Protein profiling in plasma for biomarkers of seizure.

PURPOSE: A biochemical way to measure seizures would greatly benefit epilepsy research and clinical follow-up. Short-term biomarkers like lactate exist, and interest in biomarkers representative of longer-term seizure burden is growing. In this exploratory study, we aimed to identify markers in blood plasma that differentiate persons with recent seizures from persons with epilepsy and long-standing seizure freedom. METHODS: A proteomic analysis was performed on plasma samples of 120 persons with seizures using the Olink Neuro-exploratory panel. Participants were selected from a regional biobank study in Västra Götaland (Sweden) and categorized into two groups: recent seizure and seizure-free. The panel contained 92 proteins linked to neurological diseases and processes, and levels of these proteins were compared between the patient groups to identify potential markers of seizure activity. RESULTS: We identified significant differences in protein levels between the recent seizure and seizure-free patient groups for Cadherin-15 [(CDH15; p = 0.008)], Latent transforming growth factor beta-binding protein 3 [(LTBP3; p = 0.002)], Phosphoethanolamine/phosphocholine phosphatase 1 [(PHOSPHO1; p = 0.011)], and Progestagen associated endometrial protein [(PAEP; p = 0.0005)]. CONCLUSION: The findings in this study present CDH15, LTBP3, PHOSPHO1 and PAEP as candidate markers of seizure activity. Further confirmatory studies are needed.

Neurofilament light, glial fibrillary acidic protein, and tau in a regional epilepsy cohort: High plasma levels are rare but related to seizures.

OBJECTIVE: Higher levels of biochemical blood markers of brain injury have been described immediately after tonic-clonic seizures and in drug-resistant epilepsy, but the levels of such markers in epilepsy in general have not been well characterized. We analyzed neurofilament light (NfL), glial fibrillary acidic protein (GFAP), and tau in a regional hospital-based epilepsy cohort and investigated what proportion of patients have levels suggesting brain injury, and whether certain epilepsy features are associated with high levels. METHODS: Biomarker levels were measured in 204 patients with an epilepsy diagnosis participating in a prospective regional biobank study, with age and sex distribution correlating closely to that of all patients seen for epilepsy in the health care region. Absolute biomarker levels were assessed between two patient groups: patients reporting seizures within the 2 months preceding inclusion and patients who did not have seizures for more than 1 year. We also assessed the proportion of patients with above-normal levels of NfL. RESULTS: NfL and GFAP, but not tau, increased with age. Twenty-seven patients had abnormally high levels of NfL. Factors associated with such levels were recent seizures (p = .010) and epileptogenic lesion on radiology (p = .001). Levels of NfL (p = .006) and GFAP (p = .032) were significantly higher in young patients (<65 years) with seizures ≤2 months before inclusion compared to those who reported no seizures for >1 year. NfL and GFAP correlated weakly with the number of days since last seizure (NfL: rs = -.228, p = .007; GFAP: rs = -.167, p = .048) in young patients. NfL also correlated weakly with seizure frequency in the last 2 months (rs = .162, p = .047). SIGNIFICANCE: Most patients with epilepsy do not have biochemical evidence of brain injury. The association with seizures merits further study; future studies should aim for longitudinal sampling and examine whether individual variations in NfL or GFAP levels could reflect seizure activity.

CSF biomarkers in patients with epilepsy in Alzheimer's disease: a nation-wide study.

Alzheimer's disease is the most common neurodegenerative dementia. A subset of Alzheimer's disease patients develop epilepsy. The risk is higher in young-onset Alzheimer's disease, but pathophysiological mechanisms remain elusive. The purpose of this study was to assess biomarkers reflecting neurodegeneration in Alzheimer's disease patients with and without epilepsy. By cross-referencing the largest national laboratory database with Swedish National Patient Register, we could identify CSF biomarker results from 17901 Alzheimer's disease patients, and compare levels of neurofilament light, glial fibrillary acidic protein, total tau, phosphorylated tau and amyloid beta 42 in patients with (n = 851) and without epilepsy. The concentrations of total tau and phosphorylated tau were higher in Alzheimer's disease patients with epilepsy than Alzheimer's disease patients without epilepsy and amyloid beta 42 levels were significantly lower in Alzheimer's disease patients with epilepsy. No differences in the levels of neurofilament light and glial fibrillary acidic protein were observed. Our study suggests that epilepsy is more common in Alzheimer's disease patients with more pronounced Alzheimer's pathology, as determined by the CSF biomarkers. Further studies are needed to investigate the biomarker potential of these CSF markers as predictors of epilepsy course or as indicators of epileptogenesis in Alzheimer's disease.

Blood biomarkers in epilepsy.

Robust and accessible biomarkers are greatly needed in epilepsy. Diagnostic and prognostic precision in the clinic needs to improve, and there is a need for objective quantification of seizure burden. In recent years, there have been advances in the development of accessible and cost-effective blood-based biomarkers in neurology, and these are increasingly studied in epilepsy. However, the field is in its infancy and specificity and sensitivity for most biomarkers in most clinical situations are not known. This review describes advancements regarding human blood biomarkers in epilepsy. Examples of biochemical markers that have been shown to have higher blood concentrations in study subjects with epilepsy include brain proteins like S100B or neuronal specific enolase, and neuroinflammatory proteins like interleukins, and tumor necrosis factor-alpha. Some of the blood biomarkers also seem to reflect seizure duration or frequency, and levels decrease in response to treatment with antiseizure medication. For most biomarkers, the literature contains seemingly conflicting results. This is to be expected in an emerging field and could reflect different study populations, sampling or analysis techniques, and epilepsy classification. More studies are needed with emphasis put on the classification of epilepsy and seizure types. More standardized reporting could perhaps decrease result heterogeneity and increase the potential for data sharing and subgroup analyses.

Brain injury markers in new-onset seizures in adults: A pilot study.

BACKGROUND: Biochemical markers of brain pathology could potentially contribute to diagnosis and prediction in epilepsy. We describe levels of five brain injury markers in adults with new-onset seizures, and assess group differences in patients with a single seizure, epilepsy, and poststroke epilepsy. METHODS: In this prospective observational study, adults with new-onset seizures were recruited at Sahlgrenska University Hospital, Sweden, and concentrations of glial fibrillary acidic protein (GFAP), neurofilament light (NfL), microtubule-associated protein tau (tau), S100 calcium-binding protein (S100B), and neuron-specific enolase (NSE) were measured. Participants were categorized as epilepsy, poststroke epilepsy (PSE), or single seizure (no additional seizures). Patients were followed until a diagnosis of epilepsy or PSE, or for at least two years in single seizure cases. RESULTS: The cohort included 23 (37%) individuals with a single seizure, 24 (39%) with epilepsy, and 15 (24%) with PSE. The concentrations of S100B were higher in patients with epilepsy and PSE than in single seizures (p = 0.0023 and p = 0.0162, respectively). The concentrations of NfL were higher in patients with PSE than in single seizures (p=0.0027). After age-normalization, levels of S100B were higher in patients with epilepsy and levels of NfL were higher in patients with PSE (p = 0.0021 and p = 0.0180). CONCLUSION: Levels of S100B and NfL were higher in patients with epilepsy or PSE than patients with single seizures. Further studies are needed to investigate the biomarker potential of brain injury markers as predictors of epilepsy course or indicators of epileptogenesis.

Quantitative proteomic analysis to identify differentially expressed proteins in patients with epilepsy.

There is a great need for biomarkers in epilepsy, particularly markers of epileptogenesis. A first seizure will lead to epilepsy in 20-45 % of cases, but biomarkers that can identify these individuals are missing. The purpose of this study was to identify potential biomarkers of epilepsy/epileptogenesis in a cohort of adults with new-onset seizures, using quantitative proteomic analysis. Plasma was collected from 55 adults with new-onset seizures and sufficient follow-up to identify epilepsy. After a follow up period of two years, 63.6 % of the cohort had a diagnosis of epilepsy, whereas 36.4 % of patients only had a single seizure. Plasma proteins were extracted and labelled with tandem mass tags, then analyzed using mass spectrometry approach. Proteins that were up- or downregulated by ≥20 % and with a p-value of <0.05 were considered as differentially expressed and were also annotated to their processes and pathways. Several proteins were differentially expressed in the epilepsy group compared to controls. A total of 1075 proteins were detected, out of which 41 proteins were found to be significantly dysregulated in epilepsy patients. Many of these have been identified in experimental studies of epilepogenesis. We report plasma proteome profiling in new-onset epilepsy in a pilot study with 55 individuals. The identified proteins could be involved in pathways associated with epileptogenesis. The results should be seen as hypothesis-generating and targeted, confirmatory studies are needed.

Amyloid precursor protein-b facilitates cell adhesion during early development in zebrafish.

Understanding the biological function of amyloid beta (Aß) precursor protein (APP) beyond its role in Alzheimer's disease is emerging. Yet, its function during embryonic development is poorly understood. The zebrafish APP orthologue, Appb, is strongly expressed during early development but thus far has only been studied via morpholino-mediated knockdown. Zebrafish enables analysis of cellular processes in an ontogenic context, which is limited in many other vertebrates. We characterized zebrafish carrying a homozygous mutation that introduces a premature stop in exon 2 of the appb gene. We report that appb mutants are significantly smaller until 2 dpf and display perturbed enveloping layer (EVL) integrity and cell protrusions at the blastula stage. Moreover, appb mutants surviving beyond 48 hpf exhibited no behavioral defects at 6 dpf and developed into healthy and fertile adults. The expression of the app family member, appa, was also found to be altered in appb mutants. Taken together, we show that appb is involved in the initial development of zebrafish by supporting the integrity of the EVL, likely by mediating cell adhesion properties. The loss of Appb might then be compensated for by other app family members to maintain normal development.

Cardiomyopathy with lethal arrhythmias associated with inactivation of KLHL24.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disorder, yet the genetic cause of up to 50% of cases remains unknown. Here, we show that mutations in KLHL24 cause HCM in humans. Using genome-wide linkage analysis and exome sequencing, we identified homozygous mutations in KLHL24 in two consanguineous families with HCM. Of the 11 young affected adults identified, 3 died suddenly and 1 had a cardiac transplant due to heart failure. KLHL24 is a member of the Kelch-like protein family, which acts as substrate-specific adaptors to Cullin E3 ubiquitin ligases. Endomyocardial and skeletal muscle biopsies from affected individuals of both families demonstrated characteristic alterations, including accumulation of desmin intermediate filaments. Knock-down of the zebrafish homologue klhl24a results in heart defects similar to that described for other HCM-linked genes providing additional support for KLHL24 as a HCM-associated gene. Our findings reveal a crucial role for KLHL24 in cardiac development and function.

Importance of Toxicokinetics to Assess the Utility of Zebrafish Larvae as Model for Psychoactive Drug Screening Using Meta-Chlorophenylpiperazine (mCPP) as Example.

The number of new psychoactive substances (NPS) increases rapidly, harming society and fuelling the need for alternative testing strategies. These should allow the ever-increasing number of drugs to be tested more effectively for their toxicity and psychoactive effects. One proposed strategy is to complement rodent models with zebrafish (Danio rerio) larvae. Yet, our understanding of the toxicokinetics in this model, owing to the waterborne drug exposure and the distinct physiology of the fish, is incomplete. We here explore the toxicokinetics and behavioral effects of an NPS, meta-chlorophenylpiperazine (mCPP), in zebrafish larvae. Uptake kinetics of mCPP, supported by toxicokinetic modeling, strongly suggested the existence of active transport processes. Internal distribution showed a dominant accumulation in the eye, implying that in zebrafish, like in mammals, melanin could serve as a binding site for basic drugs. We confirmed this by demonstrating significantly lower drug accumulation in two types of hypo-pigmented fish. Comparison of the elimination kinetics between mCPP and previously characterized cocaine demonstrated that drug affinities to melanin in zebrafish vary depending on the structure of the test compound. As expected from mCPP-elicited responses in rodents and humans, zebrafish larvae displayed hypoactive behavior. However, significant differences were seen between zebrafish and rodents with regard to the concentration-dependency of the behavioral response and the comparability of tissue levels, corroborating the need to consider the organism-internal distribution of the chemical to allow appropriate dose modeling while evaluating effects and concordance between zebrafish and mammals. Our results highlight commonalities and differences of mammalian versus the fish model in need of further exploration.

From the Cover: Zebrafish Larvae Are Insensitive to Stimulation by Cocaine: Importance of Exposure Route and Toxicokinetics.

Zebrafish (Danio rerio) larvae have been suggested as vertebrate model to complement or even replace mammals for rapidly assessing behavioral effects of psychoactive drugs. Yet, divergent responses have been reported in mammals and fish despite the conservation of many drug targets. Cocaine, eg, acts as stimulant in mammals but no such response has been documented for zebrafish larvae. We hypothesized that differences in exposure routes (inhalation or injection in mammals vs waterborne in fish) may be a reason for differences in behavioral responses. We characterized cocaine toxicokinetics by liquid chromatography-mass spectrometry and found its rapid uptake into larvae. We used Matrix-assisted laser desorption ionization-mass spectrometry imaging for the first time to characterize internal distribution of cocaine in zebrafish larvae. Surprisingly, eyes accumulated the highest amount of cocaine and retained most of it even after 48 h depuration. We attribute this to trapping by pigment melanin, a thus far little explored mechanism that may also be relevant for other basic drugs. Cocaine also reached the brain but with levels similar to those in trunk indicating simple passive diffusion as means of distribution which was supported by toxicokinetic models. Although brain levels covered those known to cause hyperactivity in mammals, only hypoactivity (decreased locomotion) was recorded in zebrafish larvae. Our results therefore point to cocaine's anesthetic properties as the dominant mechanism of interaction in the fish: upon entry through the fish skin and gills, it first acts on peripheral nerves rapidly overriding any potential stimulatory response in the brain.

ß-Amyloid precursor protein-b is essential for Mauthner cell development in the zebrafish in a Notch-dependent manner.

Amyloid precursor protein (APP) is a transmembrane glycoprotein that has been the subject of intense research because of its implication in Alzheimer's disease. However, the physiological function of APP in the development and maintenance of the central nervous system remains largely unknown. We have previously shown that the APP homologue in zebrafish (Danio rerio), Appb, is required for motor neuron patterning and formation. Here we study the function of Appb during neurogenesis in the zebrafish hindbrain. Partial knockdown of Appb using antisense morpholino oligonucleotides blocked the formation of the Mauthner neurons, uni- or bilaterally, with an aberrant behavior as a consequence of this cellular change. The Appb morphants had decreased neurogenesis, increased notch signaling and notch1a expression at the expense of deltaA/D expression. The Mauthner cell development could be restored either by a general decrease in Notch signaling through γ-secretase inhibition or by a partial knock down of Notch1a. Together, this demonstrates the importance of Appb in neurogenesis and for the first time shows the essential requirement of Appb in the formation of a specific cell type, the Mauthner cell, in the hindbrain during development. Our results suggest that Appb-regulated neurogenesis is mediated through balancing the Notch1a signaling pathway and provide new insights into the development of the Mauthner cell.

Oral dosing in adult zebrafish: proof-of-concept using pharmacokinetics and pharmacological evaluation of carbamazepine.

BACKGROUND AND METHODS: We describe a method for obtaining pharmacokinetics (PK) and pharmacology data from adult zebrafish in terms of mg/kg using a novel method of oral administration. Using carbamazepine (CBZ) as a test drug, we employed dried blood spot (DBS) cards to enable drug quantification for PK; and we evaluated the pharmacological anxiolytic effect using novel tank test. RESULTS: The PK study confirmed the presence of CBZ in both blood and brain and the behavioural study showed dose dependent anxiolytic effect. The reproducibility of oral dosing was confirmed by the fact that the results obtained in both the experiments had negligible errors. CONCLUSIONS: This report enables a novel approach for optimizing the utility of zebrafish in drug discovery and drug delivery research.

Transition metal free hydrolysis/cyclization strategy in a single pot: synthesis of fused furo N-heterocycles of pharmacological interest.

A transition metal free tandem two-step strategy has been developed involving hydrolysis of 2-chloro-3-alkynyl quinoxalines/pyrazines followed by in situ cyclization of the corresponding 2-hydroxy-3-alkynyl intermediates in a single pot leading to fused furo N-heterocycles as potential inhibitors of sirtuins. A representative compound showed promising pharmacological properties in vitro and in vivo.

Oral gabapentin suppresses pentylenetetrazole-induced seizure-like behavior and cephalic field potential in adult zebrafish.

We report the effect of orally administered gabapentin (GBP) on pentylenetetrazole (PTZ)-induced seizure-like activity in adult zebrafish. Zebrafish were pretreated with vehicle or GBP using a novel method of precise oral administration, followed by an intraperitoneal administration of PTZ. Behavioral assessment was carried out using locomotion-based video-tracking analysis and seizure score assignment using visual observation. Cephalic field potential recordings of the zebrafish brain were conducted using an electrical data acquisition system. Orally administered GBP significantly suppressed the seizure-like locomotor activity and strong slow-wave (~3Hz) activity in the cephalic field potential caused by PTZ. This work is the first report of the activity of an orally delivered anticonvulsant in adult zebrafish. Our study provides behavioral and physiological evidence in support of an adult zebrafish model for studying seizures including excitotoxic brain injury and a novel in vivo framework for the evaluation of pharmacological modulators of epilepsy.

Selective multicolour imaging of zebrafish muscle fibres by using fluorescent organic nanoparticles.

Glowing fish: Fluorescent organic nanoparticles (FONs) were prepared from pyrene-containing guanine analogues. FONs are able to penetrate into human cells and zebrafish embryos; they are nontoxic, biocompatible and were found to be specific for muscular tissues. This study demonstrates for the first time the possibility of using FONs for in vivo, whole-body, fluorescence imaging of zebrafish.