Kynurenic Acid and Its Analogs Are Beneficial Physiologic Attenuators

molecules

Article Kynurenic Acid and Its Analogs Are Beneﬁcial Physiologic Attenuators in Bdelloid Rotifers

Zsolt Datki 1,* , Zita Galik-Olah 1 , Zsuzsanna Bohar 2,3, Denes Zadori 3, Ferenc Fulop 4,5 , Istvan Szatmari 4,5, Bence Galik 6, Janos Kalman 1 and Laszlo Vecsei 2,3 1 Department of Psychiatry, Faculty of Medicine, University of Szeged, Kalvaria sgt. 57, H-6725 Szeged, Hungary; [email protected] (Z.G.-O.), [email protected] (J.K.) 2 MTA-SZTE Neuroscience Research Group, Semmelweis u. 6, H-6725 Szeged, Hungary; [email protected] (Z.B.); [email protected] (L.V.) 3 Department of Neurology, Interdisciplinary Excellence Centre, Faculty of Medicine, University of Szeged, Semmelweis u. 6, H-6725 Szeged, Hungary; [email protected] 4 Institute of Pharmaceutical Chemistry, Interdisciplinary Excellence Centre, Faculty of Pharmacy, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary; [email protected] (F.F.), [email protected] (I.S.) 5 MTA-SZTE Stereochemistry Research Group Eötvös u. 6, H-6720 Szeged, Hungary 6 Department of Clinical Molecular Biology, Medical University of Bialystok, ul.Jana Kilinskiego 1, 15-089 Bialystok, Poland; [email protected] * Correspondence: [email protected] or [email protected]; Tel.: +36-709442289

Received: 6 May 2019; Accepted: 6 June 2019; Published: 10 June 2019

Abstract: The in vivo investigation of kynurenic acid (KYNA) and its analogs is one of the recent exciting topics in pharmacology. In the current study we assessed the biological effects of these molecules on bdelloid rotifers (Philodina acuticornis and Adineta vaga) by monitoring changes in their survival and phenotypical characteristics. In addition to longitudinal (slowly changing) markers (survival, number of rotifers alive and body size index), some dynamic (quickly responding) ones (cellular reduction capacity and mastax contraction frequency) were measured as well. KYNA and its analogs increased longevity, reproduction and growth, whereas reduction capacity and energy-dependent muscular activity decreased conversely. We found that spermidine, a calorie restriction mimetic, exerted similar changes in the applied micro-invertebrates. This characterized systemic profile evoked by the above-mentioned compounds was named beneficial physiologic attenuation. In reference experiments, using a stimulator (cyclic adenosine monophosphate) and a toxin (sodium azide), all parameters changed in the same direction (positively or negatively, respectively), as expected. The currently described adaptive phenomenon in bdelloid rotifers may provide holistic perspectives in translational research.

Keywords: kynurenic acid; metabolism; physiology; bdelloid rotifer; survival; mastax

1. Introduction The kynurenine (KYN) pathway plays an important role in several biological systems affected by aging, via modulating the cellular changes related to oxidative stress, mitochondrial dysfunction, inflammation, cognitive and immune responses [1]. KYNs are endogenous intermediates of L-tryptophan degradation, a pathway in which serotonin, nicotinic acid and its derivatives, e.g., nicotinamide adenine dinucleotide (NAD+) is produced [2]. KYNs became the focus of scientific attention in the 1980s with the discovery of some intermediates having primary neuroactive and protective properties [3] as broad-spectrum ionotropic glutamate receptor antagonists [4,5]. Elevated concentrations of kynurenic acid (KYNA) in cerebrospinal fluid were observed in aged populations [6],

Molecules 2019, 24, 2171; doi:10.3390/molecules24112171 www.mdpi.com/journal/molecules Molecules 2019, 24, x 2 of 10 Molecules 2019, 24, 2171 2 of 10 concentrations of kynurenic acid (KYNA) in cerebrospinal fluid were observed in aged populations [6], while in the invertebrate Caenorhabditis elegans the depletion of KYNA improved learning and whilememory in [7]. the This invertebrate moleculeCaenorhabditis inhibits the overexcitati elegans theon depletionof glutamatergic of KYNA transmission; improved furthermore, learning and it memorymay influence [7]. This nutrition molecule and inhibits metabolism, the overexcitation via the regu of glutamatergiclation of food-dependent transmission; behavioral furthermore, plasticity it may influence[8,9]. In C. nutrition elegans the and level metabolism, of serotonin via the increases regulation during of food-dependent feeding behavior, behavioral even in plasticitythe absence [8, 9of]. Infood.C. elegansThe tryptophanthe level ofhydroxylase-mutant serotonin increases animals, during feeding which behavior,thus lack evenserotonin, in the showed absence reduced of food. Thefeeding tryptophan rates even hydroxylase-mutant in the presence of animals,food [10], which proving thus a key lack role serotonin, of the serotonergic showed reduced system feeding in the ratesregulation even inof themetabolism. presence of food [10], proving a key role of the serotonergic system in the regulation of metabolism.The well-known rotifer-aging model [11], also used in our laboratory, applies to bdelloid species,The which well-known have rotifer-aginga complex nervous model [11system], also main usedly in based our laboratory, on serotonergic applies toregulation bdelloid species,[12,13]. whichThus, they have seem a complex to be adequate nervous for system investigations mainly based of the on relationship serotonergic between regulation KYNA, [12,13 its]. analogs Thus, they and seemphysiology. to be adequate Bdelloid for rotifers investigations are widely of the used relationship microinvertebrate between KYNA, models its in analogs the research and physiology. fields of Bdelloidecotoxicology rotifers [14] are widelyand aging used microinvertebrate[15]. They are eutelic models (grown in the research by hypertrophy) fields of ecotoxicology animals with [14] andwell-defined aging [15 ].viability They are markers eutelic and (grown anatomical by hypertrophy) parameters. animals In our with previous well-defined work [16], viability we developed markers anda complex anatomical in vivo parameters. experimental In our viability previous assay work for [16 ],high-throughput we developed a complexscreening,in vivoby measuringexperimental the viabilitysurvival, assaynumber for of high-throughput rotifers alive (NRA), screening, body by size measuring index (BSI), the survival,cellular reduction number ofcapacity rotifers (CRC) alive (NRA),and mastax body contraction size index (BSI),frequency cellular (MCF). reduction All these capacity characteristics (CRC) and mastaxwere applied contraction in the frequency current (MCF).experiments All these as well. characteristics were applied in the current experiments as well. AsAs KYNA does not not penetrate penetrate the the blood-brain-barri blood-brain-barrier,er, several several analogs analogs have have been been synthesized synthesized to toimprove improve the the efficacy efficacy in preclinical in preclinical studies studies [17,18]. [17 ,Our18]. aims Our were aims to were test toKNYA test KNYAand its andfive analogs its five analogs(Figure (Figure1) for 1the) for first the firsttime timein rotifers in rotifers and and to tomerge merge the the previous previous separate separate assaysassays intointo aa comprehensivecomprehensive physiologicalphysiological modelmodel forfor furtherfurther complexcomplex screeningscreening ofof bioactivebioactive andand pharmaceuticalpharmaceutical molecules.molecules. The The elevated survival of individuals in the population and the reverse changechange ofof thethe NRA-BSINRA-BSI pairpair comparedcompared toto thethe CRC-MCFCRC-MCF pairpair showedshowed that KYNAKYNA and some analogs had special, beneficialbeneficial physiologicphysiologic attenuationattenuation eeffectsffects onon ourour microinvertebrates.microinvertebrates. Survival, NRA and BSIBSI areare longitudinallongitudinal respondingresponding markersmarkers ofof thethe statusstatus ofof individual physiology in contrast to CRC and MCF, MCF, whichwhich areare quickly changing ones. ones. Our Our work work presents presents a anew new systemic systemic property, property, a holistic a holistic influence influence of ofKYNA KYNA and and its its SZR73 SZR73 and and SZR81 SZR81 derivatives, derivatives, in in increasing increasing survival, survival, reproduction reproduction and growth inin contrastcontrast toto cellularcellular reductionreduction andand mastaxmastax activityactivity inin bdelloids.bdelloids.

FigureFigure 1.1. Formulas of kynurenic acidacid (KYNA)(KYNA) andand itsits SZRSZR analogs.analogs.

2.2. Results and Discussion KYNAKYNA hashas a keya key role role in numerous in numerous biological biological species species and their and environment. their environment. The natural The abundance natural ofabundance this biomolecule of this isbiomolecule essential in is plants essential [19], in animalplants feed[19], [in20 ],animal in amphibians feed [20], as in pheromones amphibians [21 as] andpheromones moreover [21] in ﬁsh and as moreover lymphocyte-activators in fish as lymphocyte-activators [22] with potential aggravating [22] with propertiespotential aggravating [23]. propertiesRotifers [23]. are recent models that have gained scientiﬁc acceptance in pharmaceutical screening [24,25]. Our aim was to study the physiological impacts of KYNA and its analogs (SZRs) on Philodina acuticornis

Molecules 2019, 24, x 3 of 10

MoleculesRotifers2019, 24 ,are 2171 recent models that have gained scientific acceptance in pharmaceutical screening3 of 10 [24,25]. Our aim was to study the physiological impacts of KYNA and its analogs (SZRs) on Philodina acuticornis and Adineta vaga in a holistic approach. First, we investigated the effects of these Adineta vaga andmolecules on thein asurvival holistic of approach. bdelloids. First, Population we investigated kinetics, the influenced effects of theseby the molecules analogs, onwere the survivalmeasured of bdelloids.after one-time Population feeding in kinetics, a self-limiting influenced system. by the We analogs, found that were the measuredsurvival of after P. acuticornis one-time feedingsignificantly in a self-limiting increased in system. the presence We found (100 that µM; the Figure survival 2A) ofof P.KYNA acuticornis (155%),significantly SZR73 (182%), increased SZR81 in µ the(236%) presence or SZR104 (100 M;(136%) Figure compared2A) of KYNA to the (155%),untreated SZR73 control (182%), (100%). SZR81 These (236%) molecules or SZR104 significantly (136%) comparedelevated the to thenumber untreated of individuals, control (100%). where Thesethe level molecules of significance significantly was determined elevated the based number on the of individuals,effect of sodium-azide where the (NaN level3; of 100 significance µM) toxin, which was determined induced the based shortest on survival the effect (33%; ofsodium-azide nine days) in (NaNour experiments.3; 100 µM) toxin, The most which effective induced agent the shortestwas SZR81. survival In A. (33%; vaga, ninethe efficacy days) in of our the experiments. drugs was Themeasured most e ffunderective the agent same was experimental SZR81. In A. conditions. vaga, the eThefficacy A. vaga of the populations drugs was exhibited measured significant under the samesurvival experimental in the presence conditions. (100 TheµM;A. Figure vaga populations 2B) of KYNA exhibited (144%), significant SZR73 (167%), survival SZR81 in the (211%) presence or (100SZR104µM; Figure(122%),2 B)after of KYNAone-time (144%), feeding. SZR73 On day (167%), nine, SZR81 the number (211%) of or rotifers SZR104 was (122%), significantly after one-time higher feeding.in KYNA, On SZR73, day nine, SZR81 the or number SZR104 oftreated rotifers groups was than significantly in the untreated higher incontrol. KYNA, SZR81 SZR73, also SZR81 showed or SZR104the most treated powerful groups impact than in in these the untreated species. control.This suggested SZR81 alsothat showedKYNA and the mostits analogs powerful had impactpositive in theseeffects species. on survival This suggested(Figure 2), thatexcept KYNA for SZR106 and its (P. analogs acuticornis had: positive91%) and e ffSZR109ects on ( survivalP. acuticornis (Figure: 82%;2), exceptA. vaga for: 78%). SZR106 Although (P. acuticornis their: 91%)efficacy and was SZR109 differen (P. acuticornist, the tendencies: 82%; A. did vaga not: 78%). show Although any species their effispecificity.cacy was different, the tendencies did not show any species specificity.

FigureFigure 2.2. SurvivalSurvival kinetics kinetics of of rotifer rotifer populations populations treated treated (100 (100 µM)µ M)with with KYNA KYNA or its or analogs. its analogs. The Thenumber number and and survival survival of ofindividuals individuals significantly significantly in increasedcreased inin the the three three trea treatedted groups groups compared compared to to untreateduntreated controls. controls. KYNAKYNA andand itsits SZR73SZR73 andand SZR81 analogs had the most beneficial beneficial effect effect on on both both PhilodinaPhilodina acuticornis acuticornis( A(A))and andAdineta Adineta vagavaga ((BB)) species.species. The two different different bd bdelloidelloid rotifer rotifer genera genera show show similarsimilar kinetics kinetics under under thethe samesame treatments.treatments. The error bars repr representesent SEM. SEM. One-way One-way ANOVA ANOVA with with BonferroniBonferronipost post hochoctest test waswas usedused for statistical analys analysis,is, the the level level of of significance significance is is***;### ***; ### p < p0.001< 0.001 (*, (*,significant significant difference difference in in number number of of rotifers rotifers fr fromom untreated untreated controls controls on on day day nine; nine; # # significant significant didifferencefference from from untreated untreated control control in in survival survival of of animals; animals;n n= = 1212 wells).

Thereafter,Thereafter, wewe studiedstudied howhow thesethese advantageousadvantageous moleculesmolecules influencedinfluenced the physiological and and viabilityviability characteristics characteristics of microinvertebrates.of microinvertebrates. In our In experiments, our experiments, four previously four previously described parametersdescribed (NRA,parameters BSI, CRC (NRA, and BSI, MCF) CRC [16 ,and26] wereMCF) measured [16,26] were on day measured six, since on the day survival six, since curves the survival of the untreated curves controlof the populationuntreated control (Figure population3) reached (Figure their maximum 3) reached number their maximum of rotifers numb then.er The of above-mentionedrotifers then. The values,above-mentioned related to physiologically values, related acuteto physiologically reactions, were acute presented reactions, relative were presented to the untreated relative controlto the valuesuntreated (100%), control which values were (100%), converted which to the were zero conver pointted on theto the “x” zero axis ofpoint the graphs.on the “x” In theaxis presence of the ofgraphs. the basic In molecule,the presence KYNA of (Figure the basic3A), themolecu samele, pronounced KYNA (Figure characteristic 3A), the profile same was pronounced observed incharacteristic both animal profile species. was We observed explored in howboth theanimal analogs species. influenced We explored the rotifers how the in analogs comparison influenced to the untreatedthe rotifers controls. in comparison The significant to the untreated reverse changecontrols. of The the significant NRA-BSI pair reverse compared changeto of the theCRC-MCF NRA-BSI pairpair showed compared that to KYNA the CRC-MCF and some analogspair showed have anthat interesting KYNA and contradictory some analogs effect. have SZR73 an interesting (Figure3B) andcontradictory SZR81 (Figure effect.3C) significantlySZR73 (Figure enhanced 3B) and these SZR81 opposing (Figure e ffects. 3C) The significantly degree of change enhanced induced these by SZR104opposing (Figure effects.3D) The was degree approximately of change the induced same asby thatSZR104 of KYNA (Figure treatment. 3D) was approximately The SZR106 (Figure the same3E) effasect that showed of KYNA a similar treatment. profile The to the SZR106 base molecule (Figure 3E) where effect all theshowed measured a similar parameters profile significantlyto the base changed.molecule We where found all thatthe SZR109measured (Figure parameters3F) was sign theificantly least e ff changed.ective; only We the found NRA that and SZR109 CRC of (FigureA. vaga populations exhibited significant alterations compared to the controls, but these changes were not so pronounced. Molecules 2019, 24, x 4 of 10

3F) was the least effective; only the NRA and CRC of A. vaga populations exhibited significant Molecules 2019, 24, 2171 4 of 10 alterations compared to the controls, but these changes were not so pronounced.

FigureFigure 3.3. BiologicalBiological characteristics of of rotifers rotifers under under treatment treatment (100 (100 µM)µM) with with KYNA KYNA or or its its analogs analogs showingshowing contradictorycontradictory profiles. profiles. On On day day six, six, three three experimental- experimental- (number (number of rotifersof rotifers alive alive (NRA), (NRA), body sizebody index size (BSI),index mastax(BSI), mastax contraction contraction frequency frequency (MCF)) (MCF)) and one and chemical one chemical cellular cellular reduction reduction capacity (CRC)capacity parameter (CRC) parameter of individual of individual rotifers rotifers and the and populations the populations were were monitored. monitored. KYNA KYNA (A )(A and) and its SZRits SZR analogs analogs (B– (EB)– hadE) had antagonistic antagonistic profiles profiles for for the the following following characteristics: characteristics: NRA-BSI NRA-BSI compared compared to CRC-MCFto CRC-MCF pairs, pairs, with with the exception the exception of SZR109 of SZR109 (F). Both (F).Philodina Both Philodinaand Adineta and speciesAdineta show species significant show disignificantfferences differences from their from untreated their untreate controlsd (showingcontrols (showing as 0 value as 0 on value the xonaxis the inx axis graphs). in graphs). The errorThe barserror represent bars represent SEM. One-waySEM. One-way ANOVA ANOVA with Bonferroni with Bonferronipost hoc posttest hoc was test used was for used statistical for statistical analysis, the levels of significance are * p 0.05, ** p 0.01 and *** p 0.001. (*, significant difference from analysis, the levels of significance≤ are * p ≤ 0.05,≤ ** p ≤ 0.01 and≤ *** p ≤ 0.001. (*, significant difference untreatedfrom untreated controls, controls, where where the corresponding the correspondi datang data of 100% of 100% is the is the 0 value 0 value on on the thex axis); x axis); NRA, NRA,n =n =24 wells;24 wells; BSI, BSI,n = 50 n randomly= 50 randomly selected selected individuals individuals from 24from wells; 24 CRC,wells;n CRC,= 24 wells;n = 24 MCF, wells;n =MCF,50 randomly n = 50 selectedrandomly individuals selected individuals from 24 wells). from 24 wells).

TheThe mostmost commoncommon propertyproperty of the above-describedabove-described inin vivo vivo physiologicalphysiological parameters parameters was was the the alterationalteration inin metabolism metabolism [15 [15,27],,27], since since all theseall thes processese processes had relativelyhad relatively high-energy high-energy demands demands [28,29 ]. The[28,29]. NRA The was NRA influenced was influenced by the fecundity by the fecundity of the individuals of the individuals in the population, in the population, while the while BSI the was BSI the resultwas the of result cellular of growthcellular ingrowth the euthelic in the euthelic rotifers ro [30tifers]. Both [30]. characteristics Both characteristics are associated are associated with activewith proteinactive protein synthesis, synthesis, which which is the is basis the ofbasis anabolic of anab processesolic processes [31]. [31]. The The survival survival of individualsof individuals in thein population,the population, together together with with the elevationthe elevation of NRA of NRA and and BSI, BSI, adequately adequately imply imply the the great great viability viability of theof species,the species, which which was was positively positively enhanced enhanced by by KYNA KYNA and and some some of of its itsanalogs. analogs. CRC CRC referred referred to to the the cellularcellular reductionreduction capacity capacity in in the the population, population, which which is ais NADHa NADH dependent dependent biochemical biochemical reaction reaction [32 ]. According[32]. According to the to academic the academic literature, literature, a certain a cert degreeain degree of attenuation of attenuation in these in these processes processes may may have beneficialhave beneficial effects effects on viability on viability [33,34 [33,34].]. Decreasing Decreasing cellular cellular NADH NADH levels levels is ais protectivea protective approach approach in biologicalin biological systems systems [35,36 [35,36].]. As another As another rotifer-related rotifer-related indicator, indicator, active muscleactive functionmuscle function was necessary was fornecessary the maintenance for the maintenance of mastax activity of mastax [16 ],activity which was[16], onewhich of thewas most one energy-consumingof the most energy-consuming operations in theseoperations model in animals these [model31]. In ouranimals experiments, [31]. In our the CRCexperiments, and MCF the were CRC reduced and MCF in individuals were reduced where in the NRAindividuals and BSI where were elevatedthe NRA inand the BSI presence were elevated of KYNA in andthe relatedpresence molecules. of KYNA Theand reducedrelated molecules. parameters mayThe indicatereduced aparameters slowed metabolism, may indicate being a slowed the molecular metabolism, basis ofbeing the caloriethe molecular restriction-induced basis of the positivecalorie impactsrestriction-induced [37,38]. In positive rotifers, impacts the calorie [37,38]. restriction In rotifers, and the chemical calorie redoxrestriction modulation and chemical were ableredox to synergisticallymodulation were enhance able to the synergistically physiological parametersenhance the andphysiological longevity [parameters15]. Our experimental and longevity (survival, [15]. NRA,Our experimental BSI and MCF) (survival, and redox NRA, (CRC) BSI and parameters MCF) and were redox appropriate (CRC) parameters to assess were the inappropriate vivo changes to inassess metabolism. the in vivo changes in metabolism. InIn orderorder toto gaingain aa deeperdeeper understandingunderstanding of the inverse inverse alteration alteration profile profile of of the the investigated investigated KYNA-molecules,KYNA-molecules, we we examined examined which which reference reference treatments treatments showed showed the samethe same pattern pattern regarding regarding rotifer physiologyrotifer physiology (Figures 4(Figures and5). A4 stimulator,and 5). A stimulator, a toxin and a calorietoxin and restriction a calorie mimetic restriction were mimetic applied inwere our experiments.applied in our Cyclic experiments. adenosine Cyclic monophosphate adenosine (cAMP;monophosphate 100 µM), (cAMP; as a potential 100 µM), stimulator as a potential [39], had no effect on survival (P. acuticornis: 99%; A. vaga: 98%), while at the same time the number of rotifers significantly increased (Figure4). However, the NaN 3 toxin [40] decreased survival (P. acuticornis: 82%; Molecules 2019, 24, x 5 of 10

stimulator [39], had no effect on survival (P. acuticornis: 99%; A. vaga: 98%), while at the same time the number of rotifers significantly increased (Figure 4). However, the NaN3 toxin [40] decreased Molecules 2019, 24, 2171 5 of 10 survival (P. acuticornis: 82%; A. vaga: 33%) and the total number of individuals. The reference point (Figures 2 and 4), day nine, was the lowest survival measured in the A. vaga population (Figure 4B). A.Spermidine vaga: 33%) (100 and theµM), total a well-known number of individuals.calorie restriction The reference mimetic point[41], significantly (Figures2 and increased4), day nine, survival was the(P. lowest acuticornis survival: 181%; measured A. vaga in: the179%)A. vaga andpopulation other parameters, (Figure4B). similarly Spermidine to KYNA (100 µ M),and asome well-known of its caloriederivatives. restriction Henceforth, mimetic we [41 tested], significantly how these increased profile-reference survival molecules (P. acuticornis affected: 181%; theA. physiological vaga: 179%) andcharacteristics other parameters, of rotifers. similarly All to KYNAfour parameters and some ofwere its derivatives. positively Henceforth,elevated by we cAMP tested howinduced these profile-referencestimulation (Figure molecules 5A) in both affected species. the physiological In NaN3 toxicity characteristics (Figure 5B) ofmeasurements, rotifers. Allfour the parameters parameters wereof interest positively showed elevated uniform by cAMP decrease. induced Antagonistic stimulation profiles (Figure were5A) inobserved both species. in spermidine-treated In NaN 3 toxicity (Figureanimals5B) (Figure measurements, 5C), similarly the parameters to groups oftreated interest with showed KYNA uniform or its analogs. decrease. The Antagonistic elevated NRA-BSI profiles wereand observedthe decreased in spermidine-treated CRC-MCF pairs animals appeared (Figure simultaneously5C), similarly with to groups prolonged treated survival. with KYNA The orsignificant its analogs. increase The elevated of slow-changing, NRA-BSI andtime-consuming the decreased physiological CRC-MCF pairs markers appeared (survival, simultaneously NRA, BSI), withtogether prolonged with the survival. alteration The of significant dynamic increase changing of slow-changing,indicators (CRC time-consumingand MCF) formed physiological a specific markersphysiological (survival, profile NRA, that BSI), we togethernamed beneficial with the alterationphysiologic of attenuation dynamic changing (BPA). ‘Beneficial’ indicators (CRCrefers and to MCF)the positive formed changes a specific in physiological survival, NRA profile and that BSI, we while named ‘attenuation’ beneficial physiologicmeans the non-toxic attenuation type (BPA). of ‘Beneficial’decrease refersas in tothe the case positive of changesCRC and in survival,MCF. The NRA measured and BSI, whilecharacteristics ‘attenuation’ describe means the the non-toxicenergy-dependent type of decrease life quality, as in covering the case ofboth CRC the and chronic MCF. and The acute measured alterations. characteristics Since, the describe exact themechanism energy-dependent of action lifeof KYNA quality, and covering its analogs both theis as chronic yet unclear and acute in rotifers, alterations. our objective Since, the in exactthis mechanismpresent work of actionwas to of describe KYNA and the itsBPA analogs phenomenon. is as yet unclear We assumed in rotifers, that ourits molecular objective in background this present workmight was be torelated describe to serotonergic the BPA phenomenon. regulation [11] We assumedand redox that modulation its molecular [15,38] background in these particular might be relatedbdelloids. to serotonergic regulation [11] and redox modulation [15,38] in these particular bdelloids.

FigureFigure 4. 4.Survival Survival kineticskinetics ofof rotiferrotifer populationspopulations treatedtreated (100 µM)µM) with stimulator, toxin toxin or or calorie calorie restrictionrestriction mimetic. mimetic. The The number number and survivaland survival of individuals of individuals significantly significantly increased underincreased spermidine under treatment,spermidine compared treatment, to untreated compared controls. to untreated The cyclic controls. adenosine The monophosphate cyclic adenosine (cAMP) monophosphate had a positive eff(cAMP)ect on thehad reproduction a positive effect of individuals; on the reproduction however, of it hadindividuals; no pronounced however, influence it had no on pronounced the survival

ofinfluence animals. on NaN the 3survivalwas toxic of animals. to both PhilodinaNaN3 was acuticornis toxic to both(A )Philodina and Adineta acuticornis vaga ( (BA)) species.and Adineta The vaga two di(ffBerent) species. rotifer The genera two different show similar rotifer kinetics genera under show the similar same kinetics treatments. under The the error same bars treatments. represent SEM.The One-wayerror bars ANOVA represent with SEM. Bonferroni One-waypost ANOVA hoc test was with used Bonferroni for statistical post hoc analysis, test was the levelused offor significance statistical isanalysis, ***; ### pthe< 0.001 level (*,of significantsignificance di ffiserence ***;### in p number< 0.001 (*, of rotiferssignificant from difference untreated in controls number on of day rotifers nine; #from significant untreated difference controls from on untreatedday nine; controls# significant in survival difference of animals; from untreatedn = 12 wells). controls in survival of animals; n = 12 wells). The BPA model includes different changes, hence, we developed a summary mathematical formula, called the BPA index (BPAi). The parameters described above were integrated into BPAi, thus, it was possible to compare the molecules which potentially modulate physiological processes. BPAi is a relative number, presented in percentage value (Figure6). The experimental reference molecule for the BPA model was spermidine. BPAi values of this polyamine compound relative to KYNA (100%) were 208% in P. acuticornis and 206% in A. vaga. These species showed 99% similarity regarding spermidine efficacy compared to KYNA. The analogs were compared to KYNA (Figure6).

Molecules 2019, 24, x 5 of 10

Figure 4. Survival kinetics of rotifer populations treated (100 µM) with stimulator, toxin or calorie restriction mimetic. The number and survival of individuals significantly increased under spermidine treatment, compared to untreated controls. The cyclic adenosine monophosphate (cAMP) had a positive effect on the reproduction of individuals; however, it had no pronounced

Moleculesinfluence 2019, 24, onx the survival of animals. NaN3 was toxic to both Philodina acuticornis (A) and Adineta vaga6 of 10 (B) species. The two different rotifer genera show similar kinetics under the same treatments. The Figureerror bars5. Biological represent characteristics SEM. One-way of ANOVA rotifers withtreate Bonferronid (100 µM) post with hoc teststimulator, was used toxin for statisticalor calorie restrictionanalysis, mimeticthe level havingof significance contradictory is ***;### profile. p < 0.001 On day(*, significant six, three differenceexperimental- in number (NRA, ofBSI, rotifers MCF) Moleculesandfrom2019 one untreated, 24 chemical, 2171 controls (CRC) onparameter day nine; of# significantindividual difference rotifers and from po untreatedpulations controls were monitored. in survival ofAll 6 of 10 animals; n = 12 wells). parameters show significant differences from their untreated controls (shown as zero value on the x axis in graphs) in a positive direction evoked by cAMP (A) in contrast to NaN3 (B). Spermidine (C) has an antagonistic profile related to the NRA-BSI compared to CRC-MCF pairs. These three types of treatments show different biological profiles (stimulation, toxicity and antagonism) in our rotifer models. The error bars represent SEM. One-way ANOVA with Bonferroni post hoc test was used for statistical analysis, the levels of significance are * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001. (*, significant difference from untreated controls, where the corresponding data of 100% is the zero value on the x axis); NRA, n = 24 wells; BSI, n = 50 randomly selected individuals from 24 wells; CRC, n = 24 wells; MCF, n = 50 randomly selected individuals from 24 wells).

FigureThe BPA 5. Biological model characteristicsincludes different of rotifers changes, treated (100hence,µM) we with developed stimulator, toxina summary or calorie restrictionmathematical formula,mimetic called having the BPA contradictory index (BPAi). profile. The On parameters day six, three described experimental- above (NRA, were BSI, integrated MCF) and into one BPAi, thus,chemical it was possible (CRC) parameter to compare of individualthe molecules rotifers which and populationspotentially weremodulate monitored. physiological All parameters processes. BPAishow is a significantrelative number, differences presented from their untreatedin percenta controlsge value (shown (Figure as zero 6). value The on experimental the x axis in graphs) reference moleculein a positive for the direction BPA model evoked was by cAMPspermidine. (A) in contrast BPAi values to NaN 3of(B this). Spermidine polyamine (C) compound has an antagonistic relative to KYNAprofile (100%) related were to 208% the NRA-BSI in P. acuticornis compared and to CRC-MCF 206% in A. pairs. vaga These. These three species types showed of treatments 99% showsimilarity regardingdifferent spermidine biological efficacy profiles (stimulation,compared to toxicity KYNA. and The antagonism) analogs were in our compared rotifer models. to KYNA The (Figure error 6). barsOur representpresent work SEM. One-waydescribed ANOVA a novel with and Bonferroni complex postapplication hoc test was of rotifers used for using statistical them analysis, as in vivo the levels of significance are * p 0.05, ** p 0.01 and *** p 0.001. (*, significant difference from models to investigate the indicators≤ of metabo≤ lic modulation.≤ We demonstrated the impacts of KYNAuntreated and its controls, SZR-analogs where thein a corresponding system applying data of bd 100%elloid is therotifers, zero value which on theshowedx axis); the NRA, experimentaln = 24 efficacywells; of BSI,thesen = agents50 randomly on microscopic selected individuals living creatures. from 24 wells; CRC, n = 24 wells; MCF, n = 50 randomly selected individuals from 24 wells).

Figure 6. Beneficial physiologic attenuation index (BPAi) of KYNA or its analogs (100 µM). KYNA Figure 6. Beneficial physiologic attenuation index (BPAi) of KYNA or its analogs (100 µM). is the reference molecule (100% BPA) for SZR analogs. SZR73 and SZR81 were the best compounds basedKYNA on their is physiologicalthe reference influences molecule on(100%Philodina BPA) acuticornis for SZR (PA)analogs. and AdinetaSZR73 vagaand (AV)SZR81 species. were the All monitoredbest compounds rotifer-specific based parameters on their werephysiological integrated intoinfluences a comprehensive on Philodina mathematical acuticornis formula, (PA) and calculatingAdineta percentile vaga (AV) beneficial species. physiologic All monitored attenuation rotifer-spec (BPA) data.ific parameters were integrated into a comprehensive mathematical formula, calculating percentile beneficial physiologic Ourattenuation present work (BPA) described data. a novel and complex application of rotifers using them as in vivo models to investigate the indicators of metabolic modulation. We demonstrated the impacts of KYNA and3. Materials its SZR-analogs and Methods in a system applying bdelloid rotifers, which showed the experimental efficacy of these agents on microscopic living creatures. 3.1. Materials 3. Materials and Methods The analogs (SZR73, SZR81, SZR104, SZR106, SZR109) of KYNA (cat# K3375, Sigma Aldrich, St. 3.1.Louis, Materials MO, USA) were prepared in the Institute of Pharmaceutical Chemistry, Interdisciplinary Excellence Centre, University of Szeged, Szeged, Hungary. In our system we separately applied the The analogs (SZR73, SZR81, SZR104, SZR106, SZR109) of KYNA (cat# K3375, Sigma Aldrich, St. Louis, MO, USA) were prepared in the Institute of Pharmaceutical Chemistry, Interdisciplinary Excellence Centre, University of Szeged, Szeged, Hungary. In our system we separately applied the Molecules 2019, 24, 2171 7 of 10

following reference molecules: cAMP (cat# A9501, Sigma Aldrich) as a stimulant, NaN3 (cat# 822335, Merck Kenilworth, NJ, USA) as a toxin and spermidine (cat# S2626, Sigma Aldrich) as a well-known calorie restriction mimetic.

3.1.1. The Invertebrate Models The experiments were performed on invertebrate bdelloid rotifers P. acuticornis and A. vaga; therefore, according to the current international regulations, no specific ethical permission was needed. They were obtained from a Hungarian aquavaristique with origins from an agricultural farm in Szarvas, Hungary. The species have been maintained in a standard laboratory environment for six years. Our measurements were carried out in accordance with globally accepted norms: Animals (Scientific Procedures) Act, 1986, associated guidelines, EU Directive 2010/63/EU for animal experiments, and the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). Animal studies comply with the ARRIVE guidelines. The rotifers were cultured following the methods in our previous publication [16] with the following modification: The fibrillar alga-based environmental matrix was changed to ethanol- and distilled water-washed organic hemp yarn (Emil Lux GMBH & Co. KG, Wermelskirchen, Germany).

3.1.2. Treatment and Monitoring Populations were harvested based on our previous study [16] in half-area 96 well-plates (cat# 3695, Costar, Corning Inc., New York, NY, USA) for the experiments. The flasks were placed at –75 ◦C for 3 min for rapid cooling and release of attached live rotifers from the hemp-matrix. The cooled medium (~ 4 ◦C) was poured off (together with numbed rotifers) and the flasks were washed again 2+ 2+ + + 2+ with cooled (4 ◦C) standard medium (mg/L): Ca 31.05; Mg 17.6; Na 0.9; K 0.25; Fe 0.001; HCO3− 153.097; SO4− 3; Cl− 0.8; F− 0.02; H2SiO3 3.3 (pH = 7.5). The healthy animals were allowed to attach to the well bottom. The starting number of rotifers (mixed ages/sizes) per well (100 µL) was 10 2. Each treatment agent was dissolved (stock solution; 10 mM) in standard medium [16]. ± At the beginning of the measurements, we applied the feeding solution (600 µg/mL homogenate), which was heat-inactivated and filtered (Whatman filter with 10 um pore, cat no: 6728-5100) baker’s yeast (EU-standard granulated instant form, cat# 2-01-420674/001-Z12180/HU). In all the treatments (final drug concentration: 100 µM/compound), the one-time treatment and feeding were administered simultaneously. To characterize and to compare the effects of the compounds on rotifer populations and individuals, five assays were used: Survival of individuals in population (n = 12 wells), NRA (n = 24 wells), BSI (n = 50 randomly selected individuals from 24 wells), CRC (n = 24 wells) and MCF (n = 50 randomly selected individuals from 24 wells). The comparison reference point in experiments on survival kinetics was day nine, which was the shortest survival time measured in the A. vaga population (Figure4B). This relative time point was applied uniformly during all survival experiments. These recordings are longitudinal in contrast to the other characteristics, which are the end point (day six) experiments. Usually, day six is the maximum point of the kinetic curve of controls followed by the decrease of NRA in this self-limiting closed system. During survival and NRA experiments we monitored the animals daily using an inverted light microscope (Leitz Labovert FS, Germany). Active motion and general movement within the body defined the living individuals. The rotifers were considered dead after loss of the telescopic reflex, abnormal morphology of the body and the appearance of fragmentation or amorphous granules in the soma. The BSI was monitored and calculated daily by the following formula: BSI (%) = maximal ’length width’ of body (µm). Animals × were photographed (digital camera) under the microscope by taking serial images per entity. The CRC was detected by the EZ4U Cell Proliferation Assay (cat# BI-5000, Biomedica, Vienna). To avoid toxicity, 20 diluted XTT solution was used. The plates were incubated for 24 h without direct light at room × temperature. The sum reduction capacity of rotifers was measured. The absorbance was measured by a microplate-reader set at 492 nm with 630 nm as a reference. The readings were normalized to the number of animals/well. To evaluate the energy dependent muscular activity of the individuals we Molecules 2019, 24, 2171 8 of 10 recorded the MCF (contraction per sec). The mastax is part of the digestive system and its function is to shred the food by periodically opening and closing.

3.1.3. Statistics

To calculate the percentile value of the BPA index (BPAi%), the following formula was used: P [(Tmax*√ R)]*[NRA%(>100) + BSI%(>100)]D6/[CRC%(<100) + MCF%(<100)]D6.Tmax is the maximal time of survival of individuals in the population and the ‘R’ is the number of all rotifers until Tmax. Statistical analysis was performed with SPSS 23.0 (SPSS Inc, Chicago, IL, USA) using one-way ANOVA with Bonferroni post hoc test. The error bars represent the standard error of the mean (SEM). The different levels of significance are indicated as follows: * p 0.05, ** p 0.01 and *** p 0.001. ≤ ≤ ≤ Author Contributions: Conceptualization, Z.D. and L.V.; Methodology, Z.D. and Z.G.O.; Validation, Z.D.; Z.G.O.; B.G. and Z.B.; Formal analysis, Z.D.; B.G. and Z.G.O.; Investigation, Z.D.; Z.G.O.; Z.B.; D.Z.; F.F. and S.I.; Resources, Z.D.; V.L.; Z.B.; F.F.; S.I. and J.K.; Data curation, Z.D. and L.V.; Writing—original draft preparation, Z.D. and Z.G.O.; Writing—review and editing, Z.D.; Z.G.O.; Z.B.; D.Z. and L.V.; Visualization, Z.D. and B.G.; Supervision, Z.D. and L.V.; Project administration, Z.D.; Z.G.O.; Z.B.; D.Z.; F.F. and Sz.I.; Funding acquisition, V.L.; D.Z. and J.K. Funding: This research was funded by the project GINOP 2.3.2-15-2016-00034 and by the Ministry of Human Capacities, Hungary grant 20391-3/2018/FEKUSTRAT. DZ was supported by János Bolyai Research Scholarship of the Hungarian Academy of Sciences. Acknowledgments: The authors wish to thank Anna Szentgyorgyi MA, a professional in EFLT (English Foreign Language Teaching) and to Jennifer Tusz, a native speaker of English for proofreading the manuscript, and to Ilona Hatvani. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Sas, K.; Szabó, E.; Vécsei, L. Mitochondria, Oxidative Stress and the Kynurenine System, with a Focus on Ageing and Neuroprotection. Molecules 2018, 23, 191. [CrossRef][PubMed] 2. Jones, S.P.; Guillemin, G.J.; Brew, B.J. The kynurenine pathway in stem cell biology. Int. J. Tryptophan. Res. 2013, 6, 57–66. [CrossRef][PubMed] 3. Stone, T.W. Kynurenines in the CNS: From endogenous obscurity to therapeutic importance. Prog. Neurobiol. 2001, 64, 185–218. [CrossRef] 4. Moroni, F.; Cozzi, A.; Sili, M.; Mannaioni, G. Kynurenic acid: A metabolite with multiple actions and multiple targets in brain and periphery. J. Neural. Transm. (Vienna) 2012, 119, 133–139. [CrossRef][PubMed] 5. Tuboly, G.; Tar, L.; Bohar, Z.; Safrany-Fark, A.; Petrovszki, Z.; Kekesi, G.; Vecsei, L.; Pardutz, A.; Horvath, G. The inimitable kynurenic acid: The roles of different ionotropic receptors in the action of kynurenic acid at a spinal level. Brain Res. Bull 2015, 112, 52–60. [CrossRef] 6. Kepplinger, B.; Baran, H.; Kainz, A.; Ferraz-Leite, H.; Newcombe, J.; Kalina, P. Age-related increase of kynurenic acid in human cerebrospinal fluid–IgG and beta2-microglobulin changes. Neurosignals 2005, 14, 126–135. [CrossRef][PubMed] 7. Vohra, M.; Lemieux, G.A.; Lin, L.; Ashrafi, K. Kynurenic acid accumulation underlies learning and memory impairment associated with aging. Genes Dev. 2018, 1, 14–19. [CrossRef] 8. Stone, T.W. Development and therapeutic potential of kynurenic acid and kynurenine derivatives for neuroprotection. Trends Pharmacol. Sci. 2000, 21, 149–154. [CrossRef] 9. Lemieux, G.A.; Ashrafi, K. Neural Regulatory Pathways of Feeding and Fat in Caenorhabditis elegans. Annu. Rev. Genet 2015, 49, 413–438. [CrossRef] 10. Cunningham, K.A.; Hua, Z.; Srinivasan, S.; Liu, J.; Lee, B.H.; Edwards, R.H.; Ashrafi, K. AMP-activated kinase links serotonergic signaling to glutamate release for regulation of feeding behavior in C. elegans. Cell Metab. 2012, 16, 113–121. [CrossRef] 11. Snell, T.W.; Johnston, R.K.; Gribble, K.E.; Mark Welch, D.B. Rotifers as experimental tools for investigating aging. Invertebr. Reprod. Dev. 2015, 59, 5–10. [CrossRef][PubMed] 12. Leasi, F.; Pennati, R.; Ricci, C. First description of the serotonergic nervous system in a bdelloid rotifer: Macrotrachela quadricornifera Milne 1886 (Philodinidae). Zoologischer Anzeiger 2009, 248, 47–55. [CrossRef] 13. Leasi, F.; Ricci, C. The role of serotonin in a bdelloid life cycle. Hydrobiologia 2011, 662, 141–147. [CrossRef] Molecules 2019, 24, 2171 9 of 10

14. Snell, T.W.; Johnston, R.K.; Matthews, A.B. Freshwater toxicity testing using rehydrated Philodina sp. (Rotifera) as test animals. Environ. Toxicol. 2017, 32, 2267–2276. [CrossRef][PubMed] 15. Macsai, L.; Olah, Z.; Bush, A.I.; Galik, B.; Onody, R.; Kalman, J.; Datki, Z. Redox Modulating Factors Affect Longevity Regulation in Rotifers. J. Gerontol. A Biol. Sci. Med. Sci. 2019, 74, 811–814. [CrossRef][PubMed] 16. Olah, Z.; Bush, A.I.; Aleksza, D.; Galik, B.; Ivitz, E.; Macsai, L.; Janka, Z.; Karman, Z.; Kalman, J.; Datki, Z. Novel in vivo experimental viability assays with high sensitivity and throughput capacity using a bdelloid rotifer. Ecotoxicol. Environ. Saf. 2017, 144, 115–122. [CrossRef][PubMed] 17. Most, J.; Tosti, V.; Redman, L.M.; Fontana, L. Calorie restriction in humans: An update. Ageing Res. Rev. 2016, 39, 36–45. [CrossRef] 18. Veres, G.; Fejes-Szabó, A.; Zádori, D.; Nagy-Grócz, G.; László, A.M.; Bajtai, A.; Mándity, I.; Szentirmai, M.; Bohár, Z.; Laborc, K.; et al. A comparative assessment of two kynurenic acid analogs in the formalin model of trigeminal activation: A behavioral, immunohistochemical and pharmacokinetic study. J. Neural. Transm. (Vienna) 2017, 124, 99–122. [CrossRef] 19. Turski, M.P.; Turska, M.; Zgrajka, W.; Bartnik, M.; Kocki, T.; Turski, W.A. Distribution, synthesis, and absorption of kynurenic acid in plants. Planta Med. 2011, 77, 858–864. [CrossRef] 20. Turski, M.P.; Zgrajka, W.; Siwicki, A.K.; Paluszkiewicz, P. Presence and content of kynurenic acid in animal feed. J. Anim. Physiol. Anim. Nutr. (Berl) 2015, 99, 73–78. [CrossRef] 21. Mariano, D.O.; Yamaguchi, L.F.; Jared, C.; Antoniazzi, M.M.; Sciani, J.M.; Kato, M.J.; Pimenta, D.C. Pipa carvalhoi skin secretion profiling: Absence of peptides and identification of kynurenic acid as the major constitutive component. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2015, 167, 1–6. [CrossRef][PubMed] 22. Małaczewska, J.; Siwicki, A.K.; Wójcik, R.; Turski, W.A.; Kaczorek, E. The in vitro effect of kynurenic acid on the rainbow trout (Oncorhynchus mykiss) leukocyte and splenocyte activity. Pol. J. Vet. Sci. 2014, 17, 453–458. [CrossRef][PubMed] 23. Kaczorek, E.; Szarek, J.; Mikiewicz, M.; Terech-Majewska, E.; Schulz, P.; Małaczewska, J.; Wójcik, R.; Siwicki, A.K. Effect of feed supplementation with kynurenic acid on the morphology of the liver, kidney and gills in rainbow trout (Oncorhynchus mykiss Walbaum, 1792), healthy and experimentally infected with Yersinia ruckeri. J. Fish Dis. 2017, 40, 873–884. [CrossRef][PubMed] 24. Snell, T.W.; Johnston, R.K.; Matthews, A.B.; Zhou, H.; Gao, M.; Skolnick, J. Repurposed FDA-approved drugs targeting genes influencing aging can extend lifespan and healthspan in rotifers. Biogerontology 2018, 19, 145–157. [CrossRef][PubMed] 25. Chuluunbaatar, B.; Béni, Z.; Dékány, M.; Kovács, B.; Sárközy, A.; Datki, Z.; Mácsai, L.; Kálmán, J.; Hohmann, J.; Ványolós, A. Triterpenes from the Mushroom Hypholoma lateritium: Isolation, Structure Determination and Investigation in Bdelloid Rotifer Assays. Molecules 2019, 24, 301. [CrossRef][PubMed] 26. Datki, Z.; Olah, Z.; Hortobagyi, T.; Macsai, L.; Zsuga, K.; Fulop, L.; Bozso, Z.; Galik, B.; Acs, E.; Foldi, A.; et al. Exceptional in vivo catabolism of neurodegeneration-related aggregates. Acta Neuropathol. Commun. 2018, 6, 6. [CrossRef] 27. Ma, S.; Gladyshev, V.N. Molecular signatures of longevity: Insights from cross-species comparative studies. Semin Cell Dev. Biol. 2017, 70, 190–203. [CrossRef] 28. Poeggeler, B.; Sambamurti, K.; Siedlak, S.L.; Perry, G.; Smith, M.A.; Pappolla, M.A. A novel endogenous indole protects rodent mitochondria and extends rotifer lifespan. PLoS ONE 2010, 5, 10206. [CrossRef] 29. Smith, R.L.; Soeters, M.R.; Wüst, R.C.I.; Houtkooper, R.H. Metabolic Flexibility as an Adaptation to Energy Resources and Requirements in Health and Disease. Endocr. Rev. 2018, 39, 489–517. [CrossRef] 30. Marotta, R.; Uggetti, A.; Ricci, C.; Leasi, F.; Melone, G. Surviving starvation: Changes accompanying starvation tolerance in a bdelloid rotifer. J. Morphol. 2012, 2731, 1–7. [CrossRef] 31. Jazwinski, S.M.; Jiang, J.C.; Kim, S. Adaptation to metabolic dysfunction during aging: Making the best of a bad situation. Exp. Gerontol. 2018, 107, 87–90. [CrossRef][PubMed] 32. Comley, J.C.W.; Turner, C.H. Potential of a soluble tetrazolium/formazan assay for the evaluation of filarial viability. Int. J. Parasitol. 1990, 20, 251–255. [CrossRef] 33. López-Lluch, G.; Navas, P. Calorie restriction as an intervention in ageing. J. Physiol. 2016, 594, 2043–2060. [CrossRef][PubMed] 34. Bock, M.J.; Jarvis, G.C.; Corey, E.L.; Stone, E.E.; Gribble, K.E. Maternal age alters offspring lifespan, fitness, and lifespan extension under caloric restriction. Sci. Rep. 2019, 9, 3138. [CrossRef][PubMed] Molecules 2019, 24, 2171 10 of 10

35. Lin, S.-J.; Ford, E.; Haigis, M.; Liszt, G.; Guarente, L. Calorie restriction extends yeast life span by lowering the level of NADH. Genes Dev. 2004, 18, 12–16. [CrossRef][PubMed] 36. Evans, C.; Bogan, K.L.; Song, P.; Burant, C.F.; Kennedy, R.T.; Brenner, C. NAD+ metabolite levels as a function of vitamins and calorie restriction: Evidence for different mechanisms of longevity. BMC Chem. Biol. 2010, 10, 2. [CrossRef] 37. Trubitsyn, A.G. The Lag of the Proliferative Aging Clock Underlies the Lifespan-Extending Effect of Calorie Restriction. Curr. Aging Sci. 2015, 8, 220–226. [CrossRef] 38. Latta, L.C.; Tucker, K.N.; Haney, R.A. The relationship between oxidative stress, reproduction, and survival in a bdelloid rotifer. BMC Ecol. 2019, 19, 7. [CrossRef] 39. Wang, Z.; Zhang, L.; Liang, Y.; Zhang, C.; Xu, Z.; Zhang, L.; Fuji, R.; Mu, W.; Li, L.; Jiang, J.; et al. Cyclic AMP Mimics the Anti-ageing Effects of Calorie Restriction by Up-Regulating Sirtuin. Sci. Rep. 2015, 5, 12012. [CrossRef] 40. Ye, J.; Jiang, Z.; Chen, X.; Liu, M.; Li, J.; Liu, N. Electron transport chain inhibitors induce microglia activation through enhancing mitochondrial reactive oxygen species production. Exp. Cell Res. 2016, 340, 315–326. [CrossRef] 41. Shintani, H.; Shintani, T.; Ashida, H.; Sato, M. Calorie Restriction Mimetics: Upstream-Type Compounds for Modulating Glucose Metabolism. Nutrients 2018, 10, 1821. [CrossRef][PubMed]

Sample Availability: Samples of the SZR compounds are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).