A cell-based model of ?-synucleinopathy for screening compounds with therapeutic potential of Parkinson's disease

Abstract

Aim: To develop a cell‐based model by stable transfection of SH‐SY5Y with mutant A53T human α‐synuclein, recapitulating neurotoxicity of α‐synuclein overexpression. Methods: The overexpression of mutant α‐synuclein was analyzed by Western blotting, immunocytochemistry, and RT‐PCR. Cell viability was processed when treated with different concentrations of 1‐methyl‐4‐phenyl‐pyridinium (MPP+) and exogenous dopamine (DA) for 24,48, and 72 h by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Early apoptosis and late apoptosis/necrosis were analyzed by flow cytometry using Annexin V and propidium iodide double staining, respectively. DNA was isolated and applied to agarose gel for electrophoresis; the typical DNA “ladder” represented severe apoptosis. We also used this model to screen 99 compounds with therapeutic potential by MTT assay. Results: One of the stably‐transfected clones overexpressed exogenous genes on both the protein level and the transcriptive level. Significant differences in cytotoxicity were found between the pcDNA3.1(+) group and the pcDNA3. 1(+)‐hmα‐synuclein group in the presence of the same concentration of MPP+ and DA within the same incubation time. The level of either early apoptosis or late apoptosis/necrosis was remarkably increased in transfected cells compared with the control after treatment with 100 μmol/L MPP+ for 24 h. In addition, the presence of the typical DNA “ladder” was observed in the pcDNA3.1(+)‐hmα‐synuclein group when treated with 200 μmol/L MPP+ for 48 h. After the screening experiment, 12 of the 99 compounds were found to decrease DA‐induced cytotoxicity on cell viability. Conclusion: We established a cell‐based model which is useful for studying the function of aL‐synuclein and screening compounds with therapeutic potential. In addition, it was identified that cells overexpressing A53T mutant α‐synuclein were significantly vulnerable against MPP+ or dopamine exposures.

Molecular and Neurochemical Mechanisms in PD Pathogenesis

“…The level of both early apoptosis and late apoptosis/necrosis remarkably increased in transfected cells expressing the A53T mutant alpha-synuclein compared to the control cells after treatment with MPP? for 24 h (Zhao et al 2007). Alphasynucleins harboring mutations that cause familial Parkinson's disease, such as A30P and A53T, exhibit increased nuclear targeting in cell culture, which promotes neurotoxicity (Kontopoulos et al 2006).…”

Current Screening Methodologies in Drug Discovery for Selected Human Diseases

“…An example of drug discover for AD is screening compounds that avoid the in vitro neuron damage triggered by extracellular Aβ [169]. In the case of PD, cell culture is treated with 1-methyl-4-phenylpyridinium (MPP+), which can specifically induce dopaminergic neuron death [170], or with rotenone which triggers mitochondrial dysfunction [171]. One marine-derived compound, Secalonic acid A, present in marine fungi such as Aspergillus ochraceus and Paecilomyces sp., protects against MPP+-induced neurotoxicity in cell line models [172].…”

Phenotypic Screens Targeting Neurodegenerative Diseases

“…Twelve protective compounds were found from Fraxinus sielboldiana blume of the Oleaceae family, which is widely distributed in East Asia. 40…”

“…Twelve protective compounds were found from Fraxinus sielboldiana blume of the Oleaceae family, which is widely distributed in East Asia. 40 Another model system for evaluating the toxicity of aggregated protein is yeast, in which toxicity can be conveniently observed via the change of cell growth measured by the colony size. In one such study, with α-synuclein overexpression and FeCl another study, a yeast model with a high expression level of α-synuclein was used for screening.…”