Role of ACSBG1 in the Biochemical Pathology of X-Linked Adrenoleukodystrophy (X-ALD)

X-linked adrenoleukodystrophy (X-ALD) is a severe, progressive neurodegenerative disorder characterized by central demyelination (cerebral form) or distal axonopathy (spinal cord form).  Adrenal gland insufficiency and testicular dysfunction are also present. Biochemically, X-ALD patients have elevated levels of saturated very long-chain fatty acids (YLCF A; containing ≥22 carbons) in plasma and most tissues. X-ALD is caused by mutations in the ABeDI gene, and the ABCD 1 protein, like elevated YLCFA levels, can be found in most tissues. These facts raise the question: Why is pathology in X-ALD confined to the nervous system, adrenal glands, and testis? An enzyme of fatty acid metabolism-the "bubblegum" fatty acyl-CoA synthetase  (ACSBG 1 )-may provide insight. Fruit flies with the bubblegum mutation exhibited neurodegeneration and elevated VLCFA levels. In mammals, ACSBGI is found only in cell types affected pathologically in X-ALD (neurons, cortisol-producing adrenocortical cells, and testosterone-producing testicular Leydig cells). No direct biochemical link between ACSBG 1 and X-ALD has yet been found.

Thus, the primary hypothesis to be tested is that ACSBG 1 performs cellular functions that are essential for manifestation of the pathological biochemical abnormalities in X-ALD. Preliminary studies in both cultured cells and ACSBG I knockout mice indicate that GABAA receptors, which are also found in cell types that express ACSBG I, require this enzyme for normal structure and function. Other preliminary data are also consistent with impairment in steroidogenesis when ACSBG 1 is deficient.

Therefore, we propose: 1) To elucidate the role of ACSBG 1 in GABA" receptor function: 2) To elucidate the role of ACSBG I in steroidogenesis: and 3) To establish a functional correlation between ACSBG I and X-ALD. Neuron-derived cell lines with stable knockdown of ACSBG 1 expression (produced via RNA interference) and ACSBG I knockout mice are already available. In addition, a Leydig cell line with stable ACSBG I knockdown will be produced. Distribution, function. and palmitoylation of GABA..\ receptors, and the role of lipid rafts will be investigated in control and ACSBG 1- deficient cultured cells. Behavioral studies will be done to assess GABAA receptor function in control and ACSBG I knockout mice. Mouse tissues will be used to assess GABA" receptor number, morphology, and ligand binding. Steroidogenesis will be investigated in the mouse MAI 0 Leydig cell line; effects of ACSBG I deficiency on hormone-stimulated progesterone synthesis and concomitant transient increases in fatty acid activation will be examined. The role of ACSBG I in neurosteroid production by Neur02a cells will be assessed, and cholesterol ester synthesis and mobilization will be evaluated in both cell types. The steroidogenic potential of brain, adrenal gland, and testis will be investigated in control and knockout mice. Transient knockdown of ABCD I in ACSBG I-deficient cells will be used to evaluate ACSBG I 's contribution to X-ALD abnormalities. Effects on both GABA" receptors and steroidogenesis will be assessed.

Results of these studies will enhance our understanding of the role of ACSBG I in GABAergic neurotransmission and steroidogenesis, and reveal whether any of these cell-specific functions help explain why pathologic manifestations in X-ALD are limited to cells that express ACSBGI.