Direct evaluation of neuroaxonal degeneration with the causative genes of neurodegenerative diseases in Drosophila using the automated axon quantification system, MeDUsA

Abstract

Drosophila is an excellent model organism for studying human neurodegenerative diseases (NDs). However, there is still almost no experimental system which could directly observe the degeneration of neurons and automatically quantify axonal degeneration. In this study, we created MeDUsA (a ‘method for the quantification of degeneration using fly axons’), a standalone executable computer program based on Python that combines a pre-trained deep-learning masking tool with an axon terminal counting tool. This software automatically quantifies the number of retinal R7 axons in Drosophila from a confocal z-stack image series. Using this software, we were able to directly demonstrate that axons were degenerated by the representative causative genes of NDs for the first time in Drosophila. The fly retinal axon is an excellent experimental system that is capable of mimicking the pathology of axonal degeneration in human NDs. MeDUsA rapidly and accurately quantifies axons in Drosophila photoreceptor neurons. It enables large-scale research into axonal degeneration, including screening to identify genes or drugs that mediate axonal toxicity caused by ND proteins and diagnose the pathological significance of novel variants of human genes in axons.

Drosophilamodel to clarify the pathological significance of OPA1 in autosomal dominant optic atrophy

“…To assess the functional impact of this novel human gene variant, we engineered transgenic Drosophila lines expressing both wild type and mutant SART1 under the control of a UAS promoter. Our MeDUsA analysis suggested that the variant may confer a gain-of-toxic-function (Nitta et al, 2023). Moreover, we identified heterozygous loss-of-function mutations in DHX9 as potentially causative for a newly characterized neurodevelopmental disorder.…”

“…To this end, we performed an RNAi experiment using the GMR-Gal4 driver and evaluated the number of retinal axons projecting to the second optic ganglion medulla (Figure 3A). To compare the number of retinal axon terminals–R7 axons–between genotypes, we used a previously developed automated method, MeDUsA ( me thod for the quantification of d egeneration us ing fly a xons) (Nitta et al, 2023). We have assessed the extent of their reduction from the total axonal terminal count, thereby determining the degree of axonal terminal degeneration (Nitta et al, 2023; Richard et al, 2022).…”

“…To compare the number of retinal axon terminals–R7 axons–between genotypes, we used a previously developed automated method, MeDUsA ( me thod for the quantification of d egeneration us ing fly a xons) (Nitta et al, 2023). We have assessed the extent of their reduction from the total axonal terminal count, thereby determining the degree of axonal terminal degeneration (Nitta et al, 2023; Richard et al, 2022). Our results showed that the dOPA1 knockdown caused axonal degeneration one day after eclosion, further decreasing one week later (Figure 3B–E, quantification in 3F).…”

“…Drosophila photoreceptors type R7/8 extend their retinal axons from the retina directly into the brain, forming synapses in the second optic ganglion medulla via the chiasma (Figure 1A), having potential application as a model for mammalian photoreceptors and RGCs. Based on this, we aimed to develop an experimental model for observing and quantifying degeneration in the Drosophila retinal axon (Nitta et al, 2023; Richard et al, 2022).…”

“…In this study, we defined the retinal axons of Drosophila as analogous to the human optic nerve. Based on this, we aimed to develop an experimental model for observing and quantifying degeneration in the Drosophila retinal axon (Nitta et al, 2023; Richard et al, 2022).…”