Parvalbumin Interneuron Impairment Leads to Synaptic Transmission Deficits and Seizures in SCN8A Epileptic Encephalopathy.

SCN8A epileptic encephalopathy (EE) is a severe epilepsy syndrome resulting from de novo mutations in the voltage-gated sodium channel Na v 1.6, encoded by the gene SCN8A . Na v 1.6 is expressed in both excitatory and inhibitory neurons, yet previous studies have primarily focused on the impact SCN8A mutations have on excitatory neuron function, with limited studies on the importance of inhibitory interneurons to seizure onset and progression. Inhibitory interneurons are critical in balancing network excitability and are known to contribute to the pathophysiology of other epilepsies. Parvalbumin (PV) interneurons are the most prominent inhibitory neuron subtype in the brain, making up about 40% of inhibitory interneurons. Notably, PV interneurons express high levels of Na v 1.6. To assess the role of PV interneurons within SCN8A EE, we used two mouse models harboring patient-derived SCN8A gain-of-function mutations, Scn8a D/+ , where the SCN8A mutation N1768D is expressed globally, and Scn8a W/+ -PV, where the SCN8A mutation R1872W is selectively expressed in PV interneurons. Expression of the R1872W SCN8A mutation selectively in PV interneurons led to the development of spontaneous seizures in Scn8a W/+ -PV mice and seizure-induced death, decreasing survival compared to wild-type. Electrophysiology studies showed that PV interneurons in Scn8a D/+ and Scn8a W/+ -PV mice were susceptible to depolarization block, a state of action potential failure. Scn8a D/+ and Scn8a W/+ -PV interneurons also exhibited increased persistent sodium current, a hallmark of SCN8A gain-of-function mutations that contributes to depolarization block. Evaluation of synaptic connections between PV interneurons and pyramidal cells showed an increase in synaptic transmission failure at high frequencies (80-120Hz) as well as an increase in synaptic latency in Scn8a D/+ and Scn8a W/+ -PV interneurons. These data indicate a distinct impairment of synaptic transmission in SCN8A EE, potentially decreasing overall cortical network inhibition. Together, our novel findings indicate that failure of PV interneuron spiking via depolarization block along with frequency-dependent inhibitory synaptic impairment likely elicits an overall reduction in the inhibitory drive in SCN8A EE, leading to unchecked excitation and ultimately resulting in seizures and seizure-induced death.

Emergence of selectivity and specificity in a coarse-grained model of the nuclear pore complex with sequence-agnostic FG-Nups.

The role of hydrophobicity of phenylalanine-glycine nucleoporins (FG-Nups) in determining the transport of receptor-bound cargo across the nuclear pore complex (NPC) is investigated using Langevin dynamics simulations. A coarse-grained, minimal model of the NPC, comprising a cylindrical pore and hydrophobic-hydrophilic random copolymers for FG-Nups was employed. Karyopherin-bound receptor-cargo complexes (Kaps) were modeled as rigid, coarse-grained spheres without (inert) and with (patchy) FG-binding hydrophobic domains. With a sequence-agnostic description of FG-Nups and the absence of any anisotropies associated with either NPC or cargo, the model described tracer transport only as a function of FG-Nup hydrophobicity, f. The simulations showed the emergence of two important features of cargo transport, namely, NPC selectivity and specificity. NPC selectivity to patchy tracers emerged due to hydrophobic Kap-FG interactions and despite the sequence-agnostic description of FG-Nups. Furthermore, NPC selectivity was observed only in a specific range of FG-hydrophobic fraction, 0.05 ≤ f ≤ 0.20, resulting in specificity of NPC transport with respect to f. Significantly, this range corresponded to the number fraction of FG-repeats observed in both S. cerevisiae and H. sapiens NPCs. This established the central role of the FG-hydrophobic fraction in determining NPC transport, and provided a biophysical basis for conservation of the FG-Nup hydrophobic fraction across evolutionarily distant NPCs. Specificity in NPC transport emerged from the formation of a hydrogel-like network inside the pore with a characteristic mesh size dependent on f. This network rejected cargo for f > 0.2 based on size exclusion, which resulted in enhanced translocation probability for 0.05 ≤ f ≤ 0.20. Extended brush configurations outside the pore resulted in entropic repulsion and exclusion of inert cargo in this range. Thus, our minimal NPC model exhibited a hybrid cargo translocation mechanism, with aspects of both virtual gate and selective-phase models, in this range of FG-hydrophobic fraction.

EpiPro, a Novel, Synthetic, Activity-Regulated Promoter That Targets Hyperactive Neurons in Epilepsy for Gene Therapy Applications.

Epileptogenesis is characterized by intrinsic changes in neuronal firing, resulting in hyperactive neurons and the subsequent generation of seizure activity. These alterations are accompanied by changes in gene transcription networks, first with the activation of early-immediate genes and later with the long-term activation of genes involved in memory. Our objective was to engineer a promoter containing binding sites for activity-dependent transcription factors upregulated in chronic epilepsy (EpiPro) and validate it in multiple rodent models of epilepsy. First, we assessed the activity dependence of EpiPro: initial electrophysiology studies found that EpiPro-driven GFP expression was associated with increased firing rates when compared with unlabeled neurons, and the assessment of EpiPro-driven GFP expression revealed that GFP expression was increased ~150× after status epilepticus. Following this, we compared EpiPro-driven GFP expression in two rodent models of epilepsy, rat lithium/pilocarpine and mouse electrical kindling. In rodents with chronic epilepsy, GFP expression was increased in most neurons, but particularly in dentate granule cells, providing in vivo evidence to support the "breakdown of the dentate gate" hypothesis of limbic epileptogenesis. Finally, we assessed the time course of EpiPro activation and found that it was rapidly induced after seizures, with inactivation following over weeks, confirming EpiPro's potential utility as a gene therapy driver for epilepsy.

Validation of traditional claims of anti-arthritic efficacy of trans-Himalayan snow mountain garlic (Allium ampeloprasum L.) extract using adjuvant-induced arthritis rat model: A comparative evaluation with normal garlic (Allium sativum L.) and dexamethasone.

ETHNOPHARMACOLOGICAL RELEVANCE: Snow Mountain Garlic (SMG) (Allium ampeloprasum L.) is a wild trans-Himalayan member of the genus Allium, valued for its anti-inflammatory and anti-arthritic properties in the mountain folk medicinal system (Sowa-Rigpa). Despite its age-old medicinal usage by traditional therapists and the native population for various ailments including rheumatism, there is no scientific validation of its phyto-pharmaceutical merits. AIM OF THE STUDY: The present pre-clinical study compared the in-vivo anti-arthritic effects of SMG with reported efficacy doses of normal garlic (Allium sativum L.) extract and dexamethasone in a complete Freund's adjuvant (CFA)-induced arthritis rat model. MATERIALS AND METHODS: The female Wistar rats were immunized by the subplannter injection of CFA into the right hind footpad. Aqueous extracts of SMG and normal garlic were administered orally at a dose of 250 mg/kg and 500 mg/kg for 28 days. Dexamethasone was used as positive control drug. Behavioral parameters including paw markers, arthritis index, joint stiffness, body weight change, etc. were measured. Also, the changes in histopathological indices, hematological profile, inflammatory mediators, and serum cytokines level was determined. RESULTS: Treatment of rats with SMG extracts significantly (p < 0.001) prevented the reduction in body weight and hematological changes as well as ameliorated clinical symptoms such as arthritic index, joint stiffness, arthritis score, edema, hyperalgesia, and histopathological indices. This was associated with a significant reduction in the serum levels of RF, CRP, anti-CCP, and proinflammatory cytokines exhibiting strong anti-arthritic potential. SMG extracts could also significantly down regulate the NF-κB, COX-2, and iNOS expression in the ankle joint tissues. CONCLUSIONS: The present study is the first attempt to validate the phyto-pharmaceutical efficacy of this folk garlic variety from the trans-Himalayan region. Overall, SMG extract showed remarkable preventive anti-inflammatory and anti-arthritic activities which were closely comparable to therapeutic effects of dexamethasone and at par or even better than normal garlic w.r.t. several study parameters.

Carbon Dots Conjugated Antibody as an Effective FRET-Based Biosensor for Progesterone Hormone Screening.

In this work, carbon dots (CDs) were synthesized by a one-step hydrothermal method using citric acid and ethylene diamine, and covalently functionalized with antibodies for the sensing of progesterone hormone. The structural and morphological analysis reveals that the synthesized CDs are of average size (diameter 8-10 nm) and the surface functionalities are confirmed by XPS, XRD and FT-IR. Further graphene oxide (GO) is used as a quencher due to the fluorescence resonance energy transfer (FRET) mechanism, whereas the presence of the analyte progesterone turns on the fluorescence because of displacement of GO from the surface of CDs effectively inhibiting FRET efficiency due to the increased distance between donor and acceptor moieties. The linear curve is obtained with different progesterone concentrations with 13.8 nM detection limits (R2 = 0.974). The proposed optical method demonstrated high selectivity performance in the presence of structurally resembling interfering compounds. The PL intensity increased linearly with the increased progesterone concentration range (10-900 nM) under the optimal experimental parameters. The developed level-free immunosensor has emerged as a potential platform for simplified progesterone analysis due to the high selectivity performance and good recovery in different samples of spiked water.

Forebrain epileptiform activity is not required for seizure-induced apnea in a mouse model of Scn8a epilepsy.

Sudden unexpected death in epilepsy (SUDEP) accounts for the deaths of 8-17% of patients with epilepsy. Although the mechanisms of SUDEP are essentially unknown, one proposed mechanism is respiratory arrest initiated by a convulsive seizure. In mice, we have previously observed that extended apnea occurs during the tonic phase of seizures. Although often survived, tonic seizures became fatal when breathing did not immediately recover postictally. We also found that respiratory muscles were tonically contracted during the apnea, suggesting that muscle contraction could be the cause of apnea. In the present study, we tested the hypothesis that pyramidal neurons of the motor cortex drive motor units during the tonic phase, which produces apnea. Mice harboring the patient-derived N1768D point mutation of an Scn8a allele were crossed with transgenic mice such that inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADD) receptors were selectively expressed in excitatory forebrain neurons. We then triggered audiogenic and hippocampal (HC) stimulated seizures under control conditions and when excitatory forebrain neurons were inhibited with the synthetic ligand Clozapine-N-Oxide (CNO). We found that inhibition with CNO was sufficient to increase seizure threshold of HC stimulated, but not audiogenic, seizures. In addition, regardless of seizure type, CNO nearly eliminated epileptiform activity that occurred proximal to the tonic phase; however, the seizure behaviors, notably the tonic phase and concomitant apnea, were unchanged. We interpret these results to indicate that while cortical neurons are likely critical for epileptogenesis and seizure initiation, the behavioral manifestations of tonic seizures are generated by neural circuitry in the mid- and/or hindbrain.

A comparative study of in-vitro and in-silico anti-candidal activity and GC-MS profiles of snow mountain garlic vs. normal garlic.

AIM: The study aimed to profile the volatile phytocomposition of snow mountain garlic (SMG) compared to normal garlic and investigate the anti-Candida efficacy against clinically relevant multi-drug resistant isolates of Candida species. METHODS AND RESULTS: Herein, SMG has shown significantly superior fungicidal power at 2x-MIC dose against C. albicans and C. glabrata in killing kinetic evaluation unlike the fungistatic effect of normal garlic. GC-MS headspace-based profiling of SMG showed 5 unique volatile compounds and a 5-fold higher content of saponins than normal garlic. In an in-silico analysis, cholesta-4,6-dien-3-ol,(3-beta) was uniquely identified in SMG as a potential inhibitor with high binding affinity to the active site of exo-1,3-betaglucan synthase, an established anti-candida drug target crucial for the biofilm matrix formation, thus suggesting a plausible anti-Candida mechanism. CONCLUSION: The in-vitro and in-silico studies have demonstrated the Candida-cidal and anti-biofilm activities of SMG, distinguishing it from the Candida-static efficacy of normal garlic. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that identifies several phytochemical signatures of SMG along with a potential anti-Candida compound, that is cholesta-4,6-dien-3-ol,(3-beta)-, which appears worthy of detailed studies in the future to explore the utility of SMG as a fungal phytotherapy agent, especially against drug-resistant Candida sp.

Astrocyte reactivity in a mouse model of SCN8A epileptic encephalopathy.

OBJECTIVE: SCN8A epileptic encephalopathy is caused predominantly by de novo gain-of-function mutations in the voltage-gated sodium channel Nav 1.6. The disorder is characterized by early onset of seizures and developmental delay. Most patients with SCN8A epileptic encephalopathy are refractory to current anti-seizure medications. Previous studies determining the mechanisms of this disease have focused on neuronal dysfunction as Nav 1.6 is expressed by neurons and plays a critical role in controlling neuronal excitability. However, glial dysfunction has been implicated in epilepsy and alterations in glial physiology could contribute to the pathology of SCN8A encephalopathy. In the current study, we examined alterations in astrocyte and microglia physiology in the development of seizures in a mouse model of SCN8A epileptic encephalopathy. METHODS: Using immunohistochemistry, we assessed microglia and astrocyte reactivity before and after the onset of spontaneous seizures. Expression of glutamine synthetase and Nav 1.6, and Kir 4.1 channel currents were assessed in astrocytes in wild-type (WT) mice and mice carrying the N1768D SCN8A mutation (D/+). RESULTS: Astrocytes in spontaneously seizing D/+ mice become reactive and increase expression of glial fibrillary acidic protein (GFAP), a marker of astrocyte reactivity. These same astrocytes exhibited reduced barium-sensitive Kir 4.1 currents compared to age-matched WT mice and decreased expression of glutamine synthetase. These alterations were only observed in spontaneously seizing mice and not before the onset of seizures. In contrast, microglial morphology remained unchanged before and after the onset of seizures. SIGNIFICANCE: Astrocytes, but not microglia, become reactive only after the onset of spontaneous seizures in a mouse model of SCN8A encephalopathy. Reactive astrocytes have reduced Kir 4.1-mediated currents, which would impair their ability to buffer potassium. Reduced expression of glutamine synthetase would modulate the availability of neurotransmitters to excitatory and inhibitory neurons. These deficits in potassium and glutamate handling by astrocytes could exacerbate seizures in SCN8A epileptic encephalopathy. Targeting astrocytes may provide a new therapeutic approach to seizure suppression.

Targeted Augmentation of Nuclear Gene Output (TANGO) of Scn1a rescues parvalbumin interneuron excitability and reduces seizures in a mouse model of Dravet Syndrome.

Dravet Syndrome (DS) is a severe developmental and epileptic encephalopathy typically caused by loss-of-function de novo mutations in the SCN1A gene which encodes the voltage-gated sodium channel isoform NaV1.1. Decreased NaV1.1 expression results in impaired excitability of inhibitory interneurons and seizure onset. To date, there are no clinically available treatments for DS that directly address the core mechanism of disease; reduced NaV1.1 expression levels in interneurons. Recently, Targeted Augmentation of Nuclear Gene Output (TANGO) of SCN1A by the antisense oligonucleotide (ASO) STK-001, was shown to increase Scn1a mRNA levels, increase NaV1.1 protein expression, reduce seizures, and improve survival in the Scn1a+/- mouse model of DS. However, it remains unknown whether STK-001 treatment rescues the reduced intrinsic excitability of parvalbumin-positive (PV) inhibitory interneurons associated with DS. In this study, we demonstrate that STK-001 treatment reduces seizures, prolongs survival, and rescues PV interneuron excitability in Scn1a+/- mice to levels observed in WT littermates. Together, these results support the notion that TANGO-mediated augmentation of NaV1.1 levels directly targets and rescues one of the core disease mechanisms of DS.

Somatostatin-Positive Interneurons Contribute to Seizures in SCN8A Epileptic Encephalopathy.

SCN8A epileptic encephalopathy is a devastating epilepsy syndrome caused by mutant SCN8A, which encodes the voltage-gated sodium channel NaV1.6. To date, it is unclear if and how inhibitory interneurons, which express NaV1.6, influence disease pathology. Using both sexes of a transgenic mouse model of SCN8A epileptic encephalopathy, we found that selective expression of the R1872W SCN8A mutation in somatostatin (SST) interneurons was sufficient to convey susceptibility to audiogenic seizures. Patch-clamp electrophysiology experiments revealed that SST interneurons from mutant mice were hyperexcitable but hypersensitive to action potential failure via depolarization block under normal and seizure-like conditions. Remarkably, GqDREADD-mediated activation of WT SST interneurons resulted in prolonged electrographic seizures and was accompanied by SST hyperexcitability and depolarization block. Aberrantly large persistent sodium currents, a hallmark of SCN8A mutations, were observed and were found to contribute directly to aberrant SST physiology in computational modeling and pharmacological experiments. These novel findings demonstrate a critical and previously unidentified contribution of SST interneurons to seizure generation not only in SCN8A epileptic encephalopathy, but epilepsy in general.SIGNIFICANCE STATEMENTSCN8A epileptic encephalopathy is a devastating neurological disorder that results from de novo mutations in the sodium channel isoform Nav1.6. Inhibitory neurons express NaV1.6, yet their contribution to seizure generation in SCN8A epileptic encephalopathy has not been determined. We show that mice expressing a human-derived SCN8A variant (R1872W) selectively in somatostatin (SST) interneurons have audiogenic seizures. Physiological recordings from SST interneurons show that SCN8A mutations lead to an elevated persistent sodium current which drives initial hyperexcitability, followed by premature action potential failure because of depolarization block. Furthermore, chemogenetic activation of WT SST interneurons leads to audiogenic seizure activity. These findings provide new insight into the importance of SST inhibitory interneurons in seizure initiation, not only in SCN8A epileptic encephalopathy, but for epilepsy broadly.

Adrenergic Mechanisms of Audiogenic Seizure-Induced Death in a Mouse Model of SCN8A Encephalopathy.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death amongst patients whose seizures are not adequately controlled by current therapies. Patients with SCN8A encephalopathy have an elevated risk for SUDEP. While transgenic mouse models have provided insight into the molecular mechanisms of SCN8A encephalopathy etiology, our understanding of seizure-induced death has been hampered by the inability to reliably trigger both seizures and seizure-induced death in these mice. Here, we demonstrate that mice harboring an Scn8a allele with the patient-derived mutation N1768D (D/+) are susceptible to audiogenic seizures and seizure-induced death. In adult D/+ mice, audiogenic seizures are non-fatal and have nearly identical behavioral, electrographical, and cardiorespiratory characteristics as spontaneous seizures. In contrast, at postnatal days 20-21, D/+ mice exhibit the same seizure behavior, but have a significantly higher incidence of seizure-induced death following an audiogenic seizure. Seizure-induced death was prevented by either stimulating breathing via mechanical ventilation or by acute activation of adrenergic receptors. Conversely, in adult D/+ mice inhibition of adrenergic receptors converted normally non-fatal audiogenic seizures into fatal seizures. Taken together, our studies show that in our novel audiogenic seizure-induced death model adrenergic receptor activation is necessary and sufficient for recovery of breathing and prevention of seizure-induced death.

Postictal Death Is Associated with Tonic Phase Apnea in a Mouse Model of Sudden Unexpected Death in Epilepsy.

OBJECTIVE: Sudden unexpected death in epilepsy (SUDEP) is an unpredictable and devastating comorbidity of epilepsy that is believed to be due to cardiorespiratory failure immediately after generalized convulsive seizures. METHODS: We performed cardiorespiratory monitoring of seizure-induced death in mice carrying either a p.Arg1872Trp or p.Asn1768Asp mutation in a single Scn8a allele-mutations identified from patients who died from SUDEP-and of seizure-induced death in pentylenetetrazole-treated wild-type mice. RESULTS: The primary cause of seizure-induced death for all mice was apnea, as (1) apnea began during a seizure and continued for tens of minutes until terminal asystole, and (2) death was prevented by mechanical ventilation. Fatal seizures always included a tonic phase that was coincident with apnea. This tonic phase apnea was not sufficient to produce death, as it also occurred during many nonfatal seizures; however, all seizures that were fatal had tonic phase apnea. We also made the novel observation that continuous tonic diaphragm contraction occurred during tonic phase apnea, which likely contributes to apnea by preventing exhalation, and this was only fatal when breathing did not resume after the tonic phase ended. Finally, recorded seizures from a patient with developmental epileptic encephalopathy with a previously undocumented SCN8A likely pathogenic variant (p.Leu257Val) revealed similarities to those of the mice, namely, an extended tonic phase that was accompanied by apnea. INTERPRETATION: We conclude that apnea coincident with the tonic phase of a seizure, and subsequent failure to resume breathing, are the determining events that cause seizure-induced death in Scn8a mutant mice. ANN NEUROL 2021;89:1023-1035.

Peri-Ictal Autonomic Control of Cardiac Function and Seizure-Induced Death.

Sudden unexpected death in epilepsy (SUDEP) accounts for the deaths of 8-17% of patients with epilepsy. Although the mechanisms of SUDEP are unknown, one proposed mechanism is abnormal control of the heart by the autonomic nervous system (ANS). Our objective was to determine whether the broad changes in ictal heart rate experienced by mouse models of SUDEP are (1) due to the ANS and (2) contribute to seizure-induced death. Seizures were induced by electrical stimulation of the hippocampus of a mouse carrying the human SCN8A encephalopathy mutation p.Asn1768Asp (N1768D; "D/+ mice"). Using standard autonomic pharmacology, the relative roles of the parasympathetic and sympathetic nervous systems on heart rate changes associated with seizures were determined. All induced seizures had pronounced ictal bradycardia and postictal tachycardia. Seizure susceptibility or severity were unchanged by the pharmacological agents. Administration of Atropine, a muscarinic antagonist, eliminated ictal bradycardia, while carbachol, a muscarinic agonist, had no effect on ictal bradycardia, but reduced postictal tachycardia. Sotalol, an adrenergic ß-receptor antagonist, had no effect on ictal bradycardia, but did suppress postictal tachycardia. Isoproterenol, a ß-receptor agonist, had no effect on either ictal bradycardia or postictal tachycardia. Administration of the α1-receptor antagonist prazosin increases the incidence of seizure-induced death in D/+ mice. Although postictal heart rate was lower for these fatal seizures in the presence of prazosin, rates were not as low as that recorded for carbachol treated mice, which all survived. Both ictal bradycardia and postictal tachycardia are manifestations of the ANS. Bradycardia is mediated by a maximal activation of the parasympathetic arm of the ANS, and tachycardia is mediated by parasympathetic inactivation and sympathetic activation. While the changes in heart rate during seizures are profound, suppression of postictal heart rate did not increase seizure mortality.

The Role of the Persistent Sodium Current in Epilepsy.

Voltage-gated sodium channels (VGSCs) are foundational to excitable cell function: Their coordinated passage of sodium ions into the cell is critical for the generation and propagation of action potentials throughout the nervous system. The classical paradigm of action potential physiology states that sodium passes through the membrane only transiently (1-2 milliseconds), before the channels inactivate and cease to conduct sodium ions. However, in reality, a small fraction of the total sodium current (1%-2%) remains at steady state despite prolonged depolarization. While this persistent sodium current (INaP) contributes to normal physiological functioning of neurons, accumulating evidence indicates a particularly pathogenic role for an elevated INaP in epilepsy (reviewed previously1). Due to significant advances over the past decade of epilepsy research concerning the importance of INaP in sodium channelopathies, this review seeks to summarize recent evidence and highlight promising novel anti-seizure medication strategies through preferentially targeting INaP.