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Poon 20et 20al 202006 20Neurobiology 20of 20Aging 2027 7 201020 1034

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Poon 20et 20al 202006 20Neurobiology 20of 20Aging 2027 7 201020 1034 Powered By Docstoc
					                                                     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: dabcns@uky.edu (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
[45]. 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 [70]. It was proposed that there is
                                                                                 2.1. Subjects
a correlation between mitochondrial oxidant production and
longevity in mammalian species [70]. 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 [55]).                            (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 [103]. 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 [37], 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 [91]. 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 [76], causing diminished mitochondrial bioenergetics                      intake to about 60% compared to the ‘ad libitum’ fed controls
diminishment in aged brains [11]. Such increased oxida-                          of the same age [43]. 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 [65].                                                        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 [85]. 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 [84]. 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 [84]. 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 [84]. 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 [22]. 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 [26].
                                                                                   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 [51], 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 [20], 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 [14]
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 [92]. 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 [14]
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 [75]. 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 [41]. 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) [46], 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
[12]. 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 [40], 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 [7], 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 [8]. 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 [81].
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 [53].
    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 [101]. Inhi-             brains.
bition of eIF-5A induces apoptosis [102], 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 [50]. 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 [14] 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 [105].            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 [106]. Moreover, inhibition of IMPCH can retard
decreased level of UNP1 could implicate less inactive forms               cell growth [100], 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           [67], 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
rats [72,73], suggesting abnormal ATP F1 regulation in aged            References
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