CHD2 variants are a risk factor for photosensitivity in epilepsy

Photosensitivity is a heritable abnormal cortical response to flickering light, manifesting as particular electroencephalographic changes, with or without seizures. Photosensitivity is prominent in a very rare epileptic encephalopathy due to de novo CHD2 mutations, but is also seen in epileptic encephalopathies due to other gene mutations. We determined whether CHD2 variation underlies photosensitivity in common epilepsies, specific photosensitive epilepsies and individuals with photosensitivity without seizures. We studied 580 individuals with epilepsy and either photosensitive seizures or abnormal photoparoxysmal response on electroencephalography, or both, and 55 individuals with photoparoxysmal response but no seizures. We compared CHD2 sequence data to publicly available data from 34 427 individuals, not enriched for epilepsy. We investigated the role of unique variants seen only once in the entire data set. We sought CHD2 variants in 238 exomes from familial genetic generalized epilepsies, and in other public exome data sets. We identified 11 unique variants in the 580 individuals with photosensitive epilepsies and 128 unique variants in the 34 427 controls: unique CHD2 variation is over-represented in cases overall (P = 2·17 × 10−5). Among epilepsy syndromes, there was over-representation of unique CHD2 variants (3/36 cases) in the archetypal photosensitive epilepsy syndrome, eyelid myoclonia with absences (P = 3·50 × 10−4). CHD2 variation was not over-represented in photoparoxysmal response without seizures. Zebrafish larvae with chd2 knockdown were tested for photosensitivity. Chd2 knockdown markedly enhanced mild innate zebrafish larval photosensitivity. CHD2 mutation is the first identified cause of the archetypal generalized photosensitive epilepsy syndrome, eyelid myoclonia with absences. Unique CHD2 variants are also associated with photosensitivity in common epilepsies. CHD2 does not encode an ion channel, opening new avenues for research into human cortical excitability

Identification of GSK-3 as a potential therapeutic entry point for epilepsy

In view of the clinical need for new antiseizure drugs (ASDs) with novel modes of action, we used a zebrafish seizure model to screen the anticonvulsant activity of medicinal plants used by traditional healers in the Congo for the treatment of epilepsy, and identified a crude plant extract that inhibited pentylenetetrazol (PTZ)-induced seizures in zebrafish larvae. Zebrafish bioassay-guided fractionation of this anticonvulsant Fabaceae species, Indigofera arrecta, identified indirubin, a compound with known inhibitory activity of glycogen synthase kinase (GSK)-3, as the bioactive component. Indirubin, as well as the more potent and selective GSK-3 inhibitor 6-bromoindirubin-3'-oxime (BIO-acetoxime) were tested in zebrafish and rodent seizure assays. Both compounds revealed anticonvulsant activity in PTZ-treated zebrafish larvae, with electroencephalographic recordings revealing reduction of epileptiform discharges. Both indirubin and BIO-acetoxime also showed anticonvulsant activity in the pilocarpine rat model for limbic seizures and in the 6-Hz refractory seizure mouse model. Most interestingly, BIO-acetoxime also exhibited anticonvulsant actions in 6-Hz fully kindled mice. Our findings thus provide the first evidence for anticonvulsant activity of GSK-3 inhibition, thereby implicating GSK-3 as a potential therapeutic entry point for epilepsy. Our results also support the use of zebrafish bioassay-guided fractionation of antiepileptic medicinal plant extracts as an effective strategy for the discovery of new ASDs with novel mechanisms of action

Development and validation of zebrafish seizure models for drug discovery

Tatiana Afrikanova 14.00 Validation of an animal model constitutes a large partof successful drug development. In this thesis we describe the development andvalidation of in vivo models of seizures in zebrafish larvae. The process of building an animal model forneurological disorders consists of several steps of development and systematicevaluation of their quality and relevance. Based on a hypothesis aboutbrain-behavior relationships, a model is developed and tested. Validation of a model is a scientific method toimprove the confidence in a model. Validity is defined as the agreement betweena test score and the quality it is believed to measure. Validity is restrictedto a specific use of a model, and must always be open for discussion andre-evaluation. Most animal models are evaluated based on the criteria proposedby Willner: construct, predictive and face validity. Other important factorsare animal welfare, reliability and replicability as well as cross-speciessimilarity. Epilepsy is one of the most common neurologicaldisorders with a high disabling risk; the main symptom of epilepsy is theoccurrence of seizures. An epileptic seizure (according to ILAE) is a transientoccurrence of signs and/or symptoms due to abnormal excessive or synchronousneuronal activity in the brain. Epilepsy is defined as a disorder of the braincharacterized by en enduring predisposition to generate epileptic seizures andby the neurobiologic, cognitive, physiological, and social consequences of thiscondition. There are cases reported where seizures stop occurringspontaneously (self-resolving). Still, in the majority of patients, adequateseizure control or even seizure freedom is achieved with the help ofantiepileptic drugs (AEDs). To date, there are several generations of AEDs onthe market with different or multiple mechanisms of action available,nevertheless, about one third of people diagnosed with epilepsy do not respondto the prescribed treatment. Some of the non-responders can be cured bysurgical intervention removing the brain area involved in seizure generation.Other alternative treatments include ketogenic diet and vagus nervestimulation. To start with, epilepsy involves excessive and/orhighly synchronized neuronal firing, which is otherwise a normal function ofthe brain. Therefore, due to the complexity of disease it s not possible tomodel it in a biochemical in vitro assay. The use of the existing -predominantly rodent -models of seizures and epilepsy is limited due to theassay costs, timing and labour-intensity despite the value of the data theyprovide. The constant trade of validity against resourceconsumption leads us to a different approach in model building. It is possibleto use smaller vertebrates to increase the assay s throughput. On the otherhand, the decrease in construct validity in this case can be partially compensated by the convenience of geneticmanipulation, visual transparency and automation solutions. Assuming that asimpler organism feels less pain, ethical considerations also support zebrafishresearch, as the zebrafish larvae are not even considered laboratory animalsbefore day 6 of life. Zebrafish video tracking haverecently emerged as an attractive in vivo model for epilepsy.Seven-day-old zebrafish larvae exposed to the GABAA antagonistpentylenetetrazol (PTZ) exhibit increased locomotor activity, seizure-likebehavior, and epileptiform electrographic activity. A previous study showedthat 12 out of 13 AEDs suppressed PTZ-mediated increases in larval movement,indicating the potential utility of zebrafish as a high-throughput in vivo modelfor AED discovery. However, a question remained as to whether an AED-induceddecrease in locomotion is truly indicative of anticonvulsant activity, as somedrugs may impair larval movement through other mechanisms such as generaltoxicity or sedation. We therefore carried out a study in PTZ-treated zebrafishlarvae, to directly compare the ability of AEDs to inhibit seizure-likebehavioral manifestations with their capacity to suppress epileptiformelectrographic activity. We re-tested the 13 AEDs of which 12 were previouslyreported to inhibit convulsions in the larval movement tracking assay, administeringconcentrations that did not, on their own, impair locomotion. In parallel, wequantified the parameters of open-field recordings on larval brains aftertreatment with each AED. For 10 of AEDs tested we obtained the same response inboth the behavioral and electrographic assays. Overall our data correlate wellwith those reported in the literature for acute rodent PTZ tests, indicatingthat the larval zebrafish brain is more discriminatory than previously thoughtin its response to AEDs with different modes of action. Our results underscorethe validity of using the zebrafish larval locomotor assay as a rapidfirst-pass screening tool in assessing the anticonvulsant and/or proconvulsantactivity of compounds, but also highlight the importance of performing adequatevalidation when using in vivo models. The other question explored was ifzebrafish could be used as a model organism for treatment-resistant seizures.Refractory seizures affect about a third of patients suffering from epilepsy.To fulfill the need for new medications targeting these difficult-to-treatcases, chronic rodent models offer the opportunity to study mechanisms of drugresistance. The use of such models, however, has been rather limited, as allhave proven to be time-consuming and labor-intensive. In this study, wecharacterize the allylglycine (AG) seizure model, a simple in vivo modelshowing a high level of treatment resistance. (D,L)-Allylglycine inhibitsglutamic acid decarboxylase the key enzyme in GABA biosynthesis leading toGABA depletion, seizures and neuronal damage. We performed a side-by-sidecomparison of mouse and zebrafish acute AG treatment including biochemical,electrographic and behavioral assessment. Interestingly, seizure progressionrate and GABA depletion kinetics were comparable in both species. Fivemechanistically diverse AEDs (valproate, diazepam, levetiracetam, phenytoin andtopiramate) were used. All tested AEDs showed a limited protective effect(mainly mortality delay) at doses close to TD50 (doseinducing motor impairment in 50% of animals) in mice; experiments in fishdemonstrated a complete protective effect ofVPA, while other AEDs were only partially or not active in the assays run.Having demonstrated cross-species similarities, we conclude that the use of AGin zebrafish could allow for high-throughput drug screening and thereforepropose it as a convenient model in the context of treatment-resistantseizures. Normal 0 21 false false false EN-US X-NONE X-NONE <w:LsdException Locked="false" Priority="19" SemiHidden="false" UnhideWhenUsed="false" QFormat="true" Name="Subtle Em

Rotavirus NSP5 phosphorylation is up-regulated by interaction with NSP2

We have previously shown that a number of isoforms of the non-structural rotavirus protein NSP5 are found in virus-infected cells. These isoforms differ in their level of phosphorylation which, at least in part, appears to occur through autophosphorylation. NSP5 co-localizes with another non-structural protein, NSP2, in the viroplasms of infected cells where virus replication takes place. We now show that NSP5 can be chemically cross-linked in living cells with the viral polymerase VP1 and NSP2. Interaction of NSP5 with NSP2 was also demonstrated by coimmunoprecipitation of NSP2 and NSP5 from extracts of UV-treated rotavirus-infected cells. In addition, in transient transfection assays, NSP5 phosphorylation in vivo was enhanced by coexpression of NSP2. An NSP5 C-terminal domain deletion mutant, was completely unable to be phosphorylated either in the presence or absence of NSP2. However, a 33 aa N-terminal deletion mutant of NSP5 was shown to become hyperphosphorylated in vivo and to be insensitive to NSP2 activation, suggesting a regulatory role for this domain in NSP5 phosphorylation and making it a candidate for the interaction with NSP2. These mutants also allow a preliminary mapping of NSP5 autophosphorylation activity

Analysis of antibiotic resistance markers in Chlamydia trachomatis clinical isolates obtained after ineffective antibiotic therapy

Antibiotic resistance markers were analyzed in C. trachomatis clinical isolates obtained after ineffective therapy of urogenital chlamydiasis with fluoroquinolones and macrolides. Heterotypical resistance to fluoroquinolones and macrolides was detected in all clinical isolates. No significant mutations in the target genes were detected.status: publishe