Abstract
Neuronal models of neurodegenerative diseases such as Parkinson’s Disease (PD) are extensively studied in pathological and therapeutical research with neurite outgrowth being a core feature. Screening of neurite outgrowth enables characterization of various stimuli and therapeutic effects after lesion. In this study, we describe an autonomous computational assay for a high throughput skeletonization approach allowing for quantification of neurite outgrowth in large data sets from fluorescence microscopic imaging. Development and validation of the assay was conducted with differentiated SH-SY5Y cells and primary mesencephalic dopaminergic neurons (MDN) treated with the neurotoxic lesioning compound Rotenone. Results of manual annotation using NeuronJ and automated data were shown to correlate strongly (-value for SH-SY5Y cells and -value for MDN). Pooled linear regressions of results from SH-SY5Y cell image data could be integrated into an equation formula (; for normalized results) with y depicting automated and x depicting manual data. This automated neurite length algorithm constitutes a valuable tool for modelling of neurite outgrowth that can be easily applied to evaluate therapeutic compounds with high throughput approaches.
Highlights
Parkinson’s Disease (PD) is a neurodegenerative movement disorder that is characterized by multifactorial cellular dysfunction of dopaminergic neurons [1]
We present a fully automated neurite outgrowth quantification assay able for high throughput screening
Our automated neurite outgrowth quantification algorithm allows for analysis of dense neurite structures in a high throughput manner and is designed for annotation of two-dimensional immunocytochemical microscopy images of neuronal cultures
Summary
Parkinson’s Disease (PD) is a neurodegenerative movement disorder that is characterized by multifactorial cellular dysfunction of dopaminergic neurons [1]. Several impairments in cellular processes involved in protein homeostasis and mitochondrial function contribute to PD pathogenesis [2,3]. Pathological aSYN accumulation, impaired mitochondrial dynamics and high levels of reactive oxygen species cause dysfunction of mitochondria resulting in higher oxidative stress, which contributes to neuronal death [2,5,6]. Neuroinflammation is another feature of PD pathology with important implications on neuronal health. Activated microglia cause neuronal death via different pathways. There is much new evidence in PD research, the exact pathogenesis remains still unclear
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