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A bacterial metabolite induces glutathione-tractable proteostatic damage, proteasomal disturbances, and PINK1-dependent autophagy in C. elegans.

Martinez BA, Kim H, Ray A, Caldwell GA, Caldwell KA - Cell Death Dis (2015)

Bottom Line: To determine remitting counter agents, we investigated several established antioxidants and found that glutathione (GSH) can significantly protect against metabolite-induced proteostasis disruption.In addition, GSH protects against the toxicity of MG132 and can compensate for the combined loss of both pink-1 and the E3 ligase pdr-1, a Parkin homolog.These studies mechanistically advance our understanding of a putative environmental contributor to neurodegeneration and factors influencing in vivo neurotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL, USA.

ABSTRACT
Gene-by-environment interactions are thought to underlie the majority of idiopathic cases of neurodegenerative disease. Recently, we reported that an environmental metabolite extracted from Streptomyces venezuelae increases ROS and damages mitochondria, leading to eventual neurodegeneration of C. elegans dopaminergic neurons. Here we link those data to idiopathic disease models that predict loss of protein handling as a component of disorder progression. We demonstrate that the bacterial metabolite leads to proteostatic disruption in multiple protein-misfolding models and has the potential to synergistically enhance the toxicity of aggregate-prone proteins. Genetically, this metabolite is epistatically regulated by loss-of-function to pink-1, the C. elegans PARK6 homolog responsible for mitochondrial maintenance and autophagy in other animal systems. In addition, the metabolite works through a genetic pathway analogous to loss-of-function in the ubiquitin proteasome system (UPS), which we find is also epistatically regulated by loss of PINK-1 homeostasis. To determine remitting counter agents, we investigated several established antioxidants and found that glutathione (GSH) can significantly protect against metabolite-induced proteostasis disruption. In addition, GSH protects against the toxicity of MG132 and can compensate for the combined loss of both pink-1 and the E3 ligase pdr-1, a Parkin homolog. In assessing the impact of this metabolite on mitochondrial maintenance, we observe that it causes fragmentation of mitochondria that is attenuated by GSH and an initial surge in PINK-1-dependent autophagy. These studies mechanistically advance our understanding of a putative environmental contributor to neurodegeneration and factors influencing in vivo neurotoxicity.

No MeSH data available.


Related in: MedlinePlus

GSH attenuates enhanced α-synuclein proteotoxicity and proteasomal dysfunction associated with the metabolite. Nematodes were exposed to the bacterial metabolite chronically for all neurodegeneration assays as described in the Figure 1 legend whereas animals were exposed to the metabolite semi-acutely when they expressed alpha synuclein in bodywall muscle cells, as described in the Figure 2 legend. RNAi was performed in a worm strain whereby RNAi knockdown would occur only in dopaminergic neurons (cell-autonomous RNAi).33 (a) Animals expressing α-syn in the dopaminergic neurons were assessed for neurodegeneration in the context of 1 mM GSH. Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test. (b) Animals expressing α-syn in the bodywall muscle cells were assessed for apparent aggregate density in the context of 1 mM GSH. Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test. (c) Animals bearing dopaminergic overexpression of SID-1 (a dsRNA transporter) in a mutant background for sid-1, used to selectively target RNAi to dopaminergic neurons in an α-syn background were exposed to 1 mM IPTG plates and either empty vector control (EV) or RNAi treatment paradigms affecting the UPS at 6 days post hatching. RNAi was initiated at the L4 larval stage to exclude potential developmental defects. Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test. (d) Animals were treated with 10 μM MG132 with 1 mM GSH in the context of neurodegeneration through enhanced α-syn toxicity elicited by the S. ven metabolite. EtAc and 0.1% DMSO were used where appropriate to serve as solvent controls. Animals were placed on MG132 concentrations at the larval L4 stage to exclude the possibility of developmental defects. Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ****P<0.0001 was assessed by two-way ANOVA with Tukey's post hoc test. (e) Animals were treated with 10 μM MG132 (using 0.1% DMSO as a solvent control) and metabolite as described in (a) in the context of cell-autonomous RNAi reduction of the GSH synthesis enzyme gcs-1 or empty vector control (EV). Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test. (f) Animals were treated with 10 μM MG132 as described in (a) in conjunction with 1 mM buthionine sulfoximine (BSO) in the context of neurodegeneration through enhanced α-syn toxicity elicited by the S. ven metabolite. EtAc and 0.1% DMSO were used where appropriate to serve as solvent controls. Data represented as mean±S.D; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test
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fig3: GSH attenuates enhanced α-synuclein proteotoxicity and proteasomal dysfunction associated with the metabolite. Nematodes were exposed to the bacterial metabolite chronically for all neurodegeneration assays as described in the Figure 1 legend whereas animals were exposed to the metabolite semi-acutely when they expressed alpha synuclein in bodywall muscle cells, as described in the Figure 2 legend. RNAi was performed in a worm strain whereby RNAi knockdown would occur only in dopaminergic neurons (cell-autonomous RNAi).33 (a) Animals expressing α-syn in the dopaminergic neurons were assessed for neurodegeneration in the context of 1 mM GSH. Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test. (b) Animals expressing α-syn in the bodywall muscle cells were assessed for apparent aggregate density in the context of 1 mM GSH. Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test. (c) Animals bearing dopaminergic overexpression of SID-1 (a dsRNA transporter) in a mutant background for sid-1, used to selectively target RNAi to dopaminergic neurons in an α-syn background were exposed to 1 mM IPTG plates and either empty vector control (EV) or RNAi treatment paradigms affecting the UPS at 6 days post hatching. RNAi was initiated at the L4 larval stage to exclude potential developmental defects. Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test. (d) Animals were treated with 10 μM MG132 with 1 mM GSH in the context of neurodegeneration through enhanced α-syn toxicity elicited by the S. ven metabolite. EtAc and 0.1% DMSO were used where appropriate to serve as solvent controls. Animals were placed on MG132 concentrations at the larval L4 stage to exclude the possibility of developmental defects. Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ****P<0.0001 was assessed by two-way ANOVA with Tukey's post hoc test. (e) Animals were treated with 10 μM MG132 (using 0.1% DMSO as a solvent control) and metabolite as described in (a) in the context of cell-autonomous RNAi reduction of the GSH synthesis enzyme gcs-1 or empty vector control (EV). Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test. (f) Animals were treated with 10 μM MG132 as described in (a) in conjunction with 1 mM buthionine sulfoximine (BSO) in the context of neurodegeneration through enhanced α-syn toxicity elicited by the S. ven metabolite. EtAc and 0.1% DMSO were used where appropriate to serve as solvent controls. Data represented as mean±S.D; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test

Mentions: We previously demonstrated that the metabolite increases ROS in C. elegans lysates.25 To determine whether oxidative damage may be a component of protein mishandling we treated animals to antioxidants and then measured dopaminergic neurodegeneration or α-synuclein accumulation. Three antioxidants: ascorbic acid,9 uric acid,8 and probucol7, 18 (Supplementary Figures S3a, c, and d) did not attenuate neurotoxicity whereas melatonin10 and GSH20 attenuated neurotoxicity (Supplementary Figure S3b,Figure 3a). Only GSH supplementation suppressed enhanced aggregate formation in C. elegans bodywall muscle cells (Figure 3b; Supplementary Figures S3e and f).


A bacterial metabolite induces glutathione-tractable proteostatic damage, proteasomal disturbances, and PINK1-dependent autophagy in C. elegans.

Martinez BA, Kim H, Ray A, Caldwell GA, Caldwell KA - Cell Death Dis (2015)

GSH attenuates enhanced α-synuclein proteotoxicity and proteasomal dysfunction associated with the metabolite. Nematodes were exposed to the bacterial metabolite chronically for all neurodegeneration assays as described in the Figure 1 legend whereas animals were exposed to the metabolite semi-acutely when they expressed alpha synuclein in bodywall muscle cells, as described in the Figure 2 legend. RNAi was performed in a worm strain whereby RNAi knockdown would occur only in dopaminergic neurons (cell-autonomous RNAi).33 (a) Animals expressing α-syn in the dopaminergic neurons were assessed for neurodegeneration in the context of 1 mM GSH. Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test. (b) Animals expressing α-syn in the bodywall muscle cells were assessed for apparent aggregate density in the context of 1 mM GSH. Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test. (c) Animals bearing dopaminergic overexpression of SID-1 (a dsRNA transporter) in a mutant background for sid-1, used to selectively target RNAi to dopaminergic neurons in an α-syn background were exposed to 1 mM IPTG plates and either empty vector control (EV) or RNAi treatment paradigms affecting the UPS at 6 days post hatching. RNAi was initiated at the L4 larval stage to exclude potential developmental defects. Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test. (d) Animals were treated with 10 μM MG132 with 1 mM GSH in the context of neurodegeneration through enhanced α-syn toxicity elicited by the S. ven metabolite. EtAc and 0.1% DMSO were used where appropriate to serve as solvent controls. Animals were placed on MG132 concentrations at the larval L4 stage to exclude the possibility of developmental defects. Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ****P<0.0001 was assessed by two-way ANOVA with Tukey's post hoc test. (e) Animals were treated with 10 μM MG132 (using 0.1% DMSO as a solvent control) and metabolite as described in (a) in the context of cell-autonomous RNAi reduction of the GSH synthesis enzyme gcs-1 or empty vector control (EV). Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test. (f) Animals were treated with 10 μM MG132 as described in (a) in conjunction with 1 mM buthionine sulfoximine (BSO) in the context of neurodegeneration through enhanced α-syn toxicity elicited by the S. ven metabolite. EtAc and 0.1% DMSO were used where appropriate to serve as solvent controls. Data represented as mean±S.D; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test
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Related In: Results  -  Collection

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fig3: GSH attenuates enhanced α-synuclein proteotoxicity and proteasomal dysfunction associated with the metabolite. Nematodes were exposed to the bacterial metabolite chronically for all neurodegeneration assays as described in the Figure 1 legend whereas animals were exposed to the metabolite semi-acutely when they expressed alpha synuclein in bodywall muscle cells, as described in the Figure 2 legend. RNAi was performed in a worm strain whereby RNAi knockdown would occur only in dopaminergic neurons (cell-autonomous RNAi).33 (a) Animals expressing α-syn in the dopaminergic neurons were assessed for neurodegeneration in the context of 1 mM GSH. Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test. (b) Animals expressing α-syn in the bodywall muscle cells were assessed for apparent aggregate density in the context of 1 mM GSH. Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test. (c) Animals bearing dopaminergic overexpression of SID-1 (a dsRNA transporter) in a mutant background for sid-1, used to selectively target RNAi to dopaminergic neurons in an α-syn background were exposed to 1 mM IPTG plates and either empty vector control (EV) or RNAi treatment paradigms affecting the UPS at 6 days post hatching. RNAi was initiated at the L4 larval stage to exclude potential developmental defects. Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test. (d) Animals were treated with 10 μM MG132 with 1 mM GSH in the context of neurodegeneration through enhanced α-syn toxicity elicited by the S. ven metabolite. EtAc and 0.1% DMSO were used where appropriate to serve as solvent controls. Animals were placed on MG132 concentrations at the larval L4 stage to exclude the possibility of developmental defects. Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ****P<0.0001 was assessed by two-way ANOVA with Tukey's post hoc test. (e) Animals were treated with 10 μM MG132 (using 0.1% DMSO as a solvent control) and metabolite as described in (a) in the context of cell-autonomous RNAi reduction of the GSH synthesis enzyme gcs-1 or empty vector control (EV). Data represented as mean±S.D.; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test. (f) Animals were treated with 10 μM MG132 as described in (a) in conjunction with 1 mM buthionine sulfoximine (BSO) in the context of neurodegeneration through enhanced α-syn toxicity elicited by the S. ven metabolite. EtAc and 0.1% DMSO were used where appropriate to serve as solvent controls. Data represented as mean±S.D; n=30 animals analyzed per treatment in 3–4 replicates. ***P<0.001 was assessed by two-way ANOVA with Tukey's post hoc test
Mentions: We previously demonstrated that the metabolite increases ROS in C. elegans lysates.25 To determine whether oxidative damage may be a component of protein mishandling we treated animals to antioxidants and then measured dopaminergic neurodegeneration or α-synuclein accumulation. Three antioxidants: ascorbic acid,9 uric acid,8 and probucol7, 18 (Supplementary Figures S3a, c, and d) did not attenuate neurotoxicity whereas melatonin10 and GSH20 attenuated neurotoxicity (Supplementary Figure S3b,Figure 3a). Only GSH supplementation suppressed enhanced aggregate formation in C. elegans bodywall muscle cells (Figure 3b; Supplementary Figures S3e and f).

Bottom Line: To determine remitting counter agents, we investigated several established antioxidants and found that glutathione (GSH) can significantly protect against metabolite-induced proteostasis disruption.In addition, GSH protects against the toxicity of MG132 and can compensate for the combined loss of both pink-1 and the E3 ligase pdr-1, a Parkin homolog.These studies mechanistically advance our understanding of a putative environmental contributor to neurodegeneration and factors influencing in vivo neurotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL, USA.

ABSTRACT
Gene-by-environment interactions are thought to underlie the majority of idiopathic cases of neurodegenerative disease. Recently, we reported that an environmental metabolite extracted from Streptomyces venezuelae increases ROS and damages mitochondria, leading to eventual neurodegeneration of C. elegans dopaminergic neurons. Here we link those data to idiopathic disease models that predict loss of protein handling as a component of disorder progression. We demonstrate that the bacterial metabolite leads to proteostatic disruption in multiple protein-misfolding models and has the potential to synergistically enhance the toxicity of aggregate-prone proteins. Genetically, this metabolite is epistatically regulated by loss-of-function to pink-1, the C. elegans PARK6 homolog responsible for mitochondrial maintenance and autophagy in other animal systems. In addition, the metabolite works through a genetic pathway analogous to loss-of-function in the ubiquitin proteasome system (UPS), which we find is also epistatically regulated by loss of PINK-1 homeostasis. To determine remitting counter agents, we investigated several established antioxidants and found that glutathione (GSH) can significantly protect against metabolite-induced proteostasis disruption. In addition, GSH protects against the toxicity of MG132 and can compensate for the combined loss of both pink-1 and the E3 ligase pdr-1, a Parkin homolog. In assessing the impact of this metabolite on mitochondrial maintenance, we observe that it causes fragmentation of mitochondria that is attenuated by GSH and an initial surge in PINK-1-dependent autophagy. These studies mechanistically advance our understanding of a putative environmental contributor to neurodegeneration and factors influencing in vivo neurotoxicity.

No MeSH data available.


Related in: MedlinePlus