D , E Representative of serial wound tissue sections from indicated genotypes . ( E ) Grayscale of hematoxylin eosin - stained sections . Insets of boxed regions show a higher magnification of region typically containing WAE ( red arrowheads , dashed indicates border with wound bed ) . Orange arrow indicates region of non - adherent residual WAE . Scale bars , 100 µm .

D, E Representative images of serial wound tissue sections from the indicated genotypes. (E) Grayscale images of hematoxylin and eosin-stained sections. Insets of the boxed regions show a higher magnification of the region typically containing WAE cells (red arrowheads, dashed line indicates border with wound bed). Orange arrow indicates region of non-adherent residual WAE cells. Scale bars, 100 µm.


( A ) BACE1 internalization kinetics after 20 minutes ( WT : 19 . 79 ± 1 . 89 % , KO : 13 . 28 ± 1 . 57 % , p WT vs KO = 0 . 017 ) 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 29 KO neurons ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 in HA - BACE1 - eGFP - overexpressing WT KO neurons . Scale bar : 5µm .

O O O O O O O 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) 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 after 20 min of HA antibody in HA - BACE1 - eGFP - overexpressing WT KO neurons , calculated as HA ( internalized ) / eGFP ( total ) signal ( WT : 0 . 50 ± 0 . 052 , KO : 0 . 23 ± 0 . 036 , p = 0 . 000 , 30 WT 36 KO neurons , N = 4 biological replicates ) . Scale bar : 5 µm . Data information : Experimental designs are depicted in top panels where analyzed BACE1 fraction is marked with red square . graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 after 20 min of HA antibody in HA - BACE1 - eGFP - overexpressing WT KO neurons , calculated as HA ( recycled ) / eGFP ( total ) signal ( WT : 0 . 29 ± 0 . 031 , KO : 0 . 53 ± 0 . 040 , p < 000 , 30 WT 32 KO neurons , N = 4 biological replicates ) . Scale bar : 5 µm . Data information : Experimental designs are depicted in top panels where analyzed BACE1 fraction is marked with red square . graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 of BACE1 after 20 min of HA antibody in HA - BACE1 - mCherry - transfected WT 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 top panels , where analyzed BACE1 fraction is marked with red square .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 of BACE1 after 20 min of HA antibody in HA - BACE1 - mCherry - transfected WT KO neurons , co - expressing either eGFP - RAB4 or eGFP - RAB4 - S22N , calculated as HA ( recycled ) / mCherry ( total ) signal ( 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 : graphs show mean ± , statistical was performed by two - way ANOVA in n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 of BACE1 after 20 min of HA antibody chase in HA - BACE1 - mCherry - transfected WT KO neurons , co - expressing either eGFP - RAB11 or eGFP - RAB11 - S22N , calculated as HA ( recycled ) / mCherry ( total ) signal ( 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 : graphs show mean ± , statistical was performed by two - way ANOVA n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 of BACE1 after 20 minutes of HA antibody uptake in HA - BACE1 - mCherry transfected WT 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 top panels where analyzed BACE1 fraction is marked with red square .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 of BACE1 after 20 minutes of HA antibody uptake in HA - BACE1 - mCherry transfected WT KO neurons , co - expressing either eGFP - RAB4 or eGFP - RAB4 - S22N , calculated as HA ( internalized ) / mCherry ( total ) signal ( 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 : graphs show mean ± , statistical was performed by 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, 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 are increased in AP - 2µ KO neurons , when compared to WT set to 100 % ( KO : 129 . 65 ± 5 . 30 % , p = 0 . 005 , N = 4 biological replicates ) . ( N ) Surface versus total BACE1 is not altered in AP - 2µ KO neurons when normalized to WT set to 100 % ( KO : 83 . 02 ± 10 . 93 % , p = 0 . 109 , N = 4 biological replicates ) . Data information : graphs show mean ± , statistical was performed by one - sample Student ' s t ‐ in ( M , N ) . n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 54 KO neurons , N = 4 biological replicates ) . Scale bar : 10 µm . Arrows indicate BACE1 accumulation within RAB5 - positive endosomes . Data information graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( A , B ) BACE1 in cortical lysates of WT AP - 2µ KO mice . in KO were normalized to WT set to 100 % ( KO : 447 . 64 ± 48 . 78 % , p < 0 . 000 , N = 7 biological replicates ) . Data information : graphs show mean ± , statistical was performed by one - sample Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 of BACE1 are significantly increased in cortex of AP - 2µ KO mice when normalized to WT set to 100 % ( KO : 162 . 714 ± 11 . 082 % , p = 0 . 005 , N = 4 , biological replicates ) . Scale bar : 100µm . Data information : graphs show mean ± , statistical was performed by one - sample Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 AP - 2µ KO neurons . Scale bars : 20 µm , 5 µm ( inserts ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Localization of BACE1 in cultured WT and AP-2µ KO neurons. Scale bars: 20 µm, 5 µm (inserts).


( F ) plot of BACE1 immunofluorescence along

(F) Line plot of BACE1 immunofluorescence along the neurites


of BACE1 colocalizing with 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


of BACE1 colocalizing with 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 : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) of BACE1 colocalizing with eGFP - RAB7 ( WT : 19 . 54 ± 1 . 91 , KO : 37 . 09 ± 3 . 13 % , p < 0 . 000 , WT = 44 KO = 43 neurons , N = 4 biological replicates ) . Scale bar : 2µm . Arrowheads indicate BACE1 accumulation within RAB7 - positive organelles . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 RAB7A in cortical sections of WT 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 : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 of WT neurons transfected with HA - BACE1 - mCherry immunostained for Cathepsin D . Channels were false color - coded to better illustrate colocalization . Scale bars : 10µm . Arrowheads indicate BACE1 accumulation in lysosomes in WT Dotted outlines neuron .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 of AP - 2µ KO neurons transfected with HA - BACE1 - mCherry immunostained for Cathepsin D . Channels were false color - coded to better illustrate colocalization . Scale bars : 10µm . Arrowheads indicate BACE1 absence in KO . Dotted outlines neuron .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 44 KO neurons , N = 4 biological replicates ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

(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 of WT AP - 2μ KO neurons transfected with BACE1 - mKeima - Red co - transfected with eBFP to reveal neuronal . Scale bars : 10µm , 2µm in zoomed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) 550nm / 438nm signal of BACE1 - mKeima - Red quantified over entire is significantly reduced in KO condition compared to WT ( WT : 0 . 59 ± 0 . 04 , KO : 0 . 48 ± 0 . 04 , p = 0 . 041 , 28 WT 25 KO neurons , N = 3 biological replicates ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( A , B ) Representative ( A ) corresponding kymographs ( B ) from time - lapse videos of control neurons transfected with HA - BACE1 - eGFP co - transfected with AP - 2μ - mCherry . Scale bars : ( A ) 2μm , 1 . 6µm inserts , ( B ) x = 2μm , y = 5s . Arrowheads in ( A ) correspond to BACE1 carriers indicated by same arrowheads in ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) Representative fluorescence images (A) and corresponding kymographs (B) from time-lapse videos of control neurons transfected with HA-BACE1-eGFP and co-transfected with AP-2μ-mCherry. Scale bars: (A) 2μm, 1.6µm inserts, (B) x=2μm, y=5s. Arrowheads in (A) correspond to BACE1 carriers indicated by the same arrowheads in (B).


( C ) Representative corresponding kymographs from time - lapse videos of WT AP - 2μ KO neurons transfected with HA - BACE1 - eGFP . Scale bar : 5µm top panels , x = 2μm , y = 5s bottom panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative fluorescence images and corresponding kymographs from time-lapse videos of WT and AP-2μ KO neurons transfected with HA-BACE1-eGFP. Scale bar: 5µm top panels, x=2μm, y=5s bottom panels.


( D ) Loss of AP - 2μ significantly decreases BACE1 motility ( WT : 37 . 42 ± 3 . 79 % , KO : 23 . 07 ± 3 . 07 % , p = 0 . 003 , WT = 31 , KO = 32 neurons , N = 4 biological replicates ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Loss of AP-2μ significantly decreases the BACE1 motility (WT: 37.42±3.79%, KO: 23.07±3.07%, p=0.003, WT=31, KO=32 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.


( E ) Loss of AP - 2μ significantly decreases anterograde retrograde BACE1 velocity ( WTAntero : 0 . 37 ± 0 . 02 μm / s , KOAntero : 0 . 22 ± 0 . 03 μm / s , pWTAntero vs pKOAntero < 0 . 000 ; WTRetro : 0 . 35 ± 0 . 02 μm / s , KORetro : 0 . 25 ± . 03 μm / s , pWTRetro vs pKORetro = 0 . 006 , WT = 35 KO = 32 neurons , N = 4 biological replicates ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Loss of AP-2μ significantly decreases the anterograde and retrograde BACE1 velocity (WTAntero: 0.37±0.02 μm/s, KOAntero: 0.22±0.03 μm/s, pWTAntero vs pKOAntero<0.000; WTRetro: 0.35±0.02 μm/s, KORetro: 0.25±.03 μm/s, pWTRetro vs pKORetro =0.006, WT=35 and KO=32 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.


( F ) Representative corresponding kymographs from time - lapse videos of WT AP - 2μ KO neurons co - transfected with HA - BACE1 - GFP AP - 2μ - mRFP . Scale bar : 5µm top panels , x = 2μm , y = 5s bottom panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Representative fluorescence images and corresponding kymographs from time-lapse videos of WT and AP-2μ KO neurons co-transfected with HA-BACE1-GFP and AP-2μ-mRFP. Scale bar: 5µm top panels, x=2μm, y=5s bottom panels.


( G ) Bi - directional mobility of BACE1 carriers in WT AP - 2μ ΚΟ neurons , co - expressing AP - 2μ - mRFP ( WT : 37 . 60 ± 2 . 20 % , KO : 25 . 61 ± 2 . 67 % , WTAP - 2μ - mRFP : 45 . 04 ± 3 . 27 % , KOAP - 2μ - mRFP : 40 . 07 ± 2 . 37 % , pWT vs KO = 0 . 005 , pKO vs KO + AP - 2μ - mRFP < 0 . 000 , pWT vs WT + AP - 2μ - mRFP = 0 . 186 , pWT + AP - 2μ - mRFP vs KO + AP - 2μ - mRFP = 0 . 570 , 43 - 53 neurons per condition , N = 4 biological replicates ) . Data information : graphs show mean ± , statistical was performed by two - way ANOVA in ( G ) . n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Bi-directional mobility of BACE1 carriers in WT and AP-2μ ΚΟ neurons, co-expressing the AP-2μ-mRFP (WT: 37.60±2.20%, KO: 25.61±2.67%, WTAP-2μ-mRFP: 45.04±3.27%, KOAP-2μ-mRFP: 40.07±2.37%, pWT vs KO=0.005, pKO vs KO+AP-2μ-mRFP<0.000, pWT vs WT+AP-2μ-mRFP=0.186, pWT+AP-2μ-mRFP vs KO+AP-2μ-mRFP=0.570, 43-53 neurons per condition, N=4 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA in (G). n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( H ) Representative kymographs of WT AP - 2μ KO neurons co - expressing HA - BACE1 - GFP tagRFP - LC3B . Scale bars : x = 2μm , y = 5s .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Representative kymographs of WT and AP-2μ KO neurons co-expressing the HA-BACE1-GFP and tagRFP-LC3B. Scale bars: x=2μm, y=5s.


( I ) Reduced bi - directional mobility of BACE1 - containing autophagosomes in AP - 2µ KO neurons . ( WT : 33 . 53 ± 4 . 19 % , KO : 19 . 45 ± 5 . 14 % , p = 0 . 000 , WT = 37 KO = 35 neurons , N = 4 biological replicates ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Reduced bi-directional mobility of BACE1-containing autophagosomes in AP-2µ KO neurons. (WT: 33.53±4.19%, KO: 19.45±5.14%, p=0.000, WT=37 and KO=35 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.


( J ) Representative kymographs of WT neurons co - expressing HA - BACE1 - GFP AP2 - αΑ WT or AP2 - αΑ Μut . Scale bar : 5µm top panels , x = 2μm , y = 5s bottom panels .

(J) Representative kymographs of WT neurons co-expressing HA-BACE1-GFP and AP2-αΑ WT or AP2-αΑ Μut. Scale bar: 5µm top panels, x=2μm, y=5s bottom panels.


( K ) Mobility of BACE1 carriers is significantly decreased in neurons overexpressing AP2 - αΑ Mut compared to neurons expressing AP2 - αΑ WT ( WT : 52 . 23 ± 4 . 16 % , Mut : 27 . 95 ± 3 . 31 , p = < 0 . 000 , WT = 26 Mut = 29 neurons , N = 4 biological replicates ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) Mobility of BACE1 carriers is significantly decreased in neurons overexpressing the AP2-αΑ Mut compared to neurons expressing the AP2-αΑ WT (WT: 52.23±4.16%, Mut: 27.95±3.31, p=<0.000, WT=26 and Mut=29 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.


( L ) Representative corresponding kymographs from time - lapse videos of WT neurons transfected with either HA - BACE1 - GFP or HA - BACE1 - LL / AA - GFP . Scale bar : 5µm top panels , x = 2μm , y = 5s bottom panels .

(L) Representative fluorescence images and corresponding kymographs from time-lapse videos of WT neurons transfected with either HA-BACE1-GFP or HA-BACE1-LL/AA-GFP. Scale bar: 5µm top panels, x=2μm, y=5s bottom panels.


( M ) Reduced mobility of BACE1 - LL / AA - positive vesicles , when compared to WT BACE1 ( WT : 38 . 97 ± 1 . 40 % , LL / AA : 19 . 89 ± 1 . 77 % , p < 0 . 000 , WT = 31 LL / AA = 33 neurons , N = 4 biological replicates ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M) Reduced mobility of BACE1-LL/AA-positive vesicles, when compared to WT BACE1 (WT: 38.97±1.40%, LL/AA: 19.89±1.77%, p<0.000, WT=31 and LL/AA=33 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.


( N ) Reduced velocity of BACE1 - LL / AA - positive vesicles , when compared to WT BACE1 ( WTAntero : 0 . 38 ± 0 . 03 μm / s , LL / AAAntero : 0 . 15 ± 0 . 03 μm / s , pWTAntero vs pLL / AAAntero < 0 . 000 ; WTRetro : 0 . 39 ± 0 . 03 μm / s , LL / AARetro : 0 . 25 ± . 03 μm / s , pWTRetro vs pLL / AARetro = 0 . 000 , WT = 31 LL / AA = 33 neurons , N = 4 biological replicates ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(N) Reduced velocity of BACE1-LL/AA-positive vesicles, when compared to WT BACE1 (WTAntero: 0.38±0.03 μm/s, LL/AAAntero: 0.15±0.03 μm/s, pWTAntero vs pLL/AAAntero<0.000; WTRetro: 0.39±0.03 μm/s, LL/AARetro: 0.25±.03 μm/s, pWTRetro vs pLL/AARetro =0.000, WT=31 and LL/AA=33 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.


( A , B ) eGFP / mCherry signal is significantly increased in mCherry - APP - P1 - eGFP - expressing KO neurons comparing to WT ( WT : 0 . 94 ± 0 . 06 % , KO : 1 . 65 ± 0 . 18 , p = 0 . 000 , WT = 39 KO = 41 neurons , N = 5 biological replicates ) . Scale bars : 2μm . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) The eGFP/mCherry signal intensity ratio is significantly increased in mCherry-APP-P1-eGFP-expressing KO neurons comparing to the WT (WT: 0.94±0.06%, KO: 1.65±0.18, p=0.000, WT=39 and KO=41 neurons, N=5 biological replicates). Scale bars: 2μm. 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.


Confocal of WT AP - 2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP APP - CTFs . Scale bars : 100 µm ( C )

O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal images of WT and AP-2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP and APP-CTFs. Scale bars: 100 µm (C)


Confocal of WT AP - 2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP APP - CTFs . Scale bars : 20µm ( D ) .

Confocal images of WT and AP-2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP and APP-CTFs. Scale bars: 20µm (D).


( E - G ) of APP - CTFs are significantly increased in AP - 2µ KO cortex compared to WT set to 100 % ( KOC99 : 156 . 65 ± 8 . 37 % , p = 0 . 004 , N = 4 biological replicates ; KOC83 : 154 . 79 ± 15 . 6 % , p = 0 . 012 ; N = 5 biological replicates ) . Data information : graphs show mean ± , statistical was performed by one - sample Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E-G) Levels of APP-CTFs are significantly increased in AP-2µ KO cortex compared to the WT set to 100% (KOC99: 156.65±8.37%, p=0.004, N=4 biological replicates; KOC83: 154.79±15.6%, p=0.012; N=5 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.


( H ) of full - ( FL ) APP are unaltered in AP - 2µ KO cortex ( KOAPP FL : 106 . 64 ± 12 . 96 % , p = 0 . 315 , N = 6 biological replicates ) . Data information : graphs show mean ± , statistical was performed by one - sample Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Levels of full-length (FL) APP are unaltered in AP-2µ KO cortex (KOAPP FL: 106.64±12.96%, p=0.315, N=6 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.


( I ) of Nicastrin ( NCT ) are unaltered in AP - 2µ KO

(I) Levels of Nicastrin (NCT) are unaltered in the AP-2µ KO cortex


( J , K ) Intracellular of Aβ1 - 40 are significantly increased in AP - 2µ KO axons compared to WT ( WTAβ1 - 40 : 1 . 82 ± 0 . 12 , KOAβ1 - 40 : 2 . 45 ± 0 . 12 , p = 0 . 000 , WT = 24 KO = 24 neurons , N = 3 biological replicates ) . Scale bars , 5µm . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J, K) Intracellular levels of Aβ1-40 are significantly increased in AP-2µ KO axons compared to the WT (WTAβ1-40: 1.82±0.12, KOAβ1-40: 2.45±0.12, p=0.000, WT=24 and KO=24 neurons, N=3 biological replicates). Scale bars, 5µm. 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.


Intracellular of Aβ1 - 42 are significantly increased in AP - 2µ KO axons ( WTAβ1 - 42 : 0 . 65 ± 0 . 08 , KOAβ1 - 42 : 0 . 91 ± 0 . 10 , p = 0 . 046 , WT = 38 KO = 38 neurons , N = 3 biological replicates ) . Scale bar : 5 µm .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Intracellular levels of Aβ1-42 are significantly increased in AP-2µ KO axons (WTAβ1-42: 0.65±0.08, KOAβ1-42: 0.91±0.10, p=0.046, WT=38 and KO=38 neurons, N=3 biological replicates). Scale bar: 5 µm.


Intracellular of Aβ1 - 42 are significantly increased in AP - 2µ KO axons ( WTAβ1 - 42 : 0 . 65 ± 0 . 08 , KOAβ1 - 42 : 0 . 91 ± 0 . 10 , p = 0 . 046 , WT = 38 KO = 38 neurons , N = 3 biological replicates ) . Scale bar : 5 µm . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Intracellular levels of Aβ1-42 are significantly increased in AP-2µ KO axons (WTAβ1-42: 0.65±0.08, KOAβ1-42: 0.91±0.10, p=0.046, WT=38 and KO=38 neurons, N=3 biological replicates). Scale bar: 5 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.


( N ) Significantly increased of Αβ1 - 42 / Αβ1 - 40 measured by ELISA in media of cultured AP - 2μ KO neurons compared to WT set to 100 % ( KO : 124 . 57 ± 10 . 97 % , p = 0 . 037 , N = 6 biological replicates ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(N) Significantly increased ratio of Αβ1-42/ Αβ1-40 measured by ELISA in the media of cultured AP-2μ KO neurons compared to the WT set to 100% (KO:124.57±10.97%, p=0.037, N=6 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.


Accumulation of intracellular Aβ1 − 42 in neurons expressing AP2 - α − WT ( WT : 1 . 14 ± 0 . 18 , WT = 28 Mut = 33 neurons , N = 4 biological replicates ) . Scale bars : 20µm , 2µm ( inserts ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Accumulation of intracellular Aβ1−42 in neurons expressing the AP2-α−WT (WT: 1.14±0.18 , WT=28 and Mut=33 neurons, N=4 biological replicates). Scale bars: 20µm, 2µm (inserts).


Accumulation of intracellular Aβ1 − 42 in neurons overexpressing AP2 - α - Mut comparing to neurons expressing AP2 - α − WT ( WT : 1 . 14 ± 0 . 18 , Mut : 2 . 06 ± 0 . 29 , p = 0 . 011 , WT = 28 Mut = 33 neurons , N = 4 biological replicates ) . Scale bars : 20µm , 2µm ( inserts ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Accumulation of intracellular Aβ1−42 in neurons overexpressing the AP2-α-Mut comparing to neurons expressing the AP2-α−WT (WT: 1.14±0.18, Mut: 2.06±0.29, p=0.011, WT=28 and Mut=33 neurons, N=4 biological replicates). Scale bars: 20µm, 2µm (inserts). 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.


Accumulation of intracellular Aβ1 - 42 is rescued upon BACE1 knock - down ( WTScr : 2 . 76 ± 0 . 20 WTshBACE1 : 2 . 52 ± 0 . 12 27 to 39 neurons per condition , N = 3 biological replicates ) . Scale bar : 10µm .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Accumulation of intracellular Aβ1-42 is rescued upon BACE1 knock-down (WTScr: 2.76±0.20 WTshBACE1: 2.52±0.12 27 to 39 neurons per condition, N=3 biological replicates). Scale bar: 10µm.


Accumulation of intracellular Aβ1 - 42 is rescued upon BACE1 knock - down in AP - 2µ KO neurons ( WTScr : 2 . 76 ± 0 . 20 , KOScr : 3 . 94 ± 0 . 21 ; WTshBACE1 : 2 . 52 ± 0 . 12 ; KOshBACE1 : 2 . 74 ± 0 . 21 ; pWTScr vs pKOScr = 0 . 000 , pKOscr vs pKOshBACE1 = 000 ; pWTscr vs pWTshBACE1 = 0 . 826 ; pWTshBACE1 vs pKOshBACE1 = 0 . 863 ; 27 to 39 neurons per condition , N = 3 biological replicates ) . Scale bar : 10µm . Data information : graphs show mean ± , statistical was performed by two - way ANOVA in ( T ) n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Accumulation of intracellular Aβ1-42 is rescued upon BACE1 knock-down in AP-2µ KO neurons (WTScr: 2.76±0.20, KOScr: 3.94±0.21; WTshBACE1: 2.52±0.12; KOshBACE1: 2.74±0.21; pWTScr vs pKOScr=0.000, pKOscr vs pKOshBACE1=000; pWTscr vs pWTshBACE1=0.826; pWTshBACE1 vs pKOshBACE1=0.863; 27 to 39 neurons per condition, N=3 biological replicates). Scale bar: 10µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA in (T) n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( U , V ) of intracellular Aβ1 − 42 are significantly upregulated in neurons overexpressing HA - BACE1 - LL / AA - GFP comparing to neurons expressing HA - BACE1 - GFP ( BACE1 : 0 . 78 ± 0 . 90 , BACE1 - LL - AA : 1 . 99 ± 0 . 33 , p = 000 , 28 - 29 neurons per condition , N = 3 biological replicates ) . Scale bar , 50µm . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(U, V) Levels of intracellular Aβ1−42 are significantly upregulated in neurons overexpressing the HA-BACE1-LL/AA-GFP comparing to neurons expressing the HA-BACE1-GFP (BACE1: 0.78±0.90, BACE1-LL-AA: 1.99±0.33, p=000, 28-29 neurons per condition, N=3 biological replicates). Scale bar, 50µm. 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.


( A , B ) AP - 2α normalized to GAPDH are significantly decreased in lysates of iPSC - derived neurons from late - onset AD carrying TREM2 p . R47H ( AD - TREM2 - 2 AD - TREM2 - 4 ) variant compared to healthy controls ( CON8 CON9 ) set to 100 % ( ADAP - 2α : 52 . 42 ± 8 . 04 % , p = 0 . 020 , N = 3 biological replicates ) . Data information : graphs show mean ± , statistical was performed by one - sample Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) AP-2α levels normalized to GAPDH are significantly decreased in lysates of iPSC-derived neurons from late-onset AD patients carrying the TREM2 p.R47H (AD-TREM2-2 and AD-TREM2-4) variant compared to healthy controls (CON8 and CON9) set to 100% (ADAP-2α: 52.42±8.04%, p=0.020, N=3 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 ) Confocal of either AP - 2 WT ( AAV - GFPCamkIIα - labelled ) or AP - 2 KO ( AAV - Cre - GFPCamkIIα - labelled ) granule neurons in dentate gyrus of 13 - week - old mice . Scale bars : 20µm , 2µm ( inserts ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Confocal images of either AP-2 WT (AAV-GFPCamkIIα-labelled) or AP-2 KO (AAV-Cre-GFPCamkIIα-labelled) granule neurons in the dentate gyrus of 13-week-old mice. Scale bars: 20µm, 2µm (inserts).


( D ) Loss of AP - 2µ significantly decreases number of spines on granule neurons ( WT : 12 . 63 ± 0 . 88 , KO : 8 . 07 ± 0 . 45 , p = 0 . 004 , N = 4 biological replicates ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Loss of AP-2µ significantly decreases the number of spines on granule neurons (WT: 12.63±0.88, KO: 8.07±0.45, p=0.004, 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.


( E , F ) Overlap coefficient between presynaptic marker Bassoon postsynaptic marker PSD95 is significantly decreased in entorhinal cortex of AP - 2µ KO mice compared to WT ( WT : 0 . 45 ± 0 . 02 , KO : 0 . 33 ± 0 . 06 , p = 0 . 034 , N = 3 biological replicates ) . Scale bar , 750nm . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E, F) Overlap coefficient between the presynaptic marker Bassoon and the postsynaptic marker PSD95 is significantly decreased in the entorhinal cortex of AP-2µ KO mice compared to the WT (WT: 0.45±0.02, KO: 0.33±0.06, p=0.034, N=3 biological replicates). Scale bar, 750nm. 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.


Lost preference for novel object ( nov ) comparing to familiar object ( fam ) in AP - 2µ KO mice tested 24h after training in NOR Representative heatmaps are shown in ( G ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Lost preference for the novel object (nov) comparing to the familiar object (fam) in AP-2µ KO mice tested 24h after training in NOR Representative heatmaps are shown in (G).


Lost preference for novel object ( nov ) comparing to familiar object ( fam ) in AP - 2µ KO mice tested 24h after training in NOR ( WTfam : 27 . 51 ± 6 . 77 , WTnov : 72 . 49 ± 6 . 77 , pWTfam vs WTnov = 0 . 000 ; KOfam : 43 . 9 ± 13 . 12 , KOnov : 59 . 91 ± 13 . 12 , pKOfam vs KOnov = 0 . 469 , N = 7 WT 8 KO mice ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Lost preference for the novel object (nov) comparing to the familiar object (fam) in AP-2µ KO mice tested 24h after training in NOR (WTfam: 27.51±6.77, WTnov: 72.49±6.77, pWTfam vs WTnov=0.000; KOfam: 43.9±13.12, KOnov: 59.91±13.12, pKOfam vs KOnov=0.469, N=7 WT and 8 KO mice). 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 ) AP - 2µ KO mice are significantly more active in open field compared to WT ( WT : 1663 ± 139 . 08 % , KO : 3976 ± 802 . 30 % , p = 0 . 020 , N = 7 WT 8 KO mice ) . Representative heatmaps are shown in ( I ) . Data information : graphs show mean ± , statistical was performed by unpaired two - Student ' s t ‐ n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I, J) AP-2µ KO mice are significantly more active in the open field compared to the WT (WT: 1663±139.08%, KO: 3976±802.30%, p=0.020, N=7 WT and 8 KO mice). Representative heatmaps are shown in (I). 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 ) Overlap coefficient between Bassoon PSD95 is rescued upon BACE1 KD in AP - 2µ KO neurons ( WTscr : 0 . 39 ± 0 . 02 ; KOscr : 0 . 24 ± 0 . 01 ; WTshBACE1 : 0 . 37 ± 0 . 02 ; KOshBACE1 : 0 . 33 ± 0 . 02 ; pWTscr vs pKOscr < 0 . 000 ; pKOscr vs pKOshBACE1 = 0 . 010 ; pWTscr vs pWTshBACE1 = 0 . 813 ; pWTshBACE1 vs pKOshBACE1 = 0 . 623 , 22 - 24 neurons for each condition , N = 3 ) . Scale bars : 10µm . Data information : graphs show mean ± , statistical was performed by two - way ANOVA n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K, L) Overlap coefficient between Bassoon and PSD95 is rescued upon BACE1 KD in AP-2µ KO neurons (WTscr: 0.39±0.02; KOscr: 0.24±0.01; WTshBACE1: 0.37±0.02; KOshBACE1: 0.33±0.02; pWTscr vs pKOscr<0.000; pKOscr vs pKOshBACE1=0.010; pWTscr vs pWTshBACE1=0.813; pWTshBACE1 vs pKOshBACE1=0.623, 22-24 neurons for each condition, N=3). Scale bars: 10µm. 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.


Neuronal loss is rescued in entorhinal cortex of AP - 2µ mice haploinsufficient for BACE1 ( AP - 2WT / BACEWT : 2 . 25 ± 0 . 08 N = 9 sections from N = 3 for each genotype ) . Scale bar : 100 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neuronal loss is rescued in the entorhinal cortex of AP-2µ mice haploinsufficient for BACE1 (AP-2WT/BACEWT: 2.25±0.08 N=9 sections from N=3 for each genotype). Scale bar: 100 μm.


Neuronal loss is rescued in entorhinal cortex of AP - 2µ KO mice haploinsufficient for BACE1 AP - 2KO / BACEWT : 1 . 51 ± 0 . 08 , AP - 2WT / BACEHET : 1 . 80 ± 0 . 07 , AP - 2KO / BACEHET : 2 . 00 ± 0 . 07 ; pAP - 2WT / BACE1WT vs pAP - 2KO / BACE1WT < 0 . 000 ; pAP - 2WT / BACE1WT vs pAP - 2WT / BACE1HET = 0 . 001 ; pAP - 2KO / BACE1WT vs pAP - 2KO / BACE1HET = 0 . 000 ; p AP - 2WT / BACE1HET vs AP - 2KO / BACE1HET = 0 . 268 , N = 9 sections from N = 3 for each genotype ) . Scale bar : 100 μm . Data information : graphs show mean ± , statistical was performed by two - way ANOVA n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neuronal loss is rescued in the entorhinal cortex of AP-2µ KO mice haploinsufficient for BACE1 AP-2KO/BACEWT: 1.51±0.08, AP-2WT/BACEHET: 1.80±0.07, AP-2KO/BACEHET: 2.00±0.07; pAP-2WT/BACE1WT vs pAP-2KO/BACE1WT<0.000; pAP-2WT/BACE1WT vs pAP-2WT/BACE1HET=0.001; pAP-2KO/BACE1WT vs pAP-2KO/BACE1HET=0.000; p AP-2WT/BACE1HET vs AP-2KO/BACE1HET=0.268, N=9 sections from N=3 for each genotype). Scale bar: 100 μm. 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.


( P , Q ) APP - CTF are rescued in entorhinal cortex of AP - 2µ KO mice haploinsufficient for BACE1 ( AP - 2WT / BACEWT : 427574 ± 24021 , AP - 2KO / BACEWT : 538895 ± 34662 , AP - 2WT / BACEHET : 389870 ± 19842 , AP - 2KO / BACEHET : 395389 ± 21853 ; pAP - 2WT / BACE1WT vs pAP - 2KO / BACE1WT = 0 . 017 ; pAP - 2KO / BACE1WT vs pAP - 2KO / BACE1HET = 0 . 001 ; p AP - 2WT / BACE1HET vs AP - 2KO / BACE1HET = 0 . 979 ; 13 - 15 sections , N = 3 mice for each genotype ) . Scale bars : 40µm . Data information : graphs show mean ± , statistical was performed by two - way ANOVA n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(P, Q) APP-CTF levels are rescued in the entorhinal cortex of AP-2µ KO mice haploinsufficient for BACE1 (AP-2WT/BACEWT:427574±24021, AP-2KO/BACEWT: 538895±34662, AP-2WT/BACEHET: 389870±19842, AP-2KO/BACEHET: 395389±21853; pAP-2WT/BACE1WT vs pAP-2KO/BACE1WT=0.017; pAP-2KO/BACE1WT vs pAP-2KO/BACE1HET=0.001; p AP-2WT/BACE1HET vs AP-2KO/BACE1HET=0.979; 13-15 sections, N=3 mice for each genotype). Scale bars: 40µm. 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.


( A - D ) Immunostaining with dopamine antibody at late 3rd instar ( 96h AEL ) in control background ( domeMESO - GFP / + ) . inset shows DomeGFP + ( B ' ) corresponding dopamine ( C ' ) in them . ( E - L ) Similar expression of dopamine at early 3rd ( 72h AEL , E - H ) early 2nd instar ( 48h AEL , I - L ) . ( D , H L ) spectral mode of iDopamine representation with blue pixels indicating low red pixels indicating high . ( M ) Quantifications of dopamine in DomeGFP + progenitors ( domeMESO - Gal4 , UAS - GFP / + , n = 14 at 48h , n = 13 at 72h n = 16 at 96h AEL ) . Data information : is stained with Hoechst in blue , intracellular Dopamine ( iDopamine ) in red , domeMESO expression in green ( representative of progenitors in medullary zone , MZ ) . iDopamine channel has been converted to spectral mode in panels D , H L . image panels show a 20µm scale bar . inset represents a 2X zoom of region of interest ( ROIs ) marked in dashed square . AEL indicates After Egg Laying . For better representation , lobe of lymph has been represented outlined in dashed lines while MZ is outlined in yellow dashed for segregation between Dome + Dome - . in M shows median with whiskers indicating maximum minimum values , . box indicates lower upper quartiles . statistical applied is One - way ANOVA with Tukey ' s multiple comparisons . ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . For better representation , lobe of lymph has been represented outlined in dashed lines while MZ is outlined in yellow dashed .

O O O O O O O B-MEASURED_VAR O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-D) Immunostaining with dopamine antibody at late 3rd instar (96h AEL) in control background (domeMESO-GFP/+). The inset shows DomeGFP+ cells (B') and the corresponding dopamine levels (C') in them. (E-L) Similar expression analysis of dopamine at early 3rd (72h AEL, E-H) and early 2nd instar (48h AEL, I-L). (D, H and L) The spectral mode of iDopamine representation with blue pixels indicating low and red pixels indicating high intensity. (M) Quantifications of dopamine levels in DomeGFP+ progenitors (domeMESO-Gal4, UAS-GFP/+, n= 14 at 48h, n=13 at 72h and n=16 at 96h AEL). Data information: DNA is stained with Hoechst in blue, intracellular Dopamine (iDopamine) in red, domeMESO expression in green (representative of progenitors in the medullary zone, MZ). The iDopamine channel has been converted to spectral mode in panels D, H and L. All image panels show a 20µm scale bar. The inset represents a 2X zoom of region of interest (ROIs) marked in white dashed square. AEL indicates After Egg Laying. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line for segregation between Dome+ and Dome- cells. The quantification in M shows median with whiskers indicating the maximum and minimum values,. box indicates the lower and upper quartiles. The statistical analysis applied is One-way ANOVA with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line.


( B - D ' ) A temporal expression profile of TH at 48 , 72 96h AEL lymph in control background ( domeMESO - Gal4 , UAS - GFP / + ) ( E - G ' ) A temporal expression profile of Ddc at 48 , 72 96h AEL lymph in control background ( domeMESO - Gal4 , UAS - GFP / + ) . ( H ) of TH ( domeMESO - Gal4 , UAS - GFP / + , n = 18 at 48h , n = 15 , at 72h n = 23 at 96h AEL ) . ( I ) of Ddc ( domeMESO - Gal4 , UAS - GFP / + , n = 17 at 48h , n = 33 at 72h n = 38 at 96h AEL ) . Data information : is stained with Hoechst in blue , iDopamine in red , Tyrosine hydroxylase ( TH ) Dopa decarboxylase ( Ddc ) in red , domeMESO ( representative of progenitors ) expression in green , AEL indicates After Egg Laying . quantifications in H , I represents median with whiskers indicating maximum minimum values , box indicates lower upper quartiles . statistical applied in H , I , is One - way ANOVA with Tukey ' s multiple comparisons ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . For better representation , lobe of lymph has been represented outlined in dashed lines while MZ is outlined in yellow dashed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D') A temporal expression profile of TH at 48, 72 and 96h AEL lymph glands in control background (domeMESO-Gal4, UAS-GFP/+) (E-G') A temporal expression profile of Ddc at 48, 72 and 96h AEL lymph glands in control background (domeMESO-Gal4, UAS-GFP/+). (H) Quantification of TH levels (domeMESO-Gal4, UAS-GFP/+, n=18 at 48h, n= 15, at 72h and n= 23 at 96h AEL). (I) Quantification of Ddc levels (domeMESO-Gal4, UAS-GFP/+, n= 17 at 48h, n= 33 at 72h and n= 38 at 96h AEL). Data information: DNA is stained with Hoechst in blue, iDopamine in red, Tyrosine hydroxylase (TH) and Dopa decarboxylase (Ddc) in red, domeMESO (representative of progenitors) expression in green, AEL indicates After Egg Laying. The quantifications in H, I represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in H, I, is One-way ANOVA with Tukey's multiple comparisons test 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line.


( J - L ) Assessment of dopamine upon knockdown of TH Ddc in progenitors using domeMESO - Gal4 , UAS - GFP . ( M ) mean of dopamine upon TH Ddc loss of function in progenitors ( domeMESO - Gal4 , UAS - GFP / + , n = 40 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 17 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 34 ) . Data information : is stained with Hoechst in blue , iDopamine in red image panels show a 20µm scale bar . quantifications in M represents median with whiskers indicating maximum minimum values , box indicates lower upper quartiles . statistical applied in M , is One - way ANOVA with Tukey ' s multiple comparisons ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . For better representation , lobe of lymph has been represented outlined in dashed

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J-L) Assessment of dopamine levels upon the knockdown of TH and Ddc in the progenitors using domeMESO-Gal4, UAS-GFP. (M) The mean intensity quantification of dopamine levels upon TH and Ddc loss of function in the progenitors (domeMESO-Gal4, UAS-GFP/+, n=40, domeMESO-Gal4, UAS-GFP; THRNAi, n= 17, domeMESO-Gal4, UAS-GFP; DdcRNAi, n= 34). Data information: DNA is stained with Hoechst in blue, iDopamine in red All image panels show a 20µm scale bar. The quantifications in M represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in M, is One-way ANOVA with Tukey's multiple comparisons test 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines


( N - P ) loss of progenitor TH Ddc function using domeMESO - Gal4 , UAS - GFP analysed at wandering 3rd instar , 120h AEL . Data information : is stained with Hoechst in blue , domeMESO ( representative of progenitors ) expression in green , P1 ( representative of mature plasmatocytes ) in red . image panels show a 20µm scale bar . AEL indicates After Egg Laying . For better representation , lobe of lymph has been represented outlined in dashed

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(N-P) The loss of progenitor TH and Ddc function using domeMESO-Gal4, UAS-GFP analysed at wandering 3rd instar, 120h AEL. Data information: DNA is stained with Hoechst in blue, domeMESO (representative of progenitors) expression in green, P1 (representative of mature plasmatocytes) in red. All image panels show a 20µm scale bar. AEL indicates After Egg Laying. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines


( Q ) Quantifications of lymph upon loss of TH ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 34 ) Ddc ( domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 56 ) when compared to stage matched control ( domeMESO - Gal4 , UAS - GFP / + , n = 90 ) . Data information : quantifications in Q represent mean with standard deviation with whiskers indicating maximum minimum values , box indicates lower upper quartiles . statistical applied in is One - way ANOVA with Tukey ' s multiple comparisons ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(Q) Quantifications of lymph gland size upon loss of TH (domeMESO-Gal4, UAS-GFP; THRNAi, n= 34) and Ddc (domeMESO-Gal4, UAS-GFP; DdcRNAi, n= 56) when compared to stage matched control (domeMESO-Gal4, UAS-GFP/+, n=90). Data information: The quantifications in Q represent mean with standard deviation with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in is One-way ANOVA with Tukey's multiple comparisons test 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( R ) Quantifications of proportions of % Dome ( green bars ) , % negative ( blue bar ) , % overlap ( Dome P1 , yellow bars ) % P1 ( red bars ) . For Dome positive ( domeMESO - Gal4 , UAS - GFP / + , n = 27 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 15 domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 18 ) , negative overlap areas ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 15 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 18 ) P1 positive ( domeMESO - Gal4 , UAS - GFP / + , n = 27 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 15 domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 18 ) . Data information : quantifications in R represents mean with whiskers indicating maximum minimum values , box indicates lower upper quartiles . statistical applied in R is Two - way Anova with Tukey ' s multiple comparisons . ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(R) Quantifications of the proportions of %Dome (green bars), %negative (blue bar), %overlap (Dome and P1, yellow bars) and %P1 (red bars). For Dome positive area (domeMESO-Gal4, UAS-GFP/+, n=27, domeMESO-Gal4, UAS-GFP; THRNAi, n= 15 and domeMESO-Gal4, UAS-GFP; DdcRNAi, n= 18), negative and overlap areas (domeMESO-Gal4, UAS-GFP; THRNAi, n= 15, domeMESO-Gal4, UAS-GFP; DdcRNAi, n= 18) and P1 positive area (domeMESO-Gal4, UAS-GFP/+, n=27, domeMESO-Gal4, UAS-GFP; THRNAi, n= 15 and domeMESO-Gal4, UAS-GFP; DdcRNAi, n= 18). Data information: The quantifications in R represents mean with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in R is Two-way Anova with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( S ) of number of PPO positive crystal upon dopamine synthesis perturbation ( domeMESO - Gal4 , UAS - GFP / + , n = 29 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 21 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 23 ) . Data information : quantifications in S represent mean with standard deviation with whiskers indicating maximum minimum values , box indicates lower upper quartiles . statistical applied in S is One - way ANOVA with Tukey ' s multiple comparisons ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(S) Quantification of the number of PPO positive crystal cells upon dopamine synthesis perturbation (domeMESO-Gal4, UAS-GFP/+, n=29, domeMESO-Gal4, UAS-GFP; THRNAi, n= 21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n= 23). Data information: The quantifications in S represent mean with standard deviation with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in S is One-way ANOVA with Tukey's multiple comparisons test 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( A - C ' ) Expression profile of dopamine activation receptor sensor ( DA1m ) in progenitors using domeMESO - Gal4 driver in a temporal manner - 48h , ( A - A ' ) , 72h , ( B - B ' ) 96h AEL , ( C - C ' ) . ( D ) of number DA1m + , % DA1m positive ( domeMESO - Gal4 ; DA1m , n = 10 at 96h , n = 16 at 72h n = 10 at 48h AEL ) . Data information : is stained with Hoechst in blue , lifeAct - RFP to mark progenitors in red , Dopamine receptor - reporter ( DA1m ) in green , image panels show a 20µm scale bar . AEL indicates After Egg Laying . in D represents median with whiskers indicating maximum minimum values , box indicates lower upper quartiles . statistical applied in is Two - way Anova with Tukey ' s multiple comparisons . ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . For better representation , lobe of lymph has been represented outlined in dashed lines while MZ is outlined in yellow dashed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C') Expression profile of dopamine activation based receptor sensor (DA1m) in the progenitors using domeMESO-Gal4 driver in a temporal manner- 48h, (A-A'), 72h, (B-B') and 96h AEL, (C-C'). (D) Quantification of the number DA1m+ cells, %DA1m positive cells (domeMESO-Gal4; DA1m, n=10 at 96h, n=16 at 72h and n=10 at 48h AEL). Data information: DNA is stained with Hoechst in blue, lifeAct-RFP to mark progenitors in red, Dopamine receptor-based reporter (DA1m) in green, All image panels show a 20µm scale bar. AEL indicates After Egg Laying. The quantification in D represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in is Two-way Anova with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line.


( E - G ' ) Temporal expression of dopamine transporter , DAT . ( H ) A plot representing trend of DAT expression at 96h AEL . ( x ) axis indicates 0µm as a beginning from region to periphery . Data information : is stained with Hoechst in blue , Dopamine transporter ( DAT ) in red , domeMESO ( representative of progenitors ) expression in green , image panels show a 20µm scale bar . AEL indicates After Egg Laying . For better representation , lobe of lymph has been represented outlined in dashed lines while MZ is outlined in yellow dashed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E-G') Temporal expression analysis of dopamine transporter, DAT. (H) A line plot representing the trend of DAT expression at 96h AEL. The distance (x) axis indicates 0µm as a line beginning from the core region to the periphery. Data information: DNA is stained with Hoechst in blue, Dopamine transporter (DAT) in red, domeMESO (representative of progenitors) expression in green, All image panels show a 20µm scale bar. AEL indicates After Egg Laying. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line.


( I - K ) loss of progenitor Dop2R DAT function using domeMESO - Gal4 , UAS - GFP analysed at wandering 3rd instar , 120h AEL . Data information : is stained with Hoechst in blue domeMESO ( representative of progenitors ) expression in green , P1 ( representative of mature plasmatocytes ) in red . image panels show a 20µm scale bar . For better representation , lobe of lymph has been represented outlined in dashed

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I-K) The loss of progenitor Dop2R and DAT function using domeMESO-Gal4, UAS-GFP analysed at wandering 3rd instar, 120h AEL. Data information: DNA is stained with Hoechst in blue domeMESO (representative of progenitors) expression in green, P1 (representative of mature plasmatocytes) in red. All image panels show a 20µm scale bar. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines


( L ) Quantifications of lymph upon loss of Dop2R DAT in progenitors ( domeMESO - Gal4 , UAS - GFP / + , n = 46 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 44 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 37 ) . Data information : in L represents mean with standard deviation with whiskers indicating maximum minimum values , box indicates lower upper quartiles . statistical applied in L is One - way ANOVA with Tukey ' s multiple comparisons ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(L) Quantifications of lymph gland size upon loss of Dop2R and DAT in the progenitors (domeMESO-Gal4, UAS-GFP/+, n= 46, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 44, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 37). Data information: The quantification in L represents mean with standard deviation with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in L is One-way ANOVA with Tukey's multiple comparisons test 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( M ) Quantifications of proportions of % Dome ( green bar ) , % negative ( blue bar ) , % overlap ( Dome P1 , yellow bar ) % P1 ( red bars ) upon loss of Dop2R DAT in progenitors . For Dome positive ( domeMESO - Gal4 , UAS - GFP / + , n = 28 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 23 ) , negative ( domeMESO - Gal4 , UAS - GFP / + , n = 28 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 23 ) , overlap ( domeMESO - Gal4 , UAS - GFP / + , n = 26 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 25 ) P1 ( domeMESO - Gal4 , UAS - GFP / + , n = 27 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 23 ) . Data information : in M represents mean , with whiskers indicating maximum minimum values , box indicates lower upper quartiles . statistical applied in M is Two - way Anova with Tukey ' s multiple comparisons . ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M) Quantifications of the proportions of %Dome (green bar), %negative (blue bar), %overlap (Dome and P1, yellow bar) and %P1 (red bars) upon loss of Dop2R and DAT in the progenitors. For Dome positive area (domeMESO-Gal4, UAS-GFP/+, n= 28, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 16, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 23), negative area (domeMESO-Gal4, UAS-GFP/+, n= 28, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 16, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 23), overlap area (domeMESO-Gal4, UAS-GFP/+, n= 26, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 16, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 25) and P1 area (domeMESO-Gal4, UAS-GFP/+, n= 27, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 16, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 23). Data information: The quantification in M represents mean, with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in M is Two-way Anova with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( N ) of number of PPO positive crystal upon dopamine sensing perturbation ( domeMESO - Gal4 , UAS - GFP / + , n = 29 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 26 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 25 ) . Data information : in N represents mean with standard deviation with whiskers indicating maximum minimum values , box indicates lower upper quartiles . statistical applied in N is One - way ANOVA with Tukey ' s multiple comparisons ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

(N) Quantification of the number of PPO positive crystal cells upon dopamine sensing perturbation (domeMESO-Gal4, UAS-GFP/+, n= 29, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 26, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 25). Data information: The quantification in N represents mean with standard deviation with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in N is One-way ANOVA with Tukey's multiple comparisons test 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( A ) A temporal of total number of nuclei across development in control background ( domeMESO - Gal4 , UAS - GFP / + , n = 37 at 48h , n = 31 at 72h , n = 19 at 96h n = 23 at 120h AEL ) . loss of dopamine modules at 48h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 19 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 30 domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 21 ) , at 72h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 22 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 24 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 33 domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 27 ) , at 96h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 21 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 18 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 20 domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 22 ) at 120h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 25 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 30 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 25 domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 31 ) . Data information : represents median with whiskers representing maximum minimum values , box indicates lower upper quartiles . statistical is Two - way Anova with Tukey ' s multiple comparisons while control comparison , domeMESO - Gal4 , UAS - GFP / + , for different time points was done by One - way ANOVA with Tukey ' s multiple comparisons . ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . AEL indicates After Egg

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) A temporal analysis of total number of nuclei across development in control background (domeMESO-Gal4, UAS-GFP/+, n= 37 at 48h, n= 31 at 72h, n= 19 at 96h and n= 23 at 120h AEL). The loss of dopamine modules at 48h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=19, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=16, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=30 and domeMESO-Gal4, UAS-GFP; DATRNAi, n=21), at 72h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=24, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=33 and domeMESO-Gal4, UAS-GFP; DATRNAi, n=27), at 96h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=18, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=20 and domeMESO-Gal4, UAS-GFP; DATRNAi, n=22) and at 120h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=25, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=30, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=25 and domeMESO-Gal4, UAS-GFP; DATRNAi, n=31). Data information: The quantification represents median with whiskers representing maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis is Two-way Anova with Tukey's multiple comparisons test while the control comparison, domeMESO-Gal4, UAS-GFP/+, for different time points was done by One-way ANOVA with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. AEL indicates After Egg Laying


( B ) of total Dome + nuclei of control ( domeMESO - Gal4 , UAS - GFP / + , n = 37 at 48h , n = 28 at 72h , n = 19 at 96h n = 23 at 120h AEL ) . loss of dopamine modules at 48h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 22 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 30 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 21 ) , at 72h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 22 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 24 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 32 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 27 ) , at 96h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 21 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 17 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 20 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 22 ) at 120h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 24 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 30 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 25 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 30 ) . ( C ) A temporal of Dome - nuclei in control ( domeMESO - Gal4 , UAS - GFP / + , n = 37 at 48h , n = 28 at 72h , n = 18 at 96h , n = 23 , at 120h AEL ) . perturbation of dopamine modules in progenitors at 48h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 22 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 30 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 21 ) , at 72h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 22 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 24 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 32 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 27 ) , at 96h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 21 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 17 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 20 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 22 ) at 120h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 24 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 30 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 25 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 30 ) . Data information : represents median with whiskers representing maximum minimum values , box indicates lower upper quartiles . statistical applied is Two - way Anova with Tukey ' s multiple comparisons while control comparison , domeMESO - Gal4 , UAS - GFP / + , for different time points was done by One - way ANOVA with Tukey ' s multiple comparisons . ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . AEL indicates After Egg

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of total Dome+ nuclei counts of control animals (domeMESO-Gal4, UAS-GFP/+, n= 37 at 48h, n= 28 at 72h, n= 19 at 96h and n=23 at 120h AEL). The loss of dopamine modules at 48h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=16, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=30, domeMESO-Gal4, UAS-GFP; DATRNAi, n=21), at 72h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=24, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=32, domeMESO-Gal4, UAS-GFP; DATRNAi, n=27), at 96h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=17, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=20, domeMESO-Gal4, UAS-GFP; DATRNAi, n=22) and at 120h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=24, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=30, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=25, domeMESO-Gal4, UAS-GFP; DATRNAi, n=30). (C) A temporal analysis of Dome- nuclei counts in the control animals (domeMESO-Gal4, UAS-GFP/+, n= 37 at 48h, n= 28 at 72h, n= 18 at 96h, n= 23, at 120h AEL). The perturbation of the dopamine modules in the progenitors at 48h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=16, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=30, domeMESO-Gal4, UAS-GFP; DATRNAi, n=21), at 72h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=24, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=32, domeMESO-Gal4, UAS-GFP; DATRNAi, n=27), at 96h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=17, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=20, domeMESO-Gal4, UAS-GFP; DATRNAi, n=22) and at 120h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=24, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=30, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=25, domeMESO-Gal4, UAS-GFP; DATRNAi, n=30). Data information: The quantification represents median with whiskers representing maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied is Two-way Anova with Tukey's multiple comparisons test while the control comparison, domeMESO-Gal4, UAS-GFP/+, for different time points was done by One-way ANOVA with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. AEL indicates After Egg Laying