Etude du role potentiel de l'Endopeptidase Neutre dans la maladie d'Alzheimer, et, Caracterisation de NL1, une nouvelle metallopeptidase a zinc chez la souris

An emerging family of metallopeptidases presenting important similarity with Neutral Endopeptidase (Neprilysin) has recently been shown to be involved in the maturation and inactivation of various bioactive peptides.;We first studied the potential role of Neprilysin in Alzheimer's disease. This important neurodegenerative disease is characterized by the deposition and accumulation of toxic amyloid beta peptide (Abeta) in brain tissues forming senile plaques and causing neuronal cell death. Abeta also forms deposit in the walls of small arteries of leptomeninges and cortex causing cerebral amyloid angiopathy (CAA).;To demonstrate the involvement of Neprilysin down regulation in AD we localized the enzyme in various brain sections of AD and control cases by RT-PCR and immunocytochemistry. We examined normal and diseased human brain regions with Abeta deposition including hippocampus, temporal lobe cortex and leptomeninges and provided convincing evidence for down-regulation of Neprilysin expression in these areas of AD brains, consistent with its putative involvement in both AD and CAA.;The second aim of the research project was to further characterize molecularly and functionally the zinc metallopeptidase NL1 which had been recently identified in the laboratory. The experimental strategy employed consisted to: (1) Study the ND mRNA expression pattern at tissular and cellular level. (2) Identify new peptide substrates for NL1 and compare its activity with Neprilysin (protein purification, and enzymatic characterization). (3) Generate homozygous NL1-/- mice by gene targeting technology.;We demonstrated that NL1 is strongly expressed in mature testis, more precisely in the round and elongated spermatids of seminiferous tubules at all spermiogenesis maturational stages. We also showed that NL1 is moderately expressed in several tissues including brain and pituitary. Moreover, we have established that NL1 is the first member of the family to be processed in the Golgi and secreted in the extracellular space where it degrades several bioactive peptides. Using specific inhibitors and site directed mutagenesis we have identified paired basic residues important for the processing and the secretion of NL1. Finally, using homologous recombination we've disrupted NL1's gene locus. The Nl1-/- mice were shown to be viable and healthy but present reduced fertility. Therefore, we've determined that NL1 plays an important role in processes related to reproduction. (Abstract shortened by UMI.).