Alpha-Synuclein Strain Variability in Body-First and Brain-First Synucleinopathies

Abstract

Pathogenic alpha-synuclein (asyn) aggregates are a defining feature of neurodegenerative synucleinopathies, which include Parkinson's disease, Lewy body dementia, pure autonomic failure and multiple system atrophy. Early accurate differentiation between these synucleinopathies is challenging due to the highly heterogeneous clinical profile at early prodromal disease stages. Therefore, diagnosis is often made in late disease stages when a patient presents with a broad range of motor and non-motor symptoms easing the differentiation. Increasing data suggest the clinical heterogeneity seen in patients is explained by the presence of distinct asyn strains, which exhibit variable morphologies and pathological functions. Recently, asyn seed amplification assays (PMCA and RT-QuIC) and conformation-specific ligand assays have made promising progress in differentiating between synucleinopathies in prodromal and advanced disease stages. Importantly, the cellular environment is known to impact strain morphology. And, asyn aggregate pathology can propagate trans-synaptically along the brain-body axis, affecting multiple organs and propagating through multiple cell types. Here, we present our hypothesis that the changing cellular environments, an asyn seed may encounter during its brain-to-body or body-to-brain propagation, may influence the structure and thereby the function of the aggregate strains developing within the different cells. Additionally, we aim to review strain characteristics of the different synucleinopathies in clinical and preclinical studies. Future preclinical animal models of synucleinopathies should investigate if asyn strain morphology is altered during brain-to-body and body-to-brain spreading using these seeding amplification and conformation-specific assays. Such findings would greatly deepen our understanding of synucleinopathies and the potential link between strain and phenotypic variability, which may enable specific diagnosis of different synucleinopathies in the prodromal phase, creating a large therapeutic window with potential future applications in clinical trials and personalized therapeutics.

Keywords: Lewy body disorders; animal models; oligothiophene ligands; peripheral biomarkers; seed amplification assay; synucleinopathies.

Figure 1

Asyn strain variability in the LBD asyn origin and connectome (SOC) model. Upper panel: Schematic representation of the strain hypothesis in LBD as described by the SOC model. Physiological unfolded monomeric protein can misfold into pathogenic β-sheet-rich subunit conformations that are capable of seeding other pathogenic subunit conformers or monomers, ultimately elongating into mature fibrillary asyn. Monomeric asyn can adopt various β-sheet-rich conformations depending on the cellular milieu where the misfolding occurs. Therefore, distinct fibrillary asyn conformers or 'strains', composed of different subunits, can arise depending on the cellular environment, determined by the neuron-type and its location (body or brain). Each distinct asyn strain may affect different parts of the brain and autonomic connectome in varying degrees, contributing to the heterogenous clinical representation of body-first and brain-first LBDs. Lower panel: Schematic representation of two LBD-subtypes as predicted by the SOC model. Numbers indicate progression of pathology from initiation (1) to more advanced disease stages (4–6). In body-first LBD, asyn arises in the ANS (usually the gut) from where it spreads bilaterally via overlapping vagal (full line) and sympathetic (dashed line) connections to the brain and to other peripheral organs causing autonomic dysfunction prior to dopaminergic deficit. Bilateral invasion of pathology in the DMV causes subsequent bilateral involvement of the LC causing RBD, and bilateral nigrostriatal neurodegeneration with symmetric parkinsonism as disease further progresses. In brain-first LBD, asyn arises unilaterally in the amygdala or olfactory bulb, after which it spreads to the unilateral SN causing unilateral neurodegeneration and asymmetric parkinsonism. Upon further progression to the LC, RBD may occur post motor deficit. Inevitably, pathology spreads to peripheral organs, causing autonomic dysfunction at advanced disease stages. Due to the low level of homotypic connections in the brain, initial predominant unilateral pathology and associated neurodegeneration is initially confined to a single hemisphere for some time in brain-first synucleinopathy. In contrast, bilateral invasion of pathology in the brain of body-first cases causes a more severe disease progression, including more severe hyposmia and cognitive decline. Body-first and brain-first LBD mainly predict disease progression in PD patients with and without pre-motor RBD. Furthermore, most DLB and all PAF patients can be categorized as having body-first LBD. Although cellular vulnerability and the presence of concomitant AD pathology may influence the spatial distribution of pathology and clinical profile in these LBDs compared to typical body-first PD. Abbreviations: SN, substantia nigra; LC, locus coeruleus; DMV, dorsal motor nucleus of the vagus; RBD, REM-sleep behavior disorder; DLB, dementia with Lewy bodies (LBs); PAF, pure autonomic failure (Created with Biorender).

Figure 2

Alpha-synuclein strains may change morphology during trans-synaptic spread along the body-brain axis, resulting in a mix of different conformers in body-first and brain-first LBDs. Monomeric asyn can adopt different β-sheet-rich conformations depending on which conformation is favored by the cellular milieu, which is defined by a certain cell-type and organ where the seeding takes place. The changing cellular environment during trans-synaptic spread along the body-brain axis may give rise to the formation of different asyn conformers throughout the connectome, and even within a single organ. Thus, despite strain characteristics appear to be linked to a certain LBD-subtype, heterogeneity within a subtype may be caused by different dominating strains in their “strain cloud”. The numbered squares indicate the stages of pathology progression throughout the ANS in body-first (left) and brain-first (right) LBD. We speculate that body-first LBD may give rise to a more variable strain cloud, since pathology can initiate in any peripheral organ and enter the brain via parasympathetic as well as sympathetic connections. This might explain why body-first LBDs exhibit distinct disease profiles within (i.e., typical body-first PD, DLB, and PAF). Importantly, propagation of pathology may occur bidirectionally, possible creating even more strain variations (Created with Biorender).

Figure 3

Asyn aggregation in a SAA reaction. Monomeric recombinant asyn substrate is added to the wells of a multi-well plate, along with the amyloid-selective fluorescent dye Thioflavin T and the biospecimen to be tested for the presence of asyn seeds. After an initial lag phase, the monomeric asyn can form de novo aggregates (so-called oligomers) that further elongate to form amyloid type β-sheet-rich fibrils during the exponential phase (the de novo pathway is marked in blue). The presence of structurally different asyn seeds, e.g., from PD and MSA patients (Shahnawaz et al., 2020) lead to structurally different asyn conformers, called strains, that can be detected in real time due to the ability of dyes (such as Thioflavin T and K114) that emit fluorescence when bound to amyloid structures. Distinct strains present different conformations and subsequently, altered affinity for binding of the fluorescent molecules. That leads to variations of maximum fluorescent values. The de novo aggregation kinetics are usually characterized by an extended lag phase when compared to the seeded aggregation and this difference forms the basis for diagnosing a biospecimen positive or negative for the presence of asyn seeds.

Figure 4

Working principle of LCO-mediated asyn-strain characterization in LBDs. (A) The strain hypothesis applied to body-first and brain-first LBDs: monomeric asyn may misfold into any pathogenic conformer (influenced by the cellular milieu at the disease onset site), which is responsible for determining which LBD-subtype a patient develops: typical body-first PD (with pre-motor iRBD), DLB, PAF, or brain-first PD (without pre-motor RBD). Due to bidirectional trans-synaptic spread, we hypothesize that each LBD is characterized by a subtype-specific mix of oligomeric and protofibrillar species, the subtype-specific “asyn cloud”, responsible for its phenotype. Furthermore, the cellular environment in a certain organ may favor one conformer over the other from a patients' asyn cloud. Consequently, the spectral fingerprint of pathology in peripheral biopsies likely differs across organs (such as skin and gut) and LBD-subtypes. A combination of multiple fluorescent LCO-ligands on gut and skin biopsies may enable more accurate stratification in pre-motor disease stages, limiting late and misdiagnosis, paving the way for more accurate prognosis and personalized treatment. Furthermore, we speculate that DLB-subtypes may have a more similar cloud distribution, compared to MSA, since asyn in MSA is mainly confined to oligodendrocytes and not neurons/axons. (B) Histology and LCO data in the gut (myenteric plexus) and amygdala of a gut- and brain-first wild-type rodent model, respectively. Immunohistochemistry (IHC) against phosphorylated asyn (Ab51253) is unable to differentiate between gut and brain pathology. In contrast, the LCO assay using the hFTAA-ligand shows a clear difference in the spectrum of gut- and brain-derived aggregates with a more typical two-peak hFTAA spectrum in the brain, indicating gut and brain pathology possess different structural characteristics despite using the same seeds for disease initiation (preliminary data). Scalebar: 50 μm.