Figure 1A . Intracellular survival of GAS strains 188 , 188SLO - ( SLO - ) , and 188NADase - ( NADase - ) . Intracellular CFU were quantified at 2 h , 4 h , 6 h , 8 h , and 24 h , and intracellular survival was calculated as the percent viable CFU compared to 2 h . B . Intracellular survival of GAS strain 188SLO - that was partially complemented from plasmid piSLO ( SLO - ( piSLO ) ) , strain 188 and 188SLO - that contained the empty complementation vector , 188 ( pSIV4 ) and SLO - ( pSIV4 ) , respectively . C . Intracellular survival of GAS strain JRS4 , JRS4SLO - ( SLO - ) , and JRS4NADase - ( NADase - ) . Experiments were performed in triplicate and values represent the mean of three independent experiments ± SD . * , P < 0 . 001 .

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

Figure 1A. Intracellular survival of GAS strains 188, 188SLO- (SLO-), and 188NADase- (NADase-). Intracellular CFU were quantified at 2 h, 4 h, 6 h, 8 h, and 24 h, and intracellular survival was calculated as the percent viable CFU compared to 2 h.B. Intracellular survival of GAS strain 188SLO- that was partially complemented from plasmid piSLO (SLO-(piSLO)), strain 188 and 188SLO- that contained the empty complementation vector, 188(pSIV4) and SLO-(pSIV4), respectively.C. Intracellular survival of GAS strain JRS4, JRS4SLO- (SLO-), and JRS4NADase- (NADase-). Experiments were performed in triplicate and values represent the mean of three independent experiments ± SD. *, P<0.001.


Figure 2A . Confocal microscopy of the association between EGFP - LC3 and GAS strain 188 , 188SLO - ( SLO - ) , 188SLS - ( SLS - ) , or 188SLO - SLS - ( SLO - SLS - ) . Immunofluorescent staining distinguished intracellular ( Alexa - 568 , red ) from extracellular ( Alexa - 568 and Alexa - 660 , red and blue , respectively ) GAS . Scale bar = 10 µm . The percent of intracellular GAS that were associated with EGFP - LC3 at 3 h post - infection is shown for each strain . B . Electron microscopy of GAS strains 188 , 188SLO - , and 188SLO - SLS - at 3 h post - infection . Arrowheads indicate the presence of double or multiple membranes ( 188 and 188SLO - ) or single membranes ( 188SLO - SLS - ) partially or completely surrounding bacteria . Vacuolar compartments associated with 188 and 188SLO - also contain cytosolic material . C . Intracellular survival of GAS strains 188 , 188SLO - , 188SLS - , and 188SLO - SLS - . * , P < 0 . 05 . D . Intracellular survival of 188 or 188SLO - in the presence or absence of Beclin1 knockdown .

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

Figure 2A. Confocal microscopy of the association between EGFP-LC3 and GAS strain 188, 188SLO- (SLO-), 188SLS- (SLS-), or 188SLO-SLS- (SLO-SLS-). Immunofluorescent staining distinguished intracellular (Alexa-568, red) from extracellular (Alexa-568 and Alexa-660, red and blue, respectively) GAS. Scale bar = 10 µm. The percent of intracellular GAS that were associated with EGFP-LC3 at 3 h post-infection is shown for each strain.B. Electron microscopy of GAS strains 188, 188SLO-, and 188SLO-SLS- at 3 h post-infection. Arrowheads indicate the presence of double or multiple membranes (188 and 188SLO-) or single membranes (188SLO-SLS-) partially or completely surrounding bacteria. Vacuolar compartments associated with 188 and 188SLO- also contain cytosolic material.C. Intracellular survival of GAS strains 188, 188SLO-, 188SLS-, and 188SLO-SLS-. *, P<0.05.D. Intracellular survival of 188 or 188SLO- in the presence or absence of Beclin1 knockdown.


Figure 3A . Intracellular survival of GAS strains 188 , 188NADase - , and 188NADase ( G330D ) . Deletion of the gene encoding NADase or inactivation of NADase by the amino acid substitution G330D resulted in a 20 - to 40 - fold reduction in intracellular survival compared to parent strain 188 . * , P < 0 . 001 . NADase western blot ( B ) and activity measurements ( C ) of culture supernatants from GAS strains 188 , 188NADase - , and 188NADase ( G330D ) .

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

Figure 3A. Intracellular survival of GAS strains 188, 188NADase-, and 188NADase(G330D). Deletion of the gene encoding NADase or inactivation of NADase by the amino acid substitution G330D resulted in a 20- to 40-fold reduction in intracellular survival compared to parent strain 188. *, P<0.001.NADase western blot (B)and activity measurements (C) of culture supernatants from GAS strains 188, 188NADase-, and 188NADase(G330D).


Figure 4A . Confocal microscopy demonstrating the association between ubiquitin ( green ) and intracellular GAS ( red ) at 30 min post - infection in keratinocytes infected with GAS strain 188 , 188SLO - , 188NADase - , or 188SLO ( Y255A ) . Extracellular GAS were stained blue and red for identification as described in Figure 2 . Scale bar = 10 µm . The percentage of intracellular GAS that were associated with ubiquitin is indicated for each strain , based on quantification of at least 100 bacteria in three independent experiments . B . Hemolytic activity of culture supernatants of GAS strain 188 , 188SLO - , and 188SLO ( Y255A ) . The amino acid substitution Y255A in SLO abrogates the ability of SLO to porate the host cell membrane , demonstrated by the failure of culture supernatants from 188SLO ( Y255A ) to lyse erythrocytes despite producing wild - type amounts of the variant SLO protein . Inset , corresponding Western blot for SLO in culture supernatants from these strains . . C . NADase activity in cell culture supernatants and in the cytosol of keratinocytes infected with GAS strain 771 , 771SLO - , 771SLO ( Y255A ) , or 771NADase - . Intracellular NADase activity due to translocation by extracellular GAS was identical between 771 and 771SLO ( Y255A ) , but reduced in 771SLO - . D . The intracellular survival of 188SLO ( Y255A ) was significantly lower than that of its parental strain , 188 , and similar to that of 188SLO - . * , P < 0 . 01 . E . Confocal microscopy demonstrating the association between galectin 8 ( green ) and intracellular GAS ( red ) in keratinocytes infected for 30 min with GAS strain 188 , 188SLO - , or 188 SLO - SLS - . Extracellular GAS ( GAS ( out ) ) were stained blue and red for identification as described in Figure 2 . Scale bar = 10 µm . The percentage of intracellular GAS that were associated with galectin 8 is indicated for each strain , based on quantification of at least 100 bacteria in three independent experiments . 188SLO - SLS - was never associated with galectin 8 ( 0 % ) .

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

Figure 4A. Confocal microscopy demonstrating the association between ubiquitin (green) and intracellular GAS (red) at 30 min post-infection in keratinocytes infected with GAS strain 188, 188SLO-, 188NADase-, or 188SLO(Y255A). Extracellular GAS were stained blue and red for identification as described in Figure 2. Scale bar = 10 µm. The percentage of intracellular GAS that were associated with ubiquitin is indicated for each strain, based on quantification of at least 100 bacteria in three independent experiments.B. Hemolytic activity of culture supernatants of GAS strain 188, 188SLO-, and 188SLO(Y255A). The amino acid substitution Y255A in SLO abrogates the ability of SLO to porate the host cell membrane, demonstrated by the failure of culture supernatants from 188SLO(Y255A) to lyse erythrocytes despite producing wild-type amounts of the variant SLO protein. Inset, corresponding Western blot for SLO in culture supernatants from these strains.. C. NADase activity in cell culture supernatants and in the cytosol of keratinocytes infected with GAS strain 771, 771SLO-, 771SLO(Y255A), or 771NADase-. Intracellular NADase activity due to translocation by extracellular GAS was identical between 771 and 771SLO(Y255A), but reduced in 771SLO-.D. The intracellular survival of 188SLO(Y255A) was significantly lower than that of its parental strain, 188, and similar to that of 188SLO-. *, P<0.01.E. Confocal microscopy demonstrating the association between galectin 8 (green) and intracellular GAS (red) in keratinocytes infected for 30 min with GAS strain 188, 188SLO-, or 188 SLO-SLS-. Extracellular GAS (GAS(out)) were stained blue and red for identification as described in Figure 2. Scale bar = 10 µm. The percentage of intracellular GAS that were associated with galectin 8 is indicated for each strain, based on quantification of at least 100 bacteria in three independent experiments. 188SLO-SLS- was never associated with galectin 8 (0%).


Figure 5SLO and NADase inhibit lysosomal fusion to GAS - containing autophagosomes in oropharyngeal keratinocytes . A . Confocal microscopy of keratinocytes infected with GAS strain 188 , 188SLO - , or 188NADase - demonstrating the association of the lysosomal marker LAMP - 1 ( red ) with GAS ( blue ) contained within EGFP - LC3 ( green ) - positive compartments at 1 h , 3 h , and 6 h post - infection . Scale bar = 10 µm . B . Quantification of the percent of GAS within EGFP - LC3 - positive compartments that are co - localized with LAMP - 1 at 1 h , 3 h , and 6 h . Data represent mean values from three independent experiments in which at least 100 intracellular GAS were quantified for each time point in each experiment .

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

Figure 5SLO and NADase inhibit lysosomal fusion to GAS-containing autophagosomes in oropharyngeal keratinocytes.A. Confocal microscopy of keratinocytes infected with GAS strain 188, 188SLO-, or 188NADase- demonstrating the association of the lysosomal marker LAMP-1 (red) with GAS (blue) contained within EGFP-LC3 (green)-positive compartments at 1 h, 3 h, and 6 h post-infection. Scale bar = 10 µm. B. Quantification of the percent of GAS within EGFP-LC3-positive compartments that are co-localized with LAMP-1 at 1 h, 3 h, and 6 h. Data represent mean values from three independent experiments in which at least 100 intracellular GAS were quantified for each time point in each experiment.


Figure 1 ( B ) Representative immunoblot of Nrxn1β - Fc variants harvested from HEK293 cell media and subjected to heparinase treatment ( ' Heps ' ) . Samples were analyzed by reducing SDS / PAGE and immunoblotting with antibodies against the Fc - tag and the 3G10 epitope ( to detect the HS stub which reveal HS after heparinase digestion ) . ( C ) Quantification of 3G10 ( reflecting HS ) signal normalized to that of Fc ( Nrxn1 ) . Bar graph shows means ± SEM of 3 independent experiments .

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

Figure 1(B) Representative immunoblot of Nrxn1β-Fc variants harvested from HEK293 cell media and subjected to heparinase treatment ('Heps'). Samples were analyzed by reducing SDS/PAGE and immunoblotting with antibodies against the Fc-tag and the 3G10 epitope (to detect the HS stub which reveal HS after heparinase digestion). (C) Quantification of 3G10 (reflecting HS) signal normalized to that of Fc (Nrxn1). Bar graph shows means ±SEM of 3 independent experiments.


Figure 2HEK293 cells : ( B ) Representative immunoblots ( of three independent experiments ) with antibodies against HA , the 3G10 epitope ( for the HS stub ) and V5 . HEK293 cells : ( D ) Representative immunoblots ( of three independent experiments ) analyzed as described for ( B ) . ( F - H ) Primary cortical neurons : Cortical neurons were infected with control lentivirus ( empty vector ) or lentivirus expressing FLAG - tagged CA10 , and cell lysates subjected to immunoprecipitation for neurexin and heparinase treatment . ( F ) Samples were analyzed by immunoblotting using antibodies against neurexins , 3G10 ( for the HS stub ) and FLAG ( for CA10 ) . Stars indicate non - neurexin bands ( see results ) . ( G ) Quantification of low molecular weight neurexin ( LMW ; white arrowhead ) in relation to total neurexin amounts [ upper band ( line ) + LMW ] . Data shown as mean ± SEM ( n = 3 independent experiments ) ; * , p < 0 . 05 by 2 - way ANOVA and Holm - Sidak tests . ( H ) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band . Data shown as mean ± SEM of biological replicates . * , p < 0 . 05 by 2 - way ANOVA and Holm - Sidak tests ( n = 3 ) . ( I - K ) Mouse brains : Neurexins and CA10 were immunoprecipitated from total mouse brain lysates and subject to heparinase treatment . ( I ) Samples were analyzed by immunoblotting using antibodies against neurexins , the 3G10 epitope ( for the HS stub ) and CA10 . Stars indicate non - neurexin bands ( see results ) . ( J ) Quantification of low molecular weight neurexin ( LMW ; white arrowhead ) in relation to total neurexin amounts [ upper band ( line ) + LMW ] . Data shown as mean ± SEM ( n = 3 independent experiments ) ; * , p < 0 . 05 by 2 - way ANOVA and Holm - Sidak tests . ( K ) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band . Data shown as mean ± SEM of 3 biological replicates . * , p < 0 . 05 by 2 - way ANOVA and Holm - Sidak tests .

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

Figure 2HEK293 cells: (B) Representative immunoblots (of three independent experiments) with antibodies against HA, the 3G10 epitope (for the HS stub) and V5.HEK293 cells: (D) Representative immunoblots (of three independent experiments) analyzed as described for (B).(F-H) Primary cortical neurons: Cortical neurons were infected with control lentivirus (empty vector) or lentivirus expressing FLAG-tagged CA10, and cell lysates subjected to immunoprecipitation for neurexin and heparinase treatment. (F) Samples were analyzed by immunoblotting using antibodies against neurexins, 3G10 (for the HS stub) and FLAG (for CA10). Stars indicate non-neurexin bands (see results). (G) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total neurexin amounts [upper band (line) + LMW]. Data shown as mean ±SEM (n=3 independent experiments); *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests. (H) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Data shown as mean ±SEM of biological replicates. *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests (n=3).(I-K) Mouse brains: Neurexins and CA10 were immunoprecipitated from total mouse brain lysates and subject to heparinase treatment. (I) Samples were analyzed by immunoblotting using antibodies against neurexins, the 3G10 epitope (for the HS stub) and CA10. Stars indicate non-neurexin bands (see results). (J) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total neurexin amounts [upper band (line) + LMW]. Data shown as mean ±SEM (n=3 independent experiments); *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests. (K) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Data shown as mean ±SEM of 3 biological replicates. *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests.


Figure 3 ( B ) Representative immunoblot of the indicated proteins harvested from HEK293 cell media and subjected to heparinase treatment ( ' Heps ' ) . Samples were analyzed by reducing SDS / PAGE and immunoblotting with antibodies against Fc ( for Nrxn1β ) , the 3G10 epitope ( for the HS stub ) and V5 ( for CA10 ) . Input samples were analyzed separately for CA10 to ensure equal expression ( lower panel ) . ( C ) Quantification of the 3G10 epitope signal , normalized to Fc ( Nrxn1β ) . Data shown are mean ± SEM of 3 independent replicates . * , p < 0 . 05 ; by Mann - Whitney tests comparing each Nrxn1 mutant with and without CA10 ( n = 3 ) . No significance difference was observed for S & T > G ( p = 0 . 1143 ) , GIGG ( p = 0 . 9714 ) or DIRR ( p = 0 . 4857 ) variants . ( E ) representative immunoblots analyzed using antibodies against the Fc - tag ( for Nrxn1β - Fc ) the 3G10 epitope ( for the HS stub ) and V5 ( CA10 - V5 ) , as indicated . CA10 in input samples is shown for demonstration of equal expression . ( F , Quantification of the 3G10 epitope signal normalized to Fc ( for Nrxn1β - Fc ) signals , Data shown are mean ± SEM of 3 biological replicates . * , p < 0 . 05 ; * * * , p < 0 . 001 by 2 - way ANOVA and Holm - Sidak tests . ( G ) show representative immunoblots analyzed using antibodies against the HA tag ( Nrxn1α - HA ) , the 3G10 epitope ( for the HS stub ) and V5 ( CA10 - V5 ) , as indicated . CA10 in input samples is shown for demonstration of equal expression . H ) Quantification of the 3G10 epitope signal normalized to HA ( for Nrxn1α - HA ) signals , respectively . Data shown are mean ± SEM of 3 biological replicates . * , p < 0 . 05 ; * * * , p < 0 . 001 by 2 - way ANOVA and Holm - Sidak tests .

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

Figure 3(B) Representative immunoblot of the indicated proteins harvested from HEK293 cell media and subjected to heparinase treatment ('Heps'). Samples were analyzed by reducing SDS/PAGE and immunoblotting with antibodies against Fc (for Nrxn1β), the 3G10 epitope (for the HS stub) and V5 (for CA10). Input samples were analyzed separately for CA10 to ensure equal expression (lower panel). (C) Quantification of the 3G10 epitope signal, normalized to Fc (Nrxn1β). Data shown are mean ±SEM of 3 independent replicates. *, p< 0.05; by Mann-Whitney tests comparing each Nrxn1 mutant with and without CA10 (n=3). No significance difference was observed for S&T>G (p=0.1143), GIGG (p=0.9714) or DIRR (p=0.4857) variants. (E) representative immunoblots analyzed using antibodies against the Fc-tag (for Nrxn1β-Fc) the 3G10 epitope (for the HS stub) and V5 (CA10-V5), as indicated. CA10 in input samples is shown for demonstration of equal expression. (F, Quantification of the 3G10 epitope signal normalized to Fc (for Nrxn1β-Fc) signals, Data shown are mean ±SEM of 3 biological replicates. *, p< 0.05; ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests.(G) show representative immunoblots analyzed using antibodies against the HA tag (Nrxn1α-HA), the 3G10 epitope (for the HS stub) and V5 (CA10-V5), as indicated. CA10 in input samples is shown for demonstration of equal expression. H) Quantification of the 3G10 epitope signal normalized to HA (for Nrxn1α-HA) signals, respectively. Data shown are mean ±SEM of 3 biological replicates. *, p< 0.05; ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests.


Figure 4 ( D ) Comparison of 1H - 13C HSQC spectra of Nrxn1 stalk ( red ) and Nrxn1 stalk / CA10 ( black ) at a 2 : 1 molar ratio ( 50 mM phosphate buffer , pH 7 . 4 , 0 . 5 mM d10 - DTT , 10 % D2O ) . Nrxn1 residues with stronger chemical shift changes are indicated blue boxes . Asterisk ( * ) indicates an impurity from glycerol .

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

Figure 4(D) Comparison of 1H-13C HSQC spectra of Nrxn1 stalk (red) and Nrxn1 stalk/CA10 (black) at a 2:1 molar ratio (50 mM phosphate buffer, pH 7.4, 0.5 mM d10-DTT, 10% D2O). Nrxn1 residues with stronger chemical shift changes are indicated blue boxes. Asterisk (*) indicates an impurity from glycerol.


Figure 5 ( B - C ) Extracted ion chromatograms ( EIC ) of DDILVASAECPSDDE peptides and its glycosylated forms , from Nrxn1β expressed in HEK293 cells . ( D ) Quantification , expressed as % of total occupancy of each glycosylation form , upon control and CA10 expression conditions . Bar graph shows means ± SEM of 4 biological replicates ; * , p < 0 . 05 by Mann - Whitney tests comparing the occupancy of each glycosylation with and without CA10 ( n = 4 biological replicates ) .

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

Figure 5(B-C) Extracted ion chromatograms (EIC) of DDILVASAECPSDDE peptides and its glycosylated forms, from Nrxn1β expressed in HEK293 cells.(D) Quantification, expressed as % of total occupancy of each glycosylation form, upon control and CA10 expression conditions. Bar graph shows means ±SEM of 4 biological replicates; *, p<0.05 by Mann-Whitney tests comparing the occupancy of each glycosylation with and without CA10 (n=4 biological replicates).


Figure 6 ( A ) Immunoblot α - showing neurexin isoform distribution in cortex , hippocampus , midbrain and cerebellum dissected from 8 - week - old mice . ( B ) Quantification of lower molecular weight neurexin ( LMW ; white arrowhead ) in relation to total levels of α - neurexins [ upper band ( line ) + LMW ] . Bar graphs show mean ± SEM of 3 independent replicates * * * , p < 0 . 001 by 2 - way ANOVA and Holm - Sidak tests , comparing to levels in cerebellum . ( C ) α - Neurexin immunoprecipitated from forebrain ( Fb ) and cerebellum ( Cb ) using an anti - pan - neurexin antibody was heparinase - treated ( ' Heps ' ) and analyzed by immunoblotting with antibodies against neurexin and the 3G10 monoclonal ( to detect the HS stub ) . Stars indicate non - neurexin bands ( see results ) . ( D ) Quantification of low molecular weight neurexin ( LMW ; white arrowhead ) in relation to total levels of α - neurexins [ upper band ( line ) + LMW ] . Bar graphs show mean ± SEMof 3 biological replicates . * * * , p < 0 . 001 by 2 - way ANOVA and Holm - Sidak tests comparing forebrain and cerebellum . ( E ) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band . Bar graphs show mean ± SEM of 3 biological replicates . * * * , p < 0 . 001 by 2 - way ANOVA and Holm - Sidak tests comparing forebrain and cerebellum .

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

Figure 6(A) Immunoblot α-showing neurexin isoform distribution in cortex, hippocampus , midbrain and cerebellum dissected from 8-week-old mice. (B) Quantification of lower molecular weight neurexin (LMW; white arrowhead) in relation to total levels of α-neurexins [upper band (line) + LMW]. Bar graphs show mean ± SEM of 3 independent replicates***, p<0.001 by 2-way ANOVA and Holm-Sidak tests, comparing to levels in cerebellum. (C) α-Neurexin immunoprecipitated from forebrain (Fb) and cerebellum (Cb) using an anti-pan-neurexin antibody was heparinase-treated ('Heps') and analyzed by immunoblotting with antibodies against neurexin and the 3G10 monoclonal (to detect the HS stub). Stars indicate non-neurexin bands (see results). (D) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total levels of α-neurexins [upper band (line) + LMW]. Bar graphs show mean ±SEMof 3 biological replicates. ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests comparing forebrain and cerebellum. (E) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Bar graphs show mean ±SEM of 3 biological replicates. ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests comparing forebrain and cerebellum.


Figure 7 ( A - B ) Cell surface binding to endogenous neurexins . ( A ) Recombinant Fc - tagged LRRTM2 was added to day 10 hippocampal neurons expressing CA10 or control ( empty vector ) by lentiviral transduction . Surface - bound protein was quantified as the Fc signal of and normalized to a staining for endogenous neurexin ( n = 4 cultures ; three fields imaged and averaged per well ) . Scatchard analysis for LRRTM2 revealed an apparent Kd of 29 . 5 nM and a Bmax of 0 . 49 nM for control and Kd 35 . 3 nM , Bmax 0 . 28 nM upon expression of CA10 ; significant ( p = 0 . 0006 ) by non - linear regression . Data shown as mean ± SEM of biological replicates ( n = 4 ) . ( B ) Increasing amounts of recombinant , Fc - tagged Nlgn1 was added to immature hippocampal neurons expressing CA10 or control ( empty vector ) by lentiviral transduction , and analyzed as in ( A ) . The apparent Kd for Nlgn1 was 186 nM and Bmax 1 . 65 nM for control and Kd 223 nM , Bmax 1 . 49 nM when CA10 was overexpressed . The difference was not significant ( p = 0 . 20 ) by non - linear regression . Data shown as mean ± SEM of biological replicates ( n = 4 ) . ( C - D ) Artificial synapse formation assays . ( C ) Confocal images of hippocampal neurons transduced to express CA10 - FLAG or control ( empty vector ) and co - cultured with HEK293 cells expressing GFP , Nlgn1 - YFP , LRRTM2 - YFP or TrkC - YFP . Cells were stained with antibodies against the synaptic marker SV2 ( red ) and somatodendritic MAP2 ( blue ) ; GFP / YFP is shown in green . Scale bar 20 μm . ( D ) Quantifications of SV2 signal overlapping GFP / YFP - positive cells . Data shown as means ± SEM with the n , reflecting number of independent cover slips analyzed , indicated in each bar with the total number of cells imaged in parenthesis . Comparisons were made by one - way ANOVA with Holm - Sidak ' s posthoc test for multiple comparisons between the indicated groups ( GFP condition was omitted from analysis due to unequal variance ; * * * , p < 0 . 001 . ( E - F ) Analysis of cerebellin ( Cbln ) - induced artificial synapse formation . ( E ) Confocal images of hippocampal neurons transduced to express CA10 - FLAG or control ( empty vector ) and co - cultured exposed to anti - V5 - coupled beads pre - incubated with recombinant V5 - tagged Cbln1 or empty anti - V5 beads . Cells were stained with antibodies against the synaptic marker synapsin ( green ) , somatodendritic MAP2 ( blue ) ; beads are colored red and indicated by arrowheads . Scale bar 10 μm . ( F ) Quantifications of the synapsin signal overlapping red beads . Data shown as means ± SEM ( n = 3 independent experiments , each data point corresponding to 58 - 171 analyzed beads ) , with comparisons made by two - way ANOVA with Holm - Sidak ' s posthoc test for multiple comparisons between the indicated groups .

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

Figure 7(A-B) Cell surface binding to endogenous neurexins. (A) Recombinant Fc-tagged LRRTM2 was added to day 10 hippocampal neurons expressing CA10 or control (empty vector) by lentiviral transduction. Surface-bound protein was quantified as the Fc signal of and normalized to a staining for endogenous neurexin (n=4 cultures; three fields imaged and averaged per well). Scatchard analysis for LRRTM2 revealed an apparent Kd of 29.5 nM and a Bmax of 0.49 nM for control and Kd 35.3 nM, Bmax 0.28 nM upon expression of CA10; significant (p=0.0006) by non-linear regression. Data shown as mean ± SEM of biological replicates (n=4). (B) Increasing amounts of recombinant, Fc-tagged Nlgn1 was added to immature hippocampal neurons expressing CA10 or control (empty vector) by lentiviral transduction, and analyzed as in (A). The apparent Kd for Nlgn1 was 186 nM and Bmax 1.65 nM for control and Kd 223 nM, Bmax 1.49 nM when CA10 was overexpressed. The difference was not significant (p=0.20) by non-linear regression. Data shown as mean ± SEM of biological replicates (n=4).(C-D) Artificial synapse formation assays. (C) Confocal images of hippocampal neurons transduced to express CA10-FLAG or control (empty vector) and co-cultured with HEK293 cells expressing GFP, Nlgn1-YFP, LRRTM2-YFP or TrkC-YFP. Cells were stained with antibodies against the synaptic marker SV2 (red) and somatodendritic MAP2 (blue); GFP/YFP is shown in green. Scale bar 20 μm. (D) Quantifications of SV2 signal overlapping GFP/YFP-positive cells. Data shown as means ±SEM with the n, reflecting number of independent cover slips analyzed, indicated in each bar with the total number of cells imaged in parenthesis. Comparisons were made by one-way ANOVA with Holm-Sidak's posthoc test for multiple comparisons between the indicated groups (GFP condition was omitted from analysis due to unequal variance; ***, p<0.001.(E-F) Analysis of cerebellin(Cbln)-induced artificial synapse formation. (E) Confocal images of hippocampal neurons transduced to express CA10-FLAG or control (empty vector) and co-cultured exposed to anti-V5-coupled beads pre-incubated with recombinant V5-tagged Cbln1 or empty anti-V5 beads. Cells were stained with antibodies against the synaptic marker synapsin (green), somatodendritic MAP2 (blue); beads are colored red and indicated by arrowheads. Scale bar 10 μm. (F) Quantifications of the synapsin signal overlapping red beads. Data shown as means ±SEM (n=3 independent experiments, each data point corresponding to 58-171 analyzed beads), with comparisons made by two-way ANOVA with Holm-Sidak's posthoc test for multiple comparisons between the indicated groups.


Figure 1 ( A ) Representative image of a typical young WT cortico - hippocampal slice ( 350 µm ) from co - culture ( 14 DIV ) stained with DAPI ( gray ) to visualize nuclei ( left ) . Dentate gyrus ( DG ) , cornus ammonis 1 ( CA1 ) and cortical ( CTX ) regions are indicated . An example of the co - culture dish is shown on the right . Co - culture includes 2 young WT ( * , young co - culture ) and 2 old APPPS1 ( + , old co - culture ) brain slices . ( B ) Immunofluorescence analysis of young WT and old APPPS1 slices cultured alone ( young alone , old alone ) or in co - culture ( young co - culture , old co - culture ) at 7 days in vitro ( DIV ) stained with propidium iodide ( PI , red ) reveals reduced cell viability in old brain slices compared to young . ( C ) Quantitative analysis of PI positive cells in young WT and old APPPS1 slices cultured alone ( white and black bars , respectively ) or in co - culture ( grey and white squared bars , respectively ) at 7 and 14 DIV . The values are expressed as percentages of PI positive cells from the total number of DAPI positive cells . The values represent mean ± SEM from 3 independent experiments , each experiment including 2 independent slice culture dishes ( n . s . = not significant ; * * * P < 0 . 001 ) . ( D - F ) Immunofluorescence analysis of young WT and old APPPS1 slices cultured alone or in co - culture at 7 DIV and immunostained using neuronal ( NeuN ) , astrocytic ( GFAP ) and microglial ( CD68 ) markers ( green ) reveals reduced NeuN and GFAP and increased CD68 immunosignal . Scale bar : 75

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

Figure 1(A) Representative image of a typical young WT cortico-hippocampal slice (350 µm) from co-culture (14 DIV) stained with DAPI (gray) to visualize nuclei (left). Dentate gyrus (DG), cornus ammonis 1 (CA1) and cortical (CTX) regions are indicated. An example of the co-culture dish is shown on the right. Co-culture includes 2 young WT (*, young co-culture) and 2 old APPPS1 (+, old co-culture) brain slices.(B) Immunofluorescence analysis of young WT and old APPPS1 slices cultured alone (young alone, old alone) or in co-culture (young co-culture, old co-culture) at 7 days in vitro (DIV) stained with propidium iodide (PI, red) reveals reduced cell viability in old brain slices compared to young.(C) Quantitative analysis of PI positive cells in young WT and old APPPS1 slices cultured alone (white and black bars, respectively) or in co-culture (grey and white squared bars, respectively) at 7 and 14 DIV. The values are expressed as percentages of PI positive cells from the total number of DAPI positive cells. The values represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (n.s.= not significant; ***P < 0.001).(D-F) Immunofluorescence analysis of young WT and old APPPS1 slices cultured alone or in co-culture at 7 DIV and immunostained using neuronal (NeuN), astrocytic (GFAP) and microglial (CD68) markers (green) reveals reduced NeuN and GFAP and increased CD68 immunosignal. Scale bar: 75 µm


Figure 2 ( A ) Immunofluorescence analysis of the old APPPS1 slice in co - culture using antibody 6E10 to detect amyloid plaques ( green ) and Thiazine Red ( TR , red ) to visualize fibrillar amyloid cores . At 7 DIV , the majority of amyloid plaques contain 6E10 and TR positive dense core surrounded by 6E10 positive halo of diffuse Aβ . In contrast , at 14 DIV , the majority of amyloid plaques are core - only plaques co - stained with 6E10 and TR without detectable 6E10 positive halo . Lower panels are higher magnification images of boxed regions . Scale bars : 50 µm . ( B ) Quantitative analysis of core - only plaques in old APPPS1 slices cultured alone ( white bars ) or in co - culture ( gray bars ) at 7 and 14 DIV reveals an increased number of core - only plaques upon co - culturing of old APPPS1 brain slices together with young WT slices . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques . The values represent mean ± SEM from 3 independent experiments , each experiment including 3 independent slice culture dishes ( * * * P < 0 . 001 ) . ( C ) Western blot analysis of Aβ levels ( triplicates ) in the old APPPS1 tissue cultured alone or in co - culture with the young WT ( 14 DIV ) using 2D8 antibody . ( D ) Quantification of Western blot signals reveals decreased levels of Aβ upon co - culturing compared to old tissue cultured alone . The values are normalized to the levels of Aβ in the old tissue cultured alone and represent mean ± SEM from 2 independent experiments , each experiment was performed in triplicates ( * * P < 0 . 01 ) .

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

Figure 2(A) Immunofluorescence analysis of the old APPPS1 slice in co-culture using antibody 6E10 to detect amyloid plaques (green) and Thiazine Red (TR, red) to visualize fibrillar amyloid cores. At 7 DIV, the majority of amyloid plaques contain 6E10 and TR positive dense core surrounded by 6E10 positive halo of diffuse Aβ. In contrast, at 14 DIV, the majority of amyloid plaques are core-only plaques co-stained with 6E10 and TR without detectable 6E10 positive halo. Lower panels are higher magnification images of boxed regions. Scale bars: 50 µm.(B) Quantitative analysis of core-only plaques in old APPPS1 slices cultured alone (white bars) or in co-culture (gray bars) at 7 and 14 DIV reveals an increased number of core-only plaques upon co-culturing of old APPPS1 brain slices together with young WT slices. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques. The values represent mean ± SEM from 3 independent experiments, each experiment including 3 independent slice culture dishes (***P < 0.001).(C) Western blot analysis of Aβ levels (triplicates) in the old APPPS1 tissue cultured alone or in co-culture with the young WT (14 DIV) using 2D8 antibody.(D) Quantification of Western blot signals reveals decreased levels of Aβ upon co-culturing compared to old tissue cultured alone. The values are normalized to the levels of Aβ in the old tissue cultured alone and represent mean ± SEM from 2 independent experiments, each experiment was performed in triplicates (**P < 0.01).


Figure 3 ( A , B ) Immunofluorescence analysis of the old APPPS1 slice in co - culture ( 14 DIV ) using antibodies 6E10 ( green ) and CD68 ( red ) . Nuclei were counterstained using DAPI ( blue ) . Image of boxed region in A is depicted at higher magnification in B and reveals clustering of several CD68 positive cells around amyloid plaques . Scale bars : 100 µm ( A ) and 10 µm ( B ) . ( C ) Quantitative analysis of plaque - associated CD68 positive cells in co - culture samples at 7 and 14 DIV reveals a trend towards increased number of CD68 positive cells . The values represent mean ± SEM from at least 3 independent experiments , including total of at least 6 independent slice culture dishes ( n . s . = not significant ) . ( D ) Quantitative analysis of co - culture samples at 7 and 14 DIV reveals a decrease in plaque size . The values represent mean ± SEM from at least 3 independent experiments , including total of at least 6 replicates ( * * * P < 0 . 001 ) . ( E ) Quantitative analysis of plaques surrounded by CD68 positive cells ( clustered plaques ) in co - culture samples at 7 and 14 DIV reveals an increase in the number of clustered plaques . The values are expressed as percentages of clustered plaques from the total number of amyloid plaques . The values represent mean ± SEM from at least 3 independent experiments , including total of at least 6 independent slice culture dishes ( * * * P < 0 . 001 ) . ( F ) Immunofluorescence analysis of the old APPPS1 slice in co - culture ( 14 DIV ) treated with cytochalasin D ( CytoD ) and vehicle control ( Ctr ) and immunostained with M3 . 2 ( green ) and CD68 ( red ) . Inhibition of phagocytosis by CytoD blocks amyloid plaque clearance . Scale bar : 50 µm . ( G ) Quantitative analysis of core - only plaques in the old APPPS1 slice in co - culture ( 14 DIV ) treated with CytoD and Ctr reveals a decreased number of core - only plaques upon CytoD treatment . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques . The values represent mean ± SEM from 3 independent experiments , including total of 6 independent slice culture dishes ( * * * P < 0 . 001 ) .

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

Figure 3(A, B) Immunofluorescence analysis of the old APPPS1 slice in co-culture (14 DIV) using antibodies 6E10 (green) and CD68 (red). Nuclei were counterstained using DAPI (blue). Image of boxed region in A is depicted at higher magnification in B and reveals clustering of several CD68 positive cells around amyloid plaques. Scale bars: 100 µm (A) and 10 µm (B).(C) Quantitative analysis of plaque-associated CD68 positive cells in co-culture samples at 7 and 14 DIV reveals a trend towards increased number of CD68 positive cells. The values represent mean ± SEM from at least 3 independent experiments, including total of at least 6 independent slice culture dishes (n.s.= not significant).(D) Quantitative analysis of co-culture samples at 7 and 14 DIV reveals a decrease in plaque size. The values represent mean ± SEM from at least 3 independent experiments, including total of at least 6 replicates (***P < 0.001).(E) Quantitative analysis of plaques surrounded by CD68 positive cells (clustered plaques) in co-culture samples at 7 and 14 DIV reveals an increase in the number of clustered plaques. The values are expressed as percentages of clustered plaques from the total number of amyloid plaques. The values represent mean ± SEM from at least 3 independent experiments, including total of at least 6 independent slice culture dishes (***P < 0.001).(F) Immunofluorescence analysis of the old APPPS1 slice in co-culture (14 DIV) treated with cytochalasin D (CytoD) and vehicle control (Ctr) and immunostained with M3.2 (green) and CD68 (red). Inhibition of phagocytosis by CytoD blocks amyloid plaque clearance. Scale bar: 50 µm.(G) Quantitative analysis of core-only plaques in the old APPPS1 slice in co-culture (14 DIV) treated with CytoD and Ctr reveals a decreased number of core-only plaques upon CytoD treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques. The values represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (***P < 0.001).


Figure 4Either young WT ( A , B ) or old APPPS1 ( C - E ) tissue was treated with clodronate ( Clo ) to remove CD68 positive cells or vehicle Ctr from 1 until 7 DIV . Treatment was stopped and subsequently old ( A , B ) or young ( C - E ) tissue was added to the culture as schematically indicated in A and C . and analyzed 14 days after . Removal of CD68 positive cells in either young WT or old APPPS1 tissue prevents amyloid plaque clearance in the co - culture model . ( A ) Immunofluorescence analysis of the old APPPS1 slice co - cultured with the young WT slice pre - treated with Clo and Ctr and immunostained with CD68 ( red ) and M3 . 2 ( green ) . Scale bar : 50 µm . ( B ) Quantitative analysis of core - only plaques in the old APPPS1 tissue co - cultured with the young WT tissue pre - treated with Clo and Ctr as indicated in A reveals a decreased number of core - only plaques upon Clo treatment . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , including total of 6 independent slice culture dishes ( * * * P < 0 . 001 ) . ( C ) Immunofluorescence analysis of the old APPPS1 slice treated with Clo and Ctr and subsequently co - cultured with the young WT slice and immunostained with CD68 ( red ) and M3 . 2 ( green ) . Scale bar : 50 µm . ( D ) Area of CD68 positive cells ( CD68 coverage ) in the old APPPS1 tissue treated with Clo and Ctr and subsequently co - cultured with the young WT tissue as indicated in C . CD68 coverage is reduced upon Clo treatment . The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments , including total of 6 independent slice culture dishes ( * * * P < 0 . 001 ) . ( E ) Quantitative analysis of core - only plaques in the old APPPS1 tissue treated with Clo and Ctr and subsequently co - cultured with the young WT tissue as indicated in C reveals a decreased number of core - only plaques upon Clo treatment . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , including total of 6 independent slice culture dishes ( * * P < 0 . 01 ) .

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

Figure 4Either young WT (A, B) or old APPPS1 (C-E) tissue was treated with clodronate (Clo) to remove CD68 positive cells or vehicle Ctr from 1 until 7 DIV. Treatment was stopped and subsequently old (A, B) or young (C-E) tissue was added to the culture as schematically indicated in A and C. and analyzed 14 days after. Removal of CD68 positive cells in either young WT or old APPPS1 tissue prevents amyloid plaque clearance in the co-culture model.(A) Immunofluorescence analysis of the old APPPS1 slice co-cultured with the young WT slice pre-treated with Clo and Ctr and immunostained with CD68 (red) and M3.2 (green). Scale bar: 50 µm.(B) Quantitative analysis of core-only plaques in the old APPPS1 tissue co-cultured with the young WT tissue pre-treated with Clo and Ctr as indicated in A reveals a decreased number of core-only plaques upon Clo treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (***P < 0.001).(C) Immunofluorescence analysis of the old APPPS1 slice treated with Clo and Ctr and subsequently co-cultured with the young WT slice and immunostained with CD68 (red) and M3.2 (green). Scale bar: 50 µm.(D) Area of CD68 positive cells (CD68 coverage) in the old APPPS1 tissue treated with Clo and Ctr and subsequently co-cultured with the young WT tissue as indicated in C. CD68 coverage is reduced upon Clo treatment. The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (***P < 0.001).(E) Quantitative analysis of core-only plaques in the old APPPS1 tissue treated with Clo and Ctr and subsequently co-cultured with the young WT tissue as indicated in C reveals a decreased number of core-only plaques upon Clo treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (**P < 0.01).


Figure 5 ( A ) Immunofluorescence analysis of the old APPPS1 slice in co - culture with the young CX3CR1 + / GFP slice ( 14 DIV ) and immunostained using GFP ( green ) , CD68 ( red ) and M3 . 2 ( blue ) reveals no co - localization of young , GFP positive microglial cells with CD68 positive cells surrounding amyloid plaques . A GFP antibody was used to amplify the signal of GFP - expressing young microglial cells . ( B ) Immunofluorescence analysis of the old APPPS1 / CX3CR1 + / GFP slice in co - culture with the young WT slice ( 14 DIV ) and immunostained using GFP ( green ) , CD68 ( red ) and M3 . 2 ( blue ) reveals co - localization of old , GFP positive microglial cells with CD68 positive cells surrounding amyloid plaques . A GFP antibody was used to amplify the signal of GFP - expressing old microglial cells . Scale bar : 10 µm ( A and B ) . ( C ) 3D reconstruction of the old APPPS1 / CX3CR1 + / GFP slice in co - culture with the young WT slice ( 10 DIV ) and immunostained using GFP ( green ) and M3 . 2 ( red ) . GFP positive old microglial cells surrounding amyloid plaques exhibit intracellular M3 . 2 positive immunoreactivity indicative of Aβ uptake . Overview at low magnification is shown in the left panel . Scale bar : 15 µm . A detailed plaque is shown at higher magnification in the right panels . Scale bar : 10 µm . ( D ) Immunofluorescence analysis of young CX3CR1 + / GFP and old APPPS1 / CX3CR1 + / GFP slices freshly cut ( 0 DIV ) , cultured alone or in co - culture ( 10 DIV ) and immunostained using GFP ( green ) shows alterations in microglial morphology of old cells upon culturing . Upper - left panels are enlargements of boxed regions . A GFP antibody was used to amplify the signal of GFP - expressing old microglial cells . Scale bars : 50 µm . ( E ) Quantitative analysis of amoeboid microglial cells in young CX3CR1 + / GFP ( white bars ) and old APPPS1 / CX3CR1 + / GFP ( green bars ) brain slices freshly cut ( 0 DIV ) , cultured alone or in co - culture ( 10 DIV ) . The values are expressed as percentages of amoeboid microglia from the total number of microglial cells . The values represent mean ± SEM from 3 independent experiments , each experiment including at least 250 cells per condition ( * P < 0 . 05 , * * * P < 0 . 001 ) .

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

Figure 5(A) Immunofluorescence analysis of the old APPPS1 slice in co-culture with the young CX3CR1+/GFP slice (14 DIV) and immunostained using GFP (green), CD68 (red) and M3.2 (blue) reveals no co-localization of young, GFP positive microglial cells with CD68 positive cells surrounding amyloid plaques. A GFP antibody was used to amplify the signal of GFP-expressing young microglial cells.(B) Immunofluorescence analysis of the old APPPS1/CX3CR1+/GFP slice in co-culture with the young WT slice (14 DIV) and immunostained using GFP (green), CD68 (red) and M3.2 (blue) reveals co-localization of old, GFP positive microglial cells with CD68 positive cells surrounding amyloid plaques. A GFP antibody was used to amplify the signal of GFP-expressing old microglial cells. Scale bar: 10 µm (A and B).(C) 3D reconstruction of the old APPPS1/CX3CR1+/GFP slice in co-culture with the young WT slice (10 DIV) and immunostained using GFP (green) and M3.2 (red). GFP positive old microglial cells surrounding amyloid plaques exhibit intracellular M3.2 positive immunoreactivity indicative of Aβ uptake. Overview at low magnification is shown in the left panel. Scale bar: 15 µm. A detailed plaque is shown at higher magnification in the right panels. Scale bar: 10 µm.(D) Immunofluorescence analysis of young CX3CR1+/GFP and old APPPS1/CX3CR1+/GFP slices freshly cut (0 DIV), cultured alone or in co-culture (10 DIV) and immunostained using GFP (green) shows alterations in microglial morphology of old cells upon culturing. Upper-left panels are enlargements of boxed regions. A GFP antibody was used to amplify the signal of GFP-expressing old microglial cells. Scale bars: 50 µm.(E) Quantitative analysis of amoeboid microglial cells in young CX3CR1+/GFP (white bars) and old APPPS1/CX3CR1+/GFP (green bars) brain slices freshly cut (0 DIV), cultured alone or in co-culture (10 DIV). The values are expressed as percentages of amoeboid microglia from the total number of microglial cells. The values represent mean ± SEM from 3 independent experiments, each experiment including at least 250 cells per condition (*P < 0.05, ***P < 0.001).


Figure 6 ( A ) Old APPPS1 tissue was cultured alone ( old alone , white bars ) or incubated with conditioned media ( CM ) collected from young WT slices ( old alone + young CM , grey bars ) . Analysis included co - culture of old APPPS1 and young WT brain slices ( old co - culture , black bars ) as a positive control and old APPPS1 tissue incubated with conditioned media collected from old APPPS1 slices ( old alone + old CM , white squared bars ) as a negative control . Quantitative analysis at 7 , 11 and 14 DIV reveals increased numbers of core - only plaques upon incubation of the old APPPS1 tissue with conditioned media collected from young WT slices , fully recapitulating core - only plaque numbers observed in the co - culture . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , each experiment including at least 2 independent slice culture dishes ( n . s . = not significant , * * P < 0 . 01 , * * * P < 0 . 001 ) . ( B ) Old APPPS1 tissue was incubated with conditioned media collected from young WT slices previously treated with clodronate ( old alone + young CM + Clo , grey bars ) and vehicle control ( old alone + young CM + Ctr , white bars ) . Quantitative analysis at 7 , 11 and 14 DIV reveals decreased numbers of core - only plaques upon incubation of the old APPPS1 tissue with conditioned media collected from young WT slices pre - treated with Clo . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , each experiment including at least 2 independent slice culture dishes ( n . s . = not significant , * * * P < 0 . 001 ) . ( C ) Old APPPS1 tissue was incubated with conditioned media collected from cultured WT primary microglial cells ( old alone + MG CM , grey bar ) or with unconditioned slice culture media as a control ( old alone + Ctr , white bar ) . Quantitative analysis of core - only plaques at 14 DIV reveals enhanced amyloid plaque clearance upon incubation of the old APPPS1 tissue with conditioned media collected from cultured primary microglia . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , each experiment including 2 independent slice culture dishes ( * P < 0 . 05 ) .

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

Figure 6(A) Old APPPS1 tissue was cultured alone (old alone, white bars) or incubated with conditioned media (CM) collected from young WT slices (old alone + young CM, grey bars). Analysis included co-culture of old APPPS1 and young WT brain slices (old co-culture, black bars) as a positive control and old APPPS1 tissue incubated with conditioned media collected from old APPPS1 slices (old alone + old CM, white squared bars) as a negative control. Quantitative analysis at 7, 11 and 14 DIV reveals increased numbers of core-only plaques upon incubation of the old APPPS1 tissue with conditioned media collected from young WT slices, fully recapitulating core-only plaque numbers observed in the co-culture. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, each experiment including at least 2 independent slice culture dishes (n.s.= not significant, **P <0.01, ***P < 0.001).(B) Old APPPS1 tissue was incubated with conditioned media collected from young WT slices previously treated with clodronate (old alone + young CM + Clo, grey bars ) and vehicle control (old alone + young CM + Ctr, white bars). Quantitative analysis at 7, 11 and 14 DIV reveals decreased numbers of core-only plaques upon incubation of the old APPPS1 tissue with conditioned media collected from young WT slices pre-treated with Clo. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, each experiment including at least 2 independent slice culture dishes (n.s.= not significant, ***P < 0.001).(C) Old APPPS1 tissue was incubated with conditioned media collected from cultured WT primary microglial cells (old alone + MG CM, grey bar) or with unconditioned slice culture media as a control (old alone + Ctr, white bar). Quantitative analysis of core-only plaques at 14 DIV reveals enhanced amyloid plaque clearance upon incubation of the old APPPS1 tissue with conditioned media collected from cultured primary microglia. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (*P < 0.05).


Figure 7 ( A - F ) Immunofluorescence analysis of the old APPPS1 slice ( 14 DIV ) treated with vehicle Ctr in A , anti - inflammatory factors IL - 10 and TGF - β in B and C and pro - inflammatory factors IL - 6 , IL - 12 / p40 and GM - CSF in D , E and F , respectively and immunostained with CD68 ( red ) and M3 . 2 ( green ) . In contrast to increased number of core - only plaques upon GM - CSF treatment , other factors tested did not induce amyloid plaque clearance , resulting in a better preservation of the M3 . 2 positive halos of Aβ surrounding dense core of amyloid plaques . Scale bar : 50 µm .

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

Figure 7(A-F) Immunofluorescence analysis of the old APPPS1 slice (14 DIV) treated with vehicle Ctr in A, anti-inflammatory factors IL-10 and TGF-β in B and C and pro-inflammatory factors IL-6, IL-12/p40 and GM-CSF in D, E and F, respectively and immunostained with CD68 (red) and M3.2 (green). In contrast to increased number of core-only plaques upon GM-CSF treatment, other factors tested did not induce amyloid plaque clearance, resulting in a better preservation of the M3.2 positive halos of Aβ surrounding dense core of amyloid plaques. Scale bar: 50 µm.


Figure 8 ( A ) CD68 coverage in the old APPPS1 tissue ( 14 DIV ) treated with GM - CSF and vehicle Ctr . CD68 coverage is increased upon treatment with GM - CSF . The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments , each experiment including 2 independent slice culture dishes ( * P < 0 . 05 ) . ( B ) Quantitative analysis of core - only plaques in the old APPPS1 tissue ( 14 DIV ) treated with GM - CSF and Ctr reveals an increased number of core - only plaques upon treatment with GM - CSF . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , each experiment including 3 independent slice culture dishes ( * * * P < 0 . 001 ) . ( C ) CD68 coverage in the old APPPS1 tissue ( 14 DIV ) incubated with conditioned media collected from young WT slices ( young CM ) or with non - conditioned slice culture media ( Ctr ) . CD68 coverage is increased upon incubation of the APPPS1 tissue with the young CM . The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments , each experiment including 2 independent slice culture dishes ( * * P < 0 . 01 ) . ( D ) Immunofluorescence analysis of the old APPPS1 slice cultured alone ( old alone ) or in co - culture with young GM - CSF - / - brain slices ( old co - culture ) at 14 DIV and immunostained with CD68 ( red ) and M3 . 2 ( green ) reveals an increased number of core - only plaques upon co - culturing with young GM - CSF - / - brain slices . Scale bar : 50 µm . ( E ) Quantitative analysis of core - only plaques in the old APPPS1 slice cultured alone or in co - culture with young GM - CSF - / - brain slices ( 14 DIV ) reveals an increased number of core - only plaques upon co - culturing of old APPPS1 brain slices together with young GM - CSF - / - brain slices . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques . The values represent mean ± SEM from 3 independent experiments , each experiment including 3 independent slice culture dishes ( * * * P < 0 . 001 ) . ( F ) CD68 coverage in the old APPPS1 tissue ( 14 DIV ) cultured alone or in co - culture with young GM - CSF - / - brain slices . CD68 coverage is increased upon co - culturing of old APPPS1 brain slices together with young GM - CSF - / - brain slices . The values are normalized to CD68 coverage of the old slice cultured alone and represent mean ± SEM from 3 independent experiments , each experiment including 2 independent slice culture dishes ( * P < 0 . 05 ) .

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

Figure 8(A) CD68 coverage in the old APPPS1 tissue (14 DIV) treated with GM-CSF and vehicle Ctr. CD68 coverage is increased upon treatment with GM-CSF. The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (*P < 0.05).(B) Quantitative analysis of core-only plaques in the old APPPS1 tissue (14 DIV) treated with GM-CSF and Ctr reveals an increased number of core-only plaques upon treatment with GM-CSF. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, each experiment including 3 independent slice culture dishes (***P < 0.001).(C) CD68 coverage in the old APPPS1 tissue (14 DIV) incubated with conditioned media collected from young WT slices (young CM) or with non-conditioned slice culture media (Ctr). CD68 coverage is increased upon incubation of the APPPS1 tissue with the young CM. The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (**P < 0.01).(D) Immunofluorescence analysis of the old APPPS1 slice cultured alone (old alone) or in co-culture with young GM-CSF-/- brain slices (old co-culture) at 14 DIV and immunostained with CD68 (red) and M3.2 (green) reveals an increased number of core-only plaques upon co-culturing with young GM-CSF-/- brain slices. Scale bar: 50 µm.(E) Quantitative analysis of core-only plaques in the old APPPS1 slice cultured alone or in co-culture with young GM-CSF-/- brain slices (14 DIV) reveals an increased number of core-only plaques upon co-culturing of old APPPS1 brain slices together with young GM-CSF-/- brain slices. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques. The values represent mean ± SEM from 3 independent experiments, each experiment including 3 independent slice culture dishes (***P < 0.001).(F) CD68 coverage in the old APPPS1 tissue (14 DIV) cultured alone or in co-culture with young GM-CSF-/- brain slices. CD68 coverage is increased upon co-culturing of old APPPS1 brain slices together with young GM-CSF-/- brain slices. The values are normalized to CD68 coverage of the old slice cultured alone and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (*P < 0.05).


Figure 9 ( A ) Immunofluorescence analysis of the old APPPS1 slice ( 14 DIV ) treated with GM - CSF and vehicle Ctr and immunostained with CD68 ( red ) , cell proliferation marker Ki67 ( green ) and M3 . 2 ( blue ) . Scale bar : 50 µm . ( B ) Quantitative analysis of Ki67 and CD68 double positive microglial cells in the old APPPS1 tissue ( 14 DIV ) treated with GM - CSF and Ctr reveals increased microglial proliferation upon treatment with GM - CSF . The values are expressed as percentages of CD68 and Ki67 double positive microglial cells from the total number of CD68 positive cells and represent mean ± SEM from 2 independent experiments , each experiment including 4 independent slice culture dishes ( * * * P < 0 . 001 ) . ( C ) Immunofluorescence analysis of the old APPPS1 tissue cultured alone or in co - culture with young WT brain slices ( 14 DIV ) and immunostained with CD68 ( red ) , Ki67 ( green ) and M3 . 2 ( blue ) Scale bar : 50 µm . ( D ) Quantitative analysis of Ki67 and CD68 double positive microglial cells in the old APPPS1 tissue cultured alone or in co - culture with young brain slices ( 14 DIV ) reveals increased microglial proliferation upon co - culturing of old and young brain slices . The values are expressed as percentages of CD68 and Ki67 double positive microglial cells from the total number of CD68 positive cells and represent mean ± SEM from 3 independent experiments , each experiment including 2 independent slice culture dishes ( * * P < 0 . 01 ) . ( E ) Old APPPS1 tissue was incubated with conditioned media collected from young WT slices and in addition treated with proliferation inhibitor AraC ( young CM + AraC ) or vehicle Ctr ( young CM + Ctr ) . Immunofluorescence analysis was performed at 14 DIV using CD68 ( red ) and M3 . 2 ( green ) . Scale bar : 50 µm . ( F ) Quantitative analysis of core - only plaques in the old APPPS1 tissue ( 14 DIV ) incubated with conditioned media from young WT slices and in addition treated with AraC or Ctr reveals a decreased number of core - only plaques upon AraC treatment . The values are expressed as percentages of core - only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments , each including 2 independent slice culture dishes ( * * * P < 0 . 001 ) .

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

Figure 9(A) Immunofluorescence analysis of the old APPPS1 slice (14 DIV) treated with GM-CSF and vehicle Ctr and immunostained with CD68 (red), cell proliferation marker Ki67 (green) and M3.2 (blue). Scale bar: 50 µm.(B) Quantitative analysis of Ki67 and CD68 double positive microglial cells in the old APPPS1 tissue (14 DIV) treated with GM-CSF and Ctr reveals increased microglial proliferation upon treatment with GM-CSF. The values are expressed as percentages of CD68 and Ki67 double positive microglial cells from the total number of CD68 positive cells and represent mean ± SEM from 2 independent experiments, each experiment including 4 independent slice culture dishes (***P < 0.001).(C) Immunofluorescence analysis of the old APPPS1 tissue cultured alone or in co-culture with young WT brain slices (14 DIV) and immunostained with CD68 (red), Ki67 (green) and M3.2 (blue) Scale bar: 50 µm.(D) Quantitative analysis of Ki67 and CD68 double positive microglial cells in the old APPPS1 tissue cultured alone or in co-culture with young brain slices (14 DIV) reveals increased microglial proliferation upon co-culturing of old and young brain slices. The values are expressed as percentages of CD68 and Ki67 double positive microglial cells from the total number of CD68 positive cells and represent mean ± SEM from 3 independent experiments, each experiment including 2 independent slice culture dishes (**P < 0.01).(E) Old APPPS1 tissue was incubated with conditioned media collected from young WT slices and in addition treated with proliferation inhibitor AraC (young CM + AraC) or vehicle Ctr (young CM + Ctr). Immunofluorescence analysis was performed at 14 DIV using CD68 (red) and M3.2 (green). Scale bar: 50 µm.(F) Quantitative analysis of core-only plaques in the old APPPS1 tissue (14 DIV) incubated with conditioned media from young WT slices and in addition treated with AraC or Ctr reveals a decreased number of core-only plaques upon AraC treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, each including 2 independent slice culture dishes (***P < 0.001).


Figure 1A . Hcp release assay . HA - tagged Hcp ( HcpHA ) release was assessed by separating cells ( C ) and cell - free culture supernatant ( S ) fractions from 109 wild - type ( WT ) , ΔtssM cells or ΔtssM cells carrying the AHT - inducible FLAG - tagged tssM - borne plasmid ( tssM + ) treated ( bulgecin ) or not ( NT ) with bulgecin A prior to tssM gene induction . Proteins were separated by 12 . 5 % - acrylamide SDS - PAGE and the periplasmic TolB protein ( control for cell lysis ) , HcpHA and FLTssM were immunodetected using anti - TolB ( middle panel ) , anti - HA ( lower panel ) and anti - FLAG ( upper panel ) antibodies . Molecular weight markers ( in kDa ) are indicated on the left . The experiment was performed in duplicate and a representative result is shown . B . Hcp release assay . HA - tagged Hcp ( HcpHA ) release was assessed by separating cells ( C ) and cell - free culture supernatant ( S ) fractions from 109 wild - type ( WT ) cells before washing cells ( before wash ) and after washing and growth ( after wash ) in absence ( NT ) or presence ( bulgecin ) of bulgecin A . Proteins were separated by 12 . 5 % - acrylamide SDS - PAGE and the periplasmic TolB protein ( control for cell lysis ) and HcpHA were immunodetected using anti - TolB ( upper panel ) and anti - HA ( lower panel ) antibodies . Molecular weight markers ( in kDa ) are indicated on the left . The experiment was performed in duplicate and a representative result is shown .

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

Figure 1A. Hcp release assay. HA-tagged Hcp (HcpHA) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 wild-type (WT), ΔtssM cells or ΔtssM cells carrying the AHT-inducible FLAG-tagged tssM-borne plasmid (tssM+) treated (bulgecin) or not (NT) with bulgecin A prior to tssM gene induction. Proteins were separated by 12.5%-acrylamide SDS-PAGE and the periplasmic TolB protein (control for cell lysis), HcpHA and FLTssM were immunodetected using anti-TolB (middle panel), anti-HA (lower panel) and anti-FLAG (upper panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in duplicate and a representative result is shown.B. Hcp release assay. HA-tagged Hcp (HcpHA) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 wild-type (WT) cells before washing cells (before wash) and after washing and growth (after wash) in absence (NT) or presence (bulgecin) of bulgecin A. Proteins were separated by 12.5%-acrylamide SDS-PAGE and the periplasmic TolB protein (control for cell lysis) and HcpHA were immunodetected using anti-TolB (upper panel) and anti-HA (lower panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in duplicate and a representative result is shown.


Figure 2Hcp release assay . FLAG - tagged Hcp ( HcpFL ) release was assessed by separating cells ( C ) and cell - free culture supernatant ( S ) fractions from 109 cells of the indicated strains . Proteins were separated by 12 . 5 % - acrylamide SDS - PAGE and TolB and HcpFL were immunodetected using anti - TolB ( upper panel ) and anti - FLAG ( lower panel ) antibodies . Molecular weight markers ( in kDa ) are indicated on the left . The experiment was performed in triplicate and a representative result is shown .

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

Figure 2Hcp release assay. FLAG-tagged Hcp (HcpFL) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 cells of the indicated strains. Proteins were separated by 12.5%-acrylamide SDS-PAGE and TolB and HcpFL were immunodetected using anti-TolB (upper panel) and anti-FLAG (lower panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.


Figure 3A . Hcp release assay . FLAG - tagged Hcp ( HcpFL ) release was assessed by separating cells ( C ) and cell - free culture supernatant ( S ) fractions from 109 WT , ΔmltE cells or ΔmltE cells producing wild - type ( mltE + ) or E64Q mutant ( mltEE64Q ) VSV - G - tagged MltE ( MltEV ) from arabinose - inducible plasmids . Proteins were separated by 12 . 5 % - acrylamide SDS - PAGE and TolB , HcpFL and MltEV were immunodetected using anti - TolB ( upper panel ) , anti - FLAG ( middle panel ) and anti - VSV - G ( lower panel ) antibodies , respectively . Molecular weight markers ( in kDa ) are indicated on the left . The experiment was performed in triplicate and a representative result is shown . B . Anti - bacterial activity . E . coli K - 12 prey cells ( W3110 gfp + , kanR ) were mixed with the indicated attacker cells , spotted onto Sci - 1 inducing medium ( SIM ) agar plates and incubated for 4 hours at 37 ° C . The image of a representative bacterial spot and the average and standard deviation ( n = 3 ) of the relative fluorescence of the bacterial mixture ( in arbitrary unit , AU ) are shown in the upper graph . The number of recovered E . coli prey cells ( counted on selective kanamycin medium ) is indicated in the lower graph ( in log10 of colony - forming unit ( cfu ) ) . The black , dark grey and light grey circles indicate values from three independent assays , and the average is indicated by the bar . The experiment was performed in triplicate and a representative result is shown . Asterisks indicate significant differences compared to the wild - type attacker strain ( NS , non significant ; * * * , p < 0 . 001 ) .

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

Figure 3A. Hcp release assay. FLAG-tagged Hcp (HcpFL) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 WT, ΔmltE cells or ΔmltE cells producing wild-type (mltE+) or E64Q mutant (mltEE64Q) VSV-G-tagged MltE (MltEV) from arabinose-inducible plasmids. Proteins were separated by 12.5%-acrylamide SDS-PAGE and TolB, HcpFL and MltEV were immunodetected using anti-TolB (upper panel), anti-FLAG (middle panel) and anti-VSV-G (lower panel) antibodies, respectively. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.B. Anti-bacterial activity. E. coli K-12 prey cells (W3110 gfp+, kanR) were mixed with the indicated attacker cells, spotted onto Sci-1 inducing medium (SIM) agar plates and incubated for 4 hours at 37°C. The image of a representative bacterial spot and the average and standard deviation (n=3) of the relative fluorescence of the bacterial mixture (in arbitrary unit, AU) are shown in the upper graph. The number of recovered E. coli prey cells (counted on selective kanamycin medium) is indicated in the lower graph (in log10 of colony-forming unit (cfu)). The black, dark grey and light grey circles indicate values from three independent assays, and the average is indicated by the bar. The experiment was performed in triplicate and a representative result is shown. Asterisks indicate significant differences compared to the wild-type attacker strain (NS, non significant; ***, p < 0.001).


Figure 4A - B . Bacterial two - hybrid assay . BTH101 reporter cells producing the indicated proteins or domains fused to the T18 or T25 domain of the Bordetella adenylate cyclase were spotted on X - Gal indicator plates . The blue color of the colony reflects the interaction between the two proteins . TolB and Pal are two proteins known to interact but unrelated to the T6SS or the MltE proteins . The experiment was performed in triplicate and a representative result is shown . C . Co - immunoprecipitation assay . The solubilized lysates from 2 × 1010 E . coli K - 12 W3110 cells co - producing the indicated FLAG - tagged TssMP variants ( exported in the periplasm ) and VSV - G - tagged MltE protein ( Total , T ) were subjected to immune precipitation on anti - FLAG - coupled agarose beads . The immunoprecipitated material ( IP ) was subjected to 12 . 5 % - acrylamide SDS - PAGE and immunodetected with anti - FLAG ( upper panel , TssM domains ) and anti - VSV - G ( lower panel , MltE ) antibodies . Molecular weight markers ( in kDa ) are indicated on the left . The experiment was performed in triplicate and a representative result is shown .

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

Figure 4A-B. Bacterial two-hybrid assay. BTH101 reporter cells producing the indicated proteins or domains fused to the T18 or T25 domain of the Bordetella adenylate cyclase were spotted on X-Gal indicator plates. The blue color of the colony reflects the interaction between the two proteins. TolB and Pal are two proteins known to interact but unrelated to the T6SS or the MltE proteins. The experiment was performed in triplicate and a representative result is shown.C. Co-immunoprecipitation assay. The solubilized lysates from 2×1010 E. coli K-12 W3110 cells co-producing the indicated FLAG-tagged TssMP variants (exported in the periplasm) and VSV-G-tagged MltE protein (Total, T) were subjected to immune precipitation on anti-FLAG-coupled agarose beads. The immunoprecipitated material (IP) was subjected to 12.5%-acrylamide SDS-PAGE and immunodetected with anti-FLAG (upper panel, TssM domains) and anti-VSV-G (lower panel, MltE) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.


Figure 5A . Remazol brilliant blue assay . The absorbance of supernatants from the reaction containing purified and RBB - labelled E . coli peptidoglycan and the indicated protein ( 50 µg ) were measured at = 595 nm after incubation for 0 . 5 or 4 hours at 37 ° C . The results shown are the average and standard deviation from triplicate reactions ( n = 3 ) . Asterisks indicate significant differences compared to the buffer ( NS , non significant ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ) . The supernatant of the reaction after 4 hours of incubation is shown on bottom . B . Peptidoglycan hydrolysis . The decrease of the absorbance of the M . luteus peptidoglycan suspension in presence of the indicated protein ( 50 µg ) was measured at = 600 nm at 37 ° C over time . The experiment was performed in triplicate and a representative result is shown .

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

Figure 5A. Remazol brilliant blue assay. The absorbance of supernatants from the reaction containing purified and RBB-labelled E. coli peptidoglycan and the indicated protein (50 µg) were measured at =595 nm after incubation for 0.5 or 4 hours at 37°C. The results shown are the average and standard deviation from triplicate reactions (n=3). Asterisks indicate significant differences compared to the buffer (NS, non significant; **, p < 0.01; ***, p < 0.001). The supernatant of the reaction after 4 hours of incubation is shown on bottom.B. Peptidoglycan hydrolysis. The decrease of the absorbance of the M. luteus peptidoglycan suspension in presence of the indicated protein (50 µg) was measured at =600 nm at 37°C over time. The experiment was performed in triplicate and a representative result is shown.


Figure 6A - B . Co - immunoprecipitation assay . The solubilized lysates from 2 × 1010 EAEC wild - type or ∆ mltE cells producing FLAG - tagged TssM ( FLTssM ) and / or HA - tagged TssJ ( TssJHA , panel A ) or TssL ( TssLHA , panel B ) ( Total , T ) were subjected to immune precipitation on anti - FLAG - coupled agarose beads . The immunoprecipitated material ( IP ) was subjected to 12 . 5 % - acrylamide SDS - PAGE and immunodetected with anti - FLAG ( upper panel , TssM ) and anti - HA ( lower panel , TssJ or TssL ) antibodies . Molecular weight markers ( in kDa ) are indicated on the left . The experiments were performed in triplicate and a representative result is shown . C . BS3 cross - linking assay . 2 × 109 cells of the indicated strain producing FLAG - tagged TssM ( with the exception of ∆ tssM cells ) were treated ( + ) or not ( - ) with the BS3 cross - linker agent . After the cross - linking reaction , cells were boiled in Laemmli buffer and total proteins were subjected to 7 % - acrylamide SDS - PAGE and immunodetected with anti - FLAG antibodies . The TssM protein ( FLTssM ) and its complexes ( * , TssM - TssJ ; * * , TssM - TssL ) are indicated on the right as well as the TssM dimer ( arrow ) . Molecular weight markers ( in kDa ) are indicated on the left . The experiment was performed in triplicate and a representative result is shown . D . Fluorescence microscopy . Recordings showing TssM localization using the chromosomally - encoded sfGFP - tssM fusion in wild - type ( WT ) or ΔmltE cells or ΔmltE cells producing the wild - type ( mltE + ) or catalytic variant ( mltEE64Q ) MltE protein . Scale bars are 1 μm . The experiment was performed in triplicate and a representative result is shown .

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

Figure 6A-B. Co-immunoprecipitation assay. The solubilized lysates from 2×1010 EAEC wild-type or ∆mltE cells producing FLAG-tagged TssM (FLTssM) and/or HA-tagged TssJ (TssJHA, panel A) or TssL (TssLHA, panel B) (Total, T) were subjected to immune precipitation on anti-FLAG-coupled agarose beads. The immunoprecipitated material (IP) was subjected to 12.5%-acrylamide SDS-PAGE and immunodetected with anti-FLAG (upper panel, TssM) and anti-HA (lower panel, TssJ or TssL) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiments were performed in triplicate and a representative result is shown.C. BS3 cross-linking assay. 2×109 cells of the indicated strain producing FLAG-tagged TssM (with the exception of ∆tssM cells) were treated (+) or not (-) with the BS3 cross-linker agent. After the cross-linking reaction, cells were boiled in Laemmli buffer and total proteins were subjected to 7%-acrylamide SDS-PAGE and immunodetected with anti-FLAG antibodies. The TssM protein (FLTssM) and its complexes (*, TssM-TssJ; **, TssM-TssL) are indicated on the right as well as the TssM dimer (arrow). Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.D. Fluorescence microscopy. Recordings showing TssM localization using the chromosomally-encoded sfGFP-tssM fusion in wild-type (WT) or ΔmltE cells or ΔmltE cells producing the wild-type (mltE+) or catalytic variant (mltEE64Q) MltE protein. Scale bars are 1 μm. The experiment was performed in triplicate and a representative result is shown.


Figure 1B Ub chain interaction of the LUBEL - NZF and UBA2 domains . Immobilized GST - NZF or GST - UBA2 was incubated with Lys ( K ) 48 - , K 63 - linked , or linear di - Ub ( Ub2 ) chains and bound Ub chains were subjected to immunoblotting using anti - Ub antibody or a linear linkage - specific Ub antibody ( anti - linear Ub antibody ) . Loading of GST - fusion proteins was visualized by Ponceau S staining .

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

Figure 1B Ub chain interaction of the LUBEL-NZF and UBA2 domains. Immobilized GST-NZF or GST-UBA2 was incubated with Lys (K) 48-, K 63-linked, or linear di-Ub (Ub2) chains and bound Ub chains were subjected to immunoblotting using anti-Ub antibody or a linear linkage-specific Ub antibody (anti-linear Ub antibody). Loading of GST-fusion proteins was visualized by Ponceau S staining.