Interrogating the role of SGPL1 in adrenal/gonadal development and acute steroidogenesis

Defects in the gene, sphingosine-1-phosphate lyase, SGPL1 are associated with a rare disease affecting the adrenal glands, kidneys, neurological system and skin. Affected children have a reduction in SGPL1, a key enzyme of an important lipid system, the sphingolipids, which are present throughout the body. Sphingolipids have many important functions in the body, smaller sphingolipids have a role in signalling whilst larger sphingolipids form important components of the body's cells. Deficiency of SGPL1 results in a block in the system, likely to cause certain sphingolipids to accumulate.

Our group primarily investigates inherited causes of adrenal disease. The adrenal glands produce cortisol, an essential steroid hormone in maintaining the body's appropriate response during times of physical and emotional stress. A significant proportion of patients with SGPL1 deficiency have abnormal adrenal function, furthermore, a third of male patients with the condition also have abnormal testicular function. The testes also produce a steroid hormone, testosterone. Patients as a consequence present with impaired testosterone production and disorders of sexual development. I will be conducting a range of cell experiments to try and understand the disease mechanism in the adrenals and testes, to hasten discovery of new therapeutic targets.

In this project I will investigate how SGPL1 deficiency affects sphingolipid accumulation in a human adrenal cell model and a mouse testis cell model deficient in SGPL1 (an equivalent human model is not currently available). Steroid hormone production in adrenal and testicular cells relies on common biological processes. I will be using these two models to assess how the disturbance of sphingolipid metabolism affects signalling pathways known to be key in steroid hormone production.

Sphingolipids may also play a significant role in the development of the adrenal and testis as evidenced by the very early disease presentations of our patients soon after birth. Current work is focused on the effect of SGPL1 on foetal mouse adrenal and testicular development in an SGPL1 deficient mouse model. In this project for the first time, together with our collaborators, I will be using a 3-dimensional human foetal adrenal model to assess the effect of SGPL1 deficiency on the steroid producing abilities of the human foetal adrenal. Similarly, I will use a 3-dimensional human foetal testis model to further investigate the impact of SGPL1 deficiency not only on the steroid hormone producing cells of the human testis but also the cell populations responsible for fertility. Whilst testicular disease can present in early life for some of our patients, it is also possible that disease can develop over time for others. These studies will help inform the clinical screening for affected patients to assess the need for hormone supplementation and consideration of future fertility.

Whilst rare, SGPL1 deficiency is extremely debilitating and can significantly affect life expectancy. Understanding the disease mechanism in the adrenal and testicular condition may crucially provide insights into which biological pathways may be disrupted in other body systems affected, for instance the kidneys and nervous system. The sphingolipid pathway is, importantly, amenable to therapeutic manipulation and this work will hasten the discovery of new therapeutic targets for treatment in adrenal and testicular disease. Furthermore, as strategies to modify the activity of SGPL1 are being explored in therapies for several conditions, understanding the impact of this genetic defect in human disease will provide important insights into the potential effects of altering this lipid system.

Sphingosine -1-phosphate lyase insufficiency syndrome, secondary to biallelic loss-of-function mutations in SGPL1, is associated with steroid resistant nephrotic syndrome, neurological disease, ichthyosis and multiple endocrinopathy including primary adrenal insufficiency (PAI) and primary testicular failure. There is significant accumulation of the sphingolipids, sphingosine and sphingosine-1-phosphate (S1P), with reduced cortisol production in SGPL1 deficient human adrenal (H295R) cells.

I aim to test the hypothesis that the adrenal and gonadal disease manifest from disturbed sphingolipid metabolism using 2 steroidogenic cell lines, our CRISPR engineered SGPL1 KO H295R and murine Leydig (MA-10) cell lines with sphingolipid profiling by LCMS.

The sphingolipid intermediate, sphingosine is postulated to attenuate the activity of Steroidogenic factor 1 (SF-1) critical for adrenal/gonadal development and acute steroidogenesis. I will assay SF-1 functional activity in the SGPL1 deficient cell lines and expression of known SF-1 transcriptional targets. S1P, meanwhile exerts its effects through five G protein coupled S1P receptors (S1PR1-5). I will investigate the effects of S1P accumulation on S1PR expression and the impacts on downstream signalling pertinent to steroidogenesis. I will also determine the impact on key components of the mitochondrial associated membrane integral to the initial steps of steroidogenesis.

Current work is focused on adrenal and gonadal development using an Sgpl1 -/- mouse model. In this project, together with collaborators from the MRC Centre for Reproductive Health, Edinburgh, I will use an ex vivo human foetal testicular organotypic (hanging-drop) model to investigate effects of SGPL1 deficiency not only on the Leydig cell population but also the Sertoli and germ cell populations of the developing human testis. I will also assay the impact of SGPL1 deficiency on steroidogenesis of the human foetal adrenal using the same model.