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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 and rapamycin promote the accumulation of LC3-II and the formation of LC3 positive puncta which is inhibited by 3-MA.(A) Immunoblot analysis of LC3 and HSP70. Cells were treated with 17-AAG (AAG, 50 nM), rapamycin (20 µM), or 3-methyladenine (3-MA, 10 mM) or with a combination of 17-AAG and 3-MA for 7 h and 24 h, respectively. Cell lysates were prepared and immunoblot analysis was carried out with antibodies against the individual proteins as indicated on the right. Co, untreated control. (B) OLN-A53T cells were either untreated (Co, a–c), or treated for 24 h with 50 nM 17AAG (AAG, d–f), or with a combination of 50 nM 17AAG and 10 mM 3-methyladenine (3-MA, g–i), or with 20 µM rapamycin (Rapa, j–l), and then subjected to indirect immunofluorescence staining using antibodies against α-synuclein (SNL-4, a,d,g,j; red) and LC3 (b,e,h,k, green). In (c,f,i,l) the overlays with DAPI staining are shown. Scale bar, 25 µm. (C) Confocal images of cells treated with 17-AAG as in (B, d–f) are shown. Arrow heads indicate colocalization of LC3 staining with α-synuclein. Scale bar, 5 µm.
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pone-0008753-g007: 17-AAG and rapamycin promote the accumulation of LC3-II and the formation of LC3 positive puncta which is inhibited by 3-MA.(A) Immunoblot analysis of LC3 and HSP70. Cells were treated with 17-AAG (AAG, 50 nM), rapamycin (20 µM), or 3-methyladenine (3-MA, 10 mM) or with a combination of 17-AAG and 3-MA for 7 h and 24 h, respectively. Cell lysates were prepared and immunoblot analysis was carried out with antibodies against the individual proteins as indicated on the right. Co, untreated control. (B) OLN-A53T cells were either untreated (Co, a–c), or treated for 24 h with 50 nM 17AAG (AAG, d–f), or with a combination of 50 nM 17AAG and 10 mM 3-methyladenine (3-MA, g–i), or with 20 µM rapamycin (Rapa, j–l), and then subjected to indirect immunofluorescence staining using antibodies against α-synuclein (SNL-4, a,d,g,j; red) and LC3 (b,e,h,k, green). In (c,f,i,l) the overlays with DAPI staining are shown. Scale bar, 25 µm. (C) Confocal images of cells treated with 17-AAG as in (B, d–f) are shown. Arrow heads indicate colocalization of LC3 staining with α-synuclein. Scale bar, 5 µm.

Mentions: The contribution of macroautophagy to the degradation of α-synuclein in OLN-A53T cells was further confirmed by using the selective inhibitor of macroautophagy, 3-methyladenine (3-MA). In cells incubated in the presence of 17-AAG (50 nM) and 3-MA (10 mM) simultaneously for 24 h, α-synuclein positive aggregates remained to be present throughout the cytoplasm, and thus the aggregate clearing effect of 17-AAG was abolished (Fig. 6C). Immunoblot analysis of cell extracts depicted that application of 3-MA (10 mM) alone or in combination with 17-AAG (50 nM; 7–24 h) prevented (after 7 h) or reduced (after 24 h) the formation of LC3-II, while the induction of HSP70 was not affected (Fig. 7A). Additionally as demonstrated by indirect immunofluorecence staining, in cells treated for 24 h with 17-AAG (50 nM) or rapamycin (20 µM) alone, LC3 positive puncta had been formed abundantly and were seen throughout the cytoplasma (Fig. 7B). In contrast thereto in control cells and cells treated for 24 h with 3-MA (10 mM) and 17-AAG (50 nM) in combination, LC3 immunoreactivity was rarely seen (Fig. 7B). Also, confocal microscopy indicates that in cells after treatment with 17-AAG, α-synuclein immunoreactivity occasionally was detectable in close proximity or in colocalization with LC3-positive vesicles (Fig. 7C).


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 and rapamycin promote the accumulation of LC3-II and the formation of LC3 positive puncta which is inhibited by 3-MA.(A) Immunoblot analysis of LC3 and HSP70. Cells were treated with 17-AAG (AAG, 50 nM), rapamycin (20 µM), or 3-methyladenine (3-MA, 10 mM) or with a combination of 17-AAG and 3-MA for 7 h and 24 h, respectively. Cell lysates were prepared and immunoblot analysis was carried out with antibodies against the individual proteins as indicated on the right. Co, untreated control. (B) OLN-A53T cells were either untreated (Co, a–c), or treated for 24 h with 50 nM 17AAG (AAG, d–f), or with a combination of 50 nM 17AAG and 10 mM 3-methyladenine (3-MA, g–i), or with 20 µM rapamycin (Rapa, j–l), and then subjected to indirect immunofluorescence staining using antibodies against α-synuclein (SNL-4, a,d,g,j; red) and LC3 (b,e,h,k, green). In (c,f,i,l) the overlays with DAPI staining are shown. Scale bar, 25 µm. (C) Confocal images of cells treated with 17-AAG as in (B, d–f) are shown. Arrow heads indicate colocalization of LC3 staining with α-synuclein. Scale bar, 5 µm.
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pone-0008753-g007: 17-AAG and rapamycin promote the accumulation of LC3-II and the formation of LC3 positive puncta which is inhibited by 3-MA.(A) Immunoblot analysis of LC3 and HSP70. Cells were treated with 17-AAG (AAG, 50 nM), rapamycin (20 µM), or 3-methyladenine (3-MA, 10 mM) or with a combination of 17-AAG and 3-MA for 7 h and 24 h, respectively. Cell lysates were prepared and immunoblot analysis was carried out with antibodies against the individual proteins as indicated on the right. Co, untreated control. (B) OLN-A53T cells were either untreated (Co, a–c), or treated for 24 h with 50 nM 17AAG (AAG, d–f), or with a combination of 50 nM 17AAG and 10 mM 3-methyladenine (3-MA, g–i), or with 20 µM rapamycin (Rapa, j–l), and then subjected to indirect immunofluorescence staining using antibodies against α-synuclein (SNL-4, a,d,g,j; red) and LC3 (b,e,h,k, green). In (c,f,i,l) the overlays with DAPI staining are shown. Scale bar, 25 µm. (C) Confocal images of cells treated with 17-AAG as in (B, d–f) are shown. Arrow heads indicate colocalization of LC3 staining with α-synuclein. Scale bar, 5 µm.
Mentions: The contribution of macroautophagy to the degradation of α-synuclein in OLN-A53T cells was further confirmed by using the selective inhibitor of macroautophagy, 3-methyladenine (3-MA). In cells incubated in the presence of 17-AAG (50 nM) and 3-MA (10 mM) simultaneously for 24 h, α-synuclein positive aggregates remained to be present throughout the cytoplasm, and thus the aggregate clearing effect of 17-AAG was abolished (Fig. 6C). Immunoblot analysis of cell extracts depicted that application of 3-MA (10 mM) alone or in combination with 17-AAG (50 nM; 7–24 h) prevented (after 7 h) or reduced (after 24 h) the formation of LC3-II, while the induction of HSP70 was not affected (Fig. 7A). Additionally as demonstrated by indirect immunofluorecence staining, in cells treated for 24 h with 17-AAG (50 nM) or rapamycin (20 µM) alone, LC3 positive puncta had been formed abundantly and were seen throughout the cytoplasma (Fig. 7B). In contrast thereto in control cells and cells treated for 24 h with 3-MA (10 mM) and 17-AAG (50 nM) in combination, LC3 immunoreactivity was rarely seen (Fig. 7B). Also, confocal microscopy indicates that in cells after treatment with 17-AAG, α-synuclein immunoreactivity occasionally was detectable in close proximity or in colocalization with LC3-positive vesicles (Fig. 7C).

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