Combined treatment of Aβ immunization with statin in a mouse model of Alzheimer's disease

Abstract

We evaluated the therapeutic efficacy of combined treatment of Aβ-immunization with simvastatin in an Alzheimer mouse model at age 22 months. DNA prime-adenovirus boost immunization induced modest anti-Aβ titers and simvastatin increased the seropositive rate. Aβ-KLH was additionally administered to boost the titers. Irrespective of simvastatin, the immunization did not decrease cerebral Aβ deposits but increased soluble Aβ and tended to exacerbate amyloid angiopathy in the hippocampus. The immunization increased cerebral invasion of leukocytes and simvastatin counteracted the increase. Thus, modest anti-Aβ titers can increase soluble Aβ and simvastatin may reduce inflammation associated with vaccination in aged Alzheimer mouse models.

Fig. 1

Simvastatin treatment and immunization schedule.

Fig. 2

Levels of buffer-soluble Aβ in the hippocampus increased in 22-month-old AD model mice subjected to Aβ vaccination. The amounts of buffer-soluble Aβ42 (A) and Aβ40 (B) in the hippocampus and neocortex were determined by Aβ42 and Aβ40 specific ELISA, respectively. The amounts of buffer soluble Aβ42 and Aβ40 in the vaccine-only and vaccine-plus-statin groups increased in the hippocampus as compared with the PBS group (*P < 0.05). The values shown are the mean ± SEM. *P < 0.05

Fig. 3

Aβ load in the brain by immunohistochemistry and ELISA. Ten months after the initial immunization, mice were euthanized at 22 months of age and Aβ deposits in the brain were visualized by anti-Aβ 6E10 antibody (A). Scale bars 500 µm. Average percentages of areas showing Aβ immunoreactivity measured by morphometry in the hippocampus and neocortex are shown (B). There is no difference between any groups in the Aβ load. The values shown are the mean ± SEM.

Fig. 4

CD45-positive cells in the brain by immunohistochemistry and immunoblot analysis. For detection of migratory leukocytes/microglia, brain sections were subjected to immunohistochemistry using anti-CD45 antibody (A). Scale bars 1 mm. Average percentages of areas showing Aβ immunoreactivity measured by morphometry in the hippocampus and neocortex are shown (B). The CD45 immunoreactive areas in both hippocampus (P < 0.01) and neocortex (P < 0.05) in the mice received vaccine only are greater than those in the PBS group, while simvastatin treatment counteracts the increase of CD45-immunoreactivity induced by vaccination in the hippocampus (P < 0.05) but not in the neocortex (P = 0.13). Levels of CD45 and GAPDH in the cerebral homogenates were determined by immunoblotting using anti-CD45 and anti-GAPDH antibodies, respectively (D). The bar graph represents densitometric quantification of CD45 after normalization with GAPDH (means ± SEM) (C). The mean of hippocampal CD45 levels in the vaccine-only group is greater than those in the PBS (P = 0.01) and vaccine-plus-statin groups (P = 0.05).

Fig. 5

Quantification of microglia and astrocytes in the hippocampus and neocortex. Brain sections were stained with anti-CD11b and anti-GFAP antibodies for detection of activated microglia and reactive astrocytes, respectively. Average percentages of areas showing immunoreactivity measured by morphometry in the hippocampus and neocortex are shown for CD11b (A) and GFAP (B). No differences in immunoreactivity were found between groups. The values shown are the mean ± SEM.

Fig. 6

Levels of cytokines in the hippocampus and neocortex. Levels of cytokines in the tissue lysates were determined by multiplex cytokine/chemokine array analysis. The bar graphs represent levels of cytokines in the hippocampus (A) and neocortex (B). Mean concentrations ± SE are expressed in picograms per milligram of brain protein. Levels of IL-2 in the vaccine-only group are lower than those in the PBS group (P < 0.01 for the hippocampus and P < 0.05 for the neocortex). Levels of IL-10 in the statin-only group are greater than those in the PBS group (P < 0.05).

Fig. 7

No correlation between the antibody titers and Aβ load. Correlation analyses were carried out between anti-Aβ antibody titers and Aβ load (6E10-immunoreactive areas) in the hippocampus (A) and neocortex (B) in the vaccine-only and vaccine-plus-statin groups. The anti-Aβ titers were not correlated with Aβ load in the hippocampus (r2 = 0.017, P = 0.834 for the vaccine-only group; r2 = 0.295, P = 0.344 for the vaccine-plus-statin group) and neocortex (r2 = 0.416, P = 0.240 for the vaccine-only group; r2 = 0.278, P = 0.351 for the vaccine-plus-statin group).

Fig. 8

Quantification of blood vessels bearing amyloid deposits in the hippocampus. Brain sections were stained with thioflavin S for detection of amyloid. Hippocampal blood vessels positive for thioflavin fluorescence were indicated by arrows (A). Scale bars 100 µm. The numbers of blood vessels positive for thioflavin fluorescence per brain section are shown (B). On average, the vaccine-only group had more thioflavin-positive blood vessels in the hippocampus than the 3 other groups but the differences are not significant. The values shown are the mean ± SEM.