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Our understanding of Alzheimer's disease pathogenesis is currently limited by difficulties in obtaining live neurons from patients and the inability to model the sporadic form of the disease. It may be possible to overcome these challenges by reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). Here we reprogrammed primary fibroblasts from two patients with familial Alzheimer's disease, both caused by a duplication of the amyloid-P precursor protein gene (1) (APP termed [APP.sup.Dp]), two with sporadic Alzheimer's disease (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines. Neurons from differentiated cultures were purified with fluorescence-activated cell sorting and characterized. Purified cultures contained more than 90% neurons, clustered with fetal brain messenger RNA samples by microarray criteria, and could form functional synaptic contacts. Virtually all cells exhibited normal electrophysiological activity. Relative to controls, iPSC-derived, purified neurons from the two [APP.sup.Dp] patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-β(1-40), phospho-tau(Thr231) and active glycogen synthase kinase-3β (aGSK-3β). Neurons from [APP.sup.Dp] and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with β-secretase inhibitors, but not γ-secretase inhibitors, caused significant reductions in phospho-Tau(Thr231) and aGSK-3β levels. These results suggest a direct relationship between APP proteolytic processing, but not amyloid-β, in GSK-3β activation and tau phosphorylation in human neurons. Additionally, we observed that neurons with the genome of one sAD patient exhibited the phenotypes seen in familial Alzheimer's disease samples. More generally, we demonstrate that iPSC technology can be used to observe phenotypes relevant to Alzheimer's disease, even though it can take decades for overt disease to manifest in patients., Alzheimer's disease is a common neurodegenerative disorder, defined post mortem by the increased presence of amyloid plaques and neurofibrillary tangles in the brain (2). Amyloid plaques are extracellular deposits consisting [...]

Selective lowering of Aβ42 levels (the 42-residue isoform of the amyloid-β peptide) with small-molecule γ-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer's [...]

Increased A[beta]42 production has been linked to the development of Alzheimer disease. We now identify a number of compounds that raise A[beta]42. Among the more potent A[beta]42-raising agents identified are fenofibrate, an antilipidemic agent, and celecoxib, a COX-2-selective NSAID. Many COX-2-selective NSAIDs tested raised A[beta]42, including multiple COX-2-selective derivatives of two A[beta]42-lowering NSAIDs. Compounds devoid of COX activity and the endogenous isoprenoids FPP and GGPP also raised A[beta]42. These compounds seem to target the [gamma]-secretase complex, increasing [gamma]-secretase-catalyzed production of A[beta]42 in vitro. Short-term in vivo studies show that two A[beta]42-raising compounds increase A[beta]42 levels in the brains of mice. The elevations in A[beta]42 by these compounds are comparable to the increases in A[beta]42 induced by Alzheimer disease-causing mutations in the genes encoding amyloid [beta] protein precursor and presenilins, raising the possibility that exogenous compounds or naturally occurring isoprenoids might increase A[beta]42 production in humans., Author(s): Thomas Kukar [1, 5]; Michael Paul Murphy [1, 4, 5]; Jason L Eriksen [1, 5]; Sarah A Sagi [2, 5]; Sascha Weggen [2, 4]; Tawnya E Smith [1]; Thomas [...]

Research has been conducted on gene preselin 1 required for intramembrane proteolysis of amyloid precursor protein. Preselin's functions as a scaffold coupling beta-catenin phosphorylation via kinase activities independent of Axin/casein kinase (sub)alpha complex are discussed.

Research has been conducted on the N-CoR corepressor complex. Results indicate that interleukin-1(beta) is responsible for the nuclear export of this complex and leads to the NF-(kappa)B -regulated gene subset derepression.

To differentiate multiple activities of presenilin 1 (PS1), we generated transgenic mice expressing two human PS1 alleles: one with the aspartate to alanine mutation at residue 257 (hPS1D257A) that impairs the proteolytic activity of PS1, and the other deleting amino acids 340-371 of the hydrophilic loop sequence (hPS1[DELTA]cat) essential for [beta]-catenin interaction. We show here that although hPS1[DELTA]cat is fully competent in rescuing the PS1-null lethal phenotype, hPS1D257A does not exhibit developmental activity. hPS1D257A also leads to the concurrent loss of the proteolytic processing of Notch and [beta]-amyloid precursor protein (APP) and the generation of [beta]-amyloid peptides (A[beta]). Further, by measuring the levels of endogenous A[[beta].sub.x-40] and A[[beta].sub.x-42] in primary neuronal cultures, we confirmed the concept that PS1 is indispensable for the production of secreted A[beta].

Author(s): Sascha Weggen [1]; Jason L. Eriksen [2]; Pritam Das [2]; Sarah A. Sagi [1]; Rong Wang [3]; Claus U. Pietrzik [1]; Kirk A. Findlay [2]; Tawnya E. Smith [2]; [...]

The amyloid [beta]-protein precursor gives rise to the amyloid [beta]-protein, the principal constituent of senile plaques and a cytotoxic fragment involved in the pathogenesis of Alzheimer disease. Here we show that amyloid [beta]-protein precursor was proteolytically cleaved by caspases in the C terminus to generate a second unrelated peptide, called C31. The resultant C31 peptide was a potent inducer of apoptosis. Both caspase-cleaved amyloid [beta]-protein precursor and activated caspase-9 were present in brains of Alzheimer disease patients but not in control brains. These findings indicate the possibility that caspase cleavage of amyloid [beta]-protein precursor with the generation of C31 may be involved in the neuronal death associated with Alzheimer disease., Author(s): Daniel C. Lu [1, 2, 5]; Shahrooz Rabizadeh [1, 3, 5]; Sreeganga Chandra [2]; Rana F. Shayya [2]; Lisa M. Ellerby [1, 4]; Xin Ye [1]; Guy S. Salvesen [...]

A study was conducted to investigate the sub-cellular localization and mechanisms of presinilins in regulating the processing of the beta-amyloid precursor protein to the amyloid beta-protein which is thought to play a crucial role in the pathogenesis of Alzheimer's disease. The results of the study, which are discussed, will be important for designing therapeutic compounds to selectively inhibit the production of highly amyloidogenic 42-residue form of the amyloid beta-protein.

Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes increase the production of the highly amyloidogenic 42-residue form of amyloid [Beta]protein (A[[Beta].sub.42]) in a variety of cell lines and transgenic mice. To elucidate the molecular mechanism of this effect, wild-type (wt) or mutant PS1 and PS2 genes were stably transfected into Chinese hamster ovary cells expressing endogenous or transfected [Beta]-amyloid precursor protein (APP). By immunoprecipitation/Western blot analysis, APP was consistently found to coimmunoprecipitate with PS1 or PS2 proteins. Several distinct PS1, PS2, or APP antibodies precipitated PS-APP complexes that were detectable by blotting with either APP or PS antibodies. Importantly, complex formation could be detected at endogenous protein levels in nontransfected cells. In various Chinese hamster ovary cell lines, the amounts of APP coprecipitated by PS antibodies were proportional to the expression levels of both APP and PS. APP-PS complexes also were recovered from human 293 and HS683 cells. Full maturation of APP was not required for the interaction; most APP molecules complexed with PS were solely N-glycosylated. Treatment of cells with brefeldin A or incubation at 20 [degrees] C did not block complex formation, suggesting that the association between APP and PS occurs in part in the endoplasmic reticulum. Complex formation was detected for both wt and mutant PS and APP proteins. Deletion of the APP C-terminal domain did not abrogate complex formation, suggesting that the interaction does not occur in the cytoplasmic domains of the proteins. Our results demonstrate that wt and mutant PS1 and PS2 proteins form complexes with APP in living cells, strongly supporting the hypothesis that mutant PS interacts with APP in a way that enhances the intramembranous proteolysis of the latter by a [Gamma]-secretase cleaving at A[[Beta].sub.42].

In mature hippocampal neurons, beta-amyloid precursor protein (beta-PP) is present mainly on the membrane surface of axons and perikarya, and in terminal axons it is located internally from where it is transported back to the perikaryon. Alzheimer's disease is a neurodegenerative disease in which there is abnormal deposition of amyloid beta-protein in the parenchyma of the brain and on the walls of meningeal and cortical blood vessels. Proteolysis of beta-PP results in the formation of amyloid beta-protein.

The use of beta-amyloid precursor protein (beta-APP) cDNA constructs having a variety of deletions and substitutions helps analyze the sorting signals in the beta-APP cytoplasmic tail. The polarized sorting of beta-APP and its proteolytic products in MDCK cells are regulated by two signals that are independent of each other. One of the two pathways localizes both surface-bound beta-APP and part of the secreted amyloid-beta protein, being influenced partly by cytoplasmic signals and the tyrosine residue 653 of beta-APP. Beta-APP is oriented toward a basolateral sorting pathway in the second mechanism, which involves specialized signals within the ectodomain of beta-APP.

Secreted beta-amyloid precursor protein (beta-APP) promotes the function of mitogen-activated protein kinase and increases the microtubule-associated protein tau phosphorylation in Alzheimer disease. Beta-APP does not enhance tau phosphorylation in ras dominant inhibitory cell line. The data suggests a ligation between the tau phosphorylation phase and secreted beta-APP levels.

A study of the sorting of beta-amyloid precursor protein (beta-APP) in Madin Darby canine kidney cells, epithelial cells which have physiologically unique apical and basolateral plasma membranes, formed part of analyses of beta-APP trafficking and processing. Polarized secretion was evident in the proteolytic processing products of beta-APP. The polarized sorting of beta-APP is followed by the production of the amyloidogenic Abeta-peptide.

beta/A4 peptide generation was decreased by 50 to 80% when cells were treated with phorbol dibutyrate which stimulates protein kinese c and/or okadaic acid, which restricts protein phosphates 1 and 2A. beta/A4 was generated in cells in which different types of amyloid protein precursor (APP) connected with familial Alzheimer disease, one of which displayed enhanced beta/A4 peptide generation in cultured cells, were lowered by phorbol dibutyrate. Increasing the APP metabolism by a nonamyloidgenic secretory pathway will lower beta/A4 peptide generation in a model which has been developed.

The utilization of amyloid beta-protein as a neurite outgrowth substrate in a well-established cultured peripheral sensory neuron model was investigated. Six synthetic peptides were used on cultured rat peripheral sensory neuron tissues and subjected to antibody blocking by polyclonal antisera. The normal physiological role of amyloid beta-protein as a complexing brain extracellular matrix was established.

A study was done on the expression of the beta-amyloid precursor protein (betaAPP) during retinoic acid induced neuronal differentiation of P19 murine embryonal carcinoma cells. It was shown that betaAPP increases selectively and progressively during neuronal differentiation but are not processed significantly by secretory cleavage. The protein is thus retained in the membrane and serve as potential substrates for amyloidogenesis in Alzheimer's disease.

The amyloid beta-peptide (A-beta) fragment that is linked to Alzheimer's disease has been identified in 4K and truncated form from cultures of primary, untransfected and beta-amyloid precursor protein-transfected brain cells. The production of A-beta in soluble form in vitro and in vivo as a part of normal metabolism shows that existing concepts of Alzheimer's disease pathology must be re-evaluated. Moreover, the new knowledge of A-beta's functions may lead to drugs that prevent or treat Alzheimer's disease by stopping the secretion of A-beta.

Presenilin 1 (PS1) is required for the proteolytic processing of Notch and the [Beta]-amyloid precursor protein (APP), molecules that play pivotal roles in cell-fate determination during development and Alzheimer's disease pathogenesis, respectively. In addition, PS1 interacts with [Beta]-catenin and promotes its turnover through independent mechanisms. Consistent with this activity, we report here that PS1 is important in controlling epidermal cell proliferation in vivo. PS1 knockout mice that are rescued through neuronal expression of human PS1 transgene develop spontaneous skin cancers. PSl-null keratinocytes exhibit higher cytosolic [Beta]-catenin and [Beta]-catenin/lymphoid enhancer factor-1/T cell factor ([Beta]-catenin/ LEF)-mediated signaling. This effect can be reversed by reintroducing wild-type PS1, but not a PS1 mutant active in Notch processing but defective in [Beta]-catenin binding. Nuclear [Beta]-catenin protein can be detected in tumors. Elevated [Beta]-catenin/LEF signaling is correlated with activation of its downstream target cyclin D1 and accelerated entry from [G.sub.1] into S phase of the cell cycle. This report demonstrates a function of PS1 in adult tissues, and our analysis suggests that deregulation of [Beta]-catenin pathway contributes to the skin tumor phenotype.

In addition to its documented role in the proteolytic processing of Notch-1 and the [Beta]-amyloid precursor protein, presenilin 1 (PS1) associates with [Beta]-catenin. In this study, we show that this interaction plays a critical role in regulating [Beta]-catenin/T Cell Factor/Lymphoid Enhancer Factor-1 (LEF) signaling. PS1 deficiency results in accumulation of cytosolic [Beta]-catenin, leading to a [Beta]-catenin/LEF-dependent increase in cyclin D1 transcription and accelerated entry into the S phase of the cell cycle. Conversely, PS1 specifically represses LEF-dependent transcription in a dose-dependent manner. The hyperproliferative response can be reversed by reintroducing PS1 expression or overexpressing axin, but not a PS1 mutant that does not bind [Beta]-catenin (PS1[Delta]cat) or by two different familial Alzheimer's disease mutants. In contrast, PS1[Delta]cat restores Notch-1 proteolytic cleavage and A[Beta] generation in PS1-deficient cells, indicating that PS1 function in modulating [Beta]-catenin levels can be separated from its roles in facilitating [Gamma]-secretase cleavage of [Beta]-amyloid precursor protein and in Notch-1 signaling. Finally, we show an altered response to Wnt signaling and impaired ubiquitination of [Beta]-catenin in the absence of PS1, a phenotype that may account for the increased stability in PS1-deficient cells. Thus, PS1 adds to the molecules that are known to regulate the rapid turnover of [Beta]-catenin. Key words: presenilin * [Beta]-catenin * Notch-1 * [Beta]-amyloid precursor protein * cyclin D1

Recent biophysical experiments have proposed the possibility that amyloid fibril formation may be somewhat responsible for age-related neurodegenerative disorders. This observation was prompted by the discovery that abnormal protein aggregation characterizes majority of these diseases. Some genetic studies have even supported findings that fibrillization contributes to cell death. It is hoped that this new amyloid hypothesis will pave the way for the discovery of therapeutic approaches for the treatment of neurodegenerative diseases.