a , Confocal micrograph of HeLa cells expressing GFP - LC3 and mCherry - galectin - 8 , stained for NDP52 1 h after infection with S . Typhimurium . The lower right panel contains a fluorescence line scan along the yellow line in the merge inset . Arrowheads , bacteria shown in insets .

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a, Confocal micrograph of HeLa cells expressing GFP-LC3 and mCherry-galectin-8, stained for NDP52 1 h after infection with S. Typhimurium. The lower right panel contains a fluorescence line scan along the yellow line in the merge inset. Arrowheads, bacteria shown in insets.


b , Percentage of GFP - LC3 - positive S . Typhimurium at 1 h p . i . in HeLa cells treated with the indicated siRNAs .

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b, Percentage of GFP-LC3-positive S. Typhimurium at 1 h p.i. in HeLa cells treated with the indicated siRNAs.


c , Percentage of bacteria positive for NDP52 . HeLa cells expressing the indicated NDP52 variants fused to YFP were infected with S . Typhimurium .

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c, Percentage of bacteria positive for NDP52. HeLa cells expressing the indicated NDP52 variants fused to YFP were infected with S. Typhimurium.


d , Percentage of bacteria positive for the indicated markers . HeLa cells , either wild type or expressing YFP - galectin - 8 as indicated , were infected with S . Typhimurium . Ubiquitin was detected by antibody staining .

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d, Percentage of bacteria positive for the indicated markers. HeLa cells, either wild type or expressing YFP-galectin-8 as indicated, were infected with S. Typhimurium. Ubiquitin was detected by antibody staining.


e , HeLa cells treated with the indicated siRNAs were infected with S . Typhimurium and stained for NDP52 at the indicated time points . Fluorescent bacteria were counted by microscopy at the indicated time points . Graphs , representing at least two independent repeats , show mean and s . d . of duplicate coverslips for which > 200 bacteria were counted . * P 0 . 05 , Student ' s t - test . Scale bar , 10 µm .

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e, HeLa cells treated with the indicated siRNAs were infected with S. Typhimurium and stained for NDP52 at the indicated time points. Fluorescent bacteria were counted by microscopy at the indicated time points. Graphs, representing at least two independent repeats, show mean and s.d. of duplicate coverslips for which >200 bacteria were counted. *P  0.05, Student's t-test. Scale bar, 10 µm.


( A ) Representative transmission electron microscope ( TEM ) image showing the presence of EVs in the CSF in two independent experiments .

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(A) Representative transmission electron microscope (TEM) image showing the presence of EVs in the CSF in two independent experiments.


( B ) NanoSightquantification of the amount of particles in the CSF 0 , 1 , 2 , 4 and 6 hours after i . p . LPS injection ( n = 3 - 5 ) .

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(B) NanoSightquantification of the amount of particles in the CSF 0, 1, 2, 4 and 6 hours after i.p. LPS injection (n=3-5).


( C ) Size distribution of the EVsin vivo in the CSF before ( black ; n = 5 ) and 6 h after ( grey ; n = 3 ) LPS treatment determined by NanoSight analysis .

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(C) Size distribution of the EVsin vivo in the CSF before (black; n=5) and 6 h after (grey; n=3) LPS treatment determined by NanoSight analysis.


( D - G ) Quantitative real - time polymerase chain reaction analysis of miR - 1a ( D ) , miR - 9 ( E ) , miR - 146a ( F ) , and miR - 155 ( G ) ( n = 4 ) . RNA was isolated from pooled CSF ( 50 µl ) from different mice ( n = 3 ) . Data are displayed as mean ± SEM and analyzed by Student ' s t - test . Significance levels are indicated on the graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 .

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(D-G) Quantitative real-time polymerase chain reaction analysis of miR-1a (D), miR-9 (E), miR-146a (F), and miR-155 (G) (n=4). RNA was isolated from pooled CSF (50 µl) from different mice (n=3). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.


( A - B ) In vitro quantification ( A ) and size distribution ( B ) of EVs isolated from conditioned medium of primary CPE cells grown in transwell system after 12 h in the absence ( black ) or presence ( grey ) of LPS ( n = 5 ) determined by NanoSight analysis .

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(A-B) In vitro quantification (A) and size distribution (B) of EVs isolated from conditioned medium of primary CPE cells grown in transwell system after 12 h in the absence (black) or presence (grey) of LPS (n=5) determined by NanoSight analysis.


( C - E ) TaqMan qPCR assay for the quantification of miR - 9 ( C ) , miR - 146a ( D ) and miR - 155 ( E ) on the exosomal pellet isolated from conditioned medium of primary CPE cells grown in a transwell system and stimulated for 12 h with LPS ( n = 3 ) .

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(C-E) TaqMan qPCR assay for the quantification of miR-9 (C), miR-146a (D) and miR-155 (E) on the exosomal pellet isolated from conditioned medium of primary CPE cells grown in a transwell system and stimulated for 12 h with LPS (n=3).


( F - I ) Quantification of the miRNAs miR - 1a ( F ) , miR - 9 ( G ) , miR - 146a ( H ) and miR - 155 ( I ) by TaqMan qPCR assay from primary CPE cells grown in a transwell system without or with LPS stimulation ( n = 3 ) . Significance levels are indicated on the graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 .

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(F-I) Quantification of the miRNAs miR-1a (F), miR-9 (G), miR-146a (H) and miR-155 (I) by TaqMan qPCR assay from primary CPE cells grown in a transwell system without or with LPS stimulation (n=3). Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.


( A ) In vitro quantification of EVs isolated from conditioned medium of LPS - stimulated primary CPE cells grown in a transwell system in the absence or presence of the exosome inhibitor GW4869 ( n = 3 ) .

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(A) In vitro quantification of EVs isolated from conditioned medium of LPS-stimulated primary CPE cells grown in a transwell system in the absence or presence of the exosome inhibitor GW4869 (n=3).


( B - D ) TaqMan assay quantification of the miRNAs miR - 9 ( B ) , miR - 146a ( C ) and miR - 155 ( D ) in supernatant of LPS - stimulated primary CPE cells grown in a transwell system and either left untreated or pretreated with GW4869 to inhibit exosome secretion ( n = 3 ) . miR - 1a levels were below detection limit .

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(B-D) TaqMan assay quantification of the miRNAs miR-9 (B), miR-146a (C) and miR-155 (D) in supernatant of LPS-stimulated primary CPE cells grown in a transwell system and either left untreated or pretreated with GW4869 to inhibit exosome secretion (n=3). miR-1a levels were below detection limit.


( E - H ) TaqMan assay quantification of the miRNAs miR - 1a ( E ) , miR - 9 ( F ) , miR - 146a ( G ) and miR - 155 ( H ) in cell lysate of LPS - stimulated primary CPE cells grown in a transwell system left untreated or treated with GW4869 to inhibit exosome secretion ( n = 3 ) . Data are displayed as mean ± SEM and analyzed by Student ' s t - test . Significance levels are indicated on the graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 .

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(E-H) TaqMan assay quantification of the miRNAs miR-1a (E), miR-9 (F), miR-146a (G) and miR-155 (H) in cell lysate of LPS-stimulated primary CPE cells grown in a transwell system left untreated or treated with GW4869 to inhibit exosome secretion (n=3). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.


( A ) Representative confocal images of CD63 , RAB5 and ANXA2 ( red ) in the choroid plexus ( CP ) 0 , 4 and 8 h after LPS treatment . Hoechst ( blue ) was used to stain the nucleus . The dotted line indicates the ependymal cells that line the ventricle and the square boxes indicate the zoomed insert images displayed at the right corner of each image . Scalebar = 100 µm .

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(A) Representative confocal images of CD63, RAB5 and ANXA2 (red) in the choroid plexus (CP) 0, 4 and 8 h after LPS treatment. Hoechst (blue) was used to stain the nucleus. The dotted line indicates the ependymal cells that line the ventricle and the square boxes indicate the zoomed insert images displayed at the right corner of each image. Scalebar = 100 µm.


( B - C ) Representative TEM images showing the presence of MVBs in the CPE cells before ( B ) and 6 hours after ( C ) LPS administration in vivo . White arrow heads point to exosomes present in MVBs ( n = 3 ) . Scale bar = 9 µm .

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(B-C) Representative TEM images showing the presence of MVBs in the CPE cells before (B) and 6 hours after (C) LPS administration in vivo. White arrow heads point to exosomes present in MVBs (n=3). Scale bar = 9 µm.


( D - F ) Quantification of amount of MVBs per cell section ( D ) , amount of exosomes per MVB ( E ) , and amount of exosomes per cell section ( F ) , based on TEM analysis .

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(D-F) Quantification of amount of MVBs per cell section (D), amount of exosomes per MVB (E), and amount of exosomes per cell section (F), based on TEM analysis.


( G - J ) Quantitative real - time polymerase chain reaction ( qPCR ) analysis of miR - 1a ( G ) , miR - 9 ( H ) , miR - 146a ( I ) , and miR - 155 ( J ) . Data are presented as relative expression normalized with housekeeping miRs by TaqMan qPCR assay ( 0 h , n = 4 ; 1 h , n = 5 ; 6 h , n = 5 ; 24 h , n = 3 ) .

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(G-J) Quantitative real-time polymerase chain reaction (qPCR) analysis of miR-1a (G), miR-9 (H), miR-146a (I), and miR-155 (J). Data are presented as relative expression normalized with housekeeping miRs by TaqMan qPCR assay (0 h, n=4; 1 h, n=5; 6 h, n=5; 24 h, n=3).


( K ) NanoSight analysis of CSF isolated from LPS - injected mice followed by icv injection of vehicle or GW4869 , a neutral sphingomyelinase inhibitor that inhibits exosome secretion ( n = 8 ) .

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(K) NanoSight analysis of CSF isolated from LPS-injected mice followed by icv injection of vehicle or GW4869, a neutral sphingomyelinase inhibitor that inhibits exosome secretion (n = 8).


( L ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the choroid plexus of mice injected with LPS and then icv injected with vehicle ( black ) or GW4869 ( grey ) , a neutral sphingomyelinase inhibitor that inhibits exosome secretion ( n = 4 ) .

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(L) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the choroid plexus of mice injected with LPS and then icv injected with vehicle (black) or GW4869 (grey), a neutral sphingomyelinase inhibitor that inhibits exosome secretion (n = 4).


( M ) NanoSight analysis of supernatant of choroid plexus explants from PBS - or LPS - injected mice ( n = 6 ) . Data are displayed as mean ± SEM and analyzed by Student ' s t - test . Significance levels are indicated on the graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 ; * * * , 0 . 0001 ≤ P < 0 . 001 .

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(M) NanoSight analysis of supernatant of choroid plexus explants from PBS- or LPS-injected mice (n = 6). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01; ***, 0.0001 ≤ P < 0.001.


( A - B ) Representative confocal images of choroid plexus ( CP ) on brain sections from naive mice and four hours after LPS injection ( n = 3 ) . Brain sections were stained for TLR4 ( A ) and TNFR1 ( B ) ( red ) , pan - cytokeratine ( panCK ; green ) and the nuclei were stained with Hoechst ( blue ) . The ependymal cells aligning the ventricles are marked with a dotted line . Scalebar = 100 µm .

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(A-B) Representative confocal images of choroid plexus (CP) on brain sections from naive mice and four hours after LPS injection (n=3). Brain sections were stained for TLR4 (A) and TNFR1 (B) (red), pan-cytokeratine (panCK; green) and the nuclei were stained with Hoechst (blue). The ependymal cells aligning the ventricles are marked with a dotted line. Scalebar = 100 µm.


( C ) NanoSight analysis of EVs in the CSF in sham - operated ( black ) relative to CLP - treated ( grey ) mice 10 hours after surgery .

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(C) NanoSight analysis of EVs in the CSF in sham-operated (black) relative to CLP-treated (grey) mice 10 hours after surgery.


( D ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the choroid plexus of sham - operated mice ( black ) and mice subjected to CLP ( grey ) 10 hours after surgery . ( E ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the CSF of sham - operated mice ( black ) and mice subjected to CLP ( grey ) 10 hours after surgery .

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(D) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the choroid plexus of sham-operated mice (black) and mice subjected to CLP (grey) 10 hours after surgery. (E) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the CSF of sham-operated mice (black) and mice subjected to CLP (grey) 10 hours after surgery.


( F ) NanoSight analysis of EVs in CSF of control ( black ) and TNF - injected ( 25 µg / 20 g ; grey ) mice 6 hours after TNF challenge .

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(F) NanoSight analysis of EVs in CSF of control (black) and TNF-injected (25 µg/20 g; grey) mice 6 hours after TNF challenge.


( G ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the choroid plexus of control mice ( black ) and in mice injected with TNF ( grey ) 6 hours after injection . ( H ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the CSF of control mice ( black ) and on mice 6 hours after TNF injection ( grey ) . Data are displayed as mean ± SEM and analyzed by Student ' s t - test . Scalebar 100 µm . Significance levels are indicated on the graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 ; * * * , 0 . 0001 ≤ P < 0 . 001 .

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(G) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the choroid plexus of control mice (black) and in mice injected with TNF (grey) 6 hours after injection. (H) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the CSF of control mice (black) and on mice 6 hours after TNF injection (grey). Data are displayed as mean ± SEM and analyzed by Student's t-test. Scalebar 100 µm. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01; ***, 0.0001 ≤ P < 0.001.


( A ) Representative confocal image of brain parenchyma , four hours after intracerebroventricular injection of PKH26 labelled EVs ( red ) . Astrocytes are stained with GFAP ( white ) , nuclei with Hoechst ( blue ) , and pan - cytokeratin ( panCK ; green ) was used to label the choroid plexus . The dotted line shows the ventricular border and the white arrow heads point to EVs that crossed the ependymal cell layer .

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(A) Representative confocal image of brain parenchyma, four hours after intracerebroventricular injection of PKH26 labelled EVs (red). Astrocytes are stained with GFAP (white), nuclei with Hoechst (blue), and pan-cytokeratin (panCK; green) was used to label the choroid plexus. The dotted line shows the ventricular border and the white arrow heads point to EVs that crossed the ependymal cell layer.


( B - C ) Representative confocal images showing the uptake of double - labelled EVs ( Ribogreen ; green , PKH26 , red ) by astrocytes stained with GFAP ( white ) ( B ) and microglia cells stained with IBA1 ( white ) ( C ) incubated on mixed cortical cultures . Boxed areas are shown as zoomed images on right . Cell nuclei are stained with Hoechst ( blue ) . The in vivo and in vitro uptake experiments were performed 3 and 2 times ( n = 3 ) , respectively . Scalebar 100 = µm .

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(B-C) Representative confocal images showing the uptake of double-labelled EVs (Ribogreen; green, PKH26, red) by astrocytes stained with GFAP (white) (B) and microglia cells stained with IBA1 (white) (C) incubated on mixed cortical cultures. Boxed areas are shown as zoomed images on right. Cell nuclei are stained with Hoechst (blue). The in vivo and in vitro uptake experiments were performed 3 and 2 times (n=3), respectively. Scalebar 100 = µm.


( A - B ) qPCR gene expression analysis of miRNA target genes Mapk3 , Notch1 , Dicer , Tab2 , Sox2 , Calm2 , Smad2 , Smad5 , Dnmt3 and Irak1 in mixed cortical cultures incubated with EVs isolated from CSF from untreated ( black ) or LPS treated ( grey ) mice ( A ) and in brain tissue ( B ) before ( black ) and 8 h after LPS injection ( grey ) ( n = 3 ) .

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(A-B) qPCR gene expression analysis of miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 in mixed cortical cultures incubated with EVs isolated from CSF from untreated (black) or LPS treated (grey) mice (A) and in brain tissue (B) before (black) and 8 h after LPS injection (grey) (n=3).


( C ) qPCR gene expression analysis of inflammatory genes Il - 1β , Tnf , Il - 6 , iNos and iκbα by qPCR in mixed cortical cultures ( left ; in vitro ) incubated with EVs isolated from CSF from untreated ( black ) or LPS treated ( grey ) mice and in brain tissue ( right ; in vivo ) before ( black ) and 8 h after LPS injection ( grey ) ( n = 3 ) .

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(C) qPCR gene expression analysis of inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα by qPCR in mixed cortical cultures (left; in vitro) incubated with EVs isolated from CSF from untreated (black) or LPS treated (grey) mice and in brain tissue (right; in vivo) before (black) and 8 h after LPS injection (grey) (n=3).


( D ) Cytokine analysis ( IL - 6 , IL - 1β and TNF ) of supernatant of mixed cortical cultures ( left ; in vitro ) incubated with EVs isolated from untreated ( black ; n = 3 ) or LPS treated ( grey ; n = 3 ) mice and of CSF ( right ; in vivo ) from untreated ( black ; n = 3 ) and LPS treated mice ( grey ; n = 6 ) .

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(D) Cytokine analysis (IL-6, IL-1β and TNF) of supernatant of mixed cortical cultures (left; in vitro) incubated with EVs isolated from untreated (black; n=3) or LPS treated (grey; n=3) mice and of CSF (right; in vivo) from untreated (black; n=3) and LPS treated mice (grey; n=6).


( E - F ) qPCR gene expression analysis of inflammatory genes Il - 1β , Tnf , Il - 6 , iNos and iκbα ( E ) and miRNA target genes Mapk3 , Notch1 , Dicer , Tab2 , Sox2 , Calm2 , Smad2 , Smad5 , Dnmt3 and Irak1 ( F ) in mixed cortical cultures incubated with CD63 - depleted EVs isolated from untreated mice ( black ) and mice treated with LPS for 6 h ( grey ) ( n = 3 ) .

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(E-F) qPCR gene expression analysis of inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα (E) and miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 (F) in mixed cortical cultures incubated with CD63-depleted EVs isolated from untreated mice (black) and mice treated with LPS for 6 h (grey) (n=3).


( G - H ) qPCR gene expression analysis of miRNA target genes Mapk3 , Notch1 , Dicer , Tab2 , Sox2 , Calm2 , Smad2 , Smad5 , Dnmt3 and Irak1 ( G ) and inflammatory genes Il - 1β , Tnf , Il - 6 , iNos and iκbα ( H ) in brain tissue from LPS - injected mice icv injected with vehicle ( black ) or GW4869 ( grey ; n = 7 ) . Data are displayed as mean ± SEM and analyzed by Student ' s t - test . Significance levels are indicated on the graphs : * , 0 . 01 ≤ P < 0 . 05 .

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(G-H) qPCR gene expression analysis of miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 (G) and inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα (H) in brain tissue from LPS-injected mice icv injected with vehicle (black) or GW4869 (grey; n=7). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05.


A ) Double fluorescence in situ hybridization using Nrg3 ( green ) and Gad1 ( red ) specific probes ; DAPI was used as a counter stain . Nrg3 is present in Gad1 - positive ( filled arrowheads ) and - negative neurons . ( A ' ) and ( A ' ' ) show higher magnifications of boxed areas indicated in ( A ) . Nrg3 - negative neurons are indicated by open arrowheads ; note that many but not all neurons express Nrg3 .

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A) Double fluorescence in situ hybridization using Nrg3 (green) and Gad1 (red) specific probes; DAPI was used as a counter stain. Nrg3 is present in Gad1-positive (filled arrowheads) and -negative neurons. (A') and (A'') show higher magnifications of boxed areas indicated in (A). Nrg3-negative neurons are indicated by open arrowheads; note that many but not all neurons express Nrg3.


B ) Comparison of mRNA levels of Nrg3 , Nrg1β type III ( Nrg1β − III ) , Nrg2β , Nrg1β type II ( Nrg1β − II ) , Nrg2α and Nrg1β type I ( Nrg1β − I ) in the cortex ( Cx ) , hippocampus ( HC ) and striatum ( Str ) of juvenile mice ( P30 - 60 ) by quantitative RT - PCR . Data represent means ± SD of 4 biological replicates . 2 - way - ANOVA with Bonferroni ' s multiple comparisons test was performed to assess statistical significance ( * * * * P < 0 . 0001 , * * * P < 0 . 001 , ns = not significant ) .

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B) Comparison of mRNA levels of Nrg3, Nrg1β type III (Nrg1β−III), Nrg2β, Nrg1β type II (Nrg1β−II), Nrg2α and Nrg1β type I (Nrg1β−I) in the cortex (Cx), hippocampus (HC) and striatum (Str) of juvenile mice (P30-60) by quantitative RT-PCR. Data represent means ± SD of 4 biological replicates. 2-way-ANOVA with Bonferroni's multiple comparisons test was performed to assess statistical significance (****P<0.0001, ***P<0.001, ns=not significant).


D ) Detection of Nrg3 by western blot analysis in fractions from brain lysates : S1 , crude lysate ; S2 , cytosol and light membranes ; S2 ' , cytosol ; LM , light membranes ; P2 ' , crude synaptosomes ; S3 ' , synaptosomal cytoplasm ; SV , synaptic vesicles ; P3 , synaptosomal membranes ; SPM , synaptic plasma membranes .

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D) Detection of Nrg3 by western blot analysis in fractions from brain lysates: S1, crude lysate; S2, cytosol and light membranes; S2', cytosol; LM, light membranes; P2', crude synaptosomes; S3', synaptosomal cytoplasm; SV, synaptic vesicles; P3, synaptosomal membranes; SPM, synaptic plasma membranes.


E , F ) Immunohistochemical analysis in the hippocampus of wildtype ( E , E ' , E ' ' ) and ErbB4 mutant mice ( F ) at 2 month of age using Nrg3 - , ErbB4 - and parvalbumin ( Parv ) - specific antibodies . The white box in ( E ) is displayed magnified in ( E ' , E ' ' ) . In control mice , Nrg3 is enriched on dendrites of ErbB4 + PV interneurons , compared to the surrounding neuropil ( E ' , E ' ' ) . In ErbB4 mutants , the Nrg3 enrichment on PV interneurons is not apparent ( F ) .

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E, F) Immunohistochemical analysis in the hippocampus of wildtype (E,E',E'') and ErbB4 mutant mice (F) at 2 month of age using Nrg3-, ErbB4- and parvalbumin (Parv)-specific antibodies. The white box in (E) is displayed magnified in (E',E''). In control mice, Nrg3 is enriched on dendrites of ErbB4+ PV interneurons, compared to the surrounding neuropil (E',E''). In ErbB4 mutants, the Nrg3 enrichment on PV interneurons is not apparent (F).


G ) Nrg3 , ErbB4 and GluA4 were identified by immunohistology and co - localized in synaptic puncta on dendrites of interneurons in the stratum radiatum of the hippocampal CA1 . Note that ( G ) displays Nrg3 / ErbB4 , ( G ' ) Nrg3 / GluA4 , and ( G ' ' ) ErbB4 / GluA4 signals of the same triple - stained image ; false colors were assigned for better signal visualization .

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G) Nrg3, ErbB4 and GluA4 were identified by immunohistology and co-localized in synaptic puncta on dendrites of interneurons in the stratum radiatum of the hippocampal CA1. Note that (G) displays Nrg3/ErbB4, (G') Nrg3/GluA4, and (G'') ErbB4/GluA4 signals of the same triple-stained image; false colors were assigned for better signal visualization.


H ) Quantification of GluA4 puncta present on ErbB4 + dendrites in the CA1 stratum radiatum of adult wildtype ( wt ) and Nrg3 - / - mice ( age P90 - 120 ) . Data are presented as box plots with Tukey ' s whiskers and outliers ; means are indicated by a Plus symbols . n = 65 ( wt ) and n = 62 ( Nrg3 - / - ) from 5 animals each . Unpaired t - test ( 2 - tailed ) with Welch ' s correction was performed to assess statistical significance ( * * * * P < 0 . 0001 ) .

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H) Quantification of GluA4 puncta present on ErbB4+ dendrites in the CA1 stratum radiatum of adult wildtype (wt) and Nrg3-/- mice (age P90-120). Data are presented as box plots with Tukey's whiskers and outliers; means are indicated by a Plus symbols. n=65 (wt) and n=62 (Nrg3-/-) from 5 animals each. Unpaired t-test (2-tailed) with Welch's correction was performed to assess statistical significance (****P<0.0001).


A ) His6 - tagged EGF domains of Nrg1β and Nrg3 were added to neuronal cultures at the indicated concentrations . Western blot analyses of ErbB4 immunoprecipitates using antibodies against phospho - tyrosine ( pY ) and ErbB4 , and of whole lysates using antibodies against pErk1 / 2 and pAkt

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A) His6-tagged EGF domains of Nrg1β and Nrg3 were added to neuronal cultures at the indicated concentrations. Western blot analyses of ErbB4 immunoprecipitates using antibodies against phospho-tyrosine (pY) and ErbB4, and of whole lysates using antibodies against pErk1/2 and pAkt


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C , D ) Neurons from Nrg3 - / - ; ErbB4 - / - ; vGAT - Cre mice were transduced with a lentivirus expressing either ( C ) ErbB4wt or ( D ) ErbB4kd ( kinase dead ErbB4 ) in a Cre - dependent manner , and with a second virus at a low titer expressing Nrg3 / SypCFP . Immunocytochemical analysis of ErbB4 and SypCFP ; ( C , C ' ) and ( D , D ' ) each show the same image , ( C ' , D ' ) show the SypCFP signal

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C, D) Neurons from Nrg3-/-;ErbB4-/-;vGAT-Cre mice were transduced with a lentivirus expressing either (C) ErbB4wt or (D) ErbB4kd (kinase dead ErbB4) in a Cre-dependent manner, and with a second virus at a low titer expressing Nrg3/SypCFP. Immunocytochemical analysis of ErbB4 and SypCFP; (C,C') and (D,D') each show the same image, (C',D') show the SypCFP signal


E - G ) Quantification of Synapse preference towards ErbB4 + neurons ( E ) , ErbB4 enrichment ( F ) and SypCFP enrichment ( G ) in synapses on neurons expressing wildtype ErbB4 ( wt ) or ErbB4kd ( kd )

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E-G) Quantification of Synapse preference towards ErbB4+ neurons (E), ErbB4 enrichment (F) and SypCFP enrichment (G) in synapses on neurons expressing wildtype ErbB4 (wt) or ErbB4kd (kd)


H ) Nrg3 - / - neuron cultures were transfected with a plasmid that strongly overexpresses Nrg3 and GFP and immunostained with antibodies against ErbB4 , GFP and GluA ; ( H ) shows the triple stained image , while ( H ' ) and ( H ' ' ) only show ErbB4 and GluA signals , respectively

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H) Nrg3-/- neuron cultures were transfected with a plasmid that strongly overexpresses Nrg3 and GFP and immunostained with antibodies against ErbB4, GFP and GluA; (H) shows the triple stained image, while (H') and (H'') only show ErbB4 and GluA signals, respectively


I ) Co - cultures of Nrg3 - / - neurons with HEK293 cells expressing Nrg3 . Cultures were stained with antibodies against Nrg3 , ErbB4 and GluA . Note that Nrg3 , ErbB4 and GluA are enriched at the contact sites between Nrg3 - expressing HEK293 cells and ErbB4 + neurons

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I) Co-cultures of Nrg3-/- neurons with HEK293 cells expressing Nrg3. Cultures were stained with antibodies against Nrg3, ErbB4 and GluA. Note that Nrg3, ErbB4 and GluA are enriched at the contact sites between Nrg3-expressing HEK293 cells and ErbB4+ neurons


C ) Example traces of evoked excitatory postsynaptic currents ( eEPSCs ) averaged over 50 sweeps . D , E ) Quantification of the peak amplitude of the first eEPSC ( D ) and of the paired pulse ratio ( E )

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C) Example traces of evoked excitatory postsynaptic currents (eEPSCs) averaged over 50 sweeps. D, E) Quantification of the peak amplitude of the first eEPSC (D) and of the paired pulse ratio (E)


F - I ) sEPSC recordings from CA1 pyramidal ( F , H ) and PV neurons ( G , I ) in acute slices . sEPSC frequencies ( F , G ) and amplitudes ( H , I ) are

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F-I) sEPSC recordings from CA1 pyramidal (F,H) and PV neurons (G, I) in acute slices. sEPSC frequencies (F,G) and amplitudes (H,I) are shown


TRF1 immunofluorescence of the lungs . Notice the absence and presence of TRF1 signal in the carcinomas and surrounding healthy tissue of Trf1Δ / Δ mice , respectively .

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TRF1 immunofluorescence of the lungs. Notice the absence and presence of TRF1 signal in the carcinomas and surrounding healthy tissue of Trf1Δ/Δ mice, respectively.


Analysis of Trf1 excision by PCR . Notice the completed excision in carcinomas of Trf1lox / lox lungs .

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Analysis of Trf1 excision by PCR. Notice the completed excision in carcinomas of Trf1lox/lox lungs.


Detection of β - galactosidase activity in the lungs as a surrogate marker of oncogenic K - RasG12V expression .

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Detection of β-galactosidase activity in the lungs as a surrogate marker of oncogenic K-RasG12V expression.


Tumor growth curve of Trf1 + / + K - Ras + / G12Vp53 + / + and Trf1Δ / ΔK - Ras + / G12Vp53 + / + measured by computed tomography ( CT ) . Quantification of the number and size of Trf1 + / + K - Ras + / G12Vp53 + / + and Trf1Δ / ΔK - Ras + / G12Vp53 + / + carcinomas at death point .

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Tumor growth curve of Trf1+/+K-Ras+/G12Vp53+/+ and Trf1Δ/ΔK-Ras+/G12Vp53+/+ measured by computed tomography (CT).Quantification of the number and size of Trf1+/+K-Ras+/G12Vp53+/+ and Trf1Δ/ΔK-Ras+/G12Vp53+/+carcinomas at death point.


Percentage of tumors that have deleted Trf1 quantified by TRF1 immunofluorescence after mice had been sacrificed . Post - mortem analysis of Trf1 deletion in each tumor revealed that none of the Trf1lox / lox K - Ras + / G12V p53 + / + ones had excised Trf1 .

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Percentage of tumors that have deleted Trf1 quantified by TRF1 immunofluorescence after mice had been sacrificed. Post-mortem analysis of Trf1 deletion in each tumor revealed that none of the Trf1lox/lox K-Ras+/G12V p53+/+ ones had excised Trf1.


Tumor growth curve and tumor growth slope of Trf1 + / + K - Ras + / G12V p53 − / − and Trf1 − / − K - Ras + / G12V p53 − / − measured by CT .

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Tumor growth curve and tumor growth slope of Trf1+/+ K-Ras+/G12V p53−/− and Trf1−/− K-Ras+/G12V p53−/− measured by CT.


Tumor maximal section of Trf1 + / + K - Ras + / G12Vp53 − / − and Trf1Δ / ΔK - Ras + / G12Vp53 − / − lungs measured by histological analysis before death point by CT .

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Tumor maximal section of Trf1+/+K-Ras+/G12Vp53−/− and Trf1Δ/ΔK-Ras+/G12Vp53−/−lungs measured by histological analysis before death point by CT.


Maximum 18F - FDG - glucose uptake by Trf1 + / + K - Ras + / G12Vp53 − / − and Trf1Δ / ΔK - Ras + / G12Vp53 − / − tumors 22 weeks after infection by positron emission tomography ( PET ) . Representative PET - CT image of Trf1 + / + K - Ras + / G12Vp53 − / − and Trf1Δ / ΔK - Ras + / G12Vp53 − / − lungs .

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Maximum 18F-FDG-glucose uptake by Trf1+/+K-Ras+/G12Vp53−/− and Trf1Δ/ΔK-Ras+/G12Vp53−/−tumors 22 weeks after infection by positron emission tomography (PET).Representative PET-CT image of Trf1+/+K-Ras+/G12Vp53−/− and Trf1Δ/ΔK-Ras+/G12Vp53−/−lungs.


Survival curve of Trf1 + / + K - Ras + / G12V p53 − / − and Trf1 − / − K - Ras + / G12V p53 − / − mice .

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Survival curve of Trf1+/+ K-Ras+/G12V p53−/− and Trf1−/− K-Ras+/G12V p53−/− mice.


Percentage of cells showing γH2AX foci in carcinomas of the indicated genotypes ( left panel ) . Representative images of γH2AX immunohistochemistry ( right panel ) .

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Percentage of cells showing γH2AX foci in carcinomas of the indicated genotypes (left panel). Representative images of γH2AX immunohistochemistry (right panel).


Percentage of cells showing 3 or more γH2AX and RAP1 colocalizing foci ( TIFs ) ( left panel ) . Representative images of γH2AX and RAP1 double immunofluorescence ( right panel ) . Yellow arrowheads : colocalization of γH2AX and RAP1 .

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Percentage of cells showing 3 or more γH2AX and RAP1 colocalizing foci (TIFs) (left panel). Representative images of γH2AX and RAP1 double immunofluorescence (right panel). Yellow arrowheads: colocalization of γH2AX and RAP1.


Percentage of active caspase - 3 ( AC3 ) - positive cells ( left panel ) . Representative images of AC3 immunohistochemistry ( right panel ) .

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Percentage of active caspase-3 (AC3)-positive cells (left panel). Representative images of AC3 immunohistochemistry (right panel).


Percentage of Ki67 - positive cells in the carcinomas of the indicated genotypes ( left panel ) . Representative images of Ki67 immunohistochemistry ( right panel ) .

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Percentage of Ki67-positive cells in the carcinomas of the indicated genotypes (left panel). Representative images of Ki67 immunohistochemistry (right panel).


Percentage of pH3 - positive cells in the carcinomas of the indicated genotypes ( left panel ) . Representative images of pH3 immunohistochemistry ( right panel ) . Red arrowheads : pH3 - positive cells .

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Percentage of pH3-positive cells in the carcinomas of the indicated genotypes (left panel). Representative images of pH3 immunohistochemistry (right panel). Red arrowheads: pH3-positive cells.


Percentage of giant nuclei in the carcinomas of the indicated genotype . Percentage of anaphase bridges out of total anaphases in the carcinomas of the indicated genotypes . Representative images of giant nuclei , multilobulated nuclei , anaphase bridges , and multipolar mitoses . Red arrowheads indicate the corresponding mitotic aberrations indicated in the image .

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Percentage of giant nuclei in the carcinomas of the indicated genotype.Percentage of anaphase bridges out of total anaphases in the carcinomas of the indicated genotypes.Representative images of giant nuclei, multilobulated nuclei, anaphase bridges, and multipolar mitoses. Red arrowheads indicate the corresponding mitotic aberrations indicated in the image.


A - C The latency ( A ) , volume ( B ) , and weight ( C ) of subcutaneous tumors generated by control and Trf1 - downregulated K - RasG12V - transformed lung cells in athymic mice . D Representative images of the subcutaneous tumors .

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A-C The latency (A), volume (B), and weight (C) of subcutaneous tumors generated by control and Trf1-downregulated K-RasG12V-transformed lung cells in athymic mice.D Representative images of the subcutaneous tumors.


E Trf1 expression levels measured by qPCR in the injected cell line and in the generated subcutaneous tumors .

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E Trf1 expression levels measured by qPCR in the injected cell line and in the generated subcutaneous tumors.


F - H Number of Ki67 - positive ( F ) , number of γH2AX - positive ( G ) , and number of active caspase - 3 - positive ( H ) cells per field in the subcutaneous tumors .

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F-H Number of Ki67-positive (F), number of γH2AX-positive (G), and number of active caspase-3-positive (H) cells per field in the subcutaneous tumors.


I Representative images of aberrant giant nuclei and anaphase bridges in the Trf1 - downregulated subcutaneous tumors compared to the normal nuclei of control tumors .

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I Representative images of aberrant giant nuclei and anaphase bridges in the Trf1-downregulated subcutaneous tumors compared to the normal nuclei of control tumors.


J TRF1 immunofluorescence shows the downregulation of Trf1 in lung tumors of the mice intravenously injected with control and Trf1 - downregulated cells .

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J TRF1 immunofluorescence shows the downregulation of Trf1 in lung tumors of the mice intravenously injected with control and Trf1-downregulated cells.


K Tumor area measured in the lungs of the mice intravenously injected with control and Trf1 - downregulated cells . L Representative images of the lungs colonized by control and Trf1 - downregulated cells , respectively .

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K Tumor area measured in the lungs of the mice intravenously injected with control and Trf1-downregulated cells.L Representative images of the lungs colonized by control and Trf1-downregulated cells, respectively.


M - O Number of Ki67 - positive ( M ) , number of γH2AX - positive ( N ) , and number of active caspase - 3 - positive ( O ) cells per field in the lung tumors .

M-O Number of Ki67-positive (M), number of γH2AX-positive (N), and number of active caspase-3-positive (O) cells per field in the lung tumors.


P Trf1 expression levels measured by qPCR in the A549 cell line infected either with sh - scrambled or sh - Trf1 .

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P Trf1 expression levels measured by qPCR in the A549 cell line infected either with sh-scrambled or sh-Trf1.


Trf1 expression levels in the indicated tissues of wild - type and Trf1lox / lox hUBC - CreERT2 mice subjected to a tamoxifen - containing diet for 7 weeks .

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Trf1 expression levels in the indicated tissues of wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.


Representative images of TRF1 immunofluorescence and quantification of the percentage of TRF1 - positive cells in skin and intestine sections of wild - type and Trf1lox / lox hUBC - CreERT2 mice subjected to a tamoxifen - containing diet for 7 weeks .

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Representative images of TRF1 immunofluorescence and quantification of the percentage of TRF1-positive cells in skin and intestine sections of wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.


Survival curve of wild - type and Trf1lox / lox hUBC - CreERT2 mice subjected to a tamoxifen - containing diet for 7 weeks .

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Survival curve of wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.


Quantification of the histological alterations observed in tamoxifen - treated Trf1lox / lox hUBC - CreERT2 mice and 4 months after tamoxifen retrieval compared to their wild - type counterparts .

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Quantification of the histological alterations observed in tamoxifen-treated Trf1lox/lox hUBC-CreERT2 mice and 4 months after tamoxifen retrieval compared to their wild-type counterparts.


Quantification of blood cell populations in wild - type and Trf1lox / lox hUBC - CreERT2mice subjected to a tamoxifen - containing diet for 7 weeks and after 3 weeks and 4 months of tamoxifen retrieval .

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Quantification of blood cell populations in wild-type and Trf1lox/lox hUBC-CreERT2mice subjected to a tamoxifen-containing diet for 7 weeks and after 3 weeks and 4 months of tamoxifen retrieval.


Trf1 expression levels in blood , intestine , skin , and bone marrow of Trf1lox / lox hUBC - CreERT2 mice subjected to a tamoxifen - containing diet either for 7 weeks or for 6 months and after 3 weeks tamoxifen retrieval compared to wild - type mice .

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Trf1 expression levels in blood, intestine, skin, and bone marrow of Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet either for 7 weeks or for 6 months and after 3 weeks tamoxifen retrieval compared to wild-type mice.


Quantification of TRF1 levels by immunofluorescence in lung tumor - derived cell line treated with DMSO , with 10 μM ETP - 47228 ( 24 h ) , and with 10 μM ETP - 47037 ( 48 h ) . Representative images are shown to the right .

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Quantification of TRF1 levels by immunofluorescence in lung tumor-derived cell line treated with DMSO, with 10 μM ETP-47228 (24 h), and with 10 μM ETP-47037 (48 h). Representative images are shown to the right.


Quantification of γH2AX levels by immunofluorescence in lung tumor - derived cell line treated with DMSO , with ETP - 47228 ( 24 h ) , and with ETP - 47037 ( 48 h ) . Representative images are shown to the right .

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Quantification of γH2AX levels by immunofluorescence in lung tumor-derived cell line treated with DMSO, with ETP-47228 (24 h), and with ETP-47037 (48 h). Representative images are shown to the right.


Quantification of telomere - induced foci ( TIFs ) by double immunofluorescence with anti - RAP1 and anti - γH2AX antibodies . Representative images are shown to the right . White arrowheads : colocalization of γH2AX and RAP1 .

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Quantification of telomere-induced foci (TIFs) by double immunofluorescence with anti-RAP1 and anti-γH2AX antibodies. Representative images are shown to the right. White arrowheads: colocalization of γH2AX and RAP1.


Effect of different ETP - 47228 and ETP - 47037 concentrations during 24 h on proliferation in lung tumor - derived cell line relative to the growth of DMSO - treated cells .

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Effect of different ETP-47228 and ETP-47037 concentrations during 24 h on proliferation in lung tumor-derived cell line relative to the growth of DMSO-treated cells.


Tumor growth quantification in allograft model ETP - 47037 or with ETP - 47228 .

Tumor growth quantification in allograft model ETP-47037 or with ETP-47228.


Quantification of tumor growth relative to initial tumor size . Representative CT images are shown to the right . The white arrowhead indicates a tumor within a highly inflammatory region .

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Quantification of tumor growth relative to initial tumor size. Representative CT images are shown to the right. The white arrowhead indicates a tumor within a highly inflammatory region.


Quantification of TRF1 levels by immunofluorescence in intestine and lung tumors samples of mice treated with vehicle or with ETP - 47037 for 10 days . Representative images are shown to the right ( n = 4 ) .

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Quantification of TRF1 levels by immunofluorescence in intestine and lung tumors samples of mice treated with vehicle or with ETP-47037 for 10 days. Representative images are shown to the right (n = 4).


Number of cells showing γH2AX foci in intestine and lung tumors samples of mice treated with vehicle or with ETP - 47037 for 10 days . Representative images are shown to the right ( n = 4 ) .

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Number of cells showing γH2AX foci in intestine and lung tumors samples of mice treated with vehicle or with ETP-47037 for 10 days. Representative images are shown to the right (n = 4).


A - C Quantification of the number of ( A ) Ki67 - , ( B ) pan - nuclear p - H3 pattern - , and ( C ) foci p - H3 pattern - positive cells in untreated and ETP - 470037 - treated lungcarcinomas . The data represent the mean values obtained for three mice in each group . Error bars represent standard errors . D Representative Ki67 and p - H3 images .

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A-C Quantification of the number of (A) Ki67-, (B) pan-nuclear p-H3 pattern-, and (C) foci p-H3 pattern-positive cells in untreated and ETP-470037-treated lungcarcinomas. The data represent the mean values obtained for three mice in each group. Error bars represent standard errors.D Representative Ki67 and p-H3 images.


E Representative H & amp ; amp ; E images of intestine samples corresponding to untreated and ETP - 47037 - treated animals . High - magnification images are shown to the right indicating the presence of normal mitosis , giant multinucleated and aberrant mitotic figures .

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E Representative H&amp;E images of intestine samples corresponding to untreated and ETP-47037-treated animals. High-magnification images are shown to the right indicating the presence of normal mitosis, giant multinucleated and aberrant mitotic figures.


F Representative H & amp ; amp ; E images of bone marrow and skin samples corresponding to untreated and ETP - 47037 - treated animals . High - magnification images are shown indicating the presence of necrosis , hemosiderosis , multinucleated cells , and giant nuclei . Bone marrow showed moderated aplasia .

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F Representative H&amp;E images of bone marrow and skin samples corresponding to untreated and ETP-47037-treated animals. High-magnification images are shown indicating the presence of necrosis, hemosiderosis, multinucleated cells, and giant nuclei. Bone marrow showed moderated aplasia.


G Telomere length in untreated and ETP - 47037 - treated lung tumor samples . Representative images are shown to the right .

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G Telomere length in untreated and ETP-47037-treated lung tumor samples. Representative images are shown to the right.


A Location of missense variants of PDIA1 and ERp57 identified in ALS cases . PDI primary structure : catalytic a and a ' domains containing the active site motif CXXC sequence ( dark purple and dark green ) , non - catalytic domains b and b ' containing ligand binding sites ( light purple and light green ) , and x - linker region ( light gray ) . The constructs used in this study contained a V5 - tag ( orange ) at the C - terminus that was inserted previous to the ER - retention signal ( dark gray ) .

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A Location of missense variants of PDIA1 and ERp57 identified in ALS cases. PDI primary structure: catalytic a and a' domains containing the active site motif CXXC sequence (dark purple and dark green), non-catalytic domains b and b' containing ligand binding sites (light purple and light green), and x-linker region (light gray). The constructs used in this study contained a V5-tag (orange) at the C-terminus that was inserted previous to the ER-retention signal (dark gray).


B Expression of PDI variants in zebrafish . Zebrafish embryos at 1 - 2 cell stage were injected with sense mRNA coding for the indicated PDIs ( PDIA1WT , PDIA1D292N and PDIA1R300H : 200 pg / embryo ; ERp57WT , ERp57D217N and ERp57Q481K : 80 pg / embryo ) . Protein expression was confirmed by Western blot analysis using anti - V5 antibody in total embryo extracts at 24 h post - fertilization ( hpf ) . Actin was used as a loading control . In the left panel 80 µg protein and in the right panel 120 µg protein extract were used .

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B Expression of PDI variants in zebrafish. Zebrafish embryos at 1-2 cell stage were injected with sense mRNA coding for the indicated PDIs (PDIA1WT, PDIA1D292N and PDIA1R300H: 200 pg/embryo; ERp57WT, ERp57D217N and ERp57Q481K: 80 pg/embryo). Protein expression was confirmed by Western blot analysis using anti-V5 antibody in total embryo extracts at 24 h post-fertilization (hpf). Actin was used as a loading control. In the left panel 80 µg protein and in the right panel 120 µg protein extract were used.


C and D , Motoneuron defects induced in zebrafish embryos after expression of the indicated ALS - linked PDI mutants and wild - type controls ( PDIA1WT and PDIA1R300H : 80 pg mRNA / embryo ; ERp57WT , ERp57D217N , ERp57D217N and ERp57Q481K : 30 pgtba mRNA / embryo ) . The most frequent global phenotypes induced by PDI injection are shown in lateral views of embryos at 24 hpf ( left column ) . Black arrows indicate the presence of curly tail and / or shorter axis phenotypes ( see details in Appendix Table S1 ) . Axon motoneuron morphology was visualized using confocal microscopy in lateral views of the trunk region in transgenic Tg ( Huc : Kaede ) zebrafish at 36 hpf ( middle and right columns ) . Images in the right column correspond to magnification views of the rectangular regions depicted in left and middle columns . Asterisks and arrows point to examples of increased axonal branching and reduced axonal length , respectively .

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C and D, Motoneuron defects induced in zebrafish embryos after expression of the indicated ALS-linked PDI mutants and wild-type controls (PDIA1WT and PDIA1R300H: 80 pg mRNA/embryo; ERp57WT, ERp57D217N, ERp57D217N and ERp57Q481K: 30 pgtba mRNA/embryo). The most frequent global phenotypes induced by PDI injection are shown in lateral views of embryos at 24 hpf (left column). Black arrows indicate the presence of curly tail and/or shorter axis phenotypes (see details in Appendix Table S1). Axon motoneuron morphology was visualized using confocal microscopy in lateral views of the trunk region in transgenic Tg(Huc:Kaede) zebrafish at 36 hpf (middle and right columns). Images in the right column correspond to magnification views of the rectangular regions depicted in left and middle columns. Asterisks and arrows point to examples of increased axonal branching and reduced axonal length, respectively.


C and D , Motoneuron defects induced in zebrafishembryos after expression of the indicated ALS - linked PDI mutants and wild - type controls ( PDIA1WT and PDIA1R300H : 80 pg mRNA / embryo ; ERp57WT , ERp57D217N , ERp57D217N and ERp57Q481K : 30 pgtba mRNA / embryo ) . The most frequent global phenotypes induced by PDI injection are shown in lateral views of embryos at 24 hpf ( left column ) . Black arrows indicate the presence of curly tail and / or shorter axis phenotypes ( see details in Appendix Table S1 ) . Axonmotoneuronmorphology was visualized using confocalmicroscopy in lateral views of the trunk region in transgenic Tg ( Huc : Kaede ) zebrafish at 36 hpf ( middle and right columns ) . Images in the right column correspond to magnification views of the rectangular regions depicted in left and middle columns . Asterisks and arrows point to examples of increased axonal branching and reduced axonal length , respectively .

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C and D, Motoneuron defects induced in zebrafishembryos after expression of the indicated ALS-linked PDI mutants and wild-type controls (PDIA1WT and PDIA1R300H: 80 pg mRNA/embryo; ERp57WT, ERp57D217N, ERp57D217N and ERp57Q481K: 30 pgtba mRNA/embryo). The most frequent global phenotypes induced by PDI injection are shown in lateral views of embryos at 24 hpf (left column). Black arrows indicate the presence of curly tail and/or shorter axis phenotypes (see details in Appendix Table S1). Axonmotoneuronmorphology was visualized using confocalmicroscopy in lateral views of the trunk region in transgenic Tg(Huc:Kaede) zebrafish at 36 hpf (middle and right columns). Images in the right column correspond to magnification views of the rectangular regions depicted in left and middle columns. Asterisks and arrows point to examples of increased axonal branching and reduced axonal length, respectively.


E and F Quantification of motoneuron axon length and axon branching in 36hpf Tg ( Huc : Kaede ) embryos injected with the indicated PDIA1 ( D ) and ERp57 ( E ) mutants .

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E and F Quantification of motoneuron axon length and axon branching in 36hpf Tg(Huc:Kaede) embryos injected with the indicated PDIA1 (D) and ERp57 (E) mutants.


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G Touch - evoked escape responses of 48 hpf zebrafish embryos injected with ALS - linked PDI mutants ( Kabashi et al , 2011 ; Kabashi et al , 2009 ) . The number of touches necessary to evoke an escape response ( top ) and speed ( bottom , in mm / s ) of the escape response was determined for each condition . The total number of analyzed embryos is indicated in parenthesis . Experiments of D - F were performed in selected animals sharing normal overall embryo morphology and viability , to avoid unspecific effects of axial shortening or curvature in motoneuron morphology and / or animal behavior . Statistical analyses were performed using one - way ANOVA and Bonferroni ´ s post hoc tests . Mean ± SEM with only statistically significant p values are shown : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . Abbreviations : A ( anterior ) , D ( dorsal ) , P ( posterior ) , V ( ventral ) . Scale bars represent 500 m ( C and D : a , d . g , j ) , 200 m ( C and D : b , e , h , k ) , 100 m ( C and D : c , f , i , l ) , and 25 µm ( G - H ) .

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G Touch-evoked escape responses of 48 hpf zebrafish embryos injected with ALS-linked PDI mutants (Kabashi et al, 2011; Kabashi et al, 2009). The number of touches necessary to evoke an escape response (top) and speed (bottom, in mm/s) of the escape response was determined for each condition. The total number of analyzed embryos is indicated in parenthesis. Experiments of D-F were performed in selected animals sharing normal overall embryo morphology and viability, to avoid unspecific effects of axial shortening or curvature in motoneuron morphology and/or animal behavior. Statistical analyses were performed using one-way ANOVA and Bonferroni´s post hoc tests. Mean ± SEM with only statistically significant p values are shown: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. Abbreviations: A (anterior), D (dorsal), P (posterior), V (ventral). Scale bars represent 500 m (C and D: a,d.g,j), 200 m (C and D: b,e,h,k), 100 m (C and D: c,f,i,l), and 25 µm (G-H).


A NSC34cells were transiently transfected with expression vectors for V5 - tagged wild - type and mutant PDIs . After 48 h , overexpressed PDI variants was assessed under reducing conditions in an 8 % SDS - PAGE . An antibody detecting total PDIA1 ( endogenous mousePDIA1 and exogenous humanV5 - tagged PDIA1 ) ( first panel ) . A second antibody detects only humanPDIA1 , therefore only V5 - tagged PDIA1 appears ( second panel ) . A mouse and human specific antibody was used to detect total ERp57 ( third panel ) . Anti - V5 was used to detect the exogenous PDI variants . Anti - β - actin was used as a loading control .

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A NSC34cells were transiently transfected with expression vectors for V5-tagged wild-type and mutant PDIs. After 48 h, overexpressed PDI variants was assessed under reducing conditions in an 8% SDS-PAGE. An antibody detecting total PDIA1 (endogenous mousePDIA1 and exogenous humanV5-tagged PDIA1) (first panel). A second antibody detects only humanPDIA1, therefore only V5-tagged PDIA1 appears (second panel). A mouse and human specific antibody was used to detect total ERp57 (third panel). Anti-V5 was used to detect the exogenous PDI variants. Anti-β-actin was used as a loading control.


B and C NSC34 cells were transiently transfected with the indicated PDIs constructs together with a GFP expression plasmid . Cell were then differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation . Increased neurite outgrowth is indicated with white arrow heads . B Quantification of the average primary neurite lengths was performed , all cells from three independent experiments were compiled . A minimum of 100 cells per experiment was analyzed . In addition , C the percentage of cells with neurites was quantified in the three independent experiments ( right panel ) .

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B and C NSC34 cells were transiently transfected with the indicated PDIs constructs together with a GFP expression plasmid. Cell were then differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation. Increased neurite outgrowth is indicated with white arrow heads. B Quantification of the average primary neurite lengths was performed, all cells from three independent experiments were compiled. A minimum of 100 cells per experiment was analyzed. In addition, C the percentage of cells with neurites was quantified in the three independent experiments (right panel).


D Primary rat ventral spinal cordneurons were prepared and after 4 daysin vitro ( DIV ) transfected with GFP alone or together with the indicated PDIs constructs . Cells were fixed at 10 DIV and SMI32 staining was performed to identify motoneurons . Images were taken and the total outgrowth of GFP and SMI32 positive cells was quantified . Results are compiled from 3 independent experiments .

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D Primary rat ventral spinal cordneurons were prepared and after 4 daysin vitro (DIV) transfected with GFP alone or together with the indicated PDIs constructs. Cells were fixed at 10 DIV and SMI32 staining was performed to identify motoneurons. Images were taken and the total outgrowth of GFP and SMI32 positive cells was quantified. Results are compiled from 3 independent experiments.


E Human motoneurons were differentiated from the human embryonic stem cell ( ESC ) HuESC 3 Hb9 : : GFP line . Differentiated neurons were transduced with lentivirus expressing GFP alone or together with PDIs - expressing plasmids . Transduced cells were cultured for another 10 days . To identify motoneurons after lentiviral transduction , immunocytochemistry analyses were performed . Quantification of neurite outgrowth in the different experimental conditions of GFP and HB9 positive neurons were obtained . Neurite number and assessment of the length of each neurite was performed in a similar manner as for primary rat motoneurons ( see methods ) . Four independent experiments were performed .

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E Human motoneurons were differentiated from the human embryonic stem cell (ESC) HuESC 3 Hb9::GFP line. Differentiated neurons were transduced with lentivirus expressing GFP alone or together with PDIs-expressing plasmids. Transduced cells were cultured for another 10 days. To identify motoneurons after lentiviral transduction, immunocytochemistry analyses were performed. Quantification of neurite outgrowth in the different experimental conditions of GFP and HB9 positive neurons were obtained. Neurite number and assessment of the length of each neurite was performed in a similar manner as for primary rat motoneurons (see methods). Four independent experiments were performed.


F NSC34 cell lines stably knocked down for PDIA1 or ERp57 were differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation . The percentage of cells with neurites was quantified in the three independent experiments . A minimum of 100 cells per experiment was analyzed . Statistical analyses were performed using one - way ANOVA and Bonferroni ´ s post hoc tests . Mean ± SEM with only statistically significant p values are shown : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 .

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F NSC34 cell lines stably knocked down for PDIA1 or ERp57 were differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation. The percentage of cells with neurites was quantified in the three independent experiments. A minimum of 100 cells per experiment was analyzed.Statistical analyses were performed using one-way ANOVA and Bonferroni´s post hoc tests. Mean ± SEM with only statistically significant p values are shown: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.


A PDI mutants form abnormal disulfide - dependent protein complexes . NSC34 cells were transiently transfected with expression vectors for V5 - tagged wild - type and mutant PDIA1 . After 48 h , differential disulfide - dependent interactions / aggregations of overexpressed PDI variants was assessed under reducing ( + DTT ) and non - reducing ( - DTT ) conditions in an 8 % SDS - PAGE . Anti - V5 was used for detection in Western blot .

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A PDI mutants form abnormal disulfide-dependent protein complexes. NSC34 cells were transiently transfected with expression vectors for V5-tagged wild-type and mutant PDIA1. After 48 h, differential disulfide-dependent interactions/aggregations of overexpressed PDI variants was assessed under reducing (+DTT) and non-reducing (-DTT) conditions in an 8% SDS-PAGE. Anti-V5 was used for detection in Western blot.


B Analysis of the PDIA1 structure to model the effects of the R300H mutation . The close association between Arg300 located to the b ' domain of PDIA1 with Trp396 located to the a ' domain adjacent to the active site motif CGHC ( designated as AS in yellow ) is shown in comparison to the mutated version of PDIA1R300H highlighting the same residues . A potential stabilization of the interaction between the b ' and a ' domain is shown that may be caused by the interaction of the imidazole rings of mutated His300 with Trp396 .

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B Analysis of the PDIA1 structure to model the effects of the R300H mutation. The close association between Arg300 located to the b' domain of PDIA1 with Trp396 located to the a' domain adjacent to the active site motif CGHC (designated as AS in yellow) is shown in comparison to the mutated version of PDIA1R300H highlighting the same residues. A potential stabilization of the interaction between the b' and a' domain is shown that may be caused by the interaction of the imidazole rings of mutated His300 with Trp396.


C ALS - linked PDIA1 variants were generated as recombinant proteins and then analyzed by circular dichroism ( CD ) . Averages for CD spectroscopic scans of PDIA1WT and mutants are shown .

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C ALS-linked PDIA1 variants were generated as recombinant proteins and then analyzed by circular dichroism (CD). Averages for CD spectroscopic scans of PDIA1WT and mutants are shown.


D Averages for CD spectroscopic thermal denaturation of recombinant PDIA1WT and mutants .

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D Averages for CD spectroscopic thermal denaturation of recombinant PDIA1WT and mutants.


E Representative electrophoresis analysis of Proteinase K - treated recombinant PDIA1 variants . Mass spectrometric analysis of Proteinase K digested samples from total sample and from in gel trypsin digested protein samples ( arrows indicate band 1 and band 2 ) indicated differences in the removal of the x - linker region . Bottom panel : Ratios of band 1 to band 2 were quantified in 4 independent experiments .

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E Representative electrophoresis analysis of Proteinase K-treated recombinant PDIA1 variants. Mass spectrometric analysis of Proteinase K digested samples from total sample and from in gel trypsin digested protein samples (arrows indicate band 1 and band 2) indicated differences in the removal of the x-linker region. Bottom panel: Ratios of band 1 to band 2 were quantified in 4 independent experiments.


F Representative fluorescence spectra of recombinant PDIA1b ' xWT fragments and the equivalent ALS - linked mutants ( n = 6 ) . Ratios from the two peak areas and change compared to PDIA1 b ' xWT . Both mutants analyzed show a significant shift in peak position compared to wild - type , but in opposite directions , suggesting that PDIA1D292N shifts equilibrium towards the capped version ( x region over the binding pocket ) and the PDIA1R300H mutant towards the uncapped version ( Nguyen et al , 2008 ) .

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F Representative fluorescence spectra of recombinant PDIA1b'xWT fragments and the equivalent ALS-linked mutants (n=6). Ratios from the two peak areas and change compared to PDIA1 b'xWT. Both mutants analyzed show a significant shift in peak position compared to wild-type, but in opposite directions, suggesting that PDIA1D292N shifts equilibrium towards the capped version (x region over the binding pocket) and the PDIA1R300H mutant towards the uncapped version (Nguyen et al, 2008).


G The activity of recombinant PDIA1 was measured in vitro using a BPTI refolding assay following by mass spectrometry analysis . The percentages of different BPTI species was calculated ( 6H , fully reduced ; 1S , one disulfide bond ; 2S , two disulfide bonds ; 3S , three disulfide bonds ) at time point 2 . 5 min in four independent experiments .

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G The activity of recombinant PDIA1 was measured in vitro using a BPTI refolding assay following by mass spectrometry analysis. The percentages of different BPTI species was calculated (6H, fully reduced; 1S, one disulfide bond; 2S, two disulfide bonds; 3S, three disulfide bonds) at time point 2.5 min in four independent experiments.


H Measurement of H2O2 levels at the ER lumen of living NSC34 . Left panel : Reduced PDIs can be oxidized by oxidized ERO1Lα that then transfers electrons to molecular oxygen ( O2 ) , generating hydrogen peroxide ( H2O2 ) as a product of PDIs activity . Right panel : NSC34 cells were transiently co - transfected with ER luminal HyPer sensor and indicated PDIs . After 48 h the 490 / 420 nm fluorescence ratio was recorded for 2 min under basal conditions . Means and SEM derived from all cells per condition ( n = 55 - 74 ) monitored in four independent experiments are shown .

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H Measurement of H2O2 levels at the ER lumen of living NSC34. Left panel: Reduced PDIs can be oxidized by oxidized ERO1Lα that then transfers electrons to molecular oxygen (O2), generating hydrogen peroxide (H2O2) as a product of PDIs activity. Right panel: NSC34 cells were transiently co-transfected with ER luminal HyPer sensor and indicated PDIs. After 48 h the 490/420 nm fluorescence ratio was recorded for 2 min under basal conditions. Means and SEM derived from all cells per condition (n=55-74) monitored in four independent experiments are shown.


I HEK293T cells were transfected with expression vectors for V5 - tagged wild - type and mutant PDIA1 , as well as empty vector . After 48 h , V5 - tagged proteins were immunoprecipitated and eluted with V5 peptide . The interaction with endogenous ERO1Lα was analyzed by western blot . The inputs and elutions are shown as control . Right panel : Quantification of the degree of interaction is presented .

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I HEK293T cells were transfected with expression vectors for V5-tagged wild-type and mutant PDIA1, as well as empty vector. After 48 h, V5-tagged proteins were immunoprecipitated and eluted with V5 peptide. The interaction with endogenous ERO1Lα was analyzed by western blot. The inputs and elutions are shown as control. Right panel: Quantification of the degree of interaction is presented.


A PDI mutants form abnormal disulfide - dependent protein complexes . NSC34 cells were transiently transfected with expression vectors for V5 - tagged wild - type and mutant ERp57 . After 48 h , differential disulfide - dependent interactions / aggregations of overexpressed PDI variants was assessed under reducing ( + DTT ) and non - reducing ( - DTT ) conditions in an 8 % SDS - PAGE . Anti - V5 was used for detection in Western blot .

A PDI mutants form abnormal disulfide-dependent protein complexes. NSC34 cells were transiently transfected with expression vectors for V5-tagged wild-type and mutant ERp57. After 48 h, differential disulfide-dependent interactions/aggregations of overexpressed PDI variants was assessed under reducing (+DTT) and non-reducing (-DTT) conditions in an 8% SDS-PAGE. Anti-V5 was used for detection in Western blot.


B ALS - linked ERp57 variants were generated as recombinant proteins and then analyzed by circular dichroism ( CD ) . Averages for CD spectroscopic scans of recombinant ERp57WT and mutants are shown .

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B ALS-linked ERp57 variants were generated as recombinant proteins and then analyzed by circular dichroism (CD). Averages for CD spectroscopic scans of recombinant ERp57WT and mutants are shown.


C A thermal denaturation curve of ERp57WT and mutants was performed .

C A thermal denaturation curve of ERp57WT and mutants was performed.


D Representative electrophoresis of Proteinase K treated ERp57 recombinant proteins .

D Representative electrophoresis of Proteinase K treated ERp57 recombinant proteins.


E Surface Plasmon Resonance was performed to monitor the affinity of recombinant ERp57 for recombinant CRT P - domain . KD values are expressed as percentage of ERp57WT . Values are derived from four independent experiments . For absolute KD values please see Appendix Table S2 .

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E Surface Plasmon Resonance was performed to monitor the affinity of recombinant ERp57 for recombinant CRT P-domain. KD values are expressed as percentage of ERp57WT. Values are derived from four independent experiments. For absolute KD values please see Appendix Table S2.


FHEK293T NSC34 were transfected with expression vectors for V5 - tagged wild - type and mutant ERp57 , as well as empty vector . After 48 h , V5 - tagged proteins were immunoprecipitated and eluted with V5 peptide . The interaction with endogenous calnexin ( CNX ) and calreticulin ( CRT ) was analyzed by Western blot . The inputs and elutions are shown . Right panel : Quantification of the degree of interaction is presented .

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FHEK293T NSC34 were transfected with expression vectors for V5-tagged wild-type and mutant ERp57, as well as empty vector. After 48 h, V5-tagged proteins were immunoprecipitated and eluted with V5 peptide. The interaction with endogenous calnexin (CNX) and calreticulin (CRT) was analyzed by Western blot. The inputs and elutions are shown. Right panel: Quantification of the degree of interaction is presented.


G The gain of an N - glycosylation site of ERp57D217N was predicted after the analysis of the protein sequence since the change of Asp217 to an Asn creates the NXT / S consensus sequence . Neuro2a cells were transfected with expression vectors for V5 - tagged wild - type and mutant ERp57 , as well as empty vector and treated with 1 µg / ml tunicamycin ( Tm ) for 20 h to inhibit N - glycosylation . Alternatively , protein extracts were digested with PNGase F and the possible removal of the N - glycosylation was analyzed by Western blot using anti - V5 antibody .

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G The gain of an N-glycosylation site of ERp57D217N was predicted after the analysis of the protein sequence since the change of Asp217 to an Asn creates the NXT/S consensus sequence. Neuro2a cells were transfected with expression vectors for V5-tagged wild-type and mutant ERp57, as well as empty vector and treated with 1 µg/ml tunicamycin (Tm) for 20 h to inhibit N-glycosylation. Alternatively, protein extracts were digested with PNGase F and the possible removal of the N-glycosylation was analyzed by Western blot using anti-V5 antibody.


A ERp57flox / flox mice were crossed with Nestin Cre transgenic mice to generate nervous system specific ERp57 deficient animals . The levels of ERp57 protein in the spinal cord were monitored by Western blot . ERp57WT ( n = 4 ) , ERp57Nes + / - ( n = 5 ) and ERp57Nes - / - ( n = 4 ) mice . HSP90 levels were determined as a loading control . Right panel : Quantification of ERp57 levels was performed relative to Hsp90 levels .

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A ERp57flox/flox mice were crossed with Nestin Cre transgenic mice to generate nervous system specific ERp57 deficient animals. The levels of ERp57 protein in the spinal cord were monitored by Western blot. ERp57WT (n=4), ERp57Nes+/- (n=5) and ERp57Nes-/- (n=4) mice. HSP90 levels were determined as a loading control. Right panel: Quantification of ERp57 levels was performed relative to Hsp90 levels.


B Body weight measurements were performed for indicated time points in ERp57WT ( n = 50 ) , ERp57Nes + / - ( n = 32 ) and ERp57Nes - / - ( n = 19 ) mice .

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B Body weight measurements were performed for indicated time points in ERp57WT (n=50), ERp57Nes+/- (n=32) and ERp57Nes-/- (n=19) mice.


C Rotarod performance was performed ERp57WT ( n = 20 ) , ERp57Nes + / - ( n = 15 ) and ERp57Nes - / - ( n = 8 ) mice .

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C Rotarod performance was performed ERp57WT (n=20), ERp57Nes+/- (n=15) and ERp57Nes-/- (n=8) mice.


D Hanging test performance was assessed in ERp57WT ( n = 41 ) , ERp57Nes + / - ( n = 32 ) and ERp57Nes - / - ( n = 12 ) mice .

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D Hanging test performance was assessed in ERp57WT (n=41), ERp57Nes+/- (n=32) and ERp57Nes-/- (n=12) mice.


E Kaplan - Meier survival curve for ERp57Nes - / - mice ( N = 19 ) that prematurely died or had to be sacrificed because of health reasons between the ages 22 and 73 days . Mean survival of this subgroup of animals was 57 days . ERp57WT ( n = 50 ) and ERp57Nes + / - ( n = 32 ) mice are shown as a reference .

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E Kaplan-Meier survival curve for ERp57Nes-/- mice (N = 19) that prematurely died or had to be sacrificed because of health reasons between the ages 22 and 73 days. Mean survival of this subgroup of animals was 57 days. ERp57WT (n=50) and ERp57Nes+/- (n=32) mice are shown as a reference.


F Histological analysis of NeuN and GFAP staining was performed in spinal cord tissue from ERp57WT and ERp57Nes - / - mice in three animals per group using indirect immunofluorescence . The nucleus was stained with Hoechst . Representative images from one mouse per group are shown . Scale bar : 50 μm .

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F Histological analysis of NeuN and GFAP staining was performed in spinal cord tissue from ERp57WT and ERp57Nes-/- mice in three animals per group using indirect immunofluorescence. The nucleus was stained with Hoechst. Representative images from one mouse per group are shown. Scale bar: 50 μm.


G Stereological analysis of the spinal cord from ERp57WT ( n = 4 ) , ERp57Nes + / - ( n = 4 ) and ERp57Nes - / - ( n = 4 ) mice . Alternate series of sections from the spinal cord of the mice were either stained for Nissl ( top row images ) or processed for immunohistochemistry for the cholinergic cell marker Choline Acetyl Transferase ( ChAT , bottom row images ) . The nucleoli of the motoneurons , as stained in the Nissl series , were counted inside the motoneurons pools previously defined using the adjacent ChAT series ( contours not shown ) . Cell densities of the three genotypes are shown on the right plots . No significant differences were found between the genotypes . Scale bar represents 200 μm and 50 μm on large and inset images , respectively .

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G Stereological analysis of the spinal cord from ERp57WT (n = 4), ERp57Nes+/- (n = 4) and ERp57Nes-/- (n = 4) mice. Alternate series of sections from the spinal cord of the mice were either stained for Nissl (top row images) or processed for immunohistochemistry for the cholinergic cell marker Choline Acetyl Transferase (ChAT, bottom row images). The nucleoli of the motoneurons, as stained in the Nissl series, were counted inside the motoneurons pools previously defined using the adjacent ChAT series (contours not shown). Cell densities of the three genotypes are shown on the right plots. No significant differences were found between the genotypes. Scale bar represents 200 μm and 50 μm on large and inset images, respectively.


H Representative images of the spinal cord myelination of ERp57WT and ERp57Nes - / - mice .

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H Representative images of the spinal cord myelination of ERp57WT and ERp57Nes-/- mice.


A Representative EMG recordings of ERp57WT ( n = 16 ) , ERp57Nes - / + ( n = 12 ) and ERp57Nes - / - ( n = 5 ) mice is presented . The presence of positive sharp waves ( PSW ) in ERp57Nes + / - and ERp57Nes - / - mice indicated muscle denervation .

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A Representative EMG recordings of ERp57WT (n=16), ERp57Nes-/+ (n=12) and ERp57Nes-/- (n=5) mice is presented. The presence of positive sharp waves (PSW) in ERp57Nes+/- and ERp57Nes-/- mice indicated muscle denervation.


B Analysis of NMJ and muscle morphologies were performed from ERp57WT ( n = 5 ) , ERp57Nes + / - ( n = 5 ) and ERp57Nes - / - ( n = 4 ) . Quantifications of endplate width , measuring the most ventral region of the diaphragms every 134 µm considering both sides of the innervation profile . Twenty to forty measurements per diaphragm were obtained per animal .

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B Analysis of NMJ and muscle morphologies were performed from ERp57WT (n=5), ERp57Nes+/- (n=5) and ERp57Nes-/- (n=4). Quantifications of endplate width, measuring the most ventral region of the diaphragms every 134 µm considering both sides of the innervation profile. Twenty to forty measurements per diaphragm were obtained per animal.


C Whole - mounted diaphragms from ERp57WT and ERp57Nes - / - mice were co - stained with anti - neurofilament ( red ) and α - BTX to reveal the postsynaptic densities ( green ) . 3D reconstructions ( lower panel ) of higher magnification . ERp57WT NMJs are fully innervated pretzel - like , whereas ERp57Nes - / - NMJs display less complex postsynaptic densities and an incomplete or aberrantly distributed innervation pattern ( Movies EV4 - 7 ) .

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C Whole-mounted diaphragms from ERp57WT and ERp57Nes-/- mice were co-stained with anti-neurofilament (red) and α-BTX to reveal the postsynaptic densities (green). 3D reconstructions (lower panel) of higher magnification. ERp57WT NMJs are fully innervated pretzel-like, whereas ERp57Nes-/- NMJs display less complex postsynaptic densities and an incomplete or aberrantly distributed innervation pattern (Movies EV4-7).


D Representative 3D reconstructions of z - stacks showing the five categories in which NMJs morphologies were grouped : Pretzel , fragmented , O shaped , C shaped , and dismantled NMJs . Histogram of the distribution frequency of > 200 different NMJ per animal is shown .

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D Representative 3D reconstructions of z-stacks showing the five categories in which NMJs morphologies were grouped: Pretzel, fragmented, O shaped, C shaped, and dismantled NMJs. Histogram of the distribution frequency of >200 different NMJ per animal is shown.


E Quantitative morphometry of > 60 NMJs in the different genotypes analyzed .

E Quantitative morphometry of >60 NMJs in the different genotypes analyzed.


A The levels of ERp57 protein in the muscle were monitored by Western blot . ERp57WT ( n = 4 ) , ERp57Nes + / - ( n = 5 ) and ERp57Nes - / - ( n = 4 ) mice . HSP90 was determined as loading control .

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A The levels of ERp57 protein in the muscle were monitored by Western blot. ERp57WT (n = 4), ERp57Nes+/- (n = 5) and ERp57Nes-/- (n = 4) mice. HSP90 was determined as loading control.