dAcsl, the Drosophila ortholog of acyl-CoA synthetase long-chain family member 3 and 4, inhibits synapse growth by attenuating bone morphogenetic protein signaling via endocytic recycling

Abstract

Fatty acid metabolism plays an important role in brain development and function. Mutations in acyl-CoA synthetase long-chain family member 4 (ACSL4), which converts long-chain fatty acids to acyl-CoAs, result in nonsyndromic X-linked mental retardation. ACSL4 is highly expressed in the hippocampus, a structure critical for learning and memory. However, the underlying mechanism by which mutations of ACSL4 lead to mental retardation remains poorly understood. We report here that dAcsl, the Drosophila ortholog of ACSL4 and ACSL3, inhibits synaptic growth by attenuating BMP signaling, a major growth-promoting pathway at neuromuscular junction (NMJ) synapses. Specifically, dAcsl mutants exhibited NMJ overgrowth that was suppressed by reducing the doses of the BMP pathway components, accompanied by increased levels of activated BMP receptor Thickveins (Tkv) and phosphorylated mothers against decapentaplegic (Mad), the effector of the BMP signaling at NMJ terminals. In addition, Rab11, a small GTPase involved in endosomal recycling, was mislocalized in dAcsl mutant NMJs, and the membrane association of Rab11 was reduced in dAcsl mutant brains. Consistently, the BMP receptor Tkv accumulated in early endosomes but reduced in recycling endosomes in dAcsl mutant NMJs. dAcsl was also required for the recycling of photoreceptor rhodopsin in the eyes, implying a general role for dAcsl in regulating endocytic recycling of membrane receptors. Importantly, expression of human ACSL4 rescued the endocytic trafficking and NMJ phenotypes of dAcsl mutants. Together, our results reveal a novel mechanism whereby dAcsl facilitates Rab11-dependent receptor recycling and provide insights into the pathogenesis of ACSL4-related mental retardation.

Keywords: BMP signaling; Drosophila; mental retardation; neuromuscular junction; synapse; vesicle trafficking.

Figure 1.

Morphological NMJ phenotypes of dAcsl mutants. A, B, Representative confocal images of NMJ4 from abdominal segments A3 to A6 costained with anti-HRP (green) and anti-CSP (magenta) in wild-type control (A) and dAcsl^KO/dAcsl⁰⁵⁸⁴⁷ mutants (B). The NMJ terminals look similar across different abdominal segments of wild-type. In dAcsl mutants, however, the NMJs innervating the anterior segment muscles (i.e., A3) with shorter axons were overgrown with more satellite boutons, whereas those innervating the posterior segment muscles (i.e., A6) with longer axons were dystrophic. Scale bar, 10 μm. C, D, Quantitative results of the total bouton number (C) and satellite bouton number (D). n > 18 NMJs for all genotypes. ***p < 0.001 (Student's t test). Error bars indicate SEM.

Figure 2.

dAcsl inhibits synaptic growth presynaptically. A, NMJ4 synapses of abdominal segment A3 were double-labeled with anti-HRP (green) and anti-CSP (magenta) to reveal presynaptic membranes and synaptic vesicles, respectively. Representative NMJ4 synapses of different genotypes are as follows: wild-type, dAcsl^KO/dAcsl⁰⁵⁸⁴⁷, neuronal rescue by dAcsl and human ACSL4 (elav-Gal4/+; dAcsl^KO/dAcsl⁰⁵⁸⁴⁷; UAS-dAcsl/+ and elav-Gal4/+; dAcsl^KO/dAcsl⁰⁵⁸⁴⁷; UAS-ACSL4/+), muscular rescue (dAcsl^KO/dAcsl⁰⁵⁸⁴⁷; Mhc-Gal4/UAS-ACSL4), and glial rescue by human ACSL4 (dAcsl^KO/dAcsl⁰⁵⁸⁴⁷; repo-Gal4/UAS-ACSL4). Pan-neural expression of dAcsl or human ACSL4 driven by elav-Gal4 rescued the NMJ overgrowth phenotype of dAcsl mutants, but muscular and glial expression of ACSL4 did not. Scale bar, 10 μm. B, C, Quantitative results of the total bouton number (B) and satellite bouton number (C) in different genotypes. n > 18 for all genotypes. *p < 0.05 (one-way ANOVA with Tukey post hoc tests). ***p < 0.001 (one-way ANOVA with Tukey post hoc tests). Error bars indicate SEM.

Figure 3.

Synaptic overgrowth in dAcsl mutants is caused by elevated BMP signaling. A, Confocal images of representative NMJ4 terminals from different genotypes: w¹¹¹⁸, dAcsl^KO/dAcsl⁰⁵⁸⁴⁷, wit^A12/wit^B11, dAcsl^KO/dAcsl⁰⁵⁸⁴⁷; wit^A12/wit^B11, dAcsl^KO/dAcsl⁰⁵⁸⁴⁷; wit^A12/+, dAcsl^KO/dAcsl⁰⁵⁸⁴⁷; tkv⁷/+, dAcsl⁸/dAcsl⁰⁵⁸⁴⁷, and dAcsl⁸/dAcsl⁰⁵⁸⁴⁷; dad^J1e4/+. dAcsl; wit double mutants exhibited dystrophic NMJs identical to those observed in wit mutants. Heterozygous wit^A12 or tkv⁷ mutations reversed the synaptic overgrowth of dAcsl mutants. The weaker dAcsl⁸/dAcsl⁰⁵⁸⁴⁷ mutants showed a mild increase in bouton number and satellite bouton formation. Heterozygous dad^J1e4 mutation resulted in a significant increase in the synaptic bouton number in dAcsl⁸/dAcsl⁰⁵⁸⁴⁷ mutants. Scale bar, 10 μm. B, Quantifications of the total bouton number of various genotypes. n > 18 for all genotypes. ***p < 0.001 (one-way ANOVA with Tukey post hoc tests). Error bars indicate SEM. C, NMJ4 synaptic boutons were colabeled with anti-pMad (green) and anti-CSP (magenta) in wild-type, dAcsl^KO/dAcsl⁰⁵⁸⁴⁷, and ubiquitous expression of human ACSL4 in dAcsl mutant background (dAcsl^KO/dAcsl⁰⁵⁸⁴⁷; tub-Gal4/UAS-ACSL4). pMad intensity was markedly increased in dAcsl mutant NMJs, and the increased pMad intensity was completely rescued by ubiquitous expression of human ACSL4. Scale bar, 3 μm. D, A fluorescence-based reporter TIPF for activated Tkv receptors was neuronally expressed by nSyb-Gal4 in control, gbb⁴/gbb^D20, and dAcsl^KO/dAcsl⁰⁵⁸⁴⁷ mutants. To preserve the YFP fluorescence of the TIPF reporter, samples were processed without detergent. Right column images, Single confocal slices of that shown in the middle column. Arrowheads indicate TIPF puncta. Scale bars: middle, 5 μm; right, 1 μm. E, Quantification of the level of total Tkv receptors by anti-GFP staining in different genotypes. F, Quantification of the normalized intensity of TIPF against anti-HRP staining in different genotypes. n > 12 for each genotype. ***p < 0.001 (one-way ANOVA with Tukey post hoc tests). Error bars indicate SEM.

Figure 4.

Localization of Rab11 is altered at dAcsl NMJ synapses. A, Representative confocal images of NMJ4 from control (left) and dAcsl^KO/dAcsl⁰⁵⁸⁴⁷ mutants (right). YFP-Rab5 (early endosomal) and Spinster-GFP (late endosomal/lysosomal) were expressed presynaptically by elav-Gal4 (green). Neuronal membranes were labeled by anti-HRP (magenta). The staining patterns of these endosomal markers were largely normal at dAcsl mutant NMJs. Scale bar, 5 μm. B, C, Images represent single confocal slices of NMJ4 colabeled with anti-HRP (red), and recycling endosome markers anti-Rab11 (green) and anti-Bchs (blue) in wild-type, dAcsl^KO/dAcsl⁰⁵⁸⁴⁷ mutants (B), and neuronally rescued larvae (C; dAcsl^KO/dAcsl⁰⁵⁸⁴⁷; elav-Gal4/UAS-ACSL4). The staining pattern of Rab11 and Bchs was altered in dAcsl mutant boutons (B), and the alteration was fully rescued by presynaptic expression of human ACSL4 (C). Scale bar, 3 μm. D, E, Confocal images showing the localization of Rab11 at synaptic boutons of wild-type and dAcsl^KO/dAcsl⁰⁵⁸⁴⁷ as related to Brp (D) and Nwk (E). Scale bar, 2 μm. F, Quantification of normalized intensity of Rab11 within the anti–HRP-positive area. n > 18 NMJs for each genotype. ***p < 0.001 (one-way ANOVA with Tukey post hoc tests). Error bars indicate SEM. G, Western results of larval brain extracts of wild-type and dAcsl^KO/dAcsl⁰⁵⁸⁴⁷ detected with antibodies against Rab11, dAcsl, and Rab7. Syntaxin and actin were used as loading controls.

Figure 5.

dAcsl affects Tkv trafficking from early endosomes to recycling endosomes. A–C, Single-slice confocal images showing colocalization between Tkv-GFP puncta (green) and endosomal markers (magenta): anti-Rab5 (A), anti-Rab11 (B), and anti-Hook (C) in genetically matched control (nSyb-Gal4/UAS-Tkv-GFP) and dAcsl mutants (dAcsl^KO UAS-Tkv-GFP/dAcsl⁰⁵⁸⁴⁷ nSyb-Gal4). All images were processed by deconvolution. GFP was localized to the presynaptic membrane and discrete intrabouton puncta in both control and mutant boutons. In dAcsl mutants, the percentage of Tkv-GFP-positive puncta costained with early endosomal Rab5 was increased (A), but that with the recycling endosomal Rab11 was reduced (B). Arrowheads indicate examples of colocalization of GFP spots with endosomal markers. Few Tkv-GFP puncta colocalized with the late endosomal Hook in both control and mutant synapses (C). Scale bar, 2 μm. D, Quantification of the percentages of Tkv-GFP puncta positive for various endosomal markers. n > 12 animals for each genotype. ***p < 0.001 (Student's t test). Error bars indicate SEM. E, Single-slice confocal images showing double-staining of dAcsl-Myc (blue) with Rab11, Rab5, Dor, and Tkv-GFP (green) in NMJ boutons, defined by anti-HRP (red) staining. Motoneuron specific expression of dAcsl-Myc and Tkv-GFP was driven by OK6-Gal4. Arrowheads indicate colocalization of dAcsl with Rab11, Rab5, and Tkv-GFP. Scale bar, 1 μm.

Figure 6.

dAcsl synergistically interacts with rab11 to inhibit BMP signaling. A, Representative confocal images of NMJ4 stained with anti-HRP (green) and anti-CSP (magenta) in wild-type, rab11^93Bi, mad^K00237, mad^K00237; rab11^93Bi, rab11^93Bi/+, dAcsl⁸/dAcsl⁰⁵⁸⁴⁷, dAcsl⁸/dAcsl⁰⁵⁸⁴⁷; rab11^93Bi/+, and dAcsl⁸/dAcsl⁰⁵⁸⁴⁷; rab11^93Bi mutants. rab11 mutants showed more boutons. Synaptic undergrowth in rab11; mad double mutants were indistinguishable from that of mad single mutants (top). Synaptic overgrowth in dAcsl⁸/dAcsl⁰⁵⁸⁴⁷ mutants was enhanced by rab11 mutations (bottom). B, Quantification of the total bouton number in the various genotypes. C, NMJ4 synaptic boutons double-labeled with anti-pMad (green) and anti-CSP (magenta) in wild-type, rab11^93Bi, dAcsl⁸/dAcsl⁰⁵⁸⁴⁷, and dAcsl⁸/dAcsl⁰⁵⁸⁴⁷; rab11^93Bi double mutants. Scale bar, 3 μm. D, Quantification of normalized pMad intensities at NMJ synapses of different genotypes. The number of larvae analyzed is indicated in the columns. *p < 0.05 (one-way ANOVA with Tukey post hoc tests). **p < 0.01 (one-way ANOVA with Tukey post hoc tests). ***p < 0.001 (one-way ANOVA with Tukey post hoc tests). Error bars indicate SEM.

Figure 7.

Membrane association of Rab11 is reduced in dAcsl mutants. A, Ultrastructure of wild-type and dAcsl^KO/dAcsl⁰⁵⁸⁴⁷ NMJ 6/7 boutons of the abdominal segments A2 and A3. The subsynaptic reticulum (SSR) is organized into meshwork membrane folds surrounding the presynaptic bouton. Arrowheads indicate electron dense membranes. T-bars are observed at some active zones (asterisks). No abnormal membrane structures were observed in dAcsl mutants. Scale bar, 500 nm. B, Western results of subcellular fractions of wild-type, dAcsl^KO/dAcsl⁰⁵⁸⁴⁷, and ubiquitous rescue (dAcsl^KO/dAcsl⁰⁵⁸⁴⁷; Tub-Gal4/UAS-ACSL4) larval brains probed with antibodies against Rab11, Rab5, and CSP. Total proteins were fractionated into membrane-associated proteins (M) and cytosolic proteins (C). C, Quantification of the fractions of membrane-associated and cytosol Rab11 in the larval brains. n > 3 for all genotypes. *p < 0.05 (one-way ANOVA with Tukey post hoc tests). Error bars indicate SEM.

Figure 8.

dAcsl promotes recycling of endocytosed rhodopsin to the rhabdomere in adult eyes. A, Cross sections of adult eyes from wild-type and dAcsl knockdown flies (GMR-Gal4/UAS-dAcsl RNAi 1) stained with anti-Rh1. Arrows indicate ERPs. Only the six peripheral rhabdomeres contain Rh1. In dark-reared flies, Rh1 is mostly concentrated in the rhabdomeres. Upon light exposure for 5 h (0 h recovery after light exposure), many ERPs were detected in the cell bodies of both wild-type and dAcsl knockdown photoreceptors. Scale bar, 5 μm. B, Quantification of ERP numbers per rhabdomere at different recovery time points after light exposure in wild-type, dAcsl knockdown by two independent RNAi lines driven by GMR-Gal4, and dAcsl^KO photoreceptors. C, Quantification of the fraction of ERPs disappeared in 2 h recovery in different genotypes. Three sets of independent data were averaged. ***p < 0.001 (one-way ANOVA with Tukey post hoc tests). Error bars indicate SEM. D, Western results showed that Rh1 levels in both wild-type and dAcsl knockdown flies were constant during the experiments. Two visual signaling proteins,PLC and the PDZ-domain scaffold protein INAD, were used as loading controls.

Figure 9.

A model for the function of dAcsl in BMP receptor trafficking at NMJs. Upon Gbb binding, presynaptic BMP receptors are activated on the presynaptic membrane. After endocytosis, the activated BMP receptors in the early endosomes are either sorted to recycling endosomes for Rab11-dependent recycling back to the presynaptic membrane or transported to late endosomes, and finally to lysosomes for degradation. dAcsl mutations disrupt recycling of activated Tkv from early endosomes to recycling endosomes probably by decreasing the membrane association of Rab11, leading to an elevation of activated BMP receptors at the presynaptic membrane and in the early endosomes. Starbursts indicate BMP receptors in phosphorylated, activated form. EE, early endosomes; LE, late endosomes; Lys, lysosome; RE, recycling endosomes.