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. 2009 May;174(5):1891–1909. doi: 10.2353/ajpath.2009.080680

Figure 2.

Figure 2

Characterization of LMP in PDMP-treated syn-overexpressing cells. P123H β-syn-overexpressing cells were transfected with vector, wild-type α-syn, or A53T α-syn. Cells were then treated with or without PDMP (25 μmol/L) for the indicated times, followed by assessment of lysosomal leakage. In some experiments, vector-transfected cells, wild-type β-syn-overexpressing cells, and P123H β-syn-overexpressing cells were also analyzed. A: Lucifer Yellow staining at 24 hours. The fluorescence was increased by PDMP treatment in P123H β-syn-overexpressing cells transfected with A53T α-syn (c--h) and to a lesser extent in vector-transfected cells (a and b). Boxes in c and d are equivalent to e and f, respectively. In g and h, cells were fixed in 4% paraformaldehyde and nuclei were labeled with DAPI. Arrowheads indicate lysosomal inclusion bodies (e--h). Note that Lucifer Yellow is accumulated in inclusions before PDMP treatment (e and g), whereas it is leaked into cytoplasm after PDMP treatment (f and h). Ba: Quantification of Lucifer Yellow redistribution. The number of Lucifer Yellow-positive cells in their cytosols at 48 hours of treatment with PDMP was calculated as a percentage of the total number of cells. Data are shown as means ± SD (n = 4). *P < 0.05, **P < 0.01 versus PDMP-untreated cells. In b, time course quantification of Lucifer Yellow redistribution was performed. For P123H β-syn-overexpressing cells transfected with vector, wild-type α-syn, or A53T α-syn, the number of Lucifer Yellow-positive cells in their cytosols was evaluated at 6, 12, 24, and 48 hours. Data are shown as means ± SD (n = 4). *P < 0.05, **P < 0.01 versus PDMP-untreated cells. C: Immunoblot analyses of cathepsins B and D, LAMP-2, and HSP70 for cytosolic and lysosome-enriched fractions (each 10 μg) derived from P123H β-syn cells transfected with either control vector or A53T α-syn. A typical blot for each molecule is presented in a. Equivalent protein loading in each lane was confirmed by immunoblotting with anti-actin antibody. In b, the band intensity corresponding to each molecule was corrected with that of actin. The data are shown as % r. intensity of P123H β-syn-overexpressing cells transfected with vector (PDMP-treated cells for the cytosolic fractions and PDMP-untreated cells for the lysosome-enriched fractions) and are expressed as the mean value ± SD (n = 4). *P < 0.05, **P < 0.01 versus PDMP-untreated cells. D: Double immunofluorescence/LSCM of cathepsin B and β-syn (a), cathepsin D and β-syn (b), and cathepsin D and LAMP-2 (c) for P123H β-syn-overexpressing cells transfected with A53T α-syn. Cells were treated with (d–f, j–l, p–r) or without (a–c, g–i, m–o) PDMP for 24 hours. Arrowheads indicate inclusion bodies. Immunoreactivities of cathepsins B and D were co-localized with those of P123H β-syn (a and b) and LAMP-2 (c) in the lysosomal inclusion bodies before PDMP treatment, but were redistributed into the cytosols after PDMP treatment (a–c). In c, the immunoreactivity of LAMP-2 was retained in the lysosomal inclusions even after PDMP treatment (q). Nuclei are simultaneously stained with DAPI in the merged figures (c, f, i, l, o, and r). Similar results were observed in three independent experiments. Scale bars: 20 μm (Aa–Ad); 5 μm (Ae and Af); 10 μm (Ag, Ah, D).