Navigating through the complexities of synucleinopathies: Insights into pathogenesis, heterogeneity, and future perspectives.

The aggregation of alpha-synuclein (aSyn) represents a neuropathological hallmark observed in a group of neurodegenerative disorders collectively known as synucleinopathies. Despite their shared characteristics, these disorders manifest diverse clinical and pathological phenotypes. The mechanism underlying this heterogeneity is thought to be due to the diversity in the aSyn strains present across the diseases. In this perspective, we will explore recent findings on aSyn strains and discuss recent discoveries about Lewy bodies' composition. We further discuss the current hypothesis for aSyn spreading and emphasize the emerging biomarker field demonstrating promising results. A comprehension of these mechanisms holds substantial promise for future clinical applications. This understanding can pave the way for the development of personalized medicine strategies, specifically targeting the unique underlying causes of each synucleinopathy. Such advancements can revolutionize therapeutic approaches and significantly contribute to more effective interventions in the intricate landscape of neurodegenerative disorders.

A small-molecule microtubule-stabilizing agent safely reduces Aß plaque and tau pathology in transgenic mouse models of Alzheimer's disease.

INTRODUCTION: Intraneuronal inclusions composed of tau protein are found in Alzheimer's disease (AD) and other tauopathies. Tau normally binds microtubules (MTs), and its disengagement from MTs and misfolding in AD is thought to result in MT abnormalities. We previously identified triazolopyrimidine-containing MT-stabilizing compounds that provided benefit in AD mouse models and herein describe the characterization and efficacy testing of an optimized candidate, CNDR-51997. METHODS: CNDR-51997 underwent pharmacokinetic, pharmacodynamic, safety pharmacology, and mouse tolerability testing. In addition, the compound was examined for efficacy in 5XFAD amyloid beta (Aß) plaque mice and PS19 tauopathy mice. RESULTS: CNDR-51997 significantly reduced Aß plaques in 5XFAD mice and tau pathology in PS19 mice, with the latter also showing attenuated axonal dystrophy and gliosis. CNDR-51997 was well tolerated at doses that exceeded efficacy doses, with a good safety pharmacology profile. DISCUSSION: CNDR-51997 may be a candidate for advancement as a potential therapeutic agent for AD and/or other tauopathies. Highlights There is evidence of microtubule alterations (MT) in Alzheimer's disease (AD) brain and in mouse models of AD pathology. Intermittent dosing with an optimized, brain-penetrant MT-stabilizing small-molecule, CNDR-51997, reduced both Aß plaque and tau inclusion pathology in established mouse models of AD. CNDR-51997 attenuated axonal dystrophy and gliosis in a tauopathy mouse model, with a strong trend toward reduced hippocampal neuron loss. CNDR-51997 is well tolerated in mice at doses that are meaningfully greater than required for efficacy in AD mouse models, and the compound has a good safety pharmacology profile.

α-Synuclein Conformations in Plasma Distinguish Parkinson's Disease from Dementia with Lewy Bodies.

Spread and aggregation of misfolded α-synuclein (aSyn) within the brain is the pathologic hallmark of Lewy body diseases (LBD), including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). While evidence exists for multiple aSyn protein conformations, often termed "strains" for their distinct biological properties, it is unclear whether PD and DLB result from aSyn strain differences, and biomarkers that differentiate PD and DLB are lacking. Moreover, while pathological forms of aSyn have been detected outside the brain ( e.g., in skin, gut, blood), the functional significance of these peripheral aSyn species is unclear. Here, we developed assays using monoclonal antibodies selective for two different aSyn species generated in vitro - termed Strain A and Strain B - and used them to evaluate human brain tissue, cerebrospinal fluid (CSF), and plasma, through immunohistochemistry, enzyme-linked immunoassay, and immunoblotting. Surprisingly, we found that plasma aSyn species detected by these antibodies differentiated individuals with PD vs. DLB in a discovery cohort (UPenn, n=235, AUC 0.83) and a multi-site replication cohort (Parkinson's Disease Biomarker Program, or PDBP, n=200, AUC 0.72). aSyn plasma species detected by the Strain A antibody also predicted rate of cognitive decline in PD. We found no evidence for aSyn strains in CSF, and ability to template aSyn fibrillization differed for species isolated from plasma vs. brain, and in PD vs. DLB. Taken together, our findings suggest that aSyn conformational differences may impact clinical presentation and cortical spread of pathological aSyn. Moreover, the enrichment of these aSyn strains in plasma implicates a non-central nervous system source.

Highly tunable bimane-based fluorescent probes: design, synthesis, and application as a selective amyloid binding dye.

Small molecule fluorescent probes are indispensable tools for a broad range of biological applications. Despite many probes being available, there is still a need for probes where photophysical properties and biological selectivity can be tuned as desired. Here, we report the rational design and synthesis of a palette of fluorescent probes based on the underexplored bimane scaffold. The newly developed probes with varied electronic properties show tunable absorption and emission in the visible region with large Stokes shifts. Probes featuring electron-donating groups exhibit rotor effects that are sensitive to polarity and viscosity by "intramolecular charge transfer" (ICT) and twisted intramolecular charge transfer (TICT) mechanisms, respectively. These properties enable their application as "turn-on" fluorescent probes to detect fibrillar aggregates of the α-synuclein (αS) protein that are a hallmark of Parkinson's disease (PD). One probe shows selective binding to αS fibrils relative to soluble proteins in cell lysates and amyloid fibrils of tau and amyloid-ß. Finally, we demonstrate the diagnostic potential of the probe in selectively detecting αS fibrils amplified from PD with dementia (PDD) patient samples.

ScISOr-ATAC reveals convergent and divergent splicing and chromatin specificities between matched cell types across cortical regions, evolution, and in Alzheimer's Disease.

Multimodal measurements have become widespread in genomics, however measuring open chromatin accessibility and splicing simultaneously in frozen brain tissues remains unconquered. Hence, we devised Single-Cell-ISOform-RNA sequencing coupled with the Assay-for-Transposase-Accessible-Chromatin (ScISOr-ATAC). We utilized ScISOr-ATAC to assess whether chromatin and splicing alterations in the brain convergently affect the same cell types or divergently different ones. We applied ScISOr-ATAC to three major conditions: comparing (i) the Rhesus macaque (Macaca mulatta) prefrontal cortex (PFC) and visual cortex (VIS), (ii) cross species divergence of Rhesus macaque versus human PFC, as well as (iii) dysregulation in Alzheimer's disease in human PFC. We found that among cortical-layer biased excitatory neuron subtypes, splicing is highly brain-region specific for L3-5/L6 IT_RORB neurons, moderately specific in L2-3 IT_CUX2.RORB neurons and unspecific in L2-3 IT_CUX2 neurons. In contrast, at the chromatin level, L2-3 IT_CUX2.RORB neurons show the highest brain-region specificity compared to other subtypes. Likewise, when comparing human and macaque PFC, strong evolutionary divergence on one molecular modality does not necessarily imply strong such divergence on another molecular level in the same cell type. Finally, in Alzheimer's disease, oligodendrocytes show convergently high dysregulation in both chromatin and splicing. However, chromatin and splicing dysregulation most strongly affect distinct oligodendrocyte subtypes. Overall, these results indicate that chromatin and splicing can show convergent or divergent results depending on the performed comparison, justifying the need for their concurrent measurement to investigate complex systems. Taken together, ScISOr-ATAC allows for the characterization of single-cell splicing and chromatin patterns and the comparison of sample groups in frozen brain samples.

MSUT2 regulates tau spreading via adenosinergic signaling mediated ASAP1 pathway in neurons.

Inclusions comprised of microtubule-associated protein tau (tau) are implicated in a group of neurodegenerative diseases, collectively known as tauopathies, that include Alzheimer's disease (AD). The spreading of misfolded tau "seeds" along neuronal networks is thought to play a crucial role in the progression of tau pathology. Consequently, restricting the release or uptake of tau seeds may inhibit the spread of tau pathology and potentially halt the advancement of the disease. Previous studies have demonstrated that the Mammalian Suppressor of Tauopathy 2 (MSUT2), an RNA binding protein, modulates tau pathogenesis in a transgenic mouse model. In this study, we investigated the impact of MSUT2 on tau pathogenesis using tau seeding models. Our findings indicate that the loss of MSUT2 mitigates human tau seed-induced pathology in neuron cultures and mouse models. In addition, MSUT2 regulates many gene transcripts, including the Adenosine Receptor 1 (A1AR), and we show that down regulation or inhibition of A1AR modulates the activity of the "ArfGAP with SH3 Domain, Ankyrin Repeat, and PH Domain 1 protein" (ASAP1), thereby influencing the internalization of pathogenic tau seeds into neurons resulting in reduction of tau pathology.

O-GlcNAc forces an α-synuclein amyloid strain with notably diminished seeding and pathology.

Amyloid-forming proteins such α-synuclein and tau, which are implicated in Alzheimer's and Parkinson's disease, can form different fibril structures or strains with distinct toxic properties, seeding activities and pathology. Understanding the determinants contributing to the formation of different amyloid features could open new avenues for developing disease-specific diagnostics and therapies. Here we report that O-GlcNAc modification of α-synuclein monomers results in the formation of amyloid fibril with distinct core structure, as revealed by cryogenic electron microscopy, and diminished seeding activity in seeding-based neuronal and rodent models of Parkinson's disease. Although the mechanisms underpinning the seeding neutralization activity of the O-GlcNAc-modified fibrils remain unclear, our in vitro mechanistic studies indicate that heat shock proteins interactions with O-GlcNAc fibril inhibit their seeding activity, suggesting that the O-GlcNAc modification may alter the interactome of the α-synuclein fibrils in ways that lead to reduce seeding activity in vivo. Our results show that posttranslational modifications, such as O-GlcNAc modification, of α-synuclein are key determinants of α-synuclein amyloid strains and pathogenicity.

α-Synuclein aggregates amplified from patient-derived Lewy bodies recapitulate Lewy body diseases in mice.

Extraction of α-Synuclein (αSyn) aggregates from Lewy body disease (LBD) brains has been widely described yet templated fibrillization of LB-αSyn often fails to propagate its structural and functional properties. We recently demonstrated that aggregates amplified from LB-αSyn (ampLB) show distinct biological activities in vitro compared to human αSyn preformed fibrils (hPFF) formed de novo. Here we compare the in vivo biological activities of hPFF and ampLB regarding seeding activity, latency in inducing pathology, distribution of pathology, inclusion morphology, and cell-type preference. Injection of ampLB into mice expressing only human αSyn (male Thy1:SNCA/Snca-/- mice) induced pathologies similar to those of LBD subjects that were distinct from those induced by hPFF-injection or developing spontaneously with aging. Importantly, αSyn aggregates in ampLB-injected Thy1:SNCA/Snca-/- mice maintained the unique biological and conformational features of original LB-αSyn. These results indicate that ampLB-injection, rather than conventional PFF-injection or αSyn overexpression, faithfully models key aspects of LBD.

A microtubule stabilizer ameliorates protein pathogenesis and neurodegeneration in mouse models of repetitive traumatic brain injury.

Tau pathogenesis is a hallmark of many neurodegenerative diseases, including Alzheimer's disease (AD). Although the events leading to initial tau misfolding and subsequent tau spreading in patient brains are largely unknown, traumatic brain injury (TBI) may be a risk factor for tau-mediated neurodegeneration. Using a repetitive TBI (rTBI) paradigm, we report that rTBI induced somatic accumulation of phosphorylated and misfolded tau, as well as neurodegeneration across multiple brain areas in 7-month-old tau transgenic PS19 mice but not wild-type (WT) mice. rTBI accelerated somatic tau pathology in younger PS19 mice and WT mice only after inoculation with tau preformed fibrils and AD brain-derived pathological tau (AD-tau), respectively, suggesting that tau seeds are needed for rTBI-induced somatic tau pathology. rTBI further disrupted axonal microtubules and induced punctate tau and TAR DNA binding protein 43 (TDP-43) pathology in the optic tracts of WT mice. These changes in the optic tract were associated with a decline of visual function. Treatment with a brain-penetrant microtubule-stabilizing molecule reduced rTBI-induced tau, TDP-43 pathogenesis, and neurodegeneration in the optic tract as well as visual dysfunction. Treatment with the microtubule stabilizer also alleviated rTBI-induced tau pathology in the cortices of AD-tau-inoculated WT mice. These results indicate that rTBI facilitates abnormal microtubule organization, pathological tau formation, and neurodegeneration and suggest microtubule stabilization as a potential therapeutic avenue for TBI-induced neurodegeneration.

A Novel Brain PET Radiotracer for Imaging Alpha Synuclein Fibrils in Multiple System Atrophy.

Abnormal α-synuclein (α-syn) aggregation characterizes α-synucleinopathies, including Parkinson's disease (PD) and multiple system atrophy (MSA). However, no suitable positron emission tomography (PET) radiotracer for imaging α-syn in PD and MSA exists currently. Our structure-activity relationship studies identified 4-methoxy-N-(4-(3-(pyridin-2-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)phenyl)benzamide (4i) as a PET radiotracer candidate for imaging α-syn. In vitro assays revealed high binding of 4i to recombinant α-syn fibrils (inhibition constant (Ki) = 6.1 nM) and low affinity for amyloid beta (Aß) fibrils in Alzheimer's disease (AD) homogenates. However, [3H]4i also exhibited high specific binding to AD, progressive supranuclear palsy, and corticobasal degeneration tissues as well as PD and MSA tissues, suggesting notable affinity to tau. Nevertheless, the specific binding to pathologic α-syn aggregates in MSA post-mortem brain tissues was significantly higher than in PD tissues. This finding demonstrated the potential use of [11C]4i as a PET tracer for imaging α-syn in MSA patients. Nonhuman primate PET studies confirmed good brain uptake and rapid washout for [11C]4i.

TRIM11 protects against tauopathies and is down-regulated in Alzheimer's disease.

Aggregation of tau into filamentous inclusions underlies Alzheimer's disease (AD) and numerous other neurodegenerative tauopathies. The pathogenesis of tauopathies remains unclear, which impedes the development of disease-modifying treatments. Here, by systematically analyzing human tripartite motif (TRIM) proteins, we identified a few TRIMs that could potently inhibit tau aggregation. Among them, TRIM11 was markedly down-regulated in AD brains. TRIM11 promoted the proteasomal degradation of mutant tau as well as superfluous normal tau. It also enhanced tau solubility by acting as both a molecular chaperone to prevent tau misfolding and a disaggregase to dissolve preformed tau fibrils. TRIM11 maintained the connectivity and viability of neurons. Intracranial delivery of TRIM11 through adeno-associated viruses ameliorated pathology, neuroinflammation, and cognitive impairments in multiple animal models of tauopathies. These results suggest that TRIM11 down-regulation contributes to the pathogenesis of tauopathies and that restoring TRIM11 expression may represent an effective therapeutic strategy.

Identification of small molecules and related targets that modulate tau pathology in a seeded primary neuron model.

Alzheimer's disease (AD) is characterized by the presence of tau protein inclusions and amyloid beta (Aß) plaques in the brain, with Aß peptides generated by cleavage of the amyloid precursor protein (APP) by BACE1 and γ-secretase. We previously described a primary rat neuron assay in which tau inclusions form from endogenous rat tau after seeding cells with insoluble tau isolated from the human AD brain. Here, we used this assay to screen an annotated library of ∼8700 biologically active small molecules for their ability to reduce immuno-stained neuronal tau inclusions. Compounds causing ≥30% inhibition of tau aggregates with <25% loss of DAPI-positive cell nuclei underwent further confirmation testing and assessment of neurotoxicity, and non-neurotoxic hits were subsequently analyzed for inhibitory activity in an orthogonal ELISA that quantified multimeric rat tau species. Of the 173 compounds meeting all criteria, a subset of 55 inhibitors underwent concentration-response testing and 46 elicited a concentration-dependent reduction of neuronal tau inclusions that were distinct from measures of toxicity. Among the confirmed inhibitors of tau pathology were BACE1 inhibitors, several of which, along with γ-secretase inhibitors/modulators, caused a concentration-dependent lowering of neuronal tau inclusions and a reduction of insoluble tau by immunoblotting, although they did not decrease soluble phosphorylated tau species. In conclusion, we have identified a diverse set of small molecules and related targets that reduce neuronal tau inclusions. Notably, these include BACE1 and γ-secretase inhibitors, suggesting that a cleavage product from a shared substrate, such as APP, might affect tau pathology.

Seeding the aggregation of TDP-43 requires post-fibrillization proteolytic cleavage.

Despite the strong evidence linking the transactive response DNA-binding protein 43 (TDP-43) aggregation to the pathogenesis of frontotemporal lobar degeneration with TDP-43, amyotrophic lateral sclerosis and several neurodegenerative diseases, our knowledge of the sequence and structural determinants of its aggregation and neurotoxicity remains incomplete. Herein, we present a new method for producing recombinant full-length TDP-43 filaments that exhibit sequence and morphological features similar to those of brain-derived TDP-43 filaments. We show that TDP-43 filaments contain a ß-sheet-rich helical amyloid core that is fully buried by the flanking structured domains of the protein. We demonstrate that the proteolytic cleavage of TDP-43 filaments and exposure of this amyloid core are necessary for propagating TDP-43 pathology and enhancing the seeding of brain-derived TDP-43 aggregates. Only TDP-43 filaments with exposed amyloid core efficiently seeded the aggregation of endogenous TDP-43 in cells. These findings suggest that inhibiting the enzymes mediating cleavage of TDP-43 aggregates represents a viable disease-modifying strategy to slow the progression of amyotrophic lateral sclerosis and other TDP-43 proteinopathies.

O-GlcNAc modification forces the formation of an α-Synuclein amyloid-strain with notably diminished seeding activity and pathology.

The process of amyloid fibril formation remains one of the primary targets for developing diagnostics and treatments for several neurodegenerative diseases (NDDs). Amyloid-forming proteins such α-Synuclein and Tau, which are implicated in the pathogenesis of Alzheimer's and Parkinson's disease, can form different types of fibril structure, or strains, that exhibit distinct structures, toxic properties, seeding activities, and pathology spreading patterns in the brain. Therefore, understanding the molecular and structural determinants contributing to the formation of different amyloid strains or their distinct features could open new avenues for developing disease-specific diagnostics and therapies. In this work, we report that O-GlcNAc modification of α-Synuclein monomers results in the formation of amyloid fibril with distinct core structure, as revealed by Cryo-EM, and diminished seeding activity in seeding-based neuronal and rodent models of Parkinson's disease. Although the mechanisms underpinning the seeding neutralization activity of the O-GlcNAc modified fibrils remain unclear, our in vitro mechanistic studies indicate that heat shock proteins interactions with O-GlcNAc fibril inhibit their seeding activity, suggesting that the O-GlcNAc modification may alter the interactome of the α-Synuclein fibrils in ways that lead to reduce seeding activity in vivo. Our results show that post-translational modifications, such as O-GlcNAc modification, of α-Synuclein are key determinants of α-Synuclein amyloid strains and pathogenicity. These findings have significant implications for how we investigate and target amyloids in the brain and could possibly explain the lack of correlation between amyloid burden and neurodegeneration or cognitive decline in some subtypes of NDDs.

Data-driven neuropathological staging and subtyping of TDP-43 proteinopathies.

TAR DNA-binding protein-43 (TDP-43) accumulation is the primary pathology underlying several neurodegenerative diseases. Charting the progression and heterogeneity of TDP-43 accumulation is necessary to better characterise TDP-43 proteinopathies, but current TDP-43 staging systems are heuristic and assume each syndrome is homogeneous. Here, we use data-driven disease progression modelling to derive a fine-grained empirical staging system for the classification and differentiation of frontotemporal lobar degeneration due to TDP-43 (FTLD-TDP, n=126), amyotrophic lateral sclerosis (ALS, n=141) and limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC) with and without Alzheimer’s disease (n=304). The data-driven staging of ALS and FTLD-TDP complement and extend previously described human-defined staging schema for ALS and behavioural variant frontotemporal dementia. In LATE-NC individuals, progression along data-driven stages was positively associated with age, but negatively associated with age in individuals with FTLD-TDP. Using only regional TDP-43 severity, our data driven model distinguished individuals diagnosed with ALS, FTLD-TDP or LATE-NC with a cross-validated accuracy of 85.9%, with misclassifications associated with mixed pathological diagnosis, age and genetic mutations. Adding age and SuStaIn stage to this model increased accuracy to 92.3%. Our model differentiates LATE-NC from FTLD-TDP, though some overlap was observed between late-stage LATE-NC and early-stage FTLD-TDP. We further tested for the presence of subtypes with distinct regional TDP-43 progression patterns within each diagnostic group, identifying two distinct cortical-predominant and brainstem-predominant subtypes within FTLD-TDP and a further two subcortical-predominant and corticolimbic-predominant subtypes within ALS. The FTLD-TDP subtypes exhibited differing proportions of TDP-43 type, while there was a trend for age differing between ALS subtypes. Interestingly, a negative relationship between age and SuStaIn stage was seen in the brainstem/subcortical-predominant subtype of each proteinopathy. No subtypes were observed for the LATE-NC group, despite aggregating AD+ and AD-individuals and a larger sample size for this group. Overall, we provide an empirical pathological TDP-43 staging system for ALS, FTLD-TDP and LATE-NC, which yielded accurate classification. We further demonstrate that there is substantial heterogeneity amongst ALS and FTLD-TDP progression patterns that warrants further investigation in larger cross-cohort studies.

Immunotherapy targeting the C-terminal domain of TDP-43 decreases neuropathology and confers neuroprotection in mouse models of ALS/FTD.

Effective therapies are urgently needed to safely target TDP-43 pathology as it is closely associated with the onset and development of devastating diseases such as frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). In addition, TDP-43 pathology is present as a co-pathology in other neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Our approach is to develop a TDP-43-specific immunotherapy that exploits Fc gamma-mediated removal mechanisms to limit neuronal damage while maintaining physiological TDP-43 function. Thus, using both in vitro mechanistic studies in conjunction with the rNLS8 and CamKIIa inoculation mouse models of TDP-43 proteinopathy, we identified the key targeting domain in TDP-43 to accomplish these therapeutic objectives. Targeting the C-terminal domain of TDP-43 but not the RNA recognition motifs (RRM) reduces TDP-43 pathology and avoids neuronal loss in vivo. We demonstrate that this rescue is dependent on Fc receptor-mediated immune complex uptake by microglia. Furthermore, monoclonal antibody (mAb) treatment enhances phagocytic capacity of ALS patient-derived microglia, providing a mechanism to restore the compromised phagocytic function in ALS and FTD patients. Importantly, these beneficial effects are achieved while preserving physiological TDP-43 activity. Our findings demonstrate that a mAb targeting the C-terminal domain of TDP-43 limits pathology and neurotoxicity, enabling clearance of misfolded TDP-43 through microglia engagement, and supporting the clinical strategy to target TDP-43 by immunotherapy. SIGNIFICANCE STATEMENT: TDP-43 pathology is associated with various devastating neurodegenerative disorders with high unmet medical needs such as frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Thus, safely and effectively targeting pathological TDP-43 represents a key paradigm for biotechnical research as currently there is little in clinical development. After years of research, we have determined that targeting the C-terminal domain of TDP-43 rescues multiple patho-mechanisms involved in disease progression in two animal models of FTD/ALS. In parallel, importantly, our studies establish that this approach does not alter the physiological functions of this ubiquitously expressed and indispensable protein. Together, our findings substantially contribute to the understanding of TDP-43 pathobiology and support the prioritization for clinical testing of immunotherapy approaches targeting TDP-43.

Post-translational modifications of soluble α-synuclein regulate the amplification of pathological α-synuclein.

Cell-to-cell transmission and subsequent amplification of pathological proteins promote neurodegenerative disease progression. Most research on this has focused on pathological protein seeds, but how their normal counterparts, which are converted to pathological forms during transmission, regulate transmission is less understood. Here we show in cultured cells that phosphorylation of soluble, nonpathological α-synuclein (α-Syn) at previously identified sites dramatically affects the amplification of pathological α-Syn, which underlies Parkinson's disease and other α-synucleinopathies, in a conformation- and phosphorylation site-specific manner. We performed LC-MS/MS analyses on soluble α-Syn purified from Parkinson's disease and other α-synucleinopathies, identifying many new α-Syn post-translational modifications (PTMs). In addition to phosphorylation, acetylation of soluble α-Syn also modified pathological α-Syn transmission in a site- and conformation-specific manner. Moreover, phosphorylation of soluble α-Syn could modulate the seeding properties of pathological α-Syn. Our study represents the first systematic analysis how of soluble α-Syn PTMs affect the spreading and amplification of pathological α-Syn, which may affect disease progression.