Both gain- and loss-of-function variants of KCNA1 are associated with paroxysmal kinesigenic dyskinesia.

KCNA1 is the coding gene for Kv1.1 voltage-gated potassium-channel α subunit. Three variants of KCNA1 have been reported to manifest as paroxysmal kinesigenic dyskinesia (PKD), but the correlation between them remains unclear due to the phenotypic complexity of KCNA1 variants as well as the rarity of PKD cases. Using the whole exome sequencing followed by Sanger sequencing, we screen for potential pathogenic KCNA1 variants in patients clinically diagnosed with paroxysmal movement disorders and identify three previously unreported missense variants of KCNA1 in three unrelated Chinese families. The proband of one family (c.496G>A, p.A166T) manifests as episodic ataxia type 1, and the other two (c.877G>A, p.V293I and c.1112C>A, p.T371A) manifest as PKD. The pathogenicity of these variants is confirmed by functional studies, suggesting that p.A166T and p.T371A cause a loss-of-function of the channel, while p.V293I leads to a gain-of-function with the property of voltage-dependent gating and activation kinetic affected. By reviewing the locations of PKD-manifested KCNA1 variants in Kv1.1 protein, we find that these variants tend to cluster around the pore domain, which is similar to epilepsy. Thus, our study strengthens the correlation between KCNA1 variants and PKD and provides more information on genotype-phenotype correlations of KCNA1 channelopathy.

Guiding Epilepsy Surgery with an LRP1-Targeted SPECT/SERRS Dual-Mode Imaging Probe.

Accurate identification of the resectable epileptic lesion is a precondition of operative intervention to drug-resistant epilepsy (DRE) patients. However, even when multiple diagnostic modalities are combined, epileptic foci cannot be accurately identified in ∼30% of DRE patients. Inflammation-associated low-density lipoprotein receptor-related protein-1 (LRP1) has been validated to be a surrogate target for imaging epileptic foci. Here, we reported an LRP1-targeted dual-mode probe that is capable of providing comprehensive epilepsy information preoperatively with SPECT imaging while intraoperatively delineating epileptic margins in a sensitive high-contrast manner with surface-enhanced resonance Raman scattering (SERRS) imaging. Notably, a novel and universal strategy for constructing self-assembled monolayer (SAM)-based Raman reporters was proposed for boosting the sensitivity, stability, reproducibility, and quantifiability of the SERRS signal. The probe showed high efficacy to penetrate the blood-brain barrier. SPECT imaging showed the probe could delineate the epileptic foci clearly with a high target-to-background ratio (4.11 ± 0.71, 2 h). Further, with the assistance of the probe, attenuated seizure frequency in the epileptic mouse models was achieved by using SPECT together with Raman images before and during operation, respectively. Overall, this work highlights a new strategy to develop a SPECT/SERRS dual-mode probe for comprehensive epilepsy surgery that can overcome the brain shift by the co-registration of preoperative SPECT and SERRS intraoperative images.

Functionalized PEG-PLA nanoparticles for brain targeted delivery of ketoconazole contribute to pregnane X receptor overexpressing in drug-resistant epilepsy.

OBJECTIVE: To develop a functionalized PEG-PLA nanoparticle system containing ketoconazole (KCZ) to overcome the overactivity of pregnane X receptor (PXR) for the treatment of drug-resistant epilepsy (DRE). SIGNIFICANCE: KCZ was developed as a therapy strategy for DRE limited by its lethal hepatotoxicity and minute brain concentration. KCZ-incorporated nanoparticles modified with angiopep-2 (NPs/KCZ) could reduce adverse effects of KCZ and achieve epileptic foci-targeted drug delivery. METHODS: NPs/KCZ was prepared by thin-film hydration method and characterized in vitro and in vivo. The efficacy evaluation of NPs/KCZ was evaluated in a kainic acid (KA)-induced mice model of epilepsy with carbamazepine (CBZ) treatment. RESULTS: The mean particle size and Zeta potential of NPs/KCZ were 17.84 ± 0.33 nm and - 2.28 ± 0.12 mV, respectively. The drug-loading (DL%) of KCZ in nanoparticles was 8.96 ± 0.12 % and the entrapment efficiency (EE%) was 98.56 ± 0.02 %. The critical value of critical micelle concentration was 10-3.3 mg/ml. No obvious cytotoxicity was found in vitro. The behavioral and electrographic seizure activities were obviously attenuated in NPs/KCZ+CBZ group. The CBZ concentration of brain tissues in mice treated with NPs/KCZ+CBZ was significantly increased than those treated with CBZ alone (P = 0.0028). A significantly decreased expression level of PXR and its downstream proteins was observed in NPs/KCZ+CBZ group compared with that in the control and CBZ group (All P < 0.05). CONCLUSION: Our results showed that NPs/KCZ achieved the epileptic foci-targeted delivery of KCZ and ameliorated the efficacy of CBZ on DRE by attenuating the overactivity of PXR.

Tailoring Materials for Epilepsy Imaging: From Biomarkers to Imaging Probes.

Excising epileptic foci (EF) is the most efficient approach for treating drug-resistant epilepsy (DRE). However, owing to the vast heterogeneity of epilepsies, EF in one-third of patients cannot be accurately located, even after exhausting all current diagnostic strategies. Therefore, identifying biomarkers that truly represent the status of epilepsy and fabricating probes with high targeting specificity are prerequisites for identifying the "concealed" EF. However, no systematic summary of this topic has been published. Herein, the potential biomarkers of EF are first summarized and classified into three categories: functional, molecular, and structural aberrances during epileptogenesis, a procedure of nonepileptic brain biasing toward epileptic tissue. The materials used to fabricate these imaging probes and their performance in defining the EF in preclinical and clinical studies are highlighted. Finally, perspectives for developing the next generation of probes and their challenges in clinical translation are discussed. In general, this review can be helpful in guiding the development of imaging probes defining EF with improved accuracy and holds promise for increasing the number of DRE patients who are eligible for surgical intervention.

Imaging epileptic foci in mouse models via a low-density lipoprotein receptor-related protein-1 targeting strategy.

BACKGROUND: In the setting of drug-resistant epilepsy (DRE), the success of surgery depends on the ability to accurately locate the epileptic foci to be resected or disconnected. However, the epileptic foci in a considerable percentage of the DRE patients cannot be adequately localised. This warrants the need for a reliable imaging strategy to identify the "concealed" epileptic regions. METHODS: Brain specimens from DRE patients and kainate-induced epileptic mouse models were immuno-stained to evaluate the integrity of the blood-brain barrier (BBB). The expression of low-density lipoprotein receptor-related protein-1 (LRP1) in the epileptic region of DRE patients and kainate models was studied by immunofluorescence. A micellar-based LRP1-targeted paramagnetic probe (Gd3+-LP) was developed and its ability to define the epileptic foci was investigated by magnetic resonance imaging (MRI). FINDINGS: The integrity of the BBB in the epileptic region of DRE patients and kainate mouse models were demonstrated. LRP1 expression levels in the epileptic foci of DRE patients and kainate models were 1.70-2.38 and 2.32-3.97 folds higher than in the control brain tissues, respectively. In vivo MRI demonstrated that Gd3+-LP offered 1.68 times higher (P < 0.05) T1-weighted intensity enhancement in the ipsilateral hippocampus of chronic kainite models than the control probe without LRP1 specificity. INTERPRETATION: The expression of LRP1 is up-regulated in vascular endothelium, activated glia in both DRE patients and kainate models. LRP1-targeted imaging strategy may provide an alternative strategy to define the "concealed" epileptic foci by overcoming the intact BBB. FUNDING: This work was supported by the National Natural Science Foundation, Shanghai Science and Technology Committee, Shanghai Municipal Science and Technology, Shanghai Municipal Health and Family Planning Commission and the National Postdoctoral Program for Innovative Talents.

An electric-field-responsive paramagnetic contrast agent enhances the visualization of epileptic foci in mouse models of drug-resistant epilepsy.

For patients with drug-resistant focal epilepsy, excision of the epileptogenic zone is the most effective treatment approach. However, the surgery is less effective in the 15-30% of patients whose lesions are not distinct when visualized by magnetic resonance imaging (MRI). Here, we show that an intravenously administered MRI contrast agent consisting of a paramagnetic polymer coating encapsulating a superparamagnetic cluster of ultrasmall superparamagnetic iron oxide crosses the blood-brain barrier and improves lesion visualization with high sensitivity and target-to-background ratio. In kainic-acid-induced mouse models of drug-resistant focal epilepsy, electric-field changes in the brain associated with seizures trigger breakdown of the contrast agent, restoring the T1-weighted magnetic resonance signal, which otherwise remains quenched due to the distance-dependent magnetic resonance tuning effect between the cluster and the coating. The electric-field-responsive contrast agent may increase the probability of detecting seizure foci in patients and facilitate the study of brain diseases associated with epilepsy.

Activation of Kir2.3 Channels by Tenidap Suppresses Epileptiform Burst Discharges in Cultured Hippocampal Neurons.

BACKGROUND & OBJECTIVE: Tenidap, a selective human inwardly rectifying potassium (Kir) 2.3 channel opener, has been reported to have antiepileptic effect in the pilocarpine temporal lobe epilepsy rat model in our previous study. However, the effect of tenidap on neurons and its relationship with the epileptiform bursting charges in neuron is still required to be explored. METHODS: In this study, cyclothiazide (CTZ) induced cultured hippocampal neuron epileptic model was used to study the antiepileptic effect of tenidap and the relationship between Kir2.3 channel and the neuronal epileptiform burst. RESULTS: Patch clamp recording showed that both acute (2h) and chronic (48h) CTZ pre-treatment all significantly induced robust epileptiform burst activities in cultured hippocampal neurons, and tenidap acutely application inhibited this highly synchronized abnormal activities. The effect of tenidap is likely due to increased activity of Kir2.3 channels, since tenidap significantly enhanced kir current recorded from those neurons. In addition, neurons overexpressing Kir2.3 channels, by transfection with Kir2.3 plasmid, showed a significant large increase of the Kir current, prevented CTZ treatment to induce epileptiform burst discharge. CONCLUSION: Our current study demonstrated that over activation of Kir2.3 channel in hippocampal neurons could positively interference with epileptiform burst activities, and tenidap, as a selective Kir2.3 channel opener, could be a potential candidate for seizure therapy.

The anticonvulsant and neuroprotective effects of kir2.3 activation in PTZ-induced seizures and the kainic acid model of TLE.

PURPOSE: To elucidate the role of activating the inwardly rectifying K+ channel 2.3 (Kir2.3) in acute seizure and chronic epilepsy, we investigated the effect of a Kir2.3 agonist (tenidap) on epileptic and electrophysiological activities in mice. Neuronal excitability and damage were also evaluated. METHODS: A Pentylenetetrazole (PTZ)-induced acute seizure model and a kainic acid (KA)-induced temporal epilepsy model were used in adult mice. The mice were given tenidap 30 min before PTZ injection or were given tenidap for 7 days after entering the chronic stage of the KA model. Video monitoring and EEG recordings were performed for comparisons. Immunofluorescence of c-fos was detected in the PTZ model, and Nissl staining was performed in the KA model. RESULTS: Tenidap intervention significantly reduced the duration and severity of PTZ-induced acute seizures, which conformed with the power-spectrum analyses of the EEG and the quantification of spikes on EEG. C-fos expression representing neuronal excitability was also reduced with tenidap pretreatment. However, the latency time to seizure onset was unaltered. Seven days of tenidap treatment in the chronic KA model significantly attenuated seizure and spike frequencies compared to the same animal before administration. Nissl staining showed reduced hilar neuron loss in the tenidap-intervention group but showed no difference in the width of the granule cell layer. CONCLUSION: To our knowledge, few studies have reported the relevance of Kir2.3 to epilepsy. The present data suggested that activation of Kir2.3 exerts an anticonvulsant effect in acute seizures and the chronic stage of TLE, which makes this channel a potent therapeutic target.

Blood concentration, efficacy, and adverse events of phenobarbital: A prospective study in rural China.

OBJECTIVE: This study evaluated the relationship between blood concentration of phenobarbital (PB) and its efficacy as well as adverse events in people with epilepsy in rural China. METHODS: People with epilepsy being treated with PB monotherapy were recruited and followed up for averagely 2.5 years. Data of clinical characteristics were collected using a standardized questionnaire by face-to-face interviews both at baseline and follow-up. Plasma concentration of PB was detected by the high-performance liquid chromatography. RESULTS: Data on treatment response and PB blood concentration was obtained from 225 subjects. Among them, 119 (52.9%) were recognized as effective cases and 106 (47.1%) as ineffective cases. In the effective group, the blood concentration of 95% subjects ranged from 1.22 µg/ml to 41.36 µg/ml with a median at 13.18 µg/ml (IQR = 8.32-20.19 µg/ml). The PB concentration of 95% of the subjects in the ineffective group ranged from 2.73 µg/ml to 70.16 µg/ml with a median at 19.80 µg/ml (IQR = 11.30-30.40 µg/ml), which was significantly higher than that of the effective group (p < 0.001). Multivariate logistic regression analysis showed that PB concentration ≥26.38 µg/ml was related to a 4.5-fold (95% confidence interval [CI], 1.85-11.08) higher risk of inefficacy. A receiver operation characteristic curve was performed to determine the cutoff value of concentration for PB efficacy at 19.02 µg/ml. SIGNIFICANCE: Blood concentration may be an important indicator for clinical decision making when PB monotherapy cannot achieve a good efficacy and more attention should be paid on it in clinical practice especially in resource-poor settings.