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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.

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.

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.