Abstracts

Rationale: Reelin is a molecule secreted into the extracellular matrix that is important for neuronal migration and dendritic development. It binds apolipoprotein E receptor 2 and very low density lipoprotein receptor expressed on neurons or radial glia. Binding and internalization of reelin leads to phosphorylation of disabled-1 (Dab1), an adaptor protein, activating downstream signaling and Dab1 degradation. Evidence implicates the reelin/Dab1 pathway in dentate granule cell (DGC) development. Mice with reelin, Dab1, or reelin receptor mutations have markedly abnormal DGC layers as well as DGCs with abnormal dendrites, including hilar basal dendrites (HBDs) similar to those seen in experimental mesial temporal lobe epilepsy (mTLE). DGC layer abnormalities in mice with deficient reelin signaling also resemble DGC layer dispersion seen in human mTLE. Previous work from our laboratory demonstrates that reelin expression decreases markedly post-status epilepticus (SE; Gong et al., J. Neurosci, 2007). These findings led us to hypothesize that HBDs and ectopic cells in experimental mTLE result from the loss of reelin/Dab1 signaling. Methods: We crossed mice with two floxed Dab1 conditional knock-in (cki) alleles (Dab1 ^cki/cki) with a nestin-CreERT2/ROSA-YFP line and used tamoxifen to conditionally delete Dab1 in postnatal DGC progenitors at P7-8 or P28-35. In rats, we unilaterally injected a retroviral Dab1 overexpression vector (RV-CAG-Dab1-IRES-mCh) into the dentate gyrus (DG) post-SE (P56 SE, P60 injection) to determine its effect on aberrant migration and HBDs compared with a control vector (RV-CAG-GFP) injected into the contralateral DG. Lastly, we injected a Dab1 shRNA (RV-pU6-shDab1-CAG-GFP-WPRE) viral vector bilaterally into the DG at P32. All animals were sacrificed at 2 months of age. Results: In conditional Dab1 deficient mice, Prox1 (a DGC marker) and YFP staining revealed an increase in hilar ectopic cells. Homozygotes were more severely affected than heterozygotes. Staining for neurofilament-M revealed axonal disorganization in Dab1 deficient mice. In rats injected with the Dab1 overexpression vector post-SE, labeled cells were often improperly oriented and displayed more abnormal dendrites than on the control side, but fewer appeared in ectopic locations than cells expressing control vector. Expression of Dab shRNA in DGC progenitors led to markedly decreased dendritic arborization of cells located in the DGC layer. Furthermore, numerous labeled glia were evident in these animals, in both the hilus and DGC layer, which were not seen in controls. Conclusions: Aberrant neurogenesis is a proposed cause of seizures and cognitive deficits common in mTLE, and this work supports an important role for Dab1 signaling in both neuronal migration and dendrite formation. Additionally, our shRNA data suggest that intact reelin signaling through Dab1 is necessary for directing DGC progenitors to a neuronal, rather than glial, phenotype. We are currently pursuing in vitro neural progenitor cell assays to further address these possibilities.