Monoamine oxidase B is associated with γ-secretase in cultured neurons and in brain
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POSTER PRESENTATION Open Access Monoamine oxidase B is associated with g-secretase in cultured neurons and in brain Sophia Schedin Weiss, Birgitta Wiehager, Bengt Winblad, Susanne Frykman, Lars Tjernberg * From Molecular Neurodegeneration: Basic biology and disease pathways Cannes, France. 10-12 September 2013 Background Monoamine oxidase B (MAOB) is a mitochondrial outer membrane protein that degrades monoamines such as dopamine. Thus, MAOB inhibitors are used to treat for instance Parkinson’s disease patients. MAOB inhibitors, however, have neuroprotective properties that are inde- pendent on the monoamine-degrading capabilities and it is possible that MAOB have additional unrevealed functions. In an affinity pulldown/mass spectrometric screen for potential regulators of APP processing, we found MAOB to be associated with g-secretase in membranes prepared from brain, and data from co-immunoprecipitation experiments supported this finding. siRNA experiments showed that reduced levels of MAOB resulted in reduced Ab production, with only minor effects on the release of Notch intracellular domain (manuscript in preparation). In the present study, we use the proximity ligation assay (PLA), which gives a fluorescent signal when two target proteins are less than 40 nm apart, to study the MAOB- g-secretase interaction in primary neurons and brain. Materials and methods PLA was performed on fixed and permeabilized mouse primary hippocampal neurons and deparaffinized thin sections from human brain cortex. The protocol from Olink Bioscience was essentially followed. Validated rabbit anti-MAOB and mouse anti-PS1 loop antibodies were used in the first incubation step, followed by secondary antibodies conjugated to oligonucleotide strands (MINUS and PLUS) in the second step. Reagents were from Olink Bioscience, and images were acquired using a Zeiss 510 confocal microscope (40x oil immersion objective). For immunohistochemistry, thin sections (7 μm) of formalde- hyde fixed, paraffin embedded human frontal cortex were de-waxed and rehydrated. The sections were heated to 120°C for efficient epitope retrieval and stained for MAOB which was visualized using the DAB/H 2 O 2 method. Micro- graphs were acquired with a brightfield microscope. Results A clear interaction between g-secretase and MAOB was observed in the primary neurons. The neuronal interaction was also supported by PLA studies on human brain sec- tions. Immunohistochemistry using two different MAOB antibodies showed intense staining of neurons in human brain frontal cortex and hippocampus. The neuronal stain- ing was more intense in AD than in control brains. Alto- gether, these data support that the interaction between MAOB and g-secretase in neurons may be an interesting target for AD treatment. Conclusions By several independent techniques, we have shown that monoamine oxidase B is associated to g-secretase in neu- rons as well as in brain. Interestingly, siRNA mediated gene silencing of MAOB in cells reduces Ab production, but not Notch processing. Thus, we conclude that interfer- ing with the g-secretase-MAOB interaction could be a therapeutic strategy for lowering Ab levels in human brain. Acknowledgements We thank Alzheimerfonden and Dainippon Sumitoma Pharma for their generous support. Published: 4 October 2013 doi:10.1186/1750-1326-8-S1-P69 Cite this article as: Weiss et al.: Monoamine oxidase B is associated with g-secretase in cultured neurons and in brain. Molecular Neurodegeneration 2013 8(Suppl 1):P69. NVS/Karolinska Institutet, Stockhom, Sweden Weiss et al. Molecular Neurodegeneration 2013, 8(Suppl 1):P69 http://www.molecularneurodegeneration.com/content/8/S1/P69 © 2013 Weiss et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Transcript of Monoamine oxidase B is associated with γ-secretase in cultured neurons and in brain
POSTER PRESENTATION Open Access
Monoamine oxidase B is associated withg-secretase in cultured neurons and in brainSophia Schedin Weiss, Birgitta Wiehager, Bengt Winblad, Susanne Frykman, Lars Tjernberg*
From Molecular Neurodegeneration: Basic biology and disease pathwaysCannes, France. 10-12 September 2013
BackgroundMonoamine oxidase B (MAOB) is a mitochondrial outermembrane protein that degrades monoamines such asdopamine. Thus, MAOB inhibitors are used to treat forinstance Parkinson’s disease patients. MAOB inhibitors,however, have neuroprotective properties that are inde-pendent on the monoamine-degrading capabilities and itis possible that MAOB have additional unrevealedfunctions.In an affinity pulldown/mass spectrometric screen for
potential regulators of APP processing, we foundMAOB to be associated with g-secretase in membranesprepared from brain, and data from co-immunoprecipitationexperiments supported this finding. siRNA experimentsshowed that reduced levels of MAOB resulted inreduced Ab production, with only minor effects on therelease of Notch intracellular domain (manuscript inpreparation).In the present study, we use the proximity ligation assay
(PLA), which gives a fluorescent signal when two targetproteins are less than 40 nm apart, to study the MAOB-g-secretase interaction in primary neurons and brain.
Materials and methodsPLA was performed on fixed and permeabilized mouseprimary hippocampal neurons and deparaffinized thinsections from human brain cortex. The protocol fromOlink Bioscience was essentially followed. Validated rabbitanti-MAOB and mouse anti-PS1 loop antibodies wereused in the first incubation step, followed by secondaryantibodies conjugated to oligonucleotide strands (MINUSand PLUS) in the second step. Reagents were from OlinkBioscience, and images were acquired using a Zeiss 510confocal microscope (40x oil immersion objective). For
immunohistochemistry, thin sections (7 μm) of formalde-hyde fixed, paraffin embedded human frontal cortex werede-waxed and rehydrated. The sections were heated to120°C for efficient epitope retrieval and stained for MAOBwhich was visualized using the DAB/H2O2 method. Micro-graphs were acquired with a brightfield microscope.
ResultsA clear interaction between g-secretase and MAOB wasobserved in the primary neurons. The neuronal interactionwas also supported by PLA studies on human brain sec-tions. Immunohistochemistry using two different MAOBantibodies showed intense staining of neurons in humanbrain frontal cortex and hippocampus. The neuronal stain-ing was more intense in AD than in control brains. Alto-gether, these data support that the interaction betweenMAOB and g-secretase in neurons may be an interestingtarget for AD treatment.
ConclusionsBy several independent techniques, we have shown thatmonoamine oxidase B is associated to g-secretase in neu-rons as well as in brain. Interestingly, siRNA mediatedgene silencing of MAOB in cells reduces Ab production,but not Notch processing. Thus, we conclude that interfer-ing with the g-secretase-MAOB interaction could be atherapeutic strategy for lowering Ab levels in human brain.
AcknowledgementsWe thank Alzheimerfonden and Dainippon Sumitoma Pharma for theirgenerous support.
Published: 4 October 2013
doi:10.1186/1750-1326-8-S1-P69Cite this article as: Weiss et al.: Monoamine oxidase B is associated withg-secretase in cultured neurons and in brain. MolecularNeurodegeneration 2013 8(Suppl 1):P69.
NVS/Karolinska Institutet, Stockhom, Sweden
Weiss et al. Molecular Neurodegeneration 2013, 8(Suppl 1):P69http://www.molecularneurodegeneration.com/content/8/S1/P69
© 2013 Weiss et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.
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