Abstract
Granule cell dispersion (GCD) is a common pathological feature observed in the hippocampus of patients with Mesial Temporal Lobe Epilepsy (MTLE). Pathomechanisms underlying GCD remain to be elucidated, but one hypothesis proposes aberrant reactivation of neurodevelopmental migratory pathways, possibly triggered by febrile seizures. This study aims to compare the proteomes of basal and dispersed granule cells in the hippocampus of eight MTLE patients with GCD to identify proteins that may mediate GCD in MTLE. Quantitative proteomics identified 1,882 proteins, of which 29% were found in basal granule cells only, 17% in dispersed only and 54% in both samples. Bioinformatics analyses revealed upregulated proteins in dispersed samples were involved in developmental cellular migratory processes, including cytoskeletal remodeling, axon guidance and signaling by Ras homologous (Rho) family of GTPases (P < 0.01). The expression of two Rho GTPases, RhoA and Rac1, was subsequently explored in immunohistochemical and in situ hybridization studies involving eighteen MTLE cases with or without GCD, and three normal post mortem cases. In cases with GCD, most dispersed granule cells in the outer-granular and molecular layers have an elongated soma and bipolar processes, with intense RhoA immunolabeling at opposite poles of the cell soma, while most granule cells in the basal granule cell layer were devoid of RhoA. A higher percentage of cells expressing RhoA was observed in cases with GCD than without GCD (P < 0.004). In GCD cases, the percentage of cells expressing RhoA was significantly higher in the inner molecular layer than the granule cell layer (P < 0.026), supporting proteomic findings. In situ hybridization studies using probes against RHOA and RAC1 mRNAs revealed fine peri- and nuclear puncta in granule cells of all cases. The density of cells expressing RHOA mRNAs was significantly higher in the inner molecular layer of cases with GCD than without GCD (P = 0.05). In summary, our study has found limited evidence for ongoing adult neurogenesis in the hippocampus of patients with MTLE, but evidence of differential dysmaturation between dispersed and basal granule cells has been demonstrated, and elevated expression of Rho GTPases in dispersed granule cells may contribute to the pathomechanisms underpinning GCD in MTLE.
Highlights
Temporal lobe epilepsy is the most common form of pharmacoresistant epilepsy in adults (Engel, 1998)
Granule Cell Pathology Observed in Cases Submitted for Proteomics In mesial form of temporal lobe epilepsy (MTLE) cases with granule cell dispersion (GCD), a thick band of neuronal nuclei (NeuN)-positive cells was observed in the dentate granule cell layer (Figure 1A)
The granule cell layer in the hippocampus of four MTLE cases with no GCD (Figure 1F) and healthy post mortem controls contained round, tightly packed NeuN-positive cells, and no immunopositive cells were observed in the molecular layer
Summary
Temporal lobe epilepsy is the most common form of pharmacoresistant epilepsy in adults (Engel, 1998). Up to 80% of patients with a mesial form of temporal lobe epilepsy (MTLE) have structural abnormalities in the hippocampus (de Tisi et al, 2011; Blumcke et al, 2017). The exact cause of GCD in human MTLE is unknown; animal models of MTLE with or without hippocampal sclerosis have shown that seizures can displace the migration of newlygenerated (Parent et al, 2006; Hester and Danzer, 2013) and mature DGCs to CA4 and molecular layer (Murphy and Danzer, 2011; Koyama et al, 2012; Chai et al, 2014). Low levels of reelin transcript and protein have been reported in the hippocampus of patients with MTLE (Haas et al, 2002; Haas and Frotscher, 2010) and animal models of MTLE (Heinrich et al, 2006), possibly as a consequence of elevated methylation at the promotor region of RELN (Kobow et al, 2009) or loss of reelin-synthesizing neurons in hippocampus (Haas et al, 2002; Orcinha et al, 2016)
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