Brain Research 1689 (2018) 30–44

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Novel Drosophila model for mitochondrial diseases by targetingof a solute carrier protein SLC25A46
Kojiro Suda a, Ibuki Ueoka a, Yumiko Azuma b,c, Yuuka Muraoka a, Hideki Yoshida a, Masamitsu Yamaguchi a,⇑a

Department of Applied Biology and The Center for Advanced Insect Research, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
Department of Neurology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Japanc
Department of Molecular Pathobiology of Brain Diseases, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566,Japanba r t i c l e

i n f oArticle history:
Received 10 January 2018
Received in revised form 21 March 2018
Accepted 23 March 2018
Available online 28 March 2018
Keywords:
Mitochondrial disease
Solute carrier
Drosophila
NeuronNeuromuscular junction
ROS

a b s t r a c t
Mutations in SLC25A46 gene have been identified in mitochondrial diseases that are sometimes classified
as Charcot-Marie-Tooth disease type 2, optic atrophy and Leigh syndrome. Human SLC25A46 functions as
a transporter across the outer mitochondrial membrane. However, it is still unknown how the neurodegeneration occurring in these diseases relates to the loss of SLC25A46 function. Drosophila has CG5755(dSLC25A46) as a single human SLC25A46 homolog. Here we established pan-neuron specific dSLC25A46
knockdown flies, and examined their phenotypes. Neuron specific knockdown of dSLC25A46 resulted in
an impaired motility in both larvae and adults. Defects at neuromuscular junctions (NMJs), such as
reduced synaptic branch length, decreased number and size of bouton, reduced density and size of active
zone were also observed with the dSLC25A46 knockdown flies. Mitochondrial hyperfusion in synapse at
NMJ, accumulation of reactive oxygen species and reduction of ATP were also observed in the dSLC25A46
knockdown flies. These results indicate that depletion of SLC25A46 induces mitochondrial defects accompanied with aberrant morphology of motoneuron and reduction of active zone that results in defect inlocomotive ability. In addition, it is known that SLC25A46 mutations in human cause optic atrophy andknockdown of dSLC25A46 induces aberrant morphology of optic stalk of photoreceptor neurons in third
instar larvae. Morphology and development of optic stalk of photoreceptor neurons in Drosophila are precisely regulated via cell proliferation and migration. Immunocytochemical analyses of subcellular localization of dSLC25A46 revealed that dSLC25A46 localizes not only in mitochondria, but also in plasma
membrane. These observations suggest that in addition to the role in mitochondrial function, plasma
membrane-localized dSLC25A46 plays a role in cell proliferation and/or migration to control optic stalkformation. The dSLC25A46 knockdown fly thus recapitulates most of the phenotypes in mitochondrial disease patients, providing a useful tool to study these diseases.
Ó 2018 Elsevier B.V. All rights reserved.

1. Introduction
Solute carriers (SLCs) consists of the largest family of membranetransport proteins in human (Hediger et al., 2004). Phylogenetic
analyses have identified 384 unique protein sequences forming
52 distinct SLC families (Rask-Andersen et al., 2013). SLCtransporters localize in the plasma membrane or in intracellularcompartment membranes. SLCs regulate the transport of various
substrates including inorganic ions, purines, nucleotides, aminoacids, sugars, fatty acids, neurotransmitters, and drug molecules
across biological membranes (Hediger et al., 2004). Defects in SLCs
Abbreviations: SLC, solute carrier; CNS, central nervous system; NMJ, neuromuscular junction; ROS, reactive oxygen species.
⇑ Corresponding author.
E-mail address: myamaguc@kit.ac.jp (M. Yamaguchi).
https://doi.org/10.1016/j.brainres.2018.03.0280006-8993/Ó 2018 Elsevier B.V. All rights reserved.

by mutations or genetic variants, have been suggested asunderlying factors in various human diseases such as diabetes,
cancer, psychiatric disorders and neurodevelopmental disorders(Rask-Andersen et al., 2013).
Out of fifty-two SLC families, SLC25 family proteins commonly
localize in the mitochondria (Hediger et al., 2004). Out of fortysix SLC25 family proteins, only SLC25A46 protein is reported tointeract with Mitofusin2. SLC25A46 protein is predicted to havethe typical structure of full-size SLC proteins, including two Solcar
repeats, each containing two transmembrane domains. SLC25A46
is the most highly expressed in nervous system (Haitina et al.,2006). Other studies predicted SLC25A46 to function as a transporter across the outer mitochondrial membrane (Palmieri,2013). Dominant or recessive mutations in the SLC25A46 gene inpatients showed cerebellar atrophy, peripheral neuropathy andK. Suda et al. / Brain Research 1689 (2018) 30–44

optic atrophy (Abrams et al., 2015; Charlesworth et al., 2016). Thisdisorder spectrum was defined as hereditary motor sensory neuropathy Type VIB (HMSN6B) that is classified as one of the axonal
Charcot-Marie-Tooth (CMT) disease, in Online Mendelian Inheritance in Man (OMIM) (http://www.omim.org/entry/616505).
Other studies showed that SLC25A46 mutations also cause multiple
neuropathies including optic atrophy, Leigh syndrome, progressive
myoclonic ataxia and lethal congenital pontocerebellar hypoplasia(Charlesworth et al., 2016p. 25; Janer et al., 2016; Nguyen et al.,2017; Wan et al., 2016).
Previous functional study with cultured non-neuron cells
revealed the role for SLC25A46 in mitochondrial fission, that is
likely mediated by protein networks distinct from the
Mitofusin2-Optic atrophy protein1 (MFN2-OPA1) fusion complex(Abrams et al., 2015). More recent studies based on cultured
non-neuron cell models suggested that SLC25A46 plays other roles
in mitochondrial dynamics by controlling contacts between endoplasmic reticulum (ER) and mitochondria or by regulating MFN1/2
oligomerization (Janer et al., 2016; Steffen et al., 2017). Other studies with SLC25A46 knockout mice demonstrated predicted phenotypes, including impaired motility, abnormal mitochondria in
Purkinje cells and optic atrophy (Li et al., 2017). However, pathological mechanism of diseases caused by SLC25A46 is not fully
understood and there is no effective treatment or therapy for
CMT disease with mitochondrial defect.
Drosophila has a single homolog for the human SLC25A46 gene
called CG5755, which is located on the region 36E4 of the fly 2L
chromosome. Because Drosophila CG5755 and human SLC25A46
are highly conserved, we designated the Drosophila CG5755 asdSLC25A46. In the present study, we have established a Drosophila
mitochondrial disease model including CMT targeted todSLC25A46. Drosophila has been established as an excellent model
for studying various human diseases (Chow and Reiter, 2017). Taking account of the advantages of Drosophila as a useful modelorganism, establishment of Drosophila models for mitochondrial
disease will contribute to understanding of its pathological mechanism. Our previous studies and other studies revealed thatmutants or knockdown of Drosophila homolog of the CMT causing
genes exhibits deficit in motor abilities of adult flies as well as
aberrant morphology of peripheral neurons. These phenotypiccharacteristics are reminiscent of human CMT patients, suggestingthat this is a useful fly model for studying CMT (Bharadwaj et al.,2016; Kyotani et al., 2016; Niehues et al., 2016; Storkebaum
et al., 2009). Here, we found that pan-neuron-specific knockdown
of dSLC25A46 results in an impaired motility in both larvae andadults. Defects at neuromuscular junctions (NMJs) were also
observed with the dSLC25A46 knockdown flies. Mitochondrial
hyperfusion in synapse at NMJ, accumulation of reactive oxygen
species and reduction of ATP were also observed in the knockdown
flies. These results indicate that depletion of SLC25A46 induces
mitochondrial defects accompanied with aberrant morphology of
motoneuron and reduction of active zone that results in defect inlocomotive ability. In addition, it is known that SLC25A46 mutations in human cause optic atrophy and knockdown of dSLC25A46
induces aberrant morphology of photoreceptor neurons in third
instar larvae. These observations suggest that in addition to therole in mitochondrial function, plasma membrane-localizeddSLC25A46 plays a role in controlling optic stalk formation.
2. Results
2.1. Comparison of the aa sequence of human SLC25A46, andDrosophila CG5755
A Blast search with human SLC25A46 identified CG5755 in Drosophila. The amino acid sequence of Drosophila CG5755 was

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retrieved from UniProt and compared with that of human SLC25
family members using FASTA and BLAST. According to this comparison, high conservation was found between Drosophila CG5755 andhuman SLC25A46, showing 21% identity and 60% similarity. Other
SLC25 subfamily proteins in humans were less similar to Drosophila CG5755. Human SLC25A46 encodes protein consisting of
418 aa (Fig. 1). The specific protein domains Solcar1 and Solcar2,
each containing two transmembrane domains are both highly conserved between CG5755 and SLC25A46, showing 40% identity (75%
similarity) and 43% identity (78% similarity), respectively (Fig. 1).
In this study, we referred to Drosophila CG5755 as Drosophila
SLC25A46 (dSLC25A46).
2.2. Specificity of the anti-dSLC25A46 antibody and evaluation ofknockdown efficiency
We performed qRT-PCR to examine knockdown efficiency. The
results showed that mRNA levels of dSLC25A46 reduced to 50.1%
and 30.0% in flies carrying w, UAS-dSLC25A46-IR142–242/ w; +; elavGAL4/+, w; UAS-dSLC25A46-IR207–350/+; elav-GAL4/+, respectively(Fig. 2A), suggesting that dSLC25A46 is effectively knocked downin these flies. We raised an antibody against a peptide corresponding to amino acid residues 423–436 of dSLC25A46 for immunological studies. In order to confirm the specificity of this antibody, we
carried out Western blotting analyses with extracts of larval brain
carrying w; UAS-GFP-IR/+; elav-GAL4/ +, w, UAS-dSLC25A46-IR142–242/
w; +; elav-GAL4/+, w; UAS-dSLC25A46-IR207–350/+; elav-GAL4/+, w;
UAS-dSLC25A46-IR207–350; elav-GAL4/+. In these flies, doublestranded RNA for GFP or dSLC25A46 is specifically expressed in all
neurons. Extracts of twenty larval CNS from each strain were prepared and subjected to the Western blotting analyses. Whole procedures were repeated twice. A single band corresponding to an
apparent molecular size of 50 kDa was detected (Fig. 2B). The
molecular size of this band is similar to that predicted for the aasequence of dSLC25A46 (50.78 kDa). The intensity of this band
was quantified and statistically evaluated. The band was reduced
to 52.2% in knockdown flies carrying w, UAS-dSLC25A46-IR142–242/
w; +; elav-GAL4/+ compared to its intensity in control flies carrying
w; UAS-GFP-IR/+; elav-GAL4/+ (Fig. 2C). The intensity was reduced to46.9% in flies carrying a single copy of UAS-dSLC25A46-IR207–350 (w;UAS-dSLC25A46-IR207–350/+; elav-GAL4/+) and to 31.4% in those carrying two copies of UAS-dSLC25A46-IR207–350 (w; UAS-dSLC25A46IR207–350; elav-GAL4/+) (Fig. 2C). These reductions of the 50 kDa band
intensities were statistically significant. These data indicate that theantibody is highly specific to the dSLC25A46 protein and further
confirming that dSLC25A46 is effectively knocked down in flies carrying w, UAS-dSLC25A46-IR142–242/w; +; elav-GAL4/+, w; UASdSLC25A46-IR207–350/+; elav-GAL4/+, w; UAS-dSLC25A46-IR207–350;
elav-GAL4/+.
2.3. Subcellular localization of dSLC2546 on larval salivary glandTo further evaluate the specificity of anti-dSLC25A46 antibody,
we performed competition experiments with the immunizing peptide (Fig. S1). The dSLC25A46 signals in the cytoplasm, nucleus andplasma membrane were all decreased (Fig. S1C, D) compared tocontrol (Fig. S1A, B). Furthermore, a flip-out experiment was
employed to make a somatic clone expressing dSLC25A46 dsRNAin the salivary glands. In RNAi clones marked with GFP, the intensity of dSLC25A46 signals was extensively reduced (Fig. 3A–D),
thus confirming that the anti-dSLC25A46 signals in the cytoplasm,
nucleus and plasma membrane truly represent dSLC25A46 protein.
Double-immunostaining of larval salivary gland with antidSLC25A46 and specific marker antibodies for each of mitochondria, endoplasmic reticulum (ER), Golgi apparatus and plasma
membrane were performed. The anti-Discs large (Dlg) antibody

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K. Suda et al. / Brain Research 1689 (2018) 30–44

Fig. 1. Sequence Alignment of human SLC25A46 and Drosophila SLC25A46. Identical amino acids are dark shaded, and similar or related amino acids are light shaded. Solcar1
and Solcar2 are boxed.

Fig. 2. Knockdown efficiency in larval brains. (A) dSLC25A46 mRNA levels in larval brains. In elav> UAS-dSLC25A46-IR larvae, the mRNA levels of dSLC25A46 were lower than incontrol larvae. The mRNA levels were normalized to GAPDH mRNA. (B) dSLC25A46 protein levels in larval brains analyzed by Western blotting. Proteins were extracted fromlarval CNS carrying elav-GAL4 > UAS-GFP-IR (w; UAS-GFP-IR/+; elav-GAL4/+), elav-GAL4 > UAS-dSLC25A46-IR142–242 (w, UAS-dSLC25A46-IR142–242/w; +; elav-GAL4/+), elav-GAL4 >UAS-dSLC25A46-IR207–350 (w; UAS-dSLC25A46-IR207–350/+; elav-GAL4/+), and elav-GAL4 > UAS-dSLC25A46-IR207–350x2 (w; UAS-dSLC25A46-IR207–350; elav-GAL4/+). The blots wereprobed with a rabbit anti-dSLC25A46 antibody. The band with an apparent molecular weight of 50 kDa corresponds to dSLC25A46 protein. (C) Quantification of the 50 kDa
band with ImageJ. a-tubulin was used as a loading control. *p < 0.05, biological replication n = 2.

specifically marks plasma membrane (Huu et al., 2014). The
dSLC25A46 signals merge with Dlg signals, indicating localizationof dSLC25A46 in plasma membrane (Fig. 3E–H). To test localizationof dSLC25A46 in mitochondria, the mitochondria protein HSP60(Soltys and Gupta, 1996) was labeled with anti-HSP60 antibody.
The dSLC25A46 signals clearly merge with HSP60 signals as

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Fig. 3. Immunocytochemistry for dSLC25A46 in the larval salivary gland. Female flies with hs-flp; Act5C > FRT y FRT > GAL4, UAS-GFP were crossed with male flies carrying
UAS-dSLC25A46-IR207–350. After flipping-out, third instar larval salivary glands were stained with anti-dSLC25A46 antibody (A). GFP clone (B) and DAPI signals (C) are also
shown. Third instar larval salivary glands from Canton S (E-T) were stained with anti-dSLC25A46 antibody (E, I, M, Q), anti-Dlg IgG (F), anti-HSP60 IgG (J), anti-KDEL IgG (N),anti-KDEL receptor IgG (R), and DAPI (G, K, O, S). Merged confocal images are shown in (D, H, L, P, T). Bars indicate 20 lm (A–D), 10 lm (E–H) and 5 lm (I–P).

expected (Fig. 3I–L). The anti-KDEL antibody has been commonlyused as the ER marker (Charroux and Royet, 2013) which labels
the sequence Lys-Asp-Glu-Leu (KDEL), or a closely relatedsequence, that is present at the carboxyl terminus of ER soluble
and some membrane proteins (Munro and Pelham, 1987;
Pelham, 1990, 1988; Teasdale and Jackson, 1996). The dSLC25A46

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K. Suda et al. / Brain Research 1689 (2018) 30–44

signals at least partially merge with KDEL signals, suggesting somefraction of dSLC25A46 localize on ER (Fig. 3 M–P). The anti-KDEL
receptor antibody has been used as marker of Golgi apparatus(Huu et al., 2014). KDEL receptor is an integral membrane protein
which mediates the retrieval of solute resident proteins from Golgi
apparatus to the ER (Lewis and Pelham, 1992, 1990; Tang et al.,
1993; Teasdale and Jackson, 1996). The dSLC25A46 signals merge
with KDEL receptor signals (Fig. 3Q–T). These results revealed thatdSLC25A46 localizes not only in mitochondria, but also in plasma
membrane and other subcellular organelles such as ER and Golgi
apparatus.
2.4. Knockdown of dSLC25A46 causes locomotor defects in both larval
and adult stages
Flies carrying UAS-dSLC25A46-IR were crossed with the elavGAL4 driver strain to specifically knockdown dSLC25A46 in Drosophila pan-neurons. To examine the effect of dSLC25A46 knockdown
on locomotor abilities, a crawling assay for the larval stage and aclimbing assay for the adult stage were performed. In the larvalstage, dSLC25A46 knockdown larvae exhibited decreases in motion
speed. The average speed of knockdown larvae reduced by 0.4 mm/
s in knockdown flies carrying w, UAS-dSLC25A46-IR142–242/w; +;elav-GAL4/+ compared with its speed in control flies carrying
w/+; +; elav-GAL4/+ (Fig. 4A). The average speed was reduced to
0.7 mm/s in flies carrying a single copy of UAS-dSLC25A46-IR207–350(w; UAS-dSLC25A46-IR207–350/+; elav-GAL4/+) and to 0.8 mm/s inthose carrying two copies of UAS-dSLC25A46-IR207–350 (w; UASdSLC25A46-IR207–350; elav-GAL4/+) (Fig. 4A). These reductions were
statistically significant. The defect in locomotor abilities ofneuron-specific dSLC25A46 knockdown flies was also observed at
the adult stage (Fig. 4B). The knockdown flies (w, UASdSLC25A46-IR142–242/w; +; elav-GAL4/+ and w; UAS-dSLC25A46IR207–350/+; elav-GAL4/+) started to exhibit a decrease in mobility
at day 3 after eclosion. At 28 days old, the climbing ability of theseflies reduced considerably to 30% and continued to decrease compared with that of control flies carrying w; UAS-GFP-IR/+; elavGAL4/+ (Fig. 4B). These results indicated that dSLC25A46 has animportant function in regulating locomotion.

2.5. dSLC25A46 plays an important role in synapse structure formation
at neuromuscular junctions in third instar larvae
The above-mentioned results on locomotor defects suggested
that dSLC25A46 might have a function in motor neurons. Therefore,
we analyzed the morphology of motor neuron presynaptic andpostsynaptic terminals at NMJs in dSLC25A46 knockdown larvae(Fig. 5A). Drosophila larval NMJ is a well-established system forstudying synaptic functions and morphology (Keshishian et al.,1996). Termini of larval segmental nerves form a series of varicosities where they contact and innervate muscles. These ‘‘boutons”
contain active zones where neurotransmitters are released andhave a stereotyped beads-on-a-string pattern.
The NMJ branch length was reduced to 36.5% in knockdown
flies carrying w, UAS-dSLC25A46-IR142–242/w; +; elav-GAL4/+ compared with its length in control flies carrying w; UAS-GFP-IR/+;elav-GAL4/+ (Fig. 5B). The length was reduced to 75.3% in flies
carrying a single copy of UAS-dSLC25A46-IR207–350 (w; UASdSLC25A46-IR207–350/+; elav-GAL4/+) and to 62.2% in those carrying two copies of UAS-dSLC25A46-IR207–350 (w; UAS-dSLC25A46IR207–350; elav-GAL4/+) (Fig. 5B). These reductions were statistically significant.
The number of 1b boutons was reduced to 52.0% in knockdown
flies carrying w, UAS-dSLC25A46-IR142–242/w; +; elav-GAL4/+ compared with its length in control flies carrying w; UAS-GFP-IR/+;elav-GAL4/+ (Fig. 5C). The bouton size was reduced to 60.4% in flies
carrying a single copy of UAS-dSLC25A46-IR207–350 (w; UASdSLC25A46-IR207–350/+; elav-GAL4/+) and to 73.2% in those carrying
two copies of UAS-dSLC25A46-IR207–350 (w; UAS-dSLC25A46-IR207–350;
elav-GAL4/+) (Fig. 5C). These reductions were statisticallysignificant.
The size of 1b boutons was reduced to 46.2% in knockdown flies
carrying w, UAS-dSLC25A46-IR142–242/w; +; elav-GAL4/+ comparedwith its length in control flies carrying w; UAS-GFP-IR/+;elav-GAL4/+ (Fig. 5D). The bouton size was reduced to 96.5% in flies
carrying a single copy of UAS-dSLC25A46-IR207–350 (w; UASdSLC25A46-IR207–350/+; elav-GAL4/+) and to 42.4% in those carrying
two copies of UAS-dSLC25A46-IR207–350 (w; UAS-dSLC25A46-IR207–350;
elav-GAL4/+) (Fig. 5D). Except for the data with flies carrying a single

Fig. 4. Locomotor dysfunction in dSLC25A46 knockdown flies. (A) Crawling assay. Knockdown of dSLC25A46 in pan-neurons exerted negative effects on the crawling ability oflarvae. Quantified data of average crawling speed of flies carrying elav-GAL4/+ (w/+; +; elav-GAL4/+, n = 104), elav-GAL4 > UAS-dSLC25A46-IR142–242 (w, UAS-dSLC25A46-IR142–242/w;+; elav-GAL4/+, n = 104), elav-GAL4 > UAS-dSLC25A46-IR207–350 (w; UAS-dSLC25A46-IR207–350/+; elav-GAL4/+, n = 103), and elav-GAL4 > UAS-dSLC25A46-IR207–350x2 (w; UASdSLC25A46-IR207–350; elav-GAL4/+, n = 76). The knockdown larvae crawled more slowly than the control larvae. ****p < 0.001. (B) Climbing assay. Knockdown of dSLC25A46 in panneurons causes a decrease in climbing ability in adults. *p < 0.05. Technical replication n = 5. elav-GAL4 > UAS-GFP-IR (w; UAS-GFP-IR/+; elav-GAL4/+, n = 100), elav-GAL4 > UASdSLC25A46-IR142–242 (w, UAS-dSLC25A46-IR142–242/w; +; elav-GAL4/+, n = 251), and elav-GAL4 > UAS-dSLC25A46-IR207–350 (w; UAS-dSLC25A46-IR207–350/+; elav-GAL4/+,
n = 240).

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Fig. 5. Knockdown of dSLC25A46 causes abnormal morphology of synapse at NMJs in muscle 4 of third instar larvae. (A) Images show NMJs that were double-stained with
anti-HRP (green) and anti-Dlg (red). NMJs of flies carrying elav-GAL4 > UAS-GFP-IR (w; UAS-GFP-IR/+; elav-GAL4/+) (a–c), elav-GAL4 > UAS-dSLC25A46-IR207–350x2 (w; UASdSLC25A46-IR207–350; elav-GAL4/+) (d–f) are shown. Length of synaptic branches are measured from white arrowhead to white arrow. Scale bars indicate 10 lm (a–f). (B–D)
The quantified data. In dSLC25A46 knockdown larvae, length of synaptic branches is reduced (B), number and size of bouton are decreased, (C, D) compared with the control
larvae (*p < 0.05 and **p < 0.01, ****p < 0.001). elav-GAL4 > UAS-GFP-IR (w; UAS-GFP-IR/+; elav-GAL4/+, n = 10), elav-GAL4 > UAS-dSLC25A46-IR142–242 (w, UAS-dSLC25A46-IR142–242/w;+; elav-GAL4/+, n = 13), elav-GAL4 > UAS-dSLC25A46-IR207–350 (w; UAS-dSLC25A46-IR207–350/+; elav-GAL4/+, n = 13), and elav-GAL4 > UAS-dSLC25A46-IR207–350x2(w; UAS-dSLC25A46-IR207–350; elav-GAL4/+, n = 9).

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K. Suda et al. / Brain Research 1689 (2018) 30–44

copy of UAS-dSLC25A46-IR207–350, these reduction were statisticallysignificant.
These results indicate that dSLC25A46 plays an important role inregulating synapse structure formation at NMJs, and locomotor
dysfunction might be caused by aberrant morphology of motor
neuron presynaptic and postsynaptic terminals in third instar
larvae.
2.6. dSLC25A46 plays an important role in active zone structureformation at neuromuscular junctions in third instar larvae
In presynaptic membrane, synaptic vesicles fuse at a specialized
membrane domain called the active zone. Neurotransmitters are
released at active zones. Therefore, we analyzed the morphologyof active zone at NMJs in dSLC25A46 knockdown larvae (Fig. 6A).
The Bruchpilot (Brp), which is a central compartment of electrondense cytomatrix at active zone, serves as a marker for active zone.
In flies carrying two copies of UAS-dSLC25A46-IR207–350 (w; UASdSLC25A46-IR207–350; elav-GAL4/+), the density and size of active
zones reduced to 72.0%, 73.6% respectively compared with control
larvae carrying w; UAS-GFP-IR/+; elav-GAL4/+ (Fig. 6B, C). These
reductions were statistically significant. The density and size ofactive zones of other knockdown flies appeared to be decreased,
although the difference was not statistically significant. The reduction with single copy knockdown flies was not statistically significant, probably due to the less efficient knockdown of dSLC25A46(Fig. 6B, C). The number of active zones is not different between
dSLC25A46 knockdown and control flies (Fig. 6D). These results
suggest that dSLC25A46 plays an important role in regulating active
zone structure formation at NMJs, and neuronal dysfunction might
be caused by decreased synaptic vesicles containing
neurotransmitters.
2.7. dSLC25A46 plays an important role in regulating dynamics ofmitochondria at neuromuscular junctions in third instar larvae
SLC25A46 has function in mitochondrial fission in cultured cells(Abrams et al., 2015). Therefore, we analyzed the morphology of
mitochondria at NMJs in dSLC25A46 knockdown larvae (Fig. 7A).
Mitochondria in NMJ are detected by overexpressed GFPtargeted to mitochondria (Pilling et al., 2006). In flies carrying
UAS-dSLC25A46-IR142–242 or UAS-dSLC25A46-IR207–350 (w, UASdSLC25A46-IR142–242/w; +; D42-GAL4, UAS-mitoGFP/+ or w/UASdSLC25A46-IR207–350/+; D42-GAL4, UAS-mitoGFP/+), the density of
mitochondria increased to 141.4%, 153.1% respectively and the sizeof mitochondria also increased to 139.5%, 155.4% respectively compared with control larvae carrying w; UAS-beta gal-IR/+; D42-GAL4,
UAS-mitoGFP/+ (Fig. 7B, C). The number of mitochondria is not different between dSLC25A46 knockdown and control flies (Fig. 7D).
These results suggest that hyperfused mitochondria accumulate
in NMJ by the lack of mitochondrial dynamics or mitochondria
are morphologically elongated under the lack of dSLC25A46 conditions. Accumulation of the defective mitochondria at NMJs mightcause neuronal dysfunction and aberrant morphology of motor
neuron presynaptic and postsynaptic terminals in third instar
larvae.
2.8. dSLC25A46 knockdown increases reactive oxygen species incentral nerves system of third instar larvae
The above-mentioned results on mitochondrial aberrant morphology suggested that dSLC25A46 might have a role in mitochondrial function. Therefore, we analyzed the ROS level as oxidative
stress marker of mitochondria in larval central nervous system(CNS) (Fig. 8A). The intensity of ROS signal was increased to170.6% in knockdown flies carrying w, UAS-dSLC25A46-IR142–242/w;+; elav-GAL4/+ compared with its intensity in control flies carrying
w; UAS-GFP-IR/+; elav-GAL4/+ (Fig. 8B). The intensity was increased
to 134.0% in flies carrying a single copy of UAS-dSLC25A46-IR207–350(w; UAS-dSLC25A46-IR207–350/+; elav-GAL4/+) and to 282.9% in thosecarrying two copies of UAS-dSLC25A46-IR207–350 (w; UAS-dSLC25A46IR207–350; elav-GAL4/+) (Fig. 8B). Except for the data with flies carrying a single copy of UAS-dSLC25A46-IR142–242, these increases were
statistically significant. These results suggest that mitochondrial
function is defective in dSLC25A46 knockdown flies.
2.9. dSLC25A46 knockdown decreases level of ATP at in larval central
nerves system
To further confirm the mitochondrial defect in dSLC25A46
knockdown flies, we analyzed the ATP level as an indicator of mitochondrial dysfunction. The ATP level was decreased to 43.2% inknockdown larvae carrying w, UAS-dSLC25A46-IR142–242/w; +; elavGAL4/+ compared with its level in control flies carrying w; UASGFP-IR/+; elav-GAL4/+ (Fig. 9). The ATP level was decreased to
48.0% in flies carrying a single copy of UAS-dSLC25A46-IR207–350(w; UAS-dSLC25A46-IR207–350/+; elav-GAL4/+). In flies carrying two
copies of UAS-dSLC25A46-IR207–350 (w; UAS-dSLC25A46-IR207–350;
elav-GAL4/+), the level of ATP reduced to 35.0% compared to control
larvae carrying w; UAS-GFP-IR/+; elav-GAL4/+ (Fig. 9). These results
taken together with those on ROS production indicate that mitochondrial function is defective in dSLC25A46 knockdown larvae.
Accumulation of the defective mitochondria at NMJs very likely
cause neuronal dysfunction and aberrant morphology of motor
neuron.
2.10. Knockdown of dSLC25A46 induces axonal targeting aberrations
and abnormal morphology of photoreceptor neuron
It is known that SLC25A46 mutations in human cause optic atrophy. Therefore, we analyzed the morphology of photoreceptor neuron in dSLC25A46 knockdown larvae. In Drosophila eye, it is wellknown that the projection of axons from photoreceptor cell neurons innervate the centers of the brain to produce visual connections. The differentiating photoreceptor cell neurons of thedeveloping Drosophila eye form an axonal bundle, that targets into
the different layers of the brains. Each ommatidium of the Drosophila eye is composed of eight photoreceptors (R1–R8). R1–R6 neurons innervates lamina and R7–R8 neurons further extends into
medulla of the brain. The Chaoptin is widely used as a marker
for retinal axons and their projections to the brain. In the control
eye imaginal disc, photoreceptor cell neurons visualized by antiChaoptin antibody innervate both medulla and lamina in the brain
and forms a characteristic ‘‘inverted cap” like orientation(Fig. 10A). In flies carrying w, UAS-dSLC25A46-IR142–242/w; +;elav-GAL4/+, w; UAS-dSLC25A46-IR207–350/+; elav-GAL4/+, w;
UAS-dSLC25A46-IR207–350; elav-GAL4/+, R1-R6 photoreceptor cell
neurons project to lamina normally, while aberrations in projection of R7-R8 into medulla were observed in 43%, 31% and 33% ofeye discs, respectively (Fig. 10B). In addition, the quantificationof optic stalk length and diameter clearly showed that the ratioof diameter per length of knockdown larvae (w; UAS-dSLC25A46IR207–350; elav-GAL4/+) was increased to 247% compared to that ofcontrol larvae (Fig. 10C). This increase was statistically significant.
The ratio of single copy knockdown flies was not significantly
increased, probably due to the less efficient knockdown ofdSLC25A46 (Fig. 10C). These results suggest that dSLC25A46 plays
a role in axonal targeting and morphology of photoreceptor
neuron.

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37

Fig. 6. Knockdown of dSLC25A46 induces reduced active zone at NMJs in muscle 4 of third instar larvae. (A) Images show NMJs that were double-stained with anti-HRP(green) and anti-Brp (red). NMJs of flies carrying elav-GAL4 > UAS-GFP-IR (w; UAS-GFP-IR/+; elav-GAL4/+) (a–c), elav-GAL4 > UAS-dSLC25A46-IR207–350x2 (w; UAS-dSLC25A46IR207–350; elav-GAL4/+) (d–f) are shown. Scale bars indicate 5 lm (a–f). (B–D) The quantified data. In dSLC25A46 knockdown larvae, the density and size of active zones are
reduced (B, C) compared with the control larvae (*p < 0.05 and **p < 0.01). The number of active zones is not different between dSLC25A46 knockdown and control flies (D).
elav-GAL4 > UAS-GFP-IR (w; UAS-GFP-IR/+; elav-GAL4/+, n = 6), elav-GAL4 > UAS-dSLC25A46-IR142–242 (w, UAS-dSLC25A46-IR142–242/w; +; elav-GAL4/+, n = 9), elav-GAL4 > UASdSLC25A46-IR207–350 (w; UAS-dSLC25A46-IR207–350/+; elav-GAL4/+, n = 8), and elav-GAL4 > UAS-dSLC25A46-IR207–350x2 (w; UAS-dSLC25A46-IR207–350; elav-GAL4/+, n = 8).

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K. Suda et al. / Brain Research 1689 (2018) 30–44

Fig. 7. Knockdown of dSLC25A46 causes abnormal mitochondrial dynamics at NMJs in muscle 4 of third instar larvae. (A) Images show NMJs that were double-stained withmito-GFP (green) and anti-Dlg (red). NMJs of flies carrying D42-GAL4 > UAS-beta gal-IR, UAS-mitoGFP (w; UAS-beta gal-IR/+; D42-GAL4, UAS-mitoGFP/+) (a–c), D42-GAL4 > UASdSLC25A46-IR142–242, UAS-mitoGFP (w, UAS-dSLC25A46-IR142–242/w; +; D42-GAL4, UAS-mitoGFP/+) (d–f) are shown. Scale bars indicate 5 lm (a–f). (B–D) The quantified data. In
dSLC25A46 knockdown larvae, the density and size of mitochondria are increased (B, C) compared with the control larvae (*p < 0.05 and **p < 0.01). The number of active zones
is not different between dSLC25A46 knockdown and control flies (D). D42-GAL4 > UAS-beta gal-IR, UAS-mitoGFP (w; UAS-beta gal-IR/+; D42-GAL4, UAS-mitoGFP/+, n = 5), D42GAL4 > UAS-dSLC25A46-IR142–242, UAS-mitoGFP (w, UAS-dSLC25A46-IR142–242/w; +; D42-GAL4, UAS-mitoGFP/+, n = 13), D42-GAL4 > UAS-dSLC25A46-IR207–350, UAS-mitoGFP (w/
UAS-dSLC25A46-IR207–350/+; D42-GAL4, UAS-mitoGFP/+, n = 10).

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39

Fig. 8. dSLC25A46 knockdown increases reactive oxygen species in central nerves system of third instar larvae. (A) Third instar larval CNS were stained with CM-H2DCFDA.
Images of elav-GAL4 > UAS-GFP-IR (w; UAS-GFP-IR/+; elav-GAL4/+) (a) and elav-GAL4 > UAS-dSLC25A46-IR207–350x2 (w; UAS-dSLC25A46-IR207–350; elav-GAL4/+) (b) are shown.
Scale bars indicate 100 lm (B) The quantified data. In dSLC25A46 knockdown larvae, ROS signals are increased. *p < 0.05. elav-GAL4 > UAS-GFP-IR (w; UAS-GFP-IR/+; elav-GAL4/+, n = 4), elav-GAL4 > UAS-dSLC25A46-IR142–242 (w, UAS-dSLC25A46-IR142–242/w; +; elav-GAL4/+, n = 4), elav-GAL4 > UAS-dSLC25A46-IR207–350 (w; UAS-dSLC25A46-IR207–350/+; elavGAL4/+, n = 4), and elav-GAL4 > UAS-dSLC25A46-IR207–350x2 (w; UAS-dSLC25A46-IR207–350; elav-GAL4/+, n = 8).

Fig. 9. dSLC25A46 knockdown decreases level of ATP at in larval central nerves system. In dSLC25A46 knockdown larvae, ATP level are decreased. *p < 0.05. n = 3. elav-GAL4 >UAS-GFP-IR (w; UAS-GFP-IR/+; elav-GAL4/+), elav-GAL4 > UAS-dSLC25A46-IR142–242 (w, UAS-dSLC25A46-IR142–242/w; +; elav-GAL4/+), elav-GAL4 > UAS-dSLC25A46-IR207–350 (w;UAS-dSLC25A46-IR207–350/+; elav-GAL4/+), and elav-GAL4 > UAS-dSLC25A46-IR207–350x2 (w; UAS-dSLC25A46-IR207–350; elav-GAL4/+).

2.11. Knockdown of dSLC25A46 does not affect in the lifespan of adultflies
We analyzed the effect of neuron-specific dSLC25A46
knockdown on the survival of flies. The numbers of knockdown
flies carrying w, UAS-dSLC25A46-IR142–242/w; +; elav-GAL4/+, w;
UAS-dSLC25A46-IR207–350/+; elav-GAL4/+ and control flies carrying
w; UAS-GFP-IR/+; elav-GAL4/+ that were still alive were recorded
every three days until the last flies died (Fig. 11). There is no significant difference in the median lifespan of control and dSLC25A46
knockdown flies (Log-rank test, n = 100). Therefore, dSLC25A46
knockdown does not affect in the lifespan of adult flies.

3. Discussion
In order to clarify whether or not disruption of the physiological
functions of dSLC25A46 are important for the development of neurodegeneration, we have here established fly models in which thedSLC25A46 gene is knocked down. We revealed that neuronspecific knockdown of dSLC25A46 induced locomotor defects inboth larval and adult stages, defects in motoneurons and mitochondria at NMJs and aberrant morphology of photoreceptor neurons, that are accompanied with accumulation of ROS andreduction of ATP. These observations indicate that decreased levelof dSLC25A46 induces the mitochondrial dysfunction as a primary

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K. Suda et al. / Brain Research 1689 (2018) 30–44

Fig. 10. Knockdown of dSLC25A46 induces axonal targeting aberrations and abnormal morphology of photoreceptor neuron. (A) Immunostaining of photoreceptor neurons
with anti-Chaoptin IgG. White arrow indicates the medulla region where R1–R6 innervate, and white arrowhead indicates the medulla region where R7 and R8 extend (a).
Red arrowheads indicate aberrations of R7 and R8 axon targeting in medulla (b). Measured diameters of optic stalk are indicated by blue. Measured length of optic stalk (fromdotted line to arrow) are indicated by magenta. The bars indicate 50 lm. (B, C) The quantified data. (B) In dSLC25A46 knockdown larvae, R1–R6 photoreceptor cell neurons
project to lamina normally, while aberrations in projection of R7–R8 into medulla were observed. (C) In dSLC25A46 knockdown larvae, ratio of diameter per length are
increased. elav-GAL4 > UAS-GFP-IR (w; UAS-GFP-IR/+; elav-GAL4/+), elav-GAL4 > UAS-dSLC25A46-IR142–242 (w, UAS-dSLC25A46-IR142–242/w; +; elav-GAL4/+), elav-GAL4 > UASdSLC25A46-IR207–350 (w; UAS-dSLC25A46-IR207–350/+; elav-GAL4/+), and elav-GAL4 > UAS-dSLC25A46-IR207–350x2 (w; UAS-dSLC25A46-IR207–350; elav-GAL4/+).

effect. Accumulation of defective mitochondria likely occurs bylack of mitochondrial dynamics. This would then cause not only
aberrant neuronal morphology but also reduction of active zone,
an indicator of neuronal dysfunction in NMJs that finally resultsin defects in locomotive ability. In contrast, knockdown of Marf
which is a Drosophila homolog of MFN2, another CMT causing gene,induces mitochondrial fragmentation with neuronal defect(Altanbyek et al., 2016; Sandoval et al., 2014). Therefore, proper
regulation of mitochondrial dynamics appears to be importantfor proper neuronal function. Previous studies established otheranimal models targeting of SLC25A46. In zebrafish knockdown
model, axon tracts in motoneurons were shortened. Mitochondria
in motoneurons are not located in proper area and also appear tobe aggregated, but they display normal swimming ability(Abrams et al., 2015). Mouse SLC25A46 knockout model displayed

impaired motility, frequently fused and aggregated mitochondria
in Purkinje cells and optic atrophy (Li et al., 2017). Phenotypes
induced by decreased level of SLC25A46 are slightly different
among different animal models. Functional analyses with various
model organisms might be necessary to reveal the pathogenesisof human diseases caused by SLC25A46.
To eliminate the possible off-target effects of the used RNAi
constructs, we examined two different dSLC25A46 inverted repeat
constructs (UAS-dSLC25A46-IR142–242 and UAS-dSLC25A46-IR207–350).
Target sequences for these two RNAi lines were designed tohave no off-target effects (VDRC and online dsCheck software
http://dscheck.rnai.jp). When these transgenic fly lines werecrossed with the elav-GAL4 line to specifically express dSLC25A46
dsRNA in neuronal tissues, each of independent fly strains showedessentially the same phenotype. These results thus demonstrate

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41

dfig4 and knockdown of dSLC25A46, these genes could involve incommon pathological mechanisms of neurodegeneration, which
opens the possibility of future pathway-oriented therapy for CMT.
4. Experimental procedure
4.1. Fly stocks

Fig. 11. Viability assay. The percentage of surviving flies carrying elav-GAL4 > UASGFP-IR (w; UAS-GFP-IR/+; elav-GAL4/+), elav-GAL4 > UAS-dSLC25A46-IR142–242 (w,UAS-dSLC25A46-IR142–242/w; +; elav-GAL4/+), and elav-GAL4 > UAS-dSLC25A46-IR207–350(w; UAS-dSLC25A46-IR207–350/+; elav-GAL4/+) is shown. dSLC25A46 knockdown does
not affect in the lifespan of adult flies. (Log-rank test, n = 100).

that the phenotypes observed with the neuron-specific dSLC25A46knockdown flies are not due to an off-target effect but rather to a
reduction in dSLC25A46 protein levels.
It is known that SLC25A46 mutations in human cause optic atrophy. According to previous studies, optic stalk morphology anddevelopment in Drosophila are precisely regulated via cell proliferation and migration. Previous study identified the Focal adhesion
kinase56D (Fak56D) gene as an important component required for
optic stalk formation (Murakami et al., 2007). Fak56D is a Drosophila homolog of mammalian focal adhesion kinase (FAK; also
known as Ptk2), that is a main regulatory component of focal contacts (Mitra et al., 2005; Parsons, 2003). Mutation in Fak56D causes
random distribution of cells that could not be arranged into a tubular structure and lose tendency to migrate along optic stalk. This
could lead to broadened and shortened optic stalk (Murakami
et al., 2007). In this study, immunocytochemical analyses of subcellular localization of dSLC25A46 revealed that dSLC25A46 localizes not only in mitochondria, but also in plasma membrane.
These observations suggest that in addition to the role in mitochondrial function, plasma membrane-localized dSLC25A46 plays
a role in cell proliferation and/or migration to control optic stalkformation. Although human SLC25A46 is reported to be mainly
localized in mitochondria (Abrams et al., 2015), these studies havebeen limited to cultured cells. The present in vivo study in Drosophila suggests the possibility that human SLC25A46 may be localized not only in mitochondria but also in plasma membranes inhuman optic axons.
In conclusion, our dSLC25A46 knock-down Drosophila model
recapitulates most of the phenotypes in mitochondrial disease
patients, providing a useful tool to study these diseases. Considering the successful use of Drosophila model to identify genetic interactants with the causing genes for various neurodegenerative
diseases (Azuma et al., 2014; Shimamura et al., 2014), extensive
genetic screen with the neuron-specific dSLC25A46-knockdown
flies will allow us to identify genes and signaling pathways thatare related to dSLC25A46 function in neuron. These genes and pathways could be promising targets for therapy of mitochondrial diseases including CMT. Therefore, the neuron-specific dSLC25A46knockdown flies established in this study could be a useful model
to search novel therapeutic target of both of these syndromes andcandidate substances for therapy. Moreover, given the similar phenotypes between CMT fly models established by knockdown(Kyotani et al., 2016) or deletion (Bharadwaj et al., 2016, p. 4) ofFlies were reared on standard food containing 0.65% agar, 10%
glucose, 4% dry yeast, 5% cone flour, and 3% rice bran at 25 °C. Canton S was used as the wild type. w UAS-dSLC25A46-IR142–242; +; +and w; UAS-dSLC25A46-IR207–350; + (CG5755) were obtained fromthe Vienna Drosophila Resource Center (VDRC). The RNAi of thesestrain was targeted to the region corresponding to amino acid residues (aa) 142–242 or 207–350 of Drosophila SLC25A46. The fly line
carrying w; P{UAS-GFP.dsRNA.R}143, w; P[GAL4-elav.L]3 was
obtained from the Bloomington Drosophila Stock Center (BDSC) at
Indiana University. In addition, w; UAS-beta-gal-IR; + (VDRC) andw; P[GawB]D42 P[w+mC = UAS-mitoGFP.AP]3/P[GawB]D42 P[w+mC =UAS-mitoGFP.AP]3 (BDSC) were also used to study the mitochondrial localization in neuromuscular junctions.
4.2. Comparison of amino acid sequences of human SLC25A46, andDrosophila SLC25A46
The amino acid (aa) sequence of Drosophila SLC25A46 was
retrieved from UniProt (http://www.uniprot.org). The identity
and similarity of human SLC25A46 and Drosophila SLC25A46 werecompared using FASTA and BLAST.
4.3. Production of rabbit anti-dSLC25A46 antibody
The dSLC25A46 peptide Cys-NKSSTTKSPSISSI-OH, corresponding to aa423-436 of dSLC25A46 was conjugated to keyhole limpet
hemocyanin and mixed with Freund’s complete adjuvant to provide a suspension. The underlined N-terminal residue Cys was an
added residue. Then the suspension was injected subcutaneously
into a rabbit (Japanese White) kept under specific pathogen-free
conditions. The rabbit was then boosted with inoculations of an
immunogen of the same quality once a week for 7 weeks, and a terminal bleed was performed to collect the maximum amount ofserum (Scrum Inc.).
4.4. Flip out experiments
RNAi clones in Drosophila larval salivary gland were generated
with a flip-out system (Sun and Tower, 1999). Female flies with
hs-flp; Act5C > FRT y FRT > GAL4, UAS-GFP were crossed with maleflies carrying UAS-dSLC25A46-IR207–350. Clones were evaluated by
the presence of green fluorescent protein (GFP) expressed undercontrol of the Act5C promoter. Flip-out was induced by heat shock(60 min at 37 °C) at 24–48 h after egg laying.
4.5. Immunostaining with anti-dSLC25A46 antibody
For immunohistochemistry, larval salivary glands were dissected, and fixed in 4% paraformaldehyde/PBS for 20 min at 25°C. After washing with PBS containing 0.3% Triton X-100, the samples were blocked with PBS containing 0.15% Triton X-100 and 10%
normal goat serum for 30 min at 25 °C. Then the samples wereincubated with diluted primary antibodies in PBS containing
0.15% Triton X-100 and 10% normal goat serum for 16 h at 4 °C.
The following antibodies were used as primary antibodies: rabbit
anti- dSLC25A46 antibody (1:50), mouse anti-Dlg (1:400) (Developmental Studies of Hybridoma Bank, DSHB), mouse anti-KDEL

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K. Suda et al. / Brain Research 1689 (2018) 30–44(1:800) (Enzo Life Sciences), mouse anti-KDEL receptor (1:300)(Abcam), mouse anti-HSP60 (1:200) (Enzo Life Sciences). Afterwashing with PBS containing 0.3% Triton X-100, samples wereincubated with secondary antibodies labeled with Alexa 594(1:400) and 488 (1:400) for 3 h at 25 °C. After washing with PBS
containing 0.3% Triton X-100, the samples were stained with DAPI
(0.5 lg/ml)/PBS/0.1% Triton X-100. After washing with PBST and
PBS, samples were then mounted in the Vectashield (Vector laboratories) and inspected by a confocal laser scanning microscopy(Olympus Fluoview FV10i).
4.6. Western blot analysis
Protein extracts from the CNS of twenty larvae carrying w; UASGFP-IR/+; elav-GAL4/+, w, UAS-dSLC25A46-IR142–242/w; +; elav-GAL4/+, w; UAS-dSLC25A46-IR207–350/+; elav-GAL4/+, w; UAS-dSLC25A46IR207–350; elav-GAL4/+ were prepared. In brief, twenty larval CNS
were boiled at 95 °C for 2 min in 0.1 M Tris-HCl (pH 7.6) and complete Mini, EDTA-free (Roche Diagnostics) and homogenized insample buffer containing 50 mM Tris-HCl (pH 6.8), 2% sodium
dodecyl sulfate (SDS), 10% glycerol, 0.1% bromophenol blue and1.2% b-mercaptoethanol. The homogenates were then boiled at
95 °C for 5 min followed by centrifugation. The supernatants containing protein extracts were electrophoretically separated on
SDS-polyacrylamide gels containing 6% acrylamide and then transferred to polyvinylidene difluoride membranes (Bio-Rad). The blotted membranes were preincubated with TBS/0.1% Tween 20
containing 0.3% skim milk for 1 h at 25 °C and incubated with rabbit anti-dSLC25A46 antibody (1:500) for 16 h at 4 °C. After
repeated washing, the membrane was incubated with horseradish
peroxidase (HRP)-conjugated anti-rabbit IgG (1:4000) in for 1 h at
25 °C. Antibody binding was detected using ECL-advance Western
blotting detection reagents, and images were analyzed with ImageJ(NIH, USA). To ensure equal protein loading in each lane, the membranes were also probed with a rabbit anti-a-tubulin antibody at
the same time and HRP-conjugated anti-rabbit IgG. For detectionof a-tubulin in some cases, mouse anti-a-tubulin monoclonal antibody (1:8000, Sigma) and HRP-conjugated anti-mouse IgG(1:10,000, GE Healthcare) were also used as the primary and secondary antibodies, respectively.
4.7. RNA isolation and quantitative RT-PCR (qRT-PCR)
Ten larval CNS were dissected and kept in liquid nitrogen. Then,
total RNA was isolated using a RNeasy Lipid Tissue Mini Kit (Qiagen). The synthesis of cDNA was then performed using a PrimeScriptTM RT Reagent Kit (Takara). Finally, a Bio-Rad CFX96TM
Real-Time System was used in order to perform qRT-PCR withSYBRÒ Premix Ex TaqTM (Tli RNase H Plus) (Takara) and targetspecific primers. The data were analyzed by a standard curvebased method calculated using CFX ManagerTM software. Specificity of primers was tested using melting curves created with
CFX ManagerTM software. GAPDH mRNA was used as an internal
control. Drosophila whole genomic nucleotide sequences used forprimer design were based on the FlyBase online database. Primer
sequences are listed below.dSLC25A46 forward primer: 50 -TCGAATGATGCAGAGGAGAATG-30 dSLC25A46 reverse primer: 50 -CTCAGTGGTAAGCATGGCTATAA-30
GAPDH forward primer: 50 -GGAGCCACCTATGACGAAATC-30
GAPDH reverse primer: 50 -TCGAACACAGACGAATGGG-30
4.8. Crawling assay
The crawling assay was performed as described previously(Nichols et al., 2012) with some modifications. Female larvae inthe early third instar stage were collected and washed with PBS

to remove food traces. Then larvae were transferred to agar plates
containing 2% agar at a density of two or three larvae per plate. Larval movement was recorded using a digital camera for 1 min. The
recorded videos were converted into AVI type using a MOV to
AVI converter (Pazera Jacek, Poland) and then analyzed using ImageJ (NIH, USA) with a wrMTrck plugin (developed by Dr. Jesper
Søndergaard Pedersen) to track larval movement and draw motion
paths.

4.9. Climbing assay
The climbing assay was performed as described previously withminor modifications (Kyotani et al., 2016). Newly eclosed adultfemale flies were collected and separated into vials at a maximum
density of 20 flies per vial. Flies were then transferred to a conicaltube without anesthesia. After that, the tubes were tapped to collect the flies to the bottom, and the length of time required for
adult flies to climb was recorded for 5 s. These procedures wererepeated five times and recorded using a digital camera. For allof the climbing experiments, the height to which each fly climbed
was scored as follows; 0 (<2 cm), 1 (between 2 and 4 cm), 2(between 4 and 6 cm), 3 (between 6 and 8 cm), 4 (between 8 and10 cm), and 5 (more than 10 cm). The climbing assays were performed every seven days from day 1 after enclosion.

4.10. Visualization of neuromuscular junctions
For the visualization of NMJs, third instar larvae were dissectedin HL3 saline and fixed with 4% paraformaldehyde in PBS for 30
min at 25 °C. After being washed, the samples were blocked with
PBS containing 0.15% Triton X-100 and 10% NGS for 30 min at 25°C and incubated with primary antibodies in PBS containing
0.15% Triton X-100 and 10% NGS for 16 h at 4 °C. The following
antibodies were used; rabbit anti-GFP IgG (1:200, Medical & Biological Laboratories [MBL], 598), mouse anti-Dlg (1:500, DSHB,
4F3), mouse anti-Brp IgG (1:200, DSHB, nc82). After being washedwith PBS containing 0.3% Triton X-100, samples were incubatedwith secondary antibodies labeled with Alexa 594 (1:400) and
488 (1:400) or, 594 (1:400) and FITC-conjugated goat anti-HRP
IgG (1:400) for 3 h at 25 °C. After extensive washing with PBS containing 0.3% Triton X-100, samples were mounted with Vectashield(Vector Laboratories) for laser confocal microscope or ProLong Diamond (Invitrogen) for super resolution microscope. Confocal
microscopic images were taken with a confocal laser scanningmicroscope (Olympus Fluoview FV10i) and processed with MetaMorph software (Molecular devices). Images of samples were also
taken with a super resolution microscope (N-SIM, Nikon)

4.11. Visualization of photoreceptor neuron
For immunohistochemistry, larval eye imaginal discs were dissected, and fixed in 4% paraformaldehyde/PBS for 15 min at 25 °C.
After washing with PBS containing 0.3% Triton X-100, the samples
were blocked with PBS containing 0.15% Triton X-100 and 10% normal goat serum for 30 min at 25 °C, and incubated with diluted primary antibodies in PBS containing 0.15% Triton X-100 and 10%
normal goat serum for 16 h at 4 °C. mouse anti-Chaoptin (1:100,
DSHB 24B10) antibody was used. After washing with PBS containing 0.3% Triton X-100, samples were incubated with secondary
antibodies labeled with either Alexa 488 or Alexa 594 (1:400, Invitrogen) for 3 h at 25 °C. After extensive washing with PBS containing 0.3% Triton X-100, samples were mounted in the Vectashield(Vector laboratories) and analyzed by a confocal laser scanningmicroscopy (Olympus Fluoview FV10i).

K. Suda et al. / Brain Research 1689 (2018) 30–44

43

4.12. ROS detection

Competing financial interests

Levels of ROS were detected by CM-H2DCFDA (Thermo Fisher
Scientific). In 1% CM-H2DCFDA /PBS, larval CNS were shaken
15sec. After 10 min incubation and extensive washing with PBS,
samples were mounted in the Vectashield (Vector laboratories)
and inspected by a confocal laser scanning microscopy (Olympus
Fluoview FV10i).

The authors declare no competing financial interests in relation
to the work described.

4.13. ATP measurement
CellTiter-Glo (Promega) were used for measuring ATP level.
After homogenizing ten larval CNS in 100lL of ATP assay buffer(Abcam), samples centrifuged (4°C, 5 min at 13,000g). The supernatant was then removed into the microtubule, 10mL of cold trichloroacetic acid (Abcam) and keep the sample on ice for 15 min.
After centrifugation at 12,000g for 5 min, 10 mL neutralization
solution (ab204708, Abcam) was added and allowed to rest for 5
min on ice. 50 lL samples and 50 lL Celltiter-Glo were mixed toeach well. Luminescence was read on Lumat LB 9507 (Berthold
Technologies).

4.14. Lifespan assay
The parent flies were placed at 28 °C, and newly eclosed adultfemale flies were separated into vials at a low density (20 flies
per vial) and maintained in a humidified, temperature controlled
incubator at 25 °C and 60% humidity in a 12 h light and 12 h darkcycle. The flies were then transferred to new tubes containing fresh
food, and deaths were scored every three days. Survival rate wasdetermined by the percentage of surviving flies from day to day
until all flies died.

4.15. Immunizing peptide competition experiments
Immunostaining methods are essentially same as Section 4.5.
The rabbit anti-dSLC25A46 antibody was pretreated with 20 lg
of blocking dSLC25A46 peptide (Cys-NKSSTTKSPSISSI-OH). The
samples were incubated with the pretreated rabbit antidSLC25A46 antibody (1:50 dilution) in PBS containing 0.15% Triton
X-100 and 1% bovine serum albumin for 16 h at 4 °C.

4.16. Data analysis
GraphPad Prism 6 was used to analyze the statistics of all
results except for the climbing assay. In the Western blot analysis,
crawling assay, NMJ and photoreceptor neuron visualization, ROS
detection, and ATP measurement, the P-values were calculated
by using unpaired Welch’s t-test (one-tailed). For the statistics ofclimbing assay, the SPSS software (IBM) was used. All data areshown as means ± SEM. Lifespan assay results were evaluatedusing a log-rank test.

Acknowledgements
We thank the Bloomington Drosophila Stock Center, Vienna Drosophila Genetic Resource Center, and Kyoto Stock Center for flylines. This study was partially supported by the JSPS Core-to-Core
Program, Asia-Africa Science Platforms, JSPS Program for Advancing Strategic International Networks to Accelerate the Circulationof Talented Researchers (Grant No. S2802). JSPS KAKENHI Grant
Number 16K07346.

Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at https://doi.org/10.1016/j.brainres.2018.03.
028.
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