Beyond Glycolysis: Aldolase A is a Novel Effector in Reelin Mediated Dendritic Development.

Reelin, a secreted glycoprotein, plays a crucial role in guiding neocortical neuronal migration, dendritic outgrowth and arborization, and synaptic plasticity in the adult brain. Reelin primarily operates through the canonical lipoprotein receptors apolipoprotein E receptor 2 (Apoer2) and very low-density lipoprotein receptor (Vldlr). Reelin also engages with non-canonical receptors and unidentified co-receptors; however, the effects of which are less understood. Using high-throughput tandem mass tag LC-MS/MS-based proteomics and gene set enrichment analysis, we identified both shared and unique intracellular pathways activated by Reelin through its canonical and non-canonical signaling in primary murine neurons during dendritic growth and arborization. We observed pathway crosstalk related to regulation of cytoskeleton, neuron projection development, protein transport, and actin filament-based process. We also found enriched gene sets exclusively by the non-canonical Reelin pathway including protein translation, mRNA metabolic process and ribonucleoprotein complex biogenesis suggesting Reelin fine-tunes neuronal structure through distinct signaling pathways. A key discovery is the identification of aldolase A, a glycolytic enzyme and actin binding protein, as a novel effector of Reelin signaling. Reelin induced de novo translation and mobilization of aldolase A from the actin cytoskeleton. We demonstrated that aldolase A is necessary for Reelin-mediated dendrite growth and arborization in primary murine neurons and mouse brain cortical neurons. Interestingly, the function of aldolase A in dendrite development is independent of its known role in glycolysis. Altogether, our findings provide new insights into the Reelin-dependent signaling pathways and effector proteins that are crucial for actin remodeling and dendritic development. Significance: Reelin is an extracellular glycoprotein and exerts its function primarily by binding to the canonical lipoprotein receptors Apoer2 and Vldlr. Reelin is best known for its role in neuronal migration during prenatal brain development. Reelin also signals through a non-canonical pathway outside of Apoer2/Vldlr; however, these receptors and signal transduction pathways are less defined. Here, we examined Reelin's role during dendritic outgrowth in primary murine neurons and identified shared and distinct pathways activated by canonical and non-canonical Reelin signaling. We also found aldolase A as a novel effector of Reelin signaling, that functions independently of its known metabolic role, highlighting Reelin's influence on actin dynamics and neuronal structure and growth.

Tight control of the APP-Mint1 interaction in regulating amyloid production.

Generation of amyloid-ß (Aß) peptides through the proteolytic processing of the amyloid precursor protein (APP) is a pathogenic event in Alzheimer's disease (AD). APP is a transmembrane protein and endocytosis of APP mediated by the YENPTY motif is a key step in Aß generation. Mints, a family of cytosolic adaptor proteins, directly bind to the YENPTY motif of APP and facilitate APP trafficking and processing. Here, we generated and examined two Mint1 mutants, Tyr633Ala of Mint1 (Mint1Y633A) that enhanced APP binding, and Tyr549Ala and Phe610Ala mutant (Mint1Y549A/F610A), that reduced APP binding. We investigated how perturbing the APP-Mint1 interaction through these Mint1 mutants alter APP and Mint1 cellular dynamics and Mint1's interaction with its other binding partners. We found that Mint1Y633A increased binding affinity specifically for APP and presenilin1 (catalytic subunit of γ-secretase), that subsequently enhanced APP endocytosis in primary murine neurons. Conversely, Mint1Y549A/F610A exhibited reduced APP affinity and Aß secretion. The effect of Mint1Y549A/F610A on Aß release was greater compared to knocking down all three Mint proteins supporting the APP-Mint1 interaction is a critical factor in Aß production. Altogether, this study highlights the potential of targeting the APP-Mint1 interaction as a therapeutic strategy for AD.

Human APOER2 Isoforms Have Differential Cleavage Events and Synaptic Properties.

Human apolipoprotein E receptor 2 (APOER2) is a type I transmembrane protein with a large extracellular domain (ECD) and a short cytoplasmic tail. APOER2-ECD contains several ligand-binding domains (LBDs) that are organized into exons with aligning phase junctions, which allows for in-frame exon cassette splicing events. We have identified 25 human APOER2 isoforms from cerebral cortex using gene-specific APOER2 primers, where the majority are exon-skipping events within the N-terminal LBD regions compared with six identified in the heart. APOER2 undergoes proteolytic cleavage in response to ligand binding that releases a C-terminal fragment (CTF) and transcriptionally active intracellular domain (ICD). We tested whether the diversity of human brain-specific APOER2 variants affects APOER2 cleavage. We found isoforms with differing numbers of ligand-binding repeats generated different amounts of CTFs compared with full-length APOER2 (APOER2-FL). Specifically, APOER2 isoforms lacking exons 5-8 (Δex5-8) and lacking exons 4-6 (Δex4-6) generated the highest and lowest amounts of CTF generation, respectively, in response to APOE peptide compared with APOER2-FL. The differential CTF generation of Δex5-8 and Δex4-6 coincides with the proteolytic release of the ICD, which mediates transcriptional activation facilitated by the Mint1 adaptor protein. Functionally, we demonstrated loss of mouse Apoer2 decreased miniature event frequency in excitatory synapses, which may be because of a decrease in the total number of synapses and/or VAMP2 positive neurons. Lentiviral infection with human APOER2-FL or Δex4-6 isoform in Apoer2 knockout neurons restored the miniature event frequency but not Δex5-8 isoform. These results suggest that human APOER2 isoforms have differential cleavage events and synaptic properties.SIGNIFICANCE STATEMENT Humans and mice share virtually the same number of protein-coding genes. However, humans have greater complexity of any higher eukaryotic organisms by encoding multiple protein forms through alternative splicing modifications. Alternative splicing allows pre-mRNAs transcribed from genes to be spliced in different arrangements, producing structurally and functionally distinct protein variants that increase proteomic diversity and are particularly prevalent in the human brain. Here, we identified 25 distinct human APOER2 splice variants from the cerebral cortex using gene-specific APOER2 primers, where the majority are exon-skipping events that exclude N-terminal ligand-binding regions of APOER2. We show that some of the APOER2 variants have differential proteolytic properties in response to APOE ligand and exhibit distinct synaptic properties.

Single molecule, long-read Apoer2 sequencing identifies conserved and species-specific splicing patterns.

Apolipoprotein E receptor 2 (Apoer2) is a synaptic receptor in the brain that binds disease-relevant ligand Apolipoprotein E (Apoe) and is highly alternatively spliced. We examined alternative splicing (AS) of conserved Apoer2 exons across vertebrate species and identified gain of exons in mammals encoding functional domains such as the cytoplasmic and furin inserts, and loss of an exon in primates encoding the eighth LDLa repeat, likely altering receptor surface levels and ligand-binding specificity. We utilized single molecule, long-read RNA sequencing to profile full-length Apoer2 isoforms and identified 68 and 48 unique full-length Apoer2 transcripts in the mouse and human cerebral cortex, respectively. Furthermore, we identified two exons encoding protein functional domains, the third EGF-precursor like repeat and glycosylation domain, that are tandemly skipped specifically in mouse. Our study provides new insight into Apoer2 isoform complexity in the vertebrate brain and highlights species-specific differences in splicing decisions that support functional diversity.

FOXR1 regulates stress response pathways and is necessary for proper brain development.

The forkhead box (Fox) family of transcription factors are highly conserved and play essential roles in a wide range of cellular and developmental processes. We report an individual with severe neurological symptoms including postnatal microcephaly, progressive brain atrophy and global developmental delay associated with a de novo missense variant (M280L) in the FOXR1 gene. At the protein level, M280L impaired FOXR1 expression and induced a nuclear aggregate phenotype due to protein misfolding and proteolysis. RNAseq and pathway analysis showed that FOXR1 acts as a transcriptional activator and repressor with central roles in heat shock response, chaperone cofactor-dependent protein refolding and cellular response to stress pathways. Indeed, FOXR1 expression is increased in response to cellular stress, a process in which it directly controls HSPA6, HSPA1A and DHRS2 transcripts. The M280L mutant compromises FOXR1's ability to respond to stress, in part due to impaired regulation of downstream target genes that are involved in the stress response pathway. Quantitative PCR of mouse embryo tissues show Foxr1 expression in the embryonic brain. Using CRISPR/Cas9 gene editing, we found that deletion of mouse Foxr1 leads to a severe survival deficit while surviving newborn Foxr1 knockout mice have reduced body weight. Further examination of newborn Foxr1 knockout brains revealed a decrease in cortical thickness and enlarged ventricles compared to littermate wild-type mice, suggesting that loss of Foxr1 leads to atypical brain development. Combined, these results suggest FOXR1 plays a role in cellular stress response pathways and is necessary for normal brain development.

ApoER2: Functional Tuning Through Splicing.

Alternative splicing occurs in over 95% of protein-coding genes and contributes to the diversity of the human proteome. Apolipoprotein E receptor 2 (apoER2) is a critical modulator of neuronal development and synaptic plasticity in the brain and is enriched in cassette exon splicing events, in which functional exons are excluded from the final transcript. These alternative splicing events affect apoER2 function, as individual apoER2 exons tend to encode distinct protein functional domains. Although several apoER2 splice variants have been characterized, much work remains to understand how apoER2 splicing events modulate distinct apoER2 activities, including ligand binding specificity, synapse formation and plasticity. Additionally, little is known about how apoER2 splicing events are regulated. Often, alternative splicing events are regulated through the combinatorial action of RNA-binding proteins and other epigenetic mechanisms, however, the regulatory pathways corresponding to each specific exon are unknown in most cases. In this mini-review, we describe the structure of apoER2, highlight the unique functions of known isoforms, discuss what is currently known about the regulation of apoER2 splicing by RNA-binding proteins and pose new questions that will further our understanding of apoER2 splicing complexity.

A rare autism-associated MINT2/APBA2 mutation disrupts neurexin trafficking and synaptic function.

MINT2/APBA2 is a synaptic adaptor protein involved in excitatory synaptic transmission. Several nonsynonymous coding variants in MINT2 have been identified in autism spectrum disorders (ASDs); however, these rare variants have not been examined functionally and the pathogenic mechanisms are unknown. Here, we examined the synaptic effects of rat Mint2 N723S mutation (equivalent to autism-linked human MINT2 N722S mutation) which targets a conserved asparagine residue in the second PDZ domain of Mint2 that binds to neurexin-1α (Nrxn1α), a presynaptic cell-adhesion protein implicated in ASDs. We show the N723S mutation impairs Nrxn1α stabilization and trafficking to the membrane while binding to Nrxn1α remains unaffected. Using time-lapse imaging in primary mouse neurons, we found that the N723S mutant had more immobile puncta at neuronal processes compared to Mint2 wild type. We therefore, reasoned that the N723S mutant may alter the co-transport of Nrxn1α at axonal processes to presynaptic terminals. Indeed, we found the N723S mutation affected Nrxn1α localization at presynaptic terminals which correlated with a decrease in Nrxn-mediated synaptogenesis and miniature event frequency in excitatory synapses. Together, our data reveal Mint2 N723S leads to neuronal dysfunction, in part due to alterations in Nrxn1α surface trafficking and synaptic function of Mint2.

CLASP2 Links Reelin to the Cytoskeleton during Neocortical Development.

The Reelin signaling pathway plays a crucial role in regulating neocortical development. However, little is known about how Reelin controls the cytoskeleton during neuronal migration. Here, we identify CLASP2 as a key cytoskeletal effector in the Reelin signaling pathway. We demonstrate that CLASP2 has distinct roles during neocortical development regulating neuron production and controlling neuron migration, polarity, and morphogenesis. We found downregulation of CLASP2 in migrating neurons leads to mislocalized cells in deeper cortical layers, abnormal positioning of the centrosome-Golgi complex, and aberrant length/orientation of the leading process. We discovered that Reelin regulates several phosphorylation sites within the positively charged serine/arginine-rich region that constitute consensus GSK3ß phosphorylation motifs of CLASP2. Furthermore, phosphorylation of CLASP2 regulates its interaction with the Reelin adaptor Dab1 and this association is required for CLASP2 effects on neurite extension and motility. Together, our data reveal that CLASP2 is an essential Reelin effector orchestrating cytoskeleton dynamics during brain development.

Differential splicing and glycosylation of Apoer2 alters synaptic plasticity and fear learning.

Apoer2 is an essential receptor in the central nervous system that binds to the apolipoprotein ApoE. Various splice variants of Apoer2 are produced. We showed that Apoer2 lacking exon 16, which encodes the O-linked sugar (OLS) domain, altered the proteolytic processing and abundance of Apoer2 in cells and synapse number and function in mice. In cultured cells expressing this splice variant, extracellular cleavage of OLS-deficient Apoer2 was reduced, consequently preventing γ-secretase-dependent release of the intracellular domain of Apoer2. Mice expressing Apoer2 lacking the OLS domain had increased Apoer2 abundance in the brain, hippocampal spine density, and glutamate receptor abundance, but decreased synaptic efficacy. Mice expressing a form of Apoer2 lacking the OLS domain and containing an alternatively spliced cytoplasmic tail region that promotes glutamate receptor signaling showed enhanced hippocampal long-term potentiation (LTP), a phenomenon associated with learning and memory. However, these mice did not display enhanced spatial learning in the Morris water maze, and cued fear conditioning was reduced. Reducing the expression of the mutant Apoer2 allele so that the abundance of the protein was similar to that of Apoer2 in wild-type mice normalized spine density, hippocampal LTP, and cued fear learning. These findings demonstrated a role for ApoE receptors as regulators of synaptic glutamate receptor activity and established differential receptor glycosylation as a potential regulator of synaptic function and memory.

Microtubule plus-end tracking protein CLASP2 regulates neuronal polarity and synaptic function.

Microtubule organization and dynamics are essential during axon and dendrite formation and maintenance in neurons. However, little is known about the regulation of microtubule dynamics during synaptic development and function in mammalian neurons. Here, we present evidence that the microtubule plus-end tracking protein CLASP2 (cytoplasmic linker associated protein 2) is a key regulator of axon and dendrite outgrowth that leads to functional alterations in synaptic activity and formation. We found that CLASP2 protein levels steadily increase throughout neuronal development in the mouse brain and are specifically enriched at the growth cones of extending neurites. The short-hairpin RNA-mediated knockdown of CLASP2 in primary mouse neurons decreased axon and dendritic length, whereas overexpression of human CLASP2 caused the formation of multiple axons, enhanced dendritic branching, and Golgi condensation, implicating CLASP2 in neuronal morphogenesis. In addition, the CLASP2-induced morphological changes led to significant functional alterations in synaptic transmission. CLASP2 overexpression produced a large increase in spontaneous miniature event frequency that was specific to excitatory neurotransmitter release. The changes in presynaptic activity produced by CLASP2 overexpression were accompanied by increases in presynaptic terminal circumference, total synapse number, and a selective increase in presynaptic proteins that are involved in neurotransmitter release. Also, we found a smaller increase in miniature event amplitude that was accompanied by an increase in postsynaptic surface expression of GluA1 receptor localization. Together, these results provide evidence for involvement of the microtubule plus-end tracking protein CLASP2 in cytoskeleton-related mechanisms underlying neuronal polarity and interplay between microtubule stabilization and synapse formation and activity.

The Reelin receptors Apoer2 and Vldlr coordinate the patterning of Purkinje cell topography in the developing mouse cerebellum.

The adult cerebellar cortex is comprised of reproducible arrays of transverse zones and parasagittal stripes of Purkinje cells. Adult stripes are created through the perinatal rostrocaudal dispersion of embryonic Purkinje cell clusters, triggered by signaling through the Reelin pathway. Reelin is secreted by neurons in the external granular layer and deep cerebellar nuclei and binds to two high affinity extracellular receptors on Purkinje cells-the Very low density lipoprotein receptor (Vldlr) and apolipoprotein E receptor 2 (Apoer2). In mice null for either Reelin or double null for Vldlr and Apoer2, Purkinje cell clusters fail to disperse. Here we report that animals null for either Vldlr or Apoer2 individually, exhibit specific and parasagittally-restricted Purkinje cell ectopias. For example, in mice lacking Apoer2 function immunostaining reveals ectopic Purkinje cells that are largely restricted to the zebrin II-immunonegative population of the anterior vermis. In contrast, mice null for Vldlr have a much larger population of ectopic Purkinje cells that includes members from both the zebrin II-immunonegative and -immunopositive phenotypes. HSP25 immunoreactivity reveals that in Vldlr null animals a large portion of zebrin II-immunopositive ectopic cells are probably destined to become stripes in the central zone (lobules VI-VII). A small population of ectopic zebrin II-immunonegative Purkinje cells is also observed in animals heterozygous for both receptors (Apoer2(+/-): Vldlr(+/-)), but no ectopia is present in mice heterozygous for either receptor alone. These results indicate that Apoer2 and Vldlr coordinate the dispersal of distinct, but overlapping subsets of Purkinje cells in the developing cerebellum.

Contribution of the Reelin signaling pathways to nociceptive processing.

The reeler gene encodes Reelin, a secreted glycoprotein that binds to the very-low-density lipoprotein receptor (Vldlr) and apolipoprotein E receptor 2 (Apoer 2), and induces Src- and Fyn-mediated tyrosine phosphorylation of the intracellular adaptor protein Disabled-1 (Dab1). This Reelin-Dab1 signaling pathway regulates neuronal positioning during development. A second Reelin pathway acts through Apoer 2-exon 19 to modulate synaptic plasticity in adult mice. We recently reported positioning errors in reeler dorsal horn laminae I-II and V, and the lateral spinal nucleus. Behavioral correlates of these positioning errors include a decreased mechanical and increased thermal sensitivity in reeler mice. Here we examined mice with deletions or modifications of both the Reelin-Dab1 signaling pathway and the Reelin-Apoer 2-exon 19 pathway on a Vldlr-deficient background. We detected reeler-like dorsal horn positioning errors only in Dab1 mutant and Apoer 2/Vldlr double mutant mice. Although Dab1 mutants, like reeler, showed decreased mechanical and increased thermal sensitivity, neither the single Vldlr or Apoer 2 knockouts, nor the Apoer 2-exon 19 mutants differed in their acute pain sensitivity from controls. However, despite the dramatic alterations in acute 'pain' processing in reeler and Dab1 mutants, the exacerbation of pain processing after tissue injury (hindpaw carrageenan injection) was preserved. Finally, we recapitulated the reeler dorsal horn positioning errors by inhibiting Dab1 phosphorylation in organotypic cultures. We conclude that the Reelin-Dab1 pathway differentially contributes to acute and persistent pain, and that the plasticity associated with the Reelin-Apoer 2-exon 19 pathway is distinct from that which contributes to injury-induced enhancement of 'pain' processing.

The Pafah1b complex interacts with the reelin receptor VLDLR.

Reelin is an extracellular protein that directs the organization of cortical structures of the brain through the activation of two receptors, the very low-density lipoprotein receptor (VLDLR) and the apolipoprotein E receptor 2 (ApoER2), and the phosphorylation of Disabled-1 (Dab1). Lis1, the product of the Pafah1b1 gene, is a component of the brain platelet-activating factor acetylhydrolase 1b (Pafah1b) complex, and binds to phosphorylated Dab1 in response to Reelin. Here we investigated the involvement of the whole Pafah1b complex in Reelin signaling and cortical layer formation and found that catalytic subunits of the Pafah1b complex, Pafah1b2 and Pafah1b3, specifically bind to the NPxYL sequence of VLDLR, but not to ApoER2. Compound Pafah1b1(+/-);Apoer2(-/-) mutant mice exhibit a reeler-like phenotype in the forebrain consisting of the inversion of cortical layers and hippocampal disorganization, whereas double Pafah1b1(+/-);Vldlr(-/-) mutants do not. These results suggest that a cross-talk between the Pafah1b complex and Reelin occurs downstream of the VLDLR receptor.

ApoE receptor 2 controls neuronal survival in the adult brain.

A central pathogenic feature of neurodegenerative diseases and neurotrauma is the death of neurons. A mechanistic understanding of the factors and conditions that induce the dysfunction and death of neurons is essential for devising effective treatment strategies against neuronal loss after trauma or during aging. Because Apolipoprotein E (ApoE) is a major risk factor for several neurodegenerative diseases, including Alzheimer's disease , a direct or indirect role of ApoE receptors in the disease process is likely. Here we have used gene targeting in mice to investigate possible roles of ApoE receptors in the regulation of neuronal survival. We demonstrate that a differentially spliced isoform of an ApoE receptor, ApoE receptor 2 (Apoer2), is essential for protection against neuronal cell loss during normal aging. Furthermore, the same splice form selectively promotes neuronal cell death after injury through mechanisms that may involve serine/threonine kinases of the Jun N-terminal kinase (JNK) family. These findings raise the possibility that ApoE and its receptors cooperatively regulate common mechanisms that are essential to neuronal survival in the adult brain.

Functional dissection of Reelin signaling by site-directed disruption of Disabled-1 adaptor binding to apolipoprotein E receptor 2: distinct roles in development and synaptic plasticity.

The Reelin signaling pathway controls neuronal positioning in human and mouse brain during development as well as modulation of long-term potentiation (LTP) and behavior in the adult. Reelin signals by binding to two transmembrane receptors, apolipoprotein E receptor 2 (Apoer2) and very-low-density lipoprotein receptor. After Reelin binds to the receptors, Disabled-1 (Dab1), an intracellular adaptor protein that binds to the cytoplasmic tails of the receptors, becomes phosphorylated on tyrosine residues, initiating a signaling cascade that includes activation of Src-family kinases and Akt. Here, we have created a line of mutant mice (Apoer2 EIG) in which the Apoer2 NFDNPVY motif has been altered to EIGNPVY to disrupt the Apoer2-Dab1 interaction to further study Reelin signaling in development and adult brain. Using primary neuronal cultures stimulated with recombinant Reelin, we find that normal Reelin signaling requires the wild-type NFDNPVY sequence and likely the interaction of Apoer2 with Dab1. Furthermore, examination of hippocampal, cortical, and cerebellar layering reveals that the NFDNPVY sequence of Apoer2 is indispensable for normal neuronal positioning during development of the brain. Adult Apoer2 EIG mice display severe abnormalities in LTP and behavior that are distinct from those observed for mice lacking Apoer2. In Apoer2 EIG slices, LTP degraded to baseline within 30 min, and this was prevented in the presence of Reelin. Together, these findings emphasize the complexity of Reelin signaling in the adult brain, which likely requires multiple adaptor protein interactions with the intracellular domain of Apoer2.

F-spondin interaction with the apolipoprotein E receptor ApoEr2 affects processing of amyloid precursor protein.

A recent study showed that F-spondin, a protein associated with the extracellular matrix, interacted with amyloid precursor protein (APP) and inhibited beta-secretase cleavage. F-spondin contains a thrombospondin domain that we hypothesized could interact with the family of receptors for apolipoprotein E (apoE). Through coimmunoprecipitation experiments, we demonstrated that F-spondin interacts with an apoE receptor (apoE receptor 2 [ApoEr2]) through the thrombospondin domain of F-spondin and the ligand binding domain of ApoEr2. Full-length F-spondin increased coimmunoprecipitation of ApoEr2 and APP in transfected cells and primary neurons and increased surface expression of APP and ApoEr2. Full-length F-spondin, but none of the individual F-spondin domains, increased cleavage of APP and ApoEr2, resulting in more secreted forms of APP and ApoEr2 and more C-terminal fragments (CTF) of these proteins. In addition, full-length F-spondin, but not the individual domains, decreased production of the beta-CTF of APP and Abeta in transfected cells and primary neurons. The reduction in APP beta-CTF was blocked by receptor-associated protein (RAP), an inhibitor of lipoprotein receptors, implicating ApoEr2 in the altered proteolysis of APP. ApoEr2 coprecipitated with APP alpha- and beta-CTF, and F-spondin reduced the levels of APP intracellular domain signaling, suggesting that there are also intracellular interactions between APP and ApoEr2, perhaps involving adaptor proteins. These studies suggest that the extracellular matrix molecule F-spondin can cluster APP and ApoEr2 together on the cell surface and affect the processing of each, resulting in decreased production of Abeta.

Reelin modulates NMDA receptor activity in cortical neurons.

Reelin, a large protein that regulates neuronal migration during embryonic development, activates a conserved signaling pathway that requires its receptors, very low-density lipoprotein receptor and apolipoprotein E receptor 2, the cytoplasmic adaptor protein Disabled-1 (Dab1), and Src family kinases (SFK). Reelin also markedly enhances long-term potentiation in the adult hippocampus, suggesting that this developmental signaling pathway can physiologically modulate learning and behavior. Here, we show that Reelin can regulate NMDA-type glutamate receptor activity through a mechanism that requires SFKs and Dab1. Reelin mediates tyrosine phosphorylation of and potentiates calcium influx through NMDA receptors in primary wild-type cortical neurons but not in Dab1 knock-out neurons or in cells in which Reelin binding to its receptors is blocked by a receptor antagonist. Inhibition of SFK abolishes Reelin-induced and glutamate-dependent enhancement of calcium influx. We also show that Reelin-induced augmentation of Ca2+ entry through NMDA receptors increases phosphorylation and nuclear translocation of the transcription factor cAMP-response element binding protein. Thus, Reelin may physiologically modulate learning and memory by modulating NMDA receptor functions.

Modulation of synaptic plasticity and memory by Reelin involves differential splicing of the lipoprotein receptor Apoer2.

Apolipoprotein E receptor 2 (Apoer2), a member of the LDL receptor gene family, and its ligand Reelin control neuronal migration during brain development. Apoer2 is also essential for induction of long-term potentiation (LTP) in the adult brain. Here we show that Apoer2 is present in the postsynaptic densities of excitatory synapses where it forms a functional complex with NMDA receptors. Reelin signaling through Apoer2 markedly enhances LTP through a mechanism that requires the presence of amino acids encoded by an exon in the intracellular domain of Apoer2. This exon is alternatively spliced in an activity-dependent manner and is required for Reelin-induced tyrosine phosphorylation of NMDA receptor subunits. Mice constitutively lacking the exon perform poorly in learning and memory tasks. Thus, alternative splicing of Apoer2, a novel component of the NMDA receptor complex, controls the modulation of NMDA receptor activity, synaptic neurotransmission, and memory by Reelin.

The apoE receptor apoER2 is involved in the maintenance of efficient synaptic plasticity.

ApoER2 is one of the major receptors for ApoE in the brain, and has been shown to be involved not only in lipoprotein endocytosis, as other members of the LDL receptor family of receptors, but also in various cellular functions such as signalling and cellular guidance. By using a model of synaptic plasticity in mice lacking none, one or two alleles of the apoER2 gene, we investigated the implication of such a receptor deficiency on the remodelling process. Our results indicate that animals lacking apoER2 express higher levels of brain APP, as well as both key amyloid peptides, while apoE levels are slightly lower. Following entorhinal cortex lesioning, apoE levels increase in the deafferented hippocampus, while a delay in the increase of APP was observed. Hippocampal amyloid levels are also increased in response to the lesion, and highly potentiated by the complete absence of apoER2 gene. The results suggest a significant role for apoER2 in signalling various proteins in response to massive deafferentation and may participate in maintaining efficient synaptic plasticity and dendritic remodelling.