Direct fluorescent labeling of NF186 and NaV1.6 in living primary neurons using bioorthogonal click chemistry.

The axon initial segment (AIS) is a highly specialized neuronal compartment that regulates the generation of action potentials and maintenance of neuronal polarity. Live imaging of the AIS is challenging due to the limited number of suitable labeling methods. To overcome this limitation, we established a novel approach for live labeling of the AIS using unnatural amino acids (UAAs) and click chemistry. The small size of UAAs and the possibility of introducing them virtually anywhere into target proteins make this method particularly suitable for labeling of complex and spatially restricted proteins. Using this approach, we labeled two large AIS components, the 186 kDa isoform of neurofascin (NF186; encoded by Nfasc) and the 260 kDa voltage-gated Na+ channel (NaV1.6, encoded by Scn8a) in primary neurons and performed conventional and super-resolution microscopy. We also studied the localization of epilepsy-causing NaV1.6 variants with a loss-of-function effect. Finally, to improve the efficiency of UAA incorporation, we developed adeno-associated viral (AAV) vectors for click labeling in neurons, an achievement that could be transferred to more complex systems such as organotypic slice cultures, organoids, and animal models.

In vitro effects of eslicarbazepine (S-licarbazepine) as a potential precision therapy on SCN8A variants causing neuropsychiatric disorders.

BACKGROUND AND PURPOSE: Variants in SCN8A, the NaV 1.6 channel's coding gene, are characterized by a variety of symptoms, including intractable epileptic seizures, psychomotor delay, progressive cognitive decline, autistic features, ataxia or dystonia. Standard anticonvulsant treatment has a limited impact on the course of disease. EXPERIMENTAL APPROACH: We investigated the therapeutic potential of eslicarbazepine (S-licarbazepine; S-lic), an enhancer of slow inactivation of voltage gated sodium channels, on two variants with biophysical and neuronal gain-of-function (G1475R and M1760I) and one variant with biophysical gain-of-function but neuronal loss-of-function (A1622D) in neuroblastoma cells and in murine primary hippocampal neuron cultures. These three variants cover the broad spectrum of NaV 1.6-associated disease and are linked to representative phenotypes of mild to moderate epilepsy (G1475R), developmental and epileptic encephalopathy (M1760I) and intellectual disability without epilepsy (A1622D). KEY RESULTS: Similar to known effects on NaV 1.6 wildtype channels, S-lic predominantly enhances slow inactivation on all tested variants, irrespective of their particular biophysical mechanisms. Beyond that, S-lic exhibits variant-specific effects including a partial reversal of pathologically slowed fast inactivation dynamics (A1622D and M1760I) and a trend to reduce enhanced persistent Na+ current by A1622D variant channels. Furthermore, our data in primary transfected neurons reveal that not only variant-associated hyperexcitability (M1760I and G1475R) but also hypoexcitability (A1622D) can be modulated by S-lic. CONCLUSIONS AND IMPLICATIONS: S-lic has not only substance-specific effects but also variant-specific effects. Personalized treatment regimens optimized to achieve such variant-specific pharmacological modulation may help to reduce adverse side effects and improve the overall therapeutic outcome of SCN8A-related disease.

Optically pumped magnetometers detect altered maximal muscle activity in neuromuscular disease.

Optically pumped magnetometers (OPM) are quantum sensors that enable the contactless, non-invasive measurement of biomagnetic muscle signals, i.e., magnetomyography (MMG). Due to the contactless recording, OPM-MMG might be preferable to standard electromyography (EMG) for patients with neuromuscular diseases, particularly when repetitive recordings for diagnostic and therapeutic monitoring are mandatory. OPM-MMG studies have focused on recording physiological muscle activity in healthy individuals, whereas research on neuromuscular patients with pathological altered muscle activity is non-existent. Here, we report a proof-of-principle study on the application of OPM-MMG in patients with neuromuscular diseases. Specifically, we compare the muscular activity during maximal isometric contraction of the left rectus femoris muscle in three neuromuscular patients with severe (Transthyretin Amyloidosis in combination with Pompe's disease), mild (Charcot-Marie-Tooth disease, type 2), and without neurogenic, but myogenic, damage (Myotonia Congenita). Seven healthy young participants served as the control group. As expected, and confirmed by using simultaneous surface electromyography (sEMG), a time-series analysis revealed a dispersed interference pattern during maximal contraction with high amplitudes. Furthermore, both patients with neurogenic damage (ATTR and CMT2) showed a reduced variability of the MMG signal, quantified as the signal standard deviation of the main component of the frequency spectrum, highlighting the reduced possibility of motor unit recruitment due to the loss of motor neurons. Our results show that recording pathologically altered voluntary muscle activity with OPM-MMG is possible, paving the way for the potential use of OPM-MMG in larger studies to explore the potential benefits in clinical neurophysiology.

A SCN8A variant associated with severe early onset epilepsy and developmental delay: Loss- or gain-of-function?

SCN8A, encoding the voltage-gated sodium channel subunit NaV1.6, has been associated with a wide spectrum of neuropsychiatric disorders. Missense variants in SCN8A which increase the channel activity can cause a severe developmental and epileptic encephalopathy (DEE). One DEE variant (p.(Arg223Gly)) was described to cause a predominant loss-of-function (LOF) mechanism when expressed in neuroblastoma cells, which is not consistent with the genotype-phenotype correlations in this gene. To resolve this discrepancy and understand the pathophysiological mechanism of this variant, we performed comprehensive electrophysiological studies in both neuroblastoma cells and primary hippocampal neuronal cultures. Although we also found that p.(Arg223Gly) significantly decreased Na+ current density and enhanced fast inactivation compared to the wild type (WT) channel in transfected neuroblastoma cells (both LOF mechanisms), it also caused a strong hyperpolarizing shift of steady-state activation and accelerated the recovery from fast inactivation (both gain-of-function (GOF) mechanisms). In cultured neurons transfected with mutant vs. WT NaV1.6 channels, we found more depolarized resting membrane potentials and a decreased rheobase leading to enhanced action potential firing. We conclude that SCN8A p.(Arg223Gly) leads to a net GOF resulting in neuronal hyperexcitability and a higher firing rate, fitting with the central role of GOF mechanisms in DEE.

Frame-based and robot-assisted insular stereo-electroencephalography via an anterior or posterior oblique approach.

OBJECTIVE: There is an increasing interest in stereo-electroencephalography (SEEG) for invasive evaluation of insular epilepsy. The implantation of insular SEEG electrodes, however, is still challenging due to the anatomical location and complex functional segmentation in both an anteroposterior and ventrodorsal (i.e., superoinferior) direction. While the orthogonal approach (OA) is the shortest trajectory to the insula, it might insufficiently cover these networks. In contrast, the anterior approach (AOA) or posterior oblique approach (POA) has the potential for full insular coverage, with fewer electrodes bearing a risk of being more inaccurate due to the longer trajectory. Here, the authors evaluated the implantation accuracy and the detection of epilepsy-related SEEG activity with AOA and POA insular trajectories. METHODS: This retrospective study evaluated the accuracy of 220 SEEG electrodes in 27 patients. Twelve patients underwent a stereotactic frame-based procedure (frame group), and 15 patients underwent a frameless robot-assisted surgery (robot group). In total, 55 insular electrodes were implanted using the AOA or POA considering the insular anteroposterior and ventrodorsal functional organization. The entry point error (EPE) and target point error (TPE) were related to the implantation technique (frame vs robot), the length of the trajectory, and the location of the target (insular vs noninsular). Finally, the spatial distribution of epilepsy-related SEEG activity within the insula is described. RESULTS: There were no significant differences in EPE (mean 0.9 ± 0.6 for the nonsinsular electrodes and 1.1 ± 0.7 mm for the insular electrodes) and TPE (1.5 ± 0.8 and 1.6 ± 0.9 mm, respectively), although the length of trajectories differed significantly (34.1 ± 10.9 and 70.1 ± 9.0 mm, repsectively). There was a significantly larger EPE in the frame group than in the robot group (1.5 ± 0.6 vs 0.7 ± 0.5 mm). However, there was no group difference in the TPE (1.5 ± 0.8 vs 1.6 ± 0.8 mm). Epilepsy-related SEEG activity was detected in 42% (23/55) of the insular electrodes. Spatial distribution of this activity showed a clustering in both anteroposterior and ventrodorsal directions. In purely insular onset cases, subsequent insular lesionectomy resulted in a good seizure outcome. CONCLUSIONS: The implantation of insular electrodes via the AOA or POA is safe and efficient for SEEG implantation covering both anteroposterior and ventrodorsal functional organization with few electrodes. In this series, there was no decrease in accuracy due to the longer trajectory of insular SEEG electrodes in comparison with noninsular SEEG electrodes. The results of frame-based and robot-assisted implantations were comparable.

Tetraparesis and sensorimotor axonal polyneuropathy due to co-occurrence of Pompe disease and hereditary ATTR amyloidosis.

INTRODUCTION/AIMS: Hereditary transthyretin amyloidosis with polyneuropathy (hATTRPN) is an autosomal dominant multi-organ disorder manifesting in the third to fifth decade with the key clinical features of distal and painful sensory loss of the lower limbs and autonomic dysregulation. Motor neuropathy and cardiomyopathy evolve in the course of the disease. Pompe disease is an autosomal recessive disease leading to decreased levels of lysosomal enzyme acid α-glucosidase and proximal muscle weakness. We report the clinical features and diagnostic workup in the rare case of a patient with ATTR amyloidosis and late-onset Pompe disease, both genetically confirmed. METHODS: We performed a detailed clinical assessment, exome sequencing, and biochemical measurements. RESULTS: The patient presented with a distal, painful hypaesthesia of both legs, a cardiomyopathy, and a muscle weakness in the form of a girdle-type pattern of the arms and legs at the beginning and a spreading to distal muscle groups in the course of disease. DISCUSSION: This study highlights the importance of searching for co-occurrence of rare monogenetic neuromuscular diseases, especially in cases in which all clinical features can be readily explained by a single gene defect.

In vitro neuronal network activity as a new functional diagnostic system to detect effects of Cerebrospinal fluid from autoimmune encephalitis patients.

The intent of this study was to investigate if cerebrospinal fluid (CSF) from autoimmune encephalitis (AE) patients regulates in vitro neuronal network activity differentially to healthy human control CSF (hCSF). To this end, electrophysiological effects of CSF from AE patients or hCSF were measured by in vitro neuronal network activity (ivNNA) recorded with microelectrode arrays (MEA). CSF from patients with either N-methyl-D-aspartate-receptor-antibody (pCSFNMDAR, n = 7) or Leucine-rich-glioma-inactivated-1-Ab (pCSFLGI1, n = 6) associated AE suppressed global spiking activity of neuronal networks by a factor of 2.17 (p < 0.05) or 2.42 (p < 0.05) compared to hCSF. The former also suppressed synchronous network bursting by a factor of 1.93 (p < 0.05) in comparison to hCSF (n = 13). As a functional diagnostic test, this parameter reached a sensitivity of 86% for NMDAR-Ab- and 100% for LGI1-Ab-associated AE vs. hCSF at a specificity of 85%. To explore if modulation at the NMDAR influences effects of hCSF or pathological CSF, we applied the NMDAR-antagonist 2-Amino-5-phosphono-pentanoic acid (AP5). In CSF from NMDAR-Ab-associated AE patients, spike rate reduction by AP5 was more than 2-fold larger than in hCSF (p < 0.05), and network burst rate reduction more than 18-fold (p < 0.01). Recording ivNNA might help discriminating between functional effects of CSF from AE patients and hCSF, and thus could be used as a functional diagnostic test in AE. The pronounced suppression of ivNNA by CSF from NMDAR-Ab-associated AE patients and simultaneous antagonism at the NMDAR by AP5, particularly in burst activity, compared to hCSF plus AP5, confirms that the former contains additional ivNNA-suppressing factors.

Neuronal mechanisms of mutations in SCN8A causing epilepsy or intellectual disability.

Ion channel mutations can cause distinct neuropsychiatric diseases. We first studied the biophysical and neurophysiological consequences of four mutations in the human Na+ channel gene SCN8A causing either mild (E1483K) or severe epilepsy (R1872W), or intellectual disability and autism without epilepsy (R1620L, A1622D). Only combined electrophysiological recordings of transfected wild-type or mutant channels in both neuroblastoma cells and primary cultured neurons revealed clear genotype-phenotype correlations. The E1483K mutation causing mild epilepsy showed no significant biophysical changes, whereas the R1872W mutation causing severe epilepsy induced clear gain-of-function biophysical changes in neuroblastoma cells. However, both mutations increased neuronal firing in primary neuronal cultures. In contrast, the R1620L mutation associated with intellectual disability and autism-but not epilepsy-reduced Na+ current density in neuroblastoma cells and expectedly decreased neuronal firing. Interestingly, for the fourth mutation, A1622D, causing severe intellectual disability and autism without epilepsy, we observed a dramatic slowing of fast inactivation in neuroblastoma cells, which induced a depolarization block in neurons with a reduction of neuronal firing. This latter finding was corroborated by computational modelling. In a second series of experiments, we recorded three more mutations (G1475R, M1760I, G964R, causing intermediate or severe epilepsy, or intellectual disability without epilepsy, respectively) that revealed similar results confirming clear genotype-phenotype relationships. We found intermediate or severe gain-of-function biophysical changes and increases in neuronal firing for the two epilepsy-causing mutations and decreased firing for the loss-of-function mutation causing intellectual disability. We conclude that studies in neurons are crucial to understand disease mechanisms, which here indicate that increased or decreased neuronal firing is responsible for distinct clinical phenotypes.

Neuro-Sweet syndrome - a rare differential diagnosis in aseptic meningoencephalitis.

Acute febrile neutrophilic dermatosis (Sweet's syndrome) is a dermatological entity, which may be associated with malignancies, drugs, and infections and which is characterized by high fever, elevated neutrophils, and tender erythematous skin lesions. Involvement of the nervous system - Neuro-Sweet syndrome (NSS) - is rare, manifesting most commonly with an encephalitic syndrome in addition to fever and dermal lesions. Here, we report an unusual case of NSS in a Caucasian male patient in the setting of B-cell-lymphocytosis, with encephalitis preceding dermal lesions. Symptoms resolved completely in response to corticoids. NSS is a rare, but important differential diagnosis in the work-up of febrile aseptic meningoencephalitis unresponsive to anti-infectious treatment. Due to its rarity and clinical variability, diagnosis of NSS might be challenging. Knowledge of this entity may facilitate proper diagnosis and differentiation from conditions with similar clinical presentation, especially Neuro-Behçet's disease. It may further lead to early detection of a potentially underlying malignancy and help in initiating adequate therapy.

Histopathological Findings in Brain Tissue Obtained during Epilepsy Surgery.

BACKGROUND: Detailed neuropathological information on the structural brain lesions underlying seizures is valuable for understanding drug-resistant focal epilepsy. METHODS: We report the diagnoses made on the basis of resected brain specimens from 9523 patients who underwent epilepsy surgery for drug-resistant seizures in 36 centers from 12 European countries over 25 years. Histopathological diagnoses were determined through examination of the specimens in local hospitals (41%) or at the German Neuropathology Reference Center for Epilepsy Surgery (59%). RESULTS: The onset of seizures occurred before 18 years of age in 75.9% of patients overall, and 72.5% of the patients underwent surgery as adults. The mean duration of epilepsy before surgical resection was 20.1 years among adults and 5.3 years among children. The temporal lobe was involved in 71.9% of operations. There were 36 histopathological diagnoses in seven major disease categories. The most common categories were hippocampal sclerosis, found in 36.4% of the patients (88.7% of cases were in adults), tumors (mainly ganglioglioma) in 23.6%, and malformations of cortical development in 19.8% (focal cortical dysplasia was the most common type, 52.7% of cases of which were in children). No histopathological diagnosis could be established for 7.7% of the patients. CONCLUSIONS: In patients with drug-resistant focal epilepsy requiring surgery, hippocampal sclerosis was the most common histopathological diagnosis among adults, and focal cortical dysplasia was the most common diagnosis among children. Tumors were the second most common lesion in both groups. (Funded by the European Union and others.).

Human Cerebrospinal fluid promotes long-term neuronal viability and network function in human neocortical organotypic brain slice cultures.

Pathophysiological investigation of CNS-related diseases, such as epilepsy or neurodegenerative disorders, largely relies on histological studies on human post mortem tissue, tissue obtained by biopsy or resective surgery and on studies using disease models including animal models, heterologous expression systems or cell culture based approaches. However, in general it remains elusive to what extent results obtained in model systems can be directly translated to the human brain, calling for strategies allowing validation or even primary investigation in live human CNS tissue. In the work reported here, we prepared human organotypic slice cultures from access tissue of resective epilepsy surgery. Employing different culture conditions, we systematically compared artificial culturing media versus human cerbrospinal fluid (hCSF) obtained from patients with normal pressure hydrocephalus (NPH). Presented data demonstrates sustained cortical neuronal survival including not only maintenance of typical cellular electrophysiological properties and activity, such as robust action potential generation and synaptic connectivity, but also preservation of tonic and phasic network activity up to several weeks in vitro. As clearly delineated by immunocytochemistry, single cell patch clamp and extracellular recordings, we find that in contrast to artificial culturing media, hCSF significantly enhances neuron viability and maintenance of network activity.

Homozygous mutation in TXNRD1 is associated with genetic generalized epilepsy.

Increased oxidative stress has been widely implicated in the pathogenesis in various forms of human epilepsy. Here, we report a homozygous mutation in TXNRD1 (thioredoxin reductase 1) in a family with genetic generalized epilepsy. TXNRD1 is an essential selenium-containing enzyme involved in detoxification of reactive oxygen species (ROS) and redox signaling. The TXNRD1 mutation p.Pro190Leu affecting a highly conserved amino acid residue was identified by whole-exome sequencing of blood DNA from the index patient. The detected mutation and its segregation within the family - all siblings of the index patient were homozygous and the parents heterozygous - were confirmed by Sanger sequencing. TXNRD1 activity was determined in subcellular fractions from a skeletal muscle biopsy and skin fibroblasts of the index patient and the expression levels of the mutated protein were assessed by 75Se labeling and Western blot analysis. As result of the mutation, the activity of TXNRD1 was reduced in the patient's fibroblasts and skeletal muscle (to 34±3% and 16±8% of controls, respectively). In fibroblasts, we detected reduced 75Se-labeling of the enzyme (41±3% of controls). An in-depth in vitro kinetic analysis of the recombinant mutated TXNRD1 indicated 30-40% lowered kcat/Se values. Therefore, a reduced activity of the enzyme in the patient's tissue samples is explained by (i) lower enzyme turnover and (ii) reduced abundance of the mutated enzyme as confirmed by Western blotting and 75Se labeling. The mutant fibroblasts were also found to be less resistant to a hydrogen peroxide challenge. Our data agree with a potential role of insufficient ROS detoxification for disease manifestation in genetic generalized epilepsy.

Impact of Genetic Polymorphisms of ABCB1 (MDR1, P-Glycoprotein) on Drug Disposition and Potential Clinical Implications: Update of the Literature.

ATP-binding cassette transporter B1 (ABCB1; P-glycoprotein; multidrug resistance protein 1) is an adenosine triphosphate (ATP)-dependent efflux transporter located in the plasma membrane of many different cell types. Numerous structurally unrelated compounds, including drugs and environmental toxins, have been identified as substrates. ABCB1 limits the absorption of xenobiotics from the gut lumen, protects sensitive tissues (e.g. the brain, fetus and testes) from xenobiotics and is involved in biliary and renal secretion of its substrates. In recent years, a large number of polymorphisms of the ABCB1 [ATP-binding cassette, sub-family B (MDR/TAP), member 1] gene have been described. The variants 1236C>T (rs1128503, p.G412G), 2677G>T/A (rs2032582, p.A893S/T) and 3435C>T (rs1045642, p.I1145I) occur at high allele frequencies and create a common haplotype; therefore, they have been most widely studied. This review provides an overview of clinical studies published between 2002 and March 2015. In summary, the effect of ABCB1 variation on P-glycoprotein expression (messenger RNA and protein expression) and/or activity in various tissues (e.g. the liver, gut and heart) appears to be small. Although polymorphisms and haplotypes of ABCB1 have been associated with alterations in drug disposition and drug response, including adverse events with various ABCB1 substrates in different ethnic populations, the results have been majorly conflicting, with limited clinical relevance. Future research activities are warranted, considering a deep-sequencing approach, as well as well-designed clinical studies with appropriate sample sizes to elucidate the impact of rare ABCB1 variants and their potential consequences for effect sizes.

Incipient preoperative reorganization processes of verbal memory functions in patients with left temporal lobe epilepsy.

We previously reported nonlinear correlations between verbal episodic memory performance and BOLD signal in memory fMRI in healthy subjects. The purpose of the present study was to examine this observation in patients with left mesial temporal lobe epilepsy (mTLE) who often experience memory decline and need reliable prediction tools before epilepsy surgery with hippocampectomy. Fifteen patients with left mTLE (18-57years, nine females) underwent a verbal memory fMRI paradigm. Correlations between BOLD activity and neuropsychological data were calculated for the i) hippocampus (HC) as well as ii) extrahippocampal mTL structures. Memory performance was systematically associated with activations within the right HC as well as with activations within the left extrahippocampal mTL regions (amygdala and parahippocampal gyrus). As hypothesized, the analyses revealed cubic relationships, with one peak in patients with marginal memory performance and another peak in patients with very good performance. The nonlinear correlations between memory performance and activations might reflect the compensatory recruitment of neural resources to maintain memory performance in patients with ongoing memory deterioration. The present data suggest an already incipient preoperative reorganization process of verbal memory in non-amnesic patients with left mTLE by simultaneously tapping the resources of the right HC and left extrahippocampal mTL regions. Thus, in the preoperative assessment, both neuropsychological performance and memory fMRI should be considered together.

A homozygous splice-site mutation in CARS2 is associated with progressive myoclonic epilepsy.

OBJECTIVE: We report a consanguineous family with 2 affected individuals whose clinical symptoms closely resembled MERRF (myoclonus epilepsy with ragged red fibers) syndrome including severe myoclonic epilepsy, progressive spastic tetraparesis, progressive impairment of vision and hearing, as well as progressive cognitive decline. METHODS: After excluding the presence of pathogenic mitochondrial DNA mutations, whole-exome sequencing of blood DNA from the index patient was performed. Detected homozygous mutations and their cosegregation were confirmed by Sanger sequencing. CARS2 (cysteinyl-tRNA synthetase 2, mitochondrial) messenger RNA analysis was performed by reverse transcription PCR and sequencing. RESULTS: We identified a homozygous c.655G>A mutation in the CARS2 gene cosegregating in the family. The mutation is localized at the last nucleotide of exon 6 and thus is predicted to cause aberrant splicing. Analysis of the CARS2 messenger RNA showed that the presence of the mutation resulted in removal of exon 6. This leads to an in-frame deletion of 28 amino acids in a conserved sequence motif of the protein involved in stabilization of the acceptor end hairpin of tRNA(Cys). CONCLUSION: CARS2 is a novel disease gene associated with a severe progressive myoclonic epilepsy most resembling MERRF syndrome.

Analysis of ELP4, SRPX2, and interacting genes in typical and atypical rolandic epilepsy.

Rolandic epilepsy (RE) and its atypical variants (atypical rolandic epilepsy, ARE) along the spectrum of epilepsy-aphasia disorders are characterized by a strong but largely unknown genetic basis. Two genes with a putative (ELP4) or a proven (SRPX2) function in neuronal migration were postulated to confer susceptibility to parts of the disease spectrum: the ELP4 gene to centrotemporal spikes and SRPX2 to ARE. To reexamine these findings, we investigated a cohort of 280 patients of European ancestry with RE/ARE for the etiological contribution of these genes and their close interaction partners. We performed next-generation sequencing and single-nucleotide polymorphism (SNP)-array based genotyping to screen for sequence and structural variants. In comparison to European controls we could not detect an enrichment of rare deleterious variants of ELP4, SRPX2, or their interaction partners in affected individuals. The previously described functional p.N327S variant in the X chromosomal SRPX2 gene was detected in two affected individuals (0.81%) and also in controls (0.26%), with some preponderance of male patients. We did not detect an association of SNPs in the ELP4 gene with centrotemporal spikes as previously reported. In conclusion our data do not support a major role of ELP4 and SRPX2 in the etiology of RE/ARE.

Episodic itch in a case of spinal glioma.

BACKGROUND: Itch is a frequent complaint reported by patients and is usually ascribed to dermatological or metabolic causes. In neurological disorders, however, it is a very unusual symptom and thus its neurological aetiology is likely to be overlooked. There are only very few reports about permanent itch related to lesions of the central nervous system. To our knowledge we report the first case of episodic itch associated with a central nervous lesion. CASE PRESENTATION: A 74-year-old female suffered from long-standing episodes of itch of the dermatomes C2 to C6 on the right side that was refractory to any treatment. On occurrence it propagated in a proximal to distal fashion. Between the episodes the patient was asymptomatic. MRI of the cervical spine uncovered a spinal glioma that matched the location of the symptoms. Treatment with gabapentin led to a prompt reduction of the symptoms. CONCLUSION: Patients with intractable pruritus and dermatomal presentation ought to undergo neurological examination and spinal cord imaging. Thus, ongoing frustrating and sometimes even harmful treatment trials could be avoided.

Regulation of ClC-2 gating by intracellular ATP.

ClC-2 is a voltage-dependent chloride channel that activates slowly at voltages negative to the chloride reversal potential. Adenosine triphosphate (ATP) and other nucleotides have been shown to bind to carboxy-terminal cystathionine-ß-synthase (CBS) domains of ClC-2, but the functional consequences of binding are not sufficiently understood. We here studied the effect of nucleotides on channel gating using single-channel and whole-cell patch clamp recordings on transfected mammalian cells. ATP slowed down macroscopic activation and deactivation time courses in a dose-dependent manner. Removal of the complete carboxy-terminus abolishes the effect of ATP, suggesting that CBS domains are necessary for ATP regulation of ClC-2 gating. Single-channel recordings identified long-lasting closed states of ATP-bound channels as basis of this gating deceleration. ClC-2 channel dimers exhibit two largely independent protopores that are opened and closed individually as well as by a common gating process. A seven-state model of common gating with altered voltage dependencies of opening and closing transitions for ATP-bound states correctly describes the effects of ATP on macroscopic and microscopic ClC-2 currents. To test for a potential pathophysiological impact of ClC-2 regulation by ATP, we studied ClC-2 channels carrying naturally occurring sequence variants found in patients with idiopathic generalized epilepsy, G715E, R577Q, and R653T. All naturally occurring sequence variants accelerate common gating in the presence but not in the absence of ATP. We propose that ClC-2 uses ATP as a co-factor to slow down common gating for sufficient electrical stability of neurons under physiological conditions.

Mutation in the mitochondrial tRNA(Ile) gene causes progressive myoclonus epilepsy.

PURPOSE: The group of the rare progressive myoclonic epilepsies (PME) include a wide spectrum of mitochondrial and metabolic diseases. In juvenile and adult ages, MERRF (myoclonic epilepsy with ragged red fibres) is the most common form. The underlying genetic defect in most patients with the syndrome of MERRF is a mutation in the tRNALys gene, but mutations were also detected in the tRNAPhe gene. METHOD: Here, we describe a 40 year old patient with prominent myoclonic seizures since 39 years of age without a mutation in the known genes who underwent intensive clinical, genetic and functional workup. RESULTS: The patient had a slight mental retardation and a severe progressive hearing loss based on a defect of the inner ear on both sides. Ictal electroencephalography (EEG) showed bilateral occipital and generalized spikes and polyspikes induced and aggravated by photostimulation. A cranial magnetic resonance imaging (cMRI) detected a global cortical atrophy of the brain and mild periventricular white matter lesions. The electromyography (EMG) was normal but the muscle biopsy showed abundant ragged red fibres. Sequencing of the mitochondrial DNA from the skeletal muscle biopsy revealed a novel heteroplasmic mutation (m.4279A>G) in the tRNAIle gene which was functionally relevant as tested in single skeletal muscle fibre investigations. CONCLUSION: Mutations in tRNAIle were described in patients with chronic progressive external ophthalmoplegia (CPEO), prominent deafness or cardiomyopathy but, up to now, not in patients with myoclonic epilepsy. The degree of heteroplasmy of this novel mitochondrial DNA mutation was 70% in skeletal muscle but only 15% in blood, pointing to the diagnostic importance of a skeletal muscle biopsy also in patients with myoclonic epilepsy.

Hereditary channelopathies in neurology.

Ion channelopathies are caused by malfunction or altered regulation of ion channel proteins due to hereditary or acquired protein changes. In neurology, main phenotypes include certain forms of epilepsy, ataxia, migraine, neuropathic pain, myotonia, and muscle weakness including myasthenia and periodic paralyses. The total prevalence of monogenic channelopathies in neurology is about 35:100,000. Susceptibility-related mutations further increase the relevance of channel genes in medicine considerably. As many disease mechanisms have been elucidated by functional characterization on the molecular level, the channelopathies are regarded as model disorders for pathogenesis and treatment of non-monogenic forms of epilepsy and migraine. As more than 35% of marketed drugs target ion channels, there is a high chance to identify compounds that counteract the effects of the mutations.