Objective: To generate disease-representative zebrafish models using CRISPR-Cas9 technique.

To characterise the zebrafish models by performing phenotypic characterization, neurobiological experiments, lipidomic and RNASeq studies.

To test targeted therapies for the Kennedy-pathway-related disorders using the characterised zebrafish models.

Background: Hereditary Spastic Paraplegia (HSP) comprises a broad and heterogeneous group of inherited neurodegenerative and neurodevelopmental disorders. Among the identified spastic paraplegia genes (SPGs), 19 are involved in membrane phospholipid metabolism. The Kennedy pathway is a major biosynthetic route for phosphatidylethanolamine (PE), in which EPT1 encodes ethanolaminephosphotransferase catalyzing the conversion of CDP-ethanolamine to PE. Biallelic loss-of-function mutations in EPT1 have been associated with SPG81.

Method: CRISPR-Cas9 technique was used to create ept1 ex5 knock-out mutants and crispants. A series of phenotypic characterization, RNA sequencing and lipidomic profiling on these zebrafish models were conducted in the zebrafish model.

Results: The ept1 ex5 knock-out mutants harbored an 8-bp deletion in the catalytic domain and were viable, exhibiting a 60% reduction in ept1 mRNA levels. Compared to wild-type controls, homozygous knockouts displayed significant impairments in locomotion, as well as reduced body length and head width. Transcriptomic analysis revealed downregulation of genes involved in light response, synaptic activity, and lipid metabolism, whereas genes associated with immune signaling and neuroinflammation were upregulated. Lipidomic profiling showed a reduction in total PE and phosphatidylcholine (PC) levels in knockouts, with plasmenyl-PE being most severely affected. Through crispant analysis, we found that both ept1 ex5 and cept1b ex4 crispants exhibited significantly impaired locomotion; however, only ept1 ex5 crispants displayed reductions in body length and head width.

Conclusion: The ept1 knock-out mutants and crispants recapitulate the movement disorder and developmental delay observed in SPG81 patients, making them valuable models for disease research. Moreover, our findings suggest that neuroinflammation may play a crucial role in HSP pathogenesis and could serve as a potential therapeutic target.

Figure 1. Background and introduction

Figure 2. Results

Figure 3. Results

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MDS Abstracts - https://www.mdsabstracts.org/abstract/using-zebrafish-model-to-investigate-complex-hereditary-spastic-paraplegia-caused-by-ept1-variants/