Abstract
Background:
The increased interest in research on DNA damage in neurodegeneration has created a need for the development of tools dedicated to the analysis of DNA damage in neurons. Double-stranded breaks (DSBs) are among the most detrimental types of DNA damage and have become a subject of intensive research. DSBs result in DNA damage foci, which are detectable with the marker γH2AX. Manual counting of DNA damage foci is challenging and biased, and there is a lack of open-source programs optimized specifically in neurons. Thus, we developed a new, fully automated application, SimplySmart_v1, for DNA damage quantification and optimized its performance specifically in primary neurons cultured in vitro.
Results:
Compared with control neurons, SimplySmart_v1 accurately identifies the induction of DNA damage with etoposide in primary neurons. It also accurately quantifies DNA damage in the desired fraction of cells and processes a batch of images within a few seconds. SimplySmart_v1 was also capable of quantifying DNA damage effectively regardless of the cell type (neuron or NSC-34). The comparative analysis of SimplySmart_v1 with other open-source tools, such as Fiji, CellProfiler and a focinator, revealed that SimplySmart_v1 is the most 'user-friendly' and the quickest tool among others and provides highly accurate results free of variability between measurements. In the context of neurodegenerative research, SimplySmart_v1 revealed an increase in DNA damage in primary neurons expressing abnormal TAR DNA/RNA binding protein (TDP-43).
Conclusions:
These findings showed that SimplySmart_v1 is a new and effective tool for research on DNA damage and can successfully replace other available software.
Keywords:
Analysis; DNA damage foci; DNA damage in neurons; Neurodegeneration; Neuronal culture; Python application.