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Summary

X-Linked adrenoleukodystrophy (X-ALD) results from a mutation in the ABCD1 gene. This gene codes for a protein that imports very-long-chain fatty acids (VLCFAs) into the peroxisome for degradation via β-oxidation (β-O). Previous research has labeled the loss of function of the ABCD1 protein, and thus, the build-up of VLCFAs as the primary cause of neurodegeneration. However, VLCFA levels do not correlate with disease severity. Therefore, we aim to investigate the downstream effects of a lack of β-O acetyl-CoA (a-CA) product and how this may impact functioning of specific molecules involved in peroxisome-mitochondria interaction. The goal of the present proposal is to investigate how the products of VLCFA β-O are shuttled to the mitochondria from the peroxisome, how lack of product affects the functioning of metabolic and transport proteins Cit2, Cat2, and Pex34, and whether the function of these proteins is altered in X-ALD. We will first examine the relationship between behavioral and cellular markers of disease severity and peroxisomal a-CA levels, both in vivo with an established Drosophila ALD model and in vitro with human fibroblast cell cultures. Peroxisomal a-CA is hypothesized to negatively correlate with behavioral and cellular markers of disease severity in Drosophila and human fibroblasts. Next, we will focus our attention on Cit2 and Cat2 functioning and their relationship with diminished a-CA by creating fibroblast groups with Cit2 and Cat2 genes knocked out. We will then measure fatty acid β-O in the peroxisome. Mitochondrial integrity (MI) will also be measured via CO2 levels and mitochondrial membrane potential (MMP). If lack of a-CA impairs Cit2 and Cat2, mitochondrial function will be impaired and further contribute to disease phenotype. Finally, we will look to establish a correlational relationship between Pex34 gene silencing and molecular function/severity in ALD fibroblasts by measuring peroxisomal a-CA levels in a cell culture of human fibroblasts. We aim to show that a-CA is diminished in ALD-Pex34 knockdowns (KD) and that this causes a chain reaction leading to mitochondrial dysfunction and overall neurodegeneration. 

Background

X-linked adrenoleukodystrophy (X-ALD) is a progressive and often fatal neurodegenerative disease that affects the white matter of the brain and spinal cord (Gordon et al. 2018). The cause of X-ALD is an autosomal recessive mutation in the ABCD1 gene which codes for the ABCD1 transporter protein residing in the peroxisomal membrane (Gordon et al. 2018). ABCD1 is responsible for the import of very-long-chain fatty acids (VLCFAs) into the peroxisome for degradation via β-O (Gordon et al. 2018; Kawaguchi et al. 2018). However, the mutations that result in X-ALD are loss of function, and as a result VLCFAs, are not imported into the peroxisome for degradation, resulting in several toxic effects (Kawaguchi et al. 2018). First, due to a lack of VLCFAs being degraded, there a lack of product that would normally be produced by β-O of VLCFAs, which include a-CA (a-CA) and medium-chain fatty acids (MCFAs) (Gordon et al. 2018). Additionally, the accumulation of VLCFAs in fibroblasts of X-ALD patients is a well-known biomarker for this disease and thought to be the first sign of a mutated ABCD1, since they cannot enter the peroxisome to be degraded and have been shown to be substrates for further elongation (Ofman et al. 2010). However, the levels of VLCFAs in cells and tissues do not correlate with phenotype nor with disease severity (Gordon et al. 2018; Stradomska and Tylki-Szymańska 2018; Schirinzi et al. 2019). Therefore, a novel hypothesis of the downstream effects of mutated ABCD1 is that the lack of product of VLCFA peroxisomal β-O is causative of disease rather than a buildup of the VLCFA/VLCFA-CoA substrate (Gordon et al. 2018; Stradomska and Tylki-Szymańska 2018; Schirinzi et al. 2019). Additionally, it has been shown that β-O of fatty acids in the mitochondria and function of the citric acid cycle (CAC) is coupled with β-O in the peroxisome (Violante et al. 2013; Violante et al. 2019; van Roermund et al. 1995; Shai et al. 2018). Indeed, a-CA, a product of peroxisomal β-O, has been shown to be shuttled from the peroxisome to the mitochondria. This is important given the fact that the peroxisomal membrane is impermeable to a-CA and NAD(H), another product of β-O (van Roermund et al. 1995). One of the pathways implicated in shuttling a-CA from the peroxisome to the mitochondria involves conversion of a-CA into citrate by citrate synthase (Cit2) (van Roermund et al. 1995; Visser et al. 2007) in the peroxisome; another pathway involves the conversion of a-CA to acetylcarnitine by carnitine transferase (Cat2) (van Roermund et al. 1995; Visser et al. 2007) in the peroxisome. Then, both citrate and acetylcarnitine seem to be shuttled to the mitochondria by citrate acetyl transferase protein (van Roermund et al. 1995; Visser et al. 2007) or the glyoxylate cycle where they can be converted back into a-CA (Visser et al. 2007). It remains an open question whether citrate and acetylcarnitine are lacking in X-ALD patients because there is a lack of a-CA product from β-O of VLCFAs not occurring. Additionally, Pex34 is a peroxisome-mitochondria tethering protein found on the peroxisomal membrane (Shai et al. 2018). Pex34 has been previously found to be involved in transporting a-CA from the peroxisome to the mitochondria (Shai et al. 2018). However, further investigation is needed to assess how these pathways and contact proteins interact as well as how a lack of product might affect their function. Lack of product from peroxisomal β-O of VLCFAs might cause downstream damage to the mitochondria, among other possibly impacted organelles and cellular functions. The goal of the present proposal is to investigate how the products of VLCFA β-O are shuttled to the mitochondria, the implication of a lack of product on the functioning of Cit2, Cat2, and Pex34, and how the functioning of these proteins are affected in X-ALD.

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