Seizure-induced increase in microglial cell population in the developing zebrafish brain.

Epilepsy is a chronic brain disorder characterized by unprovoked and recurrent seizures, of which 60% are of unknown etiology. Recent studies implicate microglia in the pathophysiology of epilepsy. However, their role in this process, in particular following early-life seizures, remains poorly understood due in part to the lack of suitable experimental models allowing the in vivo imaging of microglial activity. Given the advantage of zebrafish larvae for minimally-invasive imaging approaches, we sought for the first time to describe the microglial responses after acute seizures in two different zebrafish larval models: a chemically-induced epileptic model by the systemic injection of kainate at 3 days post-fertilization, and the didys552 genetic epilepsy model, which carries a mutation in scn1lab that leads to spontaneous epileptiform discharges. Kainate-treated larvae exhibited transient brain damage as shown by increased numbers of apoptotic nuclei as early as one day post-injection, which was followed by an increase in the number of microglia in the brain. A similar microglial phenotype was also observed in didys552-/- mutants, suggesting that microglia numbers change in response to seizure-like activity in the brain. Interestingly, kainate-treated larvae also displayed a decreased seizure threshold towards subsequent pentylenetetrazole-induced seizures, as shown by higher locomotor and encephalographic activity in comparison with vehicle-injected larvae. These results are comparable to kainate-induced rodent seizure models and suggest the suitability of these zebrafish seizure models for future studies, in particular to elucidate the links between epileptogenesis and microglial dynamic changes after seizure induction in the developing brain, and to understand how these modulate seizure susceptibility.

l-Isoaspartyl Methyltransferase Deficiency in Zebrafish Leads to Impaired Calcium Signaling in the Brain.

Isomerization of l-aspartyl and l-asparaginyl residues to l-isoaspartyl residues is one type of protein damage that can occur under physiological conditions and leads to conformational changes, loss of function, and enhanced protein degradation. Protein l-isoaspartyl methyltransferase (PCMT) is a repair enzyme whose action initiates the reconversion of abnormal l-isoaspartyl residues to normal l-aspartyl residues in proteins. Many lines of evidence support a crucial role for PCMT in the brain, but the mechanisms involved remain poorly understood. Here, we investigated PCMT activity and function in zebrafish, a vertebrate model that is particularly well-suited to analyze brain function using a variety of techniques. We characterized the expression products of the zebrafish PCMT homologous genes pcmt and pcmtl. Both zebrafish proteins showed a robust l-isoaspartyl methyltransferase activity and highest mRNA transcript levels were found in brain and testes. Zebrafish morphant larvae with a knockdown in both the pcmt and pcmtl genes showed pronounced morphological abnormalities, decreased survival, and increased isoaspartyl levels. Interestingly, we identified a profound perturbation of brain calcium homeostasis in these morphants. An abnormal calcium response upon ATP stimulation was also observed in mouse hippocampal HT22 cells knocked out for Pcmt1. This work shows that zebrafish is a promising model to unravel further facets of PCMT function and demonstrates, for the first time in vivo, that PCMT plays a pivotal role in the regulation of calcium fluxes.

Hydrophobic-interaction chromatography for purification of influenza A and B virus.

Options for hydrophobic-interaction chromatography (HIC) for purification of cell culture-derived influenza A and B virus have been assessed using a 96-well plate format using a semi-high-throughput approach. Follow-up experiments at preparative scale were used to characterize dynamic binding capacity, viral hemagglutinin protein (HA protein) recovery, and the influence of influenza virus (IV) strains on yield and contamination levels. Virus recoveries of up to 96% with a residual DNA level of about 1.3% were achieved. To achieve DNA contamination levels required for manufacturing of influenza vaccines for human use, a purification train comprising clarification, inactivation, concentration, column-based anion-exchange chromatography (AEC), and HIC was used in a final set-up. AEC using strong quaternary ammonium ligands was applied as an orthogonal method for DNA depletion by adsorption. Subsequently, HIC (with polypropylene glycol as functional group) was used to reversibly bind virus particles for capture and to remove residual contaminating DNA and proteins (flow-through). This two-step chromatographic process, which requires neither a buffer exchange step nor nuclease treatment had a total virus particle yield for IV A/PR/8/34 (H1N1) of 92%. The protein and the DNA contamination level could be reduced to 42% and at least 1.0%, respectively. With 17.2 µg total protein and 2.0 ng DNA per monovalent dose, this purity level complies with the limits of the European Pharmacopeia for cell culture-derived human vaccines. Overall, the presented downstream process represents a valuable alternative to existing virus purification schemes. Furthermore, it utilizes only off-the-shelf materials and is a simple as well as an economic process for production of cell culture-derived viruses and viral vectors.