Neuronal Reprograming of Protein Homeostasis by Calcium-Dependent Regulation of the Heat Shock Response

<div><p>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 <i>C. elegans</i> that a moderate increase in physiological cholinergic signaling at the neuromuscular junction (NMJ) induces the calcium (Ca²⁺)-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 <i>gei-11</i>, a Myb-family factor and proposed regulator of the L-type acetylcholine receptor (AChR). This, in-turn, activates the voltage-gated Ca²⁺ 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 Ca²⁺-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.</p></div

Ca²⁺-dependent kinases required for activation of the HSR and folding enhancement.

<p>(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 <i>gei-11</i> with calmodulin <i>cal-1</i>, <i>cal-2</i>, or <i>cal-4</i>; or Ca²⁺-dependent kinase <i>unc-43</i>, <i>pkc-1</i>, <i>pkc-3</i>, or <i>gsk-3</i>, prevented suppression of Q35 aggregation (±SD). % of foci are relative to Q35 in vector RNAi; Student t-test <i>p</i><0.001. (C) Real-time qPCR analysis of <i>hsp-70</i> levels in wt animals upon double RNAi of <i>gei-11</i> with the indicated genes (±SD). Data are relative to vector-treated wt animals. <i>gei-11</i> levels were 0.23±0.101 upon RNAi, relative to vector sample.</p

Knockdown of <i>gei-11</i> suppresses polyQ aggregation and toxicity.

<p>(A) <i>gei-11</i> 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 <i>gei-11</i>-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 <i>gei-11</i> RNAi, and >5 for the controls. (C) Motility assay for 6 day old Q35 and wt animals fed with vector, <i>gei-11</i> or <i>yfp</i> RNAi, measured in body-length-per-second and relative to wt speed in vector control (100%) (±SEM, Student t-test **<i>p</i><0.01, ***<i>p</i><0.001).</p

EGL-19- and RYR-mediated Ca²⁺ influx are components of the proteostasis rescue mechanism.

<p>Ca²⁺ relevance for <i>gei-11</i> effect on (A) Q35 aggregation (B) and <i>hsp-70</i> (<i>C12C8.1, F44E5.4</i>) upregulation, tested by employing a hypomorphic mutant <i>egl-19(n582)</i>, a weak hypermorph <i>egl-19(n582ad952)</i>, a hypermorph <i>egl-19(ad695), egl-19</i> RNAi (control RNAi in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003711#pgen.1003711.s008" target="_blank">Table S1</a>) or the specific EGL-19 antagonist Nemadipine A (0.75 µM, DMSO). Student t-test *<i>p</i><0.05, ***<i>p</i><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 <i>gei-11</i> RNAi, but were less efficient in Q35<i>;egl-19(n582)</i> hypomorphic mutant animals. Treatment with the RYR antagonist DS (DMSO) together with <i>gei-11</i> RNAi prevented suppression of Q35 aggregation. Student t-test ***<i>p</i><0.001, ns/not significant; data relative to vector control in respective compound % solvent (±SD). (D) Real-time qPCR analysis of <i>hsp-70</i> (<i>C12C8.1, F44E5.4</i>) levels: RYR agonists Ryr (50 nM) and 4-CmC (10 µM) up-regulated <i>hsp-70</i> in wt animals but not in mutant <i>egl-19(n582</i>) animals. Chaperone induction by <i>gei-11</i> RNAi was prevented in the RYR mutant (<i>unc-68(kh30)</i>) and by co-treatment with DS (±SD). <i>gei-11</i> levels were 0.27±0.150 upon RNAi, relative to vector sample. (E) Model for <i>gei-11</i> modulation of proteostasis in BWM. [a] Knockdown of <i>gei-11</i> 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.</p

Modulation of AChR and GABA_R can restore post-synaptic folding.

<p>(A) At the <i>C. elegans</i> NMJ, the functional balance between GABA_R 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 <i>unc-38(e264)</i> is a control for AChR-mediated effect. (C) Reduction in GABA_R 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 <i>unc-49</i> or <i>unc-47</i> RNAi, and by inhibition of GABA in <i>unc-47(gk192)</i> or <i>unc-30(e191)</i> mutant backgrounds. (E) Incubation with 50–200 mM GABA (in water) suppressed Q35 aggregation. GABA at 50 mM abolished the suppressor effect of <i>gei-11</i>, by “re-balancing” the GABAergic-cholinergic signaling. (F) Real-time qPCR analysis of <i>hsp-70</i> (<i>C12C8.1, F44E5.4</i>) levels in 5 day old wt animals upon treatment with ACh, levamisole or the GABA_R antagonist Lindane, or upon decrease in GABAergic signaling by either RNAi or mutant backgrounds of <i>unc-47(gk192)</i>, <i>unc-49(e407)</i> or <i>unc-30(e191)</i>. Student t-test **<i>p</i><0.01 and ***<i>p</i><0.001; data and statistics are relative to Q35;vector control (±SD) (RNAi controls: <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003711#pgen.1003711.s008" target="_blank">Table S1</a>).</p

<i>gei-11</i> knockdown effect through regulation of cholinergic receptors at the NMJ.

<p>(A) Real-time qPCR analysis of AChR subunits <i>unc-29</i>, <i>unc-38</i>, <i>unc-63</i>, <i>lev-1</i> and <i>acr-16</i>, and GABA_R<i>unc-49</i>, in 6 day old wt animals fed with <i>gei-11</i> RNAi. Data are normalized to the levels of each gene on vector-treated wt animals (±SD). (B) Suppression of Q35 aggregation by <i>gei-11</i> RNAi was abolished by co-treatment with L-AChR (<i>unc-38, unc-63, unc-29</i>) but not with N-AChR (<i>acr-16</i>) subunits RNAi (±SD). Individual RNAi controls are shown in light grey (also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003711#pgen.1003711.s008" target="_blank">Table S1</a>). (C) Cholinergic sensitivity assay: 5 day old animals treated with <i>gei-11</i> or vector RNAi were scored for paralysis on 1 mM levamisole plates (±SD). L-AChR mutant animals <i>unc-38(e264)</i>, <i>unc-63(x26)</i> and <i>unc-29(e1072)</i> were used as controls. Two-way ANOVA and Bonferroni test ***<i>p</i><0.001 relative to vector control. (D) AChR antagonist dTBC (2.5 mM in water) prevented suppression of Q35 aggregation by <i>gei-11</i> RNAi (±SD). Q35;<i>unc-38(e264)</i> is a control for AChR-dependent effect. Student t-test ***<i>p</i><0.001. (E) Real-time qPCR analysis of AChR subunits <i>unc-29</i>, <i>unc-38</i> and <i>unc-63</i> upon muscle-specific <i>gei-11</i> RNAi (<i>rde-1(ne219);m</i>RDE-1, 6 days old), relative to vector control (±SD). (F) Cholinergic sensitivity assay: 5 day old wt, <i>rde-1(ne219);m</i>RDE-1 and <i>rde-1(ne219)</i> animals treated with <i>gei-11</i> or vector RNAi were scored for paralysis on 1 mM levamisole plates (±SD). Two-way ANOVA and Bonferroni test ***<i>p</i><0.001, **<i>p</i><0.01, *<i>p</i><0.05 relative to vector control. (G) Aggregation quantification upon <i>gei-11</i> RNAi in Q35, Q35;<i>rde-1(ne219);m</i>RDE-1 (muscle-specific RNAi) and Q35;<i>rde-1(ne219)</i> (impaired RNAi); shown as a relative % to Q35;vector (±SD). Student t-test ***<i>p</i><0.001, ns/not significant.</p

1-EBIO potentiates residual CFTR-mediated Cl⁻ secretion in CF rectal biopsies.

<p>(A–F) Summary of effects of bumetanide (100 µM, basolateral) (A,B), CFTR_inh-172 (20 µM, basolateral) (C,D) and 1-EBIO (500 µM, basolateral) (E,F) on cAMP-mediated (IBMX/forskolin) and cholinergic (CCH) activation of equivalent short circuit current (I_sc') in rectal biopsies from control subjects, CF patients with no detectable Cl⁻ secretion (CF_absent) and CF patients with residual Cl⁻ secretion (CF_residual). All experiments were performed in the presence of amiloride and indomethacin. Only lumen-negative peak responses or plateau responses are shown for cholinergic (CCH) activation. Data are presented as mean±SEM. n = 4–26 individuals per group. *<i>P</i><0.001, ^†<i>P</i><0.01 and ^¶<i>P</i><0.05.</p

<i>CFTR</i> genotypes of CF patients with no detectable Cl⁻ secretion (CF_absent) and residual Cl⁻ secretion (CF_residual) in native rectal epithelia.

<p><i>CFTR</i> genotypes of CF patients with no detectable Cl⁻ secretion (CF_absent) and residual Cl⁻ secretion (CF_residual) in native rectal epithelia.</p

1-EBIO potentiates residual CFTR-mediated Cl⁻ secretion in CF rectal biopsies.

<p>(A–C) Original recordings of effects of cAMP-mediated (IBMX/forskolin) and cholinergic (CCH) activation, and effects of 1-EBIO (500 µM, basolateral) on transepithelial voltage (V_te) and resistance (R_te) in rectal tissues from a control subject (A), a CF patient with no detectable Cl⁻ secretion (CF_absent; R1158X/2183AA>G) (B), and a CF patient with residual Cl⁻ secretion (CF_residual; F508del/Y161C), as evidence by lumen-negative V_te responses (C). Experiments were performed in presence of amiloride and indomethacin. 1-EBIO potentiated cAMP-mediated and cholinergic Cl⁻ secretion in control and CF_residual rectal tissues, but did not induce Cl⁻ secretion in the CF_absent tissue.</p

1-EBIO activates CFTR-mediated basal and cholinergic Cl⁻ secretion in human rectal biopsies.

<p>(A,B) Original recordings of effects of 1-EBIO (500 µM, basolateral) on basal and carbachol-induced (CCH) transepithelial voltage (V_te) and transepithelial resistance (R_te) across rectal biopsies from a control subject (A) and a CF patient carrying two severe <i>CFTR</i> mutations (R1158X/2183AA>G). (B) Experiments were performed in the presence of amiloride and indomethacin. Lumen-positive V_te responses reflect K⁺ secretion and lumen-negative responses reflect Cl⁻ secretion. R_te was determined from V_te downward deflections obtained by pulsed current injection. (C) Summary of effects of 1-EBIO on basal equivalent short-circuit current (I_sc') in rectal biopsies from control subjects and CF patients with no detectable Cl⁻ secretion (CF_absent). (D,E) Effects of 1-EBIO on CCH-induced peak (open bars) and plateau (closed bars) I_sc' responses in control (D) and CF_absent rectal tissues (E). (F,G) Effect of CFTR_inh-172 on 1-EBIO-induced Cl⁻ secretion (lumen-negative I_sc') under basal conditions (F) and on carbachol-induced (CCH) Cl⁻ secretion in the presence of 1-EBIO (G) in rectal biopsies from control subjects. Data are presented as mean±SEM. n = 7–13 individuals per group. * <i>P</i><0.001 and ^†<i>P</i><0.01.</p