Pathogenesis of Parkinson's disease: emerging role of molecular chaperones

Pathogenesis of Parkinson’s disease:emerging role of molecular chaperonesRina Bandopadhyay1 and Jacqueline de Belleroche2

1 Reta Lila Weston Institute of Neurological Studies, Institute of Neurology, University College London, 1 Wakefield Street, London,

WC1N 1PJ, UK2 Neurogenetics Group, Department of Cellular and Molecular Neuroscience, Division of Neuroscience and Mental Health, Faculty

of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK

Review

Glossary

Chaperone-mediated autophagy (CMA): proteins with a consensus peptide

sequence are recognised by the binding of a Hsc70 co-chaperone complex and

are then degraded by the lysosomes. CMA is very selective for proteins and

does not degrade organelles.

Farnesylation: addition of a farnesyl group to the –SH group of cysteine residue

in a protein causes it to become membrane-bound, and this process is involved

in cellular signalling.

Lewy bodies: abnormal aggregates of several proteins within the nerve cells,

identified microscopically by eosin staining, and immunohistochemically by

ubiquitin and a-synuclein.

Mono-ubiquitinated proteins: these are not degraded by the UPS but are

implicated in other cellular processes.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP): a neurotoxin that de-

stroys dopaminergic neurons, widely used to mimic symptoms of human PD in

a mouse model.

Parkinsonism: a movement disorder that has a similar clinical phenotype to PD

but presents different pathological characteristics.

Reactive astrocytes: in response to injury, normal astrocytes undergo

morphological changes and express increased levels of glial fibrillary acidic

protein and other proteins.

Ubiquitin proteasome system (UPS): a molecular system in which misfolded

Several neurodegenerative diseases, including Parkin-son’s disease (PD) are associated with protein misfoldingand the formation of distinct aggregates, resulting in aputative pathological protein load on the nervous system.A variety of factors cause proteins to aggregate, includingaggregation-prone sequences, specific mutations,protein modifications and also dysregulation of theprotein degradation machinery. Molecular chaperonesare responsible for maintaining normal protein homeo-stasis within the cell by assisting protein folding andmodulating protein-degrading pathways. Here, wereview the fundamental mechanisms of neurodegenera-tion occurring in PD involving a-synuclein fibrillisationand aggregation, endoplasmic reticulum stress, ubiquitinproteasome systems, autophagy and lysosomal degra-dation. Molecular chaperones serve a neuroprotectiverole in many of these pathways, and we discuss recentevidence indicating that these proteins might provide thebasis for new therapeutic approaches.

Common mechanisms of neurodegeneration andneuroprotective strategies for Parkinson’s disease usingmolecular chaperonesA common feature of neurodegenerative disorders is thebuild up of misfolded or abnormal proteins, some of whichmight have a propensity to aggregate. Accumulation ofmisfolded, proteolytically cleaved, abnormally modified(by oxidation, phosphorylation, nitration) or mutatedproteins occurs in a wide range of neurodegenerative dis-eases, including for example, Alzheimer’s disease (AD),Parkinson’s disease (PD), amyotrophic lateral sclerosis,Huntington’s disease (HD) and fronto-temporal dementia.Proteindamageand inappropriateaccumulation representsa potentially pathological load on the nervous system. Awide range of cellular defence mechanisms operate to coun-teract this effect, including antioxidant proteins, superoxidedismutase 1 (SOD1), glutathione, molecular chaperonessuch as the heat shock proteins (HSPs) that reduce aggrega-tion and promote folding ofmisfolded proteins, the unfoldedprotein response (UPR) that increases the production ofendoplasmic reticulum (ER) chaperones and suppressestranslation, and the ubiquitin–proteasomal degradationpathway that targets proteins for removal.

There is considerable evidence to support the involve-ment of these processes in PD (Box 1), for example,

Corresponding author: Bandopadhyay, R. ([email protected]).

1471-4914/$ – see front matter � 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2

mutations in two genes involved in the ubiquitin–protea-some pathway PARK2/parkin (encoding E3 ubiquitinligase) andUCH-L1 (encoding ubiquitin C-terminal ligase)are causal in familial forms of PD, and theUPR is activatedin PD tissue and in experimentalmodels of PD (reviewed inRef. [1]). Although the UPR serves a neuroprotective rolethrough the production of ER chaperones and the suppres-sion of translation, it is also responsible for promotingapoptotic cell death following sustained UPR activation.Initial evidence indicates that antioxidants, HSPs (HSP70,HSP104, HSP27) and specific targeting of components ofthe UPR have beneficial effects in experimental models ofPD. These and other associated targets clearly have enor-mous potential for development of treatments for PD. Inthis review, we focus on molecular chaperones and PD,where there is strong evidence for oxidative stress andprotein aggregation in disease pathology in both familial(Table 1) and idiopathic PD.

a-synuclein is fundamental to both sporadic andfamilial PDOne of the hallmarks of PD are protein inclusions known asLewy bodies (LBs), which are found within the substantianigra (SN) and also more extensively in other brain regions[2]. The major component of LBs is now known to bea-synuclein (a-syn) [3] (Figure 1a). This emerged after

proteins are tagged with several ubiquitin molecules and are subsequently

degraded.

009.11.004 Available online 23 December 2009 27

mailto:[email protected]

http://dx.doi.org/10.1016/j.molmed.2009.11.004

Box 1. Parkinson’s disease: clinical features and

neuropathology

PD is the most common and most debilitating movement disorder,

and typically affects the elderly population. The cardinal clinical

symptoms of PD are rigidity, bradykinesia, resting tremor and

postural instability. These symptoms arise as a result of a progressive

and selective loss of dopaminergic neurons from the SN of the brain,

leading to dopamine depletion in the striatal nerve terminals. In

addition to motor symptoms, various non-motor symptoms also arise

Table 1. PD-causing genes and susceptibility factors associated with heat shock proteins and the UPS

Park loci Gene/protein Forms of PD Putative function Association

with UPS

Presence in LBs

Park1/4 SNCA/a-synuclein Early-onset autosomal

dominant and sporadic PD

Synaptic vesicle function Yes Yes

Park2 parkin/Parkin Juvenile and early-onset

autosomal recessive

and sporadic PD

E3 ubiquitin ligase Yes Yes

Park5 UCH-L1/UCH-L1 Late-onset autosomal dominant Ubiquitin C-terminal hydrolase Yes Yes

Park6 Pink-1/PINK-1 Autosomal recessive Protein kinase, mitochondrial

function

Putative Yes

Park7 DJ-1/DJ-1 Early-onset autosomal recessive Oxidative stress, chaperone Putative Yes, in minority

Park8 LRRK2/dardarin Late-onset and sporadic PD GTPase, protein kinase Putative Small minority

Park9 ATP13A2/ATP13A2 Juvenile autosomal recessive

(Kufor–Rakeb syndrome)

and early-onset PD

Lysosomal type 5 P-type ATPase Putative Yes, in minority

Park 13 OMI/HTRA2/HTRA2 Sporadic PD Mitochondrial serine protease ? In small minority

PD susceptibility

gene

GBA/GBA Unclear, recessive for GD Associated with lysosomal

function

? ?

Abbreviations: GD, Gaucher’s disease. Gene definitions: ATP13A2, lysosomal P-type transmembrane cation-transporting ATPase; GBA, glucocerebrosidase; PINK1; PTEN-

induced putative kinase1; LRRK2, leucine-rich repeat kinase 2; Omi/HTRA2, HTRA serine peptidase2; UCH-L1; ubiquitin C-terminal hydrolase-L1 (reviewed in Refs. [69–71]).

Review Trends in Molecular Medicine Vol.16 No.1

the identification of the A53T mutation in a-syn that wascausal in an autosomal dominant extended Italian–Greekfamily [4]. To date, three separate missense mutations(A30P, E46K, A53T) [4–6] and duplications/triplicationsin the gene encoding a-syn lead to autosomal dominant PD[7,8] through a presumed toxic aggregation of a-syn. Geno-mic multiplications lead to a corresponding increase in a-syn mRNA and protein levels [9] that directly correlatewith age of onset, disease duration and severity [10]. The

with the progression of PD. The neuropathological hallmarks of PD

are the presence of eosinophilic, proteinaceous, filamentous inclu-

sions called LBs and dystrophic Lewy neurites in the SN and

extranigral regions [2]. As yet there is no known permanent cure for

PD. Existing therapies are restricted to tackling the motor dysfunc-

tions but become less efficacious with time and do nothing to halt or

regress the progression of the disease.

Figure 1. Presence of various Park loci proteins in LBs and Lewy neurites (LNs). a-

synuclein is the main protein deposited in all LBs, which can also contain several

other proteins such as parkin, which is present in the majority of LBs (�90%) and

LRRK2, which is present only in a small minority. (a) A melanised neuron in

substantia nigra (SN or s. nigra) harbouring an a-synuclein-positive LB (arrow);

(b) the same case showing the amygdala, with several LBs (black arrows) and LNs

(green arrows) that are immunopositive for a-syn; (c) LB (arrow) in SN,

immunopositive for Parkin; (d) Pale staining of a LB in SN (arrow), with a

LRRK2 antibody. LBs and LNs were visualised using diaminobenzidine

immunohistochemistry (dark brown colour precipitate). Scale bar: 10 mm in

(a,b,d) and 30 mm in (c).

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strong gene-dosage effect provides compelling evidence fora causal role for elevated a-syn levels in PD. Additionally,a-syn promoter polymorphisms are associated with spora-dic PD and could be as a result of differences in transcrip-tion [11].

a-syn is a 140 amino acid protein that has a tendency tomisfold and subsequently aggregate. It comprises an N-terminal region with seven imperfect repeats of aKTKEGV consensus motif that acts as the membranebinding domain, a central hydrophobic non-amyloid betacomponent domain and an acidic C-terminal tail [12]. Theproperty of aggregation is attributed to the central hydro-phobic domain, whereas the acidic C-terminal domainprevents aggregation. The structural plasticity of a-synallows it to adopt various conformations, frommonomers topathological oligomers and filamentous forms [13](Figure 2). The three point mutations A30P, E46K andA53T localise to the amphipathic N-terminal region. Invitro, the A53T mutant demonstrates accelerated fibrilformation, whereas the A30P mutant forms spherical oli-gomers but no visible inclusion bodies [14]. To date, therelevance of LBs as either disease-causing or as a protec-tive factor is a matter of intense debate. In vivo evidencegathered from brain autopsy cases expressing the A53Tmutation indicates a high burden of a-syn pathology in

Figure 2. Factors affecting a-synuclein fibrillisation. The adverse and neuroprotective factors that influence a-synuclein are shown in orange and green boxes, respectively.

Monomeric a-synuclein exists as a random structure. A variety of factors, including oxidative stressors (including environmental toxins), membrane association, altered pH,

inhibition of proteolysis, a-synuclein mutations and multiplications, promote misfolding of a-synuclein and the formation of oligomers and/or protofibrils, which are rich in

b-sheet structures. These oligomers form the basis of stable higher-order aggregates which can form amyloid-like fibrils. Mutations in Park loci, such as parkin, and UCHL-1

can disrupt UPS function. It has been proposed that the fibrils finally aggregate to form LBs. HSP27/HSP70 can reverse the formation of higher-order aggregates (green

box). DJ-1 might act as a chaperone, and PINK1 might act separately or cooperatively with other proteins to inhibit a-synuclein fibrillisation (dashed black lines/arrows are

putative pathways).


neurites but decreased LB load [15]. This supports the viewthat LBs are neuroprotective. Indeed, recent evidenceshowing elevated levels of soluble oligomeric forms of a-syn in brain samples from cases with dementia and LBssupports the pathogenic role of these soluble forms insynucleinopathies [16].

A variety of agents linked to sporadic PD – includingoxidative stressors, changes in cellular pH [17], exposure topesticides and herbicides – all promote a-syn fibrillisation(Figure 2). Ageing-associated increases in a-syn proteinlevels have been observed in humans and non-humanprimates [18]. Dopamine in conjunction with a-syn canalso promote apoptosis in cultured cells [19]. Accordingly,oxidation products of dopamine (quinones) have beenshown to react with a-syn to form adducts that inhibit

Box 2. Oxidative stress and mitochondrial dysfunction

contribute to PD pathogenesis

Both oxidative stress and mitochondrial dysfunction are known to

feature prominently in PD pathogenesis. Oxidative stress, caused by

an overproduction of reactive oxygen species (ROS), might

influence mitochondrial dysfunction by oxidising proteins in the

mitochondria that in turn result in further production of ROS

(reviewed in Refs [21,22]). Dopamine and its metabolism can cause

the generation of ROS and compromise mitochondrial function.

Substantial experimental evidence links exposure of pesticides and

environmental factors such as paraquat, rotenone and MPTP to PD

through the production of oxidative stress or by inhibiting

mitochondrial complex 1 activity (reviewed in [23]).

fibrillisation but prolong the lifetime of protofibrils [20].These processes are summarised in Box 2 [21–23].

Post-translational modifications of a-syn have beendetected in post-mortem samples fromPDbrains, and thesecanalter toxicity in in vitroand in vivomodel systems.Thesemodifications includeC-terminal truncation, ubiquitylationand phosphorylation [24]. Nitrated a-syn is present in LBsin PD and in insoluble fractions of affected brain regions insynucleinopathies in humans [25]. A recent in vitro studyhas indicated that phospho-serine-129 (S129) a-syn levelsare upregulated by proteasomal inhibition [26], whereasadenoviral injection of wild type a-syn and S129 phospho-mutants into the SN of rats results in similar levels ofneurotoxicity [27]. By contrast, again in rat models,mutations preventing phosphorylation of S129 a-synexacerbates a-syn-induced toxicity associated withenhanced formation of aggregates [28,29]. Further exper-imental evidence will be necessary to establish whether a-syn phosphorylation is relevant for disease pathogenesis.Clearly accumulation of aberrant a-syn species is causal inPD pathogenesis and therapeutic interventions disablingthese species are warranted (recently reviewed in Ref. [30]).

Heat shock proteins play a role in sporadic and familialPDHSPs play a substantive role in PD pathology. Extensivecolocalisation with a-syn in LBs has been demonstrated forseveral HSPs, including HSP27 (also known as HSPB1),HSP70, HDJ-2, HDJ-1, HSP110, HSP60 and HSP90 [31].

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Box 3. Endoplasmic reticulum stress and the unfolded

protein response

The endoplasmic reticulum (ER) is a major site for synthesis and

maturation of secreted and membrane-bound proteins. These

processes are sensitive to perturbations in redox state, Ca2+ and

energy state that affect the protein folding capacity of the ER (ER

stress) leading to the accumulation of misfolded, unfolded,

damaged, aggregated or mutated proteins in the ER [43]. Under

these conditions, the unfolded protein response (UPR) is activated

to reduce the effect of this stress.

This is achieved in three ways through:

i. Decreasing the protein load by targeting proteins for proteaso-

mal degradation. Misfolded proteins are first translocated from

the ER lumen to the cytosol, polyubiqutinated by E3 ubiquiting

ligase and degraded in the proteasome.

ii. Increasing the capacity of molecular chaperones to fold and

transport proteins by increased expression of protein chaper-

ones.

iii. Through translational suppression.

The accumulation of unfolded proteins leads to the dissociation of

the ER chaperone GRP78 from three ER stress sensors located in the

ER membrane:

� IRE1 (inositol-requiring enzyme 1).

� PERK [pancreatic ER kinase (PKR)-like ER kinase].

� ATF6 (activated transcription factor 6).

This leads to their activation which triggers the UPR. When the

stress is prolonged, the cells switch from this pro-survival

programme to a pro-apoptotic programme, in which several pro-

apoptotic pathways are activated leading to cell death. For example,

increased expression of the pro-apoptotic transcription factor

(CHOP) is mediated through the PERK pathway (as shown in the

6OHDA and MPTP model) [44]. Activation of cell death also occurs

through the ASK1/JNK and caspase pathways (caspase-12 in

rodents, caspase-4 in man).


HSP27 mRNA and protein levels are elevated in LB dis-eases [32]. HSPs are also found associated with the dopa-mine transporter DAT, Parkin, proteasomal subunits,ubiquitin and UCH-L1 in PD. Cell culture studies haveclearly demonstrated the protective effects of small HSPsagainst a-syn toxicity, and HSP27 has been shown toreduce a-syn-induced cell death in neuronal cell lines[33] and primary cultures of midbrain dopaminergic cells,and furthermore to reduce aggregation in cell culture [32].Another small HSP, aB-crystallin (also known as HSPB5),also reduces a-syn toxicity and aggregation through itseffects on partially folded monomers to prevent amyloidfibril formation [32,34,35]. Similarly, HSP70 strongly inhi-bits a-syn fibril formation [36] and protects against dopa-minergic neuronal cell loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model (see Glossary)of PD using adenovirus vectors for HSP70 delivery [37].

In transgenic models overexpressing wild type ormutant a-syn, variable phenotypes are reported [38]. How-ever, as occurs in cell culture, viral delivery of aHSP70prevents dopaminergic cell loss in vivo in a-syn-overex-pressing flies [39] and in anMPTPmousemodel [37] of PD.Torsin A, a homologue of yeast HSP104, colocalises with a-syn in LBs, and high expression has been detected inmelanised dopaminergic cells of the brain in SN parscompacta, DG, CA3 and cerebellum. Codon (CAG) deletionin the gene encoding Torsin A (DYT1) has recently beenshown to cause the hereditary form of the neurologicalmovement disorder dystonia [40]. Using a rat model of PDin which A30P a-syn is administered intracerebrally usinga lentiviral vector, Hsp104 was shown to antagonise a-synfibrillisation and aggregation and to reduce dopaminergicdegeneration at 6 weeks after treatment [41]. HSP104 alsoinduces endogenous HSP27 [42] in a lentiviral model of HDwhere both are neuroprotective. Multiple HSPs have thepotential to reduce PD pathogenesis and even to act syner-gistically, but the future challenge will lie in the devel-opment of an efficient delivery system for the recombinantprotein(s) or drugs that induce HSPs.

Role of ER stress in PD: pathways of cell death andneuroprotectiona-syn has also been shown to be involved in ER stress. SeeBox 3 for a summary of ER-stress [43,44]. Successfulneuroprotective strategies include the promotion of thebeneficial effects of the UPR (e.g. induction of proteinchaperones and suppression of translation initiation)and inhibition of apoptosis mediated through caspase12, a key ER-resident pro-apoptotic cysteine protease acti-vated by ER stress. Knockdown of caspase 12 using RNAinterference and caspase inhibitors (e.g. z-VAD) enhancesurvival in cell lines expressing the PD-associatedmutation in a-syn (A53T) [45]. Similarly, maintaininglevels of phosphorylated eukaryotic translation initiationfactor 2 subunit a (eIF2a) induced by ER stress, using adephosphorylation inhibitor, salubrinal [46], enhances cellsurvival. As cells expressing A53T a-syn have increasedintracellular ROS levels accompanied by mitochondrialcytochrome c release with increased caspase 9 and 3activity, the effect of combining a pan-caspase inhibitor,z-VAD, with salubrinal was found to improve survival [45],

30

indicating that both ER stress and mitochondrial dysfunc-tion together contribute to cell death.

ER stress markers are known to be induced by mito-chondrial toxins, e.g. 6-hydroxydopamine, 1-methyl-4-phe-nylpyridinium (MPP+), rotenone and paraquat, whichcause degeneration of dopaminergic neurons in the SNand are used to model PD [47–50]. Cytoplasmic andnuclear markers of the three ER sensor pathways (PERK,IRE1 and ATF-6) are induced/activated in PC12 pheochro-mocytoma cells bymitochondrial toxins [47]. Cells in whichthe gene encoding PERK has been knocked out show anincreased susceptibility to cell death from these toxins,indicating the neuroprotective properties of the PERKpathway, which are linked to phosphorylation of eIF2a

and translational suppression [47]. These studies in exper-imental models of PD clearly demonstrate that ER stresssensor pathways are recruited and play a beneficial role.

Another component of the ER that plays a significantrole as a molecular chaperone during oxidative stress isprotein disulphide isomerase (PDI). PDI catalyses theformation of disulphide bonds important in maintainingthe structure and function of proteins such as the antiox-idant protein, copper–zinc-dependent superoxide dismu-tase (SOD1). More importantly, PDI can prevent thedeleterious effects of disulphide bond formation betweenprotein monomers which underlies the formation of aggre-gates of SOD1 that occur in amyotrophic lateral sclerosis.PDI is elevated in several neurodegenerative conditions


including PD. However, in PD brain, PDI is S-nitrosylatedat a critical thiol group that is necessary for its activity,potentially reducing its neuroprotective effects by leadingto the accumulation of misfolded proteins [51]. Support forthis concept has come from studies using a range of cellstress models, including the overexpression of the Paelreceptor (Pael-R) that accumulates in PD and serves as asubstrate for the E3 ubiquitin ligase, Parkin [52].

Protein degradation pathways in PD: UPS andlysosomal–autophagic degradationMultiple pathways can degrade a-syn – these include theUPS, the lysosomal degradation system via chaperone-mediated autophagy (CMA) [53,54] and macroautophagy.The major site of protein degradation in UPS is the 26Sproteasome-mediated protein degradation pathway, whichis profoundly inhibited by a-syn protofibrils [55]. Similarlyin neuronal cells and in primary ventral midbrain neurons,PD-linked mutant forms of a-syn reduce cellular protea-somal degradation, whereas surprisingly the wild typeprotein shows little effect [56]. However, current evidencesuggests that the major pathways for a-syn degradationare CMA and macroautophagy. Two lysosomal chaperonesHSC70 and HSP90 facilitate the transfer of protein fromcytosol to lysosomes [57]. a-syn protein has CMA recog-nition motifs and therefore could be a CMA substrate, andthis is supported by the finding that aberrant a-synproteins accumulate with CMA dysfunction [58,59]. Incontrast to CMA activated by a-syn overexpression whichis thought to be neuroprotective, activation of macroauto-phagy by a-syn overexpression is detrimental to neurons[58]. Furthermore, an autophagy enhancer, trehalose,accelerates the clearance of a-syn and when combined withrapamycin, an inhibitor of autophagy, has an additiveprotective effect [60].

Ageing brain is particularly vulnerable to dysfunction ofthe autophagy–lysosomal pathway [61] which is relevantto late-onset diseases such as PD. Therefore, failure in anyof these systems can cause altered steady-state levels in a-syn protein concentrations within the cell cytosol, whichmight also favour a-syn fibrillisation. Although our knowl-edge of autophagy has expanded recently, experimentalevidence suggests that dysfunctional autophagic systemscontribute to the pathophysiology of PD. Future exper-iments should aim to firmly establish the role of both thebeneficial and toxic components of autophagy in neurode-generation which might lead to potential therapies.

Mutations in UCH-L1 and parkin impair the UPSIn 1998, Leroy et al. identified a heterozygous mutation inthe gene encoding the ubiquitin C-terminal ligase UCH-L1in an autosomal dominant PD family [62]. However, laterstudies did not unequivocally clarify its role in familial PD,although a meta-analysis suggested that genetic variabil-ity in this gene could play a role in late-onset idiopathic PD[63]. UCH-L1 is highly expressed in neurons and belongs toa family of deubiquitinating enzymes and forms an obli-gatory component of the UPS. The I193 M mutationdecreases the hydrolytic activity of UCH-L1 in vitro andreduces the recycling of ubiquitin. The UCH-L1 enzymenot onlymaintains ubiquitin homeostasis but in its dimeric

form can also serve as a ligase formonoubiquitinated a-syn[64,65] (see Glossary). In addition, UCH-L1 has beenshown to be a component of LBs. In response to proteasomeinhibition, overexpressed UCH-L1 in cell culture causesaggresome formation and its colocalisation with HSP70.UCH-L1 is also associated with Parkin and a-syn, but notin all inclusions [66]. UCH-L1 can also exist in a mem-brane-associated form and a specific post-translationalmodification (farnesylation) promotes this interaction[67]. In vitro transfection of this modified form of UCH-L1 increases a-syn concentrations within the cell,suggesting that unmodified UCH-L1 is crucial in main-taining a-syn levels.

Mutations in the PARK2 gene were originally identifiedin families with autosomal recessive juvenile PD [68], andsubsequently have been shown to be relatively common infamilies with early-onset autosomal recessive PD account-ing for up to 50% of these pedigrees. The frequency ofmutations is also high in sporadic cases with disease onsetage below 20 years. Mutations also affect several differentethnic populations but the prevalence rates can vary(reviewed in Ref. [69]).

The PARK2 gene encodes the protein Parkin which is alarge protein (465 amino acids) withmultiple domains thatinclude an N-terminal ubiquitin-like domain, two RING(‘really interesting new gene’) domains and an IBR (‘in-between ring’) domain. Parkin functions as an E3-ubiqui-tin ligase and covalently tags ubiquitin to target proteinsfor degradation by the UPS system (reviewed in Refs[70,71]). Mutant Parkin causes impaired degradation ofits substrates, leading to accumulation of toxic products,and eventually cell death. Some of the putative Parkinsubstrates are glycosylated forms of a-syn, synphilin-1,CDCrel1 and 2, Pael-R, cyclin E, tubulin, synaptotagminand misfolded dopamine transporter (See review [71]).Parkin might also cooperate with other molecular chaper-ones and co-chaperone proteins such as HSP70 and the E3ubiquitin ligase C-terminus of Hsc70 interacting protein(CHIP), and this can enhance its E3 ligase activity [72].Interestingly, cases with homozygous parkinmutations donot contain LBs, suggesting that Parkin is essential for LBformation (see review [70]). However, Parkin protein is acomponent of LBs in sporadic PD [73] (Figure 1c). Pael-R, aParkin substrate, also localises prominently in LBs and ispresent in dopaminergic neurons [74,75]. Despite severalputative Parkin substrates having been described in theliterature, it is still unclear which are the most pathologi-cally relevant. Overall, the function of Parkin is to protectneurons by aiding protein degradation by the UPS, andtherefore restoring Parkin levels might form a basis for PDtherapy.

Lysosomal P-type transmembrane cation-transportingATPase and glucocerebrosidase target lysosomalpathways of degradationLysosomal P-type transmembrane cation-transportingATPase (ATP13A2), the gene product ofPARK9, maintainsan ideal pH in lysosomes. Loss of function, through reces-sively inherited mutations in this gene, causes Kufor–

Rakeb syndrome (KRS) [76]. KRS mutations lead to local-isation of ATP13A2 in ER and its enhanced proteasomal

31


degradation [76]. The substrate specificity of ATP13A2 isunknown. A recent study has demonstrated that ATP13A2can antagonise toxicity induced by a-syn overexpression inyeast, the nematode Caenorhabditis elegans and also inprimary dopaminergic neuronal cell cultures [77]. Inaddition, ATP13A2 knockdown in C. elegans causesenhanced a-syn misfolding [77]. These experiments ele-gantly demonstrate that a-syn and ATP13A2 can interactfunctionally and also show a putative link between lyso-somal dysregulation and PD pathogenesis. Of note, degra-dation of aggregation-prone huntingtin and mutated a-synproteins are compromised in a transgenic mouse model oflysosomal storage disease [78].

The glucocerebrosidase (GBA) gene encodes a lysosomalenzyme that catalyses the breakdown of the glycolipidglucosylceramide to ceramide and glucose. Mutations inGBA are most relevant in the lysosomal storage diseaseGaucher’s disease, and several homozygous and compoundheterozygote mutations have been identified. Recent stu-dies have linked these mutations in GBA to sporadic PD[79,80], suggesting that they could be risk factors. In C.elegans, ceramide metabolism has been associated with a-syn inclusion formation [81]. Asa-syn is partly degraded bychaperone-mediated autophagy, it is tempting to speculatethat mutations in GBA or ATP13A2 might cause insuffi-cient clearance of a-syn through lysosomes and thereforemight cause an imbalance in a-syn cellular homeostasis.

PD gene mutations in proteins that can function as orinteract with molecular chaperones: DJ-1, LRRK2 andPINK1Recently, evidence has emerged suggesting that genes thatare mutated in PD not only play a role as protein chaper-ones affecting a-syn homeostasis but also regulate mito-chondrial functions and counteract oxidative stress.

DJ-1 might play a role as a molecular chaperone

Homozygous loss-of-functionmutations in the PARK7 (DJ-1) gene are responsible for early-onset, L-DOPA-responsiveparkinsonism [82]. DJ-1 is a redox-sensitive protein thatpossesses antioxidant, RNA binding, antiapoptotic andchaperone properties in a variety of model systems. DJ-1has structural similarities to Hsp31, a molecular chaper-one in Escherichia coli [83]. Exogenous DJ-1 overexpres-sion protects against wild type a-syn and A53T a-syntoxicity by upregulation of Hsp70 in vitro [84,85]. Inprimary dopaminergic cell cultures, HSP70 upregulationis necessary for DJ-1-mediated suppression of a-syn aggre-gation, but not neurotoxicity [86]. By contrast, DJ-1 cansuppress an early step in the formation of a-syn aggregatesand prevents the formation of a-syn oligomers and proto-fibrils which can be achieved by promoting their degra-dation suggesting that DJ-1 can protect against a-syntoxicity via multiple mechanisms [87]. The anti-aggrega-tion effect on a-syn is dependent on the oxidation state ofDJ-1 at cysteine 106 (C106), although further oxidation ofCys and Met residues leads to a loss of its secondarystructure and diminished chaperone activity [88]. Theoxidation of C106 to cysteine–sulphinic acid allows DJ-1to translocate to mitochondria and this is necessary forneuroprotection and proper mitochondria function [89].

32

In the human brain, DJ-1 is expressed ubiquitously, ispresent in neurons and is upregulated in reactive astro-cytes [90,91] (see Glossary). Although DJ-1 is not a com-ponent of the majority of LBs, it does, however, colocalisewith microtubule-associated protein tau in AD, fronto-temporal lobar degeneration with tau pathology and pro-gressive supranuclear palsy cases [92], suggesting that itmight play a role in several neurodegenerative disordersthrough its putative chaperone role. It is noteworthy thatthe point mutation in DJ-1, which results in L166P, dis-rupts its dimer formation and the protein is rapidlydegraded by the UPS [82]. Additionally, DJ-1 and itsmutants colocalise with HSP70 and CHIP in the cellcytosol, and then DJ-1 translocates to mitochondria uponexposure to oxidative stress where it associates with themitochondrial chaperone GRP75 [93]. Taken together, itappears that DJ-1 might function as a redox-sensitivechaperone to prevent proteins from misfolding, includinga-syn, and that it also has a function in the mitochondriaand these functions could be crucial for neuroprotection.

LRRK2

Mutations in the gene encoding leucine-rich repeat serine/threonine-protein kinase 2 (LRRK2/PARK8) are associatedwith autosomal dominant and sporadic forms of PD [94,95].LRRK2/dardarin is a large protein (2527 amino acids) withseveral distinct regions comprising ankyrin-like, leucine-rich repeat, Roc, Cor, GTPase, kinase and WD40 domains.Mutations occur in all domains. LRRK2 is a Ser/Thr kinasewhich is capable of autophosphorylation. Mutations in theLRRK2 kinase domain increase kinase activity, as hasbeen shown using generic substrates [96]. A recent in vitrostudy showing that LRRK2 can phosphorylate recombi-nant a-syn at S129 and that the most common mutation inthe PARK8 gene, the glycine to serine conversion at aminoacid position 2019, enhances this activity [97] lends sup-port to the hypothesis that a-syn is a likely candidate forLRRK2 phosphorylation [98]. Interestingly, overexpres-sion of the G2019S LRRK2 mutant in differentiatedhuman neuroblastoma cells causes an increase in autop-hagic vacuoles and significantly decreases neurite length[99]. In line with this, LRRK2 localises in aminority of LBs[96] (Figure 1d).

Recent in vitro and in vivo studies have shown thatLRRK2 and HSP90 associate to form a complex [100,101].Inhibition of HSP90 with geldanamycin disrupts thisassociation and promotes LRRK2 degradation in cell cul-tures. CHIP, a co-chaperone of HSP90 binds, ubiquitinatesand promotes the UPS degradation of LRRK2 [102].Furthermore, HSP90 and CHIP can act cooperatively tomaintain the concentration of LRRK2 within cells [103].Identification of in vivo targets of LRRK2 kinase andGTPase activities and other protein–protein interactionsshould reveal the contribution of LRRK2/PARK8 to thepathogenic mechanisms of sporadic PD.

PTEN-induced kinase 1

Homozygous, autosomal recessive mutations have beenidentified in the PTEN-induced kinase 1 (PINK1) genein early-onset PD patients [104], and haploinsufficiencyof PINK1 is a likely susceptibility factor in sporadic PD


[105]. PINK1 protein has an N-terminal mitochondria-targeting sequence and a large Ser/Thr protein kinasedomain. It localises to mitochondria and is importantfor maintaining mitochondrial morphology. PINK1 isalso a functional kinase because it can autophosphorylate[106]. PINK1 phosphorylates the mitochondrial chaperonetumour necrosis factor receptor-associated protein(TRAP1/HSP75) and promotes cell survival [107]. Further-more, PINK1 and Parkin act in a common pathway that iscrucial for mitochondrial morphology, with PINK1 actingupstream of Parkin, as demonstrated inDrosophilamodels[108,109]. Another recent study has shown that Parkin,PINK1 and DJ-1 can function cooperatively in a ubiquitinE3 ligase complex in vitro and in the human brain topromote ubiquitination and degradation of Parkin sub-strates, synphilin-1 and Parkin, and that their PD-patho-genic mutations impair the E3 ligase activity [110].Furthermore, PINK1 knockdown increases ROS pro-duction, reduces mitochondrial membrane potential andelevates apoptosis, as shown in human foetal mesencepha-lic stem cells [111], suggesting that PINK1 knockdownmight influence several pathogenic processes in PD.

PINK1 has been shown to interact with Cdc37 (a mol-ecular co-chaperone) which functions with Hsp90 topromote proper folding of kinase proteins [112], andPINK1mutants inhibit this interaction. PINK-1 phosphor-ylates HTRA2, a mitochondrial protease, and regulates itsprotease activity [113] (Table 1). As with LRRK2, identi-fication of in vivo phosphorylation targets of PINK-1should increase our understanding of the involvement ofmitochondria in PD.

Figure 3. Pathogenic pathways in neurodegeneration and sites of action of gene muta

pathways and molecular chaperones (green boxes) involved in several neurodegenerat

filled circle are inhibitory, promoting neuroprotection, and the HSC70 involvement in au

mutations/risk factors (blue boxes, with mutations indicated by *) that are associated wit

PD, increased oxidative stress, ER-stress-increased aggregation and increased cell deat

aggregation and impair mitochondrial function and potentially impair chaperone activit

proteins are indicated by green boxes and green lines. HSP40 interacts with HSP70 (thro

HSP70. Gene names are defined in Table 1.

Concluding remarks and putative treatment strategiesfor PDThere have been considerable advances in our understand-ing of the pathways of neurodegeneration that are commonto many diseases. Oxidative stress and mitochondrial dys-function, ER stress and proteinmisfolding and aggregationall contribute to cell death through apoptotic and non-apoptotic pathways (Figure 3). These are major com-ponents of PD pathogenesis where a-syn is the predomi-nant protein that accumulates in PD. Elucidating the roleof endogenous molecular chaperones has shown theirimportance in preventing protein aggregation and theirroles in protein removal and degradation by the UPR, UPSand lysosomal autophagy pathways.

The importance of these well-established basic path-ways in PD and the role of molecular chaperones is high-lighted by the numerous examples of PD-linkedmutations/risk factors that target these pathways (Figure 3), wherethe consequences of mutations are indicated. In PD,increased oxidative stress, ER stress-increased aggrega-tion and increased cell death occur.Mutations impair UPS,impair lysosomal activity, increase a-syn aggregation andimpair mitochondrial function and potentially impair cha-perone activity throughout the cell in multiple compart-ments. Molecular chaperones play key roles at multiplesites by stabilising protein conformation and facilitatingthe trafficking of toxic protein intermediates for degra-dation.

More recently, there has been speculation that multipleadditional cellular compartments influence a-syn fibrilli-sation, notably from studies of the mitochondrial actions of

tions and heat shock proteins in PD. A generalised scheme showing pathogenic

ive disorders. Blue arrows represent stimulatory responses, green lines that end in

tophagy, which is stimulatory, is indicated by a green arrow. Sites of action of gene

h PD in cellular pathways of oxidative stress and protein aggregation are shown. In

h occur. Mutations impair UPS function, impair lysosomal activity, increase a-syn

y throughout the cell in multiple compartments. Sites of intervention of heat shock

ugh its J domain) and functions as a co-chaperone regulating the ATPase activity of

33

Box 4. Putative treatment strategies

The pathogenesis of PD is complex and it is now appreciated that

treatment for PD will require novel disease-modifying strategies

together with the conventional drugs that are already used to

control motor symptoms. Novel strategies should be directed at

controlling the toxic forms of a-syn species that lead to aggregation,

either by inhibiting their formation in the first instance or by

increasing their clearance. Three putative disease-modifying strate-

gies for PD include:

1. Stabilisation of a-syn levels – by reducing the levels of a-syn

oligomers and preventing toxic post-translational modifications.

This could be achieved by the use of kinase inhibitors to inhibit

phosphorylation of a-syn (reviewed in Ref. [30]). Limiting ROS

production caused by increased oxidative stress and mitochon-

drial toxins could also be beneficial in preventing a-syn from

forming oligomers or protofibrils. Here, the endogenous neuro-

protective functions of DJ-1, PINK1 and Parkin could also be

exploited.

2. HSPs and molecular chaperones – these could be recruited as the

first line of defence against toxic stimuli such as oxidative stress,

ER stress and mitochondrial toxins, and could be developed as

potential therapies. This might help to maintain a-syn in its

native conformation.

3. Encourage a-syn protein clearance – by stimulating the degrada-

tion pathways. This could be achieved by stimulating autophagic

pathways with trehalose and rapamycin [58]. Maintaining UPS

function through UCH-L1 and Parkin might also be another viable

treatment option.

The main challenge for the future will be to direct targeted

delivery of such treatments without hampering the normal function

of the proteins. However, age and disease duration also need to be

taken into account as these factors might also affect treatment

outcomes.

Box 5. Outstanding questions

� Which is the most toxic a-synuclein species?

� What is the contribution of autophagy in the pathogenesis of PD?

� What are the in vivo functional interactors of Parkin, DJ-1, PINK1

and LRRK2?


DJ-1, PINK-1 and Parkin. This is important in linkingtogether the two main features of PD pathology: LBs andmitochondrial dysfunction (Figure 3). Mitochondrial dys-function leads to the accumulation of ROS, which promotesprotein aggregation causing further mitochondrial dys-function, resulting in a vicious cycle. Taken together, thesefindings have important implications for novel treatmentstrategies for PD, in which molecular chaperones could beutilised as modulators of protein aggregation and neuro-degeneration (Box 4). Although progress has been made,much is yet to be understood about the roles of molecularchaperones in the mechanisms underlying the pathogen-esis of PD (Box 5).

AcknowledgementsR.B. is funded by the Reta Lila Weston Institute of Neurologicaldisorders. J.B. thanks the Parkinson’s Disease Society for fundingresearch on molecular chaperones in PD.

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Pathogenesis of Parkinson's disease: emerging role of molecular chaperones