JIA, Q. et al. (2019) International Worm Meeting "Mitochondrial H2O2 promotes neuropeptide release."

JIA, Q., Sieburth, D.

[International Worm Meeting, 2019]

Reactive oxygen species (ROS) are chemically reactive molecules that are formed during oxidative phosphorylation and function as important messengers in cellular signal transduction and homeostasis. We found that secretion of the neuropeptide-like protein, FLP-1, from the AIY interneurons promotes protection from oxidative stress-induced lethality and its release from AIY is positively and cell autonomously regulated by mitochondria-generated H2O2. FLP-1::Venus fusion proteins adopt a punctate pattern of fluorescence in the AIY axons that co-localize with dense core vesicle (DCV) and mitochondrial markers. Acute (10 minute) exposure to the mitochondrial ROS generator, juglone, H2O2, or mutations of trx-2/thioredoxin, which increase fluorescence of the H2O2 sensor mito-HyPer at release sites, lead to the depletion of FLP-1::Venus containing DCVs in AIY axons and an increase in FLP-1::Venus coelomocyte fluorescence. Mutations that impair mitochondrial trafficking to release sites (ric-7 mutants) or conversion of superoxide to H2O2 (sod-2 mutants) block the increase in FLP-1::Venus secretion induced by juglone or trx-2 mutations but not by H2O2. ric-7, sod-2 and trx-2 function cell autonomously in AIY to regulate FLP-1 secretion, and the catalytic activity and mitochondrial targeting of TRX-2 and SOD-2 are essential for their functions in regulating FLP-1 secretion. In addition, unc-31/CAPS or pkc-1/protein kinase C function cell autonomously in AIY to promote juglone or H2O2-induced FLP-1::Venus secretion without altering H2O2 levels. PKC-1 contains a conserved motif containing a cysteine residue (Cys524) that is predicted to be sulfenylated by H2O2. pkc-1 transgenes carrying a C524S substitution restore baseline but not juglone or H2O2 induced FLP-1::Venus secretion to pkc-1 mutants. Finally, FLP-1 may confer protection from oxidative stress-induced lethality by activating the antioxidant transcription factor SKN-1 in the intestine. Together, our data suggests that endogenously produced mROS trigger the PKC-1 dependent secretion of FLP-1 from AIY which in turns activates the anti-oxidant response in the intestine through inter-tissue signaling.

Jia Q et al. (2021) Nat Commun "Mitochondrial hydrogen peroxide positively regulates neuropeptide secretion during diet-induced activation of the oxidative stress response."

Sieburth D, Jia Q

[Nat Commun, 2021]

Mitochondria play a pivotal role in the generation of signals coupling metabolism with neurotransmitter release, but a role for mitochondrial-produced ROS in regulating neurosecretion has not been described. Here we show that endogenously produced hydrogen peroxide originating from axonal mitochondria (mtH₂O₂) functions as a signaling cue to selectively regulate the secretion of a FMRFamide-related neuropeptide (FLP-1) from a pair of interneurons (AIY) in C. elegans. We show that pharmacological or genetic manipulations that increase mtH₂O₂ levels lead to increased FLP-1 secretion that is dependent upon ROS dismutation, mitochondrial calcium influx, and cysteine sulfenylation of the calcium-independent PKC family member PKC-1. mtH₂O₂-induced FLP-1 secretion activates the oxidative stress response transcription factor SKN-1/Nrf2 in distal tissues and protects animals from ROS-mediated toxicity. mtH₂O₂ levels in AIY neurons, FLP-1 secretion and SKN-1 activity are rapidly and reversibly regulated by exposing animals to different bacterial food sources. These results reveal a previously unreported role for mtH₂O₂ in linking diet-induced changes in mitochondrial homeostasis with neuropeptide secretion.

Arroyo A et al. (2006) FEBS Lett "Coenzyme Q is irreplaceable by demethoxy-coenzyme Q in plasma membrane of Caenorhabditis elegans."

Arroyo A, Navas P, Gavilan A, Padilla S, Ruiz-Ferrer M, Santos-Ocana C, Rodriguez-Aguilera JC

[FEBS Lett, 2006]

A procedure was developed to isolate fractions enriched in plasma membrane from Caenorhabditis elegans. Coenzyme Q(9) (Q(9)) was found in plasma membrane isolated from either wild-type or long-lived qm30 and qm51 clk-1 mutant strains of Caenorhabditis elegans, along with dietary coenzyme Q(8) (Q(8)) and the biosynthetic intermediate demethoxy-Q(9) (DMQ(9)). NADH was able to reduce both Q(8) and Q(9), but not DMQ(9). Our results indicate that DMQ(9) cannot achieve the same redox role of Q(9) in plasma membrane, suggesting that proportion of all these Q isoforms in plasma membrane must be an important factor in establishing the clk-1 mutant phenotype.

Jonassen T et al. (2003) J Biol Chem "Reproductive fitness and quinone content of Caenorhabditis elegans clk-1 mutants fed coenzyme Q isoforms of varying length."

Larsen PL, Clarke CF, Jonassen T, Davis DE

[J Biol Chem, 2003]

Caenorhabditis elegans clk-1 mutants lack coenzyme Q(9) and accumulate the biosynthetic intermediate demethoxy-Q(9). A dietary source of ubiquinone (Q) is required for larval growth and development of the gonad and germ cells. We considered that uptake of the shorter Q(8) isoform present in the Escherichia coli food may contribute to the Clk phenotypes of slowed development and reduced brood size observed when the animals are fed Q-replete E. coli. To test the effect of isoprene tail length, N2 and clk-1 animals were fed E. coli engineered to produce Q(7), Q(8), Q(9), or Q(10). Wild-type nematodes showed no change in reproductive fitness regardless of the Q(n) isoform fed. clk-1(e2519) fed the Q(9) diet showed increased egg production; however, this diet did not improve reproductive fitness of the clk-1(qm30) animals. Furthermore, animals with the more severe clk-1(qm30) allele become sterile and their progeny inviable when fed Q(7)-containing bacteria. The content of Q(7) in the mitochondria of clk-1 animals was decreased relative to Q(8), suggesting less effective transport of Q(7) to the mitochondria, impaired retention, or decreased stability. Additionally, regardless of E. coli diet, clk-1(qm30) animals contain a dysfunctional dense form of mitochondria. The gonads of clk-1(qm30) worms fed Q(7)-containing food were severely shrunken and disordered. The differential fertility of clk-1 mutant nematodes fed Q isoforms may result from changes in Q localization, altered recognition by Q-binding proteins, and/or potential defects in mitochondrial function resulting from the mutant CLK-1 polypeptide itself.

Walker MW et al. (2005) J Biomol Screen "Use of Caenorhabditis elegans G{alpha}q chimeras to detect G-protein-coupled receptor signals."

Smith KE, Branchek TA, Walker MW, Jones KA, Gerald C, Tamm J, Zhong H, Vaysse P

[J Biomol Screen, 2005]

G-protein-coupled receptors (GPCRs) activate heterotrimeric G-proteins (G(i)-, G(s)-, G(q)-, or G(12)-like) to generate specific intracellular responses, depending on the receptor/G-protein coupling. The aim was to enable a majority of GPCRs to generate a predetermined output by signaling through a single G-protein-supported pathway. The authors focused on calcium responses as the output, then engineered Galpha(q) to promote promiscuous receptor interactions. Starting with a human Galpha(q) containing 5 Galpha(z) residues in the C-terminal receptor recognition domain (hGalpha(q/z5)), they evaluated agonist-stimulated calcium responses for 33 diverse GPCRs (G(i)-, G(s)-, and G(q)-coupled) and found 20 of 33 responders. In parallel, they tested Caenorhabditis elegans Galpha(q) containing 5 or 9 C-terminal Galpha(z) residues (cGalpha(q/z5), cGalpha(q/z9)). Signal detection was enhanced with cGalpha(q/z5) and cGalpha(q/z9) (yielding 25/33 and 26/33 responders, respectively). In a separate study of Galpha(s)-coupled receptors, the authors compared hGalpha(q/s5) versus hGalpha(q/s9), cGalpha(q/s9), andcGalphaq/s21 and observed optimal function with cGalpha(q/s9). Cotransfection of an engineered Galpha(q) "cocktail" (cGalpha(q/z5) plus cGalpha(q/s9)) provided a powerful and efficient screening platform. When the chimeras included N-terminal myristoylation sites (to promote membrane localization), calcium responses were sustained or improved, depending on the receptor. This approach toward a "universal functional assay" is particularly useful for orphan GPCRs whose signaling pathways are unknown.

Gaur R et al. (2007) J Biosci "Diet-dependent depletion of queuosine in tRNAs in Caenorhabditis elegans does not lead to a developmental block."

Gaur R, Tuck S, Bjork GR, Varshney U

[J Biosci, 2007]

Queuosine (Q),a hypermodified nucleoside,occurs at the wobble position of transfer RNAs (tRNAs)with GUN anticodons.In eubacteria,absence of Q affects messenger RNA (mRNA)translation and reduces the virulence of certain pathogenic strains.In animal cells,changes in the abundance of Q have been shown to correlate with diverse phenomena including stress tolerance,cell proliferation and tumour growth but the function of Q in animals is poorly understood.Animals are thought to obtain Q (or its analogues) as a micronutrient from dietary sources such as gut micro flora.However,the difficulty of maintaining animals under bacteria-free conditions on Q-deficient diets has severely hampered the study of Q metabolism and function in animals.In this study,we show that as in higher animals, tRNAs in the nematode Caenorhabditis elegans are modified by Q and its sugar derivatives.When the worms were fed on Q-deficient Escherichia coli ,Q modification was absent from the worm tRNAs suggesting that C.elegans lacks a de novo pathway of Q biosynthesis.The inherent advantages of C.elegans as a model organism,and the simplicity of conferring a Q-deficient phenotype on it make it an ideal system to investigate the function of Q modification in tRNA.

Jonassen T et al. (2002) J Biol Chem "Development and fertility in Caenorhabditis elegans clk-1 mutants depend upon transport of dietary coenzyme Q8 to mitochondria."

Marbois BN, Jonassen T, Faull KF, Clarke CF, Larsen PL

[J Biol Chem, 2002]

The Caenorhabditis elegans clk-1 mutants lack coenzyme Q, and instead accumulate the biosynthetic intermediate demethoxy-Q(9) (DMQ(9)). clk-1 animals grow to reproductive adults, albeit slowly, if supplied with Q(8)-containing Escherichia coli. However, if Q is withdrawn from the diet, clk-1 animals either arrest development as young larvae or become sterile adults depending upon the stage at the time of the withdrawal. To understand this stage-dependent response to a Q-less diet, the quinone content was determined during development of wild-type animals. The quinone content varies in the different developmental stages in wild-type fed Q(8)-replete E. coli. The amounts peak at the second larval stage, which coincides with the stage of arrest of clk-1 larvae fed a Q-less diet from hatching. Levels of the endogenously synthesized DMQ(9) are high in the clk1(qmM30)-arrested larvae and sterile adults fed Q-less food. Comparison of quinones from animals fed a Q-replete or a Q-less diet establishes that the Q(8) present is assimilated from the E. coli. Furthermore, this E. colispecific Q(8) is present in mitochondria isolated from fertile clk-1(qm30) adults fed a Q-replete diet. These results suggest that the uptake and transport of dietary Q(8) to mitochondria prevent the arrest and sterility phenotypes of clk-1 mutants and that DMQ is not functionally equivalent to Q.

Santos-Ocana C et al. (2002) J Biol Chem "Uptake of exogenous coenzyme Q and transport to mitochondria is required for bc1 complex stability in yeast coq mutants."

Clarke CF, Santos-Ocana C, Padilla S, Navas P, Do TQ

[J Biol Chem, 2002]

Coenzyme Q (Q) is an essential component of the mitochondrial respiratory chain in eukaryotic cells but also is present in other cellular membranes where it acts as an antioxidant. Because Q synthesis machinery in Saccharomyces cerevisiae is located in the mitochondria, the intracellular distribution of Q indicates the existence of intracellular Q transport. In this study, the uptake of exogenous Q(6) by yeast and its transport from the plasma membrane to mitochondria was assessed in both wild-type and in Q-less coq7 mutants derived from four distinct laboratory yeast strains. Q(6) supplementation of medium containing ethanol, a non-fermentable carbon source, rescued growth in only two of the four coq7 mutant strains. Following culture in medium containing dextrose, the added Q(6) was detected in the plasma membrane of each of four coq7 mutants tested. This detection of Q(6) in the plasma membrane was corroborated by measuring ascorbate stabilization activity, as catalyzed by NADH-ascorbate free radical reductase, a transmembrane redox activity that provides a functional assay of plasma membrane Q(6). These assays indicate that each of the four coq7 mutant strains assimilate exogenous Q(6) into the plasma membrane. The two coq7 mutant strains rescued by Q(6) supplementation for growth on ethanol contained mitochondrial Q(6) levels similar to wild type. However, the content of Q(6) in mitochondria from the non-rescued strains was only 35 and 8%, respectively, of that present in the corresponding wild-type parental strains. In yeast strains rescued by exogenous Q(6), succinate-cytochrome c reductase activity was partially restored, whereas non-rescued strains contained very low levels of activity. There was a strong correlation between mitochondrial Q(6) content, succinate-cytochrome c reductase activity, and steady state levels of the cytochrome c(1) polypeptide. These studies show that transport of extracellular Q(6) to the mitochondria operates in yeast but is strain-dependent. When Q biosynthesis is disrupted in yeast strains with defects in the intracellular transport of exogenous Q, the bc(1) complex is unstable. These results indicate that delivery of exogenous Q(6) to mitochondria is required fore activity and stability of the bc(1) complex in yeast coq mutants.

Saiki R et al. (2008) Aging Cell "Altered bacterial metabolism, not coenzyme Q content, is responsible for the lifespan extension in Caenorhabditis elegans fed an Escherichia coli diet lacking coenzyme Q."

Saiki R, Clarke CF, Furukawa S, Larsen PL, Bixler T, Lunceford AL, Lee W, Dang P

[Aging Cell, 2008]

Coenzyme Qn is a fully substituted benzoquinone containing a polyisoprene tail of distinct numbers (n) of isoprene groups. C. elegans fed E. coli devoid of Q(8) have a significant life span extension when compared to C. elegans fed a standard "Q-replete"E. coli diet. Here we examine possible mechanisms for the life span extension caused by the Q-less E. coli diet. A bioassay for Q uptake shows that a water-soluble formulation of Q(10) is effectively taken up by both clk-1 mutant and wild-type nematodes, but does not reverse life span extension mediated by the Q-less E. coli diet, indicating that life span extension is not due to the absence of dietary Q per se. The enhanced longevity mediated by the Q-less E. coli diet cannot be attributed to dietary restriction, different Qn isoforms, reduced pathogenesis or slowed growth of the Q-less E. coli, and in fact requires E. coli viability. Q-less E. coli have defects in respiratory metabolism. C. elegans fed Q-replete E. coli mutants with similarly impaired respiratory metabolism due to defects in complex V also show a pronounced life span extension, although not as dramatic as those fed the respiratory deficient Q-less E. coli diet. The data suggest that feeding respiratory incompetent E. coli, whether Q-less or Q-replete, produces a robust life extension in wild-type C. elegans. We believe the fermentation-based metabolism of the E. coli diet is an important parameter of C. elegans longevity.

Ryoichi Saiki et al. (2007) International Worm Meeting "Investigation of the effect of Q-less E. coli diet on Caenorhabditis elegans life span."

Tarra Wong, Pamela L. Larsen, Ryoichi Saiki, Catherine F. Clarke, Adam L. Lunceford, Wendy Lee

[International Worm Meeting, 2007]

Coenzyme Qn is a fully substituted benzoquinone containing a polyisoprene tail of distinct numbers (n) of isoprene groups. Q is an essential component of respiratory chain and a lipid soluble antioxidant. C. elegans fed E. coli devoid of Q₈ have a significant life span extension when compared to C. elegans fed a Q-replete E. coli diet. Extended life spans are also observed in C. elegans clk-1 mutants which have defects in Q biosynthesis and rely on a dietary supply of Q. These results suggest that the lack of Q is the important parameter affecting life span. Here we examine possible mechanisms for the life span extension caused by the Q-less E. coli diet. We show that supplementation of the Q-less E. coli diet with a water-soluble formulation of Q₁₀ (NovaSOL® Q), did not reverse the life span extension. Rescue of the clk-1 Q auxotrophy and Q₁₀ content in isolated mitochondria indicate that the Q₁₀ supplied in this manner is being assimilated. Thus the life span extension mediated by the Q-less E. coli diet cannot be attributed to the absence of Q in the diet. We show the enhanced life span mediated by the Q-less diet cannot be attributed to dietary restriction, altered isoprenoid tail lengths of the Qn isoform provided, slowed growth of the Q-less E. coli, or to reduced pathogenicity of the Q-less E. coli and in fact depends on the viability of the Q-less E. coli mutants. Q-less E. coli have defects in respiratory metabolism and fail to grow on media containing succinate as the carbon source. C. elegans fed Q-replete E. coli mutants with impaired respiratory metabolism due to defects in the ATP synthase (complex V) also show a pronounced life span extension, although feeding the Q-less E. coli diet mediates a more pronounced life span extension. Thus, respiratory deficiency of the E. coli diet is an important parameter of worm life span extension. C. elegans ttx-3 mutants, harboring defects in the gene encoding a LIM homeodomain transcription factor, cannot distinguish the Q-less from the Q-replete respiratory defective E. coli. The difference in life span extension mediated by the two respiratory deficient E. coli diets depends on food seeking behavior exhibited by N2 worms. The data suggest that the life span extension of N2 worms fed Q-less E.coli diet is caused by a combination of respiratory deficiency of the E. coli and the food seeking behavior of C. elegans. This work was supported by NIA Grant AG19777 and by the Ellison Medical Foundation.