Ictal EEG Source Imaging With Supplemental Electrodes.

INTRODUCTION: Noninvasive brain imaging tests play a major role in guiding decision-making and the usage of invasive, costly intracranial electroencephalogram (ICEEG) in the presurgical epilepsy evaluation. This study prospectively examined the concordance in localization between ictal EEG source imaging (ESI) and ICEEG as a reference standard. METHODS: Between August 2014 and April 2019, patients during video monitoring with scalp EEG were screened for those with intractable focal epilepsy believed to be amenable to surgical treatment. Additional 10-10 electrodes (total = 31-38 per patient, "31+") were placed over suspected regions of seizure onset in 104 patients. Of 42 patients requiring ICEEG, 30 (mean age 30, range 19-59) had sufficiently localized subsequent intracranial studies to allow comparison of localization between tests. ESI was performed using realistic forward boundary element models used in dipole and distributed source analyses. RESULTS: At least partial sublobar concordance between ESI and ICEEG solutions was obtained in 97% of cases, with 73% achieving complete agreement. Median Euclidean distances between ESI and ICEEG solutions ranged from 25 to 30 mm (dipole) and 23 to 38 mm (distributed source). The latter was significantly more accurate with 31+ compared with 21 electrodes (P < 0.01). A difference of ≤25 mm was present in two thirds of the cases. No significant difference was found between dipole and distributed source analyses. CONCLUSIONS: A practical method of ictal ESI (nonuniform placement of 31-38 electrodes) yields high accuracy for seizure localization in epilepsy surgery candidates. These results support routine clinical application of ESI in the presurgical evaluation.

Intact speech perception after resection of dominant hemisphere primary auditory cortex for the treatment of medically refractory epilepsy: illustrative case.

BACKGROUND: In classic speech network models, the primary auditory cortex is the source of auditory input to Wernicke's area in the posterior superior temporal gyrus (pSTG). Because resection of the primary auditory cortex in the dominant hemisphere removes inputs to the pSTG, there is a risk of speech impairment. However, recent research has shown the existence of other, nonprimary auditory cortex inputs to the pSTG, potentially reducing the risk of primary auditory cortex resection in the dominant hemisphere. OBSERVATIONS: Here, the authors present a clinical case of a woman with severe medically refractory epilepsy with a lesional epileptic focus in the left (dominant) Heschl's gyrus. Analysis of neural responses to speech stimuli was consistent with primary auditory cortex localization to Heschl's gyrus. Although the primary auditory cortex was within the proposed resection margins, she underwent lesionectomy with total resection of Heschl's gyrus. Postoperatively, she had no speech deficits and her seizures were fully controlled. LESSONS: While resection of the dominant hemisphere Heschl's gyrus/primary auditory cortex warrants caution, this case illustrates the ability to resect the primary auditory cortex without speech impairment and supports recent models of multiple parallel inputs to the pSTG.

Bilateral temporal lobe epilepsy: How many seizures are required in chronic ambulatory electrocorticography to estimate the laterality ratio?

OBJECTIVE: This study was undertaken to measure the duration of chronic electrocorticography (ECoG) needed to attain stable estimates of the seizure laterality ratio in patients with drug-resistant bilateral temporal lobe epilepsy (BTLE). METHODS: We studied 13 patients with drug-resistant BTLE who were implanted for at least 1 year with a responsive neurostimulation device (RNS System) that provides chronic ambulatory ECoG. Bootstrap analysis and nonlinear regression were applied to model the relationship between chronic ECoG duration and the probability of capturing at least one seizure. Laterality of electrographic seizures in chronic ECoG was compared with the seizure laterality ratio from Phase 1 scalp video-electroencephalographic (vEEG) monitoring. The Kaplan-Meier estimator was used to evaluate time to seizure laterality ratio convergence. RESULTS: Seizure laterality ratios from Phase 1 scalp vEEG monitoring correlated poorly with those from RNS chronic ECoG (r = .31, p = .30). Across the 13 patients, average electrographic seizure frequencies ranged from 1.4 seizures/month to 5.1 seizures/day. A 50% probability of recording at least one electrographic seizure required 9.1 days of chronic ECoG, and 90% probability required 44.3 days of chronic ECoG. A median recording duration of 150.9 days (5 months), corresponding to a median of 16 seizures, was needed before confidence intervals for the seizure laterality ratio reliably contained the long-term value. The median recording duration before the point estimate of the seizure laterality ratio converged to a stationary value was 236.8 days (7.9 months). SIGNIFICANCE: RNS chronic ECoG overcomes temporal sampling limitations intrinsic to inpatient Phase 1 vEEG evaluations. In patients with drug-resistant BTLE, approximately 8 months of chronic RNS ECoG are needed to precisely estimate the seizure laterality ratio, with 75% of people with BTLE achieving convergence after 1 year of RNS recording. For individuals who are candidates for unilateral resection based on seizure laterality, optimized recording duration may help avert morbidity associated with delay to definitive treatment.

Analysis of morphological characteristics of IDH-mutant/wildtype brain tumors using whole-lesion phenotype analysis.

BACKGROUND: Although IDH-mutant tumors aggregate to the frontotemporal regions, the clustering pattern of IDH-wildtype tumors is less clear. As voxel-based lesion-symptom mapping (VLSM) has several limitations for solid lesion mapping, a new technique, whole-lesion phenotype analysis (WLPA), is developed. We utilize WLPA to assess spatial clustering of tumors with IDH mutation from The Cancer Genome Atlas and The Cancer Imaging Archive. METHODS: The degree of tumor clustering segmented from T1 weighted images is measured to every other tumor by a function of lesion similarity to each other via the Hausdorff distance. Each tumor is ranked according to the degree to which its neighboring tumors show identical phenotypes, and through a permutation technique, significant tumors are determined. VLSM was applied through a previously described method. RESULTS: A total of 244 patients of mixed-grade gliomas (WHO II-IV) are analyzed, of which 150 were IDH-wildtype and 139 were glioblastomas. VLSM identifies frontal lobe regions that are more likely associated with the presence of IDH mutation but no regions where IDH-wildtype was more likely to be present. WLPA identifies both IDH-mutant and -wildtype tumors exhibit statistically significant spatial clustering. CONCLUSION: WLPA may provide additional statistical power when compared with VLSM without making several potentially erroneous assumptions. WLPA identifies tumors most likely to exhibit particular phenotypes, rather than producing anatomical maps, and may be used in conjunction with VLSM to understand the relationship between tumor morphology and biologically relevant tumor phenotypes.

The Interleukin-1 Receptor-Associated Kinase 4 Inhibitor PF-06650833 Blocks Inflammation in Preclinical Models of Rheumatic Disease and in Humans Enrolled in a Randomized Clinical Trial.

OBJECTIVE: To investigate the role of PF-06650833, a highly potent and selective small-molecule inhibitor of interleukin-1-associated kinase 4 (IRAK4), in autoimmune pathophysiology in vitro, in vivo, and in the clinical setting. METHODS: Rheumatoid arthritis (RA) inflammatory pathophysiology was modeled in vitro through 1) stimulation of primary human macrophages with anti-citrullinated protein antibody immune complexes (ICs), 2) RA fibroblast-like synoviocyte (FLS) cultures stimulated with Toll-like receptor (TLR) ligands, as well as 3) additional human primary cell cocultures exposed to inflammatory stimuli. Systemic lupus erythematosus (SLE) pathophysiology was simulated in human neutrophils, dendritic cells, B cells, and peripheral blood mononuclear cells stimulated with TLR ligands and SLE patient ICs. PF-06650833 was evaluated in vivo in the rat collagen-induced arthritis (CIA) model and the mouse pristane-induced and MRL/lpr models of lupus. Finally, RNA sequencing data generated with whole blood samples from a phase I multiple-ascending-dose clinical trial of PF-06650833 were used to test in vivo human pharmacology. RESULTS: In vitro, PF-06650833 inhibited human primary cell inflammatory responses to physiologically relevant stimuli generated with RA and SLE patient plasma. In vivo, PF-06650833 reduced circulating autoantibody levels in the pristane-induced and MRL/lpr murine models of lupus and protected against CIA in rats. In a phase I clinical trial (NCT02485769), PF-06650833 demonstrated in vivo pharmacologic action pertinent to SLE by reducing whole blood interferon gene signature expression in healthy volunteers. CONCLUSION: These data demonstrate that inhibition of IRAK4 kinase activity can reduce levels of inflammation markers in humans and provide confidence in the rationale for clinical development of IRAK4 inhibitors for rheumatologic indications.

Accuracy of omni-planar and surface casting of epileptiform activity for intracranial seizure localization.

OBJECTIVE: Intracranial electroencephalography (ICEEG) recordings are performed for seizure localization in medically refractory epilepsy. Signal quantifications such as frequency power can be projected as heatmaps on personalized three-dimensional (3D) reconstructed cortical surfaces to distill these complex recordings into intuitive cinematic visualizations. However, simultaneously reconciling deep recording locations and reliably tracking evolving ictal patterns remain significant challenges. METHODS: We fused oblique magnetic resonance imaging (MRI) slices along depth probe trajectories with cortical surface reconstructions and projected dynamic heatmaps using a simple mathematical metric of epileptiform activity (line-length). This omni-planar and surface casting of epileptiform activity approach (OPSCEA) thus illustrated seizure onset and spread among both deep and superficial locations simultaneously with minimal need for signal processing supervision. We utilized the approach on 41 patients at our center implanted with grid, strip, and/or depth electrodes for localizing medically refractory seizures. Peri-ictal data were converted into OPSCEA videos with multiple 3D brain views illustrating all electrode locations. Five people of varying expertise in epilepsy (medical student through epilepsy attending level) attempted to localize the seizure-onset zones. RESULTS: We retrospectively compared this approach with the original ICEEG study reports for validation. Accuracy ranged from 73.2% to 97.6% for complete or overlapping onset lobe(s), respectively, and ~56.1% to 95.1% for the specific focus (or foci). Higher answer certainty for a given case predicted better accuracy, and scorers had similar accuracy across different training levels. SIGNIFICANCE: In an era of increasing stereo-EEG use, cinematic visualizations fusing omni-planar and surface functional projections appear to provide a useful adjunct for interpreting complex intracranial recordings and subsequent surgery planning.

Real-world experience with direct brain-responsive neurostimulation for focal onset seizures.

OBJECTIVE: The RNS System is a direct brain-responsive neurostimulation system that is US Food and Drug Administration-approved for adults with medically intractable focal onset seizures based on safety and effectiveness data from controlled clinical trials. The purpose of this study was to retrospectively evaluate the real-world safety and effectiveness of the RNS System. METHODS: Eight comprehensive epilepsy centers conducted a chart review of patients treated with the RNS System for at least 1 year, in accordance with the indication for use. Data included device-related serious adverse events and the median percent change in disabling seizure frequency from baseline at years 1, 2, and 3 of treatment and at the most recent follow-up. RESULTS: One hundred fifty patients met the criteria for analysis. The median reduction in seizures was 67% (interquartile range [IQR] = 33%-93%, n = 149) at 1 year, 75% (IQR = 50%-94%, n = 93) at 2 years, 82% (IQR = 50%-96%, n = 38) at ≥3 years, and 74% (IQR = 50%-96%, n = 150) at last follow-up (mean = 2.3 years). Thirty-five percent of patients had a ≥90% seizure frequency reduction, and 18% of patients reported being clinically seizure-free at last follow-up. Seizure frequency reductions were similar regardless of patient age, age at epilepsy onset, duration of epilepsy, seizure onset in mesial temporal or neocortical foci, magnetic resonance imaging findings, prior intracranial monitoring, prior epilepsy surgery, or prior vagus nerve stimulation treatment. The infection rate per procedure was 2.9% (6/150 patients); five of the six patients had an implant site infection, and one had osteomyelitis. Lead revisions were required in 2.7% (4/150), and 2.0% (3/150) of patients had a subdural hemorrhage, none of which had long-lasting neurological consequences. SIGNIFICANCE: In this real-world experience, safety was similar and clinical seizure outcomes exceeded those of the prospective clinical trials, corroborating effectiveness of this therapy and suggesting that clinical experience has informed more effective programming.

Mesial temporal resection following long-term ambulatory intracranial EEG monitoring with a direct brain-responsive neurostimulation system.

OBJECTIVE: To describe seizure outcomes in patients with medically refractory epilepsy who had evidence of bilateral mesial temporal lobe (MTL) seizure onsets and underwent MTL resection based on chronic ambulatory intracranial EEG (ICEEG) data from a direct brain-responsive neurostimulator (RNS) system. METHODS: We retrospectively identified all patients at 17 epilepsy centers with MTL epilepsy who were treated with the RNS System using bilateral MTL leads, and in whom an MTL resection was subsequently performed. Presumed lateralization based on routine presurgical approaches was compared to lateralization determined by RNS System chronic ambulatory ICEEG recordings. The primary outcome was frequency of disabling seizures at last 3-month follow-up after MTL resection compared to seizure frequency 3 months before MTL resection. RESULTS: We identified 157 patients treated with the RNS System with bilateral MTL leads due to presumed bitemporal epilepsy. Twenty-five patients (16%) subsequently had an MTL resection informed by chronic ambulatory ICEEG (mean = 42 months ICEEG); follow-up was available for 24 patients. After MTL resection, the median reduction in disabling seizures at last follow-up was 100% (mean: 94%; range: 50%-100%). Nine patients (38%) had exclusively unilateral electrographic seizures recorded by chronic ambulatory ICEEG and all were seizure-free at last follow-up after MTL resection; eight of nine continued RNS System treatment. Fifteen patients (62%) had bilateral MTL electrographic seizures, had an MTL resection on the more active side, continued RNS System treatment, and achieved a median clinical seizure reduction of 100% (mean: 90%; range: 50%-100%) at last follow-up, with eight of fifteen seizure-free. For those with more than 1 year of follow-up (N = 21), 15 patients (71%) were seizure-free during the most recent year, including all eight patients with unilateral onsets and 7 of 13 patients (54%) with bilateral onsets. SIGNIFICANCE: Chronic ambulatory ICEEG data provide information about lateralization of MTL seizures and can identify additional patients who may benefit from MTL resection.

Responsive neurostimulation for regional neocortical epilepsy.

OBJECTIVE: Surgical resection of seizure-producing brain tissue is a gold standard treatment for drug-resistant focal epilepsy. However, several patient-specific factors can preclude resective surgery, including a spatially extensive ("regional") seizure-onset zone (SOZ). For such patients, responsive neurostimulation (RNS) represents a potential treatment, but its efficacy has not been investigated in this population. METHODS: We performed a multicenter retrospective cohort study of patients (N = 30) with drug-resistant focal epilepsy and a regional neocortical SOZ delineated by intracranial monitoring who were treated with the RNS System for at least 6 months. RNS System leads were placed at least 1-cm apart over the SOZ, and most patients were treated with a lead-to-lead stimulation pathway. Five patients underwent partial resection of the SOZ concurrent with RNS System implantation. We assessed change in seizure frequency relative to preimplant baseline and evaluated correlation between clinical outcome and stimulation parameters. RESULTS: Median follow-up duration was 21.5 months (range 6-52). Median reduction in clinical seizure frequency was 75.5% (interquartile range [IQR] 40%-93.9%). There was no significant difference in outcome between patients treated with and without concurrent partial resection. Most patients were treated with low charge densities (1-2.5 µC/cm2 ), but charge density, interlead distance, and duration of treatment were not significantly correlated with outcome. SIGNIFICANCE: RNS is a feasible and effective treatment in patients with drug-resistant regional neocortical seizures. Prospective studies in larger cohorts are necessary to determine optimal lead configuration and stimulation parameters, although our results suggest that lead-to-lead stimulation and low charge density may be effective in some patients.

Distinct signals and immune cells drive liver pathology and glomerulonephritis in ABIN1[D485N] mice.

We report that TLR7, IL-6, and the adaptive immune system are essential for autoimmunity and glomerulonephritis but not for liver pathology in mice expressing the ubiquitin-binding-defective ABIN1[D485N] mutant. The blood and organs of ABIN1[D485N] mice have exceptionally high numbers of patrolling monocytes (pMo), which develop independently of IL-6 and the adaptive immune system. They are detectable in the blood months before autoimmunity and organ pathology are seen and may have diagnostic potential. The splenic pMo, inflammatory monocytes (iMo), and neutrophils of ABIN1[D485N] mice expressed high levels of mRNAs encoding proteins released during NETosis, which together with the high numbers of monocyte-derived dendritic cells (MoDCs) may drive the liver pathology in ABIN1[D485N] mice, and contribute to the pathology of other organs. The splenic iMo of ABIN1[D485N] mice displayed high expression of mRNAs encoding proteins controlling cell division and were actively dividing; this may underlie the increased pMo and MoDC numbers, which are derived from iMo. An orally active IRAK4 inhibitor suppressed all facets of the disease phenotype and prevented the increase in pMo numbers.

Gauging seizure risk.

The current paradigm for treatment of epilepsy begins with trials of antiepileptic drugs, followed by evaluation for resective brain surgery in drug-resistant patients. If surgery is not possible or fails to control seizures, some patients benefit from implanted neurostimulation devices. In addition to their therapeutic benefit, some of these devices have diagnostic capability enabling recordings of brain activity with unprecedented chronicity. Two recent studies using different devices for chronic EEG (i.e., over months to years) yielded convergent findings of daily and multiday cycles of brain activity that help explain seizure timing. Knowledge of these patient-specific cycles can be leveraged to gauge and forecast seizure risk, empowering patients to adopt risk-stratified treatment strategies and behavioral modifications. We review evidence that epilepsy is a cyclical disorder, and we argue that implanted monitoring devices should be offered earlier in the treatment paradigm. Chronic EEG would allow pharmacologic treatments tailored to days of high seizure risk-here termed chronotherapy-and would help characterize long timescale seizure dynamics to improve subsequent surgical planning. Coupled with neuromodulation, the proposed approach could improve quality of life for patients and decrease the number ultimately requiring resective surgery. We outline challenges for chronic monitoring and seizure forecasting that demand close collaboration among engineers, neurosurgeons, and neurologists.

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a dual role in myddosome formation and Toll-like receptor signaling.

Toll-like receptors (TLRs) form part of the host innate immune system, in which they act as sensors of microbial and endogenous danger signals. Upon TLR activation, the intracellular Toll/interleukin-1 receptor domains of TLR dimers initiate oligomerization of a multiprotein signaling platform comprising myeloid differentiation primary response 88 (MyD88) and members of the interleukin-1 receptor-associated kinase (IRAK) family. Formation of this myddosome complex initiates signal transduction pathways, leading to the activation of transcription factors and the production of inflammatory cytokines. To date, little is known about the assembly and disassembly of the myddosome and about the mechanisms by which these complexes mediate multiple downstream signaling pathways. Here, we isolated myddosome complexes from whole-cell lysates of TLR-activated primary mouse macrophages and from IRAK reporter macrophages to examine the kinetics of myddosome assembly and disassembly. Using a selective inhibitor of IRAK4's kinase activity, we found that whereas TLR cytokine responses were ablated, myddosome formation was stabilized in the absence of IRAK4's kinase activity. Of note, IRAK4 inhibition had only a minimal effect on NF-κB and mitogen-activated protein kinase (MAPK) signaling. In summary, our results indicate that IRAK4 has a critical scaffold function in myddosome formation and that its kinase activity is dispensable for myddosome assembly and activation of the NF-κB and MAPK pathways but is essential for MyD88-dependent production of inflammatory cytokines. Our findings suggest that the scaffold function of IRAK4 may be an attractive target for treating inflammatory and autoimmune diseases.

Effect of neurostimulation on cognition and mood in refractory epilepsy.

Epilepsy is a common, debilitating neurological disorder characterized by recurrent seizures. Mood disorders and cognitive deficits are common comorbidities in epilepsy that, like seizures, profoundly influence quality of life and can be difficult to treat. For patients with refractory epilepsy who are not candidates for resection, neurostimulation, the electrical modulation of epileptogenic brain tissue, is an emerging treatment alternative. Several forms of neurostimulation are currently available, and therapy selection hinges on relative efficacy for seizure control and amelioration of neuropsychiatric comorbidities. Here, we review the current evidence for how invasive and noninvasive neurostimulation therapies affect mood and cognition in persons with epilepsy. Invasive therapies include vagus nerve stimulation (VNS), deep brain stimulation (DBS), and responsive neurostimulation (RNS). Noninvasive therapies include trigeminal nerve stimulation (TNS), repetitive transcranial magnetic stimulation (rTMS), and transcranial direct current stimulation (tDCS). Overall, current evidence supports stable cognition and mood with all neurostimulation therapies, although there is some evidence that cognition and mood may improve with invasive forms of neurostimulation. More research is required to optimize the effects of neurostimulation for improvements in cognition and mood.

The mechanism of activation of IRAK1 and IRAK4 by interleukin-1 and Toll-like receptor agonists.

We have developed the first assays that measure the protein kinase activities of interleukin-1 receptor-associated kinase 1 (IRAK1) and IRAK4 reliably in human cell extracts, by employing Pellino1 as a substrate in conjunction with specific pharmacological inhibitors of IRAK1 and IRAK4. We exploited these assays to show that IRAK4 was constitutively active and that its intrinsic activity towards Pellino1 was not increased significantly by stimulation with interleukin-1 (IL-1) in IL-1R-expressing HEK293 cells, Pam3CSK4-stimulated human THP1 monocytes or primary human macrophages. Our results, in conjunction with those of other investigators, suggest that the IL-1-stimulated trans-autophosphorylation of IRAK4 is initiated by the myeloid differentiation primary response gene 88-induced dimerization of IRAK4 and is not caused by an increase in the intrinsic catalytic activity of IRAK4. In contrast with IRAK4, we found that IRAK1 was inactive in unstimulated cells and converted into an active protein kinase in response to IL-1 or Pam3CSK4 in human cells. Surprisingly, the IL-1-stimulated activation of IRAK1 was not affected by pharmacological inhibition of IRAK4 and not reversed by dephosphorylation and/or deubiquitylation, suggesting that IRAK1 catalytic activity is not triggered by a covalent modification but by an allosteric mechanism induced by its interaction with IRAK4.

Chronic ambulatory electrocorticography from human speech cortex.

Direct intracranial recording of human brain activity is an important approach for deciphering neural mechanisms of cognition. Such recordings, usually made in patients with epilepsy undergoing inpatient monitoring for seizure localization, are limited in duration and depend on patients' tolerance for the challenges associated with recovering from brain surgery. Thus, typical intracranial recordings, similar to most non-invasive approaches in humans, provide snapshots of brain activity in acute, highly constrained settings, limiting opportunities to understand long timescale and natural, real-world phenomena. A new device for treating some forms of drug-resistant epilepsy, the NeuroPace RNS® System, includes a cranially-implanted neurostimulator and intracranial electrodes that continuously monitor brain activity and respond to incipient seizures with electrical counterstimulation. The RNS System can record epileptic brain activity over years, but whether it can record meaningful, behavior-related physiological responses has not been demonstrated. Here, in a human subject with electrodes implanted over high-level speech-auditory cortex (Wernicke's area; posterior superior temporal gyrus), we report that cortical evoked responses to spoken sentences are robust, selective to phonetic features, and stable over nearly 1.5 years. In a second subject with RNS System electrodes implanted over frontal cortex (Broca's area, posterior inferior frontal gyrus), we found that word production during a naming task reliably evokes cortical responses preceding speech onset. The spatiotemporal resolution, high signal-to-noise, and wireless nature of this system's intracranial recordings make it a powerful new approach to investigate the neural correlates of human cognition over long timescales in natural ambulatory settings.

Multicentric Castleman's disease with voltage-gated potassium channel antibody-positive limbic encephalitis: a case report.

BACKGROUND: Castleman's disease is a rare lymphoproliferative disorder which occurs in localized and multicentric forms and can mimic lymphoma. Despite its well-known association with certain autoimmune diseases, including paraneoplastic pemphigus and myasthenia gravis, Castleman's disease has not previously been associated with limbic encephalitis. CASE PRESENTATION: We report the case of a 47-year old Caucasian man who presented with subacute onset of constitutional symptoms, diffuse lymphadenopathy, and stereotyped spells involving olfactory aura, nausea, disorientation, and unresponsiveness. He was found to have focal dyscognitive seizures of temporal lobe origin, cerebrospinal fluid with lymphocytic pleocytosis, hyponatremia, and serum positive for voltage-gated potassium channel antibodies, consistent with limbic encephalitis. An extensive infectious workup was unrevealing, but lymph node biopsy revealed multicentric Castleman's disease. His symptoms improved with antiepileptic drugs and immunotherapy. CONCLUSION: This case highlights the clinical diversity of voltage-gated potassium channel autoimmunity and expands the association of Castleman's disease and autoimmune syndromes to include limbic encephalitis. Clinicians should be aware that paraneoplastic disorders of the central nervous system can be related to underlying hematologic disorders such as Castleman's disease.

Interleukin 1/Toll-like receptor-induced autophosphorylation activates interleukin 1 receptor-associated kinase 4 and controls cytokine induction in a cell type-specific manner.

IRAK4 is a central kinase in innate immunity, but the role of its kinase activity is controversial. The mechanism of activation for IRAK4 is currently unknown, and little is known about the role of IRAK4 kinase in cytokine production, particularly in different human cell types. We show IRAK4 autophosphorylation occurs by an intermolecular reaction and that autophosphorylation is required for full catalytic activity of the kinase. Phosphorylation of any two of the residues Thr-342, Thr-345, and Ser-346 is required for full activity, and the death domain regulates the activation of IRAK4. Using antibodies against activated IRAK4, we demonstrate that IRAK4 becomes phosphorylated in human cells following stimulation by IL-1R and Toll-like receptor agonists, which can be blocked pharmacologically by a dual inhibitor of IRAK4 and IRAK1. Interestingly, in dermal fibroblasts, although complete inhibition of IRAK4 kinase activity does not inhibit IL-1-induced IL-6 production, NF-κB, or MAPK activation, there is complete ablation of these processes in IRAK4-deficient cells. In contrast, the inhibition of IRAK kinase activity in primary human monocytes reduces R848-induced IL-6 production with minimal effect on NF-κB or MAPK activation. Taken together, these studies define the mechanism of IRAK4 activation and highlight the differential role of IRAK4 kinase activity in different human cell types as well as the distinct roles IRAK4 scaffolding and kinase functions play.

Optogenetic delay of status epilepticus onset in an in vivo rodent epilepsy model.

Epilepsy is a devastating disease, currently treated with medications, surgery or electrical stimulation. None of these approaches is totally effective and our ability to control seizures remains limited and complicated by frequent side effects. The emerging revolutionary technique of optogenetics enables manipulation of the activity of specific neuronal populations in vivo with exquisite spatiotemporal resolution using light. We used optogenetic approaches to test the role of hippocampal excitatory neurons in the lithium-pilocarpine model of acute elicited seizures in awake behaving rats. Hippocampal pyramidal neurons were transduced in vivo with a virus carrying an enhanced halorhodopsin (eNpHR), a yellow light activated chloride pump, and acute seizure progression was then monitored behaviorally and electrophysiologically in the presence and absence of illumination delivered via an optical fiber. Inhibition of those neurons with illumination prior to seizure onset significantly delayed electrographic and behavioral initiation of status epilepticus, and altered the dynamics of ictal activity development. These results reveal an essential role of hippocampal excitatory neurons in this model of ictogenesis and illustrate the power of optogenetic approaches for elucidation of seizure mechanisms. This early success in controlling seizures also suggests future therapeutic avenues.

Proteomic characterization of the dynamic KSR-2 interactome, a signaling scaffold complex in MAPK pathway.

KSR-1 is a scaffold protein that is essential for Ras-induced activation of the highly conserved RAF-MEK-ERK kinase module. Previously, we identified a close homolog of KSR-1, called KSR-2, through structural homology-based data mining. In order to further understand the role of KSR-2 in MAPK signaling, we undertook a functional proteomics approach to elucidate the dynamic composition of the KSR-2 functional complex in HEK-293 cells under conditions with and without TNF-alpha stimulation. We found nearly 100 proteins that were potentially associated with KSR-2 complex and 43 proteins that were likely recruited to the super molecular complex after TNF-alpha treatment. Our results indicate that KSR-2 may act as a scaffold protein similar as KSR-1 to mediate the MAPK core (RAF-MEK-ERK) signaling but with a distinct RAF isoform specificity, namely KSR-2 may only mediate the A-RAF signaling while KSR-1 is responsible for transducing signals only from c-RAF. In addition, KSR-2 may be involved in the activation of many MAPK downstream signaling molecules such as p38 MAPK, IKAP, AIF, and proteins involved in ubiquitin-proteasome, apoptosis, cell cycle control, and DNA synthesis and repair pathways, as well as mediating crosstalks between MAPK and several other signaling pathways, including PI3K and insulin signaling. While interactions with these molecules are not known for KSR-1, it's reasonable to hypothesize that KSR-1 may also play a similar role in mediating these downstream signaling pathways.

hKSR-2 inhibits MEKK3-activated MAP kinase and NF-kappaB pathways in inflammation.

Kinase suppressor of ras (KSR) and MEKK3 (MAP kinase kinase kinase) are integral members of the MAP kinase pathway. We have recently identified a new isoform of the KSR family named human kinase suppressor of ras-2 (hKSR-2), and demonstrated that hKSR-2 negatively regulates Cot, a MAP3K family member which is important in inflammation and oncogenesis [P.L. Channavajhala, L. Wu, J.W. Cuozzo, J.P. Hall, W. Liu, L.L. Lin, Y. Zhang, J. Biol. Chem. 278 (2003) 47089-47097]. In this report, we provide evidence that hKSR-2 also regulates the activity of MEKK3 (another MAP3K family member) in HEK-293T cells. We demonstrate that hKSR-2 is a negative regulator of MEKK3-mediated activation of MAP kinase (specifically ERK and JNK) and NF-kappaB pathways, and concurrently inhibits MEKK3-mediated interleukin-8 production. We find that while hKSR-2 blocks MEKK3 activation, it has little to no effect on other members of the MAP3K family, including MEKK4, TAK1, and Ras-Raf, suggesting that its effects are selective.