( c ) Automated microscopy . rodent cortical neurons were transfected with mApple TDP43 - EGFP , survival was determined by repeated imaging at regular intervals . last time at which was noted to be alive ( red arrows ) was used as time of . that survive entire of experiment ( cyan arrow ) were censored . Scale bar , 25 μm .

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(c) Automated fluorescence microscopy. Primary rodent cortical neurons were transfected with mApple and TDP43-EGFP, and survival was determined by repeated imaging at regular intervals. The last time at which the cell was noted to be alive (red arrows) was used as the time of death. Cells that survive the entire length of the experiment (cyan arrow) were censored. Scale bar, 25 μm.


( d , e ) Cumulative risk of over time for neurons transfected with EGFP , TDP43 ( WT ) - EGFP ( d ) or TDP43A315T - neurons ( e ) . Supplementary Table 1 lists number of neurons , hazard ratios , 95 % CI P values for each condition , as determined by Cox proportional hazards . Results were pooled from 12 wells per condition , with experiments performed in duplicate .

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(d,e) Cumulative risk of death over time for neurons transfected with EGFP, TDP43(WT)-EGFP (d) or TDP43A315T-neurons (e). Supplementary Table 1 lists the number of neurons, hazard ratios, 95% CI and P values for each condition, as determined by Cox proportional hazards analysis. Results were pooled from 12 wells per condition, with experiments performed in duplicate.


f , g ) Linear regression of calculated hazard ( ± s . e . m . ) as a function of total TDP43 ( WT ) - EGFP ( R2 = 0 . 9353 ) ( f ) or TDP43A315T - EGFP ( R2 = 0 . 9574 ) ( g ) level . total TDP43 level was determined by quantitative immunocytochemistry using TDP43 - specific antibodies ( Supplementary Fig . 3 ) normalized to amount of TDP43 in nontransfected neurons . Dashed lines , reference ( HR = 1 ) .

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f,g) Linear regression of calculated hazard ratio (± s.e.m.) as a function of total TDP43(WT)-EGFP (R2 = 0.9353) (f) or TDP43A315T-EGFP (R2 = 0.9574) (g) level. The total TDP43 level was determined by quantitative immunocytochemistry using TDP43-specific antibodies (Supplementary Fig. 3) and normalized to the amount of TDP43 in nontransfected neurons. Dashed lines, reference (HR = 1).


( a ) neurons were labeled with 35S - labeled methionine , TDP43 was immunoprecipitated using anti - TDP43 antibodies ( IP - TDP43 ) or nonspecific IgG antibodies ( IP - IgG ) . ( b ) Log - normal plot of normalized TDP43 . TDP43 half - life was determined by fitting a first - order exponential curve to data ( R2 = 0 . 8984 ) . Values were pooled from five independent experiments .

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(a) Primary neurons were labeled with 35S-labeled methionine, and TDP43 was immunoprecipitated using anti-TDP43 antibodies (IP-TDP43) or nonspecific IgG antibodies (IP-IgG). (b) Log-normal plot of normalized TDP43 levels. TDP43 half-life was determined by fitting a first-order exponential curve to the data (R2 = 0.8984). Values were pooled from five independent experiments.


( c ) Potential confounders in half - life analyses . Top , total ideally decreases over time in a predictable manner ( top right panel ) . Middle , toxicity reduces amount of measured , potentially shortening calculated half - life ( middle right panel ) . Bottom , aggregation might also shorten half - life estimations if cannot be immunoprecipitated ( black arrows , bottom right panel ) or lengthen it if is more stable successfully immunoprecipitated ( gray arrows , bottom right panel ) .

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(c) Potential confounders in half-life analyses. Top, total protein ideally decreases over time in a predictable manner (top right panel). Middle, toxicity reduces the amount of measured protein, potentially shortening the calculated half-life (middle right panel). Bottom, protein aggregation might also shorten half-life estimations if the protein cannot be immunoprecipitated (black arrows, bottom right panel) or lengthen it if the protein is more stable and successfully immunoprecipitated (gray arrows, bottom right panel).


( d ) OPL . cortical neurons were transfected with EGFP TDP43 ( WT ) - Dendra2 , photoconverted then imaged by AFM . Dead ( arrows ) were excluded at time of . Pre , before photoconversion . ( e ) half - life of TDP43 ( WT ) - Dendra2 was determined by fitting a first - order exponential curve to data ( R2 = 0 . 9558 ) . ( f ) Including with prolonged TDP43 ( WT ) - Dendra2 half - life ( pink , R2 = 0 . 9003 ; * P 0 . 0001 , F 15 . 34 , extra sum - of - squares F - ) . Excluding that died over 36 - h experiment also prolonged half - life ( cyan , R2 = 0 . 7171 ; * * P 0 . 0001 , extra sum - of - squares F - ) . Error bars represent s . e . m . Values were pooled from eight wells per condition , with experiments performed in quadruplicate .

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(d) OPL. Primary cortical neurons were transfected with EGFP and TDP43(WT)-Dendra2, photoconverted then imaged by AFM. Dead cells (arrows) were excluded at the time of death. Pre, before photoconversion. (e) The half-life of TDP43(WT)-Dendra2 was determined by fitting a first-order exponential curve to the data (R2 = 0.9558). (f) Including cells with aggregates prolonged TDP43(WT)-Dendra2 half-life (pink line, R2 = 0.9003; *P 0.0001, F 15.34, extra sum-of-squares F-test). Excluding cells that died over the 36-h experiment also prolonged half-life (cyan line, R2 = 0.7171; **P 0.0001, extra sum-of-squares F-test). Error bars represent s.e.m. Values were pooled from eight wells per condition, with experiments performed in quadruplicate.


( a ) Scatter plot depicting half - life measurements for expressing TDP43 ( WT ) - Dendra2 ( cyan , n = 206 ) TDP43A315T - Dendra2 ( red , n = 251 ) . * P = 0 . 0029 , U = 11 , 797 , Mann - Whitney .

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(a) Scatter plot depicting half-life measurements for individual cells expressing TDP43(WT)-Dendra2 (cyan, n = 206) and TDP43A315T-Dendra2 (red, n = 251). *P = 0.0029, U = 11,797, Mann-Whitney test.


b ) Probability density plot for single - half - life measurements , demonstrating a reduced half - life of TDP43A315T - Dendra2 ( red , n = 251 ) , compared to TDP43 ( WT ) - Dendra2 ( cyan , n = 206 ; * * P = 0 . 0007 , two - sided Kolmogorov - Smirnov ) . Values were pooled from eight wells per condition , with experiments performed in quadruplicate .

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b) Probability density plot for single-cell half-life measurements, demonstrating a reduced half-life of TDP43A315T-Dendra2 (red, n = 251), compared to TDP43(WT)-Dendra2 (cyan, n = 206; **P = 0.0007, two-sided Kolmogorov-Smirnov test). Values were pooled from eight wells per condition, with experiments performed in quadruplicate.


( c , d ) Probability density plots of single - half - life measurements for TDP43 ( WT ) - Dendra2 ( c ) TDP43A315T - Dendra2 ( d ) . At 0 . 5 μM , three of compounds significantly reduced TDP43 ( WT ) - Dendra2 half - life compared to vehicle control ( DMSO , n = 110 ; FPZ , n = 96 , f * P = 1 × 10 − 4 ; MTM , n = 129 , m * P = 2 × 10 − 7 ; NCP , n = 111 , n * P = 2 × 10 − 5 , two - sided Kolmogorov - Smirnov ) . compounds also reduced TDP43A315T - Dendra2 half - life , in comparison to vehicle control ( DMSO , n = 120 ; FPZ , n = 155 , f * P = 2 × 10 − 9 ; MTM , n = 152 , m * P = 0 . 004 ; NCP , n = 111 , n * P = 0 . 005 , two - sided Kolmogorov - Smirnov ) . Colored hash marks represent values from neurons in b - d . Values were obtained from eight wells per condition , with experiments performed in triplicate .

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

(c,d) Probability density plots of single-cell half-life measurements for TDP43(WT)-Dendra2 (c) and TDP43A315T-Dendra2 (d). At 0.5 μM, all three of the compounds significantly reduced TDP43(WT)-Dendra2 half-life compared to vehicle control (DMSO, n = 110; FPZ, n = 96, f* P = 1 × 10−4; MTM, n = 129, m* P = 2 × 10−7; NCP, n = 111, n* P = 2 × 10−5, two-sided Kolmogorov-Smirnov test). The compounds also reduced TDP43A315T-Dendra2 half-life, in comparison to vehicle control (DMSO, n = 120; FPZ, n = 155, f* P = 2 × 10−9; MTM, n = 152, m* P = 0.004; NCP, n = 111, n* P = 0.005, two-sided Kolmogorov-Smirnov test). Colored hash marks represent values from individual neurons in b-d. Values were obtained from eight wells per condition, with experiments performed in triplicate.


( a ) Each of compounds induced LC3 - II in neurons , as determined by western blotting . At 5 μM but not 0 . 5 μM , compounds significantly increased LC3 - II / GAPDH ( b ) LC3 - II / LC3 - I ( c ) ratios ( nonsignificant ( NS ) , P > 0 . 05 ; 0 . 05 * P 0 . 01 , * * P 0 . 01 ; F = 13 . 74 ( in b ) 9 . 74 ( in c ) , one - way ANOVA with Dunnett ' s post - ) . Error bars represent s . e . m . Values were pooled from five independent experiments .

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(a) Each of the compounds induced LC3-II in primary neurons, as determined by western blotting. At 5 μM but not 0.5 μM, the compounds significantly increased LC3-II/GAPDH (b) and LC3-II/LC3-I (c) ratios (nonsignificant (NS), P > 0.05; 0.05 *P 0.01, **P 0.01; F = 13.74 (in b) and 9.74 (in c), one-way ANOVA with Dunnett's post-test). Error bars represent s.e.m. Values were pooled from five independent experiments.


( d ) Optical labeling of LC3 - Dendra2 in rodent cortical neurons . Scale bars , 25 μm .

(d) Optical pulse labeling of LC3-Dendra2 in rodent primary cortical neurons. Scale bars, 25 μm.


( e ) change in red over time was used to calculate LC3 - Dendra2 half - life , as in Figure 2 ( DMSO , R2 = 0 . 7333 ; Beclin , R2 = 0 . 6968 , FPZ , R2 = 0 . 8752 ; MTM , R2 = 0 . 8447 ; NCP , R2 = 0 . 8357 ; * P 0 . 0001 for global trend , F = 12 . 6 , extra sum - of - squares F - ) . Error bars represent s . e . m . (

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(e) The change in red fluorescence intensity over time was used to calculate LC3-Dendra2 half-life, as in Figure 2 (DMSO, R2 = 0.7333; Beclin, R2 = 0.6968, FPZ, R2 = 0.8752; MTM, R2 = 0.8447; NCP, R2 = 0.8357; *P 0.0001 for global trend, F = 12.6, extra sum-of-squares F-test). Error bars represent s.e.m. (


. ( f ) median single - half - life of LC3 - Dendra2 was significantly reduced by beclin expression or treatment with 0 . 5 μM NCP , MTM or FPZ ( * P 0 . 01 , * * P 0 . 001 , Kruskal Wallis statistic = 52 . 56 , Kruskal - Wallis with Dunn ' s ) .

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. (f) The median single-cell half-life of LC3-Dendra2 was significantly reduced by beclin expression or treatment with 0.5 μM NCP, MTM or FPZ (*P 0.01, **P 0.001, Kruskal Wallis statistic = 52.56, Kruskal-Wallis with Dunn's test).


( g ) Probability density plot of LC3 - Dendra2 half - life measurements from neurons treated with vehicle control ( DMSO , n = 140 ) , NCP ( n = 128 ) , MTM ( n = 91 ) , FPZ ( n = 77 ) or those expressing beclin ( n = 101 ) . n * , P = 6 × 10 − 4 ; m * , P = 1 × 10 − 10 ; f * , P = 5 × 10 − 9 ; b * , P = 2 × 10 − 3 ; two - sided Kolmogorov - Smirnov ) . Colored hash marks represent values from neurons . Values were pooled from eight wells per condition , with experiments performed in triplicate .

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(g) Probability density plot of LC3-Dendra2 half-life measurements from individual neurons treated with vehicle control (DMSO, n = 140), NCP (n = 128), MTM (n = 91), FPZ (n = 77) or those expressing beclin (n = 101). n*, P = 6 × 10−4; m*, P = 1 × 10−10; f*, P = 5 × 10−9; b*, P = 2 × 10−3; two-sided Kolmogorov-Smirnov test). Colored hash marks represent values from individual neurons. Values were pooled from eight wells per condition, with experiments performed in triplicate.


( a ) FPZ ( n = 490 ) MTM ( n = 580 ) but not NCP ( n = 523 ) lowered total TDP43 ( WT ) - EGFP in comparison to vehicle ( DMSO , n = 1 , 007 ) .

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(a) FPZ (n = 490) and MTM (n = 580) but not NCP (n = 523) lowered total TDP43(WT)-EGFP levels in comparison to vehicle (DMSO, n = 1,007).


( b ) Histogram of normalized , log - transformed TDP43 ( WT ) - EGFP .

(b) Histogram of normalized, log-transformed TDP43(WT)-EGFP levels.


( c ) Each compound also reduced TDP43A315T - EGFP ( DMSO , n = 983 ; FPZ , n = 550 ; MTM , n = 542 ; NCP , n = 493 ) .

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(c) Each compound also reduced TDP43A315T-EGFP levels (DMSO, n = 983; FPZ, n = 550; MTM, n = 542; NCP, n = 493).


( d ) Histogram of single - TDP43 ( A315T ) - EGFP . Asterisks in b d denote median values for exposed to vehicle ( gray ) , FPZ ( green ) , MTM ( blue ) or NCP ( purple ) . For FPZ , MTM NCP versus DMSO in a d , P 1 × 10 − 3 by two - sided Kolmogorov - Smirnov .

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(d) Histogram of single-cell TDP43(A315T)-EGFP levels. Asterisks in b and d denote median values for cells exposed to vehicle (gray), FPZ (green), MTM (blue) or NCP (purple). For FPZ, MTM and NCP versus DMSO in a and d, P 1 × 10−3 by two-sided Kolmogorov-Smirnov test.


e ) No compound reduced TDP43 ( WT ) - EGFP inclusions 48 h after transfection ( DMSO , n = 360 ; FPZ , n = 194 ; MTM , n = 234 ; NCP , n = 203 ) .

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e) No compound reduced TDP43(WT)-EGFP inclusions 48 h after transfection (DMSO, n = 360; FPZ, n = 194; MTM, n = 234; NCP, n = 203).


( f ) FPZ MTM but not NCP reduced TDP43A315T - EGFP inclusions at 48 h ( DMSO , n = 327 ; FPZ , n = 226 ; MTM , n = 262 ; NCP , n = 253 ) . ( g ) No compound affected TDP43 ( WT ) - EGFP localization 24 h after transfection ( DMSO , n = 256 ; FPZ , n = 212 ; MTM , n = 250 ; NCP , n = 241 ) . ( h , i ) Each compound ( h ) prevented TDP43A315T - EGFP mislocalization ( DMSO , n = 233 ; FPZ , n = 268 ; MTM , n = 263 ; NCP , n = 270 ) reduced TDP43 ( A315T ) - EGFP ( DMSO , n = 125 ; FPZ , n = 102 ; MTM , n = 121 ; NCP , n = 144 . * P 0 . 05 ; NS , P > 0 . 05 ) ( i ) . For a , c e - i , * P 0 . 05 ; nonsignificant ( NS ) , P > 0 . 05 , one - way ANOVA with Dunnett ' s . Error bars represent s . e . m . Values in a - d were pooled from 8 - 12 wells per condition from six experiments , values in e - h were pooled from eight wells per condition from three experiments , values in i were pooled from five wells per condition from two experiments .

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(f) FPZ and MTM but not NCP reduced TDP43A315T-EGFP inclusions at 48 h (DMSO, n = 327; FPZ, n = 226; MTM, n = 262; NCP, n = 253). (g) No compound affected TDP43(WT)-EGFP localization 24 h after transfection (DMSO, n = 256; FPZ, n = 212; MTM, n = 250; NCP, n = 241). (h,i) Each compound (h) prevented TDP43A315T-EGFP cytoplasmic mislocalization (DMSO, n = 233; FPZ, n = 268; MTM, n = 263; NCP, n = 270) and reduced cytoplasmic TDP43(A315T)-EGFP levels (DMSO, n = 125; FPZ, n = 102; MTM, n = 121; NCP, n = 144. *P 0.05; NS, P > 0.05) (i). For a, c and e-i, *P 0.05; nonsignificant (NS), P > 0.05, one-way ANOVA with Dunnett's test. Error bars represent s.e.m. Values in a-d were pooled from 8-12 wells per condition from six experiments, values in e-h were pooled from eight wells per condition from three experiments, and values in i were pooled from five wells per condition from two experiments.


( a , b ) neurons transfected with TDP43 ( WT ) - EGFP ( a ) or TDP43A315T - EGFP ( b ) were treated with autophagy inducers survival determined by AFM . Data were obtained from eight wells per condition , with experiments performed in triplicate . Supplementary Table 2 lists number of neurons per condition , hazard ratios , 95 % CI P values , as determined by Cox hazards . ( c ) Autophagic induction improved survival in TDP43M337V HB9 - positive human iPSC - derived MNs . ( d ) FPZ MTM also reduced risk of in MAP2 - positive human iPSC - derived MNs Values in c d were pooled from 18 wells per condition , with experiments performed in triplicate . ( e ) Astrocytes differentiated from human iPSCs exhibit mutant TDP43 - related toxicity that is mitigated by FPZ MTM . Values were pooled from 18 wells per condition , performed in triplicate . Supplementary Table 3 lists number of human iPSC - derived neurons astrocytes per condition , hazard ratios , 95 % CI P values , as determined by Cox hazards .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O 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 neurons transfected with TDP43(WT)-EGFP (a) or TDP43A315T-EGFP (b) were treated with autophagy inducers and survival determined by AFM. Data were obtained from eight wells per condition, with experiments performed in triplicate. Supplementary Table 2 lists the number of neurons per condition, hazard ratios, 95% CI and P values, as determined by Cox hazards analysis. (c) Autophagic induction improved survival in TDP43M337V HB9-positive human iPSC-derived MNs. (d) FPZ and MTM also reduced the risk of death in MAP2-positive human iPSC-derived MNs Values in c and d were pooled from 18 wells per condition, with experiments performed in triplicate. (e) Astrocytes differentiated from human iPSCs exhibit mutant TDP43-related toxicity that is mitigated by FPZ and MTM. Values were pooled from 18 wells per condition, performed in triplicate. Supplementary Table 3 lists the number of human iPSC-derived neurons and astrocytes per condition, hazard ratios, 95% CI and P values, as determined by Cox hazards analysis.


a ) EGF enhances colocalization of phosphorylated ( P ) - bRAF , P - MEK P - ERK with LC3 . Immunofluorescence ( IF ) showing colocalization of P - bRAF ( green ) , P - MEK ( green ) P - ERK ( green ) with LC3 ( red ) in NIH / 3T3 in presence or absence of EGF ( 10 min as shown in scheme ) . bars represent mean ± s . e . m . * * P0 . 01 , * * * P0 . 001 ; Student ' s t - , 60 analysed from two experiments . Scale bars , 10 μm .

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a) EGF enhances colocalization of phosphorylated (P)-bRAF, P-MEK and P-ERK with LC3. Immunofluorescence (IF) showing colocalization of P-bRAF (green), P-MEK (green) and P-ERK (green) with LC3 (red) in NIH/3T3 cells in presence or absence of EGF (10 min as shown in scheme). The bars represent mean±s.e.m. **P0.01, ***P0.001; Student's t-test, 60 cells analysed from two experiments. Scale bars, 10 μm.


( b ) P - ERK colocalizes with pre - autophagosomal autophagosomal structures . IF depicting colocalization of P - ERK ( green or red ) with LC3 ( red ) , ATG5 - ATG12 ( red ) , ATG16 ( green ) , vps34 ( red ) , WIPI1 ( red ) , WIPI2 ( red ) P - ULK1 ( red ) in EGF - treated NIH / 3T3 . Scale bars , 10 μm . bars represent mean ± s . e . m . 50 analysed from two experiments . ( c ) Growth factors in serum maintain P - ERK / LC3 colocalization . IF depicting colocalization of P - ERK ( green ) with LC3 ( red ) in 2 h serum - fed NIH / 3T3 .

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(b) P-ERK colocalizes with pre-autophagosomal and autophagosomal structures. IF depicting colocalization of P-ERK (green or red) with LC3 (red), ATG5-ATG12 (red), ATG16 (green), vps34 (red), WIPI1 (red), WIPI2 (red) and P-ULK1 (red) in EGF-treated NIH/3T3 cells. Scale bars, 10 μm. The bars represent mean±s.e.m. 50 cells analysed from two experiments. (c) Growth factors in serum maintain P-ERK/LC3 colocalization. IF depicting colocalization of P-ERK (green) with LC3 (red) in 2 h serum-fed NIH/3T3 cells.


( a - c ) Native merged or with colocalization highlighted in pixels are shown . Nuclei are blue ( DAPI ) . Scale bar , 10 μm .

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(a-c) Native merged images or images with colocalization highlighted in white pixels are shown. Nuclei are blue (DAPI). Scale bar, 10 μm.


( a ) P - total MEK ERK are enriched in APh in vivo . Immunoblots for indicated in homogenate ( Hom ) , APh , APL Lys fractions from livers of fed mice , n = 4 .

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(a) P- and total MEK and ERK are enriched in APh in vivo. Immunoblots for indicated proteins in homogenate (Hom), APh, APL and Lys fractions from livers of fed mice, n=4.


( b ) EGF enhances enrichment of P - MEK ERK in APh fractions in vitro . Immunoblots for indicated in Hom , APh APL fractions from NIH / 3T3 in presence or absence of EGF ( 10 min ) . bars represent mean ± s . e . m . * P0 . 05 , * * P0 . 01 ; Student ' s t - , n = 3 .

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(b) EGF enhances enrichment of P-MEK and ERK in APh fractions in vitro. Immunoblots for indicated proteins in Hom, APh and APL fractions from NIH/3T3 cells in presence or absence of EGF (10 min). The bars represent mean±s.e.m. *P0.05, **P0.01; Student's t-test, n=3.


( c ) P - total MEK ERK localize on to / extra - luminal face of APh in vivo . Left ; model depicting localization of MEK ERK on extra - luminal face of autophagic vesicles ability of trypsin to degrade extra - luminal MEK ERK , right ; immunoblots for indicated in APh fractions from mice livers untreated ( − ) or treated with increasing amounts of trypsin for 15 min . bars represent mean ± s . e . m . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 compared with corresponding trypsin - untreated value ; ANOVA - Bonferroni post hoc , n = 3 . p44 , p42 forms of ERK , LC3 - I - associated LC3 - II , 37 kDa 25 kDa forms of cathepsin ( Cath ) B are indicated .

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

(c) P- and total MEK and ERK localize on to the cytoplasmic/extra-luminal face of APh in vivo. Left; model depicting localization of MEK and ERK on the extra-luminal face of autophagic vesicles and the ability of trypsin to degrade extra-luminal MEK and ERK, and right; immunoblots for indicated proteins in APh fractions from mice livers untreated (−) or treated with increasing amounts of trypsin for 15 min. The bars represent mean±s.e.m. *P0.05, **P0.01, ***P0.001 compared with corresponding trypsin-untreated value; ANOVA-Bonferroni post hoc test, n=3. The p44, p42 forms of ERK, LC3-I and membrane-associated LC3-II, and 37 kDa and 25 kDa forms of cathepsin (Cath) B are indicated.


( a ) EGF enhances LC3 - II content in hepatocytes . Immunofluorescence ( IF ) depicting LC3 - II in RALA hepatocytes in presence or absence of EGF ( 10 min ) . Native / inverted are shown . Scale bar , 5 μm . Bars represent mean ± s . e . m . * * * P0 . 001 compared with control ( Con ) ; Student ' s t - , 50 from two experiments .

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(a) EGF enhances nuclear LC3-II content in hepatocytes. Immunofluorescence (IF) depicting nuclear LC3-II in RALA hepatocytes in presence or absence of EGF (10 min). Native/inverted images are shown. Scale bar, 5 μm. Bars represent mean±s.e.m. ***P0.001 compared with control (Con); Student's t-test, 50 cells from two experiments.


( b ) EGF enhances P - ERK / LC3 - II colocalization . IF depicting P - ERK ( green ) / LC3 - II ( red ) colocalization in untreated ( Con ) or EGF - treated NIH / 3T3 . Native ( top ) / with colocalization in pixels ( bottom ) are shown . Scale bar , 5 μm . Bars represent mean ± s . e . m . * * * P0 . 001 compared with Con ; Student ' s t - , 50 from two experiments .

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(b) EGF enhances nuclear P-ERK/LC3-II colocalization. IF depicting P-ERK (green)/LC3-II (red) colocalization in untreated (Con) or EGF-treated NIH/3T3 cells. Native images (top)/images with colocalization in white pixels (bottom) are shown. Scale bar, 5 μm. Bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test, 50 cells from two experiments.


( c ) Adipogenic differentiation increases LC3 - II . depict LC3 - II in 3T3 - L1 preadipocytes in maintenance or differentiation medium ( 2 h ) . Scale bar , 5 μm . Bars represent mean ± s . e . m . * * P0 . 01 compared with Con ; Student ' s t - , 50 from two experiments .

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(c) Adipogenic differentiation increases nuclear LC3-II. Images depict nuclear LC3-II in 3T3-L1 preadipocytes in maintenance or differentiation medium (2 h). Scale bar, 5 μm. Bars represent mean±s.e.m. **P0.01 compared with Con; Student's t-test, 50 cells from two experiments.


( d ) P - ERK / LC3 - II colocalization in serum - fed . IF showing P - ERK ( green ) / LC3 ( red ) colocalization in 2 h serum - fed NIH / 3T3 . Native ( top ) / with colocalization in pixels ( bottom ) are shown . Scale bar , 5 μm .

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(d) Nuclear P-ERK/LC3-II colocalization in serum-fed cells. IF showing P-ERK (green)/LC3 (red) colocalization in 2 h serum-fed NIH/3T3 cells. Native images (top)/images with colocalization in white pixels (bottom) are shown. Scale bar, 5 μm.


( e ) LC3 interacts with ERK in vivo . Immunoblots showing co - immunoprecipitation of LC3 with ERK , MEK bRAF in homogenate ( Hom ) ( e , top ) , of LC3 with P - total ERK in fractions from mouse livers ( e , bottom ) .

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(e) LC3 interacts with ERK in vivo. Immunoblots showing co-immunoprecipitation of LC3 with ERK, MEK and bRAF in homogenate (Hom) (e, top), and of LC3 with P- and total ERK in nuclear fractions from mouse livers (e, bottom).


( f ) Blocking transport decreases EGF - induced increase in LC3 - II . LC3 IF ( red ) in EGF - treated NIH / 3T3 pre - exposed ( 30 min ) or not to WGA . Bars represent mean ± s . e . m . * * * P0 . 001 compared to with ; Student ' s t - , 60 from n = 3 . Scale bar , 10 μm .

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(f) Blocking nuclear transport decreases EGF-induced increase in nuclear LC3-II. LC3 IF (red) in EGF-treated NIH/3T3 cells pre-exposed (30 min) or not to WGA. Bars represent mean±s.e.m. ***P0.001 compared to with; Student's t-test, 60 cells from n=3. Scale bar, 10 μm.


( g ) Blocking transport decreases ERK content . ERK IF ( green ) in EGF - treated NIH / 3T3 pre - exposed ( 30 min ) or not to WGA . Bars represent mean ± s . e . m . * * * P0 . 001 compared with Con ; Student ' s t - .

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(g) Blocking nuclear transport decreases nuclear ERK content. ERK IF (green) in EGF-treated NIH/3T3 cells pre-exposed (30 min) or not to WGA. Bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test.


( h ) Blocking transport does not modify P - ERK / LC3 - II colocalization . IF depicting P - ERK ( green ) / LC3 ( red ) colocalization in EGF - treated NIH / 3T3 pre - exposed or not to WGA . For ( g ) ( h ) : Scale bar , 10 μm , bars are mean ± s . e . m . 50 from n = 2 . Nuclei are blue ( DAPI ) . Arrows indicate LC3 , ERK or colocalization .

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(h) Blocking nuclear transport does not modify P-ERK/LC3-II colocalization. IF depicting nuclear P-ERK (green)/LC3 (red) colocalization in EGF-treated NIH/3T3 cells pre-exposed or not to WGA. For (g) and (h): Scale bar, 10 μm, bars are mean±s.e.m. 50 cells from n=2. Nuclei are blue (DAPI). Arrows indicate LC3 puncta, ERK or colocalization.


( a ) Atg7 − / − livers display decreased ERK phosphorylation . Immunoblots for indicated in liver homogenate ( Hom ) ( Cyt ) fractions of control ( Con ) liver - specific Atg7 − / − mice are shown . bars represent mean ± s . e . m . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 compared with Con ; Student ' s t - , n = 4 .

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(a) Atg7−/− livers display decreased ERK phosphorylation. Immunoblots for the indicated proteins in liver homogenate (Hom) and cytosolic (Cyt) fractions of control (Con) and liver-specific Atg7−/− mice are shown. The bars represent mean±s.e.m. *P0.05, **P0.01, ***P0.001 compared with Con; Student's t-test, n=4.


( b ) Atg7 − / − livers display decreased ERK2 dimers . Immunoblots show indicated in Hom from Con Atg7 − / − livers . bars represent mean ± s . e . m . * P0 . 05 , * * * P0 . 001 compared to Con ; Student ' s t - , n = 4 - 5 . Arrows indicate p42 monomers dimers .

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(b) Atg7−/− livers display decreased ERK2 dimers. Immunoblots show the indicated proteins in Hom from Con and Atg7−/− livers. The bars represent mean±s.e.m. *P0.05, ***P0.001 compared to Con; Student's t-test, n=4-5. Arrows indicate p42 monomers and dimers.


( c ) Atg7 − / − brown adipose tissues ( BAT ) display decreased ERK phosphorylation . Immunoblots for indicated in Hom from Con Atg7 − / − BAT . bars represent mean ± s . e . m . * * P0 . 01 compared with Con ; Student ' s t - , n = 4 - 6 .

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(c) Atg7−/− brown adipose tissues (BAT) display decreased ERK phosphorylation. Immunoblots for indicated proteins in Hom from Con and Atg7−/−BAT. The bars represent mean±s.e.m. **P0.01 compared with Con; Student's t-test, n=4-6.


( d ) Atg7 − / − livers exhibit decreased ERK phosphorylation . Immunoblots show indicated in homogenate ( Hom , lane 1 ) fractions ( lanes 2 - 5 ) from Con Atg7 − / − livers . bars represent mean ± s . e . m . * * * P0 . 001 compared with Con ; Student ' s t - , n = 4 - 5 .

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(d) Atg7−/− livers exhibit decreased nuclear ERK phosphorylation. Immunoblots show the indicated proteins in homogenate (Hom, lane 1) and nuclear fractions (lanes 2-5) from Con and Atg7−/− livers. The bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test, n=4-5.


( e ) LC3 C terminus glycine - deleted ( ΔG ) mutants exhibit decreased ERK phosphorylation . Immunoblots for indicated in total lysates from CFP - LC3 - CFP - LC3ΔG - transfected NIH / 3T3 exposed or not to EGF ( 10 min ) . bars represent mean ± s . e . m . * * * P0 . 001 compared with Con ; Student ' s t - , n = 8 .

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(e) LC3 C terminus glycine-deleted (ΔG) mutants exhibit decreased ERK phosphorylation. Immunoblots for indicated proteins in total lysates from CFP-LC3- and CFP-LC3ΔG-transfected NIH/3T3 cells exposed or not to EGF (10 min). The bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test, n=8.


( a ) Atg5 − / − MEFs display reduced ERK phosphorylation during nutrient deprivation . of P - ERK normalized to total ERK ( detected by immunoblotting ) in WT Atg5 − / − MEFs cultured in absence of serum for indicated times . bars represent mean ± s . e . m . * P0 . 05 compared with corresponding WT value ; * * * P0 . 001 , * * * * P0 . 0001 compared with 10 min serum - starved Atg5 − / − MEFs ; ANOVA - Bonferroni post hoc , n = 3 .

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(a) Atg5−/− MEFs display reduced ERK phosphorylation during nutrient deprivation. Quantification of P-ERK levels normalized to total ERK (detected by immunoblotting) in WT and Atg5−/− MEFs cultured in the absence of serum for indicated times. The bars represent mean±s.e.m. *P0.05 compared with corresponding WT value; ***P0.001, ****P0.0001 compared with 10 min serum-starved Atg5−/− MEFs; ANOVA-Bonferroni post hoc test, n=3.


( b ) Serum - deprived Atg5 − / − MEFs display decreased ERK p90RSK phosphorylation . Immunoblots for indicated in total lysates from 2 h serum - deprived WT Atg5 − / − MEFs . bars represent mean ± s . e . m . * P0 . 05 , * * * P0 . 001 compared with Con ; Student ' s t - , n = 3 .

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(b) Serum-deprived Atg5−/− MEFs display decreased ERK and p90RSK phosphorylation. Immunoblots for the indicated proteins in total lysates from 2 h serum-deprived WT and Atg5−/− MEFs. The bars represent mean±s.e.m. *P0.05, ***P0.001 compared with Con; Student's t-test, n=3.


( c ) Atg5 − / − MEFs display decreased ERK activity . Immunoblots for phosphorylated HA ( haemagglutinin ) - tagged Elk1 in total lysates from 2 h serum - deprived WT Atg5 − / − MEFs exposed or not to EGF ( 10 min ) .

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(c) Atg5−/− MEFs display decreased nuclear ERK activity. Immunoblots for phosphorylated HA (haemagglutinin)-tagged Elk1 in total lysates from 2 h serum-deprived WT and Atg5−/− MEFs cells exposed or not to EGF (10 min).


( d ) Atg5 − / − MEFs display decreased Elk1 - driven gene expression . Elk1 - driven ZFP36 mRNA from 2 hserum - deprived WT Atg5 − / − MEFs exposed or not to EGF ( 10 min ) . bars represent mean ± s . e . m . * * * P0 . 001 compared with Con ; Student ' s t - , n = 3 . (

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(d) Atg5−/− MEFs display decreased Elk1-driven gene expression. Elk1-driven ZFP36 mRNA levels from 2 hserum-deprived WT and Atg5−/− MEFs cells exposed or not to EGF (10 min). The bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test, n=3. (


( e ) Decreased ERK phosphorylation in Atg5 − / − MEFs occurs independently of changes in ERK phosphatases . Immunoblots for indicated in 2 h serum - deprived WT MEFs transfected with scrambled siRNA ( scr ) , Atg5 − / − MEFs transfected with scr or siRNAs against MKP3 or PP2A in presence or absence of EGF . bars represent mean ± s . e . m . , n = 3 .

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(e) Decreased ERK phosphorylation in Atg5−/− MEFs occurs independently of changes in ERK phosphatases. Immunoblots for the indicated proteins in 2 h serum-deprived WT MEFs transfected with scrambled siRNA (scr), and Atg5−/− MEFs transfected with scr or siRNAs against MKP3 or PP2A in the presence or absence of EGF. The bars represent mean±s.e.m., n=3.


( a ) Mutations in FRS on ERK2 decrease colocalization of ERK2 with ATG5 - ATG12 , LC3 WIPI1 . Immunofluorescence ( IF ) showing colocalization ( depicted as pixels by ' colocalization finder application ' ) of WT - ERK2 - HA , FRS ERK2 mutants ( L198A - , L232A - , L198A / L232A - , Y261A - ERK2 - HA ) or common docking ( CD ) mutant ( D319N - ERK2 - HA ) with ATG5 - ATG12 ( panels 1 - 6 ) , LC3 ( panels 7 - 12 ) or WIPI1 ( panels 13 - 18 ) in EGF - treated NIH / 3T3 . ERK2 is stained in red , autophagy are stained in green . Scale bar , 10 μm . bars represent mean ± s . e . m . * * P0 . 01 , * * * * P0 . 0001 compared with WT - ERK2 - transfected ; Student ' s t - , 50 analysed from n = 2 . (

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(a) Mutations in FRS on ERK2 decrease colocalization of ERK2 with ATG5-ATG12, LC3 and WIPI1. Immunofluorescence (IF) showing colocalization (depicted as white pixels by 'colocalization finder application') of WT-ERK2-HA, FRS ERK2 mutants (L198A-, L232A-, L198A/L232A-, Y261A-ERK2-HA) or common docking (CD) mutant (D319N-ERK2-HA) with ATG5-ATG12 (panels 1-6), LC3 (panels 7-12) or WIPI1 (panels 13-18) in EGF-treated NIH/3T3 cells. ERK2 is stained in red, and autophagy proteins are stained in green. Scale bar, 10 μm. The bars represent mean±s.e.m. **P0.01, ****P0.0001 compared with WT-ERK2-transfected cells; Student's t-test, 50 cells analysed from n=2. (


( b ) Mutations in FRS on ERK2 decrease colocalization of ERK2 with nuclearATG5 - ATG12 or LC3 . IF showing colocalization ( pixels ) of WT - ERK2 - HA ( red ) , FRS ERK2 mutants ( L198A - , L232A - , L198A / L232A - , Y261A - ERK2 - HA ) or common docking ( CD ) mutant ( D319N - ERK2 - HA ) with ATG5 - ATG12 ( panels 1 - 6 ) or LC3 - II ( panels 7 - 12 ) in EGF - treated NIH / 3T3 . Scale bar , 5 μm . bars represent mean ± s . e . m . * * P0 . 01 , * * * P0 . 001 , * * * * P0 . 0001 compared with WT - ERK2 - transfected ; Student ' s t - , 50 analysed from n = 2 . Arrows depict colocalization .

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(b) Mutations in FRS on ERK2 decrease colocalization of ERK2 with nuclearATG5-ATG12 or LC3. IF showing colocalization (white pixels) of WT-ERK2-HA (red), FRS ERK2 mutants (L198A-, L232A-, L198A/L232A-, Y261A-ERK2-HA) or common docking (CD) mutant (D319N-ERK2-HA) with ATG5-ATG12 (panels 1-6) or LC3-II (panels 7-12) in EGF-treated NIH/3T3 cells. Scale bar, 5 μm. The bars represent mean±s.e.m. **P0.01, ***P0.001, ****P0.0001 compared with WT-ERK2-transfected cells; Student's t-test, 50 cells analysed from n=2. Arrows depict colocalization.


( c ) ATG5 - ATG12 conjugation is required for ERK phosphorylation . Immunoblots for P - ERK , total ERK β - actin in NIH / 3T3 transfected with WT ATG5 or conjugation - defective ATG5 K130R mutant treated with EGF ( 10 min ) . bars represent mean ± s . e . m . * P0 . 05 compared with WT ATG5 - transfected ; Student ' s t - , n = 3 .

(c) ATG5-ATG12 conjugation is required for ERK phosphorylation. Immunoblots for P-ERK, total ERK and β-actin in NIH/3T3 cells transfected with WT ATG5 or the conjugation-defective ATG5 K130R mutant and treated with EGF (10 min). The bars represent mean±s.e.m. *P0.05 compared with WT ATG5-transfected cells; Student's t-test, n=3.


a ) ATG4B depletion increases steady - state LC3 - II . Immunoblots for indicated in NIH / 3T3 transfected with scrambled siRNAs ( scr ) or siRNAs against ATG4B ( siATG4B ) in presence / absence of EGF . Bars represent mean ± s . e . m . * P0 . 05 , * * * P0 . 001 compared with scr ; Student ' s t - , n = 4 .

a) ATG4B depletion increases steady-state LC3-II levels. Immunoblots for indicated proteins in NIH/3T3 cells transfected with scrambled siRNAs (scr) or siRNAs against ATG4B (siATG4B) in presence/absence of EGF. Bars represent mean±s.e.m. *P0.05, ***P0.001 compared with scr; Student's t-test, n=4.


( b ) siATG4B exhibit increased autophagic vesicles . Representative electron depicting autophagic vesicles ( indicated by black arrows ) in scr siATG4B NIH / 3T3 .

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(b) siATG4B cells exhibit increased autophagic vesicles. Representative electron micrographs depicting autophagic vesicles (indicated by black arrows) in scr and siATG4B NIH/3T3 cells.


( c ) ATG4B depletion does not increase autophagic flux in serum - fed . Immunoblots for indicated in lysates from scr or siATG4B NIH / 3T3 in presence / absence of lysosomal inhibitors , ammonium chloride leupeptin ( Inh ) for 2 h . Scale bars , 1 μm . Bars represent mean ± s . e . m . , n = 3 .

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(c) ATG4B depletion does not increase autophagic flux in serum-fed cells. Immunoblots for indicated proteins in lysates from scr or siATG4B NIH/3T3 cells in presence/absence of lysosomal inhibitors, ammonium chloride and leupeptin (Inh) for 2 h. Scale bars, 1 μm. Bars represent mean±s.e.m., n=3.


( d ) siATG4B display increased LC3 - II . for LC3 - II ( red ) in scr or siATG4B NIH / 3T3 in presence / absence of EGF ( 10 min ) . Scale bar , 5 μm . Bars represent mean ± s . e . m . * * * P0 . 001 compared with scr ; Student ' s t - , 60 from n = 2 .

(d) siATG4B cells display increased nuclear LC3-II. Images for LC3-II (red) in scr or siATG4B NIH/3T3 cells in presence/absence of EGF (10 min). Scale bar, 5 μm. Bars represent mean±s.e.m. ***P0.001 compared with scr; Student's t-test, 60 cells from n=2.


( e ) ATG4B deficiency increases P - ERK / LC3 - II colocalization . Immunofluorescence ( IF ) for P - ERK ( green ) / LC3 ( red ) colocalization in scr or siATG4B NIH / 3T3 in presence / absence of EGF ( 10 min ) . Scale bars , 10 μm . Bars represent mean ± s . e . m . * * P0 . 01 , * * * P0 . 001 compared with scr ; Student ' s t - , 60 from n = 2 .

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(e) ATG4B deficiency increases P-ERK/LC3-II colocalization. Immunofluorescence (IF) for P-ERK (green)/LC3 (red) colocalization in scr or siATG4B NIH/3T3 cells in presence/absence of EGF (10 min). Scale bars, 10 μm. Bars represent mean±s.e.m. **P0.01, ***P0.001 compared with scr; Student's t-test, 60 cells from n=2.


( f ) ATG4B - deficient display increased P - ERK / LC3 colocalization . IF depicting P - ERK ( green ) / LC3 ( red ) colocalization in nuclei of scr or siATG4B NIH / 3T3 in presence / absence of EGF . Scale bars , 5 μm . Bars represent mean ± s . e . m . * * P0 . 01 , * * * P0 . 001 compared with scr ; Student ' s t - , 60 from n = 2 .

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(f) ATG4B-deficient cells display increased nuclear P-ERK/LC3 colocalization. IF depicting P-ERK (green)/LC3 (red) colocalization in nuclei of scr or siATG4B NIH/3T3 cells in presence/absence of EGF. Scale bars, 5 μm. Bars represent mean±s.e.m. **P0.01, ***P0.001 compared with scr; Student's t-test, 60 cells from n=2.


( g ) ATG4B deficiency augments EGF - induced MEK ERK phosphorylation . Immunoblots for P - total MEK ERK , LC3 GAPDH in total lysates from scr or siATG4B NIH / 3T3 in presence / absence of EGF . Bars represent mean ± s . e . m . , n = 3 .

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(g) ATG4B deficiency augments EGF-induced MEK and ERK phosphorylation. Immunoblots for P- and total MEK and ERK, LC3 and GAPDH in total lysates from scr or siATG4B NIH/3T3 cells in presence/absence of EGF. Bars represent mean±s.e.m., n=3.


h ) ATG4B deficiency increases P - ERK . Native ( top ) / inverted ( bottom ) showing P - ERK content . P - ERK in untreated ( Con ) / EGF - treated scr or siATG4B NIH / 3T3 is shown . Scale bars , 5 μm . Bars represent mean ± s . e . m . * * P0 . 01 , * * * P0 . 001 compared with scr ; Student ' s t - , 60 from n = 2 . Nuclei are blue ( DAPI ) .

h) ATG4B deficiency increases nuclear P-ERK levels. Native (top)/inverted images (bottom) showing nuclear P-ERK content. P-ERK fluorescence in untreated (Con)/EGF-treated scr or siATG4B NIH/3T3 cells is shown. Scale bars, 5 μm. Bars represent mean±s.e.m. **P0.01, ***P0.001 compared with scr; Student's t-test, 60 cells from n=2. Nuclei are blue (DAPI).


Fate of Purkinje in mice with Purkinje - - specific knockout of Ccp1 . Ccp1flox / flox L7 - cre mice show a massive but incomplete degeneration of Purkinje ( calbindin - stained ; red ) at 4 months , while Purkinje are degenerated at 18 months . Scale bar : 500 µm .

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Fate of Purkinje cells in mice with Purkinje-cell-specific knockout of Ccp1. Ccp1flox/flox L7-cre mice show a massive but incomplete degeneration of Purkinje cells (calbindin-stained; red) at 4 months, while all Purkinje cells are degenerated at 18 months. Scale bar: 500 µm.


Fate of Purkinje in mice with combinatorial knockout alleles . Knockout of Ccp1 ( pcd - mouse ; Ccp1 - / - ) results in a complete degeneration of Purkinje ( calbindin - stained ; red ) , while additional knockout of Ttll1 selectively in Purkinje ( L7 - cre ) protects entire Purkinje layer from degeneration up to 18 months . Scale bar : 500 µm .

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Fate of Purkinje cells in mice with combinatorial knockout alleles. Knockout of Ccp1 (pcd-mouse; Ccp1-/-) results in a complete degeneration of Purkinje cells (calbindin-stained; red), while the additional knockout of Ttll1 selectively in the Purkinje cells (L7-cre) protects the entire Purkinje cell layer from degeneration up to 18 months. Scale bar: 500 µm.


Nissl of Purkinje layer of brains from various knockout mice shown in ( C ) confirms absence of Purkinje in Ccp1 - / - , presence of a wild - type - like Purkinje - density in Ccp1 - / - Ttll1flox / flox L7 - cre mice . Scale bar : 20 µm . Purkinje layer ( PC ) granule layer ( GC ) are indicated .

Nissl staining of the Purkinje cell layer of the brains from various knockout mice shown in (C) confirms the absence of Purkinje cells in Ccp1-/-, and the presence of a wild-type-like Purkinje-cell density in Ccp1-/- Ttll1flox/flox L7-cre mice. Scale bar: 20 µm. Purkinje cell layer (PC) and granule cell layer (GC) are indicated.


Immunoblot of tissue extracts from cerebella of Ttll1 - / - wild type mice , probed with polyE , anti - ∆ 2 - tubulin 12G10 antibodies . Increasing amounts of extracts were loaded for detection of polyglutamylated with polyE .

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Immunoblot of tissue extracts from cerebella of Ttll1-/- and wild type mice, probed with the polyE, anti-∆2-tubulin and 12G10 antibodies. Increasing amounts of extracts were loaded for detection of polyglutamylated proteins with polyE.


Analyses of tubulin polyglutamylation ( antibody : polyE ) in extracts of different brain regions of wild type , Ccp1 - / - , Ccp6 - / - Ccp1 - / - Ccp6 - / - mice with immunoblot . In 3 - old mice , hyperglutamylation is observed in cerebral cortex , cerebellum hippocampus of Ccp1 - / - Ccp1 - / - Ccp6 - / - mice . In 2 - 5 - month - old mice , hyperglutamylation is restricted to cerebellum in Ccp1 - / - mice , however it persists in three brain regions of Ccp1 - / - Ccp6 - / - mice ( complete in Fig . EV1 ; α - β - tubulin bands indicated ) .

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Analyses of tubulin polyglutamylation (antibody: polyE) in extracts of different brain regions of wild type, Ccp1-/-, Ccp6-/- and Ccp1-/-Ccp6-/- mice with immunoblot. In 3-weeks old mice, hyperglutamylation is observed in cerebral cortex, cerebellum and hippocampus of Ccp1-/- and Ccp1-/-Ccp6-/- mice. In 2- and 5-month-old mice, hyperglutamylation is restricted to the cerebellum in Ccp1-/- mice, however it persists in all three brain regions of the Ccp1-/-Ccp6-/- mice (complete analysis in Fig. EV1; α- and β-tubulin bands indicated).


Nissl - stained frontal sections of wild type , Ccp1 - / - , Ccp6 - / - Ccp1 - / - Ccp6 - / - mice at 1 5 months , showing - dependent thinning ( arrow heads ) of cortex in Ccp1 - / - Ccp6 - / - mice ( complete in Appendix Fig . S2A ) .

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Nissl-stained frontal sections of wild type, Ccp1-/-, Ccp6-/- and Ccp1-/-Ccp6-/- mice at 1 and 5 months, showing age-dependent thinning (arrow heads) of the cortex in Ccp1-/-Ccp6-/- mice (complete analysis in Appendix Fig. S2A).


of thickness of motor somatosensory cortex of 1 - 5 - month - old mice shown in Appendix Fig . S2 . single values shown in Appendix Fig . S2B were averaged , values from each genotype - specific group were tested against average values of remaining three genotypes ( Appendix Fig . S2C ) . Only cortical thickness of 5 - month - old Ccp1 - / - Ccp6 - / - mice is significantly decreased ( mean ± ; Mann - Whitney t - ) .

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Quantification of the thickness of motor and somatosensory cortex of 1- and 5-month-old mice shown in Appendix Fig. S2. The single values shown in Appendix Fig. S2B were averaged, and values from each genotype-specific group were tested against the average values of the remaining three genotypes (Appendix Fig. S2C). Only the cortical thickness of 5-month-old Ccp1-/-Ccp6-/- mice is significantly decreased (mean ±SEM; Mann-Whitney t-test).


Immunohistochemistry of brain sections with anti - MAP2 antibody for visualization of neurons ( layer V ) apical dendritic ( layer IV ) with anti - GFAP for reactive astrocytes ( layer V ) . complete is shown in Fig . EV2A , EV3A . Scale bars : 50 µm . Quantifications of number of MAP2 - positive neurons in layer V , of number of apical dendrites in layer IV ( Fig . EV2B ) reveal reduced neuron number in layer V , reduced dendritic density in layer IV of 5 - month - old Ccp1 - / - Ccp6 - / - mice . of number of reactive astrocytes in layer V ( Fig . EV3B ) shows a significant increase over wild type in Ccp1 - / - in Ccp6 - / - mice , however a much stronger increase is seen in Ccp1 - / - Ccp6 - / - mice . Complete analyses are shown in Fig . EV2C , EV3C ( mean ± ; student ' s t - ) .

Immunohistochemistry of brain sections with anti-MAP2 antibody for visualization of neurons (layer V) and apical dendritic (layer IV) and with anti-GFAP for reactive astrocytes (layer V). The complete analysis is shown in Fig. EV2A, EV3A. Scale bars: 50 µm. Quantifications of number of MAP2-positive neurons in layer V, and of number of apical dendrites in layer IV (Fig. EV2B) reveal reduced neuron number in layer V, and reduced dendritic density in layer IV of 5-month-old Ccp1-/-Ccp6-/- mice. Quantification of the number of reactive astrocytes in layer V (Fig. EV3B) shows a significant increase over wild type in Ccp1-/- and in Ccp6-/- mice, however a much stronger increase is seen in Ccp1-/-Ccp6-/- mice. Complete analyses are shown in Fig. EV2C, EV3C (mean ±SEM; student's t-test).


Immunohistochemistry of brain sections stained with SMI - 32 antibody ( a representative section of one brain per genotype group is shown , complete in Appendix Fig . S3 ) . Neuronal SMI - 32 labelling indicates increased axonal damage in cortex of Ccp1 - / - Ccp6 - / - mice at 5 months . Scale bar : 100 µm .

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Immunohistochemistry of brain sections stained with SMI-32 antibody (a representative section of one brain per genotype group is shown, complete analysis in Appendix Fig. S3). Neuronal SMI-32 labelling indicates increased axonal damage in the cortex of Ccp1-/-Ccp6-/- mice at 5 months. Scale bar: 100 µm.


Representative electron microscopy of sections of wild type Ccp1 - / - Ccp6 - / - cerebral cortices with myelinated axons . In Ccp1 - / - Ccp6 - / - sections , several myelinated axons show signs of degeneration , such as accumulation of organelles densification ( arrows ) , or axonal lysis ( stars ) . Note that only some axons in observed sections displayed these Ccp1 - / - Ccp6 - / - , while such figures were never observed in wild type cortices . Scale bar : 1 µm .

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Representative electron microscopy images of sections of wild type and Ccp1-/-Ccp6-/- cerebral cortices with myelinated axons. In Ccp1-/-Ccp6-/- sections, several myelinated axons show signs of degeneration, such as accumulation of cellular organelles and cytoplasmic densification (arrows), or axonal lysis (stars). Note that only some axons in the observed sections displayed these phenotypes Ccp1-/-Ccp6-/-, while such figures were never observed in wild type cortices. Scale bar: 1 µm.


Fate of Purkinje in 1 - month - old wild type , spastin - / - , Ccp1 - / - Ccp1 - / - spastin - / - mice visualized with anti - calbindin antibody . Ccp1 - / - Ccp1 - / - spastin - / - mice show almost complete loss of Purkinje - layer . Scale bar : 500 µm .

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Fate of Purkinje cells in 1-month-old wild type, spastin-/-, Ccp1-/- and Ccp1-/-spastin-/- mice visualized with anti-calbindin antibody. Ccp1-/- and Ccp1-/-spastin-/- mice show almost complete loss of the Purkinje-cell layer. Scale bar: 500 µm.


Nissl shows complete loss of Purkinje in Ccp1 - / - Ccp1 - / - spastin - / - mice . Scale bar : 20 µm . Purkinje layer ( PC ) granule layer ( GC ) are indicated .

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Nissl staining shows the complete loss of Purkinje cells in Ccp1-/- and Ccp1-/-spastin-/- mice. Scale bar: 20 µm. Purkinje cell layer (PC) and granule cell layer (GC) are indicated.


Calbindin of sections showing axons of Purkinje as in ( A ) . While wild type spastin - / - show normal axons , Ccp1 - / - Ccp1 - / - spastin - / - mice show axonal swellings ( arrow heads ) in remaining Purkinje . Scale bars : 20 µm .

Calbindin staining of sections showing axons of Purkinje cells as in (A). While wild type and spastin-/- show normal axons, Ccp1-/- and Ccp1-/-spastin-/- mice show axonal swellings (arrow heads) in the remaining Purkinje cells. Scale bars: 20 µm.


Immunoblot of extracts from Ccp1flox / floxCcp6flox / flox hippocampal neurons without , or after transduction with lentivirus expressing either GFP or GFP - 2A - cre after 4 days in culture . Note specific increase of polyglutamylation ( polyE ) in neurons transduced with GFP - 2A - cre lentivirus .

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Immunoblot of cell extracts from Ccp1flox/floxCcp6flox/flox hippocampal neurons without, or after transduction with lentivirus expressing either GFP or GFP-2A-cre after 4 days in culture. Note the specific increase of polyglutamylation (polyE) in neurons transduced with GFP-2A-cre lentivirus.


kymographs of one representative set of experiments . Mitochondria movements ( Movie S1 ) were plotted in kymographs analyzed as described in ( A ) . Scale bars : 60 sec , 20 µm . Analyses of ( D ) run ( E ) speed distribution , represented as scatter dot plots with each point representing a single run extracted from kymographs shown in ( C ) . Each group represents pool of measurement of multiple neurons made in experiment . black indicates median of distribution with whiskers at interquartile range . Numerical value of medians is indicated below each plot . ( F ) Overall motility of mitochondria represented as fraction of time mitochondria spent in movement relative to total number of mitochondria during recording time of 60 s . Values ( below plots ) from one set of experiment ( C ) show a decrease of about 50 % in Ccp1 - / - Ccp6 - / - conditions , while ( G ) mitochondria density is unaltered . ( H - K ) Statistical analyses of mitochondria transport parameters of experiments shown in ( D - G ; Fig . EV4A - D ) . Each bar represents mean ( ± ) of medians of single experiments for run ( H ) speed ( I ) distributions , average ( ± ) mitochondria motility ( J ) density ( K ) of seven experimental sets . Values of fold change between control Ccp1 - / - Ccp6 - / - conditions are indicated below graphs . Green bars are for anterograde , red for retrograde transport events . Significance was tested using unpaired t - . Average run ( H ) as well as speed ( I ) showed no significant differences in these two parameters between control Ccp1 - / - Ccp6 - / - conditions . ( J ) average of overall motility reveals an about 50 % decrease in overall mitochondria motility , while mitochondria density ( K ) is unaltered between control Ccp1 - / - Ccp6 - / - conditions .

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All kymographs of one representative set of experiments. Mitochondria movements (Movie S1) were plotted in kymographs and analyzed as described in (A). Scale bars: 60 sec, 20 µm. Analyses of (D) run length and (E) speed distribution, represented as scatter dot plots with each point representing a single run extracted from the kymographs shown in (C). Each group represents the pool of measurement of multiple neurons made in the experiment. The black line indicates the median of the distribution with whiskers at interquartile range. Numerical value of the medians is indicated below each plot. (F) Overall motility of mitochondria represented as fraction of time mitochondria spent in movement relative to the total number of mitochondria during the recording time of 60 s. Values (below plots) from one set of experiment (C) show a decrease of about 50% in Ccp1-/-Ccp6-/- conditions, while (G) mitochondria density is unaltered. (H-K) Statistical analyses of mitochondria transport parameters of experiments shown in (D-G; Fig. EV4A-D). Each bar represents the mean (±SEM) of medians of single experiments for run length (H) and speed (I) distributions, and average (±SEM) mitochondria motility (J) and density (K) of all seven experimental sets. Values of fold change between control and Ccp1-/-Ccp6-/- conditions are indicated below the graphs. Green bars are for anterograde, and red for retrograde transport events. Significance was tested using unpaired t-test. Average run length (H) as well as speed (I) showed no significant differences in these two parameters between control and Ccp1-/-Ccp6-/- conditions. (J) The average of overall motility reveals an about 50% decrease in overall mitochondria motility, while mitochondria density (K) is unaltered between control and Ccp1-/-Ccp6-/- conditions.


( A ) Optically evoked auditory brainstem response in an AAV - CatCh - injected gerbil in response to a 1 ms of ~ 35 mW ( mean of 1000 stimulus presentations at 10 Hz repetition rate ) .

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(A) Optically evoked auditory brainstem response in an AAV-CatCh-injected gerbil in response to a 1 ms laser pulse of ~35 mW (mean of 1000 stimulus presentations at 10 Hz repetition rate).


( B - D ) Catch - eYFP - expressing SGNs in apical cochlear turn identified by parvalbumin expression ( B ) CatCh - eYFP ( C ) . inlay ( D ´ ) of merged immunostaining ( D ) indicates lack of CatCh - EYFP signal in inner hair ( dashed ) . Scale bar : 20 µm .

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(B-D) Catch-eYFP-expressing SGNs in the apical cochlear turn identified by parvalbumin expression (B) and CatCh-eYFP (C). The inlay (D´) of the merged immunostaining (D) indicates the lack of CatCh-EYFP signal in inner hair cells (dashed white line). Scale bar: 20 µm.


( I ) oCI inserted into cochlea ( dashed black lines ) via a cochleostomy in basal cochlear turn ( black , solid ) . round window niche is highlighted by a dashed , round window by a solid . SA : stapedial artery .

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(I) oCI inserted into the cochlea (dashed black lines) via a cochleostomy in the basal cochlear turn (black, solid line). The round window niche is highlighted by a dashed white line, the round window by a solid white line. SA: stapedial artery.


( J ) 3D X - ray tomography reconstruction of a 16 - channel oCI implanted in a gerbil cochlea via round window . Cables μLEDs are marked in blue , most apical µLED is indicated by black arrowhead ; basilar is marked in green . µLEDs have a of 60 × 60 µm .

(J) 3D X-ray tomography reconstruction of a 16-channel oCI implanted in a gerbil cochlea via the round window. Cables and μLEDs are marked in blue, the most apical µLED is indicated by the black arrowhead; the basilar membrane is marked in green. µLEDs have a size of 60×60 µm.


( A - D ) Exemplar STCs in response to SGN illumination with a single μLED ( A ) , a block of four neighboring μLEDs ( B ) μLEDs of an oCI inserted via round window in a CatCh - transduced gerbil ( C ) with µLEDs in a wildtype gerbil ( WT ; D ) . μLEDs were spaced by 250 μm .

(A-D) Exemplar STCs in response to SGN illumination with a single μLED (A), a block of four neighboring μLEDs (B) and all μLEDs of an oCI inserted via the round window in a CatCh-transduced gerbil (C) and with all µLEDs in a wildtype gerbil (WT; D). μLEDs were spaced by 250 μm.


( E ) Acoustic auditory brainstem responses ( average of 1000 stimuli at a stimulation rate of 10 Hz ) upon click stimulation from 20 - 100 dB SPL before ( left ) after deafening ( right ; star : threshold ) . ( F ) Optically evoked auditory brainstem responses before ( left ) after deafening ( right ) , elicited by ~ 35 mW pulses of 1 ms duration , delivered by a - coupled fiber ( 1000 stimuli presented at 10 Hz ) , recorded from same . Optogenetic activation of auditory system was still possible after deafening . Differences in amplitude waveform before after deafening are likely due to different positioning of recording electrodes optical fiber .

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(E) Acoustic auditory brainstem responses (average of 1000 stimuli at a stimulation rate of 10 Hz) upon click stimulation from 20-100 dB SPL before (left) and after deafening (right; star: threshold). (F) Optically evoked auditory brainstem responses before (left) and after deafening (right), elicited by ~35 mW pulses of 1 ms duration, delivered by a laser-coupled fiber (1000 stimuli presented at 10 Hz), recorded from the same animal. Optogenetic activation of the auditory system was still possible after deafening. Differences in amplitude and waveform before and after deafening are likely due to different positioning of recording electrodes and the optical fiber.


( E ) Acoustic auditory brainstem responses ( average of 1000 stimuli at a stimulation rate of 10 Hz ) upon click stimulation from 20 - 100 dB SPL before ( left ) after deafening ( right ; star : threshold ) .

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(E) Acoustic auditory brainstem responses (average of 1000 stimuli at a stimulation rate of 10 Hz) upon click stimulation from 20-100 dB SPL before (left) and after deafening (right; star: threshold).


( G - H ) Exemplar STCs in response to SGN illumination with µLEDs in a CatCh - transduced , kanamycin - deafened gerbil before ( G ) after ( H ) sacrifice .

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(G-H) Exemplar STCs in response to SGN illumination with all µLEDs in a CatCh-transduced, kanamycin-deafened gerbil before (G) and after (H) sacrifice.


( I ) Maximum strength of ICC responses ( mean ± standard deviation ) evoked by oCI stimulation with 1 ( indiv . ; n = 129 / 77 µLEDs in N = 9 / 3 hearing / deaf gerbils ) , 4 ( block ; n = 62 / 27 blocks in N = 9 / 3 hearing / deaf gerbils ) , 16 ( ; n = 24 / 9 oCIs in N = 9 / 3 hearing / deaf gerbils ) active µLEDs , with a - coupled optical fiber ( ~ 35 mW ; n = 9 / 3 fiber stimulations in N = 9 / 3 hearing / deaf gerbils ) in CatCh - transduced gerbils ( One - way ANOVA ( F - value : 73 . 25 , 4 degrees of freedom , p = 5 . 19 * 10 - 45 ) post - hoc multiple comparison ; indiv . vs . block : p = 9 . 9 * 10 - 9 ; block vs . : p = 0 . 0096 ; vs . fiber : p = 0 . 0016 ; CatCh vs . WT : p = 9 . 9 * 10 - 9 ; * * : p < 0 . 01 , * * * : p < 0 . 001 , n . s . = non - significant ) . oCI stimulation with µLEDs has also been performed in WT ( n = 7 / 7 oCIs , N = 2 / 2 hearing / deaf gerbils ) in sacrificed CatCh - transduced ( post - mortem ; n = 5 / 1 oCIs in N = 5 / 1 hearing / deaf gerbils ) . Brown dots indicate data recorded from deafened . ( J ) Number of active electrodes ( d ´ > 1 ; mean ± standard deviation ) upon oCI stimulation with 1 ( n = 129 / 77 µLEDs in N = 9 / 3 hearing / deaf gerbils ) , 4 ( n = 62 / 27 blocks in N = 9 / 3 hearing / deaf gerbils ) , 16 µLEDs ( n = 24 / 9 oCIs in N = 9 / 3 hearing / deaf gerbils ) , with optical fiber stimulation ( n = 9 / 3 fiber stimulations in N = 9 / 3 hearing / deaf gerbils ) in CatCh - transduced gerbils ( One - way ANOVA ( F - value : 30 . 18 , 4 degrees of freedom , p = 1 . 42 * 10 - 21 ) post - hoc multiple comparison ; indiv . vs . block : p = 6 . 8 * 10 - 5 ; block vs . : p = 0 . 006 ; vs . fiber : p = 0 . 25 ; CatCh vs . WT : p = 9 . 9 * 10 - 9 ; * * : p < 0 . 01 , * * * : p < 0 . 001 , n . s . = non - significant ) . Responses to oCI stimulation with µLEDs were also observed in hearing WT ( n = 3 oCIs in 2 gerbils ) . No active electrodes were found in response to 4 / 7 oCI stimulations in 2 / 2 hearing / deaf wildtype gerbils ) . ( K ) Thresholds ( d ´ = 1 , measured at best electrode ( BE ) ) for ICC activation evoked with 16 active µLEDs on an oCI ( n = 24 / 9 in N = 9 / 3 hearing / deaf gerbils ) with a - coupled optical fiber ( n = 9 / 3 in N = 9 / 3 hearing / deaf gerbils ; p = 0 . 012 , Wilcoxon rank sum ) . Central horizontal indicates median , lower , upper horizontal lines indicate minimum maximum values , box edges indicate 25th 75th percentile , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Maximum strength of ICC responses (mean ± standard deviation) evoked by oCI stimulation with 1 (indiv.; n = 129/77 µLEDs in N = 9/3 hearing/deaf gerbils), 4 (block; n = 62/27 blocks in N = 9/3 hearing/deaf gerbils), and 16 (all; n = 24/9 oCIs in N = 9/3 hearing/deaf gerbils) active µLEDs, and with a laser-coupled optical fiber (~35 mW; n = 9/3 fiber stimulations in N = 9/3 hearing/deaf gerbils) in CatCh-transduced gerbils (One-way ANOVA (F-value: 73.25, 4 degrees of freedom, p = 5.19*10-45) and post-hoc multiple comparison test; indiv. vs. block: p = 9.9*10-9; block vs. all: p = 0.0096; all vs. fiber: p = 0.0016; all CatCh vs. all WT: p = 9.9*10-9;**: p < 0.01, ***: p < 0.001, n.s. = non-significant). oCI stimulation with all µLEDs has also been performed in WT animals (n = 7/7 oCIs, N = 2/2 hearing/deaf gerbils) and in sacrificed CatCh-transduced animals (post-mortem; n = 5/1 oCIs in N = 5/1 hearing/deaf gerbils). Brown dots indicate data recorded from deafened animals. (J) Number of active electrodes (d´ > 1; mean ± standard deviation) upon oCI stimulation with 1 (n = 129/77 µLEDs in N = 9/3 hearing/deaf gerbils), 4 (n = 62/27 blocks in N = 9/3 hearing/deaf gerbils), and 16 µLEDs (n = 24/9 oCIs in N = 9/3 hearing/deaf gerbils), and with optical fiber stimulation (n = 9/3 fiber stimulations in N = 9/3 hearing/deaf gerbils) in CatCh-transduced gerbils (One-way ANOVA (F-value: 30.18, 4 degrees of freedom, p = 1.42*10-21) and post-hoc multiple comparison test; indiv. vs. block: p = 6.8*10-5; block vs. all: p = 0.006; all vs. fiber: p = 0.25; all CatCh vs. all WT: p = 9.9*10-9;**: p < 0.01, ***: p < 0.001, n.s. = non-significant). Responses to oCI stimulation with all µLEDs were also observed in hearing WT animals (n = 3 oCIs in 2 gerbils). No active electrodes were found in response to 4/7 oCI stimulations in 2/2 hearing/deaf wildtype gerbils). (K) Thresholds (d´ = 1, measured at the best electrode (BE)) for ICC activation evoked with 16 active µLEDs on an oCI (n = 24/9 in N = 9/3 hearing/deaf gerbils) and with a laser-coupled optical fiber (n = 9/3 in N = 9/3 hearing/deaf gerbils; p = 0.012, Wilcoxon rank sum test). Central horizontal line indicates the median, lower, upper horizontal lines indicate the minimum and maximum values, and box edges indicate the 25th and 75th percentile, respectively.


( I ) Maximum strength of ICC responses ( mean ± standard deviation ) evoked by oCI stimulation with 1 ( indiv . ; n = 129 / 77 µLEDs in N = 9 / 3 hearing / deaf gerbils ) , 4 ( block ; n = 62 / 27 blocks in N = 9 / 3 hearing / deaf gerbils ) , 16 ( ; n = 24 / 9 oCIs in N = 9 / 3 hearing / deaf gerbils ) active µLEDs , with a - coupled optical fiber ( ~ 35 mW ; n = 9 / 3 fiber stimulations in N = 9 / 3 hearing / deaf gerbils ) in CatCh - transduced gerbils ( One - way ANOVA ( F - value : 73 . 25 , 4 degrees of freedom , p = 5 . 19 * 10 - 45 ) post - hoc multiple comparison ; indiv . vs . block : p = 9 . 9 * 10 - 9 ; block vs . : p = 0 . 0096 ; vs . fiber : p = 0 . 0016 ; CatCh vs . WT : p = 9 . 9 * 10 - 9 ; * * : p < 0 . 01 , * * * : p < 0 . 001 , n . s . = non - significant ) . oCI stimulation with µLEDs has also been performed in WT ( n = 7 / 7 oCIs , N = 2 / 2 hearing / deaf gerbils ) in sacrificed CatCh - transduced ( post - mortem ; n = 5 / 1 oCIs in N = 5 / 1 hearing / deaf gerbils ) . Brown dots indicate data recorded from deafened .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Maximum strength of ICC responses (mean ± standard deviation) evoked by oCI stimulation with 1 (indiv.; n = 129/77 µLEDs in N = 9/3 hearing/deaf gerbils), 4 (block; n = 62/27 blocks in N = 9/3 hearing/deaf gerbils), and 16 (all; n = 24/9 oCIs in N = 9/3 hearing/deaf gerbils) active µLEDs, and with a laser-coupled optical fiber (~35 mW; n = 9/3 fiber stimulations in N = 9/3 hearing/deaf gerbils) in CatCh-transduced gerbils (One-way ANOVA (F-value: 73.25, 4 degrees of freedom, p = 5.19*10-45) and post-hoc multiple comparison test; indiv. vs. block: p = 9.9*10-9; block vs. all: p = 0.0096; all vs. fiber: p = 0.0016; all CatCh vs. all WT: p = 9.9*10-9;**: p < 0.01, ***: p < 0.001, n.s. = non-significant). oCI stimulation with all µLEDs has also been performed in WT animals (n = 7/7 oCIs, N = 2/2 hearing/deaf gerbils) and in sacrificed CatCh-transduced animals (post-mortem; n = 5/1 oCIs in N = 5/1 hearing/deaf gerbils). Brown dots indicate data recorded from deafened animals.


( A ) Exemplar STCs in response to SGN stimulation with four blocks of four µLEDs inserted via round window , from apical to basal direction ( A - D ) . markers define best electrode ( BE ) , measurement of spread of excitation ( SoE ) is indicated in left panel . In STCs where more than one peak was detected , dorsal - ventral - most electrodes have been considered as boundaries of STC in order to avoid underestimation of SoE .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Exemplar STCs in response to SGN stimulation with four blocks of four µLEDs inserted via the round window, from apical to basal direction (A-D). White markers define the best electrode (BE), and measurement of the spread of excitation (SoE) is indicated in the left panel. In STCs where more than one peak was detected, the dorsal- and ventral-most electrodes have been considered as the boundaries of the STC in order to avoid underestimation of the SoE.


( C ) Spectral spread of excitation upon SGN stimulation with µLED - oCIs ( blocks of 4 µLEDs , solid lines ; mean ± standard error of mean ) , acoustic ( black dashed ) , - coupled optical ( blue dashed ) , as well as mono - bipolar electrical stimulation with a clinical - style eCI ( dashed dotted red lines , respectively ) . Average data from stimulation via optical eCIs are replotted from Dieter et al ( Dieter et al , 2019 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Spectral spread of excitation upon SGN stimulation with µLED-based oCIs (blocks of 4 µLEDs, solid lines; mean ± standard error of the mean), acoustic (black dashed line), laser-coupled optical fibers (blue dashed line), as well as mono- and bipolar electrical stimulation with a clinical-style eCI (dashed and dotted red lines, respectively). Average data from stimulation via optical fibers and eCIs are replotted from Dieter et al (Dieter et al, 2019).


Figure 1b . Genotyping of AS WT CDK12 clones . Ethidium bromide - stained agarose gel visualizing PCR products from genomic of AS ( AS - PCR ) WT ( WT - PCR ) CDK12 HCT116 their digest with BslI enzyme ( indicated as AS - BslI WT - BslI ) . Primer positions BslI restriction sites are depicted at Expanded View Fig . 1a . Numbers on left right indicate marker fragment sizes , respectively .

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

Figure 1b. Genotyping of AS and WT CDK12 clones. Ethidium bromide-stained agarose gel visualizing PCR products from genomic DNA of AS (AS-PCR) and WT (WT-PCR) CDK12 HCT116 cells and their digest with BslI enzyme (indicated as AS- BslI and WT- BslI). Primer positions and BslI restriction sites are depicted at Expanded View Fig. 1a. Numbers on the left and right indicate DNA marker and DNA fragment sizes, respectively.


Figure 1d . Effect of CDK12 inhibition on phosphorylation of CTD of RNAPII . Western blot analyses of by indicated antibodies in AS CDK12 HCT116 treated with 5 µM 3 - MB - PP1 for indicated times . Long short exp . = long ( 4 - 14 min ) short ( 10 - 60 s ) exposures , respectively . FUS tubulin are loading controls . A representative image from 3 replicates is shown .

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

Figure 1d. Effect of CDK12 inhibition on phosphorylation of the CTD of RNAPII. Western blot analyses of protein levels by the indicated antibodies in AS CDK12 HCT116 cells treated with 5 µM 3-MB-PP1 for indicated times. Long and short exp.= long (4-14 min) and short (10-60 s) exposures, respectively. FUS and tubulin are loading controls. A representative image from 3 replicates is shown.


Figure 1e , f . Inhibition of CDK12 in AS CDK12 HCT116 results in down - regulation of CDK12 - dependent HR genes . Graph shows RT - qPCR of relative of mRNAs of described genes in AS CDK12 HCT116 ( e ) WT CDK12 HCT116 ( f ) treated for indicated times with 3 - MB - PP1 . mRNA were normalized to HPRT1 mRNA expression mRNA of untreated control ( CTRL ) were set to 1 . n = 3 replicates , error bars indicate standard error of mean ( ) .

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

Figure 1e, f. Inhibition of CDK12 in AS CDK12 HCT116 cells results in down-regulation of CDK12-dependent HR genes. Graph shows RT-qPCR analysis of relative levels of mRNAs of described genes in AS CDK12 HCT116 (e) and WT CDK12 HCT116 (f) cells treated for indicated times with 3-MB-PP1. mRNA levels were normalized to HPRT1 mRNA expression and the mRNA levels of untreated control (CTRL) cells were set to 1. n=3 replicates, error bars indicate standard error of the mean (SEM).


Figure 2b . CDK12 kinase activity is needed for G1 / S progression in arrested by serum starvation . Flow cytometry profiles of control ( - 3 - MB - PP1 ) or inhibitor ( + 3 - MB - PP1 ) treated from experiment depicted in Fig . 2a . red arrow points to onset of G1 / S progression defect in 3 - MB - PP1 - treated . To better visualize G1 / S delay in presence of inhibitor 24 h time point is also shown . n = 3 replicates ; representative result is shown .

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

Figure 2b. CDK12 kinase activity is needed for G1/S progression in cells arrested by serum starvation . Flow cytometry profiles of control (-3-MB-PP1) or inhibitor (+3-MB-PP1) treated cells from the experiment depicted in Fig. 2a. The red arrow points to the onset of the G1/S progression defect in 3-MB-PP1-treated cells. To better visualize the G1/S delay in the presence of the inhibitor the 24 h time point is also shown. n=3 replicates; representative result is shown.


Figure 2b . CDK12 kinase activity is needed for G1 / S progression in arrested by serum

Figure 2b. CDK12 kinase activity is needed for G1/S progression in cells arrested by serum starvation


Figure 2d . CDK12 peak in G0 / G1 phase of cycle . Western blots show of at indicated time points after release of serum starved AS CDK12 HCT116 . Corresponding cycle phases are depicted above time points . A representative Western blot from 3 replicates is shown .

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

Figure 2d. CDK12 protein levels peak in the G0/G1 phase of the cell cycle. Western blots show levels of proteins at indicated time points after the release of serum starved AS CDK12 HCT116 cells. Corresponding cell cycle phases are depicted above time points. A representative Western blot from 3 replicates is shown.


Figure 2f , g . Inhibition of CDK12 in early G1 perturbs normal cycle progression . of ( % ) in cycle phases from flow cytometry profiles of propidium iodide ( f ) BrdU ( g ) labeled upon addition of 3 - MB - PP1 at indicated time points after serum addition in experiment depicted in Fig . 2e . CTRL in Fig . 2g = control sample without 3 - MB - PP1 . n = 3 replicates , representative result is shown .

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

Figure 2f, g. Inhibition of CDK12 in early G1 perturbs normal cell cycle progression. Quantification of cells (%) in cell cycle phases from flow cytometry profiles of propidium iodide (f) and BrdU (g) labeled cells upon addition of 3-MB-PP1 at indicated time points after serum addition in the experiment depicted in Fig. 2e. CTRL in Fig. 2g = control sample without 3-MB-PP1. n=3 replicates, representative result is shown.


Figure 2h . Short - term CDK12 inhibition does not activate damage checkpoints . Western blot analyses of phosphorylation of depicted damage response markers upon inhibition of CDK12 for indicated times . CPT corresponds to 5 µM camptothecin . A representative Western blot from 3 replicates is shown . FUS is a loading control .

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

Figure 2h. Short-term CDK12 inhibition does not activate DNA damage checkpoints. Western blot analyses of phosphorylation of depicted DNA damage response markers upon inhibition of CDK12 for indicated times. CPT corresponds to 5 µM camptothecin. A representative Western blot from 3 replicates is shown. FUS is a loading control.


Figure 3a . CDK12 inhibition results in differential expression of a subset of genes . Comparison of log2 fold - changes versus log2 mean expression in 3 ' end - seq data shows differentially regulated genes after inhibition of CDK12 . Down - ( log2 fold - change < - 1 ) up - regulated ( log2 fold - change > 1 ) genes are shown in blue red , respectively .

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

Figure 3a. CDK12 inhibition results in differential expression of a subset of genes. Comparison of log2 fold-changes versus log2 mean expression in 3'end RNA-seq data shows differentially regulated genes after inhibition of CDK12. Down- (log2 fold-change < -1) and up-regulated (log2 fold-change >1) genes are shown in blue and red, respectively.


Figure 3d . Validation of - seq for select replication genes by RT - qPCR . Graph shows relative of mRNAs of described genes in serum arrested released ( 0 h G0 / G1 ) AS CDK12 HCT116 either treated ( 3 - MB - PP1 ) or not ( CTRL ) with inhibitor for indicated times after release . mRNA were normalized to B2M mRNA expression mRNA for each gene at time of release ( 0 h ) was set as 1 . n = 3 replicates , error bars indicate .

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

Figure 3d. Validation of RNA-seq for select DNA replication genes by RT-qPCR. Graph shows relative levels of mRNAs of described genes in serum arrested and released (0 h G0/G1) AS CDK12 HCT116 cells either treated (3-MB-PP1) or not (CTRL) with the inhibitor for indicated times after the release. mRNA levels were normalized to B2M mRNA expression and mRNA levels for each gene at the time of release (0 h) was set as 1. n=3 replicates, error bars indicate SEM.


Figure 3e . of replication factors are dependent on CDK12 kinase activity . Western blot analyses of expression by depicted antibodies in serum synchronized released ( 0 h ) either treated or not with 3 - MB - PP1 for indicated times after release . FUS is a loading control . A representative Western blot of 3 replicates is shown .

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

Figure 3e. Protein levels of core DNA replication factors are dependent on the CDK12 kinase activity. Western blot analyses of protein expression by the depicted antibodies in serum synchronized and released (0 h) cells either treated or not with 3-MB-PP1 for the indicated times after the release. FUS is a loading control. A representative Western blot of 3 replicates is shown.


Figure 3f . CDK12 inhibition affects loading of CDC6 CDT1 replication factors to . Western blotting analyses of association of indicated replication factors in serum synchronized released AS CDK12 HCT116 treated or not with 3 - MB - PP1 for indicated times . Histone H2A serves as a loading control of fractions . A = asynchronous , 0 h = time of release . A representative Western blot of 3 replicates is shown .

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

Figure 3f. CDK12 inhibition affects loading of CDC6 and CDT1 DNA replication factors to chromatin. Western blotting analyses of chromatin association of the indicated DNA replication factors in serum synchronized and released AS CDK12 HCT116 cells treated or not with 3-MB-PP1 for the indicated times. Histone H2A serves as a loading control of chromatin fractions. A=asynchronous cells, 0 h=time of release. A representative Western blot of 3 replicates is shown.


Removal of CDK12 inhibitor in early G1 / S rescues replication gene expression cycle progression . RT - qPCR of replication gene mRNA RT - qPCR were performed 7 , 12 15 h post - release , respectively . CTRL = control samples without 3 - MB - PP1 . In b , n = 3 error bars indicate . In c , d representative from 3 biological replicates are shown .

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

Removal of CDK12 inhibitor in early G1/S rescues replication gene expression and cell cycle progression. RT-qPCR of replication gene mRNA RT-qPCR were performed 7, 12 and 15 h post-release, respectively. CTRL=control samples without the 3-MB-PP1. In b, n=3 and error bars indicate SEM. In c, d representative images from 3 biological replicates are shown.


Removal of CDK12 inhibitor in early G1 / S rescues cycle progression . Western blotting of , Western blotting were performed 7 , 12 15 h post - release , respectively . CTRL = control samples without 3 - MB - PP1 . In b , n = 3 error bars indicate . In c , d representative from 3 biological replicates are shown .

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

Removal of CDK12 inhibitor in early G1/S rescues cell cycle progression. Western blotting of , protein levels Western blotting were performed 7, 12 and 15 h post-release, respectively. CTRL=control samples without the 3-MB-PP1. In b, n=3 and error bars indicate SEM. In c, d representative images from 3 biological replicates are shown.


Removal of CDK12 inhibitor in early G1 / S rescues replication cycle progression . flow cytometry analyses of cycle progression , flow cytometry analyses were performed 7 , 12 15 h post - release , respectively . CTRL = control samples without 3 - MB - PP1 . In b , n = 3 error bars indicate . In c , d representative from 3 biological replicates are shown .

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

Removal of CDK12 inhibitor in early G1/S rescues replication and cell cycle progression. flow cytometry analyses of cell cycle progression, flow cytometry analyses were performed 7, 12 and 15 h post-release, respectively. CTRL=control samples without the 3-MB-PP1. In b, n=3 and error bars indicate SEM. In c, d representative images from 3 biological replicates are shown.


Figure 4e . Rescued loading of CDC6 CDT1 on after removal of CDK12 inhibitor . Western blot analyses of fractions of serum starved AS CDK12 HCT116 treated with 3 - MB - PP1 for 6 or 9 h or with inhibitor washed off after 1 h of treatment . CTRL corresponds to not treated with inhibitor at time of serum addition . were harvested either 6 or 9 h after serum addition . Histone H2A serves as a loading control of fractions studied replication factors are indicated . A representative image of three replicates is shown .

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

Figure 4e. Rescued loading of CDC6 and CDT1 on chromatin after removal of CDK12 inhibitor. Western blot analyses of chromatin fractions of serum starved AS CDK12 HCT116 cells treated with 3-MB-PP1 for 6 or 9 h or with the inhibitor washed off after 1 h of treatment. CTRL corresponds to cells not treated with the inhibitor at the time of the serum addition. All cells were harvested either 6 or 9 h after the serum addition. Histone H2A serves as a loading control of chromatin fractions and studied DNA replication factors are indicated. A representative image of three replicates is shown.


Figure 4f . Inhibition of CDK12 kinase activity in cycling leads to decreased numbers of actively replicating . Asynchronous AS CDK12 HCT116 were grown for 24 48 h in presence or absence of 3 - MB - PP1 replicating BrdU - stained were quantified by FACS analyses . CTRL = control samples without 3 - MB - PP1 . A representative image of 3 replicates is shown .

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

Figure 4f. Inhibition of CDK12 kinase activity in cycling cells leads to decreased numbers of actively replicating cells. Asynchronous AS CDK12 HCT116 cells were grown for 24 and 48 h in the presence or absence of 3-MB-PP1 and replicating BrdU-stained cells were quantified by FACS analyses. CTRL=control samples without the 3-MB-PP1. A representative image of 3 replicates is shown.


Figure 4g , h . Prolonged CDK12 inhibition causes chromosomal aberrations in . Specific chromosomal aberrations in treated with 3 - MB - PP1 ( 24 or 48 h ) , 4 mM hydroxyurea ( 5 h ) or control solvent ( CTRL ) were identified by microscopy . A representative image from 3 biological replicates is shown ( g ) . Total numbers of chromosomal aberrations per hundred of representative replicate in g is quantified ( h ) .

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

Figure 4g, h. Prolonged CDK12 inhibition causes chromosomal aberrations in cells. Specific chromosomal aberrations in cells treated with 3-MB-PP1 (24 or 48 h), 4 mM hydroxyurea (5 h) or control solvent (CTRL) were identified by microscopy. A representative image from 3 biological replicates is shown (g). Total numbers of chromosomal aberrations per hundred cells of the representative replicate in g is quantified (h).