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(12) United States Patent (10) Patent No.: US 6,323,177 B1 Curran Et Al

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(12) United States Patent (10) Patent No.: US 6,323,177 B1 Curran Et Al USOO6323177B1 (12) United States Patent (10) Patent No.: US 6,323,177 B1 Curran et al. (45) Date of Patent: Nov. 27, 2001 (54) INTERACTION OF REELIN WITH VERY DeSilva et al., Genome Res., 7:157-164, 1997. LOW DENSITY LIPOPROTEIN (VLDL) Howell et al., EMBO J., 16:121-132, 1997. RECEPTOR FOR SCREENING AND THERAPES Howell et al., Nature, 389:733–736, 1997. Sheldon et al., Nature, 389: 730–733, 1997. (75) Inventors: Thomas Curran; Gabriella D'Arcangelo, both of Memphis, TN Oshima et al., 1996; Chae et al., Neuron, 18:29–42, 1997. (US) Kim et al., J. Biol. Chem., 271:8373-8380, 1996. Novak et al., J. Biol. Chem..., 271:11732–11736, 1996. (73) Assignee: St. Jude Children's Research Strittmatter and Roses, Proc. Natl. Acad. Sci. USA, Hospital, Memphis, TN (US) 92:4725-4727, 1995. (*) Notice: Subject to any disclaimer, the term of this Miao et al., Proc. Natl. Acad. Sci. USA, 91:11050-11054, patent is extended or adjusted under 35 1994. U.S.C. 154(b) by 0 days. Takahashi et al., Proc. Natl. Acad. Sci. USA, 89:9252–9256, 1992. (21) Appl. No.: 09/334,220 Chae, et al., Neuron, 18:29–42, 1997. (22) Filed: Jun. 16, 1999 Bergeyck, et al., “A panel of monoclonal antibodies against reelin, the extracellular matrix protein defective in reeler (51) Int. Cl. .......................... C07K 14/00; G01N 33/53; mutant mice’, Journal of Neuroscience Methods, 1998, GO1N 33/566 82(1):17-24. (52) U.S. Cl. ................................. 514/8; 435/7.1; 435/7.2; D'Arcangelo, et al., “Reelin Is a Secreted Glycoprotein 435/348; 435/325; 530/350 Recognized by the CR-50 Monoclonal Antibody', The (58) Field of Search .............................. 530/350; 435/7.1, Journal of Neuroscience, 1997, 17(1):23–31. 435/72,325, 348; 514/8 Primary Examiner Prema Mertz (56) References Cited Assistant Examiner Joseph F. Murphy PUBLICATIONS (74) Attorney, Agent, or Firm-Darby & Darby Del Rio et al., Nature, 385:70-74, 1997. (57) ABSTRACT Borrell et al., J. Neurosci., 19:1345-1358, 1999. Howell et al., Genes and Dev., 13:643–648, 1999. Interaction between Reelin and the very low density lipo Trommsdorff et al., J. Biol. Chem..., 273:33556-33560, 1999. protein (VLDL) receptor has been discovered. This allows D’Arcangelo and Curran, Reeler: New Tales on an Old development of a convenient assay System for receptor Mutant Mouse, BioEssays, In press, 1998. binding that is adaptable for Screening modulators (agonists Rice et al., Development 125:3719-3729, 1998. and antagonists) of the interaction between Reelin and the Price and Sisodia, Ann. Rev. Neurosci., 21:479-505, 1998. VLDL receptor or similar receptors. Royaux et al., Genomic Organization of the Mouse Reelin Gene, Genomics, In press, 1997. 17 Claims, 1 Drawing Sheet U.S. Patent Nov. 27, 2001 US 6,323,177 B1 F. G. 1 Reelin O-oooooooooooooooo VLD LR Dab 1 US 6,323,177 B1 1 2 INTERACTION OF REELIN WITH VERY molecular marker that allowed cloning of the reeler gene, LOW DENSITY LIPOPROTEIN (VLDL) which was named reelin (Reln). Reelin is expressed during RECEPTOR FOR SCREENING AND normal brain development (DArcangelo et al., Nature, THERAPES 374:719–723, 1995); reelin mRNA is very large (approximately 12 kb), and it contains an open reading frame of 10,383 bp. The reelin gene is composed of 65 exons The work leading to the present invention was Supported, spread over a region of approximately 450 kb (Royaux et al., in part, by National Institute of Health Grants NS 36558. Genomic Organization of the Mouse Reelin Gene, Thus, the United States Government has certain rights in the Genomics, In press, 1997). Using the mouse cDNA as a invention. probe, the human reelin gene was also cloned and Sequenced FIELD OF THE INVENTION (DeSilva et al., Genome Res., 7:157–164, 1997). The pre dicted protein encoded by the human gene is 94% identical The present invention relates to the identification of an to the mouse gene indicating an extremely high degree of interaction between Reelin and the very low density lipo Sequence conservation. This implies that the mouse and protein (VLDL) receptor. This allows development of a human genes are functionally very similar. convenient assay System for receptor binding that is adapt 15 During development, reelin is expressed at high levels in able for Screening modulators (agonists and antagonists) of the central nervous system, liver and kidney (Ikeda and the interaction between Reelin and the VLDL receptor or Terashima, Dev. Dyn., 210:157–172. 1997). In the develop Similar receptorS. ing central nervous System, Sites of high expression are the cerebral cortex, hippocampus, cerebellum, olfactory bulb, BACKGROUND OF THE INVENTION spinal cord and retina (D'Arcangelo et al., Supra, 1995; Ogawa et al., Neuron, 14:899-912, 1995; Schiffmann et al., The complexity of the human brain presents a challenge Eur. J. Neurosci., 9:1055–1071, 1997; Alcantara et al., to Scientists Striving for an understanding of the molecular 1998). In the embryonic mouse brain, reelin is expressed basis of neuronal development and function. Nevertheless, mainly by Cajal-Retzius cells in the marginal Zone of the advances in genome technologies have led to the identifi 25 developing neocortex and hippocampus, and by granule cation of Several genes, mutated in neurological disorders neurons in the external germinal layer of the cerebellum that are important for brain development and function. (D’Arcangelo et al., Supra, 1995; Ogawa et al., Supra, 1995; Furthermore, the ability to disrupt genes in the mouse Miyata et al., J. Comp. Neurol., 372:215–228, 1996). germline provides an opportunity to develop models and to Similarly, reelin is expressed in the marginal Zone of the test the contributions of Specific genes to neurobiological prenatal human neocortex (Meyer and Goffinet, J. Comp. processes. The information obtained from these Studies is Neurol., 197:29-40, 1998). In adult mice, reelin is expressed relevant to human diseases because cellular and molecular predominantly in the brain, but also in the Spinal cord, mechanisms are largely conserved between mice and spleen, thymus, liver, kidney, and gonads (DArcangelo et humans. al., Supra, 1995; Ikeda and Terashima, Supra, 1997). Several 35 populations of cells, distinct from those that express reelin Reelin in the embryonic cortex, maintain a high level of reelin Neurological abnormalities affecting behavior or locomo expression throughout adult life (Alcantara et al. 1998). tion have been identified frequently in inbred mouse Strains. Reelin expression is particularly high in the olfactory bulb, One mutant mouse that has been extensively Studied because in the hippocampus, and in the entorhinal cortex, which of its remarkable neurological phenotype is the reeler mouse 40 projects to the hippocampus. Although the expression of (reviewed in D'Arcangelo and Curran, Reeler: New Tales on reelin in embryonic brain is clearly linked to the determi an Old Mutant Mouse, BioEssays, In press, 1998; de Rou nation of neuronal positioning and the maturation of den vroit and Goffinet, Adv. Anat. Embryol. Cell Biol., drites and axonal branches during development, the Signifi 150: 1-108, 1998). Reeler is an autosomal recessive muta cance of reelin expression in the adult has not yet been tion in which homozygous mutant mice exhibit ataxia, 45 clearly established. However, the discovery and character tremors, imbalance and a typical reeling gait that becomes ization of other molecules involved in reelin function Sug apparent two weeks after birth. These behavioral defects are gest that reelin may be important for physiological and asSociated with Severe hypoplasia of the cerebellum which pathophysiological processes in the adult brain as well as lacks foliation. In addition, reeler mice exhibit neuronal other organs. ectopia in laminated brain Structures including the cerebral 50 The reelin open reading frame (DArcangelo et al., Supra, and cerebellar cortices, and the hippocampus. Neurons in 1995) predicts a novel protein of 3,461 amino acids brain Stem nuclei are also abnormally positioned. This (approximately 385 kDa). At the N terminus, Reelin con anatomical phenotype indicates that the reeler gene controls tains a cleavable Signal peptide and a Small region of cell positioning at the end of the migratory phase of brain Similarity with F-spondin, a protein Secreted by floor plate development. Correct neuronal positioning is essential for 55 cells in the developing neural tube. At the C terminus of the establishment of appropriate Synaptic connectivity and Reelin there is a stretch of positively charged amino acids. proper brain function. This is emphasized by the findings The main body of the protein comprises a Series of eight that in reeler brain the axonal branching, and the Synaptic internal repeats of 350-390 amino acids, each containing density of cerebellar and hippocampal neurons are aberrant two related Subdomains that flank a pattern of conserved (Del Rio et al., Nature, 385:70–74, 1997; Borrell et al., J. 60 cysteine residues known as an EGF-like motif. These Neurosci., 19:1345–1358, 1999). This abnormal connectiv cysteine-rich regions resemble those found in other extra ity results in altered expression of genes such as the NMDA cellular proteins, whereas the flanking Subdomains appear to receptor Subunit genes (Wanatabe et al, Neurosci. Res., be unique to Reelin. Expression Studies in transfected mam 26:335-343, 1996). malian cells and cultured neurons demonstrate that, as A mutant mouse carrying a novel allele of reeler was 65 predicted by its structural features, Reelin it is indeed an isolated by insertional mutagenesis (Miao et al., Proc. Natl. extracellular glycoprotein (DArcangelo et al., J. Neurosci., Acad. Sci. USA, 91:11050-11054, 1994). This provided a 17:23–31, 1997). US 6,323,177 B1 3 4 Recently, a signal transduction adaptor molecule, Dis of Amyloid Precursor-like Protein 1, J.
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