Vol. 47 No. 3/2000

725–733

QUARTERLY

Review

Alzheimer’s disease: Its origin at the membrane, evidence and questions*.

Rene Buchet1,½ and S³awomir Piku³a2

1Laboratoire de Physico-Chimie Biologique, Université Claude Bernard-Lyon 1, UFR de Chimie-Biochimie, CNRS UMR 5013, F-69622 Villeurbanne, France; 2Department of Cellular Biochemistry, M. Nencki Institute of Experimental Biology, L. Pasteura 3, 02-093 Warszawa, Poland Received: 04 July, 2000; accepted: 03 August, 2000

Key words: b- precursor protein, membrane , lipid composition, membrane microdomains, Alzheimer’s disease

Numerous results on membrane lipid composition from different regions of auto- psied Alzheimer’s disease in comparison with corresponding fractions isolated from control brains revealed significant differences in serine- and ethanolamine-con- taining glycerophospholipid as well as in glycosphingolipid content. Changes in mem- brane lipid composition are frequently accompanied by alterations in membrane flu- idity, hydrophobic mismatch, lipid signaling pathways, transient formation and disap- pearance of lipid microdomains, changes in membrane permeability to cations and variations of other membrane properties. In this review we focus on possible implica- tions of altered membrane composition on b-amyloid precursor protein (APP) and on proteolysis of APP leading eventually to the formation of neurotoxic b-amyloid (Ab) peptides, the major proteinaceous component of extracellular senile plaques, directly involved in Alzheimer’s disease pathogenesis.

Alzheimer’s disease is a progressive neuro- formed by cytoskeletal protein tau in the degenerative disorder that is clinically charac- neuronal cell body, and neuropil threads in terized by the presence of extracellular senile dendrites, as well as by synapse and selective plaques, intracellular neurofibrillary tangles neuronal cell loss [1–3]. b-Amyloid (Ab) pep-

*75th Anniversary of Membrane Lipid Bilayer Concept. .Work in the Authors’ laboratories is supported by a grant from C.N.R.S. (R.B.) and statutable funds from the Nencki Institute of Experimental Biology (S.P.). ½Tel.: (33 47) 243 1320; fax: (33 47) 243 1543; e-mail: [email protected] Abbreviations: Ab, b-amyloid; APP, b-amyloid precursor protein; sAPP, secreted b-amyloid precursor protein. 726 R. Buchet and S³. Piku³a 2000 tides represent the major component of senile ganization [13]. In this review, we will discuss plaques (so-called amyloid plaques) and are about possible effects of lipid compositions on generated during endoproteolysis of a large i) APP endoproteolysis and on ii) ion channel transmembrane protein, b-amyloid precursor properties of Ab peptides in relation to their protein (APP). The detailed etiopathology of neurotoxicity. Alzheimer’s disease is still unclear and is likely to be multifactorial with various genetic and environmental causes at distinct levels BASIC CHARACTERISTICS OF for each individual [4]. Several hypotheses b-AMYLOID PRECURSOR PROTEIN have been proposed to explain the degenera- tion of in Alzheimer’s disease, includ- Ab peptides, the major components of amy- ing the “amyloid cascade hypothesis“ which loid plaques, are produced during endopro- states that an increased level of 42 to 43 teolysis of a large type-I transmembrane pro- amino acid long Ab peptides (due to mis-sense tein, so-called b-amyloid precursor protein mutations in the APP and presenilin , (APP). This protein is derived by differential age-related defects or environment) leads to splicing of a single transript located on aggregation of these peptides and formation the long arm of . In this re- of amyloid plaques, resulting in progressive spect, trisomy of chromosome 21 (Down syn- neurodegeneration [5]. In addition to this hy- drome) leads to the overexpression of APP pothesis, several factors were invoked to con- and to the formation of precocious senile tribute to the progressive neurodegenerative plaques. The predominant isoforms, APP770, disease such as: selective vulnerability of cho- APP751, and APP695 (numbers indicate the linergic neurons in the basal forebrain, mito- number of amino-acid residues in each iso- chondrial dysfunction, oxidative stress, viral form), are expressed with some tissue speci- agents, toxic material deposits, deficiency of ficity [1]. The two longer isoforms of APP, particular nutrients, overstimulation of excit- APP751 and APP770, contain a 56 amino acid atory amino acid receptors, and altered phos- long ectodomain homologous to the Kunitz pholipid metabolism [6–9]. Especially the lat- family of serine inhibitors. It has ter factor gained recently the attention of in- been postulated that the secreted form of APP vestigators due to the observations that the (sAPP, see next paragraph) could function as development of Alzheimer’s disease is accom- a circulating protease inhibitor [14]. In addi- panied by changes in the levels of neuronal tion, the secreted and membrane forms of membrane phospholipids: phosphatidyl- APP may be involved in neurite adhesion, serine, phosphatidylethanolamine, and phos- neurite extension via a neurotropic effect and phatidylinositol [10, 11]. In addition, Gins- may play a protective role against excitoxicity berg et al. [12] found that decreased ratio of [1]. The secondary structure of the secreted plasmalogen to nonplasmalogen ethanol- and membrane forms of APP is largely a-heli- amine glycerophospholipids in the temporal cal, since these proteins contain 40–45% of cortex of Alzheimer’s disease individuals may a-helix and only 15–20% of b-sheet structures result in membrane instability. This would fol- [15]. low the development of the so-called hydro- phobic mismatch in the membranes, i.e., the difference in length between the hydrophobic PROCESSING OF b-AMYLOID part of membrane spanning proteins, such as PRECURSOR PROTEIN APP, and the hydrophobic region of the mem- brane lipid bilayer. Such a hydrophobic mis- Newly synthesized APP matures in the se- match can strongly affect protein and lipid or- cretory pathway by the addition of O-glycosyl Vol. 47 Alzheimer’s disease and membranes 727 and N-glycosyl residues as well as tyrosine sul- pathway [16]. In the major processing path- fation in the trans-Golgi network [14]. At least way of APP, i.e., the nonamyloidogenic path- two distinct pools of APP appear to be present way, this protein is cleaved within the Ab do- in primary neuronal cultures: the major pool main (between Lys16 and Leu17; numbering of APP is characterized by a short half-life in according to the primary sequence of Ab pep- the range of 30–60 min and a minor full- tides) preventing the formation of Ab pep- length, transmembrane pool of APP by a lon- tides. During this cleavage by the protease ger half-life [4]. It has been proposed that the called a-secretase, a soluble ectodomain of major pool of APP with the rapid turnover is APP (sAPPa) is released and a 10-kDa in part secreted out of the cell in the form of C-terminal fragment (p3CT) remains within soluble APP (sAPPa), while the minor pool of the membrane (Fig. 1). This cleavage may oc- full-length APP remains at the surface of the cur in the post-Golgi compartment [14], at the neurite. The latter localization of APP is con- surface of a neuronal cell [17] or within spe- sistent with a role for this protein in the stabi- cific membrane microdomains, caveolae [18], lization of cell–matrix or cell–cell interaction suggesting that distinct pools of APP may co- [4]. Further processing of APP via the exist within the cell [4, 19]. The soluble pep- nonamyloidogenic or amyloidogenic path- tide derived from APP, sAPPa, is detected in ways is depicted in Fig. 1. APP is more likely plasma and cerebrospinal fluid and may have to be cleaved after O-glycosylation, indicating neuroprotective roles [1, 14]. In the amylo- that the cleavage of APP occurs either idogenic pathway that is a minor route, APP is throughout the Golgi complex (site of cleaved by b-secretase at the N-terminus of the glycosylation) or in compartments subse- Ab domain, producing a soluble protein quent to trans-Golgi in the APP processing (sAPPb) shorter than sAPPa and a C-terminal p3 p3CT APP A4CT A42b A40b

C C C cytosol membrane g g cut a cut b cut g cut

a b N N N

sAPPa sAPPb Figure 1. Schematic representation of APP isoforms and their processing by a-, b- and g-secretases. APP is processed via either the nonamyloidogenic (toward the formation of less neurotoxic p3 fragment) or amyloidogenic pathways (toward the formation of more neurotoxic Ab42 or Ab40 fragments). In the nonamyloidogenic pathway a-secretase cleaves APP isoforms within the Ab domain releasing a large soluble fragments of APP (sAPPa) and a membrane-bound fragment (p3CT). Then, eventually, p3CT fragment can be cleaved by a g-secretase, releasing the C-terminal p3 peptide. In the amyloidogenic pathway, b-secretase produces the membrane-bound A4CT fragment and releases the soluble sAPPb. Further processing of the A4CT peptide by g-secretase generates the Ab40 or Ab42 peptides [1]. The filled rectangle within APP indicates the Ab42 peptide and its approximate location within the membrane bilayer. The Greek letters a, b and g beside the rectangle show the cleavage sites for the respective secretases. 728 R. Buchet and S³. Piku³a 2000 peptide residing in the membrane (A4CT). since they are good candidates for drug design The A4CT fragment is the precursor for Ab to prevent the Ab peptide formation. Al- peptides (Fig. 1). b-Secretase cleaves APP ei- though a-secretase has not been isolated yet, ther within the endocytic pathway following this protease appears to be an integral mem- reinternalization of cell-surface APP or within brane protein that is inhibited by hydroxamic the endoplasmic reticulum and Golgi. The ex- acid-based zinc metalloproteinase inhibitors istence of various sites of action of b-secretase [22]. Moreover, Vassar et al. [23] have cloned is consistent with the existence of distinct a transmembrane aspartic protease, BACE, pools of APP within the nervous system [4, which has characteristics of b-secretase. Inde- 19]. The membrane-bound fragments of APP, pendently, other groups purified and cloned A4CT and p3CT (Fig. 1), can be cleaved by b-secretase obtaining the same sequence as g-secretase, an unusual protease that seems to for BACE (also called Asp 2) [24–26]. Re- cut within the transmembrane domain of cently, Lin et al. [27] isolated a human aspar- C-terminal fragments of APP [20], releasing tic protease, memapsine 2, which cleaves at respectively the 40 and 42 amino acid long Ab the b-secretase site of APP. In the case of peptides (Ab40 and Ab42) and the shorter p3 g-secretase, several authors reported that peptide [21]. The Ab40 peptide is the major presenilins are either essential for g-secretase type of Ab peptides secreted into normal hu- activity or are themselves g-secretases [18, man cerebrospinal fluid, while the more 28–31]. Recently, presenilin I was identified pathological Ab42 peptide is the minor spe- as a g-secretase, an intramembrane aspartyl cies. protease [32]. It has to be stressed that a-sec- Recently, Kosik [18] noted that the precise retase, b-secretase and presenilin (alias g-sec- site of cleavage of APP by g-secretase could be retase) are all transmembrane proteins, im- related to changes of cholesterol content plying that subtle changes in lipid composi- within the membrane. More precisely, as APP tion could modulate their activities. transits from the endoplasmic reticulum to the plasma membrane, the cholesterol con- tent within membrane increases, inducing an ROLE OF MEMBRANE increase of membrane flexibility and thick- MICRODOMAINS IN APP ness. It has been suggested that unusual mem- PROCESSING brane flexibility could permit differential ac- cess of g-secretase to the cleavage site when The localization of APP and its fragments in APP is in the endoplasmic reticulum, Golgi or distinct membrane microdomains is still con- plasma membrane [18]. Indeed, it has already troversial due to the differences in procedures been observed that the preferred site for the used to isolate these microdomains. It has production of the Ab42 peptide is in the been reported that APP does not colocalize to endoplasmic reticulum/intermediate com- a detectable extent with glycosylphosphati- partment, while the preferred site for the pro- dylinositol (GPI)-anchored proteins and ino- duction of the Ab40 peptide is Golgi appara- sitol 1,4,5-triphosphate receptor, suggesting tus and beyond [18]. that APP is not present in detergent-insoluble membrane domains with caveolae-like proper- ties [33]. The results of similar investigations SECRETASES ARE MEMBRANE indicated that APP is indeed not present in PROTEINS abundance in caveolae or caveolae-like do- mains [34]. This is in accordance with the fact The identification of proteases involved in that caveolae are not abundant structures in APP processing is of particular importance the nervous system. Although these results Vol. 47 Alzheimer’s disease and membranes 729 are convincing, it has been suggested that a phosphatidylserine [47]. It was proposed that part of APP can be transported to caveolae- membrane perturbation by aggregated Ab like domains from other membrane domains peptides constitutes the molecular basis of the for further processing [34]. In line with this peptide neurotoxicity [43]. One likely mecha- observation, it has been found that APP is nism of neurotoxicity of Ab peptides is the for- present in a detergent-insoluble glycolipid-en- mation of calcium-permeable, zinc-sensitive riched fraction, but does not behave as a typi- ion channels from aggregated Ab peptides cal detergent-insoluble membrane protein [48, 49]. [35]. APP was also localized to a unique cho- lesterol-rich domain different from caveolae or caveolae-like domain [34], and it has been CONCLUDING REMARKS reported that depletion of cholesterol content inhibits the production of the Ab peptide [36]. On the basis of the observation that mem- These results would suggest that APP process- brane lipids are targets for oxidative damage ing may occur in specific membrane micro- and that membrane lipid composition domains [37]. In addition, Ab peptides have changes during aging, leading to progressive been found in the detergent-insoluble mem- membrane defects, the origin of sporadic Alz- brane compartment [38]. Consistent with heimer’s disease could be at the lipid level of these results, it was inferred that presenilin neuronal [10–12]. The content (alias g-secretase) is present in deter- of phosphatidylethanolamine and phospha- gent-insoluble membrane microdomains [39]. tidylinositol, but not that of phosphatidyl- choline, decreases significantly in the Alzhei- mer’s brains in comparison with control THE EFFECTS OF b-AMYLOID brains [10, 11]. It has been suggested that oxi- PEPTIDES ON MEMBRANE dative stress could contribute to the selective STRUCTURE AND PERMEABILITY loss of these phospholipid classes. Altered AS RELATIONED TO THEIR membrane composition could also affect sig- NEUROTOXICITY nal transduction and induce neurodegene- ration [11]. In addition, lipid composition of The mechanism of neurotoxicity of the Ab plasma membranes of neuronal cells could peptides is not yet completely established. It modify the stability and function of the minor is based on the conformation of the peptides. pool APP bound to cell surface, as well as APP Aggregated Ab peptides contain more processing. Oxidative stress may also perturb intermolecular b-sheet structures than the sol- the structural integrity of intracellular uble ones. The correlation between neuro- organelles, altering the processing of the ma- toxicity, aggregation and b-sheet structures jor pool of APP within trans-Golgi, or within has been observed for different types of Ab the subsequent endosomal, lysosomal and peptides in neuronal cells [40–43]. Aggrega- endoplasmic reticulum compartments. tion of Ab peptides into insoluble fibers ap- Changes in lipid composition could affect the pears to be a nucleation event which can be in- function of secretases within the membrane duced by various types of factors and mole- and their activities towards APP. For exam- cules. For example, there are numerous ple, depletion of cholesterol inhibits the gener- pieces of evidence that even lipids may induce ation of the Ab peptides [36]. Membrane sta- Ab peptides aggregation, as has been de- bility depends on the plasmalogen/non- scribed in the case of interactions of Ab pep- plasmalogen ratio. Plasmalogen deficiency tides with phosphatidylinositol [44], ganglio- could, therefore, cause membrane instability side-containing membranes [45, 46] or with [12], contributing to cell death, either inde- 730 R. Buchet and S³. Piku³a 2000 pendently or cooperatively with amylo- 6. Flint Beal, M.F. (2000) Energetics in the idogenesis [12]. It is also possible that various pathogenesis of neurodegenerative diseases. classes of lipids may favor the conversion of Trends Neurosci. 23, 298–304. soluble Ab peptides into aggregated ones. 7. Auld, D.S., Kar, S. & Quirion, R. (1998) b-Amy- Phosphatidylinositol [44], phosphatidylserine loid peptides as direct cholinergic neuro- [47], phosphatidylglycerol [47], and ganglio- modulators: A missing link? Trends Neurosci. side [45, 46] favor the conversion of soluble Ab peptides into the aggregated Ab form. In 21, 43–49. contrast, myo-inositol [44] stabilizes soluble 8. Neve, R.L. & Robakis, N.K. (1998) Alzheimer’s Ab micelles, while phosphatidylcholine [47] disease: A re-examination of the amyloid hy- slows down the aggregation process. These ob- pothesis. Trends Neurosci. 21, 15–19. servations suggest that subtle lipid changes within the membranes of neuronal cells could 9. Racchi, M. & Govoni, S. (1999) Rationalizing a induce the aggregation process and produc- pharmacological intervention on the amyloid tion of ion channels by Ab peptides, inducing precursor protein metabolism. Trends Phar- their neurotoxicity. Finally, it appears from macol. Sci. 20, 418–423. the accumulated experimental evidence that 10. Prasad, M.R., Lovell, M.A., Yatin, M., Dhillon, membrane lipid composition, which can be af- H. & Markesbery, W.R. (1998) Regional mem- fected during aging, could contribute to the brane phospholipid alterations in Alzheimer’s pathology of sporadic Alzheimer’s disease. disease. Neurochem. Res. 23, 81–88. 11. Wells, K., Farookui, A.A., Liss, L. & Horrocks, L.A. 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