NFYB-1 regulates mitochondrial function and longevity via lysosomal prosaposin

Abstract

Mitochondria are multidimensional organelles whose activities are essential to cellular vitality and organismal longevity, yet underlying regulatory mechanisms spanning these different levels of organization remain elusive1,2,3,4,5. Here we show that Caenorhabditis elegans nuclear transcription factor Y, beta subunit (NFYB-1), a subunit of the NF-Y transcriptional complex6,7,8, is a crucial regulator of mitochondrial function. Identified in RNA interference (RNAi) screens, NFYB-1 loss leads to perturbed mitochondrial gene expression, reduced oxygen consumption, mitochondrial fragmentation, disruption of mitochondrial stress pathways, decreased mitochondrial cardiolipin levels and abolition of organismal longevity triggered by mitochondrial impairment. Multi-omics analysis reveals that NFYB-1 is a potent repressor of lysosomal prosaposin, a regulator of glycosphingolipid metabolism. Limiting prosaposin expression unexpectedly restores cardiolipin production, mitochondrial function and longevity in the nfyb-1 background. Similarly, cardiolipin supplementation rescues nfyb-1 phenotypes. These findings suggest that the NFYB-1–prosaposin axis coordinates lysosomal to mitochondria signalling via lipid pools to enhance cellular mitochondrial function and organismal health.

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Fig. 1: Mitochondrial regulation during ARD and upon recovery.
Fig. 2: NFYB-1 regulates mitochondrial function and longevity.
Fig. 3: Multi-omics analyses indicate that NFYB-1 regulates organellar functions and stress response.
Fig. 4: Lysosomal SPP-8 rescues mitochondrial function.

Data availability

RNA-seq data have been deposited in Gene Expression Omnibus (GEO) under accession code GSE127917. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013233. Source data for Extended Data Figs. 1, 2 and 4 are presented with the paper.

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Acknowledgements

We thank the CGC (University of Minnesota), A. Trifunovic (University of Cologne), C. Haynes (University of Massachusetts), A. Dillin (University of California, Berkeley) and SunyBiotech for strains. Our proteomics bioinformatic and microscopy cores for assistance (MPI-AGE), T. Vicar and T. Langer (MPI-AGE) for manuscript comments, C. Geisen and C. Calabrese for scientific input, and C. Latza for lipidomics. This work was funded by the Max Planck Society.

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Contributions

A. Antebi and R.G.T. designed experiments and wrote the paper. R.G.T. carried out all experiments. I.A. performed proteomics and multi-omics analysis. A. Annibal performed lipidomic analysis and cardiolipin staining. I.S. constructed and generated spp-8 reporter, and R.L. performed co-localization and LysoTracker stainning experiments. A.L.W. gave technical assistance and B.G. aided with ARD induction and formulated the ARD recovery RNAi screen idea.

Corresponding author

Correspondence to Adam Antebi.

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Peer review information Primary Handling Editor: Pooja Jha.

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Extended data

Extended Data Fig. 1 NFYB-1 regulates mitochondrial function and longevity (Related to Fig. 2).

a, Mini lifespan screen of WT and isp-1(qm150) upon RNAi knockdown of candidate genes from egg on in comparison to luci RNAi control bacteria (N = 1, n = 120 per condition). b, Representative image of mKate2::NFYB-1 nuclear (nuc) localization in various tissues of AL day 1 adults, scale bar = 10 μm. c, Representative immunoblot of mKate2::NFYB-1 levels over age in AL WT (day 1, 3, 5, 9 and 13 as mentioned, n = 200 worms per repeat per condition), with quantitation and histone loading control. d-e, nfyb-1(cu13) mutations leads to a decrease in pcco-1::gfp expression in AL day 1 at 25 °C. d, Representative images of ca. n = 10–20 worms, scale bar = 75 μm, e, pcco-1::gfp expression levels normalised to (Time of Flight) TOF quantitated using biosorter (n ≥ 400). f, nfyb-1(cu13) mutation results in a decrease in relative basal oxygen consumption rate (OCR) compared to WT, and in germline-less glp-4(bn2);nfyb-1(cu13) compared to glp-4(bn2) animals at 25 °C, upon ARD recovery (n = 900 worms). g, Kaplan–Meier survival curves of WT, nfyb-1(cu13) upon ARD recovery shows that nfyb-1 loss leads to decrease in survival (n = 120 per repeat, per condition). h, i, Percentage decrease in mean and median survival of nfyb-1(cu13) in comparison to WT in AL and upon ARD recovery. j, cco-1 mRNA levels are reduced in nfyb-1(cu13). k, mRNA levels of nfyb-1 mitochondrial regulated genes, l, loss of nfyb-1 does not affect mRNA levels of atfs-1, dve-1 and skn-1, j-l quantitation by qPCR at AL day 1 (n ≥ 1000). b-l, All data represent N = 3 independent biological replicates, n = total worms per condition from three replicates unless stated otherwise. Error bar shows mean± s.e.m., statistics determined by c, j, l one-way ANOVA and e, f, k two-sided t-test, ns: not significant, *P < 0.5, **P < 0.01, ***P < 0.001. a, g-i, Two-sided Mantel–Cox log-rank test, refer to Supplementary Table 2 for statistics. d-f, ARD day 10 worm recovered for 1day. Source Data

Extended Data Fig. 2 NFYB-1 partially regulates UPRmt factors and MCSR (Related to Fig. 2).

a–c, nfyb-1(cu13) loss leads to reduction in nuclear localization of pdve-1::dve-1::gfp upon mitochondrial stress induced by exposure to cco-1i (pointed by white arrow), AL day 1 adults a, microscopy images scale bar = 10 μm, b, quantitation of pdve-1::dve-1::gfp expression using biosorter (n ≥ 400) and c, representative immunoblot of pdve-1::dve-1::gfp, with quantitation of DVE-1::GFP and histone loading control (N = 4, n = 100 per repeat, per condition). d, Kaplan–Meier survival curve of isp-1(qm150) and isp-1(qm150); nfyb-1(cu13) shows that dve-1i does not further reduce isp-1;nfyb-1 lifespan (n = 120 per repeat, per condition). e, nfyb-1(cu13) mutation reduces nuclear localization of patfs-1::atfs-1::gfp upon mitochondrial stress induced by hsp-6i, L3-L4 larvae, scale bar = 10 μm (n ~ 30). f, nfyb-1(cu13) shows no significant (ns) effect on publ-5::gfp expression upon exposure to cco-1i or control luci, day 1 adult (n ≥ 400). g, nfyb-1(cu13) loss only partially reduces phsp-6::gfp expression upon cco-1i induced mitochondrial stress, day 1 adult (n ≥ 400). h, i nfyb-1(cu13) reduces phsp-16::gfp expression upon exposure to hsp-6i from day 1 to day 3, showing a requirement for MCSR. h, microscopy images, scale bar = 75 μm, i, quantitation of phsp-16::gfp expression using biosorter (n ≥ 200). j, k, nfyb-1(cu13) leads to a reduction in lipids by BODIPY staining from day 1 to day 3. j, microscopy images, scale bar = 10 μm, k, quantitation of expression using biosorter (n ≥ 200). a-k, under all WT conditions, cco-1i and hsp-6i induce the mitochondrial stress response. b, d, e-g, i, k All data represent N = 3 independent biological replicates, n = total worms per condition from three replicates unless stated otherwise. Error bar shows mean ± s.e.m, b, e-g, i, k statistics determined by one-way ANOVA and c, two-sided t-test. *P < 0.5, **P < 0.01, ***P < 0.001. d, Two-sided Mantel–Cox log-rank test, refer to Supplementary Table 2 for statistics. Source Data

Extended Data Fig. 3 NFYB-1 omics analyses, ER genes only weakly modulate mitochondrial longevity (Related to Fig. 3).

a, Workflow of transcriptomics and proteomics analysis for indicated genotypes on AL day 1 adult worms. Heat maps represent log2-fold change of differentially expressed transcripts and proteins (P < 0.05). b, Averaged PCA (Principal Component Analysis) of most variable differentially expressed genes in transcriptomics and proteomics of day 1 adults for genotypes WT, nfyb-1(cu13), isp-1(qm150) and isp-1(qm150);nfyb-1(cu13), (Omics: transcriptomics N = 3, proteomics N ≥ 5 independent biological replicates. n ≥ 5000 worms per condition per repeat). c, Mean lifespan of isp-1(qm150) and isp-1(qm150);nfyb-1(cu13) upon RNAi knockdown of differentially expressed proteins or luciferase control RNAi (luci) (n = 120 per repeat, per condition), spp-8i shows the most prominent rescue of lifespan. d, nfyb-1(cu13) elevates expression of UPRER marker, phsp-4::gfp, which is dependent on UPRER factors ire-1 and xbp-1, day 1 adult, quantitation by biosorter (n ≥ 400). e, Kaplan–Meier survival curve of isp-1(qm150);nfyb-1(cu13) upon RNAi knockdown of UPRER factors show no significant regulation of lifespan (N = 2 independent biological replicates, n = 120 per repeat, per condition). c, d, All data represent N = 3 independent biological replicates unless stated otherwise, n = total worms per condition from three replicates unless stated otherwise. Error bar shows mean ± s.e.m, d, statistics determined by one-way ANOVA, *P < 0.5, **P < 0.01, ***P < 0.001. c, e, Two-sided Mantel–Cox log-rank test, refer Supplementary Table 2 for statistics.

Extended Data Fig. 4 Lysosomal SPP-8 suppresses NFYB-1 dependent mitochondrial dysfunction (Related to Fig. 4).

a, spp-8i modestly increases WT and nfyb-1(cu13) lifespan (N = 2, n = 120 per repeat, per condition). b, spp-8 mRNA levels are unaffected by nfya-1 and nfyc-1 knockdown AL day 1, quantitated by qPCR (n ≥ 1000). c, skn-1 and dve-1 knockdown diminished elevated spp-8 mRNA in nfyb-(cu13) AL day 1, quantitated by qPCR (n ≥ 1000). d, e, Representative immunoblot of 1 kb pspp-8::NLS::GFP, shows skn-1 dependent regulation of spp-8 in nfyb-1(cu13) compared to AL WT day 1 (n = 100 per repeat, per condition) with quantitation and actin loading control. f, g, spp-8i rescues the reduced mitochondrial circularity and cross-sectional area of nfyb-1(cu13) mutants in AL day 5 (n ~ 30), quantitated47. h, spp-8i has no effect on phsp-4::gfp expression in indicated genotypes, quantitation by biosorter, AL day 1 (n ≥ 400). i, spp-8i has no effect on nfyb-1(cu13) regulated ER genes, quantitated by qPCR, AL day 1 (n ≥ 1000). j, k, Confocal microscopy images show endogenously tagged mKate2::NFYB-1 in intestine, co-localizes with LMP-1::GFP, AL day 1. Percentage colocalization as measured using Manders Colocalizaton Coefficient (Costes significance test was done for colocalization, refer method section for detail), AL day 1, scale bar = 10 μm (n ~ 30). l, nfyb-1(cu13) mutation leads to less acidified lysosomes in comparison WT, stained with LysoTracker Red in AL day 1 (n ~ 30). b-l, All data represent N = 3 independent biological replicates, n = total worms per condition from three replicates unless stated otherwise. Error bar shows mean ± s.e.m, statistics determined by b-c, e-h, one-way ANOVA, i and l, two-sided t-test ns: not significant, *P < 0.5, **P < 0.01, ***P < 0.001. a, Two-sided Mantel–Cox log-rank test, refer Supplementary Table 2 for statistics. Source Data

Extended Data Fig. 5 NFYB-1 regulates specific lipid species via SPP-8 (Related to Fig. 4).

a, Absolute levels of respective ceramides species normalized to protein levels detected by targeted lipidomic analysis of day 1 isp-1(qm150) and isp-1(qm150);nfyb-1(cu13) on control and upon spp-8i in AL day 1 (N = 4, n ≥ 5000 per repeat per condition) (box plot represents line at median, using minimum and maximum of all of the data, error bar shows standard deviation above and below the mean of the data). b, Abundance of exogenously supplemented CER (ceramides) and CL (cardiolipins) as detected by Mass-spectrometry in AL day 1 (N = 1, n ≥ 1000). c, spp8i increases nonyl-acridine orange staining in AL day 1, indicating increased cardiolipin levels, quantitation by biosorter (n ≥ 400). d, Relative fold change in mRNA levels of cardiolipin synthesis genes measured by qPCR in indicated genotypes AL day1 (n ≥ 1000), e, Cardiolipin (CL) synthesis pathway. c-d, All data represent N = 3 independent biological replicates, n = total worms per condition from three replicates unless stated otherwise. Error bar shows mean ± s.e.m, statistics determined by a and c, one-way ANOVA and d, two-sided t-test ns: not significant, *P < 0.5, **P < 0.01, ***P < 0.001. f, NFYB-1 coordinates organellar activities: Transcription factor NFYB-1 promotes nuclear localization of UPRmt factors DVE-1 and ATFS-1, maintains cardiolipin levels and facilitates the mitochondria to cytosolic stress response (MCSR). Concurrently NFYB-1 limits ER associated genes, ER stress and lysosomal prosaposin. This state results in normal mitochondrial function and longevity. nfyb-1 loss (NFYB-1 KO) activates ER stress, partially disrupts UPRmt factors, and MCSR. NFYB-1 activates lysosomal prosaposin/SPP-8, leading to fragmented mitochondria, reduction of oxygen consumption, altered ceramide (CER) and cardiolipin (CL) levels, resulting in abolishment of mitochondrial longevity.

Supplementary information

Supplementary Information

Supplementary Tables 1–7

Reporting Summary

Source data

Source Data Extended Data Fig. 1c

Supplementary Fig 1c: Immunoblots of WT; mKate2::NFYB-1

Source Data Extended Data Fig. 2c

Supplementary Fig. 2c: Immunoblots of pdve-1::DVE-1::GFP

Source Data Extended Data Fig. 4d

Supplementary Fig 4d: Immunoblots of 1 kb pspp-8::NLS::GFP (4X NLS)

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Tharyan, R.G., Annibal, A., Schiffer, I. et al. NFYB-1 regulates mitochondrial function and longevity via lysosomal prosaposin. Nat Metab 2, 387–396 (2020). https://doi.org/10.1038/s42255-020-0200-2

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