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Numerous pathological changes of subcellular structures are characteristic hallmarks of neurodegeneration. The main research has focused to mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomal networks as well as microtubular system of the cell. The sequence of specific organelle damage during pathogenesis has not been answered yet. Exposition to rotenone is used for simulation of neurodegenerative changes in SH-SY5Y cells, which are widely used for in vitro modelling of Parkinson´s disease pathogenesis. Intracellular effects were investigated in time points from 0 to 24 h by confocal microscopy and biochemical analyses. Analysis of fluorescent images identified the sensitivity of organelles towards rotenone in this order: microtubular cytoskeleton, mitochondrial network, endoplasmic reticulum, Golgi apparatus and lysosomal network. All observed morphological changes of intracellular compartments were identified before αS protein accumulation. Therefore, their potential as an early diagnostic marker is of interest. Understanding of subcellular sensitivity in initial stages of neurodegeneration is crucial for designing new approaches and a management of neurodegenerative disorders.