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Neuronal reprograming of protein homeostasis by calcium-dependent regulation of the heat shock response.

Silva MC, Amaral MD, Morimoto RI - PLoS Genet. (2013)

Bottom Line: This protective effect on muscle cell protein homeostasis was identified in an unbiased genome-wide screening for modifiers of protein aggregation, and is triggered by downregulation of gei-11, a Myb-family factor and proposed regulator of the L-type acetylcholine receptor (AChR).The release of calcium into the cytoplasm of muscle cells activates Ca(2+)-dependent kinases and induces HSF-1-dependent expression of cytoplasmic chaperones, which suppress misfolding of metastable proteins and stabilize the folding environment of muscle cells.This demonstrates that the heat shock response (HSR) can be activated in muscle cells by neuronal signaling across the NMJ to protect proteome health.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, Rice Institute for Biomedical Research, Northwestern University, Evanston, Illinois, United States of America.

ABSTRACT
Protein quality control requires constant surveillance to prevent misfolding, aggregation, and loss of cellular function. There is increasing evidence in metazoans that communication between cells has an important role to ensure organismal health and to prevent stressed cells and tissues from compromising lifespan. Here, we show in C. elegans that a moderate increase in physiological cholinergic signaling at the neuromuscular junction (NMJ) induces the calcium (Ca(2+))-dependent activation of HSF-1 in post-synaptic muscle cells, resulting in suppression of protein misfolding. This protective effect on muscle cell protein homeostasis was identified in an unbiased genome-wide screening for modifiers of protein aggregation, and is triggered by downregulation of gei-11, a Myb-family factor and proposed regulator of the L-type acetylcholine receptor (AChR). This, in-turn, activates the voltage-gated Ca(2+) channel, EGL-19, and the sarcoplasmic reticulum ryanodine receptor in response to acetylcholine signaling. The release of calcium into the cytoplasm of muscle cells activates Ca(2+)-dependent kinases and induces HSF-1-dependent expression of cytoplasmic chaperones, which suppress misfolding of metastable proteins and stabilize the folding environment of muscle cells. This demonstrates that the heat shock response (HSR) can be activated in muscle cells by neuronal signaling across the NMJ to protect proteome health.

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gei-11 knockdown effect through regulation of cholinergic receptors at the NMJ.(A) Real-time qPCR analysis of AChR subunits unc-29, unc-38, unc-63, lev-1 and acr-16, and GABARunc-49, in 6 day old wt animals fed with gei-11 RNAi. Data are normalized to the levels of each gene on vector-treated wt animals (±SD). (B) Suppression of Q35 aggregation by gei-11 RNAi was abolished by co-treatment with L-AChR (unc-38, unc-63, unc-29) but not with N-AChR (acr-16) subunits RNAi (±SD). Individual RNAi controls are shown in light grey (also see Table S1). (C) Cholinergic sensitivity assay: 5 day old animals treated with gei-11 or vector RNAi were scored for paralysis on 1 mM levamisole plates (±SD). L-AChR mutant animals unc-38(e264), unc-63(x26) and unc-29(e1072) were used as controls. Two-way ANOVA and Bonferroni test ***p<0.001 relative to vector control. (D) AChR antagonist dTBC (2.5 mM in water) prevented suppression of Q35 aggregation by gei-11 RNAi (±SD). Q35;unc-38(e264) is a control for AChR-dependent effect. Student t-test ***p<0.001. (E) Real-time qPCR analysis of AChR subunits unc-29, unc-38 and unc-63 upon muscle-specific gei-11 RNAi (rde-1(ne219);mRDE-1, 6 days old), relative to vector control (±SD). (F) Cholinergic sensitivity assay: 5 day old wt, rde-1(ne219);mRDE-1 and rde-1(ne219) animals treated with gei-11 or vector RNAi were scored for paralysis on 1 mM levamisole plates (±SD). Two-way ANOVA and Bonferroni test ***p<0.001, **p<0.01, *p<0.05 relative to vector control. (G) Aggregation quantification upon gei-11 RNAi in Q35, Q35;rde-1(ne219);mRDE-1 (muscle-specific RNAi) and Q35;rde-1(ne219) (impaired RNAi); shown as a relative % to Q35;vector (±SD). Student t-test ***p<0.001, ns/not significant.
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pgen-1003711-g002: gei-11 knockdown effect through regulation of cholinergic receptors at the NMJ.(A) Real-time qPCR analysis of AChR subunits unc-29, unc-38, unc-63, lev-1 and acr-16, and GABARunc-49, in 6 day old wt animals fed with gei-11 RNAi. Data are normalized to the levels of each gene on vector-treated wt animals (±SD). (B) Suppression of Q35 aggregation by gei-11 RNAi was abolished by co-treatment with L-AChR (unc-38, unc-63, unc-29) but not with N-AChR (acr-16) subunits RNAi (±SD). Individual RNAi controls are shown in light grey (also see Table S1). (C) Cholinergic sensitivity assay: 5 day old animals treated with gei-11 or vector RNAi were scored for paralysis on 1 mM levamisole plates (±SD). L-AChR mutant animals unc-38(e264), unc-63(x26) and unc-29(e1072) were used as controls. Two-way ANOVA and Bonferroni test ***p<0.001 relative to vector control. (D) AChR antagonist dTBC (2.5 mM in water) prevented suppression of Q35 aggregation by gei-11 RNAi (±SD). Q35;unc-38(e264) is a control for AChR-dependent effect. Student t-test ***p<0.001. (E) Real-time qPCR analysis of AChR subunits unc-29, unc-38 and unc-63 upon muscle-specific gei-11 RNAi (rde-1(ne219);mRDE-1, 6 days old), relative to vector control (±SD). (F) Cholinergic sensitivity assay: 5 day old wt, rde-1(ne219);mRDE-1 and rde-1(ne219) animals treated with gei-11 or vector RNAi were scored for paralysis on 1 mM levamisole plates (±SD). Two-way ANOVA and Bonferroni test ***p<0.001, **p<0.01, *p<0.05 relative to vector control. (G) Aggregation quantification upon gei-11 RNAi in Q35, Q35;rde-1(ne219);mRDE-1 (muscle-specific RNAi) and Q35;rde-1(ne219) (impaired RNAi); shown as a relative % to Q35;vector (±SD). Student t-test ***p<0.001, ns/not significant.

Mentions: In C. elegans, two types of ACh receptors, each with distinct subunit composition and pharmacology, are expressed at the NMJ [34]–[36]. To establish the specificity of gei-11 downregulation on the expression of NMJ AChR subtypes, we monitored the expression of the L-type (levamisole-sensitive) AChR subunits unc-29, unc-38, unc-63 and lev-1, and the N-type (nicotine-sensitive) homomeric AChR, acr-16. The effect of gei-11 knockdown increased the expression of only the three essential subunits of the L-AChR (unc-29, unc-38, unc-63) by approximately 3-fold, and had no effect on the expression of N-AChR acr-16 (Figure 2A). Likewise, gei-11 RNAi did not affect the expression of the NMJ GABA receptor (GABARunc-49, Figure 2A). These results suggest that gei-11 has a highly selective effect on the regulation of the L-type of cholinergic receptors.


Neuronal reprograming of protein homeostasis by calcium-dependent regulation of the heat shock response.

Silva MC, Amaral MD, Morimoto RI - PLoS Genet. (2013)

gei-11 knockdown effect through regulation of cholinergic receptors at the NMJ.(A) Real-time qPCR analysis of AChR subunits unc-29, unc-38, unc-63, lev-1 and acr-16, and GABARunc-49, in 6 day old wt animals fed with gei-11 RNAi. Data are normalized to the levels of each gene on vector-treated wt animals (±SD). (B) Suppression of Q35 aggregation by gei-11 RNAi was abolished by co-treatment with L-AChR (unc-38, unc-63, unc-29) but not with N-AChR (acr-16) subunits RNAi (±SD). Individual RNAi controls are shown in light grey (also see Table S1). (C) Cholinergic sensitivity assay: 5 day old animals treated with gei-11 or vector RNAi were scored for paralysis on 1 mM levamisole plates (±SD). L-AChR mutant animals unc-38(e264), unc-63(x26) and unc-29(e1072) were used as controls. Two-way ANOVA and Bonferroni test ***p<0.001 relative to vector control. (D) AChR antagonist dTBC (2.5 mM in water) prevented suppression of Q35 aggregation by gei-11 RNAi (±SD). Q35;unc-38(e264) is a control for AChR-dependent effect. Student t-test ***p<0.001. (E) Real-time qPCR analysis of AChR subunits unc-29, unc-38 and unc-63 upon muscle-specific gei-11 RNAi (rde-1(ne219);mRDE-1, 6 days old), relative to vector control (±SD). (F) Cholinergic sensitivity assay: 5 day old wt, rde-1(ne219);mRDE-1 and rde-1(ne219) animals treated with gei-11 or vector RNAi were scored for paralysis on 1 mM levamisole plates (±SD). Two-way ANOVA and Bonferroni test ***p<0.001, **p<0.01, *p<0.05 relative to vector control. (G) Aggregation quantification upon gei-11 RNAi in Q35, Q35;rde-1(ne219);mRDE-1 (muscle-specific RNAi) and Q35;rde-1(ne219) (impaired RNAi); shown as a relative % to Q35;vector (±SD). Student t-test ***p<0.001, ns/not significant.
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pgen-1003711-g002: gei-11 knockdown effect through regulation of cholinergic receptors at the NMJ.(A) Real-time qPCR analysis of AChR subunits unc-29, unc-38, unc-63, lev-1 and acr-16, and GABARunc-49, in 6 day old wt animals fed with gei-11 RNAi. Data are normalized to the levels of each gene on vector-treated wt animals (±SD). (B) Suppression of Q35 aggregation by gei-11 RNAi was abolished by co-treatment with L-AChR (unc-38, unc-63, unc-29) but not with N-AChR (acr-16) subunits RNAi (±SD). Individual RNAi controls are shown in light grey (also see Table S1). (C) Cholinergic sensitivity assay: 5 day old animals treated with gei-11 or vector RNAi were scored for paralysis on 1 mM levamisole plates (±SD). L-AChR mutant animals unc-38(e264), unc-63(x26) and unc-29(e1072) were used as controls. Two-way ANOVA and Bonferroni test ***p<0.001 relative to vector control. (D) AChR antagonist dTBC (2.5 mM in water) prevented suppression of Q35 aggregation by gei-11 RNAi (±SD). Q35;unc-38(e264) is a control for AChR-dependent effect. Student t-test ***p<0.001. (E) Real-time qPCR analysis of AChR subunits unc-29, unc-38 and unc-63 upon muscle-specific gei-11 RNAi (rde-1(ne219);mRDE-1, 6 days old), relative to vector control (±SD). (F) Cholinergic sensitivity assay: 5 day old wt, rde-1(ne219);mRDE-1 and rde-1(ne219) animals treated with gei-11 or vector RNAi were scored for paralysis on 1 mM levamisole plates (±SD). Two-way ANOVA and Bonferroni test ***p<0.001, **p<0.01, *p<0.05 relative to vector control. (G) Aggregation quantification upon gei-11 RNAi in Q35, Q35;rde-1(ne219);mRDE-1 (muscle-specific RNAi) and Q35;rde-1(ne219) (impaired RNAi); shown as a relative % to Q35;vector (±SD). Student t-test ***p<0.001, ns/not significant.
Mentions: In C. elegans, two types of ACh receptors, each with distinct subunit composition and pharmacology, are expressed at the NMJ [34]–[36]. To establish the specificity of gei-11 downregulation on the expression of NMJ AChR subtypes, we monitored the expression of the L-type (levamisole-sensitive) AChR subunits unc-29, unc-38, unc-63 and lev-1, and the N-type (nicotine-sensitive) homomeric AChR, acr-16. The effect of gei-11 knockdown increased the expression of only the three essential subunits of the L-AChR (unc-29, unc-38, unc-63) by approximately 3-fold, and had no effect on the expression of N-AChR acr-16 (Figure 2A). Likewise, gei-11 RNAi did not affect the expression of the NMJ GABA receptor (GABARunc-49, Figure 2A). These results suggest that gei-11 has a highly selective effect on the regulation of the L-type of cholinergic receptors.

Bottom Line: This protective effect on muscle cell protein homeostasis was identified in an unbiased genome-wide screening for modifiers of protein aggregation, and is triggered by downregulation of gei-11, a Myb-family factor and proposed regulator of the L-type acetylcholine receptor (AChR).The release of calcium into the cytoplasm of muscle cells activates Ca(2+)-dependent kinases and induces HSF-1-dependent expression of cytoplasmic chaperones, which suppress misfolding of metastable proteins and stabilize the folding environment of muscle cells.This demonstrates that the heat shock response (HSR) can be activated in muscle cells by neuronal signaling across the NMJ to protect proteome health.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, Rice Institute for Biomedical Research, Northwestern University, Evanston, Illinois, United States of America.

ABSTRACT
Protein quality control requires constant surveillance to prevent misfolding, aggregation, and loss of cellular function. There is increasing evidence in metazoans that communication between cells has an important role to ensure organismal health and to prevent stressed cells and tissues from compromising lifespan. Here, we show in C. elegans that a moderate increase in physiological cholinergic signaling at the neuromuscular junction (NMJ) induces the calcium (Ca(2+))-dependent activation of HSF-1 in post-synaptic muscle cells, resulting in suppression of protein misfolding. This protective effect on muscle cell protein homeostasis was identified in an unbiased genome-wide screening for modifiers of protein aggregation, and is triggered by downregulation of gei-11, a Myb-family factor and proposed regulator of the L-type acetylcholine receptor (AChR). This, in-turn, activates the voltage-gated Ca(2+) channel, EGL-19, and the sarcoplasmic reticulum ryanodine receptor in response to acetylcholine signaling. The release of calcium into the cytoplasm of muscle cells activates Ca(2+)-dependent kinases and induces HSF-1-dependent expression of cytoplasmic chaperones, which suppress misfolding of metastable proteins and stabilize the folding environment of muscle cells. This demonstrates that the heat shock response (HSR) can be activated in muscle cells by neuronal signaling across the NMJ to protect proteome health.

Show MeSH
Related in: MedlinePlus