Search

Mesial temporal lobe epilepsy (MTLE) is a chronic neurological disorder affecting almost 40% of adult patients with epilepsy. Hippocampal sclerosis (HS) is a common histopathological abnormality found in patients with MTLE. HS is characterised by extensive neuronal loss in different hippocampus sub-regions. In this study, we used laser microdissection-based microproteomics to determine the protein abundances in different regions and layers of the hippocampus dentate gyrus (DG) in an electric stimulation rodent model which displays classical HS damage similar to that found in patients with MTLE. Our results indicate that there are differences in the proteomic profiles of different layers (granule cell and molecular), as well as different regions, of the DG (ventral and dorsal). We have identified new signalling pathways and proteins present in specific layers and regions of the DG, such as PARK7, RACK1, and connexin 31/gap junction. We also found two major signalling pathways that are common to all layers and regions: inflammation and energy metabolism. Finally, our results highlight the utility of high-throughput microproteomics and spatial-limited isolation of tissues in the study of complex disorders to fully appreciate the large biological heterogeneity present in different cell populations within the central nervous system.

Mesial temporal lobe epilepsy (MTLE) is the most frequent type of epilepsy in adults and it is usually refractory to clinical treatments. In most patients with MTLE a characteristic histopathological lesion is observed, including hippocampal sclerosis (HS). The subiculum is an important area which connects the hippocampus with the enthorrinal cortex. In this study aims to understand the molecular role of the subiculum in MTLE in the classical model of pilocarpine using RNA-seq (RNA sequencing)

The Wistar Audiogenic Rat (WAR) is a model whose rats are predisposed to develop seizures following acoustic stimulation. We aimed to establish the transcriptional profile of the WAR model, searching for genes that help in understanding the molecular mechanisms involved in the predisposition and seizures expression of this strain. RNA-Seq of the corpora quadrigemina of WAR and Wistar rats subjected to acoustic stimulation revealed 64 genes differentially regulated in WAR. We validated twelve of these genes by qPCR in stimulated and naive (non-stimulated) WAR and Wistar rats. Among these, Acsm3 was upregulated in WAR in comparison with both control groups. In contrast, Gpr126 and Rtel1 were downregulated in naive and stimulated WAR rats in comparison with the Wistar controls. Qdpr was upregulated only in stimulated WAR rats that exhibited audiogenic seizures. Our data show that there are genes with differential intrinsic regulation in the WAR model and that seizures can alter gene regulation. We identified new genes that might be involved in the epileptic phenotype and comorbidities of the WAR model.

Orientadores: Íscia Teresinha Lopes Cendes, André Schwambach Vieira Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: A epilepsia do lobo temporal mesial (ELTM) é uma doença neurológica crônica caracterizada pelo desenvolvimento de crises epiléticas e por anormalidades histopatológicas nas estruturas mediais do lobo temporal, principalmente a esclerose hipocampal (EH). O perfil de expressão gênica de tecido específico fornece informações biológicas relevantes sobre mecanismos moleculares potencialmente envolvidos em fenômenos biológicos complexos. Recentemente, devido à heterogeneidade da expressão gênica em diferentes subconjuntos de células, fica mais claro o quanto é importante a delimitação de regiões especificas, especialmente no sistema nervoso central. Utilizamos a técnica de RNAseq para determinar o perfil de expressão gênica no giro denteado (GD) do hipocampo e Cornu Ammonis 3 (CA3) em um modelo animal de epilepsia do lobo temporal induzido por pilocarpina. O sequenciamento de alto rendimento do RNA foi realizado a partir do tecido microdissecado do hipocampo, região dorsal e ventral do GD e CA3, assim como a intermediária de CA3, no modelo de pilocarpina em ratos Wistar. Avaliamos o perfil de expressão gênica e os processos biológicos enriquecidos a partir dos genes diferencialmente expressos. Os resultados indicam a presença de muitos genes regulados diferencialmente, dos quais, alguns foram específicos para cada sub-região do hipocampo nos ratos tratados com pilocarpina. No entanto, identificamos alguns importantes processos biológicos de sinalização que foram enriquecidos em todas os grupos celulares estudados e nas áreas examinadas, como, inflamação, navegação axonal, atividade neuronal e função sináptica. Os dados apresentados destacam a importância da análise de alto rendimento do transcriptoma no estudo de doenças complexas, como a epilepsia, e a vantagem de obter áreas delimitadas de tecido para analisar plenamente a grande heterogeneidade biológica de diferentes populações de células no sistema nervoso central. Nossos resultados sugerem uma predominância na região dorsal relacionada a atividade elétrica neuronal e orientação axonal, além de identificar uma interação entre vários componentes moleculares que levam à epileptogênese neste modelo animal que exibe dano hipocampal generalizado Abstract: Mesial temporal lobe epilepsy (MTLE) is a chronic neurological disorder characterized by the development of seizures and by histopathological abnormalities in the mesial temporal lobe structures, mainly hippocampal sclerosis (HS). It is well known that gene expression profile of specific tissue provides relevant biological information about molecular mechanisms potentially involved in complex biological phenomena. Recently, it has been recognized that due to the marked heterogeneity of gene expression in a different subset of cells, it is important to take sub-regional specificities when studying gene expression, especially in the central nervous system. We used RNA sequencing analysis to determine the profile of gene expression in the hippocampus dentate gyrus (DG) and Cornu Ammonis 3 (CA3) in an animal model of mesial temporal lobe epilepsy induced by pilocarpine. The high-throughput RNA sequencing was performed in tissue obtained from the dorsal and ventral DG and CA3, as well as the intermediate CA3, by laser-microdissection in Wistar rats. We evaluated the gene expression profile and the enriched signaling biological processes. Our results indicate that there were many differentially regulated genes, some of which were specific to each sub-regions of the hippocampus in the pilocarpine rats. However, we identified some major signaling biological processes that were enriched in all layers and areas examined, such as inflammation, axon guidance, neuronal activity and synaptic function. The data presented highlights the importance of high-throughput transcriptome analysis in the study of complex disorders such as epilepsy, and the advantage of obtaining tissue from delimitated areas to fully appreciate the large biological heterogeneity of different cell populations within the central nervous system. Our results suggest a predominance of the dorsal region in the neuronal electrical activity and axonal guidance. Furthermore, we identified an interaction among several molecular components leading to the epileptogenesis in this animal model that displays widespread hippocampal damage Doutorado Fisiopatologia Médica Doutor em Ciências FAPESP 2013/8635-5 CNPQ