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.

Clinical and molecular outcomes from the 5-Year natural history study of SSADH Deficiency, a model metabolic neurodevelopmental disorder.

BACKGROUND: Succinic semialdehyde dehydrogenase deficiency (SSADHD) represents a model neurometabolic disease at the fulcrum of translational research within the Boston Children's Hospital Intellectual and Developmental Disabilities Research Centers (IDDRC), including the NIH-sponsored natural history study of clinical, neurophysiological, neuroimaging, and molecular markers, patient-derived induced pluripotent stem cells (iPSC) characterization, and development of a murine model for tightly regulated, cell-specific gene therapy. METHODS: SSADHD subjects underwent clinical evaluations, neuropsychological assessments, biochemical quantification of γ-aminobutyrate (GABA) and related metabolites, electroencephalography (standard and high density), magnetoencephalography, transcranial magnetic stimulation, magnetic resonance imaging and spectroscopy, and genetic tests. This was parallel to laboratory molecular investigations of in vitro GABAergic neurons derived from induced human pluripotent stem cells (hiPSCs) of SSADHD subjects and biochemical analyses performed on a versatile murine model that uses an inducible and reversible rescue strategy allowing on-demand and cell-specific gene therapy. RESULTS: The 62 SSADHD subjects [53% females, median (IQR) age of 9.6 (5.4-14.5) years] included in the study had a reported symptom onset at ∼ 6 months and were diagnosed at a median age of 4 years. Language developmental delays were more prominent than motor. Autism, epilepsy, movement disorders, sleep disturbances, and various psychiatric behaviors constituted the core of the disorder's clinical phenotype. Lower clinical severity scores, indicating worst severity, coincided with older age (R= -0.302, p = 0.03), as well as age-adjusted lower values of plasma γ-aminobutyrate (GABA) (R = 0.337, p = 0.02) and γ-hydroxybutyrate (GHB) (R = 0.360, p = 0.05). While epilepsy and psychiatric behaviors increase in severity with age, communication abilities and motor function tend to improve. iPSCs, which were differentiated into GABAergic neurons, represent the first in vitro neuronal model of SSADHD and express the neuronal marker microtubule-associated protein 2 (MAP2), as well as GABA. GABA-metabolism in induced GABAergic neurons could be reversed using CRISPR correction of the pathogenic variants or mRNA transfection and SSADHD iPSCs were associated with excessive glutamatergic activity and related synaptic excitation. CONCLUSIONS: Findings from the SSADHD Natural History Study converge with iPSC and animal model work focused on a common disorder within our IDDRC, deepening our knowledge of the pathophysiology and longitudinal clinical course of a complex neurodevelopmental disorder. This further enables the identification of biomarkers and changes throughout development that will be essential for upcoming targeted trials of enzyme replacement and gene therapy.

Generation and characterization of six human induced pluripotent stem cell lines (hiPSCs) from three individuals with SSADH Deficiency and CRISPR-corrected isogenic controls.

Succinic Semialdehyde Dehydrogenase Deficiency (SSADHD) is an ultra-rare autosomal recessive neurometabolic disorder caused by ALDH5A1 mutations presenting with autism and epilepsy. Here, we report the generation and characterization of human induced pluripotent stem cells (hiPSCs) derived from fibroblasts of three unrelated SSADHD patients - one female and two males with the CRISPR-corrected isogenic controls. These individuals are clinically diagnosed and are being followed in a longitudinal clinical study.

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.

Glioma Cells Expressing High Levels of ALDH5A1 Exhibit Enhanced Migration Transcriptional Signature in Patient Tumors.

Accumulating data shows that altered metabolic activity contributes to glioma development. Recently, modulation of SSADH (succinic semialdehyde dehydrogenase) expression, implicated in the catabolism of GABA neurotransmitter, was shown to impact glioma cell properties, such as proliferation, self-renewal and tumorigenicity. The purpose of this study was to investigate the clinical significance of SSADH expression in human gliomas. Using public single-cell RNA-sequencing data from glioma surgical resections, we initially grouped cancer cells according to ALDH5A1 (Aldehyde dehydrogenase 5 family member A1) expression, which encodes SSADH. Gene ontology enrichment analysis of genes differentially expressed between cancer cells expressing high or low levels of ALDH5A1, highlighted enrichment in genes implicated in cell morphogenesis and motility. In glioblastoma cell lines, ALDH5A1 knockdown inhibited cell proliferation, induced apoptosis and reduced their migratory potential. This was accompanied by a reduction in the mRNA levels of the adherens junction molecule ADAM-15 and deregulation in the expression of EMT biomarkers, with increased CDH1 and decreased vimentin mRNA levels. Evaluation of SSADH expression in a cohort of 95 gliomas using immunohistochemistry showed that SSADH expression was significantly elevated in cancer tissues compared to normal brain tissues, without any significant correlation with clinicopathological characteristics. In summary, our data show that SSADH is upregulated in glioma tissues irrespective of the histological grade and its expression sustains glioma cell motility.

Mechanistic roles of the neighbouring cysteine in enhancing nucleophilicity of catalytic residue in a two-cysteine succinic semialdehyde dehydrogenase.

Succinic semialdehyde dehydrogenase (SSADH) catalyses the conversion of succinic semialdehyde into succinic acid and two electrons are transferred to NAD(P)+ to yield NAD(P)H. Our previous work has already reported the catalytic role of Cys289 of two-cysteine SSADH from Acinetobacter baumannii (AbSSADH). However, the mechanistic role of the neighbouring conserved Cys291 and Glu255 remains unexplored. In this study, the functional roles of Cys291 and Glu255 in AbSSADH catalysis have been characterized. Results demonstrated that the E255A activity was almost completely lost, ~ 7000-fold lower than the wild-type (WT), indicating that Glu255 is very crucial and directly involved in AbSSADH catalysis. However, the C291A and C291S variants activity and catalytic turnover (kcat ) decreased ~ 2-fold and 9-fold respectively. To further characterize the functional roles of Cys291, we employed two pH-dependent methods; pre-steady-state burst amplitude and NADP-enzyme adduct formation. The results showed that the pKa values of catalytic Cys289 measured for the WT and C291A reactions were 7.8 and 8.7-8.8, respectively, suggesting that Cys291 can lower the pKa of Cys289 and consequently trigger the deprotonation of a Cys289 thiol. In addition, the Cys291 also plays a role in disulfide/sulfhydryl redox regulation for AbSSADH activity. Hence, we demonstrated for the first time the dual functions of Cys291 in enhancing the nucleophilicity of the catalytic Cys289 and regulating a disulfide/sulfhydryl redox switch for AbSSADH catalysis. The mechanistic insights into the nucleophilicity enhancement of the catalytic cysteine of AbSSADH might be applicable to understanding how the microenvironment increases cysteine reactivity in other enzymes in the aldehyde dehydrogenase superfamily.

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.

Understanding the Molecular Mechanisms of Succinic Semialdehyde Dehydrogenase Deficiency (SSADHD): Towards the Development of SSADH-Targeted Medicine.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare genetic disorder caused by inefficient metabolic breakdown of the major inhibitory neurotransmitter, γ-aminobutyric acid (GABA). Pathologic brain accumulation of GABA and γ-hydroxybutyrate (GHB), a neuroactive by-product of GABA catabolism, leads to a multitude of molecular abnormalities beginning in early life, culminating in multifaceted clinical presentations including delayed psychomotor development, intellectual disability, hypotonia, and ataxia. Paradoxically, over half of patients with SSADHD also develop epilepsy and face a significant risk of sudden unexpected death in epilepsy (SUDEP). Here, we review some of the relevant molecular mechanisms through which impaired synaptic inhibition, astrocytic malfunctions and myelin defects might contribute to the complex SSADHD phenotype. We also discuss the gaps in knowledge that need to be addressed for the implementation of successful gene and enzyme replacement SSADHD therapies. We conclude with a description of a novel SSADHD mouse model that enables 'on-demand' SSADH restoration, allowing proof-of-concept studies to fine-tune SSADH restoration in preparation for eventual human trials.

Development of a Quality-of-Life Survey for Patients With Succinic Semialdehyde Dehydrogenase Deficiency, a Rare Disorder of GABA Metabolism.

Succinic semialdehyde dehydrogenase deficiency (SSADHD), a rare disorder of GABA metabolism, presents with significant neurodevelopmental morbidity. Although there is a growing interest in the concept of quality of life through patient reports as a meaningful outcome in rare disease clinical trials, little is known about the overall impact of SSADHD from the patient/family perspective. The purpose of this study was to determine issues related to quality of life and patient/family experience through a focus group discussion with family caregivers of patients with SSADHD. The discussion included the input of 5 family caregivers, and highlighted concerns related to physical function, cognitive and intellectual function, psychological and behavioral function, social function, and family impact. These themes represent appropriate starting points in the development of a quality-of-life survey that may serve as a meaningful clinical tool in future studies of SSADHD.

Enzyme Replacement Therapy for Succinic Semialdehyde Dehydrogenase Deficiency: Relevance in γ-Aminobutyric Acid Plasticity.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare inborn metabolic disorder caused by the functional impairment of SSADH (encoded by the ALDH5A1 gene), an enzyme essential for metabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA). In SSADHD, pathologic accumulation of GABA and its metabolite γ-hydroxybutyrate (GHB) results in broad spectrum encephalopathy including developmental delay, ataxia, seizures, and a heightened risk of sudden unexpected death in epilepsy (SUDEP). Proof-of-concept systemic SSADH restoration via enzyme replacement therapy increased survival of SSADH knockout mice, suggesting that SSADH restoration might be a viable intervention for SSADHD. However, before testing enzyme replacement therapy or gene therapy in patients, we must consider its safety and feasibility in the context of early brain development and unique SSADHD pathophysiology. Specifically, a profound use-dependent downregulation of GABAA receptors in SSADHD indicates a risk that any sudden SSADH restoration might diminish GABAergic tone and provoke seizures. In addition, the tight developmental regulation of GABA circuit plasticity might limit the age window when SSADH restoration is accomplished safely. Moreover, given SSADH expressions are cell type-specific, targeted instead of global restoration might be necessary. We therefore describe 3 key parameters for the clinical readiness of SSADH restoration: (1) rate, (2) timing, and (3) cell type specificity. Our work focuses on the construction of a novel SSADHD mouse model that allows "on-demand" SSADH restoration for the systematic investigation of these key parameters. We aim to understand the impacts of specific SSADH restoration protocols on brain physiology, accelerating bench-to-bedside development of enzyme replacement therapy or gene therapy for SSADHD patients.

Postmortem Analyses in a Patient With Succinic Semialdehyde Dehydrogenase Deficiency (SSADHD): II. Histological, Lipid, and Gene Expression Outcomes in Regional Brain Tissue.

This study has extended previous metabolic measures in postmortem tissues (frontal and parietal lobes, pons, cerebellum, hippocampus, and cerebral cortex) obtained from a 37-year-old male patient with succinic semialdehyde dehydrogenase deficiency (SSADHD) who expired from SUDEP (sudden unexplained death in epilepsy). Histopathologic characterization of fixed cortex and hippocampus revealed mild to moderate astrogliosis, especially in white matter. Analysis of total phospholipid mass in all sections of the patient revealed a 61% increase in cortex and 51% decrease in hippocampus as compared to (n = 2-4) approximately age-matched controls. Examination of mass and molar composition of major phospholipid classes showed decreases in phospholipids enriched in myelin, such as phosphatidylserine, sphingomyelin, and ethanolamine plasmalogen. Evaluation of gene expression (RT2 Profiler PCR Arrays, GABA, glutamate; Qiagen) revealed dysregulation in 14/15 GABAA receptor subunits in cerebellum, parietal, and frontal lobes with the most significant downregulation in ∊, θ, ρ1, and ρ2 subunits (7.7-9.9-fold). GABAB receptor subunits were largely unaffected, as were ionotropic glutamate receptors. The metabotropic glutamate receptor 6 was consistently downregulated (maximum 5.9-fold) as was the neurotransmitter transporter (GABA), member 13 (maximum 7.3-fold). For other genes, consistent dysregulation was seen for interleukin 1ß (maximum downregulation 9.9-fold) and synuclein α (maximal upregulation 6.5-fold). Our data provide unique insight into SSADHD brain function, confirming astrogliosis and lipid abnormalities previously observed in the null mouse model while highlighting long-term effects on GABAergic/glutamatergic gene expression in this disorder.

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.

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.

Kinetic and structural insights into enzymatic mechanism of succinic semialdehyde dehydrogenase from Cyanothece sp. ATCC51142.

As a ubiquitous enzyme, succinic semialdehyde dehydrogenase contributes significantly in many pathways including the tricarboxylic acid cycle and other metabolic processes such as detoxifying the accumulated succinic semialdehyde and surviving in nutrient-limiting conditions. Here the cce4228 gene encoding succinic semialdehyde dehydrogenase from Cyanothece sp. ATCC51142 was cloned and the homogenous recombinant cce4228 protein was obtained by Ni-NTA affinity chromatography. Biochemical characterization revealed that cce4228 protein displayed optimal activity at presence of metal ions in basic condition. Although the binding affinity of cce4228 protein with NAD+ was about 50-fold lower than that of cce4228 with NADP+, the catalytic efficiency of cce4228 protein towards succinic semialdehyde with saturated concentration of NADP+ is same as that with saturated concentration of NAD+ as its cofactors. Meanwhile, the catalytic activity of cce4228 was competitively inhibited by succinic semialdehyde substrate. Kinetic and structural analysis demonstrated that the conserved Cys262 and Glu228 residues were crucial for the catalytic activity of cce4228 protein and the Ser157 and Lys154 residues were determinants of cofactor preference.

Biochemical pathways to α-ketoglutarate, a multi-faceted metabolite.

α-Ketoglutarate (AKG) also known as 2-oxoglutarate is an essential metabolite in virtually all organisms as it participates in a variety of biological processes including anti-oxidative defence, energy production, signalling modules, and genetic modification. This keto-acid also possesses immense commercial value as it is utilized as a nutritional supplement, a therapeutic agent, and a precursor to a variety of value-added products such as ethylene and heterocyclic compounds. Hence, the generation of KG in a sustainable and environmentally-neutral manner is a major ongoing research endeavour. In this mini-review, the enzymatic systems and the metabolic networks mediating the synthesis of AKG will be described. The importance of such enzymes as isocitrate dehydrogenase (ICDH), glutamate dehydrogenase (GDH), succinate semialdehyde dehydrogenase (SSADH) and transaminases that directly contribute to the formation of KG will be emphasized. The efficacy of microbial systems in providing an effective platform to generate this moiety and the molecular strategies involving genetic manipulation, abiotic stress and nutrient supplementation that result in the optimal production of AKG will be evaluated. Microbial systems and their components acting via the metabolic networks and the resident enzymes are well poised to provide effective biotechnological tools that can supply renewable AKG globally.

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.

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.

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.

Interaction of Bacillus subtilis GabR with the gabTD promoter: role of repeated sequences and effect of GABA in transcriptional activation.

Bacillus subtilis is able to use γ-aminobutyric acid (GABA) found in the soil as carbon and nitrogen source, through the action of GABA aminotransferase (GabT) and succinic semialdehyde dehydrogenase (GabD). GABA acts as molecular effector in the transcriptional activation of the gabTD operon by GabR. GabR is the most studied member of the MocR family of prokaryotic pyridoxal 5'-phosphate (PLP)-dependent transcriptional regulators, yet crucial aspects of its mechanism of action are unknown. GabR binds to the gabTD promoter, but transcription is activated only when GABA is present. Here, we demonstrated, in contrast with what had been previously proposed, that three repeated nucleotide sequences in the promoter region, two direct repeats and one inverted repeat, are specifically recognized by GabR. We carried out in vitro and in vivo experiments using mutant forms of the gabTD promoter. Our results showed that GABA activates transcription by changing the modality of interaction between GabR and the recognized sequence repeats. A hypothetical model is proposed in which GabR exists in two alternative conformations that, respectively, prevent or promote transcription. According to this model, in the absence of GABA, GabR binds to DNA interacting with all three sequence repeats, overlapping the RNA polymerase binding site and therefore preventing transcription activation. On the other hand, when GABA binds to GabR, a conformational change of the protein leads to the release of the interaction with the inverted repeat, allowing transcription initiation by RNA polymerase.

Proteomic characterization and comparison of ram (Ovis aries) and buck (Capra hircus) spermatozoa proteome using a data independent acquisition mass spectometry (DIA-MS) approach.

Fresh semen is most commonly used in an artificial insemination of small ruminants, because of low fertility rates of frozen sperm. Generally, when developing and applying assisted reproductive technologies, sheep and goats are classified as one species. In order to optimize sperm cryopreservation protocols in sheep and goat, differences in sperm proteomes between ram and buck are necessary to investigate, which may contribute to differences in function and fertility of spermatozoa. In the current work, a data-independent acquisition-mass spectrometry proteomic approach was used to characterize and make a comparison of ram (Ovis aries) and buck (Capra hircus) sperm proteomes. A total of 2,109 proteins were identified in ram and buck spermatozoa, with 238 differentially abundant proteins. Proteins identified in ram and buck spermatozoa are mainly involved in metabolic pathways for generation of energy and diminishing oxidative stress. Specifically, there are greater abundance of spermatozoa proteins related to the immune protective and capacity activities in ram, while protein that inhibit sperm capacitation shows greater abundance in buck. Our results not only provide novel insights into the characteristics and potential activities of spermatozoa proteins, but also expand the potential direction for sperm cryopreservation in ram and buck.