Journal: Cellular and Molecular Life Sciences

Article Title: The BACE1-generated C-terminal fragment of the neural cell adhesion molecule 2 (NCAM2) promotes BACE1 targeting to Rab11-positive endosomes

doi: 10.1007/s00018-022-04575-w

Figure Lengend Snippet: Shedding of BACE1 from the cell surface is increased and BACE1 activity is reduced in NCAM2−/− mice. A Total BACE1 visualized in cultured NCAM2 + / + and NCAM2−/− hippocampal neurons. Graph shows mean + SEM labeling intensities of BACE1 in dendrites of neurons ( n = 60). *p, Mann–Whitney test. Bar, 10 µm. B Total BACE1 levels in brain homogenates (hom.) and soluble BACE1 levels in soluble protein fractions (soluble) from 2-day-old and 2-month-old NCAM2 + / + , NCAM2+/− and NCAM2−/− mice as detected by Western blot analysis with BACE1-ED antibodies. Actin served as a loading control. NCAM2-ID labeling shows higher levels of NCAM2 in the adult brain. Glycosylated (*) and non-glycosylated (**) BACE1 are enriched in brain homogenates from 2-day-old and 2-month-old mice, respectively. Bands corresponding to soluble BACE1-ED in the soluble protein fraction are ~ 6 kDa smaller than full-length BACE1. A BACE1-immunoreactive band at ~ 110 kDa most likely represents dimers of BACE1-ED . Lower molecular weight degradation products of BACE1 are also detected in the soluble protein fraction. Graph shows mean + SEM change (in fold) of the ratio of soluble vs total BACE1 levels relative to 2-month-old NCAM2 + / + brains set to 100% ( n = 6). * p , one sample t test. C Total levels of Sez6 levels in brain homogenates and soluble Sez6 levels in soluble protein fractions from 2-month-old NCAM2 + / + , NCAM2+/− and NCAM2−/− mice as detected by Western blot analysis. GAPDH served as a loading control. Graph shows mean ± SEM change (in fold) in the ratio of soluble and total Sez6 levels relative to + / + brains set to 100% ( n = 8 (−/−), 4 (+/−)). * p , one sample t test, compared to + / + . D Dot blot analysis of Aβ42 levels in brain homogenates from 5–6 months-old NCAM2 + / + , NCAM2+/− and NCAM2−/− mice. Samples from each animal were analyzed in triplicates. Graph shows mean ± SEM change (in fold) in Aβ42 levels relative to + / + brains set to 100% ( n = 6 mice per group). * p , one sample t -test, compared to + / +

Article Snippet: BACE1 inhibitor (β-Secretase Inhibitor IV, cat# sc-222304), which inhibits BACE1 (IC 50 = 15 nM) and BACE2 (IC 50 = 0.23 μM), was from Santa Cruz Biotechnology.

Techniques: Activity Assay, Cell Culture, Labeling, MANN-WHITNEY, Western Blot, Control, Molecular Weight, Dot Blot

Journal: Cellular and Molecular Life Sciences

Article Title: The BACE1-generated C-terminal fragment of the neural cell adhesion molecule 2 (NCAM2) promotes BACE1 targeting to Rab11-positive endosomes

doi: 10.1007/s00018-022-04575-w

Figure Lengend Snippet: NCAM2-ID and BACE1 co-localize in endosomes. A Distribution of NCAM2-ID and BACE1 immunoreactivity in a cultured hippocampal neuron transfected with GFP to visualize its morphology. Note overlapping accumulations of NCAM2-ID and BACE1 in the shaft (arrows) and spine (arrowhead) in the dendrite. Bars, 10 µm (low magnification), 5 µm (high magnification). B A confocal slice through a soma showing NCAM2-ED-negative intracellular accumulations of NCAM2-ID immunoreactivity co-localizing with BACE1 (arrows). Bar, 5 µm. C The grayscale image shows NCAM2-ID/BACE1-ID proximity ligation (PL) products (black aggregates) in a cultured hippocampal neuron. NCAM2-ID/BACE1-ID PL products are visible in the soma (arrowheads) and in the dendritic shaft (arrows). Co-labeling for synaptophysin shows that the dendritic PL-labeled proteins are detectable in the vicinity of synaptophysin. Bar, 10 µm. D Co-localization of internalized BACE1 and NCAM2-ID accumulations (arrows) in a CHO cell co-transfected with BACE1 and NCAM2. Accumulations of internalized BACE1 are seen as red clusters of BACE1 detected after surface membrane permeabilization (surf. + int. BACE1). These clusters do not overlap with clusters of BACE1 detected before permeabilizing membranes (surf. BACE1). Bars, 10 μm (low magnification), 5 µm (high magnification). E Scheme of the subcellular fractionation protocol used to produce fractions analyzed in F . F Western blot analysis of the brain homogenate (BH) and fractions obtained as shown in E . Note that BACE1 and the ~ 32 kDa NCAM2 fragment detected with NCAM2-ID antibodies, which was previously described as the cleavage product of BACE1 , are present in fraction P100 enriched in transport vesicles. This fragment is not detectable in fraction S100 containing soluble proteins. Graphs show the enrichment of this fragment relative to full-length NCAM2 levels (NCAM2 fragment/FL) and relative to BACE1 levels (NCAM2 fragment/BACE1) in corresponding fractions (mean ± SEM, n = 3) normalized to the enrichment in BH set to 1. * p , ANOVA with Tukey’s multiple comparisons test

Article Snippet: BACE1 inhibitor (β-Secretase Inhibitor IV, cat# sc-222304), which inhibits BACE1 (IC 50 = 15 nM) and BACE2 (IC 50 = 0.23 μM), was from Santa Cruz Biotechnology.

Techniques: Cell Culture, Transfection, Ligation, Labeling, Membrane, Fractionation, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: The BACE1-generated C-terminal fragment of the neural cell adhesion molecule 2 (NCAM2) promotes BACE1 targeting to Rab11-positive endosomes

doi: 10.1007/s00018-022-04575-w

Figure Lengend Snippet: BACE1 is involved in proteolytic processing of NCAM2 in hippocampal neurons. A Schematic diagram showing locations of the domains recognized by antibodies against NCAM2-ED, NCAM2-ID, and HA tag (only in overexpressed NCAM2) used in this study. Orange arrow denotes the BACE1 cleavage site. B Lysates and medium from cultured hippocampal neurons treated with vehicle (0.1% DMSO) or BACE inhibitor analyzed by Western blot with anti-NCAM2-ED antibodies. Levels of NCAM2 are increased in lysates and reduced in the culture medium of neurons treated with the inhibitor. Graph shows NCAM2 levels in lysates ( n = 14) and the ratios of medium/lysate levels ( n = 8) from the inhibitor-treated neurons relative to levels in vehicle-treated neurons set to 100%. Means ± SEM are indicated. * p , one sample t test compared to the vehicle level. C NCAM2-ED and NCAM2-ID labeling in hippocampal neurons co-transfected with GFP and BACE1 siRNA, BACE2 siRNA or control siRNA. Bar = 20 μm. Graphs show mean + SEM NCAM2-ED and NCAM2-ID labeling intensities along dendrites of transfected neurons and their ratios ( n > 46 neurons per group) normalized to the mean of control siRNA-transfected neurons set to 100%. * p , one-way ANOVA with Dunnett’s multiple comparisons test. D NCAM2-ED and NCAM2-ID labeling of CHO cells co-transfected with GFP and NCAM2 and treated with the BACE inhibitor or vehicle (0.1% DMSO). Bar = 20 μm. Graphs show mean + SEM NCAM2-ED and NCAM2-ID labeling intensities and their ratios ( n > 173 cells per group) normalized to the mean of GFP-transfected cells set to 100%. * p , unpaired t -test. E NCAM2-ID and HA tag labeling in CHO cells co-transfected with HA-NCAM2 and GFP or BACE1-FLAG. Note the strongly reduced HA tag labeling in BACE1 co-transfected cells. Bar, 20 μm. Graph shows mean + SEM ratios of the HA tag and NCAM2-ID labeling intensities in CHO cells co-transfected with GFP, BACE1 or BACE2 ( n > 188 cells per group) normalized to the means of GFP-transfected cells set to 100%. * p , one-way ANOVA and Dunnett’s multiple comparisons test

Article Snippet: BACE1 inhibitor (β-Secretase Inhibitor IV, cat# sc-222304), which inhibits BACE1 (IC 50 = 15 nM) and BACE2 (IC 50 = 0.23 μM), was from Santa Cruz Biotechnology.

Techniques: Cell Culture, Western Blot, Labeling, Transfection, Control

Journal: Cellular and Molecular Life Sciences

Article Title: The BACE1-generated C-terminal fragment of the neural cell adhesion molecule 2 (NCAM2) promotes BACE1 targeting to Rab11-positive endosomes

doi: 10.1007/s00018-022-04575-w

Figure Lengend Snippet: NCAM2-ED is not required for targeting of NCAM2-ID to BACE1. A NCAM2-ID and BACE1 distribution in a CHO cell co-transfected with NCAM2ΔED and BACE1. Graph shows the distribution of labeling intensities along the dashed line. Bar, 10 µm. B Schematic diagram showing the method of BiFC. Reconstitution of the fluorescent protein Venus from VN and VC fragments fused to the C-termini of NCAM2ΔED and BACE1 results in fluorescence. C BiFC signals in CHO cells co-transfected with BACE1-VC and NCAM2ΔED-VN and cherry fluorescent protein. Bar, 20 μm. Note BiFC signals at the plasma membrane and cytoplasm. D BiFC staining in a cultured hippocampal neuron co-transfected with BACE1-VC, NCAM2ΔED-VN, and cherry fluorescent protein, and co-labelled for synaptophysin. Arrowheads show BiFC in the soma (low magnification image; bar, 10 µm), dendrites (i) and axon (ii) (high magnification images; bar, 5 µm). Axonal BiFC signals co-localize with synaptophysin accumulations. E Co-localization of internalized BACE1 and NCAM2-ID accumulations (arrows) in a CHO cell co-transfected with BACE1 and NCAM2ΔED. Accumulations of internalized BACE1 are seen as red clusters of BACE1 detected after the permeabilization of membranes (surf. + int. BACE1), which do not overlap with clusters of BACE1 detected before permeabilizing membranes (surf. BACE1). Bars, 10 μm (low magnification), 5 µm (high magnification)

Article Snippet: BACE1 inhibitor (β-Secretase Inhibitor IV, cat# sc-222304), which inhibits BACE1 (IC 50 = 15 nM) and BACE2 (IC 50 = 0.23 μM), was from Santa Cruz Biotechnology.

Techniques: Transfection, Labeling, Fluorescence, Clinical Proteomics, Membrane, Staining, Cell Culture

Journal: Cellular and Molecular Life Sciences

Article Title: The BACE1-generated C-terminal fragment of the neural cell adhesion molecule 2 (NCAM2) promotes BACE1 targeting to Rab11-positive endosomes

doi: 10.1007/s00018-022-04575-w

Figure Lengend Snippet: Inhibition of endocytosis does not reduce the interaction of NCAM2 and BACE1. A BACE1 and NCAM2-ID in CHO cells co-transfected with BACE1 and NCAM2ΔED and treated with dynasore or vehicle (0.1% DMSO). Graph shows mean + SEM and Pearson’s coefficient of BACE1 and NCAM2-ID co-localization ( n > 20 cells). B, C BiFC in CHO cells co-transfected with BACE1-VC and NCAM2ΔED-VN ( B ) or NCAM2-VN ( C ). Cells were either treated with dynasore or vehicle (0.1% DMSO), and co-labelled for NCAM2-ID and BACE1. Graph shows mean + SEM BiFC fluorescence intensities. n = 50 (DMSO) and 22 (dynasore) cells in B , n = 13 (DMSO) and 13 (dynasore) cells in C . Bars = 20 μm

Article Snippet: BACE1 inhibitor (β-Secretase Inhibitor IV, cat# sc-222304), which inhibits BACE1 (IC 50 = 15 nM) and BACE2 (IC 50 = 0.23 μM), was from Santa Cruz Biotechnology.

Techniques: Inhibition, Transfection, Fluorescence

Journal: Cellular and Molecular Life Sciences

Article Title: The BACE1-generated C-terminal fragment of the neural cell adhesion molecule 2 (NCAM2) promotes BACE1 targeting to Rab11-positive endosomes

doi: 10.1007/s00018-022-04575-w

Figure Lengend Snippet: NCAM2ΔED associates with BACE1 in Rab11-positive recycling endosomes. A NCAM2-ID labeling in a cultured hippocampal neuron co-transfected with Rab11-DsRed and NCAM2ΔED. High magnification images show examples of NCAM2-ID accumulations in Rab11-DsRed positive endosomes (arrowheads) in dendrites (i) and axon (ii). Bar = 10 µm (low magnification), 5 µm (high magnification). B BiFC in cultured hippocampal neurons co-transfected with cherry, NCAM2ΔED-VN and BACE1-VC and labelled for Rab11. Arrowheads show examples of BiFC co-localized with Rab11 in the soma (low magnification image) and along dendrites (high magnification image). Bar, 10 µm (low magnification), 5 µm (high magnification). C BiFC in CHO cells co-transfected with Rab11-DsRed, BACE1-VC and NCAM2-VN or NCAM2ΔED-VN. Bar, 10 µm

Article Snippet: BACE1 inhibitor (β-Secretase Inhibitor IV, cat# sc-222304), which inhibits BACE1 (IC 50 = 15 nM) and BACE2 (IC 50 = 0.23 μM), was from Santa Cruz Biotechnology.

Techniques: Labeling, Cell Culture, Transfection

Journal: Cellular and Molecular Life Sciences

Article Title: The BACE1-generated C-terminal fragment of the neural cell adhesion molecule 2 (NCAM2) promotes BACE1 targeting to Rab11-positive endosomes

doi: 10.1007/s00018-022-04575-w

Figure Lengend Snippet: NCAM2ΔED promotes targeting of BACE1 to Rab11-positive endosomes. A NCAM2-ID and BACE1 labeling in CHO cells co-transfected with BACE1, Rab11-DsRed and full-length NCAM2, NCAM2ΔED or empty pcDNA3 vector. Graphs show mean + SEM of BACE1 levels in endosomes relative to the total BACE1 levels and percentages of BACE1 in endosomes ( n > 36). *p, one-way ANOVA and Dunnett’s multiple comparisons test, compared to pcDNA3. Bars, 10 µm (low magnification), 5 µm (high magnification). B BACE1 levels in cultured hippocampal neurons co-transfected with Rab11-DsRed and NCAM2ΔED or empty pcDNA3 vector. Note higher co-localization of BACE1 with Rab11-positive endosomes in NCAM2ΔED overexpressing neurons (arrows). Graphs show mean + SEM of levels of BACE1 in Rab11-positive endosomes relative to total BACE1 levels and percentages of BACE1 in Rab11-positive endosomes ( n = 78). * p , Mann–Whitney test. Bar, 10 µm (low magnification), 5 µm (high magnification)

Article Snippet: BACE1 inhibitor (β-Secretase Inhibitor IV, cat# sc-222304), which inhibits BACE1 (IC 50 = 15 nM) and BACE2 (IC 50 = 0.23 μM), was from Santa Cruz Biotechnology.

Techniques: Labeling, Transfection, Plasmid Preparation, Cell Culture, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences

Article Title: The BACE1-generated C-terminal fragment of the neural cell adhesion molecule 2 (NCAM2) promotes BACE1 targeting to Rab11-positive endosomes

doi: 10.1007/s00018-022-04575-w

Figure Lengend Snippet: NCAM2 reduces cell surface levels of BACE1. A, B Cell surface and total BACE1 visualized in cultured NCAM2 + / + and NCAM2−/− hippocampal neurons ( A ). Higher magnification of dendrites is shown in B . Note higher cell surface BACE1 levels in NCAM2−/− neurons ( A, B ), and higher numbers of intracellular BACE1 accumulations (arrows) in dendrites of NCAM2 + / + neurons ( B ). Graphs show mean + SEM ratios of cell surface and total BACE1 labeling intensities along dendrites ( A ) and densities of intracellular BACE1 accumulations ( B ). * p , unpaired t test ( n = 120). Bar, 20 µm ( A ), 5 µm ( B ). C Cell surface and total BACE1 visualized in CHO cells co-transfected with BACE1 and pcDNA3 (control), full-length NCAM2 or NCAM2ΔED and co-labelled for NCAM2-ID. Note reduced cell surface BACE1 levels in NCAM2 and NCAM2ΔED co-transfected cells. Graph shows mean ± SEM ratios of cell surface and total BACE1 levels. * p , one-way ANOVA and Dunnett’s multiple comparisons test, compared to pcDNA3 ( n = 37 (pcDNA3), 34 (NCAM2), 46 (NCAM2ΔED)). D Labeling of the cell surface bound pool of BACE1 antibody (surface BACE1) and combined cell surface bound and internalized pool of BACE1 antibody (surface + internalized BACE1) in cultured NCAM2 + / + and NCAM2−/− hippocampal neurons incubated with antibodies against the extracellular domain of BACE1 for 30 min at 37 °C. Graph shows mean + SEM ratios of the cell surface and combined pools ( n = 180 (+ / +), 193 (−/−))

Article Snippet: BACE1 inhibitor (β-Secretase Inhibitor IV, cat# sc-222304), which inhibits BACE1 (IC 50 = 15 nM) and BACE2 (IC 50 = 0.23 μM), was from Santa Cruz Biotechnology.

Techniques: Cell Culture, Labeling, Transfection, Control, Incubation

Journal: Scientific Reports

Article Title: Effects and possible mechanisms of action of acacetin on the behavior and eye morphology of Drosophila models of Alzheimer’s disease

doi: 10.1038/srep16127

Figure Lengend Snippet: In vitro human BACE-1 inhibitory activity of three isolated compounds (acacetin, maslinic acid, and oleanolic acid), pure organic acacetin, and two BACE-1 inhibitors IV and epigallocatechin gallate using a fluorescence resonance energy transfer-based enzyme assay.

Article Snippet: BACE-1 inhibitor IV and Acid red were supplied by Merck (Darmstadt, Germany) and Amresco (Cochran Road Solon, OH, USA), respectively.

Techniques: In Vitro, Activity Assay, Isolation, Fluorescence