Cellular and molecular outcomes of glutamine supplementation in the brain of succinic semialdehyde dehydrogenase-deficient mice.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) manifests with low levels of glutamine in the brain, suggesting that central glutamine deficiency contributes to pathogenesis. Recently, we attempted to rescue the disease phenotype of aldh5a1 -/- mice, a murine model of SSADHD with dietary glutamine supplementation. No clinical rescue and no central glutamine improvement were observed. Here, we report the results of follow-up studies of the cellular and molecular basis of the resistance of the brain to glutamine supplementation. We first determined if the expression of genes involved in glutamine metabolism was impacted by glutamine feeding. We then searched for changes of brain histology in response to glutamine supplementation, with a focus on astrocytes, known regulators of glutamine synthesis in the brain. Glutamine supplementation significantly modified the expression of glutaminase (gls) (0.6-fold down), glutamine synthetase (glul) (1.5-fold up), and glutamine transporters (solute carrier family 7, member 5 [slc7a5], 2.5-fold up; slc38a2, 0.6-fold down). The number of GLUL-labeled cells was greater in the glutamine-supplemented group than in controls (P < .05). Reactive astrogliosis, a hallmark of brain inflammation in SSADHD, was confirmed. We observed a 2-fold stronger astrocyte staining in mutants than in wild-type controls (optical density/cell were 1.8 ± 0.08 in aldh5a1 -/- and 0.99 ± 0.06 in aldh5a1 +/+ ; P < .0001), and a 3-fold higher expression of gfap and vimentin. However, glutamine supplementation did not improve the histological and molecular signature of astrogliosis. Thus, glutamine supplementation impacts genes implicated in central glutamine homeostasis without improving reactive astrogliosis. The mechanisms underlying glutamine deficiency and its contribution to SSADHD pathogenesis remain unknown and should be the focus of future investigations.

Transcriptome analysis in mice treated with vigabatrin identifies dysregulation of genes associated with retinal signaling circuitry.

Vigabatrin (VGB; γ-vinyl-GABA) is an antiepileptic drug that elevates CNS GABA via irreversible inactivation of the GABA catabolic enzyme GABA-transaminase. VGB's clinical utility, however, can be curtailed by peripheral visual field constriction (pVFC) and thinning of the retinal nerve fiber layer (RNFL). Earlier studies from our laboratory revealed disruptions of autophagy by VGB. Here, we tested the hypothesis that VGB administration to animals would reveal alterations of gene expression in VGB-treated retina that associated with autophagy. VGB (140 mg/kg/d; subcutaneous minipump) was continuously administered to mice (n = 6 each VGB/vehicle) for 12 days, after which animals were euthanized. Retina was isolated for transcriptome (RNAseq) analysis and further validation using qRT-PCR and immunohistochemistry (IHC). For 112 differentially expressed retinal genes (RNAseq), two databases (Gene Ontology; Kyoto Encyclopedia of Genes and Genomes) were used to identify genes associated with visual function. Twenty four genes were subjected to qRT-PCR validation, and five (Gb5, Bdnf, Cplx9, Crh, Sox9) revealed significant dysregulation. IHC of fixed retinas verified significant down-regulation of Gb5 in photoreceptor cells. All of these genes have been previously shown to play a role in retinal function/circuitry signaling. Minimal impact of VGB on retinal autophagic gene expression was observed. This is the first transcriptome analysis of retinal gene expression associated with VGB intake, highlighting potential novel molecular targets potentially related to VGB's well known ocular toxicity.

Longitudinal metabolomics in dried bloodspots yields profiles informing newborn screening for succinic semialdehyde dehydrogenase deficiency.

Analyses of 19 amino acids, 38 acylcarnitines, and 3 creatine analogues (https://clir.mayo.edu) were implemented to test the hypothesis that succinic semialdehyde dehydrogenase deficiency (SSADHD) could be identified in dried bloodspots (DBS) using currently available newborn screening methodology. The study population included 17 post-newborn SSADHD DBS (age range 0.8-38 years; median, 8.2 years; 10 M; controls, 129-353 age-matched individuals, mixed gender) and 10 newborn SSADHD DBS (including first and second screens from 3 of 7 patients). Low (informative) markers in post-newborn DBS included C2- and C4-OH carnitines, ornithine, histidine and creatine, with no gender differences. For newborn DBS, informative markers included C2-, C3-, C4- and C4-OH carnitines, creatine and ornithine. Of these, only creatine demonstrated a significant change with age, revealing an approximate 4-fold decrease. We conclude that quantitation of short-chain acylcarnitines, creatine, and ornithine provides a newborn DBS profile with potential as a first tier screening tool for early detection of SSADHD. This first tier evaluation can be readily verified using a previously described second tier liquid chromatography-tandem mass spectrometry method for γ-hydroxybutyric acid in the same DBS. More extensive evaluation of this first/second tier screening approach is needed in a larger population.

Correction to: Post-mortem tissue analyses in a patient with succinic semialdehyde dehydrogenase deficiency (SSADHD). I. Metabolomic outcomes.

Upon publication, it was noted that five of the on-line supplementary figures had incorrect figure: figure legend associations. These were supplementary Figs. 6, 7, 14, 15, and 23.

Post-mortem tissue analyses in a patient with succinic semialdehyde dehydrogenase deficiency (SSADHD). I. Metabolomic outcomes.

Metabolomic characterization of post-mortem tissues (frontal and parietal cortices, pons, cerebellum, hippocampus, cerebral cortex, liver and kidney) derived from a 37 y.o. male patient with succinic semialdehyde dehydrogenase deficiency (SSADHD) was performed in conjunction with four parallel series of control tissues. Amino acids, acylcarnitines, guanidino- species (guanidinoacetic acid, creatine, creatinine) and GABA-related intermediates were quantified using UPLC and mass spectrometric methods that included isotopically labeled internal standards. Amino acid analyses revealed significant elevation of aspartic acid and depletion of glutamine in patient tissues. Evidence for disruption of short-chain fatty acid metabolism, manifest as altered C4OH, C5, C5:1, C5DC (dicarboxylic) and C12OH carnitines, was observed. Creatine and guanidinoacetic acids were decreased and elevated, respectively. GABA-associated metabolites (total GABA, γ-hydroxybutyric acid, succinic semialdehyde, 4-guanidinobutyrate, 4,5-dihydroxyhexanoic acid and homocarnosine) were significantly increased in patient tissues, including liver and kidney. The data support disruption of fat, creatine and amino acid metabolism as a component of the pathophysiology of SSADHD, and underscore the observation that metabolites measured in patient physiological fluids provide an unreliable reflection of brain metabolism.

Temporal metabolomics in dried bloodspots suggests multipathway disruptions in aldh5a1-/- mice, a model of succinic semialdehyde dehydrogenase deficiency.

Succinic semialdehyde dehydrogenase (SSADH) deficiency (SSADHD; OMIM 271980) is a rare disorder featuring accumulation of neuroactive 4-aminobutyric acid (GABA; γ-aminobutyric acid, derived from glutamic acid) and 4-hydroxybutyric acid (γ-hydroxybutyric acid; GHB, a short-chain fatty acid analogue of GABA). Elevated GABA is predicted to disrupt the GABA shunt linking GABA transamination to the Krebs cycle and maintaining the balance of excitatory:inhibitory neurotransmitters. Similarly, GHB (or a metabolite) is predicted to impact ß-oxidation flux. We explored these possibilities employing temporal metabolomics of dried bloodspots (DBS), quantifying amino acids, acylcarnitines, and guanidino- metabolites, derived from aldh5a1+/+, aldh5a1+/- and aldh5a1-/- mice (aldehyde dehydrogenase 5a1 = SSADH) at day of life (DOL) 20 and 42 days. At DOL 20, aldh5a1-/- mice had elevated C6 dicarboxylic (adipic acid) and C14 carnitines and threonine, combined with a significantly elevated ratio of threonine/[aspartic acid + alanine], in comparison to aldh5a1+/+ mice. Conversely, at DOL 42 aldh5a1-/- mice manifested decreased short chain carnitines (C0-C6), valine and glutamine, in comparison to aldh5a1+/+ mice. Guanidino species, including creatinine, creatine and guanidinoacetic acid, evolved from normal levels (DOL 20) to significantly decreased values at DOL 42 in aldh5a1-/- as compared to aldh5a1+/+ mice. Our results provide a novel temporal snapshot of the evolving metabolic profile of aldh5a1-/- mice while highlighting new pathomechanisms in SSADHD.

Gamma-Hydroxybutyrate content in dried bloodspots facilitates newborn detection of succinic semialdehyde dehydrogenase deficiency.

Increased gamma-hydroxybutyric acid in urine and blood are metabolic hallmarks of succinic semialdehyde dehydrogenase deficiency, a defect of 4-aminobutyric acid metabolism. Here, we examined the hypothesis that succinic semialdehyde dehydrogenase deficiency could be identified via measurement of gamma-hydroxybutyric acid in newborn and post-newborn dried bloodspots. Quantitation of gamma-hydroxybutyric acid using liquid chromatography-tandem mass spectrometry in twelve archival newborn patient dried bloodspots was 360 ±â€¯57 µM (mean, standard error; range 111-767), all values exceeding the previously established cutoff for newborn detection of 78 µΜ established from 2831 dried bloodspots derived from newborns, neonates and children. Gamma-hydroxybutyric acid in post-newborn dried bloodspots (n = 19; ages 0.8-38 years) was 191 ±â€¯65 µM (mean, standard error; range 20-1218), exceeding the aforementioned GHB cutoff for patients approximately 10 years of age or younger. Further, gamma-hydroxybutyric acid in post-newborn dried bloodspots displayed a significant (p < .0001) inverse correlation with age. This preliminary study suggests that succinic semialdehyde dehydrogenase deficiency may be identified in newborn and post-newborn dried bloodspots via quantitation of gamma-hydroxybutyric acid, while forming the platform for more extensive studies in affected and unaffected dried bloodspots.

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.

Metabolomic analyses of vigabatrin (VGB)-treated mice: GABA-transaminase inhibition significantly alters amino acid profiles in murine neural and non-neural tissues.

The anticonvulsant vigabatrin (VGB; SabrilR) irreversibly inhibits GABA transaminase to increase neural GABA, yet its mechanism of retinal toxicity remains unclear. VGB is suggested to alter several amino acids, including homocarnosine, ß-alanine, ornithine, glycine, taurine, and 2-aminoadipic acid (AADA), the latter a homologue of glutamic acid. Here, we evaluate the effect of VGB on amino acid concentrations in mice, employing a continuous VGB infusion (subcutaneously implanted osmotic minipumps), dose-escalation paradigm (35-140 mg/kg/d, 12 days), and amino acid quantitation in eye, visual and prefrontal cortex, total brain, liver and plasma. We hypothesized that continuous VGB dosing would reveal numerous hitherto undescribed amino acid disturbances. Consistent amino acid elevations across tissues included GABA, ß-alanine, carnosine, ornithine and AADA, as well as neuroactive aspartic and glutamic acids, serine and glycine. Maximal increase of AADA in eye occurred at 35 mg/kg/d (41 ±â€¯2 nmol/g (n = 21, vehicle) to 60 ±â€¯8.5 (n = 8)), and at 70 mg/kg/d for brain (97 ±â€¯6 (n = 21) to 145 ±â€¯6 (n = 6)), visual cortex (128 ±â€¯6 to 215 ±â€¯19) and prefrontal cortex (124 ±â€¯11 to 200 ±â€¯13; mean ±â€¯SEM; p < 0.05), the first demonstration of tissue AADA accumulation with VGB in mammal. VGB effects on basic amino acids, including guanidino-species, suggested the capacity of VGB to alter urea cycle function and nitrogen disposal. The known toxicity of AADA in retinal glial cells highlights new avenues for assessing VGB retinal toxicity and other off-target effects.

Rett syndrome (MECP2) and succinic semialdehyde dehydrogenase (ALDH5A1) deficiency in a developmentally delayed female.

BACKGROUND: We present a patient with Rett syndrome (RTT; MECP2) and autosomal-recessive succinic semialdehyde dehydrogenase deficiency (SSADHD; ALDH5A1 (aldehyde dehydrogenase 5a1 = SSADH), in whom the current phenotype exhibits features of SSADHD (hypotonia, global developmental delay) and RTT (hand stereotypies, gait anomalies). METHODS: γ-Hydroxybutyric acid (GHB) was quantified by UPLC-tandem mass spectrometry, while mutation analysis followed standard methodology of whole-exome sequencing. RESULTS: The biochemical hallmark of SSADHD, GHB was increased in the proband's dried bloodspot (DBS; 673 µM; previous SSADHD DBSs (n = 7), range 124-4851 µM); control range (n = 2,831), 0-78 µM. The proband was compound heterozygous for pathogenic ALDH5A1 mutations (p.(Asn418IlefsTer39); maternal; p.(Gly409Asp); paternal) and a de novo RTT nonsense mutation in MECP2 (p.Arg255*). CONCLUSION: The major inhibitory neurotransmitter, γ-aminobutyric acid (GABA), is increased in SSADHD but normal in RTT, although there are likely regional changes in GABA receptor distribution. GABAergic anomalies occur in both disorders, each featuring an autism spectrum phenotype. What effect the SSADHD biochemical anomalies (elevated GABA, GHB) might play in the neurodevelopmental/epileptic phenotype of our patient is currently unknown.

Emerging role of proprotein convertase subtilisin/kexin type-9 (PCSK-9) in inflammation and diseases.

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is most recognized serine protease for its role in cardiovascular diseases (CVD). PCSK9 regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by selectively targeting hepatic LDL receptors (LDLR) for degradation, thereby serving as a potential therapeutic target for CVD. New pharmacological agents under development aim to lower the risk of CVD by inhibiting PCSK9 extracellularly, although secondary effects of this approach are not yet studied. Here we review the history of PCSK9 and rationale behind developing inhibitors for CVD. Importantly, we summarized the studies investigating the role and impact of modulated PCSK9 levels in inflammation, specifically in sepsis, rheumatoid arthritis and other chronic inflammatory conditions. Furthermore, we summarized studies that investigated the interactions of PCSK9 with pro-inflammatory pathways, such as scavenger receptor CD36 and thrombospondin 1 (TSP-1) in inflammatory diseases. This review highlights the conflicting role that PCSK9 plays in different inflammatory disease states and postulates that any unwanted effects of PCSK9 inhibition in early clinical testing should critically be examined.

Age-related phenotype and biomarker changes in SSADH deficiency.

Objective: Succinic Semialdehyde Dehydrogenase (SSADH) deficiency is a disorder of elevated gamma-amino butyric acid (GABA) and gamma hydroxybutyric acid (GHB) and a complex neuropsychiatric profile. Adult reports suggest worsening epilepsy and high SUDEP risk. Methods: Subjects with confirmed SSADH deficiency were recruited into a longitudinal study. Plasma thyroid hormone and total GABA/GHB were quantified by standard clinical chemistry methodologies and mass spectrometry, respectively. Results: A total of 133 subjects with SSADH deficiency are enrolled in the registry; 49 participated in the longitudinal study. The age range of the population is 8 weeks to 63 years (median 7.75 year; 44% male). There is a significant difference in proportions among the age groups in subjects affected with hypotonia, compulsive behavior, sleep disturbances, and seizures. Epilepsy is present in 50% of the total population, and more prevalent in subjects 12 years and older (P = 0.001). The median age of onset for absence seizures was 2 years, and 12 years for generalized tonic-clonic seizures (P < 0.01). The SUDEP rate in adults was 12% (4/33). There was a significant age-dependent negative correlation between GABA and T3 levels. Interpretation: There is an age-dependent association with worsening of epilepsy, behavioral disturbances including obsessive-compulsive behavior, and sleep disturbances with age in SSADH deficiency. There is a high risk of SUDEP. We have observed more absence seizures in younger patients, compared to tonic-clonic in the older cohort, which correlates with age-related changes in GABA and GHB concentration and thyroid function, as well as the natural history of seizures in the murine model.

Preclinical tissue distribution and metabolic correlations of vigabatrin, an antiepileptic drug associated with potential use-limiting visual field defects.

Vigabatrin (VGB; (S)-(+)/(R)-(-) 4-aminohex-5-enoic acid), an antiepileptic irreversibly inactivating GABA transaminase (GABA-T), manifests use-limiting ocular toxicity. Hypothesizing that the active S enantiomer of VGB would preferentially accumulate in eye and visual cortex (VC) as one potential mechanism for ocular toxicity, we infused racemic VGB into mice via subcutaneous minipump at 35, 70, and 140 mg/kg/d (n = 6-8 animals/dose) for 12 days. VGB enantiomers, total GABA and ß-alanine (BALA), 4-guanidinobutyrate (4-GBA), and creatine were quantified by mass spectrometry in eye, brain, liver, prefrontal cortex (PFC), and VC. Plasma VGB concentrations increased linearly by dose (3 ± 0.76 (35 mg/kg/d); 15.1 ± 1.4 (70 mg/kg/d); 34.6 ± 3.2 µmol/L (140 mg/kg/d); mean ± SEM) with an S/R ratio of 0.74 ± 0.02 (n = 14). Steady state S/R ratios (35, 70 mg/kg/d doses) were highest in eye (5.5 ± 0.2; P < 0.0001), followed by VC (3.9 ± 0.4), PFC (3.6 ± 0.3), liver (2.9 ± 0.1), and brain (1.5 ± 0.1; n = 13-14 each). Total VGB content of eye exceeded that of brain, PFC and VC at all doses. High-dose VGB diminished endogenous metabolite production, especially in PFC and VC. GABA significantly increased in all tissues (all doses) except brain; BALA increases were confined to liver and VC; and 4-GBA was prominently increased in brain, PFC and VC (and eye at high dose). Linear correlations between enantiomers and GABA were observed in all tissues, but only in PFC/VC for BALA, 4-GBA, and creatine. Preferential accumulation of the VGB S isomer in eye and VC may provide new insight into VGB ocular toxicity.

Involvement of adipose tissue inflammation and dysfunction in virus-induced type 1 diabetes.

The etiopathogenesis of type 1 diabetes (T1D) remains poorly understood. We used the LEW1.WR1 rat model of Kilham rat virus (KRV)-induced T1D to better understand the role of the innate immune system in the mechanism of virus-induced disease. We observed that infection with KRV results in cell influx into visceral adipose tissue soon following infection prior to insulitis and hyperglycemia. In sharp contrast, subcutaneous adipose tissue is free of cellular infiltration, whereas ß cell inflammation and diabetes are observed beginning on day 14 post infection. Immunofluorescence studies further demonstrate that KRV triggers CD68+ macrophage recruitment and the expression of KRV transcripts and proinflammatory cytokines and chemokines in visceral adipose tissue. Adipocytes from naive rats cultured in the presence of KRV express virus transcripts and upregulate cytokine and chemokine gene expression. KRV induces apoptosis in visceral adipose tissue in vivo, which is reflected by positive TUNEL staining and the expression of cleaved caspase-3. Moreover, KRV leads to an oxidative stress response and downregulates the expression of adipokines and genes associated with mediating insulin signaling. Activation of innate immunity with Poly I:C in the absence of KRV leads to CD68+ macrophage recruitment to visceral adipose tissue and a decrease in adipokine expression detected 5 days following Poly (I:C) treatment. Finally, proof-of-principle studies show that brief anti-inflammatory steroid therapy suppresses visceral adipose tissue inflammation and protects from virus-induced disease. Our studies provide evidence raising the hypothesis that visceral adipose tissue inflammation and dysfunction may be involved in early mechanisms triggering ß cell autoimmunity.