Accumulation of syn proteins 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 24 hours, followed by immunoblot analysis and RT-PCR. A: Immunoblot analysis of syn proteins. The soluble and insoluble fractions of cell extracts (10 μg) were analyzed by immunoblotting using anti-β-syn, anti-α-syn, and anti-actin antibodies. Recombinant α- and β-syn proteins are used as positive controls. a: The blots are representative of at least three experiments. In b, the band intensities corresponding to α- and β-syn were quantified and corrected with the intensities of actin bands. The data are shown as % r. intensity of the value obtained from P123H β-syn-overexpressing cells transfected with either vector or wild-type α-syn without PDMP treatment and are expressed as the mean value ± SD (n = 3). *P < 0.05 versus PDMP-untreated cells. B: RT-PCR analysis of β- and α-syn mRNAs. Cyclophilin mRNA was used as an internal control. Similar results were obtained in three independent experiments and there were no significant effects of PDMP treatment (not shown).

Decreased expression of lysosomal membrane proteins 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 24 hours, followed by various analyses. A: Immunoblot analysis of lysosomal membrane proteins. a: Cell extracts (10 μg) were analyzed by immunoblot using anti-ATP13A2 (top), anti-LAMP-2 (middle), and anti-actin (bottom). In b, the band intensities corresponding to ATP13A2 and LAMP-2 were quantified and corrected with the intensities of actin bands. The data are shown as % r. intensity of the value obtained from P123H β-syn-overexpressing cells transfected with vector and are expressed as the mean value ± SD (n = 4). *P < 0.05 versus PDMP-untreated cells. In c, HEK293 cells transfected with either vector or ATP13A2 were used as controls. The blots are representative of three experiments. B: Effects of proteasome inhibitors on PDMP-induced down-regulation of lysosomal membrane proteins. a: P123H β-syn-overexpressing cells transfected with A53T α-syn were treated with or without PDMP in the absence or presence of MG132 (10 μmol/L). Cell extracts (10 μg) were analyzed by immunoblot using anti-ATP13A2 (top), anti-LAMP-2 (middle), and anti-actin (bottom). In b, quantification was performed. The data are shown as % r. intensity of the value obtained from P123H β-syn-overexpressing cells transfected with A53T α-syn and are expressed as the mean value ± SD (n = 4). *P < 0.05, versus MG132-untreated cells. C: RT-PCR analysis of ATP13A2 and LAMP-2 mRNAs. Representative figures are shown in a. Cyclophilin mRNA was used as an internal control. In b, the band intensities corresponding to ATP13A2 and LAMP-2 were quantified and corrected with the intensities of cyclophilin (cyclophil.) bands. The data are shown as % r. intensity of the value obtained from P123H β-syn-overexpressing cells transfected with vector without PDMP treatment and are expressed as the mean value ± SD (n = 4). *P < 0.05 versus PDMP-untreated cells.

PDMP-induced lysosomal pathology is ameliorated by addition of gangliosides. P123H β-syn-overexpressing cells were transfected with A53T α-syn. Cells were then treated with or without PDMP (25 μmol/L) in the presence of either ganglioside mixtures (G. Mix.) (50 μg/ml) or vehicle (0.1% methanol) for 24 hours, followed by various analyses. A: Quantification of Lucifer Yellow redistribution. The number of Lucifer Yellow-positive cells in their cytosols 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 G. Mix.-untreated cells. B: Quantification of LDH release. LDH release was calculated as a percentage of the total cellular LDH. Data are shown as means ± SD (n = 4). *P < 0.05, **P < 0.01 versus G. Mix.-untreated cells. C: Immunoblot analysis of the autophagy regulatory molecules and syn proteins. a: The soluble fractions of cell extracts (10 μg) were analyzed using immunoblot for LC3B, Atg5-Atg12, p-mTOR, t-mTOR, p-p70 S6 kinase, t-p70 S6 kinase, β-syn, α-syn, and actin. In b, the intensities of the bands of these molecules were quantified and corrected with that of actin. For LC3B, the ratio of LC3B-II relative to LC3B-I was calculated and is expressed as the mean value ± SD (n = 4). For Atg5-Atg12, β-syn, and α-syn, the data are shown as % r. intensity of the value obtained from the cells without PDMP treatment and are expressed as the mean value ± SD (n = 4). *P < 0.05 versus G. Mix.-untreated cells.

Increased cell death 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 24 hours, followed by various cell death assays. In some experiments, vector-transfected cells, wild-type β-syn-overexpressing cells, and P123H β-syn-overexpressing cells were also analyzed. Cells were also treated with etoposide (40 μmol/L) or 0.5% DMSO for 24 hours as a positive control for apoptosis. A: TUNEL staining at 24 hours. In a, P123H β-syn-overexpressing cells transfected with vector (a, d), wild-type α-syn (b, e), or A53T α-syn (c, f–h), were treated with either PDMP (d–f, h) or etoposide (40 μmol/L) (g) for 24 hours, followed by TUNEL staining. Areas in f are equivalent to h. In all of the figures, nuclei were simultaneously stained with DAPI. In b, P123H β-syn-overexpressing cells transfected with A53T α-syn were treated without or with PDMP, 0.5% DMSO, or etoposide for 24 hours. Cell extracts (10 μg) were analyzed by immunoblot analysis using anti-cleaved caspase-3. The filter was reprobed with anti-actin antibody. B: Cell death assay at 24 hours. a: LDH release was calculated as a percentage of the total cellular LDH. Data are shown as means ± SD (n = 4). *P < 0.05, **P < 0.01 versus PDMP-untreated cells. b: WST-1 assay was performed by measurement of the absorbance values at 450 nm. Cell survival was calculated as described in the Materials and Methods. Data are shown as means ± SD (n = 4). *P < 0.05, **P < 0.01 versus PDMP-untreated cells. C: Effects of cathepsin inhibitors and autophagy modulators on LDH release at 24 hours. In a, P123H β-syn-overexpressing cells transfected with either vector or A53T α-syn were treated without or with PDMP in the absence or presence of either cathepsin B inhibitor (0.1 μmol/L) or cathepsin D inhibitor (0.5 μmol/L). In b, cells were treated with or without PDMP in the absence or presence of either 3-MA (10 mmol/L) or rapamycin (rap) (0.2 μmol/L). Data are shown as means ± SD (n = 4). *P < 0.05, **P < 0.01 versus PDMP-treated cells. Scale bars: 100 μm (Aa–Af); 5 μm (Ag and Ah).

Altered expression of the autophagy regulatory molecules 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 24 hours, followed by various analyses. Vector-transfected cells were also analyzed. A: Immunoblot analyses of the autophagy regulatory molecules. Cell extracts (10 μg) were analyzed using immunoblot for LC3B, Beclin-1, Atg5-Atg12, phospho (p)-mTOR, total (t)-mTOR, phospho (p)-p70 S6 kinase, total (t)-p70 S6 kinase, p-4E-BP1, t-4E-BP1, and actin. Representative figures are shown in a. In b, the band intensities corresponding to LC3B-I, LC3B-II, p62, Beclin-1, p-p70 S6 kinase, and t-p70 S6 kinase were quantified and corrected with the intensities of actin bands. The ratio of LC3B-II relative to LC3B-I and that of p-p70 S6 kinase relative to t-p70 S6 kinase were calculated, respectively, and are expressed as the mean value ± SD (n = 4). For p62 and Beclin-1, the data are shown as % r. intensity of the value obtained from vector-transfected cells without PDMP treatment and are expressed as the mean value ± SD (n = 4). *P < 0.05 versus PDMP-untreated cells. B: RT-PCR analysis of P62, Beclin-1, Atg5, and mTOR mRNAs. Representative figures are shown in a. Cyclophilin mRNA was used as an internal control. In b, the band intensities corresponding to p62 and Beclin-1 were quantified and corrected with the intensities of cyclophilin (cyclophil.) bands. The data are shown as % r. intensity of the value obtained from P123H β-syn-overexpressing cells transfected with vector without PDMP treatment and are expressed as the mean value ± SD (n = 4). *P < 0.05 versus PDMP-untreated cells. C: Double immunofluorescence/LSCM of LC3B and β-syn. Vector-transfected cells (a--c) and P123H β-syn-overexpressing cells transfected with A53T α-syn were treated without (d--f) or with (g--i) PDMP, followed by immunostaining for LC3B (green; a, d, and g) and β-syn (red; b, e, and h). Nuclei are simultaneously stained with DAPI in the merged figures (c, f, and i). Small boxes in d and g are equivalent to j and k, respectively. Arrowheads indicate lysosomal inclusion bodies. Similar results were observed in three independent experiments. D: Triple immunofluorescence/LSCM of LC3B, P62, and β-syn. P123H β-syn-overexpressing cells transfected with A53T α-syn were treated with PDMP, followed by immunostaining for LC3B (green; a), β-syn (red; b), and P62 (blue; c). d: Three molecules were co-localized in some inclusion bodies after PDMP treatment. Arrowheads indicate lysosomal inclusion bodies. Similar results were observed in three independent experiments. Scale bars: 20 μm (Ca--Ci); 10 μm (Cj, Ck, D).

Compromised lysosomal activities 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 24 hours, followed by various evaluations. In some experiments, vector-transfected cells, wild-type β-syn-overexpressing cells, and P123H β-syn-overexpressing cells were also analyzed. A: Double immunofluorescence/LSCM of GM2 and β-syn for P123H β-syn-overexpressing cells transfected with A53T α-syn.. All figures are shown at the same magnification. Arrowheads indicate the inclusion formation. Note that the anti-GM2 immunoreactivity is remarkably diminished by treatment with PDMP (d--f) but not with L-PDMP (g--i). Similar results were observed in three independent experiments. B: Immunoblot analysis of the anti-GM2 immunoreactivities. In a, cell extracts (10 μg) were analyzed by immunoblot using anti-GM2. Equivalent protein loading in each lane was confirmed by immunoblotting with anti-actin antibody. In b, the band intensities corresponding to GM2/GM2AP were quantified and corrected with those of actin. The data are shown as a percentage of the relative intensity (% r. intensity) obtained from P123H β-syn-overexpressing cells transfected with vector and are expressed as the mean value ± SD (n = 4). *P < 0.05 versus PDMP-untreated cells. In c, cell extracts of P123H β-syn-overexpressing cells transfected with A53T α-syn were delipidated as described in Materials and Methods. In d, the band intensities corresponding to GM2/GM2AP were quantified and corrected with the intensities of actin bands. The data are shown as % r. intensity of the value obtained from PDMP-untreated/nondelipidated cells and are expressed as the mean value ± SD (n = 4). *P < 0.05, **P < 0.01 versus PDMP-untreated cells. C: Measurement of lysosomal activities. Cell extracts (10 μg) were incubated at 37°C with fluorogenic cathepsin B substrate to measure the activity of cathepsin B. Released fluorescence (excitation, 380 nm; emission, 460 nm) was monitored every 5 minutes up to 60 minutes. Fluorogenic intensity of each time point was plotted and the slope was calculated. The data are expressed as arbitrary units (A.U.) and shown as the mean value ± SD (n = 4). *P < 0.05, **P < 0.01 versus PDMP-untreated cells. D: LysoSensor staining to evaluate lysosomal activities. In a, P123H β-syn-overexpressing cells transfected with A53T α-syn and vector-transfected cells treated without (a, b) or with (c, d) PDMP were stained with LysoSensor Green DND-153. In b, the intensities of LysoSensor green fluorescence corresponding to a were measured by software FV10-ASW 1.7 (Olympus, Tokyo, Japan). The data are shown as % r. intensity of the fluorescence of vector-transfected cells without PDMP treatment and are expressed as the mean value ± SD (n = 4). *P < 0.05, **P < 0.01 versus PDMP-untreated cells. Scale bars: 20 μm (A); 10 μm (D).

Ultrastructural analyses of PDMP-treated syn-overexpressing cells. Electron microscopic analyses were performed for P123H β-syn-overexpressing cells transfected with A53T α-syn, followed by treatment with (Ad–Af, Ba–Bd) or without (Ab and Ac) PDMP treatment for 24 hours. Vector-transfected cells were also analyzed as a control (Aa). A: At a low magnification, P123H β-syn-overexpressing cells transfected with A53T α-syn exhibited various sizes of electron-dense inclusion clusters (b and e), whereas fewer lysosomal structures were found in vector-transfected cells (a). c: At a higher magnification, some large electron-dense inclusions were composed of multilamellar myelinosomes whose membranes were tightly packed with each other, whereas other small electron-dense inclusions made a fusion with relatively light dense structures of unknown origins. f: After treatment with PDMP, the tightly packed structures of multilamellar myelinosomes became so loose as to give rise to a gap between each membrane. d: Further, empty inclusion bodies, the contents of which were probably discharged into the cytoplasm, were frequently encountered. Small boxes in b and e are equivalent to c and f, respectively. In g–i, morphometric analysis was performed as described in the Materials and Methods. g: The numbers of lysosomal inclusions were higher in both P123H β-syn-overexpressing cells transfected with A53T α-syn compared with vector-transfected cells. However, there were no significant changes of the numbers of lysosomes before and after PDMP treatment. h: Similar results were obtained when the lysosomal structures were categorized into two groups according to their diameters; small: 400 nm to ∼1 μm, and big: more than 1 μm. In i, the numbers of empty giant inclusion and loose lamellar structures of inclusions were significantly increased after PDMP treatment in P123H β-syn-overexpressing cells transfected with A53T α-syn and to a lesser extent in vector-transfected cells. Data are shown as means ± SD (n = 50 cells). *P < 0.05, **P < 0.01 versus PDMP-untreated cells. B: In PDMP-treated P123H β-syn-overexpressing cells transfected with A53T α-syn, there was extensive formation of autophagic vacuoles (b, arrow) and unclosed double-membrane structures (b, arrowheads). Furthermore, mitochondria (d, arrowheads) and ER (d, asterisks) were frequently swollen and distorted. Small boxes in a and c are equivalent to b and d, respectively. In both A and B, the electron micrograph is representative of at least three independent experiments. Scale bars: 4 μm (Aa, Ab, Ad, Ae, Ba, Bc); 1 μm (Ac, Af, Bb, Bd).

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).