iPLA2-VIA is required for healthy aging of neurons, muscle, and the female germline in Drosophila melanogaster

Abstract

Neurodegenerative disease (ND) is a growing health burden worldwide, but its causes and treatments remain elusive. Although most cases of ND are sporadic, rare familial cases have been attributed to single genes, which can be investigated in animal models. We have generated a new mutation in the calcium-independent phospholipase A2 (iPLA2) VIA gene CG6718, the Drosophila melanogaster ortholog of human PLA2G6/PARK14, mutations in which cause a suite of NDs collectively called PLA2G6-associated neurodegeneration (PLAN). Our mutants display age-related loss of climbing ability, a symptom of neurodegeneration in flies. Although phospholipase activity commonly is presumed to underlie iPLA2-VIA function, locomotor decline in our mutants is rescued by a transgene carrying a serine-to-alanine mutation in the catalytic residue, suggesting that important functional aspects are independent of phospholipase activity. Additionally, we find that iPLA2-VIA knockdown in either muscle or neurons phenocopies locomotor decline with age, demonstrating its necessity in both neuronal and non-neuronal tissues. Furthermore, RNA in situ hybridization shows high endogenous iPLA2-VIA mRNA expression in adult germ cells, and transgenic HA-tagged iPLA2-VIA colocalizes with mitochondria there. Mutant males are fertile with normal spermatogenesis, while fertility is reduced in mutant females. Mutant female germ cells display age-related mitochondrial aggregation, loss of mitochondrial potential, and elevated cell death. These results suggest that iPLA2-VIA is critical for mitochondrial integrity in the Drosophila female germline, which may provide a novel context to investigate its functions with parallels to PLAN.

About the Authors:

Surya Jyoti Banerjee

Roles Formal analysis, Investigation, Methodology, Supervision, Writing – review & editing

¶‡ These authors are co-first authors on this work.

Affiliation: Department of Biology, Yeshiva University, New York, NY, United States of America

ORCID logo https://orcid.org/0000-0001-7196-4315

Adina Schonbrun

Roles Investigation, Methodology, Writing – review & editing

¶‡ These authors are co-first authors on this work.

Affiliation: Department of Biology, Yeshiva University, New York, NY, United States of America

ORCID logo https://orcid.org/0000-0002-0831-5053

Sogol Eizadshenass

Roles Investigation

Affiliation: Department of Biology, Yeshiva University, New York, NY, United States of America

Shimshon Benji

Roles Investigation

Affiliation: Department of Biology, Yeshiva University, New York, NY, United States of America

Yaakov Tzvi Cantor

Roles Investigation

Affiliation: Department of Biology, Yeshiva University, New York, NY, United States of America

Liam Eliach

Roles Investigation

Affiliation: Department of Biology, Yeshiva University, New York, NY, United States of America

Matthew Lubin

Roles Investigation

Affiliation: Department of Biology, Yeshiva University, New York, NY, United States of America

Zev Narrowe

Roles Investigation

Affiliation: Department of Biology, Yeshiva University, New York, NY, United States of America

Jeremy Purow

Roles Investigation

Affiliation: Department of Biology, Yeshiva University, New York, NY, United States of America

Benjamin Shulman

Roles Investigation

Affiliation: Department of Biology, Yeshiva University, New York, NY, United States of America

Leib Wiener

Roles Investigation

Affiliation: Department of Biology, Yeshiva University, New York, NY, United States of America

Josefa Steinhauer

Roles Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – original draft, Writing – review & editing

* E-mail: [email protected]

Affiliation: Department of Biology, Yeshiva University, New York, NY, United States of America

ORCID logo https://orcid.org/0000-0002-0960-5271

Introduction

As global population demographics have shifted toward older age, neurodegenerative disease (ND) has become an increasing health burden worldwide [1]. Treatment has been confounded by the fact that loss of neurons in ND, which leads to dementia and reduced motor control, is associated with numerous cytopathologies, including DNA damage and epigenetic changes, mitochondrial and lysosomal dysfunction, Ca+2 dysregulation, disrupted RNA and protein homeostasis, as well as inflammation [1, 2]. Thus, there is a pressing need to better understand the underlying drivers of ND.

Parkinson’s disease is the second most common ND, affecting ~1% of individuals over the age of 60 [3]. Most cases of Parkinson’s disease are sporadic, presumably arising from a complex interplay between genotype and environment. In some cases, though, environmental toxins have been identified as direct causative agents. For example, contamination of the synthetic opioid MPPP (1-methyl-4-phenyl-4-propionoxy-piperidine) with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), which is metabolized to MPP+ (1-methyl 4-pyridinium) in vivo, caused a cluster of acute cases of parkinsonism amongst opiate users [4]. The herbicide paraquat and the pesticide rotenone also have been linked to the disease [5, 6]. These toxins inhibit the electron transport chain and promote oxidative stress, suggesting that mitochondrial dysfunction is central, and potentially causative in some cases, to the disease pathology [7, 8].

Several inherited disorders that bear striking resemblance to Parkinson’s disease have permitted identification of genetic contributors, the so-called PARK genes [9]. Many of these genes encode products that can localize to mitochondria and affect mitochondrial activity and quality control, again suggesting a key role for mitochondrial integrity in the disease and positioning it as a potential therapeutic target [7, 10]. PLA2G6/PARK14 encodes the group 6A calcium-independent phospholipase A2 (iPLA2-VIA, also called iPLA2-β). In 2006, this gene was linked to a group of severe NDs, including infantile neuroaxonal dystrophy and neurodegeneration with brain iron accumulation, and three years later was found to be associated with an autosomal recessive dystonia-parkinsonism [11–14].

The iPLA2-VIA enzyme, like other PLA2s, hydrolyzes fatty acyl chains from the sn-2 position of glycerophospholipids [15]. Acting within the Lands Cycle of deacylation and reacylation, PLA2s promote phospholipid remodeling and repair [16]. Acyl chain remodeling seems to be especially important for the mitochondrial phospholipid cardiolipin (CL), which is highly susceptible to oxidative damage due to its proximity to the electron transport chain. In the absence of remodeling, accumulation of damaged CL acyl chains is thought to reduce respiratory function and promote oxidative stress and apoptosis [17, 18]. iPLA2-VIA can localize to mitochondria in several mammalian cell types and has been implicated in CL remodeling [19–25]. Moreover, PLA2G6 mutant animal models display features of ND along with mitochondrial abnormalities, suggesting the possibility that PLA2G6-associated neurodegeneration (PLAN) arises from mitochondrial dysfunction in the absence of CL repair [26–29]. However, dramatic CL molecular changes have not been found consistently in PLA2G6 mutant models [26, 27, 30], raising questions of how important iPLA2-VIA is for CL remodeling and whether this molecular process underlies PLAN.

iPLA2-VIA hydrolyzes other phospholipids in addition to CL and can generate lipid signaling mediators [31]. iPLA2-VIA also bears near its N-terminus 8–9 ankyrin repeats, which can serve as protein-protein interaction domains, although critical interactors have yet to be characterized [32]. It has been observed in a variety of subcellular locations and has been implicated in numerous cellular activities, including cell cycle progression, vesicle trafficking, Ca+2 homeostasis, ER stress, and apoptosis [15, 21, 31, 33–35]. The relationships between iPLA2-VIA’s molecular, cellular, and physiological functions currently are unclear. We investigated iPLA2-VIA in Drosophila melanogaster by determining its endogenous expression pattern and generating a new null mutant. iPLA2-VIA is expressed ubiquitously at low levels in imaginal tissues. Null mutants are viable and show no synthetic lethality or sterility with two key phosphatidylcholine (PC) metabolizing enzymes. Consistent with a number of recent reports, null iPLA2-VIA mutants show a striking decline in locomotor ability with age [26, 36, 37], which surprisingly can be rescued by a catalytic-dead iPLA2-VIA transgene. Whereas prior reports have focused on neurons, for the purpose of modeling ND, we show here that the locomotor decline can be phenocopied with either neuronal-specific or muscle-specific knockdown, indicating the importance of this gene in multiple adult tissue types. Furthermore, high endogenous expression is observed in both male and female adult germ cells, and transgenic HA-tagged iPLA2-VIA-PB localizes prominently to mitochondria there. Although the mammalian homolog has been implicated in male fertility [38], we observe normal fertility and spermatogenesis in male iPLA2-VIA mutant Drosophila. Still, female mutants have reduced fertility, with age-dependent abnormal mitochondrial aggregation, reduced mitochondrial potential, and elevated cell death in the germline. Taken together, our results demonstrate that iPLA2-VIA activity in both neurons and muscles protects from age-related locomotor decline but implicate putative non-catalytic mechanisms in its protective function. We also show that iPLA2-VIA is important for mitochondrial integrity in the Drosophila female germline.

Results

A null iPLA2-VIA mutation does not interact genetically with phospholipid metabolism genes

The Drosophila melanogaster iPLA2-VIA (CG6718) locus encodes four predicted transcripts and two protein isoforms, which differ by 13 amino acids at their N termini (Fig 1A). Both Drosophila isoforms are highly similar to mammalian iPLA2-VIA, with a conserved ankyrin repeat region, catalytic residues, and a 1-9-14 calmodulin binding motif (Fig 1B, [32], the Drosophila PA isoform shares 48% similarity and the PB isoform 47.5% similarity with the shorter human iPLA2-VIA protein, and they are 50.4% and 49.9% similar to the longer human isoform, respectively). To generate a genomic mutation in Drosophila iPLA2-VIA, we excised the P element EY5103 and isolated a 1.4 kb deletion, called iPLA2-VIAΔ23, that spans the transcription initiation site as well as both predicted translation start codons (Fig 1A). Reverse transcription (RT)—PCR confirmed the absence of full-length iPLA2-VIA mRNA in our mutant, while the EY5103 insertion line retains a low level of iPLA2-VIA transcript (Fig 1C, see also [36]). The null mutants are homozygous and hemizygous viable at room temperature (Table 1) but have reduced lifespans (not shown), as reported in previous studies [26, 36, 39].

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Fig 1. A new mutant allele of Drosophila iPLA2-VIA.

(A) Map of the CG6718 iPLA2-VIA gene (light blue) showing the insertion site of the EY5103 P element (triangle), predicted transcripts with start codons (green arrows), and the 1.4 kb Δ23 deletion (breakpoints indicated by the dark blue lines). (B) Domain mapping of primary structures demonstrates high homology between Drosophila and human iPLA2-VIA. Colors are as in [32], with red indicating the ankyrin repeats (Ank; the ninth ankyrin repeat in the shorter mammalian iPLA2-VIA isoform is disrupted by additional residues in the fly proteins and in the longer mammalian iPLA2-VIA isoform, indicated here in orange), green indicating the oxyanion hole that coordinates the substrate during catalysis (Oxy), pink indicating the catalytic site (Cat, GTSTG) and catalytic aspartate residue (Cat D), and blue indicating the 1-9-14 calmodulin binding motif (CaM). (C) Whole fly RT-PCR demonstrates the absence of full-length iPLA2-VIA mRNA in the Δ23 mutant. The EY5103 insertion line retains low levels of iPLA2-VIA mRNA.

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Table 1. iPLA2-VIAΔ23 mutants are viable.

https://doi.org/10.1371/journal.pone.0256738.t001

Early studies implicated iPLA2-VIA in phospholipid homeostasis [33]. For example, overexpressing the phosphatidylcholine (PC) synthesis enzyme CDP phosphocholine cytidylyltransferase (Pcyt1) in mammalian cells led to compensatory upregulation of iPLA2-VIA expression and activity, to catabolize the excess PC [40, 41]. To explore whether this role is conserved at the organismal level in Drosophila, we used the EP line GS15374 to overexpress Pcyt1 with the ubiquitously expressed tubulin-GAL4 driver. After confirming Pcyt1 overexpression by RT-PCR (S1A Fig, bottom gel panel), we examined iPLA2-VIA mRNA levels. In whole flies, iPLA2-VIA mRNA expression does not increase when Pcyt1 is overexpressed (S1A Fig). We confirmed this result using RT-qPCR (S1E Fig). Additionally, no lethality is observed when Pcyt1 is overexpressed in the iPLA2-VIAΔ23 mutant (Table 2A), indicating that iPLA2-VIA is not necessary for survival upon Pcyt1 overexpression. We also tested for genetic interactions between our iPLA2-VIAΔ23 mutation and a pcyt1 loss of function allele that reduces PC levels [42], reasoning that removing iPLA2-VIA from the pcyt1 mutant might partially suppress its phenotype by allowing more PC to accumulate. However, neither the lethality nor the sterility of pcyt116919 mutants is suppressed by the absence of iPLA2-VIA (Table 2B–2D), and iPLA2-VIA mRNA levels are not reduced in pcyt1 mutant flies (S1B Fig, RT-qPCR shown in S1F Fig). Sws/NTE is a phospholipase in the same family as iPLA2-VIA and is known to be important for PC homeostasis [43]. Loss of iPLA2-VIA does not enhance the lethality of sws4 mutants (Table 3), suggesting that iPLA2-VIA does not compensate for Sws/NTE in processes that affect viability. Finally, iPLA2-VIA mRNA expression is unchanged in whole flies mutant for the ethanolamine kinase easily shocked (eas), a key enzyme in phosphatidylethanolamine (PE) synthesis (S1C Fig, RT-qPCR shown in S1G Fig, [44]). All together, our results do not point to a major unique role for Drosophila iPLA2-VIA in phospholipid housekeeping, although our results do not preclude important roles in specific tissues. Publicly available high-throughput expression data confirm that pcyt1, sws, and eas all show moderate to high expression throughout the organism, in embryonic, larval, and adult stages (http://flybase.org).

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Table 2. iPLA2-VIA and pcyt1 do not show synthetic effects on viability or fertility.

https://doi.org/10.1371/journal.pone.0256738.t002

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Table 3. iPLA2-VIA and sws do not show synthetic loss of viability.

https://doi.org/10.1371/journal.pone.0256738.t003

iPLA2-VIA in neurons and muscles maintains locomotor ability with age, via a partially catalytic-independent activity

Because iPLA2-VIA is associated with ND in humans, we tested our mutant flies for their climbing ability, which is known to decline with age under conditions of neurodegeneration [45]. In accord with other reports, iPLA2-VIAΔ23 mutants exhibit climbing defects after 20 days of age, in contrast to background control and isogenic control animals (Fig 2A and 2B, light bars [26, 36, 39]). Ubiquitous expression of a C-terminally HA-tagged iPLA2-VIA-PB wild-type cDNA transgene rescues the climbing ability of the mutant, confirming the locus-specificity of the defect (Fig 2C and 2D, dark bars). We additionally constructed an iPLA2-VIA-PB cDNA transgene in which the catalytic serine is replaced with an alanine residue (called iPLA2-VIA-PB SA), a well characterized alteration that abrogates catalytic activity [32, 46, 47]. Surprisingly, the catalytic-dead transgene also rescues the climbing defect of mutant flies, suggesting that iPLA2-VIA function is at least partially independent of catalytic activity (Fig 2C and 2D, yellow bars, [32]). Although the wild-type and catalytic-dead transgenes are identical apart from the catalytic serine, and they are inserted into the same genomic locus, expression of the catalytic-dead protein appears ~50% weaker than the wild-type protein on a Western blot (S2 Fig). This might indicate reduced stability of the mutant protein. Thus, the lower rescuing activity of the catalytic-dead transgene may be due to reduced protein levels and/or to functions associated with the catalytic residue.

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Fig 2. Impaired climbing ability in aged iPLA2-VIAΔ23 mutants can be rescued by a catalytic dead iPLA2-VIA-PB transgene and phenocopied with neuronal or muscle knockdown.

(A-B) Male (A, light blue) and female (B, light pink) iPLA2-VIAΔ23 mutant adults have normal climbing ability until 20 days of age at room temperature. After 20 days, climbing ability declines compared to yw (black) and isogenic controls (dark blue in A, and dark red in B). Newly eclosed isogenic control males (A, dark blue) show slightly reduced climbing ability compared to yw males. (C-D) Tubulin-GAL4 driven expression of wild-type UAS-iPLA2-VIA-HA in males (C, dark blue bars) and females (D, dark red bars) at 26°C rescues the climbing defect of aged iPLA2-VIAΔ23 mutants at 15 and 20 days of age. 26°C was used to increase GAL4 activity in rescue and knockdown experiments. Note that mutant flies show climbing defects by 15 days at this higher temperature. In this experiment, mutants carry the GAL4 driver without the resue transgene (light blue bars in C, and light pink bars in D). Tubulin-GAL4 driven expression of a UAS-iPLA2-VIA-SA transgene carrying a serine-to-alanine mutation in the catalytic residue at 26°C also partially rescues the climbing defect (yellow bars), although rescue is weaker than with the wild-type transgene. Wild-type and SA transgenes were transformed into the same genomic locus using the phi-C31 system. (E-F) Ubiquitous expression of UAS-driven double stranded RNA targeting iPLA2-VIA (HMS01544) with tubulin-GAL4 at 26°C phenocopies the climbing defect in male (E) and female (F) flies (gray bars, compared to GAL4-only controls, black bars). Knockdown flies die by 35 and 40 days of age in males and females, respectively. (G-H) Expressing HMS01544 in neurons only with elav-GAL4 at 26°C weakly phenocopies the climbing defect in male (G) and female (H) flies (gray bars, compared to GAL4-only controls, black bars). (I-J) Expressing HMS01544 in muscles only with DJ667-GAL4 produces a strong phenocopy of the age-induced climbing defect in male (I) and female (J) flies, similar to that resulting from knockdown of pink1 (HMS02204, red bars). Note that pink1 knockdown flies also have moderately reduced climbing ability at 10 days of age compared to iPLA2-VIA knockdown flies, but this was not statistically significant compared to GAL4-only controls. Number of groups (first number) and number of flies (second number) assayed for each condition is shown on the graphs. Bars represent the average climbing index for each condition. Error bars are standard deviations. Differences assessed by unpaired t-test, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0005.

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Ubiquitous RNAi knockdown of iPLA2-VIA using tubulin-GAL4 phenocopies the mutant in both males (Fig 2E, gray bars) and females (Fig 2F, gray bars). Knocking down iPLA2-VIA only in neurons with the elav-GAL4 driver produces a climbing defect that is markedly weaker than in the ubiquitous knockdown (Fig 2G and 2H), suggesting a requirement in additional tissues other than neurons. In accord with this, RNAi knockdown of iPLA2-VIA with the muscle-specific driver DJ667-GAL4 also reproduces the climbing defect (Fig 2I and 2J, gray bars). In comparison to knockdown of pink1, a gene required for maintenance of muscle integrity with age (Fig 2I and 2J, red bars, [48, 49]), iPLA2-VIA knockdown is comparable at 20 days in males and even stronger by 30 days of age in both males and females. Thus, iPLA2-VIA is required in neurons and muscle, and possibly other tissues, for maintenance of normal locomotor activity with age. Publicly available high throughput transcriptomic data show moderate to strong expression also in gut, fat body, and heart, all tissues known to affect aging (http://flybase.org; [50]).

Drosophila iPLA2-VIA is expressed strongly in adult germline but is not required for spermatogenesis

To explore further the endogenous expression of Drosophila iPLA2-VIA, we performed RNA in situ hybridization. We observe weak mRNA expression in wild-type imaginal tissues and no detectable expression in wild-type larval brains (Fig 3A–3F). Strikingly, strong expression is seen in the germ cells of both male and female adults (Fig 3G and 3H). Because iPLA2-VIA has been implicated previously in mammalian male fertility [38], we examined male fertility in our mutants. Surprisingly, iPLA2-VIAΔ23 mutant males are fertile, even when they are aged (Fig 4A and 4B).

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Fig 3. iPLA2-VIA mRNA is highly expressed in the adult germline.

In situ hybridization to endogenous iPLA2-VIA mRNA (purple) in w1118 animals shows weak ubiquitous expression in wild-type wing imaginal discs (A) and eye-antennal discs (C). Sense probes (B, D) were used as negative controls. Expression is negligible in larval brains (E, compare to sense probe control in F). Strong expression is seen in both female (G) and male (H) adult germlines. Expression in the male germline (H) is strongest in mitotic spermatogonia (boxed region shown at higher magnification in the inset, magenta arrow indicates spermatogonial region) and moderate in primary spermatocytes (inset, gray arrow) and meiotic spermatids (arrowhead). Scale bars: 100 μm. Positive controls for in situ hybridization are shown in S3 Fig.

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Fig 4. Male fertility and spermatogenesis are normal in iPLA2-VIAΔ23 mutants.

(A-B) Young (A, 4–5 day old) and aged (B, 20–21 day old) iPLA2-VIAΔ23 mutant males (light blue bars) show no fertility defect compared to isogenic controls (dark blue bars). Bars depict average number of adult progeny produced by 10 individual males over the course of four days, error bars represent standard deviation. (C-J) Testis squashes reveal normal post-meiotic mitochondrial Nebenkerne (phase-dark structures, indicated by the magenta arrow in C) in iPLA2-VIAΔ23 mutants (D), tazΔ161 mutants (E), and tazΔ161 iPLA2-VIAΔ23 double mutants (F), and each Nebenkern is paired with a similarly sized nucleus (phase-light structures, indicated by the magenta arrowhead in C). In slightly later spermatogenic cysts (G-J), mitochondrial derivatives (indicated by magenta arrows in G) elongate normally in all genotypes. Scale bars: 20 μm. (K-N) Phalloidin staining reveals the individualization complexes (ICs) of the maturing spermatids. In iPLA2-VIAΔ23 mutants, ICs are normal (L), in contrast with tazΔ161 mutants, in which ICs are highly disorganized (M). tazΔ161 iPLA2-VIAΔ23 double mutants show disorganized ICs (N), like tazΔ161 single mutants. Scale bar: 20 μm. (O) IC quantification shows no rescue of the tazΔ161 individualization phenotype by iPLA2-VIAΔ23 mutation. Number of testes (first number) and number of individualization complexes (second number) quantified for each genotype are indicated on the graph. Bars depict the average number of normal ICs for each genotype, error bars represent standard deviation. Differences assessed by unpaired t-test, *****p < 10−5, n.s. indicates not significant.

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Loss of function mutations in the enzyme Tafazzin (Taz), a mitochondrially-localized transacylase responsible for most of the CL remodeling activity in cells, lead to male infertility with defects in spermatid individualization in Drosophila [23, 51]. Previous results implicated iPLA2-VIA in this pathway, with reduced iPLA2-VIA expression compensating for loss of taz in male fertility [23]. We therefore closely examined several aspects of spermatogenesis in tazΔ161 or iPLA2-VIAΔ23 single mutants, as well as in tazΔ161 iPLA2-VIAΔ23 double mutants. In both single mutants and the double mutant, developmentally programmed mitochondrial aggregation is normal in post-meiotic spermatids (Fig 4C–4F), and mitochondrial derivatives begin elongation normally (Fig 4G–4J). At the individualization stage, the actin-rich individualization complexes (ICs) can be detected by phalloidin staining (Fig 4K, [52]). No IC defects are seen in iPLA2-VIAΔ23 mutants (Fig 4L). In contrast, ICs are highly disorganized in tazΔ161 single mutants (Fig 4M, [23]) and in tazΔ161 iPLA2-VIAΔ23 double mutants (Fig 4N), with no rescue of the taz mutant phenotype by removal of iPLA2-VIA (Fig 4O). We also tested the fertility of both tazΔ161 iPLA2-VIAΔ23 double mutant males (n = 8) and tazΔ161 iPLA2-VIAEY5103 double mutant males (n = 5) in individual crosses to yw females and found both to be completely sterile (0 progeny produced over the course of five days). Therefore, as the iPLA2-VIA mutant neither shows similar defects to the taz mutant nor rescues it, our data do not support the idea that iPLA2-VIA is a critical unique player in CL remodeling during Drosophila spermatogenesis.

Drosophila iPLA2-VIA mutants have reduced female fertility with abnormal mitochondrial aggregation in the germline

Consistent with strong iPLA2-VIA expression in the female germline (Fig 3G), there is a significant decrease in female fertility in iPLA2-VIAΔ23 homozygotes (Fig 5A), and the same effect is seen in hemizygotes, supporting the locus-specificity of the phenotype (Fig 5B). This is accompanied by a decrease in number of eggs laid (Fig 5C and 5D) but not maternal lethality in embryos or larvae (S4 Fig), suggesting that iPLA2-VIA is necessary before fertilization, during oogenesis. Although major developmental defects are not evident in iPLA2-VIAΔ23 mutant ovaries, we observe striking mitochondrial aggregation in the germlines of aged mutant females. The mitochondrially targeted fluorescent reporter transgene Psqh-mito-EYFP, which consists of YFP fused to the mitochondrial localization signal of human cytochrome c oxidase 8A, decorates a diffuse mitochondrial network in the nurse cells of young females, in both null mutants and controls (Fig 6A and 6B). By three weeks of age, mitochondria aggregate abnormally in the mutant, despite maintaining their normal diffuse distribution in background control and heterozygous genotypes (Fig 6C and 6D). Aggregation is especially noticeable in mid-oogenesis stages 8–9. In younger egg chambers, mitochondria are less diffuse even in control genotypes, making the mutant phenotype harder to distinguish (for example, see Fig 7A’). To quantify mitochondrial aggregation, we used ImageJ to outline the mito-YFP signal areas. In egg chambers with a normal diffuse mitochondrial network, the outlined mito-YFP signal is extensive and complex, but in egg chambers with aggregated mitochondria, the outlines separating areas of signal from background are sparser (Fig 6E and 6F). Taking the raw integrated density of the mito-YFP outlines in the area of the nurse cells in ImageJ reveals a highly significant quantitative reduction in the intricacy of the mito-YFP network in mutant germlines at mid-oogenesis compared to controls, in females three weeks of age and older (Fig 6G and 6H). Although iPLA2-VIAΔ23 mutant flies do not survive to six weeks of age [26, 36, 39], neither control genotype nor mito-YFP stock homozygotes show mitochondrial aggregation even at this late age (Figs 6I and S5A and S5B).

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Fig 5. Female fertility is reduced in iPLA2-VIAΔ23 mutants.

(A) Young (<1 week old) iPLA2-VIAΔ23 homozygous mutant females (light pink bars) produce fewer adult progeny compared to isogenic controls (dark red bars). Bars depict average number of adult progeny produced by 10 individual females over the course of four days, error bars represent standard deviations. (B) Hemizygous mutant females (iPLA2-VIAΔ23/Df(3L)BSC282) produce fewer adult progeny compared to isogenic controls. Dark red bars represent average number of adult progeny produced by 10 individual females over the course of four days, error bars are standard deviations. (C-D) Egg laying by iPLA2-VIAΔ23 mutants is reduced compared to isogenic controls in young (C, one week old) and aged (D, 18–19 day old) females. Dark red bars represent average number of eggs produced by 20 individual females over the course of four days, error bars are standard deviations. Differences assessed by unpaired t-test, n.s. not significant, *p < 0.1, **p < 0.02, ***p < 0.001, ****p < 0.0001.

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Fig 6. Germ cells from iPLA2-VIAΔ23 mutant females have abnormal mitochondrial distribution, reduced mitochondrial potential, and elevated apoptosis.

(A-B) Psqh-mito-EYFP (white) was used to observe the mitochondrial distribution in female germ cells from young (<1 week old) iPLA2-VIAΔ23 mutants (Psqh-mito-EYFP, iPLA2-VIAΔ23/iPLA2-VIAΔ23) and heterozygous controls (Psqh-mito-EYFP, iPLA2-VIAΔ23/revertantΔ11). Nuclei are stained with DAPI (magenta). Magnified views of the boxed regions are shown in the insets. (C-D) Mito-YFP labeled mitochondria appear clumpy in germ cells from aged (3 week old) iPLA2-VIAΔ23 female flies (D) but not controls (C, Psqh-mito-EYFP/revertantΔ11; for Psqh-mito-EYFP, iPLA2-VIAΔ23/revertantΔ11 heterozygotes see quantification in G and H). (E-H) Mito-YFP signal within the area of the germline nurse cells was outlined using ImageJ (white in E-F, yellow delimiting outlines indicate region of interest, i.e., the nurse cells) and the raw integrated density of the mito-YFP outline signal was used to quantify clumpiness (G-H). Homozygous iPLA2-VIAΔ23 mutants (dark red, Psqh-mito-EYFP, iPLA2-VIAΔ23/iPLA2-VIAΔ23, n = 39) show significantly reduced mito-YFP outline signal compared to heterozygotes (gray, Psqh-mito-EYFP, iPLA2-VIAΔ23/revertantΔ11, n = 43) and isogenic controls (black, Psqh-mito-EYFP/revertantΔ11, n = 19) at 3 weeks (G) and 4 weeks old (H; black, Psqh-mito-EYFP/revertantΔ11, n = 42; gray, Psqh-mito-EYFP, iPLA2-VIAΔ23/revertantΔ11, n = 67; dark red, Psqh-mito-EYFP, iPLA2-VIAΔ23/iPLA2-VIAΔ23, n = 50), indicating increased clumpiness. Bars represent averages and standard deviations for each condition. Differences assessed by unpaired t-test, ****p < 0.0001, *****p < 10−6. (I) Controls (Psqh-mito-EYFP/revertantΔ11) do not show mito-YFP clumpiness even at 6 weeks of age, while iPLA2-VIAΔ23 mutant females do not survive to 6 weeks. (J) Ovaries from 4 week old iPLA2-VIAΔ23 females (dark red bar) show reduced JC-1 fluorescence compared to age matched controls (black, Psqh-mito-EYFP/revertantΔ11; gray, Psqh-mito-EYFP, iPLA2-VIAΔ23/revertantΔ11) using plate-based fluorimetry. In each experiment, the red (595 nm) JC-1 fluorescence of the heterozygote or the mutant is expressed as a percentage of the fluorescence of the control, and 3–4 biological replicates are averaged. Error bars are standard deviations. Antimycin A was used as a mitochondrial poison to demonstrate the specificity of the JC-1 signal. (K) By 5 weeks of age, iPLA2-VIAΔ23 mutants show increased levels of germline apoptosis, marked by cleaved caspase-3 staining (green) and fragmentation of the nurse cell nuclei (DAPI, magenta), quantified in M. (L-M) The percentage of ovarioles containing dying egg chambers (green bars) is elevated in iPLA2-VIAΔ23 mutants compared to isogenic controls at 5 weeks of age (M, >1300 ovarioles counted for each genotype across four biological replicates) but not 4 weeks (L, >900 ovarioles counted for each genotype across three biological replicates). Dying egg chambers were identified by their fragmented nurse cell nuclei and cleaved caspase-3 staining, as shown in (K). Statistical comparison by two proportion z-test, n.s. not significant, ****p < 0.0001. Scale bars: 20 μm.

https://doi.org/10.1371/journal.pone.0256738.g006

[Figure omitted. See PDF.]

Fig 7. iPLA2-VIA-PB protein localizes to mitochondria in female and male germ cells.

(A-C) In the female germline, HA-tagged wild-type iPLA2-VIA-PB (green, grayscale shown in A”-C”, expressed with NGT40-GAL4) strongly colocalizes with a mitochondrial marker (A, red, Psqh-mito-EYFP, arrowheads) and shows minimal colocalization with Golgi (B, red, anti-Golgin 84) and ER (C, red, anti-Calnexin 99A) markers. Individual channels for mitochondria, Golgi, and ER shown in A’, B’, C’, respectively. (D-F) In male germ cells, iPLA2VIA-PB (green, expressed with bam-GAL4-VP16) also colocalizes more strongly with a mitochondrial marker (D, red, UAS-mCherry-mitoOMM) than Golgi (E, red) or ER (F, red) markers. Scale bars: 20 μm.

https://doi.org/10.1371/journal.pone.0256738.g007

Because mitochondrial aggregation can be a sign of damage [53, 54], we examined mitochondrial potential in mitochondrial preparations from lysed ovaries using the potential-sensitive fluorescent dye JC-1. Aged iPLA2-VIAΔ23 mutant ovaries have reduced JC-1 fluorescence compared to controls, indicating loss of mitochondrial potential (Fig 6J). By five weeks of age, germ cells of the mutant undergo apoptosis, with fragmented nuclei and cleaved caspase-3 staining, in contrast to controls (Fig 6K–6M).

To confirm further that mitochondrial aggregation is independent of the mito-YFP transgene, we examined another mitochondrial marker, immunofluorescence for the ATP-5A protein. We again saw mitochondrial aggregation in germ cells from aged iPLA2-VIAΔ23 mutant females and not in those from isogenic control flies (S5C and S5D Fig), but we were not able to quantify it due to the variability of the staining. We noticed, though, that on average germ cells from aged iPLA2-VIAΔ23 mutant females have lower ATP-5A signal intensity than those from control flies (S5E and S5F Fig), which might indicate that damaged mitochondria in the mutant are subject to quality control mechanisms involving degradation. This might be specific to the endogenous respiratory complex proteins, including ATP-5A, as the mito-YFP marker does not show this lowered signal in the mutant.

iPLA2-VIA-PB localizes to mitochondria in germ cells

Because iPLA2-VIA has been implicated in numerous different activities in a variety of cellular compartments, we examined the subcellular localization of our HA-tagged wild-type UAS-iPLA2-VIA-PB transgene, which can rescue the climbing defect of the mutant (Fig 2C and 2D). Following expression in female germ cells with NGT40-GAL4, the wild-type transgene colocalizes most strongly with a mitochondrial marker (Fig 7A–7C), consistent with the mitochondrial defect in the mutant. The transgene also colocalizes strongly with a mitochondrial marker in male germ cells, when expressed using bam-GAL4 (Fig 7D–7F). We additionally examined localization in somatic cells using the tubulin-GAL4 driver and the large polyploid cells of the larval fat body and salivary gland [55]. Interestingly, colocalization in these tissues is strongest with a Golgi marker (S6A and S6D Fig), suggesting that subcellular localization of iPLA2-VIA is regulated differently in distinct cell types and possibly is correlated with function.

Discussion

iPLA2-VIA in phospholipid metabolism

Tight regulation of iPLA2-VIA levels and activity is important for cell cycle dependent phospholipid accumulation [56] and PC homeostasis [40, 41] in several mammalian cultured cell lines. However, genetic perturbation of the rate-limiting PC synthesis enzyme Pcyt1 in whole flies does not cause obvious changes in iPLA2-VIA mRNA expression (S1 Fig), nor do we find evidence of genetic interactions between iPLA2-VIA and pcyt1 in viability or fertility (Table 2). To our knowledge, this is the first report on a possible genetic interaction between iPLA2-VIA and pcyt1 in an intact organism. iPLA2-VIA also shows no genetic redundancy for viability with sws/NTE, a conserved paralog important for PC homeostasis (Table 3, [43]). We see no global downregulation of iPLA2-VIA mRNA in an eas mutant with reduced PE levels either (S1 Fig, [57]). iPLA2-VIA mRNA expression is very low in wild-type imaginal discs (Fig 3), despite substantial cell proliferation there [58], and iPLA2-VIA null mutants are developmentally viable (Table 1). Altogether, our data suggest that iPLA2-VIA does not play unique roles in phospholipid accumulation in Drosophila, consistent with recent mass spectrometry analysis of another iPLA2-VIA knockout mutant [39].

iPLA2-VIA in male fertility and CL remodeling

We expected that, like the mouse ortholog, Drosophila iPLA2-VIA would be important for male fertility, in accord with its high expression in the male germline (Fig 3, [38]). Furthermore, it was reported to interact genetically with the mitochondrially-localized CL remodeling enzyme Taz in Drosophila male fertility [23]. We observed iPLA2-VIA colocalization with mitochondria in male germ cells (Fig 7), as has been documented in cultured mammalian cells [19, 21]. Surprisingly, however, iPLA2-VIA mutant males are fully fertile, with normal spermatid mitochondrial morphogenesis and, unlike the taz mutant, normal spermatid individualization (Fig 4). Moreover, we find no evidence that iPLA2-VIA loss of function mutations interact genetically with taz, in contrast to a prior report [23]. Our genetic data in the testis therefore may argue against the idea that iPLA2-VIA is a major unique player in CL remodeling. This is consistent with mass spectrometry results showing minimal CL changes in iPLA2-VIA hypomorphic mutant heads or whole flies [26, 30] and with recent studies indicating that Taz can remodel CL independently of phospholipases [59].

iPLA2-VIA in female fertility

iPLA2-VIA mRNA is highly expressed in the wild-type female germline (Fig 3), and iPLA2-VIA mutant females have reduced fertility compared to controls (Fig 5). Despite normal ovariole morphology, germ cells in aged iPLA2-VIA mutant females have abnormally aggregated mitochondria with decreased membrane potential, and they eventually die by apoptosis (Fig 6). In HeLa cells, pharmacological disruption of mitochondrial membrane potential induces aggregation via stabilization of the mitochondrially targeted kinase PINK1 and recruitment of its substrate and partner Parkin, an E3 ubiquitin ligase. Ubiquitinated Parkin substrates on the outer mitochondrial surface recruit the autophagy receptor p62/SQSTM1, which promotes aggregation, possibly for sequestration before degradation by mitophagy [53, 54]. Intriguingly, mitochondrial aggregation in Drosophila female germ cells has been observed in pink1 and parkin mutants, similarly to iPLA2-VIA mutants [60, 61]. This might suggest a parallel mechanism for mitochondrial damage detection and aggregation, independent of the PINK1-Parkin pathway, that perhaps relies on another mitophagy receptor, for example BNIP3, which is known to be active in the female germline [62, 63]. It is tempting to propose that in the absence of iPLA2-VIA, PINK1, or Parkin, mitochondrial damage accumulates and leads to aggregation. Eventually, persistent mitochondrial damage may overwhelm cytoprotective mechanisms like aggregation and mitophagy, leading to apoptosis. We thus hypothesize that iPLA2-VIA protects germline mitochondria from accumulated injury that can result in apoptosis, as in other contexts [19, 22, 25]. A direct effect is supported by the localization of iPLA2-VIA to mitochondria in germ cells (Fig 7). Reduced mitochondrial membrane potential also has been observed in brains of an iPLA2-VIA hypomorph [26], possibly suggesting a mechanistic connection between the germline and neuronal phenotypes.

iPLA2-VIA in neuromuscular degeneration

Our iPLA2-VIA mutants show a severe decline in locomotor ability with age, like other Drosophila and mouse mutant models (Fig 2, [26, 36, 64–67]). Phenocopy by RNAi knockdown in neurons is consistent with the idea that iPLA2-VIA acts autonomously to protect against neurodegeneration [26, 36]. Our data suggest that iPLA2-VIA also is important in other tissues to prevent locomotor decline, notably muscle, in accord with a report of muscle degeneration in two human PLAN patients [68]. The strength of the phenocopy with muscle-specific knockdown is comparable to that seen for pink1 (Fig 2). PINK1 and its partner parkin are well-established disease loci for autosomal recessive parkinsonism in humans [10]. As discussed above, PINK1 accumulates on the outer mitochondrial membrane under stress conditions and recruits Parkin to activate quality control mechanisms, including mitophagy, that maintain the overall integrity of the cellular mitochondrial network [69]. In their absence, accumulation of damaged mitochondria leads to degeneration of neurons, as well as muscle in Drosophila [48, 49]. The parkinsonism in human patients, as well as the locomotor defects and germline mitochondrial aggregation in flies, suggest some similarities between loss of iPLA2-VIA and loss of pink1 or parkin [60, 61]. Notably, however, Drosophila pink1 and parkin mutants display locomotor defects at earlier time points, as well as other indications of muscle degeneration, including crushed thoraces and wing posture abnormalities, not seen in iPLA2-VIA mutants [48, 49]. Additionally, the normal fertility and spermatid mitochondrial morphology in iPLA2-VIA mutant males stand in contrast to the highly penetrant male germline defects of pink1 and parkin mutants [48, 49, 70].

Although many studies have implicated iPLA2-VIA in mitochondrial maintenance via catalytic removal of damaged phospholipid acyl chains, our data show that a transgene with a serine-to-alanine mutation in the catalytic site can rescue the climbing defect of the mutant, consistent with another report in which a human iPLA2-VIA transgene carrying the analogous catalytic site mutation rescued bang-sensitivity and lifespan defects of iPLA2-VIA mutants [39]. This suggests that phospholipase activity is not the only important aspect in iPLA2-VIA mediated cytoprotection, possibly explaining the observation that mutations associated with PLA2G6-associated dystonia-parkinsonism do not disrupt catalytic activity [47]. iPLA2-VIA’s non-catalytic activities remain to be characterized and might depend on its ankyrin motifs, which are predicted to mediate protein-protein interactions [32]. Moreover, while loss of iPLA2-VIA is associated with aberrant mitochondrial morphology in neurons, it also is accompanied by defects in numerous other processes, including Ca+2 homeostasis, ER membrane equilibrium, and vesicle trafficking [26–28, 34, 39]. Thus, as with other forms of neurodegeneration, further investigation is necessary to determine which defects are drivers and which are passengers in PLAN.

Materials and methods

Drosophila strains

Drosophila were raised on standard media at 23°C. yw (BDSC_6599), P[EPgy2]iPLA2-VIAEY5103 (BDSC_15947), Df(3L)BSC394 (BDSC_24418), Df(3L)BSC282 (BDSC_23667), pcyt116919 (BDSC_7319), sws4 (BDSC_28121), HMS01544 (BDSC_36129), UAS-mCherry-mitoOMM (BDSC_66532 and BDSC_66533), Psqh-mito-EYFP (BDSC_7194), DJ667-GAL4 (BDSC_8171), and pink1-RNAi (HMS02204, BDSC_41671) were from Bloomington Drosophila Stock Center; P[GSV6]GS15374 (DGGR_206187) was from Kyoto Drosophila Genomics and Genetic Resource Center; tubulin-GAL4 and elav-GAL4 were gifts from J. Treisman; bam-GAL4-VP16 was a gift from Y. Yamashita; NGT40-GAL4 was a gift from R. Lehmann; easKO and isogenic controls were gifts from the Jan lab [44]; tazΔ161 mutants were a gift from M. Schlame and M. Ren [71].

The iPLA2-VIAΔ23 mutant was generated by excision of the P element EY5103, which is marked with mini-w+. The location of EY5103 was confirmed by inverse PCR before excision. Over 450 w- lines were screened for lesions in the region of interest by PCR with primers flanking the insertion site (F: 5’-CCGTGCTCTGTGGAATCAGT-3’, R: 5’-GCGCCAAAGCTAGAATTCCG-3’). Mutant line Δ23 was identified in the PCR screen, and the genomic region of iPLA2-VIA was sequenced to confirm the lesion. Several precise excision lines from the screen were kept as isogenic controls and confirmed by sequencing to be wild-type in the iPLA2-VIA gene region. This sequencing also confirmed that the same polymorphisms were present in the Δ23 line and the precise excision lines. Revertant line Δ11 was used as the isogenic control in all experiments described here. All recombinant chromosomes made with the iPLA2-VIAΔ23 allele were confirmed by PCR.

RT-PCR

Whole fly RNA was prepared from mixed sex samples (5–15 flies per sample, flies < 1 week old) with Trizol reagent (Invitrogen) according the manufacturer’s protocol. RNA preparations were treated with DNase I to remove genomic DNA (New England Biolabs) and quantified using NanoDrop (ThermoFisher). cDNAs were reverse transcribed using Superscript III (Invitrogen) according to the manufacturer’s protocol, from an equal mass of RNA for each sample per experiment (1–1.5 μg).

Qualitative PCRs were 25 cycles, unless noted otherwise. Experimental (iPLA2-VIA) and control primers (actin) were included in the same tube to control for pipetting. All experiments were repeated in triplicate. Quantifications were performed using Bio-Rad ImageLab 5.0. PCR primers were as follows:

[Figure omitted. See PDF.]

Quantitative PCRs were performed on cDNA samples diluted 6x, using SYBR green + regular ROX mix (MCLab, HSM-400). Reactions were run on an Applied Biosystems 7300 Real-Time PCR System. Primers were:

[Figure omitted. See PDF.]

Climbing assays

Climbing tests were performed as in [72]. Groups of 6–13 male or female flies were tapped to the bottom of a fresh food vial and given 20 s to climb 6 cm, into a new empty vial placed on top of the old one. Each group was given five climbing trials per assay. Each fly in the group was given one point for every success, and the total number of points for the group was divided by the number of flies in the group to yield the climbing index. Climbing indices were averaged for at least 6 groups per condition and plotted with standard deviations. Climbing indices for each condition were verified for normal distribution around the average. Statistical comparisons by unpaired t-tests.

Transgene generation

The wild-type iPLA2-VIA-PB transgene was generated by PCR from publicly available iPLA2-VIA cDNA (https://dgrc.bio.indiana.edu/, clone RE23733) using Platinum Pfx polymerase (Life Technologies, 11708013) and cloned into pUASg-HA.attB (http://www.flyc31.org/) or pPWH (https://dgrc.bio.indiana.edu/vectors/Catalog?product_category=3) using the Gateway system (Life Technologies). PCR primers were: 5’-(CACC)ATGGCGTGGATGGCGTTAG-3’ and 5’-AATCCGTCTTGCATGCGATTTTAG-3’. To introduce the serine-to-alanine mutation, an upstream PCR product was generated with primers 5’-TCTGTACCGGTCGCCGGTG-3’ and 5’-TAGAATTCCGCCAGTAGCGGTGCCGG-3’, and a downstream PCR product was generated with primers 5’-ATTGCCGGCACCGCTACTGGCGGAATT-3’ and 5’-CATGGCGTCTAGAGTCGGGTTGT-3’. The upstream and downstream PCR products were annealed and used as template to generate a PCR product spanning the entire segment. The mutated segment was cloned to replace the corresponding wild-type segment using the unique SgrA1 and Xba1 restriction sites. PCR products were cleaned with QIAquick PCR Purification kit (Qiagen 28104), digested DNA was cleaned with QIAquick Gel Extraction kit (Qiagen 28704), and plasmid DNA was prepared with QIAprep Spin Miniprep kit (Qiagen 27104) and Qiagen Plasmid Midi kit (Qiagen 12143). All constructs were verified by sequencing (Eton Bioscience) before injecting into flies (Genetivision). The VK37 ΦC31 landing site was used for both UAS-iPLA2-VIA-PB WT and UAS-iPLA2-VIA-PB SA.

RNA in situ hybridizations

iPLA2-VIA cDNA plasmid (https://dgrc.bio.indiana.edu/, clone RE23733) was linearized with BamH1 (sense) or Not1 (antisense) restriction digest, and digoxigenin-labeled RNA probes were transcribed using a digoxigenin-UTP in vitro transcription labeling mix (Roche) and either T7 or T3 RNA polymerase (Roche). Probe was carbonate-treated to reduce size. Control probes (S3 Fig) for wg and cycB were transcribed similarly, from PCR products made from genomic DNA. The PCR primers were: 5’-CAGGAATTCGACGGGAACCA-3’ and 5’-TAATACGACTCACTATAGGGAGAGTGCGTTTGGTGTCCTTGAC-3’ for cycB; 5’-GTGAAGTGCAAGCTGTGTCG-3’ and 5’-TAATACGACTCACTATAGGGAGAAAACGACAGCATGAGGAGGG-3’ for wg.

For imaginal discs, protocol was adapted from [73]. Third instar larvae were dissected in PBS and fixed in 4% formaldehyde/PBT (PBT is PBS/0.1% Tween-20) for 1 h at 4°C. Tissues were washed once in 50% PBT:methanol and once in 100% methanol before overnight storage in 100% methanol at -20°C. After two ethanol washes, tissues were incubated in 1:1 xylenes:ethanol for 1 h at 4°C. Tissues were washed again twice in ethanol and once in 50% methanol, and then incubated in 80% acetone for 10 min at -20°C. Tissues were washed twice in PBT and re-fixed in 4% formaldehyde/PBT for 20 min at 4°C. After three PBT washes, tissues were equilibrated into hybridization buffer and hybridized overnight at 65°C with digoxigenin-labeled RNA probes. The next day, tissues were equilibrated back into PBT, washed extensively, and incubated with alkaline phosphatase conjugated anti-digoxigenin antibody (1:1000, Roche) for 1 h at room temperature. After several PBT washes, tissues were washed in AP buffer and color was developed using NBT and BCIP substrates. Hybridization and color development were performed as in [74]. Larval and imaginal tissues were mounted in 80% glycerol.

For ovaries, protocol was performed as in [74].

For testes, protocol was performed as in [75]. Stained testes were mounted in 60% glycerol:PBS.

Tissue staining and immunofluorescence

For phalloidin staining, testes were dissected in PBS and fixed in 5% formaldehyde/PBX (PBS/0.1% Triton X-100) for 20 min at room temperature, washed in PBX for 15–20 min, and stained with rhodamine-phalloidin (1:200, Sigma-Aldrich) and DAPI (1:4000, Roche) for 20 min at room temperature. Following three washes in PBX, tissues were mounted in Fluoromount G (Southern Biotechnology).

For antibody staining, testes were fixed and washed as above. Testes were blocked in PBS + 5% normal donkey serum (Jackson ImmunoResearch) + 1% Triton X-100 before the primary antibody incubation. Primary antibody incubations were performed overnight (in PBX + 5–10% normal donkey serum) at 4°C, washed three times in PBX, and incubated with secondary antibodies, DAPI, and rhodamine-phalloidin for 2 hours at room temperature.

Larvae were dissected in PBS and fixed in 4% formaldehyde for 30 min at 4°C. Tissues were blocked in PBS + 5% normal donkey serum before the primary antibody incubation. For ovary antibody staining, adult females were mated to males on live yeast paste for 1–2 days before dissections. Ovaries were dissected in PBS and combed open, fixed in 5% formaldehyde for 13 min at room temperature, rinsed in PBX, washed 3–4 times in PBX, and blocked in PBS + 5% normal donkey serum + 1% Triton X-100 before primary antibody incubation. Antibody incubations were performed as above.

Primary antibodies used were mouse anti-ATP-5A (1:1000, Abcam, ab14748), mouse anti-Calnexin 99A (1:2, Developmental Studies Hybridoma Bank, ER marker), mouse anti-Golgin 84 (1:2, Developmental Studies Hybridoma Bank, Golgi marker), rat anti-HA (1:100, Roche, 3F10), rabbit anti-cleaved caspase 3 (1:100, Cell Signaling Technology, 9664), and rabbit anti-GFP (1:5000, Life Technologies, A6455). Secondary antibodies were Alexa Fluor® 488-AffiniPure Donkey Anti-Rat or Anti-Rabbit IgG and Cy3-AffiniPure Donkey Anti-Mouse IgG or Anti-Rat IgG (1:200, Jackson ImmunoResearch).

Images were captured using an Olympus IX-81 motorized inverted microscope with XM-10 monochrome camera (lenses: 10x/0.3 NA, 20x/0.75 NA, 40x oil/1.3 NA, 60x oil/1.35 NA), Zeiss LSM510 Confocal (lenses: 10x/0.3 NA, 20x/0.8 NA, 40x/0.75 NA, 40x oil/1.30 NA), or Zeiss LSM800 Confocal (lenses: 10x/0.3 NA, 20x/0.8 NA, 40x oil/1.3 NA). ICs were scored as described in [76].

Testis squashes

Internal reproductive organs were dissected from newly eclosed males in PBS, placed in a drop of PBS on a coverslip and punctured. A glass microscope slide was placed on top of the coverslip to squash the testes, and phase-contrast images were collected within 30 min of dissection using an Olympus IX-71 inverted microscope with Nikon Digital Sight camera with DS-U2 controller (20x lens/0.40 NA).

Fertility tests

For male fertility tests, ten males of each genotype were mated individually to two 3–4 day old yw virgin females at 23°C. The males were discarded after one day, and the females were transferred to fresh vials each day for three more days and discarded on day 5. Progeny per vial were counted and averaged.

For homozygous female fertility tests, ten virgin females of each genotype were mated individually to two yw males at 23°C. All flies were 3 days old at the time of mating. For hemizygous female fertility tests, virgin females less than one week old were mated to 1-day old w1118 males at 23°C. The males were discarded after one day, and the females were transferred to fresh vials each day for three more days and discarded on day 5. Progeny per vial were counted and averaged.

For egg laying tests, 20 virgin mutant or control females were mated individually to one 7-day old w1118 male each at 23°C. Young females were 7 days old at the time of mating; aged females were 18–19 days old at the time of mating. The males were discarded after one day, and the females were transferred to fresh vials each day for three more days and discarded on day 5. Eggs per vial were counted daily.

Maternal lethality tests were conducted by mating 20–25 young (<10 day old) iPLA2-VIAΔ23 or isogenic control females to control males and transferring them to cages with grape juice plates and live yeast paste overnight at 23°C. After 20 hours of egg-laying, 90–120 eggs from each genotype were collected per experiment and moved to new plates at 23°C. Larvae were collected and counted every 24 hours at 23°C. Third instar larvae were moved to vials, and pupae were counted at 23°C. The results of three experiments were averaged.

Mito-YFP quantification

iPLA2-VIAΔ23 mutant (Psqh-mito-EYFP, iPLA2-VIAΔ23/iPLA2-VIAΔ23), heterozygote (Psqh-mito-EYFP, iPLA2-VIAΔ23/revertantΔ11), and control females (Psqh-mito-EYFP/revertantΔ11) expressing Psqh-mito-EYFP were aged in groups of 10–20 flies at 23°C. Females were mated to males on live yeast paste 1–2 days before dissection. Ovaries were dissected and stained with anti-GFP antibodies as described above. Every stage 8 or 9 egg chamber from each sample was photographed using identical confocal settings and 40x oil objective. After drawing an ROI around the nurse cell area and removing background signal with the same threshold, each image was converted to binary in ImageJ. Each binary image was converted to outlines, and the raw integrated density was measured for the ROI.

JC-1 fluorimetry

18–30 ovaries per sample were dissected from age-matched iPLA2-VIAΔ23 mutant, heterozygote, and control flies (genotypes and aging conditions as above). Tissues were immediately transferred to cold 1x PBS. Sigma mitochondrial isolation kit (Sigma, MITOISO1) was used to extract mitochondria and perform the JC-1 assay according to the manufacturer’s protocol, as follows. After discarding the PBS, each tissue sample was rinsed twice with cooled 1x Extraction Buffer using a glass Pasteur pipet. In a 3 ml glass homogenizer, the lysate was prepared in 1 mL of cooled 1x Extraction Buffer containing 2 mg/ml albumin and transferred to a microcentrifuge tube. The lysate was spun at 1000 x g for 5 minutes at 4°C to remove cell debris, and the transferred supernatant was spun at 13,000 x g for 10 minutes at 4°C to obtain the mitochondrial pellet. The pellet was resuspended in cooled 1x Extraction Buffer (without albumin) by pipetting, and the two centrifugation steps were repeated to get the mitochondria-enriched pellet, which was resuspended in cooled 1x Storage Buffer and stored on ice for immediate use or at 4°C for a maximum of 24 hours. Mitochondrial protein concentration was determined by Bradford assay using a Beckman DU800 spectrophotometer. Sample concentrations were equilibrated with storage buffer. JC-1 assays were performed in clear bottom 96-well plates using a Beckman Coulter DTX800 spectrofluorometer. Each JC-1 reaction mixture of 100 μl was prepared with the same amount of mitochondrial sample (5–7 μg of mitochondrial protein per reaction), 1 μl of the JC-1 solution (final concentration 10 μg/ml) and 0.5 μl of the electron transport chain complex III inhibitor Antimycin A (final concentration 200 μg/ml) or 0.5 μl of the vehicle DMSO. Three technical replicates were prepared for each sample along with a blank lacking mitochondrial sample. After 10 minutes in the dark at room temperature, fluorescence intensity was measured using excitation wavelength of 485 nm, emission wavelength of 595 nm, and integration time of 1000 ms. The average fluorescence per mg of protein of each biological sample was calculated across technical replicates and converted to relative percentage of the isogenic control. For each genotype, 3–4 biological replicates were averaged.

Western blot

An equal number of adult flies for each sample were collected and frozen in liquid N2. Frozen flies were homogenized in lysis buffer with Tris pH 7.5, NaCl, EDTA, NP40, DTT, NaF, and protease inhibitor cocktail (Roche), centrifuged to remove carcasses, and mixed with Laemmli buffer. Protein preps were run on a 10% SDS-PAGE gel, transferred to nitrocellulose (Bio-Rad), and blotted with mouse anti-HA (1:1000, Covance, MMS-101P) and mouse anti-beta-tubulin (1:2000, BioLegend, 903401). Secondary antibody was IRDye 800CW donkey anti-mouse IgG (Li-Cor, 925–32212). Quantifications were performed using Li-Cor Odyssey software.

Supporting information

S1 Fig. iPLA2-VIA mRNA levels are unchanged by genetic perturbation of phospholipid metabolism genes.

(A) Whole fly reverse transcription (RT) PCR shows no upregulation of iPLA2-VIA mRNA expression when Pcyt1 is overexpressed using tubulin-GAL4. Control siblings lack the tubulin-GAL4 driver. pcyt1 mRNA upregulation is shown in the bottom panel of the gel image. (B-C) Whole fly RT-PCR shows no downregulation of iPLA2-VIA expression in either the pcyt116919 mutant compared to w1118 (B) or in the easKO mutant compared to isogenic controls (C). Experiments were performed in triplicate. Quantifications were taken with Bio-Rad ImageLab 5.0, shown below representative gel images. Graphs show the ratio of iPLA2-VIA mRNA normalized to internal control actin mRNA in each mutant genotype compared to the control genotype, averaged across three biological replicates. Error bars are standard deviations. (E-G) Whole fly RT-qPCR confirms that iPLA2-VIA mRNA levels are not significantly different from controls when (E) Pcyt1 is overexpressed using tubulin-GAL4, (F) in pcyt116919 mutants, or (G) in easKO mutants. iPLA2-VIA mRNA abundance was normalized to rp49 mRNA abundance (2-ΔCt) and averaged across three biological replicates. Error bars are standard deviations. Statistical analysis by unpaired t-test (A, C, E-G) or single factor ANOVA (B), n.s. not significant.

https://doi.org/10.1371/journal.pone.0256738.s001

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S2 Fig. Transgenic catalytic-dead iPLA2-VIA is expressed at lower levels than transgenic wild-type iPLA2-VIA.

Five male and five female adult flies were collected for each of the following genotypes: tubulin-GAL4 > UAS-iPLA2-VIA-PB-WT; tubulin-GAL4 > UAS-iPLA2-VIA-PB-SA; tubulin-GAL4 alone. All three samples were processed in parallel, run on a denaturing SDS-PAGE gel, and blotted with both anti-HA to detect transgenic iPLA2-VIA-HA and with anti-beta-tubulin as a loading control. Representative blot shown in (A), quantification of three biological replicates shown in (B). iPLA2-VIA protein levels were normalized to beta-tubulin levels in each lane. Biological replicates are indicated by the gray circles, the average normalized protein levels are represented by the black bars, and error bars are standard deviations.

https://doi.org/10.1371/journal.pone.0256738.s002

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S3 Fig. Control RNA in situ hybridizations.

(A) Riboprobe antisense to wg transcript (arrows) was used to demonstrate the fidelity of imaginal disc in situ hybridizations. (B) Riboprobe antisense to cycB transcript was used to demonstrate fidelity of testis in situ hybridizations. Arrows indicate staining in primary spermatocytes, and arrowheads indicate meiotic spermatids. (C) The cycB riboprobe also revealed specific expression in the larval brain (arrows). Scale bars: 100 μm.

https://doi.org/10.1371/journal.pone.0256738.s003

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S4 Fig. No maternal effect lethality in iPLA2-VIAΔ23 mutants.

Young (<10 day old) homozygous iPLA2-VIAΔ23 females or isogenic control females were mated to control males and allowed to lay eggs on grape juice plates for 20 hours at 23°C. First instar larvae hatched from isolated eggs were counted (black bars). Second instar larvae molted from isolated first instars were counted (dark gray bars). Third instar larvae molted from isolated second instars were counted (medium gray bars). Pupae were counted from isolated third instars (light gray bars). The entire experiment was repeated three times. Bars represent the average percentage of individuals that progress to each stage in the three experiments. Error bars are standard deviations. No developmental lethality is observed in progeny from iPLA2-VIAΔ23 mutant mothers compared to control mothers.

https://doi.org/10.1371/journal.pone.0256738.s004

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S5 Fig. Mitochondrial aggregation is characteristic of aged iPLA2-VIAΔ23 mutant germ cells.

(A-B) Neither the parental stock carrying homozygous Psqh-mito-EYFP (A) nor iPLA2-VIAΔ23 heterozygotes (B, Psqh-mito-EYFP, iPLA2-VIAΔ23/revertantΔ11) show mitochondrial clumping even at 6 weeks of age (white, mito-YFP; magenta, DAPI). (C-D) Mitochondrial clumping also is observed with another marker, immunofluorescence to ATP-5A protein in iPLA2-VIAΔ23 mutants (D) but not in isogenic controls (C) at five weeks of age (white, anti-ATP-5A; magenta, DAPI). Magnified views of the boxed regions are shown in the insets. (E-F) Additionally, the ATP-5A signal is weaker in iPLA2-VIAΔ23 mutants (dark red bars) than in age-matched controls (dark gray bars) at four (E) and five (F) weeks of age, possibly indicating mitochondrial degradation in the mutant. Bars represent averages, error bars are standard deviations. Statistical comparison by unpaired t-test. Scale bars: 20 μm.

https://doi.org/10.1371/journal.pone.0256738.s005

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S6 Fig. iPLA2-VIA-PB localizes to Golgi in somatic larval tissues.

HA-tagged wild-type iPLA2-VIA-PB transgenic protein appears in puncta (green, A”-F”, expressed with tubulin-GAL4) that colocalize with a Golgi marker (red, anti-Golgin 84) in larval fat body (A) and salivary glands (D). Colocalization with an ER marker (red, B, E, anti-Calnexin 99A) and a mitochondrial marker (red, C, F, UAS-mCherry-mitoOMM) is weak or undetectable in these larval tissues. Scale bars: 20 μm.

https://doi.org/10.1371/journal.pone.0256738.s006

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S1 File. Raw gel and blot images.

https://doi.org/10.1371/journal.pone.0256738.s007

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Acknowledgments

We are grateful to the Bloomington Drosophila Stock Center, Kyoto Drosophila Genomics and Genetic Resource Center, Developmental Studies Hybridoma Bank, Drosophila Genomics Resource Center, Jessica Treisman, Yukiko Yamashita, Ruth Lehmann, the Jan lab, Michael Schlame, and Mindong Ren for fly stocks. We are grateful, as always, to Flybase, for its continual support of the Drosophila community. Sarah Liberow, Nicole Soussana, and Eliezer Heller assisted with some of the experiments. Critical comments on the manuscript were provided by Jessica Treisman and Rebecca Delventhal.

Citation: Banerjee SJ, Schonbrun A, Eizadshenass S, Benji S, Cantor YT, Eliach L, et al. (2021) iPLA2-VIA is required for healthy aging of neurons, muscle, and the female germline in Drosophila melanogaster. PLoS ONE 16(9): e0256738. https://doi.org/10.1371/journal.pone.0256738

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