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.

Focal cortical dysplasia: a practical guide for neurologists.

Focal cortical dysplasia (FCD) is a malformation of cortical development characterised by disruption of cortical cytoarchitecture. Classification of FCDs subtypes has initially been based on correlation of the histopathology with relevant clinical, electroencephalographic and neuroimaging features. A recently proposed classification update recommends a multilayered, genotype-phenotype approach, integrating findings from histopathology, genetic analysis of resected tissue and presurgical MRI. FCDs are caused either by single somatic activating mutations in MTOR pathway genes or by double-hit inactivating mutations with a constitutional and a somatic loss-of-function mutation in repressors of the signalling pathway. Mild malformation with oligodendroglial hyperplasia in epilepsy is caused by somatic pathogenic SLC35A2 mutations. FCDs most often present with drug-resistant focal epilepsy or epileptic encephalopathy. Most patients respond to surgical treatment. The use of mechanistic target of rapamycin inhibitors may complement the surgical approach. Treatment approaches and outcomes have improved with advances in neuroimaging, neurophysiology and genetics, although predictors of treatment response have only been determined in part.

Precision Therapy for Epilepsy Related to Brain Malformations.

Malformations of cortical development (MCDs) represent a range of neurodevelopmental disorders that are collectively common causes of developmental delay and epilepsy, especially refractory childhood epilepsy. Initial treatment with antiseizure medications is empiric, and consideration of surgery is the standard of care for eligible patients with medically refractory epilepsy. In the past decade, advances in next generation sequencing technologies have accelerated progress in understanding the genetic etiologies of MCDs, and precision therapies for focal MCDs are emerging. Notably, mutations that lead to abnormal activation of the mammalian target of rapamycin (mTOR) pathway, which provides critical control of cell growth and proliferation, have emerged as a common cause of malformations. These include tuberous sclerosis complex (TSC), hemimegalencephaly (HME), and some types of focal cortical dysplasia (FCD). TSC currently represents the best example for the pathway from gene discovery to relatively safe and efficacious targeted therapy for epilepsy related to MCDs. Based on extensive pre-clinical and clinical data, the mTOR inhibitor everolimus is currently approved for the treatment of focal refractory seizures in patients with TSC. Although clinical studies are just emerging for FCD and HME, we believe the next decade will bring significant advancements in precision therapies for epilepsy related to these and other MCDs.

Focal cortical dysplasia: an update on diagnosis and treatment.

INTRODUCTION: Focal cortical dysplasias (FCDs) represent the most common etiology in pediatric drug-resistant focal epilepsies undergoing surgical treatment. The localization, extent and histopathological features of FCDs are considerably variable. Somatic mosaic mutations of genes that encode proteins in the PI3K-AKTmTOR pathway, which also includes the tuberous sclerosis associated genes TSC1 and TSC2, have been implicated in FCD type II in a substantial subset of patients. Surgery is the principal therapeutic option for FCD-related epilepsy. Advanced neurophysiological and neuroimaging techniques have improved surgical outcome and reduced the risk of postsurgical deficits. Pharmacological MTOR inhibitors are being tested in clinical trials and might represent an example of personalized treatment of epilepsy based on the known mechanisms of disease, used alone or in combination with surgery. AREAS COVERED: This review will critically analyze the advances in the diagnosis and treatment of FCDs, with a special focus on the novel therapeutic options prompted by a better understanding of their pathophysiology. EXPERT OPINION: Focal cortical dysplasia is a main cause of drug-resistant epilepsy, especially in children. Novel, personalized approaches are needed to more effectively treat FCD-related epilepsy and its cognitive consequences.

Definitions and classification of malformations of cortical development: practical guidelines.

Malformations of cortical development are a group of rare disorders commonly manifesting with developmental delay, cerebral palsy or seizures. The neurological outcome is extremely variable depending on the type, extent and severity of the malformation and the involved genetic pathways of brain development. Neuroimaging plays an essential role in the diagnosis of these malformations, but several issues regarding malformations of cortical development definitions and classification remain unclear. The purpose of this consensus statement is to provide standardized malformations of cortical development terminology and classification for neuroradiological pattern interpretation. A committee of international experts in paediatric neuroradiology prepared systematic literature reviews and formulated neuroimaging recommendations in collaboration with geneticists, paediatric neurologists and pathologists during consensus meetings in the context of the European Network Neuro-MIG initiative on Brain Malformations (https://www.neuro-mig.org/). Malformations of cortical development neuroimaging features and practical recommendations are provided to aid both expert and non-expert radiologists and neurologists who may encounter patients with malformations of cortical development in their practice, with the aim of improving malformations of cortical development diagnosis and imaging interpretation worldwide.

Subacute neocortical stimulation (SNCS) and its effects on epileptic activity in adults and children diagnosed with focal cortical dysplasia (FCD).

BACKGROUND: Chronic intracranial electrical stimulation is now widely used as treatment for drug resistant epilepsy. Subacute neocortical stimulation (SNCS) can also be performed during EEG recordings with intracranial electrodes (iEEG), but its diagnostic value remains largely unknown. METHODS: We assessed the effects of SNCS on the frequency of seizures and epileptiform discharges (EDs) during 290 h of iEEG- from 12 patients (6 adults, 6 children) with epilepsy secondary to focal cortical dysplasia (FCD). RESULTS: In 9/12 patients, SNCS periods showed decreased seizure-frequency (Median -73 %, p = 0.0093). At baseline, incidence of EDs were correlated with seizure-frequency (Spearman r = 0.59). However, this correlation disappeared during SNCS and a significant change in the incidence of EDs was observed. In addition, there was a trend towards greater reduction in seizure-frequency during SNCS in patients who underwent surgery. CONCLUSION: In summary, SNCS can reduce seizure-frequency and changes ED-frequency. The variability in ED changes may be explained by different effects of SNCS depending on electrode location. The magnitude of seizure reduction during SNCS suggests that this technique could contribute to preoperative assessment in epilepsy surgery.

Low Frequency Microstimulation Is Locally Excitatory in Patients With Epilepsy.

Deep brain stimulation (DBS) could become a palliative treatment for patients with drug-resistant epilepsy for which surgery cannot be proposed. The objective of this study was to perform microstimulation to measure the effects of DBS in epilepsy locally at the level of a few neurons, with microelectrode recordings, for the first time in patients with epilepsy. Microelectrode recordings were performed before, during and after microstimulation in nine patients with refractory epilepsy. Neuronal spikes were successfully extracted from multi-unit recordings with clustering in six out of seven patients during hippocampal and in one out of two patients during cortical dysplasia microstimulation (1 Hz, charge-balanced biphasic waveform, 60 µs/ph, 25 µA). The firing rates increased in four out of the six periods of microstimulation that could be analyzed. The firing rates were found higher than before microstimulation in all eight periods with increases reaching significance in six out of eight periods. Low-frequency microstimulation was hence sufficient to induce neuronal excitation lasting beyond the stimulation period. No inhibition was observed. This report presents the first evidence that microstimulation performed in epileptic patients produced locally neuronal excitation. Hence neuronal excitation is shown here as the local mechanism of action of DBS. This local excitation is in agreement with epileptogenic effects of low-frequency hippocampal macrostimulation.

Temporal requirement of dystroglycan glycosylation during brain development and rescue of severe cortical dysplasia via gene delivery in the fetal stage.

Congenital muscular dystrophies (CMDs) are characterized by progressive weakness and degeneration of skeletal muscle. In several forms of CMD, abnormal glycosylation of α-dystroglycan (α-DG) results in conditions collectively known as dystroglycanopathies, which are associated with central nervous system involvement. We recently demonstrated that fukutin, the gene responsible for Fukuyama congenital muscular dystrophy, encodes the ribitol-phosphate transferase essential for dystroglycan function. Brain pathology in patients with dystroglycanopathy typically includes cobblestone lissencephaly, mental retardation, and refractory epilepsy; however, some patients exhibit average intelligence, with few or almost no structural defects. Currently, there is no effective treatment for dystroglycanopathy, and the mechanisms underlying the generation of this broad clinical spectrum remain unknown. Here, we analysed four distinct mouse models of dystroglycanopathy: two brain-selective fukutin conditional knockout strains (neuronal stem cell-selective Nestin-fukutin-cKO and forebrain-selective Emx1-fukutin-cKO), a FukutinHp strain with the founder retrotransposal insertion in the fukutin gene, and a spontaneous Large-mutant Largemyd strain. These models exhibit variations in the severity of brain pathology, replicating the clinical heterogeneity of dystroglycanopathy. Immunofluorescence analysis of the developing cortex suggested that residual glycosylation of α-DG at embryonic day 13.5 (E13.5), when cortical dysplasia is not yet apparent, may contribute to subsequent phenotypic heterogeneity. Surprisingly, delivery of fukutin or Large into the brains of mice at E12.5 prevented severe brain malformation in Emx1-fukutin-cKO and Largemyd/myd mice, respectively. These findings indicate that spatiotemporal persistence of functionally glycosylated α-DG may be crucial for brain development and modulation of glycosylation during the fetal stage could be a potential therapeutic strategy for dystroglycanopathy.

[How must we manage epileptic encephalopathies in infants? Conclusions]. / Como debemos abordar las encefalopatias epilepticas del lactante? Conclusiones.

Epileptic encephalopathies are defined as epileptic syndromes in which the epileptic activity per se (in the form of frequent seizures or the presence of interictal epileptiform activity) contributes to a cognitive and behavioural disorder that is more important than could be expected from the causation of the disorder. Their aetiological diagnosis is fundamental to allow an early treatment to be established. We propose a diagnostic algorithm for patients with epileptic encephalopathy with onset during the first year of life, which includes management coordinated with electroencephalographic studies, neuroimaging, and screening for metabolic and genetic disorders.

TITLE: Como debemos abordar las encefalopatias epilepticas del lactante? Conclusiones.Las encefalopatias epilepticas se definen como los sindromes epilepticos en los que la actividad epileptica per se (en forma de crisis frecuentes o presencia de actividad epileptiforme intercritica) contribuye a un trastorno cognitivo y conductual mayor de lo esperable por la etiologia del trastorno. Es fundamental un diagnostico etiologico de ellas que nos permita un tratamiento precoz. Proponemos un algoritmo diagnostico para los pacientes con encefalopatia epileptica de inicio en el primer año de vida, en el que se incluye el abordaje coordinado de estudios electroencefalograficos, neuroimagen, y cribado de trastornos metabolicos y geneticos.

Focal Cortical Dysplasia: Gene Mutations, Cell Signaling, and Therapeutic Implications.

Focal cortical dysplasias (FCDs) are malformations of cortical development (MCDs) that are highly associated with medication-resistant epilepsy and are the most common cause of neocortical epilepsy in children. FCDs are a heterogeneous group of developmental disorders caused by germline or somatic mutations that occur in genes regulating the PI3K/Akt/mTOR pathway-a key pathway in neuronal growth and migration. Accordingly, FCDs are characterized by abnormal cortical lamination, cell morphology (e.g., cytomegaly), and cellular polarity. In some FCD subtypes, balloon cells express proteins typically seen in neuroglial progenitor cells. Because recurrent intractable seizures are a common feature of FCDs, epileptogenic electrophysiological properties are also observed in addition to local inflammation. Here, we will summarize the current literature regarding FCDs, addressing the current classification system, histopathology, molecular genetics, electrophysiology, and transcriptome and cell signaling changes.

Recurrent de novo BICD2 mutation associated with arthrogryposis multiplex congenita and bilateral perisylvian polymicrogyria.

Autosomal dominantly inherited mutations of BICD2 are associated with congenital-onset spinal muscular atrophy characterised by lower limb predominance. A few cases have also showed upper motor neuron pathology, including presenting with features resembling hereditary spastic paraplegia. The age-of-onset for the published families is usually at birth but also included cases with childhood- and adult-onset disease. In this report we described two isolated probands that presented in utero with features associated with reduced fetal movements. Both cases were diagnosed at birth with arthrogryposis multiplex congenita (AMC) and hypotonia. Other variable features included congenital fractures, hip dislocation, micrognathia, respiratory insufficiency, microcephaly and bilateral perisylvian polymicrogyria. Patient 1 is 4 years of age and stable, but shows significant motor developmental delay and delayed speech. Patient 2 passed away at 7 weeks of age. Through next generation sequencing we identified the same missense substitution in BICD2 (p.Arg694Cys) in both probands. Sanger sequencing showed that in both cases the mutation arose de novo. The in utero onset in both cases suggests that the p.Arg694Cys substitution may have a more deleterious effect on BICD2 function than previously described mutations. Our results broaden the phenotypes associated with BICD2 mutations to include AMC and cortical malformations and therefore to a similar phenotypic spectrum to that associated with its binding partner DYNC1H1.

Hyaline protoplasmic astrocytopathy in the setting of tuberous sclerosis.

Hyaline protoplasmic astrocytopathy is a rare disorder marked by an accumulation of protein material in the cytoplasm of astrocytic cells, mostly in the cortex. The finding has been described in Aicardi syndrome (agenesis of the corpus callosum, chorioretinal lacunae and infantile spasms) as well as in patients with pharmacoresistant epilepsy and in association with focal cortical dysplasia, polymicrogyria and nodular heterotopia. This report describes the first case of this entity described in a patient with tuberous sclerosis. The patient was a 3-year-old boy who presented at age 2months with medically intractable seizures. Has mother has a tuberous sclerosis 2 (TSC 2) gene abnormality and a diagnosis of tuberous sclerosis. On imaging, he was noted to have multiple lesions in the left parietal and temporal lobes consistent with focal cortical dysplasia and a subependymal nodule. He additionally had two hypopigmented lesions on the skin. He underwent resection of the left parietal lobe 32months after seizure onset. Histopathologic examination showed eosinophilic cytoplasmic inclusions within astrocytes in the cortex and superficial white matter focally accompanied by a disordered cortical architecture with dysmorphic neurons and balloon cells, consistent with focal cortical dysplasia classified as type IIb according to International League Against Epilepsy classification criteria (ILAE type IIb). At the time of most recent follow-up, 93months postoperatively, he is still experiencing seizures with overall worthwhile improvement while on seizure medication.

Lesion guided stereotactic radiofrequency thermocoagulation for palliative, in selected cases curative epilepsy surgery.

INTRODUCTION: Resective epilepsy surgery is an established treatment option in patients with pharmacoresistant, lesion related epilepsy. Yet, if the presurgical work-up proves multi-focal organization of the epileptogenic zone, or the area of intended resection is close to eloquent brain areas, patients may decide against resections because of an unfavorable risk-benefit-ratio. We assess if lesion guided cortical stereotactic radiofrequency thermocoagulation (L-RFTC) is a potential surgical alternative in these patients. METHODS: We performed seven procedures of L-RFTC. Three patients had monofocal epilepsy arising close to eloquent structures; in four, invasive pre-surgical workup documented monofocal seizure onset but strong interictal epileptic activity also independent and distant from the seizure onset zone. L-RFTC was restricted to the lesional area (=seizure onset site). RESULTS: 12 to 37 months after RFTC worthwhile seizure improvement was achieved in 6 patients. One patient became seizure free following complete coagulation of a focal cortical dysplasia, two had had 1-2 auras under tapered but not under continued medication. In one patient only subclinical seizures persisted. In one patient hypermotor seizures were transformed into milder short tonic seizures and another one had a seizure reduction by 50%. Only one patient did not profit at all. One patient developed a persisting neurological deficit. SIGNIFICANCE: In patients with complex epileptogenic zones L-RFTC can lead to worthwhile seizure reduction. This qualifies this procedure as a palliative surgical technique with potential good risk-benefit ratio. In patients with small focal cortical dysplasias L-RFTC may even allow minimal-invasive surgery with curative intention.

Homing of allogeneic nestin-positive hair follicle-associated pluripotent stem cells after maternal transplantation in experimental model of cortical dysplasia.

An embryo has the capability to accept allo- or xeno-geneic cells, which probably makes it an ideal candidate for stem cell transplantation of various cerebral cortex abnormalities, such as cortical dysplasia. The aim of this study was to determine hair follicle-associated pluripotent (HAP) stem cells homing into various organs of mother and fetus. Cells were obtained, analyzed for immunophenotypic features, and then labelled with CM-Dil; nestin(+)HAP stem cells or media phosphate-buffered saline (PBS) were intravenously delivered on day 16 of gestation in BALB/c mice, which intraperitoneally received methylazoxymethanol (MAM) one day in advance, and homing was assessed at 24 h after cell injection. Flow cytometry and immunocytochemistry manifested positive expression of nestin in HAP stem cells. For both mother and fetus, brain, lungs, liver, and spleen were the host organs for cell implants. For the brain, the figure was considerably higher in fetus, 4.05 ± 0.5% (p ≤ 0.05 vs. mother). MAM-injected mice had a downward trend for SDF-1α and CXCR4 (p ≤ 0.05 vs. control), but HAP stem cells group showed an upward trend for CXCR4 (p ≤ 0.05 vs. MAM). We conclude the HAP stem cells show homing potential in experimental cortical dysplasia, which may permit these cells to be a target in future work on prenatal therapy of neural disorders.

Diagnostic methods and treatment options for focal cortical dysplasia.

Our inability to adequately treat many patients with refractory epilepsy caused by focal cortical dysplasia (FCD), surgical inaccessibility and failures are significant clinical drawbacks. The targeting of physiologic features of epileptogenesis in FCD and colocalizing functionality has enhanced completeness of surgical resection, the main determinant of outcome. Electroencephalography (EEG)-functional magnetic resonance imaging (fMRI) and magnetoencephalography are helpful in guiding electrode implantation and surgical treatment, and high-frequency oscillations help defining the extent of the epileptogenic dysplasia. Ultra high-field MRI has a role in understanding the laminar organization of the cortex, and fluorodeoxyglucose-positron emission tomography (FDG-PET) is highly sensitive for detecting FCD in MRI-negative cases. Multimodal imaging is clinically valuable, either by improving the rate of postoperative seizure freedom or by reducing postoperative deficits. However, there is no level 1 evidence that it improves outcomes. Proof for a specific effect of antiepileptic drugs (AEDs) in FCD is lacking. Pathogenic mutations recently described in mammalian target of rapamycin (mTOR) genes in FCD have yielded important insights into novel treatment options with mTOR inhibitors, which might represent an example of personalized treatment of epilepsy based on the known mechanisms of disease. The ketogenic diet (KD) has been demonstrated to be particularly effective in children with epilepsy caused by structural abnormalities, especially FCD. It attenuates epigenetic chromatin modifications, a master regulator for gene expression and functional adaptation of the cell, thereby modifying disease progression. This could imply lasting benefit of dietary manipulation. Neurostimulation techniques have produced variable clinical outcomes in FCD. In widespread dysplasias, vagus nerve stimulation (VNS) has achieved responder rates >50%; however, the efficacy of noninvasive cranial nerve stimulation modalities such as transcutaneous VNS (tVNS) and noninvasive (nVNS) requires further study. Although review of current strategies underscores the serious shortcomings of treatment-resistant cases, initial evidence from novel approaches suggests that future success is possible.

Genomic variants and variations in malformations of cortical development.

Malformations of cortical development (MCDs) are a common cause of neurodevelopmental delay and epilepsy and are caused by disruptions in the normal development of the cerebral cortex. Several causative genes have been identified in patients with MCD. There is increasing evidence of role of de novo mutations, including those occurring post fertilization, in MCD. These somatic mutations may not be detectable by traditional methods of genetic testing performed on blood DNA. Identification of the genetic cause can help in guiding families in future pregnancies. Research has highlighted how elucidation of key molecular pathways can also allow for targeted therapeutic interventions.

Focal cortical dysplasia.

Focal cortical dysplasias are among the most common causes of intractable epilepsy in children. As the neuropathology of these conditions has been better clarified, the nomenclature has undergone numerous revisions. Their recognition has grown with the use of neuroimaging, and recent advances in imaging technology will further improve detection. Clinical, electroencephalographic, and imaging findings are often diagnostic, so it is imperative for the clinician to recognize the characteristic patterns. Treatment for developmental and behavioral disability remains largely symptomatic, and epilepsy medications are often ineffective. Epilepsy surgery, however, can be successful in selected patients. The basic science underlying the development of focal cortical dysplasias may lead to novel therapeutic approaches in the future.

Modulation of paroxysmal activity in focal cortical dysplasia by centromedian thalamic nucleus stimulation.

Stimulation of the centromedian thalamic nucleus (CM) was performed during presurgical depth recordings in a patient with drug-resistant partial epilepsy related to premotor focal cortical dysplasia. Low- and high-frequency stimulation of the ipsilateral CM reproducibly suppressed the interictal spikes and fast rhythms. This is the first time that the effects of CM stimulation on interictal focal paroxysmal activity have been observed in humans using depth recordings. These results need further confirmation, but suggest that the CM is a worthwhile stimulation target for alternative treatment in selected cases of drug-resistant nonsurgical epilepsy.