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. 2020 Dec;36(12):1552-1557.
doi: 10.1007/s12264-020-00562-y. Epub 2020 Aug 16.

Pgant4 and Tango1 Mediate Anoxia and Reoxygenation Injury

Qingqing Du  1   2 , Nastasia K H Lim  3   4   5 , Yiling Xia  1   2 , Wangchao Xu  6 , Qichao Zhang  4   7 , Liyao Zhang  4   7 , Fude Huang  8   9   10 , Wenan Wang  11   12   13
Affiliations

Pgant4 and Tango1 Mediate Anoxia and Reoxygenation Injury

Qingqing Du et al. Neurosci Bull. 2020 Dec.
No abstract available

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Figures

Fig. 1
Fig. 1
Genetic reduction of Pgant4 increases anoxia resistance and decreases active caspase-3 levels in the brain of Drosophila exposed to anoxia-reoxygenation. A List of Drosophila deficiency lines in a genetic screen for increased resistance to anoxia-reoxygenation. The Bloomington (BL) stock number and the deleted region in the chromosome are indicated. BL stock numbers 6965, 7675, and 7681 (indicated by asterisks) exhibit reduced mortality in the anoxia-reoxygenation assay. A hundred flies were screened for each line. B Relative chromosomal map of four deficient lines used to identify the Pgant4 gene. C (i) Deficient lines BL numbers 6965, 7787, and 6507, but not 9599, show increased resistance to anoxia-reoxygenation, compared with w1118 flies (Ctrl). (ii) Cumulative deaths 5 days after anoxia exposure (for clearer comparison between groups). D (i) Cumulative death rates of Ctrls and flies with different combinations of the Pgant4f02186 allele and Pgant4rev. (ii) Cumulative deaths 5 days after anoxia exposure. E (i) Cumulative death rates of Ctrl and Gal4 flies ([Gal4]actin-2/+ and [Gal4]elav/+) just expressing the transcription factor Gal4, and (ii) Ctrls (as in E i) and flies with Pgant4 knocked down ubiquitously (Pgant4v7286/[Gal4]actin-2 and Pgant4v46910/[Gal4]actin-2) or just in neurons (Pgant4v7286/[Gal4]elav), and UAS-transgenic control flies (Pgant4v7286/+ and Pgant4v46910/+). The UAS flies harbor the transgene for expressing double-stranded RNA against Pgant4, but this expression requires Gal4. Notably, the cumulative death rate of [Gal4]actin-2/+ is significantly higher than that of Ctrl; this might be due to the homozygous lethal insertion of the [Gal4]actin-2 transgene into a locus unrelated to Pgant4. In C–E, the cumulative death rates of flies exposed to 3 h of anoxia before recovery in normoxic conditions are analyzed over the next 5 days (represented as cumulative death percentage). Error bars, ± SEM; n = 100–200 flies/group; *P < 0.05, **P < 0.01 vs Ctrl. F, Above, representative images of active caspase-3 immunofluorescence in Ctrl, Pgant4 homozygous mutants (Pgant4f02186/f02186), and revertant Pgant4 (Pgant4rev/rev) flies after 3 h of anoxia exposure and 3 h recovery (white squares, areas used to quantify the active caspase-3 level in each hemisphere of the fly brain; images represent 5–10 fly heads/group; scale bars, 50 µm). Below, fluorescence intensity relative to Ctrl, Error bars, ± SEM; * P < 0.05, ** P < 0.01, one-way ANOVA
Fig. 2
Fig. 2
Anoxia-reoxygenation markedly increases the glycosylated and total Tango1, which is suppressed by downregulation of Pgan4. A Immunoblots showing the specificity and recognition of Drosophila Tango1 by anti-MIA3 antibody. The anti-MIA3 antibody detects a band at 250 kDa, the correct molecular weight for Tango1, but with a different density in control w1118 (Ctrl) flies, Pgant4 homozygous mutants (Pgant4f02186/f02186), with Tango1 knocked down in neurons (Tango1RNAi), and flies with Tango1 overexpression (Tango1OE). Tubulin served as loading control; n = 30–40 fly heads/group. B Representative immunoblots and levels of o-glycosylated Tango1 (Glyc-Tango1) and total Tango1 (Tango1) in Ctrl (left) and Pgant4f02186/f02186 (right) flies after exposure to anoxia for 0, 1, or 3 h, or exposure to 3 h of anoxia before recovery in 3 h under normoxic conditions. Tubulin served as loading control. Each experiment was repeated at least three times; n = 30–40 fly heads/group. C Cumulative death rates of Ctrl, [Gal4]elav (Elav/+), Tango1RNAi, and Tango1OE flies. Data for Ctrl flies is the same as in Fig. 1D. D (i) Cumulative death rates of Ctrl, deficient (9672/+, 9615/+, and 6465/+) and transposon-insertion mutants of Tango1 (Tango1k14206A/+ and Tango1k14206B/+). (ii) Cumulative deaths 5 days after anoxia exposure. In C and D, n = 100–200 flies/group. E Above, representative images of active caspase-3 immunofluorescence after 3 h of anoxia and 3 h recovery in Ctrl, Tango1RNAi, and Tango1OE flies (white squares, areas used to quantify caspase-3 levels in each hemisphere; scale bars, 50 µm), Below, fluorescence intensity relative to Ctrl. n = 5–10 fly heads/group); error bars, ± SEM; *P < 0.05, **P < 0.01, *** P < 0.001, one-way ANOVA
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