Endocrine Abstracts

Introduction: 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. Sphingolipid accumulation is implicated in mitochondrial pathology.

Objective: To investigate the impact of SGPL1 deficiency on mitochondrial morphology/ function.

Methods: Cell lines: Primary cell cultures of dermal fibroblasts from patients with SGPL1 deficiency (Patient 1 - p.F545del; PAI, later onset renal/neurological compromise; Patient 2 - p.S65Rfs*6G; PAI, early onset renal/neurological compromise) and a CRISPR SGPL1- knockout HeLa cell line. The following were investigated: steroidogenic capacity (ELISA of cortisol from progesterone stimulated fibroblasts); mitochondrial architecture (confocal microscopy); oxidative phosphorylation rate (Seahorse XF Extracellular Flux Analyser) and expression levels of fusion and fission genes, MFN1/2 and DRP1 respectively (RT-qPCR).

Results: Cortisol production was significantly reduced in patient fibroblasts vs controls (p.F545del; P<0.05; p.S65Rfs*6G; P<0.001, n=3). 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 vs 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, hyper-fused mitochondria whereas p.S65Rfs*6G had rounded, fragmented mitochondria. Accordingly, MFN1/MFN2 expression were upregulated in SGPL1-KO- and p.F545del fibroblasts (P<0.0001; n=3) and downregulated 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 in SGPL1 deficiency leads to disruption of mitochondrial morphology/function with a reduction in mitochondrial volume and an impact on steroidogenesis. Decreased DRP1 expression suggests an imbalance tilted towards reduced fission. The degree of SGPL1 deficiency or other genetic modifiers may account for differences seen. Further work is required to characterize the potential multi-systemic effects of mitochondrial dysfunction in SGPL1 deficiency.