Are synucleinopathies prion-like disorders?

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Are synucleinopathies prion-like disorders? Elodie Angot*, Jennifer A Steiner*, Christian Hansen, Jia-Yi Li, Patrik Brundin Lancet Neurol 2010; 9: 1128–38 Published Online September 15, 2010 DOI:10.1016/S14744422(10)70213-1 *These authors contributed equally Neuronal Survival Unit, Wallenberg Neuroscience Centre, Lund University, Lund, Sweden (E Angot PhD, J A Steiner PhD, C Hansen PhD, J-Y Li MD, P Brundin MD) Correspondence to: Patrik Brundin, Neuronal Survival Unit, Wallenberg Neuroscience Centre, Lund University, BMC A10, 221 84 Lund, Sweden [email protected]

A shared neuropathological feature of idiopathic Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy is the development of intracellular aggregates of α-synuclein that gradually engage increasing parts of the nervous system. The pathogenetic mechanisms underlying these neurodegenerative disorders, however, are unknown. Several studies have highlighted similarities between classic prion diseases and these neurological proteinopathies. Specifically, identification of Lewy bodies in fetal mesencephalic neurons transplanted in patients with Parkinson’s disease raised the hypothesis that α-synuclein, the main component of Lewy bodies, could be transmitted from the host brain to a graft of healthy neurons. These results and others have led to the hypothesis that a prion-like mechanism might underlie progression of synucleinopathy within the nervous system. We review experimental findings showing that misfolded α-synuclein can transfer between cells and, once transferred into a new cell, can act as a seed that recruits endogenous α-synuclein, leading to formation of larger aggregates. This model suggests that strategies aimed at prevention of cell-tocell transfer of α-synuclein could retard progression of symptoms in Parkinson’s disease and other synucleinopathies.

Introduction Protein misfolding is central to the pathogenesis of both prion diseases and Parkinson’s disease and other related neurodegenerative disorders. Some neurodegenerative disorders, such as Parkinson’s disease, can be characterised by intracytoplasmic inclusions, named Lewy bodies.1 A prion-like spread of misfolded α-synuclein (the predominant protein in Lewy bodies)2 has been proposed to contribute to the propagation of Lewy bodies throughout the nervous system during progression of Parkinson’s disease.3–9 How do synucleinopathies compare with prion diseases? The distinguishing feature of prion diseases resides in the nature of the pathogen that spreads not only within the affected organism but also between and within human beings and animals. Unlike conventional pathogenic organisms, such as viruses, bacteria, or yeast, a protein was identified as the infectious and pathogenetic agent.10 Although there is no evidence so far to support interindividual or interspecies transmission of synucleinopathies, we discuss current evidence that α-synuclein could act in a prion-like manner to transfer between cells within an organism. Hence, we use the term prion-like to indicate that α-synuclein might share some features with prions, albeit without infectivity properties.

PrPSc

PrPc

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Figure 1: Model of templating and cell-to-cell transfer of prion proteins Exogenous PrPSc (red), acting as a seed, recruits intracellular PrPC (black) and converts its three-dimensional structure into an elongated PrPSc, thus extending the amyloid aggregate. The aggregates transfer intercellularly via an unknown mechanism.

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In this Personal View, we first briefly introduce current knowledge on the mechanisms of spread of prion disease within and between animals. Next, we review the central role of α-synuclein in Parkinson’s disease and other Lewy body-related disorders and discuss evidence suggesting that α-synuclein could indeed be classified as a protein with prion-like abilities. Furthermore, we identify gaps in knowledge that must be filled in order to state definitively that α-synuclein spreads within the nervous system in a prion-like manner in synucleinopathies. Finally, we discuss future research directions and their implications for the development of novel therapeutic strategies.

Prion propagation Prion diseases in animals, such as scrapie and bovine spongiform encephalopathy, are characterised by non-viral spread of diseased proteins from one animal to another. The disease-causing pathogens can be transmitted infectiously within and between species. Additionally, several familial and sporadic forms of human prion disease are well characterised, such as fatal familial insomnia and Creutzfeldt-Jakob disease.11 Once a human being or animal acquires a prion disease, prognosis is poor; patients with Creutzfeldt-Jakob disease typically die within a year after disease onset.12 Prion diseases are progressive and incurable, and the prions themselves are fairly resistant to methods that destroy viruses and bacteria.10,13 Genetic, dominantly inherited human prion diseases are caused by mutations in the prion protein (PrP) gene (PRNP).14–16 PrP is expressed ubiquitously and its functions are under active investigation;17,18 mice without PrP have grossly normal development but are resistant to scrapie.19–21 The typical cellular form of PrP is designated PrPc, to distinguish it from the alternatively folded and disease-related scrapie isoform (PrPSc). These two isoforms share the same aminoacid sequence but differ in their secondary structures: PrPc is rich in α-helices, whereas β-sheets dominate the structure of PrPSc.22,23 Additionally, PrPSc assumes different structures according to which prion disease it causes.11,24 www.thelancet.com/neurology Vol 9 November 2010

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During sporadic, genetic, or infectious means of disease acquisition, once the change in conformation that creates PrPSc takes place, this protein isoform acts as a template (also termed a seed), recruiting PrPc into aggregates and causing its conversion to PrPSc (figure 1). Details of this transition from PrPc to PrPSc are currently unclear, but antibody and computational studies could pave the way for ultrastructural determinations of intermediate PrP structures.25,26 Accumulation of aggregates of PrPSc and subsequent breakage of the amyloid chains results in generation of more seeds, which cause accelerated accumulation of PrPSc within cells and propagation between cells, leading to widespread accumulation of PrPSc and, thus, disease progression.24,27 The ability of the amyloid aggregates to break is vital; brittle prion strains are most toxic, because they contribute extra free ends for recruitment of PrPc and amyloid extension.24,27 Transfer of PrPSc from one cell to another can take place via various processes. Mechanisms mediated by receptors, exosomes, and tunnelling nanotubes have all been suggested to have a role.28–33 For example, neuronal LDL-receptor-related protein 1 mediates endocytosis of PrPSc from the extracellular space.31 Active research into exosomes has provided new insight into protein transfer mechanisms. Both PrPSc and PrPc can be released from non-neuronal29 and neuronal cells28,32,33 and may be associated with exosomes. Exosomes are small secreted vesicles that derive from the endosomal pathway.34 Thus, as a first step, proteins endocytosed at the plasma membrane are internalised in endocytic vesicles that fuse with early endosomes in the cytoplasm. During maturation of early endosomes, the molecules they contain are sorted into smaller vesicles that bud from the endosomal membrane into the lumen. When these multiple intralumenal vesicles are secreted extracellularly through exocytosis, they are called exosomes. Once in the extracellular space, exosomes can be taken up by neighbouring cells, probably after fusion with the outer membrane of the recipient cell.34 Exosomes carrying PrPSc are infectious in vitro and in vivo29,33 and have been proposed as a vehicle for propagation of PrPSc from one cell to another.35 Tunnelling nanotubes constitute another nonconventional intercellular communication pathway that has been implicated in PrPSc propagation.30 These nanotubes are thin extensions of surface membrane that connect cells over long distances. They are formed either by actin-driven protrusion from one cell to another and subsequent membrane fusion or during cell division.36 Tunnelling nanotubes mediate spread of PrPSc between co-cultured neuronal cells and from bone-marrow-derived dendritic cells to primary neurons.30 In bovine spongiform encephalopathy, ingested PrPSc transfers from the gut to the lymphoid system, then to the peripheral nervous system, and finally to the CNS.37 In this disease, tunnelling www.thelancet.com/neurology Vol 9 November 2010

nanotubes have been suggested to participate in PrPSc propagation not only within neurons in the CNS but also from the lymphoid system to the peripheral nervous system.30

Similarities of prion diseases and neurodegenerative diseases The lesions that characterise Alzheimer’s disease histopathologically are senile plaques (which consist of extracellular aggregated amyloid β peptides) and neurofibrillary tangles (composed of intracellular polymers of tau protein). Echoing the disease-associated conformations of PrPSc, aggregation-prone amyloid β (derived from amyloid precursor protein) adopts β-sheet structures typically seen in all amyloid plaques.38 In view of these commonalities, several research groups have reported prion-like transmission for both amyloid β and tau.39–42 One group showed that brain extracts from a patient with Alzheimer’s disease—when injected into the brains of transgenic mice that expressed amyloid precursor protein—could induce aggregation and deposition of amyloid β.39 Implantation of amyloid-β-contaminated steel wires into the hippocampus and cortex of transgenic mice is sufficient to transmit amyloid β.40 Similarly, healthy mice that express nonmutant human tau develop pathogenic tau inclusions in their brains after injection with brain extracts from mutant human tau-expressing mice.42 Findings of studies also indicate that aggregates of misfolded tau can move from the extracellular space into cultured cells and between cells.41 Taken together, these results suggest that exogenous aggregates of amyloid β or tau can induce aggregation or formation of inclusions containing endogenously expressed amyloid β or tau, possibly leading to subsequent spreading of pathologies to neighbouring cells.

Symptoms and neuropathology of synucleinopathies Similar to prion diseases and Alzheimer’s disease, disorders characterised by Lewy body formation—such as Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy—involve aggregation of the misfolded protein α-synuclein. Although these synucleinopathies share the presence of Lewy bodies and some symptoms, they also have many differences.43 The most common synucleinopathy, Parkinson’s disease, is characterised by loss of pigmented dopamine neurons in the substantia nigra pars compacta and by the cardinal symptoms of bradykinesia, rigidity, and resting tremor. Although traditional thinking emphasises motor deficits in Parkinson’s disease, many non-motor symptoms have been highlighted, such as loss of olfaction, constipation, sleep disturbances, impotence, and cognitive dysfunction.44 The average survival of an affected individual from the first signs of Parkinson’s disease is two to three decades. 1129

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Misfolded and post-translationally modified α-synuclein is the primary proteinaceous component of Lewy bodies and Lewy neurites,2 the typical intracytoplasmic inclusions that develop in the cell body and neurites, respectively, of affected neurons. Whether Lewy bodies and Lewy neurites are toxic or neuroprotective is unclear and the subject of much debate.45 Lewy bodies and Lewy neurites are found in patients with Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy but are distributed according to different patterns.43 As described in detail below, Lewy bodies are present in brainstem neurons early in Parkinson’s disease and then in the cortex as disease progresses.46–48 Patients with dementia with Lewy bodies survive for about 5–8 years and show early dementia, concurrent with akinetic and rigid motor symptoms.43 Dopaminergic neurons in the substantia nigra and cholinergic neurons in the nucleus basalis of Meynert both degenerate in people with this disorder. Lewy bodies are more widespread and abundant than in other synucleinopathies, and amyloid plaques are also present in the brain of these patients.43 Individuals with multiple system atrophy are diagnosed on the basis of a combination of ataxia, parkinsonism, and autonomic dysfunction due to neuron loss in the olivopontocerebellar, striatonigral, brainstem, and autonomic systems. The disease is characterised by cytoplasmic inclusions found in glia and occasionally in

neurons. Multiple system atrophy has been classified into separate subtypes depending on the predominance of symptoms.49 The disease course is shorter than that of people with idiopathic Parkinson’s disease, with most patients living less than a decade after diagnosis.

In-vivo evidence for α-synuclein spread α-synuclein is an abundant protein in the brain, found in most cellular compartments and enriched at presynaptic terminals.50,51 It is believed to have a role in vesicular transport and neurotransmitter release, although its exact functions are unknown.2 The gene encoding human α-synuclein (SNCA) can carry rare mutations or can be duplicated or triplicated, changes which are all linked to dominant forms of inherited Parkinson’s disease and parkinsonism.52–56 α-synuclein is present in the cerebrospinal fluid and plasma of both controls and patients with neurodegenerative diseases,57–59 suggestive of exocytosis. Concentrations of α-synuclein in neuronal cell bodies increase during healthy ageing in human beings and monkeys.60 Thus, a raised cytoplasmic concentration of α-synuclein in neuronal cell bodies, both as a result of ageing and of gene duplications and triplications, seems to be a disease risk factor.

Dual-hit hypothesis Braak and co-workers46–48 have suggested that α-synuclein pathology spreads throughout the nervous system in

Neocortex Cognitive decline

Midbrain Motor symptoms Olfactory bulb Anosmia

Gut Constipation

Caudal brainstem Autonomic symptoms

Figure 2: Dual-hit hypothesis of propagation of synucleinopathy during Parkinson’s disease Parkinson’s disease-associated neuropathology originates in the gut (first hit) or the nose (olfactory bulb; second hit) and then propagates to the caudal brainstem and the temporal lobe. Lewy body pathology then ascends to midbrain structures and cortical areas. Blue arrows depict the proposed ascending progression of Parkinson’s disease pathology. Boxes indicate affected systems and main associated symptoms.

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A

Dissection of ventral mesencephalic tissue One to eight donor embryos used as a source of tissue

Transplant preparation Fresh or hibernated tissue is homogenised into cell suspension or small tissue pieces

Grafting procedure Stereotactic injection in caudate and/or putamen Three to eight injection tracts per striatum

Immunosuppression Ciclosporin for 6 months or long-term triple drug therapy (ciclosporin, azathioprine, prednisolone) to prevent rejection

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Figure 3: Grafting of neurons into brains of patients with Parkinson’s disease (A) Summary of the procedure used to prepare long-term grafts.65–67 (B) Immunostaining with α-synuclein antibodies of sections from a 16-year-old graft (left panel). Lewy bodies (arrows) in the graft are similar to those seen in the substantia nigra of the host (right panel).

Parkinson’s disease according to a stereotypic pattern following long unmyelinated axons of known anatomical pathways (figure 2). According to this so-called dual-hit hypothesis,61,62 Parkinson’s disease pathology originates in the nose and foregut after inhalation of an unknown neurotropic pathogen and subsequent swallowing of nasal mucus in saliva. Among many theories and hypotheses, Braak and colleagues speculated that “unconventional pathogens with prion-like properties” might induce spreading of Parkinson’s disease pathology.61,62 After crossing the epithelium, this pathogenic agent could get access to and be transported in an anterograde direction along axons of neurons projecting from the olfactory epithelium to the temporal lobe, and retrogradely from the enteric epithelium via sympathetic fibres in the vagus nerve to the CNS (figure 2).61,62 www.thelancet.com/neurology Vol 9 November 2010

Lewy bodies and Lewy neurites have been detected in tufted neurons47 and mitral cells47,63 in the olfactory bulb of patients with Parkinson’s disease; mitral cells receive direct input from neurons of the olfactory epithelium. Lewy body pathology is also apparent all along the olfactory pathway (anterior olfactory nucleus, olfactory tubercle, and cortices),64 even though the olfactory epithelium itself seems devoid of α-synuclein aggregates.65 In the foregut, Lewy bodies and Lewy neurites have been found in enteric nerve cell plexa in patients with Parkinson’s disease.66 After reaching the CNS via nasal and gastric routes, Lewy pathology has been proposed to ascend from the medulla oblongata to midbrain structures, including the substantia nigra, and finally to cortical areas, along a network of neurons interconnecting all these regions (figure 2).46–48 In view of the direct anatomical connection 1131

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between the olfactory system and the temporal lobe of the neocortex, temporal structures would be presumed to display Lewy bodies and Lewy neurites earlier if inhalation were the predominant mode of pathogen entry. Thus, oral ingestion might be the main method of pathogen access to the body. Moreover, the topography of Lewy pathology could be correlated with the extent and severity of symptoms. The chronology of appearance of Parkinson’s disease symptoms can be described briefly as follows: the presymptomatic phase occurs first, and it is loosely defined by non-motor symptoms such as olfactory deficits, sleep disturbances, constipation, and erectile dysfunction, all of which can occasionally first arise at later disease stages;67,68 the motor symptom phase, occurs later, and includes bradykinesia and rigidity; and finally, the late complication phase is characterised by cognitive decline and psychiatric symptoms.69 During the early non-motor phase of Parkinson’s disease, Lewy bodies and Lewy neurites are restricted to the peripheral enteric system,66 the olfactory bulb, and the caudal brainstem.47 Next, Lewy pathology appears in the midbrain, especially in the substantia nigra pars compacta, within the period of the onset of motor symptoms, and eventually it reaches the neocortex at later stages characterised by cognitive impairment (figure 2).47 In the following sections, we discuss arguments for and against the Braak hypothesis.

Lewy pathology in neural grafts In some patients with Parkinson’s disease, transplantation of embryonic dopamine neurons was started more than two decades ago in an attempt to restore dopaminergic neurotransmission (figure 3A).70–75 Findings of histological studies on post-mortem tissue revealed Lewy bodies within the transplanted dopaminergic neurons in eight patients who were operated on in four centres worldwide and who died more than a decade after surgery (figure 3B).73–75 The Lewy bodies in the grafts shared classic features with those in the substantia nigra of the host, including α-synuclein and ubiquitin immunoreactivity. Further studies characterised Lewy pathology in transplanted neurons, with detection of α-synuclein phosphorylated on serine 129,75,76 characteristic β-sheet structures stained by thioflavine S,76,77 and α-synuclein fibrils revealed by electron microscopy.77 Tyrosine hydroxylase is rarely expressed in grafted neurons containing Lewy bodies, although the fact that Lewy bodies are seen in grafted cells containing neuromelanin suggests that they are present in catecholaminergic (presumed dopaminergic) neurons. These observations— together with a reported decrease of dopamine transporter immunoreactivity,73,74,76 and a reduction in intensity of tyrosine hydroxylase immunostaining74,76 in cells with Lewy pathology within a transplant from a patient who survived 14 years after transplantation— could indicate that the subset of transplanted cells 1132

carrying Lewy bodies is functionally impaired. In one individual, Lewy bodies were noted in 1·9% and 5·0% of pigmented neurons, which were grafted (into the right and left striatum) 12 and 16 years, respectively, before death.77 40% of transplanted dopaminergic neurons showed cytoplasmic α-synuclein staining in the 12-year-old grafts versus 80% in the 16-year-old grafts.75 In other studies, cell bodies in 18-month-old grafts were devoid of any immunoreactivity to α-synuclein whereas neurons in 4-year-old and 16-year-old transplants showed cytoplasmic α-synuclein staining and α-synuclein-positive spherical aggregates, respectively.76 Taken together, these observations suggest that Lewy body development in the grafted neurons is a gradual process, and that whole Lewy bodies do not move between cells.76,77 Prion-like spread of misfolded α-synuclein, which might act as a seed and lead to progressive accumulation of α-synuclein, could result in formation of Lewy bodies in transplanted neurons.5,78

Animal models of synucleinopathy propagation Researchers have reported a mouse model79 that recapitulates some aspects of the pathological staging described by Braak and collaborators in patients with Parkinson’s disease.46,47 The model is based on intragastric administration of rotenone, an inhibitor of complex I of the mitochondrial respiratory chain. Rotenone is a widely used pesticide that has been linked to Parkinson’s disease in epidemiological studies.80–82 It induces formation of large perinuclear α-synuclein inclusions in cultured cells expressing human α-synuclein.83 Chronic intravenous84 or intraperitoneal85 administration of rotenone in rats has been reported to trigger α-synuclein aggregation in nigral neurons, probably directly, in view of the capacity of rotenone to cross the blood–brain barrier. In the new mouse model, there is progressive development of α-synuclein inclusions, starting in the enteric nervous system and then arising sequentially in spinal cord, caudal brainstem, and the substantia nigra79—ie, the same structures that show Lewy pathology in Parkinson’s disease. One possible explanation for these results lies in the induction of α-synuclein aggregation in enteric nerves by intragastric administration of the toxin, and then propagation of the pathology to vulnerable CNS areas via an unknown mechanism that might include intercellular transfer of α-synuclein aggregates. The first experimental evidence for intercellular transfer of α-synuclein in vivo came from a study on mouse cortical neuronal stem cells grafted to the hippocampus of transgenic mice overexpressing human α-synuclein.86 At 1–4 weeks after grafting, 2·5–15% of transplanted cells showed human α-synuclein staining, indicating that α-synuclein can transfer from the host brain to a graft of proliferating neuronal progenitors. These results bear some resemblance to clinical www.thelancet.com/neurology Vol 9 November 2010

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findings with neural grafts described above, but differ in the sense that no large aggregates resembling Lewy bodies were seen and that proliferating rather than post-mitotic cells were grafted. Finally, in a report of transgenic mice that overexpress human α-synuclein under the control of a neuronal promoter, α-synuclein accumulation was detected not only in neurons but also in glia.87 Because the promoter was neuron-specific, glial cells were devoid of human α-synuclein mRNA. These results suggest that glial cells that do not express the α-synuclein transgene take up α-synuclein released by neurons. The low basal expression level of α-synuclein in glia88 raises the question of the origin of glial α-synuclein inclusions characterising multiple system atrophy. Propagation of α-synuclein from neurons to glial cells, perhaps relying on prion-like properties of α-synuclein, could account for the appearance of widespread glial α-synuclein inclusions during progression of multiple system atrophy.

In-vitro studies

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Natively unfolded monomer Release α-synuclein Self-aggregation

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Structure of α-synuclein During the oligomerisation of α-synuclein into fibrils, the structure of α-synuclein undergoes major modifications that could underlie its toxic effects. Accumulating evidence suggests that α-synuclein oligomers, and possibly protofibrils, are the toxic species that cause cell death.89 Therefore, deeper understanding of the structure of α-synuclein and its changes during oligomerisation could give fundamental insights into the pathogenesis of synucleinopathies. α-synuclein is natively unfolded at low concentrations but, on accumulation, it self-aggregates into soluble oligomers and can eventually form insoluble fibrillar aggregates90–94 with a typical amyloid nature (figure 4A).95 As with PrPSc and amyloid β, α-synuclein fibrils have unbranched morphology and an antiparallel β-sheet structure,95 bind thioflavine S93 and Congo red, and are resistant to proteolysis.95 Similar to PrPSc propagation, α-synuclein fibrillisation is a nucleation-dependent process starting with a lag-phase, during which soluble α-synuclein oligomers assemble and form a nucleus.96 Aggregates of α-synuclein then grow rapidly around this nucleus during the growth or elongation phase, until they reach a thermodynamic equilibrium with monomers in a steady-state phase.96 Moreover, aggregation of recombinant α-synuclein monomers can be seeded by addition of α-synuclein aggregates acting as exogenous nuclei.96 The three mutations of α-synuclein recorded in patients with Parkinson’s disease—Ala53Thr, Ala30Pro, and Glu46Lys—accelerate this process, indicating a probable role for α-synuclein misfolding and aggregation in familial Parkinson’s disease.90,94,97

Possible mechanisms of release and uptake α-synuclein can access the extracellular space, consistent with its reported presence in cerebrospinal fluid and www.thelancet.com/neurology Vol 9 November 2010

Figure 4: Putative mechanisms of α-synuclein prion-like propagation (A) The different species of α-synuclein assemblies coexist in a highly dynamic equilibrium. (B) According to the prion-like hypothesis, a donor cell releases α-synuclein into the extracellular space via exocytosis or during its death. Next, α-synuclein enters a recipient cell via passive membrane translocation or endocytosis. Alternative mechanisms include exosomal release or transport along tunnelling nanotubes. Once in the recipient cell, the transferred α-synuclein protein (blue) recruits the endogenous α-synuclein protein (red), induces misfolding, and seeds aggregation. Green stars indicate possible targets for disease-modifying drugs to reduce, stop, or delay systematic spread of α-synuclein throughout the human body.

plasma in human beings,57,58 and in the medium of several neuronal culture models.57,98 The mechanism by which α-synuclein is released from cells is unclear, but exocytosis is likely to underlie this secretion (figure 4B), given that the process is inhibited at low temperature and α-synuclein can be detected in the lumen of vesicles isolated from rat brain or neuroblastoma cells.98,99 Furthermore, α-synuclein translocation to vesicles and subsequent vesicle release increase under conditions that promote its misfolding and could be part of a cellular quality-control system aimed at removal of damaged proteins.99 Furthermore, researchers have suggested that exosomes could have a role in secretion of α-synuclein by cultured neuroblastoma cells.100 After release of α-synuclein, the next steps are crucial. What are the mechanisms by which cells take up extracellular α-synuclein? Similar to infection of new cells by PrPSc, extracellular α-synuclein can be internalised by surrounding cells (figure 4B). Whereas recombinant α-synuclein monomers have been suggested to translocate passively across plasma membranes of neuroblastoma cells,101,102 an endocytic process is probably needed for internalisation of larger order α-synuclein assemblages.102 Uptake of recombinant α-synuclein 1133

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oligomers and fibrils is indeed inhibited at low temperatures and by expression of a dominant negative mutant of dynamin 1, a neuron-specific GTPase important for endocytosis, suggesting that uptake takes place via an endocytic process.102 In co-cultured neuronal cells, transmitted α-synuclein proteins colocalise with the endosomal GTPases Rab5a and Rab7,86 and in particular, Rab5a has been suggested to be vital for α-synuclein endocytosis.103 In rat dopaminergic cells, some internalised aggregates of α-synuclein are degraded by a Rab11a-dependent endosome-lysosome pathway and others are resecreted through exocytosis.104 In addition to α-synuclein transfer between neuronal cells, glial cells might take up α-synuclein derived from neurons via an endocytic mechanism that is blocked by expression of a mutant form of dynamin 1.87 Moreover, Lee and colleagues87 showed formation of Lewy body-like aggregates after uptake of human α-synuclein into glial cells. Thus, primary astrocytes co-cultured with neuroblastoma cells overexpressing human α-synuclein develop inclusions of human α-synuclein, which stain with thioflavine S and contain some proteins found in Lewy bodies, such as ubiquitin, the 20S proteasome α-subunit, and the chaperone protein HSP/HSC70.87 Taken together, the mechanism by which cells take up α-synuclein could be tightly linked with the putative next steps of inclusion formation.

Potential seeding effect of α-synuclein If intercellular transmission of aggregated α-synuclein is governed by a prion-like mechanism, the ultimate step would be the seeding effect—ie, recruitment of unfolded α-synuclein proteins endogenously expressed by the acceptor cell and their conversion into misfolded forms that will aggregate around the nucleus of transmitted α-synuclein (figure 4B). A few studies have looked at the seeding effect of recombinant α-synuclein. Oligomers of α-synuclein, thought to be the toxic species,89 seed the aggregation of α-synuclein proteins endogenously expressed by neuroblastoma cells105 and by primary cortical neurons105,106 in a time-dependent and concentration-dependent manner.106 In other studies,107,108 artificial techniques (such as cationic liposome-based transduction) to load cells with exogenous fibrillised α-synuclein proteins are needed for the seeding process to take place.

Arguments against synucleinopathies being prion-like disorders As described in the previous section, evidence that α-synuclein can transfer between co-cultured neurons in vitro is compelling, and studies have provided some insight into underlying mechanisms. Nonetheless, the subsequent seeding activity of transferred α-synuclein is a crucial prerequisite for propagation of α-synuclein pathology to take place during progression of Parkinson’s disease and other Lewy body-related 1134

neurodegenerative disorders. So far, in-vitro studies in which seeding has been shown have used highly artificial techniques for introduction of α-synuclein into cells,107,108 which are not compatible with physiological conditions. Similarly, studies that have shown seeding of α-synuclein in vivo—and even more importantly, animal work on α-synuclein intercellular transfer—are currently sparse. So far, published data are restricted to the study mentioned above,86 in which proliferative neuronal stem cells are grafted into the hippocampus of transgenic mice overexpressing human α-synuclein. To date, no data are available to show that post-mitotic dopaminergic neurons—the primary cell type affected in Parkinson’s disease—can take up α-synuclein released from donor neurons in an experimental animal model, or that transferred α-synuclein can trigger formation of Lewy bodies. Even if future research establishes that α-synuclein fulfils all criteria for a prion-like protein in culture and in animals, the relevance of these discoveries in relation to the pathogenesis of synucleinopathies will still be questionable. The hypothesis that misfolded α-synuclein accumulation is the central force driving the neurodegenerative process is not adopted unanimously in the field, and the correlation proposed by Braak and collaborators46–48 between the spread of Lewy pathology and the temporal pattern of development of symptoms has been questioned.109 Thus, α-synuclein misfolding and aggregation might alternatively be considered an epiphenomenon of a pathological neuronal microenvironment. For example, differences in distribution of Lewy bodies in the synucleinopathies could be explained by selective neuronal vulnerability in each disease, underlying the characteristic temporal patterns of progression of pathology. Alternatively, neuroinflammation, which leads to formation of intraneuronal α-synuclein inclusions in animals110,111 and has been detected in post-mortem analysis of brains of patients with Parkinson’s disease,112 could lie upstream of α-synuclein aggregation in the cascade of pathogenic events leading to progression of Parkinson’s disease. What makes prion-like propagation of α-synuclein an especially interesting hypothesis is that it can account for the sequence of appearance of symptoms and the findings on neuropathological changes in grafts of patients with Parkinson’s disease.73–75 Thus, this hypothesis remains a highly attractive and promising research axis. Could interindividual infectivity be another feature shared by PrPSc and misfolded α-synuclein? A paucity of epidemiological evidence, or at least case reports, suggesting that Parkinson’s disease can spread from one person to another, however, argues against this notion. How do the prion-like properties of α-synuclein differ from those of PrPSc? Under what conditions might α-synuclein act as an infectious agent? These questions still need to be answered. By contrast with the instability www.thelancet.com/neurology Vol 9 November 2010

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of PrPSc assemblies, which readily break and generate further free ends that act as seeds,23,24 the possible relative stability of α-synuclein fibrils might account for both non-infectivity of α-synuclein and the typical disease course of the synucleinopathies (from several years to decades), versus the fairly quick (within 1 year) duration of prion disease. Alternatively, major differences in structure and cellular localisation between α-synuclein and PrP could account for the varying kinetics of conversion from normal to misfolded protein, and hence could affect the rate of disease progression. Similarly, variability in disease duration of the synucleinopathies (5–8 years for dementia with Lewy bodies after diagnosis, less than 10 years for multiple system atrophy, decades for Parkinson’s disease) might also rely on different forms of misfolded α-synuclein, with various stabilities or intermediate species along the conversion process of normal into pathogenic protein, even if no evidence for such heterogeneity in α-synuclein proteins has been reported so far. In summary, the hypothesis that progression of synucleinopathies relies on the same mechanisms as prion disorders is highly attractive. However, it has potential drawbacks and alternatives, which are currently under investigation.

Conclusions and future perspectives Connections between in-vitro studies and clinical observations are important but in need of improvement and extension. What are the next steps for basic science research that will bring more insight to human disease and, specifically, to synucleinopathies? As described above, exocytosis and endocytosis seem to have important roles in cell-to-cell transfer of misfolded α-synuclein. However, in view of the similarities between PrPSc and α-synuclein, other mechanisms of cell-to-cell transfer reported to contribute to PrPSc propagation should also be considered. Both exosomes and tunnelling nanotubes might contribute to intercellular transfer of PrPSc.28–30,32,33 A minute fraction of α-synuclein released from cultured neuroblastoma cells is associated with extracellular vesicles99 and exosomal secretion has been reported.100 The relevance of α-synuclein-containing exosomes in other cell types, notably astrocytes (reported to secrete exosomes),113 are worth examining both in cell culture and in vivo. Furthermore, researchers revealed the presence of α-synuclein within tunnelling nanotubes that were interconnecting co-cultured glioblastoma cells,114 warranting further investigation into whether tunnelling nanotubes can convey α-synuclein from one neuron to another. As discussed above, the dual-hit theory suggests that a pathogenic agent with prion-like properties penetrates the body by the nasal or gastric route, or both, and then leads to propagation of Lewy bodies and Lewy neurites from the gut to the CNS.61,62 Clearly, misfolded α-synuclein is a relevant candidate. Pathogenic forms www.thelancet.com/neurology Vol 9 November 2010

Search strategy and selection criteria We searched PubMed from January, 1994, to July, 2010, with the search terms: “alpha-synuclein”, “synuclein”, “Parkinson disease”, “prion”, “scrapie”, “Creutzfeldt-Jakob”, “dementia with lewy bodies”, “multiple systems atrophy”, “prion-like”, “Alzheimer disease”, “Braak hypothesis”, “non-motor symptoms”, “neural grafting”, “rotenone”, “fibrillization”, “cell-to-cell transfer”, “exocytosis”, “endocytosis”, “seeding”, “amyloid”, “tau”, “nanotube”, “exosome”, “biomarker”, and “English”. We largely targeted publications from the past 10 years but did not exclude commonly referenced, highly regarded, and relevant older publications. Furthermore, we examined the search results obtained above and selected the most relevant for this Personal View.

of α-synuclein are unlikely to enter the organism through direct nasal inhalation. Instead, contact with substances present in our environment—eg, specific pesticides, which are known to be risk factors for Parkinson’s disease80–82 and cause α-synuclein aggregation83—could be an initial event that triggers α-synuclein misfolding in the periphery. As a result, by using exposure to a peripheral toxin and exploiting all the aforementioned knowledge about the putative prion-like mechanism of α-synuclein spread, development of animal models that faithfully reproduce all the characteristics of Parkinson’s disease might be possible. One alternative could be to manipulate selectively and transgenically the α-synuclein gene in the periphery and cause a regional synucleinopathy outside the CNS. Early gastrointestinal dysfunction and aggregates of α-synuclein in enteric nervous system ganglia have been reported in transgenic mice carrying an artificial chromosome with Parkinson’s disease-linked human mutations (Ala53Thr or Ala30Pro) of the α-synuclein gene.115 These models might mimic not only Parkinson’s disease-related neuropathological changes and neurodegenerative events but also motor and non-motor symptoms, and, thus, they would be invaluable for assessment of the efficacy of new drugs for Parkinson’s disease at early preclinical stages. All current treatments for Parkinson’s disease, from pharmaceutical to surgical, only alleviate motor symptoms;116 to date, no disease-modifying treatment has been able to stop or greatly delay neurodegeneration. Although early treatment with rasagiline—a monoamine oxidase type B inhibitor—might slow progression of Parkinson’s disease slightly, further clinical investigations are needed to assess the efficacy of this drug.117 Accumulating evidence supporting prion-like properties of α-synuclein indicates that cell-to-cell transfer of this protein might be worth targeting. Intercellular transfer of misfolded proteins followed by permissive templating could be a common pathogenetic mechanism in several cerebral proteinopathies,3,4,6,8 1135

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making this approach to new treatments even more wide-reaching. Putative novel therapies that inhibit transfer of disease-related proteins should ideally be initiated even before the individual has met current diagnostic criteria for Parkinson’s disease—ie, at the very beginning of progression of Lewy pathology. These limitations will require development of biomarkers for early Parkinson’s disease—eg, brain imaging or biological fluid biomarkers currently under development for differential diagnosis118 and identification of a set of non-motor symptoms (eg, olfactory or gastrointestinal dysfunction) with high predictive value for Parkinson’s disease. The theory of prion-like spread of misfolded α-synuclein suggests the process will take place in three steps: release, uptake, and seeding. Inhibition of α-synuclein spread could interfere with one or another of these processes (figure 4B). Exocytosis and endocytosis inhibitors might be interesting candidates to investigate, because both of these cellular mechanisms are proposed to participate in α-synuclein intercellular transfer,98,99,102,103 but, of course, other treatments specific to exosomes, tunnelling nanotubes, or receptors should be tested. In conclusion, research into the mechanisms of α-synuclein cell-to-cell transfer in combination with other ongoing strategies could lead to promising advances in clinical treatment of synucleinopathies. Contributors EA and JAS wrote the manuscript and prepared the figures, and CH, JYL, and PB edited the paper. Conflicts of interest PB has received honoraria from Pfizer, H Lundbeck A/S, Roche, and Orion Pharma, consultancy fees from Teva Pharmaceutical Industries and H Lundbeck A/S, and research support from Teva. The other authors have no conflicts of interest. Acknowledgments We thank the Human Frontier Science Program, the ERA-net Neuron program MIPROTRAN, the Michael J Fox Foundation for Parkinson’s Research, the Swedish Brain Foundation, the Swedish Parkinson Foundation, the Ragnar and Thorsten Söderbergs Foundation, Anna-Lisa Rosenberg Foundation, and the Swedish Research Council. The funding sources had no role in preparation of this paper. References 1 Uversky VN. α-synuclein misfolding and neurodegenerative diseases. Curr Protein Pept Sci 2008; 9: 507–40. 2 Spillantini MG, Schmidt ML, Lee VM, Trojanowski JQ, Jakes R, Goedert M. α-synuclein in Lewy bodies. Nature 1997; 388: 839–40. 3 Aguzzi A. Cell biology: beyond the prion principle. Nature 2009; 459: 924–25. 4 Aguzzi A, Rajendran L. The transcellular spread of cytosolic amyloids, prions, and prionoids. Neuron 2009; 64: 783–90. 5 Angot E, Brundin P. Dissecting the potential molecular mechanisms underlying α-synuclein cell-to-cell transfer in Parkinson’s disease. Parkinsonism Relat Disord 2009; 15 (suppl 3): S143–47. 6 Frost B, Diamond MI. Prion-like mechanisms in neurodegenerative diseases. Nat Rev Neurosci 2010; 11: 155–59. 7 Olanow CW, Prusiner SB. Is Parkinson’s disease a prion disorder? Proc Natl Acad Sci USA 2009; 106: 12571–72. 8 Brundin P, Melki R, Kopito R. Prion-like transmission of protein aggregates in neurodegenerative diseases. Nat Rev Mol Cell Biol 2010; 11: 301–07. 9 Goedert M, Clavaguera F, Tolnay M. The propagation of prion-like protein inclusions in neurodegenerative diseases. Trends Neurosci 2010; 33: 317–25.

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10 11 12

13 14

15

16

17

18

19

20 21

22

23

24

25

26

27

28

29

30 31

32

33

34 35

Prusiner SB. Novel proteinaceous infectious particles cause scrapie. Science 1982; 216: 136–44. Prusiner SB. Shattuck lecture: neurodegenerative diseases and prions. N Engl J Med 2001; 344: 1516–26. Appleby BS, Appleby KK, Crain BJ, Onyike CU, Wallin MT, Rabins PV. Characteristics of established and proposed sporadic Creutzfeldt-Jakob disease variants. Arch Neurol 2009; 66: 208–15. Diener TO, McKinley MP, Prusiner SB. Viroids and prions. Proc Natl Acad Sci USA 1982; 79: 5220–24. Hsiao K, Baker HF, Crow TJ, et al. Linkage of a prion protein missense variant to Gerstmann-Straussler syndrome. Nature 1989; 338: 342–45. Medori R, Tritschler HJ, LeBlanc A, et al. Fatal familial insomnia, a prion disease with a mutation at codon 178 of the prion protein gene. N Engl J Med 1992; 326: 444–49. Jackson WS, Borkowski AW, Faas H, et al. Spontaneous generation of prion infectivity in fatal familial insomnia knockin mice. Neuron 2009; 63: 438–50. Steele AD, Zhou Z, Jackson WS, et al. Context dependent neuroprotective properties of prion protein (PrP). Prion 2009; 3: 240–49. Si K, Choi YB, White-Grindley E, Majumdar A, Kandel ER. Aplysia CPEB can form prion-like multimers in sensory neurons that contribute to long-term facilitation. Cell 2010; 140: 421–35. Prusiner SB, Groth D, Serban A, et al. Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies. Proc Natl Acad Sci USA 1993; 90: 10608–12. Sailer A, Bueler H, Fischer M, Aguzzi A, Weissmann C. No propagation of prions in mice devoid of PrP. Cell 1994; 77: 967–68. Weissmann C, Bueler H, Fischer M, Sailer A, Aguzzi A, Aguet M. PrP-deficient mice are resistant to scrapie. Ann N Y Acad Sci 1994; 724: 235–40. Riek R, Hornemann S, Wider G, Billeter M, Glockshuber R, Wüthrich K. NMR structure of the mouse prion protein domain PrP(121-321). Nature 1996; 382: 180–82. Wille H, Bian W, McDonald M, et al. Natural and synthetic prion structure from X-ray fiber diffraction. Proc Natl Acad Sci USA 2009; 106: 16990–95. Tanaka M, Collins SR, Toyama BH, Weissman JS. The physical basis of how prion conformations determine strain phenotypes. Nature 2006; 442: 585–89. Yam AY, Gao CM, Wang X, Wu P, Peretz D. The octarepeat region of the prion protein is conformationally altered in PrP(Sc). PLoS One 2010; 5: e9316. Bergasa-Caceres F, Rabitz HA. Low entropic barrier to the hydrophobic collapse of the prion protein: effects of intermediate states and conformational flexibility. J Phys Chem A 2010; 114: 6978–82. Colby DW, Giles K, Legname G, et al. Design and construction of diverse mammalian prion strains. Proc Natl Acad Sci USA 2009; 106: 20417–22. Alais S, Simoes S, Baas D, et al. Mouse neuroblastoma cells release prion infectivity associated with exosomal vesicles. Biol Cell 2008; 100: 603–15. Février B, Vilette D, Archer F, et al. Cells release prions in association with exosomes. Proc Natl Acad Sci USA 2004; 101: 9683–88. Gousset K, Schiff E, Langevin C, et al. Prions hijack tunnelling nanotubes for intercellular spread. Nat Cell Biol 2009; 11: 328–36. Jen A, Parkyn CJ, Mootoosamy RC, et al. Neuronal low-density lipoprotein receptor-related protein 1 binds and endocytoses prion fibrils via receptor cluster 4. J Cell Sci 2010; 123: 246–55. Veith NM, Plattner H, Stuermer CA, Schulz-Schaeffer WJ, Bürkle A. Immunolocalisation of PrPSc in scrapie-infected N2a mouse neuroblastoma cells by light and electron microscopy. Eur J Cell Biol 2009; 88: 45–63. Vella LJ, Sharples RA, Lawson VA, Masters CL, Cappai R, Hill AF. Packaging of prions into exosomes is associated with a novel pathway of PrP processing. J Pathol 2007; 211: 582–90. Simons M, Raposo G. Exosomes: vesicular carriers for intercellular communication. Curr Opin Cell Biol 2009; 21: 575–81. Février B, Vilette D, Laude H, Raposo G. Exosomes: a bubble ride for prions? Traffic 2005; 6: 10–17.

www.thelancet.com/neurology Vol 9 November 2010

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36

37 38 39

40

41 42

43 44

45

46

47

48

49 50

51 52

53

54 55

56

57

58

59

60

Davis DM, Sowinski S. Membrane nanotubes: dynamic longdistance connections between animal cells. Nat Rev Mol Cell Biol 2008; 9: 431–36. Mabbott NA, MacPherson GG. Prions and their lethal journey to the brain. Nat Rev Microbiol 2006; 4: 201–11. Cohen AS, Connors LH. The pathogenesis and biochemistry of amyloidosis. J Pathol 1987; 151: 1–10. Meyer-Luehmann M, Coomaraswamy J, Bolmont T, et al. Exogenous induction of cerebral β-amyloidogenesis is governed by agent and host. Science 2006; 313: 1781–84. Eisele YS, Bolmont T, Heikenwalder M, et al. Induction of cerebral β-amyloidosis: intracerebral versus systemic Aβ inoculation. Proc Natl Acad Sci USA 2009; 106: 12926–31. Frost B, Jacks RL, Diamond MI. Propagation of tau misfolding from the outside to the inside of a cell. J Biol Chem 2009; 284: 12845–52. Clavaguera F, Bolmont T, Crowther RA, et al. Transmission and spreading of tauopathy in transgenic mouse brain. Nat Cell Biol 2009; 11: 909–13. Halliday GM, McCann H. The progression of pathology in Parkinson’s disease. Ann N Y Acad Sci 2010; 1184: 188–95. Barone P, Antonini A, Colosimo C, et al. The PRIAMO study: a multicenter assessment of nonmotor symptoms and their impact on quality of life in Parkinson’s disease. Mov Disord 2009; 24: 1641–49. Olanow CW, Perl DP, DeMartino GN, McNaught KStP. Lewy-body formation is an aggresome-related process: a hypothesis. Lancet Neurol 2004; 3: 496–503. Braak H, Del Tredici K, Bratzke H, Hamm-Clement J, Sandmann-Keil D, Rüb U. Staging of the intracerebral inclusion body pathology associated with idiopathic Parkinson’s disease (preclinical and clinical stages). J Neurol 2002; 249 (suppl 3): III/1–5. Braak H, Del Tredici K, Rüb U, de Vos RA, Jansen Steur EN, Braak E. Staging of brain pathology related to sporadic Parkinson’s disease. Neurobiol Aging 2003; 24: 197–211. Braak H, Ghebremedhin E, Rüb U, Bratzke H, Del Tredici K. Stages in the development of Parkinson’s disease-related pathology. Cell Tissue Res 2004; 318: 121–34. Stefanova N, Bücke P, Duerr S, Wenning GK. Multiple system atrophy: an update. Lancet Neurol 2009; 8: 1172–78. Uéda K, Fukushima H, Masliah E, et al. Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proc Natl Acad Sci USA 1993; 90: 11282–86. Jakes R, Spillantini MG, Goedert M. Identification of two distinct synucleins from human brain. FEBS Lett 1994; 345: 27–32. Polymeropoulos MH, Lavedan C, Leroy E, et al. Mutation in the α-synuclein gene identified in families with Parkinson’s disease. Science 1997; 276: 2045–47. Krüger R, Kuhn W, Müller T, et al. Ala30Pro mutation in the gene encoding α-synuclein in Parkinson’s disease. Nat Genet 1998; 18: 106–08. Singleton AB, Farrer M, Johnson J, et al. α-synuclein locus triplication causes Parkinson’s disease. Science 2003; 302: 841. Chartier-Harlin M-C, Kachergus J, Roumier C, et al. α-synuclein locus duplication as a cause of familial Parkinson’s disease. Lancet 2004; 364: 1167–69. Zarranz JJ, Alegre J, Gómez-Esteban JC, et al. The new mutation, E46K, of α-synuclein causes Parkinson and Lewy body dementia. Ann Neurol 2004; 55: 164–73. El-Agnaf OM, Salem SA, Paleologou KE, et al. α-synuclein implicated in Parkinson’s disease is present in extracellular biological fluids, including human plasma. FASEB J 2003; 17: 1945–47. El-Agnaf OM, Salem SA, Paleologou KE, et al. Detection of oligomeric forms of α-synuclein protein in human plasma as a potential biomarker for Parkinson’s disease. FASEB J 2006; 20: 419–25. Noguchi-Shinohara M, Tokuda T, Yoshita M, et al. CSF α-synuclein levels in dementia with Lewy bodies and Alzheimer’s disease. Brain Res 2009; 1251: 1–6. Chu Y, Kordower JH. Age-associated increases of α-synuclein in monkeys and humans are associated with nigrostriatal dopamine depletion: is this the target for Parkinson’s disease? Neurobiol Dis 2007; 25: 134–49.

www.thelancet.com/neurology Vol 9 November 2010

61 62 63 64

65

66

67 68 69

70

71

72

73

74

75

76 77

78

79

80 81

82

83

84

85

Hawkes CH, Del Tredici K, Braak H. Parkinson’s disease: a dual-hit hypothesis. Neuropathol Appl Neurobiol 2007; 33: 599–614. Hawkes CH, Del Tredici K, Braak H. Parkinson’s disease: the dual hit theory revisited. Ann N Y Acad Sci 2009; 1170: 615–22. Daniel SE, Hawkes CH. Preliminary diagnosis of Parkinson’s disease by olfactory bulb pathology. Lancet 1992; 340: 186. Ubeda-Bañon I, Saiz-Sanchez D, de la Rosa-Prieto C, Argandoña-Palacios L, Garcia-Muñozguren S, Martinez-Marcos A. alpha-Synucleinopathy in the human olfactory system in Parkinson’s disease: involvement of calcium-binding protein- and substance P-positive cells. Acta Neuropathol 2010; 119: 723–35. Duda JE, Shah U, Arnold SE, Lee VM, Trojanowski JQ. The expression of α-, β-, and γ-synucleins in olfactory mucosa from patients with and without neurodegenerative diseases. Exp Neurol 1999; 160: 515–22. Braak H, de Vos RA, Bohl J, Del Tredici K. Gastric α-synuclein immunoreactive inclusions in Meissner’s and Auerbach’s plexuses in cases staged for Parkinson’s disease-related brain pathology. Neurosci Lett 2006; 396: 67–72. Chaudhuri KR, Naidu Y. Early Parkinson’s disease and non-motor issues. J Neurol 2008; 255 (suppl 5): 33–38. Park A, Stacy M. Non-motor symptoms in Parkinson’s disease. J Neurol 2009; 256 (suppl 3): 293–98. Maetzler W, Liepelt I, Berg D. Progression of Parkinson’s disease in the clinical phase: potential markers. Lancet Neurol 2009; 8: 1158–71. Mendez I, Vinuela A, Astradsson A, et al. Dopamine neurons implanted into people with Parkinson’s disease survive without pathology for 14 years. Nat Med 2008; 14: 507–09. Björklund A, Dunnett SB, Brundin P, et al. Neural transplantation for the treatment of Parkinson’s disease. Lancet Neurol 2003; 2: 437–45. Olanow CW, Kordower JH, Lang AE, Obeso JA. Dopaminergic transplantation for Parkinson’s disease: current status and future prospects. Ann Neurol 2009; 66: 591–96. Kordower JH, Chu Y, Hauser RA, Freeman TB, Olanow CW. Lewy body-like pathology in long-term embryonic nigral transplants in Parkinson’s disease. Nat Med 2008; 14: 504–06. Kordower JH, Chu Y, Hauser RA, Olanow CW, Freeman TB. Transplanted dopaminergic neurons develop PD pathologic changes: a second case report. Mov Disord 2008; 23: 2303–06. Li JY, Englund E, Holton JL, et al. Lewy bodies in grafted neurons in subjects with Parkinson’s disease suggest host-to-graft disease propagation. Nat Med 2008; 14: 501–03. Chu Y, Kordower JH. Lewy body pathology in fetal grafts. Ann N Y Acad Sci 2010; 1184: 55–67. Li JY, Englund E, Widner H, et al. Characterization of Lewy body pathology in 12- and 16-year-old intrastriatal mesencephalic grafts surviving in a patient with Parkinson’s disease. Mov Disord 2010; 25: 1091–96. Brundin P, Li JY, Holton JL, Lindvall O, Revesz T. Research in motion: the enigma of Parkinson’s disease pathology spread. Nat Rev Neurosci 2008; 9: 741–45. Pan-Montojo F, Anichtchik O, Dening Y, et al. Progression of Parkinson’s disease pathology is reproduced by intragastric administration of rotenone in mice. PLoS One 2010; 5: e8762. Elbaz A, Clavel J, Rathouz PJ, et al. Professional exposure to pesticides and Parkinson disease. Ann Neurol 2009; 66: 494–504. Gorell JM, Johnson CC, Rybicki BA, Peterson EL, Richardson RJ. The risk of Parkinson’s disease with exposure to pesticides, farming, well water, and rural living. Neurology 1998; 50: 1346–50. Hatcher JM, Pennell KD, Miller GW. Parkinson’s disease and pesticides: a toxicological perspective. Trends Pharmacol Sci 2008; 29: 322–29. Lee HJ, Shin SY, Choi C, Lee YH, Lee SJ. Formation and removal of α-synuclein aggregates in cells exposed to mitochondrial inhibitors. J Biol Chem 2002; 277: 5411–17. Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT. Chronic systemic pesticide exposure reproduces features of Parkinson’s disease. Nat Neurosci 2000; 3: 1301–06. Cannon JR, Tapias V, Na HM, Honick AS, Drolet RE, Greenamyre JT. A highly reproducible rotenone model of Parkinson’s disease. Neurobiol Dis 2009; 34: 279–90.

1137

Personal View

86

Desplats P, Lee HJ, Bae EJ, et al. Inclusion formation and neuronal cell death through neuron-to-neuron transmission of α-synuclein. Proc Natl Acad Sci USA 2009; 106: 13010–15. 87 Lee HJ, Suk JE, Patrick C, et al. Direct transfer of α-synuclein from neuron to astroglia causes inflammatory responses in synucleinopathies. J Biol Chem 2010; 285: 9262–72. 88 Miller DW, Johnson JM, Solano SM, Hollingsworth ZR, Standaert DG, Young AB. Absence of α-synuclein mRNA expression in normal and multiple system atrophy oligodendroglia. J Neural Transm 2005; 112: 1613–24. 89 Waxman EA, Giasson BI. Molecular mechanisms of α-synuclein neurodegeneration. Biochim Biophys Acta 2009; 1792: 616–24. 90 Conway KA, Harper JD, Lansbury PT. Accelerated in vitro fibril formation by a mutant α-synuclein linked to early-onset Parkinson disease. Nat Med 1998; 4: 1318–20. 91 El-Agnaf OM, Jakes R, Curran MD, Wallace A. Effects of the mutations Ala30 to Pro and Ala53 to Thr on the physical and morphological properties of α-synuclein protein implicated in Parkinson’s disease. FEBS Lett 1998; 440: 67–70. 92 Giasson BI, Uryu K, Trojanowski JQ, Lee VM. Mutant and wild type human α-synucleins assemble into elongated filaments with distinct morphologies in vitro. J Biol Chem 1999; 274: 7619–22. 93 Hashimoto M, Hsu LJ, Sisk A, et al. Human recombinant NACP/α-synuclein is aggregated and fibrillated in vitro: relevance for Lewy body disease. Brain Res 1998; 799: 301–06. 94 Narhi L, Wood SJ, Steavenson S, et al. Both familial Parkinson’s disease mutations accelerate α-synuclein aggregation. J Biol Chem 1999; 274: 9843–46. 95 Conway KA, Harper JD, Lansbury PT Jr. Fibrils formed in vitro from α-synuclein and two mutant forms linked to Parkinson’s disease are typical amyloid. Biochemistry 2000; 39: 2552–63. 96 Wood SJ, Wypych J, Steavenson S, Louis JC, Citron M, Biere AL. α-synuclein fibrillogenesis is nucleation-dependent: implications for the pathogenesis of Parkinson’s disease. J Biol Chem 1999; 274: 19509–12. 97 Choi W, Zibaee S, Jakes R, et al. Mutation E46K increases phospholipid binding and assembly into filaments of human α-synuclein. FEBS Lett 2004; 576: 363–68. 98 Lee HJ, Patel S, Lee SJ. Intravesicular localization and exocytosis of α-synuclein and its aggregates. J Neurosci 2005; 25: 6016–24. 99 Jang A, Lee HJ, Suk JE, Jung JW, Kim KP, Lee SJ. Non-classical exocytosis of α-synuclein is sensitive to folding states and promoted under stress conditions. J Neurochem 2010; 113: 1263–74. 100 Emmanouilidou E, Melachroinou K, Roumeliotis T, et al. Cell-produced α-synuclein is secreted in a calcium-dependent manner by exosomes and impacts neuronal survival. J Neurosci 2010; 30: 6838–51. 101 Ahn KJ, Paik SR, Chung KC, Kim J. Amino acid sequence motifs and mechanistic features of the membrane translocation of α-synuclein. J Neurochem 2006; 97: 265–79.

1138

102 Lee HJ, Suk JE, Bae EJ, Lee JH, Paik SR, Lee SJ. Assembly-dependent endocytosis and clearance of extracellular α-synuclein. Int J Biochem Cell Biol 2008; 40: 1835–49. 103 Sung JY, Kim J, Paik SR, Park JH, Ahn YS, Chung KC. Induction of neuronal cell death by Rab5A-dependent endocytosis of α-synuclein. J Biol Chem 2001; 276: 27441–48. 104 Liu J, Zhang JP, Shi M, et al. Rab11a and HSP90 regulate recycling of extracellular α-synuclein. J Neurosci 2009; 29: 1480–85. 105 Danzer KM, Haasen D, Karow AR, et al. Different species of α-synuclein oligomers induce calcium influx and seeding. J Neurosci 2007; 27: 9220–32. 106 Danzer KM, Krebs SK, Wolff M, Birk G, Hengerer B. Seeding induced by α-synuclein oligomers provides evidence for spreading of α-synuclein pathology. J Neurochem 2009; 111: 192–203. 107 Luk KC, Song C, O’Brien P, et al. Exogenous α-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells. Proc Natl Acad Sci USA 2009; 106: 20051–56. 108 Waxman EA, Giasson BI. A novel, high-efficiency cellular model of fibrillar α-synuclein inclusions and the examination of mutations that inhibit amyloid formation. J Neurochem 2010; 113: 374–88. 109 Jellinger KA. Formation and development of Lewy pathology: a critical update. J Neurol 2009; 256 (suppl 3): 270–79. 110 Choi DY, Liu M, Hunter RL, et al. Striatal neuroinflammation promotes Parkinsonism in rats. PLoS One 2009; 4: e5482. 111 Gao HM, Kotzbauer PT, Uryu K, Leight S, Trojanowski JQ, Lee VM. Neuroinflammation and oxidation/nitration of α-synuclein linked to dopaminergic neurodegeneration. J Neurosci 2008; 28: 7687–98. 112 Hirsch EC, Hunot S. Neuroinflammation in Parkinson’s disease: a target for neuroprotection? Lancet Neurol 2009; 8: 382–97. 113 Fauré J, Lachenal G, Court M, et al. Exosomes are released by cultured cortical neurones. Mol Cell Neurosci 2006; 31: 642–48. 114 Agnati LF, Guidolin D, Baluska F, et al. A new hypothesis of pathogenesis based on the divorce between mitochondria and their host cells: possible relevance for the Alzheimer’s disease. Curr Alzheimer Res 2010; 7: 307–22. 115 Kuo YM, Li Z, Jiao Y, et al. Extensive enteric nervous system abnormalities in mice transgenic for artificial chromosomes containing Parkinson disease-associated α-synuclein gene mutations precede central nervous system changes. Hum Mol Genet 2010; 19: 1633–50. 116 Olanow CW, Stern MB, Sethi K. The scientific and clinical basis for the treatment of Parkinson disease. Neurology 2009; 72: S1–136. 117 Olanow CW, Rascol O, Hauser R, et al. A double-blind, delayed-start trial of rasagiline in Parkinson’s disease. N Engl J Med 2009; 361: 1268–78. 118 Eller M, Williams DR. Biological fluid biomarkers in neurodegenerative parkinsonism. Nat Rev Neurol 2009; 5: 561–70.

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