448 Establishing the efficacy of naturally occurring endocannabinoid-like substance in an in vitro model of Fragile X Tremor/Ataxia Syndrome (FXT/AS)

Abstract

OBJECTIVES/GOALS: FXT/AS is a devastating, rare neurological syndrome that negatively impacts movement and cognition and is suspected to induce mitochondria dysfunction. Currently, no effective pharmacological treatments for FXTAS exist. The goal is to restore mitochondrial viability using endocannabinoid-like compounds in a cell culture model of FXTAS. METHODS/STUDY POPULATION: To establish a cell model of mitochondrial dysfunction, fibroblast baby hamster kidney (BHK-21) cell lines were treated with glucose oxidase (GluOx) at varying concentrations and times. Mitochondrial viability was assessed by the colorimetric Janus B Green Assay, which stains the mitochondria and enables assessment of cell numbers and the presence of oxygen in anchorage-dependent cell culture. Upon establishing this model of mitochondrial dysfunction, we next investigated the ability of three novel mitochondrial antioxidants (e.g., macamides) to protect mitochondrial viability. RESULTS/ANTICIPATED RESULTS: GluOx treatment of BHK-21 cells caused a dose- and time-dependent increase in oxidative stress. The data demonstrated significant disruption in the morphology of BHK-21 cells at a high glucose concentration, i.e., 40 nM, between 2 and 24 hours post-exposure. The morphology data were confirmed by the Janus B Green colorimetric assay. In examining the effects of glucose on mitochondrial viability, we demonstrated that at 15, 30, 35, and 40 nM, glucose significantly decreased mitochondria viability compared to the untreated, with 40 nM having the greatest effect. Under these conditions of mitochondrial dysfunction, co-incubation of the cells with the 0.5 uM MAM69 macamide attenuated the GluOx-induced increase in oxidative stress, with 0.5 uM MAM69 alone showing no effect on mitochondria viability. DISCUSSION/SIGNIFICANCE: This study illustrates the efficacy of macamides, natural occurring endocannabinoid like-compound, as novel prognostic and therapeutic candidates in the treatment of mitochondrial dysfunction that is associated with FXTAS. The use of BHK-21 fibroblast cells provides a rapid screening model to test for pharmacological therapeutic efficacy.