NEURITE OUTGROWTH CELLPROFILER PIPELINE MANUAL

These can be subjected to several neurotoxin-based screening experiments for PD, which are especially suitable for modelling effects of oxidative stress using Rotenone, 1-methyl-4-phenylpyridinium ( MPP +), 6-hydroxydopamine (6-OHDA). Simple culturing techniques and cell amplification enable the generation of a large number of neuronal cells with dopaminergic phenotypes. An advantage of the widely used SH-SY5Y cells is that they can be differentiated into neuronal-like cells exhibiting dopaminergic but also cholinergic or noradrenergic phenotypes. Differentiated dopaminergic cell models comprise immortalized cell lines ( SH-SY5Y, LUHMES, PC12), primary dopaminergic neurons and induced pluripotent stem cells (iPSC). Characterization of pathological events demands dissection of the complex PD pathogenesis into more simple molecular models enabling precise manipulation of contributing factors and introduction of therapeutic approaches. To better analyze the multifactorial character of PD and other neurodegenerative diseases cellular analysis systems can be employed. Although there is much new evidence in PD research, the exact pathogenesis remains still unclear. By additional secretion of tumor necrosis factor (TNF) and activation of complement astrocytes are converted into neurotoxic phenotypes contributing to neuroinflammation. Release of proinflammatory cytokines, such as IL-1β, amplifies local inflammation and causes neuronal death. Activated microglia cause neuronal death via different pathways. Neuroinflammation is another feature of PD pathology with important implications on neuronal health. Pathological aSYN accumulation, impaired mitochondrial dynamics and high levels of reactive oxygen species cause dysfunction of mitochondria resulting in higher oxidative stress, which also contributes to neuronal death.

Abnormalities in protein aggregation, intracellular protein trafficking and protein disposal can result in α-Synuclein (aSYN) misfolding and aggregation as found in Lewy Bodies and Lewy Neurites in the human brain. Several impairments in cellular processes involved in protein homeostasis and mitochondrial function contribute to PD pathogenesis. Parkinson’s Disease (PD) is a neurodegenerative movement disorder that is characterized by multifactorial cellular dysfunction of dopaminergic neurons. 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.

NEURITE OUTGROWTH CELLPROFILER PIPELINE MANUAL

x + 0.09191 for normalized results) with y depicting automated and x depicting manual data.

Pooled linear regressions of results from SH-SY5Y cell image data could be integrated into an equation formula ( y = 0.5410 Results of manual annotation using NeuronJ and automated data were shown to correlate strongly ( R 2-value 0.9077 for SH-SY5Y cells and R 2-value 0.9297 for MDN). 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. 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. Screening of neurite outgrowth enables characterization of various stimuli and therapeutic effects after lesion. 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.

Neurite outgrowth cellprofiler pipeline