Figures and data
Mitochondrial stress in GABAergic neurons is sufficient to prolong the organismal lifespan.
(A and B) Lifespan analysis of systemic isp-1(sRNAi) and spg-7(sRNAi) animals. (C) GABA-neuron-specific RNAi system. The gRNAi system is based on the rde-1 mosaic mutant background, which carries a loss-of-function mutation in rde-1, thereby preventing RNAi 48, 50, 51, 53, 56, 131. This system also involves eri-1 and lin-15 loss-of-function mutant backgrounds, resulting in hyperactivated RNAi sensitivity in all neurons 48, 132. Restricted feeding RNAi to GABAergic neurons is achieved by exclusive RDE-1 expression in GABAergic neurons, along with the SID-1 dsRNA channel protein 48. (D and E) Lifespan analysis of isp-1(gRNAi) and spg-7(gRNAi) animals. (F) GABAergic neuron-specific isp-1 RNAi through in vivo transcription of sense and antisense RNAs in GABAergic neurons under the sid-1(eq9) null mutant background. (G) Lifespan analysis of sid-1(qt9) and sid-1(qt9)+Pgaba::isp-1 dsRNA animals. The significance of the lifespan curves (A, B, D, E, and G) was assessed using a Log-rank (Mantel-Cox) test. (H and I) The levels of lipofuscin in isp-1(gRNAi) and spg-7(gRNAi) animals at the 9-day-old adult stage were compared to those in control animals. (H) Representative images of lipofuscin fluorescence in the whole body of animals under the indicated conditions. Bar, 50 µm. (I) Quantification of lipofuscin fluorescence intensity. Each dot represents a single animal. ****P < 0.0001; one-way ANOVA test. Data are expressed as means ± SEM. Created with https://www.biorender.com/.
© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.
Mitochondrial stress in GABAergic neurons enhances stress resistance
(A) A diagram illustrating the paraquat and thermal stress experimental procedures is provided. (B-D) The survival rate of animals cultured in indicated concentrations of paraquat for 24 hours was measured at 3, 6, and 9-day-old adult stages. (E-G) The survival rate after incubation at 35°C for 5 hours was measured in animals at 3, 6, and 9-day-old adult stages. At least 30 animals were tested for each set. Statistical significance is indicated as follows: (H-I) Survival rates of animals at the 3-day-old adult stage after 100 mM paraquat treatment for 24 hours (H) and 24 hours after incubation at 35°C for 5 hours (I) were measured. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001; one-way ANOVA test. Data are expressed as means ± SEM.
Reproductive effects of mitochondrial perturbation in GABAergic neurons.
(A) A diagram of the C. elegans hermaphrodite reproductive system was presented, showing the distal gonad with the mitotic area for germ cell proliferation, the meiotic germ area for meiosis, and an area undergoing germ cell apoptosis, the proximal area with oogenesis, the spermatheca for sperm storage, and the uterus for fertilized egg storage. (B) Sterility in isp-1(gRNAi) animals compared to control animals. (C) The total number of embryos in fertile isp-1(gRNAi) and spg-7(gRNAi) animals. (D) The daily brood size in fertile isp-1(gRNAi) and spg-7(gRNAi) animals. (E) Representative images of dissected distal gonad arms stained with DAPI from 2-day-old isp-1(gRNAi) and control animals. The dashed lines indicate the endpoint of the mitotic area. (F) Quantitative analysis of germ cell numbers in the mitotic regions of isp-1(gRNAi) and control animals. (G) A violin plot is presented, displaying the median (solid line) and quartiles (dashed lines), illustrating the apoptotic germ cells in the loop regions of isp-1(gRNAi) and control animals. (H) Representative images of dissected proximal gonad stained with DAPI from 2-day-old isp-1(gRNAi) and control animals. The arrowheads indicate stained oocyte chromosomes. (I) Quantitative analysis of endomitotic nuclei in diakinesis oocytes in the proximal gonad of isp-1(gRNAi) and control animals. Statistical significance is indicated as follows: *P < 0.05, ***P < 0.0005, ****P < 0.0001; two-tailed Mann–Whitney test. Each dot represents an individual worm (C and D) and gonad arm (F, G, and I). Additionally, each dot indicates an individual group (B and I), animal (C and D), and gonad (F and G). Data are expressed as means ± SEM except (G). Created with https://www.biorender.com/.
© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.
GABAergic neuronal mitochondrial stress alters the mitochondrial homeostasis in peripheral tissues of animals.
(A) Representative images of mitochondrial membrane potential in isp-1(gRNAi) and spg-7(gRNAi) animals after MitoTracker CMXRos dye staining at 3-day-old adults. (B and C) Quantitative analysis of MitoTracker CMXRos fluorescent intensity measured by microscopy with a 100x magnification in the indicated animals at 3-day-old adult stage (B) and in the anterior intestine region assessed by microscopy with a 400x magnification (C). (D) The ATP bioluminescence assay measured ATP levels in the whole-body extracts of isp-1(gRNAi) and spg-7(gRNAi) animals. (E) Representative image of MitoTracker FM Green fluorescence in animals at 3-day-old adults to visualize mitochondrial mass. (F) Quantitative MitoTracker FM Green fluorescence intensity in isp-1(gRNAi) and spg-7(gRNAi) animals (G) Relative mtDNA copy number analyzed by the qPCR method in isp-1(gRNAi) and spg-7(gRNAi) animals. (H) Relative transcript levels of mitochondrial DNA polymerase gamma polg-1 gene expression measured by qPCR in isp-1(gRNAi) animals at the 2-day-old adult stage. (I) Representative images of isp-1(gRNAi) and spg-7(gRNAi) animals stained with H2DCF-DA dye to measure ROS levels in the whole body. (J) Quantitative analysis of H2DCF fluorescent intensity monitored by a spectrophotometer in isp-1(gRNAi) and spg-7(gRNAi) animals at 3-day-old adults. (K) Quantitative fluorescence intensity in the anterior intestine of animals after H2DCF-DA staining with a 400x magnification. Statistical significance is indicated as follows: *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001; one-way ANOVA test for (B, C, D, F, G, J, and K) and two-tailed student’s t-test for (H). Each dot indicates an individual worm (B, C, F, J, and K). Data are expressed as means ± SEM. Bars, 50 µm.
GABAergic neuronal mitochondria stress elevated the DAF-16/FoxO pathway.
(A) RT-qPCR analysis to assess the relative transcript levels of mitoUPR, SKN-1/Nrf, and DAF-16/FoxO signaling target genes in isp-1(gRNAi) animals compared to control EV(gRNAi) animals. The expression levels of each gene were normalized to the reference gene act-2 which encodes actin. (B) RT-qPCR analysis was conducted to assess the relative transcript levels of sod-3 in sid-1(qt9) null mutants expressing isp-1 dsRNA in GABAergic neurons. (C and D) Representative images and the quantification of Psod-3::GFP expression, a reporter for DAF-16/FoxO activity, in sid-1(qt9) control animals and sid-1(qt9)+ Pgaba::isp-1 dsRNA animals. Each dot indicates an individual worm. (E) RT-qPCR analysis was performed to assess the relative transcript levels of sod-3 and dlk-1 in daf-16(mgDf47) null mutants, both with and without isp-1 gRNAi treatment. Statistical significance is indicated as follows: **P < 0.005, ***P < 0.0005, ****P < 0.0001; two-tailed student’s t-test (B); two-tailed Mann–Whitney test (D); two-way ANOVA (A and E). Data are expressed as means ± SEM.
GABAergic neuronal mitochondria stress requires DAF-16/FoxO to trigger the non-cell-autonomous effects.
(A) Lifespan analysis to evaluate the role of daf-16 in the extended lifespan induced by Pgaba::isp-1 dsRNA. The significance of the lifespan curves was assessed using a Log-rank (Mantel-Cox) test. (B and C) Stress tolerance assays. In a daf-16(mgDf47) mutant background, Pgaba::isp-1 dsRNA expression failed to increase resistance to heat stress (B) or paraquat stress (C) compared to control EV gRNAi. (D and E) Representative images (D) and quantification (E) of mitochondrial membrane potential assessed by MitoTracker Red CMXRos dye staining at 3-day-old adults. (F and G) Representative images (F) and quantification (G) of MitoTracker FM Green fluorescent staining at 3-day-old adults to monitor mitochondria mass. (H and I) Relative mtDNA copy number (H) and polg-1 gene expression level (I) were analyzed by qPCR in EV(gRNAi) and isp-1(gRNAi) under daf-16(mgDf47) null mutant background. (J) Comparison of the mean number of eggs laid each day between daf-16(mgDf47)+isp-1(gRNAi) and control daf-16(mgDf47); EV(gRNAi) animals. (K) The total number of embryos in fertile daf-16(mgDf47)+isp-1(gRNAi) and control daf-16(mgDf47); EV(gRNAi) animals. Each dot represents an individual animal (E, G, J, and K). Statistical significance is indicated as follows: *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001; a one-way ANOVA test for B and C, and a two-tailed Mann–Whitney test for E, G, H, I, J, and K. Data are expressed as means ± SEM. Bars, 50 µm.
Non-cell autonomous effects of GABAergic neuronal mitochondrial stress were not additively enhanced by the loss of GABA signaling.
(A) Lifespan analysis was conducted on animals with indicated genetic backgrounds to investigate the relationship between GABA signaling and mitochondrial stress in GABAergic neurons. (B and C) Paraquat and thermal stress assays were performed on unc-25 null mutants with and without GABAergic neuronal mitochondrial stress induced by Pgaba::isp-1 dsRNA expression to elucidate their relationship. (D) Assessment of the fluorescence intensity of the Psod-3::GFP reporter, indicative of DAF-16/FoxO activity, in the specified mutants. (E) Evaluation of aldicarb sensitivity in response to the expression of isp-1 dsRNAs in GABAergic neurons. Each dot represents an individual group in B, D, and E and individual animal in D. *P < 0.05, **P < 0.005, ***P < 0.005, ****P < 0.001; Log-rank (Mantel-Cox) test for A; one-way ANOVA test for B, C, D, and E. Data were expressed as means ± SEM.
FLP-13 neuropeptides modulate organismal lifespan and stress tolerance, without expanding the non-cell autonomous effects of GABAergic neuronal mitochondrial stress.
(A and B) Lifespan assays were conducted to evaluate the role of unc-31 (A) and flp-13 in regulating organismal longevity. (C and D) Quantitative analysis of the thermal (C) and paraquat (D) stress resistance assays was performed after double gRNAi, induced by feeding worms a 50:50 mixture of two bacterial strains expressing either EV, flp-13, or isp-1 dsRNAs. *P < 0.05, **P < 0.005, ***P < 0.005, ****P < 0.001; Log-rank (Mantel-Cox) test for A and B; one-way ANOVA test for H and I. Data were expressed as means ± SEM.
(Related to Figure 1). Global induction of mitoUPR reporter upon isp-1 dsRNAs expression in GABAergic neurons of wild-type animals.
(A) Representative image showing the increased expression of Phsp-6::gfp transgene in the animals with transcription of isp-1 sense and antisense RNAs in vivo under the GABAergic neuron-specific promoter. Note that the N2 wild-type background allowed the systemic RNAi effects on peripheral tissues. (B) Quantitation of the Phsp-6::GFP fluorescence intensity in the intestine showing the non-cell-autonomous effects of in vivo transcription of isp-1 dsRNAs in GABAergic neurons. Each dot represents an individual animal. Data were expressed as means ± SEM. A two-tailed Mann–Whitney test.
(Related to Figure 2). Continuous gRNAi against spg-7 over three generations led to altered organismal stress resistance.
(A) A diagram showing the experimental procedure of paraquat and thermal stress response assays in spg-7(gRNAi) animals after continuous RNAi for three generations. (B and C) Quantification of paraquat stress assays in spg-7(gRNAi) animals at 1-day or 9-day-old adult stages. (D and E) Quantification of thermal stress assays in spg-7(gRNAi) animals at the L4 and 9-day-old adult stages. Animals were exposed to 35 °C for 5 hours. Data were expressed as means ± SEM. *P.<0.05, **P.<0.005, ***P.<0.005, ****P.<0.001; two-tailed student’s t-test. Created with https://www.biorender.com/.
© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.
(Related to Figure 3). Non-cell autonomous effects of GABA neuronal mitochondrial stress on the reproductive system.
(A) Diameter of the mitotic area in the distal gonad in isp-1(gRNAi) animals. (B) Hatching rates of embryos in isp-1(GABA-RNAi) and spg-7(GABA-RNAi) animals. *P.<0.05, **P.<0.005; a two-tailed Mann–Whitney test for A; ne-way ANOVA test for B. Data were expressed as means ± SEM.
(Related to Figure 6). The role of DAF-16 in the non-cell autonomous effects of GABA neuronal mitochondrial stress.
(A) Quantification of the Phsp-6::GFP intensity in the indicated sRNAi strains. (B and C) Lifespan
(B) and paraquat tolerance (C) assays of isp-1 sRNAi under the daf-16(mgDf47) null mutant background. (D) Lifespan was tested after gRNAi induction by feeding GABAergic neuronal-specific RNAi strain (XE1375) worms with a 50:50 mixture of bacteria producing either EV, isp-1, or daf-16 dsRNAs.*P.<0.05, **P.<0.005, ***P.<0.005, ****P.<0.001; a two-tailed Mann–Whitney test was used for A; Log-rank (Mantel-Cox) test for B and D; one-way ANOVA test for C. Data were expressed as means ± SEM.
(Related to Figure 7): Assessing the impact of mitochondrial stress in GABAergic neurons on lifespan in unc-25 null mutants.
(A) Repeated lifespan analysis of unc-25(qt9) mutants with and without isp-1(sRNAi). The significance was analyzed by a Log-rank (Mantel-Cox) test.
(Related to Figure 8). The effects of single GABA-RNAi inhibition against flp-13 and isp-1 on lifespan and stress response.
(A) Representative images of Pflp-13::GFP expression. GABA neurons are visualized by mCherry derived by unc-47 promoter. Arrowheads indicate the cell body of GABA neurons on the ventral nerve cord with GFP expression. Arrows indicate the cell bodies only with mCherry signals. The dashed boxes are magnified below. (A, B, and C) Lifespan and stress tolerance were tested after gRNAi induction by feeding GABAergic neuronal-specific RNAi strain (XE1375) worms with bacteria producing either isp-1 or flp-13 dsRNAs alone for each condition. (B) The survival rate after incubation at 35°C for 5 hours was measured in animals at the 3-day-old adult stage. At least 30 animals were tested for each set. (C) The survival rate of animals cultured in indicated concentrations of paraquat for 24 hours was measured at the 3-day-old adult stage. (D) Lifespan assay of isp-1(gRNAi) and flp-13(gRNAi) animals. **P < 0.005, ****P < 0.001; one-way ANOVA test for B and C; a Log-rank (Mantel-Cox) test for D. Data were expressed as means ± SEM.
