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Advanced Neurology Drosophila Sirtuin 1 and Alzheimer’s disease

qRT-PCR was performed using the Step One Plus system After secondary antibody incubation, the brain tissues
(Applied Biosystems, USA). Relative quantification was were washed 3 times in 0.1% PBST for 15 min each and
conducted using the “Delta-Delta Ct” (ΔΔct) method, mounted in DABCO (Sigma-Aldrich, USA), an antifade
normalized with the endogenous gene RP49. Data are medium. The samples were analyzed using a fluorescence
presented as mean ± standard error of the mean (SEM). microscope, and a total of 20 third-instar larval brains
Relative mRNA levels were analyzed by one-way analysis were examined for each genotype.
of variance (ANOVA) followed by Tukey’s test, using
GraphPad Prism 5.0 software. Table 1 provides details of 2.11. Statistical analysis
the primers used for qRT-PCR. All data are presented as the mean ± SEM, with the
number of biological replicates indicated as “n.” For all
2.10. Immunostaining of larval brain experiments, the significance between genotypes was
The immunostaining of the larval brain was performed assessed using one-way ANOVA with Tukey’s test in
by selecting third-instar larvae of the desired genotype. GraphPad Prism 5.0. Images were created with Adobe
Larval brains were dissected in 1X PBS and then fixed Photoshop 7.0®. Histograms were analyzed in GraphPad
in 4% paraformaldehyde (PFA) for 20 min at room Prism 5.0, with significance indicated as follows: ns, non-
temperature (RT). The dissected brain tissues were significant; *P < 0.05, **P < 0.01, ***P < 0.0001.
washed 3 times in 1% PBST (1X PBS, 1% Triton X-100) 3. Results
for 15 min each and then blocked in a 4% bovine serum
albumin solution in 1X PBS for 2 h at RT. The tissues 3.1. Overexpression of Sirt1 modulates the
were subsequently incubated overnight at 4°C in a AD-related pathologies in Drosophila
blocking solution containing primary antibodies: rabbit- AD model flies in Drosophila showed various AD-related
anti-P-JNK (1:100, Cat #V7931, Promega), mouse- pathologies in flies, including rough eyes, reduced or
anti-Drosophila Notch intracellular domain (NICD, loss of survival, and impaired locomotor and phototaxis
1:100, Cat#C17.9C6, DSHB), mouse-anti-Drosophila behaviors. To investigate the effect of Sirt1 overexpression
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Delta extracellular domain (1:50, Cat#C594.9B, DSHB), and its genetic interaction with AD-associated genes (Aβ ,
42
and rabbit-anti β-Amyloid (1-43) (1:50, Cat#E8C2D, Tau, and Appl) in fruit flies, we conducted genetic crosses
Cell Signaling Technology, USA). Following primary between fly lines overexpressing and downregulating
antibody incubation, brain tissues were washed 3 times in Sirt1 with AD model flies in Drosophila and examined
0.1% PBST for 15 min each, then blocked with blocking any phenotypic alterations. The phenotypic changes
solution at RT for 1 h, and subsequently incubated with indicated possible genetic interactions between Sirt1
secondary antibodies: AF-488 Goat-anti-Rabbit IgG and AD-associated genes. In the present study, we used
(1:150, Cat# A11008, Invitrogen), AF-488 Goat-anti- UAS-Aβ (Human), UAS-Tau , and UAS-Appl RNAi AD
2
WT
Mouse IgG (1:150, Cat# A11001, Invitrogen), and Anti- model flies as well as UAS-Sirt1 to overexpress and UAS-
Mouse Cy3 IgG (1:150, Cat# C2181, Sigma) for 2 h at RT. Sirt1 RNAi to downregulate Sirtuin1 in Drosophila. The UAS

Table 1. The primers used for quantitative real‑time reverse transcription‑quantitative polymerase chain reaction
Gene Forward primer sequence Reverse primer sequence
Sirt1 5′ TTTGCCCGCGAGATATATCC 3′ 5′ GCCCTTGGTCTCCAGCATT 3′
Aβ 5′ CGAGCGATTGCTGTTGGA 3′ 5′ TCCCGACCGCTTCTGTTC 3′
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Tau 5′CAATAGCAACACCACTTCGGATAG 3′ 5′ CGTATCTGCTGTTTGGAAACTGA 3′
Appl 5′ CCCAGATTGCCGTTCTCTGT 3′ 5′ TGTGGGACCCGGTTGTCTTCT 3′
Grim 5′ TGGATGCTGGGATCTTTTGG 3′ 5′ CGCTGGCTCGAACTGTAGCT 3′
Reaper 5′ CGGGAGTCACAGTGGAGATTC 3′ 5′ GGTCTTCGGATGACATGAAGTG 3′
Hid 5′ GAGTGCCCCGCAAATCTTC 3′ 5′ CCGTGCGGAAAGAACACAT 3′
DIAP1 5′ TTGGTTTGGCTGGGCTTATT 3′ 5′ GGCTTGGAGTGCCATCGA 3′
JNK 5′ ATCAGCTCCATGACCAGGTAGAC 3′ 5′ACTTGGATCACGACAGAATGTCC 3′
Notch 5′ CGATGCGTTGCCAAAATG 3′ 5′ CAAAGGACACTTGCACGAGATG 3′
Delta 5′GCTTCACGAATCCCATCCA 3′ 5′ TCGACGATCAGCGAGAAGGT 3′
RP49 5′ GCAAGCCCAAGGGTATCGA 3′ 5′ ACCGATGTTGGGCATCAGA 3′

Volume 3 Issue 4 (2024) 4 doi: 10.36922/an.4291