Cross-talk of membrane lipids and Alzheimer-related proteins

Alzheimer’s disease (AD) is neuropathologically characterized by the combined occurrence of extracellular β-amyloid plaques and intracellular neurofibrillary tangles in the brain. While plaques contain aggregated forms of the amyloid β-peptide (Aβ), tangles are formed by fibrillar forms of the microtubule associated protein tau. All mutations identified so far to cause familial forms of early onset AD (FAD) are localized close to or within the Aβ domain of the amyloid precursor protein (APP) or in the presenilin proteins that are essential components of a protease complex involved in the generation of Aβ. Mutations in the tau gene are not associated with FAD, but can cause other forms of dementia. The genetics of FAD together with biochemical and cell biological data, led to the formulation of the amyloid hypothesis, stating that accumulation and aggregation of Aβ is the primary event in the pathogenesis of AD, while tau might mediate its toxicity and neurodegeneration.The generation of Aβ involves sequential proteolytic cleavages of the amyloid precursor protein (APP) by enzymes called β-and γ-secretases. Notably, APP itself as well as the secretases are integral membrane proteins. Thus, it is very likely that membrane lipids are involved in the regulation of subcellular transport, activity, and metabolism of AD related proteins.Indeed, several studies indicate that membrane lipids, including cholesterol and sphingolipids (SLs) affect Aβ generation and aggregation. Interestingly, APP and other AD associated proteins, including β-and γ-secretases can, in turn, influence lipid metabolic pathways. Here, we review the close connection of cellular lipid metabolism and AD associated proteins and discuss potential mechanisms that could contribute to initiation and progression of AD.     

The role of membrane trafficking in the processing of amyloid precursor protein and production of amyloid peptides in Alzheimer's disease

Alzheimer's disease (AD) is characterized by progressive accumulation of misfolded proteins, which form senile plaques and neurofibrillary tangles, and the release of inflammatory mediators by innate immune responses. β-Amyloid peptide (Aβ) is derived from sequential processing of the amyloid precursor protein (APP) by membrane-bound proteases, namely the β-secretase, BACE1, and γ-secretase. Membrane trafficking plays a key role in the regulation of APP processing as both APP and the processing secretases traffic along distinct pathways. Genome wide sequencing studies have identified several AD susceptibility genes which regulate membrane trafficking events. To understand the pathogenesis of AD it is critical that the cell biology of APP and Aβ production in neurons is well defined. This review discusses recent advances in unravelling the membrane trafficking events associated with the production of Aβ, and how AD susceptible alleles may perturb the sorting and transport of APP and BACE1. Mechanisms whereby inflammation may influence APP processing are also considered.     

Intracellular trafficking of the β-secretase and processing of amyloid precursor protein

The main component of the amyloid plaques found in the brains of those with Alzheimer's disease (AD) is a polymerized form of the β-amyloid peptide (Aβ) and is considered to play a central role in the pathogenesis of this neurodegenerative disorder. Aβ is derived from the proteolytic processing of the amyloid precursor protein (APP). Beta site APP-cleaving enzyme, BACE1 (also known as β-secretase) is a membrane-bound aspartyl protease responsible for the initial step in the generation of Aβ peptide and is thus a prime target for therapeutic intervention. Substantive evidence now indicates that the processing of APP by BACE1 is regulated by the intracellular sorting of the enzyme and, moreover, perturbations in these intracellular trafficking pathways have been linked to late-onset AD. In this review, we highlight the recent advances in the understanding of the regulation of the intracellular sorting of BACE1 and APP and illustrate why the trafficking of these cargos represent a key issue for understanding the membrane-mediated events associated with the generation of the neurotoxic Aβ products in AD.     

Membrane trafficking pathways in Alzheimer's disease

Membrane proteins are constantly being trafficked in cells and the relevant proteins in Alzheimer's disease (AD), such as the amyloid precursor protein (APP) and its processing enzymes, are not exempted from that. Molecular cell biologists have been endeavoring to ascertain a roadmap for APP processing and trafficking in various cell types including neurons. This has led to the identification of numerous regulatory sorting mechanisms, protein-protein interactions and lipidic microenvironments that largely define how and where the substrate APP meets its processing enzymes. However, the cell biology of tau, and the formation of neurofibrillary tangles, has long been regarded as a separate field. Nonetheless, recent progress is bringing both worlds together in a new paradigm on how Aβ toxicity and tau are physiologically connected. Here, we discuss an update of our current appraisal on how membrane trafficking may play an important role in the pathogenesis of the disease and how this could be exploited for effective therapy.     

Retromer Regulates Postendocytic Sorting of β-Secretase in Polarized Madin-Darby Canine Kidney Cells

β-Amyloid (Aβ) peptides are generated from the successive proteolytic processing of the amyloid precursor protein (APP) by the β-APP cleaving enzyme (BACE or β-secretase) and the γ-secretase complex. Initial cleavage of APP by BACE leads into the amyloidogenic pathway, causing or exacerbating Alzheimer's disease. Therefore, their intracellular traffic can determine how easily and frequently BACE has access to and cleaves APP. Here, we have used polarized Madin-Darby canine kidney (MDCK) cells stably expressing APP and BACE to examine the regulation of their polarized trafficking by retromer, a protein complex previously implicated in their endosome-to-Golgi transport. Our data show that retromer interacts with BACE and regulates its postendocytic sorting in polarized MDCK cells. Depleting retromer, inhibiting retromer function, or preventing BACE interaction with retromer, alters trafficking of BACE, which thereby increases its localization in the early endocytic compartment. As a result, this slows endocytosis of apically localized BACE, promoting its recycling and apical-to-basolateral transcytosis, which increases APP/BACE interaction and subsequent cleavage of APP toward generation and secretion of Aβ peptides.     

Presenilins: how much more than γ-secretase?!

AD (Alzheimer's disease) is a neurodegenerative disease characterized by a gradual loss of neurons and the accumulation of neurotoxic Aβ (amyloid β-peptide) and hyperphosphorylated tau. The discovery of mutations in three genes, PSEN1 (presenilin 1), PSEN2 (presenilin 2) and APP (amyloid precursor protein), in patients with FAD (familial AD) has made an important contribution towards an understanding of the disease aetiology; however, a complete molecular mechanism is still lacking. Both presenilins belong to the γ-secretase complex, and serve as the catalytic entity needed for the final cleavage of APP into Aβ. PSEN only functions within the γ-secretase complex through intra- and inter-molecular interactions with three other membrane components, including nicastrin, Aph-1 (anterior pharynx defective-1) and Pen-2 (PSEN enhancer-2). However, although the list of γ-secretase substrates is still expanding, other non-catalytic activities of presenilins are also increasing the complexity behind its molecular contribution towards AD. These γ-secretase-independent roles are so far mainly attributed to PSEN1, including the transport of membrane proteins, cell adhesion, ER (endoplasmic reticulum) Ca(2+) regulation and cell signalling. In the present minireview, we discuss the current understanding of the γ-secretase-independent roles of PSENs and their possible implications in respect of AD.     

Autoreactive-Aβ antibodies promote APP β-secretase processing

Several prior investigations of Alzheimer's disease (AD) patients have indicated naturally occurring autoantibodies against amyloid-β (Aβ) species are produced. Although many studies have focused on the relative concentrations or binding affinities of autoantibodies against Aβ-related proteins in AD and aging, data regarding their functional properties are limited. It is generally believed that these antibodies act to aid in clearance of Aβ. However, as antibodies which bind to Aβ also typically bind to the parent amyloid precursor protein (APP), we reasoned that certain Aβ-targeting autoantibodies may bind to APP thereby altering its conformation and processing. Here we show for the first time, that naturally occurring Aβ-reactive autoantibodies isolated from AD patients, but not from healthy controls, promote β-secretase activity in cultured cells. Furthermore, using monoclonal antibodies to various regions of Aβ, we found that antibodies generated against the N-terminal region, especially Aβ(1-17) , dose dependently promoted amyloidogenic processing of APP viaβ-secretase activation. Thus, this property of certain autoantibodies in driving Aβ generation could be of etiological importance in the development of sporadic forms of AD. Furthermore, future passive or active anti-Aβ immunotherapies must consider potential off-target effects resulting from antibodies targeting the N-terminus of Aβ, as co-binding to the corresponding region of APP may actually enhance Aβ generation.     

Hormonal control of cerebral amyloidogenesis in Alzheimer's diseases

Estrogen reduces the risk of Alzheimer disease (AD) in postmenopausal women, β‐amyloid (Aβ) burden in animal models of AD, and secretion of Aβ from neuronal cultures. The biological basis for these effects remains unknown. Aβ is proteolytically derived from the β‐amyloid precursor protein (βAPP) within the secretory pathway by distinct enzymatic activities known as β‐ and gamma‐secretase. Aggregated Aβ peptides are found predominantly within extraneuronal space and are believed to initiate toxic and inflammatory cascades leading to neuronal death. The major population of secreted Aβ peptides is generated within the trans‐Golgi‐network (TGN), also the major site of βAPP residence in neurons at steady state. Utilizing cell‐free systems derived from both neuroblastoma cells and primary neurons, we demonstrate that 17β‐estradiol (17β‐E2) stimulates formation of vesicles containing βAPP, from the TGN. Accelerated βAPP trafficking precludes maximal Aβ generation within the TGN. 17β‐E2 appears to modulate TGN phospholipid levels, particularly those of phosphatidylinositol, and recruit soluble trafficking factors, such as Rab11, to the TGN. Together, these results suggest that estrogen may exert its anti‐Aβ effects by regulating βAPP trafficking within the late secretory pathway. These results suggest a novel mechanism through which 17β‐E2 may act in estrogen‐responsive tissues and illustrate how altering the kinetics of a protein's transport can influence its metabolic fate.     

Sphingolipid storage impairs autophagic clearance of Alzheimer-associated proteins

Recent work from our laboratory demonstrates that the accumulation of sphingolipids (SLs) decreases the capacity of cells to clear potentially amyloidogenic fragments of the amyloid precursor protein (APP) during autophagy. APP is a type I membrane protein and could undergo sequential proteolytic processing by β- and γ-secretase resulting in the generation of the amyloid β-peptide (Aβ). Genetic, molecular and biochemical evidence indicates that the accumulation of toxic Aβ aggregates plays a critical role in the degeneration of neurons during the pathogenesis of Alzheimer disease (AD). Thus, SL storage could promote the accumulation of Ab in endosomal and lysosomal compartments and thereby induce characteristic cytopathological changes of AD.     

Sphingolipid storage impairs autophagic clearance of Alzheimer-associated proteins

Recent work from our laboratory demonstrates that the accumulation of sphingolipids (SLs) decreases the capacity of cells to clear potentially amyloidogenic fragments of the amyloid precursor protein (APP) during autophagy. APP is a type I membrane protein and could undergo sequential proteolytic processing by β- and γ-secretase resulting in the generation of the amyloid β-peptide (Aβ). Genetic, molecular and biochemical evidence indicates that the accumulation of toxic Aβ aggregates plays a critical role in the degeneration of neurons during the pathogenesis of Alzheimer disease (AD). Thus, SL storage could promote the accumulation of Ab in endosomal and lysosomal compartments and thereby induce characteristic cytopathological changes of AD.     

The amyloid precursor protein and its homologues: structural and functional aspects of native and pathogenic oligomerization

Over the last 25 years, remarkable progress has been made not only in identifying key molecules of Alzheimer's disease but also in understanding their meaning in the pathogenic state. One hallmark of Alzheimer pathology is the amyloid plaque. A major component of the extracellular deposit is the amyloid-β (Aβ) peptide which is generated from its larger precursor molecule, i.e., the amyloid precursor protein (APP) by consecutive cleavages. Processing is exerted by two enzymes, i.e., the β-secretase and the γ-secretase. We and others have found that the self-association of the amyloid peptide and the dimerization and oligomerization of these proteins is a key factor under native and pathogenic conditions. In particular, the Aβ homodimer represents a nidus for plaque formation and a well defined therapeutic target. Further, dimerization of the APP was reported to increase generation of toxic Aβ whereas heterodimerization with its homologues amyloid precursor like proteins (APLP1 and APLP2) decreased Aβ formation. This review mainly focuses on structural features of the homophilic and heterophilic interactions among APP family proteins. The proposed contact sites are described and the consequences of protein dimerization on their functions and in the pathogenesis of Alzheimer's disease are discussed.     

N-cadherin enhances APP dimerization at the extracellular domain and modulates Aβ production

Sequential processing of amyloid precursor protein (APP) by β- and γ-secretase leads to the generation of amyloid-β (Aβ) peptides, which plays a central role in Alzheimer's disease pathogenesis. APP is capable of forming a homodimer through its extracellular domain as well as transmembrane GXXXG motifs. A number of reports have shown that dimerization of APP modulates Aβ production. On the other hand, we have previously reported that N-cadherin-based synaptic contact is tightly linked to Aβ production. In the present report, we investigated the effect of N-cadherin expression on APP dimerization and metabolism. Here, we demonstrate that N-cadherin expression facilitates cis-dimerization of APP. Moreover, N-cadherin expression led to increased production of Aβ as well as soluble APPβ, indicating that β-secretase-mediated cleavage of APP is enhanced. Interestingly, N-cadherin expression affected neither dimerization of C99 nor Aβ production from C99, suggesting that the effect of N-cadherin on APP metabolism is mediated through APP extracellular domain. We confirmed that N-cadherin enhances APP dimerization by a novel luciferase-complementation assay, which could be a platform for drug screening on a high-throughput basis. Taken together, our results suggest that modulation of APP dimerization state could be one of mechanisms, which links synaptic contact and Aβ production.     

Huntingtin associated protein 1 regulates trafficking of the amyloid precursor protein and modulates amyloid beta levels in neurons

J. Neurochem. (2012) 122, 1010-1022. ABSTRACT: Amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease. It is axonally transported, endocytosed and sorted to different cellular compartments where amyloid beta (Aβ) is produced. However, the mechanism of APP trafficking remains unclear. We present evidence that huntingtin associated protein 1 (HAP1) may reduce Aβ production by regulating APP trafficking to the non-amyloidogenic pathway. HAP1 and APP are highly colocalized in a number of brain regions, with similar distribution patterns in both mouse and human brains. They are associated with each other, the interacting site is the 371-599 of HAP1. APP is more retained in cis-Golgi, trans-Golgi complex, early endosome and ER-Golgi intermediate compartment in HAP1-/- neurons. HAP1 deletion significantly alters APP endocytosis and reduces the re-insertion of APP into the cytoplasmic membrane. Amyloid precursor protein-YFP(APP-YFP) vesicles in HAP1-/- neurons reveal a decreased trafficking rate and an increased number of motionless vesicles. Knock-down of HAP1 protein in cultured cortical neurons of Alzheimer's disease mouse model increases Aβ levels. Our data suggest that HAP1 regulates APP subcellular trafficking to the non-amyloidogenic pathway and may negatively regulate Aβ production in neurons.     

The interaction of beta-amyloid protein with cellular membranes stimulates its own production

Gradual changes in steady-state levels of beta amyloid peptides (Aβ) in brain are considered an initial step in the amyloid cascade hypothesis of Alzheimer's disease. Aβ is a product of the secretase cleavage of amyloid precursor protein (APP). There is evidence that the membrane lipid environment may modulate secretase activity and alters its function. Cleavage of APP strongly depends on membrane properties. Since Aβ perturbs cell membrane fluidity, the cell membrane may be the location where the neurotoxic cascade of Aβ is initiated. Therefore, we tested effects of oligomeric Aβ on membrane fluidity of whole living cells, the impact of exogenous and cellular Aβ on the processing of APP and the role of GM-1 ganglioside. We present evidence that oligoAβ_(1-40) stimulates the amyloidogenic processing of APP by reducing membrane fluidity and complexing with GM-1 ganglioside. This dynamic action of Aβ may start a vicious circle, where endogenous Aβ stimulates its own production. Based on our novel findings, we propose that oligoAβ_(1-40) accelerates the proteolytic cleavage of APP by decreasing membrane fluidity.     

Β-site APP-cleaving enzyme 1 trafficking and Alzheimer's disease pathogenesis

β-Site APP-cleaving enzyme (BACE1) cleaves the amyloid precursor protein (APP) at the β-secretase site to initiate the production of Aβ peptides. These accumulate to form toxic oligomers and the amyloid plaques associated with Alzheimer's disease (AD). An increase of BACE1 levels in the brain of AD patients has been mostly attributed to alterations of its intracellular trafficking. Golgi-associated adaptor proteins, GGA sort BACE1 for export to the endosomal compartment, which is the major cellular site of BACE1 activity. BACE1 undergoes recycling between endosome, trans-Golgi network (TGN), and the plasma membrane, from where it is endocytosed and either further recycled or retrieved to the endosome. Phosphorylation of Ser498 facilitates BACE1 recognition by GGA1 for retrieval to the endosome. Ubiquitination of BACE1 C-terminal Lys501 signals GGA3 for exporting BACE1 to the lysosome for degradation. In addition, the retromer mediates the retrograde transport of BACE1 from endosome to TGN. Decreased levels of GGA proteins and increased levels of retromer-associated sortilin have been associated with AD. Both would promote the co-localization of BACE1 and the amyloid precursor protein in the TGN and endosomes. Decreased levels of GGA3 also impair BACE1 degradation. Further understanding of BACE1 trafficking and its regulation may offer new therapeutic approaches for the treatment of Alzheimer's disease.     

Lipoprotein receptors and cholesterol in APP trafficking and proteolytic processing,implications for Alzheimer's disease

Amyloid-β (Aβ) peptide accumulation in the brain is central to the pathogenesis of Alzheimer's disease (AD). Aβ is produced through proteolytic processing of a transmembrane protein, β-amyloid precursor protein (APP), by β- and γ-secretases. Mounting evidence has demonstrated that alterations in APP cellular trafficking and localization directly impact its processing to Aβ. Members of the low-density lipoprotein receptor family, including LRP, LRP1B, SorLA/LR11, and apoER2, interact with APP and regulate its endocytic trafficking. Additionally, APP trafficking and processing are greatly affected by cellular cholesterol content. In this review, we summarize the current understanding of the roles of lipoprotein receptors and cholesterol in APP trafficking and processing and their implication for AD pathogenesis and therapy.     

Altering APP proteolysis: increasing sAPPalpha production by targeting dimerization of the APP ectodomain

One of the events associated with Alzheimer's disease is the dysregulation of α- versus β-cleavage of the amyloid precursor protein (APP). The product of α-cleavage (sAPPα) has neuroprotective properties, while Aβ1-42 peptide, a product of β-cleavage, is neurotoxic. Dimerization of APP has been shown to influence the relative rate of α- and β- cleavage of APP. Thus finding compounds that interfere with dimerization of the APP ectodomain and increase the α-cleavage of APP could lead to the development of new therapies for Alzheimer's disease. Examining the intrinsic fluorescence of a fragment of the ectodomain of APP, which dimerizes through the E2 and Aβ-cognate domains, revealed significant changes in the fluorescence of the fragment upon binding of Aβ oligomers--which bind to dimers of the ectodomain--and Aβ fragments--which destabilize dimers of the ectodomain. This technique was extended to show that RERMS-containing peptides (APP(695) 328-332), disulfiram, and sulfiram also inhibit dimerization of the ectodomain fragment. This activity was confirmed with small angle x-ray scattering. Analysis of the activity of disulfiram and sulfiram in an AlphaLISA assay indicated that both compounds significantly enhance the production of sAPPα by 7W-CHO and B103 neuroblastoma cells. These observations demonstrate that there is a class of compounds that modulates the conformation of the APP ectodomain and influences the ratio of α- to β-cleavage of APP. These compounds provide a rationale for the development of a new class of therapeutics for Alzheimer's disease.     

Cellular Membrane Fluidity in Amyloid Precursor Protein Processing

The senile plaque is a pathologic hallmark of Alzheimer's disease (AD). Amyloid-β peptide (Aβ), the main constituent of senile plaques, is neurotoxic especially in its oligomeric form. Aβ is derived from the sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases in the amyloidogenic pathway. Alternatively, APP can be cleaved by α-secretases within the Aβ domain to produce neurotrophic and neuroprotective α-secretase-cleaved soluble APP (sAPPα) in the nonamyloidogenic pathway. Since APP and α-, β-, and γ-secretases are membrane proteins, APP processing should be highly dependent on the membrane composition and the biophysical properties of cellular membrane. In this review, we discuss the role of the biophysical properties of cellular membrane in APP processing, especially the effects of phospholipases A₂ (PLA₂s), fatty acids, cholesterol, and Aβ on membrane fluidity in relation to their effects on APP processing.     

Aging impact on amyloid precursor protein neuronal trafficking

Neurons live a lifetime. Neuronal aging may increase the risk of Alzheimer's disease. How does neuronal membrane trafficking maintain synapse function during aging? In the normal aged brain, intraneuronal beta-amyloid (Aβ) accumulates without Alzheimer's disease mutations or risk variants. However, do changes with neuronal aging potentiate Aβ accumulation? We reviewed the membrane trafficking of the amyloid precursor protein in neurons and highlighted its importance in Aβ production. Importantly, we reviewed the evidence supporting the impact of aging on neuronal membrane trafficking, APP processing, and consequently Aβ production. Dissecting the molecular regulators of APP trafficking during neuronal aging is required to identify strategies to delay synaptic decline and protect from Alzheimer's disease.     

Cholesterol-depletion corrects APP and BACE1 misstrafficking in NPC1-deficient cells

Cholesterol accumulation in Niemann-Pick type C disease (NPC) causes increased levels of the amyloid-precursor-protein C-terminal fragments (APP-CTFs) and intracellular amyloid-β peptide (Aβ), the two central molecules in Alzheimer's disease (AD) pathogenesis. We previously reported that cholesterol accumulation in NPC-cells leads to cholesterol-dependent increased APP processing by β-secretase (BACE1) and decreased APP expression at the cell surface (Malnar et al. Biochim Biophys Acta. 1802 (2010) 682-691.). We hypothesized that increased formation of APP-CTFs and Aβ in NPC disease is due to cholesterol-mediated altered endocytic trafficking of APP and/or BACE1. Here, we show that APP endocytosis is prerequisite for enhanced Aβ levels in NPC-cells. Moreover, we observed that NPC cells show cholesterol dependent sequestration and colocalization of APP and BACE1 within enlarged early/recycling endosomes which can lead to increased β-secretase processing of APP. We demonstrated that increased endocytic localization of APP in NPC-cells is likely due to both its increased internalization and its decreased recycling to the cell surface. Our findings suggest that increased cholesterol levels, such as in NPC disease and sporadic AD, may be the upstream effector that drives amyloidogenic APP processing characteristic for Alzheimer's disease by altering endocytic trafficking of APP and BACE1.