Abstract Parkinson’s disease (PD) involves the generation of dopaminergic (DA) neurons in the substantia nigra, causing tremor and motor impairment. Defects in PARK2 protein are the cause of Parkinson’s disease type 2 (PARK2), also known as early-onset parkinsonism with diurnal fluctuation (EPDF) or autosomal recessive juvenile Parkinson’s disease (PDJ). In mouse model, Parkin is critical for removing dysfunctional mitochondria via autophagy. The absence of Parkin does not affect turnover under normal conditions. However, during stress conditions, Parkin helps to remove damaged mitochondria by autophagy. Innoprot has developed a novel fluorescence cell-based assay for High Content Screening to screen compounds that can promote Parkin2 mithocondrial localization without mithocondrial stress induction. In this work, we used this model to screen a library of 1200 compounds. After the screening campaign, the positive compounds were chosen for further testing, based on the strength of the initial response and the lack of cytotoxicity. Our results indicate that Parkin2 cell-based assay is a valid strategy for drug screening. Fluorescence-Based Assay Development To Screen Drugs Against Parkinson´s Disease Salado C., Kortazar D., Mella R., Roura-Ferrer M., Gámiz J. & Villacé P. Innoprot (Derio, Spain) www.innoprot.com Schematic representation of the model OMM IMM OMM IMM CCCP tGFP MTS Parkin Parkin FP602 FP602 tGFP MTS Parkin Parkin FP602 FP602 Figure 1. Schematic representation of Innoprot’s Parkin2 model in living cells. Parkin2 model is a fluorescent fusion polypeptide capable of changing its localization within the cell from the cytosol to the outer mitochondrial membrane, after CCCP addition. Parkin2 forms part of E3 ubiquitin ligase complex that mediates the targeting of proteins for degradation. CCCP is a mitochondrial proton gradient uncoupler that induces a loss of mitochondrial membrane potential (m) and therefore, promotes recruitment of Parkin2 to damaged mitochondria during the early stages of mitophagy. The second fluorescent fusion polypeptide (MTS-tGFP) allows us to visualize stability and location of mitochondria. High-content screening (HCS) in Parkin2 model uses living cells as a tool in biological research to discover and optimize new drug candidates against Parkinson’s disease. Materials and methods Cultured cells: PARK2-FP602+MTS-tGFP cell line has been obtained by a double transfection of an ex- pression vector that encodes turbo green fluorescent protein (tGFP) fused to mitochondrial targeting sequence (MTS) derived from the subunit VIII of human cytochrome C oxidase and a vector containing PARK2 protein fused to FP602 red fluorescent protein. U2OS PARK2-FP602+MTS-tGFP cell lines were cultured into 96 wells Imaging Plates (BD) at 0.25 cells/cm2 in 200 l of DMEM 10% FBS and incubated at 37ºC and 5% CO2. Image adquisition: human tagged PARK2-FP602+MTS-tGFP stably expressing cells were treated with CCCP (positive control) at 5 M for 3 hours and library compounds at 10 M for 24 hours in Optimem. After that, the nucleus was stained with Dapi and the presence of recruited Parkin to defective mito- chondria was quantified by fluorescence using image analysis algorithms and the BD Pathway 855 image platform. Cellular viability and granularity was calculated relative to the negative control. Results Figure 2. Representative data of Parkin2 redistribution screening. An initial screening of 1200 compounds were done but toxic compounds (with a cell viability under 60% with respect to control) were eliminated from the study. Negative control (DMSO) is represented in pink and positive control (CCCP, 5 M) is represented in green. Positive compounds (with an activity over 1.5 with respect to control, I-VIII) are represented in yellow. Parkin2 redistribution was quantified by measuring fluorescence granularity. Figure 3. Colocalization assay of Parkin2 and mitochondria in presence of redistribution-positive compounds (I-V