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An In Vivo Model of a-Synuclein Spread from Gut to Brain

ABSTRACT: Background: Parkinson’s disease is a progressive neurodegenerative disorder characterized by the presence of pathological aggregation of the protein -synuclein and the loss of dopaminergic neurons in the substantia nigra. There is evidence that misfolding and propagation of -synuclein aggregates through networks of interconnected neurons is responsible for the pathological spread and progressive neuron loss. However, in vivo models demonstrating such pathological progression remain elusive. Results: This study utilizes a zebrafish model in order to interrogate the mechanisms of -synuclein toxicity and spread. We describe the development of a zebrafish model of neuronal human -synuclein overexpression that causes, in young fish, behavioral and neuronal changes as well as microglia activation. In aged fish, -synuclein expression induces a slow but progressive pathological phenotype manifesting in neuron loss within the gut and the CNS. This model is further utilized to seed gut pathology by incorporating a novel method of feeding human -synuclein preformed fibrils in order to initiate protein misfolding at an early age. The combination of neuronal expression of -synuclein and the exogenous addition of misfolded protein facilitates the development of brain pathology and subsequent neuron loss in the CNS. In addition to the pathological alterations induced with the fibril feeding model, genetic changes were identified by single cell RNA sequencing. These transcriptomic changes resulted in pathway alteration that implicate neurodegenerative disease processes. Conclusion: This model of -synuclein pathology is useful for understanding mechanisms underlying disease initiation and can replicate the progressive development of pathological synuclein accumulation. It has the potential to induce neuron to neuron spread and also offers a way to explore what interventions may prevent such pathological progression.

ORGANISM(S): Danio rerio

PROVIDER: GSE316223 | GEO | 2026/01/17

REPOSITORIES: GEO

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Project description:Although-synuclein is implicated in the pathogenesis of Parkinson’s disease and related disorders, it remains unclear whether specific conformations or levels of-synuclein assemblies are toxic and how they cause progressive loss of human dopaminergic neurons. To address this issue, we used iPSC-derived dopaminergic neurons with -synuclein triplication or controls where endogenous -synuclein was imprinted into synthetic or disease-relevant conformations. We used -synuclein fibrils generated de novo or amplified from homogenates of brains affected with Parkinson’s disease (n=3) or multiple system atrophy (n=5). We found that a 2.5-fold increase in -synuclein levels in -synuclein gene triplication neurons promoted seeded aggregation in a dose and time-dependent fashion, which was associated with a further increase in -synuclein gene expression. Progressive neuronal loss was observed only in -synuclein triplication neurons seeded with brain-amplified fibrils. Transcriptomic analysis and isogenic correction of -synuclein triplication revealed that intraneuronal-synuclein levels solely and sufficiently explained vulnerability to neuronal death. Proximity-dependent biotinylation in living cells identified 56 differentially interacting proteins with endogenously assembled -synuclein including evasion of Parkinson’s disease-associated deglycase DJ-1 by aggregates triggered with brain amplified fibrils. Knockout of DJ-1 and related glyoxalase-1 in cell lines increased -synuclein aggregation. Similarly, methylglyoxal treatment or CRISPR/Cas9 knockout of DJ-1 in iPSC-derived dopaminergic neurons enhanced fibril-induced aggregation and cell death. Thus, toxicity of -synuclein strains depends on aggregate burden, which is determined by monomer levels and conformation which dictates differential interactomes. Our results define parameters for iPSC-based modellingof -synuclein pathology using brain amplified fibrils and demonstrate how Parkinson’s disease-associated genes influence the phenotypic manifestation of strains in human neurons.

Project description:Abnormal accumulation of aggregated proteins and sustained microglial activation are important contributors of neurodegenerative process in neurological diseases. Recent studies have shown that aggregation-prone proteins, such as a-synuclein, the protein implicated in Parkinson’s disease (PD), are released from neuronal cells and thus present in the extracellular fluid, pointing to the possible paracrine effects of these proteins on microglial immune responses. However, the mechanism underlying the disease-associated microglial activation and the role of neuronal proteins in this process remain unknown. Here, we show that extracellular a-synuclein released from neuronal cells is an endogenous ligand of toll-like receptor 2 (TLR2) and activates microglia, which in turn induces neurodegeneration. Interaction between neuron-released a-synuclein and TLR2 and subsequent activation of the TLR2 signaling were demonstrated comprehensively by using computational modeling of signaling network and by the experimental validation in TLR2-deficient microglia both in vitro and in vivo. In contrast to the neuron-released a-synuclein, recombinant a-synuclein proteins, including monomer, oligomer, fibril, or nitrated forms, were not able to interact or activate TLR2, suggesting that neuronal cells have a mechanism of enriching specific forms of a-synuclein capable of activating TLR2 during the process of releasing this protein. Taken together, the results suggest that both neuron-released extracellular a-synuclein and TLR2 might be novel therapeutic targets for modifying neuroinflammation in PD and related neurodegenerative diseases. We collected culture media from differentiated SH-SY5Y cells overexpressing either human a-synuclein (alpha-SCM) or beta-galactosidase (LZCM) and treat these media to primary rat microglia at the concentration of a-synuclein of 1.1M. Transcriptome analyses with microglial cells treated with either aSCM or LZCM at two different time points, 6 h and 24 h.

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An In Vivo Model of a-Synuclein Spread from Gut to Brain

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