G , H Quantitative of BrdU + ( G ) and BrdU + DCX + ( H ) cells in the DG of susceptible mice with LV - siCD39 compared with LV - GFP treatment ( n = 3 , 6 , 6 mice . For BrdU + Treatment F2 , 12 = 15 . 79 , P = 0 . 0004 ; CSDS - LV - GFP vs Ctrl - LV - GFP , P = 0 . 0012 ; CSDS - LV - siCD39 vs CSDS - LV - GFP , P = 0 . 0003 ; BrdU + DCX + Treatment F2 , 12 = 12 . 79 , P = 0 . 0011 ; CSDS - LV - GFP vs Ctrl - LV - GFP , P = 0 . 05 ; CSDS - LV - siCD39 vs CSDS - LV - GFP , P = 0 . 0003 , one - way ANOVA with Fisher ' s LSD test ) .

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

G, H Quantitative of BrdU+ (G) and BrdU+ DCX+ (H) cells in the DG of susceptible mice with LV-siCD39 compared with LV-GFP treatment (n = 3, 6, 6 mice. For BrdU+ Treatment F2, 12 = 15.79, P =0.0004; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0012; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0003; BrdU+ DCX+ Treatment F2, 12 = 12.79, P = 0.0011; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.05; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0003, one-way ANOVA with Fisher's LSD test).


Representative confocal z - stack images of LV - infected dendritic segments in the DG granule neurons from control and susceptible mice with different treatment . White arrows indicate stubby spines , yellow arrows indicate mushroom spines , and blue arrows indicate thin spines .

O O O O O O O O O O O O O O O O O O O O O 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 confocal z-stack images of LV-infected dendritic segments in the DG granule neurons from control and susceptible mice with different treatment. White arrows indicate stubby spines, yellow arrows indicate mushroom spines, and blue arrows indicate thin spines.


B - E Quantification of total ( B ) , stubby ( C ) , mushroom ( D ) and thin ( E ) spine density in DG from control and susceptible mice infected with LV - GFP or LV - siCD39 ( n = 15 segments from 4 mice / group . For total density : Treatment F2 , 42 = 3 . 12 , P = 0 . 0544 ; Ctrl - LV - GFP vs CSDS - LV - siCD39 , P = 0 . 0312 ; CSDS - LV - siCD39 vs CSDS - LV - GFP , P = 0 . 0425 ; stubby density : Treatment F2 , 42 = 10 . 93 , P = 0 . 0002 ; P = 0 . 0026 ; CSDS - LV - CD39 vs CSDS - LV - GFP , P < 0 . 0001 ; mushroom density : Treatment F2 , 42 = 0 . 34 , P = 0 . 7161 ; thin density : F2 , 42 = 1 . 00 , P = 0 . 3758 . One - way ANOVA with Fisher ' s LSD 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 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

B-E Quantification of total (B), stubby (C), mushroom (D) and thin (E) spine density in DG from control and susceptible mice infected with LV-GFP or LV-siCD39 (n = 15 segments from 4 mice/group. For total density: Treatment F2, 42 = 3.12, P = 0.0544; Ctrl - LV-GFP vs CSDS - LV-siCD39, P = 0.0312; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0425; stubby density: Treatment F2, 42 = 10.93, P=0.0002; P = 0.0026; CSDS - LV-CD39 vs CSDS - LV-GFP, P < 0.0001; mushroom density: Treatment F2, 42 = 0.34, P = 0.7161; thin density: F2, 42 = 1.00, P = 0.3758. One-way ANOVA with Fisher's LSD test).


F Representative confocal z - stack images of LV - infected dendritic segments in the DG granule neurons from control and susceptible mice with different treatment . White arrows indicate stubby spines , yellow arrows indicate mushroom spines , and blue arrows indicate thin spines .

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

F Representative confocal z-stack images of LV-infected dendritic segments in the DG granule neurons from control and susceptible mice with different treatment. White arrows indicate stubby spines, yellow arrows indicate mushroom spines, and blue arrows indicate thin spines.


G - J Quantification of total ( G ) , stubby ( H ) , mushroom ( I ) and thin ( J ) spine density in DG from control and susceptible mice treatment with ACSF or ARL67156 ( n = 11 , 13 , 10 segments from 2 , 4 , 2 mice , respectively . For total density : Treatment F2 , 31 = 4 . 474 , P = 0 . 0196 ; CSDS - ASCF vs Ctrl - ACSF , P = 0 . 0366 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0086 ; stubby density : Treatment F2 , 31 = 3 . 327 , P = 0 . 0491 ; CSDS - ASCF vs Ctrl - ACSF , P = 0 . 1026 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0179 ; mushroom density : Treatment F2 , 31 = 1 . 986 , P = 0 . 1543 ; thin density : F2 , 31 = 1 . 475 , P = 0 . 2445 . One - way ANOVA with Fisher ' s LSD 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 O O O O O O O O O O

G-J Quantification of total (G), stubby (H), mushroom (I) and thin (J) spine density in DG from control and susceptible mice treatment with ACSF or ARL67156 (n = 11, 13, 10 segments from 2, 4, 2 mice, respectively. For total density: Treatment F2, 31 = 4.474, P = 0.0196; CSDS - ASCF vs Ctrl - ACSF, P = 0.0366; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0086; stubby density: Treatment F2, 31 = 3.327, P=0.0491; CSDS - ASCF vs Ctrl - ACSF, P = 0.1026; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0179; mushroom density: Treatment F2, 31 = 1.986, P = 0.1543; thin density: F2, 31 = 1.475, P = 0.2445. One-way ANOVA with Fisher's LSD test).


a , b , Representative images of GFP - LC3 puncta ( autophagosomes ) in skeletal ( vastus lateralis ) ( a ) and cardiac muscle ( b ) from GFP - LC3 transgenic mice at serial time points after exercise . Scale bar , 20 µm . c , Quantification of data ( mean ± s . e . m . of 10 tissue sections ) in a and b . * * P 0 . 01 , * * * P 0 . 001 ( one - way ANOVA ) .

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a, b, Representative images of GFP-LC3 puncta (autophagosomes) in skeletal (vastus lateralis) (a) and cardiac muscle (b) from GFP-LC3 transgenic mice at serial time points after exercise. Scale bar, 20 µm. c, Quantification of data (mean ± s.e.m. of 10 tissue sections) in a and b. **P  0.01, ***P  0.001 (one-way ANOVA).


d , Western blot analysis of LC3 - I / II ( non - lipidated and lipidated forms of MAP1LC3 , respectively ) and p62 levels in indicated tissue from mice at rest ( - ) or after maximal exercise ( + ) . Skeletal and cardiac indicate skeletal and cardiac muscle , respectively .

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d, Western blot analysis of LC3-I/II (non-lipidated and lipidated forms of MAP1LC3, respectively) and p62 levels in indicated tissue from mice at rest (-) or after maximal exercise (+). Skeletal and cardiac indicate skeletal and cardiac muscle, respectively.


e , Co - immunoprecipitation of beclin 1 with BCL2 in muscle tissue from mice at indicated time points after exercise . IP , immunoprecipitate ; WB , western blot ; WCL , whole cell lysates .

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e, Co-immunoprecipitation of beclin 1 with BCL2 in muscle tissue from mice at indicated time points after exercise. IP, immunoprecipitate; WB, western blot; WCL, whole cell lysates.


a , Analysis of BCL2 phosphorylation ( detected by anti - BCL2 immunoprecipitation and autoradiography of 32P - labelled cells ) and beclin 1 co - immunoprecipitation with BCL2 in wild - type ( WT ) or BCL2 AAA MEFs grown in normal media or subjected to 4 h Earle ' s balanced salt solution ( EBSS ) starvation . p - BCL2 , phospho - BCL2 .

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

a, Analysis of BCL2 phosphorylation (detected by anti-BCL2 immunoprecipitation and autoradiography of 32P-labelled cells) and beclin 1 co-immunoprecipitation with BCL2 in wild-type (WT) or BCL2 AAA MEFs grown in normal media or subjected to 4 h Earle's balanced salt solution (EBSS) starvation. p-BCL2, phospho-BCL2.


b , Quantification of GFP - LC3 puncta ( autophagosomes ) in MEFs of indicated genotype in normal growth conditions or starvation conditions . Data represent mean ± s . e . m . for 100 cells per well of triplicate samples per condition . Similar results were observed in three independent experiments .

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b, Quantification of GFP-LC3 puncta (autophagosomes) in MEFs of indicated genotype in normal growth conditions or starvation conditions. Data represent mean ± s.e.m. for 100 cells per well of triplicate samples per condition. Similar results were observed in three independent experiments.


c , Representative images of GFP - LC3 puncta ( autophagosomes ) in skeletal and cardiac muscle of GFP - LC3 wild - type and GFP - LC3BCL2 AAA mice before exercise , after 80 min exercise , or after 75 % of maximal exercise capacity . Scale bar , 20 µm . d , Quantification of data ( mean ± s . d . of 4 mice per experimental group ) in c .

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c, Representative images of GFP-LC3 puncta (autophagosomes) in skeletal and cardiac muscle of GFP-LC3 wild-type and GFP-LC3BCL2 AAA mice before exercise, after 80 min exercise, or after 75% of maximal exercise capacity. Scale bar, 20 µm. d, Quantification of data (mean ± s.d. of 4 mice per experimental group) in c.


e , Western blot analysis of LC3 - I / II and p62 levels in indicated tissue from mice of indicated genotype at rest ( − ) or after maximal exercise ( + ) . Skeletal and cardiac indicate skeletal and cardiac muscle , respectively .

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

e, Western blot analysis of LC3-I/II and p62 levels in indicated tissue from mice of indicated genotype at rest (−) or after maximal exercise (+). Skeletal and cardiac indicate skeletal and cardiac muscle, respectively.


f , Co - immunoprecipitation of beclin 1 with BCL2 in muscle tissue from mice of indicated genotype at rest ( − ) or after 30 min of exercise . WCL , whole cell lysates . NS , not significant . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 , one - way ANOVA for comparison between groups ; † P 0 . 001 , two - way ANOVA for comparison of magnitude of changes between different groups in mice of different genotypes .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Co-immunoprecipitation of beclin 1 with BCL2 in muscle tissue from mice of indicated genotype at rest (−) or after 30 min of exercise. WCL, whole cell lysates. NS, not significant. *P  0.05, **P  0.01, ***P  0.001, one-way ANOVA for comparison between groups; †P  0.001, two-way ANOVA for comparison of magnitude of changes between different groups in mice of different genotypes.


a , Maximal treadmill running distance for mice of indicated genotype . Data represent mean ± s . e . m . of 5 mice per group .

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a, Maximal treadmill running distance for mice of indicated genotype. Data represent mean ± s.e.m. of 5 mice per group.


b , Representative haematoxylin and eosin ( H E ) and periodic acid - Schiff ( PAS ) staining in tibialis anterior muscle sections from mice of indicated genotype . Scale bar , 20 µm .

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b, Representative haematoxylin and eosin (H E) and periodic acid-Schiff (PAS) staining in tibialis anterior muscle sections from mice of indicated genotype. Scale bar, 20 µm.


c , d , Plasma glucose ( c ) and insulin ( d ) levels in mice of indicated genotype at rest , after 80 min exercise ( ∼ 900 m ) , or maximal exercise . Data represent combined mean ± s . e . m . for 9 - 11 mice per group from three independent cohorts ; similar results were observed in each cohort .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Plasma glucose (c) and insulin (d) levels in mice of indicated genotype at rest, after 80 min exercise (∼900 m), or maximal exercise. Data represent combined mean ± s.e.m. for 9-11 mice per group from three independent cohorts; similar results were observed in each cohort.


e , Representative images of GLUT4immunofluorescence microscopy staining in vastus lateralis muscle of mice of indicated genotype before and after maximal exercise . Scale bar , 20 µm . For b and e , similar results were observed in 3 mice per group .

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e, Representative images of GLUT4immunofluorescence microscopy staining in vastus lateralis muscle of mice of indicated genotype before and after maximal exercise. Scale bar, 20 µm. For b and e, similar results were observed in 3 mice per group.


f , Western blot analysis of AMPK phosphorylation ( p - AMPK ( Thr172 ) ) in vastus lateralis muscle lysates from mice of indicated genotype at indicated time after exercise .

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

f, Western blot analysis of AMPK phosphorylation (p-AMPK (Thr172)) in vastus lateralis muscle lysates from mice of indicated genotype at indicated time after exercise.


g , Maximal treadmill running distance for mice of indicated genotype . Data represent mean ± s . e . m . of 4 - 6 male mice per group .

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g, Maximal treadmill running distance for mice of indicated genotype. Data represent mean ± s.e.m. of 4-6 male mice per group.


h , Soleus muscle14C - deoxyglucose uptake during treadmill exercise in mice of indicated genotype . Data represent mean ± s . e . m . of 3 mice per group . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , one - way ANOVA for comparison between groups ; † P < 0 . 05 , † † P < 0 . 01 , two - way ANOVA for comparison of magnitude of changes between different groups in mice of different genotypes . NS , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Soleus muscle14C-deoxyglucose uptake during treadmill exercise in mice of indicated genotype. Data represent mean ± s.e.m. of 3 mice per group. *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA for comparison between groups; †P<0.05, ††P<0.01, two-way ANOVA for comparison of magnitude of changes between different groups in mice of different genotypes. NS, not significant.


a , b , Oral glucose tolerance test ( OGTT ) before ( a , week 0 ) and after ( b , week 4 ) 4 weeks of HFD .

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a, b, Oral glucose tolerance test (OGTT) before (a, week 0) and after (b, week 4) 4 weeks of HFD.


c , d , OGTT ( c ) and serumleptin and adiponectin levels ( d ) after 8 weeks of daily exercise . For a - d , results represent the mean ± s . e . m . for 4 - 5 mice per group . E , exercise ; NE , no exercise ; RD , regular diet . * P 0 . 05 , * * P 0 . 01 , ( c , one - way ANOVA ; d , Wilcoxon rank test ) . NS , not significant .

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c, d, OGTT (c) and serumleptin and adiponectin levels (d) after 8 weeks of daily exercise. For a-d, results represent the mean ± s.e.m. for 4-5 mice per group. E, exercise; NE, no exercise; RD, regular diet . *P  0.05, **P  0.01, (c, one-way ANOVA; d, Wilcoxon rank test). NS, not significant.


Kainic acid mouse model of TLE : Daily EEG recordings obtained from the epileptogenic focus starting from one month after KA injection and spanning the period from 2 days before to 7 days after AAV - pDyn delivery ( 2x109 gp ) .

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Kainic acid mouse model of TLE: Daily EEG recordings obtained from the epileptogenic focus starting from one month after KA injection and spanning the period from 2 days before to 7 days after AAV-pDyn delivery (2x109 gp).


higher time resolution of the indicated section of the Day + 1 in ( B ) , representing a hippocampal paroxysmal discharge ( HPD ) .

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higher time resolution of the indicated section of the Day +1 in (B), representing a hippocampal paroxysmal discharge (HPD).


A generalized seizure is depicted in Fig . EV1 D and E : The characteristic EEG features of this model , secondary generalized seizures ( bars ) and HPDs ( lines ) were reduced in number and in duration by AAV - pDyn ( red ; n = 3 ( from day 60 ) ; 7 ( till day 30 ) per time - interval ) , but not by AAV - ΔGFP or after sham treatment ( blue ; n = 4 ( day 90 ) ; 5 ( day 30 and 60 ) ; 6 ( before day 30 ) , per time - interval ) . The relatively high variability of seizure frequencies and duration is typical for this model and reflects the findings in human mTLE .

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A generalized seizure is depicted in Fig. EV1 D and E: The characteristic EEG features of this model, secondary generalized seizures (bars) and HPDs (lines) were reduced in number and in duration by AAV-pDyn (red; n = 3 (from day 60); 7 (till day 30) per time-interval), but not by AAV-ΔGFP or after sham treatment (blue; n = 4 (day 90); 5 (day 30 and 60); 6 (before day 30), per time-interval). The relatively high variability of seizure frequencies and duration is typical for this model and reflects the findings in human mTLE.


Injection of norBNI ( 20 mg / kg ; i . p . ) results in a transient reappearance of HPDs immediately and 24 hrs after application . One week after norBNI application ( washout ) suppression of HPDs was re - established . Data obtained from 4 epileptic animals before ( black ) and after ( red ) AAV - pDyn delivery ( 2x109 gp ) are depicted . *

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

Injection of norBNI (20 mg/kg; i.p.) results in a transient reappearance of HPDs immediately and 24 hrs after application. One week after norBNI application (washout) suppression of HPDs was re-established. Data obtained from 4 epileptic animals before (black) and after (red) AAV-pDyn delivery (2x109 gp) are depicted. *


EEG recordings obtained from the ipsilateral dorsal hippocampus of rats after electrical self - sustained status epilepticus ( SSSE ) before ( black ) and after ( red ) AAV - pDyn delivery ( 4x109 gp ) are depicted . Spike trains with a frequency of at least 2 . 5 Hz induced by SSSE were markedly reduced by AAV - pDyn ( n = 4 ) .

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EEG recordings obtained from the ipsilateral dorsal hippocampus of rats after electrical self-sustained status epilepticus (SSSE) before (black) and after (red) AAV-pDyn delivery (4x109 gp) are depicted. Spike trains with a frequency of at least 2.5 Hz induced by SSSE were markedly reduced by AAV-pDyn (n=4).


Spatial learning and memory was tested on the Barnes maze . Quadrant 1 ( Q1 ) contains the target hole ( red ; A ) . Unilateral injection of AAV - pDyn ( B ) or AAV - eGFP ( C ) into naïve young adult mice ( 12 weeks age ) did not influence the performance as compared to naïve controls ( D ) when tested 4 weeks after AAV injection . Mice treated two weeks after KA with AAV - pDyn ( E , H ) performed equally to age - matched naïve controls ( D , G ) 2 weeks ( E ) and 5 . 5 months ( H ) after treatment . By contrast , animals treated two weeks after KA with AAV - ΔGFP ( F , I ) gradually lost this ability . Two - way ANOVA revealed significance between AAV - ΔGFP and AAV - pDyn treated groups for interaction 2 weeks ( p = 0 . 0349 ) and 5 . 5 months ( p = 0 . 0311 ) after AAV , respectively , at each time interval and quadrant ( p < 0 . 0001 ) 2 weeks after AAV . Comparison of AAV - pDyn injected with naïve animals revealed no differences .

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Spatial learning and memory was tested on the Barnes maze. Quadrant 1 (Q1) contains the target hole (red; A). Unilateral injection of AAV-pDyn (B) or AAV-eGFP (C) into naïve young adult mice (12 weeks age) did not influence the performance as compared to naïve controls (D) when tested 4 weeks after AAV injection. Mice treated two weeks after KA with AAV-pDyn (E,H) performed equally to age-matched naïve controls (D,G) 2 weeks (E) and 5.5 months (H) after treatment. By contrast, animals treated two weeks after KA with AAV-ΔGFP (F,I) gradually lost this ability. Two-way ANOVA revealed significance between AAV-ΔGFP and AAV-pDyn treated groups for interaction 2 weeks (p = 0.0349) and 5.5 months (p = 0.0311) after AAV, respectively, at each time interval and quadrant (p < 0.0001) 2 weeks after AAV. Comparison of AAV-pDyn injected with naïve animals revealed no differences.


epileptic mice , which were not able to learn the Barnes maze task one month after KA ( J , M ) , AAV - pDyn application restored spatial memory one ( K ) and 2 months ( L ) after treatment .

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epileptic mice, which were not able to learn the Barnes maze task one month after KA (J, M), AAV-pDyn application restored spatial memory one (K) and 2 months (L) after treatment.


epileptic mice , which were not able to learn the Barnes maze task one month after Treatment with AAV - ΔGFP did not result in improved memory ( N , O ) .

epileptic mice, which were not able to learn the Barnes maze task one month after Treatment with AAV-ΔGFP did not result in improved memory (N,O).


Double - immunofluorescence labeling is depicted for pDyn and NeuN ( A - C ) or GFAP ( D - F ) in the ipsilateral dentate gyrus of KA - treated and AAV - pDyn - injected mice . Enlarged view in ( F ) represents 15 x 30 µm .

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Double-immunofluorescence labeling is depicted for pDyn and NeuN (A-C) or GFAP (D-F) in the ipsilateral dentate gyrus of KA-treated and AAV-pDyn-injected mice. Enlarged view in (F) represents 15 x 30 µm.


: mature Dyn B content ( measured by a Dyn B specific EIA ) in the dorsal hippocampus of mice treated with KA ( blue symbols ) or KA and AAV - pDyn ( red symbols ) 1 . 5 ( open symbols ; n = 6 ) and 6 ( filled symbols ; n = 3 ) months after vector treatment . Naïve animals were age matched to the 1 . 5 months after AAV group . iH stands for ipsilateral hippocampus and cH for contralateral hippocampus . * = p < 0 . 05 ; * * = p < 0 . 01 ; paired t - test was used for comparison of ipsi - and contralateral hippocampi . Two - way ANOVA was used to compare Dyn levels between the early and late time interval .

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: mature Dyn B content (measured by a Dyn B specific EIA) in the dorsal hippocampus of mice treated with KA (blue symbols) or KA and AAV-pDyn (red symbols) 1.5 (open symbols; n = 6) and 6 (filled symbols; n = 3) months after vector treatment. Naïve animals were age matched to the 1.5 months after AAV group. iH stands for ipsilateral hippocampus and cH for contralateral hippocampus.* = p<0.05; ** = p<0.01; paired t-test was used for comparison of ipsi- and contralateral hippocampi. Two-way ANOVA was used to compare Dyn levels between the early and late time interval.


Mature Dyn B content in the CSF of mice treated with KA ( blue symbols ) or KA and AAV - pDyn ( red symbols ) 1 . 5 ( open symbols ; n = 6 ) and 7 ( filled symbols ; n = 4 ) months after vector treatment . * * = p < 0 . 01 ; one - way ANOVA with Dunnett post hoc

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Mature Dyn B content in the CSF of mice treated with KA (blue symbols) or KA and AAV-pDyn (red symbols) 1.5 (open symbols; n= 6) and 7 (filled symbols; n = 4) months after vector treatment. ** = p<0.01; one-way ANOVA with Dunnett post hoc test


The microdialysis probe ( red cross ) was placed in the dentate gyrus , the stimulation electrode ( black cross ) in the entorhinal cortex . : Dyn B was quantified by EIA in the dialysate collected during different stimulation intensities . The red line represents the detection limit of the EIA . Data represent mean ± standard error of the mean * = p < 0 . 05 ; * * = p < 0 . 01 ; n = 4 ; one - way ANOVA with Tukey post hoc test .

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The microdialysis probe (red cross) was placed in the dentate gyrus, the stimulation electrode (black cross) in the entorhinal cortex. : Dyn B was quantified by EIA in the dialysate collected during different stimulation intensities. The red line represents the detection limit of the EIA. Data represent mean ± standard error of the mean * = p<0.05; ** = p<0.01; n=4; one-way ANOVA with Tukey post hoc test.


: depicts representative traces under conditions of increased KCl . B : Same as A together with Dyn A / B and C : same as A under co - application of Dyn A / B with 5 ' - GNTI . D : Burst events and E : interictal spikes were recorded from area CA1 .

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: depicts representative traces under conditions of increased KCl. B: Same as A together with Dyn A/B and C: same as A under co-application of Dyn A/B with 5'-GNTI. D: Burst events and E: interictal spikes were recorded from area CA1.


Treatment with Dyn A / B markedly reduced the amplitude and number of bursts G : but not of interictal spikes . Each color represents the last 5 minutes of each application for one slice of 4 individual patients . Data sets for each patient represent mean ± SEM for amplitude or absolute number of events .

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Treatment with Dyn A/B markedly reduced the amplitude and number of bursts G: but not of interictal spikes. Each color represents the last 5 minutes of each application for one slice of 4 individual patients. Data sets for each patient represent mean ± SEM for amplitude or absolute number of events.


In - situ hybridization for AAV - derived pDyn mRNA at 10 weeks post AAV - pDyn delivery in the left dorsal hippocampus is depicted for a single brain at five levels between 1 . 1 ( left ) and 2 . 3 ( right ) mm from bregma . The probe specifically detects the codon - optimized sequence , therefore the contralateral side represents a control devoid of the respective mRNA .

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In-situ hybridization for AAV-derived pDyn mRNA at 10 weeks post AAV-pDyn delivery in the left dorsal hippocampus is depicted for a single brain at five levels between 1.1 (left) and 2.3 (right) mm from bregma. The probe specifically detects the codon-optimized sequence, therefore the contralateral side represents a control devoid of the respective mRNA.


At 10 weeks post AAV - pDyn delivery , markers of inflammation were analyzed as follows : Relative optical densities ( ROD ) were evaluated from film autoradiographs after in - situ hybridization for mRNAs of inflammatory markers ( Interleukin - 1β ( B ) , Interleukin - 6 ( C ) , Interleukin1a receptor agonist ( D ) , Tumor Necrosis Factor - α ( E ) and Nitric Oxide Synthase - 1 ( F ) ) in 3 principal sub - regions of the hippocampus in AAV - pDyn ( n = 7 ; red ) or AAV - eGFP ( n = 5 ; blue ) injected animals . GC = granule cell layer ; CA = cornu ammonis .

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At 10 weeks post AAV-pDyn delivery, markers of inflammation were analyzed as follows: Relative optical densities (ROD) were evaluated from film autoradiographs after in-situ hybridization for mRNAs of inflammatory markers (Interleukin-1β (B), Interleukin-6 (C), Interleukin1a receptor agonist (D), Tumor Necrosis Factor-α (E) and Nitric Oxide Synthase-1 (F)) in 3 principal sub-regions of the hippocampus in AAV-pDyn (n=7; red) or AAV-eGFP (n=5; blue) injected animals. GC = granule cell layer; CA = cornu ammonis.


( A ) N - glycan composition of PD - 1 protein expressed from 293T cells by ESI - MS analysis . N - glycans of representative peaks from ESI - MS were identified by MS / MS analysis . H , mannose ; N , N - acetylglucosamine ; F , fucose . Structural formulas : blue square , N - acetylglucosamine ; red triangle , fucose ; green circle , mannose ; yellow circle , galactose .

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(A) N-glycan composition of PD-1 protein expressed from 293T cells by ESI-MS analysis. N-glycans of representative peaks from ESI-MS were identified by MS/MS analysis. H, mannose; N, N-acetylglucosamine; F, fucose. Structural formulas: blue square, N-acetylglucosamine; red triangle, fucose; green circle, mannose; yellow circle, galactose.


( B , C ) Accelerated thermal stability of PD - 1 proteins obtained from 293T cells ( green line ) , insect cells ( pink line ) or E . coli ( blue line ) is characterized in all - in - one UNcle platform . Protein unfolding is observed as an increase in barycentric mean ( B ) while small particle formation of the samples is observed as an increase in SLS intensity at 266 nm ( C ) .

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(B,C) Accelerated thermal stability of PD-1 proteins obtained from 293T cells (green line), insect cells (pink line) or E. coli (blue line) is characterized in all-in-one UNcle platform. Protein unfolding is observed as an increase in barycentric mean (B) while small particle formation of the samples is observed as an increase in SLS intensity at 266 nm (C).


( D ) SPR analysis of the binding characteristics of camrelizumab with PD - 1 expressed in 293T cells , insect cells or E . coli cell , which enabled different levels of glycosylations . The binding affinity is presented as KD . The data presented here is a representative of two independent experiments with similar results .

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(D) SPR analysis of the binding characteristics of camrelizumab with PD-1 expressed in 293T cells, insect cells or E. coli cell, which enabled different levels of glycosylations. The binding affinity is presented as KD. The data presented here is a representative of two independent experiments with similar results.


( A ) Gel filtration profiles of PD - 1 ( orange ) , camrelizumab - scFv ( blue ) , and the PD - 1 / camrelizumab - scFv complex ( red ) are analyzed by size - exclusion chromatography as indicated . The SDS - PAGE analyses are shown with peak 1 of camrelizumab - scFv , peak 2 of camrelizumab - scFv / PD - 1 complex , and peak 3 of PD - 1 .

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(A) Gel filtration profiles of PD-1 (orange), camrelizumab-scFv (blue), and the PD-1/camrelizumab-scFv complex (red) are analyzed by size-exclusion chromatography as indicated. The SDS-PAGE analyses are shown with peak 1 of camrelizumab-scFv, peak 2 of camrelizumab-scFv/PD-1 complex, and peak 3 of PD-1.


( E ) SPR analysis of the binding of camrelizumab with N58A mutated PD - 1 expressed in 293T cells .

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(E) SPR analysis of the binding of camrelizumab with N58A mutated PD-1 expressed in 293T cells.


( F ) The SPR analysis of the binding of camrelizumab with WT ( upper ) or PNGase F treated PD - 1 proteins ( lower ) expressed in 293T cells .

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(F) The SPR analysis of the binding of camrelizumab with WT (upper) or PNGase F treated PD-1 proteins (lower) expressed in 293T cells.


( G The SPR analysis of the binding of nivolumab with WT ( upper ) or N58A mutated ( lower ) PD - 1 expressed in 293T

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(G The SPR analysis of the binding of nivolumab with WT (upper) or N58A mutated (lower) PD-1 expressed in 293T cells


H ) The SPR analysis of the binding of pembrolizumab with WT ( upper ) or N58A mutated ( lower ) PD - 1 proteins .

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H) The SPR analysis of the binding of pembrolizumab with WT (upper) or N58A mutated (lower) PD-1 proteins.


( A , B ) The blocking of the binding of WT PD - 1 - mFc ( A ) or N58A mutated PD - 1 - mFc ( B ) to PD - L1 transiently expressed on the surface of 293T cells are analyzed with varied concentrations of camrelizumab . The PD - L1 expressing 293T cells stained with PBS alone is used as negative controls , whereas the cells stained with WT or N58A mutated PD - 1 - mFc proteins are used as a positive control . The frequency of PD - 1 - mFc staining positive cells in PD - L1 - GFP positive cells is labeled in the upright corner . The reduced frequency of the PD - L1 - mFc - staining - positive subpopulation compared with positive control indicates the blockage of the PD - 1 / PD - L1 interaction . The density of events at a given position in the plot is color - coded , with red representing the highest number of events at that point in the graph , while yellow , green , and blue represent progressively lower event densities . The results presented here are representative of three independent experiments with similar results .

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(A,B) The blocking of the binding of WT PD-1-mFc (A) or N58A mutated PD-1-mFc (B) to PD-L1 transiently expressed on the surface of 293T cells are analyzed with varied concentrations of camrelizumab. The PD-L1 expressing 293T cells stained with PBS alone is used as negative controls, whereas the cells stained with WT or N58A mutated PD-1-mFc proteins are used as a positive control. The frequency of PD-1-mFc staining positive cells in PD-L1-GFP positive cells is labeled in the upright corner. The reduced frequency of the PD-L1-mFc-staining-positive subpopulation compared with positive control indicates the blockage of the PD-1/PD-L1 interaction. The density of events at a given position in the plot is color-coded, with red representing the highest number of events at that point in the graph, while yellow, green, and blue represent progressively lower event densities. The results presented here are representative of three independent experiments with similar results.


( A ) Confocal microscopy of GFP - TRIM33 - overexpressing mESCs and differentiated cells . Scale bar = 5 µm . ( B ) Line scans of GFP - TRIM33 and DAPI fluorescence imaging in each cell context at the position depicted by the white line ( from left to right ) . These line scans were processed by plot profile using image J software . ( C ) FRAP assays showing recovery time of normalized fluorescence signals for GFP - TRIM33 puncta in mESCs . Photobleaching occurs at t = 5s . Data are the mean ± S . D . of three biological experiments .

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(A) Confocal microscopy of GFP-TRIM33-overexpressing mESCs and differentiated cells. Scale bar = 5 µm. (B) Line scans of GFP-TRIM33 and DAPI fluorescence imaging in each cell context at the position depicted by the white line (from left to right). These line scans were processed by plot profile using image J software. (C) FRAP assays showing recovery time of normalized fluorescence signals for GFP-TRIM33 puncta in mESCs. Photobleaching occurs at t = 5s. Data are the mean ± S.D. of three biological experiments.


( D ) Sphericity analysis and 3D imaging by NIS - Elements 3D tools of TRIM33 puncta in live mESCs . Panels show the xz , xy , and yz planes , from left to right . Scale bar = 1 µm . Data information : All data are from three biological replicates .

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(D) Sphericity analysis and 3D imaging by NIS-Elements 3D tools of TRIM33 puncta in live mESCs. Panels show the xz, xy, and yz planes, from left to right. Scale bar = 1 µm. Data information: All data are from three biological replicates.


( F ) Western blot analysis of TRIM33 interactions with PML in lysates of HEK293T cells co - transfected with plasmids encoding Flag - PML and GFP - TRIM33 . GFP - TRIM33 was immunoprecipitated with anti - GFP affinity beads and immune complexes were detected using antibodies targeting Flag and GFP . Protein inputs were detected using antibodies against Flag and GFP in the same amount of cell lysates . ( n = 3 ) . Data information : All data are from three biological replicates .

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(F) Western blot analysis of TRIM33 interactions with PML in lysates of HEK293T cells co-transfected with plasmids encoding Flag-PML and GFP-TRIM33. GFP-TRIM33 was immunoprecipitated with anti-GFP affinity beads and immune complexes were detected using antibodies targeting Flag and GFP. Protein inputs were detected using antibodies against Flag and GFP in the same amount of cell lysates. (n = 3). Data information: All data are from three biological replicates.


( G ) Lysates from mESCs and differentiated cells were immunoprecipitated with anti - PML antibody , and protein complexes and inputs were analyzed by immunoblotting with antibodies against TRIM33 and PML . ( n = 3 ) . Data information : All data are from three biological replicates .

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(G) Lysates from mESCs and differentiated cells were immunoprecipitated with anti-PML antibody, and protein complexes and inputs were analyzed by immunoblotting with antibodies against TRIM33 and PML. (n = 3). Data information: All data are from three biological replicates.


( H ) Lysates from HEK293T cells co - transfected with plasmids encoding either full length Flag - TRIM33 or the Flag - TRIM33 - RBCC domain alone and full - length HA - PML or the HA - PML - RBCC domain alone immunoprecipitated with anti - Flag affinity beads . Protein complexes were detected using antibodies against Flag and HA . The protein inputs were detected using antibodies against Flag and HA in the same amount of cell lysates . ( n = 3 ) . Data information : All data are from three biological replicates .

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(H) Lysates from HEK293T cells co-transfected with plasmids encoding either full length Flag-TRIM33 or the Flag-TRIM33-RBCC domain alone and full-length HA-PML or the HA-PML-RBCC domain alone immunoprecipitated with anti-Flag affinity beads. Protein complexes were detected using antibodies against Flag and HA. The protein inputs were detected using antibodies against Flag and HA in the same amount of cell lysates. (n = 3). Data information: All data are from three biological replicates.


( A ) Representative images of immunofluorescence staining of PML in WT and Pml null mESCs . Scale bar = 5 µm .

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(A) Representative images of immunofluorescence staining of PML in WT and Pml null mESCs. Scale bar = 5 µm.


( B ) Representative confocal microscopy images of mESCs overexpressing mCherry - PML or GFP - PML ( top ) . Representative confocal microscopy images of mESCs overexpressing mCherry - EV or GFP - EV ( bottom ) . Scale bar = 5 µm . ( C ) FRAP assays quantifying normalized fluorescence recovery of GFP - PML signal in mESCs . Photobleaching occurs at t = 5s . Data are the mean ± S . D . of four biological experiments .

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(B) Representative confocal microscopy images of mESCs overexpressing mCherry-PML or GFP-PML (top). Representative confocal microscopy images of mESCs overexpressing mCherry-EV or GFP-EV (bottom). Scale bar = 5 µm. (C) FRAP assays quantifying normalized fluorescence recovery of GFP-PML signal in mESCs. Photobleaching occurs at t = 5s. Data are the mean ± S.D. of four biological experiments.


( D , G , I , Representative images of co - localization assays with mCherry - PML and GFP - TRIM33 in mESCs ( D ) , differentiated cells ( G ) , NaAsO2 - treated mESCs ( 200 µM for 2 hr ) ( I ) Scale bar = 5 µm . ( E , H , J Line scans of GFP - TRIM33 and mCherry - PML fluorescence imaging in each cell context at the position depicted by the white line ( from left to right ) . These line scans were processed by plot profile using image J software . ( F ) Column graph of the numbers of GFP - TRIM33 puncta and mCherry - PML NBs in mESCs .

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(D, G, I, Representative images of co-localization assays with mCherry-PML and GFP-TRIM33 in mESCs (D), differentiated cells (G), NaAsO2-treated mESCs (200 µM for 2 hr) (I) Scale bar = 5 µm. (E, H, J Line scans of GFP-TRIM33 and mCherry-PML fluorescence imaging in each cell context at the position depicted by the white line (from left to right). These line scans were processed by plot profile using image J software. (F) Column graph of the numbers of GFP-TRIM33 puncta and mCherry-PML NBs in mESCs.


K ) Representative images of co - localization assays with GFP - TRIM33 in mESCs Pml null ( K ) . Scale bar = 5 µm . L ) Line scans of GFP - TRIM33 fluorescence imaging in each cell context at the position depicted by the white line ( from left to right ) . These line scans were processed by plot profile using image J software . ( M ) Column graph of the numbers of GFP - TRIM33 puncta and DAPI in mESCs .

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K) Representative images of co-localization assays with GFP-TRIM33 in mESCs Pml null (K). Scale bar = 5 µm. L) Line scans of GFP-TRIM33 fluorescence imaging in each cell context at the position depicted by the white line (from left to right). These line scans were processed by plot profile using image J software. (M) Column graph of the numbers of GFP-TRIM33 puncta and DAPI in mESCs.


( N ) Scatter plot of the numbers of GFP - TRIM33 puncta ( per cell ) in WT and Pml KO mESCs . ( n = 20 ) . ( O ) Scatter plot of the volume of GFP - TRIM33 puncta ( per cell ) in WT and Pml KO mESCs . ( n = 20 ) . Data information : In panels ( N , O , 20 individual puncta with three biological experiments were analyzed . Significant differences were determined by t - tests . ( * p < 0 . 05 , * * * * p < 0 . 0001 ) .

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(N) Scatter plot of the numbers of GFP-TRIM33 puncta (per cell) in WT and Pml KO mESCs. (n = 20). (O) Scatter plot of the volume of GFP-TRIM33 puncta (per cell) in WT and Pml KO mESCs. (n = 20). Data information: In panels (N, O, 20 individual puncta with three biological experiments were analyzed. Significant differences were determined by t-tests. (*p < 0.05, ****p < 0.0001).


( P ) Representative images of immunofluorescence staining of PML in WT and Trim33 null mESCs . Scale bar = 10 µm . ( Q ) Scatter plot of the numbers of PML NBs ( per cell ) in WT and Trim33 KO mESCs . ( n = 20 ) . ( R ) Scatter plot of the proportion of PML NBs ( per cell ) in WT and Trim33 KO mESCs . ( n = 20 ) . Data information : In panels Q and R ) , 20 individual puncta with three biological experiments were analyzed . Significant differences were determined by t - tests . ( * p < 0 . 05 , * * * * p < 0 . 0001 ) .

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(P) Representative images of immunofluorescence staining of PML in WT and Trim33 null mESCs. Scale bar = 10 µm. (Q) Scatter plot of the numbers of PML NBs (per cell) in WT and Trim33 KO mESCs. (n = 20). (R) Scatter plot of the proportion of PML NBs (per cell) in WT and Trim33 KO mESCs. (n = 20). Data information: In panels Q and R), 20 individual puncta with three biological experiments were analyzed. Significant differences were determined by t-tests. (*p < 0.05, ****p < 0.0001).


a ) COS - 7 cells expressing EGFP - tagged wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin exon 1 ( green ) for 48 h stained for endogenous mTOR ( red ) show mTOR in mutant aggregates .

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a) COS-7 cells expressing EGFP-tagged wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin exon 1 (green) for 48 h stained for endogenous mTOR (red) show mTOR in mutant aggregates.


( b ) Double - labeling immunohistochemistry of huntingtin ( EM48 ; green ) and mTOR ( red ) in brain tissue of mice expressing mutant huntingtin .

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(b) Double-labeling immunohistochemistry of huntingtin (EM48; green) and mTOR (red) in brain tissue of mice expressing mutant huntingtin.


( c ) Immunohistochemistry of brain tissue from individuals with grade III Huntington disease or unaffected controls using antibody to mTOR .

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(c) Immunohistochemistry of brain tissue from individuals with grade III Huntington disease or unaffected controls using antibody to mTOR.


( d ) Western blot of lysates from stable inducible PC12 cells 48 h after expression of wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin showing mTOR in the stacking gel ( mTOR insoluble ) in cells expressing mutant huntingtin .

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(d) Western blot of lysates from stable inducible PC12 cells 48 h after expression of wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin showing mTOR in the stacking gel (mTOR insoluble) in cells expressing mutant huntingtin.


( e ) High - molecular - mass aggregates of mTOR in both PC12 ( i ) and COS - 7 ( ii ) cells expressing mutant ( Gln74 ; Q74 ) huntingtin . Blots of cell lysates from PC12 cells were probed with antibodies to phosphorylated mTOR ( Ser2448 ; p - mTOR ; iii ) and to GFP ( iv ) ; both showed insoluble material in the stack .

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(e) High-molecular-mass aggregates of mTOR in both PC12 (i) and COS-7 (ii) cells expressing mutant (Gln74; Q74) huntingtin. Blots of cell lysates from PC12 cells were probed with antibodies to phosphorylated mTOR (Ser2448; p-mTOR; iii) and to GFP (iv); both showed insoluble material in the stack.


( f ) Cell lysates from stable inducible PC12 cells and transiently transfected COS - 7 cells expressing wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin for 48 h were immunoprecipitated using antibody to GFP and immunoblotted with antibody to mTOR . Uninduced PC12 cells with mutant huntingtin ( UI ) and untransfected COS - 7 cells ( UT ) were controls .

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(f) Cell lysates from stable inducible PC12 cells and transiently transfected COS-7 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin for 48 h were immunoprecipitated using antibody to GFP and immunoblotted with antibody to mTOR. Uninduced PC12 cells with mutant huntingtin (UI) and untransfected COS-7 cells (UT) were controls.


( g ) Brain lysates from mice expressing mutant huntingtin ( Tg ) or control ( Cont ) littermates were immunoprecipitated with antibody to mTOR and immunodetected with an antibody recognizing the polyglutamine tract ( IC2 ) . Htt ,

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(g) Brain lysates from mice expressing mutant huntingtin (Tg) or control (Cont) littermates were immunoprecipitated with antibody to mTOR and immunodetected with an antibody recognizing the polyglutamine tract (IC2). Htt, huntingtin


( a ) Reduced levels of soluble mTOR ( as a function of tubulin ) in stable inducible PC12 cells expressing mutant ( Gln74 ; Q74 ) huntingtin compared with those expressing wild - type ( Gln23 ; Q23 ) huntingtin ( four independent experiments ; P 0 . 0001 ; unpaired t - test ) .

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(a) Reduced levels of soluble mTOR (as a function of tubulin) in stable inducible PC12 cells expressing mutant (Gln74; Q74) huntingtin compared with those expressing wild-type (Gln23; Q23) huntingtin (four independent experiments; P 0.0001; unpaired t-test).


( b ) Reduced levels of soluble mTOR in brain lysates from mice expressing mutant huntingtin ( M1 , M2 ; n = 9 ) compared with age - matched wild - type littermates ( C1 , C2 ; n = 9 ; P 0 . 001 , unpaired t - test ) .

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(b) Reduced levels of soluble mTOR in brain lysates from mice expressing mutant huntingtin (M1, M2; n = 9) compared with age-matched wild-type littermates (C1, C2; n = 9; P 0.001, unpaired t-test).


( c ) COS - 7 cells transiently transfected with wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin ( green ) labeled for endogenous 4E - BP1 ( red ) .

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(c) COS-7 cells transiently transfected with wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin (green) labeled for endogenous 4E-BP1 (red).


( d ) Immunohistochemistry of brain tissue from individuals with grade III Huntington disease showed 4E - BP1 in intranuclear inclusions . No 4E - BP1 - stained inclusions were seen in controls ( data not shown ) . (

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(d) Immunohistochemistry of brain tissue from individuals with grade III Huntington disease showed 4E-BP1 in intranuclear inclusions. No 4E-BP1-stained inclusions were seen in controls (data not shown). (


( e ) Western blot of cell lysates from stable inducible PC12 cells transfected with wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin shows that cells expressing mutant huntingtin had 31 % ( ± 4 . 3 % s . e . ) less phosphorylated S6K1 ( top ) relative to total S6K1 ( bottom ) than cells expressing wild - type huntingtin . ( f ) Similarly , cells expressing mutant ( Gln74 ; Q74 ) huntingtin had 30 % ( ± 4 . 5 % s . d . ) less phosphorylated 4E - BP1 ( top ) relative to total 4E - BP1 ( bottom ) than cells expressing wild - type ( Gln23 ; Q23 ) huntingtin . Expression of wild - type and mutant huntingtin was induced for 48 h ( e , f ) . ( g ) Cells expressing mutant ( Gln74 ; Q74 ) huntingtin also had lower levels of phosphorylated forms of transiently transfected FLAG - tagged 4E - BP1 than cells expressing wild - type ( Gln23 ; Q23 ) huntingtin . The multiple bands in 4E - BP1 ( f , g ) are due to its different phosphorylated species .

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(e) Western blot of cell lysates from stable inducible PC12 cells transfected with wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin shows that cells expressing mutant huntingtin had 31% (± 4.3% s.e.) less phosphorylated S6K1 (top) relative to total S6K1 (bottom) than cells expressing wild-type huntingtin. (f) Similarly, cells expressing mutant (Gln74; Q74) huntingtin had 30% (± 4.5% s.d.) less phosphorylated 4E-BP1 (top) relative to total 4E-BP1 (bottom) than cells expressing wild-type (Gln23; Q23) huntingtin. Expression of wild-type and mutant huntingtin was induced for 48 h (e,f). (g) Cells expressing mutant (Gln74; Q74) huntingtin also had lower levels of phosphorylated forms of transiently transfected FLAG-tagged 4E-BP1 than cells expressing wild-type (Gln23; Q23) huntingtin. The multiple bands in 4E-BP1 (f,g) are due to its different phosphorylated species.


( a ) COS - 7 cells expressing wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin ( green ) were labeled for S6 phosphorylated at Ser235 and Ser236 ( red ) . Percentages of cells expressing wild - type or mutant huntingtin that did ( + agg ) or did not ( − agg ) contain aggregates were scored for negative staining for phosphorylated S6 ( p - S6 ) .

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(a) COS-7 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin (green) were labeled for S6 phosphorylated at Ser235 and Ser236 (red). Percentages of cells expressing wild-type or mutant huntingtin that did (+agg) or did not (−agg) contain aggregates were scored for negative staining for phosphorylated S6 (p-S6).


( b ) Brain sections from control ( WT ) and mice expressing mutant huntingtin ( HD ) were stained for phosphorylated S6 ( red ) and with antibody to huntingtin ( EM48 ; green ) .

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(b) Brain sections from control (WT) and mice expressing mutant huntingtin (HD) were stained for phosphorylated S6 (red) and with antibody to huntingtin (EM48; green).


( c , d ) Impaired mTOR - dependent TOP translation in cells expressing mutant huntingtin . ( c ) PC12 cells expressing wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin were transfected with luciferase constructs containing 5 ′ untranslated sequence with ( + ) or without ( − ) a TOP sequence along with β - gal . Each bar represents the difference between the luciferase activities in the presence versus the absence of rapamycin . Data shown are from one representative experiment ( of three ) done in sextuplicate ( error bars represent s . e . ; raw data are given in Supplementary Fig . 4 online ) . ( d ) COS - 7 cells were cotransfected with wild - type or mutant huntingtin , a nontypical TOP or control luciferase vector and β - gal . The experiment was done and analyzed as in c ( raw data are given in Supplementary Fig . 4 online ) ( e - g ) Relevance of mTOR sequestration to other polyglutamine expansion

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(c,d) Impaired mTOR-dependent TOP translation in cells expressing mutant huntingtin. (c) PC12 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin were transfected with luciferase constructs containing 5′ untranslated sequence with (+) or without (−) a TOP sequence along with β-gal. Each bar represents the difference between the luciferase activities in the presence versus the absence of rapamycin. Data shown are from one representative experiment (of three) done in sextuplicate (error bars represent s.e.; raw data are given in Supplementary Fig. 4 online). (d) COS-7 cells were cotransfected with wild-type or mutant huntingtin, a nontypical TOP or control luciferase vector and β-gal. The experiment was done and analyzed as in c (raw data are given in Supplementary Fig. 4 online) (e-g) Relevance of mTOR sequestration to other polyglutamine expansion diseases


( e ) COS - 7 cells expressing Xpress - tagged ataxin - 1 with 92 polyglutamine repeats ( Q92 ) for 48 h were doubly labeled with antibodies to Xpress ( green ) and to mTOR ( red ) .

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(e) COS-7 cells expressing Xpress-tagged ataxin-1 with 92 polyglutamine repeats (Q92) for 48 h were doubly labeled with antibodies to Xpress (green) and to mTOR (red).


( f ) mTOR immunohistochemistry in brains of individuals with dentatorubral - pallidoluysian atrophy ( DRPLA ) and spinocerebellar ataxia types 2 , 3 and 7 ( SCA2 , SCA3 and SCA7 ) .

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(f) mTOR immunohistochemistry in brains of individuals with dentatorubral-pallidoluysian atrophy (DRPLA) and spinocerebellar ataxia types 2, 3 and 7 (SCA2, SCA3 and SCA7).


( g ) Staining for phosphorylated S6 ( red ) in COS - 7 cells transiently transfected with mutant ( ataxin Q92 ; green ) or wild - type ( ataxin Q2 ) ataxin - 1 constructs . Results were significant and similar to those in a . Arrows in a , b , e and g show cells containing aggregates .

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(g) Staining for phosphorylated S6 (red) in COS-7 cells transiently transfected with mutant (ataxin Q92; green) or wild-type (ataxin Q2) ataxin-1 constructs. Results were significant and similar to those in a. Arrows in a, b, e and g show cells containing aggregates.


( a ) LC3 - II levels , which closely correlate with autophagic activity , were higher in mutant lines ( expressing Gln74 huntingtin ; Q74 ; lanes 3 and 4 ) than in wild - type lines ( expressing Gln23 huntingtin ; Q23 ; lanes 1 and 2 ) . Actin was used as a loading control . Data are shown for two independent clonal lines expressing mutant or wild - type huntingtin . Quantification of the band intensity is shown in the right panel .

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(a) LC3-II levels, which closely correlate with autophagic activity, were higher in mutant lines (expressing Gln74 huntingtin; Q74; lanes 3 and 4) than in wild-type lines (expressing Gln23 huntingtin; Q23; lanes 1 and 2). Actin was used as a loading control. Data are shown for two independent clonal lines expressing mutant or wild-type huntingtin. Quantification of the band intensity is shown in the right panel.


( b ) Immunofluorescence analysis of COS - 7 cells expressing wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin with antibody to LC3 showed more autophagic vacuoles in cells expressing mutant huntingtin that had aggregates ( arrow ) . COS - 7 cells without the primary antibody ( negative ) and COS - 7 cells without any treatment ( untreated ) stained for LC3 are also shown . The numbers of EGFP - positive cells expressing wild - type or mutant huntingtin with ( + agg ) and without ( − agg ) aggregates that had higher numbers of autophagic vacuoles ( > 15 - 20 vesicles per cell ) is shown in the graph . COS - 7 cells grown in starvation medium ( starvation ) or treated with rapamycin ( Rap ) for 4 h to induce autophagy had more autophagic vacuoles ( arrows ) .

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(b) Immunofluorescence analysis of COS-7 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin with antibody to LC3 showed more autophagic vacuoles in cells expressing mutant huntingtin that had aggregates (arrow). COS-7 cells without the primary antibody (negative) and COS-7 cells without any treatment (untreated) stained for LC3 are also shown. The numbers of EGFP-positive cells expressing wild-type or mutant huntingtin with (+agg) and without (−agg) aggregates that had higher numbers of autophagic vacuoles (>15-20 vesicles per cell) is shown in the graph. COS-7 cells grown in starvation medium (starvation) or treated with rapamycin (Rap) for 4 h to induce autophagy had more autophagic vacuoles (arrows).


( a ) Activation of mTOR activity with rheb increases aggregation and toxicity of mutant ( Gln74 ; Q74 ) huntingtin . COS - 7 cells were cotransfected with a mammalian expression vector encoding rheb ( or empty vector control ) and mutant huntingtin for 48 h at a 3 : 1 mass ratio ( rheb or empty vector to huntingtin ) to ensure that almost all cells expressing huntingtin also expressed rheb . The percentages of EGFP - positive cells with aggregates or apoptotic nuclear morphology ( as described previously ) were markedly higher when rheb was overexpressed . Control cells express mutant huntingtin ( green ) without rheb . The middle panels show images captured at the same comparatively low gain to distinguish aggregates from soluble EGFP . The left panels show the same fields at a similar higher gain to indicate the total number of EGFP - positive cells ( this shows that the effect seen in the middle panels is not due to loss of cells ) . Nuclear staining with DAPI ( blue ) is shown in the right panels . * * * P 0 . 0001 . Error bars represent s . e . for a representative triplicate experiment .

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(a) Activation of mTOR activity with rheb increases aggregation and toxicity of mutant (Gln74; Q74) huntingtin. COS-7 cells were cotransfected with a mammalian expression vector encoding rheb (or empty vector control) and mutant huntingtin for 48 h at a 3:1 mass ratio (rheb or empty vector to huntingtin) to ensure that almost all cells expressing huntingtin also expressed rheb. The percentages of EGFP-positive cells with aggregates or apoptotic nuclear morphology (as described previously) were markedly higher when rheb was overexpressed. Control cells express mutant huntingtin (green) without rheb. The middle panels show images captured at the same comparatively low gain to distinguish aggregates from soluble EGFP. The left panels show the same fields at a similar higher gain to indicate the total number of EGFP-positive cells (this shows that the effect seen in the middle panels is not due to loss of cells). Nuclear staining with DAPI (blue) is shown in the right panels. ***P 0.0001. Error bars represent s.e. for a representative triplicate experiment.


( b , c ) Treatment with rapamycin rescues huntingtin - induced degeneration in flies 2 d after eclosion . Photographs of ommatidia of flies expressing mutant ( Gln120 ; Q120 ) huntingtin treated with DMSO or rapamycin from the larval stage into adulthood . More rhabdomeres are visible in the ommatidia of flies treated with rapamycin . This effect is significant ( P 0 . 0001 ; Mann - Whitney U test ) . At 2 d after eclosion , there is no reduction in the number of visible rhabdomeres in wild - type untreated flies ( data not shown ) .

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(b,c) Treatment with rapamycin rescues huntingtin-induced degeneration in flies 2 d after eclosion. Photographs of ommatidia of flies expressing mutant (Gln120; Q120) huntingtin treated with DMSO or rapamycin from the larval stage into adulthood. More rhabdomeres are visible in the ommatidia of flies treated with rapamycin. This effect is significant (P 0.0001; Mann-Whitney U test). At 2 d after eclosion, there is no reduction in the number of visible rhabdomeres in wild-type untreated flies (data not shown).


Open bars , mice treated with a placebo ; filled bars , mice treated with CCI - 779 . ( a - c ) Behavioral tests in mice expressing mutant huntingtin after treatment at 16 weeks ( CCI - 779 , n = 14 ; placebo , n = 14 ) , 18 weeks ( CCI - 779 , n = 10 ; placebo , n = 9 ) , 20 weeks ( CCI - 779 , n = 9 ; placebo , n = 7 ) and 22 weeks ( CCI - 779 , n = 5 ; placebo , n = 6 ) of age . Scores are explained in Methods . Data for all time points are given in Supplementary Figure 13 online . ( a ) Tremors . Overall effect from all treated time points : P = 0 . 0006 , odds ratio ( OR ) = 0 . 21 , 95 % confidence interval ( 95 % c . i . ) = 0 . 098 - 0 . 45 . 16 weeks , P = 0 . 01 ; 18 weeks , P = 0 . 08 ; 20 weeks , P = 0 . 14 ; 22 weeks , P = 0 . 07 . ( b ) Wire maneuver . Overall effect from all treated time points : P = 0 . 02 , OR = 0 . 51 , 95 % c . i . = 0 . 29 - 0 . 92 . 16 weeks , P = 0 . 29 ; 18 weeks , P = 0 . 40 ; 20 weeks , P = 0 . 003 ; 22 weeks , P = 0 . 008 . ( c ) Grip strength . Overall effect from all treated time points : P 0 . 0001 OR = 12 . 73 , 95 % c . i . = 4 . 35 - 37 . 2 . 16 weeks , P = 0 . 1827 ; 18 weeks , P = 0 . 0412 ; 20 weeks , P = 0 . 0021 ; 22 weeks , P = 0 . 0285 .

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

Open bars, mice treated with a placebo; filled bars, mice treated with CCI-779. (a-c) Behavioral tests in mice expressing mutant huntingtin after treatment at 16 weeks (CCI-779, n = 14; placebo, n = 14), 18 weeks (CCI-779, n = 10; placebo, n = 9), 20 weeks (CCI-779, n = 9; placebo, n = 7) and 22 weeks (CCI-779, n = 5; placebo, n = 6) of age. Scores are explained in Methods. Data for all time points are given in Supplementary Figure 13 online. (a) Tremors. Overall effect from all treated time points: P = 0.0006, odds ratio (OR) = 0.21, 95% confidence interval (95% c.i.) = 0.098-0.45. 16 weeks, P = 0.01; 18 weeks, P = 0.08; 20 weeks, P = 0.14; 22 weeks, P = 0.07. (b) Wire maneuver. Overall effect from all treated time points: P = 0.02, OR = 0.51, 95% c.i. = 0.29-0.92. 16 weeks, P = 0.29; 18 weeks, P = 0.40; 20 weeks, P = 0.003; 22 weeks, P = 0.008. (c) Grip strength. Overall effect from all treated time points: P 0.0001 OR = 12.73, 95% c.i. = 4.35-37.2. 16 weeks, P = 0.1827; 18 weeks, P = 0.0412; 20 weeks, P = 0.0021; 22 weeks, P = 0.0285.


( d ) Accelerating rotarod tests in mice expressing mutant huntingtin after treatment at 4 weeks , ( CCI - 779 , n = 16 ; placebo , n = 17 ) , 14 weeks ( CCI - 779 , n = 11 ; placebo , n = 10 ) , 16 weeks ( CCI - 779 , n = 14 ; placebo , n = 14 ) and 18 weeks ( CCI - 779 , n = 10 ; placebo , n = 9 ) of age . Overall effect from all treated time points : P = 0 . 035 . 4 weeks , P = 0 . 227 ; 14 weeks , P = 0 . 012 ; 16 weeks , P = 0 . 0025 ; 18 weeks , P = 0 . 057 . CCI - 779 had no discernable effects on the performance of nontransgenic mice on any of these behavioral tests ( data not shown ) . There was no difference in the male : female ratios of the groups treated with placebo or with CCI - 779 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Accelerating rotarod tests in mice expressing mutant huntingtin after treatment at 4 weeks, (CCI-779, n = 16; placebo, n = 17), 14 weeks (CCI-779, n = 11; placebo, n = 10), 16 weeks (CCI-779, n = 14; placebo, n = 14) and 18 weeks (CCI-779, n = 10; placebo, n = 9) of age. Overall effect from all treated time points: P = 0.035. 4 weeks, P = 0.227; 14 weeks, P = 0.012; 16 weeks, P = 0.0025; 18 weeks, P = 0.057. CCI-779 had no discernable effects on the performance of nontransgenic mice on any of these behavioral tests (data not shown). There was no difference in the male:female ratios of the groups treated with placebo or with CCI-779.


( a ) Weights of mice expressing mutant huntingtin ( Tg ) and nontransgenic ( NT ) littermates treated with CCI - 779 or with a placebo . ( b ) Brain weights of mice expressing mutant huntingtin treated with CCI - 779 ( black bars ) or with a placebo ( white bars ) and nontransgenic untreated littermates ( gray bars ) at 8 weeks ( CCI - 779 , n = 3 ; placebo , n = 3 ; nontransgenic , n = 7 ; CCI - 779 versus placebo , P = 0 . 023 ) , 12 weeks ( CCI - 779 , n = 2 ; placebo , n = 3 ; nontransgenic , n = 5 ; CCI - 779 versus placebo , P = 0 . 90 ) and 16 weeks ( CCI - 779 , n = 2 ; placebo , n = 3 ; nontransgenic , n = 2 ; CCI - 779 versus placebo , P = 0 . 22 ) of age . ( c ) Ratio of brain weight to body weight of mice expressing mutant huntingtin treated with CCI - 779 ( black bars ) or with a placebo ( white bars ) and nontransgenic untreated littermates ( gray bars ) at 8 weeks ( CCI - 779 , n = 3 ; placebo , n = 3 ; nontransgenic , n = 7 ; CCI - 779 versus placebo , P = 0 . 06 ) , 12 weeks ( CCI - 779 , n = 2 ; placebo , n = 3 ; nontransgenic , n = 5 ; CCI - 779 versus placebo , P = 0 . 38 ) and 16 weeks ( CCI - 779 , n = 2 ; placebo , n = 3 ; nontransgenic , n = 2 ; CCI - 779 versus placebo , P = 0 . 56 ) of age .

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(a) Weights of mice expressing mutant huntingtin (Tg) and nontransgenic (NT) littermates treated with CCI-779 or with a placebo. (b) Brain weights of mice expressing mutant huntingtin treated with CCI-779 (black bars) or with a placebo (white bars) and nontransgenic untreated littermates (gray bars) at 8 weeks (CCI-779, n = 3; placebo, n = 3; nontransgenic, n = 7; CCI-779 versus placebo, P = 0.023), 12 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 5; CCI-779 versus placebo, P = 0.90) and 16 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 2; CCI-779 versus placebo, P = 0.22) of age. (c) Ratio of brain weight to body weight of mice expressing mutant huntingtin treated with CCI-779 (black bars) or with a placebo (white bars) and nontransgenic untreated littermates (gray bars) at 8 weeks (CCI-779, n = 3; placebo, n = 3; nontransgenic, n = 7; CCI-779 versus placebo, P = 0.06), 12 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 5; CCI-779 versus placebo, P = 0.38) and 16 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 2; CCI-779 versus placebo, P = 0.56) of age.


( a ) CCI - 779 reduces aggregate load in mice expressing mutant huntingtin . Peroxidase immunohistochemistry of brains of mice expressing mutant huntingtin at 16 weeks of age . Mice treated with CCI - 779 had fewer aggregates per unit area in the striatum than mice treated with a placebo . Overall cell staining in the tissues of mice treated with CCI - 779 was weaker than in those treated with a placebo and in nontransgenic untreated littermates ( data not shown ) . Aggregates , when visible , were smaller in mice treated with CCI - 779 than in those treated with a placebo , as apparent in the higher magnification images of single cells with aggregate ( bottom panels ) . Six coronal sections per mouse were analyzed ; these corresponded to the bregma ± 1 mm . We counted aggregates in five different high - power fields ( 60 × objective ) per slide on each side of the brain , sampling fields from the dorsal to the ventral part of the striatum just underneath the external capsule . The striata of mice treated with CCI - 779 had 70 % fewer aggregates than the striata of mice treated with a placebo ( P = 0 . 0001 , two - tailed t - test ) .

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(a) CCI-779 reduces aggregate load in mice expressing mutant huntingtin. Peroxidase immunohistochemistry of brains of mice expressing mutant huntingtin at 16 weeks of age. Mice treated with CCI-779 had fewer aggregates per unit area in the striatum than mice treated with a placebo. Overall cell staining in the tissues of mice treated with CCI-779 was weaker than in those treated with a placebo and in nontransgenic untreated littermates (data not shown). Aggregates, when visible, were smaller in mice treated with CCI-779 than in those treated with a placebo, as apparent in the higher magnification images of single cells with aggregate (bottom panels). Six coronal sections per mouse were analyzed; these corresponded to the bregma ± 1 mm. We counted aggregates in five different high-power fields (60× objective) per slide on each side of the brain, sampling fields from the dorsal to the ventral part of the striatum just underneath the external capsule. The striata of mice treated with CCI-779 had 70% fewer aggregates than the striata of mice treated with a placebo (P = 0.0001, two-tailed t-test).


( b ) CCI - 779 blocks mTOR signaling in vivo . Immunohistochemistry of mice with Huntington disease at 16 weeks of age with antibody to phosphorylated S6 , in the cortical area and striatum . Levels of phosphorylated S6 were lower in mice treated with CCI - 779 than in those treated with a placebo .

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(b) CCI-779 blocks mTOR signaling in vivo. Immunohistochemistry of mice with Huntington disease at 16 weeks of age with antibody to phosphorylated S6, in the cortical area and striatum. Levels of phosphorylated S6 were lower in mice treated with CCI-779 than in those treated with a placebo.


( A ) Western blot of autophagy proteins LC3 and p62 at various time points after infection of human NSCs with strains MR766 or Paraiba . ( B - C ) Quantitative representation of LC3 - II and p62 protein levels during ZIKV infection at indicated time points . Mean ± SEM , n = 3 biological replicates .

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(A) Western blot of autophagy proteins LC3 and p62 at various time points after infection of human NSCs with strains MR766 or Paraiba. (B-C) Quantitative representation of LC3-II and p62 protein levels during ZIKV infection at indicated time points. Mean ± SEM, n=3 biological replicates.


( D ) GFP - LC3 puncta of mock - and ZIKV - infected hNSCs . Scale bar = 100 µm .

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(D) GFP-LC3 puncta of mock- and ZIKV-infected hNSCs. Scale bar = 100 µm.


( E ) Tandem EGFP - LC3 and mCherry punctae co - localization in control and ZIKV infected hNSC . Scale bar = 100 µm , Boxed magnified images with scale bar = 50

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(E) Tandem EGFP-LC3 and mCherry punctae co-localization in control and ZIKV infected hNSC. Scale bar = 100 µm, Boxed magnified images with scale bar = 50 µm


( F ) Number of GFP - LC3 puncta in MR766 and Paraiba infected hNSCs , n = > 50 cells . Box plots show the mean and the smallest ; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group ; and solid horizontal line indicates median . * * * p < 0 . 001 by Student ' s t test .

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(F) Number of GFP-LC3 puncta in MR766 and Paraiba infected hNSCs, n =>50 cells. Box plots show the mean and the smallest; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group; and solid horizontal line indicates median. ***p < 0.001 by Student's t test.


( G ) RT - qPCR of ZIKV RNA in hNSCs incubated with vehicle or 100 nM rapamycin , 20 μM chloroquine , 10 nM bafilomycin A1 , or 5 mM 3 - methyladenine for 24 h . Mean ± SEM of 3 biological replicates . * p < 0 . 05 , * * * p < 0 . 001 by Student ' s t test .

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(G) RT-qPCR of ZIKV RNA in hNSCs incubated with vehicle or 100 nM rapamycin, 20 μM chloroquine, 10 nM bafilomycin A1, or 5 mM 3-methyladenine for 24 h. Mean ± SEM of 3 biological replicates. *p < 0.05, ***p < 0.001 by Student's t test.


( H ) GFP - LC3 puncta in ZIKV with or without rapamycin and 3 - MA treated hNSC cells . Cells were treated with or without rapamycin ( 100 nM ) or 3 - methyladenine ( 5 μM ) . Box plots show the mean and the smallest ; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group ; and solid horizontal line indicates median . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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(H) GFP-LC3 puncta in ZIKV with or without rapamycin and 3-MA treated hNSC cells. Cells were treated with or without rapamycin (100 nM) or 3-methyladenine (5 μM). Box plots show the mean and the smallest; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group; and solid horizontal line indicates median. *p < 0.05, **p < 0.01, ***p < 0.001 by Student's t test.


( I ) RT - qPCR of ZIKV RNA in infected hNSCs transfected with siRNAs targeting LC3 , BECN or ATG9A 48 h post infection . Mean ± SEM of 3 biological replicates . * p < 0 . 05 by Student ' s t test .

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(I) RT-qPCR of ZIKV RNA in infected hNSCs transfected with siRNAs targeting LC3, BECN or ATG9A 48 h post infection. Mean ± SEM of 3 biological replicates. *p < 0.05 by Student's t test.


( L ) Transmission electron microscopy of hNSCs infected by ZIKV MR766 and Paraiba strains . Mitochondria ( MT ) , endoplasmic reticulum ( ER ) , Golgi bodies ( GB ) , ribosomes ( RS ) , phagophores ( PH ) , lipid droplet ( LD ) . Scale bars , first row , 1 μm ( left ) , 500 nm ( control right ) ; 200 nm ( Right ) .

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(L) Transmission electron microscopy of hNSCs infected by ZIKV MR766 and Paraiba strains. Mitochondria (MT), endoplasmic reticulum (ER), Golgi bodies (GB), ribosomes (RS), phagophores (PH), lipid droplet (LD). Scale bars, first row, 1 μm (left), 500 nm (control right); 200 nm (Right).


Immunostaining and quantification of neural progenitor marker SOX2 ( red ) and ZIKV envelope flavivirus group antigen ( ZIKVE , green ) in the SVZ of uninfected or ZIKV Paraiba - infected wild - type mice treated with vehicle , rapamycin , or chloroquine at 6 days post - infection . n = 10 sections / group , mean ± SEM , * p < 0 . 05 , * * p < 0 . 001 . Nuclei were stained with DAPI ( blue ) . Right - most column shows enlargements of the boxed regions . STR = striatum , CC = cortical cortex , GCL = granular cell layer , LV = lateral ventricle , SGZ = subgranular zone , SVZ = subventricular zone .

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Immunostaining and quantification of neural progenitor marker SOX2 (red) and ZIKV envelope flavivirus group antigen (ZIKVE, green) in the SVZ of uninfected or ZIKV Paraiba-infected wild-type mice treated with vehicle, rapamycin, or chloroquine at 6 days post-infection. n=10 sections/group, mean ± SEM, *p < 0.05, **p < 0.001. Nuclei were stained with DAPI (blue). Right-most column shows enlargements of the boxed regions. STR = striatum, CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, SVZ = subventricular zone.


Immunostaining and quantification of neural progenitor marker SOX2 ( red ) and ZIKV envelope flavivirus group antigen ( ZIKVE , green ) in the hippocampus of uninfected or ZIKV Paraiba - infected wild - type mice treated with vehicle , rapamycin , or chloroquine at 6 days post - infection . n = 10 sections / group , mean ± SEM , * p < 0 . 05 , * * p < 0 . 001 . Nuclei were stained with DAPI ( blue ) . Right - most column shows enlargements of the boxed regions . STR = striatum , CC = cortical cortex , GCL = granular cell layer , LV = lateral ventricle , SGZ = subgranular zone , SVZ = subventricular zone .

Immunostaining and quantification of neural progenitor marker SOX2 (red) and ZIKV envelope flavivirus group antigen (ZIKVE, green) in the hippocampus of uninfected or ZIKV Paraiba-infected wild-type mice treated with vehicle, rapamycin, or chloroquine at 6 days post-infection. n=10 sections/group, mean ± SEM, *p < 0.05, **p < 0.001. Nuclei were stained with DAPI (blue). Right-most column shows enlargements of the boxed regions. STR = striatum, CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, SVZ = subventricular zone.


( E ) qRT - PCR for ZIKV RNA expression in brain , mean ± SEM , n = 3 biological replicates , * p < 0 . 05 , * * p < 0 . 001 by Student ' s t test .

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(E) qRT-PCR for ZIKV RNA expression in brain, mean ± SEM, n=3 biological replicates, *p < 0.05, **p < 0.001 by Student's t test.


( F - G ) Western blot for autophagy protein LC3 - II , p62 and quantification normalized with GAPDH , mean ± SEM , n = 3 biological replicates , * p < 0 . 05 , * * p < 0 . 001 by Student ' s t test .

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(F-G) Western blot for autophagy protein LC3-II, p62 and quantification normalized with GAPDH, mean ± SEM, n=3 biological replicates, *p < 0.05, **p < 0.001 by Student's t test.


( B ) RT - qPCR analysis of FA pathway genes at 48 h after ZIKV infection of hNSCs . Mean ± SEM of biological triplicates . * p < 0 . 05 , * * p < 0 . 01 by Student ' s t test .

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(B) RT-qPCR analysis of FA pathway genes at 48 h after ZIKV infection of hNSCs. Mean ± SEM of biological triplicates. *p < 0.05, **p < 0.01 by Student's t test.


( C - D ) RT - qPCR analysis of ZIKV mRNA 48 h after ZIKV infection of HeLa cells overexpressing FANCC ( C ) or FANCC - specific siRNA ( D ) . Mean ± SEM of biological triplicates . * * p < 0 . 01 by Student ' s t test .

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(C-D) RT-qPCR analysis of ZIKV mRNA 48 h after ZIKV infection of HeLa cells overexpressing FANCC (C) or FANCC-specific siRNA (D). Mean ± SEM of biological triplicates. **p < 0.01 by Student's t test.


( E ) Flow cytometry analysis of ZIKVE expression in mock - infected ( red ) , ZIKV - infected ( blue ) , ZIKV - infected siFANCC - transfected ( orange ) , and ZIKV - infected siE2F4 - transfected ( green ) hNSCs at 48 h post - infection .

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(E) Flow cytometry analysis of ZIKVE expression in mock-infected (red), ZIKV-infected (blue), ZIKV-infected siFANCC-transfected (orange), and ZIKV-infected siE2F4-transfected (green) hNSCs at 48 h post-infection.


( F ) ZIKV NS1 and FANCC immunostaining 48 h after ZIKV infection of hNSCs . Scale bar = 100 µm .

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(F) ZIKV NS1 and FANCC immunostaining 48 h after ZIKV infection of hNSCs. Scale bar = 100 µm.


( G ) ZIKV NS1 expression 48 h after ZIKV infection of hNSCs expressing control siRNA ( NTC ) or siFANCC - targeting siRNA . Scale bar = 100 µm . ( H - I ) ZIKV NS1 expression 48 h after ZIKV infection of hNSCs expressing control ( NTC ) or siE2F4 - targeting siRNA . Mean ± SEM of six imaging fields of biological triplicates , * * p < 0 . 01 by Student ' s t test , Scale bar = 100 µm ,

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(G) ZIKV NS1 expression 48 h after ZIKV infection of hNSCs expressing control siRNA (NTC) or siFANCC-targeting siRNA. Scale bar = 100 µm. (H-I) ZIKV NS1 expression 48 h after ZIKV infection of hNSCs expressing control (NTC) or siE2F4-targeting siRNA. Mean ± SEM of six imaging fields of biological triplicates, **p < 0.01 by Student's t test, Scale bar = 100 µm,


( J ) Immunoblot of macroautophagy ( p62 , LC3I / LC3II ) and selective autophagy ( FANCC , TOMM20 ) proteins 24 h after overexpression of FLAG - tagged ZIKV NS1 , NS2B , NS3 , NS4A , NS4B and NS5 in 293FT cells .

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(J) Immunoblot of macroautophagy (p62, LC3I/LC3II) and selective autophagy (FANCC, TOMM20) proteins 24 h after overexpression of FLAG-tagged ZIKV NS1, NS2B, NS3, NS4A, NS4B and NS5 in 293FT cells.


( K ) Analysis of E2F4 ChIP - seq datasets ( Encode ) showing direct chromatin - binding interaction between E2F4 and FA pathway genes in HeLa ( GEO : GSM935365 , Farnham lab ) , K562 ( GEO : GSM935600 , Farnham lab ) , and MCF10A ( GEO : GSM : 935400 , Struhl lab ) cell lines . H3K9ac and H3K4me3 datasets identify promoter regions . The genomic region is displayed in log scale with the scale range identified in each panel ( 0 - 30 ) . The summed interaction ( Sum ) across all three cell types is represented as a heatmap .

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(K) Analysis of E2F4 ChIP-seq datasets (Encode) showing direct chromatin-binding interaction between E2F4 and FA pathway genes in HeLa (GEO: GSM935365, Farnham lab), K562 (GEO: GSM935600, Farnham lab), and MCF10A (GEO: GSM: 935400, Struhl lab) cell lines. H3K9ac and H3K4me3 datasets identify promoter regions. The genomic region is displayed in log scale with the scale range identified in each panel (0-30). The summed interaction (Sum) across all three cell types is represented as a heatmap.


( L - M ) Heatmaps of RT - qPCR analysis of FA pathway genes ( K ) or essential selective autophagy genes ( L ) in hNSCs 48 h after transfection with control ( NTC ) or E2F4 - targeting siRNAs or after infection with ZIKV MR766 and Paraiba strains . Color patterns in heat map indicate as red showing the highest expression and blue showing the lowest expression of genes .

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

(L-M) Heatmaps of RT-qPCR analysis of FA pathway genes (K) or essential selective autophagy genes (L) in hNSCs 48 h after transfection with control (NTC) or E2F4-targeting siRNAs or after infection with ZIKV MR766 and Paraiba strains. Color patterns in heat map indicate as red showing the highest expression and blue showing the lowest expression of genes.


( A ) Representative images of mock - or ZIKV - infected human neurospheres expressing control ( NTC ) or FANCC - targeting siRNAs at 1 - 4 days post - infection . Scale bar = 250 µm . ( B - C ) Violin plot ( B ) and box plot ( C ) of relative size of neurospheres treated as described in ( A ) and measured on day 4 ( B ) or days 1 - 4 ( C ) . Box plots show the mean and the smallest ; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group ; and solid horizontal line indicates median . * * * 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative images of mock- or ZIKV-infected human neurospheres expressing control (NTC) or FANCC-targeting siRNAs at 1-4 days post-infection. Scale bar = 250 µm. (B-C) Violin plot (B) and box plot (C) of relative size of neurospheres treated as described in (A) and measured on day 4 (B) or days 1-4 (C). Box plots show the mean and the smallest; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group; and solid horizontal line indicates median. ***p < 0.001 by Student's t test.