ABSTRACT
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.
Subject(s)
Gene Expression Profiling/methods , Myelin Sheath/genetics , Myelin Sheath/metabolism , Succinate-Semialdehyde Dehydrogenase/deficiency , Animals , Brain/enzymology , Disease Models, Animal , Gene Expression Regulation, Enzymologic , Genotype , Mice , Mice, Transgenic , Models, Biological , Oligonucleotide Array Sequence Analysis , PhenotypeABSTRACT
Mice with targeted deletion of the GABA-degradative enzyme succinate semialdehyde dehydrogenase (SSADH; Aldh5a1; OMIM 271,980) manifest globally elevated GABA and regionally decreased arginine in brain extracts. We examined the hypothesis that arginine-glycine amidinotransferase catalyzed the formation of guanidinobutyrate (GB) from increased GABA by quantifying guanidinoacetate (GA), guanidinopropionate (GP) and GB in brain extracts employing stable isotope dilution gas chromatographic-mass spectrometry. GA and GB were up to 4- and 22-fold elevated, respectively, in total and regional (cerebellum, hippocampus, cortex) brain extracts derived from SSADH(-/-) mice. Corresponding analyses of urine and cerebrospinal fluid derived from SSADH-deficient patients revealed significant (P<0.05) elevations of GA and GB in urine, as well as GB levels in CSF. These data suggest that GB may be an additional marker of SSADH deficiency, implicate additional pathways of pathophysiology, and identify the second instance of elevated GB in a human inborn error of metabolism.
Subject(s)
Glycine/analogs & derivatives , Guanidines/metabolism , Propionates/metabolism , Succinate-Semialdehyde Dehydrogenase/deficiency , Animals , Brain/metabolism , Child , Child, Preschool , Female , Gas Chromatography-Mass Spectrometry , Glycine/cerebrospinal fluid , Glycine/metabolism , Glycine/urine , Guanidines/cerebrospinal fluid , Guanidines/urine , Humans , Infant , Male , Mice , Mice, Knockout , Propionates/cerebrospinal fluid , Propionates/urineABSTRACT
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, gamma-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 10(8)-10(11) viral particles by day 10 of life or (B) retro-orbital injection of 10(11) 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 (10(8) particles) at day 10; intraperitoneal (10(10) and 10(11) particles) and retro-orbital administration (10(11) particles) yielded lower survival of 11-25% (P<0.02). Under both protocols, the maximal hepatic SSADH enzyme activity was approximately 20% of SSADH(+/+) liver activity (retro-orbital > ip). At various time points postinjection, ip-treated animals (10(8) 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 (approximately 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.
