Objective: Human patients present punctual mutations and/or small deletions in genes that have been associated with Parkinson’s disease, namely, park1, park2, park6, park7, and park8. Our objective is to target the zebrafish exon counterparts to discover novel therapeutic targets.

Background: Parkinson’s disease (PD) is the second most common neurodegenerative disorder, affecting people over 60 years old. However, the disease can be manifested early in life. Most of the cases are of sporadic etiology and 10-20 % of genetic cause, with mutations occurring in only one gene following a Mendelian’s inheritance pattern. Patients experience motor and no motor symptoms that escalate as disease progresses. High conservation of PD genes is observed between humans and zebrafish, with expression patterns in the central nervous system. This animal model can be easily and cost-effectively modified at a genetic level allowing the study of a plethora of diseases.

Method: We have generated CRISPR/Cas9 loss-of-function alleles of the aforementioned PD-associated genes in zebrafish. Moreover, we have licensed a zebrafish transgenic line overexpressing the human alfa synuclein protein. To validate these models phenotypically, we have employed a standardised protocol to analyse locomotion activity responses and associated behaviours to applied surrounding stimuli. Additionally, dopaminergic neuron loss is being analysed qualitatively and quantitatively using RNA probes and antibodies. Finally, specific neurons are extracted from these models through experiments of FACS sorting to carry out an RNA-seq analysis.

Results: Validation of these models at the cellular/molecular level shows a striking reduction of tyrosine hydroxylase (th1) mRNA and protein levels, suggesting that zebrafish dopaminergic neurons are affected by our genetic disruption. Additionally, preliminary results show some differences in the locomotion behaviour of mutants compared to wild type. Lastly, on-going transcriptomic analysis of postmitotic neurons of our models could show genes differentially expressed, which could help us identify novel molecular targets and altered pathways.

Conclusion: Overall, phenotypic characterisation and a molecular signature identification could pave the ground for future development of new therapeutic strategies and drugs for Parkinson’s Disease.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/developing-zebrafish-crispr-cas9-knockout-models-of-parkinsons-disease-to-identify-novel-therapeutic-targets/