Analysis of Reelin signaling and neurodevelopmental trajectory in primary cultured cortical neurons with RELN deletion identified in schizophrenia

Reelin, an extracellular matrix protein, is secreted by Cajal-Retzius cells and plays crucial roles in the development of brain structures and neuronal functions. Reductions in Reelin cause the brain dysfunctions associated with mental disorders, such as schizophrenia. A recent genome-wide copy number variation analysis of Japanese schizophrenia patients identified a novel deletion in RELN encoding Reelin. To clarify the pathophysiological role of the RELN deletion, we developed transgenic mice carrying the RELN deletion (Reln-del) and found abnormalities in their brain structures and social behavior. In the present study, we performed an in vitro analysis of Reelin expression, intracellular Reelin signaling, and the morphology of primary cultured cortical neurons from wild-type (WT) and Reln-del mice. Reelin protein levels were lower in Reln-del neurons than in WT neurons. Dab1 expression levels were significantly higher in Reln-del neurons than in WT neurons, suggesting that Reelin signaling was decreased in Reln-del neurons. Reelin was mainly expressed in γ-aminobutyric acid (GABA)-ergic inhibitory neurons, but not in parvalbumin (PV)-positive neurons. A small proportion of Ca²⁺/calmodulin-dependent protein kinase II α subunit (CaMKIIα)-positive excitatory neurons also expressed Reelin. In comparisons with WT neurons, significant decreases were observed in neurite lengths and branch points as well as in the number of postsynaptic density protein 95 (PSD95) immunoreactive puncta in Reln-del neurons. A disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3) is a protease that inactivates Reelin by cleavage at the N-t site. The knockdown of ADAMTS-3 by short hairpin RNAs suppressed Reelin cleavage in conditioned medium and reduced Dab1 expression, indicating that Reelin signaling was enhanced in the primary cultured cortical neurons of WT and heterozygous Reln-del. Accordingly, the inhibition of ADAMTS-3 may be a potential candidate in the clinical treatment of schizophrenia by enhancing Reelin signaling in the brain.