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Downregulation of EDTP in glial cells suppresses polyglutamine protein aggregates and extends lifespan in Drosophila melanogaster
Neuroscience Letters 694 (2019) 168–175
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Research article
Downregulation of EDTP in glial cells suppresses polyglutamine protein aggregates and extends lifespan in Drosophila melanogaster
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Chengfeng Xiaoa, , Shuang Qiub a
Department of Biology, Queen’s University, Kingston, Ontario, K7L 3N6, Canada Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Xiao Ling Wei 200, Nanjing, 210094, Jiangsu, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Egg-derived tyrosine phosphatase (EDTP) Polyglutamine protein aggregates Autophagy Glial cell Drosophila melanogaster
Drosophila egg-derived tyrosine phosphatase (EDTP) is a lipid phosphatase essential for oogenesis and muscle function. Loss-of-EDTP is lethal at early developmental stages. Hypomorphic mutation of EDTP causes impaired muscle performance and shortened lifespan. Mutation of MTMR14, a mammalian homolog to EDTP, is associated with muscle fatigue in rodents and a rare genetic disease called centronuclear myopathy in humans. Despite the deleterious consequences, downregulation of MTMR14 promotes autophagy. It is proposed that selective downregulation of EDTP/MTMR14 in non-muscle tissues improves the survivorship to cellular wastes and extends lifespan. Here, we show that downregulation of EDTP in glial cells suppressed the expression of polyglutamine (polyQ) protein aggregates and improved survival. Downregulation of EDTP in glial cells also extended lifespan. These effects were not observed by targeting pan-neurons in the nervous system, suggesting the significance of tissue-specificity. Additionally, flies carrying an EDTP mutant had increased survival to prolonged anoxia and altered dynamics of polyQ expression. These data supported the proposal that selective downregulation of EDTP in non-muscle tissues improved survivorship to cellular protein aggregates and extended lifespan. Our findings suggest that EDTP/MTMR14 could be a novel molecular target for the treatment of neurodegeneration.
1. Introduction Drosophila egg-derived tyrosine phosphatase (EDTP) is a lipid phosphatase that specifically removes 3-position phosphate at the inositol rings of two phosphoinositides: phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P2). PI3P and PI(3,5)P2 are important second messengers [1]. PI3P is essential for the initiation of autophagy, a self-degradative process for the removal of damaged organelles, misfolded proteins, and protein aggregates [2,3]. PI(3,5)P2 binds to ryanodine receptors (RyR1 and RyR2) and causes calcium release from the sarcoplasmic reticulum, thus regulating contractility in skeletal and cardiac muscles [4,5]. Downregulation of MTMR14, a mammalian homolog to EDTP, leads to increased PI3P and enhanced autophagy [6]. Mutation of MTMR14 also causes an accumulation of PI(3,5)P2 which disrupts intracellular calcium homeostasis and impairs muscle performance [4,7]. The predominant phenotype of EDTP/MTMR14 mutation is muscle dysfunction such as impaired motor activities in flies [8] or a rare genetic disease called centronuclear myopathy [9]. This can be attributed to the normally abundant
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expression of EDTP/MTMR14 in muscles. Selective downregulation of EDTP/MTMR14 in non-muscular tissues would circumvent the muscle defect. In non-muscle cells, where PI (3,5)P2 has little chance of acting on RyR1 and RyR2, the PI3P-initiated autophagy could become dominant. Conceivably, one could manipulate the expression of EDTP/MTMR14 in favorable tissues for enhanced autophagy and extended lifespan. Glial cells, the major cell types that support neurons in the nervous system, are an ideal candidate for this purpose. There are at least three advantages of targeting glial cells. First, restricted downregulation of EDTP in glial cells causes no or minor disturbance to maternally-derived EDTP expression in the early stages of embryogenesis. Transcription of the repo, a glial cell-specific marker, appears to be highly restricted in glioblasts since stage 9 embryos [10]. EDTP mRNA, however, is detectable uniformly in the cytoplasm of eggs at stage 1, 5 and 11 but quickly disappears from stage 15 [11]. There is little spatial and temporal overlap of transcription between repo and EDTP in early development. Second, targeting glial cells for EDTP downregulation would have no effect on the muscle expression because these two types of cells
Corresponding author at: 116 Barrie Street, Queen’s University, Kingston, ON, K7L 3N6, Canada. E-mail address: [email protected] (C. Xiao).
https://doi.org/10.1016/j.neulet.2018.12.009 Received 2 July 2018; Received in revised form 13 November 2018; Accepted 4 December 2018 Available online 05 December 2018 0304-3940/ © 2018 Elsevier B.V. All rights reserved.
Neuroscience Letters 694 (2019) 168–175
C. Xiao, S. Qiu
2.3. Quantification of polyQ protein aggregates
are distally differentiated and distributed. Third, during adult stages, EDTP is reactivated at around day 7, reaching the peak at day 20–30 and followed by a gradual decline after [12,13]. Meanwhile, both the translation of repo and the repo-Gal4-driven expression of mCD8-GFP in glial cells persist for at least 56–63 days [14]. These allow the gliaspecific RNAi knockdown of EDTP to be most efficient in the middle and late life, the stages arguably critical for postponed senescence according to the prevailing pleiotropy theory [15]. We hypothesized that tissue-specific knockdown of EDTP in glial cells improves survival to cellular wastes and extends lifespan in Drosophila. Using the Gal4/UAS expression system [16], we directed the expression of polyglutamine (polyQ) protein aggregates in glial cells. We showed that downregulation of EDTP suppressed the expression of polyQ protein aggregates and this was associated with improved survival. In the absence of polyQ expression, RNAi knockdown of EDTP in glial cells resulted in extended lifespan. These beneficial effects were not observed by targeting pan-neurons in the nervous system. This study supported the proposal that selective downregulation of EDTP/ MTMR14 in non-muscle tissues improved survival to cellular wastes and extended lifespan.
The expression of polyQ protein aggregates in the brain was evaluated by quantifying the percent area of polyQ aggregate (% PolyQ area) in the brain. In general, image stacks were combined into a single plane. The total area of aggregate particles was measured. % PolyQ area was calculated by this formula: % PolyQ area = Cumulative area of aggregates/area of whole brain × 100%. 2.4. Lifespan experiments Adult flies were collected at 0–2 days into fresh food vials at a density of 20–25 flies per vial. They were transferred into fresh medium twice a week. Dead flies were scored during the transfer until all the flies were counted. This procedure was also used to examine the survival of flies expressing polyQ protein aggregates in glial cells, and the survival of flies having a simultaneous expression of polyQ aggregates and RNAi knockdown of EDTP in glial cells. Sample sizes are indicated in the text. 2.5. Anoxia experiments
2. Methods
Flies at 4–6 days were exposed to a prolonged anoxia (4–6 hours, generated by the continuous flow of 100% pure nitrogen gas at around 2 L/min). Dead flies were scored daily during the first week of recovery, then twice a week after.
2.1. Flies Fly strains used in this study and their sources are: repo-Gal4 (Bloomington Drosophila Stock Center (BDSC) #7415), elav-Gal4 (#8765 and #8760), D42-Gal4 (#8816), UAS-EDTP-RNAi (carrying an insertion in the chromosome II with an RNAi sequence CAGTAGTGTA ATAGTAATCAA targeting the transcripts of exon 3 of EDTP, #41633), UAS-EDTP-RNAi (in the chromosome III with a sequence CAGCTACG ACGAAGTCATCAA targeting the transcripts of exon 2, #36917); UASHttex1-Q72-eGFP [17]; UAS-hsp70-myc [18]; 20×UAS-IVSmCD8::GFP (#32194); EDTPEP2390 (#17050); EDTPKG04513 (#13605); EDTPEY22967 (#22600); and EDTPMI02389 (#34481) and w1118 (Dr. Seroude laboratory). We generated a line of UAS-Httex1-Q72-eGFP; repoGal4/M3 for this study. Flies were maintained with the standard medium (cornmeal, agar, molasses, and yeast) under regular conditions (21–23 °C, 50–60% humidity) in a light/dark (12 h/12 h) photoperiod. Male flies were used for experiments unless otherwise indicated.
2.6. Statistics Kaplan-Meier multiple group comparisons with BY adjustments were performed for the survival analysis of three curves. A two-way ANOVA with Bonferroni post-test was used for comparing the % PolyQ area. Statistics were performed using the software R [20]. P < 0.05 was considered statistically significant. 3. Results 3.1. PolyQ protein aggregates expressed in glial cells To address the potential autophagy-promoting effect of EDTP downregulation, it was necessary to establish a fly model expressing cellular protein aggregates in targeted cells. We directed the expression in glial cells of a toxic protein with polyQ tract by using repo-Gal4 [10,21] and UAS-Httex1-Q72-eGFP [17]. Each protein contained 72 tandem repeats of glutamine and a marker of enhanced green fluorescent protein (eGFP). We observed the bright fluorescence in brain tissues at day 1, 5, 10, and 19 of male flies UAS-Httex1-Q72-eGFP/+;; repo-Gal4/+ (Fig. 1 and S1). The fluorescent substrates appeared as varying-sized clumps throughout the brain. Relative large clumps were located typically in the surface region, the median septum, and the connections between the middle brain and optic lobes. Small clumps were evenly distributed. Fluorescent clumps were observed directly from freshly dissected tissues without immunohistochemistry. Collectively, we referred to these fluorescent clumps as polyQ protein aggregates. Female flies of UAS-Httex1-Q72-eGFP/+;; repo-Gal4/+ also expressed polyQ protein aggregates with the distribution characteristics similar to males (Fig. S2). There was no observable polyQ protein aggregate in the brains of control flies (UAS-Httex1-Q72-eGFP/+ and repo-Gal4/+) (Fig. S1). The expression of heat shock protein 70 [18], a soluble molecular chaperone, did not form protein aggregates in glial cells of flies repoGal4/UAS-hsp70-myc. Expression of mCD8-GFP [22], a membraneanchored protein, was evenly distributed without aggregation in the brains in repo-Gal4/20×UAS-IVS-mCD8-GFP. Thus, the formation of protein aggregates was a characteristic feature of the product containing polyQ tract. This provided a valuable opportunity to examine
2.2. Immunofluorescence The immunofluorescent experiments were performed by following a published protocol [19]. Flies and their specific antibodies were: (1) UAS-Httex1-Q72-eGFP/Y;; repo-Gal4/+ flies: primary antibody, mouse anti-GFP supernatant (12A6, DSHB) at 1:20; secondary antibody, Alexa Fluor 488 conjugated goat anti-mouse IgG (115-545-003, Jackson ImmunoResearch) at 1:500. (2) repo-Gal4/UAS-hsp70-myc flies: primary antibody, rabbit anti-c-myc (A00173, GenScript) at 1:50; secondary antibody, DyLight goat anti-rabbit IgG (111-485-144, Jackson ImmunoResearch) at 1:500. (3) repo-Gal4/20×UAS-IVS-mCD8::GFP flies: primary antibody, mouse anti-GFP supernatant (12A6, DSHB) at 1:20; secondary antibody, Alexa Fluor 488 conjugated goat anti-mouse IgG (115-545-003, Jackson ImmunoResearch) at 1:500. Notably, the immunofluorescence was unnecessary for polyQ-expressing flies because the bright fluorescence is readily observable in the freshly dissected tissues. Fresh tissues were mounted on slides with SlowFade Gold antifade reagent for confocal imaging within an hour. During that time the tissue size remained stable. Most images in the current study were taken from freshly prepared tissues without immunofluorescence. We used a Carl Zeiss LSM 710NLO laser scanning confocal/multiphoton microscope (Carl Zeiss) for imaging. The subsequent analysis was performed with ImageJ (NIH). 169
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Fig. 1. EDTP knockdown suppressed the polyQ protein aggregates. (a) Expression of polyQ protein aggregates in glial cells in three groups of flies. Shown are the brains of male flies at the ages of 1, 5, 10, 19, and 38 days. Genotypes are indicated. At day 38, images of UAS-Httex1-Q72-eGFP/+; repo-Gal4/+ are unavailable because no flies survived up to 38 days. PolyQ protein aggregates are invisible in the brains of UAS-Httex1-Q72-eGFP/+; UAS-EDTP-RNAi/+; repo-Gal4/+. Bar 200 μm. (b) Analysis of the percent area of polyQ protein aggregates (% PolyQ area) in three groups of flies. * P < 0.05; ** P < 0.01; *** P < 0.001 by two-way ANOVA with Bonferroni post-tests.
downregulation. Strikingly, polyQ protein aggregates were invisible at day 38 in flies with EDTP knockdown (Fig. 1a). Repeated tests indicated that polyQ protein aggregates became invisible at as early as day 30 (Fig. S3). Using another RNAi line with an interference sequence targeting exon 2 of EDTP (BDSC #36917), we observed persistent expression of polyQ aggregates at all tested ages (day 1, 5, 10, 19, and 38) and increased maximal survival (by around 2-fold) compared with controls. Using double RNAi for EDTP knockdown, we also observed the persistent expression of polyQ aggregates up to day 38 (Figure S4). Clearly, flies with EDTP downregulation in glial cells had improved survival to polyQ aggregates. The percent area of polyQ protein aggregates (% PolyQ area) remained relatively high and stable (26.5–33.2 %) in male flies UASHttex1-Q72-eGFP/+;; repo-Gal4/+ at day 1, 5, 10, and 19 (Fig. 1b).
the effect of EDTP downregulation. 3.2. RNAi knockdown of EDTP suppressed polyQ protein aggregates in glial cells Few males of UAS-Httex1-Q72-eGFP/+;; repo-Gal4/+ survived more than 19 days (Fig. 1a). We examined the effect of EDTP downregulation on the expression of polyQ aggregates in glial cells. EDTP knockdown was conducted with an RNAi line carrying the interference sequence targeting the transcripts of exon 3 (BDSC #41633). PolyQ aggregates were observed at day 1, 5, 10 and 19 in the brains of male flies UAS-Httex1-Q72-eGFP/+; UAS-EDTP-RNAi/+; repo-Gal4/+. These flies lived up to 38 days, which was around a 2-fold increase in the maximal survival compared with flies without EDTP 170
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Average levels of % PolyQ area were reduced at day 1, 5 and 19 but not at day 10 in UAS-Httex1-Q72-eGFP/+;; repo-Gal4/UAS-EDTP-RNAi compared with controls (Two-way ANOVA with Bonferroni post-tests). Average levels of % PolyQ area were reduced at day 1, 10 and 19 in UAS-Httex1-Q72-eGFP/+; UAS-EDTP-RNAi/+; repo-Gal4/+ compared with their controls (Two-way ANOVA with Bonferroni post-tests). At day 5, however, the average level of % PolyQ area in UAS-Httex1Q72-eGFP/+; UAS-EDTP-RNAi/+; repo-Gal4/+ was even higher than that in control (P < 0.05, Two-way ANOVA with Bonferroni posttests). These data indicated altered dynamics of polyQ expression with EDTP downregulation. Similar to males, female flies had likely reduced levels of % PolyQ area at day 1 and day 10 with the presence of RNAi (Fig. S2). In general, we observed that RNAi knockdown of EDTP in glial cells suppressed the expression of polyQ protein aggregates in male flies. 3.3. RNAi knockdown of EDTP in glial cells improved the survival to polyQ protein aggregates The median survival of UAS-Httex1-Q72-eGFP/+;; repo-Gal4/+ flies was 15 days (95% confidence interval (95% CI) 13–17 days, n = 44), which was remarkably shorter than that of repo-Gal4/+ flies (median 101 days, 95% CI 84–113 days, n = 35) (P < 0.0001, KaplanMeier multiple group comparisons) or that of UAS-Httex1-Q72-eGFP/+ flies (median 84 days, 95% CI 76–84 days, n = 62) (P < 0.0001, Kaplan-Meier multiple group comparisons) (Fig. 2a). Thus, the expression of polyQ protein aggregates in glial cells severely shortened the lifespan. Most flies of UAS-Httex1-Q72-eGFP/+;; repo-Gal4/+ died within 20 days. This was consistent with the observation that few flies survived more than 19 days for imaging. We tested the survivorship of flies with polyQ protein aggregates in the presence of EDTP-RNAi in glial cells. The median survival of UASHttex1-Q72-eGFP/+;; repo-Gal4/UAS-EDTP-RNAi (25 days, 95% CI 22–31 days, n = 50) was longer than that of UAS-Httex1-Q72-eGFP/ +;; repo-Gal4/+ flies (18 days, 95% CI 18-18 days, n = 84, tests independent of Fig. 2a) (P < 0.0001, Kaplan-Meier multiple group comparisons). The median survival of UAS-Httex1-Q72-eGFP/+; UASEDTP-RNAi/+; repo-Gal4/+ (27 days, 95% CI 25–31 days, n = 61) was also longer than that of UAS-Httex1-Q72-eGFP/+;; repo-Gal4/+ flies (P < 0.0001, Kaplan-Meier multiple group comparisons) (Fig. 2b). There was no statistical difference of median survival between flies carrying two different RNAi. Therefore, using two independent RNAi for EDTP knockdown in glial cells, we observed consistently improved survivorship to polyQ protein aggregates.
Fig. 2. EDTP knockdown improved the survivorship to polyQ protein aggregates and extended lifespan. (a) Lifespan shortened by the expression of polyQ protein aggregates in glial cells. The survival of UAS-Httex1-Q72-eGFP/ +;; repo-Gal4/+ flies (red) was markedly reduced compared with repo-Gal4/ + (grey) or UAS-Httex1-Q72-eGFP/+ (blue). Male flies were examined. *** P < 0.0001 by Kaplan-Meier multiple group comparisons. (b) EDTP knockdown in glial cells improved survivorship to polyQ protein aggregates. Two independent RNAi lines were used for EDTP knockdown. Data were collected from male flies. *** P < 0.0001 by Kaplan-Meier multiple group comparisons. (c) EDTP knockdown in glial cells extended lifespan. One RNAi line (on chromosome III) was used. *** P < 0.0001 by Kaplan-Meier multiple group comparisons.
eGFP/+; elav-Gal4/+ (Fig. 3a). PolyQ aggregates were distributed mostly in the areas of mushroom body cells (MBC), subesophageal zone (SEZ), antennal lobes (AL), and optic lobes (OL). With RNAi present, % PolyQ area likely increased at day 5 and day 22 (Fig. 3b), indicating that EDTP knockdown in neurons was less effective than in glial cells for the suppression of polyQ aggregates. Males of polyQ-expressing flies had a median lifespan of 53 days (95% CI 44–53 days, n = 27), which was shorter than UAS-Httex1-Q72-eGFP/+ (median 82 days, 95% CI 82 - 82 days, n = 71) (P < 0.0001, Kaplan-Meier multiple comparisons) or elav-Gal4/+ (median 99 days, 95% CI 99–106 days, n = 63) (P < 0.0001, Kaplan-Meier multiple comparisons) (Fig. 3c). Hence, neuronal expression of polyQ aggregates resulted in markedly reduced lifespan. With the presence of RNAi, median lifespan in UAS-Httex1Q72-eGFP/+; elav-Gal4/+; UAS-EDTP-RNAi/+ (36 days) was even shorter than the polyQ-expressing controls (P < 0.0001, Kaplan-Meier test). Thus, downregulation of EDTP in neurons failed to improve the survival to polyQ aggregates. Without polyQ expression, median lifespan was 83 days (95% CI 83–88 days, n = 92) for males of elav-Gal4/ +; UAS-EDTP-RNAi/+, 83 days (95% CI 77–83 days, n = 78) for UASEDTP-RNAi/+, and 88 days (95% CI 88–93 days, n = 57) for elavGal4/+. There was no significant difference between elav-Gal4/+; UAS-EDTP-RNAi/+ and controls (Fig. 3d). Therefore, EDTP knockdown in neurons had no effect on lifespan extension. Using another elav-Gal4 (on chromosome III, #8760), we observed polyQ aggregates expressed mainly in the antennal lobes at day 30. Once again, EDTP
3.4. RNAi knockdown of EDTP in glial cells extended lifespan A key question is whether tissue-specific downregulation of EDTP extends the lifespan in the absence of polyQ aggregates. Median lifespan of repo-Gal4/UAS-EDTP-RNAi flies was 100 days (95% CI 93–100 days, n = 106), a level significantly longer than that of repo-Gal4/+ flies (median 59 days, 95% CI 59 - 59 days, n = 51, independent tests of Fig. 2a) (P < 0.0001, Kaplan-Meier multiple group comparisons) or that of UAS-EDTP-RNAi/+ flies (median 83 days, 95% CI 77–83 days, n = 78) (P < 0.0001, Kaplan-Meier multiple group comparisons) (Fig. 2c). Therefore, in the absence of polyQ expression, RNAi knockdown of EDTP in glial cells extended lifespan. 3.5. Effect of pan-neuronal downregulation of EDTP on the survivorship to polyQ protein aggregates and lifespan extension We next explored the effect of neuronal knockdown of EDTP on the survival to polyQ aggregates. A pan-neuronal driver, elav-Gal4 (on chromosome II, #8765), was used for polyQ expression and EDTPRNAi. Compared with glial cells, neurons expressed polyQ aggregates with few large-sized clumps at day 5 and day 22 in flies of Httex1-Q72171
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Fig. 3. EDTP knockdown in pan-neurons had no effect on the improvement of survivorship and lifespan extension. (a) PolyQ aggregates expressed in the brains by elavGal4. The genotypes and ages were indicated. MBC, mushroom body cells; SEZ subesophageal zone; AL, antennal lobe; OL, optic lobe. Bar 200 μm. (b) % PolyQ area at day 5 and day 22 in two groups of flies. (c) Pan-neuronal expression of polyQ proteins shortened lifespan and RNAi knockdown of EDTP failed to improve the survivorship to polyQ aggregates. *** P < 0.0001 by Kaplan-Meier multiple group comparisons. (d) EDTP knockdown had no effect for lifespan extension.
anoxia, however, most flies recovered from the exposure. Once again, both males and females of EDTPEP2390/+ showed the best survivorship after anoxia. EDTPEP2390 was selected for next experiments. Under EDTPEP2390/+ background, the average % PolyQ area was 48.7 ± 2.6% (mean ± SEM, n = 2) at day 10 in flies of UAS-Httex1Q72-eGFP/+; EDTPEP2390/+; repo-Gal4/+. The bright fluorescence throughout the brain was seen in these flies. The average was reduced to 30.5 ± 1.8% (n = 2) at day 19 (P = 0.0283, Student’s t-test) (Fig. 4a and b). Interestingly, levels of % PolyQ area remained comparable with polyQ-expressing flies without EDTP mutation (33.0 ± 0.8% at day 10 vs 30.6 ± 1.3% at day 19) (see Fig. 1). At day 10, a higher level of polyQ aggregates was detected in flies carrying the EDTPEP2390 heterozygous allele than flies without EDTPEP2390 allele (P = 0.0281, Student’s t-test). Therefore, polyQ-expressing flies with the presence of EDTPEP2390 resulted in increased protein aggregation at day 10 compared with flies without the EDTPEP2390 allele. Notably, EDTPEP2390 allele carried a sequence of 14 × UAS in the EP element [24] with the potential to express a truncated EDTP protein. The increased polyQ expression in glia suggested that the co-existence of 14 × UAS had no competing/dilution effect on the availability of Gal4. Median lifespan of flies carrying heterozygous EDTPEP2390 was 18 days (95% CI 16–20 days, n = 103), which was comparable with that of flies carrying no EDTPEP2390 allele (median 18 days, 95% CI 16–20 days, n = 65) (P = 0.572) (Fig. 4c). Thus, EDTPEP2390 failed to improve the survivorship to polyQ aggregates expressed in glial cells.
knockdown in neurons failed to improve the survivorship to polyQ protein aggregates (Fig. S5). There was a possibility that the pan-neuronal driver elav-Gal4 targeted unfavorable cell subsets. We used D42-Gal4, a motoneuron-specific driver, to narrow down the target to a single cell type. PolyQ aggregates were observed mainly in the central region of brains at day 5 and day 22 in flies of Httex1-Q72-eGFP/+;;D42-Gal4/+ (Fig. S6). With RNAi present, % PolyQ area reduced at both day 5 and day 22. This sharply contrasted with the increased % PolyQ area in pan-neurons in the presence of RNAi (see Fig. 3b), and thus supported the notion that unfavorable cell subsets could be targeted by elav-Gal4. 3.6. Effect of EDTP mutation on the survivorship to polyQ protein aggregates To support the effect of EDTP knockdown in glial cells, we examined the potential role for EDTP mutants in the suppression of polyQ protein aggregates. We first screened several available EDTP mutants (including EDTPEP2390, EDTPKG04513, EDTPEY22967, and EDTPMI02389) under prolonged anoxia, a condition mimicking extreme hypoxia that induces autophagy [23]. Heterozygous mutants were tested to avoid the possible confounding effect of a severe motor defect found in homozygous EDTP mutant [8]. With 6 h anoxia, most flies died after exposure. Only a small proportion of EDTPEP2390/+ recovered from the acute exposure and lived for an additional life about 40–100 days (Fig. S7). With 4 h 172
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Fig. 4. Glia-specific expression of polyQ aggregates in the EDTP mutant background. (a) PolyQ aggregates expressed in glial cells of flies carrying EDTPEP2390 heterozygous allele. Extremely bright fluorescence was observed in the brains at day 10. The genotype and ages were shown. Bar 200 μm. (b) % PolyQ area at different ages. P value from the Student’s t-test. (c) EDTP mutation had no effect to improve the survivorship to polyQ aggregates. P value from Log-Rank test.
4. Discussion
HDJ2 [29–31], human Hsp70 [32], yeast Hsp104 [33], baculoviral antiapoptotic protein P35 [34], and modifiers for Spinocerebellar ataxia type 1 (SCA1) [35], are identified targets for the suppression of polyQ protein aggregates. However, there is no previous report of the complete removal of polyQ protein aggregates by targeting these molecules. Therefore, EDTP appears to be a novel molecular target for the potentially complete removal of polyQ protein aggregates in glial cells. The tissue-specificity is critical to elicit the beneficial effects of EDTP downregulation. Through targeting pan-neurons, EDTP knockdown promotes further aggregation of polyQ proteins and worsens the survivorship. If the target cells are highly restricted to motoneurons, we observe the reduced expression of polyQ aggregates again. These findings support that glial cells, and perhaps motoneurons also, are the targets favorable for the beneficial effects of EDTP downregulation. The selected EDTP mutation (EDTPEP2390) does not mimic glia-specific EDTP knockdown in improving the survivorship to polyQ aggregates. EDTPEP2390 allele does have an effect in the improvement of survivorship to prolonged anoxia. Note that this mutant could result in the expression of a truncated EDTP with Gal4 present. This might explain the potential discrepancy of improved survivorship to anoxia verse unchanged survivorship to polyQ aggregates, the former due to EDTP downregulation from a hypomorphic allele and the latter due to a compound effect of EDTP downregulation and targeted expression from the endogenous gene. Additionally, EDTP is essential for oocyte maturation and muscle functions. A global downregulation of EDTP throughout life could have profound consequences such as a promoting effect on polyQ expression. We observed that one RNAi but not the other resulted in the visual disappearance of aggregates, and that two RNAi lines caused different expression dynamics of polyQ protein aggregates. These RNAi lines carry the interference sequences specific to the transcripts of exon 2 and
We provide evidence to support a proposal that selective downregulation of EDTP in non-muscle cells, particularly glial cells, improves survival to toxic cellular wastes and extends lifespan in Drosophila. The lipid phosphatase EDTP regulates the levels of PI3P and PI(3,5) P2. The latter directly activates ryanodine receptors in the sarcoplasmic reticulum in muscles, maintaining the homeostasis of intracellular calcium and normal contractility. A predominant phenotype of EDTP/ MTMR14 mutation is the muscle defect, including impaired motor function (also termed “jumpy” phenotype) in flies, muscle fatigue in mice, and a rare genetic disease called centronuclear myopathy in humans [4,8,9]. PI3P increase and its associated consequences are secondary to muscle dysfunction [25,26]. PI3P recruits effector molecules and initiates autophagy, a process essential for the degradation and recycling of damaged organelles, denatured proteins, and other cellular wastes. The dual-function suggests that selective downregulation of EDTP in non-muscle cells could have benefits in the removal of cellular wastes and lifespan extension. Glial cells in the central nervous system are ideal candidates in this scenario. We found that the expression of polyQ protein aggregates in glial cells shortens lifespan, but clearly, EDTP downregulation in glial cells suppresses the accumulation of aggregates and greatly improves the survivorship. It is striking that polyQ aggregates became invisible at around 30 days in flies with EDTP knockdown (by one RNAi construct targeting the transcripts of exon 3). The consequence of RNAi to EDTP must be sufficient to clear the accumulated aggregates and at the same time to remove the newly synthesized product. Several proteins, including histone deacetylase (HDAC) [27], dHDJ1 (Drosophila homolog to human HSP40) [28], dTPR2 (Drosophila homolog to human tetratricopeptide repeat protein 2) [28], molecular chaperones HDJ1 and 173
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3, respectively. It is possible that these two exons have differential splicing and transcription over time in glial cells. Thus, different consequences of EDTP downregulation could be seen. Furthermore, each RNAi has its potential off-targets. Downregulation of these potential offtargets might confound the effects of EDTP knockdown, resulting in visually complete or incomplete suppression of polyQ aggregates. Although the two RNAi have differential suppression on the polyQ protein aggregates, they have similar effectiveness in the improvement of survivorship. We found a roughly 2-fold increase of maximal lifespan in flies with polyQ protein aggregates and EDTP knockdown. A doubling of survival is remarkable but might have reached the upper limit of the effect of EDTP downregulation. A complete restoration to the normal lifespan might not be expected. In the absence of polyQ protein aggregates, downregulation of EDTP in glial cells extends lifespan. This finding contrasts with the currently existing reports that loss-of-EDTP is lethal in the early development [11] and that flies homozygous for a hypomorphic EDTP mutation are short-lived with impaired motor function and reduced fecundity [8]. Apparently, targeting glial cells for EDTP knockdown causes no or little effect on the normal levels of EDTP in the eggs and muscles. Nevertheless, the effect of lifespan extension has shifted the focus from the commonly detrimental consequences to the beneficial ones. This shift is largely attributed to the selective, rather than ubiquitous, downregulation of EDTP. In conclusion, we demonstrate an approach to finetune the expression of a disease-causing gene EDTP/MTMR14 in glial cells for the suppression of polyQ protein aggregates and lifespan extension in Drosophila.
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Declaration of interest The authors declare that they have no conflict of interest. Acknowledgments The authors are grateful to Dr. R Meldrum Robertson for manuscript editing at early stages and to Dr. Laurent Seroude for providing research materials, comments, and suggestions. This study was funded by Fundamental Research Funds for the Central Universities (30918011308 to S.Q.), Natural Science Foundation of Jiangsu Province (BK20180497 to S.Q.), a grant for Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse of Nanjing University of Science and Technology (NJUST) (No. 30918014102), and the Research Start-up Grant of NJUST (to S.Q.). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.neulet.2018.12.009. References [1] T. Balla, Phosphoinositides: tiny lipids with giant impact on cell regulation, Physiol. Rev. 93 (2013) 1019–1137, https://doi.org/10.1152/physrev.00028.2012. [2] A.L. Marat, V. Haucke, Phosphatidylinositol 3-phosphates — at the interface between cell signalling and membrane traffic, EMBO J. (2016) e201593564. [3] Y. Wu, S. Cheng, H. Zhao, W. Zou, S. Yoshina, S. Mitani, H. Zhang, X. Wang, PI3P phosphatase activity is required for autophagosome maturation and autolysosome formation, EMBO Rep. 15 (2014) 973–981. [4] J. Shen, W.-M. Yu, M. Brotto, J.A. Scherman, C. Guo, C. Stoddard, T.M. Nosek, H.H. Valdivia, C.-K. Qu, Deficiency of MIP/MTMR14 phosphatase induces a muscle disorder by disrupting Ca2+ homeostasis, Nat. Cell Biol. 11 (2009) 769–776, https://doi.org/10.1038/ncb1884. [5] C.D. Touchberry, I.K. Bales, J.K. Stone, T.J. Rohrberg, N.K. Parelkar, T. Nguyen, O. Fuentes, X. Liu, C.-K. Qu, J.J. Andresen, Others, Phosphatidylinositol 3, 5-bisphosphate (PI (3, 5) P2) potentiates cardiac contractility via activation of the ryanodine receptor, J. Biol. Chem. 285 (2010) 40312–40321. [6] I. Vergne, E. Roberts, R.A. Elmaoued, V. Tosch, M.A. Delgado, T. Proikas-Cezanne, J. Laporte, V. Deretic, Control of autophagy initiation by phosphoinositide 3phosphatase jumpy, EMBO J. 28 (2009) 2244–2258, https://doi.org/10.1038/ emboj.2009.159.
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Research article
Downregulation of EDTP in glial cells suppresses polyglutamine protein aggregates and extends lifespan in Drosophila melanogaster
T
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Chengfeng Xiaoa, , Shuang Qiub a
Department of Biology, Queen’s University, Kingston, Ontario, K7L 3N6, Canada Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Xiao Ling Wei 200, Nanjing, 210094, Jiangsu, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Egg-derived tyrosine phosphatase (EDTP) Polyglutamine protein aggregates Autophagy Glial cell Drosophila melanogaster
Drosophila egg-derived tyrosine phosphatase (EDTP) is a lipid phosphatase essential for oogenesis and muscle function. Loss-of-EDTP is lethal at early developmental stages. Hypomorphic mutation of EDTP causes impaired muscle performance and shortened lifespan. Mutation of MTMR14, a mammalian homolog to EDTP, is associated with muscle fatigue in rodents and a rare genetic disease called centronuclear myopathy in humans. Despite the deleterious consequences, downregulation of MTMR14 promotes autophagy. It is proposed that selective downregulation of EDTP/MTMR14 in non-muscle tissues improves the survivorship to cellular wastes and extends lifespan. Here, we show that downregulation of EDTP in glial cells suppressed the expression of polyglutamine (polyQ) protein aggregates and improved survival. Downregulation of EDTP in glial cells also extended lifespan. These effects were not observed by targeting pan-neurons in the nervous system, suggesting the significance of tissue-specificity. Additionally, flies carrying an EDTP mutant had increased survival to prolonged anoxia and altered dynamics of polyQ expression. These data supported the proposal that selective downregulation of EDTP in non-muscle tissues improved survivorship to cellular protein aggregates and extended lifespan. Our findings suggest that EDTP/MTMR14 could be a novel molecular target for the treatment of neurodegeneration.
1. Introduction Drosophila egg-derived tyrosine phosphatase (EDTP) is a lipid phosphatase that specifically removes 3-position phosphate at the inositol rings of two phosphoinositides: phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P2). PI3P and PI(3,5)P2 are important second messengers [1]. PI3P is essential for the initiation of autophagy, a self-degradative process for the removal of damaged organelles, misfolded proteins, and protein aggregates [2,3]. PI(3,5)P2 binds to ryanodine receptors (RyR1 and RyR2) and causes calcium release from the sarcoplasmic reticulum, thus regulating contractility in skeletal and cardiac muscles [4,5]. Downregulation of MTMR14, a mammalian homolog to EDTP, leads to increased PI3P and enhanced autophagy [6]. Mutation of MTMR14 also causes an accumulation of PI(3,5)P2 which disrupts intracellular calcium homeostasis and impairs muscle performance [4,7]. The predominant phenotype of EDTP/MTMR14 mutation is muscle dysfunction such as impaired motor activities in flies [8] or a rare genetic disease called centronuclear myopathy [9]. This can be attributed to the normally abundant
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expression of EDTP/MTMR14 in muscles. Selective downregulation of EDTP/MTMR14 in non-muscular tissues would circumvent the muscle defect. In non-muscle cells, where PI (3,5)P2 has little chance of acting on RyR1 and RyR2, the PI3P-initiated autophagy could become dominant. Conceivably, one could manipulate the expression of EDTP/MTMR14 in favorable tissues for enhanced autophagy and extended lifespan. Glial cells, the major cell types that support neurons in the nervous system, are an ideal candidate for this purpose. There are at least three advantages of targeting glial cells. First, restricted downregulation of EDTP in glial cells causes no or minor disturbance to maternally-derived EDTP expression in the early stages of embryogenesis. Transcription of the repo, a glial cell-specific marker, appears to be highly restricted in glioblasts since stage 9 embryos [10]. EDTP mRNA, however, is detectable uniformly in the cytoplasm of eggs at stage 1, 5 and 11 but quickly disappears from stage 15 [11]. There is little spatial and temporal overlap of transcription between repo and EDTP in early development. Second, targeting glial cells for EDTP downregulation would have no effect on the muscle expression because these two types of cells
Corresponding author at: 116 Barrie Street, Queen’s University, Kingston, ON, K7L 3N6, Canada. E-mail address: [email protected] (C. Xiao).
https://doi.org/10.1016/j.neulet.2018.12.009 Received 2 July 2018; Received in revised form 13 November 2018; Accepted 4 December 2018 Available online 05 December 2018 0304-3940/ © 2018 Elsevier B.V. All rights reserved.
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2.3. Quantification of polyQ protein aggregates
are distally differentiated and distributed. Third, during adult stages, EDTP is reactivated at around day 7, reaching the peak at day 20–30 and followed by a gradual decline after [12,13]. Meanwhile, both the translation of repo and the repo-Gal4-driven expression of mCD8-GFP in glial cells persist for at least 56–63 days [14]. These allow the gliaspecific RNAi knockdown of EDTP to be most efficient in the middle and late life, the stages arguably critical for postponed senescence according to the prevailing pleiotropy theory [15]. We hypothesized that tissue-specific knockdown of EDTP in glial cells improves survival to cellular wastes and extends lifespan in Drosophila. Using the Gal4/UAS expression system [16], we directed the expression of polyglutamine (polyQ) protein aggregates in glial cells. We showed that downregulation of EDTP suppressed the expression of polyQ protein aggregates and this was associated with improved survival. In the absence of polyQ expression, RNAi knockdown of EDTP in glial cells resulted in extended lifespan. These beneficial effects were not observed by targeting pan-neurons in the nervous system. This study supported the proposal that selective downregulation of EDTP/ MTMR14 in non-muscle tissues improved survival to cellular wastes and extended lifespan.
The expression of polyQ protein aggregates in the brain was evaluated by quantifying the percent area of polyQ aggregate (% PolyQ area) in the brain. In general, image stacks were combined into a single plane. The total area of aggregate particles was measured. % PolyQ area was calculated by this formula: % PolyQ area = Cumulative area of aggregates/area of whole brain × 100%. 2.4. Lifespan experiments Adult flies were collected at 0–2 days into fresh food vials at a density of 20–25 flies per vial. They were transferred into fresh medium twice a week. Dead flies were scored during the transfer until all the flies were counted. This procedure was also used to examine the survival of flies expressing polyQ protein aggregates in glial cells, and the survival of flies having a simultaneous expression of polyQ aggregates and RNAi knockdown of EDTP in glial cells. Sample sizes are indicated in the text. 2.5. Anoxia experiments
2. Methods
Flies at 4–6 days were exposed to a prolonged anoxia (4–6 hours, generated by the continuous flow of 100% pure nitrogen gas at around 2 L/min). Dead flies were scored daily during the first week of recovery, then twice a week after.
2.1. Flies Fly strains used in this study and their sources are: repo-Gal4 (Bloomington Drosophila Stock Center (BDSC) #7415), elav-Gal4 (#8765 and #8760), D42-Gal4 (#8816), UAS-EDTP-RNAi (carrying an insertion in the chromosome II with an RNAi sequence CAGTAGTGTA ATAGTAATCAA targeting the transcripts of exon 3 of EDTP, #41633), UAS-EDTP-RNAi (in the chromosome III with a sequence CAGCTACG ACGAAGTCATCAA targeting the transcripts of exon 2, #36917); UASHttex1-Q72-eGFP [17]; UAS-hsp70-myc [18]; 20×UAS-IVSmCD8::GFP (#32194); EDTPEP2390 (#17050); EDTPKG04513 (#13605); EDTPEY22967 (#22600); and EDTPMI02389 (#34481) and w1118 (Dr. Seroude laboratory). We generated a line of UAS-Httex1-Q72-eGFP; repoGal4/M3 for this study. Flies were maintained with the standard medium (cornmeal, agar, molasses, and yeast) under regular conditions (21–23 °C, 50–60% humidity) in a light/dark (12 h/12 h) photoperiod. Male flies were used for experiments unless otherwise indicated.
2.6. Statistics Kaplan-Meier multiple group comparisons with BY adjustments were performed for the survival analysis of three curves. A two-way ANOVA with Bonferroni post-test was used for comparing the % PolyQ area. Statistics were performed using the software R [20]. P < 0.05 was considered statistically significant. 3. Results 3.1. PolyQ protein aggregates expressed in glial cells To address the potential autophagy-promoting effect of EDTP downregulation, it was necessary to establish a fly model expressing cellular protein aggregates in targeted cells. We directed the expression in glial cells of a toxic protein with polyQ tract by using repo-Gal4 [10,21] and UAS-Httex1-Q72-eGFP [17]. Each protein contained 72 tandem repeats of glutamine and a marker of enhanced green fluorescent protein (eGFP). We observed the bright fluorescence in brain tissues at day 1, 5, 10, and 19 of male flies UAS-Httex1-Q72-eGFP/+;; repo-Gal4/+ (Fig. 1 and S1). The fluorescent substrates appeared as varying-sized clumps throughout the brain. Relative large clumps were located typically in the surface region, the median septum, and the connections between the middle brain and optic lobes. Small clumps were evenly distributed. Fluorescent clumps were observed directly from freshly dissected tissues without immunohistochemistry. Collectively, we referred to these fluorescent clumps as polyQ protein aggregates. Female flies of UAS-Httex1-Q72-eGFP/+;; repo-Gal4/+ also expressed polyQ protein aggregates with the distribution characteristics similar to males (Fig. S2). There was no observable polyQ protein aggregate in the brains of control flies (UAS-Httex1-Q72-eGFP/+ and repo-Gal4/+) (Fig. S1). The expression of heat shock protein 70 [18], a soluble molecular chaperone, did not form protein aggregates in glial cells of flies repoGal4/UAS-hsp70-myc. Expression of mCD8-GFP [22], a membraneanchored protein, was evenly distributed without aggregation in the brains in repo-Gal4/20×UAS-IVS-mCD8-GFP. Thus, the formation of protein aggregates was a characteristic feature of the product containing polyQ tract. This provided a valuable opportunity to examine
2.2. Immunofluorescence The immunofluorescent experiments were performed by following a published protocol [19]. Flies and their specific antibodies were: (1) UAS-Httex1-Q72-eGFP/Y;; repo-Gal4/+ flies: primary antibody, mouse anti-GFP supernatant (12A6, DSHB) at 1:20; secondary antibody, Alexa Fluor 488 conjugated goat anti-mouse IgG (115-545-003, Jackson ImmunoResearch) at 1:500. (2) repo-Gal4/UAS-hsp70-myc flies: primary antibody, rabbit anti-c-myc (A00173, GenScript) at 1:50; secondary antibody, DyLight goat anti-rabbit IgG (111-485-144, Jackson ImmunoResearch) at 1:500. (3) repo-Gal4/20×UAS-IVS-mCD8::GFP flies: primary antibody, mouse anti-GFP supernatant (12A6, DSHB) at 1:20; secondary antibody, Alexa Fluor 488 conjugated goat anti-mouse IgG (115-545-003, Jackson ImmunoResearch) at 1:500. Notably, the immunofluorescence was unnecessary for polyQ-expressing flies because the bright fluorescence is readily observable in the freshly dissected tissues. Fresh tissues were mounted on slides with SlowFade Gold antifade reagent for confocal imaging within an hour. During that time the tissue size remained stable. Most images in the current study were taken from freshly prepared tissues without immunofluorescence. We used a Carl Zeiss LSM 710NLO laser scanning confocal/multiphoton microscope (Carl Zeiss) for imaging. The subsequent analysis was performed with ImageJ (NIH). 169
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Fig. 1. EDTP knockdown suppressed the polyQ protein aggregates. (a) Expression of polyQ protein aggregates in glial cells in three groups of flies. Shown are the brains of male flies at the ages of 1, 5, 10, 19, and 38 days. Genotypes are indicated. At day 38, images of UAS-Httex1-Q72-eGFP/+; repo-Gal4/+ are unavailable because no flies survived up to 38 days. PolyQ protein aggregates are invisible in the brains of UAS-Httex1-Q72-eGFP/+; UAS-EDTP-RNAi/+; repo-Gal4/+. Bar 200 μm. (b) Analysis of the percent area of polyQ protein aggregates (% PolyQ area) in three groups of flies. * P < 0.05; ** P < 0.01; *** P < 0.001 by two-way ANOVA with Bonferroni post-tests.
downregulation. Strikingly, polyQ protein aggregates were invisible at day 38 in flies with EDTP knockdown (Fig. 1a). Repeated tests indicated that polyQ protein aggregates became invisible at as early as day 30 (Fig. S3). Using another RNAi line with an interference sequence targeting exon 2 of EDTP (BDSC #36917), we observed persistent expression of polyQ aggregates at all tested ages (day 1, 5, 10, 19, and 38) and increased maximal survival (by around 2-fold) compared with controls. Using double RNAi for EDTP knockdown, we also observed the persistent expression of polyQ aggregates up to day 38 (Figure S4). Clearly, flies with EDTP downregulation in glial cells had improved survival to polyQ aggregates. The percent area of polyQ protein aggregates (% PolyQ area) remained relatively high and stable (26.5–33.2 %) in male flies UASHttex1-Q72-eGFP/+;; repo-Gal4/+ at day 1, 5, 10, and 19 (Fig. 1b).
the effect of EDTP downregulation. 3.2. RNAi knockdown of EDTP suppressed polyQ protein aggregates in glial cells Few males of UAS-Httex1-Q72-eGFP/+;; repo-Gal4/+ survived more than 19 days (Fig. 1a). We examined the effect of EDTP downregulation on the expression of polyQ aggregates in glial cells. EDTP knockdown was conducted with an RNAi line carrying the interference sequence targeting the transcripts of exon 3 (BDSC #41633). PolyQ aggregates were observed at day 1, 5, 10 and 19 in the brains of male flies UAS-Httex1-Q72-eGFP/+; UAS-EDTP-RNAi/+; repo-Gal4/+. These flies lived up to 38 days, which was around a 2-fold increase in the maximal survival compared with flies without EDTP 170
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Average levels of % PolyQ area were reduced at day 1, 5 and 19 but not at day 10 in UAS-Httex1-Q72-eGFP/+;; repo-Gal4/UAS-EDTP-RNAi compared with controls (Two-way ANOVA with Bonferroni post-tests). Average levels of % PolyQ area were reduced at day 1, 10 and 19 in UAS-Httex1-Q72-eGFP/+; UAS-EDTP-RNAi/+; repo-Gal4/+ compared with their controls (Two-way ANOVA with Bonferroni post-tests). At day 5, however, the average level of % PolyQ area in UAS-Httex1Q72-eGFP/+; UAS-EDTP-RNAi/+; repo-Gal4/+ was even higher than that in control (P < 0.05, Two-way ANOVA with Bonferroni posttests). These data indicated altered dynamics of polyQ expression with EDTP downregulation. Similar to males, female flies had likely reduced levels of % PolyQ area at day 1 and day 10 with the presence of RNAi (Fig. S2). In general, we observed that RNAi knockdown of EDTP in glial cells suppressed the expression of polyQ protein aggregates in male flies. 3.3. RNAi knockdown of EDTP in glial cells improved the survival to polyQ protein aggregates The median survival of UAS-Httex1-Q72-eGFP/+;; repo-Gal4/+ flies was 15 days (95% confidence interval (95% CI) 13–17 days, n = 44), which was remarkably shorter than that of repo-Gal4/+ flies (median 101 days, 95% CI 84–113 days, n = 35) (P < 0.0001, KaplanMeier multiple group comparisons) or that of UAS-Httex1-Q72-eGFP/+ flies (median 84 days, 95% CI 76–84 days, n = 62) (P < 0.0001, Kaplan-Meier multiple group comparisons) (Fig. 2a). Thus, the expression of polyQ protein aggregates in glial cells severely shortened the lifespan. Most flies of UAS-Httex1-Q72-eGFP/+;; repo-Gal4/+ died within 20 days. This was consistent with the observation that few flies survived more than 19 days for imaging. We tested the survivorship of flies with polyQ protein aggregates in the presence of EDTP-RNAi in glial cells. The median survival of UASHttex1-Q72-eGFP/+;; repo-Gal4/UAS-EDTP-RNAi (25 days, 95% CI 22–31 days, n = 50) was longer than that of UAS-Httex1-Q72-eGFP/ +;; repo-Gal4/+ flies (18 days, 95% CI 18-18 days, n = 84, tests independent of Fig. 2a) (P < 0.0001, Kaplan-Meier multiple group comparisons). The median survival of UAS-Httex1-Q72-eGFP/+; UASEDTP-RNAi/+; repo-Gal4/+ (27 days, 95% CI 25–31 days, n = 61) was also longer than that of UAS-Httex1-Q72-eGFP/+;; repo-Gal4/+ flies (P < 0.0001, Kaplan-Meier multiple group comparisons) (Fig. 2b). There was no statistical difference of median survival between flies carrying two different RNAi. Therefore, using two independent RNAi for EDTP knockdown in glial cells, we observed consistently improved survivorship to polyQ protein aggregates.
Fig. 2. EDTP knockdown improved the survivorship to polyQ protein aggregates and extended lifespan. (a) Lifespan shortened by the expression of polyQ protein aggregates in glial cells. The survival of UAS-Httex1-Q72-eGFP/ +;; repo-Gal4/+ flies (red) was markedly reduced compared with repo-Gal4/ + (grey) or UAS-Httex1-Q72-eGFP/+ (blue). Male flies were examined. *** P < 0.0001 by Kaplan-Meier multiple group comparisons. (b) EDTP knockdown in glial cells improved survivorship to polyQ protein aggregates. Two independent RNAi lines were used for EDTP knockdown. Data were collected from male flies. *** P < 0.0001 by Kaplan-Meier multiple group comparisons. (c) EDTP knockdown in glial cells extended lifespan. One RNAi line (on chromosome III) was used. *** P < 0.0001 by Kaplan-Meier multiple group comparisons.
eGFP/+; elav-Gal4/+ (Fig. 3a). PolyQ aggregates were distributed mostly in the areas of mushroom body cells (MBC), subesophageal zone (SEZ), antennal lobes (AL), and optic lobes (OL). With RNAi present, % PolyQ area likely increased at day 5 and day 22 (Fig. 3b), indicating that EDTP knockdown in neurons was less effective than in glial cells for the suppression of polyQ aggregates. Males of polyQ-expressing flies had a median lifespan of 53 days (95% CI 44–53 days, n = 27), which was shorter than UAS-Httex1-Q72-eGFP/+ (median 82 days, 95% CI 82 - 82 days, n = 71) (P < 0.0001, Kaplan-Meier multiple comparisons) or elav-Gal4/+ (median 99 days, 95% CI 99–106 days, n = 63) (P < 0.0001, Kaplan-Meier multiple comparisons) (Fig. 3c). Hence, neuronal expression of polyQ aggregates resulted in markedly reduced lifespan. With the presence of RNAi, median lifespan in UAS-Httex1Q72-eGFP/+; elav-Gal4/+; UAS-EDTP-RNAi/+ (36 days) was even shorter than the polyQ-expressing controls (P < 0.0001, Kaplan-Meier test). Thus, downregulation of EDTP in neurons failed to improve the survival to polyQ aggregates. Without polyQ expression, median lifespan was 83 days (95% CI 83–88 days, n = 92) for males of elav-Gal4/ +; UAS-EDTP-RNAi/+, 83 days (95% CI 77–83 days, n = 78) for UASEDTP-RNAi/+, and 88 days (95% CI 88–93 days, n = 57) for elavGal4/+. There was no significant difference between elav-Gal4/+; UAS-EDTP-RNAi/+ and controls (Fig. 3d). Therefore, EDTP knockdown in neurons had no effect on lifespan extension. Using another elav-Gal4 (on chromosome III, #8760), we observed polyQ aggregates expressed mainly in the antennal lobes at day 30. Once again, EDTP
3.4. RNAi knockdown of EDTP in glial cells extended lifespan A key question is whether tissue-specific downregulation of EDTP extends the lifespan in the absence of polyQ aggregates. Median lifespan of repo-Gal4/UAS-EDTP-RNAi flies was 100 days (95% CI 93–100 days, n = 106), a level significantly longer than that of repo-Gal4/+ flies (median 59 days, 95% CI 59 - 59 days, n = 51, independent tests of Fig. 2a) (P < 0.0001, Kaplan-Meier multiple group comparisons) or that of UAS-EDTP-RNAi/+ flies (median 83 days, 95% CI 77–83 days, n = 78) (P < 0.0001, Kaplan-Meier multiple group comparisons) (Fig. 2c). Therefore, in the absence of polyQ expression, RNAi knockdown of EDTP in glial cells extended lifespan. 3.5. Effect of pan-neuronal downregulation of EDTP on the survivorship to polyQ protein aggregates and lifespan extension We next explored the effect of neuronal knockdown of EDTP on the survival to polyQ aggregates. A pan-neuronal driver, elav-Gal4 (on chromosome II, #8765), was used for polyQ expression and EDTPRNAi. Compared with glial cells, neurons expressed polyQ aggregates with few large-sized clumps at day 5 and day 22 in flies of Httex1-Q72171
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Fig. 3. EDTP knockdown in pan-neurons had no effect on the improvement of survivorship and lifespan extension. (a) PolyQ aggregates expressed in the brains by elavGal4. The genotypes and ages were indicated. MBC, mushroom body cells; SEZ subesophageal zone; AL, antennal lobe; OL, optic lobe. Bar 200 μm. (b) % PolyQ area at day 5 and day 22 in two groups of flies. (c) Pan-neuronal expression of polyQ proteins shortened lifespan and RNAi knockdown of EDTP failed to improve the survivorship to polyQ aggregates. *** P < 0.0001 by Kaplan-Meier multiple group comparisons. (d) EDTP knockdown had no effect for lifespan extension.
anoxia, however, most flies recovered from the exposure. Once again, both males and females of EDTPEP2390/+ showed the best survivorship after anoxia. EDTPEP2390 was selected for next experiments. Under EDTPEP2390/+ background, the average % PolyQ area was 48.7 ± 2.6% (mean ± SEM, n = 2) at day 10 in flies of UAS-Httex1Q72-eGFP/+; EDTPEP2390/+; repo-Gal4/+. The bright fluorescence throughout the brain was seen in these flies. The average was reduced to 30.5 ± 1.8% (n = 2) at day 19 (P = 0.0283, Student’s t-test) (Fig. 4a and b). Interestingly, levels of % PolyQ area remained comparable with polyQ-expressing flies without EDTP mutation (33.0 ± 0.8% at day 10 vs 30.6 ± 1.3% at day 19) (see Fig. 1). At day 10, a higher level of polyQ aggregates was detected in flies carrying the EDTPEP2390 heterozygous allele than flies without EDTPEP2390 allele (P = 0.0281, Student’s t-test). Therefore, polyQ-expressing flies with the presence of EDTPEP2390 resulted in increased protein aggregation at day 10 compared with flies without the EDTPEP2390 allele. Notably, EDTPEP2390 allele carried a sequence of 14 × UAS in the EP element [24] with the potential to express a truncated EDTP protein. The increased polyQ expression in glia suggested that the co-existence of 14 × UAS had no competing/dilution effect on the availability of Gal4. Median lifespan of flies carrying heterozygous EDTPEP2390 was 18 days (95% CI 16–20 days, n = 103), which was comparable with that of flies carrying no EDTPEP2390 allele (median 18 days, 95% CI 16–20 days, n = 65) (P = 0.572) (Fig. 4c). Thus, EDTPEP2390 failed to improve the survivorship to polyQ aggregates expressed in glial cells.
knockdown in neurons failed to improve the survivorship to polyQ protein aggregates (Fig. S5). There was a possibility that the pan-neuronal driver elav-Gal4 targeted unfavorable cell subsets. We used D42-Gal4, a motoneuron-specific driver, to narrow down the target to a single cell type. PolyQ aggregates were observed mainly in the central region of brains at day 5 and day 22 in flies of Httex1-Q72-eGFP/+;;D42-Gal4/+ (Fig. S6). With RNAi present, % PolyQ area reduced at both day 5 and day 22. This sharply contrasted with the increased % PolyQ area in pan-neurons in the presence of RNAi (see Fig. 3b), and thus supported the notion that unfavorable cell subsets could be targeted by elav-Gal4. 3.6. Effect of EDTP mutation on the survivorship to polyQ protein aggregates To support the effect of EDTP knockdown in glial cells, we examined the potential role for EDTP mutants in the suppression of polyQ protein aggregates. We first screened several available EDTP mutants (including EDTPEP2390, EDTPKG04513, EDTPEY22967, and EDTPMI02389) under prolonged anoxia, a condition mimicking extreme hypoxia that induces autophagy [23]. Heterozygous mutants were tested to avoid the possible confounding effect of a severe motor defect found in homozygous EDTP mutant [8]. With 6 h anoxia, most flies died after exposure. Only a small proportion of EDTPEP2390/+ recovered from the acute exposure and lived for an additional life about 40–100 days (Fig. S7). With 4 h 172
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Fig. 4. Glia-specific expression of polyQ aggregates in the EDTP mutant background. (a) PolyQ aggregates expressed in glial cells of flies carrying EDTPEP2390 heterozygous allele. Extremely bright fluorescence was observed in the brains at day 10. The genotype and ages were shown. Bar 200 μm. (b) % PolyQ area at different ages. P value from the Student’s t-test. (c) EDTP mutation had no effect to improve the survivorship to polyQ aggregates. P value from Log-Rank test.
4. Discussion
HDJ2 [29–31], human Hsp70 [32], yeast Hsp104 [33], baculoviral antiapoptotic protein P35 [34], and modifiers for Spinocerebellar ataxia type 1 (SCA1) [35], are identified targets for the suppression of polyQ protein aggregates. However, there is no previous report of the complete removal of polyQ protein aggregates by targeting these molecules. Therefore, EDTP appears to be a novel molecular target for the potentially complete removal of polyQ protein aggregates in glial cells. The tissue-specificity is critical to elicit the beneficial effects of EDTP downregulation. Through targeting pan-neurons, EDTP knockdown promotes further aggregation of polyQ proteins and worsens the survivorship. If the target cells are highly restricted to motoneurons, we observe the reduced expression of polyQ aggregates again. These findings support that glial cells, and perhaps motoneurons also, are the targets favorable for the beneficial effects of EDTP downregulation. The selected EDTP mutation (EDTPEP2390) does not mimic glia-specific EDTP knockdown in improving the survivorship to polyQ aggregates. EDTPEP2390 allele does have an effect in the improvement of survivorship to prolonged anoxia. Note that this mutant could result in the expression of a truncated EDTP with Gal4 present. This might explain the potential discrepancy of improved survivorship to anoxia verse unchanged survivorship to polyQ aggregates, the former due to EDTP downregulation from a hypomorphic allele and the latter due to a compound effect of EDTP downregulation and targeted expression from the endogenous gene. Additionally, EDTP is essential for oocyte maturation and muscle functions. A global downregulation of EDTP throughout life could have profound consequences such as a promoting effect on polyQ expression. We observed that one RNAi but not the other resulted in the visual disappearance of aggregates, and that two RNAi lines caused different expression dynamics of polyQ protein aggregates. These RNAi lines carry the interference sequences specific to the transcripts of exon 2 and
We provide evidence to support a proposal that selective downregulation of EDTP in non-muscle cells, particularly glial cells, improves survival to toxic cellular wastes and extends lifespan in Drosophila. The lipid phosphatase EDTP regulates the levels of PI3P and PI(3,5) P2. The latter directly activates ryanodine receptors in the sarcoplasmic reticulum in muscles, maintaining the homeostasis of intracellular calcium and normal contractility. A predominant phenotype of EDTP/ MTMR14 mutation is the muscle defect, including impaired motor function (also termed “jumpy” phenotype) in flies, muscle fatigue in mice, and a rare genetic disease called centronuclear myopathy in humans [4,8,9]. PI3P increase and its associated consequences are secondary to muscle dysfunction [25,26]. PI3P recruits effector molecules and initiates autophagy, a process essential for the degradation and recycling of damaged organelles, denatured proteins, and other cellular wastes. The dual-function suggests that selective downregulation of EDTP in non-muscle cells could have benefits in the removal of cellular wastes and lifespan extension. Glial cells in the central nervous system are ideal candidates in this scenario. We found that the expression of polyQ protein aggregates in glial cells shortens lifespan, but clearly, EDTP downregulation in glial cells suppresses the accumulation of aggregates and greatly improves the survivorship. It is striking that polyQ aggregates became invisible at around 30 days in flies with EDTP knockdown (by one RNAi construct targeting the transcripts of exon 3). The consequence of RNAi to EDTP must be sufficient to clear the accumulated aggregates and at the same time to remove the newly synthesized product. Several proteins, including histone deacetylase (HDAC) [27], dHDJ1 (Drosophila homolog to human HSP40) [28], dTPR2 (Drosophila homolog to human tetratricopeptide repeat protein 2) [28], molecular chaperones HDJ1 and 173
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3, respectively. It is possible that these two exons have differential splicing and transcription over time in glial cells. Thus, different consequences of EDTP downregulation could be seen. Furthermore, each RNAi has its potential off-targets. Downregulation of these potential offtargets might confound the effects of EDTP knockdown, resulting in visually complete or incomplete suppression of polyQ aggregates. Although the two RNAi have differential suppression on the polyQ protein aggregates, they have similar effectiveness in the improvement of survivorship. We found a roughly 2-fold increase of maximal lifespan in flies with polyQ protein aggregates and EDTP knockdown. A doubling of survival is remarkable but might have reached the upper limit of the effect of EDTP downregulation. A complete restoration to the normal lifespan might not be expected. In the absence of polyQ protein aggregates, downregulation of EDTP in glial cells extends lifespan. This finding contrasts with the currently existing reports that loss-of-EDTP is lethal in the early development [11] and that flies homozygous for a hypomorphic EDTP mutation are short-lived with impaired motor function and reduced fecundity [8]. Apparently, targeting glial cells for EDTP knockdown causes no or little effect on the normal levels of EDTP in the eggs and muscles. Nevertheless, the effect of lifespan extension has shifted the focus from the commonly detrimental consequences to the beneficial ones. This shift is largely attributed to the selective, rather than ubiquitous, downregulation of EDTP. In conclusion, we demonstrate an approach to finetune the expression of a disease-causing gene EDTP/MTMR14 in glial cells for the suppression of polyQ protein aggregates and lifespan extension in Drosophila.
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Declaration of interest The authors declare that they have no conflict of interest. Acknowledgments The authors are grateful to Dr. R Meldrum Robertson for manuscript editing at early stages and to Dr. Laurent Seroude for providing research materials, comments, and suggestions. This study was funded by Fundamental Research Funds for the Central Universities (30918011308 to S.Q.), Natural Science Foundation of Jiangsu Province (BK20180497 to S.Q.), a grant for Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse of Nanjing University of Science and Technology (NJUST) (No. 30918014102), and the Research Start-up Grant of NJUST (to S.Q.). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.neulet.2018.12.009. References [1] T. Balla, Phosphoinositides: tiny lipids with giant impact on cell regulation, Physiol. Rev. 93 (2013) 1019–1137, https://doi.org/10.1152/physrev.00028.2012. [2] A.L. Marat, V. Haucke, Phosphatidylinositol 3-phosphates — at the interface between cell signalling and membrane traffic, EMBO J. (2016) e201593564. [3] Y. Wu, S. Cheng, H. Zhao, W. Zou, S. Yoshina, S. Mitani, H. Zhang, X. Wang, PI3P phosphatase activity is required for autophagosome maturation and autolysosome formation, EMBO Rep. 15 (2014) 973–981. [4] J. Shen, W.-M. Yu, M. Brotto, J.A. Scherman, C. Guo, C. Stoddard, T.M. Nosek, H.H. Valdivia, C.-K. Qu, Deficiency of MIP/MTMR14 phosphatase induces a muscle disorder by disrupting Ca2+ homeostasis, Nat. Cell Biol. 11 (2009) 769–776, https://doi.org/10.1038/ncb1884. [5] C.D. Touchberry, I.K. Bales, J.K. Stone, T.J. Rohrberg, N.K. Parelkar, T. Nguyen, O. Fuentes, X. Liu, C.-K. Qu, J.J. Andresen, Others, Phosphatidylinositol 3, 5-bisphosphate (PI (3, 5) P2) potentiates cardiac contractility via activation of the ryanodine receptor, J. Biol. Chem. 285 (2010) 40312–40321. [6] I. Vergne, E. Roberts, R.A. Elmaoued, V. Tosch, M.A. Delgado, T. Proikas-Cezanne, J. Laporte, V. Deretic, Control of autophagy initiation by phosphoinositide 3phosphatase jumpy, EMBO J. 28 (2009) 2244–2258, https://doi.org/10.1038/ emboj.2009.159.
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