Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation Annemieke T. van der Goota, Wentao Zhub, Rafael P. Vázquez-Manriquec,1, Renée I. Seinstraa, Katja Dettmerb, Helen Michelsa, Francesca Farinac, Jasper Krijnend, Ronald Melkie, Rogier C. Buijsmanf, Mariana Ruiz Silvaa, Karen L. Thijssena, Ido P. Kemad, Christian Neric, Peter J. Oefnerb, and Ellen A. A. Nollena,g,2 Departments of aGenetics and dLaboratory Medicine, University of Groningen, University Medical Centre Groningen, 9700 RB, Groningen, The Netherlands; bInstitute of Functional Genomics, University of Regensburg, 93053 Regensburg, Germany; cInstitut National de la Santé et de la Recherche Médicale, Unit 894, Laboratory of Neuronal Cell Biology and Pathology, 75014 Paris, France; eLaboratoire d’Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France; fTranslational Research Centre B.V., 5342 AC, Oss, The Netherlands; and gUniversity of Groningen, University Medical Centre Groningen, European Research Institute for the Biology of Aging, 9700 AD, Groningen, The Netherlands Edited by F. Ulrich Hartl, Max Planck Institute of Biochemistry, Martinsried, Germany, and approved July 20, 2012 (received for review February 26, 2012) Toxicity of aggregation-prone proteins is thought to play an im- Results portant role in aging and age-related neurological diseases like Knockdown of tdo-2 Suppresses Toxicity of α-Synuclein in C. elegans. Parkinson and Alzheimer’s diseases. Here, we identify tryptophan α-Synuclein is an aggregation-prone protein that is involved in 2,3-dioxygenase (tdo-2), the first enzyme in the kynurenine path- Parkinson disease and other synucleinopathies (14, 15). We have way of tryptophan degradation, as a metabolic regulator of age- previously identified 80 genes that modify α-synuclein inclusion α related -synuclein toxicity in a Caenorhabditis elegans model. formation in a C. elegans model (16). Depletion of tdo-2 also suppresses toxicity of other heterologous Toxicity of α-synuclein in this model can be measured by a β aggregation-prone proteins, including amyloid- and polyglut- progressive decline in the worms’ motility during aging (17). To amine proteins, and endogenous metastable proteins that are fi α fi search for modi ers of -synuclein toxicity, we measured the sensors of normal protein homeostasis. This nding suggests that motility of animals in which each of the 80 genes was individually tdo-2 functions as a general regulator of protein homeostasis. knocked down by RNAi (Dataset S1). We identified 10 genes that Analysis of metabolite levels in C. elegans strains with mutations increased toxicity and 3 genes that decreased toxicity on knock- in enzymes that act downstream of tdo-2 indicates that this sup- down (Fig. 1A and Dataset S1). Our data are in line with other pression of toxicity is independent of downstream metabolites in the kynurenine pathway. Depletion of tdo-2 increases tryptophan levels, studies showing that the presence of inclusions does not neces- sarily correlate with toxicity (18). The most potent suppressor of and feeding worms with extra L-tryptophan also suppresses toxicity, fi suggesting that tdo-2 regulates proteotoxicity through trypto- toxicity identi ed in this screen was C28H8.11 (NCBI accession phan. Depletion of tdo-2 extends lifespan in these worms. To- number NM_065883.3), which resulted in a 2.5-fold increase in gether, these results implicate tdo-2 as a metabolic switch of motility in day 4 adults (Fig. 1A). We named this gene tdo-2 age-related protein homeostasis and lifespan. With TDO and based on its homology to human TDO (NCBI accession number Indoleamine 2,3-dioxygenase as evolutionarily conserved human NP_005642.1) (Fig. S1A). tdo-2 encodes TDO-2, which catalyzes orthologs of TDO-2, intervening with tryptophan metabolism the first and rate-limiting step in the kynurenine pathway of may offer avenues to reducing proteotoxicity in aging and age- tryptophan degradation (Fig. 1B). Analysis of a transcriptional related diseases. reporter strain expressing GFP under the control of the tdo-2 promoter suggests that tdo-2 is mainly expressed in body wall Huntington | longevity muscle cells and the hypodermis and therefore, that it may regulate proteotoxicity through these tissues (Fig. S1B)(19). α aintaining a stable proteome is critical for an organism’s Knockdown of tdo-2 suppressed -synuclein toxicity from day 1 α Msurvival, and cells have evolved several mechanisms to cope of adulthood on without affecting expression levels of -synuclein with misfolded and aggregation-prone proteins (1). Conditions (Fig. 1C and Fig. S1 C and D). A decline in muscle function is fi such as environmental stress or aging increase protein damage, one of the rst features that can be observed during aging in – and at the same time, protein quality control systems are thought many organisms (20 23), and decreased protein quality control to be compromised, resulting over time in the accumulation of or the expression of aggregation-prone proteins can accelerate toxic aggregation-prone proteins (2). Toxicity of such aggrega- this age-dependent decline in muscle function (6, 24). We, there- tion-prone proteins is thought to play an important role in aging fore, tested whether knockdown of tdo-2 would suppress the de- and age-related diseases, such as Parkinson, Alzheimer’s, and cline in motility during aging of control animals as well. Indeed, Huntington disease (3–5). Although aging seems to be the greatest risk factor for developing neurodegenerative diseases, little is known about the metabolic and molecular processes that drive Author contributions: A.T.v.d.G. and E.A.A.N. designed research; A.T.v.d.G., W.Z., R.P.V.-M., R.I.S., K.D., H.M., F.F., J.K., R.M., M.R.S., and K.L.T. performed research; R.C.B., I.P.K., and the toxicity of aggregation-prone proteins during aging. P.J.O. contributed new reagents/analytic tools; A.T.v.d.G., W.Z., R.P.V.-M., I.P.K., C.N., Studies in the nematode Caenorhabditis elegans have identified P.J.O., and E.A.A.N. analyzed data; and A.T.v.d.G. and E.A.A.N. wrote the paper. evolutionarily conserved genetic pathways that regulate protein ho- The authors declare no conflict of interest. meostasis as well as aging, including the insulin/insulin-like growth This article is a PNAS Direct Submission. factor 1 (IGF-1) signaling (IIS), hypoxic response, and dietary re- Freely available online through the PNAS open access option. – striction pathways (6 13). 1Present address: Grupo de Investigación en Enfermedades Neurosensoriales, Instituto de Here,wedescribetheidentification of the tryptophan-con- Investigación Sanitaria IIS-La Fe, 46009 Valencia, Spain. verting enzyme tryptophan 2,3-dioxygenase (TDO-2) as a meta- 2To whom correspondence should be addressed. E-mail: [email protected]. bolic regulator of age-related protein toxicity and lifespan in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. C. elegans. 1073/pnas.1203083109/-/DCSupplemental. 14912–14917 | PNAS | September 11, 2012 | vol. 109 | no. 37 www.pnas.org/cgi/doi/10.1073/pnas.1203083109 Downloaded by guest on September 26, 2021 A which was measured by increases in kynurenine, anthranilic acid, 250 3-hydroxykynurenine, and 3-hydroxyanthranilic acid (Fig. 2E ) (% and Table S2). Deletions in kmo-1 and haao-1 did not affect the y 200 α lit motility of -synuclein worms (Fig. 2B and Table S1), nor did i ot 150 a deletion in afmd-1 encoding formamidase (Fig. S2A). Deletion m e fl α v 100 or RNAi inactivation of u-2 increased the motility of -synu- ati clein animals by 1.3-fold (Fig. 2 C and D and Table S1). When 50 Rel we then depleted tdo-2 in afmd-1, kmo-1, and haao-1 mutant 0 1 1 0 9 4 7 3 4 7 animals, we observed a strong increase in motility comparable .1 .1 .1 .3 .2 .8 -1 .9 .5 .6 .3 .2 .5 -9 -2 -9 -2 -4 -2 -2 -7 - 1 1 2 5 3 1 2 i t RNAi: A 4 k 9 4 3 2 9 6 8 0 9 2 8 x n o g 1 c- le 1 2 F d q A pr α B. A E tr- C H H H H ef m td cm gt- th- rsp 34 rab with the increase observed in WT -synuclein animals on tdo-2 90. 38. n 9 0 p 6 s 2 7 A 0 6 9 C 6 8 9 - s rb u s a m s 12 02 m T0 12 yp 34 F1 C2 D1 F5 F5 F1 C0 G1 R0 C2 RNAi (Fig. 2B, Fig. S2A, and Table S1). Similarly, depletion of K B c F C Z 71 fl Y tdo-2 increased motility in u-2 mutant animals from 1.3- up to 1.8-fold (Fig. 2C and Table S1). Knockdown of tdo-2 almost fully B C RNAi from L1 D RNAi from day 1 NH 2 YFP/ctrl RNAi syn-YFP/ctrl RNAi ctrl RNAi blocked the kynurenine pathway in all mutants, increasing the COOH YFP/ syn-YFP/ 200 tdo-2 tdo-2 tdo-2 tryptophan levels by more than fivefold (Fig. 2E and Table S2). N 60 H fi L-Tryptophan This nding showed that, in WT animals, at least 80% of tryp- 50 160 c tophan was degraded by TDO-2. Knockdown of tdo-2 reduced e n mi 0 s the levels of all other metabolites close to background levels TDO-2/ 40 s / RNAi 120 3 / d tdo-2 s (Fig. 2E and Table S2). Kynurenic acid (KA) was detected only d en n 30 in kmo-1 and flu-2 mutant animals, and its level was unaltered by y b 80 be knockdown of tdo-2 (Fig.