( A ) P . berghei sporozoite trajectories obtained with manual ( left ) and automated ( right ) tracking methods . ( B ) Image processing and automated tracking method . Shown are images illustrating each step of automated tracking : The raw data image shows the first frame of time - lapse microscopy of P . berghei sporozoites ( red ) after intradermal inoculation , followed by the same image after background subtraction and thresholding using Fiji software . Spot detection was run using ICY software and the final image shows the overlay with tracks generated using the spot tracking plugin of ICY software . Scale bars , 50 μm . ( C ) Apparent speed of sporozoites 5 to 120 min after intradermal inoculation , obtained with manual and automated tracking methods . Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually . Horizontal line indicates the median speed . No statistically significant differences between the corresponding timepoints of manual and automated tracking methods were found ( Kruskal - Wallis test ) . ( D ) Displacement of sporozoites 5 to 120 min after intradermal inoculation , obtained with manual and automated tracking methods . To normalize between tracks of different duration , displacement is displayed as final displacement per 30 seconds interval . Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually . Horizontal line indicates the median . The only statistically significant difference in sporozoite displacement between the manual and automated tracking methods was at 120 minutes ( Kruskal - Wallis test , * * * * p < 0 . 0001 ) . For panels C & D , a varying number of videos were processed for each time point : C : 5 min ( 3 videos / 37 manual tracks / 203 automated tracks ) , 10 min ( 6 videos / 112 manual tracks / 292 automated tracks ) , 20 min ( 4 videos / 95 manual tracks / 184 automated tracks ) , 30 min ( 6 videos / 63 manual tracks / 235 automated tracks ) , 60 min ( 7 videos / 77 manual tracks / 162 automated tracks ) , and 120 min ( 6 videos / 44 manual tracks / 151 automated tracks ) . D : 5 min ( 3 videos / 61 manual tracks / 203 automated tracks ) , 10 min ( 6 videos / 74 manual tracks / 292 automated tracks ) , 20 min ( 4 videos / 47 manual tracks / 184 automated tracks ) , 30 min ( 6 videos / 53 manual tracks / 235 automated tracks ) , 60 min ( 7 videos / 65 manual tracks / 162 automated tracks ) , and 120 min ( 6 videos / 48 manual tracks / 151 automated tracks ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) P. berghei sporozoite trajectories obtained with manual (left) and automated (right) tracking methods. (B) Image processing and automated tracking method. Shown are images illustrating each step of automated tracking: The raw data image shows the first frame of time-lapse microscopy of P. berghei sporozoites (red) after intradermal inoculation, followed by the same image after background subtraction and thresholding using Fiji software. Spot detection was run using ICY software and the final image shows the overlay with tracks generated using the spot tracking plugin of ICY software. Scale bars, 50 μm. (C) Apparent speed of sporozoites 5 to 120 min after intradermal inoculation, obtained with manual and automated tracking methods. Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal line indicates the median speed. No statistically significant differences between the corresponding timepoints of manual and automated tracking methods were found (Kruskal-Wallis test). (D) Displacement of sporozoites 5 to 120 min after intradermal inoculation, obtained with manual and automated tracking methods. To normalize between tracks of different duration, displacement is displayed as final displacement per 30 seconds interval. Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal line indicates the median. The only statistically significant difference in sporozoite displacement between the manual and automated tracking methods was at 120 minutes (Kruskal-Wallis test, ****p<0.0001). For panels C&D, a varying number of videos were processed for each time point: C: 5 min (3 videos/37 manual tracks/203 automated tracks), 10 min (6 videos/112 manual tracks/292 automated tracks), 20 min (4 videos/95 manual tracks/184 automated tracks), 30 min (6 videos/63 manual tracks/235 automated tracks), 60 min (7 videos/77 manual tracks/162 automated tracks), and 120 min (6 videos/44 manual tracks/151 automated tracks). D: 5 min (3 videos/61 manual tracks/203 automated tracks), 10 min (6 videos/74 manual tracks/292 automated tracks), 20 min (4 videos/47 manual tracks/184 automated tracks), 30 min (6 videos/53 manual tracks/235 automated tracks), 60 min (7 videos/65 manual tracks/162 automated tracks), and 120 min (6 videos/48 manual tracks/151 automated tracks).


( A - C ) Tracks generated through automated tracking were plotted to a common origin to visualize parasite dispersal P . berghei ( A ) , P . yoelii ( B ) and P . falciparum ( C ) at 5 , 10 , 30 and 60 min after intradermal inoculation . For each timepoint , the tracks are representative of the average displacement of the entire sporozoite population . Track numbers used for each timepoint were : 5 min , 40 tracks ; 10 min , 32 tracks ; 30 min , 25 tracks ; 60 min , 21 tracks . ( D - F ) Displacement of P . berghei ( D ) , P . yoelii ( E ) and P . falciparum ( F ) sporozoites 5 - 120 min after inoculation in box and whisker plots with whiskers displaying the 10 - 90 percentiles and values below and above the whiskers shown individually . Horizontal lines show median displacement at each time point . Percentage values in boxes indicate the fraction of tracks displacing over 75 micrometers at each time point . Within a given species , the difference in sporozoite displacement between 5 min and 120 min is statistically significant ( Kruskal - Wallis test : P . berghei p < 0 . 0001 , P . yoelii p < 0 . 0001 , P . falciparum p < 0 . 01 ) . Comparisons between species showed no statistically significant difference in displacements for any given time point , with the exception of the 10 min time point in which there was a significant difference between P . berghei and P . falciparum ( Kruskal - Wallis test , * p < 0 . 05 ) . For panels ( D - F ) , a varying number of videos were processed for each time point after inoculation : ( D ) Same dataset as used for Figure 2D : 5 min ( 3 videos / 203 tracks ) , 10 min ( 6 videos / 292 tracks ) , 20 min ( 4 videos / 184 tracks ) , 30 min ( 6 videos / 235 tracks ) , 60 min ( 7 videos / 162 tracks ) and 120 min ( 6 videos / 151 tracks ) . ( E ) 5 min ( 7 videos / 714 tracks ) , 10 min ( 16 videos / 1514 tracks ) , 20 min ( 4 videos / 415 tracks ) , 30 min ( 4 videos / 319 tracks ) , 60 min ( 4 videos / 255 tracks ) , and 120 min ( 4 videos / 180 tracks ) . ( F ) 5 min ( 6 videos / 389 tracks ) , 10 min ( 6 videos / 464 tracks ) , 20 min ( 3 videos / 184 tracks ) , 30 min ( 2 videos / 195 tracks ) , 60 min ( 2 videos / 239 tracks ) , and 120 min ( 2 videos / 103 tracks ) .

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

(A-C) Tracks generated through automated tracking were plotted to a common origin to visualize parasite dispersal P. berghei (A), P. yoelii (B) and P. falciparum (C) at 5, 10, 30 and 60 min after intradermal inoculation. For each timepoint, the tracks are representative of the average displacement of the entire sporozoite population. Track numbers used for each timepoint were: 5 min, 40 tracks; 10 min, 32 tracks; 30 min, 25 tracks; 60 min, 21 tracks. (D-F) Displacement of P. berghei (D), P. yoelii (E) and P. falciparum (F) sporozoites 5-120 min after inoculation in box and whisker plots with whiskers displaying the 10-90 percentiles and values below and above the whiskers shown individually. Horizontal lines show median displacement at each time point. Percentage values in boxes indicate the fraction of tracks displacing over 75 micrometers at each time point. Within a given species, the difference in sporozoite displacement between 5 min and 120 min is statistically significant (Kruskal-Wallis test: P. berghei p<0.0001, P. yoelii p<0.0001, P. falciparum p<0.01). Comparisons between species showed no statistically significant difference in displacements for any given time point, with the exception of the 10 min time point in which there was a significant difference between P. berghei and P. falciparum (Kruskal-Wallis test, *p<0.05). For panels (D-F), a varying number of videos were processed for each time point after inoculation: (D) Same dataset as used for Figure 2D: 5 min (3 videos/203 tracks), 10 min (6 videos/292 tracks), 20 min (4 videos/184 tracks), 30 min (6 videos/235 tracks), 60 min (7 videos/162 tracks) and 120 min (6 videos/151 tracks). (E) 5 min (7 videos/714 tracks), 10 min (16 videos/1514 tracks), 20 min (4 videos/415 tracks), 30 min (4 videos/319 tracks), 60 min (4 videos/255 tracks), and 120 min (4 videos/180 tracks). (F) 5 min (6 videos/389 tracks), 10 min (6 videos/464 tracks), 20 min (3 videos/184 tracks), 30 min (2 videos/195 tracks), 60 min (2 videos/239 tracks), and 120 min (2 videos/103 tracks).


( A ) Speed of P . berghei , P . yoelii and P . falciparum sporozoites 5 - 120 min after inoculation in box and whisker plots with whiskers displaying the 10 - 90 percentiles and values below and above the whiskers shown individually . Horizontal lines show the median speed at each time point . Comparisons among the species showed statistically significant difference in sporozoite speeds at the 20 min time point ( Kruskal - Wallis test , * * * * p < 0 . 0001 for both comparisons ) and the 120 min time point ( Kruskal - Wallis test , * * p < 0 . 006 for both comparisons ) . ( B ) Linear regression of apparent speed shows a more rapid decrease for P . berghei ( slope = - 0 . 005 ) than for P . yoelii and P . falciparum ( slopes = - 0 . 002 ) . Symbols indicate mean values + / - standard deviation . A varying number of videos were processed for each time point after inoculation , using the same data set used for Figure 3 : P . berghei ; 5 min ( 3 videos / 203 tracks ) , 10 min ( 6 videos / 292 tracks ) , 20 min ( 4 videos / 184 tracks ) , 30 min ( 6 videos / 235 tracks ) , 60 min ( 7 videos / 162 tracks ) , and 120 min ( 6 videos / 151 tracks ) . P . yoelii ; 5 min ( 7 videos / 714 tracks ) , 10 min ( 16 videos / 1514 tracks ) , 20 min ( 4 videos / 415 tracks ) , 30 min ( 4 videos / 319 tracks ) , 60 min ( 4 videos / 255 tracks ) , and 120 min ( 4 videos / 180 tracks ) . P . falciparum ; 5 min ( 6 videos / 389 tracks ) , 10 min ( 6 videos / 464 tracks ) , 20 min ( 3 videos / 184 tracks ) , 30 min ( 2 videos / 195 tracks ) , 60 min ( 2 videos / 239 tracks ) , and 120 min ( 2 videos / 103 tracks ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Speed of P. berghei, P. yoelii and P. falciparum sporozoites 5-120 min after inoculation in box and whisker plots with whiskers displaying the 10-90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median speed at each time point. Comparisons among the species showed statistically significant difference in sporozoite speeds at the 20 min time point (Kruskal-Wallis test, ****p<0.0001 for both comparisons) and the 120 min time point (Kruskal-Wallis test, **p<0.006 for both comparisons). (B) Linear regression of apparent speed shows a more rapid decrease for P. berghei (slope = -0.005) than for P. yoelii and P. falciparum (slopes = -0.002). Symbols indicate mean values +/- standard deviation. A varying number of videos were processed for each time point after inoculation, using the same data set used for Figure 3: P. berghei; 5 min (3 videos/203 tracks), 10 min (6 videos/292 tracks), 20 min (4 videos/184 tracks), 30 min (6 videos/235 tracks), 60 min (7 videos/162 tracks), and 120 min (6 videos/151 tracks). P. yoelii; 5 min (7 videos/714 tracks), 10 min (16 videos/1514 tracks), 20 min (4 videos/415 tracks), 30 min (4 videos/319 tracks), 60 min (4 videos/255 tracks), and 120 min (4 videos/180 tracks). P. falciparum; 5 min (6 videos/389 tracks), 10 min (6 videos/464 tracks), 20 min (3 videos/184 tracks), 30 min (2 videos/195 tracks), 60 min (2 videos/239 tracks), and 120 min (2 videos/103 tracks).


( A ) The number of motile and non - motile sporozoites moving in the dermis of mice was manually counted in time courses spanning 120 minutes and is displayed as a percentage of total sporozoites observed 5 min after inoculation , for P . berghei ( left panel ) , P . yoelii ( center panel ) , and P . falciparum ( right panel ) . ( B ) Fold - change in the number of motile sporozoites at 60 and 120 min post - inoculation compared to the number of motile sporozoites at 5 min . The data shown only includes complete imaging sessions over 120 min after intradermal injection of sporozoites . ( P . berghei : n = 2 , P . yoelii n = 4 , P . falciparum n = 2 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The number of motile and non-motile sporozoites moving in the dermis of mice was manually counted in time courses spanning 120 minutes and is displayed as a percentage of total sporozoites observed 5 min after inoculation, for P. berghei (left panel), P. yoelii (center panel), and P. falciparum (right panel). (B) Fold-change in the number of motile sporozoites at 60 and 120 min post-inoculation compared to the number of motile sporozoites at 5 min. The data shown only includes complete imaging sessions over 120 min after intradermal injection of sporozoites. (P. berghei: n=2, P. yoelii n=4, P. falciparum n=2).


( A ) Maximum intensity projection shows trajectories of moving P . berghei , P . yoelii and P . falciparum sporozoites ( green ) , engaging with CD31 - labeled vascular endothelia ( magenta ) . Scale bars , 25 μm . See movies EV4 - EV6 . ( B ) Lymphatic and blood vessel invasion of P . berghei , P . yoelii and P . falciparum sporozoites moving in mouse dermis . Combined invasion events observed in 4 min videos recorded at 5 , 10 and 20 min after intradermal injection of sporozoites ( left panel ) and 60 and 120 min after intradermal injection ( right panel ) . n = number of videos scored for each Plasmodium species . To view a blood vessel entry event by P . yoelii or P . falciparum , see movies EV7 and EV8 . There were no statistically significant differences in lymphatic or blood vessel entry among the three Plasmodium species at 5 to 20 min after sporozoite inoculation whereas there was a statistically significant increase in lymphatic entry by P . falciparum sporozoites compared to P . berghei and P . yoelii sporozoites at 60 to 120 min after inoculation ( Kruskal - Wallis test , p < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Maximum intensity projection shows trajectories of moving P. berghei, P. yoelii and P. falciparum sporozoites (green), engaging with CD31-labeled vascular endothelia (magenta). Scale bars, 25 μm. See movies EV4 - EV6. (B) Lymphatic and blood vessel invasion of P. berghei, P. yoelii and P. falciparum sporozoites moving in mouse dermis. Combined invasion events observed in 4 min videos recorded at 5, 10 and 20 min after intradermal injection of sporozoites (left panel) and 60 and 120 min after intradermal injection (right panel). n = number of videos scored for each Plasmodium species. To view a blood vessel entry event by P. yoelii or P. falciparum, see movies EV7 and EV8. There were no statistically significant differences in lymphatic or blood vessel entry among the three Plasmodium species at 5 to 20 min after sporozoite inoculation whereas there was a statistically significant increase in lymphatic entry by P. falciparum sporozoites compared to P. berghei and P. yoelii sporozoites at 60 to 120 min after inoculation (Kruskal-Wallis test, p<0.05).


( A ) Maximum intensity projection of video acquired 10 min after inoculation of P . falciparum sporozoites visualizes trajectories of parasites , engaging with CD31 - labeled vascular endothelia ( magenta ) . Scale bar , 50 μm . See movie EV9 . ( B ) Displacement of sporozoites 5 to 60 min after inoculation . Data is displayed in box and whisker plots with whiskers showing the 10 to 90 percentiles and values below and above the whiskers shown individually . Horizontal lines show the median at each time point . Percentage values indicate the fraction of tracks displacing over 75 microns . ( C ) Apparent speed of sporozoites 5 to 60 min after inoculation . Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually . Horizontal lines show the median speed at each time point . For panels B - C , data shown originates from the analysis of 3 - 4 videos per time point and a varying number of tracks were processed for each time point after inoculation : 5 min ( 157 tracks ) , 10 min ( 568 tracks ) , 20 min ( 286 tracks ) , 30 min ( 171 tracks ) , 60 min ( 101 tracks ) . No motile sporozoites were observed 120 min after inoculation . ( D ) Proportion of motile and non - motile sporozoites was manually counted and is displayed as percentage of sporozoites observed 5 min after inoculation . The data shown was obtained from one complete imaging session over 120 min after intradermal injection of sporozoites .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Maximum intensity projection of video acquired 10 min after inoculation of P. falciparum sporozoites visualizes trajectories of parasites, engaging with CD31-labeled vascular endothelia (magenta). Scale bar, 50 μm. See movie EV9. (B) Displacement of sporozoites 5 to 60 min after inoculation. Data is displayed in box and whisker plots with whiskers showing the 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median at each time point. Percentage values indicate the fraction of tracks displacing over 75 microns. (C) Apparent speed of sporozoites 5 to 60 min after inoculation. Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median speed at each time point. For panels B-C, data shown originates from the analysis of 3-4 videos per time point and a varying number of tracks were processed for each time point after inoculation: 5 min (157 tracks), 10 min (568 tracks), 20 min (286 tracks), 30 min (171 tracks), 60 min (101 tracks). No motile sporozoites were observed 120 min after inoculation. (D) Proportion of motile and non-motile sporozoites was manually counted and is displayed as percentage of sporozoites observed 5 min after inoculation. The data shown was obtained from one complete imaging session over 120 min after intradermal injection of sporozoites.


( E ) Sporozoite track straightness , the ratio of displacement to track length of P . falciparum sporozoites moving in mouse dermis , human skin ex vivo , and human skin graft , at 5 - 10 min post intradermal injection . Data is displayed in box and whisker plots with horizontal line showing the median and the whiskers extending to the minimum and maximum values . Comparisons showed statistically significant differences in track straightness between the human skin graft and both mouse skin ( * * * * p = 0 . 0001 ) and human skin ex vivo ( * * * * p < 0 . 0001 ) using Tukey ' s multiple comparisons test . A varying number of tracks were processed for each environment : mouse skin : 17 videos , 822 tracks ; human skin ex vivo : 12 videos , 761 tracks ; human skin graft : 9 videos , 718 tracks .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Sporozoite track straightness, the ratio of displacement to track length of P. falciparum sporozoites moving in mouse dermis, human skin ex vivo, and human skin graft, at 5-10 min post intradermal injection. Data is displayed in box and whisker plots with horizontal line showing the median and the whiskers extending to the minimum and maximum values. Comparisons showed statistically significant differences in track straightness between the human skin graft and both mouse skin (****p=0.0001) and human skin ex vivo (****p<0.0001) using Tukey's multiple comparisons test. A varying number of tracks were processed for each environment: mouse skin: 17 videos, 822 tracks; human skin ex vivo: 12 videos, 761 tracks; human skin graft: 9 videos, 718 tracks.


( F ) P . falciparum sporozoites engage with and enter blood vessels in human skin graft . Maximum intensity projection shows trajectories of moving P . falciparum sporozoites ( green ) , engaging with human CD31 - labeled vascular endothelia ( magenta ) . Scale bar , 25 μm . See movie EV10 . Graph shows lymphatic and blood vessel entry events of P . falciparum sporozoites in human skin graft . Combined invasion events observed at 5 - 20 min after intradermal injection in n number of videos . To view a blood vessel invasion event P . falciparum in human skin graft ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) P. falciparum sporozoites engage with and enter blood vessels in human skin graft. Maximum intensity projection shows trajectories of moving P. falciparum sporozoites (green), engaging with human CD31-labeled vascular endothelia (magenta). Scale bar, 25 μm. See movie EV10. Graph shows lymphatic and blood vessel entry events of P. falciparum sporozoites in human skin graft. Combined invasion events observed at 5-20 min after intradermal injection in n number of videos. To view a blood vessel invasion event P. falciparum in human skin graft,


( A ) Activity of mAb 2A10 on sporozoite speed and displacement : Data are displayed in box and whisker plots with whiskers displaying the 10 to 90 percentiles and values below and above the whiskers shown individually . Horizontal lines show the median . For the displacement data , percentage values indicate the fraction of tracks displacing over 75 microns . Statistical analysis : Mann - Whitney , p = 0 . 0001 ( speed data ) and p = 0 . 0098 ( displacement data ) . Data shown originates from the analysis of 5 videos obtained at 5 separate imaging sessions , which generated a total number of 217 ( naïve ) and 136 ( 2A10 ) sporozoite tracks .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Activity of mAb 2A10 on sporozoite speed and displacement: Data are displayed in box and whisker plots with whiskers displaying the 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median. For the displacement data, percentage values indicate the fraction of tracks displacing over 75 microns. Statistical analysis: Mann-Whitney, p=0.0001 (speed data) and p=0.0098 (displacement data). Data shown originates from the analysis of 5 videos obtained at 5 separate imaging sessions, which generated a total number of 217 (naïve) and 136 (2A10) sporozoite tracks.


( B ) Activity of mAb 2A10 on the proportion of motile sporozoites ( left panel ) and blood vessel entry ( right panel ) . Four - minute videos of mice inoculated with mAb 2A10 and naïve controls were manually counted at 10 min post P . falciparum sporozoite inoculation . 5 videos for each condition , from 5 independent imaging sessions were scored for motility . 4 videos per condition from 4 independent imaging sessions were analyzed for blood vessel entry . Statistical analysis : Percent motile , Mann - Whitney , p = 0 . 0317 ; Blood vessel entry , Fisher ' s exact test , p = 0 . 0163

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Activity of mAb 2A10 on the proportion of motile sporozoites (left panel) and blood vessel entry (right panel). Four-minute videos of mice inoculated with mAb 2A10 and naïve controls were manually counted at 10 min post P. falciparum sporozoite inoculation. 5 videos for each condition, from 5 independent imaging sessions were scored for motility. 4 videos per condition from 4 independent imaging sessions were analyzed for blood vessel entry. Statistical analysis: Percent motile, Mann-Whitney, p=0.0317; Blood vessel entry, Fisher's exact test, p=0.0163


( B ) Activity of mAb 2A10 on the proportion of motile sporozoites ( left panel ) and blood vessel entry ( right panel ) . Four - minute videos of mice inoculated with mAb 2A10 and naïve controls were manually counted at 10 min post P . falciparum sporozoite inoculation . 5 videos for each condition , from 5 independent imaging sessions were scored for motility . 4 videos per condition from 4 independent imaging sessions were analyzed for blood vessel entry . Statistical analysis : Percent motile , Mann - Whitney , p = 0 . 0317 ; Blood vessel entry , Fisher ' s exact test , p = 0 .

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( C ) Activity of human mAbs 10 and CIS43 , specific for P . falciparum CSP , and VRC01 , an isotype control human mAb specific for HIV - 1 , on the proportion of motile sporozoites ( left panel ) and blood vessel entry ( right panel ) . Four - minute videos of mice inoculated with the indicated mAbs were taken 10 min after sporozoite inoculation and manually counted . Data are pooled from 7 biological replicates , naïve and VRC01 ( n = 2 ) , naïve and mAb 10 ( n = 2 ) , VRC01 and CIS43 ( n = 3 ) . No comparisons were statistically significant ( Mann - Whitney for percent motile and Fisher ' s exact test for blood vessel entry ) .

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(C) Activity of human mAbs 10 and CIS43, specific for P. falciparum CSP, and VRC01, an isotype control human mAb specific for HIV-1, on the proportion of motile sporozoites (left panel) and blood vessel entry (right panel). Four-minute videos of mice inoculated with the indicated mAbs were taken 10 min after sporozoite inoculation and manually counted. Data are pooled from 7 biological replicates, naïve and VRC01 (n=2), naïve and mAb 10 (n=2), VRC01 and CIS43 (n=3). No comparisons were statistically significant (Mann-Whitney for percent motile and Fisher's exact test for blood vessel entry).


( A ) Analysis of the single - cell transcriptomics dataset for the mouse visual cortex ( Tasic et al , 2016 ) revealed that the expression of the metabolism - associated gene Cox6a2 is highly restricted to PV + interneurons . Cox6a2 is expressed in all subtypes of PV + interneurons ( marked in blue color ) but rarely detected in other neuronal subtypes from the mouse visual cortex . Cox6a1 , which encodes the other protein isoform of COX6A , is expressed in all subtypes .

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(A) Analysis of the single-cell transcriptomics dataset for the mouse visual cortex (Tasic et al, 2016) revealed that the expression of the metabolism-associated gene Cox6a2 is highly restricted to PV+ interneurons. Cox6a2 is expressed in all subtypes of PV+ interneurons (marked in blue color) but rarely detected in other neuronal subtypes from the mouse visual cortex. Cox6a1, which encodes the other protein isoform of COX6A, is expressed in all subtypes.


( B RT - PCR showing expression of Cox6a2 in the brain and heart .

(B RT-PCR showing expression of Cox6a2 in the brain and heart.


C ) Western - blot showing expression of Cox6a2 in the brain and heart .

C) Western-blot showing expression of Cox6a2 in the brain and heart.


( D ) Western - blot analysis showed that COX6A2 is first detected in the brain at postnatal day 0 ( P0 ) . Then , COX6A2 protein levels gradually increase until P60 .

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(D) Western-blot analysis showed that COX6A2 is first detected in the brain at postnatal day 0 (P0). Then, COX6A2 protein levels gradually increase until P60.


COX6A2 immunoreactivity is detected in PV + interneurons in different brain areas of adult mice , including the cortex ( general overview in E , and single PV + interneuron magnification in F ) White arrowheads indicate PV / Cox6a2 double positive cells .

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COX6A2 immunoreactivity is detected in PV+ interneurons in different brain areas of adult mice, including the cortex (general overview in E, and single PV+ interneuron magnification in F) White arrowheads indicate PV/Cox6a2 double positive cells.


COX6A2 immunoreactivity is detected in PV + interneurons in hippocampus ( G ) and striatum ( H ) . White arrowheads indicate PV / Cox6a2 double positive cells .

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COX6A2 immunoreactivity is detected in PV+ interneurons in hippocampus (G) and striatum (H). White arrowheads indicate PV/Cox6a2 double positive cells.


( I , J ) Quantifications showing almost 100 % of co - localization of COX6A2 + cells and PV + cells in the somatosensory ( SS ) and motor ( MT ) cortices , and the hippocampus ( HI ) of adult mice ( n animals = 4 , 2 months old , n sections = 24 SS , 16 MT , 8 HI ) .

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(I,J) Quantifications showing almost 100% of co-localization of COX6A2+ cells and PV+ cells in the somatosensory (SS) and motor (MT) cortices, and the hippocampus (HI) of adult mice (n animals=4, 2 months old, n sections=24 SS, 16 MT, 8 HI).


( K ) COX6A2 immunoreactivity in the cortex at P9 ( n animals = 2 , n sections = 12 SS , 8 MT , 4 HI ) . White arrowheads indicate Cox6a2 positive cells .

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(K) COX6A2 immunoreactivity in the cortex at P9 (n animals=2, n sections=12 SS, 8 MT, 4 HI). White arrowheads indicate Cox6a2 positive cells.


( L - N ) As soon as parvalbumin immunoreactivity becomes visible at P13 , almost 100 % of PV + cells are co - labelled with COX6A2 immunoreactivity in the SS and MT cortices , and the HI of P13 mice ( n = 2 ) .

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(L-N) As soon as parvalbumin immunoreactivity becomes visible at P13, almost 100% of PV+ cells are co-labelled with COX6A2 immunoreactivity in the SS and MT cortices, and the HI of P13 mice (n=2).


( A , B ) COX6A2 immunoreactivity is virtually restricted to PV + interneurons in the somatosensory ( SS ) and motor ( MT ) cortices , and the hippocampus ( HI ) of adult rat ( n animals = 1 , 2 . 5 months old , n sections = 12 SS , 8 MT , 4 HI ) . White arrowheads indicate PV / Cox6a2 double positive cells .

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(A,B) COX6A2 immunoreactivity is virtually restricted to PV+ interneurons in the somatosensory (SS) and motor (MT) cortices, and the hippocampus (HI) of adult rat (n animals=1, 2.5 months old, n sections=12 SS, 8 MT, 4 HI). White arrowheads indicate PV/Cox6a2 double positive cells.


( C , D ) In the SS of adult rhesus monkey , COX6A2 immunoreactivity is present exclusively in PV + cells ( n animals = 1 , 1 - year - old , n sections = 6 ) . White arrowheads indicate PV / Cox6a2 double positive cells .

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(C,D) In the SS of adult rhesus monkey, COX6A2 immunoreactivity is present exclusively in PV+ cells (n animals=1, 1-year-old, n sections=6). White arrowheads indicate PV/Cox6a2 double positive cells.


( E , F ) In adult humans , all cortical PV + interneurons from Brodmann areas 9 and 10 ( BA9 and BA10 ) are co - labelled with COX6A2 immunoreactivity . However , PV + interneurons only represent 60 % of the cells in which COX6A2 is detected ( n human subjects = 2 , 59 - and 69 - year - old , n sections = 6 BA9 , 6 BA10 ) . White arrowheads indicate PV / Cox6a2 double positive cells .

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(E,F) In adult humans, all cortical PV+ interneurons from Brodmann areas 9 and 10 (BA9 and BA10) are co-labelled with COX6A2 immunoreactivity. However, PV+ interneurons only represent 60% of the cells in which COX6A2 is detected (n human subjects=2, 59- and 69-year-old, n sections=6 BA9, 6 BA10). White arrowheads indicate PV/Cox6a2 double positive cells.


( A ) Images showing absence of COX6A2 immunoreactivity in the cortex of Cox6a2 - / - mice .

(A) Images showing absence of COX6A2 immunoreactivity in the cortex of Cox6a2-/- mice.


( B , C ) Parvalbumin immunoreactivity in PV + interneurons was significantly decreased in the somatosensory cortex of Cox6a2 - / - mice compared to wild - types ( n = 4 WT , 4 Cox6a2 - / - ; 2 - month - old ) . C : two - tailed unpaired Student ' s t - test .

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(B,C) Parvalbumin immunoreactivity in PV+ interneurons was significantly decreased in the somatosensory cortex of Cox6a2-/- mice compared to wild-types (n=4 WT, 4 Cox6a2-/-; 2-month-old). C: two-tailed unpaired Student's t-test.


( D ) In Cox6a2 - / - mice , cortical PV + interneurons showed a fewer number of synapses innervating principal neurons ( n = 3 WT , 3 Cox6a2 - / - ; 2 - month - old ) . Two - tailed unpaired Student ' s t - test .

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(D) In Cox6a2-/- mice, cortical PV+ interneurons showed a fewer number of synapses innervating principal neurons (n=3 WT, 3 Cox6a2-/-; 2-month-old). Two-tailed unpaired Student's t-test.


( E ) To analyze changes in oxidative stress in PV + interneurons , the somatosensory and motor cortices of WT and Cox6a2 - / - mice were stained with an antibody against 8 - OHdG , a marker of oxidative DNA damage . ( F ) 8 - OHdG immunoreactivity was significantly higher in PV + null in both cortices in Cox6a2 - / - mice ( n = 4 WT , 4 Cox6a2 - / - ; 2 - month - old ) . Two - tailed unpaired Mann - Whitney tests .

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(E) To analyze changes in oxidative stress in PV+ interneurons, the somatosensory and motor cortices of WT and Cox6a2-/- mice were stained with an antibody against 8-OHdG, a marker of oxidative DNA damage. (F) 8-OHdG immunoreactivity was significantly higher in PV+ null in both cortices in Cox6a2-/- mice (n=4 WT, 4 Cox6a2-/-; 2-month-old). Two-tailed unpaired Mann-Whitney tests.


( G ) The density of PNNs around PV + interneurons residing in the somatosensory and motor cortices was analyzed by measuring the immunoreactivity of WFA that stains PNNs . ( H ) WFA immunoreactivity around PV + cells was lower in both cortices in Cox6a2 - / - mice ( n = 4 WT , 4 Cox6a2 - / - ; 2 - month - old ) . Two - tailed unpaired Mann - Whitney tests .

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(G) The density of PNNs around PV+ interneurons residing in the somatosensory and motor cortices was analyzed by measuring the immunoreactivity of WFA that stains PNNs. (H) WFA immunoreactivity around PV+ cells was lower in both cortices in Cox6a2-/- mice (n=4 WT, 4 Cox6a2-/-; 2-month-old). Two-tailed unpaired Mann-Whitney tests.


( A ) PV + interneurons were sorted from cortices of PV - EGFP WT and Cox6a2 - / - mice at P15 and processed for bulk RNA sequencing ( n = 4 WT , 6 Cox6a2 - / - ; P15 ) . Heatmap shows expression for top down - ( red ) and upregulated ( blue ) genes in Cox6a2 - / - PV + interneurons relative to their WT counterparts .

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(A) PV+ interneurons were sorted from cortices of PV-EGFP WT and Cox6a2-/- mice at P15 and processed for bulk RNA sequencing (n= 4 WT, 6 Cox6a2-/-; P15). Heatmap shows expression for top down- (red) and upregulated (blue) genes in Cox6a2-/- PV+ interneurons relative to their WT counterparts.


( B ) Cox6a2 expression was effectively abolished in Cox6a2 - / - PV + interneurons . In addition , Cox6a2 knockout led to a decreased expression of Gad1 and Gad2 genes ( n = 4 WT , 6 Cox6a2 - / - ) . Two - tailed unpaired Mann - Whitney tests .

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(B) Cox6a2 expression was effectively abolished in Cox6a2-/- PV+ interneurons. In addition, Cox6a2 knockout led to a decreased expression of Gad1 and Gad2 genes (n= 4 WT, 6 Cox6a2-/-). Two-tailed unpaired Mann-Whitney tests.


( C ) Changes in expression of oxidative stress - related genes ( green ) , genes coding for AMPA receptors ( orange ) , sodium channels ( red ) , potassium channels ( magenta ) , and internal calcium signaling proteins ( blue ) . Color scale bars show normalized read counts .

(C) Changes in expression of oxidative stress-related genes (green), genes coding for AMPA receptors (orange), sodium channels (red), potassium channels (magenta), and internal calcium signaling proteins (blue). Color scale bars show normalized read counts.


( D ) Representative images of PV + interneurons from PV - CRE and PV - CRE ; Cox6a2fl / fl mice showing PercevalHR fluorescence after excitation at 930 nm and 840 nm . White arrowheads indicate PercevalHR expressing cells . Scale bar : 20 μm .

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(D) Representative images of PV+ interneurons from PV-CRE and PV-CRE;Cox6a2fl/fl mice showing PercevalHR fluorescence after excitation at 930 nm and 840 nm. White arrowheads indicate PercevalHR expressing cells. Scale bar: 20 μm.


( E ) PV + interneurons from PV - CRE ; Cox6a2fl / fl mice exhibited significantly lower ATP - to - ADP ratios than PV + interneurons from PV - CRE mice throughout the entire duration of the recordings ( 23 cells from 4 PV - CRE mice ; 15 cells from 6 PV - CRE ; Cox6a2 - / - mice ) .

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(E) PV+ interneurons from PV-CRE;Cox6a2fl/fl mice exhibited significantly lower ATP-to-ADP ratios than PV+ interneurons from PV-CRE mice throughout the entire duration of the recordings (23 cells from 4 PV-CRE mice; 15 cells from 6 PV-CRE;Cox6a2-/- mice).


( A , B ) The resting membrane potential and amplitude of action potentials in WT and Cox6a2 - / - PV + interneurons , respectively . Two - tailed unpaired Student ' s t - tests .

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(A,B) The resting membrane potential and amplitude of action potentials in WT and Cox6a2-/- PV+ interneurons, respectively. Two-tailed unpaired Student's t-tests.


( C , D ) The threshold for action potentials increased over maturation in WT but not in Cox6a2 - / - PV + interneurons . As a result , the threshold for action potentials was significantly lower in P34 - 45 Cox6a2 - / - PV + interneurons when compared to their WT counterparts . D : two - tailed unpaired Student ' s t - tests .

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(C,D) The threshold for action potentials increased over maturation in WT but not in Cox6a2-/- PV+ interneurons. As a result, the threshold for action potentials was significantly lower in P34-45 Cox6a2-/- PV+ interneurons when compared to their WT counterparts. D: two-tailed unpaired Student's t-tests.


( E , F ) Trains of action potentials were evoked by 2 sec depolarizing current pulses . The amplitude difference ( difference between the mean amplitude of 1st - 10th action potentials and 190th - 200th action potentials ) was significantly higher in P34 - 45 Cox6a2 - / - compared to WT PV + interneurons . In addition , the amplitude difference decreased with maturation in WT but not in Cox6a2 - / - PV + interneurons . F : two - tailed unpaired Mann - Whitney tests .

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(E,F) Trains of action potentials were evoked by 2 sec depolarizing current pulses. The amplitude difference (difference between the mean amplitude of 1st-10th action potentials and 190th-200th action potentials) was significantly higher in P34-45 Cox6a2-/- compared to WT PV+ interneurons. In addition, the amplitude difference decreased with maturation in WT but not in Cox6a2-/- PV+ interneurons. F: two-tailed unpaired Mann-Whitney tests.


( G ) While in WT PV + interneurons the maximal firing frequency increased during maturation , such increase was abolished by Cox6a2 knockout . Thus , maximal firing frequency reached during 2 sec depolarizing current pulses was lower in P34 - 45 Cox6a2 - / - compared to WT PV + interneurons . Two - tailed unpaired Student ' s t - tests .

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(G) While in WT PV+ interneurons the maximal firing frequency increased during maturation, such increase was abolished by Cox6a2 knockout. Thus, maximal firing frequency reached during 2 sec depolarizing current pulses was lower in P34-45 Cox6a2-/- compared to WT PV+ interneurons. Two-tailed unpaired Student's t-tests.


( H ) Recorded PV + interneurons were filled with biocytin and their dendritic morphology was reconstructed using Imaris software . Scale bars : 30 μm .

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(H) Recorded PV+ interneurons were filled with biocytin and their dendritic morphology was reconstructed using Imaris software. Scale bars: 30 μm.


( I , J ) Cox6a2 - / - PV + interneurons showed a negative correlation between age and dendritic length and between age and full branch depth ( maximal number of branches between the soma and a terminal dendritic point ) , which contrasted with the expected positive correlation between these parameters in WT PV + interneurons . Linear regression of Pearson correlation .

(I,J) Cox6a2-/- PV+ interneurons showed a negative correlation between age and dendritic length and between age and full branch depth (maximal number of branches between the soma and a terminal dendritic point), which contrasted with the expected positive correlation between these parameters in WT PV+ interneurons. Linear regression of Pearson correlation.


( K , L ) WT and Cox6a2 - / - PV + interneurons showed similar values for dendritic length and full branch depth at P22 - P33 . However , at P34 - P45 , Cox6a2 - / - PV + interneurons showed lower dendritic length and full branch depth than WT PV + interneurons . Two - way ANOVA followed by Tukey ' s multiple comparisons tests .

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(K,L) WT and Cox6a2-/- PV+ interneurons showed similar values for dendritic length and full branch depth at P22-P33. However, at P34-P45, Cox6a2-/- PV+ interneurons showed lower dendritic length and full branch depth than WT PV+ interneurons. Two-way ANOVA followed by Tukey's multiple comparisons tests.


( M , N ) While younger WT and Cox6a2 - / - PV + interneurons have similar dendritic tree complexities ( M ) , at the later stage of maturation Cox6a2 - / - PV + interneurons show a reduction in dendritic tree complexity in comparison to WT PV + interneurons ( N ) . Multiple t - tests , two - stage step - up method of Benjamini , Krieger and Yekutieli .

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(M,N) While younger WT and Cox6a2-/- PV+ interneurons have similar dendritic tree complexities (M), at the later stage of maturation Cox6a2-/- PV+ interneurons show a reduction in dendritic tree complexity in comparison to WT PV+ interneurons (N). Multiple t-tests, two-stage step-up method of Benjamini, Krieger and Yekutieli.


( B ) In the OFT , Cox6a2 - / - mice travelled a longer distance and moved faster than WT mice . Cox6a2 - / - mice also moved more often from the outer zone to the inner zone of the arena . Two - tailed unpaired Student ' s t - tests .

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(B) In the OFT, Cox6a2-/- mice travelled a longer distance and moved faster than WT mice. Cox6a2-/- mice also moved more often from the outer zone to the inner zone of the arena. Two-tailed unpaired Student's t-tests.


( D ) In the SIT , Cox6a2 - / - mice also travelled a longer distance with higher mean velocity than WT mice . Additionally , both Cox6a2 - / - and WT mice spent more time exploring the container with the stranger mouse than the empty container . Total distance moved , mean velocity , and f [ number of crossings ] outer to inner zone : two - tailed unpaired Student ' s t - test . Social interaction : two - way ANOVA followed by Tukey ' s multiple comparisons test .

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(D) In the SIT, Cox6a2-/- mice also travelled a longer distance with higher mean velocity than WT mice. Additionally, both Cox6a2-/- and WT mice spent more time exploring the container with the stranger mouse than the empty container. Total distance moved, mean velocity, and f [number of crossings] outer to inner zone: two-tailed unpaired Student's t-test. Social interaction: two-way ANOVA followed by Tukey's multiple comparisons test.


( F ) The startle responses were significantly lower in Cox6a2 - / - mice when the loud stimulus ( 120 dB ) was presented alone the first 5 times . Two - way ANOVA followed by Tukey ' s multiple comparisons tests .

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(F) The startle responses were significantly lower in Cox6a2-/- mice when the loud stimulus (120 dB) was presented alone the first 5 times. Two-way ANOVA followed by Tukey's multiple comparisons tests.


( G ) The PPI of the acoustic startle response was similar at all pre - pulses for Cox6a2 - / - and WT mice . Two - way ANOVA followed by Sidak ' s multiple comparisons tests .

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(G) The PPI of the acoustic startle response was similar at all pre-pulses for Cox6a2-/- and WT mice. Two-way ANOVA followed by Sidak's multiple comparisons tests.


RNA was extracted from young fertile adults fed ad libitum or fasted for 6 h . ddCts ( delta delta cycle thresholds ) were calculated normalizing to ama - 1 and the efficiency of the primer sets as previously described . Means + s . e . m . are depicted . lipl - 1 to lipl - 5 , n = 6 independent experiments . lipl - 6 to lipl - 8 , n = 3independent experiments . Significant t - test derived P - values are indicated . WT , wild type .

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RNA was extracted from young fertile adults fed ad libitum or fasted for 6 h. ddCts (delta delta cycle thresholds) were calculated normalizing to ama-1 and the efficiency of the primer sets as previously described. Means+s.e.m. are depicted. lipl-1 to lipl-5, n = 6 independent experiments. lipl-6 to lipl-8, n = 3independent experiments. Significant t-test derived P-values are indicated. WT, wild type.


( a ) Representative confocal images of LIPL - 1 : : GFP and LIPL - 3 : : TagRFP localization relative to the lysosomal marker PGP - 2 show that LIPL - 1 and LIPL - 3 are localized to the lysosomal - related organelle .

(a) Representative confocal images of LIPL-1::GFP and LIPL-3::TagRFP localization relative to the lysosomal marker PGP-2 show that LIPL-1 and LIPL-3 are localized to the lysosomal-related organelle.


( b ) Acid lipase activity , measured in 1 - day adult whole lysates , shows that lipl - 1lipl - 3 double mutant animals have reduced acidic lipolytic capacity . Mean ± s . d . are presented relative to wild type ( WT ) ; significant differences are indicated ; n = 4 independent experiments .

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(b) Acid lipase activity, measured in 1-day adult whole lysates, shows that lipl-1lipl-3 double mutant animals have reduced acidic lipolytic capacity. Mean±s.d. are presented relative to wild type (WT); significant differences are indicated; n = 4 independent experiments.


( c ) Oil Red O ( ORO ) staining and fatty acid methyl ester ( FAME ) analyses of wild - type and lipl - 1 ( tm1954 ) lipl - 3 ( tm4498 ) double mutant L3 larvae show that lysosomal lipases regulate cytosolic fat stores ( see Fig . 2d for adult measurements ) . Means ± s . e . m . are presented relative to wild type ; significant differences are indicated ; n = 3 independent experiments . ( d ) ORO quantification of wild - type and lipl - 1 ( tm1954 ) lipl - 3 ( tm4498 ) double mutant young adults fasted for 4 h shows that LIPL - 1 and LIPL - 3 contribute to fat mobilization following fasting . ( Mean percentage ± s . e . m . relative to fed wild type ) . Wild - type fasted worms show 20 % less ORO signal than animals fed ad libitum ( P ≤ 0 . 001 ) , whereas lipl - 1 ( tm1954 ) lipl - 3 ( tm4498 ) double mutant worms show 7 % reduction in ORO signal ( a difference that is not significant to well - fed fat levels at P ≤ 0 . 01 but it is significant at P ≤ 0 . 05 ) . n = 3 independent experiments .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Oil Red O (ORO) staining and fatty acid methyl ester (FAME) analyses of wild-type and lipl-1(tm1954) lipl-3(tm4498) double mutant L3 larvae show that lysosomal lipases regulate cytosolic fat stores (see Fig. 2d for adult measurements). Means±s.e.m. are presented relative to wild type; significant differences are indicated; n = 3 independent experiments. (d) ORO quantification of wild-type and lipl-1(tm1954) lipl-3(tm4498) double mutant young adults fasted for 4 h shows that LIPL-1 and LIPL-3 contribute to fat mobilization following fasting. (Mean percentage ± s.e.m. relative to fed wild type). Wild-type fasted worms show 20% less ORO signal than animals fed ad libitum (P≤0.001), whereas lipl-1(tm1954) lipl-3(tm4498) double mutant worms show 7% reduction in ORO signal (a difference that is not significant to well-fed fat levels at P≤0.01 but it is significant at P≤0.05). n = 3 independent experiments.


( e ) Representative transmission electron microscopy images of well - fed and 6 h - fasted wild - type and lipl - 1lipl - 3 mutant animals ( 11 , 500 × ) show that the lipl mutants have more lipid - droplet stores in ad libitum fed and fasted conditions . Quantification of vesicle number and size is depicted as mean + s . e . m . , n = 4 independent experiments .

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(e) Representative transmission electron microscopy images of well-fed and 6 h-fasted wild-type and lipl-1lipl-3 mutant animals (11,500×) show that the lipl mutants have more lipid-droplet stores in ad libitum fed and fasted conditions. Quantification of vesicle number and size is depicted as mean+s.e.m., n = 4 independent experiments.


( f ) Representative images of well - fed transgenic animals expressing LIPL - 3 : : TagRFP and ATGL : : GFP shows that LIPL - 3 does not localize to lipid droplets .

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(f) Representative images of well-fed transgenic animals expressing LIPL-3::TagRFP and ATGL::GFP shows that LIPL-3 does not localize to lipid droplets.


( g ) ORO staining and quantification of wild - type and lipl - 1 ( tm1954 ) lipl - 3 ( tm4498 ) double mutant young adults treated post - developmentally ( from L4 ) with RNAi against the essential autophagy genes lgg - 1 and lgg - 2 or vector control show that lipl - 1 lipl - 3 and the autophagy genes lgg - 1 lgg - 2 are in the same fat regulatory pathway . ( Mean percentage + s . e . m . relative to wild type on vector control is depicted . ) n = 4 independent experiments .

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(g) ORO staining and quantification of wild-type and lipl-1(tm1954) lipl-3(tm4498) double mutant young adults treated post-developmentally (from L4) with RNAi against the essential autophagy genes lgg-1 and lgg-2 or vector control show that lipl-1 lipl-3 and the autophagy genes lgg-1 lgg-2 are in the same fat regulatory pathway. (Mean percentage+s.e.m. relative to wild type on vector control is depicted.) n = 4 independent experiments.


( a ) Expression of lipl genes in well - fed mxl - 3 ( ok1947 ) and mxl - 3 ( tm2580 ) young adults normalized to same age well - fed wild - type ( WT ) worms shows that mxl - 3 loss of function is sufficient to induce lipl - 1 to 3 and lipl - 5 . Mean ddCts + s . e . m . are depicted . n = 6 independent experiments for lipl - 1 to lipl - 5 , and n = 4 independent experiments for lipl - 6 to lipl - 8 .

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(a) Expression of lipl genes in well-fed mxl-3(ok1947) and mxl-3(tm2580) young adults normalized to same age well-fed wild-type (WT) worms shows that mxl-3 loss of function is sufficient to induce lipl-1 to 3 and lipl-5. Mean ddCts+s.e.m. are depicted. n = 6 independent experiments for lipl-1 to lipl-5, and n = 4 independent experiments for lipl-6 to lipl-8.


( b ) L4 larvae were fasted and RNA was extracted at the indicated times . Mean ddCts + s . e . m . show that the response mxl - 3 orchestrates is transient . Time 0 , n = 6 independent experiments ; time 3 - 12 h , n = 4 independent experiments ; time 18 - 24 h , n = 2 independent experiments .

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(b) L4 larvae were fasted and RNA was extracted at the indicated times. Mean ddCts+s.e.m. show that the response mxl-3 orchestrates is transient. Time 0, n = 6 independent experiments; time 3-12 h, n = 4 independent experiments; time 18-24 h, n = 2 independent experiments.


( c ) Immunostainings of well - fed or 6 or 12 h fasted MXL - 3 : : GFP young adults are presented . Quantification of GFP - positive nuclei relative to total intestinal nuclei ( 4 , 6 - diamidino - 2 - phenylindole , DAPI ) of two independent experiments shows that MXL - 3 transiently delocalizes from the intestinal nuclei during fasting .

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(c) Immunostainings of well-fed or 6 or 12 h fasted MXL-3::GFP young adults are presented. Quantification of GFP-positive nuclei relative to total intestinal nuclei (4,6-diamidino-2-phenylindole, DAPI) of two independent experiments shows that MXL-3 transiently delocalizes from the intestinal nuclei during fasting.


( d ) ChIP - quantitative PCR ( ChIP - qPCR ) analysis of well - fed and 6 h fasted mixed - stage worms expressing MXL - 3 : : GFP presented as Ct in αGFP immunoprecipitated DNA normalized to input DNA and relative to a mock promoter region ( CACTAT site − 88 of ama - 1 gene ) shows that MXL - 3 vacates the lipl promoters during early fasting . Three sets of primers surrounding CACGTG target sites found up to 500 bp of the ATG of the lipl - 1 and lipl - 3 genes were used . A representative experiment is presented ; see raw data of two independent experiments in Supplementary Table S9 .

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(d) ChIP-quantitative PCR (ChIP-qPCR) analysis of well-fed and 6 h fasted mixed-stage worms expressing MXL-3::GFP presented as Ct in αGFP immunoprecipitated DNA normalized to input DNA and relative to a mock promoter region (CACTAT site −88 of ama-1 gene) shows that MXL-3 vacates the lipl promoters during early fasting. Three sets of primers surrounding CACGTG target sites found up to 500 bp of the ATG of the lipl-1 and lipl-3 genes were used. A representative experiment is presented; see raw data of two independent experiments in Supplementary Table S9.


( e ) Wild - type or lipl - 1 ( tm1954 ) lipl - 3 ( tm4498 ) double mutant animals grown on control RNAi plates were transferred as L4 larvae to control or mxl - 3 RNAi plates , incubated for 12 h at 20 ° C , and processed for total FAMEs ; as a reference , an aliquot of wild - type young adults on control RNAi bacteria was fasted for 6 h . FAME quantification ( mean percentage of fed wild type ± s . e . m . ) shows that acute mxl - 3 inactivation , as fasting , reduces fat stores in a lipl - dependent manner . n = 3 independent experiments . NSD , P - value > 0 . 05 .

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(e) Wild-type or lipl-1(tm1954) lipl-3(tm4498) double mutant animals grown on control RNAi plates were transferred as L4 larvae to control or mxl-3 RNAi plates, incubated for 12 h at 20 °C, and processed for total FAMEs; as a reference, an aliquot of wild-type young adults on control RNAi bacteria was fasted for 6 h. FAME quantification (mean percentage of fed wild type ±s.e.m.) shows that acute mxl-3 inactivation, as fasting, reduces fat stores in a lipl-dependent manner. n = 3 independent experiments. NSD, P-value>0.05.


( a ) Transcriptional levels of hlh - 30 and the mxl - 3 - dependent lipl genes measured in 5 h fasted wild - type ( WT ) or hlh - 30 ( tm1978 ) young - adult worms depicted as mean ddCt + s . e . m . relative to wild - type ad libitum - fed worms show that hlh - 30 is induced following fasting and HLH - 30 induces lysosomal lipolysis . N = 4 independent experiments for lipl data , n = 2 independent experiments for hlh - 30 mRNA . HLH - 30 deficiency fully abrogates lipl - 2 , 3 and 5 ( P0 . 0001 ) , and impairs lipl - 1 ( P0 . 01 ) transcriptional activation following fasting .

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(a) Transcriptional levels of hlh-30 and the mxl-3-dependent lipl genes measured in 5 h fasted wild-type (WT) or hlh-30(tm1978) young-adult worms depicted as mean ddCt+s.e.m. relative to wild-type ad libitum-fed worms show that hlh-30 is induced following fasting and HLH-30 induces lysosomal lipolysis. N = 4 independent experiments for lipl data, n = 2 independent experiments for hlh-30 mRNA. HLH-30 deficiency fully abrogates lipl-2, 3 and 5 (P0.0001), and impairs lipl-1 (P0.01) transcriptional activation following fasting.


( b ) Animals treated from late L3 stage with RNAi against TOR or vector control were harvested as young adults . Mean + s . e . m . of three independent experiments shows that inhibition of TOR is sufficient to induce hlh - 30 transcription .

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(b) Animals treated from late L3 stage with RNAi against TOR or vector control were harvested as young adults. Mean+s.e.m. of three independent experiments shows that inhibition of TOR is sufficient to induce hlh-30 transcription.


( c ) L3 animals carrying the rescuing construct hlh - 30P : : HLH - 30 : : mGFP : : hlh - 30 3 ′ UTR ( mGFP , monomeric GFP , and UTR , untranslated region ) , well fed or fasted for 8 h show that HLH - 30 is enriched in intestinal nuclei of fasted worms ( exposure time : well fed , 500 ms ; fasted , 100 ms ) .

(c) L3 animals carrying the rescuing construct hlh-30P::HLH-30::mGFP::hlh-30 3′UTR (mGFP, monomeric GFP, and UTR, untranslated region), well fed or fasted for 8 h show that HLH-30 is enriched in intestinal nuclei of fasted worms (exposure time: well fed, 500 ms; fasted, 100 ms).


( d ) ORO staining of wild - type or hlh - 30 ( tm1978 ) young adults fasted for 8 h reveals that hlh - 30 is required for optimal lipid mobilization on fasting . Mean percentages + s . e . m . are shown below relative to well - fed worms treated in parallel , n = 3 independent experiments .

(d) ORO staining of wild-type or hlh-30(tm1978) young adults fasted for 8 h reveals that hlh-30 is required for optimal lipid mobilization on fasting. Mean percentages+s.e.m. are shown below relative to well-fed worms treated in parallel, n = 3 independent experiments.


( e ) mxl - 3 and hlh - 30 transcriptional levels in wild - type , hlh - 30 ( tm1978 ) and mxl - 3 ( ok1947 ) mutants in the basal and fasted states show that mxl - 3 transcription is hlh - 30 independent , and hlh - 30 transcription is mxl - 3 independent . Expression is presented relative to wild - type fed animals as mean ddCt + s . e . m . No significant differences relative to wild type in the same feeding state were observed , n = 3 independent experiments .

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(e) mxl-3 and hlh-30 transcriptional levels in wild-type, hlh-30(tm1978) and mxl-3(ok1947) mutants in the basal and fasted states show that mxl-3 transcription is hlh-30 independent, and hlh-30 transcription is mxl-3 independent. Expression is presented relative to wild-type fed animals as mean ddCt+s.e.m. No significant differences relative to wild type in the same feeding state were observed, n = 3 independent experiments.


( f ) Representative ChIP - qPCR analysis of well - fed and 6 h fasted mixed stage worms expressing HLH - 30 : : GFP presented as in Fig . 3d shows that HLH - 30 occupies the lipl promoters during early fasting . See raw data of two independent experiments in Supplementary Table S9 .

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(f) Representative ChIP-qPCR analysis of well-fed and 6 h fasted mixed stage worms expressing HLH-30::GFP presented as in Fig. 3d shows that HLH-30 occupies the lipl promoters during early fasting. See raw data of two independent experiments in Supplementary Table S9.


( g ) Transcriptional levels of the mxl - 3 - dependent lipl genes in wild - type , mxl - 3 ( ok1947 ) , hlh - 30 ( tm1978 ) or mxl - 3 ( ok1947 ) ; hlh - 30 ( tm1978 ) double mutant young - adult worms show that hlh - 30 suppresses the constitutive - induction - of - the - lipl - genes phenotype of mxl - 3 mutant animals ( mean ddCt + s . e . m . relative to wild - type ad libitum - fed worms ) . n = 3 independent experiments .

(g) Transcriptional levels of the mxl-3-dependent lipl genes in wild-type, mxl-3(ok1947), hlh-30(tm1978) or mxl-3(ok1947);hlh-30(tm1978) double mutant young-adult worms show that hlh-30 suppresses the constitutive-induction-of-the-lipl-genes phenotype of mxl-3 mutant animals (mean ddCt+s.e.m. relative to wild-type ad libitum-fed worms). n = 3 independent experiments.


( a ) LRO ( lysosome - related organelle ) live Nile red - stained images of hlh - 30 ( tm1978 ) and wild - type ( WT ) L3 larvae starved for 60 h show that hlh - 30 is required for the expansion of the lysosomal compartment following fasting . Quantification as mean ± s . e . m . relative to fasted wild - type worms is also presented ; n = 3 independent experiments .

(a) LRO (lysosome-related organelle) live Nile red-stained images of hlh-30(tm1978) and wild-type (WT) L3 larvae starved for 60 h show that hlh-30 is required for the expansion of the lysosomal compartment following fasting. Quantification as mean±s.e.m. relative to fasted wild-type worms is also presented; n = 3 independent experiments.


( b ) Transcriptional levels of the autophagy genes lgg - 1 , lgg - 2 and atg - 16 . 2 in fasted wild - type and hlh - 30 ( tm1978 ) young - adult worms show that transcriptional activation of autophagy following fasting is hlh - 30 dependent ( mean ddCt + s . e . m . relative to wild - type ad libitum - fed worms ) . n = 3 independent

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(b) Transcriptional levels of the autophagy genes lgg-1, lgg-2 and atg-16.2 in fasted wild-type and hlh-30(tm1978) young-adult worms show that transcriptional activation of autophagy following fasting is hlh-30 dependent (mean ddCt+s.e.m. relative to wild-type ad libitum-fed worms). n = 3 independent experiments


( c ) The mean percentage ( + s . d . ) of L1 larvae alive after 48 h fasting in minimum media shows that hlh - 30 ( tm1978 ) mutant animals are sensitive to starvation . n = 3 independent experiments . For L4 survival see Supplementary Fig . S6g .

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(c) The mean percentage (+ s.d.) of L1 larvae alive after 48 h fasting in minimum media shows that hlh-30 (tm1978) mutant animals are sensitive to starvation. n = 3 independent experiments. For L4 survival see Supplementary Fig. S6g.


( a ) Expression analyses of the liver of C57BL / 6J 9 - week females fasted overnight ( ON ) for 10 h relative to siblings feeding ad libitum show that LipA ( mouse lysosomal acid lipase ) , Map1lc3a ( mammalian lgg - 1 / lgg - 2 ) and Tfeb ( mammalian hlh - 30 ) but not Max ( mammalian mxl - 3 ) are transcriptionally linked to nutrients in the mouse liver ( median + s . e . m . fold change ; ddCt ) . Levels of expression were normalized to ActB and Cog2 as internal controls ; all but Max differences are significant ( P0 . 05 ) , n = 3 independent experiments . Cyp4a14 is a positive control .

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(a) Expression analyses of the liver of C57BL/6J 9-week females fasted overnight (ON) for 10 h relative to siblings feeding ad libitum show that LipA (mouse lysosomal acid lipase), Map1lc3a (mammalian lgg-1/lgg-2) and Tfeb (mammalian hlh-30) but not Max (mammalian mxl-3) are transcriptionally linked to nutrients in the mouse liver (median+s.e.m. fold change; ddCt). Levels of expression were normalized to ActB and Cog2 as internal controls; all but Max differences are significant (P0.05), n = 3 independent experiments. Cyp4a14 is a positive control.


( b ) Expression analyses of control or hTFEB siRNA treated HepG2 cells incubated in EBSS minimal medium for 2 , 4 or 8 h , compared with the expression of control or hTFEB siRNA treated hepatocytes in complete media , show that LAL ( human lysosomal acid lipase ) , MAP1LC3A and TFEB but not MAX are transcriptionally linked to nutrients in human hepatocytes , and that LAL and MAP1LC3A induction under nutrient deprivation are TFEB dependent ( median dCt + s . e . m . ) . Levels of expression were normalized to ACTB as internal control ; all butMAX differences are significant ( P0 . 05 ) , n = 3 independent experiments . IGFBP is a positive control . EBSS , Earles ' s balanced salt solution .

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(b) Expression analyses of control or hTFEB siRNA treated HepG2 cells incubated in EBSS minimal medium for 2, 4 or 8 h, compared with the expression of control or hTFEB siRNA treated hepatocytes in complete media, show that LAL (human lysosomal acid lipase), MAP1LC3A and TFEB but not MAX are transcriptionally linked to nutrients in human hepatocytes, and that LAL and MAP1LC3A induction under nutrient deprivation are TFEB dependent (median dCt+s.e.m.). Levels of expression were normalized to ACTB as internal control; all butMAX differences are significant (P0.05), n = 3 independent experiments. IGFBP is a positive control. EBSS, Earles's balanced salt solution.


( a ) Wild - type ( WT ) , mxl - 3 ( ok1947 ) , hlh - 30 ( tm1978 ) or mxl - 3 ( ok1947 ) ; hlh - 30 ( tm1978 ) double mutant were used in longevity epistasis analyses , revealing that hlh - 30 suppresses the mxl - 3 extended lifespan phenotype . Animals were incubated at 20 ° C and transferred every other day until cessation of reproduction . The cumulative survival curve is depicted . Kaplan - Meier statistics , calculated using SSPS 17 , are also shown .

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(a) Wild-type (WT), mxl-3(ok1947), hlh-30(tm1978) or mxl-3(ok1947);hlh-30(tm1978) double mutant were used in longevity epistasis analyses, revealing that hlh-30 suppresses the mxl-3 extended lifespan phenotype. Animals were incubated at 20°C and transferred every other day until cessation of reproduction. The cumulative survival curve is depicted. Kaplan-Meier statistics, calculated using SSPS 17, are also shown.


( b ) Live Nile red quantification of wild - type and lipl - 1lipl - 3 double mutant worms shows that lipl - 1 and lipl - 3 contribute to the clearance of lipids from the endocytic pathway . Mean + s . e . m . signal intensity is shown as percentage relative to wild type ; significant differences are indicated ; n = 4 independent experiments .

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(b) Live Nile red quantification of wild-type and lipl-1lipl-3 double mutant worms shows that lipl-1 and lipl-3 contribute to the clearance of lipids from the endocytic pathway. Mean+s.e.m. signal intensity is shown as percentage relative to wild type; significant differences are indicated; n = 4 independent experiments.


( c , d ) Animals overexpressing LIPL - 1 or LIPL - 3 were incubated at 25 ° C and transferred every other day until cessation of reproduction . Data presented as in Fig . 7a show that activated lysosomal lipolysis extends C . elegans lifespan . a Survival presented as mean lifespan ( LS ) ± s . e . m . b Number of uncensored animals ( animals that crawled off the plate , bagged , exploded or became contaminated were censored ) .

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(c,d) Animals overexpressing LIPL-1 or LIPL-3 were incubated at 25 °C and transferred every other day until cessation of reproduction. Data presented as in Fig. 7a show that activated lysosomal lipolysis extends C. elegans lifespan. a Survival presented as mean lifespan (LS) ± s.e.m. b Number of uncensored animals (animals that crawled off the plate, bagged, exploded or became contaminated were censored).


( A ) EM analysis of cortical synapses derived from WT and MPS - IIIA mice at different ages . The size of synaptic vesicles was quantified from 400 - 500 vesicles ( taken from 5 mice for each genotype at each time point ) and expressed as the average of vesicle diameter ( nm ) . The number of synaptic vesicles per synapse was quantified from 20 different images ( taken from 5 mice for each genotype at each time point ) , normalized by the length of synaptic cleft and expressed as % of WT . The synaptic density was measured from 20 different images ( taken from 5 mice for each genotype at each time point ) as the number of synapses / area ( # / 500μm2 ) and expressed as % of WT . Arrows indicate the synaptic cleft , while asterisks indicate abnormal

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(A) EM analysis of cortical synapses derived from WT and MPS-IIIA mice at different ages. The size of synaptic vesicles was quantified from 400-500 vesicles (taken from 5 mice for each genotype at each time point) and expressed as the average of vesicle diameter (nm). The number of synaptic vesicles per synapse was quantified from 20 different images (taken from 5 mice for each genotype at each time point), normalized by the length of synaptic cleft and expressed as % of WT. The synaptic density was measured from 20 different images (taken from 5 mice for each genotype at each time point) as the number of synapses/area(#/500μm2) and expressed as % of WT.Arrows indicate the synaptic cleft, while asterisks indicate abnormal vacuoles


( B , C ) The size of the lysosomal compartment was evaluated by WB ( B ) analysis in the brain of MPS - IIIA mice at indicated ages . Quantitation of WB by densitometry analysis ( ImageJ ) was shown ( B ) .

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(B, C) The size of the lysosomal compartment was evaluated by WB (B) analysis in the brain of MPS-IIIA mice at indicated ages. Quantitation of WB by densitometry analysis (ImageJ) was shown (B).


( B , C ) The size of the lysosomal compartment was evaluated by IF ( C ) analysis in the brain of MPS - IIIA mice at indicated ages .

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(B, C) The size of the lysosomal compartment was evaluated by IF (C) analysis in the brain of MPS-IIIA mice at indicated ages.


( D ) Extracellular recordings ( fEPSPs ) in hippocampal brain slices from 6 months - old WT and MPS - IIIA mice . The left panel shows representative fEPSP traces . Summary graphs show the fEPSPs slope as a function of either the applied stimulus intensity or the prevolley amplitude . Data are the average of the values from 9 slices ( from 9 mice ) in the WT group and 6 slices ( from 5 mice ) in the MPS - IIIA . * p < 0 . 05 , repeated measure ANOVA .

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(D) Extracellular recordings (fEPSPs) in hippocampal brain slices from 6 months-old WT and MPS-IIIA mice. The left panel shows representative fEPSP traces. Summary graphs show the fEPSPs slope as a function of either the applied stimulus intensity or the prevolley amplitude. Data are the average of the values from 9 slices (from 9 mice) in the WT group and 6 slices (from 5 mice) in the MPS-IIIA. *p<0.05, repeated measure ANOVA.


( A ) Synaptic terminal endocytosis was analyzed in WT and MPS - IIIA DIV14 hippocampal neurons by quantification of incorporated FM1 - 43 dye fluorescence in ~ 300 individual boutons ( taken from 4 - 5 coverslips for each group ) . Fluorescence intensities were expressed as arbitrary units ( A . F . U . ) and displayed both as a distribution and as mean values ± s . e . m .

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(A) Synaptic terminal endocytosis was analyzed in WT and MPS-IIIA DIV14 hippocampal neurons by quantification of incorporated FM1-43 dye fluorescence in ~300 individual boutons (taken from 4-5 coverslips for each group). Fluorescence intensities were expressed as arbitrary units (A.F.U.) and displayed both as a distribution and as mean values ± s.e.m.


( B ) WT and MPS - IIIA hippocampal neurons ( DIV14 ) were transfected with v - Glut1 - pHluorin - mCherry plasmid and synaptic recycling was evaluated by the fluorescence change of the probe from 80 - 100 individual boutons ( taken from 4 - 5 coverslips for each group ) . Fluorescence intensity was quantified at each bouton during KCl perfusion , normalized to the fluorescence value obtained upon rapid alkalization ( NH4Cl perfusion ) and expressed as ∆ fluorescence ( normalized to baseline ; ΔFKCl / ΔF NH4Cl ) . Values were displayed both as fluorescence traces ( means ± s . e . m . at each time frame ) and maximum fluorescence after KCl perfusion ( means ± s . e . m . ) . Representative panels on the right show the fluorescence intensity change in control neurons ( WT ) . Pseudo - colour was applied to better reveal fluorescence changes . Note that NH4Cl alkalinizes all vesicles revealing the total ( recycling + resting ) pool in neuronal cells analyzed .

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(B) WT and MPS-IIIA hippocampal neurons (DIV14) were transfected with v-Glut1-pHluorin-mCherry plasmid and synaptic recycling was evaluated by the fluorescence change of the probe from 80-100 individual boutons (taken from 4-5 coverslips for each group). Fluorescence intensity was quantified at each bouton during KCl perfusion, normalized to the fluorescence value obtained upon rapid alkalization (NH4Cl perfusion) and expressed as ∆fluorescence (normalized to baseline; ΔFKCl/ΔF NH4Cl). Values were displayed both as fluorescence traces (means ± s.e.m. at each time frame) and maximum fluorescence after KCl perfusion (means ± s.e.m.). Representative panels on the right show the fluorescence intensity change in control neurons (WT). Pseudo-colour was applied to better reveal fluorescence changes.Note that NH4Cl alkalinizes all vesicles revealing the total (recycling + resting) pool in neuronal cells analyzed.


( C ) WT hippocampal neurons at DIV10 were treated with a lysosome inhibitor ' s cocktail for 3 days . The size of lysosomal compartment was evaluated in both treated and control untreated WT neurons by EM examination . Quantitation analysis was performed on 30 different images .

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(C) WT hippocampal neurons at DIV10 were treated with a lysosome inhibitor's cocktail for 3 days. The size of lysosomal compartment was evaluated in both treated and control untreated WT neurons by EM examination. Quantitation analysis was performed on 30 different images.


( D , E ) Endocytosis ( D ) in presynaptic boutons from treated and control untreated WT hippocampal neurons were monitored as in ( A , B ) .

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(D, E) Endocytosis (D) in presynaptic boutons from treated and control untreated WT hippocampal neurons were monitored as in (A, B).


( D , E ) exocytosis ( E ) in presynaptic boutons from treated and control untreated WT hippocampal neurons were monitored as in ( A , B ) .

(D, E) exocytosis (E) in presynaptic boutons from treated and control untreated WT hippocampal neurons were monitored as in (A, B).


( F ) EM analysis of synaptic terminals was performed in treated and control untreated WT neurons . The number of synaptic vesicles per synapse was quantified from ~ 20 different images ( taken from 3 ultrathin sections for each group ) , normalized by the length of synaptic cleft and expressed as % of WT . Arrows indicate the synaptic cleft .

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(F) EM analysis of synaptic terminals was performed in treated and control untreated WT neurons. The number of synaptic vesicles per synapse was quantified from ~20 different images (taken from 3 ultrathin sections for each group), normalized by the length of synaptic cleft and expressed as % of WT. Arrows indicate the synaptic cleft.


( A ) VAMP2 , SNAP - 25 and Syntaxin - 1 SNAREs were immunoblotted in WT and MPS - IIIA synaptosomal brain samples at indicated ages . Synapsin - 1 was also blotted as a control . Quantitation of WB was shown .

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(A) VAMP2, SNAP-25 and Syntaxin-1 SNAREs were immunoblotted in WT and MPS-IIIA synaptosomal brain samples at indicated ages. Synapsin-1 was also blotted as a control. Quantitation of WB was shown.


( B ) Confocal microscopy images of WT and MPS - IIIA hippocampal neurons ( DIV14 ) double labeled with anti - synapsin - 1 ( presynaptic marker ; red ) and either anti - VAMP2 or anti - SNAP25 antibodies ( green ) . The merges ( yellow ) of confocal images are shown . SNAP25 - synapsin - 1 and VAMP2 - synapsin - 1 co - localizations were quantified using the Manders ' Colocalization Coefficients ( MCC ) ( ImageJ ) and displayed as % ( MCC x 100 ) of Synapsin - 1 co - localizing with either SNAP25 or VAMP2 ( means ± s . e . m . from 15 different images taken from 4 - 5 coverslips for each group ) .

(B) Confocal microscopy images of WT and MPS-IIIA hippocampal neurons (DIV14) double labeled with anti-synapsin-1 (presynaptic marker; red) and either anti-VAMP2 or anti-SNAP25 antibodies (green). The merges (yellow) of confocal images are shown. SNAP25-synapsin-1 and VAMP2-synapsin-1 co-localizations were quantified using the Manders' Colocalization Coefficients (MCC) (ImageJ) and displayed as % (MCC x 100) of Synapsin-1 co-localizing with either SNAP25 or VAMP2 (means ± s.e.m. from 15 different images taken from 4-5 coverslips for each group).


( C ) VAMP2 and SNAP - 25 protein levels were quantified by immunoblot analysis in WT and MPS - IIIA hippocampal neurons ( DIV14 ) at different times after cycloheximide treatment and expressed as % of remaining protein at T0 ( 100 % ) . The proteasome was inhibited as indicated .

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(C) VAMP2 and SNAP-25 protein levels were quantified by immunoblot analysis in WT and MPS-IIIA hippocampal neurons (DIV14) at different times after cycloheximide treatment and expressed as % of remaining protein at T0 (100 %). The proteasome was inhibited as indicated.


( D ) SDS - resistant complex levels were evaluated in WT and MPS - IIIA total brain samples at indicated ages by immunoblotting of non - boiled samples with VAMP2 or Syntaxin - 1 antibodies . Quantitation of WB was shown .

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(D) SDS-resistant complex levels were evaluated in WT and MPS-IIIA total brain samples at indicated ages by immunoblotting of non-boiled samples with VAMP2 or Syntaxin-1 antibodies. Quantitation of WB was shown.


( E ) Total brain lysates were immunoprecipitated with antibodies to Syntaxin - 1 and co - immunoprecipitated VAMP2 and SNAP25 proteins were revealed by WB analysis . The levels of immunoprecipitated proteins were quantified .

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(E) Total brain lysates were immunoprecipitated with antibodies to Syntaxin-1 and co-immunoprecipitated VAMP2 and SNAP25 proteins were revealed by WB analysis. The levels of immunoprecipitated proteins were quantified.


( F ) α - Synuclein and CSPα were immunoblotted in WT and MPS - IIIA total brain lysates at indicated ages Total protein levels were quantified .

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(F) α-Synuclein and CSPα were immunoblotted in WT and MPS-IIIA total brain lysates at indicated ages Total protein levels were quantified.


( G ) α - Synuclein and CSPα were immunoblotted in WT and MPS - IIIA synaptosomal fractions at indicated ages . Hsc70 , an heat shock cognate protein , which forms together with CSPα and SGT ( small glutamine - rich tetratricopeptide repeat domain protein ) the " chaperon machine " was also blotted . Protein levels were quantified .

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(G) α-Synuclein and CSPα were immunoblotted in WT and MPS-IIIA synaptosomal fractions at indicated ages. Hsc70, an heat shock cognate protein, which forms together with CSPα and SGT (small glutamine-rich tetratricopeptide repeat domain protein) the "chaperon machine" was also blotted. Protein levels were quantified.


( A ) WB analysis of LC3 and p62 ( an autophagy substrate ) was performed on WT and MPS - IIIA brain samples at indicated ages . WB quantitation was shown .

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(A) WB analysis of LC3 and p62 (an autophagy substrate) was performed on WT and MPS-IIIA brain samples at indicated ages. WB quantitation was shown.


( B ) α - Synuclein was immunoblotted in WT and MPS - IIIA both total and synaptosomal brain fractions at indicated ages after sequential extraction with detergents with increased strength . Soluble ( Sol . ) , lowly insoluble ( L . Insol . ) and highly insoluble ( H . Insol . ) forms correspond to the protein solubilized respectively in Triton - X100 , SDS 10 % and 8M UREA .

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(B) α-Synuclein was immunoblotted in WT and MPS-IIIA both total and synaptosomal brain fractions at indicated ages after sequential extraction with detergents with increased strength. Soluble (Sol.), lowly insoluble (L. Insol.) and highly insoluble (H. Insol.) forms correspond to the protein solubilized respectively in Triton-X100, SDS 10% and 8M UREA.


( C ) Co - immunofluorescence analysis of α - synuclein with SMI - 32 in WT and MPS - IIIA hippocampal neurons ( DIV14 ) . α - Synuclein synaptic puncta present in a neurite tract of 10µm is showed in a representative enlarged image . Quantification of α - synuclein synaptic puncta was calculated from 30 different enlarged images c .

(C) Co-immunofluorescence analysis of α-synuclein with SMI-32 in WT and MPS-IIIA hippocampal neurons (DIV14). α-Synuclein synaptic puncta present in a neurite tract of 10µm is showed in a representative enlarged image. Quantification of α-synuclein synaptic puncta was calculated from 30 different enlarged images c.


( D ) Confocal analysis of α - synuclein ( green ) and LAMP1 ( red ) in WT and MPS - IIIA hippocampal neurons ( DIV14 ) . Enlarged merge images are also shown . Co - localization was quantified using the MCC coefficient ( ImageJ ) and displayed as % ( MCC x 100 ) of α - synuclein co - localizing with LAMP1 ( means ± s . e . m . from 15 different images taken from 4 - 5 coverslips for each group ) .

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(D) Confocal analysis of α-synuclein (green) and LAMP1 (red) in WT and MPS-IIIA hippocampal neurons (DIV14). Enlarged merge images are also shown. Co-localization was quantified using the MCC coefficient (ImageJ) and displayed as % (MCC x 100) of α-synuclein co-localizing with LAMP1 (means ± s.e.m. from 15 different images taken from 4-5 coverslips for each group).


( E ) CSPα was immunoblotted in WT and MPS - IIIA total brain lysates at indicated ages after sequential extraction with detergents with increased strength as in ( B ) .

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(E) CSPα was immunoblotted in WT and MPS-IIIA total brain lysates at indicated ages after sequential extraction with detergents with increased strength as in (B).


( F ) Co - immunofluorescence analysis of CSPα and SMI - 32 in DIV14 hippocampal neurons . CSPα - synaptic puncta were quantified as in ( C ) .

(F) Co-immunofluorescence analysis of CSPα and SMI-32 in DIV14 hippocampal neurons. CSPα- synaptic puncta were quantified as in (C).


( G ) CSPα protein levels were evaluated by immunoblot analysis in WT and MPS - IIIA hippocampal neurons ( DIV14 ) at different times after cycloheximide treatment and expressed as % of remaining protein at T0 ( 100 % ) . The proteasome was inhibited as indicated .

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(G) CSPα protein levels were evaluated by immunoblot analysis in WT and MPS-IIIA hippocampal neurons (DIV14) at different times after cycloheximide treatment and expressed as % of remaining protein at T0 (100 %). The proteasome was inhibited as indicated.


( H ) Palmitoylation - dependent shift in the molecular weight of CSPα was evaluated in WT and MPS - IIIA brain samples at indicated ages by immunoblotting CSPα in boiled samples prepared without exposure to sulphydryl agents ( beta - mercaptoethanol or dithiothreitol ) .

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(H) Palmitoylation-dependent shift in the molecular weight of CSPα was evaluated in WT and MPS-IIIA brain samples at indicated ages by immunoblotting CSPα in boiled samples prepared without exposure to sulphydryl agents (beta-mercaptoethanol or dithiothreitol).


( I ) The protein levels of RPN10 ( the regulatory subunit of 26S proteasome ) and ubiquitinated proteins formed by Lys - 48 ( K48 ) residue linkage ( involved in protein degradation via the proteasome ) were evaluated in WT and MPS - IIIA brain samples at indicated ages by WB analysis . WB quantitation was shown .

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(I) The protein levels of RPN10 (the regulatory subunit of 26S proteasome) and ubiquitinated proteins formed by Lys-48 (K48) residue linkage (involved in protein degradation via the proteasome) were evaluated in WT and MPS-IIIA brain samples at indicated ages by WB analysis. WB quantitation was shown.


( L ) Proteasome activity was evaluated by measuring the chymotrypsin - like activity in WT and MPS - IIIA mouse brain samples at different ages . Proteasome activity was expressed as % of WT

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(L) Proteasome activity was evaluated by measuring the chymotrypsin-like activity in WT and MPS-IIIA mouse brain samples at different ages. Proteasome activity was expressed as % of WT activity


( A ) Autophagy was monitored in WT hippocampal neurons treated with the lysosome inhibitor ' s cocktail for 3 days by immunoblot analysis with anti - LC3 antibodies . As control DIV10 WT neurons were left untreated and monitored by LC3 blot over the three days period . The LC3 - II levels quantitation was shown .

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(A) Autophagy was monitored in WT hippocampal neurons treated with the lysosome inhibitor's cocktail for 3 days by immunoblot analysis with anti-LC3 antibodies. As control DIV10 WT neurons were left untreated and monitored by LC3 blot over the three days period. The LC3-II levels quantitation was shown.