Fig 2: Proximity ligation assay (PLA) to visualize the PS1/MAO-B and ?-secretase/MAO-B interactions in mouse brain. Female adult mouse brains were fixed in formalin, and 10-µm sections were cut using a cryostat and stored at -20 °C prior to PLA assay. a Cross-section of a mouse brain showing the regions chosen for PLA imaging: cortex (blue squares) and hippocampal CA1 (red squares). b–e PLA in mouse hippocampal CA1 to visualize the association between PS1 and MAO-B (using a PS1 and an MAO-B antibody) and between active ?-secretase and MAO-B (using GTB and an MAO-B antibody) as well as negative controls (only MAO-B antibody and competition in the presence of L-685,458). f–i PLA in mouse cortex (with the same antibody and GTB combinations as in b–e). Insets are enlarged parts of the original images to clearly visualize the PLA signals (note that the small size of PLA signals makes it difficult to see them on printouts). j Quantification of the PLA signals in hippocampal CA1 and cortex for the PS1/MAO-B association. k Quantification of the PLA signals in hippocampal CA1 and cortex for the association between mature ?-secretase and MAO-B. Quantification of the number of PLA signals was done using the Duolink image tool. Mean values ± SE from at least three images in 10 experiments (traditional PLA) or three experiments (GTP-PLA) are shown (Color figure online)

Fig 3: MAO-B silencing by siRNA transfection and its effect on intracellular Aß42. The effect was investigated for three concentrations of control-siRNA/MAO-B siRNA2 (0.36, 2.9 and 29 nM). Localization of MAO-B staining by proximity ligation assay (PLA; red) in neurons, intracellular Aß42 staining (green), DAPI nuclear staining (blue) and F-actin-binding TRITC-conjugated phalloidin (gray) is illustrated for both control-siRNA-treated (a) and MAO-B siRNA2-treated (e) cells. TRITC-conjugated phalloidin staining shows no significant alterations in neuronal structures after siRNA lipofection for both control-siRNA-treated (b) and MAO-B siRNA2-treated (f) cells. Representative images of MAO-B suppression by 2.9 nM MAO-B siRNA2 and the effect on intracellular Aß42 levels in 8 DIV neurons (g, h) compared with the same concentration of control-siRNA-treated samples (c, d). MAO-B and Aß42 fluorescence signals, quantified as normalized corrected total cell fluorescence (CTCF, see Methods), are shown in box plot graphs. Data from single cells in two different experiments is shown in Additional file 1: Figure S4. Normalized data are presented as mean values ± SEM. Significance was determined using two-tailed, two-sample unequal variance t test for the same concentrations of control-siRNA (normalized as value 1.0) and MAO-B siRNA2. The decreased protein level of MAO-B (red) in MAO-B siRNA transfected neurons compared to control-siRNA transfected neurons is shown (i). The effect of MAO-B silencing on intracellular Aß42 levels (green) by MAO-B siRNA2 silencing compared with a control-siRNA-treated sample is illustrated in (j). Z-projections of maximum intensity images collected by Z-stack scanning at 0.54-µm intervals with a laser scanning confocal microscope (LSM 510 META; ZEISS) are shown, scale bar = 30 µm. **p < 0.01; ***p < 0.001. siRNA small interfering RNA

Fig 4: Immunocytochemistry in primary neurons to elucidate the MAO-B location in relation to the synapse. Primary hippocampal neurons were stained with Phalloidin to visualize fibrillar actin and the neuronal structure (white), DAPI to visualize nuclei (blue), anti-NMDAr2B to visualize the postsynaptic side of glutamatergic synapses (green) and anti-MAO-B (red). a Low-magnification overview imaged with a 20× objective, to visualize that most or all of the pyramidal neurons in our cell cultures contain NMDAr2B. b High-magnification image taken with a 60× oil immersion objective. Inset shows a zoomed part of the large image that was enriched with a high density of NMDAr-containing dendritic spines and MAO-B staining on the opposing axons. The intensity of the red channel in the inset was enhanced using image J (Color figure online)

Fig 5: MAO-B immunohistochemistry in thin sections from hippocampus CA1 and entorhinal cortex regions of Cntr and AD brains. a Hippocampus CA1 images taken at 20× magnification are shown from a Cntr case (left panel) and an AD case (right panel). Note a neuronal loss in AD and intensive staining in the remaining neurons, including fibers, and astrocyte-like cells. Examples of pyramidal neurons are marked by red arrows. Scale bar, 50 µm. b Images of CA1 regions with many pyramidal neurons taken at 40× magnification in a Cntr (left panel) and AD (right panel) brain. Note a bigger variability in the pyramidal neuron staining of AD case while neurons in the control case are more evenly stained. Red arrows point at examples of pyramidal neurons. Scale bar, 50 µm. c Entorhinal cortex, layer II image taken at 20× magnification from a Cntr (left panel) and AD (right panel) brain. Zoomed inset in the left panel shows axonal staining. Note an island-like neuronal staining in the control case that is diminished and replaced by astrocyte-like staining due to the neuronal loss and advanced neurodegeneration in AD. Scale bar, 50 µm. d Immunocytochemistry in mouse primary hippocampal neurons shows axonal, dendritic and somal locations of MAO-B. The neurons were stained for Phalloidin to visualize F-actin and the neuronal structure (white), DAPI to visualize nuclei (blue), Tau-1 to visualize axons (green) and MAO-B (red). Red zoomed inset shows MAO-B staining in a thick (apical) dendrite. Blue zoomed inset shows MAO-B staining in a narrower dendrite. Green zoomed inset shows MAO-B staining in an axon. Images were taken with a Nikon A1RSi point scanning confocal with a 60× oil immersion objective. Scale bar, 10 µm. The axonal image is the sum of a z-stack whereas all other images are single planes. AD Alzheimer disease (Color figure online)