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
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.
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
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
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
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.
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
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
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
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
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.
ATP-F1A and ATP-F1B are members of the F1 synthase This work was supported in part by grants from NIH to
(ATP F1) enzymatic complex that binds ADP, phosphate and D.A.B. [AG-05119; AG-10836] and from FIRB to V.C. and
ATP for the synthesis of ATP during oxidative phosphory- A.M.G.S. [RBNE01ZK8F].
lation. Post-transcriptionally increased levels of ATP-F1A
are observed as a function of age in the cerebral cortex of
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