The transmembrane domain of the amyloid precursor protein is required for antiamyloidogenic processing by α-secretase ADAM10

Neurotoxic amyloid β-peptides are thought to be a causative agent of Alzheimer’s disease in humans. The production of amyloid β-peptides from amyloid precursor protein (APP) could be diminished by enhancing α-processing; however, the physical interactions between APP and α-secretases are not well understood. In this study, we employed super-resolution light microscopy to examine in cell-free plasma membranes the abundance and association of APP and α-secretases ADAM10 (a disintegrin and metalloproteinase) and ADAM17. We found that both secretase molecules localize similarly closely to APP (within ≤50 nm). However, when cross-linking APP with antibodies directed against the GFP tag of APP, in confocal microscopy, we observed that only ADAM10 coaggregated with APP. Furthermore, we mapped the involved protein domain by using APP variants with an exchanged transmembrane segment or lacking cytoplasmic/extracellular domains. We identified that the transmembrane domain of APP is required for association with α-secretases and, as analyzed by Western blot, for α-processing. We propose that the transmembrane domain of APP interacts either directly or indirectly with ADAM10, but not with ADAM17, explaining the dominant role of ADAM10 in α-processing of APP. Further understanding of this interaction may facilitate the development of a therapeutic strategy based on promoting APP cleavage by α-secretases.     

Alzheimer's disease beta-secretase BACE1 is not a neuron-specific enzyme

The brains of Alzheimer's disease (AD) patients are morphologically characterized by neurofibrillar abnormalities and by parenchymal and cerebrovascular deposits of beta-amyloid peptides. The generation of beta-amyloid peptides by proteolytical processing of the amyloid precursor protein (APP) requires the enzymatic activity of the beta-site APP cleaving enzyme 1 (BACE1). The expression of this enzyme has been localized to the brain, in particular to neurons, indicating that neurons are the major source of beta-amyloid peptides in brain. Astrocytes, on the contrary, are known to be important for beta-amyloid clearance and degradation, for providing trophic support to neurons, and for forming a protective barrier between beta-amyloid deposits and neurons. However, under certain conditions related to chronic stress, the role of astrocytes may not be beneficial. Here we present evidence demonstrating that astrocytes are an alternative source of BACE1 and therefore may contribute to beta-amyloid plaque formation. While resting astroyctes in brain do not express BACE1 at detectable levels, cultured astrocytes display BACE1 promoter activity and express BACE1 mRNA and enzymatically active BACE1 protein. Additionally, in animal models of chronic gliosis and in brains of AD patients, there is BACE1 expression in reactive astrocytes. This would suggest that the mechanism for astrocyte activation plays a role in the development of AD and that therapeutic strategies that target astrocyte activation in brain may be beneficial for the treatment of AD. Also, there are differences in responses to chronic versus acute stress, suggesting that one consequence of chronic stress is an incremental shift to different phenotypic cellular states.     

Take five--BACE and the gamma-secretase quartet conduct Alzheimer's amyloid beta-peptide generation

In 1959, Dave Brubeck and Paul Desmond revolutionized modern jazz music by composing their unforgettable Take Five in 5/4, one of the most defiant time signatures in all music. Of similar revolutionary importance for biomedical and basic biochemical research is the identification of the minimal set of genes required to obtain a deadly time bomb ticking in all of us: Alzheimer's disease. It now appears that one needs to Take Five genes to produce a deadly peptide by a proteolytic mechanism, which paradoxically is otherwise of pivotal importance for development and cell fate decisions.     

The role of amyloid precursor protein processing by BACE1, the beta-secretase, in Alzheimer disease pathophysiology

Amyloid plaques, composed of the amyloid beta-protein (Abeta), are hallmark neuropathological lesions in Alzheimer disease (AD) brain. Abeta fulfills a central role in AD pathogenesis, and reduction of Abeta levels should prove beneficial for AD treatment. Abeta generation is initiated by proteolysis of amyloid precursor protein (APP) by the beta-secretase enzyme BACE1. Bace1 knockout (Bace1(-/-)) mice have validated BACE1 as the authentic beta-secretase in vivo. BACE1 is essential for Abeta generation and represents a suitable drug target for AD therapy, especially because this enzyme is up-regulated in AD. However, although initial data indicated that Bace1(-/-) mice lack an overt phenotype, the BACE1-mediated processing of APP and other substrates may be important for specific biological processes. In this minireview, topics range from the initial identification of BACE1 to the fundamental knowledge gaps that remain in our understanding of this protease. We address pertinent questions such as putative causes of BACE1 elevation in AD and discuss why, nine years since the identification of BACE1, treatments that address the underlying pathological mechanisms of AD are still lacking.     

Modulation of amyloid precursor protein processing by synthetic ceramide analogues

Previous studies suggest that membrane lipids may regulate proteolytic processing of the amyloid precursor protein (APP) to generate amyloid-beta peptide (Abeta). In the present study, we have assessed the capacity for a series of structurally related synthetic ceramide analogues to modulate APP processing in vitro. The compounds tested are established glucosylceramide synthase (GS) inhibitors based on the d-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) structure. PDMP and related compounds PPMP and EtDO-P4 inhibited Abeta secretion from Chinese hamster ovary cells expressing human APP (CHO-APP) with approximate IC₅₀ values of 15, 5, and 1 μM, respectively. A trend for reduced secretion of the APP alpha-secretase product, sAPPalpha, was also observed in PDMP-treated cells but not in PPMP- or ETDO-P4-treated cells, whereas levels of the cellular beta-secretase product APP C-terminal fragment, CTFbeta, were increased by both PDMP and PPMP but unaltered with EtDO-P4 treatment. Our data also revealed that EtDO-P4 inhibits endogenous Abeta production by human neurons. In conclusion, this study provides novel information regarding the regulation of APP processing by synthetic ceramide analogues and reveals that the most potent of these compounds is EtDO-P4.     

Inhibition of amyloid beta-peptide production by blockage of beta-secretase cleavage site of amyloid precursor protein

Amyloid beta-peptide (Abeta) is implicated as the major causative agent in Alzheimer's disease (AD). Abeta is produced by the processing of the amyloid precursor protein (APP) by BACE1 (beta-secretase) and gamma-secretase. Many inhibitors have been developed for the secretases. However, the inhibitors will interfere with the processing of not only APP but also of other secretase substrates. In this study, we describe the development of inhibitors that prevent production of Abeta by specific binding to the beta-cleavage site of APP. We used the hydropathic complementarity (HC) approach for the design of short peptide inhibitors. Some of the HC peptides were bound to the substrate peptide (Sub W) corresponding to the beta-cleavage site of APP and blocked its cleavage by recombinant human BACE1 (rhBACE1) in vitro. In addition, HC peptides specifically inhibited the cleavage of Sub W, and not affecting other BACE1 substrates. Chemical modification allowed an HC peptide (CIQIHF) to inhibit the processing of APP as well as the production of Abeta in the treated cells. Such novel APP-specific inhibitors will provide opportunity for the development of drugs that can be used for the prevention and treatment of AD with minimal side effects.     

Presenilin 1 controls gamma-secretase processing of amyloid precursor protein in pre-golgi compartments of hippocampal neurons.

Mutations of presenilin 1 (PS1) causing Alzheimer's disease selectively increase the secretion of the amyloidogenic betaA4(1-42), whereas knocking out the gene results in decreased production of both betaA4(1-40) and (1-42) amyloid peptides (De Strooper et al. 1998). Therefore, PS1 function is closely linked to the gamma-secretase processing of the amyloid precursor protein (APP). Given the ongoing controversy on the subcellular localization of PS1, it remains unclear at what level of the secretory and endocytic pathways PS1 exerts its activity on APP and on the APP carboxy-terminal fragments that are the direct substrates for gamma-secretase. Therefore, we have reinvestigated the subcellular localization of endogenously expressed PS1 in neurons in vitro and in vivo using confocal microscopy and fine-tuned subcellular fractionation. We show that uncleaved PS1 holoprotein is recovered in the nuclear envelope fraction, whereas the cleaved PS fragments are found mainly in post-ER membranes including the intermediate compartment (IC). PS1 is concentrated in discrete sec23p- and p58/ERGIC-53-positive patches, suggesting its localization in subdomains involved in ER export. PS1 is not found to significant amounts beyond the cis-Golgi. Surprisingly, we found that APP carboxy-terminal fragments also coenrich in the pre-Golgi membrane fractions, consistent with the idea that these fragments are the real substrates for gamma-secretase. Functional evidence that PS1 exerts its effects on gamma-secretase processing of APP in the ER/IC was obtained using a series of APP trafficking mutants. These mutants were investigated in hippocampal neurons derived from transgenic mice expressing PS1wt or PS1 containing clinical mutations (PS1(M146L) and PS1(L286V)) at physiologically relevant levels. We demonstrate that the APP-London and PS1 mutations have additive effects on the increased secretion of betaA4(1-42) relative to betaA4(1-40), indicating that both mutations operate independently. Overall, our data clearly establish that PS1 controls gamma(42)-secretase activity in pre-Golgi compartments. We discuss models that reconcile this conclusion with the effects of PS1 deficiency on the generation of betaA4(1-40) peptide in the late biosynthetic and endocytic pathways.     

Post-translational processing of beta-secretase in Alzheimer's disease

Beta-amyloid is released into the brains of Alzheimer's patients, where it aggregates and causes damage to neurons. It is cleaved proteolytically from a large transmembrane glycoprotein amyloid precursor protein by a membrane-bound protease, known as beta-secretase identified previously as the acid protease, Asp-2. We have shown previously that beta-secretase is up-regulated by increased intracellular cholesterol, and down-regulated by cholesterol biosynthesis inhibition. Here we show using mass spectrometry that discrete changes in the glycosylation and palmitoylation of beta-secretase occur when cells expressing it are treated with statins.     

Insights into the physiological function of the β‐amyloid precursor protein: beyond Alzheimer's disease

The β‐amyloid precursor protein (APP) has been extensively studied for its role as the precursor of the β‐amyloid protein (Aβ) of Alzheimer's disease. However, the normal function of APP remains largely unknown. This article reviews studies on the structure, expression and post‐translational processing of APP, as well as studies on the effects of APP in vitro and in vivo. We conclude that the published data provide strong evidence that APP has a trophic function. APP is likely to be involved in neural stem cell development, neuronal survival, neurite outgrowth and neurorepair. However, the mechanisms by which APP exerts its actions remain to be elucidated. The available evidence suggests that APP interacts both intracellularly and extracellularly to regulate various signal transduction mechanisms.

     

Neto1 associates with the NMDA receptor/amyloid precursor protein complex

Neuropilin tolloid‐like 1 (Neto1), is a CUB domain‐containing transmembrane protein that was recently identified as a novel component of the NMDA receptor complex. Here, we have investigated the possible association of Neto1 with the amyloid precursor protein (APP)695/GluN1/GluN2A and APP695/GluN1/GluN2B NMDA receptor trafficking complexes that we have previously identified. Neto1^HA was shown to co‐immunoprecipitate with assembled NMDA receptors via GluN2A or GluN2B subunits; Neto1^HA did not co‐immunoprecipitate APP695^FLAG. Co‐immunoprecipitations from mammalian cells co‐transfected with APP695^FLAG, Neto1^HA and GluN1/GluN2A or GluN1/GluN2B revealed that all four proteins co‐exist within one macromolecular complex. Immunoprecipitations from native brain tissue similarly revealed the existence of a GluN1/GluN2A or GluN2B/APP/Neto1 complex. Neto1^HA caused a reduction in the surface expression of both NMDA receptor subtypes, but had no effect on APP695^FLAG‐ or PSD‐95α^c‐Myc enhanced surface receptor expression. The Neto1 binding domain of GluN2A was mapped using GluN1/GluN2A chimeras and GluN2A truncation constructs. The extracellular GluN2A domain does not contribute to association with Neto1^HA but deletion of the intracellular tail resulted in a loss of Neto‐1^HA co‐immunoprecipitation which was paralleled by a loss of association between GluN2A and SAP102. Thus, Neto1 is concluded to be a component of APP/NMDA receptor trafficking complexes.

     

Gamma frequency light flicker regulates amyloid precursor protein trafficking for reducing β‐amyloid load in Alzheimer's disease model

Inducing gamma oscillations with non‐invasive light flicker has been reported to impact Alzheimer''s disease‐related pathology. However, it is unclear which signaling pathways are involved in reducing amyloid load. Here, we found that gamma frequency light flicker increased anchoring of amyloid precursor protein (APP) to the plasma membrane for non‐amyloidogenic processing, and then physically interacted with KCC2, a neuron‐specific K⁺‐Cl⁻ cotransporter, suggesting that it is essential to maintain surface GABA_A receptor α1 levels and reduce β‐amyloid (Aβ) production. Stimulation with such light flicker limited KCC2 internalization and subsequent degradation via both tyrosine phosphorylation and ubiquitination, leading to an increase in surface‐KCC2 levels. Specifically, PKC‐dependent phosphorylation of APP on a serine residue was induced by gamma frequency light flicker, which was responsible for maintaining plasma membrane levels of full‐length APP, leading to its reduced trafficking to endosomes and inhibiting the β‐secretase cleavage pathway. The activated PKC from the gamma frequency light flicker subsequently phosphorylated serine of KCC2 and stabilized it onto the cell surface, which contributed to the upregulation of surface GABA_A receptor α1 levels. Together, these data indicate that enhancement of APP trafficking to the plasma membrane via light flicker plays a critical modulatory role in reduction of Aβ load in Alzheimer''s disease.     

Young microglia restore amyloid plaque clearance of aged microglia

Alzheimer′s disease (AD) is characterized by deposition of amyloid plaques, neurofibrillary tangles, and neuroinflammation. In order to study microglial contribution to amyloid plaque phagocytosis, we developed a novel ex vivo model by co‐culturing organotypic brain slices from up to 20‐month‐old, amyloid‐bearing AD mouse model (APPPS1) and young, neonatal wild‐type (WT) mice. Surprisingly, co‐culturing resulted in proliferation, recruitment, and clustering of old microglial cells around amyloid plaques and clearance of the plaque halo. Depletion of either old or young microglial cells prevented amyloid plaque clearance, indicating a synergistic effect of both populations. Exposing old microglial cells to conditioned media of young microglia or addition of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) was sufficient to induce microglial proliferation and reduce amyloid plaque size. Our data suggest that microglial dysfunction in AD may be reversible and their phagocytic ability can be modulated to limit amyloid accumulation. This novel ex vivo model provides a valuable system for identification, screening, and testing of compounds aimed to therapeutically reinforce microglial phagocytosis.     

The novel beta-secretase inhibitor KMI-429 reduces amyloid beta peptide production in amyloid precursor protein transgenic and wild-type mice

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major component of the plaques, amyloid beta peptide (Abeta), is generated from amyloid precursor protein (APP) by beta- and gamma-secretase-mediated cleavage. Because beta-secretase/beta-site APP cleaving enzyme 1 (BACE1) knockout mice produce much less Abeta and grow normally, a beta-secretase inhibitor is thought to be one of the most attractive targets for the development of therapeutic interventions for AD without apparent side-effects. Here, we report the in vivo inhibitory effects of a novel beta-secretase inhibitor, KMI-429, a transition-state mimic, which effectively inhibits beta-secretase activity in cultured cells in a dose-dependent manner. We injected KMI-429 into the hippocampus of APP transgenic mice. KMI-429 significantly reduced Abeta production in vivo in the soluble fraction compared with vehicle, but the level of Abeta in the insoluble fraction was unaffected. In contrast, an intrahippocampal injection of KMI-429 in wild-type mice remarkably reduced Abeta production in both the soluble and insoluble fractions. Our results indicate that the beta-secretase inhibitor KMI-429 is a promising candidate for the treatment of AD.     

EFhd2 is a novel amyloid protein associated with pathological tau in Alzheimer's disease

EFhd2 is a conserved calcium‐binding protein, abundant within the central nervous system. Previous studies identified EFhd2 associated with pathological forms of tau proteins in the tauopathy mouse model JNPL3, which expresses the human tau^P301L mutant. This association was validated in human tauopathies, such as Alzheimer's disease (AD). However, the role that EFhd2 may play in tauopathies is still unknown. Here, we show that EFhd2 formed amyloid structures in vitro, a capability that is reduced by calcium ions. Electron microscopy (EM) analyses demonstrated that recombinant EFhd2 formed filamentous structures. EM analyses of sarkosyl‐insoluble fractions derived from human AD brains also indicated that EFhd2 co‐localizes with aggregated tau proteins and formed granular structures. Immunohistological analyses of brain slices demonstrated that EFhd2 co‐localizes with pathological tau proteins in AD brains, confirming the co‐aggregation of EFhd2 and pathological tau. Furthermore, EFhd2's coiled‐coil domain mediated its self‐oligomerization in vitro and its association with tau proteins in JNPL3 mouse brain extracts. The results demonstrate that EFhd2 is a novel amyloid protein associated with pathological tau proteins in AD brain and that calcium binding may regulate the formation of EFhd2's amyloid structures. Hence, EFhd2 may play an important role in the pathobiology of tau‐mediated neurodegeneration.     

High‐energy compounds promote physiological processing of Alzheimer's amyloid‐β precursor protein and boost cell survival in culture

Physiological or α‐processing of amyloid‐β precursor protein (APP) prevents the formation of Aβ, which is deposited in the aging brain and may contribute to Alzheimer's disease. As such, drugs promoting this pathway could be useful for prevention of the disease. Along this line, we searched through a number of substances and unexpectedly found that a group of high‐energy compounds (HECs), namely ATP, phosphocreatine, and acetyl coenzyme A, potently increased APP α‐processing in cultured SH‐SY5Y cells, whereas their cognate counterparts, i.e., ADP, creatine, or coenzyme A did not show the same effects. Other HECs such as GTP, CTP, phosphoenol pyruvate, and S‐adenosylmethionine also promoted APP α‐processing with varying potencies and the effects were abolished by energy inhibitors rotenone or NaN₃. The overall efficacy of the HECs in the process ranged from three‐ to four‐fold, which was significantly greater than that exhibited by other physiological stimulators such as glutamate and nicotine. This suggested that the HECs were perhaps the most efficient physiological stimulators for APP α‐processing. Moreover, the HECs largely offset the inefficient APP α‐processing in aged human fibroblasts or in cells impaired by rotenone or H₂O₂. Most importantly, some HECs markedly boosted the survival rate of SH‐SY5Y cells in the death process induced by energy suppression or oxidative stress. These findings suggest a new, energy‐dependent regulatory mechanism for the putative α‐secretase and thus will help substantially in its identification. At the same time, the study raises the possibility that the HECs may be useful to energize and strengthen the aging brain cells to slow down the progression of Alzheimer's disease.