MeDUsA: A novel system for automated axon quantification to evaluate neuroaxonal degeneration

Background Drosophila is an excellent model organism for studying human neurodegenerative diseases (NDs), and the rough eye phenotype (REP) assay is a convenient experimental system for analysing the toxicity of ectopically expressed human disease genes. However, the association between REP and axonal degeneration, an early sign of ND, remains unclear. To address this question, we developed a method to evaluate axonal degeneration by quantifying the number of retinal R7 axons in Drosophila ; however, it requires expertise and is time-consuming. Therefore, there is a need for an easy-to-use software that can automatically quantify the axonal degeneration. Result We created MeDUsA (a ‘method for the quantification of degeneration using fly axons’), which is 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 axons from a confocal z-stack image series. Using this software, we have demonstrated for the first time directly that axons degenerate when the causative factors of NDs (αSyn, Tau, TDP-43, HTT) were expressed in the Drosophila eye. Furthermore, we compared axonal toxicity of the representative causative genes of NDs and their pathological alleles with REP and found no significant correlation between them. Conclusions MeDUsA rapidly and accurately quantifies axons in Drosophila eye. By simplifying and automating time-consuming manual efforts requiring significant expertise, it enables large-scale, complex research efforts on axonal degeneration, such as screening to identify genes or drugs that mediate axonal toxicity caused by ND disease proteins. ### Competing Interest Statement The authors have declared no competing interest. * MeDUsA : a method for quantification of degeneration using fly axons ND : Neurodegenerative disease REP : rough eye phenotype SCA3 : Spinocerebellar ataxia type 3 PD : Parkinson’s disease ALS : amyotrophic lateral sclerosis CNN : convolutional neural network RNAi : RNA interference SCA31 : spinocerebellar ataxia type 31 FTLD : frontotemporal lobar degeneration WD : Wallerian degeneration