A brief history of alpha-synuclein and Parkinson’s disease
Over the past two decades, the pre-synaptic protein alpha-synuclein (a-syn) has been irrefutably tied to the neurodegenerative disorder Parkinson’s disease. First, a genetic link was found to associate a-syn to Parkinson’ disease; the point mutation in
SNCA (A53T) was demonstrated to cause autosomal dominant Parkinson’s disease [
126] and several other point mutations (A30P, E46K, H50Q, G51D and A53E) have since been shown to cause familial forms of Parkinson’s disease and dementia with Lewy bodies (DLB) [
4,
79,
84,
119,
129,
167]. Later, duplication or triplication of the a-syn locus was described in several Parkinson’s disease and DLB families [
18,
62,
70,
141] and in a handful of sporadic cases [
2]. A gene dosage effect of the synuclein gene (
SNCA) has been suggested due to the observation that patients with a triplication have an earlier onset, more severe and faster progressing disease compared to those harbouring duplication of the
SNCA locus [
18].
An equally important milestone in the field was the identification of a-syn as a component of Lewy bodies in patients with both familial and sporadic forms of the disease [
6,
147], providing a direct link between different forms of the disease. Not only did this discovery draw attention to aggregated forms of a-syn as mediators of Parkinson’s disease pathogenesis, but also opened the door to the use of a-syn detection techniques for diagnosis and staging. Although this breakthrough occurred 20 years ago, research to date has not been able to describe the exact mechanism by which a-syn accumulates causes neuronal loss and leads to the development of the disease. Multiple lines of evidence now suggest that oligomeric species of a-syn, which are thought to precede the fibrillar aggregates found in Lewy bodies, are the culprits for neuronal degeneration in Parkinson’s disease [
59,
162]. In this review, we will discuss the evidence supporting the toxicity of a-syn oligomers in Parkinson’s disease and possible mechanisms for this toxicity, current methods for neuropathological analysis of a-syn oligomer deposition, recent findings indicating the prion-like spread of a-syn and possible therapeutic avenues targeting a-syn oligomers.
A-syn seeds and spreads
Neuropathological examination of Parkinson’s disease progression suggested that a-syn pathology spreads through the nervous system following stereotyped patterns [
12], which first led to the suggestion of a pathogenic agent ‘propagating’ through the nervous system. In 2008, it was shown that foetal dopaminergic neuronal grafts presented Lewy bodies 11–16 years after transplantation into Parkinson’s disease patients, suggesting a-syn pathology had spread from diseased neurons in the recipient’s brain to the grafted neurons [
75,
86]. Consistent with these findings, intracerebral injection of brain extracts containing aggregated a-syn into a-syn-transgenic mice caused the development of lesions in anatomically linked regions of the brain, motor dysfunction and neurodegeneration [
94,
107].
Recent work suggests that a-syn may be able to self-propagate between neurons in the brain, in a prion-like manner. The molecular events at each step in this process (the seeding of aggregates, the escape of aggregates from affected neurons and the entry of aggregates into naïve neurons) are only beginning to be elucidated. As in prion diseases, the ability of the molecule to self-interact and induce aggregation and conformational changes in another molecule is key, and it appears to be a feature of a-syn, demonstrated by the ability of a-syn aggregates to seed inclusions in vitro [
58,
95] and in vivo [
93,
103,
131]. A-syn oligomers display properties, including solubility and seeding ability, which make them likely candidates for mediating the spread of pathology. Small aggregate preparations produced by sonicating a-syn fibrils, known as pre-formed fibrils (PFFs), have been shown to propagate through non-transgenic mouse brain following injection into the striatum [
93], while oligomers assembled from monomeric a-syn have also been described to have similar effects [
131]. It is interesting to note that PFF treatment requires sonication, suggesting lower molecular weight species might also be implicated in their elicited effects.
In another study, injection of sarkosyl-insoluble a-syn isolated from the brains of dementia with Lewy bodies patients or fibrils produced in vitro mediated spread of a-syn pathology in the brain of non-transgenic mice [
103]. This property is not unique to aggregates introduced directly into mouse brain: Helwig et al. induced a-syn over-expression in the medulla oblongata via viral transduction and demonstrated the presence of monomeric, oligomeric and fibrillar a-syn in medullary neurons, but crucially only monomeric or oligomeric species in pontine neurons, suggesting that non-fibrillar species of a-syn are preferentially transferred when expressed in the mouse brain [
59]. In contrast to these other studies, 4-hydroxy-2-nonenal (HNE)-induced a-syn oligomers did not succeed in seeding inclusions in vivo [
42], suggesting it is a specific conformationally distinct a-syn oligomer incapable of seeding pathology.
Further work has confirmed that different ‘strains’ of a-syn aggregates have varying propensities to propagate between neurons, in addition to their seeding ability. Peelaerts et al. generated a-syn oligomers, fibrils (which by electron microscopy more closely resemble PFFs, as opposed to full-length fibrils) and ribbons (fibrils with twists), which were all capable of crossing the blood–brain barrier and distributing themselves within the central nervous system after intravenous injection in mice [
120]. While oligomers were the a-syn strain that diffused between brain regions more efficiently, fibrils and ribbons appeared to display higher toxicity [
120]. The differences in prion-like activity and toxicity resulted in distinct pathologies and neurodegenerative phenotypes in the mice. While fibrils induced Lewy body-like inclusions, cell death and motor impairment, ribbons produced glial cytoplasmic inclusions, as found in MSA brain, in addition to Lewy body-like aggregates, and a phenotype resembling aspects of both MSA and Parkinson’s disease [
120]. This inherent difference between a-syn strains in MSA and Parkinson’s disease were confirmed by Woerman et al. in cultured cells. Prion-like aggregates were isolated from MSA or Parkinson’s disease brain homogenates by phosphotungstate anion precipitation, a technique originally developed for studying infectious prion proteins [
163]. While MSA brain-derived a-syn precipitates could seed a-syn aggregation in cultured cells, those from Parkinson’s disease brain could not, demonstrating that the a-syn species mediating the prion-like spread of pathology in Parkinson’s disease is distinct from MSA.
Interestingly, the spreading capacity of a-syn seems to be related to a-syn donor and acceptor species. While Luk et al. could not detect a-syn spread after mouse a-syn injections on a mouse a-syn KO mouse, Helwig et al. detected enhanced propagation of human a-syn, expressed from an AAV vector, on a mouse a-syn KO background. This suggests that (1) endogenous mouse a-syn is required for mouse a-syn spread, (2) human a-syn may have a higher propensity to spread than mouse a-syn, as it appears to lack the requirement for endogenous seeds in mice and (3) although human a-syn can form hybrid oligomers with mouse a-syn, these hybrid oligomers seem to form inefficiently and impede progress of spread. It is interesting to note that this is highly similar to a prion-like spread, where the interspecies barrier formed between infecting and host species is crucial [
45]. This is confirmed by a recent paper by Luk et al. [
92] where they showed that mouse and human a-syn cross-seeded with reduced efficiency, due to sequence differences.
Details are just beginning to emerge of specific molecular pathways that may mediate the secretion and uptake of a-syn seeds, although specific details on the implicated a-syn strain remain unexplored. Lee et al. identified a novel protein quality control mechanism (misfolding-associated protein secretion or MAPS), whereby under conditions of proteasome dysfunction, the deubiquitinase USP19 recruits misfolded proteins to the ER to be deubiquitinated and directed into late endosomes for secretion [
83]. USP19 promoted the secretion of a-syn, suggesting that MAPS is an unconventional secretion pathway utilized by a-syn, particularly under conditions of proteasomal impairment, which has been repeatedly linked to Parkinson’s disease. It would be highly interesting to study which types of a-syn aggregates are being secreted (e.g. oligomers) and whether differential secretion exists for example among patient and control subjects. Once released by donor neurons, a-syn must be taken up by acceptor neurons in order to continue the cycle of spreading. Recently, lymphocyte-activation gene 3 (LAG3) was identified as a receptor for a-syn PFFs, allowing their entry into neurons and accelerating their spread throughout the mouse brain [
100]. Although expression of LAG3 had not previously been reported in nervous tissue, knockout of LAG3 in neurons in vitro and in mouse brain clearly reduced a-syn propagation. An altogether different mechanism for a-syn spread was recently proposed by Abounit et al.: in their study they showed that a-syn is transmitted cell-to-cell via tunnelling nanotubules, which are transient nanostructures that allow cargo exchange between cells [
1]. Lysosomal a-syn originally targeted for degradation was able to spread inside lysosomal vesicles from donor to acceptor cells, where it was then able to induce aggregation. It is tempting to speculate that owing to their smaller size and early aggregation properties, a-syn oligomers are likely candidates to be transported in this manner. These exciting new studies should inform novel drug targets in Parkinson’s disease, which if administered sufficiently early in the disease course, could prevent the devastating march of a-syn pathology through patients’ brains.
Detection of a-syn oligomers in biological fluids
The detection of a-syn oligomers in biological fluids (blood serum or plasma and cerebrospinal fluid) is of particular interest because of the potential for its use a biomarker of PD. Oligomeric detection may have uses as a diagnostic biomarker, as a biomarker of the progression of the disease, and in the future, perhaps as an index of response to novel therapies. The detection of a-syn oligomers in CSF and plasma has also proven a more reliable indicator of the presence of disease than the levels of total a-syn, which has led to contradictory findings [
46]. Several groups have employed sandwich ELISAs to detect a-syn oligomers by using the same epitope-blocking antibody for both capture and detection. The use of a blocking antibody ensures that only one antibody molecule can bind to each molecule of a-syn so that no signal is generated from monomers. In all of these studies, higher levels of a-syn oligomers were detected in the plasma or CSF of Parkinson’s disease patients compared with controls [
39,
116,
152]. This suggests that the presence of a-syn oligomers in biological fluids is a more reliable indicator of disease and measuring total levels of a-syn obscures the difference in oligomer levels because a variety of conformers are detected. Further refinements, for example increasing sensitivity, could be addressed by developing novel antibodies with higher avidities, or by adapting AS-PLA for use in biological fluids. PLA has previously been used to detect A-beta protofibrils in fluids, and it was shown to be 25-fold more sensitive than a sandwich ELISA targeting A-beta protofibrils [
69].
ELISAs utilizing conformational antibodies targeting a-syn oligomers have also been developed. Majbour et al. [
98] used an antibody that detected only aggregated (oligomeric and fibrillar) a-syn that they had previously generated [
153]. In parallel, they undertook ELISAs targeting total a-syn or a-syn phosphorylated at serine 129. When applied to CSF samples from 46 patients with Parkinson’s and 48 healthy age-matched controls, the ELISA targeting aggregated a-syn demonstrated the highest sensitivity for detecting PD patients compared with those detecting total or phosphorylated alpha-synuclein (89 vs 74% and 54%, respectively), but the lowest specificity (52 vs 74% and 54%, respectively). Similarly, Williams et al. [
159] applied two conformation-specific single chain variable domain antibody fragments (scFvs) that display sub-femtomolar sensitivity for a-syn oligomers to a phage-based capture ELISA they previously developed [
158]. Elevated levels of a-syn oligomers were found in PD patients compared to controls or AD patients in brain homogenate, CSF and serum. However, the approaches described thus far show large variability and overlap in ELISA signal between controls and PD patients. Therefore, such assays at the current time may have limited capabilities as a biomarker readout.
The emerging hypothesis of the prion-like propagation of a-syn has led to the adaptation of techniques originally developed to detect pathogenic prion protein to detect oligomers of a-syn. The protein misfolding cyclic amplification (PMCA) assay was originally developed in 2001 in the laboratory of Claudio Soto [
135] and has been shown to allow the detection of even a single pathogenic unit of prion protein [
134]. The concept is based on the seeding capacity of prion proteins and incubates a suspected pathogenic sample with the physiological form of the protein. If pathogenic forms of the protein are present, seeding and aggregation of the monomeric protein will occur. Following the generation of new aggregates, a shaking step breaks them up to form new seeds, and the cycle is repeated many times to amplify the signal. Originally, the aggregates formed as a result of the presence of seeds were detected using western blotting [
135], but this has now been adapted to allow for a more high-throughput format and thioflavin T fluorescence is used to assess the presence of aggregates. This assay design may preclude the detection of certain ‘off-pathway’ oligomers that do not aggregate further to from beta-sheet rich structures, but results thus far are promising.
Shahnawaz et al. [
139] developed a PMCA for a-syn, first using in vitro formed a-syn oligomers and subsequently adapted for use in CSF. Levels of a-syn seeds in CSF from two cohorts of PD patients from Germany and Japan (76 patients) were compared to 65 controls (patients with other neurological diseases); they found that the assay was capable of detecting PD patients with a sensitivity of 88.5% and a specificity of 96.9%. Interestingly, of four controls in which the assay detected pathogenic a-syn, two were subsequently diagnosed with PD, suggesting the potential value of the assay for early diagnosis. Furthermore, the sensitivity and specificity of the assay for identifying PD patients is much higher than any previous reports of techniques to detect a-syn oligomers in biological fluids.
An adaptation of the PMCA, real-time quaking induced conversion (RT-QuIC) assay, has also been used to detect a-syn aggregates in CSF and proved to be even more sensitive [
43]. Although the cohorts used were small (20 patients and 15 controls), the sensitivity was 95% and the sensitivity was 100%. Even more excitingly, three at-risk individuals who had confirmed REM sleep behaviour disorder all gave a positive signal in the assay, suggesting the assay may be used to detect the early stages of the disease.
Another exciting development is the finding that distinct biophysical properties (for example, proteinase K resistance) of a-syn seeds are retained after PMCA [
65]. This may make it possible to build up an a-syn ‘seed profile’ of patient biological fluids and dissect whether there are different clinical outcomes associated with different a-syn species, as has recently been observed in Alzheimer’s disease patients [
91].
The use of prion protein assays has provided the most sensitive and specific detection of pathogenic a-syn in biological fluids to date and provides hope that, following confirmation in large cohorts, a suitable biomarker may soon be able to be used in the clinic.
Conclusions
If we are to understand the contribution of a-syn oligomers to Parkinson’s disease it is of utmost importance to study oligomeric pathology and its distribution at sequential stages of the disease in the human brain. This will allow the delineation of how the presence of a-syn oligomers is associated with pathological changes and may prove to be a better correlate of disease progression and severity compared to Lewy body counts.
Furthermore, as a-syn oligomers are being pursued as a blood plasma and CSF biomarker for the presence of Parkinson’s and its progression [
39,
43,
152], there is a need to understand the relationship between the species found in biological fluids and those present in the diseased brain. Histopathological detection of a-syn oligomers is a key step towards a full understanding of whether the complement of a-syn oligomers in biological fluids is representative of those causing neurodegeneration in the brain.
The precise understanding of the formation, seeding and spread of a-syn oligomers in vivo is one of the key questions that remain in the field. Little by little, more light is thrown on this elusive aspect of Parkinson’s disease which is only now being interrogated. Different proteopathies often coincide in the diseased brain; in fact, it was recently shown that synthetic a-syn fibrils are able to induce tauopathy, possibly by a cross-seeding mechanism [
57], and so, research regarding a-syn oligomers might not only help advance the field of Parkinson’s disease, but also that of other neurodegenerative diseases.