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Journal of Neuropathology and Experimental Neurology Copyright q 2003 by the American Association of Neuropathologists Vol. 62, No. 6 June, 2003 pp. 676 684 Reelin and Disabled-1 Expression in Developing and Mature Human Cortical Neurons KIMIKO DEGUCHI, MD, PHD, KEN INOUE, MD, PHD, WILLIAM E. AVILA, BSC, MT, ASCP, DOLORES LOPEZ-TERRADA, MD, PHD, BARBARA A. ANTALFFY, BSC, CARLO C. QUATTROCCHI, MD, MICHAEL SHELDON, PHD, KATSUHIKO MIKOSHIBA, MD, PHD, GABRIELLA D’ARCANGELO, PHD, AND DAWNA L. ARMSTRONG, MD Abstract. In developing mammalian (mouse) brain, Reelin (Reln) is secreted by the Cajal-Retzius (CR) neurons in the marginal zone, binds apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (Vldlr), and induces the phosphorylation of the downstream cytoplasmic molecule disabled-1 (Dab1) in cortical plate neurons. Although this is a well-characterized signaling pathway in mice, it has not been well defined in human brain. In this paper we examined the expression of RELN, APOER2, VLDLR, and DAB1 in the developing human brain by RT-PCR. We further determined the cellular expression of the proteins RELN and DAB1 in 50 human brains ranging in age from 10 gestational weeks (GW) to 62 years using immunochemistry. We found that the pattern of expression of RELN and DAB1 in the human brain is not identical to that observed in the mouse brain. In particular, we report the novel finding that human DAB1 and RELN are coexpressed in CR neurons during cortical development and in cortical pyramidal neurons after neuronal migration is complete. Thus, in the human brain, the whole RELN signaling pathway is present within selected populations of cortical neurons throughout life. We speculate that RELN and DAB1 coexpression in these neurons is necessary for both normal cortical development and mature function. Key Words: Cajal-Retzius neuron; Disabled-1; Human brain; Reelin. INTRODUCTION The molecular basis of mammalian neocortical development is being defined in animal models, particularly in the mouse. Analysis of the reeler mouse, a spontaneous mutant with ataxia and tremors (1) and widespread defects in cortical brain structures (2–4) has been particularly informative. The reeler phenotype resulted from a disruption of the reelin gene (Reln) (5, 6), encoding a large extracellular glycoprotein (7). Reln controls the alignment of cortical neurons in the developing mouse cerebral cortex, hippocampus and cerebellum (8–10), and the positioning of autonomic neurons in the spinal cord (11, 12). In the immature mouse cortex and hippocampus, Reln, which is identified with the CR50 antibody (7, 8), is expressed in the marginal zone by Cajal-Retzius (CR) neurons, a population of early-generated cells, and it provides a positional cue that determines the development of the normal ‘‘inside-out’’ cortical layering pattern (13). A Reln signaling pathway involving 2 receptors, the very low density lipoprotein receptor (VLDLR) and From Department of Pathology (KD, WEA, DL-T, BAA, DLA), Department of Molecular Human Genetics (KI), Department of Pediatrics (CCQ, MS, GD), Division of Neuroscience (GD), Program in Developmental Biology (GD), The Cain Foundation Laboratories (CCQ, GD), Baylor College of Medicine, Houston, Texas; Program in Neuroscience (CCQ), Universite degli Studi di Brescie, Brescie, Italy; Department of Molecular Neurobiology (KM), Institute of Medical Science, University of Tokyo, Japan. Correspondence to: Dawna L. Armstrong, MD, Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. E-mail: [email protected] This study was partially supported by NIH grant H0024064 (DA) and a grant from the Gillson Longenbaugh Foundation (G.D.). apolipoprotein E receptor-2 (ApoER2) (14–16) and the intracellular adaptor protein disabled-1 (Dab1) (17, 18) is necessary for normal mouse cortical layer formation. The transduction of Reln signals requires tyrosine phosphorylation of Dab1 (19–21). Mice lacking Reln, Dab1, or both Vldlr and Apoer2, exhibit identical behavior and neuroanatomy and provide strong evidence for the involvement of these proteins in the same signaling pathway (22). The Reln-positive CR neuron is one of the first neurons to mature during early cortical development. It was initially described in the human brain (23) as a large multipolar neuron in the molecular layer of the cortex with several horizontal dendrites radiating from its soma and a descending axon that gives off numerous collaterals before becoming a thick horizontal fiber in the lower region of layer I. This classic description of the CR neuron was based on Golgi studies. Recent neurodevelopmental studies in the human, primate, and rat brain have recognized that, in addition to the early generated CR neurons, smaller, later generated layer I neurons are also RELNpositive (24–28). Thus, for the purpose of this paper we use the term ‘‘CR’’ for neurons having the classic morphology of the early generated large neurons positioned in layer I, close to the pia, and we refer to other RELNpositive neurons in layer I as ‘‘subpial granule neurons’’ (SGN). The Reln gene is highly conserved in several vertebrate species, including humans (6). In humans, disruptions of the RELN gene cause a severe form of lissencephaly associated with cerebellar hypoplasia (LCH) that resembles 676 677 REELIN AND DISABLED-1 IN HUMAN CORTEX the reeler phenotype (29). In addition, other disorders of brain development and function, such as schizophrenia and mood disorder (30–32), autism (33), polymicrogyria (34), epilepsy (35), and Alzheimer disease (36) have been associated with RELN abnormalities. Despite its potential role in such conditions, our understanding of the ontogeny of RELN expression and its function in the human brain is incomplete. In this report we describe the expression of RELN, VLDLR, APOER2, and DAB1 transcripts and the distribution of RELN and DAB1 proteins in the developing and adult human brain, providing normative data for the further investigation of human diseases. MATERIALS AND METHODS Human Brains Fifty neurological normal autopsy brains aged from 10 gestational weeks (GW) to 69 years were examined. The autopsies were performed within 24 hours (h) of death. The brains were obtained after informed consent approved by the Institutional Review Board of Baylor College of Medicine and Texas Children’s Hospital. Semiquantitative RT-PCR We isolated total RNA from 7 frozen brains (0.5 g of each tissue from human cortex, including layers I to VI) ages 15 GW, 40 GW, 3 months, 2 years, 12 years, 54 years, and 62 years using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. When the RNA was converted to the first strand cDNA by AMV reverse transcriptase (Invitrogen) using random hexamer oligonucleotides, we performed semiquantitative PCR using primers specific to RELN, DAB1, APOER2, and VLDLR. GAPDH was used as a reference for normalization. PCR conditions were optimized to be able to detect signals in the linear range of amplification; initial denaturing at 958C for 15 min, 35 cycles of 948C for 30 s, 558C for 30 s, 728C for 30 s, followed by 728C for 10 min as a final extension (HotStarTaq; Qiagen, Valencia, CA). In order to obtain DAB1 signals in adult brains we used 40 cycles of amplification. In this condition, amplification signals in the samples from 15GW to 12 years of age are in the plateau range and thus are no longer quantitative. PCR products were separated by 1.8% agarose gel electrophoresis and visualized by ethidium bromide staining. Each set of cDNA-specific primers was designed to span at least exons to avoid genomic DNA amplification. Primers employed were RELN-U1; 59-ACCAGTGGGCAGTCGATGACATCAT-39 and RELN-L1; 59-CTTCATTAGCAACATCAACCACAC-39, DAB1-U1; 59-CC AAAGGAGAACACAAACAG-39 and DAB1-L1; 59-GTCCAG CCTCAAACACAATG-39, APOER2-U1; 59-AGTGCAAATC TCAATGG-39 and APOER2-L1; 59-GGAAGGCACAGGTATT CACA-39, VLDLR-U1; 59-CCTGAATGATGCCCAAGACA-39 and VLDLR-L1; 59-CCGAGAGGAAGAAAAAGAGA-39; GAPDH-U; 59-TTAGCACCCCTGGCCAAGG-39 and GAPDHL; 59-CTTACTCCTTGGAGGCCATG-39 (Gene Checker Kit, Invitrogen). Mice Homozygous reeler mice were obtained from the crossing of heterozygous reeler mice (B6C3F3-a/a Reln rl/1) (Jackson Fig. 1. Semiquantitative RT-PCR. Each row represents amplification of GAPDH, RELN, DAB1, APOER2, and VLDLR in 7 samples of frontal cerebral cortex samples (15 GW, 40 GW, 3 months, 2 years, 12 years, 54 years, and 69 years) and 1 control (kidney). All the PCR products were obtained from 35 cycles of amplification within the linear range except for 40 cycles of amplification in DAB1. Laboratories, Bar Harbor, ME). Dab1 knockout mice were obtained from J. A. Cooper (16). Mice at 14 postnatal days were anesthestized by an intraperitoneal injection of 0.5 ml/kg of a solution containing 42.8 mg/ml ketamine, 8.6 mg/ml xylazine, and 1.4 mg/ml acepromazine. Each animal was intracardially perfused with cold phosphate buffer saline (PBS) to wash blood from vessels, followed by 4% paraformaldehyde in PBS for about 7 min. Brains were dissected and postfixed in 4% paraformaldehyde overnight at 48C. Immunocytochemistry For mouse tissue, 5-mm sagittal sections were obtained from paraffin-embedded brains. After rehydration, slides were microwaved for antigen retrieval by placing the sections in 0.1 M citric acid and microwaving on high heat for three 5-min periods. Endogenous peroxidase was blocked by 3% H2O2 (10 min at room temperature). Nonspecific antigens were blocked with 10% horse serum in PBS containing 0.5% Triton-X-100 (30 min at room temperature). A goat anti-Dab1-C terminal antibody (21) (1:200) was incubated overnight at 48C. After 3 washes in PBS (10 min each), sections were incubated with a 1:200 solution containing a secondary anti-goat biotinylated antibody and HRP-conjugated avidin for 45 min at room temperature. Finally, Nova Red chromogen (Vector Laboratories, Burlingame, CA) was used to develop the reaction. In order to obtain consistent staining, Dab1 knockout, and reeler mouse sections were mounted on the same slide. Human brain tissues, fixed in 10% buffered formalin from the frontal and temporal (in young immature brains) or frontal cortex (in older brains) were processed, embedded in paraffin, and cut in 8-mm sections, which were deparaffinized in xylene and rehydrated through ethanol to water. Microwave irradiation was used, as above, to retrieve the RELN antigen after blocking J Neuropathol Exp Neurol, Vol 62, June, 2003 678 J Neuropathol Exp Neurol, Vol 62, June, 2003 DEGUCHI ET AL REELIN AND DISABLED-1 IN HUMAN CORTEX endogenous peroxidase activity with 3.0% hydrogen peroxide in methanol for 8 min. Mouse monoclonal anti-Reln antibody CR-50 (7) and a goat polyclonal anti-Dab1 C terminus antibody were used at 1:200 dilution in the peroxidase anti-peroxidase technique. The primary antibodies were incubated from 24 to 48 h at 48C. Biotinylated secondary antibodies (Vector Laboratories) were used at dilution of 1:200 for CR-50 and 1:400 for Dab1 antibody at room temperature for 30 min. These were exposed to peroxidase-conjugated streptavidin for 45 min (Vector Laboratories) and developed with Nova Red (Vector Laboratories). Slides were counterstained with hematoxylin. Negative controls were treated the same way with omission of the primary or secondary antibodies. Immunoreactivity for each antibody was quantified as positive (1) or negative (2) by authors K.D. and D.L.A. Double Immunofluorescence for Colocalization Experiments A 3-month human brain, with no known neurologic abnormality, was fixed in 4% paraformaldehyde for 48 h, immersed in 25% sucrose for 24 h, and frozen. Fifty-mm sections were incubated with antibody mixtures mouse monoclonal CR-50 anti-Reln antibody (1;50) and a goat anti-Dab-1 C terminus antibody (1;100) in 10% normal horse serum for 24 h at 48C. The sections were rinsed with PBS containing 0.1% Triton X100 (PBS-T). Fluorescein-conjugated anti-mouse IgG (Vector Laboratories), rhodamine-tagged anti-goat IgG (Sigma, St. Louis, MO), rhodamine-conjugated anti-mouse IgG (Sigma), and fluorescein-conjugated anti-rabbit IgG were used for visualization. Sections were rinsed 4 times with PBS-T and mounted with mounting medium for fluorescence (Vector Laboratories). Fluorescent images were acquired using NIKON E 800 upright microscope equipped with proper filters, a Cool SNAPfx digital camera and MetaVue Imaging software (Universal Imaging, Downingtown, PA). RESULTS Expression of RELN, DAB1, APOER2 and VLDLR Messenger RNA in Human Cerebral Cortex We observed expression of RELN, DAB1, APOER2, and VLDLR transcripts from fetal, postnatal, and adult human cerebral cortices by RT-PCR (Fig. 1). RELN and APOER2 were contiguously present at similar levels 679 throughout life (from 15GW to 69 years), whereas the expression levels of DAB1 and VLDLR gradually decreased with age. DAB1 signal was not visible at the ages of 54 and 69 years using our standard conditions of 35 cycles of amplification. DAB1, however, was visible when we performed 40 cycles of amplifications. Double bands in the VLDLR reactions likely represent alternative splicing variants of the transcripts. These data indicate that messenger RNA for each component of the RELN signaling pathway, consisting of RELN, DAB1, APOER2, and VLDLR, is present in postnatal human brain. Specificity of the Anti-Dab1 C-Terminus Antibody Before attempting to detect DAB1 protein expression in human brain, we determined the specificity of the polyclonal anti-Dab-1 C terminal antibody by immunohistochemistry using brain sections from postnatal day 14 reeler and Dab1 knockout mice (Fig. 2a–c). Reeler mouse brain was used as an enhanced positive control because Dab1 protein is overexpressed in this mutant (8). In both mutant mice, the cortex is a disorganized structure containing no discernible cellular layers. After incubation with the anti-Dab1 antibody, reeler sections displayed intense staining, whereas Dab1 knockout mouse revealed no staining, indicating a specific immunoreactivity for Dab1. In reeler mouse a strong staining was clearly visible in many cortical neurons in the perinuclear cytoplasm. More intense signal was observed around the plasma membrane. RELN and DAB1 Expression in Layer I Neurons in Human Brain We observed RELN-positive layer I neurons in all brains from 10 GW through 62 years of age (Table). Consistent with previous studies in the mouse (7, 37), the CR-50 antibody stained the cytoplasm of cells having the appearance of classic CR neurons situated in the outer molecular layer. The stain was positive around the nucleus and into the processes. Most cells were bipolar, though some had more than 3 processes. The cytoplasm of these ← Fig. 2. a–c: Specificity of Dab1 immunoreactivity in the mouse brain. a: Anti-Dab1 C-terminus antibody shows a specific signal in cortical neurons of reeler mouse. There is an intense staining in the cytoplasm in neurons. b: Black arrow indicates a cell with intense staining in plasma membrane. c: The staining with anti-Dab1 C-terminus antibody is negative in neurons of a Dab1 (-/-) mouse. Blue: hematoxylin; Brown: Nova Red chromogen. d–f: RELN immunostaining (CR-50) in layer I of the human cortex at 15 gestational weeks (GW). There are numerous RELN-immunoreactive cells close together in the outer part of layer I (black arrows). d: At 23 GW, the size of RELN-positive cells has increased compared with those at 15 GW. There are RELNpositive neurons in both in the outer and middle parts of layer I (black arrows). The RELN-positive cells have more than 2 processes. The smaller granular cells of layer I are RELN-negative. e: At 9 years, RELN immunoreactivity is present in 2 different cells, the large CR neuron (black arrows) and the smaller SGN (open arrows) in layer I of cortex. f: At 54 years, RELN immunoreactivity is still present in a rare, large, classic type of CR neuron in the outer part of cortical layer I (arrow). g: Dab1 immunoreactivity (goat polyclonal C-terminus antibody) in layer I of the human brain at 26 GW. Large CR neurons in the molecular layer exhibit positive staining with antibody to C-terminal Dab1 (black arrows). Calibration bar: a, c–g 5 100 mm; b, h 5 50 mm. J Neuropathol Exp Neurol, Vol 62, June, 2003 680 DEGUCHI ET AL TABLE RELN and DAB1 Immunoreactivity in Human Cortex at Different Stages of Life 10–14 15–19 20–24 25–29 30–34 35–39 40GW– 2M–1Y 1–4Y Age (n) GW (3) GW (5) GW (9) GW (2) GW (3) GW (3) 1M (2) (3) (4) Layer I RELN DAB1 5–9Y 10–19Y 20–39Y 40–59Y 60–62Y (3) (3) (2) (3) (1) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 2 1 2 1 2 1 2 1 2 Layer II–VI RELN 2 DAB1 1 2 1 2 1 2 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1, positive; 2, negative; n, the number of brains examined; GW, gestational weeks; M, months; Y, years. cells at 10 GW measured 10 to 20 mm across. The diameter increased to 25 to 40 mm by 39 GW and remained that size in older brains. The intensity of the staining was variable, possibly reflecting the differences in fixation and postmortem interval of these autopsy specimens. Between 10 and 16 GW, the cells were localized very close together in the outer most part of layer I, immediately beneath the pia (Fig. 2d). The numbers of CR neurons ranged from 19 to 23 per high power field (hpf). After 20GW, the RELN-positive CR neurons were more widely separated at 3 to 6/hpf (Fig. 2e). From 20 to 26 weeks gestation, the subpial granule neurons displaced the CR neurons towards layer II of the cortex. By 40 GW, the density of the CR cells was further reduced in the molecular layer, and after the first year careful searching of several sections was required to locate the cells. Also, after 1 year of age we observed 2 different types of RELN-immunoreactive cells in the molecular layer. The first was the classic CR neuron that could be identified (with careful analysis) in all brains up to age 62 years (Fig. 2f, g, black arrow). The second population of RELN-positive cells was SGN, which appeared smaller, with a compact nucleus and scant cytoplasm (Fig. 2f, open arrow). DAB1 staining of layer I revealed unexpected reactivity in the cytoplasm and the dendrites of some classic CR neurons (Fig. 2h) in immature brains from 10 GW until 1 year of age (Table). Unlike RELN, however, DAB1 immunoreactivity was absent from layer I after 1 year of age. The SPG neurons were DAB1-negative. RELN and DAB1 Expression in Neurons of Cortical Layers II to VI In the early embryonic human brain (10 GW to 34 GW), RELN immunoreactivity was absent in layers II to VI. At 37 GW, RELN-positive staining began to appear in the nucleus and cytoplasm of immature cortical neurons in the deeper layers of the cortex (Fig. 3a). As the brain developed further, strong RELN immunoreactivity was identified in the cell body and processes of rare isolated cortical pyramidal neurons (identified by their large J Neuropathol Exp Neurol, Vol 62, June, 2003 nucleus, pyramidal shaped cytoplasm, and prominent apical dendrite) and interneurons (identified by their large nucleus, circular-shaped cytoplasm with radial rather than apical dendrites) (Fig. 3b). At some ages there was a trace of staining in most of the neurons with cytoplasm. The isolated, strongly positive RELN pyramidal neurons and interneurons were present through 62 years of age. DAB1 immunoreactivity was also identified in some neurons of layers II to VI at every age. In the most immature brains (10–20 GW), there was nuclear staining of the immature cells in some isolated segments of cortex. At 26 GW, some of the mature neurons in the middle to lower layers of the cortex exhibited pale immunoreactivity in their cytoplasm and dendrites. In the older brains, isolated parts of the cortex showed immunoreactivity in the cytoplasm of pyramidal-shaped neurons (Fig. 3c) and some small cells without processes. The DAB1 immunoreactivity was weaker in the cortical neurons than in the CR neurons. It was more readily identified in the adolescent and adult brains than in most of the immature brains. There was variability in the intensity of staining between cases of similar ages, suggesting that the condition of the patient and tissues affected the reactivity. The DAB1 expression was stronger in the colocalization experiments in which frozen tissue and fluorescent antibody were used. RELN and DAB1 Expression in the Germinal Layer In the germinal layer both RELN immunoreactivity and DAB1 immunoreactivity were negative from 10 GW to 39 GW of age in immature human brain. Occasional positive unidentified cells were seen in the population of migrating cells in the white matter in immature brains. Colocalization of RELN and DAB1 in Developing and Mature Cortex Because RELN and DAB1 immunoreactivity were detected in the same cortical layers we questioned whether the 2 proteins were coexpressed by the same neurons. We found colocalization in layer I and layers II to V. In layer I of the 3-month human brain, both CR50 and anti-DAB1 REELIN AND DISABLED-1 IN HUMAN CORTEX C-terminus antibodies stained CR neuron, defining their cytoplasm and processes (Fig. 3d, e). Each CR neuron revealed colocalization of RELN and DAB1 as shown in the merged image (Fig. 3f). Figure 3g illustrates in layers II to VI of the same brain several RELN-positive pyramidal neurons, which also coexpressed DAB1 (Fig. 3h, i). DISCUSSION Although the mouse and the human cortex share a similar basic structure, there are differences in volume, gyral formations, and function. These similarities and differences may be associated with distinct functions of the regulatory molecules that operate during development, such as the RELN signaling pathway in neuronal migration. There is a well-characterized signaling mechanism for RELN in the mouse, but the human counterpart is not well defined. In this study, we investigated the ontogeny of RELN in the human brain to document its presence, as suggested by the mouse studies, during the period of neuronal migration. We also extended the study into older brains to explore the idea that there may be other possible functions of this signaling pathway. First we investigated expression of transcripts of the RELN pathway at different ages using RT-PCR. We demonstrated that messenger RNA for the ligand, receptors, and adaptor molecule of the RELN signaling system was present in parallel at different stages of life. These findings suggested contiguous roles for the RELN signaling pathway in both developing and mature human brain and stimulated our immunohistologic studies of RELN and DAB1. Our studies of 2 of the proteins have confirmed the presence of RELN-positive neurons in the molecular layer of the fetal and embryonic human brain and identified RELN-positive neurons, in reducing numbers, in infant and older brains ages 1 to 62 years. We believe that these RELN-positive neurons in the youngest brains, based on their morphologic features, represent the CR neurons. In addition, we observed that these CR neurons also expressed DAB1 from 10 GW to 1 year after birth. Our immunofluorescence study showed coexpression of RELN and DAB1 in the CR neurons. Although there is no information to explain the physiologic function of DAB1 in these neurons, they may participate in a selfregulation autocrine function. During the stage of neuronal migration (up to 34 GW), DAB1 immunoreactivity, but not RELN, was present in many neurons in layer II to VI. Following the completion of cortical layer formation, RELN began to be expressed in selected cortical neurons, both interneurons, and pyramidal neurons. It is noteworthy that RELN expression in pyramidal neurons has not been observed in rodent brains, but only in higher primate species (37) and humans. RELN expression has been observed in interneurons in both species (38–40). DAB1 also appears to be 681 expressed in some neurons in the postnatal cortex. If RELN and DAB1 are also colocalized in these cells, as suggested by our immunofluorescence study, an autocrine function that is distinct from the regulation of cell migration may also may be operating in some cortical neurons. It will be important in the future to define the nature of this specific cell population to elucidate the function of RELN and DAB1 in postmigration pyramidal neurons. Although the source of the mRNA expression of the RELN and DAB1 in the postnatal brain could not be defined by our RT-PCR analysis, histochemistry studies suggest that the source could be from the neurons in the molecular layer and pyramidal neurons and interneurons in cortical layers II to VI. RELN has also been found in extra-CNS tissues, including mammalian blood, liver, intermediate lobe of the pituitary gland, and in the chromaffin cells of the adrenal gland (41). These non-neuronal sources of the protein also support the idea that RELN may have multiple functions. In the developing brain, RELN is secreted into the extracellular environment where, through a system of receptors and signaling molecules, it influences the function of other neurons. One major function, as revealed by the reeler mouse and human LCH patients, is that of determining the position of migrating neurons in cortical layers (8). Similarly, in the cerebellum, RELN is required for the correct positioning of Purkinje cells in a distinct cellular layer (5, 8). In the hippocampus, RELN controls the formation of the pyramidal cell layer, but it is also required for the maturation and branching of the axons of the perforant pathway arising in the entorhinal cortex (43). What could be the significance of RELN expression in the postnatal brain? We speculate that it may be important for neuronal maturation and plasticity. In this regard, a study of heterozygous reeler mice is noteworthy (44). The heterozygous mice exhibit down-regulation of Reln and glutamic acid decarboxylase 67 in interneurons and display decreased neuropil, cortical thickness, and spine density, but none of the cellular ectopia seen in the homozygous reeler mouse. Furthermore, recent studies directly demonstrate a role for Reln in the development of peripheral synapses (45). The possible function of Reln in controlling spine density and synaptic function is of interest in several cognitive disorders. For example, a reduction of cortical RELN mRNA and RELN protein has been interpreted to represent a vulnerability factor in schizophrenia (27) and bipolar disorder (28, 29). Furthermore, particular RELN gene haplotypes with a high number of trinucleotide repeats have been associated with autism (30), and RELN appears to be down-regulated in the cerebellum of autistic subjects. The intracellular signaling molecule DAB1 binds several proteins, including the b-amyloid precursor protein. This molecule has been implicated in Alzheimer disease, and so the role of the J Neuropathol Exp Neurol, Vol 62, June, 2003 682 DEGUCHI ET AL Fig. 3. a, b: RELN immunostaining (CR-50) in layers II to VI of the human cortex. a: At 36 GW, RELN-immunoreactive neurons are present in layers II to VI of the human cortex. b: At 4 years, RELN-immunoreactive pyramidal neurons and inter neurons are present in layer II to VI of the cortex. The pyramidal neurons are indicated by black arrows and show faint apical dendrites orientated towards the top of the illustration. The interneurons, indicated with the open arrows have pale large nuclei, small amounts of cytoplasm, and no apical dendrites. c: At 12 years, Dab1 immunoreactivity (goat polyclonal C-terminus antibody) in layers II to VI of the human brain. Pyramidal (black arrow) and interneurons (open arrow) in layers II to VI of cortex exhibit positive staining with antibody to Dab1 C-terminus. d–i: Colocalization of RELN and DAB1 by fluorescence immnohistochemistry. d–f: Layer I. A CR neuron and their processes in layer I represented RELN staining (d). Same cell was also immunoreactive for DAB1 (e). Merged image of RELN and DAB1 staining (f). g–i: Layers II to VI. Similarly, an isolated pyramidal neuron revealed RELN immunoreactivity (g), as well as DAB1 immunoreactivity (h). Merged image revealed colocalization of RELN and DAB1 within the same neuron (i). Calibration bars: a, b 5 100 mm; c 5 50 mm; d–i 5 20 mm. RELN pathway needs to be kept in mind in the unraveling of Alzheimer pathoetiology. In this paper we have examined the human cortex for the expression of genes or proteins of the RELN signaling pathway that are critical for the development of the mouse brain. We identified mRNAs for RELN, DAB1, APOER2, and VLDLR in the developing and in the mature human brain. We observed RELN and DAB1 protein in CR neurons in the immature brain and also in the cortex in pyramidal and interneurons of the mature cortex. The presence of these proteins after cortical neuronal migration and their colocalization within selected neurons raises the possibility that they serve an additional role in the functioning of the mature cortex. It will be critical to define the nature of this cell population to elucidate the J Neuropathol Exp Neurol, Vol 62, June, 2003 function of RELN and DAB1 in these selected postmigration pyramidal neurons. These observations are important in relation to the reports of RELN abnormalities in human disease. We believe that our paper provides normative data that will provide a baseline for the further investigation of the human cortex and its disorders in which RELN function may be deficient. ACKNOWLEDGMENTS The authors acknowledge the generous support of Tom Curran and Lakhu Keshvara for Dab1 antibodies and of Jonathan A. Cooper for Dab1 knockout mice. We thank Robert McNeil and Subhendu Chakrabort for preparation of color figures. A portion of this paper was presented at the Annual Meeting of the AANP held in Chicago, Illinois, June 2001. REELIN AND DISABLED-1 IN HUMAN CORTEX REFERENCES 1. Falconer DS. Two new mutants, ‘trembler’ and ‘reeler’, with neurological actions in the house mouse. J Genet 1951:192–201 2. Goffinet AM. Events governing organization of postmigratory neurons: Studies on brain development in normal and reeler mice. Brain Res 1984;319:261–96 3. Rakic P, Caviness VS Jr. Cortical development: View from neurological mutants two decades later. Neuron 1995;14:1101–4 4. D’Arcangelo G, Curran T. Reeler: New tales on an old mutant mouse. Bioessays 1998;20:235–44 5. D’Arcangelo G, Miao GG, Chen SC, Soares HD, Morgan JI, Curran T. 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