Page 133 - AN-3-4

Advanced Neurology Drosophila Sirtuin 1 and Alzheimer’s disease

Sirt1 is a well-studied gene in the context of aging and age- Drosophila c-Jun N-terminal kinase (JNK) Basket (Bsk)
related neurodegenerative diseases (NDDs). Evidence downregulating transgenic fly stocks: UAS-Bsk RNAi (Chr.
3,16
suggests that it plays a protective role against NDDs, cancer, ΙΙΙ) [BL#31323] is an RNAi line for Drosophila JNK (Bsk)
cardiovascular conditions, and other age-related issues. gene downregulation. Notch downregulating transgenic fly
3,16
Alzheimer’s disease (AD) is a leading cause of dementia stocks: UAS-Notch RNAi (Chr. ΙΙΙ) is an RNAi line for Notch
and cognitive decline in the elderly, 17-19 characterized by gene downregulation. Transgenic fly stocks overexpressing
two main neuropathological hallmarks: extracellular senile or downregulating AD-related gene: UAS-Aβ (Human)/
42
plaques primarily composed of amyloid-beta (Aβ) peptide, CyO (Chr. II) expresses the human Aβ gene under the
42
formed by the amyloidogenic cleavage of the APP protein UAS system; UAS-Tau (Chr. II) [BL# 51362] expresses
WT
by β and γ-secretase and intracellular neurofibrillary wild-type Tau under UAS control. UAS-Appl RNAi (Chr. III)
tangles (NFT) associated with tau, a microtubule-binding [BL# 28043] is an RNAi line for Appl downregulation.
protein. 20-24 Recombined fly stocks include the following: w;GMR-
D. melanogaster, commonly known as the “fruit fly,” has GAL4-UAS-Aβ (Human)/CyO;+/+, a stock with
42
been an ideal and powerful invertebrate model organism GMR-GAL4 and UAS-Aβ (Human)/CyO, w;GMR-GAL4-
42
for genetic research for over 120 years. In the Drosophila UAS-Tau /CyO;+/+, a stock with GMR-GAL4; and UAS-
WT
model of AD, neuronal death results in a rough eye Tau /CyO, and GMR-GAL4/+;UAS-Appl RNAi /+ (Chr. III)
WT
phenotype, impairments in learning, memory, climbing, [BL#28043], an RNAi line for Appl downregulation.
and phototaxis. 25-28 In addition, studies have shown that UAS-ArcAβ Stocks: UAS-ArcAβ42 (Chr. III) and UAS-
the upregulation of AD-associated genes (Aβ , Tau, and ArcAβ (Chr. II) express the human Aβ42 fragment of APP
42
42
42
Appl) in Drosophila induces cell death (apoptosis) through with the familial Alzheimer’s Arctic mutation under UAS
increased cellular stress. 29-31 control. UAS-ArcAβ (Chr. III) and UAS-ArcAβ (Chr. II)
42
42
Although Sirt1 has been widely studied in the context were provided by Damin Crowther, Cambridge University,
of aging, the molecular details of its interactions with UK.
AD-related genes (Tau, Aβ42,and Appl) remain unclear. In We used the UAS-ArcAβ fly strain for climbing

this study, we investigated the potential genetic interactions assays, cell death assays, real-time reverse transcription
42
between Sirt1 and the abovementioned AD-associated polymerase chain reaction (RT-PCR) analysis, and
genes in Drosophila.
immunostaining because other AD transgenic flies, such
2. Materials and methods as UAS-Aβ (Human)/CyO, UAS-Tau WT, and UAS-Sirt1, are
42
present on the second chromosome, making it unfeasible
2.1. Drosophila stocks and genetics to perform crosses between each AD-related gene and
The W 1118 strain of D. melanogaster is used as a wild- UAS-Sirt1 with elav-Gal4.
type control in experimental studies. Gal4 stocks: Pan- All flies were cultured in a biological oxygen demand
retinal GMR-GAL4 (Chr. II) drives gene expression in all (BOD) incubator at 22°C ± 1°C on standard Drosophila
cells posterior to the morphogenetic furrow (MF) in the food media, which included agar powder (regular grade
developing eye and later becomes active throughout most for bacteriology, SRL#19661), maize powder, sugar, yeast
of the pupal eye. 27,32 Pan-neuronal elav-Gal4 (Chr. X) powder (High sugar eagle instant dry yeast), nepagin (an
drives gene expression in the sensory neurons of the fly antifungal agent, methyl-p-hydroxybenzoate sodium salt
brain under the control of elav. Both GMR-GAL4 and elav- 99% extrapure, Loba Chemie#04661), and propionic acid
Gal4 flies were used as control groups in the experiment.
(antibacterial, SRL#12931).
In this study, AD-associated genes were expressed
in the fly eye using the GMR-GAL4 driver, which 2.2. Microscopy imaging
induced retinal degeneration and resulted in a rough eye Light microscopy was employed to examine the external
phenotype. AD model flies were crossed with elav-Gal4 to eye morphology of adult flies. For this experiment, 10-day-
express AD-associated genes in sensory neurons, resulting old flies of the selected genotypes were examined under
in degenerative phenotypes, including pathological a light microscope. Eye images were captured at 51.2×
morphologies and behavioral changes associated with AD. magnification under a Carl Zeiss Stemi DV4 stereo
TM
Sirt1 overexpressing/downregulating transgenic fly binocular microscope. Measurements were recorded in
stocks: UAS-Sirt1 (Chr. ΙΙ) [BL#44216] overexpresses the micrometers using TS Viwe7 (version 7.1.3.7) software.
Sirt1 gene, while UAS-Sirt1 RNAi (Chr. ΙΙΙ) [BL#31636] is an For light microscopic imaging, 40 flies of the desired
RNA interference (RNAi) line for Sirt1 downregulation. genotype were used.

Volume 3 Issue 4 (2024) 2 doi: 10.36922/an.4291