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

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. Overall design: This study investigates the impact of human alpha-synuclein (aSyn) overexpression and exogenous fibril exposure on neuronal function and transcriptomic changes in zebrafish (Danio rerio). We generated a transgenic zebrafish model expressing human aSyn in neurons using the UAS-Gal4 system (Tg[HuC:Gal4] × Tg[UAS:aSyn-T2A-eGFP]). Larvae were categorized into four groups: (1) aSyn-negative control, (2) aSyn-positive, (3) aSyn-negative + PFF-fed, and (4) aSyn-positive + PFF-fed. At 5 days post-fertilization (dpf), zebrafish larvae were fed either control diet or food enriched with sonicated human alpha-synuclein preformed fibrils (PFFs) daily until 4 weeks post-fertilization (wpf). For transcriptomic profiling, brains from 5dpf (aSyn+ and aSyn–) and 4wpf (aSyn+ fed control or PFF food) larvae were dissected, dissociated, and processed for single-cell RNA-sequencing (scRNA-seq) using the 10X Genomics Chromium platform. CellRanger and Seurat were used for alignment, clustering, and downstream differential expression analysis. In total, three biological replicates per condition were processed. The study design allows for assessment of cell-type-specific transcriptional responses to aSyn expression and PFF-induced seeding, with a focus on neuronal and microglial changes relevant to Parkinson's disease progression.