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.

Delays in latencies of median-nerve evoked magnetic fields in patients with succinic semialdehyde dehydrogenase deficiency.

OBJECTIVE: Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a genetic disorder resulting in abnormal regulation of γ-aminobutyric acid, lipid metabolism, and myelin biogenesis, leading to ataxia, seizures, and cognitive impairment. Since the myelin sheath is thinner in a murine model of SSADHD compared to a wild type, we hypothesized that this also holds for human brain. We tested whether the conduction velocity in the somatosensory pathway is accordingly delayed. METHODS: Somatosensory evoked magnetic fields (SEF) produced by transcutaneous electrical stimulation of the median nerve were measured in 13 SSADHD patients, 11 healthy and 14 disease controls with focal epilepsy. The peak latencies of the initial four components (M1, M2, M3 and M4) were measured. RESULTS: The SEF waveforms and scalp topographies were comparable across the groups. The latencies were statistically significantly longer in the SSADHD group compared to the two controls. We found these latencies for the SSADHD, healthy and disease controls respectively to be: M1: (21.9 ± 0.8 ms [mean ± standard error of the mean], 20.4 ± 0.6 ms, and 21.0 ± 0.4 ms) (p < 0.05); M2: (36.1 ± 1.0 ms, 33.1 ± 0.6 ms, and 32.1 ± 1.1 ms) (p < 0.005); M3: (62.5 ± 2.4 ms, 54.7 ± 2.0 ms, and 49.9 ± 1.8 ms) (p < 0.005); M4: (86.2 ± 2.3 ms, 78.8 ± 2.8 ms, and 73.5 ± 2.9 ms) (p < 0.005). CONCLUSIONS: The SEF latencies are delayed in patients with SSADHD compared with healthy controls and disease controls. SIGNIFICANCE: This is the first study that compares conduction velocities in the somatosensory pathway in SSADHD, an inherited disorder of GABA metabolism. The longer peak latency implying slower conduction velocity supports the hypothesis that myelin sheath thickness is decreased in SSADHD.

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.

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.

Glymphatic dysfunction coincides with lower GABA levels and sleep disturbances in succinic semialdehyde dehydrogenase deficiency.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is an inherited metabolic disorder of γ-aminobutyrate (GABA) catabolism. Cerebral waste clearance along glymphatic perivascular spaces depends on aquaporin 4 (AQP4) water channels, the function of which was shown to be influenced by GABA. Sleep disturbances are associated independently with SSADHD and glymphatic dysfunction. This study aimed to determine whether indices of the hyperGABAergic state characteristic of SSADHD coincide with glymphatic dysfunction and sleep disturbances and to explicate the modulatory effect that GABA may have on the glymphatic system. The study included 42 individuals (21 with SSADHD; 21 healthy controls) who underwent brain MRIs and magnetic resonance spectroscopy (MRS) for assessment of glymphatic dysfunction and cortical GABA, plasma GABA measurements, and circadian clock gene expression. The SSADHD subjects responded to an additional Children's Sleep Habits Questionnaire (CSHQ). Compared with the control group, SSADHD subjects did not differ in sex and age but had a higher severity of enlarged perivascular spaces in the centrum semiovale (p < 0.001), basal ganglia (p = 0.01), and midbrain (p = 0.001), as well as a higher MRS-derived GABA/NAA peak (p < 0.001). Within the SSADHD group, the severity of glymphatic dysfunction was specific for a lower MRS-derived GABA/NAA (p = 0.04) and lower plasma GABA (p = 0.004). Additionally, the degree of their glymphatic dysfunction correlated with the CSHQ-estimated sleep disturbances scores (R = 5.18, p = 0.03). In the control group, EPVS burden did not correlate with age or cerebral and plasma GABA values. The modulatory effect that GABA may exert on the glymphatic system has therapeutic implications for sleep-related disorders and neurodegenerative conditions associated with glymphatic dysfunction.

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.

Reduced evoked cortical beta and gamma activity and neuronal synchronization in succinic semialdehyde dehydrogenase deficiency, a disorder of γ-aminobutyric acid metabolism.

Succinic semialdehyde dehydrogenase deficiency is a rare autosomal recessively inherited metabolic disorder of γ-aminobutyric acid catabolism manifested by intellectual disability, expressive aphasia, movement disorders, psychiatric ailments and epilepsy. Subjects with succinic semialdehyde dehydrogenase deficiency are characterized by elevated γ-aminobutyric acid and related metabolites, such as γ-guanidinobutyric acid, and an age-dependent downregulation of cerebral γ-aminobutyric acid receptors. These findings indicate impaired γ-aminobutyric acid and γ-aminobutyric acid sub-type A (GABAA) receptor signalling as major factors underlying the pathophysiology of this neurometabolic disorder. We studied the cortical oscillation patterns and their relationship with γ-aminobutyric acid metabolism in 18 children affected by this condition and 10 healthy controls. Using high-density EEG, we recorded somatosensory cortical responses and resting-state activity. Using electrical source imaging, we estimated the relative power changes (compared with baseline) in both stimulus-evoked and stimulus-induced responses for physiologically relevant frequency bands and resting-state power. Stimulus-evoked oscillations are phase locked to the stimulus, whereas induced oscillations are not. Power changes for both evoked and induced responses as well as resting-state power were correlated with plasma γ-aminobutyric acid and γ-guanidinobutyric acid concentrations and with cortical γ-aminobutyric acid measured by proton magnetic resonance spectroscopy. Plasma γ-aminobutyric acid, γ-guanidinobutyric acid and cortical γ-aminobutyric acid were higher in patients than in controls (P < 0.001 for both). Beta and gamma relative power were suppressed for evoked responses in patients versus controls (P < 0.01). No group differences were observed for induced activity (P > 0.05). The mean gamma frequency of evoked responses was lower in patients versus controls (P = 0.002). Resting-state activity was suppressed in patients for theta (P = 0.011) and gamma (P < 0.001) bands. Evoked power changes were inversely correlated with plasma γ-aminobutyric acid and with γ-guanidinobutyric acid for beta (P < 0.001) and gamma (P < 0.001) bands. Similar relationships were observed between the evoked power changes and cortical γ-aminobutyric acid for all tested areas in the beta band (P < 0.001) and for the posterior cingulate gyrus in the gamma band (P < 0.001). We also observed a negative correlation between resting-state activity and plasma γ-aminobutyric acid and γ-guanidinobutyric acid for theta (P < 0.001; P = 0.003), alpha (P = 0.003; P = 0.02) and gamma (P = 0.02; P = 0.01) bands. Our findings indicate that increased γ-aminobutyric acid concentration is associated with reduced sensory-evoked beta and gamma activity and impaired neuronal synchronization in patients with succinic semialdehyde dehydrogenase deficiency. This further elucidates the pathophysiology of this neurometabolic disorder and serves as a potential biomarker for therapeutic trials.

Farnesol brain transcriptomics in CNS inflammatory demyelination.

BACKGROUND: Farnesol (FOL) prevents the onset of experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). OBJECTIVE: We examined the transcriptomic profile of the brains of EAE mice treated with daily oral FOL using next-generation sequencing (RNA-seq). METHODS: Transcriptomics from whole brains of treated and untreated EAE mice at the peak of EAE was performed. RESULTS: EAE-induced mice, compared to naïve, healthy mice, overall showed increased expression in pathways for immune response, as well as an increased cytokine signaling pathway, with downregulation of cellular stress proteins. FOL downregulates pro-inflammatory pathways and attenuates the immune response in EAE. FOL downregulated the expression of genes involved in misfolded protein response, MAPK activation/signaling, and pro-inflammatory response. CONCLUSION: This study provides insight into the molecular impact of FOL in the brain and identifies potential therapeutic targets of the isoprenoid pathway in MS patients.

Phenotypic Correlates of Structural and Functional Protein Impairments Resultant from ALDH5A1 Variants.

Objective: To investigate the genotype-to-protein-to-phenotype correlations of succinic semialdehyde dehydrogenase deficiency (SSADHD), an inherited metabolic disorder of γ-aminobutyric acid catabolism. Methods: 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. Results: 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). Conclusions: 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.

Succinic semialdehyde dehydrogenase deficiency in mice and in humans: An untargeted metabolomics perspective.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare neurometabolic disorder caused by disruption of the gamma-aminobutyric acid (GABA) pathway. A more detailed understanding of its pathophysiology, beyond the accumulation of GABA and gamma-hydroxybutyric acid (GHB), will increase our understanding of the disease and may support novel therapy development. To this end, we compared biochemical body fluid profiles from SSADHD patients with controls using next-generation metabolic screening (NGMS). Targeted analysis of NGMS data from cerebrospinal fluid (CSF) showed a moderate increase of aspartic acid, glutaric acid, glycolic acid, 4-guanidinobutanoic acid, and 2-hydroxyglutaric acid, and prominent elevations of GHB and 4,5-dihydroxyhexanoic acid (4,5-DHHA) in SSADHD samples. Remarkably, the intensities of 4,5-DHHA and GHB showed a significant positive correlation in control CSF, but not in patient CSF. In an established zebrafish epilepsy model, 4,5-DHHA showed increased mobility that may reflect limited epileptogenesis. Using untargeted metabolomics, we identified 12 features in CSF with high biomarker potential. These had comparable increased fold changes as GHB and 4,5-DHHA. For 10 of these features, a similar increase was found in plasma, urine and/or mouse brain tissue for SSADHD compared to controls. One of these was identified as the novel biomarker 4,5-dihydroxyheptanoic acid. The intensities of selected features in plasma and urine of SSADHD patients positively correlated with the clinical severity score of epilepsy and psychiatric symptoms of those patients, and also showed a high mutual correlation. Our findings provide new insights into the (neuro)metabolic disturbances in SSADHD and give leads for further research concerning SSADHD pathophysiology.

Autism spectrum disorder and GABA levels in children with succinic semialdehyde dehydrogenase deficiency.

AIM: To elucidate the etiological aspects of autism spectrum disorder (ASD) in succinic semialdehyde dehydrogenase deficiency (SSADHD), related to dysregulation of γ-aminobutyric acid (GABA) and the imbalance of excitatory and inhibitory neurotransmission. METHOD: In this prospective, international study, individuals with SSADHD underwent neuropsychological assessments, as well as biochemical, neurophysiological, and neuroimaging evaluations. RESULTS: Of the 29 individuals (17 females) enrolled (median age [IQR] 10 years 5 months [5 years 11 months-18 years 1 month]), 16 were diagnosed with ASD. ASD severity significantly increased with age (r = 0.67, p < 0.001) but was inversely correlated with plasma GABA (r = -0.67, p < 0.001) and γ-hydroxybutyrate levels (r = -0.538, p = 0.004), and resting motor threshold as measured by transcranial magnetic stimulation (r = -0.44, p = 0.03). A discriminative analysis indicated that an age older than 7 years 2 months (p = 0.004) and plasma GABA levels less than 2.47 µM (p = 0.01) are the threshold values beyond which the likelihood of ASD presenting in individuals with SSADHD is increased. INTERPRETATION: ASD is prevalent but not universal in SSADHD, and it can be predicted by lower levels of plasma GABA and GABA-related metabolites. ASD severity in SSADHD increases with age and the loss of cortical inhibition. These findings add insight into the pathophysiology of ASD and may facilitate its early diagnosis and intervention in individuals with SSADHD.

Establishment and validation of a clinical severity scoring system for succinic semialdehyde dehydrogenase deficiency.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is an inherited metabolic disorder with a variable phenotype and rate of progression. We aimed to develop and validate a clinical severity scoring (CSS) system applicable to the clinical setting and composed of five domains reflecting the principal manifestations of this disorder: cognitive, communication, motor, epilepsy, and psychiatry. A prospectively characterized cohort of 27 SSADHD subjects (55% females, median [IQR] age 9.2 [4.6-16.2] years) who enrolled in the SSADHD Natural History Study were included. The CSS was validated by comparison to an objective severity scoring (OSS) system based on comprehensive neuropsychologic and neurophysiologic assessments, which mirror and complement the domains of the CSS. The total CSS was sex and age-independent, and 80% of its domains lacked interdependence. With increasing age, there was a significant improvement in communication abilities (p = 0.05) and a worsening of epilepsy and psychiatric manifestations (p = 0.004 and p = 0.02, respectively). There was a significant correlation between all the CSS and OSS domain scores, as well as between the total CSS and OSS (R = 0.855, p < 0.001). Additionally, there were no significant demographic or clinical differences in the ratio of individuals in the upper quartile to the lower three quartiles of the CSS and OSS. The SSADHD CSS is validated using objective measures and offers a reliable condition-specific instrument universally applicable in clinical settings. This severity score may be utilized for family and patient counseling, genotype-phenotype correlations, biomarker development, clinical trials, and objective descriptions of the natural history of SSADHD.

The presence and severity of epilepsy coincide with reduced γ-aminobutyrate and cortical excitatory markers in succinic semialdehyde dehydrogenase deficiency.

OBJECTIVE: Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare inherited metabolic disorder caused by a defect of γ-aminobutyrate (GABA) catabolism. Despite the resultant hyper-GABAergic environment facilitated by the metabolic defect, individuals with this disorder have a paradoxically high prevalence of epilepsy. We aimed to study the characteristics of epilepsy in SSADHD and its concordance with GABA-related metabolites and neurophysiologic markers of cortical excitation. METHODS: Subjects in an international natural history study of SSADHD underwent clinical assessments, electroencephalography, transcranial magnetic stimulation (TMS), magnetic resonance spectroscopy for GABA/N-acetyl aspartate quantification, and plasma GABA-related metabolite measurements. RESULTS: A total of 61 subjects with SSADHD and 42 healthy controls were included in the study. Epilepsy was present in 49% of the SSADHD cohort. Over time, there was an increase in severity in 33% of the subjects with seizures. The presence of seizures was associated with increasing age (p = .001) and lower levels of GABA (p = .002), γ-hydroxybutyrate (GHB; p = .004), and γ-guanidinobutyrate (GBA; p = .003). Seizure severity was associated with increasing age and lower levels of GABA-related metabolites as well as lower TMS-derived resting motor thresholds (p = .04). The cutoff values with the highest discriminative ability to predict seizures were age > 9.2 years (p = .001), GABA < 2.57 µmol·L-1 (p = .002), GHB < 143.6 µmol·L-1 (p = .004), and GBA < .075 µmol·L-1 (p = .007). A prediction model for seizures in SSADHD was comprised of the additive effect of older age and lower plasma GABA, GHB, and GBA (area under the receiver operating characteristic curve of .798, p = .008). SIGNIFICANCE: Epilepsy is highly prevalent in SSADHD, and its onset and severity correlate with an age-related decline in GABA and GABA-related metabolite levels as well as TMS markers of reduced cortical inhibition. The reduction of GABAergic activity in this otherwise hyper-GABAergic disorder demonstrates a concordance between epileptogenesis and compensatory responses. These findings may furthermore inform the timing of molecular interventions for SSADHD.

Allosteric modulation of α1ß3γ2 GABAA receptors by farnesol through the neurosteroid sites.

In mammalian cells, all-trans farnesol, a 15-carbon isoprenol, is a product of the mevalonate pathway. It is the natural substrate of alcohol dehydrogenase and a substrate for CYP2E1, two enzymes implicated in ethanol metabolism. Studies have shown that farnesol is present in the human brain and inhibits voltage-gated Ca2+ channels at much lower concentrations than ethanol. Here we show that farnesol modulates the activity of γ-aminobutyric acid type A receptors (GABAARs), some of which also mediate the sedative activity of ethanol. Electrophysiology experiments performed in HEK cells expressing human α1ß3γ2 or α6ß3γ2 GABAARs revealed that farnesol increased chloride currents through positive allosteric modulation of these receptors and showed dependence on both the alcoholic functional group of farnesol and the length of the alkyl chain for activity. In silico studies using long-timescale unbiased all-atom molecular dynamics (MD) simulations of the human α1ß3γ2 GABAA receptors revealed that farnesol modulates the channel by directly binding to the transmembrane neurosteroid-binding site, after partitioning into the surrounding membrane and reaching the receptor by lateral diffusion. Channel activation by farnesol was further characterized by several structural and dynamic variables, such as global twisting of the receptor's extracellular domain, tilting of the transmembrane M2 helices, radius, cross-sectional area, hydration status, and electrostatic potential of the channel pore. Our results expand the pharmacological activities of farnesol to yet another class of ion channels implicated in neurotransmission, thus providing a novel path for understanding and treatment of diseases involving GABAA receptor dysfunction.

Farnesol induces protection against murine CNS inflammatory demyelination and modifies gut microbiome.

Farnesol is a 15­carbon organic isoprenol synthesized by plants and mammals with anti-oxidant, anti-inflammatory, and neuroprotective activities. We sought to determine whether farnesol treatment would result in protection against murine experimental autoimmune encephalomyelitis (EAE), a well-established model of multiple sclerosis (MS). We compared disease progression and severity in C57BL/6 mice treated orally with 100 mg/kg/day farnesol solubilized in corn oil to corn-oil treated and untreated EAE mice. Farnesol significantly delayed the onset of EAE (by ~2 days) and dramatically decreased disease severity (~80%) compared to controls. Disease protection by farnesol was associated with a significant reduction in spinal cord infiltration by monocytes-macrophages, dendritic cells, CD4+ T cells, and a significant change in gut microbiota composition, including a decrease in the Firmicutes:Bacteroidetes ratio. The study suggests FOL could protect MS patients against CNS inflammatory demyelination by partially modulating the gut microbiome composition.

Intestinal Dysbiosis as a component of pathophysiology in succinic semialdehyde dehydrogenase deficiency (SSADHD).

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is an inherited inborn error of the γ-aminobutyric acid (GABA) metabolism pathway. It results from mutations in the ALDH5A1 gene leading to elevated GABA, γ-hydroxybutyric acid (GHB), succinic semialdehyde (SSA), decreased glutamine and alterations in several other metabolites. The phenotype includes developmental and cognitive delays, hypotonia, seizures, neuropsychiatric morbidity and other nervous system pathologies. The composition of the intestinal flora of patients with SSADHD has not been characterized, and dysbiosis of the gut microbiome may unveil novel treatment paradigms. We investigated the gut microbiome in SSADHD using 16S ribosomal DNA sequencing and unmasked evidence of dysbiosis in both aldh5a1-deficient mice and patients with SSADHD. In the murine model, there was a reduction in α-diversity measurements, and there were 4 phyla, 3 classes, 5 orders, 9 families, and 15 genera that differed, with a total of 17 predicted metabolic pathways altered. In patients, there were changes in Fusobacterium, 3 classes, 4 orders, 11 families, and a predicted alteration in genes associated with the digestive system. We believe this is the first evaluation of microbiome structure in an IEM with a neurometabolic phenotype that is not treated dietarily.

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.

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.

Preferential accumulation of the active S-(+) isomer in murine retina highlights novel mechanisms of vigabatrin-associated retinal toxicity.

((S)-(+)/(R)-(-)) vigabatrin (SabrilR; γ-vinyl GABA), an antiepileptic irreversibly inactivating GABA-transaminase, was administered to male C57Bl6 J mice via continuous infusion (0, 40, 80 mg/kg/d) for 12 days. Our study design pooled retina, eye (minus retina), whole brain and plasma from n = 24 animals for each dose to provide n = 8 triplicates per treatment group. Hypothesizing that (S)-(+) VGB (active isomer) would preferentially accumulate in retina, we determined VGB isomers, comprehensive amino acids, and pharmacokinetic parameters. In brain, eye and plasma, the ((S)-(+)/(R)-(-)) ratio varied from 0.73 to 1.29 and 13.3 in retina, accompanied by a partition coefficient (tissue/plasma, ((S)-(+);(R)-(-))) of 5.8;0.34, 0.63;0.49, and 0.51;0.34 in retina, eye and brain, respectively. Racemic VGB (nmol/g; plasma, nmol/mL, range of means for dose) content was: retina, 25-36; eye (minus retina), 4.8-8.0; brain, 3.1-6.8 and plasma, 8.7-14.9. GABA tissue content (nmol/g) was 1246-3335, 18-64 and 2615-3200 as a function of VGB dose for retina, eye (minus retina) and brain, respectively. The retinal glial cell toxin 2-aminoadipic acid also increased with VGB dose (76-96 nmol/g). Partitioning of active (S)-(+) VGB to retina suggests the involvement of a stereospecific transporter, the identification of which could reveal new therapeutic paradigms that might mitigate VGB's well-known retinal toxicity and expand its clinical utility.