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17-AAG induces cytoplasmic alpha-synuclein aggregate clearance by induction of autophagy.

Riedel M, Goldbaum O, Schwarz L, Schmitt S, Richter-Landsberg C - PLoS ONE (2010)

Bottom Line: By blocking the lysosomal compartment with NH(4)Cl the aggregate clearing effects of 17-AAG were abolished and alpha-synuclein deposits were enlarged.Our data demonstrate for the first time that 17-AAG not only causes the upregulation of heat shock proteins, but also is an effective inducer of the autophagic pathway by which alpha-synuclein can be removed.Hence geldanamycin derivatives may provide a means to modulate autophagy in neural cells, thereby ameliorating pathogenic aggregate formation and protecting the cells during disease and aging.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Molecular Neurobiology, University of Oldenburg, Oldenburg, Germany.

ABSTRACT

Background: The accumulation and aggregation of alpha-synuclein in nerve cells and glia are characteristic features of a number of neurodegenerative diseases termed synucleinopathies. alpha-Synuclein is a highly soluble protein which in a nucleation dependent process is capable of self-aggregation. The causes underlying aggregate formation are not yet understood, impairment of the proteolytic degradation systems might be involved.

Methodology/principal findings: In the present study the possible aggregate clearing effects of the geldanamycin analogue 17-AAG (17-(Allylamino)-17-demethoxygeldanamycin) was investigated. Towards this, an oligodendroglial cell line (OLN-93 cells), stably expressing human alpha-synuclein (A53T mutation) was used. In these cells small punctate aggregates, not staining with thioflavine S, representing prefibrillary aggregates, occur characteristically. Our data demonstrate that 17-AAG attenuated the formation of alpha-synuclein aggregates by stimulating macroautophagy. By blocking the lysosomal compartment with NH(4)Cl the aggregate clearing effects of 17-AAG were abolished and alpha-synuclein deposits were enlarged. Analysis of LC3-II immunoreactivity, which is an indicator of autophagosome formation, further revealed that 17-AAG led to the recruitment of LC3-II and to the formation of LC3 positive puncta. This effect was also observed in cultured oligodendrocytes derived from the brains of newborn rats. Inhibition of macroautophagy by 3-methyladenine prevented 17-AAG induced occurrence of LC3 positive puncta as well as the removal of alpha-synuclein aggregates in OLN-A53T cells.

Conclusions: Our data demonstrate for the first time that 17-AAG not only causes the upregulation of heat shock proteins, but also is an effective inducer of the autophagic pathway by which alpha-synuclein can be removed. Hence geldanamycin derivatives may provide a means to modulate autophagy in neural cells, thereby ameliorating pathogenic aggregate formation and protecting the cells during disease and aging.

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17-AAG induces a heat shock response in OLN-A53T cells and does not impair proteasomal activity.(A) Determination of cytotoxic potential. Cells were exposed to different concentrations of 17-AAG as indicated. After 24 h, neutral red and MTT assays were carried out. Values represent the mean ± SEM of 16 microwells each of two independent experiments (n = 32). (B) Immunoblot analysis of heat shock protein induction. Cells were treated with 17-AAG (1–200 nM, 24 h), or subjected to heat shock (HS: 44°C, 30 min, 24 h recovery) or to MG-132 (MG: 1 µM, 24 h). Cell lysates were prepared and immunoblot analysis was carried out with antibodies against the individual proteins as indicated on the right. Co, untreated control. (C) Proteasomal activity was determined in cell lysates treated with 17-AAG (lysate) and in cell lysates prepared from 17-AAG treated live cells (cells). Cytoplasmic lysates were incubated with the proteasomal inhibitor MG-132 (1 µM, 60 min) as a positive control, or 17-AAG (50 nM, 60 min). Cells were treated with MG-132 (1 µM, 24 h) and 17-AAG (50 nM, 24 h). The post-glutamyl-peptidase-hydrolase activity was determined using fluorogenic substrate Z-Leu-Leu-Glu-AMC (see Materials and Methods). The cleavage of the substrate is inhibited by MG-132 but not by 17-AAG. Data are expressed as percent of the untreated control and show the mean ± SEM from 3 independent experiments. Statistical evaluation was carried out by ANOVA/Fisher's LSD: ***p≤0.01 for MG-132 versus control.
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pone-0008753-g002: 17-AAG induces a heat shock response in OLN-A53T cells and does not impair proteasomal activity.(A) Determination of cytotoxic potential. Cells were exposed to different concentrations of 17-AAG as indicated. After 24 h, neutral red and MTT assays were carried out. Values represent the mean ± SEM of 16 microwells each of two independent experiments (n = 32). (B) Immunoblot analysis of heat shock protein induction. Cells were treated with 17-AAG (1–200 nM, 24 h), or subjected to heat shock (HS: 44°C, 30 min, 24 h recovery) or to MG-132 (MG: 1 µM, 24 h). Cell lysates were prepared and immunoblot analysis was carried out with antibodies against the individual proteins as indicated on the right. Co, untreated control. (C) Proteasomal activity was determined in cell lysates treated with 17-AAG (lysate) and in cell lysates prepared from 17-AAG treated live cells (cells). Cytoplasmic lysates were incubated with the proteasomal inhibitor MG-132 (1 µM, 60 min) as a positive control, or 17-AAG (50 nM, 60 min). Cells were treated with MG-132 (1 µM, 24 h) and 17-AAG (50 nM, 24 h). The post-glutamyl-peptidase-hydrolase activity was determined using fluorogenic substrate Z-Leu-Leu-Glu-AMC (see Materials and Methods). The cleavage of the substrate is inhibited by MG-132 but not by 17-AAG. Data are expressed as percent of the untreated control and show the mean ± SEM from 3 independent experiments. Statistical evaluation was carried out by ANOVA/Fisher's LSD: ***p≤0.01 for MG-132 versus control.

Mentions: The small punctated α-synuclein aggregates in these cells do not stain with thioflavine S and thus represent a prefibrillary species [24]. Tau is not a component of the prefibrillary species. Fig. 1 demonstrates that incubation of the cells with 17-AAG (50 nM) for 24 h caused morphological changes and the clearance of these aggregates. Cells appeared more flattened and partly damaged. To further determine the cytotoxic potential of 17-AAG in OLN-A53T cells, cells were treated with 17-AAG at increasing concentrations for 24 h and cell survival was analyzed. Half maximal cytotoxicity, as determined by neutral red acid uptake or MTT assay, was observed at a concentration of approximately 300 nM, and at a concentration of 25–50 nM about 20 per cent of the cells were affected (Fig. 2A). Geldanamycin was similarly cytotoxic (not shown). After 48 h of treatment with 17-AAG (25–50 nM) no further damage was observable (Fig. 1A,c and 2A).


17-AAG induces cytoplasmic alpha-synuclein aggregate clearance by induction of autophagy.

Riedel M, Goldbaum O, Schwarz L, Schmitt S, Richter-Landsberg C - PLoS ONE (2010)

17-AAG induces a heat shock response in OLN-A53T cells and does not impair proteasomal activity.(A) Determination of cytotoxic potential. Cells were exposed to different concentrations of 17-AAG as indicated. After 24 h, neutral red and MTT assays were carried out. Values represent the mean ± SEM of 16 microwells each of two independent experiments (n = 32). (B) Immunoblot analysis of heat shock protein induction. Cells were treated with 17-AAG (1–200 nM, 24 h), or subjected to heat shock (HS: 44°C, 30 min, 24 h recovery) or to MG-132 (MG: 1 µM, 24 h). Cell lysates were prepared and immunoblot analysis was carried out with antibodies against the individual proteins as indicated on the right. Co, untreated control. (C) Proteasomal activity was determined in cell lysates treated with 17-AAG (lysate) and in cell lysates prepared from 17-AAG treated live cells (cells). Cytoplasmic lysates were incubated with the proteasomal inhibitor MG-132 (1 µM, 60 min) as a positive control, or 17-AAG (50 nM, 60 min). Cells were treated with MG-132 (1 µM, 24 h) and 17-AAG (50 nM, 24 h). The post-glutamyl-peptidase-hydrolase activity was determined using fluorogenic substrate Z-Leu-Leu-Glu-AMC (see Materials and Methods). The cleavage of the substrate is inhibited by MG-132 but not by 17-AAG. Data are expressed as percent of the untreated control and show the mean ± SEM from 3 independent experiments. Statistical evaluation was carried out by ANOVA/Fisher's LSD: ***p≤0.01 for MG-132 versus control.
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pone-0008753-g002: 17-AAG induces a heat shock response in OLN-A53T cells and does not impair proteasomal activity.(A) Determination of cytotoxic potential. Cells were exposed to different concentrations of 17-AAG as indicated. After 24 h, neutral red and MTT assays were carried out. Values represent the mean ± SEM of 16 microwells each of two independent experiments (n = 32). (B) Immunoblot analysis of heat shock protein induction. Cells were treated with 17-AAG (1–200 nM, 24 h), or subjected to heat shock (HS: 44°C, 30 min, 24 h recovery) or to MG-132 (MG: 1 µM, 24 h). Cell lysates were prepared and immunoblot analysis was carried out with antibodies against the individual proteins as indicated on the right. Co, untreated control. (C) Proteasomal activity was determined in cell lysates treated with 17-AAG (lysate) and in cell lysates prepared from 17-AAG treated live cells (cells). Cytoplasmic lysates were incubated with the proteasomal inhibitor MG-132 (1 µM, 60 min) as a positive control, or 17-AAG (50 nM, 60 min). Cells were treated with MG-132 (1 µM, 24 h) and 17-AAG (50 nM, 24 h). The post-glutamyl-peptidase-hydrolase activity was determined using fluorogenic substrate Z-Leu-Leu-Glu-AMC (see Materials and Methods). The cleavage of the substrate is inhibited by MG-132 but not by 17-AAG. Data are expressed as percent of the untreated control and show the mean ± SEM from 3 independent experiments. Statistical evaluation was carried out by ANOVA/Fisher's LSD: ***p≤0.01 for MG-132 versus control.
Mentions: The small punctated α-synuclein aggregates in these cells do not stain with thioflavine S and thus represent a prefibrillary species [24]. Tau is not a component of the prefibrillary species. Fig. 1 demonstrates that incubation of the cells with 17-AAG (50 nM) for 24 h caused morphological changes and the clearance of these aggregates. Cells appeared more flattened and partly damaged. To further determine the cytotoxic potential of 17-AAG in OLN-A53T cells, cells were treated with 17-AAG at increasing concentrations for 24 h and cell survival was analyzed. Half maximal cytotoxicity, as determined by neutral red acid uptake or MTT assay, was observed at a concentration of approximately 300 nM, and at a concentration of 25–50 nM about 20 per cent of the cells were affected (Fig. 2A). Geldanamycin was similarly cytotoxic (not shown). After 48 h of treatment with 17-AAG (25–50 nM) no further damage was observable (Fig. 1A,c and 2A).

Bottom Line: By blocking the lysosomal compartment with NH(4)Cl the aggregate clearing effects of 17-AAG were abolished and alpha-synuclein deposits were enlarged.Our data demonstrate for the first time that 17-AAG not only causes the upregulation of heat shock proteins, but also is an effective inducer of the autophagic pathway by which alpha-synuclein can be removed.Hence geldanamycin derivatives may provide a means to modulate autophagy in neural cells, thereby ameliorating pathogenic aggregate formation and protecting the cells during disease and aging.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Molecular Neurobiology, University of Oldenburg, Oldenburg, Germany.

ABSTRACT

Background: The accumulation and aggregation of alpha-synuclein in nerve cells and glia are characteristic features of a number of neurodegenerative diseases termed synucleinopathies. alpha-Synuclein is a highly soluble protein which in a nucleation dependent process is capable of self-aggregation. The causes underlying aggregate formation are not yet understood, impairment of the proteolytic degradation systems might be involved.

Methodology/principal findings: In the present study the possible aggregate clearing effects of the geldanamycin analogue 17-AAG (17-(Allylamino)-17-demethoxygeldanamycin) was investigated. Towards this, an oligodendroglial cell line (OLN-93 cells), stably expressing human alpha-synuclein (A53T mutation) was used. In these cells small punctate aggregates, not staining with thioflavine S, representing prefibrillary aggregates, occur characteristically. Our data demonstrate that 17-AAG attenuated the formation of alpha-synuclein aggregates by stimulating macroautophagy. By blocking the lysosomal compartment with NH(4)Cl the aggregate clearing effects of 17-AAG were abolished and alpha-synuclein deposits were enlarged. Analysis of LC3-II immunoreactivity, which is an indicator of autophagosome formation, further revealed that 17-AAG led to the recruitment of LC3-II and to the formation of LC3 positive puncta. This effect was also observed in cultured oligodendrocytes derived from the brains of newborn rats. Inhibition of macroautophagy by 3-methyladenine prevented 17-AAG induced occurrence of LC3 positive puncta as well as the removal of alpha-synuclein aggregates in OLN-A53T cells.

Conclusions: Our data demonstrate for the first time that 17-AAG not only causes the upregulation of heat shock proteins, but also is an effective inducer of the autophagic pathway by which alpha-synuclein can be removed. Hence geldanamycin derivatives may provide a means to modulate autophagy in neural cells, thereby ameliorating pathogenic aggregate formation and protecting the cells during disease and aging.

Show MeSH
Related in: MedlinePlus