Anti-seizure gene therapy for focal cortical dysplasia.

Focal cortical dysplasias are a common subtype of malformation of cortical development, which frequently presents with a spectrum of cognitive and behavioural abnormalities as well as pharmacoresistant epilepsy. Focal cortical dysplasia type II is typically caused by somatic mutations resulting in mammalian target of rapamycin (mTOR) hyperactivity, and is the commonest pathology found in children undergoing epilepsy surgery. However, surgical resection does not always result in seizure freedom, and is often precluded by proximity to eloquent brain regions. Gene therapy is a promising potential alternative treatment and may be appropriate in cases that represent an unacceptable surgical risk. Here, we evaluated a gene therapy based on overexpression of the Kv1.1 potassium channel in a mouse model of frontal lobe focal cortical dysplasia. An engineered potassium channel (EKC) transgene was placed under control of a human promoter that biases expression towards principal neurons (CAMK2A) and packaged in an adeno-associated viral vector (AAV9). We used an established focal cortical dysplasia model generated by in utero electroporation of frontal lobe neural progenitors with a constitutively active human Ras homolog enriched in brain (RHEB) plasmid, an activator of mTOR complex 1. We characterized the model by quantifying electrocorticographic and behavioural abnormalities, both in mice developing spontaneous generalized seizures and in mice only exhibiting interictal discharges. Injection of AAV9-CAMK2A-EKC in the dysplastic region resulted in a robust decrease (∼64%) in the frequency of seizures. Despite the robust anti-epileptic effect of the treatment, there was neither an improvement nor a worsening of performance in behavioural tests sensitive to frontal lobe function. AAV9-CAMK2A-EKC had no effect on interictal discharges or behaviour in mice without generalized seizures. AAV9-CAMK2A-EKC gene therapy is a promising therapy with translational potential to treat the epileptic phenotype of mTOR-related malformations of cortical development. Cognitive and behavioural co-morbidities may, however, resist an intervention aimed at reducing circuit excitability.

Conserved patterns across ion channels correlate with variant pathogenicity and clinical phenotypes.

Clinically identified genetic variants in ion channels can be benign or cause disease by increasing or decreasing the protein function. As a consequence, therapeutic decision-making is challenging without molecular testing of each variant. Our biophysical knowledge of ion-channel structures and function is just emerging, and it is currently not well understood which amino acid residues cause disease when mutated. We sought to systematically identify biological properties associated with variant pathogenicity across all major voltage and ligand-gated ion-channel families. We collected and curated 3049 pathogenic variants from hundreds of neurodevelopmental and other disorders and 12 546 population variants for 30 ion channel or channel subunits for which a high-quality protein structure was available. Using a wide range of bioinformatics approaches, we computed 163 structural features and tested them for pathogenic variant enrichment. We developed a novel 3D spatial distance scoring approach that enables comparisons of pathogenic and population variant distribution across protein structures. We discovered and independently replicated that several pore residue properties and proximity to the pore axis were most significantly enriched for pathogenic variants compared to population variants. Using our 3D scoring approach, we showed that the strongest pathogenic variant enrichment was observed for pore-lining residues and alpha-helix residues within 5Å distance from the pore axis centre and not involved in gating. Within the subset of residues located at the pore, the hydrophobicity of the pore was the feature most strongly associated with variant pathogenicity. We also found an association between the identified properties and both clinical phenotypes and functional in vitro assays for voltage-gated sodium channels (SCN1A, SCN2A, SCN8A) and N-methyl-D-aspartate receptor (GRIN1, GRIN2A, GRIN2B) encoding genes. In an independent expert-curated dataset of 1422 neurodevelopmental disorder pathogenic patient variants and 679 electrophysiological experiments, we show that pore axis distance is associated with seizure age of onset and cognitive performance as well as differential gain versus loss-of-channel function. In summary, we identified biological properties associated with ion-channel malfunction and show that these are correlated with in vitro functional readouts and clinical phenotypes in patients with neurodevelopmental disorders. Our results suggest that clinical decision support algorithms that predict variant pathogenicity and function are feasible in the future.

Gene variant effects across sodium channelopathies predict function and guide precision therapy.

Pathogenic variants in the voltage-gated sodium channel gene family lead to early onset epilepsies, neurodevelopmental disorders, skeletal muscle channelopathies, peripheral neuropathies and cardiac arrhythmias. Disease-associated variants have diverse functional effects ranging from complete loss-of-function to marked gain-of-function. Therapeutic strategy is likely to depend on functional effect. Experimental studies offer important insights into channel function but are resource intensive and only performed in a minority of cases. Given the evolutionarily conserved nature of the sodium channel genes, we investigated whether similarities in biophysical properties between different voltage-gated sodium channels can predict function and inform precision treatment across sodium channelopathies. We performed a systematic literature search identifying functionally assessed variants in any of the nine voltage-gated sodium channel genes until 28 April 2021. We included missense variants that had been electrophysiologically characterized in mammalian cells in whole-cell patch-clamp recordings. We performed an alignment of linear protein sequences of all sodium channel genes and correlated variants by their overall functional effect on biophysical properties. Of 951 identified records, 437 sodium channel-variants met our inclusion criteria and were reviewed for functional properties. Of these, 141 variants were epilepsy-associated (SCN1/2/3/8A), 79 had a neuromuscular phenotype (SCN4/9/10/11A), 149 were associated with a cardiac phenotype (SCN5/10A) and 68 (16%) were considered benign. We detected 38 missense variant pairs with an identical disease-associated variant in a different sodium channel gene. Thirty-five out of 38 of those pairs resulted in similar functional consequences, indicating up to 92% biophysical agreement between corresponding sodium channel variants (odds ratio = 11.3; 95% confidence interval = 2.8 to 66.9; P < 0.001). Pathogenic missense variants were clustered in specific functional domains, whereas population variants were significantly more frequent across non-conserved domains (odds ratio = 18.6; 95% confidence interval = 10.9-34.4; P < 0.001). Pore-loop regions were frequently associated with loss-of-function variants, whereas inactivation sites were associated with gain-of-function (odds ratio = 42.1, 95% confidence interval = 14.5-122.4; P < 0.001), whilst variants occurring in voltage-sensing regions comprised a range of gain- and loss-of-function effects. Our findings suggest that biophysical characterisation of variants in one SCN-gene can predict channel function across different SCN-genes where experimental data are not available. The collected data represent the first gain- versus loss-of-function topological map of SCN proteins indicating shared patterns of biophysical effects aiding variant analysis and guiding precision therapy. We integrated our findings into a free online webtool to facilitate functional sodium channel gene variant interpretation (http://SCN-viewer.broadinstitute.org).

Translating genetic and functional data into clinical practice: a series of 223 families with myotonia.

High-throughput DNA sequencing is increasingly employed to diagnose single gene neurological and neuromuscular disorders. Large volumes of data present new challenges in data interpretation and its useful translation into clinical and genetic counselling for families. Even when a plausible gene is identified with confidence, interpretation of the clinical significance and inheritance pattern of variants can be challenging. We report our approach to evaluating variants in the skeletal muscle chloride channel ClC-1 identified in 223 probands with myotonia congenita as an example of these challenges. Sequencing of CLCN1, the gene that encodes CLC-1, is central to the diagnosis of myotonia congenita. However, interpreting the pathogenicity and inheritance pattern of novel variants is notoriously difficult as both dominant and recessive mutations are reported throughout the channel sequence, ClC-1 structure-function is poorly understood and significant intra- and interfamilial variability in phenotype is reported. Heterologous expression systems to study functional consequences of CIC-1 variants are widely reported to aid the assessment of pathogenicity and inheritance pattern. However, heterogeneity of reported analyses does not allow for the systematic correlation of available functional and genetic data. We report the systematic evaluation of 95 CIC-1 variants in 223 probands, the largest reported patient cohort, in which we apply standardized functional analyses and correlate this with clinical assessment and inheritance pattern. Such correlation is important to determine whether functional data improves the accuracy of variant interpretation and likely mode of inheritance. Our data provide an evidence-based approach that functional characterization of ClC-1 variants improves clinical interpretation of their pathogenicity and inheritance pattern, and serve as reference for 34 previously unreported and 28 previously uncharacterized CLCN1 variants. In addition, we identify novel pathogenic mechanisms and find that variants that alter voltage dependence of activation cluster in the first half of the transmembrane domains and variants that yield no currents cluster in the second half of the transmembrane domain. None of the variants in the intracellular domains were associated with dominant functional features or dominant inheritance pattern of myotonia congenita. Our data help provide an initial estimate of the anticipated inheritance pattern based on the location of a novel variant and shows that systematic functional characterization can significantly refine the assessment of risk of an associated inheritance pattern and consequently the clinical and genetic counselling.

A systems approach delivers a functional microRNA catalog and expanded targets for seizure suppression in temporal lobe epilepsy.

Temporal lobe epilepsy is the most common drug-resistant form of epilepsy in adults. The reorganization of neural networks and the gene expression landscape underlying pathophysiologic network behavior in brain structures such as the hippocampus has been suggested to be controlled, in part, by microRNAs. To systematically assess their significance, we sequenced Argonaute-loaded microRNAs to define functionally engaged microRNAs in the hippocampus of three different animal models in two species and at six time points between the initial precipitating insult through to the establishment of chronic epilepsy. We then selected commonly up-regulated microRNAs for a functional in vivo therapeutic screen using oligonucleotide inhibitors. Argonaute sequencing generated 1.44 billion small RNA reads of which up to 82% were microRNAs, with over 400 unique microRNAs detected per model. Approximately half of the detected microRNAs were dysregulated in each epilepsy model. We prioritized commonly up-regulated microRNAs that were fully conserved in humans and designed custom antisense oligonucleotides for these candidate targets. Antiseizure phenotypes were observed upon knockdown of miR-10a-5p, miR-21a-5p, and miR-142a-5p and electrophysiological analyses indicated broad safety of this approach. Combined inhibition of these three microRNAs reduced spontaneous seizures in epileptic mice. Proteomic data, RNA sequencing, and pathway analysis on predicted and validated targets of these microRNAs implicated derepressed TGF-ß signaling as a shared seizure-modifying mechanism. Correspondingly, inhibition of TGF-ß signaling occluded the antiseizure effects of the antagomirs. Together, these results identify shared, dysregulated, and functionally active microRNAs during the pathogenesis of epilepsy which represent therapeutic antiseizure targets.

A novel synaptopathy-defective synaptic vesicle protein trafficking in the mutant CHMP2B mouse model of frontotemporal dementia.

Mutations in the ESCRT-III subunit CHMP2B cause frontotemporal dementia (FTD) and lead to impaired endolysosomal trafficking and lysosomal storage pathology in neurons. We investigated the effect of mutant CHMP2B on synaptic pathology, as ESCRT function was recently implicated in the degradation of synaptic vesicle (SV) proteins. We report here that expression of C-terminally truncated mutant CHMP2B results in a novel synaptopathy. This unique synaptic pathology is characterised by selective retention of presynaptic SV trafficking proteins in aged mutant CHMP2B transgenic mice, despite significant loss of postsynaptic proteins. Furthermore, ultrastructural analysis of primary cortical cultures from transgenic CHMP2B mice revealed a significant increase in the number of presynaptic endosomes, while neurons expressing mutant CHMP2B display defective SV recycling and alterations to functional SV pools. Therefore, we reveal how mutations in CHMP2B affect specific presynaptic proteins and SV recycling, identifying CHMP2B FTD as a novel synaptopathy. This novel synaptopathic mechanism of impaired SV physiology may be a key early event in multiple forms of FTD, since proteins that mediate the most common genetic forms of FTD all localise at the presynapse.

Recent advances in gene therapy for neurodevelopmental disorders with epilepsy.

Neurodevelopmental disorders can be caused by mutations in neuronal genes fundamental to brain development. These disorders have severe symptoms ranging from intellectually disability, social and cognitive impairments, and a subset are strongly linked with epilepsy. In this review, we focus on those neurodevelopmental disorders that are frequently characterized by the presence of epilepsy (NDD + E). We loosely group the genes linked to NDD + E with different neuronal functions: transcriptional regulation, intrinsic excitability and synaptic transmission. All these genes have in common a pivotal role in defining the brain architecture and function during early development, and when their function is altered, symptoms can present in the first stages of human life. The relationship with epilepsy is complex. In some NDD + E, epilepsy is a comorbidity and in others seizures appear to be the main cause of the pathology, suggesting that either structural changes (NDD) or neuronal communication (E) can lead to these disorders. Furthermore, grouping the genes that cause NDD + E, we review the uses and limitations of current models of the different disorders, and how different gene therapy strategies are being developed to treat them. We highlight where gene replacement may not be a treatment option, and where innovative therapeutic tools, such as CRISPR-based gene editing, and new avenues of delivery are required. In general this group of genetically defined disorders, supported increasing knowledge of the mechanisms leading to neurological dysfunction serve as an excellent collection for illustrating the translational potential of gene therapy, including newly emerging tools.

Progressive myoclonus epilepsy KCNC1 variant causes a developmental dendritopathy.

OBJECTIVE: Mutations in KCNC1 can cause severe neurological dysfunction, including intellectual disability, epilepsy, and ataxia. The Arg320His variant, which occurs in the voltage-sensing domain of the channel, causes a highly penetrant and specific form of progressive myoclonus epilepsy with severe ataxia, designated myoclonus epilepsy and ataxia due to potassium channel mutation (MEAK). KCNC1 encodes the voltage-gated potassium channel KV 3.1, a channel that is important for enabling high-frequency firing in interneurons, raising the possibility that MEAK is associated with reduced interneuronal function. METHODS: To determine how this variant triggers MEAK, we expressed KV 3.1bR320H in cortical interneurons in vitro and investigated the effects on neuronal function and morphology. We also performed electrophysiological recordings of oocytes expressing KV 3.1b to determine whether the mutation introduces gating pore currents. RESULTS: Expression of the KV 3.1bR320H variant profoundly reduced excitability of mature cortical interneurons, and cells expressing these channels were unable to support high-frequency firing. The mutant channel also had an unexpected effect on morphology, severely impairing neurite development and interneuron viability, an effect that could not be rescued by blocking KV 3 channels. Oocyte recordings confirmed that in the adult KV 3.1b isoform, R320H confers a dominant negative loss-of-function effect by slowing channel activation, but does not introduce potentially toxic gating pore currents. SIGNIFICANCE: Overall, our data suggest that, in addition to the regulation of high-frequency firing, KV 3.1 channels play a hitherto unrecognized role in neuronal development. MEAK may be described as a developmental dendritopathy.

In vivo CRISPRa decreases seizures and rescues cognitive deficits in a rodent model of epilepsy.

Epilepsy is a major health burden, calling for new mechanistic insights and therapies. CRISPR-mediated gene editing shows promise to cure genetic pathologies, although hitherto it has mostly been applied ex vivo. Its translational potential for treating non-genetic pathologies is still unexplored. Furthermore, neurological diseases represent an important challenge for the application of CRISPR, because of the need in many cases to manipulate gene function of neurons in situ. A variant of CRISPR, CRISPRa, offers the possibility to modulate the expression of endogenous genes by directly targeting their promoters. We asked if this strategy can effectively treat acquired focal epilepsy, focusing on ion channels because their manipulation is known be effective in changing network hyperactivity and hypersynchronziation. We applied a doxycycline-inducible CRISPRa technology to increase the expression of the potassium channel gene Kcna1 (encoding Kv1.1) in mouse hippocampal excitatory neurons. CRISPRa-mediated Kv1.1 upregulation led to a substantial decrease in neuronal excitability. Continuous video-EEG telemetry showed that AAV9-mediated delivery of CRISPRa, upon doxycycline administration, decreased spontaneous generalized tonic-clonic seizures in a model of temporal lobe epilepsy, and rescued cognitive impairment and transcriptomic alterations associated with chronic epilepsy. The focal treatment minimizes concerns about off-target effects in other organs and brain areas. This study provides the proof-of-principle for a translational CRISPR-based approach to treat neurological diseases characterized by abnormal circuit excitability.

dCas9-Based Scn1a Gene Activation Restores Inhibitory Interneuron Excitability and Attenuates Seizures in Dravet Syndrome Mice.

Dravet syndrome (DS) is a severe epileptic encephalopathy caused mainly by heterozygous loss-of-function mutations of the SCN1A gene, indicating haploinsufficiency as the pathogenic mechanism. Here we tested whether catalytically dead Cas9 (dCas9)-mediated Scn1a gene activation can rescue Scn1a haploinsufficiency in a mouse DS model and restore physiological levels of its gene product, the Nav1.1 voltage-gated sodium channel. We screened single guide RNAs (sgRNAs) for their ability to stimulate Scn1a transcription in association with the dCas9 activation system. We identified a specific sgRNA that increases Scn1a gene expression levels in cell lines and primary neurons with high specificity. Nav1.1 protein levels were augmented, as was the ability of wild-type immature GABAergic interneurons to fire action potentials. A similar enhancement of Scn1a transcription was achieved in mature DS interneurons, rescuing their ability to fire. To test the therapeutic potential of this approach, we delivered the Scn1a-dCas9 activation system to DS pups using adeno-associated viruses. Parvalbumin interneurons recovered their firing ability, and febrile seizures were significantly attenuated. Our results pave the way for exploiting dCas9-based gene activation as an effective and targeted approach to DS and other disorders resulting from altered gene dosage.

Spider toxin inhibits gating pore currents underlying periodic paralysis.

Gating pore currents through the voltage-sensing domains (VSDs) of the skeletal muscle voltage-gated sodium channel NaV1.4 underlie hypokalemic periodic paralysis (HypoPP) type 2. Gating modifier toxins target ion channels by modifying the function of the VSDs. We tested the hypothesis that these toxins could function as blockers of the pathogenic gating pore currents. We report that a crab spider toxin Hm-3 from Heriaeus melloteei can inhibit gating pore currents due to mutations affecting the second arginine residue in the S4 helix of VSD-I that we have found in patients with HypoPP and describe here. NMR studies show that Hm-3 partitions into micelles through a hydrophobic cluster formed by aromatic residues and reveal complex formation with VSD-I through electrostatic and hydrophobic interactions with the S3b helix and the S3-S4 extracellular loop. Our data identify VSD-I as a specific binding site for neurotoxins on sodium channels. Gating modifier toxins may constitute useful hits for the treatment of HypoPP.

Epilepsy Gene Therapy Using an Engineered Potassium Channel.

Refractory focal epilepsy is a devastating disease for which there is frequently no effective treatment. Gene therapy represents a promising alternative, but treating epilepsy in this way involves irreversible changes to brain tissue, so vector design must be carefully optimized to guarantee safety without compromising efficacy. We set out to develop an epilepsy gene therapy vector optimized for clinical translation. The gene encoding the voltage-gated potassium channel Kv1.1, KCNA1, was codon optimized for human expression and mutated to accelerate the recovery of the channels from inactivation. For improved safety, this engineered potassium channel (EKC) gene was packaged into a nonintegrating lentiviral vector under the control of a cell type-specific CAMK2A promoter. In a blinded, randomized, placebo-controlled preclinical trial, the EKC lentivector robustly reduced seizure frequency in a male rat model of focal neocortical epilepsy characterized by discrete spontaneous seizures. When packaged into an adeno-associated viral vector (AAV2/9), the EKC gene was also effective at suppressing seizures in a male rat model of temporal lobe epilepsy. This demonstration of efficacy in a clinically relevant setting, combined with the improved safety conferred by cell type-specific expression and integration-deficient delivery, identify EKC gene therapy as being ready for clinical translation in the treatment of refractory focal epilepsy.SIGNIFICANCE STATEMENT Pharmacoresistant epilepsy affects up to 0.3% of the population. Although epilepsy surgery can be effective, it is limited by risks to normal brain function. We have developed a gene therapy that builds on a mechanistic understanding of altered neuronal and circuit excitability in cortical epilepsy. The potassium channel gene KCNA1 was mutated to bypass post-transcriptional editing and was packaged in a nonintegrating lentivector to reduce the risk of insertional mutagenesis. A randomized, blinded preclinical study demonstrated therapeutic effectiveness in a rodent model of focal neocortical epilepsy. Adeno-associated viral delivery of the channel to both hippocampi was also effective in a model of temporal lobe epilepsy. These results support clinical translation to address a major unmet need.

Antagonizing Increased miR-135a Levels at the Chronic Stage of Experimental TLE Reduces Spontaneous Recurrent Seizures.

Mesial temporal lobe epilepsy (mTLE) is a chronic neurological disease characterized by recurrent seizures. The antiepileptic drugs currently available to treat mTLE are ineffective in one-third of patients and lack disease-modifying effects. miRNAs, a class of small noncoding RNAs which control gene expression at the post-transcriptional level, play a key role in the pathogenesis of mTLE and other epilepsies. Although manipulation of miRNAs at acute stages has been reported to reduce subsequent spontaneous seizures, it is uncertain whether targeting miRNAs at chronic stages of mTLE can also reduce seizures. Furthermore, the functional role and downstream targets of most epilepsy-associated miRNAs remain poorly understood. Here, we show that miR-135a is selectively upregulated within neurons in epileptic brain and report that targeting miR-135a in vivo using antagomirs after onset of spontaneous recurrent seizures can reduce seizure activity at the chronic stage of experimental mTLE in male mice. Further, by using an unbiased approach combining immunoprecipitation and RNA sequencing, we identify several novel neuronal targets of miR-135a, including Mef2a Mef2 proteins are key regulators of excitatory synapse density. Mef2a and miR-135a show reciprocal expression regulation in human (of both sexes) and experimental TLE, and miR-135a regulates dendritic spine number and type through Mef2. Together, our data show that miR-135a is target for reducing seizure activity in chronic epilepsy, and that deregulation of miR-135a in epilepsy may alter Mef2a expression and thereby affect synaptic function and plasticity.SIGNIFICANCE STATEMENT miRNAs are post-transcriptional regulators of gene expression with roles in the pathogenesis of epilepsy. However, the precise mechanism of action and therapeutic potential of most epilepsy-associated miRNAs remain poorly understood. Our study reveals dramatic upregulation of the key neuronal miRNA miR-135a in both experimental and human mesial temporal lobe epilepsy. Silencing miR-135a in experimental temporal lobe epilepsy reduces seizure activity at the spontaneous recurrent seizure stage. These data support the exciting possibility that miRNAs can be targeted to combat seizures after spontaneous seizure activity has been established. Further, by using unbiased approaches novel neuronal targets of miR-135a, including members of the Mef2 protein family, are identified that begin to explain how deregulation of miR-135a may contribute to epilepsy.

SCN1A variants from bench to bedside-improved clinical prediction from functional characterization.

Variants in the SCN1A gene are associated with a wide range of disorders including genetic epilepsy with febrile seizures plus (GEFS+), familial hemiplegic migraine (FHM), and the severe childhood epilepsy Dravet syndrome (DS). Predicting disease outcomes based on variant type remains challenging. Despite thousands of SCN1A variants being reported, only a minority has been functionally assessed. We review the functional SCN1A work performed to date, critically appraise electrophysiological measurements, compare this to in silico predictions, and relate our findings to the clinical phenotype. Our results show, regardless of the underlying phenotype, that conventional in silico software correctly predicted benign from pathogenic variants in nearly 90%, however was unable to differentiate within the disease spectrum (DS vs. GEFS+ vs. FHM). In contrast, patch-clamp data from mammalian expression systems revealed functional differences among missense variants allowing discrimination between disease severities. Those presenting with milder phenotypes retained a degree of channel function measured as residual whole-cell current, whereas those without any whole-cell current were often associated with DS (p = .024). These findings demonstrate that electrophysiological data from mammalian expression systems can serve as useful disease biomarker when evaluating SCN1A variants, particularly in view of new and emerging treatment options in DS.

Biological concepts in human sodium channel epilepsies and their relevance in clinical practice.

OBJECTIVE: Voltage-gated sodium channels (SCNs) share similar amino acid sequence, structure, and function. Genetic variants in the four human brain-expressed SCN genes SCN1A/2A/3A/8A have been associated with heterogeneous epilepsy phenotypes and neurodevelopmental disorders. To better understand the biology of seizure susceptibility in SCN-related epilepsies, our aim was to determine similarities and differences between sodium channel disorders, allowing us to develop a broader perspective on precision treatment than on an individual gene level alone. METHODS: We analyzed genotype-phenotype correlations in large SCN-patient cohorts and applied variant constraint analysis to identify severe sodium channel disease. We examined temporal patterns of human SCN expression and correlated functional data from in vitro studies with clinical phenotypes across different sodium channel disorders. RESULTS: Comparing 865 epilepsy patients (504 SCN1A, 140 SCN2A, 171 SCN8A, four SCN3A, 46 copy number variation [CNV] cases) and analysis of 114 functional studies allowed us to identify common patterns of presentation. All four epilepsy-associated SCN genes demonstrated significant constraint in both protein truncating and missense variation when compared to other SCN genes. We observed that age at seizure onset is related to SCN gene expression over time. Individuals with gain-of-function SCN2A/3A/8A missense variants or CNV duplications share similar characteristics, most frequently present with early onset epilepsy (<3 months), and demonstrate good response to sodium channel blockers (SCBs). Direct comparison of corresponding SCN variants across different SCN subtypes illustrates that the functional effects of variants in corresponding channel locations are similar; however, their clinical manifestation differs, depending on their role in different types of neurons in which they are expressed. SIGNIFICANCE: Variant function and location within one channel can serve as a surrogate for variant effects across related sodium channels. Taking a broader view on precision treatment suggests that in those patients with a suspected underlying genetic epilepsy presenting with neonatal or early onset seizures (<3 months), SCBs should be considered.

Activity Clamp Provides Insights into Paradoxical Effects of the Anti-Seizure Drug Carbamazepine.

A major challenge in experimental epilepsy research is to reconcile the effects of anti-epileptic drugs (AEDs) on individual neurons with their network-level actions. Highlighting this difficulty, it is unclear why carbamazepine (CBZ), a frontline AED with a known molecular mechanism, has been reported to increase epileptiform activity in several clinical and experimental studies. We confirmed in an in vitro mouse model (in both sexes) that the frequency of interictal bursts increased after CBZ perfusion. To address the underlying mechanisms, we developed a method, activity clamp, to distinguish the response of individual neurons from network-level actions of CBZ. We first recorded barrages of synaptic conductances from neurons during epileptiform activity and then replayed them in pharmacologically isolated neurons under control conditions and in the presence of CBZ. CBZ consistently decreased the reliability of the second action potential in each burst of activity. Conventional current-clamp recordings using excitatory ramp or square-step current injections failed to reveal this effect. Network modeling showed that a CBZ-induced decrease of neuron recruitment during epileptic bursts can lead to an increase in burst frequency at the network level by reducing the refractoriness of excitatory transmission. By combining activity clamp with computer simulations, the present study provides a potential explanation for the paradoxical effects of CBZ on epileptiform activity.SIGNIFICANCE STATEMENT The effects of anti-epileptic drugs on individual neurons are difficult to separate from their network-level actions. Although carbamazepine (CBZ) has a known anti-epileptic mechanism, paradoxically, it has also been reported to increase epileptiform activity in clinical and experimental studies. To investigate this paradox during realistic neuronal epileptiform activity, we developed a method, activity clamp, to distinguish the effects of CBZ on individual neurons from network-level actions. We demonstrate that CBZ consistently decreases the reliability of the second action potential in each burst of epileptiform activity. Network modeling shows that this effect on individual neuronal responses could explain the paradoxical effect of CBZ at the network level.

Spared CA1 pyramidal neuron function and hippocampal performance following antisense knockdown of microRNA-134.

OBJECTIVE: Inhibition of microRNA-134 by an oligonucleotide antagomir (ant-134) has been shown to produce powerful antiseizure effects in multiple models of epilepsy. However, to successfully translate the treatment to the clinic, it is important to assess what potential adverse effects it may have on naive brain tissue. METHODS: To investigate this, adult male Sprague-Dawley rats were treated with either ant-134 or a scrambled control sequence. Animals were later assessed for spatial navigation, before ex vivo slices were taken to assess the effects of microRNA-134 knockdown on well-defined measures of intrinsic and synaptic properties. RESULTS: Hippocampal field potential recordings determined that silencing of microRNA-134 by ant-134 injection was associated with a reduction in epileptiform activity following application of 9 mmol/L K+ . Nevertheless, rats performed normally in the novel object location test. Action potential waveforms and miniature excitatory synaptic currents recorded in CA1 pyramidal neurons were unaffected by ant-134. SIGNIFICANCE: These results demonstrate that ant-134 confers a seizure-protective effect without obvious interference with hippocampal neuronal properties or network function. These findings support further development of this novel approach to epilepsy treatment.

Personalized translational epilepsy research - Novel approaches and future perspectives: Part II: Experimental and translational approaches.

Despite the availability of more than 15 new "antiepileptic drugs", the proportion of patients with pharmacoresistant epilepsy has remained constant at about 20-30%. Furthermore, no disease-modifying treatments shown to prevent the development of epilepsy following an initial precipitating brain injury or to reverse established epilepsy have been identified to date. This is likely in part due to the polyetiologic nature of epilepsy, which in turn requires personalized medicine approaches. Recent advances in imaging, pathology, genetics, and epigenetics have led to new pathophysiological concepts and the identification of monogenic causes of epilepsy. In the context of these advances, the First International Symposium on Personalized Translational Epilepsy Research (1st ISymPTER) was held in Frankfurt on September 8, 2016, to discuss novel approaches and future perspectives for personalized translational research. These included new developments and ideas in a range of experimental and clinical areas such as deep phenotyping, quantitative brain imaging, EEG/MEG-based analysis of network dysfunction, tissue-based translational studies, innate immunity mechanisms, microRNA as treatment targets, functional characterization of genetic variants in human cell models and rodent organotypic slice cultures, personalized treatment approaches for monogenic epilepsies, blood-brain barrier dysfunction, therapeutic focal tissue modification, computational modeling for target and biomarker identification, and cost analysis in (monogenic) disease and its treatment. This report on the meeting proceedings is aimed at stimulating much needed investments of time and resources in personalized translational epilepsy research. This Part II includes the experimental and translational approaches and a discussion of the future perspectives, while the diagnostic methods, EEG network analysis, biomarkers, and personalized treatment approaches were addressed in Part I [1].

Personalized translational epilepsy research - Novel approaches and future perspectives: Part I: Clinical and network analysis approaches.

Despite the availability of more than 15 new "antiepileptic drugs", the proportion of patients with pharmacoresistant epilepsy has remained constant at about 20-30%. Furthermore, no disease-modifying treatments shown to prevent the development of epilepsy following an initial precipitating brain injury or to reverse established epilepsy have been identified to date. This is likely in part due to the polyetiologic nature of epilepsy, which in turn requires personalized medicine approaches. Recent advances in imaging, pathology, genetics and epigenetics have led to new pathophysiological concepts and the identification of monogenic causes of epilepsy. In the context of these advances, the First International Symposium on Personalized Translational Epilepsy Research (1st ISymPTER) was held in Frankfurt on September 8, 2016, to discuss novel approaches and future perspectives for personalized translational research. These included new developments and ideas in a range of experimental and clinical areas such as deep phenotyping, quantitative brain imaging, EEG/MEG-based analysis of network dysfunction, tissue-based translational studies, innate immunity mechanisms, microRNA as treatment targets, functional characterization of genetic variants in human cell models and rodent organotypic slice cultures, personalized treatment approaches for monogenic epilepsies, blood-brain barrier dysfunction, therapeutic focal tissue modification, computational modeling for target and biomarker identification, and cost analysis in (monogenic) disease and its treatment. This report on the meeting proceedings is aimed at stimulating much needed investments of time and resources in personalized translational epilepsy research. Part I includes the clinical phenotyping and diagnostic methods, EEG network-analysis, biomarkers, and personalized treatment approaches. In Part II, experimental and translational approaches will be discussed (Bauer et al., 2017) [1].

Lentiviral expression of GAD67 and CCK promoter-driven opsins to target interneurons in vitro and in vivo.

BACKGROUND: The ability to manipulate the activity of interneurons with optogenetic tools offers the possibility of interfering with diseases caused by altered neuronal inhibition and synchrony, including epilepsy and schizophrenia. To develop vectors for therapeutic approaches, targeting optogenetic constructs to interneurons is therefore a key requirement. We investigated whether the interneuron-specific promoters glutamic acid decarboxylase (GAD)67 and cholecystokinin (CCK) allowed targeted lentiviral delivery of opsins to interneurons as a whole, or specifically CCK+ interneurons. METHODS: We generated lentiviral (LV) plasmids encoding channelrhodopsin (ChR2) and halorhodopsin (NpHR) tagged with fluorophores and driven by GAD67 or CCK promoters. Adeno-associated virus (AAV) and LV vectors carrying opsins driven by pyramidal cell promoters were used as controls. We transduced neuronal cultures and rodent brain in vivo, immunostained specimens 6-8 weeks after in vivo injection and 7-14 days after in vitro transduction, and evaluated volume and specificity of expression by confocal microscopy. RESULTS: In vitro, 90% (19/21) of LV-CCK-NpHR2.0-EYFP expressing neurons were CCK+. In vivo, LV-GAD67-ChR2-mCherry was expressed in 2.6% (5/193), LV-GAD67-NpHR2.0-EYFP in approximately 15% (43/279) and LV-CCK-NpHR2.0-EYFP in 47% (9/19) of hippocampal GABA+ interneurons. GAD67 vectors expressed in larger volumes than CCK-driven constructs. AAV vector controls achieved the largest expression volumes. CONCLUSIONS: LV-CCK-NpHR2.0-EYFP may be useful for targeting CCK+ interneurons in culture. GAD67/CCK-driven lentiviral constructs are expressed in vivo, although expression is not specific for interneurons. Overall, expression levels are low compared to opsins driven by pyramidal cell promoters. A better understanding of GAD67 and CCK promoter structure or alternative techniques is required to reliably target opsins to interneurons using viral vectors.