Sphingolipid signalling mediates mitochondrial dysfunctions and reduced chronological lifespan in the yeast model of Niemann-Pick type C1

Abstract

The Niemann-Pick type C is a rare metabolic disease with a severe neurodegenerative phenotype characterized by an accumulation of high amounts of lipids (cholesterol and sphingolipids) in the late endosomal/lysosomal network. It is caused by loss-of-function point mutations in either NPC1 or NPC2, which seem to mediate proper intracellular lipid transport through endocytic pathway. In this study, we show that yeast cells lacking Ncr1p, an orthologue of mammalian NPC1, exhibited a higher sensitivity to hydrogen peroxide and a shortened chronological lifespan. These phenotypes were associated with increased levels of oxidative stress markers, decreased levels of antioxidant defences and mitochondrial dysfunctions. Moreover, we report that Ncr1p-deficient cells displayed high levels of long chain bases (LCB), and that Sch9p-phospho-T570 and Sch9p levels increased in ncr1Δ cells through a mechanism regulated by Pkh1p, a LCB-activated protein kinase. Notably, deletion of PKH1 or SCH9 suppressed ncr1Δ phenotypes but downregulation of de novo sphingolipid biosynthesis had no protective effect, suggesting that LCBs accumulation may result from an increased turnover of complex sphingolipids. These results suggest that sphingolipid signalling through Pkh1p-Sch9p mediate mitochondrial dysfunction, oxidative stress sensitivity and shortened chronological lifespan in the yeast model of Niemann-Pick type C disease.

Fig. 1. Role of Ncr1p in hydrogen peroxide resistance

S. cerevisiae BY4741 and ncr1Δ::KanMX4 cells were grown in SC-glucose medium to exponential phase (O.D._600nm=0.6) exposed to 1.5 mM H₂O₂ for 1 hour. A. Cellular viability was measured as the percentage of the colony-forming unit (treated cells vs non-stressed cells). Values are mean ± SD of at least three independent experiments. ***p<0.001, unpaired Student’s t-test. B. Protein carbonylation. Proteins were derivatized with DNPH and slot-blotted into a PVDF membrane. Immunodetection was performed using an anti-DNP antibody. Quantitative analysis of total protein carbonyl content was performed by densitometry using data taken from the same membrane. Values are mean ± SD of at least three independent experiments.****p<0.0001, **p<0.01; Two-way ANOVA and Bonferroni test. C. Lipid peroxidation. Cellular extracts were prepared and TBARS quantification was performed as described in Experimental procedures. Values are mean ± SD of at least three independent experiments. ***p<0.001; Two-way ANOVA and Bonferroni test. D. Intracellular levels of superoxide radicals were analyzed by flow cytometry, using DHE as probe. Values are mean ± SD of at least three independent experiments. ****p<0.0001, **p<0.01; Two-way ANOVA and Bonferroni test.

Fig. 2. Ncr1p deficiency decreases chronological lifespan

S. cerevisiae BY4741 and ncr1Δ::KanMX4 cells were grown in SC-glucose medium at 26 ºC to PDS phase. A. Cells were maintained in the growth medium overtime. Cellular viability was measured at 2 to 3 days intervals and was expressed as % colony forming units (aged vs day 0). Values are mean ± SD of at least three independent experiments. B. Intracellular levels of hydrogen peroxide were analyzed by flow cytometry, using DHR 123 as probe. **p<0.01, unpaired Student’s t-test. C,D. Protein carbonylation and lipid peroxidation were measured as in Fig. 1B,C. Values are mean ± SD of at least three independent experiments. *p<0.05, unpaired Student’s t-test.

Fig. 3. Cytosolic catalase activity, mitochondrial superoxide dismutase activity and glutathione levels are decreased in ncr1Δ mutant cells

S. cerevisiae BY4741 and ncr1Δ::KanMX4 cells were grown in SC-glucose medium to PDS phase. A,B. The activity of superoxide dismutase (SOD) and catalase was measured spectrophotometrically. Values are mean ± SD of at least three independent experiments. **p<0.01, unpaired Student’s t-test. C,D. The activity of superoxide dismutases (Sod1p and Sod2p) or cytosolic catalase (Ctt1p) was assessed in situ after native-PAGE. One representative experiment out of three is shown. E. Total glutathione levels. Reduced (GSH) + oxidized (GSSG) glutathione was measured as described in methods. Values are mean ± SD of at least three independent experiments. **p<0.01, unpaired Student’s t-test.

Fig. 4. Ncr1p deficiency decreases mitochondrial function and dynamics

S. cerevisiae BY4741 and ncr1Δ::KanMX4 cells were grown in SC-glucose medium. A. Oxygen consumption rates were measured in exponential (log) and PDS phase cells. Values are mean ± SD of at least three independent experiments. ****p<0.0001, *p<0.05; Two-way ANOVA and Bonferroni test. B. Cytochrome c oxidase (COX) specific-activity was measured in cells grown to PDS phase. Values are mean ± SD of at least three independent experiments. ****p<0.0001, unpaired Student’s t-test. C. Immunoblot analysis of mitochondrial porin levels in cells grown to PDS phase. For each lane the Por1p signal was normalized to the signal for the Pgk1p internal standard (value shown below each lane). A representative experiment out of three is shown. D. Cells were grown to exponential phase and fivefold serial dilutions were plated in solid medium containing glucose or glycerol as carbon source. One representative experiment out of three is shown. E. Mitochondrial membrane potential was determined by flow cytometry using cells grown to PDS phase, unlabeled (auto fluorescence) or labeled with DiOC₆(3). Representative histograms for each condition are shown in Supplemental Fig. S3. Values are mean ± SD of three independent experiments. ***p<0.001, unpaired Student’s t-test. F. S. cerevisiae BY4741 and ncr1Δ::KanMX4 cells transformed with pYX222-mtDsRed (expressing mitochondrial DsRed) were grown to exponential (log) and PDS phase. Live cells were visualized by fluorescence microscopy. One representative experiment out of three is shown. Scale bar: 5 μm.

Fig. 5. Levels of long-chain sphingoid bases

S. cerevisiae BY4741, ncr1Δ::URA3, sch9Δ and ncr1Δsch9Δ cells were grown in SC-glucose medium to the exponential (log) and post-diauxic shift (PDS) phase. Levels of indicated long chain bases were measured by HPLC-MS/MS. A. PHS - phytosphingosine; B. PHS-1-P – phytosphingosine-1-phosphate; C. DHS– dihydrosphingosine; D. DHS-1-P – dihydrosphingosine-1-phosphate. Data are expressed as pmol of lipid per total cell number (1.9×10⁹) and are mean ± SD of three independent experiments. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05; Two-way ANOVA and Bonferroni test.

Fig. 6. Ncr1p deficient cells exhibit increased levels of Sch9p and Sch9p-phospho-T570 and ncr1Δ phenotypes are suppressed by disruption of PKH1 or SCH9 but not by myriocin

S. cerevisiae BY4741, ncr1Δ::URA3, pkh1Δ, ncr1Δpkh1Δ, sch9Δ and ncr1Δsch9Δ cells were grown in SC-glucose medium to exponential (A and B) or PDS (C and D) phase. A. Immunoblot analysis of Sch9p and P-T570-Sch9p. Pgk1p was used as loading control. . A representative experiment out of three is shown. B,C. Hydrogen peroxide resistance (B) and chronological lifespan (C) were measured as in Fig. 1A and 2A, respectively. Values are mean ± SD of at least three independent experiments. ****p<0.0001, ***p<0.001 (relative to ncr1Δ); Two-way ANOVA and Bonferroni test. D. Analysis of chronological lifespan in S. cerevisiae BY4741 and ncr1Δ::KanMX4 cells treated with ethanol (vehicle) or myriocin (600 ng mL⁻¹; Myr). Values are mean ± SD of at least two independent experiments.

Fig. 7. Role of the LCB→Pkh1p→Sch9p pathway in mitochondrial dysfunction of ncr1Δ cells

S. cerevisiae BY4741, ncr1Δ::URA3, pkh1Δ, ncr1Δpkh1Δ, sch9 and ncr1Δsch9Δ cells were grown in SC-glucose medium. A. Cells were grown to exponential phase and fivefold serial dilutions were plated in solid medium containing glucose or glycerol as carbon source. One representative experiment out of three is shown. B. Oxygen consumption rates were measured in PDS phase cells. Values are mean ± SD of at least three independent experiments. ****p<0.0001 (relative to ncr1Δ); Two-way ANOVA and Bonferroni test. C. Mitochondrial membrane potential was determined by flow cytometry using PDS phase cells, unlabeled (auto fluorescence) or labeled with DiOC₆(3). Values are mean ± SD of three independent experiments. ****p<0.0001, ***p<0.001 (relative to ncr1Δ); Two-way ANOVA and Bonferroni test. D. S. cerevisiae cells transformed with pYX222-mtDsRed (expressing mitochondrial DsRed) were grown to PDS phase. Live cells were visualized by fluorescence microscopy. One representative experiment out of three is shown. Scale bar: 5 μm.