Evolutionarily conserved, alternative splicing of reelin during brain development.

Reelin is the protein defective in reeler mutant mice and plays a pivotal role in brain development. However, some uncertainties remain about the relationship between reelin and the reeler phenotype. It is generally believed that reelin, secreted by specific neuronal types such as Cajal-Retzius cells, acts at short distance via the extracellular matrix on target neurons, the response of which requires the Dab1 gene product. However, the pattern of reelin expression in some structures such as olfactory bulb, retina, and spinal cord suggests that the protein might be endowed with different functions. In the present study, we identify two uncommon, evolutionarily conserved splicing events in the 3' part of the transcript that result in different forms of the protein. First, a 6-nucleotide, brain-specific microexon is skipped in about 10% of reelin RNA. In addition, an alternative polyadenylation event involving 10-25% of reelin mRNA results in secretion of a truncated protein lacking the terminal, highly basic stretch. This alternative reelin is generally expressed in the same cells as the major form, but is almost undetectable in retina and spinal cord. Both alternative splicing events are present in mouse, rat, and man, suggesting that the corresponding reelin forms are functionally important.

Reelin, the extracellular matrix protein deficient in reeler mutant mice, is processed by a metalloproteinase.

Reelin is the extracellular protein defective in reeler mice. It is believed that reelin acts via the extracellular matrix to influence the development of nearby neurons, but the mechanism remains thus far unknown. In the present work, we present in vivo and in vitro evidence that reelin is cleaved. This processing did not occur in Relnrl-Orl mutant mice in which reelin is not secreted and was prevented in explant cultures by brefeldin treatment, suggesting that it takes place extracellularly or in a postendoplasmic reticulum compartment. Reelin cleavage was inhibited by zinc chelators known to inhibit metalloproteinases but was unaffected by inhibitors of serine, cysteine, or aspartate proteinases. Furthermore, reelin cleavage was insensitive to inhibitors of matrixins, neprilysin, meprin, and peptidyl dipeptidase A, suggesting that the processing enzyme belongs to a different enzyme family. This enzyme and the physiological meaning of reelin processing remain to be characterized further.

A panel of monoclonal antibodies against reelin, the extracellular matrix protein defective in reeler mutant mice.

Reelin, the extracellular matrix protein defective in reeler mutant mice, plays a key role during brain development. We therefore raised antibodies directed against various reelin epitopes in order to facilitate biochemical and cell biological studies of this important molecule. Homozygous reeler mice with a large deletion of most of the reelin gene were immunized with fusion proteins and carrier-coupled peptides corresponding to parts of the reelin sequence. Monoclonal antibodies were produced using classical procedures, screened using ELISA and-or western blot prepared with the antigen, and tested by immunohistochemistry and immunoprecipitation assays to detect endogenous reelin. The labeling of Cajal-Retzius cells in the embryonic mouse telencephalon was selected as criterion for positivity in immunohistochemistry. A total of 11 monoclonal antibodies were obtained, providing useful additions to the widely used antibody CR-50. Five are directed against the N-terminal part of reelin, among which three recognize the region that has significant similarity with F-spondin, and two are specific for hinge region located downstream from the F-spondin similarity region and upstream from the reelin repeats. Six antibodies are directed against the C-terminal part of reelin, among which one anti-peptide antibody recognizes the highly basic C-terminal segment. Antibodies against the N-terminal region stain well in immunohistochemistry. By comparison, the labeling of embryonic Cajal-Retzius cells with antibodies directed against the C-terminal region is weaker, suggesting that this part of the molecule might be modified or not be as readily accessible in the tissue as the N-terminus.

A truncated Reelin protein is produced but not secreted in the 'Orleans' reeler mutation (Reln[rl-Orl]).

Reelin is the protein defective in reeler mutant mice [I. Bar, C. Lambert de Rouvroit, I. Royaux, D.B. Krizman, C. Dernoncourt, D. Ruelle, M.C. Beckers, A.M. Goffinet, A YAC contig containing the reeler locus with preliminary characterization of candidate gene fragments, Genomics 26 (1995) 543-549; G. D'Arcangelo, G.G. Miao, S.C. Chen, H.D. Soares, J.I. Morgan, T. Curran, A protein related to extracellular matrix proteins deleted in the mouse mutant reeler, Nature 374 (1995) 719-723; S. Hirotsune, T. Takahara, N. Sasaki, K. Hirose, A. Yoshiki, T. Ohashi, M. Kusakabe, Y. Murakami, M. Muramatsu, S. Watanabe, K. Nakao, M. Katsuki, Y. Hayashizaki, The reeler gene encodes a protein with an EGF-like motif expressed by pioneer neurons, Nature Genet. 10 (1995) 77-83]. In the Orleans allele of reeler (symbol: Reln[rl-Orl]), a 220 nucleotide deletion is present in the 3' region of the Reelin message, resulting in a frame shift with production of a predicted protein amputated from its C-terminal amino acids. In this study, we first show that the predicted truncated protein indeed exists in Orleans reeler mice, using several anti-Reelin antibodies. Three antibodies are directed against epitopes located in the N-terminal region of the protein, namely: monoclonal antibody CR-50 [M. Ogawa, T. Miyata, K. Nakajima, K. Yagyu, M. Seike, K. Ikenaka, H. Yamamoto, K. Mikoshiba, The reeler gene-associated antigen on Cajal-Retzius neurons is a crucial molecule for laminar organization of cortical neurons, Neuron 14 (1995) 899-912] (epitope region between Reelin residues 251-407), monoclonal antibody G10 (epitope located between amino acids 199 and 244) and the polyclonal antipeptide rp4 (positions 381-399). A fourth antibody, antipeptide rp5, reacts with the C-terminal (3443-3461) Reelin sequence. In normal embryos, all four antibodies stained cells in the marginal zone with features of Cajal-Retzius cells. While N-terminal specific antibodies detected Reelin immunoreactivity in mouse embryos homozygous for the reeler-Orleans mutation, no staining was obtained with the rp5 antibody, showing the presence of a truncated protein. Moreover, although Reelin could be detected at the surface of living Cajal-Retzius cells of normal mice, it was not revealed after vital staining of embryonic cortex from Orleans reeler mice. These results indicate that the C-terminal region of Reelin is essential for its secretion and suggest that the Orleans reeler phenotype is due to defective Reelin secretion rather than to secretion of an inactive protein.

An intracisternal A-particle sequence codes for an antigen recognized by syngeneic cytolytic T lymphocytes on a mouse spontaneous leukemia.

Cytolytic T lymphocyte (CTL) clones directed against spontaneous mouse leukemia LEC have been obtained. By transfecting a cosmid library into cells which were then tested for their ability to stimulate the CTL, we identified the gene coding for the antigen recognized by one of these CTL clones. It is the gag gene of an endogenous defective retrovirus that belongs to the intracisternal A particle (IAP) family. A gag-encoded nonapeptide presented by the H-2 Dk molecule caused recognition by the anti-LEC CTL clone. Southern blot and polymerase chain reaction analyses indicated that the expression of the antigen by the LEC tumor cell line resulted from the transposition of an IAP sequence into a new genomic location.

Structure of the gene of tum- transplantation antigen P198: a point mutation generates a new antigenic peptide.

Mutagen treatment of mouse tumor cell line P815 produces tum- variants that are rejected by syngeneic mice because they express new transplantation antigens. These tum- antigens are recognized by cytotoxic T lymphocytes (CTL) but induce no detectable antibody response. By transfecting P815 cell line P1.HTR with DNA of tum- variant P198, we obtained transfectants expressing tum- antigen P198 that could be identified on the basis of their ability to stimulate anti-P198 CTL. This was repeated with DNA of a cosmid library derived from variant P198, and a cosmid carrying the sequence encoding antigen P198 was recovered from a transfectant. Gene P198 is 3 kb long and contains eight exons. It shows no homology with previously identified tum- gene P91A, nor with any gene presently recorded in the data banks. The long open reading frame codes for a 23.5-kD protein. The antigenic allele of gene P198 differs from the normal allele by a point mutation located in exon 7. This mutation causes an Ala to Thr change, and was shown by site-directed mutagenesis to be responsible for the expression of the antigen. An 11-amino acid synthetic peptide covering the sequence surrounding the tum- mutation rendered P815 cells sensitive to lysis by anti-P198 CTL. The homologous peptide corresponding to the normal sequence of the gene did not, but it was able to compete for binding to major histocompatibility complex molecule Kd. We conclude that tum- mutation P198 generates a new epitope recognized by syngeneic T cells. As observed with gene P91A, we found that a fragment of gene P198 that contained only exons 3-7, cloned in nonexpression vectors, transferred efficiently the expression of the antigen.