daf-18/PTEN mutants exhibit phenotypes typical of GABA-deficient animals.

A and B-Quantification of paralysis induced by aldicarb (A) and levamisole (B). At least four independent trials for each condition were performed (n= 25-30 animals per trial). The strain CB156 unc-25(e156) was included as a strong GABA-deficient control. C-Quantification of body shortening in response to anterior touch. Data are represented as mean ± SEM. At least four independent trials for each condition were performed (n= 10-20 animals per genotype/trial). D-(Top) Scheme of C. elegans escape response in NGM agar. After eliciting the escape response by an anterior gentle touch, the omega turns were classified as closed (head and tail are in contact) or open (no contact between head and tail). (Bottom) Quantification of % closed omega turns/ total omega turns. At least eight independent trials for each condition were performed (n= 20-25 animals per genotype/trial). E and F-Light-evoked elongation/contraction of animals expressing Channelrhodopsin (ChR2) in GABAergic (E) and cholinergic (F) motorneurons. Animals were filmed before, during and after a 5 second-pulse 470nm light stimulus (15 frames/s). The body area in each frame was automatically tracked using a custom FIJI-Image J macro. The averaged area of each animal during the first 125 frames (0-5 seg) established a baseline for normalizing light-induced body area changes. The mean body area during the light pulse was averaged and compared, to analyze statistically significant differences (n=40-55 animals per genotype). Changes in the worm area reflect changes in body length, since the width do not change (see methods and Fig.S1G). Results are presented as mean ± SEM. One-way ANOVA was used to test statistical differences in A, B, D, F and Kruskal-Wallis test was used in C and G (ns p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001).

daf-18/PTEN mutants show neurodevelopmental defects in GABAergic motor - neurons.

A- Representative image of a wild-type animal expressing mCherry in the GABAergic motor neurons. In the upper inset, commissures are shown at higher resolution. The lower inset shows a ventral view of the animal (all the processes travel through the right side of the animal body). B- Representative images of commissure defects observed in daf-18 (ok480) mutants (arrows). The defects shown are: Short, commissure length less than half of nematode width; Bridged, neighboring commissures linked by a neurite; Guidance, commissures that do not reach dorsal nerve cord; and Handedness, commissure running along the opposite side of the animal’s body. C- Quantification of GABAergic system defects. Each bar represents the mean ± SEM. One-way ANOVA (ns p > 0.05; **** p ≤ 0.0001). At least four independent trials for each condition were performed (n: 20-25 animals per genotype/trial). D-Representative image of L1 animals expressing Punc-47::mCherry in wild-type (Top) and daf-18(ok480) mutant (Bottom) backgrounds. In this larval stage, only six GABAergic DD motor neurons are born. The inset shows a typical defective (branched) commissure. E- Quantification of GABAergic system defects in L1s. Each bar represents the mean ± SEM. Two-tailed unpaired Student’s t test. (**p ≤ 0.01). At least three independent trials for each condition were performed (n: ∼20 animals per genotype/trial). F and G- Quantification of closed omega turns/total omega turns and commissure defects in GABAergic neurons of animals expressing daf-18 solely in GABAergic neurons. One-way ANOVA (ns p > 0.05; **p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). At least four independent trials for each condition were performed (n: 15-20 animals per genotype/trial) A-Anterior; P-Posterior; D-Dorsal; V-Ventral.

Dietary βHB supplementation rescues GABAergic deficits in daf-18/PTEN mutants.

Animals were exposed to βHB (20 mM) throughout development (from embryo to L4). A and B- Quantification of paralysis induced by cholinergic drugs. At least four independent trials for each condition were performed (n: 20-25 animals per genotype/trial). C- Measurement of body length in response to anterior touch. Kruskal-Wallis test (ns p > 0.05; * p ≤ 0.05). At least three independent trials for each condition were performed (n= 10-13 animals per genotype/trial). D- Quantification of closed omega turns/total omega turns during the escape response. At least six independent trials for each condition were performed (n= 20 animals per genotype/trial). E- Quantification of commissure defects in GABAergic neurons. Results are presented as mean ± SEM. Two-tailed unpaired Student’s t test (ns p > 0.05; *p ≤ 0.05). At least three independent trials for each condition were performed (n= ∼20 animals per genotype/trial). F-K- Light-evoked elongation/contraction of animals expressing ChR2 in GABAergic (F-H) and cholinergic (I-K) motorneurons. The mean body area during the light pulse was averaged and compared, to analyze statistically significant differences between treated and not treated groups in each genotype (n=25-35 animals per condition). Two-tailed unpaired Student’s t test (ns p > 0.05; *p ≤ 0.05; ** p ≤ 0.01).

Early developmental stages are critical for βHB-modulation of GABAergic signaling.

A- Animals were exposed to βHB-enriched diet during different developmental intervals: from embryo to the L1 stage (E-L1), from L1 to L2 (L1-L2), from L2 to L4 (L2-L4), or throughout development (from embryo to L4, E-L4). B and C- Quantification of closed omega turns/total omega turns. and GABAergic commissure defects in daf-18 mutants exposed to βHB at different developmental intervals. Four and six independent trials for each condition were performed in B and C, respectively (n=20-30 (n: 20-25 animals per genotype/trial). Results are presented as mean ± SEM. One-way ANOVA (ns p > 0.05; *p ≤ 0.05; ** p ≤ 0.01).

daf-18/PTEN mutants are hypersensitive to cholinergic drugs.

A- daf-18/PTEN encodes a lipid and protein phosphatase that hydrolyzes phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to phosphatidylinositol-4,5-bisphosphate (PIP2). It is the main negative modulator of PDK and AKT activity. In daf-18/PTEN mutants, AKT-1 is overactivated leading to high levels of DAF-16/FOXO phosphorylation that prevents the translocation of this transcription factor to the nucleus. B- Gene structure of daf-18. Coding sequences are represented by blue boxes. The daf-18(e1375) mutant allele inserts a 30 bp sequence in exon IV. This insertion occurs downstream of the phosphatase catalytic domain and causes a frameshift that leads to premature truncation of the protein. This e1375 mutation partially reduces DAF-18 function. The daf-18(ok480) allele contains a 956 bp deletion that removes most of exon 3 and exon 4 and is generally considered to be a null allele. C- Schematic of C. elegans neuromuscular circuit. Red indicates GABAergic motor neurons (DD/VD) and green indicates cholinergic motor neurons (VA/VB and DA/DB). The VA and VB cholinergic motor neurons send synaptic inputs to the ventral body wall muscles and to the DD GABAergic motor neurons. The release of ACh from VA/VB neurons leads to the contraction of the ventral body wall muscles and the activation of DD GABAergic motor neurons that release GABA on the opposite side of the worm, causing relaxation of the dorsal body wall muscles. Conversely, activation of the DA and DB cholinergic motor neurons produces contraction of the dorsal body wall muscles and activates the VD GABAergic motor neurons. The VD GABAergic motor neurons release GABA, causing relaxation of the ventral body wall muscles, and thus contralateral inhibition. D-F Quantification of paralysis induced by cholinergic drugs. The assays were performed in NGM plates containing 2 mM aldicarb and 0.5 mM levamisole. Each data point represents the mean percentage of animals paralyzed ± SEM . At least four independent trials with 25-30 animals for each genotype were performed. One-way analysis of variance (ANOVA) was used to test statistical differences among strains (ns p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). Strains tested are: N2 (wild-type), OAR 144 daf-18(ok480), CB1375 daf-18(e1375), GR1310 akt-1(mg144), TJ1052 age-1(hx546), VC204 akt-2(ok393), VC222 raga-1(ok386) and KQ1366 rict-1(ft7). All of these strains carry loss-of-function mutations. Furthermore, the strains denoted as “pdk-1 (lf)” and “(gf)” correspond to JT9609 pdk-1(sa680), which possesses a loss-of-function mutation, and GR1318 pdk-1(mg142), which harbors a gain-of-function mutation in the pdk-1 gene, respectively. The strain CB156 unc-25(e156) was included as a strong GABA-deficient control. G- Manual Measurement of Body length and width upon Optogenetic Stimulation of GABAergic (Left) and Cholinergic (Right) neurons. At the 2.5-second time point of light stimulation, we manually measured both the width and length of multiple animals and compared these measurements with the corresponding areas obtained from automated analysis (see Materials and Methods). The width of the worms remained relatively constant, highlighting that the alterations in body area primarily stem from changes in the animal’s length

daf-18/PTEN mutations do not affect excitatory cholinergic motor-neuron morphology

A- Representative images of animals expressing GFP in the cholinergic neurons. In the insets, the commissural processes can be appreciated with higher resolution. B- Quantification of cholinergic system defects. Each bar represents the mean ± SEM for at least four trials (∼ 20 animals per trial). Statistical significance between the strains was determined by two-tailed unpaired Student’s t test. (ns p > 0.05). A anterior; P Posterior; D Dorsal; V Ventral.

Exposure to βHB induces sod-3 expression in daf-18, but not in daf-16 mutants.

A- Representative fluorescence images (20X magnification) of worms expressing Psod-3::GFP in different genetic backgrounds (wild-type, daf-18(ok480), daf-16(mgDf50) and daf-18(ok480); daf-16(mgDf50)) upon exposure to βHB (20mM). B- Corresponding quantification of the fluorescence intensity per animal in the head. Scatter dot plot (line at the median) with the relative expression of Psod-3::GFP normalized to naïve wild-type animals. Statistical significance between conditions was determined by Kruskal-Wallis test (ns p > 0.05; ** p ≤ 0.01; **** p ≤ 0.0001, n= 40-90).

βHB does not prevent neurodevelopmental defects in AIY and HSN neurons.

A- AIY processes were visualized in transgenic animals expressing cytoplasmic GFP in AIY neurons (Pttx-3b::GFP) in wild-type and daf-18(ok480) mutant backgrounds. AIY neuronal growth defects were quantified as described before (Christensen et al., 2011). Left: Scheme of AIY morphology and location in the nematode nerve ring. Blue, pharynx; red, AIY interneurons. Pink: wild-type AIY morphology. The two interneurons meet at the dorsal midline. Light blue and yellow: denote different levels of AIY neurite truncation. Right: Percentage of animals with truncated neurites in wild-type and daf-18(ok480) mutants under exposure (or not) to βHB (20 mM). B- HSN were visualized in transgenic animals expressing GFP in serotonergic neurons (Ptph-1::GFP) in wild-type and daf-18(ok480) mutant backgrounds. HSN under-migration defects were identified as described before (Kennedy et al., 2013). Left: Schematic representation of the HSN migratory route during embryogenesis and the corresponding location of the HSN (green circle) in a young adult animal. Only one of two bilaterally symmetric HSNs is illustrated. Colors show information about the position of HSNs: Light purple: complete migration (≥ 0.4), middle purple: intermediated migration (>0.2-<0.4), dark purple: unmigrated (≤0.2). Right: Quantification of the percentage of animals with different HSN migration positions (the most under-migrated neuron of each animal is considered). Bars represent the mean values of at least three independent experiments. Note that there is no significant effect with βHB treatment compared to controls.