Clinical and Experimental Epilepsy

Dravet syndrome (DS) is a neurodevelopmental disorder caused by heterozygous loss-of-function of the gene SCN1A encoding the voltage-gated sodium channel subunit Nav1.1, and is defined by treatment-resistant epilepsy, intellectual impairment, and sudden death. However, disease mechanisms remain unclear, as previously-identified deficiency in action potential generation of Nav1.1-expressing parvalbumin-positive fast-spiking GABAergic interneurons (PV-INs) in DS (Scn1a+/-) mice normalizes during development. We used a novel approach that facilitated the assessment of PV-IN function at both early (post-natal day (P) 16-21) and late (P35-56) time points in the same mice. We confirmed that PV-IN spike generation was impaired at P16-21 in all mice (those deceased from SUDEP by P35 and those surviving to P35-56). However, unitary synaptic transmission assessed in PV-IN:principal cell paired recordings was severely dysfunctional selectively in mice recorded at P16-21 that did not survive to P35. Spike generation in surviving mice had normalized by P35-56; yet we again identified abnormalities in synaptic transmission in surviving mice. We propose that early dysfunction of PV-IN spike propagation drives epilepsy severity and risk of sudden death, while persistent dysfunction of spike propagation contributes to chronic DS pathology.

The construction of cortical circuits requires the precise formation of connections between excitatory and inhibitory neurons during early development. Multiple factors, including neurotransmitters, neuronal activity, and neuronal-glial interactions, shape how these critical circuits form. Disruptions of these early processes can disrupt circuit formation, leading to epilepsy and other neurodevelopmental disorders. Here, I will describe our work into understanding how prolonged post-natal astrocyte development in the cortex creates a permissive window for glutamate signaling that provides tonic activation of developing interneurons through Grin2D NMDA receptors. Experimental disruption of this pathway results in hyperexcitable cortical circuits and human mutations in the Grin2D gene, as well as other related molecules that regulate early life glutamate signaling, are associated with devastating epileptic encephalopathies. We will explore fundamental mechanisms linking early life glutamate signaling and later circuit hyperexcitability, with an emphasis on potential therapeutic interventions aimed at reducing epilepsy and other neurological dysfunction.

The Kappa Opioid Receptor as Potential Drug Target in TLE Over the last decades, neuropeptides and their receptors received increasing interest as drug targets for multiple purposes. Our interest focuses on the endogenous opioid system and more specifically on dynorphins and the kappa opioid receptor (KOR). Activation of KOR blocks presynaptic Calcium channels and facilitates postsynaptic Potassium release, thereby dampening signal transduction. As KORs are situated on excitatory neurons in the hippocampus, this makes them an interesting target in temporal lobe epilepsy.

Temporal lobe epilepsy is considered a neuronal microcircuit dysfunction, yet mechanisms are poorly understood. Here we will discuss recent data on cell-type specific alterations of hippocampal microcircuit function in experimental models of temporal lobe epilepsy. We will highlight the importance of leveraging on cellular heterogeneity to better understand the complexities accompanying hippocampal sclerosis.

Brain and neuronal network development depend on a complex sequence of events, which include neurogenesis, migration, differentiation, synaptogenesis, and synaptic pruning. Perturbations to any of these processes, for example associated with ion channel gene mutations (i.e., channelopathies), can underlie neurodevelopmental disorders such as neonatal and infantile epilepsies, strongly impair psychomotor development and cause persistent deficits in cognition, motor skills, or motor control. The therapeutic options available are very limited, and prophylactic therapies for patients at an increased risk of developing such epilepsies do not exist yet. By using genetic mouse models in which we controlled the activities of Kv7/M or HCN/h-channels during different developmental periods, we obtained offspring with distinct neurological phenotypes that could not simply be reversed by the re-introduction of the affected ion channel in juvenile or adult animals. The results indicate that channelopathy/mutation-specific treatments of neonatal and infantile epilepsies and their comorbidities need to be targeted to specific sensitive periods.

KCC2, best known as the neuron-specific chloride extruder that sets the strength and polarity of GABAergic Cl-currents, is a multifunctional molecule which interacts with other ion-regulatory proteins and (structurally) with the neuronal cytoskeleton. Its multiple roles in the generation and suppression of seizures have been widely studied. In my talk, I will address some fundamental issues which are relevant in this field of research: What are EGABA shifts about? What is the role of KCC2 in shunting inhibition? What is meant by “the balance between excitation and inhibition” and, in this context, by the “NKCC1/KCC2 ratio”? Is down-regulation of KCC2 following neuronal trauma a manifestation of adaptive or maladaptive ionic plasticity? Under what conditions is K-Cl cotransport by KCC2 promoting seizures? Should we pay more attention to KCC2 as molecule involved in dendritic spine formation in brain areas such as the hippocampus? Most of these points are of potential importance also in the design of KCC2-targeting drugs and genetic manipulations aimed at combating seizures.

Memory deficits are a debilitating symptom of epilepsy, but little is known about mechanisms underlying cognitive deficits. Here, we describe a Na+ channel-dependent mechanism underlying altered hippocampal dendritic integration, degraded place coding, and deficits in spatial memory. Two-photon glutamate uncaging experiments revealed that the mechanisms constraining the generation of Na+ spikes in hippocampal 1st order pyramidal cell dendrites are profoundly degraded in experimental epilepsy. This phenomenon was reversed by selectively blocking Nav1.3 sodium channels. In-vivo two-photon imaging revealed that hippocampal spatial representations were less precise in epileptic mice. Blocking Nav1.3 channels significantly improved the precision of spatial coding, and reversed hippocampal memory deficits. Thus, a dendritic channelopathy may underlie cognitive deficits in epilepsy and targeting it pharmacologically may constitute a new avenue to enhance cognition.

A variety of pharmacological treatments exist for patients suffering from focal seizures, but systemically administered drugs offer only symptomatic relief and frequently cause unwanted side effects. Moreover, available drugs are ineffective in one third of the patients. Thus, developing more targeted and effective treatment strategies is highly warranted. Neurotrophic factors are candidates for treating epilepsy, but their development has been hampered by difficulties in achieving stable and targeted delivery of efficacious concentrations within the brain. We have developed an implantable cell encapsulation system that delivers high and consistent levels of neurotrophic molecules directly to a specific brain region. The potential of this approach has been tested by delivering glial cell line-derived neurotrophic factor (GDNF) to the hippocampus of epileptic rats. In vivo studies demonstrated that these intrahippocampal implants continue to secrete GDNF and produce high hippocampal GDNF tissue levels in a long-lasting manner. Identical implants rapidly and greatly reduced seizure frequency in the pilocarpine model. This effect increased in magnitude over 3 months, ultimately leading to a reduction of spontaneous seizures by more than 90%. Importantly, these effects were accompanied by improvements in cognition and anxiety, and by the normalization of many histological alterations that are associated with chronic epilepsy. In addition, the antiseizure effect persisted even after device removal. Finally, by establishing a unilateral epileptic focus using the intrahippocampal kainate model, we found that delivery of GDNF exclusively within the focus suppressed already established spontaneous recurrent seizures. Together, these results support the concept that the implantation of encapsulated GDNF-secreting cells can deliver GDNF in a sustained, targeted, and efficacious manner. These findings may form the basis for clinical translation of this approach.

Recent human gene discovery efforts show that gain-of-function (GOF) variants in the KCNT1gene, which encodes a Na+-activated K+ channel subunit, cause severe epilepsies and other neurodevelopmental disorders. Although the impact of these variants on the biophysical properties of the channels is well characterized, the mechanisms that link channel dysfunction to cellular and network hyperexcitability and human disease are unknown. Furthermore, precision therapies that correct channel biophysics in non-neuronal cells have had limited success in treating human disease, highlighting the need for a deeper understanding of how these variants affect neurons and networks. To address this gap, we developed a new mouse model with a pathogenic human variant knocked into the mouse Kcnt1gene. I will discuss our findings on the in vivo phenotypes of this mouse, focusing on our characterization of epileptiform neural activity using electrophysiology and widefield Ca++imaging. I will also talk about our investigations at the synaptic, cellular, and circuit levels, including the main finding that cortical inhibitory neurons in this model show a reduction in intrinsic excitability and action potential generation. Finally, I will discuss future directions to better understand the mechanisms underlying the cell-type specific effects, as well as the link between the cellular and network level effects of KCNT1 GOF.

Glioblastoma cells trigger pharmacoresistant seizures that may promote tumor growth and diminish the quality of remaining life. To define the relationship between growth of glial tumors and their neuronal microenvironment, and to identify genomic biomarkers and mechanisms that may point to better prognosis and treatment of drug resistant epilepsy in brain cancer, we are analyzing a new generation of genetically defined CRISPR/in utero electroporation inborn glioblastoma (GBM) tumor models engineered in mice. The molecular pathophysiology of glioblastoma cells and surrounding neurons and untransformed astrocytes are compared at serial stages of tumor development. Initial studies reveal that epileptiform EEG spiking is a very early and reliable preclinical signature of GBM expansion in these mice, followed by rapidly progressive seizures and death within weeks. FACS-sorted transcriptomic analysis of cortical astrocytes reveals the expansion of a subgroup enriched in pro-synaptogenic genes that may drive hyperexcitability, a novel mechanism of epileptogenesis. Using a prototypical GBM IUE model, we systematically define and correlate the earliest appearance of cortical hyperexcitability with progressive cortical tumor cell invasion, including spontaneous episodes of spreading cortical depolarization, innate inflammation, and xCT upregulation in the peritumoral microenvironment. Blocking this glutamate exporter reduces seizure load. We show that the host genome contributes to seizure risk by generating tumors in a monogenic deletion strain (MapT/tau -/-) that raises cortical seizure threshold. We also show that the tumor variant profile determines epilepsy risk. Our genetic dissection approach sets the stage to broadly explore the developmental biology of personalized tumor/host interactions in mice engineered with novel human tumor mutations in specified glial cell lineages.

The occurrence of seizures at specific times of the day has been consistently observed for centuries in individuals with epilepsy. Electrophysiological recordings provide evidence that seizures have a higher probability of occurring at a given time during the night and day cycle in individuals with epilepsy – the seizure rush hour. Which mechanisms underly such circadian rhythmicity of seizures? Why don’t they occur every day at the same time? Which mechanisms may underly their occurrence outside the rush hour? I shall present a hypothesis: MORE - Molecular Oscillations and Rhythmicity of Epilepsy, a conceptual framework to study and understand the mechanisms underlying the circadian rhythmicity of seizures and their probabilistic nature. The core of the hypothesis is the existence of circa 24h oscillations of gene and protein expression throughout the body in different cells and organs. The orchestrated molecular oscillations control the rhythmicity of numerous body events, such as feeding and sleep. The concept developed here is that molecular oscillations may favor seizure genesis at preferred times, generating the condition for a seizure rush hour. However, the condition is not sufficient, as other factors are necessary for a seizure to occur. Studying these molecular oscillations may help us understand seizure genesis mechanisms and find new therapeutic targets and predictive biomarkers. The MORE hypothesis can be generalized to comorbidities and the slower multidien (week/month period) rhythmicity of seizures.

Seizure onset is a critically important brain state transition that has proved very difficult to predict accurately from recordings of brain activity. I will present new data acquired using a range of optogenetic and imaging tools to characterize exactly how cortical networks change in the build-up to a seizure. I will show how intermittent optogenetic stimulation ("active probing") reveals a latent change in dendritic excitability that is tightly correlated to the onset of seizure activity. This data relates back to old work from the 1980s suggesting a critical role in epileptic pathophysiology for dendritic plateau potentials. Our data show how the precipitous nature of the transition can be understood in terms of multiple, synergistic positive feedback mechanisms.

Seizures can emerge from multiple or large foci in temporal lobe epilepsy (TLE), complicating focally targeted strategies such as surgical resection or the modulation of the activity of specific hippocampal neuronal populations through genetic or optogenetic techniques. Here, we evaluate a strategy in which optogenetic activation of medial septal GABAergic neurons (MSGNs), which provide extensive projections throughout the hippocampus, is used to control seizures. We found that MSGNs were structurally and functionally resilient in the chronic intrahippocampal kainate mouse model of TLE, which as is often the case in human TLE patients, presents with hippocampal sclerosis. Optogenetic stimulation of MSGNs modulated oscillations across the rostral to caudal extent of the hippocampus in epileptic conditions. Chronic wireless optogenetic stimulation of MSGNs, upon electrographic detection of spontaneous hippocampal seizures, resulted in reduced seizure durations. We propose MSGNs as a novel target for optogenetic control of seizures in TLE.

Temporal lobe epilepsy is associated with memory deficits but the circuit mechanisms underlying these cognitive disabilities are not understood. We used electrophysiological recordings, open-source wire-free miniaturized microscopy and computational modeling to probe these deficits in a model of temporal lobe epilepsy. We find desynchronization of dentate gyrus interneurons with CA1 interneurons during theta oscillations and a loss of precision and stability of place fields. We also find that emergence of place cell dysfunction is delayed, providing a potential temporal window for treatments. Computation modeling shows that desynchronization rather than interneuron cell loss can drive place cell dysfunction. Future studies will uncover cell types driving these changes and transcriptional changes that may be driving dysfunction.