Abstract Temporal lobe epilepsy (TLE) is the most frequent type of focal epilepsy in adults, typically resistant to pharmacological treatment, and mostly presents with cognitive impairment and psychiatric comorbidities. The most common neuropathological hallmark in TLE patients is hippocampal sclerosis (HS). However, the underlying molecular mechanisms involved remain poorly characterized. The dentate gyrus (DG), one specific hippocampal subarea, structural and functional changes imply a key involvement of the DG in the development of TLE. In this study, a isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic technique was performed for the analysis of hippocampal DG obtained from patients with TLE-HS compared to control samples obtained from autopsy. Our proteomic data identified 5,583 proteins, of which 82 proteins were upregulated and 90 proteins were downregulated. Bioinformatics analysis indicated that differentially expressed proteins were enriched in “synaptic vesicle,” “mitochondrion,” “cell-cell adhesion,” “regulation of synaptic plasticity,” “ATP binding,” and “oxidative phosphorylation.” Protein-protein interaction network analysis found a pivotal module of 10 proteins that were related to “oxidative phosphorylation.” This study has investigated proteomic alterations in the DG region of TLE-HS patients, and paved the way for the better understanding of epileptogenesis mechanisms and future therapeutic intervention. Keywords: temporal lobe epilepsy, hippocampal sclerosis, dentate gyrus, quantitative proteomics, iTRAQ Introduction Temporal lobe epilepsy (TLE) is the most frequent type of focal (partial) epilepsy in adults ([43]1). The most common neuropathological hallmark in TLE patients is hippocampal sclerosis (HS), and seizures of TLE patients with HS (TLE-HS) are typically resistant to pharmacological treatment ([44]2). Moreover, TLE-HS patients mostly present with cognitive impairment and psychiatric comorbidities ([45]3), mandating the research for the better understanding of underlying epileptogenesis and progression mechanisms. Initial precipitating injury events, such as febrile convulsions, inflammation, or trauma may be proposed as a driving force in the development of HS-associated TLE ([46]4, [47]5). HS is characterized pathologically by the segmental loss of pyramidal neurons, granule cell dispersion (GCD), and reactive gliosis in the hippocampus ([48]2). The structural and functional changes of the dentate gyrus (DG), one specific hippocampal subarea, including GCD, mossy fiber sprouting, aberrant neurogenesis, and increased excitation, imply a key involvement of the DG in the development of TLE ([49]6, [50]7). Although the structural alterations in TLE-HS have been widely documented, the underlying molecular mechanisms involved, however, remain poorly characterized. Proteomics has been an important methodology for studying the changes of the protein profiles in physiological and pathological conditions, providing comprehensive knowledge about molecular patterns and functional pathway alterations. To date, several proteomic studies have examined the protein changes associated with TLE in the hippocampus, as opposed to specific subregions of the hippocampus. Identified differentially expressed proteins (DEPs) were related to mitochondria and bioenergetics, neuronal excitability, and cytoskeleton ([51]8–[52]10). Given the complex and heterogeneous architecture of the hippocampus, studying whole hippocampal specimens may hinder the detection of the distinct changes seen in DG that can only be explained by molecular changes occurring within DG. Additionally, to our knowledge, few previous studies have performed quantitative proteomics in the DG of TLE-HS patients. Thus, we aim to investigate the molecular pathogenesis mechanisms that may occur in the DG of TLE-HS. Isobaric tags for relative and absolute quantitation (iTRAQ) is a robust quantitative proteomic technique that can simultaneously analyze up to 8-plex samples, and have been widely applied in clinical research ([53]11). In the present study, iTRAQ-based tandem mass spectrometry was performed to undertake proteomic analysis of hippocampal DG obtained from patients with TLE-HS compared to the control samples obtained from autopsy. Our proteomic approach may shed light on the molecular changes associated with HS in DG, that could be used for a better understanding of the specific pathomechanisms of HS and identifying potential therapeutic targets. Materials and Methods Patients and Tissue Preparation The study was carried out on seven TLE patients subjected to standard hippocampectomy at the Department of Neurosurgery of Xiangya Hospital. All patients provided their comprehensive medical history, and went through a physical examination, brain magnetic resonance imaging (MRI), and electroencephalograph (EEG). The inclusion criteria of TLE were according to our previous research ([54]12). Preoperative MRI and routine postoperative histopathologic examination of resected tissue confirmed HS. Written informed consent was obtained from all enrolled participants. The study was conducted following the guideline for research involving humans, and approved by the Ethics Committee of Central South University, Xiangya School of Medicine and the affiliated Xiangya Hospital. Hippocampal samples of the control group were obtained from autopsied patients of Xiangya School of Medicine, and the exclusion criteria were patients with neuropsychiatric disorders and abnormities in the central nervous system (CNS) based on clinical history or histological examination. The resected brain tissues were snap-frozen in liquid nitrogen and then stored at −80°C. Immunohistochemical staining against NeuN (Merck-Millipore, MAB377, 1:1,000) was performed in the brain slices as described before ([55]13), and for Nissl staining, brain slices were placed under agitation in a cresyl violet staining solution. The staining results were used as localization references, an experienced