Kara R. Vogel

Defects in GABA metabolism affect selective autophagy pathways and are alleviated by mTOR inhibition.

In addition to key roles in embryonic neurogenesis and myelinogenesis, γ‐aminobutyric acid (GABA) serves as the primary inhibitory mammalian neurotransmitter. In yeast, we have identified a new role for GABA that augments activity of the pivotal kinase, Tor1. GABA inhibits the selective autophagy pathways, mitophagy and pexophagy, through Sch9, the homolog of the mammalian kinase, S6K1, leading to oxidative stress, all of which can be mitigated by the Tor1 inhibitor, rapamycin. To confirm these processes in mammals, we examined the succinic semialdehyde dehydrogenase (SSADH)‐deficient mouse model that accumulates supraphysiological GABA in the central nervous system and other tissues. Mutant mice displayed increased mitochondrial numbers in the brain and liver, expected with a defect in mitophagy, and morphologically abnormal mitochondria. Administration of rapamycin to these mice reduced mTOR activity, reduced the elevated mitochondrial numbers, and normalized aberrant antioxidant levels. These results confirm a novel role for GABA in cell signaling and highlight potential pathomechanisms and treatments in various human pathologies, including SSADH deficiency, as well as other diseases characterized by elevated levels of GABA.

Disorders of GABA metabolism: SSADH and GABA-transaminase deficiencies

Clinical disorders known to affect inherited gamma-amino butyric acid (GABA) metabolism are autosomal recessively inherited succinic semialdehyde dehydrogenase and GABA-transaminase deficiency. The clinical presentation of succinic semialdehyde dehydrogenase deficiency includes intellectual disability, ataxia, obsessive-compulsive disorder and epilepsy with a nonprogressive course in typical cases, although a progressive form in early childhood as well as deterioration in adulthood with worsening epilepsy are reported. GABA-transaminase deficiency is associated with a severe neonatal-infantile epileptic encephalopathy.

Thirty Years Beyond Discovery—Clinical Trials in Succinic Semialdehyde Dehydrogenase Deficiency, a Disorder of GABA Metabolism

This review summarizes a presentation made at the retirement Symposium of Prof. Dr. Cornelis Jakobs in November of 2011, highlighting the progress toward clinical trials in succinic semialdehyde dehydrogenase (SSADH) deficiency, a disorder first recognized in 1981. Active and potential clinical interventions, including vigabatrin, L-cycloserine, the GHB receptor antagonist NCS-382, and the ketogenic diet, are discussed. Several biomarkers to gauge clinical efficacy have been identified, including cerebrospinal fluid metabolites, neuropsychiatric testing, MRI, EEG, and measures of GABAergic function including (11xa0C)flumazenil positron emission tomography (PET) and transcranial magnetic stimulation (TMS). Thirty years after its discovery, encompassing extensive studies in both patients and the corresponding murine model, we are now running an open-label trial of taurine intervention, and are poised to undertake a phase II trial of the GABAB receptor antagonist SGS742.

Non-physiological amino acid (NPAA) therapy targeting brain phenylalanine reduction: pilot studies in PAHENU2 mice

Transport of large neutral amino acids (LNAA) across the blood brain barrier (BBB) is facilitated by the L-type amino acid transporter, LAT1. Peripheral accumulation of one LNAA (e.g., phenylalanine (phe) in PKU) is predicted to increase uptake of the offending amino acid to the detriment of others, resulting in disruption of brain amino acid homeostasis. We hypothesized that selected non-physiological amino acids (NPAAs) such as DL-norleucine (NL), 2-aminonorbornane (NB; 2-aminobicyclo-(2,1,1)-heptane-2-carboxylic acid), 2-aminoisobutyrate (AIB), and N-methyl-aminoisobutyrate (MAIB), acting as competitive inhibitors of various brain amino acid transporters, could reduce brain phe in Pahenu2 mice, a relevant murine model of PKU. Oral feeding of 5xa0% NL, 5xa0% AIB, 0.5xa0% NB and 3xa0% MAIB reduced brain phe by 56xa0% (pu2009<u20090.01), -1xa0% (pu2009=u2009NS), 27xa0% (pu2009<u20090.05) and 14xa0% (pu2009<u20090.01), respectively, compared to untreated subjects. Significant effects on other LNAAs (tyrosine, methionine, branched chain amino acids) were also observed, however, with MAIB displaying the mildest effects. Of interest, MAIB represents an inhibitor of the system A (alanine) transporter that primarily traffics small amino acids and not LNAAs. Our studies represent the first in vivo use of these NPAAs in Pahenu2 mice, and provide proof-of-principle for their further preclinical development, with the long-term objective of identifying NPAA combinations and concentrations that selectively restrict brain phe transport while minimally impacting other LNAAs and downstream intermediates.

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.

Inherited disorders of gamma-aminobutyric acid metabolism and advances in ALDH5A1 mutation identification

Inherited disorders of gamma‐aminobutyric acid (GABA) metabolism include succinic semialdehyde dehydrogenase (SSADH) and gamma‐aminobutyric acid transaminase (GABA‐T) deficiencies. The clinical features, pathophysiology, diagnosis, and management of both, and an updated list of mutations in the ALDH5A1 gene, which cause SSADH deficiency, are discussed. A database of 112 individuals (71 children and adolescents, and 41 adults) indicates that developmental delay and hypotonia are the most common symptoms arising from SSADH deficiency. Furthermore, epilepsy is present in two‐thirds of SSADH‐deficient individuals by adulthood. Research with murine genetic models and human participants, using [11C] flumazenil positron emission tomography (FMZ‐PET) and transcranial magnetic stimulation, have led to therapeutic trials, and the identification of additional disruptions to GABA metabolism. Suggestions for new therapies have arisen from findings of GABAergic effects on autophagy, with enhanced activation of the mammalian target of rapamycin (mTOR) pathway. Details of known pathogenic mutations in the ALDH5A1 gene, three of which have not previously been reported, are summarized here. Investigations into disorders of GABA metabolism provide fundamental insights into the mechanisms underlying epilepsy, and support the importance of developing biomarkers and clinical trials. Comprehensive definition of phenotypes arising as a result of deficiencies in both SSADH and GABA‐T may increase our understanding of the neurophysiological consequences of a hyper‐GABAergic state.

Brain–blood amino acid correlates following protein restriction in murine maple syrup urine disease

BackgroundConventional therapy for patients with maple syrup urine disease (MSUD) entails restriction of protein intake to maintain acceptable levels of the branched chain amino acid, leucine (LEU), monitored in blood. However, no data exists on the correlation between brain and blood LEU with protein restriction, and whether correction in blood is reflected in brain.MethodsTo address this question, we fed intermediate MSUD mice diets of 19% (standard) and 6% protein, with collection of sera (SE), striata (STR), cerebellum (CE) and cortex (CTX) for quantitative amino acid analyses.ResultsLEU and valine (VAL) levels in all brain regions improved on average 28% when shifting from 19% to 6% protein, whereas the same improvements in SE were on average 60%. Isoleucine (ILE) in brain regions did not improve, while the SE level improved 24% with low-protein consumption. Blood-branched chain amino acids (LEU, ILE, and VAL in sera (SE)) were 362-434 μM, consistent with human values considered within control. Nonetheless, numerous amino acids in brain regions remained abnormal despite protein restriction, including glutamine (GLN), aspartate (ASP), glutamate (GLU), gamma-aminobutyric acid (GABA), asparagine (ASN), citrulline (CIT) and serine (SER). To assess the specificity of these anomalies, we piloted preliminary studies in hyperphenylalaninemic mice, modeling another large neutral aminoacidopathy. Employing an identical dietary regimen, we found remarkably consistent abnormalities in GLN, ASP, and GLU.ConclusionsOur results suggest that blood amino acid analysis may be a poor surrogate for assessing the outcomes of protein restriction in the large neutral amino acidopathies, and further indicate that chronic neurotransmitter disruptions (GLU, GABA, ASP) may contribute to long-term neurocognitive dysfunction in these disorders.

Correlation of blood biomarkers with age informs pathomechanisms in succinic semialdehyde dehydrogenase deficiency (SSADHD), a disorder of GABA metabolism.

We hypothesized that blood levels of γ-aminobutyric acid (GABA) and γ-hydroxybutyric acid (GHB), biomarkers of succinic semialdehyde dehydrogenase deficiency (SSADHD), would correlate with age. GABA and GHB were quantified in plasma and red blood cells (RBCs) from 18 patients (age range 5–41xa0years; median 8). Both metabolites negatively correlated with age (Pu2009<u20090.05). Plasma and RBC GHB declined with age, reaching a nadir and approximate steady state by 10xa0years. Declining plasma GABA achieved this approximate steady state at 30–40xa0years of age. These biomarker relationships may reflect further GABA- and GHB-ergic neurotransmission imbalances that correlate with the onset of adolescent/adulthood neuropsychiatric morbidity and epilepsy in SSADHD.

Aberrant mTOR signaling and disrupted autophagy: The missing link in potential vigabatrin‐associated ocular toxicity?

Vigabatrin (VGB; γ‐vinylGABA) is a unique antiepileptic directly elevating CNS GABA via inactivation of the GABA metabolic enzyme GABA‐transaminase. VGB is effective in treating infantile spasms, a rare seizure disorder associated with significant morbidity. The potential for unexplained bilateral constriction of the visual field associated with VGB intervention can severely limit its temporal utility. Removal of this potential adverse effect with adjuvant intervention(s) would represent a significant advance in epilepsy therapeutics.

Multicompartment analysis of protein-restricted phenylketonuric mice reveals amino acid imbalances in brain

BackgroundThe mainstay of therapy for phenylketonuria (PKU) remains dietary protein restriction. Developmental and neurocognitive outcomes for patients, however, remain suboptimal. We tested the hypothesis that mice with PKU receiving protein-restricted diets would reveal disruptions of brain amino acids that shed light on these neurocognitive deficits.MethodPhenylalanine hydroxylase-deficient (PKU) mice and parallel controls (both wild-type and heterozygous) were fed custom diets containing 18, 6, and 4xa0% protein for 3xa0weeks, after which tissues (brain, liver, sera) were collected for amino acid analysis profiling.ResultsPhenylalanine (phe) was increased in all tissues (pu2009<u20090.0001) of PKU mice and improved with protein restriction. In sera, decreased tyrosine (pu2009<u20090.01) was corrected (defined as not significantly different from the level in control mice receiving 18xa0% chow) with protein restriction, whereas protein restriction significantly increased many other amino acids. A similar trend for increased amino acid levels with protein restriction was also observed in liver. In brain, the effects of protein restriction on large neutral amino acids (LNAAs) were variable, with some deficit correction (threonine, methionine, glutamine) and no correction of tyrosine under any dietary paradigm. Protein restriction (4xa0% diet) in PKU mice significantly decreased lysine, arginine, taurine, glutamate, asparagine, and serine which had been comparable to control mice under 18xa0% protein intake.ConclusionDepletion of taurine, glutamate, and serine in the brain of PKU mice with dietary protein restriction may provide new insight into neurocognitive deficits of PKU.