The immortalized and proliferative cell line SH-SY5Y is one of the most commonly used cell lines in neuroscience and neuroblastoma research. However, undifferentiated SH-SY5Y cells share few properties with mature neurons. In this study, we present an optimized neuronal differentiation protocol for SH-SY5Y that requires only two work steps and 6 days. After differentiation, the cells present increased levels of ATP and plasma membrane activity but reduced expression of energetic stress response genes. Differentiation results in reduced mitochondrial membrane potential and decreased robustness toward perturbations with 6-hydroxydopamine. We are convinced that the presented differentiation method will leverage genetic and chemical high-throughput screening projects targeting pathways that are involved in the selective vulnerability of neurons with high energetic stress levels.
Autophagic processes play a central role in cellular homeostasis. In pathological conditions, the flow of autophagy can be affected at multiple and distinct steps of the pathway. Current analyses tools do not deliver the required detail for dissecting pathway intermediates. The development of new tools to analyze autophagic processes qualitatively and quantitatively in a more straightforward manner is required. Defining all autophagy pathway intermediates in a high-throughput manner is technologically challenging and has not been addressed yet. Here, we overcome those requirements and limitations by the developed of stable autophagy and mitophagy reporter-iPSC and the establishment of a novel high-throughput phenotyping platform utilizing automated high-content image analysis to assess autophagy and mitophagy pathway intermediates.
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