Immunological features of α-synuclein in Parkinson's disease

Abstract

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized pathologically by the presence, in the brain, of intra-cellular protein inclusions highly enriched in aggregated α-synuclein (αSyn), known as Lewy bodies. The onset of PD is accompanied by a local immune reaction in regions of the brain affected by the inclusions, although the mechanism that leads to pathogenesis is far from clear. It is, however, established that disease onset and progression are characterized by sustained activation of microglia, which is linked to significant dopaminergic neuron loss in the substantia nigra. A recent body of evidence indicates that aggregated or modified αSyn can indeed trigger the activation of microglia, inducing a lethal cascade of neuroinflammation and eventually, neuronal loss, pointing at aggregated and modified forms of αSyn as a primary cause of PD pathogenesis. By releasing toxic factors, or by phagocy-tosing neighbouring cells, activated microglia and astrocytes may form a self-perpetuating cycle for neuronal degeneration. Additional findings suggest a link between αSyn and humoural-mediated mechanisms in PD. In this review, we attempt to recapitulate our current understanding of PD physiopathology focused on αSyn and its links with the immune system, as well as of novel and promising therapeutic avenues for the treatment of PD and of other synucleinopathies.

• Introduction

• Importance of inflammation processes in PD pathology

• Stimulation of microglia by αSyn

• αSyn-triggered stimulation of the innate immune system

• Other proteins up-regulated by αSyn-triggered microglial activation

• αSyn and apoptosis of immune cells

• Links between αSyn and astrocytes or oligodendrocytes

• αSyn and the humoural immune system in PD

• Expression of αSyn in immunocompetent cells

• Prospects for αSyn- and immune-based therapeutic approaches in PD

• Concluding remarks

Introduction

Parkinson's disease (PD), a progressive neurodegenerative disorder characterized by resting tremor, muscular rigidity and gait disturbances [1, 2], is pathologically characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars com-pacta (SNpc) and their termini in their dorsal stratium [3]. The pathological hallmark of PD is the presence of deposits of aggregated α-synuclein (αSyn) in intracellular inclusions known as Lewy bodies (LB) [4, 5]. Three missense mutations, A53T, A30P and E46K, as well as multiple copies of the wild-type (Wt) αSyn gene, are linked to familial PD, which is often manifested in early onset of the disease [6–9]. However, the factors contributing to sporadic PD, which represents the majority of PD cases, are not known, and in either case, the cellular and molecular mechanisms underlying the pathological actions of αSyn are not well understood.

αSyn, together with β- and 7-synucleins, belong to the expanding family of synucleins, a group of closely related, brain-enriched proteins. αSyn is a 140-amino acid protein that is highly expressed in pre-synaptic terminals, in particular in the neocortex, hippocampus and SN [10], but is also found in other regions of neurons as well as within astrocytes and oligodendroglia [11, 12]. It is known to interact with a variety of proteins [13, 14] and also with lipid vesicles [15], and it may be involved in lipid metabolism [16, 17]. In its free state αSyn is intrinsically disordered, with no well-defined structure as determined in vitro, although NMR studies have shown long-range interactions between the acidic C-ter-minal region and the amyloidogenic central region [18–20]. Interactions with acidic phospholipids membranes result in induction of helical conformation in its N-terminal region [21, 22].

The physiological functions of αSyn are still being established. Its interaction with pre-synaptic membranes suggests that one function may be the regulation of synaptic vesicle pools, including dopamine control [23]. A role as a molecular chaperone, assisting in the folding and refolding of certain synaptic proteins, was also proposed [24]. Although αSyn is normally considered as a cyto-plasmic protein, it has also been found to be present in extracellular biological fluids, including human cerebrospinal fluid and blood plasma [25, 26]. One mechanism that leads to the presence of extracellular αSyn is thought to be membrane permeability as a result of cell death, although it has also been reported that monomeric and aggregated αSyn may be secreted by an unconventional endoplasmic reticulum/Golgi-independent exocytosis pathway [26].

αSyn can self-assemble in vitro to form ordered fibrillar aggregates, characterized by a cross β-sheet structure, that are morphologically similar to the aggregates found in LB, in neuritic plaques in Alzheimer's disease (AD) as well as in deposits associated with other amyloidogenic processes (reviewed in [27]). A significant international effort has been made to elucidate the biophysical basis for the aggregation of αSyn [28, 29]. The initial phase of the aggregation process is thought to involve the formation of oligomeric species which, according to accumulating experimental evidence, are more toxic to cells than the mature fibrils into which they develop [30, 31]. These and other findings suggest a common structure-linked toxicity among pre-fibrillar species, and it has been proposed that similar mechanisms may in general contribute to pathogenesis for this group of diseases [32, 33]. Overall, many hypotheses have been put forward that propose that αSyn induces a ‘gain of toxic function’ upon aggregation [27].

Importance of inflammation processes in PD pathology

Inflammation is the first response of the immune system to pathogens. In acute conditions, it protects tissue against invading agents and promotes healing. However, when sustained chronically it can cause serious damage to the host's own tissue [34]. Although the central nervous system (CNS) has been traditionally seen as an immune-privileged organ, it has become increasingly evident that inflammation is actively involved in the pathogenesis of many degenerative diseases including multiple sclerosis (MS), AD, and PD (see references in [34]). A robust and highly localized inflammatory response mediated by reactive microglia and reactive astrocytes is prominent in affected areas of the SN in PD brains (reviewed in [34]).

Microglia are the main immunocompetent cells within the CNS [35], capable of antigen presentation to lymphocytes [36] and rapid activation in response to pathological change in the CNS [34]. Microglial cells are evenly distributed throughout the normal brain, in close proximity to neurons and astrocytes. At the site of inflammation, activated microglia change their morphology express increased levels of major histocompatibility complex (MHC) antigens and become phagocytic [37, 38]. In addition, they start releasing inflammatory cytokines that amplify the inflammatory response by activating and recruiting other cells to the brain lesion [34]. Microglia can also release potent neurotoxins, which may cause neuronal damage, and, indeed, sustained overactiva-tion of microglia has been observed in a variety of neurodegener-ative diseases [34].

Evidence of microglial attack in PD is supported by findings within three different areas of research: epidemiological studies, animal models and cells in culture [39]. Epidemiological studies that investigated the effects of using anti-inflammatory agents showed that taking ibuprofen regularly was associated with a 35% lower risk of PD [40, 41], supporting the concept that inflammatory attack is contributing to dopaminergic neuronal loss. In vivo findings show that the specific early up-regulation of SN microglia in PD correlates with disease severity and dopamine terminal loss, but not with disease duration [42, 43]. This correlation may not be unexpected if one considers that dopaminergic cells of the CNS are highly vulnerable to oxidative and inflammatory attack. Indeed, the animal models of PD currently in use are based on oxidative stress or inflammatory stimulation to the SN area (reviewed in [39]).

The animal models of PD are generally of either of two types; ‘type 1’ is based on the administration of oxidizing compounds that are preferentially taken up by dopaminergic cells (e.g. rotenone, 6-hydroxydopamine); and ‘type 2’ is based on localized administration of inflammatory agents, mainly lipopolysaccharide (LPS), 1-methyl-4-phenyl-1,2,3,4-tetrahyfropyridine (MPTP) or αSyn [39]. The MPTP model indicates that inflammation in the SN can be self-sustaining whereas the αSyn model indicates that overexpression of this endogenous protein can provide a source of inflammation [39]. In addition, the transgenic mouse models for PD that have been described utilize neuron-specific promoters to overexpress Wt or mutant αSyn locally (reviewed in [44, 45]), and have been shown to capture major features of PD such as locomotor defects, the formation of inclusion-like structures and neurotoxicity. Studies with both animal model and cells in culture have shown, albeit indirectly, that dopaminergic cells are highly sensitive to inflammatory attack [45, 46] and that microglial cells can be activated to mount such an attack [47].

Stimulation of microglia by αSyn

Several studies have demonstrated that extracellular and nigral aggregates immunoreactive to αSyn are often surrounded by activated microglia or inflammatory mediators [48, 49]. This phenomenon mirrors what has been described in AD, where amyloid plaques are usually co-localized with clusters of activated microglia [50]. Microglial cells from αSyn knockout mice have been shown to exhibit a remarkably different morphology compared to Wt cells [47]. Moreover, after activation, the microglial cells secrete elevated levels of pro-inflammatory cytokines, such as tumour necrosis factor (TNF)-α and interleukin (IL)-6 [47], indicating that αSyn plays a critical role in modulating the activation state of microglia. Still, the mechanisms underlying microglial activation in PD, and how the latter affects neuronal survival remains poorly understood. One line of investigation posits that neuronal death itself drives the microglial immune response [51–53], but others have proposed that activation could occur as a consequence of release of aggregated protein from the cytosol or within LB to the extracellular space. In such a situation, the death of dopaminergic neurons would lead to the release of protein aggregates that would in turn activate microglia, inducing a lethal cascade of neuroinflammation and neuronal demise [54–56]. Therefore, although PD is not an autoimmune disease, evidence of localized attack by microglia places it in the autotoxic category [39].

Several recent in vitro studies have focused on the effects of extracellular αSyn on microglial activation (Table 1). Zhang et al.[54] first reported that exogeneous, aggregated αSyn activates microglial cells, which then become toxic towards cultured dopaminergic neurons. This is particularly relevant, because aggregated αSyn has been shown to be secreted by exocytosis from neuronal cells [26] although it might also be released by membrane permeability from dead cells [57]. The study found that microglial phagocytosis of αSyn and activation of NADPH oxidase were critical in microglial activation induced by aggregated αSyn, and neurotoxicity [54]. The toxicity level was lower in mice null for NADPH oxidase, indicating that oxygen-free radicals generated by the activated microglia, are likely to play a significant role in neurotoxicity. It was also reported that induction of NADPH oxidase is linked to direct activation of the Mac-1 receptor, and not by αSyn internalization via a scavenger [58]. Finally, it was proposed that nigral neuronal damage, regardless of its aetiology, might release aggregated αSyn, which could then lead to persistent and progressive neuronal damage [58].

Table 1.

Activation profile of αSyn-stimulated glial cells

αSyn	Co-stimulation	Cytokines, receptors or proteins affected	Pathways involved	Stimulated cells	Effect	Ref.
Wt, A30P, E46K, A53T	IFN-gamma;	↑ ICAM-1, ↑T IL-6	P38, JNK, ERK1/2, MAPK	Human astrocytes and U-373 MG astrocytoma cells		[93]
Wt, A30P, E46K, A53T, Δ71–82	IFN-gamma;	↑ TNF-α (but only A53T w/o IFN∼y), ↑ IL-1β		Human microglia	Reduced monocytic cell viability, but only with IFN-gamma;	[57]
Wt	IFN-gamma;		P38, JNK, ERK1/2, MAPK	Human microglia	↓ Viability dopaminergic cells	[57]
Aggregated Wt		↑ Extracellular O2, ↑ Intracellular ROS, ↑ PEG2	NADPH oxidase	Rat primary mes-enchepalic neuron-glia cell culture	↓Dopamine uptake, cell loss, morphological alterations of dopaminergic cells	[54]
wt			NADPH oxidase Binding Mac-1	Rat primary mixed neu-ron-glial cell culture	↑ O2−	[58]
A30P					↑ Intracellular ROS
A53T
Aggregated, nitrated				Microglia (C57BL/6J mice)	↑ H2O2	[60]
Aggregated versus non-aggregated, nitrated Wt		↓Actin, galectin 3 and 14-3-3 sigma	NF-kB	Microglia (C57BL/6J mice)		[56]
		↑ Biliverdin reduc-tase calmodulin and ferritin light chain
		↑ Glutamate and extracellular Cys
		↓ Intracellular glutamate and intracellular Cys and GSH (No changes with unaggregated N-αSyn)
Aggregated, Nitrated Wt		↑ TNF-a	NF-kB (↑ mRNA of Tnf, Ccl2,116, II-β, Nfkb)	Microglia (C57BL/6J mice)	↑ Dopaminergic cell death (less for non-nitrated, only with aggregated αSyn)	[61]
		↑ IL-6, ↑ MCP-1, ↑ IFN-gamma;	MAPK (↑ mRNA of Fos, Raf1)
			↑ Hsp70, SOD, Peroxiredoxins 1, 4, and 5
			↓ Aconitase and ↑ calmodulin
			β-actin, L-plas-tin, α-tubulin

Several works have concluded that the mutated, disease-causing forms of αSyn are more potent stimuli of microglial activation than the Wt protein, indicating a possible molecular mechanism for the increased toxicity of the αSyn mutants linked to familial PD [57]. Likewise, it has been shown that aggregated αSyn has a stronger stimulating effect on microglia [56] than that of non-aggregated αSyn (Table 1). Recent investigations demonstrated that aggregated αSyn induces a neurotoxic inflammatory microglial phenotype that accelerates dopaminergic neuron loss [54, 56, 59, 60]. By integrating genomic and proteomic techniques, Gendelman and coworkers [61] created a fingerprint of microglial cell activation following its interactions with aggregated, nitrated N-αSyn (N-αSyn) – previously found to form oligomers through dityrosine crosslinking [62]. They observed a neuroinflammatory phenotype that was capable of mediating neuronal toxicity that correlates with human disease (Table 1). These results appear relevant because αSyn proteins nitrated at four tyrosine (Tyr) positions have been detected in LB of human brains with PD [51]. It would be interesting to pursue analogous studies with other αSyn forms that are post-translationally modified and also found in LB, e.g. C-terminally truncated, or serine (Ser)¹²⁹-phosphorylated αSyn (reviewed in [63]).

αSyn-triggered stimulation of the innate immune system

Upon activation, microglia and astrocytes can secrete neurotoxic products and inflammatory cytokines [39]. The latter ones are produced in order to communicate and orchestrate the immune response to disease, or injury, often by inducing proliferation [64]. The cytokines TNF-a, IL-1β, IL-2, IL-4, IL-6, tumour growth factor (TGF)-a, TGF-β1, TGF-p2 have all been reported to be present at higher levels in the nigrostriatal region and cerebrospinal fluid of patients with PD or dementia with LB ([64] and references therein). Activated microglia may also produce large amounts of superoxide radicals, which may be the major source of the oxidative stress believed to be largely responsible for dopaminergic cell death in PD.

A number of cytokines and metabolites have been shown to be significantly up-regulated as a result of αSyn-induced activation of microglia in vitro (Table 1), including IL-1 β, IL-6, intercellular adhesion molecule (ICAM)-1, TNF-α, interferon (IFN)-y, MCP-1, O2⁻, iROS, and PEG₂, glutamate and iCys. Activation appears to be mainly mediated by the mitogen-activated pathway (MAP) kinase, NADPH (shown for stimulation with aggregated N-αSyn), and NF-κB, pathways (Table 1). In general, disease-linked αSyn mutants show a stronger effect on cytokine release than does the Wt protein. It may also be relevant that, under some conditions, αSyn tested variants require the presence of IFN-γ in the medium to effectively induce microglial activation or cytotoxicity (Table 1), indicating a synergy between this cytokine and αSyn. Contrary to the increase in nitric oxide species (or nitric oxide synthetase) observed for LPS-stimulated neurons or microglial cells [65–67], aggregated αSyn-treatment of microglia did not seem to significantly alter nitrite levels [54] (Table 1). Interestingly, analysis of the microglia transcriptome by Gendelman and coworkers [61] after stimulation with aggregated N-αSyn, revealed a significant up-regulation of the toll-like receptor 2 (TLR-2) gene. TLRs sense the molecular signatures of microbial pathogens, and play a fundamental role in innate immune responses, inducing the expression of diverse inflammatory genes (for a review, see [68]). It therefore seems plausible that cells challenged with αSyn, or at least with certain forms of αSyn, could become hyper-responsive to inflammatory signals.

The generation of reactive oxygen species (ROS) by microglia activated by αSyn [60] (or other stimulants) can result in oxidation and nitration of proteins, DNA modification, and lipid peroxidation, leading to neurotoxicity [54]. Oxidation [62, 69] and nitration [51, 62] of αSyn can in turn, lead to the formation of more aggregates, and hence result in increased cytotoxicity. Consistent with this, Bosco et al. have shown that high levels of oxidized cholesterol metabolites in brains from PD and dementia with LB patients, accelerate the conversion of soluble αSyn into amyloid fibrils [70].

Recently, McGeer and coworkers [71] found that human microglia constitutively express ryanodine receptors (RyRs), which help to mediate the efflux of Ca²⁺ ions from intracellular stores. Elevated levels of free intracellular Ca ⁺ ([Ca ⁺]i) lead to Ca²⁺ signals that may initiate both short- and long-term cellular responses, and indeed sustained and uncontrolled [Ca²⁺]i increases can lead to cell death (for a review, see [72]). Interestingly, αSyn stimulation of microglia, in combination with IFN-γ, has been found to induce toxicity of human monocytic cells by producing neurotoxic secretions, and this toxicity can be diminished with specific RyR ligands [71].

Other proteins up-regulated by αSyn-triggered microglial activation

Reynolds et al.[61], by determining the activated microglia pro-teome profile, found that aggregated N-αSyn activation of microglia results in differential expression of several proteins (Table 1). These range from proteins involved in oxidative stress, cell adhesion, glycolysis, regulation of growth, and migration, to proteins of the cytoskeleton. It is intriguing that two of those proteins found to be particularly highly up-regulated, calmodulin and ubiquitin, have been shown to interact with αSyn with possible functional consequences. Calmodulin has been shown, in vitro, to bind to αSyn in a Ca²⁺-dependent manner [73] and to inhibit fibrillation of αSyn [74]. Several studies have reported that a fraction of αSyn found in LB is mono-ubiquitinated [75, 76], but the role of this modification remains unclear. Recently, it has been demonstrated that the ubiquitin-protein isopeptide ligase, seven in absentia homologue, directly interacts with and monoubiquiti-nates αSyn, promoting its aggregation [77, 78] and stimulating apoptosis [78]. There is also evidence implicating a role for the ubiquitin-proteasome system (UPS) in PD (reviewed in [79]), linking some parkin mutations to UPS aberrations and altered protein degradation. The role of αSyn in UPS impairment is less clear, although it has been reported that overexpression of αSyn (in particular the disease-associated mutants) or an aggregated form of Wt αSyn, can inhibit the proteasome function [80–83]. Also of interest in activated microglia expression profile are the elevated levels of Hsp70. This chaperone has been demonstrated to inhibit αSyn aggregation in vitro[84], in neuroglioma cells [85], as well as in fly [86] and mouse [85] models of PD, protecting cells from the cytotoxic effects of aggregates.

αSyn and apoptosis of immune cells

In PD patients, disturbed cellular and humoural functions in the peripheral immune system have been described, including the occurrence of auto-antibodies (AAbs) against neuronal structures and the presence of a high number of microglial cells expressing the histocompatibility leukocyte (antigen HLA-DR) in the SN [87]. In addition to cytokines, apoptosis-related proteins are elevated in the stratium of PD patients [88, 89].

While searching for a link between the CNS and the peripheral immune system in PD, Kim et al.[90] observed that αSyn was up-regulated in peripheral blood mononuclear cells at the gene level, in idiopathic PD versus non-PD controls. Moreover, by in vitro transfection with Wt, A30P and A53T αSyn genes, they found that αSyn expression is correlated to glucocorticoid-sensi-tive apoptosis, possibly caused by the enhanced expression of glucocorticoid receptor, caspase activation, CD95 (Fas) up-regu-lation and ROS production. However, the increase in ROS production by overexpression of the αSyn mutants was markedly greater than for the Wt protein. It has also been reported that overexpression of C-terminally truncated αSyn in transfected astrocytes, especially when treated with TNF-α, induces cell death by apoptosis [91].

Links between αSyn and astrocytes or oligodendrocytes

Compared to microglia, the functions of astrocytes are poorly understood. These cells migrate to a site of injury and develop hypertrophic morphology. As opposed to microglia, they are thought not to attack a pathological target, but rather to seal it off. Because they have been shown to elaborate both pro- and anti-inflammatory agents, these cells appear to have a dual role in the immune homeostasis [39]. Many ICAM-1 positive astrocytes are seen in the SN of the brains of PD patients and this phenomenon may attract reactive microglia to the area because microglia carry the counter receptor LFA-1 [92]. Indeed, αSyn is capable of stimulating astrocytes to produce IL-6 and ICAM-1 [93] (Table 1). The action of αSyn on astrocytes is believed to be through receptors, but the identity of the latter is currently unknown; however, antagonists of such putative αSyn receptors might constitute novel PD-specific anti-inflammatory agents. Finally, astrocytes have also been shown to secrete a number of neurotrophic factors that protect dopaminergic neurons in some models of PD ([39] and references therein), but the mechanisms underlying most of these functions are not yet known.

There is very little data on oligodendrocytes in PD, although Yamada et al. have reported the presence of complement-activated oligodendrocytes in the SN of PD cases [49]. As in astrocytes [94], αSyn-containing inclusions have been reported in oligodendrocytes [94, 95], both in dementia with LB and in PD.

αSyn and the humoural immune system in PD

The observation in PD patients that small numbers of CD8⁺ T lymphocytes occur in proximity to degenerating nigral neurons [48] and that components of the classical or antibody-triggered complement cascade occur in LB [49], suggests that the pathological process may involve humoural-mediated mechanisms [43]. In addition, humoural immune mechanisms can trigger microglial-mediated neuronal injury in animal models of PD [96]. To analyse the possibility that humoural immunity may play a role in initiating or regulating inflammation, Orr et al.[43] analysed the association between nigral degeneration and humoural immune markers in brain tissue from patients with idiopathic or genetic PD and controls. All the patients with PD had significant levels of immunoglobulin G (IgG), but not of IgM, binding, on dopamine neurons. Moreover, the proportion of IgG-immunopos-itive neurons showed a negative correlation with the degree of cell loss in the SN, and a positive correlation with the number of activated microglia. IgG was found to be concentrated at the cell surfaces of neurons, but also on their LB, and was shown to co-localize with αSyn. These results, in combination with the finding that activated microglia express high-affinity IgG receptors (FcγRI) in both idiopathic and genetic forms of PD, could suggest that the activation of microglia may be induced by neuronal IgG [43]. Even though the identity of the antigen or antigens responsible for IgG binding to dopamine neurons remains unknown, it is possible to argue that IgG binding to dopamine neurons in PD may result in their selective targeting and subsequent destruction by activated microglia [43].

A possible consequence of the initial microglial activation in the affected regions of PD brains is the local permeabilization of the blood-brain barrier, leading to infiltration to the affected regions by B and/or T lymphocytes, and believed to constitute a critical step in the development of autoimmune reactions [97]. To explore the possible involvement of αSyn in steps that go beyond the initiation of the local immune response in PD, Papachroni et al.[98] have assessed the presence of AAbs against all three synucleins in the peripheral blood serum of PD patients and of healthy control individuals. Although the presence of AAbs against β- and γSyn showed no correlation with PD, AAbs against αSyn were detected in 65% of all patients. Moreover, the presence of these AAbs strongly correlated with inherited forms of the disease, but not with the sporadic form. The observation that the AAbs generated are multi-epitopic, confirms that the entire αSyn molecule is auto-immunogenic, and eliminates the possibility that the observed immune reaction could be the result of cross-reactivity with another, similar antigen [98].

The question regarding the functional importance of antibodies against disease-associated neuronal proteins remains wide open. It has been demonstrated that an IgG fraction purified from the serum of PD patients causes the death of dopaminergic neurons in vivo following stereotaxic injection into the SN of experimental animals [99], and the presence of immunoglobulins in PD brain tissue could lead to the targeting of dopaminergic nigral neurons for destruction [43]. Currently, whether or not these anti-αSyn AAbs are neurotoxic, or by contrast, they have a neuroprotective role as shown in a human αSyn transgenic mouse model of PD [100], remains unknown. Future studies aimed at clarifying a role for anti-αSyn AAbs, should evaluate their potential for diagnosis and therapy of PD [98].

Expression of αSyn in immunocompetent cells

It has been reported that αSyn is also expressed in astrocytes and that its level is increased by stimulation with the pro-inflammatory cytokine IL-1 β[101]. Also, αSyn has been found to be expressed in cultured human macrophages [102]. In this case, αSyn protein (but not mRNA) levels were seen to be up-regulated by stimulation with LPS and IL-1 β[102], further supporting a role for αSyn in the inflammatory process. Macrophages are known to participate in diverse biological processes, including the phagocytosis of pathogens and debris, antigen presentation, and regulation of the immune response through cytokine production.

It has been reported that αSyn expression in peripheral blood mononuclear cells of PD patients is significantly up-regulated, compared to healthy non-PD controls [10]. In addition, protein expression of αSyn in cultured human T cells, B cells, natural killer cells and in monocytes/macrophages, have been reported [103]. Currently, it is not known whether expression, or aggregation, of αSyn in T cells is regulated by ligand activation of these cells, an important issue as it could identify a key link between acquired immunity regulation and αSyn expression.

Prospects for αSyn- and immune-based therapeutic approaches in PD

αSyn is increasingly becoming a primary target for understanding and controlling the onset and pregression of PD. As misfolding and aggregation of αSyn into specific toxic morphologies are essential for the progression of the disease, prevention of aggregate accumulation is an important potential therapeutic strategy. Interactions with protein targets, lipid vesicles, transition metals and other small molecules have all been explored [104, 105] with a view towards developing strategies to control the aggregation of αSyn and its variants. Both β- and 7Syn have been reported to be inhibitors of fibril formation by αSyn [106, 107], and short pep-tides directed at the central portion of αSyn have also been shown to inhibit aggregation and to reduce its toxicity [105]. Additionally, as mentioned, treatment with chaperone Hsp70 has been shown to inhibit αSyn fibril formation and/or to reduce the aggregates toxicity, in animal models of PD [85, 86]. Another possible therapeutic strategy to combat protein-deposition disorders, including PD, could be to produce ‘superproteins’, or more soluble versions of the aggregating proteins [108]. Such added modified proteins would reduce the tendency of their natural counterparts to aggregate, while remaining compatible with their cellular environment and their function [108].

An interesting strategy is the generation of specific anti-αSyn single-chain Fv (scFv) antibody fragments that bind either to the monomeric [109] or oligomeric [110] protein, and inhibit its aggregation. These scFvs can be generated such that they only target the toxic oligomeric form of αSyn, allowing the monomer to perform its normal function freely [110], and they can also potentially be expressed intracellularly (intrabodies) to counteract aggregation and reduce neurodegeneration, as recently shown with a neural progenitor cell line [111], and in an animal model of Huntington's disease [112].

Given that microglial activation can maintain or even aggravate the disease process, blocking inflammation or shifting the balance between pro-inflammatory and anti-inflammatory states in a controlled manner, offers one of the most promising strategies for developing palliative (and maybe preventative) therapies for PD and related disorders. Epidemiological data has identified the non-steroidal anti-inflammatory drug ibuprofen as neuroprotective for PD [113]. A variety of other, both endogenous and synthetic compounds that might suppress neuroinflammation in PD by interacting with microglia, have been identified and proposed for therapeutic use (reviewed in [113]).

Along the same lines, compounds that block other signal pathways that are switched on as a consequence of microglial activation, which may ultimately lead to neuronal apoptosis or degeneration, might also represent new targets for pharmacotherapeutic intervention.

Concluding remarks

In the last few years, it has become accepted that abnormal aggregation of αSyn is likely to be one of the primary causes of the immunological abnormalities observed in PD. The implication of αSyn in PD is supported by observations that (i) fibrillar aggregates of αSyn are the main constituents of LB, (ii) certain mis-sense mutations, as well as duplication or triplication of the αSyn gene, cause autosomal dominant PD and (iii) the principal molecular, cellular, immunological and pathophysiological aspects of PD can be recapitulated by expression of αSyn in neuronal cell lines or animal models. It is well established that onset and progression of PD are characterized by sustained activation of microglia, linked to significant dopaminergic neuron loss in the SN, and accumulated evidence has established that aggregated or modified αSyn can trigger the activation of microglia, inducing a lethal cascade of neuroinflammation and neuronal death. By releasing toxic factors, or by phagocytosing neighbouring cells, activated microglia and astrocytes may form a destructive cycle of self-perpetuating neuronal degeneration. In addition, recent findings suggest a possible link between αSyn, humoural-mediated mechanisms and the pathological events in PD. Prevention of αSyn aggregation and intervention in the mechanisms of microglial activation mechanisms appears therefore to be highly promising therapeutic targets for the treatment of PD and other synucleinopathies.