Physiological significance of proteolytic processing of Reelin revealed by cleavage-resistant Reelin knock-in mice

Abstract

Reelin is a secreted protein that plays versatile roles in neuronal development and function. The strength of Reelin signaling is regulated by proteolytic processing, but its importance in vivo is not yet fully understood. Here, we generated Reelin knock-in (PA-DV KI) mice in which the key cleavage site of Reelin was abolished by mutation. As expected, the cleavage of Reelin was severely abrogated in the cerebral cortex and hippocampus of PA-DV KI mice. The amount of Dab1, whose degradation is induced by Reelin signaling, decreased in these tissues, indicating that the signaling strength of Reelin was augmented. The brains of PA-DV KI mice were largely structurally normal, but unexpectedly, the hippocampal layer was disturbed. This phenotype was ameliorated in hemizygote PA-DV KI mice, indicating that excess Reelin signaling is detrimental to hippocampal layer formation. The neuronal dendrites of PA-DV KI mice had more branches and were elongated compared to wild-type mice. These results present the first direct evidence of the physiological importance of Reelin cleavage.Reelin is a large secreted glycoprotein that regulates many important events in mammalian brain development 1-3 . It is also involved in synaptic plasticity and the modulation of higher brain functions 1,4-8 . At the cellular level, Reelin induces the clustering of apolipoprotein E receptor 2 (ApoER2) and very-low-density lipoprotein receptor (VLDLR), which is the prerequisite for the phosphorylation of the cytosolic adaptor protein Dab1 9-11 . Phosphorylated Dab1 is quickly degraded 1,2,5 . Thus, the amount of Dab1 is indicative of the strength of Reelin signaling [12][13][14][15] . Several pathways act downstream of Dab1 phosphorylation and regulate the cytoskeleton, adhesion molecules, ion channels, and neurotransmitter release 1-3,5,16 . Accordingly, insufficient Reelin signaling could contribute to the pathogenesis of neuropsychiatric disorders, such as epilepsy 17-19 , schizophrenia 6,16,20 , and autism 3,8,16 . Furthermore, recent studies showed that a decrease or insufficiency in Reelin signaling could aggravate Alzheimer's disease (AD) 13,21-26 . Therefore, it is important to understand the molecular mechanisms by which Reelin is regulated. Of particular importance is understanding how Reelin signaling is turned off because inhibition of such a mechanism would strengthen Reelin signaling and may help improve brain disorders caused by diminished Reelin function.Secreted signaling proteins can be inactivated or disabled by a variety of mechanisms, including internalization by its receptor, sequestration by an inactive (decoy) binding partner, and degradation. Internalization via ApoER2 and VLDLR 27,28 and a dominant-negative effect of the secreted extracellular domain of ApoER2 29 play a role in limiting Reelin signaling. Reelin protein (approximately 430 kDa as a monomer) contains three specific cleavage sites (N-t, C-t, and WC; reviewed in 7 ). N-t cleavage occurs between Pro1244 and Ala1245 to generate two fragments with molecular mas...

Reelin Functions, Mechanisms of Action and Signaling Pathways During Brain Development and Maturation

“…Therefore, N-terminal processing does not prevent the early and midterm events of Reelin signaling but rather might be important to allow Dab1 protein level recovery, preventing an excessive long-term downregulation of the intracellular signal. A reduced N-terminal processing seems to have only a mild effect in vivo [ 109 ]. A knock-in mouse expressing the mutated Reelin, uncleavable at its N-terminal site, exhibits a normal structure and layer organization in the cerebral cortex and cerebellum.…”

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

“…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 ). Overall, current evidence implies full-length reelin to be more active than its processing-derived products ( Nakano et al, 2007 ; Koie et al, 2014 ; Kohno et al, 2015 ; Sato et al, 2016 ; Dlugosz et al, 2019 ; Okugawa et al, 2020 ; Turk et al, 2021 ). However, it is also possible that the cleaved fragments diffuse to more distant regions to trigger downstream events ( Jossin et al, 2007 ; Yasui et al, 2011 ).…”

“…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 the canonical reelin signaling pathway, reelin binds to receptors of the lipoprotein receptor family, apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor (VLDLR), and induces tyrosine phosphorylation of the intracellular adaptor protein Disabled-1 (Dab1) mediated by Src family kinases ( D’Arcangelo et al, 1999 ; Howell et al, 1999 ; Arnaud et al, 2003 ; Förster, 2014 ; Jossin, 2020 ; Hattori and Kohno, 2021 ). Phosphorylated Dab1 in turn activates downstream signaling cascades, primarily influencing molecules affecting the actin and microtubule cytoskeleton but also influences adhesion molecules, ion channels, and neurotransmitter release ( D’Arcangelo et al, 1999 ; Howell et al, 1999 ; Tissir and Goffinet, 2003 ; Jossin and Goffinet, 2007 ; Leemhuis and Bock, 2011 ; Bock and May, 2016 ; Ishii et al, 2016 ; Hirota and Nakajima, 2017 ; Wasser and Herz, 2017 ; Okugawa et al, 2020 ).…”

Tetrodotoxin prevents heat-shock induced granule cell dispersion in hippocampal slice cultures

“…In HEK293 cells, after internalisation, reelin was proteolytically cleaved, followed by re-secretion of the N-R2 fragment ( Hibi and Hattori, 2009 ). Thus N-R2 expression has served as an indicator for N-t cleavage ( Okugawa et al, 2020 ). Here, western blot analysis of HEK293 secreted reelin in the supernatant did not reveal any significant differences in N-R2 cleavage following heat-shock ( Figures 4F,G ).…”