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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.

Objectives Succinic semialdehyde dehydrogenase deficiency (SSADHD) is an inherited metabolic disorder characterized by an accumulation of γ‐aminobutyric (GABA). In addition to its synaptic role as an inhibitory neurotransmitter, GABA also plays an important role in myelination. We aimed to investigate the relationship between GABA and myelination abnormalities in SSADHD patients and the mouse model. Methods Brain MRIs performed on 44 individuals (23 with SSADHD and 21 healthy controls) were independently reviewed by two neuroradiologists and scored using a disease‐specific myelination scoring system. Inter‐rater reliability (IRR) was assessed by the intraclass correlation coefficient. Myelination scores of SSADHD individuals were correlated with clinical, biochemical, magnetic resonance spectroscopy, and genetic data. Additionally, we investigated the expression of myelin‐related genes in a mouse SSADHD model. Results Dysmyelination in SSADHD patients was overall mild, but significantly greater than in healthy controls (p < 0.001). In SSADHD patients, lower myelination scores were significantly correlated with younger age (R = 0.775, p < 0.001) and higher plasma GABA (R = −0.722, p < 0.001) and γ‐hydroxybutyric acid (GHB) (R = −0.683, p = 0.001). In SSADH‐deficient mice, there was reduced expression of genes encoding myelin basic protein (p = 0.001), myelin‐associated oligodendrocyte basic protein (p = 0.001), and mitochondrial aspartate transporter (p = 0.025). Interpretation Excessive GABA and GHB, which characterize SSADHD and are further pronounced in younger SSADHD individuals, may account for delayed oligodendrocyte maturation and altered myelination dynamics in this disorder. Studying the properties of dysmyelination in this unique disorder enhances our understanding of GABA's mediating role on myelination and may contribute to monitoring disease progression and managing other white‐matter neurological disorders. Trial Registration NCT03758521

The authors show that NMDA receptor activation induces a rapid protein phosphatase 1–dependent dephosphorylation and downregulation of the neuron-specific potassium-chloride co-transporter KCC2. Deficits in KCC2 activity have been observed in disease states associated with pathophysiological glutamate release. Blocking dephosphorylation reduces the glutamate-induced degradation of KCC2 and maintains hyperpolarizing GABAergic inhibition. KCC2 is a neuron-specific K + -Cl − co-transporter that maintains a low intracellular Cl − concentration that is essential for hyperpolarizing inhibition mediated by GABA A receptors. Deficits in KCC2 activity occur in disease states associated with pathophysiological glutamate release. However, the mechanisms by which elevated glutamate alters KCC2 function are unknown. The phosphorylation of KCC2 residue Ser940 is known to regulate its surface activity. We found that NMDA receptor activity and Ca 2+ influx caused the dephosphorylation of Ser940 in dissociated rat neurons, leading to a loss of KCC2 function that lasted longer than 20 min. Protein phosphatase 1 mediated the dephosphorylation events of Ser940 that coincided with a deficit in hyperpolarizing GABAergic inhibition resulting from the loss of KCC2 activity. Blocking dephosphorylation of Ser940 reduced the glutamate-induced downregulation of KCC2 and substantially improved the maintenance of hyperpolarizing GABAergic inhibition. Reducing the downregulation of KCC2 therefore has therapeutic potential in the treatment of neurological disorders.

Significance Status epilepticus (SE) is defined as a state of continuous unremitting seizures that often exhibits underlying deficits in neuronal inhibition mediated by GABA A receptors. The efficacy of neuronal inhibition is critically dependent on the activity of the K ⁺/Cl – cotransporter KCC2, which allows neurons to maintain low intracellular Cl – levels. KCC2 activity is enhanced by phosphorylation of residue serine 940, and here we show that SE leads to rapid dephosphorylation of this key regulatory residue. Moreover, we demonstrate that deficits in S940 phosphorylation directly contribute to the onset and severity of SE. Collectively, our results suggest that deficits in KCC2 activity directly contribute to the pathophysiology of SE. The K ⁺/Cl – cotransporter (KCC2) allows adult neurons to maintain low intracellular Cl – levels, which are a prerequisite for efficient synaptic inhibition upon activation of γ-aminobutyric acid receptors. Deficits in KCC2 activity are implicated in epileptogenesis, but how increased neuronal activity leads to transporter inactivation is ill defined. In vitro, the activity of KCC2 is potentiated via phosphorylation of serine 940 (S940). Here we have examined the role this putative regulatory process plays in determining KCC2 activity during status epilepticus (SE) using knockin mice in which S940 is mutated to an alanine (S940A). In wild-type mice, SE induced by kainate resulted in dephosphorylation of S940 and KCC2 internalization. S940A homozygotes were viable and exhibited comparable basal levels of KCC2 expression and activity relative to WT mice. However, exposure of S940A mice to kainate induced lethality within 30 min of kainate injection and subsequent entrance into SE. We assessed the effect of the S940A mutation in cultured hippocampal neurons to explore the mechanisms underlying this phenotype. Under basal conditions, the mutation had no effect on neuronal Cl – extrusion. However, a selective deficit in KCC2 activity was seen in S940A neurons upon transient exposure to glutamate. Significantly, whereas the effects of glutamate on KCC2 function could be ameliorated in WT neurons with agents that enhance S940 phosphorylation, this positive modulation was lost in S940A neurons. Collectively our results suggest that phosphorylation of S940 plays a critical role in potentiating KCC2 activity to limit the development of SE.

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.

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.

KCC2 is a neuron-specific K super(+)-Cl super(-) co-transporter that maintains a low intracellular Cl super(-) concentration that is essential for hyperpolarizing inhibition mediated by GABA sub(A) receptors. Deficits in KCC2 activity occur in disease states associated with pathophysiological glutamate release. However, the mechanisms by which elevated glutamate alters KCC2 function are unknown. The phosphorylation of KCC2 residue Ser940 is known to regulate its surface activity. We found that NMDA receptor activity and Ca super(2+) influx caused the dephosphorylation of Ser940 in dissociated rat neurons, leading to a loss of KCC2 function that lasted longer than 20 min. Protein phosphatase 1 mediated the dephosphorylation events of Ser940 that coincided with a deficit in hyperpolarizing GABAergic inhibition resulting from the loss of KCC2 activity. Blocking dephosphorylation of Ser940 reduced the glutamate-induced downregulation of KCC2 and substantially improved the maintenance of hyperpolarizing GABAergic inhibition. Reducing the downregulation of KCC2 therefore has therapeutic potential in the treatment of neurological disorders.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare autosomal recessive metabolic disorder due to loss-of-function mutations impairing the catabolism of γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the brain. In SSADHD, pathologic accumulation of GABA and its metabolic by-product γ-hydroxybutyrate (GHB) corresponds to a clinical syndrome dominated by developmental delay and epilepsy in half of patients with risk of sudden death in adolescence and adulthood. Brain-wide gene replacement for SSADHD is unavailable, and whether such treatment will reverse the SSADHD phenotype is unknown. We developed an inducible mouse SSADHD model, , enabling Cre-dependent restoration to evaluate gene therapy feasibility. In the absence of SSADH, mice exhibit hyperactivity and excessive serum GHB levels, culminating in death by ∼postnatal day 22, recapitulating the severe SSADHD condition. Systemic delivery of a blood-brain barrier (BBB)-penetrating adeno-associated virus (AAV) carrying a gene to mice leads to brain-wide SSADH restoration, serum GHB level reduction, normalization of hyperactivity, and substantial increase in survival. As a step toward clinical translation, we further assessed an AAV encompassing a functional native promoter (FLnP) of tethered to its human coding sequence, namely AAV-FLnP-hALDH5A1. mice were effectively rescued when treated with AAV-FLnP-hALDH5A1 packaged in the blood-brain barrier (BBB)-penetrating capsid PHP.eB. These findings provide preclinical proof that SSADH gene replacement therapy is feasible and potentially effective.

In vertebrate neuromuscular junctions (nmjs), adenosine 5'-triphosphate (ATP) is stored at the motor nerve terminals and is co-released with acetylcholine during neural stimulation. Several lines of evidence suggest that the synaptic ATP can act as a synapse-organizing factor at the nmjs, mediated by metabotropic P2Y(1) receptors. P2Y(1) receptor mRNAs in chicken and rat muscles are low in embryo but increases markedly in the adult, and decreased after denervation. The P2Y(1) receptor protein is restricted to the nmjs and co-localized with AChRs in adult muscles. The activation of P2Y(1) receptor by adenine nucleotides in cultured chick myotubes stimulated the accumulation of inositol phosphates, intracellular Ca(2+) mobilization, protein kinase C activity and phosphorylation of extracellular signal-regulated kinases. The receptor activation led to an increase in the expression of transcripts encoding AChE catalytic subunit and AChR subunits. The ATP-induced post-synaptic gene expression is possibly mediated by the activation of signaling cascades of mitogen-activated protein kinase. Therefore, a model is being proposed here that the synaptic ATP has a role of synergy with other regulatory signals, such as neuregulin, which act via their post-synaptic receptors to activate second signaling molecules locally to enhance the transcription of AChR/AChE genes specifically in the adjacent sub-synaptic nuclei during the formation and, especially, the maintenance of post-synaptic specializations at the nmjs.

Mutation accumulation in somatic cells contributes to cancer development and is proposed as a cause of aging. DNA polymerases Pol ε and Pol δ replicate DNA during cell division. However, in some cancers, defective proofreading due to acquired POLE / POLD1 exonuclease domain mutations causes markedly elevated somatic mutation burdens with distinctive mutational signatures. Germline POLE / POLD1 mutations cause familial cancer predisposition. Here, we sequenced normal tissue and tumor DNA from individuals with germline POLE / POLD1 mutations. Increased mutation burdens with characteristic mutational signatures were found in normal adult somatic cell types, during early embryogenesis and in sperm. Thus human physiology can tolerate ubiquitously elevated mutation burdens. Except for increased cancer risk, individuals with germline POLE / POLD1 mutations do not exhibit overt features of premature aging. These results do not support a model in which all features of aging are attributable to widespread cell malfunction directly resulting from somatic mutation burdens accrued during life. Healthy tissues from individuals with germline mutations in POLE or POLD1 show increased mutational burden, suggesting that normal cells are capable of tolerating high mutation rates.