Abstract
The mitochondrial unfolded protein response (UPRmt) is an evolutionarily conserved adaptive response that functions to maintain mitochondrial homeostasis following mitochondrial damage. In C. elegans, the nervous system plays a central role in responding to mitochondrial stress by releasing endocrine signals that act upon distal tissues to activate the UPRmt. The mechanisms by which mitochondrial stress is sensed by neurons and transmitted to distal tissues is not fully understood. Here, we identify a role for the conserved follicle-stimulating hormone G protein coupled receptor (GPCR), FSHR-1, in promoting UPRmt activation. Genetic deficiency of fshr-1 severely attenuates UPRmt activation and organism-wide survival in response to mitochondrial stress. FSHR-1 functions in a common genetic pathway with SPHK-1/sphingosine kinase to promote UPRmt activation, and FSHR-1 regulates the mitochondrial association of SPHK-1 in the intestine. Through tissue-specific rescue assays, we show that FSHR-1 functions in neurons to activate the UPRmt, to promote mitochondrial association of SPHK-1 in the intestine, and to promote organism-wide survival in response to mitochondrial stress. We propose that FSHR-1 functions cell non-autonomously in neurons to activate UPRmt upstream of SPHK-1 signaling in the intestine.
Introduction
The mitochondrial unfolded protein response (UPRmt) functions to maintain mitochondrial protein homeostasis in response to mitochondrial dysfunction caused by mitochondrial DNA damage, incorrect mitochondrial protein folding, or impaired oxidative phosphorylation. Failure to appropriately control and maintain protein homeostasis in the mitochondria is associated with the development of numerous diseases, neurodegeneration, and ageing (Durieuxet al. 2011; Liuet al. 2014; Pellegrinoet al. 2014; Nargundet al. 2015; Fioreseet al. 2016; Martinezet al. 2017). The UPRmt is initiated when mitochondrial proteostasis is disrupted, detection of which by mitochondrial and cytosolic factors leads to epigenetic modifications and transcriptional responses in the nucleus that restore mitochondrial function. A critical sensor and activator of the UPRmt in C. elegans and in mammals is the leucine zipper transcription factor ATSF-1/ATF5 (Fioreseet al. 2016). ATFS-1 is normally targeted to mitochondria where it is degraded, but upon mitochondrial stress, mitochondrial import is disrupted and ATFS-1 is targeted instead to the nucleus where it regulates the expression of a cascade of genes including the conserved HSP70-like chaperone, hsp-6 which is targeted to the mitochondria to restore protein folding (Nargundet al. 2012). The intestinal UPRmt can be activated by mitochondrial stress originating either cell autonomously in the intestine or cell non-autonomously in the nervous system. Mitochondrial stress in neurons activates the UPRmt in intestine through the release of neuropeptides, serotonin and/or Wnt ligands (Berendzenet al. 2016; Shaoet al. 2016; Zhanget al. 2018).
Genetic screens for additional factors that activate the UPRmt have revealed important roles for mitochondrial ceramide produced by SPTL-1/serine palmitoyltransferase and sphingolsine-1-phosphate (S1P) produced by SPHK-1/sphingosine kinase in the activation of the UPRmt (Liuet al. 2014; Kim and Sieburth 2018a). SPHK-1 recruitment to mitochondria from cytoplasmic pools may serve as an early signal to activate UPRmt (Kim and Sieburth 2018a). SPHK-1 mitochondrial recruitment is positively regulated by mitochondrial stress originating either from the intestine or from the nervous system. Neuronal mitochondrial stress activates intestinal SPHK-1 by a mechanism that involves neuropeptide but not serotonin signaling (Kim and Sieburth 2018a). Neuropeptides exert their biological functions primarily through activating G protein coupled receptors (GPCRs) on target cells to trigger downstream signaling events (Frooninckxet al. 2012). However, the specific neuropeptides and the GPCRs functioning in SPHK-1-mediated UPRmt activation have not been identified.
FSHR-1 is a GPCR containing extracellular Leucine Rich Repeats (LRRs) homologous to the follicle-stimulating hormone receptor (Powellet al. 2009). FSHR-1 plays a critical role in activating innate immunity in response to infection by pathogenic bacteria, and functions in the intestine to promote protection to pathogenic infection and to regulate anti-microbial gene expression. (Choet al. 2007; Powellet al. 2009; Milleret al. 2015). Interestingly, infection by pathogenic bacteria leads to multiple cellular responses in the intestine, including the activation of the UPRmt (Liuet al. 2014; Pellegrinoet al. 2014).
In this study, through the analysis of fshr-1 null mutants, we show that FSHR-1 positively regulates UPRmt activation in intestine and promotes the mitochondrial association of SPHK-1. Through tissue-specific rescue experiments, we find that FSHR-1 functions primarily in the nervous system, and also in the intestine, to exert its function in UPRmt activation. We propose that FSHR-1 is part of a neuroendocrine signaling network that functions to activate the UPRmt through inter-tissue signaling.
Materials and Methods
C. elegans strains
Strains used in this study were maintained at 22°C following standard methods. Young adult hermaphrodites derived from the wild type reference strain N2 Bristol were used for all experiments. The following mutant strains were used. SJ4100: zcIs13[Phsp-6::GFP], OJ4113: vjIs138[Pges-1::sphk-1::gfp], OJ4143: vjIs148[Pges-1::tomm-20::mCherry], OJ2329: vjIs208[Pgst-4::gfp], OJ997 sphk-1(ok1097), KP3397: fshr-1(ok778). The sphk-1(ok1097) and fshr-1(ok778) strains were outcrossed at least 6 times with wild type animals prior to analysis.
Molecular biology
fshr-1 cDNA was cloned from C. elegans wild type cDNA and then inserted into the pPD49.26 expression vector using standard molecular biology techniques. The following plasmids were generated pSK55[Pges-1::fshr-1], pSK56[Prab-3::fshr-1]. Sequence files of plasmids are available upon request.
Transgenic lines
Transgenic strains were generated by injecting expression constructs (10–25 ng/ul) and the co-injection marker; pJQ70 [Pofm-1::mCherry, 40 ng/ul], KP#708[Pttx-3::rfp, 40 ng/ul] or KP#1106 [Pmyo-2::gfp, 10 ng/ul] into N2 or indicated mutants using standard techniques (Melloet al. 1991). At least three lines for each transgene were tested and a representative transgene was used for further experiments. The following transgenic lines were made: vjEx1448[Prab-3::fshr-1], vjEx1449[Pges-1::fshr-1]
Microscopy and analysis
Fluorescence microscopy experiments were performed following previous methods (Kim and Sieburth 2018a). Briefly, L4 stage or young adult worms were immobilized by using 2,3-butanedione monoxime (BDM, 30 mg/mL; Sigma) in M9 buffer then mounted on 2% agarose pads for imaging. To quantify the fluorescence intensity of Phsp-6::GFP or Pgst-4::GFP, Z stacks of the intestine posterior to the vulva were selected as a representative region because of low basal expression in the absence of stress. Images were captured with the Nikon eclipse 90i microscope equipped with a Nikon PlanApo 40 x or 60x or 100x objective (NA = 1.4) and a PhotometricsCoolsnap ES2 or a Hamamatsu Orca Flash LT+ CMOS camera.
Metamorph 7.0 software (Universal Imaging/Molecular Devices) was used to capture serial image stacks, and the maximum intensity was measured (Kim and Sieburth 2018b). Intensity quantification analysis was performed on the same day to equalize the absolute fluorescence levels between samples within same experimental set.
RNA Interference
Feeding RNAi knockdown assay was performed following established protocols (Kamath and Ahringer 2003). Briefly, gravid adult animals were placed on RNAi plates seeded with HT115(DE3) bacteria transformed with L4440 vector containing a genomic fragment of the gene to be knocked down (or empty L4440 vector as a control), to collect eggs then removed after 4 hours to obtain an age-matched synchronized worm population. Young adult animals were used for subsequent assays.
Stress induction assays
For drug-induced stress, transgenic L4 animals were transferred to fresh NGM plates seeded with HB101 bacteria, then 80 ul of stock solutions dissolved in M9 buffer of paraquat were added to plates for a final concentration of 0.4 mM paraquat. 24 hours later, adults were selected for fluorescence microscopy analysis. For imaging of animals that had been subjected to RNAi-induced knockdown, L4 animals grown on RNAi plates were transferred to new RNAi plates to obtain synchronized animals and imaged 24 hours later. For Pgst-4::GFP imaging, young adult animals are incubated with 5mM arsenite or 50mM paraquat in liquid solution (in M9 buffer) for 1 hour then drug was washed out and worms were transferred onto new NGM plates for 4 hours before imaging. M9 buffer was used as a control for paraquat and arsenite treatment.
For paraquat survival assays, young adult animals were placed onto NGM plates containing 10mM paraquat for 17 hours. After 17 hours, the percentage of surviving animals was counted every 3 hours over the course of 15 hours. Survival assay were done in experimental duplicate for each biological duplicate.
Statistical Analysis
Student’s t test (two-tailed) was used to determine the statistical significance. P values less than 0.01 or 0.001 are indicated with asterisks **(p< 0.01), ***(p<0.001), respectively. Error bars in the figures indicate the standard error of the mean (±S.E.M). The exact numbers of sample size (n) are indicated in each figure.
Data Availability
Strains and plasmids used in this study are available upon request. The authors confirm that all data necessary for confirming the conclusions of the findings are present within the article, figures.
Results
FSHR-1/GPCR signaling regulates the UPRmt
Prior studies found that fshr-1 mutants have normal lifespans (Powellet al. 2009), but exhibit enhanced sensitivity to lethality caused by the mitochondrial toxin and UPRmt activator, paraquat (Milleret al. 2015). Because impairing UPRmt activation causes sensitivity to paraquat-induced lethality (Nargundet al. 2012; Gatsiet al. 2014; Liuet al. 2014; Kim and Sieburth 2018a), we speculated that FSHR-1 may positively regulate the UPRmt. To monitor UPRmt activation, we quantified intestinal fluorescence of the UPRmt transcriptional reporter, zcIs13, in which GFP is expressed under control of the hsp-6 promoter (Phsp-6::GFP). Paraquat is an oxidant that interferes with electron transport at the inner mitochondrial membrane, and has been used widely to acutely activate the UPRmt in both C. elegans and in mammalian cells (Nargundet al. 2012; Runkelet al. 2013; Fioreseet al. 2016; Kim and Sieburth 2018a). Wild type animals treated with paraquat for 24 hours exhibited a greater than ten-fold increase in Phsp-6::GFP expression in the intestine compared to non-treated controls. In contrast, fshr-1 mutants showed either no significant change or a small increase in Phsp-6::GFP expression following paraquat treatment (Figure 1A and 2A). cco-1 (aka cox-5B), encodes the cytochrome c oxidase subunit in complex IV, which is the terminal electron acceptor of the electron transport chain, and RNA interference (RNAi)-mediated cco-1 knockdown is a potent activator of the UPRmt (Nargundet al. 2012; Berendzenet al. 2016; Merkwirth et al. 2016; TIAN et al. 2016; Kim and Sieburth 2018a). cco-1 RNAi by feeding significantly increased Phsp-6::GFP expression in wild type animals but failed to increase Phsp-6::GFP expression in fshr-1 mutants (Figure 1C). fshr-1 mutants exhibited normal induction of the antioxidant reporter Pgst-4::GFP (Inoueet al. 2005; Choeet al. 2009; Ruiz-Ramoset al. 2009; Prakashet al. 2015; Wuet al. 2016) in response to treatment with either the mitochondrial ROS generator arsenite, or with paraquat (Figure 1D). Thus, FSHR-1 positively regulates UPRmt activation in response to mitochondrial stress.
FSHR-1/GPCR functions cell non-autonomously in neurons to regulate UPRmt
FSHR-1 is expressed primarily in the intestine and in a subset of neurons (Sieburthet al. 2005; Choet al. 2007). To determine in which tissue FSHR-1 functions to regulate the UPRmt, we examined Phsp-6::GFP expression in transgenic fshr-1 mutants expressing fshr-1 cDNA in either the nervous system (using the rab-3 promoter) or the intestine (using the ges-1 promoter). Pan-neuronal fshr-1 nearly fully rescued the paraquat-induced hsp-6 expression defects of fshr-1 mutants. In contrast, intestinal fshr-1 expression only partially rescued the paraquat-induced hsp-6 expression defects of fshr-1 mutants (Figure 1A). Phsp-6 expression was increased 11 fold by pan-neuronal fshr-1 compared to just 3.3 fold by intestinal fshr-1 expression (Figure 1A). Taken together, these results suggest that FSHR-1 primarily functions cell non-autonomously in the nervous system to positively regulate UPRmt activation, and intestinal fshr-1 has a minor role in contributing to UPRmt activation.
Neuronal FSHR-1 regulates baseline hsp-6 expression
Under non-stressed conditions, fshr-1 mutants exhibited a small but significant increase in Phsp-6::GFP expression in the intestine compared to wild type controls (Figure 1A and 1C). The increase in Phsp-6::GFP expression was blocked by RNAi-mediated knockdown of atfs-1 (Figure 1B), suggesting that FSHR-1 keeps UPRmt activation low under non-stressed conditions. The increase in baseline Phsp-6::GFP expression of fshr-1 mutants was restored to wild type levels by pan-neuronal, but not by intestinal fshr-1 cDNA expression (Figure 1A). Thus, neuronal FSHR-1 functions cell non-autonomously to keep UPRmt activity low in the absence of stress.
FSHR-1 and SPHK-1 function in a common pathway to activate the UPRmt
SPHK-1 functions in the intestine to activate the UPRmt in response to a variety of mitochondrial stressors, including paraquat (Kim and Sieburth 2018a). To test whether FSHR-1 functions in a common pathway with SPHK-1 to activate the UPRmt, we examined genetic interactions between sphk-1 and fshr-1 mutants. sphk-1 mutants exhibit a significant reduction in paraquat-induced expression of Phsp-6::GFP, (89% reduction) that is similar to that exhibited by fshr-1 mutants (92% reduction (Figure 2A and (Kim and Sieburth 2018a)). Double mutants lacking both sphk-1 and fshr-1 displayed defects in paraquat-induced hsp-6 induction that were no more severe than those seen in single mutants (93% reduction, Figure 2A).
sphk-1 mutants exhibit reduced survival rates when exposed to toxic levels of paraquat (Kim and Sieburth 2018a), that are similar to those of fshr-1 mutants (Figure 2B). The survival rates of double mutants lacking both sphk-1 and fshr-1 were no more severe than those of either single mutant (Figure 2B). Expression of sphk-1 cDNA in the intestine fully rescued the increased paraquat-induced lethality of sphk-1 mutants (Kim and Sieburth 2018a). In contrast, expression of fshr-1 cDNA in the nervous system fully rescued the increased paraquat-induced lethality of fshr-1 mutants. In addition, expression of fshr-1 cDNA in the intestine restored near-normal paraquat sensitivity to fshr-1 mutants (Figure 2B and (Milleret al. 2015)). Taken together, FSHR-1 and SPHK-1 function in a common pathway to activate the UPRmt and to promote organism-wide protection from mitochondrial stress-induced lethality. Moreover, FSHR-1 functions in both the nervous system and the intestine to activate the UPRmt and promote survival, whereas SPHK-1 functions exclusively in the intestine.
FSHR-1/GPCR regulates mitochondrial association of SPHK-1 in the intestine
SPHK-1 rapidly associates with intestinal mitochondria following mitochondrial stress (Kim and Sieburth 2018a). To determine whether FSHR-1 regulates stress-induced SPHK-1 mitochondrial association, we examined the mitochondrial abundance of functional SPHK-1::GFP fusion proteins before and after paraquat exposure. Wild type animals treated with paraquat exhibit a 1.8 fold increase in SPHK-1::GFP mitochondrial fluorescence intensity (Figure 3C and (Kim and Sieburth 2018a)). fshr-1 mutants exhibited three defects in SPHK-1::GFP mitochondrial association. First, fshr-1 mutants exhibited a slightly smaller 1.5 fold increase in paraquat-induced mitochondrial SPHK-1::GFP fluorescence compared to wild type controls (Figure 3C). Second, fshr-1 mutants exhibited a nearly two-fold reduction in SPHK-1::GFP mitochondrial fluorescence compared to wild type controls in the absence of paraquat (Figure 3A). Third, fshr-1 mutants exhibited a nearly two-fold reduction in SPHK-1::GFP mitochondrial fluorescence compared to wild type controls following paraquat treatment (Figure 3C). The reduction in SPHK-1::GFP mitochondrial fluorescence is likely not due to decreased expression of the sphk-1::gfp transgene, or decreased mitochondrial mass since the fluorescence intensity of the mitochondrial marker TOMM-20::mCherry expressed under the same promoter (ges-1) was not reduced in fshr-1 mutants either in the absence or presence of paraquat (Figure 3A and (Kim and Sieburth 2018a)). Thus, fshr-1 mutations impair SPHK-1 association with mitochondria under both stressed and non-stressed conditions.
Interestingly, we observed an increase in mitochondrial mass in fshr-1 mutants, as revealed by an increase in TOMM-20::mCherry mitochondrial fluorescence compared to wild type controls (Figure 3A). Factoring in the increased mitochondrial mass in fshr-1 mutants, we found an approximately 5.9 fold decrease in mitochondrial-associated SPHK-1 at baseline (Figure 3B). Thus, FSHR-1 negatively regulates mitochondrial mass in the intestine and positively regulates SPHK-1 mitochondrial association, under normal conditions and following stress.
Neuronal fshr-1 cell non-autonomously regulates mitochondrial association of SPHK-1 in intestine
To determine in which tissue FSHR-1 functions to regulate the mitochondrial association of SPHK-1, we quantified SPHK-1::GFP mitochondrial fluorescence in fshr-1 mutants expressing fshr-1 cDNA in either the nervous system or in the intestine. We found that pan-neuronal expression of fshr-1 cDNA in fshr-1 mutants restored mitochondrial SPHK-1::GFP fluorescence intensity to wild type levels, both in the absence of paraquat and following paraquat treatment. In contrast, intestinal fshr-1 cDNA expression failed to rescue the SPHK-1::GFP fluorescence defects of fshr-1 mutants in the absence of paraquat and partially restored paraquat-induced SPHK-1 mitochondrial association (Figure 3C). These results suggest that FSHR-1 functions exclusively in the nervous system to regulate SPHK-1 abundance in the intestine in the absence of stress and that FSHR-1 function in both in the nervous system and in the intestine is likely to promote paraquat-induced SPHK-1 mitochondrial association.
Discussion
The nervous system coordinates various stress responses by releasing diffusible factors that act upon distal tissues to activate cellular defense programs (Berendzenet al. 2016; Shaoet al. 2016). Here we show that the conserved GPCR FSHR-1 is part of an integrated organism-wide response to mitochondrial stress that functions to activate the UPRmt in the intestine. FSHR-1 promotes UPRmt activation in response to either acute exposure to mitochondrial toxins or to chronic mitochondrial dysfunction. FSHR-1 functions in a common pathway with SPHK-1 to promote survival in response to toxic mitochondrial stress, and to promote UPRmt activation. FSHR-1 positively regulates the mitochondrial association of SPHK-1 in the intestine in the absence of stress, as well as stress-induced SPHK-1 mitochondrial recruitment. FSHR-1 functions in the nervous system and in the intestine to promote UPRmt activation, stress-induced SPHK-1 mitochondrial recruitment and survival in the presence of mitochondrial stress. We propose a model whereby FSHR-1 activates the UPRmt by two mechanisms: 1) neuronal FSHR-1 promotes cell non-autonomous activation of the UPRmt by regulating intestinal SPHK-1 activity, and 2) intestinal FSHR-1 activates the UPRmt by a cell-autonomous mechanism that does not involve SPHK-1 (Figure 4). FSHR-1 also regulates mitochondrial homeostasis in the absence of stress by regulating mitochondrial mass, baseline UPRmt activity, and SPHK-1 mitochondrial association.
Previous studies have established an important role for FSHR-1 in activating the innate immune response following infection with pathogenic bacteria. FSHR-1 promotes both survival following infection, and the behavioral avoidance response to pathogenic bacteria (Powellet al. 2009; Milleret al. 2015), and FSHR-1 activates a number of antimicrobial and antioxidant genes in response to pathogens (Powellet al. 2009; Milleret al. 2015). FSHR-1 is proposed to not participate in detection of the pathogenic bacteria themselves, but instead to participate in the detection of cellular damage (e.g. ROS) resulting from bacterial infection (Milleret al. 2015). Pathogenic bacterial infection is also a potent activator of the UPRmt, and UPRmt activation by pathogens promotes survival by inducing the expression of innate immune genes (Pellegrinoet al. 2014). Thus, FSHR-1 may activate the innate immune response by contributing to activation of the UPRmt following infection. Interestingly, prior studies have found that expression of FSHR-1 in the nervous system or the intestine can rescue survival defects of fshr-1 mutants (Powellet al. 2009), consistent with our studies showing a function for FSHR-1 in UPRmt activation in either tissue. We speculate that FSHR-1 may be a central component in the cellular response to mitochondrial dysfunction caused by a broad array of insults (e.g. pathogenic infection, ROS, and UPRmt dysfunction).
FSHR-1 also regulates intestinal mitochondrial homeostasis in the absence of stress. fshr-1 mutants exhibit a nearly three-fold increase in mitochondrial mass in the intestine, and a corresponding reduction in mitochondrial-associated SPHK-1. FSHR signaling negatively regulates mitochondrial biogenesis in mammals (Liuet al. 2017). Enhanced mitochondria biogenesis is correlated with UPRmt activation. Inducers of mitochondria biogenesis, such as nicotinamide riboside (NR) and poly(ADP-ribose) polymerase inhibitors (PARPi) disrupt the mito-nuclear protein homeostasis resulting in UPRmt activation via the sirtuin 1 (SIRT1) pathway in mammals and C. elegans. (Mouchiroudet al. 2013). In addition, rapamycin and resveratrol, which increase mitochondrial content, also induce UPRmt activation in C. elegans (Ungvariet al. 2011; Houtkooperet al. 2013; Lerneret al. 2013). Consistent with this, fshr-1 mutants exhibit increased baseline hsp-6 expression, which is dependent upon atfs-1, suggesting a potential function for FSHR-1 in suppressing UPRmt activation by reducing mitochondrial mass and/or biogenesis. We speculate that the defects in paraquat-induced UPRmt activation in fshr-1 mutants may be due to an underlying defect in the mitochondrial association of SPHK-1 under normal conditions.
Since FSHR-1 functions to protect animals from diverse stressors, the ligand for FSHR-1 is likely to originate from the host rather than from an exogenous source (Powellet al. 2009). FSHR-1 shares homology with three human GPCRs: FSHR, TSHR and LHCGR by virtue of nine LRRs found in its extracellular domain (Dolanet al. 2007). These GPCRs are activated by the heterodimeric glycopeptide hormones FSHα/β, TSHα/β, and LHα/β, respectively. In humans, FSH induces the generation of S1P by stimulating SphK1 activity for granulosa cell proliferation (Hernandez-Coronadoet al. 2016), suggesting a potential functional conservation of ligand-activated FSHR-1 signaling. However, the worm genome does not encode obvious orthologues of any of these ligands (Powellet al. 2009; Milleret al. 2015), suggesting divergence of an ancestral FSHR-1 ligand. A number of GPCR ligands have been identified that function non-autonomously to either activate the UPRmt or the innate immunity response (Shaoet al. 2016; Kim and Ewbank 2018). Among these are a set of neuropeptide-like proteins that function in a subset of sensory/interneurons to activate the UPRmt. These peptides are proposed to function in a neuronal circuit that transduces mitochondrial stress signals originating in the nervous system to the intestine to activate the UPRmt (Shaoet al. 2016). Because of the requirement for FSHR-1 in the nervous system in activating the UPRmt, we propose that FSHR-1 functions as part of this neuroendocrine signaling network, possibly as a receptor for one or more of these peptides. Consistent with a role for FSHR-1 in regulating neuron function, FSHR-1 positively regulates synaptic transmission at motor synapses (Sieburthet al. 2005). Identifying the ligand(s) for FSHR-1 will help to clarify both the cell autonomous and cell non-autonomous mechanisms by which FSHR-1 activates the UPRmt pathway.
Acknowledgements
This work was supported by grants from the NIH National institute of Neurological Disorders and Stroke (NINDS) to D.S. (NS071085 and NS099414). Some strains were provided by the Caenorhabditis Genetics Center (CGC), which is funded by the NIH office of Research infrastructure Programs (P40 OD010440).