Downregulation of Plasma Membrane Ca2+ ATPase driven by tyrosine hydroxylase-Gal4 reduces Drosophila lifespan independently of effects in neurons

Abstract

In Drosophila melanogaster, several Gal4 drivers are used to direct gene/RNAi expression to different dopaminergic neuronal clusters. We previously developed a fly model of Parkinson's disease, in which dopaminergic neurons had elevated cytosolic Ca²⁺ due to the expression of a Plasma Membrane Ca²⁺ ATPase (PMCA) RNAi under the thyroxine hydroxylase (TH)-Gal4 driver. Surprisingly, TH-Gal4>PMCA^RNAi flies died earlier compared to controls and showed swelling in the abdominal area. Flies expressing the PMCA^RNAi under other TH drivers also showed such swelling and shorter lifespan. Considering that TH-Gal4 is also expressed in the gut, we proposed to suppress the expression specifically in the nervous system, while maintaining the activation in the gut. Therefore, we expressed Gal80 under the direction of the panneuronal synaptobrevin (nSyb) promoter in the context of TH-Gal4. nSyb-Gal80; TH-Gal4>PMCA^RNAi flies showed the same reduction of survival as TH-Gal4>PMCA^RNAi flies, meaning that the phenotype of abdomen swelling and reduced survival could be due to the expression of the PMCA^RNAi in the gut. In perimortem stages TH-Gal4>PMCA^RNAi guts had alteration in the proventriculi and crops. The proventriculi appeared to lose cells and collapse on itself, and the crop increased its size several times with the appearance of cellular accumulations at its entrance. No altered expression or phenotype was observed in flies expressing PMCA^RNAi in the dopaminergic PAM cluster (PAM-Gal4>PMCA^RNAi). In this work we show the importance of checking the global expression of each promoter and the relevance of the inhibition of PMCA expression in the gut.

Keywords: Calcium; GAL4-UAS; GAL80; dopaminergic neurons; flies; gut.

Figure 1.

The expression of PMCA^RNAi under dopaminergic drivers, with the exception of PAM-Gal4 decreased flies’ lifespan. Diagrams show the brain dopaminergic neurons in which each promoter is expressed. a, TH-Gal4 is active in several dopaminergic clusters in the anterior and posterior regions of the brain. The expression of TH-Gal4>PMCA^RNAi markedly reduced lifespan compared to controls. b and c, TH-F1-Gal4 and TH-D4-Gal4 are active in 28 and 22 neurons, respectively, and, when used to drive expression of PMCA^RNAi, also presented a lower survival rate, but higher than TH-Gal4>PMCA^RNAi flies. d, Flies expressing PMCA^RNAi under the PAM-Gal4 promoter showed only minor differences in lifespan compared to its controls (with a mean lifespan similar to most other controls). All curves were analysed with the Log-rank Mantel Cox test and each curve was replicated 2 or 3 times.

Figure 2.

TH-Gal4 was expressed in the digestive tract. a, Schematic representation of the central nervous system and digestive tract of Drosophila melanogaster (VNC: ventral nerve cord). Microscopy images of the gut from TH-Gal4/+ and TH-Gal4>mcherry flies: b-c, Bright field; d-e, mCherry fluorescence; and f-g, merge. Red fluorescence was detected only in the proventriculus and hindgut (arrows, e and g) from TH-Gal4>mcherry flies. Scale bar: 250 µm.

Figure 3.

TH-F1-Gal4 and TH-D4-Gal4, but not PAM-Gal4, also showed expression in the gut. Confocal images showing expression of EGFP driven by TH-Gal4, TH-F1-Gal4, TH-D4-Gal4 and PAM-Gal4 in brain (a-d), VNC (e-h), proventriculus (i-l), crop (m-p) and hindgut (q-t). VNC: central nervous cord. Scale bar: d, h and p, 100 µm; l, 50 µm and t, 200 µm.

Figure 4.

TH-Gal4>PMCA^RNAi flies showed a perimortem swelling of the abdomen and suppressing neuronal expression of PMCA^RNAi did not prevent the shortening of their lifespan. a, Abdominal swelling phenotype in 10 day-old TH-Gal4>PMCA^RNAi flies, compared to TH-Gal4/+ and UAS PMCA^RNAi/+ controls. b-i, Confocal images showing that nSyb-Gal80 suppresses TH-Gal4 driven EGFP expression in the brain (b,c) and the VNC (d,e), but not in the proventriculus (f,g) and hindgut (h,i). j, the lifespan curve of nSyb-Gal80;TH-Gal4>PMCA^RNAi flies was similar to that of TH-Gal4>PMCA^RNAi flies. Scale bars: 100 µm.

Figure 5.

10-days-old, but not 2-days-old, TH-Gal4>PMCA^RNAi flies presented an increase in crop size and abnormal accumulation of cells at the entrance of the crop. a-b, Bright field microscopy images of the crops from 2 (a) and 10-days-old (b) TH-Gal4/+ (control) and TH-Gal4>PMCA^RNAi flies. c, Quantification of crop sizes (mm²) showed no difference at 2 days of age, whereas at 10 days, those from TH-Gal4>PMCA^RNAi flies were about 10 times larger than those from TH-Gal4/+ controls. d-i, Confocal microscopy images of the crop entrance (dotted lines) from TH-Gal4>egfp/mCherry (d-f) and TH-Gal4>EGFP/PMCA^RNAi (g-i). The latter showed fragmented nuclei (g) and increased EGFP fluorescence (h, merge in i) compared to the former (control). Nuclei outside the dotted line belong to a different section of the intestine that was left next to the crop in the dissection. Two-Way ANOVA Tukey’s multiple comparisons test; 10-days-old TH-Gal4>PMCA^RNAi versus 10-days-old TH-Gal4/+, 2-days-old TH-Gal4/+ and 2-days-old TH-Gal4>PMCA^RNAi, ****p > 0,0001. Scale bar: 300 µm.

Figure 6.

10-days-old TH-Gal4>PMCA^RNAi flies showed shrinking and cell loss in the proventriculus. a-c, Bright field microscopy images of proventriculi (arrows) from TH-Gal4/+ (a), TH-Gal4>egfp/mCherry (b) and TH-Gal4>EGFP/PMCA^RNAi (c) 10-days-old flies. The latter exhibited perimortem collapsing of the proventriculus, which was not evident in the controls. The diameter (indicated with a red line) of the proventriculus shown were 218,48 µm (a), 220.82 µm (b) and 131.93 µm (c). d-i, Confocal images of proventriculi from TH-Gal4>egfp/mCherry (d-f) and TH-Gal4>EGFP/PMCA^RNAi (g-i) flies, which presented a decreased number of nuclei and EGFP fluorescence. f and i show mCherry fluorescence, represented in magenta (no fluorescence shown in TH-Gal4>EGFP/PMCA^RNAi flies as they do not express mCherry). Scale bar: c, 500 µm; i, 50 µm.