X-linked adrenoleukodystrophy (ALD) is the most common leukodystrophy. All ALD patients have a mutation in the ABCD1 gene that leads to a build-up of saturated very long-chain fatty acid (VLCFA) in tissues, including adrenal glands, spinal cord and brain. ALD is characterized by a striking and unpredictable clinical spectrum, even within families. In childhood, around 50% of affected boys develop adrenal disease before the age of 10 and 30-35% of affected boys develop a fatal inflammatory brain disease (cerebral ALD). If ALD is diagnosed in an early stage, cerebral ALD can be halted or reversed by a bone-marrow transplant. In adulthood, virtually all males and >80% of women develop a chronically slow progressive spinal cord disease (myeloneuropathy) for which no disease modifying therapy is available. Unfortunately, transplanted boys can still develop spinal cord disease in adulthood, because the transplant is only effective at halting the inflammatory component of the disease without addressing the underlying biochemical defect. This therapeutic gap highlights the need to develop effective treatments aimed at the normalization of VLCFA levels in the brain and spinal cord.

Using skin cells from ALD patients we have demonstrated that saturated VLCFA induce cellular stress, with prolonged exposure resulting in cell death. Remarkably, this is not observed with mono-unsaturated VLCFA (fatty acids with a double bond in the fatty acid chain). The enzyme stearoyl-CoA desaturase-1 (SCD1) coverts saturated fatty acids into mono-unsaturated fatty acids. In a previous ELA supported project we identified and characterized the drug TO901317 (an LXR agonist) as a small-molecule that activates SCD1 activity. Treatment of ALD cells with TO901317 completely corrects VLCFA levels and treatment of the ALD mouse with TO901317 added to the food resulted in a reduction in VLCFA levels in adrenals, spinal cord and brain.

Unfortunately, TO901317 (and other LXR agonists) as potential treatment for ALD have notable limitations due to off-target effects that result in serious side effects. This is largely due to LXR agonists not being specific for SCD1. We therefore hypothesize that specifically increasing SCD1 activity will circumvent the detrimental effect of LXR activation and offer a therapeutic strategy to counteract VLCFA-induced lipotoxicity in ALD.

Strategies to specifically increase SCD1, either pharmacologically or genetically have not been reported to date. Therefore, the overall goal of the proposal is to identify genetic and pharmacological regulators of SCD1 abundance and/or activity. Specifically, in this project we aim to:

• Generate cell lines in which the SCD1 enzyme is tagged with a fluorescent protein to allow monitoring of SCD1 in

1. live cells at a single cell resolution.

• Map enzymes that control SCD1 abundance using a genome-wide CRISPR/Cas9 genetic screen.
• Identify small molecules that increase SCD1 abundance and activity. Addressing these aims will increase our basic understanding of VLCFA metabolism in ALD.

Moreover, results from these experiments have the potential to inform on novel therapeutic strategies to treat ALD patients.

Project financed by ELA up to: 97 045 €