α-Synuclein: A Promising Biomarker for Parkinson’s Disease and Related Disorders

Article information

J Mov Disord. 2024;17(2):127-137

Publication date (electronic) : 2024 April 9

doi : https://doi.org/10.14802/jmd.24075

Taku Hatano ¹^,

, Ayami Okuzumi ¹

, Gen Matsumoto ²

, Taiji Tsunemi ¹

, Nobutaka Hattori ¹^,³^,

¹Department of Neurology, Juntendo University Faculty of Medicine, Tokyo, Japan

²Department of Histology and Cell Biology, Nagasaki University School of Medicine, Nagasaki, Japan

³Neurodegenerative Disorders Collaboration Laboratory, RIKEN Center for Brain Science, Saitama, Japan

Corresponding author: Taku Hatano, MD, PhD Department of Neurology, Juntendo University Faculty of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan / Tel: +81-3-58000547 / Fax: +81-3-58000542 / E-mail: thatano@juntendo.ac.jp

Corresponding author: Nobutaka Hattori, MD, PhD Department of Neurology, Juntendo University Faculty of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan / Tel: +81-3-58000547 / Fax: +81-3-58000542 / E-mail: nhattori@juntendo.ac.jp

Received 2024 March 22; Revised 2024 March 30; Accepted 2024 April 9.

Abstract

Mutations in the SNCA gene, which encodes α-synuclein (α-syn), play a key role in the development of genetic Parkinson’s disease (PD). α-Syn is a major component of Lewy bodies in PD and glial cytoplasmic inclusions in multiple system atrophy (MSA). Rapid eye movement sleep behavior disorder patients often progress to PD, dementia with Lewy bodies, or MSA, which are collectively known as α-synucleinopathies. The loss of dopaminergic neurons with Lewy bodies precedes motor dysfunction in these diseases, but the mechanisms of neurodegeneration due to α-syn aggregation are poorly understood. Monitoring α-syn aggregation in vivo could serve as a diagnostic biomarker and help elucidate pathogenesis, necessitating a simple and accurate detection method. Seed amplification assays (SAAs), such as real-time quaking-induced conversion and protein misfolding cyclic amplification, are used to detect small amounts of abnormally structured α-syn protofibrils, which are central to aggregation. These methods are promising for the early diagnosis of α-synucleinopathy. Differences in α-syn filament structures between α-synucleinopathies, as observed through transmission electron microscopy and cryo-electron microscopy, suggest their role in the pathogenesis of neurodegeneration. SAAs may differentiate between subtypes of α-synucleinopathy and other diseases. Efforts are also being made to identify α-syn from blood using various methods. This review introduces body fluid α-syn biomarkers based on pathogenic α-syn seeds, which are expected to redefine α-synucleinopathy diagnosis and staging, improving clinical research accuracy and facilitating biomarker development.

Keywords: Parkinson’s disease; Multiple system atrophy; α-Synuclein; α-Synucleinopathy; Seed amplification assays

INTRODUCTION

Mutations in the SNCA gene, which encodes the α-synuclein (α-syn) protein, as well as multiplication of this gene, are implicated in the pathogenesis of genetic Parkinson’s disease (PD) [1,2]. α-Syn has been identified as a principal component of Lewy bodies [3], and subsequent studies have shown that it is also a major constituent protein of glial cytoplasmic inclusions in multiple system atrophy (MSA) [4,5]. Furthermore, patients with rapid eye movement sleep behavior disorder (RBD) transition to PD, dementia with Lewy bodies (DLB), and MSA during their clinical course [6]. These neurodegenerative diseases characterized by α-syn aggregation are collectively termed α-synucleinopathies [7].

Pathological studies of α-synucleinopathies have revealed that a loss of 30%–75% of dopaminergic neurons with Lewy bodies precedes motor dysfunction [8]. However, the onset and progression of neurodegeneration due to α-syn aggregation remain poorly understood. Therefore, in vivo monitoring of the α-syn aggregation process in patients with α-synucleinopathy is expected to serve as a diagnostic biomarker for this disease and a tool to elucidate its pathomechanism. To achieve this goal, a simple and accurate method for detecting α-syn aggregation is necessary.

α-Syn is an amphiphilic protein with two α-helix structures. The conversion of normal α-syn to an abnormally structured form with a β-sheet structure leads to the engulfment of normally structured α-syn proteins, converting them to an abnormal structure. These conformational changes tend to result in fibrilization, forming oligomers, protofibrils, and fibrils [9]. Protofibrils, early intermediates in the fibril formation pathway of α-syn, are considered to have the potential to propagate from cell to cell, similar to prion proteins [10]. By utilizing this property, methods such as real-time quaking-induced conversion (RTQuIC) [11] and protein misfolding cyclic amplification using ultrasound waves (PMCAs) [12] have been developed to detect minute amounts of abnormally structured α-syn protofibrils (α-syn seeds), which are central to aggregation. These methods, collectively known as α-syn seed amplification assays (SAAs), have gained attention for their potential in the early diagnosis of α-synucleinopathy [13,14].

Transmission electron microscopy (TEM) analyses have revealed that α-syn filament structures amplified by SAAs differ between α-synucleinopathies [15,16]. Additionally, cryo-electron microscopy analyses have shown that the structure of α-syn seeds generated by stimulating recombinant α-syn proteins differs between patients with MSA and patients with PD [17,18]. These differences in α-syn seed structure may play a role in the pathogenesis of neurodegeneration [19], and SAAs may be useful both for differentiating α-synucleinopathy from other diseases as well as for distinguishing subtypes of α-synucleinopathy. In addition to SAAs, various groups are attempting to identify α-syn from blood using antibodies, aptamers [20], exosomes [21,22], and other methods to differentiate α-synucleinopathy. Recently, positron emission tomography (PET) ligands have been successfully used to visualize α-syn in the MSA brain in vivo [23].

This review introduces body fluid α-syn biomarkers based on pathogenic α-syn seeds. The development of accurate and useful biomarkers will have a huge impact on the disease definition of α-synucleinopathy. Biomarker-based diagnosis and PD staging are expected to undergo a paradigm shift.

α-SYNUCLEIN AGGREGATION AND PROPAGATION

α-Syn is a protein with a molecular weight of 14 kDa. Its Nterminal region, comprising amino acids 1–60, is amphiphilic, while the subsequent nonamyloidal component (NAC) region (amino acids 61–95) is hydrophobic and involved in aggregation. The segment from the NAC region to the C-terminus (amino acids 95–140) contains numerous acidic amino acids, which are believed to bind calcium and perform a chaperone-like role [24]. The N-terminal region adopts an α-helix structure, enabling membrane binding. Additionally, calcium binding at the C-terminus is reported to regulate the binding of synaptic vesicles to the membrane [25], suggesting a physiological role for α-syn in membrane transport at synapses. α-Syn is expressed in peripheral and central neurons as well as in red blood cells and platelets [9].

Under normal conditions, α-syn is believed to exist as a natively unfolded monomer but can stabilize into a tetramer of approximately 58 kDa. An imbalance between these monomeric and tetrameric states may induce α-syn aggregation [26]. However, there is controversy regarding whether α-syn may exhibit a tetrameric structure in the brain [27]. Conformationally altered α-syn with β-sheet structures tends to aggregate, engulfing normally structured α-syn molecules and converting them into an abnormal aggregating form. This process leads to fibrillation from oligomers (protofibrils) composed of a few conformationally altered α-syn molecules. Oligomers are highly toxic and induce cellular dysfunction, including synaptic, mitochondrial, and lysosomal-autophagic dysfunction [28]. They have also been reported to form pores in cell membranes, increasing membrane permeability and causing neurotoxicity [29]. Correlative light and electron microscopy analysis revealed the involvement of various membranous organelles, including mitochondria, lysosomes, and synaptic vesicles, in the formation of Lewy bodies. It has been suggested that α-syn fibrils may initiate the aggregation of these organelles, leading to the development of Lewy bodies (Figure 1) [30].

Figure 1.

Systematic distribution and pathogenic mechanisms of α-synucleinopathies. α-Synuclein possesses the native ability to bind to membrane lipids through an α-helical conformation or to assume an unstructured, fluid state within the cytosol. It also forms stable tetramers. Pathologically, β-sheet-structured α-synuclein engulfs and transforms normally configured proteins into aberrant forms, thereby hastening the process of aggregation and the genesis of fibrils. These misfolded fibrils of α-synuclein are implicated in the formation of Lewy bodies, intricately involving membranous organelles, such as mitochondria, lysosomes, autophagosomes and synaptic vesicles, in their architecture. Notably, α-synuclein aggregates are present in the central nervous system and peripheral autonomic nerves, including the submandibular glands, skin, gastrointestinal tract, and cardiac tissue. The aberrant α-synuclein conformers that initiate fibrillation can serve as nucleation sites for further fibril propagation, potentially through the blood and cerebrospinal fluid to affect cerebral and systemic functions, resulting in motor and nonmotor symptoms.

The oligomers that initiate α-syn fibrilization are thought to be the seeds of abnormal α-syn aggregation. Autopsy of brains taken from patients with PD who received transplants of aborted fetal dopaminergic neurons showed Lewy body-like α-syn aggregates in the grafts [31,32]. This suggests that α-syn seeds may propagate from the host to the grafted cells. Experimentally, several months after mouse-derived α-syn seeds were inoculated into the mouse brain, α-syn aggregates were observed across a wide area of the brain extending from the injection site [33]. Moreover, the inoculation of Lewy bodies extracted from the brains of patients with PD into the mouse brain also induced widespread α-syn aggregation extending from the injection site [34]. Thus, α-syn seeds propagate via neuronal cells and aggregate in affected neurons. The propagation of α-syn seeds cannot be suppressed even by dissection of the corpus callosum 24 hours after inoculation into the mouse striatum, suggesting a propagation speed of less than 24 hours. Furthermore, botulinum toxin can inhibit this propagation, indicating that exocytosis may be one mechanism of α-syn seed propagation [35].

In PD, α-syn aggregates spread both to the brain and to the systemic autonomic nervous system. For example, 123I-MIBG myocardial scintigraphy, which detects degeneration of cardiac sympathetic nerves, revealed cardiac autonomic neurodegeneration in patients with PD [36,37]. Additionally, α-syn aggregation in intestinal autonomic neurons occurs in the prodromal stage of PD [38]. Although the aggregation of α-syn in skin autonomic nerves is observed in PD and MSA patients, the distribution pattern differs between them [39]. Meta-analyses investigating the pathogenic factors of PD have revealed that familial history and environmental factors, such as rural living, farming, and agriculture, are important [40]. Several epidemiological studies have implicated pesticides and air pollution in pathogenesis [41-44]. Moreover, the intestinal microbiota [45,46] and appendicitis [47] are thought to contribute to α-syn aggregation. These findings suggest that α-syn aggregation may be triggered by environmental factors, leading to the spread of systemic neurons and the central nervous system along neuronal circuits (Figure 1). Based on this concept, detecting α-syn in body fluids, including cerebrospinal fluid (CSF) and blood, may be useful as a biological marker for PD.

DETECTION OF α-SYNUCLEIN

As highlighted earlier, systemic dissemination of α-syn seeds may play a role in the pathogenesis of PD. Therefore, detecting α-syn in body fluids could aid in understanding and diagnosing α-synucleinopathy. α-Syn is detectable in CSF, peripheral blood cells and platelets. Numerous studies have assessed the total amount of α-syn in spinal fluid (t-α-syn) and investigated its levels in blood. Furthermore, α-syn oligomers (o-α-syn) and Ser129-phosphorylated α-syn (pS129-α-syn) are being explored as potential biomarkers for PD.

Using enzyme-linked immunosorbent assay (ELISA), Tokuda et al. [48] demonstrated a decrease in t-α-syn in the CSF of patients with PD. This finding was supported by subsequent studies and a meta-analysis of nine reports involving 537 controls, 843 patients with PD, 130 patients with MSA, and 98 patients with progressive supranuclear palsy. The analysis confirmed that t-α-syn levels were reduced in patients with α-synucleinopathies [49,50]. o-α-Syn and pS129-α-syn, which are associated with Lewy body formation, are directly related to the pathogenesis of α-synucleinopathies. Elevated levels of o-α-syn in CSF have been reported, and a meta-analysis also indicated that CSF o-α-syn levels were greater in patients than in controls. However, their diagnostic value is limited due to their sensitivity and specificity of approximately 0.7 and 0.65, respectively [50]. Although the o-/t-α-syn ratio in CSF has shown improved sensitivity and specificity, its utility for diagnosis has not yet been established [51,52].

In a study by Lin et al. [53], plasma α-syn levels were measured in 34 controls and 80 patients with PD using an immunomagnetic reduction-based immunoassay. The results showed a significant increase in α-syn levels in PD patients, which correlated with cognitive dysfunction but not motor dysfunction. Furthermore, they measured pS129-α-syn in 122 PD patients and 68 controls using the same method and found elevated levels of pS129-α-syn in PD patients. These findings suggest that pS129-α-syn could serve as a biomarker for motor dysfunction and disease progression in PD patients [54].

ADVANCEMENTS IN SEED AMPLIFICATION ASSAYS FOR AMYLOID FIBRIL α-SYNUCLEIN DETECTION

Recent research has increasingly focused on SAAs, such as RT-QuIC and PMCA, for detecting pathologically significant α-syn seeds [55]. These seeds can be obtained from various biological samples, including CSF, blood, skin, submandibular glands, and intestines. The amyloid protofibrils of α-syn tend to aggregate and serve as a template to convert normal endogenous α-syn into abnormal proteins, thereby amplifying them. SAAs facilitate continuous in vitro aggregation reactions, using normal recombinant proteins as substrates and amyloid fibrils as seeds for amplification. This process enables the amplification of trace amounts of amyloid fibrils in patient biofluid samples, such as CSF and blood, with the presence of amyloid fibrils revealed in realtime by detecting the fluorescence intensity of thioflavin T (ThT), which specifically binds to the β-sheet structure of proteins [56]. RT-QuIC, which was initially developed by Atarashi and colleagues [11,57] at Nagasaki University for detecting abnormal prion proteins, has been clinically applied as a powerful diagnostic tool for Creutzfeldt–Jakob disease (Figures 2 and 3).

Figure 2.

Mechanism of real-time quaking-induced conversion (RT-QuIC). RT-QuIC is an innovative technique designed to amplify and visualize proteins prone to misfolding and aggregation, such as prions and α-synuclein. This assay facilitates the detection of pathogenic conformers by incubating abnormally structured α-synuclein seeds—characterized by β-sheet configurations—with recombinant α-synuclein substrates (monomers). Under controlled conditions of persistent agitation, the mixture was incubated, which promotedfibrillogenesisin vitro. Following an initial lag phase, there is a marked and rapid increase in fibril formation, which somewhat correlates with the concentration of the seeding material. However, this method faces challenges, primarily due to the variable reaction dynamics between the seeds and substrates, which complicates the standardization and quantification of results. ThT, thioflavin T.

Figure 3.

Diagnostic potential of α-synuclein seeds as biomarkers for α-synucleinopathies. α-Synuclein seeds present throughout the body hold promise as diagnostic biomarkers for neurodegenerative diseases. These seeds can be obtained from various biological samples, including cerebrospinal fluid, blood, skin, submandibular glands, and intestines, through seed amplification assays (SAAs), such as real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA). The positive detection of α-synuclein seeds facilitates the diagnosis of α-synucleinopathies. Moreover, structural analysis of α-synuclein fibrils amplified by SAAs, coupled with the morphology of aggregates induced by patient-derived seeds in cell-based assays, offers a potential avenue for distinguishing between Lewy body disease and multiple system atrophy. CSF, cerebrospinal fluid; DLB, dementia with Lewy bodies; MSA, multiple system atrophy; PD, Parkinson’s disease; ThT, thioflavin T.

The aggregation reaction in α-syn SAAs tends to depend on phenoconthe concentration of seeds, but it is currently considered mainly a qualitative evaluation method. Protocols for α-syn SAAs vary with several factors, such as the pH of the reaction buffer, incubation time, presence of detergents (e.g., sodium lauryl sulfate), and type of plate used, influencing the aggregation reaction and sensitivity of α-syn SAAs [58,59]. Consequently, most protocols are performed in-house because of the varying reactions of recombinant α-syn reported by different laboratories. The lack of uniformity in recombinant α-syn poses a challenge for standardizing protocols and reproducibility between laboratories. Future efforts are needed to standardize materials, including recombinant α-syn as a substrate, and unify protocols for RT-QuIC and PMCA [60].

Despite these challenges, there have been successive reports on the detection of α-syn seeds in CSF using SAAs, demonstrating extremely high sensitivity (0.88–0.95) and specificity (0.92– 1.00) in PD diagnosis, suggesting their potential role as diagnostic biomarkers in clinical practice [61-63]. A recent meta-analysis of 22 CSF α-syn SAA studies revealed a sensitivity and specificity of 0.88 (95% confidence interval [CI], 0.82–0.93) and 0.95 (95% CI, 0.92–0.97), respectively, for distinguishing α-synucleinopathies from nonsynucleinopathies, including tauopathies [64]. In a study on CSF α-syn SAA in the Parkinson’s Progression Markers Initiative (PPMI) cohort, the sensitivity for detecting PD was 0.88. Notably, the sensitivity increased to 0.99 in patients with typical olfactory dysfunction, decreased to 0.78 in those without hyposmia, and decreased to 0.68 in LRRK2 mutation carriers, suggesting that the CSF α-syn SAA results may reflect specific disease subtypes or mutations, such as in the SNCA or GBA genes [63].

In a meta-analysis for DLB, the diagnostic sensitivity and specificity of CSF were reported to be 0.95 and 0.96, respectively [65]. However, it has been noted that the α-syn SAA can be positively applied in biologically confirmed Alzheimer’s disease patients, especially in older patients, necessitating consideration of diagnostic bias [66].

The diagnostic sensitivity and specificity of the α-syn SAA for some patients with idiopathic RBD have been reported to be high, at 0.80 (95% CI, 0.58–0.92) and 1.00 (95% CI, 0.82– 1.00), respectively [62]. In the PPMI study, the positive detection rate using α-syn SAA in patients with RBD or hyposmia was 0.86 (Figure 3) [63].

SAA is also considered a predictive biomarker for phenoconversion. In a longitudinal study of idiopathic RBD conducted by Iranzo et al. [67] over 10 years, it was found that progression to α-synucleinopathy could be predicted with 0.9% diagnostic accuracy. According to Kaplan–Meier analysis, participants who were negative for α-syn had a lower risk of developing PD or DLB at baseline and at 2, 4, 6, 8, and 10 years than did α-syn-positive participants (log-rank test p < 0.0001; hazard ratio 0.024, 95% CI 0.003–0.177) [67]. In a prospective study of 36 patients who presented with pure autonomic failure, the levels of α-syn in the CSF were measured using a PMCA assay, and neurofilament light chain (NfL) levels were measured via ELISA. Five patientsphenoconverted to MSA, two to PD, and two to DLB. Patients who converted to MSA had lower maximum ThT fluorescence and exhibited elevated NfL levels [68]. Poggiolini et al. [69] conducted CSF α-syn RT-QuIC on 74 patients with PD, 24 with MSA, 45 with idiopathic RBD, and 55 with healthy controls, analyzing quantitative assay parameters concerning clinical data. There was no correlation between RT-QuIC quantitative parameters and PD clinical scores; however, PD patients with higher Vmax values were significantly older (p = 0.03) and had higher scores on the postural instability gait disorder part of the Movement Disorder Society-sponsored revision of the Unified Parkinson’s Disease Rating Scale (p = 0.05). In MSA patients, RT-QuIC parameters correlated with worsening clinical progression. In the longitudinally tracked cohort with idiopathic RBD, 31% of patients converted to α-synucleinopathy, and 9 out of 14 converters (64%) were RT-QuIC positive at baseline. Further research into the prevalence of SAA in the prodromal stages of PD and MSA, along with an analysis of the relationship between SAA levels, clinical manifestations, and imaging findings, could establish SAA as a viable biomarker for prognostication and assessing the severity of these conditions.

BLOOD AMYLOID FIBRILS OF α-SYNUCLEIN

The propagation mechanisms of α-syn seeds are believed to involve several processes, such as exocytosis, endocytosis, extracellular vesicles (EVs), and tunneling nanotubules [70]. EVs, small vesicles containing proteins, DNA, and RNA, are released from cells and have garnered increasing attention for their role in cellto-cell communication, particularly in the context of neurodegenerative diseases, cancer, and inflammatory conditions [71]. These vesicles are thought to facilitate the transmission of α-syn between cells and the transport of micro-RNAs, potentially playing a crucial role in the development of PD. α-Syn has been detected in exosomes derived from the brain and CSF of patients with PD and DLB [72-74]. Moreover, EVs from DLB patients can induce the formation of α-syn aggregates in mouse brains [73,75]. In PD, although the overall quantity of EVs in serum decreases, the proportion of EVs containing α-syn seeds (α-syn filaments/total EVs) increases, suggesting their involvement in disease progression [22]. This finding is further supported by studies showing enhanced propagation and aggregation of α-syn filaments in mice following intravenous administration of EVs loaded with α-syn seeds. Kluge et al. [21] highlighted that neuron-derived EVs harboring aggregating α-syn could serve as diagnostic biomarkers for PD through α-syn seeding assays. Electrochemiluminescence measurements revealed elevated levels of α-syn in neuronderived EVs from 365 individuals at risk of developing PD based on prodromal symptoms or genetic factors and 71 people with genetic or sporadic forms of PD compared with 140 controls, indicating that α-syn is a potential risk marker for PD [76].

Recent advances in detection techniques, such as aptamer DNA-PAINT, single-aggregate confocal fluorescence, and highresolution atomic force microscopy, have enabled the identification of α-syn seeds in the serum and CSF of both PD patients and controls. Notably, the shape and quantity of these seeds were more pronounced in PD patients, suggesting the presence of α-syn seeds even in individuals without PD [20].

By employing immunoprecipitation and RT-QuIC (IP/RTQuIC), we successfully differentiated PD and MSA patients from controls with high sensitivity and specificity. In contrast, MSA patients showed a lower positive rate, indicating distinct aggregation patterns between PD patients and MSA patients. The discriminative ability between patients with α-synucleinopathy and healthy controls was analyzed based on the formation rate, one of the parameters of RT-QuIC. In receiver operating characteristic analyses, the sensitivity and specificity for differentiating patients with PD from controls were 94.6% and 92.1%, respectively, with a cutoff rate of 662.4 and an area under the curve (AUC) of 0.96 (95% CI 0.95–0.99). For differentiating patients with DLB from controls, the sensitivity and specificity were 96.4% and 92.2%, respectively, with a cutoff rate of 574 and an AUC of 0.90 (95% CI 0.95–0.99). Finally, for differentiating patients with MSA from controls, the sensitivity and specificity were 64.1% and 11.0%, respectively, with a cutoff rate of 118.4 and an AUC of 0.64 (95% CI 0.49–0.79). All pathologically proven patients with Lewy body disease were positive for IP/RT-QuIC. Interestingly, there were no cases of IP/RT-QuIC-positive for PARK2 [16].

STRUCTURAL VARIABILITY OF α-SYNUCLEIN SEEDS IN α-SYNUCLEINOPATHY

The α-syn fibrils observed through TEM displayed different structures when formed by seed-amplified aggregation. We developed an assay system in which the introduction of SAA-amplified α-syn seeds into cells expressing the A53T mutant α-syn led to aggregate formation within 48 hours. This system revealed distinct aggregate morphologies when patient-derived α-syn was introduced, varying between PD, MSA, and DLB. Moreover, the injection of α-syn seeds into mouse brains resulted in different aggregate structures across the PD, MSA-cerebellar, and MSAparkinsonism types [16]. Analyses using TEM, cellular assays, and cryo-electron microscopy have shown that the structure of α-syn seeds varies with disease severity, suggesting that these structures could define the disease (Figure 3) [15,17,18,77]. The propagation pattern also differs based on the structural and pathological mutations of α-syn seeds [78,79]. Identifying α-syn seeds from blood and analyzing their structure could provide valuable biomarkers for understanding the disease state. However, it has been reported that RT-QuIC is influenced by the presence of albumin in the blood or the binding of α-syn substrates to the plate, which triggers the reaction. Therefore, improvements in the technique may be necessary to achieve consistent and reliable outcomes. For further details on the aggregation and propagation of α-syn seeds, readers are encouraged to consult a recent comprehensive review [80].

BIOLOGICAL STAGING OF PARKINSON’S DISEASE

Staging is crucial in the diagnosis and treatment of diseases, and this is particularly true for PD. To date, the staging of PD has been based on clinical symptoms. The Hoehn and Yahr classification, which is widely used, categorizes disease into six stages based on motor symptoms. However, this classification does not consider the nonmotor symptoms of PD, making it insufficient. Furthermore, pathological studies have shown that, by the time motor symptoms emerge, 50%–70% of dopaminergic neurons have already degenerated [8]. Therefore, staging based solely on clinical symptoms is challenging. The development of diseasebased biomarker assays, such as SAA, might offer the potential for more accurate, defined staging of PD [81]. Recently, two groups proposed biological staging of PD based on a combination of several biological markers [82,83].

These proposals include the detection of α-syn seeds in CSF using SAA as the S marker; imaging diagnostics such as 123I-FPCIT single photon emission computed tomography and MRI neuromelanin as markers of dopaminergic neurodegeneration (N(D) marker); and genetic markers (G marker), such as SNCA, LRRK2, and GBA, combined with clinical symptoms for diagnosis and classification. Höglinger et al. [82] proposed a clinical classification system, termed SynNeurGe (pronounced phonetically as synergy), which combines S, G, and N markers with clinical symptoms (C marker) to classify PD. Simuni et al. [83] proposed the neuronal α-synucleinopathy integrated staging system, which considers genetic background and combines S markers, D markers (dopaminergic neurodegeneration), and clinical symptoms for staging. In this system, patients with a fully penetrant SNCA variant but no S marker, N marker, or clinical symptoms are classified as stage 0. The presence of an S marker advances the patient to stage 1, and subsequent stages up to stage 6 are determined by combining D markers and clinical symptoms. Additionally, individuals at genetic risk who have not developed the disease are classified into low-risk and high-risk groups based on age and genetic risk (Figure 4) [83].

Figure 4.

The evolution of parkinson’s disease staging. Although widely used, the traditional Hoehn and Yahr scale primarily focuses on motor symptoms. This approach is inadequate for identifying Parkinson’s disease because of the heterogeneity of clinical symptoms among patients. The term “Parkinson’s disease” better reflects the diverse manifestations of the disorder. In contrast, the innovative NSD-ISS Biological Staging system enhances the Hoehn and Yahr scale by incorporating three additional criteria: the identification of α-synuclein seeds (S anchors), the assessment of dopamine neuron degeneration through imaging (D anchors), and the inclusion of Parkinson’s disease-related genes, especially SNVs (G anchors). This comprehensive approach enables a more objective evaluation of the condition, paving the way for personalized treatment strategies. DAT, dopamine transporter; NSD-ISS, neuronal α-synucleinopathy integrated staging system; SNV, single nucleotide variant; ThT, thioflavin T.

Similar to PD, other neurodegenerative diseases, such as Alzheimer’s disease and Huntington’s disease, have adopted biomarker-based staging systems, such as the amyloid/tau/neurodegeneration (ATN) classification for Alzheimer’s disease [84] and classification based on HTT CAG repeat numbers for Huntington’s disease [85]. The establishment of the ATN classification for Alzheimer’s disease has refined diagnosis and staging, accelerating the development of disease-modifying treatments [86]. Therefore, developing biological staging for PD is considered crucial for future clinical practice. However, challenges remain, such as the qualitative nature of SAAs and the inability of neuroimaging to accurately capture dopaminergic neuron loss [81]. There have been reports of improved diagnostic accuracy achieved by combining α-syn SAA with ELISA using oligomer-specific α-syn antibodies, reflecting advancements in biomarkers that may capture the pathophysiology of PD [87]. Additionally, attempts to visualize α-syn in PD using PET imaging are ongoingandcould become a robust biomarker if successful [23].

Current methods for detecting SAAs are qualitative and do not provide a measure of the degree of α-syn aggregation or its changes over time. In the future, it will be important to develop quantitative assays to address this gap. Additionally, when evaluating the effectiveness of treatments for amyotrophic lateral sclerosis, such as SOD1 antisense oligonucleotides [88], the combination of Amylyx and sodium phenylbutyrate [89], and C9orf72 antisense oligonucleotides [90], the development of surrogate markers that align with the objectives of these treatments might be necessary. This is because the biomarkers for these treatments can vary, and not all may show measurable changes.

CONCLUSIONS

In this review, we have discussed diagnostic biomarkers for α-synucleinopathy, focusing on α-syn SAAs. To date, the diagnosis and staging of PD have been based on clinical symptoms, primarily focusing on motor dysfunction. However, relying solely on clinical symptoms for inclusion criteria in clinical research is insufficient in terms of study design. The development of biomarkers could enable more accurate diagnosis and staging, potentially leading to different outcomes in clinical studies evaluating disease-modifying therapies. Although proposals for biological staging in PD have been suggested, challenges remain, such as the insufficiency of biomarkers and that neurodegenerative diseases, especially in older individuals, often present with mixed pathology [91], necessitating verification of the accuracy of biomarkers against the final pathology. However, the use of SAAs for the diagnosis and staging of PD is expected to improve the accuracy of clinical research and facilitate the development of further biomarkers. There is no doubt that the development of SAAs will provide new opportunities for the management of PD.

Notes

Conflicts of Interest

The authors have no financial conflicts of interest.

Funding Statement

This work was supported by the Japan Agency for Medical Research and Development (AMED) (20dm0107156 to T.H. and A.O., 23wm0425015 to T.H., A.O., and T.T., 21ak0101112, 23dk0207055, 23wm0425019 to T.H., 23dm0207070 to N.H.), grants-in-aid for Scientific Research (21H04820 to N.H., 21K07424 and 21K08742 to T.H., 19K16928 to A.O.) from the Japan Society for the Promotion of Science (JSPS), the Visionary Council on the Moonshot Research and Development Program (JPMJMS2024-5 to N.H.), grants-in-aid from the Research Committee of CNS Degenerative Disease, Research on Policy Planning and Evaluation for Rare and Intractable Diseases, Health, Labor, and Welfare Sciences Research Grants, the Ministry of Health, Labor, and Welfare, Japan to N.H.

Author contributions

Conceptualization: Taku Hatano, Nobutaka Hattori. Data curation: Taku Hatano. Formal analysis: Taku Hatano. Funding acquisition: Taku Hatano, Ayami Okuzumi, Taiji Tsunemi, Nobutaka Hattori. Investigation: Taku Hatano. Methodology: Taku Hatano, Ayami Okuzumi, Gen Matsumoto, Taiji Tsunemi. Project administration: Taku Hatano. Resources: all authors. Software: Taku Hatano. Supervision: Taku Hatano. Validation: all authors. Visualization: Taku Hatano. Writing—review & editing: Taku Hatano.

Acknowledgements

None

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