Heat Shock Proteins Function as Signaling Molecules to Mediate Neuron-Glia Communication During Aging

Abstract

The nervous system is primarily composed of neurons and glia, and the communication between them plays profound roles in regulating the development and function of the brain. Neuron-glia signal transduction is known to be mediated by secreted or juxtacrine signals through ligand-receptor interactions on the cell membrane. Here, we report a novel mechanism for neuron-glia signal transduction, wherein neurons transmit proteins to glia through extracellular vesicles, activating glial signaling pathways. We find that in the amphid sensory organ of Caenorhabditis elegans, different sensory neurons exhibit varying aging rates. This discrepancy in aging is governed by the crosstalk between neurons and glia. We demonstrate that early-aged neurons can transmit heat shock proteins (HSP) to glia via extracellular vesicles. These neuronal HSPs activate the IRE1-XBP1 pathway, further increasing their expression in glia, forming a positive feedback loop. Ultimately, the activation of the IRE1-XBP-1 pathway leads to the transcriptional regulation of chondroitin synthases to protect glia-embedded neurons from aging-associated functional decline. Therefore, our studies unveil a novel mechanism for neuron-glia communication in the nervous system and provide new insights into our understanding of brain aging.

Keywords: C. elegans; UPRER; aging; chondroitin; extracellular vesicles; glia; heat shock proteins; neuron.

Figure 1.. AMsh-channel and AMsh glia-embedded neurons age differently in individual animals.

(A) Schematic diagram of the amphid sensory organ of C. elegans. (1) AMsh-channel neurons; (2) AMsh glia-embedded neurons. (B) Results from chemotaxis experiments show that ASH/ADL, AWA and AWC neurons exhibit age-dependent functional decline in response to octanol, 1% 2-methylpyrazine and 0.5% benzaldehyde, respectively. D1, D6 and D9 represent the adult stage day 1, day 6, and day 9 animals. Data are shown as mean ± SD. (C to F) Function correlation analyses among ASH/ADL, AWA and AWC neurons. After the initial chemotaxis assay testing, day 6 animals were divided into two groups (Figures S1A and S1B): animals that respond to corresponding odorants were labeled as (+); and animals that did not respond to corresponding odorants were labeled as (−). The results from chemotaxis assays for different sensory neurons by using these two groups of animals were presented in C to F. Date are shown as mean ± SD. Student’s t-test, *P < 0.05, ***P < 0.001. (G) Schematic of Auxin-inducible DAF-16 degradation in target neurons . To exclusively deplete DAF-16 in ASH/ADL neurons, we generated a transgene expressing atTIR1 (derive from Arabidopsis thaliana) under an ASH/ADL-specific promoter, Pgpa-11, in daf-16(ot853) animals. daf-16(ot853) [daf-16::linker::mNeonGreen::3xFlag::AID] is a knockin strain in which a degron-tagged mNeonGreen is inserted at the C-terminus of daf-16. daf-16(ot853) without atTIR1 expression was used as a control. Adult animals grow on the NGM plates supplied with 5 μM auxin (indole-3 acetic acid, sigma). All DAF-16 depletion experiments were carried out using this setting. (H to J) Chemotaxis assays for ASH/ADL (H), AWA (I) and AWC (J) neurons using octanol, 1% 2-methylpyrazine, and 0.5% benzaldehyde, respectively, in day 1 (D1) and day 6 (D6) animals with (DAF-16 (KD)) and without (Control) DAF-16 depletion in ASH/ADL neurons. Data are shown as mean ± SD. Student’s t-test, ***P < 0.001.

Figure 2.. ASH/ADL neuronal aging activates the UPR^ER pathway in AMsh glia.

(A) Schematic of proximity labeling in AMsh glia. (1) An confocal image shows the distribution of HA-tagged TurboID in AMsh glia by immunostaining using anti-HA antibody. Scale bar: 20 μm. (2) A diagram shows the enrichment of biotinylated AMsh glial proteins from worm lysis. (B) After proximity labeling, AMsh glial proteins extracted from day 6 control and ASH/ADL DAF-16 depletion (DAF-16 (KD)) animals were subjected to HPLC-MS/MS analyses. (C) The Volcano plot of AMsh glial proteomes show proteins with significant changes in DAF-16 (KD) animals when compared with the same age control animals (day 6). log₂FC > 0.6 or log₂FC < −0.6 (FC, fold change) with −log₁₀(p-value) > 1.3 is defined as significantly upregulated (pink square) or downregulated (blue triangle), respectively. The proteins labeled on the plot are selected examples that are known downstream targets of the IRE1-XBP1 pathway. (D) Results from gene ontology analyses for proteins that are upregulated by DAF-16 (KD) in ASH/ADL. (E) Schematic diagram to illustrate the use of xbp-1 splicing as a reporter for the activation of the IRE1-XBP1 pathway. In this reporter (xbp-1s reporter), xbp-1(23bp-intron)::GFP was expressed only in AMsh glia under a AMsh-specific promoter, Pf53f4.13. GFP was fused at the C-terminal in frame with the spliced version of xbp-1 mRNA (xbp-1s) only when the non-canonical 23bp intron is removed by IRE-1. (F and G) Confocal images (F) and quantifications (G) show the expression of the xbp-1s reporter in AMsh glia in control and DAF-16(KD) animals at the adult stage day 1 (D1) and day 6 (D6). These experiments were carried out in daf-16(ot853) animals with (DAF-16 (KD)) and without (Control) atTIR1 expression in ASH/ADL neurons. The DAF-16::mNG were not expressed in AMsh glia (Figure S1F). ‘A’ indicates the anterior of animals; ‘P’ indicates the posterior of animals. Scale bar: 10μm. Data are shown as mean ± SD. N> 30. One-way ANOVA, *P < 0.05, ***P < 0.001. (H and I) Results from testing AWA (H) and AWC (I) neuronal functions by chemotaxis assays using 1% 2-methylpyrazine and 0.5% benzaldehyde, respectively. Pf53f4.13 was used to specifically express the constitutively activated XBP-1s in AMsh glia. Data are shown as mean ± SD. Student’s t-test, ***P < 0.001. (J and K) Results from testing AWA (J) and AWC (K) neuronal functions in control and DAF-16(KD) animals with and without specifically RNAi knockdown of ire-1 or xbp-1 in AMsh glia in day 1 and day 6 animals. Data are shown as mean ± SD. One-way ANOVA, ***P < 0.001.

Figure 3.. Transmission of HSP-4 from neurons to glia activates the IRE1-XBP1 pathway in AMsh glia.

(A and B) Confocal images (A) and quantifications (B) show HSP-4::mNG expressions in AMsh glia in control, DAF-16(KD), and DAF-16(KD) with ASH/ADL neuronal hsp-4 knockdown animals. gpa-11 promoter was used to drive cell-specific expression in ASH/ADL. yadIs222 [Pf53f4.13::mCherry] was used as an AMsh glia marker. Scale bar: 10μm. N>30. Data are shown as mean ± SD. One-way ANOVA, ***P < 0.001. (C and D) Results from chemotaxis assays show AWA (C) and AWC (D) neuronal functions in control and transgenes expressing hsp-4 in ASH/ADL neurons or AMsh glia. gpa-11 promoter was used to drive cell-specific expression in ASH/ADL neurons. f53f4.13 promoter was used to drive cell-specific expression in AMsh glia. Data are shown as mean ± SD. One-way ANOVA, **P < 0.01, ***P < 0.001. (E and F) Results from chemotaxis assays show AWA (E) and AWC (F) neuronal functions in control, DAF-16(KD), and DAF-16(KD) with hsp-4 knockdown in AMsh glia, ASH/ADL neurons, or both. gpa-11 promoter was used to drive cell-specific expression in ASH/ADL neurons. f53f4.13 promoter was used to drive cell-specific expression in AMsh glia. Data are shown as mean ± SD. One-way ANOVA, **P < 0.01, ***P < 0.001, ns, no significant difference. (G) Confocal images show the expression of HSP-4::mCherry or mCherry when they were specifically expressed in AMsh-channel neurons. gpa-3 promoter was used to drive cell-specific expression in AMsh-channel neurons. yadIs48 [Pf53f4.13::GFP] was used as a marker to label AMsh glia. The yellow arrows point to the cell bodies of AMsh-channel neurons. Scale bar: 10 μm. (H) Confocal images from the xbp-1s reporter (yadIs256 [Pf53f4.13::xbp-1s(23bp-intron)::GFP]) show the effects of expressing hsp-4 in ASH/ADL neurons or AMsh glia. gpa-11 promoter was used to drive cell-specific expression in ASH/ADL neurons. f53f4.13 promoter was used to drive cell-specific expression in AMsh glia. yadIs222 [Pf53f4.13::mCherry] was used as an AMsh glia marker. Scale bar: 10μm. (I) Confocal images show the expression of the xbp-1s reporter in control and DAF-16(KD) animals with or without knockdown of hsp-4 in ASH/ADL. gpa-11 promoter was used to drive cell-specific expression in ASH/ADL neurons. f53f4.13 promoter was used to drive cell-specific expression in AMsh glia. yadIs222 [Pf53f4.13::mCherry] was used as an AMsh glia marker. Scale bar: 10μm. (J) Quantification of XBP-1s::GFP fluorescence intensity in H and I. Data are shown as mean ± SD. N>30. One-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.. Neuronal HSPs are transmitted to AMsh glia through extracellular vesicles.

(A) Confocal images show that neuronal HSP-4::mCherry is co-localized with TSP-6::GFP, a extracellular vesicle marker expressed in AMsh-channel neurons, in AMsh glia. gpa-3 promoter was used to drive cell-specific expression in AMsh-channel neurons. The red arrows point to the co-localized HSP-4::mCherry and TSP-6::GFP puncta. The white asterisks indicate the diffused HSP-4::mCherry. In the upper panel, scale bar, 10μm. In the zoom in region, scale bar, 5μm. (B and C) Confocal images (B) and quantifications (C) show the signal from neuronal expressed TSP-6::GFP in AMsh glia in control and DAF-16(KD) animals. TSP-6::GFP expression was driven by the AMsh-channel neurons specific promoter gpa-3 (yadIs226 [Pgpa-3::tsp-6::GFP]). yadIs222 [Pf53f4.13::mCherry] was used as an AMsh glia marker. Scale bar: 10μm. N>30. Data are shown as mean ± SD. Student’s t-test, ***P < 0.001. (D) Schematic diagram shows that the release and uptake of extracellular vesicles (EV) are regulated by TAT-5 and DYN-1, respectively. (E and F) Confocal images (E) and quantifications (F) of yadIs226 [Pgpa-3::tsp-6::GFP] signals in control, knockdown of tat-5 in ASH/ADL, and expression of the dominant-negative DYN-1(G40E+K46A) in AMsh glia. gpa-3 promoter was used to drive cell-specific expression in AMsh-channel neurons. gpa-11 promoter was used to drive cell-specific expression in ASH/ADL neurons. f53f4.13 promoter was used to drive cell-specific expression in AMsh glia. yadIs222 [Pf53f4.13::mCherry] was used as an AMsh glia marker. Scale bar: 10μm. N>30. Data are shown as mean ± SD. One-way ANOVA, ***P < 0.001. (G and H) Results from chemotaxis assays show AWA (G) and AWC (H) neuronal functions in control, ASH/ADL tat-5 knockdown, and ASH/ADL tat-5&hsp-4 knockdown animals. gpa-11 promoter was used to drive cell-specific expression in ASH/ADL neurons. Data are shown as mean ± SD. One-way ANOVA ***P < 0.001. (I and J) Results from chemotaxis assays show AWA (I) and AWC (J) neuronal functions in control and DAF-16(KD) animals with or without expressing the dominant-negative DYN-1(G40E+K46A) in AMsh glia. Data are shown as mean ± SD. One-way ANOVA, **P < 0.01, ***P < 0.001. (K and L) Confocal images (K) and quantifications (L) show HSP-4::mNG expression in AMsh glia in control and DAF-16(KD) animals with and without expression of the dominant-negative DYN-1(G40E+K46A) in AMsh glia. gpa-11 promoter was used to drive cell-specific expression in ASH/ADL neurons. f53f4.13 promoter was used to drive cell-specific expression in AMsh glia. yadIs222 [Pf53f4.13::mCherry] was used as an AMsh glia marker. Scale bar:10μm. N>30. Data are shown as mean ± SD. One-way ANOVA, ***P < 0.001. (M) Confocal images show the expression of the xbp-1s reporter, yadIs256 [Pf53f4.13::xbp-1s(23bp-intron)::GFP], in animals with or without tat-5 knockdown in ASH/ADL neurons and in animals with knockdown of tat-5 and hsp-4 simultaneously in ASH/ADL neurons. gpa-11 promoter was used to drive cell-specific expression in ASH/ADL neurons. f53f4.13 promoter was used to drive cell-specific expression in AMsh glia. yadIs222 [Pf53f4.13::mCherry] was used as an AMsh glia marker. Scale bar: 10μm. (N) Confocal images show the expression of the xbp-1s reporter, yadIs256 [Pf53f4.13::xbp-1s(23bp-intron)::GFP], in DAF-16(KD) animals with and without expression of the dominant-negative DYN-1(G40E+K46A) in AMsh glia. gpa-11 promoter was used to drive cell-specific expression in ASH/ADL neurons. f53f4.13 promoter was used to drive cell-specific expression in AMsh glia. yadIs222 [Pf53f4.13::mCherry] was used as an AMsh glia marker. Scale bar: 10μm. (O) Quantifications of XBP-1s::GFP fluorescence intensity in M and N. N>30. Data are shown as mean ± SD. One-way ANOVA, ***P < 0.001.

Figure 5.. Expression of chondroitin in AMsh glia protects AWA/AWC neurons from aging-associated functional decline.

(A) A strategy for testing the function of DAF-16 depletion-upregulated genes in AWA/AWC functions. The genes of interest were overexpressed in AMsh glia to test their effects on AWA/AWC neuronal functions of day 1 and day 6 animals. (B and C) Results from chemotaxis assays show AWA (B) and AWC (C) neuronal functions in control and transgenes expressing mig-22 or sqv-5 in AMsh glia. f53f4.13 promoter was used to drive cell-specific expression in AMsh glia. Data are shown as mean ± SD. One-way ANOVA, ***P < 0.001. (D and E) Confocal images (D) and quantifications (E) show the expression of MIG-22::GFP in AMsh glia in control and DAF-16(KD) animals with or without xbp-1 RNAi knockdown in AMsh glia. yadIs272 [Pmig-22::mig-22::GFP] is a translational reporter for mig-22. yadIs222 [Pf53f4.13::mCherry] was used as an AMsh glia marker. Scale bar, 10μm. N>30. Data are shown as mean ± SD. One-way ANOVA, ***P < 0.001, ns, no significant difference. (F and G) Confocal images (F) and quantifications (G) show the expression of SQV-5::GFP expression in AMsh glia in control and DAF-16(KD) animals with or without xbp-1 RNAi knockdown in AMsh glia. yadCK287 [Psqv-5::sqv-5::GFP] is a knockin reporter for sqv-5 expression. yadIs222 [Pf53f4.13::mCherry] was used as an AMsh glia marker. Scale bar: 10μm. N>30. Data are shown as mean ± SD. One-way ANOVA, ***P < 0.001. (H to J) Confocal images (H) and quantifications show the expression of MIG-22::GFP (I) and SQV-5::GFP (J) in animals with or without expressing constitutively activated xbp-1s in AMsh glia. f53f4.13 promoter was used to express the constitutively activated xbp-1s in AMsh glia. yadIs222 [Pf53f4.13::mCherry] was used as an AMsh glia marker. Scale bar: 10μm. N>30. Data are shown as mean ± SD. Student’s t-test, ***P < 0.001. (K and L) Results from chemotaxis assays show AWA (K) and AWC (L) neuronal functions in control and DAF-16(KD) animals with or without specifically knockdown of mig-22 or sqv-5 in AMsh glia. Data are shown as mean ± SD. One-way ANOVA, ***P < 0.001.