APP only led to spine loss when co-expressed with BACE1.

A-B Representative images and spine density of basal dendrites from CA1 pyramidal neurons transiently expressing GFP alone or together with APP. (filler, n =21; APP, n=16). C-D Representative images and spine density of basal dendrites from CA1 pyramidal neurons transiently expressing GFP alone or together with APP and BACE1 with a ratio of 15:1. (filler, n=8; APP plus BACE1, n=5)

E-F Representative images and spine density of basal dendrites from CA1 pyramidal neurons transiently expressing GFP alone or together with BACE1. (filler, n=15; BACE1, n=15)

G Western blot and corresponding statistical analysis of APP (Y188), BACE1 and GAPDH from HEK293T cells expressing APP or APP plus BACE1 with a ratio of 15:1. GAPDH was measured as a loading control.

H Measurement of APP β-CTF (β C-Terminal Fragment) level from HEK293T cells expressing APP alone or APP and BACE1 with a ratio of 15:1. n=4.

I Western blot and corresponding statistical analysis of APP fragments (Y188) from HEK293T cells expressing APP plus BACE1 or APPMV plus BACE1.

J Measurement of APP β-CTF levels from HEK293T cells expressing APP plus BACE1 or APPMV plus BACE1. n=4.

K Measurement of Aβ40 levels from HEK293T cells expressing APP plus BACE1 or APPMV plus BACE1. n=4.

L-M Representative images and spine density of basal dendrites from CA1 pyramidal neurons transiently expressing GFP alone or together with APP and BACE1 or APPMV and BACE1. (filler, n=12; APP plus BACE1, n=14; APPMV plus BACE1, n=10)

All dendritic images were acquired from rat organotypic hippocampal slice cultures after transfection for 6-7 days. Statistics: One-way ANOVA or Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001. Error bars show SEM. Scale bars, 10 μm.

β-CTF induced spine loss independent of Aβ.

A-B Representative images and spine density of basal dendrites from CA1 pyramidal neurons transiently expressing GFP alone or together with α-CTF, β’-CTF or β-CTF. (filler, n=19; α-CTF, n=7; β’-CTF, n=8; β-CTF, n=7)

C Western blot of APP fragments from HEK293T cells expressing α-CTF-flag, β’-CTF-flag and β-CTF-flag (flag antibody).

D Western blot of APP fragments from HEK293T cells expressing β-CTF-flag treatment with vehicle or PF (flag antibody).

E Measurement of Aβ40 secreted from HEK293T cells expressing β-CTF treatment with vehicle or PF. n=4.

F-G Representative images and spine density of basal dendrites from CA1 pyramidal neurons transiently expressing GFP alone or together with β-CTF after treated with vehicle or PF. (filler, n=11; β-CTF, n=7, β-CTF plus PF, n=14)

H-I Representative images and their spine density of basal dendrites from CA1 pyramidal neurons transiently expressing GFP together with APP and BACE1 after treated with vehicle or PF. (APP plus BACE1 with vehicle, n=9; APP plus BACE1 with PF, n=14)

J Western blot of APP fragments (Y188) from HEK293T cells co-expressing APP and BACE1 after treated with vehicle or PF.

K Measurements of Aβ40 from HEK 293T cells expressing APP and BACE1 after treated with vehicle or PF. n=4.

L Measurements of Aβ40 from HEK293T cells expressing APP and BACE1 or APPΔ59 and BACE1. n=4.

M Measurements of Aβ42 from HEK293T cells expressing APP and BACE1 or APPΔ57 and BACE1. n=4.

N-O Representative images and spine density of basal dendrites from CA1 pyramidal neurons transiently expressing GFP alone or together with APPΔ59/Δ57 and BACE1. (filler, n=9; APPΔ59 and BACE1, n=7; APPΔ57 and BACE1, n=6)

All dendritic images were acquired from rat organotypic hippocampal slice cultures after transfection for 6-7 days. PF, PF03084014, a γ secretase inhibitor. Statistics: One-way ANOVA or Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001. Error bars show SEM. Scale bars, 10 μm.

Expression of β-CTF damaged synapses in mouse brains.

A Representative images of mouse CA1 pyramidal neurons infected with lentivirus expressing GFP and β-CTF. Scale bar, 100 μm.

B-C Representative images from basal dendrites and quantitation of their spine density in CA1 pyramidal neurons infected with lentivirus expressing GFP alone or GFP with β-CTF in mouse cortices. Scale bar, 10 μm. GFP only, n=10; GFP plus β-CTF, n=13.

D-F Representative recording traces and quantitations of mEPSCs from adult mouse hippocampal neurons infected with lentivirus (identified with GFP signal) and neighboring uninfected neurons. n=10.

Statistics: Two-way ANOVA or Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001. Error bars show SEM.

Expression of β-CTF damaged cognitive function in mice in the absence of plaque formation.

A Schematic diagram showing the time line for stereotactic injection of AAV, behavioral training and tests.

B Representative images showing immunofluorescence staining of expressed GFP or β-CTF-HA by AAV in mouse hippocampi (WT). Scare bars represent 1000 μm (left) and 500 μm (right).

C Images of mouse hippocampus (WT) stained with ThS and HA antibody after infected with AAV encoding β-CTF-HA. Scale bar, 100 µm.

D Representative image of ThS staining of a 5XFAD mice hippocampal slice. Scale bar, 100 μm.

E Weight of mice infected with AAV encoding GFP or β-CTF in their hippocampi. GFP, n=14; β-CTF, n=15.

F Quantitations of spontaneous alternation in Y-maze from mice infected with AAV encoding GFP or β-CTF. GFP, n=14; β-CTF, n=15.

G Schematic diagram showing fear conditioning test design.

H Quantitation of freezing in contextual and cued fear conditioning tests from mice infected with AAV encoding GFP and β-CTF in their hippocampi. GFP, n=13; β-CTF, n=15.

I Schematic diagram showing water T maze test design.

J-K The percentage of correct responses across the 5 trials of water T maze test was quantified in each day of Acquisition (J) or Reversal (K). GFP, n=14; β-CTF, n=15.

Statistics: Two-way ANOVA or Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001. Error bars show SEM.

The C-terminal YENPTY motif was necessary for β-CTF to induce endosomal dysfunction and synapse loss.

A Immunofluorescent staining of GFP (green) from dissociated rat hippocampal neurons expressing Rab5-GFP.

B Immunofluorescent staining of GFP (green) and flag (Red) from dissociated rat hippocampal neurons expressing Rab5-GFP and α-CTF-flag.

C Immunofluorescent staining of GFP (green) and flag (Red) from dissociated rat hippocampal neurons expressing Rab5-GFP and β-CTF-flag.

D Schematic diagram of the β-CTF and β-CTFmut.

E Western blot of APP fragments from HEK293T cells expressing β-CTF-flag and β-CTFmut-flag with a flag antibody.

F Quantitations of APP fragments from HEK293T cells expressing β-CTF-flag and β-CTFmut-flag. n=4.

G Measurement of Aβ40 from HEK 293T cells expressing β-CTF or β-CTFmut. n=5.

H Immunofluorescent staining of GFP (green) and flag (Red) from dissociated rat hippocampal neurons expressing Rab5-GFP and β-CTFmut-flag.

I-J Representative images and spine density of basal dendrites from CA1 pyramidal neurons transiently expressing GFP together with β-CTF or β-CTFmut for 6-7 days in rat organotypic hippocampal slice cultures. β-CTF, n=12; β-CTFmut, n=15.

Scale bar, 10 μm. Statistics: Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001. Error bars show SEM.

Spine loss induced by β-CTF was prevented by Rab5 inhibition.

A Representative images of dissociated rat hippocampal neurons infected with lenti virus encoding GFP or β-CTF. Scale bar, 100 μm.

B Volcano plot of quantitative mass spectrometry analysis showing protein omics changes in neurons infected with lenti virus encoding β-CTF v.s. GFP. Proteins with fold change >1.2 or <0.83 and p value < 0.05 are colored.

C-D Western blot and corresponding statistic analysis of Synapsin 1, GluR 1, GluR 2 in dissociated hippocampal neurons infected with lentivirus expressing GFP or β-CTF. Tuj 1 was used as an internal control.

E The Gene Ontology (GO) processes analysis of decreased proteins by Metascape.

F The Reactome Gene Sets processes analysis of decreased protein by Metascape.

G-H Immunofluorescent staining of GFP (green), flag (Red) and HA (magenta) from dissociated rat hippocampal neurons expressing Rab5-GFP and β-CTF-flag or together with Rab5S34N-HA. Scale bar, 10 μm.

I-J Representative images and measurements of spine density from basal dendrites or CA1 pyramidal neurons transiently expressing GFP and β-CTF with or without Rab5S34N-HA for 6-7 days in rat organotypic hippocampal slice cultures. Scale bar, 10 μm. (β-CTF, n=17; β-CTF plus Rab5S34N, n=11)

Statistics: Two-way ANOVA or Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001. Error bars show SEM.