Chaski, a novel Drosophila lactate/pyruvate transporter required in glia cells for survival under nutritional stress

Abstract

The intercellular transport of lactate is crucial for the astrocyte-to-neuron lactate shuttle (ANLS), a model of brain energetics according to which neurons are fueled by astrocytic lactate. In this study we show that the Drosophila chaski gene encodes a monocarboxylate transporter protein (MCT/SLC16A) which functions as a lactate/pyruvate transporter, as demonstrated by heterologous expression in mammalian cell culture using a genetically encoded FRET nanosensor. chaski expression is prominent in the Drosophila central nervous system and it is particularly enriched in glia over neurons. chaski mutants exhibit defects in a high energy demanding process such as synaptic transmission, as well as in locomotion and survival under nutritional stress. Remarkably, locomotion and survival under nutritional stress defects are restored by chaski expression in glia cells. Our findings are consistent with a major role for intercellular lactate shuttling in the brain metabolism of Drosophila.

Figure 1

Chaski protein has homology to monocarboxylate transporters. (A) Predicted membrane topology with the characteristic 12 transmembrane domains (TM1 to TM12) organized in two groups of six separated by an intracellular loop of the SLC16A family (Monocarboxylate transporters). Green aminoacids represent glycosylation sites (B). Comparison of Chaski (first line) with MCT2 from human, rat, mouse and zebrafish. A black line in top of the alignments labels TM domains. The two characteristic MFS (Major Facilitator Superfamily) domains are: MFS1 from aminoacid 135 to 315 overlapping with TM1 to 6 and MFS2 from aminoacid 685 to 869 overlapping with TM7 to TM12. The color code is depicted in the top, from red denoting the conserved aminoacids to blue representing the non-conserved aminoacids.

Figure 2

Chaski behaves as a transporter of lactate and pyruvate. (A) HEK293 cells expressing Laconic of cytosolic distribution. (B) Single cell FRET ratio traces of HEK cells that were deprived of glucose for 30 min and then exposed for 2 min to 10 mM lactate (lac). ∆T₁ (DT₁) is the amplitude of the change (%) in Laconic ratio recorded during the first lactate pulse. After lactate washout, the procedure was repeated (DT₂) in the absence (left panel) or presence (right panel) of the MCT1/2 blocker AR-C155858 (1 μM). Trace segments used to calculate DTs are in black. The bar graph summarizes ΔT₂/ΔT₁ for control (41 cells in 3 exps.) and in the presence of 1 μM AR-C15585 (44 cells in 3 exps.). (C) HEK293 expressing Laconic and V5-tagged Chk, note the cytosolic localization of Laconic and plasma membrane distribution of Chk. (D) Representative single cell traces from a HEK culture co-expressing Laconic and Chk exposed to 10 mM lactate in the absence and presence of the MCT1/2 blocker AR-C155858 (1 μM). (E) Frequency distributions of ΔT₂/ΔT₁ in control cells (44 in 3 exps.) and chk-transfected cells (87 in 3 exps.), estimated from experiments similar to that illustrated in D. The best fit of the Gaussian function y = a exp [−(X − X₀]²/2b²] to the control data is represented by the interrupted line, with parameters a = 41 ± 1, b = 0.1 ± 0.003 and X₀ = −0.02 ± 0.003, r² = 0.99. (F) chk-transfected and control cultures were exposed for 2 min to 10 mM pyruvate (pyr) or 6 mM oxamate (oxa) in the absence or presence of 1 μM AR-C155858, data are from representative cells. (G) Summary of the responses to oxamate, for control (120 cells in 3 exps.) and chk-transfected cultures (139 cells in 3 exps.). Error bars represent s.e.m.; statistic test is a non-paired student t-test. *p < 0.05.

Figure 3

Chaski is expressed in the Drosophila central nervous system. (A) Determination of chk mRNA levels of expression using quantitative RT-PCR from whole embryo and first instar larvae and brain at the indicated developmental stages. chk expression reaches a peak at late pupae stage (average of 3 independent experiments from samples in triplicate). (B) Evaluation of chk expression in different CNS cell types using the TRAP technique. chk is found enriched in glia over neurons, and underrepresented in astrocyte-like (Alrm-Gal4) over other glia subtypes, cortex glia (NP2222-Gal4); ensheating glia (NP6520-Gal4) and subperineurial glia (moody-Gal4) (3 independent experiments from samples in triplicate). (C) Diagram of the chk gene showing the genomic locations of the insertions used as reporters. (D) Cartoon of the larval brain boxing the region in the optic lobe where the confocal images were obtained in E to H. (E–H)” Analysis of chk expression in 3^er instar larval brain, using the reporters described in C. (E,F)” Representative confocal images of larval brain of animals expressing UAS-NLS-Cherry under the control of chk-Gal4^NP3576 (E’ and F’); co-labeled with anti-Elav (F, F”) and anti-Repo (E, E”) antibodies to identify expression in neurons and glial cells respectively. (G,H”) Representative confocal images of larval brains of animals expressing UAS-NLS-Cherry under the control of chk-Gal4^MI15450 (G’ and H’), co-labeled with anti-Elav (H”) and anti-Repo (G”) antibodies to identify expression in neurons and glial cells. (I,J”). Representative confocal images of larval brains of animals expressing chk^CB04412 (GFP-trap, I’ and J’), co-labeled with anti-Elav (I”) and anti-Repo (J”) antibodies (brain region boxed in K). (K) Cartoon of the larval brain boxing the region in the central brain from where the pictures in I to J” were obtained. Expression of chk is observed in both REPO and ELAV expressing cells. Error bars represent s.e.m.; statistics applied was Kruskal-Wallis for TRAP analysis and ordinary one-way anova followed by Tukey test for the expression during development. Calibration bar in E is 20 µm. At least four brains for each line were imaged; all displayed the same distribution of immunoreactivity.

Figure 4

Chaski is required for locomotion in the adult, proper synaptic function and survival under metabolic stress. (A) Evaluation of the larval frequency of contraction in chk mutants and controls, no differences were found between controls and mutants (10 to 12 larvae per genotype). (B) Quantification of the larval speed crawling in different chk mutant genotypes, no differences were found between controls and mutants (10 to 12 larvae per genotype). (C) Quantification of the climbing index of adult flies 3 to 5 days old of different chk mutant genotypes, all chk mutants show a decreased climbing compared to controls (3 independent experiments per line, each 20–25 flies of mixed gender; see Fig. S5-C for experiments in males and females separated). (D) Representative traces of evoked post-synaptic currents (EPSC) from abdominal muscle 6–7 of 3^rd segment exposed to 1 mM external calcium (left) and a plot comparing the average amplitude of the EPSC for mutants and controls. Currents were recorded under two-electrode voltage-clamp with a holding voltage of −80mV, each box represent the mean of 10 stimuli/larvae in 12 larvae/line, chk^crspr (p = 0.027), and chk^MB04207 (p = 0.011). Stimuli-artifact was removed. (E) Representative traces of EPSC recorded at different extracellular calcium concentrations: 0.1, 0.2, 0.5, 1 and 2 mM calcium from control and mutant larvae. (F) Plot of average EPSC amplitudes as a function of external calcium concentration for control and chk mutants. Data are shown as mean ± SE, 10 recordings/larvae n = 4 larvae/point. Data from control larvae (black line), and chk mutants (grey lines) were fitted to Hill-equation I = I_max*[Ca⁺²]ⁿ_H/((K_0.5)ⁿ_H + [Ca⁺²]ⁿ_H. Differences at 1 and 2 mM concentrations are statistically significant between controls and chk mutant flies (two-way anova, p < 0.05 for chk^MB04207 and p < 0.001 for chk^crspr). (G) Survival curve for control (W¹¹¹⁸, black continuous line) and chk mutant flies (grey broken or dotted lines) obtained in normal food (two independent experiments were pooled, in each 100 flies were reared in vials of 20 flies each of mixed gender). Both mutants show fitted curves significantly different from controls, p < 0.001. (H) Survival curves for control (W¹¹¹⁸, black continuous line) and chk mutant flies (grey broken or dotted lines) obtained under starvation (three independent experiments were pooled, in each 60 flies of mixed gender were reared in 3 vials with 20 flies each, see Fig. S5-A and B for experiments in females and males separated). Both mutant curves are significantly different from control, p < 0.001. The transhetorozygous mutant is also different from the control (p < 0.05) Error bars represent s.e.m.; statistic in C and F is ordinary two-way anova.

Figure 5

Expression of Chaski in glia rescues mutant climbing defects and sensitivity to starvation. (A) Chk subcellular distribution displays membrane localization. First column are representative confocal images of brain and ventral cord from control larva and larva expressing Chk-pseudoscura-HA (Chk^pso-HA) under the control of Repo-Gal4 immunostained for Repo (red, nuclear localization) and HA epitope (green, membrane localization). Second column, details from the white square in the first image immunostained for Repo (red, nuclear localization), third column same detail immunostained for HA epitope (green, membrane localization), fourth column merge of the two previous images. (B) Climbing index for WT, mutant controls and mutants expressing UAS-Chk in glia under the control of Repo-Gal4. The expression of Chk in glia rescues the climbing defects observed in mutants (three independent experiments 20–25 flies of mixed gender each, in duplicate, see rescue experiments in females and males in Fig. 5S-C), (C) Survival curves of WT, mutants and mutants expressing UAS-Chk in glia under the control of Repo-Gal4 (two independent experiments were pooled in each 60 flies were reared in vials with 20 flies each). The expression of Chk in glia rescues the decreased survival during starvation observed in chk mutants. Error bars represent s.e.m., statistic is one-way anova followed by Tukey post-hoc test. Calibration bar is 20 µm. Experiments were performed without separating males from females.