Neurobiology of Aging 27 (2006) 1020–1034 Proteomics analysis provides insight into caloric restriction mediated oxidation and expression of brain proteins associated with age-related impaired cellular processes: Mitochondrial dysfunction, glutamate dysregulation and impaired protein synthesis H. Fai Poon a , Holly M. Shepherd a , Tanea T. Reed a , Vittorio Calabrese b , Anna-Maria Giuffrida Stella b , Giovanni Pennisi c , Jian Cai d , William M. Pierce d , Jon B. Klein d,e , D. Allan Butterﬁeld a,f,g,∗ aDepartment of Chemistry, University of Kentucky, Center of Membrane Sciences, Sanders-Brown Center on Aging, 255 Bowman Hall, Lexington, KY 40506-0055, USA b Department of Chemistry, Section of Biochemistry and Molecular Biology, University of Catania, Catania, Italy c Department of Neurological Sciences, University of Catania, Catania, Italy d Department of Pharmacology, University of Louisville School of Medicine and VAMC, Louisville, Kentucky, USA Mass Spectrometry Facility, University of Kentucky, Lexington KY 40506-0050, USA e Kidney Disease Program and Core Proteomics Laboratory, University of Louisville, School of Medicine and VAMC, Louisville, Kentucky, USA f Center of Membrane Sciences, University of Kentucky, Lexington, KY, USA g Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY, USA Received 22 December 2004; received in revised form 4 May 2005; accepted 19 May 2005 Available online 5 July 2005 Abstract Age-related impairment of functionality of the central nervous system (CNS) is associated with increased susceptibility to develop many neurodegenerative diseases. Increased oxidative stress in the CNS of aged animals is manifested by increased protein oxidation, which is believed to contribute to the age-related learning and memory deﬁcits. Glutamate dysregulation, mitochondrial dysfunction and impaired protein synthesis are observed in aged brains, along with increased protein oxidation. Interestingly, all of these age-related cellular alterations can be improved by caloric restriction (CR), which can also improve the plasticity and recovery of the CNS. Although the beneﬁcial effects of CR on brains are well established, the mechanism(s) of its action remains unclear. In order to gain insight into the mechanism of CR in the brain, we located the brain regions that are beneﬁted the most from reduced oxidative stress by CR. Along with other brain regions, striatum (ST) showed signiﬁcantly decreased bulk protein carbonyl levels and hippocampus (HP) showed decreased bulk protein 3-nitrotyrosine (3-NT) levels in CR aged rats when compared to those of age matched controls. To determine which proteins were oxidatively modiﬁed in these brain regions, we used parallel proteomics approach to identify the proteins that are altered in oxidation and expression. The speciﬁc carbonyl levels of pyruvate kinase M2 (PKM2), -enolase (ENO1), inositol monophosphatase (INSP1), and F1-ATPase Chain B (ATP-F1B) were signiﬁcantly decreased in ST of aged CR rats. In contrast, the expression levels of phosphoglycerate kinase 1 (PKG1), inosine monophosphate cyclohydrolase (IMPCH) and F1-ATPase Chain A (ATP-F1A) were signiﬁcantly increased in the ST of CR rats. In the hippocampus of CR rats, the speciﬁc 3-NT levels of malate dehydrogenase (MDH), phosphoglycerate kinase 1 (PKG1) and 14-3-3 zeta protein were signiﬁcantly decreased and expression levels of DLP1 splice variant 1 (DLP1), mitochondrial aconitase (ACO2), dihydrolipoamide dehydrogenase (DLDH), neuroprotective peptide H3 (NPH3), and eukaryotic translation initiation factor 5A (eIF-5A) are increased. Moreover, an unnamed protein product (UNP1) with similar sequence to initiation factor 2 (IF-2) was decreased in the HP of CR rats. Our data support the hypothesis that CR induces a mild metabolic stress response by increasing the production of neurotrophic proteins, therefore, priming neurons against apoptosis. Moreover, our study shows that the improvement of glutamate dysregulation, mitochondrial dysfunction and protein synthesis by CR is, at ∗ Corresponding author. Tel.: +1 859 257 3184; fax: +1 859 257 5876. E-mail address: email@example.com (D.A. Butterﬁeld). 0197-4580/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2005.05.014 H.F. Poon et al. / Neurobiology of Aging 27 (2006) 1020–1034 1021 least partially, due to the CR-mediated alteration of the oxidation or the expression of PKM2, ENO1, INSP1, ATP-F1B, PKG1, IMPCH, ATP-F1A MDH, PKG1 and 14-3-3 zeta protein, DLP1, ACO2, DLDH, NPH3, eIF-5A and UNP1. This study provides valuable insights into the mechanisms of the beneﬁcial factors on brain aging by CR. © 2005 Elsevier Inc. All rights reserved. Keywords: Proteomics; Brain aging; Oxidative stress; Caloric restriction; Cellular dysfunction 1. Introduction metabolic stress response by an increased production of neu- rotrophic proteins, therefore, priming neurons against apop- Age-related impairment of functionality of the central tosis [55,66]. In order to gain insight into the beneﬁcial effects nervous system (CNS) is associated with increased suscepti- of CR on oxidative stress, we located the brain regions that bility to the development of many neurodegenerative diseases are beneﬁted the most from reduced oxidation. Then we used such as Alzheimer’s disease (AD), Parkinson’s disease (PD), non-biased based parallel proteomics approaches to identify and amyotrophic lateral sclerosis (ALS). The free radical the proteins that are altered in oxidation and expression in theory of aging postulates that free radical reactions with these brain regions of CR aged rats when compared to those biomolecules, such as proteins and lipid membranes, are in age control rats. responsible for the functional deterioration related to aging . This theory was later reﬁned to include the concept that mitochondria play a key role in aging acting as the major 2. Materials and methods source and target of oxidants . It was proposed that there is 2.1. Subjects a correlation between mitochondrial oxidant production and longevity in mammalian species . Interestingly, caloric All animal protocols were approved by the University restriction (CR) can increase life span and resistance to vari- of Catania Laboratory Animal Care Advisory Committee ous age-related disorder in rodents (Review in ). (Prot. Number 8488). Male Wistar rats purchased from Har- In the CNS, evidence shows increased oxidative stress lan (Udine, Italy) were maintained in a temperature and in aged animals when compared to that in young animals humidity-controlled room with a 12-h light:dark cycle and [37,82,83]. Mitochondrial dysfunction plays a signiﬁcant divided in two experimental groups. Control rats were fed role in this increased oxidative damage. Injured mitochon- ad libitum up to 28 months of age a certiﬁed diet prepared drial enzymes in aged animal lead to increased oxidative according to the recommendations of the AIN (In 100 g of ad stress . Mitochondria in aged animals are enlarged libitum, 53 g Dextrin–Maltose, 25 g oil mixture, 22 g casein, with vacuolization, cristae rupture and the accumulation of with additional 0.5 g methionine, 3.5 g salt mixture and 1.2 g paracrystalline inclusions , consistent with the notion vitamin mixture). The CR group, received at 12 months of that mitochondria are targets of increased oxidative stress age up to 28 months the same diet except for a lower caloric in aged brains . Also, mitochondrial decay is a main con- intake. CR regimen was accomplished starting at month 12 tributor of accelerated aging [6,9]. Age-related increase in and continued until sacriﬁce, according to the “alternate day mtDNA deletion (common deletion) was found in elderly feeding” method, which is equivalent to a reduction of caloric brains , causing diminished mitochondrial bioenergetics intake to about 60% compared to the ‘ad libitum’ fed controls diminishment in aged brains . Such increased oxida- of the same age . In brief: the food was available all the tive stress and mitochondrial dysfunction cause oxidation of time for the ad libitum fed controls, while it was given every proteins, thereby leading to their dysfunction in most cases two days and remained available for 24 h. Both groups were [1,2,4,17,20,97]. However, oxidation of proteins during aging fasted for one day before being scariﬁed. Caloric restricted and age-related neurodegenerative disorders is rather speciﬁc old rats weighed signiﬁcantly less (−57%) compared to old [1,2,18,22–24,52,84,98,99]. It is well established that CR rats fed a normal caloric diet (p < 0.001). can reduce age-related oxidative stress [55,59,77,79,96] and protein oxidation in brains [31,35,44]. Along with reduced 2.2. Sample preparation and methods employed protein oxidation, CR is also believed to contribute to the improvement of age-related learning and memory deﬁcit The brain samples were homogenized in a lysis buffer [48,71], as well as the improvement of the plasticity and (10 mM HEPES, 137 mM NaCl, 4.6 mM KCl, 1.1 mM recovery of the CNS . KH2 PO4 , 0.6 mM MgSO4 ) and containing protease inhibitor Although the beneﬁcial effects of CR on brains are leupeptin (0.5 mg/mL), pepstatin (0.7 g/mL), trypsin well established, the mechanism(s) of its action remains inhibitor (0.5 g/mL), and PMSF (40 g/mL). Homogenates unclear. Two general mechanisms were proposed: (1) CR were centrifuged at 15,800 × g for 10 min to remove debris. reduces mitochondrial metabolism, thus decreases the level The supernatant was extracted to determine the concentration of mitochondrial radical production or (2) CR induces mild by the BCA method (Pierce, IL). 1022 H.F. Poon et al. / Neurobiology of Aging 27 (2006) 1020–1034 2.3. Immunochemistry (or oxyblots) was compared between groups using statistical analysis. Levels of 3-nitrotyrosine (3-NT), 4-hydroxynonenal (HNE) and protein carbonyls were determined immunochem- 2.7. Trypsin digestion ically as previously described . Protein carbonyl levels were determined as adducts of 2,4-dinitrophenylhydrazine Samples were digested using the techniques previ- (DNPH) [3,98]. The 2,4-dinitrophenyl hydrazone (DNP) ously described . Brieﬂy, the selected protein spots adduct of the carbonyls is detected on nitrocellulose paper were excised and washed with ammonium bicarbonate using a primary rabbit antibody (Intergen) speciﬁc for DNP- (NH4 HCO3 ), then acetonitrile at room temperature. The protein adducts (1:100). HNE and 3-NT levels were deter- gel pieces were digested with 20 ng/ L modiﬁed trypsin mined in the same manner. The HNE levels were detected (Promega, Madison, WI) and incubated at 37 ◦ C overnight using a primary rabbit antibody (Alpha Diagnostics) spe- in a shaking incubator. ciﬁc for HNE-modiﬁed protein (1:8000) and the 3-NT lev- els were detected by primary rabbit antibody (Chemicon) 2.8. Mass spectrometry speciﬁc for 3-NT (1:100). The same secondary goat anti- rabbit IgG (Sigma) antibody was used in all applications. The Digests (1 L) were mixed with 1 L -cyano-4-hydroxy- resultant stain was developed by application of Sigma-Fast trans-cinnamic acid (10 mg/mL in 0.1% TFA:ACN, 1:1, v/v). (BCIP/NBT) tablets; and the line densities were quantiﬁed The mixture (1 L) was deposited onto a fast evaporation by Scion-Image software package. nitrocellulose matrix surface and analyzed with a TofSpec 2E (Micromass, UK) MALDI-TOF mass spectrometer in 2.4. Two-dimensional gel electrophoresis reﬂectron mode. The mass axis was adjusted with trypsin autohydrolysis peaks (m/z 2239.14, 2211.10, or 842.51) as Samples of the proteins in the whole brains were prepared lock masses. The MALDI spectra used for protein identi- as previously described . Brieﬂy, 200 g of protein were ﬁcation from tryptic fragments were searched against the applied to a pH 3–10 ReadyStripTM IPG strip (Bio-Rad, NCBI protein databases using the MASCOT search engine Hercules, CA) for isoelectric focusing and Linear Gradi- (http://www.matrixscience.com). Peptide mass ﬁngerprint- ent (8–16%) Precast criterion Tris–HCl gels (Bio-Rad) were ing used the assumption that peptides are monoisotopic, used to separate proteins according to their molecular weight oxidized at methionine residues and carbamidomethylated (MrW). Sypro Ruby stain was used to stain the gel for 2 h, at cysteine residues [19,22,24,25]. Up to 1 missed trypsin following which the gels were placed in deionized water cleavage was allowed. Mass tolerance of 150 ppm was the overnight for destaining. window of error allowed for matching the peptide mass val- ues. Such search results in a probability-based Mowse score 2.5. Western blotting for each spectrum to indicate the probability that the match between the database and the spectra is a random event. Such Western blotting for 2D gels was performed as previously a probability is 10(-Mowse score/10) . described . 200 g of the brain protein were incubated with 10 mM 2,4-dinitrophenyl hydrazine (DNPH) solution 2.9. Statistics (2N HCl) at room temperature (25 ◦ C) for 20 min. No derivi- tization step is preformed for the measurement of the protein The averages of speciﬁc carbonyl level, speciﬁc 3-NT 3-NT levels. The gels were prepared in the same manner as level and protein level obtained from individual CR rats for 2D-electrophoresis. The proteins from the second dimen- were compared with the averages of age-matched control sion electrophoresis gels were transferred onto nitrocellulose by Student’s t-tests. A value of p < 0.05 was considered sta- paper (Bio-Rad) using a Transblot-Blot® SD semi-Dry Trans- tistically signiﬁcant. Only the proteins in CR aged brains fer Cell (Bio-Rad) at 15 V for 2 h. The 2,4-dinitrophenyl that were signiﬁcantly different from age matched control hydrazone (DNP) adducts of the carbonyls and the 3-NT of brains assessed by the Student’s t-test were selected for iden- the brain proteins were detected immunochemically. tiﬁcation. Similar statistical analysis are usually used for proteomics data analysis [22,54,68]. Sophisticated statistical 2.6. Image analysis analysises for microarray data are not compatible for pro- teomics data [13,68]. The gels and nitrocellulose blots were scanned and saved in TIF format using a Storm 860 Scanner (Molecular Dynam- ics) and Scanjet 3300C (Hewlett Packard), respectively. 3. Results PDQuest (Bio-Rad) was the software used for matching and analysis of visualized protein spots among differential gels In order to locate the brain regions that are mostly affected and oxyblots. After completion of spot matching, the nor- by protein oxidation by CR, we measured the bulk protein 3- malized intensity of each protein spot from individual gels NT, HNE and carbonyl levels of cortex (CX), substantia nigra H.F. Poon et al. / Neurobiology of Aging 27 (2006) 1020–1034 1023 Fig. 2. Results represent the average protein-bound HNE levels in cortex Fig. 1. Results represent the average protein carbonyl levels in cortex (CX), (CX), substantia nigra (SN), septum pellucidum (SP), Striatum (ST), hip- substantia nigra (SN), septum pellucidum (SP), striatum (ST), hippocampus pocampus (HP) and cerebellum (CB) of caloric restricted aged rats (CR), (HP) and cerebellum (CB) of caloric restricted aged rats (CR), as well as the as well as the age matched controls. Error bars indicate the S.E.M. for six age-matched controls. Error bars indicate the S.E.M. for six animals in each animals in each group. Measured values are normalized to the age matched group. Measured values are normalized to the age matched control values. control values. * p < 0.05. * p < 0.05. (SN), septum pellucidum (SP), striatum (ST), hippocam- pus (HP) and cerebellum (CB). We found that CR gener- ally reduces protein oxidation in all aged rat brain regions. However, only certain brain regions of the aged rats show statistically signiﬁcant decrease in oxidative modiﬁcation by CR. Aged rats with CR show signiﬁcant decreases in pro- tein carbonyl levels in SN (32%) and ST (19%) compared to those of age matched controls (Fig. 1). With regard to protein- bound HNE levels, signiﬁcant decreased values are observed in CX (13%) and HP (12%) of aged rats with CR (Fig. 2). We also show a signiﬁcant reduction of protein 3-NT levels in SN (26%) and HP (38%) of aged rats with CR (Fig. 3). In the HP of CR rats, the speciﬁc 3-NT levels of three Fig. 3. Results represent the average protein 3-NT levels in cortex (CX), substantia nigra (SN), septum pellucidum (SP), striatum (ST), hippocampus proteins, malate dehydrogenase (MDH), phosphoglycerate (HP) and cerebellum (CB) of caloric restricted aged rats (CR), as well as the kinase 1 (PKG1) and 14-3-3 zeta protein (14-3-3Z), are sig- age matched controls. Error bars indicate the S.E.M. for six animals in each niﬁcantly decreased (Table 1). Fig. 4 shows these proteins group. Measured values are normalized to the age matched control values. * p < 0.05. on the representative 2D Western blots of the HP for CR rat and age matched control. Moreover, expression levels of four proteins were signiﬁcantly decreased in the HP of CR aged rats. These proteins are DLP1 splice variant 1 (DLP1), are yet to be discovered. Fig. 5 shows these proteins on the mitochondrial aconitase (ACO2), dihydrolipoamide dehy- representative 2D electrophoresis gels of the HP of CR rat drogenase (DLDH), neuroprotective peptide H3 (NPH3), and and aged match control. eukaryotic translation initiation factor 5A (eIF-5A). Also, In the ST of CR rats, the speciﬁc carbonyl levels of six the expression level of an unnamed protein product (UNP1) proteins were decreased. These proteins are pyruvate kinase was decreased (Table 2). This protein is indicated in the rat M2 (PKM2), -enolase (ENO1), inositol monophosphatase genome and shows a similar sequence to elongation factor (INSP1), and F1-Atpase Chain B (ATP-F1B) (Table 3). Fig. 6 2 (EF-2). However, the function and additional information indicates these proteins on the representative 2D Western Table 1 Proteins with decreased 3-NT levels in HP of CR aged rats Protein Control (n = 5) AU ± S.E.M.a CR (n = 5) AU ± S.E.M.a Fold change p-value Malate dehydrogenase (MDH) 0.418 ± 0.127 0.099 ± 0.046 4.2↓ <0.05 Phosphoglycerate kinase 1 (PKG1) 0.392 ± 0.056 0.081 ± 0.013 4.8↓ <0.001 14-3-3 Zeta protein 0.028 ± 0.010 0.010 ± 0.002 2.8↓ <0.0005 a AU: arbitrary unit obtained from the ratio of speciﬁc 3-NT level and protein level; S.E.M.: standard error of mean. 1024 H.F. Poon et al. / Neurobiology of Aging 27 (2006) 1020–1034 Fig. 4. Representative Western blots showing 3-NT modiﬁed proteins from the hippocampus (HP) of CR aged rats. Left hand blots show 3-NT Western blots of HP from the aged-matched control rats (top) and CR aged rats (bottom). Circled proteins show signiﬁcant changes in 3-NT modiﬁcation in the HP of CR aged rats. Right hand blots show expansions of the blot outlined in the box. 3-NT Western-blot from the age matched control HP is shown in the upper panel and CR aged HP in the lower panel. Table 2 Altered protein levels in HP of CR aged rats Protein Control (n = 6) CR (n = 6) Fold change p-value AU ± S.E.M.a AU ± S.E.M.a DLP1 splice variant 1 (DLP1) 466 ± 53 257 ± 48 1.8↓ <0.05 Mitochondrial aconitase (ACO2) 1839 ± 277 720 ± 247 2.6↓ <0.05 Dihydrolipoamide dehydrogenase (DLDH) 2395 ± 195 1240 ± 168 1.9↓ <0.005 Neuroprotective peptide H3 (NPH3) 2143 ± 221 994 ± 207 2.2↓ <0.01 Similar to Eukaryotic translation initiation factor 5A (eIF-5A) 5251 ± 615 2477 ± 648 2.1↓ <0.05 Unnamed protein product 1 (UNP 1) 638 ± 103 951 ± 37 1.5↑ <0.05 a AU: arbitrary unit obtained from indicating protein level; S.E.M.: standard error of mean. blots of the ST of CR rat and aged match control. More- The identiﬁcations of the proteins were performed by over, expression levels of phosphoglycerate kinase 1 (PKG1), matching the obtained mass spectrum to the spectrum in the inosine monophosphate cyclohydrolase (IMPCH) and F1- NCBI database. The Mowse score indicates the probability Atpase Chain A (ATP-F1A) were signiﬁcantly increased in that the match is a random event; high Mowse score indi- the ST of CR rats (Table 4). Fig. 7 indicates these proteins on cates the match is unlikely to be a random event. Prior results the representative 2D electrophoresis gels of the ST of CR suggest that the accuracy of protein identiﬁcation by mass rat and aged match control. spectrometry is equivalent to immunochemical identiﬁcation Table 3 Proteins with decreased speciﬁc carbonyl levels in ST of CR aged rats Protein Control (n = 6) AU ± S.E.M.a CR (n = 5) AU ± S.E.M.a Fold change p-value Pyruvate kinase M2 (PKM2) 0.454 ± 0.217 0.016 ± 0.010 28↓ 0.05 -enolase (ENO1) 0.489 ± 0.242 0.003 ± 0.001 163↓ 0.05 Inositol monophosphatase (INSP1) 1.560 ± 0.413 0.415 ± 0.133 4↓ <0.05 F1-ATPase Chain B (ATP-F1B) 2.10 ± 0.98 0.016 ± 0.007 131↓ <0.05 a AU: arbitrary unit obtained from the ratio of speciﬁc carbonyl level and protein level; S.E.M.: standard error of mean. H.F. Poon et al. / Neurobiology of Aging 27 (2006) 1020–1034 1025 Fig. 5. Representative 2D gel electrophoresis pattern of proteins from hippocampus (HP) of age matched control (top panel) and CR aged rats (bottom panel). The proteins in HP after 2D electrophoresis from the age matched control rats (top) described in this study are indicated. Panels beneath the control 2D gel show expanded regions of the 2D gel from the HP of the control rats (outlined in the box). The proteins in HP after 2D electrophoresis from the CR aged rats (bottom) described in this study are indicated. Panels beneath the CR 2D gel show expanded regions of the 2D gel from the HP of the rats (outlined in the box). Table 4 Altered proteins levels in ST of CR aged rats Protein Control (n = 6) AU ± S.E.M.a CR (n = 5) AU ± S.E.M.a Fold change p-value Phosphoglycerate kinase 1 (PGK1) 1433 ± 303 421 ± 33 3.4↓ <0.05 F1-ATPase Chain A (ATP-F1A) 1243 ± 141 556 ± 68 2.2↓ <0.005 Inosine monophosphate cyclohydrolase (IMPCH) 549 ± 191 10 ± 1 54.9↓ <0.05 a AU: arbitrary units indicating protein level; S.E.M.: standard error of mean. 1026 H.F. Poon et al. / Neurobiology of Aging 27 (2006) 1020–1034 Fig. 6. Representative 2D Western blots from striatum (ST) of age matched control (top panel) and CR aged rats (bottom panel). Circled proteins show signiﬁcant reduction in oxidative modiﬁcation in the ST of CR aged rats. Panels beneath the control 2D Western blot show expanded of regions of the 2D gel from the ST of the control rats (outlined in the box). The proteins in HP after 2D Western blot from the CR aged rats (bottom) described in this study are indicated. Panels beneath the CR 2D Western blot show expanded regions of the 2D Western blots from the ST of the CR aged rats (outlined in the box). and immunochemical identiﬁcation conﬁrmed the identiﬁca- of the age matched control. It is possible that reduction of tion . The identiﬁcation of the proteins is summarized in oxidative stress parameters could be in part due to effects Table 5. of metal ions, particularly iron ion, since elevated non-heme iron in the aged brain is attenuated by CR . Among the brain regions, HP shows a dramatic decrease in 4. Discussion protein carbonyl, HNE and 3-NT levels in CR aged rats brains even though the decrease of the protein carbonyl level is not Consistent with other laboratories [35,38,86], we show statistically signiﬁcant. The vulnerability to age-related pro- that oxidative modiﬁcation of proteins (indicated by the pro- tein oxidation and structural changes of HP in brain are well tein carbonyl, 3-NT, and HNE levels) is generally decreased established [4,35,69]. Also, CR imposes a signiﬁcant reduc- in all brain regions of CR aged rats when compared to those tion of protein oxidation in HP of rats [4,35]. Since HP in the H.F. Poon et al. / Neurobiology of Aging 27 (2006) 1020–1034 1027 Fig. 7. Representative 2D gel electrophoresis pattern of proteins from striatum (ST) of agematched control (top panel) and CR aged rats (bottom panel). The proteins in ST after 2D electrophoresis from the age matched control rats (top) described in this study are indicated. Panels beneath the control 2D gel show expanded of regions of the 2D gel from the ST of the control rats (outlined in the box). The proteins in ST after 2D electrophoresis from the CR aged rats (bottom) described in this study are indicated. Panels beneath the CR 2D gel show expanded regions of the 2D gel from the HP of the rats (outlined in the box). CR aged rats shows the most signiﬁcant decrease (38%) of is not only determined by ROS levels but is also affected by the protein 3-NT level and is relevant to Alzheimer’s disease NOS activity. Thus, 3-NT is not simply reﬂective of ROS, but (AD) pathology , we used parallel proteomic analysis to also reﬂects RNS. Moreover, we used the same technique to speciﬁcally identify the proteins that were decreased in spe- identify the proteins with reduced speciﬁc carbonyl levels or ciﬁc 3-NT levels as well as those with the altered expression. altered expression levels in ST of CR aged brains, because ST Although tyrosine nitrosylation is a formal oxidation of the is another brain region that is vulnerable to protein oxidation protein , it should be noted that nitrosylation of proteins and lipid peroxidation [34,109], and relevant to age-related 1028 H.F. Poon et al. / Neurobiology of Aging 27 (2006) 1020–1034 Table 5 Summary of proteins identiﬁed by mass spectrometry Identiﬁed protein gi accession # # Peptides % Coverage of pI, MrW Mowse Probability of a random matched matched peptides (kD) Score identiﬁcation hit malate dehydrogenase (MDH) gi|42476181 8 36 8.93, 36.1 94 3.98 × 10−10 phosphoglycerate kinase 1 (PGK1) (HP) gi|40254752 19 58 8.02, 44.9 160 1.00 × 10−16 14-3-3 zeta protein gi|13487931 16 64 4.73, 28.0 123 5.01 × 10−13 DLP1 splice variant 1 (DLP1) gi|2435480 4 42 5.64, 13.3 64 3.98 × 10−07 mitochondrial aconitase (ACO2) gi|40538860 19 27 7.87, 86.1 224 3.98 × 10−23 Dihydrolipoamide dehydrogenase (DLDH) gi|40786469 13 32 7.96, 54.6 75 3.16 × 10−08 Neuroprotective peptide H3 (NPH3) gi|8393910 10 74 5.48, 20.9 97 2.00 × 10−10 similar to Eukaryotic translation initiation gi|27672956 4 47 5.08, 17.0 62 6.31 × 10−07 factor 5A (eIF-5A) unnamed protein product 1 (UNP1) gi|56082 6 8 N/A 62 6.31 × 10−07 pyruvate kinase M2 (PKM2) gi|206205 12 28 7.15, 58.3 72 6.31 × 10−08 -enolase (ENO1) gi|22096350 20 59 5.84, 47.5 195 3.16 × 10−20 Inositol monophosphatase (INSP1) gi|14091736 9 36 5.17, 30.8 87 2.00 × 10−09 F1-ATPase Chain B (ATP-F1B) gi|6729935 17 57 4.95, 51.3 196 2.51 × 10−20 phosphoglycerate kinase 1 (PGK1) (ST) gi|40254752 16 48 8.02, 44.9 169 1.26 × 10−17 F1-ATPase Chain A (ATP-F1A) gi|6729934 16 43 8.28, 55.4 196 2.51 × 10−20 Inosine monophosphate cyclohydrolase gi|2541906 10 24 6.72, 64.7 76 2.51 × 10−08 (IMPCH) neurodegenerative disorders such as PD and Huntington’s increased protein levels of PGK1 as a function of age  disease (HD)[42,61]. maybe a compensatory response to oxidative stress. Our study Although SN of CR rats show signiﬁcant decrease of spe- shows that reduced nitration of MDH in HP of aged rats by ciﬁc carbonyl and 3-NT levels when compared to the aged CR indicates that the CR-induced activity increase is possibly match controls, limited amount of proteins are harvested from due to reduced nitration of MDH and the reduced 3-NT level SN due to the small size of SN. Therefore, parallel proteomic in HP of aged rats, indicating that CR can possibly increase analysis of SN is not possible. Also, proteomic analysis of PGK1 activity in HP of this important region in aged rats. HNE-modiﬁed protein is not performed because the major- Both of these changes result in increasing ATP availability, ity of the HNE-modiﬁed proteins are membrane proteins that synaptic plasticity, learning and memory, antioxidant defense cannot be easily dissolved in solution for 2D-electrophoresis. and neuronal recovery as well as preventing neuronal cells from glutamate toxicity. 4.1. HP ACO2 and DLDH (E3 component of pyruvate dehydroge- nase complex, and -ketoglutarate dehydrogenase) are mito- HP plays an important role in both memory and learning. chondrial matrix enzymes involved in the Kreb’s cycle. Age- Age-related increased oxidative stress in HP of aged animals related activity alteration [28–30,56,60,88,107] and oxidative causes structural alteration [4,35,69]. Moreover, synaptic loss inhibition [57,87,90] of these enzymes are observed. Also and increased protein oxidation in HP is the major pathology stress-mediated increased expression of ACO2 is observed, of AD . Consistent with the notion that CR can reduce suggesting that increased expression of ACO2 is a compen- protein oxidation in HP of aged rats [4,35], we show that the satory response to its oxidative inactivation. In our current 3-NT levels of HP is signiﬁcantly decreased in HP of CR study, we show that CR decreased the level of ACO2 and aged rats when compared to age matched control. Further- DLDH in HP of aged rats, indicating the loss of the com- more, we identiﬁed that the speciﬁc proteins that are reduced pensatory response to oxidative stress is likely due to the in 3-NT levels are MDH, PKG1 and 14-3-3 zeta protein. improvement of normal mitochondrial functions that are Along with the reduced 3-NT level, the expression of DLP1, brought about by CR-mediated oxidative stress release. ACO2, DLDH, NPH3, eIF-5A and UNP1 are also signiﬁ- Increased protein levels of PGK1 as a function of age  cantly altered in HP of CR rats. maybe a compensatory response to oxidative stress. Here, PGK1 catalyzes the conversion of 1,3-diphosphoglycerate we show that CR can reduce the 3-NT level in HP of aged to 3-phosphoglycerate in glycolysis, and MDH is located rats, indicating that CR can possibly increase PGK1 activity within the mitochondrial matrix to connect glycolysis to in HP of this important region in aged rats, thus increasing mitochondrial respiration. Both of these reactions convert ATP availability as well as preventing neuronal cells from ADP to ATP to ensure maximum glutamate accumula- glutamate toxicity. tion into presynaptic vesicles. Moreover, activity of these 14-3-3Z protein is involved in a number of cellular enzymes are decreased during aging [47,75], possibly due functions including signal transduction, protein trafﬁcking to oxidative modiﬁcation . Consistent with this notion, and metabolism and mitochondrial import [5,33]. Oxidative H.F. Poon et al. / Neurobiology of Aging 27 (2006) 1020–1034 1029 insults cause dissociation of 14-3-3 complex and activate JNK we speculate that our study may imply that EF-2 modiﬁcation and p38 pathways, thus initiating apoptosis . Here we is possibly reduced by CR, thus restoring the normal protein show that CR can reduce the oxidative modiﬁcation of 14- synthesis process in aged brains. 3-3Z, thus maintaining normal cell functions and preventing apoptosis in aged brains. 4.2. ST DLP1 is a novel dynamin-like protein expressed in rat tissues. Dynamin is involved in receptor-mediated endocy- The ST is best known for its role in movement, but it is also tosis and intercellular trafﬁcking. Since dynamin interacts involved in a variety of other cognitive processes involving with phosphatidylinositol 3-kinase (PI3K) , it is likely in executive function. ST show increased protein oxidation and signaling and in addition may possibly promote apoptosis. lipid peroxidation as a function of age [34,109]. Moreover, Moreover, altered endocytic function in dendrites in older increased oxidative stress is observed in the ST of PD and neurons is associated with the increased level of dynamin HD patients (review in [10,15,49]). These studies indicate . Taken together, increased level of dynamin or DLP1 that age-related protein oxidation plays critical roles in the alters intercellular trafﬁcking and initiates apoptosis in neu- impaired function of ST. However, CR can modulate these ronal cells. Our current study shows that CR can possibly oxidative stresses in ST [16,35] and improve dopamine func- prevent these alterations by decreasing the protein level of tion in ST of aged animals [21,32]. Consistent with these DLP1, thus protecting cells from apoptosis. studies, we show here that the protein carbonyl level of ST NPH3 is involved in various functions in the CNS, such is reduced. Also we found that the speciﬁc carbonyl levels as binding to phosphatidylethanolamine, enhancing acetyl- of INSP1, PKM2, ENO1, ATP-F1B and are decreased and choline synthesis and inhibiting Raf-kinase. This protein also the expression levels of PKG1, IMPCH, and ATP-F1A were acts as a lipid carrier and binding protein that plays a signif- signiﬁcantly increased. icant role in membrane organization , a process that is INSP1 plays a signiﬁcant role in controlling the intracel- critical to mitochondrial integrity. The mRNA and protein lular inositol level by dephosphorylating inositol monophos- levels of NPH3 are signiﬁcantly increased in aged human phate(s) to produce inositol , which can then be used to HP and senescence-accelerated mice [64,108]. Therefore, up- produce phosphatidyl inositol (PI) to activate the PI signal regulation of NPH3 as a function of age is a likely response transduction pathway . INSP1 activity is decreased in aged to oxidative stress. In our current study, CR reduces oxida- mice brains when compared to the adult mice brains . tive stress; thus, a decreased level of NPH3 in aged CR mice Decreased INSP1 activity can be brought about by modiﬁca- when compared to their age controls are observed. tions of the oxidation-sensitive thiol residues in INSP1 . eIF-5A is a translation initiation factor that is involved in In the current study, we found that oxidation of INSP1 in the initiation of protein synthesis. Since eIF-5A plays a piv- aged rat ST is reduced by CR, thus possibly improving its otal role in regulation of protein synthesis, it is an important function for the PI signal transduction pathway in aged rats determinant of cell proliferation and senescence . Inhi- brains. bition of eIF-5A induces apoptosis , indicating that the PKM2 ENO1, and PGK1 are all glycolytic enzymes eIF-5A activity is critical to normal cell functions. However, that are involved in ATP production. Activity alter- inactivation of eIF-5A by chemical modiﬁcation inhibits cell ation and oxidation of these enzymes were demonstrated growth and diminishes 30% of protein synthesis . This [23,75,80,84,93,94,104]. Here, we showed that decreased study reﬂects that eIF-5A is required for translation of spe- carbonyl level of PKM2 and ENO1 in the CR old rats could ciﬁc mRNAs selectively. Our current study shows that the lead to improved activity, and thus to improved ATP produc- impaired protein synthesis in aged brains [36,91] was possi- tion in CR old rat brain. Moreover, the abnormally increased bly improved by increased eIF-5A protein levels in the HP of level of PKG1 in aged brains  is also decreased in the the CR rats. This change thereby provides sufﬁcient protein ST of CR, suggesting CR can improve cellular metabolism, in neuronal cells for normal cell function in CR aged rats to thereby conserving energy and materials in neurons in brain compensate the impaired protein synthesis in aged brains. aging. UNP1 is identiﬁed as a protein that shares high similar- Inosine monophosphate cyclohydrolase (IMPCH) is a ity to the primary structure of elongation factor 2(EF-2), an bifunctional protein that catalyzes the last two steps of de enzyme that catalyzes the translocation step of peptide elon- novo purine biosynthesis, which is further used for mRNA gation in protein biosynthesis. UNP1 (pI = 8, Mw = 96 kDa) and DNA synthesis. The de novo synthesis of inosine shows a slightly higher molecular weight (Mw) and dra- monophosphate plays a role in the regulation of glutamate matic shift of isoelectric point (pI) when compared to EF-2 levels since glutamine is a substrate of the ﬁrst step of this (pI = 6.65, Mw = 95 kDa). Interestingly, these Mw and pI alter- process. Alteration of inosine monophosphate concentration ations are also observed in oxidatively modiﬁed protein . in aged cardio muscle implies that IMPCH is altered as a func- Therefore, UNP1 is possibly the oxidized form of EF-2 and a tion of age . Moreover, inhibition of IMPCH can retard decreased level of UNP1 could implicate less inactive forms cell growth , suggesting IMPCH plays an important role of EF-2. Since oxidative modiﬁcation of EF-2 contributes to in normal cell functions. In the current study, we show that the impaired protein synthesis in aged rat brains [36,78,91], the level of IMPCH in ST of CR rats is signiﬁcantly lower 1030 H.F. Poon et al. / Neurobiology of Aging 27 (2006) 1020–1034 Fig. 8. Simpliﬁed pathway diagram showing the involvement of proteins in CR (either in oxidative modiﬁcation or expression level) by protein cellular functions. (Insert) Venn diagram showing the involvement of CR-altered proteins in the aged impaired cellular process: glutamate regulation, mitochondrial function and the protein synthesis. Several proteins are involved in more than one of these functions. H.F. Poon et al. / Neurobiology of Aging 27 (2006) 1020–1034 1031 than that of age matched control. Since CR reduces oxidative , which the present study imply maybe due reduction in stress, most of mRNA will not be oxidatively modiﬁed and oxidative damage and changed expression of speciﬁc hip- therefore will be successfully translated. This results in less pocampal and striatal proteins. demand of mRNA for protein synthesis. Therefore, down- regulation of IMPCH might be a result of decreased demand for mRNA nucleotide and improved glutamate control caused Acknowledgements by CR mediated oxidative stress release. 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