High Doses of ANA12 Improve Phenobarbital Efficacy in a Model of Neonatal Post-Ischemic Seizures.

Phenobarbital (PB) remains the first-line medication for neonatal seizures. Yet, seizures in many newborns, particularly those associated with perinatal ischemia, are resistant to PB. Previous animal studies have shown that in postnatal day P7 mice pups with ischemic stroke induced by unilateral carotid ligation, the tyrosine receptor kinase B (TrkB) antagonist ANA12 (N-[2-[[(hexahydro-2-oxo-1H-azepin-3-yl)amino]carbonyl]phenyl]-benzo[b]thiophene-2-carboxamide, 5 mg/kg) improved the efficacy of PB in reducing seizure occurrence. To meet optimal standards of effectiveness, a wider range of ANA12 doses must be tested. Here, using the unilateral carotid ligation model, we tested the effectiveness of higher doses of ANA12 (10 and 20 mg/kg) on the ability of PB to reduce seizure burden, ameliorate cell death (assessed by Fluoro-Jade staining), and affect neurodevelopment (righting reflex, negative geotaxis test, open field test). We found that a single dose of ANA12 (10 or 20 mg/kg) given 1 h after unilateral carotid ligation in P7 pups reduced seizure burden and neocortical and striatal neuron death without impairing developmental reflexes. In conclusion, ANA12 at a range of doses (10-20 mg/kg) enhanced PB effectiveness for the treatment of perinatal ischemia-related seizures, suggesting that this agent might be a clinically safe and effective adjunctive agent for the treatment of pharmacoresistant neonatal seizures.

Case report: Off-label use of low-dose perampanel in a 25-month-old girl with a pathogenic SYNGAP1 variant.

Introduction: Preclinical studies in a mouse model have shown that SYNGAP1 haploinsufficiency results in an epilepsy phenotype with excessive GluA2-AMPA insertion specifically on the soma of fast-spiking parvalbumin-positive interneurons associated with significant dysfunction of cortical gamma homeostasis that was rescued by perampanel (PER), an AMPA receptor blocker. In this single case, we aimed to investigate the presence of dysregulated cortical gamma in a toddler with a pathogenic SYNGAP1 variant and report on the effect of low-dose PER on electroencephalogram (EEG) and clinical profile. Methods: Clinical data from physician's clinic notes; genetic testing reports; developmental scores from occupational therapy, physical therapy, speech and language therapy evaluations; and applied behavioral analysis reports were reviewed. Developmental assessments and EEG analysis were done pre- and post-PER. Results: Clinically, the patient showed improvements in the developmental profile and sleep quality post-PER. EEG spectral power analysis in our patient revealed a loss of gamma power modulation with behavioral-state transitions similar to what was observed in Syngap1+/- mice. Furthermore, the administration of low-dose PER rescued the dysfunctional cortical gamma homeostasis, similar to the preclinical study. However, as in the epileptic mice, PER did not curb epileptiform discharges or clinical seizures. Conclusion: Similar to the Syngap1+/- mice, cortical gamma homeostasis was dysregulated in the patient. This dysfunction was rescued by PER. These encouraging results necessitate further validation of gamma dysregulation as a potential translational EEG biomarker in SYNAP1-DEE. Low-dose PER can be explored as a therapeutic option through clinical trials.

A companion to the preclinical common data elements and case report forms for neuropathology studies in epilepsy research. A report of the TASK3 WG2 Neuropathology Working Group of the ILAE/AES Joint Translational Task Force.

The International League Against Epilepsy/American Epilepsy Society (ILAE/AES) Joint Translational Task Force initiated the TASK3 working group to create common data elements (CDEs) for various aspects of preclinical epilepsy research studies, which could help improve the standardization of experimental designs. This article addresses neuropathological changes associated with seizures and epilepsy in rodent models of epilepsy. We discuss CDEs for histopathological parameters for neurodegeneration, changes in astrocyte morphology and function, mechanisms of inflammation, and changes in the blood-brain barrier and myelin/oligodendrocytes resulting from recurrent seizures in rats and mice. We provide detailed CDE tables and case report forms (CRFs), and with this companion manuscript, we discuss the rationale and methodological aspects of individual neuropathological examinations. The CDEs, CRFs, and companion paper are available to all researchers, and their use will benefit the harmonization and comparability of translational preclinical epilepsy research. The ultimate hope is to facilitate the development of rational therapy concepts for treating epilepsies, seizures, and comorbidities and the development of biomarkers assessing the pathological state of the disease.

Targeting ischemia-induced KCC2 hypofunction rescues refractory neonatal seizures and mitigates epileptogenesis in a mouse model.

Neonatal seizures pose a clinical challenge in their early detection, acute management, and long-term comorbidities. They are often caused by hypoxic-ischemic encephalopathy and are frequently refractory to the first-line antiseizure medication phenobarbital. One proposed mechanism for phenobarbital inefficacy during neonatal seizures is the reduced abundance and function of the neuron-specific K+/Cl− cotransporter 2 (KCC2), which maintains chloride homeostasis and promotes GABAergic inhibition upon its phosphorylation during postnatal development. Here, we investigated whether this mechanism is causal and whether it can be rescued by KCC2 functional enhancement. In a CD-1 mouse model of refractory ischemic neonatal seizures, treatment with the KCC2 functional enhancer CLP290 rescued phenobarbital efficacy, increased KCC2 abundance, and prevented the development of epileptogenesis, as quantified by video electroencephalogram monitoring. These effects were prevented by knock-in expression of nonphosphorylatable mutants of KCC2 (S940A or T906A and T1007A), indicating that KCC2 phosphorylation regulates both neonatal seizure susceptibility and CLP290-mediated KCC2 functional enhancement. Our findings therefore validate KCC2 as a clinically relevant target for refractory neonatal seizures and provide insights for future drug development.

Novel Concepts for the Role of Chloride Cotransporters in Refractory Seizures.

Epilepsy is associated with a multitude of acquired or genetic neurological disorders characterized by a predisposition to spontaneous recurrent seizures. An estimated 15 million patients worldwide have ongoing seizures despite optimal management and are classified as having refractory epilepsy. Early-life seizures like those caused by perinatal hypoxic ischemic encephalopathy (HIE) remain a clinical challenge because although transient, they are difficult to treat and associated with poor neurological outcomes. Pediatric epilepsy syndromes are consistently associated with intellectual disability and neurocognitive comorbidities. HIE and arterial ischemic stroke are the most common causes of seizures in term neonates and account for 7.5-20% of neonatal seizures. Standard first-line treatments such as phenobarbital (PB) and phenytoin fail to curb seizures in ~50% of neonates. In the long-term, HIE can result in hippocampal sclerosis and temporal lobe epilepsy (TLE), which is the most common adult epilepsy, ~30% of which is associated with refractory seizures. For patients with refractory TLE seizures, a viable option is the surgical resection of the epileptic foci. Novel insights gained from investigating the developmental role of Cl- cotransporter function have helped to elucidate some of the mechanisms underlying the emergence of refractory seizures in both HIE and TLE. KCC2 as the chief Cl- extruder in neurons is critical for enabling strong hyperpolarizing synaptic inhibition in the brain and has been implicated in the pathophysiology underlying both conditions. More recently, KCC2 function has become a novel therapeutic target to combat refractory seizures.

Brain-Derived Neurotrophic Factor in Neonatal Seizures.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has an extensively studied classical role in neuronal growth, differentiation, survival, and plasticity. Neurotrophic, from the Greek neuro and trophos, roughly translates as "vital nutrition for the brain." During development, BDNF and its associated receptor tyrosine receptor kinase B are tightly regulated as they influence the formation and maturation of neuronal synapses. Preclinical research investigating the role of BDNF in neurological disorders has focused on the effects of decreased BDNF expression on the development and maintenance of neuronal synapses. In contrast, heightened BDNF-tyrosine receptor kinase B activity has received less scrutiny for its role in neurological disorders. Recent studies suggest that excessive BDNF-tyrosine receptor kinase B signaling in the developing brain may promote the hyperexcitability that underlies refractory neonatal seizures. This review will critically examine BDNF-tyrosine receptor kinase B signaling in the immature brain, its role in the emergence of refractory neonatal seizures, and the potential of targeting BDNF-TrkB signaling as a novel antiseizure strategy.

Intellectual and Developmental Disabilities Research Centers: A Multidisciplinary Approach to Understand the Pathogenesis of Methyl-CpG Binding Protein 2-related Disorders.

Disruptions in the gene encoding methyl-CpG binding protein 2 (MECP2) underlie complex neurodevelopmental disorders including Rett Syndrome (RTT), MECP2 duplication disorder, intellectual disabilities, and autism. Significant progress has been made on the molecular and cellular basis of MECP2-related disorders providing a new framework for understanding how altered epigenetic landscape can derail the formation and refinement of neuronal circuits in early postnatal life and proper neurological function. This review will summarize selected major findings from the past years and particularly highlight the integrated and multidisciplinary work done at eight NIH-funded Intellectual and Developmental Disabilities Research Centers (IDDRC) across the US. Finally, we will outline a path forward with identification of reliable biomarkers and outcome measures, longitudinal preclinical and clinical studies, reproducibility of results across centers as a synergistic effort to decode and treat the pathogenesis of the complex MeCP2 disorders.

TrkB agonists prevent postischemic emergence of refractory neonatal seizures in mice.

Refractory neonatal seizures do not respond to first-line antiseizure medications like phenobarbital (PB), a positive allosteric modulator for GABAA receptors. GABAA receptor-mediated inhibition is dependent upon electroneutral cation-chloride transporter KCC2, which mediates neuronal chloride extrusion and its age-dependent increase and postnatally shifts GABAergic signaling from depolarizing to hyperpolarizing. Brain-derived neurotropic factor-tyrosine receptor kinase B activation (BDNF-TrkB activation) after excitotoxic injury recruits downstream targets like PLCγ1, leading to KCC2 hypofunction. Here, the antiseizure efficacy of TrkB agonists LM22A-4, HIOC, and deoxygedunin (DG) on PB-refractory seizures and postischemic TrkB pathway activation was investigated in a mouse model (CD-1, P7) of refractory neonatal seizures. LM, a BDNF loop II mimetic, rescued PB-refractory seizures in a sexually dimorphic manner. Efficacy was associated with a substantial reduction in the postischemic phosphorylation of TrkB at Y816, a site known to mediate postischemic KCC2 hypofunction via PLCγ1 activation. LM rescued ischemia-induced phospho-KCC2-S940 dephosphorylation, preserving its membrane stability. Full TrkB agonists HIOC and DG similarly rescued PB refractoriness. Chemogenetic inactivation of TrkB substantially reduced postischemic neonatal seizure burdens at P7. Sex differences identified in developmental expression profiles of TrkB and KCC2 may underlie the sexually dimorphic efficacy of LM. These results support a potentially novel role for the TrkB receptor in the emergence of age-dependent refractory neonatal seizures.

Transcranial photoacoustic imaging of NMDA-evoked focal circuit dynamics in the rat hippocampus.

OBJECTIVE: We report the transcranial functional photoacoustic (fPA) neuroimaging of N-methyl-D-aspartate (NMDA) evoked neural activity in the rat hippocampus. Concurrent quantitative electroencephalography (qEEG) and microdialysis were used to record real-time circuit dynamics and excitatory neurotransmitter concentrations, respectively. APPROACH: We hypothesized that location-specific fPA voltage-sensitive dye (VSD) contrast would identify neural activity changes in the hippocampus which correlate with NMDA-evoked excitatory neurotransmission. MAIN RESULTS: Transcranial fPA VSD imaging at the contralateral side of the microdialysis probe provided NMDA-evoked VSD responses with positive correlation to extracellular glutamate concentration changes. qEEG validated a wide range of glutamatergic excitation, which culminated in focal seizure activity after a high NMDA dose. We conclude that transcranial fPA VSD imaging can distinguish focal glutamate loads in the rat hippocampus, based on the VSD redistribution mechanism which is sensitive to the electrophysiologic membrane potential. SIGNIFICANCE: Our results suggest the future utility of this emerging technology in both laboratory and clinical sciences as an innovative functional neuroimaging modality.

Low-Dose Perampanel Rescues Cortical Gamma Dysregulation Associated With Parvalbumin Interneuron GluA2 Upregulation in Epileptic Syngap1+/- Mice.

BACKGROUND: Loss-of-function SYNGAP1 mutations cause a neurodevelopmental disorder characterized by intellectual disability and epilepsy. SYNGAP1 is a Ras GTPase-activating protein that underlies the formation and experience-dependent regulation of postsynaptic densities. The mechanisms that contribute to this proposed monogenic cause of intellectual disability and epilepsy remain unresolved. METHODS: We established the phenotype of the epileptogenesis in a Syngap1+/- mouse model using 24-hour video electroencephalography (vEEG)/electromyography recordings at advancing ages. We administered an acute low dose of perampanel, a Food and Drug Administration-approved AMPA receptor (AMPAR) antagonist, during a follow-on 24-hour vEEG to investigate the role of AMPARs in Syngap1 haploinsufficiency. Immunohistochemistry was performed to determine the region- and location-specific differences in the expression of the GluA2 AMPAR subunit. RESULTS: A progressive worsening of the epilepsy with emergence of multiple seizure phenotypes, interictal spike frequency, sleep dysfunction, and hyperactivity was identified in Syngap1+/- mice. Interictal spikes emerged predominantly during non-rapid eye movement sleep in 24-hour vEEG of Syngap1+/- mice. Myoclonic seizures occurred at behavioral-state transitions both in Syngap1+/- mice and during an overnight EEG from a child with SYNGAP1 haploinsufficiency. In Syngap1+/- mice, EEG spectral power analyses identified a significant loss of gamma power modulation during behavioral-state transitions. A significant region-specific increase of GluA2 AMPAR subunit expression in the somas of parvalbumin-positive interneurons was identified. CONCLUSIONS: Acute dosing with perampanel significantly rescued behavioral state-dependent cortical gamma homeostasis, identifying a novel mechanism implicating Ca2+-impermeable AMPARs on parvalbumin-positive interneurons underlying circuit dysfunction in SYNGAP1 haploinsufficiency.

Rett Syndrome and CDKL5 Deficiency Disorder: From Bench to Clinic.

Rett syndrome (RTT) and CDKL5 deficiency disorder (CDD) are two rare X-linked developmental brain disorders with overlapping but distinct phenotypic features. This review examines the impact of loss of methyl-CpG-binding protein 2 (MeCP2) and cyclin-dependent kinase-like 5 (CDKL5) on clinical phenotype, deficits in synaptic- and circuit-homeostatic mechanisms, seizures, and sleep. In particular, we compare the overlapping and contrasting features between RTT and CDD in clinic and in preclinical studies. Finally, we discuss lessons learned from recent clinical trials while reviewing the findings from pre-clinical studies.

Transcranial Recording of Electrophysiological Neural Activity in the Rodent Brain in vivo Using Functional Photoacoustic Imaging of Near-Infrared Voltage-Sensitive Dye.

Minimally-invasive monitoring of electrophysiological neural activities in real-time-that enables quantification of neural functions without a need for invasive craniotomy and the longer time constants of fMRI and PET-presents a very challenging yet significant task for neuroimaging. In this paper, we present in vivo functional PA (fPA) imaging of chemoconvulsant rat seizure model with intact scalp using a fluorescence quenching-based cyanine voltage-sensitive dye (VSD) characterized by a lipid vesicle model mimicking different levels of membrane potential variation. The framework also involves use of a near-infrared VSD delivered through the blood-brain barrier (BBB), opened by pharmacological modulation of adenosine receptor signaling. Our normalized time-frequency analysis presented in vivo VSD response in the seizure group significantly distinguishable from those of the control groups at sub-mm spatial resolution. Electroencephalogram (EEG) recording confirmed the changes of severity and frequency of brain activities, induced by chemoconvulsant seizures of the rat brain. The findings demonstrate that the near-infrared fPA VSD imaging is a promising tool for in vivo recording of brain activities through intact scalp, which would pave a way to its future translation in real time human brain imaging.

Off-Label Use of Bumetanide for Brain Disorders: An Overview.

Bumetanide (BTN or BUM) is a FDA-approved potent loop diuretic (LD) that acts by antagonizing sodium-potassium-chloride (Na-K-Cl) cotransporters, NKCC1 (SLc12a2) and NKCC2. While NKCC1 is expressed both in the CNS and in systemic organs, NKCC2 is kidney-specific. The off-label use of BTN to modulate neuronal transmembrane Cl- gradients by blocking NKCC1 in the CNS has now been tested as an anti-seizure agent and as an intervention for neurological disorders in pre-clinical studies with varying results. BTN safety and efficacy for its off-label use has also been tested in several clinical trials for neonates, children, adolescents, and adults. It failed to meet efficacy criteria for hypoxic-ischemic encephalopathy (HIE) neonatal seizures. In contrast, positive outcomes in temporal lobe epilepsy (TLE), autism, and schizophrenia trials have been attributed to BTN in studies evaluating its off-label use. NKCC1 is an electroneutral neuronal Cl- importer and the dominance of NKCC1 function has been proposed as the common pathology for HIE seizures, TLE, autism, and schizophrenia. Therefore, the use of BTN to antagonize neuronal NKCC1 with the goal to lower internal Cl- levels and promote GABAergic mediated hyperpolarization has been proposed. In this review, we summarize the data and results for pre-clinical and clinical studies that have tested off-label BTN interventions and report variable outcomes. We also compare the data underlying the developmental expression profile of NKCC1 and KCC2, highlight the limitations of BTN's brain-availability and consider its actions on non-neuronal cells.