Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts

Formation of senile plaques containing the beta-amyloid peptide (A beta) derived from the amyloid precursor protein (APP) is an invariant feature of Alzheimer's disease (AD). APP is cleaved either by beta-secretase or by alpha-secretase to initiate amyloidogenic (release of A beta) or nonamyloidogenic processing of APP, respectively. A key to understanding AD is to unravel how access of these enzymes to APP is regulated. Here, we demonstrate that lipid rafts are critically involved in regulating A beta generation. Reducing cholesterol levels in N2a cells decreased A beta production. APP and the beta-site APP cleavage enzyme (BACE1) could be induced to copatch at the plasma membrane upon cross-linking with antibodies and to segregate away from nonraft markers. Antibody cross-linking dramatically increased production of A beta in a cholesterol-dependent manner. A beta generation was dependent on endocytosis and was reduced after expression of the dynamin mutant K44A and the Rab5 GTPase-activating protein, RN-tre. This inhibition could be overcome by antibody cross-linking. These observations suggest the existence of two APP pools. Although APP inside raft clusters seems to be cleaved by beta-secretase, APP outside rafts undergoes cleavage by alpha-secretase. Thus, access of alpha- and beta-secretase to APP, and therefore A beta generation, may be determined by dynamic interactions of APP with lipid rafts.     

Compartmentalization of beta-secretase (Asp2) into low-buoyant density, noncaveolar lipid rafts

Recent epidemiological studies show a reduced prevalence of Alzheimer's disease (AD) in patients treated with inhibitors of cholesterol biosynthesis. Moreover, the cholesterol-transport protein, apolipoprotein E4, and elevated cholesterol are important risk factors for AD. Additionally, in vitro and in vivo studies show that intracellular cholesterol levels can modulate the processing of amyloid precursor protein (APP) to beta-amyloid, the major constituent of senile plaques. Cholesterol plays a crucial role in maintaining lipid rafts in a functional state. Lipid rafts are cholesterol-enriched membrane microdomains implicated in signal transduction, protein trafficking, and proteolytic processing. Since APP, beta-amyloid, and the putative gamma-secretase, presenilin-1 (PS-1), have all been found in lipid rafts, we hypothesized that the recently identified beta-secretase, Asp2 (BACE1), might also be present in rafts. Here, we report that recombinant Asp2 expressed in three distinct cell lines is raft associated. Using both detergent and nondetergent methods, Asp2 protein and activity were found in a light membrane raft fraction that also contained other components of the amyloidogenic pathway. Immunoisolation of caveolin-containing vesicles indicated that Asp2 was present in a unique raft population distinct from caveolae. Finally, depletion of raft cholesterol abrogated association of Asp2 with the light membrane fraction. These observations are consistent with the raft localization of APP processing and suggest that the partitioning of Asp2 into lipid rafts may underlie the cholesterol sensitivity of beta-amyloid production.     

The beta-amyloid precursor protein is not processed by the regulated secretory pathway.

The beta-amyloid peptide is derived from a larger membrane bound protein and accumulates as amyloid in Alzheimer's diseased brains. beta-amyloid precursor protein (beta APP) proteolytically processed during constitutive secretion cannot be a source of deposited amyloid because this processing results in cleavage within the amyloidogenic peptide. To see if other secretory pathways could be responsible for generating potentially amyloidogenic molecules we tested the possibility that beta APP is targeted to the regulated secretory pathway. Stable AtT20 cell lines expressing exogenous human beta APP were genetically engineered. These cells were labeled with [35S]-methionine, and chased in the presence or absence of secretagogue. The beta APP both inside the cells and released from the cells was analyzed by immunoprecipitation and gel analysis. Quantitation of autoradiograms showed that virtually all of the synthesized beta APP was secreted by the constitutive pathway, and that no detectable (less than 1%) beta APP was targeted to the regulated secretory pathway.     

The cholesterol transporter ABCG1 modulates the subcellular distribution and proteolytic processing of beta-amyloid precursor protein

Although intracellular cholesterol levels are known to influence the proteolysis of beta-amyloid precursor protein (APP), the effect of specific genes that regulate cholesterol metabolism on APP processing remains poorly understood. The cholesterol transporter ABCG1 facilitates cholesterol efflux to HDL and is expressed in brain. Notably, the human ABCG1 gene maps to chromosome 21q22.3, and individuals with Down syndrome (DS) typically manifest with Alzheimer's disease (AD) neuropathology in their 30s. Here, we demonstrate that expression of ABCG1 enhances amyloid-beta protein (Abeta) production in transfected HEK cells in a manner that requires functional cholesterol transporter activity. ABCG1-expressing cells also exhibit increased secreted APP (sAPP)alpha and sAPPbeta secretion and display increased cell surface-associated APP. These results suggest that ABCG1 increases the availability of APP as a secretase substrate for both the amyloidogenic and nonamyloidogenic pathways. In vivo, ABCG1 mRNA levels are 2-fold more abundant in DS brain compared with age- and sex-matched normal controls. Finally, both Abeta and sAPPalpha levels are increased in DS cortex relative to normal controls. These findings suggest that altered cholesterol metabolism and APP trafficking mediated by ABCG1 may contribute to the accelerated onset of AD neuropathology in DS.     

Membrane rafts in Alzheimer's disease beta-amyloid production

Alzheimer's disease (AD), the most common age-associated dementing disorder, is pathologically manifested by progressive cognitive dysfunction concomitant with the accumulation of senile plaques consisting of amyloid-β (Aβ) peptide aggregates in the brain of affected individuals. Aβ is derived from a type I transmembrane protein, amyloid precursor protein (APP), by the sequential proteolytic events mediated by β-site APP cleaving enzyme 1 (BACE1) and γ-secretase. Multiple lines of evidence have implicated cholesterol and cholesterol-rich membrane microdomains, termed lipid rafts in the amyloidogenic processing of APP. In this review, we summarize the cell biology of APP, β- and γ-secretases and the data on their association with lipid rafts. Then, we will discuss potential raft targeting signals identified in the secretases and their importance on amyloidogenic processing of APP.     

Oxidation of cholesterol by amyloid precursor protein and beta-amyloid peptide

Alzheimer's disease (AD) is characterized by accumulation of the neurotoxic peptide beta-amyloid, which is produced by proteolysis of amyloid precursor protein (APP). APP is a large membrane-bound copper-binding protein that is essential in maintaining synaptic function and may play a role in synaptogenesis. beta-Amyloid has been shown to contribute to the oxidative stress that accompanies AD. Later stages of AD are characterized by neuronal apoptosis. However, the biochemical function of APP and the mechanism of the toxicity of beta-amyloid are still unclear. In this study, we show that both beta-amyloid and APP can oxidize cholesterol to form 7beta-hydroxycholesterol, a proapoptotic oxysterol that was neurotoxic at nanomolar concentrations. 7beta-Hydroxycholesterol inhibited secretion of soluble APP from cultured rat hippocampal H19-7/IGF-IR neuronal cells and inhibited tumor necrosis factor-alpha-converting enzyme alpha-secretase activity but had no effect on beta-site APP-cleaving enzyme 1 activity. 7beta-Hydroxycholesterol was also a potent inhibitor of alpha-protein kinase C, with a K(i) of approximately 0.2 nm. The rate of reaction between cholesterol and beta-amyloid was comparable to the rates of cholesterol-metabolizing enzymes (k(cat) = 0.211 min(-)1). The rate of production of 7beta-hydroxycholesterol by APP was approximately 200 times lower than by beta-amyloid. Oxidation of cholesterol was accompanied by stoichiometric production of hydrogen peroxide and required divalent copper. The results suggest that a function of APP may be to produce low levels of 7-hydroxycholesterol. Higher levels produced by beta-amyloid could contribute to the oxidative stress and cell loss observed in Alzheimer's disease.     

Intracellular amyloid-beta 1-42, but not extracellular soluble amyloid-beta peptides, induces neuronal apoptosis

Alzheimer disease (AD), the most frequent cause of dementia, is characterized by an important neuronal loss. A typical histological hallmark of AD is the extracellular deposition of beta-amyloid peptide (A beta), which is produced by the cleavage of the amyloid precursor protein (APP). Most of the gene mutations that segregate with the inherited forms of AD result in increasing the ratio of A beta 42/A beta 40 production. A beta 42 also accumulates in neurons of AD patients. Altogether, these data strongly suggest that the neuronal production of A beta 42 is a critical event in AD, but the intraneuronal A beta 42 toxicity has never been demonstrated. Here, we report that the long term expression of human APP in rat cortical neurons induces apoptosis. Although APP processing leads to production of extracellular A beta 1-40 and soluble APP, these extracellular derivatives do not induce neuronal death. On the contrary, neurons undergo apoptosis as soon as they accumulate intracellular A beta 1-42 following the expression of full-length APP or a C-terminal deleted APP isoform. The inhibition of intraneuronal A beta 1-42 production by a functional gamma-secretase inhibitor increases neuronal survival. Therefore, the accumulation of intraneuronal A beta 1-42 is the key event in the neurodegenerative process that we observed.     

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.     

Selective ectodomain phosphorylation and regulated cleavage of beta-amyloid precursor protein.

The beta-amyloid precursor protein (beta APP) is a highly conserved integral membrane protein expressed in most mammalian tissues and found at highest levels in the nervous system. Cerebral deposition of the amyloid beta-peptide (A beta), derived by proteolysis of beta APP, is an early and invariant feature of Alzheimer's disease. Protein phosphorylation by protein kinase C (PKC) has been found to regulate the metabolism of beta APP into nonamyloidogenic and amyloidogenic derivatives, but both the mechanism of these effects and the nature of beta APP phosphorylation are unknown. When labeled in vivo with [32P]orthophosphate, beta APP was phosphorylated only on serine residues in the N-terminal half of the extracellular domain, resulting in the secretion of phosphorylated soluble beta APP. PKC-mediated stimulation of beta APP secretion and concurrent inhibition of A beta release did not involve enhanced phosphorylation of beta APP and proceeded in the absence of cytoplasmic or extracellular phosphorylation of the precursor. The region of beta APP required for this indirect regulation by PKC was largely restricted to a 64 amino acid stretch around the secretory cleavage site. Moreover, in a truncated molecule designed to release soluble beta APP without the need for proteolytic cleavage, secretion was no longer regulated by PKC. Our data indicate that PKC-mediated pathways play a pivotal role in the control of beta APP metabolism and amyloid formation. However, in contrast to current postulates, this regulation is independent of beta APP phosphorylation and instead involves phosphorylation of other substrates that alter beta APP processing, such as beta APP-cleaving proteases.     

Amyloid beta as a regulator of lipid homeostasis

The beta-amyloid peptide (A beta) is widely considered to be the molecule that causes Alzheimer's disease (AD). Besides this pathological function of A beta, recently published data reveal that A beta also has an essential physiological role in lipid homeostasis. Cholesterol increases A beta production, and conversely A beta production causes a decrease in cholesterol synthesis. The latter appears to be mediated by the inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), a key enzyme in cholesterol synthesis, in an action similar to that of statins. Moreover, A beta regulates sphingolipid metabolism by directly activating sphingomyelinases (SMases). This review summarizes the molecular basis for the known physiological functions of A beta and amyloid precursor protein (APP), the roles of A beta and APP in lipid homeostasis and the medical implications of addressing lipid homeostasis in respect to AD. This knowledge might provide new insights for current and future therapeutic approaches to AD.     

Caspase-6 role in apoptosis of human neurons, amyloidogenesis, and Alzheimer's disease.

Neuronal cell death, neurofibrillary tangles, and amyloid beta peptide (Abeta) deposition depict Alzheimer's disease (AD) pathology, but neuronal loss correlates best with dementia. We have shown that increased production of Abeta is a consequence of neuronal apoptosis, suggesting that apoptosis activates proteases involved in amyloid precursor protein (APP) processing. Here, we investigate key effectors of cell death, caspases, in human neuronal apoptosis and APP processing. We find that caspase-6 is activated and responsible for neuronal apoptosis by serum deprivation. Caspase-6 activity precedes the time of commitment to neuronal apoptosis by 10 h, indicating possible activity without subsequent apoptosis. Inhibition of caspase-6 activity prevents serum deprivation-mediated increase of Abeta. Caspase-6 directly cleaves APP at the C terminus and generates a C-terminal fragment of 3 kDa (Capp3) and an Abeta-containing 6.5-kDa fragment, Capp6.5, that increases in serum-deprived neurons. A pulse-chase experiment reveals a precursor-product relationship between Capp6.5, intracellular Abeta, and secreted Abeta, indicating a potential alternate amyloidogenic pathway. Caspase-6 proenzyme is present in adult human brain tissue, and the p10 active caspase-6 fragment is detected in AD brain tissue. These results indicate a possible alternate pathway for APP amyloidogenic processing in human neurons and a potential implication for this pathway in the neuronal demise of AD.     

Docosahexaenoic acid reduces amyloid beta production via multiple pleiotropic mechanisms

Alzheimer disease is characterized by accumulation of the β-amyloid peptide (Aβ) generated by β- and γ-secretase processing of the amyloid precursor protein (APP). The intake of the polyunsaturated fatty acid docosahexaenoic acid (DHA) has been associated with decreased amyloid deposition and a reduced risk in Alzheimer disease in several epidemiological trials; however, the exact underlying molecular mechanism remains to be elucidated. Here, we systematically investigate the effect of DHA on amyloidogenic and nonamyloidogenic APP processing and the potential cross-links to cholesterol metabolism in vivo and in vitro. DHA reduces amyloidogenic processing by decreasing β- and γ-secretase activity, whereas the expression and protein levels of BACE1 and presenilin1 remain unchanged. In addition, DHA increases protein stability of α-secretase resulting in increased nonamyloidogenic processing. Besides the known effect of DHA to decrease cholesterol de novo synthesis, we found cholesterol distribution in plasma membrane to be altered. In the presence of DHA, cholesterol shifts from raft to non-raft domains, and this is accompanied by a shift in γ-secretase activity and presenilin1 protein levels. Taken together, DHA directs amyloidogenic processing of APP toward nonamyloidogenic processing, effectively reducing Aβ release. DHA has a typical pleiotropic effect; DHA-mediated Aβ reduction is not the consequence of a single major mechanism but is the result of combined multiple effects.     

Cholesterol accumulation in Niemann Pick type C (NPC) model cells causes a shift in APP localization to lipid rafts

It has been suggested that cholesterol may modulate amyloid-β (Aβ) formation, a causative factor of Alzheimer’s disease (AD), by regulating distribution of the three key proteins in the pathogenesis of AD (β-amyloid precursor protein (APP), β-secretase (BACE1) and/or presenilin 1 (PS1)) within lipid rafts. In this work we tested whether cholesterol accumulation upon NPC1 dysfunction, which causes Niemann Pick type C disease (NPC), causes increased partitioning of APP into lipid rafts leading to increased CTF/Aβ formation in these cholesterol-rich membrane microdomains. To test this we used CHO NPC1^−/− cells (NPC cells) and parental CHOwt cells. By sucrose density gradient centrifugation we observed a shift in fl-APP/CTF compartmentalization into lipid raft fractions upon cholesterol accumulation in NPC vs. wt cells. Furthermore, γ-secretase inhibitor treatment significantly increased fl-APP/CTF distribution in raft fractions in NPC vs. wt cells, suggesting that upon cholesterol accumulation in NPC1-null cells increased formation of APP-CTF and its increased processing towards Aβ occurs in lipid rafts. Our results support that cholesterol overload, such as in NPC disease, leads to increased partitioning of APP/CTF into lipid rafts resulting in increased amyloidogenic processing of APP in these cholesterol-rich membranes. This work adds to the mechanism of the cholesterol-effect on APP processing and the pathogenesis of Alzheimer’s disease and supports the role of lipid rafts in these processes.     

Amyloid beta.

Amyloid beta (A beta) is a 39-43 residue amyloidogenic peptide that is deposited into the extracellular amyloid plaques which characterize an Alzheimer's disease (AD) brain. A beta is derived from the amyloid precursor protein (APP) and undergoes a toxic conformational change (gain of toxic function). The length of the A beta peptide dramatically influences its properties with the longer 42 and 43 residue species being more amyloidogenic. The genetics of familial AD (FAD) supports a central role for A beta in AD since mutations in the FAD causing genes APP and the presenilins (PS1 and PS2) increase the formation of A beta 42,43. Considerable activity is directed towards A beta as a therapeutic target. These strategies aim to inhibit A beta synthesis, A beta fibril formation, its toxic actions on cells or promote its clearance from the brain.     

Alzheimer's disease and control brain contain soluble derivatives of the amyloid protein precursor that end within the beta amyloid protein region.

The 39-43 amino acid beta amyloid protein (A beta) that deposits as amyloid in the brains of patients with Alzheimer's disease (AD) is encoded as an internal sequence within a larger membrane-associated protein known as the amyloid protein precursor (APP). In cultured cells, the APP is normally cleaved within the A beta to generate a large secreted derivative and a small membrane-associated fragment. Neither of these derivatives can produce amyloid because neither contains the entire A beta. Our study was designed to determine whether the soluble APP derivatives in human brain end within the A beta as described in cell culture or whether AD brain produces potentially amyloidogenic soluble derivatives that contain the entire A beta. We find that both AD and control brain contain nonamyloidogenic soluble derivatives that end at position 15 of the A beta. We have been unable to detect any soluble derivatives that contain the entire A beta in either the AD or control brain.     

Failure of the interaction between presenilin 1 and the substrate of gamma-secretase to produce Abeta in insect cells

Aggregates of beta-amyloid peptide (Abeta) are the major component of the amyloid core of the senile plaques observed in Alzheimer's disease (AD). Abeta results from the amyloidogenic processing of its precursor, the amyloid precursor protein (APP), by beta- and gamma-secretase activities. If beta-secretase has recently been identified and termed BACE, the identity of gamma-secretase is still obscure. Studies with knock-out mice showed that presenilin 1 (PS1), of which mutations are known to be the first cause of inherited AD, is mandatory for the gamma-secretase activity. However, the proteolytic activity of PS1 remains a matter of debate. Here we used transfected Sf9 insect cells, a cellular model lacking endogenous beta- and/or gamma-secretase activities, to characterize the role of BACE and PS1 in the amyloidogenic processing of human APP. We show that, in Sf9 cells, BACE performs the expected beta-secretase cleavage of APP, generating C99. We also show that C99, which is a substrate of gamma-secretase, tightly binds to the human PS1. Despite this interaction, Sf9 cells still do not produce Abeta. This strongly argues against a direct proteolytic activity of PS1 in APP processing, and points toward an implication of PS1 in trafficking/presenting its substrate to the gamma-secretase.     

Association of gamma-secretase with lipid rafts in post-Golgi and endosome membranes

Alzheimer's disease-associated beta-amyloid peptides (Abeta) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases. There is growing evidence that cholesterol- and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major beta-secretase in neurons is a palmitoylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the gamma-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1(-/-)/PS2(-/-) and NCT(-/-) fibroblasts, gamma-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires gamma-secretase complex assembly. Biochemical evidence shows that subunits of the gamma-secretase complex and three TGN/endosome-resident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1- or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of gamma-secretase in lipid raft microdomains of post-Golgi and endosomes, organelles previously implicated in amyloidogenic processing of APP.     

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.