140 - Reduced 7-Dehydrocholesterol Reductase Activity Impairs the Glutathione Redox Status in a Neurological Cell Model

Abstract

7-Dehydrocholesterol reductase (DHCR7) serves as the penultimate enzyme in the Kandutsch-Russel pathway of cholesterol synthesis. When the function of DHCR7 is compromised its primary substrate, 7-dehydrocholesterol (7DHC), accumulates. The accumulation of 7DHC can be detrimental to many biological processes, as observed in the metabolic disorder Smith-Lemli-Opitz syndrome, in part because 7DHC is reported to be the most highly oxidizing lipid found endogenously. Despite 7DHC’s high capacity to be oxidized, very few biomarkers of oxidant stress have been associated with elevated 7DHC levels. This project aimed to determine if elevated 7DHC levels lead to oxidant stress by examining its impact on intracellular glutathione. Neuro2A cells deficient in DHCR7 accumulate approximately four-times the amount of 7DHC in a 60 hour time span, while control cells maintain basal levels. Over this same time course DHCR7-defecient cells’ glutathione levels reduce by a factor of 4.7 as compared to control cells, which were only reduced by a factor of 1.5. Additionally, the ratio of reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured every 12 hours post seeding in these cells, and at each time point the GSH/GSSG ratio was lower in DHCR7-deficient cells compared to its respective control. We also observed that when wild type Neuro2A cells are exposed to 1 µM of the DHCR7 inhibitor, AY-9944, for 24 hours the GSH/GSSG ratio was significantly decreased. These data together suggest that when the function of DHCR7 is impaired total glutathione levels are reduced and the ratio of GSH/GSSG decreases as well. These observations are coherent with an accumulation of 7DHC, as the electrophilic oxysterols derived from 7DHC oxidation could attack GSH causing a depletion of total glutathione as well as cause general cellular stress leading to elevated GSSG levels. This study demonstrates the potential for 7DHC to impact biological processes by impairing the glutathione redox status.