Amyloid beta-protein interactions with membranes and cholesterol: causes or casualties of Alzheimer's disease

Amyloid beta-protein (Aβ) is thought to be one of the primary factors causing neurodegeneration in Alzheimer's disease (AD). This protein is an amphipathic molecule that perturbs membranes, binds lipids and alters cell function. Several studies have reported that Aβ alters membrane fluidity but the direction of this effect has not been consistently observed and explanations for this lack of consistency are proposed. Cholesterol is a key component of membranes and cholesterol interacts with Aβ in a reciprocal manner. Aβ impacts on cholesterol homeostasis and modification of cholesterol levels alters Aβ expression. In addition, certain cholesterol lowering drugs (statins) appear to reduce the risk of AD in human subjects. However, the role of changes in the total amount of brain cholesterol in AD and the mechanisms of action of statins in lowering the risk of AD are unclear. Here we discuss data on membranes, cholesterol, Aβ and AD, and propose that modification of the transbilayer distribution of cholesterol in contrast to a change in the total amount of cholesterol provides a cooperative environment for Aβ synthesis and accumulation in membranes leading to cell dysfunction including disruption in cholesterol homeostasis.     

Statins reduce amyloid-beta production through inhibition of protein isoprenylation

Epidemiological evidence suggests that long term treatment with hydroxymethylglutaryl-CoA reductase inhibitors, or statins, decreases the risk for developing Alzheimer disease (AD). However, statin-mediated AD protection cannot be fully explained by reduction of cholesterol levels. In addition to their cholesterol lowering effects, statins have pleiotropic actions and act to lower the concentrations of isoprenoid intermediates, such as geranylgeranyl pyrophosphate and farnesyl pyrophosphate. The Rho and Rab family small G-proteins require addition of these isoprenyl moieties at their C termini for normal GTPase function. In neuroblastoma cell lines, treatment with statins inhibits the membrane localization of Rho and Rab proteins at statin doses as low as 200 nm, without affecting cellular cholesterol levels. In addition, we show for the first time that at low, physiologically relevant, doses statins preferentially inhibit the isoprenylation of a subset of GTPases. The amyloid precursor protein (APP) is proteolytically cleaved to generate beta-amyloid (Abeta), which is the major component of senile plaques found in AD. We show that inhibition of protein isoprenylation by statins causes the accumulation of APP within the cell through inhibition of Rab family proteins involved in vesicular trafficking. Moreover, inhibition of Rho family protein function reduces levels of APP C-terminal fragments due to enhanced lysosomal dependent degradation. Statin inhibition of protein isoprenylation results in decreased Abeta secretion. In summary, we show that statins selectively inhibit GTPase isoprenylation at clinically relevant doses, leading to reduced Abeta production in an isoprenoid-dependent manner. These studies provide insight into the mechanisms by which statins may reduce AD pathogenesis.     

Cholesterol as a causative factor in Alzheimer's disease: a debatable hypothesis

High serum/plasma cholesterol levels have been suggested as a risk factor for Alzheimer's disease (AD). Some reports, mostly retrospective epidemiological studies, have observed a decreased prevalence of AD in patients taking the cholesterol lowering drugs, statins. The strongest evidence causally linking cholesterol to AD is provided by experimental studies showing that adding/reducing cholesterol alters amyloid precursor protein (APP) and amyloid beta‐protein (Aβ) levels. However, there are problems with the cholesterol‐AD hypothesis. Cholesterol levels in serum/plasma and brain of AD patients do not support cholesterol as a causative factor in AD. Prospective studies on statins and AD have largely failed to show efficacy. Even the experimental data are open to interpretation given that it is well‐established that modification of cholesterol levels has effects on multiple proteins, not only amyloid precursor protein and Aβ. The purpose of this review, therefore, was to examine the above‐mentioned issues, discuss the pros and cons of the cholesterol‐AD hypothesis, involvement of other lipids in the mevalonate pathway, and consider that AD may impact cholesterol homeostasis.

     

The conflicting role of brain cholesterol in Alzheimer's disease: lessons from the brain plasminogen system

Retrospective clinical studies indicate that individuals chronically treated with cholesterol synthesis inhibitors, statins, are at lower risk of developing AD (Alzheimer's disease). Moreover, treatment of guinea pigs with high doses of simvastatin or drastic reduction of cholesterol in cultured cells decrease Abeta (beta-amyloid peptide) production. These data sustain the concept that high brain cholesterol is responsible for Abeta accumulation in AD, providing the scientific support for the proposed use of statins to prevent this disease. However, a number of unresolved issues raise doubts that high brain cholesterol is to blame. First, it has not been shown that higher neuronal cholesterol increases Abeta production. Secondly, it has not been demonstrated that neurons in AD have more cholesterol than control neurons. On the contrary, the brains of AD patients show a specific down-regulation of seladin-1, a protein involved in cholesterol synthesis, and low membrane cholesterol was observed in hippocampal membranes of ApoE4 (apolipoprotein E4) AD cases. This effect was also evidenced by altered cholesterol-rich membrane domains (rafts) and raft-mediated functions, such as diminished generation of the Abeta-degrading enzyme plasmin. Thirdly, numerous genetic defects that cause neurodegeneration are due to defective cholesterol metabolism. Fourthly, in female mice, the most brain-permeant statin induces neurodegeneration and high amyloid production. Altogether, this evidence makes it difficult to accept that statins are beneficial through acting as brain cholesterol-synthesis inhibitors. It appears more likely that their advantageous role arises from improved brain oxygenation.     

Role of amyloid beta in lipid homeostasis

Alzheimer's disease (AD), the most common neurodegenerative disorder, which affects more than 35 million people worldwide, is characterized by a massive accumulation of tangles and amyloid plaques. Several risk factors linked to lipid homeostasis have been identified. Apolipoprotein E (ApoE), which also has a strong impact in coronary artery disease, is besides aging the most prominent risk factor in sporadic AD. High levels of lipoproteins and cholesterol increase the risk of AD and some cholesterol lowering drugs like statins seem to correlate with a reduced risk for dementia. Moreover, cholesterol increases amyloid β (Aβ) production, which is derived from amyloid precursor protein (APP) by proteolytic processing. Beside cholesterol, other lipids that strongly modulate APP processing could be identified and interestingly the APP cleavage products itself regulate lipid homeostasis resulting in complex regulatory feedback cycles. Here, we review the mechanistic link of cholesterol and sphingolipid homeostasis and APP processing and the consequence of this bidirectional link for and in AD. Although cholesterol is the best studied brain lipid in AD, many other lipids are involved in the Aβ-lipid regulatory system and some of these lipids exceed the cholesterol effect on Aβ production [1–5]. This involvement is bidirectional. On the one hand, lipids control APP processing and, on the other hand, APP processing controls the levels of several key lipids [6, 7]. Beside the physiological function of APP processing in lipid homeostasis, under pathological conditions like AD, these regulating (feedback-) cycles are dysfunctional. Additionally, mutual influence of lipids and APP processing raises the question if altered lipid homeostasis is the cause or consequence of AD.     

Amyloid beta-peptide1-40 increases neuronal membrane fluidity: role of cholesterol and brain region

There is increasing evidence of an interaction between cholesterol dynamics and Alzheimer's disease (AD), and amyloid beta-peptide may play an important role in this interaction. Abeta destabilizes brain membranes and this action of Abeta may be dependent on the amount of membrane cholesterol. We tested this hypothesis by examining effects of Abeta1-40 on the annular fluidity (i.e., lipid environment adjacent to proteins) and bulk fluidity of rat synaptic plasma membranes (SPM) of the cerebral cortex, cerebellum, and hippocampus using the fluorescent probe pyrene and energy transfer. Amounts of cholesterol and phospholipid of SPM from each brain region were determined. SPM of the cerebellum were significantly more fluid as compared with SPM of the cerebral cortex and hippocampus. Abeta significantly increased (P < or = 0.01) annular and bulk fluidity in SPM of cerebral cortex and hippocampus. In contrast, Abeta had no effect on annular fluidity and bulk fluidity of SPM of cerebellum. The amounts of cholesterol in SPM of cerebral cortex and hippocampus were significantly higher (P < or = 0.05) than amount of cholesterol in SPM of cerebellum. There was significantly less (P < or = 0.05) total phospholipid in cerebellar SPM as compared with SPM of cerebral cortex. Neuronal membranes enriched in cholesterol may promote accumulation of Abeta by hydrophobic interaction, and such an interpretation is consistent with recent studies showing that soluble Abeta can act as a seed for fibrillogenesis in the presence of cholesterol.     

The role of cholesterol in Alzheimer's neuro-pathogenesis]

Alzheimer's Disease (AD) is the most common neurodegenerative disorder in western societies affecting up to 15 million individuals worldwide.It leads to death after a progressive memory deficit and cognitive impairment accompanied by the appearance of two pathological hallmarks in specific brain areas: neurofibrillary tangles and amyloid plaques. Cholesterol homeostasis may play a key role in AD pathogenesis and this is supported by the demonstration that cholesterol-rich membrane domain, so-called Rafts,are disorganized in affected brains. Retrospective clinical studies indicate that individuals chronically treated with cholesterol synthesis inhibitors,statins, are at lower risk of developing AD but current literature is conflicting with regard to the neuroprotective effects of statins on cognitive impairment.Before recommending statins for prevention and/or treatment of AD it is important to investigate more the role of cholesterol levels in neurodegenerative disorders.     

Apolipoprotein E and cholesterol metabolism in the pathogenesis and treatment of Alzheimer's disease

There is much evidence suggesting that there is a strong relationship between the deterioration of brain lipid homeostasis, vascular changes and the pathogenesis of Alzheimer's disease (AD). These associations include: (1). recognition that a key cholesterol transporter, apolipoprotein E type 4, acts a major genetic risk factor for both familial and sporadic AD; (2). epidemiological studies linking cardiovascular risk factors, such as hypertension and high plasma cholesterol, to dementia; (3). the discovery that small strokes can precipitate clinical dementia in cognitively normal elderly subjects; (4). the modulation of degradation of the amyloid precursor protein by cholesterol administration in cell culture and in animal models of beta-amyloid overproduction; and (5). the beneficial effect of cholesterol-lowering drugs, such as Probucol and statins, in combating common AD. The recent finding that there is a genetic association between the HMGR gene locus and sporadic AD further suggests that brain cholesterol metabolism is central to AD pathophysiology, and a potential therapeutic target for disease stabilization and primary disease prevention.     

ABCG1 influences the brain cholesterol biosynthetic pathway but does not affect amyloid precursor protein or apolipoprotein E metabolism in vivo

Cholesterol homeostasis is of emerging therapeutic importance for Alzheimer's disease (AD). Agonists of liver-X-receptors (LXRs) stimulate several genes that regulate cholesterol homeostasis, and synthetic LXR agonists decrease neuropathological and cognitive phenotypes in AD mouse models. The cholesterol transporter ABCG1 is LXR-responsive and highly expressed in brain. In vitro, conflicting reports exist as to whether ABCG1 promotes or impedes Abeta production. To clarify the in vivo roles of ABCG1 in Abeta metabolism and brain cholesterol homeostasis, we assessed neuropathological and cognitive outcome measures in PDAPP mice expressing excess transgenic ABCG1. A 6-fold increase in ABCG1 levels did not alter Abeta, amyloid, apolipoprotein E levels, cholesterol efflux, or cognitive performance in PDAPP mice. Furthermore, endogenous murine Abeta levels were unchanged in both ABCG1-overexpressing or ABCG1-deficient mice. These data argue against a direct role for ABCG1 in AD. However, excess ABCG1 is associated with decreased levels of sterol precursors and increased levels of SREBP-2 and HMG-CoA-reductase mRNA, whereas deficiency of ABCG1 leads to the opposite effects. Although functions for ABCG1 in cholesterol efflux and Abeta metabolism have been proposed based on results with cellular model systems, the in vivo role of this enigmatic transporter may be largely one of regulating the sterol biosynthetic pathway.     

Cholesterol-dependent formation of GM1 ganglioside-bound amyloid beta-protein, an endogenous seed for Alzheimer amyloid

GM1 ganglioside-bound amyloid beta-protein (GM1/Abeta), found in brains exhibiting early pathological changes of Alzheimer's disease (AD) including diffuse plaques, has been suggested to be involved in the initiation of amyloid fibril formation in vivo by acting as a seed. To elucidate the molecular mechanism underlying GM1/Abeta formation, the effects of lipid composition on the binding of Abeta to GM1-containing lipid bilayers were examined in detail using fluorescent dye-labeled human Abeta-(1-40). Increases in not only GM1 but also cholesterol contents in the lipid bilayers facilitated the binding of Abeta to the membranes by altering the binding capacity but not the binding affinity. An increase in membrane-bound Abeta concentration triggered its conformational transition from helix-rich to beta-sheet-rich structures. Excimer formation of fluorescent dye-labeled GM1 suggested that Abeta recognizes a GM1 "cluster" in membranes, the formation of which is facilitated by cholesterol. The results of the present study strongly suggested that increases in intramembrane cholesterol content, which are likely to occur during aging, appear to be a risk factor for amyloid fibril formation.     

Oxidized neprilysin in aging and Alzheimer's disease brains

Deposition of amyloid in the brain is important in the pathogenesis of Alzheimer's disease (AD), but it remains to be determined if deposition is due to increased production or decreased clearance of fibrillogenic forms of beta-amyloid (Abeta). Except for rare genetic forms of AD, there is little evidence for increased production of Abeta, but decreases in enzymes involved in the clearance of Abeta are increasingly being investigated. Neprilysin (NEP) is a major enzyme for degradation of Abeta and changes in amount or activity of NEP may play a role in Abeta deposition in AD. Since oxidative damage to proteins, including formation of adducts such as 4-hydroxynonenal (HNE), has been reported in AD, it was of interest to determine if NEP might be susceptible to oxidative modification. To address this question, monoclonal antibody immunoprecipitates of NEP were probed with polyclonal antibodies to NEP and HNE. The results showed decreased NEP in AD compared to normal controls. NEP in both AD and controls had HNE-modification and the ratio of oxidized to total NEP was greater in AD than in controls. These findings suggest that decreased NEP may contribute to Abeta deposition in AD and that age-related oxidative damage to NEP may play a role in age-related cerebral amyloidosis that is exacerbated in AD.     

Use of in Vivo Models to Study the Role of Cholesterol in the Etiology of Alzheimer's Disease

Cholesterol has been implicated in the pathogenesis of Alzheimer's disease, both through intracellular effects, and through an extracellular effect due to its physical interaction with plaque associated amyloid. Epidemiology studies have implicated high cholesterol as a risk factor for AD, and have shown that the use of cholesterol reducing agents (statins) can be protective against the disease. We, and others have shown that cholesterol levels modulate the processing of the amyloid precursor protein (APP) both in vivo and in vitro, affecting the accumulation of Abeta (A) peptides which may directly impact the risk of AD. This review describes the biology of sterols, and identifies how cholesterol may exacerbate the pathogenesis of AD. Data from in vivo and in vitro studies will then be presented to describe how treatments aimed at modulating lipid levels may be efficacious in treating AD.     

Alzheimer's disease: cholesterol, membrane rafts, isoprenoids and statins

Alzheimer''s disease (AD) is a heterogeneous neurodegenerative disorder and the most prevalent form of dementia worldwide. AD is characterized pathologically by amyloid-β plaques, neurofibrillary tangles and neuronal loss, and clinically by a progressive loss of cognitive abilities. At present, the fundamental molecular mechanisms underlying the disease are unclear and no treatment for AD is known. Epidemiological evidence continues to mount linking vascular diseases, such as hypertension and diabetes, and hypercholesterolaemia with an increased risk for developing AD. A growing amount of evidence suggests a mechanistic link between cholesterol metabolism in the brain and the formation of amyloid plaques in AD development. Cholesterol and statins clearly modulate β-amyloid precursor protein (βAPP) processing in cell culture and animal models. Statins not only reduce endogenous cholesterol synthesis but also exert other various pleiotrophic effects, such as the reduction in protein isoprenylation. Through these effects statins modulate a variety of cellular functions involving both cholesterol (and membrane rafts) and isoprenylation. Although clearly other factors, such as vascular inflammation, oxidative stress and genetic factors, are intimately linked with the progression of AD, this review focuses on the present research findings describing the effect of cholesterol, membrane rafts and isoprenylation in regulating βAPP processing and in particular γ-secretase complex assembly and function and AD progression, along with consideration for the potential role statins may play in modulating these events.     

Amyloid beta ion channel: 3D structure and relevance to amyloid channel paradigm

Alzheimer's disease (AD) is a protein misfolding disease. Early hypothesis of AD pathology posits that 39-43 AA long misfolded amyloid beta (Abeta) peptide forms a fibrillar structure and induces pathophysiological response by destabilizing cellular ionic homeostasis. Loss of cell ionic homeostasis is believed to be either indirectly due to amyloid beta-induced oxidative stress or directly by its interaction with the cell membrane and/or activating pathways for ion exchange. Significantly though, no Abeta specific cell membrane receptors are known and oxidative stress mediated pathology is only partial and indirect. Most importantly, recent studies strongly indicate that amyloid fibrils may not by themselves cause AD pathology. Subsequently, a competing hypothesis has been proposed wherein amyloid derived diffusible ligands (ADDLs) that are large Abeta oligomers (approximately >60 kDa), mediate AD pathology. No structural details, however, of these large globular units exist nor is there any known suitable mechanism by which they would induce AD pathology. Experimental data indicate that they alter cell viability by non-specifically changing the plasma membrane stability and increasing the overall ionic leakiness. The relevance of this non-specific mechanism for AD-specific pathology seems limited. Here, we provide a viable new paradigm: AD pathology mediated by amyloid ion channels made of small Abeta oligomers (trimers to octamers). This review is focused to 3D structural analysis of the Abeta channel. The presence of amyloid channels is consistent with electrophysiological and cell biology studies summarized in companion reviews in this special issue. They show ion channel-like activity and channel-mediated cell toxicity. Amyloid ion channels with defined gating and pharmacological agents would provide a tangible target for designing therapeutics for AD pathology.     

Identification of novel genes that modify phenotypes induced by Alzheimer's beta-amyloid overexpression in Drosophila

Sustained increases in life expectancy have underscored the importance of managing diseases with a high incidence in late life, such as various neurodegenerative conditions. Alzheimer's disease (AD) is the most common among these, and consequently significant research effort is spent on studying it. Although a lot is known about the pathology of AD and the role of beta-amyloid (Abeta) peptides, the complete network of interactions regulating Abeta metabolism and toxicity still eludes us. To address this, we have conducted genetic interaction screens using transgenic Drosophila expressing Abeta and we have identified mutations that affect Abeta metabolism and toxicity. These analyses highlight the involvement of various biochemical processes such as secretion, cholesterol homeostasis, and regulation of chromatin structure and function, among others, in mediating toxic Abeta effects. Several of the mutations that we identified have not been linked to Abeta toxicity before and thus constitute novel potential targets for AD intervention. We additionally tested these mutations for interactions with tau and expanded-polyglutamine overexpression and found a few candidate mutations that may mediate common mechanisms of neurodegeneration. Our data offer insight into the toxicity of Abeta and open new areas for further study into AD pathogenesis.     

High doses of simvastatin, pravastatin, and cholesterol reduce brain cholesterol synthesis in guinea pigs

Recent epidemiological studies suggest that inhibitors of 3-hydroxy-3-methyl-glutaryl CoA reductase, so-called statins, are effective in lowering the prevalence of Alzheimer's disease. Whether the effect of statins is due to a local inhibition of cholesterol synthesis in the brain or whether it is mediated by the reduced levels of cholesterol in the circulation is not known. In the present work, we tested the possibility that high doses of lipophilic and hydrophilic statins, simvastatin and pravastatin, respectively, or a diet high in cholesterol could affect cholesterol homeostasis in the brain of guinea pigs. The total brain cholesterol levels were not affected by high-dose simvastatin or pravastatin treatment. Significantly lower levels of the cholesterol precursor lathosterol and its ratio to cholesterol were found in the brains of simvastatin and pravastatin-treated animals. 24S-Hydroxycholesterol, the transportable form of cholesterol across the blood-brain barrier, was significantly lower in the brain of pravastatin-treated animals. Excessive cholesterol feeding resulted in higher serum cholesterol levels but did not affect total brain cholesterol level. However, de novo cholesterol synthesis in the brain seemed to be down-regulated, as indicated by lower absolute levels and cholesterol-related ratios of lathosterol compared with controls. The passage of deuterium-labeled cholesterol across the blood-brain barrier in one animal was found to be approximately 1%. Our results suggest that brain cholesterol synthesis in guinea pigs can be slightly, but significantly, influenced by high doses of lipophilic and hydrophilic statins as well as by high dietary cholesterol intake, while total brain cholesterol content and thus, cholesterol homeostasis is maintained.     

Oxidative metabolites accelerate Alzheimer's amyloidogenesis by a two-step mechanism, eliminating the requirement for nucleation

The process of amyloid formation by the amyloid beta peptide (Abeta), i.e., the misassembly of Abetapeptides into soluble quaternary structures and, ultimately, amyloid fibrils, appears to be at the center of Alzheimer's disease (AD) pathology. We have shown that abnormal oxidative metabolites, including cholesterol-derived aldehydes, modify Abeta and accelerate the early stages of amyloidogenesis (the formation of spherical aggregates). This process, which we have termed metabolite-initiated protein misfolding, could explain why hypercholesterolemia and inflammation are risk factors for sporadic AD. Herein, the mechanism by which cholesterol metabolites hasten Abeta 1-40 amyloidogenesis is explored, revealing a process that has at least two steps. In the first step, metabolites modify Abeta peptides by Schiff base formation. The Abeta-metabolite adducts form spherical aggregates by a downhill polymerization that does not require a nucleation step, dramatically accelerating Abeta aggregation. In agitated samples, a second step occurs in which fibrillar aggregates form, a step also accelerated by cholesterol metabolites. However, the metabolites do not affect the rate of fibril growth in seeded aggregation assays; their role appears to be in initiating amyloidogenesis by lowering the critical concentration for aggregation into the nanomolar range. Small molecules that block Schiff base formation inhibit the metabolite effect, demonstrating the importance of the covalent adduct. Metabolite-initiated amyloidogenesis offers an explanation for how Abeta aggregation could occur at physiological nanomolar concentrations.     

Cholesterol and the biology of Alzheimer's disease

Recent results implicating cholesterol metabolism in the pathophysiology of Alzheimer's disease (AD) bring cholesterol to the forefront of AD research. Research from genetics, epidemiology, and cell biology all converge, suggesting that cholesterol plays a central role in the biology of amyloid precursor protein and the toxic peptide generated by its cleavage, beta-amyloid (Abeta). The ability of cholesterol to modulate Abeta production suggests opportunities for therapeutic intervention, although the functional significance underlying the connection between cholesterol and Abeta remains to be investigated.     

Two types of Alzheimer's beta-amyloid (1-40) peptide membrane interactions: aggregation preventing transmembrane anchoring versus accelerated surface fibril formation

The 39-42 amino acid long, amphipathic amyloid-beta peptide (Abeta) is one of the key components involved in Alzheimer's disease (AD). In the neuropathology of AD, Abeta presumably exerts its neurotoxic action via interactions with neuronal membranes. In our studies a combination of 31P MAS NMR (magic angle spinning nuclear magnetic resonance) and CD (circular dichroism) spectroscopy suggest fundamental differences in the functional organization of supramolecular Abeta(1-40) membrane assemblies for two different scenarios with potential implication in AD: Abeta peptide can either be firmly anchored in a membrane upon proteolytic cleavage, thereby being prevented against release and aggregation, or it can have fundamentally adverse effects when bound to membrane surfaces by undergoing accelerated aggregation, causing neuronal apoptotic cell death. Acidic lipids can prevent release of membrane inserted Abeta(1-40) by stabilizing its hydrophobic transmembrane C-terminal part (residue 29-40) in an alpha-helical conformation via an electrostatic anchor between its basic Lys28 residue and the negatively charged membrane interface. However, if Abeta(1-40) is released as a soluble monomer, charged membranes act as two-dimensional aggregation-templates where an increasing amount of charged lipids (possible pathological degradation products) causes a dramatic accumulation of surface-associated Abeta(1-40) peptide followed by accelerated aggregation into toxic structures. These results suggest that two different molecular mechanisms of peptide-membrane assemblies are involved in Abeta's pathophysiology with the finely balanced type of Abeta-lipid interactions against release of Abeta from neuronal membranes being overcompensated by an Abeta-membrane assembly which causes toxic beta-structured aggregates in AD. Therefore, pathological interactions of Abeta peptide with neuronal membranes might not only depend on the oligomerization state of the peptide, but also the type and nature of the supramolecular Abeta-membrane assemblies inherited from Abeta's origin.