Deficient brain GABA metabolism leads to widespread impairments of astrocyte and oligodendrocyte function.

The neurometabolic disorder succinic semialdehyde dehydrogenase (SSADH) deficiency leads to great neurochemical imbalances and severe neurological manifestations. The cause of the disease is loss of function of the enzyme SSADH, leading to impaired metabolism of the principal inhibitory neurotransmitter GABA. Despite the known identity of the enzymatic deficit, the underlying pathology of SSADH deficiency remains unclear. To uncover new mechanisms of the disease, we performed an untargeted integrative analysis of cerebral protein expression, functional metabolism, and lipid composition in a genetic mouse model of SSADH deficiency (ALDH5A1 knockout mice). Our proteomic analysis revealed a clear regional vulnerability, as protein alterations primarily manifested in the hippocampus and cerebral cortex of the ALDH5A1 knockout mice. These regions displayed aberrant expression of proteins linked to amino acid homeostasis, mitochondria, glial function, and myelination. Stable isotope tracing in acutely isolated brain slices demonstrated an overall maintained oxidative metabolism of glucose, but a selective decrease in astrocyte metabolic activity in the cerebral cortex of ALDH5A1 knockout mice. In contrast, an elevated capacity of oxidative glutamine metabolism was observed in the ALDH5A1 knockout brain, which may serve as a neuronal compensation of impaired astrocyte glutamine provision. In addition to reduced expression of critical oligodendrocyte proteins, a severe depletion of myelin-enriched sphingolipids was found in the brains of ALDH5A1 knockout mice, suggesting degeneration of myelin. Altogether, our study highlights that impaired astrocyte and oligodendrocyte function is intimately linked to SSADH deficiency pathology, suggesting that selective targeting of glial cells may hold therapeutic potential in this disease.

ALDH5A1-deficient iPSC-derived excitatory and inhibitory neurons display cell type specific alterations.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a neurometabolic disorder caused by ALDH5A1 mutations presenting with autism and epilepsy. SSADHD leads to impaired GABA metabolism and results in accumulation of GABA and γ-hydroxybutyrate (GHB), which alter neurotransmission and are thought to lead to neurobehavioral symptoms. However, why increased inhibitory neurotransmitters lead to seizures remains unclear. We used induced pluripotent stem cells from SSADHD patients (one female and two male) and differentiated them into GABAergic and glutamatergic neurons. SSADHD iGABA neurons show altered GABA metabolism and concomitant changes in expression of genes associated with inhibitory neurotransmission. In contrast, glutamatergic neurons display increased spontaneous activity and upregulation of mitochondrial genes. CRISPR correction of the pathogenic variants or SSADHD mRNA expression rescue various metabolic and functional abnormalities in human neurons. Our findings uncover a previously unknown role for SSADHD in excitatory human neurons and provide unique insights into the cellular and molecular basis of SSADHD and potential therapeutic interventions.

Consensus guidelines for the diagnosis and management of succinic semialdehyde dehydrogenase deficiency.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) (OMIM #271980) is a rare autosomal recessive metabolic disorder caused by pathogenic variants of ALDH5A1. Deficiency of SSADH results in accumulation of γ-aminobutyric acid (GABA) and other GABA-related metabolites. The clinical phenotype of SSADHD includes a broad spectrum of non-pathognomonic symptoms such as cognitive disabilities, communication and language deficits, movement disorders, epilepsy, sleep disturbances, attention problems, anxiety, and obsessive-compulsive traits. Current treatment options for SSADHD remain supportive, but there are ongoing attempts to develop targeted genetic therapies. This study aimed to create consensus guidelines for the diagnosis and management of SSADHD. Thirty relevant statements were initially addressed by a systematic literature review, resulting in different evidence levels of strength according to the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) criteria. The highest level of evidence (level A), based on randomized controlled trials, was unavailable for any of the statements. Based on cohort studies, Level B evidence was available for 12 (40%) of the statements. Thereupon, through a process following the Delphi Method and directed by the Appraisal of Guidelines for Research and Evaluation (AGREE II) criteria, expert opinion was sought, and members of an SSADHD Consensus Group evaluated all the statements. The group consisted of neurologists, epileptologists, neuropsychologists, neurophysiologists, metabolic disease specialists, clinical and biochemical geneticists, and laboratory scientists affiliated with 19 institutions from 11 countries who have clinical experience with SSADHD patients and have studied the disorder. Representatives from parent groups were also included in the Consensus Group. An analysis of the survey's results yielded 25 (83%) strong and 5 (17%) weak agreement strengths. These first-of-their-kind consensus guidelines intend to consolidate and unify the optimal care that can be provided to individuals with SSADHD.

Gene replacement therapies for inherited disorders of neurotransmission: Current progress in succinic semialdehyde dehydrogenase deficiency.

Neurodevelopment is a highly organized and complex process involving lasting and often irreversible changes in the central nervous system. Inherited disorders of neurotransmission (IDNT) are a group of genetic disorders where neurotransmission is primarily affected, resulting in abnormal brain development from early life, manifest as neurodevelopmental disorders and other chronic conditions. In principle, IDNT (particularly those of monogenic causes) are amenable to gene replacement therapy via precise genetic correction. However, practical challenges for gene replacement therapy remain major hurdles for its translation from bench to bedside. We discuss key considerations for the development of gene replacement therapies for IDNT. As an example, we describe our ongoing work on gene replacement therapy for succinic semialdehyde dehydrogenase deficiency, a GABA catabolic disorder.

The continuously evolving phenotype of succinic semialdehyde dehydrogenase deficiency.

The objective of the study is to evaluate the evolving phenotype and genetic spectrum of patients with succinic semialdehyde dehydrogenase deficiency (SSADHD) in long-term follow-up. Longitudinal clinical and biochemical data of 22 pediatric and 9 adult individuals with SSADHD from the patient registry of the International Working Group on Neurotransmitter related Disorders (iNTD) were studied with in silico analyses, pathogenicity scores and molecular modeling of ALDH5A1 variants. Leading initial symptoms, with onset in infancy, were developmental delay and hypotonia. Year of birth and specific initial symptoms influenced the diagnostic delay. Clinical phenotype of 26 individuals (median 12 years, range 1.8-33.4 years) showed a diversifying course in follow-up: 77% behavioral problems, 76% coordination problems, 73% speech disorders, 58% epileptic seizures and 40% movement disorders. After ataxia, dystonia (19%), chorea (11%) and hypokinesia (15%) were the most frequent movement disorders. Involvement of the dentate nucleus in brain imaging was observed together with movement disorders or coordination problems. Short attention span (78.6%) and distractibility (71.4%) were the most frequently behavior traits mentioned by parents while impulsiveness, problems communicating wishes or needs and compulsive behavior were addressed as strongly interfering with family life. Treatment was mainly aimed to control epileptic seizures and psychiatric symptoms. Four new pathogenic variants were identified. In silico scoring system, protein activity and pathogenicity score revealed a high correlation. A genotype/phenotype correlation was not observed, even in siblings. This study presents the diversifying characteristics of disease phenotype during the disease course, highlighting movement disorders, widens the knowledge on the genotypic spectrum of SSADHD and emphasizes a reliable application of in silico approaches.

Comparative studies on substrate specificity of succinic semialdehyde reductase from Gluconobacter oxydans and glyoxylate reductase from Acetobacter aceti.

Gluconobacter oxydans succinic semialdehyde reductase (GoxSSAR) and Acetobacter aceti glyoxylate reductase (AacGR) represent a novel class in the ß-hydroxyacid dehydrogenases superfamily. Kinetic analyses revealed GoxSSAR's activity with both glyoxylate and succinic semialdehyde, while AacGR is glyoxylate specific. GoxSSAR K167A lost activity with succinic semialdehyde but retained some with glyoxylate, whereas AacGR K175A lost activity. These findings elucidate differences between these homologous enzymes.

Functional Characterization of a Spectrum of Genetic Variants in a Family with Succinic Semialdehyde Dehydrogenase Deficiency.

Succinic semialdehyde dehydrogenase (SSADH) is a mitochondrial enzyme involved in the catabolism of the neurotransmitter γ-amino butyric acid. Pathogenic variants in the gene encoding this enzyme cause SSADH deficiency, a developmental disease that manifests as hypotonia, autism, and epilepsy. SSADH deficiency patients usually have family-specific gene variants. Here, we describe a family exhibiting four different SSADH variants: Val90Ala, Cys93Phe, and His180Tyr/Asn255Asp (a double variant). We provide a structural and functional characterization of these variants and show that Cys93Phe and Asn255Asp are pathogenic variants that affect the stability of the SSADH protein. Due to the impairment of the cofactor NAD+ binding, these variants show a highly reduced enzyme activity. However, Val90Ala and His180Tyr exhibit normal activity and expression. The His180Tyr/Asn255Asp variant exhibits a highly reduced activity as a recombinant species, is inactive, and shows a very low expression in eukaryotic cells. A treatment with substances that support protein folding by either increasing chaperone protein expression or by chemical means did not increase the expression of the pathogenic variants of the SSADH deficiency patient. However, stabilization of the folding of pathogenic SSADH variants by other substances may provide a treatment option for this disease.

Phenotypic correlates of structural and functional protein impairments resultant from ALDH5A1 variants.

To investigate the genotype-to-protein-to-phenotype correlations of succinic semialdehyde dehydrogenase deficiency (SSADHD), an inherited metabolic disorder of γ-aminobutyric acid catabolism. Bioinformatics and in silico mutagenesis analyses of ALDH5A1 variants were performed to evaluate their impact on protein stability, active site and co-factor binding domains, splicing, and homotetramer formation. Protein abnormalities were then correlated with a validated disease-specific clinical severity score and neurological, neuropsychological, biochemical, neuroimaging, and neurophysiological metrics. A total of 58 individuals (1:1 male/female ratio) were affected by 32 ALDH5A1 pathogenic variants, eight of which were novel. Compared to individuals with single homotetrameric or multiple homo and heterotetrameric proteins, those predicted not to synthesize any functional enzyme protein had significantly lower expression of ALDH5A1 (p = 0.001), worse overall clinical outcomes (p = 0.008) and specifically more severe cognitive deficits (p = 0.01), epilepsy (p = 0.04) and psychiatric morbidity (p = 0.04). Compared to individuals with predictions of having no protein or a protein impaired in catalytic functions, subjects whose proteins were predicted to be impaired in stability, folding, or oligomerization had a better overall clinical outcome (p = 0.02) and adaptive skills (p = 0.04). The quantity and type of enzyme proteins (no protein, single homotetramers, or multiple homo and heterotetramers), as well as their structural and functional impairments (catalytic or stability, folding, or oligomerization), contribute to phenotype severity in SSADHD. These findings are valuable for assessment of disease prognosis and management, including patient selection for gene replacement therapy. Furthermore, they provide a roadmap to determine genotype-to-protein-to-phenotype relationships in other autosomal recessive disorders.

Human iPSC-derived neural stem cells with ALDH5A1 mutation as a model of succinic semialdehyde dehydrogenase deficiency.

BACKGROUND: Succinic semialdehyde dehydrogenase deficiency (SSADH-D) is an autosomal recessive gamma-aminobutyric acid (GABA) metabolism disorder that can arise due to ALDH5A1 mutations, resulting in severe, progressive, untreatable neurodegeneration. SSADH-D is primarily studied using simplified models, such as HEK293 cells overexpressing genes of interest, but such overexpression can result in protein aggregation or pathway saturation that may not be representative of actual underlying disease phenotypes. METHODS: We used a CRISPR/Cas9 approach to generate human iPSC cell lines bearing ALDH5A1 mutations. Through screening, two different mutant cell lines, NM_001080.3: c.727_735del (p.L243_S245del) and NM_001080.3: c.730_738del (p.A244_Q246del), were obtained. We induced iPSCs to neural stem cells and analyzed the characteristics of ALDH5A1 mutations in stem cells. RESULTS: The human iPSC and NSC cell lines presented typical stem cell-like morphology. We found changes in ALDH5A1 expression and GABA accumulation in the different cell lines. In addition, by analyzing the cDNA between the wild-type and the mutant cell lines, we found that the mutant cell lines had a splicing variant. CONCLUSIONS: iPSCs represent a promising in vitro model for SSADH-D that can be used to study early central nervous system developmental alterations and pathogenic mechanisms.

[Analysis of gene variant in an infant with succinic semialdehyde dehydrogenase deficiency].

OBJECTIVE: To explore the genetic basis for a child with succinate semialdehyde dehydrogenase deficiency. METHODS: Peripheral blood samples of the proband and his parents were collected and subjected to Sanger sequencing. High-throughput sequencing was used to verify the gene variants. Bioinformatic software was used to analyze the pathogenicity of the variant sites. RESULTS: Sanger sequencing showed that the proband carried a homozygous c.1529C>T (p.S510F) variant of the ALDH5A1 gene, for which his mother was a carrier. The same variant was not detected in his father. However, high-throughput sequencing revealed that the child and his father both had a deletion of ALDH5A1 gene fragment (chr6: 24 403 265-24 566 986). CONCLUSION: The c.1529C>T variant of the ALDH5A1 gene and deletion of ALDH5A1 gene fragment probably underlay the disease in the child. High-throughput sequencing can detect site variation as well as deletion of gene fragment, which has enabled genetic diagnosis and counseling for the family.

Gene expression analysis in epileptic hippocampi reveals a promoter haplotype conferring reduced aldehyde dehydrogenase 5a1 expression and responsiveness.

Increasing evidence indicates the pathogenetic relevance of regulatory genomic motifs for variability in the manifestation of brain disorders. In this context, cis-regulatory effects of single nucleotide polymorphisms (SNPs) on gene expression can contribute to changing transcript levels of excitability-relevant molecules and episodic seizure manifestation in epilepsy. Biopsy specimens of patients undergoing epilepsy surgery for seizure relief provide unique insights into the impact of promoter SNPs on corresponding mRNA expression. Here, we have scrutinized whether two linked regulatory SNPs (rs2744575; 4779C > G and rs4646830; 4854C > G) located in the aldehyde dehydrogenase 5a1 (succinic semialdehyde dehydrogenase; ALDH5A1) gene promoter are associated with expression of corresponding mRNAs in epileptic hippocampi (n = 43). The minor ALDH5A1-GG haplotype associates with significantly lower ALDH5A1 transcript abundance. Complementary in vitro analyses in neural cell cultures confirm this difference and further reveal a significantly constricted range for the minor ALDH5A1 haplotype of promoter activity regulation through the key epileptogenesis transcription factor Egr1 (early growth response 1). The present data suggest systematic analyses in human hippocampal tissue as a useful approach to unravel the impact of epilepsy candidate SNPs on associated gene expression. Aberrant ALDH5A1 promoter regulation in functional terms can contribute to impaired γ-aminobutyric acid homeostasis and thereby network excitability and seizure propensity.

ALDH5A1 acts as a tumour promoter and has a prognostic impact in papillary thyroid carcinoma.

Thyroid cancer is the most common endocrine carcinoma, with papillary thyroid carcinoma (PTC) accounting for 80%-90% of thyroid cancers. Accumulating studies reported that mitochondria plays an important role in the regulation of cell proliferation. ALDH5A1, may function as an oncogene or tumour suppressor in various human cancers, and the role of ALDH5A1 in PTC is still unclear. The aim of this study was to investigate the clinical significance of ALDH5A1 expression and its functions in PTC. In this present study, we studied ALDH5A1 expression on primary papillary thyroid carcinoma (PTC) in The Cancer Genome Atlas (TCGA) database. Results showed that the levels of ALDH5A1 were found positively correlated with tumour stage, metastasis, lymph node stage, and higher levels of ALDH5A1 demonstrated poor disease-free survival (DFS). Immunohistochemistry (IHC) revealed that significantly higher expression of ALDH5A1 was found in PTC tissues. On the other hand, knockdown of ALDH5A1 significantly inhibited PTC cell proliferation, migration and invasion detection found the migration and invasion of cells also were hindered when ALDH5A1 level was reduced. The knockdown of ALDH5A1 inhibited the expression of Vimentin and promoted the expression of E-cadherin. In brief, knockdown of ALDH5A1may act as a novel molecular target for the prevention and treatment of PTC. SIGNIFICANCE OF THE STUDY: The present study focused on the role and the potential mechanism of ALDH5A1 in papillary thyroid carcinoma. We demonstrated that reduced expression of ALDH5A1 might inhibit the progression of TC by inhibiting cell proliferation, migration and invasion and reversing epithelial-mesenchymal transition (EMT). The findings ensured the interaction relation between ALDH5A1 and EMT in PTC, providing a novel biological marker for PTC and enriching the potential strategies for TC treatment.

MicroRNA hsa-miR-1301-3p Regulates Human ADH6, ALDH5A1 and ALDH8A1 in the Ethanol-Acetaldehyde-Acetate Metabolic Pathway.

Alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs) are vital enzymes involved in the metabolism of a variety of alcohols. Differences in the expression and enzymatic activity of human ADHs and ALDHs correlate with individual variability in metabolizing alcohols and drugs and in the susceptibility to alcoholic liver disease. MicroRNAs (miRNAs) function as epigenetic modulators to regulate the expression of drug-metabolizing enzymes. To characterize miRNAs that target ADHs and ALDHs in human liver cells, we carried out a systematic bioinformatics analysis to analyze free energies of the interaction between miRNAs and their cognate sequences in ADH and ALDH transcripts and then calculated expression correlations between miRNAs and their targeting ADH and ALDH genes using a public data base. Candidate miRNAs were selected to evaluate bioinformatic predictions using a series of biochemical assays. Our results showed that 11 miRNAs have the potential to modulate the expression of two ADH and seven ALDH genes in the human liver. We found that hsa-miR-1301-3p suppressed the expression of ADH6, ALDH5A1, and ALDH8A1 in liver cells and blocked their induction by ethanol. In summary, our results revealed that hsa-miR-1301-3p plays an important role in ethanol metabolism by regulating ADH and ALDH gene expression. SIGNIFICANCE STATEMENT: Systematic bioinformatics analysis showed that 11 microRNAs might play regulatory roles in the expression of two alcohol dehydrogenase (ADH) and seven aldehyde dehydrogenase (ALDH) genes in the human liver. Experimental evidences proved that hsa-miR-1301-3p suppressed the expression of ADH6, ALDH5A1, and ALDH8A1 in liver cells and decreased their inducibility by ethanol.

Functional analysis of thirty-four suspected pathogenic missense variants in ALDH5A1 gene associated with succinic semialdehyde dehydrogenase deficiency.

Deficiency of succinate semialdehyde dehydrogenase (SSADH; aldehyde dehydrogenase 5a1 (ALDH5A1), OMIM 271980, 610045), the second enzyme of GABA degradation, represents a rare autosomal-recessively inherited disorder which manifests metabolically as gamma-hydroxybutyric aciduria. The neurological phenotype includes intellectual disability, autism spectrum, epilepsy and sleep and behavior disturbances. Approximately 70 variants have been reported in the ALDH5A1 gene, half of them being missense variants. In this study, 34 missense variants, of which 22 novel, were evaluated by in silico analyses using PolyPhen2 and SIFT prediction tools. Subsequently, the effect of these variants on SSADH activity was studied by transient overexpression in HEK293 cells. These studies showed severe enzymatic activity impairment for 27 out of 34 alleles, normal activity for one allele and a broad range of residual activities (25 to 74%) for six alleles. To better evaluate the alleles that showed residual activity above 25%, we generated an SSADH-deficient HEK293-Flp-In cell line using CRISPR-Cas9, in which these alleles were stably expressed. This model proved essential in the classification as deficient for one out of the seven studied alleles. For 8 out of 34 addressed alleles, there were discrepant results among the used prediction tools, and/or in correlating the results of the prediction tools with the functional data. In case of diagnostic urgency of missense alleles, we propose the use of the transient transfection model for confirmation of their effect on the SSADH catalytic function, since this model resulted in fast and robust functional characterization for the majority of the tested variants. In selected cases, stable transfections can be considered and may prove valuable.

Isocitrate dehydrogenase 1-mutated human gliomas depend on lactate and glutamate to alleviate metabolic stress.

Diffuse gliomas often carry point mutations in isocitrate dehydrogenase ( IDH1mut), resulting in metabolic stress. Although IDHmut gliomas are difficult to culture in vitro, they thrive in the brain via diffuse infiltration, suggesting brain-specific tumor-stroma interactions that can compensate for IDH-1 deficits. To elucidate the metabolic adjustments in clinical IDHmut gliomas that contribute to their malignancy, we applied a recently developed method of targeted quantitative RNA next-generation sequencing to 66 clinical gliomas and relevant orthotopic glioma xenografts, with and without the endogenous IDH-1R132H mutation. Datasets were analyzed in R using Manhattan plots to calculate distance between expression profiles, Ward's method to perform unsupervised agglomerative clustering, and the Mann Whitney U test and Fisher's exact tests for supervised group analyses. The significance of transcriptome data was investigated by protein analysis, in situ enzymatic activity mapping, and in vivo magnetic resonance spectroscopy of orthotopic IDH1mut- and IDHwt-glioma xenografts. Gene set enrichment analyses of clinical IDH1mut gliomas strongly suggest a role for catabolism of lactate and the neurotransmitter glutamate, whereas, in IDHwt gliomas, processing of glucose and glutamine are the predominant metabolic pathways. Further evidence of the differential metabolic activity in these cancers comes from in situ enzymatic mapping studies and preclinical in vivo magnetic resonance spectroscopy imaging. Our data support an evolutionary model in which IDHmut glioma cells exist in symbiosis with supportive neuronal cells and astrocytes as suppliers of glutamate and lactate, possibly explaining the diffuse nature of these cancers. The dependency on glutamate and lactate opens the way for novel approaches in the treatment of IDHmut gliomas.-Lenting, K., Khurshed, M., Peeters, T. H., van den Heuvel, C. N. A. M., van Lith, S. A. M., de Bitter, T., Hendriks, W., Span, P. N., Molenaar, R. J., Botman, D., Verrijp, K., Heerschap, A., ter Laan, M., Kusters, B., van Ewijk, A., Huynen, M. A., van Noorden, C. J. F., Leenders, W. P. J. Isocitrate dehydrogenase 1-mutated human gliomas depend on lactate and glutamate to alleviate metabolic stress.

Novel mutations in a Chinese family with two patients with succinic semialdehyde dehydrogenase deficiency.

Background: A considerable proportion of pediatric disease burden is mainly caused by inborn errors of metabolism. Succinic semi-aldehyde dehydrogenase (SSADH) deficiency is an unusual disorder of the gamma-aminobutyric acid metabolism. Till date, very few cases have been reported in China.Case presentation: Trio-WES was used to characterize the ALDH5A1 gene in two children of a Chinese family, who presented with seizures, psychomotor delay, development regression, borderline cognition, hypotonia, and harbored the compound heterozygotes NM_001080.3: c.1321G > A (p. Gly441Arg) and c.727_735del (p. Leu243_Ser245del). The former has been reported earlier (rs1041467895), whereas the latter is novel. Amino acid coding at highly conserved amino acid residues was observed to be altered by both mutations. This structural impairment influenced the enzyme structure as indicated by the in silico protein modeling. Cerebral magnetic resonance imaging of the proband and her brother showed excessive gap in the cerebrum and abnormal signals in the bilateral frontal lobe, bilateral basal ganglia, and cerebral foot. Elevated levels of Gamma-hydroxybutyric aciduria were found in their patients on urine organic acid analysis.Conclusion: Our findings contribute to the current knowledge of missense and deletion mutations associated with SSADH deficiency.

Functional dissection of gamma-aminobutyric acid metabolism in Neurospora crassa.

The GABA shunt is one of the metabolic pathways that is ubiquitous in prokaryotes and eukaryotes. γ-aminobutyric acid (GABA) in fungi is required in the stress responses, virulence and development. The number of genes encoding glutamate decarboxylase (gad), GABA transaminase (gta) and succinic semialdehyde dehydrogenase (ssadh) varies between fungal species. The genome-wide analysis in Neurospora crassa resulted in the identification of a gta and a ssadh. Disruption of either gta or ssadh decreased respiration rate and biomass accumulation, reduced growth on GABA and beta-alanine. The gta and ssadh mutants exhibited aberrant hyphal morphology and displayed differential transcription of the GABA shunt genes. In the gta mutant, protoperithecia and perithecia formation was almost completely suppressed in the presence of GABA and beta-alanine, indicating GTA requirement for the turnover of these amino acids. The strains displayed differential metabolic dysregulations in response to different nitrogen sources. The phenotypic differences between the gta and ssadh mutants could be contributed to accumulation of intermediates of the GABA shunt and/or GABA shunt-independent functions. Together, our data suggest that the GABA shunt could function as a moderate modulator of multiple biological events, including respiration, energy metabolism, carbon and nitrogen metabolism, growth, as well as sexual development in N. crassa.

Succinic Semialdehyde Dehydrogenase Deficiency: In Vitro and In Silico Characterization of a Novel Pathogenic Missense Variant and Analysis of the Mutational Spectrum of ALDH5A1.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare, monogenic disorder affecting the degradation of the main inhibitory neurotransmitter γ-amino butyric acid (GABA). Pathogenic variants in the ALDH5A1 gene that cause an enzymatic dysfunction of succinic semialdehyde dehydrogenase (SSADH) lead to an accumulation of potentially toxic metabolites, including γ-hydroxybutyrate (GHB). Here, we present a patient with a severe phenotype of SSADHD caused by a novel genetic variant c.728T > C that leads to an exchange of leucine to proline at residue 243, located within the highly conserved nicotinamide adenine dinucleotide (NAD)+ binding domain of SSADH. Proline harbors a pyrrolidine within its side chain known for its conformational rigidity and disruption of protein secondary structures. We investigate the effect of this novel variant in vivo, in vitro, and in silico. We furthermore examine the mutational spectrum of all previously described disease-causing variants and computationally assess all biologically possible missense variants of ALDH5A1 to identify mutational hotspots.

SSADH Variants Increase Susceptibility of U87 Cells to Mitochondrial Pro-Oxidant Insult.

Succinate semialdehyde dehydrogenase (SSADH) is a mitochondrial enzyme, encoded by ALDH5A1, mainly involved in γ-aminobutyric acid (GABA) catabolism and energy supply of neuronal cells, possibly contributing to antioxidant defense. This study aimed to further investigate the antioxidant role of SSADH, and to verify if common SNPs of ALDH5A1 may affect SSADH activity, stability, and mitochondrial function. In this study, we used U87 glioblastoma cells as they represent a glial cell line. These cells were transiently transfected with a cDNA construct simultaneously harboring three SNPs encoding for a triple mutant (TM) SSADH protein (p.G36R/p.H180Y/p.P182L) or with wild type (WT) cDNA. SSADH activity and protein level were measured. Cell viability, lipid peroxidation, mitochondrial morphology, membrane potential (ΔΨ), and protein markers of mitochondrial stress were evaluated upon Paraquat treatment, in TM and WT transfected cells. TM transfected cells show lower SSADH protein content and activity, fragmented mitochondria, higher levels of peroxidized lipids, and altered ΔΨ than WT transfected cells. Upon Paraquat treatment, TM cells show higher cell death, lipid peroxidation, 4-HNE protein adducts, and lower ΔΨ, than WT transfected cells. These results reinforce the hypothesis that SSADH contributes to cellular antioxidant defense; furthermore, common SNPs may produce unstable, less active SSADH, which could per se negatively affect mitochondrial function and, under oxidative stress conditions, fail to protect mitochondria.

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.