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.

Time-dependent antidepressant-like effects of reelin and ketamine in the repeated-corticosterone model of chronic stress.

There is an urgent need for novel antidepressants, given that approximately 30% of those diagnosed with depression do not respond adequately to first-line treatment. Additionally, monoaminergic-based antidepressants have a substantial therapeutic time-lag, often taking months to reach full therapeutic effect. Ketamine, an N-methyl-d-aspartate receptor (NMDAR) antagonist is the only current effective rapid-acting antidepressant, demonstrating efficacy within hours and lasting up to two weeks with an acute dose. Reelin, an extracellular matrix glycoprotein, has demonstrated rapid-acting antidepressant-like effects at 24 h, however the exact timescale of these effects has not been investigated. To determine the short and long-term effects of reelin, female Long Evans rats (n = 120) underwent a chronic corticosterone (CORT; or vehicle) paradigm (40 mg/kg, 21 days). On day 21, rats were treated with reelin (3µg; i.v.), ketamine (10 mg/kg; i.p.), both reelin and ketamine (same doses), or vehicle (saline). Behavioural and biological effects were then evaluated at 1 h, 6 h, 12 h, and 1 week after treatment. The 1-week cohort continued CORT injections to ensure the effect of chronic stress was not lost. Individually, both reelin and ketamine significantly rescued CORT-induced behaviour and hippocampal reelin expression at all timepoints. Ketamine rescued a decrease in dendritic maturity as induced by CORT. Synergistic effects of reelin and ketamine appeared at 1-week, suggesting a potential additive effect of the antidepressant-like actions. Taken together, this study provides further support for reelin-based therapeutics to develop rapid-acting antidepressant.

Whole-exome sequencing has revealed novel genetic characteristics in intracranial germ cell tumours in the Chinese.

AIMS: Intracranial germ cell tumour (IGCT) is a type of rare central nervous system tumour that mainly occurs in children and adolescents, with great variation in its incidence rate and molecular characteristics in patients from different populations. The genetic alterations of IGCT in the Chinese population are still unknown. METHODS AND RESULTS: In this study, 47 patients were enrolled and their tumour specimens were analysed by whole-exome sequencing (WES). We found that KIT was the most significantly mutated gene (15/47, 32%), which mainly occurred in the germinoma group (13/20, 65%), and less frequently in NGGCT (2/27, 7%). Copy number variations (CNVs) of FGF6 and TFE3 only appeared in NGGCT patients (P = 0.003 and 0.032, respectively), while CNVs of CXCR4, RAC2, PDGFA, and FEV only appeared in germinoma patients (P = 0.004 of CXCR4 and P = 0.027 for the last three genes). Compared with a previous Japanese cohort, the somatic mutation rates of RELN and SYNE1 were higher in the Chinese. Prognostic analysis showed that the NF1 mutation was associated with shorter overall survival and progression-free survival in IGCT patients. Clonal evolution analysis revealed an early branched evolutionary pattern in two IGCT patients who underwent changes in the histological subtype or degree of differentiation during disease surveillance. CONCLUSION: This study indicated that Chinese IGCT patients may have distinct genetic characteristics and identified several possible genetic alterations that have the potential to become prognostic biomarkers of NGGCT patients.

Molecular Mechanisms of Reelin in the Enteric Nervous System and the Microbiota-Gut-Brain Axis: Implications for Depression and Antidepressant Therapy.

Current pharmacological treatments for depression fail to produce adequate remission in a significant proportion of patients. Increasingly, other systems, such as the microbiome-gut-brain axis, are being looked at as putative novel avenues for depression treatment. Dysbiosis and dysregulation along this axis are highly comorbid with the severity of depression symptoms. The endogenous extracellular matrix protein reelin is present in all intestinal layers as well as in myenteric and submucosal ganglia, and its receptors are also present in the gut. Reelin secretion from subepithelial myofibroblasts regulates cellular migration along the crypt-villus axis in the small intestine and colon. Reelin brain expression is downregulated in mood and psychotic disorders, and reelin injections have fast antidepressant-like effects in animal models of depression. This review seeks to discuss the roles of reelin in the gastrointestinal system and propose a putative role for reelin actions in the microbiota-gut-brain axis in the pathogenesis and treatment of depression, primarily reflecting on alterations in gut epithelial cell renewal and in the clustering of serotonin transporters.

The interaction of RELN-DNMT genes involving in neurotrophin signaling pathway contributes to schizophrenia susceptibility.

OBJECTIVE: Schizophrenia belongs to a severe mental illness with complicated clinical presentations, an ill-defined pathogenesis, and no known cause. Many genetic studies imply that polygenic interaction is important in the development of schizophrenia. The main mechanism of the RELN-BDNF-CREB-DNMT signaling pathway in neurodevelopment involves RELN, brain-derived neurotrophic factor (BDNF), transcription factor cyclic adenosine monophosphate response element binding protein (CREB), DNA methyltransferase 1 (DNMT1), as well as DNA methyltransferase 3B (DNMT3B). An early case-control research on 15 polymorphisms in the RELN, CREB, BDNF, DNMT1, and DNMT3B genes was done. A single gene variation has little effect on the pathogenesis of schizophrenia, but the combination of intergenic variation loci has a bigger impact because schizophrenia is a complex polygenic disorder. The objective of the current study sought to explore the impact of genetic interactions between RELN, BDNF, CREB, DNMT1, and DNMT3B on schizophrenia in order to further highlight the genetic factors influencing the risk of schizophrenia. METHODS: Taking the case-control study design, with the Diagnostic and Statistical Manual of Mental Disorders-Fifth Edition (DSM-5) to be the evaluation norm, 134 individuals suffering from schizophrenia hospitalized in the Third People's Hospital of Zhongshan City within January 2018 to April 2020 (case group) were selected, and 64 healthy individuals (control group) from the same geographical area had been chosen as well. MassArray identified DNMT1 gene single nucleotide polymorphisms (rs2114724 and rs2228611) and DNMT3B gene SNPs (rs2424932, rs1569686, rs6119954, and rs2424908). Using the generalized multifactor dimensionality reduction (GMDR), the RELN-BDNF-CREB-DNMT pathway's gene interactions were examined for their impact on schizophrenia. RESULTS: GMDR analysis showed that the three-order interaction model RELN (rs2073559, rs2229864)-DNMT3B (rs2424908) was the optimal model (p = 0.001), with the consistency of cross-validation of 10/10 and the test accuracy of 0.8711. CONCLUSION: The interaction between the RELN (rs2073559, rs2229864)-DNMT3B (rs2424908) may be related to schizophrenia, and large sample sizes should be verified in different population.

A frameshift-deletion mutation in Reelin causes cerebellar hypoplasia in White Swiss Shepherd dogs.

Cerebellar hypoplasia is a heterogeneous neurological condition in which the cerebellum is smaller than usual or not completely developed. The condition can have genetic origins, with Mendelian-effect mutations described in several mammalian species. Here, we describe a genetic investigation of cerebellar hypoplasia in White Swiss Shepherd dogs, where two affected puppies were identified from a litter with a recent common ancestor on both sides of their pedigree. Whole genome sequencing was conducted for 10 dogs in this family, and filtering of these data based on a recessive transmission hypothesis highlighted five protein-altering candidate variants - including a frameshift-deletion of the Reelin (RELN) gene (p.Val947*). Given the status of RELN as a gene responsible for cerebellar hypoplasia in humans, sheep and mice, these data strongly suggest the loss-of-function variant as underlying these effects. This variant has not been found in other dog breeds nor in a cohort of European White Swiss Shepherds, suggesting a recent mutation event. This finding will support the genotyping of a more diverse sample of dogs, and should aid future management of the harmful allele through optimised mating schemes.

Reelin through the years: From brain development to inflammation.

Reelin was originally identified as a regulator of neuronal migration and synaptic function, but its non-neuronal functions have received far less attention. Reelin participates in organ development and physiological functions in various tissues, but it is also dysregulated in some diseases. In the cardiovascular system, Reelin is abundant in the blood, where it contributes to platelet adhesion and coagulation, as well as vascular adhesion and permeability of leukocytes. It is a pro-inflammatory and pro-thrombotic factor with important implications for autoinflammatory and autoimmune diseases such as multiple sclerosis, Alzheimer's disease, arthritis, atherosclerosis, or cancer. Mechanistically, Reelin is a large secreted glycoprotein that binds to several membrane receptors, including ApoER2, VLDLR, integrins, and ephrins. Reelin signaling depends on the cell type but mostly involves phosphorylation of NF-κB, PI3K, AKT, or JAK/STAT. This review focuses on non-neuronal functions and the therapeutic potential of Reelin, while highlighting secretion, signaling, and functional similarities between cell types.

Postnatal injection of Reelin protein into the cerebellum ameliorates the motor functions in reeler mouse.

Reelin is a large secreted protein important for brain development and functions. In both humans and mice, the lack of Reelin gene causes cerebellar hypoplasia and ataxia. Treatment against Reelin deficiency is currently unavailable. Here, we show that the injection of recombinant Reelin protein into the cerebellum of Reelin-deficient reeler mice at postnatal day 3 ameliorates the forelimb coordination and mice are noted to stand up along cage wall more frequently. A mutant Reelin protein resistant to proteases has no better effect than the wild-type Reelin. Such ameliorations were not observed when a mutant Reelin protein that does not bind to Reelin receptors was injected and the injection of Reelin protein did not ameliorate the behavior of Dab1-mutant yotari mice, indicating that its effect is dependent on the canonical Reelin receptor-Dab1 pathway. Additionally, a Purkinje cell layer in reeler mice was locally induced by Reelin protein injection. Our results indicate that the reeler mouse cerebellum retains the ability to react to Reelin protein in the postnatal stage and that Reelin protein has the potential to benefit Reelin-deficient patients.

In silico analysis of the possible crosstalk between O-linked ß-GlcNAcylation and phosphorylation sites of Disabled 1 adaptor protein in vertebrates.

Disabled 1 (Dab1) is an adaptor protein with essential functions regulated by reelin signaling and affects many biological processes in the nervous system, including cell motility, adhesion, cortical development, maturation, and synaptic plasticity. Posttranslational modifications directly guide the fates of cytoplasmic proteins to complete their functions correctly. Reciprocal crosstalk between O-GlcNAcylation and phosphorylation is a dynamic modification in cytoplasmic proteins. It modulates the functions of the proteins by regulating their interactions with other molecules in response to the continuously changeable cell microenvironment. Although Dab1 contains conserved recognition sites for phosphorylation in their N-terminal protein interaction domain, the O-ß-GlcNAcylation and phosphorylation sites of human Dab1 sequence, their reciprocal crosstalk, and potential kinases catalyzing the phosphorylation remain unknown. In this study, we determined potential thirty-seven O-ß-GlcNAcylation and sixty-seven phosphorylation sites. Conserved twenty-one residues of these glycosylated sites were also phosphorylated with various kinases, including ATM, CKI, DNAPK, GSK3, PKC, PKG, RSK, cdc2, cdk5, and p38MAPK. In addition, we analyzed these conserved sites at our constructed two- and three-dimensional structures of human Dab1 protein. Dab1 protein models were frequently composed of coil structures as well as α-helix and ß-strands. Many of these conserved crosstalk sites between O-ß-GlcNAcylation and phosphorylation were localized at the coil region of the protein model. These findings may guide biochemical, genetic, and glyco-biology based on further experiments about the Dab1 signaling process. Understanding these modifications might change the point of view of the Dab1 signaling process and treatment for pathological conditions in neurodegenerative diseases such as Alzheimer's disease.

The ZSWIM8 ubiquitin ligase regulates neurodevelopment by guarding the protein quality of intrinsically disordered Dab1.

Protein quality control (PQC) is essential for maintaining protein homeostasis and guarding the accuracy of neurodevelopment. Previously, we found that a conserved EBAX-type CRL regulates the protein quality of SAX-3/ROBO guidance receptors in Caenorhabditis elegans. Here, we report that ZSWIM8, the mammalian homolog of EBAX-1, is essential for developmental stability of mammalian brains. Conditional deletion of Zswim8 in the embryonic nervous system causes global cellular stress, partial perinatal lethality and defective migration of neural progenitor cells. CRISPR-mediated knockout of ZSWIM8 impairs spine formation and synaptogenesis in hippocampal neurons. Mechanistic studies reveal that ZSWIM8 controls protein quality of Disabled 1 (Dab1), a key signal molecule for brain development, thus protecting the signaling strength of Dab1. As a ubiquitin ligase enriched with intrinsically disordered regions (IDRs), ZSWIM8 specifically recognizes IDRs of Dab1 through a "disorder targets misorder" mechanism and eliminates misfolded Dab1 that cannot be properly phosphorylated. Adult survivors of ZSWIM8 CKO show permanent hippocampal abnormality and display severely impaired learning and memory behaviors. Altogether, our results demonstrate that ZSWIM8-mediated PQC is critical for the stability of mammalian brain development.

Reelin regulates the migration of late-born hippocampal CA1 neurons via cofilin phosphorylation.

Reelin, a large secreted glycoprotein, plays an important role in neuronal migration during brain development. The C-terminal region (CTR) of Reelin is involved in the efficient activation of downstream signaling and its loss leads to abnormal hippocampal layer formation. However, the molecular mechanism by which Reelin CTR regulates hippocampal development remains unknown. Here, we showed that the migration of late-born, but not early-born, neurons is impaired in the knock-in mice in which Reelin CTR is deleted (ΔC-KI mice). The phosphorylation of cofilin, an actin-depolymerizing protein, was remarkably decreased in the hippocampus of the ΔC-KI mice. Exogenous expression of pseudo-phosphorylated cofilin rescued the ectopic positioning of neurons in the hippocampus of ΔC-KI mice. These results suggest that Reelin CTR is required for the migration of late-born neurons in the hippocampus and that this event involves appropriate phosphorylation of cofilin.

Identification and Validation of RELN Mutation as a Response Indicator for Immune Checkpoint Inhibitor Therapy in Melanoma and Non-Small Cell Lung Cancer.

Remarkable clinical benefits in several advanced cancers are observed under the treatment of immune checkpoint inhibitor (ICI) agents. However, only a smaller proportion of patients respond to the treatments. Reelin (RELN) is frequently mutated in the cancer genome. In this study, the RELN mutation association with ICI treatment efficacy in melanoma and non-small cell lung cancer (NSCLC) was elucidated. Data from 631 melanoma and 109 NSCLC patients with both ICI treatment data and pre-treatment mutational profiles were collected. In addition, from the Cancer Genome Atlas (TCGA) project, we also obtained both tumors to explore the immunologic features behind RELN mutations. Melanoma patients with RELN mutations exhibited a favorable ICI survival benefit when compared with wild-type patients (HR: 0.66, 95% CI: 0.51-0.87, p = 0.003). A higher response rate was also noticed in RELN-mutated patients (38.9% vs. 28.3%, p = 0.017). The association of RELN mutations with a preferable immunotherapy outcome and response was further confirmed in NSCLC. Further exploration demonstrated that favorable immunocyte infiltration and immune response signaling pathways were found in patients with RELN mutations. In this study, RELN mutations were identified to connect with a better immune microenvironment and an improved ICI efficacy in melanoma and NSCLC, which provides a potential biomarker for immunological feature evaluation and immunotherapeutic outcome prediction at the molecular level.

Lipophorin receptors regulate mushroom body development and complex behaviors in Drosophila.

BACKGROUND: Drosophila melanogaster lipophorin receptors (LpRs), LpR1 and LpR2, are members of the LDLR family known to mediate lipid uptake in a range of organisms from Drosophila to humans. The vertebrate orthologs of LpRs, ApoER2 and VLDL-R, function as receptors of a glycoprotein involved in development of the central nervous system, Reelin, which is not present in flies. ApoER2 and VLDL-R are associated with the development and function of the hippocampus and cerebral cortex, important association areas in the mammalian brain, as well as with neurodevelopmental and neurodegenerative disorders linked to those regions. It is currently unknown whether LpRs play similar roles in the Drosophila brain. RESULTS: We report that LpR-deficient flies exhibit impaired olfactory memory and sleep patterns, which seem to reflect anatomical defects found in a critical brain association area, the mushroom bodies (MB). Moreover, cultured MB neurons respond to mammalian Reelin by increasing the complexity of their neurite arborization. This effect depends on LpRs and Dab, the Drosophila ortholog of the Reelin signaling adaptor protein Dab1. In vitro, two of the long isoforms of LpRs allow the internalization of Reelin, suggesting that Drosophila LpRs interact with human Reelin to induce downstream cellular events. CONCLUSIONS: These findings demonstrate that LpRs contribute to MB development and function, supporting the existence of a LpR-dependent signaling in Drosophila, and advance our understanding of the molecular factors functioning in neural systems to generate complex behaviors in this model. Our results further emphasize the importance of Drosophila as a model to investigate the alterations in specific genes contributing to neural disorders.

Specific contribution of Reelin expressed by Cajal-Retzius cells or GABAergic interneurons to cortical lamination.

The extracellular protein Reelin, expressed by Cajal-Retzius (CR) cells at early stages of cortical development and at late stages by GABAergic interneurons, regulates radial migration and the "inside-out" pattern of positioning. Current models of Reelin functions in corticogenesis focus on early CR cell-derived Reelin in layer I. However, developmental disorders linked to Reelin deficits, such as schizophrenia and autism, are related to GABAergic interneuron-derived Reelin, although its role in migration has not been established. Here we selectively inactivated the Reln gene in CR cells or GABAergic interneurons. We show that CR cells have a major role in the inside-out order of migration, while CR and GABAergic cells sequentially cooperate to prevent invasion of cortical neurons into layer I. Furthermore, GABAergic cell-derived Reelin compensates some features of the reeler phenotype and is needed for the fine tuning of the layer-specific distribution of cortical neurons. In the hippocampus, the inactivation of Reelin in CR cells causes dramatic alterations in the dentate gyrus and mild defects in the hippocampus proper. These findings lead to a model in which both CR and GABAergic cell-derived Reelin cooperate to build the inside-out order of corticogenesis, which might provide a better understanding of the mechanisms involved in the pathogenesis of neuropsychiatric disorders linked to abnormal migration and Reelin deficits.

The role of reelin in the pathological mechanism of depression from clinical to rodents.

Major depressive disorder (MDD) is a devastating mental illness and the leading cause of disability worldwide. Previous studies have suggested that synaptic plasticity in the hippocampus plays an important role in depression pathogenesis. Reelin is expressed mainly in the frontal lobe and hippocampus, and is closely associated with neurodevelopment and synaptic plasticity. However, few studies have investigated its role in MDD combining clinical trials and animal experiments. We show that in a clinical trial, plasma reelin levels decreased in patients with first-episode drug-naïve MDD and increased after treatment; further, plasma reelin levels allowed to distinguish drug-naïve patients with first-episode MDD from healthy individuals. In rats, chronic mild and unpredictable stress led to a decrease in both reelin mRNA and protein levels in the hippocampus, which could be reversed by vortioxetine. Subsequent experiments confirmed that the reelin-ApoER2-NR2A /NR2B pathway regulates hippocampal synaptic plasticity and may be involved in depression or antidepressant responses. Our work contributes to a deeper understanding of MDD pathogenesis and provides new evidence that reelin should be considered a potential therapeutic target for MDD.

Whole genome analysis in APOE4 homozygotes identifies the DAB1-RELN pathway in Alzheimer's disease pathogenesis.

The APOE-ε4 allele is known to predispose to amyloid deposition and consequently is strongly associated with Alzheimer's disease (AD) risk. There is debate as to whether the APOE gene accounts for all genetic variation of the APOE locus. Another question which remains is whether APOE-ε4 carriers have other genetic factors influencing the progression of amyloid positive individuals to AD. We conducted a genome-wide association study in a sample of 5,390 APOE-ε4 homozygous (ε4ε4) individuals (288 cases and 5102 controls) aged 65 or over in the UK Biobank. We found no significant associations of SNPs in the APOE locus with AD in the sample of ε4ε4 individuals. However, we identified a novel genome-wide significant locus associated to AD, mapping to DAB1 (rs112437613, OR = 2.28, CI = 1.73-3.01, p = 5.4 × 10-9). This identification of DAB1 led us to investigate other components of the DAB1-RELN pathway for association. Analysis of the DAB1-RELN pathway indicated that the pathway itself was associated with AD, therefore suggesting an epistatic interaction between the APOE locus and the DAB1-RELN pathway.

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OBJECTIVES: Autism is a neurodevelopment disorder with unclear etiology. High heterogeneity is one of the main issues in the etiological studies. This study explores the relationship between RELN signaling pathway related genes (RELN, VLDLR, LRP8, DAB1, CDK5, FYN) and language development of autism patients based on a cluster analysis model which is established to reduce the heterogeneity. METHODS: Autism children were recruited from 5 different medical/autism training institutes from Hunan, Shandong, and Henan provinces, and were divided into 2 parts according to the recruitment time: The first part was the training sample, which was recruited from October 2006 to May 2011, and the second part was the validation sample, which was recruited from July 2011 to May 2012. A two-step cluster analysis was performed to cluster 374 Chinese Han autism patients into different subgroups based on 2 parameters: Onset age of the first word and interval from the first word to the first phase. A Bayes discriminatory equation was established followed the cluster results. Then we used this equation to divide another 310 autism children into prior defined subgroups. After the genotyping data was screened, a single marker case-control association study was conducted. RESULTS: The cluster analysis clustered 374 samples into 3 subgroups. Onset ages of the first word in the Group A were (11.83±4.37) months and intervals from the first word to the first phase were (24.55±8.67) months; onset ages of the first word in the Group B were (12.17±3.46) months, intervals from the first word to the first phase were (7.07±3.79) months; onset ages of the first word of Group C were (30.94±7.60) months, intervals from the first word to the first phase were (4.73±4.80) months. The established equations based on the cluster analysis were YA=-14.442+0.525X1+0.810X2, YB=-4.964+0.477X1+0.264X2, YC=-19.843+1.175X1+0.241X2. Cross validated analysis showed that the false rate of the equation was 3.8%. A total of 341 single nucleotide polymorphism (SNP) in 6 genes passed the quality control. Before divided subgroups, none of these SNPs reached the significant P value (P>2.44×10-5, Bonferroni adjustment). However the result showed that rs1288502 of LRP8 in Group B was significantly different from the control group (P=6.45×10-6). CONCLUSIONS: Based on the cluster analysis of language development, we could establish a discriminatory equation to reduce heterogeneity of autism sample. The association test indicates that LRP8 genein RELN signaling pathway is related to a particular type of language development of autism patients.

Altered Balance of Reelin Proteolytic Fragments in the Cerebrospinal Fluid of Alzheimer's Disease Patients.

Reelin binds to the apolipoprotein E receptor apoER2 to activate an intracellular signaling cascade. The proteolytic cleavage of reelin follows receptor binding but can also occur independently of its binding to receptors. This study assesses whether reelin proteolytic fragments are differentially affected in the cerebrospinal fluid (CSF) of Alzheimer's disease (AD) subjects. CSF reelin species were analyzed by Western blotting, employing antibodies against the N- and C-terminal domains. In AD patients, we found a decrease in the 420 kDa full-length reelin compared with controls. In these patients, we also found an increase in the N-terminal 310 kDa fragment resulting from the cleavage at the so-called C-t site, whereas the 180 kDa fragment originated from the N-t site remained unchanged. Regarding the C-terminal proteolytic fragments, the 100 kDa fragment resulting from the cleavage at the C-t site also displayed increased levels, whilst the one resulting from the N-t site, the 250 kDa fragment, decreased. We also detected the presence of an aberrant reelin species with a molecular mass of around 500 kDa present in AD samples (34 of 43 cases), while it was absent in the 14 control cases analyzed. These 500 kDa species were only immunoreactive to N-terminal antibodies. We validated the occurrence of these aberrant reelin species in an Aß42-treated reelin-overexpressing cell model. When we compared the AD samples from APOE genotype subgroups, we only found minor differences in the levels of reelin fragments associated to the APOE genotype, but interestingly, the levels of fragments of apoER2 were lower in APOE ε4 carriers with regards to APOE ε3/ε3. The altered proportion of reelin/apoER2 fragments and the occurrence of reelin aberrant species suggest a complex regulation of the reelin signaling pathway, which results impaired in AD subjects.

The risks of RELN polymorphisms and its expression in the development of otosclerosis.

Otosclerosis (OTSC) is the primary form of conductive hearing loss characterized by abnormal bone remodelling within the otic capsule of the human middle ear. A genetic association of the RELN SNP rs3914132 with OTSC has been identified in European population. Previously, we showed a trend towards association of this polymorphism with OTSC and identified a rare variant rs74503667 in a familial case. Here, we genotyped these variants in an Indian cohort composed of 254 OTSC cases and 262 controls. We detected a significant association of rs3914132 with OTSC (OR = 0.569, 95%CI = 0.386-0.838, p = 0.0041). To confirm this finding, we completed a meta-analysis which revealed a significant association of the rs3914132 polymorphism with OTSC (Z = 6.707, p<0.0001) across different ethnic populations. Linkage analysis found the evidence of linkage at RELN locus (LOD score 2.1059) in the OTSC family which has shown the transmission of rare variant rs74503667 in the affected individuals. To understand the role of RELN and its receptors in the development of OTSC, we went further to perform a functional analysis of RELN/reelin. Here we detected a reduced RELN (p = 0.0068) and VLDLR (p = 0.0348) mRNA levels in the otosclerotic stapes tissues. Furthermore, a reduced reelin protein expression by immunohistochemistry was confirmed in the otosclerotic tissues. Electrophoretic mobility shift assays for rs3914132 and rs74503667 variants revealed an altered binding of transcription factors in the mutated sequences which indicates the regulatory role of these variations in the RELN gene regulation. Subsequently, we showed by scanning electron microscopy a change in stapes bone morphology of otosclerotic patients. In conclusion, this study evidenced that the rare variation rs74503667 and the common polymorphism rs3914132 in the RELN gene and its reduced expressions that were associated with OTSC.

Monoallelic and biallelic mutations in RELN underlie a graded series of neurodevelopmental disorders.

Reelin, a large extracellular protein, plays several critical roles in brain development and function. It is encoded by RELN, first identified as the gene disrupted in the reeler mouse, a classic neurological mutant exhibiting ataxia, tremors and a 'reeling' gait. In humans, biallelic variants in RELN have been associated with a recessive lissencephaly variant with cerebellar hypoplasia, which matches well with the homozygous mouse mutant that has abnormal cortical structure, small hippocampi and severe cerebellar hypoplasia. Despite the large size of the gene, only 11 individuals with RELN-related lissencephaly with cerebellar hypoplasia from six families have previously been reported. Heterozygous carriers in these families were briefly reported as unaffected, although putative loss-of-function variants are practically absent in the population (probability of loss of function intolerance = 1). Here we present data on seven individuals from four families with biallelic and 13 individuals from seven families with monoallelic (heterozygous) variants of RELN and frontotemporal or temporal-predominant lissencephaly variant. Some individuals with monoallelic variants have moderate frontotemporal lissencephaly, but with normal cerebellar structure and intellectual disability with severe behavioural dysfunction. However, one adult had abnormal MRI with normal intelligence and neurological profile. Thorough literature analysis supports a causal role for monoallelic RELN variants in four seemingly distinct phenotypes including frontotemporal lissencephaly, epilepsy, autism and probably schizophrenia. Notably, we observed a significantly higher proportion of loss-of-function variants in the biallelic compared to the monoallelic cohort, where the variant spectrum included missense and splice-site variants. We assessed the impact of two canonical splice-site variants observed as biallelic or monoallelic variants in individuals with moderately affected or normal cerebellum and demonstrated exon skipping causing in-frame loss of 46 or 52 amino acids in the central RELN domain. Previously reported functional studies demonstrated severe reduction in overall RELN secretion caused by heterozygous missense variants p.Cys539Arg and p.Arg3207Cys associated with lissencephaly suggesting a dominant-negative effect. We conclude that biallelic variants resulting in complete absence of RELN expression are associated with a consistent and severe phenotype that includes cerebellar hypoplasia. However, reduced expression of RELN remains sufficient to maintain nearly normal cerebellar structure. Monoallelic variants are associated with incomplete penetrance and variable expressivity even within the same family and may have dominant-negative effects. Reduced RELN secretion in heterozygous individuals affects only cortical structure whereas the cerebellum remains intact. Our data expand the spectrum of RELN-related neurodevelopmental disorders ranging from lethal brain malformations to adult phenotypes with normal brain imaging.