Simone M Crivelli1,2,3, Qian Luo1, Jo A A Stevens1, Caterina Giovagnoni1, Daan van Kruining1, Gerard Bode1, Sandra den Hoedt4, Barbara Hobo5, Anna-Lena Scheithauer6, Jochen Walter6, Monique T Mulder4, Christopher Exley7, Matthew Mold7, Michelle M Mielke8, Helga E De Vries9, Kristiaan Wouters10,11, Daniel L A van den Hove12, Dusan Berkes13, María Dolores Ledesma14, Joost Verhaagen5, Mario Losen1, Erhard Bieberich2,3, Pilar Martinez-Martinez15. 1. Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Universiteitssingel 50, 6229 ER, Maastricht, the Netherlands. 2. Department of Physiology, University of Kentucky College of Medicine, Lexington, KY, USA. 3. Veterans Affairs Medical Center, Lexington, KY, 40502, USA. 4. Department of Internal Medicine, Laboratory Vascular Medicine, Erasmus MC University Medical Center, Rotterdam, the Netherlands. 5. Laboratory for Neuroregeneration, Netherlands institute for Neuroscience, Amsterdam, the Netherlands. 6. Department of Neurology, University Hospital Bonn, University of Bonn, Bonn, Germany. 7. The Birchall Centre, Lennard-Jones Laboratories, Keele University, Staffordshire, UK. 8. Division of Epidemiology, Department of Health Science Research, and Department of Neurology, Mayo Clinic Rochester, Rochester, MN, USA. 9. Department of Molecular Cell Biology and Immunology, Amsterdam Neuroscience, Amsterdam UMC, Amsterdam, the Netherlands. 10. Department of Internal Medicine, Maastricht University Medical Centre, Maastricht, the Netherlands. 11. Cardiovascular Research Institute Maastricht (CARIM), Maastricht, the Netherlands. 12. Department of Psychiatry, Psychosomatics and Psychotherapy, University of Wuerzburg, Wuerzburg, Germany. 13. Department of Organic Chemistry, Slovak University of Technology, Radlinského 9, 81237, Bratislava, Slovak Republic. 14. Department of Molecular Neuropathology, Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Madrid, Spain. 15. Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Universiteitssingel 50, 6229 ER, Maastricht, the Netherlands. p.martinez@maastrichtuniversity.nl.
Abstract
BACKGROUND: Dysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer's disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain. METHODS: A plasmid expressing CERTL, the long isoform of CERTs, was used to study the interaction of CERTL with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERTL protein was employed to study interaction of CERTL with amyloid-β (Aβ), Aβ aggregation process in presence of CERTL, and the resulting changes in Aβ toxicity in neuroblastoma cells. CERTL was overexpressed in neurons by adeno-associated virus (AAV) in a mouse model of familial AD (5xFAD). Ten weeks after transduction, animals were challenged with behavior tests for memory, anxiety, and locomotion. At week 12, brains were investigated for sphingolipid levels by mass spectrometry, plaques, and neuroinflammation by immunohistochemistry, gene expression, and/or immunoassay. RESULTS: Here, we report that CERTL binds to APP, modifies Aβ aggregation, and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERTL, decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERTL in vivo over-expression has a mild effect on animal locomotion, decreases Aβ formation, and modulates microglia by decreasing their pro-inflammatory phenotype. CONCLUSION: Our results demonstrate a crucial role of CERTL in regulating ceramide levels in the brain, in amyloid plaque formation and neuroinflammation, thereby opening research avenues for therapeutic targets of AD and other neurodegenerative diseases.
BACKGROUND: Dysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer's disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain. METHODS: A plasmid expressing CERTL, the long isoform of CERTs, was used to study the interaction of CERTL with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERTL protein was employed to study interaction of CERTL with amyloid-β (Aβ), Aβ aggregation process in presence of CERTL, and the resulting changes in Aβ toxicity in neuroblastoma cells. CERTL was overexpressed in neurons by adeno-associated virus (AAV) in a mouse model of familial AD (5xFAD). Ten weeks after transduction, animals were challenged with behavior tests for memory, anxiety, and locomotion. At week 12, brains were investigated for sphingolipid levels by mass spectrometry, plaques, and neuroinflammation by immunohistochemistry, gene expression, and/or immunoassay. RESULTS: Here, we report that CERTL binds to APP, modifies Aβ aggregation, and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERTL, decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERTL in vivo over-expression has a mild effect on animal locomotion, decreases Aβ formation, and modulates microglia by decreasing their pro-inflammatory phenotype. CONCLUSION: Our results demonstrate a crucial role of CERTL in regulating ceramide levels in the brain, in amyloid plaque formation and neuroinflammation, thereby opening research avenues for therapeutic targets of AD and other neurodegenerative diseases.
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