Parkinson's disease (PD), the second most common age-related neurodegenerative disease, results in abnormalities in motor functioning. Many fundamental questions regarding its aetiology remain unanswered. Pathologically, it is not until 70–80% of the dopaminergic neurons from the substantia nigra pars compacta are lost before clinical symptoms are observed. Thus research into PD is complicated by this apparent paradox in that what appears to be the beginning of the disease at the clinical level is really the end point neurochemically. Consequently, we can only second guess when the disease started and what initiated it. The causation is probably complex, with contributions from both genetic and environmental factors. Intracellular proteinaceous inclusions, Lewy bodies and Lewy neurites, found in surviving dopaminergic neurons, are the key pathological characteristic of PD. Their presence points to an inability within these terminally differentiated cells to deal with aggregating proteins. Recent advances in our knowledge of the underlying disease process have come about from studies on models based on genes associated with rare hereditary forms of PD, and mitochondrial toxins that mimic the behavioural effects of PD. The reason that dopaminergic neurons are particularly sensitive may be due to the additional cellular stress caused by the breakdown of the inherently chemically unstable neurotransmitter, dopamine. In the present review, I discuss the proposal that in sporadic disease, interlinked problems of protein processing and inappropriate mitochondrial activity seed the foundation for age-related increased levels of protein damage, and a reduced ability to deal with the damage, leading to inclusion formation and, ultimately, cell toxicity.

INTRODUCTION

Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease (for reviews, see [13]). It is a slow debilitating disease generally diagnosed in the sixth decade of life. The disease is characterized by physical symptoms of tremor, rigidity, akinesia and postural instability.

The primary group of neuronal cells affected are the terminally differentiated dopaminergic neurons of the substantia nigra pars compacta. Physical symptoms are not obvious until approx. 70–80% of these cells are lost, suggesting that, in addition to the 120000 people diagnosed with PD in the U.K., and 1–1.5 million people in the U.S.A. [3], there is a large population with asymptomatic early-stage disease for which currently there is no diagnosis. As patients present with late-stage disease, it is difficult both to assess the age of disease onset and also to identify the causative agents. There is no treatment to slow the progression of underlying disease, and, consequently, therapy is primarily symptomatic and based on dopamine replacement.

We can only speculate at the present time why the dopaminergic neuron is the targeted cell type. It may relate to the inherent chemical instability of dopamine. Its highly reactive metabolites can covalently modify proteins, potentially exacerbating an underlying age-related cellular defect accrued over a long period of time in terminally differentiated neuronal cells such as damaged mitochondrial DNA.

The age of onset of PD is variable, suggesting the involvement of genetic and/or (micro)environmental factors. Individual susceptibility may simply reflect a chance event. We do not know whether sporadic disease arises through either a relatively sudden loss of cells initiated by exposures to an environmental stress or toxin followed by periods of slow decline, or a relatively linear loss of cells that is simply faster in PD patients than in non-affected individuals.

Only 5–10% of PD is associated with a classical recessive or dominant Mendelian pattern of inheritance [416]. We do not know why mutations to proteins associated with hereditary disease primarily cause problems for dopaminergic neurons, despite being expressed in many cell types. However, the functional activities of the proteins encoded by these genes have provided clues that may be helpful for understanding the aetiology of sporadic disease (Table 1).

Table 1
Phenotypic effects resulting from gene mutations associated with hereditary forms of PD

Although the majority of mutations are missense, hereditary forms of PD are also caused by gene duplication or triplication of the α-synuclein gene. *Loss of ubiquitin–protein ligase (E3) activity may result directly from a mutation or be due to an increased ability to aggregate. †UCH-L1 has been reported to be an E3 in addition to a DUB [216]. Substrates of PINK1 and LRRK2 remain to be identified.

Protein Gene locus Function Subcellular location Phenotypic effect of mutations 
α-Synuclein PARK1 and PARK4 Unknown Cytoplasm/membranes Aggregation/reduced proteasomal activity 
Parkin PARK2 E3 Cytoplasm Loss of E3 activity* 
UCH-L1 PARK5 DUB/E3† Cytoplasm Reduced DUB/E3 activity 
DJ-1 PARK7 Chaperone Mitochondrial Loss of chaperone/antioxidant activity 
PINK1 PARK6 Kinase Mitochondrial Loss of kinase activity 
LRRK2 PARK8 Kinase Membranes/mitochondrial/lysosomal Increased kinase activity 
ATP13A2 PARK9 P-type ATPase Lysosomal Loss of ATPase activity 
Protein Gene locus Function Subcellular location Phenotypic effect of mutations 
α-Synuclein PARK1 and PARK4 Unknown Cytoplasm/membranes Aggregation/reduced proteasomal activity 
Parkin PARK2 E3 Cytoplasm Loss of E3 activity* 
UCH-L1 PARK5 DUB/E3† Cytoplasm Reduced DUB/E3 activity 
DJ-1 PARK7 Chaperone Mitochondrial Loss of chaperone/antioxidant activity 
PINK1 PARK6 Kinase Mitochondrial Loss of kinase activity 
LRRK2 PARK8 Kinase Membranes/mitochondrial/lysosomal Increased kinase activity 
ATP13A2 PARK9 P-type ATPase Lysosomal Loss of ATPase activity 

Although the majority (~90%) of PD appears to be idiopathic, we cannot rule out a contribution from a complex underlying genetic component. Exposure to environmental toxins was proposed as a potential causative mechanism as a number of chemicals have been identified that cause Parkinson-like disease, e.g. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and rotenone. Mitochondria of dopaminergic neurons appear to be particularly sensitive to chemical toxins. However, a common disease-associated environmental agent has not been identified. Susceptibility could relate to different ways common environmental agents are metabolized by different individuals. In some, they may be converted into harmless metabolites, whereas in others, the metabolite may be different and relatively more toxic, especially to dopaminergic neurons. In contrast, polymorphic differences in one or more of a multitude of genes involved in protection against oxidative damage could result in relatively lower protective functioning and thus may combine to reduce an individual's ability to cope with cellular stress. The ability of these cells to deal with such damage may also be compromised by the lower levels of proteasomal activities associated with aging ([17] and reviewed in [18,19]).

Whatever the initiating event, work from many laboratories using both in vivo (e.g. [2037]) and in vitro (e.g. [3844]) model systems, on the genes associated with hereditary disease [412,1416] and environmental toxins (e.g. [4547]), suggests that the finger of guilt should be pointed at a combination of problems of protein processing and inappropriate mitochondrial activity (Figure 1).

Molecular mechanisms underlying the development of PD; problems of inappropriate mitochondrial activity and protein processing

Figure 1
Molecular mechanisms underlying the development of PD; problems of inappropriate mitochondrial activity and protein processing

Defects in protein processing and inappropriate mitochondrial activity caused by unknown environmental, genetic or age-related factors cause increases in reactive oxygen species and more protein damage. Chaperones and the UPS are recruited to aid folding and 26S proteasome-mediated protein degradation of damaged and aggregating proteins. Other stress factors such as a build-up of misfolded membrane proteins (normally processed by ERAD) can then activate the UPR, resulting in increases in factors such as chaperones and components of the UPS. The inability of the chaperone–UPS to deal with proteins may result in protein aggregation. The UPS and DUBs may not only be recruited early to remove aggregating proteins, but also be continuously recruited in an attempt to remove inclusions as they form. The burden caused by the aggregates/Lewy bodies may ultimately overwhelm the proteasomal, ERAD and chaperone systems, leading to cell death.

Figure 1
Molecular mechanisms underlying the development of PD; problems of inappropriate mitochondrial activity and protein processing

Defects in protein processing and inappropriate mitochondrial activity caused by unknown environmental, genetic or age-related factors cause increases in reactive oxygen species and more protein damage. Chaperones and the UPS are recruited to aid folding and 26S proteasome-mediated protein degradation of damaged and aggregating proteins. Other stress factors such as a build-up of misfolded membrane proteins (normally processed by ERAD) can then activate the UPR, resulting in increases in factors such as chaperones and components of the UPS. The inability of the chaperone–UPS to deal with proteins may result in protein aggregation. The UPS and DUBs may not only be recruited early to remove aggregating proteins, but also be continuously recruited in an attempt to remove inclusions as they form. The burden caused by the aggregates/Lewy bodies may ultimately overwhelm the proteasomal, ERAD and chaperone systems, leading to cell death.

PROTEIN INCLUSIONS AND PARKINSON DISEASE

Intra- and/or extra-cellular proteinaceous inclusion bodies in brain tissue are characteristic pathological markers of many neurodegenerative diseases. Aging neurons may be particularly sensitive to problems with protein processing due to a combination of their age, their unique cellular structure and their relatively large size. They have a need for energy-dependent, long-distance, anterograde and retrograde transport of proteins and subcellular organelles such as mitochondria along axons that may be affected by aggregating proteins [4850]. Consequently, disease-initiating events may primarily be localized to different regions of the cell, e.g. the synapse or axon [49]. This type of effect is observed in hereditary spastic paraplegia, where axons degenerate from their synaptic ends, and structurally abnormal mitochondria were observed in the synaptic terminals of paraplegin knockout (Spg7−/−) mice [51,52]. This latter disease can be caused by mutations to proteins that are involved in the processing or folding of mitochondrial proteins and include a component of the mitochondrial AAA (ATPase associated with various cellular activities) protease termed paraplegin, and the mitochondrial chaperone Hsp (heat-shock protein) 60 [52].

Lewy bodies and Lewy neurites are the defining intracytoplasmic inclusions found in surviving dopaminergic neurons of PD patients [53,54]. Although many inclusions are associated with a specific neurodegenerative disease, such as amyloid plaques or paired helical filaments in Alzheimer's disease, Lewy bodies are associated with a number of different neurological conditions grouped into the α-synucleinopathies [55]. They affect different regions and subpopulations of neurons in the brain, and each is associated with different clinical presentations. The α-synucleinopathies are so-named as the small 140-amino-acid protein α-synuclein (Figure 2) was identified many years ago as a major ‘core’ component of Lewy bodies [56].

Post-translational modifications of α-synuclein identified by proteomic analysis of Lewy bodies and inclusions

Figure 2
Post-translational modifications of α-synuclein identified by proteomic analysis of Lewy bodies and inclusions

Underlined sequences indicate imperfect KTKEGV repeats; the sequence indicated by the dotted line is the central hydrophobic region of α-synuclein necessary for aggregate formation; P, K and T above the sequences are mutations A30P, E46K and A53T respectively; U above the sequences indicates protein ubiquitination; PO4 indicates protein phosphorylation; arrows indicate protein truncation sites. A 22 kDa O-glycosylated isoform of α-synuclein has also been described, but the amino acid residue(s) through which sugar residues are covalently attached has not been described [114].

Figure 2
Post-translational modifications of α-synuclein identified by proteomic analysis of Lewy bodies and inclusions

Underlined sequences indicate imperfect KTKEGV repeats; the sequence indicated by the dotted line is the central hydrophobic region of α-synuclein necessary for aggregate formation; P, K and T above the sequences are mutations A30P, E46K and A53T respectively; U above the sequences indicates protein ubiquitination; PO4 indicates protein phosphorylation; arrows indicate protein truncation sites. A 22 kDa O-glycosylated isoform of α-synuclein has also been described, but the amino acid residue(s) through which sugar residues are covalently attached has not been described [114].

It is still controversial whether ‘mature’ Lewy bodies are a toxic entity in PD neurons [57,58]. There are studies to suggest that in vitro generated α-synuclein inclusions are cytoprotective in proliferating cells [41]. There is also evidence that it is small soluble fibrillar forms of α-synuclein, or smaller inclusions found at synaptic junctions, that are the more toxic entities [58]. In cultured cells, α-synuclein appears to aggregate into different types of inclusion: smaller non-fibrillar punctate inclusions that are found widely scattered are the first to form, and larger ones that are found adjacent to the nucleus containing amyloid-like fibrils [59]. The latter type display aggresome-like features as they are associated with a number of other proteins, including vimentin and γ-tubulin, and are located at the nuclear periphery [41,60]. α-Synuclein aggregates also inhibit proteasomal activity suggesting a potential for a more widespread toxic effect [40].

Probably one of the strongest lines of evidence implicating α-synuclein as a key player in the development of sporadic PD is that increased levels of α-synuclein resulting from gene duplication or triplication of the α-synuclein gene cause autosomal-dominant (late- or early-onset respectively) forms of PD [11,12]. Although mutant forms of α-synuclein are also associated with hereditary forms of PD (Table 1), there is always the possibility that the mechanism underlying disease onset caused by these mutants may be different from that caused by wild-type α-synuclein in sporadic disease.

Could the increased expression of α-synuclein cause disease due to a toxic gain-of-function not related to its ability to aggregate, or a loss of function as it aggregates? It is impossible to say for certain at the present time, as the function of α-synuclein is not known. It exists in cells either in the cytoplasm in a relatively unfolded state or membrane-bound, where it takes up a more defined structure [61,62]. Its biological function is postulated to centre on its membrane-binding capabilities [63,64]. It binds to synaptic membranes and may be involved in vesicular trafficking. It can counteract the loss of the co-chaperone, CSPα (cysteine string protein α), by mechanisms which appear to be dependent on its phospholipid-binding activity and interaction with the SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) complexes at pre-synaptic membranes [65]. Higher than normal α-synuclein concentrations may promote its binding to other subcellular membrane structures. In this regard, α-synuclein has recently been reported to be associated with mitochondrial membranes [34].

Much of the focus of PD research involving α-synuclein is centred on using in vitro or in vivo models to create the conditions that will promote Lewy body-like inclusions (Figure 1). The analysis of experimentally induced inclusions or Lewy bodies from patients could give clues to the factors that seed them and why they are toxic (e.g. [41]). Initial approaches to identify binding partners of aggregating proteins or components of Lewy bodies employed simple immunohistochemical staining procedures to look at the expression patterns of suspected constituents, e.g. those associated with hereditary disease or involved in protein processing such as parkin, UCH-L1 (ubiquitin C-terminal hydrolase L1) or chaperones [7,41,66,67] (Table 1). More recently, more sensitive MS-based analyses have been employed. These latter approaches have the advantage that they are non-biased and, potentially, all components of the inclusions can be identified simultaneously. Moreover, the changing constituent profile of the inclusion could be monitored with time. However, because of the sensitivity of MS-based approaches, it is essential that all steps are taken to remove co-purifying contaminants before MS analysis to prevent the latter from being labelled as constituents/interactants.

In a relatively early proteomic analysis, 251 proteins were found to be associated with Nonidet P40-soluble forms of α-synuclein in mouse embryonic stem cells [68]. Rotenone, an insecticide which blocks electron flow from the Fe–S centres in the mitochondrial OXPHOS (oxidative phosphorylation) complex I to coenzyme Q (Figure 3), causing Parkinsonian-like symptoms in rats, produced inclusion formation, and the differential expression of at least 51 of the α-synuclein bound proteins [68]. On a later screen, the number of interacting proteins increased to 324 [69]. Of these, 144 appeared to be common interactants with PD-related chaperone DJ-1; 306 proteins were associated with DJ-1 complexes (Table 1) [69]. However, these authors did not observe a direct interaction between DJ-1 and α-synuclein, suggesting that the interactions were mutually exclusive [69]. In contrast, others have demonstrated an interaction [70]. The ability of DJ-1 to bind competitively a large group of α-synuclein's interacting partners may provide an explanation for DJ-1's protective effect against the toxic effects of α-synuclein [7173]. It may also be interesting to compare the lists of binding partners in these screens with those proteins which appeared to make α-synuclein more toxic in yeast [74].

Example of cellular inclusions in SHSY5Y cells produced by a combination of increased levels of parkin and inhibition of proteasomal activity

Figure 3
Example of cellular inclusions in SHSY5Y cells produced by a combination of increased levels of parkin and inhibition of proteasomal activity

Proliferating or differentiated SHSY5Y dopaminergic cells cultured in the presence or absence of the proteasomal inhibitor MG132 and stained for parkin (green staining) or NFH (neurofilament H) (red staining). Arrows indicate the presence of parkin and NFH-positive inclusions in MG132-treated cells. Note the extremely large inclusions in differentiated SHSY5Y cells (yellow staining indicates the overlapping staining patterns of parkin and NFH). Figure kindly provided by Dr Helen C. Ardley.

Figure 3
Example of cellular inclusions in SHSY5Y cells produced by a combination of increased levels of parkin and inhibition of proteasomal activity

Proliferating or differentiated SHSY5Y dopaminergic cells cultured in the presence or absence of the proteasomal inhibitor MG132 and stained for parkin (green staining) or NFH (neurofilament H) (red staining). Arrows indicate the presence of parkin and NFH-positive inclusions in MG132-treated cells. Note the extremely large inclusions in differentiated SHSY5Y cells (yellow staining indicates the overlapping staining patterns of parkin and NFH). Figure kindly provided by Dr Helen C. Ardley.

MS approaches have also been employed to identify post-translational modifications of α-synuclein in Lewy bodies [68,7577]. A significant proportion of α-synuclein was found to be phosphorylated at Ser129, and ubiquitinated at Lys12, Lys21 and Lys23 [77] (Figure 2). Interestingly, although phosphorylated α-synuclein was observed in neuronal cells, it was not observed in platelets, suggesting the lack of an appropriate kinase activity; SHSY5Y cyber cells created from the isolation of mitochondria from platelets of PD patients were also functionally defective [78,79]. Why α-synuclein is post-translationally modified and whether these modifications contribute to its toxic properties is currently the subject of intense research effort (e.g. [44,80]). In vivo models have provided convincing evidence that C-terminal truncations promote oligomer formation and toxicity [81].

GRK5 (G-protein-coupled receptor kinase 5), and casein kinases 1 and 2, have been implicated as the kinases physiologically responsible for phosphorylation at Ser129 [8285]. GRK5 was associated with α-synuclein in Lewy bodies, and promoted oligomer and aggregate formation [83]. Using an in vitro model system, an S129A mutant isoform of α-synuclein was demonstrated to be less prone to form inclusions whereas an S129E mutant (a glutamate residue is used to mimic the presence of the phosphate group) produced a similar level of inclusion formation as the wild-type, suggesting that a charged group at this position promotes inclusion formation and may potentially cause further processing of α-synuclein [44].

Protein truncations at Asp115, Asp119, Asn122, Tyr133 and Asp135 residues were also observed (Figure 2) [77]. The variability of the truncation sites suggests the involvement of multiple trimming enzymes such as calpain 1, cathepsin D and the 20S proteasome [8588]. Interestingly, overexpression of parkin has previously been demonstrated to promote calpain-mediated processing of α-synuclein, and both MPTP and rotenone (mitochondrial toxins that produce Parkinsonian-like disease in model systems) affect cellular calpain activity [8991]. At the present time, it is not clear whether post-translational modification of α-synuclein such as phosphorylation at Ser129 then leads to the promotion of further modifications such as C-terminal processing or ubiquitination or vice versa.

Alterations to the interaction of α-synuclein with some of its binding partners may contribute to its toxicity. For example, phosphorylation at Ser129 reduces its affinity for synphilin-1 [44]. α-Synuclein also binds histones (particularly histone H3) preventing acetylation and causing cellular toxicity [92]. This is interesting in the light of previous observations that HDAC (histone deacetylase) 6 was shown to be involved in aggresome formation, binding misfolded ubiquitinated proteins and dyenin motors, thereby causing the former to be transported to aggresomes [93]. Subsequently, an inhibitor of the HDAC, sirtuin 2, which preferentially deacetylates α-tubulin, was found to be protective in a Drosophila model of PD characterized by overexpression of α-synuclein [35]. The preventative effects in this model did not appear to be related to an ability to prevent α-synuclein aggregation.

The role of the other proteins associated with the hereditary form of PD (Table 1) in seeding Lewy body formation is less certain. As noted above, many have often been identified in Lewy bodies using immunohistochemical approaches. A post-translationally modified ~53 kDa protein fragment of PINK1 [PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced kinase 1] was found in stress-induced inclusions, and in approx. 5–10% of Lewy bodies together with other mitochondrial proteins, suggesting that this truncated form may have a role in Lewy body formation [9496]. However, it is not currently known whether these Lewy body components promote the seeding process or are ‘engulfed’ during inclusion formation, effectively leading to lower levels of their respective cellular activities.

DEALING WITH STRESS: THE UPS (UBIQUITIN–PROTEASOME SYSTEM) AND CHAPERONES

The UPS

Post-translational modification of proteins with ubiquitin {and the ever-growing list of Ubl (ubiquitin-like) proteins [97,98]} regulates many cellular functions, making it a potential therapeutic target area for many diseases [99]. It is an energy-dependent process that operates in all eukaryotic cell types. Although the major role for protein ubiquitination is to promote the temporospatial 26S proteasomal degradation of many intracellular proteins, a number of other functions have been assigned in the last few years (see [97] for a review). These are generally indicated either by protein mono-ubiquitination or the formation of polyubiquitin chains which are not linked through ubiquitinated Lys48.

The specificity of protein ubiquitination generally resides in the interaction between an E3 ubiquitin–protein ligase and target protein. Many E3s are multicomponent protein complexes, e.g. the large family of SCF (Skp1/cullin/F-box)–E3 complexes and the anaphase-promoting complex [100,101]. Post-translational modification of substrate is often required as an aid to its recognition by an E3. Repeated ubiquitination leads to the formation of polyubiquitin chains generally linked through Lys48. Alternatively, a pre-formed ubiquitin chain assembly may be transferred [102,103]. A large number of DUBs (de-ubiquitinating enzymes) such as UCH-L1 are also found within cells. These enzymes may remove ubiquitin molecules to prevent proteasomal destruction and be part of a proofreading mechanism. Polyubiquitinated conjugates may then be directed towards the caps of the 26S proteasome where they are unfolded and then fed into the catalytic barrel-like chamber of the proteasome where a combination of three separate enzymic activities are then used to hydrolyse the proteins into small peptides.

A role for the UPS in the development of PD was proposed many years ago when it was discovered that Lewy bodies are ubiquitinated. Indeed, anti-ubiquitin antibodies are often used by pathologists to view Lewy bodies in immunohistochemical sections. Since then abnormalities in protein processing in PD has been proposed from many experimental model systems (e.g. [104,105]). Furthermore, recent observations suggest a global change to the ubiquitin system in neurodegenerative disease [106]. Whether there is a role of protein ubiquitination in seeding Lewy body formation remains to be established as proteins may be ubiquitinated or de-ubiquitinated post-aggregation. Chaperone and UPS components may target aggregating proteins in an initial protective cellular response to stress (Figure 1). They may then become entrapped as inclusions increase in size and develop into Lewy bodies. Alternatively, they may be recruited at a later stage in an attempt to disperse the Lewy body. Problems with handling misfolded proteins may be compounded by the reported age-related decrease in proteasomal activity [17]. DUBs such as UCH-L1 may then be attracted to the inclusions in an attempt to remove polyubiquitin chains.

The chaperone/UPS and PD link was strengthened when mutations to the RING (really interesting new gene)–IBR (in-between RING)–RING domain E3 parkin, the DUB UCH-L1 and the chaperone DJ-1 were associated with familial forms of PD (Table 1) [58] (although doubt has recently been cast on susceptibility associated with UCH-L1 [107]). Parkin interacts with DJ-1 (Table 1) perhaps as part of a high-molecular-mass complex, suggesting that they are components of a common processing pathway [70,108].

An obvious effect of a loss of parkin's ligase activity would be an increase in the stability of its substrates. It was therefore surprising when the levels of a number of its substrates were found to be normal in Parkin−/− mice, and the numbers of dopaminergic neurons appeared to be similar to those of wild-type mice [26,109,110]. However, increased levels of extracellular dopamine and defects in mitochondrial activity were observed, and greater currents were required to induce synaptic responses [26,109,110]. Subsequently, the level of one of its substrates, the aminoacyl-tRNA synthetase cofactor, p38/JTV-1, was found to be higher in wild-type compared with Parkin−/− mice [111]. Moreover, a proteomic screen of the knockout mice revealed other differences, many of which were proteins associated with energy metabolism, detoxification and protein processing [27]. Whether any of these latter proteins are substrates of parkin remains to be established.

Although many substrates of parkin have been described (e.g. [66,112118]), it has proved difficult to identify the key disease-related physiological targets. There are a number of potential reasons for this. First, for an endogenous E3 to target selectively an endogenous substrate when both are expressed at low levels, a high-affinity interaction is required. It may also require an appropriate subcellular localization and post-translational modification of the substrate. Experimentally, the identification of a pairing of an E3 and its physiological substrate is not always an easy process, as increasing the levels of an E3 may lead to the targeting of low-affinity non-physiological substrates. It has been known for many years that synthetic E3s can be created by fusing an E2 ubiquitin-conjugating enzyme to an interacting domain of a selected protein. The aim is to bring an E2 in close enough contact to a targeted protein to transfer the E2-bound ubiquitin [119]. Consequently, methods used to identify E3 substrates can sometimes be misleading, as they detect interactions when both proteins are expressed at high levels. Interpretation of in vitro protein ubiquitination assays based on overexpression should therefore always be interpreted with caution.

Secondly, even with in vivo approaches, degeneracy in target selection may complicate the interpretation of results. For example, many proteins such as p53 appear to be targeted by several different E3s, including the ERAD [ER (endoplasmic reticulum)-associated degradation] ligase, synoviolin/HRD1 [HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase degradation protein 1] [120]. Which E3 is selected may be dependent on factors such the post-translational modification status of the substrate. Although parkin does not promote ubiquitination-mediated degradation of α-synuclein, it does target its glycosylated isoform, αSp22 [114]. Another of its substrates is the α-synuclein-binding protein, synphilin-1 [112]. Parkin and synphilin-1 show overlapping expression patterns in Lewy bodies and readily form inclusions when expressed together with α-synuclein, and cultured in the presence of proteasome inhibitor [112,121]. However, synphilin-1 and α-synuclein are also targeted for ubiquitination by the E3, SIAH (seven in absentia homologue), which is also found in Lewy bodies [122].

The story is potentially complicated further by the observation that the ubiquitination of synphilin catalysed by parkin in vitro leads not only to Lys48-linked ubiquitin chain assembly and proteasomal-mediated degradation, but also to Lys63 ubiquitin chain assembly [123]. It can also mono-ubiquitinate p38 and PICK1 (protein that interacts with protein C-kinase 1) [118,124]. The modification of the latter did not result in its degradation but influenced the interaction with ASIC (acid-sensing ion channel) [118].

Thirdly, signalling and/or post-translational modification promoted by environmental or aging stress factors may be absent from in vivo model systems. Some of parkin's activities are also known to be cell-specific. The ubiquitination of LIM kinase (an interactant identified via a proteomic screen) increased in human BE(12)-M17 neuroblastoma cells overexpressing parkin, but not in HEK-293 (human embryonic kidney) cells [125]. LIM kinase activity prevented parkin auto-ubiquitination, as well as the ubiquitination of p38 [111,125,126].

Modulation of parkin's E3 activity results from not only familial mutations, but also decreased solubility and post-translational modification that includes S-nitrosylation and phosphorylation, and covalent modification by dopamine [127134]. These modifications influence its ligase activity and entrapment of substrates in Lewy bodies. After the initial identification that parkin was an E3, it was proposed that all mutations lost E3 activity as a direct result of the mutation [7]. Consequently, however, a number of studies have now demonstrated that loss of activity may also result from a loss of solubility [124,127,132]. We and others have observed both wild-type and mutant isoforms of parkin readily formed large aggregates in vitro, especially after the inhibition of proteasome activity [38,39,135] (Figure 3). In contrast, we have found it is more difficult to promote α-synuclein aggregation in proliferating cells, and inclusions are found in far fewer cells. It is interesting therefore that AR-JP (autosomal recessive juvenile Parkinsonism) appears to be characterized by the absence of inclusions, raising the possibility that when cells are exposed to appropriate damaging (micro)environmental conditions, parkin-based inclusions may more rapidly formed in AJ-RP neurons, leading to a more rapid cell response and earlier cell death.

So what regulates the levels of parkin? When overexpressed, parkin auto-ubiquitinates [7]. We have also found using yeast two-hybrid screens that the RING–IBR–RING domain of parkin interacts with other proteins characterized by RING or RING-like structures including the structural RING–IBR–RING homologue, HHARI (human homologue of Drosophila ariadne) [136,137]. In addition, the RING E3, Nrdp1 (neuregulin receptor degradation protein-1)/FLRF (fetal liver RING finger), regulates parkin stability and activity [138]. These observations are interesting in the light of the observation that, during apoptosis, parkin is cleaved into a 12 kDa N-terminal and a 38 kDa C-terminal RING–IBR–RING fragments [139]. Although the effect of this cleavage may be to inactivate parkin by preventing interactions through its Ubl domain and ubiquitination of its targets, the release of its C-terminal E3 active RING–IBR–RING domain may promote the ubiquitination and degradation of other RING E3s.

The UPS, ERAD, parkin, α-synuclein and stress

The UPS also plays a major role in the removal of mutant and/or misfolded membrane proteins via ERAD [140142]. When selected, misfolding or mutant proteins are ubiquitinated, retrotranslocated and degraded in the cytoplasm by the 26S proteasome. When the folding capacity of the ER is exceeded, e.g. during oxidative stress or due to the overexpression of an ER substrate, the UPR (unfolded protein response) is activated, leading to generalized down-regulation of protein synthesis, but increased expression of ERAD components and chaperone proteins [143]. One of parkin's functions is to protect against ER-stress-induced cell death caused by overexpression of one of its substrates, the PAEL-R (parkin-associated endothelin receptor-like receptor), which is also a component of Lewy bodies [113,144146].

To date, two transmembrane RING E3s have been linked to ERAD in yeast, Doa10 and Hrd1 [141,147], and gp78 (glycoprotein 78) and HRD1/synoviolin in mammals [120,148150]. There are probably others [151]. These E3s form multiprotein complexes with other membrane, and membrane-associated, proteins [147,152]. Both yeast complexes bind the cytosolic membrane-associated E2, UBC7 (ubiquitin-conjugating enzyme 7), and the Cdc48–Npl4–Ufd1 multiprotein complex via Ubx2. Additional sets of associated proteins appear to be required for targeting of substrates with cytosolic, membrane or luminal mutations; they are classified as ERAD-C, -M or -L complexes respectively [147]. α-Synuclein may interfere selectively with the ERAD system as the overexpression of α-synuclein in yeast inhibited the processing of the ERAD-L substrate, CPY*, but not that of the ERAD-M substrate sec61-2p [147,153]. This inhibitory activity may relate to the ability of α-synuclein to bind lipids, thereby influencing the assembly of the additional components that are required for ERAD-L-mediated degradation.

Chaperones and the UPS

A number of studies have demonstrated that overexpression of chaperones can protect against α-synuclein aggregation and toxicity [23,154,155]. These findings led to the proposal that increased expression of factors that aid protein folding may be protective in PD and represent a therapeutic option. One protein that forms an important link between misfolding proteins, the chaperone system and the UPS is the co-chaperone CHIP [C-terminus of Hsc70 (heat-shock cognate 70)-interacting protein] [156158]. CHIP activity has been implicated in attempts to fold many proteins associated with a number of different neurodegenerative diseases, including α-synuclein [159,160].

CHIP is not only a co-chaperone, but also a UbcH5-dependent U-box E3 [161,162]. It binds to the C-terminus of Hsc70/Hsp70 (and Hsp90) via three TPRs (tetratricopeptide repeat domains) and promotes the ubiquitination of Hsp70's client proteins [156,161163]. The ligase activity can be inhibited by the binding of co-chaperones HspBP1 (Hsp-binding protein 1) or BAG (Bcl-2-associated athanogene)-2 [164,165]. Interaction of Hsp70 with BAG-1 promotes an association with the 26S proteasome and degradation of client proteins [166].

Key to the up-regulation of Hsps caused by inhibition of the UPS and induction of cellular stress is HSF1 (heat-shock transcription factor 1) [167]. HSF1 is normally negatively regulated by binding of Hsp70 and Hsp90, maintaining HSF1 in a monomeric state [168]. CHIP activates HSF1 by binding Hsp70, resulting in HSF1 trimerization [168,169]. Interestingly, Hsp70 levels are returned to normal with the aid of CHIP by promoting the ubiquitination-mediated degradation of Hsp70 [169].

As discussed above, previous studies have demonstrated that the membrane protein PAEL-R was a substrate of parkin, and increased expression of parkin protected the cell against the ER stress by catalysing its ubiquitination and 26S proteasome-mediated degradation. Consequently, it was with great excitement that a link between parkin, CHIP, Hsp70 and PAEL-R was identified [170,171]. Under stress conditions, increased levels of CHIP enhanced the E3 activity of parkin by promoting the disassociation of Hsp70 from parkin and PAEL-R, promoting the ubiquitination of the latter by parkin.

DJ-1 may be another component of the Hsp70–CHIP–parkin complex. DJ-1 was identified as a gene associated with familial early-onset autosomal recessive PD (Table 1) [8,172,173]. It is a homodimeric mitochondrial protein that displays both antioxidant and chaperone activities, and protects against mitochondrial toxins and oxidative-induced damage both in vitro and in vivo [28,29,71,174181]. Hereditary mutations in DJ-1 are associated with loss of these activities. When mutated, it becomes unstable and is removed by the UPS [182185].

Oxidative modification of Cys106 of DJ-1 is essential to its antioxidant activity, and, in patients, it appears to exist in many different oxidized isoforms [73]. Other post-translational modifications include cleavage at Gly157 and SUMOylation at Lys130 [186,187]. Mutation of Lys130 abolished the protective role of DJ-1 against UV-induced cellular damage, indicating a crucial role for this type of modification. DJ-1 modified at Cys106 by oxidative stress, and pathogenic mutations of DJ-1, bind parkin [108]. Parkin stabilizes the protein, preventing its dimerization. However, parkin does not appear to catalyse the ubiquitination of DJ-1 [108].

DJ-1 also stabilizes Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2), the master regulator of antioxidant transcriptional responses, by preventing its association with its inhibitor protein, Keap1 (Kelch-like enoyl-CoA hydratase-associated protein 1) [188]. Binding of the latter promotes Nrf2's ubiquitination and degradation [189]. Whether CHIP and parkin have a role in this latter activity remains to be established.

How does one of the most recently identified familial genes, the GTP kinase, LRRK2 (leucine-rich repeat kinase 2)/dardarin fit into these pathways [15,190,191]? We do not know at the present time. We will not know the answer until its endogenous substrates have been identified, and its role in cellular processes such as control of dendrite length have been determined [13,15,190194]. However, it interacts with parkin [195], and, similarly to α-synuclein, it interacts with membrane structures [196,197]. The most common disease-related mutation appears to result in an increase in kinase activity [192,194]. It will be interesting to discover whether phosphorylation of the physiological substrates of PINK1 or LRRK2 regulate their UPS-mediated degradation.

REMOVAL OF PROTEIN AGGREGATES

Perhaps it is not surprising that cells have developed mechanisms to protect large multiprotein complexes once they have formed. For example, Güngör et al. [198] recently demonstrated that the LIM homeodomain complexes are protected from proteasomal degradation by interaction with stabilizing cofactors. Other large complexes such as the mitochondrial OXPHOS complex I may be protected in a similar way by some of its components.

However, cells have also developed the ability to recognize misfolding complexes with the aid of chaperones and the UPS. When the capacity of the chaperone–UPS system is exceeded, or unable to process these substrates, specific cellular mechanisms are activated in an attempt to nullify the consequences of their presence. Cells package unwanted material into structures such as aggresomes [60] (e.g. see movies in [199]). Aggregating material is subsequently transported via microtubules to the mitotic organizing centre where it is surrounded by a ‘vimentin cage’. Only when inclusions reach a certain physical size do they become problematical. In mammalian cells, autophagy may then be activated, which may lead to cell death [200,201]. The potential importance of lysosomal-mediated processing in the development of PD is highlighted by the identification of mutant isoforms of the lysosomal ATPase, ATP13A2, in a hereditary form of PD [16].

Lewy bodies are intracellular inclusions, and neurons appear incapable of removing them using the UPS or chaperone systems. So what is their fate? Perhaps Lewy bodies or autophagic remnants of Lewy bodies that are released from dying neurons may be engulfed and degraded by microglia causing localized increased immune activity and cytokine release. Although we can only speculate whether this is a factor in the development of PD, there is a growing body of evidence suggesting an immune involvement [202204]. Indeed, increased numbers of CD4+CD25+ regulatory T-cells can provide protection against the dopaminergic neurotoxin MPTP in mice [205].

LINKING ABNORMAL PROTEIN PROCESSING AND MITOCHONDRIAL ACTIVITY

A significant number of neurological diseases are associated with defective mitochondrial function (see review [206]). Early studies in PD patients have indicated that inappropriate mitochondrial activity may have a role in the development of PD. For example, cybrid cells, created from the insertion of mitochondria from PD patients into mitochondrial-depleted rat dopaminergic or SHSY5Y dopaminergic cells, generated Lewy body-like inclusions and recapitulated deficits in mitochondrial OXPHOS activity [207]. The cause of these deficits was not established.

Subsequently, many in vivo studies, on both genes linked to hereditary forms of PD and dopaminergic toxins, appear to indicate a link between abnormal protein processing and mitochondrial activity (e.g. [2528,30,31,33,36,43,46,47,49,174,179,208211] and see [206] for a review). However, it is not obvious at this stage how the two are linked, although lower proteasomal activity would probably affect mitochondrial fission and fusion, a process dependent on the UPS [212].

Recent data from in vivo, in vitro and toxicology studies suggests that dopaminergic neurons are particularly sensitive to reduced levels of mitochondrial OXPHOS complex I activity, reasons for which are not currently understood ([25,26,30,31,36,47,209,213] and see [214] for a review). Environmentally damaging conditions may cause problems with mitochondrial complex assembly and stability, and, consequently, their activity [208]. For example, oxidative damage to one or more of the 46 subunits of OXPHOS complex I may affect their interactions with binding partners producing instability and functional deficits (Figure 4) [215]. A further contributing factor could relate to the need to import the majority of the nuclear-encoded components of these complexes (Figure 4). As they are nuclear-encoded, their synthesis and delivery to mitochondria may be affected in an environment of cytoplasmic protein aggregation.

Composition of OXPHOS complexes of mitochondria

Figure 4
Composition of OXPHOS complexes of mitochondria

The five multiprotein OXPHOS complexes are situated in the inner membrane of mitochondria [I–IV and ATP synthase (F1/Fo)]. Also displayed is coenzyme Q (CoQ) and cytochrome c (CytC). The protein components of each of the complexes are either nuclear (n) or mitochondrial (m) -encoded; numbers of each are indicated. The alternative names for complexes I–IV are indicated. The transport of H+ ions across the membrane creates a proton gradient which drives the production of ATP via ATP synthase. A by-product of the electron flow (red wavy arrows) is the production of superoxide radicals (O2•−)/reactive oxygen species. The activity of complex I is inhibited by environmental toxins such as rotenone and MPTP. Components of the complex are also found to be oxidatively modified in PD brains [208]. How the assembly or removal of (damaged) nuclear-encoded components of these complexes is affected by cytoplasmic inclusions remains to be determined. (also see http://neuromuscular.wustl.edu/pathol/diagrams/mito.htm).

Figure 4
Composition of OXPHOS complexes of mitochondria

The five multiprotein OXPHOS complexes are situated in the inner membrane of mitochondria [I–IV and ATP synthase (F1/Fo)]. Also displayed is coenzyme Q (CoQ) and cytochrome c (CytC). The protein components of each of the complexes are either nuclear (n) or mitochondrial (m) -encoded; numbers of each are indicated. The alternative names for complexes I–IV are indicated. The transport of H+ ions across the membrane creates a proton gradient which drives the production of ATP via ATP synthase. A by-product of the electron flow (red wavy arrows) is the production of superoxide radicals (O2•−)/reactive oxygen species. The activity of complex I is inhibited by environmental toxins such as rotenone and MPTP. Components of the complex are also found to be oxidatively modified in PD brains [208]. How the assembly or removal of (damaged) nuclear-encoded components of these complexes is affected by cytoplasmic inclusions remains to be determined. (also see http://neuromuscular.wustl.edu/pathol/diagrams/mito.htm).

Studies on two genes associated with familial disease, PINK1 and parkin (Table 1), also suggest a link between proteins involved in protein processing, mitochondria and cellular stress. PINK1 is a mitochondrial kinase [14,30,32,33,42,210,211]. Recent data indicate that the mitochondrial-associated serine protease HtrA2 is regulated by PINK1, and that both proteins appear to be components of the p38 stress-response pathway [211]. Consequently, reduced activity of PINK1 as a result of mutation will affect mitochondrial processing of proteins by HtrA2. As overexpression of parkin protects against PINK1-induced deficits in Drosophila, and DJ-1 and parkin also interact in response to stress, then the activities of parkin, PINK1, HtrA2 and DJ-1 may be linked in the same cellular stress process [30,33,108,210].

FUTURE PERSPECTIVES

Why do we not all develop PD? Perhaps we all would if we lived long enough. Why do some develop the disease earlier than others? Perhaps it is a chance event. Alternatively, it may relate to a complex genetic susceptibility involving the activity of a number of genes whose functions are related to an individual's ability to deal with cellular stress. Why dopaminergic neurons? These cells may be particularly sensitive due to a combination of a reduced ability to deal with aggregating proteins and additional problems caused by the inherently chemically unstable neurotransmitter dopamine.

Are Lewy bodies toxic or does their presence reflect a bystander effect of some other underlying problem in the cell? Are intermediate-size α-synuclein pre-inclusion structures or ‘protofibrils’ actually the toxic entity? If they are, why does their presence not result in cell death before Lewy bodies form? Or are Lewy bodies needed to support the presence of the protofibrils in cells? Do they interfere with either proteasomal activity or mitochondrial function or both? If they do, what is the mechanism of action? Is inclusion body toxicity due to its relatively large physical size in relation to normal neuronal cytoplasmic volume and mechanically disruptive to normal cellular function? Is the cellular response to the presence of Lewy bodies an activation of macroautophagy and apoptotic cell death? What is α-synuclein's role in the development of sporadic PD? Is it a gain-of-toxic-function resulting from post-translational modifications and high levels of expression that may or may not be related to its ability to aggregate? What is the relevance of the phosphorylation, ubiquitination and limited C-terminal processing? Do these modifications affect the function of α-synuclein?

The list of questions about what leads, specifically, to the death of dopaminergic neurons eventually leading to sporadic PD appears to be endless. However, with the identification of genes involved in hereditary forms of PD, and the analysis of their function, together with information from toxicology studies, research is now focused on understanding the relationship between lower levels of proteasomal activity, inappropriate protein processing, mitochondrial activity and increased reactive oxygen species, and the presence of Lewy bodies. When we understand the details of the link between these activities, we will be a long way to understanding the underlying cause of the disease, and, most important, developing a strategy to treat it.

Understanding the link between these pathways should also help in the design of in vivo model systems that accurately replicates the process of development of sporadic disease. In addition to dopaminergic neurons, other neuronal cells are also characteristically lost in PD. The ability to recapitulate the development of sporadic PD in a model system would represent a major step forward for the identification of new therapeutic agents. Although many of the model systems currently employed replicate some aspects of the disease process, and have been instrumental in demonstrating some of the key processes that may be involved in sporadic disease, none is ideal.

The number of manuscripts relating to the causes of PD is vast. I apologize to those many distinguished authors, who have been working in the field much longer than I have, and whose references I have not quoted. I have used references that I have found most useful in my studies trying to understand the link between the UPS, protein aggregation, mitochondrial activity and PD. Many thanks to Helen Ardley, Kyla Pennington and Nicola Robinson for their help with preparing the Figures. I also thank the Parkinson's Disease Society of Great Britain for funding our work.

Abbreviations

     
  • AR-JP

    autosomal recessive juvenile Parkinsonism

  •  
  • BAG

    Bcl-2-associated athanogene

  •  
  • DUB

    de-ubiquitinating enzyme

  •  
  • ER

    endoplasmic reticulum

  •  
  • ERAD

    ER-associated degradation

  •  
  • ERAD-L

    ERAD targeting substrates with luminal mutations

  •  
  • ERAD-M

    ERAD targeting substrates with membrane mutations

  •  
  • GRK5

    G-protein-coupled receptor kinase 5

  •  
  • HDAC

    histone deacetylase

  •  
  • HRD1

    HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase degradation protein 1

  •  
  • Hsc70

    heat-shock cognate 70

  •  
  • CHIP

    C-terminus of Hsc70-interacting protein

  •  
  • HSF1

    heat-shock transcription factor 1

  •  
  • Hsp

    heat-shock protein

  •  
  • LRRK2

    leucine-rich repeat kinase 2

  •  
  • MPTP

    1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

  •  
  • Nrf2

    nuclear factor-erythroid 2 p45 subunit-related factor 2

  •  
  • OXPHOS

    oxidative phosphorylation

  •  
  • PAEL-R

    parkin-associated endothelin receptor-like receptor

  •  
  • PD

    Parkinson's disease

  •  
  • PINK1

    PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced kinase 1

  •  
  • RING

    really interesting new gene

  •  
  • IBR

    in-between RING

  •  
  • Ubl

    ubiquitin-like

  •  
  • UCH-L1

    ubiquitin C-terminal hydrolase L1

  •  
  • UPR

    unfolded protein response

  •  
  • UPS

    ubiquitin–proteasome system

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