Journal: Nature Communications

Article Title: Altered expression of Presenilin2 impacts endolysosomal homeostasis and synapse function in Alzheimer’s disease-relevant brain circuits

doi: 10.1038/s41467-024-54777-y

Figure Lengend Snippet: a Coronal hippocampal slices of APPKI, APPxPSEN2KO and APPxFADPSEN2 stained for thioflavin, labelling dense core plaques over time. b Quantification of thioflavin + area normalized to brain area for hippocampus over time and analyzed using two-way ANOVA using a Dunnett’s correction for multiple testing comparing the different conditions to APPKI. c Dual immunolabelling of Aβ plaques in coronal cortex sections using a fibrillar (fAβ) and monomeric Aβ specific antibody (82e1). d , e Average size ( d ) and number ( e ) of thioflavin + plaques calculated from ( a , c ). Statistical analysis was through a one-way ANOVA with a Dunnett’s correction for multiple testing compared to APPKI ( f ). Immunolabelling of 3 month old coronal hippocampal slices of APPKI, APPxPSEN2KO and APPxFADPSEN2 for Aβ/APP-CTF (82e1), LAMP1, full length APP and thioflavin. g Quantification of LAMP1 + area as a percent of total hippocampus area. For ( g ) a two-way ANOVA analysis with Tukey’s correction for multiple testing compared to APPKI was used. Scale bars and p -values are indicated; number of mice (N) is shown inside the bar and the symbols represent the number of regions analyzed. All graphs are mean ± SEM.

Article Snippet: Mouse anti-human Aβ (82e1, 10323, Tecan, 1:100 for IF, 1:500 for WB), anti-human Aβ fibrils (AB2287, Sigma Aldrich, 1:200 for IF), rat anti-Lamp1 (1D4B, sc19992 Santa Cruz, 1:200 for IF, 1:1000 for WB), rabbit anti-APP (Y188, Ab32136, Abcam, 1:500 for IF), rabbit anti-APP (B63.3, home-made, 1:10 000 for WB), rabbit anti-presenilin 2 (ab51249, abcam, 1:500 for IF, 1:10 000 for WB), rabbit anti-presenilin 1 (B19, home-made, 1:20 000 for WB), mouse anti-nicastrin (9C3, home-made, 1:8000 for WB), mouse anti- actin (A5441, Sigma Aldrich, 1:10 000 for WB), chicken anti-Map2 (PA1-10005, Thermo Fisher, 1:5000 for IF), rabbit anti-EEA1 (07-1820, Sigma Aldrich, 1:250 for IF), goat anti-VPS35 (ab10099, Abcam, 1:200 for IF), mouse anti-GLUA1 (MA5-27694, Invitrogen, 1:500 for IF, 1:1000 for WB), rabbit anti-pGLUA1 (p1160-845, Phosphosolutions, 1:100 for IF), guinea-pig anti-vGLUT1 (135,304, Synaptic Systems, 1:500 for IF, 1:5000 for WB), PSD 95 nanobody-647 (N3702-AF647-L, Synaptic Systems, 1: 500 for IF), mouse anti- synaptobrevin2 (104 211, Synaptic Systems, 1:500 for IF), mouse anti-ankyrin-G coupled to FL550 (75-146-FL550, NeuroMab, 1:500).

Techniques: Staining

Journal: Nature Communications

Article Title: Altered expression of Presenilin2 impacts endolysosomal homeostasis and synapse function in Alzheimer’s disease-relevant brain circuits

doi: 10.1038/s41467-024-54777-y

Figure Lengend Snippet: a Representative images of DIV14 hippocampal neurons immunostained for full length APP, APPCTF/Aβ (82e1) and LAMP1. Arrowheads in zoomed insets highlight co-localization. b Western blot of DIV14 neuronal lysates showing increased APP-CTF levels. c Quantification of a for triple overlap between LAMP1 and APP/82e1 positive puncta. d Quantification of ratio APP: APP-CTF ratio of blots from ( b ). e Representative images of immunolabeling of early (EEA1) and recycling endosomes (VPS35), and LE/Lys (LAMP1). White arrowheads in zoomed insets show overlap between three compartments. f – h Quantification of total area of ( f ) LAMP1, ( g ) EEA1 and ( h ) VPS35 as percentage of total neuronal soma area. i Representative images of lysosomal Ca 2+ measured through feeding cells with dextran coupled Alexa488 and Ca 2+ sensitive Rhodamine-coupled dextran, and quantified in ( j ) as a Rhodamine/Alexa488 ratio. k LAMP1 surface labelling combined with Phalloidin staining, quantified in ( l ). m Schematic of the dual labelled mCherry-GFP-LC3: in autophagosomes both mCherry and GFP give yellow fluorescence; whereas in lysosomes, GFP is quenched resulting in red fluorescence. Created in BioRender. Vrancx, C. (2024) https://BioRender.com/j26q595 . n Representative images of DIV14 hippocampal neurons transfected with mCherry-GFP-LC3, quantified in ( o ) as total number of mCherry+ LC3 puncta normalized to APPKI neurons and in ( p ) as total number of mCherry + /GFP + LC3 puncta. Graphs ( c – f – g – h – j – l – o – p ) were statistically analyzed using one-way ANOVA with Tukey’s correction for multiple testing compared to APPKI. All graphs are represented as mean ± SEM, with triangles and circles representing the average per neuronal culture and individual cells, respectively. Scale bars are indicated in the figure.

Article Snippet: Mouse anti-human Aβ (82e1, 10323, Tecan, 1:100 for IF, 1:500 for WB), anti-human Aβ fibrils (AB2287, Sigma Aldrich, 1:200 for IF), rat anti-Lamp1 (1D4B, sc19992 Santa Cruz, 1:200 for IF, 1:1000 for WB), rabbit anti-APP (Y188, Ab32136, Abcam, 1:500 for IF), rabbit anti-APP (B63.3, home-made, 1:10 000 for WB), rabbit anti-presenilin 2 (ab51249, abcam, 1:500 for IF, 1:10 000 for WB), rabbit anti-presenilin 1 (B19, home-made, 1:20 000 for WB), mouse anti-nicastrin (9C3, home-made, 1:8000 for WB), mouse anti- actin (A5441, Sigma Aldrich, 1:10 000 for WB), chicken anti-Map2 (PA1-10005, Thermo Fisher, 1:5000 for IF), rabbit anti-EEA1 (07-1820, Sigma Aldrich, 1:250 for IF), goat anti-VPS35 (ab10099, Abcam, 1:200 for IF), mouse anti-GLUA1 (MA5-27694, Invitrogen, 1:500 for IF, 1:1000 for WB), rabbit anti-pGLUA1 (p1160-845, Phosphosolutions, 1:100 for IF), guinea-pig anti-vGLUT1 (135,304, Synaptic Systems, 1:500 for IF, 1:5000 for WB), PSD 95 nanobody-647 (N3702-AF647-L, Synaptic Systems, 1: 500 for IF), mouse anti- synaptobrevin2 (104 211, Synaptic Systems, 1:500 for IF), mouse anti-ankyrin-G coupled to FL550 (75-146-FL550, NeuroMab, 1:500).

Techniques: Western Blot, Immunolabeling, Staining, Fluorescence, Transfection

Journal: Molecular Neurodegeneration

Article Title: Increased amyloidogenic processing of transgenic human APP in X11-like deficient mouse brain

doi: 10.1186/1750-1326-5-35

Figure Lengend Snippet: Human sAPP levels in APP23 mouse brain samples in the presence or absence of X11L . (A) Detection of total human sAPP, sAPPα and sAPPβswe along with human APP, X11L and α-tubulin in APP23 mouse brain tissue in the presence or absence of X11L. Membrane (P100 for APP) and cytoplasmic (S100 for sAPP, sAPPα, sAPPβswe, X11L and α-tubulin) fractions of brain regions composed of cerebral cortex, hippocampus and olfactory bulb from wild-type (WT), APP23 and APP23/X11L-Ko mice (5-6 months old) were analyzed for total human sAPP (sAPPα plus sAPPβswe by 10D1 antibody), human sAPPα (2B3 antibody), human sAPPβswe (6A1 antibody), human APP (10D1 antibody), X11L and α-tubulin by immunoblotting. ( B ) The densities of total human sAPP (left), sAPPα (middle) and sAPPβswe (right) bands were standardized to the density of α-tubulin, which was compared with the ratios for APP23 mice that were assigned a reference value of 1.0 (values represent the means ± S.E.). The data were analyzed by Student's t test (n = 10; **, p < 0.01).

Article Snippet: Mouse monoclonal antibodies to human Aβ 82E1 (IBL) and 6E10 (Signet COVANCE), human APP 10D1 (IBL), sAPPα 2B3(IBL), sAPPβswe 6A1 (IBL), actin (Chemicon), flotillin-1 (BD Transduction Laboratories), α-tubulin (Zymed and Santa Cruz Biotechnologies), and X11L/Mint2 (BD Transduction Laboratories) were purchased.

Techniques: Western Blot

Journal: Molecular Neurodegeneration

Article Title: Increased amyloidogenic processing of transgenic human APP in X11-like deficient mouse brain

doi: 10.1186/1750-1326-5-35

Figure Lengend Snippet: Expression of APP in APP-ibl mouse brain in the presence or absence of X11L . ( A ) Expression of human APP695swe and mouse endogenous APP695. Brain regions composed of the cerebral cortex, hippocampus and olfactory bulb from wild-type (WT), APP-ibl and APP-ibl/X11L-Ko mice (4 months old) were homogenized in 10 mM Tris-HCl [pH 7.9] containing 1% (w/v) SDS, 4 M urea, complete protease inhibitor cocktail (Roche Diagnostics), and 1 μM pepstatin. The lysates were analyzed by immunoblotting with anti-human APP (10D1), anti-APP (8717), anti-X11L and anti-α-tubulin antibodies. ( B ) The densities of human APP695swe (left) and human plus mouse APP (right) bands were standardized to the density of α-tubulin, which was compared with the ratios for APP-ibl mice (left) or wild-type mice (right), which were assigned a reference value of 1.0 (values represent means ± S.E.). The data were analyzed by Student's t test (n = 3).

Article Snippet: Mouse monoclonal antibodies to human Aβ 82E1 (IBL) and 6E10 (Signet COVANCE), human APP 10D1 (IBL), sAPPα 2B3(IBL), sAPPβswe 6A1 (IBL), actin (Chemicon), flotillin-1 (BD Transduction Laboratories), α-tubulin (Zymed and Santa Cruz Biotechnologies), and X11L/Mint2 (BD Transduction Laboratories) were purchased.

Techniques: Expressing, Protease Inhibitor, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Increased amyloidogenic processing of transgenic human APP in X11-like deficient mouse brain

doi: 10.1186/1750-1326-5-35

Figure Lengend Snippet: Generation of primary amyloidogenic fragment CTFβ in APP-ibl mice in the presence or absence of X11L . ( A ) Generation of human CTFβ in APP-ibl mice in the presence or absence of X11L. Brain regions composed of the cerebral cortex, hippocampus and olfactory bulb from APP-ibl and APP-ibl/X11L-Ko mice (4 months old) were homogenized in 10 mM Tris-HCl [pH 7.9] containing 1% (w/v) SDS, 4 M urea, complete protease inhibitor cocktail (Roche Diagnostics), and 1 μM pepstatin. The lysates were dephosphorylated as described [ , ] and analyzed by immunoblotting with anti-human Aβ (82E1) and anti-α-tubulin antibodies. ( B ) The density of human CTFβ band was standardized to the density of α-tubulin, which was compared with the ratios for APP-ibl mice that were assigned a reference value of 1.0 (values represent the means ± S.E.). The data were analyzed by Student's t test (n = 10; **, p < 0.01). ( C ) Generation of CTFα and CTFβ in APP-ibl mice in the presence or absence of X11L. Brain lysates of the cerebral cortex, hippocampus and olfactory bulb from wild-type (WT), APP-ibl and APP-ibl/X11L-Ko mice (4 months old) were dephosphorylated and analyzed by immunoblotting with anti-APP/c 8717 and anti-α-tubulin antibodies. ( D ) The densities of C99 (CTFβ), C89 (CTFβ) and C83 (CTFα) bands were standardized to the density of α-tubulin, which was compared with the ratios for APP-ibl mice that were assigned a reference value of 1.0 (values represent means ± S.E.). The data were analyzed by Student's t test (n = 10; **, p < 0.01).

Article Snippet: Mouse monoclonal antibodies to human Aβ 82E1 (IBL) and 6E10 (Signet COVANCE), human APP 10D1 (IBL), sAPPα 2B3(IBL), sAPPβswe 6A1 (IBL), actin (Chemicon), flotillin-1 (BD Transduction Laboratories), α-tubulin (Zymed and Santa Cruz Biotechnologies), and X11L/Mint2 (BD Transduction Laboratories) were purchased.

Techniques: Protease Inhibitor, Western Blot