DUSP4 appears to be a highly localized endogenous inhibitor of epileptic signaling in human neocortex.

BACKGROUND: We previously identified the Mitogen Activated Protein Kinase (MAPK) pathway as focally upregulated in brain regions with high epileptic activity and showed that inhibition of MAPK signaling reduces epileptic spiking in an animal model. Here we examined how activators and inhibitors of the MAPK pathway are expressed in human epileptic cortex and how these could contribute to the localization of epileptic signaling. METHODS: We localized gene and protein expression in human epileptic neocortical tissues based on epileptic activities from 20 patients based on long-term intracranial recordings. Follow-up mechanistic studies by depolarization of human Sh-SY5Y cell line were used to model epileptic activity in the human brain. RESULTS: A clustering algorithm of differentially expressed genes identified a unique gene expression cluster distinct from other MAPK genes. Within this cluster was dual specificity phosphatase 4 (DUSP4), a potent MAPK inhibitor. In situ hybridization studies revealed focal patches of DUSP4 mRNA in layer 2/3 brain regions associated with a dramatic reduction in MAPK signaling genes. In vitro depolarization led to the rapid and transient induction of DUSP4 protein, which, in turn, reduced MAPK activity. Activity-dependent induction of DUSP4 protein was transient and required MAPK signaling. Human epileptic brain regions with lower epileptic activity had lower MAPK activity and higher DUSP4 protein levels. DISCUSSION: DUSP4 is a highly localized, endogenous feedback inhibitor of pro-epileptogenic MAPK signaling in the human epileptic brain. Increasing DUSP4 expression could therefore be a novel therapeutic approach to prevent the development and spread of epileptic circuits. SIGNIFICANCE STATEMENT: Epilepsy is a chronic debilitating disease. Once it develops, epileptic circuits often persist throughout life. Fortunately, in focal forms of epilepsy, these circuits can remain highly localized and are amenable to surgical resections, suggesting that endogenous mechanisms restrict their spread to other brain regions. Using a high-throughput genomic analysis of human epileptic brain regions, we identified DUSP4 as an activity-dependent inhibitor of MAPK signaling expressed in focal patches surrounding human neocortical epileptic brain regions. Our results suggest that DUSP4, through local inhibition of MAPK signaling, acts as an endogenous, spatially segregated safety mechanism to prevent the spread of epileptic activity. Augmenting DUSP4 expression could be a novel disease-modifying approach to prevent or treat human epilepsy.

Central sulcus is a barrier to causal propagation in epileptic networks.

the functional interactions of different brain regions are dependent of their underlying structural connectivity. These interactions are important for normal brain activity as well as in epileptic disorders where spontaneous epileptic discharges are generated. To understand the effects of brain topography on both normal and epileptic functional networks, we evaluated propagation frequency with respect to geodesic distance and the specific role of sulcal patterns in modulating these functional connections. We implemented a multimodal approach combining Electrocorticography (ECoG) and magnetic resonance imaging (MRI) to evaluate the relationships between epileptic and non-epileptic networks as they are related to structural connectivity in the human brain. We analyzed interictal ECoG along with MRI 3D reconstructions from seven epileptic patients and found that interictal full-dataset network propagations travel to both nearby and distant cortical locations, however with longer distance the propagations occurrence attenuates dramatically. We also discovered that the central sulcus acts as a strong barrier allowing only 30% of the propagation to cross central sulcus whereas the rest of the 70% propagations are observed on the same side of the sulcus. Epileptic spike propagations, however, were more highly localized with significantly less distant spread and had further reduction in spread across sulci. This is a novel approach to explore the structural influence of brain topography on the functional network and would help understand the interaction of different brain regions and how these are altered in patients with epilepsy. This approach could assist physician decision making during epilepsy surgery by revealing an appropriate brain network and cortical interactions.

Neocortical slices from adult chronic epileptic rats exhibit discharges of higher voltages and broader spread.

Much of the current understanding of epilepsy mechanisms has been built on data recorded with one or a few electrodes from temporal lobe slices of normal young animals stimulated with convulsants. Mechanisms of adult, extratemporal, neocortical chronic epilepsy have not been characterized as much. A more advanced understanding of epilepsy mechanisms can be obtained by recording epileptiform discharges simultaneously from multiple points of an epileptic focus so as to define their sites of initiation and pathways of spreading. Brain slice recordings can characterize epileptic mechanisms in a simpler, more controlled preparation than in vivo. Yet, the intrinsic hyper-excitability of a chronic epileptic focus may not be entirely preserved in slices following the severing of connections in slice preparation. This study utilizes recordings of multiple electrode arrays to characterize which features of epileptic hyper-excitability present in in vivo chronic adult neocortical epileptic foci are preserved in brain slices. After tetanus toxin somatosensory cortex injections, adult rats manifest chronic spontaneous epileptic discharges both in the injection site (primary focus) and in the contralateral side (secondary focus). We prepared neocortical slices from these epileptic animals. When perfused with 4-Aminopyridine in a magnesium free medium, epileptic rat slices exhibit higher voltage discharges and broader spreading than control rat slices. Rates of discharges are similar in slices of epileptic and normal rats, however. Ictal and interictal discharges are distributed over most cortical layers, though with significant differences between primary and secondary foci. A chronic neocortical epileptic focus in slices does not show increased spontaneous pacemakers initiating epileptic discharges but shows discharges with higher voltages and broader spread, consistent with an enhanced synchrony of cellular and synaptic generators over wider surfaces.

Intracranial EEG analysis in tumor-related epilepsy: Evidence of distant epileptic abnormalities.

OBJECTIVE: In patients with tumor-related epilepsy (TRE), surgery traditionally focuses on tumor resection; but identification and removal of associated epileptogenic zone may improve seizure outcome. Here, we study spatial relationship of tumor and seizure onset and early spread zone (SOSz). We also perform quantitative analysis of interictal epileptiform activities in patients with both TRE and non-lesional epilepsy in order to better understand the electrophysiological basis of epileptogenesis. METHODS: Twenty-five patients (11 with TRE and 14 with non-lesional epilepsy) underwent staged surgery using intracranial electrodes. Tumors were outlined on MRI and images were coregistered with post-implantation CT images. For each electrode, distance to the nearest tumor margin was measured. Electrodes were categorized based on distance from tumor and involvement in seizure. Quantitative EEG analysis studying frequency, amplitude, power, duration and slope of interictal spikes was performed. RESULTS: At least part of the SOSz was located beyond 1.5 cm from the tumor margin in 10/11 patients. Interictally, spike frequency and power were higher in the SOSz and spikes near tumor were smaller and less sharp. Interestingly, peritumoral electrodes had the highest spike frequencies and sharpest spikes, indicating greatest degree of epileptic synchrony. A complete resection of the SOSz resulted in excellent seizure outcome. CONCLUSIONS: Seizure onset and early spread often involves brain areas distant from the tumor. SIGNIFICANCE: Utilization of epilepsy surgery approach for TRE may provide better seizure outcome and study of the intracranial EEG may provide insight into pathophysiology of TRE.

Morphology-based automatic seizure detector for intracerebral EEG recordings.

In this paper, a new seizure detection system aimed at assisting in a rapid review of prolonged intracerebral EEG recordings is described. It is based on quantifying the sharpness of the waveform, one of the most important electrographic EEG features utilized by experts for an accurate and reliable identification of a seizure. The waveform morphology is characterized by a measure of sharpness as defined by the slope of the half-waves. A train of abnormally sharp waves resulting from subsequent filtering are used to identify seizures. The method was optimized using 145 h of single-channel depth EEG from seven patients, and tested on another 158 h of single-channel depth EEG from another seven patients. Additionally, 725 h of depth EEG from 21 patients was utilized to assess the system performance in a multichannel configuration. Single-channel test data resulted in a sensitivity of 87% and a specificity of 71%. The multichannel test data reported a sensitivity of 81% and a specificity of 58.9%. The new system detected a wide range of seizure patterns that included rhythmic and nonrhythmic seizures of varying length, including those missed by the experts. We also compare the proposed system with a popular commercial system.

A novel unsupervised spike sorting algorithm for intracranial EEG.

This paper presents a novel, unsupervised spike classification algorithm for intracranial EEG. The method combines template matching and principal component analysis (PCA) for building a dynamic patient-specific codebook without a priori knowledge of the spike waveforms. The problem of misclassification due to overlapping classes is resolved by identifying similar classes in the codebook using hierarchical clustering. Cluster quality is visually assessed by projecting inter- and intra-clusters onto a 3D plot. Intracranial EEG from 5 patients was utilized to optimize the algorithm. The resulting codebook retains 82.1% of the detected spikes in non-overlapping and disjoint clusters. Initial results suggest a definite role of this method for both rapid review and quantitation of interictal spikes that could enhance both clinical treatment and research studies on epileptic patients.

An animal model to study the clinical significance of interictal spiking.

Interictal spikes (IIS) are paroxysmal discharges commonly observed in patients with epilepsy which represent an abnormally-synchronized population of hyperexcitable neurons firing as an aggregate. Due to conflicting studies on the clinical significance of IIS, research focusing on IIS has been sparse. However, recent attention on IIS has increased for patients undergoing surgery for intractable epilepsy as a means to identify epileptic foci for surgical resection. There is growing evidence that IIS are not asymptomatic as has been commonly accepted. Other than epilepsy, IIS have been associated with a wide range of behavioral and psychiatric disorders, including attention deficit disorder, anxiety disorders and psychoses. For these reasons, a well-characterized animal model of interictal spiking which accurately mimics the human phenomenon would be a valuable tool to gain, insights both into the pathophysiology of epilepsy as well as a broad variety of human neuropsychiatric diseases. Here, we review the literature on the clinical significance of IIS in humans and on animal models where IIS has been observed. We then demonstrate the utility of using tetanus toxin to generate a reproducible pattem of progressive IIS for future studies into their clinical significance.

Compressed EEG pattern analysis for critically ill neurological-neurosurgical patients.

Recent advances in continuous electroencephalogram (EEG) monitoring with digital EEG acquisition, storage, and quantitative analysis allow uninterrupted assessment of cerebral cortical activity in critically ill neurological-neurosurgical patients. Early recognition of worsening brain function can prove of vital importance as one can initiate measures aimed to prevent further brain damage. Although continuous EEG monitoring provides adequate spatial and temporal resolution and is able to continuously assess brain function in these critically ill patients, it requires a trained electroencephalographer to interpret the massive amounts of data generated. This limitation impedes the widespread use of EEG in assessing real-time brain function in critically ill patients. Here, we demonstrate the utility of a novel method of automated EEG analysis that segments and extracts EEG features, classifies and groups them according to various patterns, and then presents them in a compressed fashion. This permits real-time viewing of several hours of EEG on a single page. Examples are presented from three patients, two with recurrent seizures and one with diagnosis of subarachnoid hemorrhage. These patients illustrate the ability of this novel method to detect important real-time physiological changes in brain function that could enable early interventions aimed to prevent irreversible brain damage.

Treatment of neurocysticercosis: current status and future research needs.

Here we put forward a roadmap that summarizes important questions that need to be answered to determine more effective and safer treatments. A key concept in management of neurocysticercosis is the understanding that infection and disease due to neurocysticercosis are variable and thus different clinical approaches and treatments are required. Despite recent advances, treatments remain either suboptimal or based on poorly controlled or anecdotal experience. A better understanding of basic pathophysiologic mechanisms including parasite survival and evolution, nature of the inflammatory response, and the genesis of seizures, epilepsy, and mechanisms of anthelmintic action should lead to improved therapies.

Calcific neurocysticercosis and epileptogenesis.

Neurocysticercosis is responsible for increased rates of seizures and epilepsy in endemic regions. The most common form of the disease, chronic calcific neurocysticercosis, is the end result of the host's inflammatory response to the larval cysticercus of Taenia solium. There is increasing evidence indicating that calcific cysticercosis is not clinically inactive but a cause of seizures or focal symptoms in this population. Perilesional edema is at times also present around implicated calcified foci. A better understanding of the natural history, frequency, epidemiology, and pathophysiology of calcific cysticercosis and associated disease manifestations is needed to define its importance, treatment, and prevention.

Neuregulin-heparan-sulfate proteoglycan interactions produce sustained erbB receptor activation required for the induction of acetylcholine receptors in muscle.

Neuregulins bind to and activate members of the EGF receptor family of tyrosine kinases that initiate a signaling cascade that induces acetylcholine receptor synthesis in the postsynaptic membrane of neuromuscular synapses. In addition to an EGF-like domain, sufficient for receptor binding and tyrosine auto-phosphorylation, many spliced forms also have an IG-like domain that binds HSPGs and maintains a high concentration of neuregulin at synapses. Here, we show that the IG-like domain functions to keep the EGF-like domain at sufficiently high concentrations for a sufficiently long period of time necessary to induce acetylcholine receptor gene expression in primary chick myotubes. Using recombinant neuregulins with and without the IG-like domain, we found that IG-like domain binding to endogenous HSPGs produces a 4-fold increase in receptor phosphorylation. This enhancement of activity was blocked by soluble heparin or by pretreatment of muscle cells with heparitinase. We show that at least 12-24 h of neuregulin exposure was required to turn on substantial acetylcholine receptor gene expression and that the erbB receptors need to be kept phosphorylated during this time. The need for sustained erbB receptor activation may be the reason why neuregulins are so highly concentrated in the extracellular matrix of synapses.

Neuregulin stimulates DNA synthesis in embryonic chick heart cells.

Neuregulins are a family of growth factors that have been shown to promote the growth or differentiation of various cell types. Recently, targeted mutations of the genes for neuregulins or their putative receptors by homologous recombination resulted in embryonic lethality characterized by cardiac malformation. Here we investigate a role for neuregulin in the growth of cultured chick heart cells. Neuregulin induced the tyrosine phosphorylation of a 185-kDa protein in cultured heart cells, and it also stimulated an increase in [(3)H]thymidine incorporation and BrDU labeling in the cell cultures. Immunocytochemistry revealed that the increased DNA synthesis was primarily in mesenchymal cells and not detected in myocytes or endocardial cells. These data suggest that neuregulin may function as a paracrine signal in mesenchymal-endothelial interactions during cardiac development.

Expression patterns of transmembrane and released forms of neuregulin during spinal cord and neuromuscular synapse development.

We mapped the distribution of neuregulin and its transmembrane precursor in developing, embryonic chick and mouse spinal cord. Neuregulin mRNA and protein were expressed in motor and sensory neurons shortly after their birth and levels steadily increased during development. Expression of the neuregulin precursor was highest in motor and sensory neuron cell bodies and axons, while soluble, released neuregulin accumulated along early motor and sensory axons, radial glia, spinal axonal tracts and neuroepithelial cells through associations with heparan sulfate proteoglycans. Neuregulin accumulation in the synaptic basal lamina of neuromuscular junctions occurred significantly later, coincident with a reorganization of muscle extracellular matrix resulting in a relative concentration of heparan sulfate proteoglycans at endplates. These results demonstrate an early axonal presence of neuregulin and its transmembrane precursor at developing synapses and a role for heparan sulfate proteoglycans in regulating the temporal and spatial sites of soluble neuregulin accumulation during development.

The neuregulin precursor proARIA is processed to ARIA after expression on the cell surface by a protein kinase C-enhanced mechanism.

We have investigated how the transmembrane precursor proARIA is processed to ARIA (acetylcholine receptor-inducing activity). Pulse-chase labeling in transfected Chinese hamster ovary (CHO) cells showed that proARIA was cleaved to release ARIA into the medium. Cell surface biotin-labeling experiments demonstrated that proARIA was first expressed on the cell surface before being rapidly cleaved to release biotin-labeled ARIA into the medium. While not essential for proteolytic cleavage of proARIA, serum or phorbol-12-myristate-13-acetate (PMA), which activates protein kinase C (PKC), was needed for the efficient release of the processed ARIA. Proteolytic cleavage was blocked by brefeldin A, suggesting that processing occurred distal to Golgi compartments, and by NH4Cl, suggesting a need for intracellular acidic compartments. Serum and PMA also stimulated ARIA release from cultured sensory neurons, suggesting that a similar regulated release mechanism occurs in neurons and may be important in determining where ARIA is released in the developing nervous system.

Functional improvement in a patient with cerebral calcinosis using a bisphosphonate.

Idiopathic cerebral calcification can be associated with a progressive neurologic disorder for which there is no known treatment. This report describes a patient with a familial form of this disorder presenting in middle age with progressive Parkinson-like features along with spasticity, dystonia, and ataxia. Disodium etidronate, a bisphosphonate, produced a two-fold improvement in the rate of his speech and gait, but did not affect his spasticity, dystonia, or ataxia. Quantitative analysis of cerebral calcification did not reveal any reduction in the amount of calcification, suggesting other possible mechanisms for this clinical improvement.

Neurotrophic factors increase neuregulin expression in embryonic ventral spinal cord neurons.

Neuregulins (NRGs) are expressed in spinal cord motor neurons and accumulate at the neuromuscular junction where they may increase the synthesis of postsynaptic acetylcholine receptors and voltage-gated sodium channels. We demonstrate here that NRG expression is selectively increased in rat ventral spinal cord neurons at approximately the time that nerve-muscle synapses first form. A rapid increase in NRG mRNA and protein expression was induced in vitro in cultured rat spinal motor neurons by brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, or glial-cell-line-derived neurotrophic factor. Agrin expression was not affected by these factors over the same time course. Brain-derived neurotrophic factor, but not neurotrophin-3, selectively regulated immunoglobulin domain-containing splice variants of NRG, which are likely to be important for binding to the synaptic basal lamina. Regulation of NRG expression in motor neurons by muscle-derived neurotrophic factors may represent one portion of a reciprocal, regulatory loop that promotes neuromuscular synapse development.

ARIA can be released from extracellular matrix through cleavage of a heparin-binding domain.

ARIA, or acetylcholine receptor-inducing activity, is a polypeptide that stimulates the synthesis of acetylcholine receptors in skeletal muscle. Here we demonstrate that the ability of ARIA to induce phosphorylation of its receptor in muscle is blocked by highly charged glycosaminoglycans. ARIA constructs lacking the NH2-terminal portion, containing an immunoglobulin-like domain, are fully active and are not inhibited by glycosaminoglycans. Limited proteolysis of ARIA with subtilisin blocks the glycosaminoglycan interaction by degrading this NH2-terminal portion, but preserves the active, EGF-like domain. We also show that ARIA can be released from freshly dissociated cells from embryonic chick spinal cord and cerebellum by either heparin, high salt or limited proteolysis with subtilisin, suggesting that ARIA is bound to the extracellular matrix through charged interactions. We present a model of how ARIA may be stored in extracellular matrix at developing synapses and how its release may be mediated by local proteolysis.

Systematic approach to diagnosis and management of biphasic insulin allergy with local anti-inflammatory agents.

In this report, we present a patient with a rarely encountered form of biphasic insulin allergy refractory to a conventional desensitization procedure. We describe a systematic approach to this clinical problem with a set of insulin preparations containing antihistamine and/or corticosteroid to verify the type of hypersensitivity reaction and identify treatment options. The approach taken here may be of use for patients with similar conditions who require insulin for adequate diabetic management but who would otherwise be forced to discontinue insulin because of allergic reactions.

Conformational changes in the chicken receptor for endocytosis of glycoproteins. Modulation of ligand-binding activity by Ca2+ and pH.

Limited proteolysis, gel filtration, and circular dichroism have been used to identify at least three distinct conformational states of a proteolytic fragment containing the ligand-binding domain of the chicken receptor for endocytosis of glycoproteins. Differences in the ligand-binding activity of intact receptor brought about by changing Ca2+ concentrations and pH values can be correlated with different physical states of the binding domain present under similar conditions. An active, ligand-binding state can be detected at either pH 7.8 or 5.4, but 10-fold higher concentrations of Ca2+ are required to stabilize this state at the lower pH. In all cases, the dependence on Ca2+ concentration is second-order, suggesting that two Ca2+ ions are bound to each domain. These studies demonstrate an interdependence between the effects of Ca2+ concentration and pH on both ligand-binding activity and receptor conformation, which is important to consider when describing the binding and dissociation of ligand during endocytosis.