Murine Succinate Semialdehyde Dehydrogenase Deficiency Research Articles

Maternal glutamine supplementation in murine succinic semialdehyde dehydrogenase deficiency, a disorder of γ-aminobutyric acid metabolism.

Murine succinic semialdehyde dehydrogenase deficiency (SSADHD) manifests with high concentrations of γ-aminobutyric acid (GABA) and γ-hydroxybutyrate (GHB) and low glutamine in the brain. To understand the pathogenic contribution of central glutamine deficiency, we exposed aldh5a1-/- (SSADHD) mice and their genetic controls (aldh5a1+/+ ) to either a 4% (w/w) glutamine-containing diet or a glutamine-free diet from conception until postnatal day 30. Endpoints included brain, liver and blood amino acids, brain GHB, ataxia scores, and open field testing. Glutamine supplementation did not improve aldh5a1-/- brain glutamine deficiency nor brain GABA and GHB. It decreased brain glutamate but did not change the ratio of excitatory (glutamate) to inhibitory (GABA) neurotransmitters. In contrast, glutamine supplementation significantly increased brain arginine (30% for aldh5a1+/+ and 18% for aldh5a1-/- mice), and leucine (12% and 18%). Glutamine deficiency was confirmed in the liver. The test diet increased hepatic glutamate in both genotypes, decreased glutamine in aldh5a1+/+ but not in aldh5a1-/- , but had no effect on GABA. Dried bloodspot analyses showed significantly elevated GABA in mutants (approximately 800% above controls) and decreased glutamate (approximately 25%), but no glutamine difference with controls. Glutamine supplementation did not impact blood GABA but significantly increased glutamine and glutamate in both genotypes indicating systemic exposure to dietary glutamine. Ataxia and pronounced hyperactivity were observed in aldh5a1-/- mice but remained unchanged by the diet intervention. The study suggests that glutamine supplementation improves peripheral but not central glutamine deficiency in experimental SSADHD. Future studies are needed to fully understand the pathogenic role of brain glutamine deficiency in SSADHD.

Therapeutic relevance of mTOR inhibition in murine succinate semialdehyde dehydrogenase deficiency (SSADHD), a disorder of GABA metabolism

Aldehyde dehydrogenase 5a1-deficient (aldh5a1−/−) mice, the murine orthologue of human succinic semialdehyde dehydrogenase deficiency (SSADHD), manifest increased GABA (4-aminobutyric acid) that disrupts autophagy, increases mitochondria number, and induces oxidative stress, all mitigated with the mTOR (mechanistic target of rapamycin) inhibitor rapamycin [1]. Because GABA regulates mTOR, we tested the hypothesis that aldh5a1−/− mice would show altered levels of mRNA for genes associated with mTOR signaling and oxidative stress that could be mitigated by inhibiting mTOR. We observed that multiple metabolites associated with GABA metabolism (γ-hydroxybutyrate, succinic semialdehyde, D-2-hydroxyglutarate, 4,5-dihydrohexanoate) and oxidative stress were significantly increased in multiple tissues derived from aldh5a1−/− mice. These metabolic perturbations were associated with decreased levels of reduced glutathione (GSH) in brain and liver of aldh5a1−/− mice, as well as increased levels of adducts of the lipid peroxidation by-product, 4-hydroxy-2-nonenal (4-HNE). Decreased liver mRNA levels for multiple genes associated with mTOR signaling and oxidative stress parameters were detected in aldh5a1−/− mice, and several were significantly improved with the administration of mTOR inhibitors (Torin 1/Torin 2). Western blot analysis of selected proteins corresponding to oxidative stress transcripts (glutathione transferase, superoxide dismutase, peroxiredoxin 1) confirmed gene expression findings. Our data provide additional preclinical evidence for the potential therapeutic efficacy of mTOR inhibitors in SSADHD.

MTOR inhibitors rescue premature lethality and attenuate dysregulation of GABAergic/glutamatergic transcription in murine succinate semialdehyde dehydrogenase deficiency (SSADHD), a disorder of GABA metabolism.

Recent studies have identified a role for supraphysiological gamma-aminobutyric acid (GABA) in the regulation of mechanistic target of rapamycin (mTOR), a protein kinase with pleiotropic roles in cellular development and homeostasis, including integration of growth factors and nutrient sensing and synaptic input in neurons (Lakhani et al. 2014; Vogel et al. 2015). Aldehyde dehydrogenase 5a1-deficient (aldh5a1 -/- ) mice, the murine orthologue of human succinic semialdehyde dehydrogenase deficiency (SSADHD), manifest increased GABA that disrupts mitophagy and increases mitochondria number with enhanced oxidant stress. Treatment with the mTOR inhibitor, rapamycin, significantly attenuates these GABA-related anomalies. We extend those studies through characterization of additional rapamycin analog (rapalog) agents including temsirolimus, dual mTOR inhibitors [Torin 1 and 2 (Tor 1/ Tor 2), Ku-0063794, and XL-765], as well as mTOR-independent autophagy inducers [trehalose, tat-Beclin 1, tacrolimus (FK-506), and NF-449) in aldh5a1 -/- mice. Rapamycin, Tor 1, and Tor 2 rescued these mice from premature lethality associated with status epilepticus. XL-765 extended lifespan significantly and induced weight gain in aldh5a1 -/- mice; untreated aldh5a1 -/- mice failed to increase body mass. Expression profiling of animals rescued with Tor 1/Tor 2 and XL-765 revealed multiple instances of pharmacological compensation and/or correction of GABAergic and glutamatergic receptors, GABA/glutamate transporters, and GABA/glutamate-associated proteins, with Tor 2 and XL-765 showing optimal outcomes. Our studies lay the groundwork for further evaluation of mTOR inhibitors in aldh5a1 -/- mice, with therapeutic ramifications for heritable disorders of GABA and glutamate neurotransmission.

Cerebellar Atrophy in Human and Murine Succinic Semialdehyde Dehydrogenase Deficiency

Human succinic semialdehyde dehydrogenase deficiency, an autosomal recessive disorder of γ-aminobutyric acid (GABA) catabolism, was modeled by a murine model sharing the phenotype of ataxia and seizures. Magnetic resonance imaging (MRI) with volumetry was obtained on 7 patients versus controls, and MRI with stereology was derived in 3 murine genotypes: null, wild-type, and heterozygous mutants. All patients had T1 hypointensity and T2 hyperintensity in globus pallidus, and 5 also had similar changes in subthalamic and cerebellar dentate nuclei. There was a trend for patients to have a smaller cerebellar vermis. Homozygous null mice had significantly lower total brain and cerebellar volumes than wild-types and heterozygotes. Stereology confirmed cerebellar atrophy and was otherwise normal in multiple regions. Cerebellar volume loss is present in the murine disorder with a trend for cerebellar atrophy in patients. Reduced cerebellar volume can reflect neurodegeneration and may be related to the clinical manifestations.

Circadian distribution of generalized tonic–clonic seizures associated with murine succinic semialdehyde dehydrogenase deficiency, a disorder of GABA metabolism

Human succinic semialdehyde dehydrogenase (SSADH) deficiency is an autosomal recessive disorder of GABA metabolism associated with motor impairment and epileptic seizures. Similarly, mice with targeted deletion of the Aldh5a1 gene ( Aldh5a1 –/–) exhibit SSADH deficiency and seizures early in life. These seizures begin as absence seizures the second week of life, but evolve into generalized convulsive seizures that increase in severity and become lethal during the fourth postnatal week. The seizures are alleviated and survival is prolonged when the mutant animals are weaned onto a ketogenic diet (KD). The persistence of spontaneous, recurrent, generalized tonic–clonic seizures in KD-treated adult Aldh5a1 –/– mice allowed us to quantify their daily (circadian) distribution using a novel behavioral method based on the detection of changes in movement velocity. Adult KD-treated Aldh5a1 –/– mice exhibited a seizure phenotype characterized by fits of wild running clonus accompanied by jumping and bouncing. These hypermotor seizures were largely spontaneous and occurred daily in a nonrandom pattern. The seizure rhythm showed a peak shortly after dark phase onset (2008 hours) with near-24-hour periodicity. Age-matched wild-type littermates showed no evidence of abnormal motor behavior. These new data suggest that generalized tonic–clonic seizures in Aldh5a1 –/– mice are more frequent during a specific time of day and will provide useful information to clinicians for the treatment of seizures associated with human SSADH deficiency.

Enzymatic and metabolic evidence for a region specific mitochondrial dysfunction in brains of murine succinic semialdehyde dehydrogenase deficiency ( Aldh5a1−/− mice)

Succinic semialdehyde dehydrogenase deficiency, a rare inherited defect of γ-aminobutyrate (GABA) catabolism, presents with characteristic biochemical abnormalities in the central nervous system (CNS). These include elevated concentrations of GABA, γ-hydroxybutyrate (GHB), succinic semialdehyde (SSA), 4,5-dihydroxyhexanoic acid (DHHA) and alanine as well as decreased concentrations of glutamine. GABA degradation is coupled to Krebs cycle function in mammalian CNS (“GABA shunt”) through succinate and α-ketoglutarate. Accordingly, we hypothesized that disruption of Krebs cycle and respiratory chain function in the CNS is involved in the neuropathogenesis of this disease. For this purpose, we investigated cerebral activities of Krebs cycle and respiratory chain enzymes as well as the glutathione content in Aldh5a1 −/− mice, a recently generated mouse model for this disease. In CNS tissue of Aldh5a1 −/− mice, we found a significantly decreased glutathione content (hippocampus, cortex) and decreased activities of complexes I–IV (hippocampus) suggesting increased oxidative stress and mitochondrial dysfunction. However, specific activities of Krebs cycle and respiratory chain were not affected by GABA, GHB, SSA, or DHHA (up to 1 mmol/L). Although our results suggest hippocampal and cortical dysfunction in Aldh5a1 −/− brain, we found no evidence that accumulating key metabolites of SSADH deficiency directly induce impairment of energy metabolism.

Lipid abnormalities in succinate semialdehyde dehydrogenase ( Aldh5a1− /− ) deficient mouse brain provide additional evidence for myelin alterations

Earlier work from our laboratory provided evidence for myelin abnormalities (decreased quantities of proteins associated with myelin compaction, decreased sheath thickness) in cortex and hippocampus of Aldh5a1 − /− mice, which have a complete ablation of the succinate semialdehyde dehydrogenase protein [E.A. Donarum, D.A. Stephan, K. Larkin, E.J. Murphy, M. Gupta, H. Senephansiri, R.C. Switzer, P.L. Pearl, O.C. Snead, C. Jakobs, K.M. Gibson, Expression profiling reveals multiple myelin alterations in murine succinate semialdehyde dehydrogenase deficiency, J. Inher. Metab. Dis. 29 (2006) 143–156]. In the current report, we have extended these findings via comprehensive analysis of brain phospholipid fractions, including quantitation of fatty acids in individual phospholipid subclasses and estimation of hexose-ceramide in Aldh5a1 − /− brain. In comparison to wild-type littermates ( Aldh5a1 +/+ ), we detected a 20% reduction in the ethanolamine glycerophospholipid content of Aldh5a1 − /− mice, while other brain phospholipids (choline glycerophospholipid, phosphatidylserine and phosphatidylinositol) were within normal limits. Analysis of individual fatty acids in each of these fractions revealed consistent alterations in n-3 fatty acids, primarily increased 22:6n-3 levels (docosahexaenoic acid; DHA). In the phosphatidyl serine fraction there were marked increases in the proportions of polyunsaturated fatty acids with corresponding decreases of monounsaturated fatty acids. Interestingly, the levels of hexose-ceramide (glucosyl- and galactosylceramide, principal myelin cerebrosides) were decreased in Aldh5a1 − /− brain tissue (one-tailed t test, p = 0.0449). The current results suggest that lipid and myelin abnormalities in this animal may contribute to the pathophysiology.

Expression profiling reveals multiple myelin alterations in murine succinate semialdehyde dehydrogenase deficiency

Succinic semialdehyde dehydrogenase (SSADH) deficiency, a rare genetic defect of GABA degradation recently modelled in mice (SSADH(-/-) mice), manifests early absence seizures that evolve into generalized convulsive seizures and lethal status epilepticus in gene-ablated mice. Disrupted GABA homeostasis, in conjunction with the epileptic phenotype and increased gamma-hydroxybutyric acid (GHB), suggested that expression profiling with the U74Av2 Affymetrix system would reveal dysregulation of receptor genes associated with GABAergic and glutamatergic neurotransmission. Unexpectedly, we found significant downregulation for genes associated with myelin biogenesis and compaction, predominantly in hippocampus and cortex. These results were confirmed by: (1) myelin basic protein (MBP) immunohistochemistry; (2) western blotting of myelin-associated glycoprotein (MAG) and MBP; (3) qRT-PCR analyses of myelin-associated oligodendrocytic basic protein (MOBP), MAG, MBP and proteolipid protein (PLP) in hippocampus, cortex and spinal cord; (4) quantitation of ethanolamine and choline plasmalogens, all core myelin components; (5) evaluation of myelin content in brain sections employing toluidine blue staining; and (6) ultrastructural evaluation of myelin sheath thickness via electron microscopy. We speculate that increased GABA/GHB, acting through GABAergic systems, results in decreased levels of the neurosteroids progesterone and allopregnanolone [Gupta et al (2003) Ann Neurol 54(Supplement 6): S81-S90] and phosphorylation of mitogen-activated protein (MAP) kinase, with resulting myelin protein abnormalities primarily in the cortex of SSADH(-/-) mice.

Liver-Directed Adenoviral Gene Transfer in Murine Succinate Semialdehyde Dehydrogenase Deficiency

Murine succinate semialdehyde dehydrogenase (SSADH) deficiency (OMIM 271980; EC 1.2.1.24), a model of the corresponding human disorder, displays 100% mortality at weeks 3–4 of life, associated with lethal tonic–clonic seizures. The biochemical hallmark, γ-hydroxybutyrate (GHB), accumulates in both human and murine disorders. In the current study we evaluated rescue of the murine model with liver-directed gene therapy using the E1-deleted adenoviral vector AD:pAD-RSV-humanSSADH. Our working hypotheses were: (1) liver expresses considerable SSADH activity and therefore represents a major source of GHB output, (2) correction of liver enzyme deficiency will reduce GHB load both peripherally and in the central nervous system, and (3) SSADH expression will improve survival. SSADH−/− and SSADH+/+ mice were treated under two protocols: (A) intraperitoneal injection of 108–1011 viral particles by day 10 of life or (B) retro-orbital injection of 1011 viral particles at day 13 of life. Intravenous administration was prohibited by the small size and fragility of the mice. Maximal survival (39%; P<0.001) was achieved with intraperitoneal administration (108 particles) at day 10; intraperitoneal (1010 and 1011 particles) and retro-orbital administration (1011 particles) yielded lower survival of 11–25% (P<0.02). Under both protocols, the maximal hepatic SSADH enzyme activity was ∼20% of SSADH+/+ liver activity (retro-orbital > ip). At various time points postinjection, ip-treated animals (108 viral particles) demonstrated upward of 80% reduction in liver GHB concentrations, with little impact on brain or serum GHB levels except at 48–72 h posttreatment (∼50% reduction for both tissues). Accordingly, we harvested retro-orbitally treated animals at 72 h and observed significant reductions of 60–70% for GHB in liver, kidney, serum, and brain extracts. Histochemical analysis of liver from retro-orbitally treated mutants demonstrated substantial SSADH staining, but with variability both within tissues and between animals. Our studies provide proof-of-principle that liver-mediated gene therapy has efficacy in treating SSADH deficiency and that hepatic tissue contributes significantly to the pool of GHB within the CNS.

Murine succinate semialdehyde dehydrogenase deficiency.

Inherited succinic semialdehyde dehydrogenase (SSADH) deficiency (gamma-hydroxybutyric aciduria) is one of the few neurogenetic disorders of GABA metabolism, and one in which tonic-clonic seizures associate with increased central nervous system GABA and gamma-hydroxybutyrate (GHB). To explore pathomechanisms and develop new preclinical treatment approaches, we developed a murine knockout model of SSADH deficiency. In the absence of intervention, SSADH(-/-) mice suffer 100% mortality at week 3 to 4 of life from generalized tonic-clonic seizures. In this report, we summarize earlier studies indicating disruption of the GABA/glutamine axis in SSADH(-/-) mouse brain, effective pharmacotherapeutic approaches, preliminary gene-therapy results, and electrophysiological analyses of mutant mice. We also present new evidence for oxidative stress in SSADH(-/-) mice, significant alterations of dopamine metabolism, and abnormal neurosteroid levels in brain, potentially implicating the GABA(A) receptor in pathogenesis. In SSADH deficiency, the accumulation of two neuroactive species, GABA and GHB, is significant because GABA is one of the earliest transmitters expressed in mammals, with key roles in synaptogenesis and myelination, whereas GHB displays a vast array of pharmacological actions. The SSADH(-/-) mouse may represent a useful model in which to explore the effect of GABA and GHB accumulation on central nervous system development and function.