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, 154 (3), 927-36

Up-regulated Production and Activation of the Complement System in Alzheimer's Disease Brain

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Up-regulated Production and Activation of the Complement System in Alzheimer's Disease Brain

K Yasojima et al. Am J Pathol.

Abstract

We used reverse transcriptase-polymerase chain reaction and Western blotting techniques to measure the levels of complement mRNAs and their protein products in Alzheimer's disease (AD) brain compared with non-AD brain. mRNAs for C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9 were detected in the 11 regions of brain that were investigated. The mRNA levels were markedly up-regulated in affected areas of AD brain. In the entorhinal cortex, hippocampus, and midtemporal gyrus, which had dense accumulations of plaques and tangles, C1q mRNA was increased 11- to 80-fold over control levels, and C9 mRNA 10- to 27-fold. These levels were substantially higher than in the livers of the same cases. Western blot analysis of AD hippocampus established the presence of all of the native complement proteins as well as their activation products C4d, C3d, and the membrane attack complex. These data indicate that high levels of complement are being produced in affected areas of AD brain, that full activation of the classical complement pathway is continuously taking place, and that this activation may be contributing significantly to AD pathology.

Figures

Figure 1.
Figure 1.
Representative Polaroid photograph showing an ethidium bromide stained gel of RT-PCR products of C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9 mRNAs. The cDNA had been obtained from a total RNA extract of AD hippocampus. Electrophoretic separation was performed on a 6% polyacrylamide gel. Size markers are in lane M, with sizes shown in base pairs. See under Methods for details.
Figure 2.
Figure 2.
Bar graphs showing relative levels of complement and cyclophilin mRNAs in extracts of AD and non-AD tissue. Optical density units represent readings of ethidium bromide gel band intensities as determined on a GDS6700 image analyzer with quantitative analysis and use of NIH Image software 1.61. Values are averages with bars indicating S.E. Bars marked with a star are those where the AD levels are significantly different from the control levels (P < 0.05) after analysis of variance tests with Holm’s stepdown correction (see under Methods for details).
Figure 3.
Figure 3.
Western blot analysis from protein extracts of AD and control hippocampus compared with normal serum and the same serum activated by aggregated IgG (see under Methods for details). Lane 1, normal serum; lane 2 , normal hippocampal extract; lane 3, AD hippocampal extract; lane 4, serum activated by IgG. Positions of molecular weight markers are shown by the arrows on the right; the estimated molecular weights of the main bands detected are shown by arrows on the left. Note that strong bands are detectable for all complement proteins in the AD extract and the activated serum sample. Detectable bands were not obtained from the control hippocampal extract for C1q, C1r, C1s, C2, C5, C6, C7, C8, C9, and the membrane attack complex. By contrast, very strong bands were obtained for the activated complement fragments C3d and C4d and the membrane attack complex in AD tissue and activated serum.
Figure 4.
Figure 4.
Immunohistochemical staining of AD transentorhinal cortex (A, C, E) and cerebellum (G, I) and control transentorhinal cortex (B, D, F) and cerebellum (H, J). Staining is for C1q (A, B, G, H), C4d (C, D, I, J) and C5b-9 (E, F ). See under Methods for details. In A, the C1q antibody stains senile plaques as well as tangled neurons very weakly. In G, the C1q antibody stains diffuse amyloid deposits. In C, the C4d antibody stains senile plaques, and in I it stains diffuse amyloid deposits. In E, the anti-C5b-9 antibody stains dystrophic neurites and tangled neurons. The control sections B, D, F, H, and J show no specific staining of brain structures. Scale bar in J = 50 μm.

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