PKC downregulation upon rapamycin treatment attenuates mitochondrial disease

Abstract

Leigh syndrome is a fatal neurometabolic disorder caused by defects in mitochondrial function. Mechanistic target of rapamycin (mTOR) inhibition with rapamycin attenuates disease progression in a mouse model of Leigh syndrome (Ndufs4 knock-out (KO) mouse); however, the mechanism of rescue is unknown. Here we identify protein kinase C (PKC) downregulation as a key event mediating the beneficial effects of rapamycin treatment of Ndufs4 KO mice. Assessing the impact of rapamycin on the brain proteome and phosphoproteome of Ndufs4 KO mice, we find that rapamycin restores mitochondrial protein levels, inhibits signalling through both mTOR complexes and reduces the abundance and activity of multiple PKC isoforms. Administration of PKC inhibitors increases survival, delays neurological deficits, prevents hair loss and decreases inflammation in Ndufs4 KO mice. Thus, PKC may be a viable therapeutic target for treating severe mitochondrial disease.

Extended Data Fig. 1. Proteome and phosphoproteome analysis statistics.

a, Number of total (all), mitochondrial (mitoc) and Complex I (C-I) proteins quantified (gray) and those with significant changes among WT, KO, and KR experimental groups (ANOVA test, FDR q-value < 0.05) (red). Mitochondrial proteins annotations were extracted from mouse MitoCarta 2.0 database. b, Correlations of log₂ transformed LFQ-normalized protein abundance measurements between samples (N = 6–7 mice). c, Same as in (a) but for phosphorylation sites. d, Correlations of log₂ transformed median-normalized phosphorylation site intensity values between samples and replicates. Two technical replicates of IMAC phosphopeptide enrichment were performed for each brain sample to increase phosphoproteome coverage (N = 12–14 samples; 6–7 mice and duplicated IMAC enrichment and LC-MS/MS analysis). e, Distribution of Pearson’s r correlation values among all samples and only for technical replicates. Box plots include the median line, the box denotes the interquartile range (IQR), whiskers denote ±1.5 × IQR. f, PCA analysis of log₂ transformed median-normalized phosphorylation site intensities data (hollow/solid symbols indicates female/male samples respectively).

Extended Data Fig. 2. Global changes in respiratory chain related proteins.

a,b, Aggregated protein abundance changes in respiratory chain complexes (a) and respiratory chain assembly proteins (b). Box plots include the median line, the box denotes the interquartile range (IQR), whiskers denote ±1.5 × IQR. Sum of relative iBAQ intensities for all members of each complex or protein group were used (N = 6–7 mice). T-test significance p-values are indicated (* p < 0.05; ** p < 0.01; *** p < 0.001).

Extended Data Fig. 3. Western blot analysis of brain extracts from P30 and P50 mice treated with vehicle or rapamycin from P10 to P30 or P50.

a,b, Western blot analysis of mTORC1 and mTORC2 markers of brain lysates from P30 wild-type (WT) and Ndufs4 KO mice treated daily with vehicle (KO) or rapamycin (KR) from P10 to P30. c, Densitometry (relative to actin) of western blot data from (a) and (b) normalized to wild-type levels (N = 6 mice). d,e, Western blot analysis of PKC isoforms of brain lysates from P30 wild-type (WT) and Ndufs4 KO mice treated daily with vehicle (KO) or rapamycin (KR) from P10 to P30. f, Densitometry (relative to actin) of western blot data from (d) and (e) normalized to wild-type levels (N = 6 mice). g,h, Representative WB images and densitometry (relative to actin) normalized to wild-type (WT) levels showing relative phosphorylated and total levels of proteins involved in mTORC1 and mTORC2 (N = 4 mice). i,j, Representative WB images and densitometry (relative to actin) normalized to WT levels showing relative phosphorylated and total levels of PKC proteins (N = 4 mice). Each lane corresponds to a brain lysate from a single mouse. T-test significance p-values are indicated (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Extended Data Fig. 4. Rapamycin exerts similar effects in the brains of wild-type mice to Ndufs4 KO mice.

a, Experimental design to evaluate rapamycin-mediated effects in the brain of wild-type mice. b, Body weight gain in mice from the two experimental groups (mean ± s.d.; N = 4–6 mice). c, Total brain weight at the end of the experimental trial (N = 4–6 mice). T-test significance p-values are indicated (* p < 0.05). Box plots include the median line, the box denotes the interquartile range (IQR), whiskers denote ±1.5 × IQR. d, Comparison of rapamycin mediated changes between wild-type and knock-out mice in individual protein levels. Pearson’s correlation values are indicated. e, 2D-enrichment analysis of GO and KEGG terms comparing the effect of rapamycin between wild-type and knock-out mice in the proteome (Wilcoxon-Mann-Whitney test, FDR q-value < 0.05). f, Comparison of rapamycin mediated changes between wild-type and knock-out mice in phosphorylation sites. Pearson’s correlation values are indicated. g, 2D-enrichment analysis of GO and KEGG terms comparing the effect of rapamycin between wild-type and knock-out mice in the phosphoproteome (Wilcoxon-Mann-Whitney test, p-value < 0.01). In 2D enrichment analysis (panels (e) and (f)) most data points are close to the diagonal dashed line (i.e. identity function), indicating no differences in the effect of rapamycin on wild-type and Ndufs4 KO mice.

Extended Data Fig. 5. Correlation between rapamycin effects in Ndufs4 KO mice at the proteomic (x axis) and phosphoproteomic (y axis) levels.

Pearson’s r coefficient and goodness-of-fit test p-value of linear curve fitted line (dashed line) are indicated.

Extended Data Fig. 6. Phosphorylation changes in brain proteins of Nfus4 KO mice upon rapamycin treatment.

a, Phosphorylation sites on proteins of the mTOR complexes and associated substrates that show significant changes among experimental groups (WT: wild-type; KO: Ndufs4 KO; KR: rapamycin-treated Ndufs4 KO). b, Average (mean ± s.e.m.) changes in phosphorylation of kinase substrates upon rapamycin treatment in Ndufs4 KO mice brain. Each dot represents an individual phosphorylation site substrate. Only kinases with more than 9 substrates found are shown. c, Significant changes in phosphorylation on activity regulatory sites of specific kinases (*activation loop sites, ^#inhibitory sites). d, Significant changes in activating phosphorylation sites of the main two calcium-release channels from the endoplasmic reticulum. All box plots include the median line, the box denotes the interquartile range (IQR), whiskers denote ±1.5 × IQR. T-test significance p-values are indicated (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; N = 12–14 samples; 6–7 mice and duplicated IMAC enrichment and LC-MS/MS analysis, if no missing values are found).

Extended Data Fig. 7. Treatment of Ndufs4 KO mice with PKC inhibitors largely prevents the alopecia phenotype at weaning (~P21).

a, Wild-type mice at weaning show no hair loss. b, Untreated Ndufs4 KO mice normally exhibit alopecia (i.e. hair loss) at 21-days old due to a TLR2/4 innate immune response. In contrast, minimal hair loss was observed in 21-day old Ndufs4 KO mice treated with c,d, GF109203X and ruboxistaurin from P10 to P21. e, Some hair loss was observed in 21-day old Ndufs4 KO mice treated with rapamycin from P10 to P21.

Extended Data Fig. 8. Histological analysis of skin pathology at P21 or P30.

a, Representative images (20X zoom) of H&E staining of skin sections of P21 WT and Ndufs4 KO mice treated with vehicle, rapamycin, or ruboxistaurin from P10 to P21. Each picture corresponds to an image from an individual mouse. b, Blinded skin inflammation pathology scores from H&E staining of skin sections of P21 WT and Ndufs4 KO mice treated with vehicle, rapamycin, or ruboxistaurin from P10 to P21. Increasing scores represent increasing severity of pathology. c, Blinded hair follicle pathology scores from H&E staining of skin sections of P21 WT and Ndufs4 KO mice treated with vehicle, rapamycin, or ruboxistaurin from P10 to P21. d, Representative images (20X zoom) of H&E staining of skin sections of P30 WT and Ndufs4 KO mice treated with vehicle, rapamycin, or ruboxistaurin from P10 to P30. Each picture corresponds to an image from an individual mouse. e, Blinded skin inflammation pathology scores from H&E staining of skin sections of P30 WT and Ndufs4 KO mice treated with vehicle, rapamycin, or ruboxistaurin from P10 to P30. Increasing scores represent increasing severity of pathology. f, Blinded hair follicle pathology scores from H&E staining of skin sections of P30 WT and Ndufs4 KO mice treated with vehicle, rapamycin, or ruboxistaurin from P10 to P30. Increasing scores represent increasing severity of pathology. Each point represents the score for an individual mouse. T-test significance p-values are indicated (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; N = 6 mice). g, Cytokine levels in mouse skin (N = 3–6 mice) of P30 WT and Ndufs4 KO mice treated with vehicle, rapamycin, or ruboxistaurin from P10 to P30. Cytokine levels were measured using a cytokine array and z-score normalized. Left, violin plot of combined z-score normalized values of median abundance of individual cytokines (each point represents an individual cytokine and the dashed line indicates median z-score value for each group; N = 14). Zero mean score is indicated by a dotted line. Right, individualized information in a heatmap. T-test significance p-values are indicated for the different treatments compared to their respective wild-type or knock-out mice values (^ p < 0.1; * p < 0.05; ** p < 0.01; N = 3–6 mice).

Extended Data Fig. 9. Untreated and vehicle-treated Ndufs4 KO mice exhibit similar symptoms of disease.

a, Vehicle treatment does not alter the lifespan of Ndufs4 KO mice compared to untreated controls. Untreated vs. vehicle p-value = 0.7898, log-rank. b, Vehicle treatment does not alter the onset of clasping of Ndufs4 KO mice compared to untreated controls. c, Vehicle treatment does not alter weight gain of Ndufs4 KO mice compared to untreated controls (mean ± s.d). N = 8 mice for vehicle and N = 11 mice for untreated controls in all plots.

Fig. 1:. Rapamycin remodels the brain proteome in Ndufs4 deficient mice.

a, Experimental design (N = 6 mice for the wild-type (WT) and Ndufs4 KO (KO) groups, N = 7 mice for the rapamycin-treated Ndufs4 KO (KR) group). b, Mice body weight from the three experimental groups (mean ± s.d.). Inner box plot shows total brain weight at the end of the experimental trial (30 days) (t-test: ** p < 0.01; *** p < 0.001; N = 6–7 mice). Box plots include the median line, the box denotes the interquartile range (IQR), whiskers denote ±1.5 × IQR. c, PCA analysis of log₂ transformed normalized protein abundance data (hollow/solid symbols indicates female/male samples respectively). Side graphs indicate significantly enriched GO slim terms in the loadings for each component. d,e, Volcano plots comparing protein abundance in KO vs. WT groups (genotype effect; N = 6 mice) at the level of individual proteins (d) or representative biological terms (e). f, Mapping of protein abundance differences in individual subunits of the respiratory chain between the KO and WT groups. Assembly subunits of each mitochondrial respiratory complex are displayed at the bottom of each plot. LFQ normalized abundance data was used, except for subunits with asterisk in which intensity iBAQ values were used. Subunits with significant changes are in bold (p-value < 0.05) and underlined if FDR q-value < 0.05. In grey are subunits with no abundance information. g,h, Volcano plots comparing protein abundance in rapamycin-treated KR vs. KO groups (rapamycin effect; N = 6–7 mice) at the level of individual proteins (g) or representative biological terms (h). In (d) and (g), dotted lines indicate cut-off for significant changes: t-test FDR q-value < 0.05 and artificial within groups variance S0 = 0.1). (e) and (h) show significantly enriched annotation terms from 1D-enrichment analysis of log₂ differences between groups (Wilcoxon-Mann-Whitney test, FDR q-value < 0.05); dot size is proportional to the number of proteins within that term. i, Same as in (f) but between KR and KO groups.

Fig. 2:. Rapamycin restores the abundance level of several mitochondrial proteins in Ndufs4 KO mice.

a, Heat map and hierarchical clustering of proteins with significant changes in abundance (ANOVA test, FDR q-value < 0.05; N = 6–7 mice). b, Significantly enriched GO terms from main clusters of panel (a) (hypergeometric test, p-value < 0.001). c,d, Scatter plot of individual protein abundances (c) and enriched GO terms (d) showing significant changes (t-test for proteins and Wilcoxon-Mann-Whitney test for GO terms, FDR q-value < 0.05) between KO vs. WT and KR vs. KO groups, in the same direction (i.e. rapamycin enhances Ndufs4 KO changes) or in opposite directions (i.e. rapamycin rescues Ndufs4 KO changes).

Fig. 3:. Rapamycin reduces PKC activity in Ndufs4 KO mouse brain.

a, Volcano plot comparing the abundance of protein kinases between KO and WT groups (genotype effect). Kinases with significant changes are colored and labeled (t-test FDR q-value < 0.05; N = 6–7 mice). b, Volcano plot comparing phosphosite abundance differences between KO and WT groups (genotype effect; N = 12 samples; 6 mice and duplicated IMAC enrichment and LC-MS/MS analysis). Dotted lines indicate cut-off for significant changes: t-test FDR q-value < 0.05 and artificial within groups variance S0 = 0.1). c,d, Same as in (a) and (b) respectively, but comparing differences between KR and KO groups (rapamycin effect; N = 12–14 samples; 6–7 mice and duplicated IMAC enrichment and LC-MS/MS analysis). e, Enriched kinase motifs in significantly changing phosphosites (Fisher test, FDR q-value < 0.05). Inner graph shows overrepresented linear sequence motifs. f, Average rapamycin-induced changes in phosphorylation of kinases. Dot size is proportional to the number of phosphosites considered for each kinase. Only kinases with n ≥ 3 sites are shown. g, Kinase Substrate Enrichment Analysis (KSEA) showing kinases with significant changes in activity upon rapamycin treatment (Kolmogorov–Smirnov test, p-value < 0.05). h, Mapping of protein abundance changes in proteins involved in intracellular calcium homeostasis between the Ndufs4 KO mice treated (KR) and untreated (KO) with rapamycin. Only significant changes are colored (t-test p-value > 0.05; N = 12–14 samples; 6–7 mice and duplicated IMAC enrichment and LC-MS/MS analysis), non-quantified proteins are shown in grey.

Fig. 4:. Rapamycin attenuates inflammation in Ndufs4 KO mice brain.

a, Volcano plot showing significantly enriched signaling pathways from 1D-enrichment analysis of phosphorylation changes between KR and KO groups (Wilcoxon-Mann-Whitney test, p-value < 0.05). Relative enrichment at the protein level was used to prevent overrepresentation of multiple sites from the same protein. Dot size is proportional to the number of proteins for each annotated term. b, Cytokine abundance changes in the brain. Cytokine classification according to the Uniprot keyword “Cytokine”. Left, violin plot of combined z-score normalized values of median abundance of individual cytokines (each point represents an individual cytokine and the dashed line indicates median z-score value for each group; N = 10). Zero mean score is indicated by a dotted line. Right, individualized information in a heatmap. LFQ normalized abundance data was used, except for cytokines in italics where iBAQ values were used (N = 6–7 mice). Significant changes are indicated for the comparison between KR and KO groups. c, Western blot analysis of proteins in the NF-κB pathway using brain extracts from P30 wild-type (WT) and Ndufs4 KO mice treated daily with vehicle (KO) or rapamycin (KR) from P10 to P30. Each lane corresponds to a brain lysate from a single mouse. d, Densitometry of western blot from (c) (relative to actin) normalized to WT levels (N = 6 mice). e, Western blot analysis of proteins in the NF-κB pathway using brain extracts from P50 male wild-type mice treated daily with vehicle (WT) or rapamycin (WR) and Ndufs4 KO mice treated daily with vehicle (KO) or rapamycin (KR) from P10 to P50. Each lane corresponds to a brain lysate from a single mouse. f, Densitometry of western blot from (e) (relative to actin) normalized to WT levels (N = 4 mice). T-test significance p-values are indicated (^ p < 0.1; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Fig. 5:. Inhibition of PKC signaling improves survival of mice with complex I deficiency.

a, Representative images of untreated, PKC inhibitor-treated, and rapamycin-treated Ndufs4 KO mice at postnatal day 21 (P21). WT mice shown for reference. b, Representative images (40X zoom) of H&E staining of skin sections from ~P21 WT and Ndufs4 KO mice treated with vehicle, rapamycin (Rapa), or ruboxistaurin (Rubox) from P10 until ~P21. Yellow arrows represent abnormal features. c, Survival of Ndufs4 KO mice treated with pan-PKC inhibitors GO6983 (N = 8 mice) and GF109203X (N = 8 mice), PKC-β inhibitor ruboxistaurin (N = 11 mice), and untreated control (N = 12 mice). Untreated vs. GO6983 p = 0.0003, untreated vs. GF109203X p = 0.0004, untreated vs. ruboxistaurin p = 0.0003, log-rank test. d, Day of first observed clasping phenotype as a sign of neurodegeneration. **** p < 0.0001, one-way ANOVA with post-hoc Tukey test. e, Weights of Ndufs4 KO mice treated with vehicle or PKC inhibitors. Gray shading represents the standard deviation. f, Western blot analysis of proteins involved in the NF-κB pathway and the neuroinflammatory marker GFAP using brain extracts from P50 WT and Ndufs4 KO male mice treated daily with either vehicle or the PKC-β inhibitor ruboxistaurin from P10 until P50. Each lane corresponds to a brain lysate from a single mouse. g, Densitometry of western blot from (f) (relative to actin) normalized to WT levels (N = 3 mice). T-test significance p-values are indicated (* p < 0.05).