The GABA transaminase, ABAT, is essential for mitochondrial nucleoside metabolism

Abstract

ABAT is a key enzyme responsible for catabolism of principal inhibitory neurotransmitter γ-aminobutyric acid (GABA). We report an essential role for ABAT in a seemingly unrelated pathway, mitochondrial nucleoside salvage, and demonstrate that mutations in this enzyme cause an autosomal recessive neurometabolic disorder and mtDNA depletion syndrome (MDS). We describe a family with encephalomyopathic MDS caused by a homozygous missense mutation in ABAT that results in elevated GABA in subjects' brains as well as decreased mtDNA levels in subjects' fibroblasts. Nucleoside rescue and co-IP experiments pinpoint that ABAT functions in the mitochondrial nucleoside salvage pathway to facilitate conversion of dNDPs to dNTPs. Pharmacological inhibition of ABAT through the irreversible inhibitor Vigabatrin caused depletion of mtDNA in photoreceptor cells that was prevented through addition of dNTPs in cell culture media. This work reveals ABAT as a connection between GABA metabolism and nucleoside metabolism and defines a neurometabolic disorder that includes MDS.

Figure 1. Subjects with ABAT mutations display brain abnormalities and signatures of mitochondrial dysfunction in muscle

(A) Brain MRI images of S1 at 5 years of age. Image 1 shows a T1-weighted sagittal section demonstrating signs of global cerebral volume loss with thinning of the corpus callosum and atrophy of subcortical structures, brainstem, and cerebellar vermis. Images 2 and 3 show T2-weighted axial and coronal sections demonstrating atrophy and diffuse white matter signal abnormalities in the cerebral hemispheres, prominent atrophy of the thalamus, and noticeable signal abnormalities in the posterior limb of the internal capsule and dentate nuclei. (B) Biochemical assessment of electron transport chain activity in muscle tissue for S1. Activity is diminished relative to controls across complex I, complexes I + III, complex II, complexes II + III, and complex IV. (C) Pedigree of Subjects 1 (II-II) and 2 (II-III) shows Mendelian segregation and recessive inheritance of mutation ABAT_NM_000663.3 c.631C>T through the family. (D) Evaluation of GABA levels in the brain by in vivo proton magnetic resonance spectroscopy is shown in mM units with standard deviation. The amount of GABA in S1 and S2 (black bars) is significantly elevated over age-matched controls (grey bar).

Figure 2. shRNA knockdown of ABAT in healthy fibroblasts results in mtDNA depletion and diminished mitochondrial membrane potential

Control fibroblasts transfected with two independent shRNAs targeting ABAT show ABAT transcript levels that are roughly half of ABAT transcript levels in controls (A) and Western blotting shows ABAT protein levels in these cells are also diminshed by half (B). Likewise, fibroblasts with ABAT mRNA and protein knockdown exhibit significantly diminshed copy number of mtDNA (C) and membrane potential (D) while non-targeting shRNA shows no difference compared to untreated control. In all panels C is control, C + NT shRNA is control cells transfected with non-targeting shRNA, C + ABAT shRNA is controls cells transfected with shRNA targeting the 3’UTR of ABAT. *** indicates p-value < 1E-5. Error bars indicate standard deviation.

Figure 3. Pathogenic ABAT mutations identified in subjects with elevated GABA levels cause a quantitative loss of mtDNA copy number

(A) Three unrelated families have been identified with ABAT deficiency that have a molecular diagnosis and elevated levels of GABA in brain confirmed by MRS. S1 and S2 were affected siblings homozygous for pLeu211Phe (M1). S3 was compound heterozygous for missense mutations M2 and M3. S4 was compound heterozygous for missense mutation M4 and deletion M5. ABAT subject mutations are distributed throughout the length of the protein. (B) A first-in-kind lentiviral vector was created to simultaneously deliver an shRNA and an ORF by modifying the pGIPZ expression vector. (C) Healthy cells were transduced with constructs containing shRNA targeted to ABAT 3’UTR and ORFs containing each subject mutation. All mutations resulted in decreased mtDNA copy number. *** indicates p-value < 1E-5. Error bars indicate standard deviation.

Figure 4. G0 fibroblasts from subjects with ABAT mutations show deficits in mtDNA copy number and mitochondrial membrane potential and transduction with wild-type ABAT cDNA rescues cellular dysfunction

Fibroblasts grown in low serum media enter the G0 phase of the cell cycle and become quiescent. (A) Subjects with ABAT mutation have lower mtDNA copy number than controls when grown in normal media (NM) and their mtDNA copy number decreases to roughly half that of healthy controls when grown in low serum media (LSM). (B) Mitochondrial membrane potential is diminished compared to controls when subject cells are cycling and in G0. Subject cells transduced with wild-type ABAT ORF (ABAT-WT) show significantly increased mtDNA copy number (C) and membrane potential (D) when compared to subject cells transduced with GFP. Fibroblasts were allowed to recover from lentiviral infection and then grown in LSM for 7 days prior to harvesting for quantitation of mtDNA. ** indicates p-value < 1E-3. *** indicates p-value < 1E-5. Error bars indicate standard deviation.

Figure 5. ABAT functions in the conversion of dNDP to dNTP in the mitochondrial nucleoside salvage pathway

(A) Genes known to cause human mtDNA depletion syndromes (MDS) are shown with their function indicated as known. (B) Fibroblasts from two subjects with ABAT mutations (S1, S2) were tested in parallel with fibroblasts from individuals with MDS due to SUCLA2 mutation (homozygous p.Gly424Aspfs*18), DGUOK mutation (homozygous p.Phe256*), MEF from SUCLA2 −/− gene trap mouse and healthy control (C). Fibroblasts were synchronized in G0 by growth in low serum media (LSM) and then nucleosides (dNTP, dNDP, or dNMP) were added to replica-plated cells. ABAT and SUCLA2 mutant cells show the same level of mtDNA depletion in LSM (50% of control) and same response to media nucleoside supplementation: rescue of mtDNA depletion when either dATP/dGTP or dCTP/dTTP are added to media. (C) Fibroblasts transduced with ABAT shRNA show 50% mtDNA copy number compared to control and this is restored to 100% of control with media supplementation with dNTPs. (D–E) SUCLG1, SUCLG2, SUCLA2 and NME4 participate in the conversion of dNDP to dNTP in mitochondria. Co-IP experiments show that ABAT binds all four of these proteins. (F–K) Additionally, each possible pairwise binding between SUCLG1, SUCLG2, SUCLA2 and NME4 was tested. SUCLG1 binds SUCLG2, SUCLA2 and NME4. SUCLG2 binds NME4 but not SUCLA2. SUCLA2 also binds NME4. *** indicates p-value < 1E-5. Error bars indicate standard deviation.

Figure 6. Pharmacological inhibition of ABAT with Vigabatrin causes mtDNA depletion in photoreceptor cells that is rescued by media supplementation with dNTPs

(A) Photoreceptor cells were grown in the presence of Vigabatrin in varying doses (0 – 300 uM) and (B) Vigabatrin plus dNTPs. *** indicates p-value < 1E-5. Error bars indicate standard deviation.

Figure 7