Downregulation of the tyrosine degradation pathway extends Drosophila lifespan
- Department of Genetics, Blavatnik Institute, Harvard Medical School, United States
- Aging Institute of UPMC and the University of Pittsburgh, United States
- National Centre for Biological Sciences, Tata Institute of Fundamental Research, India
- Department of Biology, University of Konstanz, Germany
- Department of Chemistry, Princeton University, United States
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, United States
- Howard Hughes Medical Institute, United States
- Department of Neurology, Brigham and Women's Hospital, Massachusetts General Hospital, Harvard Medical School, United States
- Division of Signal Transduction, Beth Israel Deaconess Medical Center, and Department of Medicine, Harvard Medical School, United States
- Scintillon Institute, United States
- Department of Chemistry, The Scripps Research Institute, United States
Figures
Figure 1 with 1 supplement
Tyrosine is a new lifespan-dependent metabolite.
(A) Heat map showing the metabolites that significantly changed in 1-week and 4-week-old wild-type (B3) and long-lived (O1 and O3) flies. Each row represents a mean of five biological replicates. (B) Box plots of relative levels of tyrosine in 1-week and 4-week-old wild-type (B3) and long-lived (O1 and O3) flies extracted from the heat map (A). (C) Relative mRNA levels of CG1461 in 1-week-old control (B3) and long-lived (O1, O3) flies. Means ± SD. (D) Relative mRNA levels of CG1461, Faa, Hgo, CG11796, GstZ2, and Hn from 1-week and 5-week-old wild-type (OreR) flies. Means ± SD. (E) Tyrosine metabolism pathway. *p<0.05, **p<0.01, ***p<0.001.
Figure 1—figure supplement 1
Age-dependent increase of GFP-tagged CG1461.
Immunoblot analysis of GFP and tubulin in 1-week, 4-week, and 8-week CG1461(TyrAm)-GFP-tagged flies.
Figure 2 with 1 supplement
CG1461 functions as Tyrosine Aminotransferase/TAT and is necessary to degrade tyrosine.
(A) Relative mRNA levels of CG1461 in tubulin-Gal80ts, tubulin-Gal4 flies expressing either no RNAi, control RNAi or three different CG1461 RNAi for 10 days. Means ± SD. *p<0.05, ***p<0.001 (B) Relative mRNA levels of CG1461 in wild-type and CG1461-deficient flies (both backcrossed to wild-type OreR flies). Means ± SD. ***p<0.001 (C) Feeding adult flies with 5 g/L of tyrosine significantly suppresses lifespan of flies with ubiquitous adult-onset downregulation of CG1461. Arrow indicates the beginning of tyrosine feeding. p<0.001. (D) Feeding adult flies with 5 g/L of tyrosine significantly suppresses lifespan of CG1461 mutant but not wild-type or heterozygous flies. Arrow indicates the beginning of tyrosine feeding. p<0.001. Box plots of relative levels of tyrosine (E) and phenylalanine (F) in wild-type and CG1461-deficient flies fed either control or high level of tyrosine (5 g/L) diet.
Figure 2—figure supplement 1
Tyrosine supplementation increases lifespan of wild-type OreR flies and increases expression of GFP-tagged CG1461.
Lifespan of male (A) and female (B) wild-type and CG1461-deficient flies (both backcrossed to wild-type OreR flies) fed with the range of tyrosine concentrations (1X = 0.5 g/L). Arrow indicates the beginning of tyrosine feeding. (C) Immunoblot analysis of GFP and tubulin in CG1461(TyrAm)-GFP-tagged flies fed either control or high-tyrosine diet. (D) Relative mRNA levels of CG1461, CG11796, Hgo, Faa, Hn, Rp49 in male and female flies. Means ± SD. Means ± SD. *p<0.05, **p<0.01, ***p<0.001.
Figure 3 with 1 supplement
Whole-body and neuronal-specific downregulation of CG1461/Tyrosine Aminotransferase extends lifespan.
(A) Ubiquitous adult-onset expression of CG1461 RNAi-1 increases lifespan in females. p<0.0001. (B) Ubiquitous adult-onset expression of CG1461 RNAi-3 increases lifespan in females. p<0.0001. (C) Ubiquitous adult-onset expression of CG11796 RNAi increases lifespan in females. p<0.0001. (D) Ubiquitous adult-onset expression of Hgo RNAi increases lifespan in females. p<0.0001. (E) Fat body-specific adult-onset expression of CG1461 RNAi does not affect lifespan in females. (F) Intestine-specific adult-onset expression of CG1461 RNAi does not affect lifespan in females. (G) Neuronal-specific adult-onset expression of CG1461 RNAi-1 increases lifespan in females. p<0.0001. (H) Neuronal-specific adult-onset expression of CG1461 RNAi-2 increases lifespan in females. p<0.0001. (I) Neuronal-specific adult-onset expression of CG1461 RNAi-1, -2, and -3 increases lifespan in females. p<0.0001.
Figure 3—figure supplement 1
Neuronal-specific downregulation of Tyrosine Aminotransferase/TAT leads to metabolic reprogramming similar to the whole-body downregulation of TAT.
Box plots of relative levels of tyrosine (A), glucosamine (B), methylcysteine (C), and NADH (D) in elav-Gal4,tubulin-Gal80ts flies expressing either control RNAi or TAT RNAi. FDR adjusted p-value. *p<0.05, **p<0.01.
Figure 4 with 1 supplement
Downregulation of CG1461/Tyrosine Aminotransferase leads to reprogramming of metabolism related to mitochondrial function.
(A) Principal component analysis of tubulin-Gal4, tubulin-Gal80ts flies expressing either control RNAi or two different TAT RNAi. (B) Heat map showing the significantly and commonly changed metabolites in flies expressing two different TAT RNAi. Box plots of relative levels of tyrosine (C), lactate (D), glucosamine (E), nicotinamide (F), methylcysteine (G) in tubulin-Gal4,tubulin-Gal80ts flies expressing either control RNAi or two different TAT RNAi. (H) Metabolic Set Enrichment Analysis of the metabolites that changed significantly and commonly in flies expressing two different TAT RNAi. Box plots of relative levels of NADH (I), thiamine pyrophosphate (J), NADP (K) in tubulin-Gal4,tubulin-Gal80ts flies expressing either control RNAi or two different TAT RNAi.
Figure 4—figure supplement 1
Neuronal-specific downregulation of Tyrosine Aminotransferase/TAT increases levels of tyrosine-derived neurotransmitters.
Head levels of Histamine (A) and GABA (B) in CG1461/TAT wild-type (wt), heterozygous (het), and mutant (mut) flies. Head levels of DOPA (C), Octopamine (D), Dopamine (E), Tyramine (F) in elav-Gal4, tubulin-Gal80ts > control RNAi or TAT RNAi flies maintained either on the regular food or the food containing 5 mM of tyrosine for 2 days. Means ± SD. *p<0.05, **p<0.01, ***p<0.001. Feeding adult male (G) and female (H) OreR flies with 5 mM Octopamine, L-DOPA, or Tyramine starting day 14.
Figure 5 with 1 supplement
Whole-body downregulation of Tyrosine Aminotransferase/TAT elevates levels of tyrosine-derived neurotransmitters in fly heads.
Head levels of Tyrosine (A), DOPA (B), Dopamine (C), Tyramine (D), and Octopamine (E) in CG1461/TAT wild-type (wt), heterozygous (het), and mutant (mut) flies. Means ± SD. *p<0.05, **p<0.01, ***p<0.001.
Figure 5—figure supplement 1
Suppression of complex I of ETC upregulates mRNA levels of enzymes in the tyrosine degradation pathway and decreases lifespan that can be partially rescued by supplementation of tyrosine.
(A) Relative mRNA levels of Faa, CG11796, Hgo in tubulin-Gal80ts, tubulin-Gal4 flies expressing either no RNAi, control RNAi or RNAi against different subunits of mitochondrial ETC Complex I – CG9762, NP15.6, mtacp1 for 10 days. Means ± SD. (B) Relative mRNA levels of CG1461/TAT, CG11796, Hgo, Faa, and Hn in tubulin-Gal80ts, elav-Gal4 flies expressing either control RNAi or RNAi against NP15.6. Lifespans of male (C) and female (D) tubulin-Gal80ts, tubulin-Gal4 flies expressing either control RNAi or RNAi against NP15.6 fed with the range of tyrosine concentrations (1X = 0.5 g/L). Neuronal counts (E) and histology (F) of hematoxylin-stained heads from flies with or without expression of wild-type human α-synuclein with or without three different RNAi against TAT. Means ± SD. *p<0.05, **p<0.01, ***p<0.001.
Figure 6 with 1 supplement
Mitochondrial dysfunction/neurodegeneration upregulates the level of CG1461/tyrosine aminotransferase.
(A) Relative mRNA levels of CG1461/TAT in Gal80ts; tubulin-Gal4 flies expressing either no RNAi, control RNAi or RNAi against different subunits of mitochondrial ETC – CG9762, SDHC, CG18809, CG5548, NP15.6, CG8680, CG1970, CG3214, mtacp1 for 10 days. Means ± SD. (B) Relative mRNA levels of CG1461/TAT and α -synuclein in heads of flies with or without expression of wild-type human α-synuclein. Means ± SD. (C) Relative mRNA levels of CG1461/TAT in tubulin-Gal80ts, tubulin-Gal4 flies overexpressing control or p60 (inhibitory subunit of InR), dTsc1/dTsc2 (TSC complex, dTOR inhibitor) or PGC1a/Spargel. Means ± SD. (D) Immunoblot analysis of GFP and tubulin in heads of 1-week and 3-week-old flies expressing either control or TAT RNAi under pan-neuronal driver (ElavGal4) in the presence of GstD-GFP. (E) Ubiquitous adult-onset downregulation of TAT prolongs lifespan under oxidative stress (10 mM Paraquat). (F) Relative mRNA levels of CG1461/TAT in tubulin-Gal80ts, tubulin-Gal4 flies expressing either control or NP15.6 RNAi and fed with 10 mM reduced Glutathione, 100 µM mitoQ, 10 mM methyl pyruvate, 100 µM Ibedenone, or 100 µM Tigecycline. Means ± SD (G) Relative mRNA levels of CG1461/TAT, CG11796, faa, and Hgo in tubulin-Gal80ts, tubulin-Gal4 flies expressing either control or NP15.6 RNAi and fed with either control or 100 µM Tigecycline. (H) Working model. *p<0.05, **p<0.01, ***p<0.001.
Figure 6—figure supplement 1
The effect of downregulation of tyrosine aminotransferase/TAT on GFP-tagged reporters relevant to aging.
(A) Schematic diagram of mitophagy/mitochondrial biogenesis regulation by Insulin signaling, TOR, and PGC1a/Spargel. (B) Immunoblot analysis of GFP and tubulin in heads of 1-week and 3-week-old tubulin-Gal80ts, elav-Gal4 flies in the presence of GFP-CL1 (B), hsp22-GFP (C), 10XSTAT92-GFP (D), Drs-GFP (E) reporters expressing either control or TAT RNAi. (F) Relative mRNA levels of CLPX, HSP10, HSP60, and HSP22 in tubulin-Gal80ts, tubulin-Gal4 flies expressing either control RNAi or NP15.6 RNAi and fed with either control or 100 µM Tigecycline. Means ± SD.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-α-Tubulin | Sigma | T5168 | |
| Antibody | Anti-GFP | Invitrogen | A-6455 | |
| Strain, strain background (Escherichia coli) | One Shot TOP10 Chemically Competent E. coli | Thermo Fisher Scientific | C404003 | |
| Commercial assay or kit | iScript Reverse Transcription Supermix | Bio-Rad | 1708896 | |
| Commercial assay or kit | iQ SYBR Green Supermix | Bio-Rad | 1708880 | |
| Commercial assay or kit | BenchMark Prestained Protein Ladder | Invitrogen | 10748–010 | |
| Chemical compound, drug | TRIzol reagent | Invitrogen | 15596–018 | |
| Chemical compound, drug | Mifepristone | Cayman Chemical Company | 10006317 | |
| Chemical compound, drug | Methyl viologen dichloride hydrate (Paraquat) | Sigma-Aldrich | 856177 | |
| Chemical compound, drug | ProSieve EX transfer buffer | Lonza | 00200309 | |
| Chemical compound, drug | ProSieve EX running buffer | Lonza | 200307 | |
| Chemical compound, drug | L-Glutathione reduced | Sigma-Aldrich | G6013-5G | |
| Chemical compound, drug | Laemmli Sample Buffer | Bio-Rad | 1610737 | |
| Chemical compound, drug | RIPA | Cell Signaling | 9806 | |
| Chemical compound, drug | Protease Inhibitor Cocktail Tablets | Roche | 4693159001 | |
| Chemical compound, drug | Idebenone | Cayman Chemical Company | 15475 | |
| Chemical compound, drug | Mitoquinol | Cayman Chemical Company | 89950 | |
| Chemical compound, drug | Nuclease-Free Water (not DEPC-Treated) | Ambion, Inc | AM9930 | |
| Chemical compound, drug | 3,4-Dihydroxy-L-phenylalanine | Millipore Sigma | D9628 | |
| Chemical compound, drug | Octopamine hydrochloride | Millipore Sigma | O0250 | |
| Chemical compound, drug | RQ1 RNase-Free DNase | Promega | M6101 | |
| Chemical compound, drug | Tyramine | Sigma-Aldrich | T90344 | |
| Commercial assay or kit | 4–20% Mini-PROTEAN TGX Precast Protein Gels | Bio-Rad | 4561095 | |
| Commercial assay or kit | pENTR/D-TOPO Cloning Kit | Life Technologies | K2400-20 | |
| Commercial assay or kit | Gateway LR Clonase II Enzyme mix | Invitrogen | 11791–020 | |
| Genetic reagent (D. melanogaster) | white RNAi (HMS00017) | Bloomington Drosophila Stock Center | # 33623 | |
| Genetic reagent (D. melanogaster) | GFP RNAi (HMS00314) | Perrimon’s lab | ||
| Genetic reagent (D. melanogaster) | CG1461 RNAi (HMC03212) | Bloomington Drosophila Stock Center | # 51470 | |
| Genetic reagent (D. melanogaster) | CG1461 RNAi (HMS05690) | Bloomington Drosophila Stock Center | # 67830 | |
| Genetic reagent (D. melanogaster) | CG1461 RNAi (HMS05877) | Bloomington Drosophila Stock Center | # 76065 | |
| Genetic reagent (D. melanogaster) | Hgo RNAi (HMC03775) | Bloomington Drosophila Stock Center | # 55629 | |
| Genetic reagent (D. melanogaster) | CG11796 RNAi (HMC03663) | Bloomington Drosophila Stock Center | # 52923 | |
| Genetic reagent (D. melanogaster) | CG9762 RNAi (HMC06415) | Bloomington Drosophila Stock Center | # 67311 | |
| Genetic reagent (D. melanogaster) | Sdhc RNAi (HMC03497) | Bloomington Drosophila Stock Center | # 53281 | |
| Genetic reagent (D. melanogaster) | CG18809 RNAi (HMS04326) | Bloomington Drosophila Stock Center | # 56907 | |
| Genetic reagent (D. melanogaster) | CG5548 RNAi (HM05255) | Bloomington Drosophila Stock Center | # 30511 | |
| Genetic reagent (D. melanogaster) | NP15.6 RNAi (HMS01560) | Bloomington Drosophila Stock Center | # 36672 | |
| Genetic reagent (D. melanogaster) | CG8680 RNAi (HMC03434) | Bloomington Drosophila Stock Center | # 51860 | |
| Genetic reagent (D. melanogaster) | CG1970 RNAi (HMC04814) | Bloomington Drosophila Stock Center | # 57499 | |
| Genetic reagent (D. melanogaster) | CG3214 RNAi (HMS01584) | Bloomington Drosophila Stock Center | # 36695 | |
| Genetic reagent (D. melanogaster) | mtacp1 RNAi (HM05206) | Bloomington Drosophila Stock Center | # 29528 | |
| Genetic reagent (D. melanogaster) | B3 | Gift from Dr. Trudy Mackay | ||
| Genetic reagent (D. melanogaster) | O1 | Gift from Dr. Trudy Mackay | ||
| Genetic reagent (D. melanogaster) | O3 | Gift from Dr. Trudy Mackay | ||
| Genetic reagent (D. melanogaster) | OregonR | Perrimon’s lab | ||
| Genetic reagent (D. melanogaster) | tubulinGal4; tubulinGal80ts | Perrimon’s lab | ||
| Genetic reagent (D. melanogaster) | CG1461-mutant | This paper | ||
| Genetic reagent (D. melanogaster) | Actin-GeneSwitch-Gal4 | Gift from Dr. John Tower | ||
| Genetic reagent (D. melanogaster) | Whole body fat body – GeneSwitch – Gal4 | Gift from Dr. John Tower | ||
| Genetic reagent (D. melanogaster) | TIGS-2 | Gift from Dr. John Tower | ||
| Genetic reagent (D. melanogaster) | 3X Elav-GeneSwitch-Gal4 | Gift from Dr. Scott Pletcher | ||
| Genetic reagent (D. melanogaster) | ElavGal4; tubulinGal80ts | Perrimon’s lab | ||
| Genetic reagent (D. melanogaster) | UAS-dTsc1,Tsc2 | Tapon et al., 2002 | ||
| Genetic reagent (D. melanogaster) | UAS-p60 | Bloomington Drosophila Stock Center | # 25899 | |
| Genetic reagent (D. melanogaster) | UAS-Spargel | Gift from Dr. David Walker | ||
| Genetic reagent (D. melanogaster) | GstD-GFP | Gift from Dr. Dirk Bohmann | ||
| Genetic reagent (D. melanogaster) | CG1461-GFP-tagged | VDRC stock center | # 318640 | |
| Genetic reagent (D. melanogaster) | GFP-CL1 | Gift from Dr. Udai Pandey | ||
| Genetic reagent (D. melanogaster) | hsp22-GFP | Gift from Dr. John Tower | ||
| Genetic reagent (D. melanogaster) | STAT92-GFP | Bloomington Drosophila Stock Center | # 26198 | |
| Genetic reagent (D. melanogaster) | Drs-GFP | Bloomington Drosophila Stock Center | # 55707 | |
| Genetic reagent (D. melanogaster) | Xbp1-GFP | Bloomington Drosophila Stock Center | # 60730 | |
| Genetic reagent (D. melanogaster) | Syb-QF2; QUAS- α-synuclein | Gift from Dr. Mel Feany |
Additional files
-
Supplementary file 1
Lifespan counts are recorded for all experiments.
- https://cdn.elifesciences.org/articles/58053/elife-58053-supp1-v2.xlsx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/58053/elife-58053-transrepform-v2.docx
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Downregulation of the tyrosine degradation pathway extends Drosophila lifespan
eLife 9:e58053.
https://doi.org/10.7554/eLife.58053
