Safety of Anti-Reelin Therapeutic Approaches for Chronic Inflammatory Diseases.

Reelin, a large extracellular glycoprotein, plays critical roles in neuronal development and synaptic plasticity in the central nervous system (CNS). Recent studies have revealed non-neuronal functions of plasma Reelin in inflammation by promoting endothelial-leukocyte adhesion through its canonical pathway in endothelial cells (via ApoER2 acting on NF-κB), as well as in vascular tone regulation and thrombosis. In this study, we have investigated the safety and efficacy of selectively depleting plasma Reelin as a potential therapeutic strategy for chronic inflammatory diseases. We found that Reelin expression remains stable throughout adulthood and that peripheral anti-Reelin antibody treatment with CR-50 efficiently depletes plasma Reelin without affecting its levels or functionality within the CNS. Notably, this approach preserves essential neuronal functions and synaptic plasticity. Furthermore, in mice induced with experimental autoimmune encephalomyelitis (EAE), selective modulation of endothelial responses by anti-Reelin antibodies reduces pathological leukocyte infiltration without completely abolishing diapedesis. Finally, long-term Reelin depletion under metabolic stress induced by a Western diet did not negatively impact the heart, kidney, or liver, suggesting a favorable safety profile. These findings underscore the promising role of peripheral anti-Reelin therapeutic strategies for autoimmune diseases and conditions where endothelial function is compromised, offering a novel approach that may avoid the immunosuppressive side effects associated with conventional anti-inflammatory therapies.

Targeting the intracellular neurexin interactome by in vivo proximity ligation.

In a recent study, Profes, Tiroumalechetty, and colleagues used the in vivo proximity ligation technique TurboID to scrupulously characterize the interactome of the intracellular domain (ICD) of neurexin, revealing that this domain may be involved in presynaptic actin assembly by interacting with actin-associated proteins.

The CHD Protein Kismet Restricts the Synaptic Localization of Cell Adhesion Molecules at the Drosophila Neuromuscular Junction.

The appropriate expression and localization of cell surface cell adhesion molecules must be tightly regulated for optimal synaptic growth and function. How neuronal plasma membrane proteins, including cell adhesion molecules, cycle between early endosomes and the plasma membrane is poorly understood. Here we show that the Drosophila homolog of the chromatin remodeling enzymes CHD7 and CHD8, Kismet, represses the synaptic levels of several cell adhesion molecules. Neuroligins 1 and 3 and the integrins αPS2 and ßPS are increased at kismet mutant synapses but Kismet only directly regulates transcription of neuroligin 2. Kismet may therefore regulate synaptic CAMs indirectly by activating transcription of gene products that promote intracellular vesicle trafficking including endophilin B (endoB) and/or rab11. Knock down of EndoB in all tissues or neurons increases synaptic FasII while knock down of EndoB in kis mutants does not produce an additive increase in FasII. In contrast, neuronal expression of Rab11, which is deficient in kis mutants, leads to a further increase in synaptic FasII in kis mutants. These data support the hypothesis that Kis influences the synaptic localization of FasII by promoting intracellular vesicle trafficking through the early endosome.

Differential contribution of canonical and noncanonical NLGN3 pathways to early social development and memory performance.

Neuroligin (NLGN) 3 is a postsynaptic cell adhesion protein organizing synapse formation through two different types of transsynaptic interactions, canonical interaction with neurexins (NRXNs) and a recently identified noncanonical interaction with protein tyrosine phosphatase (PTP) δ. Although, NLGN3 gene is known as a risk gene for neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability (ID), the pathogenic contribution of the canonical NLGN3-NRXN and noncanonical NLGN3-PTPδ pathways to these disorders remains elusive. In this study, we utilized Nlgn3 mutant mice selectively lacking the interaction with either NRXNs or PTPδ and investigated their social and memory performance. Neither Nlgn3 mutants showed any social cognitive deficiency in the social novelty recognition test. However, the Nlgn3 mutant mice lacking the PTPδ pathway exhibited significant decline in the social conditioned place preference (sCPP) at the juvenile stage, suggesting the involvement of the NLGN3-PTPδ pathway in the regulation of social motivation and reward. In terms of learning and memory, disrupting the canonical NRXN pathway attenuated contextual fear conditioning while disrupting the noncanonical NLGN3-PTPδ pathway enhanced it. Furthermore, disruption of the NLGN3-PTPδ pathway negatively affected the remote spatial reference memory in the Barnes maze test. These findings highlight the differential contributions of the canonical NLGN3-NRXN and noncanonical NLGN3-PTPδ synaptogenic pathways to the regulation of higher order brain functions associated with ASD and ID.

NINJ1 induces plasma membrane rupture and release of damage-associated molecular pattern molecules during ferroptosis.

Ferroptosis is a regulated form of necrotic cell death caused by iron-dependent accumulation of oxidized phospholipids in cellular membranes, culminating in plasma membrane rupture (PMR) and cell lysis. PMR is also a hallmark of other types of programmed necrosis, such as pyroptosis and necroptosis, where it is initiated by dedicated pore-forming cell death-executing factors. However, whether ferroptosis-associated PMR is also actively executed by proteins or driven by osmotic pressure remains unknown. Here, we investigate a potential ferroptosis role of ninjurin-1 (NINJ1), a recently identified executor of pyroptosis-associated PMR. We report that NINJ1 oligomerizes during ferroptosis, and that Ninj1-deficiency protects macrophages and fibroblasts from ferroptosis-associated PMR. Mechanistically, we find that NINJ1 is dispensable for the initial steps of ferroptosis, such as lipid peroxidation, channel-mediated calcium influx, and cell swelling. In contrast, NINJ1 is required for early loss of plasma membrane integrity, which precedes complete PMR. Furthermore, NINJ1 mediates the release of cytosolic proteins and danger-associated molecular pattern (DAMP) molecules from ferroptotic cells, suggesting that targeting NINJ1 could be a therapeutic option to reduce ferroptosis-associated inflammation.

Caudally pronounced deficiencies in preplate splitting and migration underly a rostro-caudal progression of cortical lamination defects in the reeler brain.

In mammalian neocortex development, every cohort of newborn neurons is guided toward the marginal zone, leading to an "inside-out" organization of the 6 neocortical layers. This migratory pattern is regulated by the extracellular glycoprotein Reelin. The reeler mouse shows a homozygous mutation of the reelin gene. Using RNA in situ hybridization we could demonstrate that the Reelin-deficient mouse cortex (male and female) displays an increasing lamination defect along the rostro-caudal axis that is characterized by strong cellular intermingling, but roughly reproduces the "inside-out" pattern in rostral cortex, while caudal cortex shows a relative inversion of neuronal positioning ("outside-in"). We found that in development of the reeler cortex, preplate-splitting is also defective with an increasing severity along the rostro-caudal axis. This leads to a misplacement of subplate neurons that are crucial for a switch in migration mode within the cortical plate. Using Flash Tag labeling and nucleoside analog pulse-chasing, we found an according migration defect within the cortical plate, again with a progressive severity along the rostro-caudal axis. Thus, loss of one key player in neocortical development leads to highly area-specific (caudally pronounced) developmental deficiencies that result in multiple roughly opposite rostral versus caudal adult neocortical phenotypes.

Caspase cleavage of gasdermin E causes neuronal pyroptosis in HIV-associated neurocognitive disorder.

Despite effective antiretroviral therapies, 20-30% of persons with treated HIV infection develop a neurodegenerative syndrome termed HIV-associated neurocognitive disorder (HAND). HAND is driven by HIV expression coupled with inflammation in the brain but the mechanisms underlying neuronal damage and death are uncertain. The inflammasome-pyroptosis axis coordinates an inflammatory type of regulated lytic cell death that is underpinned by the caspase-activated pore-forming gasdermin proteins. The mechanisms driving neuronal pyroptosis were investigated herein in models of HAND, using multi-platform molecular and morphological approaches that included brain tissues from persons with HAND and simian immunodeficiency virus (SIV)-infected non-human primates as well as cultured human neurons. Neurons in the frontal cortices from persons with HAND showed increased cleaved gasdermin E (GSDME), which was associated with ß-III tubulin degradation and increased HIV levels. Exposure of cultured human neurons to the HIV-encoded viral protein R (Vpr) elicited time-dependent cleavage of GSDME and Ninjurin-1 (NINJ1) induction with associated cell lysis that was inhibited by siRNA suppression of both proteins. Upstream of GSDME cleavage, Vpr exposure resulted in activation of caspases-1 and 3. Pretreatment of Vpr-exposed neurons with the caspase-1 inhibitor, VX-765, reduced cleavage of both caspase-3 and GSDME, resulting in diminished cell death. To validate these findings, we examined frontal cortical tissues from SIV-infected macaques, disclosing increased expression of GSDME and NINJ1 in cortical neurons, which was co-localized with caspase-3 detection in animals with neurological disease. Thus, HIV infection of the brain triggers the convergent activation of caspases-1 and -3, which results in GSDME-mediated neuronal pyroptosis in persons with HAND. These findings demonstrate a novel mechanism by which a viral infection causes pyroptotic death in neurons while also offering new diagnostic and therapeutic strategies for HAND and other neurodegenerative disorders.

Plasma Neuronal Growth Regulator 1 May Link Physical Activity to Reduced Risk of Type 2 Diabetes: A Proteome-Wide Study of ARIC Participants.

Habitual physical activity (PA) impacts the plasma proteome and reduces the risk of developing type 2 diabetes (T2D). Using a large-scale proteome-wide approach in Atherosclerosis Risk in Communities study participants, we aimed to identify plasma proteins associated with PA and determine which of these may be causally related to lower T2D risk. PA was associated with 92 plasma proteins in discovery (P < 1.01 × 10-5), and 40 remained significant in replication (P < 5.43 × 10-4). Eighteen of these proteins were independently associated with incident T2D (P < 1.25 × 10-3), including neuronal growth regulator 1 (NeGR1; hazard ratio per SD 0.85; P = 7.5 × 10-11). Two-sample Mendelian randomization (MR) inverse variance weighted analysis indicated that higher NeGR1 reduces T2D risk (odds ratio [OR] per SD 0.92; P = 0.03) and was consistent with MR-Egger, weighted median, and weighted mode sensitivity analyses. A stronger association was observed for the single cis-acting NeGR1 genetic variant (OR per SD 0.80; P = 6.3 × 10-5). Coupled with previous evidence that low circulating NeGR1 levels promote adiposity, its association with PA and potential causal role in T2D shown here suggest that NeGR1 may link PA exposure with metabolic outcomes. Further research is warranted to confirm our findings and examine the interplay of PA, NeGR1, adiposity, and metabolic health.

SCO-spondin knockout mice exhibit small brain ventricles and mild spine deformation.

Reissner's fiber (RF) is an extracellular polymer comprising the large monomeric protein SCO-spondin (SSPO) secreted by the subcommissural organ (SCO) that extends through cerebrospinal fluid (CSF)-filled ventricles into the central canal of the spinal cord. In zebrafish, RF and CSF-contacting neurons (CSF-cNs) form an axial sensory system that detects spinal curvature, instructs morphogenesis of the body axis, and enables proper alignment of the spine. In mammalian models, RF has been implicated in CSF circulation. However, challenges in manipulating Sspo, an exceptionally large gene of 15,719 nucleotides, with traditional approaches has limited progress. Here, we generated a Sspo knockout mouse model using CRISPR/Cas9-mediated genome-editing. Sspo knockout mice lacked RF-positive material in the SCO and fibrillar condensates in the brain ventricles. Remarkably, Sspo knockout brain ventricle sizes were reduced compared to littermate controls. Minor defects in thoracic spine curvature were detected in Sspo knockouts, which did not alter basic motor behaviors tested. Altogether, our work in mouse demonstrates that SSPO and RF regulate ventricle size during development but only moderately impact spine geometry.

ApoER2-Dab1 disruption as the origin of pTau-associated neurodegeneration in sporadic Alzheimer's disease.

In sporadic Alzheimer's disease (sAD) specific regions, layers and neurons accumulate hyperphosphorylated Tau (pTau) and degenerate early while others remain unaffected even in advanced disease. ApoER2-Dab1 signaling suppresses Tau phosphorylation as part of a four-arm pathway that regulates lipoprotein internalization and the integrity of actin, microtubules, and synapses; however, the role of this pathway in sAD pathogenesis is not fully understood. We previously showed that multiple ApoER2-Dab1 pathway components including ApoE, Reelin, ApoER2, Dab1, pP85αTyr607, pLIMK1Thr508, pTauSer202/Thr205 and pPSD95Thr19 accumulate together within entorhinal-hippocampal terminal zones in sAD, and proposed a unifying hypothesis wherein disruption of this pathway underlies multiple aspects of sAD pathogenesis. However, it is not yet known whether ApoER2-Dab1 disruption can help explain the origin(s) and early progression of pTau pathology in sAD. In the present study, we applied in situ hybridization and immunohistochemistry (IHC) to characterize ApoER2 expression and accumulation of ApoER2-Dab1 pathway components in five regions known to develop early pTau pathology in 64 rapidly autopsied cases spanning the clinicopathological spectrum of sAD. We found that (1) these selectively vulnerable neuron populations strongly express ApoER2; and (2) multiple ApoER2-Dab1 components representing all four arms of this pathway accumulate in abnormal neurons and neuritic plaques in mild cognitive impairment (MCI) and sAD cases and correlate with histological progression and cognitive deficits. Multiplex-IHC revealed that Dab1, pP85αTyr607, pLIMK1Thr508, pTauSer202/Thr205 and pPSD95Thr19 accumulate together within many of the same ApoER2-expressing neurons and in the immediate vicinity of ApoE/ApoJ-enriched extracellular plaques. Collective findings reveal that pTau is only one of many ApoER2-Dab1 pathway components that accumulate in multiple neuroanatomical sites in the earliest stages of sAD and provide support for the concept that ApoER2-Dab1 disruption drives pTau-associated neurodegeneration in human sAD.

Liver sinusoidal endothelial cells show reduced scavenger function and downregulation of Fc gamma receptor IIb, yet maintain a preserved fenestration in the Glmpgt/gt mouse model of slowly progressing liver fibrosis.

Liver sinusoidal endothelial cells (LSECs) are fenestrated endothelial cells with a unique, high endocytic clearance capacity for blood-borne waste macromolecules and colloids. This LSEC scavenger function has been insufficiently characterized in liver disease. The Glmpgt/gt mouse lacks expression of a subunit of the MFSD1/GLMP lysosomal membrane protein transporter complex, is born normal, but soon develops chronic, mild hepatocyte injury, leading to slowly progressing periportal liver fibrosis, and splenomegaly. This study examined how LSEC scavenger function and morphology are affected in the Glmpgt/gt model. FITC-labelled formaldehyde-treated serum albumin (FITC-FSA), a model ligand for LSEC scavenger receptors was administered intravenously into Glmpgt/gt mice, aged 4 months (peak of liver inflammation), 9-10 month, and age-matched Glmpwt/wt mice. Organs were harvested for light and electron microscopy, quantitative image analysis of ligand uptake, collagen accumulation, LSEC ultrastructure, and endocytosis receptor expression (also examined by qPCR and western blot). In both age groups, the Glmpgt/gt mice showed multifocal liver injury and fibrosis. The uptake of FITC-FSA in LSECs was significantly reduced in Glmpgt/gt compared to wild-type mice. Expression of LSEC receptors stabilin-1 (Stab1), and mannose receptor (Mcr1) was almost similar in liver of Glmpgt/gt mice and age-matched controls. At the same time, immunostaining revealed differences in the stabilin-1 expression pattern in sinusoids and accumulation of stabilin-1-positive macrophages in Glmpgt/gt liver. FcγRIIb (Fcgr2b), which mediates LSEC endocytosis of soluble immune complexes was widely and significantly downregulated in Glmpgt/gt liver. Despite increased collagen in space of Disse, LSECs of Glmpgt/gt mice showed well-preserved fenestrae organized in sieve plates but the frequency of holes >400 nm in diameter was increased, especially in areas with hepatocyte damage. In both genotypes, FITC-FSA also distributed to endothelial cells of spleen and bone marrow sinusoids, suggesting that these locations may function as possible compensatory sites of clearance of blood-borne scavenger receptor ligands in liver fibrosis.

NINJ1 is activated by cell swelling to regulate plasma membrane permeabilization during regulated necrosis.

Plasma membrane permeabilization (PMP) is a defining feature of regulated necrosis. It allows the extracellular release of damage-associated molecular patterns (DAMPs) that trigger sterile inflammation. The pore forming molecules MLKL and GSDMs drive PMP in necroptosis and pyroptosis, respectively, but the process of PMP remains unclear in many other forms of regulated necrosis. Here, we identified NINJ1 as a crucial regulator of PMP and consequent DAMP release during ferroptosis, parthanatos, H2O2-induced necrosis and secondary necrosis. Importantly, the membrane-permeabilizing function of NINJ1 takes place after the metabolic death of the cells and is independent of the pore-forming molecules MLKL, GSDMD and GSDME. During ferroptosis, NINJ1 acts downstream of lipid peroxidation, which suggested a role for reactive oxygen species (ROS) in NINJ1 activation. Reactive oxygen species were however neither sufficient nor required to trigger NINJ1-dependent PMP. Instead, we found that NINJ1 oligomerization is induced by the swelling of the cell and that its permeabilizing potential still requires an addition, and yet to be discovered, activation mechanism.

Distinct neurexin isoforms cooperate to initiate and maintain foraging activity.

Neurexins are synaptic adhesion molecules that play diverse roles in synaptic development, function, maintenance, and plasticity. Neurexin genes have been associated with changes in human behavior, where variants in NRXN1 are associated with autism, schizophrenia, and Tourette syndrome. While NRXN1, NRXN2, and NRXN3 all encode major α and ß isoforms, NRXN1 uniquely encodes a γ isoform, for which mechanistic roles in behavior have yet to be defined. Here, we show that both α and γ isoforms of neurexin/nrx-1 are required for the C. elegans behavioral response to food deprivation, a sustained period of hyperactivity upon food loss. We find that the γ isoform regulates initiation and the α isoform regulates maintenance of the behavioral response to food deprivation, demonstrating cooperative function of multiple nrx-1 isoforms in regulating a sustained behavior. The γ isoform alters monoamine signaling via octopamine, relies on specific expression of NRX-1 isoforms throughout the relevant circuit, and is independent of neuroligin/nlg-1, the canonical trans-synaptic partner of nrx-1. The α isoform regulates the pre-synaptic structure of the octopamine producing RIC neuron and its maintenance role is conditional on neuroligin/nlg-1. Collectively, these results demonstrate that neurexin isoforms can have separate behavioral roles and act cooperatively across neuronal circuits to modify behavior, highlighting the need to directly analyze and consider all isoforms when defining the contribution of neurexins to behavior.

Mouse models of human CNTNAP1-associated congenital hypomyelinating neuropathy and genetic restoration of murine neurological deficits.

The Contactin-associated protein 1 (Cntnap1) mouse mutants fail to establish proper axonal domains in myelinated axons. Human CNTNAP1 mutations are linked to hypomyelinating neuropathy-3, which causes severe neurological deficits. To understand the human neuropathology and to model human CNTNAP1C323R and CNTNAP1R764C mutations, we generated Cntnap1C324R and Cntnap1R765C mouse mutants, respectively. Both Cntnap1 mutants show weight loss, reduced nerve conduction, and progressive motor dysfunction. The paranodal ultrastructure shows everted myelin loops and the absence of axo-glial junctions. Biochemical analysis reveals that these Cntnap1 mutant proteins are nearly undetectable in the paranodes, have reduced surface expression and stability, and are retained in the neuronal soma. Postnatal transgenic expression of Cntnap1 in the mutant backgrounds rescues the phenotypes and restores the organization of axonal domains with improved motor function. This study uncovers the mechanistic impact of two human CNTNAP1 mutations in a mouse model and provides proof of concept for gene therapy for CNTNAP1 patients.

Neurexin-3 subsynaptic densities are spatially distinct from Neurexin-1 and essential for excitatory synapse nanoscale organization in the hippocampus.

Proteins critical for synaptic transmission are non-uniformly distributed and assembled into regions of high density called subsynaptic densities (SSDs) that transsynaptically align in nanocolumns. Neurexin-1 and neurexin-3 are essential presynaptic adhesion molecules that non-redundantly control NMDAR- and AMPAR-mediated synaptic transmission, respectively, via transsynaptic interactions with distinct postsynaptic ligands. Despite their functional relevance, fundamental questions regarding the nanoscale properties of individual neurexins, their influence on the subsynaptic organization of excitatory synapses and the mechanisms controlling how individual neurexins engage in precise transsynaptic interactions are unknown. Using Double Helix 3D dSTORM and neurexin mouse models, we identify neurexin-3 as a critical presynaptic adhesion molecule that regulates excitatory synapse nano-organization in hippocampus. Furthermore, endogenous neurexin-1 and neurexin-3 form discrete and non-overlapping SSDs that are enriched opposite their postsynaptic ligands. Thus, the nanoscale organization of neurexin-1 and neurexin-3 may explain how individual neurexins signal in parallel to govern different synaptic properties.

Association of Differentially Altered Liver Fibrosis with Deposition of TGFBi in Stabilin-Deficient Mice.

Liver sinusoidal endothelial cells (LSECs) control clearance of Transforming growth factor, beta-induced, 68kDa (TGFBi) and Periostin (POSTN) through scavenger receptors Stabilin-1 (Stab1) and Stabilin-2 (Stab2). Stabilin inhibition can ameliorate atherosclerosis in mouse models, while Stabilin-double-knockout leads to glomerulofibrosis. Fibrotic organ damage may pose a limiting factor in future anti-Stabilin therapies. While Stab1-deficient (Stab1-/-) mice were shown to exhibit higher liver fibrosis levels upon challenges, fibrosis susceptibility has not been studied in Stab2-deficient (Stab2-/-) mice. Wildtype (WT), Stab1-/- and Stab2-/- mice were fed experimental diets, and local ligand abundance, hepatic fibrosis, and ligand plasma levels were measured. Hepatic fibrosis was increased in both Stab1-/- and Stab2-/- at baseline. A pro-fibrotic short Methionine-Choline-deficient (MCD) diet induced slightly increased liver fibrosis in Stab1-/- and Stab2-/- mice. A Choline-deficient L-amino acid-defined (CDAA) diet induced liver fibrosis of similar distribution and extent in all genotypes (WT, Stab1-/- and Stab2-/-). A hepatic abundance of Stabilin ligand TGFBi correlated very highly with liver fibrosis levels. In contrast, plasma levels of TGFBi were increased only in Stab2-/- mice after the CDAA diet but not the MCD diet, indicating the differential effects of these diets. Here we show that a single Stabilin deficiency of either Stab1 or Stab2 induces mildly increased collagen depositions under homeostatic conditions. Upon experimental dietary challenge, the local abundance of Stabilin ligand TGFBi was differentially altered in Stabilin-deficient mice, indicating differentially affected LSEC scavenger functions. Since anti-Stabilin-directed therapies are in clinical evaluation for the treatment of diseases, these findings bear relevance to treatment with novel anti-Stabilin agents.

Calcium/P53/Ninjurin 1 Signaling Mediates Plasma Membrane Rupture of Acinar Cells in Severe Acute Pancreatitis.

Ninjurin 1 (NINJ1) is a double-transmembrane cell-surface protein that might mediate plasma membrane rupture (PMR) and the diffusion of inflammatory factors. PMR is a characteristic of acinar cell injury in severe acute pancreatitis (SAP). However, the involvement of NINJ1 in mediating the PMR of acinar cells in SAP is currently unclear. Our study has shown that NINJ1 is expressed in acinar cells, and the expression is significantly upregulated in sodium-taurocholate-induced SAP. The knockout of NINJ1 delays PMR in acinar cells and alleviates SAP. Moreover, we observed that NINJ1 expression is mediated by Ca2+ concentration in acinar cells. Importantly, we found that Ca2+ overload drives mitochondrial stress to upregulate the P53/NINJ1 pathway, inducing PMR in acinar cells, and amlodipine, a Ca2+ channel inhibitor, can reduce the occurrence of PMR by decreasing the concentration of Ca2+. Our results demonstrate the mechanism by which NINJ1 induces PMR in SAP acinar cells and provide a potential new target for treatment of SAP.

Reeling from news that reelin defends the brain against Alzheimer's.

In a recent study, Lopera and colleagues investigate a person with extreme resilience to autosomal-dominant familial Alzheimer's disease, which they attribute to a rare variant in the RELN gene encoding reelin.1.

Central repeat fragment of reelin leads to active reelin intracellular signaling and rescues cognitive deficits in a mouse model of reelin deficiency.

Reelin and its receptor, ApoER2, play important roles in prenatal brain development and postnatally in synaptic plasticity, learning, and memory. Previous reports suggest that reelin's central fragment binds to ApoER2 and receptor clustering is involved in subsequent intracellular signaling. However, limitations of currently available assays have not established cellular evidence of ApoER2 clustering upon binding of the central reelin fragment. In the present study, we developed a novel, cell-based assay of ApoER2 dimerization using a "split-luciferase" approach. Specifically, cells were co-transfected with one recombinant ApoER2 receptor fused to the N-terminus of luciferase and one ApoER2 receptor fused to the C-terminus of luciferase. Using this assay, we directly observed basal ApoER2 dimerization/clustering in transfected HEK293T cells and, significantly, an increase in ApoER2 clustering in response to that central fragment of reelin. Furthermore, the central fragment of reelin activated intracellular signal transduction of ApoER2, indicated by increased levels of phosphorylation of Dab1, ERK1/2, and Akt in primary cortical neurons. Functionally, we were able to demonstrate that injection of the central fragment of reelin rescued phenotypic deficits observed in the heterozygous reeler mouse. These data are the first to test the hypothesis that the central fragment of reelin contributes to facilitating the reelin intracellular signaling pathway through receptor clustering.