Visanji et al. Acta Neuropathologica Communications 2013, 1:2 http://www.actaneurocomms.org/1/1/2

REVIEW Open Access The prion hypothesis in Parkinson's disease: Braak to the future Naomi P Visanji1, Patricia L Brooks2, Lili-Naz Hazrati2 and Anthony E Lang1*

Abstract Parkinson’s disease (PD) is a progressive neurodegenerative disorder typified by the presence of intraneuronal inclusions containing aggregated alpha synuclein (αsyn). The progression of parkinsonian pathology and clinical phenotype has been broadly demonstrated to follow a specific pattern, most notably described by Braak and colleagues. In more recent times it has been hypothesized that αsyn itself may be a critical factor in mediating transmission of disease pathology from one brain area to another. Here we investigate the growing body of evidence demonstrating the ability of αsyn to spread transcellularly and induce pathological aggregation affecting by permissive templating and provide a critical analysis of some irregularities in the hypothesis that the progression of PD pathology may be mediated by such a prion-like process. Finally we discuss some key questions that remain unanswered which are vital to determining the potential contribution of a prion-like process to the pathogenesis of PD. Keywords: Prion, Parkinson's disease, Braak staging, Alpha synuclein

Braak staging of Parkinson’s disease The Braak model of PD staging hinges on the notion Braak and colleagues devised their widely accepted staging that Lewy pathology is not random: vulnerable sites in the system of PD progression by assessing the regional distri- brain are affected in a predictable topographic sequence bution of αsyn immunoreactive structures in the brains of (Figure 1). In support of this, Braak and colleagues have 110 αsyn positive subjects (69 incidental and 41 clinically demonstrated that the cell types in the central nervous diagnosed PD patients). According to the Braak model, system (CNS) exhibiting a propensity for developing Lewy the disease process commences in the lower in pathology share common features. Specifically, projection the dorsal motor nucleus of the vagus nerve (DMV), as neurons with a long, thin, unmyelinated or poorly myelin- well as anterior olfactory structures. The disease then ated axons are particularly susceptible to developing ascends rostrally from the DMV through susceptible re- lesions [3,4]. In agreement with this, cortical and subcor- gions of the medulla, pontine tegmentum, midbrain, and tical projection neurons giving rise to long, sturdy, heavily basal forebrain, eventually reaching the cerebral cortex myelinated axons appear to be resistant to pathology [3]. (Figure 1) [1]. This is a non-random, progressive process, According to the Braak model, it is this apparent selective with specific nuclei and neuronal types giving rise to the vulnerability of specific neuronal types that accounts for development of Lewy pathology in a stereotypic pattern. the predictable regional spread of Lewy pathology ob- As the pathology advances upwards from the brainstem, served in PD. both the severity of the lesions and the clinical manifesta- Although the Braak model outlines a pathogenic process tions of the disease increase [2]. that initiates in the brainstem and advances rostrally, it is unclear if the brainstem is the site of the initial disease process. Indeed it is known that PD extends beyond the boundaries of the CNS, affecting the peripheral nervous * Correspondence: [email protected] system (PNS), and in particular the enteric nervous system 1Division of Patient Based Clinical Research, Toronto Western Research Institute, and the Edmond J. Safra Program in Parkinson’s Disease, Toronto (ENS). This largely stems from the clinical observation Western Hospital, McLaughlin Pavilion, 7th Floor Rm 7-403, 399 Bathurst that PD patients frequently have a wide range of non- Street, Toronto, Ontario M5T 2S8, Canada motor symptoms related to dysfunction of the PNS, which Full list of author information is available at the end of the article

© 2013 Visanji et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Visanji et al. Acta Neuropathologica Communications 2013, 1:2 Page 2 of 12 http://www.actaneurocomms.org/1/1/2

Figure 1 Staging of Lewy pathology according to the Braak model. Schematic summarizing the progression of Parkinson’s disease as proposed by Braak and colleagues [1]. According to the Braak model, αsyn deposits in specific brain regions and neuronal types giving rise to Lewy pathology in a stereotypic, temporal pattern that ascends caudo-rostrally from the lower brainstem (including the dorsal motor nucleus of the vagus nerve in the medulla then the coeruleus-subcoeruleus complex, , gigantocellular reticular nucleus in the medulla aand pons) through susceptible regions of the midbrain ( and the pedunculopontine tegmental nucleus) and forebrain (e.g., ) and into the cerebral cortex (e.g., anteromedial temporal mesocortex, cingulate cortex and later neocortical structures). It is hypothesized that the disease initiates in the periphery, gaining access to the CNS through retrograde transport along projection neurons from the gastrointestinal tract. As the disease progresses, the severity of lesions in the susceptible regions increases. often precede the motor symptoms of PD. In support of of the brain. Instead, they propose that the unknown this, Lewy pathology is found throughout the PNS in both pathogen gains access to the brain through the gastric clinically diagnosed PD patients [5] as well as cases of clin- system. The gastric route would fit with the early in- ically asymptomatic incidental disease (ILBD) volvement of the ENS in PD. Like olfactory impairment, [6-8]. The finding of Lewy Bodies in the brains of asymp- gastrointestinal dysfunction is common in PD and tomatic individuals without parkinsonism (i.e., ILBD) is of seems to emerge early in the disease course [11], often particular interest as these cases are thought to represent preceding clinical parkinsonism by many years [12]. presymptomatic PD in the very beginning stages of the Lewy pathology has long been known to occur in disease process [7]. the gastrointestinal tract of PD patients and is well- Braak and colleagues have put forth the notion that documented in early, mid- and late PD [13-17]. Its pres- the pathogenic process of PD begins when an environ- ence in the premotor phase of the disease has been mental insult enters the body and subsequently gains ac- supportedbyarecentstudyofthreepatientswhowere cess to the CNS, where it then spreads trans-synaptically found to have αsyn staining in bowel biopsy samples from one vulnerable brain region to the next [3,9]. This obtained 2-5 years before they presented with signs of “dual-hit” hypothesis postulates that the unidentified PD; such staining was not seen in 23 healthy controls neurotropic pathogen enters the brain through both a [16]. In accordance with the Braak model, the cells in nasal and a gastric route. The nasal route is used to ex- the ENS that are vulnerable to Lewy pathology share the plain the early involvement of olfactory structures, as key characteristics of those vulnerable in the CNS (i.e. well as the olfactory dysfunction that is common in early projection neurons with a long, thin, unmyelinated PD [3,9]. In support of this, Lewy pathology is found in axon) [18]. If the ENS is the site of induction for a po- both the anterior olfactory nucleus as well as olfactory tential PD-causing agent, a pathogenic insult entering bulb mitral cells, the projection neurons that receive in- the body via the gastrointestinal tract must then gain ac- put from the olfactory epithelium [1,10]. cess to the CNS. The visceromotor projection neurons Despite the initial involvement of the olfactory system, of the DMV give rise to preganglionic fibers that innerv- Braak et al. [3] do not consider olfactory structures to ate the ENS. Braak et al. [3] have proposed it is these be the point of departure for Lewy pathology to the rest unmyelinated vagal preganglionic neurons that could Visanji et al. Acta Neuropathologica Communications 2013, 1:2 Page 3 of 12 http://www.actaneurocomms.org/1/1/2

providetherouteforapathogentoberetrogradely like process [25,26]. Furthermore, in contrast to Braak et transported from the ENS into the CNS. This notion is al.’s thinking, the proposal was made that αsyn itself may supported by the observation that these vagal projection be the pathogenic factor underlying the spread of the dis- neurons are some of the first cells in the brain to display ease. Adding weight to this proposal, similar prion-like Lewypathology[3].Itmaythereforebethatapatho- mechanisms have been proposed for other proteins in- genic agent enters the body and gains access to the volved in several other neurodegenerative diseases, includ- gastrointestinal tract, invades vulnerable neurons in the ing Alzheimer’s, Huntington’s and amyotrophic lateral ENS and is subsequently retrogradely transported to sclerosis [27-29]. the CNS through vagal preganglionic fibers. Indeed, two recent reviews have proposed a similar mechanism by In vitro evidence which misfolded proteins in a host of neurodegenerative Prions are composed of the PrPsc protein, a misfolded diseases may spread from cell-to-cell within the ENS form of endogenous PrPc, and underlie disorders such eventually reaching the CNS [19,20]. Thus, within the as Creutzfeldt-Jakob disease, bovine spongiform enceph- ENS, misfolded proteins, including αsyn, undergo spon- alopathy and scrapie [30]. The conversion of alpha hel- taneous glycation to form advanced glycation end prod- ical PrPc to the β-sheet rich PrPsc confers an infectivity ucts (AGEs), which are excreted via exosomes. Once in that is the defining feature of a prion [31]. Indeed, as all the extracellular space, AGEs are recognized by cell other known infectious agents contain either DNA or surface receptors for advanced glycation end products RNA, the term prion, derived from the words protein- (RAGEs) which act to internalize the misfolded AGE. aceous and infectious, was given to this unique PrPsc AGE-RAGE binding activates multiple intracellular sig- protein [32]. Though the precise molecular mechanisms naling pathways including a positive feedback loop underlying the propagation of the prion protein are un- whereby there is an increased cell surface expression of known, it is universally accepted that PrPsc acts as a RAGE along with induction of oxidative stress and in- template upon which native PrPc is refolded into PrPsc. flammation. Through this positive feedback, there is Extensive in vitro studies have shown that, when incu- cell-to-cell spreading of disease throughout the enteric bated at 37°C, monomeric αsyn forms fibrils reminis- wall, and perhaps beyond. In the context of Parkinson’s cent of those contained in Lewy bodies in PD [33-35]. disease, it is proposed that once in the DMV, the patho- Furthermore, existing fibrils can act as seeds, promot- gen follows an ascending course, spreading from one ing fibrillization of surrounding monomeric αsyn [36]. susceptible cell group to the next (Figure 1). Closer inspection of the nature of the seed-induced fi- brils has revealed that addition of seeds produced from Parkinson’s disease as a prion disorder αsyn bearing the A30P mutation to wildtype monomeric Transplanted dopamine neurons in humans αsyn leads to the generation of fibrils with the same The Braak model puts forth evidence for the progressive, character as A30P fibrils [37]. Thus it would appear that stereotypic spread of an unknown pathogenic insult the assembly of wildtype fibrils induced by A30P seeds from one affected brain region to the next. Building on involves a conformational change in wildtype αsyn to this, there has been considerable interest in recent years that of A30P. This process is remarkably similar to that in the possibility that this disease progression is medi- described for the templated conversion of PrPc to PrPsc. ated in a prion-like fashion, with the spread and seeding In the same study it was noted that the addition of of sequentially involved brain areas by misfolded pro- wildtype seeds to monomeric A30P αsyn did not involve teins. The suggestion that PD may be a prion disorder a conversion to the wildtype fibril. This feature of tem- stems from the observation of Lewy body pathology in plated conversion seems to apply to the A30P αsyn mu- embryonic dopamine neurons transplanted into the pu- tation responsible for a small number of familial cases of tamen of human PD patients [21-24]. In four separate PD but not to the wildtype form of αsyn responsible for case reports, autopsies performed 10-22 years post- the majority of cases of PD. Thus, in the context of this transplantation found evidence of αsyn-positive, Lewy- review regarding PD we would define a prion-like mech- body-like inclusions in grafted human dopamine anism of disease propagation as a mechanism by which neurons. Such inclusions are not normally seen in neurons native protein with rich α-helical structure is refolded of such a young age. These inclusions stained positively for into a toxic form with high β-sheet structure trigger- thioflavin-S (indicating the presence of β-sheets) and ubi- ing the misfolding of further native protein in a self- quitin and the affected transplants showed reduced dopa- perpetuating process. A form of the protein capable of mine transporter and tyrosine hydroxylase levels. In triggering this process can be conferred from one cell to concert with the discussed evidence of a sequential staging another, thus inducing progressive neurodegeneration. of PD pathology, these observations lead to the proposal Beyond the test tube, a series of in vitro tissue culture thattheprogressionofPDmaybemediatedbyaprion- experiments have demonstrated the following features Visanji et al. Acta Neuropathologica Communications 2013, 1:2 Page 4 of 12 http://www.actaneurocomms.org/1/1/2

that make αsyn an attractive candidate as the pathogenic surrounding cells and induce a pathological aggregative factor implicated in the prion-like spread of PD path- response [42,43]. These cellular mechanisms, by which ology: 1) Fibrillar αsyn has been shown to be transported αsyn can be both released from and taken up by neurons, via axonal transport [38]. 2) Monomeric and aggregated are illustrated in Figure 2. Thus, at the in vitro level there forms of αsyn, contained in cytosolic vesicles, are re- is compelling evidence that αsyn is capable of spreading leased from neurons by an unconventional exocytotic an aggregation-inducing disease-like pathology from one mechanism [39]. 3) Aggregated forms of αsyn (fibrillar to another. and non-fibrillar oligomers) are taken up into neurons via conventional endocytosis, whereby they can move In vivo studies through the endosomal pathway and are eventually More recent studies have investigated the translation of degraded by the lysosome [40]. 4) Transduction of these findings in vivo. Desplats et al. grafted green fluor- preformed fibrillar αsyn into cells overexpressing αsyn escent protein (GFP) labelled mouse cortical neuronal results in the formation of insoluble intracellular inclu- stem cells (MCNSCs) into the of mice sions recapitulating several key features of Lewy bodies overexpressing human αsyn [43]. Using double immuno- including size, subcellular localisation, β-pleated sheet fluorescence, these authors demonstrated that seven conformation, and hyperphosphorylation and polyu- days post grafting ~2.5% of MCNSCs had taken up αsyn biquitination of constituent αsyn [41]. Furthermore, from the host tissue; 28 days post grafting this number using C-Myc tagged preformed αsyn fibrils, it has been had risen to ~15%. Although these data demonstrate demonstrated that soluble endogenous αsyn becomes host to graft transmission of αsyn in vivo, ultrastructural recruited to these insoluble intracellular inclusions even- analysis of inclusion bodies within grafted cells found no tually comprising the major component [41]. Several evidence of the fibril or aggregate formation following other studies have employed co-cultures in a wide var- infiltration by αsyn that was evident in their in vitro iety of cell lines to demonstrate the ability of exogenous studies. A more recent study mimicked more closely the αsyn, be it monomeric, oligomeric or fibrillar, to infiltrate clinical situation grafting murine mesencephalic neurons

Cell injury 1 and leaking 2 Transmembrane

Endocytosis & Exocytosis 3 Transynaptic 6

Nanotube 5

Exosome 4 release

Figure 2 Potential mechanisms of neuron-to-neuron transmission of synuclein. Alpha synuclein can be released into the extracellular space via (1) leakage from injured cells with compromised membrane integrity. Extracellular synuclein could then directly translocate the cell membrane and gain access to neighboring neurons (2). Synuclein can also be transmitted from cell-to-cell via conventional exocytosis and endocytosis (3). Synuclein can be packaged into exosomes which are released and taken up by surrounding cells (4).Tunneling nanotubes can form a direct connection between two cells potentially allowing synuclein to transfer freely from one cell to another (5). Finally, synuclein could be transmitted by direct synaptic contact (6). Visanji et al. Acta Neuropathologica Communications 2013, 1:2 Page 5 of 12 http://www.actaneurocomms.org/1/1/2

into the striatum of mice overexpressing human αsyn this study, brain homogenates from aged transgenic [42]. Six months post grafting a small number of grafted mice, containing significant levels of aggregated A53T cells were found to contain small human (i.e., host) αsyn human αsyn (the first mutation of αsyn discovered in positive punctae. These authors also demostrated that humans), were injected into the and striatum the structural form of αsyn had no effect on the ability of younger animals long before pathology normally de- for cellular uptake in vivo. Thus monomeric, oligomeric velops. 30 days post injection some level of αsyn path- and fibrillar recombinant αsyn were all found in neur- ology was evident in the vicinity of the injection sites. onal cells following a stereotaxic injection in rats. Inter- However, 90 days post injection αsyn pathology, reminis- estingly, this phenomenon of αsyn uptake is not limited cent of Lewy bodies and surrounded by astrogliosis and to neurons, but has also been demonstrated in astrocytes microgliosis, was widespread throughout the brain. both in cell culture and in vivo, whereupon it triggers an These inclusions were not apparent in age-matched con- inflammatory response in the recipient cells [44]. As in- trol animals innoculated with saline or with homoge- flammation is increasingly implicated in a host of neuro- nates prepared from younger animals. The critical role degenerative diseases, this process may represent an of host αsyn to this process was also demonstrated by alternative means by which αsyn can mediate the pa- the observed lack of pathology following innoculation in thological progression of PD. A recent study using a αsyn null mice. In addition, detailed examination dem- transgenic mouse model of synucleinopathy (TgM83), onstrated a recruitment of endogenous murine αsyn to demonstrated that inoculation of young asymptomatic the pathological inclusions further supporting a permis- mice with brain homogenates prepared from older sive templating prion-like process. Mapping studies illus- symptomatic mice accelerated the presence of both αsyn trated that it was those brain regions with the most hyperphosphorylated at serine 129 and aggregated αsyn neuronal connections to the sites of innoculation that and significantly decreased survival time [45]. Further- displayed the most severe pathology (frontal cortex and more, this hastened pathology was absent in αsyn knock- ) and that there was a relative sparing of re- out animals inoculated with the same brain homogenates gions close by but lacking direct neuronal innervation. prepared from older symtomatic transgenic mice. This Indeed, based on evidence from their studies the authors suggests a strong role for endogenous host αsyninthe suggest that the distribution of αsyn pathology following transmission of pathology from affected to unaffected sites innoculation is suggestive of a trans-synaptic spread. and is consistent with a prion-like mechanism of disease This contrasts with the recent in vitro findings of propagation. Freundt et al (2012), who found that axon to soma In the aforementioned studies there was no evidence transfer of αsyn did not require synaptic contacts [38]. of a spread of pathologic αsyn to more widespread areas Although there is now considerable in vivo evidence of of the brain distal to the sites of application. However, cell-to-cell transmission of αsyn and recruitment of en- two recent studies have expanded upon this. Angot dogenous αsyn to inclusions reminiscent of Lewy bodies, et al., (2012) used a viral vector (AAV2/6-hu αsyn) to a crucial outstanding issue is establishing whether there engineer rat nigral neurons to express human αsyn is a link between this and the process of neurode- which, over time, was transported from nigral cell bodies generation. A recent elegant study has suggested a to the terminals in the striatum [46]. These authors then causative link between progressive Lewy inclusion for- grafted rat ventral mesencephalic neurons into the mation and neurodegeneration of nigral dopaminergic striatum and observed transmission of human αsyn cells with correspoding deficit in striatal dopamine and expressed by the virally transduced nigrostriatal cells motor function [48]. The authors demonstrated a clear into the grafted tissue. Indeed, the extent of transfer of time and connectivity dependent spread of Lewy body- the virally expressed human αsyn was shown to be of a like pathology following intrastriatal injection of fibrillar much greater magnitude than that observed in previous αsyn in mice, with expression seen earliest in those brain studies examining the transfer of αsyn into grafted tis- regions highly innervated by the striatal injection site sue. Furthermore, human αsyn immunoreactivity within (cortical layers IV and V, and the olfactory bulb) and lat- the grafted cells was shown to be surrounded by a ter spread to more distally connected areas (ventral stri- strong immunoreactivity for rodent αsyn, suggesting that atum, thalamus, occipital cortex, and commisural and the human αsyn supplied by the host tissue had induced brainstem fibers). Pathology was particularly abundant a seeding effect in the rodent αsyn present in the grafted in the dopaminergic cells of the substantia nigra pars cells. This study demonstrated for the first time that compacta. Intriguingly, at 30 days post injection, ap- αsyn can induce a seeding effect in vivo following axonal proximately 30% of nigral cells contained αsyn-positive transport along the nigrostriatal pathway. However, a inclusions, which was accompanied by negligible cell more recent study by Luk et al [47] has demonstrated loss. At 100 days post injection, both the number of cells that this phenomenon can be even more widespread. In bearing αsyn-positive inclusions, the total number of Visanji et al. Acta Neuropathologica Communications 2013, 1:2 Page 6 of 12 http://www.actaneurocomms.org/1/1/2

nigral cells and striatal levels of dopamine, declined disease through the brain from these multiple and varied concommittantly. These data strongly suggest that ag- departure points. gregation of αsyn may directly precipitate the loss of Challenges also exist for the proposed “gut to brain” nigral dopaminergic neurons that is a fundamental char- spread of Lewy pathology. Rather than suggesting initial acteristic of PD. involvement of the ENS with subsequent spread to the DMV, Beach et al (2010) have advocated a "brain to gut" Challenges to reconciling Braak staging with a prion-like direction of transmission given their findings of a rostro- spread of pathology in PD caudal gradient of phosphorylated αsyn in the GI tract Clearly there is increasing evidence, both experimental and the known richest vagal innervation of the upper and clinical, demonstrating that exogenously applied gut [5]. The recent report of Del Tredici and Braak αsyn can infiltrate surrounding cells and initiate a PD- (2012) showing evidence for involvement of the spinal like pathological response. However, it remains to be cord only after involvement of the brain also raises ques- shown what initiates this process and whether it can ac- tion about the earliest involvement of at least some ENS count for disease progression in the human brain. Here regions [54]. we evaluate the prion hypothesis of PD progression tak- Another prerequisite of the Braak model is that the ing into account the current understanding of the severity of the lesions in the affected brain regions will neuroanatomy and pathology of PD and highlight some increase as the disease progresses [1]. It would therefore critical remaining questions that need to be addressed be expected that the DMV, Braak’s point of departure before accepting PD as a prion disorder. for Lewy pathology in the brain, would have more se- The conserved propagation patterns of αsyn pathology vere pathology than the rostral regions said to be af- described by the Braak model could indicate a prion-like fected later in the disease. This, however, is not true in spread of αsyn. According to Braak, vulnerable brain re- all cases. In one study, upwards of 65% of cases had gions are affected in a predictable sequence, progressing αsyn loads in the substantia nigra and in a stereotypical caudal-rostral pattern starting in the that were roughly equivalent to that found in the DMV lower brainstem. One could hypothesize that pathogenic [55]. Further still, there are reports of severe involve- αsyn is transported to anatomically connected brain re- ment of the substantia nigra without any prominent gions along the prescribed route of progression, seeding DMV pathology [56,57]. This lack of significant αsyn αsyn aggregation in each vulnerable region encountered. pathology in the DMV compared to more rostral struc- Whether the pattern and progression of pathology can tures cannot be explained by neuronal loss, as these be generalized to the predictable, sequential involvement cases had well preserved neurons. For the Braak-prion of vulnerable sites as Braak describes, however, remains hypothesis to account for these cases, pathogenic αsyn controversial. The Braak model postulates that Lewy would have to spread and seed very rapidly to all vulner- pathology in the lower brainstem is necessary for the able brain regions once the disease process is initiated, later appearance of Lewy pathology in more rostral which does not fit with the protracted disease course of structures [1]. However, this may be an artifact of their idiopathic PD. Alternatively, mechanisms underlying pre-selection of cases on the basis of involvement of seeding and subsequent aggregation and cell death may DMV. Multiple studies have challenged the Braak sta- be dissociated. ging scheme, reporting cases with inclusions throughout One possible explanation for the inconsistencies be- the brain but with preservation of medullary nuclei tween the Braak model and the conflicting reports may [8,49-51]. In fact, recent reports suggest that the Braak be that spreading of pathology can occur in both an an- system fails to classify upwards of 50% of αsyn immuno- terograde and retrograde direction. Braak and colleagues reactive cases [50,52,53]. The pathological regional het- initially hypothesized that while the olfactory system is erogeneity between PD cases suggests Braak’s proposed affected very early in the disease process, olfactory struc- pathway is not the only possible route of spread and tures are not the point of departure for Lewy pathology pathology may even emerge simultaneously in multiple to the rest of the brain, despite connections with cortical subcortical and cortical regions. Such cell autonomous and subcortical regions [1]. Subsequent observations protein misfolding would argue against a Braak model of from a growing number of groups, including Braak and PD staging. One might expect that a cell autonomous colleagues, however, have suggested that Lewy pathology process would better explain genetic causes of PD asso- may indeed progress along olfactory pathways [9,58-60]. ciated with Lewy bodies. However, that the site of initial Lerner and Bagic [26] subsequently expanded upon this emergence of disease may well be variable and perhaps by proposing a complementary hypothesis to the Braak multiple (possibly within certain anatomical constraints) model, in which they emphasize the possibility of an- leading to a heterogeneous phenotype, does not preclude terograde spread of the pathogen through the olfactory that a prion-like mechanism still might govern spread of tubercle and bulb to the brainstem, in addition to the Visanji et al. Acta Neuropathologica Communications 2013, 1:2 Page 7 of 12 http://www.actaneurocomms.org/1/1/2

retrograde spread from the dorsal motor nucleus of the It is vital to keep this issue in mind when also consid- vagus, as described by Braak et al. [1]. The addition of ering the selective susceptibility of certain cell types and this olfactory route of progression may reconcile some specific brain regions to Lewy pathology. While the discrepancies with the original Braak model. Although, Braak model describes projection neurons with a long, the bi-directional spread of a pathogen may be at odds thin, unmyelinated axons as the cell type that is prone with the Braak staging scheme, it is not inconsistent to Lewy pathology [3,4], it is not known why these neu- with the concept of neuronal spread of a pathogen in a rons are susceptible. Although many explanations could prion-like fashion. Indeed, Lerner and Bagic suggested be provided to account for differences between the hu- such a prion-like mechanism may underlie the neurode- man disease and animal models, it is interesting to note generative process in PD [26]. that the recent important study by Luk et al. [47], which A further explanation for the reported inconsistencies provides some of the strongest evidence to date for with the Braak model may be the clinical diversity of transcellular propagation of αsyn pathology, demon- PD. Different subtypes of PD may have different under- strated most marked involvement of heavily myelinated lying pathological patterns. In support of this, Halliday projections, including the internal capsule. Furthermore, et al. [61] found that PD patients with a younger age of it is not clear why some regions with Braak’s “vulner- onset and a long clinical course had pathology that fit able” cell types are affected whereas many other anatom- with the Braak model, whereas patients with older onset ically connected/neighboring regions with similar cell and shorter disease duration did not. This suggests that types are not. Such cryptic susceptibility is further clinical phenotype may predict the pattern of pathology highlighted in Braak’s detailed description of a non- that underlies PD. The fact that Braak and colleagues geographical pattern of Lewy pathology in specific sub- only selected cases in which the DMV was positive for cortical structures in earlier disease versus a more αsyn immunoreactivity may account for some of these diffuse spreading throughout the cortex in later stages apparent discrepancies, as they may have only examined [1]. It therefore seems that αsyn propagates differently a select subtype of PD. More recent large-scale neuro- according to the type of brain tissue it encounters; how- pathological studies tend to include all cases that are ever, it is not clear how or why this would be the case. positive for αsyn immunoreactivity, regardless of which To further complicate matters, according to the Braak brain regions were affected, and as a result may include model, the neuronal type prone to developing Lewy a more diverse profile of PD and its subtypes. It may pathology in PD is specific to PD and distinguishes PD therefore be that a prion-like spread of αsyn in a pattern from other synucleinopathies [3]. Therefore, the prion- predicted by the Braak model does indeed occur in PD like spread of αsyn must discriminate between cell types but only in certain subtypes of the disease. across different synucleinopathies. Furthermore, while A key outstanding issue when considering using Lewy abnormal aggregation of αsyn is the dominant patho- body pathology to define the progression of PD is regard- logical hallmark of synucleinopathies, the nature of αsyn ing whether the Lewy body itself is harmful or protective. aggregation is distinct between different disorders. For Traditionally, the presence of Lewy bodies in sites prone example, PD, Parkinson’s disease dementia (PDD) and to neurodegeneration has supported the dogma that the dementia with Lewy bodies (DLB) are characterized by Lewy body is detrimental to neuronal survival. However, αsyn deposits in neuronal Lewy bodies and Lewy in recent years many studies have suggested that Lewy neurites, whereas multiple system atrophy (MSA) is de- body formation is a protective cellular mechanism to se- fined by abnormal filamentous deposition of αsyn in the quester toxic αsyn aggregates and even aid their degrad- nuclei and cytoplasm of both oligodendrocytes and ation by an aggresome-like mechanism [62-65]. In some neurons [70-72]. If prion-like αsyn seeding plays an im- cases cells containing Lewy bodies have been described as portant role in the pathogenesis of neurodegenerative having a healthier morphology than neighboring Lewy disease then how and why αsyn behaves differently in body negative cells [66]. Furthermore, in vitro studies have different diseases must be addressed (see below). In this demonstrated that cell death can precede Lewy body for- context, using the Lewy body as a structural marker for mation [67] and in PD it has been shown that the majority the progression of the disease may not be appropriate. of cells undergoing apoptosis do not contain Lewy bodies For example Kramer et al., have demonstrated that, in [68]. As the Lewy body may in fact represent a cell that is contrast to the relatively small number of cortical Lewy resisting neurodegeneration, caution must be used in bodies, there are a number of small presynaptic αsyn ag- employing it to define disease progression. Indeed, a recent gregates in DLB, which may account for the significant study examining the relationship between nigral cell loss, cognitive impairment in this disease [63]. Similarly, distribution of Lewy bodies and disease duration in PD Milber et al found evidence of neuronal dysfunction pre- found no evidence to support a correlation between Lewy ceding Lewy pathology in the nigra in PD [73]. These body distribution or density and neurodegeneration [69]. data suggest that synaptic dysfunction can occur without Visanji et al. Acta Neuropathologica Communications 2013, 1:2 Page 8 of 12 http://www.actaneurocomms.org/1/1/2

the presence of Lewy bodies per se and that perhaps even determine whether the final disease presentation is using the Lewy body alone as a marker of pathological PD or that of another synucleinopathy. Furthermore, if progression is not sufficient to predict the spread of indeed αsyn is responsible for a prion-like propagation disease. of the disease, understanding the relatively delayed Finally, if the progression of PD can be explained by a propagation of αsyn pathology post symptom onset, as Braak-prion-like spread, a critical question that must be compared to the rapid process of conventional prion dis- addressed is what initiates the process in the first place? orders once symptomatic, could inform the development Braak’s dual-hit hypothesis postulates that an environ- of therapies to slow the progression of both PD and mental neurotropic insult is responsible [3,9]. While the prion diseases. Braak model proposes a viral pathogen, a recent study in We have reviewed considerable experimental evidence mice demonstrated that a peripherally administered en- supporting the premise that αsyn can be released from vironmental toxin (i.e. rotenone) may induce a Braak- and enter neighbouring neurons, and induce aggregation like spread of αsyn pathology in the CNS [74]. Although of native αsyn in vitro. However, there are some caveats to this has not been shown in the human condition, such these experimental settings that necessitate caution in ex- results do support the hypothesis of an environmental trapolating these data to the human brain in PD. Firstly, insult triggering the Braak’s cascade of pathology seen in not all tissue culture studies find that supplying exogenous PD. What is not clear, however, is how an environmental αsyn in the surrounding media is sufficient for infiltration insult could induce the involvement of systems outside into cells. Indeed in order to get meaningful internalisa- of Braak’s continuous chain of projection neurons that tion Luk and colleagues had to make use of cationic lipo- are presumably isolated from the environment. Perhaps somes to facilitate entry of αsyn into the cell [41]. A one of the most challenging facts to reconcile with this further caution is required in interpreting the significance theory is the very early involvement of the cardiac sym- of data demonstrating the transmission of αsyn in animal pathetic nerves seen in PD and in the presumed pre- studies employing mice genetically engineered to over- clinical state of incidental Lewy body disease [75]. express αsyn, thus presumably enhancing any host- to-graft transmission, although more recent work Other challenges to the prion hypothesis demonstrates similar changes in non-transgenic animals In addition to the issues outlined above, there are critical [48]. Furthermore, the applicability of animal studies questions in the defining of PD as a prion disorder that employing direct stereotaxic application of αsyn to the must be addressed. One substantial issue is indeed se- brain to the initiation and propagation of idiopathic PD mantic. As the word prion itself derives from the words remains to be shown. Although the recent results of Luk “protein” and “infectious”, to define PD as a prion dis- and colleagues in non-transgenic animals [48] are impres- order mediated by αsyn, we must demonstrate that αsyn sive and very promising, it must be admitted that the rele- is capable of “infectivity”. It has been suggested that, due vance of using very high concentrations of preformed to an apparent lack of microbiological transmissibility artificial αsyn fibrils to human PD is unknown. Thus, fur- and propagation within communities, all amyloidogenic ther studies are required to demonstrate unequivocally proteins capable of permissive templating be termed that enough αsyn can be released and infiltrate surround- “prionoids” [27]. It remains possible that difficulties in ing tissues, by physiological means, to induce aggregation verifying an infectious nature of αsyn may be due to the in vivo. Secondly, there is controversy in the literature re- length of time required for the process to occur in vivo. garding the structural assembly of αsyn required to induce Indeed in human post mortem studies, Lewy-like path- inclusion formation. In the many studies employing ology was not observed in grafted tissue 18 months post co-culture and those stereotaxically implanting cells graft [76]. Accumulation was apparent four years post overexpressing αsyn, the structural nature of the infiltrat- graft, but not until 10-14 years post graft was full Lewy ing αsyn is unclear [42,77]. Tissue culture studies have pathology evident [21]. There are likely several features shown that monomeric, oligomeric and fibrillar αsyn can of both the host and the "infectious" agent that influence be taken up by cells [42]. Thus, in co-culture experiments, the cell to cell transfer, effectiveness of permissive tem- and those stereotaxically implanting cells overexpressing plating, and the distribution and nature of the resulting αsyn, it could be any of these structural forms leading to pathology. For example, in classical prion diseases, the the aggregate formation within the host. It should also be biochemical nature of the prion protein PrPsc and the emphasized that much of this discussion hinges on the genetic makeup of the host (M/V at codon 129) have concept that the insoluble fibrillar protein aggregates are major influences on the nature and distribution of the responsible for cell death, whereas there is increasing evi- underlying pathology (and as a consequence the clinical dence implicating soluble oligomers as potential culprits manifestations). The same types of processes may influ- in the pathogenesis of neurodegenerative diseases such as ence the distribution of Lewy body pathology in PD and PD [78]. Thirdly, the degradation of different assemblies Visanji et al. Acta Neuropathologica Communications 2013, 1:2 Page 9 of 12 http://www.actaneurocomms.org/1/1/2

of internalised αsyn requires further evaluation since some and the clinical presence of parkinsonism. While patho- cell culture studies suggest that exogenously applied logical studies on genetic forms of PD are limited, it is monomeric αsyn is quickly degraded by the lysosome clear that at least some of these patients do not show whereas fibrillar αsyn endures [41]. Conversely, other cell classic Lewy pathology. Indeed, in the case of LRRK2 culture studies have found that oligomeric αsyn is de- mutations even within a single family, a spectrum of graded by the lysosome, and that monomeric αsyn rapidly pathology has been observed among family members and directly translocates to cellular membranes thus with manifesting PD, ranging from those completely resisting lysosomal degradation and is therefore the more lacking any synuclein pathology, to those with signifi- likely disease spreading form [40]. Finally, although the re- cant αsyn aggregation [80-84]. Furthermore a recent cent studies by Angot et al and Luk et al. [46,47] have study comparing neuronal dysfunction in relation to demonstrated a putative trans-synaptic spread of αsyn Braak stage in patients with PD, individuals with ILBD pathology in vivo, crucially, the transfer of αsyn through and controls has demonstrated that both cellular dys- the neuroanatomical pathways implied in Braak’sstaging function (loss of tyrosine hydroxylase) and neuro- scheme has not yet been shown. Indeed in the study of degeneration can precede Lewy body pathology in ILBD Luk et al. [47], stereotaxically applied αsyn was demon- [73]. These findings are in direct contrast to those of strated to travel in both an anterograde and retrograde Luk and colleagues [47] who suggest quite the reverse, manner, being evident in both the thalamus and the that synuclein pathology precedes cell loss. Clearly, fur- substantia nigra pars compacta following a striatal applica- ther studies will need to be performed to address these tion in mice. As mentioned above, such a bidirectional concerns. spread seems at odds with Braak staging in PD, although does not argue against a prion-like disease mechanism. Conclusions Future in vitro studies should be performed to investigate Recent years have seen a tremendous surge of interest in the ability of αsyn applied in the ENS or olfactory bullb to the possibility that neurodegenerative diseases, including spread along the pathways described by Braak staging. PD, could develop and progress via non-cell autono- The occurrence of Lewy bodies in transplanted fetal mous means, spreading by transcellular mechanisms nigral neurons in patients with PD has been a strong with seeding and subsequent permissive templating, in a incentive to consider a prion-like spread of pathology. prion-like fashion. If these mechanisms are indeed inte- However, not all case reports find pathology in grafted gral to the pathogenesis of PD and other diseases, this tissue [79]. In those that do, Lewy bodies are present could have important therapeutic implications, as re- “within a minority” of grafted cells [22] although it cently discussed in more detail elsewhere [85,86]. These should be noted that the proportion of transplanted possibilities include treatments designed to block the re- neurons reported to contain Lewy bodies is almost lease or uptake of the pathogenic protein, increase its identical to the proportion of Lewy body positive neu- extracellular clearance by microglia, inhibit protein as- rons in the substantia nigra in Parkinson’s disease. Des- sembly by, for example, nanomaterials [87] and increase pite this, it must be acknowledged that Lewy bodies degradation of aggregates, for example, by lysosomes. contain much more than aggregated αsyn and occur in Currently available drugs may have additional inhibitory many disorders in which αsyn is not believed to be a effects on the endocytosis mechanism that mediates the primary contributor to the overall pathogenesis. A internalization of αsyn oligomers [88]. Another approach moreplausibleexplanationmaybethatmultipleother that could be available for clinical study in the near fu- toxic factors that are known to exist in the PD brain, ture might be the use of monoclonal antibodies or such as proinflammatory cytokines, reactive oxygen immunization therapy, which might be expected to have species (supported by the extent of neuromelanization greater potential for benefit if there is indeed an extra- uncharacteristic of such young neurons [21]) and pro- cellular phase of αsyn transmission. In light of the fact apoptotic factors, may damage the cell leading to a dis- that there is currently no available therapy to slow or ruption of cellular processes and explain the presence halt the pathological progression of PD, the potential of Lewy bodies in previously healthy grafted dopamin- therapeutic implications of a therapy targeted at the ergic cells. This is not to say that αsyn cannot be impli- mechanism responsible for the transmission of path- cated in such alternate cell death mechanisms. For ology from one region to another are formidable. Thus, example, as previously discussed, αsyncanbetakenup it is vital that further in vitro and in vivo studies are into microglia and induce the production and excretion performed to validate the potential involvement of αsyn of inflammatory mediators. as a prion-like factor and tease out the mechanisms by A final challenge to the prion-like propagation of αsyn which αsyn may be responsible for disease progression pathology accounting for PD is the observation that in the human condition. However, in view of the unre- Lewy pathology is not necessary for nigral degeneration solved challenges highlighted in this review, caution Visanji et al. Acta Neuropathologica Communications 2013, 1:2 Page 10 of 12 http://www.actaneurocomms.org/1/1/2

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Lebouvier T, Neunlist M, Bruley des Varannes S, Coron E, Drouard A, advisor for Abbott, Allon Therapeutics, Astra Zenica, Avanir Pharmaceuticals, N'Guyen JM, Chaumette T, Tasselli M, Paillusson S, Flamand M, et al: Colonic Biovail, Boerhinger-Ingelheim, BMS Cephalon, Ceregene, Eisai, GSK, Lundbeck biopsies to assess the neuropathology of Parkinson's disease and its A/S, Medtronic, Merck Serono, MSD, Novartis, Santhera, Solvay, and Teva; relationship with symptoms. PLoS One 2010, 5:e12728. received grants from Canadian Institutes of Health Research, Dystonia 16. Shannon KM, Keshavarzian A, Mutlu E, Dodiya HB, Daian D, Jaglin JA, Medical Research Foundation, Michael J. Fox Foundation, National Parkinson Kordower JH: Alpha-synuclein in colonic submucosa in early untreated Foundation, Parkinson Society of Canada, and Ontario Problem Gambling Parkinson's disease. Mov Disord 2012, 27:709–715. Research Centre; received publishing royalties from Saunders, Wiley-Blackwell, 17. 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Natale G, Pasquali L, Paparelli A, Fornai F: Parallel manifestations of Author details neuropathologies in the enteric and central nervous systems. 1Division of Patient Based Clinical Research, Toronto Western Research Neurogastroenterol Motil 2011, 23:1056–1065. Institute, and the Edmond J. Safra Program in Parkinson’s Disease, Toronto 21. Kordower JH, Chu Y, Hauser RA, Freeman TB, Olanow CW: Lewy body-like Western Hospital, McLaughlin Pavilion, 7th Floor Rm 7-403, 399 Bathurst pathology in long-term embryonic nigral transplants in Parkinson's Street, Toronto, Ontario M5T 2S8, Canada. 2Tanz Centre for Research in disease. Nature medicine 2008, 14:504–506. Neurodegenerative Diseases, University of Toronto, 6 Queens Park Crescent 22. Kordower JH, Chu Y, Hauser RA, Olanow CW, Freeman TB: Transplanted West, Toronto, Ontario M5S 3H2, Canada. dopaminergic neurons develop PD pathologic changes: a second case report. Mov Disord 2008, 23:2303–2306. 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doi:10.1186/2051-5960-1-2 Cite this article as: Visanji et al.: The prion hypothesis in Parkinson's disease: Braak to the future. Acta Neuropathologica Communications 2013 1:2.

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