Neuron-specific knockdown of solute carrier protein SLC25A46a induces locomotive defects, an abnormal neuron terminal morphology, learning disability, and shortened lifespan

Abstract

Various mutations in the SLC25A46 gene have been reported in mitochondrial diseases that are sometimes classified as type 2 Charcot-Marie-Tooth disease, optic atrophy, and Leigh syndrome. Although human SLC25A46 is a well-known transporter that acts through the mitochondrial outer membrane, the relationship between neurodegeneration in these diseases and the loss-of-function of SLC25A46 remains unclear. Two Drosophila genes, CG8931 (dSLC25A46a) and CG5755 (dSLC25A46b) have been identified as candidate homologs of human SLC25A46. We previously characterized the phenotypes of pan-neuron-specific dSLC25A46b knockdown flies. In the present study, we developed pan-neuron-specific dSLC25A46a knockdown flies and examined their phenotypes. Neuron-specific dSLC25A46a knockdown resulted in reduced mobility in larvae as well as adults. An aberrant morphology for neuromuscular junctions (NMJs), such as a reduced synaptic branch length and decreased number and size of boutons, was observed in dSLC25A46a knockdown flies. Learning ability was also reduced in the larvae of knockdown flies. In dSLC25A46a knockdown flies, mitochondrial hyperfusion was detected in NMJ synapses together with the accumulation of reactive oxygen species and reductions in ATP. These phenotypes were very similar to those of dSLC25A46b knockdown flies, suggesting that dSLC25A46a and dSLC25A46b do not have redundant roles in neurons. Collectively, these results show that the depletion of SLC25A46a leads to mitochondrial defects followed by an aberrant synaptic morphology, resulting in locomotive defects and learning disability. Thus, the dSLC25A46a knockdown fly summarizes most of the phenotypes in patients with mitochondrial diseases, offering a useful tool for studying these diseases.

Keywords: Drosophila; Learning abilities; Mitochondria; Neuromuscular junction; dSLC25A46.

Fig. 1

(A) Sequence alignment of human SLC25A46 with Drosophila SLC25A46a. Identical amino acids are darkly shaded and similar or related amino acids are lightly shaded. Solcar repeat 1 and Solcar repeat 2 are boxed with red and blue lines, respectively. (B) Comparison of aa sequences of Drosophila SLC25A46a and Drosophila SLC25A46a. Identical amino acids are darkly shaded, and similar or related amino acids are lightly shaded.

Fig. 2

Knockdown efficiency in larval brains. (A) SLC25A46a protein levels in larval brains analyzed by Western immunoblotting. Proteins were extracted from larval CNS carrying elav > UAS-w-IR (w; UAS-w-IR/+; elav-GAL4/+) (a), elav > UAS-dSLC25A46a-IR_17-183 (w; UAS-dSLC25A46a-IR_17-183/+; elav-GAL4/+) (b), and elav > UAS-dSLC25A46a -IR_217-383 (w; UAS- dSLC25A46a-IR_217-383 /+; elav-GAL4/+) (c). The blotted membrane was probed with a rabbit anti-dSLC25A46a antibody. The band with an apparent molecular mass of 50 kDa corresponding to the dSLC25A46a protein is marked. The positions of size markers are also given on the left. (B) Quantification of the 50-kDa band. α-Tubulin was used as a loading control. The relative intensity of the 50-kDa band normalized to that of α-tubulin is shown as each bar. ****p < 0.0001, biological replication, n=4.

Fig. 3

Locomotor defect in pan-neuron-specific dSLC25A46a knockdown flies. (A, B) Larval crawling assay. The pan-neuron-specific knockdown of dSLC25A46a causes a decrease in the crawling ability of larvae. Quantified data on the average crawling speed of larvae are shown. (A) Male: elav > UAS-w-IR (w/Y; UAS-w-IR/+; elav-GAL4/+), elav > UAS-dSLC25A46a-IR_17-183 (w/Y; UAS-dSLC25A46a-IR_17-183/+; elav-GAL4/+) and elav > UAS-dSLC25A46a-IR_217-383 (w/Y; UAS- dSLC25A46a-IR_217-383 /+; elav-GAL4/+). (B) Female: elav > UAS-w-IR (w/w; UAS-w-IR/+; elav-GAL4/+), elav > UAS-dSLC25A46a-IR_17-183 (w/w; UAS-dSLC25A46a-IR_17-183/+; elav-GAL4/+) and elav > UAS-dSLC25A46a-IR_217-383 (w/w; UAS- dSLC25A46a-IR_217-383 /+; elav-GAL4/+). ****p < 0.0001. n > 30. (C, D) Adult climbing assay. The pan-neuron-specific knockdown of dSLC25A46a causes a decrease in the climbing ability of adult flies. Quantified data on the climbing scores of flies are shown. (A) Male: elav > UAS-w-IR (w/Y; UAS-w-IR/+; elav-GAL4/+), elav > UAS-dSLC25A46a-IR_17-183 (w/Y; UAS-dSLC25A46a-IR_17-183/+; elav-GAL4/+) and elav > UAS-dSLC25A46a-IR_217-383 (w/Y; UAS- dSLC25A46a-IR_217-383 /+; elav-GAL4/+). (B) Female: elav > UAS-w-IR (w/w; UAS-w-IR/+; elav-GAL4/+), elav > UAS-dSLC25A46a-IR_17-183 (w/w; UAS-dSLC25A46a-IR_17-183/+; elav-GAL4/+) and elav > UAS-dSLC25A46a-IR_217-383 (w/w; UAS- dSLC25A46a-IR_217-383 /+ ; elav-GAL4/+). *p < 0.05. n > 100. Technical replication, n = 5.

Fig. 4

Knockdown of dSLC25A46a in pan-neurons causes an abnormal synapse morphology at NMJs in muscle 4 of third instar larvae. (A) Confocal images showing NMJs stained with anti-HRP IgG (a, d and g) and anti-Dlg IgG (red) (b, e and h). Merged images are shown in panels c, f, and i. The NMJs of flies carrying elav > UAS-w-IR (w; UAS-w-IR/+; elav-GAL4/+) (a-c), elav > UAS-dSLC25A46a-IR_17-183 (w; UAS-dSLC25A46a-IR_17-183/+; elav-GAL4/+) (d-f), and elav > UAS-dSLC25A46a-IR_217-383 (w; UAS-dSLC25A46a-IR_217-383 /+ ; elav-GAL4/+) (g-i) are shown. The proximal region and distal region of the synapse branch are shown by arrows and arrowheads, respectively. Examples of boutons are marked in panels c, f, and i. Scale bars indicate 20 μm. (B, C, D) Quantified data. In dSLC25A46a knockdown larvae, the longest branch length (B) and total length of synaptic branches (C) were shorter and bouton numbers (D) were smaller than control larvae. *p < 0.05 and **p < 0.01. n = 12.

Fig. 5

dSLC25A46a is required for olfactory learning abilities. (A) Male. Larval preference for n-amyl acetate (AM) versus octanol (OCT) (1 = all prefer AM; −1 = all prefer OCT) in larvae carrying elav > UAS-w-IR (w/y; UAS-W-IR/+; elav-GAL4/+, n = 40), elav > UAS-dSLC25A46a-IR_17-183 (w/Y; UAS-dSLC25A46a-IR_17-183/+; elav-GAL4/+, n = 40), and elav > UAS-dSLC25A46a-IR_217-383 (w/Y; UAS- dSLC25A46a-IR_217-383 /+ ; elav-GAL4/+, n = 40) 3 min after training on AM/sucrose-containing plates (AM+) or 3 min after training on OCT/sucrose-containing plates (OCT+). ***p < 0.001. ns: p > 0.37 and ns p > 0.76 for male larvae carrying elav > UAS-dSLC25A46a-IR_17-183 (w/Y; UAS-dSLC25A46a-IR_17-183/+; elav-GAL4/+, and elav > UAS-dSLC25A46a-IR_217-383 (w/Y; UAS- dSLC25A46a-IR_217-383 /+; elav-GAL4/+ respectively. Biological replication, n = 3. (C) Normalized AM and OCT learning index for males were averaged for LI. ****p < 0.0001 (B) Female: elav > UAS-w-IR (w; UAS-W-IR/+; elav-GAL4/+, n = 40), elav > UAS-dSLC25A46a-IR_17-183 (w; UAS-dSLC25A46a-IR_17-183/+; elav-GAL4/+, n = 40), and elav > UAS-dSLC25A46a-IR_217-383 (w; UAS- dSLC25A46a-IR_217-383 /+ ; elav-GAL4/+, n = 40). **p < 0.01 and ns: p > 0.81 ns p > 0.14 for female larvae carrying elav > UAS-dSLC25A46a-IR_17-183 (w; UAS-dSLC25A46a-IR_17-183/+; elav-GAL4/+) and elav > UAS-dSLC25A46a-IR_217-383 (w; UAS- dSLC25A46a-IR_217-383 /+ ; elav-GAL4/+), respectively. Biological replication, n = 3. (D) Normalized AM and OCT LI for females were averaged for the LI. ****p < 0.0001.

Fig. 6

Knockdown of dSLC25A46a causes abnormal mitochondrial dynamics at NMJs in muscle 4 of third instar larvae. (A) Images show NMJs that were double-stained with mito-GFP (green) and anti-Dlg IgG (red). NMJs of flies carrying w; UAS-w-IR/+; elav-GAL4, UAS-mito-GFP/+ (a–d), w; UAS-dSLC25A46a-IR_17-183/+; elav-GAL4, UAS-mito-GFP/+ (e–h) and w; UAS-dSLC25A46a-IR_217-383 /+; elav-GAL4, UAS-mitoGFP/+ (i–l) are shown. Enlarged images of the region enclosed in panels c, g, and k are shown in panels d, h, and l, respectively. Examples of mitochondria are marked in panels a, e, and i. Scale bars indicate 10 μm. (B-D) Quantified data. In dSLC25A46a knockdown larvae carrying elav>mito-GFP,dSLC25A46a-IR_17-183 (w; UAS-dSLC25A46a-IR_17-183/+; elav-GAL4, UAS-mito-GFP/+, n = 12) and elav>mito-GFP,dSLC25A46a-IR_217-383 (w; UAS-dSLC25A46a-IR_217-383 /+; elav-GAL4, UAS-mito-GFP/+, n = 12), the area and size of mitochondria significantly differed from (B, C), whereas the number of mitochondria were similar to (D) in control larvae carrying elav>mito-GFP,w-IR (w; UAS-w-IR/+; elav-GAL4, UAS-mito-GFP/+, n = 12). *p < 0.05 and **p < 0.01. ns: p > 0.63 for larvae carrying elav>mito-GFP,dSLC25A46a-IR_17-183 (w; UAS-dSLC25A46a-IR_17-183/+; elav-GAL4, UAS-mito-GFP/+), and ns: p > 0.83 for larvae carrying elav>mito-GFP,dSLC25A46a-IR_217-383 (w; UAS-dSLC25A46a-IR_217-383 /+; elav-GAL4, UAS-mito-GFP/+).

Fig. 7

dSLC25A46a knockdown decreases levels of ATP in larval CNS. Quantified data on ATP levels of extracts from the brain lobes of larvae carrying elav > UAS-w-IR (w; UAS-w-IR/+; elav-GAL4/+), elav > UAS-dSLC25A46a-IR_17-183 (w; UAS-dSLC25A46a-IR_17-183/+; elav-GAL4/+), and elav > UAS-dSLC25A46a-IR_217-383 (w; UAS-dSLC25A46a-IR_217-383 /+; elav-GAL4/+). In dSLC25A46a knockdown larvae, ATP levels were decreased. *p < 0.05 and **p < 0.01. n=4.

Fig. 8

dSLC25A46a knockdown in pan-neurons increases ROS production in the CNS of third instar larvae. (A) Third instar larval CNS stained with CM-H2DCFDA. Images of elav > UAS-w-IR (w; UAS-w-IR/+; elav-GAL4/+) (a), elav > UAS-dSLC25A46a-IR_17-183 (w; UAS-dSLC25A46a-IR_17-183/+; elav-GAL4/+) (b), and elav > UAS-dSLC25A46a-IR_217-383 (w; UAS- dSLC25A46a-IR_217-383 /+; elav-GAL4/+) (c) are shown. Scale bars indicate 100 μm. (B) Quantified data. In dSLC25A46a knockdown larvae carrying elav > UAS-dSLC25A46a-IR_17-183 (w; UAS-dSLC25A46a-IR_17-183/+; elav-GAL4/,+, n = 8) and elav > UAS-dSLC25A46a-IR_217-383 (w; UAS- dSLC25A46a-IR_217-383 /+ ; elav-GAL4/+, n = 8), ROS signals were stronger than those in control larvae carrying elav > UAS-w-IR (w; UAS-W-IR/+; elav-GAL4/+, n = 8). **p < 0.01 and ***p < 0.001.

Fig. 9

Knockdown of dSLC25A46a in pan-neurons causes a shortened lifespan in adult flies. The survival rates of adult male (A) flies carrying elav > UAS-w-IR (w/y; UAS-W-IR/+; elav-GAL4/+, n = 100), elav > UAS-dSLC25A46a-IR_17-183 (w/Y; UAS-dSLC25A46a-IR_17-183/+; elav-GAL4/+, n = 100), and elav > UAS-dSLC25A46a-IR_217-383 (w/Y; UAS- dSLC25A46a-IR_217-383 /+ ; elav-GAL4/+, n = 100) and female (B) flies carrying elav > UAS-w-IR (w/w; UAS-W-IR/+; elav-GAL4/+, n = 100), elav > UAS-dSLC25A46a-IR_17-183 (w/w; UAS-dSLC25A46a-IR_17-183/+; elav-GAL4/+, n = 100), and elav > UAS-dSLC25A46a-IR_217-383 (w/w; UAS- dSLC25A46a-IR_217-383 /+ ; elav-GAL4/+, n = 100) are shown.