We developed a novel in vitro cerebral adrenoleukodystrophy (cALD) model to investigate the molecular mechanisms underlying very long chain fatty acid(VLCFA)-induced cell death.

ALD is a devastating neurometabolic disorder caused by mutations in ABCD1 resulting in toxic accumulation of VLCFA. Over half of affected males develop inflammatory cerebral phenotype or cALD. Microglia death is an early pathogenic event in cALD, but the underlying mechanisms remain unknown.

Our cALD in vitro model combines ABCD1 knockout (KO) HMC3 cell line, generated via monoclonal CRISPR/Cas9 editing, and VLCFA (C26:0) overload. Using Scalable Time-lapse Analysis of Cell death Kinetics (STACK), we evaluated death in ABCD1 KO and control overloaded with fatty acid species with/without cell death inhibitors. Targeted lipidomics evaluated lipidome changes after VLCFA overload.

ABCD1 KO have increased accumulation of C26:0 containing lipid species, including C26-lysophosphatidylcholine, compared to control. ABCD1 KO microglia have greater cell death compared to control following VLCFA, but not long chain fatty acid overload. VLCFA-induced death is chain length dependent with distinct cell death kinetics, specifically heterogeneous death onset and slower death rate compared to apoptosis and ferroptosis. Surprisingly, cell death inhibitors targeting apoptosis, ferroptosis, necroptosis or pyroptosis fail to rescue VLCFA-induced cell death in ABCD1 KO cells.

This model replicates VLCFA accumulation and lipotoxicity observed in human cALD. Absence of ABCD1 in microglia increases vulnerability to VLCFA-induced death.The lack of rescue with inhibitors of canonical cell death pathways combined with heterogenous cell death kinetics suggest that VLCFA-induced cell death may results from accumulation of toxic intermediates rather than direct activation of cell death machinery. This is an intriguing finding and warrants further, and more comprehensive evaluation.

This in vitro cALD human microglia model is an invaluable tool for elucidating the molecular mechanisms driving VLCFA-induced cell death in cALD, and identifying potential targetable pathways for future studies.