Abstracts

Rationale: Dravet syndrome (DS) is a severe genetic developmental and epileptic encephalopathy (DEE). We have previously shown metabolic dysfunction in energy-producing pathways (mitochondrial respiration and glycolysis) in a zebrafish model of DS. Lymphoblast cell lines (LCLs) have been used to uncover metabolic alterations in neurological disorders such as autism and schizophrenia, suggesting their utility as a model to study systemic defects in DEEs. The goal of this study was to evaluate mitochondrial function by direct measurement of mitochondrial respiration, fatty acid oxidation, and membrane potential in LCLs from DS patients (DS LCLs) and those from age/sex-matched healthy volunteers (Con LCLs).

Methods: DS-LCLs were derived from patients diagnosed with DS (Coriell Institute of Technology). Key parameters of mitochondrial respiration (oxygen consumption rate, OCR) and glycolysis (extracellular acidification rate, ECAR) were measured using the Extracellular Flux 96 Analyzer (Agilent Technologies). Exogenous and endogenous substrate utilization (conjugated palmitate-bovine serum albumin (BSA) or BSA alone respectively) were evaluated by measuring the intrinsic rate and capacity of cells to oxidize fatty acids (FAs). Mitochondrial membrane potential was measured using the MitoProbe tetramethyl rhodamine methyl ester (TMRM) assay for flow cytometry (Thermo Fisher Scientific).

Results: DS LCLs showed defects in both major energy-producing pathways (mitochondrial respiration and glycolysis), from decreased spare respiratory capacity (mean% Δ OCR Con LCLs ± SEM; -42.60% ± 7.24, p = 0.0001) to decreased glycolytic reserve (mean% Δ ECAR Con LCLs ± SEM; -54.77 ± 17.13, p = 0.02). DS LCLs show a significant reliance on endogenous FA utilization under substrate limitation and cellular stress when compared to Con LCLs (mean% Δ OCR Con LCLs ± SEM; +82.9 ± 23.8, p = 0.002). In addition, under stress, DS LCL respiration is also supported by exogenous FA oxidation (mean% Δ Con OCR LCLs ± SEM; +64.4 ± 20.9, p = 0.015). Baseline proton-leak respiration in DS LCLs was significantly lower (mean% Δ OCR Con LCLs ± SEM; -14.2 ± 4.9, p = 0.004), further supporting the observed increase in mitochondrial membrane potential in DS LCLs (mean% Δ TMRM fluorescence Con LCLs ± SEM; +44.9 ± 21.4, p = 0.05).

Conclusions: Cellular energetic demands, like mitochondrial membrane uncoupling and absence of FA utilization in DS LCLs, result in impaired mitochondrial function, with maximal respiratory capacity 32.3% lower than Con LCLs (± 4.4, p = 0.0001) despite having lower proton-leak respiration and greater mitochondrial membrane potential. Under cellular stress and FA exposure, DS LCLs display greater reliance on FA oxidation to overcome stressors when compared to Con LCLs. The observed shift in the metabolic profile of peripheral cells derived from patients with DS further support the continued investigation of metabolism in DS using this novel model to target specific energy-producing pathways to help discover new therapies for DS.

Funding: Dravet Syndrome Foundation grant (MP, KK)