The Age-Sensitive Efficacy of Calorie Restriction on Mitochondrial

International Journal of Molecular Sciences

Article The Age-Sensitive Efﬁcacy of Calorie Restriction on Mitochondrial Biogenesis and mtDNA Damage in Rat Liver

Guglielmina Chimienti 1,† , Anna Picca 2,3,† , Flavio Fracasso 1, Francesco Russo 4 , Antonella Orlando 4 , Giuseppe Riezzo 4, Christiaan Leeuwenburgh 5, Vito Pesce 1 and Angela Maria Serena Lezza 1,*

1 Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari Aldo Moro, Via Orabona 4, 70125 Bari, Italy; [email protected] (G.C.); ﬂ[email protected] (F.F.); [email protected] (V.P.) 2 Fondazione Policlinico Universitario “Agostino Gemelli” IRCCS, L.go F. Vito 8, 00168 Rome, Italy; [email protected] 3 Aging Research Center, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet and Stockholm University, 11330 Stockholm, Sweden 4 Laboratory of Nutritional Pathophysiology, National Institute of Gastroenterology “S. de Bellis”, Research Hospital, 70013 Castellana Grotte, Italy; [email protected] (F.R.); [email protected] (A.O.); [email protected] (G.R.) 5 Department of Aging and Geriatric Research, Institute on Aging, Division of Biology of Aging, University of Florida, Gainesville, FL 32611, USA; cleeuwen@uﬂ.edu * Correspondence: [email protected]; Tel.: +39-080-5443309 † These authors contributed equally.

Abstract: Calorie restriction (CR) is the most efficacious treatment to delay the onset of age-related changes such as mitochondrial dysfunction. However, the sensitivity of mitochondrial markers to CR Citation: Chimienti, G.; Picca, A.; and the age-related boundaries of CR efficacy are not fully elucidated. We used liver samples from Fracasso, F.; Russo, F.; Orlando, A.; ad libitum-fed (AL) rats divided in: 18-month-old (AL-18), 28-month-old (AL-28), and 32-month- Riezzo, G.; Leeuwenburgh, C.; Pesce, old (AL-32) groups, and from CR-treated (CR) 28-month-old (CR-28) and 32-month-old (CR-32) V.; Lezza, A.M.S. The Age-Sensitive counterparts to assay the effect of CR on several mitochondrial markers. The age-related decreases in Efficacy of Calorie Restriction on citrate synthase activity, in TFAM, MFN2, and DRP1 protein amounts and in the mtDNA content Mitochondrial Biogenesis and mtDNA Damage in Rat Liver. Int. J. Mol. Sci. in the AL-28 group were prevented in CR-28 counterparts. Accordingly, CR reduced oxidative 2021, 22, 1665. https://doi.org/ mtDNA damage assessed through the incidence of oxidized purines at specific mtDNA regions in 10.3390/ijms22041665 CR-28 animals. These findings support the anti-aging effect of CR up to 28 months. Conversely, the protein amounts of LonP1, Cyt c, OGG1, and APE1 and the 4.8 Kb mtDNA deletion content Academic Editor: were not affected in CR-28 rats. The absence of significant differences between the AL-32 values and Joan Roselló-Catafau the CR-32 counterparts suggests an age-related boundary of CR efficacy at this age. However, this Received: 28 December 2020 only partially curtails the CR benefits in counteracting the generalized aging decline and the related Accepted: 4 February 2021 mitochondrial involvement. Published: 7 February 2021 Keywords: rat liver; aging; calorie restriction; mitochondrial biogenesis; mtDNA damage; age- Publisher’s Note: MDPI stays neutral sensitive efficacy of CR with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction According to the impressive progression of studies investigating the process of aging, several different definitions of the process have sequentially been composed, and a set Copyright: © 2021 by the authors. of pathways and mediators has been identified as the so-called hallmarks of aging [1,2]. Licensee MDPI, Basel, Switzerland. Among these, mitochondrial dysfunction, oxidative stress, and molecular damage of mito- This article is an open access article distributed under the terms and chondria have emerged as a crucial factor. An important feature of aging is tissue-specificity conditions of the Creative Commons that has been highlighted for mitochondrial involvement [3,4], epigenetic changes [5], and Attribution (CC BY) license (https:// other alterations such as telomere length [6]. Among the various treatments aiming at creativecommons.org/licenses/by/ preventing or delaying the onset of age-related changes, calorie restriction (CR) is the oldest 4.0/). strategy proposed [7], and still represents the gold standard for the very large number of

Int. J. Mol. Sci. 2021, 22, 1665. https://doi.org/10.3390/ijms22041665 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 18

for the very large number of beneficial effects conveyed by this intervention. Indeed, recent studies on the molecular mechanisms evoked by CR have demonstrated that several pathways are positively influenced by this nutritional intervention [8–10]. Int. J. Mol. Sci. 2021, 22, 1665 Furthermore, these findings also strongly support the existence of a “plasticity”2 of 18 of aging [5,9,11,12] that can be modulated via environmental interactions as nutritional or beneficialpharmaceutical effects conveyedinterventions by this or intervention.life-style habits. Indeed, CR recenthas already studies been on the shown molecular to counteract mechanismsthe age-related evoked alterations by CR have of demonstrated mitochondrial that biogenesis several pathways and metabolism are positively in influ- several rat encedtissues by with this nutritionaltissue-specific intervention effects [[3,13,14].8–10]. Furthermore, More specifically, these findings previous also strongly studies by our supportgroup on the rat existence liver, which of a “plasticity” is deeply ofaffected aging [by5,9 ,aging11,12] thatat the can mitochondrial be modulated level, via envi- examining ronmentalorganellar interactions markers asshowed nutritional a positive or pharmaceutical effect of interventions CR on mitochondrial or life-style habits. age-related CRalterations. has already In beenfact, shownin CR-treated to counteract aged the 28-month-old age-related alterations rats, the of mitochondrialmitochondrial DNA biogenesis and metabolism in several rat tissues with tissue-specific effects [3,13,14]. More (mtDNA) content, amount of proteins involved in mitochondrial biogenesis or ROS- specifically, previous studies by our group on rat liver, which is deeply affected by aging atsensitive, the mitochondrial and the level,extent examining of mitochondrial organellar markerstranscription showed factor a positive A (TFAM) effect of CRbinding at onspecific mitochondrial mtDNA age-relatedregions presented alterations. values In fact, significantly in CR-treated different aged 28-month-old from those rats, of the age- thematched mitochondrial ad libitum-fed DNA (mtDNA) (AL) counterparts content, amount and of proteinsnot statistically involved indifferent mitochondrial from those of biogenesisyounger 18-month-old or ROS-sensitive, AL-fed and the animals extent of [15]. mitochondrial More recently, transcription the analysis factor Aof (TFAM) mitochondrial bindingbiogenesis at specific and mtDNA mtDNA damage regions in presented liver from values AL aged significantly (28-month-old) different and from extremely those aged of(32-month-old) the age-matched rats, ad allowed libitum-fed us (AL)to study counterparts a diverse and array not of statistically mitochondrial different parameters from and thoseto suggest of younger a different 18-month-old pace of AL-fedaging in animals relation [15 ].to Morethe mitochondrial recently, the analysis involvement, of mi- in the tochondrial biogenesis and mtDNA damage in liver from AL aged (28-month-old) and few animals naturally reaching longevity with respect to the majority of those becoming extremely aged (32-month-old) rats, allowed us to study a diverse array of mitochondrial parametersaged [16,17]. and With to suggest these premises, a different we pace aimed of aging at assessing in relation the to theCR mitochondrialefficacy on a large inset of volvement,mitochondrial in the markers few animals at various naturally ages reaching in rat longevity liver. For with these respect purposes, to the majority we used of animals thosefrom becomingthe very long-living aged [16,17]. hybrid With these strain premises, Fischer we 344 aimed x Brown at assessing Norway, the from CR efficacy which the 18- onmonth-old a large set ad of libitum-fed mitochondrial rats markers (AL-18) at were various chosen ages inas rat controls, liver. For to thesebe compared purposes, with the wead libitum-fed, used animals aged from (AL-28) the very and long-living extremely hybrid aged strain (AL-32) Fischer animals 344 x and Brown the Norway,CR-treated age- frommatched which counterparts the 18-month-old (CR-28 ad libitum-fedand CR-32). rats In (AL-18) these weregroups chosen of rats, as controls, we evaluated: to be The comparedactivity of with the themitochondrial ad libitum-fed, enzyme aged (AL-28) citrate and synthase, extremely the aged expression (AL-32) animalsof a set and of proteins the CR-treated age-matched counterparts (CR-28 and CR-32). In these groups of rats, we including TFAM, Lon protease (LonP1) and cytochrome c (Cyt c), the mtDNA repair evaluated: The activity of the mitochondrial enzyme citrate synthase, the expression of a set ofenzymes proteins 8-oxoG including DNA TFAM, glycosylase/apurinic Lon protease (LonP1) andor apyrimidinic cytochrome c (Cyt (AP) c), thelyase mtDNA (OGG1) and repairapurinic/apyrimidinic enzymes 8-oxoG DNA endonuclease glycosylase/apurinic 1 (APE1), or and apyrimidinic the mitochondrial (AP) lyase (OGG1)dynamics and proteins apurinic/apyrimidinicmitofusin 2 (MFN2) and endonuclease dynamin-related 1 (APE1), protein and the mitochondrial1 (DRP1). Finally, dynamics the mtDNA proteins content mitofusinand oxidative 2 (MFN2) damage and dynamin-relatedat specific regions protein were 1 also (DRP1). determined. Finally, the mtDNA content and oxidative damage at specific regions were also determined. 2. Results 2. Results 2.1.2.1. EvaluationEvaluation of of Age Age and and CR CR Effects Effects on Markers on Markers of Mitochondrial of Mitochondrial Functionality Functionality TheThe citrate citrate synthase synthase activity, activity, which which is also ais marker also a of marker mitochondrial of mitochondrial mass, was deter- mass, was mineddetermined in all five in groupsall five of ratsgroups to evaluate of rats the sensitivityto evaluate of mitochondrial the sensitivity functionality of mitochondrial to agingfunctionality and CR(Figure to aging1). and CR (Figure 1).

FigureFigure 1. 1.Citrate Citrate synthase synthase activity activity in liver in liver from from AL-18, AL- AL-28,18, AL-28, CR-28, CR-28, AL-32, AL-32, and CR-32-month-old and CR-32-month-old rats.rats. BarsBars represent represent mean mean values values and and standard standard deviation deviation (SD) (SD) of data of obtaineddata obtained from experiments from experiments performedperformed in in quadruplicate quadruplicate and and analyzed analyzed using theusing One-way the One-way ANOVA ANOVA test and Tukey’s test and multiple Tukey’s com- multiple parisoncomparison test. * ptest.< 0.05 * p versus < 0.05 AL-18 versus rats, AL-18 # p < 0.05rats, versus # p < 0.05 AL-28 versus rats. n :AL-28 Number rats. of analyzedn: Number animals of analyzed. animals.

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 18 Int. J. Mol. Sci. 2021, 22, 1665 3 of 18

AnAn age-related statisticallystatistically significantsignificant decreasedecrease in in citrate citrate synthase synthase activity activity was was ob- observedserved in in AL-28 AL-28 (− (41%)−41%) and and AL-32 AL-32 ( −(−40%)40%) ratsrats comparedcompared toto thethe AL-18AL-18 age control. No statisticallystatistically significant significant difference difference was was found between AL-28 and AL-32 groups. The CR effecteffect was demonstrated by the significantly significantly higher (+40%) enzyme activity in the CR-28 ratsrats compared compared to to the age-matched AL-28 rats value. Furthermore, Furthermore, the CR-28 activity was similarsimilar toto thatthat observed observed in in the the younger younger AL-18 AL- control18 control rats (Figurerats (Figure1). This 1). effect This was effect instead was insteadblunted blunted in the CR-32 in the animals. CR-32 animals. In fact, at In the fact, older at the age older of 32 age months of 32 no months statistical no differencestatistical differencecould be observed could be betweenobserved the between AL and the CR AL groups. and CR groups. ToTo deepen the analysis of age and CR effectseffects on mitochondrial functionality, the protein amount of TFAM TFAM and of LonP1 was dete determinedrmined in the the mitochondria mitochondria isolated from allall rat rat groups groups by by Western Western blot analyses, in which which the the protein protein from the outer outer mitochondrial mitochondrial membranemembrane VDAC was used as an internal loading control (Figure2 2).).

FigureFigure 2. 2. AmountsAmounts of of TFAM TFAM and LonP1 proteins in isol isolatedated liver mitochondria from AL-18, AL- AL-28, 28,CR-28, CR-28, AL-32, AL-32, and and CR-32-month-old CR-32-month-old rats. rats. (A). ( TFAMA). TFAM protein protein amounts amounts in isolated in isolated liver liver mitochondria mitochondriafrom different from rat groups. different In rat the gr inset,oups. a representativeIn the inset, a representative Western blot was Western carried blot out was in onecarried rat fromout ineach one of rat the from assayed each of groups. the assa Immunoreactiveyed groups. Immunoreactive bands, from topbands, to bottom,from top represent, to bottom, respectively, represent, respectively, the signals from TFAM and VDAC. The histogram shows the quantification of the the signals from TFAM and VDAC. The histogram shows the quantification of the intensity of the intensity of the bands of TFAM, normalized to the intensity of VDAC. (B). LonP1 protein amounts B inbands isolated of TFAM, liver mitochondria normalized to from the intensitydifferent ofrat VDAC. groups. ( In). the LonP1 inset, protein a representative amounts in Western isolated blot liver wasmitochondria carried out from in one different rat from rat each groups. of the In assa theyed inset, groups. a representative Immunoreactive Western bands, blot wasfrom carried top to out bottom,in one rat represent, from each respectively, of the assayed the signals groups. from Immunoreactive LonP1 and VDAC. bands, The from histogram top to bottom, shows represent, the quantificationrespectively, the of the signals intensity from of LonP1 the bands and of VDAC. LonP1, The normalized histogram to showsthe intensity the quantification of VDAC. (A of,B) the Dataintensity represent of the the bands results of LonP1, from normalizedtriplicate Wester to then intensity blot experiments of VDAC. and (A, Bwere) Data analyzed represent using the resultsthe One-wayfrom triplicate ANOVA Western test and blot Tukey’s experiments multiple and comparis were analyzedon test. usingBars represent the One-way the mean ANOVA values test and and SDTukey’s for the multiple five experimental comparison groups. test. Bars Data represent were normalized the mean valuesagainst and the SDvalue for of the the five AL-18 experimental rats, fixed as 1. * p < 0.05 versus AL-18 rats, # p < 0.05 versus AL-28 rats. n: Number of analyzed groups. Data were normalized against the value of the AL-18 rats, fixed as 1. * p < 0.05 versus AL-18 animals. rats, # p < 0.05 versus AL-28 rats. n: Number of analyzed animals.

AgingAging ledled to to a statisticallya statistically significant significant (−31%) (− decrease31%) decrease in the TFAM in the amount TFAM comparing amount comparingAL-18 control AL-18 rats control with both rats AL-28 with both and AL-2 AL-328 and animals, AL-32 whereas animals, there whereas was nothere significant was no significantdifference betweendifference the between values the of AL-28 values and of AL-28 AL-32 and groups. AL-32 As groups. an effect As of an CR, effect the of CR-28 CR, therats CR-28 had a rats TFAM had amount a TFAM significantly amount significantly higher (+39%) higher than (+39%) that of than the age-matchedthat of the age- AL matchedcounterparts AL andcounterparts not different and from not thatdifferent of the from younger that AL-18of the controls.younger Conversely,AL-18 controls. such Conversely,diet effect was such not diet visible effect anymore was not visible in the CR-32anymore animals in the that CR-32 showed animals a TFAM that showed amount a TFAMnot statistically amount differentnot statistically from that different of the age-matchedfrom that of ALthe groupage-matched and significantly AL group lower and significantly lower than that of the AL-18 rats. As for the LonP1 amount, there was a

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 18 Int. J. Mol. Sci. 2021, 22, 1665 4 of 18

statisticallythan that of significant the AL-18 ( rats.−65%) As age-related for the LonP1 decrease amount, in the there AL-28 was group a statistically with respect significant to the AL-18(−65%) value. age-related The age decrease progression in the made AL-28 the group statistically with respect significant to the decrease AL-18 value. in the The LonP1 age amount,progression with made respect the statisticallyto the AL-18 significant rats, more decrease marked in the (− LonP175%) in amount, the AL-32 with respectgroup. Interestingly,to the AL-18 there rats, morewas no marked evident (− effect75%) of in CR the on AL-32 the LonP1 group. amount Interestingly, since the there comparison was no betweenevident effectthe AL-28 of CR onvalue the LonP1and the amount CR age-matched since the comparison counterpart between did thenot AL-28 show value any significantand the CR difference age-matched and counterpart the same occurred did not showfor the any comparison significant between difference the and AL-32 the same and CR-32occurred groups. for the comparison between the AL-32 and CR-32 groups. ToTo evaluate the the effect of aging and CR on another mitochondrial metabolic function whichwhich is the initiation ofof thethe mitochondrialmitochondrial intrinsic intrinsic apoptotic apoptotic pathway, pathway, we we determined, determined, by byWestern Western blot blot experiments, experiments, the the intra-mitochondrial intra-mitochondrial amount amount of Cytof Cyt c (Figure c (Figure3). 3).

FigureFigure 3. 3. AmountsAmounts of of Cyt Cyt c c protein protein in in isolated isolated liver mi mitochondriatochondria from AL-18, AL-28, CR-28,CR-28, AL-32,AL- 32,and and CR-32-month-old CR-32-month-old rats. rats. In theIn the inset, inset, a representative a representative Western Western blot blot was was carried carried out inout one in ratone from rat from each of the assayed groups. Immunoreactive bands, from top to bottom, represent, each of the assayed groups. Immunoreactive bands, from top to bottom, represent, respectively, the respectively, the signals from Cyt c and VDAC. The histogram shows the quantification of the signals from Cyt c and VDAC. The histogram shows the quantification of the intensity of the bands of intensity of the bands of Cyt c normalized to the intensity of VDAC. All data represent the results fromCyt c triplicate normalized Western to the blot intensity experiments of VDAC. and All we datare analyzed represent using the results the One-way from triplicate ANOVA Western test and blot Tukey’sexperiments multiple and comparison were analyzed test. using Bars therepresent One-way the ANOVAmean values test andand Tukey’sSD for the multiple five comparison experimentaltest. Bars represent groups. the Data mean were values normalized and SD foragai thenst five the experimental value of the AL-18 groups. rats, Data fixed were as normalized1. * p < 0.05against versus the AL-18 value ofrats, the # AL-18p < 0.05 rats, versus fixed AL-28 as 1. rats. * p < n 0.05: Number versus of AL-18 analyzed rats, animals. # p < 0.05 versus AL-28 rats. n: Number of analyzed animals. The age-related effect on the intra-mitochondrial Cyt c amount was peculiar since there Thewas age-relateda marked (− effect44%) onstatistically the intra-mitochondrial significant decrease Cyt cpassing amount from was the peculiar AL-18 since rats tothere the wasAL-28 a marked animals, (− 44%)but such statistically decrease significant was partially decrease prevented passing fromin the the AL-32 AL-18 group rats to showingthe AL-28 a animals, small ( but−13%) such decrease decrease in was comparison partially prevented with the in AL-18 the AL-32 counterpart group showing and a a significantsmall (−13%) marked decrease (+55%) in comparison increase in withcomparison the AL-18 with counterpart the AL-28 and rats. a The significant CR effect marked was not(+55%) relevant increase at all insince comparison there were with no signif the AL-28icant differences rats. The CR in the effect Cyt was c protein not relevant amounts at betweenall since the there age-matched were no significant AL and CR differences groups at in28 theas well Cyt as c proteinat 32 months. amounts The between age-related the decreaseage-matched in Cyt AL c andamounts CR groups in the attwo 28 CR as wellgrou asps atshowed 32 months. a trend The similar age-related to that decrease observed in inCyt the c amountsAL-28 and inthe AL-32 two CRgroups groups with showed a more a trendrelevant similar decrease to that in observed the CR-28 in the animals AL-28 partiallyand AL-32 prevented groups with in the a moreolder relevantCR-32 rats. decrease in the CR-28 animals partially prevented in the older CR-32 rats. 2.2. Evaluation of Age and CR Effects on mtDNA Repair Enzymes 2.2. Evaluation of Age and CR Effects on mtDNA Repair Enzymes Since aging has been largely shown to be associated with the accumulation of Since aging has been largely shown to be associated with the accumulation of damage damageto mtDNA, to mtDNA, we measured, we measured, by Western by blot Western experiments, blot experiments, in isolated liver in isolated mitochondria liver mitochondriathe expression the of expression two major mtDNAof two major repair mtDNA enzymes, repair namely enzymes, the 8-oxoG namely DNA the glycosy-8-oxoG DNAlase/apurinic glycosylase/apurinic or apyrimidinic or apyrimidinic (AP) lyase (OGG1) (AP) lyase and apurinic/apyrimidinic (OGG1) and apurinic/apyrimidinic endonuclease endonuclease1 (APE1), both 1 involved(APE1), inboth the involved base excision in th repaire base (BER) excision which repair is the (BER) prevalent which mtDNA is the prevalentrepair mechanism mtDNA repair (Figure mechanism4). (Figure 4).

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 18 Int. J. Mol. Sci. 2021, 22, 1665 5 of 18

FigureFigure 4. 4. AmountsAmounts of of OGG1 OGG1 and and APE1 APE1 prot proteinseins in in isolated isolated liver liver mitochondria mitochondria from from AL-18, AL-18, AL-28, AL-28, CR-28,CR-28, AL-32, and CR-32-month-old rats. (A). In thethe inset,inset, aa representativerepresentative WesternWestern blot blot was was carried carriedout in one out rat in fromone rat each from of theeach assayed of the assayed groups. Immunoreactivegroups. Immunoreactive bands, from bands, top from to bottom top to represent, bottom represent,respectively, respectively, the signals the from signals OGG1 from and OGG1 VDAC. and TheVDAC. histogram The histogram shows theshows quantification the of the quantification of the intensity of the bands of OGG1 normalized to the intensity of VDAC. (B). In intensity of the bands of OGG1 normalized to the intensity of VDAC. (B). In the inset, a representative the inset, a representative Western blot was carried out in one rat from each of the assayed groups. Western blot was carried out in one rat from each of the assayed groups. Immunoreactive bands, from Immunoreactive bands, from top to bottom, represent, respectively, the signals from APE1 and VDAC.top to bottom, The histogram represent, shows respectively, the quan thetification signals of from the intensity APE1 and of VDAC.the bands The of histogram APE1 normalized shows the toquantification the intensity of of the VDAC. intensity (A,B of) Data the bands represent of APE1 the results normalized from totriplicated the intensity Western of VDAC. blot (A,B) Data experimentsrepresent the and results were from analyzed triplicated using Western the One-way blot experiments ANOVA test and and were Tukey’s analyzed multiple using the One-way comparisonANOVA test test. and Bars Tukey’s represent multiple the comparisonmean values test. and BarsSD for represent the five theexperimental mean values groups. and SD Data for the werefive experimental normalized against groups. the Data value were of the normalized AL-18 rats, against fixed theas 1. value * p < of0.05 the versus AL-18 AL-18 rats, fixedrats, # as p 1.< n: 0.05Number versus of AL-28 analyzed rats. animals. n: Number of analyzed animals.

TheThe intra-mitochondrial intra-mitochondrial protein protein amounts amounts of ofboth both enzymes enzymes had had similar similar trends trends as for as agefor ageand andCR CReffects. effects. In Inparticular, particular, OGG1 OGG1 and and APE1 APE1 did did not not show show any any statistically significantsigniﬁcant age-related changechange inin both both the the AL AL and and CR CR groups groups suggesting suggesting no no age age effect effect at all.at all.Unexpectedly, Unexpectedly, also also CR didCR not did induce not induce any signiﬁcant any significant effect on effect OGG1 on and OGG1 APE1 and amounts APE1 amountsin the CR-28 in the group CR-28 in group comparison in comparison with the age-matchedwith the age-matched AL animals, AL asanimals, well as as between well as betweenthe 32-month-old the 32-month-old groups. groups.

2.3.2.3. Evaluation Evaluation of of Age Age and and CR CR E Effectsffects on on Mitochondrial Mitochondrial Dynamics Dynamics HavingHaving previouslypreviously reported reported a markeda marked age effectage effect on the on expression the expression of proteins of involvedproteins involvedin fusion in (MFN2) fusion and(MFN2) ﬁssion and (DRP1) fission [(DRP1)16], we [16], evaluated we evaluated here the here effects the ofeffects CR in of aCR long in atime long span time on span these on markers.these markers. Therefore, Theref theore, protein the protein amounts amount of MFN2s of MFN2 and DRP1 and DRP1 were weredetermined, determined, by Western by Western blot experiments, blot experiments, in isolated in isolated liver mitochondria liver mitochondria from all from rat groups all rat groups(Figure 5(Figure). 5).

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 18 Int. J. Mol. Sci. 2021, 22, 1665 6 of 18

FigureFigure 5. 5. AmountsAmounts of of MFN2 MFN2 and and DRP1 DRP1 proteins proteins in in isolat isolateded liver liver mitochondria mitochondria from from AL-18, AL-18, AL-28, AL-28, CR-28,CR-28, AL-32, AL-32, and CR-32-month-old rats. (A). In the inset, aa representativerepresentative WesternWestern blot blot was was carried carriedout in one out rat in fromone rat each from of theeach assayed of the groups.assayed Immunoreactivegroups. Immunoreactive bands, from bands, top tofrom bottom, top to represent, bottom, represent, respectively, the signals from MFN2 and VDAC. The histogram shows the respectively, the signals from MFN2 and VDAC. The histogram shows the quantification of the quantification of the intensity of the bands of MFN2 normalized to the intensity of VDAC. (B). In intensity of the bands of MFN2 normalized to the intensity of VDAC. (B). In the inset, a representative the inset, a representative Western blot was carried out in one rat from each of the assayed groups. ImmunoreactiveWestern blot was bands, carried from out in top one to rat bottom from eachrepres ofent, the assayedrespectively, groups. the Immunoreactivesignals from DRP1 bands, and from VDAC.top to bottom The histogram represent, shows respectively, the quan thetification signals of from the intensity DRP1 and of VDAC.the bands The of histogram DRP1 normalized shows the toquantification the intensity of of the VDAC. intensity (A,B of) Data the bands represent of DRP1 the results normalized from totriplicate the intensity Western of VDAC. blot experiments (A,B) Data andrepresent were analyzed the results using from the triplicate One-way Western ANOVA blot experimentstest and Tukey’s and weremultiple analyzed comparison using thetest. One-way Bars representANOVA testthe mean and Tukey’s values and multiple SD for comparison the five experimental test. Bars representgroups. Data the were mean normalized values and against SD for thethe value five experimental of the AL-18 groups.rats, fixed Data as 1. were * p < normalized 0.05 versus against AL-18 rats, the value # p < 0.05 of the versus AL-18 AL-28 rats, fixedrats. n as: 1. Number* p < 0.05 of versus analyzed AL-18 animals. rats, # p < 0.05 versus AL-28 rats. n: Number of analyzed animals.

WeWe found a statistically significant significant age-related decrease (−43%)43%) in in MFN2 MFN2 in in the the AL- AL- 2828 rats rats compared compared to to the AL-18 controls. Such Such decrease decrease was was prevented prevented in in the AL-32 rats showingshowing a a complex complex age age effect, effect, similar similar to to that that reported reported for for Cyt Cyt c. c. However, However, different different from CytCyt c, c, CR CR had had a a very very positive positive effect effect on on the the 28-month-old 28-month-old animals, animals, preventing preventing in the CR-28 ratsrats the the age-related age-related decrease decrease of of the the AL-28 AL-28 animals and and leading to a significant significant marked increaseincrease (+52%) (+52%) in in MFN2 MFN2 compared compared to to the the AL AL age-matched age-matched counterpart. counterpart. Furthermore, Furthermore, the the MFN2MFN2 amount amount in in the the CR-28 CR-28 rats rats was was not not different different from from the the value value in in the the AL-18 AL-18 controls, controls, indicatingindicating an an efficacious efficacious prevention prevention of of the the ag age-relatede-related effect. effect. No No significant significant difference difference was was foundfound between between the the AL-32 AL-32 and and CR-32 CR-32 groups, groups, thus thus indicating indicating a a limited limited effect effect of of CR CR in in the the oldestoldest animals. animals. Moreover, Moreover, no no age-related age-related e effectffect was was found found for for MFN2 MFN2 in in the the AL-32 group comparedcompared to to the the AL-18 counterpart. Similarly, Similarly, the the amounts amounts of of DRP1 DRP1 protein protein showed a markedmarked age-related age-related decrease decrease ( −−49%)49%) in in AL-28 AL-28 rats rats compared compared to to the the AL-18 AL-18 group which waswas prevented prevented by by CR CR in in the the 28-month-old 28-month-old animals. animals. Indeed, Indeed, the the CR-28 CR-28 group group presented presented a positivea positive difference difference (+50%) (+50%) in comparison in comparison with with the theAL-28 AL-28 one, one, demonstrating demonstrating a strong a strong CR effect.CR effect. However, However, the theabsence absence of a of difference a difference between between AL-32 AL-32 and and CR-32 CR-32 as as for for the the DRP1 DRP1 proteinprotein amounts amounts further indicated a limited efficacy efficacy of CR in extended aging. Again, Again, there there waswas no no evident evident age age effect effect also also for for the the DRP1 DRP1 amounts amounts passing passing from from the theAL-18 AL-18 group group to the to AL-32the AL-32 one. one. Then, Then, we wedetermined determined the the values values of offusion fusion index index (FI) (FI) as as the the ratio ratio between MFN2/DRP1MFN2/DRP1 for for each each of of the the examined examined rats rats (Figure (Figure 6).6).

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 18 Int. J. Mol. Sci. 2021, 22, 1665 7 of 18

FigureFigure 6. 6. ScatterScatter plots plots for for values values of MF of MFN2N2 and and DRP1 DRP1 protein protein amounts amounts and of and the of fusion the fusion index index(FI) of(FI) all of the all assayed the assayed animals. animals. As for AsMFN2 for MFN2and DRP1, and symbols DRP1, symbols show the show quantification the quantification of the of the intensityintensity of of immunoreactive bandsbands ofof MFN2 MFN2 or or DRP1 DRP1 normalized normalized to to the the intensity intensity of of VDAC VDAC in isolatedin isolatedmitochondria mitochondria from each from rat. each All datarat. All represent data represent the results the fromresults triplicate from triplicate Western Western blot experiments. blot experiments.FI was calculated FI was from calculated the ratio from of MFN2 the ratio and of DRP1 MFN2 intra-mitochondrial and DRP1 intra-mitochondrial protein amounts protein for each of amounts for each of the assayed animals. The horizontal line represents the mean value. n: the assayed animals. The horizontal line represents the mean value. n: Number of analyzed animals. Number of analyzed animals. The FI values in the AL-18 group covered a narrow range spanning from 1.37 to 2.64, whereasThe inFI thevalues AL-28 in the group AL-18 all valuesgroup covered were higher a narrow ranging range from spanning 2.20 to 8.24. from The 1.37 FI to values 2.64, whereasof the CR-28 in the group AL-28 ranged group fromall values 0.99 towere 5.14 higher and were ranging quite from similar 2.20 to those8.24. The of the FI values AL-18 ofrats. the In CR-28 the AL-32 group animals, ranged thefrom FI 0.99 values to ranged5.14 and from were 0.70 quite to 2.27,similar thus to covering those of anthe interval AL-18 rats.similar In the to thatAL-32 of animals, the AL-18 the group FI values which ranged suggests from the 0.70 absence to 2.27, of thus a marked covering age an effect. interval In similarthe CR-32 to that group, of the the AL-18 FI values group ranged which from sugge 0.87sts to the 2.02 absence and were of a comparable marked age to effect. those In of thethe CR-32 AL-32 group, animals, the thus FI values supporting ranged the from idea 0.87 that to extended 2.02 and aging were (32 comparable months) mayto those be less of thesensitive AL-32 toanimals, CR than thus aging supporting (28 months). the idea that extended aging (32 months) may be less sensitiveThe to statistical CR than analysis aging (28 of months). the FI values from all the groups is presented in Figure7. TheIn the statistical comparison analysis between of the AL-18 FI values and from AL-28 all groups, the groups the higher is presented FI value in of Figure the latter 7. indicated an age-related alteration of dynamic balance. As for the CR-treated rats, the value of the CR-28 group was not different from that of the AL-18 counterpart and supported the diet efficacy in the comparison with the age-matched AL-fed counterparts. Analyzing the AL-32 and the CR-32 values of FI the absence of a significant difference between them was evident, reinforcing the idea of a reduced efficacy of the diet in the animals naturally reaching extended aging. Furthermore, the FI values of both 32-month-old groups were not different from the AL-18 counterpart, supporting the absence of a marked age effect in the oldest animals.

Figure 7. Fusion index (FI) values in isolated liver mitochondria from AL-18, AL-28, CR-28, AL-32, and CR-32-month-old rats. Bars represent the mean values and SD of the ratio, calculated for each group of rats from the individual values in Figure 6. Comparisons were made with respect to the

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 18

Figure 6. Scatter plots for values of MFN2 and DRP1 protein amounts and of the fusion index (FI) of all the assayed animals. As for MFN2 and DRP1, symbols show the quantification of the intensity of immunoreactive bands of MFN2 or DRP1 normalized to the intensity of VDAC in isolated mitochondria from each rat. All data represent the results from triplicate Western blot experiments. FI was calculated from the ratio of MFN2 and DRP1 intra-mitochondrial protein amounts for each of the assayed animals. The horizontal line represents the mean value. n: Number of analyzed animals.

The FI values in the AL-18 group covered a narrow range spanning from 1.37 to 2.64, whereas in the AL-28 group all values were higher ranging from 2.20 to 8.24. The FI values of the CR-28 group ranged from 0.99 to 5.14 and were quite similar to those of the AL-18 rats. In the AL-32 animals, the FI values ranged from 0.70 to 2.27, thus covering an interval similar to that of the AL-18 group which suggests the absence of a marked age effect. In the CR-32 group, the FI values ranged from 0.87 to 2.02 and were comparable to those of the AL-32 animals, thus supporting the idea that extended aging (32 months) may be less Int. J. Mol. Sci. 2021, 22, x FOR PEER sensitiveREVIEW to CR than aging (28 months). 8 of 18

Int. J. Mol. Sci. 2021, 22, 1665 The statistical analysis of the FI values from all the groups8 of 18 is presented in Figure 7.

value of the AL-18 rats, fixed as 1. * p < 0.05 versus AL-18 rats; # p< 0.05 versus AL-28 rats. n: Number of analyzed animals.

In the comparison between AL-18 and AL-28 groups, the higher FI value of the latter indicated an age-related alteration of dynamic balance. As for the CR-treated rats, the value of the CR-28 group was not different from that of the AL-18 counterpart and supported the diet efficacy in the comparison with the age-matched AL-fed counterparts. Analyzing the AL-32 and the CR-32 values of FI the absence of a significant difference between them was evident, reinforcing the idea of a reduced efficacy of the diet in the animals naturally reaching extended aging. Furthermore, the FI values of both 32-month- Figureold groups 7. Fusion7. Fusion indexwere (FI)index not values (FI)different in isolatedvalues liver from in mitochondriaisolated the AL-1 liver from8 AL-18,mi counterpart,tochondria AL-28, CR-28, from supporting AL-32, AL-18, AL-28, the absence CR-28, ofAL-32, a andmarked CR-32-month-old age effect rats. Barsin the represent oldest the meananimals. values and SD of the ratio, calculated for each andgroup CR-32-month-old of rats from the individual rats. values Bars in Figure represent6. Comparisons the mean were va madelues with and respect SD to of the the ratio, calculated for each groupvalue of theof AL-18rats rats,from ﬁxed the as 1.individual * p < 0.05 versus values AL-18 rats;in Figure # p< 0.05 versus6. Comparisons AL-28 rats. n: Number were made with respect to the of2.4. analyzed Effect animals. of Age and CR on mtDNA Content and Damage 2.4. EffectSince of Age mitochondrial and CR on mtDNA Contentdynamics and Damage is closely related to the maintenance of mtDNA [16,18],Since mitochondrialwe determined dynamics the is mtDNA closely related content to the maintenance in all the of rats mtDNA by [the16,18 quantitative], polymerase we determined the mtDNA content in all the rats by the quantitative polymerase chain reactionchain reaction (qPCR) (Figure (qPCR)8). (Figure 8).

Figure 8. Relative content of mitochondrial DNA (mtDNA) in liver from AL-18, AL-28, CR-28, AL-32, Figure 8. Relative content of mitochondrial DNA (mtDNA) in liver from AL-18, AL-28, CR-28, AL- and CR-32-month-old rats. Bars represent data obtained from two independent experiments con- ducted32, and in triplicateCR-32-month-old and analyzed using rats. the Bars One-way represent ANOVA testdata and obtained Tukey’s multiple from comparison two independent experiments test.conducted In the graphical in triplicate representation, and dataanalyzed have been using normalized the One-way against the value ANOVA of the AL-18 test rats,and Tukey’s multiple ﬁxedcomparison as 1, and shown test. as In the the mean graphical value and SD. representation, * p < 0.05 versus AL-18 data rats; have # p < been 0.05 versus normalized AL-28 against the value of rats.the nAL-18: Number rats, of analyzed fixed as animals. 1, and shown as the mean value and SD. * p < 0.05 versus AL-18 rats; # < 0.05An versus age-related AL-28 decrease rats. n: inNumber the mtDNA of analyzed content was animals. found in the AL-28 (−27%) and the AL-32 rats (−23%) compared with the AL-18 animals, whereas no statistical differenceAn was age-related present between decrease the AL-28 in andthe the mtDNA AL-32 groups. content CR was was very found effective in the AL-28 (−27%) and in preventing the age-related decrease in the mtDNA content only at 28 months leading tothe an AL-32 increased rats (+19%) (−23%) value compared in the CR-28 groupwith comparedthe AL-18 to the animals, AL-fed age-matched whereas no statistical difference counterpart.was present Furthermore, between there the was AL-28 no statistical and the difference AL-32 between groups. the AL-18CR was value very and effective in preventing that of the CR-28 group. As seen for other markers in the present work, CR was not so the age-related decrease in the mtDNA content only at 28 months leading to an increased (+19%) value in the CR-28 group compared to the AL-fed age-matched counterpart. Furthermore, there was no statistical difference between the AL-18 value and that of the CR-28 group. As seen for other markers in the present work, CR was not so efficacious at 32 months since there was no difference between the AL-32 value and that of the CR-32 counterpart. In order to deepen the analysis at the mtDNA damage level, we measured the relative content of the 4.8 Kb “common deletion” by qPCR (Figure 9).

Int. J. Mol. Sci. 2021, 22, 1665 9 of 18

efﬁcacious at 32 months since there was no difference between the AL-32 value and that of Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 9 of 18 the CR-32 counterpart. In order to deepen the analysis at the mtDNA damage level, we measured the relative content of the 4.8 Kb “common deletion” by qPCR (Figure9).

FigureFigure 9. 9.Relative Relative content content of the of 4.8 the Kb deletion4.8 Kb indeletion liver from in AL-18, liver AL-28,from AL-18, CR-28, AL-32, AL-28, and CR-28, AL-32, and CR- CR-32-month-old rats. Bars represent data obtained from two independent experiments conducted 32-month-oldin triplicate and analyzed rats. Bars using represent the One-way data ANOVA obtained test and from Tukey’s two multiple independent comparison test.experiments In conducted in triplicatethe graphical and representation, analyzed data using have the been One-way normalized ANOVA against the valuetest and of the Tukey’s AL-18 rats, multiple fixed as comparison test. In the1, and graphical shown as therepresentation, mean value andSD. datan: Numberhave been of analyzed norm animals.alized against the value of the AL-18 rats, fixed as 1, and shown as the mean value and SD. n: Number of analyzed animals. No significant changes were found with age nor with CR in the relative content of the 4.8 Kb deletion in all the tested groups. Finally,No significant we screened changes three specific were regions found of mtDNA with forage the nor incidence with ofCR oxidized in the relative content of thepurines, 4.8 Kb mainly deletion 8-oxo-deoxyguanosine in all the tested (8-oxodG), groups. using the oxidized purines-sensitive enzyme formamidopyrimidine DNA glycosylase (Fpg). The three regions encompassed, respectively,Finally, the we displacement screened loop three (D-loop), specific the originregion ofs the of Light-strand mtDNA for replication the incidence of oxidized purines,(Ori-L), and mainly parts of both8-oxo-deoxyguanosine the NADH dehydrogenase (8-oxodG), (ND) subunit using 1 and 2the (ND1/ND2) oxidized purines-sensitive enzyme(Figure 10 ).formamidopyrimidine DNA glycosylase (Fpg). The three regions encompassed, As shown in Figure 10, the percentage of 8-oxodG incidence was significantly different respectively,between the rat groupsthe displacement in each analyzed loop region, (D-loop), although withthe origin a similar of pattern. the Light-strand The replication (Ori-L),post-hoc test and revealed parts the of significance both the of NADH the reduced dehy presencedrogenase of 8-oxodG (ND) in CR-28 subunit rats as 1 and 2 (ND1/ND2) (Figurecompared 10). with both AL-18 and AL-28 groups (−42% CR-28 vs. AL-18 and −43% CR-28 vs. AL-28 at the D-loop; −48% CR-28 vs. AL-18 and −49% CR-28 vs. AL-28 at the Ori-L; −54%As CR-28 shown vs. AL-18 in andFigure−56% 10, CR-28 the vs. percentage AL-28 at ND1/ND2). of 8-oxodG CR showed incidence to be was significantly differenteffective at preventingbetween thethe mtDNA rat groups oxidative in damage each analyz up to 28ed months, region, while although the incidence with a similar pattern. Theof the post-hoc same damage test was revealed not different the between significance AL-32 and of CR-32the reduced rats. Oxidized presence purines of 8-oxodG in CR-28 ratswere detectedas compared already inwith AL-18 both rats withoutAL-18 showing and AL-28 an age-related groups increase (−42% in theCR-28 AL-28 vs. AL-18 and −43% animals. The comparison between the AL-32 group and the CR-32 one did not show a CR-28significant vs. difference. AL-28 Consideringat the D-loop; the specific −48% regions’ CR-28 values vs. itAL-18 is interesting and to−49% highlight CR-28 vs. AL-28 at the Ori-L;that the D-loop−54% incidenceCR-28 vs. was AL-18 the highest and among −56% the CR-28 values from vs. theAL-28 tested at regions ND1/ND2). in each CR showed to be effectivegroup, thus at supporting preventing previous the findingsmtDNA indicating oxidative this regiondamage as a hotspotup to 28 for months, oxidative while the incidence damage [17,19–21]. of the same damage was not different between AL-32 and CR-32 rats. Oxidized purines were detected already in AL-18 rats without showing an age-related increase in the AL- 28 animals. The comparison between the AL-32 group and the CR-32 one did not show a significant difference. Considering the specific regions’ values it is interesting to highlight that the D-loop incidence was the highest among the values from the tested regions in each group, thus supporting previous findings indicating this region as a hotspot for oxidative damage [17,19–21]. All the findings reported on the CR efficacy in the age-matched groups were summarized in Table 1.

Table 1. Effect of CR on mitochondrial markers.

28 Months 32 Months Mitochondrial Markers (AL vs. CR) (AL vs. CR) Citrate synthase activity + − TFAM + − LonP1 − − Cyt c − − OGG1 − − APE1 − − MFN2 + − DRP1 + −

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 10 of 18

mtDNA content + − mtDNA 4.8 Kb deletion − − Oxidized purines-specific mt-DNA damage D-loop + − Oxidized purines-specific mt-DNA damage Ori-L + − Int. J. Mol. Sci. 2021, 22, 1665 Oxidized purines-specific mt-DNA damage ND1/ND2 + − 10 of 18 +: Difference between AL and CR; −: No difference between AL and CR.

Figure 10. IncidenceIncidence of of oxidized oxidized purines purines damage damage at atthe the D-loop, D-loop, Ori-L, Ori-L, and and ND1/ND2 ND1/ND2 mtDNA mtDNA regionsregions in liver from AL-18, AL-18, AL-28, AL-28, CR CR-28,-28, AL-32, AL-32, and and CR-32-month-old CR-32-month-old rats. rats. (A ().A ).Bars Bars represent represent data obtained from two independent experiments experiments conducted conducted in in triplicate triplicate and and analyzed analyzed using using the the One-way ANOVA test and Tukey’s multiple comparison test. In the graphical representation, data One-way ANOVA test and Tukey’s multiple comparison test. In the graphical representation, data have been normalized against the value of the AL-18 rats, fixed as 1, and shown as the mean value have been normalized against the value of the AL-18 rats, fixed as 1, and shown as the mean value and SD. * p < 0.05 versus AL-18 rats; # p < 0.05 versus AL-28 rats. n: Number of analyzed animals. and(a) D-loop; SD. * p <(b 0.05) Ori-L; versus (c) AL-18ND1/ND2. rats; #(Bp).< Representative 0.05 versus AL-28 gel rats.of formamidopyrimidinen: Number of analyzed DNA animals. (a) D-loop;glycosylase (b) Ori-L;(Fpg)-treated (c) ND1/ND2. and untreated (B). Representative total DNA from gel of one formamidopyrimidine CR-28 and one AL-28 DNA rat; glycosylase5 ng total (Fpg)-treatedDNA were amplified and untreated using the total D-loop DNA primers. from one An CR-28 aliquot and of oneeach AL-28 PCR amplification rat; 5 ng total was DNA loaded were amplifiedonto agarose using ethidium the D-loop bromide-stained primers. An aliquotgel andof analyzed each PCR for amplification band intensities. was loaded onto agarose ethidium bromide-stained gel and analyzed for band intensities.

All the ﬁndings reported on the CR efﬁcacy in the age-matched groups were summarized in Table1. Int. J. Mol. Sci. 2021, 22, 1665 11 of 18

Table 1. Effect of CR on mitochondrial markers.

28 Months 32 Months Mitochondrial Markers (AL vs. CR) (AL vs. CR) Citrate synthase activity + − TFAM + − LonP1 − − Cyt c − − OGG1 − − APE1 − − MFN2 + − DRP1 + − mtDNA content + − mtDNA 4.8 Kb deletion − − Oxidized purines-specific mt-DNA damage D-loop + − Oxidized purines-specific mt-DNA damage Ori-L + − Oxidized purines-specific mt-DNA damage ND1/ND2 + − +: Difference between AL and CR; −: No difference between AL and CR.

3. Discussion In the long-lasting search for approaches able to counteract the progressive decline of aging, CR has gained a paramount relevance due to its positive effects, reported in a wide series of organisms ranging from yeast through man [22]. CR has been demonstrated to affect a large number of molecular pathways. Among these, some are particularly effective in the modulation of mitochondrial biogenesis and activity, as those sensing nutrients and/or energy levels such as the AMP-dependent kinase (AMPK) and sirtuins, in which all cooperate to the CR-induced reprogramming of mitochondrial metabolism [23]. By improving the oxidative metabolism, CR reduces the mitochondrial production of ROS and the molecular damages induced by the age-related increase of these reactive species [8–10]. Although several studies investigated the effects of CR at the mitochondrial level [3,13–15,24], a comprehensive work on the effects of this nutritional intervention on organelles is still missing. Therefore, this study sought to test the existence of boundaries to the CRs efﬁcacy in a large set of mitochondrial markers. In particular, we focused on the comparison between the effects induced by this dietary regimen in aged (28-month-old) and extremely aged (32-month-old) rats to verify whether an interplay exists between CR and the progression of aging. The analysis of the results revealed a complex picture as for the efﬁcacy of CR on mitochondrial biogenesis and mtDNA damage, which has been summarized in Table1.

3.1. Mitochondrial Markers in 28-Month-Old Rats A large number of mitochondrial markers was positively affected by CR in 28-month- old rats. Namely, here were reported age-related decreases in the citrate synthase activity, in protein amounts of TFAM, MFN2, and DRP1 and in the mtDNA content comparing the AL-18 control group with the AL-28 one. Such changes were all prevented by CR in 28-month-old rats (CR-28). These results support the anti-aging effect of CR up to this age in rat liver. Similarly, the decrease in the incidence of oxidized purines at all the three assayed mtDNA regions, induced by CR, further reinforces this view. Of note, this oxidative damage was already present in the AL-18 animals with no age-related increase in the AL-28 rats suggesting that its appearance may be identiﬁed as an early marker of aging. A particular attention needs to be placed on the ﬁve mitochondrial markers that were not affected by CR in 28-month-old rats namely the protein amounts of LonP1, Cyt c, OGG1, and APE1 and the 4.8 Kb deletion content. In fact, as for LonP1 and Cyt c, CR did not prevent the age-related decrease found in the AL-28 animals, whereas the protein amounts of OGG1 and APE1 as well as the 4.8 Kb deletion content were not affected by age nor by CR. LonP1 is responsible for the degradation of exhausted TFAM [25–27] and has been reported to decrease with aging [28] in rat liver [29] up to 27 months of age. We Int. J. Mol. Sci. 2021, 22, 1665 12 of 18

previously suggested that such aging-decreased expression of LonP1 [16] might be related to its activity in the removal of TFAM unbound to mtDNA [26]. As a possible explanation for the inefficacy of CR to prevent the age-related decrease in LonP1 expression, despite the restored TFAM expression and TFAM-binding to mtDNA specific regions (present data and Picca et al. [15]), is the CR-mediated activation of Sirt3 [30] that might have influenced also the protein amount of LonP1 via the induced deacetylation. A different hypothesis can be proposed for explaining the inefficacy of CR to prevent the age-related decrease in the amount of Cyt c. This protein is the major trigger of the mitochondrial apoptotic pathway through its release to the cytosol, thus its decreased intra-mitochondrial amount in the AL-28 rats with respect to the AL-18 value indicated an increase in the intrinsic apoptotic process with aging [16,31]. Furthermore, as for LonP1, the Cyt c amount in the CR-28 rats was still significantly lower than the value in the AL-18 animals, indicating that CR did not substantially affect the final regulation of Cyt c expression/release to cytosol and, presumably, nor the age-related increase in mitochondrial apoptotic pathway. This is in agreement with the previously reported promotion of apoptosis mediated by CR [23]. Since the apoptotic pathway can be triggered also by the accumulation of DNA damage [32,33], we determined the protein expression of two major mitochondrial BER enzymes namely OGG1 and APE1. The protein amounts of both were not affected by aging and CR passing from 18 to 28 months of age. A possible explanation is that the expression of the two BER enzymes was already increased and sufficient in the AL-18 rats to counteract the age-related increased damage to mtDNA. Indeed, an age-related increase in the activity of mitochondrial OGG1 [34,35] and mitochondrial APE1 [36] has been reported in the liver of rodents passing from 3–6-month-old animals to 20–23-month-old ones, likely induced to efficiently counteract the age-related increase in mtDNA damage. All this further supports our hypothesis that the mtDNA damages, repaired by OGG1 and APE1, appeared very early in rodents’ lifetime (they were already present in the AL-18 rats) and induced, through a mitochondrion-nucleus retrograde communication, an early increase in expression of the BER enzymes. As for the absence of a CR effect on OGG1 and APE1, we suggest that the CR-reduced presence of ROS and related oxidative damages was efficaciously counteracted by an expression of both enzymes not different from that of the age-matched AL-animals. As for the 4.8 Kb deletion content, there was no age-related increase passing from the AL-18 animals to the AL-28 ones as if also this mtDNA damage appeared quite early in rats’ lifetime and its age-related increase could have been verified only in comparison with young animals, as reported in other studies [19,37–39]. The inefficacy of CR to significantly decrease the 4.8 Kb deletion content in the CR-28 rats in comparison with the AL-28 and the AL-18 animals further indicates that the long-term CR induced a marked decrease in the presence of ROS in liver. This might have led to a sort of steady-state level of the 4.8 Kb deletion, not different from that of the AL-18 counterpart and compatible with the respective mtDNA content. Although the mtDNA repair was not reduced by aging, the age-related decrease in the mtDNA content was still present when passing from the AL-18 rats to the AL-28 ones, as previously described in different tissues of aged rodents [3,15,19,39–42]. This may be explained by the age-related increased binding of TFAM at specific mtDNA regions that were also damage hotspots [19]. This hypothesis would thus reconcile the age-related mtDNA loss with the unchanged expression and activity of the repair enzymes. In further support to this hypothesis is the absence of mtDNA loss in the CR-28 rats. Indeed, we previously demonstrated that CR prevented the age-related increase in TFAM-binding at specific mtDNA regions [15]. Therefore, according to the present and our previous data, the modulation of TFAM-binding may be among the molecular mechanisms involved in the aging- and CR-mediated regulation of mitochondrial biogenesis. This regulation by aging and CR has also been shown to be pursued through the balance of mitochondrial dynamics which is affected by aging [43] and leads to the prevalence of fusion over fission [44,45]. Therefore, we calculated the fusion index (FI) in all assayed animals (Figure6) and found values that confirmed and expanded our previous results [16]. Some novel indications can be gathered by the present Int. J. Mol. Sci. 2021, 22, 1665 13 of 18

study, namely in the AL-18 rats the FI values covered a small range, while in the AL-28 ones the FI range was much larger, with a marked age-related increase in the individual values, in the CR-28 rats the small range of values indicated a positive effect of CR in the induction of fission, with a generalized increase in the DRP1 individual amounts in comparison with those from the AL-28 group. While an efficient fission might remove the damaged organelles, a similar entity fusion might ensure the diffusion of damaged macromolecules in the mitochondrial network, thus diluting their negative effects [46]. Therefore, the CR-mediated restoration of the finely tuned-up balance of mitochondrial dynamics might induce positive consequences on the modulation of mitochondrial biogenesis, acting synergistically with the mtDNA damage retrograde communication. This is in line with the existing literature reporting that the nutrient status and metabolic alterations can modulate the balance of mitochondrial dynamics [47] and, thus, affect also the organelle biogenesis. In particular, the ROS-sensitive regulation of mitochondrial biogenesis can face only limited increases of ROS through the increase in organelles number [48] and the CR-reduced presence of ROS [15] might prevent overcoming of a threshold-like level that might lead to mitochondrial loss. The CR efficacy in decreasing the presence of ROS was proven in the present study by the reduced accumulation of oxidized purines at the mtDNA analyzed regions in the CR-28 rats that supports an overall improvement of mitochondrial metabolism, able to counteract the age-related decline. Indeed, the CR- reduced presence of ROS has been widely demonstrated also at the level of mitochondrial proteins and lipids. In particular, CR has been shown to decrease oxidative modifications of mitochondrial proteins in rat heart [49] and to modify the saturation/unsaturation index in mitochondrial membranes preventing oxidative damage and maintaining membrane fluidity [8]. Furthermore, CR has been demonstrated to decrease the production of ROS by mitochondrial respiratory complexes in both murine skeletal muscle and liver [8]. The CR beneficial effects at the ROS level also include an improved neutralization of ROS and an increased repair of ROS-damaged molecules [10]. In addition to this CR effect, changes in mitochondrial functionality are modulated by the activation of CR-sensitive AMPK and sirtuins that regulate the activity of the transcription factors peroxisome proliferator- activated receptor gamma coactivator 1-alpha (PGC-1α), and forkhead box (FoxO), with the latter involved in biogenesis, oxidative metabolism, and turnover of the mitochondria [23]. Thus, the CR-mediated positive influence on mitochondrial metabolism should interact with other pathways to modulate the final trigger of mitochondrial biogenesis, according to a model of retrograde communication to the nucleus [50].

3.2. Mitochondrial Markers in 32-Month-Old Rats As for the comparison between AL-32 and CR-32 rats, our data show a generalized absence of significant differences. This may suggest an age limitation to the efficacy of CR in counteracting the age-related decline when passing from 28 to 32 months. With regards to the age effect, though, different conclusions can be driven depending on the analyzed marker. In fact, in both 32-month-old groups, the age-related decrease in the citrate synthase activity, amounts of TFAM, LonP1, OGG1, APE1, contents of mtDNA, and of the 4.8 Kb deletion was maintained at a level which was similar to that observed in the AL-28 rats or it was partially/completely prevented as in amounts of Cyt c, MFN2, DRP1, and the incidence of oxidized purines at specific mtDNA regions. These different behaviors may be likely due to the interplay of the various pathways involving the analyzed markers. Another conclusion can be highlighted comparing both 32-month-old groups with the AL-18 and the AL-28 ones as for the MFN2 and DRP1 amounts. In fact, the age-related decrease was not present passing from the AL-18 group to both AL-32 and CR-32 groups for the two dynamics proteins. Considering that in the comparison between the AL-18 group and both the 32-month-old ones as for the other markers there was an age-related decrease similar or smaller in size than that between the AL-18 and AL-28 groups, we can drive some consequences. This may indicate that the age-related alterations of markers may have begun or been already present at 18 months, and then reached their Int. J. Mol. Sci. 2021, 22, 1665 14 of 18

lowest/highest value in the aged, 28-month-old animals with a “pace” which is typical of the aging process. Conversely, in the extremely aged animals (32-month-old), the “pace” of the process appeared slowed since the values lower than or equal to those of the AL-18 counterparts, but not lower/higher than those from the AL-28 ones, were found, despite the longer time span elapsed. The hypothesis of two different paces in the aging process [16] implies that damages, due to different causes, begin to affect physical and metabolic activities during adulthood in each rat. Such damages are efficaciously counteracted by compensatory mechanisms and the damages do not appear openly, coinciding with a stable “middle” age characterized by a successful homeostasis. In this study, we identified as rats aging “fast” those in which the balance between intervening damages and compensatory mechanisms begins to fade later in life. In these animals, constituting the larger fraction of the AL-28 group, damages exceed the threshold compatible with normal activities and an altered condition appears. Conversely, the remaining, smaller part of the examined aging population maintains the homeostatic balance between the damages and repair for a longer time and constitutes the group of animals aging “slowly”, including both AL-32 and CR-32 rats. A similar hypothesis about the different paces of aging has recently been proposed also for humans [51]. Taken as a whole, these results indicate that the extremely aged animals may have reached longevity without being significantly affected by CR and likely due to a genetic predisposition to better cope with the age-related mitochondrial dysfunction, but such hypothesis warrants future research. These novel findings can give an answer to the original and crucial question on the existence of limits to the efficacy of CR, raised by Ingram and de Cabo in their very deep study on this issue [52], allowing the identification of such limit with the age of 28 months for rat liver. Nevertheless, the conclusion that CR or any diet did not elicit beneficial consequences in those rats naturally predisposed to longevity should not affect the overall confidence in efficacy of this nutritional intervention. In fact, CR was still able to convey beneficial effects as it was able to prevent most of the age-related alterations of mitochondrial biogenesis and mtDNA damage in the liver of 28-month-old rats. This further confirms CR as a well-founded strategy to efficiently counteract the aging decline in the general population.

4. Materials and Methods 4.1. Animals The study was approved by the Institutional Animal Care and Use Committee at the University of Florida. All procedures were performed in accordance with the National Institute of Health (NIH) guidelines for the care and use of laboratory animals. Liver samples were from Fischer 344 × Brown Norway (F344BNF1) male rats obtained from the National Institute of Aging colony (Indianapolis, IN, USA) and housed individually in a temperature- (20 +/− 2 ◦C) and light-controlled environment (12-h light/dark cycle) with regular rat chow and water available ad libitum at the Department of Aging and Geriatric Research, Division of Biology of Aging, College of Medicine, University of Florida, Gainesville, FL (USA). Calorie restriction had been initiated at 3.5 months of age (10% restriction), raised to 25% restriction at 3.75 months, and kept at 40% restriction from 4 months until the end of each animal’s life, which is at 28 months (CR-28) or 32 months (CR-32) of age. Calorie-restricted animals were fed the NIH31-NIA fortiﬁed diet to ensure that they were not malnourished, whereas ad libitum-fed animals were given the NIH31 rat diet. The animals consisted of the following groups: 18-month-old ad libitum-fed (AL-18, n = 6), 28-month-old ad libitum-fed (AL-28, n = 6), 28-month-old calorie-restricted (CR-28, n = 6), 32-month-old ad libitum-fed (AL-32, n = 6), and 32-month-old calorie-restricted (CR-32, n = 6) rats. Animals were anesthetized before sacriﬁce and liver samples were immediately removed, snap-frozen in isopentane cooled by liquid nitrogen, and stored in liquid nitrogen until further analysis. Int. J. Mol. Sci. 2021, 22, 1665 15 of 18

4.2. Determination of Citrate Synthase Activity Determination of citrate synthase activity was performed as in [17]. Brieﬂy, 80 µg of total proteins puriﬁed from liver samples were incubated in the reaction buffer (0.31 mM acetyl-CoA, 100 mM Tris buffer (pH 8.1), 0.25% Triton X-100, 0.1 mM 550-dithio-bis-2- nitrobenzoic acid, and 0.5 mM oxaloacetate (1 mL) at 30 ◦C. The citrate synthase activity (µmol × min−1 × g tissue–1) was spectrophotometrically determined by measuring the rate of production of thionitrobenzoic acid (TNB) at 412 nm.

4.3. Western Immunoblotting Liver mitochondria were isolated in a medium containing 220 mM mannitol, 70 mM sucrose, 20 mM Tris–HCl, 1 mM EDTA, and 5 mM EGTA, pH 7.4, at 4 ◦C according to [16]. In addition, 10 µg of mitochondrial proteins were used for Western immunoblotting analysis. Anti-TFAM (1:50,000), anti-VDAC (1:50,000, Abcam, Cambridge, UK), anti- OGG1 (1:2500, Abcam, Cambridge, UK), anti-APE1 (1:5000, Abcam, Cambridge, UK), anti-MFN2 (1:5000, Abnova, Taipei, Taiwan), anti-DRP1 (1:2500, Abnova, Taipei, Taiwan), anti-Cyt c (1:500, Pharmingen, San Diego, CA, USA), and anti-Lon Protease (1:10,000) were used as primary antibodies. The antibodies against TFAM and Lon were custom- made and kindly donated, respectively, by Dr. H. Hinagaki (Department of Chemistry, National Industrial Research Institute of Nagoya, Nagoya-shi, Aichi, Japan) and Dr. C. Suzuki (Department of Biochemistry and Molecular Biology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA). The proteins were detected by chemiluminescence and immunoreactive bands were quantiﬁed using the Image Lab Software (BioRad Laboratories Inc., Hercules, CA, USA) and normalized against VDAC-expression.

4.4. Determination of mtDNA and mtDNA 4.8 Kb “Common Deletion” Content The quantitative real time polymerase chain reaction (qPCR) was used to determine relative contents of mtDNA and mtDNA 4.8 Kb “common deletion”. Reactions were performed via SYBR Green chemistry using 3 ng of total DNA as a template and the following primers: MtDNA for 50-GGTTCTTACTTCAGGGCCATCA-30 (nt 15,785–15,806), mtDNA rev 50-TGATTAGACCCGTTACCATCGA-30 (nt 15,868–15,847); β-actin for 50- CCCAGCCATGTACGTAGCCA-30 (nt 2181–2200), β-actin rev 50-CGTCTCCGGAGTCCATC AC-30 (nt 2266–2248); 4.8 del for 50-AAGGACGAACCTGAGCCCTAATA-30 (nt 8109–8131), 4.8 del rev 50-CGAAGTAGATGATGCGTATACTGTA-30 (nt 13,020–12,996). The mtDNA content relative to the nuclear DNA and 4.8 Kb “common deletion” content relative to mtDNA were determined, as reported in [19].

4.5. Analysis of Oxidized Purines Formamidopyrimidine DNA glycosylase (Fpg) (New England Biolabs, Beverly, MA, USA) digestion of total DNA was used to detect oxidized purines [53]. PCR ampliﬁcation of the D-loop, Ori-L, and ND1/ND2 regions of mtDNA was conducted using the respective primers pair: D-loop For 50-TCTGGTCTTGTAAACCAAAAATGA-30 (nt 15,302–15,325), D-loop Rev 50-TGGAATTTTCTGAGGGTAGGC-30 (nt 16,302–16,282); Ori-L For 50-AACCAGACCCAA ACACGAAA-30 (nt 4414–4433), Ori-L Rev 50-CTATTCCTGCTCAGGCTCCA-30 (nt 5407– 5388); ND1 For 50-AGGACCATTCGCCCTATTCT-30 (nt 3390–3409), ND1 Rev 50-CGCCAAC AAAGACTGATGAA30 (nt 4399–4380) on 5 ng of Fpg-treated and untreated total DNA. The cycling conditions were: Pre-incubation of 10 min at 95 ◦C, followed by 18 cycles of 15 s 95 ◦C, 15 s 58 ◦C, and 1 min 72 ◦C. An aliquot of each PCR ampliﬁcation was loaded on 1.3% agarose gel. Band intensities of ethidium bromide-stained bands were analyzed by the Image Lab Software (BioRad Laboratories Inc., CA, USA). To improve the graphical visualization, the ratio between Fpg-treated and untreated band intensities was expressed as a percentage of the complement to 100.4.6. statistics Int. J. Mol. Sci. 2021, 22, 1665 16 of 18

Data are expressed as the mean and standard deviation (SD). The One-way ANOVA with Tukey’s multiple comparison test were used. For all tests, the statistical signiﬁcance was set at a 5% level. Analyses were run using a speciﬁc statistical package (Stata Corp. 2005. Stata Statistical Software: Release. College Station, TX, USA).

Author Contributions: Conceptualization, G.C., A.P., F.R., A.O., G.R., C.L., V.P. and A.M.S.L.; formal analysis, G.C., A.P., F.F. and V.P.; investigation, G.C., A.P., A.O., F.R., C.L. and A.M.S.L.; writing— review and editing, G.C., A.P., V.P., C.L., G.R., F.R. and A.M.S.L. All authors have read and agreed to the published version of the manuscript. Funding: This work was partly supported by MIUR-FFABR 2018 to V.P., MIUR-FFABR 2018 to A.M.S.L., Istituto Banco di Napoli-Fondazione 2015 to A.M.S.L., UniBa Fondi d’Ateneo 2014 to A.M.S.L., UniBa Fondi d’Ateneo 2015/2016 to A.M.S.L. Institutional Review Board Statement: The study was approved by the Institutional Animal Care and Use Committee at the University of Florida. All procedures were performed in accordance with the National Institute of Health (NIH) guidelines for the care and use of laboratory animals. Informed Consent Statement: Not applicable. Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. Conflicts of Interest: The authors declare no conflict of interest.

References 1. Lopez-Otin, C.; Blasco, M.A.; Partridge, L.; Serrano, M.; Kroemer, G. The hallmarks of aging. Cell 2013, 153, 1194–1217. [CrossRef] 2. Folgueras, A.R.; Freitas-Rodriguez, S.; Velasco, G.; Lopez-Otin, C. Mouse Models to Disentangle the Hallmarks of Human Aging. Circ. Res. 2018, 123, 905–924. [CrossRef][PubMed] 3. Picca, A.; Pesce, V.; Fracasso, F.; Joseph, A.M.; Leeuwenburgh, C.; Lezza, A.M. A comparison among the tissue-speciﬁc effects of aging and calorie restriction on TFAM amount and TFAM-binding activity to mtDNA in rat. Biochim. Biophys. Acta 2014, 1840, 2184–2191. [CrossRef] 4. Wachsmuth, M.; Hubner, A.; Li, M.; Madea, B.; Stoneking, M. Age-Related and Heteroplasmy-Related Variation in Human mtDNA Copy Number. PLoS Genet. 2016, 12, e1005939. [CrossRef] 5. Guarasci, F.; D’Aquila, P.; Mandala, M.; Garasto, S.; Lattanzio, F.; Corsonello, A.; Passarino, G.; Bellizzi, D. Aging and nutrition induce tissue-speciﬁc changes on global DNA methylation status in rats. Mech. Aging Dev. 2018, 174, 47–54. [CrossRef][PubMed] 6. Khan, S.S.; Singer, B.D.; Vaughan, D.E. Molecular and physiological manifestations and measurement of aging in humans. Aging Cell 2017, 16, 624–633. [CrossRef] 7. McCay, C.M.; Crowell, M.F.; Maynard, L.A. The effect of retarded growth upon the length of life and upon the ultimate body size. J. Nutr. 1935, 10, 63–79. [CrossRef] 8. Lopez-Lluch, G.; Navas, P. Calorie restriction as an intervention in aging. J. Physiol. 2016, 594, 2043–2060. [CrossRef] 9. Picca, A.; Pesce, V.; Lezza, A.M.S. Does eating less make you live longer and better? An update on calorie restriction. Clin. Interv. Aging 2017, 12, 1887–1902. [CrossRef] 10. Duszka, K.; Gregor, A.; Guillou, H.; Konig, J.; Wahli, W. Peroxisome Proliferator-Activated Receptors and Caloric Restriction- Common Pathways Affecting Metabolism, Health, and Longevity. Cells 2020, 9, 1708. [CrossRef] 11. Longo, V.D.; Antebi, A.; Bartke, A.; Barzilai, N.; Brown-Borg, H.M.; Caruso, C.; Curiel, T.J.; de Cabo, R.; Franceschi, C.; Gems, D.; et al. Interventions to Slow Aging in Humans: Are We Ready? Aging Cell 2015, 14, 497–510. [CrossRef] 12. Akbari, M.; Kirkwood, T.B.L.; Bohr, V.A. Mitochondria in the signaling pathways that control longevity and health span. Aging Res. Rev. 2019, 54, 100940. [CrossRef][PubMed] 13. Picca, A.; Lezza, A.M. Regulation of mitochondrial biogenesis through TFAM-mitochondrial DNA interactions: Useful insights from aging and calorie restriction studies. Mitochondrion 2015, 25, 67–75. [CrossRef][PubMed] 14. Wang, H.; Arias, E.B.; Yu, C.S.; Verkerke, A.R.P.; Cartee, G.D. Effects of Calorie Restriction and Fiber Type on Glucose Uptake and Abundance of Electron Transport Chain and Oxidative Phosphorylation Proteins in Single Fibers from Old Rats. J. Gerontol. Ser. A Biol. Sci. Med. Sci. 2017, 72, 1638–1646. [CrossRef][PubMed] 15. Picca, A.; Pesce, V.; Fracasso, F.; Joseph, A.M.; Leeuwenburgh, C.; Lezza, A.M. Aging and calorie restriction oppositely affect mitochondrial biogenesis through TFAM binding at both origins of mitochondrial DNA replication in rat liver. PLoS ONE 2013, 8, e74644. [CrossRef] 16. Picca, A.; Pesce, V.; Sirago, G.; Fracasso, F.; Leeuwenburgh, C.; Lezza, A.M.S. “What makes some rats live so long?” The mitochondrial contribution to longevity through balance of mitochondrial dynamics and mtDNA content. Exp. Gerontol. 2016, 85, 33–40. [CrossRef][PubMed] Int. J. Mol. Sci. 2021, 22, 1665 17 of 18

17. Chimienti, G.; Picca, A.; Fracasso, F.; Marzetti, E.; Calvani, R.; Leeuwenburgh, C.; Russo, F.; Lezza, A.M.S.; Pesce, V. Differences in Liver TFAM Binding to mtDNA and mtDNA Damage between Aged and Extremely Aged Rats. Int. J. Mol. Sci. 2019, 20, 2601. [CrossRef] 18. Kondadi, A.K.; Anand, R.; Reichert, A.S. Functional Interplay between Cristae Biogenesis, Mitochondrial Dynamics and Mitochondrial DNA Integrity. Int. J. Mol. Sci. 2019, 20, 4311. [CrossRef] 19. Chimienti, G.; Picca, A.; Sirago, G.; Fracasso, F.; Calvani, R.; Bernabei, R.; Russo, F.; Carter, C.S.; Leeuwenburgh, C.; Pesce, V.; et al. Increased TFAM binding to mtDNA damage hot spots is associated with mtDNA loss in aged rat heart. Free Radic. Biol. Med 2018, 124, 447–453. [CrossRef] 20. Chimienti, G.; Pesce, V.; Fracasso, F.; Russo, F.; de Souza-Pinto, N.C.; Bohr, V.A.; Lezza, A.M.S. Deletion of OGG1 Results in a Differential Signature of Oxidized Purine Base Damage in mtDNA Regions. Int. J. Mol. Sci. 2019, 20, 3302. [CrossRef][PubMed] 21. Orlando, A.; Chimienti, G.; Pesce, V.; Fracasso, F.; Lezza, A.M.S.; Russo, F. An In Vitro Study on Mitochondrial Compensatory Response Induced by Gliadin Peptides in Caco-2 Cells. Int. J. Mol. Sci. 2019, 20, 1862. [CrossRef] 22. Ruetenik, A.; Barrientos, A. Dietary restriction, mitochondrial function and aging: From yeast to humans. Biochim. Biophys. Acta 2015, 1847, 1434–1447. [CrossRef] 23. Martin-Montalvo, A.; de Cabo, R. Mitochondrial metabolic reprogramming induced by calorie restriction. Antioxid. Redox Signal. 2013, 19, 310–320. [CrossRef][PubMed] 24. Gutierrez-Casado, E.; Khraiwesh, H.; Lopez-Dominguez, J.A.; Montero-Guisado, J.; Lopez-Lluch, G.; Navas, P.; de Cabo, R.; Ramsey, J.J.; Gonzalez-Reyes, J.A.; Villalba, J.M. The Impact of Aging, Calorie Restriction and Dietary Fat on Autophagy Markers and Mitochondrial Ultrastructure and Dynamics in Mouse Skeletal Muscle. J. Gerontol. Ser. A Biol. Sci. Med. Sci. 2019, 74, 760–769. [CrossRef][PubMed] 25. Matsushima, Y.; Goto, Y.; Kaguni, L.S. Mitochondrial Lon protease regulates mitochondrial DNA copy number and transcription by selective degradation of mitochondrial transcription factor A (TFAM). Proc. Natl. Acad. Sci. USA 2010, 107, 18410–18415. [CrossRef][PubMed] 26. Lu, B.; Lee, J.; Nie, X.; Li, M.; Morozov, Y.I.; Venkatesh, S.; Bogenhagen, D.F.; Temiakov, D.; Suzuki, C.K. Phosphorylation of human TFAM in mitochondria impairs DNA binding and promotes degradation by the AAA+ Lon protease. Mol. Cell 2013, 49, 121–132. [CrossRef][PubMed] 27. Bota, D.A.; Davies, K.J. Mitochondrial Lon protease in human disease and aging: Including an etiologic classiﬁcation of Lon-related diseases and disorders. Free Radic. Biol. Med. 2016, 100, 188–198. [CrossRef] 28. Ngo, J.K.; Pomatto, L.C.; Davies, K.J. Upregulation of the mitochondrial Lon Protease allows adaptation to acute oxidative stress but dysregulation is associated with chronic stress, disease, and aging. Redox Biol. 2013, 1, 258–264. [CrossRef] 29. Bakala, H.; Delaval, E.; Hamelin, M.; Bismuth, J.; Borot-Laloi, C.; Corman, B.; Friguet, B. Changes in rat liver mitochondria with aging. Lon protease-like reactivity and N(epsilon)-carboxymethyllysine accumulation in the matrix. Eur. J. Biochem. 2003, 270, 2295–2302. [CrossRef] 30. Hebert, A.S.; Dittenhafer-Reed, K.E.; Yu, W.; Bailey, D.J.; Selen, E.S.; Boersma, M.D.; Carson, J.J.; Tonelli, M.; Balloon, A.J.; Higbee, A.J.; et al. Calorie restriction and SIRT3 trigger global reprogramming of the mitochondrial protein acetylome. Mol. Cell 2013, 49, 186–199. [CrossRef] 31. Lopez-Dominguez, J.A.; Khraiwesh, H.; Gonzalez-Reyes, J.A.; Lopez-Lluch, G.; Navas, P.; Ramsey, J.J.; de Cabo, R.; Buron, M.I.; Villalba, J.M. Dietary fat and aging modulate apoptotic signaling in liver of calorie-restricted mice. J. Gerontol. Ser. A Biol. Sci. Med. Sci. 2015, 70, 399–409. [CrossRef] 32. Tann, A.W.; Boldogh, I.; Meiss, G.; Qian, W.; Van Houten, B.; Mitra, S.; Szczesny, B. Apoptosis induced by persistent single- strand breaks in mitochondrial genome: Critical role of EXOG (5’-EXO/endonuclease) in their repair. J. Biol. Chem. 2011, 286, 31975–31983. [CrossRef][PubMed] 33. Green, D.R.; Llambi, F. Cell Death Signaling. Cold Spring Harb. Perspect. Biol. 2015, 7.[CrossRef] 34. Souza-Pinto, N.C.; Croteau, D.L.; Hudson, E.K.; Hansford, R.G.; Bohr, V.A. Age-associated increase in 8-oxo-deoxyguanosine glycosylase/AP lyase activity in rat mitochondria. Nucleic Acids Res. 1999, 27, 1935–1942. [CrossRef] 35. De Souza-Pinto, N.C.; Hogue, B.A.; Bohr, V.A. DNA repair and aging in mouse liver: 8-oxodG glycosylase activity increase in mitochondrial but not in nuclear extracts. Free Radic. Biol. Med. 2001, 30, 916–923. [CrossRef] 36. Szczesny, B.; Tann, A.W.; Mitra, S. Age- and tissue-speciﬁc changes in mitochondrial and nuclear DNA base excision repair activity in mice: Susceptibility of skeletal muscles to oxidative injury. Mech. Aging Dev. 2010, 131, 330–337. [CrossRef] 37. Gadaleta, M.N.; Rainaldi, G.; Lezza, A.M.; Milella, F.; Fracasso, F.; Cantatore, P. Mitochondrial DNA copy number and mitochondrial DNA deletion in adult and senescent rats. Mutat. Res. 1992, 275, 181–193. [CrossRef] 38. Yowe, D.L.; Ames, B.N. Quantitation of age-related mitochondrial DNA deletions in rat tissues shows that their pattern of accumulation differs from that of humans. Gene 1998, 209, 23–30. [CrossRef] 39. Picca, A.; Fracasso, F.; Pesce, V.; Cantatore, P.; Joseph, A.M.; Leeuwenburgh, C.; Gadaleta, M.N.; Lezza, A.M. Age- and calorie restriction-related changes in rat brain mitochondrial DNA and TFAM binding. Age 2013, 35, 1607–1620. [CrossRef][PubMed] 40. Barazzoni, R.; Short, K.R.; Nair, K.S. Effects of aging on mitochondrial DNA copy number and cytochrome c oxidase gene expression in rat skeletal muscle, liver, and heart. J. Biol. Chem. 2000, 275, 3343–3347. [CrossRef] Int. J. Mol. Sci. 2021, 22, 1665 18 of 18

41. Pesce, V.; Cormio, A.; Fracasso, F.; Lezza, A.M.; Cantatore, P.; Gadaleta, M.N. Age-related changes of mitochondrial DNA content and mitochondrial genotypic and phenotypic alterations in rat hind-limb skeletal muscles. J. Gerontol. Ser. A Biol. Sci. Med. Sci. 2005, 60, 715–723. [CrossRef] 42. McInerny, S.C.; Brown, A.L.; Smith, D.W. Region-speciﬁc changes in mitochondrial D-loop in aged rat CNS. Mech. Aging Dev. 2009, 130, 343–349. [CrossRef] 43. Gaziev, A.I.; Abdullaev, S.; Podlutsky, A. Mitochondrial function and mitochondrial DNA maintenance with advancing age. Biogerontology 2014, 15, 417–438. [CrossRef] 44. Mai, S.; Klinkenberg, M.; Auburger, G.; Bereiter-Hahn, J.; Jendrach, M. Decreased expression of Drp1 and Fis1 mediates mitochondrial elongation in senescent cells and enhances resistance to oxidative stress through PINK1. J. Cell Sci. 2010, 123, 917–926. [CrossRef] 45. Leduc-Gaudet, J.P.; Picard, M.; St-Jean Pelletier, F.; Sgarioto, N.; Auger, M.J.; Vallee, J.; Robitaille, R.; St-Pierre, D.H.; Gouspillou, G. Mitochondrial morphology is altered in atrophied skeletal muscle of aged mice. Oncotarget 2015, 6, 17923–17937. [CrossRef] [PubMed] 46. Scheibye-Knudsen, M.; Fang, E.F.; Croteau, D.L.; Wilson, D.M., 3rd; Bohr, V.A. Protecting the mitochondrial powerhouse. Trends Cell Biol 2015, 25, 158–170. [CrossRef][PubMed] 47. Wai, T.; Langer, T. Mitochondrial Dynamics and Metabolic Regulation. Trends Endocrinol. Metab. 2016, 27, 105–117. [CrossRef] 48. Piantadosi, C.A.; Suliman, H.B. Redox regulation of mitochondrial biogenesis. Free Radic. Biol. Med. 2012, 53, 2043–2053. [CrossRef][PubMed] 49. Shinmura, K. Effects of caloric restriction on cardiac oxidative stress and mitochondrial bioenergetics: Potential role of cardiac sirtuins. Oxid. Med. Cell. Longev. 2013, 2013, 528935. [CrossRef] 50. Finley, L.W.; Haigis, M.C. The coordination of nuclear and mitochondrial communication during aging and calorie restriction. Aging Res. Rev. 2009, 8, 173–188. [CrossRef] 51. Ferrucci, L.; Gonzalez-Freire, M.; Fabbri, E.; Simonsick, E.; Tanaka, T.; Moore, Z.; Salimi, S.; Sierra, F.; de Cabo, R. Measuring biological aging in humans: A quest. Aging Cell 2020, 19, e13080. [CrossRef][PubMed] 52. Ingram, D.K.; de Cabo, R. Calorie restriction in rodents: Caveats to consider. Aging Res. Rev. 2017, 39, 15–28. [CrossRef][PubMed] 53. Pastukh, V.M.; Gorodnya, O.M.; Gillespie, M.N.; Ruchko, M.V. Regulation of mitochondrial genome replication by hypoxia: The role of DNA oxidation in D-loop region. Free Radic. Biol. Med. 2016, 96, 78–88. [CrossRef][PubMed]