Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Aug;9(8):e1003711.
doi: 10.1371/journal.pgen.1003711. Epub 2013 Aug 29.

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

M Catarina Silva  1 , Margarida D Amaral, Richard I Morimoto
Affiliations

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

M Catarina Silva et al. PLoS Genet. 2013 Aug.

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.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Knockdown of gei-11 suppresses polyQ aggregation and toxicity.
(A) gei-11 RNAi suppressed Q35 aggregation in BWM cells of 6 day old animals, shown by the diffuse fluorescent pattern in II, IV and VI, in contrast to a foci-like pattern in the vector control I, III, V. Scale bar: 0.1 mm (I–IV), 0.025 mm (V–VI). Boxed areas correspond to the magnified images below. (B) FRAP analysis shows relative fluorescence intensity recovery at each time-point post-photobleaching. Control Q35 foci (in black; vector) revealed no fluorescence recovery, while gei-11-treated animals showed complete recovery of fluorescence (in blue), analogous to the soluble Q24 control (in red). Each curve represents an average of >12 independent measurements for gei-11 RNAi, and >5 for the controls. (C) Motility assay for 6 day old Q35 and wt animals fed with vector, gei-11 or yfp RNAi, measured in body-length-per-second and relative to wt speed in vector control (100%) (±SEM, Student t-test **p<0.01, ***p<0.001).
Figure 2
Figure 2. 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 GABAR unc-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.
Figure 3
Figure 3. Rescue of proteostasis through HSF-1 activation.
(A) gei-11 RNAi (0.27±0.070 knockdown) suppressed the TS toxic phenotypes of UNC-15 (paramyosin; unc/slow movement), UNC-52 (perlecan, stiff paralysis), UNC-45 (myosin assembly, egg laying defect) and UNC-54 (myosin, paralysis). 15°C is the permissive temperature, 25°C is the restrictive temperature and 23°C is the temperature for RNAi (±SD, Student t-test **p<0.01). (B) gei-11 double knockdown with hsf-1 or hsp-70 (C12C8.1) abolished the suppressor effect on Q35 aggregation (±SD, Student t-test ***p<0.001, see Table S1). (C) Real-time qPCR analysis of chaperone (Hsp-70 family C12C8.1, F44E5.4, C30C11.4; and small Hsps hsp-16.1, hsp-12.6, hsp-16.49) levels in wt, mutant unc-29(e1072) and hsf-1(sy441) animals, treated with gei-11 RNAi (0.24±0.070 knockdown). Data are relative to wt;vector (±SD). (D) Real-time qPCR analysis of hsp-70 (C12C8.1, F44E5.4) levels in 5 day old wt animals upon co-treatment with gei-11 RNAi (0.16±0.082 knockdown) and AChR antagonist dTBC; and gei-11 RNAi in the background of AChR mutants unc-29(e1072) or unc-63(x26), relative to wt in vector control (±SD). (E) Real-time qPCR shows upregulation of hsp levels upon muscle specific gei-11 knockdown in rde-1(ne219);myo-3p-RDE-1 animals, relative to rde-1(ne219);myo-3p-RDE-1;vector (±SD). (F) Gel mobility shift analysis shows that gei-11 RNAi induced HSF-1 DNA binding (lanes 6) in a similar way to heat shock at 35°C (lanes 3). Assay performed with a [32P]HSE oligonucleotide, HSEmutant refers to a mutated oligonucleotide in the HSE (lanes 5,8), and +self refers to competition with 100-fold molar excess of unlabeled oligonucleotide (lanes 4,7). Control (lanes 2,3) refers to animals on vector RNAi.
Figure 4
Figure 4. Modulation of AChR and GABAR can restore post-synaptic folding.
(A) At the C. elegans NMJ, the functional balance between GABAR and AChR signaling regulates post-synaptic muscle function. (B) L-AChR activation with the agonist levamisole (in water) suppressed Q35 aggregation at 5 µM, but enhanced aggregation at 50 µM. Mutant AChR unc-38(e264) is a control for AChR-mediated effect. (C) Reduction in GABAR function with lindane (in 10% EtOH) suppressed Q35 aggregation at 25 µM, and enhanced aggregation at 1 mM concentration (relative to EtOH control treatment). (D) Effect on Q35 aggregation by decrease in GABA with unc-49 or unc-47 RNAi, and by inhibition of GABA in unc-47(gk192) or unc-30(e191) mutant backgrounds. (E) Incubation with 50–200 mM GABA (in water) suppressed Q35 aggregation. GABA at 50 mM abolished the suppressor effect of gei-11, by “re-balancing” the GABAergic-cholinergic signaling. (F) Real-time qPCR analysis of hsp-70 (C12C8.1, F44E5.4) levels in 5 day old wt animals upon treatment with ACh, levamisole or the GABAR antagonist Lindane, or upon decrease in GABAergic signaling by either RNAi or mutant backgrounds of unc-47(gk192), unc-49(e407) or unc-30(e191). Student t-test **p<0.01 and ***p<0.001; data and statistics are relative to Q35;vector control (±SD) (RNAi controls: Table S1).
Figure 5
Figure 5. Ca2+-dependent kinases required for activation of the HSR and folding enhancement.
(A) Cholinergic signaling at the NMJ activates muscle EGL-19 and Ca2+ flux into the cytoplasm of muscle cells, which further activates the ryanodine receptor (RYR) at the SR for muscle contraction. (B) Double knockdown of gei-11 with calmodulin cal-1, cal-2, or cal-4; or Ca2+-dependent kinase unc-43, pkc-1, pkc-3, or gsk-3, prevented suppression of Q35 aggregation (±SD). % of foci are relative to Q35 in vector RNAi; Student t-test p<0.001. (C) Real-time qPCR analysis of hsp-70 levels in wt animals upon double RNAi of gei-11 with the indicated genes (±SD). Data are relative to vector-treated wt animals. gei-11 levels were 0.23±0.101 upon RNAi, relative to vector sample.
Figure 6
Figure 6. EGL-19- and RYR-mediated Ca2+ influx are components of the proteostasis rescue mechanism.
Ca2+ relevance for gei-11 effect on (A) Q35 aggregation (B) and hsp-70 (C12C8.1, F44E5.4) upregulation, tested by employing a hypomorphic mutant egl-19(n582), a weak hypermorph egl-19(n582ad952), a hypermorph egl-19(ad695), egl-19 RNAi (control RNAi in Table S1) or the specific EGL-19 antagonist Nemadipine A (0.75 µM, DMSO). Student t-test *p<0.05, ***p<0.001, ns/not significant; data relative to vector control or control in DMSO (±SD). (C) The RYR agonists ryanodine (50 nM, EtOH) and 4-CmC (10 µM, water) suppressed Q35 aggregation in a similar way to gei-11 RNAi, but were less efficient in Q35;egl-19(n582) hypomorphic mutant animals. Treatment with the RYR antagonist DS (DMSO) together with gei-11 RNAi prevented suppression of Q35 aggregation. Student t-test ***p<0.001, ns/not significant; data relative to vector control in respective compound % solvent (±SD). (D) Real-time qPCR analysis of hsp-70 (C12C8.1, F44E5.4) levels: RYR agonists Ryr (50 nM) and 4-CmC (10 µM) up-regulated hsp-70 in wt animals but not in mutant egl-19(n582) animals. Chaperone induction by gei-11 RNAi was prevented in the RYR mutant (unc-68(kh30)) and by co-treatment with DS (±SD). gei-11 levels were 0.27±0.150 upon RNAi, relative to vector sample. (E) Model for gei-11 modulation of proteostasis in BWM. [a] Knockdown of gei-11 by RNAi leads to an increase in L-AChR expression at the NMJ (dashed line: proposed genetic interaction). [b] This causes a shift in the cholinergic/GABAergic signaling at the NMJ towards higher (thick arrow) excitatory signaling into the muscle. ACh binding to AChRs activates the VGCC EGL-19. [c] Depolarization, conformational changes and Ca2+ influx through EGL-19 triggers the opening of RYR at the SR and further release of Ca2+ into the cytosol [d]. Ca2+ activates signaling cascades to promote muscle contraction [e], HSF-1 activation [f ] and expression of cytosolic chaperones that rescue protein folding in the cytosol [g]. Dashed lines represent proposed and simplified sequence of events.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Balch WE, Morimoto RI, Dillin A, Kelly JW (2008) Adapting proteostasis for disease intervention. Science 319: 916–919. - PubMed
    1. Morimoto RI (2008) Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging. Genes Dev 22: 1427–1438. - PMC - PubMed
    1. Morimoto RI (2012) The Heat Shock Response: Systems Biology of Proteotoxic Stress in Aging and Disease. In: Press CSH, editor. Cold Spring Harbor Symposia on Quantitative Biology: Metabolism and Disease. pp. 91–99. - PubMed
    1. Gidalevitz T, Ben-Zvi A, Ho KH, Brignull HR, Morimoto RI (2006) Progressive disruption of cellular protein folding in models of polyglutamine diseases. Science 311: 1471–1474. - PubMed
    1. Gidalevitz T, Krupinski T, Garcia S, Morimoto RI (2009) Destabilizing protein polymorphisms in the genetic background direct phenotypic expression of mutant SOD1 toxicity. PLoS genetics 5: e1000399. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources

  NODES
Project 1
twitter 2