Development of a new Parkinson’s disease model using zebrafish to study alphasynuclein aggregation

Abstract

Parkinson's Disease is a neurodegenerative disease mainly caused by the dopaminergic neuron loss in the CNS, specifically substantia nigra. It progresses through three stages: preclinical, prodromal, and clinical. Patients with Parkinson’s disease experience motor symptoms like tremors, postural problems and rigidity as well as non-motor symptoms such as autonomic dysfunction, sleep disorders, and depression. Various genetic and environmental factors influence the disease's progression, making classifying patients based on disease pathology challenging. The SNCA gene encodes the alpha-synuclein protein and is a primary risk factor for Parkinson’s disease. Mutant forms of alphasynuclein can form insoluble fibrils and Lewy bodies, affecting the transmission of healthy proteins between cells. Recent hypotheses suggest that the location of disease emergence in the body (brain-first or body-first) influences disease progression and resulting pathology. Existing zebrafish models have the potential to provide insights into neurodegenerative diseases due to their ease of handling, large population size, and genetic manipulability. This study aims to investigate the relationship between the location of alpha-synuclein emergence and the progression of Parkinson’s disease. In this study, a new zebrafish model expressing the human alpha-synuclein coding sequence was developed using the Tol2 transposase-based recombination system. The initial expression vector was created using multisite gateway cloning methodology, and the constructs were validated at each step using PCR and sequencing. The final construct was co-injected with transposase mRNA into one-cell stage zebrafish embryos to facilitate the formation of a stable line. In addition, human alpha-synuclein fibril injections were performed on young and old zebrafish, either brain or gut. This way, we ensured the transmission of α-syn between the central nervous system and peripheral organs.