Molecular tools for the characterization of seizure susceptibility in genetic rodent models of epilepsy.

Epilepsy is a chronic neurological disorder characterized by abnormal neuronal activity that arises from imbalances between excitatory and inhibitory synapses, which are highly correlated to functional and structural changes in specific brain regions. The difference between the normal and the epileptic brain may harbor genetic alterations, gene expression changes, and/or protein alterations in the epileptogenic nucleus. It is becoming increasingly clear that such differences contribute to the development of distinct epilepsy phenotypes. The current major challenges in epilepsy research include understanding the disease progression and clarifying epilepsy classifications by searching for novel molecular biomarkers. Thus, the application of molecular techniques to carry out comprehensive studies at deoxyribonucleic acid, messenger ribonucleic acid, and protein levels is of utmost importance to elucidate molecular dysregulations in the epileptic brain. The present review focused on the great diversity of technical approaches available and new research methodology, which are already being used to study molecular alterations underlying epilepsy. We have grouped the different techniques according to each step in the flow of information from DNA to RNA to proteins, and illustrated with specific examples in animal models of epilepsy, some of which are our own. Separately and collectively, the genomic and proteomic techniques, each with its own strengths and limitations, provide valuable information on molecular mechanisms underlying seizure susceptibility and regulation of neuronal excitability. Determining the molecular differences between genetic rodent models of epilepsy and their wild-type counterparts might be a key in determining mechanisms of seizure susceptibility and epileptogenesis as well as the discovery and development of novel antiepileptic agents. This article is part of the Special Issue "NEWroscience 2018".

Correction: Analysis of gene variants in the GASH/Sal model of epilepsy.

[This corrects the article DOI: 10.1371/journal.pone.0229953.].

Analysis of gene variants in the GASH/Sal model of epilepsy.

Epilepsy is a complex neurological disorder characterized by sudden and recurrent seizures, which are caused by various factors, including genetic abnormalities. Several animal models of epilepsy mimic the different symptoms of this disorder. In particular, the genetic audiogenic seizure hamster from Salamanca (GASH/Sal) animals exhibit sound-induced seizures similar to the generalized tonic seizures observed in epileptic patients. However, the genetic alterations underlying the audiogenic seizure susceptibility of the GASH/Sal model remain unknown. In addition, gene variations in the GASH/Sal might have a close resemblance with those described in humans with epilepsy, which is a prerequisite for any new preclinical studies that target genetic abnormalities. Here, we performed whole exome sequencing (WES) in GASH/Sal animals and their corresponding controls to identify and characterize the mutational landscape of the GASH/Sal strain. After filtering the results, moderate- and high-impact variants were validated by Sanger sequencing, assessing the possible impact of the mutations by "in silico" reconstruction of the encoded proteins and analyzing their corresponding biological pathways. Lastly, we quantified gene expression levels by RT-qPCR. In the GASH/Sal model, WES showed the presence of 342 variations, in which 21 were classified as high-impact mutations. After a full bioinformatics analysis to highlight the high quality and reliable variants, the presence of 3 high-impact and 15 moderate-impact variants were identified. Gene expression analysis of the high-impact variants of Asb14 (ankyrin repeat and SOCS Box Containing 14), Msh3 (MutS Homolog 3) and Arhgef38 (Rho Guanine Nucleotide Exchange Factor 38) genes showed a higher expression in the GASH/Sal than in control hamsters. In silico analysis of the functional consequences indicated that those mutations in the three encoded proteins would have severe functional alterations. By functional analysis of the variants, we detected 44 significantly enriched pathways, including the glutamatergic synapse pathway. The data show three high-impact mutations with a major impact on the function of the proteins encoded by these genes, although no mutation in these three genes has been associated with some type of epilepsy until now. Furthermore, GASH/Sal animals also showed gene variants associated with different types of epilepsy that has been extensively documented, as well as mutations in other genes that encode proteins with functions related to neuronal excitability, which could be implied in the phenotype of the GASH/Sal. Our findings provide valuable genetic and biological pathway data associated to the genetic burden of the audiogenic seizure susceptibility and reinforce the need to validate the role of each key mutation in the phenotype of the GASH/Sal model.

The clinical heterogeneity of coenzyme Q10 deficiency results from genotypic differences in the Coq9 gene.

Primary coenzyme Q10 (CoQ10) deficiency is due to mutations in genes involved in CoQ biosynthesis. The disease has been associated with five major phenotypes, but a genotype-phenotype correlation is unclear. Here, we compare two mouse models with a genetic modification in Coq9 gene (Coq9(Q95X) and Coq9(R239X)), and their responses to 2,4-dihydroxybenzoic acid (2,4-diHB). Coq9(R239X) mice manifest severe widespread CoQ deficiency associated with fatal encephalomyopathy and respond to 2,4-diHB increasing CoQ levels. In contrast, Coq9(Q95X) mice exhibit mild CoQ deficiency manifesting with reduction in CI+III activity and mitochondrial respiration in skeletal muscle, and late-onset mild mitochondrial myopathy, which does not respond to 2,4-diHB. We show that these differences are due to the levels of COQ biosynthetic proteins, suggesting that the presence of a truncated version of COQ9 protein in Coq9(R239X) mice destabilizes the CoQ multiprotein complex. Our study points out the importance of the multiprotein complex for CoQ biosynthesis in mammals, which may provide new insights to understand the genotype-phenotype heterogeneity associated with human CoQ deficiency and may have a potential impact on the treatment of this mitochondrial disorder.

Spatial and temporal dynamics of primary and secondary metabolism in Phaseolus vulgaris challenged by Pseudomonas syringae.

Many defense mechanisms contribute to the plant immune system against pathogens, involving the regulation of different processes of the primary and secondary metabolism. At the same time, pathogens have evolved mechanisms to hijack the plant defense in order to establish the infection and proliferate. Localization and timing of the host response are essential to understand defense mechanisms and resistance to pathogens (Rico et al. 2011). Imaging techniques, such as fluorescence imaging and thermography, are a very valuable tool providing spatial and temporal information about a series of plant processes. In this study, bean plants challenged with two pathovars of Pseudomonas syringae have been investigated. Pseudomonas syringae pv. phaseolicola 1448A and P. syringae pv. tomato DC3000 elicit a compatible and incompatible interaction in bean, respectively. Both types of host-pathogen interaction triggered different changes in the activity of photosynthesis and the secondary metabolism. We conclude that the combined analysis of leaf temperature, chlorophyll fluorescence and green fluorescence emitted by phenolics allows to discriminate compatible from incompatible P. syringae-Phaseolus vulgaris interactions in very early times of the infection, prior to the development of symptoms. These can constitute disease signatures that would allow an early identification of emerging plagues in crops.