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The effects of dietary cholesterol on brain amyloid precursor protein (APP) processing were examined using an APP gene-targeted mouse, genetically humanized in the amyloid beta-peptide (Abeta) domain and expressing the Swedish familial Alzheimer's disease mutations. These mice express endogenous levels of APP holoprotein and abundant human Abeta. Increased dietary cholesterol led to significant reductions in brain levels of secreted APP derivatives, including sAPPalpha, sAPPbeta, Abeta1-40, and Abeta1-42, while having little to no effect on cell-associated species, including full-length APP and the COOH-terminal APP processing derivatives. The changes in levels of sAPP and Abeta in brain all were negatively correlated with serum cholesterol levels and levels of serum and brain apoE. These results demonstrate that secreted APP processing derivatives and Abeta can be modulated in the brain of an animal by diet and provide evidence that cholesterol plays a role in the modulation of APP processing in vivo. APP gene-targeted mice lacking apoE, also have high serum cholesterol levels but do not show alterations in APP processing, suggesting that effects of cholesterol on APP processing require the presence of apoE.

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Fibrillar amyloid deposits are defining pathological lesions in Alzheimer's disease brain and are thought to mediate neuronal death. Amyloid is composed primarily of a 39-42 amino acid protein fragment of the amyloid precursor protein (APP), called amyloid beta-protein (Abeta). Because deposition of fibrillar amyloid in vitro has been shown to be highly dependent on Abeta concentration, reducing the proteolytic release of Abeta is an attractive, potentially therapeutic target. Here, the turnover rate of brain Abeta has been determined to define treatment intervals over which a change in steady-state concentration of Abeta could be measured. Mice producing elevated levels of human Abeta were used to determine approximate turnover rates for Abeta and two of its precursors, C99 and APP. The t1/2 for brain Abeta was between 1.0 and 2.5 hr, whereas for C99, immature, and fully glycosylated forms of APP695 the approximate t1/2 values were 3, 3, and 7 hr, respectively. Given the rapid Abeta turnover rate, acute studies were designed using phorbol 12-myristate 13-acetate (PMA), which had been demonstrated previously to reduce Abeta secretion from cells in vitro via induction of protein kinase C (PKC) activity. Six hours after intracortical injection of PMA, Abeta levels were significantly reduced, as measured by both Abeta40- and Abeta42-selective ELISAs, returning to normal by 12 hr. An inactive structural analog of PMA, 4alpha-PMA, had no effect on brain Abeta levels. Among the secreted N-terminal APP fragments, APPbeta levels were significantly reduced by PMA treatment, whereas APPalpha levels were unchanged, in contrast to most cell culture studies. These results indicate that Abeta is rapidly turned over under normal conditions and support the therapeutic potential of elevating PKC activity for reduction of brain Abeta.

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The processing of the beta-amyloid precursor protein (APP) in vivo has been characterized in a novel animal model that recapitulates, in part, the APP genotype of a familial form of Alzheimer's disease (AD). A gene-targeting strategy was used to introduce the Swedish familial AD mutations and convert mouse Abeta to the human sequence. The mutant APP is expressed at normal levels in brain, and cleavage at the mutant beta-secretase site is both accurate and enhanced. Furthermore, human Abeta production is significantly increased to levels 9-fold greater than those in normal human brain while nonamyloidogenic processing is depressed. The results on Abeta production extend similar findings obtained in cell culture to the brain of an animal and substantiate Abeta as a etiological factor in Swedish familial AD. These animals provide several distinguishing features over others created by conventional transgenic methodologies. The spatial and temporal expression patterns of human Abeta are expected to be faithfully reproduced because the gene encoding the mutant APP remains in its normal chromosomal context. Thus, the neuropathological consequences of human Abeta overproduction can be evaluated longitudinally in the absence of potential mitigating effects of APP overexpression or presence of the mouse Abeta peptide.

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P150Glued is the largest subunit of dynactin, which binds to cytoplasmic dynein and activates vesicle transport along microtubules. We have isolated human cDNAs encoding p150Glued as well as a 135-kDa isoform; these isoforms are expressed in human brain by alternative mRNA splicing of the human DCTN1 gene. The p135 isoform lacks the consensus microtubule-binding motif shared by members of the p150Glued/Glued/CLIP-170/BIK1 family of microtubule-associated proteins and, therefore, is predicted not to bind directly to microtubules. We used transient transfection assays and in vitro microtubule-binding assays to demonstrate that the p150 isoform binds to microtubules, but the p135 isoform does not. However, both isoforms bind to cytoplasmic dynein, and both partition similarly into cytosolic and membrane cellular fractions. Sequential immunoprecipitations with an isoform-specific antibody for p150 followed by a pan-isoform antibody revealed that, in brain, these polypeptides assemble to form distinct complexes, each of which sediments at approximately 20 S. On the basis of these observations, we hypothesize that there is a conserved neuronal function for a distinct form of the dynactin complex that cannot bind directly to cellular microtubules.

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Human beta-amyloid precursor protein (beta APP) has been targeted to transgenic neurons using synapsin I promoter-based chimeric transgenes. Native human beta APP was introduced as well as beta APP containing mutations genetically linked to familial Alzheimer's disease (AD) and to hereditary cerebral hemorrhage with amyloidosis-Dutch type. In mouse brain, human beta APP RNA was up to 60% as abundant as total endogenous beta APP RNA. Human beta APP gene expression was most abundant in the CA subfields of the hippocampus and in the piriform cortex. Correct processing of human beta APP at the beta-secretase cleavage site was demonstrated in transgenic mouse brains. Despite a 40% increase in total beta APP immunoreactivity in lines expressing mutant human beta APP, no evidence of amyloid deposition was found in brains of mice up to 14 months in age. Higher levels of mutant human beta APP, increased age, or other factors may be necessary to elicit beta-amyloid-related neuropathologies in the rodent brain.

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A rapid and nearly quantitative method for the direct analysis of steady-state mRNA levels in microgram quantities of frozen mammalian brain is described. Briefly, tissue punches 0.5-1.0 mm in diameter were sampled from 250-microns-thick cryostat sections of rat brain (approximately 50-200 micrograms tissue). The samples were homogenized in 50 microliters of a denaturing gel loading buffer and applied directly to a 2.2 M formaldehyde-agarose gel for electrophoresis and subsequent RNA blot analysis. The method is extremely rapid, results in excellent recovery of intact RNA, and allows the direct assay of mRNA levels in discrete subregions of the mammalian brain.

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The phosphoprotein synapsin I is expressed exclusively in neuronal cells. We are interested in elucidating the promoter sequences involved in cell type-specific expression of the synapsin I gene. The PC12 cell line expresses the 3.4 kb and 4.5 kb synapsin I mRNAs and is used to analyze cell type-specific gene expression. A series of deletion fragments of the rat synapsin I gene promoter were fused to the promoterless reporter gene encoding bacterial chloramphenicol acetyltransferase (CAT) for transfection analysis in PC12 cells and in HeLa cells, which do not express the gene. A -349 bp to +110 bp rat synapsin I promoter fragment contains a positive regulator, shown to be 33-times more active in PC12 cells than HeLa cells. Transfection of reporter plasmids containing up to 4.4 kb of rat synapsin I gene promoter sequences exhibit significantly reduced CAT activity in PC12 cells. The reduction in CAT expression was attributed to a negative regulator located between -349 bp and -1341 bp in the rat synapsin I promoter. Our results suggest that both positive and negative-acting sequence elements regulate cell type-specific expression of the rat synapsin I gene.

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Expression of the cloned rat growth hormone gene was studied in transfected CV-1 cells. Cell lines expressing rat growth hormone synthesized and secreted the 22 kilodalton form and the 20 kilodalton variant. The results confirm that the 20 kilodalton variant arises from an alternatively spliced mRNA rather than expression from a variant gene in rat. The 5' end of the mRNA from expressing cell lines was located upstream of the normal initiation site in rat. Transcription initiated within a 5' flanking AT rich sequence. Results presented indicate that transcription from normally silent promoter or promoter-like sequences dictated rat growth hormone gene expression in transfected cells. Finally, no hormone was expressed by CV-1 cells transfected with a plasmid containing the rat growth hormone gene in the same transcriptional reading frame as the neomycin resistance gene of pSV2neo indicating an effect of cloning orientation on expression in CV-1 cells.

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The characterization of a 20 kilodalton (20 kD) variant of rat growth hormone is reported. The 20 kD variant from rat pituitary gland extracts was identified on Western immunoblots of polyacrylamide gels. It was also shown that pituitary tissue maintained in culture secretes the 20 kD form. A rat growth hormone cDNA fragment was used as a probe in S1 nuclease mapping experiments of rat pituitary poly (A) mRNA to detect the presence of two growth hormone mRNAs in the rat pituitary gland. The protected mRNAs correspond to the predicted sizes that would encode the 22 kD and 20 kD forms of growth hormone. The site of variation between the mRNAs maps to a potential alternative 3' splice site in the 5' end of exon 3 of the coding sequence. The results support the hypothesis that the 20 kD variant in rat is the product of an mRNA alternatively spliced in exon 3, as is the case for the human growth hormone.

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