Missense mutations in the beta-amyloid precursor protein gene (APP) co-segregate with a small subset of autosomal dominant familial Alzheimer's disease (FAD) cases wherein deposition of the 39-43 amino acid beta-amyloid (A beta) peptide and neurodegeneration are principal neuropathological hallmarks. To accurately examine the effect of missense mutations on APP metabolism and A beta production in vivo, we have introduced yeast artificial chromosomes (YACs) containing the entire approximately 400 kbp human APP gene encoding APP harboring either the asparagine for lysine and leucine for methionine FAD substitution at codons 670 and 671 (APP(K670N/M671L)), the isoleucine for valine FAD substitution at codon 717 (APP(V7171)) or a combination of both substitutions into transgenic mice. We demonstrate that, relative to YAC transgenic mice expressing wild-type APP, high levels of A beta peptides are detected in the brains of YAC transgenic mice expressing human APP(K670N/M671L) that is associated with a concomitant diminution in the levels of apha-secretase-generated soluble APP derivatives. Moreover, the levels of longer A beta peptides (species terminating at amino acids 42/43) are elevated in YAC transgenic mice expressing human APP(V7171). These mice should prove valuable for detailed analysis of the in vivo effects of the APP FAD mutations in a variety of tissues and throughout aging and for testing therapeutic agents that specifically alter APP metabolism and A beta production.
The binding of [3 H]methoxymethyl-3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (methoxymethyl-MTEP), a potent and selective antagonist for metabotropic glutamate (mGlu)5 receptors, was characterized in rat brain both in vitro and in vivo. Nonspecific binding, as defined with 10 M 2-methyl-6-(phenylethynyl)-pyridine (MPEP), was less than 10% of total binding in rat brain membranes. , 1997). There are eight mGluR subtypes, which are subdivided into three groups principally based on sequence homology, but also on signal transduction pathways and agonist selectivity (Nakanishi, 1992;Pin and Duvoisin, 1995). Group I mGluRs initiate cell responses through G q/11 protein coupling to phospholipase C and stimulation of phosphoinositide hydrolysis. In contrast, group II and group III mGluRs are negatively coupled via G i /G o to adenylyl cyclase and reduce forskolinstimulated increases in cAMP in recombinant expression systems. Group I receptors are selectively activated by dihydroxyphenylglycine (DHPG), group II receptors can be stimulated by (2S,2ЈR,3ЈR)-2-(2Ј,3Ј-dicarboxycyclopropyl)glycine and (ϩ)-2-aminobicyclo[3.1.0]-hexane-2,6-dicarboxylate monohydrate (LY354740), and group III receptors are selectively stimulated by L-(ϩ)-2-amino-4-phosphonobutyric acid and (R,S)-4-phosphonophenylglycine (Schoepp et al., 1999).Group I mGluRs include the mGlu1 and mGlu5 subtypes. These two receptors exhibit a regional pattern of expression in the central nervous system, suggesting distinct, functional roles for each receptor (Spooren et al., 2001). For example, expression of mGlu5 receptor protein is high-to-moderate in frontal cortex, caudate putamen, nucleus accumbens, olfacArticle, publication date, and citation information can be found at
Dosage imbalance for chromosome 21 in Down syndrome and mutations in the amyloid precursor protein (APP) and presenilin-1 (PS-1) genes in early-onset familial Alzheimer's disease (FAD) result in elevated production and deposition of amyloid-β (Aβ) peptides 1 , particularly the 42 amino acid form, Aβ1-42. One difficulty in studying the generation and deposition of Aβ, neuritic abnormalities, synaptic dysfunction, neuronal cell death and dementia that occurs in Alzheimer's disease (AD) is the paucity of small animal models. To examine effects of the FAD mutations in vivo, we transferred yeast artificial chromosomes (YACs) containing the entire genomic copy of human APP and/or PS-1 genes harboring FAD mutations into transgenic mice 2,3 . We now document that a mutant APP YAC transgenic mouse develops Aβ deposits and that this deposition is accelerated when the animals are mated to homozygosity and/or to mutant PS-1 YAC transgenic mice.Transgenic approaches can directly test whether the overexpression of wild-type (WT) or mutant APP and PS-1 genes leads to AD-type abnormalities. Over the past several years, numerous transgenic mice have been produced using cDNAbased expression of APP or PS-1 gene fragments 4 . By contrast, our efforts have been directed toward establishing a more accurate genetic model for AD by introducing entire genomic copies of WT or mutant human APP 2,3 and PS-1 genes into the mouse genome. The APP and PS-1 genomic sequences contain transcriptional regulatory elements required for proper spatial and temporal expression with appropriate splice donor and acceptor sites needed to generate the entire spectrum of alternatively spliced transcripts and protein isoforms of APP and PS-1. We previously reported on the generation of WT and mutant APP YAC transgenic mice and demonstrated altered metabolism of Aβ peptides in the brains of mutant APP YAC transgenic mice 2,3 .In the present study, we introduced a 1,000-kb YAC containing the entire human PS-1 gene harboring the H163R and/or M146L FAD mutation into mouse embryonic stem (ES) cells to generate PS-1 YAC transgenic mice. The YAC transgene 788H12 contains the entire unrearranged human PS-1 gene and ∼550 kb of upstream and ∼350 kb of downstream sequences (Fig. 1a). The M146L and H163R PS-1 FAD mutations were introduced into the 1000-kb 788h12 PS-1 YAC with neomycin expression cassettes using standard protocols for homologous recombination in yeast 2,3 . Lipid-mediated transfection of J1 ES cell suspension cultures 2 with purified PS-1 YAC DNA was used to generate 48 lines of G418 r cells. PCR and restriction analysis showed seven WT and five H163R mutant lines containing intact copies of the PS-1 YACs (data not shown). Germline transmission was obtained for three lines with the H163R mutation (F7, G9 and 2H9).Semi-quantitative RT-PCR revealed mutant human PS-1 expression in PS-1 YAC transgenic line G9 tissues at levels comparable to that of endogenous mouse PS-1 (50-70%; Fig. 1b). In addition, G9 mouse tissues expressed alternatively spliced human PS-...
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