CERT
            L reduces C16 ceramide, amyloid-β levels, and inflammation in a model of Alzheimer’s disease

SM Crivelli; Q Luo; JA Stevens; C Giovagnoni; D van Kruining; G Bode; Sandra den Hoedt; B Hobo; AL Scheithauer; J Walter; Monique Mulder; C Exley; M Mold; MM Mielke; HE de Vries; K Wouters; DLA van den Hove; D Berkes; MD Ledesma; J Verhaagen; M Losen; E Bieberich; P Martinez-Martinez

doi:10.1186/s13195-021-00780-0

CERT _L reduces C16 ceramide, amyloid-β levels, and inflammation in a model of Alzheimer’s disease

SM Crivelli, Q Luo, JA Stevens, C Giovagnoni, D van Kruining, G Bode, Sandra den Hoedt, B Hobo, AL Scheithauer, J Walter, Monique Mulder, C Exley, M Mold, MM Mielke, HE de Vries, K Wouters, DLA van den Hove, D Berkes, MD Ledesma, J VerhaagenM Losen, E Bieberich, P Martinez-Martinez

Internal Medicine

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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 CERT_L, the long isoform of CERTs, was used to study the interaction of CERT_L with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERT_L protein was employed to study interaction of CERT_L with amyloid-β (Aβ), Aβ aggregation process in presence of CERT_L, and the resulting changes in Aβ toxicity in neuroblastoma cells. CERT_L 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 CERT_L binds to APP, modifies Aβ aggregation, and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERT_L, decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERT_L 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 CERT_L 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.

Original language	English
Article number	45
Journal	Alzheimer's Research and Therapy
Volume	13
Issue number	1
DOIs	https://doi.org/10.1186/s13195-021-00780-0
Publication status	Published - Dec 2021

Bibliographical note

Funding Information:
This work was supported by grants to NMdW, SdH, MTM, JW, AR, PMM, JV, and HEV from ZonMw Memorabel program (projectnr: 733050105). PMM is also supported by the International Foundation for Alzheimer Research (ISAO) (projectnr: 14545). SMC received a travel grant support from Alzheimer Nederlands foundation (AN) to spend a month in the laboratory of EB, University of Kentucky, Lexington KY, USA. Aspects of this work were supported by the grants NIH R01AG034389, R01NS095215, and R56AG064234; VA I01BX003643 to EB. MMM was supported by R01AG049704.

Funding Information:
We thank Prof. S. Kugler Department of Neurology, University of Gottingen, for the kind gift of adeno-associated virus plasmid. We thank Geertjan van Zonneveld for the professional approach and dedication to complete the illustrations of this research paper.

Publisher Copyright:
© 2021, The Author(s).

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Cite this

Crivelli, SM., Luo, Q., Stevens, JA., Giovagnoni, C., van Kruining, D., Bode, G., den Hoedt, S., Hobo, B., Scheithauer, AL., Walter, J., Mulder, M., Exley, C., Mold, M., Mielke, MM., de Vries, HE., Wouters, K., van den Hove, DLA., Berkes, D., Ledesma, MD., ... Martinez-Martinez, P. (2021). CERT _L reduces C16 ceramide, amyloid-β levels, and inflammation in a model of Alzheimer’s disease. Alzheimer's Research and Therapy, 13(1), Article 45. https://doi.org/10.1186/s13195-021-00780-0

@article{1e20b69219b247a6a251b9c9f5243a40,

title = "CERT L reduces C16 ceramide, amyloid-β levels, and inflammation in a model of Alzheimer{\textquoteright}s disease",

abstract = "Background: Dysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer{\textquoteright}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.",

author = "SM Crivelli and Q Luo and JA Stevens and C Giovagnoni and {van Kruining}, D and G Bode and {den Hoedt}, Sandra and B Hobo and AL Scheithauer and J Walter and Monique Mulder and C Exley and M Mold and MM Mielke and {de Vries}, HE and K Wouters and {van den Hove}, DLA and D Berkes and MD Ledesma and J Verhaagen and M Losen and E Bieberich and P Martinez-Martinez",

note = "Funding Information: This work was supported by grants to NMdW, SdH, MTM, JW, AR, PMM, JV, and HEV from ZonMw Memorabel program (projectnr: 733050105). PMM is also supported by the International Foundation for Alzheimer Research (ISAO) (projectnr: 14545). SMC received a travel grant support from Alzheimer Nederlands foundation (AN) to spend a month in the laboratory of EB, University of Kentucky, Lexington KY, USA. Aspects of this work were supported by the grants NIH R01AG034389, R01NS095215, and R56AG064234; VA I01BX003643 to EB. MMM was supported by R01AG049704. Funding Information: We thank Prof. S. Kugler Department of Neurology, University of Gottingen, for the kind gift of adeno-associated virus plasmid. We thank Geertjan van Zonneveld for the professional approach and dedication to complete the illustrations of this research paper. Publisher Copyright: {\textcopyright} 2021, The Author(s).",

year = "2021",

month = dec,

doi = "10.1186/s13195-021-00780-0",

language = "English",

volume = "13",

journal = "Alzheimer's Research and Therapy",

issn = "1758-9193",

publisher = "BioMed Central Ltd.",

number = "1",

}

Crivelli, SM, Luo, Q, Stevens, JA, Giovagnoni, C, van Kruining, D, Bode, G, den Hoedt, S, Hobo, B, Scheithauer, AL, Walter, J, Mulder, M, Exley, C, Mold, M, Mielke, MM, de Vries, HE, Wouters, K, van den Hove, DLA, Berkes, D, Ledesma, MD, Verhaagen, J, Losen, M, Bieberich, E & Martinez-Martinez, P 2021, 'CERT _L reduces C16 ceramide, amyloid-β levels, and inflammation in a model of Alzheimer’s disease', Alzheimer's Research and Therapy, vol. 13, no. 1, 45. https://doi.org/10.1186/s13195-021-00780-0

TY - JOUR

T1 - CERT L reduces C16 ceramide, amyloid-β levels, and inflammation in a model of Alzheimer’s disease

AU - Crivelli, SM

AU - Luo, Q

AU - Stevens, JA

AU - Giovagnoni, C

AU - van Kruining, D

AU - Bode, G

AU - den Hoedt, Sandra

AU - Hobo, B

AU - Scheithauer, AL

AU - Walter, J

AU - Mulder, Monique

AU - Exley, C

AU - Mold, M

AU - Mielke, MM

AU - de Vries, HE

AU - Wouters, K

AU - van den Hove, DLA

AU - Berkes, D

AU - Ledesma, MD

AU - Verhaagen, J

AU - Losen, M

AU - Bieberich, E

AU - Martinez-Martinez, P

N1 - Funding Information: This work was supported by grants to NMdW, SdH, MTM, JW, AR, PMM, JV, and HEV from ZonMw Memorabel program (projectnr: 733050105). PMM is also supported by the International Foundation for Alzheimer Research (ISAO) (projectnr: 14545). SMC received a travel grant support from Alzheimer Nederlands foundation (AN) to spend a month in the laboratory of EB, University of Kentucky, Lexington KY, USA. Aspects of this work were supported by the grants NIH R01AG034389, R01NS095215, and R56AG064234; VA I01BX003643 to EB. MMM was supported by R01AG049704. Funding Information: We thank Prof. S. Kugler Department of Neurology, University of Gottingen, for the kind gift of adeno-associated virus plasmid. We thank Geertjan van Zonneveld for the professional approach and dedication to complete the illustrations of this research paper. Publisher Copyright: © 2021, The Author(s).

PY - 2021/12

Y1 - 2021/12

N2 - 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.

AB - 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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U2 - 10.1186/s13195-021-00780-0

DO - 10.1186/s13195-021-00780-0

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SN - 1758-9193

VL - 13

JO - Alzheimer's Research and Therapy

JF - Alzheimer's Research and Therapy

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