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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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Small punctated α-synuclein aggregates are removed by 17-AAG.(A) Hoffman modulation contrast images of OLN-A53T cells are shown. Scale bar, 75 µm. Cells were either untreated (a) or treated with 50 nM 17-AAG for 24 h (b) or 48 h (c). (B) Cells were subjected to indirect immunofluorescence using antibodies against α-syn (SNL-4, red). Nuclei were stained with DAPI. Cells were either untreated (a) or treated with 50 nM 17-AAG for 24 h (b). In c and d, enlargements of the respective regions indicated in a and b are shown. Scale bars, 20 µm (a, b), 2.5 µm (c, d).
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pone-0008753-g001: Small punctated α-synuclein aggregates are removed by 17-AAG.(A) Hoffman modulation contrast images of OLN-A53T cells are shown. Scale bar, 75 µm. Cells were either untreated (a) or treated with 50 nM 17-AAG for 24 h (b) or 48 h (c). (B) Cells were subjected to indirect immunofluorescence using antibodies against α-syn (SNL-4, red). Nuclei were stained with DAPI. Cells were either untreated (a) or treated with 50 nM 17-AAG for 24 h (b). In c and d, enlargements of the respective regions indicated in a and b are shown. Scale bars, 20 µm (a, b), 2.5 µm (c, d).

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)

Small punctated α-synuclein aggregates are removed by 17-AAG.(A) Hoffman modulation contrast images of OLN-A53T cells are shown. Scale bar, 75 µm. Cells were either untreated (a) or treated with 50 nM 17-AAG for 24 h (b) or 48 h (c). (B) Cells were subjected to indirect immunofluorescence using antibodies against α-syn (SNL-4, red). Nuclei were stained with DAPI. Cells were either untreated (a) or treated with 50 nM 17-AAG for 24 h (b). In c and d, enlargements of the respective regions indicated in a and b are shown. Scale bars, 20 µm (a, b), 2.5 µm (c, d).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2807466&req=5

pone-0008753-g001: Small punctated α-synuclein aggregates are removed by 17-AAG.(A) Hoffman modulation contrast images of OLN-A53T cells are shown. Scale bar, 75 µm. Cells were either untreated (a) or treated with 50 nM 17-AAG for 24 h (b) or 48 h (c). (B) Cells were subjected to indirect immunofluorescence using antibodies against α-syn (SNL-4, red). Nuclei were stained with DAPI. Cells were either untreated (a) or treated with 50 nM 17-AAG for 24 h (b). In c and d, enlargements of the respective regions indicated in a and b are shown. Scale bars, 20 µm (a, b), 2.5 µm (c, d).
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