Investigating the role of ethanolamine plasmalogen lipid in zebrafish brain by interdisciplinary lipidomics

Abstract

Neurolipidomics 1, an evolving field at the intersection between lipid research and neuroscience, aims to offer insights into brain lipid function and thus inform the diagnosis and treatment of neurological disorders. Lipids, encompassing fatty acids, cholesterol, and phospholipids play a pivotal role in glial and neuronal cell function. Dysregulated lipid metabolism has been implicated in neurodegenerative diseases including Alzheimer’s 2,3. Notably, ethanolamine plasmalogen, a sub-class of brain phospholipids, shows a close association with Alzheimer’s disease, with decreased levels correlating with cognitive decline and disease severity 4. Zebrafish ( Danio rerio), with its nervous system akin to humans, high fecundity, and short generation time is a popular model organism in neuroscience 5. With a fully sequenced genome, and transparent embryo facilitating developmental studies, Zebrafish can be easily experimentally manipulated and visualised 6. Both adult and larval stages can be studied to further enhance our understanding of brain development and aging, function, and dysfunction 7. Despite being a powerful tool for neuroscience research, the lipid composition of Zebrafish remains inadequately characterized. Specifically, there is conflicting evidence regarding the presence of plasmalogen ethanolamine (PEp) in this model 8,9. Nevertheless plasmalogen, and specifically PEp, are known to be present in fish 10. Our research aims to investigate the role of plasmalogen lipids in the brain. Therefore, the existing gap of knowledge about the lipid composition of the zebrafish brain prompted us to utilise shotgun lipidomics to characterise the lipidome of zebrafish during development and in the adult brain. Our first objective was to provide conclusive evidence of the presence of PEp in the model, legalising its use to study the effect of brain plasmalogen deficiency in vivo. We have therefore collected Zebrafish larvae at different stages of development (0, 24, 48, 72 hours, and 5 days, post-fertilisation) as well as dissected brains from adult fish (3 months, M/F). Samples were subjected to lipid extraction according to the Bligh and Dyer method. Lipid extracts were analysed by targeted ESI-HRAM-MS/MS shotgun lipidomics using an LTQ-Orbitrap XL (ThermoFisher Scientific) coupled with a TriVersa NanoMate (Advion Biosciences) following established methods 11. PEp species identification is confirmed by both MS n analyses 12 and sensitivity to mild acid hydrolysis 13. Our preliminary data indicates plasmalogen PE is present in zebrafish larvae throughout development and later in the adult brain, warranting the use of this in vivo model for studying the role of brain plasmalogen. Next steps will encompass characterising the lipidome in the aging fish and the use gene silencing to deplete plasmalogen levels. Molecular and functional readouts will be implemented to probe brain plasmalogen biochemistry and relate to function. Investigating the role of ethanolamine plasmalogen lipid in the zebrafish brain Angelo Robles, Iwan Gane, David Lamb, Emma Kenyon, Roberto Angelini. Medical School, Swansea University, Swansea, SA2 8PP, UK. Neurolipidomics 1, an evolving field at the intersection between lipid research and neuroscience, aims to offer insights into brain lipid function and thus inform the diagnosis and treatment of neurological disorders. Lipids, encompassing fatty acids, cholesterol, and phospholipids play a pivotal role in glial and neuronal cell function. Dysregulated lipid metabolism has been implicated in neurodegenerative diseases including Alzheimer’s 2,3. Notably, ethanolamine plasmalogen, a sub-class of brain phospholipids, shows a close association with Alzheimer’s disease, with decreased levels correlating with cognitive decline and disease severity 4. Zebrafish ( Danio rerio), with its nervous system akin to humans, high fecundity, and short generation time is a popular model organism in neuroscience 5. With a fully sequenced genome, and transparent embryo facilitating developmental studies, Zebrafish can be easily experimentally manipulated and visualised 6. Both adult and larval stages can be studied to further enhance our understanding of brain development and aging, function, and dysfunction 7. Despite being a powerful tool for neuroscience research, the lipid composition of Zebrafish remains inadequately characterized. Specifically, there is conflicting evidence regarding the presence of plasmalogen ethanolamine (PEp) in this model 8,9. Nevertheless plasmalogen, and specifically PEp, are known to be present in fish 10. Our research aims to investigate the role of plasmalogen lipids in the brain. Therefore, the existing gap of knowledge about the lipid composition of the zebrafish brain prompted us to utilise shotgun lipidomics to characterise the lipidome of zebrafish during development and in the adult brain. Our first objective was to provide conclusive evidence of the presence of PEp in the model, legalising its use to study the effect of brain plasmalogen deficiency in vivo. We have therefore collected Zebrafish larvae at different stages of development (0, 24, 48, 72 hours, and 5 days, post-fertilisation) as well as dissected brains from adult fish (3 months, M/F). Samples were subjected to lipid extraction according to the Bligh and Dyer method. Lipid extracts were analysed by targeted ESI-HRAM-MS/MS shotgun lipidomics using an LTQ-Orbitrap XL (ThermoFisher Scientific) coupled with a TriVersa NanoMate (Advion Biosciences) following established methods 11. PEp species identification is confirmed by both MS n analyses 12 and sensitivity to mild acid hydrolysis 13. Our preliminary data indicates plasmalogen PE is present in zebrafish larvae throughout development and later in the adult brain, warranting the use of this in vivo model for studying the role of brain plasmalogen. Next steps will encompass characterising the lipidome in the aging fish and the use gene silencing to deplete plasmalogen levels. Molecular and functional readouts will be implemented to probe brain plasmalogen biochemistry and relate to function. References: 1. Han, X. Neurolipidomics: challenges and developments. Front Biosci 12, 2601–2615 (2007). 2. Jones, L., Harold, D. & Williams, J. Genetic evidence for the involvement of lipid metabolism in Alzheimer’s disease. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1801, 754–761 (2010). 3. Kunkle, B. W. et al. Genetic meta-analysis of diagnosed Alzheimer’s disease identifies new risk loci and implicates Aβ, tau, immunity and lipid processing. Nat Genet 51, 414–430 (2019). 4. Kling, M. A. et al. Circulating ethanolamine plasmalogen indices in Alzheimer’s disease: Relation to diagnosis, cognition, and CSF tau. Alzheimers Dement 16, 1234–1247 (2020). 5. Stewart, A. M., Braubach, O., Spitsbergen, J., Gerlai, R. & Kalueff, A. V. Zebrafish models for translational neuroscience research: from tank to bedside. Trends Neurosci 37, 264–278 (2014). 6. Xi, Y., Noble, S. & Ekker, M. Modeling Neurodegeneration in Zebrafish. Curr Neurol Neurosci Rep 11, 274–282 (2011). 7. Kalueff, A. V, Stewart, A. M. & Gerlai, R. Zebrafish as an emerging model for studying complex brain disorders. Trends Pharmacol Sci 35, 63–75 (2014). 8. Van Amerongen, Y. F. et al. Zebrafish brain lipid characterization and quantification by 1H nuclear magnetic resonance spectroscopy and MALDI-TOF mass spectrometry. Zebrafish 11, 240–247 (2014). 9. Fraher, D. et al. Zebrafish Embryonic Lipidomic Analysis Reveals that the Yolk Cell Is Metabolically Active in Processing Lipid. Cell Rep 14, 1317–1329 (2016). 10. 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