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ABSTRACT: The amyloid precursor protein (APP) plays a critical role in Alzheimer's disease (AD) pathogenesis. APP is proteolytically cleaved by β- and γ-secretases to generate the amyloid β-protein (Aβ), the core protein component of senile plaques in AD. It is also cleaved by α-secretase to release the large soluble APP (sAPP) luminal domain that has been shown to exhibit trophic properties. Increasing evidence points to the development of synaptic deficits and dendritic spine loss prior to deposition of amyloid in transgenic mouse models that overexpress APP and Aβ peptides. The consequence of loss of APP, however, is unsettled. In this study, we investigated whether APP itself plays a role in regulating synaptic structure and function using an APP knock-out (APP-/-) mouse model. We examined dendritic spines in primary cultures of hippocampal neurons and CA1 neurons of hippocampus from APP-/- mice. In the cultured neurons, there was a significant decrease (~35%) in spine density in neurons derived from APP-/- mice compared to littermate control neurons that were partially restored with sAPPα-conditioned medium. In APP-/- mice in vivo, spine numbers were also significantly reduced but by a smaller magnitude (~15%). Furthermore, apical dendritic length and dendritic arborization were markedly diminished in hippocampal neurons. These abnormalities in neuronal morphology were accompanied by reduction in long-term potentiation. Strikingly, all these changes in vivo were only seen in mice that were 12-15months in age but not in younger animals. We propose that APP, specifically sAPP, is necessary for the maintenance of dendritic integrity in the hippocampus in an age-associated manner. Finally, these age-related changes may contribute to AD pathology independent of Aβ-mediated synaptic toxicity.
Molecular and Cellular Neuroscience 08/2012; 51(1-2):43-52. · 3.66 Impact Factor
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ABSTRACT: Amyloid precursor protein (APP), the parent molecule to amyloid β peptide, is part of a larger gene family with two mammalian homologues, amyloid precursor-like protein 1 (APLP1) and amyloid precursor-like protein 2 (APLP2). Initial knock-out studies demonstrated that while single APP family gene deletions produced relatively mild phenotypes, deficiency of APLP2 and one other member of the gene family resulted in perinatal lethality, suggesting vital roles masked by functional redundancy of the other homologues. Because of the importance of APP in Alzheimer's disease, the vast majority of studies to date have concentrated on the neuronal functions of APP, leaving limited data on its homologues. APLP2 is of particular interest as it contains high sequence homology with APP, is processed similarly, is expressed in overlapping spatial and temporal patterns, and is obligatory for lethality when combined with deficiency of either APLP1 or APP but does not contain the toxic amyloid β sequence. Here we sought to test the role of APLP2 on neuronal structure and function using a combined approach involving in vitro and in vivo techniques in young and aged animals. Surprisingly, we found that unlike APP, APLP2 appears not to be essential for maintenance of dendritic structure, spine density, or synaptic function. Thus, there is clear divergence in the functional redundancy between APP and APLP2.
Molecular and Cellular Neuroscience 02/2012; 49(4):448-55. · 3.66 Impact Factor
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ABSTRACT: The amyloid precursor protein (APP) is part of a larger gene family, which has been found to form homo- or heterotypic complexes with its homologues, whereby the exact molecular mechanism and origin of dimer formation remains elusive. In order to assess the cellular location of dimerization, we have generated a cell culture model system in CHO-K1 cells, stably expressing human APP, harboring dilysine-based organelle sorting motifs [KKAA-endoplasmic reticulum (ER); KKFF-Golgi], accomplishing retention within early secretory compartments. We show that APP exists as disulfide-bonded dimers upon ER retention after it was isolated from cells, and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. In contrast, strong denaturing and reducing conditions, or deletion of the E1 domain, resulted in the disappearance of those dimers. Thus we provide first evidence that a fraction of APP can associate via intermolecular disulfide bonds, likely generated between cysteines located in the extracellular E1 domain. We particularly visualize APP dimerization itself and identified the ER as subcellular compartment of its origin using biochemical or split GFP approaches. Interestingly, we also found that minor amounts of SDS-resistant APP dimers were located to the cell surface, revealing that once generated in the oxidative environment of the ER, dimers remained stably associated during transport. In addition, we show that APP isoforms encompassing the Kunitz-type protease inhibitor (KPI) domain exhibit a strongly reduced ability to form cis-directed dimers in the ER, whereas trans-mediated cell aggregation of Drosophila Schneider S2-cells was isoform independent. Thus, suggesting that steric properties of KPI-APP might be the cause for weaker cis-interaction in the ER, compared to APP695. Finally, we provide evidence that APP/APLP1 heterointeractions are likewise initiated in the ER.
Cellular and Molecular Life Sciences CMLS 11/2011; 69(8):1353-75. · 6.57 Impact Factor
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ABSTRACT: The amyloid precursor protein (APP) is one of the key proteins in Alzheimer's disease (AD), as it is the precursor of amyloid β (Aβ) peptides accumulating in amyloid plaques. The processing of APP and the pathogenic features of especially Aβ oligomers have been analyzed in detail. Remarkably, there is accumulating evidence from cell biological and structural studies suggesting that APP and its mammalian homologs, the amyloid precursor-like proteins (APLP1 and APLP2), participate under physiological conditions via trans-cellular dimerization in synaptogenesis. This offers the possibility that loss of synapses in AD might be partially explained by dysfunction of APP/APLPs cell adhesion properties. In this review, structural characteristics of APP trans-cellular interaction will be placed critically in context with its putative physiological functions focusing on cell adhesion and synaptogenesis.
Experimental Brain Research 09/2011; 217(3-4):389-95. · 2.39 Impact Factor
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Thomas L Kukar,
Thomas B Ladd,
Paul Robertson,
Sean A Pintchovski,
Brenda Moore,
Maralyssa A Bann,
Zhao Ren,
Karen Jansen-West,
Kim Malphrus, Simone Eggert,
Hiroko Maruyama,
Barbara A Cottrell,
Pritam Das,
Guriqbal S Basi,
Edward H Koo,
Todd E Golde
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ABSTRACT: γ-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid β precursor protein (APP) to release the amyloid β peptide (Aβ). Aβ is the primary component of senile plaques in Alzheimer's disease (AD), and its mechanism of production has been studied intensely. γ-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing Aβ and the APP intracellular domain (AICD), referred to as γ and ε, respectively. The heterogeneous nature of the γ cleavage that produces various Aβ peptides is highly relevant to AD, as increased production of Aβ 1-42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to Aβ) that plays a critical role in determining the final length of Aβ peptides released by γ-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of γ-secretase cleavage from 1-40 to 1-33 without significant changes to ε cleavage. These results further support a model where ε cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of γ-secretase.
Journal of Biological Chemistry 08/2011; 286(46):39804-12. · 4.77 Impact Factor
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Thomas L. Kukar,
Thomas B. Ladd,
Paul Robertson,
Sean A. Pintchovski,
Brenda Moore,
Maralyssa A. Bann,
Zhao Ren,
Karen Jansen-West,
Kim Malphrus, Simone Eggert,
Hiroko Maruyama,
Barbara A. Cottrell,
Pritam Das,
Guriqbal S. Basi,
Edward H. Koo,
Todd E. Golde
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ABSTRACT: γ-Secretase is a multi-protein intramembrane-cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the
amyloid β precursor protein (APP) to release the amyloid beta peptide (Aβ). Aβ is the primary component of senile plaques
in Alzheimers disease (AD), and its mechanism of production has been intensely studied. γ-Secretase executes multiple cleavages
within the transmembrane domain of APP with cleavages producing Aβ and the APP intracellular domain (AICD) referred to as
γ and ϵ, respectively. The heterogeneous nature of the γ-cleavage that produces various Aβ peptides is highly relevant to
AD, as increased production of Aβ 1-42 is genetically and biochemically linked to the development of AD. We have identified
an amino acid in the juxtamemembrane region of APP, lysine 624 based on APP695 numbering (position 28 relative to Aβ) that
plays a critical role in determining the final length of Aβ peptides released by γ-secretase. Mutation of this lysine to
alanine (K28A) shifts the primary site of γ-secretase cleavage from 1-40 to 1-33, without significant changes to ϵ-cleavage.
These results further support a model where ϵ-cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane
fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane
domain limit processivity of γ-Secretase
Journal of Biological Chemistry 08/2011; · 4.77 Impact Factor
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ABSTRACT: The amyloid precursor protein (APP) plays a central role in Alzheimer disease (AD) pathogenesis because sequential cleavages by beta- and gamma-secretase lead to the generation of the amyloid-beta (Abeta) peptide, a key constituent in the amyloid plaques present in brains of AD individuals. In several studies APP has recently been shown to form homodimers, and this event appears to influence Abeta generation. However, these studies have relied on APP mutations within the Abeta sequence itself that may affect APP processing by interfering with secretase cleavages independent of dimerization. Therefore, the impact of APP dimerization on Abeta production remains unclear. To address this question, we compared the approach of constitutive cysteine-induced APP dimerization with a regulatable dimerization system that does not require the introduction of mutations within the Abeta sequence. To this end we generated an APP chimeric molecule by fusing a domain of the FK506-binding protein (FKBP) to the C terminus of APP. The addition of the synthetic membrane-permeant drug AP20187 induces rapid dimerization of the APP-FKBP chimera. Using this system we were able to induce up to 70% APP dimers. Our results showed that controlled homodimerization of APP-FKBP leads to a 50% reduction in total Abeta levels in transfected N2a cells. Similar results were obtained with the direct precursor of beta-secretase cleavage, C99/SPA4CT-FKBP. Furthermore, there was no modulation of different Abeta peptide species after APP dimerization in this system. Taken together, our results suggest that APP dimerization can directly affect gamma-secretase processing and that dimerization is not required for Abeta production.
Journal of Biological Chemistry 08/2009; 284(42):28943-52. · 4.77 Impact Factor
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ABSTRACT: Understanding the intracellular transport of the beta-amyloid precursor protein (APP) is a major key to elucidate the regulation of APP processing and thus beta-amyloid peptide generation in Alzheimer disease pathogenesis. APP and its two paralogues, APLP1 and APLP2 (APLPs), are processed in a very similar manner by the same protease activities. A putative candidate involved in APP transport is protein interacting with APP tail 1 (PAT1), which was reported to interact with the APP intracellular domain. We show that PAT1a, which is 99.0% identical to PAT1, binds to APP, APLP1, and APLP2 in vivo and describe their co-localization in trans-Golgi network vesicles or endosomes in primary neurons. We further demonstrate a direct interaction of PAT1a with the basolateral sorting signal of APP/APLPs. Moreover, we provide evidence for a direct role of PAT1a in APP/APLP transport as overexpression or RNA interference-mediated knockdown of PAT1a modulates APP/APLPs levels at the cell surface. Finally, we show that PAT1a promotes APP/APLPs processing, resulting in increased secretion of beta-amyloid peptide. Taken together, our data establish PAT1a as a functional link between APP/APLPs transport and their processing.
Journal of Biological Chemistry 01/2007; 281(52):40114-23. · 4.77 Impact Factor
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Peter Soba, Simone Eggert,
Katja Wagner,
Hanswalter Zentgraf,
Katjuscha Siehl,
Sylvia Kreger,
Alexander Löwer,
Andreas Langer,
Gunter Merdes,
Renato Paro,
Colin L Masters,
Ulrike Müller,
Stefan Kins,
Konrad Beyreuther
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ABSTRACT: The amyloid precursor protein (APP) plays a central role in Alzheimer's disease, but its physiological function and that of its mammalian paralogs, the amyloid precursor-like proteins 1 and 2 (APLPs), is still poorly understood. APP has been proposed to form dimers, a process that could promote cell adhesion via trans-dimerization. We investigated the dimerization and cell adhesion properties of APP/APLPs and provide evidence that all three paralogs are capable of forming homo- and heterocomplexes. Moreover, we show that trans-interaction of APP family proteins promotes cell-cell adhesion in a homo- and heterotypic fashion and that endogenous APLP2 is required for cell-cell adhesion in mouse embryonic fibroblasts. We further demonstrate interaction of all the three APP family members in mouse brain, genetic interdependence, and molecular interaction of APP and APLPs in synaptically enriched membrane compartments. Together, our results provide evidence that homo- and heterocomplexes of APP/APLPs promote trans-cellular adhesion in vivo.
The EMBO Journal 11/2005; 24(20):3624-34. · 9.20 Impact Factor
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ABSTRACT: Amyloid precursor protein (APP) processing is of major interest in Alzheimer's disease research, since sequential cleavages by beta- and gamma-secretase lead to the formation of the 4-kDa amyloid Abeta protein peptide that accumulates in Alzheimer's disease brain. The processing of APP involves proteolytic conversion by different secretases leading to alpha-, beta-, gamma-, delta-, and epsilon-cleavages. Since modulation of these cleavages represents a rational therapeutic approach to control amyloid formation, its interference with the processing of the members of the APP gene family is of considerable importance. By using C-terminally tagged constructs of APLP-1 and APLP-2 and the untagged proteins, we have characterized their proteolytic C-terminal fragments produced in stably transfected SH-SY5Y cells. Pharmacological manipulation with specific protease inhibitors revealed that both homologues are processed by alpha- and gamma-secretase-like cleavages, and that their intracellular domains can be released by cleavage at epsilon-sites. APLP-2 processing appears to be the most elaborate and to involve alternative cleavage sites. We show that APLP-1 is the only member of the APP gene family for which processing can be influenced by N-glycosylation. Additionally, we were able to detect p3-like fragments of APLP-1 and p3-like and Abeta-like fragments of APLP-2 in the media of stably transfected SH-SY5Y cells.
Journal of Biological Chemistry 05/2004; 279(18):18146-56. · 4.77 Impact Factor
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ABSTRACT: Amyloid precursor protein (APP) processing is of major interest in Alzheimer's disease research, since sequential cleavages
by β- and γ-secretase lead to the formation of the 4-kDa amyloid Aβ protein peptide that accumulates in Alzheimer's disease
brain. The processing of APP involves proteolytic conversion by different secretases leading to α-, β-, γ-, δ-, and ϵ-cleavages.
Since modulation of these cleavages represents a rational therapeutic approach to control amyloid formation, its interference
with the processing of the members of the APP gene family is of considerable importance. By using C-terminally tagged constructs
of APLP-1 and APLP-2 and the untagged proteins, we have characterized their proteolytic C-terminal fragments produced in stably
transfected SH-SY5Y cells. Pharmacological manipulation with specific protease inhibitors revealed that both homologues are
processed by α- and γ-secretase-like cleavages, and that their intracellular domains can be released by cleavage at ϵ-sites.
APLP-2 processing appears to be the most elaborate and to involve alternative cleavage sites. We show that APLP-1 is the only
member of the APP gene family for which processing can be influenced by N-glycosylation. Additionally, we were able to detect p3-like fragments of APLP-1 and p3-like and Aβ-like fragments of APLP-2
in the media of stably transfected SH-SY5Y cells.
Journal of Biological Chemistry 04/2004; 279(18):18146-18156. · 4.77 Impact Factor
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ABSTRACT: Regulated intramembrane proteolysis (RIP) of the amyloid precursor protein (APP) produces amyloid beta-protein (Abeta), the probable causative agent of Alzheimer's disease (AD), and is therefore an important target for therapeutic intervention. However, there is a burgeoning consensus that gamma-secretase, one of the proteases that generates Abeta, is also critical for the signal transduction of APP and a growing list of other receptors. APP is a member of a gene family that includes two amyloid precursor-like proteins, APLP1 and APLP2. Although APP and the APLPs undergo similar proteolytic processing, there is little information about the role of their gamma-secretase-generated intracellular domains (ICDs). Here, we show that APLP1 and 2 undergo presenilin-dependent RIP similar to APP, resulting in the release of a approximately 6 kDa ICD for each protein. Each of the ICDs are degraded by an insulin degrading enzyme-like activity, but they can be stabilized by members of the FE65 family and translocate to the nucleus. Given that modulation of APP processing is a therapeutic target and that the APLPs are processed in a manner similar to APP, any strategy aimed at altering APP proteolysis will have to take into account possible effects on signaling by APLP 1 and 2.
Biochemistry 07/2003; 42(22):6664-73. · 3.42 Impact Factor
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ABSTRACT: Proteolytic processing of the transmembrane domain of the amyloid precursor protein (APP) is a key component of Alzheimer's disease pathogenesis. Using C-terminally tagged APP derivatives, we have identified by amino-terminal sequencing a novel cleavage site of APP, at Leu-49, distal to the gamma-secretase site. This was termed -cleavage. Brefeldin A treatment and pulse-chase experiments indicate that this cleavage occurs late in the secretory pathway. The level of -cleavage is decreased by expression of presenilin-1 mutants known to impair Abeta formation, and it is sensitive to the gamma-secretase inhibitors MDL28170 and L-685,458. Remarkably, it shares similarities with site 3 cleavage of Notch-1: membrane topology, cleavage before a valine, dependence on presenilins, and inhibition profile.
Biochemistry 03/2002; 41(8):2825-35. · 3.42 Impact Factor
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Peter Soba, Simone Eggert,
Katja Wagner,
Hanswalter Zentgraf,
Katjuscha Siehl,
Sylvia Kreger,
Alexander Löwer,
Andreas Langer,
Gunter Merdes,
Renato Paro,
Colin L Masters,
Ulrike Müller,
Stefan Kins,
Konrad Beyreuther
