PMC:1283364 / 62160-70247 JSONTXT

{"project":"2_test","denotations":[{"id":"16279840-9117892-86242806","span":{"begin":498,"end":500},"obj":"9117892"},{"id":"16279840-8938131-86242807","span":{"begin":501,"end":503},"obj":"8938131"},{"id":"T75078","span":{"begin":498,"end":500},"obj":"9117892"},{"id":"T86129","span":{"begin":501,"end":503},"obj":"8938131"}],"text":"Supporting Information\nFigure S1 Transgenic APP Expression and Suppression by Dox in the Four New Tet-Off APP Lines\nWestern blotting with human-specific antibody 6E10 reveals transgene-derived full-length APP in cortical homogenates from untreated animals (left lanes of each panel). The new lines produce exceptionally high levels of transgene expression; an equal amount of brain homogenate from a standard transgenic APP line is shown for comparison (extreme left lane, Standard Tg line C3–3; [15,63]). After 1 mo of dox treatment, transgenic protein in all four new tet-off APP lines is reduced to residual levels (+ dox; right lanes of each panel).\n(474 KB TIF).\nClick here for additional data file.\nFigure S2 Transgenic APP mRNA Is Brain-Specific\nA slot blot of mRNA harvested from various tissues in three of the four new tet-off APP lines and a nontransgenic control was used to examine transgenic mRNA expression. Hybridization is seen only in the brain; no signal above background is seen in any other tissue.\n(781 KB PSD).\nClick here for additional data file.\nFigure S3 Amyloid Pathology in the Cortex Reiterates That in the Hippocampus\nAmyloid histology was performed on sections from line 107 double transgenic mice by Hirano silver stain (top row), thioflavin-S (middle row), and Aβ immunohistochemistry (bottom row) to examine the persistence of pathology following transgene suppression. As in the hippocampus (see Figure 4 and text), the progression of amyloid pathology in the cortex worsens substantially between 6 and 9 mo of age in untreated mice. This progression is completely prevented by suppression of the transgene with dox. For comparison, normal neurohistology is shown in an age-matched single transgenic (tTA only) animal. No amyloid pathology has been detected in either APP or tTA single transgenic animals up to 15 mo of age.\n(4.8 MB PSD).\nClick here for additional data file.\nFigure S4 Diffuse Deposits Do Not Disperse During Aβ Suppression\nCampbell–Switzer silver stain was used to differentiate cored (brown) from diffuse (black) deposits in line 107 tTA/APP mice. This stain demonstrates that both types of deposit persist throughout long periods of transgene suppression. The lower panels, showing low-power images (10×) of frontal cortex from each condition, reveal little change in the extent of diffuse amyloid following up to 6 mo of Aβ suppression. High-power images (40×) in the upper panels show that the diffuse halo surrounding individual cored deposits remains relatively unchanged in treated mice. Untreated tTA single transgenic animals are shown as a negative control. Protocol for the Campbell-Switzer Alzheimer's Method was kindly shared by Robert Switzer, III (NeuroScience Associates, Knoxville, Tennessee, United States), and can be downloaded at http://www.nsalabs.com/Documents/publications/campbell-switzer_protocol.htm [64,65].\n(923 KB JPG).\nClick here for additional data file.\nFigure S5 Chronic Transgene Suppression and Arrest of Aβ Aggregate Formation in an Independent Line of Tet-Off APP Mice (CaMKIIα-tTA × tet-APPswe/ind Line 18)\n(A) The experiment presented in the text for line 107 tet-off APP was repeated with a second tet-off APP line (line 18) to control for integration site artifacts. Cortical homogenates from untreated control and dox-treated double transgenic mice were immunoblotted for full-length APP with the human-specific antibody 6E10 to confirm transgene suppression at the time of harvest. Immunostaining for endogenous superoxide dismutase (SOD1) was included as a loading control.\n(B) Quantitation of signal intensity from the Western blot in (A) shows transgenic APP levels in line 18 are suppressed by more than 98% following 3 mo of dox treatment (significant effect of group ANOVA F 2,8 = 1559.7, p \u003c 0.001). This level of suppression was equal to or better than that attained in line 107 (see Figure 3B).\n(C) Serial dilution filter trap assay was used to quantify aggregated Aβ in cortical homogenates.\n(D) Quantitation of signal intensity in the linear range of the dilution series shown in (C). Consistent with the amyloid histology shown in Figure S5, aggregate formation was significantly increased between 6 and 9 mo of age in untreated mice (significant effect of group ANOVA F 2,18 = 12.14, p \u003c 0.001). Aggregate formation was completely arrested by transgene suppression, and is identical in 9-mo-old mice treated with dox for 3 mo as in untreated animals harvested when treatment began (p \u003e 0.5, Tukey post-hoc test). *, p \u003c 0.05; **, p \u003c 0.005; ***, p \u003c 0.001 versus 9-mo-old untreated mice, Tukey post-hoc test.\n(962 KB TIF).\nClick here for additional data file.\nFigure S6 Arrest of Amyloid Progression by Chronic Transgene Suppression in Line 18 Tet-Off APP Mice\nAmyloid histology in cortical (first and third rows) and hippocampal (second and fourth rows) sections from untreated tTA/APP mice shows a dramatic progression of pathology between 6 and 9 mo of age. Suppression of transgenic APP expression arrests this progression, although without any sign of plaque clearance (6 mo + 3 mo dox). Hirano silver stain (top panels); thioflavin-S (bottom panels).\n(5.8 MB PSD).\nClick here for additional data file.\nPatient Summary\n\nBackground\nPatients with Alzheimer disease (AD) have elevated levels of a small protein called amyloid-β peptide that sticks together to form what are known as amyloid plaques in their brains. This peptide is normally made at low levels in healthy individuals, and is made when a larger protein called amyloid precursor protein (APP) is cut down in size. New treatments are now being developed that will decrease the amount of Aβ produced from APP. However, it is not clear whether lowering the production of Aβ will allow the brain to heal itself by clearing the amyloid plaques. The answer to this question may be important for deciding when Aβ-lowering drugs should be started, and may also determine how effective they are in reversing the mental symptoms of AD.\n\nWhat Did the Researchers Do and Find?\nBecause new drugs designed to lower Aβ levels are still in development, they are not available for testing in animal models of the disease. Instead, basic questions about the effectiveness of this type of treatment must be answered using systems that mimic how the drugs work. To do this, the authors created mice that produce too much APP and that develop the same amyloid lesions as do human patients with AD. Unlike normal mice, these mice also carried a “switch” gene that allowed the researchers to turn off APP by feeding the mice special food. Turning off APP in these mice had the same effect as treating them with Aβ-lowering drugs, and so the researchers were able to ask what happened to the amyloid plaques after Aβ production was shut down. They showed that lowering Aβ production prevents the amyloid lesions from getting worse as the disease progresses. This means that treatment with Aβ-lowering drugs may be able to stop the disease from filling the brain with plaques. However, the researchers also found that the amyloid lesions that had formed before treatment was started remained intact throughout the experiment.\n\nWhat Do These Findings Mean?\nThese results indicate that treatments designed to lower the production of Aβ may be an important part of future AD treatment, as this approach seems to prevents additional amyloid plaques from forming in the mouse brain. However, by itself, this strategy may not be able to rid the brain of plaques that have already formed in the brain before treatment is started. The findings suggest that early treatment may be important for this approach to succeed.\n\nWhere Can I Get More Information Online?\nMedlinePlus has several Web pages of information on Alzheimer disease:\nhttp://www.nlm.nih.gov/medlineplus/alzheimersdisease.html\nThe ADEAR Center of the US Government's National Institute on Aging also has information on Alzheimer disease:\nhttp://www.alzheimers.org/\nThe Alzheimer's Association Web site contains information on both caregiving and research:\nhttp://www.alz.org"}