Molecular (2005) 10, 251–257 & 2005 Nature Publishing Group All rights reserved 1359-4184/05 $30.00 www.nature.com/mp FEATURE REVIEW : structure, biology and roles in health and disease SH Fatemi Division of Neuroscience Research, Department of Psychiatry, University of Minnesota , University of Minnesota, Minneapolis, MN, USA

Reelin glycoprotein is a secretory serine with dual roles in mammalian : embryologically, it guides 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 . Disruption of Reelin signaling pathway by mutations and selective hypermethylation of the Reln promoter or following various pre- or postnatal insults may lead to cognitive deficits present in neuropsychiatric disorders like or . Molecular Psychiatry (2005) 10, 251–257. doi:10.1038/sj.mp.4001613 Published online 7 December 2004 Keywords: reelin; schizophrenia; autism;

Many brain participate in the early growth and composed of up to 40 or more regularly repeated development of the mammalian monomers, which form in vivo.11. Mutated Reelin, (CNS). Here, I will focus on Reelin, a glycoprotein that which lacks a CR-50 epitope, fails to form homopoly- helps guide brain development in an orderly fashion. mers, and is, thereby, unable to transduce the Reelin Changes in the level of this or its receptors or signal.11 Reelin binds several proteins as likely downstream proteins may cause abnormal corticogen- receptors, including receptor 2 esis. These changes have also been observed in a number (ApoER2), very-low-density lipoprotein receptor of neuropsychiatric disorders causing an explosion of (VLDL-R) and a3b1 protein.12–14 Reelin binding knowledge about the biology and function of Reelin to ApoER2 and VLDLR receptors induces clustering of glycoprotein. I will discuss more about this protein and the latter receptors, causing dimerization/oligomeriza- its possible involvement in health and disease. tion of the adaptor protein, disabled-1 (Dab-1), on the Reelin gene (Reln) is localized to 7 in cytosolic aspect of the plasma membrane15 with man.1 Reelin protein product has a relative molecular eventual tyrosine of Dab-1 adapter mass of 388 kDa.2,3 On SDS-PAGE, Reelin appears as protein,16 facilitating the transduction of signaling several protein bands, ranging from 410 to 330, pathway from the Reelin-producing cells (GABAergic 180 kDa, and several smaller fragments.4–7 Reelin is neurons17 or Cajal–Retzius cells of layer I)18 to down- a secreted protein with serine stream receptor sites on cortical pyramidal cells.19 In protease activity8 containing 3461 amino acids.9 vivo, Reelin is processed by cleavage at two locations, Reelin contains a signal peptide followed by an N- that is, between repeats 2 and 3 and repeats 6 and 7,20 terminal sequence and a hinge region upstream from resulting in three final fragments.21 The central Reelin eight Reelin repeats of 350–390 amino acids.9 Each fragment is composed of repeats 3–6, and is necessary Reelin repeat is composed of two subrepeats sepa- and sufficient for receptor binding to ApoER2 and rated by an EGF motif.9 The Reelin protein ends with VLDLR proteins, causing Dab-1 phosphorylation in a highly basic C-terminus composed of 33 amino neuronal cultures21 and is able to rescue the reeler acids.9 An epitope known as the CR-50 is localized in embryonic brain cultures. Furthermore, near the N-terminus10 and composed of amino acids Reelin also activates serine–threonine (P35/ 230–346 of Reelin glycoprotein.11 This epitope is Cdk5) and Src-tyrosine family (Fyn-kinase), also essential for Reelin–Reelin electrostatic interactions leading to phosphorylation of Dab-1.22–24 Phosphory- that produce a soluble string-like homopolymer, lated Dab-1 can become the substrate for various kinases, leading to a number of important events such as synaptic and plasticity,19 neuro- Correspondence: SH Fatemi, MD, PhD, Division of Neuroscience transmission22–26 and inhibition of the level of glycogen Research, Department of Psychiatry, University of Minnesota synthase-kinase 3b (GSK-3b), leading to modulation of Medical School, 420 Delaware Street, MMC 392, Minneapolis, pathways of cell survival and growth23 (Figure 1). MN 55455, USA. E-mail: [email protected] Received 31 August 2004; revised 22 September 2004; accepted Additionally, phosphorylated Dab-1 is a substrate for 23 September 2004 polyubiquitination-dependent degradation, leading to Reelin glycoprotein SH Fatemi 252

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 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 opment of and a reeling gait in the affected the proteosome pathway.27 Dab-1 degradation may be mice. Additionally, absence of Reln gene during an important factor in fine-tuning the Reelin signal and embryogenesis leads to development of a brain with responding to it in the CNS.27 multiple histologic defects including a reversal of the Recent work by Suetsugu et al28 explains the normal layering of the brain,33–35 abnormal position- mechanisms through which Reelin stimulation of ing of the neurons and aberrant orientation of cell Dab-1 affects migration of cells. Following induction bodies and nerve fibers.33–35 The reeler is of the Reelin signaling system, Dab-1 activates N- hypoplastic36 and the Purkinje cell number is re- WASP (a neuronal type of Wiskott–Aldrich syndrome duced.37 Mutations involving ApoER2, VLDL-R and protein capable of inducing long microspikes)29 a3b1 integrin receptors result in defective cortical and stimulates actin polymerization through the Arp lamination and abnormal neuronal migration.14,38 2/3 complex (actin-related proteins 2 and 3, which are Additionally, mice that lack either Reelin or both essential for initiation of actin assembly),30 causing VLDL-R and ApoER2 receptors exhibit hyperphos- formation of microspikes or filopodia. Phosphoryla- phorylation of the , resulting in dysregu- tion of Dab-1 upon Reelin stimulation and via Fyn– lation of neuronal function.13 Several Src kinase mediation causes ubiquitination of Dab-1 other reeler-like have also been de- in a Cbl-dependent manner (Casitas B lymphoma scribed, which produce various neurologic pheno- protein, a ),31 leading to inhibition of types similar to the reeler homozygous mutant (for a filopodium induction (Figure 1) and eventual arrest detailed discussion, see Fatemi39). More interestingly, in cell migration. This mechanism may also underlie several experimental paradigms and haploinsuffi- abnormal cell migration during brain development ciency in Reln gene in mice also cause decreases in observed in the reeler mouse 2,27 (Vide infra). Reelin production with resultant cortical and beha- Mutation of the gene for Reelin, as seen in vioral abnormalities.18,39–41 In the heterozygous reeler homozygous reeler mutant mice,32,33 leads to devel- mutation, there is a 50% reduction in Reelin protein

Molecular Psychiatry Reelin glycoprotein SH Fatemi 253 and mRNA, decrease in dendritic spine density in Previous work by Rodriguez et al19 showed an frontal cortex, neuropil hypoplasticity, decreased association between Reelin and its receptor a3b1 GAD67 expression and decreased GABA turnover.42 integrin with synaptic structures, raising the possibi- Additionally, the heterozygous reeler mutant mice lity of a potential role in neurotransmission. A recent exhibit decreased prepulse inhibition,40 a pheno- report by J Herz’s laboratory 57 shows that Reelin has a menon observed in schizophrenia and autism.43,44 direct effect on enhancement of long-term potentia- Prenatal human influenza viral infection in midterm tion (LTP) in hippocampus, which is abolished when pregnant mice leads to abnormal corticogenesis,18 hippocampus slice cultures are used from VLDL-R decrease in brain Reelin protein content18 and and ApoER2 knockout mice lacking the receptors for reduced prepulse inhibition.45 Finally, exposure of Reelin. These investigators further report that Reelin rat pups to 5 methoxytryptamine leads to reductions and ApoE Receptors cooperate to enhance hippocam- in brain and blood Reelin levels, and abnormal pal synaptic plasticity and learning.57 Moreover, mice corticogenesis.41 Analogies between these animal that lack the Reelin receptors ApoER2 or VLDL-R models and development of schizophrenia and autism have pronounced defects in memory formation and will be made and correlations will be discussed in LTP.57 Other behavioral and biochemical data also the following passages. show that reductions in levels of Reelin in brain or Reelin protein is present in all vertebrates and blood, following postnatal hypoxia,58 prenatal viral conserved through evolution.46 Additionally, the infection in midgestation18,45 and in heterozygous wide distribution of Reelin in the adult lamprey brain reeler mutants40 cause abnormalities in behavior such is consistent with existence of different roles for this as decrease in prepulse inhibition (PPI), increase in protein not related to development of CNS in the anxiety and decrease in memory formation. Addi- vertebrates.47 For example, Reelin expression in tionally, mutations in RELN gene have been asso- of male European starlings is highly sensitive ciated with significant learning disability, hypoplastic to testosterone, decreasing markedly in response to cerebellum, ataxia and cognitive decline in man and exogenous administration of this hormone.48 Thus, mouse.35 here, Reelin expression in the brain varies seasonally Several studies now implicate the pathological and could therefore provide a signal that could involvement of Reln gene or its protein product in modulate the seasonal affects in the incorporation of six neuropsychiatric disorders, namely, schizophre- new neurons in the song control system.48 In nia, autism, , major depression, mammals including rodents, Reelin production be- and alzheimer’s disease. Impagnatiello gins as early as day 9.5 in the embryonic mouse et al,50 used northern and western blotting and brains.2,49 The cells synthesizing Reelin are Cajal– immunocytochemistry to show reductions in Reelin Retzius cells, which act as path-finding neurons that mRNA and protein in cerebellar, hippocampal and help in early laminar organization of the cortex.2 In frontal cortices of patients with schizophrenia and the adult mammalian brain, Reelin is localized to psychotic bipolar disorder. These authors suggested layer I cortical Cajal–Retzius cells, cortical GABAer- that Reelin might be a vulnerability factor in gic in layers II–IV,50 cerebellar granule development of psychosis.50 Later, Guidotti et al,59 cells51 and hippocampal interneurons.52 Presence of confirmed and extended these observations in post- Reelin-positive cells in the adult hippocampus indi- mortem frontal cortex of additional subjects with cates that Reelin function is not restricted to embryo- schizophrenia and psychotic bipolar disorder. Reduc- nic period, but may continue throughout adult life.53 tion in Reelin was associated with significant While controversial, a recent report demonstrates decreases in decarboxylase 67 kDa coexpression of Reelin and Dab-1 in Cajal–Retzius protein, in the same postmortem brains.59 A later cells during cortical development, and in cortical immunocytochemical report,52 showed significant pyramidal cells in the adult CNS.54 reductions in Reelin immunoreactivity in schizo- It is now clearly established that Reelin protein phrenic and bipolar patients. However, these authors serves a dual purpose in mammalian brain: embry- detected similar decreases in hippocampal Reelin ologically, it guides neurons and radial glial cells to protein levels in non-psychotic bipolar and depressed their corrected positions in the developing brain.55,56 subjects, suggesting that Reelin deficiency may not be After the fetal phase of brain development, levels of limited to subjects with psychosis alone.52 Fatemi Reelin begin to decrease, reaching a plateau by late et al subsequently demonstrated significant reductions childhood and remaining constant thereafter in mice in Reelin as well as GAD65 and 67 kDa proteins in (M Araghi-Niknam, SH Fatemi, unpublished data). cerebella of subjects with schizophrenia, bipolar Moreover, Reelin is largely replaced by Reelin- disorder and major depression.60 Further confirma- expressing GABAergic interneurons that are dis- tory data relating to Reelin abnormalities, in brains of persed throughout the mammalian neocortex50 and schizophrenic patients, were demonstrated by East- hippocampus.52,53 Levels of the Reelin receptors wood et al,61 who showed a trend for reduction in ApoER2, VLDLR and a3b1 integrin and the adapter Reelin mRNA in cerebella of schizophrenic subjects; protein Dab-1, which are all essential to the these reductions in Reelin mRNA correlated nega- Reelin signaling system, remain expressed in adult tively with semaphorin 3A. The authors suggested brain.53 that these findings were consistent with an early

Molecular Psychiatry Reelin glycoprotein SH Fatemi 254 neurodevelopmental origin for schizophrenia, and 410 kDa and 330 kDa species5,4 in the autistic subjects. that the reciprocal changes in Reelin and semaphorin These biochemical data are bolstered by two associa- 3A may be indicative of a mechanism that affects the tion studies showing significant linkage between Reln balance between inhibitory and trophic factors regu- gene polymorphisms and autism.71,72 Recently, Persi- lating synaptogenesis.61 In a further study, Eastwood co et al71 described a significant association between and Harrison extended their work to superior temp- autism and Reln gene variants using case-control and oral cortex and discovered significant reductions in family based designs. They showed a significant Reelin mRNA in interstitial white matter neurons association between autistic disorder and the length (cells representing the adult remnants of the cortical of a polymorphic GGC repeat located immediately 50 subplate) in schizophrenic brains, supporting the of the Reln gene ATG initiation codon. A further link contention that the origins of schizophrenia may be to autism was also established for specific haplotypes neurodevelopmental.62 Knable et al63 analyzed mole- defined by single-base substitutions located in a cular abnormalities of the hippocampus in severe splice junction of 6 and within the coding psychiatric illness and reconfirmed that GABAergic sequence of exon 50.71 These investigators also marker Reelin was decreased in schizophrenia, showed preferential transmission of ‘long’ triplet bipolar disorder and depression attesting to reported repeat (i.e., 411 repeats) to autistic patients GABAergic dysfunction in all three disorders. Recent and correlated this phenomenon with decreases in evidence indicates that decreased expression of blood Reelin 330 kDa levels in the autistic offspring.6 Reelin as seen in schizophrenic brains may be due These authors concluded that transmission of ‘long’ to hypermethylation of the Reln gene promoter.64,65 alleles from either parent significantly enhanced the Costa and coworkers have posited the opinion that overall probability of a child being affected by alterations in chromatin remodeling related to a autism.6,71 selective upregulation of DNA-5-cytosine methyl- In a subsequent report, Zhang et al72 did not (DNMT) expression in GABAergic neu- observe any evidence for expansion or instability of rons of schizophrenic prefrontal cortex may induce a transmission of GGC repeats in the autistic subjects, hypermethylation of Reelin and GAD67 promoter but were able to confirm, using a family-based CpG islands, which subsequently downregulate their association test that larger alleles were transmitted expression.64 These authors suggest that targeting this higher than expected in the affected children indir- deficit with inhibitors of histone deacetylases ectly supporting Persico et al’s work.71 In contrast, (HDAC), may reduce the DNMT upregulation via four reports fail to detect any genetic linkage between covalent modification of nucleosomal histone tails, Reln gene polymorphisms and autism.73–76 Krebs potentially upregulating Reelin expression in schizo- et al73 performed a transmission disequilibrium test phrenic brain.64,66 Indeed, Veldic et al67 have recently analysis of the 50 UTR in 167 families shown that mRNA for DNA-methyltransferase 1, including 218 affected subjects and could not show which catalyzes the of promoter CpG any association between this GGC polymorphism of islands, is increased in cortical GABAergic interneur- the Reln gene and autism in a population of mixed ons but not in pyramidal neurons of schizophrenic European descent. Bonora and coworkers74 using a brains. Despite these biochemical findings, two recent positional candidate gene approach found novel reports fail to report any association between Reln missence variants in Reln gene with low frequency gene polymorphisms and schizophrenia.68,69 Akahane but could not support a major role for Reln in autism et al examined the polymorphic CGG repeat in the 50 in IMGSAC and German singleton families. Devlin untranslated region of the Reln gene in 150 schizo- et al75 used a large independent family-based sample phrenic and 150 controls matched for age, sex and from the NIH Collaborative Programs of Excellence in ethnicity and found no evidence for any significant Autism (CPEA) Network and could not find any association of schizophrenia with polymorphisms for significant association between Reln gene alleles and Reln or VLDLR .68 By the same token, Chen et al autism. Finally, Li et al76 also could not find any studied a single nucleotide polymorphism at the 50 evidence for an association between WNT2 and Reln promoter region of the human Reln gene in 279 Han polymorphisms and autism. However, these authors76 Chinese schizophrenic patients and 255 controls and felt that ‘association studies of DNA variations are could not demonstrate any significant associations in often ineffective in addressing functional alteration of the Reln gene polymorphisms and schizophrenia.69 gene products at the level of ’ and In a series of postmortem studies, Fatemi et al70 also suggested additional biochemical studies of brain and showed reductions in Reelin protein in several brain blood products to further assess the involvement of sites in autism. Brain levels of Reelin 410 kDa was Reln gene in autism. Despite the controversial nature reduced significantly in frontal (Area 9) and cerebel- of genetic association studies, Rakic and coworkers41 lar areas and nonsignificantly in parietal (Area 40) have developed a potential animal model for autism cortex of autistic subjects vs. controls. There was also which links prenatal serotonergic abnormalities to a trend for reduction in Reelin 410 kDa in autistic reduced brain and blood Reelin levels and abnormal children indicating that the reduced Reelin levels brain development, indicating the relevance of bio- were present from childhood.70 Measurement of chemical/neuroanatomic studies pertaining to Reelin blood Reelin levels also showed reductions in signaling system in autism.

Molecular Psychiatry Reelin glycoprotein SH Fatemi 255 Reelin mutations have also been discovered in a In conclusion, Reelin glycoprotein acts as a serine variant of lissencephaly, whereby the affected indivi- protease both during embryogenesis and in the adult duals have very low or undetectable levels of Reelin brain. Absence of Reelin during development leads to in their sera.77 Hong et al showed that the affected abnormal corticogenesis, Purkinje cell loss and ataxia. children exhibited congenital lymphoedema and Reductions in levels of Reelin during adult life may with brain showing moderate lissence- cause cognitive deficits, as seen in autism, schizo- phaly and profound .77 Assadi phrenia, bipolar disorder and lissencephaly. More- et al78 developed compound mutant mice, with disrup- over, Reelin is involved in a signaling pathway which tions in the Reln gene and PAFAH1B1 (encoding LIS underlies, memory formation, LTP and synaptic 1), which exhibited a higher incidence of hydro- plasticity. Reelin may also have other undefined roles cephalus and enhanced cortical and hippocampal in health and disease, because of its presence in layering defects, implicating involvement of both diverse areas of the body. Future, biochemical, genetic genes in normal brain development. and neuroanatomic studies will surely expand our Finally, Saez-Valero,79 measured Reelin 180 kDa knowledge about this important protein and deter- levels in CSF of 13 healthy controls, 14 frontotemp- mine its involvement in various neurodevelopmental oral dementia and 20 Alzheimer’s disease patients. disorders. They reported significant increases in CSF 180 kDa Reelin species in both dementias vs. controls suggest- Acknowledgements ing the involvement of Reelin in neurodegenerative disorders.79 In contrast, Ignatova et al7 measured CSF The work of author has been supported by Stanley Reelin in adults and children and found no correla- Medical Research Institute, March of Dimes, The tion with age or neurologic disease (Alzheimer’s Jonty Foundation and the Kunin Fund of St Paul dementia, multiple sclerosis). However, the latter Foundation. I am grateful for secretarial assistance by investigators used a scoring technique which was Ms Laurie Iversen and Danielle Johansson. 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 References 2 Reeler homozygous mutant mice (with no Reelin 1 DeSilva U, D’Arcangelo G, Braden VY, Chen J, Miso GG, Curran T production) versus Reeler heterozygous mutation40 et al. The human reelin gene: isolation, sequencing, and mapping and that seen following prenatal viral infection18 on . Genome Res 1997; 7: 157–164. where brain Reelin levels are reduced by 50%. Thus, 2 Ogawa M, Miyata T, Nakajima K, Yoguy K, Seiko M, Ikenaka K et al. 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