Reelin glycoprotein: structure, biology and roles in ... - Semantic Scholar

Molecular Psychiatry (2005) 10, 251–257 & 2005 Nature Publishing Group All rights reserved 1359-4184/05 $30.00 www.nature.com/mp

FEATURE REVIEW

Reelin glycoprotein: structure, biology and roles in health and disease SH Fatemi Division of Neuroscience Research, Department of Psychiatry, University of Minnesota Medical School, University of Minnesota, Minneapolis, MN, USA Reelin glycoprotein is a secretory serine protease with dual roles in mammalian brain: embryologically, it guides neurons and radial glial cells to their corrected positions in the developing brain; in adult brain, Reelin is involved in a signaling pathway which underlies neurotransmission, memory formation and synaptic plasticity. Disruption of Reelin signaling pathway by mutations and selective hypermethylation of the Reln gene promoter or following various pre- or postnatal insults may lead to cognitive deficits present in neuropsychiatric disorders like autism or schizophrenia. Molecular Psychiatry (2005) 10, 251–257. doi:10.1038/sj.mp.4001613 Published online 7 December 2004 Keywords: reelin; schizophrenia; autism; reeler mouse

Many brain proteins participate in the early growth and development of the mammalian central nervous system (CNS). Here, I will focus on Reelin, a glycoprotein that helps guide brain development in an orderly fashion. Changes in the level of this protein or its receptors or downstream proteins may cause abnormal corticogenesis. These changes have also been observed in a number of neuropsychiatric disorders causing an explosion of knowledge about the biology and function of Reelin glycoprotein. I will discuss more about this protein and its possible involvement in health and disease. Reelin gene (Reln) is localized to chromosome 7 in man.1 Reelin protein product has a relative molecular mass of 388 kDa.2,3 On SDS-PAGE, Reelin appears as several protein bands, ranging from 410 to 330, 180 kDa, and several smaller fragments.4–7 Reelin is a secreted extracellular matrix protein with serine protease activity8 containing 3461 amino acids.9 Reelin contains a signal peptide followed by an Nterminal sequence and a hinge region upstream from eight Reelin repeats of 350–390 amino acids.9 Each Reelin repeat is composed of two subrepeats separated by an EGF motif.9 The Reelin protein ends with a highly basic C-terminus composed of 33 amino acids.9 An epitope known as the CR-50 is localized near the N-terminus10 and composed of amino acids 230–346 of Reelin glycoprotein.11 This epitope is essential for Reelin–Reelin electrostatic interactions that produce a soluble string-like homopolymer, Correspondence: SH Fatemi, MD, PhD, Division of Neuroscience Research, Department of Psychiatry, University of Minnesota Medical School, 420 Delaware Street, MMC 392, Minneapolis, MN 55455, USA. E-mail: [email protected] Received 31 August 2004; revised 22 September 2004; accepted 23 September 2004

composed of up to 40 or more regularly repeated monomers, which form in vivo.11. Mutated Reelin, which lacks a CR-50 epitope, fails to form homopolymers, and is, thereby, unable to transduce the Reelin signal.11 Reelin binds several proteins as likely receptors, including apolipoprotein E receptor 2 (ApoER2), very-low-density lipoprotein receptor (VLDL-R) and a3b1 integrin protein.12–14 Reelin binding to ApoER2 and VLDLR receptors induces clustering of the latter receptors, causing dimerization/oligomerization of the adaptor protein, disabled-1 (Dab-1), on the cytosolic aspect of the plasma membrane15 with eventual tyrosine phosphorylation of Dab-1 adapter protein,16 facilitating the transduction of signaling pathway from the Reelin-producing cells (GABAergic neurons17 or Cajal–Retzius cells of layer I)18 to downstream receptor sites on cortical pyramidal cells.19 In vivo, Reelin is processed by cleavage at two locations, that is, between repeats 2 and 3 and repeats 6 and 7,20 resulting in three final fragments.21 The central Reelin fragment is composed of repeats 3–6, and is necessary and sufficient for receptor binding to ApoER2 and VLDLR proteins, causing Dab-1 phosphorylation in neuronal cultures21 and is able to rescue the reeler phenotype in embryonic brain cultures. Furthermore, Reelin also activates serine–threonine kinases (P35/ Cdk5) and Src-tyrosine kinase family (Fyn-kinase), also leading to phosphorylation of Dab-1.22–24 Phosphorylated Dab-1 can become the substrate for various kinases, leading to a number of important events such as synaptic and dendritic spine plasticity,19 neurotransmission22–26 and inhibition of the level of glycogen synthase-kinase 3b (GSK-3b), leading to modulation of pathways of cell survival and growth23 (Figure 1). Additionally, phosphorylated Dab-1 is a substrate for polyubiquitination-dependent degradation, leading to

Reelin glycoprotein SH Fatemi

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Figure 1 The Reelin signaling system and cognition. Extracellular Reelin glycoprotein is secreted by Cajal-Retzius cells and certain cortical and hippocampal GABAergic cells and cerebellar granule cells. Reelin can bind its receptors ApoER2, VLDLR and a3b1 integrin directly, initiating the signaling system in the effector cells i.e., cortical pyramidal cells. Reelin induction of the cascade leads to clustering of the receptors causing dimerization/oligomerization of Dab-1 protein and activation of Src-tyrosine kinase family/Fyn-kinase leading to tyrosine phosphorylation of Dab-1 protein in a positive-feedback loop. Interaction between Dab-1, N-WASP and ARP 2/3 complex, causes formation of microspikes or filopodia which are important in processes of cell migration and synaptic plasticity. Finally, phosphorylation of a subpopulation of Dab-1 molecules causes degradation of Dab-1 via ubiquitination, resulting in termination of Reelin signaling cascade. Downstream effector proteins involved in Reelin signaling path include phosphatidylinositol- 3-kinase (PI3K) and protein kinase B (PKB/ Akt), which further impact on three other important molecules, glycogen synthase kinase (GSK-3b), b-catenin and tau. The latter proteins can modulate pathways, affecting cell proliferation, apotosis and neurodegeneration respectively. Finally, Reelin has a direct effect on enhancement of long term potentiation (LTP), via direct involvement of its receptors VLDLR and ApoER. Alternately, tyrosine phorphorylation of NR2B subunit of NMDA receptor by Fyn kinase is essential for induction of LTP and modulation of synaptic plasticity, potentially converging on Reelin’s role in cognition and memory processing.

degradation of a subpopulation of Dab-1 molecules, via the proteosome pathway.27 Dab-1 degradation may be an important factor in fine-tuning the Reelin signal and responding to it in the CNS.27 Recent work by Suetsugu et al28 explains the mechanisms through which Reelin stimulation of Dab-1 affects migration of cells. Following induction of the Reelin signaling system, Dab-1 activates NWASP (a neuronal type of Wiskott–Aldrich syndrome protein capable of inducing long actin microspikes)29 and stimulates actin polymerization through the Arp 2/3 complex (actin-related proteins 2 and 3, which are essential for initiation of actin assembly),30 causing formation of microspikes or filopodia. Phosphorylation of Dab-1 upon Reelin stimulation and via Fyn– Src kinase mediation causes ubiquitination of Dab-1 in a Cbl-dependent manner (Casitas B lymphoma protein, a ubiquitin ligase),31 leading to inhibition of filopodium induction (Figure 1) and eventual arrest in cell migration. This mechanism may also underlie abnormal cell migration during brain development observed in the reeler mouse 2,27 (Vide infra). Mutation of the gene for Reelin, as seen in homozygous reeler mutant mice,32,33 leads to develMolecular Psychiatry

opment of ataxia and a reeling gait in the affected mice. Additionally, absence of Reln gene during embryogenesis leads to development of a brain with multiple histologic defects including a reversal of the normal layering of the brain,33–35 abnormal positioning of the neurons and aberrant orientation of cell bodies and nerve fibers.33–35 The reeler cerebellum is hypoplastic36 and the Purkinje cell number is reduced.37 Mutations involving ApoER2, VLDL-R and a3b1 integrin receptors result in defective cortical lamination and abnormal neuronal migration.14,38 Additionally, mice that lack either Reelin or both VLDL-R and ApoER2 receptors exhibit hyperphosphorylation of the Tau protein, resulting in dysregulation of neuronal microtubule function.13 Several other reeler-like phenotypes have also been described, which produce various neurologic phenotypes similar to the reeler homozygous mutant (for a detailed discussion, see Fatemi39). More interestingly, several experimental paradigms and haploinsufficiency in Reln gene in mice also cause decreases in Reelin production with resultant cortical and behavioral abnormalities.18,39–41 In the heterozygous reeler mutation, there is a 50% reduction in Reelin protein


and mRNA, decrease in dendritic spine density in frontal cortex, neuropil hypoplasticity, decreased GAD67 expression and decreased GABA turnover.42 Additionally, the heterozygous reeler mutant mice exhibit decreased prepulse inhibition,40 a phenomenon observed in schizophrenia and autism.43,44 Prenatal human influenza viral infection in midterm pregnant mice leads to abnormal corticogenesis,18 decrease in brain Reelin protein content18 and reduced prepulse inhibition.45 Finally, exposure of rat pups to 5 methoxytryptamine leads to reductions in brain and blood Reelin levels, and abnormal corticogenesis.41 Analogies between these animal models and development of schizophrenia and autism will be made and correlations will be discussed in the following passages. Reelin protein is present in all vertebrates and conserved through evolution.46 Additionally, the wide distribution of Reelin in the adult lamprey brain is consistent with existence of different roles for this protein not related to development of CNS in the vertebrates.47 For example, Reelin expression in brains of male European starlings is highly sensitive to testosterone, decreasing markedly in response to exogenous administration of this hormone.48 Thus, here, Reelin expression in the brain varies seasonally and could therefore provide a signal that could modulate the seasonal affects in the incorporation of new neurons in the song control system.48 In mammals including rodents, Reelin production begins as early as day 9.5 in the embryonic mouse brains.2,49 The cells synthesizing Reelin are Cajal– Retzius cells, which act as path-finding neurons that help in early laminar organization of the cortex.2 In the adult mammalian brain, Reelin is localized to layer I cortical Cajal–Retzius cells, cortical GABAergic interneurons in layers II–IV,50 cerebellar granule cells51 and hippocampal interneurons.52 Presence of Reelin-positive cells in the adult hippocampus indicates that Reelin function is not restricted to embryonic period, but may continue throughout adult life.53 While controversial, a recent report demonstrates coexpression of Reelin and Dab-1 in Cajal–Retzius cells during cortical development, and in cortical pyramidal cells in the adult CNS.54 It is now clearly established that Reelin protein serves a dual purpose in mammalian brain: embryologically, it guides neurons and radial glial cells to their corrected positions in the developing brain.55,56 After the fetal phase of brain development, levels of Reelin begin to decrease, reaching a plateau by late childhood and remaining constant thereafter in mice (M Araghi-Niknam, SH Fatemi, unpublished data). Moreover, Reelin is largely replaced by Reelinexpressing GABAergic interneurons that are dispersed throughout the mammalian neocortex50 and hippocampus.52,53 Levels of the Reelin receptors ApoER2, VLDLR and a3b1 integrin and the adapter protein Dab-1, which are all essential to the Reelin signaling system, remain expressed in adult brain.53

Previous work by Rodriguez et al19 showed an association between Reelin and its receptor a3b1 integrin with synaptic structures, raising the possibility of a potential role in neurotransmission. A recent report by J Herz’s laboratory 57 shows that Reelin has a direct effect on enhancement of long-term potentiation (LTP) in hippocampus, which is abolished when hippocampus slice cultures are used from VLDL-R and ApoER2 knockout mice lacking the receptors for Reelin. These investigators further report that Reelin and ApoE Receptors cooperate to enhance hippocampal synaptic plasticity and learning.57 Moreover, mice that lack the Reelin receptors ApoER2 or VLDL-R have pronounced defects in memory formation and LTP.57 Other behavioral and biochemical data also show that reductions in levels of Reelin in brain or blood, following postnatal hypoxia,58 prenatal viral infection in midgestation18,45 and in heterozygous reeler mutants40 cause abnormalities in behavior such as decrease in prepulse inhibition (PPI), increase in anxiety and decrease in memory formation. Additionally, mutations in RELN gene have been associated with significant learning disability, hypoplastic cerebellum, ataxia and cognitive decline in man and mouse.35 Several studies now implicate the pathological involvement of Reln gene or its protein product in six neuropsychiatric disorders, namely, schizophrenia, autism, bipolar disorder, major depression, lissencephaly and alzheimer’s disease. Impagnatiello et al,50 used northern and western blotting and immunocytochemistry to show reductions in Reelin mRNA and protein in cerebellar, hippocampal and frontal cortices of patients with schizophrenia and psychotic bipolar disorder. These authors suggested that Reelin might be a vulnerability factor in development of psychosis.50 Later, Guidotti et al,59 confirmed and extended these observations in postmortem frontal cortex of additional subjects with schizophrenia and psychotic bipolar disorder. Reduction in Reelin was associated with significant decreases in Glutamic acid decarboxylase 67 kDa protein, in the same postmortem brains.59 A later immunocytochemical report,52 showed significant reductions in Reelin immunoreactivity in schizophrenic and bipolar patients. However, these authors detected similar decreases in hippocampal Reelin protein levels in non-psychotic bipolar and depressed subjects, suggesting that Reelin deficiency may not be limited to subjects with psychosis alone.52 Fatemi et al subsequently demonstrated significant reductions in Reelin as well as GAD65 and 67 kDa proteins in cerebella of subjects with schizophrenia, bipolar disorder and major depression.60 Further confirmatory data relating to Reelin abnormalities, in brains of schizophrenic patients, were demonstrated by Eastwood et al,61 who showed a trend for reduction in Reelin mRNA in cerebella of schizophrenic subjects; these reductions in Reelin mRNA correlated negatively with semaphorin 3A. The authors suggested that these findings were consistent with an early

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neurodevelopmental origin for schizophrenia, and that the reciprocal changes in Reelin and semaphorin 3A may be indicative of a mechanism that affects the balance between inhibitory and trophic factors regulating synaptogenesis.61 In a further study, Eastwood and Harrison extended their work to superior temporal cortex and discovered significant reductions in Reelin mRNA in interstitial white matter neurons (cells representing the adult remnants of the cortical subplate) in schizophrenic brains, supporting the contention that the origins of schizophrenia may be neurodevelopmental.62 Knable et al63 analyzed molecular abnormalities of the hippocampus in severe psychiatric illness and reconfirmed that GABAergic marker Reelin was decreased in schizophrenia, bipolar disorder and depression attesting to reported GABAergic dysfunction in all three disorders. Recent evidence indicates that decreased expression of Reelin as seen in schizophrenic brains may be due to hypermethylation of the Reln gene promoter.64,65 Costa and coworkers have posited the opinion that alterations in chromatin remodeling related to a selective upregulation of DNA-5-cytosine methyltransferase (DNMT) expression in GABAergic neurons of schizophrenic prefrontal cortex may induce a hypermethylation of Reelin and GAD67 promoter CpG islands, which subsequently downregulate their expression.64 These authors suggest that targeting this deficit with inhibitors of histone deacetylases (HDAC), may reduce the DNMT upregulation via covalent modification of nucleosomal histone tails, potentially upregulating Reelin expression in schizophrenic brain.64,66 Indeed, Veldic et al67 have recently shown that mRNA for DNA-methyltransferase 1, which catalyzes the methylation of promoter CpG islands, is increased in cortical GABAergic interneurons but not in pyramidal neurons of schizophrenic brains. Despite these biochemical findings, two recent reports fail to report any association between Reln gene polymorphisms and schizophrenia.68,69 Akahane et al examined the polymorphic CGG repeat in the 50 untranslated region of the Reln gene in 150 schizophrenic and 150 controls matched for age, sex and ethnicity and found no evidence for any significant association of schizophrenia with polymorphisms for Reln or VLDLR genes.68 By the same token, Chen et al studied a single nucleotide polymorphism at the 50 promoter region of the human Reln gene in 279 Han Chinese schizophrenic patients and 255 controls and could not demonstrate any significant associations in the Reln gene polymorphisms and schizophrenia.69 In a series of postmortem studies, Fatemi et al70 also showed reductions in Reelin protein in several brain sites in autism. Brain levels of Reelin 410 kDa was reduced significantly in frontal (Area 9) and cerebellar areas and nonsignificantly in parietal (Area 40) cortex of autistic subjects vs. controls. There was also a trend for reduction in Reelin 410 kDa in autistic children indicating that the reduced Reelin levels were present from childhood.70 Measurement of blood Reelin levels also showed reductions in


410 kDa and 330 kDa species5,4 in the autistic subjects. These biochemical data are bolstered by two association studies showing significant linkage between Reln gene polymorphisms and autism.71,72 Recently, Persico et al71 described a significant association between autism and Reln gene variants using case-control and family based designs. They showed a significant association between autistic disorder and the length of a polymorphic GGC repeat located immediately 50 of the Reln gene ATG initiation codon. A further link to autism was also established for specific haplotypes defined by single-base substitutions located in a splice junction of exon 6 and within the coding sequence of exon 50.71 These investigators also showed preferential transmission of ‘long’ triplet repeat alleles (i.e., 411 repeats) to autistic patients and correlated this phenomenon with decreases in blood Reelin 330 kDa levels in the autistic offspring.6 These authors concluded that transmission of ‘long’ alleles from either parent significantly enhanced the overall probability of a child being affected by autism.6,71 In a subsequent report, Zhang et al72 did not observe any evidence for expansion or instability of transmission of GGC repeats in the autistic subjects, but were able to confirm, using a family-based association test that larger alleles were transmitted higher than expected in the affected children indirectly supporting Persico et al’s work.71 In contrast, four reports fail to detect any genetic linkage between Reln gene polymorphisms and autism.73–76 Krebs et al73 performed a transmission disequilibrium test analysis of the 50 UTR polymorphism in 167 families including 218 affected subjects and could not show any association between this GGC polymorphism of the Reln gene and autism in a population of mixed European descent. Bonora and coworkers74 using a positional candidate gene approach found novel missence variants in Reln gene with low frequency but could not support a major role for Reln in autism in IMGSAC and German singleton families. Devlin et al75 used a large independent family-based sample from the NIH Collaborative Programs of Excellence in Autism (CPEA) Network and could not find any significant association between Reln gene alleles and autism. Finally, Li et al76 also could not find any evidence for an association between WNT2 and Reln polymorphisms and autism. However, these authors76 felt that ‘association studies of DNA variations are often ineffective in addressing functional alteration of gene products at the level of gene expression’ and suggested additional biochemical studies of brain and blood products to further assess the involvement of Reln gene in autism. Despite the controversial nature of genetic association studies, Rakic and coworkers41 have developed a potential animal model for autism which links prenatal serotonergic abnormalities to reduced brain and blood Reelin levels and abnormal brain development, indicating the relevance of biochemical/neuroanatomic studies pertaining to Reelin signaling system in autism.


Reelin mutations have also been discovered in a variant of lissencephaly, whereby the affected individuals have very low or undetectable levels of Reelin in their sera.77 Hong et al showed that the affected children exhibited congenital lymphoedema and hypotonia with brain showing moderate lissencephaly and profound cerebellar hypoplasia.77 Assadi et al78 developed compound mutant mice, with disruptions in the Reln gene and PAFAH1B1 (encoding LIS 1), which exhibited a higher incidence of hydrocephalus and enhanced cortical and hippocampal layering defects, implicating involvement of both genes in normal brain development. Finally, Saez-Valero,79 measured Reelin 180 kDa levels in CSF of 13 healthy controls, 14 frontotemporal dementia and 20 Alzheimer’s disease patients. They reported significant increases in CSF 180 kDa Reelin species in both dementias vs. controls suggesting the involvement of Reelin in neurodegenerative disorders.79 In contrast, Ignatova et al7 measured CSF Reelin in adults and children and found no correlation with age or neurologic disease (Alzheimer’s dementia, multiple sclerosis). However, the latter investigators used a scoring technique which was semiquantitative and had a smaller N for each patient population.7 This disparity in levels of Reelin protein production appears similar to the scenario seen in Reeler homozygous mutant mice2 (with no Reelin production) versus Reeler heterozygous mutation40 and that seen following prenatal viral infection18 where brain Reelin levels are reduced by 50%. Thus, a similar mechanism may be operational in various neuropsychiatric disorders where Reelin production may be affected selectively by various mutations or selective hypermethylation of the Reln promoter,64,65 causing either profound (schizophrenia, autism, lissencephaly) or moderate (bipolar, depressed) cognitive deficits50,52,77,59 associated with their respective Reelin levels. The overall picture emerging from these reports suggests that Reelin deficiency may be associated not only with vulnerability to developing psychosis, but also to the development of cognitive dysfunction, a clinical symptom often observed in various neuropsychiatric disorders such as bipolar disorder,50,59 major depression,52 autism5 and lissencephaly.77 This hypothesis is also supported by animal studies19 linking Reelin-integrin interactions with synaptic plasticity. Association of ApoER2 and LDL receptor family with Reelin protein may also link certain neurodegenerative disorders such as Alzheimer’s dementia80,81 with dysregulation of Reelin signaling system. Finally, recent discovery and localization of Reelin to a number of sites such as spinal cord,82–84 subpial granular layer of fetal human cortex,85,86 developing rat striatum,87 hepatic stellate cells,88 human esophageal carcinoma,89 human and mouse odontoblasts90,91 and blood plasma cells92 will most likely expand the potential roles for this important protein in health and disease. Future studies of pertinent animal models that implicate the involvement of Reelin are also warranted.

In conclusion, Reelin glycoprotein acts as a serine protease both during embryogenesis and in the adult brain. Absence of Reelin during development leads to abnormal corticogenesis, Purkinje cell loss and ataxia. Reductions in levels of Reelin during adult life may cause cognitive deficits, as seen in autism, schizophrenia, bipolar disorder and lissencephaly. Moreover, Reelin is involved in a signaling pathway which underlies, memory formation, LTP and synaptic plasticity. Reelin may also have other undefined roles in health and disease, because of its presence in diverse areas of the body. Future, biochemical, genetic and neuroanatomic studies will surely expand our knowledge about this important protein and determine its involvement in various neurodevelopmental disorders.

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Acknowledgements The work of author has been supported by Stanley Medical Research Institute, March of Dimes, The Jonty Foundation and the Kunin Fund of St Paul Foundation. I am grateful for secretarial assistance by Ms Laurie Iversen and Danielle Johansson.

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