# α-Synuclein in Parkinson's Disease: From Bench to Bedside

Gabriele Bellini, Vanessa d'Antongiovanni, Giovanni Palermo, Luca Antonioli, Matteo Fornai, Roberto Ceravolo, Nunzia Bernardini, Pascal Derkinderen, Carolina Pellegrini

► To cite this version:

Gabriele Bellini, Vanessa d'Antongiovanni, Giovanni Palermo, Luca Antonioli, Matteo Fornai, et al. α-Synuclein in Parkinson's Disease: From Bench to Bedside. Medicinal Research Reviews, 2024, 45, pp.909 - 946. 10.1002/med.22091. hal-05085054

HAL Id: hal-05085054

https://hal.science/hal-05085054v1

Submitted on 26 May 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

REVIEW ARTICLE

OPEN ACCESS

# α-Synuclein in Parkinson's Disease: From Bench to Bedside

Gabriele Bellini<fnref n="1" />,² ID | Vanessa D'Antongiovanni³ ID | Giovanni Palermo<fnref n="1" /> | Luca Antonioli⁴ | Matteo Fornai⁴ | Roberto Ceravolo<fnref n="1" /> | Nunzia Bernardini³ | Pascal Derkinderen² | Carolina Pellegrini³ ID

**Correspondence:** Carolina Pellegrini (carolina.pellegrini@unipi.it)

**Received:** 12 May 2024 | **Revised:** 24 September 2024 | **Accepted:** 7 November 2024

**Funding:** The authors received no specific funding for this work.

**Keywords:** α-synuclein | central nervous system | clinical trials | gut-brain axis | immune cells | neurodegeneration | neuroinflammation | Parkinson's disease | preclinical evidence | spreading

## ABSTRACT

α-Synuclein (α-syn), a pathological hallmark of PD, is emerging as a bridging element at the crossroads between neuro/ immune-inflammatory responses and neurodegeneration in PD. Several evidence show that pathological α-syn accumulates in neuronal and non-neuronal cells (i.e., neurons, microglia, macrophages, skin cells, and intestinal cells) in central and peripheral tissues since the prodromal phase of the disease, contributing to brain pathology. Indeed, pathological α-syn deposition can promote neurogenic/immune-inflammatory responses that contribute to systemic and central neuroinflammation associated with PD. After providing an overview of the structure and functions of physiological α-syn as well as its pathological forms, we review current studies about the role of neuronal and non-neuronal α-syn at the crossroads between neuroinflammation and neurodegeneration in PD. In addition, we provide an overview of the correlation between the accumulation of α-syn in central and peripheral tissues and PD, related symptoms, and neuroinflammation. Special attention was paid to discussing whether targeting α-syn can represent a suitable therapeutical approach for PD.

## 1 | Introduction

Growing evidence highlights that neuroinflammatory processes contribute to the pathogenesis of Parkinson's disease (PD) [1]. In this setting, α-synuclein (α-syn), a presynaptic neuronal protein that is linked genetically and neuropathologically to PD, is emerging as a bridging element between the trigger of neuro/ immune-inflammatory responses and the progression of neuro- degeneration in PD [2]. Accumulation of misfolded and aggre- gated α-syn in neurons and non-neuronal cells of the brain and

peripheral autonomic networks, as well as in circulating exo- somes and gut, can compromise synaptic transmissions, act as prion-like protein contributing to disease spreading and trigger neurogenic/inflammatory responses in PD [3, 4]. For instance, intestinal pathological α-syn injection in mice was associated with gut-to-brain spread of α-synucleinopathy and associated neurodegeneration and behavioral deficits via vagal transmission [5]. In parallel, pathological α-syn might result in robust activa- tion of immune and inflammatory cells that, in turn, may con- tribute to systemic and central neuroinflammation associated

<footnote n="1">¹Center for Neurodegenerative Diseases, Unit of Neurology, Parkinson's Disease and Movement Disorders, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy | ²Department of Neurology, The Marlene and Paolo Fresco Institute for Parkinson's and Movement Disorders, NYU Langone Health, New York City, New York, USA | ³Unit of Histology and Embryology, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy | ⁴Unit of Pharmacology and Pharmacovigilance, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy | ⁵Department of Neurology, Nantes Université, CHU Nantes, INSERM, Nantes, France</footnote>

<footnote>Gabriele Bellini, Vanessa D'Antongiovanni, Carolina Pellegrini, and Pascal Derkinderen contributed equally to this work.</footnote>

<footnote>This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</footnote>

<footnote>© 2024 The Author(s). *Medicinal Research Reviews* published by Wiley Periodicals LLC.</footnote>
with PD [1, 6]. Of interest, recent and pioneering evidence suggests that the initial localization of pathological α-syn forms can also identify specific PD phenotypes; α-syn accumulation primarily in the brain appears to identify a “brain first” PD phenotype, while an early α-syn deposition in the gut identifies a “body first” PD phenotype characterized by early autonomic dysfunction and REM sleep behavior disorder (RBD) during the prodromal phase [7].

On these bases, the present review provides a comprehensive overview of the available knowledge on the physiological and pathological forms of α-syn and critically discusses, covering clinical and basic research aspects, how central and systemic α-syn deposition is at the crossroads between neuroinflammation and neurodegeneration in PD. In addition, we review clinical evidence showing a correlation between the accumulation of α-syn in central and peripheral tissues and PD diagnosis, duration, motor and non-motor symptoms, neurodegeneration, and neuroinflammation. Special attention was also paid to discuss whether current pharmacological strategies aimed at counteracting α-syn can represent a suitable therapeutic approach for PD.

## 2 | Physiological and Pathological Forms of α-Syn

α-Syn was first isolated in 1988 from the Torpedo electric organ and named after its localization on synaptic vesicles and nuclear envelopes [8]. The human 140-aminoacid homolog was identified a few years later in the brain [9]. These first observations on α-syn found relatively little echo until 1997 when point mutations in the six-exon gene that encodes α-syn (SNCA) mapping to chromosome 4q21 were identified as a rare cause of autosomal-dominant PD [10]. The same year, it was shown that Lewy bodies (LBs) and neurites (LNs), the pathological hallmarks of PD, were highly immunoreactive for α-syn in sporadic PD brains, thereby suggesting that α-syn was one of the main components of these intraneuronal inclusions [11].

Since 1997, a large body of work focused on the physiological and pathological roles of α-syn. α-Syn is composed of three biochemically distinct domains: (i) a highly conserved and positively charged amino-terminal repeat (residues 1–60) showing similarity with apolipoproteins, which allows the protein to fold into an amphipathic α-helix when it is bound to lipid membranes or in the presence of phospholipids [12] (ii) a central hydrophobic region (amino acids 61–95) known as non-amyloid component (NAC), which contains a 12-aminoacid stretch that is necessary and sufficient to form the proteolysis-resistant fibril core [13], and (iii) a C-terminal acidic tail (amino acids 96–140) accountable for most interactions with other proteins and small molecules that contains most of the phosphorylation sites and especially serine 129 (pS129) (Figure 1). The exact physiological functions of α-syn are still elusive, but its association with the distal reserve pool of synaptic vesicles and the deficiencies in synaptic transmissions observed in response to overexpression or suppression of the SNCA gene suggest that it plays a role in the release of neurotransmitter and synaptic homeostasis, thereby influencing neuronal plasticity and survival [14]. Recent evidence also suggests that low levels of α-syn in the nucleus of neuronal cells can influence cell transcription [15].

As a typical intrinsically disordered protein, α-syn adopts a monomeric, random coil conformation under physiological conditions [16]. It has also been suggested that physiological α-syn occurs as a stable α-helically-folded tetramer that is resistant to amyloid formation [17], but these findings are still highly debated [18]. α-syn has a natural propensity to aggregate, and this phenomenon is strongly influenced by the cellular environment [19]. For instance, lowering of pH, increasing of temperature, hormonal changes, and oxidative stress, together with increases in hydrophobicity or decreases in charge, can modify α-syn conformation, thereby influencing its misfolding and aggregation [20–22]. These processes involve the formation of non-fibrillar off-pathway and soluble transient pre-fibrillar intermediate species, regarded as oligomers and protofibrils, which eventually convert into insoluble fibrillar aggregates with distinct cross β-sheet conformation [16]: these two species seem to be more neurotoxic than mature fibrils due to their instability and smaller size [23, 24]. The increase in wild-type α-syn or mutated α-syn can promote intracellular accumulation and accelerate the fibrillation process via a nucleation-dependent mechanism [25]. The long-term consequences of β-strand α-syn aggregates are the disruption of neuronal functions with consequent neuronal death [26]. Specifically, overexpression of α-syn inhibits endoplasmic reticulum (ER)-Golgi trafficking and Golgi fragmentation, impairs mitochondrial function, blocks ubiquitin-proteasome system, and alters the lysosomal system [27, 28]. Abnormal or increased α-syn has also been associated with suppression of exo/endocytosis processes, altering the distribution of synaptic Soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins and influencing the activity of other peripheral proteins such as complexes and synapsins [29–34]. In addition to the effects of the microenvironment, α-syn post-translational modifications also have a major impact on protein conformation. A very large number of α-syn post-translational modifications have been identified so far, but the most frequent and by far the most studied is phosphorylation at serine 129. While, under physiological conditions, only a small proportion of α-syn is phosphorylated (estimated around 4%) [35], more than 90% of α-syn is phosphorylated on this residue, suggesting a role for pS129 in inclusion formation [35, 36].

Although initially considered as being purely intracellular, it is now well demonstrated that α-syn can be secreted by cells. Several mechanisms have been proposed to explain how cytosolic α-syn, either monomeric or aggregated, is conveyed into the extracellular milieu, including unconventional exocytosis [37, 38], exosomes [39, 40], and membrane nanotubes [41]. These findings were corroborated in vivo by showing that both monomeric and oligomeric α-syn could be detected in the blood and cerebrospinal fluid (CSF) of healthy and PD subjects [42, 43]. The secretion of α-syn aggregates from neuronal cells can depend on various stress cellular conditions (e.g., proteasomal and mitochondrial dysfunction, unconventional ER/Golgi-independent exocytosis) or dopamine that, besides increasing the formation of non-fibrillar α-syn oligomers, enhances the secretion process [44]. Once dispersed in the extracellular milieu, α-syn can be passively or endocytically internalized by other neuronal, glial, and immune/inflammatory cells, and a few receptors have been so far identified which can facilitate such a process, including heparan sulfate
<figure><img src="image_3.png" /><figcaption>**FIGURE 1** | Schematic illustration of α-synuclein pathway (a) α-syn structure: 1. the amphipathic N-terminal region (red), implicated in interactions with membranes. It contains the highly conserved 11-residue repeats-KTKEGV (yellow); 2. The hydrophobic NAC region (orange), has a major role in aggregation; 3. The negatively charged C-terminal region (purple), regulates α-syn interactions with other ligands. (b) In physiological status (green), α-syn can be present through unstructured soluble monomeric and tetrameric forms. In pathological status (red), α-syn aggregates into oligomers-protofibrils, and fibrils, resulting in the development of Lewy Body. Abbreviations: AA, amino acids; α-syn, α-synuclein; T, temperature. [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

proteoglycans (HSPGs) [44] and toll-like receptor 2 (TLR2) [45]. A role for the lymphocyte activation gene 3 (LAG3) has also been suggested [46], but these findings are still debated [47]. All these receptors preferentially bind α-syn in the amyloid over the monomeric state to initiate cell-to-cell transmission and/or to activate inflammatory response in microglia [44, 46, 47], while monomeric-syn seems to be predominantly internalized via a passive mechanism [48]. Several in vitro studies showed that once internalized, extracellular misfolded α-syn, either oligomeric or in the form of preformed fibrils, is capable of promoting the recruitment of endogenous soluble α-syn into insoluble deposits in a mechanism reminiscent of the nucleation capacity of prions [49, 50]. Following these in vitro findings, the transmission and propagation of α-syn along neuronal connections have been observed after injection of pathological α-syn (either obtained from diseased brain homogenates or by generating preformed fibrils from recombinant protein) in the brain or peripheral autonomic nervous systems of rodents and non-human primates [3, 51–54].

Taken as a whole, these results show that α-syn can transmit the aggregation process between cells and tissues and, therefore, confirm that it could behave like a prion or at least like a propagon [55]. They might also explain the presence of α-syn-positive inclusions in grafted neurons in PD subjects who had

received fetal neuron transplants [56, 57], and they provide a molecular basis to the spreading of PD pathology in specific stages as proposed initially by Braak and more recently by Borghammer [7, 58].

Of interest, there is evidence that peripheral α-syn can spread and contribute to brain pathology [2, 59]. Specifically, peripheral infusion of α-syn seeds may induce central nervous system (CNS) α-syn pathology. Various mechanisms have been proposed to explain CNS neuroinvasion, but retrograde axonal transport (e.g., through the vagal nerve) is thought to be the main pathway advancing α-syn pathology via anatomically interconnected pathways of susceptible cell populations [59]. Indeed, the propagation of α-syn is aligned with the stereotypical pattern of progressive LB as proposed by Braak and colleagues and with the appearance of clinical signs in the disease course [58, 60]. Indeed, in most cases of PD, the pathological process has been hypothesized to start with the α-syn accumulation in the olfactory structures or the gastrointestinal (GI) tract and, subsequently, to spread in a trans-synaptic prion-like fashion in the CNS, contributing to brain pathology [61]. Supporting this view, subdiaphragmatic vagotomy has been found to prevent the spread of misfolded α-syn from the gut to substantia Nigra pars compacta (SNpc) as well as the occurrence of parkinsonism in an environmental animal model following
injections of α-syn preformed fibrils in the gut [5, 62]. Moreover, the injection of human α-syn into the rat nodose ganglion spread to gastric vagal endings as well as to the dorsal motor nucleus of the vagus nerve [63]. A recent study has also reported that the centripetal spreading of misfolded α-syn could also be mediated by enteroendocrine cells lining the GI tract [64].

Based on this body of evidence, it has been hypothesized that the initial localization of pathological α-syn forms can also identify specific PD subtypes. In particular, α-syn accumulation primarily in the brain appears to identify a “brain first” PD phenotype, while an early α-syn deposition in the gut identifies a “body first” PD phenotype characterized by early autonomic dysfunction and RBD [7, 65, 66].

## 3 | Neuronal and Non-Neuronal α-Syn Across Neuroinflammation and Neurodegeneration

α-syn is expressed in neuronal and non-neuronal cell types, including central and enteric neurons, astrocytes, microglia, monocytes, macrophages, dendritic cells, neutrophils, NK cells, B and T cells, enterochromaffin cells (ECs) and skin cells, and its pathological forms can influence cell functions [67–72]. In particular, oligomeric, aggregated, fibrils, and phosphorylated α-syn forms can contribute to neuronal degeneration, activation of immune/inflammatory cell pathways, alterations of cell morphology and motility, impairments of gut barrier, and skin remodeling, degeneration and inflammation (Figure 2). Of note, several studies have focused on the characterization of the role of α-syn in promoting central and peripheral neuroinflammatory and neurodegenerative processes that can contribute to PD pathology. Thus, based on the recent and pioneering brain-first and body-first PD hypothesis, the most prominent data on the role of central and enteric neuronal and non-neuronal α-syn in PD pathophysiology are addressed in the following sections and represented in Figure 3.

### 3.1 | α-Syn-Driven Brain First Subtype

Pathological α-syn forms in the neuronal and non-neuronal cells of the CNS have been found to induce neuronal degeneration and synaptic dysfunction through mitochondrial dysfunction, lysosomal impairment, and membrane disturbances as well as neurogenic/inflammatory responses, respectively, that can contribute to PD onset and progression. For instance, α-syn accumulation in central immune/inflammatory cells, including microglia, can activate inflammatory signaling as well as promote T cell recruitment and maturation towards pro-inflammatory phenotype by interfering with SNARE complexes, altering the trafficking of cell-surface receptors, or interacting with TLRs and NLRP3 inflammasome (Figure 2) [67, 73–78]. In parallel, intracellular pathological α-syn can be also secreted in extracellular space and, in turn, activate neighboring neuronal and non-neuronal cells, thus creating a cell microenvironment, that, continuously fed by α-syn, contributes to the chronicization of the ongoing neuroinflammatory and neurodegenerative processes. Thus, α-syn acts as a bridging element between

neuroinflammation and neurodegeneration (Figure 3a) [71, 79, 80]. Indeed, clinical studies have documented concomitance of α-syn inclusions, neuronal death, microgliosis, astrogliosis, and peripheral immune cell infiltration, mainly T cells (CD4⁺, CD8⁺, and CD3⁺ T cells) and monocytes, in PD post-mortem brain sections [81–88]. However, these findings from post-mortem patients do not establish a causal relationship between α-syn pathology, microgliosis, astrogliosis, and immune/inflammatory cell infiltration and the clinical stage of PD.

Studies performed in animal models have allowed to better understand the putative role of α-syn as a bridging plug across neuroinflammation and neurodegeneration in PD. A recent pioneering study performed in a mouse model with selective microglial-α-syn overexpression in the substantia nigra (CX3CR1-SNCA mice) showed that α-syn deposition in microglial cells induced selectively dopamine neuronal degeneration by promoting phagocytic exhaustion and the recruitment of peripheral immune/inflammatory cells [89]. In particular, the authors showed that α-syn accumulation in microglia altered oxidative stress-related genes, increasing intracellular and extracellular ROS levels and NO production with consequent neurotoxicity and dopamine neuronal cell loss [89]. In addition, glial-derived pro-inflammatory mediators, recruited peripheral adaptive immune cells (i.e., T cells, dendritic cells, and B cells) [89, 90]. They, once recruited, can be activated by microglial cells [91]. Such events trigger a vicious cycle, that further induces the recruitment of peripheral leukocytes and microglial neurotoxicity. In accordance, CX3CR1-SNCA nigral tissues were found enriched in numerous populations of innate and adaptive immune cells, including CD8⁺ and CD4 Th1⁺ T and NK cells that originated in the periphery and infiltrated brain tissues contributing to neuroinflammation and ongoing neurodegenerative process [45, 89, 90, 92, 93]. These findings suggest that intra- and extracellular α-syn deposition in brain tissues can represent an early event in PD that contributes to dopaminergic neuronal death and inflammation by recruitment of peripheral immune/inflammatory cells. In support of this view, different studies performed in α-syn-driven animal models of PD, including transgenic mice and rats injected with preformed α-syn fibrils into the striatum, reported that microgliosis preceded neuronal death and that, infiltrating CD8⁺ T and MHC-II cells were in close proximity to neuronal and non-neuronal cells overexpressing α-syn (Figure 3a) [94–96].

Regarding the astrocytes, an interesting study using human brain homogenates from PD patients reported that α-syn is recruited and propagated from astrocytes to neurons, eliciting neural cell damage [97]. Supporting this view, a direct correlation between the pathological α-syn accumulation in astrocytes and neurotoxicity has been shown using primary culture of astrocytes overexpressing α-syn derived from A53T mice and from pluripotent stem cells from familial PD patients [98, 99]. In particular, pathological α-syn promoted the polarization of astrocytes to neurotoxic A1 phenotype, leading to the release of ROS and pro-inflammatory cytokines in the extracellular space (Figure 3a) [100]. Accordingly, Yun et al. [101], have demonstrated that the pharmacological blockage of astrocyte conversation to a neurotoxic A1 phenotype by NLY01 (glucagon-like peptide agonist) in a mouse model of sporadic PD prevented
<figure><img src="image_4.png" /><figcaption>**FIGURE 2** | Schematic representation of the effects of α-synuclein on neuronal and non-neuronal cells. (a) Neurons: α-syn aggregates, Lewy bodies or Lewy neurites, can promote ROS formation, alter cellular components, including proteins and DNA, and induce mitochondrial dysfunction, with consequent axonal transport disruption and synaptic dysfunction. In parallel, pathological α-syn can trigger the polarization of astrocytes to neurotoxic A1 phenotype, leading to the release of ROS and pro-inflammatory cytokines in the extracellular space. (b) Immune/inflammatory cells: α-syn can be produced within immune cells that express the SNCA gene (1) or its pathological forms can be internalized from the extracellular space via endocytosis (2). Once in the intracellular space, α-syn can seed and accumulate, forming oligomers, fibrils, and aggregates, triggering inflammatory pathways, including NLRP3 inflammasome signaling (5). Of note, both pathological and physiological forms of α-syn can interact with TLR-2/4 (4), promoting cellular polarization towards a pro-inflammatory phenotype as well as the recruitment and activation of other immune/inflammatory cells (5). α-syn can also interfere with MHC and SNARE complexes, thereby affecting antigen presentation and the trafficking of cell-surface receptors (6, 7). (c) Physiological α-syn in melanocytes promotes the melanin-producing process. In keratinocytes, oligomeric α-syn induces the release of pro-inflammatory cytokines, such as IL-1β and TNF. Additionally, α-syn can be internalized by fibroblasts, leading to increased mitochondrial dysfunction, alterations in the cytoskeleton, and in turn, fibroblast cell death. Abbreviations: α-syn, alpha-synuclein; IL-1β, interleukin-1β; MHC, major histocompatibility complex; NLRP3, Nod-like receptor protein type 3; ROS, reactive oxygen species; SNARE, soluble N-ethylmaleimide-sensitive-factor attachment protein receptor; TNF, tumor necrosis factor; TLR2/4, toll-like receptor 2 and 4. [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

central inflammation and neuronal death, prolonged survival and partially rescue of behavioral and neurological deficits.

## 3.2 | α-Syn-Driven Body First Subtype

Of interest, the involvement of the gut-brain axis in PD has fostered research on the characterization of the putative role of α-syn accumulation in neuronal and non-neuronal GI tract cells [102]. Using total and phospho-α-syn immunohistochemical staining, several independent neuropathology laboratories demonstrated that LBs and LNs-like structures were observed

in the enteric neurons in both the myenteric and submucosal plexus in the vast majority of PD patients [103, 104]. In addition, pathological α-syn species, including aggregated [96], hyperphosphorylated [72], and truncated forms [97] with a seeding activity [105, 106] are found in the enteric neurons in PD, thereby recapitulating the key features of pathological α-syn observed in the brain [36]. Of note, though physiological α-syn has also been detected in ECs, there are no existing studies on the presence of pathological α-syn deposits in the ECs in the gut of PD subjects.

Of interest, enteric α-syn accumulation can also promote neurogenic/immune-inflammatory responses that, in turn,
<figure><img src="image_5.png" /><figcaption>**FIGURE 3** | Schematic representation of the interplay between α-synuclein, neuroinflammation, and neurodegeneration in Parkinson's disease (a) Within the CNS, pathological α-syn species are found in neuronal and non-neuronal cells, including dopaminergic neurons in the substantia nigra, and microglial and astroglial cells, respectively, as well as in the extracellular space (1, 2, 3). The intra- and extracellular α-syn promotes neuronal death (1), spreads, in a prion-like manner, among neuronal cells (2), and induces microglial and astroglial activation (3), that, besides contributing to neurogenic/inflammatory responses, promotes the recruitment of peripheral immune/inflammatory cells (4), that further amplifying the ongoing neurodegenerative and neuroinflammatory processes. (b) The accumulation of pathological α-syn in the ENS (1), in immune cells (2) as well as in gut barrier cells (3) can promote neurogenic/inflammatory responses, compromise further intestinal barrier integrity and permeability, and favor changes in microbiota composition (4). These events can contribute to the translocation of pathogens in the bloodstreams and α-syn forms, including in EVs, that, in turn, can spread in the CNS via systemic circulation (5). In parallel, enteric α-syn can spread in the brain via nerve ascending pathways (6). Both hypothesized spreading mechanisms contribute to neurodegeneration and neuroinflammation in the CNS. Abbreviations: α-syn, alpha-synuclein; CNS, central nervous system; ENS, enteric nervous system. [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

impair intestinal barrier integrity and permeability or directly
decrease tight junction protein expression [86, 107, 108].
Increased intestinal permeability allows the passage of α-syn,
either free or enclosed in extracellular vesicles (EVs), along with
toxins, bacteria, and other molecules, into the systemic circu-
lation. This process can promote the spread of α-syn to the brain
and activate systemic immune-inflammatory cells thereby
contributing to PD pathology [109, 110]. Supporting this view,
recent studies in transgenic PD mice demonstrated that enteric
α-syn accumulation, mainly in myenteric and submucosal
neurons, bowel inflammation, and alterations of intestinal
barrier integrity and permeability occurred in the pre-
symptomatic stages of the disease before the onset of central
pathology (Figure 3b) [107, 111]. However, whether α-syn
deposition occurred also in other GI cell types, including enteric
glia, epithelial cells, and immune/inflammatory cells has not

been evaluated and, most importantly, a direct and mutual
relationship among α-syn accumulation, enteric inflammation,
gut barrier impairments, and PD pathology has not been es-
tablished. Studies in vitro have demonstrated that α-syn incu-
bation in macrophages promoted the direct activation of
canonical caspase-1-dependent inflammasome signaling and
IL-1β release, which, in turn, contributed to impaired intestinal
epithelial barrier integrity [107].

Of note, α-syn can interact with gut microbiota, promoting
changes in bacteria composition and function that could con-
tribute to gut barrier alterations and enteric inflammation
observed in PD [102]. In parallel, gut microbiota-derived factors
can influence α-syn aggregation, thus generating a vicious circle
that contributes to the progression of α-syn pathology in the gut
[102, 107, 112, 113]. However, a better understanding of the
mechanisms by which α-syn impacts gut cells is crucial for unraveling the link between α-syn, GI symptoms and PD pathology.

# 4 | α-Syn Deposition and PD Pathogenesis

## 4.1 | Central Nervous System

α-Syn is constitutively expressed in the CNS, comprising 1% of total cytosolic proteins [114]. Previous immunohistochemical analysis in normal brain tissues showed that α-syn is mainly localized in close proximity to presynaptic terminals, while neuronal soma staining is less apparent [115]. α-Syn was also detected in the glial cytoplasm of human brains of healthy subjects [116]. Of note, the expression of α-syn is influenced during neuronal development, following the determination of neuronal phenotype and in the establishment of synaptic connections [26]. Notably, physiological α-syn was not detected in the primary visual and cerebellar cortex, regarded as brain areas not prone to develop α-syn pathology [117]. Several previous studies have reported the presence of LBs in various sites of the CNS, including the hypothalamus, nucleus basalis of Meynert, substantia nigra, locus ceruleus, dorsal raphe nucleus, dorsal motor nucleus of the vagus nerve, intermediolateral nucleus of the spinal cord, and sacral autonomic nucleus [58, 118–121]. LBs are also detected in neurons of the amygdala and the cerebral cortex, particularly in deep layers (V and VI) of the limbic system [122, 123]. Of note, LBs formation has been found to advance in a caudal to rostral manner from the brainstem to the cortex over the course of the disease (Braak staging system) [58]. However, it's important to note that not all cases of α-synucleinopathy reflect the proposed Braak staging system [124]. More recent comparative analyses of healthy and PD-affected brains have revealed the presence of α-syn aggregates [58, 124–126]. In addition, the study conducted by Beach et al. demonstrated a significant correlation between LBs and motor symptoms assessed by unified Parkinson's disease rating scale (UPDRS) scores, as well as cognitive function measured by mini-mental state examination (MMSE) scores, particularly in subjects with smaller fractions of coexisting Alzheimer's disease pathology [126]. Others have also shown that the quantity of LBs in the frontal and/or limbic cortex serves as a better predictor of dementia in PD [127–129]. However, some studies have reported that around 10% of asymptomatic individuals over 60 years old exhibited extensive LB pathology without any signs of clinical parkinsonism [130, 131]. In addition, Zaccai et al. reported an α-syn-related pathology in aged individuals without a history of parkinsonism, suggesting no correlation between α-syn deposition and PD progression [132]. These conflicting results about the accumulation of α-syn in the brain of PD patients could depend on different clinical features of recruited patients (e.g., disease duration, motor/non-motor symptoms).

Two recent studies, aimed at evaluating α-syn deposition in brain tissues from post-mortem PD patients, have allowed us to better understand the spreading of pathological α-syn in PD [133, 134]. In particular, the study from Borghammer et al. showed that α-syn deposition in the olfactory bulb (OB) correlated with α-syn accumulation in the amygdala, while PD

patients with protein deposition in the brainstem or peripheral nervous system (PNS) showed less frequent α-syn in OB. This pattern suggests two distinct origins of PD (PNS and OB), that rarely are involved simultaneously [133]. The other study from Lin et al. reported that microstructural alterations measured by diffusion magnetic resonance imaging (MRI) were associated with dopaminergic neuronal loss and LNs load in the SNpc, suggesting MRI as a suitable monitoring indicator for Lewy pathology [134].

Of interest, research on genetic PD patients has provided valuable insights. In particular, some patients with the A53T mutation in the SNCA gene (a missense point mutation), exhibited diffuse α-syn deposition associated with specific clinical features (Table 1) [135–137].

## 4.2 | Peripheral α-Syn Deposition and PD

Recent studies have focused on the evaluation of α-syn deposition in peripheral tissues as a potential biomarker of PD. In particular, the presence of LBs and α-syn deposits has been well-documented in multiple peripheral tissues and organ nerves, including the retina, olfactory epithelium, sympathetic ganglia, enteric nervous system (ENS), salivary glands, cardiac sympathetic nerves, adrenal glands, pelvic ganglia, and skin [144, 145]. In addition, peripheral α-syn pathology may precede widespread central disease and contribute to the prodromal symptoms of PD, such as dysautonomia, constipation, orthostatic hypotension, urinary and erectile dysfunction, and anosmia [146]. In this section, current data about α-syn deposition in peripheral tissues, including skin, GI tract, and blood, are addressed and summarized in Tables 2–4.

### 4.2.1 | α-Syn Deposition in Skin

Hairy skin is characterized by a complex autonomic innervation pattern, including sympathetic adrenergic and cholinergic fibers in all dermal structures, few parasympathetic fibers in skin arterioles, and arrector pili muscles and sensory nerves found in all autonomic structures involved in reflex regulation mechanisms [199]. Morphological analysis has shown that total α-syn is constitutively expressed within the synaptic terminals in skin biopsies from healthy subjects. In particular, total α-syn has been detected in sweat glands, arrector pilorum muscles, and blood vessels from control subjects [200].

Using sampled skin from the chest and forearm of autopsied patients and an immunohistochemical approach, Ikemura et al. found LN in the sympathetic nerve fascicles of the dermis and subcutaneous tissue in 10 out of 14 PD patients [201]. These findings logically prompted several groups to study if the detection of pathological α-syn by immunohistochemical means in the biopsied skin could be an interesting tool for the diagnosis of PD. Since 2010, more than 30 studies on this subject have been published, and detailing these studies one by one is definitely beyond the scope of the current review. However, the main findings of these studies can be summarized as follows: (i) Skin biopsies (performed at multiple sites and with
<table><caption>TABLE 1 | (Continued)</caption><thead><tr><th rowspan="2">Study</th><th rowspan="2">Number of patients/HC</th><th colspan="2">Demographic information PD (mean or median ± SD or range)</th><th colspan="2">Disease characteristics PD (mean or median ± SD or range)</th><th rowspan="2">Sites of investigation</th><th rowspan="2">α-Syn/LBs deposition</th><th rowspan="2">To note</th></tr><tr><th>Age (years)</th><th>M/F</th><th>Disease duration (years)</th><th>Other</th></tr></thead><tbody><tr><td>Braak 2006 [140]</td><td>Autopsy 88 PD</td><td>—</td><td>—</td><td>—</td><td>MMSE 10% (range: 25-30)</td><td>Braak stages site: 1- DMNV</td><td>PD Braak's Stage/no of patients 3/14</td><td>No correlation with cognitive status</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>16% (range: 21-24)</td><td>2- Medulla oblongata and Pontin tegmentum</td><td>4/36</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>57% (range: 11-20)</td><td></td><td>5/32</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>17% (range: 0-10)</td><td>3- Midbrain</td><td>6/6 (&gt; 80% MMSE 11-20)</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>4- Basal prosencephalon and mesocortex</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>5- Neocortex</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>6- Neocortex</td><td></td><td></td></tr><tr><td>Dächsel 2007 [141]</td><td>Autopsy 9 PD 5 HC</td><td>78.1 ± 4.6 years</td><td>—</td><td>—</td><td>—</td><td>Visual cortex of the occipital lobe, putamen, amygdala, substantia nigra</td><td>Diminished α-syn mRNA levels in substantia nigra (strongly), putamen, visual cortex.</td><td>—</td></tr><tr><td>Hely 2008 [142]</td><td>Autopsy 4 PD 17 PDD</td><td>PD = 70 PDD = 79</td><td>—</td><td>—</td><td>—</td><td>Limbic/neocortex Brainstem</td><td>No change in amygdala</td><td>—</td></tr><tr><td>Beach 2009 [126]</td><td>Autopsy 87 HC 26 ILBD, 66 PD 40 DLB, 85 ADLB, 113 ADNLB 113</td><td>79.1 ± 6.9 years</td><td>46/20</td><td>10.6 ± 8.7</td><td>Last UPDRS III 41.0 ± 22.5 Last MMSE 20.3 ± 8.7</td><td>Olfactory bulb and tract, anterior medulla, anterior and mid-pons, mid-amygdala with adjacent transentorhinal area, anterior cingulate gyrus, middle temporal gyrus, middle frontal gyrus, inferior parietal lobule.</td><td>100% brainstem LB pathology consistent with the diagnosis of PD. Limbic and/or neocortical LBs prominent features in 8/17 with dementia</td><td>—</td></tr><tr><td>Beach 2010 [104]</td><td>Autopsy 23 HC, 7 ILBD 17 PD 9 DLB 19 ADLB 17 AD-NLB</td><td>79.3 ± 7.5 years</td><td>13/4</td><td>—</td><td>Last MDS-UPDRS III 30.1 ± 15.9 Last MMSE 26.5 ± 1.6</td><td>Spinal cord (C4-5), (T6-7), (L3-4), (S1-5) Middle cervical ganglia, middle ganglia of thoracic chain, vagus nerve</td><td>70%-90% in PD and DLB Most deposition in amygdala and cerebral cortex sections</td><td>Correlation with nigrostriatal degeneration</td></tr><tr><td>Quinn 2012 [143]</td><td>Autopsy 4 PD</td><td>79.5 ± 2.9</td><td>3/1</td><td>7.8</td><td>—</td><td>Middle temporal gyrus, cerebellum, superior parietal</td><td>Spinal cord 96% Sympathetic ganglia 79% The vagus nerve 71% in PD and DLB</td><td>—</td></tr></tbody></table>

(Continues)
<table><caption>TABLE 1 | (Continued)</caption><thead><tr><th rowspan="2">Study</th><th colspan="2">Demographic information<br>PD (mean or median ± SD or range)</th><th colspan="2">Disease characteristics PD (mean or median ± SD or range)</th><th rowspan="2">Sites of investigation</th><th rowspan="2">α-Syn/LBs deposition</th><th rowspan="2">To note</th></tr><tr><th>Number of patients/HC</th><th>Age (years)</th><th>M/F</th><th>Disease duration (years)</th><th>Other</th></tr></thead><tbody><tr><td rowspan="5">Milber<br>2012 [131]</td><td>8 DLB</td><td></td><td></td><td></td><td rowspan="5">gyrus, superior frontal gyrus, superior occipital gyrus</td><td rowspan="5">No differences in α-syn mRNA expression</td><td rowspan="5">In ILBD nigral neuronal density seems to be independent of the regional distribution of LP deposition (a distinct process from PD?)</td></tr><tr><td>5 AD</td><td></td><td></td><td></td><td></td></tr><tr><td>5 HC</td><td></td><td></td><td></td><td></td></tr><tr><td>Autopsy</td><td>Age at death</td><td>8.3 ± 1.6</td><td></td><td></td><td rowspan="3">Ofactory bulb, medulla, pons, midbrain, hippocampus, amygdala, striatum at the level of the nucleus accumbens, basal forebrain, 7 neocortical regions (midfrontal, anterior superior and midtemporal, inferior parietal, calcarine, and primary motor/sensory gyri)</td><td rowspan="3">Prenigral ILBD cases with Braak PD stages 1 and 2<br>In PD nigral neuron density was observed to decrease as α-Syn burden became more severe.</td></tr><tr><td>17 HC</td><td>PD</td><td>85.0 ± 1.11</td><td></td><td></td></tr><tr><td>33 ILBD</td><td></td><td></td><td></td><td></td></tr><tr><td>13 PD</td><td>ILBD</td><td>85.7 ± 0.90</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="4">Borghammer<br>2022<br>[133]</td><td>Autopsy</td><td></td><td></td><td></td><td>Sacral spinal cord, thoracic</td><td>Vantaa dataset</td><td rowspan="4">..</td></tr><tr><td>Vantaa dataset</td><td></td><td></td><td></td><td>spinal cord, DMVN, LC, SN, amygdala, transentorhinal cortex, OB</td><td>LBs predominant in brainstem and PNS (n = 16, 2 in OB)</td></tr><tr><td>27 early LBD+</td><td></td><td></td><td></td><td></td><td>LBs predominant in amygdala (n = 5, 5 in OB)</td></tr><tr><td>Tokyo dataset</td><td></td><td></td><td></td><td></td><td>Tokyo dataset</td></tr><tr><td rowspan="4">Lin [134]</td><td>Autopsy</td><td></td><td></td><td></td><td></td><td>LBs predominant in PNS (n = 9)</td><td rowspan="4">..</td></tr><tr><td>9 PD, 6 PDD</td><td>All patients</td><td>80 ± 8</td><td>8/12</td><td></td><td>LBs predominant in brainstem and PNS (n = 23, 2 in OB)</td></tr><tr><td>5 DLB</td><td></td><td></td><td></td><td></td><td>LBs predominant in amygdala and PNS (n = 27, 24 in OB)</td></tr><tr><td>10 HC</td><td></td><td></td><td></td><td></td><td>LBs predominant in LC and/or SN (n = 3, 2 in OB)</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>SN (immunohistochemistry)</td><td>No significant differences between PD and PDD/DLB in LB density and LN in SN.</td><td rowspan="4">Correlation among MRI-measured alterations dopaminergic loss and LN load</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>SN and tracts between the SN and caudate nucleus.</td><td>Increased mean diffusivity of the SN.</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>putamen, and DLPFC (MRI)</td><td>DLPFC correlated with increased LN load in SN in the PD and PDD/DLB</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

Abbreviations: ADLB, Alzheimer's disease with Lewy bodies; ADNLB, Alzheimer's disease with no Lewy bodies; CDR, clinical dementia rating scale; DLB, dementia with Lewy bodies; DLPFC, dorsolateral prefrontal cortex; DMNV, dorsal motor nucleus of the vagus nerve; ILBD, incidental Lewy body disease; LC, locus coeruleus; MHC II, major histocompatibility complex class 2; NDD, neurodegenerative disorder clinically established; OB, olfactory bulb; PD, Parkinson's disease; PSP, progressive supranuclear pulsy; SN, substantia nigra; UPDRS, unified Parkinson's disease rating scale.
<table><caption>TABLE 2 | α-Syn/LBs detection in the skin from PD patients.</caption><thead><tr><th rowspan="2">Study</th><th rowspan="2">Number of patients/HC</th><th colspan="2">Demographic information PD (mean or median ± SD or range)</th><th colspan="3">Disease characteristics PD (mean or median ± SD or range)</th><th rowspan="2">Sites of investigation</th><th rowspan="2">α-syn deposition (%) in PD patients</th><th rowspan="2">To note</th></tr><tr><th>Age (years)</th><th>M/F</th><th>Disease duration (years)</th><th>MDS-UPDRS III</th><th>Other</th></tr></thead><tbody><tr><td>Donadio 2020 [147]</td><td>25 PD 25 MSA-P</td><td>74 ± 6.7</td><td>18/7</td><td>6.8 ± 4.1 years</td><td>—</td><td>LEDD 518 ± 250 mg</td><td>C7 Thigh leg</td><td>p-syn deposits in the autonomic skin fibers of proximal and distal skin sites (100%)</td><td>..</td></tr><tr><td>Giannoccaro 2020 [148]</td><td>26 PD 13 PAF 7 DLB 13 MSA</td><td>71.7 ± 7.6</td><td>8/18</td><td>5.2 ± 4.8</td><td>27.7 ± 10.8</td><td>—</td><td>C7 Thigh leg</td><td>p-syn deposits in skin autonomic nerves around annexes and/or around blood vessels (95.2%)</td><td>..</td></tr><tr><td>Liu X 2020 [149]</td><td>90 PD 30 HC</td><td>59.81 ± 9.15</td><td>43/47</td><td>3.98 ± 3.12</td><td>33.46 ± 20.07</td><td>—</td><td>C7 thigh distal leg forearm</td><td>p-syn deposits in the single cervical site (83.3%)</td><td>..</td></tr><tr><td>Chahine 2020 [150]</td><td>59 PD 21 HC</td><td>63.1 ± 8.6 y.o.</td><td>41/18</td><td>57.7 ± 54.9 (months)</td><td>26.4 ± 11.9</td><td>LEDD 465 ± 429 mg</td><td>C7 Mid-thigh</td><td>64.1%</td><td>..</td></tr><tr><td>Wang N 2020 [151]</td><td>29 PD 21 HC</td><td>66.3 ± 7.7</td><td>16/13</td><td>5.5 ± 5.1</td><td>29.4 ± 11.2</td><td>—</td><td>Thigh, distal leg, forearm</td><td>50 µm 100% 20 µm 90% 10 µm 73%</td><td>..</td></tr><tr><td>Manne 2020 [152]</td><td>37 PD 37 HC</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Scalp</td><td>96% (by frozen samples) 75% (paraffin-embedded samples)</td><td>..</td></tr><tr><td>Mazzetti 2020 [153]</td><td>57 PD 48 HC</td><td>61.6 ± 10.8</td><td>38/19</td><td>7.1 ± 5.6</td><td>19.3 ± 12.4</td><td>—</td><td>Forearm</td><td>α-syn oligomers in synaptic terminals of autonomic fibers (89%)</td><td>..</td></tr><tr><td>Infante 2020 [154]</td><td>1 PD<sup>OH+</sup> (three biopsies in 3 consecutive years)</td><td>67</td><td>1/0</td><td>At the first biopsy 10 months</td><td>I 15 II 25 III 31</td><td>LEDD I 0 II 200 III 350</td><td>C7 thigh distal leg</td><td>I 40% (deep dermis) 5% (autonomic nerve bundles) II 31% (adrenergic autonomic nerve) III 40% (adrenergic autonomic nerve terminals)</td><td>..</td></tr></tbody></table>

(Continues)
<table><caption>TABLE 2 | (Continued)</caption><thead><tr><th rowspan="2">Study</th><th rowspan="2">Number of patients/HC</th><th colspan="2">Demographic information PD (mean or median ± SD or range)</th><th colspan="3">Disease characteristics PD (mean or median ± SD or range)</th><th rowspan="2">Sites of investigation</th><th rowspan="2">α-syn deposition (%) in PD patients</th><th rowspan="2">To note</th></tr><tr><th>Age (years)</th><th>M/F</th><th>Disease duration (years)</th><th>MDS-UPDRS III</th><th>Other</th></tr></thead><tbody><tr><td>Wang Z<br>2021 [155]</td><td>Autopsy<br>47 PD, 7 LBD<br>3 MSA,<br>17 AD,<br>8 PSP, 5 CBD</td><td>Autopsy<br>80.1 ± 7.1 y.o.<br>Biopsy<br>68.3 ± 7.3</td><td>Autopsy<br>34/13<br>Biopsy<br>12/8</td><td>—</td><td>—</td><td>—</td><td>Abdominal Scalp</td><td>29% (muscle arrector pilorum)<br>95% (assessed by RT-QuIC)<br>80% (assessed by PMCA)</td><td>..</td></tr><tr><td>Isonaka<br>2021 [156]</td><td>43 NNC<br>Biopsy<br>20 PD,<br>20 NNC</td><td>59.5 ± 13.9</td><td>21/23</td><td>11.5 ± 10.4</td><td>—</td><td>30 individuals with pathogenic mutation<br>SNCA 3, PRKN<br>10, LRRK2 7,<br>GBA 7, PARK7/<br>DJ1 3</td><td>C2 region<br>of the<br>nape of<br>the neck</td><td>95% PD<br>100% SNCA<br>100% LRRK2<br>83% GBA<br>0% biallelic PRKN</td><td>..</td></tr><tr><td>Giannoccaro<br>2022 [157]</td><td>16 HC<br>5 mutation carriers<br>without PD</td><td>71.7 ± 7.6 y.o.</td><td>8/18</td><td>5.2 ± 4.8</td><td>27.7 ± 10.8</td><td>—</td><td>C7 thigh distal leg</td><td>65% PD<br>7% PSP/CBS</td><td>..</td></tr><tr><td>Lama<br>2022 [158]</td><td>30 PD<br>(15 with pain, 15 without pain)<br>30 HC</td><td>67.8<br>(range: 54–81)</td><td>23/7</td><td>11.1 (range: 0–22)</td><td>—</td><td>—</td><td>Lateral lower leg<br>of<br>individuals<br>approximately<br>10 cm<br>above the ankle</td><td>0% (α-syn gene expression)</td><td>..</td></tr><tr><td>Nolano<br>2022 [159]</td><td>57 PD<br>43 MSA-P</td><td>63.5 ± 9.4</td><td>36/21</td><td>16.8 ± 6.4 (months)</td><td>21.4 ± 5.2</td><td>NMSS<br>41.9 ± 34.8</td><td>Thigh Leg fingertip</td><td>96% PD<br>91% MSA</td><td>p-syn deposits correlated positively with</td></tr></tbody></table>

(Continues)
<table><caption>TABLE 2 | (Continued)</caption><thead><tr><th rowspan="2">Study</th><th rowspan="2">Number of patients/HC</th><th colspan="2">Demographic information PD (mean or median ± SD or range)</th><th colspan="2">Disease characteristics PD (mean or median ± SD or range)</th><th rowspan="2">Sites of investigation</th><th rowspan="2">α-syn deposition (%) in PD patients</th><th rowspan="2">To note</th></tr><tr><th>Age (years)</th><th>M/F</th><th>Disease duration (years)</th><th>MDS-UPDRS III</th><th>Other</th></tr></thead><tbody><tr><td>Kuzkina 2023 [160]</td><td>39 PD<br>38 iRBD<br>23 HC</td><td>PD<br>65<br>(range:<br>50-79)<br>iRBD<br>65.5<br>(range:<br>55-84)</td><td>PD 33/6<br>iRBD 33/5<br>8 (range: 1-22)<br>years</td><td>21 (range: 2-47)</td><td>—</td><td>C7<br>Th10<br>Proximal<br>leg</td><td>78.4% iRBD<br>70% PD<br>7.9% HC</td><td>sudomotor function, epidermal, pilomotor and sudomotor nerve densities, and inversely with non-motor symptoms and disease progression<br>No correlation between dermal α-synuclein aggregation and duration of iRBD, RBDSQ, Non-Motor Symptoms Questionnaire, MDS-UPDRS-III</td></tr><tr><td>Kuzkina 2023 [161]</td><td>27 PD<br>18 iRBD<br>6 atypical<br>parkinsonism<br>HC 30</td><td>PD<br>64.0 ± 8.7<br>RBD<br>65.9 ± 7.0</td><td>PD 20/7<br>iRBD 15/3<br>12.4 ± 5.6</td><td>—</td><td>—</td><td>Nasal brushing<br>C7, thigh, leg</td><td>PD<br>55.5% Skin, 48.1%<br>Nasal SAA<br>iRBD<br>100 Skin %, 66.7%<br>Nasal SAA</td><td>..</td></tr></tbody></table>

Abbreviations: AD, Alzheimer disease; ADL, Alzheimer's disease with Lewy bodies; AD-NLB, Alzheimer's disease with no Lewy bodies; CBD, corticobasal degeneration; DLB, dementia with Lewy body; iRBD, idiopathic REM sleep behavior disorders patients; LEDD, levo-dopa equivalent dose; MSA-P, multiple system atrophy parkinsonian type; MMSE, minimal state examination test;

NC, nonneurodegenerative control; PAF, pure autonomic failure; PD<sup>OH+</sup>, Parkinson's disease with orthostatic hypotension; PSP, progressive supranuclear palsy; PMCA, protein misfolding cyclic amplification; RT-Quic, real-time quaking-induced conversion; SAA, seed amplification assays.
<table><caption>TABLE 3 | α-Syn/LBs detection in gastrointestinal tract from PD patients.</caption><thead><tr><th rowspan="2">Study</th><th rowspan="2">Number of patients/HC</th><th colspan="2">Demographic information PD (mean or median ± SD or range)</th><th colspan="5">Disease characteristics PD (mean or median ± SD or range)</th><th rowspan="2">Sites of investigation</th><th rowspan="2">α-Syn deposition (%) in PD patients</th><th rowspan="2">To note</th></tr><tr><th>Age (years)</th><th>M/F</th><th>Disease duration (years)</th><th>MDS-UPDRS III</th><th>MMSE</th><th>Other</th></tr></thead><tbody><tr><td>Braak</td><td>Autopsy</td><td>69.8 ± 5.9</td><td>3/2</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Stomach</td><td>100% in neurons of the submucosal</td><td>—</td></tr><tr><td>2006</td><td>5 PD</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Meissner plexus,</td><td></td></tr><tr><td>[162]</td><td>5 NNC</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>whose axons project into the gastric mucosa and terminate in direct proximity to fundic glands</td><td>Rostrocaudal gradient: submandibular gland and lower esophagus</td></tr><tr><td>Beach</td><td>Autopsy</td><td>79.3 ± 7.5</td><td>13/4</td><td>—</td><td>30.1 ± 15.9</td><td>26.5 ± 1.6</td><td>—</td><td>—</td><td>Upper third and lower third of esophagus,</td><td>Submandibular Gland: 39%</td><td>Rostrocaudal gradient: submandibular gland and lower esophagus</td></tr><tr><td>2010</td><td>23 HC</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>stomach (body), duodenum,</td><td>Esophagus: 33%</td></tr><tr><td>[104]</td><td>7 ILBD</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>jejunum, ileum, transverse colon, sigmoid colon,</td><td>Stomach: 22%</td></tr><tr><td></td><td>17 PD</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>rectum,</td><td>Duodenum: 17%</td></tr><tr><td></td><td>9 DLB</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>rectum: 8.3%</td><td>Jejunum: 8.3%</td></tr><tr><td></td><td>19 ADLB</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Ileum: 7%</td><td></td></tr><tr><td></td><td>17 AD-NLB</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Colon: 6%</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Rectum: 6%</td><td>PASH more frequently present in the myenteric plexus than in the submucosal plexus</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Del Tredici</td><td>Autopsy</td><td>72.5 ± 9.3</td><td>1/3</td><td>14 ± 2.65</td><td>—</td><td>—</td><td>—</td><td>—</td><td>One or both submandibular glands, the superior cervical ganglion, one or both vagal nerves at the level of the bifurcation of the external and internal carotid arteries, the distal esophagus and transition zone to</td><td>100% PD (mostly in esophagus, stomach, vagal nerve)</td><td>—</td></tr><tr><td>2011</td><td>3 PD</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Incidental LP experienced</td><td></td><td></td></tr><tr><td>[145]</td><td>1 ILBD</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Gastrointestinal symptoms (primarily indigestion).</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Case 2 Developed dementia and hallucinations.</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Case 3</td><td></td><td></td></tr></tbody></table>

(Continues)
<table><caption>TABLE 3 | (Continued)</caption><thead><tr><th rowspan="2">Study</th><th rowspan="2">Number of patients/HC</th><th colspan="2">Demographic information PD (mean or median ± SD or range)</th><th colspan="5">Disease characteristics PD (mean or median ± SD or range)</th><th rowspan="2">Sites of investigation</th><th rowspan="2">α-Syn deposition (%) in PD patients</th><th rowspan="2">To note</th></tr><tr><th>Age (years)</th><th>M/F</th><th>Disease duration (years)</th><th>MDS-UPDRS III</th><th>MMSE</th><th>Other</th></tr></thead><tbody><tr><td>Annerino 2012 [163]</td><td>Autopsy 13 PD 4 incidental LBD 12 HC</td><td>PD 76.3 ± 6.6 iLBD 84.3 ± 8.4</td><td>PD 9/4 iLBD 1/3</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Experienced fatigue, shortness of breath, nocturia, and constipation. Developed dementia. Case 4 Developed cognitive decline and she became bedridden.</td><td>the gastric cardia, the celiac ganglion, the thyroid gland.</td><td>100% PD-ILB</td><td>No neuronal loss in the myenteric plexus in PD α-syn was more frequent in the proximal than distal GI tract No correlation between gut pathology with brain pathology. &lt; 3% of α-syn aggregates were TH positive.</td></tr><tr><td>Gold 2013 [164]</td><td>Autopsy 10 PD 77 NNC 8 AD</td><td>77.0</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Stomach duodenum ileum transverse colon rectum</td><td>Colon</td><td>100% PD 52% HC (without LB)</td><td>..</td></tr><tr><td>Devos D 2013 [165]</td><td>19 PD 14 HC</td><td>60.8 ± 8</td><td>11/8</td><td>9.9 ± 1.3</td><td>4.7 ± 0.7</td><td>—</td><td>—</td><td>LEDD 712 ± 151 mg Rome III score 3.5 ± 0.3</td><td>Ascending colon</td><td>33% PD</td><td>No significant difference in the expression of pro-inflammatory cytokines or glial marker between</td></tr></tbody></table>

(Continues)
<table><caption>TABLE 3 | (Continued)</caption><thead><tr><th rowspan="3">Study</th><th rowspan="3">Number of patients/HC</th><th colspan="2">Demographic information PD (mean or median ± SD or range)</th><th colspan="4">Disease characteristics PD (mean or median ± SD or range)</th><th rowspan="3">Sites of investigation</th><th rowspan="3">α-Syn deposition (%) in PD patients</th><th rowspan="3">To note</th></tr><tr><th rowspan="2">Age (years)</th><th rowspan="2">M/F</th><th colspan="2">Disease duration (years)</th><th rowspan="2">MMSE</th><th rowspan="2">Other</th></tr><tr><th>Disease duration (years)</th><th>MDS-UPDRS III</th></tr></thead><tbody><tr><td>Gelpi 2014 [103]</td><td>Autopsy<br>4 PD<br>6 PDD<br>5 DLB<br>13 non Lewy body disease</td><td>PD<br>79.8 ± 8.1<br>PDD<br>79.7 ± 6.1</td><td>PD<br>1/3<br>PDD<br>3/3</td><td>PD<br>11.5 ± 9.0<br>PDD<br>18 ± 8.8</td><td>—</td><td>—</td><td>—</td><td>Brain and spinal cord, pituitary gland, vagus nerve at brainstem and thoracic level, stellate ganglion and paravertebral sympathetic ganglia of both sides, mesenteric sympathetic ganglion, adrenal glands and surrounding fat tissue, distal esophagus, stomach (cardias, corpus, and fundus), ileum, colon (transverse and descending), rectum, periprostatic plexus or uterus, urinary bladder, heart, abdominal skin, and psos</td><td>pANS: 15/15 PD/DLB (stellate and sympathetic ganglia 100%; vagus nerve 86.7%; gastrointestinal tract 86.7%; adrenal gland and/or surrounding fat 53.3%; heart 100%; genitourinary tract 13.3%)</td><td>patients with and without enteric Lewy pathology<br>α-syn aggregates was not related to the presence of inflammatory infiltrates</td></tr><tr><td>Hilton 2014 [85]</td><td>62 PD (at least 8 years priori the diagnosis)<br>161 HC</td><td>68 (range: 46-85)</td><td>42/20</td><td>—</td><td>—</td><td>—</td><td>PD α-syn+ RBD 29% Constipation 86%</td><td>Esophagus<br>Stomach small intestine<br>Large intestine<br>Gall bladder</td><td>Esophagus 0%<br>Stomach 8.6%<br>Small intestine 13.3%<br>Large intestine 13.2%</td><td>All patients with α-syn had autonomic symptoms.<br>Some of the positive gastrointestinal</td></tr></tbody></table>

(Continues)
<table><caption>TABLE 3 | (Continued)</caption><thead><tr><th rowspan="2">Study</th><th rowspan="2">Number of patients/HC</th><th colspan="2">Demographic information PD (mean or median ± SD or range)</th><th colspan="4">Disease characteristics PD (mean or median ± SD or range)</th><th rowspan="2">Sites of investigation</th><th rowspan="2">α-Syn deposition (%) in PD patients</th><th rowspan="2">To note</th></tr><tr><th>Age (years)</th><th>M/F</th><th>Disease duration (years)</th><th>MDS-UPDRS III</th><th>MMSE</th><th>Other</th></tr></thead><tbody><tr><td>Mu</td><td>Autopsy<br/>10 PD<br/>4 HC<br/>[166]</td><td>74.5 ± 4.0</td><td>8/2</td><td>15.8 ± 9.0</td><td>42.2 ± 19.8</td><td>—</td><td>—</td><td>Postural Hypotension<br/>100%<br/>Cognitive impairment 86%<br/>PD α-syn –<br/>RBD 22%<br/>Constipation<br/>65%<br/>Postural Hypotension<br/>29%<br/>Cognitive impairment 47%<br/>Dysphagia in 5/ 10 PD patients</td><td>Gall bladder 0%</td><td>biopsies showed inflammation</td><td>PD patients with dysphagia had more p-syn-immunoreactive axons in the regions that are critical for initiating the swallowing reflex</td></tr><tr><td>Visanji</td><td>22 PD<br/>7 PD > 5 years of disease<br/>[167]</td><td>Early PD<br/>64 (54–87)<br/>Later PD<br/>11 HC</td><td>Early PD<br/>12/10<br/>Later PD<br/>62 (50–75)</td><td>Early PD<br/>1 (0–3)<br/>Later PD<br/>10 (5–16)</td><td>Early PD<br/>14<br/>(5–24)<br/>Later PD<br/>14 (6–28)</td><td>—</td><td>Early PD LEDD<br/>300 (0–600) mg<br/>Later PD LEDD<br/>800 (100–2770) mg</td><td>Tongue, Pharynx, Larynx, upper esophagus</td><td>PD 100% (highest density in anterior tonsillar pillar, aryepiglottic fold, upper esophagus)<br/>HC 0%</td><td>80% early PD<br/>100% later<br/>100% HC</td><td>..</td></tr><tr><td>Beach</td><td>Autopsy<br/>5 PD<br/>5 HC<br/>[72]</td><td>77.4 ± 6.9</td><td>5 M</td><td>15.2 ± 8.0</td><td>36.8 ± 16.9</td><td>—</td><td>—</td><td>Sigmoid colon</td><td>Sigmoid colon</td><td>100% PD</td><td>..</td></tr><tr><td>Chung</td><td>38 PD<br/>13 MSA<br/>2016<br/>[168]</td><td>66.31 ± 8.25<br/>y.o.<br/>53 HC</td><td>23/15</td><td>7.7 ± 4.7</td><td>23.42 ± 9.35</td><td>—</td><td>LEDD<br/>820.4 ± 333.2 mg<br/>MMSE<br/>26.16 ± 3.48</td><td>Stomach<br/>Ascending colon<br/>Traverse colon<br/>Sigmoid colon</td><td>Stomach<br/>Stomach 36%<br/>Ascending-Colon 8%<br/>Traverse-Colon 21%</td><td>PD<br/>Traverse-Colon 8%<br/>Traverse-Colon 21%</td><td>..</td></tr></tbody></table>

(Continues)
<table><caption>TABLE 3 | (Continued)</caption><thead><tr><th rowspan="2">Study</th><th rowspan="2">Number of patients/HC</th><th colspan="2">Demographic information PD (mean or median ± SD or range)</th><th colspan="4">Disease characteristics PD (mean or median ± SD or range)</th><th rowspan="2">Sites of investigation</th><th rowspan="2">α-Syn deposition (%) in PD patients</th><th rowspan="2">To note</th></tr><tr><th>Age (years)</th><th>M/F</th><th>Disease duration (years)</th><th>MDS duration (range: 2-21)</th><th>UPDRS III</th><th>MMSE</th><th>Other</th></tr></thead><tbody><tr><td>Lee HJ<br>2018<br>[169]</td><td>35 PD<br>52 HC</td><td>67<br>(range: 50-79)</td><td>15/20</td><td>7 (range: 2-21)</td><td>23.2 ± 9.5</td><td>—</td><td>—</td><td>NMSS<br>50.92 ± 36.22<br>LEDD<br>816 ± 339 mg</td><td>Fundus<br>Antrum<br>ascending colon<br>transverse colon<br>sigmoid colon</td><td>Sigmoid colon 0%<br>34% PD<br>38% HC</td><td>No correlation between the α-syn pathology in ENS and gastrointestinal symptoms.</td></tr><tr><td>Fenyi<br>2019<br>[106]</td><td>18 PD<br>11 HC</td><td>63.7 ± 7.7</td><td>12/6</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Rectum<br>Sigmoid<br>Antrum</td><td>55% PD2/2 upper GI biopsies<br>7/12 sigmoid biopsies<br>1/4 rectal biopsies<br>1/11 HC who developed later parkinsonian feature</td><td>..</td></tr><tr><td>Chahine<br>2020<br>[150]</td><td>59 PD<br>21 HC</td><td>63.1 ± 8.6</td><td>41/18</td><td>57.7 ± 54.9</td><td>26.4 ± 11.9</td><td>—</td><td>—</td><td>LEDD<br>465 ± 429<br>SCOPA-AUT<br>12.5 ± 5.5</td><td>Sigmoid colon (also skin, submandibular gland)</td><td>14% PD</td><td>No correlation between intraindividual measures of α-syn across the various tissues</td></tr><tr><td>Tanei<br>2021<br>[170]</td><td>Autopsy<br>518 older<br>individuals.<br>Between them:<br>PD = 8<br>PDD/<br>DLB = 38</td><td>LBP+<br>M = 81.1 ± 9.5<br>F = 86.4 ± 8.5</td><td>98/80</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Brain, spinal cord, olfactory bulb and tract, the sympathetic ganglia, left heart ventricle, lower esophagus, adrenal gland.</td><td>Lower esophagus<br>92.1% PDD<br>75% PD</td><td>43.8% of older individuals exhibited LP, of whom in the esophagus.</td></tr></tbody></table>

(Continues)
<table><caption>TABLE 3 | (Continued)</caption><thead><tr><th rowspan="2">Study</th><th rowspan="2">Number of patients/HC</th><th colspan="2">Demographic information PD (mean or median ± SD or range)</th><th colspan="4">Disease characteristics PD (mean or median ± SD or range)</th><th rowspan="2">Sites of investigation</th><th rowspan="2">α-Syn deposition (%) in PD patients</th><th rowspan="2">To note</th></tr><tr><th>Age (years)</th><th>M/F</th><th>Disease duration (years)</th><th>MDS-UPDRS III</th><th>MMSE</th><th>Other</th></tr></thead><tbody><tr><td>Beach 2021 [171]</td><td>Autopsy 53 PD 111 NNC 33 ILBD</td><td>PD 79.1 ± 6.4</td><td>40/13</td><td>13.3 ± 7.3</td><td>38.8 ± 20.5</td><td>—</td><td>—</td><td>Stomach (body) and vagus nerve (adjacent to the carotid artery in the neck)</td><td>PD Stomach 81% vagus nerve 89%</td><td>No p-syn in the vagus nerve or stomach of subjects without brain p-syn</td></tr></tbody></table>

Abbreviations: ADLB, Alzheimer's disease with Lewy bodies; ADNLB, Alzheimer's disease with no Lewy bodies; DLB, dementia with Lewy bodies; DLPFC, dorsolateral prefrontal cortex; DMNV, dorsal motor nucleus of the vagus nerve; ILBD, incidental Lewy body disease; LEDD, levo-dopa equivalent dose; LTS, Lewy-type a-synucleinopathy; MHC II, major histocompatibility complex class 2; MMSE, minimal state examination test; NDD, neurodegenerative disorder clinically established; OB, olfactory bulb; PASH, phosphorylated a-synuclein histopathology; PD, Parkinson's disease; PSP, progressive supranuclear palsy; SCOPA-AUT, Scales for Outcomes in Parkinson's Disease-Autonomic questionnaire; SN, substantia nigra.

optimized staining protocols) allow to detect α-syn deposits and discriminate between PD and controls with acceptable sensitivity and an excellent specificity [202, 203]; (ii) skin biopsy of the cervical C7 paravertebral provide the best sensitivity for the detection of α-syn deposits [204]; (iii) dermal α-syn deposits is an early phenomenon as it can be observed in skin biopsies from patients with idiopathic REM sleep behavior disorder (iRBD) [205], a well-established clinical non-motor risk marker of PD. At first glance, all the above findings are rather convincing and strongly suggest that the detection of dermal pathological α-syn can be used for the diagnosis of patients with either prodromal or overt PD. The interpretation of skin α-syn staining is nevertheless delicate and alternative approaches to immunohistochemistry would be helpful. At present, the most promising approaches are based on ultrasensitive techniques such as protein misfolding cyclic amplification (PMCA) and real-time quaking-induced conversion (RT-QuIC) assays, which have been recently shown to efficiently amplify small amounts of aggregated α-syn in skin biopsies from iRBD subjects with excellent specificity and sensitivity (Table 2) [160, 206].

### 4.2.2 | α-Syn Deposition in the GI Tract

Regarding the ENS, the presence of LBs and neurites in the GI tract was described almost 40 years ago in two seminal reports [207, 208]. Subsequently, Wakabayashi et al. found LBs in the GI tract of seven consecutive autopsies performed in PD patients [209], and more recently, Beach et al. reported LB and LN in the gut of 11 of 17 PD patients [104]. In both studies, Lewy pathology was distributed in both the myenteric and submucosal plexus from the upper esophagus to the rectum following a rostrocaudal gradient [104, 209]. Remarkably, when specific histochemical procedures were used (analysis of multiple slides of thick sections of the lower esophagus), α-syn histopathology was found in 14 out of 15 PD patients, suggesting that the pathology is scattered but nearly constant in the ENS [104].

As with the skin, the accessibility of the ENS has led many groups to study the presence of α-syn deposits using routine GI biopsies. Most of the existing studies, which used immunohistochemical staining of formalin-fixed paraffin-embedded samples, have had conflicting results regarding sensitivity and specificity [210, 211]. An alternative approach with microdissection of the biopsies to perform whole-mount immunostaining has provided interesting results in terms of specificity and sensitivity, but this approach can hardly be used routinely [212, 213]. Again, just like for skin biopsies, PMCA and real-time quaking-induced conversion RT-QuIC assays might be helpful to efficiently amplify small amounts of aggregated α-syn from GI biopsies in PD subjects (Table 3) [106].

### 4.2.3 | α-Syn Deposition in the Blood

α-Syn is physiologically expressed in the blood cells, including erythrocytes and platelets (more than 99%) and peripheral blood mononuclear cells and plasma (less than 1%) [214]. However, the quantity of α-syn in the blood can be influenced by red blood cells contamination and hemolysis. Indeed, a low degree of contamination could result in a substantially increased concentration in plasma and serum of α-syn [215].
<table><caption>TABLE 4 | α-Syn detection in plasma/serum from PD patients.</caption><thead><tr><th rowspan="2">Study</th><th rowspan="2">Number of patients/HC</th><th colspan="2">Demographic information (mean or median ± SD or range)</th><th colspan="4">Demographic information (mean or median ± SD or range)</th><th rowspan="2">Thechnique for detecting α-syn</th><th rowspan="2">Results</th></tr><tr><th>Age (years)</th><th>M/F</th><th>Disease duration (years)</th><th>UPDRS III</th><th>H-Y</th><th>MMSE</th></tr></thead><tbody><tr><td>Lee 2006 [172]</td><td>105 PD<br>38 MSA<br>51 HC</td><td>64.5 ± 11.4</td><td>45/60</td><td>44.6 ± 37.7<br>months</td><td>—</td><td>2.4 ± 0.9</td><td>—</td><td>ELISA</td><td>Significantly elevated levels in PD patients compared with MSA and controls</td></tr><tr><td>Li 2007 [173]</td><td>27 PD<br>(13 EO,<br>14 LO)<br>11 HC</td><td>EOPD<br>62.8 ± 7.0<br>LOPD<br>71.9 ± 6.8</td><td>EOPD<br>5/8<br>LOPD<br>5/9</td><td>EOPD<br>18.8 ± 9.1<br>LOPD<br>9.6 ± 5.8</td><td>EOPD<br>2.8 ± 0.8<br>LOPD<br>2.4 ± 0.8</td><td>—</td><td>—</td><td>Quantitative Western Blot</td><td>Significant decrease in PD patients (especially in EOPD)</td></tr><tr><td>Duran<br>2010 [174]</td><td>53 ntPD<br>42 tPD<br>60 HC</td><td>ntPD<br>64.3 ± 1.6<br>tPD<br>65.4 ± 1.4</td><td>ntPD<br>25/28<br>tPD<br>14/28</td><td>tPD<br>9.2 ± 1.1<br>ntPD<br>H-Y</td><td>ntPD<br>1.5 ± 0.08tPD<br>2.56 ± 0.2</td><td>—</td><td>—</td><td>ELISA</td><td>Levels in the ntPD and tPD groups were similar, higher than in HC</td></tr><tr><td>Gorostidi<br>2012 [175]</td><td>134 PD<br>32 PD LRRK2<br>109 HC</td><td>PD<br>69.0 ± 10.6<br>PD LRRK2<br>68.0 ± 10.1</td><td>PD 77/57<br>PD LRRK2<br>13/19</td><td>PD<br>6.2 ± 5.3<br>PD LRRK2<br>7.5 ± 6.3</td><td>PD<br>2.0 ± 0.8<br>PD LRRK2<br>2.1 ± 0.8</td><td>—</td><td>—</td><td>ELISA</td><td>Significant decrease in plasma total α-syn levels PD</td></tr><tr><td>Caranci<br>2013 [176]</td><td>69 PD<br>110 HC</td><td>Age<br>64.6 ± 9.3</td><td>40/29</td><td>10.8 ± 7.3</td><td>median 17</td><td>—</td><td>—</td><td>ELISA</td><td>No difference between PD and HC</td></tr><tr><td>Foulds<br>2013 [177]</td><td>189 Newly diagnosed PD</td><td>61.9 ± 9.7</td><td>119/70</td><td>5.1 ± 4.1</td><td>—</td><td>—</td><td>—</td><td>ELISA</td><td>Higher in PD compared to HC at baseline.</td></tr><tr><td>Wang 2014 [178]</td><td>21 PD<br>(13 FOG+)<br>15 HC</td><td>FOG+<br>66.9 ± 6.7<br>FOG-<br>67.8 ± 4.2</td><td>FOG+<br>8/5<br>FOG-<br>6/2</td><td>FOG+<br>6.8 ± 3.8<br>FOG-<br>4.1 ± 1.6 years</td><td>FOG<br>+31.5 ± 20.7<br>FOG-<br>33.6 ± 10.5</td><td>—</td><td>—</td><td>Luminex</td><td>t-α-Syn increases over time</td></tr><tr><td>Shi 2014 [179]</td><td>267 PD<br>215 HC</td><td>66.3 ± 9.1</td><td>145/119</td><td>9.6 ± 6.6</td><td>28.4 ± 12.6</td><td>2.4 ± 0.7</td><td>28.0 ± 2.6</td><td>Antibody-coated superparamagnetic microbeads</td><td>Levels significantly decreased in FOG+ compared to controls</td></tr><tr><td>Ishii 2015 [180]</td><td>30 PD<br>58 HC</td><td>Age<br>65.7 ± 9.7</td><td>18/12</td><td>3.9 ± 3.8 years</td><td>22.1 ± 9.9</td><td>—</td><td>—</td><td>ELISA with or without HAI</td><td>Significantly lower in the PD group than HC</td></tr><tr><td>Lin 2017 [181]</td><td>80 PD<br>34 HC</td><td>69.6 ± 12.3</td><td>60/54</td><td>7.5 y ± 5.2</td><td>17.4 ± 9.6<br>(on-state)</td><td>—</td><td>24.3 ± 5.7</td><td>Immunomagnetic reduction</td><td>Significantly higher in patients with PD</td></tr></tbody></table>

(Continues)
<table><caption>TABLE 4 | (Continued)</caption><thead><tr><th rowspan="2">Study</th><th rowspan="2">Number of patients/HC</th><th colspan="2">Demographic information (mean or median ± SD or range)</th><th colspan="4">Demographic information (mean or median ± SD or range)</th><th rowspan="2">Thechnique for detecting α-syn</th><th rowspan="2">Results</th></tr><tr><th>Age (years)</th><th>M/F</th><th>Disease duration (years)</th><th>UPDRS III</th><th>H-Y</th><th>MMSE</th></tr></thead><tbody><tr><td>NG 2019 [182]</td><td>170 PD<br>51 HC</td><td>66.6 ± 9.5</td><td>99/71</td><td>5.0 ± 5.0 years</td><td>24.8 ± 12.5</td><td>—</td><td>25.4 ± 3.7</td><td>SIMOA</td><td>Significantly higher in PD than controls</td></tr><tr><td>Bougea 2019 [183]</td><td>724 PD<br>569 no-PD (meta-analysis)</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Elisa, Luminex, immunomagnetic reduction-based immunoassay</td><td>Levels significantly higher in PD patients than healthy controls</td></tr><tr><td>Si 2019 [184]</td><td>38 PD<br>(22 Tremor dominant TD;<br>16 non tremor dominant NTD)<br>21 ET<br>28 HC</td><td>PD TD<br>62.68 ± 10.62<br>PD NTD<br>62.13 ± 10.61</td><td>PD TD<br>12/10<br>PD NTD<br>8/8</td><td>PD TD<br>19.18 ± 14.04 months<br>PD NTD<br>35.81 ± 29.56 months</td><td>PD TD<br>18.27 ± 9.39<br>PD NTD<br>18.88 ± 10.94</td><td>PD TD<br>1.61 ± 0.60<br>PD NTD<br>1.72 ± 0.52</td><td>—</td><td>ExoQuick<br>Dynabeads Antibody<br>Coupling Kit Western blot-ELISA</td><td>CNS-derived exosomal α-synuclein is reduced in PD compared to ET.</td></tr><tr><td>Jiang 2020 [185]</td><td>230 PD<br>45 PDD<br>65 RBD, 21<br>DLB, 14 CSA<br>65 FTD, 35<br>PSP, 45 CBS<br>144 HC</td><td>—</td><td>PD<br>149/81<br>PDD<br>34/11</td><td>—</td><td>—</td><td>—</td><td>—</td><td>Immunoblot Electrochemiluminescence</td><td>Increased α-synuclein egress in serum neuronal exosomes precedes the diagnosis of PD, persists with disease progression and differentiates PD from atypical parkinsonism.</td></tr><tr><td>Chang 2020 [186]</td><td>48 PD<br>40 HC</td><td>67.2 ± 9.8</td><td>24/24</td><td>9.1 ± 6.5</td><td>—</td><td>2.8 ± 1.4</td><td>23.9 ± 5.8</td><td>Monoclonal antibodies with magnetic nanoparticles</td><td>Levels significantly higher in PD patients than HC</td></tr><tr><td>Shim 2020 [189]</td><td>20 PD<br>20 HC</td><td>71.4 ± 8.76</td><td>13/7</td><td>—</td><td>—</td><td>—</td><td>—</td><td>ELISA</td><td>No difference between PD and HC</td></tr><tr><td>Zhao 2020 [188]</td><td>30 PD<br>30 HC<br>28 MDD</td><td>69.8<br>(49-81)</td><td>16/14</td><td>—</td><td>—</td><td>11 H-Y1<br>16 H-Y2<br>1 H-Y3<br>2 H-Y4</td><td>—</td><td>Immuno-precipitation-mass spectrometry</td><td>PD and HC groups showed similar levels</td></tr></tbody></table>

(Continues)
<table><caption>TABLE 4 | (Continued)</caption><thead><tr><th rowspan="2">Study</th><th rowspan="2">Number of patients/HC</th><th colspan="2">Demographic information (mean or median ± SD or range)</th><th colspan="4">Demographic information (mean or median ± SD or range)</th><th rowspan="2">Thechnique for detecting α-syn</th><th rowspan="2">Results</th></tr><tr><th>Age (years)</th><th>M/F</th><th>Disease duration (years)</th><th>UPDRS III</th><th>H-Y</th><th>MMSE</th></tr></thead><tbody><tr><td>Chen 2020 [189]</td><td>60 PD<br>28 HC</td><td>62.8 ± 9.1</td><td>21/39</td><td>Median 1<br>(0.0-3.0)</td><td>Median 20.0<br>(16.0-34.0)</td><td>—</td><td>Median<br>26<br>(22.0-27.0)</td><td>Immunomagnetic reduction-based immunoassay</td><td>Higher plasma levels in PD compared to HC</td></tr><tr><td>Fan 2020 [190]</td><td>43 PD<br>24 HC</td><td>Age<br>58.4 ± 1.4</td><td>24/19</td><td>2.3 ± 0.3</td><td>31.4 ± 2.1</td><td>—</td><td>—</td><td>UPlex Assay</td><td>Higher plasma levels of α-syn in PD compared to HC</td></tr><tr><td>Niu 2020 [191]</td><td>36 Early PD<br>17<br>advanced PD<br>20 RBD<br>21 HC</td><td>65 ± 5.3</td><td>25/47</td><td>—</td><td>22.3 ± 10.3</td><td>2.0 ± 0.5</td><td>27.6 ± 2.6</td><td>Western blot Electrochemiluminescence</td><td>α-syn neuronal exosomes significantly higher in early stage PD patients compared with HCs and correlated with motor scores Longitudinally increased α-syn is associated with higher progression of motor symptoms.</td></tr><tr><td>Zou 2020 [192]</td><td>93 PD<br>85 HC</td><td>66.93 ± 9.52</td><td>53/40</td><td>4.26 ± 2.52</td><td>28.73 ± 16.04</td><td>2.8 ± 0.5</td><td>24.28 ± 2.88</td><td>Dynabeads Antibody Ligation Kit Western blot Mass Spectrometry Analysis</td><td>Increased α-syn concentrations in L1CAM exosomes in PD compared to HC.</td></tr><tr><td>Chung 2021 [193]</td><td>116 PD<br>46 HC</td><td>69.7 ± 8.4</td><td>62/54</td><td>2.82 ± 2.48</td><td>22.48 ± 9.85</td><td>—</td><td>24.17 ± 6.36</td><td>Immunomagnetic reduction-based immunoassay for EV α-syn levels</td><td>Significantly lower levels of α-syn in plasma EVs in PD patients than HC.</td></tr><tr><td>Youssef 2021 [194]</td><td>49 PD<br>48 HC</td><td>67 ± 1.2</td><td>32/17</td><td>4.0 ± 0.3</td><td>—</td><td>2.0 ± 0.1</td><td>—</td><td>ELISA (3 distinct kits)</td><td>Levels significantly increased in PD than HC with all the 3 kits</td></tr><tr><td>Agliardi 2021 [195]</td><td>32 PD<br>40 HC</td><td>69.47 ± 8.56</td><td>21/11</td><td>6.28 ± 3.63</td><td>28.52 ± 13.16</td><td>2 (1-3)</td><td>24.23 ± 2.48 (MoCA)</td><td>ExoQuick Western-blot-ELISA</td><td>Oligomeric exosomal α-syn significantly augmented in neuronal derived extravessicles and positively correlated with</td></tr></tbody></table>

(Continues)
<table><caption>TABLE 4 | (Continued)</caption><thead><tr><th rowspan="2">Study</th><th rowspan="2">Number of patients/HC</th><th colspan="2">Demographic information (mean or median ± SD or range)</th><th colspan="4">Demographic information (mean or median ± SD or range)</th><th rowspan="2">Thechnique for detecting α-syn</th><th rowspan="2">Results</th></tr><tr><th>Age (years)</th><th>M/F</th><th>Disease duration (years)</th><th>UPDRS III</th><th>H-Y</th><th>MMSE</th></tr></thead><tbody><tr><td>Dutta 2021 [196]</td><td>50 PD<br>30 MSA<br>51 HC</td><td>71.5 ± 9.5</td><td>32/19</td><td>8.4 ± 5.0</td><td>25.4 ± 15.3</td><td>2.5 ± 1.0</td><td>26.5 ± 6.2</td><td>Immuno-precipitation<br>Electrochemiluminescence<br>ELISA</td><td>disease duration and severity of PD.<br>The ratio between α-syn concentrations in oligodendrogial exosomes compared to neuronal exosomes is sensitive biomarker for distinguishing between PD and MSA</td></tr><tr><td>Zheng 2021 [197]</td><td>36 PD<br>36 HC</td><td>70.4 ± 0.64</td><td>19/17</td><td></td><td></td><td></td><td></td><td>Total Exosome<br>Isolation kit<br>(Invitrogen)<br>Western Blot</td><td>α-syn/total α-syn and oligomeric p-α-syn/total p-α-syn ratio in plasma exosomes may be applied to assist the PD diagnosis</td></tr><tr><td>Kluge 2022 [80]</td><td>30 PD<br>50 non-PD</td><td>67 (46–84)</td><td>21/9</td><td>4 (1–16)</td><td>25 ± 15</td><td>2 (1–5)</td><td></td><td>Immunoblot<br>Western-blot<br>Seeding assay</td><td>Pathological α-syn exosomal conformer detected in all PD patients</td></tr><tr><td>Okuzumi 2023 [198]</td><td>221 PD<br>39 MSA,<br>10 DLB,<br>9 RBD<br>128 NNC,<br>30 PSP,<br>25 AD, 17<br>PD PRKN</td><td>66 ± 10</td><td>98/123</td><td>6.9 ± 5.8</td><td>13 ± 10</td><td>2.1 ± 1.0</td><td></td><td>IP/RT-QuIC</td><td>High diagnostic performance for differentiating PD versus controls</td></tr></tbody></table>

Abbreviations: DLB, dementia with lewy body patients; ELISA, enzyme-linked immunosorbent assay; EO, early onset; ET, essential tremor patients; EV, extracellular vesicle; FOG, PD patients with freezing of gait; HA, heterophilic antibody inhibitor; HC, healthy control; H-Y, Hoehn and Yahr scale; IP/RTQuIC, immunoprecipitation-based real-time quaking-induced conversion; LO, late onset; MDD, major depressive disorder; MMSE, mini-mental state examination; MoCA, Montreal cognitive assessment; MSA, multiple system atrophy; nTPD, non treated PD patients; PD, Parkinson's disease with dementia patient; PD LRRK2, Parkinson disease with LRRK2 mutation carrier patient; RBD, REM sleep behavior disorder patient; SIMOA, single-molecule array; tPD, treated PD patients; UPDRS, unified Parkinson's disease rating scale.
Over the last years, several studies have focused on the assessment of α-syn deposition in the plasma, serum, and circulating EVs from PD patients, using different assays, including validated enzyme-linked immunosorbent assay (ELISA) kits, single molecule arrays (SIMOA), Luminex assay, and immunomagnetic reduction assays or RT-QuIC [173, 174, 182, 183, 198]. Most of the evidence has reported that total α-syn is significantly increased in the plasma from PD patients when compared to healthy controls [172, 174, 177, 179, 181–183, 189, 190, 194, 196, 198], while others reported no differences or lower levels of circulating α-syn in PD patients [173, 175, 176, 180, 187, 188, 193]. These conflicting findings could result from the employment of different assays, sample degradation, including hemolysis, and different clinical features of recruited patients, such as age, disease duration, and severity. However, most of recent studies have shown that the increased levels of α-syn in the blood correlated with the severity of motor dysfunctions, poorer cognitive impairment, and with an increase in circulating inflammatory mediators [176, 181, 182, 186, 189, 190, 193].

Of interest, several studies in PD patients have focused on the evaluation of α-syn species in blood-derived EVs, regarded as membrane particles of cellular origin that accumulate pathological α-syn forms and promote its spreading among cells [215]. EVs, including exosomes, microvesicles, and apoptotic bodies, are produced by almost all cell types and can be released in several body fluids, including plasma, serum, urine, and CSF [216]. In the setting of PD, most studies have evaluated EVs-derived α-syn, including exosomes and microvesicles released by neurons, in the blood [192, 197, 215]. In particular, Shi et al. reported that plasma neural-derived exosomes (L1 cell adhesion molecule-containing exosomes) from PD patients contain higher α-syn levels as compared with control subjects HC, and this pattern correlated with disease severity [179]. Jiang et al. showed that neuron-derived exosomal α-syn was increased in iRBD and early PD patients as compared to multiple system atrophy (MSA), other neurodegenerative diseases individuals and controls, suggesting that exosomal α-syn accumulation precedes PD diagnosis, persists with disease progression and in combination with clusterin predicts and differentiates PD from atypical parkinsonism [185]. Likewise, Dutta et al. tested and validated blood-based α-syn concentrations in neuronal and oligodendroglial exosomes isolated from serum or plasma of controls, PD, and MSA patients, and they observed that the assessment of α-syn in oligodendroglial-derived exosomes can discriminate among PD, controls and MSA subjects [196]. Zou et al. showed that the increase in EVs-derived α-syn correlated positively with EVs-derived Linc-POU3F3, a long noncoding RNA involved in the pathophysiological events in PD and, negatively with lysosomal enzyme β-glucocerebrosidase (GCase), activity whom reduction is associated with PD severity [192]. Of interest, increased oligomeric α-syn forms levels were detected in circulating neuronal exosomes derived from PD patients, and this pattern was found to be correlated with a decrease in SNARE proteins, including syntaxin-1A (STX-1A) and synaptobrevin2 (VAMP-2), and with disease duration and severity [195, 197]. Increased α-syn levels in plasma neuronal exosomes were detected in early PD patients compared with HCs, and this pattern correlated with UPDRS III/(I + II + III), Non-Motor Symptoms Questionnaire, and Sniffin' sticks 16 item test scores. After a mean follow-up of 22 months in early PD patients, increased plasma α-syn levels were found to be associated with a higher risk for motor

symptom progression. No significant differences in exosomal α-syn levels were observed among iRBD patients and control subjects [191]. Of interest, a recent study, assessing α-syn levels in neuronal and non-neuronal-derived EVs from blood, revealed that the detection and amplification of pathological soluble α-syn conformers in plasma neuronal EVs is highly promising as a reliable pre-mortem biomarker for PD [80, 191]. These findings provide new insights for the development of a standardized, non-invasive assay aimed at detecting pathology-associated α-syn extracted from blood. Only one study reported that circulating exosomes-derived α-syn is reduced in PD as compared to essential tremor patients and healthy subjects [217]. This discrepancy could depend on different methodological approaches, including the extraction of exosomes from blood and the method of quantification of α-syn.

## 4.3 | Summary

Clinical evidence indicates that PD patients are characterized by α-syn deposition in many regions of the CNS and in peripheral tissues, including skin, gut, blood, and circulating EVs. However, correlations between α-syn accumulation with disease duration, severity, and motor and non-motor symptoms are complex and variable. In particular, most studies in brain tissues seem to be quite dated, and, most importantly, since being from postmortem patients, they do not allow to understand α-syn initial deposition, its spreading, and its role in neurodegeneration and neuroinflammation. The more recent studies aimed at evaluating α-syn deposition in skin, gut, blood, and cirvulating EVs have better clarified the role of α-syn pathology in PD. Indeed, increased α-syn in the blood and plasma/serum EVs was found to correlate with PD severity and symptoms. In addition, an interesting study has shown that the increased circulating levels of α-syn correlated with IL-1β levels, suggesting that α-syn could contribute to immune/inflammatory responses in PD. However, several issues remain to be clarified. For instance, the exact initiation and spreading of α-syn pathology remains debated, and most importantly, evidence about the effects of pathological α-syn forms in neuroinflammatory and neurodegenerative processes in PD needs to be further investigated. Moreover, it remains to be determined whether the evaluation of the initial localization of pathological α-syn forms by several assays, including immunolabeling, RT-QuIC, or validated ELISA kits, can represent a useful diagnostic and prognostic biomarker of PD. In this context, validated assays to evaluate α-syn deposits in several tissues from PD patients still need to be implemented.

## 5 | Disease-Modifying Pharmacological Strategies Targeting Directly or Indirectly α-syn in PD

The involvement of α-syn in neuroinflammation and neurodegeneration associated with PD is fostering research on the potential therapeutic benefits resulting from the direct or indirect pharmacological blockade of α-syn. At present, several studies aimed at investigating the pharmacological role of α-syn in PD, have been performed in in vivo and in vitro experimental models as well as in patients. In this section, the available preclinical and clinical studies testing the effects of the direct and indirect blockade of α-syn are discussed and summarized in Tables 5 and 6.
<table><caption>TABLE 5 | Effects of pharmacological compounds targeting directly or indirectly α-synuclein in animal models of PD.</caption><thead><tr><th>Therapeutic approach (category)</th><th>Drug/compound</th><th>Mouse model of PD</th><th>Therapeutic effect</th><th>Study</th></tr></thead><tbody><tr><td>Targeting directly α-syn</td><td></td><td></td><td></td><td></td></tr><tr><td>Reducing α-syn production with ASOs or RNAi</td><td>ASOs or RNAi infusion</td><td><ul><li>transgenic PD mice overexpressing human α-syn</li><li>rat and non-human primate lentivirus model of PD</li></ul></td><td>↓ α-syn expression in hippocampus, cortex and substance nigra</td><td>Alacron-Aris 2018 [218], Sapru MK 2006 [219], Lewis J 2008 [220], McCormack AL 2010 [221], Gorbatyuk OS 2010 [222]</td></tr><tr><td>Inhibition of α-syn aggregation and aggregate reduction</td><td>HSP70 and HSP104 Anle138b Nanobodies (VH14*PEST and NbSyn87)</td><td><ul><li>A30P</li><li>rat lentivirus model of PD</li><li>A30P</li><li>Rotenone mouse model of PD</li><li>rats over-expressing wild-type α-syn</li></ul></td><td>↓ phosphorylated α-syn inclusions in nigrostriatal dopaminergic neurons<br>↓ α-syn oligomer accumulation; counteract neuronal degeneration and disease progression<br>remove α-syn aggregates, replenish striatal dopamine and improve motor function</td><td>Lo Bianco C 2008 [223], Moloney TC 2014 [224], Klucken J 2004 [225]<br>Wagner J 2013 [226]<br>Brundin 2017 [227]</td></tr><tr><td>Increasing degradation of extracellular α-synuclein: active and passive immunization</td><td>PRX002 MEDI1341 BAN0805</td><td><ul><li>Transgenic PD mice overexpressing human α-syn</li><li>A53T</li></ul></td><td>↓ α-syn accumulation along with an improvement in synaptic functions and motor/memory abilities</td><td>Games D 2014 [228], Schofield 2019 [229], Nordström E 2021 [230]</td></tr><tr><td>Targeting indirectly α-syn</td><td></td><td></td><td></td><td></td></tr><tr><td>Increasing GCase activity</td><td>Ambroxol hydrochloride</td><td><ul><li>Transgenic PD mice overexpressing human α-syn</li></ul></td><td>↓ total and phosphorylated α-syn in different brain regions</td><td>Migdalska-Richards A 2016 [231]</td></tr><tr><td>Inhibiting c-Abl activity</td><td>Nilotinib</td><td><ul><li>A53T</li></ul></td><td>↑ autophagic clearance of α-syn and prevent dopaminergic neural death from α-syn toxicity</td><td>Crunkhorn S. 2023 [232], L. Hebron 2013 [233]</td></tr><tr><td>Increasing degradation of intracellular α-syn aggregates</td><td>MSDC-0160</td><td><ul><li>α-syn-based Caenorhabditis elegans model</li><li>En1+/-</li></ul></td><td>↓ α-syn aggregates and ↓ neuroinflammatory processes</td><td>Ghosh A 2016 [234]</td></tr></tbody></table>

Note: ↑ reduction; ↑ increase.

Abbreviations: α-syn, α-synuclein; MPTP, 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine; PD, Parkinson's disease.
<table><caption>TABLE 6 | Current disease-modifying pharmacological strategies aimed at counteracting α-synuclein.</caption><thead><tr><th>Therapeutic approach (category)</th><th>Drug/compound</th><th>Target/action</th><th>Study type</th><th>Clinicaltrial.gov identifier</th></tr></thead><tbody><tr><td rowspan="3">Passive immunization against α-syn</td><td>RO7046015/PRX002 (prasinezumab), a humanized IgG1 monoclonal antibody (intravenous administration)</td><td>α-syn C-terminal antibody</td><td>Phase 2b recruiting patients with early PD</td><td>NCT03100149 (PASADENA) active with results [235]</td></tr><tr><td>TAK-341 or MEDI11341, humanized IgG1 monoclonal antibody (intravenous administration)</td><td>α-syn C-terminal antibody</td><td>Phase 2b recruiting patients with early PD</td><td>NCT04777331(PADOVA)</td></tr><tr><td>Lu AF82422, humanized IgG1 monoclonal antibody (intravenous administration)</td><td>α-syn C-terminal antibody</td><td>Phase 2 recruiting patients with early MSA</td><td>NCT05526391</td></tr><tr><td>Active immunization against α-syn</td><td>UB-312 (intramuscular injection)</td><td>Synthetic peptide vaccine inducing antibodies that against specifically oligomeric and fibrillar α-syn</td><td>Phase 1b, recruiting patients with PD and MSA</td><td>NCT05634876</td></tr><tr><td rowspan="2">Decreasing α-syn production</td><td>ACI-7104 (intramuscular injection)</td><td>Optimized vaccine formulation of the PD01A</td><td>Phase 2, recruiting early PD patients</td><td>NCT06015841 (VacSyn)</td></tr><tr><td>BIIB094 (intrathecal injection)</td><td>ASO drug targeting the mRNA of LRRK2</td><td>Phase 1, recruiting PD patients with or without variations in the LRRK2 gene</td><td>NCT03976349 (REASON)</td></tr><tr><td>Inhibition α-syn Aggregation</td><td>ION464 or BIIB101, (intrathecal injection)</td><td>ASO drug targeting the SNCA gene</td><td>Phase 1, recruiting MSA patients</td><td>NCT04165486 (HORIZON)</td></tr><tr><td rowspan="4">Active immunization against α-syn</td><td>UCB0599 (minzasolmin), oral administration</td><td>α-syn misfolding inhibitor</td><td>Phase 1, recruiting healthy participants</td><td>NCT05845645</td></tr><tr><td>BIIB122 (oral administration)</td><td>Inhibitor of LRRK2</td><td>Phase 2, enrolling by invitation new onset PD patients in early-start versus delayed-start</td><td>NCT05543252</td></tr><tr><td>Radotinib (oral administration)</td><td>BCR-ABL1 tyrosine kinase inhibitor</td><td>Phase 2, recruiting early PD patients</td><td>NCT04658186</td></tr><tr><td>Vodobatinib or K0706 (oral administration)</td><td>BCR-ABL1 tyrosine kinase inhibitor</td><td>Phase 2, recruiting early PD patients</td><td>NCT03655236 (PROSEEK)</td></tr></tbody></table>

(Continues)
<table><caption>TABLE 6 | (Continued)</caption><thead><tr><th>Therapeutic approach (category)</th><th>Drug/compound</th><th>Target/action</th><th>Study type</th><th>Clinicaltrial.gov identifier</th></tr></thead><tbody><tr><td rowspan="4">Regulation α-syn gene expression</td><td>Risvodetinib or IKT-148009 (oral administration)</td><td>BCR-ABL1 tyrosine kinase inhibitor</td><td>Phase 2, recruiting untreated PD patients</td><td>NCT05424276</td></tr><tr><td>CST-103, co-administered with CST-107 (oral administration)</td><td>β2-AR agonist (clenbuterol)</td><td>Phase 2, recruiting 4 subject populations with Neurodegenerative Disorders<br/>- PD with (RBD) and Depressive Symptoms<br/>- MCI with Depressive Symptoms<br/>- DLB with Cognitive Fluctuations<br/>- PDD with Cognitive Fluctuations</td><td>NCT04739423</td></tr><tr><td rowspan="2">CST-2032, administered with CST-107 (oral administration)</td><td rowspan="2">β2-AR agonist</td><td>Phase 2, recruiting patients with MCI or dementia Due to PD or AD</td><td rowspan="2">NCT05104463</td></tr><tr></tr></tbody></table>

Abbreviations: α-syn, α-synuclein; AD, Alzheimer's Disease; ASO, antisense oligonucleotide; β2-AR, beta2 adrenoceptor; DLB, Dementia with Lewy Bodies; LRRK2, leucine rich repeat kinase 2; MCI, mild cognitive impairment; MSA, multiple system atrophy; RBD, rapid eye movement (REM) sleep behavior disorder; PD, Parkinson's disease; PDD, Parkinson's Disease Dementia.

## 5.1 | Pre-Clinical Evidence

### a. Direct targeting of α-syn

i. Decrease in α-syn expression. With antisense oligonucleotides (ASOs) or with RNA interference (RNAi). In particular, studies in transgenic and lentivirus murine and non-human primate models of PD have documented a decrease of α-syn expression in the substantia nigra, hippocampus, and cortical regions after infusion with ASOs, small hairpin (shRNA) and short interfering (siRNA) [218–222];

ii. Inhibition of α-syn aggregation and/or aggregate reduction. Among these pharmacological strategies, two molecular chaperones, HSP70 and HSP104, have been found to decrease α-syn aggregates both in in vitro nigrostriatal dopaminergic neurons obtained from transgenic mice as well as in different brain regions of rat PD models [223–225]. Another similar approach is represented by a class of small molecules recently conceived to specifically target oligomeric forms of α-syn (NPT100-18A, NPT200-11, and Anle138b), orally bioavailable and brain penetrant [236, 237]. In particular, Anle138b is one of the few compounds successfully tested in several in vivo PD mouse models with promising results [226], while the other small molecules mentioned above have been tested only in in vitro dopaminergic neuronal cell cultures. Accordingly, further studies on animal models are strongly required to corroborate the therapeutic effects of these molecules.

Of interest, over the last years, nanobodies (small antibody fragments that target antigens intracellularly) have been developed. Among them, nanobody VH14*PEST and nanobody NbSyn87 resulted effective in decreasing α-syn aggregates, replenishing striatal dopamine, and improving motor functions in rats overexpressing wild-type α-syn [227]. However, despite these promising results, a significant challenge is to maintain high levels of the nanobodies in specific brain regions for prolonged periods and, therefore, current drug delivery technology would require direct delivery to affected regions using viral vectors;

iii. Increasing degradation of extracellular α-syn: active and passive immunization. Several studies reported that both active immunization (stimulation of the immune system to produce antibodies against toxic α-syn forms) and passive immunization (using anti-α-syn antibodies) have been found to promote neuroprotective effects in transgenic models of PD [238–240]. In particular, the administration of PRX002 (humanized monoclonal antibody—Prothena/Roche), MEDI1341 (antibody targeting extracellular α-syn through passive immunization—AstraZeneca/Takeda), and BAN0805 (humanized mAb targeting α-syn oligos and protofibrils) decreased central α-syn accumulation along with an improvement in synaptic functions and motor/memory abilities in transgenic and lentivirus mouse model of PD [228–230].

### b. Indirect targeting of α-syn

iv. Increasing GCase activity. Mutations of GCase (an enzyme involved in glycolipid metabolism) or a decrease in its
activity have been reported to contribute to α-syn mis-
folding in several synucleinopathies, including PD [231].
Ambroxol hydrochloride, a small molecule able to en-
hance GCase activity and consequently increase α-syn
clearance, has been demonstrated to decrease the total
and phosphorylated α-syn in different brain regions of
transgenic PD mice [241], thus suggesting that the
indirect targeting of α-syn using GCase activators could
represent a promising therapeutic strategy;

v. *Inhibiting c-Abl activity.* A hyperactivation of c-Abelson (Abl) tyrosine kinase has been reported to contribute to α-syn aggregation [232]. Nilotinib, a drug approved for the treatment of imatinib-resistant chronic myelogenous leukemia, has been shown to increase autophagic clearance of α-syn and counteract dopaminergic neural death in brain tissues from transgenic A53T mouse [232, 242]. However, no further studies on other pre-clinical PD models have been performed, and, therefore, intensive research efforts are strongly needed;

vi. *Increasing degradation of intracellular α-syn aggregates.* Rapamycin and its analogs have been shown to promote the clearance of pathological α-syn aggregates via autophagic processes. However, these compounds show limited utility since they lack specificity, also acting on other signaling pathways involved in immunosuppression; therefore, this feature limits their use to PD patients where long-term treatment would be necessary. Accordingly, researchers have investigated other compounds that are more selective and able to promote autophagy. Among them, the compound MSDC-0160 showed to exert a neuroprotective effect on midbrain and nigral dopaminergic neurons along with a decrease in α-syn aggregates and neuroinflammatory processes in toxin-induced PD mice, α-syn-based *Cae-norhabditis elegans* model and En1⁺/⁻ (genetic model) [234], suggesting that the pharmacological modulation of autophagy could represent an intriguing strategy to counteract α-syn-mediated neuroinflammation and neurodegeneration associated with PD. However, further studies are strongly required to better clarify the putative beneficial effect of MSDC-0160 on the intracellular aggregated α-syn isoforms.

**5.2 | Clinical Trials**

i. *Increasing degradation of brain extracellular α-syn: active and passive immunization.* At present, five monoclonal antibodies (PRX002, BIIB054, ABVV-0805, MEDI1341, LU AF82422) have been tested or are currently being evaluated in Phases 1–3 clinical trials in patients with synucleinopathies. The PRX002/prasinezumab (a humanized version of the C-terminal antibody 9E4) and BIIB054/Cimpanemab (NI-202, a fully human-derived monoclonal antibody directed against the N-terminal epitope of α-syn) were recently evaluated in two similar multicenter, randomized, double-blinded trials (the PASADENA—NCT03100149—and the SPARK study—NCT03318523—, respectively) [235, 243]. In these studies, early-stage PD patients (Hoehn and Yahr stage <3) with an abnormal dopamine transporter (DaT) scan

have been recruited. In this trial, neither the primary nor
secondary endpoints, represented by a reduction in the MDS-
UPDRS scale (Movement Disorder Society-Sponsored Revi-
sion of the Unified Parkinson's Disease Rating Scale) total
score and in DaT imaging over a 52-week period, respec-
tively, were met, except for a trend of slower motor pro-
gression with the lower dose of PRX002 in the PASADENA
study that led to the launch of a Phase 2b study (PADOVA—
NCT04777331) [235, 243, 244]. Another humanized mono-
clonal antibody tested in a clinical trial was ABBV-0805
(BAN0805, selective for soluble aggregated α-syn) has been
discontinued for strategic reasons although reported a
favorable tolerability profile (NCT04127695) [245]. Likewise,
MEDI1341 (TAK-341) and LU AF82422, both monoclonal
antibodies targeting the C-terminal of α-syn [229], showed
good tolerance in both healthy subject and PD patients in
completed Phase 1 clinical trials (NCT03272165 and
NCT04449484 for MEDI1341; NCT03611569 for LU
AF82422) [246, 247].

Active immunization approaches with synthetic peptides mimicking human α-syn have also been tested in PD patients in Phase 1 clinical trials, and they have shown a good safety and tolerability profile [248, 249]. In particular, the vaccine UB-312 is currently in Phase 1 clinical trial in PD patients (NCT05634876) [250, 251], but no data are currently available. The molecules Affitope PD01A and PD03A targeting α-syn aggregates have shown to be safe, well-tolerated and highly immunogenic in two Phase 1 trials conducted in early PD (NCT01568099 for PD01A and NCT02267434 for PD03A) [252–254]; thus, an optimized version of the vaccine PD01A, now called ACI-7104, is entered into a Phase 2 trial (NCT06015841).

ii. *Decrease in α-syn production.* A Phase 1 trial (REASON, NCT03976349) is currently ongoing in patients with or without a PD-related LRRK2 mutation treated with ASO intrathecal administration [255]. In particular, in this study, BIIB094 will be tested for its ability to reduce the LRRK2 expression and, consequently, the α-syn fibril formation [255]. In this regard, it is noteworthy that the complete gene inactivation of α-syn can trigger detrimental effects on neuronal and non-neuronal cells; therefore, special attention is required to avoid an excessive reduction of physiological α-syn levels [256]. An alternative approach to decrease α-syn expression seeks to interfere with the transcription of α-syn gene [257–260]. In particular, Mittal et al. demonstrated that the activation of β2-adrenoreceptors (β2-AR) reduced SNCA expression by regulating α-syn transcription, thus suggesting a potential neuroprotective effect of β2-AR. In addition, the company CuraSen Therapeutics Inc. has reported positive results from a Phase 2 clinical trial (NCT04739423) in patients with neurodegenerative conditions (PD with Rapid Eye Movement Sleep Behavior Disorder or dementia, Dementia with LBs and Mild cognitive impairment) by using a combination of β2-AR agonists: clenbuterol (CST-103) and nadolol (CST-107) [261].

iii. *Inhibition of α-syn aggregation and aggregate reduction.* Recently, the brain-penetrant molecule UCB0599 (minzasolmin), an enantiomer of NPT-200-11, moved into two Phase 1 studies (NCT05845645 and NCT04875962) to
test safety/tolerability and pharmacokinetics profile in heal-
thy participants [262]. Based on the promising results,
UCB0599 is underway in the Phase 2 clinical trials
(NCT04658186 and NCT05543252). Another similar prom-
ising small molecule called Anle138b has reached the clinical
state of development, completing Phase 1 trials in healthy
participants (NCT04208152) and patients with mild to
moderate PD (NCT04685265) [263].

Of note, molecules able to target LRRK2 are emerging as
a potential therapeutical strategy to reduce α-syn
pathology through the restoration of lysosomal func-
tions [264]. In this regard, the selective LRRK2 inhibitor,
DNL201, recently completed the Phase 1 clinical trial
(NCT04551534) and 1b studies (NCT03710707) showing a
dose-dependent impact on lysosomal function with an
acceptable safety profile [265].

iv. *Inhibiting c-Abl activity.* Nilotinib is able to inhibit c-Abl (increased in PD patients) [266] and DDR1/2 (discoidin domain receptors), thus promoting autophagic clearance of pathological α-syn [267]. Although preliminary studies showed acceptable safety and tolerability in PD patients along with favorable effects on CSF biomarkers, including brain dopamine turnover, oligomeric α-syn, and hyper-phosphorylated tau [268, 269], Nilotinib demonstrated poor penetration in the brain and no relevant symptomatic benefit on measures of disability in a successive multi-dose placebo-controlled Phase 2 study (NCT03205488) [270]; consequently, further clinical trial developments are currently lacking. Other Abl-tyrosine kinase inhibitors with better CNS penetration (Radotinib, ikt-148009 or Risvode-tinib and Vodobatinib or K0706) are currently underway in Phase 2 multicentre trials (NCT04691661, NCT05424276, and NCT03655236, respectively).

**5.3** | **Summary**

Pre-clinical studies targeting directly or indirectly pathological α-syn have provided promising results. Among them, immunotherapy and small molecules able to dissociate α-syn aggregates have raised a significant interest as potential therapeutical approaches to halt or slow down the progression of PD. Unfortunately, the results deriving from clinical trials have not shown promising results as compared to the pre-clinical ones. Indeed, most drugs targeting α-syn have been found to be safe and well tolerated, they have shown poor efficacy in halting or slowing down PD progression as well as in alleviating motor and non-motor symptoms. Encouraging results have emerged only from the PASADENA trial, showing a trend of slower motor progression in early PD patients that led to the launch of a Phase 2b study.

In summary, based on current findings, some important issues remain to be discussed: (1) the degeneration of nigral neurons can occur before α-syn deposition, and, most importantly, it remains controversial the timing of α-syn accumulation in the pathogenic cascade of PD; (2) the physiological functions of α-syn in neuronal and non-neuronal cells as well as the exact nature of disease-relevant species (e.g., oligomers vs fibrils) needs still to be clarified; (3) the recruitment of PD patients,

since most of the clinical trials include early symptomatic PD patients with already advanced nigrostriatal dopaminergic neurodegeneration; (4) poor blood-brain barrier penetration of drugs; (5) α-syn targeting approaches may require time to produce any observable effects. Based on these considerations, in vivo markers of α-syn pathology in humans able to measure target engagement should be identified. In addition, clinical studies in very early patients with a high risk of developing PD, including iRBD subjects, and the presence of pathological α-syn deposition in gut, skin, or blood should be performed to clarify whether targeting α-syn can represent the best pharmacological strategy for the treatment of PD.

**6** | Conclusions and Future Perspectives

Several studies have shown that pathological α-syn forms in the brain and in the gut can accumulate in neuronal and non-neuronal cells and induce neuronal degeneration and neuroinflammatory responses, respectively. In parallel, pathological α-syn can be secreted in extracellular space and, in turn, it can activate neighboring neuronal and non-neuronal cells as well as recruit peripheral immune/inflammatory cells [271], thus creating a sort of vicious circle that contributes to the ongoing neuroinflammatory and neurodegenerative processes. In this setting, it is conceivable that α-syn acts as a bridging plug across central and systemic neuroinflammation and neurodegeneration in PD. However, the pathological forms of α-syn mainly involved in α-syn spreading and PD pathology remain to be clarified. Available data from clinical studies, though suggest that the evaluation of α-syn especially in the blood and circulating EVs can represent a biomarker of PD, in terms of disease severity and symptoms and systemic inflammation, but does not allow to establish the causal relationship between α-syn and brain pathology and, most importantly, whether its accumulation precedes or represents a mere consequence of PD.

Regarding its pharmacological relevance, α-syn could indeed represent a promising therapeutic target for the treatment of PD. However, the clinical trials conducted so far have mostly recruited patients already diagnosed with PD, where neurodegenerative and neuroinflammatory processes have likely already been triggered, potentially impacting the effectiveness of such targeted therapies.

Thus, further clinical and experimental studies in humans,
including early, iRBD, non-movement disorder PD patients, and
in animal models are needed to better clarify the pathophysio-
logical and pharmacological role of α-syn deposition in PD
onset and progression as well as its relevance as diagnostic or
prognostic marker of PD.

Author Contributions

Gabriele Bellini, Carolina Pellegrini, and Pascal Derkinderen: con-
ceptualization. Gabriele Bellini, Carolina Pellegrini, Giovanni
Palermo, Vanessa D'Antongiovanni, Matteo Fornai: writing the orig-
inal draft. Pellegrini Carolina, Luca Antonioli, Pascal Derkinderen,
Nunzia Bernardini, Roberto Ceravolo: supervision, writing, review
and editing. All authors read and approved the final manuscript.
## Acknowledgments

The figures were created with BioRender.com. Open access publishing facilitated by Universita degli Studi di Pisa, as part of the Wiley - CRUI-CARE agreement.

## Conflicts of Interest

The authors declare no conflicts of interest.

## Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article. All the literature used for this review is listed in the bibliography.

## References

1. M. G. Tansey, R. L. Wallings, M. C. Houser, M. K. Herrick, C. E. Keating, and V. Joers, "Inflammation and Immune Dysfunction in Parkinson Disease," *Nature Reviews Immunology* 22, no. 11 (2022): 657–673, https://doi.org/10.1038/s41577-022-00684-6.

2. P. Calabresi, A. Mechelli, G. Natale, L. Volpicelli-Daley, G. Di Lazzaro, and V. Ghiglieri, "Alpha-Synuclein in Parkinson's Disease and Other Synucleinopathies: From Overt Neurodegeneration Back to Early Synaptic Dysfunction," *Cell Death & Disease* 14, no. 3 (2023): 176, https://doi.org/10.1038/s41419-023-05672-9.

3. M. Masuda-Suzukake, T. Nonaka, M. Hosokawa, et al., "Prion-Like Spreading of Pathological α-Synuclein in Brain," *Brain* 136, no. 4 (2013): 1128–1138, https://doi.org/10.1093/brain/awt037.

4. P. Brundin and R. Melki, "Prying Into the Prion Hypothesis for Parkinson's Disease," *The Journal of Neuroscience* 37, no. 41 (2017): 9808–9818, https://doi.org/10.1523/JNEUROSCI.1788-16.2017.

5. S. Kim, S. H. Kwon, T. I. Kam, et al., "Transneuronal Propagation of Pathologic α-Synuclein From the Gut to the Brain Models Parkinson's Disease," *Neuron* 103, no. 4 (2019): 627–641.e7, https://doi.org/10.1016/j.neuron.2019.05.035.

6. Q. Wang, Y. Liu, and J. Zhou, "Neuroinflammation in Parkinson's Disease and Its Potential as Therapeutic Target," *Translational Neurodegeneration* 4, no. 1 (2015): 19, https://doi.org/10.1186/s40035-015-0042-0.

7. J. Horsager, K. B. Andersen, K. Knudsen, et al., "Brain-First Versus Body-First Parkinson's Disease: A Multimodal Imaging Case-Control Study," *Brain* 143, no. 10 (2020): 3077–3088, https://doi.org/10.1093/brain/awaa238.

8. L. Maroteaux, J. Campanelli, and R. Scheller, "Synuclein: A Neuron-Specific Protein Localized to the Nucleus and Presynaptic Nerve Terminal," *The Journal of Neuroscience* 8, no. 8 (1988): 2804–2815, https://doi.org/10.1523/JNEUROSCI.08-08-20804.1988.

9. R. Jakes, M. G. Spillantini, and M. Goedert, "Identification of Two Distinct Synucleins From Human Brain," *FEBS Letters* 345, no. 1 (1994): 27–32, https://doi.org/10.1016/0014-5793(94)00395-5.

10. M. H. Polymeropoulos, C. Lavedan, E. Leroy, et al., "Mutation in the α-Synuclein Gene Identified in Families With Parkinson's Disease," *Science* 276, no. 5321 (1997): 2045–2047, https://doi.org/10.1126/science.276.5321.2045.

11. M. G. Spillantini, M. L. Schmidt, V. M. Y. Lee, J. Q. Trojanowski, R. Jakes, and M. Goedert, "α-Synuclein in Lewy Bodies," *Nature* 388, no. 6645 (1997): 839–840, https://doi.org/10.1038/42166.

12. W. S. Davidson, A. Jonas, D. F. Clayton, and J. M. George, "Stabilization of α-Synuclein Secondary Structure Upon Binding to Synthetic Membranes," *Journal of Biological Chemistry* 273, no. 16 (1998): 9443–9449, https://doi.org/10.1074/jbc.273.16.9443.

13. B. I. Giasson, I. V. J. Murray, J. Q. Trojanowski, and V. M. Y. Lee, "A Hydrophobic Stretch of 12 Amino Acid Residues in the Middle of α-Synuclein Is Essential for Filament Assembly," *Journal of Biological*

Chemistry 276, no. 4 (2001): 2380–2386, https://doi.org/10.1074/jbc.M008919200.

14. M. Sharma and J. Burré, "α-Synuclein in Synaptic Function and Dysfunction," *Trends in Neurosciences* 46, no. 2 (2023): 153–166, https://doi.org/10.1016/j.tins.2022.11.007.

15. R. Pinho, I. Paiva, K. G. Jerčić, et al., "Nuclear Localization and Phosphorylation Modulate Pathological Effects of Alpha-Synuclein," *Human Molecular Genetics* 28, no. 1 (2019): 31–50, https://doi.org/10.1093/hmg/ddy326.

16. S. Mehra, S. Sahay, and S. K. Maji, "α-Synuclein Misfolding and Aggregation: Implications in Parkinson's Disease Pathogenesis," *Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics* 1867, no. 10 (2019): 890–908, https://doi.org/10.1016/j.bbamap.2019.03.001.

17. T. Bartels, J. G. Choi, and D. J. Selkoe, "α-Synuclein Occurs Physiologically as a Helically Folded Tetramer That Resists Aggregation," *Nature* 477, no. 7362 (2011): 107–110, https://doi.org/10.1038/nature10324.

18. B. Fauvet, M. K. Mbefo, M. B. Fares, et al., "α-Synuclein in Central Nervous System and From Erythrocytes, Mammalian Cells, and *Escherichia Coli* Exists Predominantly as Disordered Monomer," *Journal of Biological Chemistry* 287, no. 19 (2012): 15345–15364, https://doi.org/10.1074/jbc.M111.318949.

19. N. Candelise, M. Schmitz, K. Thüne, et al., "Effect of the Micro-Environment on α-Synuclein Conversion and Implication in Seeded Conversion Assays," *Translational Neurodegeneration* 9, no. 1 (2020): 5, https://doi.org/10.1186/s40035-019-0181-9.

20. M. Zhu and A. L. Fink, "Lipid Binding Inhibits α-Synuclein Fibril Formation," *Journal of Biological Chemistry* 278, no. 19 (2003): 16873–16877, https://doi.org/10.1074/jbc.M210136200.

21. I. Dikiy and D. Eliezer, "Folding and Misfolding of Alpha-Synuclein on Membranes," *Biochimica et Biophysica Acta (BBA) - Biomembranes* 1818, no. 4 (2012): 1013–1018, https://doi.org/10.1016/j.bbamem.2011.09.008.

22. R. Bovenzi, G. M. Sancesario, M. Conti, et al., "Sex Hormones Differentially Contribute to Parkinson Disease in Males: A Multimodal Biomarker Study," *European Journal of Neurology* 30, no. 7 (2023): 1983–1990, https://doi.org/10.1111/ene.15801.

23. B. Winner, R. Jappelli, S. K. Maji, et al., "In Vivo Demonstration That α-Synuclein Oligomers Are Toxic," *Proceedings of the National Academy of Sciences* 108, no. 10 (2011): 4194–4199, https://doi.org/10.1073/pnas.1100976108.

24. D. Emin, Y. P. Zhang, E. Lobanova, et al., "Small Soluble α-Synuclein Aggregates Are the Toxic Species in Parkinson's Disease," *Nature Communications* 13, no. 1 (2022): 5512, https://doi.org/10.1038/s41467-022-33252-6.

25. C. Galvagnion, A. K. Buell, G. Meisl, et al., "Lipid Vesicles Trigger α-Synuclein Aggregation by Stimulating Primary Nucleation," *Nature Chemical Biology* 11, no. 3 (2015): 229–234, https://doi.org/10.1038/nchembio.1750.

26. D. Sulzer and R. H. Edwards, "The Physiological Role of α-Synuclein and Its Relationship to Parkinson's Disease," *Journal of Neurochemistry* 150, no. 5 (2019): 475–486, https://doi.org/10.1111/jnc.14810.

27. A. A. Cooper, A. D. Gitler, A. Cashikar, et al., "α-Synuclein Blocks ER-Golgi Traffic and Rab1 Rescues Neuron Loss in Parkinson's Models," *Science* 313, no. 5785 (2006): 324–328, https://doi.org/10.1126/science.1129462.

28. K. Nakamura, V. M. Nemani, F. Azarbal, et al., "Direct Membrane Association Drives Mitochondrial Fission by the Parkinson Disease-Associated Protein α-Synuclein," *Journal of Biological Chemistry* 286, no. 23 (2011): 20710–20726, https://doi.org/10.1074/jbc.M110.213538.

29. J. Burré, M. Sharma, T. Tsetsenis, V. Buchman, M. R. Etherton, and T. C. Südhof, "α-Synuclein Promotes Snare-Complex Assembly In Vivo and In Vitro," *Science* 329, no. 5999 (2010): 1663–1667, https://doi.org/10.1126/science.1195227.
30. V. M. Nemani, W. Lu, V. Berge, et al., "Increased Expression of α-Synuclein Reduces Neurotransmitter Release by Inhibiting Synaptic Vesicle Reclustering After Endocytosis," *Neuron* 65, no. 1 (2010): 66-79, https://doi.org/10.1016/j.neuron.2009.12.023.

31. K. Eguchi, Z. Taoufiq, O. Thorn-Seshold, D. Trauner, M. Hasegawa, and T. Takahashi, "Wild-Type Monomeric α-Synuclein Can Impair Vesicle Endocytosis and Synaptic Fidelity via Tubulin Polymerization at the Calyx of Held," *Journal of Neuroscience* 37, no. 25 (2017): 6043–6052, https://doi.org/10.1523/JNEUROSCI.0179-17.2017.

32. M. Atias, Y. Tevet, J. Sun, et al., "Synapsins Regulate α-Synuclein Functions," *Proceedings of the National Academy of Sciences* 116, no. 23 (2019): 11116–11118, https://doi.org/10.1073/pnas.1903054116.

33. J. Sun, L. Wang, H. Bao, et al., "Functional Cooperation of α-Synuclein and VAMP2 in Synaptic Vesicle Recycling," *Proceedings of the National Academy of Sciences* 116, no. 23 (2019): 11113–11115, https://doi.org/10.1073/pnas.1903049116.

34. P. Garcia-Reitböck, O. Anichtchik, A. Bellucci, et al., "Snare Protein Redistribution and Synaptic Failure in a Transgenic Mouse Model of Parkinson's Disease," *Brain* 133, no. 7 (2010): 2032–2044, https://doi.org/10.1093/brain/awq132.

35. H. Fujiwara, M. Hasegawa, N. Dohmae, et al., "α-Synuclein Is Phosphorylated in Synucleinopathy Lesions," *Nature Cell Biology* 4, no. 2 (2002): 160–164, https://doi.org/10.1038/ncb748.

36. J. P. Anderson, D. E. Walker, J. M. Goldstein, et al., "Phosphorylation of Ser-129 Is the Dominant Pathological Modification of α-Synuclein in Familial and Sporadic Lewy Body Disease," *Journal of Biological Chemistry* 281, no. 40 (2006): 29739–29752, https://doi.org/10.1074/jbc.M600933200.

37. A. Jang, H. J. Lee, J. E. Suk, J. W. Jung, K. P. Kim, and S. J. Lee, "Non-Classical Exocytosis of α-Synuclein Is Sensitive to Folding States and Promoted Under Stress Conditions," *Journal of Neurochemistry* 113, no. 5 (2010): 1263–1274, https://doi.org/10.1111/j.1471-4159.2010.06695.x.

38. H. J. Lee, S. Patel, and S. J. Lee, "Intravesicular Localization and Exocytosis of α-Synuclein and Its Aggregates," *Journal of Neuroscience* 25, no. 25 (2005): 6016–6024, https://doi.org/10.1523/JNEUROSCI.0692-05.2005.

39. K. M. Danzer, L. R. Kranich, W. P. Ruf, et al., "Exosomal Cell-to-Cell Transmission of Alpha Synuclein Oligomers," *Molecular Neurodegeneration* 7 (2012): 42, https://doi.org/10.1186/1750-1326-7-42.

40. E. Emmanouilidou, K. Melachroinou, T. Roumeliotis, et al., "Cell-Produced α-Synuclein Is Secreted in a Calcium-Dependent Manner by Exosomes and Impacts Neuronal Survival," *Journal of Neuroscience* 30, no. 20 (2010): 6838–6851, https://doi.org/10.1523/JNEUROSCI.5699-09.2010.

41. S. Abounit, L. Bousset, F. Loria, et al., "Tunneling Nanotubes Spread Fibrillar α-Synuclein by Intercellule Trafficking of Lysosomes," *EMBO Journal* 35, no. 19 (2016): 2120–2138, https://doi.org/10.15252/embj.201593411.

42. R. Borghi, R. Marchese, A. Negro, et al., "Full Length α-Synuclein Is Present in Cerebrospinal Fluid From Parkinson's Disease and Normal Subjects," *Neuroscience Letters* 287, no. 1 (2000): 65-67, https://doi.org/10.1016/S0304-3940(00)01153-8.

43. O. M. A. El-Agnaf, S. A. Salem, K. E. Paleologou, et al., "α-Synuclein Implicated in Parkinson's Disease Is Present in Extracellular Biological Fluids, Including Human Plasma," *FASEB Journal* 17, no. 13 (2003): 1–16, https://doi.org/10.1096/fj.03-0098fje.

44. B. B. Holmes, S. L. DeVos, N. Kfoury, et al., "Heparan Sulfate Proteoglycans Mediate Internalization and Propagation of Specific Proteo-pathic Seeds," *Proceedings of the National Academy of Sciences* 110, no. 33 (2013): E3138–E3147, https://doi.org/10.1073/pnas.1301440110.

45. C. Kim, D. H. Ho, J. E. Suk, et al., "Neuron-Released Oligomeric α-Synuclein Is an Endogenous Agonist of TLR2 for Paracrine Activation of Microglia," *Nature Communications* 4 (2013): 1562, https://doi.org/10.1038/ncomms2534.

46. X. Mao, M. T. Ou, S. S. Karuppagounder, et al., "Pathological α-Synuclein Transmission Initiated by Binding Lymphocyte-Activation Gene 3," *Science* 353, no. 6307 (2016): aah3374, https://doi.org/10.1126/science.aah3374.

47. M. Emmenegger, E. De Cecco, M. Hruska-Plochan, et al., "LAG3 Is Not Expressed in Human and Murine Neurons and Does Not Modulate α-Synucleinopathies," *EMBO Molecular Medicine* 13, no. 9 (2021): e14745, https://doi.org/10.15252/emmm.202114745.

48. K. J. Ahn, S. R. Paik, K. C. Chung, and J. Kim, "Amino Acid Sequence Motifs and Mechanistic Features of the Membrane Translocation of α-Synuclein," *Journal of Neurochemistry* 97, no. 1 (2006): 265–279, https://doi.org/10.1111/j.1471-4159.2006.03731.x.

49. L. A. Volpicelli-Daley, K. C. Luk, T. P. Patel, et al., "Exogenous α-Synuclein Fibrils Induce Lewy Body Pathology Leading to Synaptic Dysfunction and Neuron Death," *Neuron* 72, no. 1 (2011): 57–71, https://doi.org/10.1016/j.neuron.2011.08.033.

50. K. M. Danzer, D. Haasen, A. R. Karow, et al., "Different Species of α-Synuclein Oligomers Induce Calcium Influx and Seeding," *Journal of Neuroscience* 27, no. 34 (2007): 9220–9232, https://doi.org/10.1523/JNEUROSCI.2617-07.2007.

51. K. C. Luk, V. Kehm, J. Carroll, et al., "Pathological α-Synuclein Transmission Initiates Parkinson-Like Neurodegeneration in Non-transgenic Mice," *Science* 338, no. 6109 (2012): 949–953, https://doi.org/10.1126/science.1227157.

52. A. Shimozawa, M. Ono, D. Takahara, et al., "Propagation of Pathological α-Synuclein in Marmoset Brain," *Acta Neuropathologica Communications* 5, no. 1 (2017): 12, https://doi.org/10.1186/s40478-017-0413-0.

53. A. Recasens, B. Dehay, J. Bové, et al., "Lewy Body Extracts From Parkinson Disease Brains Trigger α-Synuclein Pathology and Neurodegeneration in Mice and Monkeys," *Annals of Neurology* 75, no. 3 (2014): 351–362, https://doi.org/10.1002/ana.24066.

54. C. Challis, A. Hori, T. R. Sampson, et al., "Gut-Seeded α-Synuclein Fibrils Promote Gut Dysfunction and Brain Pathology Specifically in Aged Mice," *Nature Neuroscience* 23, no. 3 (2020): 327–336, https://doi.org/10.1038/s41593-020-0589-7.

55. T. Uchihara, B. I. Giasson, and W. Paulus, "Propagation of Aβ, Tau and α-Synuclein Pathology Between Experimental Models and Human Reality: Prions, Propagons and Propaganda," *Acta Neuropathologica* 131, no. 1 (2016): 1–3, https://doi.org/10.1007/s00401-015-1517-x.

56. J. Y. Li, E. Englund, J. L. Holton, et al., "Lewy Bodies in Grafted Neurons in Subjects With Parkinson's Disease Suggest Host-to-Graft Disease Propagation," *Nature Medicine* 14, no. 5 (2008): 501–503, https://doi.org/10.1038/nm1746.

57. J. H. Kordower, Y. Chu, R. A. Hauser, T. B. Freeman, and C. W. Olanow, "Lewy Body-Like Pathology in Long-Term Embryonic Nigral Transplants in Parkinson's Disease," *Nature Medicine* 14, no. 5 (2008): 504–506, https://doi.org/10.1038/nm1747.

58. H. Braak, K. D. Tredici, U. Rüb, R. A. I. de Vos, E. N. H. Jansen Steur, and E. Braak, "Staging of Brain Pathology Related to Sporadic Parkinson's Disease," *Neurobiology of Aging* 24, no. 2 (2003): 197–211, https://doi.org/10.1016/S0197-4580(02)00065-9.

59. J. I. Ayers, M. M. Brooks, N. J. Rutherford, et al., "Robust Central Nervous System Pathology in Transgenic Mice Following Peripheral Injection of α-Synuclein Fibrils," *Journal of Virology* 91 (2017): e02095, https://doi.org/10.1128/JVI.02095-16.

60. A. Lau, R. W. L. So, H. H. C. Lau, et al., "α-Synuclein Strains Target Distinct Brain Regions and Cell Types," *Nature Neuroscience* 23, no. 1 (2020): 21–31, https://doi.org/10.1038/s41593-019-0541-x.

61. B. A. Killinger and J. H. Kordower, "Spreading of Alpha-Synuclein – Relevant or Epiphenomenon?," *Journal of Neurochemistry* 150, no. 5 (2019): 605–611, https://doi.org/10.1111/jnc.14779.
62. L. Anselmi, C. Bove, F. H. Coleman, et al., "Ingestion of Sub-threshold Doses of Environmental Toxins Induces Ascending Parkinsonism in the Rat," *npj Parkinson's Disease* 4, no. 1 (2018): 30, https://doi.org/10.1038/s41531-018-0066-0.

63. A. Ulusoy, R. J. Phillips, M. Helwig, M. Klinkenberg, T. L. Powley, and D. A. Di Monte, "Brain-to-Stomach Transfer of α-Synuclein via Vagal Preganglionic Projections," *Acta Neuropathologica* 133, no. 3 (2017): 381-393, https://doi.org/10.1007/s00401-016-1661-y.

64. R. Chandra, A. Hiniker, Y. M. Kuo, R. L. Nussbaum, and R. A. Liddle, "α-Synuclein in Gut Endocrine Cells and Its Implications for Parkinson's Disease," *JCI Insight* 2, no. 12 (2017): e92295, https://doi.org/10.1172/jci.insight.92295.

65. P. Borghammer, J. Horsager, K. Andersen, et al., "Neuropathological Evidence of Body-First vs. Brain-First Lewy Body Disease," *Neurobiology of Disease* 161 (2021): 105557, https://doi.org/10.1016/j.nbd.2021.105557.

66. P. Borghammer, "The α-Synuclein Origin and Connectome Model (SOC Model) of Parkinson's Disease: Explaining Motor Asymmetry, Non-Motor Phenotypes, and Cognitive Decline," *Journal of Parkinson's Disease* 11, no. 2 (2021): 455-474, https://doi.org/10.3233/JPD-202481.

67. Y. Pei and R. W. Maitta, "Alpha Synuclein in Hematopoiesis and Immunity," *Heliyon* 5, no. 10 (2019): e02590, https://doi.org/10.1016/j.heliyon.2019.e02590.

68. P. Chieosilapatham, C. Kiatsurayanon, Y. Umehara, et al., "Keratinocytes: Innate Immune Cells in Atopic Dermatitis," *Clinical and Experimental Immunology* 204, no. 3 (2021): 296-309, https://doi.org/10.1111/cei.13575.

69. J. T. Oliveira, V. Dakic, G. Vitória, et al., "Oligomeric α-Synuclein Induces Skin Degeneration in Reconstructed Human Epidermis," *Neurobiology of Aging* 113 (2022): 108-117, https://doi.org/10.1016/j.neurobiolaging.2022.02.010.

70. C. Galvagnion, F. R. Marlet, S. Cerri, A. H. V. Schapira, F. Blandini, and D. A. Di Monte, "Sphingolipid Changes in Parkinson L444P GBA Mutation Fibroblasts Promote α-Synuclein Aggregation," *Brain* 145, no. 3 (2022): 1038-1051, https://doi.org/10.1093/brain/awab371.

71. P. Lyra, V. Machado, S. Rota, K. R. Chaudhuri, J. Botelho, and J. J. Mendes, "Revisiting Alpha-Synuclein Pathways to Inflammation," *International Journal of Molecular Sciences* 24, no. 8 (2023): 7137, https://doi.org/10.3390/IJMS24087137.

72. T. G. Beach, A. G. chneider, F. Letournel, et al., "Multicenter Assessment of Immunohistochemical Methods for Pathological Alpha-Synuclein in Sigmoid Colon of Autopsied Parkinson's Disease and Control Subjects," *Journal of Parkinson's Disease* 6, no. 4 (2016): 761-770, https://doi.org/10.3233/JPD-160888.

73. A. Shameli, W. Xiao, Y. Zheng, et al., "A Critical Role for Alpha-Synuclein in Development and Function of T Lymphocytes," *Immunobiology* 221, no. 2 (2016): 333-340, https://doi.org/10.1016/j.imbio.2015.10.002.

74. L. Fellner, R. Irschick, K. Schanda, et al., "Toll-Like Receptor 4 Is Required for α-Synuclein Dependent Activation of Microglia and Astroglia," *GLIA* 61, no. 3 (2013): 349-360, https://doi.org/10.1002/glia.22437.

75. D. Sulzer, R. N. Alcalay, F. Garretti, et al., "T Cells From Patients With Parkinson's Disease Recognize α-Synuclein Peptides," *Nature* 546, no. 7660 (2017): 656-661, https://doi.org/10.1038/nature22815.

76. H. González, F. Contreras, and R. Pacheco, "Regulation of the Neurodegenerative Process Associated to Parkinson's Disease by CD4+ T-Cells," *Journal of Neuroimmune Pharmacology* 10, no. 4 (2015): 561-575, https://doi.org/10.1007/s11481-015-9618-9.

77. L. Qin, G. Li, X. Qian, et al., "Interactive Role of the Toll-Like Receptor 4 and Reactive Oxygen Species in LPS-Induced Microglia Activation," *GLIA* 52, no. 1 (2005): 78-84, https://doi.org/10.1002/glia.20225.

78. Y. Li, Y. Xia, S. Yin, et al., "Targeting Microglial α-Synuclein/TLRs/NF-kappaB/NLRP3 Inflammasome Axis in Parkinson's Disease," *Frontiers in Immunology* 12 (2021): 719807, https://doi.org/10.3389/fimmu.2021.719807.

79. T. Tokuda, M. M. Qureshi, M. T. Ardah, et al., "Detection of Elevated Levels of α-Synuclein Oligomers in CSF From Patients With Parkinson Disease," *Neurology* 75, no. 20 (2010): 1766-1770, https://doi.org/10.1212/WNL.0B13E3181FD613B.

80. A. Kluge, J. Bunk, E. Schaeffer, et al., "Detection of Neuron-Derived Pathological α-Synuclein in Blood," *Brain* 145, no. 9 (2022): 3058-3071, https://doi.org/10.1093/BRAIN/AWAC115.

81. V. Brochard, B. Combadière, A. Prigent, et al., "Infiltration of CD4+ Lymphocytes Into the Brain Contributes to Neurodegeneration in a Mouse Model of Parkinson Disease," *Journal of Clinical Investigation* 119, no. 1 (2009): 182-192, https://doi.org/10.1172/JCI36470.

82. V. Joers, M. G. Tansey, G. Mulas, and A. R. Carta, "Microglial Phenotypes in Parkinson's Disease and Animal Models of the Disease," *Progress in Neurobiology* 155 (2017): 57-75, https://doi.org/10.1016/J.PNEUROBIO.2016.04.006.

83. A. F. Sommer, F. Marxreiter, F. Krach, et al., "Th17 Lymphocytes Induce Neuronal Cell Death in a Human iPSC-Based Model of Parkinson's Disease," *Cell Stem Cell* 23, no. 1 (2018): 123-131.e6, https://doi.org/10.1016/J.STEM.2018.06.015.

84. T. Lebouvier, T. Chaumette, P. Damier, et al., "Pathological Lesions in Colonic Biopsies During Parkinson's Disease," *Gut* 57, no. 12 (2008): 1741-1743, https://doi.org/10.1136/gut.2008.162503.

85. D. Hilton, M. Stephens, L. Kirk, et al., "Accumulation of α-Synuclein in the Bowel of Patients in the Pre-Clinical Phase of Parkinson's Disease," *Acta Neuropathologica* 127, no. 2 (2014): 235-241, https://doi.org/10.1007/s00401-013-1214-6.

86. G. Bellini, L. Benvenuti, C. Ippolito, et al., "Intestinal Histomorphological and Molecular Alterations in Patients With Parkinson's Disease," *European Journal of Neurology* 30, no. 11 (2022): 3440-3450, https://doi.org/10.1111/ene.15607.

87. M. A. Mohamed, Z. Zeng, M. Gennaro, et al., "Astrogliosis in Aging and Parkinson's Disease Dementia: A New Clinical Study With 11C-BU99008 Pet," *Brain Communications* 4, no. 5 (2022): fcac199, https://doi.org/10.1093/braincomms/fcac199.

88. F. Weiss, A. Labrador-Garrido, N. Dzamko, and G. Halliday, "Immune Responses in the Parkinson's Disease Brain," *Neurobiology of Disease* 168 (2022): 105700, https://doi.org/10.1016/j.nbd.2022.105700.

89. S. Bido, S. Muggeo, L. Massimino, et al., "Microglia-Specific Overexpression of α-Synuclein Leads to Severe Dopaminergic Neurodegeneration by Phagocytic Exhaustion and Oxidative Toxicity," *Nature Communications* 12, no. 1 (2021): 6237, https://doi.org/10.1038/S41467-021-26519-X.

90. A. F. Pike, T. Varanita, M. A. C. Herrebout, et al., "α-Synuclein Evokes NLRP3 Inflammasome-Mediated IL-1β Secretion From Primary Human Microglia," *GLIA* 69, no. 6 (2021): 1413-1428, https://doi.org/10.1002/glia.23970.

91. S. Isik, B. Yeman Kiyak, R. Akbayir, R. Seyhali, and T. Arpaci, "Microglia Mediated Neuroinflammation in Parkinson's Disease," *Cells* 12, no. 7 (2023): 1012, https://doi.org/10.3390/cells12071012.

92. S. G. Daniele, D. Béraud, C. Davenport, K. Cheng, H. Yin, and K. A. Maguire-Zeiss, "Activation of MyD88-Dependent TLR1/2 Signaling by Misfolded α-Synuclein, a Protein Linked to Neurodegenerative Disorders," *Science Signaling* (2015): ra45, https://doi.org/10.1126/SCISIGNAL.2005965.

93. C. D. Hughes, M. L. Choi, M. Ryten, et al., "Correction to: Picomolar Concentrations of Oligomeric Alpha-Synuclein Sensitizes TLR4 to Play an Initiating Role in Parkinson's Disease Pathogenesis," *Acta Neuropathologica* 137, no. 1 (2019): 121, https://doi.org/10.1007/S00401-018-1919-7.
94. M. B. Watson, F. Richter, S. K. Lee, et al., "Regionally-Specific Microglial Activation in Young Mice Over-Expressing Human Wildtype Alpha-Synuclein," *Experimental Neurology* 237, no. 2 (2012): 318-334, https://doi.org/10.1016/j.EXPNEUROL.2012.06.025.

95. M. F. Duffy, T. J. Collier, J. R. Patterson, et al., "Correction to: Lewy Body-Like Alpha-Synuclein Inclusions Trigger Reactive Microgliosis Prior to Nigral Degeneration," *Journal of Neuroinflammation* 15, no. 1 (2018): 169, https://doi.org/10.1186/S12974-018-1202-9.

96. G. P. Williams, A. M. Schonhoff, A. Jurkuvenaite, N. J. Gallups, D. G. Standaert, and A. S. Harms, "CD4 T Cells Mediate Brain Inflammation and Neurodegeneration in a Mouse Model of Parkinson's Disease," *Brain* 144, no. 7 (2021): 2047-2059, https://doi.org/10.1093/BRAIN/AWAB103.

97. F. Cavaliere, L. Cerf, B. Dehay, et al., "In Vitro α-Synuclein Neurotoxicity and Spreading Among Neurons and Astrocytes Using Lewy Body Extracts From Parkinson Disease Brains," *Neurobiology of Disease* 103 (2017): 101-112, https://doi.org/10.1016/j.nbd.2017.04.011.

98. X. L. Gu, C. X. Long, L. Sun, C. Xie, X. Lin, and H. Cai, "Astrocytic Expression of Parkinson's Disease-Related A53T α-Synuclein Causes Neurodegeneration in Mice," *Molecular Brain* 3 (2010): 12, https://doi.org/10.1186/1756-6606-3-12.

99. A. di Domenico, G. Carola, C. Calatayud, et al., "Patient-Specific Ipsc-Derived Astrocytes Contribute to Non-Cell-Autonomous Neurodegeneration in Parkinson's Disease," *Stem Cell Reports* 12, no. 2 (2019): 213-229, https://doi.org/10.1016/j.stemcr.2018.12.011.

100. T. W. Chou, N. P. Chang, M. Krishnagiri, et al., "Fibrillar α-Synuclein Induces Neurotoxic Astrocyte Activation via RIP Kinase Signaling and NF-κB," *Cell Death & Disease* 12, no. 8 (2021): 756, https://doi.org/10.1038/s41419-021-04049-0.

101. S. P. Yun, T. I. Kam, N. Panicker, et al., "Block of A1 Astrocyte Conversion by Microglia Is Neuroprotective in Models of Parkinson's Disease," *Nature Medicine* 24, no. 7 (2018): 931-938, https://doi.org/10.1038/s41591-018-0051-5.

102. C. Pellegrini, L. Antonioli, R. Colucci, C. Blandizzi, and M. Fornai, "Interplay Among Gut Microbiota, Intestinal Mucosal Barrier and Enteric Neuro-Immune System: A Common Path to Neurodegenerative Diseases?," *Acta Neuropathologica* 136, no. 3 (2018): 345-361, https://doi.org/10.1007/s00401-018-1856-5.

103. E. Gelpi, J. Navarro-Otano, E. Tolosa, et al., "Multiple Organ Involvement by Alpha-Synuclein Pathology in Lewy Body Disorders," *Movement Disorders* 29, no. 8 (2014): 1010-1018, https://doi.org/10.1002/mds.25776.

104. T. G. Beach, C. H. Adler, L. I. Sue, et al., "Multi-Organ Distribution of Phosphorylated α-Synuclein Histopathology in Subjects With Lewy Body Disorders," *Acta Neuropathologica* 119, no. 6 (2010): 689-702, https://doi.org/10.1007/s00401-010-0664-3.

105. B. A. Killinger, Z. Madaj, J. W. Sikora, et al., "The Vermiform Appendix Impacts the Risk of Developing Parkinson's Disease," *Science Translational Medicine* 10, no. 465 (2018): eaar5280, https://doi.org/10.1126/scitranslmed.aar5280.

106. A. Fenyi, L. Leclair-Visonneau, T. Clairembault, et al., "Detection of Alpha-Synuclein Aggregates in Gastrointestinal Biopsies by Protein Misfolding Cyclic Amplification," *Neurobiology of Disease* 129, no. April (2019): 38-43, https://doi.org/10.1016/j.nbd.2019.05.002.

107. C. Pellegrini, V. D'Antongiovanni, F. Miraglia, et al., "Enteric α-Synuclein Impairs Intestinal Epithelial Barrier Through Caspase-1-Inflammasome Signaling in Parkinson's Disease Before Brain Pathology," *npj Parkinson's Disease* 8, no. 1 (2022): 9, https://doi.org/10.1038/s41531-021-00263-x.

108. T. Clairembault, L. Leclair-Visonneau, E. Coron, et al., "Structural Alterations of the Intestinal Epithelial Barrier in Parkinson's Disease," *Acta Neuropathologica Communications* 3, no. 1 (2015): 12, https://doi.org/10.1186/s40478-015-0196-0.

109. M. Camilleri and V. Chedid, "Actionable Biomarkers: The Key to Resolving Disorders of Gastrointestinal Function," *Gut* 69, no. 10 (2020): 1730-1737, https://doi.org/10.1136/gutjnl-2019-320325.

110. C. Pellegrini, M. Fornai, V. D'Antongiovanni, L. Antonioli, N. Bernardini, and P. Derkinderen, "The Intestinal Barrier in Disorders of the Central Nervous System," *Lancet Gastroenterology & Hepatology* 8, no. 1 (2023): 66-80, https://doi.org/10.1016/S2468-1253(22)00241-2.

111. L. Rota, C. Pellegrini, L. Benvenuti, et al., "Constipation, Deficit in Colon Contractions and Alpha-Synuclein Inclusions Within the Colon Precede Motor Abnormalities and Neurodegeneration in the Central Nervous System in a Mouse Model of Alpha-Synucleinopathy," *Translational Neurodegeneration* 8, no. 1 (2019): 5, https://doi.org/10.1186/s40035-019-0146-z.

112. T. R. Sampson, J. W. Debelius, T. Thron, et al., "Gut Microbiota Regulate Motor Deficits and Neuroinflammation in a Model of Parkinson's Disease," *Cell* 167, no. 6 (2016): 1469-1480.e12, https://doi.org/10.1016/j.cell.2016.11.018.

113. E. Fitzgerald, S. Murphy, and H. A. Martinson, "Alpha-Synuclein Pathology and the Role of the Microbiota in Parkinson's Disease," *Frontiers in Neuroscience* 13, no. APR (2019): 369, https://doi.org/10.3389/fnins.2019.00369.

114. P. J. Kahle, M. Neumann, L. Ozmen, et al., "Subcellular Localization of Wild-Type and Parkinson's Disease-Associated Mutant α-Synuclein in Human and Transgenic Mouse Brain," *The Journal of Neuroscience* 20, no. 17 (2000): 6365-6373, https://doi.org/10.1523/JNEUROSCI.20-17-06365.2000.

115. P. J. Kahle, "α-Synucleinopathy Models and Human Neuropathology: Similarities and Differences," *Acta Neuropathologica* 115, no. 1 (2007): 87-95, https://doi.org/10.1007/s00401-007-0302-x.

116. F. Mori, K. Tanji, M. Yoshimoto, H. Takahashi, and K. Wakabayashi, "Demonstration of α-Synuclein Immunoreactivity in Neuronal and Glial Cytoplasm in Normal Human Brain Tissue Using Proteinase K and Formic Acid Pretreatment," *Experimental Neurology* 176, no. 1 (2002): 98-104, https://doi.org/10.1006/exnr.2002.7929.

117. D. Erskine, L. Patterson, A. Alexandris, et al., "Regional Levels of Physiological α-Synuclein Are Directly Associated With Lewy Body Pathology," *Acta Neuropathologica* 135, no. 1 (2018): 153-154, https://doi.org/10.1007/s00401-017-1787-6.

118. W. A. D. H. Jager and J. Bethlem, "The Distribution of Lewy Bodies in the Central and Autonomic Nervous Systems in Idiopathic Paralysis Agitans," *Journal of Neurology, Neurosurgery & Psychiatry* 23, no. 4 (1960): 283-290, https://doi.org/10.1136/jnnp.23.4.283.

119. E. Ohama and F. Ikuta, "Parkinson's Disease: Distribution of Lewy Bodies and Monoamine Neuron System," *Acta Neuropathologica* (1976): https://doi.org/10.1007/BF00696560.

120. K. Wakabayashi and H. Takahashi, "The Intermediolateral Nucleus and Clarke's Column in Parkinson's Disease," *Acta Neuropathologica* 94, no. 3 (1997): 287-289, https://doi.org/10.1007/s004010050705.

121. K. Oyanagi, K. Wakabayashi, E. Ohama, et al., "Lewy Bodies in the Lower Sacral Parasympathetic Neurons of a Patient With Parkinson's Disease," *Acta Neuropathologica* 80, no. 5 (1990): 558-559, https://doi.org/10.1007/BF00294619.

122. H. Braak, E. Braak, D. Yilmazer, et al., "Amygdala Pathology in Parkinson's Disease," *Acta Neuropathologica* 88, no. 6 (1994): 493-500, https://doi.org/10.1007/BF00296485.

123. K. Wakabayashi, L. A. Hansen, and E. Masliah, "Cortical Lewy Body-Containing Neurons Are Pyramidal Cells: Laser Confocal Imaging of Double-Immunolabeled Sections With Anti-Ubiquitin and SMI32," *Acta Neuropathologica* 89, no. 5 (1995): 404-408, https://doi.org/10.1007/BF00307643.
124. K. A. Jellinger, "Lewy Body-Related α-Synucleinopathy in the Aged Human Brain," *Journal of Neural Transmission* 111, no. 10-11 (2004): 1219-1235, https://doi.org/10.1007/s00702-004-0138-7.

125. W. Marui, E. Iseki, T. Nakai, et al., "Progression and Staging of Lewy Pathology in Brains From Patients With Dementia With Lewy Bodies," *Journal of the Neurological Sciences* 195, no. 2 (2002): 153-159, https://doi.org/10.1016/S0022-510X(02)00006-0.

126. T. G. Beach, C. H. Adler, L. Lue, et al., "Unified Staging System for Lewy Body Disorders: Correlation With Nigrostriatal Degeneration, Cognitive Impairment and Motor Dysfunction," *Acta Neuropathologica* 117, no. 6 (2009): 613-634, https://doi.org/10.1007/s00401-009-0538-8.

127. H. Apaydin, J. E. Ahlskog, J. E. Parisi, B. F. Boeve, and D. W. Dickson, "Parkinson Disease Neuropathology," *Archives of Neurology* 59, no. 1 (2002): 102, https://doi.org/10.1001/archneur.59.1.102.

128. E. Kövari, G. Gold, F. R. Herrmann, et al., "Lewy Body Densities in the Entorhinal and Anterior Cingulate Cortex Predict Cognitive Deficits in Parkinson's Disease," *Acta Neuropathologica* 106, no. 1 (2003): 83-88, https://doi.org/10.1007/s00401-003-0705-2.

129. P. M. Mattila, J. O. Rinne, H. Helenius, D. W. Dickson, M. Röyttä, and P. M. Mattila, "Alpha-Synuclein-Immunoreactive Cortical Lewy Bodies Are Associated With Cognitive Impairment in Parkinson's Disease," *Acta Neuropathologica* 100, no. 3 (2000): 285-290, https://doi.org/10.1007/s004019900168.

130. D. W. Dickson, H. Fujishiro, A. DelleDonne, et al., "Evidence That Incidental Lewy Body Disease Is Pre-Symptomatic Parkinson's Disease," *Acta Neuropathologica* 115, no. 4 (2008): 437-444, https://doi.org/10.1007/s00401-008-0345-7.

131. J. M. Milber, J. V. Noorigian, J. F. Morley, et al., "Lewy Pathology Is Not the First Sign of Degeneration in Vulnerable Neurons in Parkinson Disease," *Neurology* 79, no. 24 (2012): 2307-2314, https://doi.org/10.1212/WNL.0b013e318278fe32.

132. J. Zaccai, C. Brayne, I. McKeith, F. Matthews, and P. G. Ince, "Patterns and Stages of α-Synucleinopathy: Relevance in a Population-Based Cohort," *Neurology* 70, no. 13 (2008): 1042-1048, https://doi.org/10.1212/01.wnl.0000306697.48738.b6.

133. P. Borghammer, M. K. Just, J. Horsager, et al., "A Postmortem Study Suggests a Revision of the Dual-Hit Hypothesis of Parkinson's Disease," *npj Parkinson's Disease* 8, no. 1 (2022): 166, https://doi.org/10.1038/s41531-022-00436-2.

134. C. P. Lin, L. E. J. Knoop, I. Frigerio, et al., "Nigral Pathology Contributes to Microstructural Integrity of Striatal and Frontal Tracts in Parkinson's Disease," *Movement Disorders* 38, no. 9 (2023): 1655-1667, https://doi.org/10.1002/mds.29510.

135. J. E. Duda, B. I. Giasson, M. E. Mabon, et al., "Concurrence of α-Synuclein and Tau Brain Pathology in the Contursi Kindred," *Acta Neuropathologica* 104, no. 1 (2002): 7-11, https://doi.org/10.1007/s00401-002-0563-3.

136. K. Markopoulou, D. W. Dickson, R. D. McComb, et al., "Clinical, Neuropathological and Genotypic Variability in SNCA A53T Familial Parkinson's Disease," *Acta Neuropathologica* 116, no. 1 (2008): 25-35, https://doi.org/10.1007/s00401-008-0372-4.

137. K. Yamaguchi, E. J. Cochran, J. R. Murrell, et al., "Abundant Neuritic Inclusions and Microvacuolar Changes in a Case of Diffuse Lewy Body Disease With the A53T Mutation in the α-Synuclein Gene," *Acta Neuropathologica* 110, no. 3 (2005): 298-305, https://doi.org/10.1007/s00401-005-1042-4.

138. C. Colosimo, A. J. Hughes, L. Kilford, and A. J. Lees, "Lewy Body Cortical Involvement May Not Always Predict Dementia in Parkinson's Disease," *Journal of Neurology, Neurosurgery & Psychiatry* 74, no. 7 (2003): 852-856, https://doi.org/10.1136/jnnp.74.7.852.

139. E. Croisier, L. B. Moran, D. T. Dexter, R. K. B. Pearce, and M. B. Graeber, "Microglial Inflammation in the Parkinsonian Substantia

Nigra: Relationship to Alpha-Synuclein Deposition," *Journal of Neuroinflammation* 2 (2005): 1-8, https://doi.org/10.1186/1742-2094-2-14.

140. H. Braak, U. Rüb, and K. Del Tredici, "Cognitive Decline Correlates With Neuropathological Stage in Parkinson's Disease," *Journal of the Neurological Sciences* 248, no. 1-2 (2006): 255-258, https://doi.org/10.1016/j.jns.2006.05.011.

141. J. C. Dächsel, S. J. Lincoln, J. Gonzalez, O. A. Ross, D. W. Dickson, and M. J. Farrer, "The Ups and Downs of α-Synuclein mRNA Expression," *Movement Disorders* 22, no. 2 (2007): 293-295, https://doi.org/10.1002/mds.21223.

142. M. A. Hely, W. G. J. Reid, M. A. Adena, G. M. Halliday, and J. G. L. Morris, "The Sydney Multicenter Study of Parkinson's Disease: The Inevitability of Dementia at 20 Years," *Movement Disorders* 23, no. 6 (2008): 837-844, https://doi.org/10.1002/mds.21956.

143. J. G. Quinn, D. T. R. Coulson, S. Brockbank, et al., "α-Synuclein mRNA and Soluble α-Synuclein Protein Levels in Post-Mortem Brain From Patients With Parkinson's Disease, Dementia With Lewy Bodies, and Alzheimer's Disease," *Brain Research* 1459, no. January (2012): 71-80, https://doi.org/10.1016/j.brainres.2012.04.018.

144. Y. Saito, A. Shioya, T. Sano, H. Sumikura, M. Murata, and S. Murayama, "Lewy Body Pathology Involves the Olfactory Cells in Parkinson's Disease and Related Disorders," *Movement Disorders* 31, no. 1 (2016): 135-138, https://doi.org/10.1002/mds.26463.

145. K. Del Tredici and J. E. Duda, "Peripheral Lewy Body Pathology in Parkinson's Disease and Incidental Lewy Body Disease: Four Cases," *Journal of the Neurological Sciences* 310, no. 1-2 (2011): 100-106, https://doi.org/10.1016/j.jns.2011.06.003.

146. N. Niemann, A. Billnitzer, and J. Jankovic, "Parkinson's Disease and Skin," *Parkinsonism & Related Disorders* 82, no. November 2020 (2021): 61-76, https://doi.org/10.1016/j.parkreldis.2020.11.017.

147. V. Donadio, A. Incensi, G. Rizzo, et al., "Skin Biopsy May Help to Distinguish Multiple System Atrophy-Parkinsonism From Parkinson's Disease With Orthostatic Hypotension," *Movement Disorders* 35, no. 9 (2020): 1649-1657, https://doi.org/10.1002/mds.28126.

148. M. P. Giannoccaro, V. Donadio, G. Giannini, et al., "Comparison of 123I-MIBG Scintigraphy and Phosphorylated α-Synuclein Skin Deposits in Synucleinopathies," *Parkinsonism & Related Disorders* 81 (2020): 48-53, https://doi.org/10.1016/j.parkreldis.2020.10.016.

149. X. Liu, J. Yang, Y. Yuan, et al., "Optimization of the Detection Method for Phosphorylated α-Synuclein in Parkinson Disease by Skin Biopsy," *Frontiers in Neurology* 11, no. September (2020): 569446, https://doi.org/10.3389/fneur.2020.569446.

150. L. M. Chahine, T. G. Beach, M. C. Brumm, et al., "In Vivo Distribution of α-Synuclein in Multiple Tissues and Biofluids in Parkinson Disease," *Neurology* 95, no. 9 (2020): E1267-E1284, https://doi.org/10.1212/WNL.0000000000010404.

151. N. Wang, J. Garcia, R. Freeman, and C. H. Gibbons, "Phosphorylated Alpha-Synuclein Within Cutaneous Autonomic Nerves of Patients With Parkinson's Disease: The Implications of Sample Thickness on Results," *Journal of Histochemistry & Cytochemistry* 68, no. 10 (2020): 669-678, https://doi.org/10.1369/0022155420960250.

152. S. Manne, N. Kondru, H. Jin, et al., "Blinded RT-QuIC Analysis of α-Synuclein Biomarker in Skin Tissue From Parkinson's Disease Patients," *Movement Disorders* 35, no. 12 (2020): 2230-2239, https://doi.org/10.1002/mds.28242.

153. S. Mazzetti, M. J. Basellini, V. Ferri, et al., "α-Synuclein Oligomers in Skin Biopsy of Idiopathic and Monozygotic Twin Patients With Parkinson's Disease," *Brain* 143, no. 3 (2020): 920-931, https://doi.org/10.1093/brain/awaa008.

154. R. Infante, C. Scaglione, A. Incensi, G. Rizzo, R. Liguori, and V. Donadio, "A Longitudinal Skin Biopsy Study of Phosphorylated Alpha-Synuclein in a Patient With Parkinson Disease and Orthostatic
Hypotension," *Journal of Neuropathology and Experimental Neurology* 79, no. 7 (2020): 813-816, https://doi.org/10.1093/jnen/nlaa048.

155. Z. Wang, K. Becker, V. Donadio, et al., "Skin α-Synuclein Aggregation Seeding Activity as a Novel Biomarker for Parkinson Disease," *JAMA Neurology* 78, no. 1 (2021): 30-40, https://doi.org/10.1001/jamaneurol.2020.3311.

156. R. Isonaka, D. S. Goldstein, W. Zhu, et al., "α-Synuclein Deposition in Sympathetic Nerve Fibers in Genetic Forms of Parkinson's Disease," *Movement Disorders* 36, no. 10 (2021): 2346-2357, https://doi.org/10.1002/mds.28667.

157. M. P. Giannoccaro, P. Avoni, G. Rizzo, et al., "Presence of Skin α-Synuclein Deposits Discriminates Parkinson's Disease From Progressive Supranuclear Palsy and Corticobasal Syndrome," *Journal of Parkinson's Disease* 12, no. 2 (2022): 585-591, https://doi.org/10.3233/JPD-212904.

158. J. Lama, E. Salabasidou, J. Volkmann, A. Kuzkina, S. Duty, and N. Üçeyler, "Proinflammatory Profile in the Skin of Parkinson's Disease Patients With and Without Pain," *PLoS One* 17, no. 10 October (2022): e0276564, https://doi.org/10.1371/journal.pone.0276564.

159. M. Nolano, G. Caporaso, F. Manganelli, et al., "Phosphorylated α-Synuclein Deposits in Cutaneous Nerves of Early Parkinsonism," *Journal of Parkinson's Disease* 12, no. 8 (2022): 2453-2468, https://doi.org/10.3233/JPD-223421.

160. A. Kuzkina, C. Panzer, A. Seger, et al., "Dermal Real-Time Quaking-Induced Conversion Is a Sensitive Marker to Confirm Isolated Rapid Eye Movement Sleep Behavior Disorder as an Early α-Synucleinopathy," *Movement Disorders* 38, no. 6 (2023): 1077-1082, https://doi.org/10.1002/mds.29340.

161. A. Kuzkina, J. Rößle, A. Seger, et al., "Combining Skin and Olfactory α-Synuclein Seed Amplification Assays (SAA)—Towards Biomarker-Driven Phenotyping in Synucleinopathies," *NPJ Parkinson's Disease* 9, no. 1 (2023): 79, https://doi.org/10.1038/s41531-023-00519-8.

162. H. Braak, R. A. I. De Vos, J. Bohl, and K. Del Tredici, "Gastric α-Synuclein Immunoreactive Inclusions in Meissner's and Auerbach's Plexuses in Cases Staged for Parkinson's Disease-Related Brain Pathology," *Neuroscience Letters* 396, no. 1 (2006): 67-72, https://doi.org/10.1016/j.neulet.2005.11.012.

163. D. M. Annerino, S. Arshad, G. M. Taylor, C. H. Adler, T. G. Beach, and J. G. Greene, "Parkinson's Disease Is Not Associated With Gastrointestinal Myenteric Ganglion Neuron Loss," *Acta Neuropathologica* 124, no. 5 (2012): 665-680, https://doi.org/10.1007/s00401-012-1040-2.

164. A. Gold, Z. T. Turkalp, and D. G. Munoz, "Enteric Alpha-Synuclein Expression Is Increased in Parkinson's Disease But Not Alzheimer's Disease," *Movement Disorders* 28, no. 2 (2013): 237-241, https://doi.org/10.1002/mds.25298.

165. D. Devos, T. Lebouvier, B. Lardeux, et al., "Colonic Inflammation in Parkinson's Disease," *Neurobiology of Disease* 50, no. 1 (2013): 42-48, https://doi.org/10.1016/j.nbd.2012.09.007.

166. L. Mu, J. Chen, S. Sobotka, et al., "Alpha-Synuclein Pathology in Sensory Nerve Terminals of the Upper Aerodigestive Tract of Parkinson's Disease Patients," *Dysphagia* 30, no. 4 (2015): 404-417, https://doi.org/10.1007/s00455-015-9612-7.

167. N. P. Visanji, C. Marras, D. S. Kern, et al., "Colonic Mucosal α-Synuclein Lacks Specificity as a Biomarker for Parkinson Disease," *Neurology* 84, no. 6 (2015): 609-616, https://doi.org/10.1212/WNL.0000000000001240.

168. S. J. Chung, J. Kim, H. J. Lee, et al., "Alpha-Synuclein in Gastric and Colonic Mucosa in Parkinson's Disease: Limited Role as a Biomarker," *Movement Disorders* 31, no. 2 (2016): 241-249, https://doi.org/10.1002/mds.26473.

169. H. J. Lee, K. W. Jung, S. J. Chung, et al., "Relation of Enteric α-Synuclein to Gastrointestinal Dysfunction in Patients With Parkinson's Disease and in Neurologically Intact Subjects," *Journal of Neurogastroenterology and Motility* 24, no. 3 (2018): 469-478, https://doi.org/10.5056/jnm17141.

170. Z. Tanei, Y. Saito, S. Ito, et al., "Lewy Pathology of the Esophagus Correlates With the Progression of Lewy Body Disease: A Japanese Cohort Study of Autopsy Cases," *Acta Neuropathologica* 141, no. 1 (2021): 25-37, https://doi.org/10.1007/s00401-020-02233-8.

171. T. G. Beach, C. H. Adler, L. I. Sue, et al., "Vagus Nerve and Stomach Synucleinopathy in Parkinson's Disease, Incidental Lewy Body Disease, and Normal Elderly Subjects: Evidence Against the 'Body-First' Hypothesis," *Journal of Parkinson's Disease* 11, no. 4 (2021): 1833-1843, https://doi.org/10.3233/jpd-212733.

172. P. H. Lee, G. Lee, H. J. Park, O. Y. Bang, I. S. Joo, and K. Huh, "The Plasma Alpha-Synuclein Levels in Patients With Parkinson's Disease and Multiple System Atrophy," *Journal of Neural Transmission* 113, no. 10 (2006): 1435-1439, https://doi.org/10.1007/s00702-005-0427-9.

173. Q. X. Li, S. S. Mok, K. M. Laughton, et al., "Plasma α-Synuclein Is Decreased in Subjects With Parkinson's Disease," *Experimental Neurology* 204, no. 2 (2007): 583-588, https://doi.org/10.1016/j.expneurol.2006.12.006.

174. R. Duran, F. J. Barrero, B. Morales, J. D. Luna, M. Ramirez, and F. Vives, "Plasma α-Synuclein in Patients With Parkinson's Disease With and Without Treatment," *Movement Disorders* 25, no. 4 (2010): 489-493, https://doi.org/10.1002/mds.22928.

175. A. Gorostidi, A. Bergareche, J. Ruiz-Martinez, et al., "α-Synuclein Levels in Blood Plasma From LRRK2 Mutation Carriers," *PLoS One* 7, no. 12 (2012): 1-6, https://doi.org/10.1371/journal.pone.0052312.

176. G. Caranci, P. Piscopo, R. Rivabene, et al., "Gender Differences in Parkinson's Disease: Focus on Plasma Alpha-Synuclein," *Journal of Neural Transmission* 120, no. 8 (2013): 1209-1215, https://doi.org/10.1007/s00702-013-0972-6.

177. P. G. Foulds, P. Diggle, J. D. Mitchell, et al., "A Longitudinal Study on α-Synuclein in Blood Plasma as a Biomarker for Parkinson's Disease," *Scientific Reports* 3 (2013): 1-6, https://doi.org/10.1038/srep02540.

178. L. Wang, U. Das, D. A. Scott, Y. Tang, P. J. McLean, and S. Roy, "α-Synuclein Multimers Cluster Synaptic Vesicles and Attenuate Recycling," *Current Biology* 24, no. 19 (2014): 2319-2326, https://doi.org/10.1016/j.cub.2014.08.027.

179. M. Shi, C. Liu, T. J. Cook, et al., "Plasma Exosomal α-Synuclein Is Likely CNS-Derived and Increased in Parkinson's Disease," *Acta Neuropathologica* 128, no. 5 (2014): 639-650, https://doi.org/10.1007/s00401-014-1314-y.

180. R. Ishii, T. Tokuda, H. Tatebe, et al., "Decrease in Plasma Levels of α-Synuclein Is Evident in Patients With Parkinson's Disease After Elimination of Heterophilic Antibody Interference," *PLoS One* 10, no. 4 (2015): e01371, https://doi.org/10.1371/journal.pone.0123162.

181. C. H. Lin, S. Y. Yang, H. E. Horng, et al., "Plasma α-Synuclein Predicts Cognitive Decline in Parkinson's Disease," *Journal of Neurology, Neurosurgery & Psychiatry* 88, no. 10 (2017): 818-824, https://doi.org/10.1136/jnnp-2016-314857.

182. A. S. L. Ng, Y. J. Tan, Z. Lu, et al., "Plasma Alpha-Synuclein Detected by Single Molecule Array Is Increased in PD," *Annals of Clinical and Translational Neurology* 6, no. 3 (2019): 615-619, https://doi.org/10.1002/acn3.729.

183. A. Bougea and E. Kapaki, "Plasma Alpha-Synuclein Levels in Patients With Parkinson's Disease: A Systematic Review and Meta-Analysis," *Neurological Sciences* 40, no. 5 (2019): 929-938.

184. X. Si, J. Tian, Y. Chen, Y. Yan, J. Pu, and B. Zhang, "Central Nervous System-Derived Exosomal Alpha-Synuclein in Serum May Be a Biomarker in Parkinson's Disease," *Neuroscience* 413 (2019): 308-316, https://doi.org/10.1016/j.neuroscience.2019.05.015.
185. C. Jiang, F. Hopfner, A. Katsikoudi, et al., "Serum Neuronal Exosomes Predict and Differentiate Parkinson's Disease From Atypical Parkinsonism," *Journal of Neurology, Neurosurgery & Psychiatry* 91, no. 7 (2020): 720-729, https://doi.org/10.1136/jnnp-2019-322588.

186. C. W. Chang, S. Y. Yang, C. C. Yang, C. W. Chang, and Y. R. Wu, "Plasma and Serum Alpha-Synuclein as a Biomarker of Diagnosis in Patients With Parkinson's Disease," *Frontiers in Neurology* 10, no. January (2020): 1-7, https://doi.org/10.3389/fneur.2019.01388.

187. K. H. Shim, S. C. Kim, Y. C. Youn, Y. H. Sung, and S. S. A. An, "Decreased Plasma α-Synuclein in Idiopathic Parkinson's Disease Patients After Adjusting Hemolysis Factor," *Molecular & Cellular Toxicology* 16, no. 4 (2020): 477-484, https://doi.org/10.1007/s13273-020-00104-7.

188. H. Zhao, S. Huang, S. Palanisamy, C. Wang, G. Rainer, and X. Zhang, "Alpha-Synuclein Dopaminylation Presented in Plasma of Both Healthy Subjects and Parkinson's Disease Patients," *PROTEOMICS – Clinical Application* 14, no. 5 (2020): https://doi.org/10.1002/prca.201900117.

189. N. C. Chen, H. L. Chen, S. H. Li, et al., "Plasma Levels of α-Synuclein, Aβ-40 and T-Tau as Biomarkers to Predict Cognitive Impairment in Parkinson's Disease," *Frontiers in Aging Neuroscience* 12, no. April (2020): 1-8, https://doi.org/10.3389/fnagi.2020.000112.

190. Z. Fan, Y. T. Pan, Z. Y. Zhang, et al., "Systemic Activation of NLRP3 Inflammasome and Plasma α-Synuclein Levels Are Correlated With Motor Severity and Progression in Parkinson's Disease," *Journal of Neuroinflammation* 17, no. 1 (2020): 1-10, https://doi.org/10.1186/s12974-019-1670-6.

191. M. Niu, Y. Li, G. Li, et al., "A Longitudinal Study on α-Synuclein in Plasma Neuronal Exosomes as a Biomarker for Parkinson's Disease Development and Progression," *European Journal of Neurology* 27, no. 6 (2020): 967-974, https://doi.org/10.1111/ene.14208.

192. J. Zou, Y. Guo, L. Wei, F. Yu, B. Yu, and A. Xu, "Long Noncoding RNA POU3F3 and α-Synuclein in Plasma L1CAM Exosomes Combined With β-Glucocerebrosidase Activity: Potential Predictors of Parkinson's Disease," *Neurotherapeutics* 17, no. 3 (2020): 1104-1119, https://doi.org/10.1007/s13311-020-00842-5.

193. C. C. Chung, L. Chan, J. H. Chen, Y. C. Hung, and C. T. Hong, "Plasma Extracellular Vesicle α-Synuclein Level in Patients With Parkinson's Disease," *Biomolecules* 11, no. 5 (2021): 1-10, https://doi.org/10.3390/biom11050744.

194. P. Youssef, W. S. Kim, G. M. Halliday, S. J. G. Lewis, and N. Dzamko, "Comparison of Different Platform Immunoassays for the Measurement of Plasma Alpha-Synuclein in Parkinson's Disease Patients," *Journal of Parkinson's Disease* 11, no. 4 (2021): 1761-1772, https://doi.org/10.3233/jpd-212694.

195. C. Agliardi, M. Meloni, F. R. Guerini, et al., "Oligomeric α-Syn and Snare Complex Proteins in Peripheral Extracellular Vesicles of Neural Origin Are Biomarkers for Parkinson's Disease," *Neurobiology of Disease* 148 (2021): 105185, https://doi.org/10.1016/j.nbd.2020.105185.

196. S. Dutta, S. Hornung, A. Kruayatidee, et al., "α-Synuclein in Blood Exosomes Immunoprecipitated Using Neuronal and Oligodendroglial Markers Distinguishes Parkinson's Disease From Multiple System Atrophy," *Acta Neuropathologica* 142, no. 3 (2021): 495-511, https://doi.org/10.1007/s00401-021-02324-0.

197. H. Zheng, Z. Xie, X. Zhang, et al., "Investigation of α-Synuclein Species in Plasma Exosomes and the Oligomeric and Phosphorylated α-Synuclein as Potential Peripheral Biomarker of Parkinson's Disease," *Neuroscience* 469 (2021): 79-90, https://doi.org/10.1016/j.neuroscience.2021.06.033.

198. A. Okuzumi, T. Hatano, G. Matsumoto, et al., "Propagative α-Synuclein Seeds as Serum Biomarkers for Synucleinopathies," *Nature Medicine* 29, no. 6 (2023): 1448-1455, https://doi.org/10.1038/s41591-023-02358-9.

199. V. Donadio, A. Incensi, V. Vacchiano, R. Infante, M. Magnani, and R. Liguori, "The Autonomic Innervation of Hairy Skin in Humans: An In Vivo Confocal Study," *Scientific Reports* 9, no. 1 (2019): 16982, https://doi.org/10.1038/s41598-019-53684-3.

200. S. Mazzetti, M. J. Basellini, V. Ferri, et al., "α-Synuclein Oligomers in Skin Biopsy of Idiopathic and Monozygotic Twin Patients With Parkinson's Disease," *Brain* 143, no. 3 (2020): 920-931, https://doi.org/10.1093/brain/awaa008.

201. M. Ikemura, Y. Saito, R. Sengoku, et al., "Lewy Body Pathology Involves Cutaneous Nerves," *Journal of Neuropathology & Experimental Neurology* 67, no. 10 (2008): 945-953, https://doi.org/10.1097/NEN.0b013e318186de48.

202. V. Donadio, A. Incensi, V. Leta, et al., "Skin Nerve-Synuclein Deposits: A Biomarker for Idiopathic Parkinson Disease," *Neurology* 82, no. 15 (2014): 1362-1369, https://doi.org/10.1212/WNL.0000000000000316.

203. K. Doppler, S. Ebert, N. Üçeyler, et al., "Cutaneous Neuropathy in Parkinson's Disease: A Window Into Brain Pathology," *Acta Neuropathologica* 128, no. 1 (2014): 99-109, https://doi.org/10.1007/s00401-014-1284-0.

204. V. Donadio, A. Incensi, G. Rizzo, et al., "Spine Topographical Distribution of Skin α-Synuclein Deposits in Idiopathic Parkinson Disease," *Journal of Neuropathology & Experimental Neurology* 76, no. 5 (2017): 384-389, https://doi.org/10.1093/jnen/nlx021.

205. K. Doppler, H. M. Jentschke, L. Schulmeyer, et al., "Dermal Phospho-Alpha-Synuclein Deposits Confirm REM Sleep Behaviour Disorder as Prodromal Parkinson's Disease," *Acta Neuropathologica* 133, no. 4 (2017): 535-545, https://doi.org/10.1007/s00401-017-1684-z.

206. M. Rossi, N. Candelise, S. Baiardi, et al., "Ultrasensitive RT-QuIC Assay With High Sensitivity and Specificity for Lewy Body-Associated Synucleinopathies," *Acta Neuropathologica* 140, no. 1 (2020): 49-62, https://doi.org/10.1007/s00401-020-02160-8.

207. S. J. Qualman, H. M. Haupt, P. Yang, and S. R. Hamilton, "Eosophageal Lewy Bodies Associated With Ganglion Cell Loss in Achalasia. Similarity to Parkinson's Disease," *Gastroenterology* 87, no. 4 (1984): 848-856, http://www.ncbi.nlm.nih.gov/pubmed/6088351.

208. W. J. Kupsky, M. M. Grimes, J. Sweeting, R. Bertsch, and L. J. Cote, "Parkinson's Disease and Megacolon: Concentric Hyaline Inclusions (Lewy Bodies) in Enteric Ganglion Cells," *Neurology* 37, no. 7 (1987): 1253-1253, https://doi.org/10.1212/WNL.37.7.1253.

209. K. Wakabayashi, H. Takahashi, S. Takeda, E. Ohama, and F. Ikuta, "Parkinson's Disease: The Presence of Lewy Bodies in Auerbach's and Meissner's Plexuses," *Acta Neuropathologica* 76, no. 3 (1988): 217-221, https://doi.org/10.1007/BF00687767.

210. A. G. Corbillé, F. Letournel, J. H. Kordower, et al., "Evaluation of Alpha-Synuclein Immunohistochemical Methods for the Detection of Lewy-Type Synucleinopathy in Gastrointestinal Biopsies," *Acta Neuropathologica Communications* 4 (2016): 35, https://doi.org/10.1186/s40478-016-0305-8.

211. S. A. Schneider, M. Boettner, A. Alexoudi, D. Zorenkov, G. Deuschl, and T. Wedel, "Can We Use Peripheral Tissue Biopsies to Diagnose Parkinson's Disease? A Review of the Literature," *European Journal of Neurology* 23, no. 2 (2016): 247-261, https://doi.org/10.1111/ene.12753.

212. T. Lebouvier, M. Neunlist, S. Bruley des Varannes, et al., "Colonic Biopsies to Assess the Neuropathology of Parkinson's Disease and Its Relationship With Symptoms," *PLoS One* 5, no. 9 (2010): e12728, https://doi.org/10.1371/journal.pone.0012728.

213. H. Pouclet, T. Lebouvier, E. Coron, et al., "A Comparison Between Rectal and Colonic Biopsies to Detect Lewy Pathology in Parkinson's Disease," *Neurobiology of Disease* 45, no. 1 (2012): 305-309, https://doi.org/10.1016/j.nbd.2011.08.014.

214. R. Barbour, K. Kling, J. P. Anderson, et al., "Red Blood Cells Are the Major Source of Alpha-Synuclein in Blood," *Neurodegenerative Diseases* 5, no. 2 (2008): 55-59, https://doi.org/10.1159/000112832.

215. C. Agliardi, F. Guerini, M. Meloni, and M. Clerici, "Alpha-Synuclein as a Biomarker in Parkinson's Disease: Focus on Neural Derived
Extracelluar Vesicles,” *Neural Regeneration Research* 17, no. 7 (2022): 1503, https://doi.org/10.4103/1673-5374.330604.

216. R. Manek, A. Moghieb, Z. Yang, et al., “Protein Biomarkers and Neuroproteomics Characterization of Microvesicles/Exosomes From Human Cerebrospinal Fluid Following Traumatic Brain Injury,” *Molecular Neurobiology* 55, no. 7 (2018): 6112–6128, https://doi.org/10.1007/s12035-017-0821-y.

217. X. Si, J. Tian, Y. Chen, Y. Yan, J. Pu, and B. Zhang, “Central Nervous System-Derived Exosomal Alpha-Synuclein in Serum May Be a Biomarker in Parkinson's Disease,” *Neuroscience* 413 (2019): 308–316, https://doi.org/10.1016/j.neuroscience.2019.05.015.

218. D. Alarcón-Arís, A. Recasens, M. Galfré, et al., “Selective α-Synuclein Knockdown in Monoamine Neurons by Intranasal Oligonucleotide Delivery: Potential Therapy for Parkinson's Disease,” *Molecular Therapy* 26, no. 2 (2018): 550–567, https://doi.org/10.1016/J.YMTHE.2017.11.015.

219. M. K. Sapru, J. W. Yates, S. Hogan, L. Jiang, J. Halter, and M. C. Bohn, “Silencing of Human Alpha-Synuclein In Vitro and in Rat Brain Using Lentiviral-Mediated RNAi,” *Experimental Neurology* 198, no. 2 (2006): 382–390, https://doi.org/10.1016/J.EXPNEUROL.2005.12.024.

220. J. Lewis, H. Melrose, D. Bumcrot, et al., “In Vivo Silencing of Alpha-Synuclein Using Naked siRNA,” *Molecular Neurodegeneration* 3, no. 1 (2008): https://doi.org/10.1186/1750-1326-3-19.

221. A. L. McCormack, S. K. Mak, J. M. Henderson, D. Bumcrot, M. J. Farrer, and D. A. Di Monte, “α-Synuclein Suppression by Targeted Small Interfering RNA in the Primate Substantia Nigra,” *PLoS One* 5, no. 8 (2010): e12122, https://doi.org/10.1371/journal.pone.0012122.

222. O. S. Gorbatyuk, S. Li, K. Nash, et al., “In Vivo RNAi-Mediated Alpha-Synuclein Silencing Induces Nigrostriatal Degeneration,” *Molecular Therapy* 18, no. 8 (2010): 1450–1457, https://doi.org/10.1038/MT.2010.115.

223. C. Lo Bianco, J. Shorter, E. Régulier, et al., “Hsp104 Antagonizes Alpha-Synuclein Aggregation and Reduces Dopaminergic Degeneration in a Rat Model of Parkinson Disease,” *Journal of Clinical Investigation* 118, no. 9 (2008): 3087–3097, https://doi.org/10.1172/JCI35781.

224. T. C. Moloney, R. Hyland, D. O'Toole, et al., “Heat Shock Protein 70 Reduces α-Synuclein-Induced Predegenerative Neuronal Dystrophy in the α-Synuclein Viral Gene Transfer Rat Model of Parkinson's Disease,” *CNS Neuroscience & Therapeutics* 20, no. 1 (2014): 50–58, https://doi.org/10.1111/cns.12200.

225. J. Klucken, Y. Shin, E. Masliah, B. T. Hyman, and P. J. McLean, “Hsp70 Reduces Alpha-Synuclein Aggregation and Toxicity,” *Journal of Biological Chemistry* 279, no. 24 (2004): 25497–25502, https://doi.org/10.1074/JBC.M400255200.

226. J. Wagner, S. Ryazanov, A. Leonov, et al., “Anle138b: A Novel Oligomer Modulator for Disease-Modifying Therapy of Neurodegenerative Diseases Such as Prion and Parkinson's Disease,” *Acta Neuropathologica* 125, no. 6 (2013): 795–813, https://doi.org/10.1007/S00401-013-1114-9.

227. P. Brundin, K. D. Dave, and J. H. Kordower, “Therapeutic Approaches to Target Alpha-Synuclein Pathology,” *Experimental Neurology* 298 (2017): 225–235, https://doi.org/10.1016/j.expneurol.2017.10.003.

228. D. Games, E. Valera, B. Spencer, et al., “Reducing C-Terminal-Truncated Alpha-Synuclein by Immunotherapy Attenuates Neurodegeneration and Propagation in Parkinson's Disease-Like Models,” *Journal of Neuroscience* 34, no. 28 (2014): 9441–9454, https://doi.org/10.1523/JNEUROSCI.5314-13.2014.

229. D. J. Schofield, L. Irving, L. Calo, et al., “Preclinical Development of a High Affinity α-Synuclein Antibody, MEDI1341, That Can Enter the Brain, Sequester Extracellular α-Synuclein and Attenuate α-Synuclein Spreading In Vivo,” *Neurobiology of Disease* 132 (2019): 104582, https://doi.org/10.1016/j.nbd.2019.104582.

230. E. Nordström, F. Eriksson, J. Sigvardson, et al., “ABBV-0805, a Novel Antibody Selective for Soluble Aggregated α-Synuclein, Prolongs

Lifespan and Prevents Buildup of α-Synuclein Pathology in Mouse Models of Parkinson's Disease,” *Neurobiology of Disease* 161 (2021): 105543, https://doi.org/10.1016/j.nbd.2021.105543.

231. A. Migdalska-Richards and A. H. V. Schapira, “The Relationship Between Glucocerebrosidase Mutations and Parkinson Disease,” *Journal of Neurochemistry* 139, no. Suppl 1 (2016): 77–90, https://doi.org/10.1111/JNC.13385.

232. S. Crunkhorn, “Suppressing c-Abl in Parkinson Disease,” *Nature Reviews Drug Discovery* 22, no. 3 (2023): 183, https://doi.org/10.1038/D41573-023-00024-W.

233. M. L. Hebron, I. Lonskaya, and C. E. H. Moussa, “Nilotinib Reverses Loss of Dopamine Neurons and Improves Motor Behavior via Autophagic Degradation of α-Synuclein in Parkinson's Disease Models,” *Human Molecular Genetics* 22, no. 16 (2013): 3315–3328, https://doi.org/10.1093/hmg/ddt192.

234. A. Ghosh, T. Tyson, S. George, et al., “Mitochondrial Pyruvate Carrier Regulates Autophagy, Inflammation, and Neurodegeneration in Experimental Models of Parkinson's Disease,” *Science Translational Medicine* 8, no. 368 (2016): https://doi.org/10.1126/SCITRANSLMED.AAG2210.

235. G. Pagano, K. I. Taylor, J. Anzures-Cabrera, et al., “Trial of Prasinezumab in Early-Stage Parkinson's Disease,” *New England Journal of Medicine* 387, no. 5 (2022): 421–432, https://doi.org/10.1056/NEJMoa2202867.

236. “Correction to: A De Novo Compound Targeting α-Synuclein Improves Deficits in Models of Parkinson's Disease,” *Brain* 146, no. 10 (2023): e92–e93, https://doi.org/10.1093/BRAIN/AWAD207.

237. D. L. Price, M. A. Koike, A. Khan, et al., “The Small Molecule Alpha-Synuclein Misfolding Inhibitor, NPT200-11, Produces Multiple Benefits in an Animal Model of Parkinson's Disease,” *Scientific Reports* 8, no. 1 (2018): 16165, https://doi.org/10.1038/s41598-018-34490-9.

238. E. Masliah, E. Rockenstein, M. Mante, et al., “Passive Immunization Reduces Behavioral and Neuropathological Deficits in an Alpha-Synuclein Transgenic Model of Lewy Body Disease,” *PLoS One* 6, no. 4 (2011): e19338, https://doi.org/10.1371/journal.pone.0019338.

239. E. J. Bae, D. H. Ho, E. Park, et al., “Lipid Peroxidation Product 4-Hydroxy-2-Nonenal Promotes Seeding-Capable Oligomer Formation and Cell-to-Cell Transfer of α-Synuclein,” *Antioxid Redox Signal* 18, no. 7 (2013): 770–783, https://doi.org/10.1089/ARS.2011.4429.

240. V. Sanchez-Guajardo, A. Annibali, P. H. Jensen, and M. Romero-Ramos, “α-Synuclein Vaccination Prevents the Accumulation of Parkinson Disease-Like Pathologic Inclusions in Striatum in Association With Regulatory T Cell Recruitment in a Rat Model,” *Journal of Neuropathology & Experimental Neurology* 72, no. 7 (2013): 624–645, https://doi.org/10.1097/NEN.0B013E31829768D2.

241. A. Migdalska-Richards, L. Daly, E. Bezard, and A. H. V. Schapira, “Ambroxol Effects in Glucocerebrosidase and α-Synuclein Transgenic Mice,” *Annals of Neurology* 80, no. 5 (2016): 766–775, https://doi.org/10.1002/ANA.24790.

242. “Correction to: Nilotinib Reverses Loss of Dopamine Neurons and Improves Motor Behavior via Autophagic Degradation of α-Synuclein in Parkinson's Disease Models,” *Human Molecular Genetics* 32, no. 1 (2023): 172–176, https://doi.org/10.1093/HMG/DDAC274.

243. A. E. Lang, A. D. Siderowf, E. A. Macklin, et al., “Trial of Cinnapemab in Early Parkinson's Disease,” *New England Journal of Medicine* 387, no. 5 (2022): 408–420, https://doi.org/10.1056/NEJMoa2203395.

244. M. Brys, L. Fanning, S. Hung, et al., “Randomized Phase I Clinical Trial of Anti-α-Synuclein Antibody BIIB054,” *Movement Disorders* 34, no. 8 (2019): 1154–1163, https://doi.org/10.1002/mds.27738.

245. H. Kalluri, C. Zadikoff, L. Rueter, O. Graff, and H. X. Randomized, “Placebo-Controlled Single Ascending Dose Study to Evaluate the Safety, Tolerability, Pharmacokinetics, and Immunogenicity of Abbv-
0805, an Anti-Alpha-Synuclein Monoclonal Antibody in Healthy Subjects," *Movement Disorders* no. Suppl 1 (2021).

246. C. Shering, M. Pomfret, R. Kubiak, et al. "Reducing α-Synuclein in Human CSF; an Evaluation of Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of MEDI1341, an α-Synuclein-Specific Antibody, in Healthy Volunteers and Parkinson's Disease Patients (P1-11.007)," *Neurology* 100, no. 17 Supplement 2 (2023): 2469, https://doi.org/10.1212/WNL.00000000000202579.

247. L. Buur, J. Wiedemann, F. Larsen, et al., "The Anti-Alpha-Synuclein Antibody Lu AF82422 Was Safe and Well Tolerated in a FIH-SAD Study in Healthy Subjects and Patients With Pd," *Movement Disorders* 37, no. suppl 2 (2022), https://www.mdsabstracts.org/abstract/the-anti-alpha-synuclein-antibody-lu-af82422-was-safe-and-well-tolerated-in-a-fih-sad-study-in-healthy-subjects-and-patients-with-pd/.

248. M. Mandler, E. Valera, E. Rockenstein, et al., "Next-Generation Active Immunization Approach for Synucleinopathies: Implications for Parkinson's Disease Clinical Trials," *Acta Neuropathologica* 127, no. 6 (2014): 861-879, https://doi.org/10.1007/s00401-014-1256-4.

249. A. Schneeberger, L. Tierney, and M. Mandler, "Active Immunization Therapies for Parkinson's Disease and Multiple System Atrophy," *Movement Disorders* 31, no. 2 (2016): 214-224, https://doi.org/10.1002/mds.26377.

250. H. J. Yu, E. Thijssen, E. van Brummelen, et al., "A Randomized First-in-Human Study With UB-312, a UBITh® α-Synuclein Peptide Vaccine," *Movement Disorders* 37, no. 7 (2022): 1416-1424, https://doi.org/10.1002/mds.29016.

251. J. T. Nimmo, A. Verma, J. C. Dodart, et al., "Novel Antibodies Detect Additional α-Synuclein Pathology in Synucleinopathies: Potential Development for Immunotherapy," *Alzheimer's Research & Therapy* 12, no. 1 (2020): 159, https://doi.org/10.1186/s13195-020-00727-x.

252. D. Volc, W. Poewe, A. Kutzelnigg, et al., "Safety and Immunogenicity of the α-Synuclein Active Immunotherapeutic PD01A in Patients With Parkinson's Disease: A Randomised, Single-Blinded, Phase 1 Trial," *Lancet Neurology* 19, no. 7 (2020): 591-600, https://doi.org/10.1016/S1474-4422(20)30136-8.

253. W. Poewe, D. Volc, K. Seppi, et al., "Safety and Tolerability of Active Immunotherapy Targeting α-Synuclein With PD03A in Patients With Early Parkinson's Disease: A Randomized, Placebo-Controlled, Phase 1 Study," *Journal of Parkinson's Disease* 11, no. 3 (2021): 1079-1089, https://doi.org/10.3233/JPD-212594.

254. W. G. Meissner, A. P. Traon, A. Foubert-Samier, et al., "A Phase 1 Randomized Trial of Specific Active α-Synuclein Immunotherapies PD01A and PD03A in Multiple System Atrophy," *Movement Disorders* 35, no. 11 (2020): 1957-1965, https://doi.org/10.1002/mds.28218.

255. M. D. Pérez-Carrión, I. Posadas, J. Solera, and V. Ceña, "LRRK2 and Proteostasis in Parkinson's Disease," *International Journal of Molecular Sciences* 23, no. 12 (2022): 6808, https://doi.org/10.3390/ijms23126808.

256. N. M. Kanaan and F. P. Manfredsson, "Loss of Functional Alpha-Synuclein: A Toxic Event in Parkinson's Disease?," *Journal of Parkinson's Disease* 2, no. 4 (2012): 249-267, https://doi.org/10.3233/JPD-012138.

257. N. Gronich, D. R. Abernethy, E. Auriel, I. Lavi, G. Rennert, and W. Saliba, "β2-Adrenoceptor Agonists and Antagonists and Risk of Parkinson's Disease," *Movement Disorders* 33, no. 9 (2018): 1465-1471, https://doi.org/10.1002/mds.108.

258. S. Searles Nielsen, A. Gross, A. Camacho-Soto, A. W. Willis, and B. A. Racette, "β2-Adrenoreceptor Medications and Risk of Parkinson Disease," *Annals of Neurology* 84, no. 5 (2018): 683-693, https://doi.org/10.1002/ana.25341.

259. F. Hopfner, M. Wod, G. U. Höglinger, et al., "Use of β2-Adrenoceptor Agonist and Antagonist Drugs and Risk of Parkinson Disease," *Neurology* 93, no. 2 (2019): e135-e142, https://doi.org/10.1212/WNL.0000000000007694.

260. B. Liu, P. Svenningsson, J. F. Ludvigsson, et al., "β2-Adrenoceptor Agonists, Montelukast, and Parkinson Disease Risk," *Annals of Neurology* 93, no. 5 (2023): 1023-1028, https://doi.org/10.1002/ana.26638.

261. S. Mittal, K. Bjornevik, D. S. Im, et al., "β2-Adrenoreceptor Is a Regulator of the α-Synuclein Gene Driving Risk of Parkinson's Disease," *Science* 357, no. 6354 (2017): 891-898, https://doi.org/10.1126/science.aaf3934.

262. J. W. Smit, P. Basile, M. K. Prato, et al., "Phase 1/1B Studies of UCB0599, an Oral Inhibitor of α-Synuclein Misfolding, Including a Randomized Study in Parkinson's Disease," *Movement Disorders* 37, no. 10 (2022): 2045-2056, https://doi.org/10.1002/mds.29170.

263. J. Levin, N. Sing, S. Melbourne, et al., "Safety, Tolerability and Pharmacokinetics of the Oligomer Modulator anle138b With Exposure Levels Sufficient for Therapeutic Efficacy in a Murine Parkinson Model: A Randomised, Double-Blind, Placebo-Controlled Phase 1A Trial," *eBioMedicine* 80 (2022): 104021, https://doi.org/10.1016/j.ebiom.2022.104021.

264. M. Madureira, N. Connor-Robson, and R. Wade-Martins, "LRRK2: Autophagy and Lysosomal Activity," *Frontiers in Neuroscience* 14 (2020), https://doi.org/10.3389/fnins.2020.00498.

265. D. Jennings, S. Huntwork-Rodriguez, A. G. Henry, et al., "Preclinical and Clinical Evaluation of the LRRK2 Inhibitor DNL201 for Parkinson's Disease," *Science Translational Medicine* 14, no. 648 (2022): https://doi.org/10.1126/scitranslmed.abj2658.

266. M. R. Karim, E. E. Liao, J. Kim, et al., "α-Synucleinopathy Associated c-Abl Activation Causes P53-Dependent Autophagy Impairment," *Molecular Neurodegeneration* 15, no. 1 (2020): 27, https://doi.org/10.1186/s13024-020-00364-w.

267. A. J. Fowler, M. Hebron, A. A. Missner, et al., "Multikinase Abl/ DDR/Src Inhibition Produces Optimal Effects for Tyrosine Kinase Inhibition in Neurodegeneration," *Drugs in R&D* 19, no. 2 (2019): 149-166, https://doi.org/10.1007/s40268-019-0266-z.

268. F. Pagan, M. Hebron, E. H. Valadez, et al., "Nilotinib Effects in Parkinson's Disease and Dementia With Lewy Bodies," *Journal of Parkinson's Disease* 6, no. 3 (2016): 503-517, https://doi.org/10.3233/JPD-160867.

269. F. L. Pagan, M. L. Hebron, B. Wilmarth, et al., "Nilotinib Effects on Safety, Tolerability, and Potential Biomarkers in Parkinson Disease," *JAMA Neurology* 77, no. 3 (2020): 309, https://doi.org/10.1001/jamaneurol.2019.4200.

270. T. Simuni, B. Fiske, K. Merchant, et al., "Efficacy of Nilotinib in Patients With Moderately Advanced Parkinson Disease," *JAMA Neurology* 78, no. 3 (2021): 312, https://doi.org/10.1001/jamaneurol.2020.4725.

271. A. S. Harms, V. Delic, A. D. Thome, et al., "α-Synuclein Fibrils Recruit Peripheral Immune Cells in the Rat Brain Prior to Neurodegeneration," *Acta Neuropathologica Communications* 5, no. 1 (2017): 85, https://doi.org/10.1186/S40478-017-0494.