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Fig. 1 | Acta Neuropathologica Communications

Fig. 1

From: The intracellular milieu of Parkinson’s disease patient brain cells modulates alpha-synuclein protein aggregation

Fig. 1

a Table summarizing the hiPSC lines employed in the study. b Immunohistochemistry performed on 75-day old hiPSC-derived midbrain spheroids revealed abundance of TH/FOXA2-positive dopaminergic neurons. c Western blot analysis of RIPA extracts revealed an increase of 14 kDa aSYN level in the SNCA and GBA spheroids. Western blot analysis was performed using antibody specific to aSYN. Data were normalized to the level of actin and presented as mean ± SD; N = 2 patients; one line per patient; *p < 0.05. Statistical analysis was performed by comparing control, SNCA and GBA using one-way ANOVA followed by Tukey’s post hoc test; **p < 0.01 and *p < 0.05. d Dot blot analysis revealed increase of aSYN fibrillar content. Dot blot analysis was performed using OC antibody specific to amyloid fibrils. Data are normalized to the control and presented as mean ± SD; N = 2 patients; one line per patient; *p < 0.05. Statistical analysis was performed using a two-tailed t-test. e µFTIR analysis further revealed changes in molecular structures in the spheroids models of synucleinopathies. Bright field image of spheroid deposited on CaF2. Black line indicates the area of µFTIR analysis. Red-green–blue maps are infrared images at specific frequencies used for structural analysis. f Box diagrams show µFTIR analysis of β-sheet structural content, total lipids measured as area ratio of lipids between 2800–3000 cm−1 and amide I (1600–1700 cm−1); lipid oxidation measured as an area ratio of peaks centered at 1740 cm−1 and amide I (1600–1700 cm−1); and lipid chain length measured as a ratio between peaks at 2854 cm−1 and 2874 cm−1, within healthy, SNCAmultiplication, Idiopathic PD and GBA midbrain spheroids. Box diagrams show mean (represented by squares), median line, interquartile range from lowest to highest, and outliers (shown as lozenges). Statistical analysis was performed by comparing all groups using one-way ANOVA followed by Bonferroni post hoc test; ***p < 0.001. g PD midbrain spheroids cellular milieus trigger aggregation of aSYN by stimulating primary nucleation of aSYN. The kinetics of 10 μM recombinant p.A53T aSYN aggregation alone and in the presence of extracts from midbrain spheroids (50 ng/μL) was monitored by ThT fluorescence in 10 mM MES pH 5.5 buffer under quiescent conditions at 37 °C. Results are presented as mean ± SD, N = 2 patients; one line per patient

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