Targeting phospholipase PLAG-15 promotes healthy aging in C. elegans via lysosomal-related genes

Abstract

Complex lipid metabolism plays a crucial role in regulating aging. We recently discovered that the phospholipid bis(monoacylglycero)phosphate (BMP) increases in aged human muscles and many mouse tissues. The phospholipase PLA2G15 is reportedly involved in BMP synthesis, however, its specific role in aging remains unknown. To elucidate the role of PLA2G15 in aging, we used Caenorhabditis elegans as a model. When silencing plag-15, the predicted worm orthologue of PLA2G15, we observed improved healthspan and lifespan extension. Semi-targeted lipidomics highlighted that instead of changes related to BMP, plag-15 RNAi led to lower levels of lysophosphatidic acid, lysophosphatidylcholine, and lysophosphatidylethanolamine. Transcriptome-guided epistasis experiments identified that the lifespan extension of plag-15 RNAi worms is regulated by transcription factors hlh-30 and elt-3, and lysosomal vitamin B12 transporter pmp-5 (human TFEB, GATA, and ABCD4 respectively). Overall, we conclude that targeting phospholipid remodeling through plag-15 could be a promising strategy to promote healthy aging.

Keywords: Lipidomics; Molecular physiology; Transcriptomics.

Graphical abstract

Figure 1

Knockdown of plag-15 results in increased lifespan and healthspan, without changes in lysosomes (A) plag-15 RNAi worms have an increased lifespan. plag-15 RNAi consists of 50% plag-15 RNAi and 50% empty vector (EV) control RNAi. Statistical analysis was performed by log rank test, see Table S1 for lifespan statistics. (B) Knockdown of plag-15 (n = 59) promotes the mobility of the day 6 adult worms compared to the control worms (n = 34). Statistical analysis was performed by t-test, error bars denote the quartiles. (C) Body size of plag-15 in arbitrary units (AU). No differences were found in worm size between EV (n = 11) and plag-15 RNAi (n = 10) worms. Statistical analysis was performed by t-test, error bars denote standard deviation. (D) Confocal images of plag-15 knockdown worms and control. Lysosensor is shown in green and lysotracker in red. No differences were found in lysosomal mass. Scale bar = 40 μm. (E) Quantification of lysosomal imaging showed no differences in lysosomal mass in control (n = 5) versus plag-15 (n = 4). See Table S1 for lifespan statistics. ∗∗∗∗p < 0.0001. Data are presented as mean ± SD.

Figure 2

Lipidome of plag-15 RNAi worms show decreased lysophosphatidyl acid (LPA) lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) compared to control worms at the L4 stage (A) Volcano plot of L4 worms, plag-15 versus empty vector (EV). Blue is the lipids that are decreased in abundance in plag-15 RNAi (271 lipid species), while red are the lipids that are increased upon plag-15 RNAi (27 lipid species). (threshold: p-value <0.05). (B) The schematic overview of the phospholipid pathway. Glycerol-3P: Glycerol 3-phosphate; 1-acyl-G3P: 1-acylglycerol-phosphate; DG: diacylglycerol; CDP-DG: cytidine diphosphate diacylglycerol; PS: Phosphatidylserine; LPS: lysophosphatidylserine; PC: phosphatidylcholine; LPA: lysophosphatidic acid; LPC: lysophosphatidylcholine; PE: phosphatidylethanolamine; LPE: lysophosphatidylethanolamine; PA: phosphatidic acid; PI: phosphatidylinositol; LPI: lysophosphatidylinositol; PG; phosphatidylglycerol: CL; cardiolipin: MLCL; monolysocardiolipin: LPG; lysophosphatidylglycerol: BMP; bis(monoacylglycero)phosphate. (C–I) Total PGs (C), total LPGs (D), total BMPs (E), total LPAs (F), total LPCs (G), heatmap of LPC species (H), total LPE (I) in L4 plag-15 RNAi worms compared to EV worms. Statistical comparison determined by using the unpaired t-test was ∗: p < 0.05; ∗∗: p < 0.01. N = 5 for each group. Data are presented as mean ± SD.

Figure 3

RNA-seq of plag-15 knockdown worms showed an increased defense response and upregulation of lysosomal genes (A) Principal Component Analysis (PCA) plot of the transcriptome showing group separation based on the lipidome in plag-15 RNAi versus empty vector (EV) control. (B) Volcano plot of the RNAseq results, (threshold non-adjusted p-value: p < 0.01). (C and D) Pathway enrichment analysis of upregulated (C) and downregulated (D) transcripts (non-adjusted p-value: p < 0.01) in plag-15 RNAi worms compared to the controls. (E) Transcription factor enrichment analysis of the up-/down-regulated transcripts. (F) Table of candidate genes including the transcription factors (TFs) that show increased transcription and lysosomal (lyso) genes that are upregulated in plag-15 RNAi worms. N = 4 for each group. Data are presented as mean ± SD.

Figure 4

Lifespan screen of candidate genes from the RNAseq data showed that lifespan extension of plag-15 RNAi is dependent on several genes (A–C) Lifespan curves of the transcription factor enrichment analysis from RNA-seq data of the plag-15 RNAi worms. (A) Lifespan curve of EV, plag-15, hlh-30 and plag-15/hlh-30 RNAi. Lifespan extension of plag-15 RNAi is dependent on hlh-30. (B) Lifespan curve of EV, plag-15, elt-3 and plag-15/elt-3 RNAi. Lifespan extension of plag-15 RNAi is dependent on elt-3. (C) Lifespan curve of EV, plag-15, efl-1 and plag-15/efl-1. Efl-1/plag-15 RNAi still extends the lifespan compared to efl-1 RNAi only. Therefore, plag-15 RNAi is not dependent on transcription factor efl-1. (D–G) Lifespan curves of the lysosomal genes that emerged from the plag-15 RNAi RNA-seq data. (D) Lifespan curve of EV, plag-15, pmp-5 and plag-15/pmp-5 RNAi. Lifespan extension of plag-15 RNAi is dependent on pmp-5. (E) Lifespan curve of EV, plag-15, spin-3 and plag-15/spin-3 RNAi. Spin-3/plag-15 RNAi still extends the lifespan compared to spin-3 RNAi only. Therefore, plag-15 RNAi is not dependent on transcription factor spin-3. (F) Lifespan curve of EV, plag-15, mrp-4 and plag-15/mrp-4. Mrp-4/plag-15 RNAi still extends the lifespan compared to mrp-4 RNAi only. Therefore, plag-15 RNAi is not dependent on transcription factor mrp-4. (G) Lifespan extension of plag-15 is dependent on mfsd-8. Mfsd-8/plag-15 RNAi still extends the lifespan compared to mfsd-8 RNAi only. Therefore, plag-15 RNAi is not dependent on transcription factor mfsd-8. Statistical comparison determined by using the log rank test was ∗p < 0.05 ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. See Table S1 for lifespan statistics. Data are presented as mean ± SD.

Figure 5

Mechanism of plag-15 RNAi-mediated lifespan extension in C. elegans Plag-15 RNAi leads to lifespan and healthspan extension in the worm C. elegans. We found that both transcription factors elt-3 and hlh-30 were necessary for the lifespan and healthspan extension in plag-15 RNAi. Furthermore, lysosomal vitamin B12 transporter pmp-5 was also required for the lifespan extension of plag-15 RNAi. By performing lipidomics, we found a reduced abundance of the lysophospholipids lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). Created with BioRender.com.