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Summary: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.Weidentify 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 Otx2GAGbinding domain offers a novel, potent therapeutic tool with which to restore cortical plasticity in the mature brain. [ABSTRACT FROM AUTHOR]

KCC2 is a neuron-specific K+-Cl− co-transporter that maintains a low intracellular Cl− concentration that is essential for hyperpolarizing inhibition mediated by GABAA 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 Ca2+ 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 γ-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 (Ser940) within the C-terminal cytoplasmic domain of KCC2. We further demonstrated that Ser940 is the major site for PKC-dependent phosphorylation for full-length KCC2 molecules when expressed in HEK-293 cells. Phosphorylation of Ser940 increased the cell surface stability of KCC2 in this system by decreasing its rate of internalization from the plasma membrane. Coincident phosphorylation of Ser940 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. [ABSTRACT FROM AUTHOR]

Phosphorylation can affect both the function and trafficking of GABAA receptors with significant consequences for neuronal excitability. Serine/threonine kinases can phosphorylate the intracellular loops between M3-4 of GABAA receptor β and γ 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 GABAA receptor subunits expressed as GST fusion proteins (3, 4). It also increases the amplitude of GABAA 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 GABAA receptors in NG108-15 cells, which have previously been shown to support CaMK-II modulation of GABAA receptors containing the β3 subunit (8). We now demonstrate that CaMK-II mediates its effects on α1β3 receptors via phosphorylation of Ser383 within the M3-4 domain of the β subunit. Ablation of β3 subunit phosphorylation sites for CaMK-II revealed that for αβγ 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, Tyr365 and Tyr367, on the γ2S subunit, and by the tyrosine kinase inhibitor genistein. These results suggested that CaMK-II is capable of directly phosphorylating GABAA receptors and activating endogenous tyrosine kinases to phosphorylate the γ2 subunit in NG108-15 cells. These findings were confirmed in a neuronal environment by expressing recombinant GABAA receptors in cerebellar granule neurons. [ABSTRACT FROM AUTHOR]

The presence of a collagenous protein (ColQ) charac- terizes the collagen-tailed forms of acetylcholinesterase and butyrylcholinesterase at vertebrate neuromuscular junctions which is tethered in the synaptic basal lamina. ColQ subunits, differing mostly by their signal se- quences, are encoded by transcripts ColQ-1 and ColQ-la, which are differentially expressed in slow and fast twitch muscles in mammals. Two distinct promoters, pColQ-1 and pColQ-la, were isolated from the upstream sequences of human COLQ gene; they showed muscle- specific expression and were activated by myogenic transcriptional elements in cultured myotubes. After in vivo DNA transfection, pColQ-1 showed strong activity in slow twitch muscle (e.g. soleus), whereas pColQ-la was preferably expressed in fast twitch muscle (e.g. tibialis). Mutation analysis of the ColQ promoters sug- gested that the muscle fiber type-specific expression pattern of ColQ transcripts were regulated by a slow upsteam regulatory element (SURE) and a fast intronic regulatory element (FIRE). These regulatory elements were responsive to a calcium ionophore and to calcineurin inhibition by cyclosporine A. The slow fiber type-specific expression of ColQ-1 was abolished by the mutation of an NFAT element in pColQ-1. Moreover, both the ColQ promoters contained N-box element that was responsible for the synapse-specific expression of ColQ transcripts. These results explain the specific expression patterns of collagen-tailed acetylcholinesterase in slow and fast muscle fibers. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Advances in molecular biology and genetics are increasingly revealing the complex etiology of autism spectrum disorder (ASD). In parallel, a number of biochemical, anatomical, and electrophysiological measures are emerging as potential disease-relevant biomarkers that could inform the diagnosis and clinical management of ASD. Rodent ASD models play a key role in ASD research as essential experimental tools. Nevertheless, there are challenges and limitations to the validity and translational value of rodent models, including genetic relevance and cognitive performance differences between humans and rodents. In this chapter, we begin with a brief history of autism research, followed by prominent examples of disease-relevant mouse models enabled by current knowledge of genetics, molecular biology, and bioinformatics. These ASD-associated rodent models enable quantifiable biomarker development. Finally, we discuss the prospects of ASD biomarker development., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

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., (© 2023 American Academy of Neurology.)

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., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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., (Copyright 2010 Elsevier Inc. All rights reserved.)

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.