Protein Aggregation in the Brain: The Molecular Basis for ...ate, and severe. The initial onset of Alz-heimer’s disease is insidious, with mem-ory loss the earliest and most frequently

INTRODUCTIONExtracellular fibrous amyloid deposits

or intracellular inclusions containing ab-normal protein fibrils characterize manyneurodegenerative diseases, includingAlzheimer’s, Parkinson’s, and Hunting-ton’s diseases, amyotrophic lateral scle-rosis, frontal temporal dementia, and thehuman prion diseases (1). Burgeoningevidence suggests that accumulation ofproteins capable of forming amyloid de-posits may represent a common patho-logical mechanism for these diverse ill-nesses (2,3). In each of these diseases,

misfolding of a particular protein canlead to its aggregation, involving a pro-cess in which monomers interact to formdimers, oligomers, and eventually insol-uble fibrillar deposits. Alzheimer’s andParkinson’s diseases, representative ex-amples that account for the majority ofcases of neurodegenerative diseases, arereviewed here with an emphasis on thefundamental importance of aggregationas the pathological trigger. For each dis-ease, we first outline the major character-istics and then present the evidence forthe crucial role of soluble oligomers

(rather than the end-stage insolublefibrils) of the aggregating protein—β-amyloid protein (Aβ) in Alzheimer’sdisease and α-synuclein in Parkinson’sdisease. Finally, we discuss future thera-peutic and diagnostic approaches, basedon current understanding of Aβ andα-synuclein pathobiology.

ALZHEIMER’S DISEASE: INCIDENCE ANDSYMPTOMS

First identified by Alois Alzheimer in1906, Alzheimer’s disease is an irre-versible, progressive brain disease thatslowly destroys memory and cognitiveskills (4). It is the most common causeof dementia, accounting for more thanhalf of all such cases, and currently af-fects more than 24 million peopleworldwide, with 4.6 million new caseseach year (5). Age is the single biggestknown risk factor, with the incidence ofthe disease increasing from one in ten ofthose over 65 to almost half of thoseover 85 (6,7). There is no strong sex orrace effect, but because women tend tolive longer than men there are more

M O L M E D 1 4 ( 7 - 8 ) 4 5 1 - 4 6 4 , J U L Y - A U G U S T 2 0 0 8 | I R V I N E E T A L . | 4 5 1

Protein Aggregation in the Brain: The Molecular Basis forAlzheimer’s and Parkinson’s Diseases

G Brent Irvine,1 Omar M El-Agnaf,2 Ganesh M Shankar,3 and Dominic M Walsh3,4

Address correspondence and reprint requests to G Brent Irvine, Division of Psychiatry and

Neuroscience, School of Medicine and Dentistry, The Queen’s University of Belfast, Whitla

Medical Building, Belfast, BT7 9BL, Northern Ireland, UK. Phone: + 44-2890-972111; Fax: +

44-2890-975870; E-mail: [email protected]; or Dominic M. Walsh, Laboratory for Neurode-

generative Research, UCD School of Biomolecular and Biomedical Science, Conway In-

stitute of Biomolecular and Biomedical Research (SO68), University College Dublin,

Belfield, Dublin 4, Republic of Ireland. Phone: + 353-1-716 6751; Fax: + 353-1-716 6890;

E-mail: [email protected].

Submitted October 11, 2007; Accepted for publication March 17, 2008; Epub (www.

molmed.org) ahead of print March 18, 2008.

1School of Medicine and Dentistry, The Queen’s University of Belfast, Belfast, Northern Ireland; 2Department of Biochemistry, Facultyof Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates; 3Department of Neurology, HarvardMedical School and Center for Neurologic Diseases, Brigham and Women’s Hospital, Boston, Massachusetts, United States of America;4The Conway Institute for Biomedical and Biomolecular Research, University College Dublin, Belfield, Dublin, Republic of Ireland

Developing effective treatments for neurodegenerative diseases is one of the greatest medical challenges of the 21st century.Although many of these clinical entities have been recognized for more than a hundred years, it is only during the past twentyyears that the molecular events that precipitate disease have begun to be understood. Protein aggregation is a common fea-ture of many neurodegenerative diseases, and it is assumed that the aggregation process plays a central role in pathogenesis.In this process, one molecule (monomer) of a soluble protein interacts with other monomers of the same protein to form dimers,oligomers, and polymers. Conformation changes in three-dimensional structure of the protein, especially the formation ofβ-strands, often accompany the process. Eventually, as the size of the aggregates increases, they may precipitate as insolubleamyloid fibrils, in which the structure is stabilized by the β-strands interacting within a β-sheet. In this review, we discuss this themeas it relates to the two most common neurodegenerative conditions—Alzheimer’s and Parkinson’s diseases.Online address: http://www.molmed.orgdoi: 10.2119/2007-00100.Irvine

women with Alzheimer’s disease. Theaverage duration of the disease is about8 years, but it can last in excess of 20years.

The disease is divided into two cate-gories based on the age of onset. Early-onset Alzheimer’s disease is extremelyrare, accounting for only ~2% of allcases. It develops between the ages of~30 and 60 years, and more than half ofall such cases are genetic, with a strongMendelian inheritance pattern (8). Late-onset Alzheimer’s disease is by far themost common form of the disease. Ittoo has a genetic predisposition but ap-pears to involve several gene polymor-phisms, some yet to be identified, thatindividually or in combination increaseone’s risk for developing Alzheimer’sdisease (9). A genetic component forlate-onset disease is supported by thefinding that the age at onset of Alz-heimer’s disease is significantly morevariable for concordant nonidenticaltwins than concordant identical twins,providing evidence that genetic back-ground strongly influences the timingof the disease (10). Genetic factors thatpredispose to Alzheimer’s disease aredifficult to identify because their inheri-tance does not cause the disease pheno-type, but rather modulates the age ofonset.

The disease progression is similar forboth early- and late-onset Alzheimer’sdisease and is arbitrarily split into threeoverlapping stages: early/mild, moder-ate, and severe. The initial onset of Alz-heimer’s disease is insidious, with mem-ory loss the earliest and most frequentlycited symptom. In a living person, thereis no precise diagnostic test that con-firms Alzheimer’s disease. A diagnosisof probable Alzheimer’s disease isachieved by excluding other conditionsthat might explain the observed symp-toms. Currently the most important di-agnostic tool for clinicians is neuropsy-chological and mental status testing. TheDiagnostic and Statistical Manual ofMental Disorders, 4th Edition (DSM-IV)criteria for diagnosing dementia requiresloss of two or more domains, including

memory, language, calculation, orienta-tion, and judgment (11). Neuropsycho-logical tests, such as the Mini-MentalState Examination (MMSE), can provideclinical insight into the patient’s cogni-tive changes. Patients are considered tohave mild cognitive impairment, notAlzheimer’s disease, if they present withmemory loss but have only minimal im-pairment in other cognitive domains andare not functionally impaired at work orhome. Thereafter, computed tomogra-phy or magnetic resonance imaging(MRI) can be used to discriminate be-tween other forms of dementia. An ex-perienced physician can diagnose Alz-heimer’s disease with up to 90%accuracy. However, a definitive diagno-sis of Alzheimer’s disease requires notonly the presence of severe dementia butalso postmortem confirmation of twohistopathological features, tangles andplaques (Figure 1A) (12).

EMERGING DIAGNOSTIC TOOLSTechniques for imaging the brain are

progressing, and both the sensitivity andspecificity for diagnosis of Alzheimer’sdisease are constantly improving. For in-stance, MRI of the hippocampal forma-tion can be used to identify atrophyfound in patients with mild cognitiveimpairment and Alzheimer’s disease, assuch atrophy is absent in the normal el-derly. Similarly, changes in the volumeof the fusiform gyrus can be used to dis-tinguish between mild cognitive impair-ment and Alzheimer’s disease, whereascommon cerebral alterations such as en-larged ventricular spaces are used tosupport the diagnosis of Alzheimer’sdisease (13). Positron emission tomogra-phy (PET) allows for visualization ofmetabolism in various regions of thebrain by using 18F-2-deoxy-2-fluoro-D-glucose (FDG) as a surrogate marker ofglucose metabolism. In patients with

4 5 2 | I R V I N E E T A L . | M O L M E D 1 4 ( 7 - 8 ) 4 5 1 - 4 6 4 , J U L Y - A U G U S T 2 0 0 8

P R O T E I N A G G R E G A T I O N I N N E U R O D E G E N E R A T I V E D I S E A S E S

Figure 1. Pathological hallmarks of Alzheimer’s and Parkinson’s diseases. (A) Tangles andplaques in Alzheimer’s disease. Neurofibrillary tangles are intraneuronal and consist ofpaired helical filaments, the subunit of which is a microtubule-associated protein calledtau that has been phosphorylated at multiple sites (dark staining structures). Amyloidplaques are extracellular and are largely composed of a ~4-kDa protein called theamyloid β-protein (Aβ) (round diffuse structures). See Acknowledgements for source in-formation on panel A. (B) Lewy bodies in Parkinson’s disease. Nerve cell with 3 Lewybodies that are double-stained for α-synuclein (brown) and ubiquitin (blue). Where onlyα-synuclein is stained, the color appears as pale reddish-brown, but where ubiquitin alsois stained, the superposition of color gives a dark black and brown appearance. Theblue staining is not seen on its own, because all the ubiquitin-immunoreactive structuresare also positive for α-synuclein. The halo of each Lewy body is strongly immunoreactivefor ubiquitin, whereas both the core and the halo of each Lewy body are immunoreac-tive for α-synuclein. Bar, 10 µm. Panel B has been reproduced from Figure 3 of Spillantiniet al. (96), © 1993–2005 by the National Academy of Sciences of the USA, all rightsreserved.

early Alzheimer’s disease, decreasedmetabolism in parieto-temporal associa-tion cortex and cingulate gyrus andmore marked changes in the medial tem-poral region and parieto-temporal asso-ciation cortex are detected. As the condi-tion progresses, abnormal PET-FDG isalso obvious in the frontal associationcortex. In addition, changes detected inregional cerebral perfusion studies bysingle photon emission computed to-mography (SPECT) can distinguish be-tween mild Alzheimer’s disease andforms of vascular dementia (14).

As will be discussed below, accumula-tion of Aβ in the brain, manifesting as β-sheet rich plaques, is a hallmark of Alz-heimer’s disease. Recently, researchers atthe University of Pittsburgh developed athioflavin T analog, Pittsburgh com-pound B (PIB), which binds β-sheet–richfibrils (15). This compound crosses theblood–brain barrier and binds amyloiddeposits in the brain parenchyma, wherebinding of PIB labeled with carbon-11can be detected by PET imaging. As onewould expect, an inverse correlation ex-ists between FDG-PET imaging of glu-cose metabolism in the parietal cortexand PIB binding (16). This novel in vivoimaging technique provides promise formore definitive diagnosis of Alzheimer’sdisease by detecting the pathognomonicAβ accumulation; following the progres-sion of Alzheimer’s disease in individualpatients; and, tracking changes in plaqueburden in response to amyloid-loweringtherapeutics (see below).

NEUROPATHOLOGICAL HALLMARKS OFALZHEIMER’S DISEASE

Microscopically, the Alzheimer brain ischaracterized by the presence of extracel-lular amyloid plaques and intraneuronalneurofibrillary tangles. Amyloid plaquesdisplay a broad range of morphologicand biochemical characteristics and con-tain numerous proteins, the principal ofwhich is Aβ (17,18). Aβ is a ~4-kDa pro-tein with a common core sequence butheterogeneous N- and C-termini. Themost common form of Aβ is 40 aminoacids long and is called Aβ40. Aβ42, a less

abundant form of this protein that differsonly by having two additional aminoacid residues at the C-terminus, is partic-ularly associated with disease (19). Com-pact, neuritic amyloid plaques containthioflavin S and Congo red–positive fib-rillar deposits with both Aβ40 and Aβ42

present (20). Diffuse plaques, on the otherhand, are not fibrillar and consist almostexclusively of Aβ42. These immature de-posits may be detected in the brains ofyoung patients with Down’s syndromebefore the manifestation of Alzheimer’sdisease–type dementia or in brain regionsthat do not display the complete extent ofAlzheimer’s disease pathology describedabove (21). As a result, diffuse plaquesare considered precursors to mature, neu-ritic plaques. Dilated, dystrophic neurites,activated microglia, and reactive astro-cytes can be found within and immedi-ately surrounding neuritic plaques (22).The processes of neurons found hereindisplay abnormal signs of enlarged lyso-somes and numerous mitochondria.

Neurons bearing neurofibrillary tan-gles, composed of hyperphosphorylatedforms of the microtubule-associated pro-tein, tau, are also frequently found proxi-mate to amyloid deposits (23,24), andtheir temporal and spatial appearancemore closely reflects disease severity thandoes the appearance of amyloid plaques(25,26). Neurofibrillary tangles are notspecific to Alzheimer’s disease, however,and are found in other disorders (for ex-ample, subacute sclerosing panencephali-tis and progressive supranuclear palsy)not associated with the cognitive dysfunc-tion and memory impairment that charac-terize Alzheimer’s disease (27). Indeed agrowing body of genetic and biochemicalevidence suggests that neurofibrillary tan-gles are downstream of Aβ. Specifically,experimental evidence suggests that ab-normal Aβ accumulation triggers taupathology (28,29), and tau has been pro-posed as an essential mediator of Aβ-in-duced neurotoxicity (30); however, thesteps connecting Aβ to tau remain un-defined. Aβ has been shown to induce thecalpain-mediated cleavage of tau, leadingto the generation of a toxic 17-kDa frag-

ment (31), and to induce abnormal tauphosphorylation at disease-relevant sites(32,33); a recent study even suggested thattau phosphorylation is the limiting factorin Aβ-induced neurotoxicity (34). Simi-larly, tau appears to play a central role inthe memory deficits apparent in certaintransgenic mouse models of Alzheimer’sdisease (35). Together, these results sug-gest that Aβ plays an initiating role in apathogenic cascade that requires alteredmetabolism of tau to result in disease.

A MOLECULAR EXPLANATION OFALZHEIMER’S DISEASE: THE AβHYPOTHESIS

Considerable genetic, animal-modeling,and biochemical data have emerged tosuggest that Aβ plays a central role in ini-tiating Alzheimer’s disease. Aβ is derivedfrom the amyloid precursor protein (APP)by the action of two aspartyl proteasescalled β- and γ-secretases (Figure 2)(36–38). APP is first cleaved by β-secretaseshedding its large ectodomain and leav-ing a membrane-bound C-terminal stub(39,40). This 99–amino acid stub is subse-quently cleaved by γ-secretase and Aβ isreleased (41) (Figure 2). Depending on theexact point of cleavage by γ-secretase, twomain forms of Aβ, comprising either 40 or42 amino acid residues, are produced. Theproportion of Aβ42 to Aβ40 formed is par-ticularly noteworthy, because the longerform of Aβ is far more prone to oligomer-ize and form fibrils than the more abun-dantly produced Aβ40 peptide. Productionof Aβ is a normal process, but in a smallnumber of individuals the overproductionof Aβ, or an increased proportion of the42–amino acid form, appears sufficient tocause early-onset Alzheimer’s disease (seeTable 1).

The evidence in support of a causativerole for Aβ in Alzheimer’s disease is asfollows:

1. Localization of the APP gene to chro-mosome 21 and the observation thatAlzheimer’s disease–like neuropathol-ogy is invariably seen in Down’s syn-drome (trisomy 21) (Table 1) (42,43).This point is further supported by de-

R E V I E W A R T I C L E


tection of a rare case of Down’s syn-drome in which the distal location ofthe chromosome 21q breakpoint leftthe patient diploid for the APP gene(44). This individual showed no signsof dementia, and amyloid depositionwas essentially absent from the brainupon death at age 78. In addition, du-plication of APP is also associated withearly-onset Alzheimer’s disease (42).

2. Synthetic Aβ peptides are toxic to hip-pocampal and cortical neurons, bothin culture and in vivo (45–47).

3. Inherited mutations in the APP genethat immediately flank or localizewithin the Aβ region and increase theamount or aggregation properties ofAβ are sufficient to precipitate early-onset Alzheimer’s disease. Mutationslying outside the Aβ domain are prox-

imate to the β- and γ-cleavage sitesand elevate Aβ production or increasethe Aβ42/Aβ 40 ratio (48–51). The fivepoint mutations that lie within the Aβsequence are clustered around the cen-tral hydrophobic core of Aβ and causean increase in steady-state levels of Aβand/or an increased propensity of theresultant Aβ to aggregate.

4. Inherited mutations within the prese-nilin 1 and 2 genes increase theAβ42/Aβ40 ratio throughout life andcause very early and aggressiveforms of Alzheimer’s disease. In thisregard, presenilin has been found tocontribute the active site of the pro-tease (γ-secretase) that generates theC-terminus of Aβ (Figure 2) (19,52).

5. In humans, Apo E, which codes forapolipoprotein E, has three commonalleles, ε2, ε3, and ε4, and genetic epi-demiological studies show that the ε4allele is a major risk factor for devel-oping late-onset Alzheimer’s disease,whereas the ε2 allele appears to beprotective. Importantly, ε4 is associ-ated with more extensive and fulmi-nant Aβ deposition than is ε2 (53,54).

6. Mice transgenic for mutant humanAPP show a time-dependent increasein extracellular Aβ and develop cer-tain neuropathological and behavioralchanges similar to those seen in Alz-heimer’s disease (55).

7. Finally, injection of synthetic Aβ intothe brains of tau transgenic mice accel-erates tau hyperphosphorylation andleads to tangle formation reminiscentof the other hallmark that character-izes Alzheimer’s disease (Figure 1A),whereas reducing endogenous expres-sion of tau ameliorates behavioraldeficits in APP transgenic mice (35).

Aβ TOXICITY: THE IMPORTANCE OFSTRUCTURE

Aβ is a natural product present in thebrains and cerebrospinal fluid (CSF) ofnormal subjects (36,56,57). The presenceof Aβ itself does not lead to neurodegen-eration, but neuronal injury ensues asa result of the ordered self-association of Aβ molecules. Within the amyloid



Figure 2. Production of Aβ by proteolytic cleavage from APP followed by association of Aβto form oligomers and fibrils, showing potential targets for anti-amyloid therapies. Aβ, thegray shaded box, is cleaved from APP by sequential action of 2 proteases; β-secretase car-ries out the initial cleavage to form the N-terminus of Aβ; γ-secretase then cleaves the C99stub to produce the C-terminus of Aβ. The parallel dotted lines represent a membrane bi-layer in which part of the C-terminal region of APP is anchored. Hence γ-secretase activityis a protease that cleaves a substrate within a membrane. Production of Aβ by secretaseaction leads to Aβ monomer, the concentration of which in the steady state is a balancebetween formation and degradation. Monomers can associate to form small oligomersthat increase in size and eventually lead to fibril formation. One anti-amyloid strategy is toinhibit the enzymatic action of either secretase (shown by a black cross). A second strat-egy is to remove soluble and deposited Aβ using antibodies (shown as semicircles).

Table 1. Genetics of early-onset Alzheimer’s disease.

Gene Age of onset, years Aβ phenotype

APP trisomy 21 50s Aβtotal production increasedAPP mutations 50s Aβtotal production increased

Aβ42/Aβ40 ratio increasedAPP triplication of APP gene 50s Aβtotal production increasedPresenilin 1 40s and 50s Aβ42/Aβ40 ratio increasedPresenilin 2 50s Aβ42/Aβ40 ratio increased

plaques that characterize Alzheimer’sdisease, Aβ is organized into fibrils 6–10nm in diameter, whereas in vitro, Aβreadily assembles into very similar amy-loid fibrils. Many studies have demon-strated that when synthetic Aβ is pre-in-cubated to form amyloid fibrils, suchpreparations are directly toxic to neurons(46,58).

One important caveat when consider-ing the activity of Aβ assemblies is thedynamic nature of the aggregation pro-cess. Initial studies clearly demonstratedthat aggregation of Aβ was essential fortoxicity, but characterization of the as-semblies used was limited and it was as-sumed that, because amyloid fibrils weredetectable, it was fibrils that mediatedthe observed toxicity. Yet this ignored thefact that in patients dying with Alz-heimer’s disease there is a relativelyweak correlation between the severity ofdementia and the density of fibrillaramyloid (59).

In contrast, robust correlations betweenthe levels of soluble Aβ and the extent ofsynaptic loss and severity of cognitiveimpairment have been demonstrated(60,61); the term soluble Aβ refers to allforms of Aβ that remain in aqueous solu-tion following high speed centrifugationof brain extracts. To date, most studies ofsoluble Aβ brain levels have employedassays that cannot identify the aggrega-tion state of the species detected (62).Thus, although one cannot attribute theeffects to a specific assembly form of Aβ,the solubility of the species in aqueousbuffer following ultracentrifugation (typi-cally >100,000g for >1 h) would indicatethat the preparations used are free of fib-rillar assemblies. Furthermore, in very re-cent studies, antibodies reported to bespecific for oligomeric, but not mono-meric or fibrillar, Aβ revealed abundantanti-oligomer reactivity in soluble ex-tracts of Alzheimer’s disease brain, butnone in age-matched controls (63).

IDENTIFICATION OF NEUROTOXIC,NONFIBRILLAR Aβ AGGREGATES

SDS-stable dimers and trimers (so-called low-n oligomers) of Aβ have been

detected in the buffer-soluble fraction ofhuman cerebral cortex and in humanCSF (57,64). Similar oligomers are alsoformed by a fibroblast cell line geneticallymanipulated to express mutant humanAPP (7PA2 cells). These cells produceand secrete significant amounts of SDS-stable low-n oligomers of Aβ that mi-grate in denaturing SDS-polyacrylamidegels with molecular weights consistentwith dimers, trimers, and occasionallytetramers (65,66). Because of the easymaintenance and fast growth rate ofthese cells, 7PA2 culture medium hasprovided a convenient tool to investigatethe biological activities of low-n Aβoligomers. This led to the discovery thatAβ oligomers can inhibit hippocampallong-term potentiation (an electrophysio-logical measure of synaptic plasticity)(67,68), impair complex learned behaviorin the live rat (69), and reduce the den-sity of dendritic spines in cultured hip-pocampal neurons (70,71).

Additional support for a role for pre-fibrillar Aβ assemblies in Alzheimer’sdisease pathogenesis comes from studiesusing synthetic Aβ peptides. The firstnonfibrillar assemblies identified wereprotofibrils; these heterogeneous struc-tures range from spherical assemblies of~5 nm diameter to short, flexible rods ofup to 200 nm in length (72,73). The prin-cipal difference between protofibrils andmature fibrils is size and relative solubil-ity; fibrils are frequently several micronslong and are often associated with otherfibrils, whereas protofibrils tend not tobe associated with other protofibrils andseldom exceed 150 nm in length. Conse-quently, unlike fibrils, they do not sedi-ment upon low-speed centrifugation(73,74). Protofibrils can be generatedunder a variety of biochemical condi-tions and appear to behave as true fibrilintermediates in that they can both formfibrils and dissociate to lower-molecular-weight species. Acute application ofprotofibrils in vivo rapidly alters synapticphysiology, whereas chronic applicationcauses cell death (75). A second soluble,nonfibrillar assembly of synthetic Aβcalled Aβ-derived diffusible ligands

(ADDLs), appear as spheres with a diam-eter ~5 nm and migrate in polyacry-lamide gels at ~4, 8, 16, and 18 kDa.ADDLs are formed only under certainspecific in vitro conditions but can causeneuronal death and block long-term po-tentiation in ex vivo preparations (76,77).A recent study reported that syntheticADDL preparations can bind excitatorysynapses (78) and cause a reduction inspine density (79) similar to the findingsobserved with soluble Aβ oligomers se-creted in cell culture.

Together these results provide com-pelling evidence that soluble nonfibril-lar forms of Aβ are potent neurotoxins.Indeed, in the human brain it is likelythat multiple Aβ assemblies that are indynamic equilibrium simultaneouslyalter neuronal, astrocytic, and microglialfunction, and that different toxic effectsmay occur virtually concurrently invarious regions of the cerebral cortex.Thus removal or neutralization of suchtoxic species is an attractive therapeuticstrategy.

CANDIDATE Aβ-BASED THERAPIES ANDDIAGNOSTICS

To date, there is no effective treatmentthat can prevent progression of Alz-heimer’s disease; available drugs canonly delay worsening of symptoms.Therefore there is urgent need for thera-pies that alter the progression of Alz-heimer’s disease. The Aβ hypothesisposits that increased steady-state levelsand consequent Aβ assembly is the pri-mary event driving Alzheimer’s diseasepathogenesis (Figure 3). The rest of thedisease process is believed to result fromthis aberrant assembly. A number of dif-ferent anti-amyloid therapies are underdevelopment; two examples are dis-cussed and illustrated in Figure 2: de-creasing the production of soluble Aβmonomer and removing soluble and de-posited Aβ.

Reduction of Aβ levels is particularlyattractive because it may be possible totitrate Aβ down to concentrations thatwill not support oligomerization. Itwould be anticipated that cell-penetrant



agents that could reduce intracellularand/or extracellular monomer levelsbelow the critical concentration neededfor oligomerization would thus preventAβ from assembling into toxic structures.The development of potent highly selec-tive inhibitors of β- and γ-secretases thatcan readily enter the brain and lower Aβproduction (Figure 2) is being activelypursued. Similarly, efforts are also ongo-ing to develop small molecules that canupregulate the enzymes that control Aβdegradation and thus lower Aβ levels byincreasing Aβ catabolism.

Anti-Aβ immunotherapy employs an-tibodies that recognize multiple differenttoxic Aβ assemblies by both directly neu-tralizing them and preventing their toxiceffect, by promoting microglial clearance,and/or by redistributing Aβ from thebrain to the systemic circulation. This ap-proach has already been shown to reducecerebral Aβ levels, decrease amyloid-associated gliosis and neuritic dystrophy,and alleviate memory impairment intransgenic mouse models of Alzheimer’sdisease (80). More importantly, Alz-heimer’s disease patients that were im-munized with aggregated Aβ showed di-minished cognitive decline and sloweddisease progression compared with pa-

tients that received placebo (81). Unfor-tunately, this phase IIa trial had to bestopped prematurely because 18 of the298 patients who had been immunizeddeveloped meningoencephalitis. Notably,in four cases that have since come to au-topsy (two affected with encephalitis andtwo not), all showed evidence of clear-ance of amyloid deposits. Thus in thefirst clinical test of the Aβ hypothesis itappears that (as in preclinical studies ofmouse models) targeted removal of corti-cal Aβ beneficially modifies Alzheimer’sdisease progression. Efforts are ongoingto develop an equally effective immu-nization protocol that avoids inductionof encephalitis. Thus there is good reasonto believe that therapies directed at pre-venting the generation of toxic Aβ as-semblies will soon come to the clinic andthat, unlike current therapies, they willactually halt further deterioration andoffer the potential of restoring normalcognitive function.

With the advancement of potentiallydisease-modifying therapies, there is anurgent need to develop methods for usein early ante mortem diagnosis. This isrequired, not only from a clinical stand-point, but also because it affects the in-tegrity of clinical trials and epidemiolog-

ical research. Currently there are at leastfour methods that have evolved fromour better understanding of the diseaseprocess: analysis of Aβ species in CSF(82); visualization of amyloid plaques byPET as discussed above (15,83); mea-surement of Aβ in peripheral blood (84);and measurement of total tau and/orphospho-tau in CSF (85,86).

Given the genetic evidence supportinga prominent role for Aβ42 in disease,many studies have investigated the diag-nostic utility of measuring Aβ42 in CSF.For Alzheimer’s disease patients, Aβ42

levels in CSF are typically reduced toaround 50% of the level found in con-trols. The mean sensitivity and specificityto discriminate between Alzheimer’s dis-ease and normal aging are both >85%(87). However, decreased CSF Aβ42 isfound in certain patients with frontotem-poral dementia and vascular dementia,and measurement of CSF Aβ42 alone isinsufficient to discriminate between Alz-heimer’s disease and these dementias(88). CSF Aβ40 is unchanged or slightlyincreased in Alzheimer’s disease (89);consequently a decrease in the ratio ofAβ42/Aβ40 in CSF has been found in Alz-heimer’s disease, and this decreaseseems more pronounced than the reduc-tion of CSF Aβ42 alone (90). Alzheimer’sdisease is also associated with a signifi-cant increase in CSF tau and phospho-tau levels (85,86), and combining mea-surement of total tau, Aβ42, andphospho-tau identifies incipient Alz-heimer’s disease in patients with mildcognitive impairment with very high ac-curacy (90,91).

The reduced level of CSF Aβ42 in Alz-heimer’s disease is believed to be causedby deposition of Aβ42 in senile plaques,hence leaving lower levels of Aβ42 to dif-fuse into CSF. Accordingly, studies havefound a strong correlation between lowAβ42 in CSF and high retention of PIB(83). Factors that may contribute to re-duced Aβ42 levels, in addition to deposi-tion in senile plaques, include formationof Aβ42 oligomers that escape ELISA de-tection and binding of Aβ42 to other pro-teins that block the antibody recognition



Figure 3. The Aβ hypothesis: an increase in the concentration of Aβ, especially the42–amino acid form, is the underlying cause for the pathological features of Alzheimer’sdisease.

of Aβ. For instance, ELISA measurementsof plasma Aβ levels in Alzheimer’s dis-ease have yielded conflicting data andthis could, in part, reflect an inability tomeasure Aβ oligomers. This possibleconfounder might differ for antibodiesused in different ELISA protocols andcould explain some of the contradictoryresults. Development of anti-Aβ dimer/oligomer-specific antibodies should obvi-ate concerns about epitope masking dueto Aβ self-association and may provide auseful system to measure Aβ dimer/oligomer levels in both CSF and plasma.Indeed, a small number of preliminarystudies suggests that measurement of Aβoligomers will be of benefit (63,92). Ifthis holds true in larger studies, onewould anticipate that combining mea-surement of disease-linked assemblyforms (oligomers) of Aβ together withmeasurement of tau in CSF and PIBbinding in brain will provide a highlyspecific and sensitive means of measur-ing both early and incipient Alzheimer’sdisease.

PARKINSON’S DISEASE: INCIDENCE,SYMPTOMS, AND DIAGNOSIS

Named after the English physicianwho first described the clinical syndromein “An Essay on the Shaking Palsy” pub-lished in 1817 [reprinted in (93)], Parkin-son’s disease is an irreversible, progres-sive neurodegenerative disease thatimpairs movement. Incidence increasesmarkedly with age; hence young-onsetParkinson’s disease, defined as occur-rence before age 40, accounts for just 5%of newly diagnosed cases (Parkinson’sDisease Society). Upon reaching the65–69 age range, 0.6% of the populationare affected, increasing to 2.6% of thoseaged 85–89 (94), making it the secondmost common neurodegenerative illnessafter Alzheimer’s disease. The majorityof cases of Parkinson’s disease are idio-pathic and sporadic. However, studieswithin the past few years indicate that asubstantial number of cases do have agenetic component. The three principalsigns of parkinsonism, the clinical phe-notype that defines the disease, are rest-

ing tremor, often beginning in one finger,bradykinesia, and rigidity. Whereas de-mentia is the defining feature of Alz-heimer’s disease, cognitive ability re-mains intact in most Parkinson’s diseasesufferers, at least in the early stages ofthe disease.

PATHOLOGY AND MECHANISM OFDISEASE

Pathologically, the most obvious brainstructure affected by Parkinson’s diseaseis a part of the substantia nigra calledthe pars compacta. Because patients suf-fering from other neurological disorderscan display parkinsonian features, a de-finitive diagnosis of Parkinson’s diseasecan be confirmed only by post mortemhistopathological examination of thesubstantia nigra for loss of pigmentedneurons and presence of Lewy bodies inremaining neurons. Identification ofLewy bodies has been facilitated by im-munostaining for particular proteins,initially for ubiquitin and more recentlyfor α-synuclein, now regarded as themajor protein constituent (95). Suchstaining reveals filaments that, whenpurified and examined by immuno-electron microscopy, can be seen to con-tain α-synuclein (96) (Figure 1B) and re-semble filaments formed when purifiedrecombinant α-synuclein is allowed toaggregate in vitro (97).

In the mid-twentieth century, ArvidCarlsson showed that dopamine acted asthe neurotransmitter in neurons of thesubstantia nigra pars compacta, work forwhich he shared the Nobel Prize in 2000.These neurons operate in a pathway thatcontrols voluntary movement. This in-volves signals being relayed from thecerebral cortex through the basal gangliaback to the cortex and then on to mus-cles. Neurons from the substantia nigrapars compacta project axons that releasedopamine in synapses on interneurons inthe striatum. As the dopamine-contain-ing neurons die, failure to complete thiscircuit results in inability to coordinatemovement. Neuromelanin, the black pig-ment that gives the substantia nigra itsname, is a byproduct from the metabolic

pathway for dopamine synthesis. Whenthe symptoms of Parkinson’s disease firstbecome apparent more than 70% of thedopamine-containing neurons have al-ready been lost, releasing their neurome-lanin and hence turning the tissue lessblack. It is now apparent, however, thatmany regions of the brain are affected inParkinson’s disease, and indeed in theearly stages it may affect only a lower re-gion of the brain stem called the medullaoblongata, spreading gradually upwardthrough the basal ganglia into the corti-cal areas (98).

One of the most obvious questionswhen considering the etiology of Parkin-son’s disease is why dopaminergic neu-rons are particularly vulnerable. Until re-cently no clear genetic links wereapparent, and one hypothesis was thatthe metabolic pathways for dopaminesynthesis might be at the root of theproblem (99). There is evidence thatdopamine metabolites can increase levelsof reactive oxygen species that damagecells, especially mitochondria; for exam-ple, neuromelanin binds heavy metalsthat can lead to free radical production(100). Another hypothesis, that postu-lated exposure to environmental toxinsas the cause, was given impetus by twoapparently diverse findings that wereboth subsequently linked to mitochondr-ial dysfunction. First, during the 1970sand 80s, some users of illegal recre-ational drugs developed parkinsonismthat was traced to the ingestion of acontaminant called 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (101).Itself relatively innocuous, this com-pound is lipid-soluble and so crosses theblood–brain barrier, where it is oxidizedby monoamine oxidase-B, present in as-trocytes, to the toxic 1-methyl-4-phenyl-pyridinium (MPP+) ion. The unique sus-ceptibility of dopaminergic neurons tothis toxin is due to it being taken up bythe dopamine transporter. Once insidethe neuron, it is concentrated in the mito-chondria, where it binds NADH dehy-drogenase, inhibiting complex I of theelectron transport chain and leading tomitochondrial dysfunction (102). Second,



epidemiological studies had suggested alink between long-term exposure to agri-cultural pesticides, such as rotenone andparaquat. Rotenone, a plant derivativeused as an insecticide, is known to be aninhibitor of NADH dehydrogenase.Chronic infusion of either rotenone orMPTP in rodents results in parkinson-ism-like behavior and pathology, includ-ing the formation of inclusion bodies,and indeed these are among the best ani-mal models for the human disease (103).It has also been suggested that exposureto paraquat may alter the expression andaggregation of α-synuclein (see below).

The view that Parkinson’s disease wasalmost entirely a sporadic disease due tometabolic dysfunction caused by envi-ronmental toxins has altered dramati-cally during the past 10 years. The firsthereditary connection was a link to theprotein α-synuclein, and so its geneticlocus on chromosome 4 became knownas PARK1. Nevertheless, mutations in thegene for α-synuclein account for only atiny percentage of Parkinson’s diseasecases. Several additional loci, namedPARK2 through PARK12, have since beenimplicated (Online Mendelian Inheri-tance in Man; Johns Hopkins University,Baltimore; MIM no. 168600, 2007). Themost promising candidates are listed inTable 2.

α-SYNUCLEINIn 1997, a mutation in the α-synuclein

gene leading to a single amino acid sub-stitution (Ala53Thr) was detected in afamily with an early-onset autosomaldominant form of Parkinson’s diseasewith high penetrance (104). Spurred bythis report, researchers searched for anddetected α-synuclein in Lewy bodies(Figure 1B) and in so doing further im-plicated α-synuclein as a major player inParkinson’s disease pathogenesis (105).Subsequently, two additional disease-causing mutations were identified in theα-synuclein gene, each causing a differ-ent single amino acid substitution(106,107).

α-Synuclein belongs to a family ofthree closely related proteins (the others

being β- and γ-synucleins) that are en-coded by three distinct genes. In humans,minor spliced variants of α-synucleinoccur, but the predominant form in thebrain is a small, soluble protein of 140residues that accounts for about 1% oftotal protein in neurons, where it is local-ized in presynaptic nerve terminals. Theprimary structure of α-synuclein is char-acterized by three distinct regions (108):

• the N-terminal region (1–60) containsseveral degenerative repeats of an11–amino acid sequence related to anα-helical lipid-binding motif ofapolipoproteins that mediates proteinbinding to phospholipid vesicles

• the central region (61–95) is composedof extremely hydrophobic amino acidresidues, and part of this region hasbeen implicated in association ofmonomers into larger aggregates thatultimately form amyloid fibrils (109)

• the C-terminal region (96–140) is hy-drophilic and rich in the amino acidresidue proline and the acidic residuesglutamate and aspartate; its size andsequence vary markedly betweenspecies, and it has been suggested toconfer chaperone activity to α-synu-clein (110)

Until recently, the function of α-synucleinremained unclear, and indeed mice defi-cient in α- or β-synuclein, or both, sur-vive with no very obvious brain defects(111). α-Synuclein lacks secondary ortertiary structure, so it belongs to thefamily of natively unfolded proteins,many of which act as chaperones. It isknown to interact with several otherproteins, whereas exposure to lipid mi-celles induces a structural change to α-helix in the N-terminal region (112).

In the presynaptic nerve terminals, α-synuclein associates with membranesof synaptic vesicles and may have a reg-ulatory role in inhibiting neurotransmit-ter release (113). Support for a chaper-one function for α-synuclein has comefrom genetically manipulated mice inwhich cysteine-string protein-α (CSPα)was deleted (114). CSPα is a synapticvesicle protein that acts as a cochaper-one for SNARE proteins. α-Synucleinappears to be able to act as an auxiliarychaperone, complementing the chaper-one activity of CSPα.

A MOLECULAR EXPLANATION OFPARKINSON’S DISEASE: THE α-SYNUCLEIN HYPOTHESIS

In an analogous manner to the in-volvement of Aβ in Alzheimer’s disease,considerable genetic, animal-modeling,and biochemical data have emerged tosuggest that α-synuclein plays a centralrole in initiating Parkinson’s disease.

1. Solutions of α-synuclein, or syntheticpeptide fragments derived from it, as-sociate into oligomers that eventuallyaggregate to produce amyloid fibrilswhose morphology resembles that offibrils purified from Lewy bodies(97,115).

2. The three mutant forms describedabove, each involving a single aminoacid substitution, are associated withautosomal dominant Parkinson’s dis-ease. The mutations increase theaggregation rate of the resultantα-synuclein (115,116).

3. Duplication or triplication of theα-synuclein gene on one chromosome,giving 50% or 100% increase in ex-pression of protein, causes parkinson-ism (117–119). Age of onset of disease



Table 2. Genetic mutations linked to hereditary forms of Parkinson’s disease.

Locus Protein Function

PARK1 α-synuclein Putative chaperone PARK2 parkin Ubiquitin E3 ligasePARK6 PINK1 Putative serine/threonine kinasePARK7 DJ-1 Putative chaperonePARK8 LRRK2 Putative serine/threonine and tyrosine kinase

and its severity are proportional togene copy number.

4. α-Synuclein is the principal con-stituent of Lewy bodies. Much of thisα-synuclein has been posttranslation-ally modified by sequence truncationat the C-terminus, phosphorylation atSer129 (120,121), or nitration (122);these modifications have been shownto increase the rate at which oligomersare formed (121,123). Dopamine andrelated catecholamines have beenshown to interact with α-synucleinand stabilize the protofibril stage ofaggregation (124,125), providing apossible explanation for the increasedsusceptibility of dopaminergic neu-rons. Levels of soluble oligomers of α-synuclein are also affected by fattyacids, being upregulated by polyun-saturated fatty acids (126).

5. α-Synuclein aggregation is also in-creased in the presence of metals andpesticides, which may be environmen-tal risk factors (127). For example,exposure to paraquat induced in-creased expression and aggregation ofα-synuclein in mice. The authors spec-ulate that these increased levels ofα-synuclein may form part of the neu-ronal response to toxic insult, but thatthe increasing protein concentration orits interaction with paraquat may leadto the production of deleterious aggre-gates (128). Iron levels are elevated insubstantia nigra from Parkinson’s dis-ease patients (129), and in vitro it hasbeen demonstrated that, in the pres-ence of iron, solutions of α-synucleingive rise to reactive oxygen speciesthat could be toxic to cells (100).

6. Oligomers of α-synuclein are toxic tosome cell cultures, including thedopaminergic SH-SY5Y human neu-roblastoma cell line (130). As is thecase for Alzheimer’s disease, there ismounting evidence that the toxicspecies is not the final fibril, butearly aggregates, which are solubleoligomers on the pathway to fibrilformation (131). A recent studydemonstrated that the affinity ofα-synuclein for a phospholipid mem-

brane is a function of its degree ofaggregation, with tightest binding byan intermediate formed during theconversion from monomeric to fibril-lar state (132).

7. Increased human α-synuclein expres-sion in transgenic flies (133) and mice(134) is accompanied by neuronal dys-function and loss of synaptic termi-nals and/or neurons, the formation oflesions similar to those found inParkinson’s disease brain, and the de-velopment of motor abnormalities.

8. Expression of mutant forms ofα-synuclein in cells promotes mito-chondrial defects and cell death andenhances susceptibility to oxidativestress. On the other hand, mice defi-cient in synuclein are resistant to toxi-city induced by MPTP and other mito-chondrial toxins (135).

Taken together, all these studies pro-vide strong evidence for a central rolefor α-synuclein in the pathogenesis ofParkinson’s disease and, as is the case forAlzheimer’s disease, recent evidence im-plicates small aggregates rather than in-soluble fibrils as the toxic species, thoughthe exact mechanism of toxicity remainsunclear (131,136).

Genetic evidence also implicates sev-eral other proteins, listed in Table 2,some of which have been found to be as-sociated with mitochondria. Becausethese subcellular organelles are also theinitial site of damage by environmentaltoxins, sporadic and hereditary Parkin-son’s disease may converge in that bothtypes of disease have at their root mito-chondrial dysfunction. The PARK2 locuscodes for the enzyme parkin, which is aubiquitin E3-ligase. Parkin can protectcells against mutant α-synuclein, andan obvious explanation might be thatα-synuclein is a parkin substrate. Thishas not been convincingly demon-strated, however; rather, a connectionwith mitochondrial function may be in-volved, because parkin-null mutants inboth fly (137) and mouse (138) modelshad compromised mitochondrial func-tion and increased oxidative damage.

PINK1 and DJ-1 have both been shownto be required for normal mitochondrialfunction, and mutations in either ofthese proteins can decrease mitochon-drial viability. Loss of PINK1 kinase ac-tivity adversely affects mitochondriaunder stressful conditions (139), whereasDJ-1 may protect against intracellularoxidative conditions (140) and also pre-vent α-synuclein aggregation (141). Mi-tochondrial deficiency will ultimatelyresult in decreased ATP production, re-ducing proteasome activity and allowingexcessive amounts of cellular proteins,including α-synuclein, to build up. In-deed, one of the other major protein con-stituents of Lewy bodies is ubiquitin,suggesting a general malfunction of theubiquitin-proteasome system. LRRK2 isa kinase (142) whose connection withParkinson’s disease pathology remainsunclear. The proteins mentioned aboveand their potential interactions in thepathways leading to Parkinson’s diseaseare illustrated in Figure 4. Whether anyparticular individual succumbs toParkinson’s pathology may depend on acomplex set of circumstances that in-clude genetic factors, environmental ex-posures, and loss of cellular protectivemechanisms. As suggested by Sulzer(143), development of pathology may re-quire “multiple hits” that involve somecombination of these factors; this mightexplain the difficulties encountered inexplaining low penetrant inheritance.

EMERGING DIAGNOSTIC TOOLSDespite progress in understanding the

underlying disease mechanism, there re-mains an urgent need to develop meth-ods for use in diagnosis of Parkinson’sdisease. To date, there is no serologicalor urine test that can confirm the diag-nosis of Parkinson’s disease. Routineblood, CSF, and urine tests yield normalresults. Neither CT nor MRI scans of thehead show abnormalities in idiopathicParkinson’s disease, but may sometimeshelp to diagnose a secondary parkinson-ism due to tumor, infarction, etc. Deoxy-glucose PET scans of the brain reveal anabnormal pattern of increased glucose



metabolism in the globus pallidus, andfluorodopa (F-dopa) PET shows loss ofstriatal dopamine characteristic ofParkinson’s disease (144). The PET/F-dopa measures dopamine functionand SPECT/B-CIT tags the dopaminetransporter. It has to be kept in mind,however, that PET/F-dopa is an indirectmeasure of striatal dopamine levels, anddopa decarboxylase activity only poorlycorrelates with nigral cell counts. Theneed for an accurate diagnostic methodis amply demonstrated by studies indi-cating that clinical diagnosis during lifeis correct only about 75% of the time,even in specialist research centers (145).This is serious not only from a clinicaldiagnostic standpoint, but also becauseit affects the efficacy of clinical trials andepidemiological research. At present, themost advanced imaging biomarker forParkinson’s disease is F-dopa. Theamyloid-binding ligand PIB (see above)has been shown to bind filaments ofα-synuclein generated in vitro but doesnot interact with sections from Parkin-

son’s brain that contain Lewy bodies,suggesting that the amyloid in these invivo lesions is less accessible (146). Be-yond ensuring optimal use of existingimaging biomarkers, it is critical to fos-ter the development of new biomarkers.Given the likely multiple etiologies ofParkinson’s disease and clear heteroge-neity in expression of the clinical mani-festations and progression, several bio-markers will probably be necessary tofully understand the disorder. To thisend, two recent approaches have yieldedsome positive indicators, though neitheris yet at the stage of providing an estab-lished diagnostic protocol. In the firstapproach, microarrays were used to de-tect changes in mRNA expression inblood cells, and a set of 22 genes wasfound to have differential expression inpatient samples compared with controlsamples (147). The second approach wasbased on high-performance liquid chro-matographic assays for a large number(many hundreds) of low-molecular-weight analytes in plasma, so-called

metabolomic profiling, that identifiedseveral potential biomarkers (148).

CURRENT AND FUTURE THERAPIESThe realization that Parkinson’s dis-

ease was related to a deficit in dopamineproduction soon led to the first effectivetreatment, because dopamine levels canbe supplemented pharmacologically.Dopamine is synthesized in neuronsfrom L-tyrosine by a two-step process.Tyrosine hydroxylase catalyses the syn-thesis of 3,4-dihydroxy-L-phenylalanine(L-dopa or levodopa); this then is decar-boxylated by dopa decarboxylase.Dopamine cannot cross the blood–brainbarrier, but its metabolic precursor,L-dopa, can. This is usually given in con-junction with carbidopa, an inhibitor ofperipheral decarboxylase that cannotcross the blood–brain barrier but pre-vents metabolism of L-dopa before itreaches its target tissue, where it is con-verted to dopamine by neuronal decar-boxylase. Other current therapies includedrugs, such as monoamine oxidase B in-hibitors that reduce dopamine metabo-lism or dopamine receptor agonists, andphysical interventions, such as deepbrain stimulation with electrical pulsesor surgical ablation of regions of thebasal ganglia. However, L-dopa remainsthe standard treatment for Parkinson’sdisease, although it has undesirable sideeffects and efficacy wanes after treatmentfor several years.

In the longer term, transplantation ofstem cells may eventually allow regen-eration of neurons, and this might bethe ultimate goal for treatment of mostneurodegenerative diseases, though inthe more immediate future other ap-proaches are required. Because amy-loidogenic proteins display toxic prop-erties only when they form oligomers,one strategy is to prevent them fromforming aggregates. Recently, simvas-tatin has been shown to reduce α-synu-clein aggregation in cell cultures, pro-viding a possible clue to a reportedepidemiological link between simvas-tatin and a reduced incidence of Parkin-son’s disease (149). Molecules that can



Figure 4. Summary of α-synuclein’s role in Parkinson’s disease pathology. Cellular α-synucleinlevels or chemical structure may be altered by overexpression, mutations, or chemicalmodifications (such as phosphorylation, nitration, oxidation, exposure to metal ions or tox-ins, or adduct formation with dopamine quinone). The increased oxidative environmentof dopaminergic neurons is likely to exacerbate some of these processes, making thesecells especially vulnerable. The end result is increased oligomer formation that may dam-age mitochondrial membranes. The amyloid-containing Lewy body is likely to be muchless toxic than this precursor, and it may be the case that cells that rapidly produce Lewybodies survive best. Other mutations in parkin, PINK1, and DJ-1 are likely to compromisethe ability of mitochondria to resist stress.

bind and disrupt fibril formation havebeen discovered for several amyloido-genic proteins. Their mode of actionseems to be insertion into the newlyforming protein assemblies, which areheld together by interactions betweenβ-strands in an incipient β-sheet, pre-venting additional β-strands from beingadded. Several peptides derived fromthe central hydrophobic region (residues68–72) of α-synuclein have been shownto prevent aggregation of the full-lengthprotein (150). A cell-permeable versionof one of these inhibitors was able to re-duce toxicity in cells transfected withmutant A53T α-synuclein (151). Theflavinoid compound, baicalein, has alsobeen shown to inhibit α-synuclein ag-gregation and indeed to disaggregateexisting fibrils (152). However, a poten-tial danger in this approach is that al-though fibril formation might be pre-vented, smaller assemblies might bestabilized, as seems to be the case withbaicalein. Because there is now good ev-idence that even low-n oligomers aretoxic (see above regarding Aβ oligomersin Alzheimer’s disease), this strategycould well be counterproductive, as itmight shift the equilibrium of aggrega-tion away from fibrils to more haz-ardous oligomeric species. Another ap-proach would be to remove synucleinusing antibodies in a manner analogousto the Alzheimer’s disease therapy dis-cussed above. Immunization of ahuman α-synuclein transgenic mouse togenerate anti–α-synuclein antibodies re-duced the deposits of α-synuclein in in-tracellular neuronal cell bodies (153).However, this approach is at least 5years behind the analogous putativeAlzheimer’s therapy, which itself hasencountered severe technical difficultiesdue to development of encephalitis.

CONCLUSIONSDuring the past few years there has

been mounting evidence that the under-lying pathology in several neurodegener-ative diseases arises from the productionof soluble oligomeric assemblies of a pro-tein characteristic of each disease. In the

case of Alzheimer’s and Parkinson’sdiseases, these proteins are Aβ andα-synuclein, respectively. It is clear thatsuch oligomers are deleterious to neuronsin their vicinity, though the molecularmechanisms by which damage occurs re-main to be established. Emerging thera-pies are likely to be based on preventingsuch assemblies from forming and/orpersisting. When oligomeric assembliesgrow in size, they can form insoluble de-posits of amyloid fibrils. Emerging diag-nostic tools are likely to include the earlydetection of these fibrils.

ACKNOWLEDGMENTSThis work was supported by Well-

come Trust grant 067660 (D.M.W.), by aMarie-Curie short-term fellowship(G.M.S.), Michael J. Fox Foundation(O.M.E.-A.), and by the Research andDevelopment Office, Health and Per-sonal Social Services, Northern Ireland(G.B.I.). Thanks to Professor PeterMaxwell and Dr. Brian Wisdom for criti-cal reading of the manuscript.

We thank Dr. Cindy Lemere for pro-viding the image shown in Figure 1A.

REFERENCES1. Ross CA, Poirier MA. (2004) Protein aggregation

and neurodegenerative disease. Nat. Med. 10(Suppl):S10–7.

2. Dobson CM. (2004) Protein chemistry: in thefootsteps of alchemists. Science 304:1259–62.

3. Kelly JW. (2006) Structural biology: proteinsdownhill all the way. Nature 442:255–6.

4. Alzheimer A. (1906) Über einen eigenartigenschweren Erkrankungsprozeß der Hirnrinde.Neurologisches Zentralblatt 23:1129–36.

5. Ferri CP, et al. (2005) Global prevalence of demen-tia: a Delphi consensus study. Lancet 366:2112–7.

6. Evans DA, et al. (1989) Prevalence of Alzheimer’sdisease in a community population of older per-sons: higher than previously reported. JAMA262:2551–6.

7. Kukull WA, Bowen JD. (2002) Dementia epide-miology. Med. Clin. North Am. 86:573–90.

8. Rossor MN, Fox NC, Freeborough PA, Harvey RJ.(1996) Clinical features of sporadic and familialAlzheimer’s disease. Neurodegeneration 5:393–7.

9. Chai CK. (2007) The genetics of Alzheimer’sdisease. Am. J. Alzheimers Dis. Other Dement.22:37–41.

10. Gatz M, et al. (2006) Role of genes and environ-ments for explaining Alzheimer disease. Arch.Gen. Psychiatry 63:168–74.

11. Kawas CH. (2003) Clinical practice: early Alz-heimer’s disease. N. Engl. J. Med. 349:1056–63.

12. Nussbaum RL, Ellis CE. (2003) Alzheimer’s dis-ease and Parkinson’s disease. N. Engl. J. Med.348:1356–64.

13. de Leon MJ, et al. (2004) MRI and CSF studies inthe early diagnosis of Alzheimer’s disease. J. In-tern. Med. 256:205–23.

14. Masdeu JC, Zubieta JL, Arbizu J. (2005) Neu-roimaging as a marker of the onset and progres-sion of Alzheimer’s disease. J. Neurol. Sci.236:55–64.

15. Klunk WE, et al. (2004) Imaging brain amyloid inAlzheimer’s disease with Pittsburgh Compound-B. Ann. Neurol. 55:306–19.

16. Mathis CA, Klunk WE, Price JC, DeKosky ST.(2005) Imaging technology for neurodegenerativediseases: progress toward detection of specificpathologies. Arch. Neurol. 62:196–200.

17. Glenner GG, Wong CW. (1984) Alzheimer’s dis-ease: initial report of the purification and charac-terization of a novel cerebrovascular amyloid pro-tein. Biochem. Biophys. Res. Commun. 120:885–90.

18. Masters CL, Simms G, Weinman NA, MulthaupG, McDonald BL, Beyreuther K. (1985) Amyloidplaque core protein in Alzheimer disease andDown syndrome. Proc. Natl. Acad. Sci. U. S. A.82:4245–9.

19. Bentahir M, et al. (2006) Presenilin clinical muta-tions can affect gamma-secretase activity by dif-ferent mechanisms. J. Neurochem. 96:732–42.

20. Iwatsubo T, Odaka A, Suzuki N, Mizusawa H,Nukina N, Ihara Y. (1994) Visualization of A beta42(43) and A beta 40 in senile plaques with end-specific A beta monoclonals: evidence that an ini-tially deposited species is A beta 42(43). Neuron13:45–53.

21. Lemere CA, Blusztajn JK, Yamaguchi H, Wis-niewski T, Saido TC, Selkoe DJ. (1996) Sequenceof deposition of heterogeneous amyloid beta-peptides and APO E in Down syndrome: impli-cations for initial events in amyloid plaque for-mation. Neurobiol. Dis. 3:16–32.

22. Meda L, Baron P, Scarlato G. (2001) Glial activa-tion in Alzheimer’s disease: the role of Abeta andits associated proteins. Neurobiol. Aging 22:885–93.

23. Wood JG, Mirra SS, Pollock NJ, Binder LI. (1986)Neurofibrillary tangles of Alzheimer diseaseshare antigenic determinants with the axonalmicrotubule-associated protein tau (τ). Proc. Natl.Acad. Sci. U. S. A. 83:4040–3.

24. Kosik KS, Joachim CL, Selkoe DJ. (1986)Microtubule-associated protein tau (τ) is amajor antigenic component of paired helical fil-aments in Alzheimer disease. Proc. Natl. Acad.Sci. U. S. A. 83:4044–8.

25. Braak H, Braak E. (1991) Neuropathologicalstageing of Alzheimer-related changes. Acta Neu-ropathol. 82:239–59.

26. Thal DR, Capetillo-Zarate E, Del Tredici K, BraakH. (2006) The development of amyloid beta pro-tein deposits in the aged brain. Sci. Aging Knowl-edge Environ. 2006:re1.



27. Iwatsubo T, Hasegawa M, Ihara Y. (1994) Neu-ronal and glial tau-positive inclusions in di-verse neurologic diseases share common phos-phorylation characteristics. Acta Neuropathol.88:129–36.

28. Gotz J, Chen F, van Dorpe J, Nitsch RM. (2001)Formation of neurofibrillary tangles in P301l tautransgenic mice induced by Abeta 42 fibrils. Sci-ence 293:1491–5.

29. Lewis J, et al. (2001) Enhanced neurofibrillary de-generation in transgenic mice expressing mutanttau and APP. Science 293:1487–91.

30. Alexander GE, Chen K, Pietrini P, Rapoport SI,Reiman EM. (2002) Longitudinal PET evalua-tion of cerebral metabolic decline in dementia:a potential outcome measure in Alzheimer’sdisease treatment studies. Am. J. Psychiatry159:738–45.

31. Park SY, Ferreira A. (2005) The generation of a17 kDa neurotoxic fragment: an alternativemechanism by which tau mediates beta-amyloid-induced neurodegeneration. J. Neurosci.25:5365–75.

32. Busciglio J, Lorenzo A, Yeh J, Yankner BA. (1995)Beta-amyloid fibrils induce tau phosphorylationand loss of microtubule binding. Neuron14:879–88.

33. Greenberg SM, Koo EH, Selkoe DJ, Qiu WQ,Kosik KS. (1994) Secreted beta-amyloid precursorprotein stimulates mitogen-activated protein ki-nase and enhances tau phosphorylation. Proc.Natl. Acad. Sci. U. S. A. 91:7104–8.

34. Leschik J, Welzel A, Weissmann C, Eckert A,Brandt R. (2007) Inverse and distinct modulationof tau-dependent neurodegeneration by prese-nilin 1 and amyloid-beta in cultured cortical neu-rons: evidence that tau phosphorylation is thelimiting factor in amyloid-beta-induced celldeath. J. Neurochem. 101:1303–15.

35. Roberson ED, et al. (2007) Reducing endogenoustau ameliorates amyloid beta-induced deficits inan Alzheimer’s disease mouse model. Science316:750–4.

36. Haass C, et al. (1992) Amyloid beta-peptide isproduced by cultured cells during normal metab-olism. Nature 359:322–5.

37. Seubert P, et al. (1992) Isolation and quantifica-tion of soluble Alzheimer’s beta-peptide from bi-ological fluids. Nature 359:325–7.

38. Shoji M, et al. (1992) Production of the Alzheimeramyloid beta protein by normal proteolytic pro-cessing. Science 258:126–9.

39. Cai H, Wang Y, McCarthy D, Wen H, BorcheltDR, Price DL, Wong PC. (2001) BACE1 is themajor beta-secretase for generation of Abeta pep-tides by neurons. Nat. Neurosci. 4:233–4.

40. Vassar R, et al. (1999) Beta-secretase cleavage ofAlzheimer’s amyloid precursor protein by thetransmembrane aspartic protease BACE. Science286:735–41.

41. Schroeter EH, et al. (2003) A presenilin dimer atthe core of the gamma-secretase enzyme: in-sights from parallel analysis of Notch 1 and

APP proteolysis. Proc. Natl. Acad. Sci. U. S. A.100:13075–80.

42. Mann DM, Yates PO, Marcyniuk B. (1984) Alz-heimer’s presenile dementia, senile dementia ofAlzheimer type and Down’s syndrome in middleage form an age related continuum of pathologi-cal changes. Neuropathol. Appl. Neurobiol.10:185–207.

43. Olson MI, Shaw CM. (1969) Presenile dementiaand Alzheimer’s disease in mongolism. Brain92:147–56.

44. Prasher VP, Farrer MJ, Kessling AM, Fisher EM,West RJ, Barber PC, Butler AC. (1998) Molecularmapping of Alzheimer-type dementia in Down’ssyndrome. Ann. Neurol. 43:380–3.

45. Busciglio J, Lorenzo A, Yankner BA. (1992) Meth-odological variables in the assessment of betaamyloid neurotoxicity. Neurobiol. Aging13:609–12.

46. Pike CJ, Walencewicz AJ, Glabe CG, Cotman CW.(1991) In vitro aging of beta-amyloid proteincauses peptide aggregation and neurotoxicity.Brain Res. 563:311–4.

47. Pike CJ, Burdick D, Walencewicz AJ, Glabe CG,Cotman CW. (1993) Neurodegeneration in-duced by beta-amyloid peptides in vitro: therole of peptide assembly state. J. Neurosci.13:1676–87.

48. Chartier-Harlin MC, et al. (1991) Early-onset Alz-heimer’s disease caused by mutations at codon717 of the beta-amyloid precursor protein gene.Nature 353:844–6.

49. Citron M, et al. (1992) Mutation of the beta-amy-loid precursor protein in familial Alzheimer’sdisease increases beta-protein production. Nature360:672–4.

50. Goate A, et al. (1991) Segregation of a missensemutation in the amyloid precursor protein genewith familial Alzheimer’s disease. Nature349:704–6.

51. Levy E, et al. (1990) Mutation of the Alzheimer’sdisease amyloid gene in hereditary cerebral hem-orrhage, Dutch type. Science 248:1124–6.

52. Kumar-Singh S, et al. (2006) Mean age-of-onsetof familial Alzheimer disease caused by prese-nilin mutations correlates with both increasedAbeta42 and decreased Abeta40. Hum. Mutat.27:686–95.

53. Corder EH, et al. (1993) Gene dose of apolipo-protein E type 4 allele and the risk of Alz-heimer’s disease in late onset families. Science261:921–3.

54. Strittmatter WJ, Saunders AM, Schmechel D, Per-icak-Vance M, Enghild J, Salvesen GS, Roses AD.(1993) Apolipoprotein E: high-avidity binding tobeta-amyloid and increased frequency of type 4allele in late-onset familial Alzheimer disease.Proc. Natl. Acad. Sci. U. S. A. 90:1977–81.

55. Games D, Buttini M, Kobayashi D, Schenk D,Seubert P. (2006) Mice as models: transgenic ap-proaches and Alzheimer’s disease. J. AlzheimersDis. 9:133–49.

56. Vigo-Pelfrey C, Lee D, Keim P, Lieberburg I,

Schenk DB. (1993) Characterization of beta-amyloid peptide from human cerebrospinalfluid. J. Neurochem. 61:1965–8.

57. Walsh DM, Tseng BP, Rydel RE, Podlisny MB,Selkoe DJ. (2000) The oligomerization of amy-loid beta-protein begins intracellularly in cellsderived from human brain. Biochemistry39:10831–9.

58. Deshpande A, Mina E, Glabe C, Busciglio J.(2006) Different conformations of amyloid betainduce neurotoxicity by distinct mechanisms inhuman cortical neurons. J. Neurosci. 26:6011–8.

59. Terry RD, et al. (1991) Physical basis of cognitivealterations in Alzheimer’s disease: synapse lossis the major correlate of cognitive impairment.Ann. Neurol. 30:572–80.

60. Lue LF, et al. (1999) Soluble amyloid beta pep-tide concentration as a predictor of synapticchange in Alzheimer’s disease. Am. J. Pathol.155:853–62.

61. McLean CA, et al. (1999) Soluble pool of Abetaamyloid as a determinant of severity of neurode-generation in Alzheimer’s disease. Ann. Neurol.46:860–6.

62. Morishima-Kawashima M, Ihara Y. (1998) Thepresence of amyloid beta-protein in the detergent-insoluble membrane compartment of human neu-roblastoma cells. Biochemistry 37:15247–53.

63. Georganopoulou DG, Chang L, Nam JM, Thax-ton CS, Mufson EJ, Klein WL, Mirkin CA. (2005)Nanoparticle-based detection in cerebral spinalfluid of a soluble pathogenic biomarker for Alz-heimer’s disease. Proc. Natl. Acad. Sci. U. S. A.102:2273–6.

64. Enya M, et al. (1999) Appearance of sodium do-decyl sulfate-stable amyloid beta-protein (Abeta)dimer in the cortex during aging. Am. J. Pathol.154:271–9.

65. Podlisny MB, Ostaszewski BL, Squazzo SL, KooEH, Rydell RE, Teplow DB, Selkoe DJ. (1995) Ag-gregation of secreted amyloid beta-protein intosodium dodecyl sulfate-stable oligomers in cellculture. J. Biol. Chem. 270:9564–70.

66. Walsh DM, Klyubin I, Fadeeva JV, Rowan MJ,Selkoe DJ. (2002) Amyloid-beta oligomers: theirproduction, toxicity and therapeutic inhibition.Biochem. Soc. Trans. 30:552–7.

67. Walsh DM, et al. (2002) Naturally secretedoligomers of amyloid beta protein potently in-hibit hippocampal long-term potentiation in vivo.Nature 416:535–9.

68. Wang Q, Walsh DM, Rowan MJ, Selkoe DJ,Anwyl R. (2004) Block of long-term potentiationby naturally secreted and synthetic amyloidbeta-peptide in hippocampal slices is mediatedvia activation of the kinases c-Jun N-terminalkinase, cyclin-dependent kinase 5, and p38mitogen-activated protein kinase as well asmetabotropic glutamate receptor type 5. J. Neu-rosci. 24:3370–8.

69. Cleary JP, Walsh DM, Hofmeister JJ, ShankarGM, Kuskowski MA, Selkoe DJ, Ashe KH. (2005)Natural oligomers of the amyloid-beta protein



specifically disrupt cognitive function. Nat. Neu-rosci. 8:79–84.

70. Calabrese B, Shaked GM, Tabarean IV, Braga J,Koo EH, Halpain S. (2007) Rapid, concurrent al-terations in pre- and postsynaptic structure in-duced by naturally-secreted amyloid-beta pro-tein. Mol. Cell Neurosci. 35:183–93.

71. Shankar GM, Bloodgood BL, Townsend M,Walsh DM, Selkoe DJ, Sabatini BL. (2007) Nat-ural oligomers of the Alzheimer amyloid-betaprotein induce reversible synapse loss by mod-ulating an NMDA-type glutamate receptor-dependent signaling pathway. J. Neurosci.27:2866–75.

72. Harper JD, Lieber CM, Lansbury PT Jr. (1997)Atomic force microscopic imaging of seeded fib-ril formation and fibril branching by the Alz-heimer’s disease amyloid-beta protein. Chem.Biol. 4:951–9.

73. Walsh DM, Lomakin A, Benedek GB, CondronMM, Teplow DB. (1997) Amyloid beta-proteinfibrillogenesis: detection of a protofibrillar inter-mediate. J. Biol. Chem. 272:22364–72.

74. Walsh DM, et al. (1999) Amyloid beta-protein fib-rillogenesis: structure and biological activity ofprotofibrillar intermediates. J. Biol. Chem.274:25945–52.

75. Hartley DM, et al. (1999) Protofibrillar interme-diates of amyloid beta-protein induce acuteelectrophysiological changes and progressiveneurotoxicity in cortical neurons. J. Neurosci.19:8876–84.

76. Lambert MP, et al. (1998) Diffusible, nonfibrillarligands derived from Abeta1-42 are potent cen-tral nervous system neurotoxins. Proc. Natl. Acad.Sci. U. S. A. 95:6448–53.

77. Wang HW, et al. (2002) Soluble oligomers of betaamyloid (1-42) inhibit long-term potentiation butnot long-term depression in rat dentate gyrus.Brain Res. 924:133–40.

78. Lacor PN, et al. (2004) Synaptic targeting by Alz-heimer’s-related amyloid beta oligomers. J. Neu-rosci. 24:10191–200.

79. Lacor PN, et al. (2007) Abeta oligomer-inducedaberrations in synapse composition, shape, anddensity provide a molecular basis for loss of con-nectivity in Alzheimer’s disease. J. Neurosci.27:796–807.

80. Schenk D, et al. (1999) Immunization with amyloid-beta attenuates Alzheimer-disease-like pathologyin the PDAPP mouse. Nature 400:173–7.

81. Gilman S, et al. (2005) Clinical effects of Abetaimmunization (AN1792) in patients with AD inan interrupted trial. Neurology 64:1553–62.

82. Andreasen N, et al. (1999) Cerebrospinal fluidbeta-amyloid(1-42) in Alzheimer disease: differ-ences between early- and late-onset Alzheimerdisease and stability during the course of dis-ease. Arch. Neurol. 56:673–80.

83. Fagan AM, et al. (2006) Inverse relation be-tween in vivo amyloid imaging load and cere-brospinal fluid Abeta42 in humans. Ann. Neu-rol. 59:512–9.

84. Irizarry MC. (2004) Biomarkers of Alzheimer dis-ease in plasma. NeuroRx 1:226–34.

85. Kahle PJ, et al. (2000) Combined assessment oftau and neuronal thread protein in Alzheimer’sdisease CSF. Neurology 54:1498–504.

86. Buerger K, et al. (2002) Differential diagnosis ofAlzheimer disease with cerebrospinal fluid levelsof tau protein phosphorylated at threonine 231.Arch. Neurol. 59:1267–72.

87. Blennow K. (2004) Cerebrospinal fluid proteinbiomarkers for Alzheimer’s disease. NeuroRx1:213–25.

88. Riemenschneider M, et al. (2002) Tau andAbeta42 protein in CSF of patients with fronto-temporal degeneration. Neurology 58:1622–8.

89. Fukuyama R, Mizuno T, Mori S, Nakajima K,Fushiki S, Yanagisawa K. (2000) Age-dependentchange in the levels of Abeta40 and Abeta42 incerebrospinal fluid from control subjects, and adecrease in the ratio of Abeta42 to Abeta40 levelin cerebrospinal fluid from Alzheimer’s diseasepatients. Eur. Neurol. 43:155–60.

90. Hansson O, Zetterberg H, Buchhave P, Andreas-son U, Londos E, Minthon L, Blennow K. (2007)Prediction of Alzheimer’s disease using the CSFAbeta42/Abeta40 ratio in patients with mild cog-nitive impairment. Dement. Geriatr. Cogn. Disord.23:316–20.

91. Herukka SK, Hallikainen M, Soininen H, PirttilaT. (2005) CSF Abeta42 and tau or phosphorylatedtau and prediction of progressive mild cognitiveimpairment. Neurology 64:1294–7.

92. Pitschke M, Prior R, Haupt M, Riesner D. (1998)Detection of single amyloid beta-protein aggre-gates in the cerebrospinal fluid of Alzheimer’spatients by fluorescence correlation spectroscopy.Nat. Med. 4:832–4.

93. Parkinson J. (2002) An essay on the shaking palsy.1817. J. Neuropsychiatry Clin. Neurosci. 14:223–36.

94. de Rijk MC, et al. (2000) Prevalence of Parkin-son’s disease in Europe: a collaborative study ofpopulation-based cohorts. Neurologic Diseasesin the Elderly Research Group. Neurology54:S21–3.

95. Shults CW. (2006) Lewy bodies. Proc. Natl. Acad.Sci. U. S. A. 103:1661–8.

96. Spillantini MG, Crowther RA, Jakes R, HasegawaM, Goedert M. (1998) alpha-Synuclein in fila-mentous inclusions of Lewy bodies from Parkin-son’s disease and dementia with Lewy bodies.Proc. Natl. Acad. Sci. U. S. A. 95:6469–73.

97. Crowther RA, Jakes R, Spillantini MG, GoedertM. (1998) Synthetic filaments assembled fromC-terminally truncated alpha-synuclein. FEBSLett. 436:309–12.

98. Braak H, Rub U, Gai WP, Del Tredici K. (2003)Idiopathic Parkinson’s disease: possible routesby which vulnerable neuronal types may be sub-ject to neuroinvasion by an unknown pathogen.J. Neural Transm. 110:517–36.

99. Jenner P. (1989) Clues to the mechanism under-lying dopamine cell death in Parkinson’s dis-ease. J. Neurol. Neurosurg. Psychiatry Suppl:22–8.

100. Turnbull S, Tabner BJ, El-Agnaf OM, Moore S,Davies Y, Allsop D. (2001) alpha-Synuclein im-plicated in Parkinson’s disease catalyses the for-mation of hydrogen peroxide in vitro. Free Radic.Biol. Med. 30:1163–70.

101. Langston JW, Ballard P, Tetrud JW, Irwin I.(1983) Chronic Parkinsonism in humans due toa product of meperidine-analog synthesis. Sci-ence 219:979–80.

102. Ramsay RR, Salach JI, Dadgar J, Singer TP.(1986) Inhibition of mitochondrial NADH dehy-drogenase by pyridine derivatives and its possi-ble relation to experimental and idiopathicparkinsonism. Biochem. Biophys. Res. Commun.135:269–75.

103. Dauer W, Przedborski S. (2003) Parkinson’s dis-ease: mechanisms and models. Neuron39:889–909.

104. Polymeropoulos MH, et al. (1997) Mutation inthe alpha-synuclein gene identified in familieswith Parkinson’s disease. Science 276:2045–7.

105. Spillantini MG, Schmidt ML, Lee VM,Trojanowski JQ, Jakes R, Goedert M. (1997)Alpha-synuclein in Lewy bodies. Nature388:839–40.

106. Kruger R, et al. (1998) Ala30Pro mutation in thegene encoding alpha-synuclein in Parkinson’sdisease. Nat. Genet. 18:106–8.

107. Zarranz JJ, et al. (2004) The new mutation,E46K, of alpha-synuclein causes Parkinson andLewy body dementia. Ann. Neurol. 55:164–73.

108. George JM, Jin H, Woods WS, Clayton DF.(1995) Characterization of a novel protein regu-lated during the critical period for song learn-ing in the zebra finch. Neuron 15:361–72.

109. Bodles AM, Guthrie DJ, Greer B, Irvine GB.(2001) Identification of the region of non-Abetacomponent (NAC) of Alzheimer’s disease amy-loid responsible for its aggregation and toxicity.J. Neurochem. 78:384–95.

110. Park SM, Jung HY, Kim TD, Park JH, Yang CH,Kim J. (2002) Distinct roles of the N-terminal-binding domain and the C-terminal-solubilizingdomain of alpha-synuclein, a molecular chaper-one. J. Biol. Chem. 277:28512–20.

111. Chandra S, et al. (2004) Double-knockout micefor alpha- and beta-synucleins: effect on synap-tic functions. Proc. Natl. Acad. Sci. U. S. A.101:14966–71.

112. Bussell R Jr, Eliezer D. (2003) A structural andfunctional role for 11-mer repeats in alpha-synuclein and other exchangeable lipid bindingproteins. J. Mol. Biol. 329:763–78.

113. Larsen KE, et al. (2006) Alpha-synuclein overex-pression in PC12 and chromaffin cells impairscatecholamine release by interfering with a latestep in exocytosis. J. Neurosci. 26:11915–22.

114. Chandra S, Gallardo G, Fernandez-Chacon R,Schluter OM, Sudhof TC. (2005) Alpha-synu-clein cooperates with CSPalpha in preventingneurodegeneration. Cell 123:383–96.

115. El-Agnaf OM, Jakes R, Curran MD, Wallace A.(1998) Effects of the mutations Ala30 to Pro and



Ala53 to Thr on the physical and morphologicalproperties of alpha-synuclein protein impli-cated in Parkinson’s disease. FEBS Lett.440:67–70.

116. Greenbaum EA, et al. (2005) The E46K mutationin alpha-synuclein increases amyloid fibril for-mation. J. Biol. Chem. 280:7800–7.

117. Chartier-Harlin MC, et al. (2004) Alpha-synucleinlocus duplication as a cause of familial Parkin-son’s disease. Lancet 364:1167–9.

118. Ibanez P, et al. (2004) Causal relation betweenalpha-synuclein gene duplication and familialParkinson’s disease. Lancet 364:1169–71.

119. Singleton AB, et al. (2003) alpha-Synuclein locustriplication causes Parkinson’s disease. Science302:841.

120. Anderson JP, et al. (2006) Phosphorylation ofSer-129 is the dominant pathological modifica-tion of alpha-synuclein in familial and sporadicLewy body disease. J. Biol. Chem. 281:29739–52.

121. Fujiwara H, et al. (2002) alpha-Synuclein isphosphorylated in synucleinopathy lesions.Nat. Cell Biol. 4:160–4.

122. Giasson BI, et al. (2000) Oxidative damagelinked to neurodegeneration by selective alpha-synuclein nitration in synucleinopathy lesions.Science 290:985–9.

123. Yamin G, Uversky VN, Fink AL. (2003) Nitra-tion inhibits fibrillation of human alpha-synu-clein in vitro by formation of soluble oligomers.FEBS Lett. 542:147–52.

124. Conway KA, Rochet JC, Bieganski RM, Lans-bury PT Jr. (2001) Kinetic stabilization of thealpha-synuclein protofibril by a dopamine-alpha-synuclein adduct. Science 294:1346–9.

125. Mazzulli JR, Armakola M, Dumoulin M, Paras-tatidis I, Ischiropoulos H. (2007) Cellularoligomerization of alpha-synuclein is deter-mined by the interaction of oxidized catecholswith a C-terminal sequence. J. Biol. Chem.282:31621–30.

126. Sharon R, Bar-Joseph I, Frosch MP, Walsh DM,Hamilton JA, Selkoe DJ. (2003) The formation ofhighly soluble oligomers of alpha-synuclein isregulated by fatty acids and enhanced inParkinson’s disease. Neuron 37:583–95.

127. Uversky VN, Li J, Bower K, Fink AL. (2002)Synergistic effects of pesticides and metals onthe fibrillation of alpha-synuclein: implicationsfor Parkinson’s disease. Neurotoxicology23:527–36.

128. Manning-Bog AB, McCormack AL, Li J, UverskyVN, Fink AL, Di Monte DA. (2002) The herbi-cide paraquat causes up-regulation and aggre-gation of alpha-synuclein in mice: paraquat andalpha-synuclein. J. Biol. Chem. 277:1641–4.

129. Zecca L, Youdim MB, Riederer P, Connor JR,Crichton RR. (2004) Iron, brain ageing and neu-rodegenerative disorders. Nat. Rev. Neurosci.5:863–73.

130. El-Agnaf OM, et al. (1998) Aggregates from mu-tant and wild-type alpha-synuclein proteinsand NAC peptide induce apoptotic cell death in

human neuroblastoma cells by formation ofbeta-sheet and amyloid-like filaments. FEBSLett. 440:71–5.

131. Volles MJ, Lansbury PT Jr. (2003) Zeroing in onthe pathogenic form of alpha-synuclein and itsmechanism of neurotoxicity in Parkinson’s dis-ease. Biochemistry 42:7871–8.

132. Smith DP, Tew DJ, Hill AF, Bottomley SP, Mas-ters CL, Barnham KJ, Cappai R. (2008) Forma-tion of a high affinity lipid-binding intermedi-ate during the early aggregation phase ofalpha-synuclein. Biochemistry 47:1425–34.

133. Feany MB, Bender WW. (2000) A Drosophilamodel of Parkinson’s disease. Nature 404:394–8.

134. Masliah E, et al. (2000) Dopaminergic loss andinclusion body formation in alpha-synucleinmice: implications for neurodegenerative disor-ders. Science 287:1265–9.

135. Klivenyi P, et al. (2006) Mice lacking alpha-synuclein are resistant to mitochondrial toxins.Neurobiol. Dis. 21:541–8.

136. Cookson MR, van der Brug M. (2007) Cell sys-tems and the toxic mechanism(s) of alpha-synu-clein. Exp. Neurol. 209:5–11.

137. Greene JC, Whitworth AJ, Andrews LA, ParkerTJ, Pallanck LJ. (2005) Genetic and genomicstudies of Drosophila parkin mutants implicateoxidative stress and innate immune responsesin pathogenesis. Hum. Mol. Genet. 14:799–811.

138. Palacino JJ, et al. (2004) Mitochondrial dysfunc-tion and oxidative damage in parkin-deficientmice. J. Biol. Chem. 279:18614–22.

139. Valente EM, et al. (2004) Hereditary early-onsetParkinson’s disease caused by mutations inPINK1. Science 304:1158–60.

140. Canet-Aviles RM, et al. (2004) The Parkinson’sdisease protein DJ-1 is neuroprotective due tocysteine-sulfinic acid-driven mitochondrial lo-calization. Proc. Natl. Acad. Sci. U. S. A.101:9103–8.

141. Zhou W, Zhu M, Wilson MA, Petsko GA, FinkAL. (2006) The oxidation state of DJ-1 regulatesits chaperone activity toward alpha-synuclein.J. Mol. Biol. 356:1036–48.

142. Zimprich A, et al. (2004) Mutations in LRRK2cause autosomal-dominant parkinsonism withpleomorphic pathology. Neuron 44:601–7.

143. Sulzer D. (2007) Multiple hit hypotheses fordopamine neuron loss in Parkinson’s disease.Trends Neurosci. 30:244–50.

144. Rueger MA, et al. (2007) Role of in vivo imagingof the central nervous system for developingnovel drugs. Q. J. Nucl. Med. Mol. Imaging51:164–81.

145. Hughes AJ, Ben-Shlomo Y, Daniel SE, Lees AJ.(1992) What features improve the accuracy ofclinical diagnosis in Parkinson’s disease: a clini-copathologic study. Neurology 42:1142–6.

146. Ye L, et al. (2008) In vitro high affinity alpha-synuclein binding sites for the amyloid imagingagent PIB are not matched by binding to Lewybodies in postmortem human brain. J. Neu-rochem. 2008, Feb 18 [Epub ahead of print]

147. Scherzer CR, et al. (2007) Molecular markers ofearly Parkinson’s disease based on gene expres-sion in blood. Proc. Natl. Acad. Sci. U. S. A.104:955–60.

148. Bogdanov M, Matson WR, Wang L, Matson T,Saunders-Pullman R, Bressman SS, Flint BealM. (2008) Metabolomic profiling to developblood biomarkers for Parkinson’s disease. Brain131:389–96.

149. Wolozin B, Wang SW, Li NC, Lee A, Lee TA,Kazis LE. (2007) Simvastatin is associated witha reduced incidence of dementia and Parkin-son’s disease. BMC Med. 5:20.

150. Bodles AM, El-Agnaf OM, Greer B, Guthrie DJ,Irvine GB. (2004) Inhibition of fibril formationand toxicity of a fragment of alpha-synuclein byan N-methylated peptide analogue. Neurosci.Lett. 359:89–93.

151. Amer DA, Irvine GB, El-Agnaf OM. (2006) In-hibitors of alpha-synuclein oligomerization andtoxicity: a future therapeutic strategy forParkinson’s disease and related disorders. Exp.Brain Res. 173:223–33.

152. Zhu M, Rajamani S, Kaylor J, Han S, Zhou F,Fink AL. (2004) The flavonoid baicalein inhibitsfibrillation of alpha-synuclein and disaggre-gates existing fibrils. J. Biol. Chem. 279:26846–57.

153. Masliah E, et al. (2005) Effects of alpha-synu-clein immunization in a mouse model ofParkinson’s disease. Neuron 46:857–68.



Documents

Protein Aggregation in the Brain: The Molecular Basis for ...ate, and severe. The initial onset of Alz-heimer’s disease is insidious, with mem-ory loss the earliest and most frequently