We have carried out an analysis of amplified DNA sequences present in a tumorigenic mouse cell line, designated 3T3DM, to determine if the presence of cellular transforming activity is correlated with the elevated expression of any of the amplified genes. These studies utilized a selection protocol that allowed for DNA sequence amplification after the introduction of each gene into non‐transformed recipient cells. Cell lines obtained from this selection protocol were assayed for tumorigenicity in nude mice. The results provided evidence that a gene, mdm2, that is amplified more than 50‐fold in the 3T3DM cell line, induces tumorigenicity when experimentally overexpressed in NIH3T3 cells and in Rat2 cells. Analysis of the predicted amino acid composition of the mdm2 product(s) revealed features similar to those that have been shown to be functionally significant in certain DNA binding proteins/transcriptional activators. These include two potential metal binding motifs and a negatively charged domain rich in acidic amino acid residues. Overall, the data support the conclusion that mdm2 represents an evolutionarily conserved gene with tumorigenic potential and a predicted role in mechanisms of cellular growth control.

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.

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 ␤-peptide (A␤) domain and expressing the Swedish familial Alzheimer's disease mutations. These mice express endogenous levels of APP holoprotein and abundant human A␤. Increased dietary cholesterol led to significant reductions in brain levels of secreted APP derivatives, including sAPP␣, sAPP␤, A␤1-40, and A␤1-42, while having little to no effect on cellassociated species, including full-length APP and the COOH-terminal APP processing derivatives. The changes in levels of sAPP and A␤ 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 A␤ 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.Alzheimer's disease (AD) 1 pathology includes extracellular amyloid deposits, intracellular neurofibrillary tangles, synaptic loss, and neuronal death (for a review, see Ref. 1). Alterations in the production or processing of APP have been implicated in the etiology of at least some forms of AD (2, 3). Multiple pathways for APP processing have been described, including a nonamyloidogenic pathway in which a putative ␣-secretase cleaves within the A␤ domain (4, 5), resulting in the formation of a secreted NH 2 -terminal fragment, sAPP␣, and a cell-associated 9-kDa COOH-terminal derivative. Another fraction of APP is processed along an amyloidogenic pathway in which cleavage by a putative ␤-secretase at the NH 2 terminus of the A␤ domain results in the formation of a secreted NH 2 -terminal fragment, sAPP␤ (6), and a cell-associated 12-kDa COOH-terminal derivative that may be the immediate precursor of A␤ (7,8). Cleavage of APP by both ␤-secretase and ␥-secretase results in formation of A␤, 40 or 42 amino acids in length (9, 10), that is found deposited in extracellular amyloid plaques in the AD brain (1, 11).In order to elucidate mechanisms of APP processing and A␤ generation in vivo, an animal model was developed by gene targeting that converted the mouse A␤ sequence to human and incorporated the Swedish familial Alzheimer's disease mutations (12). Enhanced amyloidogenic APP processing by the Swedish mutations, resulting in higher level A␤ production has been well documented in cell culture systems (13-15) and in the APP gene-targeted mice (12). These mice are well suited for investigating modulation of APP processing in vivo, because brain A␤ levels are nearly 10-fold above normal endogenous levels, thereby reducing the stringency for assays to detect A␤, particularly for the less abundant but more amyloidogenic 42-residue form. Further...

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The pathogenic mechanism linking presenilin-1 (PS-1) gene mutations to familial Alzheimer's disease (FAD) is uncertain, but has been proposed to include increased neuronal sensitivity to degeneration and enhanced amyloidogenic processing of the ␤-amyloid precursor protein (APP). We investigated this issue by using gene targeting with the Cre-lox system to introduce an FAD-linked P264L mutation into the endogenous mouse PS-1 gene, an approach that maintains normal regulatory controls over expression. Primary cortical neurons derived from PS-1 homozygous mutant knock-in mice exhibit basal neurodegeneration similar to their PS-1 wild-type counterparts. Staurosporine and A␤1-42 induce apoptosis, and neither the dose dependence nor maximal extent of cell death is altered by the PS-1 knock-in mutation. Similarly, glutamate-induced neuronal necrosis is unaffected by the PS-1P264L mutation. The lack of effect of the PS-1P264L mutation is confirmed by measures of basal-and toxin-induced caspase and calpain activation, biochemical indices of apoptotic and necrotic signaling, respectively. To analyze the influence of the PS-1P264L knock-in mutation on APP processing and the development of AD-type neuropathology, we created mouse lines carrying mutations in both PS-1 and APP. In contrast to the lack of effect on neuronal vulnerability, cortical neurons cultured from PS-1P264L homozygous mutant mice secrete A␤42 at an increased rate, whereas secretion of A␤40 is reduced. Moreover, the PS-1 knock-in mutation selectively increases A␤42 levels in the mouse brain and accelerates the onset of amyloid deposition and its attendant reactive gliosis, even as a single mutant allele. We conclude that expression of an FAD-linked mutant PS-1 at normal levels does not generally increase cortical neuronal sensitivity to degeneration. Instead, enhanced amyloidogenic processing of APP likely is critical to the pathogenesis of PS-1-linked FAD. Key words: presenilin; amyloid; plaque, neuronal necrosis; neuronal apoptosis; plaque; amyloid precursor protein; A␤; familial Alzheimer's Disease; gene targetingMutations in the ␤-amyloid precursor protein (APP), presenilin-1 (PS-1), and presenilin-2 (PS-2) genes are a leading cause of early onset familial Alzheimer's disease (FAD) and cosegregate with FAD in an autosomal dominant manner (Price et al., 1998;Selkoe, 1998). All forms of AD are characterized by loss of neurons and synapses in specific brain regions, and deposition of protein aggregates as A␤-containing amyloid plaques in the brain parenchyma, leptomeninges, and cerebrovasculature, and as tau-containing intraneuronal neurofibrillary tangles. At least two leading hypotheses have emerged for the pathogenic mechanisms of the mutations. According to the "amyloid cascade" hypothesis, APP and PS mutations promote formation from APP of the highly insoluble A␤42 variant, whose progressive aggregation triggers the amyloid and synaptic abnormalities and neuronal loss. It is supported by findings that all of the FAD-linked mutations examined so far i...