A ) ( top ) Representative image of immunofluorescence double staining for the mitochondrial protein MT - CO1 and the DDR marker yH2A . X ( scale bar = 5µm ) and ( bottom ) quantification of MT - CO1 intensity vs number of yH2A . X foci in hepatocytes from 12 months old mice . Data are mean ± S . E . M of n = 3 mice ( at least 30 cells were analyzed per mouse ) ; Asterisks denote statistical significant P < 0 . 05 using Two - tailed 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

A) (top) Representative image of immunofluorescence double staining for the mitochondrial protein MT-CO1 and the DDR marker yH2A.X (scale bar=5µm) and (bottom) quantification of MT-CO1 intensity vs number of yH2A.X foci in hepatocytes from 12 months old mice. Data are mean±S.E.M of n=3 mice (at least 30 cells were analyzed per mouse); Asterisks denote statistical significant P<0.05 using Two-tailed t-test.


B ) Representative western blots of pS6 , S6 , p21 , PGC - 1β , MT - CO1 and NDUFB8 protein expression in mouse liver tissue at 3 and 12 months of age . Data are from n = 3 mice per group ;

O O O O O O O O O O O O O O O O O O O O O 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 western blots of pS6, S6, p21, PGC-1β, MT-CO1 and NDUFB8 protein expression in mouse liver tissue at 3 and 12 months of age. Data are from n=3 mice per group;


C ) Effect of 4 months of rapamycin supplemented diet on PGC - 1β expression in liver tissue of 16 months old mice . Data are from n = 3 mice per group ;

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

C) Effect of 4 months of rapamycin supplemented diet on PGC-1β expression in liver tissue of 16 months old mice. Data are from n=3 mice per group;


D ) ( top ) Quantification of mitochondrial number per cross section and mitochondrial volume fraction ( % Vv ) in hepatocytes from 16 months old mice with or without 4 months rapamycin diet . Data are mean ± S . E . M of n = 3 mice per group ( at least 20 cells were analyzed per mouse ) ; ( bottom ) Representative electron micrographs of hepatocytes from 16 months old mice with or without 4 months rapamycin diet , ( mitochondria are labelled in pink ) . Scale bar = 5µm ;

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

D) (top) Quantification of mitochondrial number per cross section and mitochondrial volume fraction (%Vv) in hepatocytes from 16 months old mice with or without 4 months rapamycin diet. Data are mean±S.E.M of n=3 mice per group (at least 20 cells were analyzed per mouse); (bottom) Representative electron micrographs of hepatocytes from 16 months old mice with or without 4 months rapamycin diet, (mitochondria are labelled in pink). Scale bar=5µm;


E ) mtDNA copy number ( measured by q - PCR ) in mice liver tissue at 3 , 12 , 16 months and at 16 months after 4 months rapamycin diet . Data are mean ± S . E . M of n = 3 - 4 mice per group ; Asterisks denote statistical significant P < 0 . 05 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 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

E) mtDNA copy number (measured by q-PCR) in mice liver tissue at 3, 12, 16 months and at 16 months after 4 months rapamycin diet. Data are mean±S.E.M of n=3-4 mice per group; Asterisks denote statistical significant P<0.05 using One-way ANOVA.


F ) Oxygen consumption rates ( OCR ) in isolated liver mitochondria from 16 months old mice fed with or without rapamycin for 4 months , in the presence of pyruvate / malate . State III was induced by injection of ADP . State IV was induced by inhibition of the ATP synthase with oligomycin and uncoupled respiration rates were determined by injection of FCCP . Antimycin A ( AA ) was used to determine background , non - mitochondrial OXPHOS , OCR . Data are mean ± S . E . M of n = 5 mice per group ;

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

F) Oxygen consumption rates (OCR) in isolated liver mitochondria from 16 months old mice fed with or without rapamycin for 4 months, in the presence of pyruvate/malate. State III was induced by injection of ADP. State IV was induced by inhibition of the ATP synthase with oligomycin and uncoupled respiration rates were determined by injection of FCCP. Antimycin A (AA) was used to determine background, non-mitochondrial OXPHOS, OCR. Data are mean±S.E.M of n=5 mice per group;


G ) ( top ) Representative immuno - TeloFISH images of hepatocytes from 3 and 15 months old mice with or without rapamycin ( 12 months diet ) . Co - localizing foci are amplified in the right panel ( amplified images are from single Z - planes where co - localization was found ) ; ( bottom ) Dot plot graph of Telomere - associated foci ( TAF ) in 3 , 15 and 16 months old mice ( 15 and 16 months old mice were fed with rapamycin for 12 and 4 months respectively ) . Data are from n = 3 - 9 mice per group ( at least 50 cells were analyzed per mice ) ;

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 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

G) (top) Representative immuno-TeloFISH images of hepatocytes from 3 and 15 months old mice with or without rapamycin (12 months diet). Co-localizing foci are amplified in the right panel (amplified images are from single Z-planes where co-localization was found); (bottom) Dot plot graph of Telomere-associated foci (TAF) in 3, 15 and 16 months old mice (15 and 16 months old mice were fed with rapamycin for 12 and 4 months respectively). Data are from n=3-9 mice per group (at least 50 cells were analyzed per mice);


H ) ( top ) 4 months old and 15 months old micelivers [ control ( - ) or rapamycin ( + ) ] stained with Sen - - β - Gal solution ( Sen - - β - Gal activity is evidenced by blue staining ) . Data are from n = 3 mice per group ; ( bottom ) Representative image showing Sen - - β - Gal staining ( Sen - - β - Gal activity is evidenced by blue staining ) in hepatocytes and corresponding immuno - TeloFISH ( arrows represent co - localizing foci ) ; Asterisks denote statistical significant P < 0 . 05 using One - way ANOVA .

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

H) (top) 4 months old and 15 months old micelivers [control (-) or rapamycin (+)] stained with Sen--β-Gal solution (Sen--β-Gal activity is evidenced by blue staining). Data are from n=3 mice per group; (bottom) Representative image showing Sen--β-Gal staining (Sen--β-Gal activity is evidenced by blue staining) in hepatocytes and corresponding immuno-TeloFISH (arrows represent co-localizing foci); Asterisks denote statistical significant P<0.05 using One-way ANOVA.


I ) Representative western blot showing the effect of 4 months rapamycin feeding on p21 expression in 16 months old mice . Data are from n = 3 mice per group ;

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

I) Representative western blot showing the effect of 4 months rapamycin feeding on p21 expression in 16 months old mice. Data are from n=3 mice per group;


j ) Effect of 4 months rapamycin feeding on mRNA expression of the SASP components CXCL1 , CXCL5 and Inhibin A in liver tissue of 16 months old mice with or without 4 months rapamycin treatment . Data are from n = 5 mice per condition ; Asterisks denote statistical significant P < 0 . 05 using Two - tailed t - test .

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 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

j) Effect of 4 months rapamycin feeding on mRNA expression of the SASP components CXCL1, CXCL5 and Inhibin A in liver tissue of 16 months old mice with or without 4 months rapamycin treatment. Data are from n=5 mice per condition; Asterisks denote statistical significant P<0.05 using Two-tailed t-test.


K ) Mean number of TAF in hepatocytes of wild type and PGC - 1 - β - / - mice with 18 months of age . Data are mean ± S . E . M of n = 4 mice per group ; Asterisks denote statistical significant P < 0 . 05 using Two - tailed 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

K) Mean number of TAF in hepatocytes of wild type and PGC-1-β-/- mice with 18 months of age. Data are mean±S.E.M of n=4 mice per group; Asterisks denote statistical significant P<0.05 using Two-tailed t-test.


( C ) Quantification of AQR position in adult WT , cst - 1 and / or cst - 2 mutant animals . The full length between the URXs and the end of the nematode tail is divided into 10 blocks , and the percent of AQR that stopped within each block is indicated . * * * , P < 0 . 001 by Fisher ' s exact test . N = 50 - 109 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 AQR position in adult WT, cst-1 and/or cst-2 mutant animals. The full length between the URXs and the end of the nematode tail is divided into 10 blocks, and the percent of AQR that stopped within each block is indicated. ***, P < 0.001 by Fisher's exact test. N = 50 - 109.


( E - F ) Fluorescence time - lapse images of F - actin , plasma membrane and histone during AQR / QR . ap migration in WT ( E ) or cst - 1 ; cst - 2 double mutant ( F ) animals . Merged images , left ; inverted fluorescence images of F - actin , right ; white arrows , migration direction ; asterisks , nucleus ; double headed arrows , migration distance . F - actin is labeled with GFP - tagged with the actin - binding domain of Moesin ( GFP : : Moesin ABD ) , and the plasma membrane and Histone are labeled with mCherry - tagged with a myristoylation signal and a histone . The dotted lines indicate the cell periphery . Time is presented in minutes . Scale bars , 5 μm . ( G - J ) Quantification of the QR . ap migration angle ( G H ) , distance ( I ) and speed ( J ) . Each line in ( G I ) represents the measurement from one time - lapse movie . Error bars indicate SD . N = 10 - 17 . Statistical significance is based on Student ' s t - tests , * * , P 0 . 01 ; * * * , P 0 . 001 .

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

(E - F) Fluorescence time-lapse images of F-actin, plasma membrane and histone during AQR/QR.ap migration in WT (E) or cst-1; cst-2 double mutant (F) animals. Merged images, left; inverted fluorescence images of F-actin, right; white arrows, migration direction; asterisks, nucleus; double headed arrows, migration distance. F-actin is labeled with GFP-tagged with the actin-binding domain of Moesin (GFP::Moesin ABD), and the plasma membrane and Histone are labeled with mCherry-tagged with a myristoylation signal and a histone. The dotted lines indicate the cell periphery. Time is presented in minutes. Scale bars, 5 μm. (G - J) Quantification of the QR.ap migration angle (G H), distance (I) and speed (J). Each line in (G I) represents the measurement from one time-lapse movie. Error bars indicate SD. N = 10 - 17. Statistical significance is based on Student's t-tests, **, P 0.01; ***, P  0.001.


( A ) Fluorescence time - lapse images of F - actin , plasma membrane and histone during QR . ap migration in mig - 2 ( rh17 ) gain - of - function mutant animals . Merged images , left ; inverted fluorescence images of F - actin , right ; white arrows , migration direction ; asterisks , nucleus ; double headed arrow , migration distance . The dotted lines indicate the cell periphery . Time is presented in minutes . Scale bar , 5 μm . ( B - C ) Quantification of the QR . ap migration angle ( B ) and distance ( C ) . N = 10 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fluorescence time-lapse images of F-actin, plasma membrane and histone during QR.ap migration in mig-2(rh17) gain-of-function mutant animals. Merged images, left; inverted fluorescence images of F-actin, right; white arrows, migration direction; asterisks, nucleus; double headed arrow, migration distance. The dotted lines indicate the cell periphery. Time is presented in minutes. Scale bar, 5 μm. (B - C) Quantification of the QR.ap migration angle (B) and distance (C). N = 10.


( D ) Mass spectrum of a MIG - 2 peptide that was phosphorylated at Serine 139 in C . elegans . The peptide was identified from the GFP : : MIG - 2 immunoprecipitate .

O O O O O O O O O O O O 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) Mass spectrum of a MIG-2 peptide that was phosphorylated at Serine 139 in C. elegans. The peptide was identified from the GFP::MIG-2 immunoprecipitate.


( E ) In vitro phosphorylation assay of CST - 1 / 2 to MIG - 2 . Bacterially expressed His - tagged MIG - 2 or His - tagged MIG - 2S139A were incubated with the GST - tagged kinase domain of GST - CST - 1 / 2 ( 1 - 318 aa ) . The kinase may use ATP - γ - S as a phosphodonor to thiophosphorylate its substrate ; thus , the phosphorylation reaction was performed with ATP - γ - S , and anti - thiophosphate ester antibody was used to detect substrate phosphorylation ( Allen et al , 2005 ; Allen et al , 2007 ) . The final reaction was probed via immunoblotting with antibodies as indicated . Thio , thiophosphate ester .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vitro phosphorylation assay of CST-1/2 to MIG-2. Bacterially expressed His-tagged MIG-2 or His-tagged MIG-2S139A were incubated with the GST-tagged kinase domain of GST-CST-1/2 (1-318 aa). The kinase may use ATP-γ-S as a phosphodonor to thiophosphorylate its substrate; thus, the phosphorylation reaction was performed with ATP-γ-S, and anti-thiophosphate ester antibody was used to detect substrate phosphorylation (Allen et al, 2005; Allen et al, 2007). The final reaction was probed via immunoblotting with antibodies as indicated. Thio, thiophosphate ester.


( G H ) Three - dimensional ribbon ( PDB 3TH5 ) representations of the human Rac1 polypeptide which indicate the position of the T135 ( S139 in MIG - 2 ) located between the last two α - helixes ( Krauthammer et al , 2012 ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Three-dimensional ribbon (PDB 3TH5) representations of the human Rac1 polypeptide which indicate the position of the T135 (S139 in MIG-2) located between the last two α-helixes (Krauthammer et al, 2012).


( A ) Quantification of AQR position in mig - 2 ( mu28 ) null mutant animals with different rescue transgenes . N = 108 - 114 . * , P < 0 . 05 ; # # , P < 0 . 01 , phenotype enhanced . P values are based on Fisher ' s exact tests .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 AQR position in mig-2(mu28) null mutant animals with different rescue transgenes. N = 108 - 114. *, P < 0.05; ##, P < 0.01, phenotype enhanced. P values are based on Fisher's exact tests.


( B ) Quantification of AQR position in WT and knock - in animals that harbor mig - 2S139 point mutations . N = 100 - 111 . * * * , P < 0 . 001 by Fisher ' s exact 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

(B) Quantification of AQR position in WT and knock-in animals that harbor mig-2S139 point mutations. N = 100 - 111. ***, P < 0.001 by Fisher's exact test.


( C - F ) Quantification of the QR . ap migration angle ( C D ) , distance ( E ) and speed ( F ) in mig - 2S139A / E mutant animals . Each line in ( C E ) represents the measurement from one movie of QR . ap migration . Error bars indicate SD . N = 12 - 17 . * * , P 0 . 01 ; * * * , P 0 . 001 by Student ' s t tests .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - F) Quantification of the QR.ap migration angle (C D), distance (E) and speed (F) in mig-2S139A/E mutant animals. Each line in (C  E) represents the measurement from one movie of QR.ap migration. Error bars indicate SD. N = 12 -17. **, P 0.01; ***, P  0.001 by Student's t tests.


( G ) Inverted fluorescence images ( up ) and schematics ( down ) of QR . ap in WT , cst - 1 ; cst - 2 double or mig - 2 mutant animals . The plasma membrane and histones were labeled with mCherry . Scale bar , 5 μm . ( H ) Area ratio between the cell body and the nucleus in WT and mutant animals . Error bars indicate SD . N = 17 - 22 . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 by Student ' s t tests .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Inverted fluorescence images (up) and schematics (down) of QR.ap in WT, cst-1; cst-2 double or mig-2 mutant animals. The plasma membrane and histones were labeled with mCherry. Scale bar, 5 μm. (H) Area ratio between the cell body and the nucleus in WT and mutant animals. Error bars indicate SD. N = 17 - 22. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student's t tests.


( I - K ) Representative frames ( upper ) and quantification ( lower ) of F - actin enrichment at the QR . ap periphery in WT ( I ) and mig - 2S139A / E mutant animals ( J K ) . The trace starts from the rear of the QR . ap and moves along the cell periphery to the leading edge ( L ) and back to the rear ( R ) . F - actin was labeled with GFP : : Moesin ABD ; the plasma membrane and histone were marked with mCherry fluorescence . Green lines , F - actin fluorescence intensity ; red lines , plasma membrane fluorescence intensity ; dotted lines , cell periphery . Double - headed arrows indicate the F - actin enrichment region , in which the fluorescence intensity is 2 - fold higher than the background . Scale bars , 5 μm . ( L ) Quantification of F - actin enrichment ratio in WT and mig - 2 mutants . Error bars indicate SD . N = 28 - 68 . * * , P 0 . 01 ; * * * , P 0 . 001 by Student ' s t - tests .

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

(I - K) Representative frames (upper) and quantification (lower) of F-actin enrichment at the QR.ap periphery in WT (I) and mig-2S139A/E mutant animals (J  K). The trace starts from the rear of the QR.ap and moves along the cell periphery to the leading edge (L) and back to the rear (R). F-actin was labeled with GFP::Moesin ABD; the plasma membrane and histone were marked with mCherry fluorescence. Green lines, F-actin fluorescence intensity; red lines, plasma membrane fluorescence intensity; dotted lines, cell periphery. Double-headed arrows indicate the F-actin enrichment region, in which the fluorescence intensity is 2-fold higher than the background. Scale bars, 5 μm. (L) Quantification of F-actin enrichment ratio in WT and mig-2 mutants. Error bars indicate SD. N = 28 - 68. **, P  0.01; ***, P  0.001 by Student's t-tests.


( A - C ) Fluorescence images ( upper ) and quantifications ( lower ) of GFP - tagged CST - 1 ( A B ) or GFP - labeled F - actin ( C ) and mCherry tagged plasma membrane and histone in WT and mig - 2 null mutant animals . The membrane protrusions in the white boxes are enlarged 3 times at the bottom . Yellow dashed lines indicate the cell periphery . White lines indicate the line to measure the fluorescence intensity . Green lines denote the CST - 1 fluorescence intensity ; red lines denote the plasma membrane fluorescence intensity ; double - headed arrows denote the distance between the GFP signal and cell periphery . Scale bars , 5 μm . ( D ) Distance between GFP : : CST - 1 or GFP : : Moesin ABD and the plasma membrane at the leading edge in WT and mig - 2 null mutants . Error bars indicate SD . N = 9 - 14 . * * * , P 0 . 001 by 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A - C) Fluorescence images (upper) and quantifications (lower) of GFP-tagged CST-1 (A  B) or GFP-labeled F-actin (C) and mCherry tagged plasma membrane and histone in WT and mig-2 null mutant animals. The membrane protrusions in the white boxes are enlarged 3 times at the bottom. Yellow dashed lines indicate the cell periphery. White lines indicate the line to measure the fluorescence intensity. Green lines denote the CST-1 fluorescence intensity; red lines denote the plasma membrane fluorescence intensity; double-headed arrows denote the distance between the GFP signal and cell periphery. Scale bars, 5 μm. (D) Distance between GFP::CST-1 or GFP::Moesin ABD and the plasma membrane at the leading edge in WT and mig-2 null mutants. Error bars indicate SD. N = 9 - 14. ***, P  0.001 by Student's t test.


( E - F ) Fluorescence images ( left ) and quantifications ( right ) of GFP - tagged CST - 1 and mCherry tagged plasma membrane and histone in WT and mig - 2 null mutant animals . Blue lines , sites of measurements ; dotted lines , cell periphery ; Green lines ; CST - 1 fluorescence intensity ; red lines , mCherry fluorescence intensity . Scale bars , 5 μm . ( G ) Quantification of the fluorescence intensity ratio of GFP : : CST - 1 and mCherry - membrane between the leading edge and the lagging edge of AQR in WT and mig - 2 null mutant animals . Error bars indicate SD . N = 20 . * * * , P < 0 . 001 by 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E - F) Fluorescence images (left) and quantifications (right) of GFP-tagged CST-1 and mCherry tagged plasma membrane and histone in WT and mig-2 null mutant animals. Blue lines, sites of measurements; dotted lines, cell periphery; Green lines; CST-1 fluorescence intensity; red lines, mCherry fluorescence intensity. Scale bars, 5 μm. (G) Quantification of the fluorescence intensity ratio of GFP::CST-1 and mCherry-membrane between the leading edge and the lagging edge of AQR in WT and mig-2 null mutant animals. Error bars indicate SD. N = 20. ***, P < 0.001 by Student's t test.


( A ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with shRNA targeting TDP - 43 ( shTDP ) or control ( shCtrl ) together with GFP - RAB11 to visualize trafficking of recycling endosomes ( RE ) . At least four dendrite segments per neuron were live imaged at 5 Hz for 1 minute to analyze recycling endosome transport . Representative dendrite segments and kymographs of GFP - RAB11 vesicle movement . Quantitative analysis of vesicle motility ( B ) and total number ( C ) from kymographs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primary hippocampal neurons (DIV6+3) were transfected with shRNA targeting TDP-43 (shTDP) or control (shCtrl) together with GFP-RAB11 to visualize trafficking of recycling endosomes (RE). At least four dendrite segments per neuron were live imaged at 5 Hz for 1 minute to analyze recycling endosome transport. Representative dendrite segments and kymographs of GFP-RAB11 vesicle movement. Quantitative analysis of vesicle motility (B) and total number (C) from kymographs.


( D ) iPSC - derived human neurons were transduced with shRNA targeting human TDP - 43 ( shTDP ) or control ( shCtrl ) seven days after thawing for three days . TDP - 43 levels were analyzed by quantitative RT - PCR . Expression was normalized to the housekeeping gene YWHAZ and PGK1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) iPSC-derived human neurons were transduced with shRNA targeting human TDP-43 (shTDP) or control (shCtrl) seven days after thawing for three days. TDP-43 levels were analyzed by quantitative RT-PCR. Expression was normalized to the housekeeping gene YWHAZ and PGK1.


( E ) iPSC - derived human neurons ( DIV7 + 3 ) were transduced with shTDP or shCtrl and GFP - RAB11 and at least three dendrite segments per neuron analyzed as in ( a ) .

O O O O O O O O O O O O O O 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) iPSC-derived human neurons (DIV7+3) were transduced with shTDP or shCtrl and GFP-RAB11 and at least three dendrite segments per neuron analyzed as in (a).


Quantitative analysis of vesicle motility ( F ) and number ( G ) from kymographs . Mean + / - s . e . m . , n = 3 , unpaired two - tailed t - test : * denotes p < 0 . 05 , * * denotes p < 0 . 01 , * * * denotes p < 0 . 001 .

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

Quantitative analysis of vesicle motility (F) and number (G) from kymographs. Mean +/- s.e.m., n = 3, unpaired two-tailed t-test: * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001.


( A ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with shRNA targeting TDP - 43 or a control together with tagRFP ( not depicted ) . Representative images of neurons subjected to Alexa - transferrin ( Alexa - Tf ) for 20 min ( pulse ) and chased for 0 , 20 or 60 min in complete media . Scale bar represents 100 µm . ( B ) Quantification of labeled cellular Alexa - Tf normalized to cell area ( using tagRFP ) after chase period . At least ten images per condition were analyzed per experiment in three independent 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primary hippocampal neurons (DIV6+4) were transduced with shRNA targeting TDP-43 or a control together with tagRFP (not depicted). Representative images of neurons subjected to Alexa-transferrin (Alexa-Tf) for 20 min (pulse) and chased for 0, 20 or 60 min in complete media. Scale bar represents 100 µm. (B) Quantification of labeled cellular Alexa-Tf normalized to cell area (using tagRFP) after chase period. At least ten images per condition were analyzed per experiment in three independent experiments.


( C ) ELISA measurement of β2 - transferrin in CSF of control subjects ( n = 65 ) and sporadic ALS patients ( n = 55 ) , FTD - ALS patients ( n = 12 ) and behavioral variant FTD patients ( bvFTD , n = 16 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ELISA measurement of β2-transferrin in CSF of control subjects (n=65) and sporadic ALS patients (n=55), FTD-ALS patients (n=12) and behavioral variant FTD patients (bvFTD, n=16).


( D ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected shTDP or shCtrl and GFP as transfection marker . After 3 days neurons were subjected to Alexa - Tf for 20 min and intracellular Alexa - Tf movement was imaged as in Figure 1 afterwards . Quantitative analysis of Alexa - Tf movement ( E ) and vesicle number ( F ) from kymographs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primary hippocampal neurons (DIV6+3) were transfected shTDP or shCtrl and GFP as transfection marker. After 3 days neurons were subjected to Alexa-Tf for 20 min and intracellular Alexa-Tf movement was imaged as in Figure 1 afterwards. Quantitative analysis of Alexa-Tf movement (E) and vesicle number (F) from kymographs.


( A ) Primary hippocampal neurons ( DIV6 + 4 ) were transfected with myc - tagged TDP - 43 wild - type or a mutant lacking the nuclear localization signal ( ΔNLS ) or an empty vector control . Immunostaining with the indicated antibodies and DAPI to label nuclei . Scale bar represents 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 O O O O O O O O O O

(A) Primary hippocampal neurons (DIV6+4) were transfected with myc-tagged TDP-43 wild-type or a mutant lacking the nuclear localization signal (ΔNLS) or an empty vector control. Immunostaining with the indicated antibodies and DAPI to label nuclei. Scale bar represents 50 µm.


( B ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with either TDP - 43 wild - type , TDP - 43ΔNLS or an empty vector control together with GFP - RAB11 to visualize recycling endosomes and analyzed as in Figure 1 . Quantitative analysis of recycling endosome movement ( C ) and vesicle number ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primary hippocampal neurons (DIV6+3) were transfected with either TDP-43 wild-type, TDP-43ΔNLS or an empty vector control together with GFP-RAB11 to visualize recycling endosomes and analyzed as in Figure 1. Quantitative analysis of recycling endosome movement (C) and vesicle number (D).


( A ) Primary cortical neurons were transduced with shTDP or control ( DIV2 + 7 ) and subjected to quantitative proteomic analysis . Volcano plot shows up and down regulated proteins from three biological replicates . Significant changes are colored . Compare Table EV1 .

O O O O O O O O O O O O O O O O O O O O O O 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) Primary cortical neurons were transduced with shTDP or control (DIV2+7) and subjected to quantitative proteomic analysis. Volcano plot shows up and down regulated proteins from three biological replicates. Significant changes are colored. Compare Table EV1.


( C - E ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with lentivirus expressing shRNA targeting TDP - 43 or a control . Immunoblots with the indicated antibodies ( C ) . Quantification of VPS4B and TDP - 43 protein level using densitometry ( n = 4 ) . Expression was normalized to the housekeeping gene β - actin ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Primary hippocampal neurons (DIV6+4) were transduced with lentivirus expressing shRNA targeting TDP-43 or a control. Immunoblots with the indicated antibodies (C). Quantification of VPS4B and TDP-43 protein level using densitometry (n = 4). Expression was normalized to the housekeeping gene β-actin (D).


( C - E ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with lentivirus expressing shRNA targeting TDP - 43 or a control . VPS4B mRNA level were analyzed by quantitative RT - PCR . Expression was normalized to the housekeeping genes YWHAZ and PGK1 ( E ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Primary hippocampal neurons (DIV6+4) were transduced with lentivirus expressing shRNA targeting TDP-43 or a control. VPS4B mRNA level were analyzed by quantitative RT-PCR. Expression was normalized to the housekeeping genes YWHAZ and PGK1 (E).


( F ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with either shTDP or shCtrl and iRFP670 . Immunostaining with the indicated antibodies . Scale bar represents 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

(F) Primary hippocampal neurons (DIV6+3) were transfected with either shTDP or shCtrl and iRFP670. Immunostaining with the indicated antibodies. Scale bar represents 50 µm.


( G ) iPSC - derived human neurons were transduced with shRNA targeting human TDP - 43 ( shTDP ) or control ( shCtrl ) seven days after thawing for three days . VPS4B levels were analyzed by quantitative RT - PCR . Expression was normalized to the housekeeping gene YWHAZ and PGK1 ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) iPSC-derived human neurons were transduced with shRNA targeting human TDP-43 (shTDP) or control (shCtrl) seven days after thawing for three days. VPS4B levels were analyzed by quantitative RT-PCR. Expression was normalized to the housekeeping gene YWHAZ and PGK1 (n = 3).


( A - B ) Luciferase assay to analyze transcriptional regulation of VPS4B by TDP - 43 . ( B ) HEK293 cells were transfected with shRNAs targeting human TDP - 43 , a control shRNA , TDP - 43 wild - type or empty vector control together with the luciferase reporter containing the rat VPS4B promoter . VPS4B promoter - driven renilla luciferase activity was normalized to TK promoter - driven firefly luciferase . Quantification from six independent 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 O O O O O O O O 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) Luciferase assay to analyze transcriptional regulation of VPS4B by TDP-43. (B) HEK293 cells were transfected with shRNAs targeting human TDP-43, a control shRNA, TDP-43 wild-type or empty vector control together with the luciferase reporter containing the rat VPS4B promoter. VPS4B promoter-driven renilla luciferase activity was normalized to TK promoter-driven firefly luciferase. Quantification from six independent experiments.


( C - D ) ChIP assay to analyze binding of TDP - 43 to VPS4B promoter region in rat cortical neurons , HEK293 cells and human brain tissue . PCR reaction from input , negative control and TDP - 43 immunoprecipitates . Signal intensities from at least three independent experiments for each condition were quantified by densitometry and depicted as percent of input .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ChIP assay to analyze binding of TDP-43 to VPS4B promoter region in rat cortical neurons, HEK293 cells and human brain tissue. PCR reaction from input, negative control and TDP-43 immunoprecipitates. Signal intensities from at least three independent experiments for each condition were quantified by densitometry and depicted as percent of input.


( E - H ) Analysis of VPS4B in tissue of seven patients with the neuropathological diagnosis of FTLD / ALS - TDP and five healthy controls . ( E , G ) Immunofluorescence with the indicated antibodies in the superior frontal gyrus or spinal cord . Scale bar represents 50 µm . ( F , H ) Quantification of cellular VPS4B levels . At least 50 cells per case were analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) Analysis of VPS4B in tissue of seven patients with the neuropathological diagnosis of FTLD/ALS-TDP and five healthy controls. (E, G) Immunofluorescence with the indicated antibodies in the superior frontal gyrus or spinal cord. Scale bar represents 50 µm. (F, H) Quantification of cellular VPS4B levels. At least 50 cells per case were analyzed.


( A ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with either VPS4B or an empty vector control together with GFP - RAB11 to visualize recycling endosomes . Neurons were imaged as in Figure 1 to analyze recycling endosome transport . Quantitative analysis of vesicle motility ( B ) and number ( C ) from kymographs ( n = 4 ) .

(A) Primary hippocampal neurons (DIV6+3) were transfected with either VPS4B or an empty vector control together with GFP-RAB11 to visualize recycling endosomes. Neurons were imaged as in Figure 1 to analyze recycling endosome transport. Quantitative analysis of vesicle motility (B) and number (C) from kymographs (n = 4).


( D ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with the indicated combinations of shRNA targeting TDP - 43 , VPS4B and control together with GFP - RAB11 and imaged as in Figure 1A . Quantitative analysis of vesicle motility ( E ) and number ( F ) from kymographs ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primary hippocampal neurons (DIV6+3) were transfected with the indicated combinations of shRNA targeting TDP-43, VPS4B and control together with GFP-RAB11 and imaged as in Figure 1A. Quantitative analysis of vesicle motility (E) and number (F) from kymographs (n=3).


( A ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with shRNA targeting TDP - 43 ( shTDP ) or control ( shCtrl ) and surface expression levels of glycoproteins was analyzed by metabolic labeling followed by surface biotinylation and subsequent streptavidin purification . Volcano plot depicts proteins that are significantly ( p < 0 . 01 , t - test , Benjamini - Hochberg , FDR : 0 . 05 ) decreased or increased on the cell surface in three independent experiments with two technical replicates . For full dataset see Table EV2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primary hippocampal neurons (DIV6+4) were transduced with shRNA targeting TDP-43 (shTDP) or control (shCtrl) and surface expression levels of glycoproteins was analyzed by metabolic labeling followed by surface biotinylation and subsequent streptavidin purification. Volcano plot depicts proteins that are significantly (p < 0.01, t-test, Benjamini-Hochberg, FDR: 0.05) decreased or increased on the cell surface in three independent experiments with two technical replicates. For full dataset see Table EV2.


( B - C ) Primary cortical neurons ( DIV6 + 5 ) were transduced with shCtrl or shTDP . Neurons were treated with the ErbB4 ligand NRG1 ( 10 nM ) for 10 min right before cell lysis . Immunoblots with the indicated antibodies ( B ) . Quantification of total and phosho - ERBB4 ( Y1056 ) using densitometry 3 - 7 biological replicates ( C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Primary cortical neurons (DIV6+5) were transduced with shCtrl or shTDP. Neurons were treated with the ErbB4 ligand NRG1 (10 nM) for 10 min right before cell lysis. Immunoblots with the indicated antibodies (B). Quantification of total and phosho-ERBB4 (Y1056) using densitometry 3-7 biological replicates (C).


( D - E ) Primary cortical neurons ( DIV6 + 5 ) were transduced with shCtrl or shTDP . Neurons were treated with the FGFR1 ligand FGF1 ( 10 ng / ml ) for 10 min right before cell lysis . Immunoblots with the indicated antibodies ( D ) . Quantification of total and phosho - FGFR1 ( Y654 ) using densitometry 3 - 7 biological replicates ( E ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Primary cortical neurons (DIV6+5) were transduced with shCtrl or shTDP. Neurons were treated with the FGFR1 ligand FGF1 (10 ng/ml) for 10 min right before cell lysis. Immunoblots with the indicated antibodies (D). Quantification of total and phosho-FGFR1 (Y654) using densitometry 3-7 biological replicates (E).


( F ) Primary hippocampal neurons ( DIV6 + 4 ) were transduced with shTDP or shCtrl . Gene expression was analyzed by quantitative RT - PCR normalized to the housekeeping genes YWHAZ and GAPDH ( n = 3 )

O O O O O O O O O O O O O O O O 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) Primary hippocampal neurons (DIV6+4) were transduced with shTDP or shCtrl. Gene expression was analyzed by quantitative RT-PCR normalized to the housekeeping genes YWHAZ and GAPDH (n=3)


( G ) Hippocampal neurons were transfected with ErbB4 containing a luminal HA - tag ( HA / T - ErbB4 ) and shCtrl or shTDP ( DIV9 + 3 ) . Surface HA / T - ErbB4 was stained in living cells and intracellular HA / T - ErbB4 was stained after fixation and permeabilization with HA - antibodies . ( H ) Quantification of HA / T - ErbB4 surface levels . At least 10 images per condition per experiment were analyzed in three independent experiments . Scale bar represents 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 O O O O O O O O O O O O O O O O O O O O O O O O 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) Hippocampal neurons were transfected with ErbB4 containing a luminal HA-tag (HA/T-ErbB4) and shCtrl or shTDP (DIV9+3). Surface HA/T-ErbB4 was stained in living cells and intracellular HA/T-ErbB4 was stained after fixation and permeabilization with HA-antibodies. (H) Quantification of HA/T-ErbB4 surface levels. At least 10 images per condition per experiment were analyzed in three independent experiments. Scale bar represents 50 µm.


( A , B ) Primary hippocampal neurons ( DIV6 + 3 ) were transfected with shCtrl or shTDP and ErbB4 containing an N - terminal HA epitope tag and followed by thrombin cleavage site ( HA / T - ErbB4 ) . Surface HA / T - ErbB4 was stained in living cells and intracellular HA / T - ErbB4 was stained after fixation and permeabilization with two different HA - antibodies . After removing the HA - signal of surface ErbB4 ( 0 min ) using thrombin cleavage ( 5min , 1U / ml ) membrane insertion of HA / T - ErbB4 was measured at the indicated time points . At least 10 images per condition per experiment were analyzed in three independent experiments . Scale bar represents 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primary hippocampal neurons (DIV6+3) were transfected with shCtrl or shTDP and ErbB4 containing an N-terminal HA epitope tag and followed by thrombin cleavage site (HA/T-ErbB4). Surface HA/T-ErbB4 was stained in living cells and intracellular HA/T-ErbB4 was stained after fixation and permeabilization with two different HA-antibodies. After removing the HA-signal of surface ErbB4 (0 min) using thrombin cleavage (5min, 1U/ml) membrane insertion of HA/T-ErbB4 was measured at the indicated time points. At least 10 images per condition per experiment were analyzed in three independent experiments. Scale bar represents 50 µm.


Primary hippocampal neurons ( DIV6 + 4 ) were cotransfected with shTDP and GFP to visualize neuron morphology . Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two - way ANOVA with Bonferroni ' s ( B , D ) or Tukey ' s ( F , H ) post - test . Scale bar represents 100 µm . ( A , B ) TDP - 43 knockdown ( DIV6 + 5 ) significantly reduces dendrite branching compared to control : at 25 , 37 . 5 and 87 . 5 µm radius p < 0 . 05 , at 50 µm p < 0 . 01 and from 62 . 5 to 75 µm p < 0 . 001 .

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

Primary hippocampal neurons (DIV6+4) were cotransfected with shTDP and GFP to visualize neuron morphology. Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two-way ANOVA with Bonferroni's (B, D) or Tukey's (F, H) post-test. Scale bar represents 100 µm. (A, B) TDP-43 knockdown (DIV6+5) significantly reduces dendrite branching compared to control: at 25, 37.5 and 87.5 µm radius p < 0.05, at 50 µm p < 0.01 and from 62.5 to 75 µm p < 0.001.


Primary hippocampal neurons ( DIV6 + 4 ) were cotransfected with VPS4B or appropriate controls and GFP to visualize neuron morphology . Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two - way ANOVA with Bonferroni ' s ( B , D ) or Tukey ' s ( F , H ) post - test . Scale bar represents 100 µm . ( C , D ) VPS4B transfection ( DIV6 + 4 ) significantly reduces dendrite complexity : at 25 µm radius p < 0 . 05 , from 37 . 5 to 75 µm p < 0 . 001 and from 87 . 5 to 100 µm p < 0 . 01 .

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

Primary hippocampal neurons (DIV6+4) were cotransfected with VPS4B or appropriate controls and GFP to visualize neuron morphology. Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two-way ANOVA with Bonferroni's (B, D) or Tukey's (F, H) post-test. Scale bar represents 100 µm. (C, D) VPS4B transfection (DIV6+4) significantly reduces dendrite complexity: at 25 µm radius p < 0.05, from 37.5 to 75 µm p < 0.001 and from 87.5 to 100 µm p < 0.01.


Primary hippocampal neurons ( DIV6 + 4 ) were cotransfected with shTDP or appropriate controls and GFP to visualize neuron morphology . Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two - way ANOVA with Bonferroni ' s ( B , D ) or Tukey ' s ( F , H ) post - test . Scale bar represents 100 µm . ( E , F ) Two days after shRNA transfection neurons were treated with 1 nM NRG1 - beta 1 ( NRG1 ) or vehicle and analyzed at DIV6 + 5 . shCtrl vs . shTDP : at 25 and 87 . 5 µm radius p < 0 . 05 , from 37 . 5 to 75 µm p < 0 . 001 . shCtrl vs . shCtrl + NRG1 : from 37 . 5 to 62 . 5 µm p < 0 . 05 . shTDP vs . shTDP + NRG1 : no significant difference .

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

Primary hippocampal neurons (DIV6+4) were cotransfected with shTDP or appropriate controls and GFP to visualize neuron morphology. Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two-way ANOVA with Bonferroni's (B, D) or Tukey's (F, H) post-test. Scale bar represents 100 µm. (E, F) Two days after shRNA transfection neurons were treated with 1 nM NRG1-beta 1 (NRG1) or vehicle and analyzed at DIV6+5. shCtrl vs. shTDP: at 25 and 87.5 µm radius p < 0.05, from 37.5 to 75 µm p < 0.001. shCtrl vs. shCtrl + NRG1: from 37.5 to 62.5 µm p < 0.05. shTDP vs. shTDP+NRG1: no significant difference.


Primary hippocampal neurons ( DIV6 + 4 ) were cotransfected with shTDP or appropriate controls and GFP to visualize neuron morphology . Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two - way ANOVA with Bonferroni ' s ( B , D ) or Tukey ' s ( F , H ) post - test . Scale bar represents 100 µm . ( G , H ) Transfection of the indicated combinations of shCtrl , shTDP , ErbB4 or vector control ( DIV6 + 5 ) . shCtrl + Ctrl vs . shTDP + Ctrl : from 25 to 75 µm radius p < 0 . 001 , at 87 and 112 . 5 µm p < 0 . 01 and at 100 µm p < 0 . 05 . shTDP + Ctrl vs . shTDP + ErbB4 : from 25 to 100 µm radius p < 0 . 001 , at 112 . 5 µm p < 0 . 01 . shCtrl + Ctrl vs . shTDP + ErbB4 : no significant differences .

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

Primary hippocampal neurons (DIV6+4) were cotransfected with shTDP or appropriate controls and GFP to visualize neuron morphology. Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two-way ANOVA with Bonferroni's (B, D) or Tukey's (F, H) post-test. Scale bar represents 100 µm. (G, H) Transfection of the indicated combinations of shCtrl, shTDP, ErbB4 or vector control (DIV6+5). shCtrl + Ctrl vs. shTDP + Ctrl: from 25 to 75 µm radius p < 0.001, at 87 and 112.5 µm p < 0.01 and at 100 µm p < 0.05. shTDP + Ctrl vs. shTDP + ErbB4: from 25 to 100 µm radius p < 0.001, at 112.5 µm p < 0.01. shCtrl + Ctrl vs. shTDP + ErbB4: no significant differences.


( A , B ) In situ cryo - electron tomogram displaying the proximal region of a mature mother centriole ( A ) and procentriole ( B ) . Mature centriole , MC ; procentriole , PC ; mitochondria , mito ; vacuole , vac ; white dashed line , lamella edge . Scale bars , 100 nm .

(A, B) In situ cryo-electron tomogram displaying the proximal region of a mature mother centriole (A) and procentriole (B). Mature centriole, MC; procentriole, PC; mitochondria, mito; vacuole, vac; white dashed line, lamella edge. Scale bars, 100 nm.


( C ) Side view z - projection of cartwheels containing the central hub and several CIDs from a mature centriole . Central hub , CH ; cartwheel inner densities , CID . Scale bar , 20 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 O O O O O O O O O O O O O O O

(C) Side view z-projection of cartwheels containing the central hub and several CIDs from a mature centriole. Central hub, CH; cartwheel inner densities, CID. Scale bar, 20 nm.


( D ) Cross section of the cartwheel - containing regions from a mature centriole . Microtubule triplet , MTT ; spokes , SP . Scale bar , 200 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 O O O O O O O

(D) Cross section of the cartwheel-containing regions from a mature centriole. Microtubule triplet, MTT; spokes, SP. Scale bar, 200 nm.


( E ) Side view z - projection of cartwheels containing the central hub and several CIDs from a procentriole . Scale bar , 20 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 O O O

(E) Side view z-projection of cartwheels containing the central hub and several CIDs from a procentriole. Scale bar, 20 nm.


( F ) Cross section of the cartwheel - containing regions from a procentriole . Scale bar , 200 nm .

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

(F) Cross section of the cartwheel-containing regions from a procentriole. Scale bar, 200 nm.


( G ) Nine - fold symmetrized cross sections of the cartwheel - containing region from a mature centriole ( left side ) and a procentriole ( right side ) . Dashed white circle , central hub . Scale bars , 100 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 O O O O O O O O O O O O O O O O O O O O

(G) Nine-fold symmetrized cross sections of the cartwheel-containing region from a mature centriole (left side) and a procentriole (right side). Dashed white circle, central hub. Scale bars, 100 nm.


( H ) Longitudinal periodicity measurements of the central hub and CIDs . Central hub , blue ; CID , red . Mean values are displayed above the data range . Blue data points are measured distance between individual units of the central hub , red data points are measured distances between individual units of the cartwheel inner density . Mature centriole , central hub , n = 30 , mean = 4 . 1 + / - 0 . 67 ( S . D ) ; mature centriole , cartwheel inner density , n = 18 , mean = 8 . 7 + / - 1 . 6 ( S . D ) ; procentriole , central hub , n = 10 , mean = 4 . 0 + / - 0 . 66 ( S . D ) ; procentriole , cartwheel inner density , n = 5 , mean = 8 . 6 + / - 1 . 2 ( S . D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Longitudinal periodicity measurements of the central hub and CIDs. Central hub, blue; CID, red. Mean values are displayed above the data range. Blue data points are measured distance between individual units of the central hub, red data points are measured distances between individual units of the cartwheel inner density. Mature centriole, central hub, n = 30, mean = 4.1 +/- 0.67 (S.D); mature centriole, cartwheel inner density, n = 18, mean = 8.7 +/- 1.6 (S.D); procentriole, central hub, n = 10, mean = 4.0 +/- 0.66 (S.D); procentriole, cartwheel inner density, n = 5, mean = 8.6 +/- 1.2 (S.D).


( I , J ) Nine - fold symmetrized central hub z - projections , starting at the proximal end of the cartwheel and continuing distally along the cartwheel by 5 . 4 nm steps in a mature centriole ( I ) and a procentriole ( J ) . Red arrow , CID ; blue arrow ; central hub . Scale bar : 20 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 O O O O O O O O O O O O O O O O O O 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) Nine-fold symmetrized central hub z-projections, starting at the proximal end of the cartwheel and continuing distally along the cartwheel by 5.4 nm steps in a mature centriole (I) and a procentriole (J). Red arrow, CID; blue arrow; central hub. Scale bar: 20 nm.


( A , B , C ) Cryo - electron tomograms of the proximal regions of a P . tetraurelia centriole ( A ) , a N . gruberi centriole ( B ) , and a H . sapiens procentriole ( C ) . White arrows denote a broken cartwheel ; procentriole , PC ; mature centriole , MC ; Scale bar , 100 nm . Note that most N . gruberi and H . sapiens centrioles were heavily compressed during the cryo - EM preparation , as previously described ( Guichard et al . , 2010 ) . The displayed N . gruberi centriole illustrates the damage caused by compression . The periodicities of N . gruberi and H . sapiens cartwheels were measured only on regions that were not

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C) Cryo-electron tomograms of the proximal regions of a P. tetraurelia centriole (A), a N. gruberi centriole (B), and a H. sapiens procentriole (C). White arrows denote a broken cartwheel; procentriole, PC; mature centriole, MC; Scale bar, 100 nm. Note that most N. gruberi and H. sapiens centrioles were heavily compressed during the cryo-EM preparation, as previously described (Guichard et al., 2010). The displayed N. gruberi centriole illustrates the damage caused by compression. The periodicities of N. gruberi and H. sapiens cartwheels were measured only on regions that were not damaged


( D ) Cross sections from cartwheel - containing region of P . tetraurelia centrioles . Scale bar , 50 nm . ( E ) Zoomed side view of cartwheel from P . tetraurelia , displaying the central hub ( CH ) and several cartwheel inner densities ( CIDs ) , white arrow . Scale bar , 25 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 O O O O O O O O O O O O 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) Cross sections from cartwheel-containing region of P. tetraurelia centrioles. Scale bar, 50 nm. (E) Zoomed side view of cartwheel from P. tetraurelia, displaying the central hub (CH) and several cartwheel inner densities (CIDs), white arrow. Scale bar, 25 nm.


( F ) Cross section from cartwheel - containing region of N . gruberi , ( G ) Zoomed side view of cartwheel from N . gruberi displaying the central hub ( CH ) and several cartwheel inner densities ( CIDs ) , white arrow .

O O O O O O O O O O O O O O O O O O O O O O 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) Cross section from cartwheel-containing region of N. gruberi, (G) Zoomed side view of cartwheel from N. gruberi displaying the central hub (CH) and several cartwheel inner densities (CIDs), white arrow.


( H ) Cross section from cartwheel - containing region of H . sapiens centriole . ( I ) Zoomed side view of cartwheel from H . sapiens , displaying the central hub ( CH ) and several cartwheel inner densities ( CIDs ) , white arrow .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Cross section from cartwheel-containing region of H. sapiens centriole. (I) Zoomed side view of cartwheel from H. sapiens, displaying the central hub (CH) and several cartwheel inner densities (CIDs), white arrow.


( J ) Longitudinal periodicity of the central hub and CIDs in P . tetraurelia , N . gruberi , and H . sapiens . Mean values are displayed above data range . Black lines indicate the mean and the standard deviation . Blue data points are measured distance between individual units of the central hub , red data points are measured distances between individual units of the cartwheel inner density . P . tetraurelia , central hub , n = 10 , mean = 4 . 3 + / - 0 . 38 ( S . D ) ; N . gruberi , central hub , n = 10 , mean = 4 . 4 + / - 0 . 53 ( S . D ) ; H . sapiens , central hub , n = 10 , mean = 4 . 2 + / - 0 . 68 ( S . D ) ; P . tetraurelia , cartwheel inner density , n = 8 , mean = 8 . 4 + / - 1 . 3 ( S . D ) ; N . gruberi , cartwheel inner density , n = 8 , mean = 8 . 3 + / - 1 . 8 ( S . D ) ; H . sapiens , cartwheel inner density , n = 8 , mean = 8 . 1 + / - 2 . 5 ( S . D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Longitudinal periodicity of the central hub and CIDs in P. tetraurelia, N. gruberi, and H. sapiens. Mean values are displayed above data range. Black lines indicate the mean and the standard deviation. Blue data points are measured distance between individual units of the central hub, red data points are measured distances between individual units of the cartwheel inner density. P. tetraurelia, central hub, n = 10, mean = 4.3 +/- 0.38 (S.D); N. gruberi, central hub, n = 10, mean = 4.4 +/- 0.53 (S.D); H. sapiens, central hub, n = 10, mean = 4.2 +/- 0.68 (S.D); P. tetraurelia, cartwheel inner density, n = 8, mean = 8.4 +/- 1.3 (S.D); N. gruberi, cartwheel inner density, n = 8, mean = 8.3 +/- 1.8 (S.D); H. sapiens, cartwheel inner density, n = 8, mean = 8.1 +/- 2.5 (S.D).


( K Proximal protrusion length of the cartwheel beyond the microtubule triplets in C . reinhardtii , P . tetraurelia , N . gruberi , and H . sapiens . Internal cartwheel inside the microtubule barrel , dark blue ( INT ) ; external cartwheel beyond the microtubule wall , light blue ( EXT ) . Mean values are displayed above each bar plot with the black lines indicating the standard deviation ( K ) . Mature C . reinhardtii , n = 4 , external cartwheel length = 37 . 6 + / - 3 . 4 , internal cartwheel length 74 . 2 + / - 16 . 6 ; C . reinhardtii procentriole , n = 2 , external cartwheel mean length = 52 . 8 + / - 22 . 0 , internal cartwheel length 26 . 5 + / - 29 . 7 ; P . tetraurelia , n = 23 , external cartwheel mean length = 11 . 5 + / - 9 . 2 , internal cartwheel length 66 . 6 + / - 15 . 7 ; N . gruberi , n = 19 , external cartwheel mean length = 41 . 0 + / - 22 . 5 , internal cartwheel length 259 . 0 + / - 87 . 2 ; human procentriole , n = 7 , external cartwheel length 23 . 1 + / - 10 . 3 , internal cartwheel length 154 . 7 + / - 47 . 7 . Error reported is standard deviation . Scale bar , 50 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K Proximal protrusion length of the cartwheel beyond the microtubule triplets in C. reinhardtii, P. tetraurelia, N. gruberi, and H. sapiens. Internal cartwheel inside the microtubule barrel, dark blue (INT); external cartwheel beyond the microtubule wall, light blue (EXT). Mean values are displayed above each bar plot with the black lines indicating the standard deviation (K). Mature C. reinhardtii, n = 4, external cartwheel length = 37.6 +/- 3.4, internal cartwheel length 74.2 +/- 16.6; C. reinhardtii procentriole, n = 2, external cartwheel mean length = 52.8 +/- 22.0, internal cartwheel length 26.5 +/- 29.7; P. tetraurelia, n = 23, external cartwheel mean length = 11.5 +/- 9.2, internal cartwheel length 66.6 +/- 15.7; N. gruberi, n = 19, external cartwheel mean length = 41.0 +/- 22.5, internal cartwheel length 259.0 +/- 87.2; human procentriole, n = 7, external cartwheel length 23.1 +/- 10.3, internal cartwheel length 154.7 +/- 47.7. Error reported is standard deviation. Scale bar, 50 nm.


The start of each microtubule wall is delineated by a dashed white line ( L ) . Mature C . reinhardtii , n = 4 , external cartwheel length = 37 . 6 + / - 3 . 4 , internal cartwheel length 74 . 2 + / - 16 . 6 ; C . reinhardtii procentriole , n = 2 , external cartwheel mean length = 52 . 8 + / - 22 . 0 , internal cartwheel length 26 . 5 + / - 29 . 7 ; P . tetraurelia , n = 23 , external cartwheel mean length = 11 . 5 + / - 9 . 2 , internal cartwheel length 66 . 6 + / - 15 . 7 ; N . gruberi , n = 19 , external cartwheel mean length = 41 . 0 + / - 22 . 5 , internal cartwheel length 259 . 0 + / - 87 . 2 ; human procentriole , n = 7 , external cartwheel length 23 . 1 + / - 10 . 3 , internal cartwheel length 154 . 7 + / - 47 . 7 . Error reported is standard deviation . Scale bar , 50 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The start of each microtubule wall is delineated by a dashed white line (L). Mature C. reinhardtii, n = 4, external cartwheel length = 37.6 +/- 3.4, internal cartwheel length 74.2 +/- 16.6; C. reinhardtii procentriole, n = 2, external cartwheel mean length = 52.8 +/- 22.0, internal cartwheel length 26.5 +/- 29.7; P. tetraurelia, n = 23, external cartwheel mean length = 11.5 +/- 9.2, internal cartwheel length 66.6 +/- 15.7; N. gruberi, n = 19, external cartwheel mean length = 41.0 +/- 22.5, internal cartwheel length 259.0 +/- 87.2; human procentriole, n = 7, external cartwheel length 23.1 +/- 10.3, internal cartwheel length 154.7 +/- 47.7. Error reported is standard deviation. Scale bar, 50 nm.


( A ) Top view of cartwheel reconstruction from P . tetraurelia . Scale bar , 50 nm . Dark orange arrow marks the D2 - rod . Purple arrow marks part of the pinhead . Dotted yellow box with arrows denotes the central hub - focused reslice shown in panel B ; dashed white box with arrows denotes the spoke - focused reslice shown in panel D .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top view of cartwheel reconstruction from P. tetraurelia. Scale bar, 50 nm. Dark orange arrow marks the D2-rod. Purple arrow marks part of the pinhead. Dotted yellow box with arrows denotes the central hub-focused reslice shown in panel B; dashed white box with arrows denotes the spoke-focused reslice shown in panel D.


( B ) Reslice of central hub - containing region with spokes in P . tetraurelia . Scale bar , 50 nm . Dashed light yellow line denotes the zoomed view shown in panels C , black line with arrows indicates the ~ 25 nm repeat distance between D1 densities formed by merged spokes . Dark orange arrow marks the D2 - rod .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Reslice of central hub-containing region with spokes in P. tetraurelia. Scale bar, 50 nm. Dashed light yellow line denotes the zoomed view shown in panels C, black line with arrows indicates the ~25 nm repeat distance between D1 densities formed by merged spokes. Dark orange arrow marks the D2-rod.


( C ) Zoomed view displaying periodic repeats of the central hub ( CH ) and several cartwheel inner densities ( CIDs ) in P . tetraurelia . CIDs , red arrowheads and red plot profile ; CH , blue arrowheads and blue plot profile . Overlay between CID and CH peaks is plotted on the right in red and blue . Mean distance between CID peaks , red 8 . 6 + / - . 8 ( n = 5 ) . Distances between CH peaks split into two distinct populations : smaller ( within a ring - pair ) , green 3 . 1 + / - 1 . 3 ( n = 6 ) ; larger ( between ring - pairs ) , blue 5 . 5 + / - . 7 ( n = 5 ) ; average periodicity , black 4 . 2 + / - 1 . 3 ( n = 11 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Zoomed view displaying periodic repeats of the central hub (CH) and several cartwheel inner densities (CIDs) in P. tetraurelia. CIDs, red arrowheads and red plot profile; CH, blue arrowheads and blue plot profile. Overlay between CID and CH peaks is plotted on the right in red and blue. Mean distance between CID peaks, red 8.6 +/- .8 (n= 5). Distances between CH peaks split into two distinct populations: smaller (within a ring-pair), green 3.1 +/- 1.3 (n= 6); larger (between ring-pairs), blue 5.5 +/- .7 (n= 5); average periodicity, black 4.2 +/- 1.3 (n= 11).


( D ) Serial z - projections of ~ 4 nm thickness from one cartwheel repeat unit of P . tetraurelia . Left - most z - projections display the central hub , right - most projection shows the microtubule wall . Blue circles delineate one ring - pair . White arrows mark individual emanating spokes . White arrowheads mark fused spokes . White dashed line follows the tilt of the spokes . Black arrow indicates the final merged spoke ( D1 density ) . Dark orange arrow marks the D2 - rod . Scale bar , 50 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Serial z-projections of ~4 nm thickness from one cartwheel repeat unit of P. tetraurelia. Left-most z-projections display the central hub, right-most projection shows the microtubule wall. Blue circles delineate one ring-pair. White arrows mark individual emanating spokes. White arrowheads mark fused spokes. White dashed line follows the tilt of the spokes. Black arrow indicates the final merged spoke (D1 density). Dark orange arrow marks the D2-rod. Scale bar, 50 nm.


( E ) Top view of cartwheel reconstruction from C . reinhardtii . Scale bar , 50 nm . Dark orange arrow marks the D2 - rod . Purple arrow marks part of the pinhead . Dotted yellow box with arrows denotes the central hub - focused reslice shown in panel F ; dashed white box with arrows denotes the spoke - focused reslice shown in panel H .

(E) Top view of cartwheel reconstruction from C. reinhardtii. Scale bar, 50 nm. Dark orange arrow marks the D2-rod. Purple arrow marks part of the pinhead. Dotted yellow box with arrows denotes the central hub-focused reslice shown in panel F; dashed white box with arrows denotes the spoke-focused reslice shown in panel H.


( F ) Reslice of central hub - containing region with spokes in C . reinhardtii . Scale bar , 50 nm . Dashed light yellow line denotes the zoomed view shown in panel G , black line with arrows indicates the ~ 25 nm repeat distance between D1 densities formed by merged spokes . Dark orange arrow marks the D2 - rod .

(F) Reslice of central hub-containing region with spokes in C. reinhardtii. Scale bar, 50 nm. Dashed light yellow line denotes the zoomed view shown in panel G, black line with arrows indicates the ~25 nm repeat distance between D1 densities formed by merged spokes. Dark orange arrow marks the D2-rod.


( G ) Zoomed view displaying periodic repeats of the central hub ( CH ) and several cartwheel inner densities ( CIDs ) in C . reinhardtii . CIDs , red arrowheads and red plot profile ; CH , blue arrowheads and blue plot profile . Overlay between CID and CH peaks is plotted on the right in red and blue . Mean distance between CID peaks , red 8 . 8 + / - 1 . 0 ( n = 5 ) . Distances between CH peaks split into two distinct populations : smaller ( within a ring - pair ) , green 3 . 5 + / - 1 . 3 ( n = 6 ) ; larger ( between ring - pairs ) , blue 5 . 1 + / - . 4 ( n = 5 ) ; average periodicity , black 4 . 2 + / - 1 . 3 ( n = 11 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Zoomed view displaying periodic repeats of the central hub (CH) and several cartwheel inner densities (CIDs) in C. reinhardtii. CIDs, red arrowheads and red plot profile; CH, blue arrowheads and blue plot profile. Overlay between CID and CH peaks is plotted on the right in red and blue. Mean distance between CID peaks, red 8.8 +/- 1.0 (n= 5). Distances between CH peaks split into two distinct populations: smaller (within a ring-pair), green 3.5 +/- 1.3 (n= 6); larger (between ring-pairs), blue 5.1 +/- .4 (n= 5); average periodicity, black 4.2 +/- 1.3 (n= 11).


( H ) Serial z - projections of ~ 4 nm thickness from one cartwheel repeat unit of C . reinhardtii . Left - most z - projections display the central hub , right - most projection shows the microtubule wall . Blue circles delineate one ring - pair . White arrows mark individual emanating spokes . White arrowheads mark fused spokes . White dashed line follows the tilt of the spokes . Black arrow indicates the final merged spoke ( D1 density ) . Dark orange arrow marks the D2 - rod . Scale bar , 50 nm .

(H) Serial z-projections of ~4 nm thickness from one cartwheel repeat unit of C. reinhardtii. Left-most z-projections display the central hub, right-most projection shows the microtubule wall. Blue circles delineate one ring-pair. White arrows mark individual emanating spokes. White arrowheads mark fused spokes. White dashed line follows the tilt of the spokes. Black arrow indicates the final merged spoke (D1 density). Dark orange arrow marks the D2-rod. Scale bar, 50 nm.


( A ) Cryo - electron tomogram of P . tetraurelia centriole . Blue arrow denotes cartwheel , orange arrow denotes inner scaffold . White dashed box delimits the inset represented in ( D , E ) . Red lines and arrows indicate the position and the direction of the cross - sections made in ( B ) . Scale bar , 100 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 O O O O O O O O O O O O O O O O O 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) Cryo-electron tomogram of P. tetraurelia centriole. Blue arrow denotes cartwheel, orange arrow denotes inner scaffold. White dashed box delimits the inset represented in (D, E). Red lines and arrows indicate the position and the direction of the cross-sections made in (B). Scale bar, 100 nm.


( B ) Nine - fold symmetrizations of serial cross - sections taken along the proximal to distal axis in P . tetraurelia . Each section is a z - projection of 20 . 7 nm . White dashed circles delineate the structures highlighted in C . Scale bar , 60 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 O O O O O 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) Nine-fold symmetrizations of serial cross-sections taken along the proximal to distal axis in P. tetraurelia. Each section is a z-projection of 20.7 nm. White dashed circles delineate the structures highlighted in C. Scale bar, 60 nm.


( C ) Zoomed images of proximal centriole substructures from nine - fold symmetrizations of P . tetraurelia along the proximal - distal axis . Each panel corresponds to the above image from panel B . Purple arrow , pinhead ; light green arrow , triplet base ; turquoise arrow , A - C linker ; orange arrow , inner scaffold .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Zoomed images of proximal centriole substructures from nine-fold symmetrizations of P. tetraurelia along the proximal-distal axis. Each panel corresponds to the above image from panel B. Purple arrow, pinhead; light green arrow, triplet base; turquoise arrow, A-C linker; orange arrow, inner scaffold.


( D ) Side view showing the transition from pinhead to inner scaffold in P . tetraurelia .

(D) Side view showing the transition from pinhead to inner scaffold in P. tetraurelia.


( G ) Cryo - electron tomogram of C . reinhardtii centriole . Blue arrow denotes cartwheel , orange arrow denotes inner scaffold . White dashed box delimits the inset represented in ( J , K ) . Red lines and arrows indicate the position and the direction of the cross - sections made in ( H ) . Scale bar , 100 nm .

(G) Cryo-electron tomogram of C. reinhardtii centriole. Blue arrow denotes cartwheel, orange arrow denotes inner scaffold. White dashed box delimits the inset represented in (J, K). Red lines and arrows indicate the position and the direction of the cross-sections made in (H). Scale bar, 100 nm.


( H ) Nine - fold symmetrizations of serial cross - sections taken along the proximal to distal axis in C . reinhardtii . Each section is a z - projection of 20 . 7 nm . White dashed circles delineate the structures highlighted in I . Scale bar , 60 nm .

(H) Nine-fold symmetrizations of serial cross-sections taken along the proximal to distal axis in C. reinhardtii. Each section is a z-projection of 20.7 nm. White dashed circles delineate the structures highlighted in I. Scale bar, 60 nm.


( I ) Zoomed images of proximal centriole substructures from nine - fold symmetrizations of C . reinhardtii along the proximal - distal axis . Each panel corresponds to the above image from panel H . Purple arrow , pinhead ; light green arrow , triplet base ; turquoise arrow , A - C linker ; orange arrow , inner scaffold .

(I) Zoomed images of proximal centriole substructures from nine-fold symmetrizations of C. reinhardtii along the proximal-distal axis. Each panel corresponds to the above image from panel H. Purple arrow, pinhead; light green arrow, triplet base; turquoise arrow, A-C linker; orange arrow, inner scaffold.


( J , K ) Side views showing the transition from pinhead to inner scaffold in C . reinhardtii from two consecutive sections from a tomogram .

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

(J, K) Side views showing the transition from pinhead to inner scaffold in C. reinhardtii from two consecutive sections from a tomogram.


( M , N ) Positioning of the different structures along the proximal length from representative P . tetraurelia ( M ) and C . reinhardtii ( N ) centrioles . Distance between the ends of the pinhead and cartwheel regions is denoted by zone 1 ( for quantification , see Figure EV4E ) . Distance between end of the pinhead region and start of the inner scaffold region is denoted by zone

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

(M, N) Positioning of the different structures along the proximal length from representative P. tetraurelia (M) and C. reinhardtii (N) centrioles. Distance between the ends of the pinhead and cartwheel regions is denoted by zone 1 (for quantification, see Figure EV4E). Distance between end of the pinhead region and start of the inner scaffold region is denoted by zone 2


( O ) Cartwheel and A - C linker length in C . reinhardtii ( n = 5 centrioles ) , P . tetraurelia ( n = 16 centrioles ) , N . gruberi ( n = 11 centrioles ) , and H . sapiens ( n = 5 centrioles ) . Means and standard deviations of the mean are displayed above the range . A - C linker , turquoise ; cartwheel , blue ; microtubule triplets , grey .

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

(O) Cartwheel and A-C linker length in C. reinhardtii (n= 5 centrioles), P. tetraurelia (n= 16 centrioles), N. gruberi (n= 11 centrioles), and H. sapiens (n= 5 centrioles). Means and standard deviations of the mean are displayed above the range. A-C linker, turquoise; cartwheel, blue; microtubule triplets, grey.


Representative immunofluorescence ( IF ) staining of Ki67 and TDP - 43 in the regions of frontal cortices from 6 - mon - old WT and FTLD - TDP Tg mice . Nuclei were stained with 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI ; upper panel in blue ) or neural marker NeuN ( lower panel in green ) . Scale bar : 50 μm . The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions . Scale bar : 15 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunofluorescence (IF) staining of Ki67 and TDP-43 in the regions of frontal cortices from 6-mon-old WT and FTLD-TDP Tg mice. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; upper panel in blue) or neural marker NeuN (lower panel in green). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm.


Quantification of cells or neurons with Ki67 immunoreactivity and TDP - 43 mislocalization from each view of microscope . n = 9 sections per mouse , N = 5 mice per group , data are presented as mean ± SEM , * * * p = 0 . 0007 by 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

Quantification of cells or neurons with Ki67 immunoreactivity and TDP-43 mislocalization from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, ***p = 0.0007 by t-test.


Representative data of reverse - transcription PCR ( left panel ) or Western blot ( right panel ) for cell cycle - related genes and semi - quantification of the expression levels in the frontal cortices and hippocampus from the 6 - mon - old WT and FTLD - TDP Tg mice . N = 5 mice per group , data are presented as mean ± SEM ( % ) , statistical analysis by multiple t - test with FDR correction , Q = 1 % . * P < 0 . 05 , * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 and * * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 data of reverse-transcription PCR (left panel) or Western blot (right panel) for cell cycle-related genes and semi-quantification of the expression levels in the frontal cortices and hippocampus from the 6-mon-old WT and FTLD-TDP Tg mice. N = 5 mice per group, data are presented as mean ± SEM (%), statistical analysis by multiple t-test with FDR correction, Q = 1%. *P < 0.05, **P ≤ 0.01; ***P ≤ 0.001 and ****P ≤ 0.0001


Representative image of comet assay for DNA fragmentation and the quantification of cells with comet tails in the regions of frontal cortices from 6 - mon - old WT and FTLD - TDP Tg mice . Scale bar : 50 μm . n = 9 sections per mouse , N = 5 mice per group , data are presented as mean ± SEM , * p = 0 . 0114 by t - test .

O O O O O O B-MEASURED_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 O O 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 image of comet assay for DNA fragmentation and the quantification of cells with comet tails in the regions of frontal cortices from 6-mon-old WT and FTLD-TDP Tg mice. Scale bar: 50 μm. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, *p = 0.0114 by t-test.


Representative IF staining of γH2AX and TDP - 43 in the regions of frontal cortices from 6 - mon - old WT and FTLD - TDP Tg mice . Nuclei were stained with DAPI ( upper panel in blue ) or NeuN ( middle panel in green ) . Scale bar : 50 μm . The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions . Scale bar : 15 μm . Lower panel : quantification of cells or neurons with γH2AX immunoreactivity and TDP - 43 proteinopathies from each view of microscope . n = 9 sections per mouse , N = 5 mice per group , data are presented as mean ± SEM , * * * p = 0 . 0008 by 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 O 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 IF staining of γH2AX and TDP-43 in the regions of frontal cortices from 6-mon-old WT and FTLD-TDP Tg mice. Nuclei were stained with DAPI (upper panel in blue) or NeuN (middle panel in green). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Lower panel: quantification of cells or neurons with γH2AX immunoreactivity and TDP-43 proteinopathies from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, ***p = 0.0008 by t-test.


Representative IF staining of γH2AX and Ki67 in the regions of frontal cortices from WT and FTLD - TDP Tg mice . Nuclei were stained with DAPI ( upper panel ) or NeuN ( middle panel ) . Scale bar : 50 μm . Subregions , scale bar : 15 μm . Lower panel : quantification of cells or neurons with γH2AX and Ki67 immunoreactivity from each view of microscope . n = 9 sections per mouse , N = 5 mice per group , data are presented as mean ± SEM , * * * p = 0 . 0004 by 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

Representative IF staining of γH2AX and Ki67 in the regions of frontal cortices from WT and FTLD-TDP Tg mice. Nuclei were stained with DAPI (upper panel) or NeuN (middle panel). Scale bar: 50 μm. Subregions, scale bar: 15 μm. Lower panel: quantification of cells or neurons with γH2AX and Ki67 immunoreactivity from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, ***p = 0.0004 by t-test.


Top , representative western blot data of HDAC1 and TDP - 43 in extracts obtained following RIPA fractional extraction in the frontal cortices and hippocampus from 6 - mon - old of FTLD - TDP or WT mice . Bottom , semi - quantification of HDAC1 and TDP - 43 expression levels . N = 5 mice per group , data are presented as mean ± SEM ( % ) , * * * * p < 0 . 0001 by multiple t - tests .

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

Top, representative western blot data of HDAC1 and TDP-43 in extracts obtained following RIPA fractional extraction in the frontal cortices and hippocampus from 6-mon-old of FTLD-TDP or WT mice. Bottom, semi-quantification of HDAC1 and TDP-43 expression levels. N = 5 mice per group, data are presented as mean ± SEM (%), **** p < 0.0001 by multiple t-tests.


Top , representative western blot data of HDAC1 and TDP - 43 in the cytosolic and nuclear extracts . Bottom , quantification of HDAC1 and TDP - 43 levels . N = 5 mice per group , data are presented as mean ± SEM ( % ) , * * * p = 0 . 0005 ( TDP - 43 ) or 0 . 0004 ( HDAC1 ) ; * * * * p < 0 . 0001 by multiple t - tests .

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

Top, representative western blot data of HDAC1 and TDP-43 in the cytosolic and nuclear extracts. Bottom, quantification of HDAC1 and TDP-43 levels. N = 5 mice per group, data are presented as mean ± SEM (%), *** p = 0.0005 (TDP-43) or 0.0004 (HDAC1); **** p < 0.0001 by multiple t-tests.


IF staining of HDAC1 and SMI - 32 in the regions of frontal cortices from WT and FTLD - TDP Tg mice . Scale bar : 150 μm . Blocked area in left bottom picture is showed in a magnified view in the right bottom picture . Scale bar : 50 μm .

IF staining of HDAC1 and SMI-32 in the regions of frontal cortices from WT and FTLD-TDP Tg mice. Scale bar: 150 μm. Blocked area in left bottom picture is showed in a magnified view in the right bottom picture. Scale bar: 50 μm.


Nuclear HDAC1 activity assay from frontal cortices and hippocampus in 6 - mon - old of FTLD - TDP and WT mice . TSA : nuclear extracts that were treated with Trichostatin A ( TSA , an HDAC inhibitor ) as a negative control for HDAC1 - transferred fluorescent activity during the HDAC1 activity assay . N = 5 mice per group , data are presented as mean ± SEM ( % ) , * p = 0 . 0415 by 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 B-CONTROLLED_VAR O O O O O O O B-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 O O O O O O O O O O O

Nuclear HDAC1 activity assay from frontal cortices and hippocampus in 6-mon-old of FTLD-TDP and WT mice. TSA: nuclear extracts that were treated with Trichostatin A (TSA, an HDAC inhibitor) as a negative control for HDAC1-transferred fluorescent activity during the HDAC1 activity assay. N = 5 mice per group, data are presented as mean ± SEM (%), *p = 0.0415 by t-test.


Left , representative western blot data of nuclear acetylated - histone H3 ( Lys 9 / 14 ) and total - histone H3 in 6 - mon - old of WT and FTLD - TDP Tg mice . Right , quantification of nuclear acetylated - histone H3 / total - histone H3 ratio . N = 5 mice per group , data are presented as mean ± SEM ( % ) , * p = 0 . 0147 by 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

Left, representative western blot data of nuclear acetylated-histone H3 (Lys 9/14) and total-histone H3 in 6-mon-old of WT and FTLD-TDP Tg mice. Right, quantification of nuclear acetylated-histone H3/total-histone H3 ratio. N = 5 mice per group, data are presented as mean ± SEM (%), * p = 0.0147 by t-test.


Left graph : IF staining of TDP - 43 and HDAC1 during progression of TDP - 43 proteinopathies in the frontal cortices from the FTLD - TDP Tg and WT mice . Nuclei were stained with DAPI ( in blue ) . Scale bar : 50 μm . The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions . Scale bar : 15 μm . Right histogram : Quantification of co - localized TDP - 43 and HDAC1 immunoreactivity in the cytosol or nucleus in the WT or 1 - , 6 - , and 12 - month - old FTLD - TDP Tg mice . n = 9 sections per mouse , N = 5 mice per group , data are presented as mean ± SEM . * Nucleus : Tg 1m vs . Tg 6m or 12m ; # Cytosol : Tg 1m vs . Tg 6m or 12m ; @ Tg 6m vs Tg 12m . * * * * / # # # # / @ @ @ @ p < 0 . 0001 by multiple comparison .

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

Left graph: IF staining of TDP-43 and HDAC1 during progression of TDP-43 proteinopathies in the frontal cortices from the FTLD-TDP Tg and WT mice. Nuclei were stained with DAPI (in blue). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Right histogram: Quantification of co-localized TDP-43 and HDAC1 immunoreactivity in the cytosol or nucleus in the WT or 1-, 6-, and 12-month-old FTLD-TDP Tg mice. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM. *Nucleus: Tg 1m vs. Tg 6m or 12m; #Cytosol: Tg 1m vs. Tg 6m or 12m; @ Tg 6m vs Tg 12m. ****/####/@@@@ p < 0.0001 by multiple comparison.


Left graph : IF staining of γH2AX and HDAC1 in the frontal cortices from the 12 - month - old FTLD - TDP Tg and WT mice . Nuclei were stained with DAPI ( in blue ) . Arrow head : γH2AX + nucleus ; arrow : mislocalized HDAC1 . Scale bar : 50 μm . Right histogram : Quantification of γH2AX + and HDAC1 mislocalized cells in the WT or 1 - , 6 - , and 12 - month - old FTLD - TDP Tg mice . n = 4 sections per mouse , N = 5 mice per group , data are presented as mean ± SEM . * * p = 0 . 0079 , * * * * p < 0 . 0001 by multiple comparison .

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

Left graph: IF staining of γH2AX and HDAC1 in the frontal cortices from the 12-month-old FTLD-TDP Tg and WT mice. Nuclei were stained with DAPI (in blue). Arrow head: γH2AX+ nucleus; arrow: mislocalized HDAC1. Scale bar: 50 μm. Right histogram: Quantification of γH2AX+ and HDAC1 mislocalized cells in the WT or 1-, 6-, and 12-month-old FTLD-TDP Tg mice. n = 4 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM. ** p = 0.0079, **** p < 0.0001 by multiple comparison.


Left panel : flag - tagged full - length HDAC1 was overexpressed with myc - tagged TDP - 43 in HEK - 293T cells ; the cell lysates were immunoprecipitated for flag and immunoblotted for TDP - 43 and flag . Right panel : myc - tagged TDP - 43 was overexpressed with flag - tagged full - length HDAC1 in HEK - 293T cells ; the cell lysates were immunoprecipitated for myc and immunoblotted for flag and TDP - 43 .

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

Left panel: flag-tagged full-length HDAC1 was overexpressed with myc-tagged TDP-43 in HEK-293T cells; the cell lysates were immunoprecipitated for flag and immunoblotted for TDP-43 and flag. Right panel: myc-tagged TDP-43 was overexpressed with flag-tagged full-length HDAC1 in HEK-293T cells; the cell lysates were immunoprecipitated for myc and immunoblotted for flag and TDP-43.


Upper left : flag - tagged full - length HDAC1 ( b . Ⅰ ) or various truncation mutations ( b . Ⅱ - Ⅳ ) were overexpressed with myc - tagged TDP - 43 ; the catalytic domain is indicated in red . Lower panel : the western blotting of cell lysates immunoprecipitated for flag and immunoblotted for TDP - 43 .

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

Upper left: flag-tagged full-length HDAC1 (b.Ⅰ) or various truncation mutations (b.Ⅱ-Ⅳ) were overexpressed with myc-tagged TDP-43; the catalytic domain is indicated in red. Lower panel: the western blotting of cell lysates immunoprecipitated for flag and immunoblotted for TDP-43.


Upper panel : Immunoprecipitation of cytosolic HDAC1 and immunoblotting of HDAC1 and TDP - 43 in WT and FTLD - TDP Tg mice . Lower histogram : Quantification of immunoprecipitation results of HDAC1 and TDP - 43 in WT and Tg mice . N = 5 mice per group , data are presented as mean ± SEM ( % ) , * p = 0 . 0149 , * * * p = 0 . 0003 by t - test .

Upper panel: Immunoprecipitation of cytosolic HDAC1 and immunoblotting of HDAC1 and TDP-43 in WT and FTLD-TDP Tg mice. Lower histogram: Quantification of immunoprecipitation results of HDAC1 and TDP-43 in WT and Tg mice. N = 5 mice per group, data are presented as mean ± SEM (%), * p =0.0149, *** p =0.0003 by t-test.


Western - blot of HDAC1 and TDP - 43 in urea - soluble fractions . N = 5 mice per group .

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

Western-blot of HDAC1 and TDP-43 in urea-soluble fractions. N = 5 mice per group.


Escape latency of mice in Morris water maze task . * WT + Vehicle mice vs . Tg + Vehicle mice , # Tg + 5104434 mice vs . Tg + Vehicle mice . Statistical analysis by two - way ANOVA with Bonferroni ' s multiple comparisons . # P < 0 . 05 , * * * P ≤ 0 . 001 and * * * * / # # # # P ≤ 0 . 0001 Data information : N = 12 mice per group All data are presented as mean ± SEM , * * * * p < 0 . 0001 by multiple comparison .

O 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

Escape latency of mice in Morris water maze task. *WT+Vehicle mice vs. Tg+Vehicle mice, #Tg+5104434 mice vs. Tg+Vehicle mice. Statistical analysis by two-way ANOVA with Bonferroni's multiple comparisons. #P < 0.05, ***P ≤ 0.001 and ****/####P ≤ 0.0001 Data information: N = 12 mice per group All data are presented as mean ± SEM, **** p < 0.0001 by multiple comparison.


The representative searching path of mice in the probe test .

The representative searching path of mice in the probe test.


Scores of mice with respect to the number of times they crossed the hidden platform , time spent searching in the target quadrant , and the velocity of swimming in the probe test at 24 h after escape training . * * p = 0 . 009 , * * * p = 0 . 0002 by multiple comparison . Data information : N = 12 mice per group All data are presented as mean ± SEM , * * * * p < 0 . 0001 by multiple comparison .

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

Scores of mice with respect to the number of times they crossed the hidden platform, time spent searching in the target quadrant, and the velocity of swimming in the probe test at 24 h after escape training. ** p = 0.009, *** p = 0.0002 by multiple comparison. Data information: N = 12 mice per group All data are presented as mean ± SEM, **** p < 0.0001 by multiple comparison.


Scores of the discrimination index in the novel object recognition test . Scores of mice in the rotarod test . Data information : N = 12 mice per group All data are presented as mean ± SEM , * * * * p < 0 . 0001 by multiple comparison .

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

Scores of the discrimination index in the novel object recognition test.   Scores of mice in the rotarod test.   Data information: N = 12 mice per group All data are presented as mean ± SEM, **** p < 0.0001 by multiple comparison.