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The International Journal of Biochemistry & Cell Biology 40 (2008) 181–198

Available online at www.sciencedirect.com

Review

Mechanisms of A� mediated neurodegenerationin Alzheimer’s disease

Peter J. Crouch a,b,d,∗,1, Susan-Marie E. Harding c,d,1, Anthony R. White a,b,d,James Camakaris c, Ashley I. Bush d, Colin L. Masters a,d

a Department of Pathology, The University of Melbourne, Victoria 3010, Australiab Centre for Neuroscience, The University of Melbourne, Victoria 3010, Australiac Department of Genetics, The University of Melbourne, Victoria 3010, Australiad Mental Health Research Institute of Victoria, Parkville, Victoria 3052, Australia

Received 3 May 2007; received in revised form 30 June 2007; accepted 24 July 2007Available online 1 August 2007

bstract

Development of a comprehensive therapeutic treatment for the neurodegenerative Alzheimer’s disease (AD) is limited by ournderstanding of the underlying biochemical mechanisms that drive neuronal failure. Numerous dysfunctional mechanisms haveeen described in AD, ranging from protein aggregation and oxidative stress to biometal dyshomeostasis and mitochondrial failure.n this review we discuss the critical role of amyloid-� (A�) in some of these potential mechanisms of neurodegeneration. The9–43 amino acid A� peptide has attracted intense research focus since it was identified as a major constituent of the amyloideposits that characterise the AD brain, and it is now widely recognised as central to the development of AD. Familial forms of ADnvolve mutations that lead directly to altered A� production from the amyloid-� A4 precursor protein, and the degree of AD severity
orrelates with specific pools of A� within the brain. A� contributes directly to oxidative stress, mitochondrial dysfunction, impaired
synaptic transmission, the disruption of membrane integrity, and impaired axonal transport. Further study of the mechanisms of A�mediated neurodegeneration will considerably improve our understanding of AD, and may provide fundamental insights neededfor the development of more effective therapeutic strategies.© 2007 Elsevier Ltd. All rights reserved.

Keywords: Alzheimer’s disease; Amyloid-�; Amyloid-� A4 precursor protein; Neurodegeneration

Abbreviations: A�, amyloid-�; APP, amyloid-� A4 precursor protein; ABAD, amyloid-� binding alcohol dehydrogenase; AD, Alzheimer’sdisease; AFM, atomic force microscopy; ApoE, apolipoprotein E; ATP, adenosine tri-phosphate; BBB, blood–brain barrier; Ca, calcium; COX,cytochrome c oxidase; CNS, central nervous system; CSF, cerebrospinal fluid; CHO, Chinese Hampster Ovary; CQ, clioquinol; CM, conditionedmedium; Cu/Zn-SOD, copper/zinc superoxide dismutase; ECE, endothelin-converting enzyme; ERAB, endoplasmic-reticulum amyloid-� bindingprotein; ETC, electron transport chain; fAD, familial Alzheimer’s disease; IDE, insulin degrading enzyme; ICV, intracerebroventricular; LTD, long-term depression; LTP, long-term potentiation; MMP, matrix metalloproteinase; MAP, microtubule associate protein; mGluR, metabotropic glutamatereceptors; NEP, neprilysin; nAChR, nicotinic acetylcholine receptor; NFT, neurofibrillary tangle; PreP, presequence protease; ROS, reactive oxygen
species; sAD, sporadic Alzheimer’s disease; SEC/MALLS, size exclusion microscopy/multiangle laser light scattering; Tg, transgenic
∗ Corresponding author at: Department of Pathology, The University of Melbourne, Victoria 3010, Australia. Tel.: +61 3 8344 1805;ax: +61 3 8344 4004.

E-mail address: [email protected] (P.J. Crouch).1 These authors contributed equally to the work.

357-2725/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.biocel.2007.07.013

mailto:[email protected]

dx.doi.org/10.1016/j.biocel.2007.07.013

5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192. . . . .. . . . .

activity at these sites the relative A� yield from APP isaffected. Familial APP mutations increase the relativeproduction of A�42 compared to A�40 (Suzuki et al.,1994), and this may be an important factor in the devel-

Fig. 1. Secretase mediated processing of amyloid-� A4 precursor pro-tein (APP). (A) �-Secretase mediated processing cleaves APP withinthe amyloid-� (A�) domain to produce secreted sAPP� and the non-amyloidogenic C-terminal fragment C83. C83 can undergo furtherprocessing mediated by �-secretase cleavage at the C-terminal end ofthe A� domain to yeild non-amyloidogenic P3. (B) Alternate APP pro-

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction

Central to most current Alzheimer’s disease (AD)research is a small 39–43 amino acid peptide known asamyloid-� (A�). A� is an amyloidogenic cleavage prod-uct of the A� A4 precursor protein (APP), an 87 kDatransmembrane protein with apparent homology to acell-surface receptor (Kang et al., 1987). Several met-alloproteinases (secretases) are known to cleave APP,with �-secretase mediating an APP processing path-way often regarded as normal since this pathway is notdirectly implicated in the development of AD. Alter-nate APP processing by the combined activity of �-and �-secretases gives rise to the A� peptide (Fig. 1).APP is ubiquitously expressed, and cells possess theenzymatic machinery required not only to produce A�but also to degrade it, suggesting that the productionof A� from APP may serve a normal biological role.The non-pathological role for A� is yet to be estab-lished, but several reports have indicated the involvementof A�/APP in a broad range of cellular processes(Table 1).

The post-mortem AD brain is defined histopatholog-ically by the presence of amyloid deposits in affectedareas of the brain. Following its purification fromcerebrovascular amyloid deposits and the extracellu-lar amyloid plaques that characterise the AD brain(Glenner & Wong, 1984a, 1984b; Masters et al., 1985;Selkoe, Abraham, Podlisny, & Duffy, 1986), consid-erable research attention turned towards attempting toelucidate the neurotoxic mechanisms of A�. A� is nowwidely regarded as central to the development of AD,with the A� amyloid pathway an important underlying
factor that determines the peptide’s toxicity. Developingtherapeutic strategies that aim to inhibit the A� amyloidpathway is therefore an area of intense research focus(Masters & Beyreuther, 2006).
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

Evidence for the relationship between the develop-ment of AD and abnormal A� production comes from thefamilial forms of AD. Familial AD (fAD) only accountsfor ∼10% of all AD cases, but the most significant fADmutations are all associated with APP processing to yieldA�. Mutations within APP that are adjacent to the �-,�- and �-secretase cleavage sites have been identified(Chartier-Harlin et al., 1991; Citron et al., 1992; Gameset al., 1995; Goate et al., 1991), and by altering secretase

182 P.J. Crouch et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 181–198

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1822. A� oligomerisation and the toxic A� species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1843. A� turnover and accumulation in AD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1854. Potential mechanisms of A� mediated neurodegeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

4.1. Mitochondrial dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1864.2. Oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1874.3. Synaptic transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1884.4. Axonal trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1904.5. Membrane disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

cessing initiated by �-secretase cleavage at the N-terminal end of theA� domain gives rise to sAPP� and the amyloidogenic C99 fragment.Subsequent �-secretase cleavage of C99, again at the N-terminal end ofthe A� domain, in this instance gives rise to the highly amyloidogenicA�.

P.J. Crouch et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 181–198 183

Table 1Cellular pathways and processes affected by A�/APP in health and disease

Cellular pathway/process affected References

Biometal homeostasis White, Reyes, et al. (1999), Barnham, McKinstry, et al. (2003), Bellingham, Ciccotosto, et al. (2004),Bellingham, Lahiri, et al. (2004)

ROS detoxification Moreira et al. (2005), Castellani, Lee, Perry, and Smith (2006), Moreira et al. (2006)Mitochondrial dysfunction Yan et al. (1997), Casley, Land, et al. (2002), Lustbader et al. (2004), Caspersen et al. (2005), Crouch et

al. (2005)Ca homeostasis Arispe, Rojas, et al. (1993), Mark, Ashford, Goodman, and Mattson (1995)Oxidative damage Behl et al. (1994), Shearman et al. (1994), Huang, Atwood, et al. (1999), Opazo et al. (2002)Axonal transport Wirths et al. (2002), Wirths, Weis, Kayed, et al. (2007), Wirths, Weis, Szczygielski, et al. (2006)Membrane integrity Arispe, Pollard, et al. (1993), Arispe, Rojas, et al. (1993), Barnham, Ciccotosto, et al. (2003), Ciccotosto

et al. (2004), Tickler et al. (2005)S al. (200

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some of the mechanisms of A� toxicity reported overrecent years, but we first describe two important eventsthat underlie all A� toxicity; A� oligomerisation and A�accumulation.

ynaptic transmission Cullen et al. (1997), Kim et

pment of AD since A�42 tends to be more toxic andore amyloidogenic. Familial AD mutations are also

ssociated with components of the APP secretases, suchs presenilin 1 from �-secretase. It is less clear howeverhether these fAD mutations increase A� production or

he ratio of A�42 compared to A�40 (Shioi et al., 2007).Due to their abundance within amyloid deposits,

nsoluble high molecular weight A� fibrils were initiallyelieved to be the primary toxic form of A�. However,ocus shifted onto soluble low molecular weight formsfter studies indicated that the abundance of these A�pecies within the AD brain appeared to correlate bestith AD severity. McLean et al. (1999) for examplerepared phosphate buffered saline extracts from ADrain sections and demonstrated that abundance of A�pecies in the non-sedimentable fraction (including� monomers, dimers and trimers) correlated bestith markers of disease severity such as density ofeurofibrillary tangles and age at death. Numerous initro and in vivo studies now show that A� speciesoluble in diverse solvents and media solutions areubstantially more potent than insoluble A� (Croucht al., 2005; Hartley et al., 1999; Lambert et al., 1998,001; Walsh et al., 1999). Such studies therefore providencreasing evidence that A� mediated neurodegen-ration in AD is the result of toxic pools of soluble�, and that the large, extracellular aggregates of

nsoluble A� merely represent end-stage products of theisease.

In the 20 years since A� was identified as an impor-ant constituent of the amyloid deposits that characterisehe AD brain, this small peptide has received intense
esearch focus as an important factor that contributesirectly to development of the disease. Relative abun-ance of the peptide within the AD brain is central toisease development, but less certain are the mechanisms
1), Walsh et al. (2002, 2005), Bush (2003)

of A� mediated neurotoxicity. Several potential mecha-nisms have been proposed, and in this review we discussjust some of the data to indicate a direct role for A� in theneurodegeneration of AD. These mechanisms are sum-marised in Fig. 2. Our focus in this review is to present

Fig. 2. Mechanisms of A� mediated neurodegeneration in Alzheimer’sdisease. Amyloidogenic processing of APP via �- and �-secretases pro-duce A� with the potential to generate neurodegeneration by inhibitingmitochondrial activity, synaptic transmission and axonal transport, bydisrupting membranes, and by generating oxidative stress.

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2. A� oligomerisation and the toxic A� species

Central to the A� hypothesis of AD is that diseaseprogression is the result of an increased A� burden inaffected areas of the brain. Equally important to totalA� load however is the aggregation state in which A�is present. Initially produced as a soluble 4 kDa pep-tide, the amyloidogenic A� readily interacts with otherA� molecules to progressively form a wide range ofoligomers and soluble aggregates. Continued amyloido-genesis ultimately gives rise to the high molecular weightinsoluble A� fibrils that are present within amyloiddeposits of the AD brain. Amyloidogenesis is commonto several proteins associated with degenerative diseases,indicating that the mechanisms of degeneration mayshare some commonality with respect to the proteins’amyloidogenic properties (see (Glabe, 2006) for review).

Initial AD research focused on extracellular A� fibrilsas the primary toxic A� species due to their abundancewithin amyloid plaques, but this has been a contentiouspoint since relatively early reports demonstrated poorcorrelation between AD severity and amyloid plaqueburden (Braak, & Braak, 1990, 1991). Soluble A�species (monomers, dimers, trimers, etc.) became moreintensely studied in AD once it was shown that A� inthe soluble fraction of AD brain samples correlated bestwith AD severity (Lue et al., 1999; McLean et al., 1999).This has since been further refined to exclude monomericA� after reports demonstrated that when compared tosoluble A� oligomers/aggregates, A� monomers are rel-atively non-toxic. Dahlgren et al. (2002) for exampledemonstrated that A� oligomer preparations are ∼40
times more toxic towards neuronal cells grown in cul-ture compared to unaggregated A� preparations thatcontained predominantly monomeric A�. The generalconsensus in AD research at present therefore is that
Table 2Various names for toxic soluble A�

Soluble A� name References

A4 soluble dimers (A8), tetramers (A16), etc. Masters et al. (1985)Amorphous aggregates Davies et al. (1988), HuanA�-derived diffusible ligands (ADDLs) Lambert et al. (1998)�-Balls Laurents et al. (2005)�-Amy balls Westlind-Danielsson andGlobular A� oligomer Barghorn et al. (2005)Naturally secreted oligomers Walsh et al. (2002), ClearParanuclei Bitan, Vollers, et al. (2003Preamyloid Huang et al. (2000)Protofibril Harper, Lieber, and LansbSpherocylindrical miscelles Lomakin, Teplow, KirschSpherical particles Gorman, Yip, Fraser, andSpherical prefibrillar aggregates Frost, Gorman, Yip, and C

hemistry & Cell Biology 40 (2008) 181–198

soluble A� oligomer intermediates in the A� amyloido-genic pathway are the key contributors to A� mediatedneurodegeneration, and it was recently proposed thatthe large insoluble A� deposits within the AD brainmay even act as A� reservoirs for the formation oftoxic soluble oligomeric species (Haass & Selkoe, 2007).However, there is still no consensus on the specificspecies of A� that contributes most to neurodegener-ation in AD. As described below, apparent toxicity andstructure of specific A� species may be highly biaseddepending on the toxicity assays and detection methodsused.

Numerous reports now describe soluble A�oligomers/aggregates with an apparent potential tocontribute directly to the neurodegeneration seen inAD. Although a wide range of names exist for thesetoxic A� species (Table 2), all appear consistent inthat they represent soluble A� oligomers. A�-deriveddiffusible ligands (ADDLs) for example were describedinitially as small diffusable A� oligomers with anestimated molecular mass of 17–42 kDa (Lambert etal., 1998). ADDLs have been shown to be neurotoxicto neuronal organotypic cultures (Lambert et al., 1998)and subsequently shown to be potent inhibitors of hip-pocampal long-term potentiation (Walsh et al., 2002).The concentration of ADDLs in AD patients appearsto be ∼70-fold higher than in age matched controls(Gong et al., 2003), and ADDLs bound to hippocampalneurons grown in culture have recently been shown toalter neuronal morphology and decrease expression ofspecific, memory-related neuronal receptors (Lacor etal., 2007).

When attempting to identify the primary toxic A�species relative to the continuum of A� oligomerisa-tion/aggregation, a significant technical obstacle lies inthe inherent amyloidogenic properties of A�. A� can

g, Yang, Fraser, and Chakrabartty (2000)

Arnerup (2001)

y et al. (2005))

ury (1997), Walsh, Lomakin, Benedek, Condron, and Teplow (1997)ner, and Benedek (1997), Yong et al. (2002)Chakrabartty (2003)hakrabartty (2003)

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P.J. Crouch et al. / The International Journal

ligomerise rapidly in solution, the process of which istrongly influenced by factors as varied as ionic strengthf the solvent, pH, temperature, and A� concentra-ion. Many assays of A� toxicity therefore preclude anyhort-term temporal correlation between toxicity and thebundance of specific A� species. In neuronal cell cul-ure viability assays for example, A� may be includedn cell culture media for hours to days before cell via-ility is determined. Under these conditions A� mayligomerise/aggregate substantially and therefore passhrough many aggregation states, raising the possibil-ty that the A� introduced into the toxicity assay mayot remain static while measuring relative toxicity. Inddition to this, there is some debate regarding validityf the analytical methods used to characterise specific� species. Hepler et al. (2006) have recently examined

he apparent abundance of A� species in solution byomparing the more commonly used methods of gel elec-rophoresis and atomic force microscopy (AFM) withhe alternative method of size-exclusion chromatogra-hy coupled to multiangle laser light scattering detectionSEC/MALLS). They were able to demonstrate substan-ial discrepancies in the apparent molecular weight of A�pecies in solution depending on the detection methodsed.

A study that attempted to correlate the abundancef a specific A� species with A� toxicity in vivoas described recently. Lesne et al. (2006) presentedata to show cognitive decline in transgenic AD miceTg2576 mice which over-express human APP) corre-ated with a soluble A� species they called A� star 56A�*56). Using gel electrophoresis, Lesne et al. (2006)xamined endogenous A� species from the brains ofoung, mid aged, and old Tg2576 mice, and demon-trated that of all A� species detected, abundance of the6 kDa extracellular A� oligomer A�*56 was showno have the strongest inverse correlation with perfor-

ance in a modified Morris water maze test for spatialemory. When A�*56 was isolated from brains ofg2576 mice and injected into brains of 4-month-oldale rats, the rats also demonstrated decreased perfor-ance in a test for spatial memory. In a related study,

ut one that used a substantially different experimentalpproach, Crouch et al. (2005) attempted to determineoxicity relative to the continuum of A� oligomerisationsing synthetic A� and testing for its inhibitory effectowards the mitochondrial enzyme cytochrome c oxi-ase (COX). A� preparations were aged at 37 ◦C then
liquots were removed at specific time points to measureOX inhibition and relative abundance of A� oligomersy gel electrophoresis. To stabilise A� oligomers prioro electrophoresis the A� aliquots were chemically
hemistry & Cell Biology 40 (2008) 181–198 185

cross-linked using the rapid technique of photo-inducedchemical cross-linking of unmodified proteins (Fancy& Kodadek, 1999), as modified for its application toA� (Bitan, Kirkitadze, et al., 2003; Bitan, Lomakin, &Teplow, 2001; Bitan & Teplow, 2004; Bitan, Vollers,& Teplow, 2003). Of all low molecular weight A�species detected (monomer through to hexamer) the levelof COX inhibition correlated best with abundance ofdimeric A�. The studies of Lesne et al. (2006) andCrouch et al. (2005) both therefore indicated that A�toxicity was mediated by soluble oligomeric speciesof A�. Whether the different toxic A� species identi-fied (A�*56 or dimeric A�) reflects differences in thetoxicity assays employed or differences in the A� detec-tion methods used, or perhaps a combination of thetwo, remains to be determined. This is a technical chal-lenge for all current research that attempts to identify thespecies of A� primarily responsible for neurodegenera-tion and cognitive decline in AD.

3. A� turnover and accumulation in AD

Critical in AD is the accumulation of A� withinaffected areas of the brain, and developing therapeuticstrategies that aim to decrease A� load is an importantarea of AD research. Some fAD data indicates that A�accumulation may be the result of increased A� pro-duction and/or stabilisation, but less certain is whetherA� accumulation may be due to decreased A� degrada-tion or perhaps decreased A� clearance from the brain.Genetic evidence for this possibility is yet to be pro-duced, but it may explain at least some of the forms ofAD currently regarded as sporadic.

Bateman et al. (2006) examined rates of A� turnoverin the cerebrospinal fluid (CSF) of healthy 23–45-year-old men and women by infusing with radiolabelledleucine prior to collecting CSF and analysing levels oflabelled A�. They found that fractional synthesis andclearance rates per hour were 7.6% and 8.3%, respec-tively, indicating that although A� may be producedin healthy individuals, functional clearance mechanismsprevent its accumulation. While the study of Bateman etal. (2006) represents a significant breakthrough in study-ing A� turnover in vivo, no elderly or demented patientswere examined, and it is therefore still unclear whetherdecreased clearance rates contribute significantly to A�accumulation in AD.

Several proteinases have now been described with
demonstrated capacity to degrade A� in vitro and in vivo,but a genetic correlation between proteinase activitytowards A� and AD has not been identified, despite somespecific research attempts (Shibata et al., 2005). Some of

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the proteinases with demonstrated capacity to degradeA� include insulin degrading enzyme (IDE) (Qiu etal., 1998), neprilysin (NEP) (Iwata et al., 2000), andendothelin-converting enzyme (ECE) (Eckman, Reed,& Eckman, 2001) (see (Eckman & Eckman, 2005)for a recent review), but one particular family of A�-degrading proteinases recently associated with anotherimportant aspect of AD biology (decreased metal ionhomeostasis) is the matrix metalloproteinases (MMPs).There are over 20 known MMPs and their expres-sion is relatively cell-type specific (Malemud, 2006).MMP-9 has attracted the most research attention as aproteinase that may be involved in the developmentof AD since it was first described to degrade A� in1996. Backstrom, Lim, Cullen, and Tokes (1996) demon-strated that MMP-9 is synthesised within neurons ofthe human hippocampus and that when exposed to syn-thetic A�40 it can cleave the A� at several sites. It wasalso demonstrated that the cellular distribution of MMP-9 is substantially altered in the AD brain. Subsequentstudies have shown that MMP-9 levels are increasedin the amyloid-positive brains of dogs (Lim, Russell,Cullen, & Tokes, 1997), and that the levels of MMP-2and MMP-3 are increased when cells grown in culture areexposed to A� (Deb, Wenjun Zhang, & Gottschall, 2003;Jung, Zhang, & Van Nostrand, 2003). More recently,Yin et al. (2006) demonstrated that MMP-2 and MMP-9expression is increased in astrocytes surrounding amy-loid plaques in the brains of Tg AD mice, and that theseMMPs in the astrocyte conditioned medium can degradesynthetic A�40 and A�42. This finding is of considerablesignificance to the A� theory of AD because it suggeststhat although A� within the brain may be largely neu-ronal in origin, the catabolic mechanisms responsible forregulating extracellular A� are mediated by astrocytes.

4. Potential mechanisms of A� mediatedneurodegeneration

Regardless of the biochemical or genetic factors thatdetermine A� accumulation and/or oligomerisation, itis now widely recognised that A� has the potential tocontribute directly to the cognitive decline and neurode-generation that is characteristic of AD. The remainingsections of this review presents research into just someof the potential mechanisms of A� mediated neurode-generation.

4.1. Mitochondrial dysfunction

Growing evidence indicates that mitochondrialdysfunction may be an important factor in the pathophys-


iology of AD. Impaired functionality of the mitochondriais consistent with altered glucose metabolism evidentwithin the AD brain (Duara et al., 1986), and is supportedby biochemical studies on post-mortem brain samplesthat show substantially altered activity for enzymesof the mitochondrial tricarboxylic acid cycle (Bubber,Haroutunian, Fisch, Blass, & Gibson, 2005). Electronmicroscopy studies show altered mitochondrial mor-phology in affected areas of the AD brain (Baloyannis,Costa, & Michmizos, 2004), and an overall decreasein the number of mitochondria within vulnerable neu-rons (Hirai et al., 2001). Hirai et al. (2001) have alsonoted that altered levels of mitochondrial DNA and COXoccur within neurons prior to the formation of neurofib-rillary tangles and therefore proposed “mitochondriaabnormalities as the earliest cytopathological change inAD”.

The possibility that A� contributes directly to themitochondrial dysfunction seen in AD is supported byseveral lines of evidence, some of which are describedbelow. Underlying this potential mechanism of neurode-generation in AD however is an important implicationthat has not been universally accepted; that the concen-trations of intracellular A� do not reach levels sufficientto cause significant toxicity. This is based largely onobservations that APP and the secretase complexeslocalise mainly to the secretory pathway, and that mostA� produced is secreted from the cell. The intracel-lular production and retention of A� has nonethelessbeen reported (see (Hartmann, 1999) for review), andthe most compelling data to show intracellular accumu-lation of A� has come from studies on mitochondria.Yan et al. (1997) for example described an intracellu-lar interaction between A� and ERAB (endoplasmicreticulum-associated A� binding protein) due to theapparent localisation of this protein to the endoplas-mic reticulum. Subsequent studies however revealed thatERAB was a mitochondrial protein (termed ABAD;A� binding alcohol dehydrogenase) with a cDNAsequence identical to L-3-hydroxyacyl-CoA dehydro-genase (Furuta, Kobayashi, Miyazawa, & Hashimoto,1997; Yan et al., 1999). Strong data was then providedto show that A� binding to ABAD altered the enzyme’sactive site and prevented native dehydrogenase activity,and that A� binding to ABAD was evident within mito-chondria of the AD brain (Lustbader et al., 2004). Thesestudies therefore provided evidence for the presence ofa mitochondrial pool of A� within the AD brain that
also had the capacity to disrupt normal mitochondrialfunctionality. Other groups have since provided evidencefor a mitochondrial pool of A� within the brains of ADpatients, Tg AD mouse models, and neuronal cells over-

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xpressing APP (Caspersen et al., 2005; Crouch et al.,005; Manczak et al., 2006).

The possibility that mitochondrial accumulationf A� is due to secretase mediated APP process-ng within the mitochondria is supported by studieshat show APP contains a mitochondrial targetingequence, and that APP is present in mitochondria fromD brains (Anandatheerthavarada, Biswas, Robin, &vadhani, 2003; Devi, Prabhu, Galati, Avadhani, &nandatheerthavarada, 2006). Furthermore, the pres-

nce of �-secretase constituents (nicastrin, presenilin-1,PH-1 and PEN-2) and functional �-secretase activity

n isolated rat brain mitochondria has been demonstratedHansson et al., 2004), indicating that at least some of theecretase machinery required to produce A� is presentn the mitochondrial membrane. Alternatively, A� mayccumulate within the mitochondria due to decreaseditochondrial capacity to regulate A� concentrations.tahl et al. (2002) demonstrated that the mitochondriaontain a metalloproteinase they called PreP (prese-uence protease), and it was recently shown that theuman mitochondrial homologue is capable of degrad-ng A� (Falkevall et al., 2006). A decline in activityf A� degrading proteases such as PreP within brainitochondria may contribute to an AD-related increase

n mitochondrial A�, but data is yet to be presented toupport this possibility.

Most data to demonstrate the functional effects of� on mitochondrial activity come from studies where

ntact mitochondria isolated from cell cultures or tis-ue samples are exposed directly to A�. The techniquesequired to isolate and biochemically characterise mito-hondria (Trounce, Kim, Jun, & Wallace, 1996) are aowerful tool in this regard, since the potential effectsf A� can have strong implications for the decreasednergy metabolism and oxidative stress consistent withD. Several studies have now shown that direct exposure

o A� significantly impairs functionality of the mito-hondrial electron transport chain (ETC). The ETC isentral to ATP production and its constituent enzymeomplexes are a major source of reactive oxygen speciesROS) generation, particularly when activity of one orore of the enzyme complexes is inhibited. By exposing

solated mitochondria preparations to full length (Casley,anevari, Land, Clark, & Sharpe, 2002; Crouch et al.,005, 2006) or truncated (Canevari, Clark, & Bates,999; Cardoso, Santos, Swerdlow, & Oliveira, 2001;asley, Canevari, et al., 2002; Parks, Smith, Trimmer,
ennett, & Parker, 2001) forms of the A� peptide, sev-ral groups have shown that A� is a potent inhibitorf the mitochondrial ETC. The terminal ETC enzymeomplex COX is commonly implicated as the most sig-

nificant site of A� mediated inhibition, and these in vitrostudies have particular relevance to AD since analysesof AD brain samples have shown evidence for decreasedCOX activity (Chagnon, Betard, Robitaille, Cholette, &Gauvreau, 1995; Kish et al., 1992; Mutisya, Bowling, &Beal, 1994). Two studies that examined the mechanismof COX inhibition by A� indicate that the inhibition isdependent on divalent copper (Cu) at a Cu:A� molarratio of ∼0.75:1 or higher, and that the formation of anA� dimer is involved (Crouch et al., 2005, 2006). Theredox potential of the peptide’s methionine-35 residuealso appears to be involved since substitution of themethionine residue with valine or pre-oxidation of thesulphur atom of methionine-35 prevented Cu-dependentinhibition of COX by A�42 (Crouch et al., 2006).

The effects of A� on mitochondria are not limitedto impaired activity of the ETC. Exposure to increasedlevels of A� can decrease mitochondrial membranepotential and respiration rates, as well as induce mito-chondrial swelling, cytochrome c release, transition poreopening, and mitochondrial ROS output (Aleardi et al.,2005; Cardoso, Swerdlow, & Oliveira, 2002; Clementiet al., 2005; Keil et al., 2004; Kim et al., 2002; Moreira,Santos, Moreno, Rego, & Oliveira, 2002; Mungarro-Menchaca, Ferrera, Moran, & Arias, 2002; Misiti et al.,2004; Takuma et al., 2005). All of these A� mediatedeffects can have a strong impact not only on mitochon-drial functionality, but also on overall cell viability.

4.2. Oxidative stress

Oxidative stress has been implicated as a major causeof neurotoxicity in a number of neurodegenerative disor-ders including AD, and there is strong evidence linkingoxidative stress to A�. Oxidative stress occurs when freeradical production exceeds antioxidant defence systems,and oxidative damage to cellular components follows.Most free radicals within a cell originate from ROS pro-duced via the inefficient transfer of electrons to oxygenduring oxidative ATP production within the mitochon-dria. Although a relatively small organ by mass, the brainhas a disproportionately high level of oxygen consump-tion due to its high demand for ATP. It accounts for∼20% of the body’s total basal oxygen consumption(Shulman, Rothman, Behar, & Hyder, 2004) and subse-quently generates relatively high levels of ROS. Due tothe detrimental effects of ROS on normal cell functional-ity, cells have many overlapping mechanisms to prevent
or repair oxidative damage. These include antioxidantmolecules such as glutathione and ascorbate, and antiox-idant enzymes such as catalase, glutathione peroxidase,glutathione reductase and superoxide dismutase (SOD)

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(Rae, Schmidt, Pufahl, Culotta, & O’Halloran, 1999;Valko, Morris, & Cronin, 2005). Unfortunately, the brainis comprised mostly of post-mitotic tissue with lim-ited regenerative capacity and relatively poor antioxidantsystems (Cooper, 1997). The brain is also rich in macro-molecules such as unsaturated fatty acids which areprone to oxidative damage (Clarke & Sokoloff, 1999).These features make the brain particularly susceptible tooxidative stress.

A significant, consistent feature of AD is that theaffected brain is under severe oxidative stress. An earlyreport in 1986 (Martins, Harper, Stokes, & Masters,1986) described an increase in activity of enzymes fromthe hexose monophosphate pathways in post-mortemAD brain samples compared to age-matched controls,and proposed that this reflected increased oxidative stressin the AD brain. Numerous reports have since provideddirect data to show extensive oxidative damage in AD,including data on lipid peroxidation (Arlt, Beisiegel, &Kontush, 2002; Butterfield, Castegna, Lauderback, &Drake, 2002; Sayre et al., 1997), protein carbonyls toshow oxidised protein (Smith et al., 1991), and increasednucleic acid damage (Gabbita, Lovell, & Markesbery,1998; Perry et al., 2003). Furthermore, antioxidantenzymes are elevated in the hippocampus and amygdalaof AD patients (Pappolla, Omar, Kim, & Robakis, 1992;Zemlan, Thienhaus, & Bosmann, 1989).

Oxidative damage in AD may be a direct resultof A�. Markers of oxidative DNA damage, includingmitochondrial DNA damage, have been localized toamyloid plaque affected areas in the AD brain (Mecocci,MacGarvey, & Beal, 1994); the generation of lipidperoxidation products and the lipoperoxidation of mem-branes is associated with amyloid plaques in Tg mice(Matsuoka, Picciano, La Francois, & Duff, 2001); andthere is a high correlation between amyloid plaques andlipid peroxidation in AD (Lovell, Ehmann, Butler, &Markesbery, 1995). Importantly, oxidative damage inAD, possibly induced by A�, may further exacerbateA� toxicity by modulating the A� amyloid pathway.Recently Siegel, Bieschke, Powers, and Kelly (2007)demonstrated that elevated levels of 4-hydroxy-2,3-nonenal, a product of lipid peroxidation, promote A�protofibril formation by covalently modifying A�.

The mechanism of A� mediated oxidative stress maybe direct or indirect. As described above, functional-ity of the mitochondrial ETC is particularly susceptibleto inhibition by A�, and inhibition of the ETC is a
major source of ROS within the cell. A� mediatedinhibition of mitochondrial functionality may thereforerepresent an indirect source of oxidative stress. Alter-natively, oxidative stress may involve direct production

of ROS by A�. A� can cause neurotoxicity by produc-tion of ROS (Behl, Davis, Lesley, & Schubert, 1994;Shearman, Ragal, & Iversen, 1994), the mechanism ofwhich may be directly related to biometal dyshomeosta-sis evident in the AD brain (Lovell, Robertson, Teesdale,Campbell, & Markesbery, 1998). A� binds Cu ions viaits three histidine residues and aggregated A� bindsCu via a bridging histidine molecule (Curtain et al.,2001; Dong et al., 2003). Interactions between Cu andA� can result in oxidative damage as A� has the abil-ity to reduce bound Cu(II) to Cu(I) and this reactionproduces H2O2 as a by-product (Opazo et al., 2002).Generation of H2O2 by A�-Cu can result in oxidativedamage as H2O2 is diffusible through the cell membraneand can oxidize lipids and intracellular proteins. Greateroxidative damage is then induced upon the secondaryinteraction of H2O2 with A�-bound Cu(I). This gen-erates OH• (Huang, Atwood, et al., 1999) which willreact with lipids, proteins and nucleic acids resultingin extensive oxidative modifications that are often irre-versible. Interestingly, OH• can also react with A� itselfto further promote A� aggregation. Oxidative damageto A� can result in the formation of di-tyrosine cross-linking between A� peptides and subsequent covalentoligomerization (Atwood et al., 2004; Barnham et al.,2004).

Cell culture studies have provided support for A�ROS production as a potential mechanism for A�mediated neurodegeneration. When synthetic A�42 wasincubated with rat forebrain primary neuronal culturesfor 48 h cell viability was ∼65% compared to untreatedcontrols (Huang, Cuajungco, et al., 1999). This toxiceffect of A� was further enhanced by the presence ofequimolar amounts of Cu(II) as treatment with A� andCu(II) resulted in only ∼40% cell viability (Huang,Cuajungco, et al., 1999). The data were consistent withCu inducing increased ROS production since cell viabil-ity in the presence of A�-Cu was rescued by the presenceof catalase (Huang, Cuajungco, et al., 1999). Further-more, neurons depleted of essential antioxidants suchas glutathione are more susceptible to A�-Cu mediatedtoxicity (White, Bush, Beyreuther, Masters, & Cappai,1999), and A�-Cu mediated neurotoxicity can be exac-erbated by reducing agents found at high concentrationsin the brain.

4.3. Synaptic transmission

The physical basis for cognitive decline and lossof memory in AD may be a failure of neuroplastic-ity in affected regions of the brain (Mesulam, 1999;Taylor, Birch-Machin, Bartlett, & Turnbull, 1993). Neu-

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oplasticity encompasses all processes that mediate thetructural and functional organisation of neurons andynapses, but of particular relevance to A� in AD arehe opposing processes of long-term potentiation (LTP)nd long-term depression (LTD). LTP affects neuroplas-icity by increasing the chemical strength and potentialctivity of a synapse, whereas LTD decreases the chemi-al strength. Several studies have now indicated that A�an substantially alter LTP and LTD, raising the possi-ility that cognitive decline in AD may be due to a directffect of A� on synaptic transmission.

Nalbantoglu et al. (1997) studied behaviour and LTPn Tg mice that over-expressed the C-terminal 104 aminocid residues of APP. This APP fragment includes the A�omain and it was demonstrated that with ageing theseice developed increased extracellular A� immunore-

ctivity. When subjected to the Morris water maze testor spatial learning the Tg mice were considerablyess efficient in the test compared to non-Tg controls,ndicating significant cognitive impairment. Subsequentlectrophysiology assays on sections of the hippocampusemonstrated that LTP in the Tg mice was significantlympaired (Nalbantoglu et al., 1997), providing a potential

echanistic basis for the water maze results. LTD in hip-ocampal sections was the same for Tg and non-Tg mice.he possibility that decreased cognitive capacity andTP in this study was due to non-A� regions of the APP-TF could not be ruled out, but Cullen, Suh, Anwyl, andowan (1997) presented data to show that both A� and

he APP-CTF could contribute to decreased LTP. Theydministered A�40, A�42 and APP-CTF (105 amino acidragment) directly into the brains of adult rats by intrac-rebroventricular (ICV) injection, then measured LTP inivo. All three compounds significantly decreased LTP,ith A�42 and APP-CTF proving considerably moreotent than A�40 (Cullen et al., 1997). A similar studyhat used ICV injection of A� in rats demonstrated that�42 and APP-CTF could both induce LTD in vivo

Kim, Anwyl, Suh, Djamgoz, & Rowan, 2001), indicat-ng that synaptic transmission may be affected through� mediated modulation of LTP and LTD.Walsh et al. (2002) examined the effects of A� on

TP, and as per previous studies in this field they wereble to show that ICV injection of A� inhibited hip-ocampal LTP in rats. Instead of synthetic A� however,hey used “naturally secreted A� oligomers” from cellsrown in culture. CHO cells were transfected to over-xpress human APP, and the conditioned media (CM)
rom the cells was used for the ICV injections. Experi-ents that involved degradation or immunoprecipitation
f the A� prior to LTP assays indicated that inhibi-ion of LTP was due to A� oligomers in the CM. Some


experiments for example involved the preferential degra-dation of monomeric A� in the CM by incubation withinsulin degrading enzyme (IDE). IDE had previouslybeen shown to degrade A� (Qiu et al., 1998), and by incu-bating CM containing monomeric and oligomeric formsof A� with IDE it was shown that only the monomericform of A� had been degraded (Walsh et al., 2002).Inhibition of LTP by CM depleted of monomeric A�indicated that the inhibition was due to A� oligomers(Walsh et al., 2002), and this was supported by sub-sequent reports that showed LTP could be restored byinhibitors of A� oligomerisation (Walsh et al., 2005).

The effects of A� on LTP and LTD in vitro and invivo appear consistent. Less consistent are the possi-ble mechanisms through which A� may mediate theseeffects. Itoh et al. (1999) infused the brains of rats withsynthetic A� for 10–11 days then dissected the brains torecord hippocampal LTP. Consistent with other reportsthey found that A� substantially affected LTP, and theyproposed the effect was via nicotinic acetocholine recep-tors (nAChR) since treatment of hippocampal slices withnicotine had less of an effect on vehicle infused ratscompared to A� infused rats (Itoh et al., 1999). A rolefor nAChR in A� mediated inhibition of LTP was sup-ported by a later study on hippocampal slice preparationsthat showed treatment with A� induced ERK2 activa-tion, and that this response was prevented by nAChRantagonists (Dineley et al., 2001). In another study how-ever, data was presented to suggest that nAChR werenot involved (Wang, Walsh, Rowan, Selkoe, & Anwyl,2004). In this study the effects of synthetic and natu-rally secreted A� on LTP in rat hippocampal slices invitro were determined in the presence of specific pro-tein kinase inhibitors and antagonists for membranesreceptors. The data presented indicated that the A� medi-ated effects on LTP involved signalling pathways thatincluded JNK, CdK5 and p42 MAPK, but not path-ways that included ERK2 (p42 MAPK) (Wang et al.,2004). Furthermore, antagonists to metabotropic gluta-mate receptors (mGluR), but not nAChR, prevented theA� mediated effects on LTP, indicating that mGluR andnot nAChR were involved. These differences may reflectcritical differences in the experimental approaches usedby the various research groups, or they may reflect mul-tiple modes of action for A� on synaptic transmission.

An alternative potential mechanism for A� mediatedinhibition of synaptic transmission is based on the capac-ity for A� to bind Zn ions. Numerous reports have now
described the capacity for A� to bind Zn and other metalions such as Cu (Atwood et al., 2000; Curtain et al., 2001;Danielsson, Pierattelli, Banci, & Graslund, 2007; Syme,Nadal, Rigby, & Viles, 2004), with many demonstrating

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that the interaction with metals promotes A� oligomeri-sation and aggregation (Atwood et al., 1998, 2000; Bushet al., 1994; Huang et al., 1997). During synaptic trans-mission the concentration of Zn ions within the synapticcleft can reach ∼300 �M (Frederickson, Suh, Silva,Frederickson, & Thompson, 2000) where it is believedto function as a counter ion for the high concentrationsof glutamate present and quenches the response of theNMDA receptor (Frederickson, Koh, & Bush, 2005).When Lee, Cole, Palmiter, Suh, and Koh (2002) crossedTg2576 with mice deficient in Zn transporter-3 (ZnT-3),the protein that loads Zn ions into vesicles for releaseinto the synaptic cleft, the ZnT-3 deficient mice exhib-ited a 50% decrease in amyloid plaque burden comparedto Tg2576 littermates. In a similar study it was shownthat cerebral amyloid angiopathy in Tg2576 mice is abol-ished when the ZnT-3 gene is ablated (Friedlich et al.,2004). These studies indicate that synaptic Zn ions inter-act with A� in vivo, supporting the possibility that anaberrant interaction between A� and metal ions withinthe synaptic cleft contributes to impaired synaptic trans-mission (Bush, 2003).

More recently, Lacor et al. (2007) have demon-strated that A�, in this instance ADDLs, binds stronglyto hippocampal neurons grown in culture, and thatADDLs binding significantly altered neuronal morphol-ogy, including dendritic spine length and density. Itwas also shown that ADDLs binding led to a signifi-cant decrease in membrane expression of the NMDAand EphB2 receptors required for normal synaptic trans-mission. Lacor et al. (2007) suggest that their findingsprovide support for the hypothesis that soluble A�oligomers contribute directly to the loss of functionalsynapses seen in AD.

4.4. Axonal trafficking

Functionality of neurons in the brain is dependent onthe sophisticated cellular machinery that controls antero-grade and retrograde transport of organelles, mRNAsand proteins along the neuronal axons. This primarilyinvolves the microtubule network of the cell, proteinsof the dynein and kinesin superfamilies, and severalmicrotubule associated proteins (MAPs), including tau.Several lines of evidence, including increased mitochon-drial constituents in lysosomes, synaptic vesicles failingat nerve termini, and vesicle accumulation in cell bodies,indicate that axonal transport is substantially disrupted
in AD (see Gotz, Ittner, & Kins, 2006 for review).The post-mortem AD brain characteristically containsintraneuronal neurofibrillary tangles (NFTs), a majorconstituent of which is hyperphosphorylated tau (Kopke

et al., 1993; Kosik, Joachim, & Selkoe, 1986; Wood,Mirra, Pollock, & Binder, 1986). Normal functionality oftau in stabilising the microtubule network requires thattau is partially phosphorylated, but hyperphosphoryla-tion of tau leads to its loss of function and a toxic gainof function due to its capacity to sequester normal tauand other MAPs from the microtubule network. Thereis little doubt therefore that abnormal tau metabolismcontributes to the decreased axonal transport evident inAD (see Higuchi, Lee, & Trojanowski, 2002 for review),less clear is whether A� contributes to decreased axonaltransport.

Kamal, Stokin, Yang, Xia, and Goldstein (2000)demonstrated that APP binds to the axonal transportprotein kinesin-I using co-immunoprecipitation frommouse brain and sciatic nerve samples. In a subse-quent report they demonstrated that APP, �-secretaseand a constituent of the �-secretase complex, presenilin-1, all co-exist within the same axonally transportedvesicles (Kamal, Almenar-Queralt, LeBlanc, Roberts, &Goldstein, 2001). These studies therefore raised the pos-sibility that A� may be produced within the neuronalaxon, and that this pool of A� may contribute to axonalfailure. These findings however have been the subject ofsome debate, since other studies have not been able todemonstrate the co-localisation of APP, �-secretase andpresenilin-1 within the same axonal vesicles (Lazarov etal., 2005).

Regardless of whether A� is produced de novo withinspecific axonally transported vesicles, several reportshave indicated that A� within the axon can disrupt axonalfunctionality and potentially contribute to neuronaldegeneration (Wirths et al., 2002; Wirths, Weis, Kayed,Saido, & Bayer, 2007; Wirths, Weis, Szczygielski,Multhaup, & Bayer, 2006). Tg mice over-expressingboth mutant APP and presenilin-1 exhibited substan-tially increased levels of intraneuronal A�, particularlywithin somatodendritic and axonal compartments, andthis preceded the formation of amyloid plaques (Wirthset al., 2002). The accumulation of A� led to increasedaxonal degeneration (Wirths, Weis, Kayed, et al., 2007;Wirths, Weis, Szczygielski, et al., 2006). The authorsproposed that increased intraneuronal A� triggers notonly neuronal loss, but also contributes to disturbed neu-ronal trafficking. Importantly, the authors also noted thatthe observed A� mediated axonal degeneration precededNFT pathology (Wirths, Weis, Szczygielski, et al., 2006).

4.5. Membrane disruption

A mechanism of A� toxicity with considerableresearch support is A� mediated membrane disruption.

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he hydrophobic nature of A� physically predisposeshe peptide to interact with lipid bilayers in an aque-us environment, and interactions between A� andomponents of biological membranes such as lipids,hospholipids, and proteins have all been describedsee Verdier, Zarandi, & Penke, 2004 for review), withhe interaction between A� and cholesterol receivingarticular attention. Cholesterol levels affect APP pro-essing (Howland et al., 1998; Racchi et al., 1997;imons et al., 1998; Wahrle et al., 2002) as well as

he potential for A� to bind to the plasma membraneYip, Elton, Darabie, Morrison, & McLaurin, 2001),nd patients receiving medications that lower circulatingholesterol are ∼70% less likely to develop proba-le AD (Jick, Zornberg, Jick, Seshadri, & Drachman,000; Wolozin, Kellman, Ruosseau, Celesia, & Siegel,000).

Studies to examine the membrane associated mech-nisms of A� neurotoxicity often involve cell culturetudies where synthetic or purified A� preparations aredded to the medium of cells grown in culture. Neurotox-city of the A� can be determined by cell viability and/orurvival. Essentially these assays establish the effectsf extracellular A� interacting with the plasma mem-rane, and by altering specific experimental conditionsamino acid composition of the A� peptide, A� aggre-ation state, lipid composition of the membrane, etc)he mechanisms of A� mediated membrane disruptionan be determined. Studies with mouse primary corticaleurons and modified forms of synthetic A� have shownhat the methionine-35 residue contributes significantlyo toxicity of the A� peptide by affecting the capacityf the peptide to bind the plasma membrane (Barnham,iccotosto, et al., 2003; Ciccotosto et al., 2004). Sub-

tituting the native methionine-35 residue with valinencreased the peptide’s lipid membrane binding affinity

2-fold and generated a dramatic increase in A� neuro-oxicity (Ciccotosto et al., 2004). Furthermore, oxidationf the sulphur atom in methionine-35 partially decreasesinding of A� to the lipid bilayer and toxicity towardsrimary cortical neurons (Barnham, Ciccotosto, et al.,003).

Similar cell culture studies have since shown thathe neurotoxicity and lipid membrane affinity of A�s also strongly influenced by the histidine residues atositions 6, 13 and 14 (Tickler et al., 2005), since theechanism of A� neurotoxicity involves the formation

f a histidine bridge between A� peptides (Smith et
l., 2006). By using A� peptides with either the �- or-nitrogen of the histidine imidazole side chains methy-ated, Smith et al. (2006) demonstrated that neurotoxicityf the membrane-associated A� involved the formation

of an A� dimer; methylation of the histidine side chainsprevented A� dimer formation and A� neurotoxicity.Smith et al. (2006) also demonstrated that the mech-anism of neurotoxicity required the presence of Cu2+

at a Cu2+:A� molar ratio of at least 0.6:1. It was pro-posed that the mechanism of A� mediated neurotoxicityinvolved Cu-catalysed formation of ROS which resultedin lipid peroxidation and cell death. This was supportedby experiments that showed the relative toxicity of all A�peptides studied correlated with measurements of lipidperoxidation, and that the presence of catalase inhibitedA� toxicity towards primary cortical neurons (Smith etal., 2006).

The similarity between these cell culture studies andthe data presented for A� mediated inhibition of COX(Crouch et al., 2005, 2006) (where Cu-dependent inhi-bition of COX correlated with the formation of an A�dimer and required a Cu2+:A� molar ratio of ∼0.75:1or greater) is striking, but it is unlikely that these studiesrepresent a common mechanism of A� mediated neuro-toxicity. Data from the cell culture studies relate to theeffects of extracellular A� acting on the plasma mem-brane, whereas the COX studies examined the effectsof A� on the activity of an intracellular enzyme. Thisfundamental difference was manifested by data fromexperiments that used A� with methionine-35 substi-tuted to valine. Because the M35V peptide is morehydrophobic is was found to associate more stronglywith the plasma membrane in cell culture studies, andthis correlated with increased neurotoxicity (Ciccotostoet al., 2004). In the COX activity studies the methionineresidue was proposed to contribute to COX inhibitionbecause of the residue’s redox potential and not its rela-tive hydrophobicity, and substitution to valine thereforeprevented COX inhibition (Crouch et al., 2006). The dif-ferences in these assay systems in some regards placesgreater significance on the commonality of the toxic A�species. The cell culture and COX studies both identifieddimeric A� with ∼0.7 molar equivalents of bound Cu asthe toxic species. However, they also highlight the possi-bility that the relative toxicity of A� may be determinedby factors as fundamental as the peptide’s sub-cellularlocation.

An alternative mechanism of neurotoxicity due toA� mediated membrane disruption is channel forma-tion. The potential for A� to form membrane channelshas been demonstrated by incorporating A� into phos-phatidylserine liposomes which are then fused with
planar lipid bilayers (Arispe, Pollard, & Rojas, 1993;Arispe, Rojas, & Pollard, 1993). In these studiesA� formed large channels in the lipid bilayer thatwere able to generate a linear current-voltage rela-

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tionship in symmetrical solutions. Importantly, thechannels were permeable to various cations. Poorlyselective A� channels formed in the plasma membraneof neuronal cells can contribute directly to neuronalfailure by destroying the membrane potential criti-cal for generating action potentials, and by allowingincreased Ca flux directly through the A� channels,or indirectly by triggering increased Ca flux throughvoltage-sensitive Ca channels. The potential for A�to form channels in lipid bilayers has been stud-ied in greater detail more recently (Kourie, Henry,& Farrelly, 2001), other reports have shown thatlike alternative mechanisms of A� mediated toxicity,the capacity for A� to form channels in membranesis determined largely by conditions that affect A�oligomerisation and aggregation (Hirakura, Lin, &Kagan, 1999).

5. Conclusion

The development of effective therapeutic strategiesfor treating any disease condition requires an understand-ing of the underlying biological mechanisms involved.In the case of AD there is overwhelming consensusthat an aberrant accumulation of A� within affectedareas of the brain contributes directly to developmentof the disease. Research into the mechanisms of A�mediated neurodegeneration over the past 20 years hasrevealed that A� is central to dysfunctional processes asdiverse as membrane disruption, oxidative stress, synap-tic transmission, impaired mitochondrial activity, andaxonal trafficking. Studies in these areas have all iden-tified specific potential therapeutic targets for treatingAD, and collectively they have confirmed that AD isa complex disease that involves a multitude of dys-functional processes. Targeting specific dysfunctionalprocesses in isolation will no doubt lessen the burdenof the disease, but therapeutic strategies that have agreater broad-spectrum application will have a muchgreater impact. Identifying commonality in the vari-ous mechanisms of A� mediated neurodegeneration willconsiderably advance the development of therapeuticstrategies for AD. Most likely this breakthrough in ADresearch will come from studies that attempt to corre-late A� toxicity with specific A� species within thecontinuum of the peptide’s oligomerisation and amy-loidogenesis.

Acknowledgements

The authors thank Ms Gulay Filiz for her assistancein preparing the manuscript.


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