Disease Amyloid Precursor Protein Research Articles

The Alzheimer's Disease Amyloid Precursor Protein and its Neuritogenic Actions.

The Amyloid Precursor Protein (APP) is principally known and studied for its involvement in Alzheimer's disease as the source of the amyloid β peptide; however, its physiological actions within the nervous system are also important as it is involved in a range of neuronal activities, including neurogenesis, synaptic plasticity, neurite outgrowth, and neuroprotection. Of the different neuronal functions that APP can affect, some may be relevant to APP's role in Alzheimer's disease, while others can be primarily related to its physiological roles. This review will focus on APP's neuritogenic actions and surmise the key molecular mechanisms, as well as the structural and signaling requirements, which form the basis for APP's neuritogenic effects. Deciphering the normal function(s) of APP is valuable to properly understanding its role in health as well as Alzheimer's disease.

Identification of truncated C-terminal fragments of the Alzheimer's disease amyloid protein precursor derived from sequential proteolytic pathways.

Recent evidence supports the emerging hypothesis that the amyloid-β precursor protein C-terminal fragments (APP-CTFs) and dysregulations in both their qualitative and quantitative productions may actively and directly contribute to the neuronal toxicity in early phases of Alzheimer's disease (AD). These new findings revealed the urgent needs and gaps in better understanding the metabolism and full spectrum of APP-CTFs. In this study, we characterized by mass spectrometry the full patterns of APP-CTFs in different cell types and in the brain of an AD APPPS1 mouse model. In these systems, we first discovered a series of 71-80 amino acids long N-terminally truncated APP-CTFs of unknown functions. We next demonstrated that these N-terminally truncated APP-CTFs are sequentially produced by the proteolytic processing of APP-C80, by an as yet unidentified protease. Finally, these N-terminally truncated APP-CTFs are likely protein substrates recognized and processed by the γ-secretase complex, leading to the production of N-terminally truncated Aβ peptides. Together, our findings provide new insights into the metabolism of APP and offer potential new strategies to modulate the production of toxic Aβ peptides in AD.

Alzheimer's disease against peptides products of enzymatic cleavage APP protein: Biological, pathobiological and physico-chemical properties of fibrillating peptides.

Various peptides products of enzymatic cleavage of key for Alzheimer's disease Amyloid Precursor Protein (APP) are well known, but still are matter of scientific debate. The Aβ type products are especially challenging for experimental and medical research. This paper outlines several, still poorly known, biological and medical processes such as peptides biology, i.e., formation, biodistribution, translocation, transport and finally removal from brain compartments and body fluids like Intracellular Fluid (ICF), Cerebrospinal Fluid (CSF), Interstitial Fluid (ISF), blood serum or urine. In addition, the following studies concerning AD patients might prove challenging and simultaneously promising: peptides translocation through Blood-Brain - Barrier (BBB) and Blood-Cerebrospinal Fluid Barrier (BCSFB) and their removal from the brain according to a new concept of glymphatic system; - diagnostic difficulties that stem from physico-chemical properties and the nature of proteins or fibrillating peptides itself like low concentration, short half-live and from experimental-technical problems as well like high adsorption or low solubility of Aβ, tau or amylin. The study of diagnostic parameters is very important, as it may better reflect early changes before the disease develops; one such parameter is the Aβ42/Aβ40 ratio, or the ratio with the total tau concentration combination and other new biomarkers like Aβ1-38; other factors include oxidative stress and inflammation process proteins, complement factor H, alpha-2-macroglobulin, or clusterin. The study of various forms of pathological amyloid deposits that emerge in different but specific brain regions AD patients seems to be crucial as well. The composition of the first initial pathological, pre-fibrillating monomers of fibrillating peptides and their role in AD development and disease progression have been described as well. They are even more challenging for science and simultaneously might be more promising in early diagnosis for AD patients. As always in science, research leads to endless discoveries and further inquiry. Fundamental problems in this field most probably are still far from being definitively comprehended, and multiple crucial questions await better answers. What we really need is to study more and deeper into this matter.

Systematic substrate identification indicates a central role for the metalloprotease ADAM10 in axon targeting and synapse function.

Metzincin metalloproteases have major roles in intercellular communication by modulating the function of membrane proteins. One of the proteases is the a-disintegrin-and-metalloprotease 10 (ADAM10) which acts as alpha-secretase of the Alzheimer's disease amyloid precursor protein. ADAM10 is also required for neuronal network functions in murine brain, but neuronal ADAM10 substrates are only partly known. With a proteomic analysis of Adam10-deficient neurons we identified 91, mostly novel ADAM10 substrate candidates, making ADAM10 a major protease for membrane proteins in the nervous system. Several novel substrates, including the neuronal cell adhesion protein NrCAM, are involved in brain development. Indeed, we detected mistargeted axons in the olfactory bulb of conditional ADAM10-/- mice, which correlate with reduced cleavage of NrCAM, NCAM and other ADAM10 substrates. In summary, the novel ADAM10 substrates provide a molecular basis for neuronal network dysfunctions in conditional ADAM10-/- mice and demonstrate a fundamental function of ADAM10 in the brain.

Phosphorylation of FE65 Ser610 by serum- and glucocorticoid-induced kinase 1 modulates Alzheimer's disease amyloid precursor protein processing.

Alzheimer's disease (AD) is a fatal neurodegenerative disease affecting 36 million people worldwide. Genetic and biochemical research indicate that the excessive generation of amyloid-β peptide (Aβ) from amyloid precursor protein (APP), is a major part of AD pathogenesis. FE65 is a brain-enriched adaptor protein that binds to APP. However, the role of FE65 in APP processing and the mechanisms that regulate binding of FE65 to APP are not fully understood. In the present study, we show that serum- and glucocorticoid-induced kinase 1 (SGK1) phosphorylates FE65 on Ser(610) and that this phosphorylation attenuates FE65 binding to APP. We also show that FE65 promotes amyloidogenic processing of APP and that FE65 Ser(610) phosphorylation inhibits this effect. Furthermore, we found that the effect of FE65 Ser(610) phosphorylation on APP processing is linked to a role of FE65in metabolic turnover of APP via the proteasome. Thus FE65 influences APP degradation via the proteasome and phosphorylation of FE65 Ser(610) by SGK1 regulates binding of FE65 to APP, APP turnover and processing.

F-box Only Protein 2 (Fbxo2) Regulates Amyloid Precursor Protein Levels and Processing

The amyloid precursor protein (APP) is an integral membrane glycoprotein whose cleavage products, particularly amyloid-β, accumulate in Alzheimer disease (AD). APP is present at synapses and is thought to play a role in both the formation and plasticity of these critical neuronal structures. Despite the central role suggested for APP in AD pathogenesis, the mechanisms regulating APP in neurons and its processing into cleavage products remain incompletely understood. F-box only protein 2 (Fbxo2), a neuron-enriched ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans on glycoproteins, was previously implicated in APP processing by facilitating the degradation of the APP-cleaving β-secretase, β-site APP-cleaving enzyme. Here, we sought to determine whether Fbxo2 plays a similar role for other glycoproteins in the amyloid processing pathway. We present in vitro and in vivo evidence that APP is itself a substrate for Fbxo2. APP levels were decreased in the presence of Fbxo2 in non-neuronal cells, and increased in both cultured hippocampal neurons and brain tissue from Fbxo2 knock-out mice. The processing of APP into its cleavage products was also increased in hippocampi and cultured hippocampal neurons lacking Fbxo2. In hippocampal slices, this increase in cleavage products was accompanied by a significant reduction in APP at the cell surface. Taken together, these results suggest that Fbxo2 regulates APP levels and processing in the brain and may play a role in modulating AD pathogenesis.

Structural and Functional Characterization of Cargo-Binding Sites on the μ4-Subunit of Adaptor Protein Complex 4

Adaptor protein (AP) complexes facilitate protein trafficking by playing key roles in the selection of cargo molecules to be sorted in post-Golgi compartments. Four AP complexes (AP-1 to AP-4) contain a medium-sized subunit (μ1-μ4) that recognizes YXXØ-sequences (Ø is a bulky hydrophobic residue), which are sorting signals in transmembrane proteins. A conserved, canonical region in μ subunits mediates recognition of YXXØ-signals by means of a critical aspartic acid. Recently we found that a non-canonical YXXØ-signal on the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) binds to a distinct region of the μ4 subunit of the AP-4 complex. In this study we aimed to determine the functionality of both binding sites of μ4 on the recognition of the non-canonical YXXØ-signal of APP. We found that substitutions in either binding site abrogated the interaction with the APP-tail in yeast-two hybrid experiments. Further characterization by isothermal titration calorimetry showed instead loss of binding to the APP signal with only the substitution R283D at the non-canonical site, in contrast to a decrease in binding affinity with the substitution D190A at the canonical site. We solved the crystal structure of the C-terminal domain of the D190A mutant bound to this non-canonical YXXØ-signal. This structure showed no significant difference compared to that of wild-type μ4. Both differential scanning fluorimetry and limited proteolysis analyses demonstrated that the D190A substitution rendered μ4 less stable, suggesting an explanation for its lower binding affinity to the APP signal. Finally, in contrast to overexpression of the D190A mutant, and acting in a dominant-negative manner, overexpression of μ4 with either a F255A or a R283D substitution at the non-canonical site halted APP transport at the Golgi apparatus. Together, our analyses support that the functional recognition of the non-canonical YXXØ-signal of APP is limited to the non-canonical site of μ4.

Mild oxidative stress induces redistribution of BACE1 in non-apoptotic conditions and promotes the amyloidogenic processing of Alzheimer's disease amyloid precursor protein.

BACE1 is responsible for β-secretase cleavage of the amyloid precursor protein (APP), which represents the first step in the production of amyloid β (Aβ) peptides. Previous reports, by us and others, have indicated that the levels of BACE1 protein and activity are increased in the brain cortex of patients with Alzheimer’s disease (AD). The association between oxidative stress (OS) and AD has prompted investigations that support the potentiation of BACE1 expression and enzymatic activity by OS. Here, we have established conditions to analyse the effects of mild, non-lethal OS on BACE1 in primary neuronal cultures, independently from apoptotic mechanisms that were shown to impair BACE1 turnover. Six-hour treatment of mouse primary cortical cells with 10–40 µM hydrogen peroxide did not significantly compromise cell viability but it did produce mild oxidative stress (mOS), as shown by the increased levels of reactive radical species and activation of p38 stress kinase. The endogenous levels of BACE1 mRNA and protein were not significantly altered in these conditions, whereas a toxic H2O2 concentration (100 µM) caused an increase in BACE1 protein levels. Notably, mOS conditions resulted in increased levels of the BACE1 C-terminal cleavage product of APP, β-CTF. Subcellular fractionation techniques showed that mOS caused a major rearrangement of BACE1 localization from light to denser fractions, resulting in an increased distribution of BACE1 in fractions containing APP and markers for trans-Golgi network and early endosomes. Collectively, these data demonstrate that mOS does not modify BACE1 expression but alters BACE1 subcellular compartmentalization to favour the amyloidogenic processing of APP, and thus offer new insight in the early molecular events of AD pathogenesis.

P4‐291: Structural studies of Alzheimer's disease amyloid precursor protein

P4‐291: Structural studies of Alzheimer's disease amyloid precursor protein

P4‐058: Identification of engulfment adaptor protein 1 as a novel Alzheimer's disease amyloid precursor protein interacting protein

pharmacologic blockade and genetic ablation of PLD1 alters APP processing in primary cortical neurons derived from these mice by analyzing relevant APP metabolites by Western blot and ELISA. Finally, we investigated whether genetic ablation of PLD1 affects cognitive functions in the context of the J20 mouse model using hippocampal-dependent memory tests. Results: Our results indicate that genetic ablation of PLD1 causes a significant reduction in total PLD activity as well as in the levels of PA in the forebrain. PLD1 ablation, unlike that of PLD2, causes a significant decrease in the levels of both soluble and insoluble Ab. Pharmacologic inhibition of PLD1 also decreases the secretion of Ab42 in cultured cells. Remarkably, PLD1 nullizygosity ameliorates synaptic defects and cognitive deficits observed in the J20 model of AD. Conclusions: We conclude that reducing the activity of PLD1 decreases the production of Ab; thus, PLD1 may be a novel therapeutic target to disrupt the molecular pathogenesis of AD. Overall, our recent studies suggest that blocking PLD1 or PLD2 may be beneficial in AD, although these two related lipases appear to affect distinct aspects of APP biology.

Apolipoprotein E3 (ApoE3) but Not ApoE4 Protects against Synaptic Loss through Increased Expression of Protein Kinase Cϵ

Synaptic loss is the earliest pathological change in Alzheimer disease (AD) and is the pathological change most directly correlated with the degree of dementia. ApoE4 is the major genetic risk factor for the age-dependent form of AD, which accounts for 95% of cases. Here we show that in synaptic networks formed from primary hippocampal neurons in culture, apoE3, but not apoE4, prevents the loss of synaptic networks produced by amyloid β oligomers (amylospheroids). Specific activators of PKCε, such as 8-(2-(2-pentyl-cyclopropylmethyl)-cyclopropyl)-octanoic acid methyl ester and bryostatin 1, protected against synaptic loss by amylospheroids, whereas PKCε inhibitors blocked this synaptic protection and also blocked the protection by apoE3. Blocking LRP1, an apoE receptor on the neuronal membrane, also blocked the protection by apoE. ApoE3, but not apoE4, induced the synthesis of PKCε mRNA and expression of the PKCε protein. Amyloid β specifically blocked the expression of PKCε but had no effect on other isoforms. These results suggest that protection against synaptic loss by apoE is mediated by a novel intracellular PKCε pathway. This apoE pathway may account for much of the protective effect of apoE and reduced risk for the age-dependent form of AD. This finding supports the potential efficacy of newly developed therapeutics for AD.

Genomic Organization and Promoter Cloning of the Human X11α GeneAPBA1

X11α is a brain specific multi-modular protein that interacts with the Alzheimer's disease amyloid precursor protein (APP). Aggregation of amyloid-β peptide (Aβ), an APP cleavage product, is believed to be central to the pathogenesis of Alzheimer's disease. Recently, overexpression of X11α has been shown to reduce Aβ generation and to ameliorate memory deficit in a transgenic mouse model of Alzheimer's disease. Therefore, manipulating the expression level of X11α may provide a novel route for the treatment of Alzheimer's disease. Human X11α is encoded by the gene APBA1. As evidence suggests that X11α expression can be regulated at transcription level, we have determined the gene structure and cloned the promoter of APBA1. APBA1 spans over 244 kb on chromosome 9 and is composed of 13 exons and has multiple transcription start sites. A putative APBA1 promoter has been identified upstream of exon 1 and functional analysis revealed that this is highly active in neurons. By deletion analysis, the minimal promoter was found to be located between -224 and +14, a GC-rich region that contains a functional Sp3 binding site. In neurons, overexpression of Sp3 stimulates the APBA1 promoter while an Sp3 inhibitor suppresses the promoter activity. Moreover, inhibition of Sp3 reduces endogenous X11α expression and promotes the generation of Aβ. Our findings reveal that Sp3 play an essential role in APBA1 transcription.

Alzheimer's Disease Amyloid-β Links Lens and Brain Pathology in Down Syndrome

Down syndrome (DS, trisomy 21) is the most common chromosomal disorder and the leading genetic cause of intellectual disability in humans. In DS, triplication of chromosome 21 invariably includes the APP gene (21q21) encoding the Alzheimer's disease (AD) amyloid precursor protein (APP). Triplication of the APP gene accelerates APP expression leading to cerebral accumulation of APP-derived amyloid-β peptides (Aβ), early-onset AD neuropathology, and age-dependent cognitive sequelae. The DS phenotype complex also includes distinctive early-onset cerulean cataracts of unknown etiology. Previously, we reported increased Aβ accumulation, co-localizing amyloid pathology, and disease-linked supranuclear cataracts in the ocular lenses of subjects with AD. Here, we investigate the hypothesis that related AD-linked Aβ pathology underlies the distinctive lens phenotype associated with DS. Ophthalmological examinations of DS subjects were correlated with phenotypic, histochemical, and biochemical analyses of lenses obtained from DS, AD, and normal control subjects. Evaluation of DS lenses revealed a characteristic pattern of supranuclear opacification accompanied by accelerated supranuclear Aβ accumulation, co-localizing amyloid pathology, and fiber cell cytoplasmic Aβ aggregates (~5 to 50 nm) identical to the lens pathology identified in AD. Peptide sequencing, immunoblot analysis, and ELISA confirmed the identity and increased accumulation of Aβ in DS lenses. Incubation of synthetic Aβ with human lens protein promoted protein aggregation, amyloid formation, and light scattering that recapitulated the molecular pathology and clinical features observed in DS lenses. These results establish the genetic etiology of the distinctive lens phenotype in DS and identify the molecular origin and pathogenic mechanism by which lens pathology is expressed in this common chromosomal disorder. Moreover, these findings confirm increased Aβ accumulation as a key pathogenic determinant linking lens and brain pathology in both DS and AD.

Clinical News

In Down syndrome, triplication of chromosome 21 invariably includes the gene (21q21) encoding the Alzheimer's disease amyloid precursor protein, accelerating expression of this protein and leading to cerebral accumulation of amyloid precursor protein-derived amyloid- b peptides (Ab), earlyonset Alzheimer's disease neuropathology, and age-dependent cognitive sequelae.

MicroRNA-101 Regulates Amyloid Precursor Protein Expression in Hippocampal Neurons

The amyloid precursor protein (APP) and its proteolytic product amyloid beta (Abeta) are associated with both familial and sporadic forms of Alzheimer disease (AD). Aberrant expression and function of microRNAs has been observed in AD. Here, we show that in rat hippocampal neurons cultured in vitro, the down-regulation of Argonaute-2, a key component of the RNA-induced silencing complex, produced an increase in APP levels. Using site-directed mutagenesis, a microRNA responsive element (RE) for miR-101 was identified in the 3'-untranslated region (UTR) of APP. The inhibition of endogenous miR-101 increased APP levels, whereas lentiviral-mediated miR-101 overexpression significantly reduced APP and Abeta load in hippocampal neurons. In addition, miR-101 contributed to the regulation of APP in response to the proinflammatory cytokine interleukin-1beta (IL-lbeta). Thus, miR-101 is a negative regulator of APP expression and affects the accumulation of Abeta, suggesting a possible role for miR-101 in neuropathological conditions.

Alzheimer's disease amyloid-beta links lens and brain pathology in Down syndrome.

Down syndrome (DS, trisomy 21) is the most common chromosomal disorder and the leading genetic cause of intellectual disability in humans. In DS, triplication of chromosome 21 invariably includes the APP gene (21q21) encoding the Alzheimer's disease (AD) amyloid precursor protein (APP). Triplication of the APP gene accelerates APP expression leading to cerebral accumulation of APP-derived amyloid-β peptides (Aβ), early-onset AD neuropathology, and age-dependent cognitive sequelae. The DS phenotype complex also includes distinctive early-onset cerulean cataracts of unknown etiology. Previously, we reported increased Aβ accumulation, co-localizing amyloid pathology, and disease-linked supranuclear cataracts in the ocular lenses of subjects with AD. Here, we investigate the hypothesis that related AD-linked Aβ pathology underlies the distinctive lens phenotype associated with DS. Ophthalmological examinations of DS subjects were correlated with phenotypic, histochemical, and biochemical analyses of lenses obtained from DS, AD, and normal control subjects. Evaluation of DS lenses revealed a characteristic pattern of supranuclear opacification accompanied by accelerated supranuclear Aβ accumulation, co-localizing amyloid pathology, and fiber cell cytoplasmic Aβ aggregates (∼5 to 50 nm) identical to the lens pathology identified in AD. Peptide sequencing, immunoblot analysis, and ELISA confirmed the identity and increased accumulation of Aβ in DS lenses. Incubation of synthetic Aβ with human lens protein promoted protein aggregation, amyloid formation, and light scattering that recapitulated the molecular pathology and clinical features observed in DS lenses. These results establish the genetic etiology of the distinctive lens phenotype in DS and identify the molecular origin and pathogenic mechanism by which lens pathology is expressed in this common chromosomal disorder. Moreover, these findings confirm increased Aβ accumulation as a key pathogenic determinant linking lens and brain pathology in both DS and AD.

Sorting of the Alzheimer's Disease Amyloid Precursor Protein Mediated by the AP-4 Complex

Adaptor protein 4 (AP-4) is the most recently discovered and least well-characterized member of the family of heterotetrameric adaptor protein (AP) complexes that mediate sorting of transmembrane cargo in post-Golgi compartments. Herein, we report the interaction of an YKFFE sequence from the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) with the mu4 subunit of AP-4. Biochemical and X-ray crystallographic analyses reveal that the properties of the APP sequence and the location of the binding site on mu4 are distinct from those of other signal-adaptor interactions. Disruption of the APP-AP-4 interaction decreases localization of APP to endosomes and enhances gamma-secretase-catalyzed cleavage of APP to the pathogenic amyloid-beta peptide. These findings demonstrate that APP and AP-4 engage in a distinct type of signal-adaptor interaction that mediates transport of APP from the trans-Golgi network (TGN) to endosomes, thereby reducing amyloidogenic processing of the protein.

Transcriptional regulation of human FE65, a ligand of Alzheimer's disease amyloid precursor protein, by Sp1

FE65 is a neuronal-enriched adaptor protein that binds to the Alzheimer's disease amyloid precursor protein (APP). FE65 forms a transcriptionally active complex with the APP intracellular domain (AICD). The precise gene targets for this complex are unclear but several Alzheimer's disease-linked genes have been proposed. Additionally, evidence suggests that FE65 influences APP metabolism. The mechanism by which FE65 expression is regulated is as yet unknown. To gain insight into the regulatory mechanism, we cloned a 1.6 kb fragment upstream of the human FE65 gene and found that it possesses particularly strong promoter activity in neurones. To delineate essential regions in the human FE65 promoter, a series of deletion mutants were generated. The minimal FE65 promoter was located between -100 and +5, which contains a functional Sp1 site. Overexpression of the transcription factor Sp1 potentiates the FE65 promoter activity. Conversely, suppression of the FE65 promoter was observed in cells either treated with an Sp1 inhibitor or in which Sp1 was knocked down. Furthermore, reduced levels of Sp1 resulted in downregulation of endogenous FE65 mRNA and protein. These findings reveal that Sp1 plays a crucial role in transcriptional control of the human FE65 gene.

Neurons Generated from APP/APLP1/APLP2 Triple Knockout Embryonic Stem Cells Behave Normally in Vitro and in Vivo: Lack of Evidence for a Cell Autonomous Role of the Amyloid Precursor Protein in Neuronal Differentiation

Alzheimer's disease amyloid precursor protein (APP) has been implicated in many neurobiologic processes, but supporting evidence remains indirect. Studies are confounded by the existence of two partially redundant APP homologues, APLP1 and APLP2. APP/APLP1/APLP2 triple knockout (APP tKO) mice display cobblestone lissencephaly and are perinatally lethal. To circumvent this problem, we generated APP triple knockout embryonic stem (ES) cells and differentiated these to APP triple knockout neurons in vitro and in vivo. In comparison with wild-type (WT) ES cell-derived neurons, APP tKO neurons formed equally pure neuronal cultures, had unaltered in vitro migratory capacities, had a similar acquisition of polarity, and were capable of extending long neurites and forming active excitatory synapses. These data were confirmed in vivo in chimeric mice with APP tKO neurons expressing the enhanced green fluorescent protein (eGFP) present in a WT background brain. The results suggest that the loss of the APP family of proteins has no major effect on these critical neuronal processes and that the apparent multitude of functions in which APP has been implicated might be characterized by molecular redundancy. Our stem cell culture provides an excellent tool to circumvent the problem of lack of viability of APP/APLP triple knockout mice and will help to explore the function of this intriguing protein further in vitro and in vivo.

Alzheimer's disease amyloid precursor protein and amyloid beta peptides are key regulators of brain lipid composition

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