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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 15, 2002 as doi:10.1096/fj.02-0448fje. |
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,#
* Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan;
|| Laboratory of Experimental Therapeutics, Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920-0934, Japan;
Section of Pharmacology and Toxicology, Department of Pharmacy, College of Pharmacy, Kangwon National University, Chunchon 200–701;
Institute of Environment and Life Science, Hallym University, Chunchon 200–702, South Korea;
Department of Pathology and Tumor Biology, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan; and
# Core Research for Evolutional Science and Technology, JST, Japan
3Correspondence: Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan. E-mail: tnabeshi{at}med.nagoya-u.ac.jp
SPECIFIC AIMS
Although a new gene, termed klotho, is associated with the suppression of several aging phenotypes, little is known about its function in the brain. We investigated the changes in mnemonic function accompanying aging in relation to oxidative stress in klotho mutant mice.
PRINCIPAL FINDINGS
1. Impaired recognition and associative memory in klotho mutant mice
We evaluated visual recognition memory in a novel object recognition memory task that requires the hippocampus. There were no significant differences in total exploratory time and exploratory preference between wild-type and klotho mutant mice in the training session at the age of 6 or 7 wk (Fig. 1
a), suggesting that the two mice have the same levels of motivation, curiosity, and interest in exploring novel objects. For the retention session, measured 1 and 24 h after the training, one object was replaced by a novel object. A significant difference between wild-type and klotho mutant mice in preference for the novel object was observed during the retention test 24 h after the training at the age of 7 wk, whereas no difference was observed at the age of 6 wk (Fig. 1a
). A time course study revealed that klotho mutant mice at the age of 7 wk showed the same magnitude of preference for the novel object as did wild-type mice when retention was tested 1 h after the training (Fig. 1b
). Impairment of memory retention was demonstrated, however, when the test was carried out 24 h after the training by using a different batch of mice. These results suggest that long- but not short-term retention of novel object recognition memory is impaired in klotho mutant mice at the age of 7 wk.
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We then examined associative fear memory in a conditioned fear task. In contextual fear conditioning, which is hippocampus dependent, animals learn to fear in the context in which they were trained to associate conditioned and unconditioned stimuli. Wild-type and klotho mutant mice at the age of 7 wk exhibited a marked contextual freezing 1 h after the conditioning (Fig. 1c
), and there was no significant difference between the two groups of mice. However, when contextual freezing was measured 24 h later using separate groups of animals, the mutant mice showed less freezing response whereas wild-type mice showed the same level of freezing as observed 1 h after the training (Fig. 1c
). In a tone-dependent test that is hippocampus independent, the freezing response in klotho mutant mice to the conditioned stimulus was significantly lower than that in wild-type mice in retention tests performed at 1 and 24 h after conditioning (Fig. 1d
). These data suggest that hippocampus-dependent and -independent associative fear memory are both impaired in klotho mutant mice at the age of 7 wk.
2. Accumulation of lipid peroxide and oxidative DNA damage products in the klotho mutant mice
Malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) are the most sensitive markers of oxidative lipid and DNA damage, respectively. To examine the possible involvement of oxidative stress in age-associated brain dysfunction in klotho mutant mice, we measured amounts of MDA and 8-OHdG in the brain. MDA and 8-OHdG levels in the hippocampus of wild-type mice were constant from 5 to 8 wk of age (Fig. 2
a, b). In contrast, these levels in klotho mutant mice markedly increased in an age-dependent manner.
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There was no difference in Cu/Zn-superoxide dismutase activity in the hippocampus between klotho mutant and wild-type mice at the age of 5 wk. However, at the age of 6 and 8 wk, enzymatic activity in klotho mutant mice had increased by 58.6% and 49.3% over the activity in wild-type mice. (Fig. 2c
). Glutathione peroxidase activity in the hippocampus of the mutant mice increased significantly by 21.8% at the age of 6 wk (Fig. 2d
), but no difference was found at the age of 5 and 8 wk compared with the activity in wild-type mice.
3. Expression of Bcl-2-related proteins in the klotho mutant mice
Bcl-2-related proteins are important regulators of apoptosis, their expression being influenced by reactive oxygen species. Therefore, we examined the profile of the patterns of gene expression in the hippocampus of klotho mutant mice at the age of 7 wk. A semiquantitative RT-PCR revealed an increase in bax mRNA expression and a decrease in bcl-2 and bcl-XL expression in klotho mutant mice. Western blot analysis also showed that protein expression of Bax in the hippocampus of klotho mutant mice increased by 43.4%, whereas Bcl-2 and Bcl-XL expression levels were significantly decreased by 19.6% and 28.6% compared with those in wild-type mice, at the age of 7 wk.
4. Effects of 
-tocopherol on impaired recognition and associative memory in the klotho mutant mice
To examine whether oxidative stress contributes to memory deficits in klotho mutant mice, we investigated the effects of a potent antioxidant, -tocopherol, on impairment of recognition and associative memory at the age of 7 wk. -Tocopherol (150 mg/kg, p.o.) was administered from 5 wk of age until the end of the experiments. In the retention session tested 24 h after the training vehicle-treated wild-type mice showed a significant preference for exploring the novel object, whereas vehicle-treated klotho mutant mice showed no preference at all. -Tocopherol significantly increased preference for the novel object in klotho mutant mice to the level in wild-type mice. Chronic -tocopherol treatment also completely restored the deficits of associative fear memory in klotho mutant mice at the age of 7 wk. The contextual memory deficit in klotho mutant mice was significantly and completely rescued by -tocopherol treatment, whereas the same treatment had no effect in wild-type mice. -Tocopherol completely restored the deficits of tone-dependent freezing in the klotho mutant mice without affecting the freezing in wild-type mice.
In vehicle-treated klotho mutant mice, the number of MDA-positive cells, and TdT-mediated dUTP nick end labeling (TUNEL) -positive cells increased in the CA1 region of the hippocampus compared with vehicle-treated wild-type mice. TUNEL-positive cells were colocalized with the MDA-positive cells. -Tocopherol reduced the number of MDA-positive and TUNEL-positive cells in the CA1 region of the hippocampus of klotho mutant mice without affecting the staining in wild-type mice. These results suggest that an increase in oxidative stress and apoptotic cell death occurs in the pyramidal cells of the CA1 region of the hippocampus of klotho mutant mice and that chronic -tocopherol reduces oxidative stress-induced brain damage in these mutant mice.
CONCLUSIONS AND SIGNIFICANCE
Studies of the klotho mutant mouse and the klotho gene are expected to provide new insights into human aging. Notably, klotho mutant mice that are hypomorphic with regard to klotho gene expression exhibit a human aging-related phenotype, including a reduced life span (
60 days), atherosclerosis, neural degeneration, and calcification of soft tissues. In the present study, we compared mnemonic function and the levels of oxidative stress in the brain of klotho mutant mice with those in wild-type mice. The klotho mutant mice showed an impairment of visual recognition memory and associative fear memory at the age of 7 wk, whereas the animals had normal memory function at the age of 6 wk. Oxidative damage is considered a likely cause of age-associated brain dysfunction because the brain is particularly vulnerable to oxidative stress due to a relatively high rate of generation of reactive oxygen species without commensurate levels of antioxidative defense. Lipid peroxide (MDA) and DNA oxidative damage (8-OHdG) increased in the hippocampus of klotho mutant mice in an age-dependent manner from age 5 to 8 wk, whereas levels were constant in wild-type mice. In the present study, the proapoptotic Bax gene was up-regulated and the anti-death genes Bcl-2 and Bcl-XL were down-regulated in the hippocampus of klotho mutant mice at the age of 7 wk. Bcl-2 and its homologs can form homo- and heterodimers and Bax homodimers actively promote cell death unless neutralized by heterodimerization with Bcl-2 or Bcl-XL. These results suggest that enhanced oxidative stress causes increases in the pro-death/anti-death ratio of Bcl-2 family genes, leading to apoptosis in the hippocampus of the klotho mutant mice. -Tocopherol is the most prevalent and efficacious lipid-soluble antioxidant in biological systems. Previous studies have shown that -tocopherol inhibits neuronal cell death and lipid peroxidation induced by aging. In the present study we found that repeated administration of -tocopherol ameliorated memory impairment and prevented lipid peroxidation and apoptotic cell death in the hippocampus of klotho mutant mice. These results suggest that oxidative stress causes cognition impairment accompanied by aging in klotho mutant mice.
Two klotho gene transcripts encoding membrane- or secreted-type protein are generated through alternative transcriptional termination; the klotho gene is mainly expressed in specific cells and tissues (renal tubles, brain, and choroid plexus) but not in other organs (bone, lung, and skin) showing severe age-related phenotypes in klotho mutant mice. Therefore, it is proposed that Klotho proteins act as a humoral factor. It is possible that secreted Klotho proteins or metabolites have a role in the regulation of antioxidant defense.
In conclusion, we demonstrated that aging-associated oxidative damage of neuronal membranes and DNA occurs in the hippocampus and that the increased oxidative stress may cause apoptosis, followed by memory impairment in klotho mutant mice. The repeated administration of the antioxidant, -tocopherol, prevented cognition impairment, accumulation of lipid peroxide, and apoptotic cell death in the hippocampus of klotho mutant mice. Our findings support the hypothesis that oxidative stress plays a crucial role in age-associated brain dysfunction. Klotho protein may be involved in the regulation of antioxidative defense(Fig. 3
).
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0448fje; to cite this article, use FASEB J. (November 15, 2002) 10.1096/fj.02-0448fje 
2 The first two authors contributed equally to this work.
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