Sleep slow-wave oscillations trigger seizures in a genetic epilepsy model of Dravet syndrome.

Sleep is the preferential period when epileptic spike-wave discharges appear in human epileptic patients, including genetic epileptic seizures such as Dravet syndrome with multiple mutations including SCN1A mutation and GABAA receptor γ2 subunit Gabrg2Q390X mutation in patients, which presents more severe epileptic symptoms in female patients than male patients. However, the seizure onset mechanism during sleep still remains unknown. Our previous work has shown that the sleep-like state-dependent homeostatic synaptic potentiation can trigger epileptic spike-wave discharges in one transgenic heterozygous Gabrg2+/Q390X knock-in mouse model.1 Here, using this heterozygous knock-in mouse model, we hypothesized that slow-wave oscillations themselves in vivo could trigger epileptic seizures. We found that epileptic spike-wave discharges in heterozygous Gabrg2+/Q390X knock-in mice exhibited preferential incidence during non-rapid eye movement sleep period, accompanied by motor immobility/facial myoclonus/vibrissal twitching and more frequent spike-wave discharge incidence appeared in female heterozygous knock-in mice than male heterozygous knock-in mice. Optogenetically induced slow-wave oscillations in vivo significantly increased epileptic spike-wave discharge incidence in heterozygous Gabrg2+/Q390X knock-in mice with longer duration of non-rapid eye movement sleep or quiet-wakeful states. Furthermore, suppression of slow-wave oscillation-related homeostatic synaptic potentiation by 4-(diethylamino)-benzaldehyde injection (i.p.) greatly attenuated spike-wave discharge incidence in heterozygous knock-in mice, suggesting that slow-wave oscillations in vivo did trigger seizure activity in heterozygous knock-in mice. Meanwhile, sleep spindle generation in wild-type littermates and heterozygous Gabrg2+/Q390X knock-in mice involved the slow-wave oscillation-related homeostatic synaptic potentiation that also contributed to epileptic spike-wave discharge generation in heterozygous Gabrg2+/Q390X knock-in mice. In addition, EEG spectral power of delta frequency (0.1-4 Hz) during non-rapid eye movement sleep was significantly larger in female heterozygous Gabrg2+/Q390X knock-in mice than that in male heterozygous Gabrg2+/Q390X knock-in mice, which likely contributes to the gender difference in seizure incidence during non-rapid eye movement sleep/quiet-wake states of human patients. Overall, all these results indicate that slow-wave oscillations in vivo trigger the seizure onset in heterozygous Gabrg2+/Q390X knock-in mice, preferentially during non-rapid eye movement sleep period and likely generate the sex difference in seizure incidence between male and female heterozygous Gabrg2+/Q390X knock-in mice.

Heterozygous GABAA receptor ß3 subunit N110D knock-in mice have epileptic spasms.

OBJECTIVE: Infantile spasms is an epileptic encephalopathy of childhood, and its pathophysiology is largely unknown. We generated a heterozygous knock-in mouse with the human infantile spasms-associated de novo mutation GABRB3 (c.A328G, p.N110D) to investigate its molecular mechanisms and to establish the Gabrb3+/N110D knock-in mouse as a model of infantile spasms syndrome. METHODS: We used electroencephalography (EEG) and video monitoring to characterize seizure types, and a suite of behavioral tests to identify neurological and behavioral impairment in Gabrb3+/N110D knock-in mice. Miniature inhibitory postsynaptic currents (mIPSCs) were recorded from layer V/VI pyramidal neurons in somatosensory cortex, and extracellular multi-unit recordings from the ventral basal nucleus of the thalamus in a horizontal thalamocortical slice were used to assess spontaneous thalamocortical oscillations. RESULTS: The infantile spasms-associated human de novo mutation GABRB3 (c.A328G, p.N110D) caused epileptic spasms early in development and multiple seizure types in adult Gabrb3+/N110D knock-in mice. Signs of neurological impairment, anxiety, hyperactivity, social impairment, and deficits in spatial learning and memory were also observed. Gabrb3+/N110D mice had reduced cortical mIPSCs and increased duration of spontaneous oscillatory firing in the somatosensory thalamocortical circuit. SIGNIFICANCE: The Gabrb3+/N110D knock-in mouse has epileptic spasms, seizures, and other neurological impairments that are consistent with infantile spasms syndrome in patients. Multiple seizure types and abnormal behaviors indicative of neurological impairment both early and late in development suggest that Gabrb3+/N110D mice can be used to study the pathophysiology of infantile spasms. Reduced cortical inhibition and increased duration of thalamocortical oscillatory firing suggest perturbations in thalamocortical circuits.

Dravet syndrome-associated mutations in GABRA1, GABRB2 and GABRG2 define the genetic landscape of defects of GABAA receptors.

Dravet syndrome is a rare, catastrophic epileptic encephalopathy that begins in the first year of life, usually with febrile or afebrile hemiclonic or generalized tonic-clonic seizures followed by status epilepticus. De novo variants in genes that mediate synaptic transmission such as SCN1A and PCDH19 are often associated with Dravet syndrome. Recently, GABAA receptor subunit genes (GABRs) encoding α1 (GABRA1), ß3 (GABRB3) and γ2 (GABRG2), but not ß2 (GABRB2) or ß1 (GABRB1), subunits are frequently associated with Dravet syndrome or Dravet syndrome-like phenotype. We performed next generation sequencing on 870 patients with Dravet syndrome and identified nine variants in three different GABRs. Interestingly, the variants were all in genes encoding the most common GABAA receptor, the α1ß2γ2 receptor. Mutations in GABRA1 (c.644T>C, p. L215P; c.640C>T, p. R214C; c.859G>A; V287I; c.641G>A, p. R214H) and GABRG2 (c.269C>G, p. T90R; c.1025C>T, p. P342L) presented as de novo cases, while in GABRB2 two variants were de novo (c.992T>C, p. F331S; c.542A>T, p. Y181F) and one was autosomal dominant and inherited from the maternal side (c.990_992del, p.330_331del). We characterized the effects of these GABR variants on GABAA receptor biogenesis and channel function. We found that defects in receptor gating were the common deficiency of GABRA1 and GABRB2 Dravet syndrome variants, while mainly trafficking defects were found with the GABRG2 (c.269C>G, p. T90R) variant. It seems that variants in α1 and ß2 subunits are less tolerated than in γ2 subunits, since variant α1 and ß2 subunits express well but were functionally deficient. This suggests that all of these GABR variants are all targeting GABR genes that encode the assembled α1ß2γ2 receptor, and regardless of which of the three subunits are mutated, variants in genes coding for α1, ß2 and γ2 receptor subunits make them candidate causative genes in the pathogenesis of Dravet syndrome.

Impaired State-Dependent Potentiation of GABAergic Synaptic Currents Triggers Seizures in a Genetic Generalized Epilepsy Model.

Epileptic activity in genetic generalized epilepsy (GGE) patients preferentially appears during sleep and its mechanism remains unknown. Here, we found that sleep-like slow-wave oscillations (0.5 Hz SWOs) potentiated excitatory and inhibitory synaptic currents in layer V cortical pyramidal neurons from wild-type (wt) mouse brain slices. In contrast, SWOs potentiated excitatory, but not inhibitory, currents in cortical neurons from a heterozygous (het) knock-in (KI) Gabrg2+Q/390X model of Dravet epilepsy syndrome. This created an imbalance between evoked excitatory and inhibitory currents to effectively prompt neuronal action potential firings. Similarly, physiologically similar up-/down-state induction (present during slow-wave sleep) in cortical neurons also potentiated excitatory synaptic currents within brain slices from wt and het KI mice. Moreover, this state-dependent potentiation of excitatory synaptic currents entailed some signaling pathways of homeostatic synaptic plasticity. Consequently, in het KI mice, in vivo SWO induction (using optogenetic methods) triggered generalized epileptic spike-wave discharges (SWDs), being accompanied by sudden immobility, facial myoclonus, and vibrissa twitching. In contrast, in wt littermates, SWO induction did not cause epileptic SWDs and motor behaviors. To our knowledge, this is the first mechanism to explain why epileptic SWDs preferentially happen during non rapid eye-movement sleep and quiet-wakefulness in human GGE patients.

GABAA receptor ß3 subunit mutation D120N causes Lennox-Gastaut syndrome in knock-in mice.

The Lennox-Gastaut syndrome is a devastating early-onset epileptic encephalopathy, associated with severe behavioural abnormalities. Its pathophysiology, however, is largely unknown. A de novo mutation (c.G358A, p.D120N) in the human GABA type-A receptor ß3 subunit gene (GABRB3) has been identified in a patient with Lennox-Gastaut syndrome. To determine whether the mutation causes Lennox-Gastaut syndrome in vivo in mice and to elucidate its mechanistic effects, we generated the heterozygous Gabrb3+/D120N knock-in mouse and found that it had frequent spontaneous atypical absence seizures, as well as less frequent tonic, myoclonic, atonic and generalized tonic-clonic seizures. Each of these seizure types had a unique and characteristic ictal EEG. In addition, knock-in mice displayed abnormal behaviours seen in patients with Lennox-Gastaut syndrome including impaired learning and memory, hyperactivity, impaired social interactions and increased anxiety. This Gabrb3 mutation did not alter GABA type-A receptor trafficking or expression in knock-in mice. However, cortical neurons in thalamocortical slices from knock-in mice had reduced miniature inhibitory post-synaptic current amplitude and prolonged spontaneous thalamocortical oscillations. Thus, the Gabrb3+/D120N knock-in mouse recapitulated human Lennox-Gastaut syndrome seizure types and behavioural abnormalities and was caused by impaired inhibitory GABAergic signalling in the thalamocortical loop. In addition, treatment with antiepileptic drugs and cannabinoids ameliorated atypical absence seizures in knock-in mice. This congenic knock-in mouse demonstrates that a single-point mutation in a single gene can cause development of multiple types of seizures and multiple behavioural abnormalities. The knock-in mouse will be useful for further investigation of the mechanisms of Lennox-Gastaut syndrome development and for the development of new antiepileptic drugs and treatments.

Sox17 Regulates a Program of Oligodendrocyte Progenitor Cell Expansion and Differentiation during Development and Repair.

Sox17, a SoxF family member transiently upregulated during postnatal oligodendrocyte (OL) development, promotes OL cell differentiation, but its function in white matter development and pathology in vivo is unknown. Our analysis of oligodendroglial- and OL-progenitor-cell-targeted ablation in vivo using a floxed Sox17 mouse establishes a dependence of postnatal oligodendrogenesis on Sox17 and reveals Notch signaling as a mediator of Sox17 function. Following Sox17 ablation, reduced numbers of Olig2-expressing cells and mature OLs led to developmental hypomyelination and motor dysfunction. After demyelination, Sox17 deficiency inhibited OL regeneration. OL decline was unexpectedly preceded by transiently increased differentiation and a reduction of OL progenitor cells. Evidence of a dual role for Sox17 in progenitor cell expansion by Notch and differentiation involving TCF7L2 expression were found. A program of progenitor expansion and differentiation promoted by Sox17 through Notch thus contributes to OL production and determines the outcome of white matter repair.

Single Quantum Dot Tracking Reveals Serotonin Transporter Diffusion Dynamics are Correlated with Cholesterol-Sensitive Threonine 276 Phosphorylation Status in Primary Midbrain Neurons.

Serotonin transporter (SERT) terminates serotonin signaling in the brain by enabling rapid clearance of the neurotransmitter. SERT dysfunction has been associated with a variety of psychiatric disorders, including depression, anxiety, and autism. Visualizing SERT behavior at the single molecule level in endogenous systems remains a challenge. In this study, we utilize quantum dot (QD) single particle tracking (SPT) to capture SERT dynamics in primary rat midbrain neurons. Membrane microenvironment, specifically membrane cholesterol, plays a key role in SERT regulation and has been found to affect SERT conformational state. We sought to determine how reduced cholesterol content affects both lateral mobility and phosphorylation of conformationally sensitive threonine 276 (Thr276) in endogenous SERT using two different methods of cholesterol manipulation, statins and methyl-ß-cyclodextrin. Both chronic and acute cholesterol depletion increased SERT lateral diffusion, radial displacement along the membrane, mobile fraction, and Thr276 phosphorylation levels. Overall, this work has provided new insights about endogenous neuronal SERT mobility and its associations with membrane cholesterol and SERT phosphorylation status.

Sirt1 regulates glial progenitor proliferation and regeneration in white matter after neonatal brain injury.

Regenerative processes in brain pathologies require the production of distinct neural cell populations from endogenous progenitor cells. We have previously demonstrated that oligodendrocyte progenitor cell (OPC) proliferation is crucial for oligodendrocyte (OL) regeneration in a mouse model of neonatal hypoxia (HX) that reproduces diffuse white matter injury (DWMI) of premature infants. Here we identify the histone deacetylase Sirt1 as a Cdk2 regulator in OPC proliferation and response to HX. HX enhances Sirt1 and Sirt1/Cdk2 complex formation through HIF1α activation. Sirt1 deacetylates retinoblastoma (Rb) in the Rb/E2F1 complex, leading to dissociation of E2F1 and enhanced OPC proliferation. Sirt1 knockdown in culture and its targeted ablation in vivo suppresses basal and HX-induced OPC proliferation. Inhibition of Sirt1 also promotes OPC differentiation after HX. Our results indicate that Sirt1 is an essential regulator of OPC proliferation and OL regeneration after neonatal brain injury. Therefore, enhancing Sirt1 activity may promote OL recovery after DWMI.