( C ) Activity of human mAbs 10 CIS43 , specific for P . falciparum CSP , VRC01 , an isotype control human mAb specific for HIV - 1 , on proportion of motile sporozoites ( left panel ) blood vessel entry ( right panel ) . Four - minute videos of mice inoculated with indicated mAbs were taken 10 min after sporozoite inoculation manually counted . Data are pooled from 7 biological replicates , naïve VRC01 ( n = 2 ) , naïve mAb 10 ( n = 2 ) , VRC01 CIS43 ( n = 3 ) . No comparisons were statistically significant ( Mann - Whitney for motile Fisher ' s exact 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 ) of single - transcriptomics dataset for mouse visual cortex ( Tasic et al , 2016 ) revealed that expression of metabolism - associated gene Cox6a2 is highly restricted to PV + interneurons . Cox6a2 is expressed in subtypes of PV + interneurons ( marked in blue color ) but rarely detected in other neuronal subtypes from mouse visual cortex . Cox6a1 , which encodes other isoform of COX6A , is expressed in 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 brain heart .

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


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

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


( D ) Western - blot showed that COX6A2 is first detected in brain at postnatal day 0 ( P0 ) . Then , COX6A2 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 cortex ( general overview in E , single PV + interneuron magnification in F ) arrowheads indicate PV / Cox6a2 double positive .

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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 ) striatum ( H ) . arrowheads indicate PV / Cox6a2 double positive .

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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 + PV + in somatosensory ( SS ) motor ( MT ) cortices , hippocampus ( HI ) of adult mice ( n = 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 cortex at P9 ( n = 2 , n sections = 12 SS , 8 MT , 4 HI ) . arrowheads indicate Cox6a2 positive .

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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 + are co - labelled with COX6A2 immunoreactivity in SS MT cortices , 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 somatosensory ( SS ) motor ( MT ) cortices , hippocampus ( HI ) of adult rat ( n = 1 , 2 . 5 months old , n sections = 12 SS , 8 MT , 4 HI ) . arrowheads indicate PV / Cox6a2 double positive .

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

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

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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 ) showing absence of COX6A2 immunoreactivity in cortex of Cox6a2 - / - mice .

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


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

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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 - unpaired Student ' s t - .

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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 , somatosensory motor cortices of WT Cox6a2 - / - mice were stained with an antibody against 8 - OHdG , a marker of oxidative 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 - unpaired Mann - Whitney .

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

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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 Cox6a2 - / - mice at P15 processed for bulk sequencing ( n = 4 WT , 6 Cox6a2 - / - ; P15 ) . shows expression for top down - ( red ) 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 Gad2 genes ( n = 4 WT , 6 Cox6a2 - / - ) . Two - unpaired Mann - Whitney .

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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 ) , internal calcium signaling ( blue ) . Color scale bars show normalized read .

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(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 of PV + interneurons from PV - CRE PV - CRE ; Cox6a2fl / fl mice showing PercevalHR after excitation at 930 nm 840 nm . arrowheads indicate PercevalHR expressing . 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 entire duration of recordings ( 23 from 4 PV - CRE mice ; 15 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 ) resting potential amplitude of action potentials in WT Cox6a2 - / - PV + interneurons , respectively . Two - unpaired Student ' s t - .

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

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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 . amplitude difference ( difference between mean amplitude of 1st - 10th action potentials 190th - 200th action potentials ) was significantly higher in P34 - 45 Cox6a2 - / - compared to WT PV + interneurons . In addition , amplitude difference decreased with maturation in WT but not in Cox6a2 - / - PV + interneurons . F : two - unpaired Mann - Whitney .

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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 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 - unpaired Student ' s t - .

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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 their dendritic 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 dendritic between full branch depth ( maximal number of branches between soma a terminal dendritic point ) , which contrasted with expected positive correlation between these parameters in WT PV + interneurons . Linear regression of Pearson correlation .

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

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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 Cox6a2 - / - PV + interneurons have similar dendritic tree complexities ( M ) , at later stage of maturation Cox6a2 - / - PV + interneurons show a reduction in dendritic tree complexity in comparison to WT PV + interneurons ( N ) . Multiple t - , two - stage step - up method of Benjamini , Krieger 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 OFT , Cox6a2 - / - mice travelled a longer moved faster than WT mice . Cox6a2 - / - mice also moved more often from outer zone to inner zone of arena . Two - unpaired Student ' s t - .

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

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

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

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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.


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 efficiency of 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 - 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 of LIPL - 1 : : GFP LIPL - 3 : : TagRFP localization relative to lysosomal marker PGP - 2 show that LIPL - 1 LIPL - 3 are localized to 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 have reduced acidic lipolytic capacity . Mean ± s . d . are presented relative to wild type ( WT ) ; significant differences are indicated ; n = 4 independent experiments .

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

(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 ) fatty acid methyl ester ( FAME ) analyses of wild - type lipl - 1 ( tm1954 ) lipl - 3 ( tm4498 ) double mutant L3 larvae show that lysosomal lipases regulate 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 of wild - type lipl - 1 ( tm1954 ) lipl - 3 ( tm4498 ) double mutant young adults fasted for 4 h shows that LIPL - 1 LIPL - 3 contribute to fat mobilization following fasting . ( Mean ± s . e . m . relative to fed wild type ) . Wild - type fasted worms show 20 % less ORO signal than 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 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 O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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

(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 of well - fed transgenic expressing LIPL - 3 : : TagRFP ATGL : : GFP shows that LIPL - 3 does not localize to lipid droplets .

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

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

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

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

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

(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 was extracted at indicated times . Mean ddCts + s . e . m . show that 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O 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) 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 . 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 intestinal nuclei during fasting .

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

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

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

(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 grown on control RNAi plates were transferred as L4 larvae to control or mxl - 3 RNAi plates , incubated for 12 h at 20 ° C , processed for total FAMEs ; as a reference , an aliquot of wild - type young adults on control RNAi was fasted for 6 h . FAME ( mean 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 .

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

(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 of hlh - 30 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 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 5 ( P0 . 0001 ) , impairs lipl - 1 ( P0 . 01 ) transcriptional activation following fasting .

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

(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 ) 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 .

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

(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 carrying rescuing construct hlh - 30P : : HLH - 30 : : mGFP : : hlh - 30 3 ′ UTR ( mGFP , monomeric GFP , 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 ) .

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

(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 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 .

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

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

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

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

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

(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 of 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 constitutive - induction - of - - lipl - genes phenotype of mxl - 3 mutant ( mean ddCt + s . e . m . relative to wild - type ad libitum - fed worms ) . n = 3 independent experiments .

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

(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 of hlh - 30 ( tm1978 ) wild - type ( WT ) L3 larvae starved for 60 h show that hlh - 30 is required for expansion of lysosomal compartment following fasting . as mean ± s . e . m . relative to fasted wild - type worms is also presented ; n = 3 independent experiments .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O 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) 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 of autophagy genes lgg - 1 , lgg - 2 atg - 16 . 2 in fasted wild - type 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

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

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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) 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 liver of C57BL / 6J 9 - week 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 ) Tfeb ( mammalian hlh - 30 ) but not Max ( mammalian mxl - 3 ) are transcriptionally linked to nutrients in mouse liver ( median + s . e . m . fold change ; ddCt ) . of expression were normalized to ActB Cog2 as internal controls ; but Max differences are significant ( P0 . 05 ) , n = 3 independent experiments . Cyp4a14 is a positive control .

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

(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 incubated in EBSS minimal medium for 2 , 4 or 8 h , compared with expression of control or hTFEB siRNA treated hepatocytes in complete media , show that LAL ( human lysosomal acid lipase ) , MAP1LC3A TFEB but not MAX are transcriptionally linked to nutrients in human hepatocytes , that LAL MAP1LC3A induction under nutrient deprivation are TFEB dependent ( median dCt + s . e . m . ) . of expression were normalized to ACTB as internal control ; 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 mxl - 3 extended lifespan phenotype . were incubated at 20 ° C transferred every other day until cessation of reproduction . 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 of wild - type lipl - 1lipl - 3 double mutant worms shows that lipl - 1 lipl - 3 contribute to clearance of lipids from endocytic pathway . Mean + s . e . m . signal is shown as 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 ) overexpressing LIPL - 1 or LIPL - 3 were incubated at 25 ° C 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 ( that crawled off 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 of cortical synapses derived from WT MPS - IIIA mice at different ages . of synaptic vesicles was quantified from 400 - 500 vesicles ( taken from 5 mice for each genotype at each time point ) expressed as average of vesicle diameter ( nm ) . number of synaptic vesicles per synapse was quantified from 20 different ( taken from 5 mice for each genotype at each time point ) , normalized by of synaptic cleft expressed as % of WT . synaptic density was measured from 20 different ( taken from 5 mice for each genotype at each time point ) as number of synapses / ( # / 500μm2 ) expressed as % of WT . Arrows indicate 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 ) of lysosomal compartment was evaluated by WB ( B ) in brain of MPS - IIIA mice at indicated ages . Quantitation of WB by densitometry ( 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 ) of lysosomal compartment was evaluated by IF ( C ) in 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 MPS - IIIA mice . left panel shows representative fEPSP traces . Summary graphs show fEPSPs slope as a function of either applied stimulus or prevolley amplitude . Data are average of values from 9 slices ( from 9 mice ) in WT group 6 slices ( from 5 mice ) in 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 MPS - IIIA DIV14 hippocampal neurons by of incorporated FM1 - 43 dye in ~ 300 boutons ( taken from 4 - 5 coverslips for each group ) . intensities were expressed as arbitrary units ( A . F . U . ) displayed both as a distribution 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 MPS - IIIA hippocampal neurons ( DIV14 ) were transfected with v - Glut1 - pHluorin - mCherry plasmid synaptic recycling was evaluated by change of probe from 80 - 100 boutons ( taken from 4 - 5 coverslips for each group ) . was quantified at each bouton during KCl perfusion , normalized to value obtained upon rapid alkalization ( NH4Cl perfusion ) expressed as ∆ ( normalized to baseline ; ΔFKCl / ΔF NH4Cl ) . Values were displayed both as traces ( means ± s . e . m . at each time frame ) maximum after KCl perfusion ( means ± s . e . m . ) . Representative panels on right show change in control neurons ( WT ) . Pseudo - colour was applied to better reveal changes . Note that NH4Cl alkalinizes vesicles revealing total ( recycling + resting ) pool in neuronal 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 . of lysosomal compartment was evaluated in both treated control untreated WT neurons by EM examination . Quantitation was performed on 30 different .

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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 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 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 of synaptic terminals was performed in treated control untreated WT neurons . number of synaptic vesicles per synapse was quantified from ~ 20 different ( taken from 3 ultrathin sections for each group ) , normalized by of synaptic cleft expressed as % of WT . Arrows indicate 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 Syntaxin - 1 SNAREs were immunoblotted in WT 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 of WT MPS - IIIA hippocampal neurons ( DIV14 ) double labeled with anti - synapsin - 1 ( presynaptic marker ; red ) either anti - VAMP2 or anti - SNAP25 antibodies ( green ) . merges ( yellow ) of confocal are shown . SNAP25 - synapsin - 1 VAMP2 - synapsin - 1 co - localizations were quantified using Manders ' Colocalization Coefficients ( MCC ) ( ImageJ ) displayed as % ( MCC x 100 ) of Synapsin - 1 co - localizing with either SNAP25 or VAMP2 ( means ± s . e . m . from 15 different taken from 4 - 5 coverslips for each group ) .

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(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 SNAP - 25 were quantified by immunoblot in WT MPS - IIIA hippocampal neurons ( DIV14 ) at different times after cycloheximide treatment expressed as % of remaining at T0 ( 100 % ) . 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 were evaluated in WT 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 co - immunoprecipitated VAMP2 SNAP25 were revealed by WB . of immunoprecipitated 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 CSPα were immunoblotted in WT MPS - IIIA total brain lysates at indicated ages Total 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 CSPα were immunoblotted in WT MPS - IIIA synaptosomal fractions at indicated ages . Hsc70 , an heat shock cognate , which forms together with CSPα SGT ( small glutamine - rich tetratricopeptide repeat domain ) " chaperon machine " was also blotted . 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 of LC3 p62 ( an autophagy substrate ) was performed on WT 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 MPS - IIIA both total synaptosomal brain fractions at indicated ages after sequential extraction with detergents with increased strength . Soluble ( Sol . ) , lowly insoluble ( L . Insol . ) highly insoluble ( H . Insol . ) forms correspond to solubilized respectively in Triton - X100 , SDS 10 % 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 of α - synuclein with SMI - 32 in WT MPS - IIIA hippocampal neurons ( DIV14 ) . α - Synuclein synaptic present in a neurite tract of 10µm is showed in a representative enlarged image . of α - synuclein synaptic was calculated from 30 different enlarged c .

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(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 of α - synuclein ( green ) LAMP1 ( red ) in WT MPS - IIIA hippocampal neurons ( DIV14 ) . Enlarged merge are also shown . Co - localization was quantified using MCC coefficient ( ImageJ ) displayed as % ( MCC x 100 ) of α - synuclein co - localizing with LAMP1 ( means ± s . e . m . from 15 different 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 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 of CSPα SMI - 32 in DIV14 hippocampal neurons . CSPα - synaptic were quantified as in ( C ) .

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


( G ) CSPα were evaluated by immunoblot in WT MPS - IIIA hippocampal neurons ( DIV14 ) at different times after cycloheximide treatment expressed as % of remaining at T0 ( 100 % ) . 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 molecular of CSPα was evaluated in WT 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 ) of RPN10 ( regulatory subunit of 26S proteasome ) ubiquitinated formed by Lys - 48 ( K48 ) residue linkage ( involved in degradation via proteasome ) were evaluated in WT MPS - IIIA brain samples at indicated ages by WB . 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 chymotrypsin - like activity in WT 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 lysosome inhibitor ' s cocktail for 3 days by immunoblot with anti - LC3 antibodies . As control DIV10 WT neurons were left untreated monitored by LC3 blot over three days period . LC3 - II 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.


( B ) Proteasome activity was measured in treated control untreated WT neurons using pZsProSensor - 1 vector ( green ) . were co - stained with anti SMI - 32 ( blu ) . Proteasome activity was quantified by measuring green ( inversely correlated with proteasome activity ) in 10 different ( taken from 4 - 5 coverslips for each group ) expressed as fold to WT .

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(B) Proteasome activity was measured in treated and control untreated WT neurons using pZsProSensor-1 vector (green). Cells were co-stained with anti SMI-32 (blu). Proteasome activity was quantified by measuring the green fluorescence (inversely correlated with proteasome activity) in 10 different cells (taken from 4-5 coverslips for each group) and expressed as fold to WT.


( C ) Activation of calpain 2 caspase - 3 proteolytic systems was evaluated by WB measurement of of calpain 2 caspase - 3 ( both full - activated forms of ~ 17 KDa ) in treated control untreated WT neurons . Arrow indicated full - caspase - 3 .

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(C) Activation of calpain 2 and caspase-3 proteolytic systems was evaluated by WB measurement of the protein levels of calpain 2 and caspase-3 (both full-length and activated cleaved forms of ~17 KDa) in treated and control untreated WT neurons. Arrow indicated the full-length caspase-3 protein.


( D ) After 3 days - treatment lysates from treated control untreated WT neurons were immunoblotted with α - synuclein CSPα were quantified .

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(D) After 3 days-treatment cell lysates from treated and control untreated WT neurons were immunoblotted with α-synuclein and CSPα and protein levels were quantified.


( E ) Treated control untreated WT neurons were subjected to α - synuclein ( green ) LAMP1 ( red ) confocal . Enlarged merge are also shown . Co - localization was quantified using MCC coefficient ( ImageJ ) displayed as % ( MCC x 100 ) of α - synuclein co - localizing with LAMP1 ( means ± s . e . m . from 15 different taken from 4 - 5 coverslips for each group ) . were also co - stained with anti SMI - 32 .

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(E) Treated and control untreated WT neurons were subjected to α-synuclein (green) and LAMP1 (red) confocal analysis. 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). Cells were also co-stained with anti SMI-32.


( F ) Treated control untreated WT neurons were subjected to CSPα / SMI - 32 co - immunofluorescence . CSPα synaptic present in a neurite tract of 10µm is showed in a representative enlarged image . of CSPα synaptic was calculated from 30 different enlarged taken from 4 - 5 coverslips for each group .

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(F) Treated and control untreated WT neurons were subjected to CSPα/SMI-32 co-immunofluorescence. CSPα synaptic puncta present in a neurite tract of 10µm is showed in a representative enlarged image. Quantification of CSPα synaptic puncta was calculated from 30 different enlarged images taken from 4-5 coverslips for each group.


( G ) SDS - resistant in treated control untreated WT neurons were evaluated by immunoblotting of non - boiled samples with VAMP2 or SNAP - 25 antibodies . amounts of SNARE complexes were quantified .

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(G) SDS-resistant complex levels in treated and control untreated WT neurons were evaluated by immunoblotting analysis of non-boiled samples with VAMP2 or SNAP-25 antibodies. The amounts of SNARE complexes were quantified.


( A ) α - Synuclein , CSPα , VAMP2 SNAP - 25 were immunoblotted in total brain homogenates / or synaptosomal fractions of 1 . 5 month - old WT ( CSPα + / + / α - syn + / + ) , CSPα - / - , CSPα + / - , α - syn - / - , α - syn + / - CSPα + / - / α - syn + / - . Synapsin - 1 was also blotted as a control . Quantitation of WB in synaptosomal samples was shown .

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(A) α-Synuclein, CSPα, VAMP2 and SNAP-25 were immunoblotted in total brain homogenates and/or synaptosomal fractions of 1.5 month-old WT (CSPα+/+/α-syn+/+), CSPα-/-, CSPα+/-, α-syn-/-, α-syn+/- and CSPα+/-/α-syn+/-. Synapsin-1 was also blotted as a control. Quantitation of WB in synaptosomal samples was shown.


( B ) SDS - resistant were evaluated in brain homogenates derived from mice with indicated genotypes by immunoblotting of non - boiled samples with VAMP2 or Syntaxin - 1 antibodies . Quantitation of WB was shown .

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(B) SDS-resistant complex levels were evaluated in brain homogenates derived from mice with indicated genotypes by immunoblotting of non-boiled samples with VAMP2 or Syntaxin-1 antibodies. Quantitation of WB was shown.


( C ) EM of cortical synapses derived from mice with indicated genotypes . number of synaptic vesicles per synapse was quantified from 40 different ( taken from 5 different mice for each genotype ) , normalized by of synaptic cleft expressed as % of WT . of synaptic vesicles was quantified from 400 - 500 vesicles ( taken from 5 different mice for each genotype ) expressed as average of vesicle diameter ( nm ) Arrows indicate synaptic cleft .

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(C) EM analysis of cortical synapses derived from mice with indicated genotypes. The number of synaptic vesicles per synapse was quantified from 40 different images (taken from 5 different mice for each genotype), normalized by the length of synaptic cleft and expressed as % of WT. The size of synaptic vesicles was quantified from 400-500 vesicles (taken from 5 different mice for each genotype) and expressed as the average of vesicle diameter (nm) Arrows indicate the synaptic cleft.


( D ) Extracellular recordings ( fEPSPs ) in hippocampal brain slices from 1 . 5 months - old WT ( CSPα + / + / α - syn + / + ) CSPα + / - / α - syn + / - mice . left panel shows representative fEPSP traces . Summary graphs show fEPSPs slope as a function of either applied stimulus or prevolley amplitude . Data are average of values from 5 slices ( from 4 mice ) in WT group 5 slices ( from 4 mice ) in CSPα + / - / α - syn + / - . * p < 0 . 05 , repeated measure ANOVA .

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(D) Extracellular recordings (fEPSPs) in hippocampal brain slices from 1.5 months-old WT (CSPα+/+/α-syn+/+) and CSPα+/-/α-syn+/- 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 5 slices (from 4 mice) in the WT group and 5 slices (from 4 mice) in the CSPα+/-/α-syn+/-. *p<0.05, repeated measure ANOVA.


( A ) Anti - myc immunostaining in different brain regions derived from 10 - months - old MPS - IIIA mice intraventricularly injected with AAV2 / 9 vectors encoding CSPα under Synapsin - 1 promoter ( MPS - IIIA - myc - CSPα experimental group ) . As a control immunostaining was performed in brain sections from 10 - months - old MPS - IIIA mice injected with empty AAV2 / 9 vectors ( MPS - IIIA - empty experimental groups ) . To evaluate co - localization between exogenous CSPα presynaptic compartment brain sections were also co - stained with anti - myc anti - synapsin1 antibodies ( confocal are also shown ) .

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(A) Anti-myc immunostaining in different brain regions derived from 10-months-old MPS-IIIA mice intraventricularly injected with AAV2/9 vectors encoding CSPα under Synapsin-1 promoter (MPS-IIIA-myc-CSPα experimental group). As a control immunostaining was performed in brain sections from 10-months-old MPS-IIIA mice injected with empty AAV2/9 vectors (MPS-IIIA-empty experimental groups). To evaluate co-localization between exogenous CSPα and presynaptic compartment brain sections were also co-stained with anti-myc and anti-synapsin1 antibodies (confocal images are also shown).


( B ) Anti - myc WB in synaptosomal fractions derived from MPS - IIIA - myc - CSPα mice . As a control WB was performed in samples from 10 - months - old WT MPS - IIIA mice injected with empty AAV2 / 9 vectors ( WT - empty MPS - IIIA - empty experimental groups ) . Synaptosomal fractions were also blotted with anti - CSPα .

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(B) Anti-myc WB in synaptosomal fractions derived from MPS-IIIA-myc-CSPα mice. As a control WB was performed in samples from 10-months-old WT and MPS-IIIA mice injected with empty AAV2/9 vectors (WT-empty and MPS-IIIA-empty experimental groups). Synaptosomal fractions were also blotted with anti-CSPα.


( C ) VAMP2 , SNAP - 25 Syntaxin - 1 were immunoblotted in synaptosomal samples derived from three experimental groups of mice . were quantified .

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(C) VAMP2, SNAP-25 and Syntaxin-1 were immunoblotted in synaptosomal samples derived from the three experimental groups of mice. Protein levels were quantified.


( D ) amount of SNARE - complexes was detected in synaptosomal brain samples derived from three experimental groups of mice by immunoblotting of non - boiled samples with VAMP2 , SNAP - 25 or Syntaxin - 1 antibodies . amounts of SNARE complexes were quantified .

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(D) The amount of SNARE-complexes was detected in synaptosomal brain samples derived from the three experimental groups of mice by immunoblotting analysis of non-boiled samples with VAMP2, SNAP-25 or Syntaxin-1 antibodies. The amounts of SNARE complexes were quantified.


( E ) Total brain lysates derived from three experimental groups of mice were immunoprecipitated with Syntaxin - 1 antibodies co - immunoprecipitated VAMP2 SNAP - 25 were revealed by WB .

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(E) Total brain lysates derived from the three experimental groups of mice were immunoprecipitated with Syntaxin-1 antibodies and co-immunoprecipitated VAMP2 and SNAP-25 proteins were revealed by WB.


( A ) EM of cortical hippocampal synapsis derived from three experimental groups of mice . Synaptic vesicles number per synapse was quantified from ~ 40 different ( taken from 5 mice for each group ) , normalized by of synaptic cleft expressed as % of WT . synaptic density was measured from 20 different ( taken from 5 mice for each group ) as number of synapses / ( # / 500μm2 ) expressed as % of WT . Arrows indicate synaptic cleft while asterisks indicate abnormal vacuoles . Values are means ± s . e . m . * P < 0 . 05 , Student ' s t - : Either MPS - IIIA - empty or MPS - IIIA - myc - CSPα vs WT - empty . Scale bar : 0 . 2

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(A) EM analysis of cortical and hippocampal synapsis derived from the three experimental groups of mice. Synaptic vesicles number per synapse was quantified from ~40 different images (taken from 5 mice for each group), 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 group) as the number of synapses/area(#/500μm2) and expressed as % of WT. Arrows indicate the synaptic cleft while asterisks indicate abnormal vacuoles. Values are means ± s.e.m. *P<0.05, Student's t-test: Either MPS-IIIA-empty or MPS-IIIA-myc-CSPα vs WT-empty. Scale bar: 0.2 µm


( B ) Extracellular recordings ( fEPSPs ) in hippocampal brain slices from WT - empty ( n = 8 ) , MPS - IIIA - empty ( n = 5 ) MPS - IIIA - myc - CSPα mice ( n = 5 ) . upper panel shows representative fEPSP traces . Summary graph shows fEPSPs slope as a function of applied stimulus . Data are means ± s . e . m . ; * P < 0 . 05 ( WT - empty vs MPS - IIIA - empty ) , # P < 0 . 05 ( MPS - IIIA - myc - CSPα vs . MPS - IIIA - empty ) , Kruskal - Wallis ANOVA followed by Dunn ' s .

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(B) Extracellular recordings (fEPSPs) in hippocampal brain slices from WT-empty (n=8), MPS-IIIA-empty (n=5) and MPS-IIIA-myc-CSPα mice (n=5). The upper panel shows representative fEPSP traces. Summary graph shows the fEPSPs slope as a function of the applied stimulus intensity. Data are means ± s.e.m.; *P < 0.05 (WT-empty vs MPS-IIIA-empty), #P < 0.05 (MPS-IIIA-myc-CSPα vs. MPS-IIIA-empty), Kruskal-Wallis ANOVA followed by Dunn's test.


( C ) Mean travelled , maximal speed , immobility time crossing during 10 min testing in open field , divided into 5 minute intervals in WT - empty , MPS - IIIA - empty MPS - IIIA - myc - CSPα mice ( n = 9 , n = 9 , n = 7 respectively ) . , open entries open time in elevated plus maze latency to fall off wire ( * ) in wire hanging in WT - empty , MPS - IIIA - empty MPS - IIIA - myc - CSPα mice ( n = 10 , n = 8 , n = 9 respectively ) . Values are means ± s . e . m ; * P < 0 . 05 ( MPS - IIIA - empty vs . WT - empty ) , # P < 0 . 05 ( MPS - IIIA - myc - CSPα vs . MPS - IIIA - empty ) , two - way ANOVA for repeated measures for open field measures t - for plus maze wire hanging followed by Duncan post - hoc . ' average was 28 ± 2 .

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(C) Mean distance travelled, maximal speed, immobility time and line crossing during 10 min testing in the open field, divided into 5 minute intervals in WT-empty, MPS-IIIA-empty and MPS-IIIA-myc-CSPα female mice (n=9, n=9, n=7 respectively). Distance, percentage open entries and open time in the elevated plus maze and latency to fall off the wire (*body weight) in the wire hanging test in WT-empty, MPS-IIIA-empty and MPS-IIIA-myc-CSPα male mice (n=10, n=8, n=9 respectively). Values are means ± s.e.m; *P < 0.05 (MPS-IIIA-empty vs. WT-empty), #P < 0.05 (MPS-IIIA-myc-CSPα vs. MPS-IIIA-empty), two-way ANOVA for repeated measures for open field measures and t-test for the plus maze and the wire hanging followed by Duncan post-hoc test. Animals' average age was 28±2 weeks.


( D ) Neuronal was evaluated in cerebellum ( Calbindin immunostaining ) in different layers of cortex ( NeuN immunostaining of frontal cortex ) of 10 months - old mice belonging to each experimental group of mice . Number of was quantified from 20 different ( taken from 5 mice for each group ) expressed as % of WT . Values are means ± s . e . m . * P < 0 . 05 , * * P < 0 . 001 Student ' s t - . Scale bar : 50 µm .

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(D) Neuronal cell death was evaluated in the cerebellum (Calbindin immunostaining) and in different layers of cortex (NeuN immunostaining of frontal cortex) of 10 months-old mice belonging to each experimental group of mice. Number of cells was quantified from 20 different images (taken from 5 mice for each group) and expressed as % of WT. Values are means ± s.e.m. *P<0.05, **P<0.001 Student's t-test. Scale bar: 50 µm.


( E ) Kaplan - Meier survival in WT - empty ( n = 13 ) , MPS - IIIA - empty ( n = 13 ) MPS - IIIA - myc - CSPα ( n = 13 ) mice . Kaplan - Meier survival curve was analyzed with Chi - square . A P value < 0 . 05 was considered to be statistically significant . P = 0 . 00003 ( MPS - IIIA - empty vs WT - empty ) , P = 0 . 000776 ( MPS - IIIA - myc - CSPα vs MPS - IIIA - empty ) .

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(E) Kaplan-Meier survival analysis in WT-empty (n=13), MPS-IIIA-empty (n=13) and MPS-IIIA-myc-CSPα (n=13) male mice. The Kaplan-Meier survival curve was analyzed with the Chi-square test. A P value <0.05 was considered to be statistically significant. P= 0.00003 (MPS-IIIA-empty vs WT-empty), P= 0.000776 (MPS-IIIA-myc-CSPα vs MPS-IIIA-empty).


A Number of differentially up - down - regulated genes ( DEG ) in RNAi - ASCO1 seedlings as compared to wild type ( WT ) according to - seq data ( FDR < 0 . 01 , | log2FC | > = 0 . 75 ) . B Fraction of DEG found in each transcript class as defined in Araport11 gene annotation . AS stands for antisense .

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A Number of differentially up- and down-regulated genes (DEG) in RNAi-ASCO1 seedlings as compared to wild type (WT) according to the RNA-seq data (FDR < 0.01, |log2FC| >= 0.75). B Fraction of DEG found in each transcript class as defined in the Araport11 gene annotation. AS stands for antisense.


D Representative picture of 14 - day - old grown 9 days in liquid 1 / 2MS supplemented with 1 µM flg22 . scale bar representing 0 . 6cm is included in picture . E Lateral root density of WT two independent RNAi - ASCO lines 9 days after transfer in 1 / 2MS supplemented with 0 . 1 µM or 1 µM flg22 .

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D Representative picture of 14-day-old plants grown 9 days in liquid 1/2MS supplemented with 1 µM flg22. The scale bar representing 0.6cm is included in the picture. E Lateral root density of WT and two independent RNAi-ASCO lines 9 days after transfer in 1/2MS supplemented with 0.1 µM or 1 µM flg22.


D Representative picture of 14 - day - old grown 9 days in liquid 1 / 2MS supplemented with 1 µM flg22 . scale bar representing 0 . 6cm is included in picture .

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D Representative picture of 14-day-old plants grown 9 days in liquid 1/2MS supplemented with 1 µM flg22. The scale bar representing 0.6cm is included in the picture.


F Representative picture of root apical meristems after wall , in response to flg22 . TZ : Transition Zone ; QC : Quiescent Center . G Root apical meristem of WT RNAi - ASCO1 ( eg from QC to TZ in µm ) .

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F Representative picture of root apical meristems after cell wall staining, in response to flg22. TZ: Transition Zone; QC: Quiescent Center. G Root apical meristem size of WT and RNAi-ASCO1 (eg distance from QC to TZ in µm).


F Representative picture of root apical meristems after wall , in response to flg22 . TZ : Transition Zone ; QC : Quiescent Center .

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F Representative picture of root apical meristems after cell wall staining, in response to flg22. TZ: Transition Zone; QC: Quiescent Center.


B Number of genes containing at least one differential processing event ( as defined by RNAprof p . adj < 0 . 001 ) in CDS , introns , 5 ' UTR 3 ' UTR between RNAi - ASCO1 WT . Up Down fractions correspond to increase or decrease , respectively , of - seq coverage in RNAi - ASCO1 for each specified gene feature .

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B Number of genes containing at least one differential RNA processing event (as defined by RNAprof p.adj < 0.001) in CDS, introns, 5'UTR and 3'UTR between RNAi-ASCO1 and WT. Up and Down fractions correspond to increase or decrease, respectively, of RNA-seq coverage in RNAi-ASCO1 for each specified gene feature.


B Scatter plot showing respective gene expression fold change in RNAi - ASCO 35S : ASCO lines as compared to WT . Genes showing significant changes in RNAi - ASCO 35S : ASCO are highlighted as yellow blue dots , respectively .

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B Scatter plot showing the respective gene expression fold change in RNAi-ASCO and 35S:ASCO lines as compared to WT. Genes showing significant changes in RNAi-ASCO and 35S:ASCO are highlighted as yellow and blue dots, respectively.


C Scatter plots showing respective Spliced In difference ( dPSI ) in RNAi - ASCO 35S : ASCO lines as compared to WT . Genes showing significant changes in RNAi - ASCO , 35S : ASCO or in both lines are highlighted as red , green or blue dot , respectively . Gray dots represent AS events .

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C Scatter plots showing the respective Percent Spliced In difference (dPSI) in RNAi-ASCO and 35S:ASCO lines as compared to WT. Genes showing significant changes in RNAi-ASCO, 35S:ASCO or in both lines are highlighted as red, green or blue dot, respectively. Gray dots represent all AS events.


Analyses of RT - PCR products of SR34 transcripts on 8 % acrylamide gel . of of SR34 isoforms detected in gel in ( B ) RNAs were extracted from WT RNAi - ASCO1 14 - day - old . asterisk ( * ) indicates a significant difference as determined by Student ' s T ( p < 0 . 05 , n = 3 biological replicates ) . Error bars show mean + / - standard deviation .

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Analyses of RT-PCR products of SR34 transcripts on 8% acrylamide gel. Quantification of the ratio of SR34 isoforms detected in the gel in (B) RNAs were extracted from WT and RNAi-ASCO1 14-day-old plants. The asterisk (*) indicates a significant difference as determined by Student's T test (p < 0.05, n = 3 biological replicates). Error bars show mean +/- standard deviation.


Analyses of RT - PCR products of NUDT7 transcripts on 8 % acrylamide gel . of of NUDT7 isoforms detected in gel in ( E ) respectively . RNAs were extracted from WT RNAi - ASCO1 14 - day - old . asterisk ( * ) indicates a significant difference as determined by Student ' s T ( p < 0 . 05 , n = 3 biological replicates ) . Error bars show mean + / - standard deviation .

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

Analyses of RT-PCR products of NUDT7 transcripts on 8% acrylamide gel. Quantification of the ratio of NUDT7 isoforms detected in the gel in (E) respectively. RNAs were extracted from WT and RNAi-ASCO1 14-day-old plants. The asterisk (*) indicates a significant difference as determined by Student's T test (p < 0.05, n = 3 biological replicates). Error bars show mean +/- standard deviation.