scholarly journals Development of an Antiseizure Drug Screening Platform for Dravet Syndrome at the NINDS contract site for the Epilepsy Therapy Screening Program

2020 ◽  
Author(s):  
Chelsea D. Pernici ◽  
Jeffrey A. Mensah ◽  
Elizabeth J. Dahle ◽  
Kristina J. Johnson ◽  
Laura Handy ◽  
...  
Keyword(s):  
Drug Screening ◽  
Screening Program ◽  
Survival Rates ◽  
Peak Effect ◽  
Dravet Syndrome ◽  
Channel Blockers ◽  
List Type ◽  
Temperature Thresholds ◽  
Epilepsy Therapy ◽  
Screening Platform

SummaryObjectiveDravet syndrome (DS) is a rare, but catastrophic genetic epilepsy, with 80% of patients with carrying a mutation in the SCN1A gene. Currently, no anti-seizure drug (ASD) exists that adequately controls seizures. Patients with DS often present clinically with a febrile seizure and generalized tonic-clonic seizures that continue throughout life. To facilitate the development of ASDs for DS, the contract site of the NINDS Epilepsy Therapy Screening Program (ETSP) has evaluated a mouse model of DS using the conditional knock-in Scn1aA1783V/WT mouse.MethodsSurvival rates and temperature thresholds for Scn1aA1783V/WT were determined. Prototype ASDs were administered via intraperitoneal injections at the time-to-peak effect, which was previously determined, prior to the induction of hyperthermia-induced seizures. Protection was determined if ASDs significantly increased the temperature at which Scn1aA1783V/WT mice seized.ResultsApproximately 50% of Scn1aA1783V/WT survive to adulthood and all have hyperthermia-induce seizures. The results suggest that hyperthermia-induced seizures in this model of DS are highly refractory to a battery of ASDs. Exceptions were clobazam, tiagabine, and the combination of clobazam and valproic acid with add-on stiripentol, which elevated seizure thresholdsSignificanceOverall, the data demonstrate the proposed model for DS is suitable for screening novel compounds for the ability to block hyperthermia-induced seizures and heterozygous mice can be evaluated repeatedly over the course of several weeks, allowing for higher throughput screening.Key PointsScn1aA1783V/WT mice have a 50% survival rate and all have hyperthermia-induced seizures.Common DS treatments such as CLB and combinatorial therapy of CLB, VPA, and STP increase temperature thresholds in Scn1aA1783V/WT mice.Sodium channel blockers, such as CBZ and LTG, decrease temperature thresholds of Scn1aA1783V/WT mice as predicted.Scn1aA1783V/WT mice are highly pharmacoresitant to common ASDsThe Scn1aA1783V/WT may be a useful preclinical drug screening platform for the treatment of DS.

scholarly journals Development of an antiseizure drug screening platform for Dravet syndrome at the NINDS contract site for the Epilepsy Therapy Screening Program

Epilepsia ◽  
2021 ◽  
Author(s):  
Chelsea D. Pernici ◽  
Jeffrey A. Mensah ◽  
E. Jill Dahle ◽  
Kristina J. Johnson ◽  
Laura Handy ◽  
...  
Keyword(s):  
Drug Screening ◽  
Screening Program ◽  
Dravet Syndrome ◽  
Epilepsy Therapy ◽  
Screening Platform

scholarly journals Development of an Antiepileptogenesis Drug Screening Platform: Effects of Everolimus and Phenobarbital

2020 ◽  
Author(s):  
Melissa Barker-Haliski ◽  
Kevin M Knox ◽  
Dannielle K. Zierath ◽  
Zachery Koneval ◽  
Cameron Metcalf ◽  
...  
Keyword(s):  
Drug Screening ◽  
Rat Model ◽  
Screening Program ◽  
Sprague Dawley ◽  
Pharmacological Characterization ◽  
Relevant Animal Model ◽  
Dose Study ◽  
Disease Modifying ◽  
Screening Platform ◽  
Neurological Insult

Objective: The kainic acid (KA)-induced status epilepticus (SE) model in rats is an etiologically-relevant animal model of epileptogenesis. Just as in patients, who develop temporal lobe epilepsy (TLE) following SE, this rat model of KA-induced SE very closely recapitulates many of the clinical and pathological characteristics of human TLE that arise following SE or another neurological insult. Spontaneous recurrent seizures (SRS) in TLE can present after a latent period following a neurological insult (TBI, SE event, viral infection, etc.). Moreover, this rat model of TLE is ideally suited for preclinical studies to evaluate the long-term process of epileptogenesis and screen putative disease-modifying/antiepileptogenic agents. This report details the pharmacological characterization and methodological refinement of a moderate-throughput drug screening program using the post KA-induced SE model of epileptogenesis in male Sprague Dawley rats to identify potential agents that may prevent or modify the onset or severity of SRS. Specifically, we sought to demonstrate whether our protocol could prevent the development of SRS, or lead to a reduced frequency/severity of SRS. Methods: Rats were administered everolimus (2-3 mg/kg, P.O. commencing at 1, 2, or 24-hrs after SE onset) or phenobarbital (60 mg/kg, beginning 1 hr after SE onset). The rats in all studies (n=12/treatment dose/study) were then followed intermittently by video-EEG monitoring; i.e., 2-weeks on/2-weeks off, 2-weeks on epochs to determine latency to onset of SRS, and disease burden following SRS onset. Results: While there were no adverse side effects observed in any of our studies, no treatment conferred a significant disease modifying effect, nor did any agent prevent the presentation of SRS by 6 weeks post-SE onset. Conclusions: While neither phenobarbital nor everolimus administered at several time points post-SE onset prevented the development of SRS, we herein demonstrate a moderate-throughput screen for potential antiepileptogenic agents in an etiologically-relevant rodent model of TLE.

scholarly journals The Heat Sensing Trpv1 Receptor Is Not a Viable Anticonvulsant Drug Target in the Scn1a+/− Mouse Model of Dravet Syndrome

2021 ◽  
Vol 12 ◽  
Author(s):  
Vaishali Satpute Janve ◽  
Lyndsey L. Anderson ◽  
Dilara Bahceci ◽  
Nicole A. Hawkins ◽  
Jennifer A. Kearney ◽  
...  
Keyword(s):  
Mouse Model ◽  
Drug Target ◽  
Genetic Modifier ◽  
Anticonvulsant Drug ◽  
Dravet Syndrome ◽  
Seizure Threshold ◽  
Drug Resistant ◽  
Spontaneous Seizures ◽  
Temperature Thresholds ◽  
Seizure Models

Cannabidiol has been approved for the treatment of drug-resistant childhood epilepsies including Dravet syndrome (DS). Although the mechanism of anticonvulsant action of cannabidiol is unknown, emerging data suggests involvement of the transient receptor potential cation channel subfamily V member 1 (Trpv1). Pharmacological and genetic studies in conventional seizure models suggest Trpv1 is a novel anticonvulsant target. However, whether targeting Trpv1 is anticonvulsant in animal models of drug-resistant epilepsies is not known. Thus, we examined whether Trpv1 affects the epilepsy phenotype of the F1.Scn1a+/− mouse model of DS. We found that cortical Trpv1 mRNA expression was increased in seizure susceptible F1.Scn1a+/− mice with a hybrid genetic background compared to seizure resistant 129.Scn1a+/− mice isogenic on 129S6/SvEvTac background, suggesting Trpv1 could be a genetic modifier. Previous studies show functional loss of Trpv1 is anticonvulsant. However, Trpv1 selective antagonist SB-705498 did not affect hyperthermia-induced seizure threshold, frequency of spontaneous seizures or survival of F1.Scn1a+/− mice. Surprisingly, Trpv1 deletion had both pro- and anti-seizure effects. Trpv1 deletion did not affect hyperthermia-induced seizure temperature thresholds of F1.Scn1a+/−; Trpv1+/− at P14-16 but was proconvulsant at P18 as it reduced seizure temperature thresholds. Conversely, Trpv1 deletion did not alter the frequency of spontaneous seizures but reduced their severity. These results suggest that Trpv1 is a modest genetic modifier of spontaneous seizure severity in the F1.Scn1a+/− model of DS. However, the opposing pro- and anti-seizure effects of Trpv1 deletion and the lack of effects of Trpv1 inhibition suggest that Trpv1 is unlikely a viable anticonvulsant drug target in DS.

scholarly journals Dravet variant SCN1AA1783V impairs interneuron firing predominantly by altered channel activation

2021 ◽  
Author(s):  
N. Layer ◽  
L. Sonnenberg ◽  
E. Pardo González ◽  
J. Benda ◽  
H. Lerche ◽  
...  
Keyword(s):  
Voltage Dependence ◽  
Dravet Syndrome ◽  
Slice Culture ◽  
List Type ◽  
Slow Inactivation ◽  
Loss Of Function ◽  
Peak Current Density ◽  
Action Potential Firing ◽  
Channel Activation

AbstractDravet syndrome (DS) is a developmental epileptic encephalopathy mainly caused by functional NaV1.1 haploinsufficiency in interneurons (IN). Recently, a new conditional mouse model expressing the recurrent human p.A1783V missense variant has become available. Here we provide an electrophysiological characterization of this variant in tsA201 cells, revealing both altered voltage-dependence of activation and slow inactivation without reduced sodium peak current density. Simulating IN excitability in a Hodgkin-Huxley one-compartment model suggested surprisingly similar firing deficits for Scn1aA1783V and full haploinsufficiency as caused by heterozygous truncation variants. Impaired NaVA1783V channel activation was predicted to have a significantly larger impact on channel function than altered slow inactivation and is therefore proposed as the main mechanism underlying IN dysfunction. The computational model was validated in cortical organotypic slice cultures derived from conditional Scn1aA1783V mice. Pan-neuronal activation of the p.A1783V variant in vitro confirmed the predicted IN firing deficit while demonstrating normal excitability of pyramidal neurons. Taken together these data demonstrate that despite maintained physiological peak currents density LOF gating properties may match effects of full haploinsufficiency on neuronal level, thereby causing DS.HighlightsNaV1.1A1783V alters voltage-dependence of activation and slow inactivation while not affecting fast inactivation.Depolarizing and hyperpolarizing shifts of activation and slow inactivation curves result in combined channel loss of function (LOF).Simulations of NaV1.1A1783V interneuronal properties indicate reduced action potential firing rates comparable to full SCN1A haploinsufficiency, which is often found in Dravet syndrome.In silico modelling identifies impaired channel activation as the predominant mechanism of channel LOF.Panneuronal induction of Scn1a+/A1783V in a cortical slice culture model confirms restriction of loss of function and its restriction to interneurons.

scholarly journals A versatile automated high-throughput drug screening platform for zebrafish embryos

2020 ◽  
Author(s):  
Alexandra Lubin ◽  
Jason Otterstrom ◽  
Yvette Hoade ◽  
Ivana Bjedov ◽  
Eleanor Stead ◽  
...  
Keyword(s):  
High Throughput ◽  
Drug Screening ◽  
Imaging System ◽  
Cell Types ◽  
Wide Range ◽  
Stem And Progenitor Cells ◽  
Multiple Cell ◽  
Flexible Imaging ◽  
Automated Screening ◽  
Screening Platform

AbstractZebrafish provide a unique opportunity for drug screening in living animals, with the fast developing, transparent embryos allowing for relatively high throughput, microscopy-based screens. However, the limited availability of rapid, flexible imaging and analysis platforms has limited the use of zebrafish in drug screens. We have developed a easy-to-use, customisable automated screening procedure suitable for high-throughput phenotype-based screens of live zebrafish. We utilised the WiScan®Hermes High Content Imaging System to rapidly acquire brightfield and fluorescent images of embryos, and the WiSoft®Athena Zebrafish Application for analysis, which harnesses an Artificial Intelligence-driven algorithm to automatically detect fish in brightfield images, identify anatomical structures, partition the animal into regions, and exclusively select the desired side-oriented fish. Our initial validation combined structural analysis with fluorescence images to enumerate GFP-tagged haematopoietic stem and progenitor cells in the tails of embryos, which correlated with manual counts. We further validated this system to assess the effects of genetic mutations and x-ray irradiation in high content using a wide range of assays. Further, we performed simultaneous analysis of multiple cell types using dual fluorophores in high throughput. In summary, we demonstrate a broadly applicable and rapidly customisable platform for high content screening in zebrafish.

scholarly journals Testing Tuberculosis Drug Efficacy in a Zebrafish High-Throughput Translational Medicine Screen

2014 ◽  
Vol 59 (2) ◽  
pp. 753-762 ◽  
Author(s):  
Anita Ordas ◽  
Robert-Jan Raterink ◽  
Fraser Cunningham ◽  
Hans J. Jansen ◽  
Malgorzata I. Wiweger ◽  
...  
Keyword(s):  
High Throughput ◽  
Drug Screening ◽  
Drug Efficacy ◽  
Drug Uptake ◽  
Spectrometric Analysis ◽  
Major Advance ◽  
Zebrafish Model ◽  
Content Type ◽  
Screening Platform

ABSTRACTThe translational value of zebrafish high-throughput screens can be improved when more knowledge is available on uptake characteristics of potential drugs. We investigated reference antibiotics and 15 preclinical compounds in a translational zebrafish-rodent screening system for tuberculosis. As a major advance, we have developed a new tool for testing drug uptake in the zebrafish model. This is important, because despite the many applications of assessing drug efficacy in zebrafish research, the current methods for measuring uptake using mass spectrometry do not take into account the possible adherence of drugs to the larval surface. Our approach combines nanoliter sampling from the yolk using a microneedle, followed by mass spectrometric analysis. To date, no single physicochemical property has been identified to accurately predict compound uptake; our method offers a great possibility to monitor how any novel compound behaves within the system. We have correlated the uptake data with high-throughput drug-screening data fromMycobacterium marinum-infected zebrafish larvae. As a result, we present an improved zebrafish larva drug-screening platform which offers new insights into drug efficacy and identifies potential false negatives and drugs that are effective in zebrafish and rodents. We demonstrate that this improved zebrafish drug-screening platform can complement conventional models ofin vivoMycobacterium tuberculosis-infected rodent assays. The detailed comparison of two vertebrate systems, fish and rodent, may give more predictive value for efficacy of drugs in humans.

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