SEM, n ≥ 5 mice per group) (A). Surface expression levels of PD-1 were checked on OVA257-264-specific Vα2high/Vβ5+ CD8+ effector cells at day 30 (mean ± SD, n ≥ 5 mice) (B).C WT and SKAP55−/− mice were s.c. injected with MO5 melanoma cells followed by s.c. two injection of OVA257-264-pulsed DCs at day 7 and day 14. anti-PD-1 monoantibody or IgG controls were i.v. injected three times at day 7, day 10, and day 14 (50 μg/injection). The diameter and volume of tumors were measured every three days (mean ± SEM, n ≥ 5 mice).D The WT recipient mice were s.c. injected with MO5 melanoma cells followed by injection of 10 nM OVA257-264-stimulated WT or SKAP55−/− CTLs at day 8 (n ≥ 8). The diameter and volume of tumors were measured every three days (mean ± SEM, n ≥ 8 mice).
Figure 7A, B During generation of OVA257-264 peptide-specific CTLs, naïve OT-I splenocytes were stimulated with 10 nM OVA257-264 with or without CsA (10 nM) for 3 days, then washed, and further cultured in the RPMI growth medium until day 6. The CsA-treated or untreated OVA257-264 peptide-specific CTLs were incubated with OVA257-264-pulsed EL-4 cells for 4 h to assess the in vitro cytotoxicity (A, mean of triplicates ± SD) or i.v. injected into WT mice to measure in vivo cytotoxicity (B, mean ± SD, n = 4 mice).C WT mice were s.c. injected with B16-MO5 melanoma cells followed by injection of the CsA-treated or untreated OVA257-264 peptide-specific CTLs at day 9 (n ≥ 12). The diameter and volume of tumors were measured every 3 days (mean ± SEM, n ≥ 12 mice).
Figure 1C) Representative immunoblots of brain extracts of wild-type, Ttll1-/- and Ttll7-/- mice. Polyglutamylation of α-tubulin, detected with polyE and GT335, is specifically lost in Ttll1-/- brain. By contrast, β-tubulin glutamylation, which is specifically detected with β‑monoE, is absent from brains of Ttll7-/- mice. D) Representative immunoblots of extracts of wild-type, Ttll1‑/- and Ttll7-/- primary hippocampal neurons at DIV4. As in brain (C), α-tubulin polyglutamylation is specifically lost in Ttll1-/- neurons, while β-tubulin glutamylation lacks in Ttll7-/- neurons. E F,G) Immunocytochemistry of DIV4 primary hippocampal neurons from wild type, Ttll1-/- and Ttll7‑/- mice. Microtubules in Ttll1-/- cells have reduced levels of polyE signal, indicative of lower α-tubulin polyglutamylation (F), Ttll7‑/- neurons are specifically reduced in β‑monoE reactivity, indicating the lack of β‑tubulin glutamylation (G).
Figure 2A) Representative histology images of cerebella from 3-months-old mice stained with anti-calbindin antibody, which specifically labels Purkinje cells (Sequier et al, 1988). Note the presence of an intact Purkinje-cell layer in cerebella of wild-type, Ttll1-/-, Ttll7-/- and Ccp1-/-Ttll1-/- mice, in contrast to the complete absence of these cells in Ccp1-/- and Ccp1-/-Ttll7-/- mice.B) Histology of 25-day old mice with partial degeneration of Purkinje Cells. Note that both, Ccp1-/- and Ccp1-/-Ttll7-/- brains, show highly similar pattern of partial Purkinje-cell loss (red arrow heads: gaps in the Purkinje-cell layer, indicating degenerated Purkinje cells; orange arrow heads: swellings of Purkinje-cell axons, indicative of ongoing degeneration).C) Representative immunoblots of cerebellum extracts of wild-type, Ccp1-/-, Ttll7-/-, Ccp1-/-Ttll7-/-, Ttll1-/- and Ccp1-/-Ttll1-/- mice. Loss of CCP1 leads to hyperglutamylation of α- and β-tubulin as revealed with polyE and GT335 antibodies. Concomitant deletion of TTLL7 lead to a loss of glutamylation from β-tubulin (β‑monoE, GT335), while hyperglutamylation of α-tubulin persists (polyE, GT335). Concomitant loss of CCP1 and TTLL1 eliminates hyperglutamylation from both, α- and β-tubulin (polyE, GT335), while β-tubulin glutamylation is unaltered (β-monoE).
Figure 3Semi-thin cross sections of femoral quadriceps nerves stained with alkaline methylene blue. Note the smaller size of Ccp1-/- and Ccp1-/-Ttll7-/- nerves (A).Semi-thin cross sections of femoral quadriceps nerves stained with alkaline methylene blue. These sections were quantified for (B) number of myelinated axons, (C) nerve area, (D) nerve perimeter (Fig. EV2) of femoral quadriceps nerves from wild-type, Ccp1-/-, Ttll7-/-, Ccp1-/-Ttll7-/-, Ttll1-/- and Ccp1-/-Ttll1-/- mice. Scatter dot plots in which each point represents one nerve of an individual mouse are shown. The black lines indicate means ±SEM. Significance was tested with one-way ANOVA and Holm-Šídák's multiple comparisons test. Note that all three parameters are decreased for Ccp1-/- and Ccp1-/-Ttll7‑/- mice, while Ccp1-/-Ttll1-/- mice showed no difference to the Ttll1-/- control.E,F) Representative electron microscopy images of cross sections of femoral quadriceps nerves. (E) Nerves from Ttll1-/- and Ccp1-/-Ttll1-/- mice show no abnormalities of myelinated axons (green asterisks), indicating that loss of TTLL1 prevents axonal degeneration caused by the absence of CCP1. (F) Ccp1-/- and Ccp1-/-Ttll7-/- mice both show the presence of foamy macrophages (magenta arrowheads), which are signs for myelin phagocytosis during axon degeneration. This indicates that loss of TTLL7 cannot prevent the CCP1-induced degeneration of femoral quadriceps nerves.
Figure 4A) Representative images of DIV4 primary hippocampal neurons from wild type, Ttll1-/- and Ttll7-/- mice Cells were incubated with MitoTracker® (black dots) and mitochondria movements were recorded in the longest neurite (orange lines).B) Kymographs of one representative experiment representing mitochondrial movements C - F) Analysis of one single experiment shown in (B). Scatter dot plots of (C) run length and (D) speed distributions, where each point represents a single run extracted from the kymographs shown in (B). The black lines indicate the median of the distribution, with whiskers at interquartile range. Numerical values of medians are indicated below each plot. (E) A bar graph showing the overall motility of mitochondria (the fraction of time mitochondria spent in movement relative to the total number of mitochondria during the recording time of 60 s). (F) A bar graph showing mitochondrial density in analysed axons. Values of the overall motilities and mitochondrial densities are given below the bar plots.
Figure 1A) Volcano plot of the fold-change (x-axis) plotted against significance (y-axis) of lipids changing upon HF/HS feeding. Lipids are colored according to fold-change (log2, absolute) > 1 (orange), p-value <0.05 (red) or both (green). Pvalues calculated from differential expression using limmaB) Heatmap of the fold-change (log2) of each lipid in HF/HS compared to chow diet. Only lipid species detected in all mice are shown.C) Examples of different hepatic lipids within one class that are regulated in different directions in HF/HS fed as compared chow fed mice.
Figure 2A) The relative genetic variation of hepatic lipidome composition; all lipids were quantified in proportion to the total lipidome. Each lipid class is shown in a different color where differences can be observed across the strains.B) Heatmap showing correlations between different lipid species (x-axis) and the abundance of gut microbes (y-axis). Microbes were summarized at the levels of order (o_), genus (g_) or family (f_). *=p<0.05, **=p<0.01 Pvalues were calculated based on significance of regression (students test) and adjusted for multiple comparisons (FDR = 0.05).
Figure 3A) Heatmap showing correlations among hepatic lipids.B) Heatmap showing concordance between different lipid species (class listed on y-axis) and certain phenotypic traits on the x-axis.
Figure 4A) Manhattan plot of genome-wide association for the levels of hepatic Pex16 transcript, where the only significant locus appears directly surrounding the genomic location (red arrow). Significant cut-offs are shown for FDR (blue) and Bonferroni (red). The peak SNP (rs27364570) is highlighted with a dark red box. Y-axis shows the -log10(pvalue) vs x-axis showing each SNP measured. P-values for GWAS associations were calculated using FaST-LMM.B) Allelic distribution comparing GG vs TT (x-axis) for the peak SNP of the Pex16 association (rs27364570), where the abundance of each LPC species (y-axis) showed significantly different levels depending on the allele. P-values for GWAS associations were calculated using FaST-LMM. Boxplots show mean (middle line), 25-75% quantiles (colored box) and 5-95% quantiles (vertical lines)C) Correlations between expression of hepatic Pex16 with LPC species and phenotypic traits. Box color indicates bicor value, where all relationships are positive and number in each box shows p-value for each correlation. Pvalues were calculated based on significance of regression (students test) and adjusted for multiple comparisons (FDR = 0.05)
Figure 5A) Manhattan plot of genome-wide association for TAG(48:2). Red line shows Bonferroni-corrected threshold of significance calculated based on FaST-LMM pvalues.B) Locuszoom plots showing the focused genomic region (x-axis) plotted against the -log10 (p-value) of association for liver mRNA expression of Map2k6. P-values for GWAS associations were calculated using FaST-LMM(C) Correlations between hepatic expression of Map2k6 and all TAGs identified in the study. Blue represents negative correlations.E) Western blots of liver homogenate using anti-Map2k6 and anti-β-actin.F) Comparison of total hepatic TAG between Map2k6-overexpressing mice (black bar) and control mice (empty bar).G) Differences in total phospholipid (PC), total cholesterol (TC) and unesterified cholesterol (UC) between Map2k6-overexpressing mice (black bars) and control mice (empty bars).H) Body fat % of both experimental cohorts prior to (week 0) or 8 weeks on a HF/HS diet.I,J) Plasma concentration of glucose (I) and insulin (J) at the end of the 8 weeks study.
Figure 6A) Manhattan plot of genome-wide association for expression of Ifi203 in liver. Red line shows Bonferroni-corrected threshold and blue shows an FDR =0.01 pvalue of significance calculated based on FaST-LMM pvalues.B-D) Allelic variation plots showing the peak SNP for Ifi203 expression (rs31614030) at the CC or TT allele (x-axis) plotted against expression of Ifi203 (B), levels of hepatic PC(38:3) (C), and plasma insulin levels (D). Red line shows Bonferroni-corrected threshold and blue shows an FDR =0.01 pvalue of significance calculated based on FaST-LMM pvalues.E-G) Correlation between the parameters listed above showing significant relationships between hepatic Ifi203 and PC(38:3) (E), hepatic Ifi203 and plasma insulin levels (F) or PC (38:3) and plasma insulin (G). Pvalues were calculated based on significance of regression (students test) and adjusted for multiple comparisons (FDR = 0.05)Mice receiving the control virus (open bars) or shIfi203 (black bars) were analyzed for liver expression of Ifi203 (I), total PC levels in liver (J) P-values calculated using a students t-test between groups. Data represent means ± SEM (n =4-5 per group).Mice receiving the control virus (open bars) or shIfi203 (black bars) were analyzed for liver expression of Pemt (K), total liver TAG content (L), or plasma insulin levels (M). P-values calculated using a students t-test between groups. Data represent means ± SEM (n =4-5 per group).
Figure 5A. Fluorescence micrographs of two different COPI•COPII BiFC pairs are shown: Sec16pVN•α-COPVC or Sec16pVN•ε-COPVC. DSL1 cells show dispersed punctate patterns resembling ER exit site patterns. In the dsl1-5xWA mutant, cells exhibit additional bright spots. Scale bar 10 µm.B. Quantification of fluorescence intensity differences of the strains shown in (A). COPI•COPII BiFC fluorescence is significantly increased in cells carrying the dsl1-5xWA mutant as compared to DSL1 wild type. Mean values + SEM of at least nine independent experiments (n=918) are displayed (two-sample unpaired twotailed ttest).C. Time-lapse micrographs of cells expressing the β'-COPVN•Dsl3pVC BiFC pair in a dsl1-5xWA background. Cells were imaged embedded in agarose at RT with fresh PM Glc+ura medium supply throughout one budding cycle. Fluorescence signals retain the characteristic polarization pattern as seen DSL1 wild type cells (see Fig 3). Scale bar 10 μm.