Flies deficient in dPGAM5 are vulnerable to HS.

<p>(<b>A</b>) <i>PGAM5¹</i> flies are vulnerable to HS. Survival curves of adult male control (<i>y¹w¹/Y</i>) and <i>PGAM5¹</i> flies (<i>y¹</i>, <i>PGAM5¹/Y</i>) subjected to oxidative stress (0.1% H₂O₂, <i>n</i> = 66; left), starvation (<i>n</i> = 180; middle), or HS (37°C, <i>n</i> = 190; right) are shown. Control vs. <i>PGAM5¹</i> in HS, <i>p</i><0.0001 by the log-rank test. (<b>B</b>) dPGAM5 and dPINK1 act independently in HS response. Survival curves of the indicated adult male flies (day 5–15) subjected to HS are shown (control, <i>n</i> = 120; <i>PGAM5¹</i>, <i>n</i> = 112; <i>PINK1^B9</i>, <i>n</i> = 105; <i>PINK1^B9, PGAM5¹</i>, <i>n</i> = 116). <i>PINK1^B9</i> vs <i>PGAM5¹, PINK1^B9</i>, p<0.0001 by the log-rank test. The genotypes are revertant <i>PINK1^REV/Y</i> (Control), <i>PGAM5¹/Y</i> (<i>PGAM5¹</i>), <i>PINK1^B9/Y</i> (<i>PINK1^B9</i>) and <i>PGAM5¹, PINK1^B9/Y</i> (<i>PGAM5¹, PINK1^B9</i>). (<b>C</b>) Efficiency of IR-mediated <i>dPGAM5</i> knockdown. Protein expression of dPGAM5 in <i>daughterless (da)>LacZ IR</i> flies (<i>da-GAL4/+; UAS-LacZ IR/+</i>) and <i>da>dPGAM5 IR</i> flies (<i>da-GAL4/+</i>; <i>UAS-dPGAM5 IR/+</i>) was detected by immunoblotting with dPGAM5 antibody. Actin was also detected as a loading control. (<b>D</b>) Knockdown of <i>dPGAM5</i> in the whole body induces vulnerability of flies to HS. Survival curves of adult male <i>da>LacZ IR</i> flies and <i>da>dPGAM5 IR</i> flies subjected to HS are shown (<i>n</i> = 110). <i>p</i><0.0001 by the log-rank test. (<b>E</b>) Transient knockdown of <i>dPGAM5</i> prior to HS is sufficient to induce vulnerability of flies to HS. Survival curves of the indicated adult male flies subjected to HS are shown. IR RNA was induced two days prior to sustained HS by two cycles of HS pretreatment, each of which was composed of 37°C for 30 min, 25°C for 5 hr, 37°C for 30 min and 25°C for 18 hr. “Pre-HS +” and “pre-HS −” indicate flies expressing and not expressing IR RNA, respectively, when they were subjected to sustained HS. The genotypes are <i>hs-GAL4/UAS-LacZ-IR</i> (<i>hs>LacZ-IR</i>) and <i>hs-GAL4/UAS-dPGAM5-IR</i> (<i>hs>dPGAM5-IR</i>). <i>hs>LacZ-IR</i> (pre-HS +) flies (<i>n</i> = 79), <i>hs>dPGAM5-IR</i> (pre-HS +) flies (<i>n</i> = 68), <i>hs>LacZ-IR</i> (pre-HS −) flies (<i>n</i> = 50) and <i>hs>dPGAM5-IR</i> (pre-HS −) flies (<i>n</i> = 45) were examined. <i>hs>LacZ-IR</i> (pre-HS +) vs <i>hs>dPGAM5-IR</i> (pre-HS +), p<0.0001 by the log-rank test.</p

HS induces a rapid degeneration of the nuclei of the MB neurons in the absence of dPGAM5.

<p>The number of cell bodies of the KCs decreases in HS-treated <i>PGAM5¹</i> flies. Representative images of lobes (<b>A</b>–<b>D</b>) and calyxes (<b>E</b>–<b>H</b>) visualized by mCD8::GFP are shown. The genotypes are <i>c739-GAL4/+; UAS-mCD8::GFP/+</i> (Control) and <i>PGAM5¹</i>/<i>Y; c739</i>-<i>Gal4</i>/+; <i>UAS</i>-<i>mCD8::GFP</i>/+ (<i>PGAM5¹</i>). Scale bar (<b>A</b>–<b>H</b>) = 50 µm. Representative images of the KC nuclei visualized by Histone2B::ECFP (<b>I</b>–<b>L</b>; scale bar = 20 µm) and the total number of KC nuclei (<b>M</b>) are shown. The latter was calculated by summing up the number counted manually in every tenth section, and the results were shown as the mean cell number per fly ± s.e.m. [<i>n</i> = 6, Control HS (−) and (+) and <i>PGAM5¹</i> HS (−); <i>n</i> = 8, <i>PGAM5¹</i> HS (+)]. ** P<0.01, unpaired t-test (<b>M</b>). The genotypes are <i>c739-GAL4</i>/<i>UAS-Histone2B::ECFP</i> (Control) and <i>PGAM5¹</i>/<i>Y</i>; <i>c739-GAL4</i>/<i>UAS-Histone2B::ECFP</i> (<i>PGAM5¹</i>).</p

Locomotor activity is reduced in dPGAM5-deficient flies.

<p><i>PGAM5¹</i> flies are less active than control flies under both HS (37°C; <b>A</b>) and unstressed (25°C; <b>B</b>) conditions. Locomotor activity was monitored using the <i>Drosophila</i> activity monitor (DAM) system as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030265#s4" target="_blank">Materials and Methods</a>. Data are shown as the mean counts ± s.e.m of total flies examined for each genotype [Control, <i>n</i> = 77 and 79; <i>PGAM5¹</i>, n = 76 and 78; at 37°C and 25°C, respectively]. The genotypes are <i>y¹w¹/Y</i> (Control) and <i>PGAM5¹/Y</i> (<i>PGAM5¹</i>).</p

dPGAM5 exerts its protective effect against HS by preventing apoptosis in the MB.

<p>(<b>A–F</b>) TUNEL-positive KCs are detected in the MB of HS-treated <i>PGAM5¹</i> flies, but not in that of control flies. TUNEL staining of the MB of control flies (<b>A</b>) and <i>PGAM5¹</i> flies (<b>B</b>) treated with HS for 75 min is shown. Counter nuclear staining with Hoechst33258 (<b>C</b> and <b>D</b>) and merged images of TUNEL and Hoechst 33258 staining (<b>E</b> and <b>F</b>) are also shown. Scale bar (<b>A</b>–<b>F</b>) = 20 µm. The genotypes are <i>c739-GAL4/UAS-Histone2B::ECFP</i> (Control) and <i>PGAM5¹/Y; c739-GAL4/UAS-Histone2B::ECFP</i> (<i>PGAM5¹</i>). (<b>G</b>) Expression of p35 in the MB attenuates the vulnerability of <i>PGAM5¹</i> flies to HS. Survival curves of the indicated adult male flies subjected to HS are shown (<i>n</i> = 105). <i>PGAM5¹</i> vs. <i>PGAM5¹; c739>p35</i>, <i>p</i><0.0001 by the log-rank test. The genotypes are <i>c739-GAL4/+</i> (Control), <i>PGAM5¹/Y; c739-GAL4/+</i> (<i>PGAM5¹</i>) and <i>PGAM5¹/Y; c739-GAL4/+; UAS-p35/+</i> (<i>PGAM5¹; c739>p35</i>). (<b>H</b>) Expression of p35 in the MB does not affect the response of control flies to HS. Survival curves of the indicated adult male flies subjected to HS are shown (<i>n</i> = 130). The genotypes are <i>c739-GAL4/+</i> (Control) and <i>c739-GAL4/+; UAS-p35/+</i> (Control; <i>c739>p35</i>).</p

The MB gains a toxic function, rather than loses a protective function, in response to HS in the absence of dPGAM5.

<p>(<b>A</b>) Ablation of the MB. Ablation of the MB in adult flies pretreated with hydroxyurea (HU) at the larval stage was confirmed by MB-specific expression of mCD8::GFP. Scale bar = 50 µm. The genotype is <i>c739-GAL4/+; UAS-mCD8::GFP/+</i>. (<b>B</b>) Ablation of the MB attenuates the vulnerability of <i>PGAM5¹</i> flies to HS. Survival curves of adult male <i>PGAM5¹</i> flies (<i>y¹</i>, <i>PGAM5¹/Y</i>) untreated (−) or treated (+) with HU are shown (<i>n</i> = 110). <i>p</i><0.0001 by the log-rank test. (<b>C</b>) Ablation of the MB does not affect the response of control flies to HS. Survival curves of adult male control (<i>y¹w¹/Y</i>) flies untreated (−) or treated (+) with HU are shown (<i>n</i> = 110). (<b>D</b>) Suppression of neurotransmission does not decrease vulnerability of <i>PGAM5¹</i> flies to HS. Survival curves of the indicated adult male flies (day 9–15) subjected to HS are shown. <i>c739-GAL4</i> flies (<i>+/Y; c739-GAL4/+; Sb/+</i>, <i>n</i> = 91), <i>c739>shi^TS</i> flies (<i>+/Y; c739-GAL4/+; UAS- shi^TS/+</i>, <i>n</i> = 100), <i>PGAM5¹, c739-GAL4</i> flies (<i>PGAM5¹/Y; c739-GAL4/+; Sb/+</i>, <i>n</i> = 100) and <i>PGAM5¹, c739>shi^TS</i> flies (<i>PGAM5¹/Y; c739-GAL4/+; UAS-shi^TS/+</i>, <i>n</i> = 100) were examined.</p

DEVDase is activated in a subset of ORNs in an age-dependent manner.

<p>(A) Percentages of aged brains (45-days-old) with cPAPR signals are shown. mCD8::PARP::Venus expression by various <i>Or-Gal4</i> drivers revealed that DEVDase is activated in a subset of ORNs in an age-dependent manner. Number on each column indicates the number of brains examined. (B, C) Representative images of aged fly brains expressing mCD8::PARP::Venus by <i>Or42b-Gal4</i> (B) or <i>Or85a-Gal4</i> (C). Note that cPARP signal was frequently observed in the axons of Or42b neurons (B) while it was rare in axons of Or85a neurons (C). cPARP signal, mCD8::PARP::Venus expression, and nc82 staining are shown in magenta, green, and blue, respectively. Broken lines indicate outlines of ALs. Scale bar: 75 µm. Genotypes: (A) Or42b: <i>Or42b-Gal4/+;UAS-mCD8::PARP::Venus/+</i>. Or92a: <i>Or92b-Gal4/UAS-mCD8::PARP::Venus;UAS-mCD8::PARP::Venus/+</i>. Or35a: <i>Or35a-Gal4/+;UAS-mCD8::PARP::Venus/+</i>. Or47b: <i>Or47b-Gal4/+;UAS-mCD8::PARP::Venus/+</i>. Or22a: <i>Or22a-Gal4/+;UAS-mCD8::PARP::Venus/+</i>. Or85a: <i>Or85a-Gal4/+;UAS-mCD8::PARP::Venus/+</i>. Or67d: <i>Or67d-Gal4, yw/+;;UAS-mCD8::PARP::Venus/+</i>. Or42a: <i>Or42a-Gal4/+;UAS-mCD8::PARP::Venus/+</i>. Or47a: <i>Or47aGal4/+;UAS-mCD8::PARP::Venus/TM2 or TM6B</i>. Or43b: <i>Or43b-Gal4/+;UAS-mCD8::PARP::Venus/+</i>. Or69a: <i>Or69a-Gal4/+;UAS-mCD8::PARP::Venus/+</i>. Or9a: <i>Or9a-Gal4/+;UAS-mCD8::PARP::Venus/+</i>. Or67a: <i>Or67aGal4/+;UAS-mCD8::PARP::Venus/+</i>. Or59b: <i>Or59b-Gal4, w/+;UAS-mCD8::PARP::Venus/+;UAS-mCD8::PARP::Venus/+</i>. Or67b: <i>Or67b-Gal4/UAS-mCD8::PARP::Venus</i>. Or98a: <i>Or98aGal4/+;UAS-mCD8::PARP::Venus/+</i>. Or10a: <i>Or10a-Gal4/+;UAS-mCD8::PARP::Venus/TM2 or TM6B</i>. (B) <i>Or42b-Gal4/+;UAS-mCD8::PARP::Venus/+.</i> (C) <i>Or85a-Gal4/+;UAS-mCD8::PARP::Venus/+</i>.</p

Caspase Inhibition in Select Olfactory Neurons Restores Innate Attraction Behavior in Aged <i>Drosophila</i>

<div><p>Sensory and cognitive performance decline with age. Neural dysfunction caused by nerve death in senile dementia and neurodegenerative disease has been intensively studied; however, functional changes in neural circuits during the normal aging process are not well understood. Caspases are key regulators of cell death, a hallmark of age-related neurodegeneration. Using a genetic probe for caspase-3-like activity (DEVDase activity), we have mapped age-dependent neuronal changes in the adult brain throughout the lifespan of <i>Drosophila</i>. Spatio-temporally restricted caspase activation was observed in the antennal lobe and ellipsoid body, brain structures required for olfaction and visual place memory, respectively. We also found that caspase was activated in an age-dependent manner in specific subsets of <i>Drosophila</i> olfactory receptor neurons (ORNs), Or42b and Or92a neurons. These neurons are essential for mediating innate attraction to food-related odors. Furthermore, age-induced impairments of neural transmission and attraction behavior could be reversed by specific inhibition of caspase in these ORNs, indicating that caspase activation in Or42b and Or92a neurons is responsible for altering animal behavior during normal aging.</p></div

Spatio-temporal activation of DEVDase in adult <i>Drosophila</i> brains.

<p>(A) DEVDase activity detection with mCD8::PARP::Venus. Human anti-cPARP antibodies specifically recognize the N-terminal amino acid sequences of Venus that are generated by the cleavage of mCD8::PARP::Venus. (B) Percentages of brain samples with any cPARP signal at each time point are shown. “n” indicates the number of brains examined. (C, D) cPARP signals in young fly brains (1-day-old). A brain with cPARP signal near midline and the subesophageal ganglia (SOG) (C) and only near midline, without intense signals in the SOG (D). Circles of broken lines are antennal lobes (ALs). cPARP signal and mCD8::PARP::Venus expression are shown in magenta and green, respectively. Scale bar: 50 µm. (E) Schematic drawing of a <i>Drosophila</i> adult brain. The regions outlined by broken lines are ALs and SOGs. The ellipsoid body (EB) is located on the dorsal side of the AL. OL: optic lobe. (F) Graph indicating the percentage of young brains with cPARP signals. Genotypes: (B–D, F) <i>elav-Gal4;;UAS-mCD8::PARP::Venus.</i></p

Stereotyped DEVDase activation in the AL and EB structures of aged <i>Drosophila</i> brains.

<p>(A, B, D, E) Representative aged brains (45-days-old) bearing cPARP signals (DEVDase activity) in the dorso-medial side of the AL (A), the EB structure (B), the mushroom body (MB) (D) and the local interneuron (LN) of the AL (E). mCD8::PARP::Venus was expressed in most postmitotic neurons (<i>elav-Gal4;;UAS-mCD8::PARP::Venus</i>). Circles of broken lines are ALs. cPARP signal and mCD8::PARP::Venus expression are shown in magenta and green, respectively. Scale bar: 75 µm. (C) Percentages of brains with cPAPR signal in the EB and AL at each time point are shown. “n” indicates the number of brains examined. Genotypes: (A–E) <i>elav-Gal4;;UAS-mCD8::PARP::Venus.</i></p