L - N , Sequential BrdU - EdU labelling . BrdU was injected into pregnant mice at E13 . 5 , i . e . 24 hr before sacrifice at E14 . 5 , and EdU was injected into the same pregnant mice 0 . 5 hr before sacrifice at E14 . 5 . ( L ) Representative immunofluorescence for BrdU ( magenta ) , combined with EdU staining ( cyan ) and DAPI staining ( white ) , of E14 . 5 WT and 11B mouse dorsolateral neocortex rostrally . ( M , N ) Quantification of BrdU + & EdU + cells ( M ) and the percentage of BrdU + cells that are BrdU + & EdU + ( N ) in VZ and SVZ in a 200 µm - wide field of E14 . 5 WT ( white ) and 11B ( black ) mouse dorsolateral neocortex rostrally . Data information : Data are the mean of 6 - 19 ( WT ) and 6 - 26 ( 11B ) littermate embryos , which were derived from 3 - 9 separate litters . Error bars indicate SD , two - tailed unpaired Student ' s t - test , * P < 0 . 05 ; * * P < 0 . 01 P = 0 . 0049 ( M , SVZ ) ; P = 0 . 0323 ( N , SVZ ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-N, Sequential BrdU-EdU labelling. BrdU was injected into pregnant mice at E13.5, i.e. 24 hr before sacrifice at E14.5, and EdU was injected into the same pregnant mice 0.5 hr before sacrifice at E14.5. (L) Representative immunofluorescence for BrdU (magenta), combined with EdU staining (cyan) and DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. (M, N) Quantification of BrdU+ & EdU+ cells (M) and the percentage of BrdU+ cells that are BrdU+ & EdU+ (N) in VZ and SVZ in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally. Data information: Data are the mean of 6-19 (WT) and 6-26 (11B) littermate embryos, which were derived from 3-9 separate litters. Error bars indicate SD, two-tailed unpaired Student's t-test, * P < 0.05; ** P < 0.01 P = 0.0049 (M, SVZ); P = 0.0323 (N, SVZ).


H Representative immunofluorescence for Tbr1 ( cyan ) , Ctip2 ( magenta ) and Satb2 ( green ) , combined with DAPI staining ( white ) , of E18 . 5 WT and 11B mouse neocortex at the position where cortical thickness was measured ( A , red lines ) . I Quantification of zone thickness of E18 . 5 WT ( white ) and 11B ( black ) mouse neocortex at the position where cortical thickness was measured ( A , red lines ) . J Data information : Images are single optical sections Scale bar , 20 µm Data are the mean of 7 ( WT ) and 8 - 12 ( 11B ) littermate embryos , which were derived from 4 separate litters . Error bars indicate SD . Two - way ANOVA , followed by Bonferroni ' s multiple comparisons test ( B - D ) ; two - tailed unpaired Student ' s t - test P = 0 . 0287 ( I , IZ ) ; P = 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative immunofluorescence for Tbr1 (cyan), Ctip2 (magenta) and Satb2 (green), combined with DAPI staining (white), of E18.5 WT and 11B mouse neocortex at the position where cortical thickness was measured (A, red lines). I Quantification of zone thickness of E18.5 WT (white) and 11B (black) mouse neocortex at the position where cortical thickness was measured (A, red lines). J Data information: Images are single optical sections Scale bar, 20 µm Data are the mean of 7 (WT) and 8-12 (11B) littermate embryos, which were derived from 4 separate litters. Error bars indicate SD. Two-way ANOVA, followed by Bonferroni's multiple comparisons test (B-D); two-tailed unpaired Student's t-test P = 0.0287 (I, IZ); P = 0.0039


A Dorsal view of adult wildtype ( WT ) and 11B mouse brain at P56 . Orange lines illustrate the measurements of neocortex width and length ; red lines illustrate the measurements of neocortex area . B , C Quantification of neocortex width , length and area . D Data information : Scale bar , 1 mm Data are the mean of 8 ( WT ) and 8 ( 11B ) adult littermate mice . Error bars indicate SD , two - tailed unpaired Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 . P < 0 . 0001 ( B , neocortex width ) ; P < 0 . 0001 ( C )

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

A Dorsal view of adult wildtype (WT) and 11B mouse brain at P56. Orange lines illustrate the measurements of neocortex width and length; red lines illustrate the measurements of neocortex area. B, C Quantification of neocortex width, length and area. D Data information: Scale bar, 1 mm Data are the mean of 8 (WT) and 8 (11B) adult littermate mice. Error bars indicate SD, two-tailed unpaired Student's t-test , * P < 0.05, ** P < 0.01, **** P < 0.0001. P < 0.0001 (B, neocortex width); P < 0.0001 (C)


G , H Representative immunofluorescence for Ctip2 ( magenta ) , Satb2 ( green ) and Brn2 ( yellow ) , combined with DAPI staining ( white ) , of P56 adult WT and 11B mouse neocortex at the position where cortical thickness was measured ( D , red lines ) . Data information : Images are single optical sections Scale bar , 20

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

G, H Representative immunofluorescence for Ctip2 (magenta), Satb2 (green) and Brn2 (yellow), combined with DAPI staining (white), of P56 adult WT and 11B mouse neocortex at the position where cortical thickness was measured (D, red lines). Data information: Images are single optical sections Scale bar, 20 µm


IntelliCage was used to evaluate the memory flexibility of adult 11B mice . Four separate sessions were carried out : ( i ) 5 WT males together with 5 11B males ; ( ii ) 5 other WT males together with 5 other 11B males ; ( iii ) 5 WT females together with 7 11B females ; ( iv ) 4 other WT females together with 8 other 11B females . Data are the mean of the total 10 males ( WT ) , 10 males ( 11B ) , 9 females ( WT ) and 15 females ( 11B ) adult littermate mice . Data points were obtained at consecutive days , error bars indicate SD . ( A , B ) Quantification of the discrimination error rate in the first 100 visits during the drinking phase of the acquisition stage of adult wildtype ( WT ) and 11B mice ( male , A ; female , B ) in the IntelliCage . Overall place learning performance of WT and 11B mice was analysed using two - way ANOVA followed by Fisher ' s LSD test ; all data points during the acquisition phase were included . For males ( A ) , WT vs . 11B , P > 0 . 05 ; data over time , * * * * P < 0 . 0001 ; interaction , P > 0 . 05 . For females ( B ) , WT vs . 11B , P > 0 . 05 ; data over time , * * * * P < 0 . 0001 ; interaction , P > 0 . 05 . Note that ( i ) the two male sessions and the two female sessions yielded essentially the same results , and ( ii ) not shown in panels the two separate male sessions yielded essentially the same results , and the two separate female sessions yielded essentially the same results .

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

IntelliCage was used to evaluate the memory flexibility of adult 11B mice. Four separate sessions were carried out: (i) 5 WT males together with 5 11B males; (ii) 5 other WT males together with 5 other 11B males; (iii) 5 WT females together with 7 11B females; (iv) 4 other WT females together with 8 other 11B females. Data are the mean of the total 10 males (WT), 10 males (11B), 9 females (WT) and 15 females (11B) adult littermate mice. Data points were obtained at consecutive days, error bars indicate SD. (A, B) Quantification of the discrimination error rate in the first 100 visits during the drinking phase of the acquisition stage of adult wildtype (WT) and 11B mice (male, A; female, B) in the IntelliCage. Overall place learning performance of WT and 11B mice was analysed using two-way ANOVA followed by Fisher's LSD test; all data points during the acquisition phase were included. For males (A), WT vs. 11B, P > 0.05; data over time, **** P < 0.0001; interaction, P > 0.05. For females (B), WT vs. 11B, P > 0.05; data over time, **** P < 0.0001; interaction, P > 0.05. Note that (i) the two male sessions and the two female sessions yielded essentially the same results, and (ii) not shown in panels the two separate male sessions yielded essentially the same results, and the two separate female sessions yielded essentially the same results.


Expression levels of the autophagosome marker LC3B - II were evaluated in wild - type ( WT ) and Ctns − / − fibroblasts by Western blot ( WB ) . Data are representative of five different experiments with similar results .

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Expression levels of the autophagosome marker LC3B-II were evaluated in wild-type (WT) and Ctns−/− fibroblasts by Western blot (WB). Data are representative of five different experiments with similar results.


WT and Ctns − / − fibroblasts were stained using LC3B antibodies and analyzed by fluorescence microscopy . The number of LC3B - positive puncta was quantified by the ImagePro software and normalized per area arbitrary units . Results are mean ± SEM ( n = 27 WT and 24 Ctns − / − cells ) . * * * P < 0 . 001 ( unpaired t - test ) .

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WT and Ctns−/− fibroblasts were stained using LC3B antibodies and analyzed by fluorescence microscopy. The number of LC3B-positive puncta was quantified by the ImagePro software and normalized per area arbitrary units. Results are mean ± SEM (n = 27 WT and 24 Ctns−/− cells). ***P < 0.001 (unpaired t-test).


Immunofluorescence analysis of liver ( C ) and kidney ( D ) tissues from WT and Ctns − / − GFP - LC3 transgenic mice using anti - GFP antibodies . Quantification of LC3 puncta was obtained by counting the number of GFP - positive structures relative to the number of nuclei in the same field , using the ImagePro software . GFP - LC3 puncta were counted by analyzing 6 to 10 fields per tissue section ( 200 - 400 nuclei per field ) , in a total of 3 WT and 3 Ctns − / − mice expressing GFP - LC3 . Results are mean ± SEM . In ( C ) , * * P = 0 . 0029 ; in ( D ) , * * P = 0 . 0025 ( unpaired t - test ) . Scale bar : 5 μm .

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Immunofluorescence analysis of liver (C) and kidney (D) tissues from WT and Ctns−/−GFP-LC3 transgenic mice using anti-GFP antibodies. Quantification of LC3 puncta was obtained by counting the number of GFP-positive structures relative to the number of nuclei in the same field, using the ImagePro software. GFP-LC3 puncta were counted by analyzing 6 to 10 fields per tissue section (200-400 nuclei per field), in a total of 3 WT and 3 Ctns−/−mice expressing GFP-LC3. Results are mean ± SEM. In (C), **P = 0.0029; in (D), **P = 0.0025 (unpaired t-test). Scale bar: 5 μm.


Representative images of wild - type ( WT ) and Ctns − / − mouse fibroblasts transfected with the ptfLC3 vector under resting conditions ( fed ) , or after serum starvation ( Serum Starv . ) in the presence or absence of the alkalinizing drug chloroquine ( CQ ) . GFP and RFP staining was analyzed by confocal microscopy . Examples of red - only puncta ( mature autophagosomes ) are indicated with arrows . Scale bar : 5 μm . The percentage of mature autophagosomes ( red - only vesicles ) was calculated based on the ratio between the number of red - only puncta and the total number of autophagosomes ( number of green and red + red - only puncta ) . The graph is representative of three different experiments with similar results . Results are mean ± SEM ( n = 7 cells per condition ) . In addition to the experimental conditions shown in ( A ) , the Aa / serum starvation condition is included .

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

Representative images of wild-type (WT) and Ctns−/− mouse fibroblasts transfected with the ptfLC3 vector under resting conditions (fed), or after serum starvation (Serum Starv.) in the presence or absence of the alkalinizing drug chloroquine (CQ). GFP and RFP staining was analyzed by confocal microscopy. Examples of red-only puncta (mature autophagosomes) are indicated with arrows. Scale bar: 5 μm.The percentage of mature autophagosomes (red-only vesicles) was calculated based on the ratio between the number of red-only puncta and the total number of autophagosomes (number of green and red + red-only puncta). The graph is representative of three different experiments with similar results. Results are mean ± SEM (n = 7 cells per condition). In addition to the experimental conditions shown in (A), the Aa/serum starvation condition is included.


WT and Ctns − / − fibroblasts under resting conditions were treated with 100 nM bafilomycin A ( BafA ) for 2 h and LC3B - II levels were analyzed by Western blot ( WB ) .

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

WT and Ctns−/− fibroblasts under resting conditions were treated with 100 nM bafilomycin A (BafA) for 2 h and LC3B-II levels were analyzed by Western blot (WB).


Phosphorylation levels of the mTOR complex kinase 1 substrate S6K and LC3B - II levels in WT and Ctns − / − fibroblasts were measured by WB under resting conditions ( − ) , withdrawal of both amino acids and serum ( Aa / Ser . Starv . ) and subsequent recovery by replacement of starvation medium with normal cell growth medium ( Rec ) , in the presence or absence of 100 nM BafA for the indicated time .

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

Phosphorylation levels of the mTOR complex kinase 1 substrate S6K and LC3B-II levels in WT and Ctns−/− fibroblasts were measured by WB under resting conditions (−), withdrawal of both amino acids and serum (Aa/Ser. Starv.) and subsequent recovery by replacement of starvation medium with normal cell growth medium (Rec), in the presence or absence of 100 nM BafA for the indicated time.


WB analysis showing phosphorylation levels of the mTOR substrate S6K and expression of LC3B - II in resting or serum - starved WT , Ctns − / − and Ctns − / − fibroblasts expressing GFP - CTNS .

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

WB analysis showing phosphorylation levels of the mTOR substrate S6K and expression of LC3B-II in resting or serum-starved WT,Ctns−/− and Ctns−/− fibroblasts expressing GFP-CTNS.


Analysis of the expression of endogenous SQSTM1 in wild - type or Ctns − / − mouse fibroblasts under fed ( serum ) or starved ( 15 h serum starvation ) conditions . In these assays , 20 μg of total protein lysates were resolved by SDS - PAGE and analyzed by Western blot ( WB ; A ) . Equal loading was established by silver staining ( B ) . Quantification ( mean ± SEM ) of five different areas of each silver - stained lane are shown in ( C ) .

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

Analysis of the expression of endogenous SQSTM1 in wild-type or Ctns−/− mouse fibroblasts under fed (serum) or starved (15 h serum starvation) conditions. In these assays, 20 μg of total protein lysates were resolved by SDS-PAGE and analyzed by Western blot (WB; A). Equal loading was established by silver staining (B). Quantification (mean ± SEM) of five different areas of each silver-stained lane are shown in (C).


Degradation of long - lived proteins using metabolic labeling was performed as described in Supplementary Materials and Methods . The data represent the mean ± SEM of nine independent samples analyzed in three independent experiments . * P = 0 . 009 for WT and * P = 0 . 0013 for Ctns − / − , respectively . NS , not significant ( unpaired t - test ) .

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

Degradation of long-lived proteins using metabolic labeling was performed as described in Supplementary Materials and Methods. The data represent the mean ± SEM of nine independent samples analyzed in three independent experiments. *P = 0.009 for WT and *P = 0.0013 for Ctns−/−, respectively. NS, not significant (unpaired t-test).


The expression levels of the endogenous LAMP2A and LAMP1 proteins were analyzed in wild - type ( WT ) and Ctns − / − mousefibroblasts ( A ) , kidney ( B ) and liverlysosomes ( C ) by Western blot . Quantitative densitometry analysis of immunoblots obtained from different experiments was performed by calculating the ratio between LAMP2A and actin signals in each lane . Results are mean ± SEM of four independent experiments with mousefibroblasts , 5 WT and 6 Ctns − / − mousekidneys , 3 WT and 3 Ctns − / − mouselivers ( lysosomal extracts ) .

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

The expression levels of the endogenous LAMP2A and LAMP1 proteins were analyzed in wild-type (WT) and Ctns−/−mousefibroblasts (A), kidney (B) and liverlysosomes (C) by Western blot. Quantitative densitometry analysis of immunoblots obtained from different experiments was performed by calculating the ratio between LAMP2A and actin signals in each lane. Results are mean ± SEM of four independent experiments with mousefibroblasts, 5 WT and 6 Ctns−/−mousekidneys, 3 WT and 3 Ctns−/−mouselivers (lysosomal extracts).


Comparative analysis of the amount of LAMP2A in wild - type or Ctns − / − liverlysosomes was performed using two different anti - LAMP2A antibodies . 15 μg of lysosomal proteins were loaded in each lane . Ab1 , anti - LAMP2A antibody raised against the twelve amino acids of the cytosolic region of ratLAMP2A largely validated to recognize mouseLAMP2A but not other LAMP2 isoforms ( Cuervo Dice , 1996 ) . Ab2 , anti - LAMP2A Abcam Ab18528 . Decreased lysosomalLAMP2A was evident using either antibody . LAMP1 was used as a loading control . Representative of two independent experiments with similar results .

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

Comparative analysis of the amount of LAMP2A in wild-type or Ctns−/−liverlysosomes was performed using two different anti-LAMP2A antibodies. 15 μg of lysosomal proteins were loaded in each lane. Ab1, anti-LAMP2A antibody raised against the twelve amino acids of the cytosolic region of ratLAMP2A largely validated to recognize mouseLAMP2A but not other LAMP2 isoforms (Cuervo Dice, 1996). Ab2, anti-LAMP2A Abcam Ab18528. Decreased lysosomalLAMP2A was evident using either antibody. LAMP1 was used as a loading control. Representative of two independent experiments with similar results.


Immunofluorescence analyses of endogenous LAMP1 , LAMP2A ( A , B ) , LAMP2B ( C ) and total LAMP2 ( D ) localization in wild - type ( WT ) and Ctns − / − mouse fibroblasts . ( A ) Confocal microscopy analysis . White arrows represent LAMP1 - positive circular structures ( lysosomes ) which were visible in both WT and Ctns − / − cells . Colocalization of LAMP1 and LAMP2A was observed in WT fibroblasts ( white arrows , merged panel ) but rarely in Ctns − / − cells ( red arrow ) . Most LAMP2A showed a punctate pattern and lack of colocalization with LAMP1 in Ctns − / − cells ( arrowheads , lower panels ) . Scale bars : 5 μm . Inset scale bars : 2 μm .

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Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts. (A) Confocal microscopy analysis. White arrows represent LAMP1-positive circular structures (lysosomes) which were visible in both WT and Ctns−/− cells. Colocalization of LAMP1 and LAMP2A was observed in WT fibroblasts (white arrows, merged panel) but rarely in Ctns−/− cells (red arrow). Most LAMP2A showed a punctate pattern and lack of colocalization with LAMP1 in Ctns−/− cells (arrowheads, lower panels). Scale bars: 5 μm. Inset scale bars: 2 μm.


Immunofluorescence analyses of endogenous LAMP1 , LAMP2A ( A , B ) , LAMP2B ( C ) and total LAMP2 ( D ) localization in wild - type ( WT ) and Ctns − / − mouse fibroblasts . ( B ) High - resolution stochastic optical reconstruction microscopy ( STORM ) analysis of the localization of LAMP1 and LAMP2A in wild - type and Ctns − / − cells . In Ctns − / − cells , LAMP2A was detected near ( arrowheads , estimated distance > 50 nm ) but not always adjacent ( 10 - 50 nm ) to LAMP1 or at LAMP1 - negative structures ( arrows ) . Scale bar : 0 . 5 μm .

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

Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts.(B) High-resolution stochastic optical reconstruction microscopy (STORM) analysis of the localization of LAMP1 and LAMP2A in wild-type and Ctns−/− cells. In Ctns−/− cells, LAMP2A was detected near (arrowheads, estimated distance > 50 nm) but not always adjacent (10-50 nm) to LAMP1 or at LAMP1-negative structures (arrows). Scale bar: 0.5 μm.


Immunofluorescence analyses of endogenous LAMP1 , LAMP2A ( A , B ) , LAMP2B ( C ) and total LAMP2 ( D ) localization in wild - type ( WT ) and Ctns − / − mouse fibroblasts . ( C ) Immunofluorescence showing colocalization of endogenous LAMP1 and LAMP2B in both wild - type and CTNS - deficient fibroblasts ( arrows ) . Scale bar : 20 μm .

Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts. (C) Immunofluorescence showing colocalization of endogenous LAMP1 and LAMP2B in both wild-type and CTNS-deficient fibroblasts (arrows). Scale bar: 20 μm.


Immunofluorescence analyses of endogenous LAMP1 , LAMP2A ( A , B ) , LAMP2B ( C ) and total LAMP2 ( D ) localization in wild - type ( WT ) and Ctns − / − mouse fibroblasts . ( D ) Immunofluorescence analysis of LAMP1 and total LAMP2 ( antibody ABL93 DSHB ) . Arrows show colocalization . Arrowheads show LAMP2 - positive LAMP1 - negative vesicles .

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

Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts. (D) Immunofluorescence analysis of LAMP1 and total LAMP2 (antibody ABL93 DSHB). Arrows show colocalization. Arrowheads show LAMP2-positive LAMP1-negative vesicles.


Immunofluorescence analyses of endogenous LAMP2A , ER ( Grp78 / 94 ) ( A ) , which is expanded in cystinotic cells ( white arrow ) , VAMP7 - positive vesicles ( B ) and Rab11a - positive structures ( C ) was performed using wild - type or Ctns − / − fibroblasts as described in Materials and Methods . At least 2 independent experiments were performed for each vesicular trafficking or ER marker . ( B , C ) Results are expressed as the percentage of LAMP2A that colocalizes with VAMP7 or Rab11 . For Rab11 / LAMP2A colocalization , 29 wild - type and 15 Ctns − / − cells were quantified . For VAMP7 / LAMP2A colocalization ( white arrows ) , 49 WT and 57 Ctns − / − cells were analyzed . Data are expressed as mean ± SEM . * P < 0 . 001 ( unpaired t - test ) . Scale bars : 20 μm .

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

Immunofluorescence analyses of endogenous LAMP2A, ER (Grp78/94) (A), which is expanded in cystinotic cells (white arrow), VAMP7-positive vesicles (B) and Rab11a-positive structures (C) was performed using wild-type or Ctns−/− fibroblasts as described in Materials and Methods. At least 2 independent experiments were performed for each vesicular trafficking or ER marker. (B, C) Results are expressed as the percentage of LAMP2A that colocalizes with VAMP7 or Rab11. For Rab11/LAMP2A colocalization, 29 wild-type and 15 Ctns−/− cells were quantified. For VAMP7/LAMP2A colocalization (white arrows), 49 WT and 57 Ctns−/− cells were analyzed. Data are expressed as mean ± SEM. *P < 0.001 (unpaired t-test). Scale bars: 20 μm.


WT , Ctns − / − or Ctns − / − mouse fibroblasts treated for 20 h with either a combination of both leupeptin and chloroquine ( CQ / Leu ) or bafilomycin A ( BafA ) alone were fixed and immunostained with antibodies recognizing endogenous LAMP1 and LAMP2A proteins and samples were analyzed by confocal microscopy as described in Material and Methods . Lysosomal colocalization of LAMP1 and LAMP2A was evident in treated ( middle lower and bottom panels ) but not in untreated ( middle upper panels ) Ctns − / − cells . Scale bars : 5 μm . Inset scale bars : 2 μm .

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

WT,Ctns−/− or Ctns−/− mouse fibroblasts treated for 20 h with either a combination of both leupeptin and chloroquine (CQ/Leu) or bafilomycin A (BafA) alone were fixed and immunostained with antibodies recognizing endogenous LAMP1 and LAMP2A proteins and samples were analyzed by confocal microscopy as described in Material and Methods. Lysosomal colocalization of LAMP1 and LAMP2A was evident in treated (middle lower and bottom panels) but not in untreated (middle upper panels) Ctns−/− cells. Scale bars: 5 μm. Inset scale bars: 2 μm.


Lysosomes were isolated from livers of starved wild - type ( WT ) and Ctns − / − mice as described in Supplementary Materials and Methods and incubated at 37 ° C for 30 min with the CMA substrate GAPDH , in the presence or absence of ATP ( necessary for CMA ) and protease inhibitors ( Prot . Inhib . ) . A fraction of the CMA reactions was then mixed with sample buffer and boiled at 95 ° C for 5 min , followed by SDS - PAGE and GAPDH and LAMP1 immunoblotting . Quantitative densitometry analysis of CMA activity performed in independent experiments using lysosomes isolated from a total of 11 WT and 10 Ctns − / − mice . Results are mean ± SEM . * * * P = 0 . 0005 ; * * P = 0 . 0041 ; N . S . , not significant ( unpaired t - test ) .

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Lysosomes were isolated from livers of starved wild-type (WT) and Ctns−/− mice as described in Supplementary Materials and Methods and incubated at 37°C for 30 min with the CMA substrate GAPDH, in the presence or absence of ATP (necessary for CMA) and protease inhibitors (Prot. Inhib.). A fraction of the CMA reactions was then mixed with sample buffer and boiled at 95°C for 5 min, followed by SDS-PAGE and GAPDH and LAMP1 immunoblotting.Quantitative densitometry analysis of CMA activity performed in independent experiments using lysosomes isolated from a total of 11 WT and 10 Ctns−/− mice. Results are mean ± SEM. ***P = 0.0005; **P = 0.0041; N.S., not significant (unpaired t-test).


The ability of lysosomal proteases to mediate GAPDH degradation was assessed by incubating Triton X - 100 - treated WT and Ctns − / − lysosomes with GAPDH for 30 min in acidic reaction buffer and compared with input ( reaction with no lysosomes ) . Quantitative densitometry analysis of independent reactions performed with lysosomes from a total of 9 WT and 9 Ctns − / − mice shows no significant difference in protease activity . Results are mean ± SEM .

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The ability of lysosomal proteases to mediate GAPDH degradation was assessed by incubating Triton X-100-treated WT and Ctns−/− lysosomes with GAPDH for 30 min in acidic reaction buffer and compared with input (reaction with no lysosomes). Quantitative densitometry analysis of independent reactions performed with lysosomes from a total of 9 WT and 9 Ctns−/− mice shows no significant difference in protease activity. Results are mean ± SEM.


WT and Ctns − / − mouse fibroblasts were treated with either 50 μM H2O2 or 1 mM paraquat ( PQ ) for 4 h and stained with ( A ) FITC - annexin V or ( B ) propidium iodide ( PI ) and analyzed by FACS . Results from 3 different experiments are shown as mean ± SEM . In ( A ) , * P = 0 . 05 ; * * * P = 0 . 0002 . In ( B ) , * P = 0 . 046 ; * * P = 0 . 0065 . Unpaired t - test .

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WT and Ctns−/− mouse fibroblasts were treated with either 50 μM H2O2 or 1 mM paraquat (PQ) for 4 h and stained with (A) FITC-annexin V or (B) propidium iodide (PI) and analyzed by FACS. Results from 3 different experiments are shown as mean ± SEM. In (A), *P = 0.05; ***P = 0.0002. In (B), *P = 0.046; **P = 0.0065. Unpaired t-test.


WT and Ctns − / − cells were treated as described above and samples analyzed by Western blot using antibodies recognizing cleaved ( active ) caspase 3 . Data are representative of 2 independent experiments with similar results .

WT and Ctns−/− cells were treated as described above and samples analyzed by Western blot using antibodies recognizing cleaved (active) caspase 3. Data are representative of 2 independent experiments with similar results.


Cystine content in WT , Ctns − / − , cysteamine - treated Ctns − / − fibroblasts or Ctns − / − fibroblasts expressing GFP - CTNS was assessed by mass spectrometry .

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Cystine content in WT,Ctns−/−, cysteamine-treated Ctns−/− fibroblasts or Ctns−/− fibroblasts expressing GFP-CTNS was assessed by mass spectrometry.


WT , Ctns − / − , Ctns − / − fibroblasts treated with 1 mM cysteamine ( 48 h ) , or Ctns − / − fibroblasts expressing GFP - CTNS were fixed and stained with anti - LAMP1 and LAMP2A antibodies as indicated . For better visualization of LAMP1 / LAMP2A colocalization , GFP - CTNS was pseudocolored as magenta and LAMP1 was pseudocolored as green ( lower panels ) . Some lysosomes are indicated with arrows . Arrowheads indicate LAMP2A distribution to structures different from lysosomes ( only observed in Ctns − / − cells and in cysteamine - treated Ctns − / − cells ) . Rescue of the LAMP2A localization to lysosomes was observed in Ctns − / − cells expressing GFP - CTNS . Scale bar : 2 μm . Quantification of the colocalization analysis described in ( B ) . Calculation of the Pearson ' s colocalization coefficient was done by analyzing 113 WT , 251 Ctns − / − , 88 cysteamine - treated and 164 GFP - CTNS - expressing Ctns − / − cells , by using the ZEN 2010 software . Results are mean ± SEM . * * * P < 0 . 001 ; NS , not significant ( one - way ANOVA , Bonferroni ' s multiple comparisons test ) .

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

WT, Ctns−/−, Ctns−/− fibroblasts treated with 1 mM cysteamine (48 h), or Ctns−/− fibroblasts expressing GFP-CTNS were fixed and stained with anti-LAMP1 and LAMP2A antibodies as indicated. For better visualization of LAMP1/LAMP2A colocalization, GFP-CTNS was pseudocolored as magenta and LAMP1 was pseudocolored as green (lower panels). Some lysosomes are indicated with arrows. Arrowheads indicate LAMP2A distribution to structures different from lysosomes (only observed in Ctns−/− cells and in cysteamine-treated Ctns−/− cells). Rescue of the LAMP2A localization to lysosomes was observed in Ctns−/− cells expressing GFP-CTNS. Scale bar: 2 μm.Quantification of the colocalization analysis described in (B). Calculation of the Pearson's colocalization coefficient was done by analyzing 113 WT, 251 Ctns−/−, 88 cysteamine-treated and 164 GFP-CTNS-expressing Ctns−/− cells, by using the ZEN 2010 software. Results are mean ± SEM. ***P < 0.001; NS, not significant (one-way ANOVA, Bonferroni's multiple comparisons test).


Cystine content in WT , Ctns − / − and cysteamine - treated Ctns − / − mouse livers was assessed by mass spectrometry . Results are mean ± SEM ( n = 4 ) . * * * P < 0 . 001 ; * * P = 0 . 0026 ( one - way ANOVA , Bonferroni ' s multiple comparisons test ) .

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Cystine content in WT,Ctns−/− and cysteamine-treated Ctns−/− mouse livers was assessed by mass spectrometry. Results are mean ± SEM (n = 4). ***P < 0.001; **P = 0.0026 (one-way ANOVA, Bonferroni's multiple comparisons test).


Western blot analysis of LAMP2A expression in lysosomes isolated from WT , Ctns − / − and cysteamine - treated Ctns − / − mouse livers .

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Western blot analysis of LAMP2A expression in lysosomes isolated from WT,Ctns−/− and cysteamine-treated Ctns−/− mouse livers.


Lysosomes , isolated as in ( B ) , were incubated with the CMA substrate GAPDH , and CMA activity was evaluated as in Fig8 and as described in Materials and Methods . Quantitative analysis of substrate degradation is expressed as percent of the residual amount of GAPDH in the full reaction relative to its amount in the presence of protease inhibitors . Results are mean ± SEM ( n = 4 ) . * P = 0 . 0126 for WT versus Ctns − / − and * P = 0 . 0436 for WT versus Ctns − / − + cysteamine ; NS , not significant ; unpaired t - test .

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Lysosomes, isolated as in (B), were incubated with the CMA substrate GAPDH, and CMA activity was evaluated as in Fig8 and as described in Materials and Methods.Quantitative analysis of substrate degradation is expressed as percent of the residual amount of GAPDH in the full reaction relative to its amount in the presence of protease inhibitors. Results are mean ± SEM (n = 4). *P = 0.0126 for WT versus Ctns−/− and *P = 0.0436 for WT versus Ctns−/− + cysteamine; NS, not significant; unpaired t-test.


A The intrinsic fluorescence of NAD ( P ) H co - localizes extensively with mitochondria ( marked by Mitotracker CMXRos )

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A The intrinsic fluorescence of NAD(P)H co-localizes extensively with mitochondria (marked by Mitotracker CMXRos)


B 2P - FLIM detects the fraction of NAD ( P ) H bound to enzyme partners ( a2 % )

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B 2P-FLIM detects the fraction of NAD(P)H bound to enzyme partners (a2%)


a2 % values ( starved , and after insulin or R + L addition ) were recorded pixel by pixel for mouse cortical ( C neurons . Each colored line in the center panel graphs refers to a single field of view containing 2 - 15 cells , and each pair of solid and dotted lines ( same color ) refers to the same field of view before and after stimulation , respectively . Using the same raw data , the relative contributions of NADH and NADPH to the total a2 % values were calculated ( Blacker et al , 2014 ) and are shown in the right panel bar graphs . NADH and NAD ( P ) H contributed an average of 63 % and 37 % , respectively , of the total a2 % values . All statistical analyses were performed using Student ' s two - tailed unpaired t -

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

a2% values (starved, and after insulin or R+L addition) were recorded pixel by pixel for mouse cortical (C neurons. Each colored line in the center panel graphs refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view before and after stimulation, respectively. Using the same raw data, the relative contributions of NADH and NADPH to the total a2% values were calculated (Blacker et al, 2014) and are shown in the right panel bar graphs. NADH and NAD(P)H contributed an average of 63% and 37%, respectively, of the total a2% values. All statistical analyses were performed using Student's two-tailed unpaired t-tests


D a2 % values ( starved , and after insulin or R + L addition ) were recorded pixel by pixel fo human ( D ) neurons . Each colored line in the center panel graphs refers to a single field of view containing 2 - 15 cells , and each pair of solid and dotted lines ( same color ) refers to the same field of view before and after stimulation , respectively . Using the same raw data , the relative contributions of NADH and NADPH to the total a2 % values were calculated ( Blacker et al , 2014 ) and are shown in the right panel bar graphs . NADH and NAD ( P ) H contributed an average of 63 % and 37 % , respectively , of the total a2 % values . All statistical analyses were performed using Student ' s two - tailed unpaired t -

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

D a2% values (starved, and after insulin or R+L addition) were recorded pixel by pixel fo human (D) neurons. Each colored line in the center panel graphs refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view before and after stimulation, respectively. Using the same raw data, the relative contributions of NADH and NADPH to the total a2% values were calculated (Blacker et al, 2014) and are shown in the right panel bar graphs. NADH and NAD(P)H contributed an average of 63% and 37%, respectively, of the total a2% values. All statistical analyses were performed using Student's two-tailed unpaired t-tests


Wild type ( WT ) mouse cortical neurons cultured for 10 days in vitro ( A were serum - starved in Hank ' s balanced salt solution for 2 hours . Next , the cells were imaged immediately ( starved ) , after which either amino acids ( R + L ) or insulin were added . Thirty minutes later the cells were imaged again . Finally , the mTOR inhibitor , Torin1 was added , and after an additional 30 minutes , the cells were imaged for the last time . Histograms represent changes in the fraction of enzyme - bound NAD ( P ) H ( a2 % ) for each condition . Note that insulin and amino acids significantly increased the a2 % values , which was reversed by Torin1 - mediated inhibition of mTORC1 . Average data from 5 fields of view per condition are shown . Statistical analyses were performed using Student ' s two - tailed unpaired t -

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

Wild type (WT) mouse cortical neurons cultured for 10 days in vitro (A were serum-starved in Hank's balanced salt solution for 2 hours. Next, the cells were imaged immediately (starved), after which either amino acids (R+L) or insulin were added. Thirty minutes later the cells were imaged again. Finally, the mTOR inhibitor, Torin1 was added, and after an additional 30 minutes, the cells were imaged for the last time. Histograms represent changes in the fraction of enzyme-bound NAD(P)H (a2%) for each condition. Note that insulin and amino acids significantly increased the a2% values, which was reversed by Torin1-mediated inhibition of mTORC1. Average data from 5 fields of view per condition are shown. Statistical analyses were performed using Student's two-tailed unpaired t-test


human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor cells ( B ) were serum - starved in Hank ' s balanced salt solution for 2 hours . Next , the cells were imaged immediately ( starved ) , after which either amino acids ( R + L ) or insulin were added . Thirty minutes later the cells were imaged again . Finally , the mTOR inhibitor , Torin1 was added , and after an additional 30 minutes , the cells were imaged for the last time . Histograms represent changes in the fraction of enzyme - bound NAD ( P ) H ( a2 % ) for each condition . Note that insulin and amino acids significantly increased the a2 % values , which was reversed by Torin1 - mediated inhibition of mTORC1 . Average data from 5 fields of view per condition are shown . Statistical analyses were performed using Student ' s two - tailed unpaired t -

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

human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor cells (B) were serum-starved in Hank's balanced salt solution for 2 hours. Next, the cells were imaged immediately (starved), after which either amino acids (R+L) or insulin were added. Thirty minutes later the cells were imaged again. Finally, the mTOR inhibitor, Torin1 was added, and after an additional 30 minutes, the cells were imaged for the last time. Histograms represent changes in the fraction of enzyme-bound NAD(P)H (a2%) for each condition. Note that insulin and amino acids significantly increased the a2% values, which was reversed by Torin1-mediated inhibition of mTORC1. Average data from 5 fields of view per condition are shown. Statistical analyses were performed using Student's two-tailed unpaired t-test


C Raptor - depleted , WT cortical neurons were imaged by 2P - FLIM before and 30 minutes after treatment with insulin . Inhibiting mTORC1 by depleting Raptor with shRNA made mitochondria insensitive to insulin . Shown here are average data from 4 fields of view of a single replicate out of a total of 3 replicates . Western blots are representative of 3 independent assays . Statistical analyses were performed using Student ' s two - tailed unpaired t - test

C Raptor-depleted, WT cortical neurons were imaged by 2P-FLIM before and 30 minutes after treatment with insulin. Inhibiting mTORC1 by depleting Raptor with shRNA made mitochondria insensitive to insulin. Shown here are average data from 4 fields of view of a single replicate out of a total of 3 replicates. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test


A Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor cells were serum - starved in Hank ' s balanced salt solution ( HBSS ) for 2 hours . Next , amino acids ( R + L ) , insulin , insulin + R + L , or the oxidative phosphorylation inhibitor , NaN3 , were added , or the HBSS was replaced with complete medium . One hour later cellular reactive oxygen species ( ROS ) , ATP levels and oxygen consumption were measured . The data were collected from 3 independent assays in which each experimental condition contained 6 - 8 replicates . Error bars represent ± s . e . m . Statistical analyses were performed using Student ' s two - tailed unpaired t -

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A Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor cells were serum-starved in Hank's balanced salt solution (HBSS) for 2 hours. Next, amino acids (R+L), insulin, insulin + R+L, or the oxidative phosphorylation inhibitor, NaN3, were added, or the HBSS was replaced with complete medium. One hour later cellular reactive oxygen species (ROS), ATP levels and oxygen consumption were measured. The data were collected from 3 independent assays in which each experimental condition contained 6-8 replicates. Error bars represent ± s.e.m. Statistical analyses were performed using Student's two-tailed unpaired t-test


B MP - PAM imaging of wild type ( WT ) mouse cerebral cortex through an open - skull window 1 hour after topical application of amino acids ( R + L ) . A decrease in blood oxygenation of the cortical vasculature was observed , indicating elevated oxygen extraction and consumption due to upregulation of mitochondrial activity . Data were obtained from 3 mice , each of which was measured once for O2 saturation , oxygen extraction fraction ( OEF ) and cerebral metabolic rate of oxygen ( CMRO2 ) . Error bars represent ± s . e .

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B MP-PAM imaging of wild type (WT) mouse cerebral cortex through an open-skull window 1 hour after topical application of amino acids (R+L). A decrease in blood oxygenation of the cortical vasculature was observed, indicating elevated oxygen extraction and consumption due to upregulation of mitochondrial activity. Data were obtained from 3 mice , each of which was measured once for O2 saturation, oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2). Error bars represent ± s.e.m


C MP - PAM imaging of WT mouse cerebral cortex through an open - skull window beginning at baseline , which corresponded to 30 minutes after suppression of mTORC1 activity by a single topical application of 1 µM rapamycin . Baseline levels of O2 saturation and blood flow speed were recorded , after which R + L was applied to the open skull window . One hour later , O2 saturation and blood flow speed were recorded again . Data were obtained from 4 mice , each of which was measured once for O2 saturation , oxygen extraction fraction ( OEF ) and cerebral metabolic rate of oxygen ( CMRO2 ) . Note that no significant changes in O2 saturation or OEF were observed after R + L stimulation , in contrast to what was observed in the absence of rapamycin ( Fig 3B ) . Error bars represent ± s . e .

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C MP-PAM imaging of WT mouse cerebral cortex through an open-skull window beginning at baseline, which corresponded to 30 minutes after suppression of mTORC1 activity by a single topical application of 1 µM rapamycin. Baseline levels of O2 saturation and blood flow speed were recorded, after which R+L was applied to the open skull window. One hour later, O2 saturation and blood flow speed were recorded again. Data were obtained from 4 mice, each of which was measured once for O2 saturation, oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2). Note that no significant changes in O2 saturation or OEF were observed after R+L stimulation, in contrast to what was observed in the absence of rapamycin (Fig 3B). Error bars represent ± s.e.m


A Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitors ( Control ) , or otherwise identical cells expressing Flag - Raptor fused to either the plasma membrane targeting signal of H - Ras or the lysosomal targeting signal of Rheb were serum - starved in Hank ' s balanced salt solution for 2 hours , and then imaged by 2P - FLIM before and 30 minutes after addition of insulin . Each colored line refers to a single field of view containing 5 - 15 cells , and each pair of solid and dotted lines ( same color ) refers to the same field of view . Statistical analyses were performed using Student ' s two - tailed unpaired t - test ( ns : not significant ) . Expression of the Flag - Raptor fusion proteins was confirmed by western blotting ( right panel ) . Note that NiMA was supported when mTORC1 was targeted to lysosomes ( Flag - Raptor Rheb15 ) , but not to the plasma membrane ( Flag - Raptor H - Ras25 ) . Western blots are representative of 3 independent

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A Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitors (Control), or otherwise identical cells expressing Flag-Raptor fused to either the plasma membrane targeting signal of H-Ras or the lysosomal targeting signal of Rheb were serum-starved in Hank's balanced salt solution for 2 hours, and then imaged by 2P-FLIM before and 30 minutes after addition of insulin. Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test (ns: not significant). Expression of the Flag-Raptor fusion proteins was confirmed by western blotting (right panel). Note that NiMA was supported when mTORC1 was targeted to lysosomes (Flag-Raptor Rheb15), but not to the plasma membrane (Flag-Raptor H-Ras25). Western blots are representative of 3 independent assays


B Mouse cortical neurons starved in Hank ' s balanced salt solution for 2 hours were treated with AβOs , insulin or amino acids ( R + L ) for 30 minutes , and then were fixed and double labeled with anti - mTOR , and either anti - LAMP1 or Mitotracker CMXRos . Error bars represent ± s . e . m . Data are representative of 3 independent assays

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B Mouse cortical neurons starved in Hank's balanced salt solution for 2 hours were treated with AβOs, insulin or amino acids (R+L) for 30 minutes, and then were fixed and double labeled with anti-mTOR, and either anti-LAMP1 or Mitotracker CMXRos. Error bars represent ± s.e.m. Data are representative of 3 independent assays


C Reducing Bcl - xL expression in WT mouse cortical neurons does not affect NiMA . Western blots are representative of 3 independent assays . Statistical analyses were performed using Student ' s two - tailed unpaired t - test

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C Reducing Bcl-xL expression in WT mouse cortical neurons does not affect NiMA. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test


A DNA replication in mitochondrial replisomes was detected in mouse cortical neurons after a 3 - hour pulse of the thymidine analog , EdU . Cells were labeled additionally with the mitochondrial marker , Mitotracker CMXRos , and with an antibody to the neuron marker ,

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A DNA replication in mitochondrial replisomes was detected in mouse cortical neurons after a 3-hour pulse of the thymidine analog, EdU. Cells were labeled additionally with the mitochondrial marker, Mitotracker CMXRos, and with an antibody to the neuron marker, MAP2


B Quantification of EdU uptake into mouse cortical neuron replisomes showed that nutrients inhibit mtDNA replication by ~ 60 % , an effect that was blocked by AβOs . Error bars represent ± s . e . m . Statistical analyses were performed using Student " s two - tailed unpaired t - test and are representative of 3 independent

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B Quantification of EdU uptake into mouse cortical neuron replisomes showed that nutrients inhibit mtDNA replication by ~60%, an effect that was blocked by AβOs. Error bars represent ± s.e.m. Statistical analyses were performed using Student"s two-tailed unpaired t-test and are representative of 3 independent a


C WT mouse cortical neurons expressing either Flag - Raptor H - Ras25 or Flag - Raptor Rheb15 were serum - starved in AβO - containing Hank " s balanced salt solution for 2 hours . Next , the cells were pulse - labeled for 3 hours with EdU . Note that forcing mTORC1 to the plasma membrane ( Flag - Raptor H - Ras25 ) yielded an ~ 80 % increase in EdU incorporation into mtDNA in the presence of AβOs regardless of whether or not the neurons had been stimulated with insulin + R + L . In contrast , EdU uptake into mitochondrial replisomes was not stimulated by AβOs in neurons whose mTORC1 was forced to lysosomes ( Flag - Raptor - Rheb15 ) . Error bars represent ±

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C WT mouse cortical neurons expressing either Flag-Raptor H-Ras25 or Flag-Raptor Rheb15 were serum-starved in AβO-containing Hank"s balanced salt solution for 2 hours. Next, the cells were pulse-labeled for 3 hours with EdU. Note that forcing mTORC1 to the plasma membrane (Flag-Raptor H-Ras25) yielded an ~80% increase in EdU incorporation into mtDNA in the presence of AβOs regardless of whether or not the neurons had been stimulated with insulin+R+L. In contrast, EdU uptake into mitochondrial replisomes was not stimulated by AβOs in neurons whose mTORC1 was forced to lysosomes (Flag-Raptor-Rheb15). Error bars represent ±


A Unmodified ReNcell VM human neuronal progenitor cells were co - plated with comparable Ren - GFP or Ren - APPSL cells into 3 - dimensional Matrigel matrices , and differentiated into neurons for 60 days . Cytosolic GFP is produced by both Ren - GFP and Ren - APPSL cells , and the latter also produce pathogenic human APP with the Swedish and London mutations , and secrete Aβ that becomes entrapped in the Matrige Following neuronal differentiation , 2P - FLIM was used to monitor NiMA in the ReNcell VM neurons , which were discriminated from Ren - GFP and Ren - APPSL neurons because they lack GFP . Note that NiMA was observed in ReNcell VM neurons when they were co - cultured with Ren - GFP neurons , but not when they were co - cultured with Ren - APPSL neurons . Each colored line refers to a single field of view containing 5 - 15 cells , and each pair of solid and dotted lines of the same color refers to the same field of view . Statistical analyses were performed using Student ' s two - tailed unpaired t -

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A Unmodified ReNcell VM human neuronal progenitor cells were co-plated with comparable Ren-GFP or Ren-APPSL cells into 3-dimensional Matrigel matrices, and differentiated into neurons for 60 days. Cytosolic GFP is produced by both Ren-GFP and Ren-APPSL cells, and the latter also produce pathogenic human APP with the Swedish and London mutations, and secrete Aβ that becomes entrapped in the Matrige Following neuronal differentiation, 2P-FLIM was used to monitor NiMA in the ReNcell VM neurons, which were discriminated from Ren-GFP and Ren-APPSL neurons because they lack GFP. Note that NiMA was observed in ReNcell VM neurons when they were co-cultured with Ren-GFP neurons, but not when they were co-cultured with Ren-APPSL neurons. Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines of the same color refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test


B ReNcell VM cells were differentiated into neurons for 30 days in 2D cultures . Subsequent AβO exposure for 16 hours blocked NiMA , but expression of Flag - Raptor - Rheb15 , which forces mTORC1 to lysosomes , prevented NiMA inhibition by AβOs . Each colored line refers to a single field of view containing 5 - 15 cells , and each identically colored pair of solid and dotted lines refers to the same field of view . Flag - Raptor - Rheb15 expression was confirmed by western blot ting . Statistical analyses were performed using Student ' s two - tailed unpaired t - test . Western blots are representative of 3 independent

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B ReNcell VM cells were differentiated into neurons for 30 days in 2D cultures. Subsequent AβO exposure for 16 hours blocked NiMA, but expression of Flag-Raptor-Rheb15, which forces mTORC1 to lysosomes, prevented NiMA inhibition by AβOs. Each colored line refers to a single field of view containing 5-15 cells, and each identically colored pair of solid and dotted lines refers to the same field of view. Flag-Raptor -Rheb15 expression was confirmed by western blotting. Statistical analyses were performed using Student's two-tailed unpaired t-test. Western blots are representative of 3 independent assays


A Cortical mouse neurons from tau knockout ( KO ) mice were serum - starved in Hank ' s balanced salt solution for 2 hours . The cells were then imaged for NiMA , after which insulin or amino acids were immediately added , and the cells were then imaged again 30 minutes later Data information : Each colored lin refers to a single field of view containing 2 - 15 cells , and each pair of solid and dotted lines ( same color ) refers to the same field of view . Statistical analyses were performed using Student ' s two - tailed unpaired t -

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A Cortical mouse neurons from tau knockout (KO) mice were serum-starved in Hank's balanced salt solution for 2 hours. The cells were then imaged for NiMA, after which insulin or amino acids were immediately added, and the cells were then imaged again 30 minutes later Data information: Each colored lin refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test


B Cortical mouse neurons from tau KO mice were treated with AβOs for 18 hours before being serum - starved in AβO - containing Hank ' s balanced salt solution for 2 hours . The cells were then imaged for NiMA , after which insulin was immediately added , and the cells were then imaged again 30 minutes later . Note that NiMA occurred in the absence or presence of AβOs , which inhibited NiMA in wild type human neuron Data information : Each colored lin refers to a single field of view containing 2 - 15 cells , and each pair of solid and dotted lines ( same color ) refers to the same field of view . Statistical analyses were performed using Student ' s two - tailed unpaired t -

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B Cortical mouse neurons from tau KO mice were treated with AβOs for 18 hours before being serum-starved in AβO-containing Hank's balanced salt solution for 2 hours. The cells were then imaged for NiMA, after which insulin was immediately added, and the cells were then imaged again 30 minutes later. Note that NiMA occurred in the absence or presence of AβOs, which inhibited NiMA in wild type human neuron Data information: Each colored lin refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test


Wild type mouse cortical neurons depleted of Nprl with shRNA had mitochondria that were insensitive to amino acids ( R + L Each colored line refers to a single field of view containing 5 - 15 cells , and each pair of solid and dotted lines ( same color ) refers to the same field of view . Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein , a downstream target of the mTORC1 - S6K pathway . Western blots are representative of 3 independent assays . Statistical analyses were performed using Student ' s two - tailed unpaired t -

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Wild type mouse cortical neurons depleted of Nprl with shRNA had mitochondria that were insensitive to amino acids (R+L Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein, a downstream target of the mTORC1-S6K pathway. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test


Wild type mouse cortical neurons depleted o Tsc2 with shRNA had mitochondria tha were insensitive t insuli Each colored line refers to a single field of view containing 5 - 15 cells , and each pair of solid and dotted lines ( same color ) refers to the same field of view . Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein , a downstream target of the mTORC1 - S6K pathway . Western blots are representative of 3 independent assays . Statistical analyses were performed using Student ' s two - tailed unpaired t -

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Wild type mouse cortical neurons depleted o Tsc2 with shRNA had mitochondria tha were insensitive t insuli Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein, a downstream target of the mTORC1-S6K pathway. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test


( D Human fibroblasts deficient in Tsc2 ( TSC2 Δexons1 - 14 ) were imaged before and 30 minutes after insulin addition . Lack of Tsc2 made mitochondria insensitive to insulin Cultures were serum - starved in Hank ' s balanced salt solution for 2 hours before stimulation with insulin or amino acids , and were imaged before and 30 minutes after stimulation . Each colored line refers to a single field of view containing 2 - 5 cells , and each pair of solid and dotted lines of the same color refers to the same field of view Statistical analyses were performed using Student ' s two - tailed unpaired t -

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(D Human fibroblasts deficient in Tsc2 (TSC2 Δexons1-14) were imaged before and 30 minutes after insulin addition. Lack of Tsc2 made mitochondria insensitive to insulin Cultures were serum-starved in Hank's balanced salt solution for 2 hours before stimulation with insulin or amino acids, and were imaged before and 30 minutes after stimulation. Each colored line refers to a single field of view containing 2-5 cells, and each pair of solid and dotted lines of the same color refers to the same field of view Statistical analyses were performed using Student's two-tailed unpaired t-test


( E ) Re - expression of human Tsc2 rescues mitochondrial responses to insulin . Cultures were serum - starved in Hank ' s balanced salt solution for 2 hours before stimulation with insulin or amino acids , and were imaged before and 30 minutes after stimulation . Each colored line refers to a single field of view containing 2 - 5 cells , and each pair of solid and dotted lines of the same color refers to the same field of view . Re - expression of the Flag - WT Tsc2 was corroborated by indirect immun o fluorescence ( IF ) . Statistical analyses were performed using Student ' s two - tailed unpaired t - test

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(E) Re-expression of human Tsc2 rescues mitochondrial responses to insulin. Cultures were serum-starved in Hank's balanced salt solution for 2 hours before stimulation with insulin or amino acids, and were imaged before and 30 minutes after stimulation. Each colored line refers to a single field of view containing 2-5 cells, and each pair of solid and dotted lines of the same color refers to the same field of view. Re-expression of the Flag-WT Tsc2 was corroborated by indirect immunofluorescence (IF). Statistical analyses were performed using Student's two-tailed unpaired t-test


A . Immunofluorescence microscopy of control , parental and patient fibroblasts labeled for LAMP - 1 . Patient fibroblasts ( VPS41S285P / R662 * and VPS41c . 1423 - 2A > G / R662 * ) show more LAMP - 1 puncta , which are distributed throughout the cell . ( A ' ) Quantification shows a significant increase in the number of LAMP - 1 positive compartments for both patients . > 15 Cells per condition were quantified ( n = 3 ) . Scale bars , 10µm .

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A. Immunofluorescence microscopy of control, parental and patient fibroblasts labeled for LAMP-1. Patient fibroblasts (VPS41S285P/R662* and VPS41c.1423-2A>G/R662*) show more LAMP-1 puncta, which are distributed throughout the cell. (A') Quantification shows a significant increase in the number of LAMP-1 positive compartments for both patients. >15 Cells per condition were quantified (n=3). Scale bars, 10µm.


D . HeLaVPS41KO cells transfected with VPS41WT - APEX2 - V5 , VPS41S285P - APEX2 - V5 or VPS41R662 * - APEX2 - V5 constructs labeled for LAMP - 1 and V5 immunofluorescence microscopy . All VPS41 variants co - localize with LAMP - 1 , indicating that VPS41S285P and VPS41R662 * are recruited to late endosomes / lysosomes . Scale bars 10µm ; zoom of squared area , 1µm .

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D. HeLaVPS41KO cells transfected with VPS41WT-APEX2-V5, VPS41S285P-APEX2-V5 or VPS41R662*-APEX2-V5 constructs labeled for LAMP-1 and V5 immunofluorescence microscopy. All VPS41 variants co-localize with LAMP-1, indicating that VPS41S285P and VPS41R662* are recruited to late endosomes/lysosomes. Scale bars 10µm; zoom of squared area, 1µm.


H . VPS41WT / WT , VPS41WT / R662 * and VPS41S285P / R662 * primary fibroblasts incubated with Dextran - 568 for 0 . 5 , 2 , 5 , 8 and 24 hours . Colocalization of Dextran - 568 and SiR - Lysosome Cathepsin D ( SiR - Lyso ) reveals a delay in lysosomal delivery in maternal ( VPS41WT / R662 * ) and patient ( VPS41S285P / R662 * ) cells at 2 hours of Dextran uptake . After 5 hours , maternal cells show similar to control colocalization levels . Patient cells show only after 24 hours colocalization levels similar to control , indicating a delay rather than a block in late endosome - lysosome fusion . > 10 Cells per cell line were quantified .

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H. VPS41WT/WT, VPS41WT/R662* and VPS41S285P/R662* primary fibroblasts incubated with Dextran-568 for 0.5, 2, 5, 8 and 24 hours. Colocalization of Dextran-568 and SiR-Lysosome Cathepsin D (SiR-Lyso) reveals a delay in lysosomal delivery in maternal (VPS41WT/R662*) and patient (VPS41S285P/R662*) cells at 2 hours of Dextran uptake. After 5 hours, maternal cells show similar to control colocalization levels. Patient cells show only after 24 hours colocalization levels similar to control, indicating a delay rather than a block in late endosome - lysosome fusion. >10 Cells per cell line were quantified.


A . Western blot of LC3 expression levels in control and patient fibroblasts . In steady state ( 0 hours ) conditions , patient fibroblasts have a higher ratio of lipidated LC3 ( LC3II ) : LC3I than control cells . Induction of autophagy by nutrient starvation ( 2 hours incubation with minimal EBSS medium ) results in a raise in LC3II : LC3I ratio in both control and patient cells , but in VPS41S285P / R662 * fibroblasts this increase is only modest ( quantified in A ' ) ( n = 2 ) .

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A. Western blot of LC3 expression levels in control and patient fibroblasts. In steady state (0 hours) conditions, patient fibroblasts have a higher ratio of lipidated LC3 (LC3II):LC3I than control cells. Induction of autophagy by nutrient starvation (2 hours incubation with minimal EBSS medium) results in a raise in LC3II:LC3I ratio in both control and patient cells, but in VPS41S285P/R662* fibroblasts this increase is only modest (quantified in A') (n=2).


E . Immunofluorescence of HeLaWT and HeLaVPS41KO cells transfected with LC3GFP / RFP tandem construct . The increased percentage of GFP / RFP - positive compartments in HeLaVPS41KO cells indicate a block in autophagic flux ( quantified in E ' ) . > 12 Cells per cell line were quantified . Scale bars 10µm ; zoom , 1µm .

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E. Immunofluorescence of HeLaWT and HeLaVPS41KO cells transfected with LC3GFP/RFP tandem construct. The increased percentage of GFP/RFP-positive compartments in HeLaVPS41KO cells indicate a block in autophagic flux (quantified in E'). >12 Cells per cell line were quantified. Scale bars 10µm; zoom, 1µm.


A . Immunofluorescence of control , parental and patient fibroblasts labeled for LAMP - 1 and mTOR . In steady state conditions ( 0 hours ) VPS41S285P / R662 * fibroblasts show less colocalization between mTOR and LAMP - 1 . VPS41WT / WT , VPS41WT / S285P and VPS41WT / R662 * show an appropriate mTOR response upon nutrient deprivation ( 2 hours ) or restimulation ( 2 hours starvation followed by 15 minutes restimulation ) ( Appendix Fig S5A ) . > 10 Cells per cell line were quantified ( A ' ) . Quantifications are performed relative to colocalization under steady state conditions per cell line ( n = 3 ) . Scale bars , 10µm ; zoom , 1µm .

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A. Immunofluorescence of control, parental and patient fibroblasts labeled for LAMP-1 and mTOR. In steady state conditions (0 hours) VPS41S285P/R662* fibroblasts show less colocalization between mTOR and LAMP-1. VPS41WT/WT, VPS41WT/S285P and VPS41WT/R662* show an appropriate mTOR response upon nutrient deprivation (2 hours) or restimulation (2 hours starvation followed by 15 minutes restimulation) (Appendix Fig S5A). >10 Cells per cell line were quantified (A'). Quantifications are performed relative to colocalization under steady state conditions per cell line (n=3). Scale bars, 10µm; zoom, 1µm.


D . Rescue experiments of HeLaVPS41KO cells . Expression of VPS41WT - APEX2 - V5 , VPS41S285P - APEX2 - V5 or VPS41R662 * - APEX2 - V5 . Reintroduction of VPS41WT rescues TFE3 localization , whereas expression of mutant VPS41 has no effect ( quantified in D ' ) . > 83 Cells per condition were quantified ( n = 3 ) . Scale bars , 10µm .

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D. Rescue experiments of HeLaVPS41KO cells. Expression of VPS41WT-APEX2-V5, VPS41S285P-APEX2-V5 or VPS41R662*-APEX2-V5. Reintroduction of VPS41WT rescues TFE3 localization, whereas expression of mutant VPS41 has no effect (quantified in D'). >83 Cells per condition were quantified (n=3). Scale bars, 10µm.


B . Western blot on cellular or secreted secretogranin II ( SgII ) of PC12 cells VPS41KO , or VPS41KO transduced with HA - VPS41WT or HA - VPS41S285P lentivirus . Cells were washed and incubated for 30 minutes in Tyrode ' s solution containing 2 . 5 mM K + ( basal ) or 90 mM K + ( stimulated ) . No difference in secreted sgII levels was observed between VPS41WT and VPS41S285P .

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B. Western blot on cellular or secreted secretogranin II (SgII) of PC12 cells VPS41KO, or VPS41KO transduced with HA-VPS41WT or HA-VPS41S285P lentivirus. Cells were washed and incubated for 30 minutes in Tyrode's solution containing 2.5 mM K+ (basal) or 90 mM K+ (stimulated). No difference in secreted sgII levels was observed between VPS41WT and VPS41S285P.


C . Graph representing the percentage of animals with 6 normal dopaminergic neurons in the anterior region of Pdat - 1 : : GFP ; Pdat - 1 : : α - syn ( strain UA44 ) animals with heterozygous expression of hVPS41 variants . Heterozygous expression of hVPS41WT with either variant ( strains UA389 or UA390 ) significantly rescues neurons from α - synuclein - induced degeneration at day 7 whereas compound heterozygous expression ( strain UA391 ) fails to rescue neurodegeneration . At day 10 , none of the heterozygous backgrounds significantly rescues α - synuclein - induced neurodegeneration . n = 30 Adult worms for each of 3 independent experiments for the α - synuclein ( α - syn ) strain ( total of 90 worms ) and n = 90 for each independent transgenic strain ( 30 worms / trial x 3 independent transgenic lines = 270 worms ) for each of 3 independent experiments . D . Representative images of DA neurons from C . elegans expressing Pdat - 1 : : GFP ; Pdat - 1 : : α - syn , with or without hVPS41 variants , as described in ( C ) . Intact DA neurons are indicated with arrowheads and missing neurons are indicated with arrows . Scale bar 20µm .

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

C. Graph representing the percentage of animals with 6 normal dopaminergic neurons in the anterior region of Pdat-1::GFP; Pdat-1:: α-syn (strain UA44) animals with heterozygous expression of hVPS41 variants. Heterozygous expression of hVPS41WT with either variant (strains UA389 or UA390) significantly rescues neurons from α-synuclein-induced degeneration at day 7 whereas compound heterozygous expression (strain UA391) fails to rescue neurodegeneration. At day 10, none of the heterozygous backgrounds significantly rescues α-synuclein-induced neurodegeneration. n=30 Adult worms for each of 3 independent experiments for the α-synuclein (α-syn) strain (total of 90 worms) and n=90 for each independent transgenic strain (30 worms/trial x 3 independent transgenic lines = 270 worms) for each of 3 independent experiments. D. Representative images of DA neurons from C. elegans expressing Pdat-1::GFP; Pdat-1:: α-syn, with or without hVPS41 variants, as described in (C). Intact DA neurons are indicated with arrowheads and missing neurons are indicated with arrows. Scale bar 20µm.


( A ) Immunostaining of API in the vacuole of SEY6210 ( wild - type ) cell showing a dispersed pattern .

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(A) Immunostaining of API in the vacuole of SEY6210 (wild-type) cell showing a dispersed pattern.


( B - D ) Immunostaining of TVY1 ( pep4 ) cells showing API in the Cvt complex . The arrow points to a Cvt complex . Higher magnification images are shown in C and D .

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(B-D) Immunostaining of TVY1 (pep4) cells showing API in the Cvt complex. The arrow points to a Cvt complex. Higher magnification images are shown in C and D.


E ) Freeze - substitution fixation image of Cvt complex in TVY1 cell .

E) Freeze-substitution fixation image of Cvt complex in TVY1 cell.


( F ) Freeze - substitution fixation image of a Cvt complex in strain SEY6210 transformed with a 2μ plasmid encoding API grown in SD medium .

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(F) Freeze-substitution fixation image of a Cvt complex in strain SEY6210 transformed with a 2μ plasmid encoding API grown in SD medium.


( G ) Immunostaining of strain SEY6210 containing a 2μ APE1 plasmid grown in SD medium . N , Nucleus ; V , vacuole .

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(G) Immunostaining of strain SEY6210 containing a 2μ APE1 plasmid grown in SD medium. N, Nucleus; V, vacuole.


( A and B ) Immunostaining image showing Cvt vesicles ( marked with arrows in A ) in the cytosol . Arrowhead and double arrowheads show the inner and outer membrane of Cvt vesicle , respectively .

(A and B) Immunostaining image showing Cvt vesicles (marked with arrows in A) in the cytosol. Arrowhead and double arrowheads show the inner and outer membrane of Cvt vesicle, respectively.


( C ) Freeze - substitution fixation image of Cvt vesicles ( marked with arrows ) .

(C) Freeze-substitution fixation image of Cvt vesicles (marked with arrows).


( D ) Immunostaining of Cvt vesicle ( marked with arrows ) in the cytosol and Cvt body ( marked with double arrows ) in the vacuole .

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(D) Immunostaining of Cvt vesicle (marked with arrows) in the cytosol and Cvt body (marked with double arrows) in the vacuole.


( E and F ) Freeze - substitution fixation image of Cvt bodies in the vacuole ( marked with arrows ) . A Cvt vesicle is marked with an arrowhead .

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(E and F) Freeze-substitution fixation image of Cvt bodies in the vacuole (marked with arrows). A Cvt vesicle is marked with an arrowhead.


( G and H ) Freeze - substitution fixation image of Cvt vesicle contacting and fusing to a vacuole . V , Vacuole ; VM , vacuolar membrane .

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(G and H) Freeze-substitution fixation image of Cvt vesicle contacting and fusing to a vacuole. V, Vacuole; VM, vacuolar membrane.


Cvt complexes in TVY1 ( pep4 ) cells under nitrogen starvation conditions . Logarithmically growing cells in YEPD were transferred to nitrogen starvation medium ( SD [ - N ] ) at 30 ° C for 30 min ( A and B ) or 60 min ( C and D ) and prepared for electron microscopy as described in Fig . 1 . ( A ) Freeze - substitution fixation image showing a Cvt complex in an autophagosome ( marked by an arrow ) in the cytosol . ( B ) Freeze - substitution fixation image showing a Cvt complex in an autophagic body in the vacuole . ( C ) Immunostaining of a Cvt complex in an autophagic body in the vacuole . ( D ) Immunostaining of Cvt vesicles ( marked with arrows ) in the vacuole . AB , Autophagic body ; AP , autophagosome ; V , vacuole .

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Cvt complexes in TVY1 (pep4) cells under nitrogen starvation conditions. Logarithmically growing cells in YEPD were transferred to nitrogen starvation medium (SD[-N]) at 30°C for 30 min (A and B) or 60 min (C and D) and prepared for electron microscopy as described in Fig. 1. (A) Freeze-substitution fixation image showing a Cvt complex in an autophagosome (marked by an arrow) in the cytosol. (B) Freeze-substitution fixation image showing a Cvt complex in an autophagic body in the vacuole. (C) Immunostaining of a Cvt complex in an autophagic body in the vacuole. (D) Immunostaining of Cvt vesicles (marked with arrows) in the vacuole. AB, Autophagic body; AP, autophagosome; V, vacuole.


Transport of Cvt complex in SEY6210 ( wild - type ) harboring 2μ plasmid encoding API under growing and nitrogen starvation conditions . Vegetative cells grown in SD medium were observed ( A - E ) . For starvation , logarithmically growing cells in SD were transferred to SD ( - N ) for 25 min ( F - H ) or 1 h ( I - K ) in the presence of 1 mM PMSF . Samples were prepared for electron microscopy as described in Fig . 1 . ( A ) Freeze - substitution fixation image of Cvt vesicle ( marked with an arrow ) . ( B ) Immunostaining of a Cvt vesicle ( marked with an arrow ) . ( C and D ) Higher magnification image of Cvt vesicle . Arrowhead and double arrowheads show the inner and outer membrane of a Cvt vesicle , respectively . ( E ) Freeze - substitution fixation image of a membrane sac enclosing small portion of a large Cvt complex ( marked with an arrow ) . ( F ) Freeze - substitution image of the wrapping of a Cvt complex by the isolation membrane ( marked with an arrow ) . ( G ) Freeze - substitution image of a Cvt vesicle ( marked with an arrow ) fusing to a vacuole . ( H ) Freeze - substitution image of autophagic body containing a Cvt complex . ( I ) Immunostaining image depicting the wrapping of a Cvt complex . The arrow marks the enwrapping membrane . ( J ) Immunostaining of an autophagosome containing a Cvt complex . ( K ) Immunostaining of an autophagic body containing a Cvt complex in the vacuole . V , vacuole .

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Transport of Cvt complex in SEY6210 (wild-type) harboring 2μ plasmid encoding API under growing and nitrogen starvation conditions. Vegetative cells grown in SD medium were observed (A-E). For starvation, logarithmically growing cells in SD were transferred to SD(-N) for 25 min (F-H) or 1 h (I-K) in the presence of 1 mM PMSF. Samples were prepared for electron microscopy as described in Fig. 1. (A) Freeze-substitution fixation image of Cvt vesicle (marked with an arrow). (B) Immunostaining of a Cvt vesicle (marked with an arrow). (C and D) Higher magnification image of Cvt vesicle. Arrowhead and double arrowheads show the inner and outer membrane of a Cvt vesicle, respectively. (E) Freeze-substitution fixation image of a membrane sac enclosing small portion of a large Cvt complex (marked with an arrow). (F) Freeze-substitution image of the wrapping of a Cvt complex by the isolation membrane (marked with an arrow). (G) Freeze-substitution image of a Cvt vesicle (marked with an arrow) fusing to a vacuole. (H) Freeze-substitution image of autophagic body containing a Cvt complex. (I) Immunostaining image depicting the wrapping of a Cvt complex. The arrow marks the enwrapping membrane. (J) Immunostaining of an autophagosome containing a Cvt complex. (K) Immunostaining of an autophagic body containing a Cvt complex in the vacuole. V, vacuole.


Mutant analysis allows for differentiation between vacuolar delivery by Cvt vesicles and autophagosomes . ( A ) SEY6210 ( wild - type ) , cvt3 , and cvt7 yeast were grown in SD to 1 OD600 U / ml and transferred to SD ( - N ) . Aliquots were collected after 0 h ( + ) and 4 h ( − ) incubation and subjected to immunoblotting . The positions of precursor ( pro ) and mature ( m ) API are indicated . ( B ) cvt3 and cvt7 cells were pulse labeled for 10 min . After addition of cold cysteine and methionine , the cells were harvested , washed , and resuspended in either SD or SD ( - N ) and chased for the indicated times . API was recovered by immunoprecipitation , resolved on SDS polyacrylamide gels , and quantified using a phosphorimager ( STORM ; Molecular Dynamics , Sunnyvale , CA ) .

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Mutant analysis allows for differentiation between vacuolar delivery by Cvt vesicles and autophagosomes. (A) SEY6210 (wild-type), cvt3, and cvt7 yeast were grown in SD to 1 OD600 U/ml and transferred to SD (-N). Aliquots were collected after 0 h (+) and 4 h (−) incubation and subjected to immunoblotting. The positions of precursor (pro) and mature (m) API are indicated. (B) cvt3 and cvt7 cells were pulse labeled for 10 min. After addition of cold cysteine and methionine, the cells were harvested, washed, and resuspended in either SD or SD(-N) and chased for the indicated times. API was recovered by immunoprecipitation, resolved on SDS polyacrylamide gels, and quantified using a phosphorimager (STORM; Molecular Dynamics, Sunnyvale, CA).


Analysis of the Cvt complex in the cvt3 mutant under vegetative and starvation conditions . Vegetative cells were grown in SD medium at 30 ° C to log phase . For starvation , logarithmically growing cells in SD medium were transferred to SD ( - N ) for 5 h . Samples were prepared for electron microscopy as described in Fig . 1 . ( A ) Immunostaining image depicting Cvt complex in the cytosol . Cvt vesicles are not detected in this strain . ( B ) Freeze - substitution fixation image showing Cvt complex in autophagosome ( marked with an arrow ) under starvation conditions , indicating that autophagy can still proceed . AP , Autophagosome ; N , nucleus ; V , vacuole .

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Analysis of the Cvt complex in the cvt3 mutant under vegetative and starvation conditions. Vegetative cells were grown in SD medium at 30°C to log phase. For starvation, logarithmically growing cells in SD medium were transferred to SD(-N) for 5 h. Samples were prepared for electron microscopy as described in Fig. 1. (A) Immunostaining image depicting Cvt complex in the cytosol. Cvt vesicles are not detected in this strain. (B) Freeze-substitution fixation image showing Cvt complex in autophagosome (marked with an arrow) under starvation conditions, indicating that autophagy can still proceed. AP, Autophagosome; N, nucleus; V, vacuole.


A Heat map from RNA - Seq data from FACS - isolated muscle stem cells and single myofibers showing relative expression of Pax7 , Myf5 , MyoD , Myog and Myf6 .

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A Heat map from RNA-Seq data from FACS-isolated muscle stem cells and single myofibers showing relative expression of Pax7, Myf5, MyoD, Myog and Myf6.


H Colormap of Myf5 , MyoD and Myf6 peaks within 100kb of the Transcription Start Sites ( TSS ) of myokines ( supplemental table EV3 ) ranging from zero ( black ) to six peaks ( red ) occupancy . The onset of differentiation coincides with increased binding of MRFs to the regulatory domains of the myokines genes .

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H Colormap of Myf5, MyoD and Myf6 peaks within 100kb of the Transcription Start Sites (TSS) of myokines (supplemental table EV3) ranging from zero (black) to six peaks (red) occupancy. The onset of differentiation coincides with increased binding of MRFs to the regulatory domains of the myokines genes.


A Gene expression analysis of myokines ( table EV3 ) during a five day time course of myogenic differentiation going from cycling myoblasts in growth media ( Ham ' s F10 supplemented with 20 % Fetal Bovine Serum , 1 % penicillin / streptomycin , 2 . 5 ng / ml basic Fibroblast Growth Factor ) to terminally differentiated myocytes ( 2 days in differentiation media , DMEM supplemented with 5 % horse serum ) to post mitotic multinucleated myotubes ( 5 days in differentiation media ) . Gene expression was assayed in biological triplicate by microarray ( Soleimani et al . , 2018 ; Soleimani et al . , 2012a ) . Expression of each gene was averaged across three replicates and normalized to mean zero and standard deviation of one . Expression values were then truncated at + / - 3 for color display . Blue indicates lower than average expression , while red indicates higher than average expression . B Distribution of Myf6 peaks within 100kb of the TSS of the myokine genes ( table EV3 ) , similar to the analysis shown in ( A ) .

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A Gene expression analysis of myokines (table EV3) during a five day time course of myogenic differentiation going from cycling myoblasts in growth media (Ham's F10 supplemented with 20% Fetal Bovine Serum, 1% penicillin/streptomycin, 2.5 ng/ml basic Fibroblast Growth Factor) to terminally differentiated myocytes (2 days in differentiation media, DMEM supplemented with 5% horse serum) to post mitotic multinucleated myotubes (5 days in differentiation media). Gene expression was assayed in biological triplicate by microarray (Soleimani et al., 2018; Soleimani et al., 2012a). Expression of each gene was averaged across three replicates and normalized to mean zero and standard deviation of one. Expression values were then truncated at +/- 3 for color display. Blue indicates lower than average expression, while red indicates higher than average expression. B Distribution of Myf6 peaks within 100kb of the TSS of the myokine genes (table EV3), similar to the analysis shown in (A).


C - E Snapshots of the UCSC genome browser showing enrichment of Myf6 ChIP - Seq reads compared to control around EGF ( C ) , LIF and OSM ( D ) , and VEGFA ( E ) loci superimposed on reads for Histone H3 lysine 4 mono methyl ( H3K4me1 ) , Histone H3 lysine 4 tri methyl ( H3K4me3 ) and total H3 in myoblasts ( pink ) and myotubes ( green ) .

C-E Snapshots of the UCSC genome browser showing enrichment of Myf6 ChIP-Seq reads compared to control around EGF (C), LIF and OSM (D), and VEGFA (E) loci superimposed on reads for Histone H3 lysine 4 mono methyl (H3K4me1), Histone H3 lysine 4 tri methyl (H3K4me3) and total H3 in myoblasts (pink) and myotubes (green).