The Hypothesis And Experiments

In our laboratory, we carried out quantitative western blot analysis of homogenates of brain tissues from ovariectomized aged primates exposed to either soy isoflavones, or conjugated equine estrogens (Premarin™). The antibodies utilized recognize epi-

topes on the microtubule-associated protein tau, that are hyperphosphorylated in Alzheimer's disease (Binder et al., 1985; Greenberg et al., 1992). It should be emphasized that the studies are ongoing, with the full report to be published in the future.

The hypothesis underlying these experiments was that dietary intake of either Premarin™ or soy isoflavones would have neuroprotective actions which might include attenuation of AD-relevant phosphorylations of tau. Given the structural similarity between genistein, the principal soy isoflavone, and 17P-estradiol, the principal physiological estrogen (shown in Figure 8.1), it was not unreasonable to predict that the soy isoflavones might mimic the biological actions of 17P-estradiol.

The results described in this chapter were obtained from analysis of primate brain tissues archived from a study that was carried out by Dr. Tom Clarkson and his colleagues at Wake Forest University in the Department of Comparative Medicine. They set up a primate model of menopause by ovariectomizing aged macaca fascic-ularis monkeys and asked whether soy isoflavones taken through the diet were as efficacious as estrogen-replacement therapy with regard to osteoporosis and cardiovascular disease (CVD) risk factors. While part of the results of this study were published (Clarkson et al., 2001), additional data analysis is ongoing.

In brief, the ovariectomized primates were segregated into three dietary groups, all based on soy protein. One group received intact soy protein, a second group received soy protein that had been extracted of 90% of the isoflavones, and a third group received Premarin™, against the same extracted soy protein. The exact diet compositions and amounts of Premarin™ are described elsewhere (Clarkson et al., 2001). After being maintained in these dietary groups for 36 months, the monkeys were sacrificed, and their brains were dissected, sliced into 6 mm sections, frozen in liquid nitrogen, and stored at -80°C until used.

Brain samples were analyzed by quantitative western blot as follows: a piece of frontal cortex was chipped off, weighed, and pulverized under liquid nitrogen. The sample was then homogenized using a Dounce homogenizer in lysis buffer (50 mM PIPES, pH 6.9; 2 mM EGTA; 1 mM MgCl2; 0.1 mM GTP, 1 tablet of Compleat™ protease inhibitor tablet [GIBCO-BRL] per 50 ml of solution) at a 1:4 g:ml ratio of tissue:buffer. This homogenate was clarified by centrifugation at 100,000 x g for 30 min. All manipulations up to this point were carried out at 4°C. The supernatant from the clarification was diluted 1:1 with 2x SDS-PAGE sample buffer (Laemmli et al., 1976) without tracking dye, and boiled at 100oC for 5 min.

After the protein concentration was determined, 30 |g of each SDS-denatured sample were loaded on a 7.5% acrylamide gel, and electrophoresed until the tracking dye reached the bottom. The gel was then transblotted onto nitrocellulose (Towbin et al., 1979) at 130 mA overnight with cooling. Afterward, the blots were blocked for 15-30 min in 5% nonfat dry milk (NFDM) in borate buffered saline (BBS) (25 mM sodium borate, 100 mM boric acid, 75 mM NaCl), after which they were incubated in appropriately diluted primary antibody in 1% NFDM in BBS overnight with agitation. Unbound primary antibody was rinsed off with three 5 min rinses in BBS, after which the blot was agitated in peroxidase-conjugated secondary antibody diluted in 1% NFDM in BBS for 1 h at room temperature. Unbound sec ondary antibody was rinsed off with three 10 min rinses in BBS. Primary antibody binding was visualized with the Lumiglo chemiluminescence detection kit (Kirkegaard and Perry, Inc.) using Kodak xomat film. The film was densitometrically scanned using a BioRad GS-250 Molecular Imager, and the differences in immunoreactivity were quantitated using the Molecular Analyst software version 2.1 (BioRad).

The initial results obtained from the quantitative western blot analysis with the antibodies indicated that the brains of the animals that ingested soy protein containing the isoflavones had tau protein whose phosphorylation at two AD-relevant sites were attenuated relative to the control group which did not receive the isoflavones. Contrary to expectations, these phosphorylations were not affected in the brains of the animals that ingested Premarin™ (Kim et al., 2000). The densitometric data of the western blots are summarized in Figure 8.3.

Additional western blots with antibodies that recognized total tau proteins and total tubulin, the microtubule subunit, indicated that the amount of microtubule proteins was the same in all samples (data not shown). Thus, the differences in tau phosphorylations detected were normalized against unchanging amounts of tau and tubulin.

soy-

Figure 8.3 Quantitative assessment of tau phosphorylations modulated by soy versus Premarin™. Monkey brain samples were processed for SDS-PAGE and western blot as described in the text. The gel lanes were loaded with equivalent amounts of total protein (30 |J,g). After blocking, the blot was incubated overnight with PHF-1 antibody. (Courtesy of Dr. Sharon Greenberg, Albert Einstein College of Medicine of Yeshiva University.) Binding of primary antibody was detected with horse peroxidase-conjugated secondary antibody, and the Lumiglo chemiluminescent detection system (Kirkegaard and Perry, Inc.). After visualization, the x-ray film was scanned and the immunoreactivities for the different samples was quantitated by densitometry using Molecular Dynamics software. The graph was generated by scanning three lanes for each of the three dietary groups and averaging the intensities among the three samples in each group. Lane 1: brain homogenate from group 1 that ingested soy protein that had been extracted from the isoflavones (SOY-); lane 2: brain homogenate from group 2 that ingested soy protein that had not been extracted (SOY+); lane 3: brain homogenate from group 3 that ingested Premarin™ (conjugated equine estrogens, or CEE) added to the same soy protein that group 1 ingested. The error bars reflect the standard error of the mean.

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