A model system to study intracellular trafficking and processing of the Alzheimer's amyloid precursor protein

The occurrence of consensus phosphorylation sites in the intracellular domain of the Alzheimer's amyloid precursor protein (APP), coupled with observations of their in vivo phosphorylation, prompted several workers to investigate the effects that phosphorylation of such sites could have on APP metabolism and subsequent Abeta production. However, hitherto all attempts to dissect the role played by such phosphorylation events failed to reveal substantial effects. Having decided to revisit this problem, our new approach was based on the following vectors: (1) site-directed mutagenesis of the target amino acids to mimic a specific phosphorylation state, (2) expression of wild-type and mutant APP-GFP (green fluorescent protein) fusion proteins for ease of visualization, (3) controlled low level expression to avoid 'flooding' cellular pathways, and (4) the use of cycloheximide to inhibit de novo protein synthesis. Using this method we were able to detect specific differences in APP processing that were correlated with the mimicked phosphorylation state of several phosphorylation sites. New combined methodologies, like the one described here, allow for the detailed analysis of key control points in the cellular metabolism of specific proteins that are central to neurodegenerative diseases and may be under the control of specific posttranslational modifications, such as reversible phosphorylation.     

Platelet amyloid precursor protein processing: a bio-marker for Alzheimer's disease

The amyloid precursor protein (APP) in brain is processed either by an amyloidogenic pathway by beta-secretase and gamma-secretase to yield Abeta (beta-amyloid 4 kDa) peptide or by alpha-secretase within the beta-amyloid domain to yield non-amyloidogenic products. We have studied blood platelet levels of a 22-kDa fragment containing the Abeta (beta-amyloid 4 kDa) peptide, beta-secretase (BACE1), alpha-secretase (ADAM10), and APP isoform ratios of the 120-130 kDa to 110 kDa peptides from 31 Alzheimer's disease (AD) patients and 10 age-matched healthy control subjects. We found increased levels of Abeta4, increased activation of beta-secretase (BACE1), decreased activation of alpha-secretase (ADAM10) and decreased APP ratios in AD patients compared to normal control subjects. These observations indicate that the blood platelet APP is processed by the same amyloidogenic and non-amyloidogenic pathways as utilized in brain and that APP processing in AD patients is altered compared to control subjects and may be a useful bio-marker for the diagnosis of AD, the progression of disease and for monitoring drug responses in clinical trials.     

Targeting amyloid precursor protein shuttling and processing - long before amyloid beta formation

<正>Targeting early steps in amyloid-beta production:Alzheimer’s disease(AD)has a long history as theamyloid depositdisorder.Many disorders are now known to be caused by proteinβ-sheet misfolding and aggregation(e.g.,Parkinson’s disease:α-synuclein;Huntington’s disease:Huntingtin;     

Altered processing of a mutant amyloid precursor protein in neuronal and endothelial cells

Altered proteolysis of the amyloid precursor protein (APP) may play an important role in Alzheimer disease (AD). To better understand the role of mutant APP in the pathogenesis of the disease, we stably overexpressed the mutant APP717F approximately twofold vs. the endogenous wild-type gene in several cell types. The processing of APP was examined by Western blot analysis and immunoprecipitation. We observed distinctive patterns of APP metabolites among various cell lines. Neuronal and endothelial cells expressing mutant APP717F generated higher levels of large, potentially amyloidogenic carboxyl terminal fragments, which were enhanced upon treatment of the cells with leupeptin. These results suggest that mutations in the APP gene shift the protein processing towards the amyloidogenic pathway in neuronal and endothelial cells possibly involving the endosomal-lysosomal system. © 1995 Wiley-Liss, Inc.     

Effect of aluminium on expression and processing of amyloid precursor protein

The environmental agent aluminium has been extensively investigated for a potential role in the aetiology of Alzheimer's disease. Despite many investigations there is at present no definite proof for any involvement. If aluminium is involved it is possible that its action is mediated through interaction with the synthesis or processing of amyloid precursor protein (APP). The present study compared aluminium loaded IMR-32 neuroblastoma cells and rat brains with control cells and brains to determine if aluminium affected APP expression and/or processing. In the IMR-32 model system aluminium had no effect on steady-state APP mRNA levels or on the ratio of individual isoforms. It also had no quantitative or qualitative effect on APP-immunoreactive bands detected in protein extracts from conditioned medium of these cells. In total cell extracts, aluminium reduced the intensity of APP-immunoreactive bands between 120–105 kDa but had no effect on a 9 kDa band. In rat brains, aluminium had no effect on APP-immunoreactive bands from soluble or insoluble-membranous extracts. The results, in general, provide no evidence for any effect of aluminium on APP expression or processing. © 1996 Wiley-Liss, Inc.     

Alpha-secretase cleavage of the amyloid precursor protein: proteolysis regulated by signaling pathways and protein trafficking

α-secretase is the name for a metalloprotease activity, which is assumed to play a key role in the prevention of the molecular mechanisms underlying Alzheimer's disease (AD). Proteases similar to α-secretase are essential for a wide range of biological processes, such as cell adhesion and embryonic development. The molecular culprit in AD is the amyloid β peptide (Aβ), which derives from the amyloid precursor protein (APP) through sequential cleavage by the two proteases β- and γ-secretase. In contrast, α-secretase, which is the metalloprotease ADAM10, cleaves APP within the Aβ domain, thus preventing Aβ generation. Additionally, it produces a secreted APP ectodomain with neurotrophic and neuroprotective properties. An increase in α-secretase cleavage is considered a therapeutic approach for AD, but the molecular mechanisms regulating α-secretase cleavage are only partly known. Protein kinase C and mitogen-activated protein kinase constitute central signaling hubs for the regulation of α-secretase cleavage. Additionally, recent studies increasingly demonstrate that the correct spatial and temporal localization of the two membrane proteins APP and α-secretase is essential for efficient α-secretase cleavage of APP. This review highlights the role of signaling pathways and protein trafficking in the control of APP α-secretase cleavage.     

ADAMs family members as amyloid precursor protein alpha-secretases

In the non-amyloidogenic pathway, the Alzheimer's amyloid precursor protein (APP) is cleaved within the amyloid-beta domain by alpha-secretase precluding deposition of intact amyloid-beta peptide. The large ectodomain released from the cell surface by the action of alpha-secretase has several neuroprotective properties. Studies with protease inhibitors have shown that alpha-secretase is a zinc metalloproteinase, and several members of the adamalysin family of proteins, tumour necrosis factor-alpha convertase (TACE, ADAM17), ADAM10, and ADAM9, all fulfil some of the criteria required of alpha-secretase. We review the evidence for each of these ADAMs acting as the alpha-secretase. What seems to be emerging from numerous studies, including those with mice in which each of the ADAMs has been knocked out, is that there is a team of zinc metalloproteinases able to cleave APP at the alpha-secretase site. We also discuss how upregulation of alpha-secretase activity by muscarinic agonists, cholesterol-lowering drugs, steroid hormones, non-steroidal anti-inflammatory drugs, and metal ions may explain some of the therapeutic actions of these agents in Alzheimer's disease.     

TDP-43 and amyloid precursor protein processing: implications for Alzheimer's disease

正In this perspective article, I will discuss our recent publication(Hicks et al., 2020), specifically its major findings and integration with the published literature.Alzheimer 's disease(AD) is a progressive neurodegenerative     

The Arctic mutation interferes with processing of the amyloid precursor protein

The Arctic amyloid precursor protein (APP) Alzheimer mutation, is located inside the beta-amyloid (Abeta) domain. Here, hybrid APP mutants containing both the Swedish and the Arctic APP mutations were investigated. ELISA measurements of cell media showed decreased levels of both Abeta40 and Abeta42. Similar results were obtained for the Dutch and Italian mutations, whereas the Flemish mutation displayed increased amounts of Abeta40 and Abeta42. Immunoprecipitation studies revealed increased Abeta40/p3 and Abeta42/p3 ratios for the Arctic mutation. These results were further verified by quantification revealing decreased levels of alphaAPPs accompanied by increased betaAPPs levels in the media. Thus, the pathogenic effects of the Arctic mutation may not only be due to the changed properties of the peptide but also altered processing of Arctic APP.     

Decreased expression of GGA3 protein in Alzheimer's disease frontal cortex and increased co-distribution of BACE with the amyloid precursor protein

BACE initiates the amyloidogenic processing of the amyloid precursor protein (APP) that results in the production of Aβ peptides associated with Alzheimer's disease (AD). Previous studies have indicated that BACE is elevated in the frontal cortex of AD patients. Golgi-localized γ-ear containing ADP ribosylation factor-binding proteins (GGA) control the cellular trafficking of BACE and may alter its levels. To investigate a link between BACE and GGA expression in AD, frontal cortex samples from AD (N = 20) and healthy, age-matched controls (HC, N =17) were analyzed by immunoblotting. After normalization to the neuronal marker β-tubulin III, the data indicate an average two-fold increase of BACE protein (p = 0.01) and a 64% decrease of GGA3 in the AD group compared to the HC (p = 0.006). GGA1 levels were also decreased in AD, but a statistical significance was not achieved. qRT-PCR analysis of GGA3 mRNA showed no difference between AD and HC. There was a strong correlation between GGA1 and GGA3 in both AD and HC, but no correlation between BACE and GGA levels. Subcellular fractionation of AD cortex with low levels of GGA proteins showed an alteration of BACE distribution and extensive co-localization with APP. These data suggest that altered compartmentalization of BACE in AD promotes the amyloidogenic processing of APP.     

Processing of amyloid precursor protein and amyloid peptide neurotoxicity

Alzheimer's disease is characterized by the presence of two types of lesions in brain: neurofibrillary tangles and senile plaques. Intraneuronal neurofibrillary tangles are made of paired helical filaments containing hyperphosphorylated microtubule associated protein tau. Extracellular senile plaques contain a core of beta-amyloid peptide (Abeta), which is produced by cleavage of the Amyloid Precursor Protein (APP). Among the two catabolic pathways of APP, the amyloidogenic pathway producing Abeta peptides was intensively studied in different cellular models expressing human APP. Differences in APP processing and in toxicity resulting from Abeta accumulation can be observed from one cell type to another. In particular, primary cultures of neurons process APP differently compared with other cultured cells including neuronal cell lines. Neurons accumulate intraneuronal Abeta, which is neurotoxic, and in these cells, APP can be phosphorylated at specific residues. Recent studies suggest that APP phosphorylation can play an important role in its amyloidogenic processing. In addition, protein kinases that phosphorylate APP are also able to phosphorylate the neuronal protein tau. Biochemical analysis of these two proteins in primary cultures of neurons show that phosphorylation of both APP and tau can be a factor linking the two characteristic lesions of Alzheimer's disease.     

Proteolytic processing of the Alzheimer's disease amyloid precursor protein in brain and platelets

Proteolytic processing of the amyloid precursor protein by beta -and gamma-secretases results in the production of Alzheimer's disease (AD) Abeta amyloid peptides. Modulation of secretase activity is being investigated as a potential therapeutic approach. Recent studies with human brain have revealed that the beta-secretase protein, BACE, is increased in cortex of AD patients. Analysis of betaCTF (or C99), the amyloid precursor protein (APP) product of BACE cleavage that is the direct precursor to Abeta, shows it is also elevated in AD, underlying the importance of beta-secretase cleavage in AD pathogenesis. The C-terminal product of gamma-secretase cleavage of APP, epsilonCTF (or AICD), is enriched in human brain cortical nuclear fractions, a subcellular distribution appropriate for a putative involvement of APP cytosolic domain in signal transduction. Analysis of AD cortex samples, particularly that of a carrier of a familial APP mutation, suggests that processing of APP transmembrane domain generates an alternative CTF product. All these particularities observed in the AD brain demonstrate that APP processing is altered in AD. The transgenic mouse model Tg2576 seems to be a promising laboratory tool to test potential modulators of Abeta formation. Indeed, C-terminal products of alpha-, beta-, and gamma-secretase cleavage are readily detectable in the brain of these transgenic mice. Finally, the finding of the same secretase products in platelets and neurons make platelets a potentially useful and easily accessible clinical tool to monitor effects of novel therapies based on inhibition of beta- or gamma-secretase.     

Subcellular trafficking of the amyloid precursor protein gene family and its pathogenic role in Alzheimer's disease

Changes in the intracellular transport of amyloid precursor protein (APP) affect the extent to which APP is exposed to alpha- or beta-secretase in a common subcellular compartment and therefore directly influence the degree to which APP undergoes the amyloidogenic pathway leading to generation of beta-amyloid. As the presynaptic regions of neurons are thought to be the main source of beta-amyloid in the brain, attention has been focused on axonal APP trafficking. APP is transported along axons by a fast, kinesin-dependent anterograde transport mechanism. Despite the wealth of in vivo and in vitro data that have accumulated regarding the connection of APP to kinesin transport, it is not yet clear if APP is coupled to its specific motor protein via an intracellular interaction partner, such as the c-Jun N-terminal kinase-interacting protein, or by yet another unknown molecular mechanism. The cargo proteins that form a functional complex with APP are also unknown. Due to the long lifespan, and vast extent, of neurons, in particular axons, neurons are highly sensitive to changes in subcellular transport. Recent in vitro and in vivo studies have shown that variations in APP or tau affect mitochondrial and synaptic vesicle transport. Further, it was shown that this axonal dysfunction might lead to impaired synaptic plasticity, which is crucial for neuronal viability and function. Thus, changes in APP and tau expression may cause perturbed axonal transport and changes in APP processing, contributing to cognitive decline and neurodegeneration in Alzheimer's disease.     

Notch 1 interacts with the amyloid precursor protein in a Numb-independent manner

We hypothesized that the physical interaction between the amyloid precursor protein (APP) and Notch 1 (N1) may be mediating the reported cross-talk between the respective signaling pathways. Immunoprecipitation of mouse N1 (mN1) or extracellular domain truncated mN1 (mN1-TM, mimics TACE-produced membrane-bound C-terminal fragment) specifically coprecipitated APP(751). Conversely, immunoprecipitation of APP(751) specifically coprecipitated mN1, furin-generated membrane-bound mN1 C-terminal fragment (f.mN1-TM), or mN1-TM. The London mutation of APP did not affect the APP(751)/mN1 interaction. Coexpression of APP(751) and mN1 did not affect APP processing or production of mN1 intracellular domain (mNICD). The APP(751)/mN1 interaction was Numb-independent, insofar as it was observed in HEK293 cells that lack detectable levels of Numb and was unaffected by the expression of exogenous Numb or deletion of the APP cytoplasmic domain, including the Numb-binding YENPTY sequence. This interaction was unaffected even when the N-terminal 647 amino acids of APP were replaced by a sequence of secreted alkaline phosphatase. These data combined with data showing interaction between mN1-TM and APP(751) suggest that their transmebrane domains and short sequences around them are sufficient for the interaction and that APP(751) and mN1 interact in cis. Our results imply novel functions of APP and/or N1 that derive from their interaction.     

The location and trafficking routes of the neuronal retromer and its role in amyloid precursor protein transport

The retromer complex plays an important role in intracellular transport, is highly expressed in the hippocampus, and has been implicated in the trafficking of the amyloid precursor protein (APP). Nevertheless, the trafficking routes of the neuronal retromer and the role it plays in APP transport in neuronal processes remain unknown. Here we use hippocampal neuronal cultures to address these issues. Using fluorescence microscopy, we find that Vps35, the core element of the retromer complex, is in dendrites and axons, is enriched in endosomes and trans-Golgi network, and is found in APP-positive vesicles. Next, to identify the role the neuronal retromer plays in cargo transport, we infected hippocampal neurons with a lentivirus expressing shRNA to silence Vps35. By live fluorescence imaging, Vps35 deficiency was found to reduce the frequency, but not the kinetics, of long-range APP transport within neuronal processes. Supporting the interpretation that retromer promotes long-range transport, Vps35 deficiency led to increased APP in the early endosomes, in processes but not the soma. Finally, Vps35 deficiency was associated with increased levels of Aβ, a cleaved product of APP, increased colocalization of APP with its cleaving enzyme BACE1 in processes, and caused an enlargement of early endosomes. Taken together, our studies clarify the function of the neuronal retromer, and suggest specific mechanisms for how retromer dysfunction observed in Alzheimer's disease affects APP transport and processing.     

In vitro effects of metrifonate on neuronal amyloid precursor protein processing and protein kinase C level

Alteration in the processing of the amyloid precursor protein (APP) is a central event in the formation of amyloid deposits in the brains of individuals with Alzheimer's disease (AD). It has been suggested that acetylcholinesterase (AChE) inhibitors, which promote the cholinergic function and consequently improve the cognitive deficits, may also exert a neuroprotective effect by activating normal APP processing. We now report that an irreversible AChE inhibitor (metrifonate) increase the cell-associated APP level in a basal forebrain neuronal culture and also elevate the amount of APP secreted into the medium. The alterations in APP processing were accompanied by increased protein kinase C (PKC) levels. The results suggest that AChE inhibitors modulate the metabolism of APP, possibly via their stimulatory effects on PKC. Since changes in the activity and level of PKC may be involved in the pathogenesis of AD, it is concluded that the beneficial effect of metrifonate in AD therapy may be due not only to the stimulatory cholinergic function, but also to its activating effect on PKC.     

GM1 ganglioside regulates the proteolysis of amyloid precursor protein

Plaques containing amyloid beta-peptides (Abeta) are a major feature in Alzheimer's disease (AD), and GM1 ganglioside is an important component of cellular plasma membranes and especially enriched in lipid raft. GM1-bound Abeta (GM1/Abeta), found in brains exhibiting early pathological changes of AD including diffuse plaques, has been suggested to be involved in the initiation of amyloid fibril formation in vivo by acting as a seed. However, the role of GM1 in amyloid beta-protein precursor (APP) processing is not yet defined. In this study, we report that exogenous GM1 ganglioside promotes Abeta biogenesis and decreases sAPPalpha secretion in SH-SY5Y and COS7 cells stably transfected with human APP695 cDNA without affecting full-length APP and the sAPPbeta levels. We also observe that GM1 increases extracellular levels of Abeta in primary cultures of mixed rat cortical neurons transiently transfected with human APP695 cDNA. These findings suggest a regulatory role for GM1 in APP processing pathways.     

Cholinergic activity and amyloid precursor protein metabolism

With more than 4 million Alzheimer's victims nationwide, there is intense research to elucidate the relationship among the hallmarks of the disease, amyloid plaques, neurofibrillary tangles, and degeneration of the basal forebrain cholinergic neurons. There has been much debate about which of these is the primary lesion, and which develops secondarily. The correlation between plaques and tangles and dementia is not absolute, but a consistent feature of Alzheimer's disease is loss of cortical and hippocampal cholinergic function as a result of basal forebrain compromise. Additionally, factors associated with the cholinergic system have been shown to influence the processing and metabolism of the amyloid precursor, a protein that contains the amyloidogenic sequence found in plaques. In this paper, the relationship between cholinergic compromise and amyloid deposition, as well as the cholinergic system-associated factors which appear to participate in amyloid precursor protein processing, are discussed.