Glial-derived mitochondrial signals impact neuronal proteostasis and aging

Abstract

The nervous system plays a critical role in maintaining whole-organism homeostasis; neurons experiencing mitochondrial stress can coordinate the induction of protective cellular pathways, such as the mitochondrial unfolded protein response (UPR^MT), between tissues. However, these studies largely ignored non-neuronal cells of the nervous system. Here, we found that UPR^MT activation in four, astrocyte-like glial cells in the nematode, C. elegans, can promote protein homeostasis by alleviating protein aggregation in neurons. Surprisingly, we find that glial cells utilize small clear vesicles (SCVs) to signal to neurons, which then relay the signal to the periphery using dense-core vesicles (DCVs). This work underlines the importance of glia in establishing and regulating protein homeostasis within the nervous system, which can then impact neuron-mediated effects in organismal homeostasis and longevity.

Keywords: C. elegans; aging; glia; mitochondria; neurodegeneration; protein homeostasis.

Figure 1:. Glial activation of jmjd-1.2 prolongs lifespan, improves stress resistance, and induces the cell non-autonomous UPR^MT.

(A) Survival of animals expressing jmjd-1.2 in most glia (blue), compared to a control N2 population (black). P= 0.0158. (B) Survival of animals expressing jmjd-1.2 in amphid and phasmid sheath glia (orange), compared to a control N2 population. Log-rank test, P < 0.0001 (C) Survival of animals expressing jmjd-1.2 in the cephalic sheath (CEPsh) glia (red), compared to a control N2 population. P < 0.0001 (D) Representative fluorescent micrographs of UPR^MT reporter worms (hsp-6p::GFP) expressing jmjd-1.2 under the indicated promoters. Scale bar, 250 μm. (E) Fold change in GFP fluorescence per worm using a large-particle biosorter (methods), normalized to a control (hsp-6p::GFP) population (n>300 per group). One-way analysis of variance (ANOVA) Tukey’s multiple comparisons test, **P < 0.01, ****P < 0.0001. See also Figure S1G. (F) Survival of animals expressing glial jmjd-1.2 (red) under paraquat stress, as compared to a control population (black). Worms fed with daf-2 RNAi were used as a positive control (green) (n=60 per group), see also Figure S1H.

Figure 2:. Non-autonomous activation of UPR^MT in the periphery depends on cell-autonomous regulators of the pathway UPR^MT.

(A) Representative fluorescent micrograph of UPR^MT translational reporter worms (DVE-1::GFP) expressing jmjd-1.2 under the CEPsh glia (hlh-17) promoter, and the number of DVE-1+ nuclei quantified for the anterior and posterior regions using Fiji (n>30). Scale bar, 250 μm. (B), significance was assessed relative to control by unpaired Student’s t test, ****P<0.0001. (C and D) Representative fluorescent micrograph of UPR^MT reporter worms (hsp-6p::GFP) expressing jmjd-1.2 either knocked-down (C) or knocked-out (D) for key regulators in UPR^MT activation, and quantified in (E), as in Figure 1E, (n>450), One-way analysis of variance (ANOVA) Tukey’s multiple comparisons test. n.s. not significant, *P<0.05, ****P < 0.0001. See also Figure S2 (F) Survival of animals expressing jmjd-1.2 in CEPsh glia grown on either control (E.V.) or atfs-1 RNAi.

Figure 3:. Activation of jmjd-1.2 in CEPsh glia reduces protein aggregation, increases lipid content in the intestine, and triggers the UPR^MT transcriptional program.

(A) Changes in gene expression in CEPsh glia expressing jmjd-1.2, as compared to N2 control animals, are represented in a volcano plot. (B) Differentially expressed genes in glial jmjd-1.2 worms compared to their change in published datasets of mitochondrial stress induced by ETC knockdown by RNAi (cox-5B RNAi) or over-expression of jmjd-1.2 in all worm tissues (17). (C) The average change in gene expression of the indicated gene groups (see methods), as compared to N2 control animals. UPR^MT genes are shown in (D). (E) Overlap of induced genes in glial jmjd-1.2 worms with genes induced in paraquat stress. (F) Gene enrichment analysis was plotted using gProfiler (45). (G) Representative fluorescent micrographs of protein aggregation in the intestine of worms expressing jmjd-1.2 under the CEPsh glia (hlh-17) promoter. Scale bar, 250 μm. (H) Quantification of number of aggregates per worm using Fiji local extrema analysis (n<30). (I) Representative fluorescent images of lipid droplets reporter animals (DHS-3::GFP), either in control animals or animals expressing jmjd-1.2 in CEPsh glia cells. Significance was assessed relative to control by unpaired Student’s t test. n.s. not significant, ****P < 0.0001.

Figure 4:. Cell non-autonomous activation of the UPR^MT in the intestine depends on the secretion of SCVs, DCVs, and neuropeptide processing

(A) Representative fluorescent micrographs of UPR^MT reporter worms (hsp-6::GFP) for animals with mutations in the secretion of small-clear vesicles (SCV, unc-13), dense-core vesicles (DCV, unc-31), and neuropeptide processing (egl-3). Scale bar, 250 um. (B) Quantification of UPR^MT reporter worms (hsp-6::GFP) as in Figure 1E (n>200).

Figure 5:. CEPsh glia require functional UNC-13 (SCV), while neurons require UNC-31 (DCV), to activate UPR^MT in the periphery

(A) Schematic of spatial mutation strategy. Expression of the flipase FLP D5 under the CEPsh glial-specific (hlh-17p) or pan-neuronal (rgef-1p) promoters, in combination with an independent FRT (FLP recognition target) allele of a gene of interest results in a tissue-specific mutation. (B) Representative fluorescent micrographs of the indicated strains at day 1 adult animals. Scale bar, 250 um. (C & D) Median spatial profiles of the indicated animals (see methods), for depletion in CEPsh glial cells (C) or neuronal cells (D) quantified with large-particle biosorter (n>50). The integral of the 30% most posterior portion of the animals was calculated and plotted in (E). One-way analysis of variance (ANOVA) Tukey’s multiple comparisons test. n.s. not significant, ***P < 0.001, ****P < 0.0001.

Figure 6:. Glial jmjd-1.2 rescues protein aggregation in neurons in a Huntington’s disease (HD) model via SCVs.

(A) Thrashing of animals expressing the aggregating polyglutamine tract Q40, with and without glial jmjd-1.2, measured using WormTracker (n>100) (B) Chemotaxis index of worms towards benzaldehyde (n>200). (C) Filter Retardation Assay for Q40::YFP, with and without glial jmjd-1.2 (top) and its quantification (bottom). (D) Representative fluorescent micrographs of Q40::YFP for the annotated genotypes, on D5 of adulthood. See Figure S6 for images of worms at D1. (E) Quantification of Q40 signal in the head region of the animals (n>30) using Fiji. One-way analysis of variance (ANOVA) Tukey’s multiple comparisons test. n.s. not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (F) Model of communication from glial cells to peripheral tissues. CEPsh glia utilize SCVs upon UPR^MT activation to signal to neurons, which reduce protein aggregation and utilize DCVs, neuropeptide processing, and a WNT-ligand to drive protein homeostasis and metabolic changes in the periphery.