Heterotrimeric G proteins, composed of α, β, and γ subunits, can transduce a variety of signals from seven-transmembrane-type receptors to intracellular effectors. By whole-exome sequencing and subsequent mutation screening, we identified de novo heterozygous mutations in GNAO1, which encodes a Gαo subunit of heterotrimeric G proteins, in four individuals with epileptic encephalopathy. Two of the affected individuals also showed involuntary movements. Somatic mosaicism (approximately 35% to 50% of cells, distributed across multiple cell types, harbored the mutation) was shown in one individual. By mapping the mutation onto three-dimensional models of the Gα subunit in three different complexed states, we found that the three mutants (c.521A>G [p.Asp174Gly], c.836T>A [p.Ile279Asn], and c.572_592del [p.Thr191_Phe197del]) are predicted to destabilize the Gα subunit fold. A fourth mutant (c.607G>A), in which the Gly203 residue located within the highly conserved switch II region is substituted to Arg, is predicted to impair GTP binding and/or activation of downstream effectors, although the p.Gly203Arg substitution might not interfere with Gα binding to G-protein-coupled receptors. Transient-expression experiments suggested that localization to the plasma membrane was variably impaired in the three putatively destabilized mutants. Electrophysiological analysis showed that Gαo-mediated inhibition of calcium currents by norepinephrine tended to be lower in three of the four Gαo mutants. These data suggest that aberrant Gαo signaling can cause multiple neurodevelopmental phenotypes, including epileptic encephalopathy and involuntary movements.
Exposure to maternal stress (MS) and mutations in GAD1, which encodes the γ-aminobutyric acid (GABA) synthesizing enzyme glutamate decarboxylase (GAD) 67, are both risk factors for psychiatric disorders. However, the relationship between these risk factors remains unclear. Interestingly, the critical period of MS for psychiatric disorders in offspring corresponds to the period of GABAergic neuron neurogenesis and migration in the fetal brain, that is, in the late stage of gestation. Indeed, decrement of parvalbumin (PV)-positive GABAergic interneurons in the medial prefrontal cortex (mPFC) and hippocampus (HIP) has often been observed in schizophrenia patients. In the present study, we used GAD67-green fluorescent protein (GFP) knock-in mice (that is, mice in which the Gad1 gene is heterozygously deleted; GAD67+/GFP) that underwent prenatal stress from embryonic day 15.0 to 17.5 and monitored PV-positive GABAergic neurons to address the interaction between Gad1 disruption and stress. Administration of 5-bromo-2-deoxyuridine revealed that neurogenesis of GFP-positive GABAergic neurons, but not cortical plate cells, was significantly diminished in fetal brains during MS. Differential expression of glucocorticoid receptors by different progenitor cell types may underlie this differential outcome. Postnatally, the density of PV-positive, but not PV-negative, GABAergic neurons was significantly decreased in the mPFC, HIP and somatosensory cortex but not in the motor cortex of GAD67+/GFP mice. By contrast, these findings were not observed in wild-type (GAD67+/+) offspring. These results suggest that prenatal stress, in addition to heterozygous deletion of Gad1, could specifically disturb the proliferation of neurons destined to be PV-positive GABAergic interneurons.
Caenorhabditis elegans and Drosophila melanogaster are relevant models for studying the roles of glycosaminoglycans (GAG) during the development of multicellular organisms. The genome projects of these organisms have revealed the existence of multiple genes related to GAG-synthesizing enzymes. Although the putative genes encoding the enzymes that synthesize the GAG-protein linkage region have also been identified, there is no direct evidence that the GAG chains bind covalently to core proteins. This study aimed to clarify whether GAG chains in these organisms are linked to core proteins through the conventional linkage region tetrasaccharide sequence found in vertebrates and whether modifications by phosphorylation and sulfation reported for vertebrates are present also in invertebrates. The linkage region oligosaccharides were isolated from C. elegans chondroitin in addition to D. melanogaster heparan and chondroitin sulfate after digestion with the respective bacterial eliminases and were then derivatized with a fluorophore 2-aminobenzamide. Their structures were characterized by gel filtration and anion-exchange high performance liquid chromatography in conjunction with enzymatic digestion and matrix-assisted laser desorption ionization time-of-flight spectrometry, which demonstrated a uniform linkage tetrasaccharide structure of -GlcUA-Gal-Gal-Xyl-or -GlcUA-Gal-Gal-Xyl(2-O-phosphate)-for C. elegans chondroitin and D. melanogaster CS, respectively. In contrast, the unmodified and phosphorylated counterparts were demonstrated in heparan sulfate of adult flies at a molar ratio of 73:27, and in that of the immortalized D. melanogaster S2 cell line at a molar ratio of 7:93, which suggests that the linkage region in the fruit fly first becomes phosphorylated uniformly on the Xyl residue and then dephosphorylated. It has been established here that GAG chains in both C. elegans and D. melanogaster are synthesized on the core protein through the ubiquitous linkage region tetrasaccharide sequence, suggesting that indispensable functions of the linkage region in the GAG synthesis have been well conserved during evolution. Mutations affecting the genes encoding putative proteins related to GAG biosynthetic enzymes have also been described for these organisms. Mutations in the tout velu (ttv) gene of D. melanogaster cause defects in Hedgehog movement in mosaic wing discs (1, 6). The ttv gene is a putative ortholog of vertebrate EXT1, which encodes a heparan polymerase and is associated with the hereditary multiple exostoses syndrome in humans (7). The pipe gene, which affects dorsal-ventral patterning of D. melanogaster development, encodes a homolog of vertebrate HS 2-O-sulfotransferase (2, 8). The sugarless and sulfateless genes, both of which affect the fibroblast growth factor signaling during the D. melanogaster development, en-
Ethanol (EtOH) has a number of behavioral effects, including intoxication, amnesia, and/or sedation, that are thought to relate to the activation of GABA A receptors. However, GABA A receptors at different cellular locations have different sensitivities to EtOH. The present study used the "synaptic bouton" preparation where we could stimulate nerve endings on mechanically dissociated single rat hippocampal CA1 and CA3 pyramidal neurons and investigate the effects of EtOH on presynaptic and postsynaptic GABA A receptors. Low concentrations of EtOH (10 mM) had no effect on postsynaptic GABA A and glutamate receptors or voltage-dependent Na ϩ and Ca 2ϩ channels. Higher concentrations (Ն100 mM) could significantly inhibit these current responses. EtOH at 10 mM had no direct effect on inhibitory postsynaptic currents (IPSCs) and excitatory postsynaptic currents (EPSCs) evoked by focal stimulation of single boutons [evoked IPSCs (eIPSCs) and evoked EPSCs (eEPSCs)]. However, coapplication of 10 mM EtOH with muscimol decreased the amplitude of eIPSCs and eEPSCs and increased their paired-pulse ratio. The effects on eEPSCs were reversed by bicuculline. Coapplication of muscimol and EtOH significantly increased the frequency of spontaneous IPSCs and EPSCs. The EtOH effects on the postsynaptic responses and eEPSCs were similar in neurons from neonatal and mature rats. These results revealed that low concentrations of EtOH can potentiate the activation of presynaptic GABA A receptors to inhibit evoked GABA and glutamate release. These results indicate a high sensitivity of presynaptic GABA A receptor to EtOH, which needs to be accounted for when considering the cellular mechanisms of EtOH's physiological responses.
Although focal cortical malformations are considered neuronal migration disorders, their formation mechanisms remain unknown. We addressed how the γ-aminobutyric acid (GABA)ergic system affects the GABAergic and glutamatergic neuronal migration underlying such malformations. A focal freeze-lesion (FFL) of the postnatal day zero (P0) glutamic acid decarboxylase–green fluorescent protein knock-in mouse neocortex produced a 3- or 4-layered microgyrus at P7. GABAergic interneurons accumulated around the necrosis including the superficial region during microgyrus formation at P4, whereas E17.5-born, Cux1-positive pyramidal neurons outlined the GABAergic neurons and were absent from the superficial layer, forming cell-dense areas in layer 2 of the P7 microgyrus. GABA imaging showed that an extracellular GABA level temporally increased in the GABAergic neuron-positive area, including the necrotic center, at P4. The expression of the Cl– transporter KCC2 was downregulated in the microgyrus-forming GABAergic and E17.5-born glutamatergic neurons at P4; these cells may need a high intracellular Cl– concentration to induce depolarizing GABA effects. Bicuculline decreased the frequency of spontaneous Ca2+ oscillations in these microgyrus-forming cells. Thus, neonatal FFL causes specific neuronal accumulation, preceded by an increase in ambient GABA during microgyrus formation. This GABA increase induces GABAA receptor-mediated Ca2+ oscillation in KCC2-downregulated microgyrus-forming cells, as seen in migrating cells during early neocortical development.
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