( c ) Immunoblot analysis of the autophagy related protein LC3 in its cytosolic form ( LC3 - I ) and its autophagosome membrane - associated form ( LC3 - II ) in total lysates of CD4 + T cells obtained from L2A - cKO mice or their control littermates and then left unstimulated ( Rest ) or activated for 24 h with anti - CD3 and anti - CD218 ( Act ) , cultured for the final 4 h in the presence ( + ) or absence ( − ) of NH4Cl and leupeptin ( NH4Cl + Leup ) .

O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O B-SUBCELLULAR O O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O

(c) Immunoblot analysis of the autophagy related protein LC3 in its cytosolic form (LC3-I) and its autophagosome membrane-associated form (LC3-II) in total lysates of CD4+ T cells obtained from L2A-cKO mice or their control littermates and then left unstimulated (Rest) or activated for 24 h with anti-CD3 and anti-CD218 (Act), cultured for the final 4 h in the presence (+) or absence (−) of NH4Cl and leupeptin (NH4Cl+Leup).


( d ) Spleens from an L2A - cKO mouse and its control littermate ( left ) , and flow cytometry of peripheral T cells from such mice ( right ) . Numbers in quadrants ( right ) indicate percent CD4 − CD8 + T cells ( top left ) or CD4 + CD8 − T cells ( bottom right ) .

O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O

(d) Spleens from an L2A-cKO mouse and its control littermate (left), and flow cytometry of peripheral T cells from such mice (right). Numbers in quadrants (right) indicate percent CD4−CD8+ T cells (top left) or CD4+CD8− T cells (bottom right).


( e , f ) Proliferation ( e ) and IL - 2 production ( f ) by CD4 + T cells isolated from the spleen of L2A - cKO mice and their control littermates and stimulated ex vivo for 48 h with anti - CD3 and anti - CD28 ( assessed as in Fig . 2c ( e ) or Fig . 2b ( f ) ) . * P = 0 . 0062 ( e ) or 0 . 006 ( f ) ( t - test ) .

O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-TISSUE O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e,f) Proliferation (e) and IL-2 production (f) by CD4+ T cells isolated from the spleen of L2A-cKO mice and their control littermates and stimulated ex vivo for 48 h with anti-CD3 and anti-CD28 (assessed as in Fig. 2c (e) or Fig. 2b (f)). *P = 0.0062 (e) or 0.006 (f) (t-test).


( g ) Immunoblot analysis of LAMP - 2A , Itch and RCAN1 in total lysates of CD4 + T cells obtained from L2A - cKO mice ( n = 3 ( KO1 , KO2 and KO3 ) ) and their control littermates ( n = 3 ) and activated for 24 h as in e , f . * , nonspecific band .

O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O 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) Immunoblot analysis of LAMP-2A, Itch and RCAN1 in total lysates of CD4+ T cells obtained from L2A-cKO mice (n = 3 (KO1, KO2 and KO3)) and their control littermates (n = 3) and activated for 24 h as in e,f. *, nonspecific band.


( h ) Quantitative PCR analysis of Rcan1 and Itch mRNA in CD4 + T cells isolated from L2A - cKO mice and their control littermates and activated for 24 h as in e , f ; results are presented relative to those of resting cells .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O B-ORGANISM O 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) Quantitative PCR analysis of Rcan1 and Itch mRNA in CD4+ T cells isolated from L2A-cKO mice and their control littermates and activated for 24 h as in e,f; results are presented relative to those of resting cells.


( i , j ) Recall responses to OVA ( 323 - 339 ) in draining lymph nodes isolated from L2A - cKO mice and their control littermates after immunization with OVA ( 323 - 339 ) , assessed as proliferation ( i ) and IL - 2 production ( j ) ( as in e , f ) . * P = 0 . 0054 ( i ) or 0 . 0052 ( j ) ( t - test ) .

O O O O O O O O B-GENEPROD O O O O O O B-TISSUE I-TISSUE I-TISSUE O O B-GENEPROD O O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(i,j) Recall responses to OVA(323-339) in draining lymph nodes isolated from L2A-cKO mice and their control littermates after immunization with OVA(323-339), assessed as proliferation (i) and IL-2 production (j) (as in e,f). *P = 0.0054 (i) or 0.0052 (j) (t-test).


( k , l ) Recall responses to L . monocytogenes in CD4 + T cells isolated from L2A - cKO mice and their control littermates ( n = 6 per group ) previously uninfected ( UI ) or previously infected with L . monocytogenes ( Inf ) , assessed as IFN - γ production ( k ) and proliferation ( l ) ( as in e , f ) . * P = 0 . 032 ( k ) or 0 . 02 ( l ) ( t - test ) .

O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(k,l) Recall responses to L. monocytogenes in CD4+ T cells isolated from L2A-cKO mice and their control littermates (n = 6 per group) previously uninfected (UI) or previously infected with L. monocytogenes (Inf), assessed as IFN-γ production (k) and proliferation (l) (as in e,f). *P = 0.032 (k) or 0.02 (l) (t-test).


( m ) L . monocytogenes in spleen and liver of L2A - cKO mice and their control littermates ( n = 3 per genotype ) immunized with L . monocytogenes and then reinfected with a high dose of L . monocytogenes . CFU , colony - forming units . * P = 0 . 008 and * * P = 0 . 001 ( t - test ) .

O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-TISSUE O B-TISSUE O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(m) L. monocytogenes in spleen and liver of L2A-cKO mice and their control littermates (n = 3 per genotype) immunized with L. monocytogenes and then reinfected with a high dose of L. monocytogenes. CFU, colony-forming units. *P = 0.008 and **P = 0.001 (t-test).


( a ) Immunoblot analysis of LAMP - 2A in CD4 + T cells obtained from 4 - month - old or 22 - month old mice , then left resting or stimulated for 24 h with anti - CD3 plus anti - CD28 .

O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O

(a) Immunoblot analysis of LAMP-2A in CD4+ T cells obtained from 4-month-old or 22-month old mice, then left resting or stimulated for 24 h with anti-CD3 plus anti-CD28.


( b ) Immunoblot analysis of RCAN1 and Itch in CD4 + T cells obtained from 4 - month - old or 22 - month old mice ( n = 3 per group ) , then stimulated for 24 h as in a ( left ) , and quantification of protein in 22 - month old mice , presented relative to that of 4 - month - old mice , set as 100 % . * P = 0 . 044 ( t - test ) .

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-ORGANISM O O O O O 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-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

(b) Immunoblot analysis of RCAN1 and Itch in CD4+ T cells obtained from 4-month-old or 22-month old mice (n = 3 per group), then stimulated for 24 h as in a (left), and quantification of protein in 22-month old mice, presented relative to that of 4-month-old mice, set as 100%. *P = 0.044 (t-test).


( c ) Immunoblot analysis of LAMP - 2A in total lysates of naive and memory CD4 + T cells left resting or activated for 24 h as in a .

O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O

(c) Immunoblot analysis of LAMP-2A in total lysates of naive and memory CD4+ T cells left resting or activated for 24 h as in a.


( d ) Immunoblot analysis ( bottom ) of LAMP - 2A in total lysates of naive and memory CD4 + T cells obtained from 4 - or 22 - month - old mice and left resting ( R ) or activated for 24 h as in a ( A ) ; above , quantification from results below ( n = 3 mice per group ) . * P = 0 . 009 ( t - test ) .

O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-ORGANISM O O O O 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-ORGANISM O O O O O O O O O O O O O O O O

(d) Immunoblot analysis (bottom) of LAMP-2A in total lysates of naive and memory CD4+ T cells obtained from 4- or 22-month-old mice and left resting (R) or activated for 24 h as in a (A); above, quantification from results below (n = 3 mice per group). *P = 0.009 (t-test).


( e ) Electrophoretic mobility - shift assay of the binding of NFAT ( N ) and free probe ( FP ) in nuclear extracts of CD4 + T cells isolated from 3 - , 12 - or 22 - month - old mice and activated for 4 h as in a .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O

(e) Electrophoretic mobility-shift assay of the binding of NFAT (N) and free probe (FP) in nuclear extracts of CD4+ T cells isolated from 3-, 12- or 22-month-old mice and activated for 4 h as in a.


( f ) ROS production in CD4 + T cells isolated from 4 - or 22 - month - old mice ( n = 3 per group ) and left resting or activated for 12 h as in a , assessed by flow cytometry and presented as the mean fluorescence intensity ( MFI ) of a fluorogenic probe for oxidative stress ( CellROX Green ) .

O O O B-SMALL_MOLECULE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O

(f) ROS production in CD4+ T cells isolated from 4- or 22-month-old mice (n = 3 per group) and left resting or activated for 12 h as in a, assessed by flow cytometry and presented as the mean fluorescence intensity (MFI) of a fluorogenic probe for oxidative stress (CellROX Green).


( g - i ) Immunoblot analysis of LAMP - 2A ( g ) and analysis of the proliferation ( h ) and IL - 2 production ( i ) of TH1 cells obtained from 4 - or 22 - month - old mice and transduced with retrovirus expressing GFP alone ( RV - GFP ) or human LAMP - 2A and GFP ( RV - LAMP2A ) , sorted by GFP expression , cultured for 2 weeks and then stimulated for 24 - 48 h as in a ( assessed as in Fig . 2c ( h ) or Fig . 2b ( i ) ) . * P 0 . 05 ( analysis of variance with Tukey ' s post - test ) .

O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O B-ORGANISM O O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-i) Immunoblot analysis of LAMP-2A (g) and analysis of the proliferation (h) and IL-2 production (i) of TH1 cells obtained from 4- or 22-month-old mice and transduced with retrovirus expressing GFP alone (RV-GFP) or human LAMP-2A and GFP (RV-LAMP2A), sorted by GFP expression, cultured for 2 weeks and then stimulated for 24-48 h as in a (assessed as in Fig. 2c (h) or Fig. 2b (i)). *P 0.05 (analysis of variance with Tukey's post-test).


( j ) Immunoblot analysis of LAMP - 2A in total lysates of CD4 + T cells obtained from two human donors ( A and B ) and left resting or activated for 24 or 48 h as in a ( above lanes ) .

O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O

(j) Immunoblot analysis of LAMP-2A in total lysates of CD4+ T cells obtained from two human donors (A and B) and left resting or activated for 24 or 48 h as in a (above lanes).


( k ) Real - time PCR analysis of LAMP2A mRNA in naive and memory CD4 + T cells obtained from young human donors ( 26 ± 2 years of age ; n = 3 ) or old human donors ( 72 ± 6 years of age ; n = 6 ) and activated for 24 h as in a ; results were calculated by the change - in - cycle - threshold method ( 2 − ΔCt ) relative to those of the gene encoding β - actin . * P = 0 . 047 and * * P = 0 . 0033 ( Mann - Whitney ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O

(k) Real-time PCR analysis of LAMP2A mRNA in naive and memory CD4+ T cells obtained from young human donors (26 ± 2 years of age; n = 3) or old human donors (72 ± 6 years of age; n = 6) and activated for 24 h as in a; results were calculated by the change-in-cycle-threshold method (2−ΔCt) relative to those of the gene encoding β-actin. *P = 0.047 and **P = 0.0033 (Mann-Whitney).


( B ) The degree of polymerisation of Z α1 - antitrypsin after 72 hours at 37 ° C , as determined by an end - point immunoassay using the 2C1 monoclonal antibody , in varying concentrations of compound ( shown in panel A ) . Modification of the phenyl and indole heterocycle of GSK425 ( pIC50 6 . 5 ) resulted in an ~ 100 fold increase in potency and the discovery of the 2 - oxindole GSK716 ( pIC50 8 . 3 ) . Data presented as mean ± SD , n = 2 ( GSK425 ) and n = 25 ( GSK716 ) .

O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O

(B) The degree of polymerisation of Z α1-antitrypsin after 72 hours at 37°C, as determined by an end-point immunoassay using the 2C1 monoclonal antibody, in varying concentrations of compound (shown in panel A). Modification of the phenyl and indole heterocycle of GSK425 (pIC50 6.5) resulted in an ~100 fold increase in potency and the discovery of the 2-oxindole GSK716 (pIC50 8.3). Data presented as mean ± SD, n = 2 (GSK425) and n = 25 (GSK716).


( C ) GSK716 binds to Z α1 - antitrypsin with a high affinity mean pKD of 8 . 5 ± 0 . 12 ( n = 18 ) as determined by a competition binding assay with a fluorescently labelled derivative . There was a 50 - fold lower affinity for plasma - purified wild - type M α1 - antitrypsin , with a mean pKD of 6 . 8 ± 0 . 18 ( n = 10 ) . Data presented as mean ± SD .

O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-TISSUE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O 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) GSK716 binds to Z α1-antitrypsin with a high affinity mean pKD of 8.5 ± 0.12 (n = 18) as determined by a competition binding assay with a fluorescently labelled derivative. There was a 50-fold lower affinity for plasma-purified wild-type M α1-antitrypsin, with a mean pKD of 6.8 ± 0.18 (n = 10). Data presented as mean ± SD.


( D ) The compound bound to monomeric but not polymeric Z α1 - antitrypsin ( Z α1 - AT ) as reported by fluorescence polarisation of an Alexa - 488 - labelled variant of GSK716 .

O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O

(D) The compound bound to monomeric but not polymeric Z α1-antitrypsin (Z α1-AT) as reported by fluorescence polarisation of an Alexa-488-labelled variant of GSK716.


( E ) Representative curves reporting the interaction of different concentrations of GSK716 with Z α1 - antitrypsin based on changes in intrinsic tryptophan fluorescence . Based on the concentration dependence ( inset ) , the second - order rate constant of association was found to be 4 . 1 x 104 M - 1 s - 1 . ( F ) The association of GSK716 with M α1 - antitrypsin , giving a second - order rate constant of 2 . 1 x 102 M - 1 s - 1 .

O O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O

(E) Representative curves reporting the interaction of different concentrations of GSK716 with Z α1-antitrypsin based on changes in intrinsic tryptophan fluorescence. Based on the concentration dependence (inset), the second-order rate constant of association was found to be 4.1 x 104 M-1 s-1. (F) The association of GSK716 with M α1-antitrypsin, giving a second-order rate constant of 2.1 x 102 M-1 s-1.


( G ) CHO - K1 Tet - On cells expressing Z α1 - antitrypsin were induced with doxycycline ( 0 . 5μg / ml ) for 48 h . Cells were incubated with 10μM GSK716 ( or 0 . 1 % v / v DMSO for the control ) during the induction . Culture media containing either the experimental compound or DMSO were changed every 24 h . After the induction , cells were labelled for 10 minutes with 35S Met / Cys and chased at the indicated times . Culture media were collected and cells lysed in 1 % v / v NP - 40 buffer . Intracellular fractions and culture media from cells expressing Z α1 - antitrypsin were immunoprecipitated either with a mAb against total α1 - antitrypsin ( 3C11 ) or with a polymer - specific mAb ( 2C1 ) . Samples were resolved by 4 - 12 % w / v acrylamide SDS - PAGE and detected by autoradiography . ( H ) The graphs show the effect of GSK716 on intracellular and extracellular Z α1 - antitrypsin ( mean ± SEM , n = 2 )

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

(G) CHO-K1 Tet-On cells expressing Z α1-antitrypsin were induced with doxycycline (0.5μg/ml) for 48 h. Cells were incubated with 10μM GSK716 (or 0.1% v/v DMSO for the control) during the induction. Culture media containing either the experimental compound or DMSO were changed every 24 h. After the induction, cells were labelled for 10 minutes with 35S Met/Cys and chased at the indicated times. Culture media were collected and cells lysed in 1% v/v NP-40 buffer. Intracellular fractions and culture media from cells expressing Z α1-antitrypsin were immunoprecipitated either with a mAb against total α1-antitrypsin (3C11) or with a polymer-specific mAb (2C1). Samples were resolved by 4-12% w/v acrylamide SDS-PAGE and detected by autoradiography. (H) The graphs show the effect of GSK716 on intracellular and extracellular Z α1-antitrypsin (mean±SEM, n=2)


( B ) M ( above ) and Z ( below ) α1 - antitrypsin , at a concentration of 0 . 2mg / ml in PBS + 5 % v / v glycerol , were heated at a range of temperatures between 48 ° C - 65 ° C for 4 hours in the presence and absence of GSK716 , as indicated . The oligomerisation state was determined by non - denaturing PAGE electrophoresis ; the lane denoted ' M ' contains the unheated monomeric control .

O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O 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-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

(B) M (above) and Z (below) α1-antitrypsin, at a concentration of 0.2mg/ml in PBS+5% v/v glycerol, were heated at a range of temperatures between 48°C-65°C for 4 hours in the presence and absence of GSK716, as indicated. The oligomerisation state was determined by non-denaturing PAGE electrophoresis; the lane denoted 'M' contains the unheated monomeric control.


( C ) M and Z α1 - antitrypsin were subjected to equilibrium unfolding into different concentrations of guanidine hydrochloride ( gdnhcl ) in the presence and absence of GSK716 , with bis - ANS dye added to report the presence of the unfolded intermediate . The normalised fluorescence intensity data were fitted with an equation describing a three - state unfolding curve . Values shown are the mean of three independent experiments and the error bars represent ± SEM .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) M and Z α1-antitrypsin were subjected to equilibrium unfolding into different concentrations of guanidine hydrochloride (gdnhcl) in the presence and absence of GSK716, with bis-ANS dye added to report the presence of the unfolded intermediate. The normalised fluorescence intensity data were fitted with an equation describing a three-state unfolding curve. Values shown are the mean of three independent experiments and the error bars represent ±SEM.


( D ) Top panel , the association of GSK716 induced a marked quenching and blue - shift of the α1 - antitrypsin intrinsic tryptophan fluorescence spectrum with respect to unbound protein ( inset graph ) . The association of 10µM GSK716 with four α1 - antitrypsin variants that vary in their propensity to polymerise , ranging from inert ( M ) , to mild ( S ) , moderate ( Baghdad denoted ' B ' ) and severe ( Z ) . Representative progress curves of the change in intrinsic tryptophan fluorescence at 330nm for 0 . 2mg / ml protein are shown . Bottom panel , the half - time of association calculated from three such independent experiments ( error bars are ± SEM ) show a correspondence with the polymerisation propensity of the four variants . The change in Z α1 - antitrypsin fluorescence was faster than the dead - time of the apparatus ( ~ 10s ) and scaling was with reference to unbound intensity .

O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

(D) Top panel, the association of GSK716 induced a marked quenching and blue-shift of the α1-antitrypsin intrinsic tryptophan fluorescence spectrum with respect to unbound protein (inset graph). The association of 10µM GSK716 with four α1-antitrypsin variants that vary in their propensity to polymerise, ranging from inert (M), to mild (S), moderate (Baghdad denoted 'B') and severe (Z). Representative progress curves of the change in intrinsic tryptophan fluorescence at 330nm for 0.2mg/ml protein are shown. Bottom panel, the half-time of association calculated from three such independent experiments (error bars are ±SEM) show a correspondence with the polymerisation propensity of the four variants. The change in Z α1-antitrypsin fluorescence was faster than the dead-time of the apparatus (~10s) and scaling was with reference to unbound intensity.


( B ) M α1 - antitrypsin was incubated with different molar ratios of chymotrypsin and resolved by SDS - PAGE . The position of covalent α1 - antitrypsin - chymotrypsin complex ( cpx ) , native ( nat ) and cleaved ( clv ) is shown .

O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY O O O O O O

(B) M α1-antitrypsin was incubated with different molar ratios of chymotrypsin and resolved by SDS-PAGE. The position of covalent α1-antitrypsin-chymotrypsin complex (cpx), native (nat) and cleaved (clv) is shown.


( C ) The inhibitory activity of α1 - antichymotrypsin against chymotrypsin ( ach + chym ) , neuroserpin against trypsin ( neus + tryp ) , and antithrombin against thrombin ( atiii + thrombin ) was determined in the presence and absence of GSK716 . Error bars reflect ± SD of two independent experiments .

O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

(C) The inhibitory activity of α1-antichymotrypsin against chymotrypsin (ach+chym), neuroserpin against trypsin (neus+tryp), and antithrombin against thrombin (atiii+thrombin) was determined in the presence and absence of GSK716. Error bars reflect ±SD of two independent experiments.


Z α1 - antitrypsin transgenic animals were dosed with 10 , 30 or 100 mg / kg GSK716 three times a day . Total concentrations of GSK716 were determined by LC - MS / MS ( Table 2 ) . ( A ) 100mg / kg GSK716 resulted in a mean 7 - fold increase in circulating monomeric Z α1 - antitrypsin levels demonstrating robust target engagement in the liver . The observed steady state total and free compound levels of GSK716 in the transgenic Z α1 - antitrypsin mouse were well - predicted by the in silico PK model built on : ( 1 ) in vitro metabolic clearance data , ( 2 ) plasma protein binding data , ( 3 ) in vivo PK data from wild type mice and ( 4 ) a term comprising a 5μM circulating sink for drug with an affinity of 1 . 5 nM , representing the Z α1 - antitrypsin within blood . The target free drug concentration was selected based on the observed potency in the in vitro secretion assays in which the total drug approximates to the free drug in the assay . Interestingly , 30mg / kg and 10mg / kg groups also gave significant , dose - dependent increases in circulating Z α1 - antitrypsin despite free concentrations being below the cellular EC50 for secretion for much or all of the dosing period . Significance at p < 0 . 05 by a student ' s t test is denoted by * , shown as pairwise comparisons ( each animal is compared with itself , treated vs pre - treatment ) and between groups ( compound treated vs vehicle ) , with n = 7 , 8 and 8 per group for day 6 dose level 10 , 30 and 100mg / Kg respectively ; day 15 , n = 10 ; day 21 , n = 5 . Data presented as mean ± SD .

B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O B-TISSUE O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O 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-TISSUE O O O O O O O O O O O O O O B-ORGANISM 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-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Z α1-antitrypsin transgenic animals were dosed with 10, 30 or 100 mg/kg GSK716 three times a day. Total concentrations of GSK716 were determined by LC-MS/MS (Table 2). (A) 100mg/kg GSK716 resulted in a mean 7-fold increase in circulating monomeric Z α1-antitrypsin levels demonstrating robust target engagement in the liver. The observed steady state total and free compound levels of GSK716 in the transgenic Z α1-antitrypsin mouse were well-predicted by the in silico PK model built on: (1) in vitro metabolic clearance data, (2) plasma protein binding data, (3) in vivo PK data from wild type mice and (4) a term comprising a 5μM circulating sink for drug with an affinity of 1.5 nM, representing the Z α1-antitrypsin within blood. The target free drug concentration was selected based on the observed potency in the in vitro secretion assays in which the total drug approximates to the free drug in the assay. Interestingly, 30mg/kg and 10mg/kg groups also gave significant, dose-dependent increases in circulating Z α1-antitrypsin despite free concentrations being below the cellular EC50 for secretion for much or all of the dosing period. Significance at p<0.05 by a student's t test is denoted by *, shown as pairwise comparisons (each animal is compared with itself, treated vs pre-treatment) and between groups (compound treated vs vehicle), with n= 7, 8 and 8 per group for day 6 dose level 10, 30 and 100mg/Kg respectively; day 15, n=10; day 21, n=5. Data presented as mean ± SD.


A . Flow cytometry of cells isolated from heart tissue when scaled against TMRM staining and also FSC and SSC parameters of flow cytometry .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O O O O B-EXP_ASSAY O O O O O O O O O O O

A. Flow cytometry of cells isolated from heart tissue when scaled against TMRM staining and also FSC and SSC parameters of flow cytometry.


B . Cardiac troponin I ( cTnI ) and α - Sarcomeric actinin ( α - SA ) staining of the TMRM + + population ( CMs ) and Vimentin staining of the TMRM + population ( NCs ) . The scale bar represents 100 µm .

O O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O

B. Cardiac troponin I (cTnI) and α-Sarcomeric actinin (α-SA) staining of the TMRM++ population (CMs) and Vimentin staining of the TMRM+ population (NCs). The scale bar represents 100 µm.


D . EGFP expression in iPSC - like colonies derived from CMs or NCs isolated from triple transgenic mice .

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D. EGFP expression in iPSC-like colonies derived from CMs or NCs isolated from triple transgenic mice.


E . EGFP expression combined with immunofluorescence staining of SSEA - 1 and DAPI of iPSC - like colonies derived from CMs isolated from triple transgenic mice .

O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-CELL_TYPE O O O O B-ORGANISM O

E. EGFP expression combined with immunofluorescence staining of SSEA-1 and DAPI of iPSC-like colonies derived from CMs isolated from triple transgenic mice.


F . Morphological changes of isolated CMs and NCs at different time points before AP positive colony formation at day 6 . The scale bar represents 100 µm .

O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O O

F. Morphological changes of isolated CMs and NCs at different time points before AP positive colony formation at day 6. The scale bar represents 100 µm.


A . Gene profiling of CM and NC at D0 - D6 as shown clustered into 8 categories . Red indicates up - regulated genes while green indicates down - regulated genes ( n = 3 , n = 2 for NC - D6 ) .

O O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O 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-CELL_TYPE O O O O

A. Gene profiling of CM and NC at D0-D6 as shown clustered into 8 categories. Red indicates up-regulated genes while green indicates down-regulated genes (n=3, n=2 for NC-D6).


B . Expression patterns of all genes per cluster by grey lines . Black line shows the mean value .

B. Expression patterns of all genes per cluster by grey lines. Black line shows the mean value.


D . Correlative analysis representing the relationships between CM and NC reprogramming specifically at days 2 , 4 and 6 . Red indicates closely related groups , while green indicates significantly different groups .

O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O

D. Correlative analysis representing the relationships between CM and NC reprogramming specifically at days 2, 4 and 6. Red indicates closely related groups, while green indicates significantly different groups.


A . RNA expression ratio ( D2 - to - D0 ) of candidates selected from day 2 microarray data .

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

A. RNA expression ratio (D2-to-D0) of candidates selected from day 2 microarray data.


C . Ki67 , α - MHC , and DAPI staining and quantification of CMs treated with control ( Ctrl ) , or combinations of a FoxM1 up - regulation adenovirus ( F ) , Id1 up - regulation adenovirus ( I ) , Hmgb2 up - regulation adenovirus ( H ) or a small molecule Jnk3 inhibitor ( Ji ) .

O O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O B-GENEPROD O O O B-ORGANISM O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O O

C. Ki67, α-MHC, and DAPI staining and quantification of CMs treated with control (Ctrl), or combinations of a FoxM1 up-regulation adenovirus (F), Id1 up-regulation adenovirus (I), Hmgb2 up-regulation adenovirus (H) or a small molecule Jnk3 inhibitor (Ji).


D . Ki67 , α - MHC , and DAPI staining and quantification of CMs treated with Ctrl or combination of FI with Jnk3 - shRNAs in place of the small molecule ( Ji ) . The arrow head indicates Ki67 + / α - MHC + proliferated CMs .

O O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-CELL_TYPE O

D. Ki67, α-MHC, and DAPI staining and quantification of CMs treated with Ctrl or combination of FI with Jnk3-shRNAs in place of the small molecule (Ji). The arrow head indicates Ki67+/α-MHC+ proliferated CMs.


E . Morphology and quantification of FIJs - treated CMs undergoing cytokinesis by H3P and Aurora kinase B ( AURKB ) staining .

O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O B-GENEPROD O O O

E. Morphology and quantification of FIJs-treated CMs undergoing cytokinesis by H3P and Aurora kinase B (AURKB) staining.


G . RNA expression ratio of Oct4 , FoxM1 , Id1 , or Jnk3 in doxycycline - injected versus control adult CMs isolated from reprogrammable mice .

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G. RNA expression ratio of Oct4, FoxM1, Id1, or Jnk3 in doxycycline-injected versus control adult CMs isolated from reprogrammable mice.


H . Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from control or doxycycline injected mice .

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H. Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from control or doxycycline injected mice.


B . The size of hearts isolated from Ctrl or FIJs - treated mice ; heart / body weight ratio of Ctrl or FIJs - treated mice .

O O O O O B-TISSUE O O O O O O O B-ORGANISM O B-TISSUE O O O O O O O O O O B-ORGANISM O

B. The size of hearts isolated from Ctrl or FIJs-treated mice; heart/body weight ratio of Ctrl or FIJs-treated mice.


C . H3P , cTnI , and DAPI staining and quantification of heart tissues in Ctrl or FIJs - treated mice . The arrowhead indicates H3P + / cTnI + proliferated CMs and the scale bar represents 50 µm .

O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O B-ORGANISM O O O O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O

C. H3P, cTnI, and DAPI staining and quantification of heart tissues in Ctrl or FIJs-treated mice. The arrowhead indicates H3P+/cTnI+ proliferated CMs and the scale bar represents 50 µm.


E . Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from adeno - Ctrl or adeno - FIJs injected mice .

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E. Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from adeno-Ctrl or adeno-FIJs injected mice.


F . Quantification of BrdU + / EGFP + population % in isolated CMs from adeno - Ctrl or adeno - FIJs injected M / Z mice by flow cytometry .

O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O B-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY O

F. Quantification of BrdU+/EGFP+ population % in isolated CMs from adeno-Ctrl or adeno-FIJs injected M/Z mice by flow cytometry.


B . RNA expressional changes of FoxM1 , Id1 , and Jnk3 after adeno - Ctrl or adeno - FIJs treatment after MI .

O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O

B. RNA expressional changes of FoxM1, Id1, and Jnk3 after adeno-Ctrl or adeno-FIJs treatment after MI.


C . Immunofluorescence staining of BrdU or H3P and cTnI or EGFP and quantification in the heart tissues after MI . The arrow head indicates cTnI + / BrdU + , cTnI + / H3P + or EGFP + / BrdU + , EGFP + / H3P + proliferated CMs . The scale bar represents 50 µm .

O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-TISSUE O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O

C. Immunofluorescence staining of BrdU or H3P and cTnI or EGFP and quantification in the heart tissues after MI. The arrow head indicates cTnI+/BrdU+, cTnI+/H3P+ or EGFP+/BrdU+, EGFP+/H3P+proliferated CMs. The scale bar represents 50 µm.


D . Ejection fraction and fraction shortening percentage in Ctrl or FIJs - treated mice 21 days post - MI .

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D. Ejection fraction and fraction shortening percentage in Ctrl or FIJs-treated mice 21 days post-MI.


E . Catheterization in Ctrl or FIJs - treated mice 21 days post - MI under baseline or inferior vena cava occlusion condition .

O O B-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O

E. Catheterization in Ctrl or FIJs-treated mice 21 days post-MI under baseline or inferior vena cava occlusion condition.


F . Trichrome staining and quantification showing percentage of fibrotic tissue ( blue ) in Ctrl or FIJs - treated mice 21 days after MI .

O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O

F. Trichrome staining and quantification showing percentage of fibrotic tissue (blue) in Ctrl or FIJs-treated mice 21 days after MI.


A . RNA expression of mitosis checkpoint genes in CMs with FoxM1 or Id1 overexpression , Jnk3 - shRNA , or combined treatment .

O O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O

A. RNA expression of mitosis checkpoint genes in CMs with FoxM1 or Id1 overexpression, Jnk3-shRNA, or combined treatment.


B . Transcriptional expression of cyclin - dependent kinases in FoxM1 or Id1 up - regulated , Jnk3 down - regulated , or combined treated CMs .

O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-CELL_TYPE O

B. Transcriptional expression of cyclin-dependent kinases in FoxM1 or Id1 up-regulated, Jnk3 down-regulated, or combined treated CMs.


C . Real - time RT - PCR analysis of CDK inhibitor expression in CMs with FoxM1 or Id1 overexpression , Jnk3 - shRNA or combined treatment .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O

C. Real-time RT-PCR analysis of CDK inhibitor expression in CMs with FoxM1 or Id1 overexpression, Jnk3-shRNA or combined treatment.


( B ) Representative large image of a brain slice from R6 / 2 mice after 4 - week striatal infusion of fluorescent cholesterol ( bodipy - chol , green ) . In the inlet , infused striatum is shown . Hoechst ( Ho , blue ) was used to counterstain nuclei . Scale bar : 1000 µm .

O O O O O B-EXP_ASSAY O O B-TISSUE O O O O O B-ORGANISM O O O O B-TISSUE O O B-EXP_ASSAY B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O

(B) Representative large image of a brain slice from R6/2 mice after 4-week striatal infusion of fluorescent cholesterol (bodipy-chol, green). In the inlet, infused striatum is shown. Hoechst (Ho, blue) was used to counterstain nuclei. Scale bar: 1000 µm.


( C ) Cholesterol content measured by mass spectrometry in infused striatum of untreated wt ( N = 7 ) and R6 / 2 ( N = 4 ) , R6 / 2 ACSF ( N = 5 ) , R6 / 2 chol - low ( N = 5 ) , R6 / 2 chol - medium ( N = 5 ) , and R6 / 2 chol - high ( N = 5 ) mice at 12 weeks of age after 4 - week striatal cholesterol infusion . The low , medium , and high doses respectively correspond to 15 µg , 185 µg , and 369 µg of cholesterol infused in the striatum after 4 weeks . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value obtained from each animal . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

O O O B-SMALL_MOLECULE B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-ORGANISM O O O O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cholesterol content measured by mass spectrometry in infused striatum of untreated wt (N=7) and R6/2 (N=4), R6/2 ACSF (N=5), R6/2 chol-low (N=5), R6/2 chol-medium (N=5), and R6/2 chol-high (N=5) mice at 12 weeks of age after 4-week striatal cholesterol infusion. The low, medium, and high doses respectively correspond to 15 µg, 185 µg, and 369 µg of cholesterol infused in the striatum after 4 weeks. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( D ) Latency to fall ( seconds ) from an accelerating rotarod from 5 weeks ( pre - surgery ; i . e . , before pump implantation ) to 10 weeks of age in wt ( N = 23 at 5 weeks , N = 28 at 8 and 10 weeks ) , R6 / 2 ACSF ( N = 35 at 5 weeks , N = 31 at 8 weeks and N = 30 at 10 weeks ) , R6 / 2 chol - low ( N = 22 at 5 weeks , N = 14 at 8 weeks and N = 13 ) , R6 / 2 chol - medium ( N = 13 at 5 weeks , N = 9 at 8 weeks and N = 8 at 10 weeks ) , and R6 / 2 chol - high ( N = 19 at 5 weeks , N = 17 at 8 and 10 weeks ) mice . The graph shows means ± standard error for each time point . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O 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-SMALL_MOLECULE O 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-SMALL_MOLECULE O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Latency to fall (seconds) from an accelerating rotarod from 5 weeks (pre-surgery; i.e., before pump implantation) to 10 weeks of age in wt (N=23 at 5 weeks, N=28 at 8 and 10 weeks), R6/2 ACSF (N=35 at 5 weeks, N=31 at 8 weeks and N=30 at 10 weeks), R6/2 chol-low (N=22 at 5 weeks, N=14 at 8 weeks and N=13), R6/2 chol-medium (N=13 at 5 weeks, N=9 at 8 weeks and N=8 at 10 weeks), and R6/2 chol-high (N=19 at 5 weeks, N=17 at 8 and 10 weeks) mice. The graph shows means±standard error for each time point. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


Global motor activity ( E ) , in an open - field test at 6 weeks of age ( pre - surgery ) and 11 weeks of age ( 4 weeks after infusion ) of wt ( N = 16 at 9 weeks , N = 22 at 11 weeks ) , R6 / 2 ACSF ( N = 27 at 9 weeks , N = 23 at 11 weeks ) , R6 / 2 chol - low ( N = 20 at 9 weeks , N = 13 at 11 weeks ) , R6 / 2 chol - medium ( N = 9 at 9 weeks , N = 7 at 11 weeks ) , and R6 / 2 chol - high ( N = 18 at 9 weeks , N = 16 at 11 weeks ) mice . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value obtained from each animal . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Global motor activity (E), in an open-field test at 6 weeks of age (pre-surgery) and 11 weeks of age (4 weeks after infusion) of wt (N=16 at 9 weeks, N=22 at 11 weeks), R6/2 ACSF (N=27 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=20 at 9 weeks, N=13 at 11 weeks), R6/2 chol-medium (N=9 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high (N=18 at 9 weeks, N=16 at 11 weeks) mice. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


total distance traveled ( F ) , mean velocity ( G ) , stereotyped movements ( H ) , in an open - field test at 6 weeks of age ( pre - surgery ) and 11 weeks of age ( 4 weeks after infusion ) of wt ( N = 16 at 9 weeks , N = 22 at 11 weeks ) , R6 / 2 ACSF ( N = 27 at 9 weeks , N = 23 at 11 weeks ) , R6 / 2 chol - low ( N = 20 at 9 weeks , N = 13 at 11 weeks ) , R6 / 2 chol - medium ( N = 9 at 9 weeks , N = 7 at 11 weeks ) , and R6 / 2 chol - high ( N = 18 at 9 weeks , N = 16 at 11 weeks ) mice . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value obtained from each animal . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

total distance traveled (F), mean velocity (G), stereotyped movements (H), in an open-field test at 6 weeks of age (pre-surgery) and 11 weeks of age (4 weeks after infusion) of wt (N=16 at 9 weeks, N=22 at 11 weeks), R6/2 ACSF (N=27 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=20 at 9 weeks, N=13 at 11 weeks), R6/2 chol-medium (N=9 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high (N=18 at 9 weeks, N=16 at 11 weeks) mice. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


number of rearings ( I ) in an open - field test at 6 weeks of age ( pre - surgery ) and 11 weeks of age ( 4 weeks after infusion ) of wt ( N = 16 at 9 weeks , N = 22 at 11 weeks ) , R6 / 2 ACSF ( N = 27 at 9 weeks , N = 23 at 11 weeks ) , R6 / 2 chol - low ( N = 20 at 9 weeks , N = 13 at 11 weeks ) , R6 / 2 chol - medium ( N = 9 at 9 weeks , N = 7 at 11 weeks ) , and R6 / 2 chol - high ( N = 18 at 9 weeks , N = 16 at 11 weeks ) mice . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value obtained from each animal . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

number of rearings (I) in an open-field test at 6 weeks of age (pre-surgery) and 11 weeks of age (4 weeks after infusion) of wt (N=16 at 9 weeks, N=22 at 11 weeks), R6/2 ACSF (N=27 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=20 at 9 weeks, N=13 at 11 weeks), R6/2 chol-medium (N=9 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high (N=18 at 9 weeks, N=16 at 11 weeks) mice. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( J , K ) Representative track plots ( J ) generated from the open - field test from wt ( N = 16 at 9 weeks , N = 22 at 11 weeks ) , R6 / 2 ACSF ( N = 26 at 9 weeks , N = 23 at 11 weeks ) , R6 / 2 chol - low ( N = 11 at 9 weeks , N = 7 at 11 weeks ) , R6 / 2 chol - medium ( N = 11 at 9 weeks , N = 7 at 11 weeks ) , and R6 / 2 chol - high mice ( N = 18 at 9 weeks , N = 16 at 11 weeks ) and relative quantification ( K ) of the time spent in the center and in the periphery ( % ) of the arena . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J, K) Representative track plots (J) generated from the open-field test from wt (N=16 at 9 weeks, N=22 at 11 weeks), R6/2 ACSF (N=26 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=11 at 9 weeks, N=7 at 11 weeks), R6/2 chol-medium (N=11 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high mice (N=18 at 9 weeks, N=16 at 11 weeks) and relative quantification (K) of the time spent in the center and in the periphery (%) of the arena. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( L ) Discrimination index ( DI ; % ) in the novel object recognition test at 6 weeks of age ( before pump implantation ) and 11 weeks of age ( 4 weeks after cholesterol infusion ) of wt ( N = 16 at 9 weeks , N = 21 at 11 weeks ) , R6 / 2 ACSF ( N = 28 at 9 weeks , N = 23 at 11 weeks ) , R6 / 2 chol - low ( N = 21 at 9 weeks , N = 13 at 11 weeks ) , R6 / 2 chol - medium ( N = 10 at 9 weeks , N = 7 at 11 weeks ) , and R6 / 2 chol - high mice ( N = 18 at 9 weeks , N = 16 at 11 weeks ) . DI above zero indicates a preference for the novel object ; DI below zero indicates a preference for the familiar object . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value obtained from each animal . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(L) Discrimination index (DI; %) in the novel object recognition test at 6 weeks of age (before pump implantation) and 11 weeks of age (4 weeks after cholesterol infusion) of wt (N=16 at 9 weeks, N=21 at 11 weeks), R6/2 ACSF (N=28 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=21 at 9 weeks, N=13 at 11 weeks), R6/2 chol-medium (N=10 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high mice (N=18 at 9 weeks, N=16 at 11 weeks). DI above zero indicates a preference for the novel object; DI below zero indicates a preference for the familiar object. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( B ) Spontaneous EPSCs were recorded from striatal MSNs ( wt = 6 ; R6 / 2 ACSF = 5 ; R6 / 2 chol - high = 5 ) at a holding potential of - 70 mV .

O O O O B-EXP_ASSAY O O O B-TISSUE B-CELL_TYPE O O O O O O O O B-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

(B) Spontaneous EPSCs were recorded from striatal MSNs (wt=6; R6/2 ACSF=5; R6/2 chol-high=5) at a holding potential of -70 mV.


( C - D ) Average amplitude ( C ) and average frequency ( D ) of EPSCs from wt , R6 / 2 ACSF , and R6 / 2 chol - high MSNs . Data information : The data are shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value of each cell recorded Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Average amplitude (C) and average frequency (D) of EPSCs from wt, R6/2 ACSF, and R6/2 chol-high MSNs. Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the value of each cell recorded Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( E - G ) Number of excitatory synapses per volume of striatum by using FIB / SEM followed by 3D reconstruction . ( E ) Representative tissue block of striatum ( 10 µm x 15 µm x 10 µm ) used for the 3D analysis . ( F ) Representative FIB - SEM segmentation and reconstruction of excitatory synapses ( yellow ) in 200 serial sections of striatum for a total volume of 500 µm3 , of wt , R6 / 2 , R6 / 2 ACSF , and R6 / 2 chol - high mice . ( G ) Density of excitatory synapses in at least 1500 μm3 of striatal tissue from wt , R6 / 2 , R6 / 2 ACSF , and R6 / 2 chol - high mice ( N = 3 animals / group ) . Data information : The data are shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value of , the number of synapses counted / µm3 in different blocks of striatal tissue ( G ) Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O B-TISSUE O O B-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-TISSUE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O B-SUBCELLULAR O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Number of excitatory synapses per volume of striatum by using FIB/SEM followed by 3D reconstruction. (E) Representative tissue block of striatum (10 µm x 15 µm x 10 µm) used for the 3D analysis. (F) Representative FIB-SEM segmentation and reconstruction of excitatory synapses (yellow) in 200 serial sections of striatum for a total volume of 500 µm3, of wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice. (G) Density of excitatory synapses in at least 1500 μm3 of striatal tissue from wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice (N=3 animals/group). Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the value of , the number of synapses counted/µm3 in different blocks of striatal tissue (G) Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( H - I ) TEM images ( H ) and 2D profile ( I ) of pre - synaptic terminal sections contain SVs and mitochondria ( m ) . Docked vesicles are indicated by a white arrowhead ( H ) and black circle ( I ) and are defined as a vesicle with its center located within 20 nm from the presynaptic membrane . Scale bar : 100 nm .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O

(H-I) TEM images (H) and 2D profile (I) of pre-synaptic terminal sections contain SVs and mitochondria (m). Docked vesicles are indicated by a white arrowhead (H) and black circle (I) and are defined as a vesicle with its center located within 20 nm from the presynaptic membrane. Scale bar: 100 nm.


( J , K ) Quantification showing the total SVs / µm2 ( J ) and the docked SVs / µm ( K ) in R6 / 2 , R6 / 2 ACSF , and wt striatal synapses ( N = 3 animals / group ) . N = 60 excitatory synapses / group were counted . Data information : The data are shown as scatterplot graphs with means ± standard error . Each dot corresponds to the number of total SVs / µm2 ( J ) and docked SVs / µm of active zone ( K ) counted in each synapse . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-TISSUE O O O B-TISSUE B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O 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-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J, K) Quantification showing the total SVs/µm2 (J) and the docked SVs/µm (K) in R6/2, R6/2 ACSF, and wt striatal synapses (N=3 animals/group). N=60 excitatory synapses/group were counted. Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the number of total SVs/µm2 (J) and docked SVs/µm of active zone (K) counted in each synapse. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( A ) Spontaneous IPSCs were recorded from striatal MSNs ( wt = 6 ; R6 / 2 ACSF = 5 ; R6 / 2 chol - high = 5 ) at a holding potential of 0 mV .

O O O O B-EXP_ASSAY O O O B-TISSUE B-CELL_TYPE O O O O O O O O B-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

(A) Spontaneous IPSCs were recorded from striatal MSNs (wt=6; R6/2 ACSF=5; R6/2 chol-high=5) at a holding potential of 0 mV.


( B - C ) Average amplitude ( B ) and average frequency ( C ) of IPSCs from wt , R6 / 2 ACSF , and R6 / 2 chol - high MSNs . Data information : The data are shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value of each cell recorded ( B , C ) Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * * p < 0 . 01 ; * * * p < 0 . 001 ) .

O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B-C) Average amplitude (B) and average frequency (C) of IPSCs from wt, R6/2 ACSF, and R6/2 chol-high MSNs. Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the value of each cell recorded (B, C) Statistics: one-way ANOVA with Newman-Keuls post-hoc test (**p<0.01; ***p<0.001).


( D - E ) Evaluation of the number of inhibitory synapses per volume of striatum by using FIB / SEM followed by 3D reconstruction . ( D ) Representative FIB - SEM segmentation and reconstruction of inhibitory synapses ( green ) in 200 serial sections of striatum for a total volume of 500 µm3 in wt , R6 / 2 , R6 / 2 ACSF , and R6 / 2 chol - high mice . ( E ) Density of inhibitory synapses in at least 2000 µm3 of striatal tissue of wt , R6 / 2 , R6 / 2 ACSF , and R6 / 2 chol - high mice ( N = 3 mice / group ) . Data information : The data E ) are shown as scatterplot graphs with means ± standard error . Each dot corresponds to the number of synapses counted / µm3 in different blocks of tissue for each group of animals ( N = 3 animals / group ) ( E ) . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * * p < 0 . 01 ; * * * p < 0 . 001 ) .

O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O B-TISSUE O O B-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-TISSUE O O O O O O O O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O B-ORGANISM O O O 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-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-E) Evaluation of the number of inhibitory synapses per volume of striatum by using FIB/SEM followed by 3D reconstruction. (D) Representative FIB-SEM segmentation and reconstruction of inhibitory synapses (green) in 200 serial sections of striatum for a total volume of 500 µm3 in wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice. (E) Density of inhibitory synapses in at least 2000 µm3 of striatal tissue of wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice (N=3 mice/ group). Data information: The data E) are shown as scatterplot graphs with means±standard error. Each dot corresponds to the number of synapses counted /µm3 in different blocks of tissue for each group of animals (N=3 animals/group) (E). Statistics: one-way ANOVA with Newman-Keuls post-hoc test (**p<0.01; ***p<0.001).


( B ) 24S - OHC level measured by mass spectrometry in the infused and contralateral striatum of wt , R6 / 2 ‐ ACSF , and R6 / 2 chol - high mice at 12 weeks of age after a 4 - week striatal infusion of cholesterol ( N = 4 / group ) . All values are expressed as % above the mean of wt , and these data are shown as scatterplots with means ± standard error . Each dot corresponds to the value obtained from each animal .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE O O O O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O 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) 24S-OHC level measured by mass spectrometry in the infused and contralateral striatum of wt, R6/2‐ACSF, and R6/2 chol-high mice at 12 weeks of age after a 4-week striatal infusion of cholesterol (N=4/group). All values are expressed as % above the mean of wt, and these data are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from each animal.


( C - E ) Lanosterol ( C ) , lathosterol ( D ) , and desmosterol ( E ) level measured by mass spectrometry in the infused and contralateral striatum of wt , R6 / 2 ACSF , and R6 / 2 chol - high mice at 12 weeks of age after 4 - week striatal infusion of cholesterol ( N = 4 / group ) . All values are expressed as % above the mean of wt , and these data are shown as scatterplots with means ± standard error . Each dot corresponds to the value obtained from each animal .

O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-E) Lanosterol (C), lathosterol (D), and desmosterol (E) level measured by mass spectrometry in the infused and contralateral striatum of wt, R6/2 ACSF, and R6/2 chol-high mice at 12 weeks of age after 4-week striatal infusion of cholesterol (N=4/group). All values are expressed as % above the mean of wt, and these data are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from each animal.


( F - I ) Nuclear ( white arrows ) and perinuclear ( yellow arrows ) localization of endogenous SREBP2 in the striatum of wt , R6 / 2 and R6 / 2 - chol mice . Representative confocal image ( F and H ) and relative quantification ( G and I ) of SREBP2 ( red ) in wt and R6 / 2 mice and in the infused and contralateral striatum of R6 / 2 chol - high mice ( N = 4 ) . Hoechst ( Ho , blue ) was used to counterstain nuclei . Scale bar in F and H : 10 µm . Graphs in ( G and I ) represents the intensity of SREBP2 normalized on nuclei ( % ) . Data are shown as scatterplots with means ± standard error . Each dot corresponds to the value obtained from each image . Statistics : Student ' s t - test ( * * p < 0 . 01 ; * * * * p < 0 . 0001 ) .

O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O B-GENEPROD O O B-TISSUE O O O O O O O O O O O B-SMALL_MOLECULE B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O B-TISSUE O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-SUBCELLULAR 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F-I) Nuclear (white arrows) and perinuclear (yellow arrows) localization of endogenous SREBP2 in the striatum of wt, R6/2 and R6/2-chol mice. Representative confocal image (F and H) and relative quantification (G and I) of SREBP2 (red) in wt and R6/2 mice and in the infused and contralateral striatum of R6/2 chol-high mice (N=4). Hoechst (Ho, blue) was used to counterstain nuclei. Scale bar in F and H: 10 µm. Graphs in (G and I) represents the intensity of SREBP2 normalized on nuclei (%). Data are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from each image. Statistics: Student's t-test (**p<0.01; ****p<0.0001).


J , K Representative high - magnification confocal images of immunostaining against SREBP2 ( red ) and NeuN ( J ) or GFAP ( K ) ( green ) on coronal sections of brains from R6 / 2 chol - high mice . A cell in the infused striatum , positive for NeuN and GFAP respectively , is shown . Hoechst ( Ho , blue ) was used to counterstain nuclei . Scale bars : 1 µm .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-TISSUE O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O B-TISSUE O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O

J, K Representative high-magnification confocal images of immunostaining against SREBP2 (red) and NeuN (J) or GFAP (K) (green) on coronal sections of brains from R6/2 chol-high mice. A cell in the infused striatum, positive for NeuN and GFAP respectively, is shown. Hoechst (Ho, blue) was used to counterstain nuclei. Scale bars: 1 µm.


Immunolabeling of muHTT aggregates ( red ) in R6 / 2 ACSF and R6 / 2 chol - high mice ( N = 3 / group ) . Zoom of representative confocal images of immunostaining against muHTT aggregates ( red ) showing muHTT aggregates positive for EM48 antibody in the infused and contralateral striatum ( A ) and relative quantification of number ( B ) and size ( C ) of aggregates . Hoechst ( Ho , blue ) was used to counterstain nuclei . 18 images / animal were analyzed from 9 sections throughout the entire striatum . The data are shown as scatterplots with means ± standard error . Each dot corresponds to aggregates counted in all the images from 3 animals . Scale bars : 10 µm ( A ) Statistics : one ‐ way ANOVA followed by Newman - Keuls multiple comparison tests ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 ) .

B-EXP_ASSAY O B-GENEPROD B-SUBCELLULAR O O O O O O O B-TISSUE O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD B-SUBCELLULAR O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O O B-TISSUE O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunolabeling of muHTT aggregates (red) in R6/2 ACSF and R6/2 chol-high mice (N=3/group). Zoom of representative confocal images of immunostaining against muHTT aggregates (red) showing muHTT aggregates positive for EM48 antibody in the infused and contralateral striatum (A) and relative quantification of number (B) and size (C) of aggregates. Hoechst (Ho, blue) was used to counterstain nuclei. 18 images/animal were analyzed from 9 sections throughout the entire striatum. The data are shown as scatterplots with means±standard error. Each dot corresponds to aggregates counted in all the images from 3 animals. Scale bars: 10 µm (A) Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (*p<0.05; **p<0.01; ****p<0.0001).


Representative confocal images of immunostaining against muHTT ( red ) and DARPP32 ( D ) showing muHTT aggregates positive for EM48 antibody in the infused striatum in neurons or astrocytes and relative quantification ( E Hoechst ( Ho , blue ) was used to counterstain nuclei . All values are expressed as % above the mean of aggregates in the contralateral striatum of R6 / 2 chol - high . The data are shown as scatterplots with means ± standard error . Each dot corresponds to aggregates counted in all the images from 3 animals . Scale bars : 5 µm Statistics : one ‐ way ANOVA followed by Newman - Keuls multiple comparison tests ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 ) .

O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O B-TISSUE O B-CELL_TYPE O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O B-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative confocal images of immunostaining against muHTT (red) and DARPP32 (D) showing muHTT aggregates positive for EM48 antibody in the infused striatum in neurons or astrocytes and relative quantification (E Hoechst (Ho, blue) was used to counterstain nuclei. All values are expressed as % above the mean of aggregates in the contralateral striatum of R6/2 chol-high. The data are shown as scatterplots with means±standard error. Each dot corresponds to aggregates counted in all the images from 3 animals. Scale bars: 5 µm Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (*p<0.05; **p<0.01; ****p<0.0001).


Representative confocal images of immunostaining against muHTT ( red ) and GFAP ( F ) ( green ) showing muHTT aggregates positive for EM48 antibody in the infused striatum in neurons or astrocytes and relative quantification G ) . Hoechst ( Ho , blue ) was used to counterstain nuclei . All values are expressed as % above the mean of aggregates in the contralateral striatum of R6 / 2 chol - high . The data are shown as scatterplots with means ± standard error . Each dot corresponds to aggregates counted in all the images from 3 animals . Scale bars : 5 µm Statistics : one ‐ way ANOVA followed by Newman - Keuls multiple comparison tests ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 ) .

O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O B-TISSUE O B-CELL_TYPE O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O B-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative confocal images of immunostaining against muHTT (red) and GFAP (F) (green) showing muHTT aggregates positive for EM48 antibody in the infused striatum in neurons or astrocytes and relative quantification G). Hoechst (Ho, blue) was used to counterstain nuclei. All values are expressed as % above the mean of aggregates in the contralateral striatum of R6/2 chol-high. The data are shown as scatterplots with means±standard error. Each dot corresponds to aggregates counted in all the images from 3 animals. Scale bars: 5 µm Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (*p<0.05; **p<0.01; ****p<0.0001).


( H ) TEM images of EM48 pre - embedding immunogold labelling showing muHTT aggregates in the striatal neuron cell bodies of R6 / 2 ACSF mice and R6 / 2 chol - high mice ( upper panels ) . In the lower panels , muHTT aggregates are clearly visible and the magnifications show the area indicated by the black square in the upper images . Arrows indicate large muHTT aggregates with a fibrous structure in the nucleus , and arrowheads indicate single 10 - nm gold particles in MSN . Nucleus ( N ) , nucleolus ( n ) , mitochondrion ( m ) , rough endoplasmic reticulum ( rer ) , Golgi apparatus ( g ) , and white arrows indicate the nuclear envelope ( N = 3 animals / group ) . Scale bars : 700 nm and 300 nm .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-SUBCELLULAR O O B-TISSUE B-CELL_TYPE B-SUBCELLULAR I-SUBCELLULAR O O O O B-TISSUE B-ORGANISM O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-CELL_TYPE O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O

(H) TEM images of EM48 pre-embedding immunogold labelling showing muHTT aggregates in the striatal neuron cell bodies of R6/2 ACSF mice and R6/2 chol-high mice (upper panels). In the lower panels, muHTT aggregates are clearly visible and the magnifications show the area indicated by the black square in the upper images. Arrows indicate large muHTT aggregates with a fibrous structure in the nucleus, and arrowheads indicate single 10-nm gold particles in MSN. Nucleus (N), nucleolus (n), mitochondrion (m), rough endoplasmic reticulum (rer), Golgi apparatus (g), and white arrows indicate the nuclear envelope (N=3 animals/group). Scale bars: 700 nm and 300 nm.


Quantification of aggregated and total muHTT in the infused striatum of HD mice after 4 - week cholesterol infusion by TR - FRET analysis using different antibody pairs . Preliminary assessment of the sustainability of the assay in wt and R6 / 2 striata ( N = 5 / group ) using 4C9 - 4C9 and 2B7 - MW1 antibodies in combination to detect , respectively , muHTT aggregates ( J ) and total muHTT ( K ) . Quantification of muHTT aggregates ( L ) and soluble and other muHTT species ( M ) in the infused striata of R6 / 2 ACSF and R6 / 2 chol - high mice . Data in ( J - M ) are shown as scatterplots with means ± standard error . Each dot corresponds to the value obtained from one striatum . Statistics : student ' s t - test ( * p < 0 . 05 ) .

O O B-SUBCELLULAR O O B-GENEPROD O O O B-TISSUE O B-DISEASE B-ORGANISM O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O B-TISSUE O O O O B-TISSUE O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O 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-TISSUE O O O O O O O O O O O O O O O O O O

Quantification of aggregated and total muHTT in the infused striatum of HD mice after 4-week cholesterol infusion by TR-FRET analysis using different antibody pairs. Preliminary assessment of the sustainability of the assay in wt and R6/2 striata (N=5/group) using 4C9-4C9 and 2B7-MW1 antibodies in combination to detect, respectively, muHTT aggregates (J) and total muHTT (K). Quantification of muHTT aggregates (L) and soluble and other muHTT species (M) in the infused striata of R6/2 ACSF and R6/2 chol-high mice. Data in (J-M) are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from one striatum. Statistics: student's t-test (*p<0.05).


Representative confocal images showing p62 ( A , red ) in the infused and contralateral striatum on brain coronal sections from wt , R6 / 2 ACSF , and R6 / 2 chol - high mice ( N = 3 / group ) . Hoechst ( Ho , blue ) was used to counterstain nuclei . Quantification of dots for p62 ( B ) in the contralateral and infused striatum of R6 / 2 ACSF and R6 / 2 chol - high mice . 20 images from 3 sections in the middle of the striatum for each animal were acquired and analyzed . The data in ( B are shown as scatterplots with means ± standard error , and each dot corresponds to the value obtained from each image . Statistics : one ‐ way ANOVA followed by Newman - Keuls multiple comparison tests ( * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) . Scale bars in ( A 10 µm .

O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O B-TISSUE O O O O B-TISSUE O O O O B-SMALL_MOLECULE O O B-ORGANISM O O B-EXP_ASSAY O O O O O O O O B-TISSUE O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative confocal images showing p62 (A, red) in the infused and contralateral striatum on brain coronal sections from wt, R6/2 ACSF, and R6/2 chol-high mice (N=3/group). Hoechst (Ho, blue) was used to counterstain nuclei. Quantification of dots for p62 (B) in the contralateral and infused striatum of R6/2 ACSF and R6/2 chol-high mice. 20 images from 3 sections in the middle of the striatum for each animal were acquired and analyzed. The data in (B are shown as scatterplots with means±standard error, and each dot corresponds to the value obtained from each image. Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (***p<0.001; ****p<0.0001). Scale bars in (A 10 µm.


Representative confocal images showing LAMP1 ( C , green ) in the infused and contralateral striatum on brain coronal sections from wt , R6 / 2 ACSF , and R6 / 2 chol - high mice ( N = 3 / group ) . Hoechst ( Ho , blue ) was used to counterstain nuclei . Quantification of density for LAMP1 ( D ) in the contralateral and infused striatum of R6 / 2 ACSF and R6 / 2 chol - high mice . 20 images from 3 sections in the middle of the striatum for each animal were acquired and analyzed . The data in D ) are shown as scatterplots with means ± standard error , and each dot corresponds to the value obtained from each image . Statistics : one ‐ way ANOVA followed by Newman - Keuls multiple comparison tests ( * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) . Scale bars in C ) : 10 µm .

O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O B-TISSUE O O O O B-TISSUE O O O O B-SMALL_MOLECULE O O B-ORGANISM O O B-EXP_ASSAY O O O O O O O O B-TISSUE O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative confocal images showing LAMP1 (C, green) in the infused and contralateral striatum on brain coronal sections from wt, R6/2 ACSF, and R6/2 chol-high mice (N=3/group). Hoechst (Ho, blue) was used to counterstain nuclei. Quantification of density for LAMP1 (D) in the contralateral and infused striatum of R6/2 ACSF and R6/2 chol-high mice. 20 images from 3 sections in the middle of the striatum for each animal were acquired and analyzed. The data in D) are shown as scatterplots with means±standard error, and each dot corresponds to the value obtained from each image. Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (***p<0.001; ****p<0.0001). Scale bars in C): 10 µm.


Gene expression analyzed by quantitative PCR in brown adipocytes that were fed with or without 1mM asparagine as in ( A ) . n = 3 independent experiments . Data information : Data represent means ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; unpaired two - tailed Student ' s t test .

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Gene expression analyzed by quantitative PCR in brown adipocytes that were fed with or without 1mM asparagine as in (A). n=3 independent experiments. Data information: Data represent means±SEM. *P<0.05; **P<0.01; unpaired two-tailed Student's t test.


Preadipocytes bearing the shRNA targeting Pgc1α were induced differentiation with or without 1mM asparagine . Protein expression was analyzed on day 6 . n = 3 biological replicates .

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Preadipocytes bearing the shRNA targeting Pgc1α were induced differentiation with or without 1mM asparagine. Protein expression was analyzed on day 6. n=3 biological replicates.


Brown preadipocytes were infected by lentiviral shRNAs against Asns . Cells were differentiated in culture media with or without 1mM asparagine . Western blot was performed on day 6 .

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Brown preadipocytes were infected by lentiviral shRNAs against Asns. Cells were differentiated in culture media with or without 1mM asparagine. Western blot was performed on day 6.


UCP1 and PPARγ protein levels in BAT and inguinal WAT ( iWAT ) from mice in ( E ) .

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UCP1 and PPARγ protein levels in BAT and inguinal WAT (iWAT) from mice in (E).


I , J . Mice supplemented with asparagine in drinking water for 3 weeks were subjected to the indirect calorimetry analysis at both basal ( I ) and CL316 , 243 - stimulated ( J ) states . n = 5 mice each group . Data information : Data represent means ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; unpaired two - tailed Student ' s t test .

O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I,J. Mice supplemented with asparagine in drinking water for 3 weeks were subjected to the indirect calorimetry analysis at both basal (I) and CL316,243-stimulated (J) states. n=5 mice each group. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.


Rectal temperature in mice of ( A ) during cold exposure . n = 5 mice each group .

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Rectal temperature in mice of (A) during cold exposure. n=5 mice each group.


BAT and inguinal WAT ( iWAT ) gene expression analysis in mice of ( A ) .

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BAT and inguinal WAT (iWAT) gene expression analysis in mice of (A).


Hematoxylin and eosin staining of BAT , inguinal WAT ( iWAT ) and epididymal WAT ( eWAT ) of mice in ( A ) .

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Hematoxylin and eosin staining of BAT, inguinal WAT (iWAT) and epididymal WAT (eWAT) of mice in (A).


Glucose tolerance test was performed after a high - fat diet feeding for 17 weeks in mice of ( A ) . n = 5 mice each group . Serum insulin levels in mice of ( A ) . n = 5 mice each group . Serum triglyceride levels in mice of ( A ) . n = 5 mice each group . Data information : Data represent means ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; unpaired two - tailed Student ' s t test .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O B-TISSUE B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O B-ORGANISM O O O B-TISSUE B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Glucose tolerance test was performed after a high-fat diet feeding for 17 weeks in mice of (A). n=5 mice each group.   Serum insulin levels in mice of (A). n=5 mice each group.   Serum triglyceride levels in mice of (A). n=5 mice each group.   Data information: Data represent means±SEM. *P<0.05; **P<0.01; unpaired two-tailed Student's t test.


BAT and inguinal WAT ( iWAT ) UCP1 and PPARγ protein levels in mice of ( A ) .

B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O

BAT and inguinal WAT (iWAT) UCP1 and PPARγ protein levels in mice of (A).


Heat map of metabolite relative levels in brown fat cells treated with or without 1mM asparagine .

Heat map of metabolite relative levels in brown fat cells treated with or without 1mM asparagine.


Immunoblot detection of the glucose transporters , glycolysis and TCA key enzymes in primary brown fat cell line Asparagine was supplemented to cells during differentiation .

B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O

Immunoblot detection of the glucose transporters, glycolysis and TCA key enzymes in primary brown fat cell line Asparagine was supplemented to cells during differentiation.


Immunoblot detection of the glucose transporters , glycolysis and TCA key enzymes in immortalized brown fat cell line ( E ) , BAT from mice fed asparagine ( F ) and injected asparaginase ( G ) . Asparagine was supplemented to cells during differentiation .

B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O B-TISSUE O B-ORGANISM O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O

Immunoblot detection of the glucose transporters, glycolysis and TCA key enzymes in immortalized brown fat cell line (E), BAT from mice fed asparagine (F) and injected asparaginase (G). Asparagine was supplemented to cells during differentiation.


Intracellular abundance of 13C - isotopologue metabolites downstream of glycolysis in brown adipocytes . n = 5 independent experiments . Data information : Data represent means ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; unpaired two - tailed Student ' s t test .

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Intracellular abundance of 13C-isotopologue metabolites downstream of glycolysis in brown adipocytes. n=5 independent experiments. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.


C , D . Quantification of glucose ( C ) and lactate ( D ) concentration from media . The media was collected after incubated with mature brown adipocytes for 48 h . n = 3 independent experiments in ( C ) , n = 6 independent experiments in ( D ) . Data information : Data represent means ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; unpaired two - tailed Student ' s t test .

O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of glucose (C) and lactate (D) concentration from media. The media was collected after incubated with mature brown adipocytes for 48 h. n=3 independent experiments in (C), n=6 independent experiments in (D). Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.


Quantification of intracellular amino acid levels in brown fat cells ( day 6 ) treated with or without 1mM asparagine . n = 4 independent experiments . Data information : Data represent means ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; unpaired two - tailed Student ' s t test .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of intracellular amino acid levels in brown fat cells (day 6) treated with or without 1mM asparagine. n=4 independent experiments. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.


B - D . mTORC1 pathway activity was analyzed by immunoblotting for the phosphorylation of 4E - BP1 and S6K in brown adipocytes ( B ) , BAT from mice fed asparagine ( C ) or asparaginase ( D ) . ( B ) Asparagine was added to cultured media from day 4 to day 6 .

O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O B-TISSUE O B-ORGANISM O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

B-D. mTORC1 pathway activity was analyzed by immunoblotting for the phosphorylation of 4E-BP1 and S6K in brown adipocytes (B), BAT from mice fed asparagine (C) or asparaginase (D). (B) Asparagine was added to cultured media from day 4 to day 6.


Brown preadipocytes were induced to differentiation with or without 1mM asparagine . Rapamycin ( 75nM ) was added to cells for 48h . Then western blot detected protein expression on day 6 .

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Brown preadipocytes were induced to differentiation with or without 1mM asparagine. Rapamycin (75nM) was added to cells for 48h. Then western blot detected protein expression on day 6.


Brown preadipocytes were knocked down Raptor following standard induction to mature adipocytes with or without asparagine supplementation . Protein expression analysis was performed on day 6 .

B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

Brown preadipocytes were knocked down Raptor following standard induction to mature adipocytes with or without asparagine supplementation. Protein expression analysis was performed on day 6.


F . Dopaminergic neuronal cultures from control lines and glucocerebrosidase ( GBA ) N370S PD patients were immunoblotted for the presence of LAG3 . No band for LAG3 could be observed in neurons .

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F. Dopaminergic neuronal cultures from control lines and glucocerebrosidase (GBA) N370S PD patients were immunoblotted for the presence of LAG3. No band for LAG3 could be observed in neurons.


G . Using high power , high resolution laser scanning confocal microscopy , no human LAG3 signal could be detected in human neurons ( Auto - hLAG3 transduced , DOX OFF ) by two different anti - human LAG3 antibodies ( 17B4 and D2G40 ; left panel and zoomed - in insets ) whereas LAG3 was clearly detected in human neurons induced to express hLAG3 ( DOX ON ; right panel and zoomed - in insets ) . Scale bars 25 µm .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM B-GENEPROD O O O O O B-ORGANISM B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

G. Using high power, high resolution laser scanning confocal microscopy, no human LAG3 signal could be detected in human neurons (Auto-hLAG3 transduced, DOX OFF) by two different anti-human LAG3 antibodies (17B4 and D2G40; left panel and zoomed-in insets) whereas LAG3 was clearly detected in human neurons induced to express hLAG3 (DOX ON; right panel and zoomed-in insets). Scale bars 25 µm.


H . Human brain homogenates from autopsy material were immunoblotted for the presence of LAG3 . No band for LAG3 could be evidenced in any of the brain homogenates . Control samples ( tonsils and activated human T cells ) show expected bands .

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H. Human brain homogenates from autopsy material were immunoblotted for the presence of LAG3. No band for LAG3 could be evidenced in any of the brain homogenates. Control samples (tonsils and activated human T cells) show expected bands.