Hypothesis: A role for EBV-induced molecular mimicry in Parkinson's disease

Parkinsonism and Related Disorders xxx (2014) 1e10

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Point of view

Hypothesis: A role for EBV-induced molecular mimicry in Parkinson’s disease John M. Woulfe a, b, c, *, Madison T. Gray a, c, Douglas A. Gray a, c, David G. Munoz d, Jaap M. Middeldorp e a

Centre for Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, Canada Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Canada Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Canada d Department of Pathology, St. Michael’s Hospital, Toronto, Canada e Department of Pathology, VU Medical Center, Amsterdam, The Netherlands b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 October 2013 Received in revised form 18 February 2014 Accepted 22 February 2014

Current concepts regarding the pathogenesis of Parkinson’s disease support a model whereby environmental factors conspire with a permissive genetic background to initiate the disease. The identity of the responsible environmental trigger has remained elusive. There is incontrovertible evidence that aggregation of the neuronal protein alpha-synuclein is central to disease pathogenesis. A novel hypothesis of Parkinson’s pathogenesis, articulated by Braak and colleagues, implicates a pathogen acting in the olfactory mucosa and gastrointestinal tract as the inciting agent. In this point-of-view article, we hypothesize that a-synuclein aggregation in Parkinson’s disease is an Epstein-Barr virus (EBV)induced autoimmune phenomenon. Specifically, we have shown evidence for molecular mimicry between the C-terminal region of a-synuclein and a repeat region in the latent membrane protein 1 encoded by EBV. We hypothesize that, in genetically-susceptible individuals, anti-EBV latent membrane protein antibodies targeting the critical repeat region cross react with the homologous epitope on a-synuclein and induce its oligomerization. Consistent with the Braak’s proposed pattern of spread, we contend that axon terminals in the lamina propria of the gut are among the initial targets, with subsequent spread of pathology to the CNS. While at this time, we can only provide evidence from the literature and preliminary findings from our own laboratory, we hope that our hypothesis will stimulate the development of tractable experimental systems that can be exploited to test it. Further support for an EBV-induced immune pathogenesis for Parkinson’s disease could have profound therapeutic implications. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Parkinson’s disease Alpha-synuclein EpsteineBarr virus Molecular mimicry Autoimmunity

“A theory always guides research, opens the path to experiments; it is the ferment that attacks the unknown and transforms it into new knowledge that widens our intellectual horizon. Without theory, scientific research would be a futile practice, tantamount to counting the grains of dust on the road.” Ernesto Lugaro, 1898.

* Corresponding author. Dept. of Pathology, The Ottawa Hospital, Civic Campus, 1053 Carling Avenue, Ottawa, ON K1Y 4E9, Canada. Tel.: þ1 613 798 5555x13345; fax: þ1 613 761 4199. E-mail address: [email protected] (J.M. Woulfe).

1. The enigma of PD pathogenesis Parkinson’s disease is the most common age-related movement disorder. Clinically, it is characterized predominantly by motor features in the form of tremor, rigidity, akinesia, and postural instability. More recently, non-motor symptoms including rapid eye movement sleep behaviour disorder, dysautonomia, and gastrointestinal dysfunction have taken centre stage as clinical precursors to the hallmark extrapyramidal stigmata. The defining neuropathological features of PD comprise loss of dopamineproducing neurons in the substantia nigra pars compacta and the formation of proteinaceous cytoplasmic inclusions called Lewy bodies in surviving nigral neurons. Despite intensive research, the cause of PD has remained enigmatic. The existence of genetic forms

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Please cite this article in press as: Woulfe JM, et al., Hypothesis: A role for EBV-induced molecular mimicry in Parkinson’s disease, Parkinsonism and Related Disorders (2014), http://dx.doi.org/10.1016/j.parkreldis.2014.02.031

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caused by single gene mutations in rare families provides some clues to PD pathogenesis. Mutations in several genes have been described in rare families in monogenic forms of PD. In addition, a multitude of genetic risk factors have been documented. However, despite evidence for a genetic substrate underlying PD pathogenesis, current evidence supports a stressediathesis model wherein some environmental factor or factors initiate the disease process in genetically-susceptible individuals [1]. Indeed, the results of twin studies are consistent with environmental factors having a more important role in disease pathogenesis than genetic substrates [2e 5]. The identification of geographically defined clusters of PD is also consistent with an important role for environmental initiators [6]. What has become fairly certain is that the abnormal oligomerization and aggregation of the neuronal synaptic protein a-synuclein (a-syn) plays a central role in PD pathogenesis. Mutations in the gene encoding a-syn were described in 1997 [7]. The demonstration that duplications and triplications of the a-syn gene cause PD in rare pedigrees further established a primary role for a-syn by linking a-syn dosage to the development of PD [8,9]. The demonstration that a-syn was the dominant protein species in Lewy bodies, firmly established it as an important player not only in mutation carriers, but also in the far more common sporadic form of the disease [10]. The precise mechanisms by which aggregated asyn causes disease are uncertain. Current consensus supports a more important role for oligomeric or protofibrillar aggregates rather than larger microscopically visible fibrillar forms [11]. Perhaps a more important question from the perspective of upstream pathophysiological mechanisms is what causes a-syn to aggregate in PD patients in the first place? What if there were a single environmental factor, shared by the majority of the human population, which is necessary for PD initiation, but whose effects are manifest within the temporal confines of the human lifespan only in those who are endowed with a permissive genetic background? 2. Shifting paradigms: emergence of the Braak hypothesis A recent controversial hypothesis, put forward by Braak and colleagues [12,13], forms the foundation for our current staging scheme for the post-mortem neuropathological diagnosis of PD [14]. The Braak hypothesis is based on the observation that Lewy body pathology in PD is not confined to the substantia nigra but is also present in many other brain areas [15] as well as in the peripheral nervous system, most notably, the enteric nervous system (ENS), where it occurs very early in the disease process [16e18]. This is consistent with observations that gastrointestinal dysfunction precedes the onset of PD motor symptoms by decades [19,20]. In its current iteration, the Braak hypothesis envisions a-syn aggregation as a spatiotemporally dynamic process that is initiated in axon terminals of neurons in the ENS (as well as in the olfactory mucosa). From here, the process is proposed to “spread”, by analogy with the “spread” of prion protein aggregation in Creutzfeldt-Jakob disease [13,21]. This trans-neuronal spreading phenomenon occurs through a template-mediated molecular mechanism whereby misfolded a-syn is able to bind to normal a-syn and induce it to misfold. Once initiated in the ENS, a-syn aggregation “spreads” autonomously along vagal axonal projections to ultimately reach the origin of enteric innervation, the dorsal motor nucleus of the vagus. From here, the spreading process continues to higher brain centres, including the substantia nigra and ultimately, the cerebral cortex. Like other neurodegenerative proteins including prion protein, tau, and TDP-43, the ability of pathological a-syn to spread trans-synaptically along multisynatpic neuronal pathways has been convincingly demonstrated [21e25]. How this occurs is a topic of intense investigation. Interestingly, a-syn has been

identified in the extracellular space in association with exocytosed lipid vesicles called exosomes, which, following secretion are taken up with their cargo into recipient cells [23]. This mechanism has been implicated in the trans-cellular transfer of pathological a-syn and the progression of PD [26]. A key component of our hypothesis concerns the initiator of a-syn aggregation in the olfactory region and the gut. Braak postulates that this is induced by some yet-to-be identified, environmentally-derived, toxin or microbial pathogen [13]. We hypothesize that EBV, or more accurately the adaptive immune response to this common virus, represents the key pathogenic initiating event in PD. 3. EBV, a-syn, and molecular mimicry EBV is a ubiquitous human gamma herpesvirus which infects and establishes latency in 90e95% of the human adult population worldwide. Acute infection occurs in infancy or in adolescence. In the former it is usually asymptomatic whereas in the latter, it may manifest as infectious mononucleosis. The adaptive immune response to acute EBV infection is powerful and complex, characterized by the generation of humoural and cellular immune effectors targeting a variety of virally-encoded antigens [27,28]. Following acute infection, EBV establishes latency in a small proportion of Blymphocytes. Lifelong immune surveillance of latent EBV infection is mediated by both the humoural and cellular arms of the immune system. The development of EBV-induced malignancies in immunosuppressed individuals attests to the importance of this lifelong immune surveillance. In addition to antibodies against early (EA) and late lytic (VCA) viral antigens [28], antibodies to EBV-encoded latent proteins including the EpsteineBarr nuclear antigens (EBNAs) 1, 2, 3A, B, C, and LP, and the latent membrane proteins (LMPs) 1, 2A, and 2B can be found in the sera of latently-infected persons [29]. The nature of the immune response to these proteins shows considerable inter-individual variation, with a limited number of viral antigens being immunodominant [30]. Some of these viral proteins share functional and linear sequence identity with host proteins. In susceptible individuals (predisposition being endowed by polygenic genetic factors), this may result in autoreactivity targeting host tissues. Such “molecular mimicry” among microbes and target antigens may represent a mechanism for breaking down immune tolerance to self-antigens. There are clear examples of autoantibodies induced by a mimicry mechanism that are cross-reactive with EBV epitopes on EBNA 1 and 2, and EBVencoded small RNA (EBER)-bound self proteins. These persistent antibodies are considered pathogenic factors in Sjogren’s syndrome, lupus, and rheumatoid arthritis, not to mention multiple sclerosis [31e34]. It is also to be considered that during acute or persistent reactivating infection EBV can produce pro-inflammatory signals that can press the immune system to abandon tolerance and promote anti-viral responses (via MHC-II risk alleles) with possible mimicry to self-antigens, leading to autoimmune responses. In 2000, we reported that a commercially-available cocktail of four monoclonal antibodies (clone CS1-4, Sigma) recognizing the C-terminus of the EBV LMP1 protein produced intense, highly specific labelling of Lewy bodies in the substantia nigra of postmortem PD brain [35]. In addition, in normal brain, it displayed a pattern of axon terminal labeling identical to that of wild type a-syn in adjacent sections. Western blot and ELISA experiments confirmed that the CS1-4 antibody cocktail was cross-reacting with high avidity with full length human wild type a-syn. Therefore, we further characterised this cross-reaction by determining the nature of the shared epitope. Given that CS1-4 recognises a-syn both by immunohistochemistry and immunoblotting on a reducing gel, the common epitope was likely to be based on linear rather than conformational homology.

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A BLAST search revealed a region of similarity between the primary sequences of LMP-1 and a-syn, consisting of the amino acids PXDPDN (Fig. 1). In a-syn, a PVDPDN motif is found in the Cterminus. In LMP-1, the PQDPDN sequence forms part of a twelve amino-acid repeat sequence in the cytoplasmic C-terminal half. Using site-directed mutagenesis to generate molecular constructs lacking particular amino acids in the putative cross-reactive epitope, we confirmed that the DPDN motif represents the cross-reactive target in a-syn (unpublished observations). However, the “mimicry” epitope may be larger than this identical four amino acid sequence. There are nearby residues which confer additional structural homology. The “ED” and “DD” dinucleotides beginning 5 and 6 amino acids N-terminal to DPDN in a-syn and LMP1, respectively, introduce upstream negative charges. Moreover, the proline between this negatively charged dipeptide and the DPDN sequence introduces a “proline bend” common to the two epitopes (see Fig. 1). Accordingly, one of us (JM) has demonstrated that monoclonal antibodies other than CS1-4, synthesized against the LMP1 repeat region, potently crossreact with much higher avidity than the commercially-available CS1-4 monoclonal antibody cocktail (Fig. 2a). At least one of these (OT21C) produces intense immunostaining of pathological (Fig. 2b) as well as normal physiological (Fig. 2c) a-syn in tissue sections at dilutions as high as 1:5000. Does this example of molecular mimicry hold any biological significance? Our hypothesis is illustrated in Fig. 3. We hypothesize that, in genetically-susceptible individuals, an immune response is generated targeting the DDNGPQDPDN repeat region of LMP1. This results in the production of autoantibodies cross-reacting with the similar EDMPPVDPDN epitope of a-syn, culminating in its aggregation (More detail regarding the possible mechanisms by which this might occur are outlined below). Following initial immunoglobulin-initiated aggregation, the abnormal a-syn conformer itself becomes the transmissible pathogenic agent and the pathological process progresses autonomously along interconnected neuronal pathways. This hypothesis is consistent with a previously articulated concept that infections in early life, including EBV, may predispose to late onset neurodegenerative diseases [36,37]. The validity of this hypothesis is predicated on a number of factors including: 1. Evidence that immune mechanisms are operative in PD pathogenesis. 2. Epidemiological evidence for a relationship between EBV infection and PD. 3. Evidence that EBVinfected humans actually generate anti-LMP1 antibodies against the cross-reactive epitope and 4. Evidence that such cross-reacting antibodies influence the state of a-syn oligomerization. Each of these will be addressed in turn below.

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3.1. The immune system and PD A role for the adaptive immune system in “non-autoimmune” CNS disorders, including neurodegenerative diseases, is becoming increasingly appreciated [38,39]. Several lines of evidence support an important role for immune mechanisms in PD including a significant increase in the frequency of the DQB1*06 MHC class II allele [40]. Most notably, a recent genome-wide association study revealed a significant association of sporadic PD with the HLA-DR locus [41]. Antibodies to dopaminergic neurons have been described in the sera of patients with PD [42]. Remarkably, stereotactic injection of IgG from PD patients into the SN of adult rats resulted in selective immune-mediated injury of nigral dopaminergic neurons [43]. In humans with PD, there is IgG binding on nigral dopamine neurons [44]. More specifically, anti-a-syn antibodies targeting multiple epitopes of the a-syn protein have been described in PD sera as well as within the sera of non-diseased controls [45e50]. Interestingly, in at least one study, anti-a-syn antibody titres were much higher in PD patients at early disease stages than controls, but then tapered off substantially with disease progression [50]. This temporal pattern of anti-a-syn antibody titres was cited by the latter authors as evidence that these “naturally-occurring” anti-a-syn autoantibodies serve a protective role with respect to a-syn aggregation and consequent disease progression. However, in the context of the Braak hypothesis, it is equally plausible that the temporal pattern of anti-a-syn titres described in early PD reflects a pathogenic “spike” in anti-a-syn cross-reacting antibodies at disease onset, thereby initiating the disease process, with a subsequent natural tapering off of the antibody response as the disease progresses. It is notable, that the highest titre autoantibodies were directed against monomeric asyn [50]. In either case, the immune mechanisms underlying the loss of tolerance to this “self” protein are unknown. In addition to the HLA locus, there are other genetic links between immune dysfunction and PD. Mutations in the gene encoding leucine rich repeat kinase 2 (LRRK2) are a common cause of both sporadic and familial PD [51]. However, it is difficult to reconcile an important role for LRRK2 in PD pathogenesis, with the paucity of this enzyme in mammalian brain, including sites involved in PD such as the striatum and substantia nigra [52,53]. Indeed, the weight of evidence implicates LRRK2 as a more important player in immune function than in the CNS. Accordingly, single nucleotide polymorphisms in LRRK2 represent risk factors for susceptibility to leprosy [54] and modulators of the autoimmune disorder Crohn’s disease [55]. Consistent with this, LRRK2 is expressed in circulating and tissue mononuclear cells in which its

Fig. 1. Alignment of the primary amino acid sequences of human a-syn (top) and EBV-LMP1 (bottom). The cross-reactive epitopes comprise a nine amino acid sequence in the cterminus (amino acids 114e122) of a-syn and a triplet of nine amino acids within a repeat region in the c-terminal half of LMP1 (amino acids 250e291) of EBV-LMP1. The crossreactive epitope consists of an identical DPDN sequence (purple), a conserved proline (red), and a conserved negatively-charged dinucleotide (green).

Please cite this article in press as: Woulfe JM, et al., Hypothesis: A role for EBV-induced molecular mimicry in Parkinson’s disease, Parkinsonism and Related Disorders (2014), http://dx.doi.org/10.1016/j.parkreldis.2014.02.031

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Fig. 2. A) Results of enzyme-linked immunoabsorption (ELISA) assay of affinity of various anti-LMP1 monoclonal antibodies for purified human a-syn. Antibody dilutions are colour coded (box at right). MJFR1: mouse anti-a-syn (Epitomics); OT21C: Mouse IgG1 anti-EBV-LMP1 C-terminus [67,71] OT22CN: Mouse IgG1 anti-EBV-LMP1 N-terminus [67]; CS1-4: Mouse IgG1 mix anti-EBV-LMP1 C-terminus (Dakopatts); OT17A: Mouse IgG2a (S12) anti-EBV-LMP1 C-terminus [67]; K97/52-verbl: Immune rabbit serum anti-recombinant-EBVLMP1 C-terminus; K97/52-0: pre-immune rabbit serum; OT1x: mouse IgG1 anti-EBV-EBNA1. All mouse antibodies were purified and used at 2 mg/ml stock solution. Purified human a-syn courtesy Peter Lansbury, PMID: 10639120. Y-axis ¼ optical density at 450 nM (OD450) values. B, C) Section of human hippocampus from a patient with dementia with Lewy bodies immunostained using OT21C at 1:5000. There is intense immunostaining of both pathological a-syn in Lewy bodies (thick arrows) and Lewy neurites (thin arrows) in the CA2 sector (B) as well as normal synaptic a-syn in mossy fibre terminals in CA3 (C).

expression is upregulated following recognition of microbial structures [56]. Recent evidence indicates that mutant LRRK2 may disrupt B-lymphocyte function. Rossi and colleagues have described functional changes in the B-lymphocytes of LRRK2 PD patients, including alterations in B-cell MAPK signalling and CD95 expression [57]. These latter investigators are studying how such abnormalities might result in humoural immune dysregulation in PD, including the generation of autoreactive anti-neuronal antibodies (Margarida Soutos-Carneiro, personal communication). 3.2. EBV infection and PD A viral aetiology for PD has been proposed by several previous authors (reviewed in Ref. [58]. The precise mechanisms underlying this association are unknown. In many cases, direct viral infection of nigral neurons has been implicated or even demonstrated, whereas in others, virally-induced autoimmune mechanisms have been suggested. Our hypothesis is novel in that it proposes a virallyinduced autoimmune mechanism that is compatible with topographic progression of the alpha-synuclein pathology, as described by Braak. Support for the widely-acknowledged role of EBV in MS was derived in part from large epidemiological studies demonstrating an EBV seropositivity rate of 100% in MS subjects compared to the

90e95% infectivity rate in the general population [31,59,60]. In prospective studies EBV seronegative individuals seroconvert prior to the development of MS, while only 35.7% of those remaining MSfree do. Unfortunately, formal epidemiological studies examining such an association between EBV infection and PD have not been performed. However, according to data compiled by eHealthme extracted from the USA Food and Drug Administration’s Adverse Event Reporting System, as of March 9, 2013, among 13,581 subjects with clinically diagnosed PD, all showed serological evidence of EBV infection. Assuming seroprevalence rates of 90 and 95% in the general population (the lower and upper limits of the estimated rates), the expected numbers of EBV-negative PD patients are approximately 1358 and 679, respectively. A chi-squared goodness of fit analysis of the data reveals high significance (p < 0.0001) for both seroprevalence estimates. Even assuming a population seroprevalence rate of 99.9% yields a p-value less than 0.001 (Table 1). Thus there is overwhelming statistical evidence that EBV seropositivity in PD subjects is significantly higher than that in the general population. It will be interesting to determine whether these data can be replicated in a properly controlled validation cohort. Moreover, it has been stated that “Every scientific hypothesis must be testable, and the way to test it is to look for circumstances in which it does not hold” [61]. Thus, it will be important to address the incidence of PD in the EBV-negative population.

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[62]. Indeed, the author of this latter report cites our molecular mimicry article and speculates that EBV infection may have initiated an autoimmune recognition of a-syn in this patient. In this context, it is also tempting to speculate that case reports of EBVassociated acute autonomic neuropathy [65] and intestinal pseudo-obstruction [66] might be related to autoimmune crossreactivity with a-syn in axon terminals of the autonomic and enteric nervous systems, respectively. 3.3. The humoural immune response to LMP1 The nature of the immune response to EBV infection displays considerable inter-individual variability in terms of protein and epitope dominance. Studies of the immune response to EBV reveal that a small proportion of EBV-infected individuals do generate detectable antibodies against LMP1 [67]. Such antibodies targeting the LMP1 protein cross-reactive PQDPDN repeat region have been demonstrated in human sera using “pepscan” analysis in the laboratory of Dr. Middeldorp ([68] see Fig. 4). Notably, the antibody response targeting LMP1 is more common in the setting of acute EBV infection (infectious mononucleosis), suggesting that there may be a transient spike in anti-LMP1 immunity. In general, antiLMP1 immune responses are considered subdominant, for both humoural and cellular immune responses, even in patients with LMP1 expressing EBV-linked malignant diseases like Hodgkin lymphoma and nasopharyngeal carcinoma [69e71]. On the other hand, a recent study demonstrated that anti-EBV immune surveillance targeting LMP1 is important for the prevention of EBVassociated malignancies [72] and vaccination trials are underway to boost anti-LMP1 responses [73]. It is tempting to speculate that, as a surveillance strategy, a much larger proportion of latentlyinfected individuals may continually generate anti-LMP1 antibodies, but at very low levels, below the threshold of detection by methods employed in studies that have formally investigated this. Our hypothesis suggests that boosting the response to the DPDN sequence may have pernicious consequences. Fig. 3. Model for EBV-induced autoimmune initiation of a-syn aggregation. A) In the model shown in the figure, the cross-reactive immune response occurs locally after EBV-infected B-lymphocytes expressing LMP1 (lower right) home to inflammatory foci in the gut wall. A subset of genetically-susceptible individuals (upper right panel), generates immunoglobulins (red) targeting the PQDPDN repeat region of EBV-LMP1 (purple). These antibodies (Abs) cross-react with the homologous PVDPDN epitope on a-syn (blue) with loss of immune tolerance to a-syn. The resulting anti-a-syn autoantibodies are now able to bind a-syn associated with the axon terminals of enteric neurons (orange). It is also possible that the cross-reactive immune recognition of LMP1 occurs in the blood stream with anti-a-syn antibodies and plasma cells accessing mucosal a-syn from the bloodstream (not shown). B) Possible mechanism of autoantibody-induced pathology. The cross-reactive immunoglobulins (red) bind to extracellular (exosome-associated) a-syn and initiate its oligomerization. The exosomal Ab-a-syn cargo is internalized (1) by recipient neurons where Ab-mediated oligomerization of donor a-syn (blue) continues (2). The resulting Ab-induced oligomers acquire “neopathogenic” status and are now capable of recruiting, via a prion-like templating mechanism, wild type a-syn in the recipient cell (purple) into the pathological oligomers independently of the Ab (3). Thus, although Ab-induced, the process has now become an autonomous “a-syn-only” phenomenon. Pathological a-syn conformers are now transported via the endosomal-lysosomal system to the cell surface (4) where they undergo exosomal secretion. a-syn aggregation in the subsequent recipient cell will be induced by the a-syn oligomers and this will proceed along interconnected autonomic neuronal pathways to the CNS.

Case reports and case series of neurological syndromes associated with EBV infection provide anecdotal support for our hypothesis. These disorders, many of which affect the nervous system are presumably autoimmune in nature, although the identity of the autoantigen is unknown. There are several reports of parkinsonism associated with acute EBV infection [62e64]. In some of these, the topographical distribution of signal changes on brain imaging is reminiscent of the normal pattern of a-syn distribution in the brain

3.4. Cross-reactive antibodies and a-syn aggregation That immunoglobulins against intracellular antigens can induce their aggregation is nicely illustrated in studies of therapeutic immune targeting of the oncogenic protein p21Ras [74e76]. These studies elegantly demonstrated that anti-p21Ras antibodies not only bind intracellular p21Ras but, in addition, induce its aggregation to form pericentriolar aggresomes identical to those found in neurons in neurodegenerative disease. Remarkably, these antibody-induced inclusions not only stain for ubiquitin and proteasome subunits, but the cells in which they form show evidence of proteasomal inhibition, further extending the analogy to neurodegenerative cellular pathophysiology. Preliminary results in our laboratory indicate that the CS1-4 antibody cocktail targeting the a-syn cross-reactive epitope of LMP1 is indeed capable of inducing a-syn aggregation [77]. We employed a novel technique called “bimolecular fluorescence complementation (BiFC)” as a quantitative read-out for a-syn oligomerization. Briefly, we fused either the C- or N-terminal regions of full length human a-syn with complementary halves of yellow fluorescent protein respectively, thereby requiring antiparallel alignment to produce a fluorescent signal. Using flow cytometry and immunohistochemistry, we demonstrated that the CS1-4 antibody, as well as certain anti-a-syn antibodies targeting the cross-reactive epitope, but not anti-a-syn antibodies targeting other epitopes, induce a-syn oligomerization in cultured HT4 mouse neuroblastoma cells transfected with the a-syn constructs. In addition, the oligomer-inducing immunoglobulins were

Please cite this article in press as: Woulfe JM, et al., Hypothesis: A role for EBV-induced molecular mimicry in Parkinson’s disease, Parkinsonism and Related Disorders (2014), http://dx.doi.org/10.1016/j.parkreldis.2014.02.031

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Table 1 Chi-squared goodness of fit analysis of the FDA-derived data available from eHealthMe. EBV EBVþ EBV c2 seroprevalence Expected Observed Expected Observed 95% 99% 99.9%

12,902 13,445 13,567

13,581 13,581 13,581

679 136 14

0 0 0

p-Value

714.79 <0.0001 137.18 <0.0001 13.59 <0.001

internalized into cells where they co-localized with perinuclear asyn aggregates [77]. Candidate molecular mechanisms underlying this phenomenon are discussed below. 4. When, where, and how? The emergence of the Braak hypothesis has engendered growing appreciation that the development of PD is a spatiotemporally dynamic process affecting several levels of the nervous system and extending over the course of several decades. How might our hypothesis be incorporated into this complex pathogenetic paradigm? 4.1. Where? There is growing appreciation that pathological a-syn aggregation in PD is initiated in neuronal axon terminals and that it then propagates retrogradely toward neuronal cell bodies [78]. We contend that a-syn-containing axon terminals within the ENS, olfactory mucosa, and perhaps other peripheral organs represent the

initial targets of the offending cross-reactive immune response. Once initiated by the antibody, the resulting misfolded/aggregated a-syn conformer becomes the pathogenic agent itself and a-syn pathology “spreads” autonomously towards the CNS, as discussed above. Thus, we do not necessarily imply the existence of a significant autoimmune response in the CNS, (an immune privileged site). Immunoglobulin-induced effects on a-syn in the systemic organs may occur either as a result of 1) an in situ cross-reactive immune response to local tissue infiltration by EBV-infected Blymphocytes in susceptible individuals or 2) exposure of a-syncontaining axon terminals in these organs to blood-borne crossreactive antibodies. Locally restricted inflammatory conditions, induced by EBV or other pathological conditions may trigger (temporal) formation of cross-reactive antibody responses. The first of these models is somewhat reminiscent of what some groups have proposed and recently confirmed for the involvement of EBV in MS. There is strong epidemiological evidence that EBV infection is a pre-requisite for the development of MS and molecular mimicry induced by viral proteins has been implicated as an initiating event [79]. Recent studies using more sensitive techniques have demonstrated that EBV-infected lymphocytes can be detected in the brains of approximately 80% of MS patients, although this is disputed by subsequent detailed studies [80]. However, local production of EBV-encoded inflammatory molecules, such as EBER1, secreted in exosomes or as protein-RNA complexes may provide triggers for inducing breakage of tolerance, thus assisting (potentially boosting) local production of antibody responses driven by mimicry [81e83]. In our own recent study on the distribution of a-syn along the ENS [84], we have confirmed the presence of a-syn-immunoreactive ganglion cells, fibres, and axon terminals within the ENS of the

Fig. 4. A) Epitope scan (“pepscan”) analysis [68] (CS1-4, S12, OT21C and OT22CN; blue boxes). Numbers along the x-axis correspond to individual 12-mer peptides corresponding to the primary amino acid sequence of LMP1. The schematic of LMP1 at the top is aligned to indicate their respective positions along the LMP1 protein. The vertical blue lines indicate the affinity of the antibody for each of the 133 12-mer peptides. B) Example of a similar pepscan epitope analysis of human serum. Instead of anti-LMP1 monoclonal antibodies, human serum has been tested on each of the 12-mer peptides. This sample contains a high titre of antibodies against the region of LMP1 containing the a-syn cross-reacting region.

Please cite this article in press as: Woulfe JM, et al., Hypothesis: A role for EBV-induced molecular mimicry in Parkinson’s disease, Parkinsonism and Related Disorders (2014), http://dx.doi.org/10.1016/j.parkreldis.2014.02.031

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stomach, small intestine, and large intestine, as described previously by other authors. In addition however, we demonstrated that fibres and axon terminals were enriched within the lamina propria of the gut. There was a particularly dense network of a-synimmunoreactive axon terminals and fibres within the lamina propria of the vermiform appendix. The a-syn-positive innervation of the gut lamina propria could be consistent with either the “in situ” or the “blood-borne” models listed above. With respect to the former, EBV-infected B-lymphocytes are known to home to sites of tissue inflammation [85]. The gut, and particularly the appendix, is in part an immune organ characterized by the presence of lymphoid aggregates throughout its wall. Interestingly, recent data indicate that EBV-infected B-cells may accumulate at sites of gastrointestinal inflammation, providing support for our hypothesis [86,87]. The enrichment of a-syn-positive axon terminals in the lamina propria of the gut is also compatible with the “blood borne” hypothesis. The lamina propria of the gut lacks a blood-tissue barrier, permitting ready access of blood-borne immunoglobulins to a-syn-rich axon terminals. In this context, a-syn in the ENS may be vulnerable to aggregation early in the PD disease process not by virtue of its proximity to some pathogen in the gut lumen, but instead by virtue of its sheer abundance combined with its proximity to the rich, leaky vasculature investing the gut wall. 4.2. When? Canonical autoimmune neurological disorders (paraneoplastic syndromes representing the prototype), are characterized by the presence of the offending cross-reactive autoantibodies within the blood and/or csf of the affected individual throughout the course of the disease process. Consistent with this, it is conceivable that the EBV-induced anti-a-syn antibodies are present at low levels and actively promoting a-syn oligomerization throughout the PD disease process. However, with the emergence of the Braak hypothesis, it is not necessary to invoke the presence of autoimmunity throughout the course of the disease. Specifically, there is increasing evidence that, once initiated, a-syn aggregation becomes autonomous, self-propagating, and spreads along neuronal networks in a templating, “prion-like” fashion [88,89]. Accordingly, a single, transient “catastrophic event” initiating a-syn aggregation would be sufficient to jump-start the disease process. If that event is the (temporary) EBV-induced production of anti-a-syn antibodies, it may occur at any point in time during the course of EBV infection. Notably, there is ample evidence for fleeting immune responses to a variety of EBV-encoded antigens, particularly in the setting of acute (infectious mononucleosis) and reactivating (chronic) EBV infections. The origin of the anti-a-syn autoantibodies in PD patients and controls [45e50], which are polyclonal and target multiple epitopes on the a-syn protein, is unknown. We would argue that the polyclonal nature of the anti-a-syn antibodies in these patients is the result of “epitope spreading” subsequent to a more restricted, and pathogenic, immune response specifically targeting the PQDPDN epitope of a-syn in response to a spike in anti-LMP1 (PVDPDN) immunity. According to our hypothesis, this latter event represents the onset of PD. 4.3. How? Consideration of the mechanisms whereby anti-a-syn autoantibodies might induce a-syn aggregation must address: 1) how these immunoglobulins achieve access to a-syn, which is an intracellular antigen and 2) how a cross-reactive immune response might ultimately result in a-syn aggregation. In terms of access to a-syn, there are at least two possibilities: 1. Binding to extracellular a-syn and 2. Immunoglobulin internalization

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with subsequent binding to intracellular a-syn. With respect to the first possibility, under physiological conditions, small amounts of asyn are released from cells, including enteric neurons, and can be detected in human csf and plasma [90]. Postulated mechanisms of asyn release include non-canonical exocytic pathways or association with released exosomes [26]. There is evidence that extracellular misfolded and/or aggregated a-syn can re-enter neurons [91]. Thus, subsequent to extracellular immunoglobulin binding, misfolded and/or aggregated extracellular a-syn would be free to enter a neuron and act as a template for normal intracellular a-syn, thereby initiating the cascade of intraneuronal a-syn aggregation. The second of these possibilities, that a-syn autoantibodies might bind to intracellular a-syn, is germane to an on-going debate in the field of paraneoplastic autoimmune neurological disorders, particularly those targeting synaptic proteins [92]. These disorders are archetypal molecular mimicry-induced neurological autoimmune diseases. They are characterized by a diverse array of clinical syndromes resulting from autoimmune recognition of specific neuronal antigens. The autoimmunity is induced by structurally homologous antigens expressed by the systemic neoplasm. The neuronal autoantigens in some of these disorders are intracellular. Consequently, the corresponding autoantibodies were considered to be mere markers of the autoimmune phenomenon with negligible pathogenic potential. However, novel findings are challenging this assumption. For example, in autoimmune stiff-person syndrome, autoantibodies targeting their intracellular target, amphiphysin, are internalized into axon terminals where they bind to intracytoplasmic amphiphysin and disrupt its function [93]. Interestingly, in this study, anti-amphiphysin antibody binding also induced the aggregation of intracellular amphiphysin. In paraneoplastic cerebellar degeneration, autoantibodies against a Purkinje neuron intracellular antigen called “Yo” are generated [94]. A recent study demonstrated that anti-Yo autoantibodies can be taken up by Purkinje cells in cerebellar slice cultures to cause cell death, suggesting that anti-Yo antibodies may be directly involved in the pathogenesis of the disease [95]. This is consistent with the relative paucity of cerebellar inflammatory cellular infiltrates in this disease. How the autoreactive antibodies are internalized remains to be determined. However, these studies challenge the prevailing perception that autoantibodies targeting intraneuronal antigens are non-pathogenic. In the context of our hypothesis, they render conceivable the possibility that EBV-generated anti-a-syn antibodies may access a-syn within neuronal axon terminals. Our hypothesis predicts that anti-LMP1 antibodies crossreacting with the c-terminal region of a-syn somehow induce its oligomerization. How might this occur? In the case of a-syn, it is possible that IgG binding changes the intramolecular electrostatic properties of a-syn: The C-terminal region of a-syn surrounding the PVDPDN cross-reactive motif is highly negatively charged. The maintenance of this charge is essential for inhibiting protein autooligomerisation [96]. Structural studies have also shown that the region surrounding the shared epitope is involved in long-range interactions with other moieties on the same a-syn molecule. These intramolecular interactions are also essential for the autoinhibitory control of a-syn oligomerisation. Conceivably, immunoglobulin binding to the PVDPDN motif could inhibit the effect of any net charge as well as preventing the formation of tertiary structure, thereby increasing the propensity of a-syn to oligomerise. Another possibility is disruption of the native tetrameric state of a-syn: Recent studies have challenged the widely-held notion that a-syn exists predominantly or exclusively as an unstructured monomer in its native state and have provided evidence that it may exist instead in a helically-folded tetrameric form [97,98]. These authors have further postulated that the disruption of helically-folded tetramers may increase the aggregation propensity of a-syn, resulting in PD. It

Please cite this article in press as: Woulfe JM, et al., Hypothesis: A role for EBV-induced molecular mimicry in Parkinson’s disease, Parkinsonism and Related Disorders (2014), http://dx.doi.org/10.1016/j.parkreldis.2014.02.031

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is conceivable that immunoglobulin binding to the PVDPDN region of a-syn could impede the formation of, and or disrupt, native oligomeric forms of a-syn. We are well aware that there is evidence that anti-a-syn antibodies can actually subvert a-syn aggregation. Active [99] and passive [100] anti-a-syn immunization strategies have provided evidence for an anti-a-syn aggregation effect and demonstrated improvements in the behavioural and neuropathological features in mouse models of PD. Importantly, these studies confirmed that anti-a-syn antibodies could be internalized into neurons and bind intracellular a-syn. These animal studies have provided the basis for the initiation of phase I anti-a-syn vaccination trials in human subjects. However, other studies showed that anti-a-syn immunity can promote the degeneration of nigral neurons [101]. In this context, it is important to note that antibodies may have pleiotropic effects on protein aggregation. Whether they promote or inhibit multimer formation is dependent on the epitope bound by the immunoglobulin. This phenomenon was nicely illustrated by Li and co-workers who demonstrated that an immunoglobulin targeting a specific epitope of the native human prion protein resulted in the exposure of residues that may enhance its propensity to misfold [102]. Antibody-induced aggregation of p21Ras, discussed above, provides another salient example with clear relevance to neurodegenerative disease [74,75]. Finally, a role for cross-reactive antibodies in initiating PD is not necessarily predicated on a direct antibody-mediated promotion of a-syn aggregation. Whether the acute effect of this interaction is pro- or anti-aggregation may be irrelevant in the short term. However, in the long term, any factor which abnormally interacts with this aggregation-prone protein would reasonably be expected to alter its normal physiological homoeostasis. Alpha-syn levels are meticulously regulated because increased dosage results in its aggregation and PD [103]. It is plausible that, over the long term, low level autoantibody binding to intracellular and/or extracellular asyn might engage regulatory cellular feedback mechanisms, leading to an upregulation of a-syn production, with consequent aggregation. 5. Perspective and implications There is considerable evidence that environmental factors conspire with a permissive genetic background to initiate the PD pathogenic process. An important corollary of our hypothesis is that, by inducing a-syn aggregation via LMP1-induced molecular mimicry, infection with EBV is essential, but perhaps not sufficient, for the development of PD within the time-frame of a normal human lifespan. This does not discount a crucial role for genetic factors in PD pathogenesis. On the contrary, we postulate that genetic background, including single gene mutations in monogenic PD, critically modulate the temporal profile of disease onset and progression. In this context, genetic factors may dictate the nature of the immune response targeting EBV proteins (eg. HLA-DR, LRRK2 (see above)), the propensity of a-syn to aggregate (eg. SNCA), the capacity of cells to clear oligomerized or aggregated a-syn (eg. PARKIN), and the capacity of cells to deal with the consequences (oxidative stress, etc.) of aggregated a-syn (eg. DJ-1, PINK-1) etc., in the face of EBV-induced a-syn autoimmunity. In the context of our hypothesis, we view PD as one of the autoimmune “synaptic encephalitides”. These are a growing family of antibody-mediated neurological disorders caused by autoimmune recognition of neuronal synaptic proteins. They are mediated by autoantibody induced dysregulation of the target synaptic auto-antigen. Immune targeting therapies have been variably successful as treatments for these diseases. If our hypothesis is correct, the nosological classification of PD would be

transformed, at least in part. The implications of considering PD an immune-mediated synaptic encephalopathy would have obvious and profound therapeutic implications. As in other autoimmune and neurodegenerative disorders, the PD field is experiencing somewhat of a renaissance. The “genetic” and “environmental” camps are back on speaking terms. Even some of the staunchest proponents of the former camp concede an important role for environmental factors acting on a variably permissive genetic canvas. We propose that the human immune response to EBV plays a crucial role in the origin of PD. We have begun addressing this hypothesis experimentally. These studies may provide evidence for a heretofore unsuspected cause of PD with profound implications for its treatment, prevention, and eradication.

Acknowledgements The authors wish to thank Dr. Michael Schlossmacher and Julianna Tomlinson for helpful advice and for the provision of laboratory reagents and transgenic mice and to acknowledge financial support from The Parkinson Society Ottawa, The Parkinson Society Canada (2012-29), and The Ottawa Parkinson’s Research Consortium. The authors would also like to acknowledge Johnson Chen at eHealthMe for helpful advice and Hedy Juwana for performing the peptide-ELISA experiments.

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Please cite this article in press as: Woulfe JM, et al., Hypothesis: A role for EBV-induced molecular mimicry in Parkinson’s disease, Parkinsonism and Related Disorders (2014), http://dx.doi.org/10.1016/j.parkreldis.2014.02.031