Divergent Age-Dependent Conformational Rearrangement within Aß Amyloid Deposits in APP23, APPPS1, and AppNL-F Mice.

Amyloid plaques composed of fibrils of misfolded Aß peptides are pathological hallmarks of Alzheimer's disease (AD). Aß fibrils are polymorphic in their tertiary and quaternary molecular structures. This structural polymorphism may carry different pathologic potencies and can putatively contribute to clinical phenotypes of AD. Therefore, mapping of structural polymorphism of Aß fibrils and structural evolution over time is valuable to understanding disease mechanisms. Here, we investigated how Aß fibril structures in situ differ in Aß plaque of different mouse models expressing familial mutations in the AßPP gene. We imaged frozen brains with a combination of conformation-sensitive luminescent conjugated oligothiophene (LCO) ligands and Aß-specific antibodies. LCO fluorescence mapping revealed that mouse models APP23, APPPS1, and AppNL-F have different fibril structures within Aß-amyloid plaques depending on the AßPP-processing genotype. Co-staining with Aß-specific antibodies showed that individual plaques from APP23 mice expressing AßPP Swedish mutation have two distinct fibril polymorph regions of core and corona. The plaque core is predominantly composed of compact Aß40 fibrils, and the corona region is dominated by diffusely packed Aß40 fibrils. Conversely, the AßPP knock-in mouse AppNL-F, expressing the AßPP Iberian mutation along with Swedish mutation has tiny, cored plaques consisting mainly of compact Aß42 fibrils, vastly different from APP23 even at elevated age up to 21 months. Age-dependent polymorph rearrangement of plaque cores observed for APP23 and APPPS1 mice >12 months, appears strongly promoted by Aß40 and was hence minuscule in AppNL-F. These structural studies of amyloid plaques in situ can map disease-relevant fibril polymorph distributions to guide the design of diagnostic and therapeutic molecules.

Thiophene-Based Ligands for Specific Assignment of Distinct Aß Pathologies in Alzheimer's Disease.

Aggregated species of amyloid-ß (Aß) are one of the pathological hallmarks in Alzheimer's disease (AD), and ligands that selectively target different Aß deposits are of great interest. In this study, fluorescent thiophene-based ligands have been used to illustrate the features of different types of Aß deposits found in AD brain tissue. A dual-staining protocol based on two ligands, HS-276 and LL-1, with different photophysical and binding properties, was developed and applied on brain tissue sections from patients affected by sporadic AD or familial AD associated with the PSEN1 A431E mutation. When binding to Aß deposits, the ligands could easily be distinguished for their different fluorescence, and distinct staining patterns were revealed for these two types of AD. In sporadic AD, HS-276 consistently labeled all immunopositive Aß plaques, whereas LL-1 mainly stained cored and neuritic Aß deposits. In the PSEN1 A431E cases, each ligand was binding to specific types of Aß plaques. The ligand-labeled Aß deposits were localized in distinct cortical layers, and a laminar staining pattern could be seen. Biochemical characterization of the Aß aggregates in the individual layers also showed that the variation of ligand binding properties was associated with certain Aß peptide signatures. For the PSEN1 A431E cases, it was concluded that LL-1 was binding to cotton wool plaques, whereas HS-276 mainly stained diffuse Aß deposits. Overall, our findings showed that a combination of ligands was essential to identify distinct aggregated Aß species associated with different forms of AD.

A Novel Drosophila Model of Alzheimer's Disease to Study Aß Proteotoxicity in the Digestive Tract.

Amyloid-ß (Aß) proteotoxicity is associated with Alzheimer's disease (AD) and is caused by protein aggregation, resulting in neuronal damage in the brain. In the search for novel treatments, Drosophila melanogaster has been extensively used to screen for anti-Aß proteotoxic agents in studies where toxic Aß peptides are expressed in the fly brain. Since drug molecules often are administered orally there is a risk that they fail to reach the brain, due to their inability to cross the brain barrier. To circumvent this problem, we have designed a novel Drosophila model that expresses the Aß peptides in the digestive tract. In addition, a built-in apoptotic sensor provides a fluorescent signal from the green fluorescent protein as a response to caspase activity. We found that expressing different variants of Aß1-42 resulted in proteotoxic phenotypes such as reduced longevity, aggregate deposition, and the presence of apoptotic cells. Taken together, this gut-based Aß-expressing fly model can be used to study the mechanisms behind Aß proteotoxicity and to identify different substances that can modify Aß proteotoxicity.

The fluorescent ligand bTVBT2 reveals increased p-tau uptake by retinal microglia in Alzheimer's disease patients and AppNL-F/NL-F mice.

BACKGROUND: Amyloid beta (Aß) deposits and hyperphosphorylated tau (p-tau) accumulation have been identified in the retina of Alzheimer's disease (AD) patients and transgenic AD mice. Previous studies have shown that retinal microglia engulf Aß, but this property decreases in AD patients. Whether retinal microglia also take up p-tau and if this event is affected in AD is yet not described. In the current study, we use the p-tau-specific thiophene-based ligand bTVBT2 to investigate the relationship between disease progression and p-tau uptake by microglia in the retina of AD patients and AppNL-F/NL-F knock-in mice, an AD mouse model known to demonstrate extracellular Aß plaques and dystrophic neurites in the brain from 6 months of age. METHODS: Evaluation of bTVBT2 specificity and its presence within microglia was assessed by immunofluorescent staining of hippocampal sections and flat-mount retina samples from non-demented controls, AD patients, 3-, 9-, and 12-month-old AppNL-F/NL-F knock-in mice and 12- and 18-month-old wild type (WT) mice. We used ImageJ to analyze the amount of bTVBT2 inside Iba1-positive microglia. Co-localization between the ligand and p-tau variant Ser396/Ser404 (PHF-1), Aß, phosphorylated TAR DNA binding protein 43 (pTDP-43), and islet amyloid polypeptide (IAPP) in the brain and retina was analyzed using confocal imaging. RESULTS: Confocal imaging analysis showed that bTVBT2 binds to PHF-1- and AT8-positive aggregates inside retinal microglia, and not to Aß, pTDP-43, or IAPP. The density of bTVBT2-positive microglia was higher in cases with a high Aß load compared to those with a low Aß load. This density correlated with the neurofibrillary tangle load in the brain, but not with retinal levels of high molecular weight (aggregated) Aß40 or Aß42. Analysis of AppNL-F/NL-F knock-in mouse retina further showed that 50% of microglia in 3-month-old AppNL-F/NL-F knock-in mice contained bTVBT2. The percentage significantly increased in 9- and 12-month-old mice. CONCLUSION: Our study suggests that the microglial capability to uptake p-tau in the retina persists and intensifies with AD progression. These results also highlight bTVBT2 as a ligand of interest in future monitoring of retinal AD pathology.

Real-time imaging of mitochondrial redox reveals increased mitochondrial oxidative stress associated with amyloid ß aggregates in vivo in a mouse model of Alzheimer's disease.

BACKGROUND: Reactive oxidative stress is a critical player in the amyloid beta (Aß) toxicity that contributes to neurodegeneration in Alzheimer's disease (AD). Damaged mitochondria are one of the main sources of reactive oxygen species and accumulate in Aß plaque-associated dystrophic neurites in the AD brain. Although Aß causes neuronal mitochondria reactive oxidative stress in vitro, this has never been directly observed in vivo in the living mouse brain. Here, we tested for the first time whether Aß plaques and soluble Aß oligomers induce mitochondrial oxidative stress in surrounding neurons in vivo, and whether this neurotoxic effect can be abrogated using mitochondrial-targeted antioxidants. METHODS: We expressed a genetically encoded fluorescent ratiometric mitochondria-targeted reporter of oxidative stress in mouse models of the disease and performed intravital multiphoton microscopy of neuronal mitochondria and Aß plaques. RESULTS: For the first time, we demonstrated by direct observation in the living mouse brain exacerbated mitochondrial oxidative stress in neurons after both Aß plaque deposition and direct application of soluble oligomeric Aß onto the brain, and determined the most likely pathological sequence of events leading to oxidative stress in vivo. Oxidative stress could be inhibited by both blocking calcium influx into mitochondria and treating with the mitochondria-targeted antioxidant SS31. Remarkably, the latter ameliorated plaque-associated dystrophic neurites without impacting Aß plaque burden. CONCLUSIONS: Considering these results, combination of mitochondria-targeted compounds with other anti-amyloid beta or anti-tau therapies hold promise as neuroprotective drugs for the prevention and/or treatment of AD.

Neuronal Ndst1 depletion accelerates prion protein clearance and slows neurodegeneration in prion infection.

Select prion diseases are characterized by widespread cerebral plaque-like deposits of amyloid fibrils enriched in heparan sulfate (HS), a abundant extracellular matrix component. HS facilitates fibril formation in vitro, yet how HS impacts fibrillar plaque growth within the brain is unclear. Here we found that prion-bound HS chains are highly sulfated, and that the sulfation is essential for accelerating prion conversion in vitro. Using conditional knockout mice to deplete the HS sulfation enzyme, Ndst1 (N-deacetylase / N-sulfotransferase) from neurons or astrocytes, we investigated how reducing HS sulfation impacts survival and prion aggregate distribution during a prion infection. Neuronal Ndst1-depleted mice survived longer and showed fewer and smaller parenchymal plaques, shorter fibrils, and increased vascular amyloid, consistent with enhanced aggregate transit toward perivascular drainage channels. The prolonged survival was strain-dependent, affecting mice infected with extracellular, plaque-forming, but not membrane bound, prions. Live PET imaging revealed rapid clearance of recombinant prion protein monomers into the CSF of neuronal Ndst1- deficient mice, neuronal, further suggesting that HS sulfate groups hinder transit of extracellular prion protein monomers. Our results directly show how a host cofactor slows the spread of prion protein through the extracellular space and identify an enzyme to target to facilitate aggregate clearance.

Live Detection of Neural Progenitors and Glioblastoma Cells by an Oligothiophene Derivative.

There is an urgent need for simple and non-invasive identification of live neural stem/progenitor cells (NSPCs) in the developing and adult brain as well as in disease, such as in brain tumors, due to the potential clinical importance in prognosis, diagnosis, and treatment of diseases of the nervous system. Here, we report a luminescent conjugated oligothiophene (LCO), named p-HTMI, for non-invasive and non-amplified real-time detection of live human patient-derived glioblastoma (GBM) stem cell-like cells and NSPCs. While p-HTMI stained only a small fraction of other cell types investigated, the mere addition of p-HTMI to the cell culture resulted in efficient detection of NSPCs or GBM cells from rodents and humans within minutes. p-HTMI is functionalized with a methylated imidazole moiety resembling the side chain of histidine/histamine, and non-methylated analogues were not functional. Cell sorting experiments of human GBM cells demonstrated that p-HTMI labeled the same cell population as CD271, a proposed marker for stem cell-like cells and rapidly migrating cells in glioblastoma. Our results suggest that the LCO p-HTMI is a versatile tool for immediate and selective detection of neural and glioma stem and progenitor cells.

Two structurally defined Aß polymorphs promote different pathological changes in susceptible mice.

Misfolded Aß is involved in the progression of Alzheimer's disease (AD). However, the role of its polymorphic variants or conformational strains in AD pathogenesis is not fully understood. Here, we study the seeding properties of two structurally defined synthetic misfolded Aß strains (termed 2F and 3F) using in vitro and in vivo assays. We show that 2F and 3F strains differ in their biochemical properties, including resistance to proteolysis, binding to strain-specific dyes, and in vitro seeding. Injection of these strains into a transgenic mouse model produces different pathological features, namely different rates of aggregation, formation of different plaque types, tropism to specific brain regions, differential recruitment of Aß40 /Aß42 peptides, and induction of microglial and astroglial responses. Importantly, the aggregates induced by 2F and 3F are structurally different as determined by ssNMR. Our study analyzes the biological properties of purified Aß polymorphs that have been characterized at the atomic resolution level and provides relevant information on the pathological significance of misfolded Aß strains.

Mutation ∆K281 in MAPT causes Pick's disease.

Two siblings with deletion mutation ∆K281 in MAPT developed frontotemporal dementia. At autopsy, numerous inclusions of hyperphosphorylated 3R Tau were present in neurons and glial cells of neocortex and some subcortical regions, including hippocampus, caudate/putamen and globus pallidus. The inclusions were argyrophilic with Bodian silver, but not with Gallyas-Braak silver. They were not labelled by an antibody specific for tau phosphorylated at S262 and/or S356. The inclusions were stained by luminescent conjugated oligothiophene HS-84, but not by bTVBT4. Electron cryo-microscopy revealed that the core of tau filaments was made of residues K254-F378 of 3R Tau and was indistinguishable from that of Pick's disease. We conclude that MAPT mutation ∆K281 causes Pick's disease.

Contrast Enhanced Magnetic Resonance Imaging of Amyloid-ß Plaques in a Murine Alzheimer's Disease Model.

BACKGROUND: Early detection of amyloid-ß (Aß) aggregates is a critical step to improve the treatment of Alzheimer's disease (AD) because neuronal damage by the Aß aggregates occurs before clinical symptoms are apparent. We have previously shown that luminescent conjugated oligothiophenes (LCOs), which are highly specific towards protein aggregates of Aß, can be used to fluorescently label amyloid plaque in living rodents. OBJECTIVE: We hypothesize that the LCO can be used to target gadolinium to the amyloid plaque and hence make the plaque detectable by T1-weighted magnetic resonance imaging (MRI). METHODS: A novel LCO-gadolinium construct was synthesized to selectively bind to Aß plaques and give contrast in conventional T1-weighted MR images after intravenous injection in Tg-APPSwe mice. RESULTS: We found that mice with high plaque-burden could be identified using the LCO-Gd constructs by conventional MRI. CONCLUSION: Our study shows that MR imaging of amyloid plaques is challenging but feasible, and hence contrast-mediated MR imaging could be a valuable tool for early AD detection.

Thiophene-Based Ligands: Design, Synthesis and Their Utilization for Optical Assignment of Polymorphic-Disease-Associated Protein Aggregates.

The development of ligands for detecting protein aggregates is of great interest, as these aggregated proteinaceous species are the pathological hallmarks of several devastating diseases, including Alzheimer's disease. In this regard, thiophene-based ligands have emerged as powerful tools for fluorescent assessment of these pathological entities. The intrinsic conformationally sensitive photophysical properties of poly- and oligothiophenes have allowed optical assignment of disease-associated protein aggregates in tissue sections, as well as real-time in vivo imaging of protein deposits. Herein, we recount the chemical evolution of different generations of thiophene-based ligands, and exemplify their use for the optical distinction of polymorphic protein aggregates. Furthermore, the chemical determinants for achieving a superior fluorescent thiophene-based ligand, as well as the next generation of thiophene-based ligands targeting distinct aggregated species are described. Finally, the directions for future research into the chemical design of thiophene-based ligands that can aid in resolving the scientific challenges around protein aggregation diseases are discussed.

ApoE Alzheimer's Disease Aß-amyloid plaque morphology varies according to APOE isotype.

Background: The apolipoprotein E (APOE, gene; apoE, protein) ε4 allele is the most common identified genetic risk factor for typical late-onset sporadic Alzheimer's disease (AD). Each APOE ε4 allele roughly triples the relative risk for AD compared to that of the reference allele, APOE ε3. Methods: We have employed hyperspectral fluorescence imaging with an amyloidspecific, conformation-sensing probe, p-FTAA, to elucidate protein aggregate structure and morphology in fresh frozen prefrontal cortex samples from human postmortem AD brain tissue samples from patients homozygous for either APOE ε3 or APOE ε4. Results: As expected APOE ε4/ε4 tissues had significantly larger load of CAA than APOE ε3/ε3. APOE isoform-dependent morphological differences in amyloid plaques were also observed. Amyloid plaques in APOE ε3/ε3 tissue had small spherical cores and large corona while amyloid plaques in APOE ε4/ε4 tissues had large irregular and multilobulated plaques with relatively smaller corona. Despite the different morphologies of their cores, the p-FTAA stained APOE ε3/ε3 amyloid plaque cores had spectral properties identical to those of APOE ε4/ε4 plaque cores. Conclusions: These data support the hypothesis that one mechanism by which the APOE ε4 allele affects AD is by modulating the macrostructure of pathological protein deposits in brain. APOE ε4 is associated with a higher density of amyloid plaques (as compared to APOE ε3). We speculate that multilobulated APOE ε4-associated plaques arise from multiple initiation foci that coalesce as the plaques grow.

Thiophene-Based Ligands for Histological Multiplex Spectral Detection of Distinct Protein Aggregates in Alzheimer's Disease.

The aggregation and accumulation of proteins in the brain is the defining feature of many devastating neurodegenerative diseases. The development of fluorescent ligands that bind to these accumulations, or deposits, is essential for the characterization of these neuropathological lesions. We report the synthesis of donor-acceptor-donor (D-A-D) thiophene-based ligands with different emission properties. The D-A-D ligands displayed selectivity towards distinct disease-associated protein deposits in histological sections from postmortem brain tissue of individuals affected by Alzheimer's disease (AD). The ability of the ligands to selectively identify AD-associated pathological alterations, such as deposits composed of aggregates of the amyloid-ß (Aß) peptide or tau, was reduced when the chemical composition of the ligands was altered. When combining the D-A-D ligands with conventional thiophene-based ligands, superior spectral separation of distinct protein aggregates in AD tissue sections was obtained. Our findings provide the structural and functional basis for the development of new fluorescent ligands that can distinguish between aggregated proteinaceous species, as well as offer novel strategies for developing multiplex fluorescence detection of protein aggregates in tissue sections.

Self-Assembly of Chiro-Optical Materials from Nonchiral Oligothiophene-Porphyrin Derivatives and Random Coil Synthetic Peptides.

Biomimetic chiral optoelectronic materials can be utilized in electronic devices, biosensors and artificial enzymes. Herein, this work reports the chiro-optical properties and architectural arrangement of optoelectronic materials generated from self-assembly of initially nonchiral oligothiophene-porphyrin derivatives and random coil synthetic peptides. The photo-physical- and structural properties of the materials were assessed by absorption-, fluorescence- and circular dichroism spectroscopy, as well as dynamic light scattering, scanning electron microscopy and theoretical calculations. The materials display a three-dimensional ordered helical structure and optical activity that are observed due to an induced chirality of the optoelectronic element upon interaction with the peptide. Both these properties are influenced by the chemical composition of the oligothiophene-porphyrin derivative, as well as the peptide sequence. We foresee that our findings will aid in developing self-assembled optoelectronic materials with dynamic architectonical accuracies, as well as offer the possibility to generate the next generation of materials for a variety of bioelectronic applications.

Whole-brain microscopy reveals distinct temporal and spatial efficacy of anti-Aß therapies.

Many efforts targeting amyloid-ß (Aß) plaques for the treatment of Alzheimer's Disease thus far have resulted in failures during clinical trials. Regional and temporal heterogeneity of efficacy and dependence on plaque maturity may have contributed to these disappointing outcomes. In this study, we mapped the regional and temporal specificity of various anti-Aß treatments through high-resolution light-sheet imaging of electrophoretically cleared brains. We assessed the effect on amyloid plaque formation and growth in Thy1-APP/PS1 mice subjected to ß-secretase inhibitors, polythiophenes, or anti-Aß antibodies. Each treatment showed unique spatiotemporal Aß clearance, with polythiophenes emerging as a potent anti-Aß compound. Furthermore, aligning with a spatial-transcriptomic atlas revealed transcripts that correlate with the efficacy of each Aß therapy. As observed in this study, there is a striking dependence of specific treatments on the location and maturity of Aß plaques. This may also contribute to the clinical trial failures of Aß-therapies, suggesting that combinatorial regimens may be significantly more effective in clearing amyloid deposition.

Distinct Heterocyclic Moieties Govern the Selectivity of Thiophene-Vinylene-Based Ligands Towards Aß or Tau Pathology in Alzheime's Disease.

Distinct aggregated proteins are correlated with numerous neurodegenerative diseases and the development of ligands that selectively detect these pathological hallmarks is vital. Recently, the synthesis of thiophene-based optical ligands, denoted bi-thiophene-vinyl-benzothiazoles (bTVBTs), that could be utilized for selective assignment of tau pathology in brain tissue with Alzheime's disease (AD) pathology, was reported. Herein, we investigate the ability of these ligands to selectively distinguish tau deposits from aggregated amyloid-ß (Aß), the second AD associated pathological hallmark, when replacing the terminal thiophene moiety with other heterocyclic motifs. The selectivity for tau pathology was reduced when introducing specific heterocyclic motifs, verifying that specific molecular interactions between the ligands and the aggregates are necessary for selective detection of tau deposits. In addition, ligands having certain heterocyclic moieties attached to the central thiophene-vinylene building block displayed selectivity to aggregated Aß pathology. Our findings provide chemical insights for the development of ligands that can distinguish between aggregated proteinaceous species consisting of different proteins and might also aid in creating novel agents for clinical imaging of tau pathology in AD.

Medin co-aggregates with vascular amyloid-ß in Alzheimer's disease.

Aggregates of medin amyloid (a fragment of the protein MFG-E8, also known as lactadherin) are found in the vasculature of almost all humans over 50 years of age1,2, making it the most common amyloid currently known. We recently reported that medin also aggregates in blood vessels of ageing wild-type mice, causing cerebrovascular dysfunction3. Here we demonstrate in amyloid-ß precursor protein (APP) transgenic mice and in patients with Alzheimer's disease that medin co-localizes with vascular amyloid-ß deposits, and that in mice, medin deficiency reduces vascular amyloid-ß deposition by half. Moreover, in both the mouse and human brain, MFG-E8 is highly enriched in the vasculature and both MFG-E8 and medin levels increase with the severity of vascular amyloid-ß burden. Additionally, analysing data from 566 individuals in the ROSMAP cohort, we find that patients with Alzheimer's disease have higher MFGE8 expression levels, which are attributable to vascular cells and are associated with increased measures of cognitive decline, independent of plaque and tau pathology. Mechanistically, we demonstrate that medin interacts directly with amyloid-ß to promote its aggregation, as medin forms heterologous fibrils with amyloid-ß, affects amyloid-ß fibril structure, and cross-seeds amyloid-ß aggregation both in vitro and in vivo. Thus, medin could be a therapeutic target for prevention of vascular damage and cognitive decline resulting from amyloid-ß deposition in the blood vessels of the brain.

Proteophenes - Amino Acid Functionalized Thiophene-based Fluorescent Ligands for Visualization of Protein Deposits in Tissue Sections with Alzheimer's Disease Pathology.

Protein deposits composed of specific proteins or peptides are associated with several neurodegenerative diseases and fluorescent ligands able to detect these pathological hallmarks are vital. Here, we report the synthesis of a class of thiophene-based ligands, denoted proteophenes, with different amino acid side-chain functionalities along the conjugated backbone, which display selectivity towards specific disease-associated protein aggregates in tissue sections with Alzheimer's disease (AD) pathology. The selectivity of the ligands towards AD associated pathological hallmarks, such as aggregates of the amyloid-ß (Aß) peptide or tau filamentous inclusions, was highly dependent on the chemical nature of the amino acid functionality, as well as on the location of the functionality along the pentameric thiophene backbone. Finally, the concept of synthesizing donor-acceptor-donor proteophenes with distinct photophysical properties was shown. Our findings provide the structural and functional basis for the development of new thiophene-based ligands that can be utilized for optical assignment of different aggregated proteinaceous species in tissue sections.

Multiscale optical and optoacoustic imaging of amyloid-ß deposits in mice.

Deposits of amyloid-ß (Aß) in the brains of rodents can be analysed by invasive intravital microscopy on a submillimetre scale, or via whole-brain images from modalities lacking the resolution or molecular specificity to accurately characterize Aß pathologies. Here we show that large-field multifocal illumination fluorescence microscopy and panoramic volumetric multispectral optoacoustic tomography can be combined to longitudinally assess Aß deposits in transgenic mouse models of Alzheimer's disease. We used fluorescent Aß-targeted probes (the luminescent conjugated oligothiophene HS-169 and the oxazine-derivative AOI987) to transcranially detect Aß deposits in the cortex of APP/PS1 and arcAß mice with single-plaque resolution (8 µm) and across the whole brain (including the hippocampus and the thalamus, which are inaccessible by conventional intravital microscopy) at sub-150 µm resolutions. Two-photon microscopy, light-sheet microscopy and immunohistochemistry of brain-tissue sections confirmed the specificity and regional distributions of the deposits. High-resolution multiscale optical and optoacoustic imaging of Aß deposits across the entire brain in rodents thus facilitates the in vivo study of Aß accumulation by brain region and by animal age and strain.

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

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.