Reelin Is Required for Maintenance of Granule Cell Lamination in the Healthy and Epileptic Hippocampus

Abstract

One characteristic feature of mesial temporal lobe epilepsy is granule cell dispersion (GCD), a pathological widening of the granule cell layer in the dentate gyrus. The loss of the extracellular matrix protein Reelin, an important positional cue for neurons, correlates with GCD formation in MTLE patients and in rodent epilepsy models. Here, we used organotypic hippocampal slice cultures (OHSC) from transgenic mice expressing enhanced green fluorescent protein (eGFP) in differentiated granule cells (GCs) to monitor GCD formation dynamically by live cell video microscopy and to investigate the role of Reelin in this process. We present evidence that following treatment with the glutamate receptor agonist kainate (KA), eGFP-positive GCs migrated mainly toward the hilar region. In the hilus, Reelin-producing neurons were rapidly lost following KA treatment as shown in a detailed time series. Addition of recombinant Reelin fragments to the medium effectively prevented the KA-triggered movement of eGFP-positive GCs. Placement of Reelin-coated beads into the hilus of KA-treated cultures stopped the migration of GCs in a distance-dependent manner. In addition, quantitative Western blot analysis revealed that KA treatment affects the Reelin signal transduction pathway by increasing intracellular adaptor protein Disabled-1 synthesis and reducing the phosphorylation of cofilin, a downstream target of the Reelin pathway. Both events were normalized by addition of recombinant Reelin fragments. Finally, following neutralization of Reelin in healthy OHSC by incubation with the function-blocking CR-50 Reelin antibody, GCs started to migrate without any direction preference. Together, our findings demonstrate that normotopic position of Reelin is essential for the maintenance of GC lamination in the dentate gyrus and that GCD is the result of a local Reelin deficiency.

Proteomic differences in hippocampus and cortex of sudden unexplained death in childhood

“…Defects in reelin signaling are suggested in SUDC cases with hippocampal abnormalities [ 26 , 37 ]. Reelin is also related to abnormal granule cell migration and dispersion in epilepsy patients [ 21 ], after seizure induction in animal models [ 5 , 54 , 63 ], and a reelin knockout mouse model had developmental defects [ 19 , 53 ]. Further investigation is required to identify the etiologies underlying these pathway changes, including how the neuronal projections between the hippocampus and frontal cortex may influence altered signaling pathways, i.e.…”

Considering the Role of Extracellular Matrix Molecules, in Particular Reelin, in Granule Cell Dispersion Related to Temporal Lobe Epilepsy

“…Reelin is a large secreted ECM glycoprotein regulating many important processes in mammalian brain development, and dysregulation of reelin signaling has been linked to several brain diseases such as autism, schizophrenia, depression, AD, and, in particular, epilepsy ( Ishii et al, 2016 ; Hirota and Nakajima, 2017 ; Santana and Marzolo, 2017 ; Wasser and Herz, 2017 ; Armstrong et al, 2019 ; Okugawa et al, 2020 ; Orcinha et al, 2021 ). The highly conserved human reelin gene is located on chromosome 7q22 and is circa 450 kbp long ( Desilva et al, 1997 ; Manoharan et al, 2015 ).…”

“…In this process, reelin can be cleaved into different fragments, depending on which protease is active, although it is controversial which of the resulting reelin fragments is critical for the activation of the reelin signaling pathway ( Lambert de Rouvroit et al, 1999 ; Lugli et al, 2003 ; Tinnes et al, 2013 ). Thus, some studies show that the N-terminal fragment is important for its biological function ( Nakajima et al, 1997 ; Kubo et al, 2002 ; Orcinha et al, 2021 ), other studies focus on the C-terminal part ( Nakano et al, 2007 ; Kohno et al, 2015 ) of the protein, while others suggest that a dimeric central fragment is signaling competent ( Jossin et al, 2004 ; Yasui et al, 2007 ; Orcinha et al, 2021 ; Turk et al, 2021 ). Results of a recent study suggest an essential role for the dimeric central reelin fragment, but not the other reelin fragments or the monomeric central fragment, in the lipoprotein receptor-dependent activation of the canonical reelin pathway, whereas only full-length reelin is potent of stimulating both canonical and non-canonical reelin signaling cascade ( Turk et al, 2021 ).…”

“…In addition, TLE may be accompanied by GCD, reminiscent of GCD in reelin-deficient reeler mice ( Houser, 1990 ; Frotscher et al, 2003 ; Haas and Frotscher, 2010 ; Orcinha et al, 2021 ). However, homozygous reeler mice do not have spontaneous seizures but exhibit increased seizure susceptibility ( Patrylo et al, 2006 ).…”

“…In kainate-treated rodents, GCD formation was prevented in vivo by infusion of exogenous reelin into the hippocampus during epileptogenesis ( Müller et al, 2009 ). Addition of recombinant reelin to KA-treated organotypic hippocampal slice cultures could prevent GCD ( Orcinha et al, 2016 ; Orcinha et al, 2021 ). Conversely, antibody blockade of reelin function in the healthy mouse hippocampus resulted in local broadening of the GCL ( Heinrich et al, 2006 ), which could also be shown in vitro ( Orcinha et al, 2021 ).…”