Journal of Neuroscience Research 84:647–654 (2006)
Chronic Systemic D-Galactose Exposure Induces Memory Loss, Neurodegeneration, and Oxidative Damage in Mice: Protective Effects of R-a-Lipoic Acid Xu Cui,1 Pingping Zuo,2 Qing Zhang,2 Xuekun Li,2 Yazhuo Hu,1 Jiangang Long,3 Lester Packer,4 and Jiankang Liu3,5,6* 1
Institute of Gerontology and Geriatrics, Chinese PLA General Hospital, Beijing, People’s Republic of China 2 Insititute of Basic Medical Sciences, Peking Union Medical College, Beijing, People’s Republic of China 3 Institute for Nutritional Science, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, People’s Republic of China 4 University of Southern California, Los Angeles, California 5 Children’s Hospital Oakland Research Institute, Oakland, California 6 Institute for Brain Aging and Dementia, University of California, Irvine, California
Chronic systemic exposure of mice, rats, and Drosophila to D-galactose causes the acceleration of senescence and has been used as an aging model. The underlying mechanism is yet unclear. To investigate the mechanisms of neurodegeneration in this model, we studied cognitive function, hippocampal neuronal apoptosis and neurogenesis, and peripheral oxidative stress biomarkers, and also the protective effects of the antioxidant R-alpha-lipoic acid. Chronic systemic exposure of D-galactose (100 mg/kg, s.c., 7 weeks) to mice induced a spatial memory deficit, an increase in cell karyopyknosis, apoptosis and caspase-3 protein levels in hippocampal neurons, a decrease in the number of new neurons in the subgranular zone in the dentate gyrus, a reduction of migration of neural progenitor cells, and an increase in death of newly formed neurons in granular cell layer. The D-galactose exposure also induced an increase in peripheral oxidative stress, including an increase in malondialdehyde, a decrease in total anti-oxidative capabilities (T-AOC), total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-Px) activities. A concomitant treatment with lipoic acid ameliorated cognitive dysfunction and neurodegeneration in the hippocampus, and also reduced peripheral oxidative damage by decreasing malondialdehyde and increasing T-AOC and T-SOD, without an effect on GSH-Px. These findings suggest that chronic D-galactose exposure induces neurodegeneration by enhancing caspase-mediated apoptosis and inhibiting neurogenesis and neuron migration, as well as increasing oxidative damage. In addition, D-galactose-induced toxicity in mice is a useful model for studying the mechanisms of neurodegeneration and neuroprotective drugs and agents. VC 2006 Wiley-Liss, Inc.
A chronic administration with a low dose of D-galactose (D-gal) induces changes that resemble natural aging in animals, such as a shortened lifespan (Jordens et al., 1999; Cui et al., 2004), cognitive dysfunction (Shen et al., 2002; Xu and Zhao, 2002; Deng et al., 2003; Wei et al., 2005), neurodegeneration (Li et al., 1995; Cui et al., 1997; Cui et al., 2000; Zhang et al., 2005a,b), oxidative stress (Cui et al., 1998; Ho et al., 2003), decreased immune responses (Lei et al., 2003) and advanced glycation endproduct (AGE) formation (Song et al., 1999; Tian et al., 2005) and gene transcriptional changes (Li et al., 1995; Tian et al., 2005). D-gal-induced senescence acceleration has been widely used as a aging model for studying aging mechanisms and screening drugs (Song et al., 1999; Shen et al., 2002; Wei et al., 2005). Different mechanisms, including the hypothesis of increasing oxidant generation and consequent oxidative damage have been proposed (Cui et al., 2004; Zhang et al., 2005a,b). The molecular mechanisms underling D-gal-induced aging and neurodegeneration remain unclear. Aging influences the progression of neurodegeneration. For example, one of the strongest risk factors for the development of Alzheimer’s disease is age. The pos-
Key words: aging; cognitive dysfunction; neurodegeneration; neurogenesis; oxidative damage; R-a-Lipoic acid
Published online 18 May 2006 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.20899
' 2006 Wiley-Liss, Inc.
Contract grant sponsor: Overseas Scholar Grant of Chinese Academy of Sciences; Contract grant sponsor: Shanghai Natural Science Foundation Grant; Contract grant sponsor: HiSun Science and Technology. *Correspondence to: Jiankang Liu, Institute for Brain Aging and Dementia, University of California, 1261 Gillespie Neuroscience Research Facility, Irvine, CA 92697-4540. E-mail:
[email protected] Received 6 January 2006; Revised 8 February 2006; Accepted 15 February 2006
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sible pathways through which aging feeds into and modulates the development of age-associated neurodegenerative pathologies may include increased protein aggregation, decreased protein degradation mediated both by the proteasome and lysosomes, impaired calcium homeostasis, increased oxidative stress, and impaired mitochondrial dysfunction (Troulinaki and Tavernarakis, 2005). Because D-gal-induced senescence is accompanied by neurodegeneration, it would be an ideal model to study the molecular mechanisms involved with aging and ageassociated neurodegeneration. R-a-lipoic acid (a-LA) is a potent biological antioxidant and neuroprotective agent (Packer et al., 1997b, 1995). a-LA scavenges hydroxyl radical (OH), hypochlorous acid (HOCl), singlet oxygen (1O2), and nitric oxide (NO). a-LA chelates a number of transition metals to prevent the generation of hydroxyl radical (Ou et al., 1995). Dihydrolipoid acid (DHLA), the reduced form of a-LA when entering in mitochondria, is another antioxidant, scavenges OH, HOCl, NO, superoxide anion radical (O2), and peroxide hydroxyl (H2O2). In particular, DHLA recycles other antioxidants, such as vitamin C, vitamin E, glutathione, and coenzyme Q, raises the level of intracellular antioxidants (Packer et al., 1995, 1997a; Kozlov et al., 1999). In addition, a-LA ameliorates age-associated cognitive dysfunction in mice and rats (Liu et al., 2002a; Stoll et al., 1993). To elucidate the underlying molecular mechanisms of D-gal-induced neurodegeneration associated with aging, we have chronically exposed D-gal to mice, and examined spatial memory with the Morris water maze, the extent of neuronal apoptosis and neurogenesis in the hippocampus, and also the extent of oxidative stress biomarkers including malondialdehyde (MDA), total antioxidative capabilities (T-AOC), total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-Px) activities in serum. We further hypothesized that treating D-gal exposed mice to a-LA will lead to reduced signs of cognitive dysfunction and neurodegeneration. MATERIALS AND METHODS Reagents D-gal, 5-bromodeoxyuridine (BrdU), mouse anti-BrdU, and alkaline phosphatase (AP)-conjugated sheep anti-digoxigenin (DIG) were purchased from Sigma (St. Louis, MO); terminal deoxynucleotidyl transferase (TdT), from Promega (Madison, WI); mouse anti-neuron-specific nuclear protein (NeuN, MAB377), rabbit anti-active caspase-3 (AB3623), and rhodamine-conjugated rabbit anti-mouse IgG (AP160R), from Chemicon (Temecula, CA); sheep anti-BrdU and FITC-conjugated rabbit anti-sheep IgG, from Biodesign International, Saco, ME; proteinase K, from Merck, Whitehouse Station, NJ; test kits for malondialdehyde (MDA), total antioxidant capabilities (T-AOC), total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-Px), from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). R-alipoic acid (tris salt) was gift from Dr. K. Wessel (Variats Inc., Germany).
Animal Treatments and BrdU Labeling Male adult C57BL/6 mice (Laboratory Animal Center, Chinese Academy of Medical Science, Beijing, China), weighing 26–28 g at the beginning of the experiment were used. Animals were randomly divided into three groups (control, D-gal-administration, and D-gal-administration plus a-LA treatment) and maintained at 20 6 18C, 12 hr light/12 hr dark cycle with free access to food and water. D-Gal (100 mg/kg) was injected subcutaneously (s.c.) daily into mice for 7 weeks. a-LA (100 mg/kg body weight) was injected peritoneally (i.p.) daily concomitantly for 7 weeks. All control animals were given saline. The brains of a subset of animals from each group was used for hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyltransferase-mediated dUTP-DIG nick end labeling (TUNEL) and caspase-3 immunohistochemistry after 7 weeks of administration. The remaining animals were given three BrdU injections (50 mg/kg i.p.) on Day 50 at 8-hr intervals, five animals from each group were terminated 24 hr after the last injection to study progenitor cell proliferation, another five animals from each group were allowed to live for 4 weeks after the last BrdU injection to further investigate the differentiation and survival of newly born neurons in the dentate gyrus (DG) with immunofluorescent methods. Water Maze Performance At the 7th week of the D-gal administration, spatial memory was assayed with Morris water maze (Liu et al., 2002a) (JRO 4.5, China). After 3 days of training, the time required for individual mice to find the submerged platform within 2 min (escape latency) and the swimming distance were monitored by a digital camera and a computer system for four consecutive days and four trials per day. The administration of D-gal and a-LA was continued during the water maze performance. MDA, T-AOC, T-SOD, and GSH-Px Assay Blood was sampled from mice by extirpating eyes after completion of the spatial memory test. Serum was collected and stored at 708C until analyses of MDA, T-AOC, TSOD, and GSH-Px. The assays were carried out according to the manufacturer’s protocols with a UV-160A Photospectrometer (Shimadzu, Japan). Tissue Preparation for Histologic Observations Animals were anesthetized with ketamine (44 mg/kg) and xylazine (13 mg/kg) and mice brains were fixed by transcardially perfusion with saline, followed by 4% paraformaldehyde in PBS. The brains were post-fixed in the same fixative overnight for preparing paraffin or freezing sections (30% sucrose in 0.1 M PBS for 2 days after post-fixation). Serial coronal paraffin sections (5 lm-thick) were cut for H&E staining, TUNEL, and caspase-3 immunohistochemical staining. Serial coronal frozen sections were cut into 40 lm-thick sections for BrdU immunohistochemical staining and BrdU/NeuN immunofluorescent labeling to establish the neuronal phenotype. Journal of Neuroscience Research DOI 10.1002/jnr
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TUNEL Assay Brain sections were digested in 20 lg/ml proteinase K for 15 min and were then covered with 3% H2O2 to inactivate endogenous peroxidase. TdT buffer containing 200 U/ml TdT and 1 mM dUTP-DIG were applied for 2 hr at 378C. The reaction was stopped with 0.3 M sodium chloride, 0.03 M sodium citrate for 5 min, followed by washing in TBS. The sections were blocked by immersion in blocking buffer for 30 min, then incubated with AP-conjugated sheep antiDIG and developed to produce staining by using BCIP/NBT. Caspase-3 Immunohistochemistry Brain sections were incubated in 0.3% H2O2 for 10 min after being covered with 0.01 M citrate buffer for 10 min, and then incubated overnight at 48C with rabbit anti-caspase3 (1:100 dilution) followed by incubation for 30 min in 10% goat serum and 1% BSA buffer. The sections were then incubated with biotinylated anti-rabbit IgG (1:100 dilution) for 30 min followed with the avidin-biotin complex solution for 30 min To visualize caspase-3 labeling, sections were incubated in diaminobenzidine (DAB) peroxidase substrate for 4–10 min and counterstained by hematoxylin QS. BrdU Immunohistochemistry Brain sections were pretreated in 50% formamide in 2 3 SSC for 2 hr at 658C, followed by 5 min in 2 3 SSC, 30 min in 2 M HCl at 378C, and 10 min in 0.1 M borate buffer. Sections were blocked with 5% normal horse serum and then incubated overnight with monoclonal mouse antiBrdU (1:1,000). After three washes, they were incubated for 2 hr with biotinylated horse anti-mouse IgG (1:80), followed by incubation for 1 hr in an avidin-biotin-peroxidase complex solution. The chromogen was DAB. BrdU/NeuN Double Immunofluorescence Brain sections were incubated in blocking buffer (TBS containing 0.1% Triton X-100 and 5% goat serum) for 30 min at 378C, with sheep anti-BrdU and mouse anti-NeuN complex in blocking buffer for 3648 hr at 48C, with FITCconjugated rabbit anti-sheep and rhodamine-conjugated rabbit anti-mouse secondary antibodies (1:200) for 2 hr at 378C, then washed three times with TBS. Control sections were processed with omission of the primary anti-sera. Cell Counting All TUNEL positive nuclei and caspase-3 positive cells in hippocampus and BrdU immunoreactive nuclei in DG including hilus and granule cell layer (GCL) were imaged with a computer-based C-CCD camera system (BX60, Olympus, Melville, NY). The immunoreactive neurons were analyzed with Image-Pro Plus software (IPP 4.5, Media Cybernetics, North Reading, MA). Images were taken at 200 lm intervals through the region of interest, and optical stocks of 610 images were produced for the figures. Fluorescent signals were detected and imaged with a confocal laser scanning system (Radiance 2100TM, Bio-Rad, Richmond, CA) to determine the number of BrdU/NeuN double labeled mature neurons in the GCL at each section. Journal of Neuroscience Research DOI 10.1002/jnr
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Statistical Analysis Data are expressed as mean 6 SE. Differences between mean values for each group were analyzed using ANOVA with Dunnett’s post-hoc analysis by using SPSS 11.5 software (SPSS Inc., Chicago, IL), and P < 0.05 was defined as statistically significance.
RESULTS D-gal-Induced Spatial Memory Decline and the Effect of a-LA None of the mice tested had obvious health problems (e.g., weight loss, cataracts, or toxicity reaction). The escape latency in D-gal-treated mice was longer than that in control mice on Days 1, 2, and 4 (Fig. 1). The swimming distance in D-gal-administered animals was also longer than that observed in control animals (data not shown). a-LA treatment to the D-gal-administered animals resulted in a significant decrease in escape latency and swimming distance (P < 0.01) when compared with D-gal-administered group on each testing day. The a-LA treatment further resulted in a decrease in escape latency when compared to the vehicle control on Day 3 (P < 0.05). Oxidative Stress Biomarkers in Serum and the Effect of a-LA MDA level and the activities of T-AOC, T-SOD, and GSH-Px have been used as the oxidative biomarkers for D-gal-induced aging models (Cui et al., 1998, 1999, 2000, 2004; Shen et al., 2002; Ho et al., 2003; Zhang et al., 2005a,b). We showed that D-gal-administration induced an increase in the MDA level and a decrease in the activities of T-AOC, T-SOD, and GSH-Px in serum (Table I). a-LA treatment resulted in a decrease in MDA level and an increase in SOD and T-AOC activities, but no effect on GSH-Px. Neurodegeneration in the Hippocampus and Effect of a-LA We used three techniques to quantify cell death within hippocampus (the pyramidal cells in the CA1, CA2, and CA3 subfields) and DG (the granular cells in GCL and newly born cells in SGZ): counting of pyknotic nuclei in H&E-stained section, TUNEL-positive nuclei, and caspase-3 positive cells in immunohistochemical stained sections. The results showed that the percentages of pyknotic nuclei in the D-gal-treated mice (14.1 6 1.42% in hippocampus and 7.7 6 1.95% in the DG, respectively) were higher (P < 0.01) than in control (2.0 6 0.29% in hippocampus and 0.6 6 0.21% in the DG). Animals that received a-LA showed a significant decrease in the percentage of the damaged cells (2.8 6 0.38% in hippocampus and 0.7 6 0.2% in the DG; P < 0.01, respectively) with respect to D-galreceiving mice (Fig. 2a–c). Increased TUNEL positive cells were observed in the hippocampus and DG in D-gal-treated group (Fig. 2d–f). TUNEL positive cells were distributed in both
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T-SOD (NU/ml)
GSH-Px (U/ml)
T-AOC (U/ml)
MDA (nmol/ml)
169 6 3 144 6 4* 164 6 4**
162 6 8 79 6 5* 73 6 3
9.7 6 0.5 6.9 6 0.2* 9.0 6 0.2**
2.3 6 0.2 3.0 6 0.2* 2.4 6 0.2**
Data are shown as the mean 6 SE of 910 animals. *P < 0.05 versus Saline group. **P < 0.05 vs. Galactose group.
from the SGZ to the GCL. The survival number of newly born cells in the DG in D-gal-administered group was significantly decreased in comparison with control (P < 0.01). Similar to the results after 24 hr BrdU injection, and a-LA treatment effectively inhibited the decrease in survival number of newborn cells (P < 0.05, Fig. 3g).
Fig. 1. Spatial memory test using Morris water maze. Values are mean 6 SE of 910 animals. *P < 0.05, **P < 0.01, vs. Saline group. ^P < 0.01, ^^P < 0.001, vs. Galactose group.
GCL and SGZ in the DG. Figure 2j shows the percentage of TUNEL positive cells was significantly increased by D-gal-administration. a-LA treatment significantly decreased the number of TUNEL positive cells induced by D-gal exposure. Caspase-3 expression was investigated in the same area. Similar to the results of TUNEL, the percentage of caspase-3 expressed cells significantly increased in different subfields of hippocampus in D-gal-administered group. a-LA significantly reduced the D-gal-induced increase in caspase-3 expression (Fig. 2g–i,k). Proliferation of Progenitor Cells, Survival of Newly Born Cells in DG, and Effects of a-LA Most newly born cells (BrdU-labeled cell) in the DG were located at the SGZ (the border between hilus and GCL) 24 hr following BrdU injection. BrdU-labeled nuclei were irregular in shape, and many proliferating cells occurred in clusters (P < 0.05, Fig. 3a–c). The number of proliferated progenitor cells in the DG decreased significantly at 7th week of by D-gal administration (24 hr BrdU observation). Almost complete rescue of the newly born cells was found in D-gal-treated animals treated for 4 weeks with a-LA (P < 0.05, Fig. 3c). To evaluate progenitor cell survival, we studied a subset of animals that were allowed to survive for 4 weeks after BrdU administration. The number of BrdU positive cells was lower than in the shorter survival study suggesting that some of the newly born cells migrated
Differentiation of Newly Born Neuron in the GCL and Effect of a-LA In addition to the observation that a fraction of newborn cells migrated from the SGZ into the GCL, we confirmed that BrdU positive nuclei were neuronal by quantitative analysis of NeuN/BrdU double staining. We found that most of the BrdU-labeled cells became NeuN/BrdU double immuno-positive neurons, and the morphology of BrdU/NeuN double labeled nuclei displayed a marked change from irregular to large and round in shape, which is a characteristic associated with maturation (Fig. 3d–f). D-gal administration caused a significantly decrease in the number of newly formed neurons, and a-LA could effectively reverse the quantitative analysis (Fig. 3h). DISCUSSION The risk for developing a neurodegenerative disorder increases with age and may be associated with an excessive generation of ROS and oxidative stress (Finkel and Holbrook, 2000). As a main pathway of neuronal death, apoptosis is an active form of cell degeneration and is executed by caspase proteins. There is a growing consensus that ROS is a potent inducer of apoptosis, and apoptosis contributes to the loss of neurons during normal aging (Zhang and Herman, 2002). The pathophysiological role of ROS has been intensively studied in D-gal-induced aging (Yelinova et al., 1996; Shen et al., 2002; Xu and Zhao, 2002; Ho et al., 2003; Cui et al., 2004; Zhang et al., 2005b). In the present study, we demonstrated that mice treated with D-gal show cognitive deficits, neuronal apoptosis and an elevation of caspase-3 protein levels in the hippocampus. Accompanying these changes is the reduction of T-SOD, GSHPx, and T-AOC activity and high level of MDA in serum. Our data indicate that D-gal-induced mimetic aging is associated with an increase in oxidative stress and consequent damage of hippocampal neurons by triggering Journal of Neuroscience Research DOI 10.1002/jnr
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Fig. 2. Neuronal damage and apoptotic analysis. H&E staining (a–c) shows that the pyknotic nucleis in galactose-treated group (b) were significantly increased compared with saline-treated group (a) and decreased in galactose þ a-LA treatment group (c) compared with galactose alone group in the CA1 subfield of hippocampus (P < 0.01, respectively) after 7 weeks of administration. TUNEL analysis (d–f) shows that TUNEL-positive cells were significantly increased in galactose-treated group (e) compared with saline-treated group (d), but decreased in galactose þ a-LA treatment group (f) in the pyrami-
dal cells (P < 0.01) and the granular cells (P < 0.001). (j) shows the quantitative data of TUNEL-positive cell number in the hippocampus. Caspase-3 immunoreactive positive cells were significantly increased in galactose-treated group (h) compared with saline group (g) and the galactose plus a-LA treatment group (i) were significantly decreased compared with galactose alone group in the pyramidal cells (P < 0.01, k) and the granular cells (P < 0.001, k) in the hippocampus. Scare bar ¼ 25 lm (c), 50 lm (f,i).
apoptosis cascades leading to neuronal loss and cognitive dysfunction. Consistent with our findings of induction of brain aging, chronic D-gal treatment has also been shown to induce formation of advanced glycation endproduct, untrastructural aging, and changes of transcriptome response similar to generalized aging in the retinal pigment epithelium-choroid of mice (Tian et al., 2005). Recent studies demonstrated that the adult mammalian brain retains the capacity for neurogenesis, by which new neurons may be generated to replace those lost through physiological or pathological process (Eriksson et al., 1998; Van Praag et al., 1999; Nottebohm, 2002). Neurogenesis diminishes with aging, however (Kuhn et al., 1996; Kempermann et al., 2002; Bondolfi et al., 2004; Heine et al., 2004). A decrease in neurogenesis may result in neurodegeneration because of the continuous loss of neurons. Reduced neurogenesis has been shown to have a deleterious effect on learning and memory (Shors et al., 2001). Several molecular signals control the proliferation, differentiation and survival of adult endogenous progenitors including hormones, neurotransmitters and trophic factors, and oxidative stress may reduce adult hippocampal neurogenesis (Shors
et al., 2001; Herrera et al., 2003; Jin et al., 2004; Limoli et al., 2004). In this study, we showed that D-gal not only induces a downregulation of proliferation of endogenous progenitor cells in SGZ, but also a reduction of migration and survival of new neurons in GCL. The impairment of hippocampal neurogenesis is similar to that in normal aging mice (Kuhn et al., 1996; Eriksson et al., 1998). It suggests that the progenitor cells and newly born neurons might be very vulnerable to ROS accumulation, and that impaired neurogenesis in the hippocampus might exacerbate learning and memory deficit. The results of our studies suggest that the marked effect of D-gal on the survival of newly formed neurons in the adult hippocampus could contribute to the impairment of hippocampal-dependent cognitive function. Modifying oxidative pathways and reducing ageassociated neuronal degeneration and reduced neurogenesis could provide a basis for preventive and therapeutic approaches. Direct antioxidants such as chain-breaking free radical scavengers can prevent oxidative damage, prolong life span and inhibit cancer (Harman, 1961, 1968, 1982). a-LA is an antioxidant in mitochondria and a neuroprotector (Packer et al., 1997a,b). a-LA
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Fig. 3. Neurogenesis analysis (a–c) shows that BrdU immunostaining in the SGZ of DG. treated with saline (a), galactose (b) and combination of galactose with a-LA (c). BrdU-positive cells are seen at the border of the hilus and the SGZ, the nuclei of BrdU-positive cells are irregular in shape and many proliferating cells are found to be in clusters. (g) shows quantitative data of BrdU immunoreactive cell number in the SGZ at 24 hr and 4 weeks after the last BrdU (50 mg/kg, i.p. three times, at 8 hr intervals) injection, *P < 0.05, **P < 0.01, vs. saline group; ^P < 0.05, vs. galactose group. Double im-
munofluorescent staining shows that some neuronal progenitor cell (d, BrdU) in SGZ can migrate to the GCL (e, NeuN) and further form newly born neurons (f, BrdU/NeuN) at 4 weeks after the last BrdU injection. The mature nuclei change from irregular to large and round. (h) shows the quantitative data of double immunostaining in GCL treated with saline, galactose and galactose plus a-LA, *P < 0.05, **P < 0.01, vs. saline group; ^P < 0.05, vs. galactose group. Scale bar ¼ 10 lm (c); 50 lm (f).
improves memory in aged mice and rats (Moini et al., 2002; Liu and Ames, 2005). In the present study, supplementing animals with a-LA prevents memory deficits in the D-gal-induced aging mice, increases antioxidant activity and lowers lipid peroxidation in serum. Analysis of the number of pyknotic nuclei and TUNEL/caspase3 positive cells in hippocampus showed that a-LA had an important protective effect against D-gal-induced apoptosis of pyramidal and granular cells. a-LA also showed protection of neuroprogenitor proliferation, restoration of the differentiation, and survival of newly born neurons in the hippocampus. The mechanisms by which aLA may mediate these effects include:
(Cao et al., 2003; Suh et al., 2004) and reduces the extent of oxidized RNA/DNA, lipid and protein in rats (Liu et al., 2002a,b).
1. a-LA can recycle antioxidants and scavenge ROS to block oxidative pathways (Packer et al., 1997a,b). Treating old rats with a-LA can markedly increase tissue cysteine levels and subsequently restored cerebral GSH levels, and increased GSH/GSSG ratios and vitamin C in both heart and brain (Hagen et al., 2002; Moini et al., 2002; Suh et al., 2004b). 2. a-LA improves mitochondrial function and prevents mitochondrial decay. a-LA-supplemented old rats have decreased oxidative damage and increased mitochondrial membrane potential (Hagen et al., 1999, 2002; Liu et al., 2002a). 3. a-LA enhances antioxidant defense by induction of phase 2 enzymes and reduces oxidative damage. a-LA induces phase 2 antioxidant enzyme
In conclusion, the present study shows that Dgal-induced cognitive dysfunction is associated with hippocampal neurodegeneration and impairment in neurogenesis, and that deficits can be prevented by the mitochondrial antioxidant a-LA treatment. These findings suggest that D-gal induces aging through oxidative damage pathways and mitochondrial dysfunction and a-LA could be used in the treatment of cognitive impairment in aging and possibly in other neurodegenerative disorders. In addition, D-gal-induced an acceleration in the aging phenotype in animals and can be a useful model system in which to test new therapeutics. ACKNOWLEDGMENTS We are grateful to Dr. E. Head, Institute for Brain Aging and Dementia, University of California at Irvine for critical and helpful comments, and to Dr. K. Wessel in Variats (Germany) for kindly providing R-a-lipoic acid for this study. REFERENCES Bondolfi L, Ermini F, Long J, Ingram D, Jucker M. 2004. Impact of age and caloric restriction on neurogenesis in the dentate gyrus of C57BL/ 6 mice. Neurobiol Aging 25:333–340. Cao Z, Tsang M, Zhao H, Li Y. 2003. Induction of endogenous antioxidants and phase 2 enzymes by alpha-lipoic acid in rat cardiac H9C2 Journal of Neuroscience Research DOI 10.1002/jnr
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Journal of Neuroscience Research DOI 10.1002/jnr
