Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-06-16
2001-11-27
Mertz, Prema (Department: 1646)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C435S007100, C435S007200, C435S348000, C435S325000, C530S350000
Reexamination Certificate
active
06323177
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the identification of an interaction between Reelin and the very low density lipoprotein (VLDL) receptor. This allows development of a convenient assay system for receptor binding that is adaptable for screening modulators (agonists and antagonists) of the interaction between Reelin and the VLDL receptor or similar receptors.
BACKGROUND OF THE INVENTION
The complexity of the human brain presents a challenge to scientists striving for an understanding of the molecular basis of neuronal development and function. Nevertheless, advances in genome technologies have led to the identification of several genes, mutated in neurological disorders that are important for brain development and function. Furthermore, the ability to disrupt genes in the mouse germline provides an opportunity to develop models and to test the contributions of specific genes to neurobiological processes. The information obtained from these studies is relevant to human diseases because cellular and molecular mechanisms are largely conserved between mice and humans.
Reelin
Neurological abnormalities affecting behavior or locomotion have been identified frequently in inbred mouse strains. One mutant mouse that has been extensively studied because of its remarkable neurological phenotype is the reeler mouse (reviewed in D'Arcangelo and Curran, Reeler: New Tales on an Old Mutant Mouse, BioEssays, In press, 1998; de Rouvroit and Goffinet, Adv. Anat. Embryol. Cell Biol., 150:1-108, 1998). Reeler is an autosomal recessive mutation in which homozygous mutant mice exhibit ataxia, tremors, imbalance and a typical reeling gait that becomes apparent two weeks after birth. These behavioral defects are associated with severe hypoplasia of the cerebellum which lacks foliation. In addition, reeler mice exhibit neuronal ectopia in laminated brain structures including the cerebral and cerebellar cortices, and the hippocampus. Neurons in brain stem nuclei are also abnormally positioned. This anatomical phenotype indicates that the reeler gene controls cell positioning at the end of the migratory phase of brain development. Correct neuronal positioning is essential for the establishment of appropriate synaptic connectivity and proper brain function. This is emphasized by the findings that in reeler brain the axonal branching, and the synaptic density of cerebellar and hippocampal neurons are aberrant (Del Rio et al., Nature, 385:70-74, 1997; Borrell et al., J. Neurosci., 19:1345-1358, 1999). This abnormal connectivity results in altered expression of genes such as the NMDA receptor subunit genes (Wanatabe et al, Neurosci. Res., 26:335-343, 1996).
A mutant mouse carrying a novel allele of reeler was isolated by insertional mutagenesis (Miao et al., Proc. Natl. Acad. Sci. USA, 91:11050-11054, 1994). This provided a molecular marker that allowed cloning of the reeler gene, which was named reelin (Reln). Reelin is expressed during normal brain development (D'Arcangelo et al., Nature, 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 spread over a region of approximately 450 kb (Royaux et al., Genomic Organization of the Mouse Reelin Gene, Genomics, In press, 1997). Using the mouse cDNA as a probe, the human reelin gene was also cloned and sequenced (DeSilva et al., Genome Res., 7:157-164, 1997). The predicted protein encoded by the human gene is 94% identical to the mouse gene indicating an extremely high degree of sequence conservation. This implies that the mouse and human genes are functionally very similar.
During development, reelin is expressed at high levels in the central nervous system, liver and kidney (Ikeda and Terashima, Dev. Dyn., 210:157-172. 1997). In the developing central nervous system, sites of high expression are the cerebral cortex, hippocampus, cerebellum, olfactory bulb, spinal cord and retina (D'Arcangelo et al., supra, 1995; Ogawa et al., Neuron, 14:899-912, 1995; Schiffmann et al., Eur. J. Neurosci., 9:1055-1071, 1997; Alcantara et al., 1998). In the embryonic mouse brain, reelin is expressed mainly by Cajal-Retzius cells in the marginal zone of the developing neocortex and hippocampus, and by granule neurons in the external germinal layer of the cerebellum (D'Arcangelo et al., supra, 1995; Ogawa et al., supra, 1995; Miyata et al., J. Comp. Neurol., 372:215-228, 1996). Similarly, reelin is expressed in the marginal zone of the prenatal human neocortex (Meyer and Goffinet, J. Comp. Neurol., 197:29-40, 1998). In adult mice, reelin is expressed predominantly in the brain, but also in the spinal cord, spleen, thymus, liver, kidney, and gonads (D'Arcangelo et al., supra, 1995; Ikeda and Terashima, supra, 1997). Several populations of cells, distinct from those that express reelin in the embryonic cortex, maintain a high level of reelin expression throughout adult life (Alcantara et al. 1998). Reelin expression is particularly high in the olfactory bulb, in the hippocampus, and in the entorhinal cortex, which projects to the hippocampus. Although the expression of reelin in embryonic brain is clearly linked to the determination of neuronal positioning and the maturation of dendrites and axonal branches during development, the significance of reelin expression in the adult has not yet been clearly established. However, the discovery and characterization of other molecules involved in reelin function suggest that reelin may be important for physiological and pathophysiological processes in the adult brain as well as other organs.
The reelin open reading frame (D'Arcangelo et al., supra, 1995) predicts a novel protein of 3,461 amino acids (approximately 385 kDa). At the N terminus, Reelin contains a cleavable signal peptide and a small region of similarity with F-spondin, a protein secreted by floor plate cells in the developing neural tube. At the C terminus of Reelin there is a stretch of positively charged amino acids. The main body of the protein comprises a series of eight internal repeats of 350-390 amino acids, each containing two related subdomains that flank a pattern of conserved cysteine residues known as an EGF-like motif. These cysteine-rich regions resemble those found in other extracellular proteins, whereas the flanking subdomains appear to be unique to Reelin. Expression studies in transfected mammalian cells and cultured neurons demonstrate that, as predicted by its structural features, Reelin it is indeed an extracellular glycoprotein (D'Arcangelo et al., J. Neurosci., 17:23-31, 1997).
Recently, a signal transduction adaptor molecule, Disabled1 (Dab1) (Howell et al., EMBO J., 16:121-132, 1997a) that plays a key role in the Reelin pathway was identified. This discovery was prompted by the observation that mutant mice lacking Dab1 display a phenotype indistinguishable from that of reeler mice (Howell et al., Nature, 389:733-736, 1997b; Sheldon et al., Nature, 389:730-733, 1997). Dab1 is expressed in the target cells of Reelin, i.e., the neurons that go astray in the mutant mice (Rice et al., Development, 125:3719-3729, 1998). Dab1 is an intracellular molecule that is phosphorylated in the developing brain and in cultured neurons exposed to Reelin (Howell et al., Genes and Dev., 13:643-648, 1999a). These findings point to a critical signaling pathway in which Reelin, secreted by a subset of neurons, acts on other neurons that express Dab1.
The missing link in this emerging pathway is the receptor for Reelin. This receptor is predicted to be a transmembrane protein that detects the presence of Reelin in the extracellular environment and communicates this information to the intracellular environment through interactions with Dab1 (D'Arcangelo and Curran, supra, 1998). Receptor occupancy may lead to phosphorylation of Dab1, and ultimately changes in gene expression in the target cell populations that alter the morphology and properties of neurons necessary for layer formation and neuronal maturation.
Curran Thomas
D'Arcangelo Gabriella
Darby & Darby
Mertz Prema
Murphy Joseph F.
St. Jude Children's Research Hospital
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