Figure 5 ( A - C ) Mitochondrial hydrogen peroxide ( H2O2 ) measurement in 1205Lu and WM3734 with and without stable MCUA _ KD using mito - HyPer . Exemplary ratiometric images ( F505 nm / F420 nm ) are shown for all conditions ( A ) . Scale bar : 10 µm . Quantification of mito - HyPer ratio in 1205Lu ( B ) and WM3734 ( C ) under resting state ( 1205Lu ─ shCTRL : n = 217 cells from 6 biological replicates ; shMCU _ 1 : n = 197 cells from 7 biological replicates ; shMCU _ 2 : n = 212 cells from 7 biological replicates ; WM3734 ─ shCTRL : n = 104 cells from 6 biological replicates ; shMCU _ 1 : n = 107 cells from 7 biological replicates ; shMCU _ 2 : n = 114 cells from 7 biological replicates ) . Data information : All data are presented as mean ± SEM . and were measured in Ringer ' s buffer containing 0 . 5 mM Ca2 + statistical significance was determined using unpaired , two ‐ tailed Student ' s t ‐ test , ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 005 . In all statistical analyses , KD cells were compared to their respective control . ( D - F ) Cytosolic hydrogen peroxide ( H2O2 ) measurement in 1205Lu and WM3734 with and without stable MCUA _ KD using HyPer . Exemplary ratiometric images ( F505 nm / F420 nm ) are shown for all conditions ( D ) . Scale bar : 10 µm . Quantification of HyPer ratio in 1205Lu ( E ) and WM3734 ( F ) under resting state ( 1205Lu ─ shCTRL : n = 232 cells from 6 biological replicates ; shMCU _ 1 : n = 290 cells from 6 biological replicates ; shMCU _ 2 : n = 290 cells from 6 biological replicates ; WM3734 ─ shCTRL : n = 443 cells from 9 biological replicates ; shMCU _ 1 : n = 297 cells from 9 biological replicates ; shMCU _ 2 : n = 233 cells from 9 biological replicates ) . Data information : All data are presented as mean ± SEM . and were measured in Ringer ' s buffer containing 0 . 5 mM Ca2 + statistical significance was determined using unpaired , two ‐ tailed Student ' s t ‐ test , ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 005 . In all statistical analyses , KD cells were compared to their respective control . ( I - K ) Mitochondrial glutathione redox potential , measured with mito - Grx1 - roGFP2 in 1205Lu and WM3734 with and without stable MCUA _ KD . Representative ratiometric images ( F385 nm / F475 nm ) are shown for all conditions ( I ) . Scale bar : 10 µm . Quantification in 1205Lu ( J ) and WM3734 ( K ) under resting state ( 1205Lu ─ shCTRL : n = 231 cells from 10 biological replicates ; shMCU _ 1 : n = 176 cells from 7 biological replicates ; shMCU _ 2 : n = 164 cells from 7 biological replicates ; WM3734 ─ shCTRL : n = 127 cells from 7 biological replicates ; shMCU _ 1 : n = 142 cells from 7 biological replicates ; shMCU _ 2 : n = 116 cells from 7 biological replicates ) . Data information : All data are presented as mean ± SEM . and were measured in Ringer ' s buffer containing 0 . 5 mM Ca2 + statistical significance was determined using unpaired , two ‐ tailed Student ' s t ‐ test , ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 005 . In all statistical analyses , KD cells were compared to their respective control .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A-C) Mitochondrial hydrogen peroxide (H2O2) measurement in 1205Lu and WM3734 with and without stable MCUA_KD using mito-HyPer. Exemplary ratiometric images (F505 nm/F420 nm) are shown for all conditions (A). Scale bar: 10 µm. Quantification of mito-HyPer ratio in 1205Lu (B) and WM3734 (C) under resting state (1205Lu ─ shCTRL: n=217 cells from 6 biological replicates; shMCU_1: n=197 cells from 7 biological replicates; shMCU_2: n=212 cells from 7 biological replicates; WM3734 ─ shCTRL: n=104 cells from 6 biological replicates; shMCU_1: n=107 cells from 7 biological replicates; shMCU_2: n=114 cells from 7 biological replicates). Data information: All data are presented as mean ± SEM. and were measured in Ringer's buffer containing 0.5 mM Ca2+ statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005. In all statistical analyses, KD cells were compared to their respective control.(D-F) Cytosolic hydrogen peroxide (H2O2) measurement in 1205Lu and WM3734 with and without stable MCUA_KD using HyPer. Exemplary ratiometric images (F505 nm/F420 nm) are shown for all conditions (D). Scale bar: 10 µm. Quantification of HyPer ratio in 1205Lu (E) and WM3734 (F) under resting state (1205Lu ─ shCTRL: n=232 cells from 6 biological replicates; shMCU_1: n=290 cells from 6 biological replicates; shMCU_2: n=290 cells from 6 biological replicates; WM3734 ─ shCTRL: n=443 cells from 9 biological replicates; shMCU_1: n=297 cells from 9 biological replicates; shMCU_2: n=233 cells from 9 biological replicates). Data information: All data are presented as mean ± SEM. and were measured in Ringer's buffer containing 0.5 mM Ca2+ statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005. In all statistical analyses, KD cells were compared to their respective control.(I-K) Mitochondrial glutathione redox potential, measured with mito-Grx1-roGFP2 in 1205Lu and WM3734 with and without stable MCUA_KD. Representative ratiometric images (F385 nm/F475 nm) are shown for all conditions (I). Scale bar: 10 µm. Quantification in 1205Lu (J) and WM3734 (K) under resting state (1205Lu ─ shCTRL: n=231 cells from 10 biological replicates; shMCU_1: n=176 cells from 7 biological replicates; shMCU_2: n=164 cells from 7 biological replicates; WM3734 ─ shCTRL: n=127 cells from 7 biological replicates; shMCU_1: n=142 cells from 7 biological replicates; shMCU_2: n=116 cells from 7 biological replicates). Data information: All data are presented as mean ± SEM. and were measured in Ringer's buffer containing 0.5 mM Ca2+ statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005. In all statistical analyses, KD cells were compared to their respective control.


Figure 6 ( G - I ) Mitochondrial ATP , measured using mito - ATEAM in 1205Lu and WM3734 with and without stable MCUA _ KD . Exemplary ratiometric images ( FRET / CFP ) are shown for all conditions ( G ) . Scale bar : 10 µm . Quantification of basal levels in 1205Lu ( H ) and WM3734 ( I ) ( 1205Lu ─ shCTRL : n = 141 cells from 14 biological replicates ; shMCU _ 1 : n = 143 cells from 12 biological replicates ; shMCU _ 2 : n = 143 cells from 13 biological replicates ; WM3734 ─ shCTRL : n = 122 cells from 12 biological replicates ; shMCU _ 1 : n = 103 cells from 12 biological replicates ; shMCU _ 2 : n = 99 cells from 13 biological replicates ) . Data information : Statistical significance was determined using unpaired , two ‐ tailed Student ' s t ‐ test , ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 005 ; no asterisk means no statistical significance ( p > 0 . 05 ) . ( J ) Exemplary confocal microscope images of the mitochondrial network ( blue : DAPI staining of the nucleus ; green : TOMM20 staining of mitochondria ) ; scale bar : 50 µm . ( K - O ) Electron microscopy of mitochondria of stable MCUA _ KD cell lines . ( K ) Exemplary images of stable MCUA _ KD cells ' mitochondria . Scale bar : 500 nm . ( L - O ) Quantification of mitochondrial diameter ( L and N ) and intercristae distance ( M and O ) of 1205Lu ( L and M ) and WM3734 ( N and O ) cells with and without stable MCUA _ KD , presented as boxplot . The box presents the 25 % - quartile , median and 75 % - quartile , the X represents the mean and the whiskers the minimum and maximum , outliers are represented as dots ( mitochondrial diameter : 1205Lu ─ shCTRL : n = 47 technical replicates ; shMCU _ 1 : n = 44 technical replicates ; shMCU _ 2 : n = 40 technical replicates ; WM3734 ─ shCTRL : n = 35 technical replicates ; shMCU _ 1 : n = 52 technical replicates ; shMCU _ 2 : n = 38 technical replicates ; intercristace distance : 1205Lu ─ shCTRL : n = 330 technical replicates ; shMCU _ 1 : n = 285 technical replicates ; shMCU _ 2 : n = 255 technical replicates ; WM3734 ─ shCTRL : n = 352 technical replicates ; shMCU _ 1 : n = 433 technical replicates ; shMCU _ 2 : n = 357 technical replicates ) . Data information : Statistical significance was determined using unpaired , two ‐ tailed Student ' s t ‐ test , ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 005 ; no asterisk means no statistical significance ( p > 0 . 05 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(G-I) Mitochondrial ATP, measured using mito-ATEAM in 1205Lu and WM3734 with and without stable MCUA_KD. Exemplary ratiometric images (FRET/CFP) are shown for all conditions (G). Scale bar: 10 µm. Quantification of basal levels in 1205Lu (H) and WM3734 (I) (1205Lu ─ shCTRL: n=141 cells from 14 biological replicates; shMCU_1: n=143 cells from 12 biological replicates; shMCU_2: n=143 cells from 13 biological replicates; WM3734 ─ shCTRL: n=122 cells from 12 biological replicates; shMCU_1: n=103 cells from 12 biological replicates; shMCU_2: n=99 cells from 13 biological replicates). Data information: Statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).(J) Exemplary confocal microscope images of the mitochondrial network (blue: DAPI staining of the nucleus; green: TOMM20 staining of mitochondria); scale bar: 50 µm. (K-O) Electron microscopy of mitochondria of stable MCUA_KD cell lines. (K) Exemplary images of stable MCUA_KD cells' mitochondria. Scale bar: 500 nm. (L-O) Quantification of mitochondrial diameter (L and N) and intercristae distance (M and O) of 1205Lu (L and M) and WM3734 (N and O) cells with and without stable MCUA_KD, presented as boxplot. The box presents the 25 %- quartile, median and 75 %-quartile, the X represents the mean and the whiskers the minimum and maximum, outliers are represented as dots (mitochondrial diameter: 1205Lu ─ shCTRL: n=47 technical replicates; shMCU_1: n=44 technical replicates; shMCU_2: n=40 technical replicates; WM3734 ─ shCTRL: n=35 technical replicates; shMCU_1: n=52 technical replicates; shMCU_2: n=38 technical replicates; intercristace distance: 1205Lu ─ shCTRL: n=330 technical replicates; shMCU_1: n=285 technical replicates; shMCU_2: n=255 technical replicates; WM3734 ─ shCTRL: n=352 technical replicates; shMCU_1: n=433 technical replicates; shMCU_2: n=357 technical replicates). Data information: Statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).


Figure 1 ( D - E ) Western blot analysis of RIPA soluble - fraction and RIPA - insoluble , urea - soluble fraction of proteins prepared from HEK293T cells expressing empty plasmid ( control ) , FLAG - KIF5A WT , FLAG - ΔExon27 . Insoluble / soluble KIF5A fractions were detected with anti - FLAG and actin was used as a loading control , three biological replicates . Statistical analysis was performed using student t - test ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . Data represent the mean ± s . d .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(D-E) Western blot analysis of RIPA soluble-fraction and RIPA-insoluble, urea-soluble fraction of proteins prepared from HEK293T cells expressing empty plasmid (control), FLAG-KIF5A WT, FLAG-ΔExon27. Insoluble/soluble KIF5A fractions were detected with anti-FLAG and actin was used as a loading control, three biological replicates. Statistical analysis was performed using student t-test (*p < 0.05, **p<0.01, ***p<0.001). Data represent the mean ± s.d.


Figure 2 ( B ) Pull down analysis showing the association of ΔExon27 with WT KIF5A , two biological replicates .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O

Figure 2(B) Pull down analysis showing the association of ΔExon27 with WT KIF5A, two biological replicates.


Figure 5 ( A ) Sanger sequencing chromatograms of cDNA from control and patient - derived iMNs ( P1 - P3 ) showing the effect of mutations on KIF5A transcripts . While the c . 3020 + 2T > C mutation causes exon 27 skipping , the c . 2993 - 1G > A mutation results in an aberrant transcript with a splice acceptor shifted only by a single nucleotide , leading to a frameshift but containing exon 27 . The respective protein product predicted for both mutations comprise the same C - terminal aberrant 39 amino acid end . ( B ) Western blot analysis for validation of the custom mutant - specific antibody using transfected HEK293T cells . While an antibody against the total KIF5A ( lower panel ) recognized both wild type and mutant protein , the custom antibody targeting the aberrant " neopeptide " detected ΔExon27 specifically ( upper panel ) . NT : No transfection . ( C ) Mutant KIF5A proteins are endogenously expressed in iMNs , detected by using the custom mutant specific antibody directed against the C - terminal aberrant 39 amino acid sequence . CTL : Control , P1 : Patient 1 , P2 : Patient 2 , P3 : Patient 3 , two independent experiments . ( D ) Representative images of KIF5A distribution ( arrowheads ) in control and patient iMNs . Scale bar : 20 µm . ( E ) Abundance of KIF5A inclusions after normalizing to total neuron numbers . Graph represents data from n = 146 control 1 ( CTL1 ) , n = 134 control 2 ( CTL2 ) , n = 154 patient 1 ( P1 ) , n = 162 patient 2 ( P2 ) and n = 133 patient 3 ( P3 ) iMNs from 3 individual experiments . Bars indicate mean ± s . d . Statistical analysis was performed using Mann Whitney Wilcoxon Test ( * p < 0 . 05 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Sanger sequencing chromatograms of cDNA from control and patient-derived iMNs (P1-P3) showing the effect of mutations on KIF5A transcripts. While the c.3020+2T>C mutation causes exon 27 skipping, the c.2993-1G>A mutation results in an aberrant transcript with a splice acceptor shifted only by a single nucleotide, leading to a frameshift but containing exon 27. The respective protein product predicted for both mutations comprise the same C-terminal aberrant 39 amino acid end.(B) Western blot analysis for validation of the custom mutant-specific antibody using transfected HEK293T cells. While an antibody against the total KIF5A (lower panel) recognized both wild type and mutant protein, the custom antibody targeting the aberrant "neopeptide" detected ΔExon27 specifically (upper panel). NT: No transfection.(C) Mutant KIF5A proteins are endogenously expressed in iMNs, detected by using the custom mutant specific antibody directed against the C-terminal aberrant 39 amino acid sequence. CTL: Control, P1: Patient 1, P2: Patient 2, P3: Patient 3, two independent experiments.(D) Representative images of KIF5A distribution (arrowheads) in control and patient iMNs. Scale bar: 20 µm. (E) Abundance of KIF5A inclusions after normalizing to total neuron numbers. Graph represents data from n=146 control 1 (CTL1), n = 134 control 2 (CTL2), n = 154 patient 1 (P1), n = 162 patient 2 (P2) and n = 133 patient 3 (P3) iMNs from 3 individual experiments. Bars indicate mean ± s.d. Statistical analysis was performed using Mann Whitney Wilcoxon Test (*p<0.05).


Figure 1 . Full - length TLN ( but not APP ) accumulates in a distinct compartment in PS1 − / − hippocampal neurons . Double - immunofluorescence staining for endogenous TLN ( green , B36 . 1 ) and APP ( red , mAb C1 6 . 1 ) in wild - type ( top ) and PS1 − / − ( bottom ) hippocampal neurons ( 15 - d culture ) . The inset shows an overview of the neuron . In wild - type neurons TLN labeling is essentially localized to the somatodendritic plasmalemma in contrast to APP , which is found in intracellular compartments . In addition , PS1 deficiency causes TLN , but not APP , to cluster in large somatic accumulations . Bars , 10 μm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1.Full-length TLN (but not APP) accumulates in a distinct compartment in PS1−/−hippocampal neurons. Double-immunofluorescence staining for endogenous TLN (green, B36.1) and APP (red, mAb C1 6.1) in wild-type (top) and PS1−/− (bottom) hippocampal neurons (15-d culture). The inset shows an overview of the neuron. In wild-type neurons TLN labeling is essentially localized to the somatodendritic plasmalemma in contrast to APP, which is found in intracellular compartments. In addition, PS1 deficiency causes TLN, but not APP, to cluster in large somatic accumulations. Bars, 10 μm.


Figure 2 . ( A ) Western blot analysis of endogenous TLN and APP in wild - type and PS1 − / − hippocampal neurons . PS1 deficiency results in the accumulation of APP - CTF . No TLN - CTF ( estimated ± 7 kD ) was detected , neither was it detected after SFV - induced exogenous overexpression . Also , overexpression of a TLNΔE fragment did not generate a PS1 - dependent TLN intracellular domain . ( B ) Different constructs fused to the Gal4VP16 domain were used in a γ - secretase reporter assay . Control transfections ( pIPspAdApt empty vector and pIPspAdApt - APP without Gal4VP16 ) show no luciferase activity . APP - Gal4VP16 and NotchΔE - Gal4VP16 activate the luciferase gene upon cleavage and are inhibited by DAPT , L658 , 458 , or compound 32 ( 125 nM ) . No luciferase activity was detected using TLNΔE or APP in which the transmembrane domain was replaced by that of TLN ( APP / TLNTMR chimaera ) , indicating that this region is not cleaved by γ - secretase . ( C ) Left : γ - secretase inhibitors do not induce the formation of TLN accumulations . Wild - type hippocampal neurons were treated with γ - secretase inhibitors ( 125 nM , daily from d 7 - 14 post - plating ) , fixed , and immunostained for TLN and the percentage of neurons with TLN accumulations was scored . Culturing neurons for 25 d increases the size and number ( insets ) , but not the frequency of TLN accumulations ( mean ± SEM , n = 3 , 200 - 700 neurons per time point ) . Right : Western blot showing that only APP - CTF accumulates after chronic treatment with γ - secretase inhibitors . ( D ) Adenoviral reintroduction of human PS1 suppresses TLN accumulations . 30 - 35 % of PS1 − / − neurons display TLN accumulations , in contrast to < 5 % in PS1 + / + and + / − cultures as well as in cultures expressing human PS1 in a PS1 − / − background . However , suppression of TLN accumulations was obtained by adenoviral introduction of human PS1 . A clear dose - dependent inhibition was seen with increasing MOI of adenoviral ( AV ) human PS1 , but also the familial Alzheimer ' s disease - linked PS1G384A and dominant - negative PS1D257A mutants . Control adenoviral infections with eGFP did not have a significant effect . After 9 - 11 d post - infection ( 15 d post - plating ) , neurons displayed significant PS1 protein expression as demonstrated by double staining for TLN and human PS1 ( mAb 5 . 2 ) . Bars , 20 μm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2.(A) Western blot analysis of endogenous TLN and APP in wild-type and PS1−/− hippocampal neurons. PS1 deficiency results in the accumulation of APP-CTF. No TLN-CTF (estimated ± 7 kD) was detected, neither was it detected after SFV-induced exogenous overexpression. Also, overexpression of a TLNΔE fragment did not generate a PS1-dependent TLN intracellular domain.(B) Different constructs fused to the Gal4VP16 domain were used in a γ-secretase reporter assay. Control transfections (pIPspAdApt empty vector and pIPspAdApt-APP without Gal4VP16) show no luciferase activity. APP-Gal4VP16 and NotchΔE-Gal4VP16 activate the luciferase gene upon cleavage and are inhibited by DAPT, L658,458, or compound 32 (125 nM). No luciferase activity was detected using TLNΔE or APP in which the transmembrane domain was replaced by that of TLN (APP/TLNTMR chimaera), indicating that this region is not cleaved by γ-secretase.(C) Left: γ-secretase inhibitors do not induce the formation of TLN accumulations. Wild-type hippocampal neurons were treated with γ-secretase inhibitors (125 nM, daily from d 7-14 post-plating), fixed, and immunostained for TLN and the percentage of neurons with TLN accumulations was scored. Culturing neurons for 25 d increases the size and number (insets), but not the frequency of TLN accumulations (mean ± SEM, n = 3, 200-700 neurons per time point). Right: Western blot showing that only APP-CTF accumulates after chronic treatment with γ-secretase inhibitors.(D) Adenoviral reintroduction of human PS1 suppresses TLN accumulations. 30-35% of PS1−/− neurons display TLN accumulations, in contrast to <5% in PS1+/+ and +/− cultures as well as in cultures expressing human PS1 in a PS1−/− background. However, suppression of TLN accumulations was obtained by adenoviral introduction of human PS1. A clear dose-dependent inhibition was seen with increasing MOI of adenoviral (AV) human PS1, but also the familial Alzheimer's disease-linked PS1G384A and dominant-negative PS1D257A mutants. Control adenoviral infections with eGFP did not have a significant effect. After 9-11 d post-infection (15 d post-plating), neurons displayed significant PS1 protein expression as demonstrated by double staining for TLN and human PS1(mAb 5.2). Bars, 20 μm.


Figure 3 . Wild - type and PS1 − / − cortical neurons were transduced with SFV - TLN ( A ) or - APP ( B ) , pulse labeled for 15 min with [ 35S ] methionine , and chased for different time periods . ImmunoprecipitatedTLN and APP were treated with EndoH and analyzed by SDS - PAGE and phosphorimaging . The accumulation of an EndoH - resistant band indicates progressive maturation during Golgi passage ; however , the ratio of EndoH - resistant to - sensitive TLN ( A , top ) and APP ( B , top ) revealed no difference ( mean ± SEM , n = 3 ) . Instead , the half - life of overexpressed TLN ( A , bottom ) , but not full - length APP ( B , bottom ) , is significantly prolonged in neurons − / − neurons ( mean ± SEM , n = 3 ) . ( C ) Wild - type and PS1 − / − hippocampal neurons ( 12 d ) were scraped , pelleted , and treated with 10 mU EndoH , followed by Western blot detection of APP , TLN , PS1 , nicastrin ( NCT ) , synaptophysin , and ductin . TLN levels are increased almost fourfold in PS1 − / − neurons . No EndoH - sensitive TLN was detected in PS1 − / − neurons , indicating a complete maturation . APP - CTF shows a dramatic accumulation ( 44 - fold ; mean ± SEM , n = 3 ) .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3.Wild-type and PS1−/− cortical neurons were transduced with SFV-TLN (A) or -APP (B), pulse labeled for 15 min with [35S]methionine, and chased for different time periods. ImmunoprecipitatedTLN and APP were treated with EndoH and analyzed by SDS-PAGE and phosphorimaging. The accumulation of an EndoH-resistant band indicates progressive maturation during Golgi passage; however, the ratio of EndoH-resistant to -sensitive TLN (A, top) and APP (B, top) revealed no difference (mean ± SEM, n = 3). Instead, the half-life of overexpressed TLN (A, bottom), but not full-length APP (B, bottom), is significantly prolonged in neurons−/−neurons (mean ± SEM, n = 3).(C) Wild-type and PS1−/− hippocampal neurons (12 d) were scraped, pelleted, and treated with 10 mU EndoH, followed by Western blot detection of APP, TLN, PS1, nicastrin (NCT), synaptophysin, and ductin. TLN levels are increased almost fourfold in PS1−/− neurons. No EndoH-sensitive TLN was detected in PS1−/− neurons, indicating a complete maturation. APP-CTF shows a dramatic accumulation (44-fold; mean ± SEM, n = 3).


Figure 4 . TLN accumulation occurs in a compartment distinct from classic biosynthetic and endosomal pathways . ( A - F ) Early compartments . No colocalization was observed between TLN ( green in all panels ) and nuclei ( A , TOPRO - 3 ) , ER - markers BIP ( B ) and calnexin ( C ) , ERGIC - 53 ( D ) , β - COP ( E ) or GM130 ( F ) , and labeling cis - Golgi . ( G - L ) Late compartments . TLN accumulations are not accessible for exogenous biotin ( G ) . Early and recycling endosomes ( H , EEA1 ) and transferrin receptor ( I , TFR ) , late endosomes ( J , LBPA ) , and lysosomes ( K , Lamp - 2 ; L , catD ) also stained negative for TLN . Vertical sections ( x - z , arrowheads in I and J ) clearly distinguish TLN accumulations from recycling ( I , TFR ) and late endosomes ( J , LBPA ) . Bars , 10 μm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4.TLN accumulation occurs in a compartment distinct from classic biosynthetic and endosomal pathways. (A-F) Early compartments. No colocalization was observed between TLN (green in all panels) and nuclei (A, TOPRO-3), ER-markers BIP (B) and calnexin (C), ERGIC-53 (D), β-COP (E) or GM130 (F), and labeling cis-Golgi. (G-L) Late compartments. TLN accumulations are not accessible for exogenous biotin (G). Early and recycling endosomes (H, EEA1) and transferrin receptor (I, TFR), late endosomes (J, LBPA), and lysosomes (K, Lamp-2; L, catD) also stained negative for TLN. Vertical sections (x-z, arrowheads in I and J) clearly distinguish TLN accumulations from recycling (I, TFR) and late endosomes (J, LBPA). Bars, 10 μm.


Figure 5 . Correlative light immuno - EM analysis of TLN accumulations . ( A ) Insert : overview of the selected neuron as seen by immunocytochemistry . The bright signal for TLN reflects the numerous membranous accumulations seen at the ultrastructural immuno - EM level ( star ) . ( B ) Higher magnification of the boxed area in A . ( C ) High magnification of the boxed structure seen in B . 10 - nm gold particles label TLN . Note the complex composition of the internal membranes of the TLN - positive compartments . ( D ) Example of a TLN ( 15 - nm gold ) - containing structure obtained by the conventional cryosectioning technique . Note some heterogeneity in the internal membranes between C and D . Lamp - 1 ( 10 - nm gold ) is absent from the TLN - containing compartment . Arrows point to invaginations of the cytoplasm . N , nucleus ; P , plasma membrane . Bars : A , 5 μm ; B , 2 μm ; C , 500 nm ; D , 200 nm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5.Correlative light immuno-EM analysis of TLN accumulations. (A) Insert: overview of the selected neuron as seen by immunocytochemistry. The bright signal for TLN reflects the numerous membranous accumulations seen at the ultrastructural immuno-EM level (star). (B) Higher magnification of the boxed area in A. (C) High magnification of the boxed structure seen in B. 10-nm gold particles label TLN. Note the complex composition of the internal membranes of the TLN-positive compartments. (D) Example of a TLN (15-nm gold)-containing structure obtained by the conventional cryosectioning technique. Note some heterogeneity in the internal membranes between C and D. Lamp-1 (10-nm gold) is absent from the TLN-containing compartment. Arrows point to invaginations of the cytoplasm. N, nucleus; P, plasma membrane. Bars: A, 5 μm; B, 2 μm; C, 500 nm; D, 200 nm.


Figure 6 . TLN accumulations are distinct from lysosomes . A TLN ( 15 - nm gold ) - containing structure as obtained by the conventional cryosectioning technique double labeled with Lamp - 1 ( 10 - nm gold ) . The TLN - positive compartment is clearly distinct in size and from lysosomes ( L ) and lacks Lamp - 1 . P , plasma membrane . Bar , 200 nm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6.TLN accumulations are distinct from lysosomes. A TLN (15-nm gold)-containing structure as obtained by the conventional cryosectioning technique double labeled with Lamp-1 (10-nm gold). The TLN-positive compartment is clearly distinct in size and from lysosomes (L) and lacks Lamp-1. P, plasma membrane. Bar, 200 nm.


Figure 7 . ( B and C ) Double immunostaining of wild - type and PS1 − / − hippocampal neurons for TLN ( green ) and autophagic vacuole - associated Apg12p ( B ) and LC3 ( C ) . To detect both sets of pAbs , an alternative method was developed using biotinylated anti - TLN ( see Materials and methods ) . Each experiment included controls for specificity ( not depicted ) . In wild - type neurons , both Apg12p and LC3 ( B and C , top row ) were broadly distributed in cell bodies and neurites , but did not colocalize with TLN . Colocalization was only observed in multiple TLN accumulations in PS1 − / − hippocampal neurons ( second row in B and C , respectively ) . For Apg12p , coimmunostaining was also noticed in small discrete structures in the proximal dendrites ( B , bottom row ) . Bars , 10 μm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7.(B and C) Double immunostaining of wild-type and PS1−/− hippocampal neurons for TLN (green) and autophagic vacuole-associated Apg12p (B) and LC3 (C). To detect both sets of pAbs, an alternative method was developed using biotinylated anti-TLN (see Materials and methods). Each experiment included controls for specificity (not depicted). In wild-type neurons, both Apg12p and LC3 (B and C, top row) were broadly distributed in cell bodies and neurites, but did not colocalize with TLN. Colocalization was only observed in multiple TLN accumulations in PS1−/− hippocampal neurons (second row in B and C, respectively). For Apg12p, coimmunostaining was also noticed in small discrete structures in the proximal dendrites (B, bottom row). Bars, 10 μm.


Figure 8 . ( A ) Intracellular sorting and maturation of catD . Wild - type and PS1 − / − neurons were metabolically labeled and chased for the indicated time periods . Phosphorimaging of immunoprecipitated radiolabeled catD did not reveal a difference in the ratio of proteolytically processed 46 - kD intermediate vs . 52 - kD precursor ( mean ± SEM , n = 3 ) . ( B - D ) TLN localizes to autophagic vacuoles of catD − / − hippocampal neurons . Lysotracker staining revealed sparsely distributed small - sized organelles in wild - type and PS1 − / − neurons in contrast to catD − / − neurons where high numbers of large Lysotracker - positive organelles were found ( B , arrowheads ) . Although TLN accumulations in PS1 − / − were fully negative for Lysotracker , some acidic organelles tended to closely associate ( C , arrowheads , middle ) . In catD − / − neurons , TLN was clearly detected in the large acidic organelles ( C , bottom ) . Bars , 20 μm . ( D ) At the immuno - EM level , the large acidic organelles seen in catD − / − neurons represent dense autophagic vacuole - like structures ( star ) that label positive for TLN ( 10 - nm gold ) . M , mitochondrion . Bars , 200 nm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8.(A) Intracellular sorting and maturation of catD. Wild-type and PS1−/− neurons were metabolically labeled and chased for the indicated time periods. Phosphorimaging of immunoprecipitated radiolabeled catD did not reveal a difference in the ratio of proteolytically processed 46-kD intermediate vs. 52-kD precursor (mean ± SEM, n = 3).(B-D) TLN localizes to autophagic vacuoles of catD−/−hippocampal neurons. Lysotracker staining revealed sparsely distributed small-sized organelles in wild-type and PS1−/− neurons in contrast to catD−/−neurons where high numbers of large Lysotracker-positive organelles were found (B, arrowheads). Although TLN accumulations in PS1−/− were fully negative for Lysotracker, some acidic organelles tended to closely associate (C, arrowheads, middle). In catD−/− neurons, TLN was clearly detected in the large acidic organelles (C, bottom). Bars, 20 μm. (D) At the immuno-EM level, the large acidic organelles seen in catD−/−neurons represent dense autophagic vacuole-like structures (star) that label positive for TLN (10-nm gold). M, mitochondrion. Bars, 200 nm.


Figure 9 . TLN mediates phagocytic uptake of microbeads in hippocampal neurons . ( A ) Microbeads align along neurites of hippocampal neurons as demonstrated by phase contrast ( DIC ) . Already after 4 h , many beads stain positive for TLN ( arrowheads , top ) . At 48 h ( bottom ) , most TLN immunoreactivity was associated with microbeads at the expense of its typical plasma membrane staining . * , cell body . Note that microbeads were not found associated with preexisting TLN accumulations ( asterisk in top panels , 4 h ) . ( B ) Microbeads ( 24 h ) accumulate TLN and actin , as shown by phalloidin - Alexa 568 . An overview of the neuron is given in each top left inset , next to two detailed areas ( white boxes ) . The differential interference contrast ( DIC ) clearly demonstrates a complete colocalization of microbeads with TLN and phalloidin ( arrowheads ) . TOPRO - 3 ( blue ) marks the nucleus ( see also Fig . S1 , available at http : / / www . jcb . org / cgi / content / full / jcb . 200406060 / DC1 ) . ( C ) Similar as in B , but immunostained for TLN and endogenous PIP2 . The overview demonstrates the overall recruitment of PIP2 to microbeads ( arrows ) . The parallel insets show a detailed area ( white box ) with PIP2 being colocalized with TLN on individual microbeads ( arrowheads ) . Note that PIP2 occasionally colocalizes with TLN at the plasma membrane ( asterisk ) . Bars ( A - C ) , 10 μm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9.TLN mediates phagocytic uptake of microbeads in hippocampal neurons. (A) Microbeads align along neurites of hippocampal neurons as demonstrated by phase contrast (DIC). Already after 4 h, many beads stain positive for TLN (arrowheads, top). At 48 h (bottom), most TLN immunoreactivity was associated with microbeads at the expense of its typical plasma membrane staining. *, cell body. Note that microbeads were not found associated with preexisting TLN accumulations (asterisk in top panels, 4 h). (B) Microbeads (24 h) accumulate TLN and actin, as shown by phalloidin-Alexa 568. An overview of the neuron is given in each top left inset, next to two detailed areas (white boxes). The differential interference contrast (DIC) clearly demonstrates a complete colocalization of microbeads with TLN and phalloidin (arrowheads). TOPRO-3 (blue) marks the nucleus (see also Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200406060/DC1). (C) Similar as in B, but immunostained for TLN and endogenous PIP2. The overview demonstrates the overall recruitment of PIP2 to microbeads (arrows). The parallel insets show a detailed area (white box) with PIP2 being colocalized with TLN on individual microbeads (arrowheads). Note that PIP2 occasionally colocalizes with TLN at the plasma membrane (asterisk). Bars (A-C), 10 μm.


Figure 1A Proteins captured by BGL3 and RMRP antisense purifications . Proteins with an absolute log2 ( fold change ) > 1 and - log10 ( p - value ) > 1 . 3 ( compared with control purification ) were considered as proteins enriched by BGL3 specifically . C & D RNA pull - down assay for BGL3 - BARD1 / PARP1 interactions before and after irradiation . Biotinylated in vitro - transcribed LncRNA - BGL3 sense or antisense transcripts were incubated with HEK - 293T cell lysates with or without ionizing radiation ( 10 Gy ) with 1 hr recovery for in vitro streptavidin RNA pull - down assays , followed by Western blots using the indicated antibodies . HA - tagged full - length or deletion mutants of BARD1 were transfected into 293T cells . 48 hr later , BGL3 RNA pull - down assay were performed , and arrowheads indicate the bands for HA - tagged full - length or deletion mutants of BARD1HA - tagged full - length or deletion mutants of PARP1 ( H ) were transfected into 293T cells . 48 hr later , BGL3 RNA pull - down assay were performed ,

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Proteins captured by BGL3 and RMRP antisense purifications. Proteins with an absolute log2 (fold change) > 1 and -log10 (p-value) > 1.3 (compared with control purification) were considered as proteins enriched by BGL3 specifically.C&D RNA pull-down assay for BGL3-BARD1/PARP1 interactions before and after irradiation. Biotinylated in vitro-transcribed LncRNA-BGL3 sense or antisense transcripts were incubated with HEK-293T cell lysates with or without ionizing radiation (10 Gy) with 1 hr recovery for in vitro streptavidin RNA pull-down assays, followed by Western blots using the indicated antibodies.HA-tagged full-length or deletion mutants of BARD1 were transfected into 293T cells. 48 hr later, BGL3 RNA pull-down assay were performed, and arrowheads indicate the bands for HA-tagged full-length or deletion mutants of BARD1HA-tagged full-length or deletion mutants of PARP1(H) were transfected into 293T cells. 48 hr later, BGL3 RNA pull-down assay were performed,


Figure 2A BGL3 is recruited to DNA damage sites . U2OS cells were subjected to laser micro - irradiation to generate DSBs in a line pattern . The relocation kinetics of BGL3 to DSBs was monitored by RNA fluorescence in situ hybridization ( FISH ) in a time course as indicated . BGL3 intensities at the laser line were normalized into a numerical value using Nikon NIS - Elements AR software ( version 4 . 40 . 00 ) . Data are presented as mean ± SD of four biological replicates . B Chromosome aberrations induced by BGL3 depletion . Wild - type or BGL3 depleted BJ - 5ta cells were used in the metaphase spread analysis for spontaneous DNA breaks . Representative image ( left panel ) and the percentage of cells containing at least one DNA break ( right panel ) . Data are presented as mean ± SD of three biological replicates . Two - tailed Student ' s t test , * * P < 0 . 01 . C MCF - 7 cells were treated as indicated , and cell response to ionizing radiation were analyzed by colony formation assays ( left panel ) . Data are presented as mean ± SEM of four independent experiments , analyzed by two - way analysis of variance ( ANOVA ) , * * P < 0 . 01 . Knock - down efficiency of BGL3 siRNA was examined by qRT - PCR and normalized to β - Actin ( middle panel ) , data are presented as mean ± SD of three biological replicates . Two - tailed Student ' s t test , * * P < 0 . 01 . Knock - down efficiency of CtIP siRNA was examined by blotting ( right panel ) . D MCF - 7 cells were transfected with the indicated siRNAs . Cell sensitivity to camptothecin ( CPT ) , hydroxyurea ( HU ) , etoposide ( ETO ) , or ionizing radiation was determined by MTS assays . Data are presented as mean ± SD of three biological replicates . Two - tailed Student ' s t test , * P < 0 . 05 , * * P < 0 . 01 , NS : no significant difference . E BGL3 deficiency inhibits DNA damage repair . Quantification of γ - H2AX foci at indicated times after irradiation ( 2Gy ) are presented . Data shown are results of three independent experiments ( 100 cells for each experiment ) , presented as mean ± SD , two - tailed Student ' s t test , * P < 0 . 05 , * * P < 0 . 01 , NS : no significant difference .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A BGL3 is recruited to DNA damage sites. U2OS cells were subjected to laser micro-irradiation to generate DSBs in a line pattern. The relocation kinetics of BGL3 to DSBs was monitored by RNA fluorescence in situ hybridization (FISH) in a time course as indicated. BGL3 intensities at the laser line were normalized into a numerical value using Nikon NIS-Elements AR software (version 4.40.00). Data are presented as mean ±SD of four biological replicates.B Chromosome aberrations induced by BGL3 depletion. Wild-type or BGL3 depleted BJ-5ta cells were used in the metaphase spread analysis for spontaneous DNA breaks. Representative image (left panel) and the percentage of cells containing at least one DNA break (right panel). Data are presented as mean ± SD of three biological replicates. Two-tailed Student's t test, ** P<0.01.C MCF-7 cells were treated as indicated, and cell response to ionizing radiation were analyzed by colony formation assays (left panel). Data are presented as mean ± SEM of four independent experiments, analyzed by two-way analysis of variance (ANOVA), **P < 0.01. Knock-down efficiency of BGL3 siRNA was examined by qRT-PCR and normalized to β-Actin (middle panel), data are presented as mean ± SD of three biological replicates. Two-tailed Student's t test, **P < 0.01. Knock-down efficiency of CtIP siRNA was examined by blotting (right panel).D MCF-7 cells were transfected with the indicated siRNAs. Cell sensitivity to camptothecin (CPT), hydroxyurea (HU), etoposide (ETO), or ionizing radiation was determined by MTS assays. Data are presented as mean ± SD of three biological replicates. Two-tailed Student's t test, *P < 0.05, **P < 0.01, NS: no significant difference.E BGL3 deficiency inhibits DNA damage repair. Quantification of γ-H2AX foci at indicated times after irradiation (2Gy) are presented. Data shown are results of three independent experiments (100 cells for each experiment), presented as mean ± SD, two-tailed Student's t test, *P < 0.05, **P < 0.01, NS: no significant difference.


Figure 3A & B HEK293 cells integrated with HR or NHEJ reporter were transfected with the indicated siRNAs and subjected to the HR ( A ) or NHEJ assay ( B ) as described in Methods . Data presented as mean ± SD of four biological replicates , and positive cell percentages as compared with control group . Two - tailed Student ' s t test , * * P < 0 . 01 . NS : no significant difference . C - F Wild - type ( CTRL ) or BGL3 knockdown U2OS cells were subjected to micro - irradiation ; 1 hr later , cells were fixed and processed for immunostaining with indicated antibodies . Representative images of BRCA1 ( C ) , BARD1 ( D ) , RAD51 ( E ) , and RPA70 ( F ) accumulation at sites of laser - induced DNA damage are shown .

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Figure 3A&B HEK293 cells integrated with HR or NHEJ reporter were transfected with the indicated siRNAs and subjected to the HR (A) or NHEJ assay (B) as described in Methods. Data presented as mean ±SD of four biological replicates, and positive cell percentages as compared with control group. Two-tailed Student's t test, **P < 0.01. NS: no significant difference.C-F Wild-type (CTRL) or BGL3 knockdown U2OS cells were subjected to micro-irradiation; 1 hr later, cells were fixed and processed for immunostaining with indicated antibodies. Representative images of BRCA1 (C), BARD1 (D), RAD51 (E), and RPA70 (F) accumulation at sites of laser-induced DNA damage are shown.


Figure 4Parental or BGL3 - deficient U2OS cells were subjected to laser micro - irradiation to generate DSBs in a line pattern . DDR factor recruitment were examined 2 hr later . Data shown are the average of three independent experiments , and 100 cells were counted for each experiment . Data are presented as mean ± SD , analyzed by two - tailed Student ' s t test , * * P < 0 . 01 . Parental or BGL3 - deficient U2OS cells were subjected to laser micro - irradiation to generate DSBs in a line pattern . DDR factor recruitment were examined 2 hr later . Data shown are the average of three independent experiments , and 100 cells were counted for each experiment . Data are presented as mean ± SD , analyzed by two - tailed Student ' s t test , * * P < 0 . 01 . U2OS cells were transfected with the indicated siRNAs , and 48 hr later , HR efficiency were determined using the reporter assay , as described in the Methods . Data shown are the average of three independent experiments . Data are presented as mean ± SD , analyzed by two - tailed Student ' s t test , NS : no significant difference . E U2OS cells were transfected with the indicated siRNA , and RAD51 accumulation at sites of laser - induced DNA damage were examined . Data shown are the average of three independent experiments , and 100 cells were counted for each experiment . Data are presented as mean ± SD , analyzed by two - tailed Student ' s t test , NS : no significant difference . Knock - down efficiency of BARD1 siRNA was examined by blotting ( bottom left panel ) , and knock - down efficiency of BGL3 siRNA was examined by qRT - PCR and normalized to β - actin ( bottom right panel ) , data are presented as mean ± SD of three biological replicates . Two - tailed Student ' s t test , * * P < 0 . 01 .

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Figure 4Parental or BGL3-deficient U2OS cells were subjected to laser micro-irradiation to generate DSBs in a line pattern. DDR factor recruitment were examined 2 hr later. Data shown are the average of three independent experiments, and 100 cells were counted for each experiment. Data are presented as mean ± SD, analyzed by two-tailed Student's t test, **P < 0.01.Parental or BGL3-deficient U2OS cells were subjected to laser micro-irradiation to generate DSBs in a line pattern. DDR factor recruitment were examined 2 hr later. Data shown are the average of three independent experiments, and 100 cells were counted for each experiment. Data are presented as mean ± SD, analyzed by two-tailed Student's t test, **P < 0.01.U2OS cells were transfected with the indicated siRNAs, and 48 hr later, HR efficiency were determined using the reporter assay, as described in the Methods. Data shown are the average of three independent experiments. Data are presented as mean ± SD, analyzed by two-tailed Student's t test, NS: no significant difference.E U2OS cells were transfected with the indicated siRNA, and RAD51 accumulation at sites of laser-induced DNA damage were examined. Data shown are the average of three independent experiments, and 100 cells were counted for each experiment. Data are presented as mean ± SD, analyzed by two-tailed Student's t test, NS: no significant difference. Knock-down efficiency of BARD1 siRNA was examined by blotting (bottom left panel), and knock-down efficiency of BGL3 siRNA was examined by qRT-PCR and normalized to β-actin (bottom right panel), data are presented as mean ± SD of three biological replicates. Two-tailed Student's t test, **P < 0.01.


Figure 5A BGL3 promotes BARD1 - HP1γ interactions . 293T cells expressing the indicated siRNAs were irradiated ( 10 Gy ) , and BARD1 - HP1γ interactions examined at the indicated time points . B BGL3 promotes RAP80 binding to K63 ubiquitin chain in cells . 293T cells expressing the indicated siRNAs and plasmids were irradiated ( 10 Gy ) . Cell lysates were immunoprecipitated with HA beads and subjected to immunoblot with the indicated antibodies . C BGL3 promotes BARD1 - HP1γ ( up panel ) and BARD1 - RAD51 ( middle panel ) interactions in vitro . GST - HP1γ , GST - RAD51 , and FLAG - BARD1 were overexpressed and purified from cells as indicated in Methods . FLAG - BARD1 was divided into two equal parts , one part was used for BARD1 - HP1γ interaction analysis ( up panel ) , and another part was used for BARD1 - RAD51 interaction analysis ( middle panel ) . BARD1 - HP1γ ( up panel ) and BARD1 - RAD51 ( middle panel ) interactions with or without BGL3 were respectively detected by GST pull - down assays , here GST was used as a pull - down control .

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Figure 5A BGL3 promotes BARD1-HP1γ interactions. 293T cells expressing the indicated siRNAs were irradiated (10 Gy), and BARD1-HP1γ interactions examined at the indicated time points.B BGL3 promotes RAP80 binding to K63 ubiquitin chain in cells. 293T cells expressing the indicated siRNAs and plasmids were irradiated (10 Gy). Cell lysates were immunoprecipitated with HA beads and subjected to immunoblot with the indicated antibodies.C BGL3 promotes BARD1-HP1γ (up panel) and BARD1-RAD51 (middle panel) interactions in vitro. GST-HP1γ, GST-RAD51, and FLAG-BARD1 were overexpressed and purified from cells as indicated in Methods. FLAG-BARD1 was divided into two equal parts, one part was used for BARD1-HP1γ interaction analysis (up panel), and another part was used for BARD1-RAD51 interaction analysis (middle panel). BARD1-HP1γ (up panel) and BARD1-RAD51 (middle panel) interactions with or without BGL3 were respectively detected by GST pull-down assays, here GST was used as a pull-down control.


Figure 6A PARP1 inhibitor treatment abolished BGL3 recruitment to DSBs . U2OS cells were treated with DMSO , PARP inhibitor ( olaparib ) , ATM inhibitor ( KU - 55933 ) , ATR inhibitor ( VE - 821 ) or caffeine . BGL3 recruitment to DNA damage sites was assessed by RNA - FISH assays . B Quantification of the positive cells . For each group , 100 randomly selected cells were counted . Data are presented as mean ± SD of three biological replicates . Two - tailed Student ' s t test , * * P < 0 . 01 , N : no significant difference . C PARP inhibitor mainly affected BGL3 recruitment at an early time point . U2OS cells were treated with DMSO or olaparib , and BGL3 recruitment was examined following laser micro - irradiation at indicated time point . Data presented are the average of three independent experiments , and 100 cells were counted for each experiment . Two - tailed Student ' s t test , * * P < 0 . 01 , N : no significant difference . D U2OS cells were transfected with the indicated siRNAs , and BGL3 recruitment to DSBs were monitored at the indicated time point by RNA FISH . Data presented are the average of three independent experiments , and 100 cells were counted for each experiment . Two - tailed Student ' s t test , * * P < 0 . 01 , N : no significant difference .

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Figure 6A PARP1 inhibitor treatment abolished BGL3 recruitment to DSBs. U2OS cells were treated with DMSO, PARP inhibitor (olaparib), ATM inhibitor (KU-55933), ATR inhibitor (VE-821) or caffeine. BGL3 recruitment to DNA damage sites was assessed by RNA-FISH assays. B Quantification of the positive cells. For each group, 100 randomly selected cells were counted. Data are presented as mean ±SD of three biological replicates. Two-tailed Student's t test, **P < 0.01, N: no significant difference. C PARP inhibitor mainly affected BGL3 recruitment at an early time point. U2OS cells were treated with DMSO or olaparib, and BGL3 recruitment was examined following laser micro-irradiation at indicated time point. Data presented are the average of three independent experiments, and 100 cells were counted for each experiment. Two-tailed Student's t test, **P < 0.01, N: no significant difference.D U2OS cells were transfected with the indicated siRNAs, and BGL3 recruitment to DSBs were monitored at the indicated time point by RNA FISH. Data presented are the average of three independent experiments, and 100 cells were counted for each experiment. Two-tailed Student's t test, **P < 0.01, N: no significant difference.


Figure 2 ( A ) Pancreatic cancer cells ( PANC - 1 ) and normal human fibroblasts ( NHFs ) were treated with increasing concentrations of Green 1 and NSC 51046 and following treatment at the indicated time points , cells were incubated with WST - 1 cell viability dye for 4 hours . Absorbance was read at 450 nm and results were expressed as a percent of the control . Values are expressed as mean ± SD from quadruplicates ; ( i ) PANC - 1 treated with NSC 51046 ( Interaction between each concentration : * * * * p < 0 . 0001 ; Control versus column factor : * * * * p < 0 . 0001 ) ; ( ii ) NHF treated with NSC 51046 ( Interaction between each concentration : * * * * p < 0 . 0001 ; Control versus column factor : * * * * p < 0 . 0001 ) ; ( iii ) PANC - 1 treated with Green 1 ( Interaction between each concentration : p = 0 . 9955 ; Control versus column factor : * * * * p < 0 . 0001 ) ; ( iv ) NHF treated with Green 1 ( Interaction between each concentration : p = 0 . 3642 ; Control versus column factor : * p = 0 . 0370 ) ( B ) Following treatment of human acute T cell leukemia cells ( Jurkat ) with Green 1 , a trypan blue excluding assay was carried out . Cells were stained with trypan blue cell membrane impermeable dye and the numbers of trypan blue positive cells were obtained using a hemocytometer . Results are expressed as percent of trypan blue positive cells , indicating dead cells with non - intact cell membranes . Values are expressed as mean ± SD from two independent experiments .

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Figure 2(A) Pancreatic cancer cells (PANC-1) and normal human fibroblasts (NHFs) were treated with increasing concentrations of Green 1 and NSC 51046 and following treatment at the indicated time points, cells were incubated with WST-1 cell viability dye for 4 hours. Absorbance was read at 450 nm and results were expressed as a percent of the control. Values are expressed as mean ± SD from quadruplicates; (i) PANC-1 treated with NSC 51046 (Interaction between each concentration: ****p<0.0001; Control versus column factor: ****p<0.0001); (ii) NHF treated with NSC 51046 (Interaction between each concentration: ****p<0.0001; Control versus column factor: ****p<0.0001); (iii) PANC-1 treated with Green 1 (Interaction between each concentration: p = 0.9955; Control versus column factor: ****p<0.0001); (iv) NHF treated with Green 1 (Interaction between each concentration: p = 0.3642; Control versus column factor: *p = 0.0370)(B) Following treatment of human acute T cell leukemia cells (Jurkat) with Green 1, a trypan blue excluding assay was carried out. Cells were stained with trypan blue cell membrane impermeable dye and the numbers of trypan blue positive cells were obtained using a hemocytometer. Results are expressed as percent of trypan blue positive cells, indicating dead cells with non-intact cell membranes. Values are expressed as mean ± SD from two independent experiments.


Figure 3Following treatment with either Green 1 ( A ) Pancreatic cancer cells ( BxPC - 3 and PANC - 1 ) and normal human and fetal fibroblasts ( NHFs and NFFs ) were incubated with Hoechst 33342 dye to characterize nuclear morphology and detect the induction of apoptosis . Brightly stained , condensed nuclei accompanied by apoptotic bodies are indicative of apoptosis . ( B ) PANC - 1 cells were incubated with Annexin V dye to verify apoptotic induction caused by NSC 51046 . Cells were counterstained with Hoechst 33342 dye and propidium iodide ( PI ) to visualize nuclear morphology and cell death . Images shown are representative of two independent experiments at 48 hours following treatment with NSC 51046 and colchicine .

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Figure 3Following treatment with either Green 1(A) Pancreatic cancer cells (BxPC-3 and PANC-1) and normal human and fetal fibroblasts (NHFs and NFFs) were incubated with Hoechst 33342 dye to characterize nuclear morphology and detect the induction of apoptosis. Brightly stained, condensed nuclei accompanied by apoptotic bodies are indicative of apoptosis.(B) PANC-1 cells were incubated with Annexin V dye to verify apoptotic induction caused by NSC 51046. Cells were counterstained with Hoechst 33342 dye and propidium iodide (PI) to visualize nuclear morphology and cell death. Images shown are representative of two independent experiments at 48 hours following treatment with NSC 51046 and colchicine.


Figure 4 ( A ) Pancreatic cancer cells , human leukemia cells ( Jurkat ) and NHFs were incubated with Monodansylcadaverine ( MDC ) to stain autophagic vacuoles and propidium iodide ( PI ) for cell death to determine if autophagic induction led to cell death . Bright , punctate staining is indicative of autophagy , as seen in our positive control ( Tamoxifen treated cells ) . Images were taken at 400X magnification on a fluorescent microscope . ( B ) Western blot analyses of Beclin - 1 expression and LC3 conversion were conducted on PANC - 1 cells treated with 5 . 0 µM Green 1 at various time points . β - actin was measured as an internal control . Samples treated for 3 and 6 hours were compared to the early control , while the sample treated for 48 hours was compared to the 48 - hour control . Results are expressed as mean ± SD from two independent experiments for both Western blots .

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Figure 4(A) Pancreatic cancer cells, human leukemia cells (Jurkat) and NHFs were incubated with Monodansylcadaverine (MDC) to stain autophagic vacuoles and propidium iodide (PI) for cell death to determine if autophagic induction led to cell death. Bright, punctate staining is indicative of autophagy, as seen in our positive control (Tamoxifen treated cells). Images were taken at 400X magnification on a fluorescent microscope.(B) Western blot analyses of Beclin-1 expression and LC3 conversion were conducted on PANC-1 cells treated with 5.0 µM Green 1 at various time points. β-actin was measured as an internal control. Samples treated for 3 and 6 hours were compared to the early control, while the sample treated for 48 hours was compared to the 48-hour control. Results are expressed as mean ± SD from two independent experiments for both Western blots.


Figure 5 ( A ) Colchicine derivatives ( Green 1 and NSC 51046 ) were incubated with porcine tubulin for an hour in a 96 - well plate . The absorbance ( OD 340 nm ) was obtained every minute during the one hour incubation . Colchicine was used for comparison to the derivatives . ( B ) Isolated mitochondria from PANC - 1 cells were treated with Green 1 and ROS production was measured using Amplex Red substrate in the presence of horseradish peroxidase ( HRP ) . Results were compared to control untreated mitochondria , colchicine treated mitochondria and positive control , paraquat ( PQ ) . Fluorescence readings were taken in 5 min intervals for 4 hours at Ex . 560 nm and Em . 590 nm and expressed as relative fluorescence units ( RFU ) . Results are a representative of three independent experiments .

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Figure 5(A) Colchicine derivatives (Green 1 and NSC 51046) were incubated with porcine tubulin for an hour in a 96-well plate. The absorbance (OD 340 nm) was obtained every minute during the one hour incubation. Colchicine was used for comparison to the derivatives.(B) Isolated mitochondria from PANC-1 cells were treated with Green 1 and ROS production was measured using Amplex Red substrate in the presence of horseradish peroxidase (HRP). Results were compared to control untreated mitochondria, colchicine treated mitochondria and positive control, paraquat (PQ). Fluorescence readings were taken in 5 min intervals for 4 hours at Ex. 560 nm and Em.590 nm and expressed as relative fluorescence units (RFU). Results are a representative of three independent experiments.


Figure 6Following acclimatization , CD - 1 nu / nu mice were separated into two groups , one group was injected subcutaneously in the right and left hind flanks with Me2SO in PBS ( 10 µL in 200 µL PBS ) , while the second group received subcutaneous injections of Green 1 ( 10 mg / kg / day for a total volume of 10 µL Green 1 / 200 µL PBS ) three times a week for a period of one month . ( A ) Mice weights were recorded twice a week for the duration of the study . ( B ) Following the study , mice were sacrificed and their organs were obtained for histopathological assessment . Hematoxylin and Eosin staining was carried out on the tissue samples .

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Figure 6Following acclimatization, CD-1 nu/nu mice were separated into two groups, one group was injected subcutaneously in the right and left hind flanks with Me2SO in PBS (10 µL in 200 µL PBS), while the second group received subcutaneous injections of Green 1 (10 mg/kg/day for a total volume of 10 µL Green 1/200 µL PBS) three times a week for a period of one month. (A) Mice weights were recorded twice a week for the duration of the study.(B) Following the study, mice were sacrificed and their organs were obtained for histopathological assessment. Hematoxylin and Eosin staining was carried out on the tissue samples.


Figure 1 ( A ) BACE1 internalization kinetics after 20 minutes ( WT : 19 . 79 ± 1 . 89 % , KO : 13 . 28 ± 1 . 57 % , p WT vs KO = 0 . 017 ) and 40 minutes of HA antibody chase ( WT : 15 . 99 ± 2 . 04 , KO : 9 . 29 ± 1 . 33 , pWT vs KO = 0 . 010 ) is significantly decreased in AP - 2µ KO neurons ( 27 WT and 29 KO neurons ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . ( B ) Representative examples of internalized BACE1 after 5 min of HA antibody pulse in HA - BACE1 - eGFP - overexpressing WT and KO neurons . Scale bar : 5µm . ( C , D ) Internalized BACE1 levels after 20 min of HA antibody pulse in HA - BACE1 - eGFP - overexpressing WT and KO neurons , calculated as the HA ( internalized ) / eGFP ( total ) signal intensity ratio ( WT : 0 . 50 ± 0 . 052 , KO : 0 . 23 ± 0 . 036 , p = 0 . 000 , 30 WT and 36 KO neurons , N = 4 biological replicates ) . Scale bar : 5 µm . Data information : Experimental designs are depicted in the top panels where analyzed BACE1 fraction is marked with the red square . All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . ( E , F ) Recycled BACE1 levels after 20 min of HA antibody pulse in HA - BACE1 - eGFP - overexpressing WT and KO neurons , calculated as the HA ( recycled ) / eGFP ( total ) signal intensity ratio ( WT : 0 . 29 ± 0 . 031 , KO : 0 . 53 ± 0 . 040 , p < 000 , 30 WT and 32 KO neurons , N = 4 biological replicates ) . Scale bar : 5 µm . Data information : Experimental designs are depicted in the top panels where analyzed BACE1 fraction is marked with the red square . All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . Recycled levels of BACE1 after 20 min of HA antibody pulse in HA - BACE1 - mCherry - transfected WT and KO neurons , co - expressing either eGFP - RAB4 or eGFP - RAB4 - S22N pWTRAB4 vs pWTRAB4S22N > 0 . 999 , 34 - 38 neurons , N = 4 biological replicates ) . Scale bar : 5 µm . Data information : Experimental designs are depicted in the top panels , where analyzed BACE1 fraction is marked with the red square . Recycled levels of BACE1 after 20 min of HA antibody pulse in HA - BACE1 - mCherry - transfected WT and KO neurons , co - expressing either eGFP - RAB4 or eGFP - RAB4 - S22N , calculated as the HA ( recycled ) / mCherry ( total ) signal intensity ratio ( WTRAB4 : 0 . 14 ± 0 . 02 , KORAB4 : 0 . 20 ± 0 . 02 , WTRAB4 - S22N : 0 . 14 ± 0 . 02 , KORAB4S22N : 0 . 14 ± 0 . 01 , pWTRAB4 vs pKORAB4 = 0 . 033 , pKORAB4 vs pKORAB4S22N = 0 . 035 , pWTRABS22N vs pKORABS22N = 0 . 999 , pWTRAB4 vs pWTRAB4S22N > 0 . 999 , 34 - 38 neurons , N = 4 biological replicates ) . Scale bar : 5 µm . Data information : All graphs show mean ± SEM , statistical analysis was performed by two - way ANOVA in n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . Recycled levels of BACE1 after 20 min of HA antibody chase in HA - BACE1 - mCherry - transfected WT and KO neurons , co - expressing either eGFP - RAB11 or eGFP - RAB11 - S22N , calculated as the HA ( recycled ) / mCherry ( total ) signal intensity ratio ( WTRAB11 : 0 . 11 ± 0 . 01 , KORAB11 : 0 . 29 ± 0 . 02 , WTRAB11A - S25N : 0 . 14 ± 0 . 02 , KORAB11A - S25N : 0 . 19 ± 0 . 02 , pWTRAB11 vs pKORAB11 < 0 . 0001 , pWTRAB11 vs pWTRAB11A - S25N = 0 . 761 , pKORAB11 vs pKORAB11A - S25N = 0 . 001 , pWTRAB11A - S25N vs pKORAB11A - S25N = 0 . 175 , 28 - 37 neurons per condition , N = 3 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by two - way ANOVA n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . Internalized levels of BACE1 after 20 minutes of HA antibody uptake in HA - BACE1 - mCherry transfected WT and KO neurons , co - expressing either eGFP - RAB4 or eGFP - RAB4 - S22N pWTRAB4 vs pWTRAB4S22N = 0 . 450 , 27 - 37 neurons per condition , N = 3 biological replicates ) . Scale bar : 5 µm . Data information : Experimental designs are depicted in the top panels where analyzed BACE1 fraction is marked with the red square . Internalized levels of BACE1 after 20 minutes of HA antibody uptake in HA - BACE1 - mCherry transfected WT and KO neurons , co - expressing either eGFP - RAB4 or eGFP - RAB4 - S22N , calculated as the HA ( internalized ) / mCherry ( total ) signal intensity ratio ( WTRAB4 : 0 . 54 ± 0 . 05 , KORAB4 : 0 . 33 ± 0 . 02 , WTRAB4 - S22N : 0 . 63 ± 0 . 06 , KORAB11A - S25N : 0 . 50 ± 0 . 04 , pWTRAB4 vs pKORAB4 = 0 . 004 , pKORAB4 vs pKORAB4S22N = 0 . 046 , pWTRABS22N vs pKORABS22N = 0 . 182 , pWTRAB4 vs pWTRAB4S22N = 0 . 450 , 27 - 37 neurons per condition , N = 3 biological replicates ) . Scale bar : 5 µm . Data information : All graphs show mean ± SEM , statistical analysis was performed by ANOVA n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . ( L , M ) BACE1 surface levels are increased in AP - 2µ KO neurons , when compared to the WT set to 100 % ( KO : 129 . 65 ± 5 . 30 % , p = 0 . 005 , N = 4 biological replicates ) . ( N ) Surface versus total BACE1 ratio is not altered in AP - 2µ KO neurons when normalized to the WT set to 100 % ( KO : 83 . 02 ± 10 . 93 % , p = 0 . 109 , N = 4 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by one - sample Student ' s t ‐ test in ( M , N ) . n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . ( O , P ) Accumulation of endogenous BACE1 in RAB5 - positive early endosomes , quantified using Pearson ' s colocalization coefficient ( WT : 0 . 41 ± 0 . 01 , KO : 0 . 46 ± 0 . 02 , pWT vs pKO = 0 . 021 , 58 WT and 54 KO neurons , N = 4 biological replicates ) . Scale bar : 10 µm . Arrows indicate BACE1 accumulation within RAB5 - positive endosomes . Data information All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) BACE1 internalization kinetics after 20 minutes (WT: 19.79±1.89%, KO: 13.28±1.57%, p WT vs KO=0.017) and 40 minutes of HA antibody chase (WT: 15.99±2.04, KO: 9.29±1.33, pWT vs KO=0.010) is significantly decreased in AP-2µ KO neurons (27 WT and 29 KO neurons). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.(B) Representative examples of internalized BACE1 after 5 min of HA antibody pulse in HA-BACE1-eGFP-overexpressing WT and KO neurons. Scale bar: 5µm.(C, D) Internalized BACE1 levels after 20 min of HA antibody pulse in HA-BACE1-eGFP-overexpressing WT and KO neurons, calculated as the HA (internalized)/eGFP (total) signal intensity ratio (WT: 0.50±0.052, KO: 0.23±0.036, p=0.000, 30 WT and 36 KO neurons, N=4 biological replicates). Scale bar: 5 µm. Data information: Experimental designs are depicted in the top panels where analyzed BACE1 fraction is marked with the red square. All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.(E, F) Recycled BACE1 levels after 20 min of HA antibody pulse in HA-BACE1-eGFP-overexpressing WT and KO neurons, calculated as the HA(recycled)/eGFP(total) signal intensity ratio (WT: 0.29±0.031, KO: 0.53±0.040, p<000, 30 WT and 32 KO neurons, N=4 biological replicates). Scale bar: 5 µm. Data information: Experimental designs are depicted in the top panels where analyzed BACE1 fraction is marked with the red square. All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.Recycled levels of BACE1 after 20 min of HA antibody pulse in HA-BACE1-mCherry-transfected WT and KO neurons, co-expressing either eGFP-RAB4 or eGFP-RAB4-S22N pWTRAB4 vs pWTRAB4S22N>0.999, 34-38 neurons, N=4 biological replicates). Scale bar: 5 µm. Data information: Experimental designs are depicted in the top panels , where analyzed BACE1 fraction is marked with the red square.Recycled levels of BACE1 after 20 min of HA antibody pulse in HA-BACE1-mCherry-transfected WT and KO neurons, co-expressing either eGFP-RAB4 or eGFP-RAB4-S22N, calculated as the HA(recycled)/mCherry(total) signal intensity ratio (WTRAB4: 0.14±0.02, KORAB4: 0.20±0.02, WTRAB4-S22N: 0.14±0.02, KORAB4S22N:0.14±0.01, pWTRAB4 vs pKORAB4=0.033, pKORAB4 vs pKORAB4S22N=0.035, pWTRABS22N vs pKORABS22N=0.999, pWTRAB4 vs pWTRAB4S22N>0.999, 34-38 neurons, N=4 biological replicates). Scale bar: 5 µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA in n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.Recycled levels of BACE1 after 20 min of HA antibody chase in HA-BACE1-mCherry-transfected WT and KO neurons, co-expressing either eGFP-RAB11 or eGFP-RAB11-S22N, calculated as the HA(recycled)/mCherry(total) signal intensity ratio (WTRAB11: 0.11±0.01, KORAB11: 0.29±0.02, WTRAB11A-S25N: 0.14±0.02, KORAB11A-S25N: 0.19±0.02, pWTRAB11 vs pKORAB11<0.0001, pWTRAB11 vs pWTRAB11A-S25N=0.761, pKORAB11 vs pKORAB11A-S25N=0.001, pWTRAB11A-S25N vs pKORAB11A-S25N=0.175, 28-37 neurons per condition, N=3 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.Internalized levels of BACE1 after 20 minutes of HA antibody uptake in HA-BACE1-mCherry transfected WT and KO neurons, co-expressing either eGFP-RAB4 or eGFP-RAB4-S22N pWTRAB4 vs pWTRAB4S22N=0.450, 27-37 neurons per condition, N=3 biological replicates). Scale bar: 5 µm. Data information: Experimental designs are depicted in the top panels where analyzed BACE1 fraction is marked with the red square.Internalized levels of BACE1 after 20 minutes of HA antibody uptake in HA-BACE1-mCherry transfected WT and KO neurons, co-expressing either eGFP-RAB4 or eGFP-RAB4-S22N, calculated as the HA(internalized)/mCherry(total) signal intensity ratio (WTRAB4: 0.54±0.05, KORAB4: 0.33±0.02, WTRAB4-S22N: 0.63±0.06, KORAB11A-S25N: 0.50±0.04, pWTRAB4 vs pKORAB4=0.004, pKORAB4 vs pKORAB4S22N=0.046, pWTRABS22N vs pKORABS22N=0.182, pWTRAB4 vs pWTRAB4S22N=0.450, 27-37 neurons per condition, N=3 biological replicates). Scale bar: 5 µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by ANOVA n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.(L, M) BACE1 surface levels are increased in AP-2µ KO neurons, when compared to the WT set to 100% (KO: 129.65±5.30%, p=0.005, N=4 biological replicates). (N) Surface versus total BACE1 ratio is not altered in AP-2µ KO neurons when normalized to the WT set to 100% (KO: 83.02±10.93%, p=0.109, N=4 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by one-sample Student's t‐test in (M,N). n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.(O, P) Accumulation of endogenous BACE1 in RAB5-positive early endosomes, quantified using Pearson's colocalization coefficient (WT: 0.41±0.01, KO: 0.46±0.02, pWT vs pKO=0.021, 58 WT and 54 KO neurons, N=4 biological replicates). Scale bar: 10 µm. Arrows indicate BACE1 accumulation within RAB5-positive endosomes. Data information All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


Figure 2 ( A , B ) BACE1 protein levels in cortical lysates of WT and AP - 2µ KO mice . Protein levels in the KO were normalized to the WT set to 100 % ( KO : 447 . 64 ± 48 . 78 % , p < 0 . 000 , N = 7 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by one - sample Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . ( C , D ) Immunofluorescence levels of BACE1 are significantly increased in the cortex of AP - 2µ KO mice when normalized to the WT set to 100 % ( KO : 162 . 714 ± 11 . 082 % , p = 0 . 005 , N = 4 , biological replicates ) . Scale bar : 100µm . Data information : All graphs show mean ± SEM , statistical analysis was performed by one - sample Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . ( E ) Localization of BACE1 in cultured WT and AP - 2µ KO neurons . Scale bars : 20 µm , 5 µm ( inserts ) . ( F ) Line plot of BACE1 immunofluorescence along the neuritesPercentage of BACE1 puncta colocalizing with the LC3b ( WT : 21 . 75 ± 2 . 88 , KO : 30 . 28 ± 2 . 97 , p = 0 . 043 , WT = 41 , KO = 45 neurons , N = 4 biological replicates ) . Scale bar : 2µm . Arrowheads indicate BACE1 accumulation within LC3b - positive organelles . Percentage of BACE1 puncta colocalizing with the LC3b ( WT : 21 . 75 ± 2 . 88 , KO : 30 . 28 ± 2 . 97 , p = 0 . 043 , WT = 41 , KO = 45 neurons , N = 4 biological replicates ) . Scale bar : 2µm . Arrowheads indicate BACE1 accumulation within LC3b - positive organelles . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . ( I , J ) Percentage of BACE1 puncta colocalizing with the eGFP - RAB7 ( WT : 19 . 54 ± 1 . 91 , KO : 37 . 09 ± 3 . 13 % , p < 0 . 000 , WT = 44 and KO = 43 neurons , N = 4 biological replicates ) . Scale bar : 2µm . Arrowheads indicate BACE1 accumulation within RAB7 - positive organelles . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . ( K , L ) Colocalization of BACE1 and RAB7A in cortical sections of WT and AP - 2μ ΚΟ brains , quantified using Pearson ' s colocalization coefficient ( WT : 0 . 18 ± 0 . 04 , KO : 0 . 35 ± 0 . 05 , p = 0 . 038 , N = 4 biological replicates ) . Scale bar : 10 µm . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . ( M Representative fluorescence images of WT neurons transfected with HA - BACE1 - mCherry and immunostained for Cathepsin D . Channels were false color - coded to better illustrate the colocalization . Scale bars : 10µm . Arrowheads indicate BACE1 accumulation in lysosomes in the WT Dotted line outlines the neuron . N ) Representative fluorescence images of AP - 2µ KO neurons transfected with HA - BACE1 - mCherry and immunostained for Cathepsin D . Channels were false color - coded to better illustrate the colocalization . Scale bars : 10µm . Arrowheads indicate BACE1 absence in the KO . Dotted line outlines the neuron . ( O ) Lysosomal localization of BACE1 is decreased in KO neurons ( WT : 73 . 05 ± 3 . 80 , KO : 57 . 68 ± 4 . 28 , p = 0 . 008 , 45 WT and 44 KO neurons , N = 4 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . ( Q , R ) Representative fluorescence images of WT and AP - 2μ KO neurons transfected with BACE1 - mKeima - Red and co - transfected with eBFP to reveal the neuronal morphology . Scale bars : 10µm , 2µm in zoomed images . ( S ) The 550nm / 438nm signal intensity ratio of BACE1 - mKeima - Red quantified over the entire cell is significantly reduced in the KO condition compared to the WT ( WT : 0 . 59 ± 0 . 04 , KO : 0 . 48 ± 0 . 04 , p = 0 . 041 , 28 WT and 25 KO neurons , N = 3 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A, B) BACE1 protein levels in cortical lysates of WT and AP-2µ KO mice. Protein levels in the KO were normalized to the WT set to 100% (KO: 447.64±48.78%, p<0.000, N=7 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by one-sample Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.(C, D) Immunofluorescence levels of BACE1 are significantly increased in the cortex of AP-2µ KO mice when normalized to the WT set to 100% (KO: 162.714±11.082%, p=0.005, N=4, biological replicates). Scale bar: 100µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by one-sample Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.(E) Localization of BACE1 in cultured WT and AP-2µ KO neurons. Scale bars: 20 µm, 5 µm (inserts).(F) Line plot of BACE1 immunofluorescence along the neuritesPercentage of BACE1 puncta colocalizing with the LC3b (WT: 21.75±2.88, KO: 30.28±2.97, p=0.043, WT=41, KO=45 neurons, N=4 biological replicates). Scale bar: 2µm. Arrowheads indicate BACE1 accumulation within LC3b-positive organelles.Percentage of BACE1 puncta colocalizing with the LC3b (WT: 21.75±2.88, KO: 30.28±2.97, p=0.043, WT=41, KO=45 neurons, N=4 biological replicates). Scale bar: 2µm. Arrowheads indicate BACE1 accumulation within LC3b-positive organelles. Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.(I, J) Percentage of BACE1 puncta colocalizing with the eGFP-RAB7 (WT:19.54±1.91, KO: 37.09±3.13%, p<0.000, WT=44 and KO=43 neurons, N=4 biological replicates). Scale bar: 2µm. Arrowheads indicate BACE1 accumulation within RAB7-positive organelles. Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.(K, L) Colocalization of BACE1 and RAB7A in cortical sections of WT and AP-2μ ΚΟ brains, quantified using Pearson's colocalization coefficient (WT: 0.18±0.04, KO: 0.35±0.05, p=0.038, N=4 biological replicates). Scale bar: 10 µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.(M Representative fluorescence images of WT neurons transfected with HA-BACE1-mCherry and immunostained for Cathepsin D. Channels were false color-coded to better illustrate the colocalization. Scale bars: 10µm. Arrowheads indicate BACE1 accumulation in lysosomes in the WT Dotted line outlines the neuron.N) Representative fluorescence images of AP-2µ KO neurons transfected with HA-BACE1-mCherry and immunostained for Cathepsin D. Channels were false color-coded to better illustrate the colocalization. Scale bars: 10µm. Arrowheads indicate BACE1 absence in the KO. Dotted line outlines the neuron.(O) Lysosomal localization of BACE1 is decreased in KO neurons (WT: 73.05±3.80, KO: 57.68±4.28, p=0.008, 45 WT and 44 KO neurons, N=4 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.(Q, R) Representative fluorescence images of WT and AP-2μ KO neurons transfected with BACE1-mKeima-Red and co-transfected with eBFP to reveal the neuronal morphology. Scale bars: 10µm, 2µm in zoomed images.(S) The 550nm/438nm signal intensity ratio of BACE1-mKeima-Red quantified over the entire cell is significantly reduced in the KO condition compared to the WT (WT: 0.59±0.04, KO: 0.48±0.04, p=0.041, 28 WT and 25 KO neurons, N=3 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.