Characterization of an epileptic disorder of GABA metabolism through development of murine Aldh5a1 deficient neuronal stem cells
Authors: Vogel Kara R. , Ainslie Garrett Robert , Roullet Jean‐Baptiste O , Gibson K. Michael 
Published: November 2023

BackgroundSuccinic semialdehyde dehydrogenase deficiency (SSADHD) is a disorder of GABA metabolism presenting with developmental delay, motor and cognitive deficits. Patients often demonstrate co‐morbid sleep and behavioral disturbances, mood disorders, and/or seizures. The SSADHD mouse model represents a phenocopy of the human disorder and shows the hallmark biochemical increases in GABA and GHB (γ‐hydroxybutyric acid). Furthermore, SSADHD mice exhibit increased mitochondria and oxidative stress, failure to thrive, and a progressive epileptic phenotype with early demise. We sought to characterize a high‐throughput in vitro model of SSADHD for evaluation of preclinical therapeutics and to establish biomarker targets for translational work in human‐derived cell lines.MethodsWe characterized six lines of murine primary neuronal progenitors (mNSCs): two wild‐type and four SSADHD lines isolated from the cortex and hippocampus of newborn pups (postnatal P1). GHB in media was quantified by GC‐MS, mitochondrial abundance quantified by fluorescent microscopy (mitoTracker and NucBlue), and ATP levels assessed with a fluorescent plate reader assay (Promega Cytotoxicity Kit). RNA for expression analysis was isolated with Trizol, treated with DNase and assessed for gene expression. Gene expression was evaluated in both mNSCs and brain tissue from P1 and P20 mice to assess developmental characteristics.ResultsSSADHD mNSCs manifested increased levels of GHB in the cell media. The null cells also showed significantly increased mitoTracker/NucBlue fluorescence after six hours of starvation, with normal levels of mitoSox (luminescent) and cytotoxicity (fluorescence). ATP levels were higher in mutant cells, potentially corresponding to increased mitochondrial abundance, an effect attenuated by the PI3K/mTOR inhibitor XL765. Expression studies of mNSCs revealed heightened levels of Gabrb3 (GABAA Receptor subunit beta 3), an essential subunit of GABAA receptors in fetal, perinatal, and adult mammalian brain. TNFα expression was downregulated in SSADHD mNSCs.DiscussionMetabolic parameters (GHB) and oxidative stress components (mitochondrial number, ATP) verified that the NSC model faithfully reflects the in vivo SSADH null mouse model. Expression studies, encompassing evaluation in both murine brain regions and mNSCs revealed similar expression levels of Slc12a2 (NKCC1) and Slc12a5 (KCC2) across mNSCs and P1 hypothalamic tissue. Gabrb3, an epileptic marker, was elevated in deficient mNSCs. TNFα downregulation in SSADH null mNSCs supports the concept that inflammation may be a pathophysiological consideration in SSADHD, perhaps interrelated to downregulation by TNFα of cell‐surface GABAAR receptors.ConclusionThe SSADHD mNSCs reflect key metabolic and biochemical changes observed in SSADH deficient patients and mice, suggesting that further development of genetic lines of neuronal stem cells in mice or human may represent an ideal system for in vitro screening and translational characterization.Support or Funding InformationK.V. is funded by the Lily's Fund for Epilepsy Research. Research support was also obtained from the SSADH Association.