Redirection of sphingolipid metabolism drives cytoskeletal defects in SPLIS and reveals ROCK inhibition as therapy
Adam Majcher, Ranjha Khan, Kathrin Buder, Florence Bourquin, Julie D. Saba, Thorsten Hornemann
Adam Majcher, Ranjha Khan, Kathrin Buder, Florence Bourquin, Julie D. Saba, Thorsten Hornemann
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Research Article Genetics Metabolism Nephrology

Redirection of sphingolipid metabolism drives cytoskeletal defects in SPLIS and reveals ROCK inhibition as therapy

Abstract

Sphingosine-1-phosphate lyase (SPL) insufficiency syndrome (SPLIS), also known as nephrotic syndrome type 14, is an autosomal recessive multisystem disorder caused by loss-of-function mutations in SGPL1, encoding the enzyme responsible for the terminal degradation of sphingosine-1-phosphate (S1P). We investigated a patient carrying a previously undescribed c.1084T>A (p.Ser362Thr) SGPL1 variant and analyzed the metabolic and cellular consequences of SPL deficiency, using patient fibroblasts, SGPL1-KO HEK293T cells, and Sgpl1–/– and Sgpl1rosa+fl/fl mice. Metabolic stable isotope labeling revealed that SPL deficiency does not invariably result in S1P accumulation. Instead, SPL-deficient cells maintain near-normal S1P levels through (a) feedback regulation of de novo sphingolipid synthesis via the ORMDL–ceramide axis and (b) increased diversion of excess ceramides into glycosphingolipids. However, perturbation of sphingolipid homeostasis, either by exogenous sphingolipid load or disruption of compensatory regulation, induces pathological intracellular S1P accumulation. In vivo, Sgpl1–/– mice had pronounced urinary S1P excretion and renal S1P enrichment, accompanied by cytoskeletal disorganization and impaired epithelial morphogenesis. Mechanistically, we identify aberrant Rho/ROCK signaling as a key mediator of S1P-driven cytoskeletal dysregulation. Pharmacological ROCK inhibition with fasudil mitigated renal cytoskeletal defects in Sgpl1–/– and Sgpl1rosa+fl/fl mice and partially restored epithelial architecture. These findings redefine the metabolic consequences of SPL deficiency and identify S1P-driven Rho/ROCK hyperactivation as a tractable therapeutic target in SPLIS.

Authors

Adam Majcher, Ranjha Khan, Kathrin Buder, Florence Bourquin, Julie D. Saba, Thorsten Hornemann

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Figure 7

Fasudil rescues S1P induced cytoskeletal phenotypes in SPLIS fibroblasts and SGPL1-deficient HK2 cell line.

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Fasudil rescues S1P induced cytoskeletal phenotypes in SPLIS fibroblasts...
(A) Fluorescence imaging of SPLIS primary fibroblasts and control fibroblasts treated with vehicle (MeOH) or S1P (0.25 μM) for 6 hours. Cells were also treated with the ROCK inhibitor (fasudil), an S1P receptor 2 inhibitor (JTE013), or an S1P receptor 1 modulator (fingolimod; FTY720). After treatment, cells were fixed with 4% paraformaldehyde (PFA) and stained with phalloidin (actin) and DAPI (nucleus). Scale bars: 100 μm. (B) Quantification of cell contraction. Compiled images of whole wells were analyzed using CellProfiler software. Cell contraction was defined by the formula 1/log10(cell surface/nucleus surface). Values were normalized to the average of vehicle-treated cells. Data are represented as mean ± SD (n = 4). Statistical analysis was performed using a 2-tailed parametric t test. Comparison between control (yellow bar) and SPLIS fibroblasts at the same S1P concentration (orange bar) (*P < 0.05, ***P < 0.0005). Comparison between SPLIS fibroblasts treated without or with 2 concentrations of fasudil (ROCK inhibitor) (green bar and blue bar) (##P < 0.005, ###P < 0.0005). (C) Impairment of renal epithelium formation in SGPL1 KO HK2 cells after S1P supplementation. SGPL1 KO or WT HK2 cells were grown for 72 hours in the presence of increasing S1P concentrations, with or without fasudil, as indicated. Cells were fixed with 4% PFA and stained with phalloidin (actin) and DAPI (nucleus). Whole wells were imaged using fluorescence microscopy. Representative images from 3 independent replicates are shown. Scale bars: 500 μm.