Mitochondrial biogenesis is transcriptionally repressed in lysosomal lipid storage diseases
- University Medical Center Goettingen, Germany
- International Max-Planck Research School in Neuroscience, Germany
- Friedrich-Alexander University Erlangen-Nürnberg (FAU), Germany
- University of Helsinki, Biomedicum Helsinki, Finland
Figures
Figure 1 with 2 supplements
Mitochondrial genes are down-regulated in brain and liver of symptomatic NPC1 KO mice.
(a) Schematic representation of the in silico approach. The list of mitochondria-related genes was built by converting the MitoCarta proteome inventory into a transcript list. This was then crossed …
Figure 1—figure supplement 1
Behaviour of organelle-specific gene lists in NPC1 KO tissues.
(a) The average expression of 435 lysosomal genes was higher both in brain and liver of NPC1 KO, even in asymptomatic mice, and increased further upon disease onset. (b) Expression of 86 …
Figure 1—figure supplement 2
Disease progression changes in the expression of mitochondria-related genes in brain and liver of NPC1 KO mice.
Of the 1049 mitochondria-related genes analyzed, we determined how many of them were in the differentially-expressed gene (DEG) list of NPC1 KO mice brain and liver. The dot plots show all …
Figure 2
Impaired mitochondrial biogenesis and function in mouse and cellular models of Niemann-Pick disease.
The transcript levels of several nuclear-encoded and mitochondrial DNA (mtDNA)-encoded mitochondria-related genes were measured. (a) transcript levels of mitochondria-related genes are decreased in …
Figure 3 with 4 supplements
Mitochondrial function and mitochondrial mass are impaired in acid sphingomyelinase (ASM)- and NPC1-deficient patient fibroblasts.
(a,c) ASM- and NPC1-deficient fibroblasts have substantially lower O2 Consumption Rate (OCR) than controls. OCR was measured using whole cells, sequentially in basal conditions (complete medium), …
Figure 3—figure supplement 1
Validation of lysosomal defects in patient fibroblasts and causal relationship of defects to mitochondrial biogenesis and function.
The ASM-1 patient cells used in this manuscript have 5% left of acid sphingomyelinase activity, and present the expected signs of lysosomal impairment, specifically (a) decreased autophagic capacity …
Figure 3—figure supplement 2
Increased content of dysfunctional mitochondria in ASM-deficient and NPC fibroblasts.
(a) Mitochondrial mass was assessed by flow cytometry with MitoTracker Green staining. Histogram plot shows a right shift in intensity of MitoTracker Green dye in ASM and NPC patient cells relative …
Figure 3—figure supplement 3
Mitochondrial deficits in control fibroblasts treated with desipramine 40 µM for 72 hr (inhibitor of acid sphingomyelinase).
(a) Transcript levels of mitochondrial genes depicted as mean ± s.e.m., n = 3. T-test p-values *p<0.05 **p<0.01 ***p<0.001. Sterile distilled water used as vehicle control. (b) Increased …
Figure 3—figure supplement 4
Mitochondrial deficits in control fibroblasts treated with U18666A 10 µM for 72 hr (inhibitor of NPC1).
(a) Mitochondrial gene expression levels in U1866A treated cells shown as mean ± s.e.m., n = 3. T-test p-values *p<0.05 **p<0.01 ***p<0.001. DMSO used as vehicle control. (b) Increased mitochondrial …
Figure 4 with 5 supplements
Transcription factors Etv1 and Klf2 are induced in Niemann-Pick and involved in the regulation of mitochondrial biogenesis.
(a) Venn diagram illustrating the intersection between the list of transcription factors (TFs) that are significantly activated or repressed in tissues of Npc1-/- mice, and the list of TFs that are …
Figure 4—figure supplement 1
Etv1 and Klf2 levels in ASM-deficient and NPC fibroblasts.
(a) Transcript levels of ETS family members ELK1 and ETV1 in ASM-deficient fibroblasts, measured by qPCR. Plots represent mean ± s.e.m., n = 3. (b) Transcript levels of KLF2 in ASM-deficient …
Figure 4—figure supplement 2
Etv1 and Klf2 levels in desipramine-treated fibroblasts.
(a) KLF2 and ETV1 protein levels are increased and mitochondrial protein TFAM is reduced in desipramine-treated fibroblasts, as assessed by western blot. (b) Quantification of the western blot shows …
Figure 4—figure supplement 3
Increased expression of mitochondria-related genes in the absence of KLF2.
Using a publicly-available transcriptional dataset of erythroid cells obtained from KLF2 KO (n = 4) and WT (n = 4) mice, we found that the average expression of ~1000 mitochondria-related genes is …
Figure 4—figure supplement 4
Targets of KLF2 as determined by ChIP-Seq data analysis.
Graphic representation of publicly available KLF2 ChIP-Seq data, highlighting its target genes to include several mitochondrial genes. KLF2 target genes from the ChIP-Seq data were crossed with the …
Figure 4—figure supplement 5
Targets of ETV1 as determined by ChIP-ChIP data analysis.
Analysis of publicly available ETV1 ChIP-ChIP dataset with the same approach described in Figure 4—figure supplement 4 above, shows several mitochondrial genes as targets of ETV1. Schematic …
Figure 5 with 1 supplement
Silencing of ETV1 or KLF2 rescues mitochondrial biogenesis and function in Niemann-Pick fibroblasts.
Using siRNA-mediated silencing, we knocked-down Etv1 (a) or Klf2 (b) in ASM1-deficient fibroblasts, which brought the protein levels of mitochondrial protein TFAM, and of mitochondrial biogenesis …
Figure 5—figure supplement 1
Autophagy defects in Niemann Pick patients are independent of Klf2 and Etv1.
To assess if silencing of ETV1 or KLF2 in ASM-deficient fibroblasts had any impact on lysosomal function, we assessed the accumulation of autophagic substrates p62 (Sqstm1) and LC3BII, a marker of …
Figure 6
ETV1 up-regulation is dependent on KLF2 and ERK.
(a) Silencing of KLF2 in ASM-deficient fibroblasts results in reduced levels of ETV1, shown by a representative western blot of whole cell extracts, with quantification of band densities (mean ± s.e.…
Figure 7 with 1 supplement
Dynamic regulation of S1PR1 activity impacts mitochondrial biogenesis and function.
(a) Transcript levels of mitochondrial-related genes increase upon activation of S1PR1 with the agonist Sew2871 (5 µM, 16 hr; DMSO as vehicle control), as measured by qPCR. Plots show mean ± s.e.m., …
Figure 7—figure supplement 1
Transcript levels of sphingosine-1-phosphate receptor 1 (S1PR1) and sphingosine kinases 1 (SPHK1) and 2 (SPHK2) in ASM-deficient fibroblasts.
Transcript levels of S1PR1, SPHK1 and SPHK2 were measured by qPCR and depicted as mean ± s.e.m, n = 3. T-test p-values ***p<0.001.
Figure 8
S1PR1 signaling in Niemann-Pick disease.
(a) Schematic illustration of sphingosine-1-phospate (S1P) signaling. S1P is generated from sphingosine by the kinases SPHK1 (plasma membrane) and SPHK2 (endoplasmic reticulum and mitochondria), and …
Author response image 1
Tables
Table 1
Sources of organelle-specific proteomes.
https://doi.org/10.7554/eLife.39598.002| Dataset | Number of genes | Reference (source) |
|---|---|---|
| Mitochondria | 1049 | (Pagliarini et al., 2008) |
| Respiratory chain subunits | 108 | (Pagliarini et al., 2008) |
| Lysosomes | 435 | (Skon et al., 2013) |
| Peroxisomes | 254 | (Hollenhorst et al., 2007) |
| Endoplasmic reticulum | 297 | (Herzog et al., 2006) |
| Golgi (COP I) Vesicles | 86 | (Dugas et al., 2006) |
Table 2
Transcription factors with statistically enriched cis-elements in the promoters of genes encoding for subunits of mitochondrial respiratory chain.
https://doi.org/10.7554/eLife.39598.012| Transcription factor family | p-value (Fisher's exact test) | Transcription factors |
|---|---|---|
| SP1 | 1.52E-09 | SP1, SP4 |
| E2F | 2.79E-08 | E2F1, E2F2, E2F3, E2F4 |
| KLF | 0.000265 | KLF2, KLF6, KLF7, KLF15 |
| ETS | 0.000796 | ELK1, SPI1, ETV1 |
Additional files
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Supplementary file 1
Results of the promoter analysis of lysosomal genes.
The transcription factor families that passed the significance threshold (Fisher exact test p<0.01) and the respective p-values are indicated.
- https://doi.org/10.7554/eLife.39598.025
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Supplementary file 2
Mitochondrial KLF2 target genes obtained from the analysis of dataset E-MTAB-2365.
These targets represent the common genes between the complete KLF2 target list and the mitochondrial gene list.
- https://doi.org/10.7554/eLife.39598.027
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Transparent reporting form
- https://doi.org/10.7554/eLife.39598.030
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Supplementary file 3
List of the transcription factors predicted to be significantly activated in the liver and brain of NPC1 KO mice compared to WT.
The transcription factors and respective p-value is indicated. Transcription factors labelled in red were found to be significantly involved in both liver and brain of NPC1 KO, and thus selected for further analysis.
- https://doi.org/10.7554/eLife.39598.026
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Supplementary file 4
qPCR primers.
- https://doi.org/10.7554/eLife.39598.028
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Supplementary file 5
siRNA sequences.
- https://doi.org/10.7554/eLife.39598.029
