Complex biophysical changes and reduced neuronal firing in an SCN8A variant associated with developmental delay and epilepsy.

Mutations in the SCN8A gene, encoding the voltage-gated sodium channel NaV1.6, are associated with a range of neurodevelopmental syndromes. The p.(Gly1625Arg) (G1625R) mutation was identified in a patient diagnosed with developmental epileptic encephalopathy (DEE). While most of the characterized DEE-associated SCN8A mutations were shown to cause a gain-of-channel function, we show that the G1625R variant, positioned within the S4 segment of domain IV, results in complex effects. Voltage-clamp analyses of NaV1.6G1625R demonstrated a mixture of gain- and loss-of-function properties, including reduced current amplitudes, increased time constant of fast voltage-dependent inactivation, a depolarizing shift in the voltage dependence of activation and inactivation, and increased channel availability with high-frequency repeated depolarization. Current-clamp analyses in transfected cultured neurons revealed that these biophysical properties caused a marked reduction in the number of action potentials when firing was driven by the transfected mutant NaV1.6. Accordingly, computational modeling of mature cortical neurons demonstrated a mild decrease in neuronal firing when mimicking the patients' heterozygous SCN8A expression. Structural modeling of NaV1.6G1625R suggested the formation of a cation-π interaction between R1625 and F1588 within domain IV. Double-mutant cycle analysis revealed that this interaction affects the voltage dependence of inactivation in NaV1.6G1625R. Together, our studies demonstrate that the G1625R variant leads to a complex combination of gain and loss of function biophysical changes that result in an overall mild reduction in neuronal firing, related to the perturbed interaction network within the voltage sensor domain, necessitating personalized multi-tiered analysis for SCN8A mutations for optimal treatment selection.

Preferential expression of SCN1A in GABAergic neurons improves survival and epileptic phenotype in a mouse model of Dravet syndrome.

The SCN1A gene encodes the alpha subunit of a voltage-gated sodium channel (Nav1.1), which is essential for the function of inhibitory neurons in the brain. Mutations in this gene cause severe encephalopathies such as Dravet syndrome (DS). Upregulation of SCN1A expression by different approaches has demonstrated promising therapeutic effects in preclinical models of DS. Limiting the effect to inhibitory neurons may contribute to the restoration of brain homeostasis, increasing the safety and efficacy of the treatment. In this work, we have evaluated different approaches to obtain preferential expression of the full SCN1A cDNA (6 Kb) in GABAergic neurons, using high-capacity adenoviral vectors (HC-AdV). In order to favour infection of these cells, we considered ErbB4 as a surface target. Incorporation of the EGF-like domain from neuregulin 1 alpha (NRG1α) in the fiber of adenovirus capsid allowed preferential infection in cells lines expressing ErbB4. However, it had no impact on the infectivity of the vector in primary cultures or in vivo. For transcriptional control of transgene expression, we developed a regulatory sequence (DP3V) based on the Distal-less homolog enhancer (Dlx), the vesicular GABA transporter (VGAT) promoter, and a portion of the SCN1A gene. The hybrid DP3V promoter allowed preferential expression of transgenes in GABAergic neurons both in vitro and in vivo. A new HC-AdV expressing SCN1A under the control of this promoter showed improved survival and amelioration of the epileptic phenotype in a DS mouse model. These results increase the repertoire of gene therapy vectors for the treatment of DS and indicate a new avenue for the refinement of gene supplementation in this disease. KEY MESSAGES: Adenoviral vectors can deliver the SCN1A cDNA and are amenable for targeting. An adenoviral vector displaying an ErbB4 ligand in the capsid does not target GABAergic neurons. A hybrid promoter allows preferential expression of transgenes in GABAergic neurons. Preferential expression of SCN1A in GABAergic cells is therapeutic in a Dravet syndrome model.

Heat-induced seizures, premature mortality, and hyperactivity in a novel Scn1a nonsense model for Dravet syndrome.

Dravet syndrome (Dravet) is a severe congenital developmental genetic epilepsy caused by de novo mutations in the SCN1A gene. Nonsense mutations are found in ∼20% of the patients, and the R613X mutation was identified in multiple patients. Here we characterized the epileptic and non-epileptic phenotypes of a novel preclinical Dravet mouse model harboring the R613X nonsense Scn1a mutation. Scn1aWT/R613X mice, on a mixed C57BL/6J:129S1/SvImJ background, exhibited spontaneous seizures, susceptibility to heat-induced seizures, and premature mortality, recapitulating the core epileptic phenotypes of Dravet. In addition, these mice, available as an open-access model, demonstrated increased locomotor activity in the open-field test, modeling some non-epileptic Dravet-associated phenotypes. Conversely, Scn1aWT/R613X mice, on the pure 129S1/SvImJ background, had a normal life span and were easy to breed. Homozygous Scn1aR613X/R613X mice (pure 129S1/SvImJ background) died before P16. Our molecular analyses of hippocampal and cortical expression demonstrated that the premature stop codon induced by the R613X mutation reduced Scn1a mRNA and NaV1.1 protein levels to ∼50% in heterozygous Scn1aWT/R613X mice (on either genetic background), with marginal expression in homozygous Scn1aR613X/R613X mice. Together, we introduce a novel Dravet model carrying the R613X Scn1a nonsense mutation that can be used to study the molecular and neuronal basis of Dravet, as well as the development of new therapies associated with SCN1A nonsense mutations in Dravet.

Viral vector-mediated expression of NaV1.1, after seizure onset, reduces epilepsy in mice with Dravet syndrome.

Dravet syndrome (DS), an intractable childhood epileptic encephalopathy with a high fatality rate, is typically caused by loss-of-function mutations in one allele of SCN1A, which encodes NaV1.1, a 250-kDa voltage-gated sodium channel. In contrast to other epilepsies, pharmaceutical treatment for DS is limited. Here, we demonstrate that viral vector-mediated delivery of a codon-modified SCN1A open reading frame into the brain improves DS comorbidities in juvenile and adolescent DS mice (Scn1aA1783V/WT). Notably, bilateral vector injections into the hippocampus and/or the thalamus of DS mice increased survival, reduced the occurrence of epileptic spikes, provided protection from thermally induced seizures, corrected background electrocorticographic activity and behavioral deficits, and restored hippocampal inhibition. Together, our results provide a proof of concept for the potential of SCN1A delivery as a therapeutic approach for infants and adolescents with DS-associated comorbidities.

Acute effect of antiseizure drugs on background oscillations in Scn1a A1783V Dravet syndrome mouse model.

Dravet syndrome (Dravet) is a rare and severe form of developmental epileptic encephalopathy. Antiseizure medications (ASMs) for Dravet patients include valproic acid (VA) or clobazam (CLB), with or without stiripentol (STP), while sodium channel blockers like carbamazepine (CBZ) or lamotrigine (LTG) are contraindicated. In addition to their effect on epileptic phenotypes, ASMs were shown to modify the properties of background neuronal activity. Nevertheless, little is known about these background properties alterations in Dravet. Here, utilizing Dravet mice (DS, Scn1a A1783V/WT), we tested the acute effect of several ASMs on background electrocorticography (ECoG) activity and frequency of interictal spikes. Compared to wild-type mice, background ECoG activity in DS mice had lower power and reduced phase coherence, which was not corrected by any of the tested ASMs. However, acute administration of Dravet-recommended drugs, VA, CLB, or a combination of CLB + STP, caused, in most mice, a reduction in the frequency of interictal spikes, alongside an increase in the relative contribution of the beta frequency band. Conversely, CBZ and LTG increased the frequency of interictal spikes, with no effect on background spectral properties. Moreover, we uncovered a correlation between the reduction in interictal spike frequency, the drug-induced effect on the power of background activity, and a spectral shift toward higher frequency bands. Together, these data provide a comprehensive analysis of the effect of selected ASMs on the properties of background neuronal oscillations, and highlight a possible correlation between their effect on epilepsy and background activity.

Functional Investigation of a Neuronal Microcircuit in the CA1 Area of the Hippocampus Reveals Synaptic Dysfunction in Dravet Syndrome Mice.

Dravet syndrome is severe childhood-onset epilepsy, caused by loss of function mutations in the SCN1A gene, encoding for the voltage-gated sodium channel NaV1.1. The leading hypothesis is that Dravet is caused by selective reduction in the excitability of inhibitory neurons, due to hampered activity of NaV1.1 channels in these cells. However, these initial neuronal changes can lead to further network alterations. Here, focusing on the CA1 microcircuit in hippocampal brain slices of Dravet syndrome (DS, Scn1a A1783V/WT) and wild-type (WT) mice, we examined the functional response to the application of Hm1a, a specific NaV1.1 activator, in CA1 stratum-oriens (SO) interneurons and CA1 pyramidal excitatory neurons. DS SO interneurons demonstrated reduced firing and depolarized threshold for action potential (AP), indicating impaired activity. Nevertheless, Hm1a induced a similar AP threshold hyperpolarization in WT and DS interneurons. Conversely, a smaller effect of Hm1a was observed in CA1 pyramidal neurons of DS mice. In these excitatory cells, Hm1a application resulted in WT-specific AP threshold hyperpolarization and increased firing probability, with no effect on DS neurons. Additionally, when the firing of SO interneurons was triggered by CA3 stimulation and relayed via activation of CA1 excitatory neurons, the firing probability was similar in WT and DS interneurons, also featuring a comparable increase in the firing probability following Hm1a application. Interestingly, a similar functional response to Hm1a was observed in a second DS mouse model, harboring the nonsense Scn1a R613X mutation. Furthermore, we show homeostatic synaptic alterations in both CA1 pyramidal neurons and SO interneurons, consistent with reduced excitation and inhibition onto CA1 pyramidal neurons and increased release probability in the CA1-SO synapse. Together, these results suggest global neuronal alterations within the CA1 microcircuit extending beyond the direct impact of NaV1.1 dysfunction.

Novel ADNP Syndrome Mice Reveal Dramatic Sex-Specific Peripheral Gene Expression With Brain Synaptic and Tau Pathologies.

BACKGROUND: ADNP is essential for embryonic development. As such, de novo ADNP mutations lead to an intractable autism/intellectual disability syndrome requiring investigation. METHODS: Mimicking humans, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 editing produced mice carrying heterozygous Adnp p.Tyr718∗ (Tyr), a paralog of the most common ADNP syndrome mutation. Phenotypic rescue was validated by treatment with the microtubule/autophagy-protective ADNP fragment NAPVSIPQ (NAP). RESULTS: RNA sequencing of spleens, representing a peripheral biomarker source, revealed Tyr-specific sex differences (e.g., cell cycle), accentuated in females (with significant effects on antigen processing and cellular senescence) and corrected by NAP. Differentially expressed, NAP-correctable transcripts, including the autophagy and microbiome resilience-linked FOXO3, were also deregulated in human patient-derived ADNP-mutated lymphoblastoid cells. There were also Tyr sex-specific microbiota signatures. Phenotypically, Tyr mice, similar to patients with ADNP syndrome, exhibited delayed development coupled with sex-dependent gait defects. Speech acquisition delays paralleled sex-specific mouse syntax abnormalities. Anatomically, dendritic spine densities/morphologies were decreased with NAP amelioration. These findings were replicated in the Adnp+/- mouse, including Foxo3 deregulation, required for dendritic spine formation. Grooming duration and nociception threshold (autistic traits) were significantly affected only in males. Early-onset tauopathy was accentuated in males (hippocampus and visual cortex), mimicking humans, and was paralleled by impaired visual evoked potentials and correction by acute NAP treatment. CONCLUSIONS: Tyr mice model ADNP syndrome pathology. The newly discovered ADNP/NAP target FOXO3 controls the autophagy initiator LC3 (microtubule-associated protein 1 light chain 3), with known ADNP binding to LC3 augmented by NAP, protecting against tauopathy. NAP amelioration attests to specificity, with potential for drug development targeting accessible biomarkers.

Transfer of SCN1A to the brain of adolescent mouse model of Dravet syndrome improves epileptic, motor, and behavioral manifestations.

Dravet syndrome is a genetic encephalopathy characterized by severe epilepsy combined with motor, cognitive, and behavioral abnormalities. Current antiepileptic drugs achieve only partial control of seizures and provide little benefit on the patient's neurological development. In >80% of cases, the disease is caused by haploinsufficiency of the SCN1A gene, which encodes the alpha subunit of the Nav1.1 voltage-gated sodium channel. Novel therapies aim to restore SCN1A expression in order to address all disease manifestations. We provide evidence that a high-capacity adenoviral vector harboring the 6-kb SCN1A cDNA is feasible and able to express functional Nav1.1 in neurons. In vivo, the best biodistribution was observed after intracerebral injection in basal ganglia, cerebellum, and prefrontal cortex. SCN1A A1783V knockin mice received the vector at 5 weeks of age, when most neurological alterations were present. Animals were protected from sudden death, and the epileptic phenotype was attenuated. Improvement of motor performance and interaction with the environment was observed. In contrast, hyperactivity persisted, and the impact on cognitive tests was variable (success in novel object recognition and failure in Morris water maze tests). These results provide proof of concept for gene supplementation in Dravet syndrome and indicate new directions for improvement.

Reprogramming of two induced pluripotent stem cell lines from a heterozygous GRIN2D developmental and epileptic encephalopathy (DEE) patient (BGUi011-A) and from a healthy family relative (BGUi012-A).

The GLUN2D subunit of the N-methylD-aspartate receptor (NMDAR) is encoded by the GRIN2D gene. Mutations in GRIN2D have been associated with neurodevelopmental and epileptic encephalopathies. Access to patient samples harboring mutations in GRIN2D can contribute to understanding the role of NMDAR in neuronal development and function. We report the generation of induced pluripotent stem cell (iPSC) lines from a GRIN2D-developmental and epileptic encephalopathy (DEE) patient, carrying a de novo c.1999G>A heterozygous pathogenic variant, and his healthy parent. Generated lines highly expressed pluripotency markers, spontaneously differentiated into the three germ layers, retained the deficiency-causing mutation, and displayed normal karyotypes.

Developmental alterations in firing properties of hippocampal CA1 inhibitory and excitatory neurons in a mouse model of Dravet syndrome.

Dravet syndrome (Dravet) is a rare, severe childhood-onset epilepsy, caused by heterozygous de novo mutations in the SCN1A gene, encoding for the alpha subunit of the voltage-gated sodium channel, NaV1.1. The neuronal basis of Dravet is debated, with evidence favoring reduced function of inhibitory neurons, that might be transient, or enhanced activity of excitatory cells. Here, we utilized Dravet mice to trace developmental changes in the hippocampal CA1 circuit, examining the properties of CA1 horizontal stratum-oriens (SO) interneurons and pyramidal neurons, through the pre-epileptic, severe and stabilization stages of Dravet. Our data indicate that reduced function of SO interneurons persists from the pre-epileptic through the stabilization stages, with the greatest functional impairment observed during the severe stage. In contrast, opposing changes were detected in CA1 excitatory neurons, with a transient increase in their excitability during the pre-epileptic stage, followed by reduced excitability at the severe stage. Interestingly, alterations in the function of both inhibitory and excitatory neurons were more pronounced when the firing was evoked by synaptic stimulation, implying that loss of function of NaV1.1 may also affect somatodendritic functions. These results suggest a complex pathophysiological mechanism and indicate that the developmental trajectory of this disease is governed by reciprocal functional changes in both excitatory and inhibitory neurons.

Convulsive seizures and some behavioral comorbidities are uncoupled in the Scn1aA1783V Dravet syndrome mouse model.

OBJECTIVE: Dravet syndrome (Dravet) is a severe childhood epileptic encephalopathy. The disease begins with a febrile stage, characterized by febrile seizures with otherwise normal development. Progression to the worsening stage features recurrent intractable seizures and the presentation of additional nonepileptic comorbidities, including global developmental delay, hyperactivity, and motor deficits. Later in life, at the stabilization stage, seizure burden decreases, whereas Dravet-associated comorbidities persist. To date, it remains debated whether the nonepileptic comorbidities result from severe epilepsy or represent an independent phenotypic feature. METHODS: Dravet mice (DS) faithfully recapitulate many clinical aspects of Dravet. Using wild-type (WT) and DS at different ages, we monitored multiple behavioral features as well as background electroencephalogram (EEG) activity during the different stages of Dravet epilepsy. RESULTS: Behavioral tests of WT and DS demonstrated that some deficits manifest already at the pre-epileptic stage, prior to the onset of convulsive seizures. These include motor impairment and hyperactivity in the open field. Deficits in cognitive functions were detected at the onset of severe spontaneous seizures. Power spectral analysis of background EEG activity, measured through development, showed that DS exhibit normal background oscillations at the pre-epileptic stage, a marked reduction in total power during the onset of severe epilepsy, and a subsequent smaller reduction later in life. Importantly, low EEG power at the stage of severe frequent convulsive seizures correlated with increased risk for premature death. SIGNIFICANCE: Our data provide a comprehensive developmental trajectory of Dravet epilepsy and Dravet-associated comorbidities in mice, under controlled settings, demonstrating that the convulsive seizures and some nonepileptic comorbidities may be uncoupled. Moreover, we report the existence of an inverse correlation, on average, between the power of background EEG and the severity of epileptic phenotypes, suggesting that such measurements may potentially serve as a biomarker for Dravet severity.

Striatin Is Required for Hearing and Affects Inner Hair Cells and Ribbon Synapses.

Striatin, a subunit of the serine/threonine phosphatase PP2A, is a core member of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complexes. The protein is expressed in the cell junctions between epithelial cells, which play a role in maintaining cell-cell adhesion. Since the cell junctions are crucial for the function of the mammalian inner ear, we examined the localization and function of striatin in the mouse cochlea. Our results show that in neonatal mice, striatin is specifically expressed in the cell-cell junctions of the inner hair cells, the receptor cells in the mammalian cochlea. Auditory brainstem response measurements of striatin-deficient mice indicated a progressive, high-frequency hearing loss, suggesting that striatin is essential for normal hearing. Moreover, scanning electron micrographs of the organ of Corti revealed a moderate degeneration of the outer hair cells in the middle and basal regions, concordant with the high-frequency hearing loss. Additionally, striatin-deficient mice show aberrant ribbon synapse maturation. Loss of the outer hair cells, combined with the aberrant ribbon synapse distribution, may lead to the observed auditory impairment. Together, these results suggest a novel function for striatin in the mammalian auditory system.

Mitochondrial Regulation of the Hippocampal Firing Rate Set Point and Seizure Susceptibility.

Maintaining average activity within a set-point range constitutes a fundamental property of central neural circuits. However, whether and how activity set points are regulated remains unknown. Integrating genome-scale metabolic modeling and experimental study of neuronal homeostasis, we identified mitochondrial dihydroorotate dehydrogenase (DHODH) as a regulator of activity set points in hippocampal networks. The DHODH inhibitor teriflunomide stably suppressed mean firing rates via synaptic and intrinsic excitability mechanisms by modulating mitochondrial Ca2+ buffering and spare respiratory capacity. Bi-directional activity perturbations under DHODH blockade triggered firing rate compensation, while stabilizing firing to the lower level, indicating a change in the firing rate set point. In vivo, teriflunomide decreased CA3-CA1 synaptic transmission and CA1 mean firing rate and attenuated susceptibility to seizures, even in the intractable Dravet syndrome epilepsy model. Our results uncover mitochondria as a key regulator of activity set points, demonstrate the differential regulation of set points and compensatory mechanisms, and propose a new strategy to treat epilepsy.

In vivo, in vitro and in silico correlations of four de novo SCN1A missense mutations.

Mutations in the SCN1A gene, which encodes for the voltage-gated sodium channel NaV1.1, cause Dravet syndrome, a severe developmental and epileptic encephalopathy. Genetic testing of this gene is recommended early in life. However, predicting the outcome of de novo missense SCN1A mutations is difficult, since milder epileptic syndromes may also be associated. In this study, we correlated clinical severity with functional in vitro electrophysiological testing of channel activity and bioinformatics prediction of damaging mutational effects. Three patients, bearing the mutations p.Gly177Ala, p.Ser259Arg and p.Glu1923Arg, showed frequent intractable seizures that had started early in life, with cognitive and behavioral deterioration, consistent with classical Dravet phenotypes. These mutations failed to produce measurable sodium currents in a mammalian expression system, indicating complete loss of channel function. A fourth patient, who harbored the mutation p.Met1267Ile, though presenting with seizures early in life, showed lower seizure burden and higher cognitive function, matching borderland Dravet phenotypes. In correlation with this, functional analysis demonstrated the presence of sodium currents, but with partial loss of function. In contrast, six bioinformatics tools for predicting mutational pathogenicity suggested similar impact for all mutations. Likewise, homology modeling of the secondary and tertiary structures failed to reveal misfolding. In conclusion, functional studies using patch clamp are suggested as a prognostic tool, whereby detectable currents imply milder phenotypes and absence of currents indicate an unfavorable prognosis. Future development of automated patch clamp systems will facilitate the inclusion of such functional testing as part of personalized patient diagnostic schemes.

Lithium reduces the span of G protein-activated K+ (GIRK) channel inhibition in hippocampal neurons.

OBJECTIVES: Lithium (Li+ ) is one of the most widely used treatments for bipolar disorder (BD). However, the molecular and neuronal basis of BD, as well as the mechanisms of Li+ actions are poorly understood. Cellular and biochemical studies identified G proteins as being among the cellular targets for Li+ action, while genetic studies indicated an association with the KCNJ3 gene, which encodes the G protein-activated inwardly rectifying K+ (GIRK) channels. GIRK channels regulate neuronal excitability by mediating the inhibitory effects of multiple neurotransmitters and contribute to the resting potassium conductance. Here, we explored the effects of therapeutic dose of Li+ on neuronal excitability and the role of GIRK channels in Li+ actions. METHODS: Effects of Li+ on excitability were studied in hippocampal brain slices using whole-cell electrophysiological recordings. RESULTS: A therapeutic dose of Li+ (1 mM) dually regulated the function of GIRK channels in hippocampal slices. Li+ hyperpolarized the resting membrane potential of hippocampal CA1 pyramidal neurons and prolonged the latency to reach the action potential threshold and peak. These effects were abolished in the presence of tertiapin, a specific GIRK channel blocker, and at doses above the therapeutic window (2 mM). In contrast, Li+ reduced GIRK channel opening induced by GABAB receptor (GABAB R) activation, causing reduced hyperpolarization of the membrane potential, attenuated reduction of input resistance, and a smaller decrease of neuronal firing. CONCLUSIONS: A therapeutic dose of Li+ reduces the span of GIRK channel-mediated inhibition due to enhancement of basal GIRK currents and inhibition of GABAB R evoked responses, providing an important link between Li+ action, neuronal excitability, and cellular and genetic targets of BD.

A Quantitative Model of the GIRK1/2 Channel Reveals That Its Basal and Evoked Activities Are Controlled by Unequal Stoichiometry of Gα and Gßγ.

G protein-gated K+ channels (GIRK; Kir3), activated by Gßγ subunits derived from Gi/o proteins, regulate heartbeat and neuronal excitability and plasticity. Both neurotransmitter-evoked (Ievoked) and neurotransmitter-independent basal (Ibasal) GIRK activities are physiologically important, but mechanisms of Ibasal and its relation to Ievoked are unclear. We have previously shown for heterologously expressed neuronal GIRK1/2, and now show for native GIRK in hippocampal neurons, that Ibasal and Ievoked are interrelated: the extent of activation by neurotransmitter (activation index, Ra) is inversely related to Ibasal. To unveil the underlying mechanisms, we have developed a quantitative model of GIRK1/2 function. We characterized single-channel and macroscopic GIRK1/2 currents, and surface densities of GIRK1/2 and Gßγ expressed in Xenopus oocytes. Based on experimental results, we constructed a mathematical model of GIRK1/2 activity under steady-state conditions before and after activation by neurotransmitter. Our model accurately recapitulates Ibasal and Ievoked in Xenopus oocytes, HEK293 cells and hippocampal neurons; correctly predicts the dose-dependent activation of GIRK1/2 by coexpressed Gßγ and fully accounts for the inverse Ibasal-Ra correlation. Modeling indicates that, under all conditions and at different channel expression levels, between 3 and 4 Gßγ dimers are available for each GIRK1/2 channel. In contrast, available Gαi/o decreases from ~2 to less than one Gα per channel as GIRK1/2's density increases. The persistent Gßγ/channel (but not Gα/channel) ratio support a strong association of GIRK1/2 with Gßγ, consistent with recruitment to the cell surface of Gßγ, but not Gα, by GIRK1/2. Our analysis suggests a maximal stoichiometry of 4 Gßγ but only 2 Gαi/o per one GIRK1/2 channel. The unique, unequal association of GIRK1/2 with G protein subunits, and the cooperative nature of GIRK gating by Gßγ, underlie the complex pattern of basal and agonist-evoked activities and allow GIRK1/2 to act as a sensitive bidirectional detector of both Gßγ and Gα.

Dissecting the phenotypes of Dravet syndrome by gene deletion.

Neurological and psychiatric syndromes often have multiple disease traits, yet it is unknown how such multi-faceted deficits arise from single mutations. Haploinsufficiency of the voltage-gated sodium channel Nav1.1 causes Dravet syndrome, an intractable childhood-onset epilepsy with hyperactivity, cognitive deficit, autistic-like behaviours, and premature death. Deletion of Nav1.1 channels selectively impairs excitability of GABAergic interneurons. We studied mice having selective deletion of Nav1.1 in parvalbumin- or somatostatin-expressing interneurons. In brain slices, these deletions cause increased threshold for action potential generation, impaired action potential firing in trains, and reduced amplification of postsynaptic potentials in those interneurons. Selective deletion of Nav1.1 in parvalbumin- or somatostatin-expressing interneurons increases susceptibility to thermally-induced seizures, which are strikingly prolonged when Nav1.1 is deleted in both interneuron types. Mice with global haploinsufficiency of Nav1.1 display autistic-like behaviours, hyperactivity and cognitive impairment. Haploinsufficiency of Nav1.1 in parvalbumin-expressing interneurons causes autistic-like behaviours, but not hyperactivity, whereas haploinsufficiency in somatostatin-expressing interneurons causes hyperactivity without autistic-like behaviours. Heterozygous deletion in both interneuron types is required to impair long-term spatial memory in context-dependent fear conditioning, without affecting short-term spatial learning or memory. Thus, the multi-faceted phenotypes of Dravet syndrome can be genetically dissected, revealing synergy in causing epilepsy, premature death and deficits in long-term spatial memory, but interneuron-specific effects on hyperactivity and autistic-like behaviours. These results show that multiple disease traits can arise from similar functional deficits in specific interneuron types.

Genetic background modulates impaired excitability of inhibitory neurons in a mouse model of Dravet syndrome.

Dominant loss-of-function mutations in voltage-gated sodium channel NaV1.1 cause Dravet Syndrome, an intractable childhood-onset epilepsy. NaV1.1(+/-) Dravet Syndrome mice in C57BL/6 genetic background exhibit severe seizures, cognitive and social impairments, and premature death. Here we show that Dravet Syndrome mice in pure 129/SvJ genetic background have many fewer seizures and much less premature death than in pure C57BL/6 background. These mice also have a higher threshold for thermally induced seizures, fewer myoclonic seizures, and no cognitive impairment, similar to patients with Genetic Epilepsy with Febrile Seizures Plus. Consistent with this mild phenotype, mutation of NaV1.1 channels has much less physiological effect on neuronal excitability in 129/SvJ mice. In hippocampal slices, the excitability of CA1 Stratum Oriens interneurons is selectively impaired, while the excitability of CA1 pyramidal cells is unaffected. NaV1.1 haploinsufficiency results in increased rheobase and threshold for action potential firing and impaired ability to sustain high-frequency firing. Moreover, deletion of NaV1.1 markedly reduces the amplification and integration of synaptic events, further contributing to reduced excitability of interneurons. Excitability is less impaired in inhibitory neurons of Dravet Syndrome mice in 129/SvJ genetic background. Because specific deletion of NaV1.1 in forebrain GABAergic interneuons is sufficient to cause the symptoms of Dravet Syndrome in mice, our results support the conclusion that the milder phenotype in 129/SvJ mice is caused by lesser impairment of sodium channel function and electrical excitability in their forebrain interneurons. This mild impairment of excitability of interneurons leads to a milder disease phenotype in 129/SvJ mice, similar to Genetic Epilepsy with Febrile Seizures Plus in humans.

Recruitment of Gßγ controls the basal activity of G-protein coupled inwardly rectifying potassium (GIRK) channels: crucial role of distal C terminus of GIRK1.

The G-protein coupled inwardly rectifying potassium (GIRK, or Kir3) channels are important mediators of inhibitory neurotransmission via activation of G-protein coupled receptors (GPCRs). GIRK channels are tetramers comprising combinations of subunits (GIRK1-4), activated by direct binding of the Gßγ subunit of Gi/o proteins. Heterologously expressed GIRK1/2 exhibit high, Gßγ-dependent basal currents (Ibasal) and a modest activation by GPCR or coexpressed Gßγ. Inversely, the GIRK2 homotetramers exhibit low Ibasal and strong activation by Gßγ. The high Ibasal of GIRK1 seems to be associated with its unique distal C terminus (G1-dCT), which is not present in the other subunits. We investigated the role of G1-dCT using electrophysiological and fluorescence assays in Xenopus laevis oocytes and protein interaction assays. We show that expression of GIRK1/2 increases the plasma membrane level of coexpressed Gßγ (a phenomenon we term 'Gßγ recruitment') but not of coexpressed Gαi3. All GIRK1-containing channels, but not GIRK2 homomers, recruited Gßγ to the plasma membrane. In biochemical assays, truncation of G1-dCT reduces the binding between the cytosolic parts of GIRK1 and Gßγ, but not Gαi3. Nevertheless, the truncation of G1-dCT does not impair activation by Gßγ. In fluorescently labelled homotetrameric GIRK1 channels and in the heterotetrameric GIRK1/2 channel, the truncation of G1-dCT abolishes Gßγ recruitment and decreases Ibasal. Thus, we conclude that G1-dCT carries an essential role in Gßγ recruitment by GIRK1 and, consequently, in determining its high basal activity. Our results indicate that G1-dCT is a crucial part of a Gßγ anchoring site of GIRK1-containing channels, spatially and functionally distinct from the site of channel activation by Gßγ.

Reciprocal changes in phosphorylation and methylation of mammalian brain sodium channels in response to seizures.

Voltage-gated sodium (Nav) channels initiate action potentials in brain neurons and are primary therapeutic targets for anti-epileptic drugs controlling neuronal hyperexcitability in epilepsy. The molecular mechanisms underlying abnormal Nav channel expression, localization, and function during development of epilepsy are poorly understood but can potentially result from altered posttranslational modifications (PTMs). For example, phosphorylation regulates Nav channel gating, and has been proposed to contribute to acquired insensitivity to anti-epileptic drugs exhibited by Nav channels in epileptic neurons. However, whether changes in specific brain Nav channel PTMs occur acutely in response to seizures has not been established. Here, we show changes in PTMs of the major brain Nav channel, Nav1.2, after acute kainate-induced seizures. Mass spectrometry-based proteomic analyses of Nav1.2 purified from the brains of control and seizure animals revealed a significant down-regulation of phosphorylation at nine sites, primarily located in the interdomain I-II linker, the region of Nav1.2 crucial for phosphorylation-dependent regulation of activity. Interestingly, Nav1.2 in the seizure samples contained methylated arginine (MeArg) at three sites. These MeArgs were adjacent to down-regulated sites of phosphorylation, and Nav1.2 methylation increased after seizure. Phosphorylation and MeArg were not found together on the same tryptic peptide, suggesting reciprocal regulation of these two PTMs. Coexpression of Nav1.2 with the primary brain arginine methyltransferase PRMT8 led to a surprising 3-fold increase in Nav1.2 current. Reciprocal regulation of phosphorylation and MeArg of Nav1.2 may underlie changes in neuronal Nav channel function in response to seizures and also contribute to physiological modulation of neuronal excitability.