Parkinson’s Disease (PD) is the second most common neurodegenerative disorder that is clinically diagnosed based on motor symptoms (bradykinesia, tremor, rigidity, hypomimia, shuffling gait, difficulty walking, and postural instability) and non-motor symptoms (cognitive decline, autonomous disorders, disrupted sleep, and sensory disturbances) [1,2,3]. The progressive loss of dopaminergic neurons in substantia nigra pars compacta (SNpc), and the deposition of cytoplasmic protein inclusions referred to as Lewy bodies (LBs) in affected brain areas are two pathological hallmarks of PD [4]. Motor symptoms usually occur late in the neurodegenerative process in PD patients, as more than half of striatal dopaminergic neurons are lost when motor symptoms occur [3]. Non-motor symptoms may precede the development of motor symptoms, and thus, can be considered a prodromal state of PD [5]. So far, the exact pathogenesis mechanism behind PD is still unknown. It has been suggested that endogenous dopamine (DA) can be considered an endogenous pathogenic factor for PD [6]. In dopaminergic neurons, DA can undergo auto-oxidation and enzyme-catalyzed metabolism to generate deleterious oxidative DA metabolites, including reactive oxygen species (ROS), DA quinones (DAQs), 3,4-dihydroxyphenylacetaldehyde (DOPAL), and neuromelanin (NM), contributing to the dopaminergic neuron impairment [6,7].

Currently, PD is still an incurable neurodegenerative disease without any therapeutic strategy that could halt or reverse the progressive loss of dopaminergic neurons. The levodopa (L-DOPA) replenishing strategy is the current gold standard for clinical PD treatment to alleviate PD symptoms [8]. Numerous active and passive immunotherapies have exhibited promising therapeutic efficacy in preclinical studies, and many of these are currently undergoing clinical trials [9]. Therefore, an objective and reliable biomarker is urgently required to enhance the diagnostic accuracy of PD in its early stage or prodromal state and monitor its progression. Accumulated evidence suggests that the abnormal deposition and spreading of the misfolded and aggregated α-synuclein (α-Syn) proteins are the central molecular events behind PD pathogenesis [10]. The misfolded and aggregated α-Syn protein is involved in multiple cellular dysfunctions, including the disruption of the autophagy-lysosomal pathway, the dysregulation of mitochondrial function, and the disappearance of dopaminergic neurons [10,11]. The toxicity caused by the aggregated α-Syn protein is closely related to DA and DA oxidative metabolites [6,12]. The overexpression of α-Syn enhances the DA-dependent dopaminergic neuron toxicity [13]. DA-derived metabolites, such as DAQs and DOPAL, can conjugate with α-Syn protein to stabilize the deleterious oligomer form α-Syn protein and enhance α-Syn protein toxicity [14,15,16,17]. Moreover, α-Syn protein promotes the synthesis of NM, an insoluble granular pigment, in dopaminergic neurons [18]. NM generates ROS under oxidative stress, and the interaction between NM and α-Syn is identified to induce neuroinflammation and microglia activation related to α-Syn-associated DA neurodegeneration [7,19,20]. Therefore, the misfolded and aggregated form of α-syn protein could be considered a vital pathogenic biomarker in PD.

The α-Syn, encoded by the SNCA gene, is a small soluble protein comprising 140 amino acids with three distinct domains: an N-terminal domain (1–60 amino acid residues), a non-amyloid-β component (NAC) domain (61–95 amino acid residues), and a C-terminal domain (96–140 amino acid residues) [21]. The N-terminal domain is positively charged and characterized by an amphipathic lysine-rich amino terminus that serves as the membrane anchor region of α-Syn [21]. The NAC domain is hydrophobic and constitutes the amino acid segments that are essential in fibril formation and aggregation, which has been identified as the most aggregation-prone region [21]. The C-terminal domain is a negatively charged tail that does not contain secondary structures, including ten glutamate and five aspartate residues, characterized by disease-promoting regions and involved in chaperone-like activity [22].

The α-syn is abundant in the brains and exists in erythrocytes, platelets, and other tissues [23]. In the brains, α-Syn is primarily expressed in neuronal cells, located in the pre-synaptic terminal and probably bound to the membrane of synaptic vesicles [24]. It exists in the dynamic equilibrium in the unfolded form in the cytosol and an α helical-rich form when bound to membranes, which is thought to play a vital role in modulating the stability of the membrane, altering membrane trafficking via vesicular transport and promoting the assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex (SNARE) [25,26]. Native α-Syn mainly exists as an unfolded monomer, dimer, or tetramer. However, under disease conditions, the native α-Syn can aggregate and misfold into pathological α-syn oligomers, also known as aggregates, to increase membrane permeability and disrupt membrane integrity [27]. Furthermore, the α-Syn oligomers can further polymerize to form α-Syn fibrils and accumulate in the brain, peripheral tissues, and biofluids [28]. The aggregation of α-Syn fibrils is believed to be the pathological hallmark of synucleinopathies, including PD, Dementia with Lewy Bodies (DLB), and multiple system atrophy (MSA), based on the cellular predilections and neuroanatomical distributions [11,29].

The oligomeric α-Syn, the earlier stage of α-Syn aggregation, is widely distributed in multiple cortical and subcortical regions, contributing to synaptic dysregulation, dopaminergic neurodegeneration, motor deficits, and cognitive impairments in PD patients [56,57]. Therefore, detecting oligomeric α-Syn could be considered a routine strategy to distinguish PD patients from healthy individuals or related synucleinopathies in the prodromal or early stage. Nevertheless, multiple factors, such as the distribution of oligomeric α-Syn, gene mutation, post-translational modifications, and exposure to mental heavy, have positive or negative effects on the α-Syn seeding activity, which needs to be re-assessed, re-evaluated, and re-addressed in future studies for precision diagnosis.

Until now, the presence of α-Syn seeds has been detected in various biological fluids, especially CSF and peripheral blood. According to the gut-to-brain axis hypothesis, the misfolded or toxic oligomeric α-Syn originates from the periphery enteric plexus or erythrocytes. The erythrocytes-derived and periphery-derived α-Syn oligomers can spread from the gut to the brain via the enteric nervous system, vagus nerve, and glossopharyngeal nerve [58,59]. Meanwhile, previous studies have shown that the aberrant deposition of α-Syn in the intestinal tract was earlier than the loss of dopaminergic neurons and the occurrence of motor symptoms of PD [60]. Nevertheless, the presence of oligomeric α-Syn in specific body tissues and the occurrence of PD clinical features has not been fully determined, which needs to be emphasized, evaluated, and addressed in future studies to provide scientific insight for further application of α-Syn SAAs-based strategy for PD precision diagnosis.

Mutations in SNCA, including missense and multiplication mutations, can cause early-onset autosomal-dominant PD [61]. Six missense point mutations in the N-terminal domain, including A53T, A30P, A53E, E46K, H50Q, and G51D, have been strongly associated with autosomal dominant familial PD, which might be related to their capacities to influence the oligomerization or fibrillation of α-Syn protein [62,63,64,65]. PD patients affected by SNCA point mutations present a clinical phenotype similar to those with sporadic PD, with an earlier age at onset, and often suffer from dementia and autonomic disturbances [66]. Recent SAAs have demonstrated that the A53T mutation has a more robust seeding activity than the wild-type (WT) protein [67]. Moreover, PD patients with SNCA duplication exhibit a typical late-onset PD phenotype, and PD patients with SNCA triplication have been reported to cause hereditary early-onset PD phenotype with dementia [68,69]. It has been demonstrated that the α-Syn seeding activities in the CSF from PD patients with triplication were more robust than those of the WT PD patients [70]. Other PD-related genes, such as LRRK2, GBA1, DJ1, PINK1, and PRKN, can significantly influence the misfolding and aggregation of α-Syn [6]. Among them, PRKN, PINK1, and DJ-1 variants are usually found in individuals with autosomal recessive early-onset PD [71]. In contrast, variants in GBA1 and LRRK2 are widely accepted as the two most crucial genetic risk factors associated with PD [72]. The GBA variants, such as the L444P and N370S variants and LRRK2 variants, especially the G2019S variant, can remarkably promote α-Syn aggregation [72,73,74]. Recent studies highlight that PD patients carrying GBA and LRRK2 variants have higher seeding activity in CSF than those carrying PINK1 and PRKN [46]. So far, the effects of PD-related pathogenic genes and their variants on α-Syn seeding activity have not been thoroughly investigated. Genetic tests, such as next-generation sequencing, have been used to identify pathogenic variants in PD patients [75]. Hence, screening the pathogenic variants by multiple genetic tests could be considered an adjunct measurement to improve the sensitivity and accuracy of SAA.

α-Syn is subjected to extensive post-translational modification, such as truncation, phosphorylation, ubiquitination, nitration, and O-GlcNAcylation, which could influence its aggregation and cytotoxicity. The C-terminal truncated α-Syn produced by aberrant proteolysis has been detected in the brains of healthy individuals and PD patients, and it is thought to be associated with the formation of α-Syn aggregates [76]. Previous studies have shown that C-terminal truncated fragments, α-Syn (1–108) and α-Syn (1–124), aggregate faster than the full-length α-Syn protein [77]. Recent SAA has proved that the PD-related C-terminal 123–140 and 104–140 truncations of α-Syn present higher seeding activity than the WT α-Syn [67]. Further mechanistic studies revealed that C-terminal truncations promote the conversion of monomers to aggregated forms of α-Syn, enhance autocatalytic aggregation on existing fibrils, and interfere with the interactions between the N-terminal of α-Syn and the membrane [67,76]. These findings demonstrated that the truncation of α-Syn affects their seeding activity and aggregating properties. In addition, truncation, phosphorylation, ubiquitination, nitration, and O-GlcNAcylation of α-Syn have been reported to influence α-Syn aggregation and fibril formation at various degrees [78,79,80,81,82,83,84]. The effects of these post-translational modifications on the α-Syn seeding activity have not been comprehensively evaluated, which needs to be emphasized and addressed in future studies. Moreover, proteomics techniques, particularly those based on high-resolution mass spectrometry, have been rapidly developed as a preferred method to determine relative changes in post-translational modifications and protein abundance [85]. Thus, screening and monitoring post-translational modifications via multiple proteomic techniques may enhance the sensitivity and accuracy of SAA, which need to be assessed, evaluated, and addressed in future studies.

Numerous epidemiological studies have correlated exposure to metal ions with the onset and development of PD [86]. The pathogenic effects of metal in PD patients are associated with their capacities to elevate ROS levels and enhance aberrant α-Syn aggregation in the brain [87,88]. It was previously reported that divalent or trivalent metal ions, particularly aluminum (III), copper (II), iron (II), cobalt (III), and manganese (II), significantly accelerated the rate of α-Syn aggregation and fibril formation [89]. Recent studies indicated that bivalent metal ions, including zinc (II), copper (II), calcium (II), and manganese (II), accelerate the α-Syn fibrillation and form different amyloid-competent conformations, suggesting that α-Syn exposure to these mental ions have a higher seeding activity [90,91]. These α-Syn fibrils formed in the presence of different bivalent metal ions have distinct kinetics, size, morphology, secondary structure, and cytotoxicity, as well as the charge status of bivalent metal ions (measured by the native nanoelectrospray ionization ion mobility-mass spectrometry) was remarkably reduced when bound to the α-Syn [90,91]. Moreover, the bivalent metal ions have also been reported to significantly promote the aggregation of mutant α-Syn protein. For instance, in the presence of copper (II) ion, the mutants G51D α-Syn protein have higher aggregation kinetics and fibril formation rate than WT α-Syn protein [92]. Nevertheless, further studies are warranted to evaluate the pathogenic effects of other trivalent or heavy metal ions on α-Syn aggregation and fibril formation. Strategies that determine the morphology and structure of fibrils formed by α-Syn aggregates and the charge status of metal ions could be used as an adjunct measurement to SAA, which also needs to be addressed in future studies.