Copper promotes the trafficking of the amyloid precursor protein

Accumulation of the amyloid β peptide in the cortical and hippocampal regions of the brain is a major pathological feature of Alzheimer disease. Amyloid β peptide is generated from the sequential protease cleavage of the amyloid precursor protein (APP). We reported previously that copper increases the level of APP at the cell surface. Here we report that copper, but not iron or zinc, promotes APP trafficking in cultured polarized epithelial cells and neuronal cells. In SH-SY5Y neuronal cells and primary cortical neurons, copper promoted a redistribution of APP from a perinuclear localization to a wider distribution, including neurites. Importantly, a change in APP localization was not attributed to an up-regulation of APP protein synthesis. Using live cell imaging and endocytosis assays, we found that copper promotes an increase in cell surface APP by increasing its exocytosis and reducing its endocytosis, respectively. This study identifies a novel mechanism by which copper regulates the localization and presumably the function of APP, which is of major significance for understanding the role of APP in copper homeostasis and the role of copper in Alzheimer disease.     

Role of phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) in intracellular amyloid precursor protein (APP) processing and amyloid plaque pathogenesis

One of the pathological hallmarks of Alzheimer disease is the accumulation of amyloid plaques in the extracellular space in the brain. Amyloid plaques are primarily composed of aggregated amyloid β peptide (Aβ), a proteolytic fragment of the transmembrane amyloid precursor protein (APP). For APP to be proteolytically cleaved into Aβ, it must be internalized into the cell and trafficked to endosomes where specific protease complexes can cleave APP. Several recent genome-wide association studies have reported that several single nucleotide polymorphisms (SNPs) in the phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) gene were significantly associated with Alzheimer disease, suggesting a role in APP endocytosis and Aβ generation. Here, we show that PICALM co-localizes with APP in intracellular vesicles of N2a-APP cells after endocytosis is initiated. PICALM knockdown resulted in reduced APP internalization and Aβ generation. Conversely, PICALM overexpression increased APP internalization and Aβ production. In vivo, PICALM was found to be expressed in neurons and co-localized with APP throughout the cortex and hippocampus in APP/PS1 mice. PICALM expression was altered using AAV8 gene transfer of PICALM shRNA or PICALM cDNA into the hippocampus of 6-month-old APP/PS1 mice. PICALM knockdown decreased soluble and insoluble Aβ levels and amyloid plaque load in the hippocampus. Conversely, PICALM overexpression increased Aβ levels and amyloid plaque load. These data indicate that PICALM, an adaptor protein involved in clathrin-mediated endocytosis, regulates APP internalization and subsequent Aβ generation. PICALM contributes to amyloid plaque load in brain likely via its effect on Aβ metabolism.     

Soluble amyloid precursor protein induces rapid neural differentiation of human embryonic stem cells

Human embryonic stem cells (hESCs) offer tremendous potential for not only treating neurological disorders but also for their ability to serve as vital reagents to model and investigate human disease. To further our understanding of a key protein involved in Alzheimer disease pathogenesis, we stably overexpressed amyloid precursor protein (APP) in hESCs. Remarkably, we found that APP overexpression in hESCs caused a rapid and robust differentiation of pluripotent stem cells toward a neural fate. Despite maintenance in standard hESC media, up to 80% of cells expressed the neural stem cell marker nestin, and 65% exhibited the more mature neural marker β-3 tubulin within just 5 days of passaging. To elucidate the mechanism underlying the effects of APP on neural differentiation, we examined the proteolysis of APP and performed both gain of function and loss of function experiments. Taken together, our results demonstrate that the N-terminal secreted soluble forms of APP (in particular sAPPβ) robustly drive neural differentiation of hESCs. Our findings not only reveal a novel and intriguing role for APP in neural lineage commitment but also identify a straightforward and rapid approach to generate large numbers of neurons from human embryonic stem cells. These novel APP-hESC lines represent a valuable tool to investigate the potential role of APP in development and neurodegeneration and allow for insights into physiological functions of this protein.     

Ubiquilin-1 is a molecular chaperone for the amyloid precursor protein

Alzheimer disease (AD) is associated with extracellular deposition of proteolytic fragments of amyloid precursor protein (APP). Although mutations in APP and proteases that mediate its processing are known to result in familial, early onset forms of AD, the mechanisms underlying the more common sporadic, yet genetically complex forms of the disease are still unclear. Four single-nucleotide polymorphisms within the ubiquilin-1 gene have been shown to be genetically associated with AD, implicating its gene product in the pathogenesis of late onset AD. However, genetic linkage between ubiquilin-1 and AD has not been confirmed in studies examining different populations. Here we show that regardless of genotype, ubiquilin-1 protein levels are significantly decreased in late onset AD patient brains, suggesting that diminished ubiquilin function may be a common denominator in AD progression. Our interrogation of putative ubiquilin-1 activities based on sequence similarities to proteins involved in cellular quality control showed that ubiquilin-1 can be biochemically defined as a bona fide molecular chaperone and that this activity is capable of preventing the aggregation of amyloid precursor protein both in vitro and in live neurons. Furthermore, we show that reduced activity of ubiquilin-1 results in augmented production of pathogenic amyloid precursor protein fragments as well as increased neuronal death. Our results support the notion that ubiquilin-1 chaperone activity is necessary to regulate the production of APP and its fragments and that diminished ubiquilin-1 levels may contribute to AD pathogenesis.     

Amyloid precursor protein revisited: neuron-specific expression and highly stable nature of soluble derivatives

APP processing and amyloid-β production play a central role in Alzheimer disease pathogenesis. APP has been considered a ubiquitously expressed protein. In addition to amyloid-β, α- or β-secretase-dependent cleavage of APP also generates soluble secreted APP (APPsα or APPsβ, respectively). Interestingly, APPsβ has been shown to be subject to further cleavage to create an N-APP fragment that binds to the DR6 death receptor and mediates axon pruning and degeneration under trophic factor withdrawal conditions. By performing APP immunocytochemical staining, we found that, unexpectedly, many antibodies yielded nonspecific staining in APP-null samples. Screening of a series of antibodies allowed us to identify a rabbit monoclonal antibody Y188 that is highly specific for APP and prompted us to re-examine the expression, localization, and stability of endogenous APP and APPsβ in wild-type and in APPsβ knock-in mice, respectively. In contrast to earlier studies, we found that APP is specifically expressed in neurons and that its expression cannot be detected in major types of glial cells under basal or neuroinflammatory conditions. Both APPsα and APPsβ are highly stable in the central nervous system (CNS) and do not undergo further cleavage with or without trophic factor support. Our results clarify several key questions with regard to the fundamental properties of APP and offer critical cellular insights into the pathophysiology of APP.     

Lipoprotein lipase is a novel amyloid beta (Abeta)-binding protein that promotes glycosaminoglycan-dependent cellular uptake of Abeta in astrocytes

Lipoprotein lipase (LPL) is a member of a lipase family known to hydrolyze triglyceride molecules in plasma lipoprotein particles. LPL also plays a role in the binding of lipoprotein particles to cell-surface molecules, including sulfated glycosaminoglycans (GAGs). LPL is predominantly expressed in adipose and muscle but is also highly expressed in the brain where its specific roles are unknown. It has been shown that LPL is colocalized with senile plaques in Alzheimer disease (AD) brains, and its mutations are associated with the severity of AD pathophysiological features. In this study, we identified a novel function of LPL; that is, LPL binds to amyloid β protein (Aβ) and promotes cell-surface association and uptake of Aβ in mouse primary astrocytes. The internalized Aβ was degraded within 12 h, mainly in a lysosomal pathway. We also found that sulfated GAGs were involved in the LPL-mediated cellular uptake of Aβ. Apolipoprotein E was dispensable in the LPL-mediated uptake of Aβ. Our findings indicate that LPL is a novel Aβ-binding protein promoting cellular uptake and subsequent degradation of Aβ.     

Mitochondrial dysfunction in skeletal muscle of amyloid precursor protein-overexpressing mice

Inclusion body myositis, the most common muscle disorder in the elderly, is partly characterized by abnormal expression of amyloid precursor protein (APP) and intracellular accumulation of its proteolytic fragments collectively known as β-amyloid. The present study examined the effects of β-amyloid accumulation on mitochondrial structure and function of skeletal muscle from transgenic mice (MCK-βAPP) engineered to accumulate intramyofiber β-amyloid. Electron microscopic analysis revealed that a large fraction of myofibers from 2-3-month-old MCK-βAPP mice contained numerous, heterogeneous alterations in mitochondria, and other cellular organelles. [(1)H-decoupled](13)C NMR spectroscopy showed a substantial reduction in TCA cycle activity and indicated a switch from aerobic to anaerobic glucose metabolism in the MCK-βAPP muscle. Isolated muscle fibers from the MCK-βAPP mice also exhibited a reduction in cytoplasmic pH, an increased rate of ROS production, and a partially depolarized plasmalemma. Treatment of MCK-βAPP muscle cells with Ru360, a mitochondrial Ca(2+) uniporter antagonist, reversed alterations in the plasmalemmal membrane potential (V(m)) and pH. Consistent with altered redox state of the cells, treatment of MCK-βAPP muscle cells with glutathione reversed the effects of β-amyloid accumulation on Ca(2+) transient amplitudes. We conclude that structural and functional alterations in mitochondria precede the reported appearance of histopathological and clinical features in the MCK-βAPP mice and may represent key early events in the pathogenesis of inclusion body myositis.     

Beta-amyloid peptide variants in brains and cerebrospinal fluid from amyloid precursor protein (APP) transgenic mice: comparison with human Alzheimer amyloid

In this study, we report a detailed analysis of the different variants of amyloid-β (Aβ) peptides in the brains and the cerebrospinal fluid from APP23 transgenic mice, expressing amyloid precursor protein with the Swedish familial Alzheimer disease mutation, at different ages. Using one- and two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry, we identified the Aβ peptides Aβ(1-40), -(1-42), -(1-39), -(1-38), -(1-37), -(2-40), and -(3-40) as well as minor amounts of pyroglutamate-modified Aβ (Aβ(N3pE)) and endogenous murine Aβ in brains from 24-month-old mice. Chemical modifications of the N-terminal amino group of Aβ were identified that had clearly been introduced during standard experimental procedures. To address this issue, we additionally applied amyloid extraction in ultrapure water. Clear differences between APP23 mice and Alzheimer disease (AD) brain samples were observed in terms of the relative abundance of specific variants of Aβ peptides, such as Aβ(N3pE), Aβ(1-42), and N-terminally truncated Aβ(2/3-42). These differences to human AD amyloid were also noticed in a related mouse line transgenic for human wild type amyloid precursor protein. Taken together, our findings suggest different underlying molecular mechanisms driving the amyloid deposition in transgenic mice and AD patients.     

Intracellular pathways of folded and misfolded amyloid precursor protein degradation

A number of studies suggest that early events in the maturation of amyloid precursor protein (APP) are important in determining its entry into one of several alternative processing pathways, one of which leads to the toxic protein beta-amyloid (Abeta). In pulse-labeled APP expressing CHO cells two proteolytic systems can degrade newly translated APP: the proteosome and a cysteine protease. When N-glycosylation was inhibited by tunicamycin, the former system is the dominant mechanism of APP degradation. Without tunicamycin present, the cysteine protease is operational: cysteine protease inhibitors completely inhibit APP turnover in cells in which the secretory pathway is interrupted with brefeldin A or when alpha-secretase and endosomal degradation are also pharmacologically blocked. APP immunoprecipitated from cells extracted under mild conditions and labeled in the presence of tunicamycin exhibited greater sensitivity to endoproteinase glu-C (V8) or lys-C than from cells without drug. The V8 fragment missing in tunicamyin treated cells encompassed the KPI inhibitor insertion site but was distinct from the site of N-glycosylation. It is concluded that a conformational change caused by interrupted N-glycosylation shunts newly translated APP into the proteasomal degradation pathway. Pulse-labeled and chased cells showed an additional V8 fragment that was not present in pulsed-labeled cells and was not due to glycosylation since it was also present in cells labeled in the presence of brefeldin. This latter result indicates that an additional, delayed conformational alteration occurs in the endoplasmic reticulum.     

In vitro and in vivo degradation of Abeta peptide by peptidases coupled to erythrocytes

It is generally believed that amyloid β peptides (Aβ) are the key mediators of Alzheimer's disease. Therapeutic interventions have been directed toward impairing the synthesis or accelerating the clearance of Aβ. An equilibrium between blood and brain Aβ exists mediated by carriers that transport Aβ across the blood–brain barrier. Passive immunotherapy has been shown to be effective in mouse models of AD, where the plasma borne antibody binds plasma Aβ causing an efflux of Aβ from the brain. As an alternative to passive immunotherapy we have considered the use of Aβ-degrading peptidases to lower plasma Aβ levels. Here we compare the ability of three Aβ-degrading peptidases to degrade Aβ. Biotinylated peptidases were coupled to the surface of biotinylated erythrocytes via streptavidin. These erythrocyte-bound peptidases degrade Aβ peptide in plasma. Thus, peptidases bound to or expressed on the surface of erythroid cells represent an alternative to passive immunotherapy.     

Tissue transglutaminase-mediated glutamine deamidation of beta-amyloid peptide increases peptide solubility, whereas enzymatic cross-linking and peptide fragmentation may serve as molecular triggers for rapid peptide aggregation

Tissue transglutaminase (TGase) has been implicated in a number of cellular processes and disease states, where the enzymatic actions of TGase may serve in both, cell survival and apoptosis. To date, the precise functional properties of TGase in cell survival or cell death mechanisms still remain elusive. TGase-mediated cross-linking has been reported to account for the formation of insoluble lesions in conformational diseases. We report here that TGase induces intramolecular cross-linking of β-amyloid peptide (Aβ), resulting in structural changes of monomeric Aβ. Using high resolution mass spectrometry (MS) of cross-linked Aβ peptides, we observed a shift in mass, which is, presumably associated with the loss of NH3 due to enzymatic transamidation activity and hence intramolecular peptide cross-linking. We have observed that a large population of Aβ monomers contained an 0.984 Da increase in mass at a glutamine residue, indicating that glutamine 15 serves as an indispensable substrate in TGase-mediated deamidation to glutamate 15. We provide strong analytical evidence on TGase-mediated Aβ peptide dimerization, through covalent intermolecular cross-linking and hence the formation of Aβ1-40 dimers. Our in depth analyses indicate that TGase-induced post-translational modifications of Aβ peptide may serve as an important seed for aggregation.     

RAC1 inhibition targets amyloid precursor protein processing by gamma-secretase and decreases Abeta production in vitro and in vivo

beta-Amyloid peptides (Abeta) that form the senile plaques of Alzheimer disease consist mainly of 40- and 42-amino acid (Abeta 40 and Abeta 42) peptides generated from the cleavage of the amyloid precursor protein (APP). Generation of Abeta involves beta-secretase and gamma-secretase activities and is regulated by membrane trafficking of the proteins involved in Abeta production. Here we describe a new small molecule, EHT 1864, which blocks the Rac1 signaling pathways. In vitro, EHT 1864 blocks Abeta 40 and Abeta 42 production but does not impact sAPPalpha levels and does not inhibit beta-secretase. Rather, EHT 1864 modulates APP processing at the level of gamma-secretase to prevent Abeta 40 and Abeta 42 generation. This effect does not result from a direct inhibition of the gamma-secretase activity and is specific for APP cleavage, since EHT 1864 does not affect Notch cleavage. In vivo, EHT 1864 significantly reduces Abeta 40 and Abeta 42 levels in guinea pig brains at a threshold that is compatible with delaying plaque accumulation and/or clearing the existing plaque in brain. EHT 1864 is the first derivative of a new chemical series that consists of candidates for inhibiting Abeta formation in the brain of AD patients. Our findings represent the first pharmacological validation of Rac1 signaling as a target for developing novel therapies for Alzheimer disease.     

Role of endoplasmic reticulum, endosomal-lysosomal compartments, and microtubules in amyloid precursor protein metabolism of human neurons

A wide interest in amyloid precursor protein (APP) metabolism stems from the fact that increased amounts of amyloid beta peptide (Abeta), arising through proteolytic processing of APP, likely play a significant role in Alzheimer's disease. As Alzheimer's disease pathology is limited almost exclusively to the human species, we established human primary neuron cultures to address the possibility of distinctive APP processing in human CNS neurons. In the present study, we investigate the role of organelles and protein trafficking in APP metabolism. Using brefeldin A, we failed to detect APP processing into Abeta in the endoplasmic reticulum. Monensin and the lysomotropic agents, NH4Cl and chloroquine, revealed a bypass pH-dependent secretory pathway in a compartment between the endoplasmic reticulum and the medial Golgi, resulting in the secretion of full-length APP. Colchicine treatment resulting in the loss of neurites inhibited processing of APP through the secretory, but not the endosomal-lysosomal, pathway of APP metabolism. The serine protease inhibitor, leupeptin, indicates a role for lysosomes in APP, Abeta, and APP C-terminal fragment turnover. These results demonstrate that the regulation of APP metabolism in human neurons differs considerably from those reported in rodent CNS primary neuron cultures or continuously dividing cell types.     

Characterization of prefibrillar Tau oligomers in vitro and in Alzheimer disease

Neurofibrillary tangles, composed of insoluble aggregates of the microtubule-associated protein Tau, are a pathological hallmark of Alzheimer disease (AD) and other tauopathies. However, recent evidence indicates that neuronal dysfunction precedes the formation of these insoluble fibrillar deposits, suggesting that earlier prefibrillar Tau aggregates may be neurotoxic. To determine the composition of these aggregates, we have employed a photochemical cross-linking technique to examine intermolecular interactions of full-length Tau in vitro. Using this method, we demonstrate that dimerization is an early event in the Tau aggregation process and that these dimers self-associate to form larger oligomeric aggregates. Moreover, using these stabilized Tau aggregates as immunogens, we generated a monoclonal antibody that selectively recognizes Tau dimers and higher order oligomeric aggregates but shows little reactivity to Tau filaments in vitro. Immunostaining indicates that these dimers/oligomers are markedly elevated in AD, appearing in early pathological inclusions such as neuropil threads and pretangle neurons as well as colocalizing with other early markers of Tau pathogenesis. Taken as a whole, the work presented herein demonstrates the existence of alternative Tau aggregates that precede formation of fibrillar Tau pathologies and raises the possibility that these hierarchical oligomeric forms of Tau may contribute to neurodegeneration.     

Presenilin-1 and the amyloid precursor protein are transported bidirectionally in the sciatic nerve of adult rat

The amyloid precursor protein (APP) and presenilin-1 (PS-1) are not only of importance for the normal functioning of the various neurons, but also play central roles in the pathogenesis of Alzheimer’s disease (AD). Through the use of immunohistochemical and Western blot techniques, the bidirectional axonal transport of these proteins has been demonstrated in the sciatic nerve of adult rat. Double-ligation of the sciatic nerve for 6, 12 or 24 h was observed to cause a progressive accumulation of the 45 kDa presenilin-1 holoprotein and APPs with molecular masses of 116 and 94 kDa on both sites of the ligature. It is concluded that the functions of presenilin-1 and APPs are not restricted to the neuronal perikarya: they may carry information in both directions, from the cell body to the axon terminals and vice versa.     

Autophagic vacuoles are enriched in amyloid precursor protein-secretase activities: implications for beta-amyloid peptide over-production and localization in Alzheimer's disease

In Alzheimer’s disease (AD), the neuropathologic hallmarks of β-amyloid deposition and neurofibrillary degeneration are associated with early and progressive pathology of the endosomal–lysosomal system. Abnormalities of autophagy, a major pathway to lysosomes for protein and organelle turnover, include marked accumulations of autophagy-related vesicular compartments (autophagic vacuoles or AVs) in affected neurons. Here, we investigated the possibility that AVs contain the proteases and substrates necessary to cleave the amyloid precursor protein (APP) to Aβ peptide that forms β-amyloid, a key pathogenic factor in AD. AVs were highly purified using a well-established metrizamide gradient procedure from livers of transgenic YAC mice overexpressing wild-type human APP. By Western blot analysis, AVs contained APP, βCTF - the β-cleaved carboxyl-terminal domain of APP, and BACE, the protease-mediating β-cleavage of APP. β-Secretase activity measured against a fluorogenic peptide was significantly enriched in the AV fraction relative to whole-liver lysate. Compared to other recovered subcellular fractions, AVs exhibited the highest specific activity of γ-secretase based on a fluorogenic assay and inhibition by a specific inhibitor of γ-secretase, DAPT. AVs were also the most enriched subcellular fraction in levels of the γ-secretase components presenilin and nicastrin. Immunoelectron microscopy demonstrated selective immunogold labeling of AVs with antibodies specific for the carboxyl termini of human Aβ40 and Aβ42. These data indicate that AVs are a previously unrecognized and potentially highly active compartment for Aβ generation and suggest that the abnormal accumulation of AVs in affected neurons of the AD brain contributes to β-amyloid deposition.     

Increased generation of alternatively cleaved beta-amyloid peptides in cells expressing mutants of the amyloid precursor protein defective in endocytosis

The subcellular location of the secretases processing the beta-amyloid precursor protein (APP) is not established yet. We analyzed the generation of the beta-amyloid peptide (Abeta) in human embryonic kidney 293 cell lines stably expressing wild-type and noninternalizing mutants of human APP. APP lacking the entire cytoplasmic domain or with both tyrosine residues of the motif GYENPTY mutated to alanine showed at least fivefold reduced endocytosis. In these cell lines, the production of Abeta1-40 was substantially reduced, but accompanied by the appearance of two prominent alternative Abeta peptides differing at the amino-termini. Based on antibody reactivity and mobility in high-resolution gels in comparison with defined Abeta fragments, these peptides were identified as Abeta3-40 and Abeta5-40. Notably, these alternative Abeta peptides were not generated when the APP mutants were retained in the early secretory pathway by treatment with brefeldin A. These results indicate that the alternative processing is the result of APP accumulation at the plasma membrane and provide evidence of distinct beta-secretase activities. Cleavage amino-terminal to position 1 of Abeta occurs predominantly in endosomes, whereas the processing at positions 3 or 5 takes place at the plasma membrane.     

CX3CR1 protein signaling modulates microglial activation and protects against plaque-independent cognitive deficits in a mouse model of Alzheimer disease

Aberrant microglial activation has been proposed to contribute to the cognitive decline in Alzheimer disease (AD), but the underlying molecular mechanisms remain enigmatic. Fractalkine signaling, a pathway mediating the communication between microglia and neurons, is deficient in AD brains and down-regulated by amyloid-β. Although fractalkine receptor (CX3CR1) on microglia was found to regulate plaque load, no functional effects have been reported. Our study demonstrates that CX3CR1 deficiency worsens the AD-related neuronal and behavioral deficits. The effects were associated with cytokine production but not with plaque deposition. Ablation of CX3CR1 in mice overexpressing human amyloid precursor protein enhanced Tau pathology and exacerbated the depletion of calbindin in the dentate gyrus. The levels of calbindin in the dentate gyrus correlated negatively with those of tumor necrosis factor α and interleukin 6, suggesting neurotoxic effects of inflammatory factors. Functionally, removing CX3CR1 in human amyloid precursor protein mice worsened the memory retention in passive avoidance and novel object recognition tests, and their memory loss in the novel object recognition test is associated with high levels of interleukin 6. Our findings identify CX3CR1 as a key microglial pathway in protecting against AD-related cognitive deficits that are associated with aberrant microglial activation and elevated inflammatory cytokines.     

Serum albumin prevents protein aggregation and amyloid formation and retains chaperone-like activity in the presence of physiological ligands

Although serum albumin has an established function as a transport protein, evidence is emerging that serum albumin may also have a role as a molecular chaperone. Using established techniques to characterize chaperone interactions, this study demonstrates that bovine serum albumin: 1) preferentially binds stressed over unstressed client proteins; 2) forms stable, soluble, high molecular weight complexes with stressed client proteins; 3) reduces the aggregation of client proteins when it is present at physiological levels; and 4) inhibits amyloid formation by both WT and L55P transthyretin. Although the antiaggregatory effect of serum albumin is maintained in the presence of physiological levels of Ca(2+) and Cu(2+), the presence of free fatty acids significantly alters this activity: stabilizing serum albumin at normal levels but diminishing chaperone-like activity at high concentrations. Moreover, here it is shown that depletion of albumin from human plasma leads to a significant increase in aggregation under physiologically relevant heat and shear stresses. This study demonstrates that serum albumin possesses chaperone-like properties and that this activity is maintained under a number of physiologically relevant conditions.