RESUMO
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.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Deficiências do Desenvolvimento , Succinato-Semialdeído Desidrogenase , Succinato-Semialdeído Desidrogenase/deficiência , Humanos , Succinato-Semialdeído Desidrogenase/genética , Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Erros Inatos do Metabolismo dos Aminoácidos/terapia , Erros Inatos do Metabolismo dos Aminoácidos/genética , Consenso , Ácido gama-Aminobutírico/metabolismo , Guias de Prática Clínica como AssuntoRESUMO
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.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Sistema Glinfático , Imageamento por Ressonância Magnética , Transtornos do Sono-Vigília , Succinato-Semialdeído Desidrogenase , Ácido gama-Aminobutírico , Humanos , Masculino , Feminino , Ácido gama-Aminobutírico/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/fisiopatologia , Erros Inatos do Metabolismo dos Aminoácidos/complicações , Transtornos do Sono-Vigília/fisiopatologia , Sistema Glinfático/fisiopatologia , Criança , Succinato-Semialdeído Desidrogenase/deficiência , Espectroscopia de Ressonância Magnética , Adolescente , Encéfalo/diagnóstico por imagem , Encéfalo/fisiopatologia , Encéfalo/metabolismo , Aquaporina 4 , Laringoestenose/fisiopatologia , Pré-Escolar , Deficiências do DesenvolvimentoRESUMO
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.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Terapia Genética , Succinato-Semialdeído Desidrogenase , Transmissão Sináptica , Humanos , Succinato-Semialdeído Desidrogenase/deficiência , Succinato-Semialdeído Desidrogenase/genética , Terapia Genética/métodos , Erros Inatos do Metabolismo dos Aminoácidos/terapia , Erros Inatos do Metabolismo dos Aminoácidos/genética , Transmissão Sináptica/genética , AnimaisRESUMO
Traumatic brain injury (TBI) increases cerebral reactive oxygen species production, which leads to continuing secondary neuronal injury after the initial insult. Cortical parvalbumin-positive interneurons (PVIs; neurons responsible for maintaining cortical inhibitory tone) are particularly vulnerable to oxidative stress and are thus disproportionately affected by TBI. Systemic N-acetylcysteine (NAC) treatment may restore cerebral glutathione equilibrium, thus preventing post-traumatic cortical PVI loss. We therefore tested whether weeks-long post-traumatic NAC treatment mitigates cortical oxidative stress, and whether such treatment preserves PVI counts and related markers of PVI integrity and prevents pathologic electroencephalographic (EEG) changes, 3 and 6 weeks after fluid percussion injury in rats. We find that moderate TBI results in persistent oxidative stress for at least 6 weeks after injury and leads to the loss of PVIs and the perineuronal net (PNN) that surrounds them as well as of per-cell parvalbumin expression. Prolonged post-TBI NAC treatment normalizes the cortical redox state, mitigates PVI and PNN loss, and - in surviving PVIs - increases per-cell parvalbumin expression. NAC treatment also preserves normal spectral EEG measures after TBI. We cautiously conclude that weeks-long NAC treatment after TBI may be a practical and well-tolerated treatment strategy to preserve cortical inhibitory tone post-TBI.
Assuntos
Acetilcisteína , Lesões Encefálicas Traumáticas , Ratos , Animais , Acetilcisteína/farmacologia , Acetilcisteína/metabolismo , Parvalbuminas/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Estresse Oxidativo/fisiologia , Interneurônios/metabolismoRESUMO
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.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Criança , Humanos , Masculino , Feminino , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Deficiências do Desenvolvimento/genética , Fenótipo , Succinato-Semialdeído Desidrogenase/genética , Succinato-Semialdeído Desidrogenase/metabolismoRESUMO
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.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Epilepsia , Oxibato de Sódio , Humanos , Criança , Erros Inatos do Metabolismo dos Aminoácidos/complicações , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Deficiências do Desenvolvimento , Epilepsia/metabolismo , Ácido gama-Aminobutírico/metabolismo , Aminobutiratos , ConvulsõesRESUMO
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.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Succinato-Semialdeído Desidrogenase , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Animais , Criança , Deficiências do Desenvolvimento/genética , Humanos , Camundongos , Succinato-Semialdeído Desidrogenase/deficiência , Succinato-Semialdeído Desidrogenase/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Traumatic brain injury (TBI) results in a decrease in glutamate transporter-1 (GLT-1) expression, the major mechanism for glutamate removal from synapses. Coupled with an increase in glutamate release from dead and dying neurons, this causes an increase in extracellular glutamate. The ensuing glutamate excitotoxicity disproportionately damages vulnerable GABAergic parvalbumin-positive inhibitory interneurons, resulting in a progressively worsening cortical excitatory:inhibitory imbalance due to a loss of GABAergic inhibitory tone, as evidenced by chronic post-traumatic symptoms such as epilepsy, and supported by neuropathologic findings. This loss of intracortical inhibition can be measured and followed noninvasively using long-interval paired-pulse transcranial magnetic stimulation with mechanomyography (LI-ppTMS-MMG). Ceftriaxone, a ß-lactam antibiotic, is a potent stimulator of the expression of rodent GLT-1 and would presumably decrease excitotoxic damage to GABAergic interneurons. It may thus be a viable antiepileptogenic intervention. Using a rat fluid percussion injury TBI model, we utilized LI-ppTMS-MMG, quantitative PCR, and immunohistochemistry to test whether ceftriaxone treatment preserves intracortical inhibition and cortical parvalbumin-positive inhibitory interneuron function after TBI in rat motor cortex. We show that neocortical GLT-1 gene and protein expression are significantly reduced 1 week after TBI, and this transient loss is mitigated by ceftriaxone. Importantly, whereas intracortical inhibition declines progressively after TBI, 1 week of post-TBI ceftriaxone treatment attenuates the loss of inhibition compared to saline-treated controls. This finding is accompanied by significantly higher parvalbumin gene and protein expression in ceftriaxone-treated injured rats. Our results highlight prospects for ceftriaxone as an intervention after TBI to prevent cortical inhibitory interneuron dysfunction, partly by preserving GLT-1 expression.
Assuntos
Antibacterianos/administração & dosagem , Lesões Encefálicas Traumáticas/metabolismo , Ceftriaxona/administração & dosagem , Transportador 2 de Aminoácido Excitatório/metabolismo , Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Animais , Modelos Animais de Doenças , Expressão Gênica , Masculino , Córtex Motor/fisiopatologia , Parvalbuminas/metabolismo , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Estado Epiléptico/metabolismo , Estado Epiléptico/patologia , Simportadores/metabolismo , Animais , Cloretos/metabolismo , Endocitose , Técnicas de Introdução de Genes , Glutamatos/farmacologia , Camundongos , Camundongos Mutantes Neurológicos , Proteínas Mutantes/metabolismo , Mutação/genética , Fosforilação , Fosfosserina/metabolismo , Proteína Fosfatase 1/antagonistas & inibidores , Proteína Fosfatase 1/metabolismo , Simportadores/genética , Ácido gama-Aminobutírico/metabolismo , Cotransportadores de K e Cl-RESUMO
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 ⼠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.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Deficiências do Desenvolvimento , Células-Tronco Pluripotentes Induzidas , Succinato-Semialdeído Desidrogenase , Adolescente , Animais , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Camundongos , Erros Inatos do Metabolismo dos Aminoácidos/terapia , Erros Inatos do Metabolismo dos Aminoácidos/fisiopatologia , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/complicações , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Neurônios GABAérgicos/metabolismo , Ácido gama-Aminobutírico/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/etiologia , Transtornos do Neurodesenvolvimento/genética , Succinato-Semialdeído Desidrogenase/deficiência , Succinato-Semialdeído Desidrogenase/metabolismo , Succinato-Semialdeído Desidrogenase/genéticaRESUMO
Specific transfer of (orthodenticle homeobox 2) Otx2 homeoprotein into GABAergic interneurons expressing parvalbumin (PV) is necessary and sufficient to open, then close, a critical period (CP) of plasticity in the developing mouse visual cortex. The accumulation of endogenous Otx2 in PV cells suggests the presence of specific Otx2 binding sites. Here, we find that perineuronal nets (PNNs) on the surfaces of PV cells permit the specific, constitutive capture of Otx2. We identify a 15 aa domain containing an arginine-lysine doublet (RK peptide) within Otx2, bearing prototypic traits of a glycosaminoglycan (GAG) binding sequence that mediates Otx2 binding to PNNs, and specifically to chondroitin sulfate D and E, with high affinity. Accordingly, PNN hydrolysis by chondroitinase ABC reduces the amount of endogenous Otx2 in PV cells. Direct infusion of RK peptide similarly disrupts endogenous Otx2 localization to PV cells, reduces PV and PNN expression, and reopens plasticity in adult mice. The closure of one eye during this transient window reduces cortical acuity and is specific to the RK motif, as an Alanine-Alanine variant or a scrambled peptide fails to reactivate plasticity. Conversely, this transient reopening of plasticity in the adult restores binocular vision in amblyopic mice. Thus, one function of PNNs is to facilitate the persistent internalization of Otx2 by PV cells to maintain CP closure. The pharmacological use of the Otx2 GAG binding domain offers a novel, potent therapeutic tool with which to restore cortical plasticity in the mature brain.
Assuntos
Matriz Extracelular/metabolismo , Interneurônios/metabolismo , Plasticidade Neuronal/fisiologia , Fatores de Transcrição Otx/metabolismo , Córtex Visual/metabolismo , Fatores Etários , Animais , Neurônios GABAérgicos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição Otx/fisiologia , Ligação Proteica/fisiologia , Córtex Visual/citologiaRESUMO
Treatment options for inherited metabolic epilepsies are rapidly expanding with advances in molecular biology and the genomic revolution. Traditional dietary and nutrient modification and inhibitors or enhancers of protein and enzyme function, the mainstays of therapy, are undergoing continuous revisions to increase biological activity and reduce toxicity. Enzyme replacement and gene replacement and editing hold promise for genetically targeted treatment and cures. Molecular, imaging, and neurophysiologic biomarkers are emerging as key indicators of disease pathophysiology, severity, and response to therapy.
Assuntos
Epilepsia , Humanos , Criança , Epilepsia/genética , Epilepsia/terapiaRESUMO
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.
RESUMO
The activity of the neuronal-specific potassium chloride co-transporter KCC2 allows neurons to maintain low intracellular Cl(-) concentrations. These low Cl(-) concentrations are critical in mediating fast synaptic inhibition upon the activation of Cl(-)-permeable ligand-gated ion channels such as type A gamma-aminobutyric acid receptors (GABA(A)Rs). Deficits in KCC2 functional expression thus play central roles in the etiology of epilepsy and ischemia. It is emerging that KCC2 is phosphorylated on tyrosine residues, but the molecular substrates for this covalent modification within KCC2 and its functional significance remain poorly understood. Here we demonstrate that in HEK-293 cells the principal sites of tyrosine phosphorylation within KCC2 are residues 903 and 1087 (Y903/1087), which lie within the major C-terminal intracellular domain of KCC2. Phosphorylation of Y903/1087 decreases the cell surface stability of KCC2 principally by enhancing their lysozomal degradation. We further demonstrate that in cultured hippocampal neurons prolonged activation of muscarinic acetylcholine receptors (mAChRs) enhances KCC2 tyrosine phosphorylation and lysozomal degradation. Consistent with our in vitro studies, induction of status epilepticus (SE) in mice using pilocarpine, a mAChR agonist, induces large deficits in the cell surface stability of KCC2 together with enhanced tyrosine phosphorylation. Tyrosine phosphorylation of KCC2 is thus likely to play a key role in regulating the degradation of KCC2, a process that may be responsible for pathological losses of KCC2 function that are evident in SE and other forms of epilepsy.
Assuntos
Membrana Celular/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Estado Epiléptico/metabolismo , Simportadores/metabolismo , Tirosina/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Fosforilação , Pilocarpina/efeitos adversos , Receptores Muscarínicos/metabolismo , Estado Epiléptico/induzido quimicamente , Simportadores/genética , Cotransportadores de K e Cl-RESUMO
Brain plasticity is often restricted to critical periods in early life. Here, we show that a key regulator of this process in the visual cortex, Otx2 homeoprotein, is synthesized and secreted globally from the choroid plexus. Consequently, Otx2 is maintained in selected GABA cells unexpectedly throughout the mature forebrain. Genetic disruption of choroid-expressed Otx2 impacts these distant circuits and in the primary visual cortex reopens binocular plasticity to restore vision in amblyopic mice. The potential to regulate adult cortical plasticity through the choroid plexus underscores the importance of this structure in brain physiology and offers therapeutic approaches to recovery from a broad range of neurodevelopmental disorders.
Assuntos
Plexo Corióideo/citologia , Plexo Corióideo/metabolismo , Plasticidade Neuronal/fisiologia , Fatores de Transcrição Otx/metabolismo , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição Otx/genéticaRESUMO
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.
Assuntos
Hipocampo/citologia , Neurônios/metabolismo , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Simportadores/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Regulação para Baixo , Fosforilação , Proteína Fosfatase 1/metabolismo , Ratos , Serina/metabolismo , Cotransportadores de K e Cl-RESUMO
KCC2 comprises the major Cl(-) extruding mechanism in most adult neurons. Hyperpolarizing GABAergic transmission depends on KCC2 function. We recently demonstrated that glutamate reduces KCC2 function by a phosphorylation-dependent mechanism that leads to excitatory GABA responses. Here we investigated the methods by which to estimate changes in E(GABA), as well as the processes that lead to depolarizing GABA responses and their effects on neuronal excitability. We demonstrated that current-clamp recordings of membrane potential responses to GABA can determine upper and lower limits of E(GABA). We also further characterized depolarizing GABA responses, which both excited and inhibited neurons. Our analyses revealed that persistently active GABA(A) receptors contributed to loading Cl(-) during the glutamate exposure, indicating that tonic inhibition can facilitate the development of depolarizing GABA responses and increase excitability after pathophysiological insults. Finally, we demonstrated that hyperpolarizing GABA responses could temporarily switch to depolarizing responses when they coincided with an afterhyperpolarization.
Assuntos
Neurônios GABAérgicos/metabolismo , Potenciais da Membrana/fisiologia , Receptores de GABA-A/metabolismo , Simportadores/metabolismo , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismo , Cloretos/metabolismo , Humanos , Transporte de Íons/fisiologia , Cotransportadores de K e Cl-RESUMO
Phosphorylation can affect both the function and trafficking of GABA(A) receptors with significant consequences for neuronal excitability. Serine/threonine kinases can phosphorylate the intracellular loops between M3-4 of GABA(A) receptor beta and gamma subunits thereby modulating receptor function in heterologous expression systems and in neurons (1, 2). Specifically, CaMK-II has been demonstrated to phosphorylate the M3-4 loop of GABA(A) receptor subunits expressed as GST fusion proteins (3, 4). It also increases the amplitude of GABA(A) receptor-mediated currents in a number of neuronal cell types (5-7). To identify which substrate sites CaMK-II might phosphorylate and the consequent functional effects, we expressed recombinant GABA(A) receptors in NG108-15 cells, which have previously been shown to support CaMK-II modulation of GABA(A) receptors containing the beta3 subunit (8). We now demonstrate that CaMK-II mediates its effects on alpha1beta3 receptors via phosphorylation of Ser(383) within the M3-4 domain of the beta subunit. Ablation of beta3 subunit phosphorylation sites for CaMK-II revealed that for alphabetagamma receptors, CaMK-II has a residual effect on GABA currents that is not mediated by previously identified sites of CaMK-II phosphorylation. This residual effect is abolished by mutation of tyrosine phosphorylation sites, Tyr(365) and Tyr(367), on the gamma2S subunit, and by the tyrosine kinase inhibitor genistein. These results suggested that CaMK-II is capable of directly phosphorylating GABA(A) receptors and activating endogenous tyrosine kinases to phosphorylate the gamma2 subunit in NG108-15 cells. These findings were confirmed in a neuronal environment by expressing recombinant GABA(A) receptors in cerebellar granule neurons.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Subunidades Proteicas/metabolismo , Receptores de GABA-A/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Células Cultivadas , Cerebelo/citologia , Ativação Enzimática , Humanos , Camundongos , Técnicas de Patch-Clamp , Fosforilação , Subunidades Proteicas/genética , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismo , Tirosina/metabolismoRESUMO
The potassium chloride cotransporter KCC2 plays a major role in the maintenance of transmembrane chloride potential in mature neurons; thus KCC2 activity is critical for hyperpolarizing membrane currents generated upon the activation of gamma-aminobutyric acid type A and glycine (Gly) receptors that underlie fast synaptic inhibition in the adult central nervous system. However, to date an understanding of the cellular mechanism that neurons use to modulate the functional expression of KCC2 remains rudimentary. Using Escherichia coli expression coupled with in vitro kinase assays, we first established that protein kinase C (PKC) can directly phosphorylate serine 940 (Ser(940)) within the C-terminal cytoplasmic domain of KCC2. We further demonstrated that Ser(940) is the major site for PKC-dependent phosphorylation for full-length KCC2 molecules when expressed in HEK-293 cells. Phosphorylation of Ser(940) increased the cell surface stability of KCC2 in this system by decreasing its rate of internalization from the plasma membrane. Coincident phosphorylation of Ser(940) increased the rate of ion transport by KCC2. It was further evident that phosphorylation of endogenous KCC2 in cultured hippocampal neurons is regulated by PKC-dependent activity. Moreover, in keeping with our recombinant studies, enhancing PKC-dependent phosphorylation increased the targeting of KCC2 to the neuronal cell surface. Our studies thus suggest that PKC-dependent phosphorylation of KCC2 may play a central role in modulating both the functional expression of this critical transporter in the brain and the strength of synaptic inhibition.
Assuntos
Membrana Celular/metabolismo , Cloreto de Potássio/química , Proteína Quinase C/metabolismo , Simportadores/química , Sítios de Ligação , Linhagem Celular , Endocitose , Escherichia coli/metabolismo , Hipocampo/metabolismo , Humanos , Modelos Biológicos , Neurônios/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Receptores de GABA-A/química , Cotransportadores de K e Cl-RESUMO
Presynaptic motor neuron synthesizes and secretes acetylcholinesterase (AChE) at vertebrate neuromuscular junctions. In order to determine the retrograde role of muscle in regulating the expression of AChE in motor neuron, a chimeric co-culture of NG108-15 cell, a cholinergic cell line that resembles motor neuron, with chick myotube was established to mimic the neuromuscular contact in vitro. A DNA construct of human AChE promoter tagged with luciferase (pAChE-Luc) was stably transfected into NG108-15 cells. The co-culture with myotubes robustly stimulated the promoter activity as well as the endogenous expression of AChE in pAChE-Luc stably transfected NG108-15 cells. Muscle extract derived from chick embryos when applied onto pAChE-Luc-expressing NG108-15 cells induced expressions of AChE promoter and endogenous AChE. The cAMP-responsive element mutation on human AChE promoter blocked the muscle-induced AChE transcriptional activity in cultured NG108-15 cells either in co-culturing with myotube or in applying muscle extract. The accumulation of intracellular cAMP and the phosphorylation of cAMP-responsive element-binding protein in cultured NG108-15 cells were stimulated by applied muscle extract. Part of the muscle-induced signaling was mimicked by application of calcitonin gene-related peptide in cultured NG108-15 cells. These results suggest the muscle-induced neuronal AChE expression in the co-culture is mediated by a cAMP-dependent signaling.