Intersectin 1 is a component of the Reelin pathway to regulate neuronal migration and synaptic plasticity in the hippocampus Burkhard Jakoba, Gaga Kochlamazashvilia, Maria Jäpela, Aziz Gauharb, Hans H. Bockb, Tanja Maritzena, and Volker Hauckea,c,1 aDepartment of Molecular Pharmacology and Cell Biology, Leibniz-Forschungsinstitut für Molekulare Pharmakologie, 13125 Berlin, Germany; bClinic of Gastroenterology and Hepatology, Heinrich-Heine Universität Düsseldorf, 40225 Duesseldorf, Germany; and cFaculty of Biology, Chemistry, and Pharmacy, Freie Universität Berlin, 14195 Berlin, Germany. Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved April 17, 2017 (received for review March 17, 2017) Brain development and function depend on the directed and co- (13) or of the VLDLR/ApoER2-associated signaling adaptor ordinated migration of neurons from proliferative zones to their Dab1 (17, 20) mimics the loss of Reelin, whereas less severe final position. The secreted glycoprotein Reelin is an important migration defects are seen in KO mice lacking either VLDLR factor directing neuronal migration. Loss of Reelin function results or ApoER2. in the severe developmental disorder lissencephaly and is associ- Although previous studies have indicated that VLDLR and ated with neurological diseases in humans. Reelin signals via ApoER2 exhibit partially overlapping functions in Reelin signal- the lipoprotein receptors very low density lipoprotein receptor ing, others show that VLDLR and ApoER2 also serve divergent (VLDLR) and apolipoprotein E receptor 2 (ApoER2), but the exact roles in neuronal migration (15, 21). Moreover, both receptors are mechanism by which these receptors control cellular function is required independently for Reelin-mediated augmentation of poorly understood. We report that loss of the signaling scaffold hippocampal LTP (22, 23), whereas ApoER2 mediates the aug- intersectin 1 (ITSN1) in mice leads to defective neuronal migration mentation of spontaneous neurotransmission by Reelin (24). and ablates Reelin stimulation of hippocampal long-term potenti- These data suggest that additional as-yet unidentified components NEUROSCIENCE ation (LTP). Knockout (KO) mice lacking ITSN1 suffer from disper- must exist that contribute to the shared and specific functions of sion of pyramidal neurons and malformation of the radial glial Reelin receptors in neuronal migration and synaptic plasticity (15, scaffold, akin to the hippocampal lamination defects observed in 19, 21, 23). Thus, the molecular mechanisms underlying signaling VLDLR or ApoER2 mutants. ITSN1 genetically interacts with Reelin via VLDLR and ApoER2 (e.g., the mechanism by which ligand receptors, as evidenced by the prominent neuronal migration and binding to these receptors is transmitted to intracellular tyrosine radial glial defects in hippocampus and cortex seen in double-KO phosphorylation of Dab1) remain incompletely understood (6). mice lacking ITSN1 and ApoER2. These defects were similar to, In the present study, we analyzed KO mice deficient in inter- albeit less severe than, those observed in Reelin-deficient or sectin 1 (ITSN1) (25, 26), a scaffold protein highly expressed in VLDLR/ ApoER2 double-KO mice. Molecularly, ITSN1 associates neurons (27). ITSN1 is a multidomain protein comprising two with the VLDLR and its downstream signaling adaptor Dab1 to fa- Eps15 homology (EH) domains and five SH3 domains (A–E) cilitate Reelin signaling. Collectively, these data identify ITSN1 as a connected via a central helical region and a C-terminal DH-PH component of Reelin signaling that acts predominantly by facilitat- domain with guanine nucleotide-exchange activity toward Cdc42 ing the VLDLR-Dab1 axis to direct neuronal migration in the cortex (Fig. S1A). These domains provide interaction surfaces for mul- and hippocampus and to augment synaptic plasticity. tiple endocytic and signaling proteins, including dynamin, Eps15, FCHo, assembly protein 2 (AP-2), N-WASP, and Sos (27–30). Reelin signaling | hippocampus | synaptic plasticity | endocytosis | Although based on these activities, ITSN1 has been implicated in multidomain scaffold Significance he development and function of the brain depend on the Tdirected and coordinated migration of neurons from pro- – The development and function of the brain depend on the mi- liferative zones to their final position (1 3). Studies in mutant gration of neurons from the proliferative zone in which they are mice and flies have shown that neuronal migration is governed by born to their final position. Reelin, a signaling molecule implicated developmental signaling but does not depend on neuronal ac- in the developmental disorder lissencephaly and associated with tivity (2, 4, 5). Formation of the complex six-layered architecture neurologic diseases in humans, plays an important role in directing of the neocortex and the laminated structure of the hippocampus – neuronal migration and brain development. Here, we identify (1 3) involves the proper migration of neurons along radial glial intersectin 1, a large scaffold protein genetically linked to Down cells, a process guided by the extracellular Reelin glycoprotein syndrome, as a component of the Reelin signaling pathway that is (2, 6, 7). Loss of Reelin function results in the severe develop- important for brain development and Reelin-mediated augmen- mental disorder lissencephaly (8) and is associated with neuro- ’ tation of synaptic plasticity, a cellular model for learning and logical diseases, such as epilepsy (9), Alzheimer s disease (10), memory. and schizophrenia (11) in humans. Reelin binding to its recep- tors, very low density lipoprotein receptor (VLDLR) and apo- Author contributions: B.J., G.K., H.H.B., T.M., and V.H. designed research; B.J., G.K., M.J., lipoprotein E receptor 2 (ApoER2) (12, 13), induces tyrosine and T.M. performed research; A.G. and H.H.B. contributed new reagents/analytic tools; phosphorylation of the signaling adaptor protein Disabled 1 B.J., G.K., M.J., and T.M. analyzed data; and V.H. wrote the paper. (Dab1) by Src family kinases (14–17). Defective Reelin signal- The authors declare no conflict of interest. ing (e.g., in Reeler mice that lack Reelin expression) disrupts This article is a PNAS Direct Submission. neuronal migration, resulting in defective cortical layering (6, 7, Freely available online through the PNAS open access option. 13, 17), malformation of the radial glial scaffold (17, 18), and 1To whom correspondence should be addressed. Email: [email protected]. dispersion of hippocampal pyramidal and granule neurons (9, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 19). Combined genetic ablation of both VLDLR and ApoER2 1073/pnas.1704447114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1704447114 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 cell signaling (30), exocytosis/endocytosis (25, 27, 28, 31–33), de- enable learning and memory in adults (2, 6, 7). Conditional defects velopment of dendritic spines (29, 34), cortical midline connec- in Reelin signaling impair radial neuronal migration, resulting in tivity (35), and spatial learning (35), its precise physiological the ectopic location of newborn neurons in the hilar region of function in the mammalian central nervous system in vivo remains the DG (20). A similar accumulation of ectopic newborn double- incompletely understood. Here we identify ITSN1 as a component cortin (Dcx)-positive neurons in the hilus was found in ITSN1 of Reelin signaling that acts predominantly by facilitating the KO mice (Fig. 1 D and E). These data suggest that ITSN1 is re- VLDLR-Dab1 axis to direct neuronal migration in the forebrain quired for normal hippocampal cell layering, formation of the ra- and to augment synaptic plasticity. dial glial scaffold, and proper migration of newborn neurons in the hippocampus. Results In contrast, we did not detect consistent defects in our ITSN1 Hippocampal Lamination Defects in ITSN1 KO Mice. To explore the KO mice with respect to cortical midline connectivity (Fig. S2 C physiological functions of ITSN1 in vivo, we analyzed the overall and D) that were previously reported in a gene trap line with brain architecture of KO mice deficient in ITSN1 (25, 26). Nissl- different genetic background (35). Moreover, loss of ITSN1 did stained sections of hippocampi from ITSN1 KO mice showed a not affect the kinetics of presynaptic vesicle exocytosis or endo- gross dispersion of pyramidal neurons in the CA1 and CA3 areas cytic membrane retrieval as measured by fluorescence analysis of and an altered shape of the dentate gyrus (DG) (Fig. 1A), sug- pHluorin-tagged synaptic vesicle proteins (Fig. S3 A and B), levels gesting a possible defect in neuronal migration. A similar phenotype of exocytic/endocytic proteins (Fig. S3C), and the number, area, – has been observed in KO mice lacking Reelin receptors (e.g., length, and morphology of dendritic spines (Fig. S3 D H). VLDLR, ApoER2) (13). Defects in Reelin signaling are accom- panied by malformation of the radial glial scaffold in the DG (17, Intersectin Is Required for Reelin Signaling to Facilitate NMDAR- Mediated LTP. Given that pyramidal cell dispersion, malformation 18); therefore, we examined whether loss of ITSN1 affects radial of the radial glial scaffold, and defects in neuronal migration in the glial cell morphology. Indeed, in KO mice lacking ITSN1, radial hippocampus have been observed in mouse mutants