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Mesial temporal lobe epilepsy (MTLE) is a chronic neurological disorder characterized by the occurrence of seizures, and histopathological abnormalities in the mesial temporal lobe structures, mainly hippocampal sclerosis (HS). We used a multi-omics approach to determine the profile of transcript and protein expression in the dorsal and ventral hippocampal dentate gyrus (DG) and Cornu Ammonis 3 (CA3) in an animal model of MTLE induced by pilocarpine. We performed label-free proteomics and RNAseq from laser-microdissected tissue isolated from pilocarpine-induced Wistar rats. We divided the DG and CA3 into dorsal and ventral areas and analyzed them separately. We performed a data integration analysis and evaluated enriched signaling pathways, as well as the integrated networks generated based on the gene ontology processes. Our results indicate differences in the transcriptomic and proteomic profiles among the DG and the CA3 subfields of the hippocampus. Moreover, our data suggest that epileptogenesis is enhanced in the CA3 region when compared to the DG, with most abnormalities in transcript and protein levels occurring in the CA3. Furthermore, our results show that the epileptogenesis in the pilocarpine model involves predominantly abnormal regulation of excitatory neuronal mechanisms mediated by N-methyl D-aspartate (NMDA) receptors, changes in the serotonin signaling, and neuronal activity controlled by calcium/calmodulin-dependent protein kinase (CaMK) regulation and leucine-rich repeat kinase 2 (LRRK2)/WNT signaling pathways.

The hippocampus comprises several neuronal populations such as CA1, CA2, CA3, and the dentate gyrus (DG), which present different neuronal origins, morphologies, and molecular mechanisms. Laser capture microdissection (LCM) allows selectively collecting samples from target regions and eliminating unwanted cells to obtain more specific results. LCM of hippocampus neuronal populations coupled with RNA-seq analysis has the potential to allow the exploration of the molecular machinery unique to each of these subfields. Previous RNA-seq investigation has already provided a molecular blueprint of the hippocampus, however, there is no RNA-seq data specific for each of the rat hippocampal regions. Serial tissue sections covering the hippocampus were produced from frozen brains of adult male Wistar rats, and the hippocampal subfields CA1, CA2, CA3, and DG were identified and isolated by LCM. Total RNA was extracted from samples, and cDNA libraries were prepared and run on a HiSeq 2500 platform. Reads were aligned using STAR, and the DESeq2 statistics package was used to estimate gene expression. We found evident segregation of the transcriptomic profile from different regions of the hippocampus and the expression of known, as well as novel, specific marker genes for each region. Gene ontology enrichment analysis of CA1 subfield indicates an enrichment of actin regulation and postsynaptic membrane AMPA receptors genes indispensable for long-term potentiation. CA2 and CA3 transcripts were found associated with the increased metabolic processes. DG expression was enriched for ribosome and spliceosome, both required for protein synthesis and maintenance of cell life. The present findings contribute to a deeper understanding of the differences in the molecular machinery expressed by the rat hippocampal neuronal populations, further exploring underlying mechanisms responsible for each subflied specific functions.

Hippocampus is a brain region mainly composed by neuron layers described as Dentate Gyrus(DG) and Ammon’s horn(CA). This study explores the differential transcripts datasets of hippocampal layers (CA1, CA2, CA3, DG) for elucidating their distinctions. All layers were bilaterally collected from hippocampus of each rat, total RNA was extracted, and libraries for RNA-Seq in Illumina Hiseq platform were prepared according to manufacturer instructions. Sequences were aligned and quantified with the STAR Aligner/DESeq2 pipeline. Gene Onthologies were analyzed with the DAVID software. In results, CA1 neurons are expressing more genes related to circadian rhythms, postsynaptic membrane receptors that are important for manutence of excitability, learning and memory. In CA2 and CA3 were found transcripts associated to higher metabolic pathways demand and endocytosis recycling. Dentate gyrus neurons expression was related to ribosome, spliceosome, essential for protein synthesis that supports dendritic growth and maintenance of long-term potentiation. These findings yield new information into specific molecular alterations in hippocampus layers which contribute to understanding the singularity of hippocampal formation.

Alcoholism is a complex multifactorial disorder with a strong genetic influence. Although several studies have shown the impact of high ethanol intake on the striatal gene expression, few have addressed the relationship between the patterns of gene expression underlying the compulsive behaviour associated with the two major concerns in addiction: the excessive drug consumption and relapsing. In this study, we used a chronic three-bottle free-choice murine model to address striatal transcript regulation among animals with different ethanol intakes and preferences: Light Drinkers (preference for water throughout the experiment), Heavy Drinkers (preference for ethanol with a non-compulsive intake) and Inflexible Drinkers (preference for ethanol and simultaneous loss of control over the drug intake). Our aim was to correlate the intake patterns observed in this model with gene expression changes in the striatum, a brain region critical for the development of alcohol addiction. We found that the transcripts of the Lrrk2 gene, which encodes a multifunctional protein with kinase and GTPase activities, is upregulated only in Inflexible Drinkers suggesting, for the first time, that the Lrrk2 pathway plays a major role in the compulsive ethanol intake behaviour of addicted subjects.

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)

Much of the studies related to the hippocampus are aimed to elucidate the diseases which affect this structure, such as Mesial Temporal Lobe Epilepsy (MTLE) and Alzheimer’s disease (AD). However, to better understand the pathophysiology of these diseases, which are not fully elucidated, we must focus in other structures from the Hippocampal Formation, that can be involved with these mechanisms, such as the DG¹. The DG itself is also subdivided into dorsal and ventral in rodents and posterior and anterior in humans². Moreover, the neurodegenerative diseases, as others, are usually investigated using animal models, mainly rodents, due to its possibility of study the development of the diseases and not only its final stage³. However, despite its remarkable similarities with humans, it is necessary to evaluate the differences between the two species to validate the rodents as a suitable model for human pathologies. Therefore, here we present the comparative multi-OMICs analysis of the laser microdissected DG from rat and humans, intending to characterize and describe both species, validating the rat as a good animal model for human pathologies. We also present the comparative analysis of dorsal and ventral DG isolated from the rats.

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

Alcohol use disorder (AUD) is a complex multifactorial disease with heritability of ∼50% and corresponds to the state in which the body triggers a reinforcement or reward compulsive behavior due to ethanol consumption, even when faced with negative consequences. Although several studies have shown the impact of high ethanol intake on the prefrontal cortex (PFC) gene expression, few have addressed the relationship between the patterns of gene expression underlying the compulsive behaviour associated with relapsing. In this study, we used a chronic three-bottle free-choice mouse model to investigate the PFC transcriptome in three different groups of mice drinkers: 'Light drinkers' (preference for water throughout the experiment); 'Heavy drinkers' (preference for ethanol with a non-compulsive intake), and 'Inflexible drinkers' (preference for ethanol with a compulsive drinking component). Our aim was to correlate the intake patterns observed in this model with gene expression changes in the PFC, a brain region critical for the development and maintenance of alcohol addiction. We found that the Camk2a gene showed a downregulated profile only in the Inflexible when compared to the Light drinkers group, the Camk2n1 and Pkp2 genes showed an upregulated profile only in the Inflexible drinkers when compared to the Control group, and the Gja1 gene showed an upregulated profile in the Light and Inflexible drinkers when compared to the Control group. These different transcription patterns have been associated to the presence of alcohol, in the Camk2n1 and Gja1 genes; to the amount of ethanol consumed, in the Camk2a gene; and to the loss of control in the alcohol consumption, in the Pkp2 gene. Here, we provide, for the first time, the potential involvement of the Pkp2 gene in the compulsivity and loss of control over the voluntary ethanol consumption.

IKK epsilon (IKKε) is induced by the activation of nuclear factor-κB (NF-κB). Whole-body IKKε knockout mice on a high-fat diet (HFD) were protected from insulin resistance and showed altered energy balance. We demonstrate that IKKε is expressed in neurons and is upregulated in the hypothalamus of obese mice, contributing to insulin and leptin resistance. Blocking IKKε in the hypothalamus of obese mice with CAYMAN10576 or small interfering RNA decreased NF-κB activation in this tissue, relieving the inflammatory environment. Inhibition of IKKε activity, but not TBK1, reduced IRS-1Ser307 phosphorylation and insulin and leptin resistance by an improvement of the IR/IRS-1/Akt and JAK2/STAT3 pathways in the hypothalamus. These improvements were independent of body weight and food intake. Increased insulin and leptin action/signaling in the hypothalamus may contribute to a decrease in adiposity and hypophagia and an enhancement of energy expenditure accompanied by lower NPY and increased POMC mRNA levels. Improvement of hypothalamic insulin action decreases fasting glycemia, glycemia after pyruvate injection, and PEPCK protein expression in the liver of HFD-fed and db/db mice, suggesting a reduction in hepatic glucose production. We suggest that IKKε may be a key inflammatory mediator in the hypothalamus of obese mice, and its hypothalamic inhibition improves energy and glucose metabolism.

Orientadores: Iscia Teresinha Lopes Cendes, Vinicius D'Ávila Bitencourt Pascoal Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: Wistar audiogenic rat (WAR) é um modelo genético de epilepsia de crises audiogênicas desencadeadas após alta intensidade de estimulação sonora. Outro modelo genético recentemente identificado é o da epilepsia generalizada com crises de ausência (GEAS). O objetivo do presente estudo foi caracterizar o perfil de expressão gênica destas duas cepas através de uma análise em larga escala. Para os estudos de expressão foi utilizada inicialmente a tecnologia de microarranjos seguida da validação dos resultados por técnica quantitativa de PCR em tempo real. Os resultados foram analisados em ambiente R, utilizando os pacotes AFFY e RankProd do bioconductor, utilizando o algoritmo MAS 5 os array foram normalizados e calculou-se a intensidade do sinal e a detecção (presença ou ausência de expressão). Após a detecção, os transcritos que estavam ausentes foram removidos. Para a análise estatística foi utilizado o teste RankProd, que é biologicamente projetado para testar e detectar genes diferencialmente expressos em experimentos de microarranjos. Foi utilizado um valor de p ? 0,01 e pfp ? 0,05, a fim de considerar os transcritos diferencialmente expressos. No geral, nossos resultados mostram uma assinatura molecular similar nos dois modelos de ratos genéticos analisados. Houve uma sobreposição na lista de genes diferencialmente expressos encontrados em ambos os modelos, quando comparado com controles. Além disso, descobrimos que duas importantes vias moleculares para epileptogênese: neurotransmissão GABAérgica e potencialização de longo prazo pós-sináptica NMDA-dependente, foram encontrados em ambos os modelos, quando combinamos os dados dos animais WAR e GEAS. No entanto, algumas diferenças nas vias de sinalização expressas nos dois modelos também foram identificadas. Portando os resultados mostram claramente a natureza heterogênea e complexa dos mecanismos moleculares envolvidos na epileptogênese Abstract: Wistar audiogenic rat (WAR) is a genetic epilepsy model susceptible to audiogenic seizures, after high-intensity sound stimulation. Another genetic model we have recently identified is the generalized epilepsy with absence seizures (GEAS) rat. The aim of the present study was to characterize and compare the genetic profile of these two strains using gene expression analysis. Experiments were performed initially using microarray technology followed by quantitative real-time PCR. Results were analyzed in R environment using the Affy and RankProd packages from Bioconductor, using the algorithm MAS 5 we normalized the arrays and calculated the signal intensity and the detection (presence or absence of expression), after the detection, transcripts which were absent in all samples were removed. For statistical analysis we used the Rank Product test, which is biologically motivated and designed to test and detect differentially expressed genes in replicated microarray experiments. This is a simple non-parametric statistical method based on ranks of fold changes. We used a p-value ? 0.01 and a pfp ? 0.05 in order to consider a given transcript to be differentially expressed Overall, the results show a different molecular signature in the two genetic rat models analyzed, since different enriched gene ontology categories were found. However, there was some overlap in the list of genes differentially expressed found in both models when comparing to controls. In addition, we found that two important molecular pathways for epileptogenesis: GABAergic neurotransmission and: Neurophysiological process NMDA-dependent postsynaptic long-term potentiation in CA1 hippocampal neurons, were found to be present in both models when combining data from WAR and GEAS animals. In conclusion, our results clearly show the heterogeneous and intricate nature of the molecular mechanisms involved in epileptogenesis as well as the importance of studies looking at different regulatory pathways at once, in order to better appreciate this complexity Mestrado Neurociências Mestre em Ciências