SSADH Variants Increase Susceptibility of U87 Cells to Mitochondrial Pro-Oxidant Insult

Giovanna Menduti,Daniele Lettieri-Barbato,Concetta Rosa Capo,Katia Aquilano,Patrizia Malaspina,Luisa Rossi,Alessandra Vitaliti

doi:10.3390/ijms21124374

Abstract

Succinate semialdehyde dehydrogenase (SSADH) is a mitochondrial enzyme, encoded by ALDH5A1, mainly involved in γ-aminobutyric acid (GABA) catabolism and energy supply of neuronal cells, possibly contributing to antioxidant defense. This study aimed to further investigate the antioxidant role of SSADH, and to verify if common SNPs of ALDH5A1 may affect SSADH activity, stability, and mitochondrial function. In this study, we used U87 glioblastoma cells as they represent a glial cell line. These cells were transiently transfected with a cDNA construct simultaneously harboring three SNPs encoding for a triple mutant (TM) SSADH protein (p.G36R/p.H180Y/p.P182L) or with wild type (WT) cDNA. SSADH activity and protein level were measured. Cell viability, lipid peroxidation, mitochondrial morphology, membrane potential (ΔΨ), and protein markers of mitochondrial stress were evaluated upon Paraquat treatment, in TM and WT transfected cells. TM transfected cells show lower SSADH protein content and activity, fragmented mitochondria, higher levels of peroxidized lipids, and altered ΔΨ than WT transfected cells. Upon Paraquat treatment, TM cells show higher cell death, lipid peroxidation, 4-HNE protein adducts, and lower ΔΨ, than WT transfected cells. These results reinforce the hypothesis that SSADH contributes to cellular antioxidant defense; furthermore, common SNPs may produce unstable, less active SSADH, which could per se negatively affect mitochondrial function and, under oxidative stress conditions, fail to protect mitochondria.

Highlights

Succinic semialdehyde dehydrogenase (SSADH) is a mitochondrial homotetrameric enzyme, catalyzing a NAD+-dependent reaction in the catabolic pathway of γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter of central nervous system
The enzymatic activity of SSADH was measured in total cell extracts by fluorimetric analysis and, as reported in Figure 1A, the amino acid replacements in the triple mutant (TM) SSADH protein (p.G36R/p.H180Y/p.P182L) cause a strong decrease of enzyme activity when compared to the wild type (WT) protein
Our results showed that both WT SSADH and TM SSADH exert an early protection from the accumulation of 4-HNE-protein adducts

Summary

Introduction

Succinic semialdehyde dehydrogenase (SSADH) is a mitochondrial homotetrameric enzyme, catalyzing a NAD+-dependent reaction in the catabolic pathway of γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter of central nervous system. The first one, a transamination reaction, is catalyzed by GABA transaminase enzyme (GABA-T), leading to the formation of the succinic semialdehyde (SSA). Different mutations in the ALDH5A1 gene (coding for SSADH) lead to enzyme failure and cause SSADH deficiency, a rare autosomal recessive disorder of childhood and the best characterized inherited metabolic disorder of GABA catabolism [1]. Upon SSADH activity lacking, SSA is sidetracked to GHB by an alternative cytosolic reaction, catalyzed by an SSA reductase (SSAR). Both GABA and GHB are excreted with urine. When SSADH activity is missing, due to mutations of ALDH5A1 gene, GHB increases, representing the pathognomonic feature of SSADH deficiency ( called γ-hydroxybutyric aciduria)

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