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Effects of α-tocopherol on an animal model of tauopathies
Free Radical Biology & Medicine, Vol. 37, No. 2, pp. 176 – 186, 2004 Copyright D 2004 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/$-see front matter
doi:10.1016/j.freeradbiomed.2004.04.037
Original Contribution EFFECTS OF a-TOCOPHEROL ON AN ANIMAL MODEL OF TAUOPATHIES HANAE NAKASHIMA,* TAKESHI ISHIHARA,* OSAMU YOKOTA,* SEISHI TERADA,* JOHN Q. TROJANOWSKI, y VIRGINIA M.-Y. LEE, y and SHIGETOSHI KURODA * * Department of Neuropsychiatry, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan; and y The Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, and Institute on Aging, The University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA (Received 12 January 2004; Revised 26 March 2004; Accepted 28 April 2004) Available online 24 May 2004
Abstract—We have reported that transgenic (Tg) mice overexpressing human tau protein develop filamentous tau aggregates in the CNS. We overexpressed the smallest human tau isoform (T44) in the mouse CNS to model tauopathies. These tau Tg mice acquire age-dependent CNS pathologies, including insoluble, hyperphosphorylated tau and argyrophilic intraneuronal inclusions formed by tau-immunoreactive filaments. Therefore, these Tg mice are a model that can be exploited for drug discovery in studies that target amelioration of tau-induced neurodegeneration as well as for elucidating mechanisms of tau pathology in various neurodegenerative tauopathies. Oxidative stress has been implicated in the pathogenesis of various neurodegenerative diseases, including tauopathies, and many epidemiological, clinical, and basic studies have suggested the neuroprotective effects of vitamin E in neurodegenerative diseases. To elucidate the role of oxidative damage in the pathological mechanisms of these Tg mice, we fed them a-tocopherol, the major component of antioxidant vitamin E. Supplementation of a-tocopherol suppressed and/or delayed the development of tau pathology, which correlated with improvement in the health and attenuation of motor weakness in the Tg mice. These results suggest that oxidative damage is involved in the pathological mechanisms of the tau Tg mice and that treatment with antioxidative agents like a-tocopherol may prevent neurodegenerative tauopathies. D 2004 Elsevier Inc. All rights reserved. Keywords—Tauopathies, Transgenic mouse, Oxidative stress, Antioxidant, Vitamin E, a-Tocopherol, Free radicals
FTDP-17 showed that these tau gene mutations alter the level and/or functions of tau and that tau abnormalities do not require the presence of other brain lesions (e.g., amyloid plaques) to induce a neurodegenerative disorder [2– 5]. Indeed, efforts to produce animal models with tau pathologies by overexpressing a normal or mutant tau gene have shown that alteration of the level and/or functions of tau causes neurodegeneration in the mouse CNS [6 –17]. As previously reported [7], although the distribution of the tau pathology in our mice most closely resembles that found in amyotrophic lateral sclerosis/Parkinson dementia complex and progressive supranuclear palsy, as well as in some FTDP-17 syndromes, these filamentous tau aggregates share many characteristics with authentic NFTs in AD and other tauopathies. First, like highly insoluble PHF-tau in AD NFTs [18], a substantial fraction of tau proteins from the Tg mice is extracted
INTRODUCTION
Abnormal tau proteins are implicated in the mechanisms of brain degeneration in Alzheimer’s disease (AD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), Pick’s disease, and a group of neurodegenerative diseases that are collectively known as tauopathies [1]. The discovery of an autosomal dominant pathogenic tau gene mutation in FTDP-17 has led to the rapid emergence of new insights into the mechanisms underlying FTDP-17, AD, and related tauopathies, as well as opportunities to develop transgenic mouse models of these disorders. Studies on the mechanisms of Address correspondence to: Takeshi Ishihara, Department of Neuropsychiatry, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Okayama, Japan; Fax: +81-86-235-7246; E-mail: [email protected]. 176
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only with RIPA buffer and FA, despite the fact that normal tau is an extremely soluble protein. Second, the amount of insoluble tau protein progressively accumulates with age and disease progression in the Tg mice, similar to AD and other tauopathies. Third, PHF-tau proteins in human NFTs are hyperphosphorylated and so is soluble and insoluble tau recovered from Tg mice [19,20]. Fourth, although AD NFTs contain mostly PHFs, straight filaments similar to those found in the Tg mice are also present [21]. Oxidative stress has been implicated in the pathogenesis of various neurodegenerative disease conditions, including AD, vascular dementia, and Parkinson’s disease as well as in aging. Epidemiological, in vivo, and in vitro studies have shown that antioxidant agents, including vitamin E, have a preventive effect on neurodegenerative oxidative damage [22 – 32]. Actually, the current standard care for pharmacologic management of the cognitive and functional disabilities of AD consists of the combination of a cholinesterase inhibitor (e.g., donepezil) and high-dose vitamin E, and this regimen is based on the results of large-scale, double-blind, and placebocontrolled trials [33]. Vitamin E has been also recommended in the treatment of AD because of its safety and low cost. So far, studies of the effects of antioxidative agents on neurodegenerative diseases have focused mainly on hamyloid- [24,34 – 39] or a-synuclein- [40 –48] induced neurodegeneration and to a far less extent on tau-induced conditions, and there are no studies on the direct role of oxidative damage in mechanisms of tau-induced neurodegeneration in animal models of tauopathies. Thus, to elucidate the role of oxidative damage in human wildtype (WT) tau (T44) Tg mice, we treated them with atocopherol. Our study showed that routine administration of a-tocopherol to T44 Tg mice reduced or delayed development of the pathological phenotype as demonstrated histopathologically, biochemically, and clinically. These results show that oxidative damage is involved in the mechanisms of tau-induced neurodegeneration and that antioxidant treatment has a protective effect on neurodegenerative tauopathies. MATERIALS AND METHODS
Generation of mice A transgene including a cDNA of the shortest human tau isoform (T44) driven by the mouse PrP promoter and 3V untranslated sequences was used to create tau Tg mice on a B6D2/F1 background; studies characterizing three lines of T44 Tg mice generated with this transgene were described earlier [6– 8]. The heterozygous Tg mouse lines 7, 43, and 27 overexpress human tau proteins at levels approximately 5-, 10-, and 15-fold higher than endoge-
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nous mouse tau, respectively. The heterozygous line 27, with the highest levels of Tg tau, is not viable beyond 3 months, and none of the homozygous mice generated from any of these lines survive longer than 3 months. Therefore, we conducted the studies described here on 3to 9-month-old heterozygous line 7 T44 Tg mice and WT littermate control mice. Many studies and experiments have been conducted on the T44 Tg mice over the past 5 years, and we do not find gender differences among the phenotypes in these Tg mice, nor does the fact that the transgene is inherited from father or mother affect features of the phenotype in these mice or their response to the treatments here. Treatment with a-tocopherol Mice were randomly split into four different experimental groups (Table 1). Mice in the first group (Controls) were fed chow (PMI Feeds, Inc., St. Louis, MO, USA) with a conventional dietary level of a-tocopherol (49 IU/kg) throughout their lifetimes. The parents of the second group (AT-R) of mice were fed chow containing 160 IU/kg a-tocopherol (d-a-tocopherol; Eisai, Tokyo, Japan), and right after pups were born the diet for the mothers was changed to a conventional one, and the experimental mice were fed conventional chow thereafter until the conclusion of experiments. In the AT-R group supplementation with a-tocopherol was for mothers of the newborn mice only before their delivery. For transgenic mice in the third (AT(+)) and fourth (AT(++)) groups, the diet for the mothers was changed to supplemented (160 or 1500 IU/kg, respectively) right after the pups were born, and also, after the experimental mice were weaned, they were fed the same supplemented diets as their mothers received while nursing. All animals were treated in accordance with the Guidelines for Animal Experimentation of Okayama University. Immunohistochemical analyses Tau Tg and WT mice were anesthetized and perfused transcardially with 20 ml of phosphate-buffered saline followed by 20 ml of 70% ethanol in isotonic saline. A representative series of 6 Am thick paraffin sections of Tg and WT mouse brain and spinal cord was immunostained by standard streptavidin – biotin – peroxidase methods as described [7] using well-characterized antibodies: rabbit polyclonal antibodies to human recombinant tau (17026, 1:1500) [7], mouse monoclonal antibodies (MAbs) to phosphorylation-independent tau epitopes (T14, 1:500, Zymed, South San Francisco, CA, USA), mouse MAbs to phosphorylation-dependent tau epitopes (T1, 1:1000, Roche, Indianapolis, IN, USA; AT8, 1:1000, Pierce Biotechnology, Rockford, IL, USA; AT100, 1:500, Innogenetics, Gent, Belgium; AT180, 1:500, Innogenetics), mouse MAbs to the phosphorylated high-molecular-
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H. Nakashima et al. Table 1. a-Tocopherol Dose, Plasma and Brain Levels, and Mortality Ratio of Experimental Groups
Group
Controls AT-R AT(+) AT(++)
a-Tocopherol in chow (IU/kg) Parents
After birth
49 160 49 49
49 49 160 1500
a-Tocopherol level Plasma (Ag/ml) 0.960 0.932 1.408 3.013
F F F F
0.109 0.055 0.131 0.441
Mortality of Tg mice (before 3 months of age)
Brain (Ag/g) 8.900 F 9.114 F 15.721 F 26.86 F
0.381 0.062 0.250 0.097
6.90% 3.08% 0%* 0%*
Plasma concentration of a-tocopherol represents the mean F SEM (n = 5 – 7). Brain level of a-tocopherol represents the mean F SEM (n = 3). Mortality was evaluated only in Tg mice (n = 29 – 65, *p < .01).
weight subunit of neurofilament (NFH) (SMI31, 1:1000) and nonphosphorylated NFH (SMI32, 1:1000) (Sternberger Monoclonals, Lutherville, MD, USA), rabbit polyclonal antibodies to the middle-molecular-weight subunit of neurofilament (NA1216, 1:500, Affinity, Exeter, UK), and rabbit polyclonal antibodies to the lowmolecular-weight subunit of neurofilament (AB1983, 1:250, Chemicon, Temecula, CA, USA) and to 8-hydroxydeoxyguanosine (8-OHdG) derivatives (NOF, Tokyo, Japan). Axonal tau pathology in the tau Tg mice was quantified by counting the number of tau-positive spheroids with a diameter of >10 Am in 9 –12 lumbar spinal cord sections of Tg mice from each group (n = 3 – 5), and the average number of spheroids per section was used as a representative value. This histological quantification of the spheroids was done by a person who was blinded to the experimental status of the animals. As reported previously [2], the number of tau-positive spheroids in the spinal cord of the T44 mice increased with age until 6 months, and it decreased thereafter. In the older Tg mice, many vacuolar lesions of the same size as or larger than the inclusions were also observed in the spinal cord, which may reflect the degeneration of affected axons. In this experiment, the mean and SEM values of the number of spheroids at the ages of 6 and 9 months were calculated in each experimental group, and the difference in the number of spheroids among all of the groups was examined statistically by one-way ANOVA. Confocal laser scanning microscopy Double-label immunofluorescent staining was performed with a combination of primary antibodies 8-OHdG and 17026 diluted 1:50 and 1:1000, respectively. Sections were deparaffinized and nonspecific binding was blocked. Sections were first incubated in a mixture of the two primary antibodies overnight at 4jC and then in fluorescence-labeled secondary antibodies (Alexa 488-labeled anti-rabbit IgG (H+L) and Alexa 594-labeled anti-mouse IgG (H+L); Molecular Probes, Eugene, OR, USA) for 1 h. Sections were viewed with a confocal microscope (FV300; Olympus, Tokyo, Japan).
Western blot analysis of tau expressed in the CNS of Tg mice Brains and spinal cords were dissected from lethally anesthetized 6 month old Tg and WT mice, and methods similar to those described recently [6– 9,49] were used in the isolation procedures here. Briefly, brain and spinal cord tissues were sequentially extracted with ice-cold high-salt RAB buffer (0.1 M Mes, 1 mM EGTA, 0.5 mM MgSO4, 0.75 M NaCl, 0.02 M NaF, 1 mM PMSF, and 0.1% protease inhibitor cocktail (100 Ag/ml each of pepstatin A, leupeptin, TPCK, TLCK, and soybean trypsin inhibitor and 100 mM EDTA; Sigma– Aldrich, St. Louis, MO, USA), pH 7.0) followed by RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 5 mM EDTA, 0.5% sodium deoxycholate, and 0.1% SDS, pH 8.0) and finally with 70% FA. Equal amounts of samples were subsequently resolved on 7.5% SDS – PAGE gels and transferred onto nitrocellulose membranes. Western blot analysis was performed, and the bands detected by enhanced chemiluminescence (ECL) reagent (Amersham, Buckinghamshire, UK) were analyzed quantitatively. Measurement of oxidized proteins Amounts of oxidized proteins were quantified using an Oxyblot kit (Intergen, Purchase, NY, USA) as previously reported [50]. Briefly, the same amount of protein (approximately 15 Ag) from the RIPA buffer extract of each 6 month old mouse brain was reacted with dinitrophenylhydrazine (DNPH) for 20 min, followed by neutralization with a solution containing glycerol and 2-mercaptoethanol. Western blot analysis was performed using a rabbit anti-DNPH antibody as the primary antibody (1:150) and goat anti-rabbit antibody as the secondary antibody (1:300). Bands were visualized using ECL reagent and quantified. Tail suspension test The tail suspension test was performed as described previously [6,51]. Briefly, mice from each experimental group (minimum n = 4) were videotaped while being suspended by their tails for 15 s at 6 and 9
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months of age. Animals were assessed for clasping behavior. The test period was divided into 2 s segments. An animal received a score of 1 point if it displayed any abnormal movements during each time segment. An abnormal movement was defined as dystonic movements of the hind limbs or a combination of hind limbs, forelimbs, and trunk, during which the limbs were pulled into the body in a manner not observed in WT mice. Unbiased sampling was used in this test. RESULTS
Plasma concentration of a-tocopherol and mortality of mice The plasma and brain levels of a-tocopherol of each experimental group at 3 months of age are summarized in
Fig. 2. Quantification of spheroids in the mouse spinal cord of each experimental group. Quantification of spheroids is described under Materials and Methods. The mean values of the numbers of spheroids among the four groups are summarized here. The error bars represent SEM. Statistical analysis was performed using mice at 6 and 9 months by ANOVA. Significant difference between each experimental group is indicated by asterisks (*p < .01; **p < .001).
Fig. 1. Representative tau-positive spheroids in the spinal cord of tau Tg mice. The spinal cord sections were stained with 17,026, polyclonal antibodies to recombinant tau protein. (A) Shown are representative taupositive spheroids in the spinal cord of a 6 month old Tg mouse from the Control group (arrows), which were never seen in (B) WT littermates. Scale bar, 100 Am (A, B); 10 Am (A inset).
Table 1. As previously reported [52] and also shown in Table 1, the plasma and brain levels of a-tocopherol have a linear relationship. The difference in the plasma and brain levels reached statistical significance between Controls and AT(+) ( p < .05), Controls and AT(++) ( p < .001), AT(+) and AT(++) ( p < .001), but not Controls and AT-R. The plasma concentration of a-tocopherol was also measured at 6 months of age, but there was no significant difference from that at 3 months in any group. There was no difference in the plasma or brain levels of a-tocopherol between Tg and WT mice in each group at 3 and 6 months of age. As previously reported [7], a proportion of the T44 Tg mouse line used in this study died from undetermined causes (6.90%). Remarkably, however, T44 Tg mice in the AT(+) and AT(++) groups showed increased viability relative to their untreated counterparts (Fisher’s exact probability test, p < .01) (Table 1). The Tg mice weigh 20 –30% less than agematched WT littermates after 6 months of age [7], and the same tendency was observed in each group in this study. Although the weight deficit in the AT(+) and AT(++) groups was less than that of the Control and AT-R groups (f15%), there were no significant differences among all the experimental groups (data not shown).
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A reduction in the number of spheroids in the CNS of the Tg mice treated with a-tocopherol Because the major pathology found in the tau Tg mice is the presence of tau-immunopositive spheroids in the spinal cord [6– 8], we sought to determine whether the treatment with a-tocopherol would have any effect on the number of spheroids. As reported previously, the number of tau-positive spheroids in the spinal cord of the Tg mice increases with age until f6 months, and it decreases thereafter. Fig. 1 shows representative taupositive spheroids in the spinal cord of a Tg mouse from the Control group at 6 months. As shown in Fig. 2, the number of spheroids decreased significantly in the Tg mice treated with a-tocopherol (AT(+), AT(++)) compared with the Control group at the ages of both 6 and 9 months. Notably, there also is a significant reduction in the total number of spheroids in the Tg mice from the AT-R group, demonstrating that supplemental a-tocopherol in utero has a preventive effect on tau pathology in the Tg mice. As previously reported, the spheroids in the
spinal cord of Tg mice were recognized by the phosphorylation-dependent antibodies AT8 (phosphoserine 202 and phosphothreonine 205), AT100 (phosphoserine 212 and phosphothreonine 214), and AT180 (phosphothreonine 231), but there was no obvious difference in the extents of immunoreactivity with these antibodies in immunohistochemical staining among each experimental group (data not shown). a-Tocopherol treatment reduced tau-induced 8-OHdG immunoreactivity Because 8-OHdG, a product of oxidized DNA, is a good marker of oxidative stress, we studied the immunoreactivity of 8-OHdG in the brain and spinal cord of Tg and WT mice in each group with 17026 and anti-8OHdG antibodies using confocal laser scanning microscopy. As shown in Fig. 3B, there is no unequivocal staining with anti-8-OHdG antibody in the spinal cord of WT mice in the Control group, and the same results were seen in WT mice in the other groups (data not shown). In
Fig. 3. Double immunofluorescent staining of the spinal cord. The sections were stained with 17,026, a polyclonal antibody to recombinant tau protein, and mouse MAb to 8-OHdG. 17,026 and 8-OHdG immunoreactivities are indicated as red and green, respectively. (A – C) WT from the Control group, (D – F) Tg from the Control group, (G – I) Tg from the AT(++) group. All sections were from mice at the age of 6 months. White arrows indicate tau-positive spheroids.
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contrast, tau Tg mice from the Control group showed strong immunoreactivity of 8-OHdG in the nuclei in white matter and also in perikarya of motoneurons in the spinal cord (Fig. 3E). The immunoreactivity of 8OHdG was detected also in the spinal cord of a-tocopherol-treated Tg mice but to a lesser extent compared with that of Tg mice from the Control group (Fig. 3H), although obvious differences in the immunoreactivity of 8-OHdG were not detected among the AT-R, AT(+), and AT(++) groups. a-Tocopherol treatment reduced accumulation of insoluble tau protein in the CNS of Tg mice As reported previously, tau protein becomes progressively more insoluble and hyperphosphorylated with age in the tau Tg mice, as in human tauopathies [6 –8]. To determine the effect of a-tocopherol treatment on the accumulation of insoluble tau, we analyzed the solubility of tau protein in the different experimental groups by extracting brain and spinal cord samples using buffers with increasing extraction strengths. The brain and spinal cord samples from 6 month old mice from each group were sequentially extracted with RAB, RIPA buffer, and 70% FA. The three fractions were then analyzed by quantitative Western blotting with antibody 17026. As shown in Fig. 4 and reported previously [6 –8], over 90% of endogenous mouse tau from both the brain and the spinal cord of the WT mouse was primarily RAB soluble, and no tau immunoreactivity was detected in the FAsoluble fraction. At 6 months of age, f76 and f74% of total tau proteins were RAB soluble, and f1.2 and 1.7% were in the FA-soluble fraction in the brain and spinal cord of the Tg mice, respectively [6]. Although the RABsoluble tau remained relatively constant in Tg mice in all
Fig. 5. Measurement of oxidized proteins in the brains of mice. (A) Representative examples of Oxyblot using f15 Ag of protein from the neocortex of each experimental group at 6 months of age. (B) Levels of oxidized proteins in Oxyblots of RIPA buffer-extracted supernatants from neocortex of each experimental group at 6 months of age. ANOVA showed a significant reduction in oxidized proteins in the tau Tg mice from AT(+) and AT(++) groups compared with Tg mice from the Control group (*p < .01) or AT-R group (**p < .05) (n = 4).
experimental groups (Figs. 4A and 4B), the intensity of the RIPA buffer-soluble fraction on Western blotting was decreased in the spinal cord of Tg mice from the AT(++) group (Fig. 4D), and the intensity of the FA-soluble fraction was decreased in Tg mice from the AT(+) and AT(++) groups in both the brain and the spinal cord (Figs. 4E and 4F). These results are generally consistent with the immunohistochemical finding that a-tocopherol-treated Tg mice have fewer tau aggregates in the spinal cord compared with Tg mice from the Control group. Oxidative damage is reduced in a-tocopherol-treated mice
Fig. 4. Reduced accumulation of insoluble tau protein in the CNS of atocopherol-treated mice. (A) Neocortical and (B) spinal cord tissues of 6 month old mice from each group were sequentially extracted with RAB, RIPA buffer, and 70% FA, and the tau levels were determined by quantitative Western blot analysis with antibody 17,026, a polyclonal antibody to recombinant tau protein. Changes in the RIPA buffer- and FA-soluble tau as intensity on Western blotting are shown in C – F (C, E, neocortex; D, F, spinal cord). The intensity of the RIPA buffer fraction was significantly decreased in the spinal cord of Tg mice from the AT(++) group, and that of the FA-soluble fraction was significantly decreased in Tg mice from AT(+) and AT(++) groups compared with that of Tg mice from the Control group (n = 3) (*p < .05, **p < .01).
Oxidative damage was assessed in each experimental group at 6 months of age using Western blotting. Carbonyl groups on oxidized proteins were derivatized with DNPH on Oxyblots and detected using an anti-DNP antibody as described under Materials and Methods. Representative examples of Oxyblot data are shown in Fig. 5A. A drastic increase in oxidized proteins was detected in Tg mice from the Control group compared with WT mice from the Control group (f3.2-fold), but these differences diminished with a-tocopherol treatment (Fig. 5A). As shown in Fig. 5B, ANOVA revealed a significant reduction in oxidized proteins in a-tocopherol-
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treated Tg mice (AT(+), AT(++)). Therefore, both low and high doses of a-tocopherol treatment decreased levels of oxidized proteins to about the same extent in the Tg mice. a-Tocopherol-treated Tg mice showed behavioral improvements As reported previously, the T44 tau Tg mice develop progressive motor weakness as demonstrated by an impaired ability to stand on a slanted surface and by the clasping of their hindlimbs when lifted by the tail. As shown in Fig. 6, long-term a-tocopherol treatment significantly reduced the abnormal clasping behavior at 6 months of age. For example, the Tg mice from the Control group had a clasping score of f2.4 at 6 months of age, but the Tg mice from the AT(+) and AT(++) groups had clasping scores of f1 and f1.2 by the same age, respectively, suggesting a reduction in hind-limb weakness. Together with the change in viability (Table 1), our data suggest that long-term a-tocopherol treatment of tau Tg mice partially reduces motor impairments caused by overexpression of human tau protein. DISCUSSION
In the present study, we have provided evidence that overexpressed human tau protein in mouse CNS causes oxidative damage, which is reduced by long-term administration of an antioxidant, a-tocopherol. To accomplish this, we fed T44 tau Tg mice an a-tocopherol supplement for up to 9 months, and our examination of
Fig. 6. Clasping phenotype of mice. Mice were videotaped during a 15 s tail suspension test at 6 months of age and observed for clasping behavior as described under Materials and Methods. Tau Tg mice from AT(+) and AT(++) groups showed a significantly lower score than Tg mice from the Control group. Bar graphs show means F SE of five trials (n = 6) (*p < .05, **p < .01).
a-tocopherol-treated Tg mice (AT(+) and AT(++)) provided unequivocal evidence that oxidative damage plays a pathogenic role in T44 Tg mice. For example, we observed a significant reduction in the number of taupositive aggregates in the spinal cord of the Tg mice fed supplementary a-tocopherol, suggesting that oxidative damage plays an important role in tau aggregate-induced pathological mechanisms. Furthermore, this reduction in aggregate number with a-tocopherol treatment seems to correlate with the overall improvement in the health of Tg mice because survival increased in a-tocopheroltreated mice. Finally, the reduction in tau spheroids in a-tocopherol-treated Tg mice also correlated with attenuation of motor weakness in these tau Tg mice, suggesting a pathologic role of the aggregates in causing motor neuron degeneration. Although oxidative stress has long been associated with a number of neurodegenerative diseases because evidence of oxidative damage have been detected in several well-known neurological conditions, including AD [39,53– 56], Parkinson’s disease [48,57], vascular dementia [53,54,58], amyotrophic lateral sclerosis [59 – 61], Huntington’s disease [62 – 65], tardive dyskinesia [66 – 69], and multiple sclerosis [70,71], and in normal aging [72], the precise role oxidative damage plays in the pathogenesis of these neurodegenerative diseases remains unclear. As discussed above, there has been a paucity of efforts to elucidate the contribution of oxidative damage in tau-induced neurodegenerative conditions, although recent studies suggest that oxidative stress modifies the phosphorylation state of tau [73,74]. Notably, some cell culture studies have shown that both overexpression and phosphorylation of tau cause oxidative stress-induced cell degeneration. For example, overexpressed tau in primary cortical neurons, retinal ganglion cells, and neuroblastoma cells inhibits kinesin-dependent transport of peroxisomes, neurofilaments, and Golgi-derived vesicles into neurites and loss of peroxisomes makes cells vulnerable to oxidative stress and leads to degeneration [75,76]. The data from our study suggest that overexpressed tau proteins induce oxidative damage rather than the alternative possibility that oxidative damage modifies tau pathology [77 – 79], although both, and other, mechanisms may act synergistically during disease progression in these tau Tg mice. For example, as we previously reported [7], tau aggregation in the proximal axons compromised axonal transport in ventral roots, was linked to the dying back of motoneurons (gain of toxic function), reduced the numbers of microtubules (MTs), and reduced the levels of tubulin in the remaining axons of the degenerating ventral roots in the Tg mice, thereby implying that a loss of the MT-stabilizing function of tau (loss of function) leads to neurodegeneration. Based on indirect evidence from studies of human tauopathies, we
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and others have proposed that both gains of toxic functions and losses of normal tau function could be involved mechanistically in causing neurodegenerative tauopathies [3,80,81]. From the data presented here, oxidative damage should be considered one of the mechanisms caused by tau lesions. Although it seems obvious that oxidative damage to DNA is induced by tau overexpression in T44 Tg mice (Fig. 3), we cannot conclude that excess tau itself directly causes oxidative damage to DNA. As seen in the tau lesions of AD cases [82,83], tau- and 8-OHdG-positive lesions do not colocalize in T44 Tg mice. This phenomenon has been partially explained by the exclusion of DNA-containing structures from tau lesions [83], and this also suggests that oxidative damage related to tau overexpression is mediated by yet unspecified factors. As discussed above, compromised axonal transport of certain proteins by tau overexpression and consequent oxidative damage in cell bodies seen in cultural cells may occur also in T44 Tg mice. Another explanation for the difference in tau and 8-OHdG staining locations is the distribution pattern of tau in T44 Tg mice. As reported previously [7], we did transmission electron microscopy studies of spinal cord axons of T44 Tg mice. Those data show that at least 30% of axons have tau-positive aggregates, but they are not often perinuclear, although we cannot conclude that tau accumulations occur more in axons than in cell bodies in these mice. 8-OHdG stains oxidized DNA most intensely in cytoplasm and to a lesser extent in the nucleus. So it may be possible that tau-positive lesions are abundant in axons, whereas 8OHdG-positive lesions are in the cytoplasm. More work is needed to understand the detailed relationship between tau accumulation and nucleic acid damage in human tauopathies and animal models of tauopathies as well. a-Tocopherol treatment significantly lowered the number of tau-positive inclusions in the spinal cord of T44 Tg mice and also reduced the accumulation of insoluble tau and oxidized proteins, effects that are consistent with the increased survival and attenuation of motor weakness of T44 Tg mice. In the present study, we used high- and low-dose a-tocopherol supplementation. Despite substantial differences between the two doses (high:low = 9.375:1), we did not detect any obvious differences between the two dose groups in any aspect investigated in this study, suggesting that the effect of a-tocopherol is not dose dependent. It is notable that attenuation of the impaired viability and the number of tau-positive inclusions was seen in the Tg mice in the AT-R group, in which the parents of the experimental mice were fed chow containing a low-dose supplement of a-tocopherol (160 IU/kg) and the offspring were fed the conventional dietary level of atocopherol after birth. Although we cannot evaluate the
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precise influence of milk from mother mice supplemented with a-tocopherol while pregnant, it has been reported that a-tocopherol levels in plasma or brain after long-term supplementation begin to decrease right after the last supplementation and that they return to presupplementation levels within 1 or 4 weeks, respectively [52]. We measured plasma concentrations of a-tocopherol in the AT-R group mice at 3 weeks of age, at which time they were weaned from their mothers, and the values were almost the same as those of 3 month old mice (data not shown). Therefore it is plausible that there is no significant postnatal supplementation with a-tocopherol for the AT-R group of mice and that their brain levels of a-tocopherol return to baseline levels within 5– 6 weeks after birth. Thus, this remarkable result raises the issue of how early a preventive strategy against oxidative damage should be implemented to ameliorate oxidationrelated neurodegenerative diseases. Although the influence of nutrition on AD has been reported, most of these reports focused on the effects of antioxidants, including vitamin E [31,32,84 – 88], and little is known about the influence of in utero and/or postnatal nutritional conditions on the risk for developing AD late in the human life span. However, based on our data here, we speculate that there may be a threshold level of antioxidants during pregnancy and/or the postnatal period above which the risk for late life neurodegenerative disease is minimized, although further studies are needed to elucidate the plausibility of this speculation. In summary, oxidative damage is involved in mechanisms of neurodegeneration induced by tau overexpression, and the long-term administration of a-tocopherol has a protective effect on the consequences of tau overexpression, including the formation of tau pathology histochemically and biochemically as well as the behavioral sequelae thereof. Indeed, whereas a-tocopherol (vitamin E) has long been used for the standard pharmacological management of AD, which is one of the major tauopathies, and it has few side effects and little toxicity, this is the first study to demonstrate potential ameliorative effects of a-tocopherol on tau pathologies, thereby suggesting that a-tocopherol may be neuroprotective in tauopathies other than AD as well. Acknowledgments — We thank S. Fujisawa and M. Onbe for technical assistance. This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (T.I.), the Zikei Institute of Psychiatry (T.I.), the National Institutes of Health (V.M.-Y.L., J.Q.T.), as well as by grants from the Marian S. Ware Alzheimer Program (V.M.-Y.L., J.Q.T.). REFERENCES [1] Lee, V. M.-Y.; Goedert, M.; Trojanowski, J. Q. Neurodegenerative tauopathies. Annu. Rev. Neurosci. 24:1121 – 1159; 2001. [2] Foster, N. L.; Wilhelmsen, K.; Sima, A. A.; Jones, M. Z.; D’Amato, C. J.; Gilman, S. Frontotemporal dementia and parkinsonism
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AD — Alzheimer’s disease CNS — central nervous system FA — formic acid FTDP-17 — frontotemporal dementia with parkinsonism linked to chromosome 17 MAb — monoclonal antibody Tg — transgenic WT — wild type
doi:10.1016/j.freeradbiomed.2004.04.037
Original Contribution EFFECTS OF a-TOCOPHEROL ON AN ANIMAL MODEL OF TAUOPATHIES HANAE NAKASHIMA,* TAKESHI ISHIHARA,* OSAMU YOKOTA,* SEISHI TERADA,* JOHN Q. TROJANOWSKI, y VIRGINIA M.-Y. LEE, y and SHIGETOSHI KURODA * * Department of Neuropsychiatry, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan; and y The Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, and Institute on Aging, The University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA (Received 12 January 2004; Revised 26 March 2004; Accepted 28 April 2004) Available online 24 May 2004
Abstract—We have reported that transgenic (Tg) mice overexpressing human tau protein develop filamentous tau aggregates in the CNS. We overexpressed the smallest human tau isoform (T44) in the mouse CNS to model tauopathies. These tau Tg mice acquire age-dependent CNS pathologies, including insoluble, hyperphosphorylated tau and argyrophilic intraneuronal inclusions formed by tau-immunoreactive filaments. Therefore, these Tg mice are a model that can be exploited for drug discovery in studies that target amelioration of tau-induced neurodegeneration as well as for elucidating mechanisms of tau pathology in various neurodegenerative tauopathies. Oxidative stress has been implicated in the pathogenesis of various neurodegenerative diseases, including tauopathies, and many epidemiological, clinical, and basic studies have suggested the neuroprotective effects of vitamin E in neurodegenerative diseases. To elucidate the role of oxidative damage in the pathological mechanisms of these Tg mice, we fed them a-tocopherol, the major component of antioxidant vitamin E. Supplementation of a-tocopherol suppressed and/or delayed the development of tau pathology, which correlated with improvement in the health and attenuation of motor weakness in the Tg mice. These results suggest that oxidative damage is involved in the pathological mechanisms of the tau Tg mice and that treatment with antioxidative agents like a-tocopherol may prevent neurodegenerative tauopathies. D 2004 Elsevier Inc. All rights reserved. Keywords—Tauopathies, Transgenic mouse, Oxidative stress, Antioxidant, Vitamin E, a-Tocopherol, Free radicals
FTDP-17 showed that these tau gene mutations alter the level and/or functions of tau and that tau abnormalities do not require the presence of other brain lesions (e.g., amyloid plaques) to induce a neurodegenerative disorder [2– 5]. Indeed, efforts to produce animal models with tau pathologies by overexpressing a normal or mutant tau gene have shown that alteration of the level and/or functions of tau causes neurodegeneration in the mouse CNS [6 –17]. As previously reported [7], although the distribution of the tau pathology in our mice most closely resembles that found in amyotrophic lateral sclerosis/Parkinson dementia complex and progressive supranuclear palsy, as well as in some FTDP-17 syndromes, these filamentous tau aggregates share many characteristics with authentic NFTs in AD and other tauopathies. First, like highly insoluble PHF-tau in AD NFTs [18], a substantial fraction of tau proteins from the Tg mice is extracted
INTRODUCTION
Abnormal tau proteins are implicated in the mechanisms of brain degeneration in Alzheimer’s disease (AD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), Pick’s disease, and a group of neurodegenerative diseases that are collectively known as tauopathies [1]. The discovery of an autosomal dominant pathogenic tau gene mutation in FTDP-17 has led to the rapid emergence of new insights into the mechanisms underlying FTDP-17, AD, and related tauopathies, as well as opportunities to develop transgenic mouse models of these disorders. Studies on the mechanisms of Address correspondence to: Takeshi Ishihara, Department of Neuropsychiatry, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Okayama, Japan; Fax: +81-86-235-7246; E-mail: [email protected]. 176
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only with RIPA buffer and FA, despite the fact that normal tau is an extremely soluble protein. Second, the amount of insoluble tau protein progressively accumulates with age and disease progression in the Tg mice, similar to AD and other tauopathies. Third, PHF-tau proteins in human NFTs are hyperphosphorylated and so is soluble and insoluble tau recovered from Tg mice [19,20]. Fourth, although AD NFTs contain mostly PHFs, straight filaments similar to those found in the Tg mice are also present [21]. Oxidative stress has been implicated in the pathogenesis of various neurodegenerative disease conditions, including AD, vascular dementia, and Parkinson’s disease as well as in aging. Epidemiological, in vivo, and in vitro studies have shown that antioxidant agents, including vitamin E, have a preventive effect on neurodegenerative oxidative damage [22 – 32]. Actually, the current standard care for pharmacologic management of the cognitive and functional disabilities of AD consists of the combination of a cholinesterase inhibitor (e.g., donepezil) and high-dose vitamin E, and this regimen is based on the results of large-scale, double-blind, and placebocontrolled trials [33]. Vitamin E has been also recommended in the treatment of AD because of its safety and low cost. So far, studies of the effects of antioxidative agents on neurodegenerative diseases have focused mainly on hamyloid- [24,34 – 39] or a-synuclein- [40 –48] induced neurodegeneration and to a far less extent on tau-induced conditions, and there are no studies on the direct role of oxidative damage in mechanisms of tau-induced neurodegeneration in animal models of tauopathies. Thus, to elucidate the role of oxidative damage in human wildtype (WT) tau (T44) Tg mice, we treated them with atocopherol. Our study showed that routine administration of a-tocopherol to T44 Tg mice reduced or delayed development of the pathological phenotype as demonstrated histopathologically, biochemically, and clinically. These results show that oxidative damage is involved in the mechanisms of tau-induced neurodegeneration and that antioxidant treatment has a protective effect on neurodegenerative tauopathies. MATERIALS AND METHODS
Generation of mice A transgene including a cDNA of the shortest human tau isoform (T44) driven by the mouse PrP promoter and 3V untranslated sequences was used to create tau Tg mice on a B6D2/F1 background; studies characterizing three lines of T44 Tg mice generated with this transgene were described earlier [6– 8]. The heterozygous Tg mouse lines 7, 43, and 27 overexpress human tau proteins at levels approximately 5-, 10-, and 15-fold higher than endoge-
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nous mouse tau, respectively. The heterozygous line 27, with the highest levels of Tg tau, is not viable beyond 3 months, and none of the homozygous mice generated from any of these lines survive longer than 3 months. Therefore, we conducted the studies described here on 3to 9-month-old heterozygous line 7 T44 Tg mice and WT littermate control mice. Many studies and experiments have been conducted on the T44 Tg mice over the past 5 years, and we do not find gender differences among the phenotypes in these Tg mice, nor does the fact that the transgene is inherited from father or mother affect features of the phenotype in these mice or their response to the treatments here. Treatment with a-tocopherol Mice were randomly split into four different experimental groups (Table 1). Mice in the first group (Controls) were fed chow (PMI Feeds, Inc., St. Louis, MO, USA) with a conventional dietary level of a-tocopherol (49 IU/kg) throughout their lifetimes. The parents of the second group (AT-R) of mice were fed chow containing 160 IU/kg a-tocopherol (d-a-tocopherol; Eisai, Tokyo, Japan), and right after pups were born the diet for the mothers was changed to a conventional one, and the experimental mice were fed conventional chow thereafter until the conclusion of experiments. In the AT-R group supplementation with a-tocopherol was for mothers of the newborn mice only before their delivery. For transgenic mice in the third (AT(+)) and fourth (AT(++)) groups, the diet for the mothers was changed to supplemented (160 or 1500 IU/kg, respectively) right after the pups were born, and also, after the experimental mice were weaned, they were fed the same supplemented diets as their mothers received while nursing. All animals were treated in accordance with the Guidelines for Animal Experimentation of Okayama University. Immunohistochemical analyses Tau Tg and WT mice were anesthetized and perfused transcardially with 20 ml of phosphate-buffered saline followed by 20 ml of 70% ethanol in isotonic saline. A representative series of 6 Am thick paraffin sections of Tg and WT mouse brain and spinal cord was immunostained by standard streptavidin – biotin – peroxidase methods as described [7] using well-characterized antibodies: rabbit polyclonal antibodies to human recombinant tau (17026, 1:1500) [7], mouse monoclonal antibodies (MAbs) to phosphorylation-independent tau epitopes (T14, 1:500, Zymed, South San Francisco, CA, USA), mouse MAbs to phosphorylation-dependent tau epitopes (T1, 1:1000, Roche, Indianapolis, IN, USA; AT8, 1:1000, Pierce Biotechnology, Rockford, IL, USA; AT100, 1:500, Innogenetics, Gent, Belgium; AT180, 1:500, Innogenetics), mouse MAbs to the phosphorylated high-molecular-
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H. Nakashima et al. Table 1. a-Tocopherol Dose, Plasma and Brain Levels, and Mortality Ratio of Experimental Groups
Group
Controls AT-R AT(+) AT(++)
a-Tocopherol in chow (IU/kg) Parents
After birth
49 160 49 49
49 49 160 1500
a-Tocopherol level Plasma (Ag/ml) 0.960 0.932 1.408 3.013
F F F F
0.109 0.055 0.131 0.441
Mortality of Tg mice (before 3 months of age)
Brain (Ag/g) 8.900 F 9.114 F 15.721 F 26.86 F
0.381 0.062 0.250 0.097
6.90% 3.08% 0%* 0%*
Plasma concentration of a-tocopherol represents the mean F SEM (n = 5 – 7). Brain level of a-tocopherol represents the mean F SEM (n = 3). Mortality was evaluated only in Tg mice (n = 29 – 65, *p < .01).
weight subunit of neurofilament (NFH) (SMI31, 1:1000) and nonphosphorylated NFH (SMI32, 1:1000) (Sternberger Monoclonals, Lutherville, MD, USA), rabbit polyclonal antibodies to the middle-molecular-weight subunit of neurofilament (NA1216, 1:500, Affinity, Exeter, UK), and rabbit polyclonal antibodies to the lowmolecular-weight subunit of neurofilament (AB1983, 1:250, Chemicon, Temecula, CA, USA) and to 8-hydroxydeoxyguanosine (8-OHdG) derivatives (NOF, Tokyo, Japan). Axonal tau pathology in the tau Tg mice was quantified by counting the number of tau-positive spheroids with a diameter of >10 Am in 9 –12 lumbar spinal cord sections of Tg mice from each group (n = 3 – 5), and the average number of spheroids per section was used as a representative value. This histological quantification of the spheroids was done by a person who was blinded to the experimental status of the animals. As reported previously [2], the number of tau-positive spheroids in the spinal cord of the T44 mice increased with age until 6 months, and it decreased thereafter. In the older Tg mice, many vacuolar lesions of the same size as or larger than the inclusions were also observed in the spinal cord, which may reflect the degeneration of affected axons. In this experiment, the mean and SEM values of the number of spheroids at the ages of 6 and 9 months were calculated in each experimental group, and the difference in the number of spheroids among all of the groups was examined statistically by one-way ANOVA. Confocal laser scanning microscopy Double-label immunofluorescent staining was performed with a combination of primary antibodies 8-OHdG and 17026 diluted 1:50 and 1:1000, respectively. Sections were deparaffinized and nonspecific binding was blocked. Sections were first incubated in a mixture of the two primary antibodies overnight at 4jC and then in fluorescence-labeled secondary antibodies (Alexa 488-labeled anti-rabbit IgG (H+L) and Alexa 594-labeled anti-mouse IgG (H+L); Molecular Probes, Eugene, OR, USA) for 1 h. Sections were viewed with a confocal microscope (FV300; Olympus, Tokyo, Japan).
Western blot analysis of tau expressed in the CNS of Tg mice Brains and spinal cords were dissected from lethally anesthetized 6 month old Tg and WT mice, and methods similar to those described recently [6– 9,49] were used in the isolation procedures here. Briefly, brain and spinal cord tissues were sequentially extracted with ice-cold high-salt RAB buffer (0.1 M Mes, 1 mM EGTA, 0.5 mM MgSO4, 0.75 M NaCl, 0.02 M NaF, 1 mM PMSF, and 0.1% protease inhibitor cocktail (100 Ag/ml each of pepstatin A, leupeptin, TPCK, TLCK, and soybean trypsin inhibitor and 100 mM EDTA; Sigma– Aldrich, St. Louis, MO, USA), pH 7.0) followed by RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 5 mM EDTA, 0.5% sodium deoxycholate, and 0.1% SDS, pH 8.0) and finally with 70% FA. Equal amounts of samples were subsequently resolved on 7.5% SDS – PAGE gels and transferred onto nitrocellulose membranes. Western blot analysis was performed, and the bands detected by enhanced chemiluminescence (ECL) reagent (Amersham, Buckinghamshire, UK) were analyzed quantitatively. Measurement of oxidized proteins Amounts of oxidized proteins were quantified using an Oxyblot kit (Intergen, Purchase, NY, USA) as previously reported [50]. Briefly, the same amount of protein (approximately 15 Ag) from the RIPA buffer extract of each 6 month old mouse brain was reacted with dinitrophenylhydrazine (DNPH) for 20 min, followed by neutralization with a solution containing glycerol and 2-mercaptoethanol. Western blot analysis was performed using a rabbit anti-DNPH antibody as the primary antibody (1:150) and goat anti-rabbit antibody as the secondary antibody (1:300). Bands were visualized using ECL reagent and quantified. Tail suspension test The tail suspension test was performed as described previously [6,51]. Briefly, mice from each experimental group (minimum n = 4) were videotaped while being suspended by their tails for 15 s at 6 and 9
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months of age. Animals were assessed for clasping behavior. The test period was divided into 2 s segments. An animal received a score of 1 point if it displayed any abnormal movements during each time segment. An abnormal movement was defined as dystonic movements of the hind limbs or a combination of hind limbs, forelimbs, and trunk, during which the limbs were pulled into the body in a manner not observed in WT mice. Unbiased sampling was used in this test. RESULTS
Plasma concentration of a-tocopherol and mortality of mice The plasma and brain levels of a-tocopherol of each experimental group at 3 months of age are summarized in
Fig. 2. Quantification of spheroids in the mouse spinal cord of each experimental group. Quantification of spheroids is described under Materials and Methods. The mean values of the numbers of spheroids among the four groups are summarized here. The error bars represent SEM. Statistical analysis was performed using mice at 6 and 9 months by ANOVA. Significant difference between each experimental group is indicated by asterisks (*p < .01; **p < .001).
Fig. 1. Representative tau-positive spheroids in the spinal cord of tau Tg mice. The spinal cord sections were stained with 17,026, polyclonal antibodies to recombinant tau protein. (A) Shown are representative taupositive spheroids in the spinal cord of a 6 month old Tg mouse from the Control group (arrows), which were never seen in (B) WT littermates. Scale bar, 100 Am (A, B); 10 Am (A inset).
Table 1. As previously reported [52] and also shown in Table 1, the plasma and brain levels of a-tocopherol have a linear relationship. The difference in the plasma and brain levels reached statistical significance between Controls and AT(+) ( p < .05), Controls and AT(++) ( p < .001), AT(+) and AT(++) ( p < .001), but not Controls and AT-R. The plasma concentration of a-tocopherol was also measured at 6 months of age, but there was no significant difference from that at 3 months in any group. There was no difference in the plasma or brain levels of a-tocopherol between Tg and WT mice in each group at 3 and 6 months of age. As previously reported [7], a proportion of the T44 Tg mouse line used in this study died from undetermined causes (6.90%). Remarkably, however, T44 Tg mice in the AT(+) and AT(++) groups showed increased viability relative to their untreated counterparts (Fisher’s exact probability test, p < .01) (Table 1). The Tg mice weigh 20 –30% less than agematched WT littermates after 6 months of age [7], and the same tendency was observed in each group in this study. Although the weight deficit in the AT(+) and AT(++) groups was less than that of the Control and AT-R groups (f15%), there were no significant differences among all the experimental groups (data not shown).
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A reduction in the number of spheroids in the CNS of the Tg mice treated with a-tocopherol Because the major pathology found in the tau Tg mice is the presence of tau-immunopositive spheroids in the spinal cord [6– 8], we sought to determine whether the treatment with a-tocopherol would have any effect on the number of spheroids. As reported previously, the number of tau-positive spheroids in the spinal cord of the Tg mice increases with age until f6 months, and it decreases thereafter. Fig. 1 shows representative taupositive spheroids in the spinal cord of a Tg mouse from the Control group at 6 months. As shown in Fig. 2, the number of spheroids decreased significantly in the Tg mice treated with a-tocopherol (AT(+), AT(++)) compared with the Control group at the ages of both 6 and 9 months. Notably, there also is a significant reduction in the total number of spheroids in the Tg mice from the AT-R group, demonstrating that supplemental a-tocopherol in utero has a preventive effect on tau pathology in the Tg mice. As previously reported, the spheroids in the
spinal cord of Tg mice were recognized by the phosphorylation-dependent antibodies AT8 (phosphoserine 202 and phosphothreonine 205), AT100 (phosphoserine 212 and phosphothreonine 214), and AT180 (phosphothreonine 231), but there was no obvious difference in the extents of immunoreactivity with these antibodies in immunohistochemical staining among each experimental group (data not shown). a-Tocopherol treatment reduced tau-induced 8-OHdG immunoreactivity Because 8-OHdG, a product of oxidized DNA, is a good marker of oxidative stress, we studied the immunoreactivity of 8-OHdG in the brain and spinal cord of Tg and WT mice in each group with 17026 and anti-8OHdG antibodies using confocal laser scanning microscopy. As shown in Fig. 3B, there is no unequivocal staining with anti-8-OHdG antibody in the spinal cord of WT mice in the Control group, and the same results were seen in WT mice in the other groups (data not shown). In
Fig. 3. Double immunofluorescent staining of the spinal cord. The sections were stained with 17,026, a polyclonal antibody to recombinant tau protein, and mouse MAb to 8-OHdG. 17,026 and 8-OHdG immunoreactivities are indicated as red and green, respectively. (A – C) WT from the Control group, (D – F) Tg from the Control group, (G – I) Tg from the AT(++) group. All sections were from mice at the age of 6 months. White arrows indicate tau-positive spheroids.
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contrast, tau Tg mice from the Control group showed strong immunoreactivity of 8-OHdG in the nuclei in white matter and also in perikarya of motoneurons in the spinal cord (Fig. 3E). The immunoreactivity of 8OHdG was detected also in the spinal cord of a-tocopherol-treated Tg mice but to a lesser extent compared with that of Tg mice from the Control group (Fig. 3H), although obvious differences in the immunoreactivity of 8-OHdG were not detected among the AT-R, AT(+), and AT(++) groups. a-Tocopherol treatment reduced accumulation of insoluble tau protein in the CNS of Tg mice As reported previously, tau protein becomes progressively more insoluble and hyperphosphorylated with age in the tau Tg mice, as in human tauopathies [6 –8]. To determine the effect of a-tocopherol treatment on the accumulation of insoluble tau, we analyzed the solubility of tau protein in the different experimental groups by extracting brain and spinal cord samples using buffers with increasing extraction strengths. The brain and spinal cord samples from 6 month old mice from each group were sequentially extracted with RAB, RIPA buffer, and 70% FA. The three fractions were then analyzed by quantitative Western blotting with antibody 17026. As shown in Fig. 4 and reported previously [6 –8], over 90% of endogenous mouse tau from both the brain and the spinal cord of the WT mouse was primarily RAB soluble, and no tau immunoreactivity was detected in the FAsoluble fraction. At 6 months of age, f76 and f74% of total tau proteins were RAB soluble, and f1.2 and 1.7% were in the FA-soluble fraction in the brain and spinal cord of the Tg mice, respectively [6]. Although the RABsoluble tau remained relatively constant in Tg mice in all
Fig. 5. Measurement of oxidized proteins in the brains of mice. (A) Representative examples of Oxyblot using f15 Ag of protein from the neocortex of each experimental group at 6 months of age. (B) Levels of oxidized proteins in Oxyblots of RIPA buffer-extracted supernatants from neocortex of each experimental group at 6 months of age. ANOVA showed a significant reduction in oxidized proteins in the tau Tg mice from AT(+) and AT(++) groups compared with Tg mice from the Control group (*p < .01) or AT-R group (**p < .05) (n = 4).
experimental groups (Figs. 4A and 4B), the intensity of the RIPA buffer-soluble fraction on Western blotting was decreased in the spinal cord of Tg mice from the AT(++) group (Fig. 4D), and the intensity of the FA-soluble fraction was decreased in Tg mice from the AT(+) and AT(++) groups in both the brain and the spinal cord (Figs. 4E and 4F). These results are generally consistent with the immunohistochemical finding that a-tocopherol-treated Tg mice have fewer tau aggregates in the spinal cord compared with Tg mice from the Control group. Oxidative damage is reduced in a-tocopherol-treated mice
Fig. 4. Reduced accumulation of insoluble tau protein in the CNS of atocopherol-treated mice. (A) Neocortical and (B) spinal cord tissues of 6 month old mice from each group were sequentially extracted with RAB, RIPA buffer, and 70% FA, and the tau levels were determined by quantitative Western blot analysis with antibody 17,026, a polyclonal antibody to recombinant tau protein. Changes in the RIPA buffer- and FA-soluble tau as intensity on Western blotting are shown in C – F (C, E, neocortex; D, F, spinal cord). The intensity of the RIPA buffer fraction was significantly decreased in the spinal cord of Tg mice from the AT(++) group, and that of the FA-soluble fraction was significantly decreased in Tg mice from AT(+) and AT(++) groups compared with that of Tg mice from the Control group (n = 3) (*p < .05, **p < .01).
Oxidative damage was assessed in each experimental group at 6 months of age using Western blotting. Carbonyl groups on oxidized proteins were derivatized with DNPH on Oxyblots and detected using an anti-DNP antibody as described under Materials and Methods. Representative examples of Oxyblot data are shown in Fig. 5A. A drastic increase in oxidized proteins was detected in Tg mice from the Control group compared with WT mice from the Control group (f3.2-fold), but these differences diminished with a-tocopherol treatment (Fig. 5A). As shown in Fig. 5B, ANOVA revealed a significant reduction in oxidized proteins in a-tocopherol-
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treated Tg mice (AT(+), AT(++)). Therefore, both low and high doses of a-tocopherol treatment decreased levels of oxidized proteins to about the same extent in the Tg mice. a-Tocopherol-treated Tg mice showed behavioral improvements As reported previously, the T44 tau Tg mice develop progressive motor weakness as demonstrated by an impaired ability to stand on a slanted surface and by the clasping of their hindlimbs when lifted by the tail. As shown in Fig. 6, long-term a-tocopherol treatment significantly reduced the abnormal clasping behavior at 6 months of age. For example, the Tg mice from the Control group had a clasping score of f2.4 at 6 months of age, but the Tg mice from the AT(+) and AT(++) groups had clasping scores of f1 and f1.2 by the same age, respectively, suggesting a reduction in hind-limb weakness. Together with the change in viability (Table 1), our data suggest that long-term a-tocopherol treatment of tau Tg mice partially reduces motor impairments caused by overexpression of human tau protein. DISCUSSION
In the present study, we have provided evidence that overexpressed human tau protein in mouse CNS causes oxidative damage, which is reduced by long-term administration of an antioxidant, a-tocopherol. To accomplish this, we fed T44 tau Tg mice an a-tocopherol supplement for up to 9 months, and our examination of
Fig. 6. Clasping phenotype of mice. Mice were videotaped during a 15 s tail suspension test at 6 months of age and observed for clasping behavior as described under Materials and Methods. Tau Tg mice from AT(+) and AT(++) groups showed a significantly lower score than Tg mice from the Control group. Bar graphs show means F SE of five trials (n = 6) (*p < .05, **p < .01).
a-tocopherol-treated Tg mice (AT(+) and AT(++)) provided unequivocal evidence that oxidative damage plays a pathogenic role in T44 Tg mice. For example, we observed a significant reduction in the number of taupositive aggregates in the spinal cord of the Tg mice fed supplementary a-tocopherol, suggesting that oxidative damage plays an important role in tau aggregate-induced pathological mechanisms. Furthermore, this reduction in aggregate number with a-tocopherol treatment seems to correlate with the overall improvement in the health of Tg mice because survival increased in a-tocopheroltreated mice. Finally, the reduction in tau spheroids in a-tocopherol-treated Tg mice also correlated with attenuation of motor weakness in these tau Tg mice, suggesting a pathologic role of the aggregates in causing motor neuron degeneration. Although oxidative stress has long been associated with a number of neurodegenerative diseases because evidence of oxidative damage have been detected in several well-known neurological conditions, including AD [39,53– 56], Parkinson’s disease [48,57], vascular dementia [53,54,58], amyotrophic lateral sclerosis [59 – 61], Huntington’s disease [62 – 65], tardive dyskinesia [66 – 69], and multiple sclerosis [70,71], and in normal aging [72], the precise role oxidative damage plays in the pathogenesis of these neurodegenerative diseases remains unclear. As discussed above, there has been a paucity of efforts to elucidate the contribution of oxidative damage in tau-induced neurodegenerative conditions, although recent studies suggest that oxidative stress modifies the phosphorylation state of tau [73,74]. Notably, some cell culture studies have shown that both overexpression and phosphorylation of tau cause oxidative stress-induced cell degeneration. For example, overexpressed tau in primary cortical neurons, retinal ganglion cells, and neuroblastoma cells inhibits kinesin-dependent transport of peroxisomes, neurofilaments, and Golgi-derived vesicles into neurites and loss of peroxisomes makes cells vulnerable to oxidative stress and leads to degeneration [75,76]. The data from our study suggest that overexpressed tau proteins induce oxidative damage rather than the alternative possibility that oxidative damage modifies tau pathology [77 – 79], although both, and other, mechanisms may act synergistically during disease progression in these tau Tg mice. For example, as we previously reported [7], tau aggregation in the proximal axons compromised axonal transport in ventral roots, was linked to the dying back of motoneurons (gain of toxic function), reduced the numbers of microtubules (MTs), and reduced the levels of tubulin in the remaining axons of the degenerating ventral roots in the Tg mice, thereby implying that a loss of the MT-stabilizing function of tau (loss of function) leads to neurodegeneration. Based on indirect evidence from studies of human tauopathies, we
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and others have proposed that both gains of toxic functions and losses of normal tau function could be involved mechanistically in causing neurodegenerative tauopathies [3,80,81]. From the data presented here, oxidative damage should be considered one of the mechanisms caused by tau lesions. Although it seems obvious that oxidative damage to DNA is induced by tau overexpression in T44 Tg mice (Fig. 3), we cannot conclude that excess tau itself directly causes oxidative damage to DNA. As seen in the tau lesions of AD cases [82,83], tau- and 8-OHdG-positive lesions do not colocalize in T44 Tg mice. This phenomenon has been partially explained by the exclusion of DNA-containing structures from tau lesions [83], and this also suggests that oxidative damage related to tau overexpression is mediated by yet unspecified factors. As discussed above, compromised axonal transport of certain proteins by tau overexpression and consequent oxidative damage in cell bodies seen in cultural cells may occur also in T44 Tg mice. Another explanation for the difference in tau and 8-OHdG staining locations is the distribution pattern of tau in T44 Tg mice. As reported previously [7], we did transmission electron microscopy studies of spinal cord axons of T44 Tg mice. Those data show that at least 30% of axons have tau-positive aggregates, but they are not often perinuclear, although we cannot conclude that tau accumulations occur more in axons than in cell bodies in these mice. 8-OHdG stains oxidized DNA most intensely in cytoplasm and to a lesser extent in the nucleus. So it may be possible that tau-positive lesions are abundant in axons, whereas 8OHdG-positive lesions are in the cytoplasm. More work is needed to understand the detailed relationship between tau accumulation and nucleic acid damage in human tauopathies and animal models of tauopathies as well. a-Tocopherol treatment significantly lowered the number of tau-positive inclusions in the spinal cord of T44 Tg mice and also reduced the accumulation of insoluble tau and oxidized proteins, effects that are consistent with the increased survival and attenuation of motor weakness of T44 Tg mice. In the present study, we used high- and low-dose a-tocopherol supplementation. Despite substantial differences between the two doses (high:low = 9.375:1), we did not detect any obvious differences between the two dose groups in any aspect investigated in this study, suggesting that the effect of a-tocopherol is not dose dependent. It is notable that attenuation of the impaired viability and the number of tau-positive inclusions was seen in the Tg mice in the AT-R group, in which the parents of the experimental mice were fed chow containing a low-dose supplement of a-tocopherol (160 IU/kg) and the offspring were fed the conventional dietary level of atocopherol after birth. Although we cannot evaluate the
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precise influence of milk from mother mice supplemented with a-tocopherol while pregnant, it has been reported that a-tocopherol levels in plasma or brain after long-term supplementation begin to decrease right after the last supplementation and that they return to presupplementation levels within 1 or 4 weeks, respectively [52]. We measured plasma concentrations of a-tocopherol in the AT-R group mice at 3 weeks of age, at which time they were weaned from their mothers, and the values were almost the same as those of 3 month old mice (data not shown). Therefore it is plausible that there is no significant postnatal supplementation with a-tocopherol for the AT-R group of mice and that their brain levels of a-tocopherol return to baseline levels within 5– 6 weeks after birth. Thus, this remarkable result raises the issue of how early a preventive strategy against oxidative damage should be implemented to ameliorate oxidationrelated neurodegenerative diseases. Although the influence of nutrition on AD has been reported, most of these reports focused on the effects of antioxidants, including vitamin E [31,32,84 – 88], and little is known about the influence of in utero and/or postnatal nutritional conditions on the risk for developing AD late in the human life span. However, based on our data here, we speculate that there may be a threshold level of antioxidants during pregnancy and/or the postnatal period above which the risk for late life neurodegenerative disease is minimized, although further studies are needed to elucidate the plausibility of this speculation. In summary, oxidative damage is involved in mechanisms of neurodegeneration induced by tau overexpression, and the long-term administration of a-tocopherol has a protective effect on the consequences of tau overexpression, including the formation of tau pathology histochemically and biochemically as well as the behavioral sequelae thereof. Indeed, whereas a-tocopherol (vitamin E) has long been used for the standard pharmacological management of AD, which is one of the major tauopathies, and it has few side effects and little toxicity, this is the first study to demonstrate potential ameliorative effects of a-tocopherol on tau pathologies, thereby suggesting that a-tocopherol may be neuroprotective in tauopathies other than AD as well. Acknowledgments — We thank S. Fujisawa and M. Onbe for technical assistance. This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (T.I.), the Zikei Institute of Psychiatry (T.I.), the National Institutes of Health (V.M.-Y.L., J.Q.T.), as well as by grants from the Marian S. Ware Alzheimer Program (V.M.-Y.L., J.Q.T.). REFERENCES [1] Lee, V. M.-Y.; Goedert, M.; Trojanowski, J. Q. Neurodegenerative tauopathies. Annu. Rev. Neurosci. 24:1121 – 1159; 2001. [2] Foster, N. L.; Wilhelmsen, K.; Sima, A. A.; Jones, M. Z.; D’Amato, C. J.; Gilman, S. Frontotemporal dementia and parkinsonism
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AD — Alzheimer’s disease CNS — central nervous system FA — formic acid FTDP-17 — frontotemporal dementia with parkinsonism linked to chromosome 17 MAb — monoclonal antibody Tg — transgenic WT — wild type