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

Robles, Angelo Ross Andrew Villanueva; Gane, Iwan; Lamb, David; Kenyon, Emma; Angelini, Roberto

doi:10.14293/P2199-8442.1.SOP-.PWMATS.v1

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. Chen, Z., Jia, J., Wu, Y., Chiba, H. & Hui, S.-P. LC/MS analysis of storage-induced plasmalogen loss in ready-to-eat fish. Food Chem 383, 132320 (2022).

11. Nielsen, I. Ø. et al. Comprehensive Evaluation of a Quantitative Shotgun Lipidomics Platform for Mammalian Sample Analysis on a High-Resolution Mass Spectrometer. J Am Soc Mass Spectrom 31, (2020).

12. Hsu, F.-F. Mass spectrometry-based shotgun lipidomics - a critical review from the technical point of view. Anal Bioanal Chem 410, 6387–6409 (2018).

13. Han, X., Yang, K., Cheng, H., Fikes, K. N. & Gross, R. W. Shotgun lipidomics of phosphoethanolamine-containing lipids in biological samples after one-step in situ derivatization. J Lipid Res 46, (2005).

Content

Author and article information

Journal

Title: ScienceOpen Posters

Publisher: ScienceOpen

Publication date (Electronic preprint): 30 April 2024

Affiliations

[1 ] Swansea Unibersity;

[2 ] Swansea University;

Author notes

[* ]Email: 991434@ 123456swansea.ac.uk .

Author information

Angelo Ross Andrew Villanueva Robles https://orcid.org/0009-0006-5008-7281

Iwan Gane https://orcid.org/0009-0004-3758-2822

David Lamb https://orcid.org/0000-0001-5446-2997

Emma Kenyon https://orcid.org/0000-0002-3898-1866

Roberto Angelini https://orcid.org/0000-0001-5136-5921

Article

DOI: 10.14293/P2199-8442.1.SOP-.PWMATS.v1

SO-VID: f00fd839-a600-4b6f-a5b9-2d7247beb341

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This work has been published open access under Creative Commons Attribution License CC BY 4.0 , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Conditions, terms of use and publishing policy can be found at www.scienceopen.com .

History

Date received : 30 April 2024

Funding

Funded by: funder-id http://dx.doi.org/10.13039/501100001317, Swansea University;

References

Han Xianlin. Neurolipidomics: challenges and developments. Frontiers in Bioscience. Vol. 12(1)2007. IMR Press. [Cross Ref]
Jones Lesley, Harold Denise, Williams Julie. Genetic evidence for the involvement of lipid metabolism in Alzheimer's disease. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. Vol. 1801(8):754–761. 2010. Elsevier BV. [Cross Ref]
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Kling Mitchel A., Goodenowe Dayan B., Senanayake Vijitha, MahmoudianDehkordi Siamak, Arnold Matthias, Massaro Tyler J., Baillie Rebecca, Han Xianlin, Leung Yuk‐Yee, Saykin Andrew J., Nho Kwangsik, Kueider‐Paisley Alexandra, Tenenbaum Jessica D., Wang Li‐San, Shaw Leslie M., Trojanowski John Q., Kaddurah‐Daouk Rima F.. Circulating ethanolamine plasmalogen indices in Alzheimer's disease: Relation to diagnosis, cognition, and CSF tau. Alzheimer's & Dementia. Vol. 16(9):1234–1247. 2020. Wiley. [Cross Ref]
Stewart Adam Michael, Braubach Oliver, Spitsbergen Jan, Gerlai Robert, Kalueff Allan V.. Zebrafish models for translational neuroscience research: from tank to bedside. Trends in Neurosciences. Vol. 37(5):264–278. 2014. Elsevier BV. [Cross Ref]
Xi Yanwei, Noble Sandra, Ekker Marc. Modeling Neurodegeneration in Zebrafish. Current Neurology and Neuroscience Reports. Vol. 11(3):274–282. 2011. Springer Science and Business Media LLC. [Cross Ref]
Kalueff Allan V., Stewart Adam Michael, Gerlai Robert. Zebrafish as an emerging model for studying complex brain disorders. Trends in Pharmacological Sciences. Vol. 35(2):63–75. 2014. Elsevier BV. [Cross Ref]
van Amerongen Yvonne F., Roy Upasana, Spaink Herman P., de Groot Huub J.M., Huster Daniel, Schiller Jürgen, Alia A.. Zebrafish Brain Lipid Characterization and Quantification by<sup>1</sup>H Nuclear Magnetic Resonance Spectroscopy and MALDI-TOF Mass Spectrometry. Zebrafish. Vol. 11(3):240–247. 2014. Mary Ann Liebert Inc. [Cross Ref]
Fraher Daniel, Sanigorski Andrew, Mellett Natalie A., Meikle Peter J., Sinclair Andrew J., Gibert Yann. Zebrafish Embryonic Lipidomic Analysis Reveals that the Yolk Cell Is Metabolically Active in Processing Lipid. Cell Reports. Vol. 14(6):1317–1329. 2016. Elsevier BV. [Cross Ref]
Chen Zhen, Jia Jiaping, Wu Yue, Chiba Hitoshi, Hui Shu-Ping. LC/MS analysis of storage-induced plasmalogen loss in ready-to-eat fish. Food Chemistry. Vol. 383:2022. Elsevier BV. [Cross Ref]
Ferreri Carla, Ferocino Alessandra, Batani Gessica, Chatgilialoglu Chryssostomos, Randi Vanda, Riontino Maria Vittoria, Vetica Fabrizio, Sansone Anna. Plasmalogens: Free Radical Reactivity and Identification of Trans Isomers Relevant to Biological Membranes. Biomolecules. Vol. 13(5)2023. MDPI AG. [Cross Ref]
Dorninger Fabian, Berger Johannes, Honsho Masanori. Editorial: Solving the plasmalogen puzzle—From basic science to clinical application. Frontiers in Cell and Developmental Biology. Vol. 11:2023. Frontiers Media SA. [Cross Ref]

Comments

[1] Han Xianlin. Neurolipidomics: challenges and developments. Frontiers in Bioscience. Vol. 12(1)2007. IMR Press. [Cross Ref]

[2] Jones Lesley, Harold Denise, Williams Julie. Genetic evidence for the involvement of lipid metabolism in Alzheimer's disease. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. Vol. 1801(8):754–761. 2010. Elsevier BV. [Cross Ref]

[4] Kling Mitchel A., Goodenowe Dayan B., Senanayake Vijitha, MahmoudianDehkordi Siamak, Arnold Matthias, Massaro Tyler J., Baillie Rebecca, Han Xianlin, Leung Yuk‐Yee, Saykin Andrew J., Nho Kwangsik, Kueider‐Paisley Alexandra, Tenenbaum Jessica D., Wang Li‐San, Shaw Leslie M., Trojanowski John Q., Kaddurah‐Daouk Rima F.. Circulating ethanolamine plasmalogen indices in Alzheimer's disease: Relation to diagnosis, cognition, and CSF tau. Alzheimer's & Dementia. Vol. 16(9):1234–1247. 2020. Wiley. [Cross Ref]

[5] Stewart Adam Michael, Braubach Oliver, Spitsbergen Jan, Gerlai Robert, Kalueff Allan V.. Zebrafish models for translational neuroscience research: from tank to bedside. Trends in Neurosciences. Vol. 37(5):264–278. 2014. Elsevier BV. [Cross Ref]

[6] Xi Yanwei, Noble Sandra, Ekker Marc. Modeling Neurodegeneration in Zebrafish. Current Neurology and Neuroscience Reports. Vol. 11(3):274–282. 2011. Springer Science and Business Media LLC. [Cross Ref]

[7] Kalueff Allan V., Stewart Adam Michael, Gerlai Robert. Zebrafish as an emerging model for studying complex brain disorders. Trends in Pharmacological Sciences. Vol. 35(2):63–75. 2014. Elsevier BV. [Cross Ref]

[8] van Amerongen Yvonne F., Roy Upasana, Spaink Herman P., de Groot Huub J.M., Huster Daniel, Schiller Jürgen, Alia A.. Zebrafish Brain Lipid Characterization and Quantification by<sup>1</sup>H Nuclear Magnetic Resonance Spectroscopy and MALDI-TOF Mass Spectrometry. Zebrafish. Vol. 11(3):240–247. 2014. Mary Ann Liebert Inc. [Cross Ref]

[9] Fraher Daniel, Sanigorski Andrew, Mellett Natalie A., Meikle Peter J., Sinclair Andrew J., Gibert Yann. Zebrafish Embryonic Lipidomic Analysis Reveals that the Yolk Cell Is Metabolically Active in Processing Lipid. Cell Reports. Vol. 14(6):1317–1329. 2016. Elsevier BV. [Cross Ref]

[10] Chen Zhen, Jia Jiaping, Wu Yue, Chiba Hitoshi, Hui Shu-Ping. LC/MS analysis of storage-induced plasmalogen loss in ready-to-eat fish. Food Chemistry. Vol. 383:2022. Elsevier BV. [Cross Ref]

[11] Ferreri Carla, Ferocino Alessandra, Batani Gessica, Chatgilialoglu Chryssostomos, Randi Vanda, Riontino Maria Vittoria, Vetica Fabrizio, Sansone Anna. Plasmalogens: Free Radical Reactivity and Identification of Trans Isomers Relevant to Biological Membranes. Biomolecules. Vol. 13(5)2023. MDPI AG. [Cross Ref]

[12] Dorninger Fabian, Berger Johannes, Honsho Masanori. Editorial: Solving the plasmalogen puzzle—From basic science to clinical application. Frontiers in Cell and Developmental Biology. Vol. 11:2023. Frontiers Media SA. [Cross Ref]