ESPE Abstracts

Background: Loss of function mutations in SGPL1, a key component of sphingolipid metabolism, are associated with accumulation of sphingolipid intermediates giving rise to a multisystemic disease incorporating primary adrenal insufficiency (PAI) and progressive renal and neurological disease. Sphingolipids are implicated in mitochondrial apoptosis via induction of mitochondrial outer membrane permeabilization, cytosolic release of inter-membranal cytochrome c and activation of executioner caspases.

Objective and hypotheses: To investigate the impact of SGPL1 deficiency on mitochondrial morphology and function using patient derived human dermal fibroblasts and a SGPL1- knockout HeLa cell line.

Methods: Primary cell cultures of dermal fibroblasts were established from two patients with SGPL1 deficiency (Patient 1 - p.F545del; PAI, later onset renal/neurological compromise; Patient 2 - renal/neurological compromise). Mitochondrial architecture was examined by confocal microscopy with volumetric analysis using Z-stack images of stained cells. Mitochondrial oxidative phosphorylation rate was measured by Seahorse XF Extracellular Flux Analyser in control/patient fibroblasts. RT-qPCR for expression levels of genes regulating mitochondrial fusion and fission, MFN1/2 and DRP1.

Results: Total mitochondrial volume in patient fibroblasts and SGPL1-KO-HeLa cell lines vs controls was reduced: (p.F545del; P<0.05; n=20; p.S65Rfs*6G; P<0.001, n=20), SGPL1-KO-HeLa, P<0.01; n=20. Additionally, the number of fragmented mitochondria was increased in p.S65Rfs*6G compared to control (P<0.0001; n=20). The respiratory flux profile of p.F545del fibroblasts was unaltered, however, p.S65Rfs*6G fibroblasts showed a significant reduction in non-mitochondrial respiration (P<0.01, n=3, maximal respiration (P<0.05), ATP production (P<0.05) and spare respiratory capacity (P<0.05). Mitochondrial morphology differed; SGPL1-KO-HeLa and p.F545del had elongated and hyper-fused mitochondria whereas p.S65Rfs*6G had rounded, fragmented mitochondria. MFN1 and MFN2 expression were markedly upregulated in SGPL1-KO- and p.F545del fibroblasts (P<0.0001; n=3) while the opposite was seen in p.S65Rfs*6G (P<0.0001; n=3). However, DRP1 was uniformly downregulated in SGPL1-KO-HeLa and patient fibroblasts (P<0.0001, n=3).

Conclusion: Aberrant sphingolipid metabolism leads to disruption of mitochondrial morphology/function. The significant decreased DRP1 expression suggests an imbalance tilted towards reduced fission. The expression levels of fusion proteins MFN1/2 differed between cell lines; up in SGPL1-KO-HeLa and p.F545del, down in p.S65Rfs*6G fibroblasts, in keeping with the morphology observed. The degree of SGPL1 deficiency or other genetic modifiers may account for differences seen. However, importantly, in both patient fibroblasts and SGPL1-KO-HeLa cells mitochondrial volume is reduced. Further work is required to characterise mitochondrial effects of SGPL1 deficiency, including fusion-fission imbalance and effects on steroid output.