( F ) Smear plot of RNA - seq data from GFP + / F4 / 80 + / Ly6CLow cells FACS sorted from GFP - Control and GFP - CriptoMy - LOF muscles at day 5 after injury . Data show the expression level as Log2 fold - change ( FC ) [ Log2 ( GFP - Control / GFP - CriptoMy - LOF ) ] , against Log2 ( expression ) [ Log2 ( average of gene expression across all samples ] , for each individual gene . The blue lines correspond to LogFC of 1 and - 1 . ( n = 3 biological replicates ) .

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

(F) Smear plot of RNA-seq data from GFP+/F4/80+/Ly6CLow cells FACS sorted from GFP-Control and GFP-CriptoMy-LOF muscles at day 5 after injury. Data show the expression level as Log2 fold-change (FC) [Log2(GFP-Control/GFP-CriptoMy-LOF)], against Log2(expression) [Log2(average of gene expression across all samples], for each individual gene. The blue lines correspond to LogFC of 1 and -1. (n=3 biological replicates).


B . Representative pictures of double immunostaining with Laminin ( green ) and embryonic myosin heavy chain ( eMHC ; red ) at day 5 ( left panel ) and 30 ( right panel ) after cardiotoxin ( CTX ) injection . Data information : Nuclei were counterstained with DAPI ( blue ) .

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

B. Representative pictures of double immunostaining with Laminin (green) and embryonic myosin heavy chain (eMHC; red) at day 5 (left panel) and 30 (right panel) after cardiotoxin (CTX) injection. Data information: Nuclei were counterstained with DAPI (blue).


C . Minimal Feret ' s diameter distribution of Control and CriptoMy - LOF myofibers at day 5 ( top panel ) and 30 ( bottom panel ) after CTX injection . Data are mean ± SEM ( n = 5 biological replicates ; P = ns , Student ' s t - test ) .

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

C. Minimal Feret's diameter distribution of Control and CriptoMy-LOF myofibers at day 5 (top panel) and 30 (bottom panel) after CTX injection. Data are mean±SEM (n=5 biological replicates; P=ns, Student's t-test).


E . Representative pictures of double immunostaining with Laminin ( green ) and eMHC ( red ) at day 5 ( left panel ) and 30 ( right panel ) after re - injury ( CTX - II ) . Data information : Nuclei were counterstained with DAPI ( blue ) .

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

E. Representative pictures of double immunostaining with Laminin (green) and eMHC (red) at day 5 (left panel) and 30 (right panel) after re-injury (CTX-II). Data information: Nuclei were counterstained with DAPI (blue).


F . Minimal Feret ' s diameter distribution of Control and

F. Minimal Feret's diameter distribution of Control and Cripto


B . Representative pictures of immunostaining of mdx - Control and mdx - CriptoMy - LOF diaphragm sections with GFP ( green ) and CD206 ( white ; left panel ) , pSMAD3 ( red ; middle panel ) and GFP and pSMAD3 ( right panel ) . C - E . Quantification of GFP ( C ) , GFP / CD206 ( D ) and GFP / pSMAD3 ( E ) positive cells per area ( mm2 ) in mdx - Control and mdx - CriptoMy - LOF diaphragms . Data are expressed as box plots displaying minimum , first quartile , median , third quartile and maximum ( n ≥ 5 biological replicates ; * * P < 0 . 01 , Student ' s t - test ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x .

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

B. Representative pictures of immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections with GFP (green) and CD206 (white; left panel), pSMAD3 (red; middle panel) and GFP and pSMAD3 (right panel). C-E. Quantification of GFP (C), GFP/CD206 (D) and GFP/pSMAD3 (E) positive cells per area (mm2) in mdx-Control and mdx-CriptoMy-LOF diaphragms. Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n≥5 biological replicates; **P<0.01, Student's t-test). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.


F - G . Representative pictures of Picrosirius red staining of mdx - Control and mdx - CriptoMy - LOF diaphragm sections ( F ) and quantification of picrosirius red staining ( G ) . Data are expressed as percentage of stained area and are box plots displaying minimum , first quartile , median , third quartile and maximum ( n = 5 biological replicates ; * P < 0 . 05 , Student ' s t - test ) .

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

F-G. Representative pictures of Picrosirius red staining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections (F) and quantification of picrosirius red staining (G). Data are expressed as percentage of stained area and are box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; *P<0.05, Student's t-test).


H . Hydroxyproline ( HOP ) concentration in mdx - Control and mdx - CriptoMy - LOF diaphragm muscle sections . Data are expressed as HOP levels ( μg ) per muscle section volume ( mm3 ) and as box plots displaying minimum , first quartile , median , third quartile and maximum ( n = 5 biological replicates ; * * P < 0 . 01 , Student ' s t - test ) .

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

H. Hydroxyproline (HOP) concentration in mdx-Control and mdx-CriptoMy-LOF diaphragm muscle sections. Data are expressed as HOP levels (μg) per muscle section volume (mm3) and as box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; **P<0.01, Student's t-test).


Representative images of Laminin ( red ) immunostaining of mdx - Control and mdx - CriptoMy - LOF diaphragm sections of centrally nucleated myofibers . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x .

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

Representative images of Laminin (red) immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections of centrally nucleated myofibers. Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.


Representative mdx - Control and mdx - CriptoMy - LOF diaphragm sections minimal Feret ' s diameter distribution of centrally nucleated myofibers . Data are expressed as mean ± SEM ( n = 5 biological replicates ; * P < 0 . 05 ; * * * P < 0 . 001 ; Student ' s t - test )

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

Representative mdx-Control and mdx-CriptoMy-LOF diaphragm sections minimal Feret's diameter distribution of centrally nucleated myofibers. Data are expressed as mean±SEM (n=5 biological replicates; *P<0.05; ***P<0.001; Student's t-test)


Representative mdx - Control and mdx - CriptoMy - LOF diaphragm sections minimal Feret ' s diameter distribution Variance coefficient of centrally nucleated myofibers . box plots displaying minimum , first quartile , median , third quartile and

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

Representative mdx-Control and mdx-CriptoMy-LOF diaphragm sections minimal Feret's diameter distribution Variance coefficient of centrally nucleated myofibers. box plots displaying minimum, first quartile, median, third quartile and maximum


L - M . Representative images of eMHC ( red ) immunostaining of mdx - Control and mdx - CriptoMy - LOF diaphragm sections ( L ) and quantification of eMHC + myofibers per area ( mm2 , M ) . Data are expressed as box plots displaying minimum , first quartile , median , third quartile and maximum ( n = 5 biological replicates ; * P < 0 . 05 ; Student ' s t - test ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x .

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

L-M. Representative images of eMHC (red) immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections (L) and quantification of eMHC+ myofibers per area (mm2, M). Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; *P<0.05; Student's t-test). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.


A . Representative pictures of CD31 ( red ) immunostaining of Control and CriptoMy - LOF TA sections at day 5 ( left panel ) and 30 ( right panel ) after single injury ( CTX - I ) . B - D . Quantification of CD31 + capillary number per area ( mm2 ; B ) , average of capillary cross - sectional area ( µm2 ; C ) and percentage of small ( < 20 µm2 ) and large ( > 100 µm2 ) capillaries ( D ) in Control and CriptoMy - LOF injured muscles at indicated time points after single injury ( CTX - I ) . Data are mean ± SEM ( n = 5 biological replicates ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ' s t - test ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x .

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

A. Representative pictures of CD31 (red) immunostaining of Control and CriptoMy-LOF TA sections at day 5 (left panel) and 30 (right panel) after single injury (CTX-I). B-D. Quantification of CD31+ capillary number per area (mm2; B), average of capillary cross-sectional area (µm2; C) and percentage of small (<20 µm2) and large (>100 µm2) capillaries (D) in Control and CriptoMy-LOF injured muscles at indicated time points after single injury (CTX-I). Data are mean±SEM (n=5 biological replicates; *P<0.05; **P<0.01; ***P<0.001, Student's t-test). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.


E . Representative pictures of CD31 ( red ) immunostaining of Control and CriptoMy - LOF TA sections at day 5 ( left panel ) and 30 ( right panel ) after re - injury ( CTX - II ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x .

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

E. Representative pictures of CD31 (red) immunostaining of Control and CriptoMy-LOF TA sections at day 5 (left panel) and 30 (right panel) after re-injury (CTX-II). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.


F . Representative pictures of mdx - Control and mdx - CriptoMy - LOF diaphragm sections immunostained with VEcad ( red ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x .

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

F. Representative pictures of mdx-Control and mdx-CriptoMy-LOF diaphragm sections immunostained with VEcad (red). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.


G - H . Quantification of CD31 + capillary number per area ( mm2 ; G ) and average of capillary cross - sectional area ( µm2 ; H ) in Control and CriptoMy - LOF injured muscles at the indicated time points after re - injury ( CTX - II ) Data are mean ± SEM ( n = 6 biological replicates ; * P < 0 . 05 ; * * * P < 0 . 001 , Student ' s t - test ) .

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

G-H. Quantification of CD31+ capillary number per area (mm2; G) and average of capillary cross-sectional area (µm2; H) in Control and CriptoMy-LOF injured muscles at the indicated time points after re-injury (CTX-II) Data are mean±SEM (n=6 biological replicates; *P<0.05; ***P<0.001, Student's t-test).


I - J . Quantification of capillary number per area ( mm2 ; I ) and average of capillary cross - sectional area ( µm2 ; J ) in mdx - Control and mdx - CriptoMy - LOF diaphragm muscles . Data are mean ± SEM ( n = 5 biological replicates ; * * * P < 0 . 001 , Student ' s t - test ) .

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

I-J. Quantification of capillary number per area (mm2; I) and average of capillary cross-sectional area (µm2; J) in mdx-Control and mdx-CriptoMy-LOF diaphragm muscles. Data are mean±SEM (n=5 biological replicates; ***P<0.001, Student's t-test).


A . Representative pictures of double immunostaining with VEcad ( red ; left and middle panel ) and KLF4 ( green , left panel ) or TCF4 ( green , middle panel ) , and with CD31 ( red ; right panels , confocal pictures ) and TCF4 ( white , right panel ) on Control and CriptoMy - LOF TA sections at day 5 after injury . B - D . Quantification of VEcad / KLF4 ( B ) , VEcad / TCF4 ( C ) and CD31 / TCF4 ( D ) double positive cells per area ( mm2 ) in Control and CriptoMy - LOF TA sections at day 5 after injury . Data information : Nuclei were counterstained with DAPI ( blue ) .

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

A. Representative pictures of double immunostaining with VEcad (red; left and middle panel) and KLF4 (green, left panel) or TCF4 (green, middle panel), and with CD31 (red; right panels, confocal pictures) and TCF4 (white, right panel) on Control and CriptoMy-LOF TA sections at day 5 after injury. B-D. Quantification of VEcad/KLF4 (B), VEcad/TCF4 (C) and CD31/TCF4 (D) double positive cells per area (mm2) in Control and CriptoMy-LOF TA sections at day 5 after injury. Data information: Nuclei were counterstained with DAPI (blue).


E . Representative Confocal pictures of CD31 + ( red ) and PDGFRα + ( green ) immunostaining on Control and CriptoMy - LOF TA sections at day 5 after injury . F - G . Quantification of CD31 + / PDGFRα + ( F ) and CD31 - / PDGFRα + ( G ) cells per area ( mm2 ) . Data information : Nuclei were counterstained with DAPI ( blue ) .

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

E. Representative Confocal pictures of CD31+ (red) and PDGFRα+ (green) immunostaining on Control and CriptoMy-LOF TA sections at day 5 after injury. F-G. Quantification of CD31+/PDGFRα+ (F) and CD31-/PDGFRα+ (G) cells per area (mm2). Data information: Nuclei were counterstained with DAPI (blue).


H - I . Representative pictures of triple immunostaining with VEcad ( red ) , KLF4 ( green ) and pSMAD3 ( white ) on Control and CriptoMy - LOF TA sections at day 5 after injury ( H ) and quantification of VEcad / KLF4 / pSMAD3 triple positive cells per area ( mm2 ; I ) . Data information : Nuclei were counterstained with DAPI ( blue ) .

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

H-I. Representative pictures of triple immunostaining with VEcad (red), KLF4 (green) and pSMAD3 (white) on Control and CriptoMy-LOF TA sections at day 5 after injury (H) and quantification of VEcad/KLF4/pSMAD3 triple positive cells per area (mm2; I). Data information: Nuclei were counterstained with DAPI (blue).


Representative pictures of double immunostaining with VEcad ( red ) and KLF4 ( green ) on Control and CriptoMy - LOF TA sections at day 5 after re - injury ( CTX - II Data information : Nuclei were counterstained with DAPI ( blue ) .

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

Representative pictures of double immunostaining with VEcad (red) and KLF4 (green) on Control and CriptoMy-LOF TA sections at day 5 after re-injury (CTX-II Data information: Nuclei were counterstained with DAPI (blue).


Representative pictures of double immunostaining with VEcad ( red ) and KLF4 ( green ) on diaphragm sections of mdx - Control and mdx - CriptoMy - LOF Data information : Nuclei were counterstained with DAPI ( blue ) .

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

Representative pictures of double immunostaining with VEcad (red) and KLF4 (green) on diaphragm sections of mdx-Control and mdx-CriptoMy-LOF Data information: Nuclei were counterstained with DAPI (blue).


Quantification of VEcad / KLF4 double positive cells per area ( mm2 ) in Control and CriptoMy - LOF TA sections at day 5 after re - injury ( CTX -

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

Quantification of VEcad/KLF4 double positive cells per area (mm2) in Control and CriptoMy-LOF TA sections at day 5 after re-injury (CTX-II


Quantification of VEcad / KLF4 double positive cells per area ( mm2 ) in diaphragm sections of mdx - Control and mdx - CriptoMy -

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

Quantification of VEcad/KLF4 double positive cells per area (mm2) in diaphragm sections of mdx-Control and mdx-CriptoMy-LOF


Strep - Tactin pull down of GIDAnt ( strep - tagged at Gid8 C - terminus ) probing binding of Gid1057 - 292 and Gid4117 - 362 to the complex visualized with Coomassie - stained SDS - PAGE . The experiment was performed with WT and C - terminal deletion ( ∆ C ) of the substrate receptors ( ∆ 289 - 292 ( FEIA ) and ∆ 359 - 362 ( FEFA ) for Gid10 and Gid4 , respectively ) and WT and mutant ( mut , Gid5W606A / Y613A / Q649A ) GIDAnt .

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

Strep-Tactin pull down of GIDAnt (strep-tagged at Gid8 C-terminus) probing binding of Gid1057-292 and Gid4117-362 to the complex visualized with Coomassie-stained SDS-PAGE. The experiment was performed with WT and C-terminal deletion (∆C) of the substrate receptors (∆289-292 (FEIA) and ∆359-362 (FEFA) for Gid10 and Gid4, respectively) and WT and mutant (mut, Gid5W606A/Y613A/Q649A) GIDAnt.


Lysates from yeast strains expressing endogenously tagged 3xFLAG - Gid4 or 3x - FLAG - Gid10 that were grown in SD complete at 30 ° C ( no stress ) , SE complete for 19 hours ( EtOH ) , SD complete for 1 hour following 19 hour ethanol treatment ( EtOH rec . ) , 42 ° C for 1 hour ( HS ) , SD complete supplemented with 0 . 5M NaCl for 1 hour ( OS ) , or SD - N for 1 hour ( - N ) were run on an SDS - PAGE gel and immunoblotted with αFLAG ( two exposures from the same gel are shown ) and αPGK .

O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Lysates from yeast strains expressing endogenously tagged 3xFLAG-Gid4 or 3x-FLAG-Gid10 that were grown in SD complete at 30°C (no stress), SE complete for 19 hours (EtOH), SD complete for 1 hour following 19 hour ethanol treatment (EtOH rec.), 42°C for 1 hour (HS), SD complete supplemented with 0.5M NaCl for 1 hour (OS), or SD-N for 1 hour (-N) were run on an SDS-PAGE gel and immunoblotted with αFLAG (two exposures from the same gel are shown) and αPGK.


Lysates from yeast strains expressing endogenously tagged 3xFLAG - Gid4 or 3xFLAG - Gid10 that were grown at 42 ° C for one hour , and then returned to 30 ° C for the indicated timepoints were run on an SDS - PAGE gel and immunoblotted with αFLAG ( two exposures from the same gel are shown ) and αPGK .

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

Lysates from yeast strains expressing endogenously tagged 3xFLAG-Gid4 or 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG (two exposures from the same gel are shown) and αPGK.


Lysates from wildtype and ∆ Gid2 yeast strains expressing endogenously tagged 3xFLAG - Gid10 that were grown at 42 ° C for one hour , and then returned to 30 ° C for the indicated timepoints were run on an SDS - PAGE gel and immunoblotted with αFLAG and αPGK .

Lysates from wildtype and ∆Gid2 yeast strains expressing endogenously tagged 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK.


Lysates from wildtype , Gid5W606A , Y613A , Q649A , and Gid10 ∆ C strains expressing endogenously tagged 3xFLAG - Gid10 that were grown at 42 ° C for one hour , and then returned to 30 ° C for the indicated timepoints were run on an SDS - PAGE gel and immunoblotted with αFLAG and αPGK .

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

Lysates from wildtype, Gid5W606A,Y613A,Q649A, and Gid10∆C strains expressing endogenously tagged 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK.


Lysates from wildtype , ∆ Gid4 , and Gid4 ∆ C strains expressing endogenously tagged 3xFLAG - Gid10 that were grown at 42 ° C for one hour , and then returned to 30 ° C for the indicated timepoints were run on an SDS - PAGE gel and immunoblotted with αFLAG and αPGK .

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

Lysates from wildtype, ∆Gid4, and Gid4∆C strains expressing endogenously tagged 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK.


Wildtype and Gid10 overexpressing yeast strains expressing endogenously tagged Gid4 were grown for 19 hours in YPE , and transitioned to YPD for the indicated timepoints . Lysates were run on an SDS - PAGE gel and immunoblotted with αFLAG and αPGK . Points represent mean , error bars represent standard deviation ( n > 3 biological replicates ) .

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

Wildtype and Gid10 overexpressing yeast strains expressing endogenously tagged Gid4 were grown for 19 hours in YPE, and transitioned to YPD for the indicated timepoints. Lysates were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK. Points represent mean, error bars represent standard deviation (n>3 biological replicates).


In vitro competition assay probing the ability of N - terminally 2xStrep - tagged Gid4 ( 2xS - Gid4 ) to exchange with Gid10 in the GIDSR10 complex . Proteins after Strep - Tactin pull - down were visualized by Coomassie - stained SDS - PAGE .

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

In vitro competition assay probing the ability of N-terminally 2xStrep-tagged Gid4 (2xS-Gid4) to exchange with Gid10 in the GIDSR10 complex. Proteins after Strep-Tactin pull-down were visualized by Coomassie-stained SDS-PAGE.


Art2 - 3xFLAG was immunocaptured from yeast cells grown in YPD and incubated with GidSR10 , GidSR4 , GidAnt , or Gid10 for the indicated timepoints . Progress of the reaction was followed by αFLAG immunoblot .

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

Art2-3xFLAG was immunocaptured from yeast cells grown in YPD and incubated with GidSR10, GidSR4, GidAnt, or Gid10 for the indicated timepoints. Progress of the reaction was followed by αFLAG immunoblot.


Mdh2 - 3xFLAG was immunocaptured from yeast cells following growth in YPE for 19h and incubated with GidSR10 , GidSR4 , GidAnt , or Gid4 for the indicated timepoints . Progress of the reaction was followed by αFLAG immunoblot .

Mdh2-3xFLAG was immunocaptured from yeast cells following growth in YPE for 19h and incubated with GidSR10, GidSR4, GidAnt, or Gid4 for the indicated timepoints. Progress of the reaction was followed by αFLAG immunoblot.


Art2 - 3xFLAG or Art2P2S - 3xFLAG was immunocaptured from yeast cells grown in YPD and incubated with GidSR10 , or GidSR10 ∆ C for the indicated timepoints . Progress of the reaction was followed by αFLAG immunoblot .

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

Art2-3xFLAG or Art2P2S-3xFLAG was immunocaptured from yeast cells grown in YPD and incubated with GidSR10, or GidSR10∆C for the indicated timepoints. Progress of the reaction was followed by αFLAG immunoblot.


In vitro ubiquitination assay probing the ability of Gid10 and Gid4 to promote ubiquitination of Art22 - 29 N - terminus and its P2S mutant . Progress of the reaction was monitored by fluorescent scan of the gel visualizing the Art2 peptide with FAM appended to its C - terminus ( pep * ) .

In vitro ubiquitination assay probing the ability of Gid10 and Gid4 to promote ubiquitination of Art22-29 N-terminus and its P2S mutant. Progress of the reaction was monitored by fluorescent scan of the gel visualizing the Art2 peptide with FAM appended to its C-terminus (pep*).


( A - F ) Vero E6 - TMPRSS2 ( A , C , E ) or Vero - E6 cells ( B , D , F ) were infected with SARS - CoV - 2 WA1 at an MOI of 0 . 001 . Supernatants were collected twice daily for two days , daily for an additional two days , and cells and supernatants were collected for a final time point at day 7 ( for Vero E6 - TMPRSS2 cells ) or day 8 ( for Vero E6 cells ) . A , B ) Viral titer was determined by immune - focus forming assay ( focus - forming units [ FFU ] / mL ) , day 0 values are calculated based on known titer of inoculum . RNA levels were determined by RT - PCR from cells ( C , D ) or viral supernatants ( E , F ) for each time point using the indicated primer - probe sets . Note that CT is plotted inversely on the Y axes , so that lower RNA concentrations are shown lower on the axes . All data points represent four technical replicates from two independent experiments and are plotted as mean with error bars plotted as + / - SEM . Limit of detection for infectious titer is plotted as the minimum y value .

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

(A-F) Vero E6-TMPRSS2 (A, C, E) or Vero-E6 cells (B, D, F) were infected with SARS-CoV-2 WA1 at an MOI of 0.001. Supernatants were collected twice daily for two days, daily for an additional two days, and cells and supernatants were collected for a final time point at day 7 (for Vero E6-TMPRSS2 cells) or day 8 (for Vero E6 cells). A, B) Viral titer was determined by immune-focus forming assay (focus-forming units [FFU]/mL), day 0 values are calculated based on known titer of inoculum. RNA levels were determined by RT-PCR from cells (C, D) or viral supernatants (E, F) for each time point using the indicated primer-probe sets. Note that CT is plotted inversely on the Y axes, so that lower RNA concentrations are shown lower on the axes. All data points represent four technical replicates from two independent experiments and are plotted as mean with error bars plotted as +/- SEM. Limit of detection for infectious titer is plotted as the minimum y value.


( A - D ) Pooled patient samples at high ( 5 . 7x104 copies ) , medium ( 3 . 6x102 copies ) or low ( 2x101 copies ) concentrations ( A , B ) , or stocks of SARS - CoV - 2 WA1 ( C , D ) were subjected to - 80ºC storage interrupted by ( A , C ) successive freeze - thaw cycles or ( B , D ) storage at 4ºC , room temperature ( RT ) or 37ºC for the indicated time periods . N = 1 for A , B . For C and D datapoints are from 3 different viral stocks titered in duplicate and plotted as mean with error bars plotted as + / - SEM . For C raw values are plotted while values are normalized to starting titer for D . Limit of detection for infectious titer in C is indicated by a dashed line .

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

(A-D) Pooled patient samples at high (5.7x104 copies), medium (3.6x102 copies) or low (2x101 copies) concentrations (A, B), or stocks of SARS-CoV-2 WA1 (C, D) were subjected to -80ºC storage interrupted by (A, C) successive freeze-thaw cycles or (B, D) storage at 4ºC, room temperature (RT) or 37ºC for the indicated time periods. N=1 for A, B. For C and D datapoints are from 3 different viral stocks titered in duplicate and plotted as mean with error bars plotted as +/- SEM. For C raw values are plotted while values are normalized to starting titer for D. Limit of detection for infectious titer in C is indicated by a dashed line.


( A _ B ) Vero E6 - TMPRSS2 ( A ) or Vero E6 ( B ) cells were inoculated with a total of 126 clinical NP swab samples representing a range of viral RNA levels , both total E RNA ( Clinical Sample E CT detected by WHO - E primer / probe set ) and subgenomic E RNA ( CT detected by the Mills - SgE primer set developed in this study ) . Culture supernatants were harvested when cells displayed CPE or at day 7 if no CPE was observed earlier , clarified , and presence ( blue square ) or absence ( yellow circle ) of SARS - CoV - 2 determined by immuno - focus assay .

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

(A_B) Vero E6-TMPRSS2 (A) or Vero E6 (B) cells were inoculated with a total of 126 clinical NP swab samples representing a range of viral RNA levels, both total E RNA (Clinical Sample E CT detected by WHO-E primer/probe set) and subgenomic E RNA (CT detected by the Mills-SgE primer set developed in this study). Culture supernatants were harvested when cells displayed CPE or at day 7 if no CPE was observed earlier, clarified, and presence (blue square) or absence (yellow circle) of SARS-CoV-2 determined by immuno-focus assay.


( A - B ) Supernatants from the panel of infected ( A ) Vero E6 - TMPRSS2 and ( B ) Vero E6 cells were harvested when CPE was observed and the viral titer ( FFU / mL measured by focus forming assay ) was determined for the final time point of each sample that successfully isolated . Samples are colored according to the day CPE was observed and the sample was harvested . Total E RNA CT of the original clinical sample used for viral isolation is plotted on the X axis . Data shown is from the subset of samples that successfully isolated in each cell line . Limit of detection for infectious titer is 400 FFU / mL .

(A-B) Supernatants from the panel of infected (A) Vero E6-TMPRSS2 and (B) Vero E6 cells were harvested when CPE was observed and the viral titer (FFU/mL measured by focus forming assay) was determined for the final time point of each sample that successfully isolated. Samples are colored according to the day CPE was observed and the sample was harvested. Total E RNA CT of the original clinical sample used for viral isolation is plotted on the X axis. Data shown is from the subset of samples that successfully isolated in each cell line. Limit of detection for infectious titer is 400 FFU/mL.


( A ) The amount of subgenomic E RNA in the panel of clinical samples used to infect Vero E6 - TMPRSS2 cells was measured using a previously - published primer - probe set ( Wölfel - sgE )

(A) The amount of subgenomic E RNA in the panel of clinical samples used to infect Vero E6-TMPRSS2 cells was measured using a previously-published primer-probe set (Wölfel-sgE)


( B ) For a subset of 85 NP swab samples for which sufficient material remained , SARS - CoV - 2 negative - strand E RNA levels were determined by strand - specific RT - PCR with tagged primers . Data information : Culture supernatants were harvested when cells displayed CPE or at day 7 if no CPE was observed earlier , clarified , and presence ( blue square ) or absence ( yellow circle ) of infectious SARS - CoV - 2 determined by immuno - focus assay .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) For a subset of 85 NP swab samples for which sufficient material remained, SARS-CoV-2 negative-strand E RNA levels were determined by strand-specific RT-PCR with tagged primers. Data information: Culture supernatants were harvested when cells displayed CPE or at day 7 if no CPE was observed earlier, clarified, and presence (blue square) or absence (yellow circle) of infectious SARS-CoV-2 determined by immuno-focus assay.


Levels of the cellular housekeeping genes GAPDH and 18S rRNA , negative - strand E ( negE ) , and sgE relative to total levels of E RNA were measured in infected cells , clarified supernatants and virus concentrated through a sucrose cushion by ultracentrifugation . Data points represent four technical replicates from two independent experiments ( Cell and Supernatant ) or three replicates ( Concentrated Virus ) and are normalized to the total levels of E for each sample , plotted as mean with error bars plotted as + / - SEM .

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

Levels of the cellular housekeeping genes GAPDH and 18S rRNA, negative-strand E (negE), and sgE relative to total levels of E RNA were measured in infected cells, clarified supernatants and virus concentrated through a sucrose cushion by ultracentrifugation. Data points represent four technical replicates from two independent experiments (Cell and Supernatant) or three replicates (Concentrated Virus) and are normalized to the total levels of E for each sample, plotted as mean with error bars plotted as +/- SEM.


B . Blots showing expression of hTauAT ( hTau , Mr ~ 67 kDa ) and / or endogenous mouse Tau ( mTau , Mr ~ 45 - 55 kDa ) in the hippocampus of 10 month old heterozygous ( + / - ) and homozygous ( + / + ) hTauAT mice in comparison to age matched control ( Ctrl ) detected by pan - Tau antibody K9JA . Actin at 42 kDa serves as loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Blots showing expression of hTauAT (hTau, Mr ~67 kDa) and/or endogenous mouse Tau (mTau, Mr ~45-55 kDa) in the hippocampus of 10 month old heterozygous (+/-) and homozygous (+/+) hTauAT mice in comparison to age matched control (Ctrl) detected by pan-Tau antibody K9JA. Actin at 42 kDa serves as loading control.


C . Quantification of ( B ) . Ratio of hTau / mTau indicating a higher hTauAT - expression in homozygous mice ( ~ 1 . 8 ) compared to heterozygous mice ( ~ 1 . 0 ) . Each bar represents an average of n = 4 animals , error bars represent SEM .

C. Quantification of (B). Ratio of hTau / mTau indicating a higher hTauAT -expression in homozygous mice (~1.8) compared to heterozygous mice (~1.0). Each bar represents an average of n=4 animals, error bars represent SEM.


D . Quantification of mRNA levels in hippocampi of heterozygous and homozygous hTauAT 16 month old mice ( n = 4 per group ) .

D. Quantification of mRNA levels in hippocampi of heterozygous and homozygous hTauAT 16 month old mice (n = 4 per group).


E . Distribution of hTauAT visualized by the human Tau specific antibody HT7 in the hippocampal CA3 - region of hetero - and homozygous hTauATmice at the age of 14 month . Notice the mis - sorted hTauAT in cell bodies and dendrites ( arrowheads ) of pyramidal neurons of the CA3 region and immunreactivity of the axons ( mossy fibers , asterisks ) . By contrast the control shows no immunoreactivity .

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

E. Distribution of hTauAT visualized by the human Tau specific antibody HT7 in the hippocampal CA3-region of hetero- and homozygous hTauATmice at the age of 14 month. Notice the mis-sorted hTauAT in cell bodies and dendrites (arrowheads) of pyramidal neurons of the CA3 region and immunreactivity of the axons (mossy fibers, asterisks). By contrast the control shows no immunoreactivity.


F . Elevated hTauAT - expression causes Tau - pathology inside area CA3 of the hippocampus . Note the increase in Tau phosphorylation probed with the antibody against pT217 , a site upstream of the repeat domain . Arrowheads point to areas with phosphorylated and mislocalized Tau in cell somata of pyramidal neurons in area CA3 .

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

F. Elevated hTauAT -expression causes Tau-pathology inside area CA3 of the hippocampus. Note the increase in Tau phosphorylation probed with the antibody against pT217, a site upstream of the repeat domain. Arrowheads point to areas with phosphorylated and mislocalized Tau in cell somata of pyramidal neurons in area CA3.


G . Tauopathy detected by antibody AT180 specific for phosphorylation sites pT231 + pSer235 ( arrowheads denote somato - dendritic mislocalized tau ) .

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

G. Tauopathy detected by antibody AT180 specific for phosphorylation sites pT231+pSer235 (arrowheads denote somato-dendritic mislocalized tau).


H . Antibody PHF - 1 ( phosphorylation sites pSer396 + pSer404 ) illustrates pathological phosphorylation of Tau due to hTauAT expression especially in stratum lucidum ( asterisks ) of area CA3 and pyramidal neuroncell bodies ( arrows ) .

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

H. Antibody PHF-1 (phosphorylation sites pSer396+pSer404) illustrates pathological phosphorylation of Tau due to hTauAT expression especially in stratum lucidum (asterisks) of area CA3 and pyramidal neuroncell bodies (arrows).


I . Tau phosphorylation detected by antibody AT8 ( pS202 + pT205 ) in stratum pyramidale of area CA3 ( arrowhead ) . Note that immunoreactivity increases with expression level .

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

I. Tau phosphorylation detected by antibody AT8 (pS202+pT205) in stratum pyramidale of area CA3 (arrowhead). Note that immunoreactivity increases with expression level.


A . Conformational change of Tau detected by ALZ - 50 antibody immunoreactivity in stratum lucidum ( asterisks ) of area CA3 and somata in stratum pyramidale ( arrowheads ) .

A. Conformational change of Tau detected by ALZ-50 antibody immunoreactivity in stratum lucidum (asterisks) of area CA3 and somata in stratum pyramidale (arrowheads).


B . Tau aggregation confirmed by Gallyas silver staining of NFTs bearing neurons in hetero - and homozygous hTauAT mice at 14 month of age compared to non - reactive control littermate mice . In homozygous hTauAT mice the extent of neurofibrillary tangles ( NFTs ) visualized by Gallyas silver staining is enhanced compared to heterozygous hTauAT mice ( white arrowheads ) . The control shows no silver - reactive Tau aggregates .

B. Tau aggregation confirmed by Gallyas silver staining of NFTs bearing neurons in hetero- and homozygous hTauAT mice at 14 month of age compared to non-reactive control littermate mice. In homozygous hTauAT mice the extent of neurofibrillary tangles (NFTs) visualized by Gallyas silver staining is enhanced compared to heterozygous hTauAT mice (white arrowheads). The control shows no silver-reactive Tau aggregates.


C . Western blot analysis using the pan - Tau antibody K9JA indicates sarcosyl - insoluble Tau species of human hTauAT ( upper band ) and mouse Tau ( mTau , lower band ) . Note the enhanced Tau aggregation in homozygous hTauAT mice compared to heterozygous hTauAT mice and control mice . D . Quantification of ( C ) . The ratio of hTauAT / mTau indicates a stronger aggregation in the homozygous hTauAT mice ( ~ 1 . 4 ) compared to heterozygous hTauAT mice ( ~ 0 . 7 ( n = 4 animals , error bars represent SEM ) .

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

C. Western blot analysis using the pan-Tau antibody K9JA indicates sarcosyl-insoluble Tau species of human hTauAT (upper band) and mouse Tau (mTau, lower band). Note the enhanced Tau aggregation in homozygous hTauAT mice compared to heterozygous hTauAT mice and control mice.D. Quantification of (C). The ratio of hTauAT /mTau indicates a stronger aggregation in the homozygous hTauAT mice (~1.4) compared to heterozygous hTauAT mice (~0.7 (n=4 animals, error bars represent SEM).


E . Electron micrograph of CA3 region of the hippocampus illustrating typical synapse with a perforated postsynaptic density ( asterisks ) in a control animal at 12 months . ( ds ) dendriticspine , ( pt ) presynaptic terminal and ( m ) mitochondrium . F . Example of a degenerating synaptic bouton ( arrow ) containing swollen synaptic vesicles in proximity to a large terminal with a perforated postsynaptic density ( asterisks ) in a transgenic animal at 13 months . G . Electron dense neuronal cytoplasm with dark nucleoplasm and various vacuoles ( arrow ) detected in CA3pyramidal neurons ( arrow ) at 13 months in transgenic animals in proximity to normally appearing neurons ( asterisks ) , degenerating apical dendrite ( d ) . H . Neuropil of a transgenic animal ( 13 months ) with a degenerating neuritic profile ( arrow ) . I . Degenerating dendrite ( arrow ) in a transgenic animal with darkened cytoplasm and abnormally distributed mitochondria ( arrow ) . J . Degenerating dendrite containing electron - dense whorling membrane fragments ( arrow ) in proximity to normally appearing synapses ( synapse with a perforated postsynaptic density , asterisks ) .

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

E. Electron micrograph of CA3 region of the hippocampus illustrating typical synapse with a perforated postsynaptic density (asterisks) in a control animal at 12 months. (ds) dendriticspine, (pt) presynaptic terminal and (m) mitochondrium.F. Example of a degenerating synaptic bouton (arrow) containing swollen synaptic vesicles in proximity to a large terminal with a perforated postsynaptic density (asterisks) in a transgenic animal at 13 months.G. Electron dense neuronal cytoplasm with dark nucleoplasm and various vacuoles (arrow) detected in CA3pyramidal neurons (arrow) at 13 months in transgenic animals in proximity to normally appearing neurons (asterisks), degenerating apical dendrite (d).H. Neuropil of a transgenic animal (13 months) with a degenerating neuritic profile (arrow).I. Degenerating dendrite (arrow) in a transgenic animal with darkened cytoplasm and abnormally distributed mitochondria (arrow).J. Degenerating dendrite containing electron-dense whorling membrane fragments (arrow) in proximity to normally appearing synapses (synapse with a perforated postsynaptic density, asterisks).


A . Western blot analysis with pan - Tau K9JA antibody to estimate expression levels of hTauAT and endogenous Tau ( mTau ) . Slice homogenates from non - transgenic littermate ( lane 1 , Ctrl ) , heterozygouse hTauAT ( lane 2 , ( + / - ) ) or homozygouse hTauAT slices ( lane 3 , ( + / + ) ) were analyzed after three weeks in culture . Actin served as a loading control . B . Quantification of hTauAT expression levels ( n = 3 - 6 slice homogenates ) . The ratio between hTauAT and endogenous mouse Tau is shown .

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

A. Western blot analysis with pan-Tau K9JA antibody to estimate expression levels of hTauAT and endogenous Tau (mTau). Slice homogenates from non-transgenic littermate (lane 1, Ctrl), heterozygouse hTauAT (lane 2, (+/-)) or homozygouse hTauAT slices (lane 3, (+/+)) were analyzed after three weeks in culture. Actin served as a loading control.B. Quantification of hTauAT expression levels (n=3-6 slice homogenates). The ratio between hTauAT and endogenous mouse Tau is shown.


C . Immunohistochemistry with antibodies against human Tau ( TauY9 antibodies ; middle panel ) and dendritic marker MAP2 ( left panel ) in slice cultures at DIV 10 . Stratum radiatum of area CA3 is shown . Although MAP2 staining is apparent , no signal is detected by the TauY9 antibody in control slices . In contrast , in hTauAT slices human Tau is detected in both dendrites and axons ( see asterisk in merged image ) .

C. Immunohistochemistry with antibodies against human Tau (TauY9 antibodies; middle panel) and dendritic marker MAP2 (left panel) in slice cultures at DIV 10. Stratum radiatum of area CA3 is shown. Although MAP2 staining is apparent, no signal is detected by the TauY9 antibody in control slices. In contrast, in hTauAT slices human Tau is detected in both dendrites and axons (see asterisk in merged image).


D . Immunohistochemistry against phosphorylated Tau at the PHF1 - epitope in area CA3 of a control ( upper lane ) or hTauAT slice ( bottom lane ) . PHF1 staining is slightly visible in some axons in control slices . In contrast , PHF1 - phosphorylated Tau is apparent in somata , dendrites and axons of neurons in hTauAT slice cultures .

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

D. Immunohistochemistry against phosphorylated Tau at the PHF1-epitope in area CA3 of a control (upper lane) or hTauAT slice (bottom lane). PHF1 staining is slightly visible in some axons in control slices. In contrast, PHF1-phosphorylated Tau is apparent in somata, dendrites and axons of neurons in hTauAT slice cultures.


E . Higher magnification image of apical dendrites in area CA3 in a hTauAT slice after immunohistochemistry against MAP2 ( red ) and Tau ( K9JA antibody , green ) . Tau staining is seen in dendritic spines ( asterisks ) in contrast to MAP2 , which is restricted to the dendritic shaft .

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

E. Higher magnification image of apical dendrites in area CA3 in a hTauAT slice after immunohistochemistry against MAP2 (red) and Tau (K9JA antibody, green). Tau staining is seen in dendritic spines (asterisks) in contrast to MAP2, which is restricted to the dendritic shaft.


A . Slices ( DIV15 ) from control mice were loaded with the Ca + + sensitive dye Fura - 2AM . Ratiometric images are presented at baseline and after application of high potassium . Note the increase in intracellular Ca + + ( [ Ca + + ] i ) after KCl application . B . Slices ( DIV15 ) from hTauAT mice were loaded with the Ca + + sensitive dye Fura - 2AM . Ratiometric images at baseline and after application of high potassium in area CA3 are depicted . Note the increase in [ Ca + + ] i ( false color legend ) under both conditions in hTauAT slices compared to controls .

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

A. Slices (DIV15) from control mice were loaded with the Ca++ sensitive dye Fura-2AM. Ratiometric images are presented at baseline and after application of high potassium. Note the increase in intracellular Ca++ ([Ca++]i) after KCl application.B. Slices (DIV15) from hTauAT mice were loaded with the Ca++ sensitive dye Fura-2AM. Ratiometric images at baseline and after application of high potassium in area CA3 are depicted. Note the increase in [Ca++]i (false color legend) under both conditions in hTauAT slices compared to controls.


C . Electrophysiological example traces ( control = black ; hTauAT = red ) of excitatory postsynaptic field potentials ( fEPSP ) depicts examples of depolarizations that are induced by a single high potassium chloride application in stratum pyramidale ( s . p . ) of area CA3 in slices in which calcium imaging experiments were conducted . The example depolarization evokes averaged calcium influxes into neurons depicted in the calcium imaging graph . The graph shows the mean of [ Ca + + ] i changes in response to high KCl in stratum radiatum and stratum pyramidale of area CA3 in Fura2 - AM loaded hippocampal slices . After depolarization [ Ca + + ] i rises up to ~ 600 nM in hTauAT slices ( red trace , n = 6 slices , prepared from at least three animals ) , compared to ~ 300 nM in control slices ( black trace , n = 6 slices ; prepared from at least three animals ) . Note that even under resting conditions [ Ca + + ] i is elevated to ~ 120nM due to hTauAT expression .

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

C. Electrophysiological example traces (control = black; hTauAT = red) of excitatory postsynaptic field potentials (fEPSP) depicts examples of depolarizations that are induced by a single high potassium chloride application in stratum pyramidale (s.p.) of area CA3 in slices in which calcium imaging experiments were conducted. The example depolarization evokes averaged calcium influxes into neurons depicted in the calcium imaging graph. The graph shows the mean of [Ca++]i changes in response to high KCl in stratum radiatum and stratum pyramidale of area CA3 in Fura2-AM loaded hippocampal slices. After depolarization [Ca++]i rises up to ~600 nM in hTauAT slices (red trace, n=6 slices, prepared from at least three animals), compared to ~300 nM in control slices (black trace, n=6 slices; prepared from at least three animals). Note that even under resting conditions [Ca++]i is elevated to ~120nM due to hTauAT expression.


D . Quantification of the maximum [ Ca + + ] i from ratiometric images as depicted in ( A ) and ( B ) after background subtraction and selection of regions of interest ( 10 circles of fixed diameter ) in stratum radiatum border to stratum pyramidale of area CA3 . Under resting conditions [ Ca + + ] i was elevated in transgenic slice cultures ( ~ 120 nM , n = 11 ) compared to controls ( ~ 80 nM , n = 8 ) . Slices were incubated with one of the following channel blockers : Nifedipine ( NIF , VGCC ; Ctrl n = 8 ; A152T : n = 7 ) , APV ( NMDAR ; Ctrl n = 7 ; A152T n = 8 ) , ifenprodil ( IFEN , NMDAR ; Ctrl n = 8 ; A152T n = 7 ) , memantine ( MEM , NMDAR ; Ctrl / A152T n = 6 ) , 6 - cyano - 7 - nitroquinoxaline - 2 , 3 - dione ( CNQX ; AMPAR ; Ctrl n = 8 ; A152T n = 7 ) ) , tetrodotoxin ( TTX , VGNaC ; Ctrl n = 4 ; A152T n = 5 ) , tetanus neurotoxin ( TeNT , neurotransmitter release ; A152T n = 6 ) or in Ca + + free buffer ( Ctrl n = 10 ; A152T n = 7 ) . For each experiment slice preparations from at least three different mice were used . One - way ANOVA followed by Tukey ' s post - hoc test ; Ctrl : F ( 7 / 51 ) . 3 , 533 ; p = 0 . 0036 ; A152T : F ( 8 / 55 ) = 5 , 230 ; p < 0 . 0001 . For an overview of drug concentrations see EV Table 1 . E . Quantification of the maximum peak increase in intracellular calcium in the same slices than in ( D ) after high KCl stimulation . One way ANOVA followed by Tukey ' s post - hoc test Ctrl : F ( 7 / 45 ) = 4 , 080 ; p = 0 . 0015 ; A152T : F ( 8 / 53 ) = 7 , 580 ; p < 0 . 0001 ) . Error bars represent SEM ; One - Way ANOVA * p < 0 . 05 ; * * p < 0 . 01 and * * * p < 0 . 001 .

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

D. Quantification of the maximum [Ca++]i from ratiometric images as depicted in (A) and (B) after background subtraction and selection of regions of interest (10 circles of fixed diameter) in stratum radiatum border to stratum pyramidale of area CA3. Under resting conditions [Ca++]i was elevated in transgenic slice cultures (~120 nM, n=11) compared to controls (~80 nM, n=8). Slices were incubated with one of the following channel blockers: Nifedipine (NIF, VGCC; Ctrl n=8; A152T: n=7), APV (NMDAR; Ctrl n=7; A152T n=8), ifenprodil (IFEN, NMDAR; Ctrl n=8; A152T n=7), memantine (MEM, NMDAR; Ctrl/A152T n=6), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; AMPAR; Ctrl n=8; A152T n=7)), tetrodotoxin (TTX, VGNaC; Ctrl n=4; A152T n=5), tetanus neurotoxin (TeNT, neurotransmitter release; A152T n=6) or in Ca++ free buffer (Ctrl n=10; A152T n=7). For each experiment slice preparations from at least three different mice were used. One-way ANOVA followed by Tukey's post-hoc test; Ctrl: F(7/51) .3,533; p=0.0036; A152T: F(8/55)=5,230; p<0.0001.For an overview of drug concentrations see EV Table 1.E. Quantification of the maximum peak increase in intracellular calcium in the same slices than in (D) after high KCl stimulation. One way ANOVA followed by Tukey's post-hoc test Ctrl: F(7/45)=4,080; p=0.0015; A152T: F(8/53)=7,580; p<0.0001).Error bars represent SEM; One-Way ANOVA *p<0.05; **p<0.01 and ***p<0.001.


A . Glutamate content in the culture medium ( [ Glu ] e ) from either hTauAT ( n = 10 culture inserts containing 6 slices each ) or control slices ( n = 5 - 12 culture inserts ) analyzed at DIV 5 , 10 , 20 and 25 . Glutamate content from medium cultured with hTauAT slices is shown in percent of control at each time point . Increased levels were observed between DIV 5 and DIV 20 . At DIV 25 glutamate levels already declined to control levels . One - way ANOVA followed by Tukey ' s post - hoc test * p < 0 . 05 ; * * p < 0 . 01 * * * p < 0 . 001 . B . Lactate dehydrogenase ( LDH ) release ( a measure of cytotoxicity ) analyzed in hTauAT ( n = 17 - 19 culture inserts containing 6 slices each ) or control slices ( n = 15 - 16 culture inserts ) at DIV 5 , 10 , 20 and 25 . LDH release of hTauAT slices is shown in percent of control at each time point . A pronounced increase ( ~ 25 % ) in LDH release was observed due to hTauAT expression starting at DIV 10 but not at DIV 5 One - way ANOVA : * p < 0 . 05 ; * * p < 0 . 01 and * * * p < 0 . 001 .

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

A. Glutamate content in the culture medium ([Glu]e) from either hTauAT (n=10 culture inserts containing 6 slices each) or control slices (n=5-12 culture inserts) analyzed at DIV 5, 10, 20 and 25. Glutamate content from medium cultured with hTauAT slices is shown in percent of control at each time point. Increased levels were observed between DIV 5 and DIV 20. At DIV 25 glutamate levels already declined to control levels. One-way ANOVA followed by Tukey's post-hoc test *p<0.05; **p<0.01 ***p<0.001.B. Lactate dehydrogenase (LDH) release (a measure of cytotoxicity) analyzed in hTauAT (n=17-19 culture inserts containing 6 slices each) or control slices (n=15-16 culture inserts) at DIV 5, 10, 20 and 25. LDH release of hTauAT slices is shown in percent of control at each time point. A pronounced increase (~25%) in LDH release was observed due to hTauAT expression starting at DIV 10 but not at DIV 5 One-way ANOVA: *p<0.05; **p<0.01 and ***p<0.001.


C . Slice cultures from control ( left ) , hTauAT ( middle ) or hTauAT treated with ceftriaxone ( CEF ; 100 µM ; right ) stained against neuronal nuclear protein ( NeuN ) at DIV 30 . Representative neuronal cell layers are shown . hTauAT slices show neuronal loss in the granular cell layer of the dentate gyrus and the pyramidal cell layer in the region of the hilus in area CA3 ( middle panel , red arrows ) . Treatment with ceftriaxone for 30 days prevents neuronal loss in DG and area CA3 . D . NeuN positive cell bodies were counted in the DG and within the pyramidal cell layer in area CA3 and CA1 at DIV 30 in defined regions of interest ( ROI ) . The number of neurons was reduced in hTauAT slices , both within area DG and CA3 , whereas the number of neurons in the CA1 was only slightly affected . Neuronal loss was largely prevented in cultures treated with ceftriaxone ( CEF ) in dentate gyrus and area CA3 ( n = 14 - 19 slices per group and area ; prepared from at least 6 animals ) . One - way ANOVA followed by Tukey ' s post - hoc test * p < 0 . 05 ; * * p < 0 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Slice cultures from control (left), hTauAT (middle) or hTauAT treated with ceftriaxone (CEF; 100 µM; right) stained against neuronal nuclear protein (NeuN) at DIV 30. Representative neuronal cell layers are shown. hTauAT slices show neuronal loss in the granular cell layer of the dentate gyrus and the pyramidal cell layer in the region of the hilus in area CA3 (middle panel, red arrows). Treatment with ceftriaxone for 30 days prevents neuronal loss in DG and area CA3.D. NeuN positive cell bodies were counted in the DG and within the pyramidal cell layer in area CA3 and CA1 at DIV 30 in defined regions of interest (ROI). The number of neurons was reduced in hTauAT slices, both within area DG and CA3, whereas the number of neurons in the CA1 was only slightly affected. Neuronal loss was largely prevented in cultures treated with ceftriaxone (CEF) in dentate gyrus and area CA3 (n=14-19 slices per group and area; prepared from at least 6 animals). One-way ANOVA followed by Tukey's post-hoc test *p<0.05; **p<0.01


A . Representative microphotograph of spines on dendrites from control littermate mice at the age of 12 months . Apical dendrites from CA3pyramidal neurons are visualized by Golgi staining . The contrast of photomicrographs was inverted . B . Dendritic spines from heterozygous hTauATmice , seen by Golgi staining . C . Quantification of dendritic spine densities in control littermate and hTauAT mice . Note that the spine density in heterozygous hTauAT is increased ( control littermates : n = 2 animals ; heterozygous hTauATn = 4 animals ; an average of 5 dendrites of CA3pyramidal neurons were analyzed per animal ; unpaired t - test ) .

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

A. Representative microphotograph of spines on dendrites from control littermate mice at the age of 12 months. Apical dendrites from CA3pyramidal neurons are visualized by Golgi staining. The contrast of photomicrographs was inverted.B. Dendritic spines from heterozygous hTauATmice, seen by Golgi staining.C. Quantification of dendritic spine densities in control littermate and hTauAT mice. Note that the spine density in heterozygous hTauAT is increased (control littermates: n = 2 animals; heterozygous hTauATn = 4 animals; an average of 5 dendrites of CA3pyramidal neurons were analyzed per animal; unpaired t-test).


D . Input / output curve of field potential recording in the mossy fiber pathway in acute slices of 12 ± 1 month old mice demonstrating significantly enhanced basal synaptic transmission ( Multiple repeat ANOVA , F ( 9 , 126 ) = 2 . 00 , p = 0 . 044 ) in area CA3 in mice expressing hTauAT . Scale bars : 500 µV vertical bar and 20 msec horizontal bar .

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

D. Input / output curve of field potential recording in the mossy fiber pathway in acute slices of 12±1 month old mice demonstrating significantly enhanced basal synaptic transmission (Multiple repeat ANOVA,F(9, 126)=2.00, p=0.044) in area CA3 in mice expressing hTauAT. Scale bars: 500 µV vertical bar and 20 msec horizontal bar.


E . Long - term potentiation in the mossy fiber pathway demonstrates no differences in long - term plasticity between control ( black ) and hTauAT expressing mice ( red ) .

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

E. Long-term potentiation in the mossy fiber pathway demonstrates no differences in long-term plasticity between control (black) and hTauAT expressing mice (red).


F . Input / output curve of field potential recording in stratum pyramidale ( s . p . ) of area CA3 in organotypic hippocampal slices at DIV 30 . Somatic field potentials are increased in hTauAT compared to control littermates . Scale bars : 800 µV vertical bar and 20 msec horizontal bar .

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

F. Input / output curve of field potential recording in stratum pyramidale (s.p.) of area CA3 in organotypic hippocampal slices at DIV 30. Somatic field potentials are increased in hTauAT compared to control littermates. Scale bars: 800 µV vertical bar and 20 msec horizontal bar.


A . Example of electrophysiological fEPSP recording in stratum pyramidale of area CA3 in a control slice ( DIV30 ) during wash - in of 50 µm picrotoxin . This leads to blockage of GABAergic transmission followed by epileptiform discharges ( burst ) occurring approximately after 3 min . The zoom - in reveals that such an epileptiform discharge ( burst ) consists of repetitive firings . B . Similar example trace as in ( A ) recorded in a hTauAT expressing slice ( red ) demonstrating an increase in burst frequency as well as in firings per burst due to hTauAT expression . C . Quantification of bursts per minute in DIV 10 slices from control littermates and hTauAT slices demonstrating a ~ 2 - fold increase in hTauAT slices ( n = 8 slices per group ) . D . Firings per burst are increased ~ 1 . 5 - fold already at the early time point DIV10 due to hTauAT expression . E . Analysis of burst frequency in slices at DIV 30 demonstrates the progression of the pathological effect beyond that observed at DIV 10 . Burst frequency is enhanced in hTauAT expressing slices ( n = 10 slices ) . If Ceftriaxone ( CEF ) is applied to hTauAT slices the pathological burst frequency enhancement is partly prevented ( n = 5 slices ) . F . Firings per burst are strongly enhanced due to hTauAT expression in slices at DIV 30 . Ceftriaxone prevents this pathological phenotype if applied during the entire period of cultivation ( n = 6 slices ) .

O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

A. Example of electrophysiological fEPSP recording in stratum pyramidale of area CA3 in a control slice (DIV30) during wash-in of 50 µm picrotoxin. This leads to blockage of GABAergic transmission followed by epileptiform discharges (burst) occurring approximately after 3 min. The zoom-in reveals that such an epileptiform discharge (burst) consists of repetitive firings.B. Similar example trace as in (A) recorded in a hTauAT expressing slice (red) demonstrating an increase in burst frequency as well as in firings per burst due to hTauAT expression.C. Quantification of bursts per minute in DIV 10 slices from control littermates and hTauAT slices demonstrating a ~2-fold increase in hTauAT slices (n=8 slices per group).D. Firings per burst are increased ~1.5-fold already at the early time point DIV10 due to hTauAT expression.E. Analysis of burst frequency in slices at DIV 30 demonstrates the progression of the pathological effect beyond that observed at DIV 10. Burst frequency is enhanced in hTauAT expressing slices (n = 10 slices). If Ceftriaxone (CEF) is applied to hTauAT slices the pathological burst frequency enhancement is partly prevented (n = 5 slices).F. Firings per burst are strongly enhanced due to hTauAT expression in slices at DIV 30. Ceftriaxone prevents this pathological phenotype if applied during the entire period of cultivation (n = 6 slices).


G . Example of a control littermate organotypic slice culture at DIV 24 stained against NeuN ( red ) and pan - Tau ( K9JA antibody , green ) . Axonal projections ( green ) of granule cells ( red ) in the dentate gyrus are depicted . H . Hippocampal slice culture expressing hTauAT stained against NeuN ( red ) and pan - Tau ( green ) showing mossy fiber sprouting in the hilar region , CA3 and dentate gyrus ( white arrows ) .

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

G. Example of a control littermate organotypic slice culture at DIV 24 stained against NeuN (red) and pan-Tau (K9JA antibody, green). Axonal projections (green) of granule cells (red) in the dentate gyrus are depicted.H. Hippocampal slice culture expressing hTauAT stained against NeuN (red) and pan-Tau (green) showing mossy fiber sprouting in the hilar region, CA3 and dentate gyrus (white arrows).


A . Quantification of glutamate concentrations in the medium of control and hTauAT cultures in the presence of either 50 nM tetanus neurotoxin ( TeNT ) or 1 µM tetrodotoxin ( TTX ) at DIV5 . Under both conditions , glutamate levels in the culture medium of hTauAT slices decreased to control levels ( n = 6 culture inserts containing 6 slices each / group and condition ) . B . Quantification of extracellular glutamate concentrations in culture medium of either control or hTauAT slices at DIV 20 in the presence or absence of 50 µM benzyloxy - aspartic acid ( TBOA ) from DIV1 - DIV20 demonstrating a possible contribution of astrocytic glutamate dysregulation to increased extracellular glutamate in hTauAT slices ( n = 8 - 9 culture inserts containing 6 slices each / group and condition ) . One - Way ANOVA F ( 3 , 30 ) = 14 , 89 ; p < 0 .

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

A. Quantification of glutamate concentrations in the medium of control and hTauAT cultures in the presence of either 50 nM tetanus neurotoxin (TeNT) or 1 µM tetrodotoxin (TTX) at DIV5. Under both conditions, glutamate levels in the culture medium of hTauAT slices decreased to control levels (n=6 culture inserts containing 6 slices each / group and condition).B. Quantification of extracellular glutamate concentrations in culture medium of either control or hTauAT slices at DIV 20 in the presence or absence of 50 µM benzyloxy-aspartic acid (TBOA) from DIV1-DIV20 demonstrating a possible contribution of astrocytic glutamate dysregulation to increased extracellular glutamate in hTauAT slices (n=8-9 culture inserts containing 6 slices each / group and condition). One-Way ANOVA F(3,30)=14,89; p<0.001


C . Quantification of LDH release into the culture medium of either control or hTauAT slices at DIV 20 in the presence or absence of 50 µM benzyloxy - aspartic acid ( TBOA ) from DIV 1 to DIV 20 ( n = 6 - 12 culture inserts containing 6 slices each / group and condition ) . One - Way ANOVA followed by Tukey ' s post - hoc test .

O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Quantification of LDH release into the culture medium of either control or hTauAT slices at DIV 20 in the presence or absence of 50 µM benzyloxy-aspartic acid (TBOA) from DIV 1 to DIV 20 (n=6-12 culture inserts containing 6 slices each / group and condition). One-Way ANOVA followed by Tukey's post-hoc test.


D . Quantification of extracellular glutamate concentrations in either control or hTauAT cultures treated ( n = 7 - 8 culture inserts containing 6 slices each / group and condition ) with ceftriaxone from DIV 1 to DIV 25 demonstrating the preventative effect of CEF on the increase in extracellular glutamate in hTauAT slices . One - Way ANOVA F ( 3 , 25 ) = 9 . 075 ; p = 0 . 0003 ) .

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

D. Quantification of extracellular glutamate concentrations in either control or hTauAT cultures treated (n=7-8 culture inserts containing 6 slices each / group and condition) with ceftriaxone from DIV 1 to DIV 25 demonstrating the preventative effect of CEF on the increase in extracellular glutamate in hTauAT slices. One-Way ANOVA F(3,25)=9.075; p=0.0003).


E . LDH release at DIV 25 is strongly reduced due to long term treatment with ceftriaxone in hTauAT slice cultures ( n = 8 - 10 culture inserts containing 6 slices each / group and condition ) . One Way ANOVA F ( 3 , 32 ) = 7 . 685 ; p = 0 . 0005 ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. LDH release at DIV 25 is strongly reduced due to long term treatment with ceftriaxone in hTauAT slice cultures (n=8-10 culture inserts containing 6 slices each / group and condition). One Way ANOVA F(3,32)=7.685; p=0.0005).


F . Intracellular calcium rise after KCl stimulation is not distinguishable from control slices in hTauAT slices treated with ceftriaxone for 15 days ( n = 5 - 9 slices ; prepared from at least three animals ) . One way ANOVA F ( ( 3 , 25 ) = 7 . 317 ; p = 0 . 0011 ) .

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

F. Intracellular calcium rise after KCl stimulation is not distinguishable from control slices in hTauAT slices treated with ceftriaxone for 15 days (n=5-9 slices; prepared from at least three animals). One way ANOVA F((3,25)=7.317; p=0.0011).


A Representative images of eyes and lenses from 6 - 7 week old wildtype ( WT ) and nervous system - specific YME1L knockout ( NYKO ) mice . Orange dashed lines mark eye morphology . Scale bars 5 mm .

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

A Representative images of eyes and lenses from 6-7 week old wildtype (WT) and nervous system-specific YME1L knockout (NYKO) mice. Orange dashed lines mark eye morphology. Scale bars 5 mm.


Retinal sagittal cross - sections from 6 - 7 week old mice stained with hematoxylin and eosin . NFL = Nerve fiber layer , IPL = inner plexiform layer , INL = inner nuclear layer , OPL = outer plexiform layer , ONL = outer nuclear layer , R & C = Rods and cones . Scale bars 30

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

Retinal sagittal cross-sections from 6-7 week old mice stained with hematoxylin and eosin. NFL = Nerve fiber layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer, R&C = Rods and cones. Scale bars 30 µ


Quantification of nuclei in OPL ( area = 1000 µm2 ) from retina cross - sections of 6 - 7 week old WT ( n = 4 ) and NYKO ( n = 4 ) mice . Data were analyzed using unpaired t - test , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± SEM .

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

Quantification of nuclei in OPL (area = 1000 µm2) from retina cross-sections of 6-7 week old WT (n = 4) and NYKO (n = 4) mice. Data were analyzed using unpaired t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


D Immunoblot analysis of retinal lysates from 6 - 7 week old WT and NYKO mice . GFAP was used as a marker for reactive astrogliosis and SDHA as a loading control . Data were analyzed using unpaired t - test , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± SEM .

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

D Immunoblot analysis of retinal lysates from 6-7 week old WT and NYKO mice. GFAP was used as a marker for reactive astrogliosis and SDHA as a loading control. Data were analyzed using unpaired t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


E mRNA levels of proinflammatory cytokines and NF - κB target genes from 6 - 7 week old retinas ( WT , n = 5 ; NYKO , n = 5 ) . Transcript levels were normalized to Hprt mRNA levels . Data were analyzed using unpaired t - test , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± SEM .

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

E mRNA levels of proinflammatory cytokines and NF-κB target genes from 6-7 week old retinas (WT, n = 5; NYKO, n = 5). Transcript levels were normalized to Hprt mRNA levels. Data were analyzed using unpaired t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


mRNA levels of Fgf21 from 6 - 7 week old retinas ( WT , n = 5 ; NYKO , n = 5 ) . Transcript levels were normalized to Hprt mRNA levels . Data were analyzed using unpaired t - test , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± SEM .

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

mRNA levels of Fgf21 from 6-7 week old retinas (WT, n = 5; NYKO, n = 5). Transcript levels were normalized to Hprt mRNA levels. Data were analyzed using unpaired t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


G Transmission electron micrographs of optic nerves from 6 - 7 week old WT and NYKO mice . Scale bars 2 µm .

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

G Transmission electron micrographs of optic nerves from 6-7 week old WT and NYKO mice. Scale bars 2 µm.


Mean body weight of male wild type ( WT , n = 15 ) , NYKO ( n = 12 ) and heterozygous NYKO ( het NYKO , n = 7 ) mice from 4 - 32 weeks . Unpaired t - test was used for the comparison of two groups , ordinary one - way ANOVA for the comparison of three groups . *

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

Mean body weight of male wild type (WT, n = 15), NYKO (n = 12) and heterozygous NYKO (het NYKO, n = 7) mice from 4-32 weeks. Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups. *


B Body composition analysis by nuclear magnetic resonance ( NMR ) of 31 - 32 week old male WT ( n = 12 ) and NYKO ( n = 7 ) mice . Unpaired t - test was used for the comparison of two groups , ordinary one - way ANOVA for the comparison of three groups . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . Data are means ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Body composition analysis by nuclear magnetic resonance (NMR) of 31-32 week old male WT (n = 12) and NYKO (n = 7) mice. Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups. *P ≤ 0.05, *** P ≤ 0.001, ****P ≤ 0.0001. Data are means ± SEM.


C Whole - body energy expenditure per kg lean mass of 26 week old male WT ( n = 5 ) and NYKO ( n = 5 ) mice after disease onset . Unpaired t - test was used for the comparison of two groups , ordinary one - way ANOVA for the comparison of three groups .

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

C Whole-body energy expenditure per kg lean mass of 26 week old male WT (n = 5) and NYKO (n = 5) mice after disease onset. Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups.


Walking beam test of 6 - 7 week old ( WT , n = 10 ; NYKO , n = 10 ) , 17 - 18 week old ( WT , n = 8 ; NYKO , n = 9 ) and 31 - 32 week old mice ( WT , n = 12 ; NYKO , n = 19 ; het NYKO , n = 9 ) . Unpaired t - test was used for the comparison of two groups , ordinary one - way ANOVA for the comparison of three groups . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . Data are means ± SEM .

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

Walking beam test of 6-7 week old (WT, n = 10; NYKO, n = 10), 17-18 week old (WT, n = 8; NYKO, n = 9) and 31-32 week old mice (WT, n = 12; NYKO, n = 19; het NYKO, n = 9). Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups. *P ≤ 0.05, *** P ≤ 0.001, ****P ≤ 0.0001. Data are means ± SEM.


Walking beam test of 6 - 7 week old ( WT , n = 10 ; NYKO , n = 10 ) , 17 - 18 week old ( WT , n = 8 ; NYKO , n = 9 ) and 31 - 32 week old mice ( WT , n = 12 ; NYKO , n = 19 ; het NYKO , n = 9 ) . Hind limb slips per run are shown in E . Unpaired t - test was used for the comparison of two groups , ordinary one - way ANOVA for the comparison of three groups . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . Data are means ± SEM .

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

Walking beam test of 6-7 week old (WT, n = 10; NYKO, n = 10), 17-18 week old (WT, n = 8; NYKO, n = 9) and 31-32 week old mice (WT, n = 12; NYKO, n = 19; het NYKO, n = 9). Hind limb slips per run are shown in E. Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups. *P ≤ 0.05, *** P ≤ 0.001, ****P ≤ 0.0001. Data are means ± SEM.


F Representative images of 31 - 32 week old WT and NYKO mice showing aberrant positioning of hind limbs .

F Representative images of 31-32 week old WT and NYKO mice showing aberrant positioning of hind limbs.


Brain weights were monitored at 31 - 32 weeks of age ( WT , n = 15 ; NYKO , n = 14 ) . Unpaired t - test , ns = not significant . Data are means ± SEM .

Brain weights were monitored at 31-32 weeks of age (WT, n = 15; NYKO, n = 14). Unpaired t-test, ns = not significant. Data are means ± SEM.


B Nissl stainings of sagittal sections across brain regions from 31 - 32 week old WT and NYKO mice . 4x and 40x enlargements are shown in the left and right panels , respectively . Scale bars 4x = 500 µm , 40x = 50 µm .

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

B Nissl stainings of sagittal sections across brain regions from 31-32 week old WT and NYKO mice. 4x and 40x enlargements are shown in the left and right panels, respectively. Scale bars 4x = 500 µm, 40x = 50 µm.


C Morphometric analysis of different brain regions from 31 - 32 week old WT ( n = 6 ) and NYKO ( n = 6 ) mice . GCL = Granule cell layer , ML = Molecular layer . Unpaired t - test , ns = not significant . Data are means ± SEM .

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

C Morphometric analysis of different brain regions from 31-32 week old WT (n = 6) and NYKO (n = 6) mice. GCL = Granule cell layer, ML = Molecular layer. Unpaired t-test, ns = not significant. Data are means ± SEM.


Immunoblot analysis of tissue lysates ( FB = forebrain ; CB = cerebellum ) from 31 - 32 week old WT and NYKO mice , using GFAP - specific and , as a loading control , GAPDH - specific antibodies . Unpaired t - test , ns = not significant . Data are means ± SEM .

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

Immunoblot analysis of tissue lysates (FB = forebrain; CB = cerebellum) from 31-32 week old WT and NYKO mice, using GFAP-specific and, as a loading control, GAPDH-specific antibodies. Unpaired t-test, ns = not significant. Data are means ± SEM.


B Transverse semithin sections of spinal cords of WT and NYKO mice of the indicated age . Sections were stained with toluidine blue . Orange arrows indicate degenerating neurons ( Myelin dense bodies , MDBs ) . Scale bars 25 µm .

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

B Transverse semithin sections of spinal cords of WT and NYKO mice of the indicated age. Sections were stained with toluidine blue. Orange arrows indicate degenerating neurons (Myelin dense bodies, MDBs). Scale bars 25 µm.


C Myelin dense bodies ( MDBs ) in dorso - lateral tracts of 6 - 7 week old WT ( n = 4 ) and NYKO ( n = 4 ) , 17 - 18 week old WT ( n = 4 ) and NYKO ( n = 4 ) and 31 - 32 week old WT ( n = 6 - 7 ) and NYKO ( n = 5 - 6 ) mice spinal cords . Unpaired t - test , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± SEM .

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

C Myelin dense bodies (MDBs) in dorso-lateral tracts of 6-7 week old WT (n = 4) and NYKO (n = 4), 17-18 week old WT (n = 4) and NYKO (n = 4) and 31-32 week old WT (n = 6-7) and NYKO (n = 5-6) mice spinal cords. Unpaired t-test, *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


D Immunoblots of 31 - 32 week old spinal cord lysates . GAPDH was used to control for gel loading .

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

D Immunoblots of 31-32 week old spinal cord lysates. GAPDH was used to control for gel loading.


E mRNA levels of proinflammatory cytokines from spinal cords of 31 - 32 week old mice ( WT , n = 5 ; NYKO , n = 5 ) . Transcript levels were normalized to Hprt mRNA levels . Unpaired t - test , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± SEM .

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

E mRNA levels of proinflammatory cytokines from spinal cords of 31-32 week old mice (WT, n = 5; NYKO, n = 5). Transcript levels were normalized to Hprt mRNA levels. Unpaired t-test, *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


Fluorescence images of isolated cortical neurons from Yme1lfl / fl animals expressing CAG - GFP ( WT ) or CAG - Cre - IRES - GFP ( Yme1l - / - ) at DIV12 and stained with TOMM20 to visualize mitochondria . Scale bars 30 µm .

Fluorescence images of isolated cortical neurons from Yme1lfl/fl animals expressing CAG-GFP (WT) or CAG-Cre-IRES-GFP (Yme1l-/-) at DIV12 and stained with TOMM20 to visualize mitochondria. Scale bars 30 µm.


B - E Quantification of total axon length ( WT , n = 13 neurons ; Yme1l - / - , n = 13 neurons ) , total dendritic length ( WT , n = 12 neurons ; Yme1l - / - , n = 13 neurons ) , axonal branch points ( WT , n = 12 neurons ; Yme1l - / - , n = 13 neurons ) and number of dendrites ( WT , n = 12 neurons ; Yme1l - / - , n = 13 neurons ) . Individual neurons originate from two independent preparations . Unpaired t - test , ns = not significant , * P ≤ 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - E Quantification of total axon length (WT, n = 13 neurons; Yme1l-/-, n = 13 neurons), total dendritic length (WT, n = 12 neurons; Yme1l-/-, n = 13 neurons), axonal branch points (WT, n = 12 neurons; Yme1l-/-, n = 13 neurons) and number of dendrites (WT, n = 12 neurons; Yme1l-/-, n = 13 neurons). Individual neurons originate from two independent preparations. Unpaired t-test, ns = not significant, *P ≤ 0.05.


F , G Kymographs showing mitochondrial movements in representative axons . Vertical lines correspond to stationary mitochondria and diagonal lines to moving mitochondria . Mitochondria were stained using Mitotracker . In total 39 axonal segments were analyzed for WT control neurons and 37 axonal segments for Yme1l - / - neurons at DIV7 . Data are means of three independent experiments ± SEM . Unpaired t - test , ns = not significant , * P ≤ 0 . 05 .

F, G Kymographs showing mitochondrial movements in representative axons. Vertical lines correspond to stationary mitochondria and diagonal lines to moving mitochondria. Mitochondria were stained using Mitotracker. In total 39 axonal segments were analyzed for WT control neurons and 37 axonal segments for Yme1l-/- neurons at DIV7. Data are means of three independent experiments ± SEM. Unpaired t-test, ns = not significant, *P ≤ 0.05.


H , I Kymographs showing mitochondrial movements in representative axons . Vertical lines correspond to stationary mitochondria and diagonal lines to moving mitochondria . Mitochondria were stained using Mitotracker . In total 120 axonal segments were analyzed for WT control neurons and 107 axonal segments for Yme1l - / - neurons at DIV10 - 14 . Data are means of three independent experiments ± SEM . Unpaired t - test , ns = not significant , * P ≤ 0 . 05 .

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

H, I Kymographs showing mitochondrial movements in representative axons. Vertical lines correspond to stationary mitochondria and diagonal lines to moving mitochondria. Mitochondria were stained using Mitotracker. In total 120 axonal segments were analyzed for WT control neurons and 107 axonal segments for Yme1l-/- neurons at DIV10-14. Data are means of three independent experiments ± SEM. Unpaired t-test, ns = not significant, *P ≤ 0.05.


J Axonal area occupied by mitochondria in Yme1l - / - neurons relative to WT controls . In total 120 axonal segments were analyzed for WT control neurons and 107 axonal segments for Yme1l - / - neurons at DIV10 - 14 . Data are means of three independent experiments ± SEM . Unpaired t - test , ns = not significant , * P ≤ 0 . 05 .

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

J Axonal area occupied by mitochondria in Yme1l-/- neurons relative to WT controls. In total 120 axonal segments were analyzed for WT control neurons and 107 axonal segments for Yme1l-/- neurons at DIV10-14. Data are means of three independent experiments ± SEM. Unpaired t-test, ns = not significant, *P ≤ 0.05.


A , B Oxygen consumption rates of spinal cord mitochondria isolated from WT ( n = 4 ) and NYKO ( n = 4 ) mice in the presence of pyruvate - glutamate - malate ( C I ) and pyruvate - glutamate - malate + succinate ( C I + C II ) as substrates . State 3 ( substrates + ADP ) , state 4 ( + oligomycin ) , uncoupled ( + FCCP ) . Unpaired t - test , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , ns = not significant . Data are means ± SEM .

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

A, B Oxygen consumption rates of spinal cord mitochondria isolated from WT (n = 4) and NYKO (n = 4) mice in the presence of pyruvate-glutamate-malate (C I) and pyruvate-glutamate-malate + succinate (C I + C II) as substrates. State 3 (substrates + ADP), state 4 (+ oligomycin), uncoupled (+ FCCP). Unpaired t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ns=not significant. Data are means ± SEM.


C , D TEM of dorso - lateral tracts of spinal cords of 6 - 7 week old WT ( n = 4 , 1209 mitochondria ) and NYKO ( n = 4 , 1792 mitochondria ) mice , 17 - 18 week old WT ( n = 4 , 442 mitochondria ) and NYKO ( n = 4 , 724 mitochondria ) mice and 31 - 32 week old WT ( n = 3 , 764 mitochondria ) and NYKO ( n = 3 , 817 mitochondria ) mice . Scale bars , 200 nm . Unpaired t - test , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , ns = not significant . Data are means ± SEM .

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

C, D TEM of dorso-lateral tracts of spinal cords of 6-7 week old WT (n = 4, 1209 mitochondria) and NYKO (n = 4, 1792 mitochondria) mice, 17-18 week old WT (n = 4, 442 mitochondria) and NYKO (n = 4, 724 mitochondria) mice and 31-32 week old WT (n = 3, 764 mitochondria) and NYKO (n = 3, 817 mitochondria) mice. Scale bars, 200 nm. Unpaired t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ns=not significant. Data are means ± SEM.


A TEM analysis of sagittal spinal cord sections of 6 - 7 week old WT and NYKO mice . Scale bars 500 nm .

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

A TEM analysis of sagittal spinal cord sections of 6-7 week old WT and NYKO mice. Scale bars 500 nm.


B Frequency distribution of mitochondrial lengths in spinal cords of 6 - 7 week old WT ( 182 mitochondria ) and NYKO mice ( 192 mitochondria ) . Kruskal - Wallis test , * P < 0 .

O O O O O O O O O O O O O O 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 Frequency distribution of mitochondrial lengths in spinal cords of 6-7 week old WT (182 mitochondria) and NYKO mice (192 mitochondria). Kruskal-Wallis test, *P < 0.05


C TEM analysis of cerebellar sections of 28 - 32 week old WT and NYKO mice . Scale bars 1 µm . D Frequency distribution of mitochondrial aspect ratios were calculated from WT ( n = 210 ) and NYKO ( n = 230 ) mitochondria . Mann - Whitney test , * * * * P < 0 . 0001 . Data are presented as median values .

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

C TEM analysis of cerebellar sections of 28-32 week old WT and NYKO mice. Scale bars 1 µm. D Frequency distribution of mitochondrial aspect ratios were calculated from WT (n = 210) and NYKO (n = 230) mitochondria. Mann-Whitney test, ****P < 0.0001. Data are presented as median values.


Immunoblot analysis of forebrain , cerebellar and spinal cord lysates from 31 - 32 week old WT and NYKO mice ( n = 3 ) using the indicated antibodies . SDHA and LONP1 were used to control for gel loading . * indicates unspecific antibody binding .

Immunoblot analysis of forebrain, cerebellar and spinal cord lysates from 31-32 week old WT and NYKO mice (n = 3) using the indicated antibodies. SDHA and LONP1 were used to control for gel loading. * indicates unspecific antibody binding.


Immunoblot analysis of retinal and spinal cord lysates from 6 - 7 week old WT , NYKO , NOKO and NYOKO mice ( n = 3 ) using the indicated antibodies . SDHA was used as a loading control . * was not reproducibly detectable in all experiments .

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

Immunoblot analysis of retinal and spinal cord lysates from 6-7 week old WT, NYKO, NOKO and NYOKO mice (n = 3) using the indicated antibodies. SDHA was used as a loading control. * was not reproducibly detectable in all experiments.


B Frequency distribution of mitochondrial lengths in spinal cords of 6 - 7 week old NYOKO mice ( 176 mitochondria ) compared to WT Kruskal - Wallis test , ns = not significant . Data were analyzed using one - way ANOVA .

O O O O O O O O O O O O O O O O O O O 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 Frequency distribution of mitochondrial lengths in spinal cords of 6-7 week old NYOKO mice (176 mitochondria) compared to WT Kruskal-Wallis test, ns = not significant. Data were analyzed using one-way ANOVA.


Representative images of eyes and lenses from 6 - 7 week old WT and mice lacking OMA1 ( NOKO ) or both YME1L and OMA1 ( NYOKO ) in the nervous system . Orange dashed lines mark eye morphology . Scale bars 2 . 5 mm .

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

Representative images of eyes and lenses from 6-7 week old WT and mice lacking OMA1 (NOKO) or both YME1L and OMA1 (NYOKO) in the nervous system. Orange dashed lines mark eye morphology. Scale bars 2.5 mm.