Abstracts

Rationale: Aldehyde dehydrogenase family 5 member A1 (ALDH5A1) encodes the mitochondrial enzyme succinic semialdehyde dehydrogenase (SSADH) which is critical for the catabolism of the inhibitory neurotransmitter g-aminobutyric acid (GABA). Homozygous ALDH5A1 loss-of-function mutations result in SSADH deficiency (SSADHD), an autosomal recessive ultra-rare disorder characterized by intellectual disability, autism, and epilepsy. Pathologic accumulation of GABA and its metabolite g-hydroxybutyrate (GHB), and downregulation of brain GABA_A receptors. In contrast, individuals with one allelic ALDH5A1 copy variant are considered non-pathologic carriers without core SSADHD symptoms. However, emerging evidence suggests that they may exhibit subtle neurologic phenotypes. In this project, we investigated the molecular signature of SSADH haploinsufficiency in Aldh5a1 mutant mice.


Methods: We collected whole blood and microdissected brain tissues from heterozygous Aldh5a1 mutant (Het) mice and wild-type (WT) control at postnatal (P) 16 and 100 days to investigate early and mature stages of brain development. Serum GABA and GHB contents were measured via high performance liquid chromatography-tandem mass spectrometry. Cortical protein expressions of GABA_A receptor g2 subunit (Gabrg2) and glutamic acid decarboxylase isoform 67 kDa (Gad67) were measured by western blot analysis. Het mice were also treated at P14 with an adeno-associated virus (AAV) expressing a human ALDH5A1 gene, AAV-ALDH5A1. Serum biomarkers were measured at P100 after AAV-ALDH5A1 treatment.


Results: Het mice have excess brain GABA and GHB at P100 but not at P16 (in µM, GABA P16: WT=5.07±0.17, Het=6.32±0.81, p=0.2956; GABA P100: WT=3.37±0.39, Het=4.52±0.11, **p=0.0056; GHB P16: WT=8.99±0.80, Het=12.77±1.70, p=0.1382; GHB P100: WT=5.7±0.88, Het=9.71±0.71, **p=0.0069, Fig. 1A-B). Het mice have significantly reduced level of SSADH across development (P16: 57.72±0.66% WT, ****p< 0.0001; P100: 66.98±3.33% WT, ****p< 0.0001, Fig. 1C), as well as compensatory changes at mature stage in Gabrg2 (P16: 90.64±4.33% WT, p=0.1413; P100: 113.6±2.08% WT, ***p=0.0005, Fig. 1D) and Gad67 (P100: 90.73±1.73% WT, *p=0.0476, Fig. 1E) protein expressions. Upon treatment of AAV-ALDH5A1 (Fig. 2A), Het mice have normalized GABA and GHB (in µM, GABA: WT+AAV=4.96±0.72, Het+AAV=3.89±0.51, p=0.3908; GHB: WT+AAV=7.27±1.44, Het+AAV=7.13±0.51, p=0.9519, Fig. 2B-C).


Conclusions: ALDH5A1 loss-of-function carriers (or SSADH haploinsufficiency) are considered unaffected healthy individuals. In this study, we challenge this conventional idea, and test whether ALDH5A1 single allele loss of function alters GABA metabolites, GABA synthetic enzymes, and GABA_A receptor subunit protein expression. We found that heterozygous Aldh5a1 mutant mice have abnormal GABA and GHB accumulation at mature development stage, as well as altered protein expressions of GABA_A receptor g2 subunit and GABA-synthesizing enzyme Gad67, suggesting molecular mechanisms underlying a neurologic phenotype in SSADH haploinsufficiency.


Funding: SSADH Association, NIH, Translational Research Program at Boston Children's Hospital