The synthetic neuroactive steroid SGE-516 reduces status epilepticus and neuronal cell death in a rat model of soman intoxication.

Organophosphorus nerve agents (OPNAs) are irreversible inhibitors of acetylcholinesterase that pose a serious threat to public health because of their use as chemical weapons. Exposure to high doses of OPNAs can dramatically potentiate cholinergic synaptic activity and cause status epilepticus (SE). Current standard of care for OPNA exposure involves treatment with cholinergic antagonists, oxime cholinesterase reactivators, and benzodiazepines. However, data from pre-clinical models suggest that OPNA-induced SE rapidly becomes refractory to benzodiazepines. Neuroactive steroids (NAS), such as allopregnanolone, retain anticonvulsant activity in rodent models of benzodiazepine-resistant SE, perhaps because they modulate a broader variety of GABAA receptor subtypes. SGE-516 is a novel, next generation NAS and a potent and selective GABAA receptor positive allosteric modulator (PAM). The present study first established that SGE-516 reduced electrographic seizures in the rat lithium-pilocarpine model of pharmacoresistant SE. Then the anticonvulsant activity of SGE-516 was investigated in the soman-intoxication model of OPNA-induced SE. SGE-516 (5.6, 7.5, and 10mg/kg, IP) significantly reduced electrographic seizure activity compared to control when administered 20min after SE onset. When 10mg/kg SGE-516 was administered 40min after SE onset, seizure activity was still significantly reduced compared to control. In addition, all cohorts of rats treated with SGE-516 exhibited significantly reduced neuronal cell death as measured by FluoroJade B immunohistochemistry. These data suggest synthetic NASs that positively modulate both synaptic and extrasynaptic GABAA receptors may be candidates for further study in the treatment of OPNA-induced SE.

The major brain cholesterol metabolite 24(S)-hydroxycholesterol is a potent allosteric modulator of N-methyl-D-aspartate receptors.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that are critical to the regulation of excitatory synaptic function in the CNS. NMDARs govern experience-dependent synaptic plasticity and have been implicated in the pathophysiology of various neuropsychiatric disorders including the cognitive deficits of schizophrenia and certain forms of autism. Certain neurosteroids modulate NMDARs experimentally but their low potency, poor selectivity, and very low brain concentrations make them poor candidates as endogenous ligands or therapeutic agents. Here we show that the major brain-derived cholesterol metabolite 24(S)-hydroxycholesterol (24(S)-HC) is a very potent, direct, and selective positive allosteric modulator of NMDARs with a mechanism that does not overlap that of other allosteric modulators. At submicromolar concentrations 24(S)-HC potentiates NMDAR-mediated EPSCs in rat hippocampal neurons but fails to affect AMPAR or GABAA receptors (GABA(A)Rs)-mediated responses. Cholesterol itself and other naturally occurring oxysterols present in brain do not modulate NMDARs at concentrations ≤10 µM. In hippocampal slices, 24(S)-HC enhances the ability of subthreshold stimuli to induce long-term potentiation (LTP). 24(S)-HC also reverses hippocampal LTP deficits induced by the NMDAR channel blocker ketamine. Finally, we show that synthetic drug-like derivatives of 24(S)-HC, which potently enhance NMDAR-mediated EPSCs and LTP, restore behavioral and cognitive deficits in rodents treated with NMDAR channel blockers. Thus, 24(S)-HC may function as an endogenous modulator of NMDARs acting at a novel oxysterol modulatory site that also represents a target for therapeutic drug development.

Preclinical characterization of zuranolone (SAGE-217), a selective neuroactive steroid GABAA receptor positive allosteric modulator.

Zuranolone (SAGE-217) is a novel, synthetic, clinical stage neuroactive steroid GABAA receptor positive allosteric modulator designed with the pharmacokinetic properties to support oral daily dosing. In vitro, zuranolone enhanced GABAA receptor current at nine unique human recombinant receptor subtypes, including representative receptors for both synaptic (γ subunit-containing) and extrasynaptic (δ subunit-containing) configurations. At a representative synaptic subunit configuration, α1ß2γ2, zuranolone potentiated GABA currents synergistically with the benzodiazepine diazepam, consistent with the non-competitive activity and distinct binding sites of the two classes of compounds at synaptic receptors. In a brain slice preparation, zuranolone produced a sustained increase in GABA currents consistent with metabotropic trafficking of GABAA receptors to the cell surface. In vivo, zuranolone exhibited potent activity, indicating its ability to modulate GABAA receptors in the central nervous system after oral dosing by protecting against chemo-convulsant seizures in a mouse model and enhancing electroencephalogram ß-frequency power in rats. Together, these data establish zuranolone as a potent and efficacious neuroactive steroid GABAA receptor positive allosteric modulator with drug-like properties and CNS exposure in preclinical models. Recent clinical data support the therapeutic promise of neuroactive steroid GABAA receptor positive modulators for treating mood disorders; brexanolone is the first therapeutic approved specifically for the treatment of postpartum depression. Zuranolone is currently under clinical investigation for the treatment of major depressive episodes in major depressive disorder, postpartum depression, and bipolar depression.

Protein kinase a mediates activity-dependent Kv4.2 channel trafficking.

The A-type potassium channel subunit Kv4.2 influences hippocampal function through regulation of dendritic excitability, and changes in Kv4.2 surface expression alter synaptic plasticity. Recent data from our laboratory demonstrate that EGFP (enhanced green fluorescent protein)-tagged Kv4.2 channels located in dendritic spines are internalized in an activity-dependent manner after synaptic stimulation and during chemically induced long-term potentiation. However, the molecular trigger for Kv4.2 internalization remains unknown. Here we examined the role of protein kinase A (PKA) in Kv4.2 activity-dependent trafficking. In hippocampal neurons, PKA activation with forskolin or 8-Br-cAMP induced Kv4.2 internalization from dendritic spines, whereas PKA inhibition with H89 prevented AMPA-induced internalization. Furthermore, introduction of a point mutation at the C-terminal PKA phosphorylation site of Kv4.2 (S552A) prevented the AMPA-induced internalization of Kv4.2. Together, these data demonstrate that Kv4.2 activity-dependent internalization requires PKA phosphorylation of Kv4.2 at serine 522.

Small-conductance Ca2+-activated K+ channel type 2 (SK2) modulates hippocampal learning, memory, and synaptic plasticity.

Apamin-sensitive, small-conductance, Ca2+-activated K+ channels (SK channels) modulate neuronal excitability in CA1 neurons. Blocking all SK channel subtypes with apamin facilitates the induction of hippocampal synaptic plasticity and enhances hippocampal learning. In CA1 dendrites, SK channels are activated by Ca2+ through NMDA receptors and restrict glutamate-mediated EPSPs. Studies of SK channel knock-out mice reveal that of the three apamin-sensitive SK channel subunits (SK1-SK3), only SK2 subunits are necessary for the apamin-sensitive currents in CA1 hippocampal neurons. To determine the specific influence of SK2 channels on hippocampal synaptic plasticity, learning, and memory, we used gene targeting through homologous recombination in embryonic stem cells to generate transgenic mice that overexpress SK2 subunits by 10-fold (SK2+/T). In these mice, the apamin-sensitive current in CA1 neurons was increased by approximately fourfold, relative to wild-type (WT) littermates. In addition, the amplitude of synaptically evoked EPSPs recorded from SK2+/T CA1 neurons increased twice as much in response to SK channel blockade relative to EPSPs recorded from WT CA1 neurons. Consistent with this, SK2 overexpression reduced long-term potentiation after high-frequency stimulation compared with WT littermates and severely impaired learning in both hippocampus- and amygdala-dependent tasks. We conclude that SK2 channels regulate hippocampal synaptic plasticity and play a critical role in modulating mechanisms of learning and memory.

The synthetic neuroactive steroid SGE-516 reduces seizure burden and improves survival in a Dravet syndrome mouse model.

Dravet syndrome is an infant-onset epileptic encephalopathy with multiple seizure types that are often refractory to conventional therapies. Treatment with standard benzodiazepines like clobazam, in combination with valproate and stiripentol, provides only modest seizure control. While benzodiazepines are a first-line therapy for Dravet syndrome, they are limited by their ability to only modulate synaptic receptors. Unlike benzodiazepines, neuroactive steroids potentiate a wider-range of GABAA receptors. The synthetic neuroactive steroid SGE-516 is a potent positive allosteric modulator of both synaptic and extrasynaptic GABAA receptors. Prior work demonstrated anticonvulsant activity of SGE-516 in acute seizure assays in rodents. In this study, we evaluated activity of SGE-516 on epilepsy phenotypes in the Scn1a +/- mouse model that recapitulates many features of Dravet syndrome, including spontaneous seizures, premature death and seizures triggered by hyperthermia. To evaluate SGE-516 in Scn1a +/- mice, we determined the effect of treatment on hyperthermia-induced seizures, spontaneous seizure frequency and survival. SGE-516 treatment protected against hyperthermia-induced seizures, reduced spontaneous seizure frequency and prolonged survival in the Scn1a +/- mice. This provides the first evidence of SGE-516 activity in a mouse model of Dravet syndrome, and supports further investigation of neuroactive steroids as potential anticonvulsant compounds for refractory epilepsies.

Anticonvulsant profile of the neuroactive steroid, SGE-516, in animal models.

Despite the availability of multiple antiepileptic drugs (AED), failure to adequately control seizures is a challenge for approximately one third of epilepsy patients, and new therapies with a differentiated mechanism of action are needed. The neuroactive steroid, SGE-516, is a positive allosteric modulator of both gamma- and delta-containing GABAA receptors. This broad GABAA receptor activity differentiates neuroactive steroids like SGE-516 from benzodiazepines, a class of anticonvulsants which have been shown in vitro to selectively target gamma-subunit containing GABAA receptors. As a neuroactive steroid, SGE-516 has pharmacokinetic properties that are intended to allow for chronic oral dosing. We investigated the anticonvulsant activity of SGE-516 across numerous in vitro and in vivo models of seizure activity. SGE-516 dose-dependently reduced neuronal firing rates and epileptiform activity in vitro. In mice, SGE-516 protected against acute seizures in the PTZ-induced chemo-convulsant seizure model and the 6Hz psychomotor seizure model. In addition, SGE-516 demonstrated anticonvulsant activity in the mouse corneal kindling model. These data suggest that SGE-516 may have potential for development as a novel oral AED for the treatment of refractory seizures.

Small conductance Ca2+-activated K+ channels modulate synaptic plasticity and memory encoding.

Activity-dependent changes in neuronal excitability and synaptic strength are thought to underlie memory encoding. In hippocampal CA1 neurons, small conductance Ca2+-activated K+ (SK) channels contribute to the afterhyperpolarization, affecting neuronal excitability. In the present study, we examined the effect of apamin-sensitive SK channels on the induction of hippocampal synaptic plasticity in response to a range of stimulation frequencies. In addition, the role of apamin-sensitive SK channels on hippocampal-dependent memory encoding and retention was also tested. The results show that blocking SK channels with apamin increased the excitability of hippocampal neurons and facilitated the induction of synaptic plasticity by shifting the modification threshold to lower frequencies. This facilitation was NMDA receptor (NMDAR) dependent and appeared to be postsynaptic. Mice treated with apamin demonstrated accelerated hippocampal-dependent spatial and nonspatial memory encoding. They required fewer trials to learn the location of a hidden platform in the Morris water maze and less time to encode object memory in an object-recognition task compared with saline-treated mice. Apamin did not influence long-term retention of spatial or nonspatial memory. These data support a role for SK channels in the modulation of hippocampal synaptic plasticity and hippocampal-dependent memory encoding.

Reversal of disease-related pathologies in the fragile X mouse model by selective activation of GABAB receptors with arbaclofen.

Fragile X syndrome (FXS), the most common inherited cause of intellectual disability and autism, results from the transcriptional silencing of FMR1 and loss of the mRNA translational repressor protein fragile X mental retardation protein (FMRP). Patients with FXS exhibit changes in neuronal dendritic spine morphology, a pathology associated with altered synaptic function. Studies in the mouse model of fragile X have shown that loss of FMRP causes excessive synaptic protein synthesis, which results in synaptic dysfunction and altered spine morphology. We tested whether the pharmacologic activation of the γ-aminobutyric acid type B (GABA(B)) receptor could correct or reverse these phenotypes in Fmr1-knockout mice. Basal protein synthesis, which is elevated in the hippocampus of Fmr1-knockout mice, was corrected by the in vitro application of the selective GABA(B) receptor agonist STX209 (arbaclofen, R-baclofen). STX209 also reduced to wild-type values the elevated AMPA receptor internalization in Fmr1-knockout cultured neurons, a known functional consequence of increased protein synthesis. Acute administration of STX209 in vivo, at doses that modify behavior, decreased mRNA translation in the cortex of Fmr1-knockout mice. Finally, the chronic administration of STX209 in juvenile mice corrected the increased spine density in Fmr1-knockout mice without affecting spine density in wild-type mice. Thus, activation of the GABA(B) receptor with STX209 corrected synaptic abnormalities considered central to fragile X pathophysiology, a finding that suggests that STX209 may be a potentially effective therapy to treat the core symptoms of FXS.

Dendritic ion channel trafficking and plasticity.

Dendritic ion channels are essential for the regulation of intrinsic excitability as well as modulating the shape and integration of synaptic signals. Changes in dendritic channel function have been associated with many forms of synaptic plasticity. Recent evidence suggests that dendritic ion channel modulation and trafficking could contribute to plasticity-induced alterations in neuronal function. In this review we discuss our current knowledge of dendritic ion channel modulation and trafficking and their relationship to cellular and synaptic plasticity. We also consider the implications for neuronal function. We argue that to gain an insight into neuronal information processing it is essential to understand the regulation of dendritic ion channel expression and properties.

Chronic dietary alpha-lipoic acid reduces deficits in hippocampal memory of aged Tg2576 mice.

Oxidative stress may play a key role in Alzheimer's disease (AD) neuropathology. Here, the effects of the antioxidant, alpha-lipoic acid (ALA) were tested on the Tg2576 mouse, a transgenic model of cerebral amyloidosis associated with AD. Ten-month old Tg2576 and wild type mice were fed an ALA-containing diet (0.1%) or control diet for 6 months and then assessed for the influence of diet on memory and neuropathology. ALA-treated Tg2576 mice exhibited significantly improved learning, and memory retention in the Morris water maze task compared to untreated Tg2576 mice. Twenty-four hours after contextual fear conditioning, untreated Tg2576 mice exhibited significantly impaired context-dependent freezing. ALA-treated Tg2576 mice exhibited significantly more context freezing than the untreated Tg2576 mice. Assessment of brain soluble and insoluble beta-amyloid levels revealed no differences between ALA-treated and untreated Tg2576 mice. Brain levels of nitrotyrosine, a marker of nitrative stress, were elevated in Tg2576 mice, while F2 isoprostanes and neuroprostanes, oxidative stress markers, were not elevated in the Tg2576 mice relative to wild type. These data indicate that chronic dietary ALA can reduce hippocampal-dependent memory deficits of Tg2576 mice without affecting beta-amyloid levels or plaque deposition.

On the delay-dependent involvement of the hippocampus in object recognition memory.

The role of the hippocampus in object recognition memory processes is unclear in the current literature. Conflicting results have been found in lesion studies of both primates and rodents. Procedural differences between studies, such as retention interval, may explain these discrepancies. In the present study, acute lidocaine administration was used to temporarily inactivate the hippocampus prior to training in the spontaneous object recognition task. Male C57BL/6J mice were administered bilateral lidocaine (4%, 0.5 microl/side) or aCSF (0.5 microl/side) directly into the CA1 region of the dorsal hippocampus 5 min prior to sample object training, and object recognition memory was tested after a short ( 5 min) or long (24 h) retention interval. There was no effect of intra-hippocampal lidocaine on the time needed for mice to accumulate sample object exploration, suggesting that inactivation of the hippocampus did not affect sample session activity or the motivation to explore objects. Lidocaine-treated mice exhibited impaired object recognition memory, measured as reduced novel object preference, after a 24 h but not a 5 min retention interval. These data support a delay-dependent role for the hippocampus in object recognition memory, an effect consistent with the results of hippocampal lesion studies conducted in rats. However, these data are also consistent with the view that the hippocampus is involved in object recognition memory regardless of retention interval, and that object recognition processes of parahippocampal structures (e.g., perirhinal cortex) are sufficient to support object recognition memory over short retention intervals.