The Discovery of α-Synuclein in Lewy Pathology of Parkinson's Disease: The Inspiration of a Revolution.

Abstract

Spillantini MG, Schmidt ML, Lee VM, Trojanowski JQ, Jakes R, Goedert M. Alpha-synuclein in Lewy bodies. Nature 1997;388(6645):839–840. When tasked with selecting one of the most influential papers in the field since the 1950s, Spillantini et al1 describing the presence of α-synuclein (aSyn) in Lewy bodies (LBs) in Parkinson's disease (PD) was our clear first choice. Following James Parkinson's description of the Shaking Palsy in 1817, progress in understanding PD mirrored the slowness of movement that characterizes the disease itself. Almost 100 years later, in 1912, Friedrich Lewy described the inclusion bodies we now recognize as the pathological hallmark of PD and a further 85 years later, investigation of the Contursi kindred, manifesting autosomal dominant PD, identified a causative single base mutation in SNCA.2 It took just 2 months for Spillantini and colleagues to publish their landmark paper describing the presence of aSyn in LBs in idiopathic PD and dementia with Lewy bodies (DLB). In the 24 years since this short paper was published (Spillantini et al's article was less than half the length of this Viewpoint), it has catalyzed numerous advances in understanding the pathophysiology of synucleinopathies, amassing over 2,225 citations and influencing almost every aspect of research in PD and other neurodegenerative disorders. We now recognize that LBs have myriad components and a proteome of over 300 proteins.3 However, in 1997 the biochemical composition of LBs was largely unknown and the major component of the characteristic filamentous structures elusive. In aSyn, Spillantini et al1 defined a target that revolutionized our thinking, bringing a protein to the forefront of PD pathology and placing PD under the umbrella of neurodegenerative conformational proteinopathies. Indeed, expansion of the “proteinopathies” including tauopathies, prion diseases, and A-β proteinopathy reinforced the need to classify neurodegenerative diseases by the dominantly involved protein and forged the concept of proteins contributing to disease pathogenesis. The synucleinopathies were soon expanded to include multiple system atrophy (MSA), a disease previously thought to be unrelated to PD.4 To this day, the definitive diagnosis of PD, DLB, and MSA remains the post-mortem finding of aSyn pathology in the brain. Immunohistochemistry for aSyn vastly expanded our appreciation of the extent of pathology in PD and other synucleinopathies. aSyn characterized not only the classical LBs, but also Lewy neurites, together constituting “Lewy pathology”, and oligodendrocytic and astrocytic aSyn pathology was also recognized.5 Furthermore, the anatomic and cellular extent of pathology was far broader than previously recognized with traditional histopathological methods (Fig. 1). The subsequent elegant staging studies of Braak et al,6 which evaluated the topographical distribution of aSyn in 168 post-mortem brains, characterized PD into 6 progressive stages, each defined by the presence of aSyn pathology in particular brain regions. This staging, entirely reliant on the discovery of synucleinopathy in PD, now represents a major conceptual framework in our understanding of the progressive nature of PD and has lent support to the hypothesis that aSyn may have prion-like qualities (see below). Notably, Braak's work considered aSyn merely as a marker of disease stage and did not consider that aSyn itself might catalyze the spread of pathology, instead postulating a “neurotropic pathogen, probably viral” that originated in the periphery.7 Staging studies also defined incidental Lewy body disease,6 which introduced the concept of preclinical disease. In this regard, PD led the field, being the first neurodegenerative proteinopathy recognized as having a prodromal phase, later adopted in Alzheimer's disease (AD). The PD prodrome, which includes hyposmia, constipation, mood and sleep disorders among others, likely begins decades before motor symptom onset, providing an opportunity for disease modifying intervention. Without the discovery of aSyn in Lewy pathology, it is possible this tantalizing opportunity might still remain hidden. Using research criteria for prodromal PD8 we can now identify individuals with incipient disease and facilitate their enrollment in clinical trials of disease modifying therapies, providing such trials a greater likelihood of success. The ultimate goal of this approach is to halt the disease before the development of motor symptoms. Further exploration of synucleinopathy also revealed an extensive involvement of the peripheral nervous system in PD. Although peripheral LBs were recognized before a focus on aSyn, their potential contribution to disease pathogenesis was not appreciated. We now understand that peripheral aSyn immunoreactive deposits are widespread throughout the enteric nervous system, epicardial plexus, paravertebral and mesenteric sympathetic ganglia, and beyond.9 Studies in rapid eye movement behavior disorder (RBD) suggest peripheral accumulation of aSyn might precede central nervous system (CNS) involvement and may contribute to prodromal symptoms.10 These studies also further support the hypothesis that aSyn itself might drive the pathogenic progression of syncleinopathy from the periphery to the CNS first proposed in Braak's dual hit hypothesis.7 The recognition that misfolded aSyn may share the ability of the prion protein to template misfolding gained significant traction with the reporting of aSyn deposits in fetal mesencephalic grafts in PD brain.11, 12 Over the ensuing decade a multitude of studies have capitalized on an experimental paradigm whereby a proteopathic “seed” of misfolded aSyn is applied, in vitro or in vivo, to induce misfolding of native aSyn.13 The seeding material is often recombinant protein incubated, with or without shaking and/or sonication, to generate markedly toxic fibrillar forms of the protein, which may not closely resemble those found in human disease.14, 15 These studies have demonstrated that aSyn is efficiently taken up by neurons, microglia, and astrocytes, via both receptor mediated and receptor independent mechanisms.13 That which escapes lysosomal degradation can be released, particularly under conditions of stress,16 and enter neighboring cells. Inoculation studies in animals have revealed a highly predictable spread of synucleinopathy through anatomically interconnected regions17 mediated by an ability of exogenous misfolded aSyn to initiate seeding of native intracellular aSyn14 generating insoluble aggregates resembling LBs,18 associated with neuronal dysfunction and cell death.19, 20 Collectively, these studies provide compelling evidence that aSyn itself can propagate pathology in an experimental setting, and supports targeting extracellular aSyn, for example using immunotherapy, as a disease modifying strategy. However, critically, the contribution of seeding to pathogenesis in human synucleinopathies remains to be fully substantiated. Indeed, the failure of immunotherapy trials in humans to date (discussed below) casts a shadow on targeting aSyn-mediated propagation as a sole pathogenic process supporting progression in PD. Stemming from similarities between the behavior of aSyn and the prion protein, mounting evidence suggests that strains of aSyn exist that may underlie the different pathologies that typify the synucleinopathies.21 When injected into animal brains, PD- and MSA-derived extracts induce distinct pathologies that are maintained on serial propagation.22-25 Following an elegant study by Peng et al,26 a leading hypothesis posits that different strains may arise as a result of the cellular environment in which they are generated. The likely existence of multiple strains of aSyn has profound implications for the development of disease modifying therapies targeting aSyn. Resolving the precise structure of fibrillar species in different synucleinopathies, as shown already for MSA using methods such as cryo-EM,27 will be critical to the development of precision therapeutics targeting different species with high affinity. Following several descriptions of aSyn deposition in the amygdala in various neurodegenerative disorders, in 2006 Uchikado et al28 proposed that AD with amygdala restricted LBs (AD/ALB) might represent a distinct synucleinopathy. Without the ability to evaluate aSyn, this condition likely would have remained unreported, because aSyn deposits in the amygdala have a different morphology than LBs. AD/ALB, now recognized as 1 of the most frequent copathologies in neurodegenerative diseases, strengthened appreciation that copathologies are so common they are the rule rather than the exception to the rule.29 Lewy pathology is present in ~40% of AD cases, ~20% of progressive supranuclear palsy, corticobasal degeneration and sporadic Creutzfeldt-Jakob disease cases, ~15% of frontotemporal lobe dementia, and ~ 10% of MSA cases.30 Indeed, aSyn may be a driving force in neurodegenerative diseases with concomitant synucleinopathy able to influence aggregation of tau, TAR DNA-binding protein 43, amyloid-β and prion protein, and potentially expanding the applicability of drugs targeting aSyn.30 Biomarkers, to confirm the underlying diagnosis and to differentiate different synucleinopathies early in their course, are a major unmet need in movement disorders. A reliable diagnostic biomarker would assist in providing a more accurate prognosis to patients and families (eg, differentiating PD from MSA-parkinsonian type) and improve clinical trials by facilitating recruitment of appropriate candidates. Biomarkers could also validate target engagement of novel therapeutics and provide surrogates measure of disease progression. aSyn is a particularly attractive candidate for biomarker development because it (1) represents the primary pathology, (2) is found in peripheral tissues and biofluids, and (3) abnormalities may occur early in disease. An imaging agent able to visualize aSyn aggregation in living patients, similar to amyloid imaging in AD, would have a profound impact, but to date remains elusive and a multitude of studies in biofluids and peripheral tissues have yielded inconsistent results, with no clear method emerging with sufficient sensitivity and specificity to apply clinically.31 Seeding assays, initially developed for prion diseases, are now being vigorously applied to a variety of tissues and fluids in synucleinopathies.32 Capitalizing on the existence of strains of aSyn, these assays provide great promise for the development of disease specific biomarkers, and have recently been reported to distinguish PD and MSA in cerebrospinal fluid (CSF) with >95% sensitivity33 as well as detecting abnormal aSyn in isolated RBD34 and pure autonomic failure.35 As these methods can be applied in a very short time frame using existing biosamples we now anticipate rapid development in this field. The greatest impact of the work of Spillantini and colleagues would be translation into a successful disease modifying therapy. Strategies targeting aSyn currently involve reducing the production of aSyn, inhibiting aggregation, enhancing degradation, or diminishing spread using small molecules, including repurposed and new chemical entities, antisense oligonucleotides and RNA interference, active and passive immunotherapy and the use of small antibody fragments or intrabodies.36 A recent review of all clinical trials for PD identified 5 studies involving small molecule drugs targeting aSyn aggregation and 7 immunotherapy trials in progress.37 Disappointingly, the first 2 aSyn monoclonal antibody trials recently failed to meet their primary endpoints, although prasinezumab demonstrated sufficient impact on motor score progression to encourage further study in early PD. These failures may result from heterogeneity in PD and support calls for future molecular subtyping of clinical trial participants, based on the biology of their underlying disease.38 They also highlight the need not to commit to a single hypothesis of disease pathogenesis centered around extracellular aSyn, however, must be interpreted with an appreciation of shortcomings in our ability to accurately stratify patients, especially those with early or prodromal disease who are most likely to respond to disease modifying therapy, and a lack of sensitive outcome measures, which is further exacerbated by the slow progression and protracted course of PD and the availability of effective symptomatic therapies that mask evidence of disease progression.39 Future development in these domains will be critical to afford disease-modifying therapies the best chance of success. Spillantini and colleagues' discovery of aSyn in LBs revolutionized our understanding of PD pathogenesis. We appreciate the extent and staging of pathology, including a prodromal phase of the disease, have uncovered a molecular mechanism of self-propagation that likely contributes to disease progression, and appreciate the possibility of different strains that may underlie different disease phenotypes. In aSyn, we not only have a promising biomarker, but also an exciting therapeutic target. However, despite all this progress, many unanswered questions exist. Germane to these unknowns is the fact that Spillantini's work was performed in post-mortem brain. We must exercise caution in assuming that observations at autopsy reflect those in earlier disease. It remains possible that aggregation of aSyn could be an epiphenomenon unrelated to disease pathogenesis, a protective mechanism to reduce exposure of the brain to toxic forms of the protein, or finally aSyn could be a driving force in disease pathogenesis.40 Future research will reveal if we, as a field, have perhaps inappropriately sanctified aSyn, or whether aSyn is indeed the “holy grail” for disease modification we so desperately seek. (1) Manuscript: A. Writing of the First Draft, B. Review and Critique. N.P.V.: 1A G.G.K.: 1B A.E.L.: 1B The authors confirm that the approval of an institutional review board and patient informed consent was not required for this work. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines. The authors declare that there are no potential conflicts of interest or financial disclosures related to the material in the article. N.P.V. has stock ownership in Rosetta Therapeutics and has received grants from the Michael J. Fox Foundation for Parkinson's disease and philanthropic funding from the Blidner Family Foundation. G.G.K. has served as an advisor for Biogen, and has received grants from Edmond J. Safra Philanthropic Foundation, Rossy Philanthropic Foundation, the Michael J. Fox Foundation, and Parkinson Society Canada. A.E.L. has served as an advisor for Abbvie, Acorda, AFFiRis, Biogen, Denali, Janssen, Intracellular, Kallyope, Lilly, Lundbeck, Paladin, Retrophin, Roche, SPARC, Theravance, and Corticobasal Degeneration Solutions; received honoraria from Sun Pharma, Medichem, Medtronic, AbbVie, and Sunovion; and received grants from Brain Canada, Canadian Institutes of Health Research, Corticobasal Degeneration Solutions, Edmond J. Safra Philanthropic Foundation, Rossy Philanthropic Foundation, the Michael J. Fox Foundation, the Ontario Brain Institute, Parkinson Foundation, Parkinson Society Canada, and W. Garfield Weston Foundation.