The α-Synuclein Seed Amplification Technology for Parkinson’s disease and related Synucleinopathies

Abstract

Synucleinopathies are a group of neurodegenerative diseases associated to the cerebral accumulation of alpha-synuclein (αSyn) misfolded aggregates. At this time, there is no effective treatment to stop or slow down disease progression, which in part is due to the lack of an early and objective biochemical diagnosis. In the last 5 years, the seed amplification technology has emerged for high sensitive identification of these diseases, even at the preclinical stage of the illness. Much research has been done in multiple laboratories to validate the efficacy and reproducibility of this assay. This article provides a comprehensive review of this technology, including its conceptual basis and its multiple applications for disease diagnosis, understanding the disease biology and therapeutic development.

Alpha-synuclein (αSyn) and Synucleinopathies

The hallmark event in various neurodegenerative diseases (NDs) (see Glossary) is the accumulation in the brain of misfolded protein aggregates [1]. In different diseases misfolded aggregates composed of distinct proteins deposit in diverse areas of the brain resulting in a series of downstream cellular alterations, leading to brain damage and ultimately to disease [1]. Four seemingly unrelated proteins are the main culprits of most NDs, including amyloid-beta (Aβ), tau, alpha-synuclein (αSyn) and the TAR DNA-binding protein 43 (TDP-43) (Fig. 1) [1]. Other proteins accumulating in some rarer forms of NDs include huntingtin, prion protein, superoxide dismutase 1 and ataxins, implicated in Huntington’s disease, Creutzfeldt-Jakob disease (and related prion disorders), amyotrophic lateral sclerosis and spinocerebellar ataxias, respectively [2]. From the four main proteins accumulating in the brain, tau and αSyn have the remarkable ability to be responsible for various clinically-diverse NDs, often referred to as tauopathies and synucleinopathies [1, 2]. Synucleinopathies include Parkinson’s disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA) and PD dementia (PDD) (Fig. 2) [3]. It is unknown how the same protein would be implicated in sometimes very different diseases, but the current thinking is that the aggregated form of these proteins can adopt various different structures, usually referred to as conformational strains, that can target distinct areas of the brain, leading to a different clinical phenotype [1].

Figure 1: Schematic representation of the three-dimensional structure of the protein aggregates most commonly found in NDs.

The near-atomic resolution structure of the protein aggregates composed of Aβ, Tau, αSyn and TDP-43 was determined by cryogenic electron microscopy. The structures were obtained from the PDB database (Aβ: 5oqv; Tau: 6hre; αSyn: 6xyo and TDP-43: 6n37) and drawn using the UCSF ChimeraX software version 1.6.1.

Figure 2: αSyn conformational strains in synucleinopathies.

So far, αSyn has been shown to adopt two different conformational strains. One of them is found in LBs extracted from cases of PD, DLB, PDD and AD, and a different strain is found in glial cytoplasmic inclusions in the brain of MSA patients. Structures were obtained from the PDB database (LB-fold: 8A9L; MSA: 6xyo; native αSyn: 1XQ8).

Altogether synucleinopathies affect a large number of people worldwide. DLB is the second most prevalent form of dementia after AD, affecting >7 million people in the world [4]. PD is the second most prevalent ND and the fastest growing neurological disorder, estimated to affect over 10 million people [5]. Both PD and DLB are associated with the accumulation of misfolded αSyn in the form of Lewy bodies (LBs) and Lewy neurites in the cytoplasm of neurons (Fig. 2). Importantly, more than 40% of AD cases have LBs identical to those observed in PD or DLB [6]. The contribution of LBs to AD pathophysiology is currently unknown, but postmortem histopathological studies have shown that the mean age at onset of dementia symptoms was younger for participants who had AD with LBs as well as more severe clinical deterioration [7]. Moreover, recent reports have shown that AD cases having αSyn pathology in addition to amyloid plaques and neurofibrillary tangles show faster rate of progression [8, 9]. Another interesting, but less prevalent synucleinopathy is MSA, which involves the accumulation of misfolded αSyn aggregates in oligodendrocytes as glial cytoplasmatic inclusions, which are morphologically different from LBs (Fig. 2) [10]. Despite the substantial differences in neuropathological alterations, the clinical symptoms of PD and MSA are remarkably similar [11], with the exception that deterioration in MSA typically progresses faster than in PD and that MSA exhibits higher autonomic dysfunction and poorer response to Levodopa treatment than PD [12].

αSyn is a 140 amino acid protein encoded by the SNCA gene, located in chromosome 4 [13]. It is highly abundant in brain cells from the striatum, midbrain and cortex, especially in axon terminals of pre-synaptic neurons [13]. Although its biological function is not entirely known, compelling evidence suggest that it plays an important role in modulating synaptic vesicle trafficking and release of neurotransmitter during synapsis [14]. However, mice lacking αSyn showed no major changes in survival, lifespan or behavioral abnormalities, albeit, they exhibit some subtle alterations in synaptic morphology and function [15]. In its physiological form αSyn is either a monomer or a tetramer and has an intrinsically disordered structure, but in the aggregated state it adopts a rigid and highly stable intermolecular β-sheet structure [16, 17], similar to fibrillar aggregates composed by various other misfolded proteins (Fig. 2).

The mechanism of αSyn aggregation: The importance of seeding

As with all other proteins implicated in diseases of protein misfolding, αSyn aggregation follows a seeding/nucleation mechanism [1, 18] characterized by the progressive formation of oligomers, protofibrils and fibrils, which culminate in the accumulation of LBs, usually located at the perinuclear site of neurons or in glial cytoplasmatic inclusions in oligodendrocytes (Fig. 2). In a seeding/nucleation polymerization reaction, the first and rate-determining step is the formation of a nucleus, which then rapidly elongate at expenses of the monomeric protein to form large aggregates [1, 18]. The nucleus, which has the ability to serve as a seed for catalyzing further polymerization, most likely corresponds to a misfolded protein oligomer that can be highly neurotoxic even before growing into larger aggregates [19]. A typical characteristic of the seeding/nucleation processes is that addition of preformed seeds can greatly accelerate the aggregation process by recruiting the soluble native protein into the growing aggregate.

The seeding/nucleation mechanism provides a biochemical explanation for the intriguing ability of misfolded proteins to spread its abnormalities between cells and tissues [20, 21]. This first became clear in prion diseases (Box 1) in which misfolded seeds composed of the prion protein act as an infectious agent to transmit disease between individuals [20, 22]. Remarkably, in prion diseases the sole administration of small quantities of misfolded prion protein aggregates can efficiently induce disease in animal models [22]. From the time between administration of the misfolded protein to the moment in which animals show clinical symptoms there is a massive amplification of the amount of misfolded protein aggregates present in the brain. The similarities on the molecular mechanisms of aggregate formation between prion protein and αSyn, as well as other proteins implicated in NDs, led to propose that misfolded proteins in most NDs may be able to spread between cells and tissues to initiate and expand the pathogenesis [18, 23]. Much data accumulated over the past 10 years has shown that αSyn misfolded aggregates can move from cell-to-cell to spread the damage from some areas of the brain to others [20, 21].

Box 1: Prion diseases and the prion principle.

Prion diseases, also known as transmissible spongiform encephalopathies, are fatal and infectious NDs affecting humans and various species of mammals [107]. The diseases in this group include Creutzfeldt-Jakob disease in humans, scrapie in sheep, bovine spongiform encephalopathy in cattle and chronic wasting disease in deer. The investigation of the nature of the infectious agent responsible for these diseases was a subject of intense research and passionate controversy for several decades [22, 108]. Since the beginning it was clear that the infectious material has different properties to conventional microorganisms responsible for infectious diseases (e.g. viruses or bacteria). In 1982, Stanley Prusiner coined the term prion to refer to a new class of infectious agent composed exclusively of a protein [109]. The prion hypothesis was initially taken with much skepticism since it is difficult to imagine how a protein can self-propagate to transmit a disease with complex traits including strain diversity and species barriers selectivity [22, 108]. Many subsequent studies provided increasing support for the protein-only nature of prions, including landmark experiments using the PMCA technology which enabled to generate bona-fide infectious materials in the lab by in vitro cell-free prion replication [47, 108]. Today the prion hypothesis is almost universally accepted. The elucidation of the process of prion propagation in prion diseases led to the recognition of a novel biological concept (i.e. the prion principle) that has been shown to be at the root of pathological progression in several highly prevalent diseases [20]. The prion principle posits that the infectious form of the prion protein is a polymer that can self-propagate by incorporating the normal prion protein present in the cells and template their misfolding to adopt the same abnormal structure as in the initial aggregate [20]. In this way, slowly but progressively, prions grow and spread in the body until they reach a concentration that becomes toxic and induce brain damage and disease. A similar process, albeit involving a different protein in each disease, is responsible for the progression of protein misfolding and aggregation in various other diseases, including AD, PD, DLB, amyotrophic lateral sclerosis, Huntington’s disease and many others [1]. The prion biology is fascinating and has revolutionized our understanding of brain diseases with potentially high impact for the development of novel strategies for diagnosis and therapeutic intervention.

Initial clinical support for the prion-like propagation of αSyn came from the analysis of PD patients who were treated by grafts of fetal mesencephalic progenitor neurons. Upon autopsy of these patients it was seen that LBs were detected in the grafted tissue, which was interpreted as host-to-graft spreading of αSyn aggregates [24]. The prion-like spreading of αSyn also provides a molecular explanation for the Braak model of PD pathological progression, in which LB pathology follows a characteristic stereotypical and spatiotemporal progression indicative of pathology spreading through neuroanatomically connected neurons in different brain areas [25].

Experiments with cells in culture showed that addition of αSyn seeds either prepared in vitro or extracted from patients brains can be uptaken by the cells and seed the conversion of endogenous αSyn leading to the progressive accumulation of aggregates in the cells [26]. In vivo studies using animal models showed that administration of recombinant αSyn aggregates, usually referred as pre-formed fibrils or PFF, can induce αSyn pathology even in wild type mice [27, 28], leading to dysfunction of dopaminergic neurons and motor deficits. Intracerebral injection of brain extracts from MSA patients into transgenic mice expressing human mutant αSyn also resulted in pathological transmission [29, 30]. Surprisingly, a similar experiment administering PD brain extracts did not produce LB pathology or behavioral abnormalities in the recipient animals [29], however another study reported positive results in mice and monkeys [31]. It is unclear why MSA and PD αSyn aggregates have different abilities to transmit pathology in vivo, but it may reflect the more aggressive and faster progression of pathological abnormalities in MSA as compared to PD.

The emergence of αSyn-SAA: A Prion inspiration

The prion principle by which a misfolded and infectious form of the prion protein can template the exponential pathological conversion of the normal prion protein (Box 1), inspired the development of in vitro assays to amplify the process of prion replication. Typically infectious agents are propagated in research laboratories to study their biological properties. Since prions have a different composition and biology compared to conventional infectious agents, a new methodology was needed to propagate them in vitro. The first of these assays was called protein misfolding cyclic amplification (PMCA), which was able to mimic the process of prion replication to induce the conversion of a large amount of normal prion protein into the misfolded, infectious form [32]. The PMCA technique was subsequently modified to give rise to other amplification technologies, most notoriously the real-time quacking induced conversion (RT-QuIC) assay [33, 34]. Both PMCA and RT-QuIC are able to detect prions in diverse human and animal biological samples (e.g. CSF, blood, urine skin, olfactory mucosa) with sensitivities and specificities approaching 100% [34–42]. Indeed, these assays are now been routinely used for helping diagnosis of prion diseases worldwide. PMCA and RT-QuIC achieve the rapid amplification of prion seeds through a cyclical process involving elongation of the oligomeric seeds at expenses of the normal prion protein followed by a step of aggregate fragmentation to release more converting units to accelerate the reaction (Fig. 3) [43, 44]. Fragmentation is usually performed by a mechanical force, most typically sonication in the case of conventional PMCA and strong shaking in the case of RT-QuIC. The cyclical nature of these assays as well as their power of amplification makes them comparable to the polymerase chain reaction (PCR) to amplify DNA [45]. Over the past 20 years, PMCA and RT-QuIC have been extensively used by many investigators worldwide to detect prions in biological fluids and tissue samples and to study the intriguing biology of infectious prions [45, 46]. Using PMCA, it was possible to produce, for the first time, infectious prions in the test tube capable to induce a disease in wild type animals with all the typical features of prion disorders [47]. Later on, it was shown that amplified prions maintain the typical properties of these infectious agents, suggesting that PMCA might help to study in greater depth the characteristics of prions [48, 49]. PMCA and RT-QuIC are widely considered as major technological breakthroughs in the study of prion diseases.

Figure 3: Schematic representation of αSyn-SAA.

Samples of human biological fluids (e.g. CSF, blood) collected from patients affected by synucleinopathies contain seed-competent αSyn aggregates that can seed the aggregation of monomeric αSyn provided to the reaction. Cycles of incubation and fragmentation (shaking, represented by waves) are done to exponentially amplify the process of protein misfolding and aggregation. The amount of aggregates produced over time during the assay is measured in real-time by the fluorescence of an amyloid-binding dye (thioflavin T). Figure was created using Biorender.

Considering that all misfolded protein aggregates implicated in NDs utilize the prion principle for forming and spreading the pathology [1], it was natural to extend these technologies to amplify and detect misfolded protein aggregates associated with more prevalent diseases such as AD and PD. In 2014, we extended the principles of PMCA/RT-QuIC to amplify amyloid-beta (Aβ) aggregates implicated in AD [50] and in 2016, we and Green’s group introduced a similar procedure to amplify αSyn aggregates implicated in PD and other synucleinopathies [51, 52]. Caughey’s group developed assays for detection of tau seeds [53, 54]. Recently, the consensus term seed amplification assay (SAA) was introduced by a group of scientists to refer to these assays outside the prion field [55, 56]. This was in order to avoid the association of the terms PMCA and RT-QuIC with replication of infectious prions and to better represent the basis for these assays, i.e. the amplification of biological seeds. Thus, the recommendation is to reserve the terms PMCA and RT-QuIC for the prion field, while use the unified name SAA for all cyclic amplification assays involving other proteins and diseases, referring to them as for example Aβ-SAA, Tau-SAA or αSyn-SAA for the assays to detect Aβ, tau or αSyn aggregates, respectively.

The past, present and future of αSyn-SAA

The αSyn-SAA technology uses the seeding concept to amplify the amount of misfolded αSyn aggregates present in patients’ biological samples (Fig. 3). Since the first reports in 2016, there have been many articles from various different groups describing the use of αSyn-SAA to detect pathological αSyn aggregates in various matrices [8, 9, 55, 57–85]. The assay conditions have been progressively improved and the most recent methodologies require less than 24h to produce results. The large majority of the studies have focused on the use of cerebrospinal fluid (CSF), since it is a sample with a low complexity, not highly invasive to collect and it is surrounding the brain, which is the place where the largest amount of αSyn aggregates accumulate in the disease. Considering all published studies so far, few thousand samples of CSF from patients affected by PD, DLB or MSA have been tested along control samples from healthy individuals and those affected by other neurological diseases [8, 9, 51, 52, 55, 57–65, 68, 73, 75–77, 79, 84, 85]. Many of the assays reported use different experimental conditions, but the assay performance is remarkably similar [56]. In average, sensitivity and specificity values across the different studies have been between 85–95% (for meta-analysis reviews, see [86, 87]). Interestingly, studies done with the same samples provided blinded to investigators in different laboratories, reached very similar results, supporting the robustness of the assay [55, 63, 88]. Remarkably, several studies have reported the successful detection of αSyn aggregates in CSF of prodromal cases of PD or DLB, including idiopathic REM sleep behavior disorder (iRBD), mild cognitive impairment and pure autonomic failure [57, 58, 60, 65, 77, 89]. These results suggest that αSyn-SAA can be used to detect pre-symptomatic individuals on the way to develop the clinical disease. A recently published article utilized a large number of CSF samples (1123 participants) from the PPMI (Parkinson’s progression markers initiative) collection, including patients diagnosed with idiopathic PD, familial cases associated with LRKK2 and GDA mutations (including normal carriers) and subjects classified as prodromal cases [57]. This study represents the largest evaluation of αSyn-SAA for biochemical diagnosis of PD and the results obtained indicated that the assay distinguish PD patients from controls with high sensitivity and specificity. In addition the findings provided valuable information about molecular heterogeneity in different patient populations and demonstrated the ability to detect prodromal individuals, years before clinical diagnosis [57].

In the past couple of years, several groups have studied the detection of αSyn aggregates by αSyn-SAA in other samples, including skin, olfactory mucosa, submandibular gland, saliva and blood plasma [66, 68, 69, 71, 78–81, 83, 89]. Several of these reports have shown a good level of sensitivity and specificity, but more studies are needed to analyze the robustness and reproducibility of those assays. The adaptation of αSyn-SAA to easily collectable samples is a main goal for the future. This will enable the massive use of αSyn-SAA for diagnosis of PD and other synucleinopathies as well as for screening healthy individuals at risk for the disease.

Another goal for future research is to demonstrate that αSyn-SAA can provide quantitative information regarding the number of seeds present in the biological sample and to see if this correlate with the stage of the disease and can be used to monitor treatment efficacy. Several articles have shown data suggesting the assay is quantitative since the kinetic parameters of aggregation correlate with the amount of synthetic seeds spiked into biological fluids [51, 76]. However, it is unclear if the concentration of misfolded αSyn correlates with the disease progression. Based on the findings from other NDs, including AD and prion diseases, the misfolded protein is most likely the triggering event that initiate the pathological cascade in the brain, but once this process starts, the amount of protein aggregates accumulating in the brain may not correlate with the pathological damage [90–92]. Much more research is needed to understand the exact role of αSyn in PD, DLB, MSA and AD, in particularly how the quantity of αSyn changes with disease progression.

Finally, an important focus of future research is to study if αSyn-SAA faithfully amplify the structure and properties of misfolded αSyn in patients’ brain. This is irrelevant for detection and diagnosis, but it is important to use the technology to study the underlining biology of the disease and to screen molecules for therapeutic intervention. Based on findings reported in prion disease, it seems clear that the ability to replicate the structure and properties depends on the conditions used in the amplification reaction. Indeed, the infectivity and biological properties of misfolded prion protein are faithfully maintained when using the PMCA conditions, but not when using the RT-QuIC methodology [45]. As such, it is possible that some αSyn-SAA protocols may preserve the properties of αSyn seeds than others.

The multiple applications of the αSyn-SAA technology

Seed amplification assays, including PMCA and RT-QuIC for prions and αSyn-SAA, Aβ-SAA and Tau-SAA have revolutionized the study of NDs and have had a large practical impact on patient diagnosis. These technologies have multiple applications and here I will briefly outline the many areas of research that will benefit from using the αSyn-SAA technique: to understand the biology of synucleinopathies, as a disease biomarker, and to improve drug discovery and clinical trials (Table 1).

Table 1:

Multiple applications of αSyn-SAA.

Understanding disease biology	αSyn-SAA as the basis for a biological definition of PD, DLB and MSA
	Using αSyn-SAA to understand the mechanism of αSyn spreading
	Using the product of αSyn-SAA as a faithful surrogate of patients’ αSyn aggregates
	Identification of αSyn conformational strains
αSyn detection and disease diagnosis	αSyn-SAA as a biochemical test for objective diagnosis of PD, DLB and MSA
	Using αSyn-SAA to identify the subset of AD patients having αSyn pathology
	Detection of prodromal cases on the way to develop brain damage and disease
	Screening the healthy population at risk for preclinical αSyn pathology
	Identification of patients with Parkinsonism not associated to αSyn pathology
	Monitoring disease progression by αSyn-SAA
Drug discovery and clinical trials	αSyn-SAA as a screening tool to identify molecules inhibiting αSyn spreading
	Using αSyn-SAA to study the mechanism of action of drug candidates
	αSyn-SAA for patient enrollment in clinical trials
	Monitoring treatment efficacy by αSyn-SAA
	Using αSyn-SAA for personalized medicine

Using αSyn-SAA to understand the biology of synucleinopathies

A hallmark event in NDs associated with the accumulation of LBs or glial cytoplasmatic inclusions in the brain is the ability of the misfolded protein to spread the pathology through the seeding mechanism [20, 21]. Since αSyn-SAA utilizes the seeding activity as a mean to amplify the reaction, it is likely that αSyn-SAA could be used to further understand the molecular and structural basis of αSyn spreading, which will open novel targets for therapeutic intervention. The technique may also serve to generate large quantities of misfolded αSyn aggregates for biological and structural studies. The utility of αSyn-SAA to study αSyn seeding depends to a large extent on whether or not the assay allows the faithful replication of the biochemical and structural properties of αSyn aggregates. A large effort is currently being devoted by several groups to answer this important question. The basic concept behind seeding of protein misfolding and aggregation is that a preformed polymer (coming from a patient sample) templates and direct the conversion of the monomeric protein by adding it to the growing aggregate [1]. Thus, in theory seeded conversion should faithfully maintain the structure and biology of the seed. However, as stated earlier, it is likely that the specific experimental conditions used in the αSyn-SAA will play a big role on whether or not in vitro seeding keeps the seed properties. In case that some forms of αSyn-SAA do not replicate the structure of some of the seeds, it should be possible to modify the experimental conditions to achieve faithful replication of the structural properties after seeding. It is also possible that post-translational modifications which are not present in the recombinant αSyn used as substrate might be important to keep the strain properties. Finally, it is also conceivable that other brain co-factors may participate in the in vivo spreading of αSyn pathology and hence in the absence of these extra co-factors the in vitro reaction may not successfully replicate the properties of endogenous αSyn aggregates. If this is the case, αSyn-SAA might be very useful to identify those brain co-factors.

Another interesting area of research where αSyn-SAA may be useful is on the identification of αSyn conformational strains. In the case of αSyn, compelling evidence suggest the existence of at least two different conformational strains, one resulting in LB formation and producing PD or DLB and another one leading to glial cytoplasmic inclusions and resulting in MSA (Fig. 2) [61, 93, 94]. Near atomic resolution structural studies by cryo-electron microscopy have shown that the three-dimensional structure of αSyn aggregates in LBs are substantially different as those from MSA patients [17, 95]. Interestingly, αSyn-SAA can distinguish these strains by analyzing the amplification product obtained in the reaction [61, 62, 78, 89]. Using various biochemical, biological and structural techniques it was possible to differentiate the amplification product coming from PD and MSA CSF samples with high sensitivity and specificity [61]. Thus the assay may allow not only to detect αSyn pathology, but also which conformational strain a patient has. It remains to be seen if the assay fully recapitulate the strain properties of diverse seeds and also whether there are other natural αSyn strains that might be implicated in different clinical presentations of PD or DLB subjects or associated to other diseases (e.g. AD).

Finally, another important use of αSyn-SAA is the implementation of a biological definition of synucleinopathies. Currently, these diseases are diagnosed and classified based on the onset of clinical symptoms, aided by indirect imaging and biomarkers tests. The clinical symptoms of synucleinopathies are a spectrum of abnormalities including motor and cognitive impairments to different degrees in distinct diseases [96]. However, there are several other movement and cognitive disorders unrelated to the accumulation of αSyn deposits that present a similar clinical manifestation. Furthermore, it is clear that the formation and accumulation of αSyn pathology begins years before the onset of symptoms. Given the high accuracy of αSyn-SAA to identify the presence of αSyn pathology, it is now possible to advance towards a biological definition of the disease regardless of the presence and type of clinical symptoms [97, 98]. A diagnosis based on biological changes measured in live patients rather than the clinical consequences of the pathology will enable the identification of these diseases in early stages and enable therapeutic intervention before substantial damage in the brain.

Using αSyn-SAA to detect αSyn aggregates and biochemically diagnose the disease

The most obvious application of the αSyn-SAA technology is to detect minute quantities of pathological αSyn circulating in biological fluids to serve as a biomarker for disease diagnosis. As discussed above, many independent studies have confirmed the high diagnostic accuracy of αSyn-SAA in CSF samples [86, 87]. As a result, the technology is currently being used clinically for biochemical diagnosis of PD and other synucleinopathies. In order for the technology to be used more massively, it is imperative to show that the assay works robustly in less invasive samples, such as blood, urine or saliva. Several reports have shown pilot data supporting the use of αSyn-SAA in samples of skin, olfactory mucosa, saliva and blood [66, 68, 71, 78–81, 83, 88, 89]. More studies need to be done to confirm the robustness and reproducibility of these assays.

One interesting application of αSyn-SAA will be to identify in living people the subset of AD patients that have αSyn aggregates in their brains. Typically AD is associated with the cerebral accumulation of Aβ and Tau aggregates. However, compelling evidence coming from postmortem histopathological studies have shown that between 30–50% of AD patients also display abundant LBs in the brain [2, 99]. We and others have shown that αSyn-SAA can detect αSyn aggregates in a subset of AD patients [51, 85]. The role of αSyn aggregates in AD is unknown, but some studies have reported that AD subjects having LBs progress faster and to a more severe stage than patients not showing evidence for αSyn pathology [6, 9]. Indeed, LB pathology, but not Aβ or Tau, was associated with hallucinations and worse attention/executive, visuospatial and motor function [8]. Detection of αSyn-SAA signal was also observed in AD patients displaying a faster longitudinal decline. Thus, the available evidence point to an important contribution of αSyn pathology in AD.

An important practical objective in the study of NDs including synucleinopathies is the identification of the disease at the earliest possible time during the preclinical phase. It is well-established that αSyn begins to accumulate in the brain years, if not decades, before the onset of massive brain damage and clinical symptoms. As has been shown for AD, it is likely that disease-modifying therapeutic interventions will produce the maximum benefit when treatment is started early on. Various studies have shown the high accuracy of αSyn-SAA to detect αSyn pathology in prodromal cases, including iRBD, mild cognitive impairment and pure autonomic failure [57, 58, 60, 65, 77, 79]. The data shows not only that αSyn-SAA can detect αSyn pathology at the preclinical stage, but also that a substantial number of αSyn-SAA positive prodromal cases convert into clinically-diagnosed patients. Related to the above, availability of an accurate and non-invasive assay for detection of αSyn pathology may enable a massive screening of the healthy population at risk of synucleinopathies. Such a test will enable to identify people on the way to develop pathology, much before brain damage and disease.

Another significant goal in which αSyn-SAA can contribute is to identify those patients with Parkinsonism who do not have αSyn pathology. Parkinsonism is not a disease, but a syndrome [100], which can be caused by multiple processes leading to death of dopaminergic neurons, including for example exposure to toxic molecules, stroke or traumatic brain injury. There are also various diseases which lead to a similar clinical manifestation of PD, like progressive supranuclear palsy or corticobasal degeneration, both of which are caused by accumulation of Tau aggregates [100]. Unlike PD, some of these disorders may have acceptable treatments, so it is very important to identify cases in which the cause of the disease is not a synucleinopathy.

Finally, αSyn-SAA could be used to monitor disease progression in an objective biochemical manner. This will require an assay providing quantitative information. It is also necessary to show that αSyn pathology correlates with disease progression. As discussed earlier, it is possible that αSyn is just the trigger of neurodegeneration but not the cause itself. In this case the quantity of αSyn aggregates may not relate to severity of the disease.

Using αSyn-SAA for helping drug discovery and clinical trials

Since αSyn-SAA reproduces in an accelerated manner the process of αSyn spreading that occurs during the disease, the technology may serve as a primary screening assay to identify hits for therapeutic intervention. αSyn-SAAs are routinely performed in ELISA plates and have a fluorescence readout, thus making them amenable to medium or high throughput screening assays. Moreover, the most recent assays can be done in less than 24h. With some modifications, it is likely that αSyn-SAA might be improved to be able to screen hundreds of thousands compounds. A related application is the use of αSyn-SAA to study the mechanism of action of existing drug candidates. Addition of these molecules to the αSyn-SAA reaction may help to identify compounds capable to block αSyn seeding. Pre-treatment of patient derived seeds may show if the compounds are able to disrupt the aggregates and their seeding properties. The assay may also be useful as a readout to improve the potency of drugs.

One critical aspect in performing a successful clinical trial is to enroll the right patients in the study population. As discussed earlier, a set of people with symptoms of Parkinsonism do not have αSyn pathology [100]. Thus, a trial testing an anti-αSyn therapy should not include these patients. Up to know there was not any technology available to identify αSyn pathology is living patients. αSyn-SAA accomplish this with high sensitivity and specificity as illustrated by the many studies with CSF samples [86, 87], including those with histopathologically confirmed patients [52, 68, 76, 85, 101, 102]. Therefore, the seed amplification technology may have a high impact in improving patient enrollment in clinical trials, likely resulting in increasing the chances for effective molecules to produce statistically significant improvement.

The αSyn-SAA technology might also be used to monitor efficacy of treatments targeting αSyn pathology. Availability of an objective tool to measure the pharmacodynamic efficacy of drug candidates in terms of lowering the extent of αSyn pathology will certainly be very useful in clinical trials. For this purpose, a quantitative αSyn-SAA is needed, as described earlier. Finally, given the ability of αSyn-SAA to identify specific αSyn conformational strains [61], the technology might be useful in the future to help determining the best treatment for a given patient, thus contributing to personalized medicine. Furthermore, when SAA tools to detect other misfolded proteins become fully optimized and validated, a panel of seed amplification tests targeting different misfolded aggregates may be essential to treat patients with the right cocktail of drugs.

Concluding remarks

The development of assays for ultrasensitive detection of misfolded protein aggregates based on the seeding activity of these aggregates represents a major breakthrough in the diagnosis of NDs. Currently, PMCA and RT-QuIC are being routinely used in the clinic to help diagnosing human prion diseases. Similarly, SAA methods to detect misfolded αSyn aggregates are already available to patients and physicians for helping diagnosis of PD, DLB, MSA and for identifying the subset of AD patients having αSyn pathology. Although αSyn-SAA has already provided substantial advances in the field of synucleinopathies, still much more research is needed. Many open questions remain (see Outstanding Questions) and further technical improvements are needed to fully capitalize on the power of αSyn-SAA. Among the next key steps is the adaptation as well as the analytical and clinical validation of the technology to detect αSyn aggregates in more easily to collect biological fluids or tissues than CSF. Also important will be to investigate whether the αSyn-SAA output can provide information regarding the disease progression or to identify the individuals at prodromal stages who are close to develop clinical symptoms. Having a quantitative or even a semi-quantitative αSyn-SAA test will be an important goal for the future. Finally, to use the technology for studying the underlying biology of the disease will be important to understand whether the assay fully recapitulate the in vivo seeding process and the product of the assay retains the structural and biological properties of the seeds.

Outstanding questions.

• Is detection of seeding-competent misfolded αSyn a good biomarker for diagnosis of Parkinson’s disease and related synucleinopathies?

• Does αSyn-SAA enable early, sensitive and specific biochemical diagnosis of synucleinopathies?

• Can αSyn-SAA be adapted to detect the biomarker in easily collectable biological fluids?

• Does αSyn-SAA provide information about the quantity of misfolded αSyn present in a sample?

• Does misfolded αSyn produced by αSyn-SAA keep the biological and structural properties of the aggregates present in patients’ brains?

• Can αSyn-SAA be used for identification of therapeutic molecules and to increase the performance of clinical trials?

• Will αSyn-SAA enable a biological definition of Parkinson’s disease and other synucleinopathies?

Various additional SAA techniques are under development and will likely be implemented soon for detection of other misfolded protein aggregates, including Aβ, tau, TDP43, SOD1, huntingtin, etc [50, 53, 54, 103, 104]. Each of these assays is at different stages of development, but it should be possible to successfully develop them in the near future. Distinct aggregation-prone proteins behave differently and some (e.g. Aβ) are more difficult to manage experimentally than others and to obtain reproducible results. Giving the complexity and heterogeneity of NDs, it is likely that the best approach for identifying incipient brain pathology will be to test simultaneously all the main proteins accumulating in the brain. This is very relevant since it is widely accepted that many patients affected by NDs have mixed pathology in the brain, i.e. they contain aggregates composed of diverse proteins [105, 106]. Cases with mixed pathologies may show a distinct clinical presentation, most likely more severe progression, which may change the clinical course of the disease. This, in turn, has many important implications for disease management, therapeutic intervention and patient engagement in clinical trials. Having a set of SAA tests that can detect different relevant proteins, such as Aβ, tau and TDP43 among others, will be a major contribution to scientific, clinical and translational research in NDs.

Early detection of disease pathological abnormalities is crucial for therapeutic intervention. It is very clear from recent studies that the accumulation of αSyn aggregates detectable by αSyn-SAA in CSF starts years if not decades before the onset of clinical symptoms [57, 58, 77, 79]. At these early stages there is not yet brain damage and the pathology is weak. Early identification of patients may allow to attempt slowing down progression simply by introducing life-style changes or by using safe pharmacological drugs that can be administered to asymptomatic individuals. The goal in NDs in general and in PD in particular is to begin treatment at a time the brain is not irreversibly damaged. Therefore, combining an early diagnosis with a safe treatment may enable to eradicate these diseases that are devastating our society.

Highlights.

• Synucleinopathies are highly prevalent neurodegenerative diseases affecting the elderly population.

• By the time disease manifests clinically there is extensive neuronal death. Early detection of the pathology at the preclinical phase is essential for therapeutic benefit.

• The best biomarker is the abnormal αSyn protein that likely causes the brain damage. However, the quantity of this protein is high only in the brain at the end of the disease.

• Seed amplification assays (SAAs) have recently emerged to accurately detect small amounts of abnormal αSyn in human biological fluids.

• The αSyn-SAA enables highly sensitive, specific and reproducible detection of this biomarker in cerebrospinal fluid and is currently available to patients for clinical use.

• The SAA technology can help to understand disease biology, to identify new therapeutic molecules and to improve clinical trials.

Glossary

Alpha-synuclein (αSyn)

A neuronal protein that regulates synaptic vesicle trafficking and subsequent neurotransmitter release and the main constituent of Lewy bodies in Parkinson’s disease and dementia with Lewy bodies.

αSyn seed

Refers to an aggregated (likely oligomeric) form of the protein that has the ability to nucleate polymerization, speeding up the aggregation process.

Amyloid

A generic term to refer to protein aggregates characterized by a fibrillar appearance of typically 7–13 nm in diameter, an intermolecular β-sheet structure (known as cross-β) and ability to be stained by specific dyes, such as Congo red and Thioflavin S.

Conformational strains

Refer to the ability of various misfolded protein aggregates to adopt alternative structures that can faithfully propagate and produce different forms of brain damage and clinical symptoms.

Idiopathic rapid eye movement (REM) sleep behaviour disorder (iRBD)

A parasomnia characterized by dream-enacting behavior and loss of muscle atonia during REM sleep. It is a considered a prodromal stage of synucleinopathies.

Neurodegenerative diseases (NDs)

A group of human disorders characterized by progressive and chronic brain degeneration.

Lewy bodies (LBs)

Protein deposits typically found in neurons in the brain of patients affected by Parkinson’s disease (PD) and dementia with Lewy bodies (DLB).

Lewy body disease (LBD)

Refers to any disease in which LBs are prominent in the brain by neuropathological analysis. This includes PD, DLB and a subset of AD patients.

Prions

The protein-only infectious agent responsible for prion disease which propagates and transmit disease in the absence of nucleic acids.

Seed amplification assay (SAA)

Refers to a technology to achieve cyclic amplification of the process of protein misfolding and aggregation implicated in various neurodegenerative diseases. SAA is referred in the prion field as PMCA (Protein Misfolding Cyclic Amplification) or RT-QuIC (Real Time-Quaking Induced Conversion).

Synucleinopathies

A group of neurodegenerative diseases associated with the accumulation of alpha-synuclein deposits, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA)