Page 143 - AN-3-4

Advanced Neurology Drosophila Sirtuin 1 and Alzheimer’s disease

F

A B C D E

G H I

Figure 7. Downregulation of Notch and overexpression of Sirt1 modulate Alzheimer’s disease-related pathologies and RT-PCR analysis of Grim, Reaper, Hid,
and DIAP1 in Drosophila. (A-E) Light microscopy images of 10-day-old AD model flies: GMR-GAL4-UAS-Tau /+;+/+ (A); with Notch downregulation

WT
(single copy) (B); Notch downregulation (double copy) (C); Sirt1 overexpression with Notch downregulation (single copy) (D); and Notch downregulation
(double copy) (E). Scale bar indicates 100 μm (A-E). The yellow-highlighted area represents degenerated areas in the fly eye. (F) Box-and-whisker plot
displaying phototaxis activity (expressed as light preference index) of 10-day-old adult flies: GMR-GAL4/+;+/+, GMR-GAL4-UAS-Tau /+;+/+, GMR-
WT
GAL4-UAS-Tau /+;UAS-Notch RNAi /TM6B, GMR-GAL4-UAS-Tau /+;UAS-Notch RNAi /UAS-Notch RNAi , GMR-GAL4-UAS-Tau /UAS-Sirt1;UAS-Notch RNAi /
WT
WT
WT
UAS-Notch RNAi (n = 100). (G) Histogram indicating climbing activity (expressed as % climbing in 8 cm 10 s ) of 10-day-old flies: GMR-GAL4/+;+/+, GMR-
−1
GAL4-UAS-Tau /+;+/+, GMR-GAL4-UAS-Tau /+;UAS-Notch RNAi /TM6B, GMR-GAL4-UAS-Tau /+;UAS-Notch RNAi /UAS-Notch RNAi , GMR-GAL4-UAS-
WT
WT
WT
Tau /UAS-Sirt1;UAS-Notch RNAi /UAS-Notch RNAi (n = 100). (H) Histogram illustrating the expression levels of proapoptotic genes (Grim, Reaper, and Hid)
WT
as determined by RT-qPCR in the heads of 10-day-old adult flies: GMR-GAL4/+;+/+, GMR-GAL4-UAS-Tau /+;+/+, GMR-GAL4-UAS-Tau /+;UAS-
WT
WT
Notch RNAi /UAS-Notch RNAi , GMR-GAL4-UAS-Tau /UAS-Sirt1;UAS-Notch RNAi /UAS-Notch RNAi . RP49 was used as an endogenous control for normalization.
WT
(I) Histogram indicating the DIAP1 gene expression level as determined by RT-qPCR in the heads of 10-day-old adult flies: GMR-GAL4/+;+/+, GMR-
GAL4-UAS-Tau /+;+/+, GMR-GAL4-UAS-Tau /+;UAS-Notch RNAi /UAS-Notch RNAi , GMR-GAL4-UAS-Tau /UAS-Sirt1;UAS-Notch RNAi /UAS-Notch RNAi .
WT
WT
WT
RP49 was used as an endogenous control for normalization.
Abbreviations: RT-PCR: Reverse transcription polymerase chain reaction; RT-qPCR: Reverse transcription-quantitative polymerase chain reaction.
in GMR-GAL4-UAS-Tau /+;UAS-Notch RNAi /TM6B and activity was significantly improved when Sirt1 was
WT
GMR-GAL4-UAS-Tau /+;UAS-Notch RNAi /UAS-Notch RNA i, overexpressed along with Notch downregulation in the AD
WT
respectively. Furthermore, the light preference index genetic background: 10-day-old GMR-GAL4-UAS-Tau /
WT
(10.13) in GMR-GAL4-UAS-Tau /+;+/+ flies significantly UAS-Sirt1;UAS-Notch RNAi /UAS-Notch RNAi flies showed
WT
increased to 16.55 when Sirt1 was overexpressed along with 73.6% climbing activity compared to the 45% shown by
Notch downregulation in GMR-GAL4-UAS-Tau /UAS- GMR-GAL4-UAS-Tau /+;+/+ (Figure 7G).
WT
WT
Sirt1;UAS-Notch RNAi /UAS-Notch RNAi in AD model flies. Furthermore, we investigated the effects of Sirt1
In the climbing activity assay, the climbing activity overexpression and Notch downregulation on the expression
of 10-day-old AD model flies (GMR-GAL4-UAS- of proapoptotic genes (Grim, Reaper, and Hid). For this
Tau /+;+/+) significantly decreased to 45.4% compared purpose, we performed qRT-PCR analysis of these genes in
WT
to age-matched GMR-GAL4/+;+/+ control) flies with the heads of 10-day-old adult flies from both control and
a climbing activity of 80.8% (Figure 7G). The climbing experimental groups. The expression levels of Grim, Reaper,
activity increased significantly when Notch was and Hid were significantly increased by 1.61-, 2.43-, and
downregulated using UAS-Notch RNAi in heterozygous/ 6.71-fold, respectively, in AD model flies (GMR-GAL4-UAS-
homozygous conditions (i.e., 10-day-old GMR-GAL4- Tau /+;+/+) compared to control flies (1.0) (Figure 7H).
WT
UAS-Tau /+;UAS-Notch RNAi /TM6B and GMR-GAL4- However, the expression levels of Grim, Reaper, and Hid
WT
UAS-Tau /+;UAS-Notch RNAi /UAS-Notch RNAi flies showed were significantly reduced to 0.18-, 0.96-, and 2.1-fold,
WT
56.6% and 68.8% climbing activity, respectively, compared respectively, when Notch was downregulated (homozygous)
to the 45.4% climbing activity observed in GMR-GAL4- in GMR-GAL4-UAS-Tau /+;UAS-Notch RNAi /UAS-Notch RNAi
WT
UAS-Tau /+;+/+). In addition, the reduced climbing flies. Furthermore, when Sirt1 was overexpressed alongside
WT
Volume 3 Issue 4 (2024) 12 doi: 10.36922/an.4291