Understanding the molecular basis of Parkinson’s disease, identification of biomarkers and routes to therapy

565

Review

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Parkinson’s disease (PD) is a very common highly debilitating neurodegenerative disease, which affects all human populations [1,2]. It is age related, generally becoming first apparent in the sixth decade of life, affecting approximately 2% of the population over 60 years of age, and rising to 5% over 80 years of age. The major symptoms include tremor, akinesia, postural instability and bradykinesia. Although many neuronal types are affected to some degree, the problems with body movement essentially result from a loss of dopaminergic neurons in the sub-stantia nigra pars compacta [3]. It is estimated

that approximately 70% of dopaminergic cells have to be lost before clinical symptoms are observed. This suggests that the number with diagnosed disease reflects only the tip of the ice-berg, and a high percentage with asymptomatic disease in the general population. At present, the only available treatments are those to amel-iorate the symptoms of the disease; there are no treatments targeting prevention or slowing its progression.

The major pathological feature associated with sporadic PD is the presence of Lewy bodies in surviving dopaminergic neurons [4]. There is

Philip A RobinsonSection of Ophthalmology and Neuroscience, Wellcome Trust Brenner Building, Leeds Institute for Molecular Medicine, St James’s University Hospital, Leeds LS9 7TF, UK Tel.: +44 113 343 8419 Fax: +44 113 343 8603 [email protected]

These are really exciting times in the field of Parkinson’s disease research. Although the etiology of sporadic disease still remains a mystery, many of the proteins associated with hereditary disease (5–10% of all disease) have now been identified. Only time will tell whether proteins associated with hereditary disease are involved in the development of sporadic disease. The most valuable proteomic studies performed to date are, and continue to be, those aimed at identifying endogenous binding partners, substrates, post-translational modifications and cellular pathways affected by these proteins. Similar to global proteomic approaches, even these approaches have surprisingly often been characterized by the production of very long lists of proteins. Consequently, the parallel development of more refined protein–protein interactions maps has aided the chance of identifying those protein complexes and/or cellular pathways, which, when disrupted, lead to the development of disease. The knowledge gained from these studies is essential, as targeting the activities of these proteins, or the pathways they operate in, currently offers the best opportunity to develop new therapeutic strategies to treat the disease. They may include agents to modulators of kinase activities (e.g., PINK1 and LRRK2), modulators of the activity of the ubiquitin–protein ligase, Parkin, proteostasis agents to block a-synuclein filament assembly and toxicity, or promote the refolding of mutant proteins, modulators of a-synuclein transfer between cells, reagents to regulate cargo dynamics along axonal microtubule networks, stimulators of autophagy and/or modulators of cellular stress pathways. The second major challenge will be to identify biomarkers to enable population screening to identify those with asymptomatic early-stage disease. Whether the analysis of blood or urine samples will yield such a marker, remains to be determined. Success or failure will be highly dependent on adopting strict standard operating procedures for the collection, processing and storage of samples, combined with the need for the identification of the most robust methods of prefractionation of samples to remove the most abundant proteins prior to proteomic screening.

Keywords: a-synuclein • DJ-1• HtrA2 • LRRK2 • Parkin • Parkinson’s disease • PINK1

Understanding the molecular basis of Parkinson’s disease, identification of biomarkers and routes to therapyExpert Rev. Proteomics 7(4), 565–578 (2010)

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Review Robinson

still controversy whether it is the physical presence of Lewy bod-ies, per se, which ultimately results in cell death, or whether fila-mentous or oligomeric structures of its major protein constituent, a-synuclein, are the toxic entities [5]. From proteomic analyses, the constituent make-up of Lewy bodies is very complex, with more than 250 proteins being described [6–8]. Loss of activity of such a large number of components from the cell as they integrate into the inclusion makes it difficult to determine which, if any, are associated with disease onset.

Although the majority of PD is apparently sporadic, 5–10% is inherited [1]. The genes associated with many of these inher-ited autosomal dominant and recessive forms of the disease have now been identified (Table 1), although there are also a few chromosomal loci where the nature of genes still need to be described (e.g., PARK3, 10, 11 and 12) [1,9]. The list of pro-teins includes kinases (PINK1 and LRRK2), proteins regulat-ing protein folding and disposal (Parkin, ATP13A2, HtrA2 and DJ-1), anti oxidants (DJ-1), and those whose function are prov-ing difficult to determine (a-synuclein). The deubiquitylating enzyme, UCH-L1, was also reported to be a disease-susceptibil-ity gene [10,11]. However, a subsequent genetic analysis brought this conclusion into question [12]. The protein is, therefore, not included in Table 1. Many different proteomic approaches have been used to identify binding partners and substrates [13]. The cellular processes that they may regulate are numerous (Table 1). Interestingly, recent genome-wide association studies on spo-radic PD patients have identified a few susceptibility loci, which include a-synuclein, LRRK2 and tau (plus two others) [14,15]. The former two are of obvious interest in relation to hereditary disease, but the latter is also interesting in light of its relationship to the development of Alzheimer’s disease. Combined with the knowledge that aggregated a-synuclein, in the form of Lewy bodies, is also observed in other neuronal cell types in different neurodegenerative diseases – the ‘a-synucleinopathies’ [16] – these data hint at a potential common mechanism of disease onset, at least for some of the neurodegenerative diseases [17,18].

We do not yet know what triggers the initiation of sporadic dis-ease, how the disease progresses or what drives the progression. However, recent studies demonstrating that (aggregating) a-synu-clein can be internalized by cells suggests a mechanism based on cell–cell transmission [19–22]. The neurons initially affected could depend purely on chance exposure to an environmental trigger, the response to which depends on the ability of the cells, at that time, to deal with the challenge. The response to this initiating event could then be determined by a combination of other complex genetic factors that regulate, for instance, response to toxic chemicals, or reflects a neuron’s underlying ability to deal with oxidative stress.

However, a general over-riding scheme to explain the develop-ment of sporadic disease appears to revolve around problems with mitochondrial dynamics, resulting in oxidative stress [23–33], calcium flux [34–36], and protein folding, transport and/or degradation [37–40] (Figure 1; see also [41]). The problems may be exacerbated by an age-related (or genetic) inability to deal efficiently with cellular stress caused by external agents and/or dopamine [42]. How mitochondrial activity and protein processing affect one another is the subject of intense research activity. For example, the ubiquitin–proteasome system (UPS) controls mitochondrial fission and fusion [43–45], and fission followed by selective fusion is required for the removal of defective mitochondria [46,47]. Similarly, increased output of reac-tive oxygen species (ROS) from mitochondria associated with the development of disease will produce protein damage, and potentially decreased ATP output from mitochondria, thereby affecting the efficiency of the UPS and chaperone activity. Furthermore, as ROS can also act as a signaling molecule, they may activate transcription of proteins to deal with this increased stress analogous to activa-tion of the unfolded-protein response by proteins misfolding in the endoplasmic reticulum (ER) or in mitochondria [48–51].

It is essential that the schematic shown in Figure 1 should be viewed in the context of a neuron. What is not clear is which arms of the model initially drive disease progression. Damage to mitochondria or decreased proteasomal activity [37] could lead to increased levels of ROS, and activation of ROS-regulated signaling pathways [50],

and localized oxidative damage to proteins in the vicinity of the mitochondria. The size and shape of a neuron requires that sub-cellular organelles, such as mitochondria and other cargos, such as proteins, have to be transported over relatively huge dis-tances along axons [52,53]. Synaptic regions of dopaminergic cells may be particularly sensitive, as they are rich in mitochondria, a-synuclein and dopamine, an inherently unstable chemical [54–56]. a-synuclein aggre-gation could lead to reduced proteasomal activity, interference with mitochondrial dynamics and calcium flux [35], eventu-ally setting off a chain of events that would require constant retrograde transport of both aggregating a-synuclein and also ‘damaged’ or depolarized mitochondria to the cell body for macroautophagy [52,57,58].

Table 1. Proteins associated with hereditary forms of Parkinson’s disease.

Protein Mitochondrial association

Cytoskeletal association

Reported activities

a-synuclein Y† Y Chaperone/vesicular trafficking

Parkin Y† Y Ubiquitin–protein ligase

PINK1 Y Y Kinase

DJ-1 Y Y Chaperone/antioxidant

LRRK2 Y† Y Kinase

ATP13A2 N N P-type ATPase

HTRA2 Y Y Protease

Proteins demonstrating a mitochondrial association (Y), may have a mitochondrial location if overexpressed, or as a result of cellular stress (Y†), indicated in the second column. Interaction with or modulation of the cytoskeleton or cytoskeletal proteins are indicated in the third column (Y). The reported activities of each protein are indicated in the fourth column. This does not preclude the assignment of additional functions in the future (e.g., for a-synuclein). ATP13A2 is a lysosomal protein. N: No; Y: Yes.

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For efficient transport, microtubule tracks in neurons must be kept free. If transport is hindered, then the constant supply of cargos destined for the synapses could be inhibited, possibly resulting in a loss of synaptic cell–cell contact and a retraction of the axon.

A number of global proteomic approaches have been employed to identify disease-related changes in brain tissue or substantia nigra [59–61]. Major changes were consistent with the model described pre-viously, as they included increases in mitochondrial complex III and ATP synthase D chain (mitochondrial dysfunction) and L-type calcium channel d-subunit (calcium flux), and a decrease in levels of mortalin expression (protein folding) [59,60]. Interestingly, they also suggest modifications to the cellular cytoskeleton as decreased levels of the intermediate filament (NF) proteins, NF-L and NF-M [59], and an increase in profilin, an actin-binding protein, were also observed. We also recently observed increased interaction of cytoskeletal elements with mitochondria in a cellular model, which was created to overexpress a-synuclein [62]. In future screens, focus-ing specifically on changes in the expression profile of components of the cytoskeleton and/or changes to the interaction of proteins under study with components of the cytoskeleton in regions of neurons, such as synaptic extracts or axons, may prove to be par-ticularly informative, whether using in vitro or in vivo cell models.

The late presentation of sporadic disease presents its own unique problems when try-ing to develop animal models to mimic its development, as we are always presented with end-stage disease [63]. We do not know what initiates the disease process, why it is pro-gressive, whether there are complex genetic factors involved and/or if there are disease-promoting factors in the cellular microenvi-ronment. Furthermore, the animal models themselves may have a genetic background that causes a different response to environ-mental agents compared with humans, or lack interacting partners of suspect proteins. Some model systems (and particularly lower eukaryotes) may lack the orthologue of the genes associated with hereditary forms of human disease under study, such as a-synu-clein or their interaction partners (e.g., [64]). The criticism is then often raised regarding the relevance of models to sporadic disease. Any protein (whether wild type or mutant) prone to aggregate could be potentially toxic if expressed at high enough levels. In addi-tion, over expression of an ubiquitin–protein ligase or protein kinase may promote inter-action with, and subsequent modification of, low-affinity binding partners, which are not normal physiological substrates. Only rigor-ous follow-up studies including comparative proteomic profiling would provide evidence for involvement in disease development.

With some of the models based on hereditary forms of the dis-ease, phenotypes are observed [65–67]. The severity of the pheno-types are often enhanced when two or more genes associated with hereditary forms of the disease are overexpressed, knocked out and/or mutated [68–70], or additional stress factors, such as chemi-cal toxins, are included in the model systems (e.g., [71,72]). These models can also demonstrate the protective effect of some of the proteins associated with PD, counter acting the toxicity of another (e.g., Parkin and G2019S LRRK2, respectively) [73]. These model systems have often been interrogated using proteo mics technolo-gies to identify pathways that are affected [74,75]. Even if a model system, such as those based on genes associated with hereditary disease, does not accurately reproduce the phenotype of PD observed in sporadic disease, they can often provide valuable information. For example, a model based on a high-throughput screen to block the toxicity of a-synuclein in yeast demonstrated how a-synuclein may interfere with the ER-to-Golgi transport in humans when expressed at high levels [76].

In the long term, whether Lewy bodies, per se, are the toxic entity in PD, or not, eventually, the model must be able to explain their presence. In this regard, targeted loss of 26S proteasomal

Figure 1. Etiology of Parkinson’s disease. Central to the onset of Parkinson’s disease are decreased levels of UPS activity, altered mitochondrial dynamics and output of ROS. Problems with mitochondrial dynamics as a result of a cellular stress, for example, may result in an increased output of ROS, leading to increased protein damage if not dealt with by antioxidant defences, such as SOD or glutathione. In time, increased protein damage may result in inclusion/Lewy body formation. Their physical presence may, ultimately, lead to cell death. Prior attempts by the cell to remove aggregating protein may involve both autophagy and the UPS. Released aggregated a-synuclein may be taken up by adjoining cells, or engulfed by cells of the immune system. Initial problems with mitochondria may result from problems of mitochondrial fission and fusion resulting from lower levels of UPS activity (which may be age related or disease related). Defective, depolarized mitochondria may be removed from cells via mitophagy promoted by Parkin (and PINK1/DJ-1), and be dependent on binding to microtubules and transport along axons to the cell body. The stress response ensuing from any of these activities may activate kinases, such as LRRK2 or PINK1. GSH: Glutathione; ROS: Reactive oxygen species; SOD: Superoxide dismutase; UPS: Ubiquitin–proteasome system.

Inclusions(Aggresomes)

Proteindamage

(Localized)

(Syn)n+

Lewybodies

Glialcells

Endocytosis

Chemicals,parasites

Autophagy

ATP13A2 Cell death

Fission

Fusion

Parkin/PINK1/DJ1

SODGSH

ROS(H2O2, O2

-•,OH•, NO•)

(Oxidativestress)

UPS proteinprocessing

Environmentalstress

Mitochondrialactivity/dynamics


Review Robinson

function in dopaminergic neurons in mice resulted in the forma-tion of Lewy-like inclusions positive for a-synuclein and mito-chondrial components [77]. This observation is also interesting in light of the knowledge that a-synuclein is removed via both an autophagic route and proteasomal pathways, leading to the proposal that stimulation of autophagy in neurons could repre-sent a therapeutic approach, as it could lead to an increased rate of removal of these early inclusions [78,79]. Detailed proteomic analyses of these ‘early’ inclusions may also provide additional valuable information. They may identify the proteins other than a-synuclein that seed their formation, and reveal the particular post-translational modified isoform(s) of a-synuclein associated with initial inclusion formation.

Much is often made regarding the lack of inclusions associated with in vivo model systems of hereditary disease. This could be due to a number of reasons. It could reflect the possibility that more than one pathway is affected in sporadic disease, or the mechanism of cell death in hereditary disease may be dif-ferent. Other confounding factors could include the genetic background and age of the animal. For example, the rodent a-synuclein sequence is more closely equivalent to the A53T mutant isoform of human a-synuclein than wild type. The model systems antioxidant cellular defence systems may have developed differently and/or it may express different levels of chaperones or UPS components that provide protection against misfolding proteins. In lower eukaryotes that do not express genes associated with the human disease, interpretation of results could be further complicated by the additional lack of binding partners or substrates. The mechanism of toxicity caused by a protein that appears to have a normal propensity to aggregate could be different in different cell types, as well as in different species. All these factors often conspire to make the generation of in vivo model systems to mimic human disease much more difficult. However, I do not wish to portray the idea that such systems are not useful (e.g., see [25,80]). For example, they have been particularly useful for providing evidence that some of the genes associated with hereditary disease appear to interact in the same cellular pathways (e.g., [81]).

Research into the mechanisms underlying the development of PD also benefits from the knowledge that certain pesticides and herbicides can cause parkinsonian-like disease. Consequently, model systems of PD often employ these toxins, in combi-nation with other stress factors. Indeed, proteomic analyses of these models have been undertaken to identify the cellular processes they target (e.g., [82–87]). Again, proteins, such as mortalin [85,86], were identified, and other proteins were impli-cated in processes described in Figure 1, such as 75-kDa subu-nit of mitochondrial dehydrogenase, a ubiquitin-conjugating enzyme (E2M), polyubiquitin and the proteasome subunit a2 [84,87]. Interestingly, not only was mortalin demonstrated to be covalently modified by dopamine, but also UCH-L1 and DJ-1 [86]. Such a modification would be likely both to affect their function, and cause them to aggregate; we have demon-strated previously that UCH-L1 is very prone to aggregate when overexpressed and/or mutated [88].

The following sections will discuss briefly how studies on indi-vidual genes associated with hereditary disease are providing very valuable information on the pathways that may be disrupted in sporadic disease and, where appropriate, how additional proteomic approaches may prove to be beneficial.

a-synuclein a-synuclein remains the protein most convincingly linked to the development of both hereditary and sporadic forms of the disease [89]. Indeed, the knowledge that gene duplication or trip-lication of SNCA [90,91], with resultant relatively higher levels of the protein, causes late- and early-onset disease, respectively, and that a-synuclein is a major constituent of Lewy bodies, indicate that increased levels are a major factor in the development of the disease. Consequently, the aggregation of a-synuclein as a cause of toxicity has obviously generated a lot of interest in identifying agents that block aggregation or disperse inclusions or promote their autophagic removal as a treatment for PD [92,93].

However, as the normal function of a-synuclein is still not clearly understood, it is proving difficult to predict a mechanism of toxicity [94]. It is known to associate with membranes and, as noted previously, interferes with ER–Golgi transport when expressed at high concentrations [76]. A number of other functions have been proposed, including chaperone activity and regula-tion of vesicular trafficking [89,95]. However, the knowledge that knockout of the gene protects against the toxic effects of cellular stressors and the association of increased levels of expression with disease onset, indicates a gain of toxic function [96].

What is also evident is that this small 140-amino acid-residue protein undergoes significant levels of post-translational modifica-tion, and that one or more of these modifications may increase its toxicity. Consequently, therapeutic approaches targeted at inhibit-ing such modifications may reduce toxicity. A combination of pro-teomic analyses [97] were used to interrogate Lewy body inclusions, and identified ubiquitylation at Lys 12, 21 and 23, phosphorylation at Ser 129, and a host of C-terminal truncations (at Asp115, Asp119, Asn122, Tyr 133 and Asp 135) [97]. Although the enzyme(s) responsible for these truncations have not been identified, cathe-psin D has been proposed as a prime candidate [98,99]. Additional phosphorylation sites have also been identified in separate studies [100–102]. The observed ubiquitin modifications may reflect attempts by the cell to remove aggregating protein via the UPS or autophagy, while C-terminal truncations may reflect alternative routes of dis-posal, such as via lysosomes (the cellular location of cathepsin D). Of potential importance to the development of new treatments is that truncated isoforms of a-synuclein may increase its cytotoxic-ity compared with non truncated isoforms [103,104]. Other low-level post-translational modifications proposed to increase its toxicity include modification by dopamine and nitration [105–109]. Although occurring at low levels, these post-translationally modified forms could act as a seed for aggregation.

Early proteomic studies aimed at identifying binding partners of a-synuclein revealed the presence of 324 proteins [110]. In the same study, there were 306 partners immunocaptured by DJ-1 [110]. Of these, 144 were common to both. These numbers are perhaps



greater than was expected, but could reflect the capture of a number of large multiprotein complexes and/or reflect the knowledge that both proteins display chaperone-like activities [95,111]. A separate study identified more than 250 proteins [112]. Proteins included were those associated with ribosomes, RNA binding and protein synthesis, the cytoskeleton and chaperones. Interestingly, the same group performed a study 2 years previously, where they looked at the expression profiles of mitochondrial proteins extracted from the substantia nigra of brains of mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a potent mitochon-drial toxin that produces parkinsonian-like disease in humans [113]. They identified DJ-1, and noted its colocalization with a-synuclein in substantia nigra and to cytoplasmic inclusions.

The post-translational modification of a-synuclein at ser129 has been the most intensely studied to date, owing to its relatively high abundance in Lewy bodies. This phosphorylation modifica-tion appears to be cell specific, as a-synuclein is not phosphory-lated in platelets in PD [114]. Its presence in Lewy bodies has led to the suggestion that this modification may promote aggregation. By contrast, a recent study indicates that phosphorylation at S87 may protect against oligomerization [102]. As such, the kinase that promotes this activity could also represent a therapeutic target.

Although the functional consequence of ser129 phosphoryla-tion is not known, we, and others, have found that this is post-translational modification occurs at low levels in the absence of large protein aggregates [35,115]. Pull-down assays, followed by mass spectrometry analysis using phosphorylated and nonphos-phorylated peptides of this region of a-synuclein, suggested that the former interacts with mitochondrial electron transport chain components, but the unmodified peptide did not [115]. Interactants of the phosphorylated peptide also included cytoskeletal proteins and vesicular trafficking proteins. It is interesting from nonbiased proteomic analyses of a previous study, and our recent studies on the interaction of a-synuclein with mitochondria [62], that both phosphorylation of a-synuclein and overexpression of a-synuclein identified not only mitochondrial components, but also cyto-skeletal components [116]. This supports the proposal put forward in a previous article in this journal, by Fasano and Lopiano [117], that the inter action of a-synuclein with cyto skeletal components may be an important modulator of its toxicity. It could also sup-port the idea that a-synuclein could disrupt transport along axons or neurites, which is dependent on cytoskeletal interactions, as has been proposed for other neurodegenerative diseases [52]. We also noted that overexpression of a-synuclein caused a reduction in the levels of two components of OXPHOS complex 1, and a small but significant reduction in OXPHOS 1 activity [118]. How many oth-ers of the 46 component OXPHOS complex are affected by high levels of a-synuclein was not determined but could be the focus of future studies. Not only could changes to the levels of individual units be monitored, but also their post-translational modification status, using approaches such as stable isotope labeling by amino acids in cell culture combined with immunocapture.

The observation that a-synuclein is toxic in lower eukaryo-tes [76] has led to their use for various high-throughput screens and proteomic analyses; the assumption is made that the method

of toxicity is the same as in humans. Furthermore, as the A53T mutant in humans is reported to aggregate more readily, this iso-form is often used in place of wild-type a-synuclein to study toxic function. However, we have recently observed that the wild-type isoform appears to produce more changes to the mitochondrial proteome than the A53T mutant isoform, despite both being phosphorylated [62]. This may indicate that, although high con-centrations of a-synuclein affects mitochondrial function, this may not be the cause of its cytotoxicity. The gain of toxic function may be multifactorial, and properties, such as its interference with ER–Golgi transport, are more likely to be involved [76].

In vitro, we found that mammalian cells can proliferate when a-synuclein is expressed at relatively high levels. Toxicity is not observed until cells are cultured under conditions that produce further cell stress, or expressed at relatively high levels in eukaryo-tic cells not normally expressing the protein [76], or perhaps lack sufficient levels of the chaperones normally preventing such aggre-gation. The choice of cell type may be very important as, similar to the group of Pamela McLean [119], we found that the H4 neuro-glioma cell line produces sodium dodecyl sulfate (SDS)-insoluble aggregates when a-synuclein is overexpressed, but not in HEK293 or SH-SY5Y cells, for example.

Parkin/PINK1/DJ-1 (& ATP13A2)Loss of Parkin activity, associated with hereditary disease, can occur through loss of enzymic activity or misfolding leading to decreased solubility [120]. It appears that phosphorylation, both by casein kinase I and cyclin-dependent kinase 5 (cdk5), can also result in loss of activity as the consequence of reducing its solubility, and inhibition of these kinase activities can partially block aggregation of disease-related mutants [121].

A number of key observations over the last few years have aided our understanding of the mechanism of action of Parkin based on the establishment of links between it, PINK1 and DJ-1. Perhaps the first key observation for a role of Parkin in mitochondrial function came from a Drosophila model system, demonstrating morphologi-cal defects in Parkin-null animals [27]. This was followed up by the observation that overexpression of Parkin in Drosophila could rescue the phenotypic defects caused by PINK1 mutants [81,122]. More recently, Parkin was demonstrated to target depolarized mitochondria for autophagic removal (mitophagy) from cells [123], and PINK1 is required for the regulation of mitophagy [124,125]. Subsequent studies have indicated roles for Beclin 1, p62, mitofusin and VDAC1 in Parkin/PINK1-mediated mitophagy [47,126,127].

Parkin expression and activity is not restricted to one type of cell. It is widely distributed. Why it affects dopaminergic cells in particular in hereditary PD is not understood. It may be that these cells are normally subject to higher levels of cellular stress, owing to the presence of dopamine. However, Parkin is expressed in other cell types, including HEK293 cells [123]. Our nonbiased proteomic screen of protein extracts of HEK293 cells induced to overexpress Parkin, indicated interaction with a number of mitochondrial components, suggesting an interaction with mito-chondria in cells at low levels, even in the absence of cellular stress [128]. This interaction increased with the inhibition of


Review Robinson

proteasomal activity [128]. Furthermore, potential interaction with components (ANT2/ANT3) of the mitochondrial per meable transition pore suggests another potential target for Parkin in mitochondria [128]. Another nonbiased screen also identified pro-teins associated with energy metabolism, stress-related chaperones and additional components of the UPS [129].

Parkin, PINK1 and DJ-1 have recently been reported to form a ubiquitin–protein ligase complex (the PPD complex), which pro-motes the degradation of misfolded proteins [130]. However, rather surprisingly, a triple knockout of these three proteins in mice does not appear to generate a visible phenotype [131]. Perhaps, this reflects the proposal that phenotypic defects will not be observed until cells are subjected to additional stress.

Combining this information with the knowledge that Parkin promotes removal of misfolding DJ-1 via autophagy, defects in autophagy as a result of loss of ATP13A2 [132] results in an inherited form of PD, and defects with mitochondrial activity are observed in patients provides further indirect evidence for problems with the removal of defective mitochondria via axonal transport in sporadic disease.

DJ-1 is present in cells in relatively high concentrations. Its pri-mary role appears to be to act as an antioxidant [133,134], protecting neurons against oxidative stress and preventing apoptosis [135]. It has also been reported to interact with OXPHOS complex 1, maintaining its activity [136], and performs chaperone activity [111], which is regulated by the cochaperone BAG-1; loss of chaperone activity as a result of the L166P hereditary mutation was restored by BAG-1 [111]. DJ-1 also appears to protect cells against hypoxia-induced cell death [137]. Interestingly, DJ-1 also appears to bind to tyrosine hydroxylase and 4-dihydroxy-l-phenylalanine decarbox-ylase (DDC), enzymes involved in the synthesis of dopamine [138]. Similar to Parkin, it appears that a greater percentage of DJ-1 associates with the mitochondria when cells are exposed to oxida-tive stress, a translocation that was independent of the mutational status of the protein, and which provided cytoprotection [139]. Proteomic analyses have also demonstrated that DJ-1 is phospho-rylated, and that this modification can be mediated by p53 [140]. Studies of model systems also indicate it helps to maintain the integrity of lysosomes, in addition to mitochondria [141]. What is also interesting is that the activity of DJ-1 appears to be regu-lated by ERK1/2 signaling, similar to LRRK2 [141–145]. DJ-1 can be processed by caspase 6. However, whereas D149A mutation protects DJ-1 from caspase 6 cleavage, the L166P mutation does not, perhaps suggesting different mechanisms of toxicity [146]. As noted, DJ-1 interacts with many of the proteins also bound by a-synuclein [110], potentially suggesting a link between the PPD complex and a-synuclein.

LRRK2Similar to a-synuclein, there is a strong case for the involvement of LRRK2 (or its substrate[s]) in the development of both hereditary and sporadic forms of the disease. Hereditary forms of the disease are autosomal dominant, and display typical features of PD. It is a large 2527-amino acid-residue protein that is expressed in many different neuronal types [147,148], and is proposed to regulate striatal

dopamine transmission [149]. The stability of LRRK2 is regulated by the UPS [150,151]. Dimerization leads to activation of its kinase activity and autophosphorylation [152–154]. It is characterized by a number of recognizable domains, which include kinase and Ras of complex (ROC; GTPase) domains [155]. Mutations of these domains are often observed in hereditary forms of the disease, indicating their importance to protein activity [156]. Furthermore, the ROC domain is reported to regulate its interaction with the microtubule network [157]. This interaction has been reported to result in phosphorylation of b-tubulin, with mass spectrometry analysis, demonstrating Thr107 as the target residue [158]. These data, together with others, again suggest interaction and modula-tion of the cytoskeletal network by a molecule associated with the onset of PD [158,159].

The focus of research on LRRK2, at present, is to identify sub-strates of the protein (e.g., [160–168]). Of interest, one of its substrates was reported to be a-synuclein [169]. LRRK2 pull-down and mass spectrometry analysis of the interactants identified potential inter-actants that were grouped into the cellular pathways associated with the cytoskeleton, chaperones and kinases [170]. Whether these are physiological substrates involved in disease progression is not yet known. LRRK2 kinase activity, acting through ERK signal-ing pathways, has also been associated with a modest increase in transcription from SNCA, the gene encoding a-synuclein [143,144]. With particular relevance to PD, it has also been reported to mod-ulate vesicle endocytosis [168], and a role for LRRK2 in autophagy has recently been proposed [171,172].

The knowledge that the common G2019S mutation results in an activation of the kinase activity [155], and is a cause of hereditary PD with a similar pathology to that of sporadic disease, makes this a prime therapeutic target [173]. The large size of the molecule probably precludes the use of approaches, such as yeast 2 hybrid. A greater level of success is likely to be achieved by employ-ing proteomic analyses of affinity-captured interactants from cell lysates for mass spectrometry identification. Comparative phosphoproteomic analyses of animal model systems designed to express mutant isoforms of the protein at endogenous levels with wild-type expression, may aid the identification of the key substrates intimately involved in the disease process. Such studies may need to be performed in the presence of agents that promote cellular stress if the hypothesis that additional challenges, which may be age related, are required before disease onset occurs.

Omi/HtrA2HtrA2 also appears to be a susceptibility gene for PD [30]. The studies on HtrA2 are limited at present. However, a proteomic approach, COFRADIC™, has been employed to identify potential substrates of HtrA2 [174,175]. Amongst those identified, cytoskeletal proteins, again, appeared to be particularly prominent.

Identification of early biomarkers of PDSimilar to other neurodegenerative diseases [176], one of the great challenges of PD research is to identify biomarkers of early-stage disease [177–179]. Their identification would permit a rapid screen-ing procedure to be developed for the general population, and also



to monitor disease progression (see review [180]). For large-scale screening procedures, the use of an easily accessible body fluid for potential biomarkers should be pursued actively. Biomarkers may include peptide fragments, protein degradation products and post-translational modifications. However, these markers are likely to be present only in very low concentrations in body fluids, such as blood or urine [53,181–184]. This obviously sets a challenge, as the most abundant proteins are likely to mask changes in much rarer abundance peptides/proteins.

One of the major criteria for success in these types of approaches is that rigorous standard operating procedures must be put in place for human sample collection [185,186]. As yet, no systematic large-scale screen of serum or urine has been reported for PD. The reason for this partly reflects the fact that proteomic procedures needed for a successful outcome are still being refined to enable the analysis of body fluids with such a large dynamic range of protein expres-sion levels, combined with the need for the collection of samples suitable for proteomic analyses [187,188]. It probably also reflects resistance in the PD research community that there is a persuasive argument that a disease affecting specific regions of the brain will lead to the presentation of a biomarker in blood or urine [189]. If changes do occur, they are likely to be subtle, and present at very low levels. However, changes in the blood have been noted previ-ously [190]. Similarly, differences in metabolomic profiles have been reported [191].

Proteomic analysis of body fluids collected over a period of time from animal models of PD may also aid the identification of potential biomarkers, which can then be evaluated in humans for their suitability [192–194]. Of course, any response observed in animal models may be very different to that in humans.

By contrast, studies have been undertaken to look for markers in cerebrospinal fluid (CSF) in various neurodegenerative disor-ders, including PD [195–202]. Various proteins have been proposed, such as a-synuclein and autotoxin [197,200]. However, a-synuclein does not appear to be a reliable marker [200]. Post-translationally modified proteins have also been detected, such as oxidatively modified superoxide dismutase 1. In addition, proteins (e.g., apol-ipoprotein e) associated with other neurodegenerative diseases, such as Alzheimer’s disease [197,202], have also been suggested as potential biomarkers. Although, CSF sampling is not without its problems, and would not be a preferred option for routine large population screening, analysis of CSF could be particularly useful for following disease progression in individuals and to monitor changes resulting from treatment with new therapeutics.

Expert commentaryTo date, proteomic applications have demonstrated mixed success to the wider PD research community in efforts to aid the under-standing of the etiology of PD. While they have been particularly useful for monitoring changes to key proteins in disease develop-ment, such as a-synuclein, and identifying potential components of inclusions, such as Lewy bodies, the more global analyses have had limited value. Their major contribution, to date, has probably been to reinforce the proposal that problems associated with han-dling of protein disposal and mitochondrial dynamics are central

to the disease process, and that interactions of proteins intimately associated with the development of PD with the cytoskeleton are often observed. In my opinion, the more global cellular proteomic approaches should now be replaced by studies focusing on deter-mining how genes associated with hereditary disease affects or interact with these cellular activities/structures.

What is always a problem with proteomic approaches for study-ing PD is the decision as to which model systems should be used for the analysis. Unfortunately, there is no easy answer. In vitro models represent the more controlled environment systems for proteomic analysis, but may lack the cellular microenvironment critical to disease development, and the cell type chosen may not express the particular protection systems expressed in cells such as dopaminergic cells. Are primary cells a better model than estab-lished cell lines? Similar to established cell lines, primary cells must adapt to grow in culture conditions, which will be reflected by a continuing change to the cellular proteome. The longer they are in culture, the more the proteome is likely to change. In the long term, embryonic stem cells that are differentiated into dopaminergic-like cells probably offer the best in vitro model system for routine manipulation and proteomic analysis. The use of inducible pluripotent stem cells derived from hereditary and sporadic patients may be particularly useful to look for evidence of subtle background genetic influences on disease development.

By contrast, animal models offer a microenvironment more akin to that of humans, but mice are not humans, and worms do not have complex brain structures. However, they can be very useful for answering specific questions, such as do changes to the expression of proteins under study specifically affect dopamin-ergic neurons or cause some of the phenotypic changes observed in PD? Care must always be taken with comparisons between mouse studies, as different inbred strains of mice may be more or less susceptible to different environmental toxins, based on com-parative minor differences in their own genomes, and expression, for example, of different antioxidant proteins. Combining these limitations with the potential absence of other key environmen-tal factors make the creation of an accurate model of disease difficult to develop. Consequently, the model used to address a particular question always needs to be considered carefully. The analysis of human tissue may be useful, but also has limitations. The main criticism is that the cellular makeup of the tissue used for analysis is likely to be different between diseased and non-diseased tissue. Consequently, the identification of differences in protein expression in a particular cell type may be masked.

Five-year viewThe development of a treatment to halt or slow the progression of PD remains a major goal. Therapeutic agents may include modula-tors of kinase activities [203] or ubiquitin–protein ligases, proteo-stasis agents to block a-synuclein filament assembly [92,93,204,205], reagents to regulate cargo dynamics along axonal microtubule networks [206,207], or modulators of cellular stress pathways [208]. Proteomic approaches have much to offer in this regard, as they can be used to identify interactants and cellular pathways affected. To date, the study of the function and interactions of proteins


Review Robinson

associated with hereditary disease has suggested underlying prob-lems with the activities or interactions with mitochondria, the cytoskeleton and protein processing. These may relate to activities associated with changing expression patterns of proteins associated with hereditary disease, such as LRRK2 or PINK1 (phosphopro-teome screens) or Parkin (immunocapture and mass spectrom-etry analysis). Proteomic approaches should now focus on the downstream effects of these proteins, and mirror the findings to those observed in human disease. An excellent example will be to employ proteomic approaches to aid the identification of key target(s) of Parkin on depolarized mitochondria, and to under-stand the mechanism of how this interaction promotes mitophagy, and how the activities of PINK1 and DJ-1 regulate this activity [125]. Similarly, the improving sensitivity of proteomic approaches will aid PD research to look at more subtle changes in the com-position and post-translational modification status of multipro-tein complexes implicated in the development of PD, such as the OXPHOS 1 multiprotein complex of mitochondria [118], either in animal models or human tissue.

As a group interested in the application of proteomic tech-nologies to study the molecular basis of PD, we have found the development of software packages that rapidly convert lists of

proteins generated by mass spectrometry analysis into cellular pathways and interaction networks invaluable. As these develop in sophistication, it is likely that changes in model systems of PD will be more rapidly interpreted to identify key targets for therapeutic development.

Over the next few years, it is likely that serum-depletion meth-ods, combined with the latest advances in mass spectrometry technologies, will offer the best-ever opportunity to look for dis-ease-associated biomarkers in blood or urine. However, to avoid problems of the past, it is imperative that improvements in sam-ple handling methodologies are put in place, and that nationally agreed rigorous standard operating procedures are employed for sample collection, processing and storage.

Financial & competing interests disclosureThe author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Key issues

• Many of the genes associated with hereditary forms of Parkinson’s disease have been identified. They appear to function in protein processing and/or mitochondrial activity.

• In vitro and in vivo model systems have been developed based on the overexpression or knockout of genes associated with hereditary genes, allowing comparative proteomic analyses. Interactions with the cytoskeleton appear to be a common feature.

• Improved methods and refinement of existing bioinformatic analyses are needed to convert the lists of proteins produced from the analysis of many model systems into affected pathways to enable therapeutic targets to be identified more rapidly.

• Increased a-synuclein expression is implicated both in hereditary and sporadic Parkinson’s disease. Proteomic analysis has identified a series of post-translational modifications in inclusions. The aim will be to identify the enzyme(s) catalyzing the post-translational modifications, as they will represent therapeutic targets.

• More than 250 potential interactants of a-synuclein have potentially been identified. The interactions that promote the seeding of inclusions need to be identified. The analysis of experimentally derived ‘early’ Lewy-like inclusions in animal models may aid this aim.

• Parkin, PINK1 and DJ-1 operate in the same pathway; they can interact to create an ubiquitin–protein ligase complex. Substrates will need to be identified in stressed cells.

• Parkin binds to depolarized mitochondria, promoting mitophagy; PINK1 stabilizes this interaction. Proteomic approaches should aid the identification of interacting mitochondrial proteins or complexes.

• (Phospho)proteomic analyses of cellular models of LRRK2 and PINK1 should aid the identification of key substrates, as well as the effects caused by modulating these post-translational modifications.

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Review Robinson

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•• ExampleofhowproteomicscanbeusedtoinvestigatechangestomultiproteincomplexesassociatedwiththeonsetofParkinson’sdisease.

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•• DemonstrationthatParkinpromotesmitophagyofdepolarizedmitochondria.

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128 Davison EJ, Pennington K, Hung CC et al. Proteomic analysis of increased Parkin expression and its interactants provides

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• LinksthreegenesassociatedwithhereditaryParkinson’sdiseaseintoonepathway.

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• AlterationstoautophagyandmitochondriaresultingfromlossofDJ-1.

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• ExampleofremodelingofthecytoskeletonbyproteinsassociatedwithParkinson’sdisease.

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• LinksautophagytotheactivityofLRRK2.

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Review Robinson

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Understanding the molecular basis of Parkinson’s disease, identification of biomarkers and routes to therapy