A protocol to detect neurodegeneration in Drosophila melanogaster whole-brain mounts using advanced microscopy

Abstract

Summary Drosophila melanogaster is an excellent model organism to study neurodegeneration. Assessing evident neurodegeneration within the fly brain involves the laborious preparation of thin-sectioned H&E-stained heads to visualize brain vacuole degeneration. Here, we present an advanced microscopy-based protocol, without the need for sectioning, to detect vacuole degeneration within whole fly brains by applying commonly used stains to reveal the brain parenchyma. This approach preserves the whole-brain architecture and enables rapid, reproducible, and quantitative analyses of vacuole-like degeneration associated with specific brain regions. For complete details on the use and execution of this protocol, please refer to Behnke et al. (2021) .

Restoration of the GTPase activity and cellular interactions of Gα o mutants by Zn 2+ in GNAO1 encephalopathy models

“…S12). However, this degeneration was very modest as compared to that observed, e.g., in Drosophila models of Alzheimer’s disease ( 47 ), the finding that may be aligned with the fact that the G203R mutant flies did not display any signs of spontaneous epilepsy. Given the neonatal lethality of GNAO1[G203R]/+ mice ( 9 ), we thus establish the first viable animal model of G203R encephalopathy with this Drosophila line capable of recapitulating some of the clinical manifestations of the disease.…”

“…Measurement of the life span was performed as described ( 90 ); A total of 110 males and 140 females of each genotype and on each supplemented food were monitored. Adult brain degeneration was assessed following ( 47 ) using costaining with Alexa Fluor 488 phalloidin (1:100; Thermo Fisher Scientific) and DAPI (1:1000) after fixation in 4% paraformaldehyde/0.5% Triton X-100 in PBS. Fluorescent images ( Z -stacks) were acquired with a Zeiss LSM 800 Airyscan confocal microscope.…”

Heparan sulfate modified proteins affect cellular processes central to neurodegeneration and modulatepresenilinfunction

“…We, therefore, examined the capacity of reduced heparan sulfate biosynthesis to affect neurodegeneration in the brain of shPsn3 RNAi-expressing animals. Adult flies expressing shPsn3 RNAi under the control of elav-Gal4 were aged for 7 or 30 days prior to phalloidin and Hoechst staining of adult heads to visualize actin and nuclei in whole brain preparations 51 . Serial confocal sections of the entire brain were taken to identify vacuoles, areas of acellularity produced by neuronal loss (Figure 4A).…”

“…A SecureSeal TM imaging spacer was applied to each microscope slide, and brains were mounted anterior side up with SlowFade TM Gold Antifade Mountant (without DAPI). A more detailed version of this procedure can be found in 51 . Full brain samples were imaged nonsequentially on an Olympus FV3000 confocal microscope at a step size of 2 µm.…”

Targeting the “hallmarks of aging” to slow aging and treat age-related disease: fact or fiction?

“…Studies in Drosophila melanogaster, for instance, can also draw from a rich set of aging-associated phenotypic changes, which can be utilized collectively as a proxy to measure aging. This includes, but is not limited to, analyses of molecular alterations (e.g., bulk changes in transcriptome [172][173][174][175], proteome [176,177] and metabolome [178,179]; single-cell transcriptomic changes [180]); neuromorphological changes (e.g., neurodegeneration [181]), behavioral assessments (e.g., learning and memory [182][183][184], locomotor activity [185,186], circadian rhythm and sleep patterns [187,188]), an assessment of muscle structure and function (e.g., changes in muscle morphology and integrity [189]), analyses of changes in heart function (e.g., assessment of cardiac performance [190,191]) and gut homeostasis (e.g., histopathological analyses of epithelial dysplasia and barrier function [91]).…”